Mutations in the apically located kidney Na-K-2Cl cotransporter NKCC2 cause type I Bartter syndrome, a life-threatening kidney disorder. We previously showed that transport from the ER represents the limiting phase in NKCC2 journey to the cell surface. Yet very little is known about the ER quality control components specific to NKCC2 and its disease-causing mutants. Here, we report the identification of Golgi alpha1, 2-mannosidase IA (ManIA) as a novel binding partner of the immature form of NKCC2. ManIA interaction with NKCC2 takes place mainly at the cis-Golgi network. ManIA coexpression decreased total NKCC2 protein abundance whereas ManIA knock-down produced the opposite effect. Importantly, ManIA coexpression had a more profound effect on NKCC2 folding mutants. Cycloheximide chase assay showed that in cells overexpressing ManIA, NKCC2 stability and maturation are heavily hampered. Deleting the cytoplasmic region of ManIA attenuated its interaction with NKCC2 and inhibited its effect on the maturation of the cotransporter. ManIA-induced reductions in NKCC2 expression were offset by the proteasome inhibitor MG132. Likewise, kifunensine treatment greatly reduced ManIA effect, strongly suggesting that mannose trimming is involved in the enhanced ERAD of the cotransporter. Moreover, depriving ManIA of its catalytic domain fully abolished its effect on NKCC2. In summary, our data demonstrate the presence of a ManIA-mediated ERAD pathway in renal cells promoting retention and degradation of misfolded NKCC2 proteins. They suggest a model whereby Golgi ManIA contributes to ERAD of NKCC2, by promoting the retention, recycling, and ERAD of misfolded proteins that initially escape protein quality control surveillance within the ER. The sodium balance and its regulation by the kidney, by regulating the extra-cellular volume, is a key determinant of long-term blood pressure (BP) control, as illustrated by rare monogenic syndromes affecting renal salt handling and considerably altering BP ,2. Indee3Man9GlcNAc2) to a nascent protein [N-glycans are then further trimmed by specific mannosidases such as Golgi α1,2-mannosidases in the cis-Golgi [N-glycans within the medial- and trans-Golgi compartments [N-glycosylated proteins are targeted to their final destination. When the folding process fails, the terminal mannose residues from the core glycan structure are progressively trimmed, and defective proteins are translocated across the membrane for cytosolic proteasome degradation through mechanisms known as ERAD . Theradation ,37. Neveradation . Both wiradation ,40. Underadation ,40. Evenradation ,40. Simiradation , ROMK [4radation  and NCC radation , we haveradation ,45,46. Eradation . The typradation ,47. Consradation ,47,48. Fradation . Likewisradation ,50. Simiradation ,52,53. Aradation ,52. Moreradation ,54,55,56radation ,58,59,60Yeast two-hybrid (Y2H) screening was performed as described previously in detail , using aN-terminus region (ManIA-ΔNter) was generated also from WT MAnIA-V5 plasmid by QuikChange site-directed mutagenesis method  using forward primer 5′ GAGGGCAAAGATCAAAGAGTAGATGACCCATGCTTGGAAT 3′ and reverse primer 5′ ATTCCAAGCATGGGTCATCTACTCTTTGATCTTTGCCCTC 3′. All mutations and truncations were confirmed by sequencing.Generation of WT Myc-NKCC2, unglycosylated Myc-NKCC2 (N442Q/N452Q) and WT EGFP-NKCC2 was previously described 2Q and WT,51. The 2. Human embryonic kidney (HEK) 293 cells were grown in DMEM media complemented with 10% fetal bovine serum and 1% penicillin/streptomycin. For plasmid DNA transfection, cells were grown to 60–70% confluence before being transiently transfected for 5 h using Lipofectamine plus kit according to manufacturer’s instructions . For protein degradation experiments, cells were treated with MG132 (2 μM) or chloroquine (100 μM) 6 h prior to cell lysis, as previously described [Opossum kidney cells (OKP cells) were maintained in DMEM (Gibco 42430) supplemented with 10% fetal bovine serum , penicillin (100 U/mL), and streptomycin (100 U/mL) at 37 °C in a humidified atmosphere containing 5% COescribed ,61. Kifuv/v) Triton X-100 and protease inhibitors . Samples were then harvested and centrifuged at 16,000 rpm for 15 min at 4 °C. For immunoprecipitation, cells were solubilized with lysis buffer containing 0.4 M NaCl; 1.5 mM MgCl2; 10 mM Hepes, pH 7.9; 5% (v/v) glycerol; 0.5% (v/v) Nonidet P-40) and protease inhibitors . Immunoprecipitation was carried out using anti-V5 (Invitrogen) or anti-ManIA (Sigma ) antibody, and affinity purification using protein G-agarose beads . After incubation with protein G-agarose beads for 1 h at room temperature, the immunocomplex was washed in PBS (Invitrogen). The protein samples were then boiled in loading buffer, run on gradient 7.5% or 10% or 15% SDS-polyacrylamide gels, probed with primary antibodies of interest and horseradish peroxidase-conjugated secondary antibody. Proteins were visualized by enhanced chemiluminescence detection  according to the manufacturer′s instructions.After transfection, cells were washed with cold PBS before being solubilized in a lysis buffer containing 120 mM Tris/Hepes, pH 7,4; 150 mM NaCl, 5 mM EDTA, 3 mM KCl; 1% . Cells were then fixed with 2% paraformaldehyde in PBS for 20 min at room temperature before being incubated with 50 mM NH4Cl, permeabilized with 0.1% Triton X-100 for 1 min. To block no specific antibody binding, cells were incubated with DAKO (antibody diluent with background-reducing components) for 30 min. Fixed cells were incubated for 1h at room temperature with the primary antibody of interest. The primary antibodies used in this study are the following: mouse anti-V5 , mouse anti–Myc , rat anti-V5 , rabbit anti-Calnexin (Abcam), rabbit anti-Giantin (Abcam), rabbit anti-GM130 (Abcam), rabbit anti-ManIA (Abcam), The secondary antibodies used are the following: goat anti-rabbit Alexa Fluor 555 (Invitrogen), goat anti-rabbit Alexa Fluor 488 (Invitrogen), goat anti-rabbit FITC , goat anti-mouse Texas red (Invitrogen), goat anti-rat Alexa Fluor 555 (Invitrogen), goat anti-rat Alexa Fluor 488 (Invitrogen), Alexa Texas Red conjugated anti-mouse , chicken anti-mouse Alexa Fluor 647 (Invitrogen). After incubations with primary and secondary antibodies of interest, cells were then washed with PBS and mounted with Vectashield.Twenty-four–forty-eight h post-transfection, confluent cells were washed with PBSTo monitor the stability and maturation of NKCC2, cycloheximide was added at a concentration of 100 μM to OKP or HEK cells 14−16 h post-transfection with NKCC2 plasmids. For the chase period, cell lysates were collected at 0, 1, 2, and 4 h after cycloheximide treatment and analyzed by immunoblotting.ManIA siRNAs were purchased from Dharmacon as ON-TARGET plus SMART pools (L-012174-00-0005). HEK cells were first transfected with control or specific ManIA siRNAs with Lipofectamine RNAiMAX (Invitrogen) using the manufacturer′s specifications as performed previously ,53. One p < 0.05 was considered statistically significant.Data are expressed as mean ± SE. Differences between means were evaluated using paired or unpaired t test or ANOVA as appropriate. lane 3, NKCC2 protein was detected in ManIA immunoprecipitates witnessing interaction of the proteins. Importantly, the interaction engages only the immature and core-glycosylated forms of the cotransporter. Moreover, the association appears to be specific since Myc-NKCC2 protein was not recovered in control experiments (lane 2) in which cells were transfected in the absence of ManIA-V5.As previously described , to ideneriments B, lane 2lane 3). Of note, Myc-NKCC2 protein was not detected in control experiments in which immunoprecipitations were carried out using mouse anti-V5 antibodies indicating, again, that the interaction is specific (lane 2). Taken in concert, these findings clearly indicate that, similar to exogenously expressed ManIA, endogenous ManIA binds in vivo to NKCC2, an interaction that implicates only the immature form of the cotransporter.To discount the possibility that the interaction described above is simply due to aggregation and nonspecific binding of proteins because of heterologous expression systems, we next tested whether endogenous ManIA protein and NKCC2 interact in HEK cells. To address this question, we used a rabbit ManIA antibody raised against native ManIA protein. Importantly, western blot analysis, following immunoprecipitation with anti-ManIA antibody, detected a protein band around 70 kDa, corresponding to the expected size band of ManIA protein. Most importantly, immunoprecipitation of endogenous ManIA protein brought down only the immature form NKCC2, indicating therefore physical interaction between ManIA and the immature form of the cotransporter C, lane 3specific C, lane 2The association of ManIA with only the immature form of NKCC2 strongly suggests that the two proteins may interact at the ER. However, given that the N-glycan of glycoproteins at the entrance of the Golgi apparatus is still of high mannose type, it is also conceivable that NKCC2 interaction with ManIA may occur at the cis-Golgi network. To check whether ManIA interaction with NKCC2 takes place at the ER and/or the cis-Golgi, we visualized the subcellular distribution of NKCC2 and ManIA by immunofluorescence microscopy in renal cultured cells. To that end, HEK cells were first co-transfected with EGFP-NKCC2 and ManIA-V5 proteins. Of note, we have previously shown that, similar to Myc, N-terminal tagging of NKCC2 with EGFP does not alter intracellular trafficking of the cotransporter ,51. As sTo determine the functional implications of ManIA-NKCC2 interaction, we first tested the impact of ManIA knock-down on total NKCC2 protein abundance using small interference RNA (siRNA). To this end, a sequential transfection in HEK cells was performed. Cells were first transfected with siRNAs for at least 24 h, before transfection with Myc-NKCC2 plasmids. As can be seen in The interaction of ManIA only with the immature form of NKCC2 and the downregulation of the latter are likely due to changes in NKCC2 protein stability and maturation. To test this hypothesis, we analyzed the effect of ManIA on the stability and maturation of NKCC2 using cycloheximide assay. Towards that end, 14 to 16 h after transfection of HEK cells with NKCC2 singly or in combination with ManIA, cells were incubated with cycloheximide for 0, 1, 2, and 4 h to block protein synthesis and immunoblotting was used to detect changes in NKCC2 protein maturation and degradation rate. During the chase period, under control conditions, the immature form of NKCC2 was progressively converted to a more slowly migrating band, representing the mature form. As shown in lane 3). To verify whether, similar to HEK cells, the site of interaction between NKCC2 and ManIA is also the cis-Golgi network, the subcellular localization of the proteins was analyzed by confocal microscopy. As illustrated in Because protein subcellular distributions, ER quality control mechanism and protein–protein interaction phenomena may depend on the expression system used, we sought to confirm our observations by conducting the experiments in another renal cell line, OKP cells. As shown in To study the effect of ManIA on NKCC2 biogenesis in OKP cells, we checked again the effect of ManIA co-expression on the cotransporter protein levels. As show in To further elucidate the consequence of ManIA effect on NKCC2 maturation, the subcellular localization of NKCC2 proteins in the presence and absence of ManIA co-expression