Synergistic Antitumor Effect Of Obatoclax In Combination With Chemotherapeutics In Esophageal Cancer Cell Lines In Vitro

BACKGROUNDEsophageal cancer is a common malignant tumor in human, whose incidence rankes the fifth and mortality rankes fourth in our country. It is a serious threat to the people’s life and health. The early symptoms of esophageal cancer are not obvious and are easily confused with other digestive system disease. Therefore, the majority of patients when diagnosed has becomed the middle-advanced stage esophageal cancer and missed the best surgical treatment time. So the radiation and chemotherapy are the main treatment for most patients with esophageal cancer. In the clinical treatment, tumor cells often show resistance to traditional first-line chemotherapy drugs such as cisplatin, oxaliplatin and 5-fluorouracil, which result in the failure of the treatment. Therefore, the development of new drugs and new treatment options in research is particularly important.With more in-depth research of the carcinogenic factor and the signaling pathways associated with cancer, studies have found that the Bcl-2 family proteins in tumor cells are expressed abnormally, which is associated with the circumvention to apoptosis. To overcome this feature of tumor cells to evade apoptosis, a range of Bcl-2 family protein inhibitors including the development of ABT-263, ABT-199 and Obatoclax, based on the BH3 mimetic are designed. ABT-263 interacts with the Bcl-2, Bcl-xL and Bcl-w, inducing cell apoptosis in esophageal cancer cells and cycle arrest. ABT-199 designed on the basis of ABT-263 can be targeted in combination with the Bcl-2 while sparing platelets. Obatoclax is competitive to combine with the hydrophobic area of antiapoptotic protein, leading to cell apoptosis. Meanwhile, obatoclax can block autophagic degradation through inhibiting the cathepsin levels. Preclinical experimental results show that obatoclax has a definite therapeutic effect in both various blood malignant tumor including acute myeloid leukemia, chronic myelogenous leukemia and lymphoma and other solid tumor such as lung cancer, prostate cancer and colorectal cancer.A large number of studies have shown that tumor cell survival and metastasis are highly dependent on the normal protein degradation pathway. The ubiquitin proteasome system (UPS) is one of the main path-way of protein degradation in vivo eukaryotic cells, involved in cell cycle, DNA repair, cell adhesion and other life processes such as cell apoptosis. In view of the important role of proteasome system in the development of tumor, proteasome inhibitors have gradually becomed the research hotspot of anti-tumor drugs. MG-132 inhibits the activity of ubiquitin proteasome system by blocking the 26S proteasome and the class chymotrypsin in the 20S proteasome. MG-132 possesses anti-tumor activity which induces G2/M arrest in HL-60 cell and inhibits the proliferation and induces apoptosis in NRK-49F cells. In the malignant lymphocyte, MG-132 could arrest the cell cycle and induce mitochondrial depolarization to trigger apoptosis.When applied in the treatment or in the process of clinical trials, the traditional chemotherapy drugs, the Bcl-2 protein inhibitors and the proteasome inhibitor, the emergence of resistance has becomed the one of the main reasons for treatment failure or low pharmacological activity.The expression of BCL-2 protein is increased in cisplatin resistant cells, while reduction of Bcl-xL/Mcl-1 could effectively increase the sensitivity of cells to cisplatin and oxaliplatin. Meanwhile, cisplatin and oxaliplatin can induce autophagy, which is one of the important reasons for the resistance because autophagy provides raw materials and energy for tumor cell self-renewal by degradating the damaged organelles. Studies have shown that autophagy inhibitors can increase the sensitivity to cisplatin and oxaliplatin into tumor cells, which is a further evidence of the view that autophagy induced by cisplatin and oxaliplatin is related with the resistance.The overexpression of Mcl-1 is present in ABT-263 and ABT-263 resistant cells. The effect of ABT-263 on tumor cells could be increased by inhibiting the expression of Mcl-1 with the method of pharmaceutical drug interference. This indicates that the overexpression of Mcl-1 is related to the drug resistance of ABT-263 and ABT-199.Inhibition of the ubiquitin-proteasome system activates autophagy, because autophagy and the ubiquitin-proteasome system are two complementary pathways for protein degradation. Autophagy inhibitor 3-MA could not neutralize the cytotoxity caused by proteasome inhibitors, and knocking down autophagy related genes by RNA interference can increase the sensitivity of proteasome inhibitor in tumor cells. These rusults indicates that the autophagy induced by proteasome inhibitor plays an important role on protecting the cells from cell death and inhibiting