Aspergillus niger L14 derived from the marine sponge of Reniera japonica collected off Xinghai Bay (China) resulted in the isolation of two dimeric naphtho-γ-pyrones, fonsecinone A (1) and isoaurasperone A (2). Through a combination of ECD spectra and X-ray diffraction analysis, the chiral axes of compounds 1 and 2 were unambiguously determined as αR-configurations. Bioassay results indicated that these substances exhibited remarkably inhibitory effects on human pathogens Helicobacter pylori G27 and 159 with MIC values of ≤4 μg/mL, which are similar to those of the positive control, ampicillin sodium. To the best of our knowledge, this is the first report on absolute configuration of 1 and crystallographic data of 2, as well as their potent anti-H. pylori activities.A chemical investigation into endozoic fungus  Helicobacter pylori is one of the most serious pathogenic bacteria that threaten human health, since it has infected approximately half of the world’s population +) and 593 ([M + Na]+) in their ESI-MS spectra suggested that 1 and 2 are isomeric : triclinic, space group P-1 (no. 2), a = 8.0417 (7) Å, b = 11.9593 (10) Å, c = 17.1410 (15) Å, α = 97.364 (6)°, β = 100.693 (6)°, γ = 104.037 (5)°, V = 1545.4 (2) Å3, Z = 2, T = 173.01 K, μ(GaKα) = 0.558 mm−1, Dcalc = 1.364 g/cm3, 15,365 reflections were measured (7.458° ≤ 2Θ ≤ 110.172°), 5733 unique , which were used in all calculations. The final R1 was 0.0956 (I > 2σ(I)) and wR2 was 0.3083 for all data. Each molecule of fonsecinone A (1) was co-crystallized with a water molecule and an ethanol molecule , monoclinic, space group P21/c (no. 14), a = 11.7688 (5) Å, b = 13.7141 (7) Å, c = 17.5181 (8) Å, β = 109.295 (3)°, V = 2668.6 (2) Å3, Z = 4, T = 193.0 K, μ(GaKα) = 0.570 mm−1, Dcalc = 1.420 g/cm3, 27,052 reflections were measured (6.924° ≤ 2Θ ≤ 110.078°), 5071 unique , which were used in all calculations. The final R1 was 0.0508 (I > 2σ(I)) and wR2 was 0.1499 for all data.  or can be obtained from the CCDC, 12 Union Road, Cambridge CB2 1EZ, UK; Fax: +44 1223 336033; E-mail: deposit@ccdc.cam.ac.uk)CCDC Nos. 2064113 and 2064114 contain the supplementary crystallographic data for this paper. These data are freely available via γ-pyrones 1 and 2 exhibited potent antimicrobial effects on human pathogen H. pylori G27 with minimum inhibitory concentration (MIC) values of 2 μg/mL, as well as multi-drug resistant H. pylori 159 with MIC values of 2 and 4 μg/mL, respectively , followed by condensation, cyclization, dehydration and methylation [2 is the dimerization product of two molecules of rubrofusarin B at C-7 and C-10, respectively, and it has been shown to be catalyzed by the pre-anthraquinone-dimerizing cytochrome P450 (CYP) enzyme encoded by the gene aunB [1 is the dimerization product of rubrofusarin B and flavasperone at C-10 and C-9, respectively.Biosynthetically, naphtho-hylation ,27. Compene aunB   equipped with a 5 mm triple resonance (HCN) cold probe, using TMS as an internal standard. ESI-MS and HR-ESI-MS data were obtained from an Agilent 6210 LC/TOF-MS spectrometer . A suitable crystal was selected and measured on a Bruker APEX-II CCD diffractometer . The structure was solved with the ShelXT structure solution program using intrinsic phasing and refined with the ShelXL refinement package using least squares minimization . A high Reniera japonica collected at Xinghai Bay , as described in the literature [A. niger according to its morphological characteristics and molecular phylogeny based on 18S rDNA gene sequence (GenBank accession No. MF093522).Endozoic strain L14 was isolated from fresh specimens (MNP-2016) of terature , and ideFirst, strain L14 was cultured on potato dextrose agar (PDA) at 30 °C for 5 days. Then, a balanced amount of fungal colony was transferred to the culture broth in a 500 mL Erlenmeyer flask containing 300 mL sterilized potato dextrose broth (PDB); it was then shaken at 180 rpm for 3 days under 30 °C as seed broth. After that, the seed broth was inoculated to a solid rice medium in a 1000 mL Erlenmeyer flask with sterilized rice (160 g) and water (320 mL) and cultivated at 20 °C for 40 days. At the end of fermentation, all fermented material was collected and extracted with the same volume of ethyl acetate 3 times. The organic layer was concentrated under vacuum at 38 °C to obtain crude extract (approx. 50 g).3CN and H2O on a preparative HPLC column, according to our previous established method [1)  and isoaurasperone A (2)  using a semi-preparative C18 column under a gradient condition of mobile phase (CH3CN and H2O) with a flow rate of 3.0 mL/min.The ethyl acetate extract was separated into six fractions, A-F, under a gradient condition of CHd method . FractioH. pylori G27 was a clinically susceptible strain, while multi-drug resistant clinical strain H. pylori 159 was obtained from a biopsy sample of a gastritis patient. Isolation and identification of H. pylori 159 were used standard protocols on the basis of colony appearance, Gram staining and positive reactions in the rapid urease test [H. pylori strains. Incubation of strains was conducted under microaerophilic conditions  using a double-gas CO2 incubator  at 37 °C for 48 to 72 h. Three replicates were performed for every antimicrobial assay.Human pathogen stain ase test . Then, 1H. pylori activities were carried out according to broth microdilution assay [H.pylori liquid culture was diluted in BHI broth and was inoculated into each well with a final concentration of 5 × 105 CFU/mL. After being incubated in a microaerophilic atmosphere at 37 °C for 2 days, the plates were examined visually. The MIC was determined to be the lowest concentration with no turbidity in well. For quality control and comparative analysis, the conventional antibiotics metronidazole (MTZ), clarithromycin (CLR) and levofloxacin (LVX) were tested as positive control. All MIC assays were performed in triplicate at least. Antimicrobial activity testing of each compound followed antimicrobial susceptibility testing standards outlined by the document M07-A7  against strains G27 and Hp159.Anti-on assay . Briefly


This study aims to evaluate clinical outcomes of MRI-guided adaptive brachytherapy (MR-IGABT) for each brachytherapy fraction in patients with locally advanced cervical cancer (LACC).A retrospective analysis was performed on 97 consecutive patients with LACC treated with 44.0–50.4 Gy external beam radiotherapy (EBRT) ± concurrent platinum-containing chemotherapy followed by 4 × 7 Gy MR-IGABT between September 2014 and April 2019. Intracavitary (IC)/interstitial (IS)/hybrid intracavitary and interstitial (IC/IS) brachytherapy was used in MR-IGABT. Brachytherapy planning and dose reporting followed the GEC-ESTRO recommendations. Clinical outcomes including overall survival (OS), cancer-specific survival (CSS), progression-free survival (PFS), local control (LC), and treatment-related toxicity evaluated by the RTOG criteria were analyzed. Kaplan–Meier and univariable and multivariable Cox regression analyses were used to analyze the prognostic factor.90) of the high-risk clinical target volume (HR-CTV) was 91.7 Gy (range 76.7~107.2 Gy). Two-year OS, CSS, PFS, and LC were 83.5%, 84.1%, 71.1%, and 94.8%, respectively. Four patients (4.1%) suffered from grade 3 late gastrointestinal radiation toxicity, and no other grade 3 or greater radiation toxicity occurred. Initial HR-CTV was an independent factor of OS , PFS , and LC . The HR-CTV D90  was an independent factor of PFS. Age was an independent factor of LC .Median follow-up was 21.1 months. Median dose to 90% (D90, and age were significant prognostic factors.For patients with LACC, MR-IGABT was effective and safe. It showed favorable LC, OS, and minimal toxicity. Moreover, initial HR-CTV, HR-CTV D In global cancer statistics, cervical cancer ranks fourth for both incidence and mortality in women . In ChinStages IB2, IIA2, IIB, IIIA, IIIB, and IVA FIGO 2009) cervical cancers are all locally advanced cervical cancer (LACC). To treat this type of cervical cancer, the National Comprehensive Cancer Network (NCCN) guidelines recommend the external beam radiotherapy (EBRT), concurrent platinum-containing chemotherapy, and brachytherapy (category 1) . As a cr cervicalThe preferred imaging technologies for IGABT for LACC are CT and MRI. Compared with CT, advantages of MRI lie in the soft tissue contrast and in discrimination of cervical cancer from normal uterine and adjacent tissue . This heNinety-seven consecutive patients were included in this retrospective study, treated between September 2014 and April 2019. The following eligibility criteria were applied: patients with stages IB2 to IVA (FIGO 2009), who underwent the MR-IGABT (4 × 7 Gy) in our institution and did not have a previous history of malignancy. The present study was approved by the ethics committee of our institution.All patients received EBRT to the pelvis with and without concurrent platinum-containing chemotherapy as described below, followed by 4 × 7 Gy MR-IGABT. Each brachytherapy fraction was guided by T2-weighted (T2W) MRI.Seventy-five (77.3%) patients underwent concurrent platinum-containing chemotherapy, 49 (50.5%) patients were administered platinum drugs as a single agent, and 26 (26.8%) patients were administered platinum combined with paclitaxel or docetaxel  fractions used intracavitary (IC) brachytherapy. Three hundred and 21 (83%) fractions used hybrid intracavitary and interstitial (IC/IS) brachytherapy or interstitial (IS) brachytherapy alone. The IS brachytherapy alone was performed in a few brachytherapy fractions (6/388) with the obstruction of cervical canal orifice which were not appropriate to use tandem. In addition, 80 (20.6%) fractions used freehand interstitial brachytherapy, and for 90 patients (92.8%), the IC/IS or IS technique was used.in situ). T2W MRI of each brachytherapy fraction was used for the delineation of target volume and organs at risk (OARs), as referred to in the GEC-ESTRO recommendations  were performed after recovery from anesthesia , with α/β of 10 Gy for tumor and 3 Gy for OARs, was used to calculate the cumulative doses from EBRT and MR-IGABT. Dosimetric parameters were evaluated after the GEC-ESTRO recommendations  every 3 months in the first 2 years, at 6 months intervals for the next 3 years, and then annually.The RECIST guidelines (version 1.1)  were uset-test or rank-sum test. The correlations were analyzed using Pearson’s or Spearman’s correlation. The survival curves were performed using the Kaplan–Meier method. Univariable factors were evaluated using log-rank tests and Cox regression analysis. Multivariable factors were evaluated with Cox regression analysis. p < 0.05 was considered statistically significant.Statistical analysis was performed using SPSS (version 24). Continuous variables and classification variables were described as medians (ranges) and counts (percentages), respectively. Continuous variables were compared using Student’s A total of 97 consecutive patients were included in this study, treated between September 2014 and April 2019. The median age at diagnosis was 54 (range 30~79) years. The median overall treatment time (OTT) was 63 days (range 40~141 days). 