The Influence Of PBDE-209 On Proliferation Of Ovcar-3 Cells And CHO Cells In Vitro

Polybrominated diphenly ethers (PBDEs) are a class of aromatic series that contain bromine atoms, which are added to manufactured products including paints, plastics, textiles, and electronic appliances and other fields,end products widely used for PBDEs include electronics, computers, and foam padding in furniture . With the needs of the PBDE become more and more and the dose of the PBDE rise more with the development of industry. Some data showed that 80% of e-waste in developed countries were brougt to China, India and Pakistan and other countries, which also accounted for 90% of e-waste in China. For exterior air, urban PBDE concentrations were 10 times greater than rural concentrations, indicating an urban rural gradient and greater PBDE sources in urban areas, There formed collection and distribution center for dismantling e-waste in some areas in China, PBDEs and other persistent toxic pollutants continuously release to the local environment so that they result in serious pollution and endangering human health because of the original disassembling means (open burning, baking, and pickling, etc.).PBDEs have the property of low degradation, environmental stability, high fat-solubility and biological amplification functions, which can be transferred through food chain to allow the organisms of higher-level grade to be poisoned and finally bring dangers to human health. Until now, so many study about Lower brominated congeners is certain that it has chronic toxicity on the liver, the kidney and the thyroid gland upon chronic exposure, so the lower brominated congeners have been banned in United States and Europe. Although the toxicity of PBDE-209 is lower than that of the fewer brominated congeners, our country is the largest one to produce, use and export lots of PBDEs, especially PBDE-209, so PBDE-209 has been the most environmental pollution. PBDE-209 can be broken down to the lower brominated congeners and induce more toxicity. But the dispute about the PBDE-209 hazard to the human haven't stopped. Some research also proved that BDE-209 has a neural developmental toxicity, immunotoxicity, endocrine toxicity and carcinogenecity. It can also damage the reproductive system of the male mouse after exposure PBDE-209 during pregnancy.Ovarian cancer is the highest death rate cancer for women with the increased incidence. Ovarian cancer due to not yet clear, but it has attracted attention that the interaction of genetic factors and environmental factors influence ovarian cancer. PBDE-209 can affect the female reproductive system and the endocrine (hormone)。But the relationship between the rising dose of PBDE-209 and increasing incidence of the b ovarian cancer is uncertain yet. So in this study, we focus on the effluence of PBDE-209 exposure on the proliferation of ovarian cancer in vitro, choosing the OVCAR-3 cell line and CHO cell line as the model, to study how PBDE-209 affect the cell cycle disposition and the PKCαand ERK phosphorylation.PartⅠ: the effluence of PBDE-209 exposure on the proliferation of ovarian cancer and CHO cell in vitro[Objective]To observe how low dose PBDE-209 exposure affect the morphology change of the OVCAR-3 cells and the CHO cells in vitro; to measure the cell activity rate by MTT method and the Ki67 expression by immunofluorescence (IMF) after 0-100nM PBDE-209 exposure for 0-72h to the cancer cell lines; and to observe how the cell cycle disposition change by Flow Cytometry after 0-100nM PBDE-209 treatment.[Methods](1) Adherent culture OVCAR-3 and CHO cells were used as cell models, to count the cell number after plant 0-7 days, and to draw the cell growth curve to calculate the cell population doubling time (pdt);(2) 2nd -3rd passage cells were synchronized after 24h starvation before PBDE-209 treatment; MTT method was used to detect the cell proliferation rate induced by PBDE-209 and the cell morphology change under the inverted microscope were observed.(3) IMF method was used to detect the Ki67 expression after 0-100nM PBDE-209 treatment for 72h.(4) Flow cytometry was deployed to measure the cell cycle disposition after 0-100nM PBDE-209 exposure in the OVCAR-3 and CHO cells for 72h.[Results](1) The obvious difference in the growth morphology and shape was observed between the OVCAR-3 and CHO cells: the cell body of OVCAR-3 cell is oval and the contact inhibition is not obvious, with the pdt 36.74±1.29 h; The cell body of CHO cell is fusiform and growing as single cell with a poor enduring starvation, and the pdt is 40.56±1.25 h.(2) Cells proliferation in the OVCAR-3 and CHO can be observed after PBDE-209 exposure by MTT method, with a dose-dependent and time-dependent effect. 50nM PBDE-209 can induce the OVCAR-3 cells proliferation 1.53 times and 1.76 times after 48h and 72h treatment. While in the CHO cells, the proliferation rate is only 1.47 times and 1.60 times increased after 100nM PBDE-209 treatment for 48h and 72h.