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葫蘆素B誘導腫瘤細胞DNA損傷導致G2/M期細胞週期阻滯 / Cucurbitacin B induced DNA damage causes G2/M cell cycle arrest in cancer cells / Guo Jiajie.

English Abstract

Cucurbitacins are a classs of oxygenated tetracyclic triterpenoids from cucurbitaceous plants, including cucurbitacin B, cucurbitacin D, cucurbitacin E and so on. Recent studies showed that they demonstrated potent anti-cancer activities both in vitro and in vivo by inducing cycle arrest, autophagy, and apoptosis. Inhibition of STAT3 signaling pathway is generally considered as the main mechanism of Cucurbitacins’s actions. Cucurbitacin B (Cuc B) is ubiquitous cucurbitacin distributed in plantkingdom. It induces cell cycle arrest in many kinds of tumor cell lines. Cuc B could induce G2/M or S phase arrest depending the cell type while the detailed mechanisms of Cuc B induced cell cycle arrest remain to be clear. We investigated the effect of Cuc B on cell cycle in human lung cancer cell A549 cells at low-dose (from 50 to 200 nM) without affecting STAT3 pathway. Cuc B (50 to 200 nM) shows no significant cytotoxicity to A549 cells but dramatically induced G2/M phase arrest. Cuc B caused DNA double-strands breaks as indicated by the increasing expression of phosphorylation of H2AX at Ser-139 (γH2AX) and the positive result of comet assay. Cuc B treatment activated Chk1-Cdc25C-Cdk1 pathway via ATM Cuc B induced G2/M arrest could be reversed by ATM and Chk1 siRNA transfection. Cuc B also triggers ATM-activated p53-14-3-3-σ pathways, which could be reversed by ATM siRNA transfection. Furthermore, our results show that Cuc B induced high level of reactive oxygen species (ROS) formation in A549 cells, which could be inhibited by NAC. NAC pretreatment ix could block Cuc B induced γH2AX expression, and almost completely reversed G2/M phase arrest. In addition, NAC pretreatment also block Cuc B induced DNA damage response pathways. Thus, Cuc B induced DNA damage and G2/M phase arrest in A549 cells is due to increased intracellular ROS formation. Collectively, Cuc B induced DNA damage mediated by increasing intracellular ROS production, which activated ATM-Chk1-Cdc25C pathway joined with ATM-p53-14-3-3-σ branch, leading to the G2/M phase arrest. Beside, we also found that Cuc B, Cuc D, and Cuc E could induce DNA damage in MCF-7, MDA-MB-231, K562, THP-1, and B16 cells. Cuc B induced G2/M phase arrest in MCF-7, MDA-MB-231, and K562 cells. These results indicated that induction of DNA damage response might be a common effect of cucurbitacins. However, the biological significance of this effect need further studies to elucidate in future. In summary, present data provides new insights into the anti-cancer mechanisms of cucurbitacins by regulating the cell cycle arrest and thus sheds new research directions for exploring the molecular targets of Cuc B and related compounds.

Chinese Abstract

葫蘆素(Cucurbitacins)是一類高度氧化的四環三萜類化合物,依取代基的不同, 可分為 12 大類共 229 種化合物,主要分佈于葫蘆科植物中。目前研究已證實,該類化 合物在體內外都表現出良好的抗腫瘤活性,可誘導腫瘤細胞發生週期阻滯、凋亡、自噬 等現象。 葫蘆素 B(Cucurbitacin B, Cuc B)是自然界中含量較為豐富的葫蘆素類化合物, 可誘導多種腫瘤細胞週期阻滯。在不同細胞型上,Cuc B 可誘導細胞週期阻滯于 G2/M 或 S 期。我們研究了在對人肺癌 A549 細胞無明顯生長抑制條件下的 Cuc B 對人肺癌 A549 細胞周期阻滯的作用及其可能的調控機制。Cuc B 在低濃度下(50 到 200 nM), 對 A549 細胞無顯著的細胞毒性,但可顯著誘導 G2/M 期細胞阻滯。Cuc B 處理 A549 細胞 3 小時后,彗星電泳實驗中即可觀察到細胞核拖尾現象,此外,DNA 損傷的生物 標記物 γH2AX 的表達明顯升高,這提示細胞發生 DNA 雙鏈損傷。Cuc B 處理 A549 細 胞后,可激活 ATM 以及隨後的 Chk1-Cdc25C-Cdk1 信號通路。ATM 和 Chk1 的 siRNA 轉染均可逆轉 Cuc B 誘導的 G2/M 期阻滯以及 Cuc B 激活的 Chk1-Cdc25C-Cdk1 信號通 路蛋白表達的改變。Cuc B 引起的 A549 細胞週期阻滯的同時,ATM 下游的 p53-14-3-3-σ 旁路也被啟動,且也可被 ATM siRNA 所逆轉。此外,Cuc B 可以誘導細胞內活性氧 (Reactive oxygen species, ROS)的異常升高,產生的 ROS 可被 NAC 抑制。NAC 預處 理顯著抑制 Cuc B 誘導的 γH2AX 升高,以及 Cuc B 激活的 DNA 損傷響應通路,從而 逆轉 Cuc B 誘導的 G2/M 期阻滯。這提示 Cuc B 诱导的 ROS 的生成導致 DNA 損傷引起 vii 細胞週期阻滯。綜上所述,Cuc B 通過誘導 A549 細胞內 ROS 的異常升高導致 DNA 雙 鏈損傷,繼而活化 ATM-Chk1-Cdc25C 信號通路,以及 ATM-p53-14-3-3-σ 旁路,從而引 起 A549 細胞發生 G2/M 期阻滯。 此外,我們還發現葫蘆素 B、D、E 誘導 MCF-7、MDA-MB-231、K562、THP-1、 B16 細胞發生 DNA 損傷。Cuc B 可誘導 MCF-7、MDA-MB-231、K562 細胞發生 G2/M 期阻滯。這提示誘導 DNA 損傷可能是該類化合物的普遍作用,但該作用的具體的意義 仍需要更深入的研究。 綜上所述,本研究首次發現了葫蘆素類化合物誘導腫瘤細胞發生 DNA 損傷,通過 激活細胞週期檢驗點,誘導腫瘤細胞發生細胞週期阻滯,為闡明葫蘆素類化合物誘導週 期阻滯的作用機制提供了新研究方向。

Issue date





Institute of Chinese Medical Sciences




Cancer -- Treatment

癌症 -- 治療

Medicinal plants -- China -- Analysis

藥用植物 -- 中國 -- 化學分析

Materia medica -- China -- Analysis

藥物學 -- 中國 -- 化學分析



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