Antiapoptotic Protein Myeloid Cell Leukemia-1 Research Articles

Triptolide induces apoptotic cell death of human cholangiocarcinoma cells through inhibition of myeloid cell leukemia-1

BackgroundCholangiocarcinoma (CCA), a devastating neoplasm, is highly resistant to current chemotherapies. CCA cells frequently overexpress the antiapoptotic protein myeloid cell leukemia-1(Mcl-1), which is responsible for its extraordinary ability to evade cell death. Triptolide, a bioactive ingredient extracted from Chinese medicinal plant, has been shown to inhibit cell proliferation and induce apoptosis in several cancers.MethodsCCK-8 assay was performed to detect cell survival rate in vitro. DAPI staining and Flow cytometry were used to analyze apoptosis. Western blot was performed to determine the expression levels of caspase-3, caspase-7, caspase-9, PARP, and Mcl-1. Quantitative real-time PCR and immunofluorescence were used to detect the expression levels of Mcl-1. The nude mice xenograft model was used to evaluate the antitumor effect of triptolide in vivo.ResultsTriptolide reduced cell viability in cholangiocarcinoma cell lines in a dose- and time-dependent manner, with IC50 values of 12.6 ± 0.6 nM, 20.5 ± 4.2 nM, and 18.5 ± 0.7 nM at 48 h for HuCCT1, QBC939, and FRH0201 respectively. Triptolide induced apoptosis in CCA cell lines in part through mitochondrial pathway. Using quantitative real-time PCR, western blot and immunofluorescence, we have shown that triptolide downregulates Mcl-1 mRNA and protein levels. Furthermore, triptolide inhibited the CCA growth in vivo.ConclusionsTriptolide has profound antitumor effect on CCA, probably by inducing apoptosis through inhibition of Mcl-1. Triptolide would be a promising therapeutic agent for CCA.

Role of mitogen‐activated protein kinases and Mcl‐1 in apoptosis induction by withaferin A in human breast cancer cells

Withaferin A (WA), a bioactive constituent of Ayurvedic medicine plant Withania somnifera, is a potent apoptosis inducer in cancer cells but the mechanism of cell death induction is not fully characterized. The present study was undertaken to determine the role of mitogen-activated protein kinases (MAPK), including c-jun NH2 -terminal kinase (JNK), extracellular signal-regulated kinase (ERK), and p38 MAPK, and anti-apoptotic protein myeloid cell leukemia-1 (Mcl-1) in regulation of WA-induced apoptosis using human breast cancer cells. Exposure of MCF-7 (estrogen responsive) and SUM159 (triple negative) human breast cancer cells to WA resulted in increased phosphorylation of ERK, JNK, and p38 MAPK, but these effects were relatively more pronounced in the former cell line than in SUM159. Overexpression of manganese-superoxide dismutase conferred partial protection against WA-mediated hyperphosphorylation of ERK, but not JNK or p38 MAPK. Cell death resulting from WA treatment in MCF-7 cells was significantly augmented by pharmacological inhibition of ERK and p38 MAPK. Interestingly, the WA-induced apoptosis in MCF-7 cells was partially but significantly blocked in the presence of a JNK-specific inhibitor. Pharmacological inhibition of ERK or JNK had no effect on WA-induced apoptosis in SUM159 cells. The WA-treated cells exhibited induction of long and short forms of Mcl-1. RNA interference of Mcl-1 alone triggered apoptosis. Furthermore, the WA-induced cell death in MCF-7 cells was modestly but significantly augmented by knockdown of the Mcl-1 protein. These observations indicate that: MAPK have cell line-specific role in cell death by WA, and Mcl-1 induction confers modest protection against WA-induced apoptosis.

Mcl-1 as a therapeutic target in acute myelogenous leukemia (AML)

The B-cell lymphoma-2 (Bcl-2) family of proteins regulates the intrinsic, or mitochondrial pathway of apoptosis, the final common mechanism of cell death in response to a variety of physiologic and pharmacologic signals, and plays a central role in AML pathogenesis, prognosis and responsiveness to chemotherapy. Traditionally thought to be an important survival factor for multiple myeloma cells, the anti-apoptotic Bcl-2 family protein myeloid cell leukemia-1 (Mcl-1) has recently been shown in preclinical studies to be critical to the development and maintenance of AML, making it an attractive therapeutic target in this disease. Several characteristics, such as its very short half-life, distinguish Mcl-1 from other anti-apoptotic Bcl-2 family members. Additionally, Mcl-1 levels are regulated by a large number of pathways affecting its transcription, translation and degradation. A variety of approaches exploiting these features have been developed to inhibit directly or indirectly the anti-apoptotic function of Mcl-1. Many of these lend themselves well to combination therapies, leading to striking synergism, at least in preclinical models. In this brief review, we highlight some of the more promising strategies targeting Mcl-1 in AML, with a particular emphasis on rational combinations of novel agents.

