Bcl-2 and Bcl-xL play important roles in the crosstalk between autophagy and apoptosis

Autophagy and apoptosis play important roles in the development, cellular homeostasis and, especially, oncogenesis of mammals. They may be triggered by common upstream signals, resulting in combined autophagy and apoptosis. In other instances, they may be mutually exclusive. Recent studies have suggested possible molecular mechanisms for crosstalk between autophagy and apoptosis. Bcl-2 and Bcl-xL, the well-characterized apoptosis guards, appear to be important factors in autophagy, inhibiting Beclin 1-mediated autophagy by binding to Beclin 1. In addition, Beclin 1, Bcl-2 and Bcl-xL can cooperate with Atg5 or Ca(2+) to regulate both autophagy and apoptosis. Thus, Bcl-2 and Bcl-xL represent a molecular link between autophagy and apoptosis. Here, we discuss the possible roles of Bcl-2 and Bcl-xL in apoptosis and autophagy, and the crosstalk between them.     

Survivin inhibition is critical for Bcl-2 inhibitor-induced apoptosis in hepatocellular carcinoma cells

Our study aims to study the therapeutic effects of a novel Bcl-2 inhibitor, ABT-263, on hepatocellular carcinoma (HCC) and to provide primary preclinical data for future clinical trial with ABT-263. In this study we showed that Bcl-xL and survivin were up-regulated in HCC cell lines and human liver cancer tissues. Clinic used ABT-263 single treatment had no apoptotic effects on HCC cells whereas higher doses of ABT-263 did. Interestingly, the combination treatment of ABT-263 with survivin inhibitor YM-155 could result in significant apoptosis in HCC cells. Survivin inhibition through gene silencing significantly enhanced ABT-263 to induce apoptosis in HCC cells. We found that low dose of ABT-263 single treatment resulted in ERK activation and survivin up-regulation, which might be involved in the resistance of HCC cells to ABT-263 since blockade of ERK activation sensitized ABT-263-induced apoptosis. Importantly, ABT-263 and YM-155 combination treatment had no apoptotic effects on normal human hepatocytes. Taken together, these data suggest the combination treatment of Bcl-2 inhibitor and survivin inhibition may have a great potential for liver cancer therapy.     

Unidirectional crosstalk between Bcl-xL and Bcl-2 enhances the angiogenic phenotype of endothelial cells

Expression of Bcl-x(L) correlates with the clinical outcomes of patients with cancer. While the role of Bcl-2 in angiogenesis is becoming increasingly evident, the function of Bcl-x(L) in angiogenesis is unclear. Here, we showed that epidermal growth factor (EGF) induces in vitro capillary sprouting and Bcl-x(L) expression in primary endothelial cells. Bcl-x(L)-transduced human dermal microvascular endothelial cells (HDMEC-Bcl-x(L)), but not empty vector control cells, spontaneously organize into capillary-like sprouts. Searching for a mechanism to explain these responses, we observed that Bcl-x(L) induced expression of the pro-angiogenic chemokines CXC ligand-1 (CXCL1) and CXC ligand-8 (CXCL8), and that blockade of CXC receptor-2 (CXCR2) signaling inhibited spontaneous sprouting of HDMEC-Bcl-x(L). Bcl-x(L) led to Bcl-2 upregulation, but Bcl-2 did not upregulate Bcl-x(L), suggesting the existence of a unidirectional crosstalk from Bcl-x(L) to Bcl-2. EGF and Bcl-x(L) activate the mitogen-activated protein kinase/ERK pathway resulting in upregulation of vascular endothelial growth factor (VEGF), a known inducer of Bcl-2 in endothelial cells. Inhibition of VEGF receptor signaling in HDMEC-Bcl-x(L) prevented Bcl-2 upregulation and demonstrated the function of a VEGF-mediated autocrine loop. Bcl-2 downregulation by RNAi blocked CXCL1 and CXCL8 expression downstream of Bcl-x(L), and markedly decreased angiogenesis in vivo. We conclude that Bcl-x(L) functions as a pro-angiogenic signaling molecule controlling Bcl-2 and VEGF expression. These results emphasize a complex interplay between Bcl-2 family members beyond their classical roles in apoptosis.     

