β-Hydroxyisovalerylshikonin, A Novel Tyrosine Kinase Inhibitor, Inhibits Cell Growth Of Various Tumor Cell Lines Alone Or In Combination

β-Hydroxyisovalerylshikonin (β-HIVS) inhibited the growth of BCR-ABL-positive leukemia K562 and BCR-ABL-negative U937 cells with the estimated IC50 value of 0.12μ,M and 0.14 μM, respectively. At higher concentrations, β-HIVS was found to inhibit the cell growth of rat SR-3Y1, KDR/Flk-1-NIH3T3 and human epidermoid A431 cells, which expressed high levels of different species of tyrosine kinases. The estimated IC50 values for SR-3Y1 cells, KDR/Flk-1-NIH3T3 cells, and A431 cells were 1.2 uM, 2.5 uM and 11.0 uM, respectively.The tyrosine kinase activities of a receptor for EGF (EGFR) and v-Src were strongly inhibited by β-HIVS. The inhibition by β-HIVS of the activities of EGFR and v-Src was much stronger than that by shikonin. The IC50 values of β-HIVS for EGFR and v-Src were approx. 0.7 μM and 1.0μM, respectively. Higher concentration of /?-FflVS also inhibited tyrosine kinase of BCR-ABL. The estimated IC50 for BCR-ABL was closed to 4.0 μM. β--HIVS have practically no effect on PKA and weakly inhibited the activity of PKC. The results suggested that β-HIVS selectively inhibited tyrosine kinase.Combination of /MTIVS with bufalin, with VP16, or with Dox had synergistic, marginal synergistic to additive, or additive to subadditive effects on U937 cells, respectively. The above combinations showed little synergistic effects on DMS114 cells. The combination of β-HIVS and cisplatin (CDDP) showed synergistic effects on both U937 and DMS114 cells.Growth inhibition of β-HIVS and CDDP on DMS114 cells was accompanied by induction of apoptosis. Treatment of DMS114 cells with β-HIVS and CDDP together resulted in slight stimulation of protein kinases ERK1, ERK2 and p38 but no effect on JNK. Growth inhibition on DMS114 cells with β-HIVS and CDDP together was, however, unaffected by inhibitors of MEK (PD98059 and U0126) and of p38 (SB230580), suggesting that MAP kinases might not be involved in thegrowth-inhibition. Using phosphotyrosine-speciflc antibodies (PY20), we observed that tyrosine kinase activity in DMS114 cells was inhibited by treatment with β-HIVS and CDDP together. The tyrosine kinase activities of the isolated v-Src and of the isolated EGFR were also additively inhibited by the two agents together. The synergistic effects on growth of DMS114 cells by β-HIVS and CDDP combined were not simply due to the intracellular accumulation of CDDP or to the levels of DNA-adduct. Our data suggest that the synergistic effects on growth by DMS114 cells of β-HIVS and CDDP might result from the unexpected inhibition of a tyrosine kinase-dependent pathway.The unexpected inhibitory effect on cell growth was also found by the combination of β-HIVS and CDDP isomer transplatin (trans-DDP) on human epidermoid carcinoma A431 cells. By contrast, trans-DDP and β-HIVS by themselves, at the same respective concentrations used for the combined experiment, had practically no effect. The activities of both tyrosine kinases of EGFR and v-Src were strongly inhibited by combination of two agents.All the results suggested that β-HIVS is a special tyrosine kinase inhibitor and can be used alone or combined with other anticancer agents for growth-inhibition of cancer cells.