AMP‐activated protein kinase (AMPK)–induced preconditioning in primary cortical neurons involves activation of MCL‐1

Neuronal preconditioning is a phenomenon where a previous exposure to a sub-lethal stress stimulus increases the resistance of neurons towards a second, normally lethal stress stimulus. Activation of the energy stress sensor, AMP-activated protein kinase (AMPK) has been shown to contribute to the protective effects of ischaemic and mitochondrial uncoupling-induced preconditioning in neurons, however, the molecular basis of AMPK-mediated preconditioning has been less well characterized. We investigated the effect of AMPK preconditioning using 5-aminoimidazole-4-carboxamide riboside (AICAR) in a model of NMDA-mediated excitotoxic injury in primary mouse cortical neurons. Activation of AMPK with low concentrations of AICAR (0.1 mM for 2 h) induced a transient increase in AMPK phosphorylation, protecting neurons against NMDA-induced excitotoxicity. Analysing potential targets of AMPK activation, demonstrated a marked increase in mRNA expression and protein levels of the anti-apoptotic BCL-2 family protein myeloid cell leukaemia sequence 1 (MCL-1) in AICAR-preconditioned neurons. Interestingly, over-expression of MCL-1 protected neurons against NMDA-induced excitotoxicity while MCL-1 gene silencing abolished the effect of AICAR preconditioning. Monitored intracellular Ca²⁺ levels during NMDA excitation revealed that MCL-1 over-expressing neurons exhibited improved bioenergetics and markedly reduced Ca²⁺ elevations, suggesting a potential mechanism through which MCL-1 confers neuroprotection. This study identifies MCL-1 as a key effector of AMPK-induced preconditioning in neurons.

Targeting TCTP as a New Therapeutic Strategy in Castration-resistant Prostate Cancer

Heat shock protein 27 (Hsp27) is highly overexpressed in castration-resistant prostate cancer (CRPC) and an antisense inhibitor (OGX-427) is currently in phase II clinical trials. In order to understand mechanisms of action of Hsp27 and find new therapeutic targets specific of CRPC, we screened for Hsp27 client proteins. Here, we report that translationally controlled tumor protein (TCTP) is a new Hsp27 client protein involved in Hsp27 cytoprotection. We found that TCTP expression is absent or weak in normal prostate cells, moderately expressed in 18.5% of treatment naive PC, and becomes uniformly and strongly expressed in 75% of CRPC. To define TCTP function, we developed and worldwide patented a TCTP antisense oligonucleotide (ASO). Interestingly, we found that CRPC progression correlates with TCTP overexpression and loss of P53. TCTP knockdown restored P53 expression and function, suggesting that castration-sensitivity is directly linked to P53 expression. Collectively, these findings provide a new Hsp27 cytoprotection mechanism in CRPC, and preclinical proof-of-concept that combining ASO-mediated TCTP knockdown with castration and/or docetaxel therapy could serve as a novel strategy to treat CRPC, with no or little toxicity for normal prostate cells.

Anti-cancer effects of 2-oxoquinoline derivatives on the HCT116 and LoVo human colon cancer cell lines.

