Cytotoxic effects of Coptis chinensis and Epimedium sagittatum extracts and their major constituents (berberine, coptisine and icariin) on hepatoma and leukaemia cell growth.
Lin CC, Ng LT, Hsu FF, Shieh DE, Chiang LC. Clin Exp Pharmacol Physiol. 2004 Jan-Feb;31(1-2):65-9.
The present study was conducted to evaluate the cytotoxic effects of Coptis chinensis and Epimedium sagittatum extracts and their major constituents on hepatoma and leukaemia cells in vitro.
Four human liver cancer cell lines, namely HepG2, Hep3B, SK-Hep1 and PLC/PRF/5, and four leukaemia cell lines, namely K562, U937, P3H1 and Raji, were used in the present study.
Of the two crude drugs, C. chinensis exhibited the strongest activity against SK-Hep1 (IC50 = 7 microg/mL) and Raji (IC50 = 4 microg/mL) cell lines. The IC50 values for C. chinensis on HepG2, Hep3B and PLC/PRF/5 cell lines were 20, 55 and 35 microg/mL, respectively. The IC50 values for C. chinensis on K562, U937 and P3H1 cell lines were 29, 29 and 31 microg/mL, respectively.
With the exception of HepG2 and Hep3B, the E. sagittatum extract inhibited the proliferation of all cell lines (SK-Hep1, PLC/PRF/5, K562, U937, P3H1 and Raji), with IC50 values of 15, 57, 74, 221, 40 and 80 microg/mL, respectively. Interestingly, the two major compounds of C. chinensis, berberine and coptisine, showed a strong inhibition on the proliferation of both hepatoma and leukaemia cell lines, with IC50 values varying from 1.4 to 15.2 microg/mL and from 0.6 to 14.1 microg/mL, respectively. However, icariin (the major compound of E. sagittatum) showed no inhibition of either the hepatoma or leukaemia cell lines.
The results of the present study suggest that the C. chinensis extract and its major constituents berberine and coptisine possess active antihepatoma and antileukaemia activities.

Berberine-induced apoptosis of human leukemia HL-60 cells is associated with down-regulation of nucleophosmin/B23 and telomerase activity

Hsing L. Wu, Chen Y. Hsu, Wen H. Liu, Benjamin Y.M. Yung. International Journal of Cancer. Volume 81, Issue 6, pages 923–929, 11 June 1999

The steady-state level of nucleophosmin/B23 mRNA decreased during berberine-induced (25?g/ml, 24 to 96 hr) apoptosis of human leukemia HL-60 cells. A decline in telomerase activity was also observed in HL-60 cells treated with berberine. A stable clone of nucleophosmin/B23 over-expressed in HL-60 cells was selected and found to be less responsive to berberine-induced apoptosis. About 35% to 63% of control vector–transfected cells (pCR3) exhibited morphological characteristics of apoptosis, while about 8% to 45% of nucleophosmin/B23-over-expressed cells (pCR3-B23) became apoptotic after incubation with 15?g/ml berberine for 48 to 96 hr. DNA extracted from pCR3 cells contained more fragmented DNA than pCR3-B23 cells during treatment with 15?g/ml berberine for 24 to 48 hr. Our results indicate that berberine-induced apoptosis is associated with down-regulation of nucleophosmin/B23 and telomerase activity. We also suggest that nucleophosmin/B23 may play an important role in the control of the cellular response to apoptosis induction.

Apoptosis, the physiological mode of cell death, is representative of an endogenous mechanism which can be selectively triggered by cells in response to largely unknown stimuli. During apoptosis, a series of well-defined degenerative changes occur within the cell which ultimately result in degradation of the nuclear DNA into oligonucleosome chains (Wyllie, 1980) and fragmentation of the cell into neat “bite-size” pieces for efficient disposal by neighboring cells or marauding macrophages (Savill et al.,1989). According to Wyllie et al. (1980), at the morphological level, necrosis is associated with cell swelling, rupture of membranes and dissolution of an organized structure while apoptosis is characterized by cell shrinkage and chromatin condensation. Apoptosis has been described as programmed, as opposed to accidental, cell death (Wyllie, 1980).

