ApoG2, a novel inhibitor of antiapoptotic Bcl-2 family proteins, induces apoptosis and suppresses tumor growth in nasopharyngeal carcinoma xenografts

Nasopharyngeal carcinoma (NPC) is a common malignant tumor in South China. It has been reported that overexpression of antiapoptotic Bcl-2 family proteins in NPC has caused the lack of long-term efficacy of conventional therapies. Apogossypolone (ApoG2), a novel small-molecule inhibitor of antiapoptotic Bcl-2 family proteins, has been discovered as the optimized derivative of gossypol. In this study, we found that in NPC cells, ApoG2 totally blocked the antiapoptotic function of Bcl-2 family proteins without affecting the expression levels of these proteins. ApoG2 selectively inhibited proliferation of 3 NPC cell lines (C666-1, CNE-1 and CNE-2) that highly expressed the antiapoptotic Bcl-2 proteins. This inhibitory activity was associated with release of cytochrome c, activation of caspase-9 and caspase-3 and apoptosis of sensitive NPC cells. However, ApoG2 had no obvious inhibitory effect on NPC cell line HONE-1, which expressed antiapoptotic Bcl-2 and Bcl-xL at a low level. We further found that ApoG2 effectively suppressed tumor growth of NPC xenografts in nude mice and enhanced the antitumor effect of CDDP (cisplatin) on NPC cells in vitro and in vivo. Immunohistochemical results showed that the expression of CD31 decreased after ApoG2 treatment, which suggested inhibition of angiogenesis in NPC xenografts. Our findings strongly suggest that ApoG2 may serve as a novel inhibitor of Bcl-2 family proteins and, by targeting these proteins, may become a promising drug for the treatment of NPC.     

Role of Bcl-2 family of proteins in malignancy

B cell lymphoma gene-2 (Bcl-2) is the prototypic member of a growing family of proteins that play evolutionarily conserved, key regulatory roles in apoptosis. The Bcl-2 family members are characterized by the presence of one or more Bcl-2 homology domains and are comprised of both the prosurvival and proapoptotic proteins. Bcl-2 itself is a prosurvival member of the family and its aberrant expression has been linked to a variety of different cancers, including several hematological malignancies. Although the exact mechanism of action of Bcl-2 family of proteins in regulating apoptosis is still a matter of some debate, these proteins appear to act upstream of caspase activation. Many recent studies have shown the therapeutic potential of targeting Bcl-2 family members for the treatment of cancer. This article summarizes what is currently known about Bcl-2-like proteins and how the evolving understanding of the biology of these proteins is paving way for the development of novel cancer therapeutics.     

The bisphosphonate pamidronate induces apoptosis in human melanoma cells in vitro

Pamidronate belongs to the class of nitrogen-containing bisphosphonates that are potent inhibitors of bone resorption frequently used for the treatment of osteoporosis and cancer-induced osteolysis. The inhibition of osteoclasts' growth has been suggested as the main mechanism of the inhibitory effect of pamidronate on bone metastases. Recent findings indicated that bisphosphonates also have a direct apoptotic effect on other types of tumour cells. Nitrogen-containing bisphosphonates were shown to inhibit farnesyl diphosphate synthase, thus blocking the synthesis of higher isoprenoids. By this mechanism they inactivate monomeric G-proteins of the Ras and Rho families for which prenylation is a functional requirement. On the background of the known key role of G-proteins in tumorigenesis, we investigated a possible beneficial use of pamidronate in the treatment of malignant melanoma. Our results indicate that pamidronate inhibits the cell growth and induces apoptosis in human melanoma cells in vitro. Susceptibility to pamidronate did not correlate to CD95 ligand sensitivity or p53 mutational status. Furthermore it is interesting to note that overexpression of bcl-2 did not abolish pamidronate-induced apoptosis. These data suggests that pamidronate has a direct anti-tumour effect on malignant melanoma cells, independently of the Bax/Bcl-2 level.     

Significance of phosphotyrosine proteins, Bcl-2 and p53 for apoptosis in resting B-chronic lymphocytic leukemia (CLL) cells.

