Glutathione concentration may be a useful predictor of response to second-line chemotherapy in patients with ovarian cancer

BACKGROUND: No useful predictor of resistance or sensitivity to second-line chemotherapy is known for ovarian cancer. The objective of this prospective study was to determine the utility of tumor glutathione S-transferase-pi (GST-pi) expression or glutathione (GSH) concentration in predicting ovarian cancer patients' responses to second-line chemotherapy. METHODS: Tumor samples were obtained from 26 patients with relapsed epithelial ovarian cancer 3-4 weeks before the initiation of second-line chemotherapy with etoposide (daily on Days 1-5) and cisplatin (on Day 5). The expression of GST-pi in tumor samples was determined by immunohistochemical staining and Western blot analysis. GSH concentration was measured by an enzymatic assay. RESULTS: The response rate was 38.4%. The estimated 3-year survival rate for the responders (66.7%) significantly exceeded that for the nonresponders (9.1%). Expression of GST-pi by immunohistochemical staining was more frequently observed in nonresponders (2 of 10 responders vs. 11 of 16 nonresponders). Western blot analysis detected GST-pi in all cases. There was no significant difference in the relative density values of the GST-pi Western blot analysis between the two groups. The mean value of GSH concentration in nonresponders was significantly higher than in responders (18.4 +/- 9.7 vs. 7.5 +/- 8.2 microg/mg protein). GSH concentration was below the cutoff point (10.3 microg/mg protein) in all responders except one. CONCLUSIONS: Second-line chemotherapy consisting of etoposide and cisplatin is effective in the treatment of relapsed epithelial ovarian cancer. In addition, tumor concentration of GSH may be a useful predictor of the response to this therapy.     

In vitro and in vivo effects of phenolic antioxidants against cisplatin-induced nephrotoxicity

We have investigated the effect of phenolic antioxidants on cisplatin-induced cytotoxicity in vero (African Green Monkey Kidney) cells and in rat renal cortical slices in vitro, and on cisplatin-induced nephrotoxicity in rats in vivo. Incubation of cisplatin with vero cells resulted in time- and concentration-dependent cytotoxicity, as characterized by decreased tryphan blue exclusion (TBE) and increased release of lactate dehydrogenase (LDH) into the medium. Cisplatin also caused reduction of glutathione (GSH) in a concentration-dependent manner. In the rat renal cortical slices model, incubation of cisplatin for 120 min caused an increase in malondialdehyde (MDA), a decrease in GSH and inhibited p-aminohippurate (PAH) uptake in a concentration-dependent manner. Among phenolic antioxidants, isoeugenol (IG) was found to be more active against cisplatin-induced cytotoxicity in vero cells as well as in rat renal cortical slices than eugenol (EG) and dehydrozingerone (DZ). However none of the test compounds were able to arrest the reduction of the GSH content induced by cisplatin in either the vero cells or the renal cortical slice model. Administration of cisplatin (3 mg/kg) i.p. to rats resulted in significant reduction of body weight, and elevation of blood urea nitrogen (BUN) and serum creatinine. Treatment with IG 10 mg/kg i.p. 1 h before cisplatin resulted in partial but significant protection against the cisplatin-induced reduction of body weight, and elevation of BUN and serum creatinine, the protection being 34, 46, and 62%, respectively. EG and DZ (10 mg/kg, i.p.) were found to be inactive in vivo. Because IG is a potent free radical scavenger and protects against cisplatin-induced toxicitiy, the present results have many clinical implications in chemotherapy and thus warrants further investigation.     

