Effect Of Orail And STIM1on The Apoptosis Of Human Lung Adenocarcinoma Cells Treated By Curcumin

BackgroundLung cancer is the most common cancer and the first leading cause of cancer-related death in China. Over the past30years, the death rates of lung cancer in China have increased by465%, this figure looks unbelievable, but it is true. Statistics show that lung cancers have been the first leading cause of malignant tumors in China. Non small cell lung cancer (NSCLC), including squamous cell carcinoma, adenocarcinoma, and large cell carcinoma. Whose cancer cell growth and division slower and diffusion transfer later as compared with small cell carcinoma, non-small cell lung cancer accounts for about80-85%of the total number of lung cancers. In recent years, the effectiveness of chemotherapy about non-small cell lung cancer does have significant progress both from the local control rate of tumor shrinkage (single-agent response rates of10-30%, two chemotherapy drugs in the treatment response rates of35-45%),and from the ease progression to improve the symptoms. However, even with the progress of every aspect in radiation therapy, surgery and so on, how to prolong the survival time of patients still has limited progress. This reality highlights a need to develop better drugs to prevent and treat lung cancers at the early stage.Curcumin is a chemical composition which extracted from the roots of the ginger family and Araceae. It contains about3-6%of turmeric, which is diketones compounds with very rare diketone of pigmeng in the plant kingdom. Curcumin is orange yellow crystalline power, slightly bitter taste. Modern research has found that curcumin plays a role in inhibiting inflammation, anti-oxidation, anti-rheumatoid. The VITY vitamin magazine of American reported:"The main pharmacological effects of curcumin have antioxidant, anti-inflammatory, anticoagulant, lipid-lowering, anti-atherosclerotic, anti-aging, eliminate free radicals, inhibit tumor growth and so on." Curcumin has extensive pharmacological effects, which is not only the antimutagenic agent, but also the anti-cancer-promoting agent. Curcumin is classified as the third generation cancer chemopreventive drugs by American Tumor Institute. The antitumor mechanisms of curcumin are diverse, such as the inhibition of cell proliferation, induction of cell apoptosis, antioxidant and scavenging oxygen free radicals. Curcumin can inhibit non-small cell lung cancer, studies have showed that curcumin can inhibit the proliferation of non-small cell lung cancer and induce the cycle arrest or cell apoptosis. But the specific mechanism of curcumin-induced apoptosis is unclear.Calcium operated Ca2+channels (SOC) is the main channel of extracellular Ca2+influx in non-excitable cells, which is extensively present in the cell membrane. The concept of capacitative calcium influx (capacitative Ca2+entry, CCE) was firstly proposed by James Putney in1986, which is known as store-operated calcium entry (SOCE) now. SOCE refers to the decrese of ER Ca2+concentration which induces cell extracellular Ca2+influx process. Since this process has been disclosed, SOCE almost exists in all cells involved in cell secretion, growth, control, lymphocyte activation and immune and other important functions. With the development of RNA interference technology, two important elements of SOCE progress were identified: STIM1(Stromal interaction molecule1) and Orai1. SOCE mainly causes extracellular Ca2+influx, this mechanism is involved in a variety of important physiological functions, including cell proliferation, cell apoptosis, and cell differentiation and so on. 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 curcumin, however, has not yet been investigated. For this reason, this study mainly explore whether SOCE is involved in cell apoptosis process of non-small cell lung cancer induced by curcumin, and to provide theoretical support for curcumin which is developed as a tumor treatment drug.PurposeTo explore whether STIM1and Orail are participate in the apoptosis induced by curcumin of non-small cell lung cancer cell (A549cell).To explore the mechanism of STIM1and Orai1participate in the apoptosis induced by curcumin of non-small cell lung cancer.Methods1. Non-small cell lung cancer cell line A549cells were cultured in37℃incubator (5percent CO2,95percent air), which were treated with different concentrations of curcumin respectively at different time, and to detect the cell viability by MTT.2. Cells cultured for24h, and then treatment with different concentrations of curcumin for24h, detecting the apoptosis rate by flow cytometry.3. To detect the STIM1and Orail changes in mRNA levels after curcumin treatment in A549cells by fluorescence quantitative PCR method.4. The protein levels of STIM1and Orail in non-small cell lung cancer cells A549detected by Western blot. 5. Downregulate the expression of STIM1and Orail in the A549cells by the siRNA interference, and detected the protein level of STIM1and Orail by immunoblotting.6. Upregulate the expression of STIM1and Orail in the A549cells transfected with empty and wild-type STIM1and Orail plasmid. And detected the protein level of STIM1and Orai1after STIM1and Orai1overexpression by immunoblotting.7. Using the flow cytometry to detect the changes of cell apoptosis rates after STIM1and Orail interfere and overexpress.Results1. Curcumin induces the apoptosis of A549cells Non-small cell lung cancer cell A549cells were treated by different concentrations of curcumin for12、24、48h, and detected the cell viability by methyl thiazolyl tetrazolium(MTT) colorimetric determination. The results indicated that the cell viability rates significantly decreased compared with control group after treated with curcumin for24h, the difference was statistically significant(P<0.01). The cell apoptosis rates were detected by flow cytometry after treated by different concentrations of curcumin for24h, and the results suggested that the cell apoptosis rates significantly increased compared with control group after treatment of25μmol/L curcumin for24h, the difference was statistically significant(P<0.01). These results showed that curcumin can induce apoptosis of A549cells, and have concentration-dependent and time-dependent.2. Curcumin can induce the changes of expression levels about mRNA of STIM1and Orai1in A549cells After treated with curcumin, the expression levels about mRNA of STIM1and Orail in A549cells significantly decreased(P<0.01).3. The protein expression levels of STIM1and Orai1in A549treated by curcumin were changed. After treatment with different concentrations of curcumin, we found that the protein expression levels obviously descended.4. Using Western blot method to detect interference efficiency of STIM1or Orail expression after siRNA interference, showed that in A549cells the interference efficiency of STIM1siRNA-1is57%, STIM1siRNA-2is75%, and Orail siRNA-1is49%, Orail siRNA-2is50%; the cell apoptosis rates were detected by flow cytometry, the outcomes suggested that cell apoptosis increased when STIM1or Orai1interference and treatd with curcumin for24h.5. The empty plasmid and STIM1and Orail wild-type plasmid were transfected into the non-small cell lung cancer cells.Western blot analysis showed that in A549cells, the STIM1plasmid transfection efficiency was216%(figure10), the Orai1plasmid transfection ffficiency was154%(figure11); the apoptosis rates with STIM1or Orail overexpression were detected using flow cytometry method, and the results showed that the apoptosis rates descented compared with control group after treated with curcumin for24h.Conclusion1. Curcumin can induce apoptosis of non-small cell lung cancer A549cells, and the apoptosis rates of A549cells have concentration-dependent and time-dependent.2. Curcumin treatment led to the reduction of the expression levels of STIM1and Orai1mRNA and protein levels in A549cells, curcumin may be inhibit the protein levels of STIM1and Orai1by inhibit the mRNA expression, the further downregulate the protein of STIM1and Orail, eventually leading to apoptosis.3. Downregulation of STIM1and Orail enhanced apoptosis rates, overexpression of STIM1and Orail attenuated the apoptosis rates induced by curcumin, further proved that STIM1and Orail participate in curcumin-induced apoptosis of non-small cell lung cancer cell A549through the changes of STIM1and Orai1.