Signal transduction and apoptosis in B-cell chronic lymphocytic leukemia (CLL) cells with a post-germinal center (GC) phenotype were studied. Specific activation of the cells was induced by a combination of soluble anti-CD40 monoclonal antibody and interleukin-4 (CD40/IL-4) and nonspecific activation with a combination of phytohemagglutinin, phorbol-12-myristate-13-acetate and ionomycin (chemical mixture). Less than 5% of these leukemia cells entered the cell cycle after activation, as indicated by the number of cells in G0/G1 phase. The protein tyrosine phosphorylation pattern and expression of the Bcl-2 protein were specific in ex vivo CLL cells of each individual patient. Expression of the p53 protein was not detectable in these leukemia cells. Cross-linking of the CD40/IL-4 receptors on CLL cells significantly upregulated phosphotyrosine proteins and the p53 protein. In the presence of chemical mixture, downregulated phosphotyrosine proteins were detected. Alterations in Bcl-2 expression were independent of cross-linking with CD40/IL-4 or chemical mixture. A high frequency of apoptotic cells was detected in cells that had downregulated phosphotyrosine proteins and Bcl-2 protein. There was no correlation between induction of apoptosis and expression of p53 protein. Our results suggest that apoptosis in resting leukemia cells could occur prior to the cell cycle progression. Alterations in phosphotyrosine proteins and Bcl-2 but not p53 might play an important role in the regulation of apoptosis in resting G0/G1 memory post-GC B-CLL cells.     

Induction of apoptosis and chemosensitization of mesothelioma cells by Bcl-2 and Bcl-xL antisense treatment

Our study was designed to investigate the role of the anti-apoptotic proteins Bcl-2 and Bcl-xL in the chemoresistance of cells derived from malignant pleural mesothelioma. First, we determined the basal expression levels of Bcl-2 and Bcl-xL in mesothelioma cells and examined the effect of their downregulation by antisense oligonucleotides. Bcl-xL mRNA and protein could be readily detected in mesothelioma cell lines, whereas only low levels of Bcl-2 mRNA and protein were found. Preferential downregulation of either Bcl-xL alone or of Bcl-xL and Bcl-2 simultaneously was achieved by treatment with antisense oligonucleotides 4259 and 4625, respectively, whereas the expression of other apoptosis-relevant genes remained unaffected. Treatment with oligonucleotides 4259 or 4625 lowered the apoptosis threshold in ZL34 mesothelioma cells, as indicated by an increase in cell death accompanied by increased caspase-3-like activity, a decrease of the mitochondrial transmembrane potential and the cleavage of procaspase-7 and ICAD. In addition to the direct induction of apoptosis, antisense treatment sensitized ZL34 cells to the cytostatic effect of cisplatin and gemcitabine, with the combination of 4625 and cisplatin being the most effective. Our results demonstrate that Bcl-2 and Bcl-xL antisense treatment facilitates apoptosis in mesothelioma cells and suggest the use of Bcl-2/Bcl-xL bispecific antisense treatment in combination with cisplatin or gemcitabine for therapy of malignant pleural mesothelioma.     

Overexpression of Bcl-2 regulates sodium butyrate- and/or docetaxel-induced apoptosis in human bladder cancer cells both in vitro and in vivo

Sodium butyrate (NaBt), a physiologically occurring short-chain fatty acid, induces differentiation as well as apoptosis in numerous cell types, and this induction is partially regulated by Bcl-2 expression. The objectives of our study were to characterize the in vitro effects of NaBt and/or docetaxel on the growth, cell cycle and apoptosis of human bladder cancer cells, and to determine whether tumor growth in vivo is inhibited by isobutyramide, an orally bioavailable Bt analogue, and/or docetaxel by using Bcl-2-transfected human bladder cancer cell line KoTCC-1/BH and control vector only-transfected cell line KoTCC-1/C. NaBt caused a decrease in growth of both KoTCC-1/C and KoTCC-1/BH cells, however, its growth inhibitory effect was significantly greater in KoTCC-1/C cells. One mM NaBt resulted in G1 cell cycle arrest, accompanied by up-regulation of p21 (waf1/cip1) and down-regulation of cyclin D1 in KoTCC-1/C cells, whereas KoTCC-1/BH showed resistance to G1 cell cycle arrest. An amount of 5 mM NaBt induced apoptosis, accompanied by up-regulation of Bak in KoTCC-1/C cells but failed to induce apoptosis in KoTCC-1/BH cells despite substantial down-regulation of Bcl-2. Oral administration of isobutyramide significantly reduced the KoTCC-1/C tumor volume compared with the KoTCC-1/BH tumor volume in nude mice. Furthermore, docetaxel induced Bcl-2 phosphorylation in KoTCC-1/BH cells and combined treatment with isobutyramide and docetaxel synergistically inhibited the growth of KoTCC-1/BH cells both in vitro and in vivo. These findings suggest that isobutyramide therapy could be a novel therapeutic strategy for patients with bladder cancer if docetaxel is combined according to the Bcl-2 expression status.     