Comparative study of mortality and morbidity in premature infants (birth weight, < 1,250 g) with candidemia or candidal meningitis

Tissue from primary tumors was analyzed for 118 patients with urothelial cancer who subsequently received cisplatin-based chemotherapy. Immunohistochemical staining was performed for nuclear p53 reactivity; for two proposed mediators of drug resistance, metallothionein (MT) and P-glycoprotein; and for the cell proliferation marker MIB-1. For each marker, immunoreactivity was expressed as a percentage of positively staining cells, and overall intensity of staining was graded on a scale from 0 to 3. The product of these two measurements was calculated to generate a percentage-intensity index. Clinical data were obtained independently via retrospective chart review. Chemotherapy regimens containing cisplatin (cisplatin, methotrexate, and vinblastine or methotrexate, vinblastine, doxorubicin, and cisplatin) were administered for metastatic disease (n = 64), for locally advanced disease (n = 45), or as an adjuvant treatment (n = 9). The overall response rate was 56% among 99 evaluable patients, and median survival was 12.7 months. By univariate analysis, Eastern Cooperative Oncology Group performance status (P = 0.0025), tumor grade (P = 0.03), percentage of MT staining (P = 0.01), and percentage-intensity index of MT staining (P = 0.04) were significant predictors of response to chemotherapy. The first three of these were significant in a multivariate model (P = 0.05, 0.04, and 0.04, respectively). By subgroup analysis, the percentage of MT staining predicted for response in metastatic disease (P = 0.03), but not in locally advanced disease (P = 0.28). Only performance status was significantly related to overall survival (P = 0.0001, log-rank test) in the whole cohort. Overexpression of MT in the 64 patients with metastatic disease was associated with a shorter survival (P = 0.04). Expression of p53, P-glycoprotein, and MIB-1 did not predict for survival. In conclusion, overexpression of MT is associated with a poorer outcome from chemotherapy, possibly due to cisplatin resistance.     

Role of cellular glutathione and glutathione S-transferase in the expression of alkylating agent cytotoxicity in human breast cancer cells

Glutathione (GSH) and glutathione S-transferases (GSTs) play an important role in the protection of cells against toxic effects of many electrophilic drugs and chemicals. Modulation of cellular GSH and/or GST activity levels provides a potentially useful approach to sensitizing tumor cells to electrophilic anti-cancer drugs. In this study, we describe the interactions of four representative alkylating agents (AAs), melphalan, 4-hydroperoxy-cyclophosphamide (4HC), an an activated form of cyclophosphamide, 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), and cisplatin, with GSH and GST in the human breast cancer cell line MCF-7. Depletion of cellular GSH pools by approximately 80% by treatment of the cells with the GSH synthesis inhibitor buthionine sulfoximine (BSO) sensitized the tumor cells to each AA to a different extent, with dose-modifying factors of 2.39, 2.21, 1.64, and 1.27 observed for melphalan, 4HC, cisplatin, and BCNU, respectively. Treatment of the cells with the GST inhibitor ethacrynic acid (EA) failed to show any significant effects on the cytotoxicity of these AAs. However, EA did potentiate the cytotoxicity of melphalan when given in combination with BSO, an effect that may be due to a more complete depletion of cellular GSH levels by the combined modulator treatment. Following a 1-hr exposure to cytotoxic-equivalent concentrations of these AAs, GSH levels decreased substantially in the case of 4HC and BCNU, but increased by 30-50% in the case of cisplatin and melphalan. BSO pretreatment largely blocked this effect of cisplatin and melphalan on cellular GSH, while it further enhanced the GSH-depleting activity of both 4HC and BCNU. On the basis of these results, it is concluded that (a) GSH affects the cytotoxicity of different AAs to different extents, (b) basal GST expression in MCF-7 cells does not play a major role in AA metabolism, (c) EA can potentiate the enhancing effect of BSO on melphalan cytotoxicity in MCF-7 cells, and (d) depletion of cellular GSH by pretreatment with BCNU or cyclophosphamide may correspond to a useful strategy for enhancing the anti-tumor activity of other AAs given in a sequential combination.     

