Potent antitumor agent proteasome inhibitors: a novel trigger for Bcl2 phosphorylation to induce apoptosis.

The sustained cytotoxicity conferred by proteasome inhibitors against a broad spectrum of human cancer cells is mediated by a delicate mechanism of programmed cell death. Similar to microtubule disarraying agents, the cell death induced by these potent antitumor agents precedes blocking in cell cycle transition at G2-M phase. The microtubule damaging antineoplastic drugs can kill tumor cells by inducing phosphorylation of antiapoptotic proteins such as Bcl2, Bcl-xL or MCL-1. The simultaneous apoptosis with Bcl2 phosphorylation was evident in cancer cells challenged with the proteasome inhibitor, MG132. Our studies suggest that the proteasome inhibitor MG132 induced tumor cell killing is mediated through Bcl2 phosphorylation.     

Role of AIF in caspase-dependent and caspase-independent cell death

The major challenge in treating cancer is that many tumor cells carry mutations in key apoptotic genes such as p53, Bcl family proteins or those affecting caspase signaling. Such defects render treatment with traditional chemotherapeutic agents ineffective. Many studies have demonstrated the importance of caspase-independent cell death pathways in injury, degenerative diseases and tumor tissue. It is now recognized that in addition to their critical role in the production of cellular energy, mitochondria are also the source of key proapoptotic molecules involved in caspase activation. More recently, it has been discovered that in response to apoptotic stimuli, mitochondria can also release caspase-independent cell death effectors such as AIF and Endonuclease G. In this review, we examine the role of Bcl family proteins and poly(ADP-ribose) polymerase-1 signaling in the regulation of these apoptotic pathways and address the ongoing controversies in this field. Continued study of the mechanisms of apoptosis including caspase-independent death processes are likely to reveal novel therapeutic targets for the treatment of diverse human pathologies including cancer, neurodegenerative diseases and acute injuries such as stroke or myocardial infarction.     

E1A-induced apoptosis does not prevent replication of adenoviruses with deletion of E1b in majority of infected cancer cells

Apoptotic pathways are initiated as a cellular defense mechanism to eliminate adenovirus-infected cells. We have investigated how E1A-induced apoptosis interferes with viral replication in cancer cells. We found that E1B19K alone can efficiently suppress E1A-induced apoptosis in cancer cells. Viruses deleted for both E1B19K and E1B55K resulted in cellular DNA degradation. However, less than 20% of human lung cancer cells infected with a virus deleted for both E1B19K and E1B55 K had evidence of chromatin condensation and multiple-micronuclei formation (apoptotic hallmarks); these cells could not produce infectious viral particles. The majority of cancer cells infected with viruses deleted for the entire E1b gene did not undergo extended apoptosis and produced abundant viral progeny. Thus, only a fraction of cancer cells underwent apoptosis and did not allow E1b-deleted viruses to replicate, while the majority of cancer cells were resistant to E1A-induced apoptosis and could support virus-selective replication. The results of this study imply that, in addition to inhibiting E1A-induced apoptosis, E1B proteins may contribute other important roles in the viral life cycle. Our results also suggest that combining virus-induced apoptosis and selective viral replication into one vector will be a novel approach to destroy cancer cells.     

Predictive value of p53, Bcl2 and bax in the radiotherapy of head and neck cancer

Radiation is known to induce DNA damage resulting in the onset of apoptosis. The apoptosis is modulated by p53, Bcl2 and Bax proteins. High level of wild type p53 is required for radiation induced apoptosis. The p53 status, therefore, may be a crucial determinant of radiosensitivity of tumor cells. Overexpression of Bcl2, however, inhibits apoptosis via hetero- and homodimeric interaction. Bax might function as a cell death effector molecule that is neutralized by Bcl2. The aim of the present study is to investigate the correlation between p53, Bcl2, Bax and c-myc levels and the clinical response of head and neck cancer patients to radiation. The base line and 30 GY gamma radiation induced values of p53, Bcl2, Bax and c-myc were estimated by Western blot in 40 biopsies of head and neck cancers. We found that the radiosensitivity of head and neck cancer patients depends on the ratio of p53, Bcl2 and Bax protein levels. High Bcl2 levels resulted in radioresistance of cancer patients. Overexpression of Bax and c-myc may ensure the radiosensitivity of head and neck cancer patients. Our studies indicate that prediction of radiation sensitivity of tumors could be based on the simultaneous evaluation of p53, Bax and Bcl2 levels.     

