Effect of modified Jianpi Yangzheng on regulating content of PKM2 in gastric cancer cells-derived exosomes,Phytomedicine

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Effect of modified Jianpi Yangzheng on regulating content of PKM2 in gastric cancer cells-derived exosomes
Phytomedicine ( IF 6.7 ) Pub Date : 2022-06-02 , DOI: 10.1016/j.phymed.2022.154229
Jian Wu ₁ , Mengyun Yuan ₂ , Junyu Shen ₂ , Yuxuan Chen ₂ , Ruijuan Zhang ₂ , Xu Chen ₂ , Haidan Wang ₁ , Zhonghua Yin ₂ , Xingxing Zhang ₁ , Shenlin Liu ₁ , Qingmin Sun ₁

Affiliation

Background

Modified Jianpi Yangzheng decoction (mJPYZ), as an empirical decoction of Traditional Chinese medicine has been shown significantly to prolong the survival of patients with advanced stage gastric cancer. Pyruvate kinase M2 (PKM2), has attracted attention for its important role on cellular aerobic glycolysis, however, few studies focus on PKM2 non-metabolic roles in tumor progression.

Purpose

Our study aimed to investigate the potential role of gastric cancer exosomes containing PKM2 in regulating tumor-associated macrophages (TAM) and the mechanism of mJPYZ against gastric cancer.

Methods

Colony Formation Assay, flow cytometry and TUNEL staining were employed to estimate the effect of mJPYZ on gastric cancer in tumor-bearing mice and cells. Western blot analyzed apoptosis-related protein expression changes. Network pharmacology and bioinformatics predicted potential exosomes modulation of mJPYZ in gastric cancer. Exosomes were isolated and co-cultured with TAM. Diff-Quik Staining observed the TAM morphological changes when incubating with gastric cancer cells exosomes. Flow cytometry and immunofluorescence were performed to demonstrate whether exosomes PKM2 involved in TAM polarization.

Results

mJPYZ induced apoptosis of gastric cancer cells by targeting PKM2 and downregulating PI3K/Akt/mTOR axis in vivo and in vitro. Network pharmacology showed potential exosomes modulation of mJPYZ in gastric cancer. We extracted exosomes and found mJPYZ decreased the abundance of serum exosomes PKM2 in patients with advanced gastric cancer and xenograft tumor model. Additionally, we firstly detected and confirmed that PKM2 is a package protein of exosomes extracted from gastric cancer cells, and mJPYZ could diminish the content of exosomal PKM2 in gastric cancer cells. Importantly, mJPYZ reduced the delivery of exosomal PKM2 from tumor cells to macrophages, and alleviated exosomal PKM2-induced differentiation of M2-TAM in tumor microenvironment, eventually inhibited gastric cancer progression.

Conclusion

Gastric cancer exosomes containing PKM2 could lead to M2 macrophages differentiation, thereby promoting gastric cancer progression. Our findings provide a rationale for potential application of mJPYZ in the treatment of gastric cancer via PKM2.

中文翻译：

健脾养正加味对胃癌细胞源性外泌体PKM2含量的调节作用

背景

加味健脾养正汤（mJPYZ）作为一种经验性中药汤剂，已被证明可显着延长晚期胃癌患者的生存期。丙酮酸激酶 M2 (PKM2) 因其在细胞有氧糖酵解中的重要作用而引起人们的关注，然而，很少有研究关注 PKM2 在肿瘤进展中的非代谢作用。

目的

我们的研究旨在探讨含有 PKM2 的胃癌外泌体在调节肿瘤相关巨噬细胞（TAM）中的潜在作用以及 mJPYZ 抗胃癌的机制。

方法

采用集落形成测定、流式细胞术和TUNEL染色来评估mJPYZ对荷瘤小鼠和细胞中胃癌的作用。 Western blot分析了凋亡相关蛋白表达的变化。网络药理学和生物信息学预测了 mJPYZ 在胃癌中的潜在外泌体调节作用。分离外泌体并与 TAM 共培养。 Diff-Quik 染色观察了与胃癌细胞外泌体孵育时 TAM 的形态变化。进行流式细胞术和免疫荧光来证明外泌体 PKM2 是否参与 TAM 极化。

结果

mJPYZ在体内和体外通过靶向 PKM2 并下调 PI3K/Akt/mTOR 轴诱导胃癌细胞凋亡。网络药理学显示外泌体对 mJPYZ 在胃癌中的潜在调节作用。我们提取了外泌体，发现 mJPYZ 降低了晚期胃癌患者和异种移植肿瘤模型中血清外泌体 PKM2 的丰度。此外，我们首次检测并证实PKM2是从胃癌细胞中提取的外泌体的包装蛋白，mJPYZ可以减少胃癌细胞中外泌体PKM2的含量。重要的是，mJPYZ减少了外泌体PKM2从肿瘤细胞到巨噬细胞的递送，并减轻了外泌体PKM2诱导的M2-TAM在肿瘤微环境中的分化，最终抑制了胃癌的进展。