melphalan and 2-aminobicyclo(2-2-1)heptane-2-carboxylic-acid

L-type amino acid transporter 1 (LAT1), an isoform of amino acid transport system L, transports branched or aromatic amino acids essential for fundamental cellular activities, such as cellular growth, proliferation and maintenance. LAT1 has recently received attention because of its preferential and upregulated expression in a variety of human tumours which is in contrast to its limited distribution and low-level expression in normal tissues. In this study, the feasibility of using an LAT1 inhibitor as a new therapeutic agent was explored for mammary gland tumours (MGT). [(3)H]l-leucine uptake by CHM, a cell line established from MGT, and effects on cell growth were analysed in the presence or absence of two LAT1 inhibitors, namely, BCH (2-amino-2-norbornane-carboxylic acids) or melphalan (LPM). [(3)H]l-leucine uptake and cellular growth activities in CHM were inhibited in a dose-dependent manner by both LAT1 inhibitors. The inhibitory growth activities of various conventional anti-cancer drugs used for MGT treatment, including carboplatin, cyclophosphamide, doxorubicin, mitoxantrone, vinblastine and vincristine, were significantly enhanced by combining use with BCH or LPM. The findings suggest that LAT1 could be a new therapeutic target for canine MGT.

Topics: Amino Acids, Cyclic; Animals; Antineoplastic Agents; Blotting, Western; Cell Line, Tumor; Cell Proliferation; Dog Diseases; Dogs; Dose-Response Relationship, Drug; Female; Gene Expression Regulation, Neoplastic; Humans; Inhibitory Concentration 50; Large Neutral Amino Acid-Transporter 1; Mammary Neoplasms, Animal; Melphalan; Real-Time Polymerase Chain Reaction

L-type amino acid transporter 1 (LAT1), an isoform of amino acid transport system L, transports branched or aromatic amino acids essential for fundamental cellular activities such as cellular growth, proliferation and maintenance. This amino acid transporter recently has received attention because of its preferential and up-regulated expression in a variety of human tumors in contrast to its limited distribution and low-level expression in normal tissues. In this study, we explored the feasibility of using LAT1 inhibitor as a new therapeutic agent for human malignant melanomas (MM) using canine spontaneous MM as a model for human MM. A comparative study of LAT expression was performed in 48 normal tissues, 25MM tissues and five cell lines established from MM. The study observed LAT1 mRNA levels from MM tissues and cell lines that were significantly (P<0.01) higher than in normal tissues. Additionally, MM with distant metastasis showed a higher expression than those without distant metastasis. Functional analysis of LAT1 was performed on one of the five cell lines, CMeC-1. [(3)H]l-Leucine uptake and cellular growth activities in CMeC-1 were inhibited in a dose-dependent manner by selective LAT1 inhibitors (2-amino-2-norbornane-carboxylic acid, BCH and melphalan, LPM). Inhibitory growth activities of various conventional anti-cancer drugs, including carboplatin, cyclophosphamide, dacarbazine, doxorubicin, mitoxantrone, nimustine, vinblastine and vincristine, were significantly (P<0.05) enhanced by combination use with BCH or LPM. These findings suggest that LAT1 could be a new therapeutic target for MM.

Topics: Amino Acids, Cyclic; Animals; Antineoplastic Agents; Cell Line, Tumor; Cell Proliferation; Dogs; Dose-Response Relationship, Drug; Gene Expression Regulation, Neoplastic; Humans; Inhibitory Concentration 50; Large Neutral Amino Acid-Transporter 1; Melanoma; Melphalan; Neoplasm Metastasis; Pilot Projects

It has been suggested that system L (LAT1/CD98hc) is up-regulated in cancer cells, including breast tumour cells, and is therefore a promising molecular target to inhibit or limit tumour cell growth. In view of this, we have examined the effect of BCH and other inhibitors of system L on the growth of MCF-7, ZR-75-1 and MDA-MB-231 cells. Treating cells with BCH markedly inhibited the metabolism of WST-1 in a dose-dependent fashion. Similarly, melphalan and D-leucine inhibited the growth of cultured breast cancer cells whereas MeAIB, an inhibitor of system A, was without effect. The effects of BCH and melphalan on cell growth were non-additive suggesting that both compounds were acting at a single locus. The results indicate that system L is required to maintain MCF-7, ZR-75-1 and MDA-MB-231 cell growth and support the notion that LAT1/CD98hc may be a suitable target to inhibit breast cancer progression.

