Kuniyuki Katano

Increased Expression of the Copper Efflux Transporter ATP7A Mediates Resistance to Cisplatin, Carboplatin, and Oxaliplatin in Ovarian Cancer Cells

Purpose: The goal of this study was to determine the effect of small changes in ATP7A expression on the pharmacodynamics of cisplatin, carboplatin, and oxaliplatin in human ovarian carcinoma cells. Experimental Design: Drug sensitivity and cellular pharmacology parameters were determined in human 2008 ovarian carcinoma cells and a subline transfected with an ATP7A-expression vector ATP7A (2008/MNK). Drug sensitivity was determined by clonogenic assay, platinum (Pt) levels were measured by inductively coupled plasma mass spectroscopy, copper (Cu) accumulation was quantified with 64Cu, and the subcellular distribution of ATP7A was assessed by confocal digital microscopy. Results: The 1.5-fold higher expression of ATP7A in the 2008/MNK cells was sufficient to alter Cu cellular pharmacokinetics but not confer Cu resistance. In contrast, it was sufficient to render the 2008/MNK cells resistant to cisplatin, carboplatin, and oxaliplatin. Resistance was associated with increased rather than decreased whole-cell Pt drug accumulation and increased sequestration of Pt into the vesicular fraction. Cu triggered relocalization of ATP7A away from the perinuclear region, whereas at equitoxic concentrations the Pt drugs did not. Conclusions: A small increase in ATP7A expression produced resistance to all three of the clinically available Pt drugs. Whereas increased expression of ATP7A reduced Cu accumulation, it did not reduce accumulation of the Pt drugs. Under conditions where Cu triggered ATP7A relocalization, the Pt drugs did not. Thus, although ATP7A is an important determinant of sensitivity to the Pt drugs, there are substantial differences between Cu and the Pt drugs with respect to how they interact with ATP7A and the mechanism by which ATP7A protects the cell.

Cisplatin rapidly down-regulates its own influx transporter hCTR1 in cultured human ovarian carcinoma cells

Purpose: Cisplatin (DDP)-resistant cells commonly exhibit reduced drug accumulation. Previous studies have shown that the major copper (Cu) influx transporter CTR1 controls the uptake of DDP in yeast and mammalian cells. The goal of this study was to examine the effect of Cu and DDP on the level and subcellular localization of hCTR1 protein in human ovarian carcinoma cells. Experimental Design: Cultured human ovarian carcinoma A2780 cells were exposed to DDP and Cu, and the effect on hCTR1 was determined using Western blot analysis and confocal digital deconvolution microscopy. Results: Loss of hCTR1 was triggered by DDP exposure in a concentration and time-dependent manner. Exposure to 0.5 μmol/L DDP for 5 minutes reduced hCTR1 levels and exposure to DDP concentrations ≥2 μmol/L caused almost complete disappearance. The loss of hCTR1 was observed within 1 minute of the start of exposure to 2 μmol/L DDP. Treatment of cells with 100 μmol/L Cu for 5 minutes produced a smaller effect. Pretreatment of cells with 2 μmol/L DDP for 5 minutes resulted in a 50% decrease in 64Cu uptake, demonstrating that the DDP-induced loss of hCTR1 detected by Western blot analysis and imaging was functionally significant. Conclusions: DDP down-regulated the amount of its major influx transporter in cultured human ovarian carcinoma cells in a concentration- and time-dependent manner. The effect was observed at DDP concentrations within the range found in the plasma of patients being treated with DDP, and it occurred very quickly relative to the half-life of the drug.

Confocal Microscopic Analysis of the Interaction between Cisplatin and the Copper Transporter ATP7B in Human Ovarian Carcinoma Cells

Some cisplatin (DDP)-resistant cells overexpress the copper export transporter ATP7B, and cells molecularly engineered to overexpress ATP7B are resistant to DDP. The interaction of Cu with ATP7B normally triggers its relocalization from the perinuclear region to more peripheral vesicles. To investigate the interaction of DDP with ATP7B, we examined the effect of DDP on the subcellular localization of ATP7B using human ovarian carcinoma cells expressing a cyan fluorescent protein (ECFP)-tagged ATP7B (2008/ECFP-ATP7B). ATP7B expression was confirmed in 2008/ECFP-ATP7B cells by Western blotting, and its functionality was documented by showing that it rendered the cells 1.9-fold resistant to CuSO4 and 4.1-fold resistant to DDP and also reduced the accumulation of both drugs. There was greater sequestration of Pt into intracellular vesicles in the 2008/ECFP-ATP7B cells than in the 2008/ECFP cells. Confocal digital microscopy revealed that ECFP-ATP7B localized in the perinuclear region in absence of drug exposure and that both Cu and DDP triggered relocalization to more peripheral vesicular structures. A fluorescein-labeled form of DDP that retained cytotoxicity and was subject to the same mechanisms of resistance as DDP colocalized with ECFP-ATP7B in the 2008/ECFP-ATP7B cells, whereas the same fluorochrome lacking the DDP moiety did not. These results provide evidence that DDP directly interacts with ATP7B to trigger its relocalization and that ATP7B mediates resistance to DDP by sequestering it into vesicles of the secretory pathway for export from the cell.

