Breast cancer (BC) is the leading cause of death in women all over the world. Currently, combined chemotherapy with two or more agents is considered a promising anti-cancer tool to achieve better therapeutic response and to reduce therapy-related side effects. In our study, we demonstrated an antagonistic effect of cytostatic agent-cisplatin (CDDP) and histone deacetylase inhibitor: cambinol (CAM) for breast cancer cell lines with different phenotypes: estrogen receptor positive  and triple negative . The type of pharmacological interaction was assessed by an isobolographic analysis. Our results showed that both agents used separately induced cell apoptosis; however, applying them in combination ameliorated antiproliferative effect for all BC cell lines indicating antagonistic interaction. Cell cycle analysis showed that CAM abolished cell cycle arrest in S phase, which was induced by CDDP. Additionally, CAM increased cell proliferation compared to CDDP used alone. Our data indicate that CAM and CDDP used in combination produce antagonistic interaction, which could inhibit anti-cancer treatment efficacy, showing importance of preclinical testing. Breast cancer (BC) is the most frequently diagnosed type of cancer in women worldwide, with a mortality rate of 15% ,3. MolecHistone deacetylase inhibitors (HDIs) are group of compounds that receive growing attention in the treatment of cancer in monotherapy, as well as in combination with other anti-cancer drugs ,16. Thei50 = 56 uM and IC50 = 59 uM, respectively) [Cambinol (CAM), a cell-permeable β-naphtol derivative, inhibits SIRT1 and SIRT2 activity (ICctively) ,21. The ctively) . In addictively) . Additioctively) . To valictively) ,25. The ctively) .Platinum-based compounds are broadly applied for chemotherapy. Cisplatin (CDDP) is one of the derivatives of platinum used as a highly effective drug in a variety of cancers, such as sarcomas, lymphomas, and germ cell tumors and carcinomas, including BC . The CDDThe aim of this study is to investigate the anti-cancer activity of CAM and CDDP administered alone or in combination on two types of BC cell lines  using advanced pharmacokinetic isobolographic analysis.50 values ± S.E.M.) for each line was calculated based on long-probit analysis of the concentration–response relationship effects of both agents . The results are presented as median inhibitory concentrations  of CAM and CDDP administered in combination with respect to their anti-proliferative effects in four breast cancer cell lines  (The antiproliferative effect of CAM and CDDP administered in combination against BC cell lines was assessed using MTT assay. CAM and CDDP were administered in combination with 1:1 drug mixture in increasing concentrations. All BC cell lines were exposed to the CAM and CDDP mixture treatment using different ratios of IC-MB-468) .50 values for CDDP, which were 1.57 µg/mL (y = 1616x + 4.6829), 1.13 µg/mL (y = 1.0066x + 4.9479), 1.51 µg/mL (y = 0.9637x + 4.8285), and 1.57 µL/mL (y = 0.9564x + 4.8123) for MCF7, T47D, MDA-MB-231, and MDA-MB-468, respectively. Simultaneously, the equations of dose–response relationship curves (DRRCs) that allowed us to determine IC50 values for CAM were calculated: 20.31 µg/mL (y = 6.6232x − 3.6617), 16.51 µg/mL (y = 5.7965x − 2.0591), 13.44 µg/mL (y = 4.0016x + 0.485), and 15.03 µL/mL (y = 4.8046x − 0.6546) for MCF7, T47D, MDA-MB-231, and MDA-MB-468, respectively  allowed us to determinate ICectively .50 values calculated from the DRRC for the mixture of CAM and CDDP were: 32.48 µg/mL (y = 7.3866x − 6.166), 19.11 µg/mL (y = 2.1449x + 2.2517), 21.35 µg/mL (y = 4.1335x − 0.4953), and 15.71 µg/mL (y = 3.5604x + 0.7408) for MCF7, T47D, MDA-MB-231, and MDA-MB-468, respectively  and MDA-MB-468 cell lines. In T47D and MCF7 cells, after 2IC50 CAM treatment, the active caspase-3 had not been decreased meaningfully compared to IC50. In MCF7 cells, the 2IC50 decrease was not statistically significant compared to the control. This phenomenon may be explained by the fact that CAM does not activate caspase-3, whereas CDDP does, and CAM abolishes the CDDP effect on caspase-3, which leads to a decrease in active caspase-3.The impact of CAM and CDDP administered separately and in combination on induction of apoptosis was measured as a number of cells with activated caspase-3 . CAM and50 concentration. For 2IC50 concentration, there was a slight increase in the number of cells with active caspase-3 for CAM and CDDP used in combination, compared with drugs used separately. The difference in the level of active caspase-3 between CAM and CDDP used alone and combined is weak, which may confirm that only CDDP induces caspase-3 activation.Interestingly, in the MDA-MB-231 cell line, the apoptosis induction caused by CAM and CDDP used separately and both agents used in combination remained at the same level for IC50 and 2IC50 concentration led to cell arrest in phase S; however, the number of cells arrested in S-phase was slightly higher for TNBC lines. CAM abolished this effect in each BC cell line.The impact of CAM and CDDP administered separately and in combination on the cell cycle is presented in According to the cell cycle progression analysis, the cell cycle changes induced by CAM and CDDP administered separately and in combination were not dependent on the type of cell line.Sirtuins are known as key players in tumor development affecting a variety of signaling pathways ,35. AmonIn our study, we analyzed the interaction between CAM, SIRT1 and SIRT2 inhibitor, and the CDDP standard chemotherapeutic used in BC therapy. We analyzed four BC cell lines differing from each other by molecular profile . Our finIn multiple melanoma cells, CAM inhibits cell proliferation in a time- and dose-dependent manner, increasing apoptosis and arresting cells in G1 phase . In our Although the molecular mechanism of CDDP action and its cytotoxic implication is well understood, there is still need to search for appropriate drugs that will allow us to reduce its cytotoxicity while maintaining therapeutic benefits . Our finIt is obvious that synergetic interaction is the most favorable; however, drugs often interact in a non-synergistic way ,54, whicTaken together, our data indicate that CAM and CDDP used in combination produce antagonistic interaction. The type of pharmacological drug–drug interaction between CAM and CDDP disclaims potential application of combined treatment in BC cells; however, this interaction could be completely different in other cancer cell lines in which SIRT1 and SIRT2 are involved and in combination with different anti-cancer drugs.2+ and Ca2+ at 1 mg/mL as a stock solution. The drugs were diluted with culture medium to the respective concentration just before use.Cambinol (CAM) was purchased from Sigma-Aldrich . A stock solution of CAM (100 mM) was prepared in dimethyl sulfoxide (DMSO). Cisplatin (CDDP) was purchased from Sigma  and dissolved in phosphate buffered saline (PBS) without Mg® HTB-22TM), T47D (ATCC® HTB-133TM), MDA-MB-231 (ATCC® CRM-HTB-26TM), and MDA-MB-468 (ATCC® HTB-132TM) human BC cell lines were obtained from the American Type Culture Collection . T47D, MCF7, MDA-MB-231, and MDA-MB-468 cancer cells were grown in DMEM/Nutrient F-12 Ham (DMEM/F12) culture medium (Sigma) with 10% FBS (Sigma), penicillin (100 μg/mL), (Sigma) and streptomycin (100 μg/mL) (Sigma). Mycoplasma-free cultures were maintained in a humidified atmosphere with 5% CO2 at 37 °C.MCF7  or CDDP (0.01–10 µg/mL) individually or a combination of both compounds for 96 h. Then, the BC cell lines were incubated with the 3--2,5-diphenyltetrazolium bromide (MTT) solution at a concentration of 5 mg/mL (Sigma) for 3 h. During this time, MTT was metabolized by living cells to purple formazan crystals, which were solubilized in a sodium dodecyl sulfate (SDS) buffer (10% SDS in 0.01 N HCl) overnight. The optical density of the product was measured at 570 nm using an Infinite M200 Pro microplate reader . Dose–response curves were plotted to determine half-maximal inhibitory concentrations (IC50) for the CAM and CDDP using GraphPad Prism 9.0 . The results of combined treatment CAM and CDDP were analyzed according to isobolographic protocol. The drug doses were determined based on the IC50 values calculated from the previous cytotoxicity test.MCF7, T47D, MDA-MB-231, and MDA-MB-468 BC cell lines were placed on a 96-well plate  at a density of 1 × 10MCF7, T47D, MDA-MB-231, and MDA-MB-468 cell lines were treated with tested compounds individually or in combination for 48 h, and as a control, cells without treatment were used. Afterwards, cells were harvested, fixed, and permeabilized using the Cytofix/Cytoperm. All experiments were performed according to the manufacturer’s instructions of PE Active Caspase-3 Apoptosis Kit . Labeled cells were analyzed by flow cytometer FACSCalibur , operating with CellQuest software (BD Biosciences) to quantitatively assess the caspase-3 activity.MCF7, T47D, MDA-MB-231, and MDA-MB-468 cell lines were treated with tested compounds and mixture for 48 h and then fixed in 80% ethanol at −20 °C for 24 h. The experiment was conducted by utilizing PI/RNase Staining Buffer (BD Biosciences) according to the manufacturer’s instructions. Cell cycle analysis was performed using flow cytometry FACSCalibur (Becton Dickinson). Acquisition rate was 60 events per second in low acquisition mode, and 10,000 events were measured.50 values) for CAM and CDDP in each cell line, as recommended elsewhere [50 add) for drugs in combination was theoretically calculated using the method described elsewhere [50 mix values for tested drug combinations in a fixed 1:1 ratio. The particular drug concentrations (CAM and CDDP) in combination were calculated by multiplying IC50 mix values accordingly to proportions in additive drug combinations by means of the unpaired Student’s t-test with Welch’s correction, as recommended elsewhere [The pharmacodynamic nature of interactions between CAM and CDDP in four BC cell lines was analyzed by means of the type I isobolographic analysis for both parallel and nonparallel concentration–response relationship lines ,61. To slsewhere ,31. The lsewhere . The callsewhere ,63. An ilsewhere ,31.50 and IC50 mix values for CAM and CDDP administered separately or in combination at the fixed ratio of 1:1 were calculated by computer-assisted log-probit analysis according to Litchfield and Wilcoxon [50 exp values for the combination of CAM and CDDP were statistically compared to their respective theoretical additive IC50 add values by the use of unpaired Student’s t-test with Welch’s correction, according to Tallarida [p < 0.05 . Data are expressed as mean ± standard deviation of the mean (±SD).The ICWilcoxon . The expallarida . Results


