TRPC1Is Involved In Cadmium-induced Apoptosis In Human Embryo Kidney Cells

BackgroundCadmium is a heavy metal, the formation of inorganic compounds with oxygen, chlorine, sulfur and other chemical elements distributed in nature. It can accumulate in the bodies of plants and animals, and can enter the human body by food chain, followed by slow accumulation in the body. Cadmium is an environmental contaminant that has recently gained public attention due to the worldwide increase in discard of this toxic metal. Cd has been classified as a human carcinogen by the National Toxicology Program of USA. Cadmium affects a diverse range of cellular events, such as proliferation, differentiation, and apoptosis. Cadmium pollution problem has been worsening with the rapid development of modern industry, technology, cadmium apparently there are potential hazards to humans. But so far, no effective therapy for chronic cadmium poisoning. This reality highlights a need to develop new chemoprophylactic agents for prevention of cadmium poisoning at the early stage.Apoptosis is a fundamental form of cell death and plays an essential role in the development and homeostasis of multicellular organisms. Ca2+signaling pathway plays an important role in many cellular activities, including neurosecretion, skeletal muscle contraction, cell growth and differentiation. On the other hand, intracellular calcium homeostasis is very important in maintaining normally the cellular functions in response to extragenous and/or endogenous stimuli. Thus, it is not surprising that changes in Ca2+concentration in cytoplasm as well as in different intracellular organelles could be responsible for induction of apoptosis. Accumulating evidence supports the notion that increased cytoplasmic Ca2+could cause cell apoptosis, at both early and late stages of apoptotic processes.At concentrations higher than5.0μM, Cd could induce apoptosis both in vivo and in vitro in many tissues and organs such as, kidney, lung, testis and liver. Although cadmium-induced cytotoxicity and apoptosis are well studied in various cell types, the precise mechanisms are not well understood. Cadmium is reported to interfere with cell calcium homeostasis at different levels. It has been reported that cadmium increases intracellular levels of calcium ion, which mediates apoptosis in various types of cells. There is now growing evidence that members of the transient receptor potential canonical (TRPC) channels can participate in Ca2+influx pathways. It is considered to be an important ion channel for the regulation of intracellular Ca2+homeostasis. Moreover, TRPC1is important for critical processes such as cell proliferation and apoptosis. However, it is still not clear that TRPC1involved in Cadmium-induced apoptosis in Human Embryo Kidney cells. And the detailed mechanisms of Cd-induced apoptosis still remain poorly understood. Therefore, In this research we report that TRPCl play a key role in Cadmium-induced apoptosis in Human Embryo Kidney cells, HEK293.ObjectiveIn this research, we use CdCl2induced apoptosis in Human Embryo Kidney cells. We investigate the function of TRPC1on the Human Embryo Kidney cells and molecular mechanism in this model in order to provide important clue for the therapy on cadmium poisoning. Further study the specific mechanisms of cadmium on the kidney damage, emerging TRPC channels are involved in the apoptotic function makes it a potential target for the regulation of apoptosis drugs.Methods1. To establish a model of CdCl2-induced apoptosis, the HEK293cells were treated with different concentrations of CdCl2for6h using annexin V+FITC/PI staining followed by flow cytometric analysis.2. Real-time RT-PCR and Western Blot were used to detected the expression in mRNA level and protein level of TRPCs in the normal Human Embryo Kidney cells.3.TRPC1were overexpressed by transiently transfecting corresponding plasmids. Real-time RT-PCR and western blot were used to detected the expression in mRNA level and protein level of TRPC1. Flow cytometry assay was used to detect whether upregulation of TRPC1infulenced on apoptosis of HEK293cells treated with CdCl2.4. Flow cytometry assay was used to detect whether different concentrations of CdCl2influenced the cells cycle of HEK293cells.5. Inductively coupled plasma mass spectrometry(ICP-MS) was used