Poly ADP-ribose Polymerase Cleavage Research Articles

Evidence of apoptosis as an early event leading to cyclophosphamide-induced primordial follicle depletion in a prepubertal mouse model.

The mechanisms leading to ovarian primordial follicle depletion following gonadotoxic chemotherapy with cyclophosphamide and other cytotoxic drugs are currently understood through two main explanatory theories: apoptosis and over-activation. Discrepancies between the findings of different studies investigating these mechanisms do not allow to reach a firm conclusion. The heterogeneity of cell types in ovaries and their different degrees of sensitivity to damage, cell-cell interactions, periodical follicle profile differences, model age-dependent differences, and differences of exposure durations of tested drugs may partially explain the discrepancies among studies. This study used intact prepubertal mice ovaries in culture as study model, in which most follicles are primordial follicles. Histological and transcriptional analyses of ovaries exposed to the active metabolite of cyclophosphamide 4-hydroperoxycyclophosphamide (4-HC) were carried out via a time-course experiment at 8, 24, 48, and 72 h. 4-HC treated ovaries showed a significant decrease in primordial follicle density at 24 h, along with active DNA damage (TUNEL) and overexpressed apoptosis signals (cleaved-poly ADP ribose polymerase in immunohistochemistry and western blotting). Meanwhile 4-HC treatment significantly up-regulated H2ax, Casp 6, Casp 8, Noxa, and Bax in ovaries, and up-regulated Puma in primordial follicles (FISH). Our results indicated that cyclophosphamide-induced acute ovarian primordial follicle depletion was mainly related to apoptotic pathways. No evidence of follicle activation was found, neither through changes in the expression of related genes to follicle activation nor in the density of growing follicles. Further validation at protein level in 4-HC-treated prepubertal mice ovaries at 24 h confirmed these observations.

Silmitasertib in Combination With Cabozantinib Impairs Liver Cancer Cell Cycle Progression, Induces Apoptosis, and Delays Tumor Growth in a Preclinical Model.

The rising incidence of hepatocellular carcinoma (HCC) is a global problem. Several approved treatments, including immune therapy and multi-tyrosine kinase inhibitors, are used for treatment, although the results are not optimum. There is an unmet need to develop highly effective chemotherapies for HCC. Targeting multiple pathways to attack cancer cells is beneficial. Cabozantinib is an orally available bioactive multikinase inhibitor and has a modest effect on HCC treatment. Silmitasertib is an orally bioavailable, potent CK2 inhibitor with a direct role in DNA damage repair and is in clinical trials for other cancers. In this study, we planned to repurpose these existing drugs on HCC treatment. We observed a stronger antiproliferative effect of these two combined drugs on HCC cells generated from different etiologies as compared to the single treatment. Global RNA-seq analyses revealed a decrease in the expression of G2/M cell cycle transition genes in HCC cells following combination treatment, suggesting G2 phase cell arrest. We observed G2/M cell cycle phase arrest in HCC cells upon combination treatment compared to the single-treated or vehicle-treated control cells. The downregulation of CCNA2 and CDC25C following combination therapy further supported the observation. Subsequent analyses demonstrated that combination treatment inhibited 70 kDa ribosomal protein S6 kinase (p70S6K) phosphorylation, and increased Bim expression. Apoptosis of HCC cells were accompanied by increased poly (ADP-ribose) polymerase cleavage and caspase-9 activation. Next, we observed that a combination therapy significantly delayed the progression of HCC xenograft growth as compared to vehicle control. Together, our results suggested combining cabozantinib and silmitasertib would be a promising treatment option for HCC.

Novel repurposing of sulfasalazine for the treatment of adrenocortical carcinomas, probably through the SLC7A11/xCT-hsa-miR-92a-3p-OIP5-AS1 network pathway

BackgroundRecent multigenomic analysis of adrenocortical carcinomas (ACCs) identified SLC7A11/xCT as a novel biomarker. The Food and Drug Administration–approved anti-inflammatory drug, sulfasalazine (SAS), induces ferroptosis by blocking SLC7A11 expression. We hypothesize that SAS could be repurposed to target ACC cells. MethodsExpression of SLC7A11 and its association with ACC survival was analyzed using Gene Expression Profiling Interactive Analysis (GEPIA). The validated ACC cell lines NCI-H295R, ACC1, and ACC2 were grown in 2D culture. In vitro studies included the CellTiter-Glo assay to calculate viability, Western blot (WB) analysis for apoptosis and other target protein changes, reverse transcriptase polymerase chain reaction for steroidogenic enzyme changes, C11BODIPY for lipid peroxidation, and mass spectrometry for changes in lipids. ResultsThe Cancer Genome Atlas Program database analysis in GEPIA showed that SLC7A11 and linked long noncoding RNA OAP5-AS1 are highly expressed in ACC tumors versus normal adrenals (n = 77 vs 128; P < .05). This was associated with poor overall and disease-free survival with hazard ratios of 4.3 and 5.2 for SLC7A11 and 4.8 and 2.7 for OAP5-AS1, respectively. ACC cell line half-inhibitory maximum concentration values after 72-hour SAS treatment ranged from 412 nM (ACC1) to 799 nM (ACC2), and all showed cleavage of poly (ADP-ribose) polymerase, upregulation of p-Akt and p-ERK, and downregulation of GPX4 and SLC7A11 (P < .05) by WB analysis. Sphere formation, migration, and invasion assay showed inhibition, and lipid peroxidation using C11BODIPY, increase in intracellular iron, induction of oxidative stress, and significant upregulation of oxidized polyunsaturated fatty acid phospholipids (P < .05 each) by mass spectrometry suggests induction of ferroptosis. ConclusionSAS downregulates tSLC7A11 in ACCs, targets the Akt/ERK pathway and lipid metabolism, and induces cell death in vitro, warranting additional translational studies to define its therapeutic potential in ACC.

