Molecular mechanism of gossypol-induced cell growth inhibition and cell death of HT-29 human colon carcinoma cells

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Abstract

Gossypol, a male contraceptive drug, has been demonstrated to have antiproliferative and antimetastatic effects on many kinds of cancer cells in vitro. HT-29 human carcinoma cell line is one of the most susceptible cell lines to gossypol-induced cell death. Here, it is shown that treatment of HT-29 cells with gossypol not only induces cell cycle arrest on the G0/G1 phase, but also induces apoptosis. With a serial of Western blot analysis, it is revealed that gossypol-induced cell cycle arrest is involved in P21 up-regulation and cyclin D1 down-regulation; gossypol-induced apoptosis triggers down-regulation of anti-apoptosis Bcl-2 members: Bcl-XL, Bag-1 and Mcl-1, up-regulation of pro-apoptosis Bcl-2 member Bak, activation of caspase-3, -6, -7, -8, and -9, up-regulation of Apaf-1, release of cytochrome c (cyto-c) from mitochondria, and activation of both DFF45 and PARP. Taken together, gossypol-induced cell death initiates extensive alterations of cell cycle and apoptosis proteins. Gossypol-induced apoptosis of HT-29 cells is through first the mitochondrial pathway, then the death receptor pathway, and the mitochondria pathway is, at least in part, involved in cyto-c release.

Introduction

Gossypol is a polyphenolic compound (Fig. 1) originally isolated from cottonseed; it has been successfully used as a contraceptive drug for males for a long time [1]. It was first found by Tuszynski and Cossu to have anticancer effects against several tumor cell lines grown in tissue culture, and the most sensitive cell lines to gossypol-induced cell death were melanoma and colon carcinoma cells [2]. Since then more and more observations have been reported on its antiproliferative or antimetastatic activity, such as Ehrlich ascites tumor cells [3], P388 or L1210 murine leukemias [4], SW-13 adrenocortical carcinoma cells [5], murine erythroleukemia cells (clone 6A11A) [6], human glioma cell line HS 683, U373, U87, and U138 [7], hormone-dependent and hormone-independent breast carcinoma cells MCF-7 (drug-sensitive), MCF-7Adr (multidrug-resistant), MDA-MB-231 and T47D [8], [9], [10], [11], melanoma cell line SK-mel-19 and SK-mel-28 [12], promyelocytic leukemia cell line HL60 [13], [14], breast carcinoma cell line T47D and ovarian carcinoma cell line OVCAR-3 [15], [16], colon carcinoma cell line HT-29 and LoVo [17], metastatic MAT-LyLu lung cells [18], and other kinds of human or murine normal or carcinoma cells [19], [20], [21], [22], [23]. Even the milk collected from gossypol-treated dairy cows was reported to have antitumor activity [24]. It is worthy to note that the in vitro antitumor activity of gossypol and its derivatives is prevented by serum protein [25], [26], [27]. It seems that it is possible to enhance the antitumor activity of gossypol by synthesizing certain kinds of stereo-structure or structure modification derivatives [28], [29], [30]. Besides its male antifertility activity and anticancer activity, gossypol also inhibits the growth of numerous parasitic organisms and shows antiviral activity against a number of enveloped virus, including HIV virus [31].

Gossypol has been used as an anticancer drug in clinical research. For metastatic adrenal cancer, the response rate is similar to the other agents currently used for adrenal cancer, but responses were seen in patients who had failed other chemotherapeutic regimes [32]. For malignant gliomas, it shows a low, but measurable response [33], [34]. For refractory metastatic breast cancer, it shows a negligible antitumor activity [35]. There is one report indicating that gossypol is unlikely to be useful in patients with advanced cancer [36]. All these studies agreed that gossypol is tolerated and clinically safe.

