Berberine inhibits the proliferation of prostate cancer cells and induces G0/G1 or G2/M phase arrest at different concentrations

  • Authors:
    • Wei Lu
    • Shanshan Du
    • Jiaqiang Wang
  • View Affiliations

  • Published online on: December 30, 2014     https://doi.org/10.3892/mmr.2014.3139
  • Pages: 3920-3924
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Abstract

Prostate cancer is the second most common disease of the male reproductive system. Berberine is a quaternary ammonium salt that is extracted from plants. The aim of the current study was to explore the antitumor activity of berberine in prostate cancer cells and identify the underlying mechanism of its effects. PC3 human and RM‑1 mouse prostate cancer cells were treated with increasing concentrations of berberine, followed by analysis of the cell viability with an MTT assay. The results demonstrated that berberine markedly inhibited the proliferation of PC3 and RM‑1 cells, and that the inhibitory effects to PC3 and RM‑1 were enhanced in a concentration‑ and time‑dependent manner. Flow cytometry was used to analyze the cell cycle of PC3 human prostate cancer cells, and the results demonstrated that G0/G1 phase arrest was induced following treatment with 10 µM berberine (P<0.05). However, with an increased concentration of berberine (50 µM) the survival rate of PC3 cells at the G2/M phase was significantly increased compared with the cells treated with 10 µM berberine, which suggests that different cell cycle signaling pathways were activated when PC3 cells were treated with low and high concentrations of berberine. Thus, clarifying the mechanism underlying these effects in prostate cancer may provide novel molecular targets for prostate cancer therapy.

Introduction

Prostate cancer is a malignant disease that originates in the prostate gland, and it is the second most commonly observed disease of the male reproductive system (1). The incidence of prostate cancer increase with age and there are clear regional differences (24), for example the incidence is higher in the US and Europe compared with Asia (5,6). It has been reported that prostate cancer is the second most common cause of cancer-related mortality among males, with a high mortality rate (7). In China, the incidence is lower in younger males, however, the mortality rate is strongly associated with age, with the highest mortality rates observed in older males (8,9). With the rise in the number of older individuals in China, the number of mortalities associated with prostate cancer has increased in recent years (810).

Berberine is a quaternary ammonium salt and a member of the protoberberine group of isoquinoline alkaloids. It is an important ingredient with biological properties in Chinese medicine (11,12). It is predominantly found in the roots, rhizomes, stems and bark of a number of medicinal plants, including Mahonia aquifolium (Oregon grape), Phellodendron amurense (Amur cork tree), Berberis aristata (tree turmeric), Hydrastis canadensis (goldenseal), Coptis chinensis (Chinese goldthread), Tinospora cordifolia and Eschscholzia californica (Californian poppy). Berberine has a strong inhibitory effect on growth and replication in a variety of microorganisms (13,14). Berberine has primarily been used in early clinical trials to treat intestinal infections or diarrhea (15,16). In addition, it has been found that berberine has an important role in lowering blood pressure in the treatment of cardiovascular disease and diabetes (1719).

Recently, in vivo and in vitro evidence has shown that berberine has potent anti-inflammatory and antitumor effects; however, its mechanism of action has yet to be clarified (2023). In the present study, the antitumor effects of berberine were explored and the underlying mechanisms were investigated in PC3 human and RM-1 mouse prostate cancer cell lines.

Material and methods

Cells and reagents

PC3 human and RM-1 mouse prostate cancer cell lines were obtained from American Type Culture Collection (Manassas, VA, USA) and cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal calf serum (Hyclone Corporation, South Logan, UT, USA). Berberine and MTT were purchased from Sigma-Aldrich (St. Louis, MO, USA). The purity of berberine was ≥98% and it was dissolved in dimethyl sulfoxide (Sigma-Aldrich).

MTT assay

The cells were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal calf serum in an atmosphere of 5% CO2. When the cell density reached ~70%, different concentrations of berberine were added to treat the cells. In one group, the prostate cancer cells were incubated with 5, 10, 20 or 50 μM berberine for 24 h, while in the other group, the cells were incubated with 10 μM berberine for 24, 48 or 72 h. The survival rate of the cells was then measured via an MTT assay as previously described (24,25). The 96-well plates containing the cells were read on a microplate reader (Thermo Scientific, Rockford, IL, USA) with a test wavelength of 490 nm and a reference wavelength of 570 nm.

