Different concentrations of berberine result in distinct cellular localization patterns and cell cycle effects in a melanoma cell line.
OBJECTIVE
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 different 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 fluorescent molecule, and its uptake and distribution in cells can be studied by flow cytometry and epifluorescence microscopy. To test the relationships between berberine uptake, distribution and cellular effect in melanoma cells, K1735-M2 mouse and WM793 human melanoma cells were treated with different 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 flow cytometry, berberine uptake and distribution by laser scanning confocal microscopy and flow cytometry, cell cycle progression by flow cytometry, and DNA synthesis, M-phase, and mitochondrial effects by immunolabeling and epifluorescence microscopy methods.


RESULTS
In these melanoma cell lines, berberine at low doses (12.5-50 muM) is concentrated in mitochondria and promotes G1 arrest. In contrast, higher doses (over 50 muM) result in cytoplasmic and nuclear berberine accumulation, and G2 arrest. DNA synthesis is not markedly affected by low doses of berberine, but 100 muM is strongly inhibitory. Even at 100 muM, berberine inhibits cell growth with relatively little induction of apoptosis.


CONCLUSIONS
Berberine displays multiphasic effects in these malignant cell lines, which are correlated with the concentration and intracellular distribution of this alkaloid. These results help explain some of the conflicting information in the literature regarding the effects of berberine, and suggest that its use in clinical development may be more as a cytostatic agent than a cytotoxic compound.
Center of Neurosciences and Cellular Biology, Department of Zoology, University of Coimbra, P3004-597 Coimbra, Portugal.
Teresa L
TL
Serafim
Paulo J
PJ
Oliveira
Vilma A
VA
Sardao
Ed
E
Perkins
Donna
D
Parke
Jon
J
Holy
D000818:Animals; D000963:Antimetabolites; D001599:Berberine / Q000493:pharmacokinetics* / Q000494:pharmacology*; D001973:Bromodeoxyuridine; D002259:Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone / Q000494:pharmacology; D002453:Cell Cycle / Q000187:drug effects*; D016923:Cell Death / Q000187:drug effects; D002455:Cell Division / Q000187:drug effects; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D004247:DNA / Q000096:biosynthesis / Q000235:genetics; D006801:Humans; D008546:Melanoma, Experimental / Q000378:metabolism* / Q000473:pathology; D051379:Mice; D008856:Microscopy, Fluorescence; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism; D014475:Uncoupling Agents / Q000494:pharmacology
D000963:Antimetabolites; D014475:Uncoupling Agents; D001599:Berberine; D002259:Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone; D004247:DNA; D001973:Bromodeoxyuridine