to observe changes of Cadmium concentration when the normal Human Embryo Kidney cells treated with CdCl2.6. After overexpression TRPC1, Inductively coupledplasma 「mass spectrometry was used to observe changes of Cadmium concentration when the HEK293cells treated with CdCl2.7. Confocal microscopy was used to observe changes of calcium concentration when the normal Human Embryo Kidney cells treated with CdCl2.8. After overexpression TRPC1, confocal microscopy was used to observe changes of calcium concentration when the HEK293cell treated with CdCl2. Results1. CdCl2induces the apoptosis of HEK293cellsTo determine the apoptosis of HEK293at different dose and time of CdCl2, the apoptosis of HEK293was quantified by annexin V+PI assay. Compared with the control, the apoptosis of HEK293increase is correlated with the concentration and time exposure of CdCl2. Furthermore, we measure the cleavage of PARP, which is a marker of the apoptotic response, by Western Blot at different dose and time of CdCl2. A clear increase in the percentage of cleavage of PARP degradation6h after treatment with30μM CdCl2was noted, which is in coincident with the apoptosis of HEK293quantitified by annexin V+PI assay. The apoptosis of HEK293induced by CdCl2is confirmed by hoechst33342staining, where we observed condensation and fragmentation of chromatin in HEK293cells with30μM CdCl2incubated for6h. This suggests that the increase of apoptosis of HEK293by CdCl2is dose and time-dependent.2. Normal HEK293cells express six TRPC subtypesTotal RNA was isolated from HEK293cells in normoxia. The cDNAs were amplified for40cycles and PCR products (5μl) were subjected to2%agarose gel electrophoresis to analyze expression profile of TRPC in normal HEK293cells.3. The HEK293cells treated with CdCl2affect TRPC1activityTo use RT-PCR to detected the expression in mRNA level of TRPCs in the HEK293cells treated with30uM CdCl2for6h, CdCl2affect TRPC1,TRPC3,TRPC4activity. Under the HEK293cells treated with CdCl2, TRPC1,TRPC3,TRPC4expression decreased. TRPC1expression is hightest. We use Western Blot to detected the expression in protein level of TRPC1in the HEK293cells treated with30uM CdCl2for6h. Confirmed the the CdCl2impact TRPC1protein expression. The results show that the decline of TRPC1protein expression with the increase of the the CdCl2role of time. It said that CdCl2induced HEK293cells TRPC1protein expression.4. upregulation of TRPC1influenced on CdCl2-induced apoptosis of HEK293cellsTo identify TRPC1is involved in CdCl2-induced apoptosis of HEK293cells, we upregulate TRPC1expression by transiently transfecting corresponding plasmid. The efficiency of upregulation is determined by RT-PCR and western blot. The apoptosis of HEK293overexpression TRPC1was decreased significantly measured by annexin V+PI assay compared with those of the control cells.5. CdCl2infulence the cells cycle of HEK293cellsFlow cytometry assay was used to detect whether different concentrations of CdCl2infulenced the cells cycle of HEK293cells. The results showed that compared with the negative control group, HEK293cells treated with30μM CdCl2for6h no difference in cell cycle. It is said that HEK293cells treated with CdCl2do not impact cell cycle arrest.6. CdCl2can increase Cadmium concentration of HEK293cellsTo examine the Cd2+concentration in the HEK293treated with CdCl2, Inductively coupled plasma mass spectrometry was used to observe changes of Cd2+concentration when the normal HEK293cells treated with CdCl2. The results showed that CdCl2can increase Cd2+concentration of HEK293cells. Compared with those of the control cells, after overexpression TRPC1, Cd2+concentration of HEK293cells was decreased.7. CdCl2can increase calcium concentration of HEK293cellsTo examine the role of the Ca2+in the apoptosis of HEK293by CdCl2, confocal microscopy was used to observe changes of calcium concentration when the normal HEK293cells treated with CdCl2.The results showed that CdCl2can increase calcium concentration of HEK293cells. ConclusionIn conclusion, this study shows for the first time that TRPC1is involved in Cadmium-induced apoptosis in Human Embryo Kidney cells. Theses data not only provide new insights into the molecular mechanism of the Cadmium-induced apoptosis in Human Embryo Kidney cells, but also highlights the role of TRPC1in the regulation of apoptosis.