Effects Of Disodium Cantharidinate On Human Multidrug-resistant K562/AO₂ Cells In Vitro And Its Mechanisms

Objective: This experiment aimed to evaluate the reverse action to K562/AO₂ cells with disodium cantharidinate(SCA) in vitro, and further investigated possible mechanism of its role as multidrug-resistant reversal .Methods: Selecting classic multidrug-resistant cell lines- K562/AO₂ and its sensitive cell lines-K562, using MTT assay to detect SCA's cell toxicity to K562/AO₂, in order to choose SCA's nontoxic doses. Cum verapamil(VRP) for positive control, electing nontoxic doses SCA (or VRP) to reverse multidrug- resistant experiment combined with adriamycin(ADM) to K562/AO₂ and K562 cells. The level of mdr1 expression was examined by semiquantitative RT-PCR technique in K562/AO₂ cell lines treated before and after with SCA (or VRP). The amount of ADM in cells and the expression of P-glycoprotin (P-gp) and Bcl-2 protein were semi-quantitately examined by flow cytometry(FCM) in cell lines treated before and after with SCA (or VRP).Results:1 MTT assay results:Within the concentration of (0.05～10)μg/ml, SCA can inhibit proliferation of K562/AO₂ and K562 cells in vitro. The inhibition ratio was stepping up as the concentration increasing. When SCA's concentration was lower than 0.11μg/ml, SCA could not obviously proliferation inhibiting by regression analysis, and the experiment select 0.1μg/ml for nontoxic doses. IC50 values in K562/AO₂ cell decreased from (40.85±0.40)μg/ml to (27.09±0.63)μg/ml,(4.29±0.13)μg/ml treated before and after with 0.1μg/ml SCA (or 5μg/ml VRP). After being treated with the two drugs, and the multidrug resistance of K562/AO₂ cell were partially reversed. And the reversal index is respectively 1.51,9.52. There was significant difference between two drugs treatment (P<0.01). 2 RT-PCR detection results:Before being treated with SCA (or VRP), mdr1 mRNA was highly expressed in K562/AO₂ cell, and hardly expressed in K562 cell. Their mdr1 mRNA expressions was significant difference (P<0.01). After being treated 24h,48h,72h, mdr1 mRNA expressions became reducing as action time increasing, the weaker expressions was in the group with 72h. There was no significant difference in the reduced mdr1 mRNA expression between two drugs (P>0.05). 3 FCM assay results:P-gp protein was highly expressed in K562/AO₂ cell, and hardly expressed in K562 cell. The FI-value of P-gp in K562/AO₂ was 5.82±0.08. Their P-gp expressions was significant difference (P<0.01). After being treated with SCA 24h,48h,72h, P-gp expressions in K562/AO₂ cell became reducing as action time increasing, the weaker expressions was in the group with 72h, and the relative magnitude was 2.71±0.02. After being treated with VRP the same time, P-gp expressions reduced to 1.16±0.03. Both two drug reduced P-gp expression, and there was significant difference in the inhibition degree of P-gp protein expression between SCA and VRP (P<0.01). The expression of Bcl-2 protein had been examined in two cells, and there was no significant difference between two cells (P>0.05). After being treated in K562 cell with SCA (or VRP), Bcl-2 expressions became reducing as action time increasing. The weaker expression was in the group with 72h. There was significant difference in the inhibition degree of Bcl-2 protein expression after treating 24h between SCA and VRP (P<0.01). But there was no significant difference between SCA and VRP , as action time increasing (P>0.05). After being treated in K562/AO₂ cell with SCA (or VRP), Bcl-2 expressions became reducing as action time increasing. The weaker expressions was in the group with 72h, and the relative magnitude respectively was 1.59±0.05,1.67±0.08. There was no significant difference in inhibition degree of Bcl-2 protein expression between two drugs (P>0.05). The relative magnitude (mode) of amount of ADM in K562/AO₂ was (31.01±0.79)^{44.81±1.15}, after being treated 30¹20 minites simply with ADM. And incubation with SCA (or VRP), the amount of ADM in cells was stepping up as the time increasing (P<0.01). And there was significant difference in the improvement degree of the accumulation of chemotherapeutics between SCA and VRP (P<0.01), SCA was worse than VRP.Conclusions: 1 Within the concentration of (0.05¹0) μg/ml, SCA might inhibit proliferation of K562/AO₂ and K562 cells in vitro which depending on concentration. 2 Nontoxic doses SCA can partially reverse the multidrug resistance of K562/AO₂ cells to ADM, and reversal level is weaker than VRP. 3â‘ Within a certain drug concentration, SCA might reverse multidrug-resistance by inhibiting expression of mdr1 mRNA,P-gp in K562/AO₂ cells.â‘¡Within a certain drug concentration, SCA might reverse multidrug- resistance by inhibiting P-gp pumping function and improving the accumulation of chemotherapeutics in resistant cells.â‘¢Within a certain drug concentration, SCA might relieve Bax\Bak inhibition and relieve drug impairment's dead signal'inhibition by inhibiting protein expression of Bcl-2 to promote cells apoptosis. 4 Our research provides basic theory to clinical application, and provides a new choice for multidrug-resistant reversal.