90, HR-CTV D98, HR-CTV D100, intermediate-risk CTV (IR-CTV) D90, and IR-CTV D100 were 91.7 Gy (76.7~107.2 Gy), 81.7 Gy (69.2~92.5 Gy), 71.2 Gy (63.0~82.1 Gy), 67.0 Gy (60.4~75.0 Gy), and 56.6 Gy (51.6~62.3 Gy), respectively. The initial, second, third, and fourth HR-CTV (range) were 32.6 cm3 (9.3~221.0 cm3), 31.0 cm3 (10.9~115.8 cm3), 28.5 cm3 (9.9~103.3 cm3), and 29.6 cm3 (8.9~118.8 cm3), respectively.Dosimetric outcomes are presented in The median follow-up was 21.1 months (5.4~67.0 months). The initial tumor responses were 64 complete responses (CR) and 33 partial responses (PR). CR + PR were achieved in 97/97 (100%) patients. Two-year OS, CSS, PFS, and LC were 83.5%, 84.5%, 71.1%, and 94.8%, respectively. Eighteen patients have died, 17 from cervical cancer, 1 from a nontumor cause. The leading cause of the nontumor-caused death (OS = 16.0 months) was infection. This patient’s last physical examination showed positive hemoculture, without tumor recurrence, or digestive tract fistula. Therefore the death of this patient was not caused by cervical cancer or radiotherapy-related toxicity. Five patients suffered from local failures. Two-year LC was 100% for IB2, 92.3% for IIA2, 98.3% for IIB, 75% for IIIA, 93.3% for IIIB, and 75% for IVA. A total of 29 events occurred in PFS: 5 local failures (1 with pelvic metastasis), 2 pelvic metastasis, 20 distant metastasis (4 with pelvic metastasis), and 2 deaths.90, HR-CTV D98, each fraction of HR-CTV, and initial HR-CTV >40 cm3 showed a statistical difference in OS. HR-CTV D90, HR-CTV D90 ≥87 Gy, HR-CTV D98, each fraction of HR-CTV, and initial HR-CTV >40 cm3 showed a statistical difference in PFS. HR-CTV D90 ≥87 Gy, each fraction of HR-CTV, and initial HR-CTV >40 cm3, the initial tumor response showed a statistical difference in LC.Outcomes of univariable and multivariable analyses are shown in p = 0.001, HR = 1.018/cm3, 95% CI = 1.007~1.029), PFS , and LC . The HR-CTV D90  was an independent factor of PFS. Age was an independent factor of LC .Multivariable analyses show the initial HR-CTV was an independent factor of OS (Four patients (4.1%) suffered from grade 3 late gastrointestinal radiation toxicity, and no other severe acute or late radiation toxicity occurred. MRI has been recommended as gold standard imaging for cervical cancer contours, with some comparative studies previously published , 23, 24,p = 0.005), and 3-year LC of MR-IGABT was achieved in 91% of patients. Moreover, the moderate and severe late morbidity were both reduced by about 50% (p = 0.02). Kamran et al. (p = 0.036), and 2-year LC were 96% and 87% (p = 0.65), respectively. A large multicenter cohort study of Retro EMBRACE (in situ), in limited patient numbers (49 Chinese patients), with the first and the third brachytherapy fractions using MR-IGABT and other brachytherapy fractions were planned on CT imaging. Two-year OS and LC were both achieved in 90% of the patients with no severe late toxicity.Whether MR-IGABT brings satisfactory clinical outcomes for cervical cancer, it has been a research priority of many radiotherapy centers in recent years. Lindegaard et al.  comparedn et al.  compared EMBRACE  included EMBRACE  recentlyPFS is another important clinical outcome. Two-year PFS in the present study was 71.1%. A total of 20 distant metastasis occurred, which was the largest share of PFS (20/29). This was similar to other studies , 33. Pot2cc to the bladder, rectum, and sigmoid are ≤90, ≤75, and ≤75 Gy, respectively. In this study, only 2 (2%) patients had a rectum D2cc higher than 75 Gy. The interstitial-related side effect  was settled with symptomatic treatment. MR-IGABT with interstitial technique can fully conform to dose limits for OARs, which will further lead to a well- tolerated treatment.The most common radiation toxicity that occurred was grade 1 acute mucous membrane radiation toxicity (58.1%). Other incidences of radiation toxicity  were all less than 20% . Only 4 patients (4.1%) showed grade 3 late gastrointestinal radiation toxicity. After symptomatic treatment, 2 patients fell to grade 1 and 2 patients fell to grade 0. ABS guideline  recommen3) was an independent factor for worse OS, PFS, and LC. We investigated the correlations between initial HR-CTV and other dose–volume parameters. The initial HR-CTV was negatively correlated with HR-CTV D90 (p = 0.002), HR-CTV D98 (p = 0.016), and HR-CTVD100 (p = 0.006) and positively correlated with bladder D0.1cc (p = 0.047), bladder D1cc (p < 0.001), bladder D2cc (p < 0.001), rectum D0.1cc (p = 0.027), rectum D1cc (p < 0.001), rectum D2cc (p < 0.001). The initial HR-CTV showed no correlation between the dose to sigmoid and small bowel. The probable cause is the geometrical uncertainties in OARs  between patients with and without concurrent chemotherapy [median age was 53 (range 30–76) vs. 61.5 (range 33–79)]. The older age resulted in incomplete treatment, which may influence the LC.In the present study, the larger initial HR-CTV  and 100% for initial HR-CTV ≤40 cm3 versus 84.8% for initial HR-CTV >40 cm3 . In addition, we found that 2-year LC was significantly different between CR and PR , indicating the initial tumor response may influence LC.The independent prognostic factors for LC in previous studies , 38, 39 e et al.  found th90 ≥87 Gy and 96 patients (99.0%) HR-CTV D90 ≥80 Gy. The median HR-CTV D90 was 91.7 Gy, which meet the ABS . Compared with traditional IC brachytherapy alone, interstitial technique is more feasible with adequate coverage of disease in the vagina and parametrium  HR-CTV D the ABS  and NCCN the ABS . Only 1 ametrium .p = 0.004, and Cox model, p = 0.047). Although, for OTT, there was no statistical significance in OS, PFS, and LC in the present study, we found OTT was significantly different (p = 0.035) between CR and PR [median OTT was 62 days (range 40–125 days) vs. 64 days (range 54–141 days)]. In consideration of the significant difference of 2-year LC between CR and PR in this study, the longer OTT results in a poorer initial tumor response, which further impacts LC. However, there is still insufficient high-level evidence with regard to OTT effects because OTT is closely related to many factors such as tumor response, dose, fractionation, and treatment-related side effects.In the present study, 69 patients (71.1%) had OTT longer than 8 weeks, which was suggested within 8 weeks . The maiThe major limitation of this study is its retrospective nature, with 23 patients receiving EBRT in other institutions before the MR-IGABT in our center. This results in a lack of unity in the EBRT dosimetric data and treatment characteristics. In addition, a longer follow-up may be required to obtain more meaningful results.90, and age were significant prognostic factors. Future investigations and systemic treatment need to be emphasized.This retrospective study including 97 consecutive Chinese patients with LACC has shown that MR-IGABT was effective and safe. It showed favorable LC, OS, toxicity, and morbidity. Moreover, initial HR-CTV, HR-CTV DThe raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by the ethics committee of China-Japan Union Hospital of Jilin University. The patients/participants provided their written informed consent to participate in this study.YC: manuscript writing, data collection, and data analysis. YP: protocol development. NZ: manuscript writing. DH: data management. XG: data analysis. ZM: data analysis. GC: protocol development and manuscript editing. All authors listed have made a substantial, direct, and intellectual contribution to the work and approved it for publication.This work was partially supported by grants from the National Natural Science Foundation of China , Project of Science and Technology Department of Jilin Province , and Horizontal Project of Jilin University .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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


We found that both complexes exhibited a potent cytotoxic effect in a panel of cancer cell lines in monolayer cultures, as well as in a 3D model of multicellular spheroids formed from DU-145 human prostate adenocarcinoma cells. Furthermore, the complex (2) suppressed the colony formation, induced G2/M-phase arrest, and downregulated Aurora-B. The mechanism studies suggest that complex (2) stimulate the overproduction of reactive oxygen species (ROS) and triggers caspase-dependent apoptosis as a result of changes in expression of several genes related to cell proliferation and caspase-3 and -9 activation. Interestingly, we found that N-acetyl-L-cysteine, a ROS scavenger, suppressed the generation of intracellular ROS induced by complex (2), and decreased its cytotoxicity, indicating that ROS-mediated DNA damage leads the DU-145 cells into apoptosis. Overall, we highlighted that coordination of lapachol to phosphinic ruthenium(II) compounds considerably improves the antiproliferative activities of resulting complexes granting attractive selectivity to human prostate adenocarcinoma cells. The DNA damage response to ROS seems to be involved in the induction of caspase-mediated cell death that plays an important role in the complexes' cytotoxicity. Upon further investigations, this novel class of lapachol-containing ruthenium(II) complexes might indicate promising chemotherapeutic agents for prostate cancer therapy.Lapachol is a well-studied natural product that has been receiving great interest due to its anticancer properties that target oxidative stress. In the present work, two novel lapachol-containing ruthenium(II) complexes [Ru(Lap)(dppm)(bipy)]PF Prostate cancer is the second most frequent malignancy (after lung cancer) in the male population and rising incidences have been observed worldwide , 2. The The current chemotherapeutic drugs have clinically serious toxicities. Furthermore, anti-androgen drugs, designed to disrupt the function of androgens, have been linked to the onset of drug-resistant prostate cancer . Therefoin vivo (Lapachol (2-hydroxy-3-(3-methylbut-2-en-1-yl)naphthalene-1,4-dione) is a naphthoquinone, which was originally isolated from species of the Bignoniaceae family . We, aloin vivo .Historically, much attention has been given to naphtoquinones after its ability to directly target DNA topoisomerases and inhibit their activity, which results in cytotoxicity . Recogniin vivo. Furthermore, ruthenium complexes under pre-clinical and clinical studies are showing promising results, representing a possible novel class of less toxic antineoplastic chemotherapy  and [Ru(Lap)(dppm)(phen)]PF6 (2). The corresponding complexes methane (dppm), 2,2'-bipyridine (bipy), 1,10-phenanthroline (phen), and KPF6 were purchased from Sigma-Aldrich MERCK  and used as received. Lapachol (Lap) was extracted from the bark of Tabebuia aurea (Manso) S. Moore as reported in the literature  (Fluka Purum) as a support electrolyte. The Ag/AgCl (0.10 M Bu4NOH in CH2Cl2) in a Luggin capillary probe was used as reference electrode and platinum foils as working and auxiliary electrodes. The IR spectra were acquired using a FT-IR Bomem-Michelson 102 spectrometer using KBr pellets. Elemental analysis  was determined using an CHNS Element Analyzer . UV-vis spectra were recorded in a Hewlett Packard diode array—8452A spectrophotometer. Conductivity was acquired using a MeterLab CDM2300 at room temperature.The cis-[RuCl2(dppm)(N-N)], where N-N = 2,2'-bipyridine (bipy) or 1,10-phenanthroline (phen) and dppm = bis(diphenylphosphine)methane, were synthesized according to described in the literature (bipy)] (0.14 mmol), lapachol (0.21 mmol), KPF6 (0.28 mmol), and 50 µl of triethylamine were added. The reaction was maintained in reflux under argon atmosphere for 12 h. After that, the volume of the mixture was reduced to ca. 3 ml, and the complex precipitated by addition of 1 ml of water. The solid was filtered off, washed with water, diethyl ether and dried under vacuum. Yield: 0.109 g (76%). Elemental analysis for [C50H43F6N2O3P3Ru]: exp. : C, 58.85 (58.43), H, 4.40 (4.22), and N, 2.85 (2.73). ΛM = 59 Ω−1 cm2 mol−1, in 1.0 mM CH2Cl2 solution. IR (cm-1): v(C1=O) 1546, v(C4=O) 1608, v(C2–O) 1103, v(P–F) 840 and 557, v(Ru–O) 510 and v(Ru-N) 483. 