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Astemizole Sensitizes Adrenocortical Carcinoma Cells to Doxorubicin by Inhibiting Patched Drug Efflux Activity.

ABSTRACT: Adrenocortical carcinoma (ACC) presents a high risk of relapse and metastases with outcomes not improving despite extensive research and new targeted therapies. We recently showed that the Hedgehog receptor Patched is expressed in ACC, where it strongly contributes to doxorubicin efflux and treatment resistance. Here, we report the identification of a new inhibitor of Patched drug efflux, the anti-histaminergic drug astemizole. We show that astemizole enhances the cytotoxic, proapoptotic, antiproliferative and anticlonogenic effects of doxorubicin on ACC cells at concentrations of astemizole or doxorubicin that are not effective by themselves. Our results suggest that a low concentration of astemizole sensitizes ACC cells to doxorubicin, which is a component of the standard treatment for ACC composed of etoposide, doxorubicin, cisplatin and mitotane (EDPM). Patched uses the proton motive force to efflux drugs. This makes its function specific to cancer cells, thereby avoiding toxicity issues that are commonly observed with inhibitors of ABC multidrug transporters. Our data provide strong evidence that the use of astemizole or a derivative in combination with EDPM could be a promising therapeutic option for ACC by increasing the treatment effectiveness at lower doses of EDPM, which would reduce the severe side effects of this regimen.

SUBMITTER: Hasanovic A

PROVIDER: S-EPMC7460240 | biostudies-literature | 2020 Jul

REPOSITORIES: biostudies-literature

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Astemizole Sensitizes Adrenocortical Carcinoma Cells to Doxorubicin by Inhibiting Patched Drug Efflux Activity.

Hasanovic Anida A Simsir Méliné M Choveau Frank S FS Lalli Enzo E Mus-Veteau Isabelle I

Biomedicines 20200729 8

Adrenocortical carcinoma (ACC) presents a high risk of relapse and metastases with outcomes not improving despite extensive research and new targeted therapies. We recently showed that the Hedgehog receptor Patched is expressed in ACC, where it strongly contributes to doxorubicin efflux and treatment resistance. Here, we report the identification of a new inhibitor of Patched drug efflux, the anti-histaminergic drug astemizole. We show that astemizole enhances the cytotoxic, proapoptotic, antipr ...[more]

PMID: 32751066

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Project description:Angiosarcoma is a rare cancer of blood vessel-forming cells with a high patient mortality and few treatment options. Although chemotherapy often produces initial clinical responses, outcomes remain poor, largely due to the development of drug resistance. We previously identified a subset of doxorubicin-resistant cells in human angiosarcoma and canine hemangiosarcoma cell lines that exhibit high lysosomal accumulation of doxorubicin. Hydrophobic, weak base chemotherapeutics, like doxorubicin, are known to sequester within lysosomes, promoting resistance by limiting drug accessibility to cellular targets. Drug synergy between the beta adrenergic receptor (β-AR) antagonist, propranolol, and multiple chemotherapeutics has been documented in vitro, and clinical data have corroborated the increased therapeutic potential of propranolol with chemotherapy in angiosarcoma patients. Because propranolol is also a weak base and accumulates in lysosomes, we sought to determine whether propranolol enhanced doxorubicin cytotoxicity via antagonism of β-ARs or by preventing the lysosomal accumulation of doxorubicin. β-AR-like immunoreactivities were confirmed in primary tumor tissues and cell lines; receptor function was verified by monitoring downstream signaling pathways of β-ARs in response to receptor agonists and antagonists. Mechanistically, propranolol increased cytoplasmic doxorubicin concentrations in sarcoma cells by decreasing the lysosomal accumulation and cellular efflux of this chemotherapeutic agent. Equivalent concentrations of the receptor-active S-(-) and -inactive R-(+) enantiomers of propranolol produced similar effects, supporting a β-AR-independent mechanism. Long-term exposure of hemangiosarcoma cells to propranolol expanded both lysosomal size and number, yet cells remained sensitive to doxorubicin in the presence of propranolol. In contrast, removal of propranolol increased cellular resistance to doxorubicin, underscoring lysosomal doxorubicin sequestration as a key mechanism of resistance. Our results support the repurposing of the R-(+) enantiomer of propranolol with weak base chemotherapeutics to increase cytotoxicity and reduce the development of drug-resistant cell populations without the cardiovascular and other side effects associated with antagonism of β-ARs.

