Pattern-Recognition Receptor Agonist-Containing Immunopotentiator CVC1302 Boosts High-Affinity Long-Lasting Humoral Immunity.

Ideally, a vaccine should provide life-long protection following a single administered dose. In our previous study, the immunopotentiator CVC1302, which contains pattern- recognition receptor (PRR) agonists, was demonstrated to prolong the lifetime of the humoral immune response induced by killed foot-and-mouth disease virus (FMDV) vaccine. To elucidate the mechanism by which CVC1302 induces long-term humoral immunity, we used 4-hydroxy-3-nitrophenylacetyl (NP)-OVA as a pattern antigen and administered it to mice along with CVC1302, emulsified together with Marcol 52 mineral oil (NP-CVC1302). From the results of NP-specific antibody levels, we found that CVC1302 could induce not only higher levels of NP-specific antibodies but also high-affinity NP-specific antibody levels. To detect the resulting NP-specific immune cells, samples were taken from the injection sites, draining lymph nodes (LNs), and bone marrow of mice injected with NP-CVC1302. The results of these experiments show that, compared with mice injected with NP alone, those injected with NP-CVC1302 had higher percentages of NP+ antigen-presenting cells (APCs) at the injection sites and draining LNs, higher percentages of follicular helper T cells (TFH), germinal center (GC) B cells, and NP+ plasma-blasts in the draining LNs, as well as higher percentages of NP+ long-lived plasma cells (LLPCs) in the bone marrow. Additionally, we observed that the inclusion of CVC1302 in the immunization prolonged the lifetime of LLPCs in the bone marrow by improving the transcription expression of anti-apoptotic transcription factors such as Mcl-1, Bcl-2, BAFF, BCMA, Bax, and IRF-4. This research provides a blueprint for designing new generations of immunopotentiators.

Gene expression profiles of two testicular somatic cell lines respond differently to 4-nitrophenol mediating vary reproductive toxicity.

4-Nitrophenol (PNP) has been extensively used in manufacturing for several decades. Its toxic effects on the male reproductive system have been reported, but the underlying mechanisms remain unclear. In this study, we utilized two testicular somatic cell lines (TM3 and TM4 cells) to explore the possible toxic effects of PNP on the male reproductive system. The activity of the cells after exposure to different doses of PNP (0.01, 0.1, 1, 10 and 100 µM) was evaluated. PNP treatment at 10 µM significantly inhibited cell viability, and 10 µM PNP was thus selected for subsequent experiments. Although PNP (10 µM) inhibited cell proliferation, promoted cell apoptosis, and changed the cell cycle distribution and ultrastructure in both types of cells, these effects were more significant in the TM4 cells. In addition, an Agilent mouse mRNA array was used to identify the gene expression differences between the control and PNP (10 µM) exposed TM3 and TM4 cells. The microarray analysis identified 67 and 1372 differentially expressed genes mainly concentrated in endothelial cell morphogenesis and anatomical structure development in TM3 cells and associated with cardiovascular system development and circulatory system development in TM4 cells. Moreover, a pathway analysis revealed that PNP not only predominately affected meiotic recombination and meiosis in TM3 cells, but also influenced axon guidance and developmental biology in TM4 cells. These results suggest that TM3 and TM4 cells exhibit different responses to PNP, which might mediate different toxic mechanisms.

BCL-2 Inhibitor ABT-737 Effectively Targets Leukemia-Initiating Cells with Differential Regulation of Relevant Genes Leading to Extended Survival in a NRAS/BCL-2 Mouse Model of High Risk-Myelodysplastic Syndrome.