Bcl-xL promotes metastasis of breast cancer cells by induction of cytokines resistance

Metastasis is a highly complex process involving the survival of tumor cells, both in the blood stream and within specific organs. Cell-death and survival are determined by a number of gene products from an expanding family of the Bcl-2 gene, either promoting or preventing apoptosis. Furthermore, the survival of tumor cells may favor the accumulation of additional genetic alterations causing further growth and invasive opportunities which may lead to metastasis. To examine whether the prevention of cell-death influences the metastatic behavior, we transfected a human breast cancer cell line MDA-MB-435 with the Bcl-xL cDNA and then studied metastatic ability of the selected clones in vivo. Our results show that Bcl-xL-clones had a decreased tumor growth latency and an increased metastatic ability. Apoptosis-resistance to cytokines was induced in 435 cells by Bcl-xL-expression with minor modifications in their proliferation rates. These cells also showed diminished adhesion to extracellular matrix proteins and a survival advantage in suspension over 435/Neo cells. Moreover, to determine survival in blood stream and in cells lodged in the lungs, we injected 435/Bcl-xL and 435/Neo cells at 1:3 proportion i.v., and animals were killed at intervals of 15' to 16 h after injection. Tumor cells were recovered from the lungs and Southern-blot analysis revealed the presence of exogenous Bcl-xL cDNA. These results showed that 435/Bcl-xL cells had a survival advantage in circulation over 435/Neo cells. This advantage in vivo was attributable to Bcl-xL expression. We conclude that Bcl-xL expression in breast cancer cells can increase metastatic activity. This advantage could be created by inducing resistance to apoptosis against cytokines, increasing cell survival in circulation, and enhancing anchorage-independent growth.     

Unknotting the roles of Bcl-2 and Bcl-xL in cell death

The antiapoptotic Bcl-2 family proteins Bcl-2 and Bcl-xL play important roles in inhibiting mitochondria-dependent extrinsic and intrinsic cell death pathways. It seems that these two proteins have distinct functions for inhibiting extrinsic and intrinsic cell death pathways. The overexpression of Bcl-2 is able to inhibit not only apoptotic cell death but also in part nonapoptotic cell death, which has the role of cell cycle arrest in the G1 phase, which may promote cellular senescence. The overexpression of Bcl-2 may also have the ability to enhance cell death in the interaction of Bcl-xL with other factors. The overexpression of Bcl-xL enhances autophagic cell death when apoptotic cell death is inhibited in Bax(-/-)/Bak(-/-) double knockout cells. This review discusses the previously unexplained aspects of Bcl-2 and Bcl-xL functions associated with cell death, for better understanding of their functions in the regulation.     

Effect of arsenic trioxide on human hepatocellular carcinoma HepG2 cells: inhibition of proliferation and induction of apoptosis

Arsenic trioxide (As(2)O(3)), a major ingredient of Traditional Chinese Medicine (TCM), is found to be an effective anticancer drug in acute promyelocytic leukemia (APL). The present study explored the use of As(2)O(3) on human hepatocellular carcinoma by in vitro study. The study showed that the clinically achievable concentration of As(2)O(3), i.e. 2 microM, inhibited the cell proliferation of human hepatocellular carcinoma cell line, HepG2, in a time-dependent manner. The mechanistic study showed that 2 microM of As(2)O(3) acted through induction of apoptosis in which caspase-3 was activated. The results also suggested that mitochondria did not take part in As(2)O(3)-induced apoptosis.     

Epigenetic loss of CDH1 correlates with multidrug resistance in human hepatocellular carcinoma cells

Promoter CpG hypermethylation of tumor suppressor genes is an essential step in cancer progression but little is known about its effect on cancer multidrug resistance. In this study, we showed that CDH1 promoter was hypermethylated in drug resistance of a doxorubicin-induced multidrug resistant hepatocellular carcinoma cell line R-HepG2. Transfection of CDH1 cDNA into R-HepG2 cells led to increased amount of doxorubicin uptake, decreased cell viability, decreased P-glycoprotein expression and increased apoptotic population of cells exposed to doxorubicin. Proto-oncogene tyrosine-protein kinase FYN was over-expressed in R-HepG2 cells which displayed a negative correlation with the expression of CDH1. FYN was knocked down in R-HepG2 cells, leading to less drug resistance by increased cell viability, increased doxorubicin uptake and attenuated P-glycoprotein expression. Our findings identified epigenetic silencing of CDH1 in cancer cells might be a new molecular event of multidrug resistance.     

