合併Endostatin和PEDF抗血管新生因子應用於原位性肝細胞癌的基因療法

Antiangiogenic Gene Therapy on Orthotopic Hepatocellular Carcinoma Using Combination of Endostatin and Pigment Epithelium-Derived Factor

陳雪芳(Suit-Fong Chan)

指導教授 ： 黃麗華

國立臺灣大學/醫學院/微生物學研究所/碩士(2007年)

https://doi.org/10.6342/NTU.2007.00225

摘要

肝細胞癌通常有會較多血管異常增生的現象，所以很適合使用抗血管新生療法治療。然而，如果只使用單一種的抗血管新生因子對抗單一種的促血管新生因子，可能會因為腫瘤改合成另一種促血管新生因子而失效。尤以較後期的癌症，腫瘤會同時分泌多種的促血管新生因子以利於血管新生，所以在抗血管新生的策略中，最好同時能應用兩種或兩種以上的抗血管新生因子，比較能達到好的療效。

在這個研究中，我們嘗試使用Endostatin (ED)和Pigment Epithelium-Derived Factor (PEDF) 兩種抗血管新生因子治療原位性肝癌。我們應用了帶有小鼠Endostatin和人類PEDF的重組腺病毒載體。這些由重組腺病毒載體所表現的ED和PEDF在細胞內或動物體內都先經測試顯示具有生物活性。隨後我們同時打入帶有ED和帶有PEDF的重組腺病毒治療原位性肝細胞癌。動物實驗結果顯示，合併兩種不同的因子在治療腫瘤中達到加成性的效果。從組織免疫化學染色也發現，合併組相較於單一治療組可以加成性地降低腫瘤內的血管數目。抑制腫瘤的血管新生可以阻斷氧氣和養分，使腫瘤細胞凋亡。

接下來，我們也稍微觀察了兩種因子對於VEGF mRNA 表現的影響。不管是ED或PEDF都會使VEGF mRNA表現量降低；而合併治療組更放大了對於VEGF表現量的抑制。我們也意外的發現，合併治療組能誘導出比單一治療組更高量的腫瘤浸潤CD8+ T細胞。我們推論由於抗血管新生所導致的腫瘤細胞凋亡增加了腫瘤抗原的釋出，而誘導更高量的CD8+ T細胞。

關鍵字

抗血管新生 ； 基因療法 ； 原位性肝細胞癌

並列摘要

The hepatocellular carcinoma is considered as one of the suitable targets for anti-angiogenic approaches due to its highly neovascularization. However, anti-angiogenic therapies aimed at single target can be neutralized by up-regulation of other pro-angiogenic factor. The fact that redundant angiogenic factor can be up-regulated by tumors in advanced stage of carcinoma suggests that the angiostatic strategies may require a combination of multiple anti-angiogenic factor.

In this study, we assessed the therapeutic effects of combination therapy with two anti-angiogenic factors, Endostatin (ED) and Pigment Epithelium-Derived Factor (PEDF), on established orthotopic liver tumor. We used recombinant adenoviral vector carrying genes encoded mouse ED or human PEDF. The adenovirally expressed endostatin and PEDF were biologically active, as demonstrated in vitro and in vivo. We performed an in situ tumor therapy with co-administration of ED and PEDF by recombinant adenoviral vectors. The results of animal experiments indicated that the combination of both factors provided a synergistic effect on tumor regression. The immunohistochemistry staining of endothelial cell marker in tumor regions also revealed that the combined therapy synergistically reduced the tumor vascularization. The inhibition of tumor vascularization which starved tumors from oxygen and nutrients induced apoptosis in tumor regions.

Furthermore, we examined the impact of both factors on Vascular Endothelial Growth Factor (VEGF) mRNA level. A down-regulation of VEGF mRNA level was observed in response to ED or PEDF treatment. Moreover, the combined ED/PEDF treatment was able to augment the inhibition of VEGF expression. Surprisingly, the combination therapy also induced a higher level of tumor-infiltrating CD8+ T lymphocytes. We infer that the apoptosis induced by anti-angiogenesis gave rise to a bystander effect which activated higher levels of CD8+ T cells.

Based on this hypothesis, we combined immunotherapy and anti-angiogenic therapy to improve the therapeutic effect on orthotopic hepatocellular carcinoma. We performed a combination of immunotherapy and anti-angiogenic therapy by adenoviruses-mediated gene transfer of ED, PEDF, IL-12, and GM-CSF simultaneously on the orthotopic hepatocellular carcinoma. The “four-in-one” therapy exhibited a synergistic effect on tumor regression compared to the immunotherapy group (GM-CSF + IL-12) or anti-angiogenic group (ED + PEDF) at the same dose. Our findings suggest that the co-action between two different treatment strategies can more effectively reduce tumor burden compared to single treatment strategy. Hence, the combination strategy between immunotherapy and anti-angiogenic therapy renders a promising treatment for hepatocellular carcinoma.

並列關鍵字

antiangiogenic gene therapy ； Endostatin ； Pigment Epithelium-Derived Factor

參考文獻

1. Hoofnagle JH. Hepatocellular carcinoma: summary and recommendations. Gastroenterology 2004;127:S319-23.

2. Deuffic S, Poynard T, Buffat L, Valleron AJ. Trends in primary liver cancer. Lancet 1998;351:214-5.

3. Marrero JA. Hepatocellular carcinoma. Curr Opin Gastroenterol 2006;22:248-53.

4. Schafer DF, Sorrell MF. Hepatocellular carcinoma. Lancet 1999;353:1253-7.

5. Otto G, Heise M, Moench C, Herber S, Bittinger F, Schuchmann M, Hoppe-Lotichius M, Pitton M. Transarterial chemoembolization before liver transplantation in 60 patients with hepatocellular carcinoma. Transplant Proc 2007;39:537-9.