Idarubicin determine if an apoptotic mechanism was involved induction of apoptosis

Average expression of the chosen PKC isoforms in the samples from 65 AML patient and six bone marrow transplant donors is depicted in Figure 1B. There was Osthole a significant increase in the expression of PKC a , PKC b , and PKC d when comparing AML blast cells and normal BM cells. This finding suggests these kinases may play a role in AML biology. To investigate the use of enzastaurin as a cytotoxic agent against AML cells, OCI AML3 were used in a dose response study. While enzastaurin inhibits PKC b in the nanomolar range, it is in the low micromolar range where the drug has demonstrated effectiveness against a wide variety of cancer cell lines including leukemia cells .
OCI AML3 cells Idarubicin clinical trial were treated with vehicle , 1, 5, or 10 mMenzastaurin for 24, 48, and 72 h. Cell viability was assessed by trypan blue exclusion. As shown in Figure 2A, enzastaurin suppressed cell growth of cells but only at higher doses of the drug . Similar patterns of cell growth inhibition were observed with OCI AML2 and THP 1 cells . To determine if enzastaurin promotes apoptosis, HL60 and OCI AML3 were treated with 5mM enzastaurin for 24, 48, and 72 h. Cell viability was assessed by trypan blue exclusion. As shown in Figure 2B, enzastaurin potently killed HL60 cells but was less effective against the OCI AML3 cells. While roughly 50% of HL60 cells were killed after 72 h with 5 mM enzastaurin only 26% of OCI AML3 cells were killed by the drug under those conditions .
Cell death induced by enzastaurin was significant as compared to vehicle control in all cases for both cell lines . To determine if an apoptotic mechanism was involved, induction of apoptosis in enzastaurintreated cells was observed by identifying Annexin V positive cells . Enzastaurin high throughput chemical screening promoted Annexin V staining of HL60 cells and to a lesser degree OCI AML3 cells. As shown in Figure 2C, flow cytometry analysis Candesartan solubility of untreated HL60 cells and cells treated with 5 mMenzastaurin for 72 h indicate that nearly one third of cells were Annexin V positive after staining. After 72 h treatment of OCI AML3 cells with 5 mM enzastaurin, only 18% were apoptotic. These data indicate that enzastaurin has varying effects on the promotion of apoptosis in AML cell lines.
Next we investigated whether enzastaurin induced apoptosis involves a caspase dependent mechanism. Apoptosis assay measuring Annexin V positive cells revealed that pretreatment of HL60 cells with 40 mM caspase 3 inhibitor partially protected cells citizenship from treatment with 5 mM enzastaurin after 72 h . This result suggests that the mechanism of enzastaurin induced cells death involves caspase 3 but since protection with the caspase inhibitor was not complete, the process may require additional mechanisms. To determine if enzastaurin induced cell death requires suppression of PKC b, OCI AML3 cells were co treated with another PKC b inhibitor . The IC50 values for PKC a, PKC bI, and PKC bII for the Calbiochem PKC b inhibitor are 331, 21, and 5 nM, respectively. We used Calbiochem PKC b inhibitor at 200nM . Both HL60 and OCI AML3 cells express abundant levels of PKC b.