Volume-regulated chloride channel regulates cell proliferation and is involved in the possible interaction between TMEM16A and LRRC8A in human metastatic oral squamous cell carcinoma cells.

OBJECTIVES: Volume-regulated anion channels (VRACs), expressed in various cells, play an important role in cell volume regulation. Despite being physiologically defined almost half a century ago, only the molecular candidates of VRAC, TMEM16A, LRRC8A, and bestrophin-1 (BEST1), are known. Here, we aimed to explore the functional significance of VRAC in, HST-1, an oral squamous cell carcinoma (OSCC) cell line. METHODS: Cell proliferation assays, RT-PCR, Western blot, and flow cytometry were used to estimate changes in gene expression and cell proliferation. Ion channel activity was recorded using the patch-clamp technique. Specific genes were knocked-down by siRNA assays. RESULTS: VRAC, identified as a hypotonicity-induced current, was highly functional and associated with the proliferation of HST-1 cells but not of HaCaT (a normal keratinocyte) cells. The pharmacological profile of VRAC in HST-1 was similar to that reported previously. DCPIB, a specific VRAC inhibitor, completely inhibited VRAC and proliferation of HST-1 cells, eventually leading to apoptosis. VRAC in HST-1 was attenuated by the knockdown of TMEM16A and LRRC8A, while knockdown of BEST1 affected cell proliferation. In situ proximity ligation assay showed that TMEM16A and LRRC8A co-localized under isotonic conditions (300 mOsM) but were separated under hypotonic conditions (250 mOsM) on the plasma membrane. CONCLUSIONS: We have found that VRAC acts to regulate the proliferation of human metastatic OSCC cells and the composition of VRAC may involve in the interactions between TMEM16A and LRRC8A in HST-1 cells.

Genistein Suppresses v-Src-Driven Proliferative Activity by Arresting the Cell-Cycle at G2/M through Increasing p21 Level in Src-Activated Human Gallbladder Carcinoma cells.

Activated Src have been strongly implicated in the development, progression, and metastasis of many human cancers. Although soy isoflavones exhibit potential anticancer activity, effects of isoflavones on oncogenic activity of Src remains unknown. Using v-src-transfected human adenocarcinoma cells (HAG/src3-1), we investigated the functional role of Src in anti-proliferative activity of isoflavones including genistein, daidzein, glycitein and equol. The growth of HAG/neo3-5 vehicle control cells was inhibited potently by genistein and equol, but modestly by daidzein and glycitein. In contrast, Src activation conferred resistance to either daidzein, glycitein or equol, but rendered the cells more sensitive to genistein, compared to HAG/neo3-5 cells. Genistein significantly arrested HAG/src3-1 cells at G2/M, while neither daidzein, glycitein nor equol arrested the cells at any cell cycle phases. Apoptosis was not induced by either isoflavones. Genistein increased the expression levels of p53 and p21 with decreased phosphorylated p21, but did not affect the levels of major cyclin-CDK complexes. Taken together, genistein would be considered as the only isoflavone component that may potentially suppress Src-driven proliferative activity by arresting at G2/M induction through increasing the p21 levels, thus providing the mechanistic rationale for the potential use of genistein for the prevention of human cancers with activated Src.

Equol Enhances Apoptosis-inducing Activity of Genistein by Increasing Bax/Bcl-xL Expression Ratio in MCF-7 Human Breast Cancer Cells.

Anticancer activities of soy isoflavones, such as genistein and equol, a bioactive metabolite of daidzein, have been extensively studied because of possible involvement in the prevention of breast cancer. However, their interactions still remain unclear. We investigated here whether cytotoxic activity of genistein was enhanced by equol, using estrogen receptor positive MCF-7, HER2-positive SK-BR-3, and triple-negative MDA-MB-468 cell lines. Although cytotoxicity of genistein did not significantly differ between three subtypes of breast cancer cells, cytotoxic activities of genistein were significantly enhanced in combination with 50 µM equol in MCF-7 cells, but not in SK-BR-3 and MDA-MB-468 cells. In fluorescence activated cell sorting (FACS) analyses, MCF-7 cells were arrested at the G2/M by genistein but at G1/S by equol. Combination treatment arrested cells at G2/M but abolished equol-induced G1 block, indicating an antagonistic activity of genistein against equol in cell-cycle progression. Although apoptosis was not so evident with genistein alone, the combination made a drastic induction of apoptosis, accompanied by the increase of Bax/Bcl-xL expression ratio, without affecting the activities of Akt and mTOR. Taken together, these data suggest that enhancement of genistein activity by equol would be mainly mediated by augmented induction of apoptosis rather than arrest or delay of the cell cycle.

