氯化甲基汞抗胶质瘤作用及其分子机制的研究
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摘要
神经胶质瘤具有浸润性生长特点,手术难以根治,术后复发率很高,现今大多数化疗不能透过血脑屏障,限制临床应用,胶质瘤还具有辐射抗性,尽管经过手术、化放疗等综合治疗其平均存活率不足一年,胶质瘤的治疗成为当今世界公认的难题,因此寻找有效抗肿瘤靶向药物是亟待解决的问题。
我们在以往探讨甲基汞所致脑发育损伤机制的基础上,根据甲基汞的理化特性、药动学特点和多靶点细胞毒作用,提出用氯化甲基汞(MMC)治疗神经胶质瘤的新策略。本研究采用体内外实验方法探讨MMC对脑胶质瘤的抗肿瘤作用及其机制,体外实验结果:①采用MTT法检测显示,MMC对SHG44胶质瘤细胞增殖具有明显的抑制作用,呈剂量依赖关系;应用ATP生物荧光法进行MMC与As2O3抗胶质瘤作用对比研究,发现前者抑制作用更强。②流式细胞仪测定发现MMC可诱导胶质瘤细胞凋亡和坏死、干扰细胞周期进程,使SHG44胶质瘤细胞阻滞在G0/G1期和G2/M期,减少进入S期的细胞百分数。③激光扫描共聚焦显微镜发现MMC可升高胶质瘤细胞内钙浓度,影响SHG44胶质瘤细胞钙稳态。④免疫印迹和激酶分析法探讨了MMC诱导凋亡、抑制细胞周期与调PKC之间的关系,发现对照组SHG44胶质瘤细胞PKCα及PKCδ蛋白质均呈阳性表达,SHG44胶质瘤细胞给予MMC处理的实验组,PKCα蛋白表达下调,而PKCδ的表达则升高,PKC活性被抑制。在建立大鼠C6脑胶质瘤原位模型和裸鼠SHG44皮下移植胶质瘤模型的基础上,进行了MMC抗脑胶质瘤的体内实验研究,结果显示:上述两种模型荷瘤动物实验组肿瘤体积较对照组明显缩小(p<0.05)、生存期明显延长,光镜观察可见实验组胶质瘤细胞固缩、数目减少和液化坏死,电镜观察实验组胶质瘤细胞出现染色质周边化、空泡变性和细胞崩解等改变;免疫组化检测表明裸鼠SHG44皮下移植瘤接实验组PKCα阳性表达细胞数较对照组明显减少,而PKCδ阳性细胞表达数则增加。
综上可见,MMC对神经胶质瘤具有明显的抗肿瘤活性,其机制可能与氯化甲基汞升高SHG44胶质瘤细胞内钙浓度,影响胶质瘤细胞PKC亚类表达,抑制PKC活性有关,其确切的分子机制有待进一步研究。该项研究具有明显的创新性,为开发抗神经胶质瘤新药提供实验依据。
Glioma,the most common malignant brain tumor, accounts for half of all human intracranial tumors. The mortality of glioma is just below lung cancer and pancreatic cancer. In latest two decades, therapeutic effect and prognosis of glioma weren’t improved obviously. Because of its invasive growth, it can’t be resected by surgery totally. Furthermore, radiotherapy is not sufficient to glioma for its resistence to radiation. Also most of chemotherapy drug can’t permeate the blood brain barrier so they can’t work effectly. So It is very expected to develop an effectually targeted drug to treat malignant glioma.
Based on the forely study of molecular mechanisms of MMC neurotoxic effect,we learned about the feature of physical chemistry and pharmacokinetics of MMC,and its construction and feature of multi-target, we consider that MMC may have potential to treat malignant tumor of central nervous system. Our in vitro and in vivo study showed that MMC can inhibit rat brain C6 glioma cell and athymic mouse SHG44 glioma cells proliferation, the inhibition effect was possibly correlative with the glioma cells apoptosis induced by MMC.
The study as follows.