31P{1H} NMR : δ (ppm) 18.2; 6.2/14.7; 5.8/2J = 76.1/66.4, –144 . 1H NMR : δ (ppm) 8.7–6.6 , bipy (8H), and Lap (4H)), 5.4 , 4.8 , 3.1–2.8 , 1.4–1.3 . 13C{1H} NMR : δ(ppm) 196.5 (C1=O), 181.4 (C4=O), 169.6 (C2–O).To a Schlenk flask containing 40 ml of dichlorometane and 40 ml of acetone , the cis-[RuCl2(dppm)(phen)] (0.17 mmol), lapachol (0.21 mmol), KPF6 (0.28 mmol), and 50 µl of triethylamine were added. For the synthesis of this complex, the same procedure of complex (1) was used. Yield: 0.107 g (75%). Elemental analysis for [C52H43F6N2O3P3Ru]: exp. : C, 59.71 (59.37); H, 4.39 (4.12); and N, 2.95 (2.66). ΛM = 38.27 Ω−1 cm2 mol−1, in 1.0 mM CH2Cl2 solution. IR (cm−1): v(C1=O) 1547, v(C4=O) 1610, v(C2–O) 1097, v(P-F) 837, and 555, v(Ru-O) 508 and v(Ru-N) 480. 31P{1H} NMR : δ (ppm) 18.3; 7.1/14.9; 6.5/2J = 74.5/68.0, −144 . 1H NMR : δ (ppm) 9.0–6.3 , phen (8H), and Lap (4H)), 5.5 , 4.8 , 3.2–2.8 , 1.8–1.7 . 13C{1H} NMR : δ(ppm) 196.8 (C1=O), 181.8 (C4=O), 169.6 (C2–O).To a Schlenk flask containing 40 ml of dichlorometane and 40 ml of acetone , the P) were quantified using the shake-flask method /[compound] (in water). The experiments were carried out in triplicate.The octanol-water partition coefficients (log k method . Each co® CCL-185™), human hepatocellular carcinoma , and human breast adenocarcinoma  were obtained from American Type Cell Collection (ATCC). Human melanoma , Human colorectal adenocarcinoma , human prostate adenocarcinoma , human mouth fibroblast , and human prostate epithelial cells  were obtained from Rio de Janeiro Cells Bank (BCRJ). The cells A549, A-375, Caco-2, DU-145, HepG2, MDA-MB-231, and FGH were cultured in Dulbecco’s Modified Eagle’s Medium , PC-3 and PNT-2 cells were grown in Roswell Park Memorial Institute Medium . Both mediums were supplemented according to the needs of each lineage, as recommended by its cell banks with 2 mM l-glutamine (Gibco-Invitrogen®), 10% or 20% fetal bovine serum (Gibco-Invitrogen®), 1% antibiotic-antimycotic solution , and 3.7 g/L of NaHCO3 (Sigma-Aldrich). All cell lines were cultured in flasks at 37°C in 5% CO2 and 96% of relative humidity according to the procedures proposed by . After 24 h, the complexes were dissolved in DMSO and added to each well and incubated for 24 h. Dilutions of the complexes were prepared to obtain concentrations ranging from 0.3 to 100 μM. Cisplatin (Fauldcispla®) and doxorubicin hydrochloride (Fauldoxo®) were used as the reference cytotoxic drugs. DMSO (0.1% v/v) was used as the vehicle control. Following 24 h of incubation, 50 μl of Alamar blue®  was added to each well, and the plates were incubated, at 37°C, in the dark. Fluorescence reading was performed in a Synergy H1 Fluorescence Spectrophotometer (BioTek®), using excitation and emission filters at wavelengths of 560 and 590 nm, respectively. The fluorescence intensity was measured in arbitrary fluorescence units (AFU) and the AFU of untreated cells (CTL) was considered 100%. The cytotoxic effects of the compounds were estimated in terms of cell proliferation inhibition (%) and expressed as half-maximal inhibitory concentration (IC50). The cell proliferation inhibition percentage of cells exposed to treatments was calculated following: % Inhibition = 100 − (AFU of treated well/AFU of control well) × 100). The IC50 values were calculated from the cell proliferation inhibition percentage by nonlinear regression method using CompusSyn® Software.The orted by . Cells w3 cells/well) were inserted in agarose-coated 96-well plates (1.5% w/v) and cultured in complete medium plus 3% Matrigel® (Corning). DU-145-MCTSs with stable structures had formed after five days. Then, spheroids were exposed to the complexes in a range of five different concentrations varying from 0.25 to 4.0 μM and incubated for 24 h. Cisplatin (Fauldcispla®) at 25 μM was used as the reference cytotoxic drug. Negative control received the vehicle (DMSO 0.1% v/v) that was used for diluting the compounds tested. Finally, the MCTS proliferation inhibition (%) and IC50 were determined by Alamar blue® assay as described above. To investigate morphological changes other MCTSs were treated with 0.25, 0.5, 1.0, 1.5, and 2.0 μM of complex (2) and examined for 7 days. Spheroid integrity and diameter of MCTSs were analyzed by phase-contrast microscopy (Inverted Trinocular Microscope Opton TNB-O5T-PL) using AxioVision LE software .Multicellular tumor spheroids (MCTSs) were obtained according to the method reported by . Briefly6cells/well) were inserted in six-well plate and treated with different concentrations varying of 0.125 to 1.5 μM of both complexes. Cisplatin (Fauldcispla®) and doxorubicin hydrochloride (Fauldoxo®) were used as the reference cytotoxic drugs. DMSO (0.1% v/v) was used as the vehicle control. After 24 h, the cells were washed with PBS (Gibco-Invitrogen®) and harvested by trypsinization. Cells were counted using a Neubauer chamber and replated then on cell culture dishes of 21.5 cm2 in a density of 200 cells per culture dish for each treatment in triplicate. After 7 days, cells were fixed in ice-cold methanol: acetic acid: distilled water (1:1:8), air-dried, and stained with crystal violet  for 20 min. Colonies with at least 50 cells were counted. Plating efficiency (PE) of DU-145 cells was calculated by the ratio of the number of colonies observed after 7 days of incubation to the number of cells seeded (two hundred). The average of colonies in control was considered as 100%, and the number of colonies that arise after treatment of cells is called the surviving fraction (SF), obtained by:The clonogenic assay was performed in accordance with the guidelines of . The DU-™ Plus DNA Kit  according to the manufacturer’s instructions. Briefly, DU-145 cells were seeded (5.0 × 104 cells/well) into 24-well culture plates and maintained to attach at 37°C in 5% CO2. After 24 h, the culture medium was replaced by complete culture medium containing 0.25, 0.5, 1.0, and 1.5 μM of complex (2). DMSO (0.1% v/v) was used as the vehicle control. Then, after 24 h treatment, the cells were harvested and fixed with 70% ethanol at 4°C for 1 h. Cells were incubated with 0.1 mg/ml RNase and 0.5 mg/ml propidium iodide (PI) for 10 min. Samples were then filtered using 50-µm nylon mesh and the FACSCanto flow cytometer  was used to analyze the DNA histogram. Data from 10,000 cells were acquired and analyzed using the ModFit software . The results were expressed as percentage of cells in each cell cycle phase .DNA staining was performed using BD Cycletest® 488 Annexin V/PI (Thermo Fischer Scientific), and the analyses were performed according to the manufacturer’s instructions. Briefly, DU-145 cells were seeded (5.0 × 104 cells/well) in 12-well plates and maintained to attach at 37°C in 5% CO2 for 24 h. After, cells were treated with 0.25, 0.5, 1.0, and 1.5 μM of both complexes. Cisplatin (Fauldcispla®) and doxorubicin hydrochloride (Fauldoxo®) were used as the reference cytotoxic drugs. DMSO (0.1% v/v) was used as the vehicle control. Following 24 h of incubation, the cells were collected with Accutase® (Gibco-Invitrogen®), washed with PBS and resuspended in 200 μl of cold annexin-binding buffer. Next, 10 μl of Alexa Fluor® was added to Annexin V (50 μl/ml) staining buffer, and the mixture was incubated in the dark at 4°C for 15 min. Previously, the analyses, 100 μl of PI (2 μg/ml) was added, and then, the fluorescence was measured by flow cytometry in a FACSCanto flow cytometer  using the Diva software. Ten thousand events were evaluated per experiment, and cellular debris was omitted from the analysis.The measurement of cell death was performed by the guidelines of . Detecti2DCFDA, Sigma-Aldrich), as reported by (4 cells/well) into black 96-well plates and maintained to attach at 37°C in 5% CO2. After 24 h, cells were treated with 1.0 μM (~IC50) of the complex (2) for 1, 6, 12, and 24h. After that, cells were labeled with H2DCFDA solution at 5 μM in N,N-dimethylformamide (Sigma-Aldrich). After that, fluorescence intensity was measured in a Synergy H1 Fluorescence Spectrophotometer (BioTek®), at the excitation and emission wavelengths of 495 and 527 nm, respectively. Thereafter, ROS/Superoxide levels were measured using Cellular ROS/Superoxide Detection Assay Kit (Abcam®). Cells were seeded (1.5 × 104 cells/well) into black 96-well plates and maintained to attach at 37°C in 5% CO2. After 24 h, cells were treated with complex (2)  for 24 h. Negative control received the vehicle (DMSO 0.1% v/v), and positive control received pyocyanin (250 μM) as ROS inducer. Following this, cells were labeled with oxidative stress detection reagent (Green) for ROS detection and superoxide detection reagent (Orange) according to the manufacturer’s instruction. Fluorescence was measured in quadruplicates using a fluorescent microplate reader Synergy H1 (BioTek®) with standard fluorescein  and rhodamine  filter sets for ROS and superoxide determination, respectively. Protection assays using the antioxidant N-acetyl-l-cysteine (NAC) were also performed. In brief, the cells were treated with 5 mM NAC (Sigma-Aldrich) in association with complex (2)  for 24 h. Cells were labeled as described above, and the ROS levels were measured. In a new set of experiments, cells were treated with 5 mM NAC in association with cytotoxic concentrations of (2). After 24 h of treatment, the cell viability was determined by Alamar blue® assay as described above.Total intracellular ROS generation was measured using 2′,7′-dichlorodihydrofluorescein diacetate (Horted by . Briefly5 cells/well) in 24-well plates and maintained to attach at 37°C in 5% CO2 for 24 h. After, cells were treated with different concentrations of complex (2) for 24 h. DMSO (0.1% v/v) was used as the vehicle control and methyl methanesulfonate  as DNA damage inducer. Following 24 h of incubation, the cells were collected, and cell viability assay was performed using trypan blue dye. Analyzed samples showed more than 70% of viability (data not shown). Then, cells were mixed with UltraPure™ Low-Melting-Point Agarose , and added on pre-coated slides with UltraPure™ Agarose . After agarose solidification, the slides were immersed in a lysis solution  for 18 h at 4°C. After that, the slides were added with electrophoresis buffer  to the electrophoresis chamber. The electrophoresis run was set up for 20 min of an electric field of 25 V and 300 mA (0.9 V/cm). Slides were neutralized  and fixed in absolute ethanol for 5 min. Dried slides were stained with Gel Red® . The nucleoids were identified in a fluorescence microscope  using a 516 to 560 nm filter, 590 nm filter barrier, in 20× objective with an integrated digital camera. For each treatment, 100 random nucleoids were analyzed, and the tail fluorescence was measured through the Comet Assay IV™ imaging system .The comet assay was performed to evaluate DNA damage following the method described by . BrieflyPlasmid pBR322  was mixed with different concentrations (0.125–1.0 µM) of both complexes and incubated at 37°C for 18 h. Then, the loading buffer was added and the samples were analyzed by agarose gel electrophoresis for 90 min in 1% agarose gel using a Tris-acetate-EDTA (TAE) buffer , and ethidium bromide was employed for staining. Bands were visualized with a ChemiDoc MP imager .