Antitumor Potential of Guttiferone E Combined With Carboplatin Against Osimertinib-resistant H1975 Lung Cancer Through Apoptosis.

Low selectivity and high frequency of side-effects are the major problems of currently used chemotherapeutics. Among natural compounds, the polyprenylated acylphloroglucinol, guttiferone E, isolated from Brazilian red propolis, has attracted attention due to its marked anticancer properties and was evaluated here for its role against osimertinib-resistant H1975 cells (with double mutations of epidermal growth factor receptor: EGFR L858R/T790M). Guttiferone E was obtained from red propolis using established extraction procedures. Guttiferone E was tested using the H1975 cell line in in vitro (2D and 3D) cell cultures and in vivo in BALB/c athymic nude mice. Live/dead assay was also performed to support the results. Tumor tissues obtained from in vivo studies were used for western blotting. Guttiferone E reduced H1975 cell viability in a concentration-dependent manner. The IC50 values in 2D and 3D cell lines were 2.56±0.12 μM and 11.25±0.34 μM. Furthermore, at 10 mg/kg intraperitoneally, guttiferone E significantly reduced the tumor volume in tumor xenografts when used alone and in combination with carboplatin. Guttiferone E and carboplatin displayed synergistic inhibition of H1975 cells and animal tumors. Co-treatment of guttiferone E with carboplatin induced more prominent apoptosis than treatment with either drug alone. Guttiferone E treatment induced cleavage of poly-ADP ribose polymerase and induced apoptosis by significantly reducing levels of mammalian target of rapamycin, sirtuin 1, sirtuin 7, superoxide dismutase, programmed death-ligand 1, and programmed cell death 1 in tumor tissues. Our results show guttiferone E to be a promising, novel and potent antitumor drug candidate for osimertinib-resistant lung cancer with EGFR L858R/T790M mutations.

Extracellular Vesicle Inhibitors Enhance Cholix-Induced Cell Death via Regulation of the JNK-Dependent Pathway.

Vibrio cholerae is an important foodborne pathogen. Cholix cytotoxin (Cholix), produced by V. cholerae, is a novel eukaryotic elongation factor 2 (eEF2) adenosine diphosphate ribosyltransferase that causes host cell death by inhibiting protein synthesis. However, the role of Cholix in the infectious diseases caused by V. cholerae remains unclear. Some bacterial cytotoxins are carried by host extracellular vesicles (EVs) and transferred to other cells. In this study, we investigated the effects of EV inhibitors and EV-regulating proteins on Cholix-induced hepatocyte death. We observed that Cholix-induced cell death was significantly enhanced in the presence of EV inhibitors (e.g., dimethyl amiloride, and desipramine) and Rab27a-knockdown cells, but it did not involve a sphingomyelin-dependent pathway. RNA sequencing analysis revealed that desipramine, imipramine, and EV inhibitors promoted the Cholix-activated c-Jun NH2-terminal kinase (JNK) pathway. Furthermore, JNK inhibition decreased desipramine-enhanced Cholix-induced poly (ADP-ribose) polymerase (PARP) cleavage. In addition, suppression of Apaf-1 by small interfering RNA further enhanced Cholix-induced PARP cleavage by desipramine. We identified a novel function of desipramine in which the stimulated JNK pathway promoted a mitochondria-independent cell death pathway by Cholix.

Piperine enhances doxorubicin sensitivity in triple-negative breast cancer by targeting the PI3K/Akt/mTOR pathway and cancer stem cells

Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer that lacks an actionable target with limited treatment options beyond conventional chemotherapy. Therapeutic failure is often encountered due to inherent or acquired resistance to chemotherapy. Previous studies implicated PI3K/Akt/mTOR signaling pathway in cancer stem cells (CSCs) enrichment and hence chemoresistance. The present study aimed at investigating the potential effect of piperine (PIP), an amide alkaloid isolated from Piper nigrum, on enhancing the sensitivity of TNBC cells to doxorubicin (DOX) in vitro on MDA-MB-231 cell line and in vivo in an animal model of Ehrlich ascites carcinoma solid tumor. Results showed a synergistic interaction between DOX and PIP on MDA-MB-231 cells. In addition, the combination elicited enhanced suppression of PI3K/Akt/mTOR signaling that paralleled an upregulation in this pathway’s negative regulator, PTEN, along with a curtailment in the levels of the CSCs surrogate marker, aldehyde dehydrogenase-1 (ALDH-1). Meanwhile, in vivo investigations demonstrated the potential of the combination regimen to enhance necrosis while downregulating PTEN and curbing PI3K levels as well as p-Akt, mTOR, and ALDH-1 immunoreactivities. Notably, the combination failed to change cleaved poly-ADP ribose polymerase levels suggesting a pro-necrotic rather than pro-apoptotic mechanism. Overall, these findings suggest a potential role of PIP in decreasing the resistance to DOX in vitro and in vivo, likely by interfering with the PI3K/Akt/mTOR pathway and CSCs.

