探討新合成arylamide衍生物MPT0B640誘導細胞凋亡與合併etoposide呈現協同作用於人類非小細胞肺癌之體外及體內的作用機轉

MPT0B640, a novel synthetic arylamide derivative, induces cell apoptosis and displays synergistic anti-cancer activity with etoposide in human lung adenocarcinoma in vitro and in vivo

吳珏衡(Chueh-Heng Wu)

指導教授 ： 鄧哲明

共同指導教授 ： 潘秀玲(Shiow-Lin Pan)

國立臺灣大學/醫學院/藥理學研究所/碩士(2014年)

https://doi.org/10.6342/NTU.2014.10505

摘要

癌症已經蟬聯國人十大死因之首逾三十年，其中肺癌亦不論是在全球或是台灣，都位居癌症致死率之首。缺乏早期的確診工具使得疾病容易發展至末期才被偵測到，而當腫瘤已蔓延到手術無法切除的程度，則需要依靠全身性的化療藥物，或是標靶藥物進行治療。目前的標靶藥物雖然有效提升了肺癌患者的存活率，但仍然有部分病患發展出後天抗藥性，因此，新一代的抗癌藥物仍有待發展。

MPT0B640是一個具有arylamide結構的Hsp90抑制劑，本篇實驗發現其於Sulforodamine B (SRB) 和3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) 測試中，能有效抑制人類非小細胞肺癌A549細胞的增生與存活，並能在Hsp90 activity assay kit中以IC50 = 110.18 nM的低濃度抑制Hsp90的活性。觀察MPT0B640所影響的蛋白表現，發現其能夠以濃度及時間依存性的方式，降低Hsp90多方受質蛋白的表現，如抑制Akt而使Akt的活化態減少，並抑制下游蛋白p-GSK3b的表現；抑制EGFR與其磷酸化；抑制MEK，而進一步抑制K-ras突變所造成的MEK、ERK高度活化。由於PI3K/Akt與MAPK路徑會共同造成mTOR的活化，所以使用MPT0B640抑制這兩條路徑，則觀察到p-mTOR的減少；Src、FAK亦為Hsp90受質蛋白，會受到MPT0B640的作用而降解，並促使Src與FAK的活化減少。且已知Src、FAK活化會參與在細胞的移行作用，於是也進行cell migration assay，發現MPT0B640會濃度依存性地抑制A549細胞移行。在細胞週期方面，發現給予MPT0B640能夠使細胞週期被干擾，並使mitotic phase相關的蛋白累積。而觀察細胞凋亡相關的蛋白表現，發現MPT0B640能夠促進p53累積、內生性細胞凋亡因子caspase-3, -6, -7的活化與切割，以及PARP的切割。而silence p53能夠部分回復細胞存活率，所以p53在MPT0B640導致的A549細胞凋亡中也扮演一定的角色。在腫瘤異體移植之動物實驗則發現，口服給予25、50、100 mg/kg的MPT0B640，皆能較控制組，延遲腫瘤生長天數，並於腫瘤組織中看到受質蛋白Akt被抑制的情形。

另一方面，我們亦將MPT0B640與治療非小細胞肺癌的傳統化療藥物taxol、gemcitabine、etoposide合併使用，並測定combination index (CI) value，發現MPT0B640能以低濃度增強三種傳統化療藥物的細胞毒殺作用。進一步觀察DNA損傷與細胞凋亡的蛋白表現，則與藥物合併使用產生的毒殺效果一致，MPT0B640能夠最明顯地與etoposide產生協同性毒殺作用，其次是gemcitabine，最後則是taxol。於是探討MPT0B640和etoposide合併使用產生協同作用的機轉，由觀察DNA修復及細胞週期相關的蛋白表現，發現MPT0B640可能藉由抑制Chk1及其活化，來干擾etoposide作用後所產生的細胞週期停滯及DNA修復。其中，DNA修復蛋白Rad51在此具有一定重要性；過度表現Rad51的A549細胞，能夠部分重建在兩藥合併使用下的細胞存活率。最後，於動物實驗亦觀察到兩藥併用時，抑制腫瘤生長的效果被增強。總結來說，MPT0B640能夠於單用時抑制多方Hsp90受質蛋白，因而促使A549細胞凋亡，合併它藥使用時，也能以低劑量增強藥物的效果，因此，MPT0B640可望成為有潛力的肺癌治療候選藥物。