BH3-only proteins: integrated control point of apoptosis

Bcl-2 family proteins play central roles in the regulation of most, if not all, apoptotic pathways, and hence this family plays a critical role in oncogenesis. The Bcl-2 homology 3 (BH3)-only members of this family are "proapoptotic," promoting apoptosis by sensing cellular stresses: that is, they are activated or induced in response to stress stimuli. These BH3-only proteins then interfere with the function of "prosurvival" Bcl-2 family members, thereby promoting the progression of apoptosis. It has long been recognized that the induction and activation of each of the BH3-only proteins are subject to the type of stress stimulus. Although it was originally assumed that all the BH3-only proteins exert similar effects on the downstream apoptotic machinery, recent studies have uncovered their distinct functional properties, indicating the operation of more intricate, versatile control mechanisms of apoptosis. In this review, we will summarize recent findings on the diversity in the activation and function of BH3-only proteins. In particular, we focus on the overlapping and individual roles of 2 BH3-only proteins, Puma and Noxa, in the context of the apoptotic response induced by the tumor suppressor p53.     

Targeting Bcl-2 family proteins in adult T-cell leukemia/lymphoma: in vitro and in vivo effects of the novel Bcl-2 family inhibitor ABT-737

Adult T-cell leukemia/lymphoma (ATLL) is a peripheral T-cell malignancy caused by human T-lymphotropic virus type I (HTLV-1). ABT-737, a small molecule inhibitor of Bcl-2, Bcl-X(L), and Bcl-w, significantly induced apoptosis in HTLV-1 infected T-cell lines as well as in fresh ATLL cells, and synergistically enhanced the cytotoxicity and apoptosis induced by conventional cytotoxic drugs. Moreover, ABT-737 significantly inhibited the in vivo tumor growth of an ATLL mouse model. These results suggest that the use of an agent targeting anti-apoptotic bcl-2 family proteins, either alone or in combination with other conventional drugs, represents a novel promising approach for ATLL.     

In vitro and in vivo antitumor effect of 2-methoxyestradiol on human melanoma

2-methoxyestradiol (2ME(2)) is an endogenous metabolite of estradiol with estrogen-receptor-independent antitumor and antiangiogenic activity. We examined the effects of 2ME(2) on the cellular proliferation of 8 human melanoma cell lines. We show that 2ME(2) inhibited cell proliferation by inducing apoptosis and an arrest in the G(2)/M phase, and the mechanism of action involved microtubules, mitochondrial damage and caspase activation. In male SCID mice, 2ME(2) was effective in reducing primary tumor weight and the number of liver metastases after intrasplenic injection of human melanoma cells. In the metastases, we found a significantly higher rate of apoptotic cells after 2ME(2) treatment. These findings on the antitumor effect of 2ME(2) in cell culture as well as in an animal model may have implications for designing alternative treatment options for patients with advanced malignant melanoma.     

In vitro induction of apoptosis and in vivo effects of a flavone nitroderivative in murine mammary adenocarcinoma cells