autophagy is one way to overcome the drug resistence of proteasome inhibitors.These findings suggest that inhibition of antiapoptotic Bcl-2 protein expression autophagy, can overcome the limitations of cisplatin, oxaliplatin, ABT-263, ABT-199 and MG-132 in the application process of limitations in a certain range.OBJECTIVEObatoclax is similar with BH3-only protein functional, and has a strong inhibitory effect to the Bel-2 antiapoptotic proteins of known (Bcl-2, the Bcl-xL and Mcl-1). At the same time, obatoclax can block autophagic degradation through inhibiting the cathepsin levels. In this topic, we designed the combination of obatoclax with MG-132, ABT-199, ABT-263, cisplatin and oxaliplatin and determined whether these drug combination showed a synergistic antitumor effect on esophageal cancer cells by means of MTT method and CompuSyn software in vitro. Then we elucidated and explored the mechanism of synergistic effect antitumor effect in vitro. To provide reference for solving low drug activity, drug resistance and the same types of drugs combination, also construct a theoretical basis for the clinical combination of these drugs in the future.METHODS1. MTT assay was used to determine the cell cytotoxicityHuman esophageal cancer cell EC 109, CaES-17 and HKESC-1 were treated with different concentrations of a variety of compounds for 48 h. After treatment, cells were cultivated with 1 x MTT solution (0.5 mg/L) treatment for 4 h. DMSO was added and shocked to mixing. The optical density value was detected by means of an enzyme-linked immune detector at 570 nm.2. CompuSyn software was used to calculated the combination index (CI) and Dose Redution of two drugs combinationTwo drugs combined with the molar ratio was determined according to the IC50 of compounds. After doubling dilution, compounds were cultivated with human esophageal cancer cell EC 109, CaES-17 and HKESC-1 for 48 h. After treatment, cells were cultivated with 1 x MTT solution (0.5 mg/L) treatment for 4 h. DMSO was added and shocked to mixing. The optical density value was detected by means of an enzyme-linked immune detector at 570 nm. CompuSyn software was used to calculate the combination index (CI) and Dose Redution of two drugs combination.3. Trypan Blue staining method was used to explore the effect on cell viabilityEsophageal cancer cells CaES-17 and HKESC-1 were treated with obatoclax, MG-132, cisplatin and oxaliplatin alone or in combination for 48 h. Cell suspension was collected and the cells were stained with Trypan Blue.4. Flow cytometry was used to measure the effect on apoptosis after obatoclax combining with MG-132CaES-17 and HKESC-1 cells were exposed to obatoclax (0.125 μM) or MG-132 (0.3 μM) alone or obatoclax (0.125 μM) plus MG-132 (0.3 μM) for 48 h.Then harvested cells with trypis (no EDTA) and washed cells twice with PBS after centrifugation.Then washed cells with 3 mL 1 x binding buffer and resuspended cells in 100 μL 1 x binding buffer buffer,added 5 uL FITC-AnexinV, avoid light and incubate at room temperature 15min.400 μL 1 x binding buffer buffer was added to each tube. Apoptosis of cells were detected by flow cytometry within 1 h.5. Western Bloting was used to investigate the effect on the expression of the cleavage of PARP, caspase-9, phospho-Histone H3 and phospho-Aurora A/B/C after obatoclax combining with MG-132CaES-17 and HKESC-1 cells were treated with obatoclax (0.125 μM) or MG-132 (0.3 uM) alone or obatoclax (0.125 μM) plus MG-132 (0.3 μM) for 48 h, then cells were dissolved and proteins were quantified. The expression of the cleavage of PARP, caspase-9, phospho-Histone H3 and phospho-Aurora A/B/C after obatoclax combining with MG-132 of CaES-17 and HKESC-1 were detected by western bloting.6. Flow cytometry was used to determine the cell-cycle distribution after obatoclax combining with MG-132CaES-17 and HKESC-1 cells were incubated with obatoclax (0.125 μM) or MG-132 (0.3 μM) alone or obatoclax (0.125 μM) plus MG-132 (0.3 μM) for 48 h. Then harvested cells with trypsin, washed cells twice with PBS and collected cells with 75% ethanol-fixed overnight at 4℃. Cells were collected by centrifugation, washed twice with PBS, and then resuspended in 500 μL PBS containing 10 μL propidium iodide (PI) and 1.25μL RNase-A.Then incubate at 37 ℃ in the dark for 30 min.The cell-cycle distribution was analysed by flow cytometry with 1 h.7. Western Bloting was used to investigate the effect on the accumulation of ubiquitinated proteins and the expression of LC3-Ⅱ after obatoclax combining with MG-132CaES-17 and HKESC-1 cells were exposed to obatoclax (0.125 μM) or MG-132 (0.3 μM) alone or obatoclax (0.125 μM) plus MG-132 (0.3 μM) for 48 h,then cells were dissolved and proteins were quantified. The accumulation of ubiquitinated proteins and the expression of LC3-Ⅱ of CaES-17 and HKESC-1 were detected by western bloting.8. Statistical analysisThe software of GraphPad