Aulosirazole Stimulates FOXO3a Nuclear Translocation to Regulate Apoptosis and Cell-Cycle Progression in High-Grade Serous Ovarian Cancer (HGSOC) Cells.

Ovarian cancer, the fifth leading cause of cancer-related mortality in women, is the most lethal gynecological malignancy globally. Within various ovarian cancer subtypes, high-grade serous ovarian cancer is the most prevalent and there is frequent emergence of chemoresistance. Aulosirazole, an isothiazolonaphthoquinone alkaloid, isolated from the cyanobacterium Nostoc sp. UIC 10771, demonstrated cytotoxic activity against OVCAR3 cells (IC50 = 301 ± 80 nM). Using immunocytochemistry, OVCAR3 cells treated with aulosirazole demonstrated increased concentrations of phosphorylated protein kinase B and phosphorylated c-Jun N-terminal kinase with subsequent accumulation of forkhead box O3a (FOXO3a) in the nucleus. The combination of aulosirazole with protein kinase B inhibitors resulted in the most nuclear accumulation of FOXO3a aulosirazole-induced apoptosis based on cleavage of poly(ADP-ribose) polymerase, annexin V staining, and induction of caspase 3/7 activity in OVCAR3, OVCAR5, and OVCAR8. The expression of downstream targets of FOXO3a, including B-cell lymphoma 2 (BCL2) and p53-upregulator modulator of apoptosis, increased following aulosirazole treatment. Aulosirazole upregulated the FOXO3a target, cyclin-dependent kinase inhibitor 1, and increased cell-cycle arrest in the G0/G1 phase. The downregulation of FOXO3a by short hairpin RNA (shRNA) reduced the cytotoxicity after aulosirazole treatment by 3-fold IC50 (949 ± 16 nM) and eliminated its ability to regulate downstream targets of FOXO3a. These findings underscore FOXO3a as a critical mediator of aulosirazole-induced cytotoxicity. Additionally, aulosirazole was able to decrease migration and invasion while increasing cell death in 3D tumor spheroids. However, in vivo OVCAR8 tumor burden was not reduced by aulosirazole using an intraperitoneal tumor model. Given the mechanism of action of aulosirazole, this class of alkaloids represents promising lead compounds to develop treatments against FOXO3a-downregulated cancers. SIGNIFICANCE STATEMENT: Aulosirazole, an isothiazolonaphthoquinone alkaloid, exhibits potent cytotoxic effects against high-grade serous ovarian cancer by promoting forkhead box O3a (FOXO3a) nuclear accumulation and modulating downstream targets. These findings highlight the potential of aulosirazole as a promising therapeutic intervention for cancers characterized by FOXO3a downregulation.

Curzerene suppresses hepatocellular carcinoma progression through the PI3K/AKT/MTOR pathway.

Hepatocellular carcinoma (HCC) is one of the most aggressive cancers worldwide. Curzerene is a sesquiterpene and component of Curcuma rhizomes and has anti-tumor and anti-inflammatory properties. The study aimed to investigate the effects of curzerene on the malignant phenotypes and tumor growth in HCC. Various concentrations of curzerene were used to treat human HCC cells (Huh7 and HCCLM3). Cell viability, apoptosis, cell cycle, invasion, and migration were detected by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, flow cytometry, Transwell, and wound healing assays. Cell cycle-, apoptosis-, and signaling pathway-related proteins were analyzed by Western blot analysis. A mouse xenograft model was established to analyze the anti-tumor effects of curzerene in vivo. Curzerene repressed the proliferation, invasion, and migration of Huh7 and HCCLM3 cells. Curzerene also induced G2/M cycle arrest and cell apoptosis. Curzerene downregulated the CDK1, cyclin B1, PCNA, Bcl-2, matrix metallopeptidases (MMP)2, and MMP9 protein expression and upregulated the Bax, cleaved caspase3, and cleaved poly ADPribose polymerase protein expression in HCC cells. Curzerene restrained the phosphorylation of PI3K, AKT, and the Mammalian target of rapamycin (mTOR) in Huh7 and HCCLM3 cells. The in vivo data revealed that curzerene inhibited HCC tumor growth and decreased the expression of phosphorylated mTOR in xenograft mouse models. Curzerene inhibited cell malignancy in vitro and tumor growth in vivo in HCC, suggesting that curzerene may be a candidate agent for anti-HCC therapy.

