Different concentrations of berberine result in distinct cellular localization patterns and cell cycle effects in a melanoma cell line

Abstract

Purpose Natural products represent a rich reservoir of potential small molecule inhibitors exhibiting antiproliferative and tumoricidal properties. An example is the isoquinoline alkaloid berberine, which is found in plants such as goldenseal (Hydrastis canadensis). Studies have shown that berberine is able to trigger apoptosis in diVerent malignant cell lines, and can also lead to cell cycle arrest at sub-apoptotic doses. A particularly interesting feature of berberine is the fact that it is a Xuorescent molecule, and its uptake and distribution in cells can be studied by Xow cytometry and epiXuorescence microscopy. To test the relationships between berberine uptake, distribution and cellular eVect in melanoma cells, K1735-M2 mouse and WM793 human melanoma cells were treated with diVerent concentrations of berberine, and alterations in cell cycle progression, DNA synthesis, cell proliferation, and cell death measured. Methods Cell proliferation was measured by sulforhodamine B assays, cell death by Xow cytometry, berberine uptake and distribution by laser scanning confocal microscopy and Xow cytometry, cell cycle progression by Xow cytometry, and DNA synthesis, M-phase, and mitochondrial eVects by immunolabeling and epiXuorescence microscopy methods. Results In these melanoma cell lines, berberine at low doses (12.5-50 M) is concentrated in mitochondria and promotes G1 arrest. In contrast, higher doses (over 50 M) result in cytoplasmic and nuclear berberine accumulation, and G2 arrest. DNA synthesis is not markedly aVected by low doses of berberine, but 100 M is strongly inhibitory. Even at 100 M, berberine inhibits cell growth with relatively little induction of apoptosis. Conclusion Berberine displays multiphasic eVects in these malignant cell lines, which are correlated with the concentration and intracellular distribution of this alkaloid. These results help explain some of the conXicting information in the literature regarding the eVects of berberine, and suggest that its use in clinical development may be more as a cytostatic agent than a cytotoxic compound.

Berberine inhibits Rho GTPases and cell migration at low doses but induces G2 arrest and apoptosis at high doses in human cancer cells

“…Nuclear localization of berberine was clearly detected in the nuclei of cells treated with 300 μM of berberine. Hence, we were able to confirm the results of a previous study (19) demonstrating that berberine concentrates in the mitochondria in cells at a low concentration but can be translocated to nuclei at high concentrations. The differential distribution of berberine at different concentrations may determine the biological properties exerted by berberine on treated cells.…”

“…However, the mechanisms involved in the differential intracellular localization of berberine are still not fully understood at this stage. As berberine is a DNA-binding molecule (19), translocation of berberine to the nucleus may interfere with DNA and block G2 cells from entering the M phase. A previous study revealed that different concentrations of berberine resulted in distinct cellular responses which correlated with its intracellular distribution in melanoma cell lines (19).…”

“…While the effects of berberine on the cell cycle may be dependent on the cell types used, the different effects of berberine observed may also be attributed to the difference in concentration of berberine used in the studies. A recent study showed that berberine had multi-phasic effects on cancer cells and may be related to the differential intracellular distribution of berberine at different concentrations; berberine was shown to be accumulated in mitochondria at low concentrations (12.5-50 μM) but translocated to nuclei at high concentrations (>50 μM) (19).…”

Antiproliferative effect of berberine on canine mammary gland cancer cell culture

“…Previous studies indicated that BBR may lead to cell death, and hence delay cell proliferation (19,21). In addition, detailed investigations on molecular carcinogenesis provided the potential for therapeutic intervention in cancer by specifically targeting and sensitizing cancer cells to apoptosis (22,23).…”

Mitochondrial disruption occurs downstream from β-adrenergic overactivation by isoproterenol in differentiated, but not undifferentiated H9c2 cardiomyoblasts: Differential activation of stress and survival pathways

“…Cell density was evaluated by the sulforhodamine B (SRB) assay (Serafim et al, 2008, Houghton et al, 2007. H9c2 cells were seeded in 6-or 24-well plates (35,000 cells/ml), allowed to attach for 1 day and pre-incubated individually with the specific compounds: 30 minutes with 30 µM of pifithrin-alpha, 1 hour with 50 µM of LY294002 or 2 hours with 100 µM NAC, always prior to ISO treatment (24, 48 or 96 hours).…”