Kenji Mitsugi

Gefitinib, a selective EGFR tyrosine kinase inhibitor, induces apoptosis through activation of Bax in human gallbladder adenocarcinoma cells†

Although gefitinib, a selective inhibitor of epidermal growth factor receptor (EGFR) tyrosine kinase, has been clinically demonstrated to be effective for certain cancer cell types, the molecular mechanisms of the anti‐tumor activity have not been fully elucidated. In this study, we investigated the mechanism of gefitinib‐induced growth inhibition and apoptosis in HAG‐1 human gallbladder adenocarcinoma cells. Treatment of gefitinib at a dose of 1 µM resulted in a significant growth inhibition, and the cell number irreversibly declined after 72‐h incubation, with a progressive expansion of apoptotic cell population over 120‐h. Following 2‐h treatment, gefitinib significantly inhibited EGFR autophosphorylation and subsequent downstream signaling pathway through Erk and Akt, and induced accumulation of cells in the G0/G1 phase of the cell cycle at 24‐h, accompanied by a concomitant increase in p21 transcript and increased expression of p27. Gefitinib did not affect the amount of total and phosphorylated p53 at serine 15, but upregulated the expression of total Bax, with subsequent increase in p18 Bax, an active form of Bax. The expression of Bcl‐2 and Bad was unchanged. An increase in gefitinib‐induced expression of total Bax might be due to the decreased degradation of Bax, because the level of Bax mRNA has not been altered by gefitinib treatment. Gefitinib promoted the cleavage of full‐length p21 Bax into p18 Bax in mitochondrial‐enriched fraction, a characteristic feature of Bax activation toward apoptosis. Moreover, blockade of Bax by using anti‐Bax small interfering double stranded RNA (siRNA) significantly reduced gefitinib‐induced apoptosis. Taken together, these data suggest a critical role of p18 Bax in gefitinib‐induced apoptosis. J. Cell. Biochem. 97: 724–734, 2006.

In vivo comparative therapeutic study of optimal administration of 5-fluorouracil and cisplatin using a newly established HST-1 human squamous-carcinoma cell line

SummaryThe efficacy and toxicity of 5-fluorouracil (5-FU) and cisplatin (CDDP) given in a sequential combination were evaluated in nude mice transplanted with HST-1, a newly established human squamous-carcinoma cell line. 5-FU and CDDP were given i.p. for 5 days and 1 day, respectively, either as single agents or in a sequential manner separated by a 24-h interval. The treatment was repeated every 30 days. Although inhibition of tumor growth was seen in all of the treated groups after two cycles, the sequence of 5-FU followed by CDDP significantly reduced the tumor burdens throughout all three courses and was more effective than the reverse sequence or either drug alone. Neither treatment-related death nor significant hematologic or nephrologic toxicities were seen, even following three cycles of therapy. Significant weight loss was observed only in mice treated with CDDP followed by 5-FU. This sequence dependence of the activity and toxicity of the 5-FU and CDDP combination should thus be incorporated into the design of a clinical trial.

Oxaliplatin-induced allergic reaction in patients with colorectal cancer in Japan

BackgroundOxaliplatin is a platinum compound that is clinically effective for colorectal cancer (CRC), in combination with 5-fluorouracil (5-FU) and leucovorin (LV), and it is widely used for metastatic disease and for the adjuvant treatment of stage III CRC. With the increasing use of oxaliplatin in Japan, serious adverse events have been experienced other than hematologic and neurologic toxicities.MethodsIn order to clarify the clinical features of allergic reactions to oxaliplatin, we retrospectively investigated CRC patients who had received oxaliplatin-based chemotherapies.ResultsOne hundred and twenty-five CRC patients who had been treated with FOLFOX regimens (containing oxaliplatin, 5-FU, and LV) were examined, and 21 patients (17%) were found to have developed allergic reactions. Sixteen patients (13%) had grade 1/2 adverse events, classified according to the common terminology criteria for adverse events (CTC-AE) version 3.0 and 5 (4%) had grade 3/4 adverse events. The allergic reaction appeared after a median number of nine cycles (range, 2–15 cycles). Previous chemotherapy included 5-FU/LV, CPT-11, and S-1. All of the patients with allergic reactions recovered completely when treated with antiallergy drugs. Oxaliplatin was reintroduced in 11 patients, with the use of prophylactic agents; allergic reaction to the reintroduction was not observed in 8 patients and grade 1/2 allergic reactions developed in 3 patients. No correlation was identified between allergic reaction and patients’ background characteristics such as sex, history of allergy, and profile of other adverse events.ConclusionAllergic reactions to oxaliplatin remain an important issue for patients being able to safely continue effective chemotherapies; further analysis will be needed to establish methods for the prediction and prophylaxis of such reactions.