Clotrimazole inhibits growth of multiple myeloma cells in vitro via G0/G1 arrest and mitochondrial apoptosis

Patients with multiple myeloma (MM) experience relapse and drug resistance; therefore, novel treatments are essential. Clotrimazole (CTZ) is a wide-spectrum antifungal drug with antitumor activity. However, CTZ’s effects on MM are unclear. We investigated CTZ’s effect on MM cell proliferation and apoptosis induction mechanisms. CTZ’s effects on MM.1S, NCI- H929, KMS-11, and U266 cell growth were investigated using Cell Counting Kit-8 (CCK-8) assay. The apoptotic cell percentage was quantified with annexin V-fluorescein isothiocyanate/7-amino actinomycin D staining. Mitochondrial membrane potential (MMP) and cell cycle progression were evaluated. Reactive oxygen species (ROS) levels were measured via fluorescence microscopy. Expression of apoptosis-related and nuclear factor (NF)-κB signaling proteins was analyzed using western blotting. The CCK-8 assay indicated that CTZ inhibited cell proliferation based on both dose and exposure time. Flow cytometry revealed that CTZ decreased apoptosis and MMP and induced G0/G1 arrest. Immunofluorescence demonstrated that CTZ dose-dependently elevated in both total and mitochondrial ROS production. Western blotting showed that CTZ enhanced Bax and cleaved poly ADP-ribose polymerase and caspase-3 while decreasing Bcl-2, p-p65, and p-IκBα. Therefore, CTZ inhibits MM cell proliferation by promoting ROS-mediated mitochondrial apoptosis, inducing G0/G1 arrest, inhibiting the NF-κB pathway, and has the potential for treating MM.

O-310 PARP inhibitors have no toxic effect on follicle reserve, mitotic and steroidogenic function of granulosa cells in human ovary

Abstract Study question Do the PARP inhibitors have gonadotoxic effects on human ovary? Summary answer No. The PARP inhibitor drugs Olaparib and Niraparib do not appear to have any toxic effects on the human ovary. What is known already The Poly (ADP-ribose) polymerase (PARP) family has many essential functions in cellular processes, including the regulation of transcription, apoptosis, and the DNA damage response. PARP has Poly (ADP-ribose) activity, which facilitates the recruitment of other repair proteins to promote the repair of DNA single-strand breaks when DNA damage occurs. PARP inhibitors are approved for the treatment of BRCA1/2 mutated breast and ovarian cancers. No data is available regarding the gonadotoxic potentials, if any, of these drugs on the human ovary. We aimed to address this issue in the current study. Study design, size, duration A clinical translational research study that utilizes ex-vivo and in-vitro experimental models on human ovarian cortical samples, primary luteinized non-mitotic granulosa cells, and immortalized human granulosa cells. Participants/materials, setting, methods Ovarian cortical samples were obtained from young women undergoing laparoscopic surgery for benign ovarian cysts (n = 5). Human non-mitotic luteinized granulosa cells (HLGCs) obtained from 10 IVF patients during the oocyte retrieval procedure and immortalized mitotic non-luteinized granulosa cell line (HGrC1), and immortalized granulosa cell tumor cell line (COV434) were used for the experiments. Cell/tissue culture, confocal imaging, flow cytometry, immunoblotting, cell viability/proliferation and hormone assays were applied. Main results and the role of chance Treatment of ovarian tissue samples and granulosa cells with PARP inhibitors at five different therapeutic concentrations (1, 5, 10, 20, and 40 µM/mL) up to 96 hours in culture resulted in resulted in the cleavage of PARP, validating in vitro activity of the drugs. The primordial follicle reserve and steroidogenic activity of the tissue samples remained stable at the end of culture period. Similar results were obtained in the granulosa cells, which retained their viability and steroidogenic functions after exposure to the PARP inhibitors. Real-time impedance-based cell proliferation assay with xCelligence system revealed that granulosa cells continued to proliferate normally and reached log phase in the presence of PARP inhibitors without any discernible cytotoxic effect. In immunoblot analysis, the expression of steroidogenic enzymes (StAR, aromatase, 3B-HSD and 17B-HSD) was comparable to control cells and cleaved caspase-3 expression was absent in the granulosa cells exposed to PARP inhibitors. Limitations, reasons for caution Caution should be exercised before extrapolating our data because these findings were obtained in ex-vivo and in-vitro models. They need to be confirmed using in-vivo experimental models such as human ovarian tissue xenografting in nude mice. Wider implications of the findings This study provides the first reassuring molecular evidence that PARP inhibitor drugs do not appear to have a significant gonadotoxic effect on human ovary. Trial registration number not applicable

Vitamin C alleviates hyperglycemic stress in retinal pigment epithelial cells.

Retinal pigment epithelial cells (RPECs) are a type of retinal cells that structurally and physiologically support photoreceptors. However, hyperglycemia has been shown to play a critical role in the progression of diabetic retinopathy (DR), which is one of the leading causes of vision impairment. In the diabetic eye, the high glucose environment damages RPECs via the induction of oxidative stress, leading to the release of excess reactive oxygen species (ROS) and triggering apoptosis. In this study, we aim to investigate the antioxidant mechanism of Vitamin C in reducing hyperglycemia-induced stress and whether this mechanism can preserve the function of RPECs. ARPE-19 cells were treated with high glucose in the presence or absence of Vitamin C. Cell viability was measured by MTT assay. Cleaved poly ADP-ribose polymerase (PARP) was used to identify apoptosis in the cells. ROS were detected by the DCFH-DA reaction. The accumulation of sorbitol in the aldose reductase (AR) polyol pathway was determined using the sorbitol detection assay. Primary mouse RPECs were isolated from adult mice and identified by Rpe65 expression. The mitochondrial damage was measured by mitochondrial membrane depolarization. Our results showed that high glucose conditions reduce cell viability in RPECs while Vitamin C can restore cell viability, compared to the vehicle treatment. We also demonstrated that Vitamin C reduces hyperglycemia-induced ROS production and prevents cell apoptosis in RPECs in an AR-independent pathway. These results suggest that Vitamin C is not only a nutritional necessity but also an adjuvant that can be combined with AR inhibitors for alleviating hyperglycemic stress in RPECs.