1. Experimental study of inhibitory effect of MMC on human SHG44 glioma cells in vitro
1.1 Cytotoxicity effect of MMC on SHG44 glioma cells evaluated by MTT assay
The SHG44 glioma cells were cultured and divided into control group and experimental group. MTT assay was performed to evaluate the cytotoxicity effect of MMC with different density(10.00、5.00、2.50、1.25、0.63、0.31 and 0.16μmol·L~(-1))on SHG44 glioma cells for 4h,8h,16h and 32h, and then examined by MTT assay. It showed that the cell survival rates of SHG44 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 lethal effect of MMC on SHG44 glioma was strengthened with dose and time dependence.
1.2 Inhibitory effect of MMC on SHG44 glioma cells evaluated by MTT assay
Different density(10.00、5.00、2.50、1.25、0.63、0.31 and 0.16μmol·L~(-1))of MMC were observed on cultured SHG44 glioma cells for 72h. It showed that 2.50、5.00 and 10.00μmol·L~(-1) MMC could inhibit the proliferation of cultured SHG44 cells,the survival rates of MMC treated SHG44 glioma cells were 48.2±8.0%、39.6±1.1 % and 10.6±2.1% respectively, lower than those in control group significantly (P<0.05), which showed that MMC can inhibit the proliferation of SHG44 glioma cells and the inhibitory effect has dose-dependence.
1.3 Inhibitory effect of MMC on SHG44 glioma cells observed on morphology Different density(10.00、5.00、2.50、1.25、0.63、0.31 and 0.16μmol·L~(-1))of MMC were observed on cultured SHG44 glioma cells for 72h. The morphogy and growth density of SHG44 glioma cells were observed with inverted microscope.It showed that cells of control group grows well, adherence sufficiently, polygoned or spindled. The SHG44 glioma cells treated with 1.25 mol·L~(-1) MMC had decreased density, shrinked volume, contracted to round .Treated with 2.50 mol·L~(-1) MMC, SHG44 glioma cells were fewer, non- adherence, most of cells were collapsed, and the remained contracted to round.
1.4 Pathology observed
Different density(10.00、5.00、2.50、1.25、0.63、0.31 and 0.16μmol·L~(-1))of MMC were observed on cultured SHG44 glioma cells for 72h for microscopy. It showed that cells grows densityed, pantomorphiaed, density-karyotin, light-kytoplasm.The treated group,the density of SGH44 glioma cells was decreased, cell nucleus was more density-karyotin,some nuclear fragmentations can be observed.
1.5 Inhibitory effect of MMC and AS_2O_3 on SHG44 glioma cells by ATP bioluminescence assay
Different density(10.00、5.00、2.50、1.25、0.63、0.31 and 0.16μmol·L~(-1))of MMC and AS_2O_3 were observed on cultured SHG44 glioma cells for 72h.The inhibition ratio was detected by ATP bioluminescence assay.It showed that MMC has higher inhibition ratio than AS_2O_3 with dose-dependence.The difference with density on 2.5、5.0 and 10.0μmol·L~(-1) have statistical significance.(p<0.01).
1.6 Apoptosis or necrosis effect of MMC on SHG44 glioma cells observed by flow cytometry
Different density(10.00、5.00、2.50、1.25、0.63、0.31 and 0.16μmol·L~(-1))of MMC were observed on cultured SHG44 glioma cells. It showed that MMC induced cell apoptosis with dose-dependece. SHG44 glioma cells were treated with 2.5、5.0、10.0umol·L~(-1) MMC for 12h, the necrosis rates were 60.32±2.97%、75.13±0.96%、79.54±1.36% respectively, significantly higher than corresponding control group (P<0.01).The study showed that SHG44 glioma cells apoptosis can be induced by MMC.
1.7 Cell cycle effect of MMC on SHG44 glioma cells observed by flow cytometry
Different density(10.00、5.00、2.50、1.25、0.63、0.31 and 0.16μmol·L~(-1))of MMC were observed on cultured C6 glioma cells for 72h. We detected with flow cytometry and analysed data with HodFlit software. The rate of G0/G1 phase cells of treated group was higher than the control group(P<0.05), while S phase cells were lesser(P<0.05). The study showed that MMC can stop C6 glioma cells in the G0/G1 phase, interfere the progression of cell cycle,so S phase cells were less.
1.8 Inhibitory effect of MMC on SHG44 glioma cells PKC
Different density(0.16、0.31、0.63、1.25、2.50、5.00和10.00μmol·L~(-1))of MMC were observed on cultured SHG44 glioma cells.The modified Takai method was used. It showed that PKC activity of SHG44 glioma cells was decreased with increasd density of MMC(P<0.05) .
