Apoptosis induction by MEK inhibition in human lung cancer cells is mediated by Bim

AZD6244 (ARRY-142886) is an inhibitor of MEK1/2 and can inhibit cell proliferation or induce apoptosis in a cell-type dependent manner. The precise molecular mechanism of AZD6244-induced apoptosis is not clear. To investigate mechanisms of AZD6244 induced apoptosis in human lung cancer, we determined the molecular changes of two subgroups of human lung cancer cell lines that are either sensitive or resistant to AZD6244 treatment. We found that AZD6244 elicited a large increase of Bim proteins and a smaller increase of PUMA and NOXA proteins, and induced cell death in sensitive lung cancer cell lines, but had no effect on other Bcl-2 related proteins in those cell lines. Knockdown of Bim by siRNA greatly increased the IC(50) and reduced apoptosis for AZD6244 treated cells. We also found that levels of endogenous p-Thr32-FOXO3a and p-Ser253-FOXO3a were lower in AZD6244-sensitive cells than in AZD6244-resistant cells. In the sensitive cells, AZD6244 induced FOXO3a nuclear translocation required for Bim activation. Moreover, the silencing of FOXO3a by siRNA abrogated AZD6244-induced cell apoptosis. In addition, we found that transfection of constitutively active AKT up-regulated p-Thr32-FOXO3a and p-Ser253-FOXO3a expression and inhibited AZD6244-induced Bim expression in sensitive cells. These results show that Bim plays an important role in AZD6244-induced apoptosis in lung cancer cells and that the PI3K/AKT/FOXO3a pathway is involved in Bim regulation and susceptibility of lung cancer cells to AZD6244. These results have implications in the development of strategies to overcome resistance to MEK inhibitors.     

Differences in expression of pro-caspases in small cell and non-small cell lung carcinoma.

Expression of several molecular determinants of apoptosis was analyzed in 10 untreated small cell (SCLC) and 6 untreated non-small cell (NSCLC) lung carcinoma cell lines. Although SCLC lines were more prone to spontaneous apoptosis compared with NSCLC lines, the former showed higher Bcl-2 expression and a higher Bcl-2/Bax ratio. In order to understand this apparent contradiction, the expression of pro-caspases as well as calpain was analyzed in these cell lines at the protein and mRNA levels. No differences in protein level of pro-caspases-2, -3, -7, and -9 and of calpain were detected between the SCLC and the NSCLC lines, but a striking difference in pro-caspase-8 expression was noted. All 6 NSCLC, but only 2 of the 10 SCLC lines, expressed pro-caspase-8 protein. Further experiments using the RNase protection assay indicated that the lack of pro-caspase-8 expression at the mRNA level was characteristic for SCLC. Using the same experimental approach, we found that SCLC cell lines in addition to pro-caspase-8 were deficient in mRNA expression of pro-caspases-1, -4, and -10, suggesting a different caspase-activating cascade in SCLC compared with NSCLC. This first systematic characterization of pro-caspase expression in lung cancer surprisingly showed that SCLC, which are more prone to undergo spontaneous apoptosis, are deficient in several pro-caspases and have a high Bcl-2/Bax ratio. Thus, the propensity of SCLC cells to undergo apoptosis cannot be explained only by the expression of factors involved in regulation or execution of apoptosis.     

Metastatic SW620 colon cancer cells are primed for death when detached and can be sensitized to anoikis by the BH3-mimetic ABT-737

Anoikis, a Bax-dependent apoptosis triggered by detachment from the extracellular matrix, is often inhibited in metastatic cancer cells. Using a couple of isogenic human colon cancer cell lines derived either from the primary tumor (SW480) or from a lymph node metastasis (SW620), we found that only SW480 cells were sensitive to anoikis. Bim upregulation but not Mcl-1 degradation was determined to be a critical factor of anoikis initiation in SW480 cells. ERK-mediated phosphorylation targets Bim for ubiquitination and proteasomal degradation. A MEK inhibitor (PD0325901) was able to increase Bim expression in SW620 cells and to sensitize these cells to anoikis. Thus, in both cell lines anoikis is under the control of proteins of the Bcl-2 family. Most interestingly, the BH3-mimetic ABT-737 was found not only to increase the level of apoptosis in suspended SW480 cells but also to sensitize SW620 cells to anoikis. Accordingly, both cell lines cultured in suspension were found to be primed for death, as determined by the detection of Bcl-2:Bim and Bcl-xL:Bim complexes. In contrast, adherent SW480 and SW620 cells were resistant to ABT-737. This indicates that, whether or not they undergo anoikis, colon cancer cells that have detached from the extracellular matrix might go through a transient state, where they are sensitive to BH3 mimetics. This would confer to compounds such as Navitoclax or ABT-199 a therapeutic window where they could have anti-metastatic potential.     

