PKC412 Induces Apoptosis in Human Multiple Myeloma Cell Lines and Primary Myeloma Cells.

This study was conducted to explore the effect and mode of action of the protein kinase C inhibitor PKC412 on proliferation and apoptosis in human multiple myeloma cell lines (HMCL) and primary myeloma cells. MTS assay was used to measure cell proliferation of 6 HMCLs and to examine the effect of co-treatment of HMCLs with PKC412, Bortezomib or the NFkB inhibitor, SN50. PKC412-induced apoptosis was evaluated by western blot (PARP cleavage) and annexin-V-FITC / propidium iodide (PI) flow cytometry. Similarly, the level of PKC412-induced apoptosis of HMCLs co-treated with the JNK inhibitor SP600125 was also determined. PKC412 and/or Bortezomib-induced primary myeloma cell death after 72hr treatment was measured by flow cytometry by identifying CD38+/CD138+/PI positive and negative cells. PKC activity after PKC412 treatment was assessed using the Peptag PKC functional assay and expression of PKC isoforms by HMCLs was determined by western blot. c-fos expression was evaluated by both RT-PCR and western blot. Expression of c-jun and phospho-c-jun was evaluated by western blot. NFkB activation was determined using the NFkB transcriptional assay kit. Sensitivity to PKC412 varied but some activity was seen against all HMCLs after 72hr treatment at doses of 0.5μM and above. PARP cleavage was observed in all HMCLs tested at both 24 and 72hr following treatment. PKC activity of PKC412 treated HMCLs was decreased in all HMCLs tested but no correlation between PKC isoform expression and sensitivity to PKC412 was found. However, a correlation between sensitivity to PKC412 and the presence of either an activating ras or FGFR3 mutation in HMCLs was observed. When HMCLs were pre-treated with PKC412, the PMA-induced increase in c-fos mRNA was inhibited, and PKC412 down-regulated baseline c-fos mRNA expression over time. PKC412 also down-regulated c-fos protein expression in nuclear extracts of HMCLs. c-jun phosphorylation was up-regulated for 24hr after PKC412 treatment and this increase in expression and phosphorylation was inhibited by co-treatment with the JNK inhibitor SP600125. Co-treatment of the HMCL NCI-H929 with PKC412 and SP600125 partially abrogated PKC412-induced apoptosis, indicating that it is partially JNK dependent. Increased NFkB activation was observed in PKC412 treated HMCLs with the level of increase correlating with PKC412 sensitivity. Enhanced killing of HMCL was seen when NFkB activation was inhibited with either Bortezomib or SN50, suggesting the increase in activation of NFkB may be a PKC412-induced survival response. This effect was also seen when primary myeloma cells where co-treated with PKC412 and Bortezomib for 72hr. The sequence in which compounds where added when treating both primary myeloma cells and the HMCL NCI-H929 with PKC412 and Bortezomib was important, with greater killing seen when cells were treated with PKC412 for 6hr before the addition of Bortezomib. Our results demonstrate that PKC412 induces apoptosis in HMCLs which is partially dependent on JNK. Furthermore PKC412 is more effective when used in combination with NFkB inhibition. The further evaluation of PKC412 in the treatment of multiple myeloma is justified.