Elsevier

Phytomedicine

Volume 102, 20 July 2022, 154149
Phytomedicine

Original Article
ACSL4 promotes colorectal cancer and is a potential therapeutic target of emodin

https://doi.org/10.1016/j.phymed.2022.154149Get rights and content

Highlights

  • ACSL4 promotes VEGF secretion and VEGFR activation.

  • ACSL4 facilitate the proliferation and invasion of CRC cells.

  • Emodin inhibits the development of CRC in vivo and in vitro by targeting ACSL4.

  • Emodin inhibit the secretion of VEGF and reduces the expression of VEGFRs.

Abstract

Background

Colorectal cancer (CRC) is an important death-related disease in the world and new therapeutic strategies are urgently needed to reduce mortality. Several studies have demonstrated that emodin, the main ingredient of Rheum palmatum, fights cancer but its potential anti-tumor effect on CRC is still unknown.

Purpose

The present study is aimed to explore the potential anti-tumor effects of emodin against CRC and the underlying molecular mechanism.

Methods

CRC-related datasets were screened according to filter criteria in the GEO database and TCGA database. By using screened differentially expressed genes, GO, KEGG and survival analysis were carried out. The expressions of ACSL4, VEGFR1, and VEGFR2 were examined by immunohistochemistry and western blot. Then, pcDNA-ACSL4, pcDNA-VEGFR1, and pcDNA-VEGFR2 were used to overexpress ACSL4, VEGFR1, and VEGFR2, while ACSL4 siRNA was used to silence ACSL4 expression in HCT116 cells. CCK-8 assay and transwell migration assay were used to detect the cell proliferation and invasion. A docking simulation assay and an MST assay were performed to explore the potential mode of emodin binding to ACSL4. The HCT116 cells and CRC mouse model were established to investigate the effects of emodin on CRC.

Results

The ACSL4, VEGFR1, and VEGFR2 expression were upregulated in CRC tissues and ACSL4 was associated with a shorter survival time in CRC patients. ACSL4 downregulation reduced cell proliferation and invasion, while ACSL4 exhibited a positive correlation with the levels of VEGFR1, VEGFR2, and VEGF. In HCT116 cells, emodin reduced cell proliferation and invasion by inhibiting ACSL4, VEGFR1, and VEGFR2 expression and VEGF secretion. Docking simulation and MST assay confirmed that emodin can directly bind to ACSL4 target. Moreover, ACSL4 overexpression abolished the inhibitory effect of emodin on VEGF secretion and VEGFR1 and VEGFR2 expression, but VEGFR1 and VEGFR2 overexpression did not affect the inhibitory effect of emodin on ACSL4 expression and VEGF secretion. Furthermore, emodin reduced the mortality and tumorigenesis of CRC mice and reduced ACSL4, VEGFR1, VEGFR2 expression, and VEGF content.

Conclusion

Our findings indicate that emodin inhibits proliferation and invasion of CRC cells and reduces VEGF secretion and VEGFR1 and VEGFR2 expression by inhibiting ACSL4. This emodin-induced pathway offers insights into the molecular mechanism of its antitumor effect and provides a potential therapeutic strategy for CRC.

Introduction

Cancer is a major public health problem worldwide with an estimated 18.1 million new cases and 9.6 million cancer deaths in 2018 (Bray et al., 2018). In recent years, the incidence and mortality of colorectal cancer (CRC) are increasing gradually. From 2000 to 2013, the incidence of CRC increased by 22%, while the 5-year relative survival rate was 65% (Siegel et al., 2017). Although targeted drugs have advantages in the treatment of CRC, the survival benefit is not very ideal for the natural or acquired drug resistance (Xie et al., 2020). Hence, the development of new drugs for CRC treatment is urgent.

A large number of traditional Chinese medicines have anti-inflammatory activity and are rich in anti-cancer compounds, however, these traditional Chinese medicines or compounds exert direct cytotoxicity with indirect regulation of tumors. Hence, traditional medicinal extracts are considered a better resource library for anti-cancer drug therapy (Wang et al., 2012, 2020; Zou et al., 2020). Emodin is a natural anthraquinone derivative that is the main ingredient of Rheum palmatum, which has been widely used in traditional Chinese medicine to treat various diseases (Dong et al., 2016). The core of emodin is very similar to that of the anthracyclines, such as doxorubicin and daunorubicin, used in cancer treatment (Minotti et al., 2004). It has immunomodulatory and anti-inflammatory properties but its potential in cancer therapy remains needing exploration (Srinivas et al., 2007; Sun, 2008). Several studies have confirmed that emodin has cytotoxic and growth inhibitory effects on many types of tumor cells (Liu et al., 2020a, 2020b; Shrimali et al., 2013) including colorectal cancer (CRC) cells (Zhang et al., 2021). Studies have shown that emodin exhibits dose-dependent cytotoxicity in CRC cell lines, such as SW480 and HCT116 (Lee et al., 2017), and its inhibitory effect on HCT116 cells was the most obvious, which may be related to the high expression of fatty acid synthase (FASN), whereas FASN expression was lower in SNU-C2A, SNU-C5, and SW480 cells (Lee et al., 2017). However, the mechanism by which emodin inhibits CRC is inarticulate, which cannot explain its weak inhibitory effect on other CRC cells, but strong inhibitory effect on CRC in vivo, including azoxymethane/dextran sodium sulfate (AOM/DSS)-induced CRC model (Zhang et al., 2021) and xenograft tumor model (Gu et al., 2019).

