596

BCL2

B-cell CLL/lymphoma 2;BCL2;B-cell CLL/lymphoma 2

apoptosis regulator Bcl-2|protein phosphatase 1, regulatory subunit 50

18q21.3

protein-coding

Reduction in apoptosis has been associated with tumor metastases and response to chemotherapy in breast cancer. We examine the influence of apoptosis status and the expression of antiapoptotic proteins Bcl-2 and Bcl-x(L) on metastatic progression and response to therapy in an experimental model of breast cancer. We used human breast cancer cells (MDA-MB 435, MDA-MB 468 and MCF-7) to induce orthotopic xenograft tumors in nude mice. The overexpression of Bcl-2 or Bcl-x(L) influenced tumorigenicity, 468 transfectants being less tumorigenic than control (p < 0.0001). Lung metastasis appeared at day 120 in animals injected with 435/Bcl-2 or 435/Bcl-x(L) and they showed higher metastatic activity than control 435/Neo tumors (p = 0.02). In contrast, mice with 468 tumors were followed for 1 year after tumor excision, but they did not develop metastatic foci. 435/Bcl-2 and 435/Bcl-x(L) transfectant cells bound less readily to laminin (ANOVA, p < 0.0001), fibronectin (ANOVA, p < 0.0001) and collagen type-IV (ANOVA, p < 0.0001) than 435/Neo cells. The overexpression of antiapoptotic proteins in 435 transfectants rescued 20-40% of cells from anoikis at 64 hr in rocking conditions. In contrast, at this time only 5-10% of 468 and MCF-7 transfectant cells were rescued. Thus, the overexpression of the Bcl-2 or Bcl-x(L) associated with the loss of apoptosis in breast cancer cells in vivo may account for their metastatic behavior. These genes increase tumor metastasis when the oncogenic background has triggered the metastatic process, in which anoikis might determine tumor progression when the life span of the cells is extended.

Disruption of apoptosis may allow metastatic cell survival and confer resistance to chemotherapeutic drugs. We have analysed the molecular pathways that activate these survival genes in specific sites of metastasis. Estrogen receptor-negative breast cancer cell line MDA-MB435 and two metastatic sublines derived from lung (435L) and brain (435B) were analysed for the expression of members of the Bcl-2 family of apoptosis regulators. The levels of Bcl-2 were higher in the metastatic sublines than in parental cells, which correlated with the activation of Stat3, but not with the expression and/or activation of known bcl-2 transcription factors (CREB and WT1). In the brain subline, both expression of Bcl-2 and Stat3 activation were induced by epidermal growth factor and abrogated after treatment with kinase inhibitors specific for epidermal growth factor receptor or Jak2. Furthermore, transfection of 435B with a dominant-negative Stat3 markedly reduced the expression of Bcl-2 protein, whereas transient expression of a constitutively active Stat3 increased Bcl-2 in parental 435 cells. In addition, blockade of Stat3 activation by treatment with epidermal growth factor receptor and Jak2 kinase inhibitors or transfection with a dominant negative Stat3, sensitizes 435B cells to chemotherapy-induced apoptosis. Our data suggest that an increased activation of the Stat3-Bcl-2 pathway in estrogen receptor-negative metastatic breast cancer cell lines confer a survival advantage to these cells and contribute to their chemoresistance.

The majority of patients who succumb to cancer die from metastatic disease progression rather than from the primary tumor. Elucidation of the mechanisms underlying tissue-specific metastasis is essential to the development of effective therapies. The mitogen-activated protein kinase kinase (MEK) pathway is frequently activated in human tumors and has been shown to regulate genes involved in proliferation, migration, and invasion. Studies with MEK-transformed EpH4 mouse mammary epithelial cells showed that these cells are highly tumorigenic but have a limited metastatic ability. Detachment of epithelial cells from the extracellular matrix causes disruption of the actin cytoskeleton and induces apoptosis. Several metastatic breast carcinoma cell lines have been shown to be resistant to cell death following actin disruption. This death-resistant phenotype can be modeled by overexpressing the antiapoptotic Bcl-2 protein in cells. This suggests that mechanisms that regulate survival of extravasated tumor cells may enhance metastatic efficiency. Therefore, we examined whether expression of Bcl-2 in MEK-transformed EpH4 mammary epithelial cells could provide a survival advantage and promote metastasis. expression of Bcl-2 in parental EpH4 mammary epithelial cells or MEK-transformed cells was insufficient to induce increased migration, invasion, or tumor development. However, Bcl-2 expression markedly enhanced spontaneous lung metastasis from orthotopically implanted primary tumors. These results clearly show that mechanisms that regulate primary tumor development are distinct from those that promote metastasis and that assays designed to isolate genes involved in transformation may fail to identify genes that are critical regulators of metastasis.

