開發PE092002肝癌治療新藥

Newly Developed PE092002 as Molecular Therapy for Hepatocellular carcinoma (HCC)

謝玲珊(Ling-Shan Tse)

指導教授 ： 鄭幼文

臺北醫學大學/藥學院/藥學系(碩博士班)/碩士(2013年)

摘要

中文摘要

Hepatocellular carcinoma, HCC即原發性惡性肝腫瘤，為目前最常見的肝癌類型。全球男性癌症十大死因之中，肝癌排行第二名,女性則排行第六名。而根據台灣衛生署公佈2010年及2011年的十大死因之中，肝癌在所有癌症死因中排行第二名，屬台灣之國病。然而標靶治療藥物於晚期肝癌的治療成效經長期的努力雖已漸露曙光，但礙於差強人意的療效但副作用低與病患耐受性佳的特性，亦要合併使用化學藥物或是併用多重標靶藥物。如臨床上唯一被認可可以治療肝癌的藥物Sorafenib，的確可以使肝腫瘤縮小，但只有2至3%，而整體存活時間的延長也只差強人意。因此研發更有效但副作用較低的肝癌用藥並可預防早期肝癌術後復發的用藥是急需要的。根據本研究將人類肝癌細胞株 (Hep3B及HUH-7)以皮下注射的方式分別進行小鼠異位移植實驗，於引發肝腫瘤的產生後，發現雖然Hep 3B 及HUH-7皆是以人類肝癌細胞，而且從病理切片中得知兩者皆有高度的有絲分裂，但不論由外觀及腫瘤剖開後之內觀皆可以看到其腫瘤的型態有明顯的差異。由於肝臟是血管以及血流量最多之臟器之一，兩種肝癌細胞所誘發出來的腫瘤特性相較之下，Huh-7所誘發的腫瘤較適合探討血管新生之抗癌機制。因此，在進行以下實驗時，先選用了huh-7之腫瘤進行實驗。在本研究的結果顯示連續管餵給予5mg/kg的PE092002其TGD的效果就與腹腔注射4.986mg/kg Docetaxel相近；連續管餵給予1mg/kg的PE092002其TGD的效果甚至比連續管餵給予82.08mg/kg Sorafenib好。因此我們推斷PE092002在雄性小鼠的有效劑量為5mg/kg。再以現有的數據，初步計算出其ED50為8.4664 mg/kg。由於雄小鼠口服其LD50為100mg/kg，因此，雄性小鼠之治療性指數(therapeutic index(TI))為11.81。另外，在離體細胞實驗當中我們發現PE092002會使Huh-7細胞株Bcl-2 家族中的Bid表現量上升並使Bcl-2表現量下降，使之有apoptosis的現象。同時，PE092002會使Huh-7停留在G2/M期，並促進Cyclin B1及CDK1並抑制tubulin polymerization promoting protein(TPPP)的表現量，使紡錘體無法形成。另外，PE092002亦會促使MAPK Kinase(ERK1/2、p38及JNK)與AKT之蛋白質磷酸化，此作用轉機和市面上之去穩定化的藥物 (microtubule destabilizing agents)—Vinorelbine相似。與此同時，由之前的kinase panel結果得知PE092002會使insulin receptor產生抑制的效果，而在本研究當中，當Huh-7經處理PE092002後也有抑制IGF-1R之活化的情形。由於在生成的同時IGF-1R會與各種的Pro-angiogenic proteins產生交互作用， 其中一個Angiogenic growth factors為VEGF。因此PE092002除了調控細胞分裂之外可能亦具有anti-angiogensis 的作用。根據本研究結果以及綜合前人的資料，PE092002是一個令人期待的化合物。

關鍵字

肝腫瘤 ； 異位移植 ； 去穩定化的試劑

並列摘要

Abstract
Primary malignant liver tumor is the most common type of liver cancer and the lending cause of cancer-related death worldwide (male ranked second and female ranked sixth). Besides, HCC is the second largest cause of cancer-related death in Taiwan. HCC had limited treatment option. However, after long-term efforts, molecular target drugs show effectiveness against the disease. Although molecular target drugs have lower side-effect and good tolerability, they still are not effective enough to use along and have to combine with chemical drugs as a treatment. For example , Sorafenib, even FDA proved it can cure HCC and shrink the liver tumor, it has only 2-3% effectiveness. Besides the overall survival time prolonged only passably. Therefore, development of a high effective and low side-effect anti-HCC drug is must and pressing. In this study, we injected human hepatoma cell line (Hep3B ,HUH-7) subcutaneously in CB17 SCID mice to induce tumor. Although we found that both tumors produced high degree of mitosis, both tumors obviously were different in appearance and pattern. Since liver is one of organs which has the most fully blood vessels distributed and highly blood flow, with respect to the tumor induced by Hep 3B and HUH-7, HUH-7 tumor has higher angiogenic amount potential more than Hep 3B tumor. Therefore, we chose HUH-7 to continue the following study. The results showed that when mouse were orally-administered drugs PE092002 5mg/kg/day , its tumor growth delay(TGD) was similar to Docetaxel 4.986mg/kg ip once. Moreover, when mouse were orally-administered drugs PE092002 1mg/kg/day, its tumor growth delay (TGD) was much better than orally-administered drugs Sorafenib 82.08mg/kg/day. Therefore, we assumed the effective dose of PE092002 as 5mg/kg at male mice. as prior study ,we found that the LD50 of PE092002 on male mice was 100mg/kg. Then we calculated the ED50 and therapeutic index (TI) as 8.4664/mg/kg and 11.81. In our in vitro studies of PE092002 in Huh-7, we found that PE092002 could upregulate the Bid and down regulate the Bcl-2 which are in the Bcl-2 family to induce apoptosis. At the meanwhile, PE092002 could also cause G2/M arrest, upregulating Cyclin B1 and CDK1. Furthermore PE092002 could also downregulate the expression of tubulin polymerization promoting protein (TPPP) to suppress the spindle formation. PE092002 could also increase the phosphorylation of MAPK Kinase (ERK1/2、p38及JNK) and AKT in Huh-7. Those effects showed the mechanism of PE092002 is similar to the microtubule destabilizing agents—Vinorelbine. According to the Kinase panel result, PE092002 is able to suppress the insulin receptor. In our study, also found PE092002 could downregulate the phosphorylation of IGF-1R in Huh-7. Because of IGF-1R would cause interaction with some angiogenic growth factors, like VEGF. So, we also assumed PE092002 may not only be able to cause apoptosis but also may cause anti- angiogenesis effect. In conclusion all the results of PE092002, PE092002 is a prospective compound.

並列關鍵字

HCC ； xenograft ； microtubule destabilizing agents

參考文獻

參考文獻(References)

.

Alexia C, F. G., Lasfer M, Schweizer-Groyer G, Groyer A. (2004). "An evaluation of the role of insulin-like growth factors (IGF) and of type-I IGF receptor signalling in hepatocarcinogenesis and in the resistance of hepatocarcinoma cells against drug-induced apoptosis." Biochem Pharmacol. 68(6): 1003-1015.

Anelli A, A., Youngson B, Rosen PP, Bborgen PI (1995). "Mutations of the p53 in male breast cancer." camcer 75: 2233-2238.

Asnaghi L, C. A., Bevilacqua A, D'Agnano I, Gatti G, Canti G, Delia D, Capaccioli S, Nicolin A. (2004). "Bcl-2 phosphorylation and apoptosis activated by damaged microtubules require mTOR and are regulated by Akt." Oncogene. 23(34): 5781-5797.