Background

Spiropachysine A is the extracted compound of traditional Chinese ethnic medicine Pachysandra axillaries Franch. var. styiosa (Dunn) M. Cheng. Spiropachysine A is the primary active steroidal alkaloids (SAs) widely used to facilitate blood circulation and relieve pain and inflammation. Few previous studies have investigated the anti-cancer activity of Spiropachysine A to treat hepatocellular carcinoma (HCC), and its molecular mechanism remains unknown.

Purpose

This study aims to investigate the anti-cancer activity of Spiropachysine A and the underlying mechanisms by inducing methuosis in vitro and in vivo.

Methods

Here, the activity of Spiropachysine A against cancer was evaluated by the experiments with MHCC-97H cells and the xenografted mice model. The cell proliferation was examined using MTT assay, and cell morphological characteristics were observed by microscope cellular imaging. The effects of autophagy, paraptosis, and oncosis on cytoplasmic vacuolisation were detected using immunofluorescence staining, transmission electron microscopy (TEM) and western blotting (WB). The cell cycle distribution and apoptosis were analysed by flow cytometry. Hematoxylin eosin (H & E) staining was used to observe the pathological changes of the tissues.

Results

The in vitro and in vivo results indicated that Spiropachysine A could inhibit HCC cells proliferation (IC₅₀ = 2.39 ± 0.21 μM against MHCC-97H cells) and tumor growth (TGI = 32.81 ± 0.23% at 25 mg/kg and 50.32 ± 0.26% at 50 mg/kg). The morphological changes of the treated cells showed that cell proliferation inhibition caused by Spiropachysine A was associated with numerous cytoplasmic vacuolization. Mechanistically, Spiropachysine A-induced methuosis rather than autophagy or arapaptic because the autophagy flux was blocked, leading to the increased LC3-II/I value and an accumulation of selective autophagy substrate p62. And, there was no activation of the regulatory parapaptic MAPK pathway. Additionally, the TEM and Lucifer yellow (LY) accumulation data confirmed that Spiropachysine A significantly triggered methuosis instead of oncosis. Further, the study indicated that the anti-proliferative activity of Spiropachysine A was independent of PCD since no alterations in apoptosis and cell cycle arrest-related proteins were observed after Spiropachysine A treatment. Impressively, the increased expression of Rac1 was observed in Spiropachysine A-treated MHCC-97H cells and its xenograft tumours, confirming that Spiropachysine A inhibited cell proliferation and induced methuosis through Ras/Rac1 signal pathways.

Conclusions

Spiropachysine A was collectively identified as a novel methuosis inducer that suppresses HCC in vitro and in vivo. The underlying mechanisms might be involved in the Ras/Rac1 pathway. Such data predict that Spiropachysine A is a promising candidate for developing novel chemotherapeutic agents as a methuosis inducer for cancer therapy.

中文翻译：

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Spiropachysine A 通过在体外和体内诱导代谢抑制肝细胞癌增殖

背景

Spiropachysine A 是中药材的提取物。变种。styiosa (Dunn) M. Cheng。Spiropachysine A 是主要的活性甾体生物碱 (SA)，广泛用于促进血液循环、缓解疼痛和炎症。以往很少有研究探讨螺蛳苷 A 治疗肝细胞癌 (HCC) 的抗癌活性，其分子机制尚不清楚。

目的

本研究旨在通过在体外和体内诱导 mehuosis 来研究 Spiropachysine A 的抗癌活性及其潜在机制。

方法

在这里，通过对 MHCC-97H 细胞和异种移植小鼠模型的实验评估了 Spiropachysine A 对癌症的活性。MTT法检测细胞增殖情况，显微细胞成像观察细胞形态特征。使用免疫荧光染色、透射电子显微镜 (TEM) 和蛋白质印迹 (WB) 检测自噬、截肢和肿瘤对细胞质空泡形成的影响。通过流式细胞仪分析细胞周期分布和细胞凋亡。采用苏木精伊红(H&E)染色观察组织病理变化。

结果

体外和体内结果表明螺旋藻碱 A 可以抑制 HCC 细胞增殖 (IC ₅₀ = 2.39 ± 0.21 μM 针对 MHCC-97H 细胞）和肿瘤生长（TGI = 32.81 ± 0.23% 在 25 mg/kg 和 50.32 ± 0.26% 在 50 mg/kg）。处理细胞的形态学变化表明，螺旋藻A引起的细胞增殖抑制与大量细胞质空泡化有关。机制上，螺旋藻 A 诱导 mehuosis 而不是自噬或 arapaptic，因为自噬通量被阻断，导致 LC3-II/I 值增加和选择性自噬底物 p62 的积累。并且，没有激活调节性突触 MAPK 通路。此外，TEM 和路西法黄 (LY) 积累数据证实螺旋藻 A 显着引发了 mehuosis 而不是肿瘤病。更远，该研究表明，螺旋藻碱 A 的抗增殖活性与 PCD 无关，因为在螺旋藻碱 A 处理后未观察到细胞凋亡和细胞周期停滞相关蛋白的变化。令人印象深刻的是，在螺旋体 A 处理的 MHCC-97H 细胞及其异种移植肿瘤中观察到 Rac1 的表达增加，证实螺旋体 A 抑制细胞增殖并通过 Ras/Rac1 信号通路诱导变质。

结论

Spiropachysine A 被共同确定为一种在体外和体内抑制 HCC 的新型 mehuosis 诱导剂。潜在的机制可能与 Ras/Rac1 通路有关。这些数据预测，螺旋藻碱 A 是开发新型化学治疗剂作为癌症治疗的 mehuosis 诱导剂的有希望的候选者。