Masato Mutoh

Expression of multidrug resistance-related transporters in human breast carcinoma

The expression levels of mRNA for multidrug resistance 1 (MDR1) gene, multidrug resistance protein 1 (MRP1), lung resistance‐related protein (LRP) and breast cancer resistance protein (BCRP), which confer multidrug resistance in vitro, were examined in 43 untreated breast carcinoma patients, of whom 38 subsequently received doxorubicin‐based chemotherapy after surgery, in order to elucidate the roles of these genes in drug resistance in vivo. The mRNA levels were determined using a semi‐quantitative reverse‐transcription polymerase chain reaction method in breast carcinoma tissues including at least 80% carcinoma cells. The expression level of BCRP gene was low and did not vary markedly in comparison with that of MDR1, MRP1 or LRP gene. The expressions of MDR1 and MRP1 genes were correlated with each other, but the expression of BCRP or LRP gene did not correlate with that of other genes. These four gene expressions were independent of age, TNM categories and the status of progesterone or estrogen receptor. The expression levels of these four genes were not related to the relapse or prognosis of the 38 patients treated with doxorubicin‐based chemotherapy. P‐glycoprotein (P‐gp)/MDRl, MRP1 and LRP may play more important roles than BCRP in chemotherapy of human breast carcinoma.

Prognostic Value of the Cu-Transporting ATPase in Ovarian Carcinoma Patients Receiving Cisplatin-Based Chemotherapy

Purpose: A major obstacle in the treatment of ovarian carcinoma is the intrinsic/acquired resistance to cisplatin-based chemotherapy. Cu-transporting ATPase (ATP7B) has been reported to be associated with cisplatin resistance in vitro. However, the clinical significance of this transporter has not previously been addressed. Our goal was to investigate ATP7B expression in ovarian carcinoma and whether its expression correlates with prognosis and reduced responsiveness to cisplatin treatment. Experimental Design: We retrospectively examined the expression of ATP7B and p53 in primary ovarian carcinoma and its association with chemotherapeutic effect. Tissues were surgically removed from 104 ovarian carcinomas patients who received cisplatin-based chemotherapy. We performed immunohistochemical analysis of ATP7B and p53 using a monoclonal antibody against ATP7B and DO7 antibody against p53 protein in 104 ovarian carcinomas and adjacent nonneoplastic tissues. The significance of ATP7B and p53 in the prognosis of patients with ovarian carcinomas was also examined in the survival analysis of mortality follow-up data covering the period between 1988 and 2001. Furthermore, mutation analysis at the six Cu-binding domain and ATP-binding domain, which may be important for cisplatin transport, were performed using single-strand conformational polymorphism after reverse transcriptase-PCR. Results: A variable degree of cytoplasmic staining of ATP7B in tumor cells was observed in 34.6% (36 of 104 cases) of the analyzed carcinomas. ATP7B expression was not observed in adjacent nonneoplastic tissues. ATP7B positivity in poorly/moderately differentiated carcinoma was significantly higher than that in low malignant potential tumor/well-differentiated carcinoma (P = 0.0276). Patients with ATP7B-positive tumors had a significantly inferior response to chemotherapy compared with the patients with ATP7B-negative tumors (P = 0.025). The multivariate Cox regression analysis revealed that ATP7B expression (hazard ratio, 1.8; 95% confidence interval, 1.0–3.2, P = 0.048), as well as International Federation of Gynecologists and Obstetricians stage (hazard ratio, 2.0; 95% confidence interval, 1.1–3.6, P = 0.018), was prognostic for poor disease outcome after adjustment for p53 expression, grade, and residual tumor. p53 expression was detected in 31.5% (26/104 cases). No mutation was observed on the six Cu-binding domain or ATP-binding domain in human ovarian carcinomas expressing ATP7B gene. Conclusions: This study demonstrates that overexpression of ATP7B in ovarian carcinoma is correlated with unfavorable clinical outcome in patients treated with cisplatin-based chemotherapy. Therefore, ATP7B expression may be considered as a predictive marker of chemoresistance for cisplatin-based chemotherapy in patients with ovarian carcinoma. We further predict that drugs targeting ATP7B might be useful in combination with cisplatin-based regimen for the improvement of patients with ovarian carcinoma.

