Shikonin Induces Apoptosis by Inhibiting Phosphorylation of IGF-1 Receptor in Myeloma Cells.

Multiple myeloma is an incurable hematological malignancy of plasma cells despite advances in conventional chemotherapy or high dose chemotherapy with stem cell transplantation. Recent advances in the biological treatment such as thalidomide and bortezomib will offer great promise to improve the outcome of refractory or relapsed patients with multiple myeloma; therefore, more novel biologically based therapies targeting both myeloma cells and its bone marrow microenvironment are urgently required. Shikonin, a Chinese herb derived from purple gromwell, Boraginaceae Lithospermum erythrorhizon, is previously known to have anti-inflammatory, anti-tumor, and anti-pyretic properties. Several studies have shown that Shikonin induces p53-mediated cell cycle arrest and apoptosis in human malignant melanoma cells, and induces apoptosis in human myeloid leukemic HL60 cells. However, none of the studies have concerned in the effects of Shikonin on myeloma cells. In this study, we investigated the possibilities to develop Shikonin as a novel promising agent for multiple myeloma. Shikonin suppressed the cellular growth of RPMI8226 and IM9 myeloma cells, via induction of apoptosis in a dose (0–1 μM)- and time (0–24 h)-dependent manner. Apoptotic cell death was induced in both RPMI8226 and IM9 cells within 3 h for treatment with only 0.5 μM of Shikonin, and finally 78% of RPMI8226 cells and 91% of IM9 cells were dead by apoptosis within18 h. Treatment with 0.5 μM Shikonin rapidly increased the population of cells in the G₀/G₁ phase with reduction of cells in the S phase, and then induction of apoptosis was confirmed by the appearance of cells in the sub-G₁ fraction. Shikonin-induced apoptosis was in association with the loss of mitochondrial transmembrane potentials, and activation of caspase-3. Shikonin-induced apoptosis was completely blocked by the treatment with pan-caspase inhibitor (20 μM, Z-VAD), suggesting that caspase plays an important role in Shikonin-induced cell death in myeloma cells. Expression of p53 and Bax proteins was increased with down-regulation of Mcl-1 protein, but no changes were confirmed of the expression of Bcl-2 and Bcl-X_L. Recent investigations have shown that cytokines such as IL-6, insulin growth factor (IGF), VEGF, and TNF-α mediate myeloma cell growth, survival and migration. IGF-1 increases survival of myeloma cells by activating various down-stream signal transduction molecules. Recently, inhibitors of IGF-1 receptor demonstrate promising anti-multiple myeloma activity in preclinical studies. Shikonin has reported to be an inhibitor of protein tyrosine kinase such as EGFR, v-Src, and KDR/Flk-1. To address the mechanism of Shikonin-induced apoptosis in myeloma cells, we thus examined the effects of Shikonin on the phosphorylation of IGF-1 receptor. IGF-1 (100 ng/ml) stimulated the proliferation of both RPMI8226 and IM9 cells. Interestingly, Shikonin (0.5 μM) overcame IGF-1-induced cell proliferation, and inhibited proliferation of myeloma cells via induction of apoptosis. Shikonin inhibited phosphorylation of IGF-1 receptor as early as 30 min with inhibition of PI3K/Akt signaling. These results suggest that Shikonin-induced apoptosis in myeloma cells was mediated by inhibiting phosphorylation of IGF-1 receptor and modulating its down-stream signaling pathway. In conclusion, Shikonin inhibited cellular growth by inhibiting IGF-1 receptor signaling in myeloma cells, and may have a potential as a novel biologically based therapeutic agent for the patients with multiple myeloma..