本文關(guān)鍵詞：靶向ErbB2抗體H2-18與小分子抑制劑聯(lián)合應(yīng)用在Trastuzumab耐藥乳腺癌中的抗腫瘤作用及其機(jī)理　出處：《第二軍醫(yī)大學(xué)》2017年博士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 乳腺癌 Trastuzumab耐藥 ErbB2 協(xié)同作用

【摘要】：ErbB2(human epidermal growth factor receptor 2,HER2)是人表皮生長因子受體家族成員。25-30%的乳腺癌細(xì)胞及4-50%的胃癌細(xì)胞存在著ErbB2過表達(dá)。ErbB2的過表達(dá)與腫瘤的侵襲及不良預(yù)后有著密切關(guān)聯(lián)。曲妥珠單抗(Trastuzumab)是一個(gè)靶向ErbB2的人源化抗體,已獲批用于ErbB2高表達(dá)轉(zhuǎn)移性乳腺癌和胃癌的臨床治療。然而,臨床研究表明大部分ErbB2高表達(dá)的乳腺癌患者對(duì)Trastuzumab治療不產(chǎn)生反應(yīng),即使對(duì)Trastuzumab產(chǎn)生反應(yīng)的患者有半數(shù)也會(huì)在1年內(nèi)產(chǎn)生耐藥。因此,發(fā)展新的乳腺癌靶向治療策略已成為基礎(chǔ)研究和臨床治療的迫切需求。在前期研究中,我們通過篩選噬菌體抗體庫獲得了一株全新的抗ErbB2全人源單克隆抗體H2-18,其能夠強(qiáng)有力誘導(dǎo)Trastuzumab耐藥乳腺癌細(xì)胞發(fā)生程序化死亡(Programmed cell death,PCD),而Trastuzumab則不能有效誘導(dǎo)乳腺癌細(xì)胞發(fā)生PCD。我們通過乳腺癌荷瘤小鼠模型對(duì)H2-18的體內(nèi)抗腫瘤活性進(jìn)行了評(píng)價(jià),結(jié)果表明H2-18能有效抑制Trastuzumab耐藥乳腺癌生長。我們的研究進(jìn)一步提示H2-18的PCD誘導(dǎo)活性是其對(duì)Trastuzumab耐藥乳腺癌產(chǎn)生抗腫瘤活性的重要原因。ErbB2過表達(dá)乳腺癌對(duì)Trastuzumab產(chǎn)生耐藥的機(jī)制有多種,其中一個(gè)非常重要的原因是PI3K/AKT信號(hào)通路的異常激活。PTEN缺失以及PIK3CA基因突變都能夠?qū)е翽I3K/AKT信號(hào)通路異常激活。但這種異常激活可被PI3K抑制劑所抑制。GDC-0941是一個(gè)pan-PI3K的小分子抑制劑,因其在腫瘤細(xì)胞中表現(xiàn)出理想的抗腫瘤活性而備受關(guān)注。Src是一個(gè)屬于Src激酶家族的非受體酪氨酸激酶,是多條與Trastuzumab耐藥產(chǎn)生相關(guān)的細(xì)胞信號(hào)通路共同的關(guān)鍵結(jié)點(diǎn)。Saracatinib是Src的抑制劑,能夠在一定程度上使耐藥乳腺癌細(xì)胞對(duì)Trastuzumab的作用重新變得敏感。為了進(jìn)一步提高H2-18抗體在耐藥乳腺癌中的治療效果,在本研究中我們將探討H2-18與GDC-0941或Saracatinib的聯(lián)合抗腫瘤作用。首先,我們通過三維細(xì)胞生長實(shí)驗(yàn)檢測了H2-18與GDC-0941或Saracatinib聯(lián)用對(duì)ErbB2過表達(dá)的Trastuzumab敏感的乳腺癌細(xì)胞BT-474、SKBR-3和Trastuzumab耐藥的乳腺癌細(xì)胞HCC-1954、HCC-1419的體外生長抑制活性。實(shí)驗(yàn)結(jié)果顯示,聯(lián)用H2-18與GDC-0941在以上乳腺癌細(xì)胞中均能發(fā)揮比單藥更強(qiáng)的抗腫瘤作用。用CompuSynsoware軟件擬合之后發(fā)現(xiàn)H2-18與GDC-0941具有協(xié)同抗腫瘤活性。聯(lián)用H2-18與Saracatinib在這幾株乳腺癌細(xì)胞中也展現(xiàn)出類似的比單藥明顯增強(qiáng)的抗腫瘤作用。為了探討以上藥物聯(lián)用產(chǎn)生協(xié)同抗腫瘤活性的原因,我們采用了蛋白印跡法檢測不同藥物處理下BT-474和HCC-1954細(xì)胞內(nèi)ErbB2下游信號(hào)通路的改變。我們發(fā)現(xiàn),GDC-0941能夠有效抑制Akt的磷酸化,對(duì)ErK磷酸化影響較弱,且不影響p-JNK和p-c-jun。H2-18能夠明顯抑制Erk的磷酸化,促進(jìn)JNK/c-jun的磷酸化,對(duì)AKT磷酸化的抑制作用較弱。與H2-18單用相比,H2-18/GDC-0941聯(lián)用在抑制Erk活性或激活JNK/c-jun的能力上沒有明顯差別,但抑制p-Akt的能力有所增強(qiáng)。接下來我們用pAkt(S473)的Elisa試劑盒檢測細(xì)胞內(nèi)pAkt的含量。結(jié)果顯示,聯(lián)用H2-18和GDC-0941組與單用GDC-0941組在抑制p-Akt的作用上并沒有統(tǒng)計(jì)學(xué)意義的差異。相似的,聯(lián)用H2-18與Saracatinib在ErbB2下游信號(hào)通路上也沒能顯示出比單藥更強(qiáng)的作用。下一步,我們采用Annexin V/PI雙染法進(jìn)行細(xì)胞染色,并通過流式細(xì)胞術(shù)檢測藥物處理后乳腺癌細(xì)胞BT-474、SKBR-3、HCC-1954和HCC-1419的死亡情況。結(jié)果發(fā)現(xiàn),與單藥相比,H2-18與GDC-0941聯(lián)用組誘導(dǎo)以上乳腺癌細(xì)胞發(fā)生PCD的能力顯著增強(qiáng)。而聯(lián)用H2-18與Saracatinib也顯示了比單藥更強(qiáng)的誘導(dǎo)細(xì)胞PCD的能力。我們還通過PI染色用流式細(xì)胞技術(shù)來檢測藥物處理后細(xì)胞周期分布的改變。結(jié)果表明,在BT-474細(xì)胞中,與單藥相比,聯(lián)用H2-18與GDC-0941時(shí)發(fā)生G1期阻滯的細(xì)胞比例更高。而在HCC-1954細(xì)胞中,聯(lián)用H2-18與GDC-0941并未比單用GDC-0941顯現(xiàn)出更強(qiáng)的G1期阻滯作用。H2-18與Saracatinib兩藥聯(lián)用在Trastuzumab敏感的細(xì)胞株BT-474與SKBR-3細(xì)胞中較單藥組引起更明顯的G1期阻滯。而在Trastuzumab耐藥的細(xì)胞HCC-1954和HCC-1419中,聯(lián)用H2-18與Saracatinib也并未顯示出比單用Saracatinib更強(qiáng)的誘導(dǎo)G1期阻滯的作用。此外,我們用流式細(xì)胞技術(shù)檢測了藥物處理后BT-474和HCC-1954細(xì)胞內(nèi)的活性氧含量。結(jié)果顯示,在這兩株細(xì)胞中,單用H2-18與GDC-0941都能增高活性氧水平。而與單用組相比,H2-18/GDC-0941聯(lián)用組不能進(jìn)一步提高活性氧水平。類似的,H2-18/Saracatinib聯(lián)用組細(xì)胞內(nèi)活性氧水平并不比H2-18單用組高。我們利用HCC-1954乳腺癌荷瘤小鼠模型對(duì)以上藥物進(jìn)行了體內(nèi)抗腫瘤作用研究,結(jié)果表明單用H2-18與單用GDC-0941都能有效抑制移植瘤的生長,而兩者聯(lián)用后比單藥組具有顯著增強(qiáng)的抑制腫瘤生長的作用。聯(lián)用H2-18與Saracatinib的情況與H2-18/GCD-0941相似。因此,我們推斷無論是聯(lián)用H2-18與GDC-0941還是聯(lián)用H2-18與Saracatinib,它們較單藥組表現(xiàn)出更強(qiáng)抗腫瘤活性的原因可能主要由于藥物聯(lián)用可協(xié)同誘導(dǎo)腫瘤細(xì)胞發(fā)生PCD。除此之外,兩藥聯(lián)用后能同時(shí)抑制ErbB2下游關(guān)鍵的PI3K/AKT與RAS/MAPK兩條通路也發(fā)揮了一定的作用。綜上所述,H2-18與GDC-0941或Saracatinib聯(lián)合應(yīng)用在Trastuzumab耐藥的乳腺癌中均展現(xiàn)出顯著增強(qiáng)的體內(nèi)外抗腫瘤活性,提示這樣的藥物組合有望成為克服Trastuzumab耐藥的新策略。