Transitional cell carcinoma (TCC) is the most common malignant tumor of the canine urinary tract and tends to have a poor prognosis due to its invasive potential. Recent studies have reported that up to 80% of canine urothelial carcinoma has the BRAF V595E mutation, which is homologous to the human V600E mutation. Activating the BRAF mutation is an actionable target for developing effective therapeutic agents inhibiting the BRAF/mitogen-activated protein kinase (MAPK) pathway in canine cancer as well as human cancer. We established novel canine TCC cell lines from two tumor tissues and one metastatic lymph node of canine TCC patients harboring the BRAF V595E mutation. Tumor tissues highly expressed the BRAF mutant and phosphorylated extracellular signal-related kinases (ERK)1/2 proteins. The derived cell lines demonstrated activated MAPK pathways. We also evaluated the cell lines for sensitivity to BRAF inhibitors. Sorafenib, a multiple kinase inhibitor targeting RAF/vascular endothelial growth factor receptor (VEGFR), successfully inhibited the BRAF/MAPK pathway and induced apoptosis. The established canine TCC cell lines responded with greater sensitivity to sorafenib than to vemurafenib, which is known as a specific BRAF inhibitor in human cancer. Our results demonstrated that canine TCC cells showed different responses compared to human cancer with the BRAF V600E mutation. These cell lines would be valuable research materials to develop therapeutic strategies for canine TCC patients. Transitional cell carcinoma (TCC) is the most common malignant tumor of the canine urinary tract ,2. RecenNon-mutated BRAF is activated by the phosphorylation of Thr599 and Ser602 induced by RAS . HoweverIn human medicine, small molecule inhibitors vemurafenib and dabrafenib targeting the BRAF V600E mutant are approved by the Food and Drug Administration (FDA) ,17,18. PIn addition, canine TCC harboring BRAF mutations will be a unique model for human cancers with BRAF signaling activation in the field of comparative oncology. As dogs share life environments with humans, naturally occurring cancers in dogs provide beneficial insight into cancer development and disease progression in humans. Cross-species comparative studies have revealed that canine cancers were relevant to human cancers, demonstrating conserved driver oncogenes and signaling pathways ,26,27. TAlthough there have been some reports on the establishment of canine TCC cell lines ,29, the The aim of this study was to characterize novel canine TCC cell lines harboring the BRAF V595E mutation. We investigated their morphology, molecular expression, proliferation, and tumorigenicity. We also compared the anti-tumor effects of BRAF inhibitors sorafenib and vemurafenib in the canine TCC cell lines. This report suggests new research materials and therapeutic strategies for canine TCC patients.A 10-year-old Jindo dog (Patient TCC-001) and an 11-year-old Maltese (Patient TCC-002) were diagnosed with TCC and underwent surgery. The tumor tissue and metastatic sub-lumbar lymph node of patient TCC-001 and tumor tissue of patient TCC-002 were collected after surgery A. All tiTo screen for cancer driver genes, we performed whole-exome sequencing (WES) of KU-CTCC-001 . Of the All TCC cell lines proliferated faster than D17 cells, which were used as a comparison group A. KU-CTC3 but as the tumor surface scar became severe, we euthanized the NOG mice injected with KU-CTCC-002 and D17 cells seven weeks after the injections. There was no metastasis in either mouse strain. Tumor weight and volume were proportional (In vivo tumorigenicity was investigated using a xenograft mouse model. We injected the cell lines subcutaneously into athymic nude (NU(NCr)-Foxn1nu) and NOG (NOD.Cg-Prkdcscid Il2rgtm1Sug/Jic) mice. All cell lines demonstrated tumorigenicity in both strains . Signifiortional C. To invortional . In contWe examined the effect of sorafenib (BAY 43-9006), a multiple kinase inhibitor targeting RAF/VEGFR, on canine TCC cell lines. We also compared the sensitivity of sorafenib with vemurafenib (PLX4032) since vemurafenib is a specific inhibitor of BRAF mutations. G361, a human melanoma cell line harboring heterozygous BRAF V600E mutations, was used as a control. Cell viability was measured after treatment with drugs at each concentration and the half maximal inhibitory concentration (IC50) values were compared between drugs A,B. As eTransitional cell carcinoma, also called urothelial carcinoma, is the most common malignancy of the canine urinary tract, affecting tens of thousands of dogs worldwide every year . Most blIn our cases, the majority of the original tumor of patient TCC-001 was located in the urethra. However, as the tumor grew invasively, it extended to the trigone area. The tumors of patient TCC-002 were distributed widely from the bladder trigonal region to the proximal urethra including the prostate. In addition, patient TCC-002 showed severe hydronephrosis due to ureteral dilation. The trigonal mass obstructed the ureterovesical junction and induced ureteral dilation. Both patients had regional lymph node metastasis, but the metastatic lymph nodes of patient TCC-002 were not available for further experiments.There are diverse treatment methods for TCC including surgery, radiation therapy, chemotherapy, and a combination of these. In contrast to human bladder cancer, which requires complete cystectomy as a front-line treatment, surgical excision is not recommended as a front-line treatment for canine TCC. Due to the trigonal location of the tumors and the frequency of urethral involvement, complete surgical excision is difficult ,32. ExceThe canine BRAF V595E mutation, which is homologous to the human BRAF V600E mutation, is known as a common driver gene mutation in canine TCC ,4,5. GivSeveral previous studies have reported the establishment of canine TCC cell lines ,29. HoweKU-CTCC-001 and KU-CTCC-002, derived from the original tumors of patients, showed different growth characteristics. KU-CTCC-001 proliferated faster than KU-CTCC-002 in vitro. However, it showed the opposite characteristics in vivo. The tumors that were formed by KU-CTCC-002 cells had a large area of necrosis, numerous immune cells, and connective tissues in the xenograft tumor, suggesting that cancer cells significantly interacted with other cells such as fibroblasts or macrophages in vivo. In addition, as we established paired cell lines from the original tumor mass and a metastatic region, we compared them in almost all experiments we performed. KU-CTCC-001 proliferated faster than its metastatic cell line KU-CTCC-001-LM3 both in vitro and in vivo. Even though we concluded that KU-CTCC-001-LM3 did not have more oncogenic characteristics than KU-CTCC-001 based on these results, further investigation should be conducted on these relationships.3, mice injected with KU-CTCC-002 cells were euthanized before the tumor volume reached 1500 mm3, so longer monitoring may be required to detect metastasis.Moreover, to confirm whether the oncogenic characteristics of the cells changed after tumorigenesis in vivo, we dissociated the tumors derived from xenograft mice injected with KU-CTCC-001 to single cells (TCC-001-XDC) and compared the two cell lines. There was no significant difference between the two cell lines in the ratio of wild-type to mutant, MAPK pathway activation at the protein level, and in vitro evaluation of growth characteristics. There was also no metastasis of any xenografts formed by injection of the TCC cell lines. Although xenografts from KU-CTCC-001 and KU-CTCC-001-LM3 injections showed no metastatic regions until the tumor volume reached 1500 mmIn terms of comparative oncology, these TCC cell lines would be also valuable research materials. Canine invasive urethral TCC mimics that of humans in pathology, local invasion, frequency of distant metastasis, and response to chemotherapy . CurrentIn conclusion, we established novel canine TCC cell lines from two tumor tissues and one metastatic lymph node of canine TCC patients harboring BRAF V595E mutations. They all exhibited MAPK pathway activation and tumorigenicity in in vivo xenografts. In addition, we evaluated our cell lines for sensitivity to two RAF inhibitors, sorafenib and vemurafenib. In contrast to the human cell line, sorafenib showed more anti-tumor effects than vemurafenib. These cell lines would be valuable research materials for investigating therapeutic agents or studying drug mechanisms in both humans and dogs. In addition, this study suggests a new therapeutic strategy for canine TCC patients.2. The growth rate of each cell line was evaluated by counting cell numbers at 24-h intervals for 5 days. Cell doubling time was calculated from the growth rate.A 10-y-old spayed female Jindo dog (Patient TCC-001) and an 11-y-old neutered male Maltese (Patient TCC-002) came to Konkuk University Veterinary Medical Teaching Hospital  and were pathologically diagnosed with TCC . PatientBRAF V595E mutations were detected by Sanger sequencing and whole-exome sequencing (WES). Genomic DNA was extracted from the cells using a QIAamp DNA Mini Kit  and BRAF exon 15, in which the V595E mutation is located, was amplified using BRAF primers . The WES details are described in the ® Kits for Dogs  were used to analyze the STRs of genomic DNA and performed according to the manufacturer’s protocols.StockMarks2 cells were seeded in 6-well plates. The medium was changed twice a week. After two weeks, the medium was removed and stained with 0.5% crystal violet solution. The colony-covering area was measured using ImageJ software . We also conducted migration assays to evaluate migratory ability. Cells (2 × 104) were seeded in 96-well plates and incubated for 24 h to form a monolayer of cells. Wounds were made on the cells using a 96-pin Wound Maker and cell migration was monitored using the IncuCyte™ Live-Cell Imaging System . The images were collected every 2 h and analyzed by the IncuCyte software system. All experiments were performed in triplicate.To evaluate colony-forming ability, 7 × 10Proteins were extracted in cell lysis buffer containing phosphatase inhibitor cocktail, protease inhibitor, dithiothreitol, and phenylmethylsulfonyl fluoride. All samples were loaded on sodium dodecyl sulfate-polyacrylamide gels in equal amounts, electrophoresed, and transferred to membranes. After blocking with 5% skim milk-Tris-buffered saline with 0.1% Tween 20 detergent for 1h, the membranes were incubated with primary antibodies. Antibodies were purchased from Cell Signaling Technology  (MEK1/2 (#4694), p-MEK1/2 (Ser217/221) (#9154), ERK1/2 (#4696), p-ERK1/2 (Thr202/Tyr204) (#4370)), Santa Cruz Biotechnology  (BRAF(#sc-55522)); Thermo Fisher Scientific (p-BRAF (ser602) (#PA5-38412)); and Abcam  ). For detailed information on the EMT antibodies, see Supplementary Materials and Methods.6) were inoculated subcutaneously into the flanks of 6-week-old athymic nude (NU(NCr)-Foxn1nu) and NOG (NOD.Cg-Prkdcscid Il2rgtm1Sug/Jic) mice. We inoculated cells into both sides of the flanks on each mouse. Tumor volume (mm3) was measured twice a day and estimated using the equation (A × B2)/2 . Before exceeding a tumor volume of 1500 mm3, the mice were euthanized and necropsies were performed. Tumor tissues in which the cells were subcutaneously inoculated were harvested and dissociated into cells. All animal experiments in this study were performed under the guidelines of the Institutional Animal Care and Use Committee (IACUC) . Cells . After treating with 3% hydrogen peroxide to inhibit peroxidase activity, the slides were blocked in 1% bovine serum albumin and then incubated with primary antibody overnight. After incubation with a biotinylated secondary antibody, the 3,3′-diaminobenzidine substrate was applied for enzymatic detection. 4) were seeded in 96-well plates and incubated overnight at 37 °C in a humidified atmosphere of 5% CO2. The medium was replaced with that containing gradient doses of drugs. After 48 h, cell viability was examined using the CellTiter-Glo® Luminescent Cell Viability Assay . IC50 values were measured using GraphPad Prism 5  with nonlinear regression analysis. Sorafenib (BAY 43-9006) and vemurafenib (PLX4032) were purchased from Selleckchem . Each drug was serially diluted with dimethyl sulfoxide to the proper concentrations. All experiments were performed in triplicate.Cells  and the proportion of cells in the sub-G1 phase was calculated using FlowJo software .t-test. Statistical significance was indicated at * p < 0.05, ** p < 0.01, and *** p < 0.001.All data were statistically analyzed using GraphPad Prism 5 . The cell growth rates and tumor volume growth in the xenograft model B were an