Project description:BACKGROUND:Adrenocortical carcinoma (ACC) is a rare endocrine cancer with treatments limited in efficacy for metastatic disease. New molecular targeted therapies have yet to improve patient outcomes. In contrast, established treatment regimens of adrenolytics and chemotherapy have demonstrated treatment benefit, although admittedly in a minority of patients. Identification of microRNAs (miRNAs) in patients responsive to adjuvant therapy may offer a means to sensitize patients with progressive disease to existing adjuvant regimens. MATERIALS AND METHODS:Samples from primary ACC tumors of 10 Stage IV patients were examined for differentially expressed miRNAs between a "sensitive" and "resistant" cohort. Candidate microRNAs were restored via transfection in two functional ACC cell lines. Gain of function and effects on apoptosis and cell cycle were assessed. RESULTS:microRNA-431 (miR-431) was underexpressed in patients with ACC with progressive disease undergoing adjuvant therapy. Restoration of miR-431 in vitro decreased the half maximal inhibitory concentrations of doxorubicin and mitotane, with markedly increased apoptosis. We found that a reversal of epithelial-mesenchymal transition underlies the action of miR-431 with doxorubicin treatment, with Zinc Finger E-Box Binding Homeobox 1 implicated as the molecular target of miR-431 in ACC. CONCLUSION:This is the first report of the potential of miRNA therapy to sensitize ACC to current established adjuvant therapy regimens, which may mitigate the resistance underlying treatment failure in patients with advanced ACC. Effective and well-studied methods of targeted miRNA delivery in existence hints at the imminent translatability of these findings. IMPLICATIONS FOR PRACTICE:Adrenocortical carcinoma (ACC) is a rare endocrine cancer with outcomes not improving despite extensive research and new targeted therapies. Mitotane and etoposide/doxorubicin/cisplatin chemotherapy is trial validated for improved recurrence-free survival. However, a minority of patients experience sustained benefit. Significant side effects exist for this regimen, with patients often unable to attain target drug doses shown to give survival benefit. This preclinical study examines the role of microRNAs in sensitizing ACC to doxorubicin or mitotane. This study offers an important bridge between new and existing cancer treatments, offering an imminently translatable approach to the treatment of adrenocortical carcinoma.

Project description:Adrenocortical carcinoma (ACC) has a poor prognosis, and no new drugs have been identified in decades. The absence of drug development can partly be attributed to a lack of preclinical models. Both animal models and 2D cell cultures of ACC fail to accurately mimic the disease, as animal physiology is inherently different than humans, and 2D cultures fail to represent the crucial 3D architecture. Organoids and other small 3D in vitro models of tissues or tumors can model certain complexities of human in vivo biology; however, this technology has largely yet to be applied to ACC. In this study, we describe the generation of 3D tumor constructs from an established ACC cell line, NCI-H295R. NCI-H295R cells were encapsulated to generate 3D ACC constructs. Tumor constructs were assessed for biomarker expression, viability, proliferation, and cortisol production. In addition, matrix metalloproteinase (MMP) functionality was assessed directly using fluorogenic MMP-sensitive biosensors and through infusion of NCI-H295R cells into a metastasis-on-a-chip microfluidic device platform. ACC tumor constructs showed expression of biomarkers associated with ACC, including SF-1, Melan A, and inhibin α. Treatment of ACC tumor constructs with chemotherapeutics demonstrated decreased drug sensitivity compared to 2D cell culture. Since most tumor cells migrate through tissue using MMPs to break down extracellular matrix, we validated the utility of ACC tumor constructs by integrating fluorogenic MMP-sensitive peptide biosensors within the tumor constructs. Lastly, in our metastasis-on-a-chip device, NCI-H295R cells successfully engrafted in a downstream lung cell line-based construct, but invasion distance into the lung construct was decreased by MMP inhibition. These studies, which would not be possible using 2D cell cultures, demonstrated that NCI-H295R cells secreted active MMPs that are used for invasion in 3D. This work represents the first evidence of a 3D tumor constructs platform for ACC that can be deployed for future mechanistic studies as well as development of new targets for intervention and therapies.

Project description:BackgroundCalcitriol antiproliferative effects include inhibition of the oncogenic ether-à-go-go-1 potassium channel (Eag1) expression, which is necessary for cell cycle progression and tumorigenesis. Astemizole, a new promising antineoplastic drug, targets Eag1 by blocking ion currents. Herein, we characterized the interaction between calcitriol and astemizole as well as their conjoint antiproliferative action in SUM-229PE, T-47D and primary tumor-derived breast cancer cells.Methodology/principal findingsMolecular markers were studied by immunocytochemistry, Western blot and real time PCR. Inhibitory concentrations were determined by dose-response curves and metabolic activity assays. At clinically achievable drug concentrations, synergistic antiproliferative interaction was observed between calcitriol and astemizole, as calculated by combination index analysis (CI <1). Astemizole significantly enhanced calcitriol's growth-inhibitory effects (3-11 folds, P<0.01). Mean IC(20) values were 1.82 ± 2.41 nM and 1.62 ± 0.75 µM; for calcitriol (in estrogen receptor negative cells) and astemizole, respectively. Real time PCR showed that both drugs alone downregulated, while simultaneous treatment further reduced Ki-67 and Eag1 gene expression (P<0.05). Astemizole inhibited basal and calcitriol-induced CYP24A1 and CYP3A4 mRNA expression (cytochromes involved in calcitriol and astemizole degradation) in breast and hepatoma cancer cells, respectively, while upregulated vitamin D receptor (VDR) expression.Conclusions/significanceAstemizole synergized calcitriol antiproliferative effects by downregulating CYP24A1, upregulating VDR and targeting Eag1. This study provides insight into the molecular mechanisms involved in astemizole-calcitriol combined antineoplastic effect, offering scientific support to test both compounds in combination in further preclinical and clinical studies of neoplasms expressing VDR and Eag1. VDR-negative tumors might also be sensitized to calcitriol antineoplastic effects by the use of astemizole. Herein we suggest a novel combined adjuvant therapy for the management of VDR/Eag1-expressing breast cancer tumors. Since astemizole improves calcitriol bioavailability and activity, decreased calcitriol dosing is advised for conjoint administration.

Dataset Information

Astemizole Sensitizes Adrenocortical Carcinoma Cells to Doxorubicin by Inhibiting Patched Drug Efflux Activity.

Publications

Astemizole Sensitizes Adrenocortical Carcinoma Cells to Doxorubicin by Inhibiting Patched Drug Efflux Activity.

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