Accordingly, interest in the role of ferroptosis in neurodegeneration is escalating and specific evidence is rapidly emerging. One aspect that has thus far received little attention is the antioxidant transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2). This transcription factor regulates hundreds of genes, of which many are either directly or indirectly involved in modulating ferroptosis, including metabolism of glutathione, iron and lipids, and mitochondrial function. This potentially positions Nrf2 as a key deterministic component modulating the onset and outcomes of ferroptotic stress. The minimal direct evidence currently available is consistent with this and indicates that Nrf2 may be critical for protection against ferroptosis. In contrast, abundant evidence demonstrates that enhancing Nrf2 signaling is potently neuroprotective in models of neurodegeneration, although the exact mechanism by which this is achieved is unclear. Further studies are required to determine to extent to which the neuroprotective effects of Nrf2 activation involve the prevention of ferroptosis.Ferroptosis is a newly described form of regulated cell death, distinct from apoptosis, necroptosis and other forms of cell death. Ferroptosis is induced by disruption of glutathione synthesis or inhibition of glutathione peroxidase 4, exacerbated by iron, and prevented by radical scavengers such as ferrostatin-1, liproxstatin-1, and endogenous vitamin E. Ferroptosis terminates with mitochondrial dysfunction and toxic lipid peroxidation. Although conclusive identification of ferroptosis  The last few decades have witnessed a surge in the discovery of new forms of regulated cell death that have immense implications for both health and disease . FerroptFigure 1).A discriminating feature of ferroptosis is the potent capacity of lipid peroxide scavengers  to prevent ferroptotic cell death . This exFerroptosis appears to require the presence of specific highly oxidisable phosphatidylethanolamine phospholipids containing the polyunsaturated fatty acids (PUFAs) arachidonic acid and adrenic acid. Acyl-CoA synthetase long-chain family member 4 (ACSL4) is important for the synthesis of phospholipids from these PUFAs, while lysophosphatidylcholine acyltransferase 3 (LPCAT3) is important for their insertion into membrane phospholipids . PharmacAs mentioned above, the precise role of iron in ferroptosis remains unclear. The ability of the iron chelators including deferoxamine and deferiprone to salvage cells from ferroptotic death in a variety of models underscores the role of iron in triggering ferroptosis . The lipc-  .Ferroptosis can be experimentally induced by direct inhibition of Gpx4 via Ras-selective lethal small molecule 3 (RSL3) or by genetic knockdown or deletion of Gpx4 . As Gpx4rafenib)   is exquisitely positioned to modulate the onset and outcomes of ferroptosis. Nrf2 is responsible for regulating hundreds of antioxidant genes . Under nTable 1). These include genes for glutathione regulation , NADPH regeneration which is critical for Gpx4 activity , and iron regulation  .Importantly in the context of ferroptosis, almost all genes thus far implicated in ferroptosis are transcriptionally regulated by Nrf2 (abolism) . In addiabolism) . PPARγ iabolism) , and canabolism) . Hence Nabolism)   would provide some insight.The above mentioned studies in cancer cells provide empirical Ferroptosis is an iron-dependent form of regulated cell death instigated by impaired glutathione metabolism, culminating in mitochondrial failure and toxic lipid peroxidation. Emerging evidence implicates ferroptosis in neurodegeneration, both in the molecular and biochemical signatures of neurodegeneration, and in terms of functional abrogation of neuron death via specific ferroptosis inhibitors. Elucidating how ferroptosis provokes neurodegeneration will expose new therapeutic opportunities to treat these diseases. To this end, targeting the antioxidant transcription factor Nrf2 is an attractive option. Nrf2 signaling is involved in regulating mitochondrial function and impacts almost all identified molecular aspects of ferroptosis. Treatments targeting Nrf2 have been demonstrated to exert anti-ferroptotic effects in the context of cancer cells, and are beneficial in many models of neurodegeneration. The protective mechanism of Nrf2 activation in these models may involve attenuating ferroptosis via upregulation of the endogenous anti-ferroptotic machinery, however, direct evidence for this is currently lacking. The myriad of Nrf2 actions described here suggest that targeting Nrf2 is an exciting therapeutic option to attenuate ferroptosis.Several questions remain unanswered. The discovery of bonafide biomarkers of ferroptosis will be invaluable to unequivocally probe its involvement in neurodegeneration and facilitate the development of therapeutic treatments targeting ferroptosis. The continued evaluation of ferroptosis inhibitors and comparison to inhibitors of other cell death modalities in further models of neurodegeneration will help elucidate the key pathways involved. Finally, whether Nrf2 activation is directly alleviating ferroptotic stress.Taken together, the arguments presented in this review elucidate a coherent network that links Nrf2 signaling to mitochondrial function and ferroptotic cell death, and proposes the targeting of Nrf2 as a rational line of therapy for ferroptotic neurodegeneration.JL conceived the review. MA prepared the first draft. RL, KK, and TM reviewed literature and contributed to writing. JL prepared the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


However, the underlying mechanisms of metformin in the treatment of cancer are not fully understood. This systematic review aims to discuss the possible anticancer mechanism of action of metformin.The anticancer activity of metformin has been confirmed against several cancer types A search through different databases was conducted, including Medline and EMBASE.A total of 96 articles were identified of which 56 were removed for duplication and 24 were excluded after reviewing the title and abstract. A total of 12 research articles were included that describe different antiproliferative mechanisms that may contribute to the antineoplastic effects of metformin.This analysis discussed the potential anticancer activity of metformin and highlighted the importance of AMPK as a potential target for anticancer therapy. Metformin is a widely used antidiabetic drug. It is the recommended treatment for Type II diabetes. Recently published reports claimed that the antidiabetic drug can also protect patients against cancer. This has led to a widespread interest in the antidiabetic medication as a possible treatment for cancer. While studies have confirmed the anticancer potential of metformin, the way in which metformin improves cancer outcome remains unknown. Hence, the current study investigates the reported mechanisms that explain how the antidiabetic drug works as an anticancer agent. Type II diabetes mellitus is a metabolic disorder that leads to cardiovascular complications. What is more, people with diabetes have an increased risk of several common cancers  as well Metformin is known to reduce hepatic gluconeogenesis and increase skeletal muscle glucose uptake by activating AMPK, which is a cellular energy sensing enzyme that regulates cellular energy status by undergoing phosphorylation and increasing activity when ATP levels decrease and AMP levels increase. The change in the ATP:AMP ratio is used as an indicative marker of energy deficiency .There is a continual increase in the prescription and usage of metformin; data from the Quality Outcomes Framework in England show that across the whole of the last decade, prescribing of metformin has more than doubled, from 9.4 million items in 2006/2007 to 20.8 million items in 2016/2017 . PrescriIn addition to its on-label use as an antidiabetic medication, metformin has other off-label uses, such as for the treatment of polycystic ovary syndrome, hyperinsulinemia, prediabetes, obesity and metabolic syndrome. Data from the National Disease and Therapeutic Index (NDTI) database showed that the most common diagnoses with metformin use were diabetes (34·9%), followed by metabolic syndrome (20·9%), polycystic ovary syndrome (17.2%) and obesity (6.5%) .in vitro and preclinical studies confirmed the anticancer activity of metformin against several types of cancer, which promoted the initiation of more than 55 clinical trials that aimed to investigate the potential anticancer effect of metformin against endometrial, prostate, pancreas, lung and breast cancer [Interestingly, the antidiabetic drug is receiving extensive attention as a potential anticancer treatment following retrospective reports that showed improved survival rate in different cancer types for diabetic patients using metformin. The drug is gaining international interest for its potential use to treat/prevent different types of cancer, cardiovascular disease, ageing and neurological disorders . Data frt cancer .However, the underlying mechanism of action of how metformin exerts its anticancer activity is still not completely understood. Thus, the current manuscript is a systematic review of the literature that aims to analyze and characterize the different reported mechanisms of the anticancer activity of metformin.et al. [The current review was performed according to the guidelines shown in the PRISMA statement for reporting systematic reviews and meta-analyses of studies. That evaluate health care interventions: explanation and elaboration by Liberati et al. .Articles published within the last 15 years to 15 February 2019 were searched through the available databases including; Medline via PubMed and EMBASE via Elsevier. We used ‘Metformin’, ‘metformin mechanism of action’ and ‘metformin and cancer’ as the search terms. The search was restricted to English language studies including journal articles, theses and conference proceedings.M Aljofan and D Riethmacher carried out the study search, study selection, as well as quality assessment and any disagreement was solved by discussion. The quality of the studies included were assessed according to the quality of the body of evidence The GRADE approach .in vitro and/or in vivo.Experimental studies including The primary measures of interest include reported mechanism of actions of metformin other than its hypoglycemic activity. Secondary measure was the anticancer effect of metformin.The extracted data included the name of the first author; publication year; methodology and findings.in vitro analyses, which do not necessarily reflect all aspects of the organism as a whole. Some studies were not