Study on mechanism of inhibiting proliferation of head and neck cancer cells by citral, active ingredient of lemon essential oil

To explore the active substances exerting anti-tumour effect in lemon essential oil and the molecular mechanism inhibiting the proliferation of head and neck cancer cells SCC15 and CAL33, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay(MTT) was utilized to identify the active component inhibiting the proliferation of head and neck cancer cells, namely citral. The IC_(50) of citral inhibiting the proliferation of head and neck cancer cells and normal cells were also determined. In addition, a 5-ethynyl-2'-deoxyuridine(EdU) staining assay was used to detect the effect of citral on the proliferation rate of head and neck cancer cells, and a colony formation assay was used to detect the effect of citral on tumor sphere formation of head and neck cancer cells in vitro. The cell cycle arrest and apoptosis induction of head and neck cancer cells by citral were evaluated by flow cytometry, and Western blot was used to detect the effect of citral on the expression levels of cell cycle-and apoptosis-related proteins in head and neck cancer cells. The findings indicated that citral could effectively inhibit the proliferation and growth of head and neck cancer cells, with anti-tumor activity, and its half inhibitory concentrations for CAL33 and SCC15 were 54.78 and 25.23 μg·mL~(-1), respectively. Furthermore, citral arrested cell cycle at G_2/M phase by down-regulating cell cycle-related proteins such as S-phase kinase associated protein 2(SKP2), C-MYC, cyclin dependent kinase 1(CDK1), and cyclin B. Moreover, citral increased the cysteinyl aspartate-specific proteinase-3(caspase-3), cysteinyl aspartate-specific proteinase-9(caspase-9), and cleaved poly ADP-ribose polymerase(PARP). It up-regulated the level of autophagy-related proteins including microtubule associated protein 1 light chain 3B(LC3B), sequestosome 1(P62/SQSTM1), autophagy effector protein Beclin1(Beclin1), and lysosome-associate membrane protein 1(LAMP1), suggesting that citral could effectively trigger cell apoptosis and cell autophagy in head and neck cancer cells. Furthermore, the dual-tagged plasmid system mCherry-GFP-LC3 was used, and it was found that citral impeded the fusion of autophagosomes and lysosomes, leading to autophagic flux blockage. Collectively, our findings reveal that the main active anti-proliferation component of lemon essential oil is citral, and this component has a significant inhibitory effect on head and neck cancer cells. Its underlying molecular mechanism is that citral induces apoptosis and autophagy by cell cycle arrest and ultimately inhibits cell proliferation.

Paris saponin VII induces Caspase-3/GSDME-dependent pyroptosis in pancreatic ductal adenocarcinoma cells by activating ROS/Bax signaling

ObjectiveParidis Rhizoma (Chonglou in Chinese), a traditional Chinese herbal medicine, has been shown have strong anti-tumor effects. Paris saponin VII (PSVII), an active constituent isolated from Paridis Rhizoma, was demonstrated to significantly suppress the proliferation of BxPC-3 cells in our previous study. Here, we aimed to elucidate the anti-pancreatic ductal adenocarcinoma (PDAC) effect of PSVII and the underlying mechanism. MethodsCell viability was determined by CCK-8, colony formation, and cell migration assays. Cell apoptosis and reactive oxygen species (ROS) production were measured by flow cytometry with annexin V/propidine iodide (Annexin V/PI) and 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA), respectively. Pyroptosis was evaluated by morphological features, Hoechst 33342/PI staining assay, and release of lactate dehydrogenase (LDH). JC-1 fluorescent dye was employed to measure mitochondrial membrane potential. Western blotting and reverse transcription-quantitative polymerase chain reaction (RT-qPCR) were used to determine the levels of proteins or mRNAs. The effect in vivo was assessed by a xenograft tumor model. ResultsPSVII inhibited the viability of PDAC cells (BxPC-3, PANC-1, and Capan-2 cells) and induced gasdermin E (GSDME) cleavage, as well as the simultaneous cleavage of Caspase-3 and poly (ADP-ribose) polymerase 1 (PARP). Knockdown of GSDME shifted PSVII-induced pyroptosis to apoptosis. Additionally, the effect of PSVII was significantly attenuated by Z-Asp(OMe)-Glu(OMe)-Val-Asp(OMe)-fluoromethylketone (Z-DEVD-FMK), on the induction of GSDME-dependent pyroptosis. PSVII also elevated intracellular ROS accumulation and stimulated Bax and Caspase-3/GSDME to conduct pyroptosis in PDAC cells. The ROS scavenger N-acetyl cysteine (NAC) suppressed the release of LDH and inhibited Caspase-9, Caspase-3, and GSDME cleavage in PDAC cells, ultimately reversing PSVII-induced pyroptosis. Furthermore, in a xenograft tumor model, PSVII markedly suppressed the growth of PDAC tumors and induced pyroptosis. ConclusionThese results demonstrated that PSVII exerts therapeutic effects through Caspase-3/GSDME-dependent pyroptosis and may constitute a novel strategy for preventing chemotherapeutic resistance in patients with PDAC in the future.

