埃博霉素衍生物抗肿瘤作用的研究
发布时间：2018-09-09 12:25
【摘要】：埃博霉素(epothilones)是由土壤粘细菌合成的大环内酯类化合,具有很强的抗肿瘤活性。虽与临床主流抗癌药紫杉醇(taxanes)具有相似的作用机制,但在疗效、副作用和安全性等方面均优于紫杉醇,尤其是对紫杉醇耐药的肿瘤仍有很好的治疗效果。因此,埃博霉素类化合物成为新型抗肿瘤药物的开发热点。我们的前期工作利用同源重组的方法对甲基转移基团进行缺失突变,得到单甲基埃博霉素衍生物UTD2,研究表明UTD2表现出很好的抗肿瘤活性。虽然已有报道表明埃博霉素类化合物可以通过稳定微管来诱导肿瘤细胞发生凋亡,但是埃博霉素类化合物对Rho家族小GTP酶(Rho GTPases)相关通路的影响目前尚未见报道,并且从作用于微管蛋白到诱导细胞凋亡之间的分子机制仍有待阐明,同时埃博霉素类化合物不产生耐药性的机制迄今为止几乎毫无了解。 本论文通过研究埃博霉素衍生物UTD2对肌动蛋白细胞骨架关键调节因子RhoGTPases及相关信号通路的影响,进一步阐明埃博霉素类化合物的抗肿瘤分子机制。具体研究结果及结论如下： 1.采用MTT检测UTD2和Ixabepilone对乳腺癌细胞增殖的影响,结果显示：UTD2显著抑制乳腺癌细胞增殖,其抑制乳腺癌细胞生长的IC50值显著低于Ixabepilone,说明UTD2具有更好的抗肿瘤活性；微管聚合实验证明UTD2与Ixabepilone同样具有很强的促进微管蛋白聚合的能力；IC50浓度下,UTD2可以阻滞乳腺癌细胞周期于G2/M期；20nmol/L UTD2诱导MCF-7细胞早期凋亡和晚期凋亡的比率分别为19%和29%；进一步通过Western blot检测UTD2对凋亡相关蛋白表达的影响,结果显示：UTD2可以显著抑制Bcl-2的表达,同时增加Bax表达量,激活caspase-8。以上结果表明,UTD2通过稳定微管蛋白,阻滞肿瘤细胞于G2/M期,调控Bcl-2家族,激活caspase级联通路,诱导MCF-7细胞凋亡。 2.通过免疫细胞化学方法检测UTD2对MCF-7细胞骨架重组的影响,结果显示：UTD2可以促进微管蛋白的聚合,改变微管蛋白的形态和分布,进而影响微管细胞骨架的重组；MCF-7细胞经UTD2处理后,片状伪足消失,肌动蛋白丝也呈现较为混乱的分布,表明UTD2同样影响肌动蛋白细胞骨架的重组。Trans well实验结果表明UTD2可以抑制MCF-7细胞迁移和侵袭。明胶酶谱实验结果表明,UTD2对基质金属蛋白酶MMP2活性有较明显的抑制作用。以上结果说明,UTD2通过影响细胞骨架重组,进而影响细胞骨架的相关功能,如肿瘤细胞迁移、侵袭和基质金属蛋白酶活性等。 3.利用MTT法检测UTD2对人脐静脉内皮细胞(HUVEC)增殖的抑制,Trans well法检测HUVEC的运动能力,明胶酶谱分析UTD2对基质金属蛋白酶活性的影响,RT-PCR检测MMP2RNA表达水平；以鸡胚绒毛尿囊膜为模型,研究UTD2对血管新生的影响；发现UTD2可以显著抑制HUVEC的增殖、迁移和侵袭以及MMPs的活性,同时可以抑制鸡胚尿囊膜中新生血管的数目。 4.通过GST pull down和Western blot实验检测UTD2对Rho GTPases;和MAPK通路的影响,发现UTD2可以显著抑制Racl的活性,同时减少PAK和p38的磷酸化。以上结果表明UTD2通过抑制Rac/PAK/p38MAPK信号通路调控乳腺癌细胞肌动蛋白细胞骨架进而调节细胞的运动。 5.采用Focus formation assay计数转化集落的数目,结果显示经UTD2处理后Rac和Raf共同诱导产生的转化集落数目明显减少,说明UTD2抑制Rac在细胞转化实验中与Raf激酶的协同效应,同时可以影响Raf激酶通路。Western blot检测ERK1/2的磷酸化水平发现,UTD2可以使ERK1/2的磷酸化水平显著降低。以上结果说明UTD2可通过抑制Rac/Raf/ERK通路调控肿瘤细胞增殖和转化。 6.采用Western blot、双荧光素酶报告基因等实验检测UTD2对Rac下游Akt和Wnt信号通路中关键蛋白表达、活性及相关转录因子的影响,发现UTD2可以降低Akt磷酸化水平,抑制其活性,其下游信号蛋白FKHR磷酸化下游转录因子CREB均被抑制,结果表明UTD2抑制Racl的活性,减弱Akt通路信号,最终诱导肿瘤细胞凋亡。