−1 dm3 cm−1 at 260 nm (®), at 37°C.DNA minor groove interaction was assessed by examining the ability of the complexes to displace Hoechst 33258 (Thermo Fischer Scientific) from calf thymus DNA (ct-DNA) (Sigma-Aldrich). The ct-DNA solution was prepared by dilution of 2 mg of the ct-DNA in 1 ml of Tris-HCl buffer , and the concentration was determined by absorption spectrophotometric using the molar absorption coefficient 6600 molt 260 nm . Thus, t6 cells/well) into six-well plates and maintained to attach at 37°C in 5% CO2. After 24 h, the cells were treated with 1.0 μM of the complex (2). DMSO (0.1% v/v) was used as the vehicle control. After 12 h of incubation, total RNA was isolated from the cells using the RNeasy® Mini kit  according to the manufacturer’s instructions. RNA was quantified by NanoDrop spectrophotometer . The Agilent 2100 Bioanalyzer and Agilent RNA 6000 Nanochip kits were used to assess the total RNA quality as per manual instruction . The Agilent 2100 Expert software (Version B.02.07.SI532) was used with the Eukaryote Total RNA Nano assay on the Agilent 2100 bioanalyzer. No significant differences in RIN values were observed across the samples. Complementary DNA strands were synthesized using the RT2 First Strand kit , following the manufacturer’s instructions. Gene expression profiling was performed using the RT2 Profiler™ PCR Array Human Cancer Drug Targets  by qPCR. The reactions were conducted in the Applied Biosystems 7500 PCR System  thermocycler. The plate was heated to 95°C for 10 min, followed by 40 cycles of 95°C for 15 s and 60°C for 1 min, followed by the dissociation curve. The expression profile of 84 key drug targets genes was analyzed. Fold changes amplification for targeted genes was normalized to the housekeeping genes GAPDH and RPLP0 by the delta–delta cycle threshold method (ΔΔCt), and the cells treated with vehicle control (0.1% of DMSO v/v) was used as calibrator. The threshold cycle values of control wells were all within the ranges recommended by the PCR array user manual. Three independent biological replicates were performed. All experiments were performed in DNase/RNase free conditions. Genes were considered differentially regulated if the difference was ≥1.8-fold, which means that the gene expression in the compound-treated cells was at 1.8-fold that in the negative control-treated cells. Differences were considered significant at p ≤ 0.05. Statistical analysis of two groups was performed automatically according to the GeneGlobe Data Analysis Center .Briefly, DU-145 cells were seeded (1.0 × 106 cells/well) into six-well plates and maintained to attach at 37°C in 5% CO2. After 24 h, cells were treated with 0.25, 0.50, 1.0, and 1.5 μM of complex (2). DMSO (0.1% v/v) was used as the vehicle control. After 24 h of incubation, cells were collected and suspended in Western blotting lysis buffer . Cell lysates were centrifuged at 11,000g for 15 min at 4 °C, and supernatant was collected. Protein concentrations were quantified using the BCA Protein Assay Kit® (Thermo Fischer Scientific), according to the manufacturer’s instructions. For immunoblotting, equal amounts (30 μg) of total protein were electrophoresed in a NuPAGE® 4-12% Bis-Tris gel and transferred to an Invitrolon™ polyvinylidene difluoride (PVDF) membrane  at 100 V for 1 h. PVDF-membrane was washed two times (5 min each) with Tris-buffered saline plus Tween 20  and blocked in 5% non-fat milk in phosphate buffered saline with Tween®20  for 1 h at room temperature. After, the membranes were incubated 4 h at 18°C with primary antibodies against β-actin , PARP1 , cleaved PARP1 , cleaved caspase-3 , cleaved caspase-8 , cleaved caspase-9 , and aurora B , previously diluted in blocking solution to appropriate concentrations. After removing unbound primary antibodies, membranes were washed four times (7.5 min each) with TBST buffer  and incubated with the respective secondary goat anti-mouse HRP antibody  for 1 h at room temperature. Membranes were washed additional eight times (7.5 min. each) with TBST before performing the enhanced chemiluminescence step using ChemiDoc™ MP Image System . Digital images were acquired using a software Scion Imaging . The protein β-actin was used for sample loading normalization.Briefly, DU-145 cells were seeded  obtained by nonlinear regression. Differences between experimental groups were compared using analysis of variance (ANOVA) followed by the Dunnet’s or Tukey’s test (p < 0.05). All statistical analyses were performed using GraphPad Prism 8 for MacOS X .Data are presented as mean ± S.E.M. or as IC1 and 2), with the general structures represented in cis-[RuCl2(dppm)(N-N)], where N-N = 2,2'-bipyridine (bipy) or 1,10-phenanthroline (phen) and dppm = 1,1′-bis(diphenylphosphine)methane.Two novel ruthenium complexes  and (2) confirmed the coordination of the Lap ligand. The band assigned to the v(O-H) stretching vibration of Lap was not observed in all complexes, indicating the deprotonation. The vC1 = O and vC2−O of free Lap present values of 1,643 and 1,050 cm−1, respectively. In the complex (1) the vC1=O stretching vibration was shifted to lower frequencies, 1546 cm1, and the vC2−O shifted to 1103 cm−1 (−1. The same comportment was observed for complex (2).Infrared spectra of the complexes  and (2), respectively, which are higher than the precursor complexes (0.1–0.6 V)  of the bipy, phen, dppm, and Lap ligands. Also, bands around 350 to 600 nm was observed and can be assigned to metal-to-ligands charge transfer (MLCT) and, n → π* transitions of quinone carbonyl groups , 40. The1–0.6 V) . This ob31P{1H} NMR spectra of complexes (1) and (2), it was observed a typical AB spin system with chemical shifts at 18.2/6.2 and 14.7/5.8 ppm for complex (1) and 18.3/7.1 and 14.9/6.5 ppm, for complex (2), indicating the nonequivalence of the phosphorus atoms. The duplication of phosphorus signals in the 31P{1H} NMR spectra are related to the presence of isomers in solution. In one isomer the phosphorus atom from the dppm ligand is trans to oxygen in the carbonyl of the lapachol and in another, the phosphorus atom is trans to the oxygen of enol , in the range 8.7 to 6.6 ppm it was observed multiplets referent to 32H atoms of the bipy, dppm and Lap. There were observed multiplets at 5.5, 4.8, and 3.1 ppm of CH and CH2 groups of the aliphatic chains of Lap and dppm. Also, 1.5 to 1.4 ppm was observed signals attributed to the two CH3 groups of Lap  and 0.839 ± 0.007 to complex (2). The phenanthroline ligand might have granted higher lipophilicity to complex (2) which could be related to its cytotoxicity described below.Lipophilicity is an essential physicochemical feature that predicts the pharmacokinetic/pharmacodynamic profile of new drug candidates drugs . The cla® assay. 1) presented IC50 values of 0.9 to 3.3 μM and complex (2) presented IC50 values of 0.8 to 2.6 μM for cancer cell lines DU-145 and A549, respectively. The complexes also displayed higher cytotoxic activity than cisplatin (Fauldcispla®) and doxorubicin (Fauldoxo®). Doxorubicin presented IC50 values ranging from 1.9 to 3.7 μM for cancer cell lines A549 and A-375, respectively. Cisplatin presented IC50 values varying from 10.8 to 24.3 μM for cancer cell lines PC-3 and DU-145, respectively. The precursors cis-[RuCl2(dppm)(bipy)] and cis-[RuCl2(dppm)(phen)]) had been previously tested and exhibited only weak cytotoxicity (IC50 > 100 μM) , and 2.5 and 6.8 μM for the complex (2) for FGH and PNT-2 cells, respectively. In addition, the IC50 value for non-cancer cells was 2.6 and 3.2 μM for doxorubicin and 11.4 and 9.5 μM for cisplatin for FGH and PNT-2 cells, respectively.The potential of the complexes to disturb the viability of different histological types of cancer cell lines  and two non-cancer cells (FGH and PNT-2) was assessed by Alamar blue50 [non-cancer cells]/IC50 [cancer cells]. The SI is a clear manner to estimate the therapeutic index of a drug and to identify safe drug candidates for further investigations (1) and (2) were more cytotoxic and selective to prostate cancer cells DU-145. In fact, complex (2) showed higher selectivity for prostate cancer cells  when compared to prostate non-cancer cells. Furthermore, both complexes exhibit SI greater than displayed by the reference drugs cisplatin and doxorubicin. In a further round of experiments, the DU-145 was used as a cellular model, since displayed higher sensibility to treatments.The selectivity index (SI) measured for each complex has been presented in igations . Complex2) caused a decrease in the total spheroid area observed over time, which possibly resulted from cell apoptosis (50 of the complexes (1) and (2) against the DU-145 MCTSs, were 2.4 and 1.9 μM, respectively, while cisplatin presented IC50 of 62.6 μM. The complex (2) was more potent than cisplatin at the least 32-fold, showing that its cytotoxic activity is effective in the tumor spheroids of DU-145 cells.We also evaluate the complexes toward 3D cell cultures based on DU-145 cells. Interestingly, the prostate multicellular tumor spheroids (MCTSs) undergo alterations in their structure, indicating an effective permeability and, consequently cytotoxicity. The treatment of DU-145 MCTSs with complex (2) was greater than that of the complex (1) at the same concentrations. At concentrations of 0.125, 0.25, 0.5, 1.0, and 1.5 μM, the complex (1) reduced colony formation, presenting survival fractions (SF) of 65.1%, 35.1%, 18.3%, 9.0%, and 4.3%, respectively. Complex (2), at the same concentrations, presented SF of 58.0%, 17.7%, 4.3%, 0%, and 0%, respectively. No significant (p > 0.05) decrease in the number of colonies was observed in control (DMSO 0.1% v/v). Doxorubicin at 2.5 μM and cisplatin at 25 μM presented SF of 18.7% and 69.1%, respectively. At concentrations of 1.0 and 1.5 μM of complex (2), no colonies were visible at the plates (2) increased cell number at the G2/M phase after 24 h treatment with increasing concentration, followed by decreased cell number at G0/G1 and S phases in DU-145 cells , its effect on the expression of Aurora-B kinase was examined, since it is a mitotic checkpoint that performs a critical role during mitosis, guaranteeing correct chromosome segregation (2). Similar results were observed at the mRNA level as demonstrated on 2) in DU-145 prostate cells is associated with Aurora-B downregulation and cell cycle arrest in G2/M phase.To further evaluate the antiproliferative activity of complex (regation . Some stregation . As show2), the population of apoptotic cells have increased remarkably in a concentration-dependent manner  (50) showed 19.5% of apoptotic cells. Therefore, a 100-fold lower concentration of complex (2) was required for the same effect of cisplatin on DU-145 cells. To further determine whether extrinsic or intrinsic pathway the complex (2) induced apoptosis, we evaluated the expression of