Cells with defective p53-p21-pRb pathway are susceptible to apoptosis induced by p84N5 via caspase-6

Adeno-associated virus (AAV) infection triggers a DNA damage response in the cell. This response is not induced by viral proteins but by virtue of the structure of AAV ssDNA being recognized by the cell as damaged DNA. The consequence of this is the killing of cells lacking p53 activity. We have observed that cells that lack p21 or pRb activity are also sensitive to AAV-induced cell death. We report that cells respond to AAV infection by activating two DNA damage signaling cascades. The first activates the p84N5 protein, which in turn activates caspase-6, leading to cell death. The second cascade activates the p53-21-pRb pathway, which inhibits activation of the p84N5 protein and thus prevents cell death. The result of the antagonistic interaction between these two pathways is that cells that do not exhibit functional p53-p21-pRb signaling undergo apoptosis as a consequence of AAV infection. Cells with a functional p53-21-pRb pathway are refractory to AAV-induced cell death. These results show that p53, although a proapoptotic protein, together with pRb and p21 proteins, is a member of an antiapoptotic cellular mechanism. As such, these experiments reveal features that may be exploited to specifically kill cells that lack the p53-p21-pRb pathway, such as cancer cells. The use of AAV to expose these subtle characteristics of intracellular signaling further highlights the advantages of using viruses as precision tools with which to address questions of cell biology.     

特异性环氧合酶-2抑制剂SC236诱导胃癌细胞株凋亡的实验研究

目的：探讨环氧合酶-2（COX-2）特异性抑制剂SC236诱导胃癌细胞株SGC7901凋亡的机制。方法：SC236对人胃癌来源的SGC7901细胞进行凋亡诱导。以吖啶橙染色后荧光显微镜下观察凋亡细胞形态并计算凋亡指数。Western blot法检测凋亡调节蛋白Bcl-2、Bak、Bax与CED-9的表达情况。流式细胞仪检测细胞凋亡率。结果：SGC7901细胞经SC236作用后,细胞中凋亡促进因子Bak的表达水平明显增高、凋亡抑制因子Bcl-2的表达水平显著下降、而凋亡促进因子Bax和凋亡抑制因子CED-9表达水平无明显变化。细胞凋亡指数或凋亡率则随SC236浓度的增加和作用的时间延长而升高。结论：COX-2特异抑制剂SC236可通过上调凋亡促进因子Bak表达和下调凋亡抑制因子Bcl-2表达而诱导胃癌细胞株SGC7901的凋亡。     

Oncolytic viruses in radiation oncology

Oncolytic viruses are investigational cancer treatments. They are currently being assessed as single agents or in combination with standard therapies such as external beam radiotherapy - a DNA damaging agent that is a standard of care for many tumour types. Preclinical data indicate that combinations of oncolytic viruses and radiation therapy are promising, showing additional or synergistic antitumour effects in in vitro and in vivo studies. This interaction has the potential to be multifaceted: viruses may act as radiosensitizing agents, but radiation may also enhance viral oncolysis by increasing viral uptake, replication, gene expression and cell death (apoptosis, autophagy or necrosis) in irradiated cells. Phase I and II clinical trials investigating combinations of viruses and radiation therapy have been completed, paving the way for ongoing phase III studies. The aim of this review is to focus on the therapeutic potential of these combinations and to highlight their mechanistic bases, with particular emphasis on the role of the DNA damage response.     

Signal-induced site specific phosphorylation targets Bcl2 to the proteasome pathway

The oncogene derived protein Bcl2 and its family members such as Bcl-xL or Mcl-1 can confer negative control in the pathway of cellular suicide machinery. The reversible phosphorylation of the components in the apoptotic-signaling pathway is likely to be an important regulatory mechanism to control the fate of a cell. Previous reports by others and us demonstrate that phosphorylation of anti-apoptotic proteins such as Bcl2, Bcl-xL or Mcl-1 can regulate their function depending on the apoptotic trigger or cell type. Also, evidence is now accumulating that the ubiquitin proteasome pathway can play an important role in apoptosis. In order to understand whether any cross-talk exists between proteasome and Bcl2 phosphorylation pathways, studies were undertaken employing cell permeable proteasome inhibitors. When proteasomes were inactivated, enhanced accumulation of slower mobility forms of Bcl2 was clearly evident. Due to substitution of the major phosphorylation sites Ser 70, 87 to Ala, no such effect was observed. It is known that in contrary to phospho Bcl2, native Bcl2 (non-phosphoform) is unable to associate with cis-trans peptidyl prolyl isomerase Pin1-a key factor to regulate the fate of phosphoforms of Bcl2 and apoptosis. Thus the enhanced resistance to cell death exhibited by phosphorylation defective mutant Bcl2 might be attributed to its inability to associate with Pin1.     