UTD2对Wnt信号通路中β-catenin的表达和转录因子活性无显著影响,说明UTD2UTD2影响肿瘤细胞增殖和转移则不依赖Wnt通路。 7.利用MTT、流式细胞术和Western blot等方法,检测UTD2和Palitaxel在肿瘤多药耐药性方面表现不同的分子机制,结果显示：UTD2抑制MCF-7/ADR细胞生长的IC50值为78.02nmol/L; Palitaxel抑制MCF-7/ADR细胞生长的IC50值则远远大于1000nmol/L; UTD2以终浓度为100nmol/L作用MCF-7/ADR细胞24h后,MCF-7/ADR细胞早期凋亡率和晚期凋亡率分别为]4.46%和30.7%；UTD2同样可以显著降低MCF-7/ADR细胞Akt激酶磷酸化,但是对照药物Paclitaxel作用后,MCF-7/ADR细胞内的Akt蛋白磷酸化程度没有任何变化。以上结果表明,UTD2对多药耐药细胞MCF-7/ADR同样敏感,可能与UTD2阻断Akt信号通路有关。 综上所述,论文研究阐明了UTD2影响乳腺癌细胞增殖,抑制肿瘤转移和血管新生,诱导MCF-7细胞凋亡的机制；证实UTD2可通过改变Rho GTPases相关信号通路蛋白的活性,影响肌动蛋白细胞骨架并诱导肿瘤细胞凋亡。研究工作为埃博霉素类药物作用机制提供了新的例证,同时可以为埃博霉素类药物的临床应用提供指导。
[Abstract]:Epothilones is a combination of macrolides synthesized by soil Myxobacteria and has strong anti-tumor activity. Although it has the same mechanism as taxanes, it is superior to paclitaxel in efficacy, side effects and safety, especially in the treatment of paclitaxel-resistant tumors. Therapeutic effect. Therefore, Ebolomycin compounds have become a new anti-tumor drug development hotspot. Our previous work used homologous recombination method to delete the methyl transfer group mutation, obtained monomethyl Ebolomycin derivative UTD2, studies have shown that UTD2 shows good anti-tumor activity. Ebolomycin-like compounds can induce apoptosis of tumor cells by stabilizing microtubules. However, the effects of Ebolomycin-like compounds on the Rho family small GTPases-related pathways have not been reported, and the molecular mechanism from acting on tubulin to inducing apoptosis remains to be elucidated, and Ebolomycin-like compounds do not. The mechanism of drug resistance has so far been virtually unknown.
In this paper, we studied the effects of Ebolomycin derivative UTD2 on RhoGTPases, a key regulator of actin cytoskeleton, and related signaling pathways to further elucidate the anti-tumor molecular mechanism of Ebolomycin derivatives.