downstream apoptotic proteins by western blot. As shown in 2) induced apoptosis by activating the intrinsic pathway. Consequently, complex (2) was chosen for comprehensive studies of the mechanism of cell death.To assess whether the cytotoxicity of the complexes is associated with cell death, we performed Annexin-V/PI double staining. The flow cytometry analysis showed that both complexes increased the number of cells in the early and late apoptosis stages (Q2 and Q4 quadrants) in a concentration-dependently manner (2), we used the Cellular ROS/Superoxide Detection Assay . Previously, the total ROS levels were assessed after 1, 6, 12, and 24 h of incubation with 1.0 μM of complex (2) (~IC50) using H2DCFDA as a fluorescent dye to determine which time would be more appropriate for a ROS/Superoxide analysis. The treatment of 24 h showed the highest increase of ROS (2). We determined that total ROS levels in DU-145 cells were significantly higher after the treatment (2) exposure induced an oxidative stress response in the prostate tumor cells. It is demonstrably in 2). Moreover, at 1.5 μM there was observed a decrease of ROS/Superoxide levels, probably because increasing concentrations promotes cell death.To explore whether ROS are involved with the cytotoxicity of the complex (2), we used the ROS-scavenger NAC, an antioxidant that has been proven to display cytoprotective effects against ROS-induced cytotoxicity (2)  on prostate cells were investigated by comet assay, a sensitive method for DNA strand break revelation. As shown in 2) at 0.5 μM (2) gave inconsistent results with presence of nondetectable cell nuclei due the extensive cell death and DNA fragmentation during processing of DU-145 cells.Considering that ROS are known to trigger DNA damage, the genotoxicity of the complex (2) at the same concentrations. Only at the maximum tested concentration of 6.0 μM (~IC50) an increase in DNA damage was detected on PNT-2 cells (2) can enhance intracellular oxidative stress suggesting that apoptosis may be the principal mechanism of cell death triggered by ROS-mediated DNA damage.However, under the same experimental conditions, prostate normal cells showed no DNA damage after treatment with complex (2) was able to cause DNA damage, we studied the capability of these lapachol-containing ruthenium complexes to interact with ct-DNA by fluorescence and gel electrophoresis methods. DNA minor groove interaction was assessed by investigating the ability of the complexes to replace Hoechst 33258 from ct-DNA. To achieve this, the complexes were added to a DNA-Hoechst 33258 mixture to assess if the complexes compete with Hoechst and interact with DNA. If the complexes displace Hoechst from DNA, the fluorescence intensity of Hoechst decreases. Indeed, complex (2) could decrease the fluorescence of the Hoechst-DNA system by enhancing the concentration, indicating that it interacts with DNA by the minor groove and competes with Hoechst, removing it from DNA structure, thereby decreasing the fluorescence of the system (1) (2) was able of influencing the mobility of the bands of pBR322 plasmid DNA in electrophoresis in agarose gel. Modification of the DNA structure causes interference in the migration of supercoiled DNA and a slight increase in the mobility of open circular DNA where both forms comigrate. The complex (2) was found to exhibit nuclease activity followed by conversion of supercoiled (SC) into linear (L), proposing single-strand DNA cleavage at 1.0 μM (2) directly interacts with DNA.As complex (2) for 12 h by qPCR using a 96-well plate Human Cancer Drug Targets RT2 Profiler PCR Array®. To screen out inaccurate results a 1.8-fold change in gene expression was applied as the cutoff point. The assessment shows a significant change in 26 genes (30.9%) in the treated DU-145 cells compared to the untreated cells. A total of two genes were upregulated while 24 genes were downregulated (BCL2 (-4.1-fold), BIRC5 (-6.4-fold), CDK1 (-4.5-fold), CDK4 (-4.6-fold), AURKA (-5.3-fold), AURKB (-11.1-fold), and AURKC (-3.0-fold) were downregulated in the cells treated with complex (2), while the genes MDM4 (2.8-fold) and PTGS2 (2.1-fold) were upregulated.The expression of several genes associated with important cancer drug targets, such as cell proliferation, apoptosis, DNA damage repair, and epigenetics, was investigated in prostate cancer cells treated with 1.0 µM of complex (egulated Table 2.2) decreased markedly the expression of Aurora kinase genes, especially AURKB (-11.1-fold). The inhibition of this class of serine/threonine kinases leads to stop cytokinesis and promote cell growth repression. This effect may indicate that the complex (2) inhibits the progression of cell cycle, resulting in its antiproliferative effect demonstrated by inhibiting the clonogenicity of DU-145 cells.Curiously, the complex  provoked alterations in the shape of the spheroid, such as loss of circularity and consequent disaggregation. These findings are consistent with an effective permeability of the complex inside the spheroids.In 3D cell culture experiments, both complexes were also more potent in inducing cytotoxicity than cisplatin. Comparable with solid tumors isplatin . In our 2) induce caspase-dependent apoptosis on DU-145 cells as observed by externalization of phosphatidylserine, DNA fragmentation, and caspase-3 and -9 activation. Besides, our data showed that complex (2) could suppress cell proliferation that was accompanied by an accumulation of cells in the G2/M phase, which is consistent with the downregulation of Aurora-B kinase in prostate cancer cells. The G2/M checkpoint blocks cells from entering mitosis with genomic DNA damage, granting an opportunity for repair or stopping the proliferation of damaged cells (2) increased the proportion in the G2/M phase and decreased the cell proportion in G0/G1 and S phases in DU-145 cells. Further, gene and protein expression analysis showed that complex (2) led to a decrease in the expression of the Aurora kinase family, an important group of enzymes that execute an essential function in cell cycle progression especially at the mitotic stage. In the current study, we show that the treatment with complex (2) decreased the expression of Aurora-B, followed by antiproliferative effects in DU-145 cells, such as loss of its clonogenicity. Studies also revealed that Aurora-B regulates the G2/M phase transition through several key factors at the transcriptional level  induces oxidative DNA damage and inhibits Aurora-B on DU-145 cells. The principal mechanism of action is the ability of this complex to increase the generation of ROS, thereby being responsible for its ability to cause DNA damage and trigger apoptosis through the intrinsic pathway.Some studies propose that Aurora-B expression is inhibited by proteins of DNA repair system –71. The 2) could also increase general ROS and superoxide anion levels. Interestingly, the cotreatment with ROS inhibitor NAC remarkably suppressed the generation of ROS and prevented the reduction of cell viability. Additionally, complex (2) induced DNA damage on DU-145 cells and directly interacts with DNA, suggesting that this compound trigger caspase-dependent apoptosis through ROS-mediated DNA damage in DU-145 prostate cells.As mentioned above, lapachol induces the generation of ROS, which damages DNA and afterward promotes apoptosis. It is widely known that ruthenium(II) complexes could lead to apoptosis through ROS-mediated pathway in cancer cells , 74. ROS2) were evaluated at the level of mRNA expression of diverse genes. Notably, it was detected the decreasing expression of genes associated with apoptosis, such as BCL2 and BIRC5, and genes involved in the cell cycle transition, such as CDK1, CDK4, AURKA, AURKB, and AURKC.The mechanisms underlying the cytotoxic and antiproliferative activities of complex  also decreased the expression of the BIRC5 gene on DU-145 cells, which was 6.4 times less expressed than the untreated cells. BIRC5 encodes a protein called survivin, which became an attractive drug target for anticancer therapies. Studies showed that blocking the transcription of the BIRC5 gene in prostate cells inhibits the growth of xenografted PC-3 tumors in mice (2) promoted dysregulation of several genes that encode CDKs, especially CDK4, a protein commonly overexpressed in prostate cancer . Aurora-B plays a crucial role in the Chromosomal Passenger Complex (CPC), a complex necessary for binding of microtubules to the kinetochore of chromosomes, in order to ensure adequate fusion for cell division  can represent a promising candidate for modulation of AURKB expression, opening new trails for anticancer therapy.We also observed a considerable decrease in the expression of Aurora kinase genes, a family of kinases normally altered in different types of tumors, and consequently, a therapeutic target for cancer . Aurora-division . Addepaldivision  demonstr in vivo , 86, 87.MDM4 and PTGS2 genes in the treated cells. In the present data, complex (2) increased the levels of ROS/Superoxide, which could directly modulate the expression of PTGS2 through the MAPK/ERK cascade on damaged cells (PTGS2 expression is related to the cellular response to high levels of ROS in DU-145 after complex (2) exposure. The strength of complex (2) generates ROS is also associated with increased expression of MDM4, a key regulator of P53 that plays an important role in regulating genomic instability (MDM4 is in agreement with the ROS-mediated DNA damage induced by complex (2) as well as the elevated apoptosis by activating the intrinsic  pathway. Taken together, these results showed that complex (2) can regulate several genes associated with the proliferation of prostate adenocarcinoma cells. The regulation of these genes strengthens the mechanism of action of complex (2), which appears to be involved in the interruption of the cell cycle in G2/M in response to ROS-mediated DNA damage and the induction of caspase-dependent apoptosis.It was further observed the upregulation of ed cells . Therefotability . Increas1) and (2) exhibited remarkable antiproliferative activities to human prostate adenocarcinoma cells. We found that complex (2) significantly induced G2/M cell cycle arrest, induce caspase-dependent apoptosis, and provoke ROS-mediated DNA damage. Moreover, the expression of various genes, including cell division cycle and apoptosis was regulated under treatment. These encouraging results warrant further studies to enhance the knowledge of the anticancer properties of these complexes, as well as identify their pharmacokinetics/pharmacodynamics properties in an attempt to expand the field of prostate anticancer chemotherapy.In summary, our study revealed that lapachol-containing ruthenium complexes (The datasets presented in this study can be found in online repositories. The names of the repository and accession number can be found in the Conceived and designed the experiments: RG, AB and FP. Performed the synthesis and structural characterization of the novel complexes: KO and AG. Performed the cellular and molecular effects of the novel complexes: RG, PS, and AA. Analyzed the data and wrote the manuscript: RG, PS, AA, and KO. Provided the technical and financial support: RG, AB, and FP. All authors contributed to the article and approved the submitted version.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.