was compared with the distribution of an ER marker, calnexin. In full agreement with our previous reports ,46,52,61The experiments described above strongly suggest that enhanced expression of ManIA promotes ER retention and degradation of misfolded NKCC2 proteins. This mechanism implicates, in general, activation of the proteasome pathway and/or the lysosomal machinery ,63,64,65N-linked glycosylation [NKCC2 is N-glycosylated at two sites, N442 and N452, located in the long extracellular loop . Consequsylation  and givesylation ,66,67, wsylation ,66,67,68unglycosylated NKCC2. As can be seen in To further study the role of the N-Glycan in ManIA-induced down-regulation of NKCC2, we next studied the role of N-glycosylation of NKCC2 on its interaction with ManIA. To this end, we assessed the effect of mutating the two predicted glycosylation sites, N442 and N452 of NKCC2, to glutamine (Q), on ManIA interaction with the cotransporter. As expected, elimination of the two sites of N-glycosylation; (N442Q/N452Q) resulted in the complete loss of NKCC2 glycosylation with a lower band around 130 kDa remaining B, corresN-terminus on the cytoplasmic side and the C-terminus on the luminal side [N-terminus and cytoplasmic side of this Golgi mannosidase. To verify this hypothesis, we studied the influence of deleting the N-terminus of ManIA on its interaction with NKCC2 using co-immunoprecipitation assay. To this end, cells were co-transfected with NKCC2 with full length ManIA (WT ManIA) or ManIA devoid of its N-terminus and cytoplasmic region (ManIA-ΔNter) (N-terminus (lanes 3 and 5 vs. lanes 4 and 6). Hence, these results strongly suggest that the cytoplasmic tail of ManIA is involved in its interaction with the cotransporter in HEK cells.Mammalian Golgi 1,2-mannosidase IA is a type II transmembrane protein with an nal side . As mentA-ΔNter) A. Cell lterminus B. Indeedlanes 3 and 5 vs. lanes 4 and 6). Most importantly, overexpressing ManIA-ΔNter reversed the ManIA effect on the mature form of NKCC2  or Man5GlcNAc2 (M5), contributes also to triggering the degradation of misfolded glycoproteins [IB, and IC) were shown to enhance the degradation and mannose trimming of an ERAD-L substrate, NHK α1-antitrypsin, when overexpressed in cultured cells [It is now clearly established that mannose trimming by class I α1,2-mannosidases is involved in the recognition stage for the ERAD of glycoproteins since this process is greatly reduced by their inhibitors 1-deoxymannojirimycin and kifunensine ,71,72,73proteins ,75,76,77proteins ,79,80. Oproteins ,82. Anotproteins ,54. Thised cells . In the We previously provided evidence that ERAD and export from the ER constitute the major regulator of NKCC2 maturation and cell surface expression ,44,45,46The accumulation of misfolded and aggregation-prone polypeptides is harmful to cellular health ,91,92. IExhaustive exploration of the mechanisms underlying the ERAD machinery has furnished several new insights into how ERAD contributes to human health during both normal and diseased states ,92. The In summary, we found that Golgi ManIA interacts with the immature form of NKCC2 at the cis-Golgi to promote its ER retention and to accelerate its ERAD by the proteasome pathway. To the best of our knowledge, this is the first study describing a major role of the Golgi quality control mechanism in NKCC2 biogenesis. Moreover, this also is the first report providing evidence that besides luminal proteins, ManIA can be also involved in the ERAD of transmembrane proteins. Our data strongly suggest that ManIA maneuvers within a cis-Golgi-localized quality control checkpoint to serve as a backup system to ERAD surveillance in the ER, providing therefore a powerful additional network for adequate removal of unwanted misfolded NKCC2 proteins protecting therefore cells from proteotoxicity caused by the formation of protein aggregates, in particular during Bartter syndrome disease. Undeniably, ManIA cannot work in isolation to mediate the ERAD of the cotransporter. Appropriately, one may plausibly postulate that ManIA works in concert, sequentially or simultaneously, with other NKCC2 binding-proteins and ERAD components, such as OS9  and STCH


Immune-mediated cytopenias (AIC) are challenging complications following allogeneic hematopoietic stem cell transplantation (HSCT). While broad-acting immunosuppressive agents like corticosteroids are often standard of care, several novel therapies which target specific immunological pathways have recently been developed and provide hope for patients with steroid-refractory courses and may limit long-term toxicity. The successful off-label use of the plasma cell depleting anti-CD38 antibody daratumumab was published in several case reports, suggesting efficacy, i.e., in patients with antibody-mediated AIC refractory to previous B cell depletion. We want to share our experience with two children, whom we treated with daratumumab, including one fatal course with uncontrolled disease. Given the absence of substantial data from HSCT registries or prospective trials, we furthermore provide a critical review of the literature on daratumumab treatment of AIC.Patient 1 (P1), an 11-year-old girl with lipopolysaccharide-responsive and beige-like anchor protein (LRBA) deficiency who developed immune-mediated thrombocytopenia (AIT) from day +58 after HSCT, showed a complete response to daratumumab after the fourth of six total daratumumab doses. She remains transfusion independent for over a year of follow-up. Previously, her thrombocytopenia was refractory to corticosteroids, rituximab, intravenous immunoglobulins (IVIG), eltrombopag, cyclosporine A, and sirolimus. Patient 2 (P2), a 6-year-old boy with CD40 ligand (CD40L) deficiency, developed both AIT and hemolytic anemia (AIHA) after HSCT on days +58 and +83, respectively, and was also treated with daratumumab after being previously refractory to prednisolone, rituximab, and IVIG. Yet, he did neither respond to daratumumab nor the concomitantly administered methyprednisolone pulse, plasmapheresis, and eculizumab and succumbed due to refractory disease.Reviewing the literature on the use of daratumumab for refractory AIC post-HSCT, we consider daratumumab a promising agent for this life-threatening disorder: ten of the twelve patients reached transfusion independency in the literature. However, treatment failures are likely to be underreported. Thus, controlled trials are needed to explore the safety and efficacy of daratumumab in this rare post-HSCT complication. Patient 1 is an 11-year-old girl from non-consanguineous Caucasian parents with lipopolysaccharide-responsive and beige-like anchor protein (LRBA) deficiency who underwent an allogeneic HSCT for the treatment of refractory immune dysregulation.LRBA deficiency is a rare primary immunodeficiency characterized by the combination of a pathological susceptibility to infection and autoimmune phenomena including inflammatory bowel disease, diabetes mellitus, arthritis, autoimmune cytopenia, and interstitial lung disease. LRBA is an important regulator of the cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), which plays a major role in the modulation of immune responses of T cells and other immune cells . PatientP1 developed various clinical problems in the context of LRBA deficiency. The leading clinical problem had been infant-onset autoimmune enteropathy, which resulted in failure to thrive and necessity of long-term partially and finally complete parenteral nutrition and additional tube feeding since infancy. Insulin-dependent diabetes mellitus with repeated crisis-like derailments was first diagnosed at the age of 1.5 years. Since the age of 4, P1 presented with oligoarthritis of the lower extremities. In contrast to her deceased LRBA-deficient brother, she had no history of autoimmune cytopenia before HSCT was performed. Moreover, no pathologic susceptibility to infections had been noted before HSCT, and B cell differentiation was normal without the need for immunoglobulin substitution. Because of her multiple autoimmune manifestations, P1 received long-term immunosuppressive therapy with azathioprine (since 1.5 year of age), sirolimus (since 3 years of age), intermittent systemic steroid therapies during autoimmune flare-ups, abatacept , hydroxychloroquine since the age of 8 years, and intermittent anti-inflammatory therapy with ibuprofen for knee joint arthritis.The indication for allogeneic HSCT was based on the insufficient control of autoimmunity despite multimodal immunosuppressive therapy, persistent enteropathy, and failure to thrive despite parenteral nutrition and the expected aggravating natural course of the disease.2), fludarabine (4 × 40mg/m2), and anti-thymocyte globulin (3 × 15 mg/kg), the patient was grafted with bone marrow from a 10/10 HLA-matched unrelated female donor. She received 7.32 × 108 nucleated cells per kilogram of body weight, changing blood type from B rhesus positive to O rhesus positive. She experienced early complete engraftment with a leukocyte, neutrophil, and platelet engraftment on days +18, +20, and +24, respectively. Following graft-versus-host disease (GvHD) prophylaxis with methotrexate and cyclosporine A, we observed no signs of GvHD. Besides transient grade 2 mucositis, the girl had an uneventful post-transplant course. The chimerisms on days +29 and +53 in the peripheral blood showed full donor origin.Following a myeloablative preparative regimen consisting of thiotepa (1 × 8mg/kg), treosulfan  [In the first weeks of thrombocytopenia, the only bleeding signs were skin hematomas, petechia, and mild oral mucosal bleeding. Gradually, P1 started experiencing recurrent episodes of Hb-relevant bleeding , requiring multiple packed red blood cell (PRBC) transfusions. Overall, since the diagnosis of the immune-mediated thrombocytopenia, P1 received 163 platelet and 18 PRBC transfusions.2) and eltrombopag 50 to 75 mg daily, all without significant impact on her platelet count. Cyclosporine A (trough level 150–200 ng/ml), primarily administered for GvHD prophylaxis, was switched to sirolimus (trough levels 8–12 ng/ml), also without any significant response.We initiated treatment with high-dose corticosteroids  and 4 doses of intravenous immunoglobulin 1 g/kg) from day +61 onward .P2 was diagnosed with CD40L deficiency at the age of 4.5 years. He was hemizygous for a pathogenic mutation  in the At age 1 he presented with a pronounced eyelid phlegmon for which he was hospitalized. Subsequently, he showed recurrent bilateral episodes of perforating otitis media. In the 3rd and 4th year of life, radiologically confirmed pneumonia with protracted recovery occurred. At the age of 3 years, P2 experienced an onset of transient diarrhea episodes, for which no infectious triggers could be identified and was therefore judged an immune dysregulatory manifestation of his disease. From age 5 onward, P2 received prophylactic treatment by intravenous immunoglobulin substitution and cotrimoxazole.8 nucleated cells per kilogram of body weight) from a 9/10 HLA-matched unrelated male donor, following the same conditioning regimen and GvHD prophylaxis as P1. He changed blood type from O rhesus negative to O rhesus positive. During the uneventful post-transplant course, besides grade 2 mucositis, there was timely hematologic recovery with leukocyte and neutrophil engraftment on day +22 and platelet engraftment on day +20. We observed no signs of acute GvHD. The chimerisms on days +30, +43, +62, +100, and +149 in the peripheral blood and on