1. MTT was used to detect the effects of UTD2 and Ixabepilone on the proliferation of breast cancer cells. The results showed that UTD2 significantly inhibited the proliferation of breast cancer cells. The IC50 value of UTD2 was significantly lower than that of Ixabepilone, indicating that UTD2 had better antitumor activity. UTD2 could block the cell cycle of breast cancer in G2/M phase at IC50 concentration; the early and late apoptosis rates of MCF-7 cells induced by 20nmol/L UTD2 were 19% and 29% respectively; furthermore, the effect of UTD2 on the expression of apoptosis-related proteins was detected by Western blot. The results showed that UTD2 could significantly inhibit the expression of Bcl-2. These results showed that UTD2 could induce MCF-7 cell apoptosis by stabilizing tubulin, blocking G2/M phase, regulating Bcl-2 family, activating caspase cascade pathway and inducing apoptosis of MCF-7 cells.
2. The effect of UTD2 on the cytoskeleton reorganization of MCF-7 cells was detected by immunocytochemistry. The results showed that UTD2 could promote the polymerization of tubulin, change the morphology and distribution of tubulin, and then affect the cytoskeleton reorganization of MCF-7 cells. After the treatment with UTD2, the lamellar pseudopodia disappeared and the actin filaments appeared to be disordered. Trans well experiment showed that UTD2 could inhibit the migration and invasion of MCF-7 cells. Gelatin zymogram showed that UTD2 could inhibit the activity of matrix metalloproteinase MMP2 significantly. The above results showed that UTD2 could affect the cytoskeletal reorganization of MCF-7 cells by influencing the cytoskeleton reorganization, and then the effect was fine. Cytoskeletal related functions, such as tumor cell migration, invasion and matrix metalloproteinase activity.
3. MTT assay was used to detect the inhibition of UTD2 on the proliferation of human umbilical vein endothelial cells (HUVEC), Trans well assay was used to detect the motility of HUVEC, gelatin zymogram was used to analyze the effect of UTD2 on the activity of matrix metalloproteinase, and RT-PCR was used to detect the expression of MMP2RNA. It can inhibit the proliferation, migration and invasion of HUVEC and the activity of MMPs, and inhibit the number of new blood vessels in chick embryo allantoic membrane.
4. The effects of UTD2 on Rho GTPases and MAPK pathways were detected by GST pull down and Western blot. It was found that UTD2 could significantly inhibit the activity of Racl and decrease the phosphorylation of PAK and p38. The above results suggest that UTD2 regulates the cytoskeleton of actin cells in breast cancer by inhibiting the Rac/PAK/p38 MAPK signaling pathway.
5. The number of transformed colonies was counted by Focus formation assay. The results showed that the number of transformed colonies induced by Rac and Raf decreased significantly after UTD2 treatment, indicating that UTD2 inhibited the synergistic effect of Rac and Raf kinase in cell transformation experiment, and affected the Raf kinase pathway. The phosphorylation level of ERK1/2 was detected by Western blot. These results suggest that UTD2 can regulate the proliferation and transformation of tumor cells by inhibiting the Rac/Raf/ERK pathway.
6. Western blot and double luciferase reporter gene were used to detect the effect of UTD2 on the key protein expression, activity and related transcription factors of Akt and Wnt signaling pathways downstream of Rac. It was found that UTD2 could decrease the phosphorylation level of Akt and inhibit its activity. The downstream signal protein FKHR phosphorylation of CREB was inhibited. The results showed that UTD2 could inhibit the phosphorylation of Akt and Wnt signaling pathways downstream transcrip D2 inhibited Racl activity, weakened Akt signaling pathway and eventually induced tumor cell apoptosis. UTD2 had no significant effect on the expression of beta-catenin and the activity of transcription factors in Wnt signaling pathway, suggesting that UTD2 did not depend on Wnt pathway in influencing tumor cell proliferation and metastasis.
