Characterization of amino acid transport system L in HTB-41 human salivary gland epidermoid carcinoma cells.

BACKGROUND: The amino acid transport system L is a major nutrient transport system that is responsible for transport of neutral amino acids, including several essential amino acids. The current study attempted to investigate the expression and functional characterization of amino acid transport system L in HTB-41 human submaxillary salivary gland epidermoid carcinoma cells. MATERIALS AND METHODS: RT-PCR analysis, Western blot analysis and amino acid transport measurements were used. RESULTS: The HTB-41 cells expressed the L-type amino acid transporter 1 (LAT1) together with its associating protein heavy chain of 4F2 antigen (4F2hc) in the plasma membrane, whereas the HTB-41 cells did not express the L-type amino acid transporter 2 (LAT2). The uptakes of [14C]L-leucine were Na+-independent and completely inhibited by a system L selective inhibitor, 2-aminobicyclo-(2,2,1)-heptane-2-carboxylic acid (BCH). The affinity of [14C]L-leucine uptake and the inhibition profile of [14C]L-leucine uptake by various L-amino acids in the HTB-41 cells were comparable with those for the LAT1 expressed in Xenopus oocytes. CONCLUSION: The transport of neutral amino acids including several essential amino acids into the HTB-41 human submaxillary salivary gland epidermoid carcinoma cells are mediated by LAT1.

Cordyceps militaris induces the IL-18 expression via its promoter activation for IFN-gamma production.

ETHNOPHARMACOLOGICAL RELEVANCE: Cordyceps militaris, one of traditional herbal ingredient in oriental medicine, has been known to promote anticancer and immunomodulatory activities in vitro and in vivo. However, the biological mechanism of anticancer activity has been unknown. OBJECTIVE: To investigate the effect of Cordyceps militaris extract on expression of interferon gamma (IFN-gamma) through interlukin-18 (IL-18) induction and its biological mechanism in vitro and in vivo. MATERIALS AND METHODS: Mice were administrated orally with solution extracted from Cordyceps militaris. The transcription level of IL-18 and IFN-gamma production were measured by reverse transcriptase polymerase chain reaction (RT-PCR) and immunohistochemistry. RAW 264.7 cells were transiently transfected with pCATp1 and pCATp2 for IL-18 promoter functional analysis. RESULTS: Cordyceps militaris extracts treatment significantly induced level of IL-18 transcription in mouse brain and liver and enhanced IL-18 transcription level and activated the IFN-gamma production in RAW 264.7 cells. Furthermore, Cordyceps militaris extract led to increase the activity of pCATp1 construct containing the 5' franking region of IL-18 promoter in RAW 264.7 cells. CONCLUSION: Cordyceps militaris extract induced IL-18 mRNA level via enhancing of P1 promoter region result in activation of IFN-gamma production, indicating its potential as an immune activator or anticancer drug.

BCH, an inhibitor of system L amino acid transporters, induces apoptosis in cancer cells.

PURPOSE: L-Type amino acid transporter 1 (LAT1) is highly expressed in cancer cells to support their continuous growth and proliferation. We have examined the effect of 2-aminobicyclo-(2,2,1)-heptane-2-carboxylic acid (BCH), an inhibitor of system L amino acid transporters, and the mechanism by which BCH suppresses cell growth in cancer cells. METHODS: The effect of BCH and the mechanism of BCH on cell growth suppression in cancer cells were examined using amino acid transport measurement, MTT assay, DNA fragmentation analysis, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay and immunoblotting. RESULTS: BCH inhibited L-leucine transport in a concentration-dependent manner, and it inhibited cell growth in a time-dependent manner in KB human oral epidermoid carcinoma cells, Saos2 human osteogenic sarcoma cells and C6 rat glioma cells. The formation of a DNA ladder was observed, and the number of TUNEL-positive cells was increased with BCH treatment. Furthermore, the proteolytic processing of caspase-3 in KB and C6 cells and of caspase-7 in KB, Saos2 and C6 cells was increased by BCH treatment. CONCLUSION: These results suggest that the inhibition of LAT1 activity by BCH leads to apoptotic cancer cell death by inducing intracellular depletion of neutral amino acids necessary for cancer cell growth.