Active Efflux System for Cisplatin in Cisplatin‐resistant Human KB Cells

Mutants, KCP‐4 and PC‐5, resistant to an anticancer agent, cisplatin, were selected in multiple steps from human epidermoid KB carcinoma cells and human prostate PC‐3 carcinoma cells, respectively. KCP‐4 and PC‐5 were 63 and 10 fold more resistant to cisplatin than the parental cells, respectively. KCP‐4 cells exhibited increased resistance to cisplatin analogues and were also slightly cross‐resistant to melphalan, cyclophosphamide, mitomycin C and methotrexate. KCP‐4 cells were not cross‐resistant to doxorubicin, daunorubicin, vincristine or CdSO4. The accumulations of cisplatin in KCP‐4 cells and PC‐5 in medium containing 50 μM cisplatin were approximately 20% of those in the parental cells. Revertant analysis suggested that a defect in cisplatin accumulation may be related to cisplatin resistance in PC‐5 cells. The uncoupling agent of oxidative phosphorylation, 2,4‐dinitrophenol, increased the accumulation of cisplatin in KCP‐4 and cisplatin‐resistant human prostate carcinoma PC‐5 cells to nearly the same level as in their parental KB‐3‐1 and human prostate carcinoma PC‐3 cells without 2,4‐dinitrophenol, but did not increase accumulation in KB‐3‐1 and PC‐3 cells. Addition of glucose in the medium inhibited the enhancement of cisplatin accumulation in KCP‐4 cells by 2,4‐dinitrophenol. Enhanced active efflux of cisplatin from KCP‐4 cells was observed. A cell‐cell hybridization test showed that the cisplatin resistance and the accumulation defect behaved as codominant traits. These data suggest that an active efflux system for cisplatin exists in cisplatin‐resistant KCP‐4 cells.

Loss of heterozygosity of nucleotide excision repair factors in sporadic ovarian, colon and lung carcinomas: implication for their roles of carcinogenesis in human solid tumors

The deficiencies of nucleotide excision repair (NER) factors are genetic diseases, xeroderma pigmentosum (XP) increasing risk of developing cancer on sun-exposed areas of the skin. However, the abnormality of NER factors in human sporadic carcinoma remains unclear. Loss of heterozygosity (LOH) analysis for the XP, XPA, XPB, XPC, XPD, XPE, XPF, XPG and the transcription-coupled repair factor, Cockayne syndrome B (CSB) revealed that NER factors were abnormal in 62.1 % of ovarian tumors (18/29), 16.7% of colon (2/12) and 22.2% lung (2/9) carcinomas. Furthermore, 13.8% of ovarian, 8.3% of colon and 22% of lung carcinomas exhibited LOH for NER factors without LOH for tumor suppressor genes such as p53, FHIT, APC, BRCAI, BRCA2 and DCC. Although both microsatellite instability and LOH of NER factors were observed in some cases, there was no strong association between them in the present study. These observations raise the possibility that alterations of NER factors may be frequent in human sporadic carcinomas. Further study should be needed to find the direct evidence of NER gene abnormalities in human sporadic carcinoma tissues.

Involvement of free radicals in cisplatin-induced emesis in Suncus murinus

The participation of free radicals in cisplatin-induced emesis was investigated in the house musk shrew, Suncus murinus. Thiobarbituric acid (TBA) values, which indicate the degree of lipid peroxidation, in brain, liver and small intestine were increased significantly 60 min after the treatment with cisplatin (20 mg/kg, i.p.). Moreover, cisplatin (20 mg/kg, i.p.)-induced emesis was prevented by intraperitoneal injection of N-(2-mercaptopropionyl)glycine (MPG), a radical scavenging agent, with ID50 value of 130 mg/kg. However, MPG did not block the emesis induced by copper sulfate (40 mg/kg, p.o.), veratrine (0.5 mg/kg, s.c.) or serotonin (10 mg/kg, i.p.). We also investigated the effects of superoxide dismutase conjugated to polyethylene glycol and catalase, but the number of vomiting episodes and latency did not change significantly when these agents were intraperitoneally injected 30 min prior to or 20 min after the administration of cisplatin. MPG did not affect the antitumor effect of cisplatin tested in vitro. These results suggest that free radicals mediate emesis caused by cisplatin and that radical scavengers may become a new class of prophylactic drug against cancer-chemotherapeutic drug-induced emesis.

Copper‐transporting P‐Type Adenosine Triphosphatase (ATP7B) Is Expressed in Human Breast Carcinoma

This is the first report to show that a copper‐transporting P‐type adenosine triphosphatase, ATP7B, is expressed in certain breast carcinomas, and a priori knowledge of its expression is important for the choice of therapy. We investigated the hypothesis that ATP7B, which was shown to be associated with cisplatin resistance in vitro, is expressed in certain breast carcinomas. To test this hypothesis, ATP7B expression and protein level were examined in 41 breast carcinomas using RT‐PCR and immunohistochemistry. ATP7B gene/protein could be detected in 22.0% (9/41) of breast carcinomas and ATP7B gene expression was correlated well with the protein expression. In nine ATP7B‐positive tumors, adjacent normal breast tissue was similarly analyzed, revealing that ATP7B is upregulated in breast carcinoma. ATP7B gene expression in poorly differentiated carcinoma was significantly higher than that in well‐/moderately differentiated carcinoma (P=0.012). Furthermore, we found no association between the ATP7B gene/protein expression and that of MDR1, MRP1, LRP and BCRP. These findings suggested that ATP7B gene expression might be a chemoresistance marker for cisplatin in patients with poorly differentiated breast carcinoma.