OBJECTIVE: To evaluate the expression of BCL-2 and BAD genes in tissues of breast carcinoma and investigate the relationship between the expression of BCL-2 and BAD in breast cancer cells with chemosensitivity. METHODS: Immunohistochemical technique was used to detect the expression of BCL-2, BAD in 10 normal breast tissues, 10 breast fibroadenoma tissues, 40 youth human breast carcinoma tissues, 40 menopause human breast carcinoma tissues. And to detect the expression of ER, PR in 80 human breast carcinoma tissues. 20 Surgical samples of breast cancer, diagnosed by pathology, were obtained from The First Affiliated Hospital of Chongqing Medical University. The cancer sample cells were cultured separately in the incubator at 37 degrees C, 5% CO2 in vitro. The rate of inhibition of cancer cells in 4 kinds of anticancer drugs-- Epirubicin Adriamycin (EADM),5-Fluorouracil (5-Fu), Navelbine(NVB) and Diaminedichloroplatinum (DDP), were assayed by MTT method. RESULTS: The expression of BCL-2, BAD genes in young human breast carcinoma tissues were lower than that in menopause human breast carcinoma tissues (P < 0.05). There was a negative correlation between the positive expression rate of BCL-2 and histologic grade or the lymph node metastasis (P < 0.05). There was a positive correlation between the expression rates of BCL-2 and of ER, PR (P < 0.05). The expression of BAD had no relationship with the expression of ER, PR, histologic grade and the lymph node metastasis(P = NS). Sensitivity rates of 20 breast cancer cells in 0.1 x PPC within 48 h in vitro were 30% EADM,20% 5-Fu,45% NVB and 25% DDP. Respectively, the rate of inhibition of EADM,5- Fu, NVB and DDP were significantly higher in the BCL-2 negative cancer cells than in the BCL-2 positive cancer cells. A negative correlation was found between expression of BCL-2 and chemosensitivity for all the 4 anticancer drugs. The inhibition rates of EADM and NVB were significantly lower in the BAD negative cancer cells than in the BAD positive cancer cells. A positive correlation was found between expression of BAD and chemosensitivity for Epirubicin. CONCLUSION: The expression of BCL-2 and BAD can be used as prognosis factors of breast cancer. Detection of the BCL-2 protein expression level, particularly, combined with the detection of the expression of BCL-2 and BAD as well as ER and PR were helpful in confirming the prognosis of breast carcinoma. The combined detection of BCL-2 and BAD may be markers for predicting the responses to anticancer drugs.

The antiapoptotic protein Bcl-2 plays multiple roles in apoptosis, immunity, and autophagy. Its expression in tumors correlates with tumor grade and malignancy. The recapitulation of the normal developmental process of epithelial-mesenchymal transition (EMT) contributes to tumor cell plasticity. This process is also a characteristic of metastatic cells and vasculogenic mimicry. In the present study we report functional and structural interactions between Bcl-2 and the EMT-regulating transcription factor Twist1 and the relationship with metastasis and vascular mimicry. Bcl-2 and Twist1 are coexpressed under hypoxia conditions. The Bcl-2 can bind to Twist1 in vivo and in vitro. This interaction involves basic helix-loop-helix DNA binding domain within Twist1 and through two separate domains within Bcl-2 protein. Formation of the Bcl-2/Twist1 complex facilitates the nuclear transport of Twist1 and leads to transcriptional activation of wide ranges of genes that can increase the tumor cell plasticity, metastasis, and vasculogenic mimicry. Finally, nuclear expression of Bcl-2 and Twist1 is correlated with poor survival of these patients in a cohort of 97 cases of human hepatocellular carcinoma. CONCLUSION: The results describe a novel function of Bcl-2 in EMT induction, provide insight into tumor progression, and implicate the Bcl-2/Twist1 complex as a potential target for developing chemotherapeutics.

