| 摘要: | 膀胱癌是全球第10大常見的惡性腫瘤，根據統計2020年全球約有 573,000例新增病例和213,000例死亡病例。隨著醫學的發展，在美國膀胱癌患者的五年存活率至今已提高至約80%，然而其中大約25%的病人屬於容易轉移的肌肉侵襲性膀胱癌，而一旦進展為轉移型膀胱癌，即使經過化療五年存活率仍只有約5%，因此當前臨床上迫切需要開發新的治療藥物。
本研究利用來自石蒜科 (Amaryllidaceae family) 植物的生物鹼—石蒜鹼 (lycorine)，主要探討其在膀胱癌細胞中的抗癌功效以及研究潛在的藥理機制。首先，我們發現lycorine對兩種泌尿系統癌症—腎細胞癌和膀胱癌細胞都具有抑制增殖作用，並且會減少數種與細胞存活相關的蛋白激?之表現。藉由MTT assay我們證明lycorine對膀胱癌細胞具有較高之選擇性，在膀胱癌和正常膀胱細胞的IC50 分別為2.47和 8.99 μM，而對腎臟癌細胞則無選擇性。此外lycorine在膀胱癌中會造成更明顯的Sub-G1 phase堆積和凋亡相關蛋白的活化，顯示出lycorine在膀胱癌中的細胞凋亡更為顯著，因此我們便選擇膀胱癌細胞作為後續主要研究之模型。
在細胞週期分析中，我們發現lycorine會使腎臟癌細胞停滯於G2期，而在膀胱癌中則會延遲S 期的進程，同時也觀察到其會促使磷酸化ATM的增加，意味著lycorine具有DNA 損傷能力進而引發細胞週期停滯。另一方面，我們也發現lycorine不會活化細胞自噬，然而當以Atg5-knock out的方式抑制細胞自噬時，原本lycorine誘導之細胞凋亡會大幅減少，代表自噬作用仍有參與在lycorine引發的細胞凋亡中。上述研究結果表明了 lycorine在細胞凋亡、細胞週期、細胞自噬方面的藥理作用，也顯示出lycorine極具潛力發展為膀胱癌的治療藥物。
Bladder cancer is the 10th most common malignancy worldwide, with approximately 573,000 new cases and 213,000 deaths in 2020. Since the 1970s, the five-year survival rate for those diagnosed with bladder cancer in the United States has improved to be about 80%. However, 25% of patients have muscle-invasive bladder cancer (NIBC) and either present with or later develop metastases, which accompanies by a five-year survival of ~5%. Therefore, it is an urgent clinical need to develop novel therapeutic strategy to bladder cancer.
Here, we demonstrated the anticancer effect of lycorine, a natural alkaloid extracted from plants of Amaryllidaceae family, and investigated its underlying mechanisms in bladder cancer. First, we found that lycorine exhibited anti-proliferative activity in two urologic cancer cell lines: renal cell carcinoma and bladder cancer. Protein array also showed that lycorine attenuated phosphorylation of several kinases which functions as critical regulators of cell survival and proliferation. By MTT assay, we demonstrated that lycorine had better selective cytotoxicity towards bladder cancer cells, with IC50 of 2.47 μM in RT112 cells and 8.99 μM in normal bladder cells, while it showed no selectivity in renal cancer. Moreover, lycorine induced apoptosis through the evidence of sub G1 accumulation and caspase activation, and the effect was more significant in bladder cancer. So, we decided to focus on the mechanistic study in bladder cancer in later research.
In addition, lycorine promoted cell cycle arrested at G2 phase in renal cancer, while inducing S phase retardation in bladder cancer cells. In the meantime, lycorine also increased the expression of phospho-ATM. Those results implied that lycorine possessed DNA damage ability that in turns triggered cell cycle arrest. On the other hand, lycorine couldn’t induce autophagy. However, Knockout of ATG5 decreased the apoptotic effects of lycorine, suggesting that lycorine-induced apoptosis can be counteracted by inhibition of autophagy. Collectively, our findings showed that lycorine had various effects in apoptosis, cell cycle regulation and autophagy. Those results suggest that lycorine could be a novel candidate for drug discovery in bladder cancer treatment. |