Modulation of mitochondrial permeability transition pore affects multidrug resistance in human hepatocellular carcinoma cells

Multidrug resistance (MDR) is a critical problem in the chemotherapy of cancers. Human hepatocellular carcinoma (HCC) responds poorly to chemotherapy owing to its potent MDR. Chemotherapeutic drugs primarily act by inducing apoptosis of cancer cells, and defects in apoptosis may result in MDR. Mitochondrial permeability transition (mPT) is implicated as an important event in the control of cell death or survival and mPT represents a target for the development of cytotoxic drugs. This study aimed to investigate the effects of selective opener (Atractyloside glycoside, ATR) and inhibitor (Cyclosporine A, CsA) of mitochondrial permeability transition pore (mPTP) on a CDDP-resistant HCC cell line (SK-Hep1 cells). In this study, a stable MDR phenotype characterization of SK-Hep1 cell line (SK-Hep1/CDDP cells) was established and used to investigate the role of mPTP in MDR. Results suggested that ATR accelerated the decrease of mitochondrial membrane potential (ΔΨm), reduced the Bax activity, and increased the apoptosis of SK-Hep1/CDDP cells; while CsA inhibited mPTP opening, reduced and delayed the decline of mitochondrial membrane potential, and increased the Bax activity, leading to increased tolerance of SK-Hep1/CDDP cells to apoptosis induction. However, mPTP activity had no effect on the expression of MDR1 in cells,meanwhile the P-gp translocation to mitochondria was increased, and functionally activated. In conclusion, selective modulation of mPTP can affect MDR in human HCC cells. Therefore, activation of mPTP may provide a new strategy to sensitize cancer cells to chemotherapeutic drugs and to reverse the MDR in cancer cells.     

HIV-specific CD8+ T cells exhibit markedly reduced levels of Bcl-2 and Bcl-xL

Human immunodeficiency virus-specific CD8(+) T cells are highly sensitive to spontaneous and CD95/Fas-induced apoptosis, and this sensitivity may impair their ability to control HIV infection. To elucidate the mechanism behind this sensitivity, in this study we examined the levels of antiapoptotic molecules Bcl-2 and Bcl-x(L) in HIV-specific CD8(+) T cells from HIV-infected individuals. Bcl-2 expression was markedly decreased in HIV-specific CD8(+) T cells compared with CMV-specific and total CD8(+) T cells from HIV-infected individuals as well as total CD8(+) T cells from healthy donors. CD8(+) T cell Bcl-2 levels inversely correlated with spontaneous and CD95/Fas-induced apoptosis of CD8(+) T cells from HIV-infected individuals. HIV-specific CD8(+) T cells also had significantly lower levels of Bcl-x(L) compared with CMV-specific CD8(+) T cells. Finally, IL-15 induces both Bcl-2 and Bcl-x(L) expression in HIV-specific and total CD8(+) T cells, and this correlated with apoptosis inhibition and increased survival in both short- and long-term cultures. Our data indicate that reduced Bcl-2 and Bcl-x(L) may play an important role in the increased sensitivity to apoptosis of HIV-specific CD8(+) T cells and suggest a possible mechanism by which IL-15 increases their survival.     