BACKGROUND: The role of bcl-2 expression, bcl-2 inhibitor HA14-1, and matrix metalloproteinase (MMP)-2 is still unclear in nasopharyngeal carcinoma (NPC). METHODS: From 1996 to 2000, 145 patients with newly diagnosed NPC who were treated with high dose radiotherapy were enrolled. The relationship between bcl-2 expression, TNM stage, and disease-specific survival was analyzed. Furthermore, the NPC cell line HONE-1 was used to confirm the relationship between bcl-2 and cell metastasis. RESULTS: Among the 145 patients, 47 (32.4%) of them were bcl-2 positive. The expression of bcl-2 was significantly correlated with neck lymph node metastasis (p=0.006), and patients with negative bcl-2 expression had better disease-free survival (p=0.007). A Cox regression model revealed that only bcl-2 expression (p=0.023) and stage IV (p=0.043) were statistically significant in the prognosis of NPC. In vitro analysis also showed that treatment with the bcl-2 inhibitor HA14-1 exerted an inhibitory effect on migration and expression of MMP-2 in HONE-1 cells. CONCLUSIONS: Bcl-2 expression represents an important and easily assayed prognostic factor, and it may play an important role in lymph node metastasis. Inhibition of the migration mediated by MMP-2 may be a key feature for the prevention of cancer metastasis.

BACKGROUND: Regulation of apoptosis is an important mechanism during the development of tumors including non-small cell lung cancer (NSCLC). The aim of this study was to assess the relationship between p53 and bcl-2 expression and various clinicopathological features and survival in patients with NSCLC. MATERIALS AND METHODS: Histological specimens obtained from 120 patients with stage I-III NSCLC were examined immunohistochemically for p53 and bcl-2. RESULTS: Positive immunostaining for p53 was observed in 50 and for bcl-2 in 35 patients. tumors with lymph node metastasis were significantly more likely to be bcl-2-positive. However, there was no correlation between p53 immunostaining and clinicopathological parameters. Cox proportional hazard multiple regression analyses identified gender, N status and bcl-2 expression as independent prognostic factors. When advanced stage tumors or tumors with lymph node metastasis were analyzed, a more favorable survival was noted in patients with bcl-2-positive tumors than those with bcl-2-negative tumors. CONCLUSION: Bcl-2 protein expression correlates with better prognosis in patients with advanced NSCLC.

Survivin and Bcl-2 are generally considered to be inhibitors of apoptosis and are frequently overexpressed in several types of human cancers. However, their role in regulating the biological behavior of non-small cell lung carcinoma (NSCLC) remains controversial. We aimed to determine the expression of survivin and Bcl-2 and explore their correlation with clinicopathological features and prognosis. The expression of survivin and Bcl-2 proteins in 62 specimens of NSCLC tissues and 30 specimens of tumor adjacent tissues was detected using immunohistochemistry. The correlation between protein expression and clinicopathological features and prognosis was analyzed. The percentage of survivin-positive samples obtained from NSCLC tissues was 58.06% (36/62), which was significantly higher compared to that in normal lung tissues (10%, 3/30; P<0.05). Similarly, the percentage of Bcl-2-positive samples obtained from NSCLC tissues was statistically higher compared to that from normal lung tissues (51.61%, 32/62 vs. 6.67%, 2/30; P<0.05). Survivin expression was closely correlated with tumor differentiation, lymph node metastasis and TNM stage (P<0.05), while Bcl-2 expression was only associated with TNM stage (P<0.05). The expression of survivin was positively correlated with that of Bcl-2 (P<0.05). A five-year follow-up study revealed that the expression of survivin and Bcl-2 was negatively correlated with post-operative survival duration. Our findings suggest that survivin and Bcl-2 may act synergistically in the occurrence, development, invasion and metastasis of NSCLC, both of which are up-regulated in NSCLC tissues. The co-expression of survivin and Bcl-2, which is closely related to malignancy, may serve as a biomarker for predicting prognosis.

