Bcl-2 Transfectants Research Articles

Abstract 988: Targeting immunological and apoptotic cell death to improve therapeutic efficacy in melanoma

Abstract Apoptotic and immunological cell death are the two major pathways that can lead to demise of cancer cells. Targeting both pathways by combining chemotherapy and checkpoint inhibitors has proven to be more effective in treating lung cancer but not in melanoma, due to the fact that melanoma cells often express high levels of antiapoptotic proteins Bcl-2 ( 60%) which contribute to their resistance to chemotherapy. We have previously shown that melanoma cell lines with high Bcl-2 expression , regardless of their BRAF mutation, do not possess high PD-L1 ( AACR abstract #4747 2019). This is also seen in melanoma tissue obtained from patients. Here, we attempt to explore whether there is any relationship between Bcl-2 and PD-L1 in melanoma cells using knock in and knock out methods. Two melanoma cells derived from patients ( mel1220 which possess high levels of Bcl-2 and Mel-F which possess high levels of PD-L1 ) and their BRAF/MEK counterparts, Mel-1220 BMR and MelF BMR, respectively, were used for the study. Interestingly, cell lines with high Bcl-2 , their BMR counterparts showed no changes in Bcl-2 levels, However, in cell lines with high PD-L1 expression, the levels of PD-L1 is even higher when they become BMR. This could explain why after failure to BRAF/MEK inhibitor , the clinical response to anti PD-1 check point inhibitors varies. Next, we transfected PD-L1 using the SP-GFP-CD274-NU Vector ( purchased from Addgene )into the BcL-2 over expressing cell lines. Three clones were selected. Two clones ( 1220.PD-L1/1 and 1220 PD-L1/3) showed only slight decreased Bcl-2 expression ( less than 1 fold) and in one clone ( 1220.PD-L1/2), there is no effect seen. Furthermore, there is no discernable change in other antiapoptotic proteins ( Bcl-xl, BAX, Mcl-2 survivin, NOXA and PUMA ) by westernblot noted. Similar results were obtained with their BMR counterparts. Total knock down of Bcl-2 using CRISPR knockout ( kit purchased from Origene) results in cell death. In contrast, transfection of Bcl2 into PD-L1 overexpression results in decreased PD-L1 expression by 2-4 fold both by PCR and by westernblot in both clones. however , there is no discernable changes on surface PD-L1 by flow cytometery. Transfection of BCl-2 also does not affect other BCL-2 family proteins. Similar results were obtained with their BMR counterparts. From these data, it appears that the presence of Bcl-2 ( RNA/protein) could affect intrinsic PD-L1 expression, however, the surface PD-L1 which is the key to bind PD-1 did not change. On the other hand, the presence of PD-L1 RNA/protein has no discernable effect on BcL-2 expression. Whether partial knock down BCl-2 or treatment with Bcl-2 inhibitor can lead to increased PD-L1 expression is not known and is under investigation. Overall, it appears that combination of Bcl-2 inhibitor and check point inhibitor may be more effective in melanoma patients with high Bcl-2 expression. (Supported by VA merit review award; 1BX003328) Citation Format: Niramol Savaraj, Min You, Ying-Ying Li, Chunjing Wu, Medhi Wangpaichitr, Saurez Miguel, Alfred Chicco, Macus T. Kuo, Lynn G. Feun. Targeting immunological and apoptotic cell death to improve therapeutic efficacy in melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 988.

Effects of BCL2 transfection on the cell cycle and proliferation of human GES-1 cells.