During transformation, myelodysplastic syndromes (MDS) are characterized by reducing apoptosis of bone marrow (BM) precursors. Mouse models of high risk (HR)-MDS and acute myelogenous leukemia (AML) post-MDS using mutant NRAS and overexpression of human BCL-2, known to be poor prognostic indicators of the human diseases, were created. We have reported the efficacy of the BCL-2 inhibitor, ABT-737, on the AML post-MDS model; here, we report that this BCL-2 inhibitor also significantly extended survival of the HR-MDS mouse model, with reductions of BM blasts and lineage negative/Sca1+/KIT+ (LSK) cells. Secondary transplants showed increased survival in treated compared to untreated mice. Unlike the AML model, BCL-2 expression and RAS activity decreased following treatment and the RAS:BCL-2 complex remained in the plasma membrane. Exon-specific gene expression profiling (GEP) of HR-MDS mice showed 1952 differentially regulated genes upon treatment, including genes important for the regulation of stem cells, differentiation, proliferation, oxidative phosphorylation, mitochondrial function, and apoptosis; relevant in human disease. Spliceosome genes, found to be abnormal in MDS patients and downregulated in our HR-MDS model, such as Rsrc1 and Wbp4, were upregulated by the treatment, as were genes involved in epigenetic regulation, such as DNMT3A and B, upregulated upon disease progression and downregulated upon treatment.

Efonidipine Exerts Cerebroprotective Effect by Down-regulation of TGF-ß/SMAD-2-Dependent Signaling Pathway in Diabetic Rats.

Calcium overload and hyperglycemia are risks of stroke onset in diabetics. Our study was designed to elucidate the beneficial role of calcium channel blockers by targeting voltage-gated calcium channels in diabetes-associated cerebrovascular complications. Diabetes was induced using the neonatal streptozotocin rat model. After confirmation of diabetes, middle cerebral artery occlusion (MCAO) was carried out. The pre-treatment with 1 mg/kg/day efonidipine was administered for the period of 4 weeks. After 24 h of ischemic induction surgery, the neurological score was determined, and blood was collected for determination of biochemical parameters. Treatment with efonidipine showed a significant reduction in post-ischemic brain infract volume, brain hemisphere weight difference, neurological score, Na+-K+ ATPase activity, serum CK-MB, and LDH levels in normoglycemic and hyperglycemic MCAO-induced animals. While no significant changes in glucose and lipid levels were observed by treatment, efonidipine significantly decreased the levels of malondialdehyde, acetylcholine esterase, and nitrite levels and increased the levels of antioxidant markers in both normoglycemic and hyperglycemic MCAO animals. TGF-ß and VEGF were found to be down-regulated after treatment with efonidipine in gene expression study. In conclusion, the study data supports the cerebroprotective role of efonidipine in diabetic animals possibly through TGF-ß/SMAD-2 signaling pathway.

Apoptotic Ablation of Platelets Reduces Atherosclerosis in Mice With Diabetes.

OBJECTIVE: People with diabetes are at a significantly higher risk of cardiovascular disease, in part, due to accelerated atherosclerosis. Diabetic subjects have increased number of platelets that are activated, more reactive, and respond suboptimally to antiplatelet therapies. We hypothesized that reducing platelet numbers by inducing their premature apoptotic death would decrease atherosclerosis. Approach and Results: This was achieved by targeting the antiapoptotic protein Bcl-xL (B-cell lymphoma-extra large; which is essential for platelet viability) via distinct genetic and pharmacological approaches. In the former, we transplanted bone marrow from mice carrying the Tyr15 to Cys loss of function allele of Bcl-x (known as Bcl-xPlt20) or wild-type littermate controls into atherosclerotic-prone Ldlr+/- mice made diabetic with streptozotocin and fed a Western diet. Reduced Bcl-xL function in hematopoietic cells significantly decreased platelet numbers, exclusive of other hematologic changes. This led to a significant reduction in atherosclerotic lesion formation in Bcl-xPlt20 bone marrow transplanted Ldlr+/- mice. To assess the potential therapeutic relevance of reducing platelets in atherosclerosis, we next targeted Bcl-xL with a pharmacological strategy. This was achieved by low-dose administration of the BH3 (B-cell lymphoma-2 homology domain 3) mimetic, ABT-737 triweekly, in diabetic Apoe-/- mice for the final 6 weeks of a 12-week study. ABT-737 normalized platelet numbers along with platelet and leukocyte activation to that of nondiabetic controls, significantly reducing atherosclerosis while promoting a more stable plaque phenotype. CONCLUSIONS: These studies suggest that selectively reducing circulating platelets, by targeting Bcl-xL to promote platelet apoptosis, can reduce atherosclerosis and lower cardiovascular disease risk in diabetes. Graphic Abstract: A graphic abstract is available for this article.