included in the review for reasons including subject duplication, article language and no/limited access to articles. Another limitation is that some of the reported mechanisms have not yet been reconfirmed. This may be in part due to the fact that this topic, anticancer activity of metformin, is relatively new and that most of these studies were published within the last decade or so.In this review, we aimed to analyze the reported mechanisms of antitumor action of metformin, thus, we only included studies with The database search produced 96 articles and no other articles were identified using other sources. A total of 56 articles were removed for duplication and 24 articles were excluded after reviewing the title and abstract. Full text of the 16 remaining articles was obtained and reviewed for the final inclusion. Three review articles and one irrelevant study were further excluded. Thus, a total of 12 studies were included in the current review. in vitro, in vivo and epidemiological and observational studies reported that metformin may be an effective treatment or adjuvant for cancer therapy. Initially, in an observational study, Evans et al. reported a link between the use of metformin and a reduced risk of cancers and cancer-related deaths [Mounting evidence from d deaths . The stuin vitro studies that have reported the putative anticancer effect of metformin including Wu et al., which reported the effect using iterative genomic screening in C. elegans, identified a genetic pathway linking metformin to the inhibition of cancer growth and lifespan extension [For instance, xtension . Also, Zxtension .et al. in 2018, which studied the effect of metformin in genetic mouse models of pancreatic cancer, claimed that metformin can inhibit tumor growth. They treated the mice with a daily dose of metformin starting at 6 weeks of age. They showed that mice treated with metformin presented a prolonged overall survival, and decreased tumor volume and tumor weight [Results from animal studies such as that of Chen r weight . What iset al. presented a study using human data that examined the anti-angiogenic effects of metformin on patients with endometrioid carcinoma, which is considered the most common histologic type of endometrial carcinoma and of uterine malignancy overall [Hanawa  overall ,31. The  overall .et al. aimed to investigate the molecular antineoplastic mechanism of metformin on epithelial cells [In 2006, a study by Zakikhani al cells . The autal cells .However, instead of enhancing insulin or IGF-I-stimulated growth, metformin acted as a growth inhibitor, an action that the authors claimed to be associated with a suppression of p70S6K phosphorylation. Furthermore, they examined the effect of metformin on cellular proliferation using several cancer cell lines including the LKB1 null HeLa cell lines. Except, for LKB1 null HeLa, dose–response treatment on several cell lines resulted in growth inhibition, suggesting that the antiproliferative activity of metformin is most likely achieved via LKB1 signaling, which aligns with growth inhibition seen on the untransformed MCF-10A human breast epithelial cells ,33.Furthermore, the authors investigated whether the metabolic actions of metformin require AMPK activity similar to that in other tissues such as muscles or hepatocytes. They not only found that metformin stimulates AMPK phosphorylation, but also that the phosphorylation was achieved in a dose-dependent manner. To confirm these findings and to determine whether activation of AMPK by metformin is required for the antiproliferative effects of metformin, the authors used siRNA against AMPK α1 subunit. Interestingly, the AMPKα1 siRNA reduced the stimulatory effect of metformin on AMPK phosphorylation and that the reduction was correlated with the levels of reduced AMPKα1.This study suggests that metformin’s activation of the AMPK pathway is not confined to hepatocytes but can be observed in epithelial cells as well. In conclusion, the authors claimed that metformin’s antiproliferative activity was achieved via an LBK-dependent pathway.et al. studied the antiproliferative antisurvival activity of metformin against six triple-negative breast cancer cell lines, and found that metformin induced growth inhibition on all of these cell lines [Deng ll lines .The study showed that metformin significantly reduced both tyrosine and serine phosphorylation of Stat3 (P-Stat3 at Tyr705 or Ser727), reduced P-mTOR and induced P-AMPK/AMPK. Also, the study showed that metformin inhibits Stat3 activation, either directly or indirectly, through a time- and dose-dependent manner.To further explore the role of Stat3 in metformin action, in one experiment they transfected these cell lines with constitutively active Stat3, and in another experiment knocked down Stat3 expression using a lentiviral system.Interestingly, constitutively active Stat3 expression reduced metformin-associated changes in cell growth and apoptosis as well as cell signaling in all of the tested triple-negative cancer cells. Whereas, specific knockdown of Stat3 expression enabled metformin to significantly induce more growth inhibition in each of the Stat3-knockdown cells compared with the control.In order to investigate the association between the anticancer activity of metformin and mTOR, the authors compared the growth inhibition and apoptotic effects of metformin alone or combined with the selective Stat3 inhibitor S3I-201 to that of the mTOR inhibitor rapamycin. The study found that both metformin and S3I-201 alone induced apoptosis, while the combination was even more potent; however, rapamycin treatment alone induced mild growth inhibition, but failed to significantly enhance the metformin-associated growth inhibition when combined with metformin, which indicates that metformin’s effect was not due to its effect upon mTOR.et al., the study claims that the metformin activation of Stat3 and hence, its antitumor activities are independent of mTOR.The study concluded that the anticancer activity of metformin is achieved via the direct or indirect activation of Stat3. Unlike Zakikhani et al. investigated the effect of nutritional environment on the anticancer activity of metformin using renal cancer cells. The authors examined whether poor outcome of metformin in renal cancer cells may be influenced by the nutrient condition [Since cancer cells encounter nutrient limitations, a study by Liu ondition . In thisThe study indicated that cellular treatment with metformin under normal condition resulted in a significant suppression of cell growth, but that changing the cellular environment from normal to glucose-deprived reversed the metformin-induced growth suppression and that metformin appears to promote cell growth under this condition.et al., which investigated the antiproliferative effect of metformin using triple-negative MDA-MB-231 breast cancer cells. The study suggested that treatment with metformin did not inhibit the growth of MDA-MB-231 cells cultured in hyperglycemic conditions. However, MDA-MB-231 growth was inhibited by metformin when normoglycemic conditions were used for cell culture.This finding was aligned with that of Zordoky Unlike previous reports, Liu and colleagues suggested that metformin activation of AMPK is not affected by the environment, and that treatment with metformin did not produce obvious apoptosis in renal cancer cells under either normal or glucose-deprived conditions. However, several reports have found the exact opposite and that low glucose environment was reported to increase AMPK activation ,18,34–366 A498 cells into nude mice, which was supposed to mimic an in vivo form of renal cancer, and restricted their dietary intake to resemble the effect of glucose deprivation. Food starvation alone slowed, but did not suppress, tumor growth. However, the study suggested that under glucose starvation, metformin treatment did not suppress the tumor growth, but in fact increased its volume.To further test the effect of nutritional environment/food starvation on anticancer activity of metformin, the authors injected 1 × 10in vivo analysis of Ki-67 revealed that metformin treatment promoted cellular proliferation in food-starved nude mice and that metformin treatment may increase renal cancer cell proliferation under glucose deprivation. In conclusion, they suggested that the antiproliferative effects of metformin in cancer cells are highly dependent on the glucose concentration in the extracellular environment.In addition, the authors tested the expression of proliferation marker, Ki-67, and reported that metformin treatment suppressed the expression of Ki-67 under normal conditions, but induced its expression under glucose deprivation. Additionally, the et al. investigated the anticancer mechanism of action of metformin on different human ovarian cancer cells [A study by Ma er cells . The stuAlso, the combination of low glucose and metformin was shown to enhance the expression of cytosolic cytochrome c and reduce the mitochondrial membrane potential in cancer cells compared with normal glucose with or without metformin, indicating that cell apoptosis is triggered by combination of low glucose and metformin, via the mitochondria-associated pathway. These findings suggested that mitochondrial dysregulation plays an important role in apoptosis that was induced by the combination of low glucose and metformin.Additionally, metformin and low glucose treatments resulted in a significant increase in the expression of Noxa, which is a pro-apoptotic protein associated with mitochondrial damage, and a substantial decrease in the expression of the anti-apoptotic protein, Bcl-2. Intriguingly, the treatment combination of low glucose and metformin induced the phosphorylation of the ASK1-dependent pathway, which is a key regulatory component of Noxa expression ,37,38.To determine the involvement of ASK1 in the subcellular localization of Noxa protein, cells were pretreated with NQDI-1, a pharmacological inhibitor of ASK1. The results showed that the total amount of Noxa and the level translocated to the mitochondria were significantly decreased, and the expression of Bcl-2 was increased, suggesting that ASK1 plays an essential role in low glucose and metformin-induced subcellular localization of Noxa.To further confirm the role of ASK1, knockdown of ASK1 expression using siRNA reduced the loss of mitochondrion membrane potential and apoptotic ratio in ovarian cancer cells treated with the combination of low glucose and metformin, which suggests that low glucose and metformin-induced mitochondrial damage are the consequence of ASK1/Noxa pathway activation.Furthermore, the study shows that ASK1 activation is associated