Research section
Indole-3-carbinol and 3,3′-diindolylmethane induce apoptosis in human prostate cancer cells

https://doi.org/10.1016/S0278-6915(03)00004-8Get rights and content

Abstract

Cruciferous vegetables contain glucobrassicin which, during metabolism, yields indole-3-carbinol (I3C). In a low pH environment I3C is converted into polymeric products, among which 3,3′-diindolylmethane (DIM) is the main one. The apoptotic effects of I3C and DIM were exhibited in human breast cancer cells. The objectives of this study were: (a) examination of the potential effects of I3C and DIM on the proliferation and induction of apoptosis in human prostate cancer cell lines with different p53 status; (b) to try to characterise the mechanism(s) involved in these effects. Our results indicate that both indole derivatives suppress the growth of these cells in a dose- and time-dependent manner, by inducing apoptosis. It appears that these indolic compounds may offer effective means against prostate cancer. Induction of apoptosis was p53-independent. Moreover, the indole derivatives employed did not affect the levels of bcl-2, bax and fasL.

Introduction

Epidemiological studies have shown that diets rich in fruits and vegetables and low in fat and proteins are associated with a lower risk of cancer (Dragsted et al., 1993, Tavani, and Vecchia, 1995). A number of studies have demonstrated a decreased incidence of various cancers (including prostate cancer) in humans consuming large amounts of cruciferous vegetables, such as broccoli, Brussels sprouts, cabbage and cauliflower (Cohen et al., 2000). These vegetables contain glucobrassicin, which undergoes hydrolysis by myrosinase upon crushing, or by cooking (Bradfield, and Bjeldanes, 1987, Verhoeven et al., 1997). The main hydrolysis product of glucobrassicin is indole-3-carbinol (I3C). In a low pH environment, I3C is converted into many polymeric products, among which 3,3′-diindolylmethane (DIM) is the main one (Fenwick et al., 1982, Bradfield, and Bjeldanes, 1987). It was shown that these products have apoptotic properties. The apoptotic effect of I3C and DIM has been demonstrated in human breast cancer cells. I3C suppressed MCF-7 cell growth, in part by induction of apoptosis (programmed cell death), which was independent of p53 and bax expression. It may also be due to its conversion into a more potent compound, DIM (Fares et al., 1998, Ge et al., 1996, Ge et al., 1999). Animal studies have shown that mice and rats fed indolic compounds had a significantly lower incidence of both carcinogen-induced (Wattenberg, and Loub, 1978, Srivastava, and Shukla, 1998) and spontaneous tumours (Bradlow et al., 1991, Kojima et al., 1994), and the time intervals for the appearance of cancer was longer.

Recently it has been reported that I3C inhibits the proliferation of PC3, a poorly differentiated human prostate cancer cell line. Induction of G1 cell cycle arrest, leading to apoptosis, was also observed in those cells, treated with 30–100 μm of I3C (Chinni et al., 2001).

Prostate cancer is the most common diagnosed malignancy (accounting for 29% of the newly diagnosed cancers) and the second leading cause of male death in Western industrialized countries (Tang and Porter, 1997, Nupponen, and Visakorpi, 1999, Cookson, 2001). Age, race and family history are known risk factors for this disease; however, recently it has been found that nutritional and hormonal risk factors are also involved in prostate cancer (Chan et al., 1998). Mortality from prostate cancer results from metastases to the bones and lymph nodes and progression from androgen-dependent to androgen-independent prostatic growth (Bruckheimer and Kyprianou, 2000). Androgen withdrawal causes involution of the prostate gland, as a result of inhibition of cellular proliferation and stimulation of apoptosis of the androgen-dependent cells. Although androgen withdrawal remains the only effective therapy for men with advanced disease, in approximately 80% of the patients, progression to the lethal and untreatable stage of androgen-independence eventually occurs (Dorkin, and Neal, 1997, Abate-Shen, and Shen, 2000). The increasing incidence of prostate cancer in men all over the world, has led to the performance of intense investigations, searching for compounds having efficient suppressive effect against this type of cancer. These studies examined mainly apoptotic properties of the compounds investigated on human prostate cancer cells, under in vitro and in vivo conditions.

The purposes of our current study were to examine the potential beneficial effects of 3,3′-diindolylmethane on proliferation and induction of apoptosis in prostate cancer cells (both well and poorly differentiated) in vitro, and characterisation of the pathways involved in these apoptotic effects.

Section snippets

Materials

I3C was purchased from Sigma (Israel). 3,3′-diindolylmethane was purchased from Designed Nutritional Products (USA). Cell culture media and reagents were obtained from Biological Industries (Beit Haemek, Israel). Anti-bcl-2, anti-bax, anti-p53 and anti-actin monoclonal antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-PARP monoclonal antibody was purchased from Oncogene (USA). Anti-mouse IgG HRP antibody was purchased from Amersham Life Science (Arlington

Effect of I3C and DIM on inhibition of cell proliferation

In this study we used the prostate cancer cell lines LNCaP (with wild-type p53), DU145 (p53 mutant) and PC3 (deficient in p53 gene) to examine the effects of I3C and DIM on cell proliferation, in vitro. The prostate cancer cell lines were treated with I3C (10–400 μm) or DIM (10–100 μm) for 48 h. Cells were trypsinised and the population density was counted. The results indicated that I3C (Fig. 1A) and DIM (Fig. 1B) had an inhibitory effect on the growth of all cell lines and that this effect

Discussion

In this study we have demonstrated that the indole derivatives found in crucifers, I3C and DIM, induce apoptosis in three human prostate cancer cells, LNCaP, DU145 and PC3. We have previously shown that these indole derivatives have a suppressive effect on the growth of breast cancer cell lines (Ge et al., 1996, Fares et al., 1998). The IC50 values for DIM on growth inhibition of prostate cancer cells were similar to those found for breast cancer cells (20–40 and 17–30 μm, respectively) (Ge et

References (35)

  • C. Abate-Shen et al.

    Molecular genetics of prostate cancer

    Gene Development

    (2000)
  • C.A. Bradfield et al.

    High-performance liquid chromatographic analysis of anticarcinogenic indoles in brassica oleracea

    Journal of Agricultural and Food Chemistry

    (1987)
  • M.M. Bradford

    A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding

    Analytical Biochemistry

    (1976)
  • H.L. Bradlow et al.

    Effects of dietary indole-3-carbinol on estradiol metabolism and spontaneous mammary tumours in mice

    Carcinogenesis

    (1991)
  • E.M. Bruckheimer et al.

    Apoptosis in prostate carcinogenesis

    Cell Tissue Research

    (2000)
  • S.R. Chinni et al.

    Indole-3-carbinol (I3C) induced cell growth inhibition, G1 cell cycle arrest and apoptosis in prostate cancer cells

    Oncogene

    (2001)
  • J.H. Cohen et al.

    High consumption of vegetables associated with reduced risk of prostate cancer

    Evidence-based Oncology

    (2000)
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