Topics: Amino Acid Transport System L; Amino Acids, Cyclic; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Dose-Response Relationship, Drug; Female; Fusion Regulatory Protein 1, Heavy Chain; Humans; Large Neutral Amino Acid-Transporter 1; Leucine; Melphalan

An in vitro model of acquired melphalan resistance was developed by serial incubation of an MCF-7 human breast cancer cell line in increasing concentrations of melphalan. The resulting derivative cell line, Me1R MCF-7, was 30-fold resistant to melphalan. Uptake studies demonstrated decreased initial melphalan accumulation in Me1R MCF-7 cells. Inverse-reciprocal plots of initial melphalan uptake revealed a 4-fold decrease in the apparent Vmax of Me1R MCF-7 compared with WT MCF-7 (516 amol cell-1 min-1 vs 2110 amol cell-1 min-1 respectively) as well as a decrease in the apparent Kt (36 microM vs 70 microM respectively). Two amino acid transporters have previously been identified as melphalan transporters: system L, which is sodium-independent and inhibited by 2-amino-bicyclo[2,2,1]heptane-2-carboxylic acid (BCH), and system ASC which is sodium dependent and unaffected by BCH. At low concentrations of melphalan (3-30 microM), 1mM BCH competition eliminated the differences between the two cell lines, thus implicating an alteration of the system L transporter in the transport defect in the resistant cells. Me1R MCF-7 cells were also evaluated for glutathione-mediated detoxification mechanisms associated with melphalan resistance. There was no difference between Me1R MCF-7 and WT MCF-7 in glutathione content, glutathione-S-transferase activity and expression of pi class glutathione S-transferase RNA. In addition, buthionine sulfoximine did not reverse melphalan resistance in Me1R MCF-7 cells. Therefore, Me1R MCF-7 cells provide an in vitro model of transport-mediated melphalan resistance in human breast cancer cells.

Topics: Amino Acid Transport Systems; Amino Acids; Amino Acids, Cyclic; Breast Neoplasms; Carrier Proteins; Dose-Response Relationship, Drug; Drug Resistance; Glutathione; Glutathione Transferase; Humans; Melphalan; Time Factors; Tumor Cells, Cultured

In human tumor cells freshly obtained from patients with breast cancer, ovarian cancer, or adenocarcinoma of unknown etiology and in normal human bone marrow cells, the cell-to-medium ratio (intracellular/extracellular concentration) in vitro of 5.42 microM melphalan rose rapidly to levels of 6-17 after 35 min at 37 degrees C in Dulbecco's phosphate-buffered saline containing bovine serum albumin and glucose. Only patient C (breast cancer) had received chemotherapy. In all cells studied, L amino acids (1 mM) such as leucine, glutamine, tyrosine, and methionine reduced the cell-to-medium ratio of melphalan at 3 and 35 min. There was a good correlation between the reduction of melphalan transport at 35 min in the heterogeneous nucleated bone marrow cell population by amino acids and their effect on melphalan cytotoxicity in the CFU-C system. Aminoisobutyric acid (A1B), a specific substrate of the A system of amino acid transport, at a concentration between 1 and 50 mM had no significant effect on melphalan uptake at 3 min in any of the human cells studied except those of patient C. At 35 min A1B (10 or 50 mM) significantly reduced the intracellular melphalan concentration in normal bone marrow cells and tumor cells from patients B and C. At 2 mM, 2-aminobicyclo-(2, 2,1)-heptane-2-carboxylic acid (BCH), a specific substrate of the L system of amino acid transport, reduced the cell-to-medium ratio to 70% of control at 3 and 35 min in human bone marrow cells. In tumor cells from patients A, B, D, and F, 2 mM BCH had no significant effect on melphalan uptake at 3 min; it slightly decreased uptake in tumor cells from patient C. At 35 min, 2 mM BCH significantly reduced melphalan transport in tumor cells from patients C and F only. The lack of a BCH-suppressible component to melphalan uptake into human tumor cells freshly obtained from previously untreated patients contrasts with the presence of this component in murine L1210 leukemia cells, murine P388 leukemia cells, and human tumor cell lines. This suggests that minor differences in melphalan transport may exist amongst species and also between human tumor cells which are freshly obtained and cell lines maintained in culture.

Topics: Adenocarcinoma; Amino Acids; Amino Acids, Cyclic; Animals; Biological Transport; Bone Marrow; Breast Neoplasms; Cell Line; Cell Survival; Chromatography, Thin Layer; Colony-Forming Units Assay; Culture Media; Female; Humans; Kinetics; Melphalan; Mice; Neoplasms