Reversal Effect And Molecular Mechanism Of Ligustrazine Derivates In K562/A02 Cells

Multidrug resistance (MDR) of tumor cells is a major obstacle in chemotherapeutic treatment of cancer. It contributes significantly to the insensitivity of cancer cells to cytotoxic killing by chemotherapeutic drugs. MDR is mediated through transporter-based or non-transporter-based processes. Transporter-based MDR is largely due to altered functions and expressions of transport proteins of the ATP-binding cassette family. These cell membrane proteins which include P-glycoprotein (P-gp), multidrug resistance protein (MRP), breast cancer resistance protein (BCRP) and so on act by transporting the drugs out of cells through active transport. Increased expressions of these proteins in cancer cells thus causes reduced concentration of the drugs within cancer cells. Non-transporter-based MDR on the other hand act by changing the activities of several cellular enzymes such as glutathione S-transferases (GSTs), topoisomerases and apoptosis cascades, which may alter the metabolism or targeting of anticancer agents.Ligustrazine is a component of the Chinese traditional medicinal herb Chuanxiong(Ligusticum chuanxiong Hort), which is widely used for the treatment of cardiovascular diseases in China. Ligustrazine has recently been suggested to be useful for treatment of cancer as an adjuvant agent to counter multidrug resistance (MDR). It can enhance the chemosensitivity effects of Adr on human hepatocellular carcinoma cells. On the other hand, pharmacokinetics studies have shown that the half-live of ligustrazine in vivo is low. To improve the biavailability of ligustrazine for in vivo use in cancer treatment, a series of novel ligustrazine derivates were synthesized. In this study, we screened the derivates and investigated the effect of DLJ14, typical one of the ligustrazine derivates with high reversal potential, on MDR in human chronic myeloid leukemia cell line K562 and its subline K562/A02 cells in response to chemotherapeutic agent adriamycin (Adr).The resistant fold, reversal fold and cell viability was assessed by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-etrazolium bromide assay. The intracellular accumulation of Adr in K562/A02 cells was determined by its autofluorescence. The expression of P-gp was assayed by western blot and its mRNA was determined by Realtime-PCR. The activity of glutathione S-transferase(GST)-related enzymes and glutathione(GSH) level were assayed by chemical colormetry method. The expression of GSTπ, c-jun NH2-terminal kinase (JNK) and p-JNK activation were determined by western blotting. Real-time PCR was used to assay GSTπmRNA level.The IC50 value of Adr toward K562/A02 cells was higher than that toward K562 cells, the resistant fold was reached 133.3. Ligustrazine derivates DLJ14, DLJ21 and DLJ29 could enhance the sensitivity of K562/A02 cells to cytotoxic killing by therapeutic drug Adr, and the reversal fold of each compound at the concentration of 20μmol/L was 22.46,7.28 and 9.16. DLJ14 showed a weaker inhibition as well as the strongest reversal potential, compared with Verapamil(VP). The elevated accumulation of Adr in K562/A02 cells as well as in K562 cells was observed after treatment with DLJ14.In P-gp-mediated mechanism study, the intracellular accumulation of Rh123 in untreated K562/A02 cells was only 33.64% of that in K562 cells. But after induction by DLJ14 it was apparently increased in K562/A02 cells while no significant alternation was seen in K562 cells. DLJ14 treatment stimulated ATPase activity in K562/A02 cells as well. The released Pi content which represented ATPase activity was significantly decreased in K562/A02 cells and no significant differences in K562 cells in the presence of different concentrations of DLJ14. Following exposure of K562/A02 to DLJ14, the ATP levels were depleted using luciferin-luciferase assay for ATP intracellular levels. However, there were no effects on the expression of P-gp after 72h incubation with DLJ14. And the result of Realtime-PCR showed that only the highest dose 20μmol/L of DLJ14 slightly reduced Mdrl gene expression.In GSTπ-mediated mechanism study, results showed that the activity of GST and GPX was higher in K562/A02 cells than in K562 cells. And DLJ14 at different concentrations(5,10,20μmol/L) caused a reduction in the activity of these enzymes, 3.26%,11.57%,20.77% of GST activity and 18.80%,32.83%,42.61% of GPX acitivity, respectively. GSH level, lower in K562/A02 than in K562 cells due to the high activity of GST and GPX was elevated by 4.86%,18.69%,31.59% by treating with DLJ14(5,10,20μmol/L) for 72h. Moreover, dramatic down regulation of GSTπexpression was observed after incubation with DLJ14 for 72h as well as its mRNA level. However, JNK1 protein expression was not significantly altered in either cell and DLJ14-treatment in the cells at 20μmol/L caused a weak increase of JNK phosphorylation/activation in both cells. Interestingly, the presence of Adr(10μmol/L) resulted in marked increase of JNK phosphorylation in response of the cells to DLJ14. These results suggest that DLJ14 could significantly enhance JNK activation of the cells in response to Adr treatment.Ligustrazine derivate DLJ14 significantly reduced multidrug resistance of K562/A02 cells with much lower anti-proliferation effect than Verapamil. And the mechanism may lie on both P-gp- and GSTπ-mediated pathways. It suggests that DLJ14 has the potential to be developed as a useful chemoadjuvant agent for cancer treatment.