A prospective pilot study of extended (D3) and superextended para-aortic lymphadenectomy (D4) in patients with T3 or T4 gastric cancer managed by total gastrectomy☆

BACKGROUND Japanese surgeons have been actively performing extended lymphadenectomy (D2, removal of perigastric nodes and nodes along the left gastric, common hepatic, celiac and splenic arteries; or D3, D2 plus removal of nodes in the hepatoduodenal ligament, in the retropancreatic space and along the vessels of the transverse mesocolon). In recent years interest has expanded to superextended lymphadenectomy (D4) of nodes around abdominal aorta (para-aortic lymph nodes from aortic hiatus to aortic bifurcation). Because the therapeutic value of this D4 procedure remains controversial, we initiated a prospective study to compare D3 and D4 lymphadenectomy. METHODS Seventy patients with T3 or T4 gastric cancer and without macroscopic metastasis to the para-aortic nodes treated by potentially curative total gastrectomy were randomized to D4 (group A, n = 35) and D3 (group B, n = 35) lymphadenectomies. RESULTS Metastases to para-aortic nodes were found in 4 patients. Postoperative survival after D4 resection was not statistically significant between the groups. Postoperative morbidity for group A was greater. In group A 4 patients had postoperative retention of intra-abdominal fluid (lymphorrhea) and 4 others had prolonged diarrhea. One patient in each group died of postoperative complications. CONCLUSIONS Surgical treatment of microscopic disease in grossly normal para-aortic lymph nodes may generate occasional long-term survivors. Selecting appropriate candidates who might benefit from D4 resections needs to be refined. On the basis of this study, a nationwide study should be considered.

Serum concentration of CD44 variant 6 and its relation to prognosis in patients with gastric carcinoma

The expression of variant isoforms of CD44 is correlated with the ability of tumor cells to metastasize in some clinical carcinomas. In this study, the serum concentration of soluble splice isoforms of CD44 that shared exon variant 6 (sCD44v6) were measured and the histologic expression of CD44v6 in tumors from patients with gastric carcinoma examined.

Expression of phosphorylated Akt (pAkt) in gastric carcinoma predicts prognosis and efficacy of chemotherapy

BackgroundThe Akt signaling pathway controls the survival and growth of human cancers. We investigated the expression of phosphorylated Akt (pAkt) in patients with gastric cancer.MethodsThe expression of pAkt was immunohistochemically examined in 140 gastric cancer patients who underwent a gastrectomy. The expression of pAkt was evaluated based on staining intensity, and staining was classified as negative or positive. We examined the expression of pAkt and its association with the clinicopathological findings, prognosis, depth of invasion, the expression of p53, and efficacy of oral fluorouracil chemotherapy after surgery.ResultsExpression of pAkt was positive in 81 (58%) patients and negative in 59 (42%) patients. There were no significant correlations between pAkt expression and the clinicopathological findings. The prognosis of patients with pAkt-negative tumors was superior to that of patients with pAkt-positive tumors, and the difference was significant for T3/T4 gastric cancer (P < 0.05). Among the patients with T3/T4 gastric cancer, postoperative oral fluorouracil treatment was effective in those who were pAkt-positive. Multivariate analysis revealed that pAkt expression and lymph node metastasis were independent prognostic factors. In 88 patients with T3 gastric carcinoma who had undergone curative surgery, in whom we studied the prognostic impact of a combined analysis of pAkt and p53 expression, patients with both pAkt- and p53-positive tumors showed a significantly poorer prognosis than patients with either or both pAkt- and p53-negative tumors (P < 0.05).ConclusionOur results indicate that pAkt expression may be useful for predicting the prognosis and efficacy of fluorouracil treatment in patients with gastric cancer.

Expression of p53 and p21 are independent prognostic factors in patients with serosal invasion by gastric carcinoma.