day +149 in the bone marrow aspirate showed full donor origin.P2 underwent HSCT at the age of 6 years and was grafted with bone marrow , and continued GvHD prophylaxis with cyclosporine A. This regimen initially resulted in a transient recovery of P2’s platelet count . Further immunological studies revealed the presence of anti-E, heat, and cold autoantibodies as well as free platelet-reactive antibodies, in P2’s serum, providing immunological correlates of AIHA and AIT.During the following prednisolone tapering, the patient’s AIT relapsed on day +83 with a dropping platelet count to 9 × 102/dose, a total of weekly 6 doses) starting day +87. Due to severe thrombocytopenia with platelet counts of 2–9 × 103/μl, our patient received daily single-donor apheresis platelets.Since steroid treatment was refractory, we administered high-dose IVIG (1 g/kg/dose) in combination with rituximab  on day +184 with the rationale to deplete antibody-producing plasma cells after an unavailing B cell depletion. Upon the first administration of daratumumab, P2 developed transient and uncomplicated bronchial obstruction and vomiting. The following doses on days +192 und +200 were tolerated well.On day +190, P2 experienced another acute flare of autoimmune hemolyis. Given the rapid dynamics of the AIHA, we initiated additional antibody reduction by plasmapheresis, which was performed on days +190 to +192 and +195 to +199. The patient required daily PRBC transfusions.Despite the high frequency of plasmapheresis, the elimination of RBC antibodies was not sufficient enough and starting from day +199 P2 developed signs of a hypovolemic shock and aggravated hemolysis with Hb levels <3 g/dl. He received a salvage therapy by methylprednisolone pulses (20 mg/kg/day for 3 days), three doses of IVIG (1 g/kg/day), and one dose of eculizumab (25 mg/kg). Without any response to this therapy, P2 deceased from cardiac arrest due to acute heart failure most likely related to pulmonary embolism.To our knowledge, eight previous reports presented data on ten patients –12. A sen=2), myelodysplastic syndrome (n=3), severe aplastic anemia (n=2), primary myelofibrosis (n=1), or a primary immunodeficiency disease (n=4). Six patients received peripheral blood stem cells (PBSC), three whole bone marrow, and in three cases the stem cell source was not reported. Five patients were transplanted from a 10/10 and three from a 9/10 unrelated donor. Three patients were transplanted from a family donor. The four patients with PRCA and one patient each with AIT and ES received an ABO mismatch graft. Chimerism was 100% donor in 6 and >90% in the other six patients. Excluding patients with PRCA, the onset of AIC was at a median of day +127 (range +58 to +359), and the first dose of daratumumab was administered at a median of 189 days (range 63–503 days) after AIC diagnosis. In patients with PRCA, the first daratumumab was given at days +206, +390, and +411, and at 2.5 years after HSCT. In all cases, daratumumab was administered once weekly at a dose of 16mg/kg. The median number of infusions was 6 (range 2–11). Patients had previously received two to nine different therapies, most commonly corticosteroids, rituximab, and bortezomib , three with AIHA, two with AIT, and three with Evans syndrome (ES). Five patients were children  and seven adults . The primary disease leading to HSCT was acute leukemia (AIC is a challenging complication following allogeneic HSCT, mostly occurring in patients with a non-malignant primary disease , 14. OthBesides the fusion protein abatacept blocking T cell activation via CTLA-4, and the anti-C5 antibody eculizumab preventing terminal complement activation, daratumumab, an anti-CD38 antibody targeting plasma cells, crystalizes out as a promising treatment option  for refrThe data presented demonstrate that daratumumab treatment of refractory post-HSCT AIC can be effective and life-saving. Yet, single-case reports of variable detail have a low evidence level and it is likely that treatment failures are underreported . Long-te


This review summarizes the recent advances in understanding the contribution of SOCE-mediated intracellular Ca2+ signaling to gastroesophageal cancers. It assesses the pathophysiological role of each component in SOCE machinery, such as Orais and STIMs in the cancer cell proliferation, migration, and invasion as well as stemness maintenance. Lastly, it discusses efforts towards development of more specific and potent SOCE inhibitors, which may be a new set of chemotherapeutic drugs appearing at the horizon, to provide either targeted therapy or adjuvant treatment to overcome drug resistance for gastroesophageal cancers.Gastroesophageal cancers, including tumors occurring in esophagus and stomach, usually have poor prognosis and lack effective chemotherapeutic drugs for treatment. The association between dysregulated store-operated calcium entry (SOCE), a key intracellular Ca Gastroesophageal (GE) cancers are malignancies that occur in upper gastrointestinal track, including esophageal, gastric and gastroesophageal junction, and are usually presented with poor prognosis . GastricH. pylori infection in old ages  family of receptor tyrosine kinases (RTKs) have been approved as a successful approach for lung, breast and other cancers. EGFR/RTK has four members including EGFR (HER1), HER2, HER3, and HER4. EGFR is a 170 kDa transmembrane receptor on cell membrane. Upon activation, EGFR triggers activation of MAPK, STAT5, and Ras-Raf-MEK pathways resulting in a cascade signaling of cell proliferation and survival . As a maInhibiting EGFR/RTKs pathways can be achieved either by monoclonal antibody (mAb) or downstream by tyrosine kinase inhibitor (TKI). Several mAbs and more than 20 TKIs have been approved by FDA. For example, cetuximab and panitumumab specifically bind EGFR and trastuzumab and pertuzumab target HER2. Afatinib is a pan-HER family TKI, but selectively and irreversibly inhibits EGFR, HER2, and HER4, and blocks transphosphorylation of HER3. While many of these mAbs and TKIs have been approved by FDA for treatment of lung, colon, breast, or head and neck cancers, their therapeutic benefits for EC are still unclear . For exa2+ entry (SOCE), which is a key intracellular Ca2+ signaling pathway in GE cancers. We will summarize the recent advances in understanding pathophysiological role of each component of the SOCE machinery in GE cancer cell proliferation, migration, and invasion, as well as stemness maintenance. Lastly, we will discuss efforts towards development of more specific and potent SOCE inhibitors, which appear to be a new set of chemotherapeutic drugs appearing at the horizon to provide either targeted therapy or adjuvant treatment to overcome drug resistant in GE cancers.The failure in all above mentioned Phase III trials for GE cancer patients suggests that new targeted therapies, either alone or combined with inhibiting EGFR/RTK, should be considered. In this review, we intent to discuss an emerging new drug target, known as the store-operated Ca2+ is a vital second messenger in the cells and controls multiple cellular processes including cell growth, cell migration, cell death, cell cycle, autophagy and downstream genes transcription  receptor or ryanodine receptor (RyR) mediates Ca2+ stores such as endoplasmic/sarcoplasmic reticulum (ER/SR) (2+-ATPase (SERCA) pumps Ca2+ from cytosol back to ER/SR. Plasma membrane (PM) Ca2+-ATPase drives Ca2+ from cytosol to extracellular space (2+ uniporter (MCU) controls mitochondrial Ca2+ uptakes. Fourthly, PM Ca2+ channels or transporters mediate Ca2+ influx from extracellular space into cytosol, which include voltage-gated Ca2+channel, transient receptor potential channel (TRP), SOCE channel, Na+/Ca2+ exchanger (NCX) and purinergic receptor, etc. Since these Ca2+ channels or transporters contain extracellular domains as good drug targets, they received great attention for chemotherapy researchers. Among them, SOCE channel has been actively investigated.Cacription . Thus, itastasis . The int+ spikes , waves a+ spikes . They ar (ER/SR) . Secondlar space . Thirdly2+ entry (CCE)  current. The identification of SOCE machinery has been a long journey and hitherto, a consensus view is that SOCE machinery is complex with various tissue and cell specific components.SOCE was first reported about three decades ago in the name of capacitative Cary (CCE) . In this2+ store . This ER2+ content  and Orai  molecules . STIM1 a content . Orai1 l content . Upon ERl region . Then, tsol [39] . Both Ord repair . Our stud repair  and eotad repair .2+ permeable channels are also involved in the SOCE pathway. TRPC channels are tetrameric molecules with six transmembrane domains on each subunit and located on PM. In human cells, there are 6 TRPC channels divided into 2 families according to their biological functions: 1) TRPC1, TRPC4, TRPC5; 2) TRPC3, TRPC6, TRPC7  family of Ca6, TRPC7 . Their a6, TRPC7 . TRPCs a6, TRPC7 . The int6, TRPC7 . TRPCs m6, TRPC7 . While n6, TRPC7 . It has 6, TRPC7 , vascula6, TRPC7 , smooth 6, TRPC7 , and end6, TRPC7 . TRPC4-m6, TRPC7 , human a6, TRPC7 , both mo6, TRPC7 , human g6, TRPC7 , human c6, TRPC7  and huma6, TRPC7 . TRPC3 m6, TRPC7 . TRPC6-m6, TRPC7 .Alteration in SOCE has been observed in many diseases. While genetic mutations in Orai1 or STIM1 were found in immune disorders, skeletal muscle myopathy and heart hypertrophy , changes2+ oscillations and, thus, controls rampant cell proliferation in ESCC cells. Interestingly, an essential trace mineral nutrient zinc is able to inhibit Orai1-mediated SOCE in ESCC cells, which has been linked to its cancer prevention function  stimulates SOCE and results in the activation of downstream NF-κB signaling pathway. It is well known that LPS is an enriched component of the outer membrane of gram-negative bacteria H. pylori, which is a major risk factor for GC and triggers chronic inflammation responses . Additionally, its anti-cancer effects have been reported in both ESCC and pancreatic ductal adenocarcinoma .50 of BTP-2 is 17 µM as best. Our studies showed that treatment of RP4010 resulted in reduction of intracellular Ca2+ oscillations, and caused cell cycle arrest at G0/G1 phase in several cultured human ESCC cell lines. This inhibitory effect on cell proliferation is caused due to decreasing nuclear translocation of nuclear factor kappa B (NF-κB). Moreover, it dramatically inhibits tumor growth in xenograft ESCC nude mice without observable adverse side effect, evidenced by normal histology results in all vital organs. Therefore, it remains to be a promising chemotherapy drug for GE cancers. Further, mechanistic study on the exact inhibitory effect on SOCE machinery and continue clinical evaluation are warranted.Compared with other reported SOCE channel inhibitors, RP4010 is more potent in blocking SOCE in ESCC cells . In MTT 2+ overload  or is acquired after therapy. Accumulating evidence suggests that SOCE plays a significant role in drug resistance. The expression of Orai1 and STIM1 as well as SOCE are enhanced in therapy resistant ovary cancer cells . SOCE isoverload , autophaoverload , upreguloverload . Moreoveoverload .Cancer stem cells (CSC) are a subpopulation of tumor cells with capabilities of proliferating, self-renewing and differentiating. They are more resistant to chemotherapy drugs or radiation, which often leads to treatment failure and subsequent tumor recurrence  Bao et .et al showed that integrin α7 (ITGA7) is characterized as a functional CSC marker in ESCC  . While s in