MK591 (Quiflapon), a 5-lipoxygenase inhibitor, kills pancreatic cancer cells via downregulation of protein kinase C-epsilon.

Pancreatic tumors and cell lines derived from them exhibit elevated expression of 5-lipoxygenase (5-Lox), whereas non-tumor glands or normal cells do not exhibit this overexpression. Arachidonic acid stimulates pancreatic cancer cell growth via metabolic conversion through the 5-Lox pathway, and inhibition of 5-Lox activity decreases the viability of pancreatic cancer cells. However, the downstream signaling mechanisms through which 5-Lox exerts its effects on the survival of pancreatic cancer cells remain to be elucidated. The effects of 5-Lox inhibition on cell proliferation, apoptosis, and invasive potential were investigated in pancreatic cancer cells. The protein expression was analyzed by Western blot. Apoptosis was analyzed by Annexin-V binding assay and by detecting the degradation of chromatin-DNA to nucleosomal fragments. The protein kinase C-epsilon (PKCε) activity was measured by an immunoprecipitation-kinase assay. The in vivo effects of MK591 were evaluated in pancreatic tumor xenograft model. MK591, a specific inhibitor of 5-Lox activity, killed pancreatic cancer cells via induction of apoptosis, involving externalization of phosphatidylserine, cleavage of PARP (poly-ADP ribose polymerase) and degradation of chromatin DNA to nucleosomes. MK591 effectively blocked in vitro invasion and soft-agar colony formation by pancreatic cancer cells and decreased pancreatic tumor growth in nude mice xenografts. Furthermore, inhibition of 5-Lox downregulated K-Ras and inhibited phosphorylation of c-Raf and ERKs. Interestingly, 5-Lox inhibition induced apoptosis in pancreatic cancer cells without the inhibition of Akt but the protein level of PKCε was dramatically downregulated. Furthermore, inhibition of 5-Lox decreased the phosphorylation of Stat3 at Serine-727. Pre-treatment of pancreatic cancer cells with peptide activators of PKCε prevented apoptosis induced by 5-Lox inhibition, suggesting that the mechanism by which 5-Lox inhibition causes cell death in pancreatic cancer involves downregulation of PKCε. The combination of low doses of MK591 and gemcitabine synergistically reduced the oncogenic phenotype and killed pancreatic cancer cells by inducing apoptosis. These findings indicate that inhibition of 5-Lox interrupts an Akt-independent, PKCε-dependent survival mechanism in pancreatic cancer cells and suggest that metabolism of arachidonic acid through the 5-Lox pathway plays an integral part in the survival of pancreatic cancer cells via signaling through PKCε, an oncogenic, pro-survival serine/threonine kinase.

Sequence-dependent effects of hematoporphyrin derivatives (HPD) photodynamic therapy and cisplatin on lung adenocarcinoma cells

BackgroundHematoporphyrin derivatives (HPD)-Photodynamic therapy (PDT) in combination with cisplatin (DDP) is an effective anticancer strategy. However, whether the order of combination affects efficacy has not been studied. MethodsThe human lung adenocarcinoma (LUAD) A549 cells were used as the study subjects. After A549 cells were treated with a single medication (PDT/DDP) or a sequential combination (PDT + DDP / DDP + PDT), the cell viability was assayed using the cell counting kit-8 method. Hoechst staining, Annexin-V/propidium iodide (PI) double staining, western blotting, and a real-time quantitative polymerase chain reaction (RT-qPCR) were performed to examine the mechanisms behind the combined effects. ResultsA synergistic impact between HPD-PDT and DDP was found. The cell viability in the PDT+DDP group was significantly lower than in the DDP+PDT group. A significant apoptotic profile and a high apoptotic rate were seen in the PDT + DDP group. The western blot showed that the expression levels of Bcl2-associated x(Bax) and cleaved-poly ADP-ribose polymerase (PARP) increased, and those of B-cell lymphoma-2 (Bcl-2) and Caspase-9 decreased in the PDT + DDP group. At the same time, the RT-qPCR revealed the upregulation of Bax and PARP mRNA and the downregulation of Bcl-2 and Caspase-9 mRNA. ConclusionThe order of the combination therapy (PDT + DDP / DDP + PDT) was important. The HPD-PDT followed by DDP significantly inhibited LUAD cell viability, which may be related to the mitochondrial apoptotic pathway.

Cymensifin A: a promising pharmaceutical candidate to defeat lung cancer via cellular reactive oxygen species-mediated apoptosis.