To evaluate whether the expression of p53 andthat of p21 are independent prognostic factors inpatients with advanced gastric cancer, we investigatedclinicopathological factors and the expression of p53 and p21 in 158 patients with gastric cancerthat had invaded the serosa and who had undergonecurative gastrectomy. In multivariate survival analysisof 156 surviving patients, we evaluated the size of the tumor, lymph node metastasis, venousinvasion, lymph node dissection, expression of p53, andexpression of p21 as independent prognostic factors.Moreover, we divided patients into four groups according to the expression of p53 and p21 in theirtumors [group A, p53-/p21-, N = 40(one died within 30 days of surgery); group B, p53-/p21-, N = 23; group C,p53+/p21+, N = 58; and group D,p53+/p21+, N = 37 (one died within 30 days of surgery)]. The 5- and 10-yearsurvival rates of 39 patients in group A were 71.7% and64.3%, those of 23 patients in group B were 81.4% and81.4%, those of 58 patients in group C were 35.6% and 30.2%, and those of 36 patients ingroup D were 67.9% and 60.7%. The prognosis of patientsin group C was poorer than that of patients in the otherthree groups. This result indicates that the evaluation of the expression of both p53 andp21 expression might provide prognostic information thatis more accurate than that provided by evaluation of theexpression of p53 alone.

Topoisomerase I expression in tumors as a biological marker for CPT-11 chemosensitivity in patients with colorectal cancer.

Irinotecan (CPT-11) is used as a first- and second-line chemotherapy for advanced or recurrent colorectal cancer (CRC). However, only 20%–30% of patients show an objective response to CPT-11 and the drug has severe toxicities, such as delayed-onset diarrhea, neutropenia, nausea, and vomiting. It is important to select patients who will demonstrate sensitivity to CPT-11 treatment to avoid unnecessary drug toxicities and to introduce anticancer treatment benefits to CRC patients. DNA topoisomerase I (Topo I) is essential for vital cellular processes such as DNA replication, transcription, translation, recombination, and repair. This article reviews the possibility of assessing Topo I protein expression in tumors as a biological marker for CPT-11 treatment in CRC.

Correlation between the lymphocytic infiltration of tumors and the proliferative activity of cancer cells from surgically treated esophageal carcinoma

To evaluate the correlation among the biological behavior of cancer cells, the local immune response of the host, and survival of patients with esophageal carcinoma, we investigated the proliferative activity of cancer cells by immunostaining with the Ki-67 monoclonal antibody in 95 cases of surgically resected esophageal squamous cell carcinoma and compared them with the extent of lymphocytic infiltration of the tumor (LI) in the same specimens. The mean Ki-67 score of 43 tumors with low-grade LI was 46.7% and it was not different from that of 52 tumors with high-grade LI (49.4%, p = 0.441). A significant correlation between the Ki-67 score and the clinicopathological characteristics of tumors (depth of tumor invasion, lymph node metastasis, and stage) was detected. However, in the same stage, the survival of patients with a low Ki-67 score was not different from that of patients with a high Ki-67 score. On the other hand, even no significant correlation between the extent of LI and the clinicopathological characteristics of tumors was found; the 5-year survival rate of 17 patients with high-grade LI (13.9%) was significantly better than that of 15 patients with low-grade LI (0%, p = 0.07) in stage III. These facts indicate that proliferative activity might correlate with the tumor progression, however, when the survival is compared in the same stage, the local immune response such as the extent of lymphocytic infiltration of the tumor might be the better prognostic factor than proliferative activity in patients with esophageal cancer.

Clinicopathologic significance of the expression of mutated p53 protein and the proliferative activity of cancer cells in patients with esophageal squamous cell carcinoma.

BACKGROUND The aim of this study was to investigate the relation between the expression of mutated p53 protein and the proliferative activity of cancer cells in esophageal squamous cell carcinoma. In addition, the clinical and biologic significance of p53 status and the proliferative activity of cancer cells were evaluated in these patients. STUDY DESIGN Samples of esophageal tumors from 94 patients were subjected to immunohistochemical staining with a monoclonal antibody against p53 and with the monoclonal antibody Ki-67. The immunoreactivity against p53 and the proliferative activity of cancer cells were compared with the clinicopathologic findings in each sample. Prognostic factors including p53 status and Ki-67 labeling index (LI; percentage of Ki-67-immunostained cells) were evaluated for 81 surviving patients by univariate and multivariate analysis. RESULTS The mean Ki-67 LI of 50 p53-positive patients was higher than that of 44 p53-negative patients (p = 0.009). The Ki-67 LI increased according to the progression of tumors. Overexpression of mutated p53 protein was observed in 40.9% of tumors that invaded to the submucosa, and this percentage was not significantly changed in tumors with invasion to the adventitia. Metastases to the regional lymph nodes were observed in 3 of 22 patients with tumors that invaded to the submucosa, and these 3 tumors had both over-expression of mutated p53 protein and high Ki-67 LI. In 81 surviving patients, only lymph node metastasis (p = 0.045) and the curability of tumors (p < 0.001) were identified as independent prognostic factors by multivariate analysis. CONCLUSIONS Overexpression of mutated p53 protein is detected in the early stage of esophageal cancer. This mutated p53 protein may not play an important role for tumor invasion. When tumor invasion is limited to the submucosa, cancer cells that overexpress mutated p53 protein may acquire high proliferative activity. Such cells might have considerable potential for metastasis to the lymph nodes.