Tangeretin and nobiletin induce G1 cell cycle arrest but not apoptosis in human breast and colon cancer cells

Abstract

Tangeretin and nobiletin are citrus flavonoids that are among the most effective at inhibiting cancer cell growth in vitro and in vivo. The antiproliferative activity of tangeretin and nobiletin was investigated in human breast cancer cell lines MDA-MB-435 and MCF-7 and human colon cancer line HT-29. Both flavonoids inhibited proliferation in a dose- and time-dependent manner, and blocked cell cycle progression at G1 in all three cell lines. At concentrations that resulted in significant inhibition of proliferation and cell cycle arrest, neither flavonoid induced apoptosis or cell death in any of the tumor cell lines. To test the ability of arrested cells to recover, cells that were incubated with tangeretin and nobiletin for 4 days were then cultured in flavonoid-free medium for an additional 4 days. Cells resumed proliferation similar to untreated control within a day of flavonoid removal. Cell cycle distribution was similar to that of control within 4 days of flavonoid removal. These data indicate that, in these cell lines at concentrations that inhibit proliferation up to 80% over 4 days, tangeretin and nobiletin are cytostatic and significantly suppress proliferation by cell cycle arrest without apoptosis. Such an agent could be expected to spare normal tissues from toxic side effects. Thus, tangeretin and nobiletin could be effective cytostatic anticancer agents. Inhibition of proliferation of human cancers without inducing cell death may be advantageous in treating tumors as it would restrict proliferation in a manner less likely to induce cytotoxicity and death in normal, non-tumor tissues.

Introduction

Cytotoxic anticancer drug efficacy is contingent upon differences in drug sensitivity between normal and tumor tissue (i.e., therapeutic index). The therapeutic index of these drugs is often small, with little difference between the dose required for optimal antitumor activity (effective dose) and that which produces unacceptable toxicities in normal tissues (toxic dose) [1]. The ideal anticancer agent would exert minimal adverse effects on normal tissues with maximal capacity to kill tumor cells and/or inhibit tumor growth (i.e., with large therapeutic indices). Targeting tumor-associated molecules is one way to enhance therapeutic index [2]. However, a molecularly targeted agent has maximal antitumor effect and minimal host toxicity only when its target is the single cause of disease or is only found in tumor tissues [3]. Another approach is to target a physiological process essential to tumor growth such as cellular proliferation, but not processes involved in survival. With this approach, tumor cell proliferation can be reduced or halted while cell viability is relatively unimpaired (cytostasis). Most normal tissues would be less functionally impeded by cytostatic agents, thus increasing the gap between effective and toxic doses. Even if affected, cell viability and the capacity to quickly return to normal function would remain more intact in non-tumor tissues [4]. With less dependence on biochemical differences between tumor and normal cells, cytostatic agents potentially have an important role to play in anticancer chemotherapy.

Because they are inherently low in toxicity, naturally occurring constituents of the human diet such as flavonoids (low molecular weight polyphenols ubiquitous in plants) are of particular interest as potential sources of compounds with cytostatic activity [5], [6]. There is a correlation between diets high in fruits and vegetables and decreased risk of cancer [7], [8]. High consumption of fruits and vegetables is significantly protective against risk of cancers arising in the lung, colon, ovary, esophagus, breast, cervix, bladder, pancreas, oral cavity and stomach [9]. Flavonoids have demonstrated good potential as anticancer agents by their antiproliferative activity against human tumor cell lines both in vitro[10], [11] and as xenografts in immunocompromised mice [12], [13].

Two polymethoxylated flavones, 4′,5,6,7,8-pentamethoxyflavone (tangeretin) and 3′,4′,5,6,7,8-hexamethoxyflavone (nobiletin), are among the most effective citrus flavonoids at inhibition of human cancer cell proliferation [14]. Tangeretin and nobiletin inhibited proliferation of human cancer cell lines derived from squamous cell carcinoma [15], [16], gliosarcoma [16], leukemia [17], [18], melanoma [19], [20], colorectal cancer [21], gastric carcinoma [18], and lung carcinoma [18]. In addition, they have been reported to decrease the incidence of chemically induced tumors in mice and rats [22], [23], [24]. Although the chemopreventive and antiproliferative mechanisms of action may differ, the in vivo tumor preventive activity of tangeretin and nobiletin demonstrate the potential for their safe pharmacological use.

At the cellular and molecular level, flavonoids have a broad range of effects and interactions that contribute to their chemopreventive, anticarcinogenic and antiproliferative activities. The modulation of cellular processes such as cell cycle and apoptosis contributes to their antiproliferative effects [10], [25]. At the molecular level, flavonoids interact with receptors, enzymes, and kinases [26], [27], [28], [29]. These cellular and molecular effects also mediate the antiproliferative effect of tangeretin and nobiletin in human cancers. Both flavonoids have been shown to induce cell cycle arrest and apoptosis in tumor cell lines [17], [21], [30], [31], [32]. Even though flavonoids are considered non-toxic constituents of the diet, their antiproliferative action reportedly relies on induction of cell death. There is little evidence to date that tangeretin and nobiletin can significantly inhibit tumor cell proliferation in a purely cytostatic, non-cytotoxic manner. This mode of action could potentially avoid complications of toxicity to normal tissues.

In this study, tangeretin and nobiletin inhibited proliferation of human breast and colon cancer cells in the absence of cytotoxicity. Inhibition of proliferation of human cancers without inducing cell death may be advantageous in treating human tumors in their natural environment, as it would restrict proliferation in a manner less likely to cause cytotoxicity and induce death in non-tumor tissues.