Experimental Study Of Inhibitory Effect Of Methylmercury Chloride On Rat Glioma Cells In Vitro And In Vivo

Glioma, the most common primary brain tumor, account for approximately 40% of all human intracranial tumors. The prognosis of patients with malignant glioma is poor, the life expectancy is usually less than 1 year from the time of diagnosis. Therefore, how to improve the therapy effect is a focus topic of glioma research work. At present, the treatment strategy of glioma is still a cosmopolitan difficult problem. Though surgery is preferred, the remnant invading cells will proliferate and cause recurrence besides the neuro-dysfunction consequently. Radiotherapy is usually recommended for gliomas while it's effect is unsatisfied. Traditional chemotherapy is often of little therapeutic value because the drug can hardly pass the blood-brain barrier. Therefore, searching and developing an ideal chemotherapy drug for malignant gliomas is mandatory. Our team have been engaged in the research work of the neurotoxic effect of Methylmercury Chloride (MMC) with lots of scientific practices for over 20 years, and we consider that MMC, the neurotrophic toxicant, as an new anti-glioma drug which have the potential to treat or even cure the malignant tumor of central nervous system. To confirm the new viewpoint, experimental study of its anti-glioma effect (C6 glioma cell and U251 glioma cell) in vivo and vitro was performed. The research work of this experiment listed as follow:1. Experimental study of inhibitory effect of MMC on rat C6 glioma cells in vitro 1.1 Inhibitory effect of MMC on the proliferation of cultured rat C6 glioma cells The rat C6 glioma cells were cultivated in vitro and divided into control group and MMC-treated group. MTT assay was performed to evaluate the proliferation inhibitory effect of MMC with different concentrations(10.00,5.00,2.50,1.25,0.63,0.31,0.16å’Œ0.08μmol·L^-1)on cultured rat C6 glioma cells. Results showed that 1.25, 2.50, 5.00 and 10.00μmol·L^-1 MMC could inhibit the proliferation of cultured rat C6 glioma cells in vitro, the viability of MMC treated C6 glioma cells is 10.5%±0.6%,17.2%±4.2%,59.0%±1.7% and 74.6%±3.9% of control group respectively, and were significantly lower than those in control group (P<0.05), which indicated that MMC could inhibit the proliferation of C6 glioma cells and the inhibitory effect was in a dose-dependent manner.1.2 Cell morphology of C6 glioma cells exposed to MMC observed under inverted light microscopeCell morphology of C6 glioma cells exposed to MMC were observed under inverted light microscope. C6 glioma cells in control group showed adherent, cells grew densely and exhibited fusiform or polygon shape. C6 cells sticked to the walls of culture plate firmly. 1.25 mol·L^-1 MMC treated group showed that the shapes of C6 cells was irregular and cell volume began to reduce. The number of the treated cells was less than that of control, some round cells were found under light microscope. 2.50 mol·L^-1 MMC treated group showed that the number of C6 glioma cells was significant lesser than that of control, and C6 cells could not stick to the walls of culture plate firmly. Most cells died and broke down, and the left cells became round cells which were easily detached from the walls of culture plate and floated in the culture medium.1.3 C6 glioma cell apoptosis observed by HE stainC6 glioma cells were treated with different concentration(s10.00,5.00,2.50, 1.25,0.63,0.31,0.16å’Œ0.08μmol·L-1)MMC for 72h, and observed with optical microscopy by HE staining. C6 glioma cells in control group showed that cells grew densely and exhibited fusiform or polygon shape, cytoplasm showed pink color and cell nucleus took on blue. MMC treated group showed that cell volume began to reduce, cell number was less than that of control, some diffused distribution round cells were found, cytoplasm showed dense and dark stained, cell nucleus took on hyperchromatism of dark blue, nuclear fragmentation could be found and take on apoptosis morphology characters.1.4 Cytotoxicity effect of MMC on C6 glioma cells evaluated by MTT assayC6 glioma cells