Inhibitiory Effect On The Proliferation Of Human Renal Carcinoma Cell Line GRC-1 By Blockers Of Chloride Channel

Renal cell carcinoma is most common in tumors of kidney. Its attack rate ranks second among all urinary tumors. Renal carcinoma cell contains a natural multi-drug-resistance gene (mdr-1) and protein P170, the latter can pump the intracellular drug out . Consequently, chemotherapy has a very low efficiency on renal carcinoma , no matter single or combinally used. Meanwhile, renal carcinoma is insensitive to radiotherapy, and has no actual effect to immunotherapy. Up to now, the surgery resection is still the only curing method . The treatment of postoperative andterminal patients has already become one of the difficulties in clinic.Chloride channels are found in variety of plasma membranes and intracellular compartment membranes. The CIC-type chloride channels make up a independent protein family, which is expressed in almost all the tissues of the organism, involved in many physiological functions , associated with many profiles and processes of the cell, such as pH, volume, excitability , proliferation and differentiation etc. Therefore, the chloride channels in kidney is very important for physiological functions. Three of the eight CIC-type chloride channels: C1C-K1 , C1C-K2 , C1C-5 , have been shown to be associated with kidney diseases in human and mice. However, whether chloride channels are related with renal carcinoma has not been reported yet. The present experiment is aimed to study the inhibitory effects of NPPB and NFA, chloride channels blockers, on human renal carcinoma cell line GRC~1, so as to add some theoretic support for pharmacotherapy in renal carcinoma. Methods Part one: Cell Morphology Observation1. We adopted inverted microscope to observe the cells growth of the human renal carcinoma cell line GRC-1.2. The cell ultrastructure was observed under electronicmicroscope.Part two: Cell function studies1. Cell viability evaluation( MTT assay)2. Cell cycle analysis, (flow cytometry)3. Intracellular calcium concentration changes detection (Confocal microscope with Fluo-3/AM as indicator) . Results1. Under invert microscope, the cell volume of GRC-1 cell was decreased , the intercellular gaps were enlarged, and the cell proliferation was inhibited.2. The ultrastructure of human renal carcinoma cell line GRC-1 was observed under the electronic microscope, and the result showed that NPPB or NFA can induce the apoptosis of human renal carcinoma cell line GRC-1by.3. MTT assay showed that the proliferation of the human renal carcinoma cell line GRC-1 could be significantly inhibited by 50 n M/L, lOOu M/L NPPB or NFA.4. From FCM we found that the most cells of human renal carcinoma cell line GRC-1 was inhibited by NPPB or NFA at G0/G1 phase, Suggesting that chloride channel blockers may act at or before the G1/S checkpoint.5. Confocal observation showed that when treated with 100uM/L NPPB or NFA, the intracellular calcium fluorescence intensity of human renal carcinoma cell line GRC-1 was significantly lower than that of the controls.ConclusionThe results suggest that chloride channels blocker can significantly inhibit the growth of human renal carcinoma cell line GRC-1 cells. One of the possible mechanisms is that chloride channels may act at or before the G1/S checkpoint to stagnate the human renal carcinoma cell line GRC-1 cells at G0/G1 phase; NPPB or NFA can decrease the intracellular calcium concentrationand induce the apoptosis of human renal carcinoma cell line GRC-1 cells.