The present study demonstrated the anti-tumor effects of the quinoline derivative [5-(3-chloro-oxo-4-phenyl-cyclobutyl)-quinoli-8-yl-oxy] acetic acid hydrazide (CQAH) against colorectal carcinoma. Substantial apoptotic effects of CQAH on HCT116 and LoVo human colon cancer cell lines were observed. Apoptosis was identified based on cell morphological characteristics, including cell shrinkage and chromatin condensation as well as AnnexinV/propidium iodide double staining followed by flow cytometric analysis and detection of apoptosis-associated proteins by western blot analysis. CQAH induced caspase-3 and PARP cleavage, reduced the expression of the anti-apoptotic proteins myeloid cell leukemia-1 and B-cell lymphoma (Bcl) extra large protein and elevated the expression of the pro-apoptotic protein Bcl-2 homologous antagonist killer. In addition, pharmacological inhibition of c-Jun N-terminal kinase (JNK), but not extracellular signal-regulated kinase or p38, significantly reduced CQAH-mediated cell death as well as cleavage of caspase-3 and PARP. Co-treatment of CQAH with the commercial chemotherapeutics 5-fluorouracil and camptothecin-11 significantly improved their efficacies. Comparison of the apoptotic effects of CQAH with those of two illustrated structure-activity associations for this compound type, indicating that substitution at position-4 of the azetidine phenyl ring is pivotal for inducing apoptosis. In conclusion, the results of the present study indicated CQAH and its analogues are potent candidate drugs for the treatment of colon carcinoma.

Expression of the enteroviral capsid protein VP1 in the islet cells of patients with type 1 diabetes is associated with induction of protein kinase R and downregulation of Mcl-1

Immunohistochemical staining reveals that the enteroviral capsid protein VP1 is present at higher frequency in the insulin-containing islets of patients with recent-onset type 1 diabetes than in controls. This is consistent with epidemiological evidence suggesting that enteroviral infection may contribute to the autoimmune response in type 1 diabetes. However, immunostaining of VP1 is not definitive since the antibody widely used to detect the protein (Clone 5D8/1) might also cross-react with additional proteins under some conditions. Therefore, we sought to verify that VP1 immunopositivity correlates with additional markers of viral infection. Antigen immunoreactivity was examined in formalin-fixed, paraffin-embedded, pancreases from two different collections of type 1 diabetes and control cases: a historical collection from the UK and the nPOD (network of Pancreatic Organ donors with Diabetes) cohort from the USA. VP1 immunoreactivity was present in ~20% of insulin-containing islets of both cohorts under stringent conditions but was absent from insulin-deficient islets. The presence of VP1 was restricted to beta cells but only a minority of these contained the antigen. The innate viral sensor, protein kinase R (PKR) was upregulated selectively in beta cells that were immunopositive for VP1. The anti-apoptotic protein myeloid cell leukaemia sequence-1 (Mcl-1) was abundant in beta cells that were immunonegative for VP1 but Mcl-1 was depleted in cells containing VP1. The presence of immunoreactive VP1 within beta cells in type 1 diabetes is associated with a cellular phenotype consistent with the activation of antiviral response pathways and enhanced sensitivity to apoptosis. However, definitive studies confirming whether viral infections are causal to beta cell loss in human diabetes are still awaited.

Exposure to the Viral By-Product dsRNA or Coxsackievirus B5 Triggers Pancreatic Beta Cell Apoptosis via a Bim / Mcl-1 Imbalance

The rise in type 1 diabetes (T1D) incidence in recent decades is probably related to modifications in environmental factors. Viruses are among the putative environmental triggers of T1D. The mechanisms regulating beta cell responses to viruses, however, remain to be defined. We have presently clarified the signaling pathways leading to beta cell apoptosis following exposure to the viral mimetic double-stranded RNA (dsRNA) and a diabetogenic enterovirus (Coxsackievirus B5). Internal dsRNA induces cell death via the intrinsic mitochondrial pathway. In this process, activation of the dsRNA-dependent protein kinase (PKR) promotes eIF2α phosphorylation and protein synthesis inhibition, leading to downregulation of the antiapoptotic Bcl-2 protein myeloid cell leukemia sequence 1 (Mcl-1). Mcl-1 decrease results in the release of the BH3-only protein Bim, which activates the mitochondrial pathway of apoptosis. Indeed, Bim knockdown prevented both dsRNA- and Coxsackievirus B5-induced beta cell death, and counteracted the proapoptotic effects of Mcl-1 silencing. These observations indicate that the balance between Mcl-1 and Bim is a key factor regulating beta cell survival during diabetogenic viral infections.