We investigated the effects of BCL2 transfection on the cell cycle and proliferation of GES-1 cells. A pcDNA3-BCL2 plasmid was used to transfect GES-1 cell line human gastric epithelial cells. Clones were obtained by G418 screening. BCL2-positive cells were identified by fluorescence immunohistochemistry. The pcDNA3-BCL2 vectors carrying the NeoR gene were transfected into GES-1 cells, while the empty plasmid was transfected into the same cells as controls. BCL2-positive clones were screened by neomycin 418 (G418). Flow cytometry was used to detect the cell cycle. Hematoxylin and eosin (H&E) staining revealed morphological changes, and the effects of BCL2 transfection on cell proliferation were analyzed by cell counting and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. The plasmid pcDNA3-BCL2 was identified by restriction enzyme digestion. Different degrees of BCL2 gene expression were detected in all seven clones. BCL2 was expressed mainly in the cytoplasm and the nuclear membrane. There were significantly more S-phase cells in the transfection group than in the controls. The morphology did not change after H&E staining. Cell growth was faster than in the controls after transfection for 6 days. At 24, 48, and 72 h after transfection, the A values were 4.15 ± 0.31, 5.98 ± 0.56, and 8.94 ± 0.79; those of the controls were 3.01 ± 0.20, 4.76 ± 0.52, and 7.69 ± 0.84; there was a significant difference between the two groups (P < 0.05). BCL2 transfection increased GES-1 cells in the S phase; the GES-1 cells were stable and BCL2 expression was high, which promoted cell proliferation.

Waltonitone induces apoptosis through mir-663-induced Bcl-2 downregulation in non-small cell lung cancer.

Our previous study reported that waltonitone treatment inhibited proliferation and induced apoptosis of lung cancer cells. However, the mechanism of waltonitone-induced toxicity remains unclear. In the present study, we treated H460 and H3255 lung cancer cells using different concentration of waltonitone (0, 10, 20, 30μmol/L). We observed that waltonitone inhibited cell viability and induced apoptosis in a concentration dependent manner, with upregulation of caspase-3 cleavage. We also observed upregulation of miR-663, a potential tumor suppressor, after waltonitone treatment. Suppression of miR-663 function using miR-663 inhibitor partly alleviated cell toxicity induced by waltonitone. In addition, both waltonitone treatment and transfection of miR-663 mimic upregulated Bcl-2 mRNA and protein expression. Bcl-2 transfection alleviated waltonitone-induced toxicity. Furthermore, transfection of miR-663 inhibitor upregulated Bcl-2 levels in both cell lines. In summary, the present study demonstrated that waltonitone induced apoptosis of lung cancer cells through, at least partly, miR-663-induced Bcl-2 downregulation.

BH4 domain of bcl-2 protein is required for its proangiogenic function under hypoxic condition

Beyond its classical role as apoptosis inhibitor, bcl-2 protein promotes tumor angiogenesis and the removal of N-terminal bcl-2 homology (BH4) domain abrogates bcl-2-induced hypoxia-inducible factor 1 (HIF-1)-mediated vascular endothelial growth factor (VEGF) expression in hypoxic cancer cells. Using M14 human melanoma cell line and its derivative clones stably overexpressing bcl-2 wild-type or deleted of its BH4 domain, we found that conditioned media (CM) from cells expressing BH4-deleted bcl-2 protein showed a reduced capability to increase in vitro human endothelial cells proliferation and differentiation, and in vivo neovascularization compared with CM from cells overexpressing wild-type bcl-2. Moreover, xenografts derived from cells expressing bcl-2 lacking BH4 domain showed a reduction of metastatic potential compared with tumors derived from wild-type bcl-2 transfectants injection. Stably expressing the Flag-tagged N-terminal sequence of bcl-2 protein, encompassing BH4 domain, we found that this domain is sufficient to enhance the proangiogenic HIF-1/VEGF axis under hypoxic condition. Indeed, lacking of BH4 domain abolishes the interaction between bcl-2 and HIF-1α proteins and the capability of exogenous bcl-2 protein to localize in the nucleus. Moreover, when endoplasmic reticulum-targeted bcl-2 protein is overexpressed in cells, this protein lost the capability to synergize with hypoxia to induce the proangiogenic HIF-1/VEGF axis as shown by wild-type bcl-2 protein. These results demonstrate that BH4 domain of bcl-2 is required for the ability of this protein to increase tumor angiogenesis and progression and indicate that bcl-2 nuclear localization may be required for bcl-2-mediated induction of HIF-1/VEGF axis.

