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Archive > Volume 17 Issue 1 > 2025,17(1):127-138. DOI:https://doi.org/10.1016/j.chmed.2024.04.002 Prev Next

華蟾素通過下調(diào)胸苷酸合成酶發(fā)揮抗肝癌作用的物質(zhì)基礎(chǔ)揭示

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Material basis revelation of anti-hepatoma effect of Huachansu (Cinobufacini) through down-regulation of thymidylate synthase

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    摘要:
    目的：肝細(xì)胞性肝癌 （HCC） 已成為世界范圍內(nèi)癌癥死亡的主要原因。華蟾素是從蟾蜍科中華大蟾蜍的干燥皮膚中分離出的活性物質(zhì)。目前已廣泛應(yīng)用于癌癥的臨床治療，本研究旨在闡明華蟾素誘導(dǎo)的胸苷酸合成酶（TYMS）下調(diào)的物質(zhì)基礎(chǔ)。 方法：利用超高效液相色譜-串聯(lián)質(zhì)譜（UPLC-MS/MS）對華蟾素的主要生物活性成分進(jìn)行鑒定。采用CCK-8法和克隆形成實(shí)驗(yàn)檢測腫瘤細(xì)胞活力。通過實(shí)時熒光定量PCR和/或蛋白質(zhì)印跡法檢測TYMS和γ-H2AX水平。通過使用小干擾RNA（siRNA）轉(zhuǎn)染明確抑制TYMS是否能增強(qiáng)華蟾素對人肝癌細(xì)胞生長的抑制作用。 結(jié)果：首先，我們從華蟾素中鑒定出21種主要生物活性成分。CCK-8檢測結(jié)果顯示，華蟾素及其蟾蜍二烯酸內(nèi)酯類成分（蟾毒靈、蟾毒它靈、華蟾蜍精、去乙酰華蟾蜍精、沙蟾毒精、遠(yuǎn)華蟾毒精和酯蟾毒配基）以劑量依賴性的方式顯著抑制肝癌HepG2和SK-HEP-1細(xì)胞的增殖。進(jìn)一步機(jī)制研究表明，華蟾素顯著抑制TYMS蛋白水平，該酶是DNA合成所必須的胸苷酸唯一的從頭合成來源。進(jìn)一步研究顯示抑制TYMS可誘導(dǎo)HCC細(xì)胞周期阻滯和DNA損傷，此外，華蟾素顯著增加γ-H2AX表達(dá)，這表明DNA損傷發(fā)生。加之，敲低TYMS能夠增加華蟾素對人肝癌細(xì)胞增殖的抑制作用。實(shí)時熒光定量PCR結(jié)果表明，華蟾素對TYMS的轉(zhuǎn)錄水平?jīng)]有影響，蛋白酶體抑制劑MG132可以逆轉(zhuǎn)華蟾素誘導(dǎo)的TYMS抑制，蛋白質(zhì)合成抑制劑環(huán)己酰亞胺（CHX）和華蟾素聯(lián)用能夠進(jìn)一步抑制TYMS的蛋白水平，上述結(jié)果表明華蟾素促進(jìn)肝癌細(xì)胞中TYMS發(fā)生蛋白酶體依賴性降解。更重要的是，我們證明了華蟾素中蟾蜍二烯酸內(nèi)酯類活性成分，如蟾毒靈、蟾毒它靈、華蟾蜍精、去乙酰華蟾蜍精、沙蟾毒精、遠(yuǎn)華蟾毒精和酯蟾毒配基，也能夠顯著抑制TYMS的水平，這揭示了華蟾素抑制TYMS的物質(zhì)基礎(chǔ)。5-氟尿嘧啶（5-FU）是一種TYMS抑制劑，我們進(jìn)一步評估了華蟾素與5-FU聯(lián)合治療的效果，結(jié)果表明華蟾素與5-FU聯(lián)合用藥具有協(xié)同或拮抗作用。因此，在臨床上，應(yīng)注意華蟾素與5-FU聯(lián)合使用的劑量。 結(jié)論：華蟾素通過蛋白酶體依賴途徑降解TYMS抑制肝癌細(xì)胞生長并誘導(dǎo)DNA損傷。蟾蜍二烯酸內(nèi)酯類成分是華蟾素抑制TYMS的物質(zhì)基礎(chǔ)。