Flow cytometric analysis

The levels of apoptosis of the PC3 cells were tested by Annexin V/propidium iodide (PI) dual staining according to the manufacturer’s instructions (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA). Briefly, the cells were washed in phosphate-buffered saline and treated with different concentrations of berberine (10 and 50 μM)for 24 or 48 h followed by resuspension in a binding buffer (10 mM HEPES-NaOH pH 7.4; 25 mM CaCl2 and 144 mM NaCl). Subsequently, Annexin V (0.1 μg/μl) and PI (0.05 μg/μl) staining dyes were added and the cells were incubated in the dark for 30 min on ice. The cells were then subjected to fluorescence-activated cell sorting (FACS) analysis; the samples were tested by the BD FACSCalibur Cell Sorting system (BD Biosciences, San Jose, CA, USA).

Statistical analysis

SPSS statistical package version 11.5 (SPSS, Inc., Chicago, IL, USA) was used to analyze the data. All experiments were performed at least three times and results are presented as the mean ± standard error of the mean. P<0.01 was considered to indicate a statistically significant difference.

Results

Antitumor effects of berberine on prostate cancer cells increase with concentration

In order to explore whether berberine exhibited antitumor effects on prostate cancer cells, the PC3 human and RM-1 mouse prostate cancer cell lines were used as cellular models. The structure of berberine is shown in Fig. 1. Different concentrations of berberine were used to treat PC3 and RM-1 cells for 24 h. As shown in Fig. 2, the survival rates of PC3 and RM-1 cells as detected by an MTT assay were markedly reduced when treated with increasing concentrations of berberine. The results demonstrated that berberine has an antitumor effect and that the inhibitory effects increased in a concentration-dependent manner.

Berberine kills prostate cancer cells in a time-dependent manner

The PC3 and RM-1 cells were treated for different times with berberine at a concentration of 10 μM. The MTT assay (Fig. 3) revealed that the survival rate of prostate cancer cells was decreased in a time-dependent manner. Here, untreated cells were used as negative controls and all of the samples were analyzed in duplicates.

Berberine induces the apoptosis of human prostate cancer PC3 cells

PC3 human prostate cancer cells were treated with 10 μM or 50 μM berberine for 24 h. As shown in Fig. 4, the apoptosis rates of the PC3 cells treated with berberine were significantly higher compared with those of the untreated cells, and the apoptosis rate increased in a concentration-dependent manner.

Berberine induces cell-cycle arrest in the G0/G1 and G2/M phases

In order to elucidate the mechanism underlying the effects of berberine, cell-cycle analysis of PC3 cells was performed using a FACS assay. PC3 cells were treated with 10 μM berberine for 24 or 48 h, and cell-cycle analysis was performed using a PI staining method (Fig. 5). As shown in Fig. 6, the distribution of PC3 cells in the cell cycle phases was analyzed. The results demonstrated that the treatment of cells with 10 μM berberine induced G0/G1 phase arrest (P<0.05). However, with an increased berberine concentration of 50 μM, the survival rates of the PC3 cells in the G2/M phase (28.4%; data not shown) were significantly increased compared with those treated with 10 μM berberine (13.2%; data not shown), which suggests that different signaling pathways associated with the cell cycle were activated when PC3 cells were treated with low and high concentrations of berberine.

Discussion

Prostate cancer is one of the most common types of malignancies in males, which significantly affects quality of life (1,26). In the late stages of prostate cancer, treatment primarily consists of chemotherapy. However, the chemotherapeutic drugs that are currently used are expensive and have serious side-effects, hence, it is useful to identify novel natural compounds with low toxicity for the treatment of prostate cancer. Berberine is an important compound that is extracted in traditional Chinese medicine (27). It has an key role in clinical trials and in the anti-inflammatory response, and has antitumor effects (2830). In the present study, we further explored the mechanism of anti-tumor activity of beberine on human and mouse prostate cancer cells. Berberine is primarily used in the clinical treatment of intestinal infections. Recent studies have found that berberine may have an important role in the treatment of diabetes and cardiovascular diseases in addition to antitumor activity.