In vitro schedule-dependent interaction between paclitaxel and oxaliplatin in human cancer cell lines

PurposeIn order to define the most effective administration schedule of the combination of paclitaxel and oxaliplatin, we investigated the in vitro interaction between these drugs in a panel of three human cancer cell lines (AZ-521 gastric adenocarcinoma cell line, HST-1 tongue squamous carcinoma cell line, and KSE-1 esophageal squamous carcinoma cell line).Materials and methodsCytotoxic activity was determined by the WST-1 assay. Different administration schedules of the two drugs were compared and evaluated for synergism, additivity, or antagonism with a quantitative method based on the median-effect principle of Chou and Talalay. Cell cycle perturbation and apoptosis were evaluated by flow cytometry.ResultsSimultaneous treatment of cells with paclitaxel and oxaliplatin showed greater than additive effects. Upon 24-h sequential exposure, the sequence of paclitaxel followed by oxaliplatin showed synergistic effects in AZ-521 and HST-1 cells, and greater than additive effects in KSE-1 cells, while the opposite sequence yielded marked antagonistic effects in all three cell lines. Flow cytometric analysis indicated that paclitaxel induced G2/M arrest with subsequent induction of apoptosis in the sub-G1 phase. Apoptosis was most prominent when paclitaxel preceded oxaliplatin, which produced apoptosis in the majority of treated cells (75%). By contrast, the reverse sequence yielded only 39% induction of apoptotic cells, the rate being not different from those induced by each drug singly.ConclusionsOur findings suggest that the interaction of paclitaxel and oxaliplatin is highly schedule-dependent and that the sequential administration of paclitaxel followed by oxaliplatin should thus be incorporated into the design of a clinical trial.

Suppression of anoikis by v-Src but not by activated c-H-Ras in human gallbladder epithelial cells

Detachment of anchorage‐dependent normal epithelial cells from their substratum causes the type of apoptosis known as anoikis, whereas malignant cells can proliferate independently of anchorage. Because src and ras oncogenes are activated in many human cancers, we investigated their role and downstream signaling pathways in anoikis resistance, using HAG‐1 human epithelial cells transfected with v‐src or activated H‐ras. Consequently, anchorage‐dependent mock‐ or ras‐transfected cells underwent anoikis. In contrast, anchorage‐independent v‐Src‐transformed cells did not exhibit such apoptotic features. Focal adhesion kinase (FAK), a transducer of integrin, was only activated in v‐Src‐transformed cells. Herbimycin A, an Src kinase inhibitor, reduced tyrosyl phosphorylation of FAK and reversed resistance to anoikis. However, both protein kinase C (PKC) and phophatidylinositol‐3 (PI‐3) kinase inhibitors failed to induce anoikis. These data suggest that the ability of activated Src to prevent anoikis may be mediated by Src to a downstream signaling pathway involving FAK, but not Ras, PI‐3 kinase, or PKC.

Small Cell Carcinoma of the Tonsil Treated with Irinotecan and Cisplatin: A Case Report and Literature Review

We report a rare case of extrapulmonary small cell carcinoma arising in the palatine tonsil treated by combined chemotherapy with irinotecan/cisplatin following irradiation therapy. This chemotherapy regimen was recently found to be effective for small cell lung carcinoma. Our case is the first report of combined irinotecan/cisplatin chemotherapy to treat extrapulmonary small cell carcinoma of the oropharynx.

Phase I study of CPT-11 and bolus 5-FU/l-leucovorin in patients with metastatic colorectal cancer

BackgroundIrinotecan (CPT-11) and bolus 5-fluorouracil (5-FU)/leucovorin (LV) administered weekly for 4 weeks every 42 days (Saltz regimen) is active but substantially toxic in patients with metastatic colorectal cancer (CRC). The efficacy and toxicity of this regimen, however, have not been determined in Japanese patients with metastatic CRC.MethodsWe investigated the maximum tolerated dose (MTD), dose-limiting toxicity (DLT), and recommended phase II dose (RD) for CPT-11 given i.v. (90-min infusion) and bolus 5-FU plus biologically active l-LV administered weekly for 3 weeks every 28 days (modified Saltz regimen) in Japanese patients with metastatic CRC. Eighteen patients with measurable disease, Eastern Cooperative Oncology Group (ECOG) performance status of 2 or less, and adequate organ functions were enrolled.ResultsAt dose level 2 (CPT-11, 100 mg/m2; 5-FU, 400 mg/m2; and l-LV, 25 mg/body), 1 of 6 patients had DLT (febrile neutropenia). At dose level 3 (CPT-11, 100 mg/m2; 5-FU, 500 mg/m2; and l-LV, 25 mg/body), 2 of 6 patients had DLT (febrile neutropenia and grade 4 neutropenia lasting more than 4 days). To determine whether level 3 was the MTD level, an additional 3 patients were treated at this level, but no DLT was observed. Consequently, 2 of 9 patients had DLT at level 3, this level thus being considered as the RD. At this level, grade 3–4 neutropenia was common but manageable. Nonhematological toxicities were mild. Seven partial responses were observed in the 18 enrolled patients (response rate [RR], 39%), and 8 patients (44%) experienced stable disease.ConclusionThis CPT-11/5-FU/l-LV regimen administered weekly for 3 weeks every 28 days has substantial antitumor activity, with manageable toxicities. A multicenter phase II study is currently underway.

