替莫唑胺衍生物的抗腫瘤機(jī)制研究
發(fā)布時(shí)間：2019-05-28 14:22
【摘要】：替莫唑胺(Temozolomide,TMZ)是一個(gè)口服安全有效的咪唑并四嗪類烷化劑藥物,容易透過血腦屏障,且骨髓抑制作用較弱,已作為一線的抗神經(jīng)膠質(zhì)瘤的化療藥物在臨床使用,而且對(duì)黑色素瘤等腫瘤具有很好的效果。然而,研究發(fā)現(xiàn) 06-甲基鳥嘌呤-DNA 甲基轉(zhuǎn)移酶(06-methyl guanine-DNA methyltransferase,MGMT)可以修復(fù)TMZ導(dǎo)致的DNA損傷,同時(shí)TMZ發(fā)揮抗腫瘤作用需要功能正常錯(cuò)配修復(fù)(mismatch repair,MMR)系統(tǒng)。因此,MGMT高表達(dá)和MMR缺陷的腫瘤細(xì)胞對(duì)TMZ不敏感。此外,像其它化療藥物一樣,TMZ使用一段時(shí)間后也可產(chǎn)生獲得性耐藥,耐藥的主要機(jī)制與MGMT和MMR相關(guān),從而限制了TMZ的臨床應(yīng)用。因此,開發(fā)可以克服TMZ耐藥問題的TMZ衍生物具有重要的意義。前期通過MTT實(shí)驗(yàn)篩選出了可以抑制TMZ耐藥腫瘤細(xì)胞生長(zhǎng)的TMZ衍生物377和465,它們對(duì)MMR缺陷的結(jié)腸癌細(xì)胞HCT116的IC50值分別為44.23和25.37μM,對(duì)MGMT高表達(dá)的神經(jīng)膠質(zhì)瘤細(xì)胞T98G的IC50值分別為62.5和33.09μM。先前研究報(bào)道TMZ可以引起MGMT低表達(dá)和MMR功能正常的腫瘤細(xì)胞發(fā)生G2/M期阻滯,導(dǎo)致DNA發(fā)生雙鏈斷裂,進(jìn)而誘導(dǎo)細(xì)胞凋亡,最終發(fā)揮抗腫瘤作用。因此,我們檢測(cè)了衍生物對(duì)HCT116和T98G細(xì)胞的作用,發(fā)現(xiàn)20μM的465就可以引起HCT116細(xì)胞發(fā)生G2/M期周期阻滯,100μM 377和50μM 465可以引起T98G細(xì)胞發(fā)生G2/M期周期阻滯;100μM 377和465可以引起T98G細(xì)胞發(fā)生DNA雙鏈斷裂,并導(dǎo)致細(xì)胞發(fā)生凋亡。此前實(shí)驗(yàn)也發(fā)現(xiàn)50μM 465可以導(dǎo)致HCT116細(xì)胞發(fā)生DNA雙鏈斷裂和細(xì)胞凋亡。比較有意思的是,我們發(fā)現(xiàn)465在蛋白和mRNA水平均可以下調(diào)HCT116細(xì)胞的MGMT表達(dá)。因此,我們進(jìn)一步研究了 465下調(diào)MGMT的作用機(jī)制。研究報(bào)道多種因素影響MGMT基因的表達(dá),例如MGMT啟動(dòng)子發(fā)生高度甲基化可以導(dǎo)致染色體壓縮,隔絕轉(zhuǎn)錄因子,抑制轉(zhuǎn)錄。因此我們通過Bisulfite Sequencing PCR實(shí)驗(yàn)檢測(cè)50μM465處理HCT116細(xì)胞48h后,MGMT的啟動(dòng)子甲基化的影響。結(jié)果發(fā)現(xiàn)HCT116細(xì)胞內(nèi)MGMT的啟動(dòng)子甲基化水平較高,而465并沒有影響MGMT的啟動(dòng)子甲基化水平。研究報(bào)道轉(zhuǎn)錄因子p53可以直接結(jié)合在MGMT啟動(dòng)子序列上,從而抑制其轉(zhuǎn)錄。我們發(fā)現(xiàn)50μM 465處理HCT116細(xì)胞48h后,細(xì)胞內(nèi)野生型p53的表達(dá)增加,接著我們通過Chromatin Immunoprecipitation Assay實(shí)驗(yàn)證明465處理HCT116細(xì)胞后增加了野生型p53與MGMT啟動(dòng)子的結(jié)合。