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Astragalus saponins modulates colon cancer development by regulating calpain-mediated glucose-regulated protein
Yue Wang, Kathy K Auyeung, Xiaoyu Zhang
1Center for Cancer and Inflammation Research, School of Chinese Medicine, Hong Kong Baptist University, 7 Baptist University Road, Kowloon Tong, Hong Kong, China. jksko@hkbu.edu.hk.
Background:
Glucose-regulated proteins (GRP) are induced in the cancer microenvironment to promote tumor survival, metastasis and drug resistance. AST was obtained from the medicinal plant Astragalus membranaceus, which possesses anti-tumor and pro-apoptotic properties in colon cancer cells and tumor xenograft. The present study aimed to investigate the involvement of GRP in endoplasmic reticulum (ER) stress-mediated apoptosis during colon cancer development, with focus on the correlation between AST-evoked regulation of GRP and calpain activation.
Methods:
The effects of AST on GRP and apoptotic activity were assessed in HCT 116 human colon adenocarcinoma cells. Calpain activity was examined by using a fluorescence assay kit. Immunofluorescence staining and immunoprecipitation were employed to determine the localization and association between calpains and GRP. GRP78 gene silencing was performed to confirm the importance of GRP in anticancer drug activities. The modulation of GRP and calpains was also studied in nude mice xenograft.
Results:
ER stress-mediated apoptosis was induced by AST, as shown by elevation in both spliced XBP-1 and CHOP levels, with parallel up-regulation of GRP. The expression of XBP-1 and CHOP continued to increase after the peak level of GRP was attained at 24 h. Nevertheless, the initial increase in calpain activity as well as calpain I and II protein level was gradually declined at later stage of drug treatment. Besides, the induction of GRP was partly reversed by calpain inhibitors, with concurrent promotion of AST-mediated apoptosis. The knockdown of GRP78 by gene silencing resulted in higher sensitivity of colon cancer cells to AST-induced apoptosis and reduction of colony formation. The association between calpains and GRP78 had been confirmed by immunofluorescence staining and immunoprecipitation. Modulation of GRP and calpains by AST was similarly demonstrated in nude mice xenograft, leading to significant inhibition of tumor growth.
Conclusions:
Our findings exemplify that calpains, in particular calpain II, play a permissive role in the modulation of GRP78 and consequent regulation of ER stress-induced apoptosis. Combination of calpain inhibitors and AST could exhibit a more pronounced pro-apoptotic effect. These results help to envisage a new therapeutic approach in colon cancer by targeting calpain and GRP.
Insights
Astragalus membranaceus (AST) induces endoplasmic reticulum (ER) stress and apoptosis in colon cancer by modulating glucose-regulated proteins (GRP) and calpains. Combining AST with calpain inhibitors enhances anti-cancer effects.
Area of Science:
- Oncology
- Molecular Biology
- Pharmacology
Background:
- Glucose-regulated proteins (GRP) are implicated in cancer progression, promoting tumor survival and drug resistance.
- Astragalus membranaceus (AST) exhibits anti-tumor and pro-apoptotic properties against colon cancer.
- Endoplasmic reticulum (ER) stress is a key pathway in colon cancer development.
Purpose of the Study:
- To investigate the role of GRP in ER stress-mediated apoptosis during colon cancer.
- To explore the correlation between AST-induced GRP regulation and calpain activation.
- To evaluate the therapeutic potential of targeting GRP and calpains in colon cancer.
Main Methods:
- Assessed AST effects on GRP and apoptosis in HCT 116 colon cancer cells.
- Measured calpain activity using fluorescence assays.
- Utilized immunofluorescence and immunoprecipitation to study GRP and calpain interactions.
- Performed GRP78 gene silencing and in vivo xenograft studies in nude mice.
Main Results:
- AST induced ER stress-mediated apoptosis, up-regulating GRP, spliced XBP-1, and CHOP.
- Calpain activity initially increased but declined later; calpain inhibitors promoted AST-induced apoptosis.
- GRP78 knockdown sensitized colon cancer cells to AST, reducing colony formation.
- AST modulated GRP and calpains in vivo, significantly inhibiting tumor growth.
Conclusions:
- Calpains, particularly calpain II, play a role in modulating GRP78 and ER stress-induced apoptosis.
- Combining calpain inhibitors with AST demonstrates a synergistic pro-apoptotic effect.
- Targeting calpain and GRP presents a novel therapeutic strategy for colon cancer.