Loss of Bcl-2 During the Senescence Exacerbates the Impaired Angiogenic Functions in Endothelial Cells by Deteriorating the Mitochondrial Redox State

Ageing is an important risk factor for ischemic cardiovascular diseases, although its underlying molecular mechanisms remain to be elucidated. Here, we report a crucial role of Bcl-2 in the impaired angiogenic functions in senescent endothelial cells (ECs) by modulating the mitochondrial redox state. Cellular senescence impaired angiogenic functions in ECs without attenuating the mitogen-activated protein kinase or Akt signaling, and vascular endothelial growth factor receptor 2 or Tie-2 expressions. We identified that Bcl-2 expression was markedly reduced in 3 independent models for senescent ECs, and pharmacological inhibition, as well as small interfering RNA-mediated gene silencing of Bcl-2, significantly impaired the angiogenic functions in young ECs. Bcl-2 has an antioxidative role by locating the glutathione at mitochondria, and we found that mitochondrial oxidative stress was significantly augmented in senescent ECs, in association with reduced mitochondria-associated glutathione. Transfection of Bcl-2 in senescent ECs significantly reduced the mitochondrial oxidative stress, restored the mitochondrial membrane potential, and improved the angiogenic capacity. Furthermore, gene transfer of Bcl-2 using adenovirus significantly improved the in vivo angiogenesis in the Matrigel plugs implanted into aged mice, whereas the Bcl-2 inhibitor reduced the angiogenesis in the Matrigel plugs implanted into young mice. Together, Bcl-2 plays a crucial role in the regulation of the mitochondrial redox state in ECs, and, thus, loss of Bcl-2 during the senescence exacerbates the impaired angiogenesis by augmenting the mitochondrial oxidative stress.

Protective effects of bone marrow mesenchymal stem cells with stable expression of Bcl-2 on ischemia- hypoxia of hippocampal neurons

The protective effect of bone marrow mesenchymal stem cells (MSCs) with stable expression of Bcl-2 on the in vitro ischemia-hypoxia of hippocampal neurons was investigated in the present study. Mouse Bcl-2 cDNA was amplified by RT-PCR followed by Lentivirus-mediated gene transfection into MSCs. Puromycin resistance was detected to screen MSCs undergoing successful transfection, and the mRNA expression of Bcl-2 was determined by RT-PCR. Serum and oxygen deprivation was performed in normal MSCs and those undergoing transfection aiming to mimic ischemia injury in vitro, and flow cytometry was used to detect the apoptosis rate of these cells. Neurogenic induction of Bcl-2 expressing MSCs was carried out followed by detection of neuron markers. The rat hippocampal neurons were isolated from neonate rats. The Bcl-2 expressing MSCs and hippocampal neurons were cocultured, and the protective effect of Bcl-2 expressing MSCs on the ischemia-hypoxia of hippocampal neurons was observed. Following transfection, MSCs had stable and efficient expression of Bcl-2 after screening. When compared with normal MSCs, the number of apoptotic Bcl-2 expressing cells was significantly decreased and that of survival cells markedly increased after serum and oxygen deprivation. In addition, the Bcl-2 expressing MSCs had the potent differentiation into neurons. In the coculture system, Bcl-2 expressing MSCs could remarkably reduce the apoptosis rate of hippocampal neurons after ischemia-hypoxia (P < 0.01). The MSCs undergoing Bcl-2 transfection had stable Bcl-2 expression, which can protect them from serum and oxygen deprivation induced apoptosis and protect hippocampal neuron against ischemia-hypoxia induced apoptosis in vitro. Furthermore, the Bcl-2 transfecion does not affect the differentiation of MSCs into neurons.

Bcl-2 regulates HIF-1alpha protein stabilization in hypoxic melanoma cells via the molecular chaperone HSP90.

