Differential Expression of PKC Isoforms and Their Tumoricidal Activity in Two Macrophage Cell Lines: Involvement of Nitric Oxide-dependent Mechanisms

Abstract

Objective: To investigate the role of PKC isoforms in the regulation of LPS-triggered tumoricidal activity in macrophages and further elucidate its signal mechanisms. Methods: Two macrophage cell lines (P388D1 and RAW264.7) were stimulated by LPS alone, or with long-term of PMA pretreatment. Then cytotoxicities to P815 cells (by MTT assay) and IL-1, TNF-α (by ELISA) and nitric oxide (NO) production (by Griess reagent) in supernatants were measured. Western blot for PKC isoforms after long-term PMA pretreatment was analyzed. Results: RAW264.7 cells were stimulated with LPS to kill target tumor cells P815, whereas P388D1 cells failed to develop such an ability. Down-regulation of PKC isoforms by chronic treatment with PMA significantly inhibited the LPS-induced cytotoxicity in RAW264.7 cells. In unstimulated state, Western blotting with rabbit antiserum specific for the PKCα, β1, β2, δ or e showed all 5 isoforms were detected in P388D1 cells, while only PKCα, PKCβ1 and PKCδ were detected in RAW264.7 cells. Exposure of the cells to long-term of PMA treatment significantly down-regulated the expression of PKCα, PKCβ1 and PKCδ in RAW264.7 cells. But in P388D1 cells, although PKCα, PKCδ and PKCe were down-regulated, the expression of PKCβ1 and PKCβ2 could not be regulated. Comparing with LPS-induced IL-1, TNF- and NO production by the two macrophage cell lines, P388D1 failed to produce NO. In RAW264.7 cells, LPS-induced NO production and antitumor activity was attenuated by the addition of L-NAME, an iNOS inhibitor. Conclusion: The results indicated a critical role of PKCβ in LPS-induced antitumor activity and this cytotoxicity is mainly due to PKC-β mediated NO production by RAW264.7 cells, but not a direct cytotoxic activity.