Mass spectrometric detection was done using triple quadrupole mass spectrometry using the ESI interface operating in a positive ionization mode. The developed method was validated over a linearity range of 1–500 ng mL−1 with detection and a lower quantification limit of 0.5 ng mL−1 and 1 ng mL−1. The method accuracy ranged from 95.89–98.78% (inter-day) & 93.39–97.68% (intra-day) with a precision range of 6.09–8.12% for inter-day & 7.55–9.87% for intra-day, respectively. The PGZ shows the highest Cmax of 495.03 ng mL−1 in plasma and the lowest Cmax, 24.50 ± 2.71 ng mL−1 in bone. The maximum Tmax of 5.00 ± 0.49 h was observed in bone and a minimum of 1.01 ± 0.05 h in plasma. The AUC∞) and lowest in brain (166.93 ± 15.70 &167.12 ± 16.77 ng h−1 mL−1), and the T1/2 was highest in plasma (5.62 ± 0.74 h) and lowest in kidney (2.78 ± 0.19). The developed method was successfully used to measure the PGZ pharmacokinetic and tissue distribution. Further, the developed method could be utilized for validating target organ (adipose tissue) specific delivery of PGZ (nano-formulations) in addition to conventional dosage forms.In the present study, a sensitive LC-MS/MS method was developed and validated to measure pioglitazone (PGZ) concentrations in rat plasma and tissues. The chromatographic separation was achieved by using a YMC Pro C The developed method was investigated for target and off-target distribution of pioglitazone and could be applied to validate the site-specific delivery systems. Pioglitazone (PGZ) and rosiglitazone (RGZ) were approved in the year 1999 by USFDA for the management of T2DM.4,5 Although these drugs were found to be safe on the hepatic system, later some serious side effects associated with RGZ  and PGZ , were reported, resulting in WHO restriction of RGZ and a label change for PGZ for risk of bladder cancer.4,6–11 The above off-target side effects associated with RGZ and PGZ are reported to be due to the activation of PPAR-γ (target receptor) in the non-adipose tissues such as kidney, bone, brain, and heart.11–17 To overcome RGZ and PGZ, off-target side effects, researches are employing nano-drug delivery systems  to achieve target-specific delivery of these drugs to adipose tissue.18 In this context, it becomes apparent to develop sensitive analytical methods to measure the concentration of glitazones in different matrices including, blood, adipose, brain, bone, heart, etc. to demonstrate the site-specific delivery of novel drug delivery systems.15,19–22 Further, the reported methods have their individual advantages such as short run time, simple sample treatment technique, requiring low plasma volume and improved method sensitivity.19,20,23 However, it is significant to have individual methods with all the advantages. Hence, in the present study, we have developed and validated a sensitive liquid chromatography-tandem mass spectrometry method (LC-MS/MS) for measuring PGZ concentration in rat plasma and tissues . Further, the method can be used for validating site-specific delivery systems of PGZ in addition to conventional dosage forms.Glitazones or thiazolidinediones are a class of insulin-sensitizers used in the treatment of type-2 diabetes mellitus (T2DM).22.1PGZ (purity 99.98%) and RGZ (purity 99.96%)  were obtained in the form of a gift sample from Wallace Pharmaceutical Pvt. Ltd., Goa, India. LC-MS grade formic acid and acetonitrile were procured from Sisco Research Lab Pvt. Ltd., Mumbai, India. Other reagents were of analytical grade from SD Fine Chemicals, Mumbai, India. The ultrapure water was obtained using the Milli-Q RO system, Millipore, India.2.2−1) was prepared in acetonitrile/plasma/tissue homogenate (10% w/v). Working stock solution of PGZ  was prepared by diluting the standard stock solution with acetonitrile/plasma/tissue homogenate. For quality control, the PGZ was prepared at a concentration of 450, 100, 15 and 1 ng mL−1 in acetonitrile/plasma/tissue homogenate to represent high-quality control (HQC), medium quality control (MQC), low-quality control (LQC), and lower limit quality control (LLQC) samples, respectively. In all these samples, the concentration of IS was 100 ng mL−1.For the linearity curve generation, the standard stock solution of PGZ (1 mg mL2.3−1) was added and the volume was made up to 2 mL with acetonitrile. The tubes were vortexed for 30 seconds, followed by centrifugation  for 15 min at 5000 rpm. After centrifugation, the upper supernatant acetonitrile layer was separated for LC-MS/MS analysis.A simple protein precipitation method was adopted and optimized for the extraction of PGZ from plasma and tissue homogenate. To the plasma (200 μL)/tissue homogenate  taken in 2 mL eppendorf tube a 100 μL of IS  interface, and SIL – 20 AC autosampler.18 column  with a mobile phase consisting of formic acid (0.1% v/v) and acetonitrile  at a flow rate of 0.7 mL min−1 and injection volume of 10 μL. Mass spectrometric detection was done on a triple quadrupole mass spectrometry using the ESI interface operating in a positive ionization mode. The control of the instrument and acquisition of data was carried using Lab solution software . The block temperature of 350 °C and desolvation temperature of 250 °C was maintained with a detector voltage and CID gas set at 1.3 kV and 230 kPa, respectively. For nebulization and collision, nitrogen (99.95%) and argon gases (99.99%) were used. Multiple reaction monitoring (MRM) mode was used for the quantification of PGZ. MRM transitions of m/z 357.95 → 135.15 and m/z → 94.05 with the collision energy of −30 eV and −47 eV were chosen for quantification of PGZ and IS, respectively , and the blood sample (∼1 mL) was collected from retro-orbital plexus under mild isoflurane anesthesia at 0, 0.5, 1, 3, 5, 7 and 24 h in a time staggered manner as shown in Table SI.25 The pharmacokinetic data were analyzed using PK solver software.Twelve male Wistar rats (200 ± 20 g) were procured from the in-house animal facility of JSS College of Pharmacy, Ooty. All the rats were housed in standard laboratory conditions  with normal pellet diet  and water ad libitum. The study protocol was prior approved by the Institutional Animal Ethics Committee (IAEC) . The overnight fasted animals (8–10 h) were orally administered with PGZ (10 mg kg33.1The various mobile phase compositions used for LC-MS/MS method development are given in Table SII.3.2−1 in both plasma and tissue samples with a correlation coefficient (R2) ranging from 0.992–0.999. Further, the % CV, LOD, and LLOQ for this method in all the biological samples was found to be <2%, 0.5, and 1.0 ng mL−1, respectively (−1) in six replicates were found to be within limits (−1) in six replicates show that the PGZ is stable after 3-freeze–thaw cycles at −70 ± 2 °C (% RSD −5.04 to 7.25%) and at 25 °C for 24 h in both tissue matrix (% RSD −7.35 to 11.94%) and stock solution (% RSD −1.47 to 6.91%)  . The obt3.3Cmax (in plasma), Tmax (in bone), AUC(0–24 h) (in plasma), AUCα)(0– (in plasma), and T1/2 (in plasma) values of 495.03 ± 0.74 ng mL−1, 5.00 ± 0.49 h, 1056.58 ± 65.78 ng h−1 mL−1, 1069.38 ± 77.50 ng h−1 mL−1 and 5.62 ± 0.74 h, respectively, and the lowest Cmax (in bone), Tmax (in adipose), AUC(0–24 h) (in brain), AUCα)(0– (in brain), and T1/2 (in kidney) values of 24.50 ± 2.71 ng mL−1, 1.03 ± 0.04 h, 166.93 ± 15.70 ng h−1 mL−1, 167.12 ± 16.77 ng h−1 mL−1 and 2.78 ± 0.19 h, respectively. These pharmacokinetic variations indicate the non-specific tissue distribution of PGZ.The pharmacokinetic data of PGZ is given in 4+ of m/z 357.95 for PGZ and 358.00 for IS  had a more efficient intense mass resolution in positive mode with a protonated molecular ion [M + H]0 for IS . Moreove18 column  with the mobile phase consists of 0.1% formic acid and acetonitrile  at the flow rate of 0.7 mL min−1. The retention time of PGZ and IS were found to be 2.45 and 2.46 min, respectively, with a total run time of 4.00 min  and off-target tissues . Table SIII−1 and 1.0 ng mL−1, respectively for accurate determination of PGZ in plasma and tissues so that the method can be used for validating adipose tissue-specific delivery systems of PGZ in addition to conventional dosage forms.The separation of PGZ and IS was performed on a YMC Pro C4.00 min . The opt5etc., in addition to the conventional dosage form of PGZ.A rapid and sensitive LC-MS/MS method has been developed and validated for the determination of PGZ in different biological matrices. Compared with the previous methods, the present method has significant advantages and can estimate PGZ in various tissues. The present method can be adopted for validating adipose tissue specific delivery systems of PGZ such as solid lipid nanoparticles, dendrimers, carbon nanomaterials, metallic nanoparticles, T2DMType-2 diabetes mellitusPGZPioglitazoneRGZRosiglitazoneLC-MS/MSLiquid chromatography-tandem mass spectrometryISInternal standardHQCHigh quality controlMQCMedium quality controlLQCLow-quality controlLLQCLower limit quality controlESIElectro-spray ionizationMRMMultiple reaction monitoringIAECInstitutional animal ethics committeeThe present work was funded by DST-Nanomission (DST/NM/NB/2018/227 (G), and (C)), Govt. of India, New Delhi.Ms Kusuma Kumari conceptualized, analyzed, and majorly contributed in writing manuscript. Dr Praveen supervised, proof-read the manuscript. Mr Ravi Kiran contributed in research work and proof-read. Dr Narenderan helped in analytical work and interpretation of the data. Dr Vishwanath, Dr Babu & Dr Krishnaveni provided valuable insights and proof-read the manuscript.The authors reported no potential conflict of interest.RA-011-D1RA01126J-s001


