Extracellular ATP is a dynamic signaling molecule that modulates myriad of cellular functions through P2 purinergic receptors activation and is cytotoxic to a variety of cells at high concentration. But the mechanism of this extracellular ATP/ATP analogs- elicited cytotoxicity is not fully understood. In this study we aim to investigate whether there is differential sensitivity towards induction of apoptosis by ATP analogs (2\'-Me ATP and 3\'-Me ATP) and its effect on receptor mediated or extrinsic and mitochondria mediated or intrinsic apoptotic signaling pathways. Our findings demonstrated that the IC50 values for 2\'-Me ATP and 3\'-Me ATP were 3mM and 2mM, respectively, in Hep2, and SiHa cells. The downregulation of anti-apoptotic proteins Bcl-2 and Bcl-xL, along with a significant increase in the expression of the pro-apoptotic protein Bax (p<0.05), indicated the involvement of both pro- and anti-apoptotic factors in HeP2 cells, whereas in SiHa cells, a downregulation of anti-apoptotic proteins Bcl-2 and Bcl-xL was observed, whereas the expression level of the pro-apoptotic protein Bax remained unaffected. Furthermore, an upregulation of p53 and apoptosis-inducing factor (AIF) was observed in HeP2 cells (p<0.05) whereas, an upregulation of p53 was observed while no change was seen on the level of apoptosis inducing factor (AIF) was observed in SiHa cells. Additionally, there was a notable rise in caspase-3 and -9 activities, PARP cleavage, and the release of cytochrome c (p<0.05) from the mitochondria to the cytosol in both cells. Collectively, our study suggests that 3\'-Me ATP induces apoptosis in Hep2 and SiHa cells through the intrinsic mitochondrial pathway.

UNASSIGNED: MEK inhibitors (MEKi) lack monotherapy efficacy in most RAS-mutant cancers. BCL-xL is an anti-apoptotic protein identified by a synthetic lethal shRNA screen as a key suppressor of apoptotic response to MEKi.
UNASSIGNED: We conducted a dose escalation study (NCT02079740) of the BCL-xL inhibitor navitoclax and MEKi trametinib in patients with RAS-mutant tumors with expansion cohorts for: pancreatic, gynecologic (GYN), non-small cell lung cancer (NSCLC), and other cancers harboring KRAS/NRAS mutations. Paired pretreatment and day 15 tumor biopsies and serial cell-free (cf)DNA were analyzed.
UNASSIGNED: A total of 91 patients initiated treatment, with 38 in dose escalation. Fifty-eight percent had ≥3 prior therapies. A total of 15 patients (17%) had colorectal cancer, 19 (11%) pancreatic, 15 (17%) NSCLC, and 32 (35%) GYN cancers. The recommended phase II dose (RP2D) was established as trametinib 2 mg daily days 1 to 14 and navitoclax 250 mg daily days 1 to 28 of each cycle. Most common adverse events included diarrhea, thrombocytopenia, increased AST/ALT, and acneiform rash. At RP2D, 8 of 49 (16%) evaluable patients achieved partial response (PR). Disease-specific differences in efficacy were noted. In patients with GYN at the RP2D, 7 of 21 (33%) achieved a PR and median duration of response 8.2 months. No PRs occurred in patients with colorectal cancer, NSCLC, or pancreatic cancer. MAPK pathway inhibition was observed in on-treatment tumor biopsies. Reductions in KRAS/NRAS mutation levels in cfDNA correlated with clinical benefit.
UNASSIGNED: Navitoclax in combination with trametinib was tolerable. Durable clinical responses were observed in patients with RAS-mutant GYN cancers, warranting further evaluation in this population.

B-cell lymphoma-extra-large (BCL-xL) regulates apoptosis and is an attractive anticancer therapeutic target. However, BCL-xL inhibition also kills mature platelets, hampering clinical development. Using an innovative prodrug strategy, we have developed pelcitoclax (APG-1252), a potent, dual BCL-2 and BCL-xL inhibitor. Aims of this study were to characterize the antitumor activity and safety of pelcitoclax and explore its underlying mechanisms of action (MOA).
Cell line-derived xenograft and patient-derived xenograft (PDX) models were tested to evaluate antitumor activity and elucidate MOA. Subjects (N = 50) with metastatic small-cell lung cancer and other solid tumors received intravenous pelcitoclax once or twice weekly. Primary outcome measures were safety and tolerability; preliminary efficacy (responses every 2 cycles per RECIST version 1.1) represented a secondary endpoint.
Pelcitoclax exhibited strong BAX/BAK‒dependent and caspase-mediated antiproliferative and apoptogenic activity in various cancer cell lines. Consistent with cell-based apoptogenic activity, pelcitoclax disrupted BCL-xL:BIM and BCL-xL:PUMA complexes in lung and gastric cancer PDX models. Levels of BCL-xL complexes correlated with tumor growth inhibition by pelcitoclax. Combined with taxanes, pelcitoclax enhanced antitumor activity by downregulating antiapoptotic protein myeloid cell leukemia-1 (MCL-1). Importantly, pelcitoclax was well tolerated and demonstrated preliminary therapeutic efficacy, with overall response and disease control rates of 6.5% and 30.4%, respectively. Most common treatment-related adverse events included transaminase elevations and reduced platelets that were less frequent with a once-weekly schedule.
Our data demonstrate that pelcitoclax has antitumor activity and is well tolerated, supporting its further clinical development for human solid tumors, particularly combined with agents that downregulate MCL-1.

