Activation of prodeath Bcl-2 family proteins and mitochondrial apoptosis pathway by sanguinarine in immortalized human HaCaT keratinocytes.
Sanguinarine, derived from the root of Sanguinaria canadensis and other poppy fumaria species, possesses strong antimicrobial, anti-inflammatory, and antioxidant properties. We earlier showed that sanguinarine kills human epidermoid carcinoma A431 cells via an induction of apoptosis [N. Ahmad et al., Clin. Cancer Res., 6: 1524-1528, 2000]. In this study, using immortalized human keratinocytes (HaCaT cells), we provide information about mechanism of the antiproliferative effect of sanguinarine. Sanguinarine [0.1 (M-2 (M)] treatment to HaCaT cells was found to inhibit in a dose-dependent manner the cell proliferation and induce apoptosis, as measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and ELISA, respectively. Sanguinarine treatment also resulted in a significant cleavage of poly(ADP-ribose) polymerase in HaCaT cells. Because mitochondrial pathway is critical for the regulation of apoptosis, we studied the involvement and regulation of mitochondrial events in sanguinarine-mediated apoptosis of HaCaT cells. As shown by the immunoblot analysis, our data clearly demonstrated that sanguinarine treatment to HaCaT cells resulted in a dose-dependent (a) increase in the level of Bax with a concomitant decrease in Bcl-2 levels and (b) increase in Bax/Bcl-2 ratio. Sanguinarine also resulted in significant increases in the proapoptotic members of Bcl-2 family proteins, i.e., Bak and Bid. This was accompanied by increase in (a) protein expression of cytochrome c and apoptotic protease-activating factor-1 and (b) activity and protein expression of caspase-3, caspase-7, caspase-8, and caspase-9. Taken together, our data showed the involvement of mitochondrial pathway and Bcl-2 family proteins during sanguinarine-mediated apoptosis of immortalized keratinocytes. We suggest that sanguinarine could be developed as a drug for the management of hyperproliferative skin disorders, including skin cancer.
Adhami
Vaqar Mustafa
VM
Department of Dermatology, University of Wisconsin, Madison, Wisconsin 53706, USA.
Aziz
Moammir Hasan
MH
Mukhtar
Hasan
H
Ahmad
Nihal
N
D000470:Alkaloids / Q000627:therapeutic use*; D000975:Antioxidants / Q000494:pharmacology; D017209:Apoptosis; D053119:Benzophenanthridines; D053148:Caspase 3; D053179:Caspase 7; D053181:Caspase 8; D053453:Caspase 9; D020169:Caspases / Q000096:biosynthesis; D002455:Cell Division; D045744:Cell Line, Tumor; D002470:Cell Survival; D045304:Cytochromes c / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors / Q000627:therapeutic use*; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D015151:Immunoblotting; D007546:Isoquinolines; D015603:Keratinocytes / Q000378:metabolism; D008928:Mitochondria / Q000378:metabolism; D008954:Models, Biological; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism*; D051028:bcl-2-Associated X Protein
D000470:Alkaloids; D000975:Antioxidants; C494826:BAX protein, human; D053119:Benzophenanthridines; D004791:Enzyme Inhibitors; D007546:Isoquinolines; D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D051028:bcl-2-Associated X Protein; D045304:Cytochromes c; C005705:sanguinarine; D011065:Poly(ADP-ribose) Polymerases; C505407:CASP3 protein, human; C505520:CASP7 protein, human; C505530:CASP8 protein, human; C506433:CASP9 protein, human; D053148:Caspase 3; D053179:Caspase 7; D053181:Caspase 8; D053453:Caspase 9; D020169:Caspases