Background: The upgrade of natural products for cancer treatment is essential since current anticancer drugs still pose severe side effects. Cymensifin A (Cym A) isolated from an orchid Cymbidium ensifolium has shown its potential to induce the death of several cancer cells; however, its underlying molecular mechanisms are hitherto unknown. Methods: Here, we conducted a set of in vitro preliminary tests to assess the cytotoxic effects of Cym A on non-small-cell lung cancer (NSCLC) cells (A549, H23, H292, and H460). A flow cytometry system and Western blot analyses were employed to unveil molecular mechanisms underlying cancer cell apoptosis caused by Cym A. Results: Cym A at 25-50μM caused the death of all NSCLC cells tested, and its cytotoxicity was comparable to cisplatin, a currently used anticancer drug. The compound induced apoptosis of all NSCLC cells in a dose-dependent manner (5-50μM), proven by flow cytometry, but H460 cells showed more resistance compared to other cells tested. Cym A-treated H460 cells demonstrated increased reactive oxygen species (ROS) and downregulated antioxidants (catalase, superoxide dismutase, and thioredoxin). The compound also upregulated the tumor suppressor P53 and the pro-apoptotic protein BAX but downregulated pro-survival proteins (BCL-2 and MCL-1) and deactivated survival signals (AKT and ERK) in H460 cells. Cym A was proven to trigger cellular ROS formation, but P53 and BAX were 2-fold more activated by Cym A compared to those treated with hydrogen peroxide. Our findings also supported that Cym A exerted its roles in the downregulation of nuclear factor erythroid 2-related factor 2 (a regulator of cellular antioxidant activity) and the increased levels of cleaved poly (ADP-ribose) polymerase (PARP) and cleaved caspase 3/7 during apoptosis. Conclusion: We propose that Cym A induces lung cancer cell death via ROS-mediated apoptosis, while the modulation of cellular ROS/antioxidant activity, the upregulation of P53 and BAX, the downregulation or deactivation of BCL-2, MCL-1, AKT, and ERK, and the increased cleavage of PARP and caspase 3/7, were the elucidated underlying molecular mechanisms of this phytochemical. The compound can be a promising candidate for future anticancer drug development.

Repurposing the diuretic benzamil as an anti-osteosarcoma agent that acts by suppressing integrin/FAK/STAT3 signalling and compromising mitochondrial function.

Osteosarcoma is the most common primary bone malignancy among children and adolescents. We investigated whether benzamil, an amiloride analogue and sodium-calcium exchange blocker, may exhibit therapeutic potential for osteosarcoma in vitro. MG63 and U2OS cells were treated with benzamil for 24 hours. Cell viability was evaluated with the MTS/PMS assay, colony formation assay, and flow cytometry (forward/side scatter). Chromosome condensation, the terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) assay, cleavage of poly-ADP ribose polymerase (PARP) and caspase-7, and FITC annexin V/PI double staining were monitored as indicators of apoptosis. Intracellular calcium was detected by flow cytometry with Fluo-4 AM. The phosphorylation and activation of focal adhesion kinase (FAK) and signal transducer and activator of transcription 3 (STAT3) were measured by western blot. The expression levels of X-linked inhibitor of apoptosis protein (XIAP), B-cell lymphoma 2 (Bcl-2), B-cell lymphoma-extra large (Bcl-xL), SOD1, and SOD2 were also assessed by western blot. Mitochondrial status was assessed with tetramethylrhodamine, ethyl ester (TMRE), and intracellular adenosine triphosphate (ATP) was measured with BioTracker ATP-Red Live Cell Dye. Total cellular integrin levels were evaluated by western blot, and the expression of cell surface integrins was assessed using fluorescent-labelled antibodies and flow cytometry. Benzamil suppressed growth of osteosarcoma cells by inducing apoptosis. Benzamil reduced the expression of cell surface integrins α5, αV, and β1 in MG63 cells, while it only reduced the expression of αV in U2OS cells. Benzamil suppressed the phosphorylation and activation of FAK and STAT3. In addition, mitochondrial function and ATP production were compromised by benzamil. The levels of anti-apoptotic proteins XIAP, Bcl-2, and Bcl-xL were reduced by benzamil. Correspondingly, benzamil potentiated cisplatin- and methotrexate-induced apoptosis in osteosarcoma cells. Benzamil exerts anti-osteosarcoma activity by inducing apoptosis. In terms of mechanism, benzamil appears to inhibit integrin/FAK/STAT3 signalling, which triggers mitochondrial dysfunction and ATP depletion.

Design, Synthesis, and Anti-Leukemic Evaluation of a Series of Dianilinopyrimidines by Regulating the Ras/Raf/MEK/ERK and STAT3/c-Myc Pathways.