Neonatology in Europe. Training, accreditation, and research

PURPOSE: To correlate cellular glutathione content and gamma-glutamyl cysteine synthetase (gamma GCS) mRNA expression with cisplatin sensitivity in a panel of seven head and neck squamous cancer cell lines. METHODS: Cisplatin IC50 was determined for each cell line using a sodium tetreazolium (XTT) assay. Cellular glutathione content was measured by using a previously reported enzymic method. gamma GCS mRNA expression was measured using an RNase protection assay. RESULTS: Total cellular glutathione was an excellent predictor of cisplatin sensitivity in this series of cell lines. The IC50 for cisplatin in the cell line with the highest glutathione concentration was approximately 90 times higher than in the cell line with the lowest glutathione concentration. Regression analysis showed a highly statistically significant positive correlation between cisplatin IC50 and cellular glutathione (coefficient of determination R2 = 0.81, P = 0.0012). Some-what surprisingly, in contrast to previous studies in ovarian cancer, gamma GCS mRNA expression in these cell lines was not significantly predictive of either total cellular glutathione or cisplatin sensitivity (R2 = 0.005, P = 0.84). As expected, treatment of resistant cell lines with buthionine sulfoximine resulted in decreased cellular glutathione and enhanced cisplatin sensitivity. CONCLUSIONS: Our results suggest that glutathione may be an important determinant of cisplatin sensitivity in clinical head and neck cancer. Since cisplatin is the most active chemotherapy drug for the treatment of this disease, this correlation may have important clinical relevance. The lack of correlation between glutathione level and gamma GCS expression suggests that salvage or alternate synthetic pathways may be critical in these cells.     

Acquired resistance to cisplatin and doxorubicin in a small cell lung cancer cell line is correlated to elevated expression of glutathione-linked detoxification enzymes

A human small cell lung cancer cell line, U-1906, developed altered functional properties upon continuous in vitro cultivation. Cells obtained at late (U-1906 L) and early (U-1906 E) passages of cultivation differ in drug resistance to the cytostatic therapeutic agents cisplatin and doxorubicin. The U-1906 L cells are 1.6-fold and 1.3-fold more resistant to cisplatin and doxorubicin respectively, than are the U-1906 E cells. In the more resistant U-1906 L cells, the total glutathione (GSH plus GSSG) level is 40% lower, whereas the activities of GSH-linked enzymes such as GSH peroxidase and GSH transferases are significantly higher. Quantitative analysis with isoenzyme-specific ELISAs demonstrated increased concentrations of all three of the measurable GSTs, M1-1, M3-3 and P1-1, in the more resistant cells. The intracellular protein expression patterns of the U-1906 E and the U-1906 L cells are very similar as revealed by two-dimensional denaturing electrophoresis, but show significant alterations in the concentrations of some components. Two 35 kDa proteins of different pI values, the concentrations of which are increased in the U-1906 L cells, were both identified as glyceraldehyde-3-phosphate dehydrogenase, either by N-terminal or by internal amino acid sequence analysis. The present study demonstrates that the increased resistance of the U-1906 L cells may involve multiple detoxification mechanisms and that the contribution of the GSH-linked detoxification can be ascribed to the elevation of cytosolic GST isoenzymes, GSH peroxidase and glutathione reductase, rather than to the intracellular GSH concentrations.     

Factors determining the weaning outcome

OBJECTIVE: To identify the clinicopathological and chemoresistant factors predicting the response to neoadjuvant chemotherapy and the patient prognosis in high-risk cervical carcinomas. METHODS: We retrospectively reviewed 47 patients with locally advanced or bulky cervical carcinoma treated with two courses of intraarterial infusion of cisplatin, doxorubicin, mitomycin C, and 5-fluorouracil (5-FU), followed by radical hysterectomy at our hospital between 1988 and 1995. Expressions of the chemoresistance-related proteins, such as P-glycoprotein, glutathione S-transferase pi (GST-pi), and proliferating cell nuclear antigen (PCNA) in the tumor cells, were examined by immunohistochemistry using pretreatment biopsy specimens. These results were compared with the chemotherapeutic response, which was evaluated by magnetic resonance imaging (MRI) and histopathology. Outcome of the patients was also studied. RESULTS: Chemotherapeutic effect of either complete (CR) or partial (PR) response on MRI was obtained in 36 of the 47 (86%) patients. Poor response to chemotherapy was significantly correlated with P-glycoprotein expression (P < 0.005) and low PCNA labeling (P < 0. 05), but not GST-pi expression in the tumor cells. Independent prognostic factors for patient survival were parametrial involvement and lymph node metastasis. Neither the expression of GST-pi nor PCNA was correlated with the patient survival. CONCLUSION: Assessment of the expression of P-glycoprotein and PCNA is potentially useful for the prediction of tumor response to neoadjuvant chemotherapy for cervical carcinomas.     

