替莫唑胺衍生物的抗肿瘤机制研究
发布时间：2019-05-28 14:22
【摘要】：替莫唑胺(Temozolomide,TMZ)是一个口服安全有效的咪唑并四嗪类烷化剂药物,容易透过血脑屏障,且骨髓抑制作用较弱,已作为一线的抗神经胶质瘤的化疗药物在临床使用,而且对黑色素瘤等肿瘤具有很好的效果。然而,研究发现 06-甲基鸟嘌呤-DNA 甲基转移酶(06-methyl guanine-DNA methyltransferase,MGMT)可以修复TMZ导致的DNA损伤,同时TMZ发挥抗肿瘤作用需要功能正常错配修复(mismatch repair,MMR)系统。因此,MGMT高表达和MMR缺陷的肿瘤细胞对TMZ不敏感。此外,像其它化疗药物一样,TMZ使用一段时间后也可产生获得性耐药,耐药的主要机制与MGMT和MMR相关,从而限制了TMZ的临床应用。因此,开发可以克服TMZ耐药问题的TMZ衍生物具有重要的意义。前期通过MTT实验筛选出了可以抑制TMZ耐药肿瘤细胞生长的TMZ衍生物377和465,它们对MMR缺陷的结肠癌细胞HCT116的IC50值分别为44.23和25.37μM,对MGMT高表达的神经胶质瘤细胞T98G的IC50值分别为62.5和33.09μM。先前研究报道TMZ可以引起MGMT低表达和MMR功能正常的肿瘤细胞发生G2/M期阻滞,导致DNA发生双链断裂,进而诱导细胞凋亡,最终发挥抗肿瘤作用。因此,我们检测了衍生物对HCT116和T98G细胞的作用,发现20μM的465就可以引起HCT116细胞发生G2/M期周期阻滞,100μM 377和50μM 465可以引起T98G细胞发生G2/M期周期阻滞;100μM 377和465可以引起T98G细胞发生DNA双链断裂,并导致细胞发生凋亡。此前实验也发现50μM 465可以导致HCT116细胞发生DNA双链断裂和细胞凋亡。比较有意思的是,我们发现465在蛋白和mRNA水平均可以下调HCT116细胞的MGMT表达。因此,我们进一步研究了 465下调MGMT的作用机制。研究报道多种因素影响MGMT基因的表达,例如MGMT启动子发生高度甲基化可以导致染色体压缩,隔绝转录因子,抑制转录。因此我们通过Bisulfite Sequencing PCR实验检测50μM465处理HCT116细胞48h后,MGMT的启动子甲基化的影响。结果发现HCT116细胞内MGMT的启动子甲基化水平较高,而465并没有影响MGMT的启动子甲基化水平。研究报道转录因子p53可以直接结合在MGMT启动子序列上,从而抑制其转录。我们发现50μM 465处理HCT116细胞48h后,细胞内野生型p53的表达增加,接着我们通过Chromatin Immunoprecipitation Assay实验证明465处理HCT116细胞后增加了野生型p53与MGMT启动子的结合。为进一步确认465处理HCT116细胞后,增加的野生型p53可以抑制MGMT表达,我们用50μM 465处理转染p53 shRNA干扰质粒的HCT116细胞。结果发现465处理细胞后,野生型p53被敲低的细胞内MGMT的表达量上升。进一步说明465可以上调野生型p53表达进而降低MGMT表达。此外,为了探究TMZ衍生物377和465对不同p53背景细胞内MGMT表达的影响,我们使用不同浓度的377和465处理T98G(突变型p53)和H1299(p53-/-)细胞48h,结果发现50μM 377和465不影响这两种细胞内MGMT的表达,而100μM377和465可以下调T98G细胞内MGMT表达以及10OμM465可以降低H1299细胞内MGMT表达。这些结果说明,不同p53背景细胞内MGMT对TMZ衍生物的敏感度不同。同时有研究报道MGMT启动子序列上有多处转录因子Sp1的结合位点,Sp1可以直接结合在MGMT启动子序列上,促进转录进行。我们发现50μM465处理HCT116细胞48h后,细胞内Sp1的表达降低。随后通过Electrophoretic Mobility Shift Assay检测Sp1与Sp1结合位点探针的结合情况,发现465处理HCT116细胞后,细胞核内Sp1蛋白与探针形成阻滞减少,这初步说明465可以下调Sp1表达,影响HCT116细胞中Sp1与MGMT的启动子结合,从而影响MGMT表达。综上所述,TMZ衍生物377和465可以引起MGMT高表达的神经胶质瘤细胞T98G和MMR缺陷的结肠癌细胞HCT116发生G2/M期周期阻滞,进而DNA双链断裂,最终引起细胞发生凋亡。衍生物465在转录水平下调HCT116细胞内MGMT表达,其发挥作用不是通过影响MGMT的启动子甲基化水平,而是通过转录因子野生型p53和Sp1与MGMT启动子的结合进而调控MGMT的表达。此外,不同p53背景细胞内MGMT对TMZ衍生物377和465的敏感度不同。
[Abstract]:temozolide (TMZ) is an oral safe and effective medetomidine and a four-class alkylating agent, is easy to penetrate the blood-brain barrier, and has weak bone marrow-inhibiting effect, and has been used in clinical use as a line-based chemotherapy medicine for resisting glioma, But also has good effect on the tumor such as melanoma and the like. However, the study found that the 06-methylornithine-DNA methyltransferase (MGMT) can repair the DNA damage caused by TMZ, and the TMZ plays an anti-tumor role, which requires a normal mismatch repair (MMR) system. Thus, MGMT high expression and MMR-deficient tumor cells are not sensitive to TMZ. In addition, as with other chemotherapeutic agents, TMZ can also produce acquired drug resistance after a period of time, and the main mechanism of drug resistance is related to MGMT and MMR, thus limiting the clinical application of TMZ. Therefore, it is of great significance to develop TMZ derivatives which can overcome the resistance of TMZ. TMZ derivatives 377 and 465, which can inhibit the growth of TMZ-resistant tumor cells, were selected by MTT assay in the early stage, and the IC50 values of the colon cancer cells HCT116 of the MMR deficiency were 44.23 and 25.37. m The IC50 values of T98G for glioma cells with high MGMT expression were 62.5 and 33.09. m u.M, respectively. Therefore, we have detected the effect of the derivative on the cells of the HCT116 and T98G, and it is found that the 20. m u.M of 465 can cause the G2/ M phase block of the HCT116 cell, and the 100. m u.M 377 and 50. m u.M 465 may cause a G2/ M phase block to occur in the T98G cell;100. m And lead to the apoptosis of the cells. It was also found that 50. m u.M of 465 could