title: Mechanism of Mitf inhibition and morphological differentiation effects of hirsein A on B16 melanoma cells revealed by DNA microarray.
abstract: BACKGROUND
  We have previously reported that hirsein A inhibits melanogenesis in B16 melanoma cells by downregulating the Mitf gene expression.
  
  
  OBJECTIVE
  In this study, microarray was employed to determine the transcriptional response of B16 cells to hirsein A (HA) treatment and to find out the mechanism underlying Mitf downregulation.
  
  
  METHODS
  DNA microarray, spotted with 265 genes for melanogenesis and signal transduction, was performed using the total RNA isolated from B16 cells treated with HA. Validation of the results was done using real-time PCR. In addition, real-time PCR using primers for Mda-7 gene and F-actin staining were performed. Transfection experiments were performed to knockdown the expression of the Mc1r gene to evaluate its role in the cell morphological change observed.
  
  
  RESULTS
  As expected, the expressions of the Mitf-regulated melanosome transport genes and the Mc1r gene were downregulated. Furthermore, the expressions of the MAPK pathway intermediates were either up- or downregulated. Genes associated with cell differentiation, such as Gadd45b, were upregulated and prompted us to determine the expression of the Il-24 (Mda-7) gene using real-time PCR. There was an increase in the Mda-7 mRNA expression in B16 and HMV-II melanoma cells, and in human melanocytes. To better visualize the cell morphology, F-actin staining was performed and the results showed an increase in the dendrite outgrowth in HA-treated cells. Silencing the Mc1r gene did not cause a change in the B16 cell morphology observed in cells treated with HA.
  
  
  CONCLUSIONS
  This study demonstrated that HA downregulates Mitf gene expression by regulating the expressions of the MAPK signaling pathway intermediates. In addition, the inhibited Mc1r gene expression also contributed to the overall Mitf downregulation but does not play a role in the observed change in B16 cell morphology. HA surprisingly can regulate genes associated with differentiating cells (Mda-7) suggesting a role for HA in the melanoma cell differentiation induction. While the exact molecular mechanism by which HA promotes cell differentiation remain to be determined, it is clear that HA can downregulate Mitf expression and promote cell differentiation and has the potential to be used in the development of therapy for melanoma.
authors.affiliation: Graduate School of Life and Environmental Sciences, University of Tsukuba, Tsukuba, Ibaraki, Japan.
authors.forename: Myra O
authors.initials: MO
authors.lastname: Villareal
authors.forename: Junkyu
authors.initials: J
authors.lastname: Han
authors.forename: Kenjiro
authors.initials: K
authors.lastname: Ikuta
authors.forename: Hiroko
authors.initials: H
authors.lastname: Isoda
mesh_terms: D000199:Actins / Q000378:metabolism; D000818:Animals; D000972:Antineoplastic Agents, Phytogenic / Q000494:pharmacology*; D002454:Cell Differentiation / Q000187:drug effects* / Q000235:genetics; D045744:Cell Line, Tumor; D048430:Cell Shape / Q000187:drug effects* / Q000235:genetics; D004224:Diterpenes / Q000494:pharmacology*; D020869:Gene Expression Profiling / Q000379:methods*; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D007378:Interleukins / Q000235:genetics / Q000378:metabolism; D020935:MAP Kinase Signaling System / Q000187:drug effects; D008543:Melanins / Q000096:biosynthesis; D008546:Melanoma, Experimental / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D051379:Mice; D051739:Microphthalmia-Associated Transcription Factor / Q000037:antagonists & inhibitors* / Q000378:metabolism; D020411:Oligonucleotide Array Sequence Analysis; D034622:RNA Interference; D060888:Real-Time Polymerase Chain Reaction; D044102:Receptor, Melanocortin, Type 1 / Q000235:genetics / Q000378:metabolism; D015203:Reproducibility of Results; D014158:Transcription, Genetic / Q000187:drug effects; D014162:Transfection; D000099180:Interleukin-24
chemical_list: D000199:Actins; D000972:Antineoplastic Agents, Phytogenic; D004224:Diterpenes; D007378:Interleukins; D008543:Melanins; D051739:Microphthalmia-Associated Transcription Factor; D044102:Receptor, Melanocortin, Type 1; D000099180:Interleukin-24; C497235:Mitf protein, mouse; C550879:hirsein A