Loss of the bcl-2 phosphorylation loop domain increases resistance of human leukemia cells (U937) to paclitaxel-mediated mitochondrial dysfunction and apoptosis.

The impact of ectopic expression of an N-terminal phosphorylation loop deletant Bcl-2 protein (Bcl-2Delta32-80) on the response of U937 monoblastic leukemia cells to paclitaxel was examined. In contrast to recent findings in HL-60 cells (Fang et al., Cancer Res. 58, 3202, 1998), U937 cells overexpressing Bcl-2Delta32-80 were significantly more resistant than those overexpressing full-length protein to caspase-3 and -9 activation, PARP degradation, and apoptosis induced by paclitaxel (500 nM; 18 h). Bcl-2Delta32-80 was also more effective than its full-length counterpart in opposing paclitaxel-mediated mitochondrial dysfunction, e.g., loss of mitochondrial membrane potential (Deltapsim) and cytochrome c release into the cytoplasm. Enhanced resistance of U937/Bcl-2Delta32-80 cells to paclitaxel was observed primarily in the G2M population. Together, these findings demonstrate that deletion of the Bcl-2 phosphorylation loop domain increases resistance of U937 leukemia cells to paclitaxel-mediated mitochondrial damage and apoptosis and suggest that factors other than, or in addition to, phosphorylation contribute to Bcl-2-related cytoprotectivity against paclitaxel in this model system.     

The Cyclin-Dependent Kinase Inhibitor p21CIP1/WAF1 Blocks Paclitaxel-Induced G2M Arrest and Attenuates Mitochondrial Injury and Apoptosis in p53-Null Human Leukemia Cells

The functional significance of the cyclin-dependent kinase inhibitor (CDKI) p21Cip1/WAF1 in paclitaxel-mediated lethality was examined in p53-null human leukemia cells (U937 and Jurkat). In these cells, paclitaxel exposure failed to induce p21Cip1/Waf1 expression. Nevertheless, stable expression of U937 cells with a p21Cip1/WAF1 antisense construct blocked paclitaxel-induced G2M arrest and significantly, albeit modestly, increased mitochondrial injury, caspase activation, apoptosis, and loss of clonogenic potential. These protective effects were less than those observed in cells exposed to the antimetabolite ara-C. Consistent with these results, enforced expression of p21Cip1/WAF1 in Jurkat cells transfected with a construct driven by a doxycycline-responsive promoter increased the percentage of cells arrested in G2M, but attenuated paclitaxel-mediated mitochondrial injury and apoptosis. Unexpectedly, enforced expression of p21Cip1/WAF1 diminished paclitaxel-mediated inactivation of ERK, and reduced paclitaxel-induced activation of JNK as well as Bcl-2 phosphorylation. Together, these findings suggest that the CDKI p21Cip1/WAF1 modestly but significantly protects p53-null human leukemia cells from paclitaxel-mediated lethality, and raise the possibility that p21Cip1/WAF1-associated perturbations in signal transduction pathways as well as Bcl-2 phosphorylation status may play a role in this phenomenon.     

BimS-induced apoptosis requires mitochondrial localization but not interaction with anti-apoptotic Bcl-2 proteins

Release of apoptogenic proteins such as cytochrome c from mitochondria is regulated by pro- and anti-apoptotic Bcl-2 family proteins, with pro-apoptotic BH3-only proteins activating Bax and Bak. Current models assume that apoptosis induction occurs via the binding and inactivation of anti-apoptotic Bcl-2 proteins by BH3-only proteins or by direct binding to Bax. Here, we analyze apoptosis induction by the BH3-only protein Bim(S). Regulated expression of Bim(S) in epithelial cells was followed by its rapid mitochondrial translocation and mitochondrial membrane insertion in the absence of detectable binding to anti-apoptotic Bcl-2 proteins. This caused mitochondrial recruitment and activation of Bax and apoptosis. Mutational analysis of Bim(S) showed that mitochondrial targeting, but not binding to Bcl-2 or Mcl-1, was required for apoptosis induction. In yeast, Bim(S) enhanced the killing activity of Bax in the absence of anti-apoptotic Bcl-2 proteins. Thus, cell death induction by a BH3-only protein can occur through a process that is independent of anti-apoptotic Bcl-2 proteins but requires mitochondrial targeting.     