關鍵字

非小細胞肺癌 ； 細胞凋亡 ； 協同作用

並列摘要

Lung cancer has been the leading cause of cancer-related death worldwide. Although current targeted therapy improves the survival rate of lung cancer patients, some of them, however, develop secondary resistance. Therefore, new drugs and mechanisms of anti-cancer therapies are urgently needed to be discovered.

The anti-cancer activities and mechanisms of action of a newly synthesized Hsp90 inhibitor, MPT0B640, were evaluated in this study. First, SRB and MTT assays revealed the antiproliferative activity and cytotoxicity of MPT0B640 in human lung adenocarcinoma A549 cells. Using Hsp90 activity assay kit, we discovered that MPT0B640 exerted a potent inhibitory effect on Hsp90 activity (IC50 = 110.18 nM). Next, MPT0B640 induced multiple Hsp90 client protein degradation. For example, MPT0B640 inhibited Akt and its activation, and further inhibited the activation of Akt downstream molecule, GSK3b. And then, depletion of MEK by MPT0B640 led to the abrogation of MEK/ERK hyperactivation. With a simultaneous inhibition of PI3K/Akt and MAPK by MPT0B640, we observed a decrease in mTOR phosphorylation. Src and FAK were also degraded upon MPT0B640 treatment. And the reduced phosphorylated form of Src and FAK were also observed. Because Src and FAK activations are known to be involved in cell migration, we investigated the migratory ability of MPT0B640-treated A549 cells, and found that cell migration was inhibited by MPT0B640. Next, we examined the effect of MPT0B640 on cell cycle. By disrupting cell cycle, MPT0B640 led to the mitotic protein accumulation in 24 hrs. With respect to A549 cell apoptosis, we found p53 accumulation, caspase-3, -6, -7 cleavage and PARP cleavage induced by MPT0B640. Moreover, A549 cells with transient p53 knockdown, displayed a partial reverse in cell viability, indicating the role of p53 in MPT0B640-induced A549 cell apoptosis. Finally, mice orally administrating 25, 50, 100 mg/kg MPT0B640, showed tumor growth delay in A549 tumor xenograft. And the suppression of a Hsp90 client, Akt, was seen in tumor xenograft tissues.

We next combined MPT0B640 with taxol, gemcitabine, etoposide, and evaluated the drug combinatorial effect, respectively. Cell viability assay and protein analysis showed that MPT0B640 enhanced the cytotoxicities of three conventional chemotherapeutic agents. Since the strongest synergistic effect (CI = 0.6) on MPT0B640 and etoposide combining group, we further discovered the drug combinatorial mechanisms on DNA damage response, and found that MPT0B640 enhanced etoposide cytotoxicty by disrupting the etoposide-induced cell cycle arrest and DNA repair mechanism. MPT0B640-induced depletion of checkpoint protein, Chk1, abrogated the checkpoint activation, and interrupted with G2/M related protein. Rad51, a homologous recombination protein which involved in DNA DSBs repair, was also inhibited in combination treatment. Cells overexpressing Rad51 became more resistant to MPT0B640 + etoposide treatment. Finally, the synergistic effect of MPT0B640 + etoposide on tumor growth inhibition was found in A549 xenograft models. And DNA damage markers were elevated in combination group in tumor xenograft tissues. Taken together, MPT0B640 may become a novel candidate for targeted therapy or a new strategy in combination with conventional drugs for lung cancer treatment.

並列關鍵字

NSCLC ； apoptosis ； synergism

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