ESCC . The kno2+ signaling compared with non-stem cancer cells (2+ channels (VOC) are a cluster of transmembrane proteins located on the cell membrane, which have been reported to regulate cell proliferation and migration in the cancer cells  enhances tumor migration and invasion by up-regulating expression of CSC, ALDH1, and CD133 in glioma cells . Transie2+ channels in gastroesophageal CSC. Regardless the answer, it could be a promising treatment to combine metformin with SOCE inhibitors for a superior anti-cancer effect with reduced drug resistance.In order to overcome cancer reoccurring issue, many clinical trials have been conducted focusing on elimination of CSC. Mithramycin, a selective inhibitor of transcription factor Sp1, is on phase 1 clinical trial for esophageal neoplasms with the identifier number of NCT01624090. Metformin, a widely used diabetes drug, has been administrated in combination with chemotherapy in several clinical trials to treat multiple cancers, including colon cancer (NCT01440127), ovarian, fallopian tube, and primary peritoneal cancer (NCT01579812). Both drugs may not show huge effects on diminishing the tumor size, but they significantly decrease the recurrence of tumor. Currently, there is no report on whether mithramycin or metformin alter the expression of SOCE, voltage-gated or other Ca2+ plays a vital role in the EGFR signaling pathway, SOCE-mediated signaling pathway may crosstalk with EGFR pathway. We speculate that combined inhibitors for both SOCE and EGFR pathways could achieve better anti-cancer effects than single agent alone for GE cancer. While the function of SOCE in drug resistant and CSC in GE cancers remains unclear, it is reasonable to foresee that a similar association exist as that in other cancers. Compared with parental cancer cells, the drug resistance GE cancer cells and CSC may contain different SOCE components or ratio, which may present distinct SOCE channel properties. Targeting the SOCE channels, specific and potent SOCE blockers could be used as a new chemotherapy for GE cancers. Moreover, combining SOCE inhibitors with other chemotherapy drugs targeting both normal tumor cells and CSC may enhance treatment efficiency and prevent tumor re-occurrences. In the combined treatment, how to synergize the drugs and reduce the drug resistance? How to decrease the side effect of drugs and obtain a better prognosis? Further investigation is required along those lines.In this review, we summarized the recent studies on the role of SOCE in GE cancers. Apparently, SOCE plays multiple important roles in cancer cells proliferation, migration, invasion, metastasis and stemness maintenance. EGFR-targeting therapy has been used in many other cancers but with limited benefits for GE cancer patients. Since Ca


Nigella sativa extract, powder and seed oil and its main components, thymoquinone and α-hederin, have showed potent anticancer and chemosensitizing effects against various types of cancer, such as liver, colon, breast, renal, cervical, lung, ovarian, pancreatic, prostate and skin tumors, through the modulation of various molecular signaling pathways. Herein, the purpose of this review was to highlight the anticancer activity of Nigella sativa and it constitutes, focusing on different in vitro, in vivo and clinical studies and projects, in order to underline their antiproliferative, proapoptotic, cytotoxic and antimetastatic effects. Particular attention has been also given to the synergistic effect of Nigella sativa and it constitutes with chemotherapeutic drugs, and to the synthesized analogs of thymoquinone that seem to enhance the chemo-sensitizing potential. This review could be a useful step towards new research on N. sativa and cancer, to include this plant in the dietary treatments in support to conventional therapies, for the best achievement of therapeutic goals.In recent times, scientific attention has been paid to different foods and their bioactive components for the ability to inhibit the onset and progress of different types of cancer.  Cancer is one of the major threats of modern life, being the second cause of death after myocardial infarction worldwide ,2. The mNigella sativa (N. sativa) can be a valid tool for health promotion, due to its low toxicity and multiple mechanisms of action [N. sativa have antimicrobial and anti-inflammatory activity, immune stimulant properties, together with anticancer, antioxidant, hypoglycemic, spasmolytic and bronchodilator capacities [N. sativa, has shown to exert antiproliferative effects on different cancer cell lines, such as ovarian, colon, larynx, breast, leukemia, lung and osteosarcoma [Natural compounds are non-nutritive secondary plant compounds with health-promoting and disease preventive properties, mainly found in fruits, vegetables, grain, herbs, spice and other plant foods. Among them, the use of herbals in medicine is widespread and is growing dramatically because of their nourishing and synergistic actions that make them an excellent treatment choice. Among others, f action . For exapacities ,13,14,15osarcoma .N. sativa and its main compounds  is the second most common cancer in Europe and its onset is strictly correlated with several risk factors, such as high-fat diet poor in fiber, high alcohol consumption, red meat, obesity, smoking, lack of physical exercise, diabetes, older age and several genetic and epigenetic alterations. Protective effects have been found for necrosis . TQ has necrosis ,46. In anecrosis . Furthernecrosis . Moreovenecrosis .N. sativa and its main constituents  prevented oxidative DNA damage and inhibited malondialdehyde (MDA) liver contents [Regarding in vivo studies, in azoxymethane (AOM)-treated Sprague-Dawley rats, contents , while icontents ; finallycontents .N. sativa and TQ have strong antioxidant properties and have been found to exert chemopreventive effects in this kind of cancer. For example, in human hepatocellular carcinoma HepG2 cells alcoholic extracts of N. sativa decreased cell viability and increased apoptotic cell death through the improvement of the antioxidant condition [N. sativa, exerted cytotoxic effect in HepG2 cells and together with carvacrol, it protected liver cells via antioxidant activity and repression of proinflammatory cytokines, such as TNF-α and IL-1β [Hepatocellular carcinoma is the most common primary liver cancer and numerous strategies have been used for the treatment of this disease. ondition . Similarondition ,54. TQ aondition . Moreovend IL-1β .N. sativa and TQ suppressed the EGFR/ERK1/2 signaling pathway and augmented ERK and p38 phosphorylation by modulating PCNA, c-fos and Bcl-2, and protected liver from oxidative stress injury by enhancing the levels of antioxidant enzymes, and decreasing the concentrations of alkaline phosphatase, alanine trans aminase, total bilirubin and total NOx [N. sativa ethanolic extract exerted antitumor effect via reducing the serum levels of alpha-fetoprotein, TNF-α, IL-6 levels NO and iNOS activity in DENA-treated rats [Regarding in vivo studies, in diethylnitrosamine (DENA)-treated rats otal NOx ,57. Inteted rats .Finally, in N-Nitrosodiethylamine (NDEA) treated rats, TQ exerted antiproliferative activities and inhibited hepatic tumorigenesis by regulating cell cycle at the G1/S phase and modulating some specific proteins , CDK4 and Cyclin E) .The rate of mortality associated with lung cancer is high in the world. The natural source has recently received much attention for their chemotherapeutic effects against this type of cancer. In this context, in human lung carcinoma A549, NCI-H460 and NCI-H146 cells, TQ promoted apoptosis, by elevating Bax/Bcl-2 ratio, p53 and caspase 3 and 9, arrested cell cycle and suppressed cell viability, invasion and migration, by phosphorylating of ERK1/2 and suppressing cyclin D1, PCNA, MMP2 and MMP9 expression, and inhibited neoangiogenesis by decreasing proangiogenic cytokines ENA-78, Gro-alpha ,62. InteTo the best of our knowledge, there is only one in vivo study on TQ in lung cancer: in monocrotaline (MCT) treated rats, the intake of TQ significantly increased cancer apoptosis and inhibited pulmonary arterial remodeling via p38MAPK/NF-κβ signaling pathway .N. sativa oil extract induced a antiproliferative effect, by upregulating p21 expression, suppressing histone deacetylase activity, decreasing MCP-1, TNF-α, IL-1b and Cox-2 via NF-kβ modulation [Pancreatic cancer is one of the main causes of death among male cancer in the world. TQ is a potential candidate for the development of novel therapies against this cancer. For example, in human adenocarcinoma FG/COLO357 and CD18/HPAF81 cells, TQ was able to promote apoptosis, decrease motility and migration by reducing MUC4 and Bcl-xL expression via the activation of c-Jun NH2-terminal kinase and p38 pathways , while idulation . Additiodulation ,68.N. sativa seed extracts and TQ have shown to exert antitumor activities against cervical cancer. For example, in human cervical adenocarcinoma HeLa cells the extract of different solvents  mediated apoptosis via increasing p53, caspase-3, -8 and -9, and downregulating Bcl-2 and Bcl-XL expression [pression , while Tpression . Interespression ,72, whilpression . Furtherpression . Recentlpression .N. sativa and TQ exert anticancer activities also on blood tumors. For example, in immortalized line of human T lymphocytes (Jurkat cells), and in an acute lymphocyte leukemic cell line (CEMss cells) TQ played proapoptotic activity via downregulation of ubiquitin-like, containing PHD and RING finger domains, 1 (UHRF1) and cyclic nucleotide phosphodiesterase (PDE)1A, arrested cell cycle by targeting p73-dependent pathway and stimulated caspases 3 and 8, Bax, DNA fragmentation, ROS generation and reduced Bcl-2 through mitochondrial-induced apoptosis [N. sativa seed oil has shown anticancer activities by degrading α/β tubulin with the upregulation of the tumor suppressor p73 and by inducing apoptosis, but had no effect on α/β tubulin protein expression in normal human fibroblast cells [poptosis ,77. Simist cells . Moreovest cells . TQ modust cells .A recent study also suggested that TQ could be used as an epigenetic drug that induces both histone post-translational modifications and DNA methylation regulating several tumor suppressor genes (TSGs) with subsequent apoptosis. Specifically, in Jurkat cells TQ suppressed cell growth, by upregulating some TSGs, that are usually suppressed in cancer, such as DLC1, PPAR, ST7, FOXO6, TET2, CYP1B1, SALL4 and DDIT3 along with activating several proapoptotic genes, such as RASL11B, RASD1, GNG3, BAD and BIK . MoreoveIn addition, TQ derivatives, bound to terpene residues, showed more potent antiproliferative effect than the native compound through the induction of apoptosis associated with DNA laddering, the reduction of mitochondrial membrane potential and the raise in ROS in human HL-60 leukemia cells . SimilarRecent studies suggested that TQ is a potential anticancer agent in renal cancer, exerting an antiproliferative effect by (i) reducing angiogenesis, (ii) preventing interaction between HSP90 and HIF-1 α, (iii) degrading HIF-1 α protein, (iv) changing glucose, lactate, and ATP levels as a result of anaerobic metabolic disturbance, (v) decreasing mitochondrial membrane potential and consequent cytochrome c release into cytoplasm, (vi) arresting cell cycle at sub-G1, (vii) inducing ROS mediated apoptosis through the modulation of the expression of antiapoptotic proteins such as Bcl-2 and c-FLIP, or and pro-oncogenic JAK2/STAT3 