Selective and synergistic activity of L-S,R-buthionine sulfoximine on malignant melanoma is accompanied by decreased expression of glutathione-S-transferase

L-buthionine-S,R-sulfoximine (BSO) selectivley inhibits glutathione (GSH) synthesis. Malignant melanoma may be uniquely dependent on GSH and its linked enzymes, glutathione S-transferase (GST) and GSH-peroxidase, for metabolism of reactive orthoquinones and peroxides produced during melanin synthesis. We compared the in vitro effects of BSO on melanoma cell lines and fresh melanoma specimens (n = 118) with breast and ovarian cell lines and solid tumors (n = 244). IC50 values (microM) for BSO on melanoma, breast and ovarian tumor specimens were 1.9, 8.6, and 29, respectively. The IC90 for melanoma was 25.5 microM, a level 20-fold lower than steady state levels achieved clinically. The sensitivity of individual specimens of melanoma correlated with their melanin content (r = 0.63). BSO synergistically enhanced BCNU activity against melanoma cell lines and human tumors. We followed GSH levels, GST enzyme activity, GST isoenzyme profiles and mRNA levels after BSO. BSO (50 microM) treatment for 48 hr resulted in a 95% decrease in ZAZ and M14 melanoma cell line GSH levels, and a 60% decrease in GST enzyme activity. GST-mu protein and mRNA levels were significantly reduced in both cell lines. GST-pi expression was unaffected. These data suggest that BSO action on melanoma may be related to GSH depletion, diminishing the capacity to scavenge toxic metabolites produced during melanin synthesis. We report here for the first time that BSO enhancement of alkylator action may be related in part to down regulation of GST. BSO may be a clinically useful adjunct in the treatment of malignant melanoma.     

Effect of aldose reductase inhibition on glutathione redox status in erythrocytes of diabetic patients

Diabetic patients undergo a chronic oxidative stress. This phenomenon is demonstrated by low levels of reduced glutathione (GSH) levels. The NADPH used by glutathione reductase for the reduction of oxidized glutathione (GSSG) to GSH is also used by aldose reductase for the reduction of glucose to sorbitol through the polyol pathway. The competition for NADPH could be responsible for the decreased glutathione levels found in non-insulin-dependent diabetic patients. For this purpose, we investigated the effect of polyol pathway inhibition on the glutathione redox status in these patients. We measured GSH and GSSG levels in erythrocytes of non-insulin-dependent diabetic patients (n = 15) before and after 1 week of treatment with placebo, followed by 1 week of treatment with an aldose reductase inhibitor (tolrestat 200 mg/dl). We found lower GSH levels (7.7 +/- 1.4 mumol/g hemoglobin [Hb]), higher GSSG levels (0.35 +/- 0.09 mumol/g Hb), and lower GSH/GSSG ratios (23.9 +/- 7.7) in diabetics compared with controls (n = 15; 9.8 +/- 0.8 mumol/g Hb, P < .001; 0.17 +/- 0.02, P < .001; and 58.3 +/- 9.1, P < .001, respectively). We did not demonstrate any statistical difference after 1 week of treatment with placebo. In contrast, the treatment with tolrestat induced a significant increase in GSH (8.9 +/- 0.7 mumol/g Hb, P < .01), a decrease in GSSG (0.25 +/- 0.06 mumol/g Hb, P < .02), and an increase in the GSH/GSSG ratio (37.3 +/- 8.4, P < .01). These data strongly support the hypothesis that the polyol pathway plays an important role in the impairment of the glutathione redox status in diabetic patients.     

