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Updated: Sep 25, 2025
Malachite Green Assay for the Discovery of Heat-Shock Protein 90 Inhibitors
Published on: January 20, 2023
Study on molecular mechanism of benzo (ɑ) pyrene on CMA by HSP90ɑ and HIF-1ɑ
Shasha Zhang1, Tingting Liu2, Mengdi Zhang3
1School of Pharmacy, Inner Mongolia Medical University, PR China; Jungar Banner Central Hospital, Erdos, Inner Mongolia Autonomous Region, PR China.
Objective:
The effects of benzo (α) pyrene (BaP) on chaperone mediated autophagy (CMA) through heat shock protein 90 (HSP90) and hypoxia- inducible factor-1 (HIF-1) are studied by RNA interference and subcutaneous tumor formation technique in nude mice.
Methods:
40 nude mice that were inoculated with the silenced HSP90ɑ A549 cells line under the armpits of the forelimbs were divided into 4 groups, and were intragastrically administered with 1.80 mg/kg/d BaP-corn oil solutionfor for 60d (except the Control group), and the growth curves of nude mice and transplanted tumors were recorded. The size and morphological changes of tumors were observed by small animal imaging technique. qPCR, Western blot and Immunohistochemistry were used to detect the expression of HSP90ɑ, HSC70 and Lamp-2A. A549 cells were treated with 0.1 μmol/L, 1 μmol/L and 10 μmol/L BaP for 24 h, EPO and HIF-1ɑ concentration and HIF-1ɑ protein expression were detected by Elisa and Western blot; A549 cells were treated with 10 μmol/L BaP and HIF-1ɑ inhibitor for 24 h, qPCR, Western blot and Immunofluorescence methods were used to detect the expression of HSP90ɑ, HSC70 and Lamp-2A.
Results:
The weight of nude mice and transplanted tumors silenced HSP90ɑ was reduced by BaP; the expression of HSP90ɑ, HSC70, Lamp-2A mRNA and proteins in transplanted tumor tissues silenced HSP90ɑ were reduced by BaP; the total number of bioluminescence photons of transplanted tumors silenced HSP90ɑ were reduced by BaP. The concentration of EPO and HIF-1ɑ and the expression of HIF-1ɑ protein in A549 cells was increased by 10 μmol/L BaP; with HIF-1ɑ inhibitors treated, HSP90ɑ, HSC70, Lamp-2A mRNA and proteins expression and the fluorescence intensity of HSP90ɑ were decreased of A549 cells.
Conclusions:
The growth of transplanted tumor in nude mice is promoted by BaP, and is inhibited when HSP90ɑ was silenced. BaP promotes the occurrence of CMA by promoting the expression of HSP90ɑ and HIF-1ɑ, which are vital regulatory genes of BaP activation of CMA.
Insights
Benzo (α) pyrene (BaP) promotes tumor growth by activating chaperone-mediated autophagy (CMA) via heat shock protein 90 (HSP90) and hypoxia-inducible factor-1 (HIF-1). Silencing HSP90ɑ inhibits BaP-induced tumor growth and CMA activation.
Area of Science:
- Cellular Biology
- Molecular Oncology
- Environmental Toxicology
Background:
- Benzo (α) pyrene (BaP) is a polycyclic aromatic hydrocarbon found in environmental pollutants.
- Chaperone-mediated autophagy (CMA) is a selective degradation pathway crucial for cellular homeostasis.
- Heat shock protein 90 (HSP90) and hypoxia-inducible factor-1 (HIF-1) are key regulators in cellular stress responses and cancer progression.
Purpose of the Study:
- To investigate the role of Benzo (α) pyrene (BaP) in regulating chaperone-mediated autophagy (CMA).
- To elucidate the involvement of heat shock protein 90 (HSP90) and hypoxia-inducible factor-1 (HIF-1) in BaP-induced CMA.
- To assess the therapeutic potential of targeting HSP90ɑ in BaP-driven tumor growth.
Main Methods:
- Utilized a xenograft nude mouse model with silenced HSP90ɑ A549 cells.
- Administered BaP intragastrically and monitored tumor growth and morphology using imaging techniques.
- Analyzed the expression of HSP90ɑ, HSC70, and Lamp-2A via qPCR, Western blot, and immunohistochemistry.
- Investigated the effects of BaP and HIF-1ɑ inhibition on EPO, HIF-1ɑ, HSP90ɑ, HSC70, and Lamp-2A expression in A549 cells.
Main Results:
- BaP administration increased the weight and bioluminescence of transplanted tumors, which was attenuated by HSP90ɑ silencing.
- BaP treatment upregulated the expression of HSP90ɑ, HSC70, and Lamp-2A in tumor tissues.
- BaP exposure elevated EPO and HIF-1ɑ levels and protein expression in A549 cells, effects reversed by HIF-1ɑ inhibitors.
Conclusions:
- BaP promotes transplanted tumor growth in nude mice, an effect inhibited by HSP90ɑ silencing.
- BaP activates CMA by upregulating HSP90ɑ and HIF-1ɑ expression.
- HSP90ɑ and HIF-1ɑ are critical regulatory factors in BaP-mediated activation of CMA.
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