| BACKGROUDEsophageal cancer is a highly aggressive tumor and sixth leading cause of cancer-related death in the world. Globally, there were482,300new esophageal cancer cases and406,800deaths in2008. Mortality rates are close to incidence rates, due to the relatively late stage of diagnosis and the poor efficacy of treatment. The long-term prognosis is grim with overall five-year survival rates less than10%. Better knowledge of the signaling pathways connected to carcinogenesis, tumor growth and metastasis can probably provide potential novel molecular-targeted therapy in esophageal cancer treatment.The major mode of programmed cell death, apoptosis, was first recognized morphologically. The nuclei and cytoplasm, including the mitochondria, shrink in cells undergoing apoptosis, and the cell contents typically become encased in ’apoptotic bodies’ surrounded by plasma membrane. In response to’eat me’signals on their surface, these apoptotic bodies are rapidly engulfed by nearby phagocytic cells and digested in their lysosomes.The ability of cancer cells to evade apoptosis is a hallmark in human cancers, but also an important reason for the failure of many cancer therapies. Apoptosis occurs via activation of two different pathways, the extrinsic pathway, triggered by the activation of the cell-surface death receptors, and the intrinsic pathway, followed by the perturbation of mitochondrial membrane integrity. Members of the Bcl-2family are essential factors in the control of the intrinsic apoptosis pathway. At present, more than30proteins of Bcl-2family have been identified, the anti-apoptotic proteins (e.g., Bcl-2, Mcl-1, Bcl-XL) and two fractions of pro-apoptotic proteins that include multidomain Bak and Bax and the BH3-only proteins (e.g., Bad, Bim, Bid, Puma, Noxa). Noticeably, aberrant expression of Bcl-2family members such as down-regulation of Bax and up-regulation of Bcl-2and Bcl-XL has been found in esophageal cancer. Since the Bcl-2family members are very important to regulate cell death, targeting the anti-apoptotic Bcl-2proteins is an attractive approach in esophageal cancer therapy.A new series of small molecules that mimetic BH3-only proteins have been developed, constituting a new class of potentially useful anticancer drugs in recent decades. Among the BH3-mimetics, obatoclax (GX15-070) is an indole bipyrrole compound that can inhibit all known anti-apoptotic Bcl-2family members. This agent is proposed to operate like a BH3mimetic, competitively inhibiting the binding of pro-apoptotic proteins to the hydrophobic groove of anti-apoptotic proteins. Obatoclax has been under investigation in phase I and II clinical trials for the treatment of hematological malignancies and solid tumors, however, the molecular mechanisms of its anti-tumor activity have still not been fully resolved. Obatoclax was shown to induce apoptosis at concentrations that resulted in disruption of Bak from Mcl-1and cytochrome c release. However, obatoclax was also cytotoxic in cells deficient for the apoptosis effectors Bax and Bak, suggesting the existence of additional target mechanisms. Many of the compounds under investigation as anti-tumor agents act at multiple steps in the cell cycle, however, the relationship between cell cycle progression and inhibitory effect of obatoclax on cell viability remains obscure. While obatoclax was reported to induce an S-phase arrest in acute leukemia cells, the precise mechanisms have not been explored. In this study, we sought to investigate the effects of obatoclax on cell viability in relation to cell cycle progression in esophageal cancer cells. Moreover, we explored the mechanisms underlying the action of obatoclax on cell cycle progression. We anticipate that this study will bring new insight into the anti-cancer mechanism of obatoclax.OBJECTIVE:To investigate the effects of obatoclax on cell viability in relation to cell cycle progression in human esophageal cancer cells, and explored the mechanisms underlying the action of obatoclax on cell cycle progression.METHODS:1. CellTiter-Glo Luminescent assay was used to test cell viabilityVarious concentrations of obatoclax were incubated with EC109, CaES-17and HKESC-1for24h. CellTiter-Glo Luminescent assay was used to test cell viability2. Flow cytometry was used to determine the cell-cycle distribution1) EC109, CaES-17and HKESC-1cells were incubated for24h with different concentration of obatoclax;2) EC109, CaES-17and HKESC-1cells were treated with obatoclax alone or combination with MAPK/Erk kinase (MEK) inhibitor U0126, p38inhibitor SB203580, and JNK inhibitor Ⅱ for24h;3) After transfection with control siRNA, p21wafl/Cipl siRNA, Bak siRNA, Bax siRNA and p38siRNA, EC109, CaES-17and HKESC-1cells were treated with obatoclax for24h;4) The above cells were collected with75%ethanol-fixed overnight. Cells were harvested and washed once with PBS. Fixed cells were resuspended in500μL1×PBS containing50μg/mL propidium iodide (PI),10μg/mL RNase-A, and then incubate for30min at37℃in the dark. The distribution of cell cycle was analyzed by flow cytometer within1h.3. Flow cytometry was used to determine the extent of apoptosis Various concentrations of obatoclax were incubated with EC109, CaES-17and HKESC-1for24h. Then harvested cells with trypsin (no EDTA) and washed cells twice with cold PBS after centrifugation, and then resuspended cells in100μL1×Binding Buffer, according to the FITC Annexin V Apoptosis Detection Kit, added5uL of FITC Annexin V, gently vortexed the cells and incubate for15min at RT (25℃) in the dark.400μL