Production of anti-cardiolipin antibody in AKR/J mice with streptozocin-induced insulitis and diabetes

We herein report that anti-cardiolipin antibodies (ACA) were detected in AKR/J mice treated with multiple low doses of streptozocin (STZ)-induced insulitis and diabetes. Daily intraperitoneal (i.p.) injections of 40 mg/kg body wt. of STZ for five consecutive days in the AKR/J mice resulted in hyperglycemia and mononuclear cell infiltrations of islets (insulitis). ACA appeared on day 14, when hyperglycemia began to occur, at a rate of 13.3% (4/30). The rate increased to 83.3% (25/30) on day 21, when diabetes developed, and then fell to 10% (3/30) on day 28. Neither the diabetic AKR/J mice treated with a single high dose of STZ (200 mg/kg body wt.) nor the non-diabetic insulitis free Balb/c mice and B10.S(9R) mice treated with multiple low doses of STZ (40 mg/kg body wt.) produced ACA. The IgG subclass of the ACA belonged mainly to IgG2a. These findings suggest that ACA are produced in association with the development of insulitis, but not induced by either hyperglycemia or STZ.

Phase I/II study of a 3‐week cycle of irinotecan and S‐1 in patients with advanced colorectal cancer

The combination of an oral fluoropyrimidine derivative, S‐1, and irinotecan is expected to be a promising regimen for advanced colorectal cancer. This study was performed to determine the maximum tolerated dose (MTD) and recommended dose (RD) of irinotecan combined with S‐1 in a 3‐week cycle regimen and to observe the safety and efficacy for patients with previously untreated advanced colorectal cancer. Eighty milligrams per m2 of S‐1 was given orally for 14 consecutive days and escalated doses of irinotecan were administered on days 1 and 8 every 3 weeks in the phase I trial. Forty patients were treated at the RD during the phase II trial. Forty‐three patients were enrolled between February 2005 and March 2007. The dose‐limiting toxicity was diarrhea and abdominal pain. The MTD of irinotecan was 100 mg/m2 and the RD was determined to be 80 mg/m2 of irinotecan combined with 80 mg/m2 of S‐1. The phase II trial showed that 22 of 40 patients achieved a complete or partial response and eight had stable disease. The overall response rate was 55.0%. The median progression‐free survival time and median survival time were 6.7 and 21 months, respectively. There were no treatment‐related deaths. The main toxicities were leukopenia, neutropenia, anorexia and diarrhea. This study suggests the combination of irinotecan and S‐1 repeated every 3 weeks is tolerable and effective for patients with previously untreated advanced colorectal cancer. (Cancer Sci 2010; 101: 2591–2595)

In vitro sequence-dependent interaction between nedaplatin and paclitaxel in human cancer cell lines

PurposeTo define the most effective combination schedule of paclitaxel and nedaplatin, a new platinum derivative, we investigated the in vitro interaction between these drugs in AZ-521 and NUGC-4 gastric adenocarcinoma and KSE-1 esophageal squamous carcinoma cell lines.Materials and methodsCytotoxic activity was determined by the WST-1 assay. Different treatment schedules of the two drugs were compared and evaluated for synergism, additivity, or antagonism using a quantitative method based on the median-effect principle of Chou and Talalay. Cell-cycle perturbation and apoptosis were evaluated by means of flow cytometry.ResultsUpon 24-h sequential exposure, the sequence paclitaxel followed by nedaplatin induced greater than additive effects in all of the cell lines, with synergistic interactions in NUGC-4 and KSE-1 cells. By contrast, antagonistic effects were observed with the reverse sequence. Simultaneous treatment resulted in either a synergistic or antagonistic effect, depending on the cell line. Therefore, the sequence paclitaxel followed by nedaplatin appears most active, at least in these three cell lines. Flow cytometric analyses at IC50 indicated that paclitaxel induced G2/M arrest with subsequent induction of apoptosis (56%) in the sub-G1 phase. When paclitaxel preceded nedaplatin, apoptosis was most prominent (70%) with pronounced G2/M arrest. By contrast, the reverse sequence yielded only 28% induction of apoptotic cells, with almost identical cell-cycle distribution patterns to those observed with nedaplatin alone, indicating that the activity of paclitaxel is abolished by pretreatment with nedaplatin.ConclusionsOur findings suggest that the interaction of nedaplatin and paclitaxel is highly schedule dependent and that the sequential administration of paclitaxel followed by nedaplatin should be thus incorporated into the design of a clinical trial.