[Abstract]:ErbB2 (human epidermal growth factor receptor 2, HER2) is a member of the human epidermal growth factor receptor family. The ErbB2 overexpression exists in the breast cancer cells of 25-30% and the gastric cancer cells of 4-50%. The overexpression of ErbB2 is closely related to the invasion and poor prognosis of the tumor. Trastuzumab (Trastuzumab) is a humanized antibody targeting ErbB2, and has been approved for the clinical treatment of ErbB2 high expression of metastatic breast cancer and gastric cancer. However, clinical studies show that most ErbB2 patients with high expression of breast cancer do not respond to Trastuzumab treatment. Even half of patients who respond to Trastuzumab respond to drug resistance within 1 years. Therefore, the development of new targeted treatment strategies for breast cancer has become an urgent need for basic research and clinical treatment. In our previous study, we have obtained a new anti ErbB2 humanized monoclonal antibody H2-18 by screening phage antibody library, which can strongly induce PCD Trastuzumab resistant breast cancer cells (Programmed cell death, PCD), but not Trastuzumab can effectively induce the occurrence of breast cancer cell PCD. We evaluated the in vivo antitumor activity of H2-18 through a breast cancer bearing mice model. The results showed that H2-18 could effectively inhibit the growth of Trastuzumab resistant breast cancer. Our study further suggests that the PCD induced activity of H2-18 is an important reason for its antitumor activity in Trastuzumab resistant breast cancer. There are many mechanisms for ErbB2 over expression of Trastuzumab resistance to breast cancer, and one of the most important reasons is the abnormal activation of the PI3K/AKT signaling pathway. The deletion of PTEN and the mutation of PIK3CA gene can cause abnormal activation of the PI3K/AKT signaling pathway. However, this abnormal activation can be suppressed by PI3K inhibitors. GDC-0941 is a small molecule inhibitor of pan-PI3K, which has attracted much attention because of its ideal antitumor activity in tumor cells. Src is a non receptor tyrosine kinase belonging to the Src kinase family, which is a key node of a number of cell signaling pathways associated with Trastuzumab resistance. Saracatinib is an inhibitor of Src, which can, to a certain extent, make drug resistant breast cancer cells more sensitive to the role of Trastuzumab. In order to further improve the therapeutic effect of H2-18 antibody in drug-resistant breast cancer, we will explore the combined anti-tumor effect of H2-18 and GDC-0941 or Saracatinib in this study. First, we used three-dimensional cell growth assay to detect the inhibitory effect of H2-18 combined with GDC-0941 or Saracatinib on ErbB2 overexpressing Trastuzumab sensitive breast cancer cells BT-474, SKBR-3 and Trastuzumab resistant breast cancer cells HCC-1954 and HCC-1419 in vitro. The results showed that both H2-18 and GDC-0941 could play a stronger anti-tumor effect than single drug in the breast cancer cells. After fitting with CompuSynsoware software, it was found that H2-18 and GDC-0941 have synergistic antitumor