Expression of glutathione S-transferase-pi in operative specimens as marker of chemoresistance in patients with ovarian cancer

OBJECTIVE: To study the relationship between the expression of glutathione S-transferase-pi (GST-pi) in operative specimens and chemoresistance in patients with ovarian cancer. METHODS: The expression of GST-pi in 87 epithelial ovarian cancer tissues and 30 normal ovarian epithelial tissues was determined with labelled streptavidin biotin method (LSAB). All the patients had not received chemotherapy before operation. We used Chi-Square and Cox-Mantel test to analyze the relativity between the expression of GST-pi and clinical pathological data, chemotherapeutic response, prognosis in patients with ovarian cancer. RESULTS: (1) 59 (67.8%) of 87 ovarian cancer tissue were demonstrated to be positive expression with GST-pi, but all 30 normal ovarian epithelial tissue were negative. (2) There was no direct correlation between the expression of GST-pi and clinical pathological data. (3) 43 (43/59) of GST-pi positive cases were chemoresistant, while only 3 (3/28) of GST-pi negative ones were chemoresistant. (4) The difference in the chemotherapeutic response between the two groups was obviously significant (P < 0.005). The survival period of the patients with GST-pi positive expression was also obviously shorter than that of those with GST-pi negative expression (P = 0.004). CONCLUSION: These results strongly suggest that GST-pi expression in epithelial ovarian cancer tissues is closely related to chemoresistance clinically and it may be served as a useful marker to predict the prognosis of patients.     

Toxicity of cytostatic drugs to normal bone marrow cells in vitro

Metastatic melanomas are often resistant to chemotherapy. To study whether the p53 mutational status affects chemosensitivity, we compared the responses to chemotherapy of four melanoma cell lines containing the wild-type p53 and four cell lines carrying the mutant p53. Cisplatin, at 10 microM, virtually killed all the cells in the wild-type p53 cell lines, while 57-95% of the cells in the mutant p53 cell lines survived (P = 0.005). After treatment with 100 nM of vincristine, on average 18% of the wild-type p53 melanoma cells survived compared with 55% of the mutant p53 cells (P = 0.04). After treatment with 40 nM, 200 nM or 1 microM of camptothecin the survival rates were, on average, 16%, 8% and 4% for the wild-type p53 melanoma cells, compared with 89%, 67% and 38% for the mutant p53 cells, respectively (P = 0.00004, P = 0.003 and P = 0.04, respectively). The anticancer agents were not toxic to normal melanocytes at doses inducing cytotoxicity in wild-type p53 melanoma cells. The main mechanism of cytotoxicity appears to be drug-induced apoptosis. Cisplatin, camptothecin and vincristine all induced apoptosis in wild-type p53 melanoma cells, but not in mutant p53 cells. Our results suggest that chemotherapy-induced apoptosis in melanoma cells is p53 dependent, and mutation of the p53 gene is an indicator of drug resistance in melanoma.     

Receptor mediated effects of adenosine and caffeine

The inhibition of glutathione (GSH) synthesis by L-buthionine-SR-sulfoximine (BSO) causes aggravation of hepatotoxicity of paraquat (PQ), an oxidative-stress inducing substance, in mice. On the other hand, synthesis of metallothionein (MT), a cysteine-rich protein having radical scavenging activity, is induced by PQ, and the induction by PQ is significantly enhanced by pretreatment of mice with BSO. The purpose of present study is to examine whether generation of reactive oxygens is involved in the induction of MT synthesis by PQ under inhibition of GSH synthesis. Administration of PQ to BSO-pretreated mice increased hepatic lipid peroxidation and frequency of DNA single strand breakage followed by manifestation of the liver injury and induction of MT synthesis. Both vitamin E and deferoxamine prevented MT induction as well as lipid peroxidation in the liver of mice caused by administration of BSO and PQ. In cultured colon 26 cells, both cytotoxicity and the increase in MT mRNA level caused by PQ were significantly enhanced by pretreatment with BSO. Facilitation of PQ-induced reactive oxygen generation was also observed by BSO treatment. These results suggest that reactive oxygens generated by PQ under inhibition of GSH synthesis may stimulate MT synthesis. GSH depletion markedly increased reactive oxygen generation induced by PQ, probably due to the reduced cellular capability to remove the radical species produced.     