with accumulation of reactive oxygen species (ROS) and further activates downstream signaling including JNK. The analysis of the role of ROS in the activation of ASK1 and JNK in low glucose environment and metformin treatment demonstrated that the inhibition of ROS decreased the phosphorylation level of ASK1 in cells treated with the combination of low glucose and metformin. These results suggest that the accumulation of ROS may have been involved in the low glucose and metformin-induced loss of mitochondrion membrane potential.Also, the pretreatment of cancer cells with the ROS scavenger NAC reduced several effects seen with the combination of low glucose and metformin, including a significant decrease in the loss of mitochondrion membrane potential, a decrease in the mitochondrial localization of Noxa and a significant inhibition of caspase 3 activity in cancer cells, suggesting that ROS accumulation is associated with Noxa-mediated mitochondrial damage.in vivo, and showed that metformin decreased tumor volume and weight in a dose-dependent manner as well as effectively induced ASK1 phosphorylation.Of note, the study suggested that the combination of low glucose and metformin activate endoplasmic reticulum stress through ROS that triggers endoplasmic reticulum (ER) stress-associated apoptosis through ROS/ASK1/JNK pathway. Thus, the study confirmed the antitumor effect of metformin in ovarian cancer cells in vivo.Finally, they claimed that the treatment with metformin alone effectively increased the expression of Grp78, GADD153 and cleaved caspase 3, suggesting that metformin treatment in the xenograft model could induce ER stress and cell apoptosis. In conclusion, the study suggested that metformin induces ER stress and cell apoptosis, and showed that ASK1 plays an important role in the antitumor effect of metformin et al. investigated the effect of metformin on sphingosine kinases. These bioactive lipids are a singular group that is believed to regulate tumor progression [A recent study by Hart gression , includigression ,39,40. Cgression ,40.First, the authors wanted to determine whether S1P and SPHK1 promote ovarian cancer, cancer cell migration and invasion. The results of wound closure assays demonstrated that exogenous S1P promoted migration of ovarian cancer cells and that this effect was attenuated by pretreatment with metformin. Also, they confirmed that S1P promoted invasion of the ovarian cancer cell line and that metformin treatment reduced this effect.Second, direct evaluation in ovarian cancer cell lines (in which endogenous levels of SPHK1 are nearly undetectable) of the effect of SPHK1 overexpression on several hallmarks of cancer growth, showed that cells with ectopic SPHK1 expression had a higher rate of migration and proliferated more rapidly than control transfected cells, confirming that SPHK1 could indeed promote tumorigenicity.To determine whether metformin modulates the S1P rheostat in ovarian cancer, analysis of serum metabolites from patients with stage III/IV ovarian cancer was performed. This demonstrated that ovarian cancer patients who were using metformin for the treatment of Type II diabetes mellitus had significantly lower serum S1P levels than patients not using metformin.in vitro experiment that aimed to investigate the effects of metformin on the sphingolipid rheostat, where an ovarian cancer cell line was treated with metformin or control. The results confirmed that metformin-treated cells had markedly reduced S1P and sphingosine levels while ceramide levels were increased. Based on these findings the authors claimed that metformin shifts the rheostat toward reduced S1P production.These results were further supported in an Lu and colleagues used human gastric cancer AGS cells to investigate the antiproliferative effect of metformin on cancer cells and potentially determine the underlying apoptotic mechanisms . After tIn order to investigate the molecular pathway involved in the anticancer activity of metformin, and whether or not AMPK is involved, AGS cells were treated with an AMPK inhibitor, compound C and then p-AMPK (indicative of AMPK activation) and cell viability were analyzed.The results demonstrated that compound C suppressed phosphorylation of AMPK and significantly reversed the effect of metformin on cell viability compared with metformin treatment only. This suggests that, for AGS cells, modulated AMPK signaling mediates metformin-induced apoptosis. Further analyses of the phosphorylation of AKT (p-AKT), mTOR (p-mTOR) and p70S6K (p-p70S6K) demonstrated that metformin decreased the phosphorylation of AKT, mTOR and p70S6K without affecting protein expression, which indicates that metformin enhances apoptosis potentially by targeting AMPK and AKT/mTOR pathway in AGS cells.Based on different analyses the authors claimed several potential theories of how metformin induces apoptosis. For instance, the study claimed that the apoptotic mechanism of metformin may involve ERK, JNK and p38 MAPK-regulated pathways in AGS cells, or through an increase in mitochondrion ROS, or through an intrinsic signaling that induces mitochondria-mediated caspase-dependent apoptosis.et al. investigated the possible antitumor mechanism of metformin on human esophageal cells [in vitro by measuring the mRNA and protein levels of PELP1 in different ESCC cell lines. The data from both of these tests confirmed that not only the DNA copy, but also the mRNA and protein levels of PELP1 are increased in human ESCC.The recently published study by Wang al cells . The stuThe study suggested that PELP1 plays an important role in ESCC recurrence and elevated levels of PELP1 in ESCC lead to poor prognosis. This led the researchers to investigate the role of metformin on PELP1 using two ESCC cell lines that were treated with different concentrations of metformin over different time periods. The results indicated that metformin downregulated the levels of PELP1 protein and mRNA in a dose- and time-dependent manner.The study claimed that cellular treatment with metformin upregulates miR-497, which is an miRNA that was shown to be significantly downregulated in cancer tissues . InteresWhat is more, RT-qPCR results indicated that the mRNA levels of PELP1 and miR-497 were down- and upregulated by metformin, respectively. Further analyses showed that the levels of PELP1 in tumors carried by metformin-treated mice were significantly reduced and the levels of GSDMD, which is a characteristic of pyroptosis, were significantly increased. GSDMD interacts with membrane phospholipids to form pores in the plasma membrane that eventually leads to pyroptosis, which is a nontraditional programed cell death characterized by pore-formation on the plasma membrane resulting in cell swelling and plasma membrane disruption.in vitro and in vivo findings, the study claimed that metformin exerts its anticancer activity by the induction of nontraditional programed cell death in ESCC through targeting the miR-497-PELP1 axis.Based on both the et al., the recent study by Tseng and colleagues also supports the notion that metformin is likely to exert its anticancer activity via miRNA regulation [Like Wang gulation . The stuThe results showed a significant reduction on the migration ability and motility of human melanoma cell lines A2058 and A375 after a 3-day treatment with 5 mM of metformin. Also, flow cytometry analysis of melanoma cell cycle after metformin treatment indicated that the number of cells at the S and G2/M phases increased and the number of cells at the G0/G1 phase decreased compared with those in the control group. Likewise, a significant increase in the population of apoptotic cells after 5 mM metformin treatment was noticed. These results indicated that melanoma cell growth could be suppressed after metformin treatment through impairing cell cycle progression and inducing cellular apoptosis.Analyses from the next gene sequencing indicated that metformin may produce different gene expression in different cell lines. For example, metformin treatment resulted in an increase in 41 types of miRNAs expression and a reduction of 35 types of miRNAs expression in A2058 cells, but the results were different in A375 cells where the treatment resulted in a significant upregulation of 27 types of miRNAs and downregulated 28 types of miRNAs.However, based on the fact that metformin suppressed the growth of melanoma cells, it should have upregulated tumor-suppressive miRNAs and downregulated oncogenic miRNAs. Thus, out of the many upregulated/downregulated miRNAs, the study selected miR-192-5p and miR584-3p, which were previously verified to suppress tumors in human cancers to decipher metformin anticancer activity.Interestingly, an overexpression of miR-192-5p and miR584-3p on melanoma cell growth was observed that resulted in a clear suppression of colony formation and invasion abilities as well as proliferation, which were partly improved after miR-192-5p and miR584-3p inhibitor transfection.EFEMP1 and SCAMP3, which were significantly decreased after transfection with miR192-5p and miR-584-3p mimics, respectively.Furthermore, microarray analyses identified several potential target genes for miR-192-5p and miR-584-3p including the two oncogenes EFEMP1 and SCAMP3 knockdown on melanoma cell growth. They found that EFEMP1 and SCAMP3 knockdown had considerably suppressed cell colony formation, proliferation and substantially induced cell cycle arrest at G2/M and increased the sub-G1 population. Furthermore, cell invasion and migration were clearly suppressed by EFEMP1 and were not changed by SCAMP3 knockdown. In conclusion, the results suggested that metformin treatment suppressed the motility and growth of melanoma cells due to direct modulation of miR-192-5p-EFEMP1 and miR-584-3p-SCAMP3 axes in melanoma cells.They used siRNA to investigate the effects of Metformin (dimethylbiguanide) is the recommended first-line treatment for Type II Diabetes Mellitus . GeneralIn addition to its effect against hyperglycemia, the use of metformin was linked to possible reduction in risk of cancer and cancer-related mortality and that patients with diabetes using metformin were protected against different types of cancer, such as glioma, endometrial, breast, colon and gastric cancer .in vitro studies demonstrated that treatment with metformin was capable of inhibiting cellular metastasis of EC109 esophageal squamous cell carcinoma cells [While the antitumor mechanisms of metformin are not yet known, several reports have confirmed the antiproliferative ability of metformin against different types of cancer through different mechanisms . For exama cells , MG63 anma cells , and SiHma cells .Furthermore, in a dose- and