Differential Anticancer Activity of Pterostilbene Against Three Subtypes of Human Breast Cancer Cells.

Although pterostilbene, a natural analog of resveratrol, has potent antitumor activity against several human cancer types, the possible inhibitory mechanisms against subtypes of human breast cancer with different hormone receptor and human epidermal growth factor receptor 2 (HER2) status remain unknown. We investigated the anticancer activity of pterostilbene using three subtypes of breast cancer cell lines. Pterostilbene treatment exhibited a dose-dependent antiproliferative activity, with the greatest growth inhibition observed in triple-negative MDA-MB-468 cells. Although pterostilbene arrested cell-cycle progression at the G0/G1 phase regardless of breast cancer subtype, its apoptosis-inducing activity was highly apparent in MDA-MB-468 cells. Pterostilbene induced strong and sustained activation of extracellular signal-regulated kinase (ERK) 1/2, with concomitant cyclin D1 suppression and p21 up-regulation, and inhibited the phosphorylation of AKT and mammalian target of rapamycin (mTOR), followed by subsequent up-regulation of BAX without affecting B-cell lymphoma-extra large (BCL-xL). Oral administration of pterostilbene significantly suppressed tumor growth in nude mice xenotransplanted with MDA-MB-468 cells. These data suggest a potential role of pterostilbene for prevention and treatment of human breast cancer, especially of triple-negative breast cancer.

Mechanism of the Anticancer Effect of Lycopene (Tetraterpenoids).

Increasing evidence suggests that lycopene, a major carotenoid detected in human plasma, may be preventive against the formation and the development of different types of human cancers including prostate, breast, and lung cancer. Experimental studies demonstrated that lycopene inhibits the growth of various cancer cells of different organs and prevent chemically induced carcinogenesis in animal models. Although the excellent antioxidant property of lycopene is most likely the basis for its preventive role toward cancer, the direct anticancer activities of lycopene through multiple mechanisms are disclosed, including regulation of growth factor signaling, cell cycle arrest and/or apoptosis induction, and changes in antioxidant and phase II detoxifying enzymes. The anti-inflammatory activity of lycopene is also considered as an important determinant that suppresses the promotion and progression of carcinogenesis. Moreover, lycopene inhibits cell invasion, angiogenesis, and metastasis. Importantly, those activities have been shown to be exhibited at the physiologically attainable concentration in humans. Although the preclinical data strongly suggest an antitumor activity of lycopene, a number of epidemiological and intervention studies indicate that there is still no clear clinical evidence that supports its use for the prevention of those cancers. More well controlled clinical intervention trials are needed to further clarify the exact role of lycopene in the cancer prevention. Nonetheless, because of its multiple tumor-inhibitory activities, lycopene still remains to be an attractive and promising carotenoid that will potentially contribute to the prevention and treatment of human cancers. This chapter reviews data on the cancer preventive activities of lycopene, possible mechanisms involved, and the relationship between lycopene consumption and human cancer risk.

Antiproliferative and apoptosis-inducing activity of nobiletin against three subtypes of human breast cancer cell lines.

UNLABELLED: Although nobiletin has a potent antitumor activity against several types of human cancers, its inhibitory effects and possible mechanisms of action on breast cancer cells with different hormone receptor and HER2 status remains unknown. MATERIALS AND METHODS: Using hormone receptor-positive MCF-7, HER2-positive SK-BR-3, and triple-negative MDA-MB-468 cell lines, we investigated the antitumor mechanisms of nobiletin. RESULTS: Nobiletin exhibited dose- and time-dependent antitumor activity against these different subtypes of cell lines, with the greatest inhibition observed against the MDA-MB-468 cell line. Nobiletin induced cell-cycle arrest at the G0/G1 phase by suppressing ERK1/2 activity, with concomitant cyclin-D1 suppression and p21 up-regulation. Nobiletin induced apoptotic cell death by reducing Bcl-xL expression, without affecting Bax levels, and inhibited the activity of AKT and downstream mTOR in MDA-MB-468 cells, but not in other cell lines. CONCLUSION: The predominant anticancer activity of nobiletin in MDA-MB-468 cells suggests a potential role of nobiletin for the prevention of triple-negative breast cancer.