Bcl-xL represents a therapeutic target in Philadelphia negative myeloproliferative neoplasms.

Myeloproliferative neoplasms are divided into essential thrombocythemia (ET), polycythemia vera (PV) and primary myelofibrosis (PMF). Although ruxolitinib was proven to be effective in reducing symptoms, patients rarely achieve complete molecular remission. Therefore, it is relevant to identify new therapeutic targets to improve the clinical outcome of patients. Bcl-xL protein, the long isoform encoded by alternative splicing of the Bcl-x gene, acts as an anti-apoptotic regulator. Our study investigated the role of Bcl-xL as a marker of severity of MPN and the possibility to target Bcl-xL in patients. 129 MPN patients and 21 healthy patients were enrolled in the study. We analysed Bcl-xL expression in leucocytes and in enriched CD34+ and CD235a+ cells. Furthermore, ABT-737, a Bcl-xL inhibitor, was tested in HEL cells and in leucocytes from MPN patients. Bcl-xL was found progressively over-expressed in cells from ET, PV and PMF patients, independently by JAK2 mutational status. Moreover, our data indicated that the combination of ABT-737 and ruxolitinib resulted in a significantly higher apoptotic rate than the individual drug. Our study suggests that Bcl-xL plays an important role in MPN independently from JAK2 V617F mutation. Furthermore, data demonstrate that targeting simultaneously JAK2 and Bcl-xL might represent an interesting new approach.

Impact of elevated anti-apoptotic MCL-1 and BCL-2 on the development and treatment of MLL-AF9 AML in mice.

Many acute myeloid leukaemias (AMLs) express high levels of BCL-2 and MCL-1, especially after therapy. To test the impact of these anti-apoptotic proteins on AML development and treatment, we used haemopoietic reconstitution to generate MLL-AF9 AMLs expressing BCL-2 or Mcl-1 transgenes. AMLs with elevated BCL-2 or MCL-1 had a higher proportion of mature myeloid cells but, like conventional MLL-AF9 AMLs, were readily transplantable. Short-term cell lines established from multiple primary AMLs of each genotype were tested in vitro for susceptibility to chemotherapeutics currently used for treating AML (daunorubicin, etoposide, cytarabine); the proteasome inhibitor bortezomib; CDK7/9 inhibitors; and BH3 mimetics, which bind and inhibit pro-survival proteins. The BH3 mimetics tested, alone and in combination with the other drugs, were: ABT-737 which, like its clinical counterpart navitoclax, targets BCL-2, BCL-XL and BCL-W; BCL-2-specific ABT-199 (venetoclax); BCL-XL-specific A-1331852; and S63845, a new MCL-1-specific BH3 mimetic. As single agents, daunorubicin and bortezomib had the greatest efficacy. Elevated MCL-1 or BCL-2 reduced sensitivity to daunorubicin but, surprisingly, not to bortezomib. MCL-1 markedly enhanced resistance to ABT-737 and ABT-199 but not S63845, and BCL-2 increased resistance to S63845 but not to ABT-737 or ABT-199. Notable synergies were achieved by combining BH3 mimetics with daunorubicin: S63845 increased the sensitivity of both MCL-1 and BCL-2 overexpressing MLL-AF9 AMLs, and ABT-737 aided in killing those overexpressing BCL-2. Synergy between daunorubicin and ABT-199 was also apparent in vivo, although not curative. Impressive synergistic responses were achieved for human MLL-fusion AML cell lines treated with daunorubicin plus either ABT-737, ABT-199 or S63845, and with ABT-199 plus S63845, with or without daunorubicin. Our data suggest that AML patients may benefit from combining conventional cytotoxic drugs with BH3 mimetics targeting BCL-2 or MCL-1 or, if tolerated, both these agents.

Targeting the Sphingosine 1-Phosphate Axis Exerts Potent Antitumor Activity in BRAFi-Resistant Melanomas.