Identification of the genes and their products that are involved in responses to growth stimuli is essential for understanding normal cell growth and death. During the past decade, numerous regulatory factors which control the balance between a cycling and quiescent state have been identified. These factors include proto-oncogenes and negative and positive regulatory growth factors. Other proliferation-associated molecules are being studied to determine their potential role in cell-growth regulation (Valdez et al.,1994; Chou and Yung, 1995). Nucleophosmin/B23, also called protein B23, NO38 or numatrin (Schmidt-Zachmann et al.,1987; Yung et al.,1985), is a major nucleolar phosphoprotein that displays a number of activities. These include a potential role as a positive regulator of cell proliferation. Nucleophosmin/B23 is significantly more abundant in tumor and proliferating cells than in normal resting cells (Chan et al.,1989). Nucleophosmin/B23 mRNA is 50- and 5-fold higher in Novikoff hepatoma and hypertrophic rat liver, respectively, compared to normal rat liver (Chan et al.,1989). Nucleophosmin/B23 is localized in granular regions of the nucleolus (Peculis and Gall, 1992), associated with pre-ribosomal particles (Yung et al.,1985), and forms hexamers (Yung and Chan, 1987) which may be essential for the assembly of ribosomes. Our previous results have shown that nucleophosmin/B23 translocates from nucleoli to nucleoplasm during the stationary phase of growth (Yung et al.,1990b) or during treatment with certain anti-tumor drugs, particularly DNA intercalators (Yung et al.,1985, 1990a; Wu et al.,1995). Valdez et al. (1994) have demonstrated that nucleophosmin/B23 binds to amino acid sequence 24–56 of protein p120, a cell cycle–related protein. In light of many potential roles, it is conceivable that nucleophosmin/B23 is involved in the regulation of cell proliferation.

Normal diploid somatic cells lose approximately 50 to 200 bp of telomeric DNA/mean population doubling due to the inability of DNA polymerase to completely replicate the end parts of linear chromosomes (Harley et al.,1990). In contrast, germ cell lines and most immortal cell lines maintain their telomeres at a constant length, irrespective of the number of divisions they undergo, due to the activity of telomerase, a ribonucleoprotein that synthesizes and adds telomeric repeats onto chromosomal termini. Since telomerase activity is absent in most normal human somatic cells and tissues that have limited replicative spans (Kim et al.,1994) but is activated during cellular immortalization (Counter et al.,1992), a telomere hypothesis of cell aging and immortalization has been proposed, in which the attrition of telomeric sequences ultimately interferes with the expression of genes required for continued cell growth (Allsopp et al.,1992). Strong correlative support for this hypothesis was also provided by the observation of telomerase activity in 98% of established immortal cell lines and 90% of tumors tested and the absence of telomerase activity in over 50 normal human somatic tissues (Kim et al.,1994). Telomerase is down-regulated during terminal differentiation and in quiescent cells (Sharma et al.,1995; Holt et al.,1996). Further, there is evidence that telomere length controls the life span of cells and, hence, cellular senescence (Harley et al.,1990; Allsopp et al.,1992; Bodnar et al.,1998).

Berberine, an alkaloid, originated from Chinese herbal medicine, where it is used as an antibiotic; its anti-bacterial activity has been demonstrated against many species (Ghosh et al.1985). The drug was subsequently screened for anti-cancer activity following evidence of anti-neoplastic properties (Zhang, 1990). We have shown that berberine induces apoptosis in human leukemia HL-60 cells (Kuo et al.1995) and have observed differential dose-dependent effects on the cell cycle and induction of apoptosis in BALB/c3T3 cells (Yang et al.,1996). To better understand the induction of apoptosis by berberine, attempts have been made to determine the effects of berberine on nucleophosmin/B23 mRNA expression and telomerase activities. Our results show that nucleophosmin/B23 mRNA and telomerase activity are down-regulated during berberine-induced apoptosis of HL-60 cells. Whether there is a link between down-regulation of nucleophosmin/B23 and berberine-induced apoptosis thus becomes an important question. For this reason, we have investigated the influence of nucleophosmin/B23 over-expression on berberine-induced apoptosis. We report here that over-expression of nucleophosmin/B23 decreases the responsiveness of HL-60 cells to berberine-induced apoptosis.

Berberine & STAT3
Berberine, an alkaloid derivative from Berberis vulgaris L., has been used extensively in traditional Chinese medicine to treat diarrhoea and diabetes, but the underlying mechanisms for treating diabetes are not fully understood. Recent studies suggested that berberine has many beneficial biological effects, including anti-inflammation. Because type 1 diabetes is caused by T cell-mediated destruction of β cells and severe islet inflammation, we hypothesized that berberine could ameliorate type 1 diabetes through its immune regulation properties. Here we reported that 2 weeks of oral administration of berberine prevented the progression of type 1 diabetes in half of the NOD mice and decreased Th17 and Th1 cytokine secretion. Berberine suppressed Th17 and Th1 differentiation by reducing the expression of lineage markers. We found that berberine inhibited Th17 differentiation by activating ERK1/2 and inhibited Th1 differentiation by inhibiting p38 MAPK and JNK activation. Berberine down-regulated the activity of STAT1 and STAT4 through the suppression of p38 MAPK and JNK activation, and it controlled the stability of STAT4 through the ubiquitin-proteasome pathway. Our findings indicate that berberine targets MAPK to suppress Th17 and Th1 differentiation in type 1 diabetic NOD mice. This study revealed a novel role of ERK in Th17 differentiation through down-regulation of STAT3 phosphorylation and RORγt expression.
Cui, G., Qin, X., Zhang, Y., Gong, Z., Ge, B. & Zang, Y.Q.Berberine Differentially Modulates the Activities of ERK, p38 MAPK, and JNK to Suppress Th17 and Th1 T Cell Differentiation in Type 1 Diabetic Mice. Journal of Biological Chemistry, 2009 No.284, Pp.28420-29. DOI:10.1074/jbc.M109.012674