Expression of inhibitor of apoptosis proteins in small- and non-small-cell lung carcinoma cells

Small-cell lung cancer (SCLC) and non-small-cell lung cancer (NSCLC) cells both initiate apoptotic signaling, resulting in caspase activation, after treatment with anti-cancer agents. However, in contrast to SCLC cells, NSCLC cells do not fully execute apoptosis. The apoptotic process in NSCLC cells seems to be blocked downstream of caspase activation, thus the failure of NSCLC cells to execute apoptosis could result from inhibition of active caspases by inhibitor of apoptosis proteins (IAPs). Here we investigate the mRNA and protein expression of IAPs in a panel of SCLC and NSCLC cell lines. The NSCLC cell lines had a stronger cIAP-2 expression at both mRNA and protein levels, while the SCLC cell lines had a higher level of XIAP protein. Expression of cIAP-1, cIAP-2, and XIAP, the most potent caspase inhibitors, was further investigated in three lung carcinoma cell lines after treatment with 8 Gy of ionizing radiation or etoposide (VP16). In response to treatment, the level of IAPs was not altered in a way that explained the differences in cellular chemo- and radiosensitivity. The intracellular localization of IAPs was analyzed in untreated and treated lung cancer cells. Surprisingly, we found that cIAP-2 was mainly detected in the mitochondrial fraction, although the function of this protein in mitochondria is unknown. No major relocalization of IAPs was observed after treatment. Taken together, these results indicate that IAPs alone are not the main factor responsible for the resistance of NSCLC cells to treatment.     

CSE1L/CAS, a microtubule-associated protein, inhibits taxol (paclitaxel)-induced apoptosis but enhances cancer cell apoptosis induced by various chemotherapeutic drugs

CSE1L/CAS, a microtubule-associated, cellular apoptosis susceptibility protein, is highly expressed in various cancers. Microtubules are the target of paclitaxel-induced apoptosis. We studied the effects of increased or reduced CAS expression on cancer cell apoptosis induced by chemotherapeutic drugs including paclitaxel. Our results showed that CAS overexpression enhanced apoptosis induced by doxorubicin, 5-fluorouracil, cisplatin, and tamoxifen, but inhibited paclitaxel-induced apoptosis of cancer cells. Reductions in CAS produced opposite results. CAS overexpression enhanced p53 accumulation induced by doxorubicin, 5-fluorouracil, cisplatin, tamoxifen, and etoposide. CAS was associated with alpha-tubulin and beta-tubulin and enhanced the association between alpha-tubulin and beta-tubulin. Paclitaxel can induce G2/M phase cell cycle arrest and microtubule aster formation during apoptosis induction, but CAS overexpression reduced paclitaxel-induced G2/M phase cell cycle arrest and microtubule aster formation. Our results indicate that CAS may play an important role in regulating the cytotoxicities of chemotherapeutic drugs used in cancer chemotherapy against cancer cells.     

紫杉醇对NK-92MI细胞杀伤功能及凋亡的影响

探讨紫杉醇对人自然杀伤细胞系NK-92MI细胞株杀伤活性及凋亡的影响。用不同浓度的紫杉醇处理NK.92MI细胞后,测定NK-92MI细胞的增殖率和对靶细胞的杀伤作用;同时检测NK·92MI细胞凋亡率及凋亡相关基因Bcl-2、Ba】【的表达。结果表明,紫杉醇可以抑制NK-92MI细胞增殖并且降低其杀伤活性,机制可能是通过诱导NK.92MI细胞的凋亡,而后者与Bcll2/Ba】【基因表达增高有关。     

Roles of Bim in apoptosis of normal and Bcr-Abl-expressing hematopoietic progenitors