BackgroundHypoxia-Inducible Factor 1 (HIF-1) is a transcription factor that is a critical mediator of the cellular response to hypoxia. Enhanced levels of HIF-1α, the oxygen-regulated subunit of HIF-1, is often associated with increased tumour angiogenesis, metastasis, therapeutic resistance and poor prognosis. It is in this context that we previously demonstrated that under hypoxia, bcl-2 protein promotes HIF-1/Vascular Endothelial Growth Factor (VEGF)-mediated tumour angiogenesis.Methodology/Principal FindingsBy using human melanoma cell lines and their stable or transient derivative bcl-2 overexpressing cells, the current study identified HIF-1α protein stabilization as a key regulator for the induction of HIF-1 by bcl-2 under hypoxia. We also demonstrated that bcl-2-induced accumulation of HIF-1α protein during hypoxia was not due to an increased gene transcription or protein synthesis. In fact, it was related to a modulation of HIF-1α protein expression at a post-translational level, indeed its degradation rate was faster in the control lines than in bcl-2 transfectants. The bcl-2-induced HIF-1α stabilization in response to low oxygen tension conditions was achieved through the impairment of ubiquitin-dependent HIF-1α degradation involving the molecular chaperone HSP90, but it was not dependent on the prolyl hydroxylation of HIF-1α protein. We also showed that bcl-2, HIF-1α and HSP90 proteins form a tri-complex that may contribute to enhancing the stability of the HIF-1α protein in bcl-2 overexpressing clones under hypoxic conditions. Finally, by using genetic and pharmacological approaches we proved that HSP90 is involved in bcl-2-dependent stabilization of HIF-1α protein during hypoxia, and in particular the isoform HSP90β is the main player in this phenomenon.Conclusions/SignificanceWe identified the stabilization of HIF-1α protein as a mechanism through which bcl-2 induces the activation of HIF-1 in hypoxic tumour cells involving the β isoform of molecular chaperone HSP90.

PKC-δ Promotes Renal Tubular Cell Apoptosis Associated with Proteinuria

Proteinuria may contribute to progressive renal damage by inducing tubulointerstitial inflammation, fibrosis, and tubular cell injury and death, but the mechanisms underlying these pathologic changes remain largely unknown. Here, in a rat kidney proximal tubular cell line (RPTC), albumin induced apoptosis in a time- and dose-dependent manner. Caspase activation accompanied albumin-induced apoptosis, and general caspase inhibitors could suppress this activation. In addition, Bcl-2 transfection inhibited apoptosis and attenuated albumin-induced Bax translocation to mitochondria and cytochrome c release from the organelles, further confirming a role for the intrinsic pathway of apoptosis in albuminuria-associated tubular apoptosis. We observed phosphorylation and activation of PKC-delta early during treatment of RPTC cells with albumin. Rottlerin, a pharmacologic inhibitor of PKC-delta, suppressed albumin-induced Bax translocation, cytochrome c release, and apoptosis. Moreover, a dominant-negative mutant of PKC-delta blocked albumin-induced apoptosis in RPTC cells. In vivo, we observed activated PKC-delta in proteinuric kidneys of streptozotocin-induced diabetic mice and in kidneys after direct albumin overload. Notably, albumin overload induced apoptosis in renal tubules, which was less severe in PKC-delta-knockout mice. Taken together, these results suggest that activation of PKC-delta promotes tubular cell injury and death during albuminuria, broadening our understanding of the pathogenesis of progressive proteinuric kidney diseases.

Goniothalamin-induced oxidative stress, DNA damage and apoptosis via caspase-2 independent and Bcl-2 independent pathways in Jurkat T-cells