pathway and (viii) preventing migration, invasion and epithelial–mesenchymal transition (EMT) via autophagy-dependent AMPK/mTOR signaling pathway, in several human renal carcinoma cells, such as Caki-1, Caki-2, A498, 786-O and ACHN cells ,85,86. AN. sativa were effective in inducing significant morphological changes in human renal adenocarcinoma ACHN cells but not in normal renal epithelial GP-293 cells, stimulating apoptosis and decreasing cell viability in a time and dose dependent manner [Regarding bladder cancer, the total and fraction extracts of t manner . Similart manner ,90,91; it manner .To the best of our knowledge, two in vivo studies were performed on mice: the first highlighted the anticarcinogenic mechanism of TQ in tumor xenograft nude mice by blocking EMT through the downregulation of N-cadherin, β-catenin and vimentin proteins and suppressed lung metastasis , the latThere are three main types of skin cancer: squamous cell carcinoma, basal cell carcinoma and melanoma, whose epidemiological characteristics, clinical classifications and treatment methods are different . Since sThe most common cause of death from gynecologic malignancies is ovarian cancer since many women with epithelial ovarian cancers are diagnosed with advanced, metastatic disease characterized by carcinomatosis and abdominal ascites. In the last decade, TQ has showed an antiproliferative effect in in vitro and in vivo models against ovarian cancer. In murine ID8-NGL ovarian cancer cells, treatment with TQ decreased cell proliferation with the induction of apoptosis by increasing p-p65 and reducing Ki67, PCNA and break downing PARP; it also suppressed the NF-κβ pathway and the expression of TNF-α and IL-1β . MoreoveRegarding in vivo studies, TQ has been shown to suppress tumor cell growth in a mouse model of ovarian cancer cells by inhibiting NF-κβ activation , while iProstate cancer is the most common cancer in men and the range of clinical behavior varies from a microscopic, well-differentiated tumor to an aggressive, invasive cancer, which finally results in metastases. TQ is a potential candidate that regulates several major signaling pathways and crucial oncogenic molecules that play a central role in prostate cancer initiation, progression, invasion, metastasis and angiogenesis stage. For example, it turned EMT by raising E-cadherin expression and reducing vimentin and Slug expression, via downregulation of the TGF-β/Smad2/3 signaling pathway, and promoted apoptosis by modulating PI3K-AKT pathway in human prostate carcinoma DU145 and PC3 cells ,99. In hN. sativa and TQ in other types of cancer. For example, TQ decreased chemoresistance and angiogenesis through blocking NF-κβ activation, DNA-binding activity, survivin and VEGF and increased the expression of cleaved caspase-3 and Smac in human osteosarcoma cells SaOS-2121; these results were confirmed in an in vivo study on male athymic BALB/c nu/nu mice treated with TQ, where it was able to prevent tumor angiogenesis and inhibit osteosarcoma growth through decreasing the expression of inhibitor of apoptosis proteins, VEGF, prosurvival molecules, such as survivin and XIAP, and proliferation markers, including CD34 and Ki-67 [N. sativa has shown chemopreventive potential in fibrous histiosarcoma by decreasing malignant tumor sizes, incidences and multiplicities in carcinogens-treated rats [Many in vitro and in vivo studies have also confirmed the anticancer activities of nd Ki-67 . Moreovend Ki-67 . Moreoveted rats .It has been reported that TQ showed anticancer activity also in oral cancer, by downregulating p38β MAPK in oral T28 cancer cells, by increasing the expression of the proapoptotic proteins Bad and Bid and activating p53 and caspase 3 cleavage . It was N. sativa seed oil has shown anticancer activities also in brain U87 cancer cells through the degradation of α/β tubulin with the upregulation of the tumor suppressor p73 gene and induction of apoptosis [poptosis . In addipoptosis .Finally, TQ acted as a potent agent in the treatment of head and neck cancer by exerting antiproliferative and radio sensitizing properties through the decrease of colony formation and the induction of apoptosis in tongue carcinoma SCC25, CAL27 and HNSCC cells .N. sativa and its main components in different types of cancers.A wide variety of chemotherapeutic drugs have been developed for the treatment of different malignancies since the 1940s. Despite the advent of numerous highly efficient cytotoxic agents, the overall rate of cancer-related death is barely reduced during last 70 years. Several major drawbacks have been identified in anticancer chemotherapy, such as non-specific cytotoxicity, which results in bone marrow suppression and other organ toxicity and the development of drug resistance. Indeed, in aggressive malignant neoplasm, a highly variable sensitivity to therapeutics can be observed and some of cell lines can develop resistance to the treatment; the combined effects of bioactive compounds and conventional therapy should be higher compared to a single compound, slowing the development of resistance .An increased drug efflux, alteration of the molecular targets, increased repair of drug-induced DNA damage, activation/suppression of signaling pathways lead to an upregulation of survival molecules and avoidance of apoptosis, the most prominent reasons of tumor cell challenge against chemotherapy ,112. TheAnother recent study reported that synergistic effects of TQ and Vitamin D3 or 5-FU could control colon cancer progression in azoxymethane-induced rat model, by arresting the cell cycle in the G1 phase. Specifically, the combined treatment showed a remarkable antitumor effect by dramatically reducing tumor growth and large aberrant crypts loci via Wnt pathway, by decreasing β-catenin, NF-κβ, COX-2, iNOS, VEGF and HSP-90 and increasing DKK-1, CDNK-1A, TGF-β1, TGF-β/RII, smad4 and GPx in the early stage of CRC ,118. TQ In case of leukemia, TQ combined with doxorubicin significantly inhibited cell growth in multidrug-resistant, HL-60 and Jukart cells, by increasing Bax/Bcl-2 mRNA ratio and increasing ROS ,121. MorN. sativa oil and TQ exerted protective effect in long term cancer chemotherapy with cisplatin [In bladder cancer, in adult male Wistar rat isplatin , while tisplatin . In addiisplatin ,125.Moreover, TQ and Zoledronic combination raised the caspase 3/7 activity in prostate PC-3 cancer cells, but not in DU-145 cell line and showed also significant cytotoxic activity by DNA fragmentation and apoptosis induction ; interesThe treatment with TQ and cisplatin or diosgenin decreased cell viability and increased apoptosis, reduced cell growth and proliferation in oral squamous cell carcinoma cell line UMSCC-14C, by upregulating the expression of p53 and caspase 9 and downregulating the expression of Bcl 2 ,127.Finally, TQ showed synergism with radiation in the treatment of head and neck squamous cell carcinoma in SCC25 and CAL27 HNSCC lines due to its antiproliferative and radio sensitizing properties .N. sativa and its constituents with anticancer drug and/or other bioactive molecule in various types of cancer.N. sativa [N. saliva seed significantly improved the treatment outcome: the combination of TQ with other cytotoxic drugs, including L-asparaginase, administered at dose 40 mg/kg 2 times orally for 6 months led to a remission rate of 92%; at the same time TQ increased survival rate and exerted non-toxic effects in healthy subjects [ClinicalTrials.gov Identifier: NCT03208790) [To the best of our knowledge, few clinical studies have been performed with . sativa ,129,130.subjects . FurtherN. sativa have contributed to the discovery of new anticancer strategies and its major constituents such as TQ are promising candidates to remarkably combat cancer progression. Their effectiveness to prevent different processes related to cancer, such as proliferation, migration and invasion, angiogenesis and metastasis were here described and reported (N. sativa and its constituents in suitable combinations with existing chemotherapeutic agents for effective treatment of many cancers. Moreover, it should be considered the specific cellular and molecular targets of various constituents of N. sativa. It is known that N. sativa can act both as a ROS inducer at relatively high concentration and as an antioxidant at low concentration. For well understanding these differential cellular effects, further in vivo and in silico studies should be performed at both the genomic and proteomic level, especially considering that the data obtained in vitro are generally difficult to translate in in vivo conditions as recently reported [Despite the efforts, cancer still remains undefeated in the history of mankind with an annual death rate of over a million. Natural products like reported . Moreovereported .N. sativa in order to highlight its potency and poor bioavailability. These issues can be resolved by synthesizing various analogs of N. sativa and its constituents and formulating those into different delivery systems in adequate preclinical and clinical studies, where N. sativa or its derivatives can potentiate the antitumorigenic potential of various conventional and well-established anticancer agents.Therefore, more studies are recommended to improve the pharmacodynamics and bioavailability of


Nausea and vomiting are common gastrointestinal complaints that can be triggered by diverse emetic stimuli through central and/or peripheral nervous systems. Both nausea and vomiting are considered as defense mechanisms when threatening toxins/drugs/bacteria/viruses/fungi enter the body either via the enteral  or parenteral routes, including the blood, skin, and respiratory systems. While vomiting is the act of forceful removal of gastrointestinal contents, nausea is believed to be a subjective sensation that is more difficult to study in nonhuman species. In this review, the authors discuss the anatomical structures, neurotransmitters/mediators, and corresponding receptors, as well as intracellular emetic signaling pathways involved in the processes of nausea and vomiting in diverse animal models as well as humans. While blockade of emetic receptors in the prevention of vomiting is fairly well understood, the potential of new classes of antiemetics altering postreceptor signal transduction mechanisms is currently evolving, which is also reviewed. Finally, future directions within the field will be discussed in terms of important questions that remain to be resolved and advances in technology that may help provide potential answers.  