were treated with different concentrations(10.00,5.00,2.50,1.25,0.63,0.31 and 0.16μmol·L-1)MMC for 4h,8h,16h and 32h, and then examine the A value by MTT assay. Result showed that the cell viabilities of C6 glioma cells treated with 2.50, 5.00 and 10.00μmol·L-1 MMC for 4h, 8h, 16h and 32h were significantly lower than those in control group (P<0.05), the cytotoxicity effect was in a dose and time dependent manner.1.5 C6 glioma cells apoptosis induced by MMCC6 glioma cells were treated with different concentration(s10.00,5.00,2.50,1.25,0.63,0.31 and 0.16μmol·L-1)MMC for 24h, and flow cytometry was used to assess the effect of MMC treatment on cell apoptosis. Result showed that C6 glioma cells were treated with 0.31, 0.63, 2.50, 5.00 and 10.00μmol·L-1 MMC for 24h, the apoptosis/necrosis rates were significantly higher than those in control group (P<0.05), the normal C6 glioma cell rate significantly lower than those in control group (P<0.05), which indicated that MMC can induce C6 glioma cells apoptosis.1.6 Effects of MMC on cell cycle of C6 glioma cellsC6 glioma cells were treated with different concentration(s10.00,5.00,2.50, 1.25,0.63,0.31 and 0.16μmol·L-1)MMC for 72h, and flow cytometry was used to assess the effect of MMC treatment on cell cycle. Result showed that C6 glioma cells were treated with 1.25, 2.50 and 5.00μmol·L-1 MMC for 72h, the rates of G0/G1 phase cell significantly increased compared with control group(P<0.05), while cells into S phase were decreased(P<0.05), which indicated that MMC can block the cell in the G0/G1 phase, decrease cell into S phase, and interfere the cell cycle progression.1.7 Effects of MMC on reactive oxygen species(ROS) level of C6 glioma cells The ROS was measured by 2', 7'- Dichlorodihydrofluorescein Diacetate (DCFH-DA), assessed by flow cytometry. Results showed that the ROS level increased in C6 glioma cells with the MMC exposure time prolonged. For 5.00 and 10.00μmol·L-1MMC treated groups, the ROS level peaked at 12 hour and decreased at 24 hour but still kept high level, for 1.25 and 2.50μmol·L-1MMC treated groups, the ROS level increased continuously. Group comparison showed that the ROS level increased in each exposure time with MMC concentration increase.1.8 Effects of MMC on mitochondrial trans-membrance potential (MMP) of C6 glioma cellsThe MMP of MMC-treated C6 glioma cell was labeled with Rhodamine123 (Rh-123), and assessed by flow cytometry. Results showed that MMP decreased in each MMC treated group with the exposure time prolonged, and increased in the same exposure time with MMC concentration increase. MMC displayed a dose and time-dependent effect on MMP.1.9 Effects of MMC on calcium of C6 glioma cellsProbed by Fluo-3/AM, calcium changes were assessed by laser scanning confocal microscope (LSCM). Results showed that MMC can cause immediate increasing of calcium level and the effect was dose-dependent.1.10 Effects of MMC on Bcl-2 protein expression of C6 glioma cellsThe expression of Bcl-2 protein was studied by western blot. Results showed that Bcl-2 protein expression decreased with MMC concentration increased and exposure time prolonged, the effect was dose-dependent and time-dependent.Our in vitro study showed that MMC has cytotoxicity effect on cultured rat C6 glioma cells in vitro; can inhibit its proliferation; induce cell apoptosis; block the cell in the G0/G1 phase, decrease cell into S phase, and interfere the cell cycle progression; increase ROS level; destroying MMP; inhibiting the expression of Bcl-2. Overall, we consider that MMC as an new anti-glioma drug which have the potential to treat or even cure the malignant tumor of central nervous system, while its molecular mechanism of those effect needs to be further explored.2. Experimental study of inhibitory effect of MMC on rat C6 glioma cells and athymic mouse U251 glioma cells in vivo2.1 Experimental study of inhibitory effect of MMC on rat C6 glioma cells in vivo2.1.1 Establish Wistar rat C6 glioma brain tumor model2.1.2 Observation of general status and survival time of C6 glioma model ratsAt 7th day after C6 glioma cells implantation, all the rats in both