Abstract C41: Targeting the antiapoptotic protein Mcl-1 in glioblastoma multiforme

Abstract Glioblastoma multiforme (GBM) is the most aggressive and malignant tumour of the central nervous system. It is characterised by a rapid clinical course and extremely unfavourable prognosis. GBM cells are highly resistant to apoptosis induced by anti-tumour drugs and radiotherapy, resulting in cancer progression. A critical determinant of a cell's susceptibility to undergo apoptosis is the balance of pro-apoptotic and antiapoptotic proteins within a cell. Both commercially bought GBM cells lines (A172 and U87) and a cell line recently derived from a GBM patient (MZ294), were resistant to apoptosis induction upon treatment with temozolomide (TMZ; 150 μM) and TNF-related apoptosis inducing ligand (TRAIL; 100 ng/ml) as assessed by MTT cell survival assay and Hoechst staining. However, they were shown to undergo apoptosis after treatment with r-roscovitine (20 μM) in a time dependent manner. R-roscovitine, a cyclin-dependent kinase inhibitor, has previously been shown to induce apoptosis in multiple cell types via downregulation of the anti-apoptotic protein myeloid cell leukemia-1 (Mcl-1). We also saw that the induction of apoptosis in MZ294 cells was associated with a downregulation of Mcl-1 protein expression. Western blot analysis of resections from GBM patients demonstrated a negative correlation between the expression of the anti-apoptotic protein Mcl-1 and progression free survival times of patients after treatment, highlighting an important role for Mcl-1 in GBM progression. Since Mcl-1 was shown to be an important survival protein in GBM patient samples and cell lines, we assessed if the downregulation of Mcl-1 could sensitize MZ294 cells to TMZ and TRAIL and further augment r-roscovitine-induced apoptosis. Accordingly, MZ294 cells were transfected with a shRNA targeting Mcl-1 and subsequently treated with TMZ (150 μM), TRAIL (100 ng/ml) or r-roscovitine (20 μM) for 48 h. Knockdown of Mcl-1 alone increased the percentage of MZ294 cells undergoing apoptosis and enhanced their sensitivity to TMZ, TRAIL and r-roscovitine treatment. We next assessed if the pharmacological targeting of Mcl-1, a more clinically relevant approach, could also yield similar results. Co-treatment of MZ294 cells with r-roscovitine (20 μM) and TRAIL (100 ng/ml) or r-roscovitine (20 μM) and TMZ (150 μM) for 48 h synergistically increased the induction of apoptosis when compared to either drug treatment alone. Finally and perhaps most crucially similar observations were noted upon treatment of MZ294 neurospheres, the cancer stem cell population of this cell line. Collectively this body of work highlights that the efficacy of potential therapies for GBM patients can be increased if the critical levels of Mcl-1 expression are concomitantly reduced. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the Second AACR International Conference on Frontiers in Basic Cancer Research; 2011 Sep 14-18; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2011;71(18 Suppl):Abstract nr C41.

Wogonin and related natural flavones are inhibitors of CDK9 that induce apoptosis in cancer cells by transcriptional suppression of Mcl-1

The wogonin-containing herb Scutellaria baicalensis has successfully been used for curing various diseases in traditional Chinese medicine. Wogonin has been shown to induce apoptosis in different cancer cells and to suppress growth of human cancer xenografts in vivo. However, its direct targets remain unknown. In this study, we demonstrate for the first time that wogonin and structurally related natural flavones, for example, apigenin, chrysin and luteolin, are inhibitors of cyclin-dependent kinase 9 (CDK9) and block phosphorylation of the carboxy-terminal domain of RNA polymerase II at Ser2. This effect leads to reduced RNA synthesis and subsequently rapid downregulation of the short-lived anti-apoptotic protein myeloid cell leukemia 1 (Mcl-1) resulting in apoptosis induction in cancer cells. We show that genetic inhibition of Mcl-1 or CDK9 expression by siRNA is sufficient to mimic flavone-induced apoptosis. Pull-down and in silico docking studies demonstrate that wogonin directly binds to CDK9, presumably to the ATP-binding pocket. In contrast, wogonin does not inhibit CDK2, CDK4 and CDK6 at doses that inhibit CDK9 activity. Furthermore, we show that wogonin preferentially inhibits CDK9 in malignant compared with normal lymphocytes. Thus, our study reveals a new mechanism of anti-cancer action of natural flavones and supports CDK9 as a therapeutic target in oncology.

