Berberine-induced apoptosis in human prostate cancer cells is initiated by reactive oxygen species generation

https://doi.org/10.1016/j.taap.2007.12.027Get rights and content

Abstract

Phytochemicals show promise as potential chemopreventive or chemotherapeutic agents against various cancers. Here we report the chemotherapeutic effects of berberine, a phytochemical, on human prostate cancer cells. The treatment of human prostate cancer cells (PC-3) with berberine induced dose-dependent apoptosis but this effect of berberine was not seen in non-neoplastic human prostate epithelial cells (PWR-1E). Berberine-induced apoptosis was associated with the disruption of the mitochondrial membrane potential, release of apoptogenic molecules (cytochrome c and Smac/DIABLO) from mitochondria and cleavage of caspase-9,-3 and PARP proteins. This effect of berberine on prostate cancer cells was initiated by the generation of reactive oxygen species (ROS) irrespective of their androgen responsiveness, and the generation of ROS was through the increased induction of xanthine oxidase. Treatment of cells with allopurinol, an inhibitor of xanthine oxidase, inhibited berberine-induced oxidative stress in cancer cells. Berberine-induced apoptosis was blocked in the presence of antioxidant, N-acetylcysteine, through the prevention of disruption of mitochondrial membrane potential and subsequently release of cytochrome c and Smac/DIABLO. In conclusion, the present study reveals that the berberine-mediated cell death of human prostate cancer cells is regulated by reactive oxygen species, and therefore suggests that berberine may be considered for further studies as a promising therapeutic candidate for prostate cancer.

Introduction

The incidence of cancer is a growing health problem around the world particularly the prostate cancer is one of the leading causes of cancer-related deaths in men worldwide (Jemal et al., 2007). In the United States, one of nine men over the age of 65 years is diagnosed with prostate cancer (Jemal et al., 2007, Bosland et al., 2002). As surgery and current chemotherapeutic options seem to be inadequate in curing or controlling prostate cancer, there is an urgent need for the identification of alternative chemopreventive and chemotherapeutic strategies. The botanicals from traditional medicinal plants show promise in this area as their potential chemopreventive or chemotherapeutic actions in prostate cancer have been indicated by epidemiologic and experimental studies (Hong and Sporn, 1997, Kucuk, 2002, Barnes, 2001). Out of more than one hundred prescription drugs in use for cancer treatment, approximately 74% of these drugs were discovered by investigating a folklore claim (Craig, 1997, Craig, 1999). Chemically berberine is an isoquinoline alkaloid and is found in the roots, rhizome and stem bark of a number of medicinal plants (e.g., Berberis vulgaris, B. aquifolium, B. aristata and Tinospora cordifolia etc.) The potential medicinal importance of berberine has been described in the Ayurvedic, Unani and Chinese systems of medicine since time immemorial (Sathyavathi et al., 1987). Berberine has been shown to possess anti-inflammatory activities in vivo (Iizuka et al., 2000) and preliminary studies have been conducted to determine its anti-carcinogenic activity in skin (Nishino et al., 1986). It has been shown to inhibit activator protein-1, a key transcription factor in inflammation and carcinogenesis, in human cell lines (Fukuda et al., 1999a, Fukuda et al., 1999b), inhibit cyclooxygenase-2 transcriptional activity in human colon cancer cells (Lin et al., 1999, Fukuda et al., 1999a, Fukuda et al., 1999b), and inhibit DNA topoisomerase II (Kim et al., 1998). We have shown previously that in vitro treatment of human prostate cancer cells with berberine induces G1 phase cell cycle arrest and apoptosis which was associated with the activation of caspases (Mantena et al., 2006). As the induction of apoptosis is believed to be an important mechanism by which berberine may inhibit proliferation of cancer cells, an understanding of the mechanism of berberine-induced cancer cell death is essential for its further development as a clinically useful chemopreventive or chemotherapeutic agent because this knowledge could lead to identification of mechanism-based biomarkers potentially useful in future clinical trials.

Prostate cancer progresses from an androgen-responsive to an androgen-unresponsive state, and at the time of clinical diagnosis, most prostate cancers represent a mixture of androgen-responsive and androgen-unresponsive cancer cells (Gleave et al., 1998). Mortality among prostate cancer patients generally occurs from the abnormal proliferation and invasion of these androgen-unresponsive cells (Denmeade et al., 1996), but it is hard to identify those agents that can eradicate these tumor cells without incurring toxic responses in normal cells. Using primarily PC-3 (androgen unresponsive) human prostate cancer cells as a model, we provide experimental evidences that the berberine-induced apoptosis in human prostate cancer cells is initiated by generation of reactive oxygen species (ROS) leading to disruption of mitochondrial membrane potential, release of apoptogenic molecules from mitochondria to the cytosol and that leads to the activation of caspase-3 and PARP proteins. Under the conditions used in this study, treatment of berberine did not affect the levels of ROS and did not induce apoptosis in non-neoplastic human prostate epithelial cells (PWR-1E).

Section snippets

Chemicals and antibodies

The berberine chloride, N-acetyl-l-cysteine, DCFH-DA, allopurinol, xanthine and other chemicals employed in this study were of analytical grade and purchased from Sigma Chemical Co. (St. Louis, MO). The Annexin V-conjugated AlexaFluor 488 apoptosis and JC-1 mitochondrial membrane potential detection kits were purchased from Molecular Probes Inc. (Eugene, OR). The primary antibodies against caspase-9, cleaved caspase-9,-3, PARP and β-actin were purchased from Cell Signaling Technology (Beverly,

Berberine induces apoptosis in PC-3 cells

We have shown previously that treatment of human prostate carcinoma cells, LNCaP, DU145 and PC-3, with berberine results in inhibition of cell proliferation and induction of apoptosis. In contrast this effect of berberine was not observed on normal human prostate epithelial cells (PWR-1E) (Mantena et al., 2006). Based on our previous studies, the IC50 values with berberine for both PC-3 and LNCaP cells in vitro were 30 μM (Mantena et al., 2006). Here, we sought to determine the chemotherapeutic

Discussion

A considerable attention has been focused on identifying naturally occurring chemopreventive or chemotherapeutic botanicals capable of inhibiting, retarding or reversing the carcinogenic process including the prostate cancer development. The evaluation of ancient herbal medicines may provide the better strategies for the treatment of prostate cancer, which remains the leading cause of cancer-related deaths in US (Jemal et al., 2007). According to the American Cancer Society, the prostate cancer

References (36)

  • WolfB.B. et al.

    Suicidal tendencies: apoptotic cell death by caspase family proteinases

    J. Biol. Chem.

    (1999)
  • ZorattiM. et al.

    The mitochondrial permeability transition

    Biochim. Biophys. Acta

    (1995)
  • ZweierJ.L. et al.

    Determination of the mechanism of free radical generation in human aortic endothelial cells exposed to anoxia and reoxygenation

    J. Biol. Chem.

    (1994)
  • American Cancer Society

    Cancer facts and figures 2007

    (2007)
  • BoslandM.C. et al.

    Chemoprevention strategy for prostate cancer

    Eur. J. Cancer Chemoprev.

    (2002)
  • BarnesS.

    Role phytochemicals in prevention and treatment of prostate cancer

    Epidemiol. Rev.

    (2001)
  • CraigW.J.

    Health-promoting properties of common herbs

    Am. J. Clin. Nutr.

    (1999)
  • CromptonM.

    The mitochondrial permeability transition pore and its role in cell death

    Biochem. J.

    (1999)
  • Cited by (0)

    View full text