BRAF inhibitors (BRAFi) are used to treat patients with melanoma harboring the V600E mutation. However, resistance to BRAFi is inevitable. Here, we identified sphingosine 1-phosphate (S1P) receptors as regulators of BRAFV600E-mutant melanoma cell-autonomous resistance to BRAFi. Moreover, our results reveal a distinct sphingolipid profile, that is, a tendency for increased very long-chain ceramide species, in the plasma of patients with melanoma who achieve a response to BRAFi therapy as compared with patients with progressive disease. Treatment with BRAFi resulted in a strong decrease in S1PR1/3 expression in sensitive but not in resistant cells. Genetic and pharmacologic interventions, that increase ceramide/S1P ratio, downregulated S1PR expression and blocked BRAFi-resistant melanoma cell growth. This effect was associated with a decreased expression of MITF and Bcl-2. Moreover, the BH3 mimetic ABT-737 improved the antitumor activity of approaches targeting S1P-metabolizing enzymes in BRAFi-resistant melanoma cells. Collectively, our findings indicate that targeting the S1P/S1PR axis could provide effective therapeutic options for patients with melanoma who relapse after BRAFi therapy.

Increased bioavailability of efonidipine hydrochloride nanosuspensions by the wet-milling method.

The aim of this study was to improve the oral bioavailability of a practically insoluble drug, efonidipine hydrochloride (EFH), by agglomeration in acid solution/gastric fluid. The EFH nanosuspension was prepared by the wet-milling method with F68 as a dispersing agent, SDS as an auxiliary stabilizer and l-arginine as a pH adjusting agent. The EFH nanosuspension have been prepared in industrial scale-up. The dissolution rate of the EFH nanosuspension was greater than that of bulk EFH. An in vitro intestinal permeability study showed a clear increase in the apparent permeability of different intestinal segments compared with bulk EFH. Also, a pharmacokinetic study showed that the Cmax and AUC0-24h of the nanosuspensions were approximately 1.76-fold and 2.2-fold greater than that of bulk EFH, respectively, and there was no significant difference compared with commercial tablets. It appears that wet-milling offers an effective approach to improve the dissolution rate and oral absorption of this practically insoluble drug.

BCL2 Regulates Differentiation of Intestinal Fibroblasts.

BACKGROUND: Fibrosis in patients with Crohn's disease (CD) results from an imbalance toward excessive fibrous tissue formation driven by fibroblasts. Activation of fibroblasts is linked to the B-cell lymphoma 2 (BCL2) family, which is involved in the induction of apoptosis. We investigated the impact of BCL2 repression on fibrogenesis. METHODS: The model of dextran sodium sulfate (DSS)-induced chronic colitis and the heterotopic transplantation model of fibrosis were used. Following the administration of the BCL2 antagonist (ABT-737, 50 mg/kg/d), collagen layer thickness and hydroxyproline (HYP) content were determined. Fibroblasts were stimulated with the BCL2 antagonist (0.01-100 µM). BCL2, alpha smooth muscle actin (αSMA), and collagen I (COL1A1) were determined by quantitative polymerase chain reaction (qPCR), immunofluorescence microscopy (IF), and western blot (WB). mRNA expression pattern was determined by next-generation sequencing (NGS). RESULTS: Collagen layer thickness was significantly decreased in both DSS-induced chronic colitis and the transplantation model of fibrosis upon BCL2 antagonist administration compared with vehicle. Decreased HYP content confirmed the preventive effects of the BCL2 antagonist on fibrosis. In vitro, a significant increase in PI+/annexin V+ human colonic fibroblasts was determined by fluorescence-activated cell sorting upon treatment with high-dose BCL2 antagonist; at a lower dose, αSMA, COL1A1, and TGF were decreased. NGS, IF, and qPCR revealed decreased expression and nuclear translocation of GATA6 and SOX9, known for reprogramming fibroblasts. CONCLUSION: BCL2 antagonist administration partially prevented fibrogenesis in both fibrosis models. The BCL2 antagonist reduced the expression of TGFß-induced factors involved in differentiation of myofibroblasts, and therefore might represent a potential treatment option against CD-associated fibrosis.