BACKGROUND: Loco-regional spread of disease causes high morbidity and is associated with the poor prognosis of malignant oral tumors. Better understanding of mechanisms underlying the establishment of lymph node metastasis is necessary for the development of more effective therapies for patients with oral cancer. The aims of this work were to evaluate a possible correlation between endothelial cell Bcl-2 and lymph node metastasis in patients with oral squamous cell carcinoma (OSCC), and to study signaling pathways that regulate Bcl-2 expression in lymphatic endothelial cells. METHODS: Endothelial cells were selectively retrieved from paraffin-embedded tissue sections of primary human OSCC from patients with or without lymph node metastasis by laser capture microdissection. RT-PCR was used to evaluate Bcl-2 expression in tumor-associated endothelial cells and in tumor cells. In vitro, mechanistic studies were performed to examine the effect of vascular endothelial growth factor (VEGF)-C on the expression of Bcl-2 in primary human lymphatic endothelial cells. RESULTS: We observed that Bcl-2 expression is upregulated in the endothelial cells of human oral tumors with lymph node metastasis as compared to endothelial cells from stage-matched tumors without metastasis. VEGF-C induced Bcl-2 expression in lymphatic endothelial cells via VEGFR-3 and PI3k/Akt signaling. Notably, OSCC cells express VEGF-C and induce Bcl-2 in lymphatic endothelial cells. CONCLUSIONS: Collectively, this work unveiled a mechanism for the induction of Bcl-2 in lymphatic endothelial cells and suggested that endothelial cell Bcl-2 contributes to lymph node metastasis in patients with oral squamous cell carcinoma.

PURPOSE: Oropharyngeal squamous cell carcinoma (OPSCC) associated with human papilloma virus (HPV) is rapidly growing in incidence. Despite better prognosis than OPSCC associated with traditional risk factors, treatment failure still occurs in a significant proportion of patients. We had identified the antiapoptotic protein Bcl2 as a marker for poor outcome in advanced OPSCC treated with concurrent chemoradiation. To determine whether Bcl2 and HPV together might further characterize treatment response, we examined whether the prognostic value of Bcl2 was independent of HPV status. EXPERIMENTAL DESIGN: Pretreatment tumor biopsies from 68 OPSCC patients were tested for HPV by in situ hybridization and were immunostained for Bcl2 to evaluate relations with disease-free (DFS) and overall survival following platin-based concurrent chemoradiation. Median follow-up among surviving patients was 47 months (range, 10-131 months). RESULTS: Bcl2 and HPV independently predicted DFS and overall survival. Hazard ratios (with 95% confidence interval) for positive versus negative status in bivariate Cox proportional hazard analysis of DFS were 6.1 (1.8-21) for Bcl2 and 0.11 (0.035-0.37) for HPV. Only 1 of 32 HPV-positive/Bcl2-negative tumors recurred. Pretreatment Bcl2 expression was specifically associated with distant metastasis; five of six distant metastases occurred in the <40% of patients whose primary tumors were Bcl2 positive. CONCLUSIONS: Independent of HPV status, pretreatment Bcl2 expression identifies a subset of OPSCC patients having increased risk of treatment failure, particularly through distant metastasis, after concurrent chemoradiation. Considering HPV and Bcl2 together should help in devising better personalized treatments for OPSCC.

OBJECTIVE: This study assesses TRAIL-R2 (TNF-related apoptosis-inducing ligand receptor 2) and BCL2 (B cell CLL/lymphoma 2) expression as well as CpG island methylation within the TRAIL-R2 promoter in ovarian serous tumors and primary and metastatic serous EOC (epithelial ovarian cancer). METHODS: RNA and DNA were obtained from women with normal ovarian tissues (n = 18), ovarian serous cystadenoma tumors (n = 11) and serous EOC (n = 16) using Trizol(R). Quantitative PCR was performed to quantify the relative levels of TRAIL-R2 and BCL2. The methylation frequency of the TRAIL-R3 promoter was assessed using a methylation-specific PCR assay after DNA bisulfite conversion. Differences between the groups were evaluated using the chi (2), Mann-Whitney U or Kruskal-Wallis tests, as indicated. RESULTS: We identified TRAIL-R2 and BCL2 mRNA expressed in all ovarian tumor groups, and there were significant differences between the groups. Both genes had low expression levels in ovarian serous cystadenoma and primary EOC tumors when compared with metastatic EOC. Methylation of the TRAIL-R2 promoter was frequently observed in all groups; however, there were no statistically significant associations. CONCLUSIONS: Primary EOC is associated with lower TRAIL-R2 and BCL2 expression levels, while metastatic EOC is associated with higher expression of these genes. Promoter DNA methylation was not related to this finding, suggesting there are other mechanisms involved in transcriptional control.