Major apoptotic mechanisms and genes involved in apoptosis

As much as the cellular viability is important for the living organisms, the elimination of unnecessary or damaged cells has the opposite necessity for the maintenance of homeostasis in tissues, organs and the whole organism. Apoptosis, a type of cell death mechanism, is controlled by the interactions between several molecules and responsible for the elimination of unwanted cells from the body. Apoptosis can be triggered by intrinsically or extrinsically through death signals from the outside of the cell. Any abnormality in apoptosis process can cause various types of diseases from cancer to auto-immune diseases. Different gene families such as caspases, inhibitor of apoptosis proteins, B cell lymphoma (Bcl)-2 family of genes, tumor necrosis factor (TNF) receptor gene superfamily, or p53 gene are involved and/or collaborate in the process of apoptosis. In this review, we discuss the basic features of apoptosis and have focused on the gene families playing critical roles, activation/inactivation mechanisms, upstream/downstream effectors, and signaling pathways in apoptosis on the basis of cancer studies. In addition, novel apoptotic players such as miRNAs and sphingolipid family members in various kind of cancer are discussed.     

Impact of viral oncogenesis on responses to anti-cancer drugs and irradiation

The view that chemical or physical oncogenesis and tumor therapy resistance represent different parts of common cellular alterations gained considerable attractiveness, because it explains the inherent unreponsiveness of many tumors. Viruses are potent oncogenes and are causally linked to approximately one-fifth of all human malignancies. Whether viral oncogenesis exerts comparable effects was less clear. Recent progress in experimental research provided ample evidence that viruses affect response of tumor cells toward anti-cancer drugs and irradiation. Resistance to cytostatic drugs and radiation develops by alterations at the drug-target sites (i.e., DNA or specific target proteins), upstream (i.e., detoxification mechanisms), or downstream of them (i.e., programmed cell death). Viruses interfere with specific cellular genes at these three levels. Viral proteins induce the expression and expression of drug resistance genes, that is, MDR1, DHFR, or CAD. Viral interactions with the tumor suppressor genes (p53, pRB) abrogate cell cycle arrests and disturb DNA repair of drug- and radiation-induced DNA lesions. The readiness to commit cellular suicide (apoptosis) is also affected by viral genes. The connection between viral oncogenesis and the response of tumor cells to treatment adds a new dimension to tumor biology and may have important consequences for oncological treatment modalities in the future.     

Getting back on track, or what to do when apoptosis is de-railed: recoupling oncogenes to the apoptotic machinery

Programmed cell death or apoptosis is of fundamental importance to cancer as it both limits tumorigenesis and is also triggered by many cancer chemotherapeutics. Many oncogenes that deregulate the cell cycle also trigger apoptosis, so eliminating cells that are proliferating inappropriately. To acquire a complete neoplastic phenotype, cancer cells often acquire mutations that compromise the apoptotic process, allowing these cells to escape both normal growth constraints but to also become resistant to many anti-cancer drugs and leading to the emergence of drug-resistant malignancies. Thus discovering how oncogenes are coupled to apoptosis and how these links are compromised in cancer is central to both understanding cancer progression and developing new therapies to counter chemo-resistant cancers. This review will consider how oncogenes activate apoptosis and how this response is subverted in cancer cells with a focus on the proposed therapeutic strategies that exploit these changes.     

Inhibition of BCL11B expression leads to apoptosis of malignant but not normal mature T cells

The B-cell chronic lymphocytic leukemia (CLL)/lymphoma 11B gene (BCL11B) encodes a Krüppel-like zinc-finger protein, which plays a crucial role in thymopoiesis and has been associated with hematopoietic malignancies. It was hypothesized that BCL11B may act as a tumor-suppressor gene, but its precise function has not yet been elucidated. Here, we demonstrate that the survival of human T-cell leukemia and lymphoma cell lines is critically dependent on Bcl11b. Suppression of Bcl11b by RNA interference selectively induced apoptosis in transformed T cells whereas normal mature T cells remained unaffected. The apoptosis was effected by simultaneous activation of death receptor-mediated and intrinsic apoptotic pathways, most likely as a result of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) upregulation and suppression of the Bcl-xL antiapoptotic protein. Our data indicate an antiapoptotic function of Bcl11b. The resistance of normal mature T lymphocytes to Bcl11b suppression-induced apoptosis and restricted expression pattern make it an attractive therapeutic target in T-cell malignancies.     