Aspirin restores ABT-737-mediated apoptosis in human renal carcinoma cells.

Aspirin is a novel chemopreventive agent against malignancy. However, outcomes of aspirin monotherapy of renal cell carcinoma (RCC) are inconsistent across studies. ABT-737, an BH3 mimetic inhibitor, is also a promising antitumor drug. Cancer cells including those from RCC, that have high levels of Mcl-1, are refractory to ABT-737-induced apoptosis. We here investigated how aspirin treatment modulates the ABT-737-induced apoptosis. Using the in vitro model of human 786-O cells, we showed that aspirin had sensitized cells to ABT-737 induced apoptosis. Such aspirin-induced changes of ABT-737 resistance was accompanied by a host of biochemical events like protein phosphatase 2A (PP2A) activation, AKT dephosphorylation, Mcl-1/FLICE inhibiting protein (FLIP)/XIAP downregulation, and Bax mitochondrial redistribution. The PP2A inhibitor, okadaic acid, was able to reverse the apirin-induced apoptotic changes. Apart from the aspirin treatment, Mcl-1 silencing also rendered cells vulnerable to ABT-737 induced apoptosis. Since PP2A, Akt, and Mcl-1 play critical roles in RCC malignancy and treatment resistance, our present study showed that aspirin, an alternative adjuvant agent, had recalled ABT-737 sensitivity in the RCC cells through processes involving the PP2A/Akt/Mcl-1 axis.

Synergistic cytotoxicity of a prostate cancer-specific immunotoxin in combination with the BH3 mimetic ABT-737.

In many tumors, including prostate cancer, anti-apoptotic members of the Bcl-2 family are overexpressed and cause cell death resistance, which is a typical hallmark of cancer. Different therapeutic approaches, therefore, aim to restore the death mechanisms for enhanced apoptosis. Our recombinant immunotoxin D7(VL-VH)-PE40 is composed of the scFv D7(VL-VH) against the prostate-specific membrane antigen (PSMA) on the surface of prostate cancer cells and of the cytotoxic domain of the bacterial toxin Pseudomonas Exotoxin A (PE40). Since Pseudomonas Exotoxin A-based immunotoxins are known to preferentially inhibit the expression of the anti-apoptotic protein Mcl-1, the rationale was to test our immunotoxin in combination with the BH3 mimetic ABT-737, which specifically inhibits Bcl-2, Bcl-xl, and Bcl-w for enhanced induction of apoptosis in prostate cancer cells. The immunotoxin showed high and specific binding and cytotoxicity against PSMA expressing prostate cancer cells marked by a direct inhibition of Mcl-1. The combination of the immunotoxin with a subtoxic concentration of ABT-737 caused additive or even synergistic effects, which were based on an enhanced apoptosis induction as detected by poly(ADP-ribose) polymerase (PARP) and Caspase-3 cleavage in Western blot. Our study shows that the combination therapy of immunotoxin plus ABT-737 is a promising approach for the future treatment of advanced prostate cancer to improve therapeutic efficacy and to reduce adverse side effects.

Cooperation of IRAK1/4 inhibitor and ABT-737 in nanoparticles for synergistic therapy of T cell acute lymphoblastic leukemia.

T cell acute lymphoblastic leukemia (T-ALL) is caused by clonal expansion of variant T cell progenitors and is considered as a high risk leukemia. Contemporary single chemotherapy has a limited effect due to dynamic and versatile properties of T-ALL. Here IRAK1/4 inhibitor and ABT-737 were co-encapsulated into polyethylene glycol modified poly (lactic-co-glycolic acid) nanoparticles (IRAK/ABT-NP) to enhance synergistic therapy of T-ALL. The formulation was optimized to achieve high drug loading using Box-Behnken design and response surface methodology. The optimal parameter comprised 2.98% polymer in acetonitrile, a ratio of oil phase to water phase of 1:8.33, and 2.12% emulsifier concentration. High drug loading and uniform spherical shape was achieved. In vitro release study showed sustained release of IRAK1/4 inhibitor for 72 hours as well as sustained release of ABT-737 for more than 120 hours. Uptake efficiency of IRAK/ABT-NP and induced apoptotic T-ALL fraction by IRAK/ABT-NP were much higher than the IRAK1/4 and ABT-737 combined solution. IC50 of IRAK/ABT-NP was two-fold lower than free drug combination in Jurkat cells. Additionally, we conducted in vivo experiments in which IRAK/ABT-NP exhibited greater cytotoxicity toward T-ALL cells, the capacity to significantly restore white blood cell number in peripheral blood, and improved survival time of T-ALL mouse model compared to the IRAK1/4 and ABT-737 combined solution.