In the present study, the inhibitory effects of berberine on prostate cancer and its mechanisms were investigated. PC3 human and RM-1 mouse prostate cancer cells were used as cellular models. The results of the MTT assay revealed that berberine significantly inhibited the proliferation of PC3 and RM-1 cells in a concentration- and time-dependent manner. Cell cycle distribution results showed that berberine induced G0/G1 phase arrest and apoptosis in PC3 prostate cancer cells, which is consistent with the results described in other studies (3133).

Berberine inhibits the growth of a variety of tumor cells, and its antitumor mechanisms include the induction of cell cycle arrest and apoptosis (3437). The current study determined that different signaling pathways were activated when prostate cancer cells were treated with low or high concentrations of berberine. The results demonstrated that G0/G1 phase arrest was induced at a lower concentrarion of berberine, and G2/M phase arrest was observed with an increased berberine concentration of 50 μM. Different proteins were expressed at different cell cycle stages, and these proteins are associated with different signalling pathways. However, the regulatory mechanisms by which berberine induced G2/M phase arrest remain unclear. In addition, Liu et al (38) found that high concentrations of berberine could induce G2/M phase arrest in human osteosarcoma cells, and that this was not dependent on p53 function.

In conclusion, the results of the present study have provided a rationale for the development of berberine-based therapies to treat malignant tumors and have aided in the elucidation of the mechanism underlying the antitumor activity of berberine in prostate cancer cells.

References

1 

Xiao H, Tan F, Goovaerts P, et al: Multilevel factors associated with overall mortality for men diagnosed with prostate cancer in florida. Am J Mens Health. 8:316–326. 2013. View Article : Google Scholar : PubMed/NCBI

2 

Gerritse FL, Meulenbeld HJ, Roodhart JM, et al: Analysis of docetaxel therapy in elderly (≥70 years) castration resistant prostate cancer patients enrolled in the Netherlands Prostate Study. Eur J Cancer. 49:3176–3183. 2013. View Article : Google Scholar : PubMed/NCBI

3 

Damaschke NA, Yang B, Bhusari S, Svaren JP and Jarrard DF: Epigenetic susceptibility factors for prostate cancer with aging. Prostate. 73:1721–1730. 2013. View Article : Google Scholar : PubMed/NCBI

4 

No authors listed. Summaries for patients. Effect of age, tumor risk, and comorbidity on prostate cancer survival. Ann Intern Med. 158:709–717. 2013. View Article : Google Scholar

5 

Greenberg DC, Wright KA, Lophathanon A, Muir KR and Gnanapragasam VJ: Changing presentation of prostate cancer in a UK population - 10 year trends in prostate cancer risk profiles in the East of England. Br J Cancer. 109:2115–2120. 2013. View Article : Google Scholar : PubMed/NCBI

6 

Carter HB: American Urological Association (AUA) guideline on prostate cancer detection: process and rationale. BJU Int. 112:543–547. 2013. View Article : Google Scholar : PubMed/NCBI

7 

Lamy S, Wilmart JF, Hein T, Scheiden R and Capesius C: Prostate cancer in Luxembourg from 1982 to 2006. Incidence and mortality. Survival of a hospital cohort. Bull Soc Sci Med Grand Duche Luxemb. 6–19. 2013.(In French).

8 

Peng P, Gong YM, Bao PP, et al: Estimates and prediction of prostate cancer incidence, mortality and prevalence in China, 2008. Zhonghua Liu Xing Bing Xue Za Zhi. 33:1056–1059. 2012.(In Chinese).

