MDR1 inhibition increases sensitivity to doxorubicin and etoposide in adrenocortical cancer

in Endocrine-Related Cancer
Correspondence should be addressed to L J Hofland: l.hofland@erasmusmc.nl
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Chemotherapy for adrenocortical carcinoma (ACC) has limited efficacy and is accompanied by severe toxicity. This lack of effectiveness has been associated with high tumoral levels of the multidrug resistance (MDR) pump P-glycoprotein (P-gp), encoded by the MDR1 gene. In this study, effects of P-gp inhibition on the sensitivity of ACC cells to cytotoxic drugs were evaluated. MDR1 mRNA and P-gp expression were determined in human adrenal tissues and cell lines. H295R, HAC15 and SW13 cells were treated with mitotane, doxorubicin, etoposide, cisplatin and streptozotocin, with or without the P-gp inhibitors verapamil and tariquidar. Cell growth and surviving fraction of colonies were assessed. MDR1 mRNA and P-gp protein expression were lower in ACCs than in adrenocortical adenomas (P < 0.0001; P < 0.01, respectively). MDR1 and P-gp expression were positively correlated in ACC (P < 0.0001, ρ = 0.723). Mitotane, doxorubicin, cisplatin and etoposide dose dependently inhibited cell growth in H295R, HAC15 and SW13. Tariquidar, and in H295R also verapamil, increased the response of HAC15 and H295R to doxorubicin (6.3- and 7.5-fold EC50 decrease in H295R, respectively; all P < 0.0001). Sensitivity to etoposide was increased in H295R and HAC15 by verapamil and tariquidar (all P < 0.0001). Findings were confirmed when assessing colony formation. We show that cytotoxic drugs, except streptozotocin, used for ACC treatment, inhibit ACC cell growth and colony formation at clinically achievable concentrations. P-gp inhibition increases sensitivity to doxorubicin and etoposide, suggesting that MDR1 is involved in sensitivity to these drugs and could be a potential target for cytotoxic treatment improvement in ACC.

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  • Supplementary Table 1. Primers and probes used for real time quantitative PCR.
  • Supplementary Figure 1. Effects of cytotoxic drugs with or without the P-gp inhibitors verapamil (V) or tariquidar (T) on the colony size in H295R (left panel), HAC15 (middle panel), and SW13 (right panel). Data are presented as percentage change compared to vehicle treated control. When combination of cytotoxic drugs with verapamil (+V) or tariquidar (+T) were examined, the control was set as verapamil (10 #x00B5;M) or tariquidar (1 #x00B5;M) monotherapy, respectively. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001 vs vehicle treated control or as stated by the lines. MIT, mitotane; DOX, doxorubicin; ETO, etoposide; CIS, cisplatin. Values represent mean #x00B1; SEM.

 

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    MDR1 mRNA expression in adrenal cell lines (A) and adrenal tissues (B) and P-glycoprotein expression in human adrenocortical tissues (C). Correlation between MDR1 mRNA expression and P-glycoprotein expression in ACCs (D). Representative example of P-gp immunohistochemical staining in adrenocortical carcinoma as determined by immunohistochemistry (E). Sections were blinded and independently evaluated by two investigators. Microscopic magnification 40×, 200×, and 400×. Lines represent medians. ρ represents Spearman’s rank correlation coefficient. *P < 0.05, **P < 0.01, ****P < 0.0001. ACA, adrenocortical adenoma; ACC, adrenocortical carcinoma; IRS, immunoreactivity score; NA, normal adrenals; ND, not detectable.

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    Dose–response curves of mitotane, doxorubicin, etoposide, cisplatin and streptozotocin on total DNA measurement, as a measure of cell amount, in H295R (left panel), HAC15 (middle panel) and SW13 (right panel) after 7 days of treatment. Values represent mean ± s.e.m. and are shown as percentage of control.

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    Effects of mitotane (MIT), doxorubicin (DOX), etoposide (ETO) and cisplatin (CIS), with or without the P-gp inhibitors verapamil (10 µM) or tariquidar (1 µM) on cell growth in three human ACC cell lines H295R (left panel), HAC15 (middle panel) and SW13 (right panel) after 7 days of treatment. Gray solid lines represent the effect of monotherapy. Dotted lines represent the combination of verapamil (black dotted lines) or tariquidar (gray dotted lines) with cytotoxic therapy. Data are presented as percentage of vehicle treated control. When combination of cytotoxic drugs with verapamil (+V) or tariquidar (+T) were examined, the control was set as verapamil (10 µM) or tariquidar (1 µM) monotherapy, respectively. Values represent mean ± s.e.m. and are shown as percentage of control.

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    Effects of cytotoxic drugs with or without the P-gp inhibitors verapamil (V) or tariquidar (T) on the surviving fraction of colonies in H295R (left panel), HAC15 (middle panel) and SW13 (right panel). Data are presented as percentage change compared to vehicle treated control. When combination of cytotoxic drugs with verapamil (+V) or tariquidar (+T) were examined, the control was set as verapamil (10 µM) or tariquidar (1 µM) monotherapy, respectively. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 vs vehicle treated control or as stated by the lines. CIS, cisplatin; DOX, doxorubicin; ETO, etoposide; MIT, mitotane. Values represent mean ± s.e.m.

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