MiR-29a down-regulation in ALK-positive anaplastic large cell lymphomas contributes to apoptosis blockade through MCL-1 overexpression

AbstractAlthough deregulated expression of specific microRNAs (miRNAs) has been described in solid cancers and leukemias, little evidence of miRNA deregulation has been reported in ALK-positive (ALK+) anaplastic large cell lymphomas (ALCL). These tumors overexpress the major antiapoptotic protein myeloid cell leukemia 1 (MCL-1), a situation that could compensate for the lack of BCL-2. We report that ALK+ ALCL cell lines and biopsy specimens (n = 20) express a low level of miR-29a and that this down-modulation requires an active NPM-ALK kinase. Murine models (transgenic mice and mouse embryonic fibroblast [MEF] cells), which allow conditional NPM-ALK fusion protein expression, showed an increase of miR-29a expression in the absence of NPM-ALK. Concordant results were observed after the abolition of NPM-ALK kinase activity (siALK or PF-2341066) in NPM-ALK+ ALCL cell lines. In addition, we showed that low expression of miR-29a, probably through methylation repression, plays an important regulatory role in MCL-1 overexpression that could promote tumor cell survival by inhibiting apoptosis. Enforced miR-29a expression was found to modulate apoptosis through inhibition of MCL-1 expression in ALCL cell lines and in a xenografted model, with a concomitant tumor growth reduction. Thus, synthetic miR-29a represents a potential new tool to affect tumorigenesis in these lymphomas.

Abstract 4216: Antitumor effect of Nexrutine, a phellodendron amurense bark extract, in multiple myeloma

Abstract Multiple myeloma (MM), a clonal malignancy of plasma cells, is the second most common adult hematological malignancy with approximately 15,000 new cases diagnosed annually. Despite recent advances in its clinical management, morbidity and mortality associated with MM remain high and there is a need for continuing development of novel anti-myeloma agents. Nexrutine, a non-toxic herbal extract from the bark of Chinese tree Phellodendron amurense, has been shown to exert significant anti-tumor effects in animal models of prostate cancer and is currently being evaluated in phase I/II clinical trials in patients with prostate cancer. In this study, we investigated the potential anti-tumor effect of nexrutine in MM. Exposure to nexrutine (3-100µg/ml) reduced viability of murine 5TGM1 and human RPMI 8226 MM cells in a dose- and time-dependent manner, independent of cell cycle disruption. Loss of cell viability was due primarily to apoptosis as indicated by >2-fold increase in annexin V+, 7AAD- cells and increased DNA fragmentation detectable by 24 hours. Previous studies showed that down-regulation of short-lived anti-apoptotic protein myeloid cell leukemia-1 (Mcl-1), in part by caspase-dependent cleavage of the full-length protein (Mcl-1L) to generate smaller (pro-apoptotic) Mcl-1 fragments, plays a major role in execution of apoptosis in MM cells. Nexrutine-induced apoptosis in MM cells was associated with reduced levels of Mcl-1L, caspase activation as indicated by a large increase in the active 17kDa cleavage product of procaspase 3 and an increase in pro-apoptotic Mcl-1 cleavage product. Nexrutine also inhibited the mammalian target of rapamycin (mTOR) and this was accompanied by accumulation of LC3-II, a marker of autophagosomes, in autophagy-competent MM cells. However, treatment of MM with cells with either pepstatin A and E64D that target lysosomal proteases involved in autophagy or with chloroquine, a lysosomotrophic autophagy inhibitor also resulted in cytotoxicity and these autophagy inhibitors did not rescue nexrutine-induced loss of viability in MM cells suggesting that autophagy plays a role constitutively in MM cell survival. Finally, we evaluated potential in vivo anti-tumor efficacy of nexrutine in the well-characterized preclinical model of MM in which 5TGM1 cells are inoculated intravenously in syngeneic naïve female C57BL/KaLwRij mice. Administration of nexrutine (400µg/mouse p.o. by gavage 6 days/week for 5 weeks) to tumor-bearing mice reduced serum levels of the monoclonal IgG2bκ paraprotein, a surrogate for tumor progression and overall tumor burden. Collectively, our results indicate an anti-myeloma effect of nexrutine and support further extensive preclinical testing and early phase clinical trial of this non-toxic herbal extract in MM. Our data also indicate that a combination of nexrutine and one or more autophagy inhibitors would result in enhanced anti-tumor effect in MM. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4216. doi:10.1158/1538-7445.AM2011-4216

Caffeine-suppressed ATM pathway leads to decreased p53 phosphorylation and increased programmed cell death in gamma-irradiated leukaemic molt-4 cells