BACKGROUND: We explored the mechanisms of apoptosis after Bcl-2 protein downmodulation in metastatic LNCaP-LN3 cells (LN3). METHODS: LNCaP, LNCaP-Pro5 (Pro5) and LN3 cells were cultured in 5% charcoal-stripped serum (CSS) or in R1881 (synthetic androgen) and bicalutamide (synthetic anti-androgen) and growth inhibition was assessed. expression levels of androgen receptor (AR) and Bcl-2 were determined. LN3 cells were transfected with small interfering RNA Bcl-2 (siRNA Bcl-2) or control siRNA oligonucleotides. Rates of apoptosis and proliferation were obtained. Cytochrome c localization in treated and control cells was assessed +/- cyclosporine A (CsA). Caspases 9, 3, and poly (ADP-ribose) polymerase cleavage (PARP) were measured upon downmodulation of Bcl-2; and cell growth inhibition in vitro after Bcl-2 modulation combined with docetaxel chemotherapy was determined. RESULTS: LN3 cells maintained growth under castrate conditions in vitro. AR protein amplification did not explain castrate-resistant LN3 cell growth. Bcl-2 protein levels in LN3 cells were significantly higher than in Pro5 cells, and were effectively downmodulated by siRNA Bcl-2. Subsequently increased apoptosis and decreased proliferation mediated by cytochrome c was noted and this was reversed by CsA. siRNA Bcl-2-transfected LN3 cells exhibited elevated levels of caspases 9, 3, and PARP cleavage. Exposure of LN3 cells to docetaxel led to increased apoptosis, and simultaneous downmodulation of Bcl-2 substantially enhanced this effect. CONCLUSIONS: Downmodulation of Bcl-2 in metastatic castrate-resistant LNCaP-LN3 cells led to apoptosis via a cytochrome c-dependent pathway that was enhanced with docetaxel treatment.

BACKGROUND: To test the hypothesis that modulation of Bcl-2 with 13-cis retinoic acid (CRA)/interferon-alpha2b (IFN) with paclitaxel (TAX), or mitoxantrone, estramustine and vinorelbine (MEV) will have clinical activity in men with metastatic castrate-resistant prostate cancer (CRPC). METHODS: 70 patients were treated with either MEV (Arm A) in a 3-week cycle or CRA/IFN/TAX with an 8-week cycle (Arm B). Patients were assessed for response, toxicity, quality of life (QOL), and the effect of treatment on Bcl-2 levels in peripheral blood mononuclear cells (PBMC). RESULTS: The PSA response rates were 50% and 23%, measurable disease response rates (CR+PR) 14% and 15%, and median overall survival 19.4 months and 13.9 months on Arm A and Arm B respectively. Transient grade 4 neutropenia occurred in 18 and 2 patients, and grade 3 to 4 thrombosis in 7 patients and 1 patient in Arm A and Arm B respectively. Patients on Arm B reported a clinically significant decline in QOL between baseline and week 9/10 (.71 s.d.), and a significantly lower level of QOL than Arm A (p = 0.01). As hypothesized, Bcl-2 levels decreased with CRA/IFN therapy only in Arm B (p = 0.03). CONCLUSIONS: Treatment with MEV was well tolerated and demonstrated clinical activity in patients with CRPC. Given the adverse effect of CRA/IFN/TAX on QOL, the study of other novel agents that target Bcl-2 family proteins is warranted. The feasibility of measuring Bcl-2 protein in a cooperative group setting is hypothesis generating and supports further study as a marker for Bcl-2 targeted therapy. TRIAL REGISTRATION: CLINICAL TRIALS REGISTRATION NUMBER: CDR0000067865.