Experimental Study Of Therapy Effect Of Methylmercury Chloride On Lung Cancer And Its Mechanism

The incidence of Lung Cancer raised up year after year, now it became the prime malignant tumor. About 80% of patients who has been diagnosed for the first time were belong to the advanCED stage. Chemotherapy was the primary therapy, especially for the SCLC. However the drug used in chemotherapy, of which remission rate was low, side effect was high, and could not penetrate the brain-blood barrier easily, so it could not control brain metastases. To find a drug which could make high remission rate, low side effect, and could penetrate the brain-blood barrier easily has been the urgent priority. This study used the methylmercury chloride, which can penetrate both cellular membrane and the brain-blood barrier easily, to investigate its inhibitory effect on NCI-H446 cell, interference of the cell cycle, induction of apoptosis, and its mechanisms.1. Experimental study of inhibitory effect of MMC on NCI-H446 lung cancer cell in vitro1.1 The inhibitory effect of MMC on NCI-H446 lung cancer cell1.1.1 Density and morphologic changes of NCI-H446 lung cancer cellMorphologic change of NCI-H446 lung cancer cell under inverted microscope showed that in control group the cell adherence tight, cell turns to fusiform, cell grown intensive. In the DDP,As2O3,MMC groups, as well as the concentration increases, the cell which was round and float in the culture fluid, increased gradually. The inhibitory effect of MMC and DDP were same in the different concentration group to a great extent, but of which As2O3 was lower.Both the method of MTT and morphologic change under inverted microscope showed that MMC could inhibit both the Lewis and NCI-H44 lung cancer cell, and exits dose dependent and time dependence.1.1.2 Inhibitory effect of MMC on NCI-H446 lung cancer cell assessed by MTT assayTo assess the inhibitory effect of different concentration of MMC on NCI-H446 lung cancer cell using the method of MTT, and compared with DDP and As2O3.The results showed the inhibitory effect of MMC on NCI-H446 lung cancer cell was rather high, the OD rates grown down gradually, when the concentration increases, the inhibitory effect showed dose dependence and time dependence, compared with the control group , which was significant difference.The inhibitory effect of DDP and As2O3 on NCI-H446 lung cancer cell were both effective. There was non-significant difference between DDP and MMC, and the inhibitory effect of both were stronger than that of As2O3, in each concentration at 24h, 48h, 72h. The OD rate was significant difference treated with concentration of 10μM (P<0.05).The inhibitory rate of MMC was 3.07%, at 24h, in the density of 2.5μM, as well as, the rate of inhibitory effect of DDP is 1.89%. The rate of inhibitory effect of DDP was little lower than that of MMC, but there is non-significant difference between them.There was no inhibitory effect of As2O3 in the concentration of 2.5μM at 24h, 48h, 72h. The rate of inhibitory effect of As2O3 is 1.07% in the concentration of 5μM at 24h. Producing a marked effect, the density of As2O3 was lower; the time of which was little slowly than that of DDP and MMC.1.2 Interference of MMC on NCI-H446 cell cycle:The results showed: After treated with MMC of 1.25,2.5,5.0,10.0,20.0μM for 48h, the number of NCI-H446 cell in the stage of splitting decreases, the concentration increased, the number of cell decreased further. In the concentration of 20.0μM, the number of NCI-H446 cell was 1.01%. At the same time the number of cell increases gradually in the stage of G0/G1, presenting a blockage in the stage of G0/G1.After treated with As2O3 of 1.25,2.5,5.0,10.0,20.0μM for 48h, the number of NCI-H446 cell in the stage of splitting increased, the concentration increased, the number of cell decreased further. In the concentration of 20.0μM, the number of cell was 2.87%. At the same time the number of cell increased gradually in the stage of G2/M, presenting a blockage in the stage of G2/M.After treated with DDP of 1.25,2.5,5.0,10.0,20.0μM for 48h, the number of NCI-H446 cell in the stage of splitting decreased, the concentration increased, the number of cell decreases further. In the density of 20.0μM, the number of cell is 2.38%. At the same time the number of cell in the stage of G0/G1 increased gradually, presenting a blockage in the stage of G0/G1.Among of MMC, As2O3, DDP, the inhibition effect of MMC is the most significant on cells in the stage of splitting.1.3 MMC induCED apoptosis in NCI-H446 cell.1.3.1 To detect apoptosis by FCMThe results showed: in different concentration of 1.25,2.5,5.0,10.0,20.0μM, after treated with MMC, As2O3, DDP at 24h,as well as concentration increased, the rate of apoptosis increased. In the group of MMC, above 10.0μM, concentration increased, the rate of apoptosis increased as well, in 10.0μM, the rate of apoptosis reaching the max is 74.43%, above 10.0μM, concentration increased, the rate of apoptosis decreased. We could see the same regularity in the As2O3 and DDP. The rate of apoptosis reaching the max, in the group of As2O3, was 56.20%, which DDP was 60.50%, both of them were lower than that of MMC (P<0.05), DDP was a little higher than As2O3, but has non-significant difference. 