Summary Ionising radiation (IR) is one of the main treatment modalities in oncology. However, we still search for substances which can radio-sensitize tumour cells. In this study we used caffeine, a non-specific ataxia-telangiectasia mutated kinase (ATM) inhibitor, and studied its effect on the activation of the proteins involved in cell cycle control and the induction of apoptosis in human T-lymphocyte leukaemic MOLT-4 cells (p53 wt). We evaluated the expression of the tumour-suppressor p53 (itself and phosphorylated on Ser 15 and Ser 392 ), the cell cycle regulator p21, and the anti-apoptotic protein myeloid cell leukemia 1 (Mcl-1). After treatment with 2 mM caffeine, the cells were irradiated by 1 or 3 Gy, lysed and the proteins detected by Western-blotting. Apoptosis was determined by flow-cytometric annexin V/propidium iodine detection. Irradiation by 1 or 3 Gy induced p53 phosphorylation at Ser 15 and Ser 392 after 2 h with maximum after 4 h. Adding caffeine significantly inhibited Ser 15 phosphorylation, which is ATM-dependent but surprisingly also Ser 392 phosphorylation, which is ATM-independent, suggesting that caffeine might have another cellular target (protein kinase). Similarly, caffeine caused a substantial decrease in p21 in combination with both doses of IR and also Mcl-1 was down-regulated. Three days after irradiation, caffeine significantly increased induction of apoptosis. The ATM/p53 pathway was suppressed by caffeine, which led to increased apoptosis accompanied by a p53-independent decrease in Mcl-1. It also caused down-regulation of p21, which possibly contributed to the shortened cell cycle arrest necessary for effective DNA repair and thus impeded radio-resistance. Caffeine promotes the cytotoxic effect of ionising radiation and provides a possible platform for the development of new anti-cancer therapeutics known as radio-sensitizers.

Potent in vitro and in vivo antitumor activity of sorafenib against human intrahepatic cholangiocarcinoma cells

Intrahepatic cholangiocarcinoma (ICC) is rising in clinical importance due to the increasing incidence worldwide, poor prognosis, and suboptimal response to therapies. New effective therapeutic approaches are needed for improvement of treatment outcome. A recent study showed that sorafenib, a multikinase inhibitor that acts predominantly through inhibition of Raf kinase and vascular endothelial growth factor (VEGF) and platelet-derived growth factor (PDGF) receptors, exhibited potent antitumor activity in a preclinical model of cholangiocarcinoma cells. We tested the in vitro and in vivo antitumor activity of sorafenib against human ICC cell lines. Treatment of ICC cells with sorafenib resulted in inhibition of proliferation and induction of apoptosis in the cell lines. In the cells treated with sorafenib, phosphorylation of mitogen-activated protein kinase kinase (MEK) and mitogen-activated protein kinase (MAPK) and also interleukin-6-induced phosphorylation of signal transducer and activator of transcription 3 (STAT3) were inhibited in a dose-dependent manner. Down-regulation of the anti-apoptotic protein myeloid cell leukemia-1 (Mcl-1) paralleled the reduced phosphorylation of STAT3. However, sorafenib induced no significant change in the cell cycle distribution and the expression levels of cyclin D1 and p27(Kip1) in the cells. For the in vivo antitumor activity, oral administration of sorafenib significantly inhibited the growth of subcutaneous tumors established in immunodeficient mice at doses of 10, 30, and 100 mg/kg. Moreover, administration of sorafenib (30 mg/kg) to animals with peritoneally disseminated ICC resulted in significantly prolonged survival compared with that of untreated animals (76 vs. 43 days in treated and vehicle-treated mice, respectively). These results indicate that sorafenib is a potent agent that may provide a new therapeutic option for human ICC.

Homoharringtonine reduced Mcl-1 expression and induced apoptosis in chronic lymphocytic leukemia

AbstractHomoharringtonine (HHT) is a plant alkaloid that inhibits the elongation phase of translation that is currently in clinical trials. Because the intrinsically short-lived antiapoptotic protein myeloid cell leukemia-1 (Mcl-1) has been reported to support the survival of chronic lymphocytic leukemia (CLL) cells, we hypothesized that inhibition of protein synthesis by HHT would decrease Mcl-1 expression and induce apoptosis in CLL. In primary CLL cells, HHT induced significant apoptosis independent of the prognostic characteristics of the patients. This was associated with inhibition of translation and decreased Mcl-1 levels in CLL cells. Mcl-1 reduction was evident as early as 2 hours and continued to decrease in the next 6-8 hours, whereas cell death started in 2 hours and continued to increase for 24 hours. Reduction of the Mcl-1 level was due to translation inhibition and proteasome degradation rather than to transcription inhibition or caspase cleavage. HHT and the transcription inhibitor SNS-032 induced synergistic cell killing. Although stromal cells induced Mcl-1 expression and protected CLL cells from the toxicity of fludarabine, this induction was reversed by HHT, which overcame stromal cell–mediated protection. Thus, these results provide a rationale for clinical development of HHT in CLL as single agent or in combinations.