We explored the in vitro and in vivo mechanism of antitumor action of the synthetic flavonoid 2′‐nitroflavone on LM3 murine mammary adenocarcinoma cells. In vitro assays showed that 2′‐nitroflavone increased the population of LM3 hypodiploid cells and produced a typical ladder of DNA fragmentation. Apoptotic cell death was also characterized by the activation of caspase‐8, ‐9 and ‐3, by an increment in the expression levels of the proapoptotic protein Bax and by the release of cytochrome c to cytosol. The in vivo effect of 2′‐nitroflavone on tumor growth was studied in BALB/c mice injected subcutaneously with LM3 cells. Results showed that tumor volume and weight were significantly reduced at doses of 10 and 40 mg/kg of 2′‐nitroflavone, respectively. Apoptotic cells were identified by TUNEL assay in tumor slices from mice treated with 10 mg/kg of 2′‐nitroflavone. Western blot analysis of tumor lysate supernatants from treated mice revealed an upregulation of the total levels of Bax and Fas receptor. In addition, administration of 40 mg/kg of 2′‐nitroflavone to nontumor‐bearing mice showed no histopathological effects on different organ tissues. This is the first report of the in vivo growth inhibitory effect of 2′‐nitroflavone as an apoptotic agent likely useful for mammary adenocarcinoma treatment. © 2009 UICC     

Bcl-2 promotes malignant progression in a PDGF-B-dependent murine model of oligodendroglioma

A significant subset of gliomas arises after activation of the proproliferative platelet-derived growth factor (PDGF) pathway. The progression of low-grade gliomas to more malignant tumors may be due to oncogenic cellular programs combining with those suppressing apoptosis. Antiapoptotic genes are overexpressed in a variety of cancers, and the antiapoptotic gene, BCL2, is associated with treatment resistance and tumor recurrence in gliomas. However, the impact of antiapoptotic gene expression to tumor formation and progression is unclear. We overexpressed Bcl-2 in a PDGFB-dependent mouse model of oligodendroglioma, a common glioma subtype, to assess its effect in vivo. We hypothesized that the antiapoptotic effect would complement the proproliferative effect of PDGFB to promote tumor formation and progression to anaplastic oligodendroglioma (AO). Here, we show that coexpression of PDGFB and Bcl-2 results in a higher overall tumor formation rate compared to PDGFB alone. Coexpression of PDGFB and Bcl-2 promotes progression to AO with prominent foci of necrosis, a feature of high-grade gliomas. Median tumor latency was shorter in mice injected with PDGFB and Bcl-2 compared to those injected with PDGFB alone. Although independent expression of Bcl-2 was insufficient to induce tumors, suppression of apoptosis (detected by cleaved caspase-3 expression) was more pronounced in AOs induced by PDGFB and Bcl-2 compared to those induced by PDGFB alone. Tumor cell proliferation (detected by phosphohistone H3 activity) was also more robust in high-grade tumors induced by PDGFB and Bcl-2. Our results indicate that suppressed apoptosis enhances oligodendroglioma formation and engenders a more malignant phenotype.     

Protease inhibitors restore radiation-induced apoptosis to Bcl-2-expressing lymphoma cells.

The proteasome pathway is important for the turnover of many regulatory proteins. This pathway has recently become a target for antitumor agents and several research groups have demonstrated that inhibitors with specificities for the proteasome are potent apoptosis-inducing agents. Many mechanisms by which proteasome inhibitors exert their effects have been suggested, including inhibition of NF-kappa B activity and stabilization of the p53 tumor suppressor protein. We investigated the ability of inhibitors with specificities for the proteasome and for another protein degradation enzyme, calpain, to sensitize a murine B-cell lymphoma with constitutive NF-kappa B1 homodimer activity and high expression of Bcl-2 protein to radiation-induced apoptosis. Protease inhibitors tested were calpain inhibitor I, calpain inhibitor II, calpeptin, MG132, and Lactacystin. All five inhibitors induced apoptosis and sensitized cells to radiation despite the maintenance of Bcl-2 protein levels throughout the course of treatment. An electrophoretic migration shift assay for NF-kappa B1 activity provided evidence that reversal of NF-kappa B activity was not required for induction of cell death; however, p53 levels were elevated for all inhibitors tested. HL-60 cells, devoid of p53, could not be sensitized to radiation by MG132 treatment, suggesting that p53 was important for cell death induced by combined treatment with protease inhibitors and radiation. We concluded that protease inhibitors are capable of overcoming the protective effects of Bcl-2 to induce apoptosis and suggest that protease inhibitor treatment, when combined with ionizing radiation, leads to p53-mediated apoptosis.     