Nausea and vomiting are essential protective defense processes by which humans as well as vomit-competent animals tend to avoid ingestion and/or digestion of potentially toxic substances. Nausea is the unpleasant sensation of having the urge to vomit, whereas vomiting (emesis) is a physical event and is the forceful expulsion of intestinal and gastric contents through the mouth . VomitinNausea and vomiting can be triggered by several mechanisms, as depicted in In this article, we will initially describe the underlying neuroanatomical, neurochemical, and receptor basis of diverse causes of nausea and vomiting along with corresponding, currently available agonist/antagonist antiemetics, followed by their potential intracellular signaling cascades. Finally, we will discuss the potential future treatments for nausea and vomiting caused by different stimuli. Understanding the circuitry and cellular mechanisms of nausea and vomiting helps us in developing new medications for the prevention and treatment of these debilitating symptoms. The gut–brain axis is a continuous, bidirectional neural and endocrine communication network between the gastrointestinal tract and the brain that is crucial to gastrointestinal regulation . The gutDifferent stimuli initiate the emetic reflex universally coordinated by the dorsal vagal complex in the brainstem, which is composed of the area postrema, nucleus of the solitary tract, and dorsal motor nucleus of the vagus . The areVagal afferent pathways are major peripheral emetic nerves that prominently participate in vomiting in response to emetic stimuli in the gastrointestinal tract . Indeed,2+)-dependent manner [3 and substance P neurokinin NK1 receptors, respectively), which are present on the vagal afferents initiate nausea and vomiting [In the gastrointestinal tract, enterochromaffin cells synthesize > 90% of the body’s 5-hydroxytryptamine , as well as large amounts of substance P (SP), which are both fundamental to gastrointestinal motility, nausea, and vomiting ,25. Chemt manner ,26,27. Fvomiting . Since 5vomiting . Neuronsvomiting . While tvomiting ,32,33.Currently, significant knowledge exists on the neuroanatomy and neurochemistry of vomiting. Despite being closely associated, central pathways of nausea and vomiting appear to be at least partly different . Our curBrain imaging as a biomarker of nausea has facilitated noninvasive identification of the activation sequence of brain structures involved in the nausea pathway . The latSeveral established animal models are the mainstay of preclinical nausea and vomiting research in both academia and pharmaceutical industry, including the dog, cat, ferret, house musk shrew , and leaIn non-emetic animals, particularly rodents that lack a vomiting reflex and the capacity to vomit, behaviors such as pica ) and gaping can accompany or follow the response to emetic stimuli  . Pica an3), neurokinin 1 (NK1), dopamine D2 and D3, opioid mu and kappa, muscarinic M1, and histamine H1, many of which are located both in the periphery  and the brainstem dorsal vagal complex  [3 and transient receptor potential vanilloid type 1 receptor (TRPV1), many emetic receptors belong to the G-protein-coupled family of receptors (GPCRs).Exogenous stimuli, including emetogenic cancer chemotherapy, bacterial toxins in the gut, viruses and fungi infection, food poisoning, epigastric radiation, diverse drugs, and motion, evoke vomiting, which is frequently, but not always, accompanied by nausea . They acostrema) ,12. Per 1–7), which are trans-membrane G-protein-coupled receptors, except for the 5-HT3 receptor, belonging to the cys-loop ligand-gated ion channel family. The 7 families are divided further into subfamilies, and to date a total of 14 5-HT receptor subtypes have been described. Although basic evidence indicate that serotonin 5-HT1–4 receptors could be involved in the modulation of the emetic reflex, to date application of clinical evidence support a major emetic role for 5-HT3 receptors [3 receptor activation induces fast excitatory postsynaptic potentials and rapid depolarization of serotonergic neurons [2+ concentration that causes the release of different emetic neurotransmitters and/or peptides  is involved in vomiting was deduced from drugs that interfere with its synthesis, storage, release, and metabolism long before the discovery of selective tools that modulate specific serotonergic receptor subtypes . Serotoneceptors . Being a neurons , leading, itself . Signific player ,51. In fd emesis ,51,52. 1, NK2, and NK3, which belong to the superfamily of G protein-coupled receptors. These receptors are recognized with moderate selectivity by endogenous substance P, neurokinin A, and neurokinin B, with respective preferential selectivity for NK1, NK2, and NK3 receptors. Although their affinity differences are not large and cross-interaction can occur across neurokinin receptors, substance P appears to be the endogenous ligand for NK1 receptors and has a major role in the induction of vomiting, particularly during the delayed phase of cancer chemotherapy-evoked emesis [1 receptor agonist GR73632  causes vomiting in least shrews in a dose-dependent fashion [1Rs and substance P are expressed in central and peripheral emetic loci, including the brainstem dorsal vagal complex emetic nuclei, as well as vagal afferents, enteric neurons, and enterochromaffin cells of the gastrointestinal tract [1Rs by substance P excites neurons in the area postrema of dogs and subsequently evokes vomiting [1Rs leads to blockade of vomiting evoked by the NK1R selective agonist GR73632 in the least shrew [1Rs play a major role, and peripheral NK1Rs play a minor role, in the induction of vomiting [1R antagonists  are recommended for the suppression of the second phase of vomiting evoked by highly emetogenic cancer chemotherapeutics [3 receptor antagonists plus dexamethasone [1R antagonists behave as broad-spectrum antiemetic [Substance P is one member of structurally related mammalian family of tachykinin peptides, including neurokinin A, neurokinin B, and the N-terminally extended forms of neurokinin A such as neuropeptide K and neurokinin g . Mammalid emesis ,18. In fd emesis  but not d emesis . In factd emesis . This is fashion . Both neal tract ,56. Morevomiting , whereasst shrew . In factvomiting . In the apeutics . In factethasone ,51,52,60tiemetic  and are tiemetic ,63; howetiemetic .1-like (D1 and D5) and dopamine D2-like  receptors [2 and D3 receptors in the mediation of vomiting [2  and/or D3  receptors preferring agonists elicit emesis in vomit-competent species, which can be attenuated by their corresponding competitive antagonists, whereas dopamine D1/D4/D5 receptor-selective agonists are devoid of emetic properties [Dopamine is a monoamine proemetic neurotransmitter and produces its diverse physiological effects through activation of two classes of membrane-bound G-protein-coupled receptors, namely dopamine Deceptors . Surfeiteceptors . The diseceptors . To datevomiting . In factoperties ,68,69,702/3), and/or their mRNA are well distributed in the emetic reflex arc, including the dorsal vagal complex , vagal nerves, gastrointestinal tract, and the enteric nervous system [2-like receptor antagonists such as phenothiazines , butyrophenones , and benzamides  are employed in the clinic for prevention of vomiting caused by diverse emetic stimuli, including: uremia, radiation sickness, viral gastroenteritis, postoperative nausea, and vomiting, as well as prophylaxis for cancer patients receiving chemotherapy with low emetogenic potential, or as a recue antiemetic in patients who have breakthrough emesis [Dopamine, its proemetic receptors  repeated exposure to rotary stimulation habituated animals to motion sickness and elevated histamine N-methyltransferase expression in their brainstem; and (iii) elevation of histamine N-methyltransferase gene expression in the dorsal vagal complex inhibited the motion sickness, whereas introduction of recombinant lentiviral vectors expressing rat histamine N-methyltransferase-shRNA into the DVC to reduce the expression of histamine N-methyltransferase promoted motion sickness. Additionally, increased histamine tissue levels in the hypothalamus and brainstem nuclei such as the vestibular nucleus and the dorsal vagal complex are considered the primary reason for induction of motion sickness [1 receptor mRNA, and H1 receptor protein expression in their hypothalamus and brainstem, which can be significantly reduced by pretreatment with hesperidin [1 receptor blockers such as dimenhydrinate and diphenhydramine are commonly used antiemetic agents against nausea and vomiting secondary to motion sickness [1 receptor blockers have anticholinergic properties that block muscarinic receptors, which may also contribute to their antiemetic effects [The physiological effects of histamine are mediated via four histamine receptors, including H, and H4 . Signifivomiting . In factvomiting . In addivomiting . Concomisickness ,87,88,89speridin , a bioflsperidin . Histamisickness ,92,93. I effects . Histami effects . 2 receptors as well as 5-HT3 receptors present in the area postrema. (ii) Opioid µ or κ receptor agonists can inhibit intestinal motility that can lead to gut distension and increased emptying time, resulting in stimulation of visceral mechanoreceptors and chemoreceptors, which are also associated to nausea and vomiting. These emetic signals from the gastrointestinal tract to the brainstem nucleus of the solitary tract occurs through visceral afferents, which may further stimulate the already described serotonergic signaling pathway of vagal afferents [1 and cholinergic systems [Though the exact physiological mechanisms underpinning opioids-induced nausea and vomiting are not fully understood, activation of opiate receptors in the area postrema, vestibular apparatus and the gastrointestinal tract are implicated . It appe systems ,14. Thes systems . Both em systems ,99. Ther systems , and gen systems .1, Y2, Y4, Y5, and Y6), which mediate a large variety of physiological and pathophysiological processes [The neuropeptide Y hormone receptor family are activated by the three endogenous ligands: neuropeptide Y, peptide YY (PYY), and the pancreatic polypeptide processes ,101,102.rocesses ,101,102.rocesses . PYY , while abdominal vagotomy can abolish the effect [2 receptor agonists, PYY (3–36), and obinepitide cause nausea in humans [In dogs and rats, high affinity binding sites for PYY are highly localized in the brainstem area postrema . Periphe neurons  and myen neurons . Peptide neurons . In the 31020028  has been31020028 . In addie effect . The anoe effect . The Y2 n humans ,113,114.2+-sensing receptor, or by ruthenium red, an antagonist of transient receptor ankyrin-1 [Furthermore, the common foodborne mycotoxin deoxynivalenol triggers vomiting and elevates plasma levels of peptide YY(3–36) and 5-HT in the mink, which were inhibited by NPS-2143, an antagonist of the Cankyrin-1 . GDF15 is a cytokine expressed in a variety of tissues and is secreted into circulation in response to diverse stimuli, targeting activation of hindbrain glial-derived neurotrophic factor receptor alpha-like (GFRAL), which has been identified as the GDF15 receptor . Cisplat2 [Eicosanoid lipids, prostaglandins, and leukotrienes are arachidonic acid metabolites derived from cell membrane phospholipids, which are released by phospholipase A2 ,125. The2 . Since t2 . 2 (PGI2), prostaglandin D2 (PGD2), prostaglandin E2 (PGE2), and prostaglandin F2α (PGF2α) [2 (PGE2) is the most abundant prostaglandin [The major types of prostaglandins include prostaglandin I (PGF2α) . Among taglandin . There aaglandin . 