The difference with density on 5.0 and 10.0μmol·L~(-1) have statistical significance.(p<0.01).
1.9 Expression effected of MMC on subgroup PKC of SHG44 glioma cells detected by immunoblot
1.9.1 Expression of subgroup PKC of SHG44 glioma cells Immunoblot was used to detect the expression of subgroup PKC of SHG44 glioma cells.After extracted subgroup PKC from endochylema and cell membrane ,we separated PKCα(80KD),PKCβⅠ(77KD),PKCγ(79KD), PKCδ(76KD) by electrophoretic, and analyzed the expression of PKC by immunoblot. It was found that PKCαand PKCδin SHG44 glioma cells were positive.
1.9.2 Expression effected of MMC on PKCαand PKCδin SHG44 glioma cells Immunoblot was used to detect the expression of PKCαat protein level. The expression of PKCαwas decreased and PKCδwas increased with MMC increased.
1.10 Effects on calcium of MMC in SHG44 glioma cells
Fluo/AM was used for indicator assessed calcium changes with laser scanning confocal microscope (LSCM). we observed the basic-fluorescence intensity, then added MMC of different concentration MMC every 200s and made their final density to 1.25、2.50、5.00、10.00μmol·L~(-1) It showed that MMC can increas the level of calcium .
In vitro study we found that MMC has cytotoxicity effect on cultured SHG44 glioma cells .It can inhibit proliferation, induce cell apoptosis, block the cell in the G0/G1 phase, decrease cell into S phase, and interfere the cell cycle progression. It can also inhibit the activity of PKCαand PKCδ. We found that PKCαand PKCδwas over-expressed in SHG44 glioma cells. Expression of PKCαprotein was decreased while PKCδwas increased with MMC increased. we consider that MMC has the potential to treat glioma as an new anti-glioma drug, but its molecular mechanism needs to be further studyed.
2.Study of MMC on rat C6 glioma cells and SHG44 glioma cells in nude mice in vivo
2.1 Study of MMC on rat C6 glioma cells in vivo
2.1.1 The establishment of Wistar rat C6 glioma model
2.1.2.The general status, survival time and tumor volume determination of tumor-bearing mice after administration. At a week after C6 glioma cells implantation, the experimental groups appear abnormal phenomenon including weakness, paralysis, epilepsy like tremor, hyperspasmia, rotation along the long axis, et al. which was taken as a successful model.Daily oral administration of 10mg ? kg -1BW MMC for 5 days,control group received the same volume of saline. 4 rats in control group dead after tumor cell implantation (1 dead on 14th day, 1 dead on 18th day, and 1dead on 21st day), in the experimental group 1 dead on 17th day, At 24th day after C6 glioma cells implantation. Get the rat brain tissue by decapitation . The tumor volume of control group and MMC treated group were (0.1352±0.0675) cm3 and (0.028±0.0064) cm3 respectively. Brain tumor volume of treated group was significantly smaller than of control group (p <0.05).
2.1.3 Tumor pathology study of two groups
By observation with electron microscopy the C6 glioma cells of control group were of high density and very few of necrosis , while in the treated group the density of cells is lower than that in the control group, Cell numbers reduced,the size of cell becomes smaller, apoptosis of nuclear pyknosis morphological, part of the region of the liquefaction. Sectioned the brain through the tumor region and observed with optical and electron microscopy, cell apoptosis was assessed by TUNEL assay, in the treated group apoptosis was significantly higher. With TEM in the treated group the volume,tumor cell nuclear increased, is irregular in shape, mainly euchromatin, prominent nucleoli, condensation of chromatin, around, and nuclear fragmentation, degeneration and other changes.
2.2 Study the effect of MMC on athymic mouse SHG44 glioma cells in vivo
2.2.1 Observed the general status and tumor growth of treated Tumor-bearing mice
At 14th day after SHG44 glioma cells implantation. in nude mice ,tumor volume of the control group: 160.87+61.64mm3 ,of the experimental group: 12.69+6.19 mm3, subcutaneous tumor size in experimental group smaller than the control group(p <0.01).
2.2.2 Tumor pathology study of two groups
Histological sections indicated that the SHG44 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 SHG44 glioma cells were less in quantity. High power magnification showed modality change of apoptosis with dense chromatin and karyopyknosis. Some areas present colliquation necrosis.