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.
Meeran, S.M., Katiyar, S. & Katiyar, S.K. Berberine-induced apoptosis in human prostate cancer cells is initiated by reactive oxygen species generation. Toxicol Appl Pharmacol. 2008 May 15;229(1):33-43.

Berberine & Breast Cancer
Huanglian (Coptidis rhizoma), a widely used herb in traditional Chinese medicine, has been shown recently to possess anticancer activities. However, the molecular mechanism underlying the anticancer effect of the herb is poorly understood. Specifically, whether huanglian extract affects the expression of cancer-related genes has not been defined. This study used DNA microarray technology to examine the effect of the herbal extract on expression of the common genes involved in carcinogenesis in two human breast cancer cell lines, the ER-positive MCF-7 and ER-negative MDA-MB-231 cells. Treatment of the cancer cells with huanglian extract markedly inhibited their proliferation in a dose- and time-dependent manner. The growth inhibitory effect was much more profound in MCF-7 cell line than that in MDA-MB-231 cells. DNA microarray assay revealed that treatment with huanglian dramatically increased the mRNA expression of interferon-ß (IFN-ß) and tumour necrosis factor-{alpha} in MCF-7 cells. Quantitative analysis by real-time PCR or western blotting confirmed the upregulation of the two genes (especially IFN-ß) in MCF-7 cells, but not in MDA-MB-231 cells. Addition of neutralizing antibody against IFN-ß to culture medium markedly inhibited the huanglian-induced antiproliferative effect, confirming the involvement of IFN-ß in the huanglian's effect and also suggesting an autocrine pathway for the action of IFN-ß in this setting. Given that IFN-ß is among the most important anticancer cytokines, the upregulation of this gene by huanglian is, at least in part, responsible for its antiproliferative effect. The results of this study implicate huanglian as a promising herb for chemoprevention and chemotherapy of certain cancers.

Kang , J.X., Liu, J., Wang, J., He, C. & Li, F.P. The extract of huanglian, a medicinal herb, induces cell growth arrest and apoptosis by upregulation of interferon-ß and TNF-{alpha} in human breast cancer cells. Carcinogenesis 2005 26(11):1934-1939; doi:10.1093/carcin/bgi154

Inhibitory effect of berberine on the motivational effects of ethanol in mice.
It is believed that drug-induced rewarding effects play an important role in the development of substance dependence. Recently, berberine was reported to inhibit the rewarding effects of drugs of abuse such as cocaine, morphine, and nicotine. Berberine is also demonstrated to modulate the activity of several neurotransmitter systems like, dopamine, nitric oxide, serotonin, and NMDA, which are implicated in rewarding effects of ethanol. Hence, we hypothesized that berberine may modulate the ethanol-induced rewarding effects. Therefore, we studied the effect of berberine on locomotor sensitization, conditioned place preference (CPP), and ethanol drinking preference in mice. The results revealed that acute administration of berberine (2.5, 5, and 10 mg/kg, i.p.) dose dependently reduced locomotor stimulant effect of acute ethanol and expression of sensitization to locomotor stimulant effect of ethanol. Further, pretreatment with berberine (2.5, 5, and 10 mg/kg, i.p.) prior to each dose of ethanol, blocked the development as well as expression of sensitization to locomotor stimulant effect of ethanol. In another set of experiment, treatment with berberine (5 and 10 mg/kg, i.p.) reduced the induction and expression of ethanol-induced CPP in mice. In addition, berberine in these doses also reduced preference to ethanol drinking over water, but did not alter the general reward. In conclusion, the results of the present study revealed that berberine attenuates ethanol-induced rewarding effects in mice and that could be attributed to its neuro-modulatory action.

Bhutada P, Mundhada Y, Bansod K, et al. Prog Neuropsychopharmacol Biol Psychiatry. 2010 Dec 1;34(8):1472-9. Epub 2010 Aug 14.

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