Flavonoids regulate cell death-related cellular signaling via ROS in human colon cancer cells

Highlights

• Catechins and naringenin showed considerable cytotoxicity in colon cancer cells.

• Catechins and naringenin induce cell death via upregulation of cell death-related genes.

• The pro-apoptotic and autophagic activity of catechins and naringenin was ROS dependent.

• Catechin and naringenin induce G2/M cell cycle arrest in cancer cells.

• Catechins and naringenin can suppress overexpressed CAMs in colon cancer cells.

Abstract

Although several studies investigated effects of flavonoids on proliferation and apoptosis, yet no study has correlated the cellular damage caused by reactive oxygen species (ROS) production, cell death related pathways and cell adhesion molecules (CAM) expression with cell survival. Here, we investigate cytotoxic effect of catechin, epicatechin and naringenin on colon cancer cells. While especially naringenin demonstrated most significant inhibition of colon cancer cell viability, high concentrations treatment did not exhibit more pronounced effect in colon epithelial cells. In addition, these flavonoids caused excessive ROS generation resulting in the impairment of lipid and protein, followed by the induction of apoptosis and autophagy. In details, mechanism studies revealed that elevated ROS production leads to caspase activation and pretreated NAC, an antioxidant, blocks catechins and naringenin induced apoptosis and autophagy. PKC activity assay data also showed that catechin and naringenin treatment decreases PKC activity that leads to cell death. Moreover, studied flavonoids, especially naringenin, induced G₂/M cell cycle arrest in a ROS-independent manner. Furthermore, the studied flavonoids suppressed or decreased the expression of the cancer progression and metastasis-related cell adhesion molecules. Taken together, our results indicate that studied flavonoids suppress colon cancer cell growth via inducing cell death in ROS dependent manner.

Introduction

Colon cancer is the third-most common cancer worldwide and its incidence has risen dramatically especially in developed countries [1]. A previous study indicated that dietary habits might responsible majority of colon cancer cases, and consumption of flavonoids is suggested to help in prevention of colorectal cancer [2,3]. Flavonoids are the most researched natural products for cancer prevention activity and have a wide variety of biochemical and pharmacological characteristics [4]. Flavonoids mediate their anticancer effects by targeting various signal transduction pathways, that modulate variety of cellular processes, such as cell cycle, apoptosis, DNA repair, glycolysis, invasion, and metastasis [5,6]. Current therapies for colon cancer, such as chemotherapy and radiation, have vital therapeutic value but the major obstacle in the clinical applications is non-specific toxicity [7]. For this reason, identification of new therapeutic agents, which may reduce the cancer patients’ mortality with less side effects, has a high priority.

Catechin and epicatechin, which are belong to flavanols subgroup, are the most abundant flavonoids in tea, and studies documented that catechins have therapeutic effects [8,9]. The hydroxylation pattern of catechin takes a critical effect on the their activity, especially in the protein kinase inhibition, which can lead to anti-proliferative activities [10]. Several other studies documented that catechins has therapeutic effects on cancer cells by inhibiting invasive cancer growth [[11], [12], [13]]. Naringenin, one of the most abundant flavonoids in grapefruit, presents wide antitumor activity such as inhibiting cell proliferation, inducing cell cycle arrest or apoptosis, reversing drug resistance, blocking subcellular signal transduction and promoting DNA repair [14]. A recent study showed that naringenin presents the anti-cancer effect in human pancreatic cancer by combining with hesperetin via down regulation of FAK pathway [15]. Another recent study demonstrated that naringenin exerts an anticancer effect in breast cancer cell line via arresting cell cycle, and it also affects the mitochondrial-mediated apoptosis in vivo [16]. Furthermore, others indicated that naringenin inhibited the migration and invasion prostate cancer cells by altering expression of genes involved in EMT (epithelial-to-mesenchymal transition) [17].

A study demonstrated that phenolic compounds exert their anticancer effect via their prooxidant properties, which increases ROS formation [18]. On the other hand, antioxidant capacities of many flavonoids have been also described both in vivo and in vitro [19]. Under normal conditions, while cellular defense enzymes maintain the balance of ROS content, excessive intracellular ROS in the presence of a therapeutic chemical ensures higher oxidative stress and leads to ROS and oxidative damage of molecules-induced cell death mechanisms such as apoptosis and autophagy [7]. Furthermore, ROS affects cell cycle progression depending on the amount and duration of ROS exposure. Activation of growth factor-induced signaling cascades with low ROS levels causes increased cell cycle progression or to growth arrest with the prolonged exposure [20]. In addition, earlier studies indicated that flavonoids have effects on inhibition of kinase activity, such as protein kinase CK2 [21], and matrix metalloproteinases [22] in tumor cells. Moreover, several studies have shown a straight relation of different PKC isoforms with flavonoid and ROS signaling [23,24]. Another study documented that the total ROS level and activity of PKC was down-regulated after quercetin treatment. They suggested quercetin modulates the PKC signal by decreasing oxidative stress and its cancer inhibiting activity via induction of apoptosis [25]. Despite knowledge on effect of flavonoids on many pathways, the effects on ROS-dependent PKC-mediated cell adhesion molecules (CAMs), are not known in detail. Jin et al. observed that flavonoids from Citrus unshiu Marc. (FCM) hinder TNF-induced cancer cell adhesion in HUVECs cells by inhibiting VCAM-1 via inhibition of PKC. They also suggested that the FCM has anti-metastatic activity by inhibiting adhesion molecules and invasion [26]. These findings led this group to hypothesis that indicated flavonoid would inhibit human colon cancer cell viability through inducing ROS-mediated cell death, and would correlate altered PKC and CAM expressions. The objective was to explain the ROS-mediated effects of catechin, epicatechin, and naringenin in colon cell lines with particular emphasis on apoptosis, autophagy, cell cycle, PKC, and CAMs.