2.2.3 PKCα, PKCδprotein expression influenced by MMC on SHG44 glioma examined by immunohistochemistry
2.2.3.1 PKCα, PKCγprotein positive expression in SHG44 glioma Cells Expression of PKCα, PKCγ(sub-group of PKC protein protein ) was positive in SHG44 glioma cells by immunohistochemistry. The cytoplasm and (or) nuclei stained brown in positive cells.
2.2.3.2 Expression effected of MMC on subgroup PKC of SHG44 glioma cells The different density of MMC treated SHG44 glioma cells .Compared with the control group, PKCαexpression of the treated group is gradually decreased, while PKCδexpression is increased with dose increased. Positive cells gradually were upgraded ,while tumor cells were increased in density。
2.2.4 Assaying of HG in tumor
The control group were 0.0030+0.0020ug/g , while the treated group were2.2692+0.1289ug/g(P<0.05).
Our in vivo study showed that MMC can inhibit rat brain C6 glioma cell and athymic mouse subcutaneous SHG44 glioma cells proliferation, the inhibition effect was possibly correlative with the glioma cells apoptosis induced by MMC. The MMC can inhibit the activity of PKC of SHG44 glioma cell, downgrade the express of PKCα,and increase PKCδprotein expression. Overall, we consider that MMC have the potential to influence the express of subgroup of PKC,induce cells apoptosis and interfere cell cycle ,while its molecular mechanism of those effect needs to be further explored.
Creative point of this study:
1. we have new idea of the MMC used for treatment of glioma according to the feature of physical chemistry and biology. Based on in vitro study of SHG44 glioma cells, and in vivo study of setting up two animal model, for the first time we proved that MMC have proliferation inhibiting and killing effect on SHG44 glioma cells, the glioma cells apoptosis can be induced by MMC with interfering cell cycle. Our research made a good foundation for clinical application. (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 we made use of the method of immunoblot and immunohistochemistry to prove that the MMC can regulate subgroup of glioma cells protein kinase C.we also found that the PKCαand PKCδof SHG44 glioma cells were positive. Based on the results of in vitro and in vivo study, we found that the MMC can influence the subgroup of protein kinase C to induce apoptosis of glioma cells and interfere cell cycle, this maybe the most important mechanism of anti-glioma effect.
我们在以往探讨甲基汞所致脑发育损伤机制的基础上,根据甲基汞的理化特性、药动学特点和多靶点细胞毒作用,提出用氯化甲基汞(MMC)治疗神经胶质瘤的新策略。本研究采用体内外实验方法探讨MMC对脑胶质瘤的抗肿瘤作用及其机制,体外实验结果:①采用MTT法检测显示,MMC对SHG44胶质瘤细胞增殖具有明显的抑制作用,呈剂量依赖关系;应用ATP生物荧光法进行MMC与As2O3抗胶质瘤作用对比研究,发现前者抑制作用更强。②流式细胞仪测定发现MMC可诱导胶质瘤细胞凋亡和坏死、干扰细胞周期进程,使SHG44胶质瘤细胞阻滞在G0/G1期和G2/M期,减少进入S期的细胞百分数。③激光扫描共聚焦显微镜发现MMC可升高胶质瘤细胞内钙浓度,影响SHG44胶质瘤细胞钙稳态。④免疫印迹和激酶分析法探讨了MMC诱导凋亡、抑制细胞周期与调PKC之间的关系,发现对照组SHG44胶质瘤细胞PKCα及PKCδ蛋白质均呈阳性表达,SHG44胶质瘤细胞给予MMC处理的实验组,PKCα蛋白表达下调,而PKCδ的表达则升高,PKC活性被抑制。在建立大鼠C6脑胶质瘤原位模型和裸鼠SHG44皮下移植胶质瘤模型的基础上,进行了MMC抗脑胶质瘤的体内实验研究,结果显示:上述两种模型荷瘤动物实验组肿瘤体积较对照组明显缩小(p<0.05)、生存期明显延长,光镜观察可见实验组胶质瘤细胞固缩、数目减少和液化坏死,电镜观察实验组胶质瘤细胞出现染色质周边化、空泡变性和细胞崩解等改变;免疫组化检测表明裸鼠SHG44皮下移植瘤接实验组PKCα阳性表达细胞数较对照组明显减少,而PKCδ阳性细胞表达数则增加。
综上可见,MMC对神经胶质瘤具有明显的抗肿瘤活性,其机制可能与氯化甲基汞升高SHG44胶质瘤细胞内钙浓度,影响胶质瘤细胞PKC亚类表达,抑制PKC活性有关,其确切的分子机制有待进一步研究。该项研究具有明显的创新性,为开发抗神经胶质瘤新药提供实验依据。
Glioma,the most common malignant brain tumor, accounts for half of all human intracranial tumors. The mortality of glioma is just below lung cancer and pancreatic cancer. In latest two decades, therapeutic effect and prognosis of glioma weren’t improved obviously. Because of its invasive growth, it can’t be resected by surgery totally. Furthermore, radiotherapy is not sufficient to glioma for its resistence to radiation. Also most of chemotherapy drug can’t permeate the blood brain barrier so they can’t work effectly. So It is very expected to develop an effectually targeted drug to treat malignant glioma.