Bcr-Abl kinase is known to reverse apoptosis of cytokine-dependent cells due to cytokine deprivation, although it has been controversial whether chronic myeloid leukemia (CML) progenitors have the potential to survive under conditions in which there are limited amounts of cytokines. Here we demonstrate that early hematopoietic progenitors (Sca-1(+) c-Kit(+) Lin(-)) isolated from normal mice rapidly undergo apoptosis in the absence of cytokines. In these cells, the expression of Bim, a proapoptotic relative of Bcl-2 which plays a key role in the cytokine-mediated survival system, is induced. In contrast, those cells isolated from our previously established CML model mice resist apoptosis in cytokine-free medium without the induction of Bim expression, and these effects are reversed by the Abl-specific kinase inhibitor imatinib mesylate. In addition, the expression levels of Bim are uniformly low in cell lines established from patients in the blast crisis phase of CML, and imatinib induced Bim in these cells. Moreover, small interfering RNA that reduces the expression level of Bim effectively rescues CML cells from apoptosis caused by imatinib. These findings suggest that Bim plays an important role in the apoptosis of early hematopoietic progenitors and that Bcr-Abl supports cell survival in part through downregulation of this cell death activator.     

Importance of Bcl-2-family proteins in murine hematopoietic progenitor and early B cells

Mitochondrial apoptosis regulates survival and development of hematopoietic cells. Prominent roles of some Bcl-2-family members in this regulation have been established, for instance for pro-apoptotic Bim and anti-apoptotic Mcl-1. Additional, mostly smaller roles are known for other Bcl-2-members but it has been extremely difficult to obtain a comprehensive picture of the regulation of mitochondrial apoptosis in hematopoietic cells by Bcl-2-family proteins. We here use a system of mouse ‘conditionally immortalized’ lymphoid-primed hematopoietic progenitor (LMPP) cells that can be differentiated in vitro to pro-B cells, to analyze the importance of these proteins in cell survival. We established cells deficient in Bim, Noxa, Bim/Noxa, Bim/Puma, Bim/Bmf, Bax, Bak or Bax/Bak and use specific inhibitors of Bcl-2, Bcl-X_L and Mcl-1 to assess their importance. In progenitor (LMPP) cells, we found an important role of Noxa, alone and together with Bim. Cell death induced by inhibition of Bcl-2 and Bcl-X_L entirely depended on Bim and could be implemented by Bax and by Bak. Inhibition of Mcl-1 caused apoptosis that was independent of Bim but strongly depended on Noxa and was completely prevented by the absence of Bax; small amounts of anti-apoptotic proteins were co-immunoprecipitated with Bim. During differentiation to pro-B cells, substantial changes in the expression of Bcl-2-family proteins were seen, and Bcl-2, Bcl-X_L and Mcl-1 were all partially in complexes with Bim. In differentiated cells, Noxa appeared to have lost all importance while the loss of Bim and Puma provided protection. The results strongly suggest that the main role of Bim in these hematopoietic cells is the neutralization of Mcl-1, identify a number of likely molecular events during the maintenance of survival and the induction of apoptosis in mouse hematopoietic progenitor cells, and provide data on the regulation of expression and importance of these proteins during differentiation along the B cell lineage.Subject terms: Apoptosis, Immune cell death     

Lovastatin-induced up-regulation of the BH3-only protein, Bim, and cell death in glioblastoma cells

The mechanism of lovastatin-induced cell death was examined in three established human glioblastoma cell lines; U87, U251, and U138. Changes in potential modifiers of apoptosis, including Bcl-2 family proteins and MAP kinase targets after such lovastatin treatment, were evaluated. Lovastatin (5 microm) treatment causes extensive cell death in two of the cell lines, U87 and U251; but only minimal in a third, U138. Lovastatin-induced death occurs in correlation with significantly increased levels of the BH3-only protein, Bim. The up-regulation of Bim levels was directly associated with an increased incidence of apoptosis. Lovastatin treatment in U87 cells results in activation of targets of three major mitogen-activating protein kinase cascades including Erk1/2, JNK and p38. Changes in levels of Bim, as well as increase phosphorylation of Erk1/2, c-jun, and p38 are all prevented by co-incubation of lovastatin and the isoprenylation metabolite, geranylgeranyl pyrophosphate. Inhibition of the MAP kinase pathways failed to block the increased expression of Bim expression or cell death. Further elucidation of the mechanisms of lovastatin-induced up-regulation of Bim and apoptosis in glioblastoma cells are important in determining a potential role for lovastatin as a chemotherapy agent.     

