Introduction

Previously, we have reported a withanolide-type steroid, named tubocapsenolide A (TA), which shows potent anti-proliferative activity in several cancer cell lines. However, its inhibitory effect on the Janus kinase/signal transducer and activator of transcription 3 (JAK/STAT3) pathway and therapeutic potential on osteosarcoma have not been reported.

Objectives

In the present study, we aimed to investigate the effect and molecular mechanism of TA in osteosarcoma.

Methods

The biological functions of TA in U2OS cells were investigated using colony formation, 5-ethynyl-20-deoxyuridine (EDU) staining, and cell cycle/apoptosis assays. The interaction between TA and Src homology 2 phosphatase 2 (SHP-2) was detected by enzyme activity and validated by target-identification methods such as drug affinity responsive target stability (DARTS), cellular thermal shift assay (CETSA), and biolayer interferometry (BLI). The in vivo anti-tumor efficacy of TA was analyzed in the xenograft tumor model. Western blotting analysis was performed to detect the protein expression levels.

Results

TA exhibited antitumor activity against osteosarcoma both in vitro and in vivo by regulating the JAK/STAT3 signaling pathway. Mechanically, TA interacted with SHP-2 directly and activated its phosphatase activity. Importantly, protein tyrosine phosphatase (PTP) inhibitor, SHP-2 inhibitor, and SHP-2 siRNA could reverse the inhibitory effect of TA on the JAK/STAT3 signaling pathway and restored the TA-induced cell death.

Conclusion

TA activated the phosphatase activity of SHP-2, which resulted in the inhibition of the JAK/STAT3 pathway and contributed to the antitumor efficacy of TA. Collectively, these findings suggested that TA could serve as a novel therapeutic agent for the treatment of osteosarcoma.

中文翻译：

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通过 SHP-2 介导的 tubocapsenolide A 对 JAK/STAT3 通路的失活抑制骨肉瘤细胞增殖

介绍

以前，我们报道了一种名为 tubocapsenolide A (TA) 的 withanolide 类固醇，它在几种癌细胞系中显示出有效的抗增殖活性。然而，其对 Janus 激酶/信号转导和转录激活因子 3 (JAK/STAT3) 通路的抑制作用和对骨肉瘤的治疗潜力尚未见报道。

目标

在本研究中，我们旨在探讨 TA 在骨肉瘤中的作用及其分子机制。

方法

使用集落形成、5-乙炔基-20-脱氧尿苷 (EDU) 染色和细胞周期/细胞凋亡测定研究了 TA 在 U2OS 细胞中的生物学功能。通过酶活性检测TA和Src同源2磷酸酶2（SHP-2）之间的相互作用，并通过药物亲和反应靶稳定性（DARTS）、细胞热位移测定（CETSA）和生物层干涉仪等靶标鉴定方法进行验证。 BLI)。在异种移植肿瘤模型中分析了TA的体内抗肿瘤功效。进行蛋白质印迹分析以检测蛋白质表达水平。

结果

TA通过调节 JAK/STAT3 信号通路在体外和体内均表现出抗骨肉瘤的抗肿瘤活性。在机械上，TA 直接与 SHP-2 相互作用并激活其磷酸酶活性。重要的是，蛋白酪氨酸磷酸酶 (PTP) 抑制剂、SHP-2 抑制剂和 SHP-2 siRNA 可以逆转 TA 对 JAK/STAT3 信号通路的抑制作用并恢复 TA 诱导的细胞死亡。

结论

TA激活了SHP-2的磷酸酶活性，从而抑制了JAK/STAT3通路，有助于TA的抗肿瘤功效。总的来说，这些发现表明 TA 可以作为治疗骨肉瘤的新型治疗剂。