The circadian clock coordinates biological and physiological functions to day/night cycles. The perturbation of the circadian clock increases cancer risk and affects cancer progression. Here, we studied how BMAL1 knockdown (BMAL1-KD) by shRNA affects the epithelial–mesenchymal transition (EMT), a critical early event in the invasion and metastasis of colorectal carcinoma (CRC). In corresponding to a gene set enrichment analysis, which showed a significant enrichment of EMT and invasive signatures in BMAL1_high CRC patients as compared to BMAL1_low CRC patients, our results revealed that BMAL1 is implicated in keeping the epithelial–mesenchymal equilibrium of CRC cells and influences their capacity of adhesion, migration, invasion, and chemoresistance. Firstly, BMAL1-KD increased the expression of epithelial markers  but decreased the expression of Twist and mesenchymal markers (N-cadherin and vimentin) in CRC cell lines. Finally, the molecular alterations after BMAL1-KD promoted mesenchymal-to-epithelial transition-like changes mostly appeared in two primary CRC cell lines  compared to the metastatic cell line SW620. As a consequence, migration/invasion and drug resistance capacities decreased in HCT116 and SW480 BMAL1-KD cells. Together, BMAL1-KD alerts the delicate equilibrium between epithelial and mesenchymal properties of CRC cell lines, which revealed the crucial role of BMAL1 in EMT-related CRC metastasis and chemoresistance. Colorectal cancer (CRC) is one of the most common cancers and represents the fourth leading cause of cancer death worldwide. In 2019, there were 145,600 new cases and approximately 51,020 deaths in the United States . SeveralThe circadian timing system (CTS) coordinates organismal behavior, including physiology and metabolism, over 24 h environmental cycles. In mammals, the suprachiasmatic nuclei (SCN) of the hypothalamus allows the coordination of circadian rhythms via peripheral molecular clocks composed of about twenty genes located in each cell of the organism. The circadian clock genes compose a complex network of auto-regulatory transcription/translation feedback loops, such as positive (BMAL1 and CLOCK) or negative (PER and CRY) loops and also feedback loops (REV-ERB α/β and ROR α/β/γ) to regulate different biological rhythm progressions . The cirAs a main positive loop element which initiates circadian oscillation, BMAL1 is considered an essential and non-redundant component of circadian clocks. Among canonical clock genes, only BMAL1 knockout results in complete loss of circadian rhythm in both SCN and peripheral tissues , which cp = 6.04 × 10−10, Disruption of the circadian clock plays a crucial role in carcinogenesis, in particular in human CRC ,12,13. Dp < 0.001, FUCA1, SGCB, IL15, WIPF1, TGFB1, ECM2, ADM12, POSTN, PRRX1, VIM, BASP1, and EMP3) allowed for the discrimination of the subgroup of BMAL1-dependent CRC patients  allowed for the discrimination of subgroups of BMAL1-dependent CRC patients  showed a significant enrichment of epithelial–mesenchymal transition signature in BMAL1_high CRC patients as compared to BMAL1_low CRC patients  and a metastatic cell line, SW620, were transduced with lentiviruses encoding a scrambled shRNA (shScr) or a shRNA-targeting BMAL1 (shBMAL1). SW480 and SW620 cell lines are derived from the primary tumor and a metastasis site of the same patient, respectively. Previous results from our laboratory showed that BMAL1 mRNA and protein substantially decreased in the three stable BMAL1 knockdown (BMAL1-KD) cell lines compared to their proper control, thereby modulating different cellular activities such as proliferation, apoptosis, and senescence .We here investigated whether BMAL1-KD could also be involved in the EMT process of colon cancer. As a main component of the cell–cell adhesion junctions, E-cadherin plays a central role in maintaining epithelium integrity ,23,24. SRecruitment of β-catenin and others Catenins to E-cadherin at the cell membrane is essential in the adhesion complexes that play a crucial role for maintaining cell–cell integrity and the epithelial architecture. Disrupting E-cadherin/β-catenin complex, which conducts to a nuclear translocation of β-catenin, affects not only the cell adhesive repertoire, but also Wnt-signaling activation ,26,27. TWestern blot on cytoplasmic and nuclear extracts of BMAL1-KD and control CRC cell lines were applied to analyze the localization of β-catenin in the different cell fractions E. ConsisDownregulation of E-cadherin is considered as a major hallmark of EMT as restoring E-cadherin expression is sufficient for reversal of the transformed phenotype . IncreasFurthermore, quantitative RT-PCR revealed a significant decrease in the expression of the mesenchymal marker N-cadherin in SW480 and SW620 BMAL1-KD cells, this gene being expressed at very low levels in HCT116 cell lines C. Flow cCells undergoing EMT or its reverse process MET, exhibited profound morphological changes. As BMAL1-KD leans the epithelial–mesenchymal balance of the colon carcinoma cell toward epithelial properties, we investigated the cell morphology of the three CRC cell lines by phase contrast microscopy A. HCT116Besides modified cell–cell junctions, epithelialization is associated also with actin cytoskeletal re-organization from stress fibers toward a cortical actin ring ,29. We tp > 0.05).As EMT promotes invasiveness of cancer cells , a scratFor the in vitro Matrigel invasion assay, cells were added to Matrigel-coated Boyden chambers and allowed to migrate across the matrix along a serum gradient B. In agrNext, we further investigated whether BMAL1-KD could influence the expression of cell adhesion molecules, such as integrin β1 (CD29), integrin alpha5 (CD49e), and CD44, that were involved in the matrix–cell adhesion ,32 and tIn addition to the migratory and invasive abilities, recent studies have shown that the EMT phenotype is closely related to chemoresistance of cancer cells ,35,36,37Epithelial-to-mesenchymal transition (EMT) is a key process driving CRC metastasis and chemoresistance. In this report, we provided evidence, for the first time, that BMAL1-KD promotes mesenchymal-to-epithelial transition (MET)-like changes of colorectal carcinoma cell lines. Consistently, several typical mesenchymal markers, such as N-cadherin and vimentin, as well as the EMT transcription factor Twist decreased, while several classic epithelial markers, such as E-cadherin, CK-20, and EpCAM, increased. Among all the molecular markers implicated in EMT, E-cadherin is referred to as the “caretaker” of the epithelial phenotype, and its loss is considered a key feature of the EMT ,23,24. FKRAS, BRAF, PIK3CA, CTNNB1, BRCA2, CDKN2A, P53, APC) present in the different CRC cell lines. All these distinct mutations, as well as the different molecular circadian clock statuses, provide unique pathological backgrounds in CRC cell lines, which could produce various cell fates after BMAL1-KD. In our study, HCT116, SW480, and SW620 cells were all mtKRAS cell lines, suggesting that our results may be valid in a context of mutated KRAS, which accounts for 35-45% of all metastatic CRC [Acquisition of mesenchymal phenotypes destabilizes cell–cell adhesive junctions and affects cell migratory and invasive properties . Thus, tatic CRC . FurtherBy repressing the expression of the EMT-TF Twist as well as increasing EpCAM and E-cadherin at the plasma membrane of CRC cells, BMAL1-KD promoted MET-like changes, supporting the conclusion that the circadian transcription factor BMAL1 plays a pivotal role in CRC metastasis progression, regulating the balance between EMT and MET. In a previous study, we observed that BMAL1-KD enhanced PI3K/AKT/mTOR signaling that triggered different CRC cell fates  . While nIn recent years, circadian biology is becoming a critical factor for improving drug efficacy and diminishing drug toxicity in cancer treatment (chronotherapy) . The thiIn summary, our study showed that the circadian transcription factor BMAL1 plays crucial functions in CRC metastasis progression, promoting EMT, which is a key step in the tumors’ invasiveness and chemoresistance. Thus, therapeutic targeting of BMAL1 could be considered as a potential strategy to enhance the efficacy of chemotherapy and improve outcomes for patients with metastatic CRC.t-test between groups of patients with an alpha error below 0.05.The TCGA consortium RNAsequencing data matrix of Z-scores from the CRC cohort  were dowHuman colon cancer cell lines HCT116, SW480, and SW620 were grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with GlutaMAX  and 10% fetal bovine serum .One short hairpin RNAs (shRNAs) against human BMAL1 and a control scrambled sequence (Scr) were cloned separately into a lentiviral vector (pLKO-shBMAL1-GFP-puro and pLKO-Scr-GFP-puro), as described previously .Lentivirus particles were prepared and used to transduce human colon cancer cell lines HCT116, SW480, and SW620 as previously described ,67.CRC cell cultures were scrapped in RIPA buffer containing protease inhibitors. Then, cytoplasmic and nuclear fractions of different cell lines were extracted by using NE-PER™ Nuclear and Cytoplasmic Extraction Reagents  according to the manufacturer’s manual. The different extracts were stored at −80° C until Western blot analysis.t-test was used for statistical analysis.Western blot analysis was performed with sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) as previously described . Antibodt-test was used for statistical analysis.Quantitative real-time PCR was performed as previous described by using LightCycler 480 SYBR Green I master kit  . Primerst-test was used for statistical analysis.Cells were detached with Accutase  and cell surface expression of molecules was analyzed by flow cytometry. Briefly, cells were permeabilized or not with BD Cytofix/Cytoperm reagent , and 105 cells were suspended in DMEM medium supplemented with 1% FCS. Indirect stainings were performed for cell surface expression of EpCAM , E-cadherin , N-cadherin , and intracellular expression of Cytokeratin-20  and vimentin , using species-specific secondary antibodies conjugated to phycoerythrin. Phycoerythrin (PE)-conjugated antibodies against CD29 , CD49e , and CD44  were used to perform direct stainings of cell surface expression markers. After 3 washes, 10,000 cells were analyzed on a LSR FortessaTM flow cytometer , and the data were analyzed using FlowJo software . The experiment was repeated at least three times. The Immunofluorescence stainings were performed as previously described  using anMigration was assessed using a wound healing assay (scratch assay). Briefly, CRC cells were cultured in 6-well plates for 24 h to achieve 100% confluence followed by starvation in serum-free DMEM medium containing 10 μg/mL mitomycin C  for 2 h to completely inhibit cell proliferation. After generating a wound in the cell monolayer with a sterile 200 μL pipette tip, detached cells and cellular debris were removed three times by washing with culture medium. The cells were fed with fresh growth medium and the width of the gap between the invasion fronts was measured using Image-J software  at time points from 0 to 72 h after scratching to calculate the rate of wound closure.Invasion assays were performed in 24-well Transwell chambers with 8 µm pore polycarbonate filter inserts . CRC cells were serum starved and exposed to mitomycin C before seeding (5 × 104) on Matrigel-coated inserts  in 100 μL of serum-free medium inserts. The lower chambers were filled with 0.5 mL of complete media with 10% FBS. After 24 h and/or 48 h, assays were terminated by scraping the top of the membrane to remove non-migratory cells. The cells on the lower surface of the insert were fixed in 4% paraformaldehyde and stained with 4’,6-Diamidino-2-Phenylindole, Dihydrochloride  and counted. Quantification of cells was carried out by counting 10 random fields using a 10X objective. Assays were performed in triplicates.2. Oxaliplatin was added to the cell culture medium at various concentrations (ranging from 12.5 to 75 μM). Untreated cells were used as control. After 48 h of treatment, 20 μL of 5 mg/mL MTT  solution was added to each well and incubated at 37 °C for 3 h. Then, the supernatant was removed and 100 μL of 1:1 solution of DMSO and methanol were added to dissolve the formazan precipitate. Absorbance of the solution was read using a microplate reader  at 540 nm, with a background correction at 655 nm. The results were analyzed using GraphPad PRISM software  and were plotted as the mean ± SEM of the absorbance for each dose of drug tested from three independent experiments.The viability of CRC cells to Oxaloplatin (LOH) was measured using the MTT assay. Cells were seeded in quadruplicate at 3 × 103 per well in 96-well plates and incubated overnight at 37 °C in a humidified environment containing 5% COt-test or two-way ANOVA, and p < 0.05 was considered to be a statistically significant result. Error bars represent standard error of the mean (±SEM) of independent experiments.The data were analysis by GraphPad Prism 8.0.1. Statistically significant differences were determined by