In this study, using a conscious guinea pig model of cough, we investigated whether PGE2 can sensitize the cough reflex via central actions and, if so, via which mechanisms.Cough hypersensitivity is a major characteristic feature associated with several types of cough, including chronic cough, but its underlying mechanisms remain to be fully understood. Inflammatory mediators, such as prostaglandin EAll drugs were administered by intracerebroventricular (i.c.v.) route and whole-body plethysmograph set-up was used for both induction, using aerosolized citric acid (0.2 M), and recording of cough. Immunohistochemistry was performed to confirm the expression of NaV 1.8 channels in the nucleus tractus solitarius (nTS).2 and the non-selective EP1/EP3 agonist, sulprostone, dose-dependently enhanced the citric acid-induced cough . Pretreatment with the EP1 antagonist, ONO-8130, did not affect the sulprostone-induced cough sensitization, whilst the EP3 antagonist, L-798,106, dose-dependently inhibited this effect (P ≤ 0.05). Furthermore, treatment with either the EP2 agonist, butaprost or the EP4 agonist, L-902,688, had no effect on cough sensitization. Additionally, pretreatment with either the TRPV1 antagonist, JNJ-17203212 or the TRPA1 antagonist, HC-030031, alone or in combination, nor with the NaV 1.1, 1.2, 1.3, 1.4, 1.6 and 1.7 channel blocker, tetrodotoxin, had any effect on the cough. In contrast, pretreatment with the NaV 1.8 antagonist, A-803467, dose-dependently inhibited this effect (P ≤ 0.05). Furthermore, NaV 1.8 channels were shown to be expressed in the nTS.We show that both PGE2 sensitizes the cough reflex centrally via EP3 receptor-dependent activation of NaV 1.8 but independently of TRPV1,TRPA1 and TTX-sensitive sodium channel activation. These results indicate that PGE2 plays an important role in central sensitization of the cough reflex and suggest that central EP3 receptors and/or NaVv 1.8 channels may represent novel antitussive molecular targets.Collectively, our findings show that PGE Cough, particularly that of a chronic nature, continues to be a challenging clinical condition with a high global prevalence leading to a significant economic expenditure and utilization of health care resources , 2. CougThe role of the brainstem and higher brain regions in cough is particularly poorly understood, compared to that of the airways, possibly due to difficulty of access, complexity of the neuroanatomy and lack of appropriate animal models –9. NotwiThere is a growing body of evidence suggesting that airway and central neuroplastic changes, driven by inflammation, are important drivers of cough. Interestingly, many of the inflammatory mediators involved in hyperalgesia, such as substance P (SP), nerve growth factor (NGF) and bradykinin (BK) also induce and/or sensitize the cough reflex –17 partl2 (PGE2), a major mediator of pain, has been demonstrated, paradoxically, to have both proinflammatory and protective bronchodilator effects in the respiratory system [2 can both induce and sensitize the cough reflex to citric acid and/or capsaicin [2 in cough induced via these modalities [Prostaglandin Ey system , particuy system , 25. Morapsaicin , 26, 27.dalities , 29.2 are mediated predominantly through E-prostanoid (EP) receptors, which are classified into four distinct subtypes: EP1, EP2, EP3 and EP4 [2-sensitization and induction of cough at the airway level [2 can sensitize the cough reflex via a central action has not been previously addressed.The actions of PGE and EP4 , 31. EP ay level . However2 in peripheral induction and sensitization of the cough reflex, we proposed that PGE2 also sensitizes the cough reflex centrally. In this study, and using a conscious guinea pig model of cough, we investigated [2 administered centrally can sensitize the cough reflex and if so, [Based on our previous findings with inflammatory mediators and the evidence supporting the role of PGEstigated  whether All the experimental protocols were approved by the Animal Ethics Committee in the Health Sciences Center at Kuwait University. In addition, all the experiments were conducted in accordance with international and Kuwait University guidelines and complied with the EU Directive 2010/63/EU for animal experiments and the National Institutes of Health Guide for the care and use of laboratory animals .Conscious, unrestrained adult Dunkin-Hartley guinea pigs, both males and females, weighing 400–600 g were used for all the experiments in this study. The in- house bred animals were generously supplied by the Animal Resources Center of Faculty of Medicine at Kuwait University and were housed in a fully controlled room in the animal facility maintained at 21–25 °C, relative humidity of 50% and 12-h light/dark cycle. Following cannulae implantation, all animals were placed individually in plastic cages on sawdust bedding to avoid removal or damage to the implanted cannula. The animals had unrestricted access to standard diet and tap water supplemented with multivitamins.The surgical implantation procedures were performed as previously described . Guinea 2  was initially prepared by dissolving the drug in absolute ethanol and dilutions were made in artificial cerebrospinal fluid (ACSF) to yield the desired final concentrations. Sulprostone, butaprost, L-902,688, ONO-8130, L-798,106, A-803467 , JNJ-17203212 (Tocris Bioscience) and HC-030031 (Sigma Aldrich) were initially dissolved in dimethyl sulfoxide (DMSO) and subsequent dilutions were made in ACSF to yield the desired final concentrations as shown in Table PGEThe drugs were administered to conscious and unrestrained guinea pigs on day 8 after cannula implantation as previously described , 32, 33.2-induced sensitization of the cough reflex is shown in Fig. The citric acid challenge and the assessment of cough were conducted using the Buxco whole body plethysmography (WBP) system  as previously described , 34. FifDue to the scarcity of data for central expression of NaV 1.8 channels, a qualitative IHC studies were conducted to detect the expression of NaV 1.8 channels in the brainstem nucleus, nTS, in a few guinea pigs. The guinea pigs were anesthetized with ketamine hydrochloride 80 mg/kg  and xylazine hydrochloride 6 mg/kg  administered intramuscularly (i.m.) and perfused transcardially with freshly prepared 4% paraformaldehyde. Briefly, the heart was exposed and perfused with 150 mL of heparinized saline followed by 800 mL of 4% paraformaldehyde . The brain was dissected out and post fixed for 48 h in the same fixative. Five mm length of medulla oblongata tissue starting from obex towards the spinal cord was processed for paraffin section cutting. Five-micron thick serial sections were cut and mounted on Poly-L-lysine coated glass slides. Serial brain sections were stained for cresyl violet staining and double immunofluorescence staining for NaV 1.8 and NeuN (marker for Neuron). Selected sections were incubated in a cocktail of polyclonal rabbit anti-NaV1.8  and monoclonal mouse anti-NeuN-1  antibodies overnight. The sections were then washed in PBS and treated with a cocktail of goat anti-rabbit DyLight-594 and horse anti-mouse DyLight 488 secondary antibodies . Sections were washed and mounded with vectashield mounting media. Sections were observed and photographed in the confocal microscope.2) inhalation. The CO2 gas was delivered using a compressed gas cylinder, pressure regulator and flowmeter. The flow rate of CO2 was adjusted to 5 L/min and maintained until breathing, all muscle activity and signs of life are completely absent. This was followed by cervical dislocation to ensure death.At the end of each experiment, animals were sacrificed by carbon dioxide  and GraphPad Prism . The data were analyzed blindly and expressed as mean cough ± standard error of the mean (SEM) and represent the number of coughs during the 20-min period of assessment. All treatment groups were initially tested for normality using Shapiro–Wilk test of normality. Normally distributed data were analyzed by parametric one-way analysis of variance (ANOVA), followed by Dunnett's multiple comparisons test. Nonparametric data were analyzed using nonparametric Kruskal–Wallis ANOVA followed by Dunn’s multiple comparison. At P ≤ 0.05, differences were considered statistically significant. All the groups in each experiment were time-matched to ensure all treatment groups were exposed to the same experimental conditions.2; i.c.v.  resulted in a dose-dependent increase in citric acid-induced cough response compared to vehicle treated guinea pigs (n = 12). The mean cough ± SEM were the following: 5.25 ± 1.53 (0.3 mg/ml PGE2), 7.58 ± 1.28 (0.6 mg/ml PGE2), 16.31 ± 3.16 (1 mg/ml PGE2) and 2.75 ± 0.94 (vehicle). Both 0.6 mg/ml and 1 mg/ml resulted in a significant increase in citric acid-induced cough . The data are presented in Fig. 2 at concentration of 1 mg/ml resulted in the highest enhancement of the citric acid-induced cough without any overt unwanted effects, this dose was used in the subsequent experiments in this study involving the use of PGE2.Treatment of guinea pigs with PGETreatment of guinea pigs with the non-selective EP1/EP3 agonist, sulprostone; i.c.v.  resulted in a dose-dependent increase in citric acid-induced cough response compared to vehicle treated guinea pigs (n = 9). The mean cough ± SEM were the following: 6.00 ± 2.20 (0.1 mg/ml sulprostone), 8.29 ± 1.90 (0.3 mg/ml sulprostone), 10.33 ± 1.33 (1 mg/ml sulprostone) and 3.44 ± 1.30 (vehicle). Sulprostone at 1 mg/ml caused a significant increase in citric acid-induced cough . The data are presented in Fig. Pretreatment of guinea pigs with the EP1 antagonist, ONO-8130; i.c.v.  did not affect the sulprostone-enhancement of citric acid-induced cough response compared to vehicle pretreated guinea pigs (n = 10). The mean cough ± SEM were the following: 15.71 ± 2.92 (1 mg/ml ONO-8130), 16.83 ± 4.45 (5 mg/ml ONO-8130) and 15.80 ± 5.67 (vehicle). The data are presented in Fig. Pretreatment of guinea pigs with the EP3 antagonist, L-798,106; i.c.v.  resulted in a dose-dependent inhibition of the sulprostone-enhancement of citric acid-induced cough response compared to vehicle pretreated guinea pigs (n = 10). The mean cough ± SEM were the following: 8.29 ± 1.69 , 6.20 ± 1.88  and 16.30 ± 3.49 (vehicle). L-798,106 at 5 mg/ml but not 2.5 mg/ml significantly reduced the sulprostone-enhancement of citric acid-induced cough response by 62% (P = 0.022). The data are presented in Fig. Treatment of guinea pigs with the selective EP2 agonist, butaprost; i.c.v.  did not affect the citric acid-induced cough response compared to vehicle treated guinea pigs (n = 11). The mean cough ± SEM were the following: 2.50 ± 1.56 (0.3 mg/ml butaprost), 2.60 ± 1.10 (1 mg/ml butaprost) and 2.27 ± 0.93 (vehicle). The data are presented in Fig. Treatment of guinea pigs with the selective EP4 agonist, L-902,688; i.c.v.  did not affect the citric acid-induced cough response compared to vehicle treated guinea pigs n = 7). The mean cough ± SEM were the following: 2.33 ± 1.29 , 2.38 ± 0.92  and 2.29 ± 1.25 (vehicle). The data are presented in Fig. . The mea2-enhancement of citric acid-induced cough response compared to vehicle pretreated guinea pigs (n = 10). The mean cough ± SEM were the following: 11.00 ± 4.55 (0.4 mg/ml JNJ-17203212), 10.67 ± 2.62 (1.3 mg/ml JNJ-17203212) and 12.10 ± 1.92 (vehicle). The data are presented in Fig. Pretreatment of guinea pigs with the TRPV1 channel antagonist, JNJ-17203212; i.c.v.  did not affect the PGE2-enhancement of citric acid-induced cough response compared to vehicle pretreated guinea pigs (n = 8). The mean cough ± SEM were the following: 11.57 ± 3.60 (0.02 mg/ml HC-030031), 13.00 ± 3.48 (0.05 mg/ml HC-030031) and 14.25 ± 2.29 (vehicle). The data are presented in Fig. Pretreatment of