Leptin-leptin receptor are involved in angiogenesis in human hepatocellular carcinoma

Recent evidence in vitro and in vivo indicates that leptin, an adipose tissue-secreted hormone which is involved in the regulation of satiety, metabolic rate and thermogenesis, is implicated in angiogenesis. However, the role of leptin-mediated angiogenesis in hepatic carcinogenesis has not yet been completely elucidated. In this study, we have correlated microvascular density and leptin/leptin receptor (Ob-R) expression in endothelial and tumor cells with the histopathological type in human hepatocellular carcinoma (HCC). For this purpose, specimens of 40 primary HCC were submitted to immunohistochemical investigation using anti-CD31, anti-leptin and anti-Ob-R antibodies. Poorly-differentiated HCC had a higher degree of vascularization than other stages and leptin/Ob-R expression in both tumor and endothelial cells increased in parallel with the grade of malignancy and was highly correlated with the degree of angiogenesis. In the chick embryo chorioallantoic membrane in vivo assay, HCC biopsy specimens induced a strong angiogenic response, which was counteracted by an anti-leptin antibody. Taken together, these findings indicate that leptin/Ob-R correlate with angiogenesis and tumor progression in patients with HCC and that an anti-leptin antibody exerts an angiostatic activity in HCC.     

Towards the next generation of dual Bcl-2/Bcl-xL inhibitors

Structural modifications of the left-hand side of compound 1 were identified which retained or improved potent binding to Bcl-2 and Bcl-x_L in in vitro biochemical assays and had strong activity in an RS4;11 apoptotic cellular assay. For example, sulfoxide diastereomer 13 maintained good binding affinity and comparable cellular potency to 1 while improving aqueous solubility. The corresponding diastereomer (14) was significantly less potent in the cell, and docking studies suggest that this is due to a stereochemical preference for the R_S versus S_S sulfoxide. Appending a dimethylaminoethoxy side chain (27) adjacent to the benzylic position of the biphenyl moiety of 1 improved cellular activity by approximately three-fold, and this activity was corroborated in cell lines overexpressing Bcl-2 and Bcl-x_L.     

P-glycoprotein enhances radiation-induced apoptotic cell death through the regulation of miR-16 and Bcl-2 expressions in hepatocellular carcinoma cells

P-glycoprotein (Pgp), an efflux pump, was confirmed the first time to regulate the expressions of miR/gene in cells. Pgp is known to be associated with multidrug resistance. RHepG2 cells, the multidrug resistant subline of human hepatocellular carcinoma HepG2 cells, expressed higher levels of Pgp as well as miR-16, and lower level of Bcl-2 than the parental cells. In addition, RHepG2 cells were more radiation sensitive and showed more pronounced radiation-induced apoptotic cell death than the parental cells. Mechanistic analysis revealed that transfection with mdr1 specific antisense oligos suppressed radiation-induced apoptosis in HepG2 cells. On the other hand, ectopic mdr1 expression enhanced radiation-induced apoptosis in HepG2 cells, SK-HEP-1 cells, MiHa cells, and furthermore, induced miR-16 and suppressed its target gene Bcl-2 in HepG2 cells. Moreover, the enhancement effects of Pgp and miR-16 on radiation-induced apoptosis were counteracted by overexpression of Bcl-2. The Pgp effect on miR-16/Bcl-2 was suppressed by Pgp blocker verapamil indicating the importance of the efflux of Pgp substrates. The present study is the first to reveal the role of Pgp in regulation of miRNA/gene expressions. The findings may provide new perspective in understanding the biological function of Pgp.     

Noxa/Bcl-2 protein interactions contribute to bortezomib resistance in human lymphoid cells

Previous studies have suggested that the BH3 domain of the proapoptotic Bcl-2 family member Noxa only interacts with the anti-apoptotic proteins Mcl-1 and A1 but not Bcl-2. In view of the similarity of the BH3 binding domains of these anti-apoptotic proteins as well as recent evidence that studies of isolated BH3 domains can potentially underestimate the binding between full-length Bcl-2 family members, we examined the interaction of full-length human Noxa with anti-apoptotic human Bcl-2 family members. Surface plasmon resonance using bacterially expressed proteins demonstrated that Noxa binds with mean dissociation constants (K(D)) of 3.4 nm for Mcl-1, 70 nm for Bcl-x(L), and 250 nm for wild type human Bcl-2, demonstrating selectivity but not absolute specificity of Noxa for Mcl-1. Further analysis showed that the Noxa/Bcl-2 interaction reflected binding between the Noxa BH3 domain and the Bcl-2 BH3 binding groove. Analysis of proteins expressed in vivo demonstrated that Noxa and Bcl-2 can be pulled down together from a variety of cells. Moreover, when compared with wild type Bcl-2, certain lymphoma-derived Bcl-2 mutants bound Noxa up to 20-fold more tightly in vitro, pulled down more Noxa from cells, and protected cells against killing by transfected Noxa to a greater extent. When killing by bortezomib (an agent whose cytotoxicity in Jurkat T-cell leukemia cells is dependent on Noxa) was examined, apoptosis was enhanced by the Bcl-2/Bcl-x(L) antagonist ABT-737 or by Bcl-2 down-regulation and diminished by Bcl-2 overexpression. Collectively, these observations not only establish the ability of Noxa and Bcl-2 to interact but also identify Bcl-2 overexpression as a potential mechanism of bortezomib resistance.     

