Maintenance of the Expression of c-FLIPL by Hsp70 to Resist Licochalcone A-Induced Anti-Colorectal Cancer Effect through ERK-Mediated Autophagy Induction

Maintenance of the Expression of c-FLIP_L by Hsp70 to Resist Licochalcone A-Induced Anti-Colorectal Cancer Effect through ERK-Mediated Autophagy Induction

Tianpeng Li^1,†, Ting Li^1,†, Hongbin Zhang², Chunyan Liu³, Min Li⁴, Chu Wang⁴, Yuanyuan Zheng⁴, Lihua Zhang⁵, Xiaoyi Long⁶, Shaoqing Shi⁷, Yun Long^8,*, Wei Chen^1,*

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¹ Institute of Basic Medicine and Forensic Medicine, Medical Imaging Key Laboratory of Sichuan Province, North Sichuan Medical College, 637000 Nanchong, Sichuan, China

² Department of Pediatric Surgery, the First Affiliated Hospital of Kunming Medical University, 650032 Kunming, Yunnan, China

³ Institute of School Health, Yunnan Center for Disease Control and Prevention, 650032 Kunming, Yunnan, China

⁴ Department of Respiratory Medicine, the First Affiliated Hospital of Kunming Medical University, 650032 Kunming, Yunnan, China

⁵ Department of General Medicine, the First Affiliated Hospital of Kunming Medical University, 650032 Kunming, Yunnan, China

⁶ Department of Pathophysiology, Institute of Basic Medicine and Forensic Medicine, North Sichuan Medical College, 637000 Nanchong, Sichuan, China

⁷ Scientific Research Laboratory Center, the First Affiliated Hospital of Kunming Medical University, 650032 Kunming, Yunnan, China

⁸ Department of General Medicine, Kunming Yan’an Hospital, 650051 Kunming, Yunnan, China

^*Correspondence: longyun0514@126.com (Yun Long); chengwei197959@163.com (Wei Chen)
^†These authors contributed equally.

Front. Biosci. (Landmark Ed) 2023, 28(12), 325; https://doi.org/10.31083/j.fbl2812325

Submitted: 17 April 2023 | Revised: 4 June 2023 | Accepted: 9 June 2023 | Published: 1 December 2023

(This article belongs to the Special Issue Targeting of Multiple Predictive Biomarkers for Checkpoint Immunotherapy)

This is an open access article under the CC BY 4.0 license.

Abstract

Background: The mortality rate of colorectal cancer (CRC) ranks second worldwide. Previous research had indicated that licochalcone A (LA) was a flavonoid in licorice with diverse anticancer effects. We explored the underlying mechanisms of LA-triggered anticancer activity in CRC. Methods: Thiazolyl Blue (MTT) experiment and EdU staining were utilized to evaluate cell proliferation. Meanwhile, cells were stained by Annexin V/PI to investigate apoptosis through flow cytometry assay. Moreover, expressions of proteins were detected by immunoblotting, and the level of related mRNA was investigated using real-time quantitative PCR. Results: LA selectively suppressed the proliferation and triggered apoptosis of CRC cells. Strikingly, LA induced cytoprotective autophagic activities since the suppression of autophagy significantly strengthened LA-induced cytotoxicity and FLICE inhibitory protein (c-FLIP ${}_{\text{L}}$ ) degradation, meanwhile reversing LA-mediated heat shock protein 70 (Hsp70) upregulation. Moreover, autophagy-mediated Hsp70 upregulation resisted LA-induced anticancer effects since the suppression of Hsp70 strengthened LA-triggered cytotoxicity and c-FLIP ${}_{\text{L}}$ degradation. Furthermore, LA greatly activated extracellular signal-regulated protein kinases (ERK) and p38. However, blocking of ERK, but not p38, significantly boosted LA-triggered cell death and c-FLIP ${}_{\text{L}}$ downregulation. Suppression of ERK also reversed LA-mediated autophagic induction. Conclusions: LA increased Hsp70 expression depending on ERK-mediated autophagy, which protected CRC cells from LA-induced anticancer activities.

Keywords

colorectal cancer

licochalcone A

Hsp70

ERK

apoptosis

autophagy

Funding

81960156/National Natural Science Foundation of China

H2018002/High Level Reserve Talents in Health Science

2020YGC01/Medical edible flowers technology innovation team of universities in Yunnan province

202001AY070001-189/Kunming Medical University Applied basic Research Joint Project

202201AT070043/Kunming Medical University Applied basic Research Joint Project

MIKLSP202004/Opening Project of Medical Imaging Key Laboratory of Sichuan Province

Figures

Fig. 1.

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Fig. 1.

The suppression of licochalcone A (LA) on colorectal cancer (CRC) cell proliferation is significantly greater than its toxicity to normal cells. (A) different thickness of LA was used to treat SW620, HCT116, HT29 and NCM460 cells. After 48 h, cell proliferations were detected by Thiazolyl Blue (MTT) experiment. (B) SW620 cells were cultured with diverse thickness LA, and then cell viability was tested by MTT experiment at 24, 48, 72, and 96 h. (C) SW620 cells were cultured with LA (0, 5, and 10 µM) for 7 days; then crystal violet staining was carried out. (D) SW620 cells were cultured with 20 µM LA for 8 h; next, EdU staining was performed. Scale bar 100 µm. (E) SW620 and HCT116 were cultured with LA (0, 10, 20, 30, 40, and 50 µM) for 48 h in cultures with serum or not, and then cell viabilities were measured by MTT experiment. (F) SW620 and HCT116 cells were cultured with LA (20 µM); next, flow cytometric assay was utilized to examine the cell cycle at 8 h. N = 3. *p $<$ 0.05, **p $<$ 0.01, ***p $<$ 0.001.

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