Acaulospora mellea ZZ successfully colonized the plant roots. AM inoculation significantly reduced the concentrations of DTPA-Cd, -Pb, and -Zn in soil, and the concentrations of Cd, Pb, and Zn in plants, indicating that AM fungi substantially facilitated heavy metal immobilization. Both B-nZVI and S-nZVI, ranging from 50 mg/kg to 1000 mg/kg, did not impede plant growth, and generally enhanced the phytoextraction of heavy metals. XRD, EDS and mapping analyses showed that S-nZVI was more susceptible to oxidation than B-nZVI, and thus had more effective immobilization effects on heavy metals. Low concentrations of nZVI  and AM inoculation had synergistic effects on heavy metal immobilization, reducing the concentrations of Pb and Cd in roots and enhancing root Zn accumulation. In conclusion, our results showed that AM inoculation was effective in immobilizing heavy metals, whereas nZVI had a low phytotoxicity, and they could jointly contribute to the phytoremediation of heavy metal-contaminated soils with sweet sorghum.Soil pollution with heavy metals has attracted increasing concern, which calls for the development of new remediation strategies. The combination of physical, chemical, and biological techniques can achieve more efficient remediation. However, few studies have focused on whether nanomaterials and beneficial microbes can be jointly used to facilitate phytoremediation. Therefore, we studied the role of nano-zero-valent iron (nZVI) and arbuscular mycorrhizal (AM) fungi in the phytoremediation of an acidic soil polluted with Cd, Pb and Zn, using sweet sorghum. X-ray diffraction (XRD), energy dispersive X-ray spectroscopy (EDS), and mapping analyses were conducted to explore the mechanisms of metal immobilization by nZVI. The results showed that although both bare nZVI (B-nZVI) and starch-stabilized nZVI (S-nZVI) inhibited root mycorrhizal colonization,  The concentrations of heavy metals in soil have increased sharply in the past three decades, posing potential risks for the environment and human health . The maiDue to its high specific surface area, high surface activity and reduction ability, nano-zero-valent iron (nZVI) can effectively remove soil heavy metal(-loid) pollution  ,4,5. nZVw/w) and AM fungi altered metal(loid) uptake and translocation by maize plants. Therefore, to fill the knowledge gap, it is necessary to study the combined effect of nZVI and AM fungi in the phytoremediation of heavy metal-contaminated soil.Arbuscular mycorrhizal (AM) fungi widely occur in the soil, which can form symbiosis with the majority of land plants. AM fungi can provide plants with mineral nutrients  and improve their adaptability to environmental stress, including soil contamination ,13. MoreSweet sorghum is a widely planted biomass energy crop that can be used for bioethanol production. Sweet sorghum can adapt to environmental stresses  . PreviouAcaulospora mellea ZZ) were investigated in a multi-metal-contaminated agricultural soil. Our aim is to select the appropriate concentration and type of nZVI to assist sweet sorghum (inoculated with or without AM fungi) for the phytoremediation of heavy metal-contaminated soil.We hypothesize that nZVI and AM fungi may synergistically enhance the phytoremediation of heavy metal-contaminated soil with sweet sorghum. Here, for the first time, the effects of a series of concentrations of nZVI (bare or starch stabilized nZVI) and an AM fungus (The soil was sampled from the surface layer (0–20 cm) of an abandoned paddy field contaminated with Cd, Pb and Zn, which was described in our recent study . The soiAcaulospora mellea ZZ was selected for this experiment. In our previous results, A. mellea ZZ significantly enhanced plant growth and tolerance in copper-contaminated soil [ted soil  and salited soil . The AM ted soil .2/g. The scanning electron microscopy (SEM) image, transmission electron microscopy (TEM) image and X-ray diffraction (XRD) pattern of S-nZVI are shown in 2/g. The TEM image and XRD pattern are shown in Starch-stabilized nZVI (S-nZVI) is synthesized according to the borohydride reduction method described in our previous study . The aveA. mellea ZZ, (2) two nZVI treatments, i.e., S-nZVI and B-nZVI, and (3) different nZVI concentrations, i.e., 0, 50, 100, 200, 500, and 1000 mg/kg, respectively. Four replicates were set for each treatment. An appropriate amount of each nZVI was mixed into the soil to obtain the target concentration. After that, 1 kg of soil mixture was put into each pot. For the inoculation treatment, 50 g AM inoculum was mixed into each pot. In the non-inoculation treatment, each pot received an equal amount of sterile AM inoculum. A control filtrate of 10 mL AM inoculum was added to all pots to provide similar microbial communities [In order to study the single and combined effects of AM fungi and nZVI, a three-factor experiment was designed, including (a) two inoculation treatments, i.e., inoculation and non-inoculation with AM fungus, munities .A widely grown variety  of sweet sorghum was used as the test plant. Twenty surface-sterilized seeds were grown in each pot, and 8 seedlings were retained one week after emergence. The pots were randomly placed in a plant growth chamber at about 25–30 °C with a photoperiod of 12/12 h , and a relative humidity of 50–60%. During plant growth, pots were weighed every three days and deionized water was added when necessary to maintain the soil moisture at about 20%.3:HClO4, 4:1, v/v) in a graphite digestion instrument . ICP-OES  was used to measure the element concentrations of the contaminants Cd, Pb, and Zn, as well as the nutrients Ca, Cu, Fe, Mg, Mn, and P. Diethylenetriaminepentaacetic acid (DTPA) solution  was used to extract available metals based on the method described previously [The seedlings were harvested on the 60th day after emergence. Shoots and roots were sampled separately, and the fresh weight was measured after washing with tap water and distilled water. Fresh lateral root samples were taken for root colonization evaluation, based on the procedure described in our previous study . Fresh leviously  and deteMycorrhizal response (%) is calculated to estimate the difference in elements absorbed by AM plants relative to non-AM plants (Equation (1)) :(1)Elemep < 0.05). Three–way ANOVA was performed to test the main effect and interaction effect between three independent factors , while two–way ANOVA was tested to analyze the interactions between AM inoculation and each nZVI, or between nZVI type and concentration. The Pearson correlation coefficient was calculated to analyze the correlation between different parameters.Data were submitted to SPSS 22.0 for analysis of variance and significance. The results are expressed as mean ± standard deviation (SD). Duncan’s multiple range test was used to compare the differences between different treatments 2, which were not observed in B-nZVI. AM inoculation changed the state of the iron element. For example, the compounds such as Fe3(PO4)H2O, PbZnP2O4, PbFe3(P2O7)2, and ZnAs2 were only observed in non-inoculation treatments.After plant harvest, Fef S-nZVI . There wEDS and mapping analyses confirmed the occurrence of Pb, Cd and/or Zn on the surfaces of S-nZVI and B-nZVI . AM inocMycorrhizal responses of heavy metal concentrations and uptake in plants are summarized in In this study, AM inoculation effectively immobilized a variety of heavy metals, evidenced by the decreased metal concentrations in plant roots and shoots . This caUnexpectedly, AM inoculation did not significantly increase plant biomass, which may be ascribed to several reasons. First, the effects of AM fungi on plant P acquisition from the soil highly vary with soil fertility, particularly the available P . Plant gOur results show that nZVI, at all the concentrations, had no phytotoxicity but rather promoting effects on plant growth, confirming that nZVI is non-toxic to sweet sorghum. Some previous studies have found that nZVI exhibits “low-concentration promotion and high-concentration inhibition effects” on plant growth ,9,39. OuB-nZVI and S-nZVI showed different effects on heavy metal accumulation in plants, which vary depending on nZVI type and concentration, and the mycorrhizal status. In most cases, both nZVI alone did not influence shoot Zn, Cd and Pb concentrations a but som0) larger than Fe  through reduction and precipitation, while metal(loid)s with E0 smaller than Fe  cannot be reduced, but only sorbed or complexed [0 slightly larger than nZVI and can be immobilized by nZVI via sorption and precipitation, which may partly explain why nZVI decreased root Pb concentration but did not decrease Zn and Cd accumulation s depends to a large extent on the characteristics of the metal(loid)s . There momplexed ,44. Pb hmulation .Soil pH influences the immobilization of metal(loid)s by nZVI . The pH A. melllea ZZ to the combined pollution by Cd, Pb and Zn. This is similar to our previous findings that A. melllea ZZ is tolerant to various pollutants [2+ and reactive oxygen species [AM colonization rates can reach higher than 70%, indicating the tolerance of llutants ,47,48. Fllutants ,49,50. Pllutants ,51, and llutants . Possibl species ,53, and  species . Here, oA. melllea ZZ to nZVI toxicity, and their combined applications. Both AM fungi and appropriate nZVI can benefit plant nutrient uptake and growth, and safe crop production in contaminated soils [Despite nZVI type and concentration, the AM colonization rate of inoculated plants reached as high as 20%, even in the roots exposed to 1000 mg/kg nZVI, indicating a high tolerance of ed soils ,13,54,55Irrespective of the inhibited AM colonization by nZVI, we found some synergistic effects of nZVI and AM inoculation on metal immobilization. For example, the plants in combination treatments generally had the lowest root Pb and Cd concentrations . In factMore importantly, nZVI can induce phytotoxicity even at environmentally relevant concentrations ,57,58,59We tested the single and joint effects of AM fungus and nZVI (S-nZVI and B-nZVI) on the phytoremediation of an acidic soil with realistic environmental pollution, using sweet sorghum. We found that the remediation effects depended on AM fungi and the type and dosage of nZVI. AM inoculation alone effectively immobilized heavy metals in soil and decreased heavy metal accumulation in plants. Both S-nZVI and B-nZVI at all test concentrations (50–1000 mg/kg) showed no phytotoxicity to sweet sorghum, and enhanced the phytoextraction of heavy metals and the immobilization of heavy metals (particularly Pb) on their surface. AM inoculation successfully colonized plant roots, irrespective of the fungitoxicity of nZVI. Overall, low concentrations of nZVI  and AM inoculation had a synergistic effect on heavy metal immobilization, indicating their combined applications in the future phytoremediation of heavy metal-contaminated soil with sweet sorghum.