Based on the forely study of molecular mechanisms of MMC neurotoxic effect,we learned about the feature of physical chemistry and pharmacokinetics of MMC,and its construction and feature of multi-target, we consider that MMC may have potential to treat malignant tumor of central nervous system. Our in vitro and in vivo study showed that MMC can inhibit rat brain C6 glioma cell and athymic mouse SHG44 glioma cells proliferation, the inhibition effect was possibly correlative with the glioma cells apoptosis induced by MMC.
The study as follows.
1. Experimental study of inhibitory effect of MMC on human SHG44 glioma cells in vitro
1.1 Cytotoxicity effect of MMC on SHG44 glioma cells evaluated by MTT assay
The SHG44 glioma cells were cultured and divided into control group and experimental group. MTT assay was performed to evaluate the cytotoxicity effect of MMC with different density(10.00、5.00、2.50、1.25、0.63、0.31 and 0.16μmol·L~(-1))on SHG44 glioma cells for 4h,8h,16h and 32h, and then examined by MTT assay. It showed that the cell survival rates of SHG44 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 lethal effect of MMC on SHG44 glioma was strengthened with dose and time dependence.
1.2 Inhibitory effect of MMC on SHG44 glioma cells evaluated by MTT assay
Different density(10.00、5.00、2.50、1.25、0.63、0.31 and 0.16μmol·L~(-1))of MMC were observed on cultured SHG44 glioma cells for 72h. It showed that 2.50、5.00 and 10.00μmol·L~(-1) MMC could inhibit the proliferation of cultured SHG44 cells,the survival rates of MMC treated SHG44 glioma cells were 48.2±8.0%、39.6±1.1 % and 10.6±2.1% respectively, lower than those in control group significantly (P<0.05), which showed that MMC can inhibit the proliferation of SHG44 glioma cells and the inhibitory effect has dose-dependence.
1.3 Inhibitory effect of MMC on SHG44 glioma cells observed on morphology Different density(10.00、5.00、2.50、1.25、0.63、0.31 and 0.16μmol·L~(-1))of MMC were observed on cultured SHG44 glioma cells for 72h. The morphogy and growth density of SHG44 glioma cells were observed with inverted microscope.It showed that cells of control group grows well, adherence sufficiently, polygoned or spindled. The SHG44 glioma cells treated with 1.25 mol·L~(-1) MMC had decreased density, shrinked volume, contracted to round .Treated with 2.50 mol·L~(-1) MMC, SHG44 glioma cells were fewer, non- adherence, most of cells were collapsed, and the remained contracted to round.
1.4 Pathology observed
Different density(10.00、5.00、2.50、1.25、0.63、0.31 and 0.16μmol·L~(-1))of MMC were observed on cultured SHG44 glioma cells for 72h for microscopy. It showed that cells grows densityed, pantomorphiaed, density-karyotin, light-kytoplasm.The treated group,the density of SGH44 glioma cells was decreased, cell nucleus was more density-karyotin,some nuclear fragmentations can be observed.
