Role of GRP78 inhibiting artesunate-induced ferroptosis in KRAS mutant pancreatic cancer cells

Abstract

Objective:

To investigate the exact role of GRP78 in artesunate-induced ferroptosis in KRAS mutant pancreatic cancer cells.

Methods:

Artesunate-induced KRAS mutant human pancreatic cancer cells (AsPC-1 and PaTU8988) ferroptosis was confirmed by fluorescent staining experiments and CCK8. Western blot and short-hairpin RNA-based knockdown assays were conducted to detect GRP78 activity and its role in artesunate-induced ferroptosis.

Results:

Artesunate induced AsPC-1 and PaTU8988 cell death in ferroptosis manner, rather than necrosis or apoptosis. In addition, artesunate increased the mRNA and protein levels of GRP78 in a concentration-dependent manner in AsPC-1 and PaTU8988 cells. Knockdown GRP78 enhanced artesunate-induced ferroptosis of pancreatic cancer cells in vitro and in vivo.

Conclusion:

Combining artesunate with GRP78 inhibition may be a novel maneuver for effective killing of KRAS mutant pancreatic ductal adenocarcinoma cells.

Keywords:

• ferroptosis
• GRP78
• artesunate
• pancreatic cancer

Acknowledgments

This study was supported by grants from the National Natural Science Foundation of China (grant number 81502663), the Social Development Foundation of Jiangsu Province (grand number BE2018691), Young Medical Talents of Jiangsu (grand number QNRC20168339), Postgraduate Innovation Project of Jiangsu Province (grand number KYCX17_1802), Six talent peals project of Jiangsu Province (grand number WSW-039), and Six for one project of Jiangsu Province (grand numbers LGY2018093).

Abbreviation list

GRP78, 78-kDa glucose-regulated protein; CCK8, Cell Counting Kit-8; RCD, regulated cell death; GPX4, glutathione peroxidase 4; DMEM, dulbecco’s modified eagle medium; MDA, malondialdehyde; DMSO, dimethylsulfoxide; BCA, bicinchoninic acid; PBS, phosphate buffer saline; BSA, bovine serum albumin; PDAC, pancreatic ductal adenocarcinoma.

Ethics approval and consent to participate

All animal studies were approved by the Committee on the Use of Live Animals for Teaching and Research of the Jiangsu University.

Supplementary material

Figure S1 (A) AsPC-1 and PaTU8988 were treated DMSO (control), ART (20 µM), ART (20 µM) + Fer-1 for 24 hrs. The level of MDA was assayed. (B) The protein expression level of GAPDH in AsPC-1 and PaTU8988 treated with or without artesunate. ART represents for artesunate. *P<0.05 relative to control. Statistical analysis was performed using Student’s t-test.

Author contributions

All authors contributed to data analysis, drafting and revising the article, gave final approval of the version to be published, and agree to be accountable for all aspects of the work.

Disclosure

The authors report no conflicts of interest in this work.