Hemiasterlin衍生物BF65與Akt抑制劑MK-2206協同抑制卵巢癌細胞生長

Abstract

合併用藥在癌症治療上扮演重要的角色，常結合分子標靶藥物與毒殺細胞藥物的共同使用藉由降低劑量以達到降低毒性及副作用的目的，提高藥物抗癌作用。訊息傳導路徑PI3K/Akt/mTOR的異常會導致細胞不正常的生長及癌化的發生；在癌症細胞中此路徑的過度活化亦容易導致腫瘤細胞對化學治療產生抗藥性。 本論文測試一系列海綿胜肽成分hemiasterlin的全合成衍生物，包括BF65、BA32、LC15、LC17、LC20、LC22、BA35與BA36。實驗結果發現大部份化合物當具有(R)(S)(S)組態對於卵巢癌細胞(SKOV3)及前列腺癌細胞(PC-3)都有生長抑制的作用，並使細胞週期停滯於有絲分裂期(mitosis phase);其中，以BF65具有最強的抑制細胞生長作用。實驗發現BF65可擾亂細胞骨架微管的形成，並造成抗細胞凋亡蛋白Bcl-2的磷酸化，Mcl-1表現下降、PARP斷裂，而產生細胞凋亡，並可藉由抑制Akt、ERK、p38來抑制細胞保護作用，抑制細胞生長與增生，並且在長時間處理後造成細胞DNA的損傷。 MK-2206為Akt (又稱蛋白質激酶B，PKB)的抑制劑，可抑制Akt的磷酸化，並抑制蛋白質生合成及癌細胞生長。本論文將BF65與MK-2206合併使用，發現MK-2206可以提升BF65抑制微管聚合的作用及提高SKOV-3卵巢癌細胞的死亡率，且此併用組合可同時造成細胞週期G0/G1及G2/M的停滯。進一步探究其分子作用機轉發現，此組合可抑制Akt及其下游受質mRNA轉錄抑制因子p70S6K與4EBP1的磷酸化，進而阻礙蛋白質的生合成。此外，MK-2206可以提高因BF65所造成的Bcl-2磷酸化，而BF65則可降低MK-2206誘導Bcl-2蛋白的效應。另外，兩者併用對於Mcl-1下降、PARP斷裂、DNA損傷標記 r-H2AX的產生都有明顯的協同作用。而兩者併用可更進一步的破壞微管蛋白，而BF65對於p38、ERK的抑制能力可以解決MK-2206由於抑制Akt而造成MAPK路徑活化回饋。這些機制可能是BF65與MK-2206合併使用能產生協同作用，提高細胞死亡率的原因。

The concept of combination treatment plays an important role in cancer therapy. Molecular targeted agents are frequently combined with traditional cytotoxic drugs at lower doses than those required for single treatment to reduce the toxicity and side effects, and to enhance the anticancer effect. Abnormal activation of the PI3K/Akt/mTOR signaling pathway may lead to aberrant cell growth and contribute to tumorigenesis. In addition, overactivation of this signaling pathway is also associated with resistance to chemotherapy. In this study, we screened a series of synthetic hemiasterlin derivatives including BF65, BA32, LC15, LC17, LC20, LC22, BF35 and BA36, and discovered that compounds in the (R)(S)(S) configuration could inhibit the growth of an ovarian cancer cell line SKOV3 and a prostate cancer cell line PC-3, and arrest cells in mitosis. BF65 is the most potent hemiasterlin derivative among all the compounds tested. BF65 disrupted microtubule assembly, induced phosphorylation and inactivation of the pro-survival protein Bcl-2, decreased the level of another pro-survival protein Mcl-1 and caused PARP cleavage, leading to apoptosis. BF65 also inhibited ERK, p38 and Akt, which can stimulate cell proliferation upon activation, and induced DNA damage in SKOV3 cells after long-term treatment. MK-2206 is an allosteric inhibitor of Akt (also called serine/threonine kinase B, PKB) and can inhibit Akt phosphorylation/activation, protein synthesis and cancer cell growth. We show here that combination treatment of BF65 and MK-2206 synergistically reduced SKOV3 viability. MK-2206 enhanced microtubule depolymerization effect of BF65 and the combination treatment resulted in G0/G1 and G2/M cell cycle arrest at the same time. Study of the underlying molecular mechanisms revealed that BF65/MK-2206 inhibited phosphorylation of Akt and its downstream effectors p-P70S6K and p-4EBP1, thereby downregulated protein synthesis. Furthermore, MK-2206 potentiated Bcl-2 phosphorylation induced by BF65, and BF65 caused phosphorylation and inactivation of Bcl-2 induced by MK-2206. The combination also caused a significant reduction in Mcl-1 protein, more serious PARP cleavage, and induction of the DNA damage marker r-H2AX. BF65/MK-2206 combination also caused more significant microtubule disruption and the inhibitory effect of BF65 on p38 and ERK blocked the activation of MAPK pathway resulting from a feedback of Akt inhibition by MK-2206. These molecular changes may explain why BF65 can synergize with MK-2206 to inhibit ovarian cancer cell growth and to enhance cytotoxicity.