HSP90 inhibitor (NVP-AUY922) enhances the anti-cancer effect of BCL-2 inhibitor (ABT-737) in small cell lung cancer expressing BCL-2.

Small cell lung cancer (SCLC) cannot be efficiently controlled using existing chemotherapy and radiotherapy approaches, indicating the need for new therapeutic strategies. Although ABT-737, a B-cell lymphoma-2 (BCL-2) inhibitor, exerts anticancer effects against BCL-2-expressing SCLC, monotherapy with ABT-737 is associated with limited clinical activity because of the development of resistance and toxicity. Here, we examined whether combination therapy with ABT-737 and heat shock protein 90 (HSP90) inhibitor NVP-AUY922 exerted synergistic anticancer effects on SCLC. We found that the combination of ABT-737 and NVP-AUY922 synergistically induced the apoptosis of BCL-2-expressing SCLC cells. NVP-AUY922 downregulated the expression of AKT and ERK, which activate MCL-1 to induce resistance against ABT-737. The synergistic effect was also partly due to blocking NF-κB activation, which induces anti-apoptosis protein expressions. However, interestingly, targeting BCL-2 and MCL-1 or BCL2 and NF-κB did not induce the cytotoxicity. In conclusion, our study showed that combination of BCL2 inhibitor with HSP90 inhibitor increased activity in in vitro and in vivo study in only BCL-2 expressing SCLC compared to either single BCL2 inhibitor or HSP inhibitor. The enhanced activity might be led by blocking several apoptotic pathways simultaneously rather than a specific pathway.

ABT-737 potentiates cisplatin-induced apoptosis in human osteosarcoma cells via the mitochondrial apoptotic pathway.

ABT-737 is a BH-3 mimetic that inhibits Bcl-2 and induces apoptosis of cancer cells, which has potential for anticancer therapies. Studies have shown that Bcl-2 expression in human osteosarcoma (OS) cells plays a significant role in tumor progression; however, its effects on OS cell apoptosis are still unknown. Therefore, we examined whether ABT-737 was effective in eliminating human U-2OS cells, either alone or in combination with the chemotherapy drug cisplatin [cis-diamminedichloroplatinum (II); DDP]. Furthermore, we studied the molecular mechanisms of ABT-737 in combination with DDP to induce apoptosis. To analyze the role of ABT-737 and/or DDP on osteosarcoma progression, CCK-8 viability assay, flow cytometry, Hoechst 33258 staining, and western blots were performed. Combined use of ABT-737 and DDP synergistically suppressed cell viability and induced apoptosis in human U-2OS cells when compared with either compound treated alone at low doses. We found that the combination of ABT-737 and DDP upregulated the expression of the pro-apoptotic protein Bax and downregulated the expression of the pro-survival protein Bcl-2, resulting in a change in the Bax/Bcl-2 ratio, release of cytochrome c, and activation of the mitochondrial apoptotic pathway, which resulted in caspase-9 and caspase-3 activation and PARP cleavage. Our results demonstrated that ABT-737 alone has a nominal influence on human U-2OS cells when treated within the clinically administered range, but when combined with DDP, it can inhibit the proliferation of human U-2OS cells by inducing apoptosis via the mitochondrial apoptotic pathway.

Protein kinase A activation by the anti-cancer drugs ABT-737 and thymoquinone is caspase-3-dependent and correlates with platelet inhibition and apoptosis.