Functional screening identifies a microRNA,miR‐491 that induces apoptosis by targeting Bcl‐XL in colorectal cancer cells

MicroRNAs (miRNAs) are a class of small noncoding RNAs that negatively regulate expression of target mRNA. They are involved in many biological processes, including cell proliferation, apoptosis and differentiation, and considered as new therapeutic targets for cancers. In our study, we performed a gain‐of‐function screen using 319 miRNAs to identify those affecting cell proliferation and death in human colorectal cancer cells (DLD‐1). We discovered a number of miRNAs that increased or decreased cell viability in DLD‐1. They included known oncogenic miRNAs such as miR‐372 and miR‐373, and tumor suppressive miRNAs such as miR‐124a, but also some for which this information was novel. Among them, miR‐491 markedly decreased cell viability by inducing apoptosis. We demonstrated that Bcl‐X_L was a direct target of miR‐491, and its silencing contributed to miR‐491‐induced apoptosis. Moreover, treatment of miR‐491 suppressed in vivo tumor growth of DLD‐1 in nude mice. Our study provides a new regulation of Bcl‐X_L by miR‐491 in colorectal cancer cells, and suggests a therapeutic potential of miRNAs for treating colorectal cancer by targeting Bcl‐X_L.     

Viruses - seeking and destroying the tumor program

DNA viruses have enormous utility in cancer research, both as tools for tumor target discovery as well as agents for lytic cancer therapies. This is because there is a profound functional overlap between the DNA viral and tumor cell programs. DNA viruses encode proteins that elicit growth deregulation in infected cells similar to that engendered by mutations in tumor cells. Evolution has refined viral proteins to target the critical cellular hubs that regulate growth. Thus, viral proteins are discriminating biochemical probes that can be used to identify and characterize novel tumor targets. Moreover, the overlap between the DNA viral and tumor programs can also be exploited for the development of lytic cancer therapies. Discovering whether tumor cells selectively complement the replication of viral mutants can reveal novel oncolytic viral therapies, as well as unexpected tumor properties. For example, altered RNA export was recently uncovered as a novel tumor cell property that underlies ONYX-015 replication, a promising oncolytic adenoviral therapy. A perspective is provided on how adenovirus could be systematically exploited to map the requisite role, or indeed the redundancy, of cellular pathways that act in an integrated program to elicit pathological replication. This knowledge has important applications for the rational design of the next generation of oncolytic viruses, as well as the discovery of efficacious combination cancer therapies.     

Functional similarity between adenovirus e1b 19k gene and bcl2 oncogene - mutant complementation and suppression of cell-death induced by DNA-damaging agents

Adenovirus mutants defective in a 19 kD protein (19K gene) encoded by the early region E1B induce DNA fragmentation in a manner similar to the internucleosomal DNA fragmentation observed during programmed cell death (apoptosis) induced by a number of chemical, physical and biological stimuli. Zn++ ions effectively suppressed DNA fragmentation induced by a 19K mutant consistent with their inhibition of DNA fragmentation during apoptosis. Since the cellular proto-oncogene Bcl2 has been shown to suppress DNA fragmentation and the resulting programmed cell death, we have carried out a functional complementation analysis to determine whether the E1B 19 kD protein is functionally similar to the Bcl2 protein. Our results indicate that the DNA fragmentation induced by the 19K mutant can be efficiently suppressed by stable expression of human Bcl2 protein. Further, the 19 kD and Bcl2 proteins also function similarly to suppress DNA fragmentation and cell death induced by the DNA damaging agents cisplatin and UV.     

溶瘤病毒促进肿瘤细胞自噬的研究进展

自噬是一种正常的细胞生理活动,当正常细胞内存在异常蛋白、损坏的细胞器或处于饥饿状态时被激活,蛋白质和细胞器被捕获后送入到溶酶体降解,再将降解产物作为新合成蛋白和细胞器的底物加以循环利用,从而维持细胞内的平衡;溶瘤病毒（oncolytic virus,OV）是自然存在的或由人工生物技术合成的具有高度肿瘤靶向性及溶瘤性等特点的病毒。作为一种新颖的抗癌治疗方法其作用机制尚未明确。近年来研究发现OV引起肿瘤细胞死亡涉及到细胞自噬相关的机制。为了推进利用OV作为抗癌药物的研制,本文就OV诱导肿瘤细胞发生自噬的机制予以综述。      