lead to DNA double-strand breaks and cell apoptosis in the HCT116 cells. Interestingly, we found 465 that the MGMT expression of the HCT116 cells could be down-regulated at both the protein and the mRNA levels. Therefore, we have further studied 465 down-regulation of the mechanism of the MGMT. A variety of factors have been reported to affect the expression of the MGMT gene, for example, the high methylation of the MGMT promoter can lead to chromosome compression, isolation of transcription factors, and inhibition of transcription. Therefore, the effect of 50. m. M465 on the methylation of the promoter of the MGMT was detected by the Bisulfite Sequencing PCR assay for 48 h after the treatment of the HCT116 cell. The results showed that the methylation level of MGMT in HCT116 cells was high, and 465 did not affect the methylation level of the promoter of MGMT. The study reported that the transcription factor p53 could be directly bound to the MGMT promoter sequence to inhibit its transcription. We found that after 48 h of the 50. m u.M 465 treatment of the HCT116 cells, the expression of the wild-type p53 in the cells was increased, followed by the addition of the combination of the wild-type p53 with the MGMT promoter after the treatment of the HCT116 cells by the Chromatin Immunopreservation Assay. In order to further confirm 465 treatment of the HCT116 cells, the increased wild-type p53 could inhibit MGMT expression, and we treated the HCT116 cells transfected with the p53 shRNA interference plasmid with 50.mu. M 465. As a result, the expression of MGMT in wild-type p53 was increased after the cells were treated with 465 cells. Further instructions 465 may increase the expression of wild-type p53 and, in turn, decrease the expression of MGMT. In addition, in order to investigate the effects of TMZ derivatives 377 and 465 on the expression of MGMT in different p53 background cells, we used different concentrations of 377 and 465 to process T98G (mutant p53) and H1299 (p53-/-) cells for 48 h, and it was found that 50.mu. M 377 and 465 did not affect the expression of MGMT in both cells, in addition, that expression of MGMT in the T98G cell and the expression of MGMT in the H1299 cell can be reduced by 100. m These results indicate that the sensitivity of the MGMT to the TMZ derivative in different p53 background cells is different. At the same time, it is reported that the binding site of multiple transcription factors Sp1 on the MGMT promoter sequence, Sp1 can be directly bound to the MGMT promoter sequence to promote transcription. We found that the expression of Sp1 in the cells decreased after 48 h of treatment with 50. m The binding of Sp1 with the Sp1 binding site probe was then detected by the Electrosurgical Mobility Shift Assay. After the treatment of the HCT116 cells, the expression of Sp1 in the nucleus and the probe formation was reduced. This preliminary explanation 465 could downregulate the expression of Sp1 and affect the binding of Sp1 to the promoter of the MGMT in the HCT116 cells, thereby affecting the expression of the MGMT. In conclusion, the TMZ derivatives 377 and 465 can cause the G2/ M phase block of the MGMT high-expression glioma cell T98G and the MMR-deficient colon cancer cell HCT116, and then the DNA double-strand breaks, resulting in the apoptosis of the cells. The derivative 465 down-regulates the MGMT expression in the HCT116 cell at the transcription level, and its role is not to regulate the expression of the MGMT by the binding of the transcription factor wild-type p53 and Sp1 to the MGMT promoter by affecting the promoter methylation level of the MGMT. In addition, the sensitivity of MGMT to TMZ derivatives 377 and 465 in different p53 background cells is different.