The BCL2 family of proteins regulate apoptosis by controlling mitochondrial outer membrane permeability. However, the effects on mitochondrial structure and bioenergetics have also been reported. Here we comprehensively characterized the effects of BCL2 and BCL(X)L on cellular energetics in MCF7 breast cancer cells using time-lapse confocal single-cell imaging and mitochondrial and cytosolic FRET reporters. We found that BCL2 and BCL(X)L increase the metabolic robustness of MCF7 cells, and that this was associated with increased mitochondrial NAD(P)H and ATP levels. Experiments with the F1F0 synthase inhibitor oligomycin demonstrated that BCL2 and in particular BCL(X)L, while not affecting ATP synthase activity, more efficiently coupled the mitochondrial proton motive force with ATP production. This metabolic advantage was associated with an increased resistance to nutrient deprivation and enhanced clonogenic survival in response to metabolic stress, in the absence of profound effects on cell death. Our data suggest that a primary function of BCL(X)L and BCL2 overexpression in tumor cells is to increase their resistance to metabolic stress in the tumor microenvironment, independent of cell death signaling.

Fragment-based lead discovery (FBLD) is one of the most efficient methods to develop new drugs. We present here a new computational protocol called High-Throughput Supervised Molecular Dynamics (HT-SuMD), which makes it possible to automatically screen up to thousands of fragments, representing therefore a new valuable resource to prioritise fragments in FBLD campaigns. The protocol was applied to Bcl-XL, an oncological protein target involved in the regulation of apoptosis through protein-protein interactions. Initially, HT-SuMD performances were validated against a robust NMR-based screening, using the same set of 100 fragments. These independent results showed a remarkable agreement between the two methods. Then, a virtual screening on a larger library of additional 300 fragments was carried out and the best hits were validated by NMR. Remarkably, all the in silico selected fragments were confirmed as Bcl-XL binders. This represents, to date, the largest computational fragments screening entirely based on MD.

BACKGROUND Osteoarthritis (OA) is a common disorder in the elderly. OA influences the daily life of patients and has become a worldwide health problem. It is still unclear whether the pathogenesis mechanism is different between males and females. This study investigated the differentially expressed genes (DEGs) and explored the different signaling pathways of OA between males and females. MATERIAL AND METHODS Data sets of GSE55457, GSE55584, and GSE12021 were retrieved from Gene Expression Omnibus to conduct DEGs analysis. Enrichment analysis of Kyoto Encyclopedia of Genes and Genomes pathway and Gene Ontology term was performed using the Database for Annotation, Visualization and Integrated Discovery (DAVID) bioinformatics tool. The protein interaction network was constructed in Cytoscape 3.7.2. qRT-PCR was then performed to validate the expression of hub genes in OA patients and healthy people. RESULTS In total, 4 co-upregulated and 10 co-downregulated genes were identified. We found that enriched pathways were different between males and females. BCL2L1, EEF1A1, EEF2, HNRNPD, and PABPN1 were considered as hub genes in OA pathogenesis in males, while EEF2, EEF1A1, RPL37A, FN1 were considered as hub genes in OA pathogenesis in females. Consistent with the bioinformatics analysis, the qRT-PCR analysis also showed that the gene expression of BCL2L1, HNRNPD, and PABPN1 was significantly lower in male OA patients. In contrast, EEF2, EEF1A1, and RPL37A were significantly lower in female OA patients. CONCLUSIONS The DEGs identified may be involved in different OA disease progression mechanisms between males and females, and they are considered as treatment targets or prognosis markers for males and females. The pathogenesis mechanism is sex-dependent.