1.3.2 To detect apoptosis by TUNEL assayThe method of TUNEL was used, after the action of MMC, As2O3, and DDP at 48h, to detect apoptosis in NCI-H446 cells. Final concentration of 0μM is the control group, as well as, final concentration of 1.25,2.5,5.0,10.0,20.0μM, is the experiment groups. The results showed that concentration increased, the rate of apoptosis increases. The result future to improve that MMC introducing apoptosis in NCI-H446cell is stronger than As2O3 and DDP.1.4 Changes in mRNA level and protein expression of apoptosis-related genes1.4.1 To detect the protein level change of Bcl-2, Bax, Caspase-3,PARP by RT-PCRRT-PCR method was used, in 10μM, after the action of MMC, to detect the expressed protein of Bcl-2, Bax, Caspase-3, PARP in NCI-H446 cells.The results showed: the gene of Bax which could promote apoptosis: the mRNA level of Bax in the group of MMC was higher than the control group, and exits time dependence, at 24h it can get the maximum.The gene of Bcl-2 which could inhibit apoptosis: the mRNA level of Bcl-2 in the group of MMC was lower than the control group, and exits time dependence, at 24h it can get the maximum.The prolease of Caspase-3 which could promote apoptosis: the mRNA level of Caspase-3 mRNA in the group of MMC is higher than the control group, and exits time dependence, at 24h it can get the maximum.1.4.2 To detect the expressed protein level of Bcl-2, Bax, Caspase-3,PARP by immunocytochemical methodImmunocytochemical method was used, in1.25,2.5,5.0,10.0, and 20.0μM, after the action of MMC, As2O3, DDP at 24h, to detect the expressed protein of Bcl-2, Bax, Caspase-3, PARP in NCI-H446cells.The positive cell is Buffy, the expressed protein of Bcl-2, Bax, Caspase-3 located intracytoplasm, PARP leis in cellular nucleus. The dose of MMC, As2O3,DDP increases, the expression of the protein of Bcl-2 and PARP grows down gradually, as well as, the expression of the protein of Bax and Caspase-3, grows up gradually.1.4.3 To detect the expressed protein level of Bcl-2, Bax, Caspase-3,PARP by Western blotThe method of Western blot was used, in 1.25,2.5,5.0,10.0, and 20.0μM, after the action of MMC for 24hours, to detect the expressed protein of Bcl-2, Bax, Caspase-3, PARP in NCI-H446cells.Bcl-2: The dose of MMC increases, the expression of the protein of Bcl-2 grows down gradually.Bax: The dose of MMC increases, the expression of the protein of Bax grows up gradually.Caspase-3: The protein of Caspase-3 can be seen in each group at 32KD.A splitting fragment which can be seen at 17KD in each experiment group, on the contrary, can not be seen at 17KD in the control group. It shows that, after the action of MMC, the proenzyme of Caspase-3 could be acted and splitted.PARP: A distinctive strap which can be seen from 5μM.The result of experiment in vitro shows that MMC could inhabit the cell proliferation of NCI-H446 cell and introduce its apoptosis. The mechanism may be:to promote the expression of mRNA, protein of Bax; to inhabit the expression of mRNA, protein of Bcl-2; to promote the expression of mRNA, protein of Caspase-3, and to promote the activation of its enzyme precursor; after the activation of Caspase-3, it can Shear PARP, make it lose its function of proenzym, can not repair the damaged DNA, introduce cell death at last.2. Experimental study of inhibitory effect of MMC on SCLC in vivo.When the NCI-H446 mice-transplanted tumor models set up, has been turned into two groups at random. In the experiment groups, using MMC 10mg/kg in administration, every other day, 10 days later,withdraw the drug for one week, the rats was to put to death, measuring the length of tumor, and calculating the volume, in the control groups, using the same voluminal Sodium Chloride. The volume of tumor= the length of tumor×the square of the short of tumor×0.5236.The volume of tumor exits significant difference compared with Sodium Chloride group (P<0.01).To investigate the mechanism, we observe the form of cancer cell through H E scanning. The results showed, in the experiment group, cancer cell population decrease, caryon concentrate, NCI-H446 apoptosis gather near the karyolemma of chromatin. The method of TUNEL was used to detect the apoptosis, the AI of E exits significant difference compared with control (P<0.01).The experiment in vivo showed that MMC could inhibit NCI-H446 cell multiplication. The mechanism may be that MMC could introduce cell apoptosis.The experiments can be proved that MMC could inhibit NCI-H446 cell, and introduce apoptosis. The mechanism could be that MMC can block the cell cycle and introduce apoptosis, through regulating the apoptosis gene, and interacting the process of cancer cell cycle.The results of those trails will give a new path in the exploitation of drugs used in lung cancer chemotherapy, especially for the SCLC, and provide the evidence of its feasibility.