Poly ADP-ribose glycohydrolase (PARG) is responsible for the catabolism of PARP-synthesized PAR to free ADP-ribose. Inhibition of PARG leads to DNA repair interruption and consequently induces cell death. This study aims to evaluate the effect of a PARG inhibitor (PARGi) on epithelial ovarian cancer (OC) cell lines, alone and in combination with a PARP inhibitor (PARPi) and/or Cisplatin.PARG mRNA levels were studied in three different OC datasets: TCGA, Hendrix, and Meyniel. PARG protein levels were assessed in 100 OC specimens from our bio-bank. The therapeutic efficacy of PARGi was assessed using cell migration and clonogenic formation assays. Flow cytometry was used to evaluate the cell apoptosis rate and the changes in the cell cycle.PARG protein was highly expressed in 34% of the OC tumors and low expression was found in another 9%. Similarly, Hendrix, Meyneil and TCGA databases showed a significant up-regulation in PARG mRNA expression in OC samples as compared to normal tissue . The use of PARGi leads to decreased cell migration. PARGi in combination with PARPi or Cisplatin induced decreased survival of cells as compared to each drug alone. In the presence of PARPi and Cisplatin, PARG knockdown cell lines showed significant G2/M cell cycle arrest and cell death induction.PARG inhibition appears as a complementary strategy to PARP inhibition in the treatment of ovarian cancer, especially in the presence of homologous recombination defects. Ovarian cancer (OC) is the most lethal gynecologic malignancy, with an estimated 313 959 new cases and 207 252 deaths worldwide in 2020 . Hence, BRCA1 or BRCA2 genes are present in approximately 20% of patients with newly diagnosed OC  arising from loss of homologous recombination (HR) function secondary to epigenetic perturbations such as aberrant methylation (5-31% in ovarian cancer), somatic mutations (<5%) and other abnormalities of the following HR repair genes: TM, ATR, BARD1, BLM, BRIP1, CDK12, CHEK1, CHEK2, FANCA, FANCC, FANCD2, FANCE, FANCF, FANCI, FANCL, FANCM, MRE11, NBN, PALB2, RAD50, RAD51, RAD51B, RAD51C, RAD51D, RAD52, RAD54L, and RPA1  formation is one of the earliest events in the mechanism of DNA damage repair and is catalyzed by PARP (Poly ADP-ribose polymerase) enzymes –15. Addih occurs . There a cancers –24. More cancers , 25, 26  cancers –29. Despto PARPi , 22, 30. in vivo , which c and three-dimensional cancer organoids (ORG-KB2P26S.1), using DNA repair-focused shRNA and CRISPR-based libraries, confirmed PARG as a synthetic lethal gene, and loss of this gene represents a major resistance mechanism for PARPi (Poly ADP-ribose glycohydrolase (PARG) is responsible for the catabolism of PARP-synthesized PAR to free ADP-ribose , 32. Lik, 36. Onl as the f. This in–43. Morefunction . Anotherfunction  in BRCA1or PARPi .Given the high rate of HR defects in OC, we hypothesize that inhibiting PARG may be an effective alternative therapeutic strategy for targeting specific OC cancer cells that are dependent on this activity. In addition, PARGi might increase the cytotoxicity of DNA damaging agents and may be useful against diverse ovarian malignancies, including PARPi-resistant tumors. In this study, we aimed to assess the expression of PARG in OC cells and evaluate the effect of PARGi on OC cell lines, alone and in combination with PARPi and Cisplatin.The study was approved by the Jewish General Hospital Research Ethics Board and all patients participating in this study gave informed consent in accordance with the JGH ethics committee regulations (protocol #15-070).https://www.oncomine.org). Using this database, we investigated PARG mRNA levels in normal ovarian tissue and ovarian cancer cases.Oncomine ™ database categorized patients according to different datasets, based on variations in gene expression patterns derived from different cDNA microarrays analysis. Ovarian cancer RNA-seq expression data were obtained from browser website  on ice. Additionally, OVCAR3, SNU251, SKOV3, A2780PAR , A2780CR (Cisplatin resistant), and primary tumor cell lines were harvested  and then lysed in 500μL of radio-immunoprecipitation assay (RIPA) buffer . Protein concentration was determined using bicinchoninic acid assay (BCA) kit  using a spectrophotometer at 570nm.In total, 100 tumor samples were analyzed including 20 ascites cell pellets, 62 primary tumors and 18 omental metastases. Snap-frozen tumor tissues were minced and lysed in lysis buffer , PARG  and β-actin  were diluted in 0.1% Tween-PBS/5% Milk and put in presence of the membrane overnight at 4°C. After 3 washing (0.1%Tween-PBS1X), membranes were exposed to secondary anti-rabbit-horseradish peroxidase  or anti-mouse HRP  for 1 hour at room temperature. Immunoblotting proteins were visualized using horseradish peroxidase (HRP)-conjugated secondary antibodies, and antigen-antibody complexes were detected using the Clarity™ Western ECL Substrate kit .Protein lysates (10-25μg) were separated electrophoretically on a 7.5 to 10% denaturing SDS-polyacrylamide gels and transferred to 0.2μm nitrocellulose membranes. Primary antibodies specific for Cell lines : OVCAR3 (#HTB-161), SNU-251 (#CVCL-5040) and SKOV3 (#HTB-77) were purchased from ATCC. A2780PAR and A2780CR cells were provided by Dr. Seftor .BRCA1 genes, and SNU-251demonstrates a homozygous 1815 G>A BRCA1 mutation (All the cell lines were authenticated by short tandem repeat (STR) profiling by the DNA sequencing and analysis core of the University of Colorado . All celmutation , 52. OVCPatient tumor-derived ovarian cancer cells labeled GOC31 and GOC17 were isolated in our laboratory from two high-grade serous OC specimens obtained fresh at surgery. Primary cell lines were grown in OSE medium supplemented with 20% FBS and growth factors . The cells were routinely passaged every 4 to 6 days. All cells were maintained at 37°C, in a 5% CO2, 95% air atmosphere incubator.The PARGi  was purchased from Tocris . OlapariSKOV3 cells were used to generate stable cell lines with PARG knockdown. Cells were cultured to 90% confluence and transfected with lentiviral constructs expressing shRNA targeting PARG  . Twelve Cells were grown to near confluence in 6-well adherent cell culture flat bottom plates . A ‘‘wound’’ was then inflicted to the cells in triplicate in each well using a sterile 200-KL pipette tip. The cells were then carefully rinsed with phosphate-buffered saline to remove any floating cells. Medium containing various concentrations of PARGi was then added. Pictures were taken of all “wounds” under an optical microscope (Olympus CKX41) at different time points , and the “wound” mean width was measured at three cross-sections along the length of the “wound”, using Photoshop CS3 Extended version . “Wound” closure was then calculated as a percentage value over time. At the completion of the wound healing assay, cells from the 6-well plates were collected for protein extraction and Western blotting.The clonogenic assay was used to determine survival fraction of cells. Briefly, 500–800 cells were plated in 6-well flat bottom cell culture plates . 24 hours after plating, cells were washed, and fresh medium was added in the presence or absence of increasing doses of PARGi alone and in combination with Olaparib and Cisplatin. Media containing the drug was refreshed on day 4. Colonies were fixed and stained after 7-10 days of treatment with 1.5 ml of 6% glutaraldehyde and 0.5% crystal violet and colonies were counted using the GelCount Optronix. The surviving fraction (SF) and Plating Efficiency (PE) of cells were calculated as follows :The interaction between PARGi, Olaparib and Cisplatin was assessed using the multiple drug effects analysis method of Chou and Talalay . This me6 cells were seeded in flat bottom cell culture plates . 24 hours after plating, fresh medium was added in the presence or absence of 2μM Olaparib or/and 1μg/mL Cisplatin. After 48 hr treatment, Hoechst 33342 was added for 30 minutes, then adherent cells were collected using trypsin-EDTA by centrifugation at 10000 rpm for 5 min and washed twice with ice cold PBS. During the last spin, 5ul PI was added for every ml of hypotonic buffer , and incubated on ice in the dark (at least 20 min). Stained cells were analyzed  with a FACS Fortessa flow cytometer . ModFit LT software  was used to analyze the percentage of cells at different phases. Cells treated with DMSO  were used as control.Cell cycle analysis was performed by propidium iodide (PI) staining for DNA content and flow cytometry analysis. For this experiment we used SKOV3-shVector and SKOV3-shPARG1305 cell lines. Briefly, 10Apoptosis was assessed by Annexin V/PI assay using flow cytometry, according to the manufacturer’s protocol (eBioscience™ Ann exin V Apoptosis Detection Kit eFluor™ 450). Apoptotic cells were determined using the FACS Fortessa  .Results are shown as means ± standard deviations of three independent experiments. The difference between groups was analyzed using Student’s t-test, and a p-value <0.05 was considered statistically significant.We initially evaluated the PARG mRNA expression in normal ovarian samples compared to high grade serous adenocarcinoma samples using two different datasets of ONCOMINE database: the TCGA dataset (586 cases)  and primary cell lines derived from patient tumors (GOC31 and GOC17). Baseline characteristics of the study population are displayed in BRCA1 protein was at a very low level in SNU251 cells.Western blot analysis showed high expression level of PARG protein in 34% and low expression in 9% of the tumors (BRCA proficient (SKOV3) and BRCA deficient (SNU251) cell lines. Results indicate that the migration of both cell types was inhibited by PARGi reaching a maximum at 48 hours, at which time the ‘‘wound’’ of SKOV3 cells remained 42% (2μM) and 53% (5μM) open as compared with 33% in untreated cells at the same time, suggesting slower cell mobility  were treated with increasing doses of PARGi (0.1–10μM), alone and in combination with olaparib (0.5μM) or cisplatin (0.5μg/mL). Decreased survival of OC cells was shown with combination treatment (PARGi+Olaparib/PARGi+Cisplatin) as compared to single treatments   and wild type BRCA proficient  ovarian cancers. Results of this study suggest that PARGi reduces cell migration and suppresses formation of clones in BRCA proficient and deficient ovarian cell lines. In addition, knocking down PARG promotes G2/M arrest and cell death when cells are exposed to PARPi as well as DNA damaging agents (Cisplatin).The cell lines used in this study represented both In order to spread and disseminate throughout the body, ovarian cancer cells must migrate and invade through extracellular matrix, intravasate into blood circulation, attach to a distant site, and finally extravasate to form distant foci; cell migration is a key property for the development of this process. In this study, we observed the inhibitory effect of PARGi on cell migration in a time- and concentration-dependent manner, in support of our conclusion that the inhibitory effect of PARGi on cell migration is genuine and is not only due to cell death.The mechanisms by which PARG inhibition affects cancer cells remain elusive with various reported potential mechanisms; (1) HeLa-derived PARG deficient cells exhibited enhanced sensitivity to radiotherapy, caused by defects in the repair of single and double-strand breaks and in mitotic spindle checkpoint, leading to alteration of progression of mitosis ; (2) PARAll five commercial cell lines we investigated in our study are widely used in ovarian cancer research. However, only OVCAR3 is undoubtedly of high grade serous ovarian cancer origin. Although SKOV3 line is frequently cited as “serous’’, it has been only vaguely reported in the original paper as “adenocarcinoma cell line derived from the ascitic fluid of ovarian cancer patient” . In addiPARPi introduction has made considerable progress in the clinical outcomes of ovarian cancer. The recognition that certain molecular pathways including the PAR metabolism are critical to carcinogenesis has triggered a revolution in ovarian cancer drug development. However, PARPi resistance continues to be a significant challenge and it is well-recognized that the failure of PARPi