    Abstract:
    Objective: Hepatocellular carcinoma (HCC) is a leading cause of mortality worldwide. Huachansu (Cinobufacini) is active extract isolated from the dry skin of Bufo Bufo gargarizans. It has now been widely used in clinical treatment of cancer, this study is to clarify the material basis of down-regulation of thymidylate synthase (TYMS) induced by Huachansu. Methods: Our study utilized UPLC-MS/MS to identify major bioactive components from Huachansu. Cell Counting Kit 8 (CCK-8) assay and clone formation assay were used to examine the cell viability of tumor cells. TYMS and c-H2AX level were detected by using quantitative real-time RT-PCR and/or western blotting. Small interfering RNA (siRNA) transfection was used to explore whether inhibition of TYMS could enhance the suppressive effect of Huachansu on cell growth of HCC cells. Results: In our study, firstly, we identify 21 major bioactive components from Huachansu. CCK-8 assay results showed that Huachansu and its bioactive bufadienolides (Bufalin, Bufotalin, Cinobufotalin, Desacetylcinobufagin, Arenobufagin, Telocinobufagin, and Resibufogenin) significantly inhibited the proliferation of HepG2 and SK-HEP-1 cells in a dose- and time-dependentmanner. Further molecularmechanistic investigation demonstrates that Huachansu significantly suppresses thymidylate synthase (TYMS), the enzyme which provides the sole de novo source of thymidylate for DNA synthesis. The inhibition of TYMS could lead to cell-cycle block and DNA damage of HCC cells. Furthermore, we identified that Huachansu markedly increased c-H2AX expression, which indicated the presence of DNA damage. Moreover, we confirmed that transfection of cells with small interfering RNA specific to TYMS could increase the suppressive effects of Huachansu on the HCC cells proliferation. Quantitative RT-PCR analysis showed that Huachansu treatment had no effect on the transcription level ofTYMS. Furthermore, proteasomal inhibitorMG132 could blockTYMS inhibition induced by Huachansu, and concomitant administration of protein synthesis inhibitor cycloheximide (CHX) with Huachansu could further suppress the protein level of TYMS, indicating that Huachansu promotes proteasome-dependent degradation of TYMS in liver cancer cells. More importantly, the bioactive bufadienolides of Huachansu such as Bufalin, Bufotalin, Cinobufotalin, Desacetylcinobufagin, Arenobufagin, Telocinobufagin, and Resibufogenin could also significantly restrain the protein level of TYMS, revealing the material basis of inhibition of TYMS exposed to Huachansu. 5-Fluorouracil (5-FU) is a TYMS inhibitor, we also evaluate the effects of the combined treatment of Huachansu with 5-FU, the results show that interactions between Huachansu and 5-FU are synergistic or antagonistic. Thus, in clinical, attention should be paid to the dosage of Huachansu in combination with 5-FU. Conclusion: Huachansu inhibits the growth and induces DNA damage of human HCC cells through proteasome-dependent degradation of TYMS, bioactive bufadienolides are the material basis of downregulation of TYMS induced by Huachansu.

    關(guān)鍵詞:
    DNA損傷;肝細(xì)胞性肝癌;華蟾素;物質(zhì)基礎(chǔ);胸苷酸合成酶;蛋白酶體降解

    Keywords:


    Project Supported:
    This work has been financially supported by National Natural Science Foundation of China Grant (No. 82204693), Taishan scholar engineering youth expert program of Shandong Province (No. tsqn202103110), Young and Creative Team for Talent Introduction of Shandong Province (No. 10073004), Binzhou Medical University Scientific Research Fund for High-level Talents(NO. BY2020KYQD13, 2019KYQD06), Standard study on Chinese medicinal materials and Decoction pieces in Shandong Province (No. 2020-201).

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