Although the long-term survival rate for leukemia has made significant progress over the years with the development of chemotherapeutics, patients still suffer from relapse, leading to an unsatisfactory outcome. To discover the new effective anti-leukemia compounds, we synthesized a series of dianilinopyrimidines and evaluated the anti-leukemia activities of those compounds by using leukemia cell lines (HEL, Jurkat, and K562). The results showed that the dianilinopyrimidine analog H-120 predominantly displayed the highest cytotoxic potential in HEL cells. It remarkably induced apoptosis of HEL cells by activating the apoptosis-related proteins (cleaved caspase-3, cleaved caspase-9 and cleaved poly ADP-ribose polymerase (PARP)), increasing apoptosis protein Bad expression, and decreasing the expression of anti-apoptotic proteins (Bcl-2 and Bcl-xL). Furthermore, it induced cell cycle arrest in G2/M; concomitantly, we observed the activation of p53 and a reduction in phosphorylated cell division cycle 25C (p-CDC25C) / Cyclin B1 levels in treated cells. Additionally, the mechanism study revealed that H-120 decreased these phosphorylated signal transducers and activators of transcription 3, rat sarcoma, phosphorylated cellular RAF proto-oncogene serine / threonine kinase, phosphorylated mitogen-activated protein kinase kinase, phosphorylated extracellular signal-regulated kinase, and cellular myelocytomatosis oncogene (p-STAT3, Ras, p-C-Raf, p-MEK, p-MRK, and c-Myc) protein levels in HEL cells. Using the cytoplasmic and nuclear proteins isolation assay, we found for the first time that H-120 can inhibit the activation of STAT3 and c-Myc and block STAT3 phosphorylation and dimerization. Moreover, H-120 treatment effectively inhibited the disease progression of erythroleukemia mice by promoting erythroid differentiation into the maturation of erythrocytes and activating the immune cells. Significantly, H-120 also improved liver function in erythroleukemia mice. Therefore, H-120 may be a potential chemotherapeutic drug for leukemia patients.

Effects of metalation on the PDT activity of arene ruthenium porphyrin-based photosensitizers on prostate cancer

SignificanceProstate cancer is a major health issue (second most common cancer in men worldwide). Despite therapeutic advances and early detection, many side effects are often observed during treatment. Photodynamic therapy (PDT) is a promising therapy to reduce side effects, many 2nd and 3rd generation of photosensitizer (PS) are poorly soluble in aqueous media, limiting their attractiveness. ApproachWe have developed and tested 3rd generation of PS vectorized by arene-ruthenium (Ru) complexes coordinated to tetrapyridylporphin (tpp-2H), Zn-tetrapyridylporphin (tpp-Zn) or Co-tetrapyridylporphin (tpp-Co). ResultsTwo of the three complexes show a good efficacy. The compounds localize into the cytoplasm. An analysis of the apoptotic process shows a cleavage of poly-ADP ribose polymerase (PARP) and a strong induction of DNA fragmentation following irradiation. ConclusionsArene-ruthenium tetrapyridylporphin complexes are excellent candidates for PDT application. The addition of the metal shows a decrease (tpp-Zn) or no effects (tpp-Co), compared to the metal free photosentizer (tpp-2H).

Pharmacological p38 MAPK inhibitor SB203580 enhances AML stem cell line KG1a chemosensitivity to daunorubicin by promoting late apoptosis, cell growth arrest in S-phase, and miR-328-3p upregulation

Acute myeloid leukaemia (AML) is characterized by uncontrolled proliferation of myeloid progenitor cells and impaired maturation, leading to immature cell accumulation in the bone marrow and bloodstream, resulting in hematopoietic dysfunction. Chemoresistance, hyperactivity of survival pathways, and miRNA alteration are major factors contributing to treatment failure and poor outcomes in AML patients. This study aimed to investigate the impact of the pharmacological p38 mitogen-activated protein kinase (MAPK) inhibitor SB203580 on the chemoresistance potential of AML stem cell line KG1a to the therapeutic drug daunorubicin (DNR). KG1a and chemosensitive leukemic HL60 cells were treated with increasing concentrations of DNR. Cell Titer-Glo®, flow cytometry, phosphokinase and protein arrays, Western blot technology, and reverse transcription-quantitative polymerase chain reaction (RT-qPCR) were employed for assessment of cell viability, half-maximal inhibitory concentration (IC50) determination, apoptotic status detection, cell cycle analysis, apoptosis-related protein and gene expression monitoring. Confocal microscopy was used to visualize caspase and mitochondrial permeability transition pore (mPTP) activities. Exposed at various incubation times, higher DNR IC50 values were determined for KG1a cells than for HL60 cells, confirming KG1a cell chemoresistance potential. Exposed to DNR, late apoptosis induction in KG1a cells was enhanced after SB203580 pretreatment, defined as the combination treatment. This enhancement was confirmed by increased cleavage of poly(ADP-ribose) polymerase, caspase-9, caspase-3, and augmented caspase-3/-7 and mPTP activities in KG1a cells upon combination treatment, compared to DNR. Using phosphokinase and apoptosis protein arrays, the combination treatment decreased survival Akt phosphorylation and anti-apoptotic Bcl-2 expression levels in KG1a cells while increasing the expression levels of the tumor suppressor p53 and cyclin-dependent kinase inhibitor p21, compared to DNR. Cell cycle analysis revealed KG1a cell growth arrest in G2/M-phase caused by DNR, while combined treatment led to cell growth arrest in S-phase, mainly associated with cyclin B1 expression levels. Remarkably, the enhanced KG1a cell sensitivity to DNR after SB203580 pretreatment was associated with an increased upregulation of miR-328-3p and slight downregulation of miR-26b-5p, compared to DNR effect. Altogether, these findings could contribute to the development of a new therapeutic strategy by targeting the p38 MAPK pathway to improve treatment outcomes in patients with refractory or relapsed AML.