group showed signs of neurological distress, such as weakness, paralysis, epilepsy like tremor, hyperspasmia, rotation along the long axis, et al.2.1.3 Tumor pathology studyThe tumor volume of control group and MMC treated group were 231.24±215.10mm³ and 7.56±3.81mm³ respectively, the tumor volume of MMC treated group was significantly less than control group (p<0.05). Get the rat brain tissue by decapitation at 24th day after C6 glioma cells implantation. Sectioned the brain through the tumor region and observed with optical and electron microscopy, cell apoptosis was assessed by TUNEL assay. Histological sections indicated that the glioma cells of control group were of high density and proliferation activity, while the sections of MMC treated group revealed that the tumor had areas of necrosis, the glioma cells were less in quantity. High power magnification showed modality change of apoptosis with dense chromatin, karyopyknosis and karyorrhexis, these change could be further confirmed by observation with electron microscopy.2.1.4 MMC concentration in brain tissueUsed the atomic absorption spectrophotometer (Varian Spector-AA200) to detected the concentration of MMC in brain tissues by cyanide atomic absorption protocol. The detection result of MMC concentration in the rats brain tissue was 1.95±0.57μg/g in MMC treated group and less than 0.010μg/g in control group, the result indicated that MMC can pass through the blood brain barrier.2.1.5 Survival time of C6 glioma model rats5 rats in control group dead after tumor cell implantation (1 dead on 13th day, 2 dead on 15th day, 1 dead on 19th day and 1dead on 21st day), 1 rat in MMC treated group dead on 18th day. All the rest rats were killed on 24th day. The survival time of experiment group was significantly prolonged.2.2 Experimental study of inhibitory effect of MMC on athymic mouse U251 glioma cells in vivo2.2.1 Establish athymic mouse U251 subcutaneous tumor model2.2.2 Observation of general status and tumor growth of U251 model athymic mouseAt 14th day after U251 glioma cells implantation, the athymic mouse in control group showed obviously subcutaneous lump, and with activity dullness and body weight decrease. The tumor volume of MMC treated group was significantly less than control group (p<0.05).2.2.3 Tumor pathology studySectioned the tumor tissue and observed with optical and electron microscopy. Histological sections indicated that the U251 glioma cells of control group were of high density and proliferation activity, while the sections of MMC treated group revealed that the tumor had areas of necrosis, the U251 glioma cells were less in quantity. High power magnification showed modality change of apoptosis with dense chromatin, karyopyknosis and karyorrhexis, these change could be further confirmed by observation with electron microscopy.Our in vivo study showed that MMC can inhibit rat brain C6 glioma cell and athymic mouse subcutaneous U251 glioma cells proliferation, the inhibition effect was possibly correlative with the glioma cells apoptosis induced by MMC. Overall, we consider that MMC as an new anti-glioma drug which have the potential to treat or even cure the malignant tumor of central nervous system, while its molecular mechanism of those effect needs to be further explored.Creative point of this experiment:1. It the first time at home and abroad that we take study of the MMC used for treatment of glioma in according to the special neurotoxic effect of MMC. (author's director applied the national patent of"The new application of MMC used for treatment of intracranial malignant tumor as an anti-tumor drug"and was approved with patent No. ZL200410010827.7)2. For the first time at home and abroad to explore the mechanism of glioma cells apoptosis induced by MMC with mitochondrial pathway. Our research showed that MMC can increase ROS level; destroying MMP; inhibiting the expression of Bcl-2.3. We proved the anti-glioma effect of low dose MMC by in vitro and in vivo study, and the results of this study will provide us a new idea for finding anti-glioma drugs and experimental basis for clinical application of MMC.