The deubiquitinase Usp27x stabilizes the BH3‐only protein Bim and enhances apoptosis

Bim is a pro‐apoptotic Bcl‐2 family member of the BH3‐only protein subgroup. Expression levels of Bim determine apoptosis susceptibility in non‐malignant and in tumour cells. Bim protein expression is downregulated by proteasomal degradation following ERK‐dependent phosphorylation and ubiquitination. Here, we report the identification of a deubiquitinase, Usp27x, that binds Bim upon its ERK‐dependent phosphorylation and can upregulate its expression levels. Overexpression of Usp27x reduces ERK‐dependent Bim ubiquitination, stabilizes phosphorylated Bim, and induces apoptosis in PMA‐stimulated cells, as well as in tumour cells with a constitutively active Raf/ERK pathway. Loss of endogenous Usp27x enhances the Bim‐degrading activity of oncogenic Raf. Overexpression of Usp27x induces low levels of apoptosis in melanoma and non‐small cell lung cancer (NSCLC) cells and substantially enhances apoptosis induced in these cells by the inhibition of ERK signalling. Finally, deletion of Usp27x reduces apoptosis in NSCLC cells treated with an EGFR inhibitor. Thus, Usp27x can trigger via its proteolytic activity the deubiquitination of Bim and enhance its levels, counteracting the anti‐apoptotic effects of ERK activity, and therefore acts as a tumour suppressor.     

Tyrosine-phosphorylated caveolin-1 (Tyr-14) increases sensitivity to paclitaxel by inhibiting BCL2 and BCLxL proteins via c-Jun N-terminal kinase (JNK)

Paclitaxel, an anti-microtubule agent, is an effective chemotherapeutic drug in breast cancer. Nonetheless, resistance to paclitaxel remains a major clinical challenge. The need to better understand the resistant phenotype and to find biomarkers that could predict tumor response to paclitaxel is evident. In estrogen receptor α-positive (ER(+)) breast cancer cells, phosphorylation of caveolin-1 (CAV1) on Tyr-14 facilitates mitochondrial apoptosis by increasing BCL2 phosphorylation in response to low dose paclitaxel (10 nM). However, two variants of CAV1 exist: the full-length form, CAV1α (wild-type CAV1 or wtCAV1), and a truncated form, CAV1β. Only wtCAV1 has the Tyr-14 region at the N terminus. The precise cellular functions of CAV1 variants are unknown. We now show that CAV1 variants play distinct roles in paclitaxel-mediated cell death/survival. CAV1β expression is increased in paclitaxel-resistant cells when compared with sensitive cells. Expression of CAV1β in sensitive cells significantly reduces their responsiveness to paclitaxel. These activities reflect an essential role for Tyr-14 phosphorylation because wtCAV1 expression, but not a phosphorylation-deficient mutant (Y14F), inactivates BCL2 and BCLxL through activation of c-Jun N-terminal kinase (JNK). MCF-7 cells that express Y14F are resistant to paclitaxel and are resensitized by co-treatment with ABT-737, a BH3-mimetic small molecule inhibitor. Using structural homology modeling, we propose that phosphorylation on Tyr-14 enables a favorable conformation for proteins to bind to the CAV1 scaffolding domain. Thus, we highlight novel roles for CAV1 variants in cell death; wtCAV1 promotes cell death, whereas CAV1β promotes cell survival by preventing inactivation of BCL2 and BCLxL via JNK in paclitaxel-mediated apoptosis.     

ABT-737 Induces Bim Expression via JNK Signaling Pathway and Its Effect on the Radiation Sensitivity of HeLa Cells

ABT-737 is a BH3 mimetic small molecule inhibitor that can effectively inhibit the activity of antiapoptotic Bcl-2 family proteins including Bcl2, Bcl-xL and Bcl-w, and further enhances the effect of apoptosis by activating the proapoptotic proteins (t-Bid, Bad, Bim). In this study, we demonstrate that ABT-737 improved the radiation sensitivity of cervical cancer HeLa cells and thereby provoked cell apoptosis. Our results show that ABT-737 inhibited HeLa cell proliferation and activated JNK and its downstream target c-Jun, which caused the up-regulation of Bim expression. Blockade of JNK/c-Jun signaling pathway resulted in significant down-regulation of ABT-737-induced Bim mRNA and protein expression level. Also, ABT-737 could evoke the Bim promoter activity, and enhance the radiation sensitivity of HeLa cells via JNK/c-Jun and Bim signaling pathway. Our data imply that combination of ABT-737 and conventional radiation therapy might represent a highly effective therapeutic approach for future treatment of cervical cancer.