arises due to an inability to induce apoptosis at a cellular level. In this study, it has been shown that different OC cell lines responds better when PARG is silenced, suggesting that PARGi can maximize the benefit of chemotherapy and delay the process of chemo-resistance. By knocking down PARG, the ratio of pro-apoptotic Bcl-2 family members  was favored to anti-apoptotic Bcl-2 family (Bcl-2 and Bcl-XL) members, with increased cell apoptosis as indicated by flow cytometry analysis and western blot.BRCA2-mutated mouse mammary tumors by restoring PARP1 signaling. Be at variance with these findings, in the current study we observed a synergistic interaction between PARGi and PARPi in all cell lines. This dissimilar interaction might be explained by the difference in cell lines and drug doses used in the studies: while we chose SKOV3, A2780PAR, A2780CR and OVCAR3 cell lines to represent BRCA wild type serous ovarian cancer, and SNU251 which is an endometroid ovarian cancer cell line that was previously reported to carry a nonsense mutation at amino acid 1815 of BRCA1. Pillay et al. assembled a panel of six serous ovarian cell lines, 3 are reported to have BRCA1/2 mutation: Kuramochi (BRCA2mutant), OVSAHO (BRCA2mutant), COV362 (BRCA1mutant) and 3 BRCA wild type cell lines: COV318, CAOV3, and OVCAR3. Lastly, Gogola’s group used two types of in vitro cultures that they derived from BRCA2 -/-; Trp53-/- mouse mammary tumors from K14cre;Trp53F/F;BRCA2F/F (KB2P) mice: two-dimensional (2D) tumor cell lines  and three-dimensional (3D) cancer organoids (ORG-KB2P26S.1). In regard to treatment protocol, while “PDD00017273” was used in all studies, the doses used in our study were remarkably different from the others. Unlike Gogola and Pillay who used one fixed dose of 1µM in all experiments, we used a wider range of doses, and we showed an increased effect of the treatment in the higher doses .Our observations validate the potential anti-tumor role of PARGi in the treatment of ovarian cancer which was shown recently by Pillay et al. . By emplAnother added value of our study include: 1- this study opens a window to the potential clinical benefit of PARGi as we report on high expression of PARG in ovarian cancer cells using novel analysis of online databases and in patient derived samples. 2-our results show that PARGi also inhibits cancer cells migration in addition to capability to induce cell death.BRCA1 deficient cell-line (SNU-251), however in further studies it will be interesting to evaluate the influence of BRCA2 mutation and evaluate xenograft models.Limitations include: the use of commercial cell lines can differ from real patient’s tumors which are often more heterogeneous. We used This study shows that in ovarian cancer, PARG inhibition reduces cell migration, suppresses clone formation, and promotes G2/M cell cycle arrest and cell death, alone and in combination with PARPi and Cisplatin. PARG inhibitors suitable for clinical evaluation are not yet available. Our results, however, support the potential use of PARG inhibitors as viable, complementary strategy to induce cell lethality and invasion arrest in ovarian cancer and potentially other HR-deficient cancers.The 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 Medical/Biomedical Research Ethics and Committee of the CIUSSS West-Central Montreal research ethics board at the Jewish General Hospital, Montreal, Canada. The patients/participants provided their written informed consent to participate in this study.EM, TB, LK, RK, and FR have performed clonogenic and proliferation assay. EM, AY, OR, and LK did transfection studies and analyzed the data. VL-O carried out in silica analysis and contribute to the analysis of the results. EM, TB, and AK have carried out apoptosis and cell cycle experiments. EM and VL-O wrote the manuscript and analyzed the results. WG, SL, and SS obtained all the clinical samples and revised and edited the manuscript. AY and WG designed and supervised the study and helped drafting the manuscript. All authors made substantial contributions to the conception and design, data acquisition, data analysis and revision of the intellectual content of the manuscript. In addition, each author has agreed to be accountable for the accuracy and integrity of this research work. All authors contributed to the article and approved the submitted version.This work was made possible in part by grants from the Montreal-Israel Cancer Research Foundation, Gloria’s Girls and the Susan and Jonathan Fund. The funding bodies had no role in the design of the study, collection, analysis, interpretation of data, or in writing the 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.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 also quantified the cytotoxic index (CyI90 = C Max/IC90) to compare drug efficacy. The main findings of this study are that idarubicin emerged as the most cytotoxic agent to most of the tested CRC cell lines . Gemcitabine seemed to be the most efficient chemotherapy for SW48. Interestingly, the most commonly used cytotoxic agents in the systemic and intra-arterial treatment of colorectal liver metastasis (CRLM)  showed very limited cytotoxicity to all the cell lines.To treat colorectal liver metastases, intra-arterial chemotherapies may complete therapeutic arsenal. Drugs using intra-arterially are very heterogeneous. The aim of this study was to select the most efficient drug on a panel of colorectal cancer (CRC) cell lines  exposed for 30 min to 12 cytotoxic agents  at different concentrations. The effect on cell viability was measured using the WST-1 cell viability assay. For each drug and cell line, the IC Colorectal cancer (CRC) represents the third most common type of cancer in the world and the second in terms of cancer mortality . SynchroNowadays, systemic treatments in metastastic CRC combine 2 or 3 drugs such as FOLFOX , FOLFIRI , or FOLFIRINOX  with targeted therapies  depending on the RAS status ,5,6,7. SIntra-arterial (IA) therapies have been developed for a long time to treat liver metastases ,12,13,14Among IA techniques, HAIC has been the most widely investigated in CRLM, based on two different regimens: FUDR-based  or oxaliOther IA techniques are easier to implement in routine clinical practice, but efforts should be made to optimize the treatment regimen. Surprisingly, drugs used for IA therapies vary a lot .However, to our knowledge, drug selection has never been based on any cytotoxicity screening in CRC cell lines. Yet, it might be useless to reach higher intra-tumoral concentration thanks to IA injection if the drug is ineffective against the tumor cells. Varela et al. studied doxorubicin pharmacokinetics (PK) after intra-arterial treatment of hepatocellular carcinoma (HCC)  and showThe present study aimed to screen drugs (currently or not currently used on CRC) on a panel of CRC cell lines with a short exposure time (30 min), in order to select the most efficient one to design further clinical trials of IA treatments for CRLM. The assessment of drug mechanism was however outside the scope of this study.Topoisomerase II inhibitors had a strong effect over SW620. Regarding doxorubicin and epirubicin, Caco-2, HCT116 and HT29 (at high concentrations for the latter) were the most resistant cell lines a,b. WithRegarding 5-FU, HCT116 and Caco-2 were quickly resistant a. ComparA dose-effect relationship was strongly and quickly observed with cisplatin and oxaliplatin . With ciThe alkylating antibiotic, mitomycin C, led to more than 90% of cell death from the first 3 dilutions for SW48, SW480, SW620, and HT29 . HCT 116Irinotecan led to 90% of cell death at high concentrations , but wasThe antitumoral antibiotic, streptozocin, had almost no cytotoxicity effect, except at Cmax and at the first dilution .The cell viability curve of taxane (paclitaxel)  showed aThe statistical analysis (Anova and post-hoc test (LSD-Fisher)) compared cell viability for each drug; control versus any concentration is available in 50 and IC90, respectively) are represented in 50 and IC90.The cytotoxic agent concentrations inducing 50% and 90% of cell death (IC90) was determined for each agent and cell line by calculating the ratio of maximal concentration to IC90. The values are represented in 90 was obtained by idarubicin for all the studied cell lines, except for SW48, over which gemcitabine had the highest CyI90  for each cell lines. Idarubicin stood out as the anticancer-drug with the most significant different CyI90 in comparison with the other cytotoxic agents over Caco-2 (p < 0.001), HT29 (p < 0.001), SW480 (p = 0.020), and SW 620 (p < 0.001) (p = 0.140)   were reported directly in For the CyI< 0.001) a–d. For = 0.140) e. Gemcit= 0.490) f. The siChemotherapeutic agents for IA treatment of CRLM are very heterogeneous. The rationale for drug choice has never been based on any cytotoxicity study. In the present study, we tested 12 anticancer drugs after short exposure time, in keeping with pharmacokinetics (PK) data observed during IA therapies , as suggThe biphasic curves for anthracyclines and the cell growth obtained with low dose of paclitaxel may need an additional study on the mechanisms of response. Nevertheless, according to our results, paclitaxel did not seem to be significant over the CRC cell lines.Boulin et al., who used a similar methodology  on 3 HCC cell lines, also found idarubicin to be the best cytotoxic agent for TACE in HCC treatment . IdarubiResponse to standard oncologic treatment is limited in CRC and there is a great potential to improve treatment efficacy by the molecularly-guided repurposing of targeted drugs. De facto, molecular classification of CRC has evolved in recent years, resulting in the four biologically distinct consensus molecular subtypes (CMS): CMS1 MSI (microsatellite instability)-immune, CMS2 epithelial and canonical, CMS3 epithelial and metabolic, and CMS4 mesenchymal . The CMSCaco-2, HCT 116, SW480, and SW620 belong to CMS 4 , associated with the most aggressive disease and the worst survival. For all these CRC cell lines, idarubicin is the best candidate in our study. HT29 belongs to CMS 3, i.e., the metabolic group. Idarubicin is also the best candidate with a short exposure time. As for SW48 which is classified into CMS 1, i.e., an MSI-immune subtype, gemcitabine appears as the most interesting drug compared with other drugs. Our results are thus in line with those of Sveen et al.  who repo50 compatible with CIAH [50 on HT29 was 200 µg/mL, which was similar to that obtained in the present study (202 ± 57 µg/mL). Kornmann et al. studied in-vitro effect of oxaliplatin and gemcitabine with a 2-h, 4-h, and 24-h exposure time on CRC cell lines  and on fresh liver metastases [50 values for oxaliplatin and gemcitabine were <10 µg/mL and 100 µg/mL after a 2-h exposure on HT29. In the present study, the IC50 values on HT29 after a 30-min exposure were 40.9 ± 41 µg/mL for oxaliplatin and 793 µg/mL for gemcitabine, which clearly highlighted the time-dependent effect of these two drugs. A concentration dependence is also known for gemcitabine [90 of cytotoxic agents over CRC cell lines. Yet, the stronger the cytotoxicity index, the higher the chance to find an effective agent for locoregional therapies. In addition, colorectal adenocarcinomas are supposed to be sensitive to anthracyclines and other topoisomerase II inhibitors. The Caco-2 resistance was well studied and related to two mechanisms: MDR (Multi Drug Resistance) system and a confluence-dependent resistance [A comparison of the present results with the literature was carried out based on the few available studies. Three studies evaluated whether oxalipatin, irinotecan, and gemcitabine could have ICith CIAH ,40,41. Otastases . The rescitabine . No studsistance . Our ressistance .Some limitations to our study must be acknowledged. First, the cell viability analysis methodology was based on WST-1, which is a metabolic marker less sensitive than crystal violet (cell marker) used by Boulin et al. . HoweverAnother perspective could be to test “combined chemotherapies”.According to our results, we plan to propose a phase II trial with idarubicin in MSI-mCRC after failure of two systemic treatment lines.CRC is a