Ginsenoside Rh 2 and Rh 3 induce differentiation of HL-60 cells into granulocytes: modulation of protein kinase C isoforms during differentiation by ginsenoside Rh 2

Abstract

Ginsenoside Rh 1 or Rh 2 differentiated B16 melanoma or F9 teratocarnoma to phenotypic normal melanocyte-like cells or parietal endoderm-like cells. Ginsenoside Rh 3 and Rh 4 were recently isolated from Panax ginseng, but their biochemical and pharmacological effects remain unidentified. The present study investigated whether the ginsenoside Rh group (G-Rh 1, -Rh 2, -Rh 3 and -Rh 4) having similar structures induce differentiation of HL-60 cells and whether protein kinase C (PKC) is involved in differentiation by ginsenoside. Differentiation was assessed by Wright–Giemsa stain and nitroblue tetrazolium reduction. G-Rh 2 and G-Rh 3 induced differentiation of HL-60 cells into morphologically and functionally granulocytes but G-Rh 1 and G-Rh 4 did not. G-Rh 2 and G-Rh 3 arrested the cell cycle at the G1/S phase, consistent with the ability to induce differentiation in a decreasing order of retinoic acid>G-Rh 2>G-Rh 3. During differentiation by G-Rh 2, Ca 2+/phospholipid-dependent PKC activity was increased in both the cytosol and total cell extract and Ca 2+/phospholipid-dependent phosphorylation of 38 and 200 kDa endogenous proteins increased, while phosphorylation of 60, 64, 66 and 97 kDa proteins was Ca 2+/phospholipid-independent. When cytosolic PKC isoforms were analyzed by immunoblotting, no significant change was observed in the α level, however, the immunoreactive 60 kDa band of a similar mass to the PKC catalytic fragment appeared following treatment with G-Rh 2. The β isoform was gradually increased with prolonged treatment. The γ isoform was not detected in the cytosol of untreated cells, whereas a small amount was detected 5 days after treatment. It is concluded that G-Rh 2 and G-Rh 3 can induce differentiation of HL-60 cells into granulocytes and modulation of PKC isoform levels may contribute to differentiation of HL-60 cells by G-Rh 2.