Goniothalamin (GTN) isolated from Goniothalamus sp. has been demonstrated to induce apoptosis in a variety of cancer cell lines including Jurkat T leukemia cells. However, the mechanism of GTN-induced apoptosis upstream of mitochondria is still poorly defined. In this study, GTN caused a decrease in GSH with an elevation of reactive oxygen species as early as 30min and DNA damage as assessed by Comet assay. Analysis using topoisomerase II processing of supercoiled pBR 322 DNA showed that GTN caused DNA damage via a topoisomerase II-independent pathway suggesting that cellular oxidative stress may contribute to genotoxicity. A 12-fold increase of caspase-2 activity was observed in GTN-treated Jurkat cells after 4h treatment and this was confirmed using Western blotting. Although the caspase-2 inhibitor Z-VDVAD-FMK inhibited the proteolytic activity of caspase-2, apoptosis ensued confirming that caspase-2 activity was not crucial for GTN-induced apoptosis. However, GTN-induced apoptosis was completely abrogated by N-acetylcysteine further confirming the role of oxidative stress. Since cytochrome c release was observed as early as 1h without any appreciable change in Bcl-2 protein expression, we further investigated whether overexpression of Bcl-2 confers resistance in GTN-induced cytotoxicity. Using a panel of Jurkat Bcl-2 transfectants, GTN cytotoxicity was not abrogated in these cells. In conclusion, GTN induces DNA damage and oxidative stress resulting in apoptosis which is independent of both caspase-2 and Bcl-2.

Divergent pathways for TNF and C2-ceramide toxicity in HTC hepatoma cells

We previously showed that, in the rat hepatoma cell line HTC, TNF brings about a non-caspase-dependent, apoptosis-like process requiring NADPH oxidase activity, an iron-mediated pro-oxidant status, and a functional acidic vacuolar compartment. This process may thus involve mechanisms such as autophagy or relocation of lysosomal enzymes, perhaps secondary to the formation of ceramide by acidic sphingomyelinase. Here we investigated whether ceramide formation contributes to the apoptogenic process. HTC cells were found to be sensitive to exogenous ceramide and significantly protected against TNF by desipramine, an inhibitor of lysosomal acid sphingomyelinase. However, Bcl-2 transfection and Bcl-xL upregulation by dexamethasone significantly diminished the apoptogenic effect of ceramide but not that of TNF, suggesting that ceramide is not directly involved in TNF toxicity. Moreover, Bcl-xL silencing precluded dexamethasone-induced protection against ceramide and, by itself, induced massive death, demonstrating the strict dependence of HTC cells on Bcl-xL for survival also under standard culture conditions.

Bcl-2 Small Interfering RNA Sensitizes Cisplatin-resistant Human Lung Adenocarcinoma A549/DDP Cell to Cisplatin and Diallyl Disulfide

Bcl-2 is overexpressed in a variety of human tumors and is involved in tumorigenesis and chemoresistance. In this study, we investigated the inhibitory effect of the hairpin Bcl-2 small interfering (si)RNA on the expression of the Bcl-2 gene in the cisplatin (DDP)-resistant human lung adenocarcinoma cell line A549/DDP, and the effect of Bcl-2 siRNA on drug sensitization in A549/DDP cells. Bcl-2 siRNA and negative siRNA plasmids were constructed and stably transfected into A549/DDP cells. Reverse transcription-polymerase chain reaction, immunofluorescence microscopy and Western blot analysis were used to detect the target gene expression. Spontaneous cell apoptosis was detected by acridine orange and ethidium bromide staining. Drug sensitivity of the cells to DDP and diallyl disulfide (DADS) was analyzed by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay and flow cytometry. Expression levels of Bcl-2 mRNA and protein in siRNA stable transfectants were clearly reduced compared with negative siRNA transfectants and untreated cells. MTT results indicated that Bcl-2 transfectants had a higher cell inhibition rate after treatment with 0.2-200 microg/ml DDP or 50-200 microM DADS. Flow cytometry revealed increased apoptosis in Bcl-2 siRNA cells. After the addition of 20 microg/ml DDP or 100 microM DADS, siRNA targeting of the Bcl-2 gene specifically down-regulated gene expression in A549/DDP cells, increased spontaneous apoptosis, and sensitized cells to DDP and DADS.