Mcl-1 Stability Determines Mitotic Cell Fate of Human Multiple Myeloma Tumor Cells Treated with the Kinesin Spindle Protein Inhibitor ARRY-520

Kinesin spindle protein (KSP/Eg5) inhibitors are novel anticancer agents that have thus far shown only modest activity in the clinic. Understanding how to identify patients who may be most sensitive to treatment is clearly needed to improve the development of these molecules. We studied four multiple myeloma cell lines treated with the KSP inhibitor ARRY-520 to identify factors important for initiating apoptosis while cells are arrested in mitosis. The majority (three of four) of cell lines underwent mitotic arrest, with apoptosis occurring in mitosis within 24 to 30 hours. The remaining line (NCI H929) is temporally refractory to ARRY-520 treatment, undergoing mitotic slippage and subsequently peaking in apoptotic markers after 72 hours of treatment, while most cells are in interphase. Interestingly, loss of the antiapoptotic protein myeloid cell leukemia 1 (Mcl-1) coincided with mitotic cell death. Stabilization of Mcl-1 resulted in a delayed onset of apoptosis, whereas enforced downregulation of Mcl-1 increased cell death in response to KSP inhibition. Thus, variation in responses to KSP inhibition is governed by a balance between survival proteins and spindle checkpoint integrity. Cells relying on short-lived survival proteins during mitosis are more likely to undergo apoptosis in response to KSP inhibition. We propose that patients with hematologic malignancies, which rely on Mcl-1, would therefore be good candidates for treatment with KSP inhibitors.

Mechanism by Which Mcl-1 Regulates Cancer-Specific Apoptosis Triggered by mda-7/IL-24, an IL-10–Related Cytokine

Melanoma differentiation-associated gene-7/interleukin-24 (mda-7/IL-24), a cytokine belonging to the IL-10 family, selectively induces apoptosis in cancer cells without harming normal cells by promoting an endoplasmic reticulum (ER) stress response. The precise molecular mechanism by which the ER stress response culminates in cell death requires further clarification. The present study shows that in prostate carcinoma cells, the mda-7/IL-24-induced ER stress response causes apoptosis by translational inhibition of the antiapoptotic protein myeloid cell leukemia-1 (Mcl-1). Forced expression of Mcl-1 blocked mda-7/IL-24 lethality, whereas RNA interference or gene knockout of Mcl-1 markedly sensitized transformed cells to mda-7/IL-24. Mcl-1 downregulation by mda-7/IL-24 relieved its association with the proapoptotic protein Bak, causing oligomerization of Bak and leading to cell death. These observations show the profound role of the Bcl-2 protein family member Mcl-1 in regulating cancer-specific apoptosis induced by this cytokine. Thus, our studies provide further insights into the molecular mechanism of ER stress-induced cancer-selective apoptosis by mda-7/IL-24. As Mcl-1 is overexpressed in the majority of prostate cancers, mda-7/IL-24 might provide an effective therapeutic for this disease.

Mcl-1 Functions as Major Epidermal Survival Protein Required for Proper Keratinocyte Differentiation

