Therapeutic arginine depletion in small cell lung cancer : preclinical activity, mechanisms and resistance

Abstract

Small cell lung cancer (SCLC) is an aggressive malignancy, which is characterized by rapid doubling time, good initial response to chemoradiotherapy, but early recurrence with distant metastasis, resulting in extremely poor overall prognosis. Current chemotherapeutic regimens in SCLC are still the time-honored etoposide/cisplatin in the first-line and topotecan in the second-line, which lack specificity with serious adverse events. Therefore, there is an urgent need to explore novel drugs to improve the clinical outcomes of SCLC. Arginine is a non-essential amino acid to human, but certain tumor cells cannot synthesize arginine endogenously. Arginine depletion has become a potential targeted therapy for auxotrophic tumors including human hepatocellular carcinoma, melanoma, leukemia and mesothelioma. BCT-100, a pegylated recombinant human arginase, can catalyze arginine to ornithine, leading to arginine depletion in tumor cells. Our aim is to investigate the preclinical activity and mechanism of BCT-100 in SCLC in vitro and in vivo. In addition, the BCT-100 acquired resistant cell lines derived from SCLC were employed to explore the underlying resistance mechanism to BCT-100 in SCLC. In the first part, most (7/9) of the SCLC cell lines tested were sensitive to BCT-100 treatment. H446, H510A and H526 were relatively sensitive to BCT-100 treatment and chosen for further study on the underlying mechanisms in vitro. BCT-100 induced cytotoxicity via arginine depletion accompanying with oxidative stress and cell cycle arrest in sensitive cell lines. Hydrogen peroxide (H2O2) and superoxide (O2-) were increased and glutathione (GSH) was decreased, accounting for oxidative stress triggered by BCT-100. Co-treatment with N-acetyl-L-cysteine (NAC) reversed the cytotoxic effect mediated by ROS generation, while buthionine sulfoximine (BSO) enhanced the apoptosis induced by BCT-100 treatment. Besides, mitochondrial membrane depolarization (MMD) led to cytochrome c and smac releasing from mitochondria to cytoplasm, suggesting mitochondrial dependent apoptosis. The G1 cell cycle arrest related biomarkers cyclin A2, cyclin B1, CDK2 and CDK4 were down-regulated in a time-dependent manner upon BCT-100 treatment. AKT-mTOR signaling pathway was activated and co-incubation with mTOR inhibitor rapamycin potentiated the cytotoxic effect of BCT-100. In the second part, H446 and H510A cells were employed to establish xenograft models in mice. BCT-100 suppressed the tumor growth and prolonged the median survival time significantly in a dose-dependent manner in both xenografts, confirming the in vitro activity observed in earlier part of study. Apoptosis and cell cycle arrest were observed to explain antitumor effect of BCT-100 in vivo. In the third part, BCT-100 acquired resistant cells (H446-BR and H526-BR) were obtained after long-term exposure. Compared with parental cells, resistant cells exhibited more aggressive migration features, anoikis resistance and epithelial-mesenchymal transition (EMT) phenotype. Contactin-1 (CNTN-1) was fished out by gene chip assay as it was up-regulated in resistant cells, and silencing CNTN-1 re-sensitized H446-BR and H526-BR to BCT-100 treatment. Besides, knockdown of CNTN-1 attenuated the EMT progression through inhibiting AKT signaling pathway. In summary, BCT-100 was shown to have anticancer effect in SCLC via arginine depletion, oxidative stress, cell cycle arrest and apoptosis. CNTN-1 enhanced the acquired drug resistance through induction of EMT progression by AKT signaling pathway.