Proteasome inhibitors and Smac mimetics cooperate to induce cell death in diffuse large B-cell lymphoma by stabilizing NOXA and triggering mitochondrial apoptosis

Copy number gains and increased expression levels of cellular Inhibitor of Apoptosis protein (cIAP)1 and cIAP2 have been identified in primary diffuse large B-cell lymphoma (DLBCL) tissues. Second mitochondria-derived activator of caspases (Smac) mimetics were designed to antagonize IAP proteins. However, since their effect as single agents is limited, combination treatment represents a strategy for their clinical development. Therefore, we investigated the Smac mimetic BV6 in combination with proteasome inhibitors and analyzed the molecular mechanisms of action. We discovered that BV6 treatment sensitizes DLBCL cells to proteasome inhibition. We show a synergistic decrease in cell viability and induction of apoptosis by BV6/Carfilzomib (CFZ) treatment, which was confirmed by calculation of combination index (CI) and Bliss score. BV6 and CFZ acted together to trigger activation of BAX and BAK, which facilitated cell death, as knockdown of BAX and BAK significantly reduced BV6/CFZ-mediated cell death. Activation of BAX and BAK was accompanied by loss of mitochondrial membrane potential (MMP) and activation of caspases. Pretreatment with the caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (zVAD.fmk) rescued BV6/CFZ-induced cell death, confirming caspase dependency. Treatment with CFZ alone or in combination with BV6 caused accumulation of NOXA, which was required for cell death, as gene silencing by siRNA or Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9-mediated NOXA inactivation inhibited BV6/CFZ-induced cell death. Together, these experiments indicate that BV6 and CFZ cooperatively induce apoptotic cell death via the mitochondrial pathway. These findings emphasize the role of Smac mimetics for sensitizing DLBCL cells to proteasome inhibition with important implications for further (pre)clinical studies.     

Incretin mimetics as a novel therapeutic option for hepatic steatosis.

BACKGROUND: Fat accumulation in the liver or non-alcoholic fatty liver disease (NAFLD) is regarded as a key pathogenic factor and component of the metabolic syndrome. It was reported that administration of the incretin mimetic exenatide reversed hepatic steatosis in an obese mouse model. We had the opportunity to study the effect of additional exenatide administration on liver fat content in a patient with type 2 diabetes. CASE REPORT: A 59-year-old male with poorly controlled type 2 diabetes was treated with exenatide in addition to metformin monotherapy. Following 44 weeks of exenatide therapy, mean the liver fat measured by liver spectroscopy declined from 15.8% to 4.3%. This dramatic decrease in liver fat was accompanied by significant beneficial changes in several cardiovascular disease risk factors and improvement of all liver enzymes, in particular alanine aminotransferase, the most important marker of liver steatosis. CONCLUSION: This case report suggests that the incretin mimetic exenatide decreases hepatic fat accumulation and may play a role in the future treatment of NAFLD, and the associated insulin resistance and cardiovascular risk factors in an ever-growing high-risk population.     

Immunohistochemical analysis of Smac/DIABLO expression in human carcinomas and sarcomas

Second mitochondria-derived activator of caspases (Smac/DIABLO) is released from mitochondria into the cytosol during apoptosis, promoting caspase activation by neutralizing the inhibition of inhibitor of apoptosis proteins (IAPs) on caspases. Alteration of apoptosis is essential for cancer development, and cancer cell death by radiation and chemotherapy is largely dependent upon apoptosis. In this study, archival tissues of 100 carcinomas and 50 sarcomas from various origins were analyzed by immunohistochemistry for the expression of Smac/DIABLO. Smac/DIABLO immunoreactivity was seen in 62 of 100 (62%) carcinomas, including 42 of 60 stomach carcinomas, 7 of 10 colorectal carcinomas, 4 of 10 lung carcinomas, 7 of 10 ovarian carcinomas, and 2 of 10 prostate carcinomas. Smac/DIABLO is expressed in 11 of 50 (22%) sarcomas, including 2 of 8 malignant schwannomas, 5 of 11 rhabdomyosarcomas, 2 of 7 malignant fibrous histiocytomas, 1 of 6 leiomyosarcomas, 0 of 8 angiosarcomas, 0 of 8 liposarcomas, and 1 of 2 Ewing's sarcomas. These data demonstrated that Smac/DIABLO expression levels vary depending on the individual cancer types. Furthermore, the present study showed that many human cancers do not express Smac/DIABLO, and suggest that lack of Smac/DIABLO expression in the cancer cells may inhibit apoptosis, thereby promoting their survival.     

