ABSTRACT
Previous studies have demonstrated that treating cultured cells with cisplatin (CDDP) up-regulated the expression of glutathione (GSH) and its de novo rate-limiting enzyme glutamate-cysteine ligase (GCL), which consists of a catalytic (GCLC) and a modifier (GCLM) subunit. It has also been shown that many CDDP-resistant cell lines exhibit high levels of GCLC/GCLM and GSH. Because the GSH system is the major intracellular regulator of redox conditions that serve as an important detoxification cytoprotector, these results have been taken into consideration that elevated levels of GCL/GSH are responsible for the CDDP resistance. In contrast to this context, we demonstrated here that overexpression of GSH by transfection with an expression plasmid containing the GCLC cDNA conferred sensitization to CDDP through up-regulation of human copper transporter (hCtr) 1, which is also a transporter for CDDP. Depleting GSH levels in these transfected cells reversed CDDP sensitivity with concomitant reduction of hCtr1 expression. Although rates of copper transport were also up-regulated in the transfected cells, these cells exhibited biochemical signature of copper deficiency, suggesting that GSH functions as an intracellular copper-chelator and that overexpression of GSH can alter copper metabolism. More importantly, our results reveal a new role of GSH in the regulation of CDDP sensitivity. Overproduction of GSH depletes the bioavailable copper pool, leading to up-regulation of hCtr1 and sensitization of CDDP transport and cell killing. These findings also have important implications in that modulation of the intracellular copper pool may be a novel strategy for improving chemotherapeutic efficacy of platinum-based antitumor agents.
Subject(s)
Cation Transport Proteins/genetics , Cisplatin/toxicity , Glutathione/metabolism , Up-Regulation/drug effects , Biological Transport/drug effects , Buthionine Sulfoximine/pharmacology , Catalytic Domain , Cation Transport Proteins/metabolism , Cell Line, Tumor , Cisplatin/metabolism , Copper/metabolism , Copper/pharmacology , Copper Transporter 1 , Down-Regulation/drug effects , Drug Resistance, Neoplasm/drug effects , Drug Screening Assays, Antitumor , Fluorescent Antibody Technique , Gene Expression Regulation, Neoplastic/drug effects , Glutamate-Cysteine Ligase/metabolism , Humans , Models, Biological , RNA, Messenger/genetics , RNA, Messenger/metabolism , RNA, Small Interfering/metabolism , TransfectionABSTRACT
PURPOSE: Proliferative activity and suppression of apoptosis of cancer cells are important to tumor progression in hepatocellular carcinoma (HCC). Recently, the expressions of inducible nitric oxide synthase (iNOS) and survivin mRNA have been reported to correlate with suppression of apoptosis in some tumors. However, the clinical importance of expression of these genes in HCC progression remains unclear. In the present study, the correlation between the expression of iNOS and survivin mRNA and the occurrence of spontaneous apoptosis and proliferative activity of cancer cells and prognostic importance of expression of these genes in HCC were investigated. EXPERIMENTAL DESIGN: Tissues were obtained by surgical resection of livers from 61 patients with HCC and 8 without HCC. Expressions of iNOS and survivin mRNA were evaluated using the reverse transcription-PCR in 61 tumors, 61 adjacent histologically noncancerous livers, and 8 normal livers. Apoptotic cancer cells and the proliferative activity of cancer cells were detected by immunohistochemistry. RESULTS: iNOS mRNA expression was detected in 34 of 61 (55.7%) HCCs, 19 of 61 (31.1%) noncancerous liver tissues adjacent to carcinoma, and none of the 8 normal livers. In addition, survivin mRNA was detected in 19 of 61 (31.1%) HCCs, none of 61 noncancerous liver tissues, and none of the 8 normal livers. iNOS mRNA expression did not correlate with the proliferative activity of cancer cells or with the occurrence of apoptosis in HCCs. In contrast, survivin mRNA expression strongly correlated with a high proliferative activity of cancer cells and a low apoptotic index. Disease-specific survivals did not differ between patients with iNOS-positive or -negative HCCs. Although, the disease-specific survival of patients with survivin-positive HCCs was significantly poorer than that of patients with survivin-negative HCCs. CONCLUSIONS: These results indicate that iNOS may not correlate with cancer cell-proliferative activity or apoptosis; survivin, however, may not only suppress apoptosis but also accelerate cancer cell-proliferative activity and play an important role in tumor progression in HCC.
Subject(s)
Carcinoma, Hepatocellular/metabolism , Liver Neoplasms/metabolism , Microtubule-Associated Proteins/genetics , Nitric Oxide Synthase/genetics , RNA, Messenger/metabolism , Adult , Aged , Aged, 80 and over , Apoptosis , Carcinoma, Hepatocellular/genetics , Carcinoma, Hepatocellular/pathology , Cell Division , DNA Primers/chemistry , Female , Humans , Immunoenzyme Techniques , Inhibitor of Apoptosis Proteins , Ki-67 Antigen/metabolism , Liver Diseases/genetics , Liver Diseases/metabolism , Liver Neoplasms/genetics , Liver Neoplasms/pathology , Male , Microtubule-Associated Proteins/metabolism , Middle Aged , Neoplasm Proteins , Neoplasm Staging , Nitric Oxide Synthase/metabolism , Nitric Oxide Synthase Type II , RNA, Neoplasm/analysis , Reverse Transcriptase Polymerase Chain Reaction , Survival Rate , Survivin , Tumor Cells, CulturedABSTRACT
BACKGROUND AND OBJECTIVES: In the process of tumor invasion, cancer cells secrete enzymes including heparanase that degrade extracellular matrices and basement membranes of blood vessels. In the present study, the clinical importance of enhanced expression of heparanase messenger RNA (mRNA) in hepatocellular carcinoma (HCC) was evaluated. METHODS: Fresh tumor and noncancerous liver tissue adjacent to a tumor were obtained from 50 patients with HCC immediately after hepatic resection. The expression levels of heparanase mRNA were analyzed quantitatively, using the real-time reverse transcriptase-polymerase chain reaction (RT-PCR) method, and were compared with clinicopathological findings. RESULTS: Relative heparanase mRNA expression level in HCC was significantly lower than that of noncancerous liver tissue (P < 0.001). Tumor heparanase expression did not correlate with tumor differentiation, tumor stage, or patient prognosis. CONCLUSIONS: Enhanced heparanase mRNA expression may not be a good biological marker in HCC. J. Surg. Oncol. 2002;81:148-154.
