Natural Product Dl Research On Glioma Cell Proliferation And Chemotaxis

Our previous screening study has found that a natural fraction DL could inhibit the cell viability of a variety of tumor cell lines, of which human malignant glioma cell U87 is most sensitive to DL. The present study thus investigated the anti-glioma effect of DL and further investigated its action mechanism. The effect of DL at the non-cytotoxic concentration on the FPR function and migration of U87 cells was also investigated.MTT results showed that DL (5μg/mL,10μg/mL,30μg/mL,60μg/mL and 100μg/mL) significantly inhibited the cell viability of many glioma cells including human glioma cell lines U87, TJ899, TJ905, U251 and rat glioblastoma C6 cell line in a concentration-and time-dependant manner. Glioblastom cells treated with DL (30μg/mL,60μg/mL and 100μg/mL) for 24 h displayed marked morphological changes. Cells became round and extensive cytoplasmic vacuoles were observed.Further study of the DL-induced cell death in U87 found that, fluorescence microscopic studies of cells stained with AO/EB confirmed that DL (30μg/mL,60μg/mL and 100μg/mL) caused a dramatic increase in vacuolization upon exposure and the cells became round. There were no signs of classic apoptotic changes, such as cell shrinkage or membrane bleb formation. DNA flow-cytometric analysis revealed that treatment of U87 cells with DL for 24 h increased the cells in G2/M phase. The population in sub-G1 phase, which is characteristic of apoptosis, only slightly increased.To clarify the mechanism underlying glioma cell death upon DL (30μg/mL,60μg/mL and 100μg/mL) exposure, DL in U87 cell lines was evaluated. DL induced a concentration-dependent increase in ROS production, but pre-incubation with N-acetylcysteine (NAC) and rotenone, resulted in partial inhibition of DL-induced ROS generation. Furthermore, DL (30μg/mL,60μg/mL and 100μg/ML) could decrease mitochondrial membrane potential, and pre-incubation with NAC and rotenone could not necessarily attenuation of the loss of MMP. Moreover, DL could induced U87 cells DNA damage. DL could stimulate intracellular calcium increase which is mainly from extracellular and could be blocked by pertussis toxin. These results suggest the involvement of the Gi protein in this process. It was also found that non-cytotoxic concentrations of DL were able to significantly induce the migration of U87 cells to its higher concentrations, which helped a lot to exert its cytotoxic effect. DL-induced U87 cell migration was mainly chemotaxis with slight chemokinetics in a pertussis toxin (PTX)-sensitive manner.In addition, it was found that DL could interfered with the function and signaling of FPR. Non-cytotoxic concentrations of DL could markedly inhibit fMLF-induced U87 cell chemotaxis, calcium mobilization and VEGF mRNA expression.In summary, the results demonstrated that DL could significantly inhibit the growth of a variety of glioma cells. The death mechanism induced by DL might be at least partly due to an increase in intracellular reactive oxygen species. It was also found that non-cytotoxic concentrations of DL markedly interfered with the function and signaling of FPR. What is more, non-cytotoxic concentrations DL could induce U87 cells chemotaxis to its high concentrations. Taken together, DL has great potential to be developed into an effective antigioma agent.