time-dependent manner, metformin was shown to induce apoptosis in various cancer cell lines including SKOV3, A2780 and ES2 ovarian cancer cells , CAL 27,et al., who showed activation of the AMPK pathway by metformin is not confined to hepatocytes but can be observed in epithelial cells as well [Metformin-stimulated activation of AMPK in cells other than hepatocytes will result in control of cellular proliferation , which i as well .et al. who suggested that in tissues other than liver and muscles, the α1 isoform, which was shown to be associated with cellular proliferation, is physiologically more important than the metabolism and energy demand associated α2 isoform [Furthermore, the result of AMPK activation in epithelial cells, such as in breast cancer tissue, may lead to reduced proliferation, general reduction of mRNA translation and protein synthesis. These findings are supported by several reports including a study by Stapleton  isoform . Further isoform ,56.in vivo study showed metformin to have a reduced antitumor activity on control diet mice compared with high-energy diet mice associated with hyperinsulinemia and accelerated tumor growth [et al. suggested that low glucose environment can directly influence the result of AMPK activation and that the activation of AMPK promotes renal cancer cell proliferation under stressful metabolic conditions (On the contrary, it was reported that pretreatment with an AMPK antagonist inhibited prostate cancer cell proliferation ,58. Simir growth . Moreovenditions . Similaret al., which showed that the combination of metformin and glucose withdrawal were quite lethal to cancer cells [However, the observed survival in a low glucose environment may be due to the Warburg effect, where cancer cells reprogram their metabolism to promote growth, survival and proliferation . AMPK acer cells .et al., reported that glucose deprivation enhanced the antitumor effect of metformin, which aligns with earlier reports that showed the synergistic effect of combining low glucose with metformin-induced AMPK activation [In contrast, Ma tivation ,66. Howeet al. proved that metformin activates ROS and induces ER-dependent apoptosis [2+ [However, a number of reports including that by Yang poptosis . Furtherpoptosis , hypoxiapoptosis , viral ipoptosis  and ER-Cosis [2+  lead to osis [2+ , indicatosis [2+ .in vitro and some have confirmed it in vivo. Metformin can directly act on cancer cells by targeting the AMPK pathway in tumor cells that control metabolism, angiogenesis, inflammation and cancer stem cells [All of the studies included in the current report showed that metformin was able to, directly and indirectly, inhibit different cancer types em cells , or by iem cells .The studies reported a number of interesting potential mechanisms and external factors that could explain metformin’s antitumor effect. Of note, both the cancer cell sensitivity to metformin and the anticancer mechanism of actions reported were shown to be cell dependent, thus the different and sometimes conflicting mechanisms described might be due to the physiological differences between different cells. However, all of the reported theories of anticancer effect of metformin revolve around or are linked to AMPK activation and the external factors are mostly glucose related. Therefore, we can speculate that the antiglycemic activity of metformin represents an important means of its anticancer activity and that it is likely to exert its anticancer effect by targeting AMPK in cancer tissues.in vitro and in vivo, as well as the large number of clinical trials that aim to further investigate its efficacy as a potential anticancer adjuvant or treatment, reflect the potential that this drug can offer and warrants the need to decipher the exact mechanisms of its anticancer activity.The current study is a systematic review of the literature that investigates metformin’s antiproliferative mechanisms. While the current findings provide an insight into the anticancer mechanisms of metformin, it also highlights the importance of AMPK as a potential target for anticancer therapy. In conclusion, the safety profile, the route of administration and the long history of use make metformin an ideal candidate for repurposing to include other uses as well. However, the significant efficacy of metformin to inhibit cancer growth proliferation Metformin is a relatively safe drug that has been used as an antidiabetic medication for several decades, which makes it a good candidate for repurposing. Currently, the drug has several off-label uses including the treatment of symptoms of polycystic ovarian syndrome. While we believe that metformin will have new indications/uses, either off-label or in-label, added to the existing ones, the indications are unlikely to include anticancer as a monotherapy. Nonetheless, metformin may be more suited as an adjuvant or as a combination to anticancer regimen. Further studies are needed to identify cellular targets of metformin that could be utilized in anticancer treatments, as well as the role of its glucose lowering ability as a potential mechanism or contributing factor. Additionally, future research should focus on investigating metformin’s tissue distribution in normal and cancer cells, which will probably help in better understanding its anticancer mechanisms and improve its potential anticancer usage.Metformin is a widely used antidiabetic drug that has been reported to have antiproliferative activity against several cancers.The antiproliferative mechanism of metformin remains unclear.This study investigates the reported mechanisms of action that may contribute to metformin’s antiproliferative activity.


Ulnar impaction syndrome (UIS) is a common source of ulnar-sided wrist pain, yet not all cases of radiographic ulnar impaction are symptomatic. We retrospectively analyze clinical or radiologic factors that affect prognoses of conservative treatment for idiopathic UIS. A total of 114 patients who had been diagnosed with UIS were treated with 6 weeks of short arm orthosis followed by formal physiotherapy for 6 weeks, with lifestyle modification to limit aggravating movements. The response to treatment, including pain numeric rating scale on an ulnar provocation test, grip strength, Disability of the Arm, Shoulder, and Hand score (DASH), was assessed at 24-week follow-up. For the 24-week follow-up, 29 patients (25%) underwent ulnar shortening osteotomy due to persistent symptoms after conservative treatment, and 18 (16%) patients had pain scores of greater than 5, but they had not undergone surgery. After controlling for confounding variables, female gender (odds ratio (OR) 1.39), duration of symptom (OR 1.27), high pain NRS score on provocation test (OR 1.45), and enhanced carpal or distal ulna bone on MRI (OR 1.82) were associated with a higher likelihood of treatment failure. Knowledge of the factors offers physicians insight into predicting prognoses and helps patients set realistic expectations. It is a degenerative condition that occurs secondary to excessive load across the ulno-carpal joint2. The condition is termed idiopathic UIS when patients have congenital or dynamic positive ulnar variance with wrist pronation and forceful grip without any history of fracture or premature physeal arrest3. Patients complain of ulnar-sided wrist pain and occasionally swelling and loss of wrist motion and forearm rotation5. Although this condition is usually associated with positive ulnar variance, it is not unusual to see a patient with radiographic evidence of ulnar impaction but who has minimal or no symptoms, and not all cases of ulnar impaction are symptomatic6. Furthermore, there is little information known about the prognostic factors and natural history of UIS7.Ulnar impaction syndrome (UIS) is a common source of ulnar-sided wrist pain10. However, few studies have investigated the correlations between radiologic findings and the clinical symptom severity and prognosis of UIS. Clarity regarding these radiologic findings and their correlations with the clinical symptoms and prognoses would be useful for individualizing and quantifying each patient’s condition.Radiologic evaluations including magnetic resonance imaging (MRI) are frequently performed for patients with UIS because it allows for earlier detection of an abnormality in the triangular fibrocartilage complex (TFCC), cartilage, or bone marrow of carpal bones; it is also helpful in extensive differential diagnosis for patients with ulnar-sided wrist painLittle information is currently available to analyze unsatisfactory outcomes after conservative treatment for idiopathic UIS, and knowledge of these factors will offer physicians insight into predicting prognoses and help determine the proper indication for surgical treatment for UIS. The purpose of this study was to analyze the factors that affect clinical symptoms and prognoses of conservative treatment for UIS. We hypothesized that certain demographic, clinical, and radiologic features would be associated with unsatisfactory outcomes.A total of 198 patients diagnosed with UIS at an urban tertiary referral hospital between June 2013 and May 2015 were retrospectively enrolled in this study; the diagnosis had been based on clinical symptoms and physical examination with radiographic confirmation of positive ulnar variance. The inclusion criteria for the diagnosis were (1) a history of ulnar wrist pain that worsened with pronation and ulnar deviation of the wrist during such activities as opening a jar, squeezing a wet towel, typing, or changing the gearshift in an automobile for more than three months; (2) no history of trauma of the forearm and wrist; (3) a positive provocation test ; and (4) plain radiograph with positive ulnar variance in neutral or pronation power-grip position. Patients underwent complete wrist examinations to rule out other sources of pain such as pisotriquetral arthritis, distal radioulnar joint arthrosis, extensor carpi ulnaris subluxation or tendonitis, or neuritis of the dorsal cutaneous branch of the ulnar nerve. The exclusion criteria were radiographic evidence of an old fracture of the forearm or wrist or radiographic evidence of congenital anomalies such as a Madelung deformity; based on these, 181 patients were approached for study. Of those, 123 patients had undergone MRI to identify abnormality in the TFCC, cartilage, or bone marrow of carpal bones. Among these patients, nine (7%) were lost to follow-up before six months, leaving 114 for analysis here , with 0 indicating no pain and 10 indicating the worst pain imaginable. We also analyzed patients’ self-reported outcomes with the Disabilities of the Arm, Shoulder and Hand (DASH) score; the DASH is a self-administered, upper-extremity specific questionnaire that consists of 30 items on physical functions, social-role function, symptoms, work, sleep, and confidence12. The items are rated on a five-point Likert-type scale where 1 is without difficulty or no symptom and 5 is unable to engage in activity or very severe symptoms. Thus, the DASH provides a best possible score of 0 and a worst possible score of 100. The DASH is user friendly, reliable, and valid for a range of upper extremity disorders14. A study investigator who was a trained nurse checked all returned questionnaires for completion, and the participants were assisted in completing the missing items.Patients returned for their functional assessments at 12 (11–14) and 24 (22–26) weeks after conservative interventions  with positive ulno-carpal stress test or surgical intervention at six- month follow-up with conservative treatment. The decision to undergo surgery was mutual between patient and surgeon.2 of 0.15) for a regression with five main predictors.A post hoc power analysis indicated that the sample consisting of 114 patients would provide 90% statistical power with an α of 0.05 for a medium effect size  and the outcome variables . We tested these relationships using an independent t test or a one-way ANOVA for categorical potential predictors and a correlation coefficient for continuous predictor variables. We selected bivariate predictors with p < 0.1 on bivariate analysis as candidates for the multivariable linear regression model to prevent model over-fitting, dummy coding the categorical variables and considering the largest subgroup to be the reference group.All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.The mean pain NRS  and DASH  scores exhibited significant clinical improvement at the 24 week follow-up , symptom duration , high pain NRS score on ulnar provocation test , and enhanced carpal or distal ulna bone  were associated with a higher likelihood of treatment failure.In cases of idiopathic ulnar impaction syndrome, nonoperative treatment should be provided initially because not all cases of radiographic ulnar impaction are symptomatic, and it is crucial to identify predictive factors related to the clinical symptoms and prognoses of UIS; however, few reports suggest specific clinical and radiologic features as predictive factors for conservative treatment of idiopathic UIS. The current study was an attempt to determine which preoperative clinical or radiologic features were associated with poor outcomes of conservative treatment for UIS. We found that female gender, long symptom duration, high pain NRS score on ulnar provocation test, and enhancement of carpal or distal ulna bone on MRI were associated with a higher likelihood of treatment failure after conservative treatment for idiopathic UIS.7. Long symptom duration was also reported to be associated with poor outcomes after ulnar shortening osteotomy15. The sex difference in treatment outcomes could in part be explained by higher physical vulnerability16 or pain sensitivity17 in women; musculoskeletal pain and disability were found to be more prevalent18 and more severe in women19. The ulno-carpal stress test is reportedly highly sensitive but not specific for UIS; other ulno-capal pathology including lunotriquetral ligament injury or isolated ulno-carpal arthritis will produce positive findings21. However, our results suggests that pain NRS scores on ulno-carpal stress test in the presence of a history of ulnar wrist pain worsened by pronation or ulnar deviation of the wrist and plain radiograph with positive ulnar variance had significant prognostic value for conservative treatment of idiopathic UIS.In terms of patient demographic or clinical factors, female gender, longer symptom duration, and initial high pain NRS score on ulnar provocation test were associated with a higher likelihood of treatment failure after conservative management of idiopathic UIS. The natural history of ulno-carpal abutment is thought to be one of persistent or progressive derangement, and a long persistence of symptoms may be attributable to pathologic change to the ulno-carpal joint20, suggesting that the diagnosis of UIS is made on clinical ground and supported by radiographic studies. However, MRI findings are often a precursor to plain radiographic findings22 and our results suggest that carpal or distal ulna bone enhancement is distinctive and helps to predict the prognosis. Subchondral bone marrow edema is an indirect sign of chondromalacia and an early UIS finding22. One previous study showed that the signal intensity often returns to normal after ulnar-shortening osteotomy6.In terms of radiologic factors, patients with the signal enhancement of carpal or distal ulna on MRI had higher pain NRS and DASH scores, and this was the only significant prognostic factor; almost none of the radiologic factors including degree of ulnar plus variance, type of TFCC lesion, or arthritis of ulno-carpal or distal radio-ulnar joint had a significant effect on the treatment failure for idiopathic UIS. These results are consistent with previous findings that diagnostic MRI findings and values are limited in TFCC injury24. The conservative treatment protocols with 3- to 4-month duration might be not sufficient for patients with a long symptom duration or bone enhancement on MRI. Second, surgical management is not a precise indicator of the efficacy of conservative treatment because the decision for surgery is multifactorial and not determined entirely by symptom abatement via treatment. Thus, we defined treatment failure as persistence of pain NRS score greater than 5 with positive ulno-carpal stress test result or surgical intervention at six-month follow-up. Furthermore, we only used one questionnaire to evaluate the patients’ functional outcomes, but the minimal clinically important differences or responses after conservative treatment for idiopathic UIS could differ across functional assessments, and another functional assessment might have resulted in different conclusions. Third, nine (7%) patients were lost to follow-up before the six-month evaluation, and there were some missing responses and questionnaires in our cohort. Finally, our subjects were limited to one ethnic population drawn from an urban area, and therefore, their characteristics and the study results may not be representative of general populations.There were a number of limitations to our study. There were no follow-up data on functional outcomes beyond 24 weeks of conservative treatment. Some patients whose symptoms recurred likely had surgery whereas others could have exhibited improvement in objective findings and outcome scores after the study time period. In addition, there is no consensus about the best way of conservative management for UIS although several conservative interventions are routinely used in clinical practiceAfter 24-week follow-up, approximately 60% of patients showed improvements in both objective and patient-reported outcomes after conservative treatment for idiopathic UIS. However, 25% patients underwent ulnar shortening osteotomy due to persistent symptoms after conservative treatment, and 16% patients who had not undergone surgery had pain NRS scores of greater than 5 on ulnocarpal provocation test at the last follow-up. Our study suggests that female gender, long symptom duration, initial high pain NRS score on ulnar provocation test, and enhancement of carpal or distal ulna bone on MRI were associated with a higher likelihood of treatment failure. Although the outcomes are not modifiable, knowledge of the factors that affect unsatisfactory outcomes offer physicians insight into predicting prognoses and interpreting treatment outcomes and can help patients set realistic expectations and increase satisfaction.


Longitudinal-zonation hypotheses generally predict gradual changes in fish composition from upstream to downstream due to changes in habitat conditions, but largely disregard downstream effects on upstream segments. Floodplains of large rivers represent areas of high connectivity during flood periods and can act as stable refuges in dry seasons, which may attenuate deterministic constraints imposed by local conditions on fish assemblages in surrounding habitats. In this study, we investigated the effects of proximity to large rivers on taxonomic- and functional-diversity patterns of stream-fish assemblages in an extensive region of Central Amazonia. We sampled 31 headwater-stream reaches in nine catchments in the Purus and Madeira Rivers interfluve between December 2014 and March 2015. Ninety seven fish species from seven orders and 19 families were captured. The results indicate that distance to large rivers is more important than distance among sites and local conditions in explaining functional and taxonomic diversity of stream-fish assemblages at large spatial scales. We also found a decrease in taxonomic and functional richness towards headwaters, mainly related to the loss of benthic and sedentary species along the distance gradient. These species may be favored by the proximity to refuge areas and high resource availability near the floodplain. In contrast, upstream assemblages were mainly occupied by small-sized, nektonic species with higher dispersal capacity, highly dependent of allochthonous resources. Downstream effects could be detected for many kilometers upstream in hydrographic catchments and this reinforces the crucial role of connectivity between fluvial habitats in maintenance of stream-fish diversity patterns in the region. Longitudinal fish zonation occurs when assemblages undergo changes along the linear gradient that connects upstream and downstream portions of a river network . In geneProximity to large rivers increases dispersal opportunities between habitats and availability of allochthonous resources, amplifying the role of mass-effects and source-sink dynamics in local and regional fish assembly ,7,13,16.In a functional perspective, the river-continuum hypothesis predicts that headwater systems should be primarily occupied by small-sized and nektonic fish species mainly dependent on allochthonous resources, while downstream systems should harbor larger species predominantly supported by autochthonous production, especially for benthic fish guilds ,12,23. HBeta-diversity indices are useful tools to infer changes in species composition between sites and to identify the ecological gradients that structure these changes. Several methods have been proposed to partition the two main components of dissimilarity between habitats: species turnover and richness differences/nestedness ,29. SpecIn this study, we investigate