Anti-proliferative and apoptosis-inducing activity of lycopene against three subtypes of human breast cancer cell lines.

Although lycopene, a major carotenoid component of tomatoes, has been suggested to attenuate the risk of breast cancer, the underlying preventive mechanism remains to be determined. Moreover, it is not known whether there are any differences in lycopene activity among different subtypes of human breast cancer cells. Using ER/PR positive MCF-7, HER2-positive SK-BR-3 and triple-negative MDA-MB-468 cell lines, we investigated the cellular and molecular mechanism of the anticancer activity of lycopene. Lycopene treatment for 168 consecutive hours exhibited a time-dependent and dose-dependent anti-proliferative activity against these cell lines by arresting the cell cycle at the G0 /G1 phase at physiologically achievable concentrations found in human plasma. The greatest growth inhibition was observed in MDA-MB-468 where the sub-G0 /G1 apoptotic population was significantly increased, with demonstrable cleavage of PARP. Lycopene induced strong and sustained activation of the ERK1/2, with concomitant cyclin D1 suppression and p21 upregulation in these three cell lines. In triple negative cells, lycopene inhibited the phosphorylation of Akt and its downstream molecule mTOR, followed by subsequent upregulation of proapoptotic Bax without affecting anti-apoptotic Bcl-xL. Taken together, these data indicate that the predominant anticancer activity of lycopene in MDA-MB-468 cells suggests a potential role of lycopene for the prevention of triple negative breast cancer.

Differential anti-tumor activities of curcumin against Ras- and Src-activated human adenocarcinoma cells.

Although curcumin has been studied as a potential anticancer drug targeting multiple signaling molecules, the role of oncogenic Src and Ras in curcumin sensitivity remains unknown. Using HAG-1 human adenocarcinoma cells transfected with either activated Src or Ras, we investigated here the functional role of these oncogenes in curcumin sensitivity. Activation of either Src or Ras did not confer resistance to curcumin, compared to vehicle-transfected cells. Curcumin enhanced Erk1/2 predominantly in Ras-activated cells, but inhibited Akt and its downstream molecules (mTOR and S6K1) regardless of these oncogene activations. The sub-G0/G1 apoptotic populations were substantially increased with demonstrable cleavage of PARP, but this increase was most prominent in Src-activated cells. Suppression of Bcl-xL level and enhanced expression of Bax were demonstrated in Src-activated, but not Ras-activated cells. By contrast, drastic increases of G2/M cell populations were seen in Ras-activated cells rather than Src-activated cells, suggesting a potential role of Ras/Erk1/2 activation in curcumin-induced G2/M arrest. These data indicate that curcumin-induced growth inhibition would be mediated mainly by G2/M arrest in Ras-driven cells but by apoptosis induction in Src-driven cells, providing a mechanistic rationale for the potential use of curcumin in the treatment of human cancers with activated Src or Ras.

Antiproliferative and apoptosis-inducing activity of curcumin against human gallbladder adenocarcinoma cells.

AIM: Curcumin has potent antitumor activity against many types of human cancers. However, the inhibitory effects and possible mechanisms of curcumin on gallbladder cancer remains to be determined. MATERIALS AND METHODS: Using HAG-1 human gallbladder adenocarcinoma cells, we investigated the effects of curcumin on cell proliferation, apoptosis, cell-cycle perturbation, and signal proteins for survival, proliferation, and apoptosis. RESULTS: Curcumin exhibited dose-dependent antitumor activity against HAG-1 cells, arresting the cells in G2/M phase, with progressive expansion of the apoptotic cell population. Upon curcumin treatment, AKT activation was substantially suppressed, with subsequent reduction of activities of mammalian target of rapamycin (mTOR) and its downstream molecules S6 kinase-1 (S6K1) and elF4E-binding protein-1 (4E-BP1), but constitutive activity of extracellular signal-regulated kinase (ERK1/2) was clearly enhanced. Curcumin reduced the expression and phosphorylation of anti-apoptotic Bcl-2, but did not affect the expressions of pro-apoptotic Bax and anti-apoptotic nuclear factor (NF-κB). CONCLUSION: These results suggest that curcumin induces G2/M arrest and apoptosis through multiple mechanisms involving enhanced mitogen-activated protein (MAP) kinase activity, reduced AKT-mTOR activity, and reduced Bcl-2 function. These data provide a mechanistic rationale for the potential use of curcumin in the treatment of gallbladder cancer.