N-acetylcysteine improves cytotoxic activity of cirrhotic rat liver-associated mononuclear cells

Liver cirrhosis, which is associated with decreased plasma and hepatic glutathione (GSH), has been reported to cause the suppression of NK activity in peripheral blood mononuclear cells. Since low GSH levels in lymphocytes are known to alter lymphocyte function, we examined the correlation between intracellular GSH levels and the cytotoxic activity of liver-associated mononuclear cells (liver MNC). We show here that rat liver contains a highly active population of NK cells (CD3- NKR-P1 + cells) that kill Yac-1 in vitro and that the cytotoxic activity of this NK population is directly proportional to liver MNC GSH. This proportionality is independent of the methods used to alter GSH level. Thus, in vitro treatment of liver MNC with buthionine sulfoximine to lower GSH levels lowers the cytotoxic activity. MNC from cirrhotic liver, in which implanted tumor cells grow faster, have both low GSH levels and low cytotoxicity, and supplementation of cirrhotic liver MNC with N-acetylcysteine raises GSH levels and increases cytotoxicity. These findings suggest a physiologic mechanism, i.e. decreased GSH, may be causally associated with the increased incidence of hepatoma in cirrhotic individuals and the increased growth of hepatoma cells in cirrhotic animals. Thus, we suggest that the GSH is important to the optimal functioning of the hepatic immunity that protects against hepatoma development.     

Coordinated induction of MRP/GS-X pump and gamma-glutamylcysteine synthetase by heavy metals in human leukemia cells

We recently reported that GS-X pump activity, as assessed by ATP-dependent transport of the glutathione-platinum complex and leukotriene C4, and intracellular glutathione (GSH) levels were remarkably enhanced in cis-diamminedichloroplatinum(II) (cisplatin)-resistant human leukemia HL-60 cells (Ishikawa, T., Wright, C. D., and Ishizuka, H. (1994) J. Biol. Chem. 269, 29085-29093). Now, using Northern hybridization and RNase protection assay, we provide evidence that the multidrug resistance-associated protein (MRP) gene, which encodes a human GS-X pump, is expressed at higher levels in cisplatin-resistant (HL-60/R-CP) cells than in sensitive cells, whereas amplification of the MRP gene is not detected by Southern hybridization. Culturing HL-60/R-CP cells in cisplatin-free medium resulted in reduced MRP mRNA levels, but these levels could be induced to rise within 30 h by cisplatin and heavy metals such as arsenite, cadmium, and zinc. The increased levels of MRP mRNA were closely related with enhanced activities of ATP-dependent transport of leukotriene C4 (LTC4) in plasma membrane vesicles. The glutathione-platinum (GS-Pt) complex, but not cisplatin, inhibited ATP-dependent LTC4 transport, suggesting that the MRP/GS-X pump transports both LTC4 and the GS-Pt complex. Expression of gamma-glutamylcysteine synthetase in the cisplatin-resistant cells was also co-induced within 24 h in response to cisplatin exposure, resulting in a significant increase in cellular GSH level. The resistant cells exposed to cisplatin were cross-resistant to melphalan, chlorambucil, arsenite, and cadmium. These observations suggest that elevated expression of the MRP/GS-X pump and increased GSH biosynthesis together may be important factors in the cellular metabolism and disposition of cisplatin, alkylating agents, and heavy metals.     

