Theranostics 2020; 10(8):3562-3578. doi:10.7150/thno.39944

Research Paper

The failure of DAC to induce OCT2 expression and its remission by hemoglobin-based nanocarriers under hypoxia in renal cell carcinoma

Lu Chen1*, Zeyang Wang1*, Qingwen Xu1, Yuxi Liu1, Le Chen1, Suhang Guo1, Hua Wang2, Kui Zeng1, Junqing Liu3, Su Zeng1✉, Lushan Yu1✉

1. Institute of Drug Metabolism and Pharmaceutical Analysis, Zhejiang Province Key Laboratory of Anti-Cancer Drug Research, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou 310058, China.
2. Department of Urology, Cancer Hospital of Zhejiang Province, Hangzhou 310022, China
3. The First Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou 310022, China
*These authors contributed equally to this work.

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Citation:
Chen L, Wang Z, Xu Q, Liu Y, Chen L, Guo S, Wang H, Zeng K, Liu J, Zeng S, Yu L. The failure of DAC to induce OCT2 expression and its remission by hemoglobin-based nanocarriers under hypoxia in renal cell carcinoma. Theranostics 2020; 10(8):3562-3578. doi:10.7150/thno.39944. Available from https://www.thno.org/v10p3562.htm

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Abstract

Background: Human organic cation transporter 2 (OCT2) is the most abundant and important uptake transporter involved in the renal excretion of cationic drugs. Abnormal hypermethylation- mediated silencing of OCT2 results in oxaliplatin resistance in renal cell carcinoma (RCC). The epigenetic activation of OCT2 by decitabine (DAC) reversed this resistance in normoxic conditions. Given the hypoxic characteristic of RCC, it is still unclear whether hypoxia promotes DAC resistance and is involved in the regulation of OCT2.

Methods: The mRNA and protein expression of OCT2 was determined by qRT-PCR and Western blotting. MSRE-qPCR and BSP were used to examine methylation modifications at the OCT2 promoter. The ChIP-qPCR analysis was performed to detect the abundance of histone modification and HIF-1α. The accumulation of DAC and 5-mC were detected using LC-MS, and the amount of 5-hmC was determined by dot blot analysis. To understand the role of hypoxia in the regulation of equilibrative nucleoside transporter 1 (ENT1) expression, the HIF-1α KO cell model was constructed. The re-emulsion method was used for the construction of H-NPs, an oxygen nanocarrier based on hemoglobin, to alleviate the drug resistance of DAC under hypoxia.

Results: DAC was unable to upregulate OCT2 expression in hypoxic conditions because of the hypermethylation and low H3K4me3 modification in its promoter region. Hypoxia-mediated repression of human ENT1, which was markedly suppressed in RCC, resulted in a decrease in the cellular accumulation of DAC. Besides, hypoxia-induced upregulation of histone deacetylase HDAC9, which impaired the enrichment of H3K27ac modification in the OCT2 promoter, led to the transcriptional repression of OCT2. H-NPs could attenuate the hypoxia-induced loss of DAC activity and sensitize RCC cells to the sequential combination therapy of DAC and oxaliplatin.

Conclusions: Hypoxia-mediated repression of ENT1 led to the inability of DAC to upregulate the expression of OCT2 under hypoxia. H-NPs could alleviate resistance to oxaliplatin and DAC in RCC cells under hypoxia and may have potential clinical applications.

Keywords: renal cell carcinoma, hypoxia, OCT2, ENT1, nanoparticles