探討 KMUP-1 在人類肝癌細胞中抗血管新生與抗增生之藥理作用及機轉

磷酸二酯酶抑制劑 (phosphodiesterase) 已被證實在許多種的腫瘤細胞中，可以藉由許多不同的訊息傳遞路徑，而達到具有抗血管新生 (anti-angiogenesis) 及抗增生的效果。在過去的研究中已證實 KMUP-1 具有磷酸二酯酶抑制的作用；在本次的研究當中，利用 KMUP-1 此藥理特性來研究投予 KMUP-1 至人類肝癌細胞後，是否也具有抗血管新生以及抗增生的作用。首先，利用細胞生長試驗 (MTT assay) 初步觀察 KMUP-1 對於腫瘤細胞生長抑制的程度；結果指出，KMUP-1 不論在正常氧壓 (normoxic condition) 或著低氧狀態 (hypoxic condition) 下都有抑制細胞生長的作用，不過此作用在正常氧壓下比較顯著。利用流式細胞儀分析細胞生長週期發現，不論在正常氧壓或著低氧狀態下，肝癌細胞在投予 KMUP-1 後會有大量細胞進行細胞凋亡，其效果與濃度和時間呈現正相關性 (concentration- and time-dependent)；此ㄧ結果則在給予 sGC 抑制劑 (10 μM ODQ) 或 cyclic GMP 競爭性抑制劑 (10 μM (RP)-8-pCPT-cGMP) 之後會被拮抗，顯示KMUP-1 造成細胞凋亡 (apoptosis) 是依賴 sGC/cyclic GMP/PKG 路徑的調節。另外，自細胞週期分析中發現 KMUP-1 還具有抑制細胞週期進行的作用，能使肝癌細胞的細胞週期被終止在G0/G1 時期。以西方點墨法分析指出，在正常氧壓及低氧情形下，KMUP-1 可以增加具有將細胞終止在 G0/G1 時期之蛋白質 p21CIP1/WAF1 及 p27KIP1 表現；同時也能夠抑制細胞要自 G0/G1 時期轉進 S 期時必要的蛋白質 cyclin D、CDK6 和 CDK4 的表現。 Hypoxia-inducible factor-1 (HIF-1) 是ㄧ個由HIF-1α 和HIF-1β 組成的異構雙體。在低氧狀況下，HIF-1α 會趨於穩定並能使腫瘤細胞利於存活及生長；除此之外，HIF-1α 對於處在低氧狀態下的腫瘤細胞之血液供應是ㄧ個關鍵的轉錄因子，因為其可以活化血管內皮生長因子 (VEGF) 表現，進ㄧ步促使血管新生，而讓腫瘤細胞可以獲得足夠的氧氣和養分。西方點墨法分析指出，低氧下，肝癌細胞在給予KMUP-1 後，肝癌細胞中HIF-1α 和血管內皮生長因子蛋白質的表現皆有顯著的被抑制。總而言之，KMUP-1 可以藉由活化cyclin kinase inhibitors (CKIs) 和抑制 cyclin/CDK 複合體的表現達到抑制腫瘤細胞生長；同時也能藉由依賴 sGC/cGMP/PKG 路徑的調節引起腫瘤細胞凋亡；同時在低氧下亦能抑制調控血管新生的轉錄因子HIF-1α 及血管內皮生長因子的表現。因此，本研究證實了KMUP-1 在肝癌細胞中的確具有抗腫瘤細胞增生和抗血管新生的作用。

關鍵字

KMUP-1 ；抗增生

並列摘要

Phosphodiesterase inhibitor had been proved on its anti-angiogenesis and anti-proliferation effects through numerous signal transduction pathway in various cancer cells. In this study, KMUP-1, a phosphodiesterase inhibitor, was investigated on its anti-angiogenesis and anti-proliferation effects in human liver cancer cell line Hep G2 . The cell growth inhibition ability of KMUP-1 was determined by MTT assay. The results indicated that KMUP-1, possessed a higher cell growth inhibition ability in normoxic condition than in hypoxic condition. Using flow cytometry techniques, we found that KMUP-1 induced apoptosis in a time- and concentration dependent manners under normoxic and hypoxic state, and these effects was dependent on sGC/cyclic GMP/PKG signal pathway. Notably, the effects were dramatically changed after 48 and 72 hrs exposured to KMUP-1. In addition, we also found that KMUP-1 could arrest cell cycle progression at G0/G1 phase. Western blotting analysis indicated that KMUP-1-induced cell cycle arrest may depend on increasing p21CIP1/WAF1 and p27KIP1 protein expression and on decreasing cyclin D/CDK6 or cyclin D/CDK4 complex expression. Hypoxia-inducible factor-1 alpha (HIF-1α), a component of HIF-1, is expressed in survived human tumors and renders cells grown under hypoxia condition. Besides, HIF-1 is a key transcription factor that regulate the blood supply through activity on the expression of vascular endothelial growth factor (VEGF), which promotes the angiogenesis. cause significant decrease on HIF-1α and VEGF protein expression. In summary, KMUP-1 could not only activate cyclin kinase inhibitors (CKIs) expression and inhibit cyclin D/CDK4 or 6 complex level for arresting hepatoma cells growth but also cause cell apoptosis dependent on sGC/cGMP/PKG signal pathway both under normoxic and hypoxic condition. In addition, KMUP-1 can reduce HIF-1α and VEGF expression under hypoxic condition. These findings implicate that KMUP-1 may be useful for anti-angiogenesis and anti-proliferation therapy in human liver cancer.The result from Western blotting analysis also showed that KMUP-1 could