Sphingolipids and the sphingosine kinase inhibitor, SKI II, induce BCL‐2‐independent apoptosis in human prostatic adenocarcinoma cells

Elevated BCL-2 is one mechanism of therapeutic resistance in prostate cancer (PC), and new approaches are needed to overcome such resistance. We evaluated the effects of BCL-2 over-expression in human prostatic adenocarcinoma cells on their susceptibility to sphingolipids (SLs) and to the sphingosine kinase (SpK) inhibitor, SKI II. In survival assays, no significant differences were observed in the responses to sphingosine or ceramide among parental PC-3 cells lacking detectable BCL-2 and BCL-2 over-expressing PC-3 transfectants; similarly, the responses to dimethyl-sphingosine (DMSP) of parental LNCaP cells and a BCL-2 over-expressing LNCaP transfectant were equivalent. SKI II induced protracted, BCL-2-independent survival loss in both PC-3 and LNCaP parental/transfectant pairs; in contrast, DMSP induced rapid cell shrinkage, caspase activation and caspase-dependent DNA fragmentation. DMSP-induced DNA fragmentation and loss of mitochondrial membrane potential were equivalent in BCL-2 transfectants and parental PC-3 cells and were not associated with BCL-2 downregulation. DMSP-mediated cytotoxicity was not associated with the enhanced production of reactive oxygen intermediates. SL analyses of parental and transfectant PC-3 cells did not reveal increased levels of sphingosine-1-phosphate in the BCL-2 transfectants; further, there only a modest early shift, corresponding to apoptotic onset, in pro- versus anti-apoptotic SLs in response to DMSP treatment. Thus, in contrast to the inhibitory effects of BCL-2 on apoptosis induced by various agents in tumor cells, SKI II and selected pro-apoptotic SLs appear atypical in their independence from such inhibition, and may have merits as new candidates for treatment of AI PC.

Bcl-2 Localized at the Nuclear Compartment Induces Apoptosis after Transient Overexpression

Bcl-2 is the best characterized member of a large family of proteins that regulate apoptosis. Although it is established that Bcl-2 localized at the mitochondria functions as an anti-apoptotic protein, the function of Bcl-2 at the nucleus remains unclear. Recently we showed that nuclear compartment-associated Bcl-2 inhibits transcription factor activation. Based on this observation, we hypothesized that presence of Bcl-2 at the nucleus may induce rather than protect cells from apoptosis. Here we investigated the putative apoptotic role of nuclear compartment-associated Bcl-2. Additionally, we examined the role of the Bcl-2 BH4 domain in mediating binding to FKBP38, the Bcl-2 mitochondrial chaperone. Our results demonstrate a novel, pro-apoptotic function for nuclear Bcl-2 and identify the Bcl-2 BH4 domain as a key regulator in mediating Bcl-2/FKBP38 binding. These results indicate that Bcl-2 has a dual role as both a protector and a killer and that the ability to switch roles depends on Bcl-2 subcellular localization.

Bcl-2 overexpression disrupts the morphology of PC12 cells through reduced ERK activation

Bcl-2 has been hypothesized to regulate many cellular functions in addition to its well-characterized role in the prevention of programmed cell death. To understand the role of Bcl-2 in regulating cell morphology and to explore the mechanism of this effect, we examined the effects of Bcl-2 overexpression on the morphology of PC12 cells in culture. We demonstrate that the overexpression of Bcl-2 in PC12 cells results in altered cell morphology and reduced actin expression. Analysis of extracellular signal-regulated kinase (ERK) 1/2 phosphorylation reveals that the morphological changes seen after bcl-2 transfection are associated with reduced ERK activation. Treatment of control (mock-transfected) PC12 cells with the mitogen-activated ERK-activating kinase (MEK) inhibitor PD98059 converts their flat, process-bearing morphology into the rounded, process-free morphology of bcl-2-transfected cells, further confirming the association of ERK activation with altered cell shape. In conclusion, the present study describes a novel function of Bcl-2 in regulating cell shape through reduced ERK activation.

The Role of Bcl-2 in Apoptosis Induced by khat (Catha Edulis) in Acute Myeloid Leukemia Cell Lines.

