Open Access

ARTICLE

SPINK1 contributes to proliferation and clonal formation of HT29 cells through Beclin1 associated enhanced autophagy

by NA HU^1,2,#, SHIQING ZHANG^2,3,#, AQUAN JIN², LIANYING GUO², ZHENYUN QU², JUN WANG^2,*

1 Department of Pathology, Northwest Women’s and Children’s Hospital, Xi’an, 710061, China
2 Department of Pathophysiology, College of Basic Medical Sciences, Dalian Medical University, Dalian, 116000, China
3 Department of Respiratory and Critical Care Medicine, Guizhou Provincial People’s Hospital, Guiyang, 550000, China

* Corresponding Author: JUN WANG. Email:
# These authors contributed equally to this work

Oncology Research 2022, 30(2), 89-97. https://doi.org/10.32604/or.2022.027058

Received 11 October 2022; Accepted 17 December 2022; Issue published 05 January 2023

Abstract

We aimed to explore the molecular mechanism that were involved in SPINK1-induced proliferation and clonogenic survival of human colorectal carcinoma (CRC) HT29 cells. Initially, we generated HT29 cells either permanently silencing or overexpressing SPINK1 protein. The results showed that SPINK1 overexpression (OE) significantly stimulated the proliferation and clonal formation of HT29 cells at the varied time points. Secondly, we found SPINK1 OE enhanced the ratio of LC3II/LC3I and the level of autophagy-related gene 5 (ATG5), whereas SPINK1 knockdown (Kd) reversed the above outcome under normal culturing and/or fasting condition in the cells, indicating its role in autophagy enhancement. Moreover, LC3-GFP-transfected SPINK1-OE HT29 cells strengthened the fluorescence intensity compared with the untransfected control. Chloroquine (CQ) significantly decreased the level of autophagy in both control and SPINK1-OE HT29 cells. The autophagy inhibitors, CQ and 3-Methyladenine (3-MA), remarkably inhibited the proliferation and colony formation of SPINK1-OE HT29 cells, while ATG5 upregulation resulted in the growth of the cells, suggesting the important function of autophagy in cell’s growth. Thirdly, SPINK1-induced autophagy was independently of mTOR signaling as p-RPS6 and p-4EBP1 were activated in SPINK1-OE HT29 cells. Instead, Beclin1 up and down regulation were clearly observed in SPINK1-OE and SPINK1 Kd HT29 cells, respectively. Moreover, Beclin1 silencing apparently reduced autophagy in SPINK1-OE HT29 cells, indicating that SPINK1-induced autophagy was closely associated with Beclin1. Collectively, SPINK1-promoted proliferation and clonal formation of HT29 cells were closely associated with Beclin1 associated enhanced autophagy. The above findings would open a new window for probing the role of SPINK1-related autophagic signaling in the pathogenesis of CRC.

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