Cell death induction by CTL: perforin/granzyme B system dominantly acts for cell death induction in human hepatocellular carcinoma cells

Cell death induction by cytotoxic T lymphocytes (CTLs) is an important thesis for the understanding of tumor immunotherapy. In the current study we investigated the molecular machinery of CTL-induced cell death in human hepatocellular carcinoma cell lines (HCC lines). CTLs prepared from human peripheral blood induced cell death in all tested HCC lines. As the CTL-induced death system, the effectiveness of Fas ligand/Fas and/or Perforin/Granzyme B systems has been suggested, whereas cell death induction by CTLs was shown independently on Fas expression in the current study. Using various tetrapeptide inhibitors for caspase and its associated factor, we additionally demonstrated that inhibitors for caspase 3 (Ac-DEVD-CHO) and caspase 8/granzyme B (Ac-IETD-CHO) suppressed CTL-induced cell death, but an inhibitor for Fas-activated serine proteinase, which acts for the caspase 3 activator, did not, suggesting that CTL-induced cell death was initiated by the Perforin/Granzyme B system, rather than the Fas ligand/Fas system. On the basis of our current results, we report here that the Perforin/Granzyme B system acts dominantly for the cell death induction of HCC lines.     

VEGF is essential for the growth and migration of human hepatocellular carcinoma cells

Vascular endothelial growth factor (VEGF) plays a crucial role in tumor angiogenesis. VEGF induces new vessel formation and tumor growth by inducing mitogenesis and chemotaxis of normal endothelial cells and increasing vascular permeability. However, little is known about VEGF function in the proliferation, survival or migration of hepatocellular carcinoma cells (HCC). In the present study, we have found that VEGF receptors are expressed in HCC line BEL7402 and human HCC specimens. Importantly, VEGF receptor expression correlates with the development of the carcinoma. By using a comprehensive approaches including TUNEL assay, transwell and wound healing assays, migration and invasion assays, adhesion assay, western blot and quantitative RT-PCR, we have shown that knockdown of VEGF165 expression by shRNA inhibits the proliferation, migration, survival and adhesion ability of BEL7402. Knockdown of VEGF165 decreased the expression of NF-κB p65 and PKCα while increased the expression of p53 signaling molecules, suggesting that VEGF functions in HCC proliferation and migration are mediated by P65, PKCα and/or p53.     

Glycine inhibits angiogenic signaling in human hepatocellular carcinoma cells

Hepatocellular carcinoma (HCC) is a highly vascularized tumor with limited susceptibility to chemotherapy. Modern targeted therapies are aimed at specific properties of this neoplasm. Glycine is a simple non-essential amino acid with potential antiangiogenic effects. In this study, the amino acid’s effect on angiogenic signaling in an in vitro model of HCC was evaluated. HepG2 and Huh7 cells were treated with glycine-free DMEM supplemented with 0, 0.01, 0.1, 1.0, 2.0, 5.0 and 10 mM glycine. The direct effects of glycine on the viability of HCC cells were monitored using MTT assay. To detect angiogenic signaling, mRNA and protein levels of vascular endothelial growth factor (VEGF-A) were measured using RT-PCR and Western Blot assays. To determine whether or not glycine receptors (GlyR) played a significant role, the specific antagonist, strychnine, was used as a direct inhibitor. Western Blotting was performed to show the presence of GlyR. While there was no direct pro- or antiproliferative effect of either glycine or strychnine in both cell lines, glycine was shown to significantly decrease VEGF-A expression on mRNA and protein level up to 63 % in both cell lines. This effect was blunted by the presence of strychnine. GlyR was also identified in both cell lines. Glycine decreases GlyR-dependent, VEGF-A-mediated, angiogenic signaling in human HCC and thus might be a promising additive to chemotherapy treatment strategies for highly vascularized tumors.     