The herbal drug khat (Catha edulis Forsk.) is habitually used in Eastern Africa and the Middle East. The pharmacological activity of khat is due to its alkaloid fraction which initiates a rapid release of monoamine neurotransmitters from nerve terminals when ingested. Extracts from khat have been shown to induce cell death caspase-dependent apoptosis in human leukaemia cells. In this study, apoptotic sensitivity is correlated with endogenous protein expression of the anti-apoptogen Bcl-2, as well as transfection enforced over expression of Bcl-2. Exposure of a panel of human leukaemia cell lines to khat extract resulted in the development of similar apoptotic morphotypes. The sensitivity of the cell lines to khat induced apoptosis revealed a trend of inverse correlation with endogenous levels of Bcl-2. Stable or transient transfection of Bcl-2 inhibited khat-induced apoptosis, and the threshold for khat-induced apoptosis was lowered by blocking Bcl-2 synthesis with small interfering RNA. Together, these data suggest Bcl-2 modulation as a possible mechanistic pathway through which khat-induced apoptosis may occur.

Bcl-2 Promotes Invasion and Lung Metastasis by Inducing Matrix Metalloproteinase-2

Bcl-2 is involved in the progression of human malignancies, but the precise role and mechanism of Bcl-2 for tumor invasion and metastasis remains unclear. In this study, we have investigated the role and mechanism of Bcl-2 on tumor cell invasion and metastasis by using Bcl-2 overexpressing non-small cell lung cancer cells. Matrix metalloproteinases (MMPs) are important proteins involved in the processes of tumor invasion and metastasis. In vitro Matrigel invasion assays showed that Bcl-2 overexpression increased tumor cell invasion by 15-fold. Moreover, Bcl-2 overexpression enhanced in vivo lung metastasis by 4-fold. Consistent with its effect on invasion and metastasis, Bcl-2 overexpression induced not only MMP-2 mRNA and its protein expression, but this also activated the pro-MMP-2 protein to its active form. To explore the induction mechanism of MMP-2 by Bcl-2, we investigated the effects of Bcl-2 overexpression on MMP-2 transcriptional regulation. Nuclear run-on assays showed a 6-fold increase in the transcription rate of MMP-2 mRNA in the Bcl-2 transfectants (H157/Bcl-2) compared with that of the H157/vector control cells (H157/C). Overexpression of Bcl-2 induced the nuclear transcription factor activator protein 1 family, including the c-Jun, JunD, c-Fos, FosB, and Fra-1 proteins. Reporter assays combined with deletion mutagenesis analysis and gel shift assays showed the involvement of activator protein 1 in the activation of MMP-2 promoter activity by Bcl-2. Taken together, we have shown that Bcl-2 promotes tumor invasion and lung metastasis by inducing MMP-2 gene expression through the combined action of transcriptional and posttranslational mechanisms.

Bcl-2 Rescues Ceramide- and Etoposide-induced Mitochondrial Apoptosis through Blockage of Caspase-2 Activation

Recent studies indicate that caspase-2 is involved in the early stage of apoptosis before mitochondrial damage. Although the activation of caspase-2 has been shown to occur in a large protein complex, the mechanisms of caspase-2 activation remain unclear. Here we report a regulatory role of Bcl-2 on caspase-2 upstream of mitochondria. Stress stimuli, including ceramide and etoposide, caused caspase-2 activation, mitochondrial damage followed by downstream caspase-9 and -3 activation, and cell apoptosis in human lung epithelial cell line A549. When A549 cells were pretreated with the caspase-2 inhibitor benzyloxycarbonyl-Val-Asp(-OMe)-Val-Ala-Asp(-OMe)-fluoromethyl ketone or transfected with caspase-2 short interfering RNA, both ceramide- and etoposide-induced mitochondrial damage and apoptosis were blocked. Overexpression of Bcl-2 prevented ceramide- and etoposide-induced caspase-2 activation and mitochondrial apoptosis. Furthermore, caspase-2 was activated when A549 cells were introduced with Bcl-2 short interfering RNA or were treated with Bcl-2 inhibitor, which provided direct evidence of a negative regulatory effect of Bcl-2 on caspase-2. Cell survival was observed when caspase-2 was inhibited in Bcl-2-silencing cells. Blockage of the mitochondrial permeability transition pore and caspase-9 demonstrated that Bcl-2-modulated caspase-2 activity occurred upstream of mitochondria. Further studies showed that Bcl-2 was dephosphorylated at serine 70 after ceramide and etoposide treatment. A protein phosphatase inhibitor, okadaic acid, rescued Bcl-2 dephosphorylation and blocked caspase-2 activation, mitochondrial damage, and cell death. Taken together, ceramide and etoposide induced mitochondria-mediated apoptosis by initiating caspase-2 activation, which was, at least in part, regulated by Bcl-2.