9 

Zhang L, Wu S, Guo LR and Zhao XJ: Diagnostic strategies and the incidence of prostate cancer: reasons for the low reported incidence of prostate cancer in China. Asian J Androl. 11:9–13. 2009. View Article : Google Scholar

10 

Song FJ, Zhang BL, He M, et al: Trend analysis of the incidence of prostate cancer in Tianjin between 1981 and 2004. Zhonghua Yi Xue Za Zhi. 90:2811–2814. 2010.(In Chinese). PubMed/NCBI

11 

Wang JR, Tanaka T, Zhang H, Kouno I and Jiang ZH: Formation and conformation of baicalin-berberine and wogonoside-berberine complexes. Chem Pharm Bull (Tokyo). 60:706–711. 2012. View Article : Google Scholar

12 

Philogène BJ, Arnason JT, Towers GH, et al: Berberine: A naturally occurring phototoxic alkaloid. J Chem Ecol. 10:115–123. 1984. View Article : Google Scholar : PubMed/NCBI

13 

Li B, Zhu WL and Chen KX: Advances in the study of berberine and its derivatives. Yao Xue Xue Bao. 43:773–787. 2008.(In Chinese). PubMed/NCBI

14 

Chin LW, Cheng YW, Lin SS, et al: Anti-herpes simplex virus effects of berberine from Coptidis rhizoma, a major component of a Chinese herbal medicine, Ching-Wei-San. Arch Virol. 155:1933–1941. 2010. View Article : Google Scholar : PubMed/NCBI

15 

Derosa G and Maffioli P: Alkaloids in the nature: pharmacological applications in clinical practice of berberine and mate tea. Curr Top Med Chem. 14:200–206. 2014. View Article : Google Scholar

16 

Zhang Q, Piao XL, Piao XS, Lu T, Wang D and Kim SW: Preventive effect of Coptis chinensis and berberine on intestinal injury in rats challenged with lipopolysaccharides. Food Chem Toxicol. 49:61–69. 2011. View Article : Google Scholar

17 

Yao J, Kong W and Jiang J: Learning from berberine: Treating chronic diseases through multiple targets. Sci China Life Sci. Oct 30–2013.(Epub ahead of print). View Article : Google Scholar : PubMed/NCBI

18 

Doggrell SA: Berberine - a novel approach to cholesterol lowering. Expert Opin Investig Drugs. 14:683–685. 2005. View Article : Google Scholar : PubMed/NCBI

19 

Affuso F, Mercurio V, Fazio V and Fazio S: Cardiovascular and metabolic effects of Berberine. World J Cardiol. 2:71–77. 2010. View Article : Google Scholar : PubMed/NCBI

20 

Chappell WH, Abrams SL, Franklin RA, et al: Ectopic NGAL expression can alter sensitivity of breast cancer cells to EGFR, Bcl-2, CaM-K inhibitors and the plant natural product berberine. Cell Cycle. 11:4447–4461. 2012. View Article : Google Scholar : PubMed/NCBI

21 

Yu M, Tong X, Qi B, et al: Berberine enhances chemosensitivity to irinotecan in colon cancer via inhibition of NFκB. Mol Med Rep. 9:249–254. 2014.

22 

Pierpaoli E, Arcamone AG, Buzzetti F, Lombardi P, Salvatore C and Provinciali M: Antitumor effect of novel berberine derivatives in breast cancer cells. Biofactors. 39:672–679. 2013. View Article : Google Scholar : PubMed/NCBI

23 

Hur JM and Kim D: Berberine inhibited radioresistant effects and enhanced anti-tumor effects in the irradiated-human prostate cancer cells. Toxicol Res. 26:109–115. 2010. View Article : Google Scholar : PubMed/NCBI

24 

Bernas T and Dobrucki J: Mitochondrial and nonmitochondrial reduction of MTT: interaction of MTT with TMRE, JC-1, and NAO mitochondrial fluorescent probes. Cytometry. 47:236–242. 2002. View Article : Google Scholar : PubMed/NCBI

25 

Sylvester PW: Optimization of the tetrazolium dye (MTT) colorimetric assay for cellular growth and viability. Methods Mol Biol. 716:157–168. 2011. View Article : Google Scholar : PubMed/NCBI

26 

Zhou ZR, Zhu XD, Xia J, et al: Short-term versus long-term hormone therapy plus radiotherapy or prostatectomy for prostate cancer: a systematic review and meta-analysis. J Cancer Res Clin Oncol. 139:783–796. 2013. View Article : Google Scholar : PubMed/NCBI