Anticancer drug development based on modulation of the Bcl-2 family core apoptosis mechanism

Cancer cells generally maintain their survival by suppressing apoptosis. Mitochondrial mechanisms are involved in most forms of apoptosis (referred to as mitochondrial apoptosis), and the Bcl-2 family controls apoptosis at the mitochondrion via a balance of the effects of pro- and antiapoptotic members. Antiapoptotic molecules such as Bcl-2 and Bcl-x_L are often overexpressed in cancer cells and their inhibition is an attractive target for selective killing of tumor cells via induction of apoptosis. Reduction of the levels of these proteins with antisense molecules has shown encouraging experimental and clinical results and there has been some success in developing small-molecule inhibitors. These are likely to be the most productive drug development approaches in the near future. However, growing understanding of the molecular mechanisms involved has identified other potential targets. Cardiolipin is important for the proapoptotic activity of Bcl-2 family members such as Bid, Bax and Bak, and modulation of its metabolism and translocation in mitochondrial membranes could potentially have a strong influence on apoptosis. Post-translational modifications strongly influence the activity of Bcl-2 family members. Several molecules have been identified that bind to Bcl-2 family members and could be intracellular control mechanisms. These mechanisms may yield several drug development targets for the induction of apoptosis. Further research will qualify these targets and, in the longer term, could lead to a more specific means of inducing apoptosis in cancer cells.     

Kinetin riboside preferentially induces apoptosis by modulating Bcl-2 family proteins and caspase-3 in cancer cells

Here, we demonstrate that kinetin riboside (KR), a cytokinin analog, induces apoptosis in HeLa and mouse melanoma B16F-10 cells. KR disrupted the mitochondrial membrane potential and induced the release of cytochrome c and activation of caspase-3. Bad were upregulated while Bcl-2 was down-regulated under KR exposure. A tumor growth in mice was dramatically suppressed by KR. In contrast, human skin fibroblast CCL-116 and bovine primary fibroblast cells show resistances to KR and no significant changes in Bad, Bcl-X(L,) and cleaved PARP were observed. Our data suggest that KR selectively induces apoptosis in cancer cells through the classical mitochondria dependent apoptosis pathway.     

Temozolomide induces senescence but not apoptosis in human melanoma cells

Temozolomide (TMZ), a DNA alkylating agent used in the treatment of melanoma, is believed to mediate its effect by addition of a methyl group to the O(6) position of guanine in DNA. Resistance to the agent may be in part due to the activity of O(6)-methylguanine-DNA methyl transferase (MGMT). In the present study, we show that sensitivity of melanoma cells to TMZ was dependent on their p53 status and levels of MGMT. Analysis of the mechanisms underlying reduced viability showed no evidence for induction of apoptosis even though marked levels of apoptosis was seen in TK6 lymphoma cells. Sensitivity of melanoma cells was associated with p53-dependent G2/M cell cycle arrest and induction of senescence. To verify the role of p53, the assays were repeated in presence of pifithrin-alpha, an inhibitor of p53. This resulted in increased viability of melanoma cells with wild-type p53 and reversed G2/M cell cycle arrest. Paradoxically, apoptosis was increased in melanoma but decreased as expected in TK6 lymphoma cells. These results are consistent with the view that TMZ is relatively ineffective against melanoma due to defective apoptotic signalling resulting from activation of p53. The nature of the defects in apoptotic signalling remains to be explored.     

Anti-proliferative and proapoptotic effects of (-)-epigallocatechin-3-gallate on human melanoma: possible implications for the chemoprevention of melanoma

