探討 Metformin 和Aspirin 的結合對腫瘤的形成和成長反應的影響

Abstract

背景與研究目的：近年的研究發現 Metformin 有抗腫瘤的效應；而Aspirin 運用其抗發炎能力和對其路徑下游的代謝反應，也有抗腫瘤的效果。依照之前研究發現 Metformin 可以抑制 PI3K/AKT/mTOR 代謝路徑上的效果，而在有些的實驗上 Aspirin 可以減緩腫瘤細胞成長速度，原因也可能是作用在 PI3K/AKT/mTOR得效果，本研究探討合併使用了 Metformin 和 Aspirin 是否可以減緩早期低細胞數的腫瘤成長速度，以達到減緩少量細胞發生後的實質腫瘤體積的體積，和早期的轉移的發生。 材料與方法：實驗中利用腫瘤細胞株為 4T1；小鼠品系為BALB/cByJNarl。進行動物和細胞實驗，細胞實驗上先用MTT觀察藥物對細胞活性的影響，再用western探討可能的機制，實驗分成 Control 組、 DMSO 組、Aspirin 2mM 組、Metformin10mM 組、Metformin 20mM 組、Metformin 10mM+Aspirin 2mM 組、Metformin 20mM+Aspirin 2mM 組觀察細胞活性和 PI3K/AKT/mTOR 蛋白表現，動物實驗部份，利用 BALB/cByJNarl 母鼠先餵食含 Metformin 和 Aspirin 的水 10 天後種殖癌細胞當作原位腫瘤，接著依照時間點的不同種殖第 2 次細胞當作不同時間轉移，並觀察轉移的發生率和腫瘤的大小。 結果：本實驗的結果顯示在細胞實驗同時加入了 Metformin 和 Aspirin 會比只單純加入 Metformin 更能降低細胞活性，而且 Aspirin 可以增強 Metformin 的抑制癌細胞活性，而在 western 中 Metformin 和 Aspirin 會比只單純加入 Metformin 或Aspirin 增加抑制了 PDK1 蛋白質活性，動物實驗中腫瘤的成長體積上，有餵兩種藥的組別腫瘤體積比對照組的腫瘤體積還要小，且也產生了統計上的顯著差距，而生存率分析上面有餵藥的小鼠生存率，會比控制組還要長也有顯著差距。而在第二顆腫瘤上有餵藥的發生率是比沒有餵藥的還要低，而且腫瘤生長速度也比較慢，這邊也使用 NCBI 上的微陣列資料分析，經過分析後比對出有跟 PDK1有關的基因，發現代謝路徑上 PI3K/AKT/mTOR 基因表現有達顯著差異，動物實驗上預防轉移的結果與微陣列上的分析符合，免疫染色上，控制組的 ki-67 染色表現量比用藥組還要高，而 PI3K 和 PDK1 的染色對照組也比實驗組高，總和上述結果是符合腫瘤大小表現。 結論：合併 Metformin 和 Aspirin 的藥物治療確實會比只單純只含有一種藥物的治療有效，而在細胞實驗結果原因是 Aspirin 可能會增強 Metformin 抑制癌細胞的成長，而 PI3K/AKT/mTOR 蛋白質在合併 Metformin 和 Aspirin 也會被抑制，動物實驗中合併 Metformin 和 Aspirin 藥物治療會降低原位腫瘤的形成和第二顆腫瘤的發生機率。

Background and Purpose: Recently studies have shown that Metformin has an anti-tumor effect. Aspirin has anti-inflammatory abilities and can influence downstream metabolic reactions, and it also has an anti-tumor effect. According to previous studies, Metformin can also inhibit the effects of PI3K/AKT/mTOR metabolic pathways. And Aspirin can slow the growth rate of tumor cells in some experiments and may also be affected by PI3K/AKT/mTOR. The main purpose of this study is to investigate the effect of antitumor growth rate abilities in early stage cancer and early stage metastasis when treated with Metformin and Aspirin. Materials and Methods: Female BALB/cByJNarl strain mice and cancer cell lines 4T1 were used in this study. In vitro study, the viability of the cells and their probability mechanisms cells were tested by using MTT assay after drug treatment. Western blotting, respectively the experiment was grouped by the following protocols: control group, DMSO group, Aspirin 2mM group, Metformin 10mM group, Metformin 20mM group, Metformin 10mM+Aspirin 2mM group and Metformin 20mM+Aspirin 2mM group, to test the cell’s viability and PI3K/AKT/mTOR protein expression. In vivo study, mice were treated with drugs 10 days before the start of the experiment. The first implanted tumor was referred to as the in situ tumor. And the later implanted was referred to as the metastasis tumor. The probability of the metastasis tumor formatting and tumor growth rate were observed. Results: The in vitro study shows that the Metformin combined with Aspirin treatment can inhibit a cell viability more than just a solely Metformin treatment. In the resort of western blotting, Metformin combination with Aspirin treatment can down regulate the PDK1 expression more than just using Metformin or Aspirin alone. In vivo study, animals’ drug-treated tumor volumes are statistically smaller than that of the control group. The survival rate of the treatment group had a significantly higher difference from that of the control group. In the metastasis model, the treatment group’s second tumor formatting rates and growth rates were lower than of the control group. This discrepancy in growth and formatting rates can be explained by the used Microarray data analysis, where a PDK1-related gene was chosen. The Microarray data demonstrated a significant lower expression of the PI3K/AKT/mTOR gene expression in tumor bearing mice. This delay in metastasis result matches the Microarray analysis. Immunohistochemistry staining showed that the control group’s ki-67, PI3K, and PDK1 expression was higher than that of treatment groups. Conclusion: In vitro study demonstrated that combine Metformin and Aspirin treatment has more effect than treatment with Metformin or Aspirin alone. The reason might be that Aspirin can enhance the Metformin antitumor ability. The expression of PI3K/AKT/mTOR protein is down regulated in combine treatment. In vivo study Metformin and Aspirin combine treatment can significantly reduce in situ tumor growth rate and the second tumor formation.