how proximity to large rivers determines the taxonomic and functional composition of fish assemblages in headwater systems in an extensive region of Central Amazonia. The region is characterized by a highly dynamic landscape where headwater streams can dry completely during drought months, and/or receive inputs from floodplains in the high-water season, depending on their distance to large rivers. We hypothesized that fish assemblages in similar-sized streams would differ in taxonomic and functional richness as a function of distance to large-river floodplain systems. This pattern should be determined mainly by the greater occurrence of species that benefit from the proximity to refuge areas and from the higher productivity of the floodplain, such as detritivorous benthic and less dispersive species. In contrast, fish assemblages in streams further from large rivers should be dominated by species with better dispersal abilities and sustained by allochthonous food sources. We expected distance to the large rivers to be an important factor structuring stream-fish assemblages, and that streams similarly positioned in the drainage network would share a greater number of species and functional traits, regardless of local conditions and watercourse-distances among streams.Sampling was undertaken along 700 km of the Madeira-Purus interfluve, in southwestern Amazonas State, Brazil. The region is covered by dense rainforest with mean annual precipitation varying from 2000 to 2400 mm  Fig 1)..Fig 1). st to 3rd order streams) in nine catchments along the Madeira-Purus interfluve that drain to the Madeira or Amazon Rivers. The dense network is mainly non-hierarchical and the sub-basins can be short and flow directly to large rivers or be extensive and receive little influence from the floodplain of large rivers. Sampling was carried out in the first months of the rainy season, between December 2014 and March 2015, when the streams returned to normal flow and the water level remained stable for months, allowing rapid recolonization of formerly dry stream channels. During the inundation phase, small streams overflow and vast areas are flooded, including parts of the road, making access difficult. These limitations make it hard to obtain more accurate information about the extent of flooding in terrestrial habitats. All stream segments sampled are permanent sampling plots of the Research Program in Biodiversity (PPBio) field-site network (http://ppbio.inpa.gov.br/sitios/br319).We sampled 31 headwater-stream reaches , coarse litter , fine litter (organic silt) or roots (fine roots from riparian vegetation). Flow velocity was estimated as the average time that a floating object took to travel 1 m, replicated three times. For statistical analyses, we use the average values of channel width, depth, flow velocity and the percentage of each substrate type of each stream reach. For details regarding the environmental-sampling protocol see , ,  and 42]42]. All Each fish species was functionally characterized with respect to locomotor ability, habitat use and food intake. Body mass and morphometric measurements were taken from 3–10 adult individuals per species and then combined in 13 ecomorphological traits  on the functional-distance matrix to obtain the coordinates of the species in multidimensional space. To define the number of dimensions that best describes the functional space, we used the framework developed by , which aTo evaluate how functional space varies between fish-assemblages, we computed the functional-richness index (FRic), which represents the amount of functional space filled by all species within the assemblage, indicating the range of trait combinations or niche occupation . To inveWe calculated the variation in taxonomic species composition  among the 31 stream reaches by means of pairwise comparisons using Sørensen-based index developed by  for use Variation in functional diversity between stream reaches (hereafter Fβsør) was calculated from pair-wise intersections between convex hulls in a multidimensional functional space created with the first four PCA axes using the Sørensen dissimilarity index. Fβsør was decomposed into functional turnover (Fβsim) and functional nestedness (Fβnes), following the framework proposed by . FunctioTo test the effect of distance to large rivers on diversity patterns, we measured the distance between each stream reach and the nearest large river (Madeira or Amazon Rivers) following the watercourse. The distances were calculated by summing the lengths of segments between each stream reach and the nearest large river in a GIS hydrological framework developed by  using QGTo evaluate the effect of watercourse distance on taxonomic and functional patterns, we calculated the network distance between each pair of stream reaches using the QNEAT3 extension for QGIS Network Analysis in the GIS hydrological framework developed by . We alsoAll statistical analyses were carried out using R software . The relSimilarities within catchments could cause autocorrelation, so we also provide a partial Mantel test to examine the relationships between the beta diversity components and the predictor variables, including a catchment identity matrix as confounding factor to ensure that significant correlations are not determined by autocorrelation effects due to greater similarities within catchments. The catchment identity matrix consists of a dissimilarity matrix where pairwise comparison of stream reaches from the same catchment had value equal to zero and comparisons between streams of different catchment have had a value equal to one. The analysis results are given in 2 were calculated using the package “MuMIn” [I values using the residuals of linear mixed-models to evaluated if the significance of the relationship that we found is affected by spatial autocorrelation and the results are given in To evaluate how local conditions interact with distance to large rivers to determine functional characteristics of stream-fish assemblages, we summarized the effects of local conditions with a Principal Component Analyses (PCA) for the standardized abiotic variables and retained the first axis as a descriptive variable, which represented 52% of variation in local conditions. The loading values for each variable are given in  “MuMIn” . We alsoCopella callolepis, Apistogramma agassizii, Crenuchus spilurus and Hemigrammus melanochrous, in decreasing order of participation). Most species found in the streams were small-sized fishes typical of headwater systems and almost 75% of species had adult standard length less than 15 cm.A total of 97 fish species from seven orders, 19 families and 57 genera were captured. Characiformes was the richest order with 52 species, followed by Siluriformes (14 species) and Gymnotiformes (12 species); the remaining 19 species were from four orders (We found a significant positive correlation between taxonomic beta diversity (Tβsør) and distance to large river, mainly associated with the richness component (Tβrich) . The parFew species occurred continuously along the connection gradient to large rivers, indicating a strong pattern of substitution between catchments , and theIn accordance with the Mantel tests, the LMM results indicated that distance to large rivers had a negative effect on richness of stream-fish assemblages ; Fig 4. There was a significant correlation between the functional beta-diversity index (Fβsør) and difference in distance to large rivers , indicatst PCA) , body transversal surface (Bsf) and aspect ratio of the pectoral fin (PFar), and distance to large rivers, indicating that assemblages from streams far from large rivers are mainly composed by smaller-sized species with greater swimming ability that inhabit the upper and middle layers of the water column . Furtherst PCA) . That axst PCA) .The results corroborated our hypothesis that, at large spatial scales, differences in connection to large rivers are more important than watercourse distance and local conditions in determining functional and taxonomic dissimilarity between stream-fish assemblages. The longitudinal fish zonation that we found is mainly determined by the increase in taxonomic and functional richness close to large rivers and cannot be attributed to an increase in local habitat availability, since all streams are positioned at the headwaters of their respective catchments and are of similar physical dimensions. Similarity within catchments could result in autocorrelation, but the fact that catchments similarly positioned in relation to large rivers, such as catchments 1 and 9, share species and richness patterns is strong evidence of structuring processes unrelated to geographical proximity and the significance values of tests are not affected by autocorrelation, because there was no significant autocorrelation in the residuals of the linear mixed models . This reProximity to floodplains seems to be one of the main factors favoring the constitution of assemblages with higher species richness close to large rivers, since flood pulses allow dispersal that increases the lateral connectivity and the heterogeneity of resources and habitats ,22,52–54Functional changes in fish assemblages along the longitudinal gradient have been frequently reported, mainly related to changes in local conditions ,5,12,55.Microcharacium, Axelrodia, Tythocharax, Xenurobrycon, Nemuroglanis and Priocharax, were predominant in the assemblages of the most isolated streams. Miniature species rarely live longer than one year in the wild and low nutrient levels in their natural habitats may limit body development of these lineages, which do not exceed 26 mm standard length as adults [Hoplerythrinus, Synbranchus, Pterygoplichthys, Lepthoplosternum and Gymnotus, did not occur in the stream reaches furthest from the large rivers. These species are occasionally caught in low-order streams [Small-sized or miniature species, such as those of the genera s adults . The lacs adults . Large-b streams , 42 but Our results highlight the importance of connectivity between small catchments and river-floodplain systems for the maintenance of stream-fish assemblage composition, diversity and functional characteristics. The fact that the drainages studied are exposed to seasonal droughts may amplify the role of dispersal in structuring of local assemblages and select species capable of reaching favorable environments throughout the catchment –59. The S1 Text(PDF)Click here for additional data file.S2 Text(PDF)Click here for additional data file.S1 Table(XLS)Click here for additional data file.S2 TableMore details about functional-trait calculations can be found in (XLS)Click here for additional data file.