heterogeneous disease mainly related to the heterogeneity of genomic instability. We chose different cell lines to represent the different molecular characteristics of CRC  or MicroSatellite Instability (MSI)) coming from primary or metastatic site and located at different places in the colon.2 in RPMI , 1640 were supplemented with 10% decomplemented FBS (20% FBS for Caco-2 cells), 1% L-glutamine and 1% penicilline-streptomicine. All cell lines were tested to be mycoplasma-free.Six human CRC cell lines  were selected from the American Type Culture Collection (ATCC). Their characteristics and their respective CMS groups  are summA total of 13 molecules were tested, currently used in CRC or not. Twelve cytotoxic agents, all of which are routinely used in colorectal cancer for systemic treatment . We included chemotherapeutic agents used for intra-arterial treatments and idarubicin, the best candidate after in-vitro screening on HCC cell lines . We selen = 4) were tested during the same experience. Each experience was repeated three times (N = 3), as previously described for a total period of four months.All the manipulations were carried out by two independent operators with, respectively, 4 and 27 years of experience in cell cultures and experimental studies. Cell lines were detached (Day 0) with a mixture of 0.05% trypsine/0.5 mM EDTA  after rinsing with Dulbecco’s Phosphate Buffered Saline (PBS). Cells were seeded onto a 96-well tissue culture-plate, 10,000 cells per well, and cultured for 24 h. At Day 1, the culture medium was changed to lower the FBS level to 0.5%. Drugs with the maximal concentration were deposited in the first column of the plate and we attended to successive three-fold dilutions with fresh RPMI containing 0.5% FBS. The last column (Column 12) of the plate contained drug-free medium. The cells were exposed to drugs for 30 min at 37 °C before being washed twice or thrice if the drugs were viscous or stained with RPMI (0.5% FBS). The cells were then put back in the incubator for 72 h. For each condition , replicates (2), as recommended by the WST-1manufacturer.At Day 4, to evaluate the effect of drugs on cell viability, we performed spectrophotometric quantification by using the WST-1 Cell Viability Assay . Ten µL WST-1 per well were added to 100 µL of medium. After 2 h of incubation at 37 °C, the plate was shacked and absorbance was read on a microplate reader  at 440 nm incubation in a humidified atmosphere  obtained from the mean of each quadruplicates was calculated. After normalization with the control (100% of viability), the viability curves were generated for the six cell lines treated by each of the 12 drugs, the lipiodol and the bevacizumab  non-linear solver (Curve Fitting Toolbox) estimated the parameters of the dose response function for each of the cell lines and drug conditions. Based on the fitted curve, we extracted the IC50 and IC90, which correspond to the drug concentration (mg/mL) required to obtain 50% and 90% of cell death, respectively. Finally, we calculated the CyI90 cytotoxicity index, corresponding to the ratio of the maximal drug concentration to IC90 (CyI90 = C Max/IC90), according to Boulin et al. [90 of 1000 means that the drug kills 90% of the cells even when diluted (1:1000). The IC50, IC90, and CyI90 values are represented in 90 was tested with a one-way ANOVA. Whenever there was a significant mean difference between the drugs, an additional post-hoc test (Fisher’s LSD) was conducted. A p value < 0.05 was considered statistically significant. Results were reported as mean ± standard deviation for each cell lines.The dose-response data were fitted by a four-parameter logistic nonlinear regression model (Equation (1)), modified from the original Hill equation (1)OD(c)n et al. . For exa® Software; 13.4.0.14; Palo Alto, CA, USA).All the statistical analyses were performed with Statistica  in CRC have a very limited cytotoxicity effect on CRC cell lines after short exposure time (30 min). On the contrary, idarubicin  exhibits a strong cytotoxicity profile . These results argue for further trials of idarubicin  for the intra-arterial treatment of CRC liver metastases in patients with unresectable CRLM. In the same way, the poor patient prognosis associated with CMS4 warrants additional studies to pursue the potential for clinical testing of HSP90 inhibitor repositioning and combination therapy with idarubicin in colorectal cancer.


This paper provides information concerning the activity and expression levels of three sulfurtransferases (STRs): rhodanese , 3-mercaptopyruvate sulfurtransferase  and cystathionine γ-lyase  in various cell lines. Since very limited data are available in the scientific literature on this subject, the available data are included in this paper. These shortages often force the researchers to carry out their own screening tests that allow them to choose an appropriate model for their further studies. This work supplements the existing deficiencies in this area and presents the activity and expression of STRs in the eight most frequently chosen cell lines: the mouse mammary gland cell line , mouse mammary gland tumor , mouse fibroblast , mouse melanoma , human colorectal adenocarcinoma , human embryonic kidney , human osteosarcoma  and rat myocardium . Changes in STRs activity are directly related to the bioavailability of cysteine and the sulfane sulfur level, and thus the present authors also measured these parameters, as well as the level of glutathione (its reduced (GSH) and oxidized (GSSG) form) and the [GSH]/[GSSG] ratio that determines the antioxidant capacity of the cells. STRs demonstrate diverse functionality and clinical relevance; therefore, we also performed an analysis of genetic variation of STRs genes that revealed a large number of polymorphisms. Although STRs still provide challenges in several fields, responding to them could not only improve the understanding of various diseases, but may also provide a way to treat them. Rhodanase , 3-mercaptopyruvate sulfurtransferase  and cystathionine γ-lyase CTH, EC: 4.4.1.1) belong to the enzyme superfamily known as sulfurtransferases (STRs). The enzymes are widespread in the world of prokaryotic and eukaryotic organisms, but, in recent years, they have rapidly become a subject of our growing interest; here, the trigger was the discovery that they were involved in the multi-enzyme pathway, which can generate hydrogen sulfide from cysteine, homocysteine and their disulfides .4.1.1 be,6.- (as TST and MPST do), but produces sulfur-donor molecules for STRs /[GSSG] concentration ratio—the parameters that are very often determined along with the activity and expression of STRs.www.surveymonkey.com, accessed on 22 February 2018). The questionnaire listed 80 cell lines diverse in species, tissue origin, and disease occurrence. The cell lines included in this study represent 10% of the most frequently chosen records from scientists employed by the Jagiellonian University, willing to participate in the survey. The present investigators also present data and references for five additional cell lines , and Naglu−/− line , since there are data from the scientific literature addressing this topic of interest.The cell lines presented in this paper were selected based on responses to an anonymous questionnaire from the free online survey tool, Survey Monkey (v/v) fetal bovine serum and 100 U/mL penicillin and 100 μg/mL streptomycin. To make the complete growth medium of the NMuMG cells, the addition of insulin was necessary . For experimental procedures cell lines were maintained in exponential growth and were refed 48 h prior to harvest. They were maintained at 37 °C, 5% CO2, 95% humidity.All experiments were performed using cell lines obtained from American Type Culture Collection (ATCC). We used eight cell lines: mouse mammary gland cell line , mouse mammary gland tumor (4T1—ATCC: CRL-2539)—this tumor mimics an animal stage IV human breast cancer—mouse fibroblast , mouse melanoma , human colorectal adenocarcinoma , human embryonic kidney , human osteosarcoma  and rat myocardium . All cell lines were grown in Dulbecco’s Modified Eagle’s Medium supplemented with 10% (6 cells) were suspended in 0.1 M phosphate buffer, pH 7.5, in the proportion 1 mln cells/0.04 mL of the buffer and sonicated 15 s at 4 °C . After centrifugation at 1600× g, at 4 °C for 10 min, the supernatant was used for determinations of protein content, sulfane sulfur levels and sulfurtransferases activity. For determinations of low-molecular-weight, sulfur-containing compounds using reverse-phase, high-performance liquid chromatography (RP-HPLC), the pellets were suspended in 250 μL 0.9% NaCl/1 mM BPDS/70% perchloric acid (PCA) and sonicated for 15 s at 4 °C . The sediment was separated by centrifugation at 1600× g for 10 min, and supernatant was saved at −80 °C until used for RP-HPLC analyses.The pellets of cells . After equilibration at 37 °C, 2.5 μL of lactate dehydrogenase  was added, and the decrease in absorbance was measured at 340 nm. The difference between the initial value of absorbance (before LDH addition) and the lowest value (after LDH addition) corresponded to the amount of pyruvate formed in the reaction catalyzed by MPST. The MPST activity was expressed as nmoles of pyruvate produced during one-minute-incubation at 37 °C per 1 mg of protein.The MPST activity was assayed according to the method of Valentine and Frankenfeld  with somg for 10 min. After that, 25 μL of supernatant was transferred to 625 μL of 0.194 mM NADH solution and kept at 37 °C. Control samples (without 24 mM cystathionine) were prepared in the same way as the examined samples. The measurement (absorbance at 340 nm) was conducted for 180 s; after 10 s, 25 μL of LDH (9.06 IU) was added. The difference between the initial value of absorbance (before LDH addition) and the lowest value (after LDH addition) corresponded to the amount of α-ketobutyrate formed in the reaction catalyzed by CTH. The enzyme activity was expressed as nmoles of α-ketobutyrate formed during a one-minute-incubation at 37 °C per 1 mg of protein.The CTH activity was determined according to Matsuo and Greenberg’s method , which w- (thiocyanate) per 1 mg of protein.Sulfane sulfur level was determined by the method of Wood  with somTotal protein content was determined by the method of Lowry et al. . The cryThe levels of the reduced (GSH) and oxidized (GSSG) glutathione, cysteine and cystine in the incubation mixtures were determined using the RP-HPLC method of Dominic et al.  with modTotal RNA was extracted from the cells using TRI reagent, according to the protocol provided by the manufacturer . The extracted RNA was suspended in ribonuclease free-water and quantified by measuring the absorbance at 260 nm. After the procedure, the purity of the obtained RNA was determined by the spectrophotometric analysis (A260 nm/A280 nm). The integrity of the achieved RNA was confirmed by the separation of the 28S and 18S rRNA bands in 2.0% agarose-gel electrophoresis. The RNA solutions were stored at −80 °C until further use.TM Reverse Transcriptase Kit according to the manufacturer’s protocol . For a reverse transcription reaction, 3 µg of total RNA was mixed with 1 μL of Oligo (dT)15 primer (0.5 μg/reaction) and water pretreated with diethylpyrocarbonate (DEPC-H2O), and then incubated for 5 min at 70 °C. After preincubation, other components were added to this mixture: 4 μL of GoScriptTM 5× concentrated reaction buffer, 3 μL of MgCl2 , 1 μL of deoxyribonucleotide triphosphates , 1 μL of RNase inhibitor (20 U/μL), and 1 μL of GoScriptTM Reverse Transcriptase (160 U/μL) in a total volume of 20 μL. The mixture was first incubated for 5 min at 25 °C, then for 60 min at 42 °C, and, finally, for 15 min at 70 °C. If necessary, the solutions of complementary DNA (cDNA) were stored at −20 °C.Total RNA from the cell samples was reverse-transcribed using a GoScript2O. In each the case, a similar reaction was also performed in the mixture without DNA (the negative control) in order to confirm the specificity of the obtained reaction products. The temperature profile of PCR amplification for these genes, as well as gene-specific primer sequences, are collected in TM MP