Store-operated Calcium Entry Is Involved In Sodium Butyrate-induced Apoptosis In Colon Cancer Cells

BackgroundColon cancer is the third most common cancer and the fourth leading cause of cancer-related death in the world, with an estimated incidence of1,233,700new cases and a mortality of608,700deaths annually based on the statistics for the year2008. Despite the recent advent of targeted therapy (e.g. cetuximab and bevacizumab) and the improvement of other treatment modalities, the prognosis for patients with metastatic colon cancer remains limited. This reality highlights a need to develop new chemoprophylactic agents for prevention of colon cancer at the early stage.Short-chain fatty acids have been shown to inhibit progression of colorectal cancer. They are naturally produced via the process of anaerobic microbial fermentation of dietary fibers within the colon. Among them, butyric acid has received the greatest attention due to its potent and pleiotropic anticancer effects. Sodium butyrate inhibits cell cycle progression, induces apoptosis and promotes differentiation in various types of cancer cells, including colorectal cancer, lymphoma cancer and breast cancer. However, the mechanism of the action remains elusive. It was suggested that the anticancer effect of sodium butyrate is mediated, at least in part, through inhibition of histone deacetylase. Ca2+signaling pathway plays an important role in many cellular activities, including neurosecretion, skeletal muscle contraction, cell growth and differentiation. Accumulating evidence supports the notion that increased cytoplasmic Ca2+could cause cell apoptosis, at both early and late stages of apoptotic processes. Store-operated Ca2+entry (SOCE) refers to the phenomenon that depletion of intracellular Ca2+stores activates Ca2+channels in the plasma membrane as a compensatory mechanism to refill the internal Ca2+store, stromal interaction molecule1(STIM1) and Orail are two proteins essential for SOCE. STIM1has a single transmembrane domain and an EF-hand Ca2+binding domain as a sensor of the ER luminal Ca2+. Through redistribution, STIM1binds to and transmits signal to Orail, the pore forming subunit of the store-operated Ca2+(SOC) channel. This interaction results in extracellular Ca2+influx. The SOC channel-mediated Ca2+release from the ER and extracellular Ca2+influx could in turn cause apoptosis. Therefore, since SOCE is one of the major mechanisms for Ca2+entry in non-excitable cells, we hypothesized that SOCE is involved in apoptosis induced by sodium butyrate, however, has not yet been investigated. Here, we report that both intracellular and extracellular Ca2+play a key role in sodium butyrate-induced apoptosis in the colon cancer cell line, HCT-116. Moreover, the influx of extracellular Ca2+is mediated through SOCE.MethodsCell culture, Conventional and Real-Time Reverse Transcription-Polymerase Chain Reaction, Western Blots, Annexin V/PI assay for apoptosis, Morphological assessment of apoptosis, RNA Interference, Immunofluorescence Imaging.Results 1. NaB induces the apoptosis of HCT-116cellsThe apoptosis of HCT-116induced by NaB is confirmed by hoechst33342staining, where we observed condensation and fragmentation of chromatin in HCT-116cells with lOmM NaB incubated for24h. To determine the apoptosis of HCT-116at different dose and time of NaB, the apoptosis of HCT-116was quantified by annexin V+PI assay. Compared with the control, the apoptosis of HCT-116increase is correlated with the concentration and time exposure of NaB. This suggests that the increase of apoptosis of HCT-116by NaB is dose and time-dependent. Furthermore, we measure the cleavage of PARP, which is a marker of the apoptotic response, by western blot at different dose and time of NaB. A clear increase in the percentage of cleavage of PARP degradation24h after treatment with10mM NaB was noted, which is in coincident with the apoptosis of HCT-116quantitified by annexin V+PI assay.2. Ca2+is involved in NaB-induced apoptosis of HCT-116cellsTo examine the role of the Ca2+in the apoptosis of HCT-116by NaB, extracellular Ca2+was chelated by0.5mM EGTA. Compare with those of NaB-treated alone, preincubation with EGTA exhibited a significantly attenuation of the apoptosis of HCT-116. Furthermore, chelated the intercellular Ca2+with10μM BAPTA-AM have shown same results. These data suggest that the extracellular Ca2+influx implicated in the apoptosis of the HCT-116induced by NaB.3. SOCE is involved in NaB-induced apoptosis of HCT-116cellsTo identify the SOCE is involved in NaB-induced apoptosis in HCT-116cells, HCT-116was preincubated with5μM SKF96365and75μM2-APB which were the pharmacologic inhibitor of SOCE. The apoptosis of HCT-116was decreased significantly measured by annexin V+PI assay compared with those of the control cells. Meantime the percentage of the cleavage of PARP degradation in the pretreated cells with SKF96365or2-APB decreased compared with the control cells treated by NaB only.In order to confirm the involvment of SOCE in the apoptosis of HCT-116induced by NaB, STIM1, a key molecule of SOCE located mostly in the ER, was first downregulated. Follwing the downrgulation of STIM1, NaB-induced apoptosis was also antenuated. The efficiency of downregulation is determined by real-time PCR and western blot The apoptosis of HCT-116was quantitified by annexin V+PI assay and the percentage of cleavage of PARP degradation decreased significantly after the the downregulation of STIM1.4. The apoptosis of HCT-116induced by NaB are independent on the expression of STIMlTo determine whether the apoptosis of HCT-116induced by NaB is dependent of the transcriptional activation of STIM1, just like the way NaB works on other proapoptosis genes in cancer cell apoptosis, we measured the levels of protein STIM1in HCT-116at different dose and time under NaB treatment. Immunoblotting with anti-STIM1antibody showed no difference in the protein of STIM1in treated cells in comparison to those of the control cells. This result indicated that the role of NaB is independent of the expression of STIM1.5. NaB triggers the translocation and colocalization of STIM1and OrailWe investigated the change of STIM1localization followed by NaB-treatment. The treatment of NaB triggered a redistribution of STIMl into puncta. Moreover, STIM1and Orail were colocalized in the same puncta that have previously been characterized. These results indicate the treatment by NaB induced the translocation of STIM1and Orail, which suggests that the apoptosis induced by NaB is correlated with the redistribution of STIM1and Orail.ConclusionIn conclusion, this study shows for the first time that extracellular Ca2+influx, which is mediated by SOC channels in a STIM1-dependent manner, is involved in sodium butyrate-induced apoptosis in colon cancer cells. Theses data not only provide new insights into the molecular mechanism of the pro-apoptotic action of sodium butyrate in colon cancer cells, but also highlights the role of SOCE in the regulation of apoptosis.