Otsuki Hideo, Kimura Toru, Yamaga Takashi, Kosaka Takeo, Suehiro Jun-Ichi, Sakurai Hiroyuki
Department of Pharmacology and Toxicology, Kyorin University School of Medicine, Mitaka City, Tokyo, Japan.
Department of Urology, Keio University School of Medicine, Shinjuku-ku, Tokyo, Japan.
Prostate. 2017 Feb;77(2):222-233. doi: 10.1002/pros.23263. Epub 2016 Oct 3.
Leucine stimulates cancer cell proliferation through the mTOR pathway, therefore, inhibiting leucine transporters may be a novel therapeutic target for cancer. L-type amino acid transporter (LAT) 1, a Na -independent amino acid transporter, is highly expressed in many tumor cells. However, leucine transporter(s) in different stages of prostate cancer, particularly in the stages of castration resistance with androgen receptor (AR) expression, is unclear.
LNCaP and DU145 and PC-3 cell lines were used as a model of androgen dependent, and metastatic prostate cancer. A new "LN-cr" cell line was established after culturing LNCaP cells for 6 months under androgen-free conditions, which is considered a model of castration resistant prostate cancer (CRPC) with androgen AR expression. The expression of leucine transporters was investigated with quantitative PCR and immunofluorescence. Uptake of C Leucine was examined in the presence or absence of BCH (a pan-LAT inhibitor), JPH203 (an LAT1-specific inhibitor), or Na . Cell growth was assessed with MTT assay. siRNA studies were performed to evaluate the indispensability of y LAT2 on leucine uptake and cell viability in LN-cr.
Cell viability showed a 90% decrease in the absence of leucine in all four cell lines. LNCaP cells principally expressed LAT3, and their leucine uptake was more than 90% Na -independent. BCH, but not JPH203, inhibited leucine uptake, and cell proliferation (IC :15 mM). DU145 and PC-3 cells predominantly expressed LAT1. Leucine uptake and cell growth were suppressed by BCH or JPH203 in a dose-dependent manner (IC : ∼20 mM, IC : ∼5 µM). In LN-cr cells, Na -dependent uptake of leucine was 3.8 pmol/mgprotein/min, while, Na -independent uptake was only 0.52 (P < 0.05). Leucine uptake of LN-cr was largely (∼85%) Na -dependent. y LAT2 expression was confirmed in LN-cr. Knockdown of y LAT2 lead to significant leucine uptake inhibition (40%) and cell growth inhibition (20%).
New CRPC cell line with increased expression of y LAT2 as a leucine transporter was established in vitro. Anti-leucine transporter therapy could be an important option against prostate cancer. Prostate 77:222-233, 2017. © 2016 Wiley Periodicals, Inc.
亮氨酸通过mTOR途径刺激癌细胞增殖，因此，抑制亮氨酸转运蛋白可能是一种新型的癌症治疗靶点。L型氨基酸转运蛋白（LAT）1是一种不依赖钠的氨基酸转运蛋白，在许多肿瘤细胞中高表达。然而，前列腺癌不同阶段，尤其是伴有雄激素受体（AR）表达的去势抵抗阶段的亮氨酸转运蛋白尚不清楚。
使用LNCaP、DU145和PC-3细胞系作为雄激素依赖型和转移性前列腺癌的模型。在无雄激素条件下培养LNCaP细胞6个月后建立了新的“LN-cr”细胞系，该细胞系被认为是具有雄激素AR表达的去势抵抗性前列腺癌（CRPC）模型。通过定量PCR和免疫荧光研究亮氨酸转运蛋白的表达。在存在或不存在BCH（一种泛LAT抑制剂）、JPH203（一种LAT1特异性抑制剂）或钠的情况下检测¹⁴C亮氨酸的摄取。用MTT法评估细胞生长。进行siRNA研究以评估γLAT2在LN-cr细胞中对亮氨酸摄取和细胞活力的不可或缺性。
在所有四种细胞系中，亮氨酸缺失时细胞活力下降90%。LNCaP细胞主要表达LAT3，其亮氨酸摄取超过90%不依赖钠。BCH而非JPH203抑制亮氨酸摄取和细胞增殖（IC₅₀：15 mM）。DU145和PC-3细胞主要表达LAT1。BCH或JPH203以剂量依赖性方式抑制亮氨酸摄取和细胞生长（IC₅₀：20 mM，IC₅₀：5 μM）。在LN-cr细胞中，亮氨酸的钠依赖性摄取为3.8 pmol/mg蛋白/分钟，而不依赖钠的摄取仅为0.52（P < 0.05）。LN-cr细胞的亮氨酸摄取很大程度上（~85%）依赖钠。在LN-cr细胞中证实了γLAT2的表达。敲低γLAT2导致亮氨酸摄取显著抑制（40%）和细胞生长抑制（20%）。
体外建立了具有增加的γLAT2作为亮氨酸转运蛋白表达的新CRPC细胞系。抗亮氨酸转运蛋白疗法可能是对抗前列腺癌的重要选择。《前列腺》77:222 - 233, 2017。© 2016威利期刊公司。
