The anti-estrogen receptor drug, tamoxifen, is selectively Lethal to P-glycoprotein-expressing Multidrug resistant tumor cells.

BACKGROUND: P-glycoprotein (P-gp), a member of the ATP Binding Cassette B1 subfamily (ABCB1), confers resistance to clinically relevant anticancer drugs and targeted chemotherapeutics. However, paradoxically P-glycoprotein overexpressing drug resistant cells are "collaterally sensitive" to non-toxic drugs that stimulate its ATPase activity. METHODS: Cell viability assays were used to determine the effect of low concentrations of tamoxifen on the proliferation of multidrug resistant cells (CHORC5 and MDA-Doxo400), expressing P-gp, their parental cell lines (AuxB1 and MDA-MB-231) or P-gp-CRISPR knockout clones of AuxB1 and CHORC5 cells. Western blot analysis was used to estimate P-gp expression in different cell lines. Apoptosis of tamoxifen-induced cell death was estimated by flow cytometry using Annexin-V-FITC stained cells. Oxidative stress of tamoxifen treated cells was determined by measuring levels of reactive oxygen species and reduced thiols using cell-permeant 2',7'-dichlorodihydrofluorescein diacetate (H2DCFDA) and 5,5-dithio-bis-(2-nitrobenzoic acid) DTNB, respectively. RESULTS: In this report, we show that P-gp-expressing drug resistant cells (CHORC5 and MDA-Doxo400) are collaterally sensitive to the anti-estrogen tamoxifen or its metabolite (4-hydroxy-tamoxifen). Moreover, P-gp-knockout clones of CHORC5 cells display complete reversal of collateral sensitivity to tamoxifen. Drug resistant cells exposed to low concentrations of tamoxifen show significant rise in reactive oxygen species, drop of reduced cellular thiols and increased apoptosis. Consistent with the latter, CHORC5 cells expressing high levels of human Bcl-2 (CHORC5-Bcl-2) show significant resistance to tamoxifen. In addition, the presence of the antioxidant N-acetylcysteine or P-gp ATPase inhibitor, PSC-833, reverse the collateral sensitivity of resistant cells to tamoxifen. By contrast, the presence of rotenone (specific inhibitor of mitochondria complex I) synergizes with tamoxifen. CONCLUSION: This study demonstrates the use of tamoxifen as collateral sensitivity drug that can preferentially target multidrug resistant cells expressing P-gp at clinically achievable concentrations. Given the widespread use of tamoxifen in the treatment of estrogen receptor-positive breast cancers, this property of tamoxifen may have clinical applications in treatment of P-gp-positive drug resistant breast tumors.

Knockout of P-glycoprotein abolish the collateral sensitivity of CHORC5 multidrug resistant cells.

Multidrug resistant tumor cells show collaterally sensitive to a range of non-toxic drugs. In this report, we describe the isolation of several P-glycoprotein-knockout cell clones, using CRISPR/Cas9, from Chinese hamster multidrug resistant model cell line and its parental cells (e.g., CHORC5 and AuxB1, respectively). All three P-glycoprotein-knockout clones of CHORC5 cells show complete loss of resistance to anti-cancer drugs (e.g., colchicine and doxorubicin), while gaining resistance to well characterized collateral sensitivity drugs (e.g., verapamil, progesterone and NSC73306). A correlation between P-glycoprotein and Sorcin expression levels and a possible role for the latter in low grade resistance to colchicine and doxorubicin was observed. Furthermore, we show that P-glycoprotein expression is necessary for the ROS-mediated mechanism of collateral sensitivity. However, expectantly, P-glycoprotein-knockout clones of CHORC5 cells revealed a dramatic increase in the accumulation of Rhodamine 123, Mito tracker red and doxorubicin, but not Hoechst 33342. The latter findings and their significance to P-glycoprotein collateral sensitivity remain to be determined.

The phenothiazine, trifluoperazine, is selectively lethal to ABCB1-expressing multidrug resistant cells.

P-glycoprotein, member of the B-subfamily of the ATP-binding cassette (ABC) superfamily (e.g., ABCB1), has been demonstrated to confer resistance to clinically relevant anticancer drugs. Paradoxically, ABCB1-expressing multidrug resistant (MDR) cells are hypersensitivity or collateral sensitivity to non-toxic drugs. In this report, we demonstrate the capacity of trifluoperazine (TFP), a calmodulin inhibitor, to confer a collateral sensitivity onto ABCB1-overexpressing MDR cells. We show TFP-induced collateral sensitivity to be linked to ABCB1 expression and ATPase activity, as such phenotype is abolished in ABCB1-knockout MDR cells (CHORC5ΔABCB1 clones A1-A3) or with inhibitors of ABCB1 ATPase. TFP-induced collateral sensitivity is mediated by apoptotic cell death, due to enhanced oxidative stress. The findings in this study show for first time the use TFP as a collateral sensitivity drug, at clinically relevant concentrations. Moreover, given the use of trifluoperazine in the treatment for symptoms of schizophrenia and the role of ABCB1 transporter in tissue blood barriers and other physiologic functions, the finding in this study may have implications beyond cancer chemotherapy.

Down-regulation of ABCB1 by collateral sensitivity drugs reverses multidrug resistance and up-regulates enolase I.

The emergence of drug resistance remains an obstacle in the clinical treatment of cancer. Recent developments in the studies of drug resistance have identified compounds such as verapamil and tamoxifen that specifically target ABCB1-expressing multidrug-resistant (MDR) cells, through an ATP-dependent ROS-generating mechanism. In this report, we demonstrate that treatment of ABCB1-expressing MDR cells (CHORC5 or MDA-Doxo400) or individual clones of the latter with sub-lethal concentrations of tamoxifen or verapamil down-regulates ABCB1 protein and mRNA expression in surviving clones. Consequently, tamoxifen- and verapamil-treated cells show increased sensitivity to chemotherapeutic drugs (e.g., colchicine and doxorubicin) and decreased sensitivity to collateral sensitivity drugs (e.g., verapamil and tamoxifen). Importantly, we show for the first time that down-regulation of ABCB1 expression resulting from tamoxifen treatment and CRISPR-knockout of ABCB1 expression up-regulate α-enolase (enolase I) protein levels and activity. These findings demonstrate a possible effect of ABCB1 expression on the metabolic homeostasis of MDR cells. Moreover, given the use of tamoxifen to prevent the recurrence of oestrogen receptor-positive breast cancer, the findings of this study may be clinically important in modulating activity of other drugs.