The enzyme long-chain acyl-CoA synthetase 4 (ACSL4), which is also involved in fatty acid metabolism, has attracted much attention recently. Its role in tumor treatment has been reviewed (Rossi Sebastiano and Konstantinidou, 2019). A systematic analysis showed that in a variety of tumors, high levels of ACSLs are associated with poor prognosis. Overexpression of ACSL4 in vitro increased the growth, invasion, and anchorage-independent growth ability of ACSL4-negative MCF-7 cells, and ACSL4 overexpression in MCF-7 cells promoted tumor growth in nude mice (Wu et al., 2013). Knockdown of ACSL4 reduced the proliferation and invasion of breast cancer cells (Belkaid et al., 2017), and the reduction of ACLS4 expression significantly inhibited tumor growth in an orthotopic xenograft mouse model of hepatic carcinoma (Xia et al., 2017). A study by Chen et al. (2016) showed that three major ACSL isotypes were found to be upregulated in CRC, including ACSL1, ACSL4, and ACSL6. Compared with that in empty vector control cells, overexpression of ACSL4 in CRC cells promoted cell proliferation, invasion, and proliferation. Further, ACSL4 silencing, mediated by shRNA, reduced cell proliferation in CRC cells (Sanchez-Martinez et al., 2017). Although the above studies suggest that ACSL4 is generally upregulated in cancers (such as CRC, breast cancer, and hepatic carcinoma) (Tang et al., 2018), it is still unclear how the dysregulation of ACSL4 expression influences the development of CRC. In the current study, we hypothesized that emodin inhibits CRC by regulating ACSL4 expression. This study supplements the mechanism of ACSL4 in the development of CRC, and the experimental evidence that ACSL4 is a therapeutic target for emodin, which is a candidate drug for the treatment of CRC.

Section snippets

Gene expression omnibus (GEO) and the cancer genome atlas (TCGA) database analysis

GEO data download and analysis: After downloading the gene expression matrix of GSE89076, the information and DNA expression data of 23 patients with colon cancer tissues and adjacent normal tissues were extracted. The selection condition for differentially expressed genes between colon cancer tissues and adjacent normal tissues was p < 0.05. The log2FC values of the genes in the samples were used to draw a volcano map. The differentially expressed genes were used for Kyoto Encyclopedia of

ACSL4 is highly expressed in CRC patients

DNA microarray analysis data from colon cancer tissues and adjacent normal tissues from 23 CRC patients (Fig. 1A) showed that the gene expression levels of ACSL4, VEGFA, and VEGFB in cancer tissues were higher than those in adjacent normal tissues (Fig. 1B). These abnormally expressed genes are enriched in multiple signaling pathways. Of these, the PI3K-Akt signaling pathway, which is ranked first, has been reported to promote the development of various cancers (Hoxhaj and Manning, 2020). ACSL4

Discussion

In this study, we explored the possible mechanism by which emodin inhibits CRC progression by targeting ACSL4. In addition to confirming that ACSL4 is highly expressed in human CRC samples and promotes the proliferation of colon cancer cells in vitro, we provide evidence that emodin inhibits the secretion of VEGF and reduces the expression of VEGFRs by targeting ACSL4, thereby inhibiting the development of CRC in vivo and in vitro.

We first analyzed the gene expression profile of CRC patients

CRediT authorship contribution statement

Guoliang Dai: Conceptualization, Investigation, Writing – original draft, Project administration. Dong Wang: Investigation. Shitang Ma: Methodology, Formal analysis. Shengwei Hong: Formal analysis. Kang Ding: Resources. Xiying Tan: Writing – review & editing. Wenzheng Ju: Conceptualization, Validation, Supervision, Writing – review & editing, Funding acquisition.

Declaration of Competing Interest

The authors declare that there are no conflicts of interest.

Acknowledgments

The authors are grateful for the financial support provided by the Natural Science Foundation of Jiangsu Province (grant NO. BK20211394).

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