為進(jìn)一步確認(rèn)465處理HCT116細(xì)胞后,增加的野生型p53可以抑制MGMT表達(dá),我們用50μM 465處理轉(zhuǎn)染p53 shRNA干擾質(zhì)粒的HCT116細(xì)胞。結(jié)果發(fā)現(xiàn)465處理細(xì)胞后,野生型p53被敲低的細(xì)胞內(nèi)MGMT的表達(dá)量上升。進(jìn)一步說明465可以上調(diào)野生型p53表達(dá)進(jìn)而降低MGMT表達(dá)。此外,為了探究TMZ衍生物377和465對(duì)不同p53背景細(xì)胞內(nèi)MGMT表達(dá)的影響,我們使用不同濃度的377和465處理T98G(突變型p53)和H1299(p53-/-)細(xì)胞48h,結(jié)果發(fā)現(xiàn)50μM 377和465不影響這兩種細(xì)胞內(nèi)MGMT的表達(dá),而100μM377和465可以下調(diào)T98G細(xì)胞內(nèi)MGMT表達(dá)以及10OμM465可以降低H1299細(xì)胞內(nèi)MGMT表達(dá)。這些結(jié)果說明,不同p53背景細(xì)胞內(nèi)MGMT對(duì)TMZ衍生物的敏感度不同。同時(shí)有研究報(bào)道MGMT啟動(dòng)子序列上有多處轉(zhuǎn)錄因子Sp1的結(jié)合位點(diǎn),Sp1可以直接結(jié)合在MGMT啟動(dòng)子序列上,促進(jìn)轉(zhuǎn)錄進(jìn)行。我們發(fā)現(xiàn)50μM465處理HCT116細(xì)胞48h后,細(xì)胞內(nèi)Sp1的表達(dá)降低。隨后通過Electrophoretic Mobility Shift Assay檢測(cè)Sp1與Sp1結(jié)合位點(diǎn)探針的結(jié)合情況,發(fā)現(xiàn)465處理HCT116細(xì)胞后,細(xì)胞核內(nèi)Sp1蛋白與探針形成阻滯減少,這初步說明465可以下調(diào)Sp1表達(dá),影響HCT116細(xì)胞中Sp1與MGMT的啟動(dòng)子結(jié)合,從而影響MGMT表達(dá)。綜上所述,TMZ衍生物377和465可以引起MGMT高表達(dá)的神經(jīng)膠質(zhì)瘤細(xì)胞T98G和MMR缺陷的結(jié)腸癌細(xì)胞HCT116發(fā)生G2/M期周期阻滯,進(jìn)而DNA雙鏈斷裂,最終引起細(xì)胞發(fā)生凋亡。衍生物465在轉(zhuǎn)錄水平下調(diào)HCT116細(xì)胞內(nèi)MGMT表達(dá),其發(fā)揮作用不是通過影響MGMT的啟動(dòng)子甲基化水平,而是通過轉(zhuǎn)錄因子野生型p53和Sp1與MGMT啟動(dòng)子的結(jié)合進(jìn)而調(diào)控MGMT的表達(dá)。此外,不同p53背景細(xì)胞內(nèi)MGMT對(duì)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.
【學(xué)位授予單位】：昆明理工大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R96
，
本文編號(hào)：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.
【學(xué)位授予單位】：昆明理工大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R96
，
本文編號(hào)：2487131
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