Prism 5.0 was used to analysing data and mapping. Tukey HSD test was used to compare data between three or more groups. Test level a=0.05, P<0.05 indicates significant difference.RESULTS1. As shown in MTT assay, obatoclax, MG-132, ABT-199, ABT-263, cisplatin and oxaliplatin had significant cytotoxicity with human esophageal cancer cell EC 109, CaES-17 and HKESC-1 when treated alone.The IC50 of obatoclax for EC109, CaES-17 and HKESC-1 was 0.21 ±0.06 μM, 0.12±0.03 μM and 0.13±0.01 μM, respectively. The IC50 of MG-132 for EC 109, CaES-17 and HKESC-1 was 0.24±0.05 μM,0.32±0.04 μM and 0.29±0.07 μM, respectively. The IC50 of ABT-199 for EC109, CaES-17 and HKESC-1 was 12.89±1.15 μM,14.82±2.11 μM and 9.01±1.36 μM, respectively. The IC50 of ABT-263 for EC109, CaES-17 and HKESC-1 was 10.75±1.02 uM,8.21±1.57 uM and 6.49±1.11 μM, respectively. The IC50 of cisplatin for EC 109, CaES-17 and HKESC-1 was 6.05±0.95 μM,3.33±0.92 μM and 2.34±0.80 μM, respectively. The IC50 of oxaliplatin for CaES-17 and HKESC-1 was 5.84±1.01 μM and 5.95±0.89 μM, respectively.2. By calculating the Combination Index and Dose Recdution of two drugs combination, we confirmed there was a significant synergistic antitumor effect on esophageal cancer cell lines EC 109, CaES-17 and HKESC-1 when combined obatoclax with MG-132 or cisplatin in vitro. Meanwhile there was a significant synergistic antitumor effect on esophageal cancer cell lines CaES-17 and HKESC-1 when combined obatoclax with oxaliplatin in vitro.While there was no significant synergistic antitumor effect on esophageal cancer cell lines EC 109, CaES-17 and HKESC-1 when combined obatoclax with ABT-199 or ABT-263 in vitro.3. The cell viability in the cells esposed with obatoclax plus MG-132, cisplatin and oxaliplatin were significantly increased compared to the cells treated wiih drug alone.4. The combination of obatoclax with MG-132 resulted in the increase of apoptosis on esophageal cancer cell lines CaES-17 and HKESC-1.The results of Annexin V positive cells detected by flow cytometry indicated that obatoclax alone did not induce apoptosis, but the use of MG-132 alone induced apoptosis. After two drug combination, the Annexin V positive rate was significantly increased significantly, in which there was a statistically difference compared to the drug alone group.The cleavage of PARP and Caspase-9 were detected by means of Western bloting. When exposed with obatoclax plus MG-132, the cleavage of PARP was significantly increased compared the drug alone group in esophageal cancer cell lines CaES-17 and HKESC-1.As for the cleavage of Caspase-9, drug combination resulted in a significant increase on esophageal cancer cell lines CaES-17, but there was no obvious change on esophageal cancer cell lines HKESC-1. Those results suggested the combination of obatoclax with MG-132 resulted in the increase of apoptosis on esophageal cancer cell lines CaES-17 and HKESC-1.5. Obatoclax combined with MG132 arresed esophageal cancer cell lines CaES-17 and HKESC-1 in the G2/M phase.We evaluated the effects of drug combination on cell cycle by flow cytometry. Our results showed that the percentage of sub-G1 phase on sophageal cancer cell lines CaES-17 and HKESC-1 was significantly increased, which futher illustrated the drug combination increased the apoptosis. We also found the percentage of G2/M phase in cells CaES-17 and HKESC-1 was significantly upregulated after obatoclax combining whit MG-132. While S phase cell ratio was decresed and the G1/G0 cell proportion showed no obvious impacts.From the view of protein molecules, we found the expression of phospho-Histone H3 and phospho-Aurora A/B/C in the cells treated with obatoclax plus MG-132 were significantly increased compared to the cells treated with obatoclax or MG-132 alone.Collectively, our data showed obatoclax combined with MG132 resulted in the arrest of G2/M phase in cells CaES-17 and HKESC-1.6. The accumulation of ubiquitinated proteins and the expression of LC3-Ⅱ in the cells treated with obatoclax plus MG-132 were significantly increased (P<0.05) compared to the cells treated with obatoclax or MG-132 alone.When cells were treated with obatoclax and MG-132 alone, the accumulation of ubiquitinated proteins and the expression of LC3-Ⅱ were upregulated. After two drug combination, the accumulation of ubiquitinated proteins and the expression of LC3-II were significantly increased.CONCLUSIONS1. Obatoclax combined MG-132, cisplatin and oxaliplatin showed a significant synergistic effect on esophageal cancer cell lines CaES-17 and HKESC-1.2. Obatoclax combined with MG-132 showed a significant synergistic effect on esophageal cancer cell lines CaES-17 and HKESC-1, by inducing apoptosis and G2/M phage cell cycle arrest.3. The accumulation of ubiquitinated proteins and the expression of LC3-II in esophageal cancer cells CaES-17 and HKESC-1 were synergistically increased when obatoclax combined with MG-132.