Introduction

Trichosanthin (TK) is a glycoprotein extracted from the Chinese medicinal herb Trichosanthes kirilowi, which has anti-virus and anti-tumor activity. However, the target and detailed mechanism of TK remains elusive.

Objectives

We aimed to identify novel antitumor targets of TK in lung adenocarcinoma and study its anti-tumor mechanism.

Methods

We utilized a Lewis lung carcinoma mouse model to evaluate the inhibition of TK on tumor growth. CCK8 assay was utilized to calculate IC₅₀ of trichosanthin on A549 and H1299. In-vitro cellular assays and in-vivo xenograft mice studies were used to investigate MICU3 overexpression and TK treatment on tumor growth. Fluo-4 dye and JC-1 staining was used to measure the mitochondrial calcium levels and membrane potential. H&E and immunohistochemistry staining were applied the asses the effect of TK on tumor and microenvironment. RNA sequencing was applied to analyze transcriptome changes in TK-treated and MICU3-overexpressed tumor cells. The influence of trichosanthin on DNMT3B expression and MICU3 methylation were detected by qPCR and Western blotting. Transcriptional activity of the MICU3 gene was measured by ChIP-PCR and luciferase assays.

Results

Trichosanthin ihibited the tumor growth in vivo, resulting cancer cell growth inhibition and cell death, with almost no effect on normal cells. IC₅₀ of trichosanthin in A549 and H1299 cells were 62.8 μg/ml and 39.7 μg/ml, respectively. Mitochondrial Calcium Uptake Family complex MICU3 was shown to associated with favorable prognosis and was upregulated upon trichosanthin treatment, along with reduces tumor cell growth and migration, and increased cell death both in vitro and in vivo. Increased mitochondrial calcium level was observed in MICU3 overexpression cells. Pathway analysis of RNA-seq data revealed that cytokine and receptor pathways were enriched in MICU3-overexpressing cells. Trichosanthin decreased DNMT3B expression and altered MICU3 methylation while increased FOSL2 expression and reduced methylation that correlated with increased transcription of the MICU3 gene.

Conclusion

Trichosanthin elicits antitumor activity in lung adenocarcinoma via repressing DNMT3B and increasing FOSL2, which in turn induces MICU3-mediated mitochondrial calcium influx and tumor cell death.

中文翻译：

---

毛孢生素通过 MICU3 介导的线粒体钙内流引发抗肿瘤活性

 介绍

毛竹红素 （TK） 是从中药 Trichosanthes kirilowi 中提取的糖蛋白，具有抗病毒和抗肿瘤活性。然而，TK 的目标和详细机制仍然难以捉摸。

 目标

我们旨在确定 TK 在肺腺癌中的新抗肿瘤靶点并研究其抗肿瘤机制。

 方法

我们利用 Lewis 肺癌小鼠模型来评估 TK 对肿瘤生长的抑制作用。CCK8 法计算毛孢烷素对 A549 和 H1299 的 IC₅₀。体外细胞测定和体内异种移植小鼠研究用于研究 MICU3 过表达和 TK 治疗对肿瘤生长的影响。Fluo-4 染料和 JC-1 染色测定线粒体钙水平和膜电位。应用 H&E 和免疫组化染色评估 TK 对肿瘤和微环境的影响。应用 RNA 测序分析 TK 处理和 MICU3 过表达肿瘤细胞的转录组变化。通过 qPCR 和 Western blotting 检测毛孢子烷对 DNMT3B 表达和 MICU3 甲基化的影响。通过 ChIP-PCR 和荧光素酶测定测量 MICU3 基因的转录活性。

 结果

Trichosanthin 抑制体内肿瘤生长，导致癌细胞生长抑制和细胞死亡，对正常细胞几乎没有影响。毛孢烷素在 A549 和 H1299 细胞中的 IC₅₀ 分别为 62.8 μg/ml 和 39.7 μg/ml。线粒体钙摄取家族复合物 MICU3 被证明与良好的预后相关，并且在毛孢生素处理后上调，同时减少肿瘤细胞生长和迁移，并在体外和体内增加细胞死亡。在 MICU3 过表达细胞中观察到线粒体钙水平升高。RNA-seq 数据的通路分析显示，细胞因子和受体通路在 MICU3 过表达细胞中富集。毛孢生红素降低 DNMT3B 表达并改变 MICU3 甲基化，同时增加 FOSL2 表达并减少与 MICU3 基因转录增加相关的甲基化。

 结论

Trichosanthin 通过抑制 DNMT3B 和增加 FOSL2 在肺腺癌中引发抗肿瘤活性，进而诱导 MICU3 介导的线粒体钙内流和肿瘤细胞死亡。