Bortezomib-Induced Anti-Colon Cancer Effects And Targeted Sensitization:A Mechanism Study

BackgroundThe incidence of colon cancer, a common gastrointestinal tumor, has been increasing annually in our country. Although significant improvements have been achieved in surgery and/or chemotherapy treatments in the past 20 years, there has been no major improvement in the patients’ overall survival. Thus, the searching for novel and efficient agents against colon cancer is urgently needed.Bortezomib, also known as PS-341 or Velcade, is a potent and highly selective proteasome inhibitor. Studies have shown that bortezomib exerts broad anti-tumor activities. United States Food and Drug Administration has approved bortezomib for the treatment of refractory or relapsed multiple myeloma (MM) and mantle cell lymphoma (MCL), based on its highly favorable results from clinical trials in these patients. Meanwhile, this drug is currently undergoing evaluation in several other solid tumors including colon cancers. However, studies have found that bortezomib as a single agent has somehow limited effects in above cancers, probably due to chemo-resistance or other unknown mechanisms. Thus, bortezomib chemo-sensitization strategies are developing.Autophagy is an evolutionarily conserved caspase-independent process, which is responsible for the degradation and recycling of long-lived proteins and cytoplasmic-damaged organelles. Studies have showed that autophagy is a pro-survival factor in cancer cells, probably due to its anti-apoptosis ability. AMPK is the main intracellular energy sensor and balancer. Recent studies have confirmed other important functions of AMPK activation. It is involved in regulating of a number key cellular functions including cell apoptosis, oxidative stress and autophagy. However, whether AMPK’s function can be regulated by bortezomib is not fully understood.Our premilary data in cultured colon cancer cells demonstrated that bortezomib inhibited colon cancer survival and proliferation. The aim of this study is to investigate the relationship between mPTP opening and cytochrome c release and cell apoptosis. Also, we will confirm that AMPK signaling activate autophagy in cultured colon cancer cells. Inhibition of this pathway using pharmacological or siRNA strategies enhanced bortezomib-induced colon cancer cell apoptosis.Our study was divided into three parts. Part one is aim to investigate the mechanism of bortezomib-induced anti-colon cancer effects. Part two is aim to confirm bortezomib treatment induces protective autophagy. Part three is aim to demonstrate TAK1-AMPK signaling activates autophagy in cultured colon cancer cell.Part one Mechanism of Bortezomib-induced anti-colon cancer effectsObject:To observe the bortezomib-induced anti-colon cancer effects and investigate its possible mechanism.Method:After bortezomib treatment (100 nmol/L), cell viability was measured by MTT assay, cell apoptosis and cell cycle were also detected by flow cytometry, cell death was analyzed by Trypan blue staining, expression level of Cyto-C was examined by Western blots, and MMP level was assessed by JC-10 staining.Result:Compared with the control group, cells viability decreased and cells death increased in colon cell lines HT-29, HCT-116 and SW-620 (p<0.05). In cell line HT-29 alone, cell apotosis and the percentage of cells in G1 phase increased, MMP level decreased significantly (p<0.05), and the expression level of Cyto-C increased compared with the control group. Compared with bortezomib alone, cell survival and MMP level increased (p<0.05), but the expression level of Cyto-C decreased after SFA or CSA treatment.Conclusion:Bortezomib can inhibit colon cancer cell viability and induce cell cycle G1 arrest, cell death and apoptosis. The mPTP-Cyto-C pathway may be the core mechanism of the bortezomib-induced colon cell apoptosis. So highly selective proteasome inhibitors, including bortezomib are potent to be new anti-colon cancer strategy in the future.Part two Bortezomib induce protective autophagy in colon cancerObject:To investigate the relationship between bortezomib-induced autophagy and chemo-resistance in colon cancerMethod:HT-29 cell was treated with vehicle (0.1% DMSO) or bortezomib (100 nmol/L) for 12 or 24 hours, autophagy induction was analyzed by LC3B puncta immuno-fluorescence, expression levels of LC3B-I, LC3B-II, Beclin-1, p62, phospho-and total Ulkl were examined by Western blots. HT-29 cell was treated with bortezomib (100 nmol/L) in the presence or absence of autophagy inhibitors 3-MA (1 mmol/L) and chloroquine (400 μmol/L), cell viability was analyzed by MTT assay after 48 hours, and the percentage of Annexin V positive (apoptotic) cells were also recorded.Result:Compared with the control group, bortezomib induced significant autophagy induction in HT-29 cell, as the expressions of beclin-1, LC3B-II and p-Ulkl were increased but the expressions of LC3B-I and p62 decreased after bortezomib stimulation. Meanwhile, both 3-MA and chloroquine facilitated bortezomib-induced HT-29 cell death and apoptosis (p<0.05).Conclusion:These results indicate that bortezomib-induced autophagy serves as a pro-survival and resistance factor, and autophagy inhibition could enhance bortezomib-induced apoptosis and cytotoxic effect in colon cancer cell line.Part three Boterzomib induce protective autophagy through TAK1-AMPK pathwayObject:To investigate the relationship between bortezomib-induced TAK1-AMPK-autophagy pathway and chemo-resistance in colon cancerMethod:HT-29 cell was treated with vehicle (0.1% DMSO) or bortezomib (100 nmol/L) for 12 or 24 hours, phospho- and total level of AMPK, TAK1 and ACC were tested by Western blots. AMPKa-shRNA-transfected or scramble shRNA (Ctrl shRNA)-transfected stable HT-29 cells were treated with bortezomib (100 nmol/L) in the presence or absence of AMPK inhibitor compound C (CC,10 μmol/L) for 24 hours, phospho-and total level of AMPK, ACC, Ulkl as well as tubulin and LC3B were examined by Western blots, LC3B puncta positive cells were recorded and percentage was calculated. AMPKa-shRNA-transfected or scramble shRNA (Ctrl shRNA)-transfected stable HT-29 cells were treated with with bortezomib (100 nmol/L) in the presence or absence of AMPK inhibitor compound C (CC,10 μmol/L) for 48 hours, cell viability was analyzed by MTT assay, cell apoptosis was detected by Annexin V assay. TAKl-shRNA-transfected or scramble shRNA (Ctrl shRNA)-transfected stable HT-29 cells were treated with bortezomib (100 nmol/L) for 24 hours, phospho-and total level of AMPK as well as tubulin, Beclin-1, TAK1 and p62 were examined by Western blots. After 48 hours, cell viability was analyzed by MTT assay, cell apoptosis was detected by Annexin V assay.Result:Compared with the control group, we observed a significant TAK1-AMPK activation (AMPK/ACC/TAK1 phosphorylation) after bortezomib stimulation in HT-29 cells. AMPK stable knockdown by RNAi and AMPK inhibitor compound C largely alleviated bortezomib-activated autophagy, compound C and AMPK knockdown similarly enhanced bortezomib-induced survival loss and cell apoptosis (p<0.05). TAK1 stable knockdown by RNAi largely alleviated AMPK activation (AMPK phosphorylation) and bortezomib-activated autophagy (Beclin-1 decreased and p62 increased). TAK1 knockdown enhanced bortezomib-induced survival loss and cell apoptosis (p<0.05).Conclusion:These results indicate that TAK1-AMPK activation by bortezomib mediates autophagy induction and TAK1 or AMPK inhibition enhances bortezomib-induced cytotoxicity probably through facilitating cell apoptosis in cultured colon cancer cell. But the interaction between TAK1 and AMPK deserves further investigated.