27 

Huang ZJ, Zeng Y, Lan P, Sun PH and Chen WM: Advances in structural modifications and biological activities of berberine: an active compound in traditional Chinese medicine. Mini Rev Med Chem. 11:1122–1129. 2011. View Article : Google Scholar

28 

Pierpaoli E, Arcamone AG, Buzzetti F, Lombardi P, Salvatore C and Provinciali M: Antitumor effect of novel berberine derivatives in breast cancer cells. Biofactors. 39:672–679. 2013. View Article : Google Scholar : PubMed/NCBI

29 

Goto H, Kariya R, Shimamoto M, et al: Antitumor effect of berberine against primary effusion lymphoma via inhibition of NF-κB pathway. Cancer Sci. 103:775–781. 2012. View Article : Google Scholar : PubMed/NCBI

30 

Wang Z, Chen Z, Yang S, et al: Berberine ameliorates collagen-induced arthritis in rats associated with anti-inflammatory and anti-angiogenic effects. Inflammation. 37:1789–1798. 2014. View Article : Google Scholar : PubMed/NCBI

31 

Barzilai A and Yamamoto K: DNA damage responses to oxidative stress. DNA Repair (Amst). 3:1109–1115. 2004. View Article : Google Scholar

32 

Muralimanoharan SB, Kunnumakkara AB, Shylesh B, et al: Butanol fraction containing berberine or related compound from nexrutine inhibits NFkappaB signaling and induces apoptosis in prostate cancer cells. Prostate. 69:494–504. 2009. View Article : Google Scholar :

33 

Choi MS, Oh JH, Kim SM, et al: Berberine inhibits p53-dependent cell growth through induction of apoptosis of prostate cancer cells. Int J Oncol. 34:1221–1230. 2009.PubMed/NCBI

34 

Kim JB, Lee KM, Ko E, et al: Berberine inhibits growth of the breast cancer cell lines MCF-7 and MDA-MB-231. Planta Med. 74:39–42. 2008. View Article : Google Scholar : PubMed/NCBI

35 

Chidambara Murthy KN, Jayaprakasha GK and Patil BS: The natural alkaloid berberine targets multiple pathways to induce cell death in cultured human colon cancer cells. Eur J Pharmacol. 688:14–21. 2012. View Article : Google Scholar : PubMed/NCBI

36 

Yan L, Yan K, Kun W, et al: Berberine inhibits the migration and invasion of T24 bladder cancer cells via reducing the expression of heparanase. Tumor Biol. 34:215–221. 2013. View Article : Google Scholar

37 

Letasiova S, Jantova S, Cipak L and Muckova M: Berberine-antiproliferative activity in vitro and induction of apoptosis/necrosis of the U937 and B16 cells. Cancer Lett. 239:254–262. 2006. View Article : Google Scholar

38 

Liu Z, Liu Q, Xu B, et al: Berberine induces p53-dependent cell cycle arrest and apoptosis of human osteosarcoma cells by inflicting DNA damage. Mutat Res. 662:75–83. 2009. View Article : Google Scholar : PubMed/NCBI

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Spandidos Publications style
Lu W, Du S and Wang J: Berberine inhibits the proliferation of prostate cancer cells and induces G0/G1 or G2/M phase arrest at different concentrations. Mol Med Rep 11: 3920-3924, 2015.
APA
Lu, W., Du, S., & Wang, J. (2015). Berberine inhibits the proliferation of prostate cancer cells and induces G0/G1 or G2/M phase arrest at different concentrations. Molecular Medicine Reports, 11, 3920-3924. https://doi.org/10.3892/mmr.2014.3139
MLA
Lu, W., Du, S., Wang, J."Berberine inhibits the proliferation of prostate cancer cells and induces G0/G1 or G2/M phase arrest at different concentrations". Molecular Medicine Reports 11.5 (2015): 3920-3924.
Chicago
Lu, W., Du, S., Wang, J."Berberine inhibits the proliferation of prostate cancer cells and induces G0/G1 or G2/M phase arrest at different concentrations". Molecular Medicine Reports 11, no. 5 (2015): 3920-3924. https://doi.org/10.3892/mmr.2014.3139