Abstract
Introduction: Adrenocortical carcinoma (ACC) is a rare endocrine malignancy, with very poor prognosis as a majority of the patients have advanced disease at the time of diagnosis. Currently, adjuvant therapy for most patients consists of either mitotane (M) alone or in combination with multi-drug chemotherapeutics such as etoposide (E), doxorubicin (D), and cisplatin (P), known as the Italian protocol (IP; EDPM). This multi-drug treatment regimen, however, carries significant toxicity potential for patients. One way to improve toxicity profiles with these drugs in combination is to understand where their synergy occurs and over what dosing range so that lower dose regimens could be applied in combination with equal or improved efficacy. We hypothesize that a better understanding of the synergistic effects as well as the regulation of steroidogenic enzymes during combination therapy may provide more optimized combinational options with good potency and lower toxicity profiles. Methods: Two human ACC cell lines, NCI-H295R (hormonally active) and SW13 (hormonally inactive), were grown in 2D culture in appropriate growth medium. The viability of the cells after treatment with varying concentrations of the drugs (E, D, and P) either alone or in combinations with M was determined using the CellTiter Glow assay after 72 h, and the combination index for each was calculated using Compusyn by the Chou–Talalay method. The expression levels of enzymes associated with steroidogenesis were evaluated by RT-PCR in NCI-H295R. Results: When both cell lines were treated with M (ranging 25–50 μM), +E (ranging 18.75–75 μM), and +D (ranging 0.625–2.5 μM) we observed a synergistic effect (CI < 1) with potency equivalent to the full Italian protocol (IP), whereas combining M + P + D had an antagonistic effect (CI > 1) indicating the negative effect of adding cisplatin in the combination. Comparing the hormonally active and inactive cell lines, M + P + E was antagonistic in NCI-H295R and synergistic in SW13. Treatment of NCI-H295R cells with antagonistic combinations (M + P + D, M + P + E) resulted in a significant decrease in the levels of steroidogenic enzymes STAR, CYP11A1, and CYP21A2 compared to IP (p < 0.05) while M + E + D resulted in increased expression or no significant effect compared to IP across all genes tested. Conclusions: The synergistic effect for M + E + D was significant and equivalent in potency to the full IP in both cell lines and resulted in a steroidogenic gene expression profile similar to or better than that of full IP, warranting further evaluation. Future in vivo evaluation of the combination of M + E + D (with removal of P from the IP regimen) may lower toxicity while maintaining anticancer efficacy in ACC.
Highlights
Adrenocortical carcinoma (ACC) is a rare endocrine malignancy, with very poor prognosis as a majority of the patients have advanced disease at the time of diagnosis
Italian pro-were treated multiple concentrations of chemotherapeutics and the doxorubicin, viability was astocol,with we first evaluated the effect of single drugs, namely mitotane, cisplatin, and etoposide
Our results shown with multiple concentrations of chemotherapeutics and the viability was assessed in Figure 1 showed a dose-dependent decrease in viability with increasing concentrations using Cell
Summary
Adrenocortical carcinoma (ACC) is a rare endocrine malignancy, with very poor prognosis as a majority of the patients have advanced disease at the time of diagnosis. Adjuvant therapy for most patients consists of either mitotane (M) alone or in combination with multi-drug chemotherapeutics such as etoposide (E), doxorubicin (D), and cisplatin (P), known as the Italian protocol (IP; EDPM). This multi-drug treatment regimen, carries significant toxicity potential for patients. We hypothesize that a better understanding of the synergistic effects as well as the regulation of steroidogenic enzymes during combination therapy may provide more optimized combinational options with good potency and lower toxicity profiles. Even though the exact anti-cancer mechanism of action of mitotane is not clear, inhibition of 11-β hydroxylation and P450 side-chain cleavage leading to blockage of cortisol synthesis in the mitochondria, and mitochondrial-mediated intracellular stress, are known to play an important role in the effectiveness of mitotane treatment in ACC [8,9]
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