The Anti-proliferation Effect Of Ligustrazine Piperazine Derivate Dlj14and Adriamycin Combinational Treatment On MCF-7/A Cells In Vitro And In Vivo

Ligustrazine (TMP) is an alkaloid isolated and purified from Chuanxiong rhizome of the genus Artemisia of Umbelliferae plant. It is an active component of the Chinese traditional medicinal herb Chuanxiong. Currently, it has been reported that Ligustrazine has the multi-drug resistance reversal activity aganist tumor cells. However, because the Methyl group of TMP can be easily oxidized, TMP has shortcomings of short half-life and low bioavailability. Aimed to overcome the above shortcomings and improve the effects on tumor treatment by synergizing with disseveration, a series of novel ligustrazine derivates were synthesized. We made the derivates screen to get DLJ14with the best multi-drug resistance reversal activity. Previous study showed that DLJ14could reverse the multidrug resistance on Adriamycin (Adr)-resistant human myelogenous leukemia cells (K562/A02) and Adr-resistant human breast cancer cells (MCF-7/A) in vitro or in vivo. However, the molecular mechanisms underlying the enhancement effects of DLJ14and Adr combinational treatment on cancer cells have not been thoroughly elucidated. In this study, we investigated the effects and action mechanisms of DLJ14and Adr combinational treatment in MCF-7/A cells.Firstly, MTT assay in vitro showed the combined DLJ14and Adr treatment strongly inhibited the cell proliferation compared with Adr alone. In vivo, DLJ14(4mg/kg,8mg/kg) or Adr (1mg/kg) treatment alone doesn’t obviously inhibit tumor growth compared with control treatment. And DLJ14(4mg/kg,8mg/kg) and Adr (lmg/kg) combinational treatment shows the significant inhibition rate. Cell cycle analysis showed that combination of DLJ14with Adr arrested MCF-7/Adr cells in G2/M phase, which was due to the increase of the expression of P53and P21and the decrease of Cyclin A expression. Hoechst33258staining and Annexin V-FITC/Propidium iodide (PI) double staining assay suggested that the combined DLJ14and Adr treatment showed shrinked peripheral nucleus and the increase population of late apoptotic cells in comparison with Adr alone. The ratio of Bax/Bcl-2expression and the expression of cleaved caspase-9and caspase-3proteins were increased. JC-1assay and western blotting analysis showed that the combined DLJ14and Adr treatment significantly decreased mitochondria membrane potential and increased the release of cytochrome c from mitochondrial into cytosol in comparison with Adr alone. Furthermore, the combination of DLJ14with Adr could suppress the survival pathway, as shown by the decreased expression of EGFR and PI3K and the decreased phosphorylation of EGFR and Akt, without affection on total Akt. Overall, DLJ14and Adr combination treatment may inhibit the proliferation of Adr-resistant human breast cancer cells MCF-7/A through inhibition of the EGFR/PI3K/Akt survival pathway and induction of apoptosis via the mitochondrial-mediated apoptosis pathway.Secondly, we explored the action mechanisms of the reversal activity of DLJ14on ABC superfamily resistant-related protein P-gp and BCRP. The cellular acculumation of Rh123demonstrated that the combined DLJ14and Adr treatment, compared with Adr alone, significantly enhanced the fluorescence intensity of Rh123, that is to say, significantly increased the cellular acculumation of Rh123. The cellular ATP content and Ca2+-Mg2+ATPase activity were decreased obviously by the combined DLJ14and Adr treatment in comparison with Adr alone. Furthermore, the expression of ERK, P-ERK, P-gp and BCRP proteins was decreased significantly by the combined DLJ14and Adr treatment. Overall, DLJ14and Adr combinational treatment may inhibit the function and protein expression of P-gp and BCRP to exert the reversal action. In conclusion, DLJ14and Adr combination treatment may inhibit the proliferation of Adr-resistant human breast cancer cells through inhibition of the EGFR/PI3K/Akt survival pathway and induction of apoptosis via the mitochondrial-mediated apoptosis pathway, and may inhibit the function and protein expression of P-gp and BCRP to reverse the resistance of MCF-7/A to adriamycin.