DNA damage in cancer development: special implications in viral oncogenesis

DNA lesions arise from a combination of physiological/metabolic sources and exogenous environmental influences. When left unrepaired, these alterations accumulate in the cells and can give rise to mutations that change the function of important proteins (i.e. tumor suppressors, oncoproteins), or cause chromosomal rearrangements (i.e. gene fusions) that also result in the deregulation of key cellular molecules. Progressive acquisition of such genetic changes promotes uncontrolled cell proliferation and evasion of cell death, and hence plays a key role in carcinogenesis. Another less-studied consequence of DNA damage accumulating in the host genome is the integration of oncogenic DNA viruses such as Human papillomavirus, Merkel cell polyomavirus, and Hepatitis B virus. This critical step of viral-induced carcinogenesis is thought to be particularly facilitated by DNA breaks in both viral and host genomes. Therefore, the impact of DNA damage on carcinogenesis is magnified in the case of such oncoviruses via the additional effect of increasing integration frequency. In this review, we briefly present the various endogenous and exogenous factors that cause different types of DNA damage. Next, we discuss the contribution of these lesions in cancer development. Finally, we examine the amplified effect of DNA damage in viral-induced oncogenesis and summarize the limited data existing in the literature related to DNA damage-induced viral integration. To conclude, additional research is needed to assess the DNA damage pathways involved in the transition from viral infection to cancer. Discovering that a certain DNA damaging agent increases the likelihood of viral integration will enable the development of prophylactic and therapeutic strategies designed specifically to prevent such integration, with an ultimate goal of reducing or eliminating these viral-induced malignancies.     

Resveratrol modulation of signal transduction in apoptosis and cell survival: a mini-review

BACKGROUND: There is mounting evidence in the literature that resveratrol is a promising natural compound for prevention and treatment of a variety of human cancers. This overview summarizes recent studies of the major apoptosis and survival pathways regulated by resveratrol. BIOLOGICAL MECHANISMS: Apoptosis or programmed cell death is a key regulator of tissue homeostasis during normal development and also in adult organism under various conditions including adaptive responses to cellular stress. For example, tissue homeostasis is maintained by tight control of signaling events regulating cell death and survival. Thus, uncontrolled proliferation or failure to undergo cell death is involved in pathogenesis and progression of many human diseases, for example in tumorigenesis or in cardiovascular disorders. Moreover, current cancer therapies primarily act by triggering apoptosis programs in cancer cells. THERAPEUTIC APPLICATIONS: Natural products such as resveratrol have gained considerable attention as cancer chemopreventive or cardioprotective agents and also because of their antitumor properties. Among its wide range of biological activities, resveratrol has been reported to interfere with many intracellular signaling pathways, which regulate cell survival or apoptosis. DISCUSSION: Further insights into the signaling network and interaction points modulated by resveratrol may provide the basis for novel drug discovery programs to exploit resveratrol for the prevention and treatment of human diseases.     

Interchangeable binding of Bcl10 to TRAF2 and cIAPs regulates apoptosis signaling

Bcl10 was identified as a candidate gene responsible for low grade B cell lymphomas of mucosa-associated lymphoid tissue. Overexpression of Bcl10 in cultured cells was reported to promote apoptosis, however, the mechanism of regulation of apoptosis mediated by Bcl10 has not been demonstrated. In the present study, we analysed the apoptosis signaling pathway mediated by Bcl10, focusing on phosphorylation of Bcl10 and the dynamic interaction with its binding partners during apoptosis. Previously, we have demonstrated that Bcl10 potentially interacts with the other apoptosis regulator, TNF receptor associated factor-2 (TRAF2) and inhibitor of apoptosis proteins (cIAPs). The present results showed that the complex formation of these molecules was regulated by phosphorylation of Bcl10, that is, phosphorylation of Bcl10 resulted in binding of Bcl10 to cIAPs and the dissociation of it from TRAF2. Moreover, hyperphosphorylation of Bcl10 enhanced apoptosis, suggesting that changes in the binding partners of Bcl10 were correlated to the promotion of apoptosis as mediated by Bcl10. Indeed, the mutant which was deleted from the binding site of Bcl10 for cIAPs, could not induce apoptosis. These findings indicate that Bcl10 is a mediator of apoptosis signaling, by switching over binding to cIAPs from TRAF2 through the events of Bcl10 phosphorylation.