1.5 Inhibitory effect of MMC and AS_2O_3 on SHG44 glioma cells by ATP bioluminescence assay
Different density(10.00、5.00、2.50、1.25、0.63、0.31 and 0.16μmol·L~(-1))of MMC and AS_2O_3 were observed on cultured SHG44 glioma cells for 72h.The inhibition ratio was detected by ATP bioluminescence assay.It showed that MMC has higher inhibition ratio than AS_2O_3 with dose-dependence.The difference with density on 2.5、5.0 and 10.0μmol·L~(-1) have statistical significance.(p<0.01).
1.6 Apoptosis or necrosis effect of MMC on SHG44 glioma cells observed by flow cytometry
Different density(10.00、5.00、2.50、1.25、0.63、0.31 and 0.16μmol·L~(-1))of MMC were observed on cultured SHG44 glioma cells. It showed that MMC induced cell apoptosis with dose-dependece. SHG44 glioma cells were treated with 2.5、5.0、10.0umol·L~(-1) MMC for 12h, the necrosis rates were 60.32±2.97%、75.13±0.96%、79.54±1.36% respectively, significantly higher than corresponding control group (P<0.01).The study showed that SHG44 glioma cells apoptosis can be induced by MMC.
1.7 Cell cycle effect of MMC on SHG44 glioma cells observed by flow cytometry
Different density(10.00、5.00、2.50、1.25、0.63、0.31 and 0.16μmol·L~(-1))of MMC were observed on cultured C6 glioma cells for 72h. We detected with flow cytometry and analysed data with HodFlit software. The rate of G0/G1 phase cells of treated group was higher than the control group(P<0.05), while S phase cells were lesser(P<0.05). The study showed that MMC can stop C6 glioma cells in the G0/G1 phase, interfere the progression of cell cycle,so S phase cells were less.
1.8 Inhibitory effect of MMC on SHG44 glioma cells PKC
Different density(0.16、0.31、0.63、1.25、2.50、5.00和10.00μmol·L~(-1))of MMC were observed on cultured SHG44 glioma cells.The modified Takai method was used. It showed that PKC activity of SHG44 glioma cells was decreased with increasd density of MMC(P<0.05) .
The difference with density on 5.0 and 10.0μmol·L~(-1) have statistical significance.(p<0.01).
1.9 Expression effected of MMC on subgroup PKC of SHG44 glioma cells detected by immunoblot
1.9.1 Expression of subgroup PKC of SHG44 glioma cells Immunoblot was used to detect the expression of subgroup PKC of SHG44 glioma cells.After extracted subgroup PKC from endochylema and cell membrane ,we separated PKCα(80KD),PKCβⅠ(77KD),PKCγ(79KD), PKCδ(76KD) by electrophoretic, and analyzed the expression of PKC by immunoblot. It was found that PKCαand PKCδin SHG44 glioma cells were positive.
1.9.2 Expression effected of MMC on PKCαand PKCδin SHG44 glioma cells Immunoblot was used to detect the expression of PKCαat protein level. The expression of PKCαwas decreased and PKCδwas increased with MMC increased.
1.10 Effects on calcium of MMC in SHG44 glioma cells
Fluo/AM was used for indicator assessed calcium changes with laser scanning confocal microscope (LSCM). we observed the basic-fluorescence intensity, then added MMC of different concentration MMC every 200s and made their final density to 1.25、2.50、5.00、10.00μmol·L~(-1) It showed that MMC can increas the level of calcium .
In vitro study we found that MMC has cytotoxicity effect on cultured SHG44 glioma cells .It can inhibit proliferation, induce cell apoptosis, block the cell in the G0/G1 phase, decrease cell into S phase, and interfere the cell cycle progression. It can also inhibit the activity of PKCαand PKCδ. We found that PKCαand PKCδwas over-expressed in SHG44 glioma cells. Expression of PKCαprotein was decreased while PKCδwas increased with MMC increased. we consider that MMC has the potential to treat glioma as an new anti-glioma drug, but its molecular mechanism needs to be further studyed.