Involvement of the epidermal growth factor receptor in the modulation of multidrug resistance in human hepatocellular carcinoma cells in vitro

Background

The synergic action of KHCO₃ and D-ribose is tested on A72 and HTB-126 cell lines proliferation using K:D-Rib solution. Altered Na⁺/K⁺ ATPase expression and activity were shown in patients with cancer. Studies in human epithelial-derived malignancies indicate that K⁺ depletion also occurs, contributing to the increased intracellular Na⁺/K⁺ ratio [1]. D-ribose transformed to piruvate, enters into the Krebs's cycle and has a key role on energetic metabolism. The up-regulation of glycolysis in tumor cells is already well known and it is the rationale of F¹⁸-FDG PET diagnostic technique. D-ribose is synthesized by the non-oxidative transketolase PPP reaction.

Results

Results with different K:D-Rib concentrations show that MTT salt interferes with K:D-Rib solution and therefore this method is not reliable. The UV/VIS measurements show that K:D-Rib solutions reduce MTT salt to formazan in absence of cells. Cell proliferation has then been evaluated analysing the digital photos of the Giemsa stained cells with MCID? software. At 5 mM K:D-Rib concentration, the cell growth arrests between 48 h and 72 h; in fact the cell number after 48 h is around the same with respect to the control after 72 h. In case of HTB-126 human cancer cells, the growth rate was valuated counting the splitting times during 48 days: control cells were split sixteen times while 5 mM treated cells eleven times. Most relevant, the clonogenic assay shows that nine colonies are formed in the control cells while only one is formed in the 5 mM and none in 10 mM treated cells.

Conclusions

The K:D-Rib solution has an antioxidant behaviour also at low concentrations. Incubation with 5 mM K:D-Rib solution on A72 cells shows a cytostatic effect at 5 mM, but it needs more than 24 h of incubation time to evidence this effect on cell proliferation. At the same concentration on human HTB-126 cells, K:D-Rib solution shows a clear replication slowing but the cytostatic effect at 10 mM K:D-Rib solution only. Results on A72 cells indicate the K⁺ uptake could be determinant either to arrest or to slow down cell growth.     

Grb2 dominantly associates with dynamin II in human hepatocellular carcinoma HepG2 cells.

The two SH3 domains and one SH2 domain containing adaptor protein Grb2 is an essential element of the Ras signaling pathway in multiple systems. The SH2 domain of Grb2 recognizes and interacts with phosphotyrosine residues on activated tyrosine kinases, whereas the SH3 domains bind to several proline-rich domain-containing proteins such as Sos1. To define the difference in Grb2-associated proteins in hepatocarcinoma cells, we performed coprecipitation analysis using recombinant GST-Grb2 fusion proteins and found that several protein components (p170, p125, p100, and p80) differently associated with GST-Grb2 proteins in human Chang liver and hepatocarcinoma HepG2 cells. Sos1 and p80 proteins dominantly bind to Grb2 fusion proteins in Chang liver, whereas p100 remarkably associate with Grb2 in HepG2 cells. Also GST-Grb2 SH2 proteins exclusively bound to the p46(Shc), p52(Shc), and p66(Shc) are important adaptors of the Ras pathway in HepG2 cells. The p100 protein has been identified as dynamin II. We observed that the N-SH3 and C-SH3 domains of Grb2 fusion proteins coprecipitated with dynamin II besides Sos1. These results suggest that dynamin II may be a functional molecule involved in Grb2-mediated signaling pathway on Ras activation for tumor progression and differentiation of hepatocarcinoma cells.