Chemotherapy-induced thrombocytopenia is a common bleeding risk in cancer patients and limits chemotherapy dose and frequency. Recent data from mouse and human platelets revealed that activation of protein kinase A/G (PKA/PKG) not only inhibited thrombin/convulxin-induced platelet activation but also prevented the platelet pro-coagulant state. Here we investigated whether or not PKA/PKG activation could attenuate caspase-dependent apoptosis induced by the anti-cancer drugs ABT-737 (the precursor of navitoclax) and thymoquinone (TQ), thereby potentially limiting chemotherapy-induced thrombocytopenia. This is particularly relevant as activation of cyclic nucleotide signalling in combination chemotherapy is an emerging strategy in cancer treatment. However, PKA/PKG-activation, as monitored by phosphorylation of Vasodilator-stimulated phosphoprotein (VASP), did not block caspase-3-dependent platelet apoptosis induced by the compounds. In contrast, both substances induced PKA activation themselves and PKA activation correlated with platelet inhibition and apoptosis. Surprisingly, ABT-737- and TQ-induced VASP-phosphorylation was independent of cAMP levels and neither cyclases nor phosphatases were affected by the drugs. In contrast, however, ABT-737- and TQ-induced PKA activation was blocked by caspase-3 inhibitors. In conclusion, we show that ABT-737 and TQ activate PKA in a caspase-3-dependent manner, which correlates with platelet inhibition and apoptosis and therefore potentially contributes to the bleeding risk in chemotherapy patients.

B-cell lymphoma 2 inhibitor ABT-737 induces Beclin1- and reactive oxygen species-dependent autophagy in Adriamycin-resistant human hepatocellular carcinoma cells.

ABT-737, a B-cell lymphoma 2 homology 3 mimetic, not only induces cell apoptosis by inhibiting the interaction of B-cell lymphoma 2 and Bax but also induces cell autophagy by interrupting the interaction of B-cell lymphoma 2 and Beclin1. Several recent studies have reported that ABT-737 has antitumor efficacy in diverse cancers. However, another study showed that hepatocellular carcinoma cells with high B-cell lymphoma 2 expression were resistant to ABT-737 compared to hepatocellular carcinoma cells with low B-cell lymphoma 2 expression. It was also found that ABT-737-induced autophagy is crucial for drug resistance. Here, we observed that of B-cell lymphoma 2 expression in Adriamycin-resistant human hepatocellular carcinoma HepG2/ADM cells is higher than that in human hepatocellular carcinoma HepG2 cells. Therefore, we further confirmed the mechanism and effect of autophagy induced by ABT-737 on apoptosis in HepG2/ADM cells with high B-cell lymphoma 2 expression. Our results showed that ABT-737 induced apoptosis and autophagy in time- and dose-dependent manner in HepG2/ADM cells, and this ABT-737-induced autophagy was Beclin1-dependent. In addition, we demonstrated that ABT-737 induced reactive oxygen species-mediated autophagy, and the reactive oxygen species-inhibitor N-acetyl-l-cysteine suppressed the reactive oxygen species-induced autophagy and ABT-737-induced increase in HepG2/ADM cell apoptosis. Furthermore, autophagy inhibitors increased HepG2/ADM cell apoptosis. In conclusion, our study further confirms that Beclin1- and reactive oxygen species-dependent autophagy induced by ABT-737 also plays a protective function in HepG2/ADM cells, which show B-cell lymphoma 2 expression higher than that in HepG2 cells.

Right inferior frontal cortex activity correlates with tolcapone responsivity in problem and pathological gamblers.