Treatment with gossypol inhibits DNA synthesis of the cells [9], causes DNA breaks [30] or DNA fragmentation [14], [37]. The other target of gossypol is mitochondria. Ultrastructurally it caused marked swelling and vacuolization of mitochondria; there was an almost complete loss of cristae but the outer membrane was retained, upon cells being treated with gossypol [15], [20]. Various kinds of molecular targets have been investigated for gossypol-induced antiproliferative or antimetastatic activity. It was proposed that gossypol-induced DNA fragmentation or cell death resulted from its inhibition of protein kinase C activity [13], [37], [38]. Additionally, gossypol is a potent inhibitor of arachidonate 5- and 12-lipoxygenases [39]. In rat spermatocytes, gossypol-induced cell death is coupled with up-regulation of c-fos [40] and down-regulation of c-myc [41]. Most importantly, treatment of cancer cells with gossypol resulted in cell cycle arrest on G0/G1 phase [16], [35], [42], [43], [44]. The mechanism is that transforming growth factor-β is activated [42], [43] or P53 and P21 are up-regulated, and cyclin D1 and Rb are inhibited. It has been known that these four proteins are pivotal in controlling the progress of the cell cycle [35], [44].

Apoptosis is a major process for cell death. Little is known about the molecular mechanism of gossypol-induced apoptosis. There is only a brief report in HT-29 and LoVo cells indicating that gossypol-induced apoptosis may not be involved in the regulation of P53 but possibly associated with the regulation of Bcl-2 and Bax expression [17].

Since HT-29 is one of the most sensitive cell lines to gossypol-induced cytotoxicity, in the present study, the expression of molecules involved in mitosis and apoptosis pathways was examined. The results presented here demonstrate that gossypol-induced cell growth inhibition and cell death of HT-29 cells not only includes cell cycle arrest, but also includes apoptosis. HT-29 cell cycle arrest is triggered by up-regulation of P21 and down-regulation of cyclin D1; HT-29 cell apoptosis is through anti-apoptosis Bcl-2 member down-regulation, extensive caspase, DFF, and PARP activation.

Section snippets

Cells and reagents

Human colon cancer cell line HT-29 was obtained from National Cancer Institute (NCI), which was maintained in RPMI-1640 medium (Biofluids) supplemented with 10% (v/v) heated-inactivated bovine serum (Gibco, BRL) plus 1% glutamine (Gibco, BRL) at 37° in 5% (v/v) CO2. The expression of apoptosis cascade components was detected by using the following antibodies: anti-Bcl-2 mAb (Oncogene) for Bcl-2, anti-Bcl-XL mAb (Transduction Laboratories) for Bcl-XL, anti-Bax rabbit polyclonal antibody

Induction of cell cycle arrest and apoptosis of HT-29 cells by gossypol

To confirm gossypol-induced cell cycle arrest as reported [17], FACScan analysis was performed at only 24 hr after HT-29 cells were exposed to 5, 10 and 20 μM of gossypol. As shown in Fig. 2, 10 μM of gossypol caused more than 70% of the cells resting on G0/G1 phase of the cell cycle; at 20 μM concentration, almost all the cells died (data not shown). To quantify gossypol-induced apoptosis of HT-29 cells, Annexin V-Propidium Iodide assay was conducted. It was observed that as low as 1.0 μM of

Discussion

Van Poznak et al. recently proposed a model regarding gossypol-induced cell cycle arrest of breast cancer cells, which connected the relation of P53, P21, cyclin D1 and Rb to cell cycle [35]; alteration of any of these pivotal molecules would affect cell cycle. Inconsistent with previous report in HT-29 cells [17], the change of P53 was not observed in this experiment. The possible explanation is that P53 was not influenced by treatment with gossypol. Rb alterations have been observed in breast

Acknowledgements

The authors thank Dr. Karen Creswell in our Flow Cytometry Core Facility for her excellent assistance in operating FACScan, Karen Kreutzer in Division of Hematology/Oncology, University of Michigan for kindly helping with preparation of the manuscript.

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