Smac合成肽靶向下调凋亡抑制蛋白XIAP提高胰腺癌细胞化疗敏感性

目的:探讨包含目的蛋白功能结构域的Smac合成肽能否增强胰腺癌细胞的化疗药物敏感性及其作用机制.方法:化学合成SmacN7细胞可穿透融合多肽,共沉淀实验观察SmacN7细胞穿透肽与胰腺癌Panc-1细胞内的XIAP的相互作用,应用流式细胞术检测SmacN7细胞穿透肽与顺铂、5-FU联用对Panc-1细胞凋亡的影响,MTT法检测SmacN7细胞穿透肽应用前后Panc-1细胞的化疗药物敏感性.结果:SmacN7融合多肽能与内源性XIAP结合,明显下调Panc-1细胞XIAP表达水平,显著增强顺铂或5-FU诱导的Panc-1细胞凋亡,使其对顺铂、5-FU的药物半数抑制浓度(IC50)分别降低1.98倍、2.62倍.结论:应用包含目的蛋白功能结构域的Smac合成肽能靶向下调胰腺癌Panc-1细胞XIAP表达,显著提高其化疗敏感性,为胰腺癌的生物治疗协同化疗提供了新思路.     

Inhibitor of apoptosis proteins as targets for anticancer therapy

Cell death by apoptosis plays a critical role in regulating the subtle balance between cell death and proliferation to maintain tissue homeostasis. Accordingly, tipping the balance in either direction may cause human disease. Too little cell death may promote tumor formation and progression. In addition, killing of cancer cells by current therapies is largely due to induction of apoptosis in tumor cells. Since a hallmark of human cancers is their resistance to apoptosis, there is a demand to develop novel strategies that restore the apoptotic machinery in order to overcome cancer resistance. Inhibitor of apoptosis proteins (IAPs) block apoptosis at the core of the apoptotic machinery by inhibiting caspases. Elevated levels of IAPs are found in many human cancers and have been associated with poor prognosis. Recent insights into the role of IAPs have provided the basis for various exciting developments that aim to modulate the expression or function of IAPs in human cancers. Targeting IAPs (e.g., by antisense approaches or small-molecule inhibitors) presents a promising novel approach to either directly trigger apoptosis or to potentiate the efficacy of cytotoxic therapies in cancer cells. Thus, inhibition of IAPs such as X chromosome-linked IAP may prove to be a successful strategy to overcome apoptosis resistance of human cancers that deserves further exploitation.     

Differential role of RIP1 in Smac mimetic-mediated chemosensitization of neuroblastoma cells

