Aims

Hsp90 is regarded as an important therapeutic target in cancer treatment. Client proteins of Hsp90 like Beclin-1, PI3K, and AKT, are associated with tumor development, poor prognosis, and resistance to cancer therapies. This study aims to analyze the role of Gedunin, an Hsp-90 inhibitor, in mediation of crosstalk between apoptosis and autophagy by targeting Beclin-1:Bcl-2 interaction, and ER stress.

Main methods

A549 cells were treated with different concentrations of gedunin, and inhibitory rate was evaluated by MTT assay. Effect of gedunin on generation of reactive oxygen species, mitochondrial membrane potential, and chromatin condensation was studied by staining methods like DCFH-DA, MitoTracker, and DAPI. Expression of EGFR, PIK3CA, AKT, marker genes for apoptosis and autophagy were studied using semi-quantitative RT-PCR. Interaction study of Hsp90:Beclin-1:Bcl-2 was done by immunoprecipitation analysis. Protein expression of autophagy and apoptosis markers along with Grp78, Hsp70, and Hsp90 was analyzed by immunoblotting.

Key findings

Gedunin exerts cytotoxic effects, causes increase in ROS generation, downregulates mitochondrial membrane potential and induces loss in DNA integrity. mRNA expression analysis revealed that gedunin sensitized A549 cells towards apoptosis by downregulating EGFR, PIK3CA, AKT, and autophagy. Gedunin also inhibited interaction between Hsp90:Beclin-1:Bcl-2, leading to downregulation of autophagy (Beclin-1, Atg5-12 complex, and LC3) and antiapoptotic protein Bcl-2, which may result in ER stress-induced apoptosis. Moreover, Hsp90 inhibition by gedunin did not cause upregulation of Hsp70 expression.

Significance

Gedunin induces apoptosis in lung cancer cells by disrupting Hsp90:Beclin-1:Bcl-2 interaction and autophagy downregulation, thus making gedunin a good drug lead for targeting lung cancer.

中文翻译：

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Hsp90抑制剂葛根素通过破坏Hsp90：Beclin-1：Bcl-2相互作用并下调自噬而导致A549肺癌细胞凋亡。

目的

Hsp90被认为是癌症治疗中的重要治疗靶标。Hsp90的客户蛋白（如Beclin-1，PI3K和AKT）与肿瘤的发展，预后不良和对癌症疗法的抵抗力有关。这项研究旨在通过靶向Beclin-1：Bcl-2相互作用和内质网应激分析Hsp-90抑制剂Gedunin在细胞凋亡和自噬之间的串扰中的作用。

主要方法

用不同浓度的葛根素处理A549细胞，并通过MTT测定评估抑制率。通过DCFH-DA，MitoTracker和DAPI等染色方法研究了葛根素对活性氧生成，线粒体膜电位和染色质凝聚的影响。的表达EGFR，PIK3CA，AKT使用半定量RT-PCR进行了研究，标记基因的细胞凋亡和自噬。Hsp90：Beclin-1：Bcl-2的相互作用研究通过免疫沉淀分析进行。通过免疫印迹分析自噬和凋亡标记以及Grp78，Hsp70和Hsp90的蛋白质表达。

主要发现

格丁宁发挥细胞毒性作用，引起ROS产生增加，下调线粒体膜电位，并导致DNA完整性下降。mRNA表达分析表明，葛根素可通过下调EGFR，PIK3CA，AKT和自噬来使A549细胞凋亡。葛根素还抑制Hsp90：Beclin-1：Bcl-2之间的相互作用，导致自噬（Beclin-1，Atg5-12复合物和LC3）和抗凋亡蛋白Bcl-2的下调，这可能导致内质网应激诱导的细胞凋亡。此外，葛根素对Hsp90的抑制作用不会引起Hsp70表达的上调。

意义

葛根素通过破坏Hsp90：Beclin-1：Bcl-2相互作用和自噬下调来诱导肺癌细胞凋亡，从而使葛根素成为靶向肺癌的良好药物。