埃博霉素衍生物抗腫瘤作用的研究
發(fā)布時間：2018-09-09 12:25
【摘要】：埃博霉素(epothilones)是由土壤粘細菌合成的大環(huán)內(nèi)酯類化合,具有很強的抗腫瘤活性。雖與臨床主流抗癌藥紫杉醇(taxanes)具有相似的作用機制,但在療效、副作用和安全性等方面均優(yōu)于紫杉醇,尤其是對紫杉醇耐藥的腫瘤仍有很好的治療效果。因此,埃博霉素類化合物成為新型抗腫瘤藥物的開發(fā)熱點。我們的前期工作利用同源重組的方法對甲基轉移基團進行缺失突變,得到單甲基埃博霉素衍生物UTD2,研究表明UTD2表現(xiàn)出很好的抗腫瘤活性。雖然已有報道表明埃博霉素類化合物可以通過穩(wěn)定微管來誘導腫瘤細胞發(fā)生凋亡,但是埃博霉素類化合物對Rho家族小GTP酶(Rho GTPases)相關通路的影響目前尚未見報道,并且從作用于微管蛋白到誘導細胞凋亡之間的分子機制仍有待闡明,同時埃博霉素類化合物不產(chǎn)生耐藥性的機制迄今為止幾乎毫無了解。 本論文通過研究埃博霉素衍生物UTD2對肌動蛋白細胞骨架關鍵調(diào)節(jié)因子RhoGTPases及相關信號通路的影響,進一步闡明埃博霉素類化合物的抗腫瘤分子機制。具體研究結果及結論如下： 1.采用MTT檢測UTD2和Ixabepilone對乳腺癌細胞增殖的影響,結果顯示：UTD2顯著抑制乳腺癌細胞增殖,其抑制乳腺癌細胞生長的IC50值顯著低于Ixabepilone,說明UTD2具有更好的抗腫瘤活性；微管聚合實驗證明UTD2與Ixabepilone同樣具有很強的促進微管蛋白聚合的能力；IC50濃度下,UTD2可以阻滯乳腺癌細胞周期于G2/M期；20nmol/L UTD2誘導MCF-7細胞早期凋亡和晚期凋亡的比率分別為19%和29%；進一步通過Western blot檢測UTD2對凋亡相關蛋白表達的影響,結果顯示：UTD2可以顯著抑制Bcl-2的表達,同時增加Bax表達量,激活caspase-8。以上結果表明,UTD2通過穩(wěn)定微管蛋白,阻滯腫瘤細胞于G2/M期,調(diào)控Bcl-2家族,激活caspase級聯(lián)通路,誘導MCF-7細胞凋亡。 2.通過免疫細胞化學方法檢測UTD2對MCF-7細胞骨架重組的影響,結果顯示：UTD2可以促進微管蛋白的聚合,改變微管蛋白的形態(tài)和分布,進而影響微管細胞骨架的重組；MCF-7細胞經(jīng)UTD2處理后,片狀偽足消失,肌動蛋白絲也呈現(xiàn)較為混亂的分布,表明UTD2同樣影響肌動蛋白細胞骨架的重組。Trans well實驗結果表明UTD2可以抑制MCF-7細胞遷移和侵襲。明膠酶譜實驗結果表明,UTD2對基質(zhì)金屬蛋白酶MMP2活性有較明顯的抑制作用。以上結果說明,UTD2通過影響細胞骨架重組,進而影響細胞骨架的相關功能,如腫瘤細胞遷移、侵襲和基質(zhì)金屬蛋白酶活性等。 3.利用MTT法檢測UTD2對人臍靜脈內(nèi)皮細胞(HUVEC)增殖的抑制,Trans well法檢測HUVEC的運動能力,明膠酶譜分析UTD2對基質(zhì)金屬蛋白酶活性的影響,RT-PCR檢測MMP2RNA表達水平；以雞胚絨毛尿囊膜為模型,研究UTD2對血管新生的影響；發(fā)現(xiàn)UTD2可以顯著抑制HUVEC的增殖、遷移和侵襲以及MMPs的活性,同時可以抑制雞胚尿囊膜中新生血管的數(shù)目。 4.通過GST pull down和Western blot實驗檢測UTD2對Rho GTPases;和MAPK通路的影響,發(fā)現(xiàn)UTD2可以顯著抑制Racl的活性,同時減少PAK和p38的磷酸化。以上結果表明UTD2通過抑制Rac/PAK/p38MAPK信號通路調(diào)控乳腺癌細胞肌動蛋白細胞骨架進而調(diào)節(jié)細胞的運動。 5.采用Focus formation assay計數(shù)轉化集落的數(shù)目,結果顯示經(jīng)UTD2處理后Rac和Raf共同誘導產(chǎn)生的轉化集落數(shù)目明顯減少,說明UTD2抑制Rac在細胞轉化實驗中與Raf激酶的協(xié)同效應,同時可以影響Raf激酶通路。Western blot檢測ERK1/2的磷酸化水平發(fā)現(xiàn),UTD2可以使ERK1/2的磷酸化水平顯著降低。以上結果說明UTD2可通過抑制Rac/Raf/ERK通路調(diào)控腫瘤細胞增殖和轉化。 6.采用Western blot、雙熒光素酶報告基因等實驗檢測UTD2對Rac下游Akt和Wnt信號通路中關鍵蛋白表達、活性及相關轉錄因子的影響,發(fā)現(xiàn)UTD2可以降低Akt磷酸化水平,抑制其活性,其下游信號蛋白FKHR磷酸化下游轉錄因子CREB均被抑制,結果表明UTD2抑制Racl的活性,減弱Akt通路信號,最終誘導腫瘤細胞凋亡。UTD2對Wnt信號通路中β-catenin的表達和轉錄因子活性無顯著影響,說明UTD2UTD2影響腫瘤細胞增殖和轉移則不依賴Wnt通路。 7.利用MTT、流式細胞術和Western blot等方法,檢測UTD2和Palitaxel在腫瘤多藥耐藥性方面表現(xiàn)不同的分子機制,結果顯示：UTD2抑制MCF-7/ADR細胞生長的IC50值為78.02nmol/L; Palitaxel抑制MCF-7/ADR細胞生長的IC50值則遠遠大于1000nmol/L; UTD2以終濃度為100nmol/L作用MCF-7/ADR細胞24h后,MCF-7/ADR細胞早期凋亡率和晚期凋亡率分別為]4.46%和30.7%；UTD2同樣可以顯著降低MCF-7/ADR細胞Akt激酶磷酸化,但是對照藥物Paclitaxel作用后,MCF-7/ADR細胞內(nèi)的Akt蛋白磷酸化程度沒有任何變化。以上結果表明,UTD2對多藥耐藥細胞MCF-7/ADR同樣敏感,可能與UTD2阻斷Akt信號通路有關。 綜上所述,論文研究闡明了UTD2影響乳腺癌細胞增殖,抑制腫瘤轉移和血管新生,誘導MCF-7細胞凋亡的機制；證實UTD2可通過改變Rho GTPases相關信號通路蛋白的活性,影響肌動蛋白細胞骨架并誘導腫瘤細胞凋亡。研究工作為埃博霉素類藥物作用機制提供了新的例證,同時可以為埃博霉素類藥物的臨床應用提供指導。
[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.
【學位授予單位】：大連理工大學
【學位級別】：博士
【學位授予年份】：2014
【分類號】：R96
本文編號：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.
【學位授予單位】：大連理工大學
【學位級別】：博士
【學位授予年份】：2014
【分類號】：R96
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