Imaging system with Image Lab Software, version 6.0, Bio-Rad).The expressions of the three genes  were analyzed by PCR. As a reference , gene-encoding glyceraldehyde 3-phosphate dehydrogenase  was used. The amplification of cDNA samples was performed in a 12.5 μL reaction volume containing 1 μL of synthesized cDNA, 10 μM of each of gene-specific primer pair, 2 U/μL Taq DNA polymerase in 10 mM buffer Tris-HCl at pH 8.8, and 10 mM of each dNTPs and DEPC-H2S). H2S plays a role in protection of neurons against oxidative stress and stimulates an increase in γ-glutamylcysteine synthetase, and thereby an increase in the level of GSH. On the other hand, diminished CTH expression entails a decrease in the level of cysteine, glutathione (GSH), taurine and H2S in the cells [2S-generating enzymes  in the cells may causes a decrease/increase in H2S bioavailability and indicates the possible genetic background of H2S-geneting enzyme regulation [We performed an analysis of genetic variations of the TST-, MPST- and CTH-encoding genes . Some inhe cells ,31. The gulation . Therefogulation  and/or igulation . MoreoveNaglu−/− cell lines (the cellular model of mucopolysaccharidosis IIIB). Therefore, to expand this knowledge, the expression levels of the genes encoding TST, MPST and CTH were investigated in selected cell lines. The obtained data are presented in In the scientific literature, the expression data of tested STRs are presented only in a few cell lines. Jurkowska et al.  showed tThe performed gene expression analysis confirmed that the expression of all three STRs took place in all investigated cell lines: the mouse mammary gland cells MNuMG, mouse mammary gland tumor 4T1, mouse fibroblast MEF, mouse melanoma B16-F1, human colorectal adenocarcinoma Caco-2, human embryonic kidney cells HEK, human osteosarcoma MG-63, and rat myocardium H9c2. Taking into consideration the species tested, the highest TST and CTH expression level was observed in the mouse cell lines, while the MPST expression was the highest in the rat H9c2 cell line . Based oNaglu−/− cell line—the line derived from mice with mutation B6.129S6-Naglutm1Efn/J in the gene encoding the N-alpha-glucosaminidase [2S from cysteine, homocysteine and their disulfides [The STRs activity analysis confirmed the presence of the activity of TST, MPST and CTH in all the tested and some additional cell lines; the latter were added to minidase , as wellNaglu−/− cells (2 nmole/mg protein), and this is likely a biochemical cellular response to the mutation existing in this line (the mutation likely causes an increase in the sulfate levels originating from undigested disaccharide—heparan sulfate) [Naglu−/− cell line (p < 0.05), it can be concluded that, in the MG-63, SH-SY5Y and H9c2 cells, the low level of GSH and sulfane sulfur-containing compounds (Naglu−/− and Caco-2 cells and high activity of TST (the Caco-2 cells)  ,40. L-cyell line . Furtherompounds  correspoompounds  and loweompounds . In contompounds  were corompounds  and the ompounds . The low2 cells)  were als2 cells) . The mos2 cells)  determinll lines  was corrll lines . MeanwhiThe scientific literature presents a hypothesis postulating a decrease in STRs activity and the level of sulfane sulfur-containing compounds in neoplastic cell lines ,37,42. TOur studies of the STRs activity and expression levels and directly related parameters  provide researchers with a choice of cellular research models without the need to conduct their own large-scale screening studies. The presented investigation results confirm the expression and activity of STRs in all the tested cell lines and demonstrate differences in the activity and the level of STRs mRNA depending on the species and origin the cells. Taking into consideration the species tested, the greatest TST and CTH expression was observed in the mouse cell lines, while the MPST expression was the highest in the rat H9c2 cell line . Among t


Three-dimensional (3D) cell culture methods are identified for simulating the biological microenvironment and demonstrating more similarity to in vivo circumstances. Anaplastic thyroid carcinoma (ATC) is a lethal endocrine malignancy. Despite different treatment approaches, no improvement in the survival rate of the patients has been shown. In this study, we used the 3D in vitro ATC model to investigate the cytotoxic effect of BI-847325 anticancer drug in two-dimensional (2D)- and 3D- cultured cells.′,6-diamidino-2-phenylindole) staining. Survival of alginate-encapsulated cells was evaluated by CFSE  staining. The population doubling times of C643 and SW1736 cell lines cultured in 2D monolayer as well as in 3D system were calculated. The cytotoxic effect of BI-847325 on 2D- and 3D- cultured cell lines was assessed for 24–72 h by MTT  assay. Finally, the 3D culture results were compared with the 2D culture method.Human ATC cell lines, C643 and SW1736, were cultured in one percentage (w/v) sodium alginate. Spheroids were incubated in medium for one week. The reproducibility of the fabrication of alginate beads was evaluated. Encapsulation of the cells in alginate was examined by DAPI (450) values of BI-847325 were higher in 3D culture compared to 2D culture. The cytotoxicity data indicated that 3D in vitro models were more resistant to chemotherapy agents.The half-maximal inhibitory concentration (ICThe findings of this study are beneficial for developing in vitro ATC 3D models to analyze the efficacy of different chemotherapy drugs and formulations. Thyroid cancer is the most frequent endocrine malignancy with the highest growing incidence rate between the other types of solid tumors in the United States –3. AnaplIn general, cancer investigators rely on 2D cell culture in vitro experiments and animal models to understand the complex biological processes of tumor formation, progression and, its treatment , 9. In r2, nutrients, metabolic wastes, growth factors, etc. Therefore, hydrogel scaffolds are used in different pharmaceutical and biomedical fields  assay. The C643 and SW1736 cell lines were seeded in 96-well plates at a concentration of 5000 cells/well. After 24 h cells were treated with 0.125–8 μM doses of BI-847325 for 24–72 h. The viability of the cells was evaluated by the metabolic conversion of the tetrazolium dye to insoluble formazan. MTT (5 mg/ml) was added to the 96-well plates at 10 μl/well, and the plates were then incubated for three hours in 5% CO2 at 37 °C incubator. The medium was removed cautiously with a pipette to avoid the disruption of beads. To dissolve the formazan salt, 500 μl of 0.1 M HCl  in 2-propanol  was added. Plates were placed in the incubator for 45 min to allow the formazan to diffuse out of the alginate hydrogel and entirely be dissolved in the solvent [50 values were calculated via the Reed–Muench method [The effect of BI-847325 treatment on encapsulated ATC cell lines was also examined by MTT assay. Cells were treated with BI-847325 (1–64 μM) for 24 h as described earlier , and cur solvent . The absh method , 32.p < .05 (*); p < .01 (**); p < .001 (***); p < .0001 (****). IC50 values were calculated by nonlinear regression using GraphPad Prism v5 software. Statistical comparisons of population doubling times were carried out using the unpaired t-test method in each case (n = 3).The data show the mean of four independent experiments±SD. Statistical analysis of MTT results were performed using One-way ANOVA followed by Tukey’s post hoc tests. Statistical significances are expressed as n = 20), their size was measured using ImageJ software  which indicates appropriate reproducibility in spheroids formation. Encapsulation and survival of cell lines are shown in Fig. First, to evaluate the reproducibility of the fabrication of alginate spheroids  indicating the same growth rate of ATC cell lines in 2D and 3D cultures.As shown in Fig. RAS mutation and SW1736 with BRAFV600E mutation [50 remained constant after 48 h and IC50 values of BI-847325 on C643 and SW1736 were at 2 μM and 4 μM, respectively  for 24–72 h prevented proliferation in the two cell lines Fig. . In both50 values in the 3D method demonstrated that C643 cell line is more sensitive towards BI-847325 compared to the SW1736 cell line.Interestingly, similar to 2D cell culture, the IC50 values in 3D cell culture system may be associated with multiple mechanisms, including a reduced penetration of chemotherapy agents because of the simulation of key factors of natural tumor microenvironment such as physiological gradients and presence of ECM, enhanced pro-survival signaling pathways such as MAPK pathway due to upregulation of genes involving in anticancer drug resistance [The results of this study are the first report of 3D model for a more accurate assessment of the effect of chemotherapy drugs in ATC. These findings indicate that 3D in vitro models demonstrate more resistance to anticancer drugs. Increased ICsistance , 37–41 (Furthermore, CSCs cultured in the 2D system may reduce their stemness characteristics, while CSCs cultured in 3D may be more resistant to anticancer drugs , 42, 43.50 values were significantly higher in the 3D cell culture system compared to the 2D one for both cell lines (Table 50 values of BI-847325 for C643 and SW1736 were at 15 μM and 34 μM concentrations, respectively. The higher IC50 values in 3D cell cultures demonstrate the resistance potential gained by cells while growing in the 3D system which simulates the natural physiological conditions. This evidence supports the higher resistance rate of cultured cells in the 3D system to chemotherapy drugs as reported previously [BI-847325 cytotoxicity on ATC C643 and SW1736 cell lines was measured in 2D and 3D cell culture systems. The study has revealed that the concentration ranges used in the 2D cell culture method were not appropriate to exhibit significant cytotoxicity in the 3D cell culture system . Based oeviously , 23.In a similar study, higher viability rates following treatment with Paclitaxel were observed in ovarian cancer cells cultured in 3D compared to 2D cell culture . In anot50 values are higher in 3D cell culture system compared to the 2D cell culture method. Like what observed in 2D culture, the obtained IC50 in the 3D culture for SW1736 was higher than that for C643 similar to conventional 2D culture. This observation indicates the resistant nature of this cell line which is probably due to more stemness properties of ATC SW1736 cell line. The elevated IC50 values confirm the Lei and colleagues report about 3D matrix-driven CSCs theory in which 3D culture of breast cancer cell line, MCF-7, resulted in more tumorigenesis and elevated anticancer drug resistance [On the other hand, the subpopulation of malignant cells in the tumor, known as CSCs, is responsible for cancer recurrence and different drug resistance mechanisms –48. We hsistance , 23, 50.In order to understand the difference between cytotoxic responses of chemotherapeutic drugs in 2D- and 3D- based culture systems, Godudu et al. examined the uptake efficiency of Doxorubicin inside the alginate-encapsulated lung cancer cell lines . DoxorubCollectively, 3D cell culture systems such as alginate-encapsulated cells more closely mimic the key factors of the natural tumor microenvironment such as molecular concentration gradients and important cellular interactions including cell-cell and cell-matrix communications. However, additional cellular investigations such as xenograft tumors are required to promote the predictive value of 3D cell culture in the response of ATC cells to anticancer drugs in vivo.In this study, the cytotoxicity data indicated that 3D in vitro models display more resistance to chemotherapy agents. ATC tumors are unresponsive to the current anticancer drugs. Having to do with 3D in vitro ATC models designed by the current research, it would be feasible to analyze the effect of different anticancer drugs and diverse signaling pathways affected by the chemotherapy agents and formulations. In terms of the future application of the proposed model, this culture system can be modified to support the multicellular 3D cell culture. Moreover, the attachment of different peptides can improve alginate scaffold according to extracellular matrix properties.