ABSTRACT
The active metabolite of vitamin D(3), 1α,25(OH)(2)D(3) , displays anticancer effects by regulating cell cycle and apoptosis in many cancer cells. However, it has not been determined whether 1α,25(OH)(2)D(3) increases the susceptibility of cancer cells to NK cells. Here, we investigated the anticancer effect of 1α,25(OH)(2)D(3) in human melanoma cell lines by investigating enhancement of NK susceptibility and elucidating the mediator of NK cytotoxicity. 1α,25(OH)(2)D(3)-resistant melanoma cells (G-361 and SK-MEL-5) treated with 1α,25(OH)(2)D(3) showed higher susceptibility to NK cells with up-regulation of Fas expression. Furthermore, G-361 cells treated with 1α,25(OH)(2)D(3) showed significantly increased caspase activity. In addition to Fas up-regulation, expression of heat shock protein 60 (Hsp60) was elevated by 1α,25(OH)(2) D(3) . Increased expression of Hsp60 by 1α,25(OH)(2)D(3) was related to not only up-regulation of Fas expression but also to NK susceptibility of G-361 cells. Taken together, our data suggest that 1α,25(OH)(2)D(3) acts as an anticancer agent by increasing expression of Fas on the surface of melanoma cells through Hsp60 induction and strengthens caspase sensitivity to Fas-mediated apoptotic pathway by NK cells. 1α,25(OH)(2)D(3) treatment may therefore have a preventive role in melanoma occurrence or potentiate the anticancer effects of NK-cell immune therapy.
Subject(s)
Calcitriol/pharmacology , Chaperonin 60/metabolism , Killer Cells, Natural/immunology , Killer Cells, Natural/metabolism , Melanoma/drug therapy , fas Receptor/metabolism , Apoptosis/immunology , Blotting, Western , Caspase 3/metabolism , Caspase 8/metabolism , Cell Line, Tumor , Fas Ligand Protein/metabolism , Flow Cytometry , Humans , Melanoma/immunology , Melanoma/metabolism , RNA Interference , RNA, Small InterferingABSTRACT
B7-H4 is a recently discovered B7 family member that has inhibitory effects on T-cell immunity. However, the reverse signalling mechanism of the B7-H4-expressing cells remains unclear. Previous work has shown that B7-H4 expression was enhanced on B cells following Epstein-Barr virus (EBV) infection, and engagement of cell-surface-expressed B7-H4 induces cell death of EBV-transformed B cells. Here we found that B7-H4 was constitutively expressed on EBV-positive lymphoma cells, Raji and IM-9 cells, but was not expressed on EBV-negative lymphoma cells (Ramos). Engagement of B7-H4 significantly reduced cell growth of Raji and IM-9 cells and resulted in cell cycle arrest at G0-G1 phase in a dose- and time-dependent manner. To clarify the mechanism of cell cycle arrest via activation of B7-H4, cell cycle regulatory factors were examined by reverse transcription-polymerase chain reaction and immunoblotting. We found that B7-H4 triggered down-regulation of CDK4/6 and up-regulation of p21 expression at both protein and RNA levels. Furthermore, CDK2 and cyclin E/D expression was down-regulated by B7-H4 triggering. Additionally, the down-regulation of phospho-AKT and phospho-cyclin E were clearly detected in B7-H4-activated Raji cells, but the phosphorylation of p53 was constitutively maintained. These results indicate that B7-H4-mediated signalling on EBV-positive B-cell lymphoma cells modulates the cell cycle through down-regulation of the AKT pathway. Consequently, B7-H4 may be a new potential target for use in EBV-positive lymphoma therapy.