activity. The combined use of H2-18 and Saracatinib in these breast cancer cells also showed a similar antitumor effect that was significantly enhanced by a single drug. In order to explore the cause of synergistic antitumor activity of the above drug combination, we used Western blot to detect the changes of downstream ErbB2 signaling pathway in BT-474 and HCC-1954 cells treated with different drugs. We found that GDC-0941 can effectively inhibit the phosphorylation of Akt, which has a weak effect on the phosphorylation of ErK, and does not affect p-JNK and p-c-jun. H2-18 can significantly inhibit the phosphorylation of Erk, promote the phosphorylation of JNK/c-jun, and the inhibition of AKT phosphorylation is weak. Compared with H2-18 alone, there was no significant difference in the ability of H2-18/GDC-0941 to inhibit Erk activity or to activate JNK/c-jun, but the ability to inhibit p-Akt was enhanced. Then we used the pAkt (S473) Elisa kit to detect the content of pAkt in the cells. The results showed that there was no significant difference in the inhibition of p-Akt between the combined H2-18 and GDC-0941 group and the single GDC-0941 group. Similarly, the use of H2-18 and Saracatinib in the downstream signal pathway of ErbB2 also did not show a stronger effect than the single drug. Next, we used Annexin V/PI double staining to detect cell death and detect the death of BT-474, SKBR-3, HCC-1954 and HCC-1419 after treatment with flow cytometry. The results showed that compared with the single drug, the ability to induce PCD in the breast cancer cells was significantly enhanced by the combination of H2-18 and GDC-0941. The combined use of H2-18 and Saracatinib also showed a stronger ability to induce cell PCD than single drug. We also used PI staining to detect the changes in cell cycle distribution after treatment with flow cytometry. The results showed that in BT-474 cells, compared with the single drug, the proportion of cells with G1 block at G1 was higher than that of GDC-0941. In HCC-1954 cells, the combination of H2-18 and GDC-0941 did not show a stronger G1 phase blocking effect than the single use of GDC-0941. The combined use of H2-18 and Saracatinib two drugs in Trastuzumab sensitive cell lines, BT-474 and SKBR-3 cells, resulted in a more pronounced G1 phase block than in the single drug group. In Trastuzumab resistant cells HCC-1954 and HCC-1419, H2-18 and Saracatinib also did not show a stronger effect than G1 alone on induction of G1 phase arrest. In addition, we used flow cytometry to detect the content of reactive oxygen species in BT-474 and HCC-1954 cells after treatment. The results showed that both H2-18 and GDC-0941 could increase the level of reactive oxygen species in both of the two cells. Compared with the single use group, the H2-18/GDC-0941 combined group could not further improve the activity of reactive oxygen species. Similarly, the level of intracellular reactive oxygen species in the H2-18/Saracatinib combined group was not higher than that of the H2-18 single use group. We used the HCC-1954 breast cancer bearing mice model to study the antitumor effect of the above drugs. The results showed that H2-18 alone and GDC-0941 alone can effectively inhibit the growth of transplanted tumor.
【學(xué)位授予單位】：第二軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類號(hào)】：R737.9

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5 葛廣哲;樹，

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