我們已證實經檳榔子誘導自體吞噬的成分（ANE 30-100K）長期刺激後會使不同的腫瘤細胞自體吞噬活性與抗壓性增強，使其無論是在體外無血清的環境或裸鼠體內均有較佳的生長，若其自體吞噬作用受到抑制，無論在體內或體外的生長優勢均會受到抑制。最近我們更觀察到經檳榔子成分刺激過的食道癌細胞在裸鼠體內的生長，對CQ或3-MA的治療明顯地比未經刺激的細胞更為敏感。本計畫除了採用氯?與3-甲基腺嘌呤外，亦將採用atg5 shRNA來達成特異性的自體吞噬抑制效果；並且使用口腔癌細胞進行同樣的分析，期能提供更完整的證據支持經檳榔成分長期刺激後的腫瘤細胞，對自體吞噬的抑制特別敏感。 Autophagy inhibition (AI) has been highlighted as a new strategy for treating various types of cancers; however, due to the dual roles of autophagy in tumor development, which types of cancers are suitable for such treatment remain currently to be confirmed. Our previous studies revealed that tumor cells chronically stimulated with the 30-100 kDa fraction of areca-nut-extract (ANE 30-100K) exhibited stronger resistance against stressed environments including serum-free (SF) conditions and cisplatin (DDP) through elevation of higher autophagy activity. In addition, the growth of esophageal carcinoma CE81T/VGH cells in both culture medium and nude mice was significantly enhanced by chronic ANE 30-100K stimulation (CAS), which could be attenuated by autophagy inhibitors chloroquine (CQ) and 3-methyladenine (3-MA). Meanwhile, both inhibitors synergistically enhanced anti-tumor effects of DDP on CAS-treated cells both in vitro and in vivo. These findings raised a possibility that CAS-treated tumor cells could become autophagy addicted and more sensitive to AI compared to naïve cells. Our recent data supported this possibility: the growth of CAS-treated CE81T/VGH cells in nude mice was more sensitive to either CQ or 3-MA monotherapy than control CE81T/VGH cells. This project firstly is to compare the in vitro and in vivo effects of both single and combined treatments of AI and DDP on CAS-treated and untreated CE81T/VGH cells, as well as investigation of the underlying mechanism of DDP resistance. In addition to CQ and 3-MA, we will knock down the expression of autophagy-related atg5 gene by using short hairpin RNA (shRNA) strategy to achieve specific AI purpose, because studies have shown that some non-specific activities of CQ and 3-MA are irrelevant to AI. As the carcinogenic areca nut (AN) is still popular in our country and closely correlated with incidences of esophageal and oral squamous cell carcinomas, we also plan to use the same strategies to address whether therapeutic advantages of AI observed in CE81T/VGH cells can be applied to oral squamous cell carcinoma SCC15 cells in this project. Finally, this project is expected to provide translational reference especially for carcinoma cells encountered with CAS.
