Elsevier

Life Sciences

Volume 306, 1 October 2022, 120859
Life Sciences

Pt3R5G inhibits colon cancer cell proliferation through inducing ferroptosis by down-regulating SLC7A11

https://doi.org/10.1016/j.lfs.2022.120859Get rights and content

Abstract

Aims

Colon cancer (CC) is a prevalent malignancy worldwide and is one of the most easily altered cancers by dietary regulation. Petunidin 3-O-[rhamnopyranosyl-(trans-p-coumaroyl)]-5-O-(β-D-glucopyranoside) (Pt3R5G) isolated and purified from Lycium ruthenicum Murray, which exhibits highly efficient antioxidant activity and specific anticancer effects, is the flavonoids compound. We aimed to study the effect of Pt3R5G on CC cells and elucidate the potential underlying mechanisms.

Main methods

Cell proliferation was measured by the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) and colony formation assays. Cell cycle, cell apoptosis and reactive oxygen species (ROS) analysis were performed by flow cytometry. RNA-sequencing was performed to elucidate the potential underlying mechanisms. The lipid peroxidation level of cells was detected by malondialdehyde (MDA) assay. The mitochondrial morphology of cells was inspected using a transmission electron microscope. Additionally, we overexpressed SLC7A11 to perform rescue experiments. In vivo, xenograft mice assay was performed to verify the effect of Pt3R5G on the growth of colon cancer.

Key findings

Pt3R5G reduced the cell activity by blocking the cell cycle in G0/G1 phase, inducing the apoptosis and ferroptosis in RKO cells. The overexpressed of SLC7A11, a significantly down-regulated expression gene caused by Pt3R5G, rescued the cell proliferation inhibition and ferroptosis process. Furthermore, Pt3R5G inhibited tumor growth in nude mice. Our study suggests that Pt3R5G inhibits RKO cell proliferation through mainly reducing ferroptosis by down-regulated SLC7A11.

Significance

As a potential therapeutic drug, Pt3R5G showed efficient anticancer activity through a variety of pathways.

Introduction

Colorectal cancer (CRC) is the third most common cancer in both sexes combined, accounting for 10.2 % of the total cancer deaths, ranked second for 9.2 % mortality [1], [2]. Current treatments for colorectal cancer, including chemotherapy, radiotherapy and surgery are utilized to palliate symptoms and prolong life, but all of which are associated with a high risk of complications and are not always successful [3]. The risk of developing colorectal cancer is increased by smoking, alcohol intake and excessive intake of red meat. By contrast, consumption of milk, whole grains, fresh fruits and vegetables decreases this risk [4]. Digestive tract cancers, especially colon cancer, is amenable to dietary modification. Previous research showed that colorectal cancer is affected by diets rich in fruits and vegetables [5], [6], [7]. Plant foods contain a variety of components, including micronutrients, polyunsaturated fatty acids and phytochemicals such as flavonoids with potent bioactivity [8]. Recently, the consumer perception is that phytochemicals with antioxidant properties in fruits and vegetables might prevent some processes involved in the development of cancer, such as protecting DNA from oxidative damage [9].

Anthocyanins are polyphenolic pigments that belong to the flavonoids in fruits and vegetables that render them vivid red to blue. Six anthocyanidins are predominant in nature which represent ∼90 % of all anthocyanins identified to date, including Pelargonidin, Cyanidin, Peonidin, Delphinidin, Petunidin and Malvidin [10]. Anthocyanins have attracted the attention of the scientific community because anthocyanins have anti-inflammatory and anti-carcinogenic activity, cardiovascular disease prevention, obesity control, and diabetes alleviation properties [11], [12], [13], [14], [15]. Anthocyanin is a kind of potential phytochemical that shows chemopreventive effects against oxidative stress-induced cell damage. Anthocyanins promote the expression of phase II enzymes regulated by antioxidant response element (ARE) through the Nrf2 pathway, thus inhibiting the activity of caspase-3 and protecting normal cells from oxidative stress [16]. Moreover, their potential antitumor effects are reported to be based on a wide variety of biological activities including antioxidants; inhibiting proliferation by modulating signal transduction pathways, inducing cell cycle arrest and stimulating apoptosis or autophagy of cancer cells [17]. Delphinidin protected HaCaT keratinocytes and mouse skin against UVB-mediated oxidative stress and apoptosis [18]. Anthocyanins from Java plum fruit extract suppressed proliferation in HCT-116 cells and increased apoptosis in both HCT-116 cells [19]. Anthocyanidins inhibited epithelial–mesenchymal transition through a TGF-β/Smad2 signaling pathway in glioblastoma cells [20]. Above all, anthocyanins have anti-cancer activity by interfering with a variety of signaling pathways.

