| MDR (multidrug-resistance) is a major obstacle in the chemotheraputy of cancer. Numerous mechanisms are known to contribute to MDR, including overexpression of drug efflux pumps, increased activity of DNA repair mechanisms, alteration of drug target enzymes, overexpression of enzymes involved in drug detoxification and elimination, and alterations at the level of apoptosis control of cancer cells. The present study focused on the reversing MDR effect of two compounds by inhibiting P-glycoprotein, and the mechanism of 5-bromotetrandrine enhancing the sensitivity of doxorubicin-induced apoptosis in intrinsic resistant human hepatic cancer Bel7402 cells.The cytotoxity of two derivatives of Tetrandrine W6 and W18 on resistant cells and their parent cells (such as KBv200 and KB, MCF-7/Dox and MCF-7, A549/Taxol and A549) was determined by MTT assay. Resistant cell lines and their parent cell lines had similar sensitivity to W6和W18, which shown that the two compounds seems not to be the substrates of drug efflux pumps. The expression of P-glycoprotein, MRP land BCRP in the six cell lines mentioned above and intrinsic resistant Bel7402 cells were detected by Western blot analysis. P-glycoprotein significantly overexpressed in KBv200 and MCF-7/Dox cells, and slightly overexpressed in A549/Taxol cells compared with other cell lines. MRP1 significantly overexpressed in required resistant A549/Taxol and its parent A549 cells compared with other cells. The expression of BCRP was relatively low in all cell lines, but the level of BCRP was higher in MCF-7/Dox and A549/Taxol than that of other cells. Nontoxic concentrations of W6 or W18 (0.25μM,0.5μM,1.OμM) obviously enhanced the sensitivity of KBv200 and MCF-7/Dox cells to vincristine (VCR), doxorubicin (Dox) and taxol which were the substrates of P-glycoprotein in a dose-dependent manner. 1.0μM W6 or W18 nearly completely reversed the KBv200 and MCF-7/Dox cells to the three anticancer drugs. The MDR reversal effect of W6 or W18 at 1.0μM was better than that of 10μM verapamil (Vrp). Though W6 and W18 also enhanced the sensitivity of A549/Taxol to the three anticancer drugs, the MDR reversal activity was low. However, 1.0μM W6 or W18 had no chemosensitising effect on A549 cells to VCR and Dox which are the substrates of MRP 1 and BCRP (R482G and R482T) respectively. It seems that the MDR reversal activities of W6 and W18 depended on the levels of over-expressing P-glycoprotein. Therefore the MDR reversal effect of the two derivatives based on Tetrandrine was possibly through the inhibition of P-glycoprotein.Flow cytometry determined the effect of the two compounds on the drug efflux function of P-glycoprotein. W6 or W18 (0.25μM,0.5μM,1.0μM) were able to inhibit the drug efflux function of P-glycoprotein, and significantly increased the accumulation of Dox in KBv200 and MCF-7 cells in a concentration dependent manner. Chemiluminescence method further determined the mechanism of the two compounds inhibiting function of P-glycoprotein. W6 (12.5μM,25μM,50μM) inhibited basal activity of recombinant human Pgp ATPase in a concentration dependent manner, and also inhibited the increased activity of recombinant human Pgp ATPase stimulated by Vrp (200μM) which is the reversible competitive substrate of P-glycoprotein. W18 (12.5μM,25μM,50μM) had no effect on the basal activity of recombinant human Pgp ATPase, but also inhibited the increased activity of recombinant human Pgp ATPase stimulated by Vrp (200μM) too. These results further suggest that the two compounds were not the substrates of P-glycoprotein, and they inhibited the drug efflux effect by inhibiting the activity of Pgp ATPase. Western blot analysis detected the effect of the two derivatives based on Tetrandrine on the expression of P-glycoprotein in KBv200 cells. W6 or W18 (1.OμM) was able to reduce the expression of P-glycoprotein in a time dependent manner. RT-PCR determined whether the two compounds reduced the expression of P-glycoprotein by regulating its transcription. W6 or W18 (1.OμM) had no effect on the levels of mRNA of Pgp as well as MRP1, BCRP and LRP. The mechanism of the two compounds reducing the over-expression might be through other pathways.Because alterations at the level of apoptosis control of cancer cells play a very important roll in MDR, Western blot analysis detected the effect of the two compounds on the key proteins involved in apoptosis regulation. Compared with its parent KB cells, PI3K/Akt pathway was low active and MAPK/Erkl/2 pathway was highly active in resistant KBv200 cells. W6 or W18 (1.OμM)had no effect on the PI3K/Akt pathway, but reduced the level of p-Erk1/2 to inhibit the activation MAPK/Erk1/2 survival pathway. Compared with its parent KB cells, pro-apoptotic protein p53 slightly reduced, pro-apoptotic protein Bax significantly reduced, anti-apoptotic protein XIAP obviously increased, and anti-apoptotic protein Bcl-2 did not change in KBv200 cells. W6 or W18 (1.0μM) had no effect on these proteins. It is concluded that the two derivatives of Tetrandrine W6 and W18 reversed MDR in vitro by inhibiting P-glycoprotein and MAPK/Erk1/2 survival pathway.The present study also investigated the mechanism of 5-bromotetrandrine enhancing the sensitivity of doxorubicin-induced apoptosis in intrinsic resistant human hepatic cancer Bel7402 cells.5-bromotetrandrine (BrTet) was shown to overcome multi-drug resistance (MDR) in vitro and in vivo by inhibiting the overexpression and efflux function of P-glycoprotein in our previous study. The purpose of the present study was to evaluate the effect of BrTet on the sensitivity of doxorubicin (Dox) induced apoptosis in intrinsic resistant human hepatic cancer Bel7402 cells. The cells were treated with non-cytotoxic concentrations of BrTet (1μM,2μM,4μM) or the positive control drug verapamil (Vrp) (10μM) for 24 h followed by a low dose Dox (3μM) for 24 h. The results showed that BrTet pretreatment followed by Dox led to typical apoptotic characters as indicated by morphologic changes, DNA fragmentation and changes in cell cycle, while the same dose of BrTet, Vrp and Dox alone did not induce apoptosis in Bel7402 cells. In addition, the pretreatment of BrTet or Vrp followed by Dox induced activation of caspase-3, release of cytochrome c and AIF from mitochondria into cytosol, loss of mitochondrial transmembrane potential(ΔΨm) and elevation of Bax/Bcl-2 ratio, with no effect on activation of caspase-8 and the expression of Fas/FasL. In conclusion, BrTet pretreatment enhanced the sensitivity of Dox to induce apoptosis by causing loss ofΔΨm and elevating the ratio of Bax/Bcl-2, eventually activated mitochondrial apoptotic pathway.In summary, the present study evaluated the MDR reversal activity of two Tetrandrine derivatives W6 and W18. They were shown to inhibit drug efflux function and overexpression of P-glycoprotein, and inhibit the highly activated MAPK/Erk1/2 survival pathway to sensitize several resistant cancer cells to anti-cancer drugs in vitro. Other mechanism and MDR reversal activity would be worthy to be further investigated. In addition, the present study also shown that BrTet pretreatment enhanced the sensitivity of Dox to induce apoptosis by causing loss ofΔΨm and elevating the ratio of Bax/Bcl-2, eventually activated mitochondrial apoptotic pathway. These findings further support the potential of BrTet to be used in clinical trail of cancer treatment. |
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