Failures of self-regulation in problem and pathological gambling (PPG) are thought to emerge from failures of top-down control, reflected neurophysiologically in a reduced capacity of prefrontal cortex to influence activity within subcortical structures. In patients with addictions, these impairments have been argued to alter evaluation of reward within dopaminergic neuromodulatory systems. Previously we demonstrated that augmenting dopamine tone in frontal cortex via use of tolcapone, an inhibitor of the dopamine-degrading enzyme catechol-O-methyltransferase (COMT), reduced delay discounting, a measure of impulsivity, in healthy subjects. To evaluate this potentially translational approach to augmenting prefrontal inhibitory control, here we hypothesized that increasing cortical dopamine tone would reduce delay discounting in PPG subjects in proportion to its ability to augment top-down control. To causally test this hypothesis, we administered the COMT inhibitor tolcapone in a randomized, double-blind, placebo-controlled, within-subject study of 17 PPG subjects who performed a delay discounting task while functional MRI images were obtained. In this subject population, we found that greater BOLD activity during the placebo condition within the right inferior frontal cortex (RIFC), a region thought to be important for inhibitory control, correlated with greater declines in impulsivity on tolcapone versus placebo. Intriguingly, connectivity between RIFC and the right striatum, and not the level of activity within RIFC itself, increased on tolcapone versus placebo. Together, these findings support the hypothesis that tolcapone-mediated increases in top-down control may reduce impulsivity in PPG subjects, a finding with potential translational relevance for gambling disorders, and for behavioral addictions in general.

Cyclin-dependent kinase 2 inhibitor SU9516 increases sensitivity of colorectal carcinoma cells Caco-2 but not HT29 to BH3 mimetic ABT-737.

Colorectal carcinoma (CRC) that represents one of the major causes for cancer-related death in humans is often associated with over-expression of anti-apoptotic proteins of Bcl-2 family. The aim of presented study was to determine the effect of ABT-737 inhibitor of anti-apoptotic proteins Bcl-2, Bcl-XL and Bcl-w as well as cyclin-dependent kinase 2 (CDK2) inhibitor SU9516 alone and in combination with ABT-737 on survival of colorectal cell lines HT29 and Caco-2. We have shown that both Caco-2 and HT29 cells that are relatively resistant to ABT-737 are also partially sensitive to SU9516, which increased sensitivity of Caco-2 but not HT29 cells to ABT-737. Increased sensitivity of Caco-2 cells to ABT-737 after addition of SU9516 correlated well with SU9516-induced decrease of Mcl-1 expression while we have not observed downregulation of Mcl-1 after the treatment of HT29 cells with SU9516. Instead of this, we have shown that treatment of HT29 cells with SU9516 is associated with decreased expression of tumour suppressor protein p53. Our findings provide a rationale for clinical use of Bcl-2 family inhibitors in combination with CDK2 inhibitors for treatment of Mcl-1-dependent colorectal tumours associated with expression of Bcl-2, Bcl-XL and Bcl-w proteins. In addition, we have shown potential of CDK2 inhibitors for treatment of tumours expressing R273H mutant p53.

Epothilone B induces apoptosis and enhances apoptotic effects of ABT-737 on human cancer cells via PI3K/AKT/mTOR pathway.

PURPOSE: Epothilone B and its derivatives are tested in multiple clinical trials. Epothilone B induces neurotoxic effect in clinical trials; however, low-dose epothilone B regimen can promote neuroprotection and neurogenesis. Thus, the study of new combination chemotherapy regimen incorporating low-dose epothilone B with other chemotherapeutic agents might help to develop epothilone B-based approaches to cancer treatment and avoid the neurotoxicity of epothilone B. METHODS: Cell proliferation was assessed by SRB cell viability assay. Apoptosis was analyzed by propidium iodide (PI) staining. Mitochondrial membrane depolarization was evaluated using JC-1 staining. The expression of proteins was detected by western blotting. RESULTS: In this study, we demonstrated that the combination of ABT-737 and low-dose epothilone B showed synergistic anti-proliferation effects on human cancer cells. In addition, epothilone B + ABT-737 synergy was through mitochondria-mediated apoptosis pathway. Furthermore, combination treatment markedly induced the activation of caspase-3 and the cleavage of PARP. The activation of PI3K/Akt/mTOR pathway is associated with resistance to epothilone B. Our data showed that epothilone B plus ABT-737 resulted in a blockade of the PI3K/AKT/mTOR signaling pathway. CONCLUSIONS: These data indicate that ABT-737 may be a pertinent sensitizer to epothilone B, and the strategy of combining epothilone B with ABT-737 appears to be an attractive option for overcoming the resistance and neurotoxicity of epothilone B.