(3) The Ki67 positive rate increased by dose-dependent effect after PBDE-209 exposure for 72h by IMF method. The Ki67 positive rate is about 82.14%,85.42% and 92.16% after 25nM, 50nM and 100nM PBDE-209 treatment for 72h in OVCAR-3 cells. However, in the CHO cells, the positive rate of Ki67 is lower than that of OVCAR-3 cells.(4) Cell cycle disposition in the OVCAR-3 and CHO cells can be changed by PBDE-209 exposure for 72h detected by Flow cytometry, with G0/G1 phase percentage decrease and G2/M phase(OVCAR-3)and S phase (CHO) percentage increase. In the OVCAR-3 cells, 100nM PBDE-209 induce the G0/G1 phase cells to decrease from 80.25% to 72.35%, and the G2/M phase cells to increase from 3.95% to11.90%. While in the CHO cells, PBDE-209 make G0/G1 phase cells percentage of the HeLa cells 5.10% increase, and induce the S phase cells percentage to rise 5.20%.[Conclusion]0-100nM PBDE-209 can make the G0/G1 cell cycle phase decrease and G2/M cell cycle phase(OVCAR-3)and S cell cycle phase (CHO)increase to induce OVCAR-3 and CHO cells to proliferate, with a dose-dependent and time-dependent effect.Part II: The effluence of PBDE-209 on the PKCαand ERK phosphorylation in the OVCAR-3 and CHO cells[Objective]To observe the role of PKCαand ERK in the period of the PBDE-209 induced proliferation in the OVCAR-3 and CHO cells.[Methods]Western blot method was used to observe the phosphorylation of PKCαand ERK after 0-100nM PBDE-209 exposure for 15min or 50nM PBDE-209 treatment for 0-120min in the OVCAR-3 and CHO cells.[Results] PBDE-209 can activate the phosphorylation of PKCαand ERK in the OVCAR-3 and CHO cells, with a dose-dependent and time-dependent effect, although a little difference can be observed in the dose point or the time point when reach the peak.(1) Dose-dependent effect experiment: in the OVCAR-3 cells, 50nM PBDE-209 can induce the PKCαreach peak and lasted util 100nM, and only 25nM PBDE-209 treatment can activate ERK phosphorylation, and reach maximum at 50nM; in the CHO cells, a little PBDE-209 exposure can activate PKCαand ERK phosphorylation and continue to increase, until reach peak at 50nM. In the mean while, 25nM PBDE-209 can also induce CHO cells PKCαand ERK phosphorylation, and 50nM dose make PKCαmaximum; and the ERK phosphorylation peak continue until 100nM.(2) Time-dependent effect experiment: 50nM PBDE-209 exposure for 30min can induce OVCAR-3 cells'PKCαphosphorylation, and 15min treatment can activate ERK, until 120min. In the CHO cells, obvious PKCαphophorylation can be observed after 5min PBDE-209 exposure, and reach peak at 120min point. In the CHO cells, 50nM PBDE-209 exposure for 15min can activate ERK, and reach the maximum at 60min point.[Conclusion]PBDE-209 can induce the phosphorylation of PKCαand ERK in the period of the PBDE-209 induced proliferation in the OVCAR-3 and CHO cells.PartⅢ: The effluence of special inhibitor of PKCαand ERK on the proliferation induced by PBDE-209 in the OVCAR-3 , CHO cells[Objective]To observe the effulence of PKCαspecial inhibitor G?6976 or ERK special inhibitor PD98059 on the proliferation induced by PBDE-209 in the OVCAR-3 and CHO cells, and also to observe the effect of G(?)6976 or PD98059 on the PKCαand ERK activated by PBDE-209.[Methods](1) IMF was used to observe the Ki67 expression after the co-treatment of 50nM or 100nM PBDE-209 with 1μM G(?)6976 or 20μM PD98059 for 72h in the OVCAR-3 and CHO cells.(2) Flow Cytometry was deployed to measure the apoptosis rate after 50nM or 100nM PBDE-209 with 1μM G(?)6976 or 20μM PD98059 for 72h in the OVCAR-3 and CHO cells.(3) Western Blot was used to observe the effluence of 1μM G(?)6976 or 20μM PD98059 pretreatment for 30min in the OVCAR-3 and CHO cells on PKCαand ERK phosphorylation induced by PBDE-209.[Results](1) We can observe from the IMF experiment that G(?)6976 or PD98059 can decrease the Ki67 expression, and the effect of G(?)6976 is more than that of PD98059. The Ki67 expression increase when PBDE-209 co-operate with G(?)6976 or PD98059, the effect of 100nM was stronger than that of 50nM.(2) From the Flow cytometry experiment, we know that G(?)6976 or PD98059 can induce cell apoptosis in some extent, and the apoptosis rate induced by G(?)6976 in the OVCAR-3 and CHO cells is 9.6% and 17.5%, with statistical significance when compared with the negative group; and the PD98059 induced apoptosis rate is 3.25% and 14.9%. However, PBDE-209 can partly antagonize the apoptosis induced by G(?)6976 or PD98059, with 5.25% and 11.9% decrease when pretreatment with G(?)6976 in the OVCAR-3 and CHO cells, and 2.6% and 12.1% apoptosis rate decrease was detected when pretreatment with PD98059.(3) We can observe from the Western Blot experiment that PBDE-209 can activate the phosphorylation of PKCαand ERK in the OVCAR-3 and CHO cells, with a dose-dependent, and the effect of G(?)6976 is more than that of PD98059. G(?)6976 or PD98059 can decrease the phosphorylation of PKCαand ERK compared with the negative group. However, PBDE-209 can decrease the phosphorylation of PKCαand ERK in the OVCAR-3 and CHO cells when pretreatment with G(?)6976 or PD98059.[Conclusion]PBDE-209 can partly antagonize the apoptosis induced by G(?)6976 or PD98059, and to improve deeply that PBDE-209 can promote cells proliferation. In this period, PKCαand ERK have encouragement use.