Over the past decade, researchers have investigated various programmed cell deaths such as apoptosis, necrosis and autophagy. Recently, ferroptosis has gradually gained attention [21]. Discovered by Scott J Dixon et al. in 2012, ferroptosis is an iron-dependent form of nonapoptotic cell death which was triggered by the oncogenic RAS-selective lethal small molecule “erastin” [22]. As a novel death phenotype which is distinct from apoptosis (chromatin condensation), necrosis (cell membrane rupture and content release) and autophagy (double-layer autophagic vacuole formation), ferroptosis is characterized by the overwhelming, iron-dependent accumulation of lethal lipid ROS and leads to a decrease in the density of the mitochondrial membrane rather than morphological changes [23], [24], [25]. General ROS production is not related to ferroptosis because the compounds that induce cytosolic or mitochondrial ROS do not induce ferroptosis [26], [27].

Ferroptosis has recently been implicated in multiple diseases and is functional as a tumor suppression mechanism [28], [29]. Most antitumor drugs exert their anticancer effects through a variety of molecular regulatory mechanisms reported to date. For example, Bing Yuan et al. found that dihydroartemisinin inhibited the proliferation, colony formation and induced ferroptosis of lung cancer cells by inhibiting PRIM2/SLC7A11 axis [30]. Understanding the mechanisms underlying the anticancer effects of these effective natural pharmaceutical ingredients is highly valuable for fully exploring the potential drugs and for developing new ideas for tumor therapy.

In this study, the flavonoids compound, Pt3R5G was isolated and purified from the medicinal fruit Lycium ruthenicum Murray, which exhibited highly efficient antioxidant activity [31] (Fig. 1A). However, the antitumor properties of Pt3R5G are not well characterized. Our data suggested that Pt3R5G was a type of potential phytochemical that showed antitumor effects.

Section snippets

Pt3R5G, plasmids construction and transfection

An anthocyanin compound with over 97 % purity, Petunidin 3-O-[rhamnopyranosyl-(trans-p-coumaroyl)]-5-O-[β-D-glucopyranoside] (Pt3R5G) was isolated and purified from fruits of Lycium ruthenicum Murray, kindly provided by Prof. CAO Youlong from National Wolfberry Engineering Research Center, Yinchuan. Pt3R5G was dissolved in phosphate-buffered saline (PBS) to 1000 μM concentration and stored at −80 °C. The SLC7A11 overexpression vector and control were purchased from GeneChem (Shanghai Genechem

Pt3R5G suppressed RKO cell growth

To investigate whether Pt3R5G has an effect on the cell viability in RKO cells, we determined the relative cell viability of RKO cells treated with Pt3R5G employing the MTT assay. We found that Pt3R5G (125–500 μg/mL, 48 h) treatment resulted in significant growth inhibition of RKO cells. Pt3R5G treatment resulted in 9 %, 51 %, 74 %, 77 %, 78 %, 80 % and 80 % decrease in cell viability at 125, 150, 200, 250, 300, 400 and 500 μg/mL, respectively (Fig. 1B). The IC50 value of Pt3R5G for RKO cells

Discussion

Epidemiological analysis suggests that the increased incidence of colon cancer points to the influence of dietary patterns, obesity and lifestyle factors [34]. The American Institute for Cancer Research/World Cancer Research Fund (AICR/WCRF) report indicated that red meat, processed meat, alcoholic beverages and body fat increased the risk of colon cancer, while exercise had a protective effect [35]. Studies have shown an association between flavonoid consumption and a reduced risk of CRC [36].

