Paris saponin VII induces Caspase-3/GSDME-dependent pyroptosis in pancreatic ductal adenocarcinoma cells by activating ROS/Bax signaling

doi:https://doi.org/10.1016/j.chmed.2024.04.004

Archive > Volume 17 Issue 1 > 2025,17(1):94-107. DOI:https://doi.org/10.1016/j.chmed.2024.04.004 Prev Next

重樓皂苷VII激活ROS/Bax通路誘導(dǎo)胰腺導(dǎo)管腺癌細(xì)胞Caspase-3/GSDME依賴性焦亡

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Paris saponin VII induces Caspase-3/GSDME-dependent pyroptosis in pancreatic ductal adenocarcinoma cells by activating ROS/Bax signaling

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摘要:

目的：重樓是傳統(tǒng)中藥，被證實(shí)具有良好的抗腫瘤效果。在前期研究中，我們從重樓中分離出活性成分重樓皂苷VII，證實(shí)其能顯著抑制BxPC-3細(xì)胞的增殖。本研究旨在闡明重樓皂苷VII對胰腺導(dǎo)管腺癌（PDAC）的抑制作用及其機(jī)制。方法：通過CCK-8法、集落形成和細(xì)胞遷移實(shí)驗(yàn)測定細(xì)胞活力。采用Annexin V/PI和DCFH-DA染色流式細(xì)胞術(shù)分別檢測細(xì)胞凋亡和活性氧（ROS）的產(chǎn)生。通過形態(tài)學(xué)、Hoechst 33342/PI染色和乳酸脫氫酶（LDH）釋放實(shí)驗(yàn)檢測細(xì)胞焦亡。使用JC-1熒光染料測定線粒體膜電位變化。利用Western blotting和RT-qPCR確定蛋白質(zhì)和mRNA水平。通過異種移植瘤模型評估了重樓皂苷VII的體內(nèi)抑瘤作用。結(jié)果：重樓皂苷VII抑制了PDAC細(xì)胞（BxPC-3、PANC-1和Capan-2細(xì)胞）的增殖，誘導(dǎo)了gasdermin E（GSDME）以及Caspase-3和PARP的裂解。敲低GSDME可使重樓皂苷VII誘導(dǎo)的細(xì)胞焦亡轉(zhuǎn)變?yōu)榈蛲觥４送?，Z-DEVD-FMK顯著減弱了重樓皂苷VII誘導(dǎo)的GSDME依賴性細(xì)胞焦亡。重樓皂苷VII還可增加細(xì)胞內(nèi)ROS的累積，并激活Bax和Caspase-3/GSDME通路在PDAC細(xì)胞中引發(fā)細(xì)胞焦亡。ROS清除劑NAC可抑制LDH的釋放，同時抑制PDAC細(xì)胞中Caspase-9、Caspase-3和GSDME的裂解，最終逆轉(zhuǎn)重樓皂苷VII誘導(dǎo)的細(xì)胞焦亡。此外，在異種移植瘤模型中，重樓皂苷VII也可顯著抑制PDAC腫瘤的生長并在腫瘤中誘導(dǎo)細(xì)胞焦亡。結(jié)論：以上結(jié)果表明，重樓皂苷VII通過誘導(dǎo)Caspase-3/GSDME依賴性細(xì)胞焦亡發(fā)揮抗腫瘤作用，這可能在未來成為預(yù)防PDAC患者化療耐藥性的新策略。

Abstract:

Objective: Paridis Rhizoma (Chonglou in Chinese), a traditional Chinese herbal medicine, has been shown have strong anti-tumor effects. Paris saponin VII (PSVII), an active constituent isolated from Paridis Rhizoma, was demonstrated to significantly suppress the proliferation of BxPC-3 cells in our previous study. Here, we aimed to elucidate the anti-pancreatic ductal adenocarcinoma (PDAC) effect of PSVII and the underlying mechanism. Methods: Cell viability was determined by CCK-8, colony formation, and cell migration assays. Cell apoptosis and reactive oxygen species (ROS) production were measured by flow cytometry with annexin V/propidine iodide (Annexin V/PI) and 2',7'- dichlorodihydrofluorescein diacetate (DCFH-DA), respectively. Pyroptosis was evaluated by morphological features, Hoechst 33342/PI staining assay, and release of lactate dehydrogenase (LDH). JC-1 fluorescent dye was employed to measure mitochondrial membrane potential. Western blotting and reverse transcription-quantitative polymerase chain reaction (RT-qPCR) were used to determine the levels of proteins or mRNAs. The effect in vivo was assessed by a xenograft tumor model. Results: PSVII inhibited the viability of PDAC cells (BxPC-3, PANC-1, and Capan-2 cells) and induced gasdermin E (GSDME) cleavage, as well as the simultaneous cleavage of Caspase-3 and poly (ADP-ribose) polymerase 1 (PARP). Knockdown of GSDME shifted PSVII induced pyroptosis to apoptosis. Additionally, the effect of PSVII was significantly attenuated by Z-Asp(OMe)-Glu(OMe)-Val-Asp(OMe)-fluoromethylketone (Z-DEVD-FMK), on the induction of GSDME-dependent pyroptosis. PSVII also elevated intracellular ROS accumulation and stimulated Bax and Caspase-3/GSDME to conduct pyroptosis in PDAC cells. The ROS scavenger N-acetyl cysteine (NAC) suppressed the release of LDH and inhibited Caspase-9, Caspase-3, and GSDME cleavage in PDAC cells, ultimately reversing PSVII-induced pyroptosis. Furthermore, in a xenograft tumor model, PSVII markedly suppressed the growth of PDAC tumors and induced pyroptosis. Conclusion: These results demonstrated that PSVII exerts therapeutic effects through Caspase-3/GSDMEdependent pyroptosis and may constitute a novel strategy for preventing chemotherapeutic resistance in patients with PDAC in the future.

關(guān)鍵詞:

Caspase-3;gasdermin E;胰腺導(dǎo)管腺癌;重樓皂苷VII;焦亡;活性氧

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Project Supported:

This work was supported by the National Natural Science Foundation of China (No. 81973192).

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