Research Article
Effects of quercetin combined with anticancer drugs on metastasis-associated factors of gastric cancer cells: in vitro and in vivo studies
Introduction
Gastric cancer (GC) is the fifth most commonly diagnosed malignancy and the third leading cause of cancer mortality worldwide [1]. Although surgery is a crucial treatment for GC, chemotherapy and chemoradiation are necessary for patients to improve outcomes with resectable malignant tumors [2]. Many chemotherapeutic regimens have already demonstrated efficacy in GC [3]. One of the compounds used in those regimens is irinotecan, a water-soluble derivative of the plant alkaloid camptothecin. Irinotecan (also known as CPT-11) is actually a prodrug with lower toxicity and little biological activity, but its active metabolite, 7-ethyl-10-hydroxycamptothecin (SN-38), is a potent inhibitor of DNA topoisomerase I (Topo I) [4]. Once Topo I is trapped by anticancer drugs, double-strand DNA damage and subsequent induction of apoptosis occur [5]. High-dose irinotecan may cause adverse effects, including gastrointestinal toxicity (massive diarrhea, nausea, vomiting, or abdominal cramps) and hematological toxicity (neutropenia) [6]. In addition, irinotecan generates high levels of oxidative stress in cells [7], which leads to cell dysfunction and unpredictable impacts. Determining how to optimize the efficacy of the drug while reducing its side effects is still being investigated.
Angiogenesis plays a critical role in malignant tumor growth and metastasis and is modulated by proangiogenic and antiangiogenic factors [8]. Vascular endothelial growth factor (VEGF) is one of the molecules that is involved in physiological and pathological angiogenesis [9], [10]. Moreover, tumor cells express specific VEGF receptors (VEGFRs) to mediate VEGF signaling. Among three classical VEGFRs, VEGFR2 (also known as KDR) is the predominant tyrosine kinase (RTK) that regulates VEGF signaling and drives VEGF-mediated angiogenesis [11]. On top of that, the angiopoietin (Ang)-Tie system is another important mechanism contributing to the angiogenic process. Recent findings indicated that a subset of monocytes expressing Tie2, known as Tie2-expressing monocytes (TEMs), has an essential role in tumor angiogenesis [12], [13]. There are more TEMs penetrating into tumors when stimulated by Ang-2, leading to further tumor vascularization [14].
GC cells with more mesenchymal states present increased motility and invasiveness owing to decreased cell–cell adhesion, which is defined as the epithelial-mesenchymal transition (EMT), an indispensable process in cancer metastasis and progression [15]. E-cadherin is the key mediator of cell–cell adhesion [16], [17]. Down-regulation of E-cadherin expression in human tumors is commonly caused by methylation of its promoter or up-regulation of its transcriptional repressors, such as SNAIL, SLUG, and SIP1, resulting in higher incidences of metastasis and tumor recurrence [18]. Additionally, E-cadherin is a transmembrane glycoprotein, with a cytoplasmic domain binding to β-catenin and p120-catenin. Therefore, loss of E-cadherin expression brings about the liberation of β-catenin which can migrate into nuclei and there induce expressions of EMT-inducing transcriptional factors [19].
Quercetin (3,4′,5′,5,7-pentahydroxyflavone) is a common dietary flavonoid widely found in vegetables, fruits, and nuts [20]. Treatment of AGS and MKN28 human GC cells with quercetin gave rise to apoptotic effects through activation of the mitochondrion pathway [21]. Quercetin also enhances the efficacy of anticancer drugs via reducing P-glycoprotein expression, suppressing drug transporter, and down-regulating ABCB1 gene expression [22], [23]. Apart from this, a previous study revealed that a combination of quercetin and irinotecan had synergistic effects on reducing the peritoneal cavity tumor number in Ehrlich ascites tumor-bearing mice [24]. However, at present, no study has investigated the combined effects of quercetin with irinotecan or SN-38 on GC. We hypothesized that the combination of quercetin and irinotecan may work more effectively at decreasing cancer cell metastasis-related genes and protein expression and consequently slowing the development of GC. In vitro and xenograft animal studies were performed to evaluate the possible efficacy of quercetin in combination with low-dose irinotecan on the metastasis of GC.
Section snippets
Cell culture
The AGS human gastric adenocarcinoma cell line (purchased from ATCC, Manassas, VA, USA) was maintained in RPMI1640 medium supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine, and 10 μg/mL gentamicin.
Cell viability
After AGS cells were seeded overnight at a density of 5×103 cells per well in 96-well plates, cells were exposed to quercetin (6.25, 12.5, 25, 50, and 100 μM), SN-38 (2.5, 5, 25, 50, and 100 nM), and a combination of quercetin and SN-38 for 48 h. Dimethyl sulfoxide (DMSO) at a final
Effects of the combination of quercetin and SN-38 on AGS cell survival and apoptosis
Cell viability was determined using alamarBlue®. Treatment with quercetin or SN-38 resulted in growth inhibition of AGS cells in dose-dependent manners. The 50% inhibitory concentration (IC50) values of quercetin and SN-38 against AGS cells were estimated to be 50 μM and 25 nM, respectively (Fig. 1A and B). The cell survival rate in the group treated with 5 nM SN-38 in combination with 12.5 μM quercetin (S5 + Q12.5) did not statistically significantly differ from the group treated with 25 nM
Discussion
Quercetin appears to be a potent chemopreventive agent in a wide range of cancers, such as lung, breast, prostate, and colon cancers. Even though the relationship between GC and quercetin is still ambiguous, our results demonstrated that a combination of quercetin and a low dose of the irinotecan metabolite, SN-38, had comparable effects in inhibiting tumor cell growth and enhancing apoptosis as those of high-dose SN-38. The subsequent xenograft animal model also showed that metastasis-related
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