Expression Of Store-operated Calcium Entry And Its Functional Proteins Trpc, Orai And Stim1 In Non-small Cell Lung Cancer

BACKGROUND: Lung cancer, which appears the highest morbidity and mortality among all human tumor types, is one of the leading diseases harmful to human health. Futher study on the pathogenetic mechanim of lung cander is needed to establish novel diagnostic or treatment strategies for this lethal disease. Calcium is an important messenger mediating various cellular activities such as proliferation, differentiation, and gene transcription. Calcium influx through store-operated calcium channels (SOCC), termed calcium store-operated calcium influx (SOCE), is an important approach regulating the cytoplasmic concentration of calcium. SOCC are thought to be constituted by the transient receptor potential canonical (TRPC) and store-operated Ca²⁺-release-actived Ca²⁺ channel (CRAC/ORAI) family members including TRPC 1-7 and ORAI 1-3. Stromal-interacting molecules, i.e., STIM1 play pivatol roles in transducing the calcium store depletion signal and activating SOCC. Over the past decade of years, the studies of SOCC and its associated functional proteins were mainly in the nervous, blood, lymphatic, cardiovascular system and urinary systems. Recently, emerging evidence has revealed that abnormal SOCE was also associated with the progresses of many kinds of tumors, i.e., hepatoma, breast cancer and prostatic carcinoma; however, it remains unclear whether SOCE plays a role in the development of non-small cell lung cancer (NSCLC). OBJECTIVES: To determine the presence of SOCE and the expression of its functional proteins TRPC, ORAI, and STIM1 in NSCLC tissues and cells, with the aim to provide molecular basis for further identification of their functional importance in the pathogenetic mechanisms of lung cancer in future study.METHODS: Fluorescent quantitative PCR and western blotting were respectively used to detect the mRNA and protein expression of TRPC, ORAI, and STIM1 in NSCLC tissues, normal lung tissues and non-small cell lung cancer cells (A549 and SPC-A-1). Intracedular Ca²⁺ concentration ([Ca²⁺]_i) in NSCLC cells was measured via Ca²⁺-sensitive dye, fura 2-based fluorescent microscopy, and SOCE was assessed by monitoring the increase of [Ca²⁺]_i caused by restoration of extracellular Ca²⁺ after perfusing the cells in Ca²⁺-free physiological salt solutions containing cyclopiazonic acid and nifedipine.RESULTS: 1. Among the seven known members of TRPC family, we found TRPC1, TRPC3, TRPC4 and TRPC6 mRNA were selectively expressed in human NSCLC tissues, while TRPC2, TRPC5, and TRPC7 mRNA were not detectable. The relative abundance of the expressed TRPC mRNA was TRPC1≈TRPC6>>TRPC3>TRPC4. 2. The mRNA levels of all expressed TRPC in NSCLC tissues exhibited a lower trend comparing to normal lung tissues; however, only the reduction of TRPC6 mRNA was found statiscally significant. 3. In NSCLC cells, TRPC1 and/or TRPC6 were the one mainly expressed, followed by lower expression of TRPC3 and TRPC4; neither TRPC2, TRPC5, or TRPC7 was detectable. 4. Western blotting demonstrated the protein expresseion of TRPC1 and TRPC6, which were found mainly expressed in NSCLC tissues and cells. The expression of TRPC6 protein in NSCLC tissues was significant less in comparison with normal lung tissues. The amount of TRPC1 protein in the two types of tissues was not different. 5. ORAI1, ORAI2, ORAI3 and STIM1 mRNA were found expressed in NSCLC tissues, with no difference in their expression levels comparing to normal lung tissues. 6. Fluorescent microscopic calcium quantification system demonstrated the presence of SOCE in NSCLC cell lines as well as primary cells.CONCLUSION: We demonstrated the presence of SOCE in human NSCLC cells. NSCLC tissues and cells selectively express TRPC1, 3, 4 and 6 of TRPC members, with TRPC1 and TRPC6 as the one mainly expressed; NSCLC tissues express all ORAI family members (ORAI1³) and STIM1. The individual role of these expressed molecules in constituting SOCC, mediating SOCE in NSCLC cells and in the development of NSCLC need to be further investigated. The fact of decreased expression of TRPC6 in NSCLC tissues indicates that it may be a novel molecular marker for NSCLC dianosis and treatment.