Genetic diagnosis of cardiovascular diseases and the therapeutic application

We have examined the effect of neutralizing TGF-beta antibodies on cisplatin-mediated cytotoxicity against MDA-231 human breast tumor cell spheroids. These tridimensional in vitro systems have been shown to recapitulate the drug sensitivity pattern of tumor cells in vivo. MDA-231 tumor cell spheroids exhibit higher protein levels of the cyclin-dependent kinase (Cdk) inhibitors p21 and p27 and >10-fold lower Cdk2 activity compared to adherent cell monolayers, as well as pRb hypophosphorylation, a predominant G1 population, and a cisplatin 1-h IC50 of approximately 100 microM. Treatment of MDA-231 cells in monolayer with cisplatin for 1 h, subsequently grown as spheroids, increased steady-state TGF-beta1 mRNA levels, secretion of active TGF-beta, cellular Cdk2 activity, pRb phosphorylation, and p21 protein levels, while downregulating p27. Accumulation of cells in G2M and progression into S were noted 48 h after treatment with 100 microM cisplatin. We tested whether drug-induced upregulation of TGF-beta1 and p21, perhaps by preventing cell cycle progression, were protective mechanisms against drug-mediated toxicity by using neutralizing anti-TGF-beta antibodies. Anti-TGF-beta antibodies diminished the induction of p21, enhanced the activation of Cdk2, and facilitated progression into S and G2M following cisplatin treatment. This resulted in a >twofold enhancement of drug-induced DNA fragmentation and a shift in the cisplatin 1-h IC50 from 100 to <10 microM. These data suggest that tumor cell TGF-beta1 may protect from DNA damage and that postchemotherapy administration of TGF-beta inhibitors may facilitate progression beyond G1/S, potentially increasing the efficacy of cytotoxic chemotherapy.     

Transfection of glutathione S-transferase (GST)-pi antisense complementary DNA increases the sensitivity of a colon cancer cell line to adriamycin, cisplatin, melphalan, and etoposide

The goal of this study was to demonstrate that glutathione S-transferase (GST)-pi is directly involved in the intrinsic and acquired resistance of cancer cells to anticancer drugs. To this end, GST-pi antisense cDNA was transfected into the cultured human colon cancer cell line M7609, which expresses an innately high level of GST-pi and shows intrinsic drug resistance, and into an M7609 strain with acquired resistance to Adriamycin (ADR;i.e., M7609/ADR cells). The changes in the sensitivity of these transfectants to various anticancer drugs were investigated. The intracellular concentrations of GST-pi in M7609/anti-1 cells and M7609/anti-2 cells, two clones that were established by transfection of GST-pi antisense cDNA into M7609 cells, were decreased to approximately half of those detected in the parent cells (M7609) and in the control cells transfected with vector alone (M7609/pLJ). The sensitivities of the antisense transfectants in relation to ADR, cisplatin, melphalan, and etoposide were increased -3.3-fold, 2.3-fold, 2.2-fold, and 2.1-fold, respectively, compared with those of M7609 and M7609/pLJ. On the other hand, the sensitivities of the antisense transfectants to Taxol, vincristine, 5-fluorouracil, and mitomycin C were not significantly changed. Similarly, the transfection of antisense cDNA into M7609/ADR cells resulted in the reduction of intracellular GST-pi concentration (by about half) and an increased sensitivity to ADR (4.4-fold), but no increase in 5-fluorouracil sensitivity. Thus, GST-pi is considered to be a multidrug resistance factor that is responsible for both the intrinsic and acquired resistance of cancer cells to anticancer drugs such as ADR, cisplatin, melphalan, and etoposide.     

Glutathione-doxorubicin conjugate expresses potent cytotoxicity by suppression of glutathione S-transferase activity: comparison between doxorubicin-sensitive and -resistant rat hepatoma cells