We explored the potential of Smac mimetics, which antagonize Inhibitor of Apoptosis (IAP) proteins, for chemosensitization of neuroblastoma (NB). Here, we report that Smac mimetics, e.g. BV6, prime NB cells for chemotherapeutics including the topoisomerase II inhibitor doxorubicin (DOX) and vinca alkaloids such as Vincristine (VCR), Vinblastine (VBL) and Vinorelbine (VNR). Additionally, BV6 acts in concert with DOX or VCR to suppress long-term clonogenic growth. While BV6 causes rapid downregulation of cellular IAP (cIAP)1 protein and nuclear factor-kappaB (NF-κB) activation, DOX/BV6- or VCR/BV6-induced apoptosis occurs independently of NF-κB or TNFα signaling, since overexpression of dominant-negative IκBα superrepressor or the Tumor Necrosis Factor (TNF)α-blocking antibody Enbrel fail to block cell death. Mechanistic studies reveal that Receptor-interacting protein (RIP)1 is required for DOX/BV6-, but not for VCR/BV6-induced apoptosis, since transient or stable knockdown of RIP1 or the pharmacological RIP1 inhibitor necrostatin-1 significantly reduce apoptosis. By comparison, VCR/BV6-mediated apoptosis critically depends on the mitochondrial pathway. VCR/BV6 cotreatment causes phosphorylation of BCL-2 during mitotic arrest, enhanced activation of BAX and BAK and loss of mitochondrial membrane potential (MMP). Additionally, overexpression of BCL-2 profoundly suppresses VCR/BV6-induced apoptosis. Thus, BV6 sensitizes NB cells to chemotherapy-induced apoptosis via distinct initial signaling mechanisms depending on the chemotherapeutic drug. These findings provide novel mechanistic insights into Smac mimetic-mediated chemosensitization of NB.     

Calorie restriction and sirtuins revisited

Calorie or dietary restriction (CR) has attracted attention because it is the oldest and most robust way to extend rodent life span. The idea that the nutrient sensors, termed sirtuins, might mediate effects of CR was proposed 13 years ago and has been challenged in the intervening years. This review addresses these challenges and draws from a great body of new data in the sirtuin field that shows a systematic redirection of mammalian physiology in response to diet by sirtuins. The prospects for drugs that can deliver at least a subset of the benefits of CR seems very real.     

The role of NF-κB and Smac/DIABLO proteins in the treatment response and survival of acute myeloid leukemia patients

IntroductionThe misbalance between a family of inhibitor of apoptosis proteins (IAP), regulated by the nuclear factor kappa B (NF-κB) and their natural antagonist second mitochondrial-derived activator of caspases/direct IAP binding protein with low pI (Smac/DIABLO) are important to biology of acute myeloid leukemia (AML).Material and methodsThe aim of the study was to assess NF-κB and Smac/DIABLO proteins expression in blasts of 109 newly diagnosed AML patients using the multicolor flow cytometry and evaluate their influence on AML patients outcome.ResultsExpression of NF-κB and of Smac/DIABLO proteins were found in 95% and 98% of the patients, respectively. A negative correlation between Smac/DIABLO and NF-κB was observed. Age < 60 years old as well as higher Smac/DIABLO expression were associated with a higher probability of complete response achievement in the multivariate analysis. Longer overall survival (OS) in the univariate and multivariate analyses was influenced by age < 60 years old, a favorable or intermediate-risk karyotype and high Smac/DIABLO expression. Additionally, in the survival analysis of the subgroups, the patients aged < 60 years old, with high Smac/DIABLO expression, lower NF-κB expression and < 50% of bone marrow blasts who were treated with standard treatment had better OS.ConclusionsLower NF-κB and higher Smac/DIABLO expression may influence AML patients outcome.     

Caloric Restriction and Caloric Restriction Mimetics: Current Status and Promise for the Future

Dietary caloric restriction is the most reproducible means of extending longevity and maintaining health and vitality. It has been shown to be relevant to a wide rage of species, including primates. Examination of key markers of the calorically restricted phenotype, such as plasma insulin, dehydroepiandrosterone sulfate, and body temperature, suggest that they may predict longevity in humans as well. However, most people would be unwilling or unable to adopt the 30% to 40% reduction in food intake necessary to achieve optimal health and longevity benefits. For this reason, a number of laboratories are pursuing caloric restriction mimetics: ways to achieve the benefits of restriction without eating less. This approach will undoubtedly remain a major focus of biogerontolgy for the foreseeable future.