of1xBinding Buffer was added to each tube. Stained cells were analyzed within1h and ratios of apoptosis were analyzed by Flow Cytometry.4. Western blotting was used to determine G1/G0checkpoint correlative proteins and MAPK signaling proteins expression1) EC109, CaES-17and HKESC-1cells were treated in growth media with the obatoclax at IC50for0,1/4,1/2,1,3,6,12and24h, follow by dissolving cells and quantifying proteins. Then, the expression of G1/G0checkpoint correlative proteins p221waf1/Cip1, phospho-p21waf1/Cip1, CDK2, CDK4, CDK6and MAPK signaling proteins p38, phospho-p38, Erk, phospho-Erk, JNK, and phospho-JNK were determined by western blotting.2) EC109, CaES-17and HKESC-1cells were treated with obatoclax alone or combination with MAPK/Erk kinase (MEK) inhibitor U0126, p38inhibitor SB203580, and JNK inhibitor Ⅱ were used to inhibit Erkl/2, p38, and JNK phosphorylation for24h, respectively. Then, G1/G0checkpoint correlative proteins p21wafl/Cipl was detected by western blotting.5. RNA interferenceThe expression of Bak, Bax, p21waf1/Cip1, and p38were lowered using predesigned target-specific siRNA oligonucleotides. Bak, Bax, p21waf1/Cip1, or p38siRNA were transfected into cells using HiPerFect Transfection Reagent according to the manufacturer’s instruction. After48h later. cells were incubated for24h with different concentration of obatoclax.6. Statistical analysisData analysis and mapping are based on the software of GraphPad Prism5.0(data analysis and mapping software); all the datas were statistically treated by the SPSS13.0statistical analysis software package.RESULTS:1. obatoclax reduced cell viability in human esophageal cancer cells which was measured by CellTiter-Glo Luminescent method Obatoclax reduced cell viability in EC109, CaES-17and HKESC-1cells, with IC50values of1.0±0.1μM,0.3±0.1μM, and0.9±0.2μM, respectively.2. Obatoclax reduced cell viability by inducing G1/G0-phase arrest in human esophageal cancer cells (EC109, CaES-17and HKESC-1) EC109, CaES-17and HKESC-1cells were treated with obatoclax (1/4IC50、1/2IC50and IC50). Obatoclax significantly induced G1/G0-phase arrest. There was significant increase in the proportion of cells at G1/G0-phase between cells treated with obatoclax even at1/4IC50concentration and control cells. Moreover, with the increasing concentrations of the drug, proportion of cells at G1/G0-phase gradually increased. 3. Obatoclax failed to cause apoptosis in human esophageal cancer cells (EC109, CaES-17and HKESC-1).EC109, CaES-17and HKESC-1cells were treated with obatoclax (1/2IC50and IC50), obatoclax could not cause apoptosis in cells. From the flow cytometry results of Annexin V staining cells, obatoclax at indicated concentrations (1/2IC50and IC50for each cell line) did not increase apoptosis in EC109, CaES-17and HKESC-1cells, with no significant difference compared with the control group. At the same time, obatoclax failed to induce PARP cleavage in EC109, CaES-17and HKESC-1cells, which is indicative of apoptosis. In contrast, cisplatin, which was used as a positive control for apoptosis induction, obviously induced PARP cleavage in EC109, CaES-17and HKESC-1cells. At the tested concentrations (1/2IC50and IC50), obatoclax neither induced PARP cleavage nor increased the Annexin V-positive population, suggesting G1/G0-phase arrest rather than apoptosis accounts for most of the reduction of cell proliferation produced by obatoclax.4. Obatoclax reduced cell viability by inducing G1/G0-phase arrest not via Bak and Bax signaling pathway in human esophageal cancer cells (EC109, CaES-17and HKESC-1).Double knockdown of Bak and Bax did not block obatoclax-induced G1/G0-phase arrest, suggesting other targets rather than Bak/Bax contributed to cell cycle arrest induced by obatoclax.5. Obatoclax reduced cell viability by induces G1/G0-phase arrest via p38/p21waf1/Cip1signaling pathway in human esophageal cancer cells.With regard to the expression of proteins involved in G1/G0-phase, obatoclax up-regulated cyclin-dependent kinase (CDK) inhibitor p21waf1/Cip1expression. P21waf1/Cip1expression was gradually increased in a time-dependent manner. On the contrary, it did not alter the expression of phospho-p21waf1/Cip1CDK2, CDK4and CDK6proteins.The mechanism study found that down-regulation of p21waf1/Cip1significantly attenuated obatoclax-induced G1/G0-phase arrest in EC109, CaES-17and HKESC-1cells. These findings suggested that p21waf1/Cip1was implicated in the cell cycle arresting effect of obatoclax. Although obatoclax increased phosphorylation of Erk, p38, and JNK, only inhibition of p38completely reversed obatoclax-induced G1/G0-phase arrest. In consistent with this finding, inhibition of p38completely blocked obatoclax-promoted p21waf1/Cip1expression, whereas inhibition of Erk or JNK did not attenuated obatoclax-induced p21waf1/Cip1expression. Moreover, down-regulation of p38significantly attenuated obatoclax-induced G1/G0-phase arrest in EC109, CaES-17and HKESC-1cells. These findings suggested that p38was implicated in the cell cycle arresting effect of obatoclax.CONCLUSION:1. Obatoclax reduced cell viability in human esophageal cancer cells.2. Obatoclax reduced cell viability by inducing G1/G0-phase arrest, whereas it did not induce apoptosis. Moreover, it is suggested that other targets rather than Bak/Bax contributed to cell cycle arrest induced by obatoclax.3. Obatoclax reduced cell viability by inducing G1/G0-phase arrest via p38/p21waf1/Cip1signaling pathway in human esophageal cancer cells. |
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