Retina is the highest oxygen-demanding and vascularized tissue in the body. Retinal development and function require proper vascularization and blood vessel function and integrity. Dll4 is most prominently expressed in the endothelium of angiogenic blood vessels and in quiescent arteries and capillaries in all tissues and organs of the mammalian species, and it is the key regulator of blood vessel sprouting.  Dll4 is a transmembrane protein that acts as a ligand for Notch receptors 1 and 4. Genetic deletion of Dll4 causes severe abnormalities in embryonic and postnatal vascular development. Deletion of even a single Dll4 allele results in almost complete embryonic lethality due to severe vascular abnormalities, the phenomenon called haploinsufficiency indicating the critical role of Dll4/Notch in vascular development. Dll4/Notch pathway interplays at multiple levels with other signaling pathways including VEGF, Wnt/Fzd, and genes controlling vascular toning. Multiple studies of the effects of Dll4 inhibition were performed in the developing retina to elucidate the key functions of Dll4 in normal and pathological angiogenesis. Several genetic approaches and therapeutic molecules were tested to evaluate the biological and therapeutic effects of acute and prolonged Dll4 inhibition in the eye and oncology.  All current studies demonstrated that Dll4 controls blood vessel sprouting, growth, and remodeling in normal and pathological conditions as well as arterial-venous differentiation. Genetic and therapeutic Dll4 modulation studies show that Dll4 inhibition can promote blood vessel sprouting and might be useful to stimulate vessel growth in the ischemic retina and Dll4 is the key modulator of the postangiogenic vascular remodeling that ultimately defines vascular patterning. In. In29]. In the developing superficial retinal vasculature and all other forming vascular beds, Dll4 specifically expressed at the tips of the sprouting blood vessels . Under tHowever, Dll4 is expressed not only in “tip” and stalk cells during active angiogenesis, it is also expressed at particularly high levels in the vascular endothelium of mature arteries and arterioles, and to a lesser extent in capillaries, but not veins , 33 Fig. MoreoveVEGF-A is the main growth factor controlling endothelial cell proliferation and blood vessel outgrowth , 39, whiIt was also found that the degree of vascular abnormalities and tissue side effects depend on the potency of Dll4 inhibition, namely, on the dose and reagent affinity   that mimTo evaluate the therapeutic potential of Dll4 inhibition in ocular vascular diseases, Dll4-Fc chimeric recombinant protein (a soluble Dll4/Notch inhibitor) and an anti-Dll4-blocking antibody were utilized in the murine model of OIR in multiple settings. In OIR, Dll4/Notch inhibition results in a more rapid revascularization of the ischemic portions of the retina , while sThere are several severe retinal vascular disorders besides ROP causing blindness that are characterized by blood vessel loss at the initial stages and severe retinal ischemia, and as a result, excessive pathological neovascularization on the terminal phases that include diabetic retinopathy (DR)  that priAlthough the role of Dll4 in sprouting angiogenesis has been extensively studied, until recently, very little was known about the function of the Dll4/Notch pathway in postangiogenic and mature blood vessels, where Dll4 is expressed in the arteries.COIN/+mice or pharmacological inhibition of Dll4/Notch signaling using Dll4-Fc inhibited the normal developmental pruning of capillaries in the developing retinal vasculature, as well as blood vessel regression following the acute exposure to hyperoxia in OIR [Genetic or pharmacological modulation of Dll4/Notch signaling affects postangiogenic blood vessel remodeling and regression, a process that plays a critical role in determining the patterning and density of blood vessels in mature tissues. Specifically, constitutive or conditional genetic deletion of Dll4 in ROSA26–CreERT2+/−; Dll4a in OIR .Genetic deletion of a negative feedback regulator of the Notch pathway, Notch-regulated ankryrin repeat protein (Nrarp) , resultsIn OIR, blood vessel regression is preceded by the excessive vasoconstriction of the immature capillaries and loss of blood flow, followed by rapid synchronous apoptosis in the capillaries . In someEffects of Dll4/Notch signaling modulation were largely distinct from changes produced by modulation of VEGF-A activity, at both morphological and gene expression levels, indicating that these two pathways independently regulate postangiogenic blood vessel remodeling and homeostasis .From the therapeutic perspectives, importantly, attenuation of Dll4/Notch signaling maintained blood flow and prevented capillary regression without inducing the gross morphological changes in the postangiogenic vasculature that were evident following administration of VEGF-A . NoteworConditional genetic deletion of another transcription factor, negative-feedback regulator of Notch signaling pathway, recombination signal-binding protein Jkappa (RBP-J) in the adult mice, strikingly induced spontaneous angiogenesis in the multiple tissues, including retina and cornea . That daRecent data also suggest that Dll4/Notch signaling is involved in arteriogenic differentiation that further limits angiogenic sprouting and maintains arterial endothelium in nonproliferative state , 58.Dll4/Notch signaling pathway modulates angiogenic sprouting and blood vessel growth, and Dll4/Notch inhibition can promote therapeutic angiogenesis in the ischemic tissues and at the same time over exuberate pathological angiogenesis in solid tumors showing anticancer effect. Dll4/Notch inhibition can also maintain capillary perfusion and prevent pathological blood vessel regression, for example, induced by exposure to hyperoxia. A number of diseases, such as retinal ischemic disorders, cerebral, and cardiac ischemia, are characterized by vascular insufficiency resulting in reduced tissue perfusion . In thes


Epithelial ovarian carcinoma is the most predominant type of ovarian carcinoma, the deadliest gynecologic malignancy. It is typically diagnosed late when the cancer has already metastasized. Transcoelomic metastasis is the most predominant mechanism of dissemination from epithelial ovarian carcinoma, although both hematogenously and lymphogenously spread metastases also occur. In this review, we describe molecular mechanisms known to regulate organ-specific metastasis from epithelial ovarian carcinoma. We begin by discussing the sites colonized by metastatic ovarian carcinoma and rank them in the order of prevalence. Next, we review the mechanisms regulating the transcoelomic metastasis. Within this chapter, we specifically focus on the mechanisms that were demonstrated to regulate peritoneal adhesion—one of the first steps in the transcoelomic metastatic cascade. Furthermore, we describe mechanisms of the transcoelomic metastasis known to regulate colonization of specific sites within the peritoneal cavity, including the omentum. Mechanisms underlying hematogenous and lymphogenous metastatic spread are less comprehensively studied in ovarian cancer, and we summarize mechanisms that were identified to date. Lastly, we discuss the outcomes of the clinical trials that attempted to target some of the mechanisms described in this review. Ovarian carcinoma is the fifth leading cause of death from female cancers  and compHGSOC metastases spread via several distinct routes, including transcoelomic, hematogenous, and lymphogenous, with the former being the most predominant ,6. TransThese patients are typically treated by surgery and neoadjuvant chemotherapy (NACT) consisting of a combination of a platinum drug and a taxane. The Gynecologic Oncology Group defined optimal debulking as residual implants less than 1 cm . OptimalFor these reasons, the mechanisms regulating peritoneal metastasis from EOC are studied most extensively with the aim of identifying ways of preventing re-colonization of mesothelial linings and blocking or retarding the growth of intraperitoneal lesions using novel targeted molecular therapies that could be applied either alone or in conjunction with conventional chemotherapies. The hematogenous route of metastatic colonization also contributes to formation of intraperitoneal metastases ,24. The In this review, we focus on the molecular mechanisms currently known to underlie the formation and development of organ-specific metastases from EOC.Several studies assessed the patterns of metastatic spread from EOC at relapse, as well as at autopsy. These studies demonstrated that, although EOC typically colonized a wide variety of organs and tissues, there was not one single metastatic site that was always involved by metastasis in all studied cases. The main site of metastasis was the peritoneum, including the parietal and visceral peritoneum and omentum, which was involved in 77% of cases on average among several reports  is linked with the propensity to seed either mainly peritoneal or peritoneal and distant metastases. Ninety-six percent of all cases belonging to HGSOC carry mutations in the sequence ,38,39. Nype TP53 , althougype TP53 .In summary, the studies showed that metastases from ovarian carcinoma predominantly formed locally within the peritoneal cavity ; however, a large number of cases also presented with distant metastases, most commonly at lymph nodes, liver, and lung.Transcoelomic dissemination is thought to be the major route via which EOC metastasis spreads . PeritonProgression of ovarian carcinoma is often accompanied by the presence of the malignant ascites, a fluid that accumulates within the peritoneal cavity ,45. It wThe peritoneal mesothelium is a monolayer of mesothelial cells that lines the abdominal cavity . DissemiMesothelial cells produce hyaluronan, which is thought to serve as a protective layer preventing attachment of the malignant EOC cells . InflammThe majority of EOC cases affect the elderly, as the median age at diagnosis is 63 . Thus, sEOC cells can co-opt mesothelial cells in order to facilitate peritoneal adhesion. It was demonstrated that EOC cells can secrete exosomes enriched for CD44 molecule (CD44). These exosomes could be internalized by mesothelial cells, resulting in the secretion of matrix metallopeptidase 9 (MMP-9), which helps EOC with cell invasion . MesotheNumerous in vitro and in vivo studies used different experimental approaches to show that disseminating ovarian cancer cells themselves express a number of membranous receptors and adhesion molecules that facilitate their adhesion to mesothelial cells expressing ligands for these receptors .The first interaction reported to facilitate peritoneal adhesion was between hyaluronan (HA) expressed by mesothelial cells and CD44 expressed by EOC cells ,61. It wOther molecular interactions supporting peritoneal adhesion are fostered by a glycoprotein on the surface of EOC cells, ovarian carcinoma antigen CA125 (MUC16), and mesothelin expressed on the surface of mesothelial cells, both of which were demonstrated by in vitro studies using EOC cell lines ,67.A series of in vitro studies that utilized EOC cell lines and mesothelial cells demonstrated that neuropilin-1 expressed in mesothelial cells can interact with a glycoprotein expressed in EOC cells, the L1 adhesion molecule (L1CAM) .3CR1) expressed in EOC cells can interact with its ligand CX3CL1 expressed (in its transmembrane form) by peritoneal mesothelial cells [Both in vitro studies of EOC and mesothelial cell co-cultures and in vivo studies of short-term adhesion and survival xenograft studies showed that a chemokine receptor fractalkine  occurring in the peritoneal microenvironment of EOC, which involved stimulation of expression of a calcium-dependent receptor, P-selectin, on mesothelial cells