Cisplatin-based chemotherapy is the primary treatment for metastatic bladder urothelial carcinoma (UC). Most patients inevitably encounter drug resistance and resultant disease relapse. Reduced apoptosis plays a critical role in chemoresistance. Trifluoperazine (TFP), an antipsychotic agent, has demonstrated antitumor effects on various cancers. This study investigated the efficacy of TFP in inhibiting cisplatin-resistant bladder UC and explored the underlying mechanism. Our results revealed that cisplatin-resistant UC cells (T24/R) upregulated the antiapoptotic factor, B-cell lymphoma-extra large (Bcl-xL). Knockdown of Bcl-xL by siRNA resensitized cisplatin-resistant cells to the cisplatin cytotoxic effect. TFP (10-45 μM) alone elicited dose-dependent cytotoxicity, apoptosis, and G0/G1 arrest on T24/R cells. Co-treatment of TFP potentiated cisplatin-induced cytotoxicity in T24/R cells. The phenomenon that TFP alleviated cisplatin resistance to T24/R was accompanied with concurrent suppression of Bcl-xL. In vivo models confirmed that TFP alone effectively suppressed the T24/R xenograft in nude mice. TFP co-treatment enhanced the antitumor effect of cisplatin on the T24/R xenograft. Our results demonstrated that TFP effectively inhibited cisplatin-resistant UCs and circumvented cisplatin resistance with concurrent Bcl-xL downregulation. These findings provide a promising insight to develop a therapeutic strategy for chemoresistant UCs.

Deregulation of the normal cellular apoptotic function is a fundamental element in the etiology of most cancers and the anti-apoptotic B cell lymphoma 2 (BCL‑2) protein family is known to play crucial role in the regulation of this function. Overexpression of this protein family has been implicated in some cancers, such that agents that could inhibit their over-activity are now being explored for anticancer drug development. A number of studies have revealed the anticancer potential of Morinda lucida-derived extracts and compounds. In search of more inhibitors of this anti-apoptotic protein family from plant resources, 47 compounds, identified in Morinda lucida Benth (Rubiaceae) were screened for their inhibitory activities against BCL-XL, BCL-2, and MCL-1 by molecular docking using BINDSURF, while binding interactions of the top compounds were viewed with PyMOL. Druglikeness and Absorption-Distribution-Metabolism-Excretion (ADME) parameters of the top 6 compounds from docking study were evaluated using SuperPred webserver. Results revealed that out of the 47 compounds, 2 triterpenes (ursolic acid and oleanolic acid) and 4 phytosterols (cycloartenol, campesterol, stigmasterol, and β-sitosterol) have higher binding affinities for the selected BCL-2 proteins, compared to known standard inhibitors; these compounds also fulfill oral drugability of Lipinski rule of five. Therefore, since these Morinda lucida-derived phytosterols and triterpenes show high binding affinity toward the selected anti-apoptotic proteins and exhibited good drugability characteristics, they qualify for further study on drug development against cancers characterized by overexpression of this family of protein.

To study the effect of integrin αvβ6 on the proliferation and apoptosis of thyroid carcinoma cells.
The experiment was conducted on 3 groups : the control group, the positive observation group (in which the ανβ6 on the surface of the thyroid carcinoma cell line SW579 was blocked by monoclonal antibody 10D5) and the negative observation group (in which the ανβ6 was dealt with the negative placebo of 10D5-the IgG2a). Cell proliferation was detected by MTT assay, apoptosis by flow cytometry and the protein levels in Caspase-3, CyclinB1 and Bcl-xl as well as the protein levels in ERK, p-ERK, JNK, p-JNK, p38 and p-p38 were detected by Western blot.
The cell survival rates of the control group and the negative observation group were prominently higher than those of the positive observation group, following decrease in the apoptosis rates, and the differences were statistically significant (p<0.05). The protein levels in CyclinB1 and Bcl-x1 of the control group and the negative observation group were prominently higher than those of the positive observation group, whereas the levels in Caspase-3 were decreased; the differences were statistically significant (p<0.05). The protein levels in p-ERK, p-JNK and p-p38 of the control group and the negative observation group were prominently higher than those of the positive observation group, while the protein levels of ERK, JNK and p38 showed no difference.
Integrin ανβ6 can mediate the MAPK signal pathway of the cells and regulate the expression of CyclinB1 and the apoptosis-related proteins like Bcl-x1 and Caspase-2, thus affecting the process of the proliferation and apoptosis of thyroid carcinoma cells.

Myclobutanil is a conazole class fungicide widely used as an agrichemical. It is approved for use on fruit, vegetables and seed commodities in the EU and elsewhere to control fungi such as Ascomycetes, Fungi Imperfecti and, Basidiomycetes. Its widespread use has raised the issue of possible health risks for agrarian communities and the general population, which can be exposed to residues present in food and drinking water. The toxicities identified include adverse effects on liver and kidney and on the development of male reproductive organs. Since the liver is the first-line organ in the defense against xenobiotics, toxic effects on hepatic metabolism cause degeneration, necrosis, and tissue hypertrophy. Therefore, we investigated myclobutanil\'s effects on the human liver cell line HepG2. We found that myclobutanil increases the amount of fatty acids in these hepatic cells, as evaluated with Oil Red O staining, and progressively reduces cell viability from 1ppm to 500ppm. Analysis of biomarkers such as Bcl-xL/Bak and Mcl-1/Bak confirmed activation of cell death pathways at low doses. Therefore, myclobutanil may play an important role in the pathogenesis and progression of chronic hepatocellular diseases in humans.