2 or PGF2α for abortion cause nausea and vomiting in up to ~20% patients and is worse in patients already suffering from hyperemesis gravidarum, an extreme form of nausea and vomiting of pregnancy [2, 17-phenyl-omega-trinor prostaglandin E2, misoprostol, and sulprostone), FP (prostaglandin F2α and fluprostenol), and IP (iloprost and cicaprost) receptor agonists and failed to induce emesis in the Suncus murinus [2 is able to induce emesis in ferrets [2), TP (U46619), DP (BW245C), and FP , but the IP agonist cicaprost was not effective in evoking emesis [2, 20-hydroxyPGE2, and PGF2α) evoke robust vomiting in the least shrew model of vomiting [A direct relationship between prostaglandins and nausea and vomiting is being established. In humans, elevated serum PGE2 levels is associated with increased episodes of nausea and vomiting in pregnancy . Intraveregnancy ,131. In regnancy  and Suncregnancy ,134, whi ferrets . Further ferrets , was als ferrets . Anotherg emesis . In addivomiting . These f1 and NK2, adrenaline, etc.; (iii) maintaining the normal physiological functions of organs and systems; and (iv) regulation of the hypothalamic–pituitary–adrenal axis [Corticosteroids block the biosynthesis of prostaglandins by inhibiting the phospholipase A2 and cyclooxygenase enzymes  and helpnal axis . Of eicosanoids, the emetic role of the leukotriene family has been neglected. In least shrews, cysteinyl leukotrienes  but not its precursor leukotrienes  induce vomiting with the different emetogenic efficacy (LTC4 = LTD4 > LTE4) . LTC4-inThe platinum-based chemotherapeutic agents such as cisplatin, carboplatin, and oxaliplatin are regularly prescribed in the treatment of cancers . Nausea Prevention: Various antiemetic guidelines have been developed for the prophylactic treatment of chemotherapy- and radiotherapy-induced nausea and emesis [3 receptor antagonist with an NK1 receptor antagonist plus the corticosteroid dexamethasone) has revolutionized prophylactic antiemetic therapy against both the acute and delayed phases of vomiting caused by highly emetogenic chemotherapeutic regimens, neither complete emesis protection in all cancer patients, nor significant nausea protection has yet been achieved [d emesis ,51,52,60achieved ,156.3 receptor antagonists the most commonly prescribed antiemetics for RINV prophylaxis [3 receptor antagonists such as ondansetron, granisetron, and palonosetron appear to be effective in suppression both “acute” and “delayed” phases of RINV for both nausea  and vomiting  [1 receptor antagonist aprepitant, the antinausea  and antiemetic  effects were in a similar range during both phases [Radiation-induced nausea and vomiting (RINV) are also common side effects of radiotherapy, which can affect up to 50–80% of patients ,158. Whiphylaxis . In factctively) ,161. Wheh phases ,161.Postoperative nausea and vomiting and postdischarge nausea and vomiting (PDNV) remain common and distressing complications of surgery. The routine use of opioid analgesics for perioperative pain management is a major contributing factor to both PONV and PDNV after surgery . Opioids3 receptor function in vitro, which could also contribute to nausea and vomiting [Clinical studies also indicate that PONV can be caused by inhalational anesthetics , which appear to have equal potency for evoking PONV, as well as nitrous oxide (laughing gas) usage . Nitrousvomiting . Signifivomiting . 1 receptor antagonist aprepitant exerts improved antiemetic efficacy against both acute and delayed PONV, when compared with the 5-HT3 receptor antagonist ondansetron [2/3 receptor antagonist droperidol, corticosteroid dexamethasone, and a 5-HT3 receptor antagonist ondansetron, in conjunction with an NK1 receptor antagonist aprepitant, significantly reduces cumulative episodes of emesis through 48 h postoperatively, compared with discussed triple therapy prophylaxis, alone [Prevention: The NKansetron . In patiansetron . In facts, alone .9-THC), have been used as a second line prophylactic antiemetic medication for patients receiving cancer chemotherapeutics [9-THC (dronabinol) and nabilone (cesamet) have been approved by the FDA for this purpose. Unlike most antiemetics, cannabinoid agonists are classified as agonist antiemetics, and their broadspectrum antiemetic efficacy is mediated via activation of cannabinoid CB1 receptors [9-THC produces its antiemetic effects via activation of both central and peripheral CB1 receptors in the brainstem dorsal vagal complex and the gastrointestinal tract [9-THC can attenuate both the acute and delayed phases of chemotherapy-evoked vomiting [9-THC and related cannabinoid agonists dose-dependently suppress vomiting caused by diverse emetogens, including the selective agonists of serotonin 5-HT3 [2 [1 [9-THC and other cannabinoids may have better antiemetic efficacy than dopamine D2 receptor antagonist antiemetics  [9-THC with 5-HT3 antagonists such as tropisetron or ondansetron do not substantially improve D9-THC’s antiemetic efficacy in either animals [The cannabis plant (marijuana) and its main psychoactive component, delta-9-tetrahydrocannabinol  , additio animals  or patie animals ,176. 9-THC or related cannabinoids result in changes to the endocannabinoid system which can dysregulate stress and anxiety responses, thermoregulation, the transient receptor potential vanilloid system, and several neurotransmitters systems, which could be potential mechanisms for induction of CHS [However, use of large doses of the cannabis plant or related synthetic cannabinoids on a long-term basis can cause a paradoxical hyperemetic effect known as cannabinoid hyperemesis syndrome (CHS). The CHS is characterized by cyclical episodes of nausea and vomiting ,178. Pron of CHS . 2/3 receptor antagonists such as haloperidol is also effective in treating acute CHS symptoms [3 receptor (5-HT and 2-Methyl-5-HT), dopaminergic D2/3 receptor (apomorphine and quinpirole), and cholinergic M1 receptor (pilocarpine and McN-A-343), as well as the selective neurokinin NK1 receptor agonist GR73632, the selective L-Type Ca2+ channel agonist FPL64176 and the sarcoplasmic endoplasmic reticulum calcium ATPase (SERCA) inhibitor thapsigargin [2+ channels, by increasing intracellular Ca2+ through vanilloid receptors [Prevention: Typical antiemetics appear to be ineffective for the treatment of CHS, but anxiolytic and sedative drugs such as benzodiazepines , along with hot showers (or hot baths), seem to be consistently useful in reducing CHS symptoms . Some stsymptoms . The hotsymptoms . Moreovesymptoms ,182,183.symptoms . RTX can1 receptors in the dorsal vagal complex and gastrointestinal tract [1 receptors also have a similar distribution and are extensively colocalized with TRPV1 receptors, thus suggesting a functional interaction [1 and TRPV1 receptor agonists have the capacity to completely abolish cisplatin-induced emesis in least shrews at doses that are ineffective when used individually [1 receptor expression and receptor internalization also occurs in rat tissues following systemic capsaicin treatment [1 receptor antagonist class of antiemetics possess broad-spectrum antiemetic efficacy, and, as such, aprepitant has been used successfully in the treatment of CHS when all other common antiemetics failed [TRPV1 receptors are found widely throughout the body, often in proximity to cannabinoid CBal tract . In factal tract . Cannabieraction . Indeed,vidually . The TRPvidually . Exposurvidually . By bindvidually . Downregreatment . The subs failed . Cessatis failed .1 receptor antagonists  are the most effective drugs [Motion sickness is a common physiological response to real or virtual motion. Motion sickness occurs in both humans and animals during travel by sea, automobile, airplane, and in space, as well as under some other situations such as simulators, the cinema and video games . Structuve drugs  against ve drugs . Bacteria, viruses, and even fungi are often regarded as primitive organisms and are classically considered as individualistic, solely focused on self-preservation and proliferation. However, recent research has shed light on innumerous intrinsic processes, illustrating how these microorganisms exhibit sophisticated intracellular and signaling behaviors. Only in the last few decades was it even considered feasible that bacteria, viruses, and fungi could exhibit commensalism with a human host or delay their pathological properties until conditions are deemed adequate . SpecifiStaphylococcus aureus and Bacillus cereus. Staphylococcus aureus (Staph aureus) induces vomiting following consumption of their enterotoxins, producing what is known as food poisoning. Specifically, Staph aureus is known to possess staphylococcal enterotoxins, which are believed to function as in vivo immune response inhibitors [3 receptor antagonist granisetron, or surgical vagotomy, can suppress SEA-induced vomiting in house musk shrews [2+ via release of intracellular stored Ca2+ from the endoplasmic reticulum, which has been shown to be sensitive to nitric oxide synthase inhibitors [2+-dependent release of 5-HT in the gastrointestinal tract is thought to stimulate vagal afferent serotonergic 5-HT3 receptors, which subsequently activates the dorsal vagal complex emetic nuclei in the brainstem to evoke vomiting [Two of the most notable bacterial species responsible for emesis are unquestionably hibitors ,198. Somhibitors . In facthibitors . It appek shrews . Furtherhibitors . The Ca2vomiting .Bacillus cereus (B. cereus), most known for causing vomiting and diarrhea after eating reheated rice or other starchy foods, induces emesis by means of a heat stable peptide known as cereulide [B. cereus in the small intestine, emesis can be evoked by ingestion of cereulide, which accumulates in contaminated foods [B. cereus produces such similar symptomology to Staph aureus food poisoning is because the evoked mechanism responsible is believed to be very similar, though provoked by cereulide. Cereulide is a potassium ionophore and is structurally like valinomycin [Staph aureus cereulide can activate vagal afferents [3 receptors present on vagal gastrointestinal afferents or indirectly activates these receptors following release of 5-HT from enterochromaffin cells [3 receptor antagonist ondansetron, or vagotomy, prevents the emetic effects of cereulide [ereulide ,202. Whied foods . The reainomycin . Like vafferents . Cereulifferents  and rhesfferents . The exain cells . In factereulide .2+-dependent release of hormone which evokes emesis [Fungi, on the other hand, induce vomiting by producing various mycotoxins, which range from generating only mild symptoms to causing fatality . For inss emesis . Lastly, we will review a few viral signaling mechanisms responsible for emesis. Viruses have developed their own ion channels to subvert their host cell’s basal ion-gradients to facilitate their replication and pathogenesis. As a result, viral proteins create holes in cellular membranes  through which ions and low-molecular-weight compounds can enter freely.S. aureus and Bacillus cereus), RV infection also induces secretion of 5-HT from enterochromaffin cells, which plays a key role in vomiting after stimulation of vagal afferent nerves and activation of the dorsal vagal complex emetic nuclei in the brainstem. In fact, suppression of vomiting following rotavirus or noravirus infections with 5-HT3 receptor antagonists such as ondansetron supports the latter hypothesis [Rotaviruses: Ten different rotavirus (RV) species (A–J) have been classified and are a leading cause of severe, dehydrating gastroenteritis in children under the age of five. Rotavirus infections still cause >600,000 deaths per year despite global introduction of vaccinations . As withpothesis .2+ concentration in a human midgut carcinoid enterochromaffin cell line in a dose- and time-dependent manner [2+ levels [2+ in human intestinal cells and in human intestinal enteroids by stimulating phospholipase C (PLC)-mediated inositol 1,4,5-trisphosphate (IP3) production and subsequent opening of IP3-sensitive calcium cannels in the endoplasmic reticulum membrane [2+ stores, which activates stromal interaction protein 1, an endoplasmic calcium sensor, which, in turn, activates extracellular Ca2+ influx through store-operated calcium entry channels in the plasma membrane, primarily through Orai1 [The nonstructural protein (NSP4) of the RV enterotoxin has a major role in the induction of vomiting following rotavirus infection. Indeed, purified NSP4 has been shown to not only evoke release of serotonin from enterochromaffin cells but also increase intracellular Cat manner . NSP4 ha+ levels . Moreovemembrane ,211. Thigh Orai1 ,211. Thugh Orai1 ,212.2+ levels [2+ homeostasis, which is believed to play a major role in the emetogenic reaction seen with infection of this virus [2+ mobilization is involved in the release of 5-HT via activation of the enteric nervous system and extrinsic vagal afferents to the brain, which leads to vomiting [Other viruses have also developed viroporins such as 2B/2BC (polio virus and coxsackievirus), human tlymphotropic virus 1, sindbis virus 6K, polyomavirus agnoprotein, influenza A (virus APB1-F2), and severe acute respiratory syndrome (SARS) Coronavirus (ORF3a), all of which modulate cytosolic Ca+ levels . Porcineis virus . This isvomiting ,215. Simvomiting ,215. Coronaviruses: Are a group of related RNA viruses that causes diseases in mammals and birds, including respiratory tract infections, neurological, and gastrointestinal disorders . The firBased upon the mechanisms of emetic actions of cancer chemotherapeutic agents (cisplatin) and rotaviruses ,209 discAmong the various functions of gut microbiota is their ability to resist colonization of pathogenic bacteria . AntibioAs mentioned above, the SARS–Cov-2 virus is now known not only to affect the respiratory system but the gastrointestinal system, as well , leading2+). Cytoplasmic Ca2+ concentration is the dominant element in determining the amount of transmitter to be released from nerve terminals [2+ mobilization can be an important aspect of vomit induction, since it is involved in both triggering the quantity of neurotransmitter released coupled with receptor activation, as well as postreceptor excitation–contraction coupling mechanisms [2+ imaging performed in vitro in the brainstem slice preparation suggest that emetic agents evoke direct excitatory effects on cytosolic Ca2+ signals in vagal afferent terminals in the nucleus of the solitary tract which potentiate local neurotransmitter release [Per our previous reviews ,229, amoerminals . Thus, Cchanisms . Studies release ,233,234.2+ levels both via mobilization of intracellular Ca2+ stores from the endoplasmic reticulum and extracellular Ca2+ influx resulting from direct opening of diverse ion channels present on the cell membrane or indirectly via signal transduction pathways following G protein-coupled receptor activation [3 receptor is a Ca2+-permeable ligand-gated ion channel [3 receptors can evoke an influx of extracellular Ca2+ that is sensitive to selective antagonists of both 5-HT3 receptors  and L-type calcium channels (LTCCs) such as verapamil, nimodipine, or nitrendipine [3 receptor Ca2+-permeable ion channels are involved in extracellular Ca2+ influx induced by 5-HT3 receptor activation. Moreover, 5-HT3 receptor stimulation and the evoked extracellular Ca2+ entry trigger release of intracellular Ca2+ from ryanodine-sensitive sarco/endoplasmic reticulum calcium stores via a process often referred to as Ca2+-induced Ca2+ release [2+ [1 receptors also increases the cytosolic levels of calcium, it does so via different signaling mechanisms, including an initial intracellular Ca2+ release to deplete intracellular stored calcium from the sarco/endoplasmic reticulum through activation of the phospholipase C (PLC)/inositol trisphosphate pathway (see later) and subsequent extracellular calcium influx through plasma–membrane-bound calcium channels, including store-operated Ca2+ entry and LTCCs [1 receptor selective agonist GR73632 induces vomiting in least shrews by an initial depletion of stored sarco/endoplasmic calcium and subsequent extracellular Ca2+ influx mainly through opening of LTCCs and, to a smaller extent, via SOCE [2 [1 [1 [2+ that involve extracellular Ca2+ influx and/or Ca2+ release from intracellular pools. Receptor activation by corresponding agonists can increase cytosolic Cativation . The ser channel . Cell liendipine ,238,239.nd LTCCs ,242,243.via SOCE . Other e SOCE [2 ,246, choCE [2 [1 ,248, his[2 [1 [1 ,249 and [2 [1 [1 ,251 rece[2 [1 [1 ,253, pro[2 [1 [1 , rotavir[2 [1 [1 ,209, fun[2 [1 [1 , and bac[2 [1 [1 ,256, als2+ entry in electrically excitable cells such as neurons and muscle [2+ mobilization is an important facet in the mediation of emesis. In fact, LTCCs have been shown to be present on the enterochromaffin cells of guinea pig and human small intestinal crypts [2+ evoked by emetic stimuli such as radiation or FPL64176 evoke release of emetic neurotransmitters such as serotonin from these cells [LTCCs serve as the principal route of Cad muscle ,258. FPLd muscle ,244. In d muscle ,244. Thel crypts  and incrse cells .2+ entry through LTCCs cause inhibition of excitation-coupled contraction [3 , neurokinin NK1 (GR73632), dopamine D2/3 (apomorphine or quinpirole), and muscarinic M1 (McN-A-343) receptors [2+ mobilization and the emetic receptors-driven downstream signaling pathways involving key molecules [Blockade of extracellular Catraction . In the eceptors . In addieceptors . Likewiseceptors  and propeceptors . The mecolecules .2+-ATPase (SERCA) pump transports the free cytosolic Ca2+ into the lumen of sarco/endoplasmic reticulum to counterbalance the cytosolic intracellular Ca2+ release from the sarco/endoplasmic reticulum into the cytoplasm via inositol 1,4,5-trisphosphate- (IP3Rs) and ryanodine receptors (RyRs) present on the wall of sarco/endoplasmic reticulum [2+ mobilizing agent thapsigargin is a selective SERCA inhibitor, which increases cytosolic Ca2+ concentration via an initial intracellular release of stored Ca2+ from the sarco/endoplasmic reticulum into the cytosol, followed by store-operated extracellular Ca2+ entry (SOCE) [2+ [The sarco/endoplasmic reticulum Caeticulum ,266,267.y (SOCE) ,269,270.OCE) [2+ ,272,273.OCE) [2+ . 3Rs-mediated intracellular Ca2+-release through pharmacological use of their respective antagonists dantrolene and 2-APB in vomiting has been carried out in our laboratory, and the attained results help to support the involvement of these intracellular Ca2+ release channels in emesis. In fact, in the least shrew emesis model, we have demonstrated vomiting evoked by either the 5-HT3R agonist 2-methyl-5HT or the LTCC agonist FPL64176 were insensitive to 2-APB, but, in contrast, both the frequency and percentage of shrews vomiting were dose-dependently suppressed by dantrolene [1R agonist GR73632-induced emesis; however, the RyR inhibitor dantrolene did not affect the evoked vomiting . Thus, RyRs and IP3Rs can be differentially modulated by diverse emetogens, and suppression of Ca2+ release from sarco/endoplasmic reticulum through IP3Rs and RyRs may provide additional targets for suppression of nausea and vomiting. SOCE inhibitors such as MRS1845 also provide some antiemetic effect in such studies [2+ entry in the PLC-coupled GPCRs signaling.The role of RyRs- and IPntrolene . On the ntrolene . Moreove studies ,275,276.Historically, studies focusing on intracellular emetic signaling mechanisms are limited to enhancement of cyclic adenosine monophosphate (cAMP) levels after inhibition of its metabolism by phosphodiesterase inhibitors . HoweverThe cAMP–PKA signaling pathway is a universal mediator of signal transduction that relays extracellular stimuli, including hormones or neurotransmitters, to intracellular targets. PKA is a downstream signal in the adenylyl cyclase/cAMP pathway that phosphorylates target proteins such as nuclear receptors and transcription factors, other kinases and phosphatases, G-protein coupled receptors, and ion channels . The eme1 receptor agonist GR73632, ii) LTCC activator FPL64176, (iii) the SERCA inhibitor, a Ca2+ signaling amplifier, thapsigargin, (iv) the 5-HT3 receptor agonist 2-Methyl-5-HT [ERK1/2 are members of the mitogen activated protein kinase superfamily, and their signaling pathway is implicated in a diverse array of cellular functions, including cell differentiation, proliferation, and inflammatory responses. ERK1/2 phosphorylation has been used to indicate neuronal activation . Our pubhyl-5-HT ,275,276,hyl-5-HT , and  serotonin 5-HT3R-evoked vomiting mediated by its selective agonist 2-methyl-5-HT, (ii) thapsigargin-induced emesis, (iii) substance P neurokinin NK1R-induced vomiting evoked by its selective agonist GR73632, and (iv) vomiting caused by the selective LTCC agonist FPL64176 [CaMKII is a serine/threonine-specific protein kinase that is regulated by the CaFPL64176 ,289,290.1 receptor agonist, GR73632 [PKC consists of a family of 10 serine/threonine kinases, which are dependent on phosphatidylserine, calcium, and diacylglycerol for activity (Black and Black 2021). Our published studies demonstrate that phosphorylation of protein kinase Cα/βII (PKCα/βII) in least shrew brainstem is associated with vomiting evoked by the: (i) chemotherapeutic agent, cisplatin ,286,  selective LTCC agonist FPL64176 [The Akt/GSK-3 pathway is a critical signaling pathways that is dysregulated in numerous human cancers. The Akt cascade acts in cancer process by regulating apoptosis, cell cycle, metabolism, and cells’ longevity. Akt phosphorylation has been observed in the least shrew brainstem following vomiting evoked by the: (i) substance P selective NKFPL64176 ,289,290.FPL64176 . In factFPL64176 ,292.3 , neurokinin NK1 (GR73632), dopamine D2/3 (apomorphine or quinpirole), and muscarinic M1 (MCN-A343 or pilocarpine) receptors, as well as the LTCC agonist FPL64176 and the SERCA inhibitor thapsigargin [The GSK-3 enzyme is a constitutively active multifunctional serine–threonine kinase that affects diverse physiological processes. In fact, GSK-3 has been linked in a broad range of diseases, including neurodegeneration, inflammation, diabetes, and cancer. GSK-3 is a downstream target for Akt, which phosphorylates GSK-3 and suppresses its activity ,296,297.sigargin . In addisigargin . These dsigargin . The presence of the vomiting reflex is widespread among mammals, such as carnivores , primates , and insectivores  ,300. The3 and substance P neurokinin NK1 receptor antagonists combined with the glucocorticoid dexamethasone, which protect up to 80–90% of cancer patients from vomiting who were prescribed highly emetogenic cytotoxic chemotherapeutic regimens such as cisplatin. Experimental use of selective receptor antagonist antiemetics has led to the current body of knowledge, but future development of antiemetics probably lies in development of drugs that target intracellular emetic signals. In addition, bacteria, viruses, and fungi enter cells by known and unknown mechanisms and hijack hosts’ intracellular signaling mechanisms to evoke vomiting, and drugs that block membrane-bound receptors may not be useful. With the multifaceted and comprehensive knowledge reviewed here, further studies are required to ascertain the “cross talks” between intracellular nauseogenic and emetogenic signaling pathways caused by diverse specific and nonspecific emetogens. Additional studies should involve potential antiemetic compounds targeting common or specific intracellular emetic signaling pathways, alone or in combination with conventional antiemetics of choice.Although systematic research in the area of nausea and vomiting began in the late 1950s, the main impetus in this field of research has been development of receptor selective antiemetics such as the first- and second-generation serotonin 5-HT