Melanoma accounts for only about 4% of all skin cancer cases but most of skin cancer-related deaths. Standard systemic therapies such as interferon (IFN) have not been adequately effective in the management of melanoma. Therefore, novel approaches are needed for prevention and treatment of this disease. Chemoprevention by naturally occurring agents present in food and beverages has shown benefits in certain cancers including nonmelanoma skin cancers. Here, employing 2 human melanoma cell lines (A-375 amelanotic malignant melanoma and Hs-294T metastatic melanoma) and normal human epidermal melanocytes (NHEM), we studied the antiproliferative effects of epigallocatechin-3-gallate (EGCG), the major polyphenolic antioxidant present in green tea. EGCG treatment was found to result in a dose-dependent decrease in the viability and growth of both melanoma cell lines. Interestingly, at similar EGCG concentrations, the normal melanocytes were not affected. EGCG treatment of the melanoma cell lines resulted in decreased cell proliferation (as assessed by Ki-67 and PCNA protein levels) and induction of apoptosis (as assessed cleavage of PARP, TUNEL assay and JC-1 assay). EGCG also significantly inhibited the colony formation ability of the melanoma cells studied. EGCG treatment of melanoma cells resulted in a downmodulation of anti-apoptotic protein Bcl2, upregulation of proapoptotic Bax and activation of caspases -3, -7 and -9. Furthermore, our data demonstrated that EGCG treatment resulted in a significant, dose-dependent decrease in cyclin D1 and cdk2 protein levels and induction of cyclin kinase inhibitors (ckis) p16INK4a, p21WAF1/CIP1 and p27KIP1. Our data suggest that EGCG causes significant induction of cell cycle arrest and apoptosis of melanoma cells that is mediated via modulations in the cki-cyclin-cdk network and Bcl2 family proteins. Thus, EGCG, alone or in conjunction with current therapies, could be useful for the management of melanoma.     

Suppression of oncogenic NRAS by RNA interference induces apoptosis of human melanoma cells

The majority of human melanomas harbor activating mutations in either the BRAF or NRAS gene. To date, the role of oncogenic NRAS in melanoma remains poorly defined and no current therapies are directed at specifically suppressing oncogenic NRAS in human melanoma tumors. The aim of our study, therefore, was to investigate the effects of suppressing oncogenic NRAS in human melanoma cell lines in vitro. Using both small interfering RNA- and plasmid based-RNA interference techniques, oncogenic NRAS was specifically suppressed in 2 human melanoma cell lines, 224 and BL, which harbor a codon 61 CAA (glutamine) to CGA (arginine) NRAS mutation. Suppression of oncogenic NRAS in these cell lines resulted in increased apoptosis. Furthermore, in 224 cells we demonstrated decreased phosphorylation of extracellular signal-regulated kinase (ERK) and Akt, and reduced expression of NF-kappaB and cyclin D1 in the N-Ras signaling pathway. In contrast, RNA interference directed at wild-type (WT) NRAS had no significant effect on apoptosis of 224 cells or 2 human melanoma cell lines (A375 and 397) containing WT NRAS but a codon 600 GTG (valine) to GAG (glutamate) mutation in BRAF. These data suggest that oncogenic NRAS is important for avoidance of apoptosis in melanomas that harbor the codon 61 NRAS mutation and emphasizes oncogenic NRAS as a therapeutic target in patients with tumors that harbor this mutation.     

Cytokine-mediated protection of human dendritic cells from prostate cancer-induced apoptosis is regulated by the Bcl-2 family of proteins

Prostate cancer is the most common cancer in men in the United States, and second in cancer-induced mortality. It is likely that tumour-induced immunosuppression is one of the reasons for low treatment efficacy in patients with advanced prostate cancer. It has been recently demonstrated that prostate cancer tissue is almost devoid of dendritic cells (DC), the major antigen-presenting cells responsible for the induction of specific antitumour immune responses. In this study, we have tested the hypothesis that prostate cancer induces progressive suppression of the DC system. We found that co-incubation of human DC with three prostate cancer cell lines led to the high levels of premature apoptosis of DC, which were significantly higher than in DC cultures co-incubated with normal prostate cells or blood leucocytes. Stimulation of DC for 24 hours with CD40 ligand (CD154), IL-12 or IL-15 prior to their co-incubation with prostate cancer cells resulted in a significant increase in DC survival in the tumour microenvironment. Furthermore, activation of DC with these cytokines was also accompanied by increased expression of the anti-apoptotic protein Bcl-x(L) in DC, suggesting a possible mechanism involved in DC protection from apoptotic death. In summary, our data demonstrate that prostate cancer induces active elimination of DC in the tumour microenvironment. Stimulation of DC by CD154, IL-12 or IL-15 leads to an increased expression of the anti-apoptotic protein Bcl-x(L) and increased resistance of DC to prostate cancer-induced apoptosis. These results suggest a new mechanism of tumour escape from immune recognition and demonstrate the cytokine-based approaches which might significantly increase the efficacy of DC-based therapies for cancer.