2.Study of MMC on rat C6 glioma cells and SHG44 glioma cells in nude mice in vivo
2.1 Study of MMC on rat C6 glioma cells in vivo
2.1.1 The establishment of Wistar rat C6 glioma model
2.1.2.The general status, survival time and tumor volume determination of tumor-bearing mice after administration. At a week after C6 glioma cells implantation, the experimental groups appear abnormal phenomenon including weakness, paralysis, epilepsy like tremor, hyperspasmia, rotation along the long axis, et al. which was taken as a successful model.Daily oral administration of 10mg ? kg -1BW MMC for 5 days,control group received the same volume of saline. 4 rats in control group dead after tumor cell implantation (1 dead on 14th day, 1 dead on 18th day, and 1dead on 21st day), in the experimental group 1 dead on 17th day, At 24th day after C6 glioma cells implantation. Get the rat brain tissue by decapitation . The tumor volume of control group and MMC treated group were (0.1352±0.0675) cm3 and (0.028±0.0064) cm3 respectively. Brain tumor volume of treated group was significantly smaller than of control group (p <0.05).
2.1.3 Tumor pathology study of two groups
By observation with electron microscopy the C6 glioma cells of control group were of high density and very few of necrosis , while in the treated group the density of cells is lower than that in the control group, Cell numbers reduced,the size of cell becomes smaller, apoptosis of nuclear pyknosis morphological, part of the region of the liquefaction. Sectioned the brain through the tumor region and observed with optical and electron microscopy, cell apoptosis was assessed by TUNEL assay, in the treated group apoptosis was significantly higher. With TEM in the treated group the volume,tumor cell nuclear increased, is irregular in shape, mainly euchromatin, prominent nucleoli, condensation of chromatin, around, and nuclear fragmentation, degeneration and other changes.
2.2 Study the effect of MMC on athymic mouse SHG44 glioma cells in vivo
2.2.1 Observed the general status and tumor growth of treated Tumor-bearing mice
At 14th day after SHG44 glioma cells implantation. in nude mice ,tumor volume of the control group: 160.87+61.64mm3 ,of the experimental group: 12.69+6.19 mm3, subcutaneous tumor size in experimental group smaller than the control group(p <0.01).
2.2.2 Tumor pathology study of two groups
Histological sections indicated that the SHG44 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 SHG44 glioma cells were less in quantity. High power magnification showed modality change of apoptosis with dense chromatin and karyopyknosis. Some areas present colliquation necrosis.
2.2.3 PKCα, PKCδprotein expression influenced by MMC on SHG44 glioma examined by immunohistochemistry
2.2.3.1 PKCα, PKCγprotein positive expression in SHG44 glioma Cells Expression of PKCα, PKCγ(sub-group of PKC protein protein ) was positive in SHG44 glioma cells by immunohistochemistry. The cytoplasm and (or) nuclei stained brown in positive cells.
2.2.3.2 Expression effected of MMC on subgroup PKC of SHG44 glioma cells The different density of MMC treated SHG44 glioma cells .Compared with the control group, PKCαexpression of the treated group is gradually decreased, while PKCδexpression is increased with dose increased. Positive cells gradually were upgraded ,while tumor cells were increased in density。
2.2.4 Assaying of HG in tumor
The control group were 0.0030+0.0020ug/g , while the treated group were2.2692+0.1289ug/g(P<0.05).
Our in vivo study showed that MMC can inhibit rat brain C6 glioma cell and athymic mouse subcutaneous SHG44 glioma cells proliferation, the inhibition effect was possibly correlative with the glioma cells apoptosis induced by MMC. The MMC can inhibit the activity of PKC of SHG44 glioma cell, downgrade the express of PKCα,and increase PKCδprotein expression. Overall, we consider that MMC have the potential to influence the express of subgroup of PKC,induce cells apoptosis and interfere cell cycle ,while its molecular mechanism of those effect needs to be further explored.
Creative point of this study:
1. we have new idea of the MMC used for treatment of glioma according to the feature of physical chemistry and biology. Based on in vitro study of SHG44 glioma cells, and in vivo study of setting up two animal model, for the first time we proved that MMC have proliferation inhibiting and killing effect on SHG44 glioma cells, the glioma cells apoptosis can be induced by MMC with interfering cell cycle. Our research made a good foundation for clinical application. (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 we made use of the method of immunoblot and immunohistochemistry to prove that the MMC can regulate subgroup of glioma cells protein kinase C.we also found that the PKCαand PKCδof SHG44 glioma cells were positive. Based on the results of in vitro and in vivo study, we found that the MMC can influence the subgroup of protein kinase C to induce apoptosis of glioma cells and interfere cell cycle, this maybe the most important mechanism of anti-glioma effect.
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