[Development trends for therapeutic antibody].

Recent developments for therapeutic antibody have provided new options for cancer treatments. Modification of antibody molecules pursuing improvement of binding efficacy for Fcgamma receptors and enabling efficient recycling of antibody have been performed. Novel constructs of antibody possessing multivalent specificity and conjugated agents have also been developed. Based on exploration of new class of target molecules for therapeutic antibody, antibodies enhancing anti-tumor immunity such as anti-CTLA-4 antibody have appeared. These advancements would achieve more effective and safer therapy for various kinds of cancers.

Effects of dietary genistein on hormone-dependent rat mammary carcinogenesis induced by ethyl methanesulphonate.

Genistein, a major soy isoflavone having weak estrogenic activities, has been suggested to reduce the risk of breast cancer incidence. However, many studies have yielded inconsistent results. We investigated the effects of dietary genistein on the development of breast cancer using ethyl methanesulphonate (EMS) chemically induced rat model of hormone-dependent mammary carcinoma. Female Wistar King A rats were orally given EMS for 12 wk and fed isoflavone-free NIH-07PLD diets with or without genistein, beginning immediately after weaning period. All EMS-treated rats fed either diet developed estrogen and/or progesterone receptor-positive mammary carcinoma by 24 wk. The addition of either low or high genistein, which produced the plasma concentrations comparable with those observed in humans consuming high soy diets, did not show any preventive activity. Soy-containing pellet food, exhibiting substantial plasma concentrations of isoflavones such as genistein, daidzein, equol, and glycitein, significantly increased the latency periods, compared to either NIH-07PLD diet with low (P = 0.027) or high (P = 0.034) genistein. Body weights, total EMS uptakes, and urinary estradiol concentrations were not significantly different among groups. These data indicate that genistein does not exert clear preventive effects and that isoflavone components other than genistein might be preventive against hormone-dependent mammary carcinogenesis.

Phase 2 study of modified irinotecan and bolus 5-fluorouracil/l-leucovorin in Japanese metastatic colorectal cancer patients.

INTRODUCTION: Using the recommended doses obtained from our previous phase 1 trial of a modified Saltz chemotherapy regimen for metastatic colorectal cancer (weekly irinotecan and bolus 5-fluorouracil/l-leucovorin for 3 weeks every 28 days), we performed the present phase 2 trial to evaluate efficacy and toxicity. METHODS: A total of 29 patients with metastatic colorectal cancer were included. Our modified Saltz regimen was administered. The primary endpoint was overall response rate. RESULTS: Of the 29 patients, 11 had previous chemotherapy. A partial response occurred in 11 patients, stable disease in 16 patients, and progressive disease in two patients. Disease control rate was 93.1%. Response rates with and without previous treatment were 18.2% and 50%, respectively. Median progression-free survival was 17.3 months. The main hematologic toxicities were leukopenia (22.6%) and neutropenia (45.2%). No treatment-related deaths occurred. CONCLUSION: Our modified Saltz regimen exhibited sufficient efficacy, feasibility, and manageable toxicity as a therapeutic option for selected colorectal cancer patients.

Phase II study of sequential treatment with S-1 and cisplatin for metastatic gastric cancer.