Rapid downregulation of the antiapoptotic Bcl-2 family protein myeloid cell leukemia 1 (Mcl-1) is required for UV-induced apoptosis, underlining an important role for Mcl-1 in epidermal pathology. To determine if Mcl-1 has a specific role in normal keratinocyte (KC) biology, Mcl-1 was downregulated in human KCs by RNAi and these KCs were induced to differentiate in organotypic raft cultures. Mcl-1 shRNA organotypic cultures showed increased levels of spontaneous premature apoptosis, implicating Mcl-1 as an essential KC survival protein. Mcl-1-downregulated cultures also had reduced granular and cornified layers, and produced lower levels of cross-linked protein and cornified envelopes. Cornification could only partially be rescued with the general caspase inhibitor z-VAD, suggesting that reduced cornification was not entirely because of premature apoptosis. Differentiation markers (K1, K10, filaggrin, loricrin, cleaved caspase-14) were normally expressed in control organotypic cultures, but were expressed at reduced levels in organotypic cultures with downregulated Mcl-1. The defect in differentiation marker expression was independent of apoptosis as it could not be rescued by z-VAD. Thus, Mcl-1 serves two important, independent functions in epidermal KCs: acting as a major survival protein by inhibiting premature apoptosis in the spinous and granular layers to promote conification, and promoting the robust induction of KC differentiation markers.

Down‐regulation of myeloid cell leukemia 1 by epigallocatechin‐3‐gallate sensitizes rheumatoid arthritis synovial fibroblasts to tumor necrosis factor α–induced apoptosis

Overexpression of the antiapoptotic protein myeloid cell leukemia 1 (Mcl-1) in rheumatoid arthritis (RA) synovial fibroblasts is a major cause of their resistance to tumor necrosis factor alpha (TNFalpha)-induced apoptosis. This study was undertaken to evaluate the efficacy of epigallocatechin-3-gallate (EGCG) in down-regulating Mcl-1 expression and its mechanism of RA synovial fibroblast sensitization to TNFalpha-induced apoptosis. EGCG effects on cultured RA synovial fibroblast cell morphology, proliferation, and viability over 72 hours were determined by microscopy and a fluorescent cell enumeration assay. Caspase 3 activity was determined by a colorimetric assay. Western blotting was used to evaluate the apoptosis mediators poly(ADP-ribose) polymerase (PARP), Mcl-1, Bcl-2, Akt, and nuclear translocation of NF-kappaB. In RA synovial fibroblasts, EGCG (5-50 microM) inhibited constitutive and TNFalpha-induced Mcl-1 protein expression in a concentration- and time-dependent manner (P<0.05). Importantly, EGCG specifically abrogated Mcl-1 expression in RA synovial fibroblasts and affected Mcl-1 expression to a lesser extent in osteoarthritis and normal synovial fibroblasts or endothelial cells. Inhibition of Mcl-1 by EGCG triggered caspase 3 activity in RA synovial fibroblasts, which was mediated via down-regulation of the TNFalpha-induced Akt and NF-kappaB pathways. Caspase 3 activation by EGCG also suppressed RA synovial fibroblast growth, and this effect was mimicked by Akt and NF-kappaB inhibitors. Interestingly, Mcl-1 degradation by EGCG sensitized RA synovial fibroblasts to TNFalpha-induced PARP cleavage and apoptotic cell death. Our findings indicate that EGCG itself induces apoptosis and further sensitizes RA synovial fibroblasts to TNFalpha-induced apoptosis by specifically blocking Mcl-1 expression and, hence, may be of promising adjunct therapeutic value in regulating the invasive growth of synovial fibroblasts in RA.

Down-regulation of Mcl-1 with antisense technology alters the effect of various cytotoxic agents used in treatment of squamous cell carcinoma of the head and neck

Antisense oligonucleotides have recently been identified as new anticancer agents. Since human head and neck cancer cells highly express the antiapoptotic protein myeloid cell leukemia-1 (Mcl-1), the aim of this study was to explore the efficacy of the Mcl-1 suppression in combination with various cytotoxic agents in the head and neck cancer cell line SCC9. After oligonucleotide transfection and/or treatment with cisplatin, 5-fluorouracil (5-FU), gemcitabine, paclitaxel or cetuximab, proliferation assays were performed to determine cell viability. The expression patterns of Mcl-1, Bax and Bak were assessed by Western blot analysis and the apoptotic cells were determined by immunohistochemistry using the M30 antibody. A combined Mcl-1 antisense oligonucleotide treatment with paclitaxel, cetuximab and gemcitabine led to a significant reduction in the viable cells. However, the combination with cisplatin and 5-FU showed only moderate synergistic cytotoxic effects. According to the cytotoxic data, distinct apoptosis rates were observed after the combined treatment with the different substances. Western blot analysis also showed a significant suppression of the Mcl-1 synthesis. Our data show that the Mcl-1 antisense oligonucleotide in combination with certain cytotoxic agents has the potential to significantly decrease cell viability in vitro.