guinea pigs with the TRPA1 channel antagonist, HC-030031; i.c.v.  did not affect the PGE2-enhancement of citric acid-induced cough response compared to vehicle pretreated guinea pigs (n = 7). The mean cough ± SEM were the following: 11.40 ± 2.50 (low-dose combination), 10.17 ± 2.66 (high-dose combination) and 10.71 ± 1.95 (vehicle). The data are presented in Fig. Pretreatment of guinea pigs with combined doses of JNJ-17203212 and HC-030031, i.c.v., at either low  or high doses  did not affect the PGE2-enhancement of citric acid-induced cough response compared to vehicle pretreated guinea pigs (n = 7). The mean cough ± SEM were the following: 15.29 ± 4.98 (0.015 µg/ml TTX), 16.60 ± 3.61 (0.1 µg/ml TTX) and 18.14 ± 4.75 (vehicle). The data are presented in Fig. Pretreatment of guinea pigs with the tetrodotoxin, TTX; i.c.v.  did not affect the PGE2-enhancement of citric acid-induced cough response compared to vehicle pretreated guinea pigs (n = 6). The mean cough ± SEM were the following: 8.80 ± 1.79 (5 mg/ml A-803467), 4.40 ± 1.97 (10 mg/ml A-803467) and 19.33 ± 5.00 (vehicle). A-803467 at 10 mg/ml significantly reduced the PGE2-enhancement of citric acid-induced cough response by 77% (P = 0.018). The data are presented in Fig. Pretreatment of guinea pigs with the NaV1.8 channel antagonist, A-803467; i.c.v.  resulted in a dose-dependent inhibition of the PGEImmunohistochemistry using a polyclonal NaV 1.8 antibody confirmed the expression of NaV 1.8 channels in the nTS. NaV 1.8 channels were clearly expressed within all the neurons of the brainstem nucleus, nTS, of guinea pigs. The data are presented in Fig. 2, via the central route, results in sensitization of the cough reflex which is mediated via EP3 receptor-dependent activation of NaV 1.8 channels but independently of TRPV1, TRPA1 or TTX-sensitive channel activation.In this study we show, using a conscious guinea pig model of cough, that acute exposure to PGEInflammatory mediators have been reported to play a role in the sensitization of cough, at both peripheral and central levels. Elucidating the mechanism by which this is achieved will not only result in better understanding of the cough mechanisms but will also result in identification of novel targets for cough therapy.2, via the i.c.v route, resulted in a dose-dependent enhancement of the citric acid-induced cough. The enhancement effect of PGE2 was observed shortly after its administration and occurs over the same time frame seen with other inflammatory mediators such as NGF and BK [2 plays a role in the central sensitization of the cough reflex, in agreement with both clinical and preclinical studies which show that PGE2  can sensitize the cough reflex/airway vagal nerves [2 is reported to be involved in the central mechanisms that underlie the sensitization of pain pathways leading to hyperalgesia. For example, both intrathecal (i.t.) and i.c.v. administration of PGE2 result in a dose-dependent enhancement of the sensitivity to pain in mice and rats [Our data show that acute exposure to PGEF and BK , 14. Thel nerves , 35, 36.and rats –40.2 acts to enhance the cough reflex has not been determined in this study. However, several studies have previously identified the nucleus tractus solitarius (nTS) as a likely site. For example, the enhanced release of several excitatory mediators of the synaptic transmission in the nTS such as glutamate and SP is proposed to result in synaptic plasticity and central sensitization of the cough reflex [The specific site in the brainstem where PGEh reflex , 41–44. h reflex , 46. Nuc2 [2. A pharmacological approach using selective agonists and also antagonists, when the selectivity of an agonist to a specific sub-type was not high, were used to rule in or out the involvement of a particular EP receptor sub-type [All four EP receptors are reportedly expressed in the brainstem/nTS and have a high affinity for PGE2 –49 and csub-type .2 enhancement of the cough response to citric acid [2, over the same dose range, suggesting that the sensitizing effects of PGE2 and sulprostone on cough were likely to be mediated via the same receptor subtype/s, namely EP1/EP3. These data are in line with that showing that both sulprostone and PGE2 induce depolarizations in the isolated guinea-pig vagus nerves, indicating that the activation of sensory nerves was also dependent on EP1/EP3 receptor subtype activation [2 [2/sulprostone mediate the enhanced citric acid-induced cough centrally via activation of EP1 and/or EP3 receptors.Sulprostone, a non-selective agent with high affinity for both EP1 and EP3 receptors, was used to explore whether EP1 and/or EP3 receptors mediate the central PGEric acid , 51. Ourtivation . Furtheration [2 , 52, 53.2 in central sensitization of the cough reflex is mediated, at least in part, via activation of EP3 receptors. This observation is in agreement with findings from a recent study, using both in vitro and in vivo approaches, that identified EP3 as the key receptor in mediating PGE2-induced sensory nerve activation as well as cough induction, when administered peripherally [2-induced central sensitization of pain. For example, activation of spinal, or supra-spinal EP3 receptors had been reported to play an important role in the development of hyperalgesia and allodynia in mice and rats [2- induced central sensitization of pain [To identify which one of the two receptors, or if indeed both, are involved in the sulprostone- enhanced cough, we used selective antagonists for EP1 and EP3 receptors. Our data show that pretreatment with the highly selective EP1 antagonist, ONO-8130, did not inhibit the sulprostone-enhanced citric acid-induced cough indicating that EP1 receptors were not involved in the central sensitization of the cough reflex. Higher doses of ONO-8130 could not be tested because of solubility limitations. In contrast, our data show that pretreatment with the highly selective EP3 antagonist, L-798,106 dose-dependently decreased the sulprostone-enhanced citric acid-induced cough. These findings clearly show that the effect of PGEpherally . Our resand rats , 52–54.  of pain . These d of pain  and alth of pain –59.2 enhancement of cough response to citric acid centrally. Our data show that acute exposure to either butaprost (FA) or L-902,688, via the i.c.v route, did not affect the cough response suggesting that neither EP2 receptors nor EP4 receptors have contributed to the PGE2-enhanced cough centrally. These findings are in line with the results of an in vitro study which reported that neither EP2 receptors nor EP4 receptors were involved in the PGE2-induced depolarization of vagal sensory nerves isolated from guinea pigs and mice [The highly selective EP2 agonist, butaprost  and EP4 agonist, L-902,688 were used to explore whether EP2 receptors and EP4 receptors, respectively, mediate the PGEand mice . Our resand mice . Moreoveand mice . Some stand mice , 59, 60.2-enhanced citric acid-induced cough. These data indicate that these channels are not involved in PGE2-induced central sensitization of the cough reflex. However, based on the small reduction that was noted with the individual drugs, as well as good evidence of positive interaction between TRPV1 and TRPA1 channels, we postulated that perhaps both channels may need to be blocked in order to see any significant degree of inhibition. Our data show that pretreatment with combined doses of JNJ-17203212 and HC-030031 (low and high doses) did not affect the PGE2-enhancement of citric acid-induced cough response thus confirming that neither TRPV1 nor TRPA1 channels are involved in PGE2- induced central sensitization of the cough reflex.We also investigated whether TRP channels, specifically TRPV1 and TRPA1, are the downstream effectors of EP3 receptor activation and central sensitization of the cough reflex. These channels have been reported to be involved in cough induced/enhanced by other inflammatory mediators as well as in EP3 receptor- mediated sensory nerve activation and peripheral cough , 61, 62.Given that many studies have reported a critical role for TRP channels in cough, both at peripheral and central level, this was a surprising finding –65. A po2 and NaVs has been investigated because PGE2 was shown to potentiate both TTX-sensitive and TTX-resistant currents in different neuronal cells [2 [Finally we investigated whether voltage gated sodium channels, tetrodotoxin (TTX)-sensitive  and the TTX-resistant channel, specifically NaV 1.8, are coupled to EP3 receptor activation and are involved in the central sensitization of the cough reflex. The link between PGEal cells –70. In aal cells , 71, 72.cells [2 –77.2-enhanced citric acid-induced cough suggesting that TTX-sensitive channels are not involved in PGE2-induced central sensitization of the cough reflex. This lack of effect was not dose related as TTX was shown to have clear pharmacological effects with comparable doses used in previous studies [Our data show that pretreatment with the potent neurotoxin, TTX, via the i.c.v. route, failed to inhibit the PGE studies , 79. Mor studies . Some, b2-enhanced citric acid-induced cough. This suggests that NaV 1.8 channels, (TTX-resistant sodium channels), are coupled to EP3 receptor activation and mediate the PGE2-induced central sensitization of the cough reflex. Our results are in agreement with a recent study reporting that both local and systemic administration of A-803467 reduced the number of coughs induced by capsaicin challenge suggesting a role of NaV 1.8 channels in peripheral cough [2 potentiates TTX-R channel function, most likely NaV 1.8 channels, in capsaicin-sensitive vagal pulmonary neurons by increasing the TTX-R conductance and increasing the voltage sensitivity; an effect that could be dependent on EP3 receptors [A-803467 is a potent and highly selective NaV 1.8 antagonist which has been widely used to investigate the role of these channels in pain and cough studies. Our data show that pretreatment with A-803467, via the i.c.v. route, dose-dependently decreased the PGEal cough . Furtheral cough –84. Inteal cough , 86. Theeceptors . However2, centrally, enhances citric acid-induced cough via EP3 receptor-dependent activation of NaV 1.8 channels but independently of TRPV1, TRPA1 and TTX-sensitive channels activation. Altogether, our findings support an important role of inflammatory mediators, specifically PGE2, in driving cough hypersensitivity and identify central EP3 receptors and NaV 1.8 channels as part of an important signaling pathway in enhanced cough. Therefore, targeting central EP3 receptors and/or NaV 1.8 channels may represent a novel approach for the treatment of cough hypersensitivity syndrome.In summary, our data show that PGE