【学位授予单位】：昆明理工大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R96
，
本文编号：2487131
[Abstract]:temozolide (TMZ) is an oral safe and effective medetomidine and a four-class alkylating agent, is easy to penetrate the blood-brain barrier, and has weak bone marrow-inhibiting effect, and has been used in clinical use as a line-based chemotherapy medicine for resisting glioma, But also has good effect on the tumor such as melanoma and the like. However, the study found that the 06-methylornithine-DNA methyltransferase (MGMT) can repair the DNA damage caused by TMZ, and the TMZ plays an anti-tumor role, which requires a normal mismatch repair (MMR) system. Thus, MGMT high expression and MMR-deficient tumor cells are not sensitive to TMZ. In addition, as with other chemotherapeutic agents, TMZ can also produce acquired drug resistance after a period of time, and the main mechanism of drug resistance is related to MGMT and MMR, thus limiting the clinical application of TMZ. Therefore, it is of great significance to develop TMZ derivatives which can overcome the resistance of TMZ. TMZ derivatives 377 and 465, which can inhibit the growth of TMZ-resistant tumor cells, were selected by MTT assay in the early stage, and the IC50 values of the colon cancer cells HCT116 of the MMR deficiency were 44.23 and 25.37. m The IC50 values of T98G for glioma cells with high MGMT expression were 62.5 and 33.09. m u.M, respectively. Therefore, we have detected the effect of the derivative on the cells of the HCT116 and T98G, and it is found that the 20. m u.M of 465 can cause the G2/ M phase block of the HCT116 cell, and the 100. m u.M 377 and 50. m u.M 465 may cause a G2/ M phase block to occur in the T98G cell;100. m And lead to the apoptosis of the cells. It was also found that 50. m u.M of 465 could lead to DNA double-strand breaks and cell apoptosis in the HCT116 cells. Interestingly, we found 465 that the MGMT expression of the HCT116 cells could be down-regulated at both the protein and the mRNA levels. Therefore, we have further studied 465 down-regulation of the mechanism of the MGMT. A variety of factors have been reported to affect the expression of the MGMT gene, for example, the high methylation of the MGMT promoter can lead to chromosome compression, isolation of transcription factors, and inhibition of transcription. Therefore, the effect of 50. m. M465 on the methylation of the promoter of the MGMT was detected by the Bisulfite Sequencing PCR assay for 48 h after the treatment of the HCT116 cell. The results showed that the methylation level of MGMT in HCT116 cells was high, and 465 did not affect the methylation level of the promoter of MGMT. The study reported that the transcription factor p53 could be directly bound to the MGMT promoter sequence to inhibit its transcription. We found that after 48 h of the 50. m u.M 465 treatment of the HCT116 cells, the expression of the wild-type p53 in the cells was increased, followed by the addition of the combination of the wild-type p53 with the MGMT promoter after the treatment of the HCT116 cells by the Chromatin Immunopreservation Assay. In order to further confirm 465 treatment of the HCT116 cells, the increased wild-type p53 could inhibit MGMT expression, and we treated the HCT116 cells transfected with the p53 shRNA interference plasmid with 50.mu. M 465. As a result, the expression of MGMT in wild-type p53 was increased after the cells were treated with 465 cells. Further instructions 465 may increase the expression of wild-type p53 and, in turn, decrease the expression of MGMT. In addition, in order to investigate the effects of TMZ derivatives 377 and 465 on the expression of MGMT in different p53 background cells, we used different concentrations of 377 and 465 to process T98G (mutant p53) and H1299 (p53-/-) cells for 48 h, and it was found that 50.mu. M 377 and 465 did not affect the expression of MGMT in both cells, in addition, that expression of MGMT in the T98G cell and the expression of MGMT in the H1299 cell can be reduced by 100. m These results indicate that the sensitivity of the MGMT to the TMZ derivative in different p53 background cells is different. At the same time, it is reported that the binding site of multiple transcription factors Sp1 on the MGMT promoter sequence, Sp1 can be directly bound to the MGMT promoter sequence to promote transcription. We found that the expression of Sp1 in the cells decreased after 48 h of treatment with 50. m The binding of Sp1 with the Sp1 binding site probe was then detected by the Electrosurgical Mobility Shift Assay. After the treatment of the HCT116 cells, the expression of Sp1 in the nucleus and the probe formation was reduced. This preliminary explanation 465 could downregulate the expression of Sp1 and affect the binding of Sp1 to the promoter of the MGMT in the HCT116 cells, thereby affecting the expression of the MGMT. In conclusion, the TMZ derivatives 377 and 465 can cause the G2/ M phase block of the MGMT high-expression glioma cell T98G and the MMR-deficient colon cancer cell HCT116, and then the DNA double-strand breaks, resulting in the apoptosis of the cells. The derivative 465 down-regulates the MGMT expression in the HCT116 cell at the transcription level, and its role is not to regulate the expression of the MGMT by the binding of the transcription factor wild-type p53 and Sp1 to the MGMT promoter by affecting the promoter methylation level of the MGMT. In addition, the sensitivity of MGMT to TMZ derivatives 377 and 465 in different p53 background cells is different.
【学位授予单位】：昆明理工大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R96
，
本文编号：2487131
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