CAT-1-mediated arginine uptake and regulation of nitric oxide synthases for the survival of human breast cancer cell lines

Abstract

Growth of the human MCF-7 breast cancer cell line is highly dependent on L-arginine. We have reported that L-arginine, released from extracellular substrates by prolactin (PRL)- and 17β-estradiol (E2)-induced carboxypeptidase-D in the cell membrane, promotes nitric oxide (NO) production for MCF-7 cell survival. Arginine uptake is mediated by members of the cationic amino acid transporter (CAT) family and may coincide with induction of nitric oxide synthase (NOS) for the production of NO. The present study investigated the CAT isoforms and PRL/E2 regulation of CAT and NOS in breast cancer cell lines. Using RT-PCR analysis, CAT-1, CAT-2A, and CAT-2B transcripts were detected in MCF-7, T47D, and MDA-MB-231 cells. The CAT-4 transcript was detected in MDA-MB-231 only. CAT-3 was not detected in any of these cells. PRL and E2 did not significantly alter levels of CAT-1 mRNA and protein, nor CAT-2A and CAT-2B mRNAs in MCF-7 and T47D cells. PRL and E2 also had no effect on the overall uptake of L-[2,3,4,5-H(3)] arginine into these cells. However, confocal immunofluorescent microscopy showed that PRL and E2 upregulated eNOS and iNOS proteins, which distributed in the cytoplasm and/or nucleus of MCF-7 cells. Knockdown of CAT-1 gene expression using small interfering RNA significantly decreased L-[2,3,4,5-H(3)]-arginine uptake, decreased viability and increased apoptosis of MCF-7 and T47D cells. In summary, several CAT isoforms are expressed in breast cancer cells. The CAT-1 isoform plays a role in arginine uptake and, together with PRL/E2-induced NOS, contribute to NO production for the survival of MCF-7 and T47D cells.