利用B型肝炎病毒x蛋白基因轉殖肝癌小鼠模式評估新型的肝癌治療策略之研究

Abstract

肝癌是全世界造成死亡的癌症第三名，且盛行率近年來在歐、美國家呈倍數成長，因此發展新的預防及治療肝癌的方法，是迫切需要的。本研究的目的為利用一種具免疫力的原位肝癌模式-B型肝炎病毒x蛋白基因轉殖肝癌小鼠模式，其自發性生成肝癌、病理和基因變化相似於人類的肝癌，於臨床前測試評估兩種新的治療藥物，包括需要仰賴癌細胞中的端粒酶活性，才能進行病毒複製的溶瘤腺病毒(Telomelysin)和生物還原藥物Tirapazamine (TPZ)。 第一個治療肝癌的藥物是溶瘤腺病毒-Telomelysin，它具有能特別導致腫瘤細胞死亡的溶瘤效力。它的鑑別能力來自於利用人類端粒酶逆轉錄酶(hTERT)啟動子來驅動病毒的複製，而肝癌組織中有很高量的端粒酶表現，正常細胞則無。在原位肝癌模式中，Telomelysin對肝癌表現出強有力的溶瘤作用，卻不影響正常肝組織，且其安全劑量為1.25×108 PFU。從多次腫瘤注射Telomelysin後也發現，病毒能複製於具免疫活性的肝癌模式中。這個臨床前研究顯示，適當劑量的Telomelysin可應用於治療人類的肝癌，且其溶瘤效力於多次注射後依舊存在。 第二種的治療肝癌的藥物為生物還原藥物-TPZ。在缺氧的情況下，TPZ可以被活化並導致DNA斷裂，故能有效地殺死缺氧環境下的細胞，而不影響正常氧氣環境的細胞。而肝臟中肝癌的血液供應特色，使得肝癌成為TACE結合TPZ組合療法的理想目標，因為TACE可以有效地阻塞肝癌的動脈供血，而非腫瘤的肝組織依然可以接受肝門靜脈的血供，這使得肝癌能缺氧並活化TPZ的細胞殺能力。在HBx基因轉殖小鼠肝癌模式中，其肝癌能經肝動脈結紮動脈產生缺氧，並於結合TPZ劑量為3 mg/kg的治療下，可以實現90％以上的肝腫瘤壞死。這樣的結果支持TPZ與TACE能結合應用，對肝腫瘤產生高度的治療成效。而臨床上的開發計劃，若能基於此臨床前研究，將使TPZ和TACE成為有效治療肝癌的方法。 總結而論，我們成功應用了具免疫能力的原位HBx基因轉殖小鼠肝癌模式，來驗證兩種藥物對於新的肝癌治療方式之可行性。結果支持此兩種方法對於未來人類肝癌治療的效用。

HCC (hepatocellular carcinoma) is the third leading cause of cancer deaths worldwide, and the incidence of HCC remains high in Asian and is increasing in USA and European. The development of new treatments for effective therapies of HCC is urgently needed. The current study aims to preclinically evaluate two new treatment reagents, including the telomerase-specific replication-competent oncolytic adenovirus (Telomelysin) and the biological reductive drugs-Tirapazamine (TPZ), in the HBx transgenic HCC mouse model. This is an immunocompetent in situ orthotopic HCC model, with HCC developed spontaneously and pathologically and genetically similar to human HCC. The first anti-HCC strategy is the oncolytic adenovirus, Telomelysin, which was developed for virus-mediated preferential lysis of tumor cells. Its selectivity derived from a human telomerase reverse transcriptase (hTERT) promoter-driven active viral replication, which occurs in HCC with high telomerase activity but not in normal cells lacking such activity. In the orthotopic HCC model, Telomelysin showed a potent oncolytic effect on HCC but spared normal liver tissue. Dose escalation analysis identified a safety dose of 1.25 x 108 PFU for this model. The effect of multiple injections of Telomelysin was also evaluated in this immunocompetent HCC model. We found that the virus replicates in HCC after a second intratumoral injection despite an immune response induced by the previous injection. This preclinical study shows that Telomelysin can be used for treatment of human HCC at an appropriate dosage and that its tumor-killing activity persists after multiple injections. The second anti-HCC strategy is a bioreductive agent, TPZ. In the absence of oxygen TPZ can be activated and induces DNA breaks and effectively kill the hypoxia cells but spare the normoxia cells. In this aspect, the HCC growing in the liver becomes an ideal target for testing the combinational therapy by TACE (transarterial chemoembolization) and TPZ. TACE can effectively choke the arterial blood supply to HCC but the non-tumor liver tissues can still receive the portal vein supply, which makes the hypoxia and also the TPZ induced cytotoxic effect specific for HCC. We have tested this hypothesis for HCC in HBx transgenic mice, which is mainly supplied by arteries with hypoxia induced by hepatic artery ligation. Combination of TPZ and hepatic artery ligation clearly increases the toxicity of TPZ against liver. Most of our efficacy study can achieve over 90% tumor necrosis at 3 mg/kg. The results supported that the anti-tumor activities of TPZ and TAE are highly synergistic to each other. A clinical development program based on this preclinical work will follow to examine if TPZ and TAE would be an effective therapy for HCC. In summary, we have successfully applied the immunocompentent orthotopic HCC model of HBx transgenic mice to test the feasibility of two novel new reagents for treatment of HCC. The restuls provided critical information for their future application to human HCC.