7. MTT, flow cytometry and Western blot were used to detect the different molecular mechanisms of UTD2 and Palitaxel in tumor multidrug resistance. The results showed that the IC50 value of UTD2 in inhibiting MCF-7/ADR cell growth was 78.02 nmol/L, the IC50 value of Palitaxel in inhibiting MCF-7/ADR cell growth was much higher than 1000 nmol/L, and the final concentration of UTD2 was 100 nmol/L. The early and late apoptosis rates of MCF-7/ADR cells were 4.46% and 30.7% respectively after 24 hours of treatment with nmol/L. UTD2 also significantly decreased the phosphorylation of Akt kinase in MCF-7/ADR cells, but the phosphorylation of Akt protein in MCF-7/ADR cells did not change after treatment with Paclitaxel. Multidrug resistant cells MCF-7/ADR are also sensitive, which may be related to UTD2 blocking Akt signaling pathway.
In summary, this paper elucidates the mechanism of UTD2 affecting the proliferation of breast cancer cells, inhibiting tumor metastasis and angiogenesis, and inducing apoptosis of MCF-7 cells. It is confirmed that UTD2 can affect actin cytoskeleton and induce apoptosis of tumor cells by altering the activity of Rho GTPases-related signaling pathway proteins. The mechanism provides new evidence and provides guidance for the clinical application of ebolycin.
【学位授予单位】：大连理工大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R96
本文编号：2232366
[Abstract]:Epothilones is a combination of macrolides synthesized by soil Myxobacteria and has strong anti-tumor activity. Although it has the same mechanism as taxanes, it is superior to paclitaxel in efficacy, side effects and safety, especially in the treatment of paclitaxel-resistant tumors. Therapeutic effect. Therefore, Ebolomycin compounds have become a new anti-tumor drug development hotspot. Our previous work used homologous recombination method to delete the methyl transfer group mutation, obtained monomethyl Ebolomycin derivative UTD2, studies have shown that UTD2 shows good anti-tumor activity. Ebolomycin-like compounds can induce apoptosis of tumor cells by stabilizing microtubules. However, the effects of Ebolomycin-like compounds on the Rho family small GTPases-related pathways have not been reported, and the molecular mechanism from acting on tubulin to inducing apoptosis remains to be elucidated, and Ebolomycin-like compounds do not. The mechanism of drug resistance has so far been virtually unknown.
In this paper, we studied the effects of Ebolomycin derivative UTD2 on RhoGTPases, a key regulator of actin cytoskeleton, and related signaling pathways to further elucidate the anti-tumor molecular mechanism of Ebolomycin derivatives.
1. MTT was used to detect the effects of UTD2 and Ixabepilone on the proliferation of breast cancer cells. The results showed that UTD2 significantly inhibited the proliferation of breast cancer cells. The IC50 value of UTD2 was significantly lower than that of Ixabepilone, indicating that UTD2 had better antitumor activity. UTD2 could block the cell cycle of breast cancer in G2/M phase at IC50 concentration; the early and late apoptosis rates of MCF-7 cells induced by 20nmol/L UTD2 were 19% and 29% respectively; furthermore, the effect of UTD2 on the expression of apoptosis-related proteins was detected by Western blot. The results showed that UTD2 could significantly inhibit the expression of Bcl-2. These results showed that UTD2 could induce MCF-7 cell apoptosis by stabilizing tubulin, blocking G2/M phase, regulating Bcl-2 family, activating caspase cascade pathway and inducing apoptosis of MCF-7 cells.
2. The effect of UTD2 on the cytoskeleton reorganization of MCF-7 cells was detected by immunocytochemistry. The results showed that UTD2 could promote the polymerization of tubulin, change the morphology and distribution of tubulin, and then affect the cytoskeleton reorganization of MCF-7 cells. After the treatment with UTD2, the lamellar pseudopodia disappeared and the actin filaments appeared to be disordered. Trans well experiment showed that UTD2 could inhibit the migration and invasion of MCF-7 cells. Gelatin zymogram showed that UTD2 could inhibit the activity of matrix metalloproteinase MMP2 significantly. The above results showed that UTD2 could affect the cytoskeletal reorganization of MCF-7 cells by influencing the cytoskeleton reorganization, and then the effect was fine. Cytoskeletal related functions, such as tumor cell migration, invasion and matrix metalloproteinase activity.