by a C–C motif chemokine ligand 4 (CCL4 or MIP-1β) secreted by the AAMs; EOC cells expressing CD24 interacted with P-selectin-expressing mesothelial cells, as demonstrated by ex vivo and in vivo studies of a syngeneic ovarian cancer model .Once metastasizing EOC cells adhered to the mesothelial monolayer, to anchor metastatic lesions, they need to disrupt the mesothelial lining and invade submesothelial parenchymal tissues consisting of the ECM, stromal cells, and cells of the organ parenchyma. EOC cells express various molecules that assist their invasion into the parenchyma of the organs and tissues to which they have adhered. Overexpression of alcohol dehydrogenase 1B (ADH1B) was demonstrated to regulate EOC cell attachment and clearance of the mesothelial lining, as well as subsequent matrix invasion . AdherenAfter breaching the mesothelial monolayer, ovarian cancer cells quickly adhere to the submesothelial matrix, which is predominantly composed of collagens type I and III, using both α2β1- and α3β1-integrins ,79. MT1-In summary, several molecular interactions between cancer and mesothelial cells establish successful cell–cell adhesion during mesothelial adhesion. Disseminating cancer cells take advantage of secreted molecules produced by mesothelial cells and can reprogram their gene expression to aid peritoneal adhesion. Likewise, aging can amplify the process of peritoneal carcinomatosis by providing more permissive conditions for cancer cell adhesion. Importantly, EOC cells themselves express proteins that enable their attachment and tissue invasion.The omentum is a peritoneal fold that connects the stomach with abdominal organs . The omeIn metastasis from EOC, omentum plays a central role as one of the major tissues hosting peritoneal metastatic lesions ,25,27,32Recent studies suggested that, as cells detach from the primary tumor and become suspended in the ascites, they undergo a metabolic shift from glycolysis to lipid metabolism, which later affords and facilitates their survival within the omental tissue . MetastaMilky spots are mainly composed of macrophages and lymphocytes, and represent initial lymphatics of the omentum that drain into lymph collectors . PreclinOnce EOC cells lodge onto the omentum, proximity to adipocytes results in upregulation of fatty-acid-binding protein 4 (FABP4) and a fatty-acid receptor CD36, followed by transfer of lipids from adipocytes to EOC cells, and induction of lipolysis in adipocytes and β-oxidation in cancer cells ,94. InteFibroblasts in omentum also play a prominent role in regulating this organ-specific metastasis. A study uncovered interaction between tumor necrosis factor alpha (TNFα), transforming growth factor alpha (TGFα), and epidermal growth factor receptor (EGFR); this TNFα–TGFα–EGFR interacting loop is thought to form between EOC cells and fibroblasts that reside in omentum, and it is suggested that it functions to promote peritoneal metastasis .Interactions between chemokine receptors expressed by cancer cells and their corresponding chemokines at the metastatic sites was suggested to regulate homing of metastasizing cells to their niches. Among these interactions, association between the C–X–C motif chemokine receptor 4 (CXCR4) and its ligand, stromal-derived factor 1, was demonstrated to regulate pro-metastatic functions of cells from several cancer types, including ovarian ,99,100. Thus, studies of the mechanisms of omental metastasis to date demonstrated important roles of the omentum itself and metastasizing EOC cells in facilitating formation and development of this major type of metastatic lesions.3CL1) and its receptor (CX3CR1), regulates dissemination of the CX3CR1-positive EOC cells to the surfaces of the CX3CL1-positive tissues, including peritoneal wall, diaphragm, liver, mesentery, and retroperitoneal kidneys [The mechanisms regulating organ-specific intraperitoneal dissemination to other organs, including peritoneal wall, viscera of the bowels, viscera of the liver, etc., are less well understood as compared to the mechanisms regulating formation of the omental metastasis. Several studies uncovered the pivotal role of the chemokine–receptor interactions in regulating these organ-specific metastases. Inhibition of CXCR4 with AMD3100 significantly reduced colonization of the colon, peritoneal wall, diaphragm, and liver . Downreg kidneys ,70.Peritoneovenous shunting is a procedure in which a shunt could be used to return the peritoneal fluid from the peritoneal cavity into veins, such as the superior vena cava or the internal jugular vein, by means of a one-way valved anastomosis ,104,105.In another patient-based study that focused on investigating the outcomes of inferior vena cava filter placement in patients with epithelial ovarian, fallopian, and primary peritoneal cancer, the authors reported that patients who underwent this procedure had significantly lower survival and significantly higher incidence of deep vein thrombosis and distant metastasis , supportExperimentally, evidence of development of the hematogenous metastasis within the omentum was recently presented . A novelIn another study, three approaches were employed to investigate the role of the hematogenous route of EOC metastases, including an intravascular tail-vein injection of ovarian cancer cells, as well as subcutaneous engraftment of murine and human tumors. Primary ovarian cancer cells were co-injected with mesenchymal stem cells subcutaneously. To promote formation of blood vessels in the tumor, human infantile hemangioma stem cells were co-injected as well. This protocol resulted in 100% engraftment rate and macroscopic ovarian metastases by the time of sacrifice . The autIn summary, many patient studies reported occurrence of the distant metastasis, which could have arrived at these sites, notably the brain, likely via the hematogenous route. Although these distant hematogenously spread metastases are not considered to be the cause of death from EOC by themselves, their presence is significantly correlated with worse survival. Presently, the peritoneal metastases from EOC is still an unsolved problem in clinical management of this disease. However, it is very likely that continuous progress in the treatment of the peritoneal metastasis and increased survival could allow for more time for development of the distant metastasis, which could become clinically relevant in long-term survivors of metastatic EOC.The International Federation of Gynecology and Obstetrics (FIGO) ovarian cancer staging states that metastatic involvement of the retroperitoneal lymph nodes indicates FIGO Stage IIIC of the disease, and colonization of the inguinal lymph nodes and lymph nodes outside of the abdominal cavity by metastases signifies Stage IVB of the disease . AlthougVascular endothelial growth factor receptor 3 (VEGFR3) is the major receptor involved in lymphangiogenesis and maintenance of the lymphatic endothelium . The ligIn summary, lymphatic involvement is correlated with worse outcomes. Patient studies demonstrated preferential colonization of the abdominal over other lymph nodes in the human body.Development of targeted therapies against ovarian carcinoma, although still mainly at the stage of characterization of new potential targets, is an actively growing field. Several proteins that were demonstrated to play a role in progression and metastasis of ovarian cancer were or currently are investigated as novel drug targets in clinical trials. The targeting agents used in these studies vary widely from small-molecule inhibitors to monoclonal antibodies, antibody–drug conjugates, and immunotherapy.ClinicalTrials.gov identifier: NCT03078400).For example, a CD44-targeting compound SPL-108 is being investigated in conjunction with paclitaxel in a phase I trial against epithelial ovarian carcinoma  that is currently in phase I is aiming to use anti-mesothelin chimeric antigen receptor (CAR) natural killer (NK) cells in epithelial ovarian carcinoma. This study is taking advantage of the new targeting technology based on the use of CAR-NK therapy consisting of chimeric antigen receptor (CAR)-expressing natural killer cells of the immune system [ClinicalTrials.gov identifier: NCT02751918). Anetumab ravtansine is an antibody–drug conjugate, in which an anti-mesothelin antibody (anetumab) is attached to a tubulin inhibitor (ravtansine) [Several clinical trials are attempting to target mesothelin. Among those, one clinical trial (e system . Anothertansine) .ClinicalTrials.gov identifier: NCT00635193) was conducted in patients with advanced ovarian cancer or those who relapsed after platinum/taxane therapy that tested volociximab in a combination with liposomal doxorubicin. Preliminary data from this trial suggest that the combination was well tolerated [As β1-integrins play a pivotal role in different mechanisms underlying progression of ovarian carcinoma, they are targeted in clinical trials in ovarian carcinoma. Volociximab, a chimeric monoclonal antibody that binds to and inhibits the functional activity of α5β1-integrins, was studied in a phase II trial as a monotherapy in patients with platinum-resistant advanced epithelial ovarian or primary peritoneal cancer. Although the agent alone did not provide sufficient clinical activity, it was well tolerated, prompting the development of improved strategies to target α5β1-integrins . A phaseolerated , while nClinicalTrials.gov identifier: NCT00073541), combinations of EGFR-targeting therapy with the standard chemotherapy are being tested in other clinical trials. In one such combination trial, gefitinib (Iressa), a small-molecule inhibitor of EGFR, is being investigated in combination with topotecan, a topoisomerase inhibitor, in patients with relapsed ovarian, peritoneal, and fallopian tube cancers .Epidermal growth factor receptor (EGFR) is overexpressed in epithelial ovarian carcinoma and its high expression is associated with poor prognosis . EGFR isClinicalTrials.gov identifier: NCT02179970). VEGFRs were studied as targets of angiogenesis in ovarian carcinoma, resulting in approval of a monoclonal antibody against VEGFA, bevacizumab (Avastin), by The Food and Drug Administration in 2014 [A small-molecule inhibitor of CXCR4, plerixafor (Mozobil), will be investigated in patients with advanced cancers, including ovarian -deficient ovarian carcinoma with the inhibitors of poly (ADP)-ribose polymerase [In summary, although many targeted therapies directed at mechanisms described in this review are yet to show any advantage in the treatment of relapsed ovarian carcinoma when given as a monotherapy, optimism still remains that their combinations with other chemotherapeutic agents will benefit the patients at terminal stages of the disease. The successes behind targeted therapies for breast cancer gene  in the intraperitoneal milieu, as well as the role of the molecular changes during aging that support peritoneal metastases. Thus, these causal aspects of the microenvironment should also be viewed as potential molecular targets for reduction of the metastatic spread and prevention of recurrences. Both hematogenous and lymphatic routes of dissemination are relatively less studied due to their perceived limited impact on the outcomes, as most EOC patients succumb to the intraperitoneal metastasis. Nonetheless, it is likely that their clinical relevance, however unfortunate for the patients, will increase as treatment of the intraperitoneal metastasis improves in the future. Therefore, understanding of the mechanisms regulating the distant metastasis is essential for ultimately blocking this deadly metastatic disease. Overall, a more comprehensive characterization of the mechanisms regulating metastatic ovarian carcinoma and therapy response is required for the development of new targeted therapies and improvement of currently used treatment regimens.