Conclusions

Pt3R5G inhibits cell proliferation through inducing ferroptosis by down-regulating SLC7A11 in colon cancer. As a potential therapeutic drug, Pt3R5G showed good anticancer effects through a variety of pathways.

Ethics statement

The animal experiment was reviewed and approved by the Biomedical Ethics Committee of the Medical Department of Xi'an Jiaotong University.

Funding

This work was supported by the Pilot project by Ningxia Academy of Agriculture and Forestry Science (QCYC-2018-05), and the Scientific Research and Sharing Platform Construction Project of Shaanxi Province (Grant Number: 2022PT-07).

CRediT authorship contribution statement

Lin Han: Investigation, Formal analysis, Writing – original draft. Yamei Yan: Resources. Meiyang Fan: Investigation, Data curation. Shanfeng Gao: Resources. Lingyu Zhang: Data curation. Xiaofan Xiong: Writing – review & editing. Rufeng Li: Methodology. Xuan Xiao: Data curation. Xiaofei Wang: Visualization. Lei Ni: Project administration. Dongdong Tong: Data curation. Chen Huang: Conceptualization, Supervision. Youlong Cao: Funding acquisition. Juan Yang: Project administration, Writing – review

Declaration of competing interest

The authors declared no conflict of interest.

Acknowledgments

The authors wish to thank Xiaoge, Zhao (Biomedical Experiment Center, Xian Jiaotong University) for technical assistance of animal experiment.

References (51)

  • D. Tsikas

    Assessment of lipid peroxidation by measuring malondialdehyde (MDA) and relatives in biological samples: analytical and biological challenges

    Anal. Biochem.

    (2017)
  • M.Z. Ma

    Xc- inhibitor sulfasalazine sensitizes colorectal cancer to cisplatin by a GSH-dependent mechanism

    Cancer Lett.

    (2015)
  • C. Yang

    Apigenin enhances apoptosis induction by 5-fluorouracil through regulation of thymidylate synthase in colorectal cancer cells

    Redox Biol.

    (2021)
  • J.R. Araujo et al.

    Chemopreventive effect of dietary polyphenols in colorectal cancer cell lines

    Nutr. Res.

    (2011)
  • S. Zeng

    Scutellarin ameliorates colitis-associated colorectal cancer by suppressing Wnt/beta-catenin signaling cascade

    Eur. J. Pharmacol.

    (2021)
  • J.P.F. Angeli

    Ferroptosis inhibition: mechanisms and opportunities

    Trends Pharmacol. Sci.

    (2017)
  • Erratum: Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries

    CA Cancer J. Clin.

    (2020)
  • F. Bray

    Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries

    CA Cancer J. Clin.

    (2018)
  • G. Binefa

    Colorectal cancer: from prevention to personalized medicine

    World J. Gastroenterol.

    (2014)
  • E.J. Kuipers

    Colorectal cancer

    Nat. Rev. Dis. Primers

    (2015)
  • M. Song et al.

    Nutrients, foods, and colorectal cancer prevention

    Gastroenterology

    (2015)
  • S.K. Jaganathan

    Chemopreventive effect of apple and berry fruits against colon cancer

    World J. Gastroenterol.

    (2014)
  • A. Rodriguez-Casado

    The health potential of fruits and vegetables phytochemicals: notable examples

    Crit. Rev. Food Sci. Nutr.

    (2016)
  • J. He et al.

    Anthocyanins: natural colorants with health-promoting properties

    Annu. Rev. Food Sci. Technol.

    (2010)
  • M.L. Ho

    Peonidin 3-glucoside inhibits lung cancer metastasis by downregulation of proteinases activities and MAPK pathway

    Nutr. Cancer

    (2010)
  • Cited by (0)

    1

    These authors share first authorship: Lin Han, Yamei Yan.

    2

    These authors share last authorship: Juan Yang, Youlong Cao, Chen Huang.

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