3. MTT assay was used to detect the inhibition of UTD2 on the proliferation of human umbilical vein endothelial cells (HUVEC), Trans well assay was used to detect the motility of HUVEC, gelatin zymogram was used to analyze the effect of UTD2 on the activity of matrix metalloproteinase, and RT-PCR was used to detect the expression of MMP2RNA. It can inhibit the proliferation, migration and invasion of HUVEC and the activity of MMPs, and inhibit the number of new blood vessels in chick embryo allantoic membrane.
4. The effects of UTD2 on Rho GTPases and MAPK pathways were detected by GST pull down and Western blot. It was found that UTD2 could significantly inhibit the activity of Racl and decrease the phosphorylation of PAK and p38. The above results suggest that UTD2 regulates the cytoskeleton of actin cells in breast cancer by inhibiting the Rac/PAK/p38 MAPK signaling pathway.
5. The number of transformed colonies was counted by Focus formation assay. The results showed that the number of transformed colonies induced by Rac and Raf decreased significantly after UTD2 treatment, indicating that UTD2 inhibited the synergistic effect of Rac and Raf kinase in cell transformation experiment, and affected the Raf kinase pathway. The phosphorylation level of ERK1/2 was detected by Western blot. These results suggest that UTD2 can regulate the proliferation and transformation of tumor cells by inhibiting the Rac/Raf/ERK pathway.
6. Western blot and double luciferase reporter gene were used to detect the effect of UTD2 on the key protein expression, activity and related transcription factors of Akt and Wnt signaling pathways downstream of Rac. It was found that UTD2 could decrease the phosphorylation level of Akt and inhibit its activity. The downstream signal protein FKHR phosphorylation of CREB was inhibited. The results showed that UTD2 could inhibit the phosphorylation of Akt and Wnt signaling pathways downstream transcrip D2 inhibited Racl activity, weakened Akt signaling pathway and eventually induced tumor cell apoptosis. UTD2 had no significant effect on the expression of beta-catenin and the activity of transcription factors in Wnt signaling pathway, suggesting that UTD2 did not depend on Wnt pathway in influencing tumor cell proliferation and metastasis.
7. MTT, flow cytometry and Western blot were used to detect the different molecular mechanisms of UTD2 and Palitaxel in tumor multidrug resistance. The results showed that the IC50 value of UTD2 in inhibiting MCF-7/ADR cell growth was 78.02 nmol/L, the IC50 value of Palitaxel in inhibiting MCF-7/ADR cell growth was much higher than 1000 nmol/L, and the final concentration of UTD2 was 100 nmol/L. The early and late apoptosis rates of MCF-7/ADR cells were 4.46% and 30.7% respectively after 24 hours of treatment with nmol/L. UTD2 also significantly decreased the phosphorylation of Akt kinase in MCF-7/ADR cells, but the phosphorylation of Akt protein in MCF-7/ADR cells did not change after treatment with Paclitaxel. Multidrug resistant cells MCF-7/ADR are also sensitive, which may be related to UTD2 blocking Akt signaling pathway.
In summary, this paper elucidates the mechanism of UTD2 affecting the proliferation of breast cancer cells, inhibiting tumor metastasis and angiogenesis, and inducing apoptosis of MCF-7 cells. It is confirmed that UTD2 can affect actin cytoskeleton and induce apoptosis of tumor cells by altering the activity of Rho GTPases-related signaling pathway proteins. The mechanism provides new evidence and provides guidance for the clinical application of ebolycin.
【学位授予单位】：大连理工大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R96
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