PURPOSE: This single-arm, phase II clinical study evaluated the efficacy and safety of sequential treatment with S-1 followed by cisplatin in patients with advanced or recurrent gastric cancer. METHODS: Fifty patients with histologically confirmed advanced or recurrent gastric cancer and an Eastern Cooperative Oncology Group performance status of 0-2 who had measurable and/or assessable lesions and gave written informed consent were enrolled. S-1 (40 mg/m(2), bid) was administered on days 1-21, and cisplatin (70 mg/m(2)) was given as an intravenous infusion on day 22 of a 35-day cycle. Treatment was continued until disease progression or intolerable adverse events. Cisplatin was administered for 6 cycles. Adverse events were assessed according to Common Terminology Criteria of Adverse Events version 3.0, and efficacy was evaluated according to the Response Evaluation Criteria in Solid Tumors version 1.0 for patients with measurable lesions and by the criteria of the Japanese Research Society for Gastric Cancer for all patients. RESULTS: Efficacy could be evaluated in 49 of the 50 enrolled patients. The median age was 62 years. Lesions were measurable in 38 patients and assessable in 11. The response rate was 44.7% in patients with measurable lesions and 40.8% overall. The progression-free survival and overall survival were, respectively, 233 days (7.8 months) and 574 days (19.0 months) in patients with measurable lesions and 192 days (6.4 months) and 402 days (13.4 months) overall. Serious adverse events (grade 3 or higher) included neutropenia (24.5%), anemia (20.4%), and anorexia (20.4%) and were safely managed. CONCLUSION: The safety and effectiveness of sequential treatment with S-1 followed by cisplatin every 35 days is equivalent to that reported for conventional chemotherapeutic regimens in patients with advanced or recurrent gastric cancer.

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 m(2) 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/m(2) and the RD was determined to be 80 mg/m(2) of irinotecan combined with 80 mg/m(2) 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.

Prediction of myocardial infarction using coronary risk scores among Japanese male workers: 3M Study.

BACKGROUND: It remains unclear how much coronary risk factors contribute to the prediction of myocardial infarction among Japanese populations. METHODS AND RESULTS: A nested case-control study of Japanese male workers aged 35 to 65 years was conducted between 1997 and 2000 in the Morbidity of Myocardial Infarction Multicenter Study in Japan (3M Study). Two hundred four myocardial infarctions were identified and two controls per case were selected by matching for age. We calculated odds ratios using a conditional logistic regression model, and constructed risk predictive models for the risk of myocardial infarction using coronary risk factors. The multivariable odds ratios (95 percent confidence intervals) of myocardial infarction were 2.02 (1.29-3.16) for high blood pressure, 2.33 (1.51-3.59) for high LDL-cholesterol, 4.16 (2.36-7.33) for low HDL-cholesterol, 1.49 (0.94-2.35) for high triglycerides, 1.46 (0.89-2.39) for high glucose, and 2.95 (1.90-4.59) for current smoking. A large reduction of the predictive value for myocardial infarction was shown after exclusion of high LDL-cholesterol (reduction of predictive value was -3.4%), further exclusion of low HDL-cholesterol (-7.1%), and further exclusion of current smoking (-16.4%). CONCLUSIONS: High LDL-cholesterol and low HDL-cholesterol as well as current smoking had high predictive values for myocardial infarction among Japanese middle-aged male workers.

Irinotecan-based combination chemotherapy for metastatic small intestinal adenocarcinoma.

Metastatic small intestinal adenocarcinoma (SIA) is rare among digestive tract malignancies, and a standard therapy has yet to be established. The present study described a patient who was treated with irinotecan-based chemotherapy. A 67-year-old woman with a long history of anemia was diagnosed as having SIA using small bowel endoscopy. Tumor invasion of the mesentery and multiple metastases to the lungs and peritoneal lymph nodes were detected. Nine courses of chemotherapy, each consisting of bolus infusion of 5-fluorouracil at 500 mg/m(2), plus infusion of irinotecan at 100 mg/m(2) with l-leucovorin at 20 mg/m(2) on days 1, 8 and 15, were administered at 4-weekly intervals postoperatively. After two courses, the metastatic nodules in the lungs showed a decrease in number and size, and this response continued for over 6 months. Adverse events were manageable during this period. The patient succumbed to the disease 12 months after the initial diagnosis. The present results therefore suggest that irinotecan-based chemotherapy is a potential treatment for metastatic SIA.

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

BACKGROUND: Oxaliplatin 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. METHODS: In order to clarify the clinical features of allergic reactions to oxaliplatin, we retrospectively investigated CRC patients who had received oxaliplatin-based chemotherapies. RESULTS: One 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. CONCLUSION: Allergic 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.

Nonmyeloablative allogeneic hematopoietic stem cell transplantation as immunotherapy for pancreatic cancer.