Bcl‐2 overexpression in melanoma cells increases tumor progression‐associated properties and in vivo tumor growth

In this study, we demonstrated that bcl-2 overexpression in human melanoma cells consistently enhanced the activity of multiple metastasis-related proteinases, in vitro cell invasion, and in vivo tumor growth. In particular, by using the M14 parental cell line, the MN8 control clone, and two bcl-2 overexpressing derivatives, we found that bcl-2 overexpressing cells exposed to hypoxia, when compared to parental cells, expressed higher level of several metalloproteases (MMPs) such as MMP-2, MMP-7, MT1-MMP, and tissue inhibitors of metalloproteases-1 and -2. Moreover, bcl-2 overexpression in melanoma cells enhanced in vitro invasion on matrigel and, in vivo tumor growth. The more aggressive behavior of bcl-2 transfectants tumors is significantly associated to an increase in MMP-2 expression as well as in a more elevated microvessel density as compared to the parental line. Taken together, our data suggest that bcl-2 plays a pivotal role in the regulation of molecules associated with the migratory and invasive phenotype, contributing, in cooperation to hypoxia, to tumor progression.

Overexpression of Bcl-2 is associated with apoptotic resistance to the G-quadruplex ligand 12459 but is not sufficient to confer resistance to long-term senescence

The triazine derivative 12459 is a potent G-quadruplex interacting agent that inhibits telomerase activity. This agent induces time- and dose-dependent telomere shortening, senescence-like growth arrest and apoptosis in the human A549 tumour cell line. We show here that 12459 induces a delayed apoptosis that activates the mitochondrial pathway. A549 cell lines selected for resistance to 12459 and previously characterized for an altered hTERT expression also showed Bcl-2 overexpression. Transfection of Bcl-2 into A549 cells induced a resistance to the short-term apoptotic effect triggered by 12459, suggesting that Bcl-2 is an important determinant for the activity of 12459. In sharp contrast, the Bcl-2 overexpression was not sufficient to confer resistance to the senescence-like growth arrest induced by prolonged treatment with 12459. We also show that 12459 provokes a rapid degradation of the telomeric G-overhang in conditions that paralleled the apoptosis induction. In contrast, the G-overhang degradation was not observed when apoptosis was induced by camptothecin. Bcl-2 overexpression did not modify the G-overhang degradation, suggesting that this event is an early process uncoupled from the final apoptotic pathway.

Antisense bcl-2 transfection up-regulates anti-apoptotic and anti-oxidant thioredoxin in neuroblastoma cells

Antisense bcl-2 therapy combined with chemotherapy has been proved to be effective in various tumors. However, the role played by antisense bcl-2 therapy alone is not clear. In this study, we compared the apoptosis and the protein profiles of antisense bcl-2 transfected human neuroblastoma SK-N-MC cells to the control cells. Flow cytometric data indicated that antisense bcl-2 transfection did not lead to more extensive apoptosis in SK-N-MC cells (14.9 +/- 3.8%) than the control cells (10.3 +/- 2.3%). The above observation was confirmed by fluorescence microscopy using Hoechst 33258 staining. However, antisense bcl-2 induced changes in the expression of various proteins as shown by proteomic comparison, which included the up-regulation of the anti-apoptotic and anti-oxidant protein thioredoxin. By western blot validation, thioredoxin was found to be up-regulated by 2.9-folds with the corresponding down-regulation of Bcl-2 by 2.1-folds. The up-regulation of thioredoxin may be a compensating mechanism for cell survival in neuroblastoma when Bcl-2 expression is suppressed, and it may to some extent attenuate the effectiveness of antisense bcl-2 therapy.