The cytotoxic mechanism of a conjugate of doxorubicin (DXR) and glutathione (GSH) via glutaraldehyde (GSH-DXR) was investigated using DXR-sensitive (AH66P) and -resistant (AH66DR) rat hepatoma cells. GSH-DXR accumulated in AH66DR cells as well as in AH66P cells without efflux by P-gp and exhibited the potent cytocidal activity against both cells compared with DXR. To examine whether thiol from GSH-DXR affected the expression of cytotoxicity, two conjugates of DXR, with modified peptides containing alanine or serine substituted for cysteine in GSH were prepared and their cytotoxicities determined. Substitution of these amino acids for cysteine resulted in an approximately two- to fourfold reduction in cytotoxic activity against both cell lines compared with the effect of GSH-DXR. Depletion of intracellular GSH by treatment of both cells with buthionine sulphoximine did not change the cytotoxic activity of DXR, BSA-DXR or GSH-DXR. By co-treating the cells with tributyltin acetate, an inhibitor of glutathione S-transferase (GST), and either DXR, BSA-DXR or GSH-DXR, the cytotoxicity was markedly increased. Interestingly, GSH-DXR showed non-competitive inhibition of GST activity and its IC50 value was 1.3 microM. These results suggested that the inhibition of GST activity by GSH-DXR must be an important contribution to the expression of potent cytotoxicity of the drug.     

Dual effect of erbB-2 depletion on the regulation of DNA repair and cell cycle mechanisms in non-small cell lung cancer cells

Overexpression of the erbB-2 tyrosine kinase receptor, p185erbB-2, is a common alteration in non-small cell lung cancer (NSCLC) and has been associated with poor prognosis and a tumor drug resistance phenotype. In this study, we have examined the consequences of erbB-2 depletion on DNA repair, cell cycle, and apoptosis using a panel of NSCLC cell lines constitutively overexpressing erbB-2 receptor. Depletion of the erbB-2 was achieved using the tyrosine kinase inhibitor CP127,374 which promotes erbB-2 degradation. Treatment with CP127,374 concentrations which deplete erbB-2 and inhibit tyrosine phosphorylation resulted in downregulation of DNA repair mechanisms and cell accumulation at G1 phase of the cell cycle. GI arrest was observed in cells with mutated p53 as well as cells lacking p53 protein, suggesting a p53-independent mechanisms. NSCLC cells which overexpress erbB-2 were more resistant to cisplatin-induced cytotoxicity in comparison to cells expressing low levels of erbB-2. Treatment with CP127,374 alone did not result in any induction of apoptosis. A combination of CP127,374 and cisplatin, however, was more potent in cell growth inhibition and induction of apoptosis compared to treatment with cisplatin alone. Together, our results further support a pivotal role of erbB-2 signaling in the regulatory balance between DNA repair, cell cycle checkpoints and apoptosis; all these mechanisms are essential determinants for tumor cell destiny following chemotherapy stress.     

What is core? Guidelines for the core curriculum in paediatrics

This multicentre, randomised, double-blind, double-dummy, parallel group study was investigated in order to compare on 3 days the efficacy and the safety of the 16 mg once a day (od) ondansetron suppository (suppository group) with the recommended ondansetron treatment, i.e. 8 mg intravenous (i.v.) ondansetron on day 1 followed by 8 mg tablet (p.o.) twice daily (i.v. + p.o. group) on days 2 and 3 in patients receiving cisplatin (> or = 50 mg/m2) containing chemotherapy. In the 420 patients included in the intent-to-treat population, 209 received the 16 mg suppository and 211 the i.v. + p.o. treatment. The number of emetic episodes and the nausea score were recorded each day. Concerning the primary criterion, both treatments provided good anti-emetic control with 87% of all patients having a complete or major response (0-2 emetic episodes) on day 1 in the suppository group and 92% in the i.v. + p.o. group (P = 0.058). The 90% confidence interval for the difference between the two treatments for complete or major control was included in the interval (-15%, 15%) and showed that the treatment groups could be regarded as equivalent. Small differences in favour of the i.v. + p.o. group were observed concerning the secondary parameters. Both treatments were well tolerated. The results of this study show that both treatments are equivalent in the prevention of cisplatin-containing chemotherapy induced emesis for the primary efficacy criteria and that the ondansetron suppository is efficient and well tolerated and is a suitable alternative to the anti-emetic treatment combining the intravenous and oral routes.