OBJECTIVE: Advanced unresectable pancreatic cancer has an extremely poor prognosis despite intensive chemotherapy. As a new therapeutic modality, we investigated nonmyeloablative allogeneic hematopoietic stem cell transplantation from a related donor. METHODS: Five patients with chemotherapy-resistant pancreatic cancer received allogeneic peripheral blood stem cell transplantation after a conditioning regimen consisting of low-dose total body irradiation and fludarabine. The prophylaxis for graft-versus-host disease consisted of mycophenolate mofetil and cyclosporine. RESULTS: The median age of the 5 patients was 54 years, and the median duration from diagnosis to nonmyeloablative allogeneic hematopoietic stem cell transplantation was 10 months. Three of the 5 patients achieved complete donor chimerism of peripheral T cells, at a median time of day 42. Acute graft-versus-host disease developed in 3 patients: grade 2 in 2 patients and grade 1 in 1. Tumor reduction was observed in 2 patients: 1 patient showed disappearance of the pancreatic tumor, and the other patient showed approximately 20% reduction of the tumor. Marked elevation of tumor necrosis factor alpha was observed as the tumor regressed. CONCLUSIONS: Although advanced pancreatic cancer progresses rapidly, some graft-versus-tumor effects and pivotal role of tumor necrosis factor alpha were suggested. To obtain the durable response, patient selection and new strategies become important.

Phase I study of the sequential administration of S-1 and cisplatin for metastatic gastric cancer.

The combination of 5-fluorouracil (5-FU) and cisplatin (CDDP) has been reported to be active against metastatic gastric cancer (MGC) and great synergy has been shown in vivo and in vitro when 5-FU precedes CDDP. The sequential combination of S-1 (tegafur, oxonic acid, 5-chloro-2,4-dihydroxypyridine) followed by CDDP for MGC was investigated. A phase I trial applying increasing doses of oral administration of S-1 (65-80 mg/m(2)) for 21 days and increasing doses of CDDP (60-80 mg/m(2)) on day 22 every 35 days was conducted in order to determine the maximum tolerated dose (MTD) and recommended phase II dose. Patients with metastatic or recurrent gastric cancer, no prior chemotherapy, measurable disease, ECOG performance status less than 3 and adequate organ functions were eligible for the study. Three patients were treated at each dose level with escalation based on toxicity. Fifteen patients were included and evaluated for dose-limiting toxicity (DLT) and MTD. DLT included NCICTC grade 3 anorexia and fatigue in patients treated at S-1 80 mg/m(2) and CDDP 80 mg/m(2) (dose level 5). The other toxicities, grade 3 or higher, included neutropenia (grade 3) and nausea/vomiting (grade 3). Non-hematological toxicities were grade 1/2 and included diarrhea, nausea and stomatitis. There was no treatment-related mortality. Therefore, the recommended dose was a combination of S-1 at 80 mg/m(2) and CDDP at 70 mg/m(2). This sequential administration of S-1 and CDDP every 35 days is tolerable and warrants a phase II trial. A multicenter phase II study is currently under way.

Schedule-dependent synergistic interaction between gemcitabine and oxaliplatin in human gallbladder adenocarcinoma cell lines.

To define the most effective combination schedule of gemcitabine and oxaliplatin (L-OHP), we investigated the in-vitro interaction between these drugs in a panel of four human gallbladder adenocarcinoma cell lines (HAG-1, GB-d1, NOZ, and G-415). Cytotoxic activity was determined by the WST-1 assay. Different 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. Simultaneous and sequential treatments of gemcitabine followed by L-OHP exhibited synergistic effects in all four cell lines, whereas the reverse sequence largely showed an antagonism. Gemcitabine exclusively arrested cells at the G0/G1 phase, and L-OHP at the G2/M phase, as measured by flow cytometric analyses. Apoptosis was most prominent when cells were treated simultaneously or in a sequence gemcitabine followed by L-OHP, producing apoptosis in treated cells (27-30%). In contrast, the reverse sequence yielded only 6-7% induction of apoptosis, the rate being not significantly different from those induced by each drug singly. Moreover, this sequence dependence was further confirmed by the experiment, which compared the number of HAG-1 cells 7 days after these combination schedules. These findings suggest that the interaction of gemcitabine and L-OHP is highly schedule dependent, with the most efficacious interaction observed in either simultaneous combination or in a sequence combination of gemcitabine followed by L-OHP.