Hiroshi Ariyama

Mice That Express Human Interleukin-8 Have Increased Mobilization of Immature Myeloid Cells, Which Exacerbates Inflammation and Accelerates Colon Carcinogenesis

BACKGROUND & AIMS Interleukin (IL)-8 has an important role in initiating inflammation in humans, attracting immune cells such as neutrophils through their receptors CXCR1 and CXCR2. IL-8 has been proposed to contribute to chronic inflammation and cancer. However, mice do not have the IL-8 gene, so human cancer cell lines and xenograft studies have been used to study the role of IL-8 in colon and gastric carcinogenesis. We generated mice that carry a bacterial artificial chromosome that encompasses the entire human IL-8 gene, including its regulatory elements (IL-8Tg mice). METHODS We studied the effects of IL-8 expression in APCmin(+/-) mice and IL-8Tg mice given azoxymethane and dextran sodium sulfate (DSS). We also examined the effects of IL-8 expression in gastric cancer in INS-GAS mice that overexpress gastrin and IL-8Tg mice infected with Helicobacter felis. RESULTS In IL-8Tg mice, expression of human IL-8 was controlled by its own regulatory elements, with virtually no messenger RNA or protein detectable under basal conditions. IL-8 was strongly up-regulated on systemic or local inflammatory stimulation, increasing mobilization of immature CD11b(+)Gr-1(+) myeloid cells (IMCs) with thioglycolate-induced peritonitis, DSS-induced colitis, and H. felis-induced gastritis. IL-8 was increased in colorectal tumors from patients and IL-8Tg mice compared with nontumor tissues. IL-8Tg mice developed more tumors than wild-type mice following administration of azoxymethane and DSS. Expression of IL-8 increased tumorigenesis in APCmin(+/-) mice compared with APCmin(+/-) mice that lack IL-8; this was associated with increased numbers of IMCs and angiogenesis in the tumors. CONCLUSIONS IL-8 contributes to gastrointestinal carcinogenesis by mobilizing IMCs and might be a therapeutic target for gastrointestinal cancers.

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.

Activated Src and Ras induce gefitinib resistance by activation of signaling pathways downstream of epidermal growth factor receptor in human gallbladder adenocarcinoma cells

Purpose: Although gefitinib, a selective inhibitor of epidermal growth factor receptor (EGFR) tyrosine kinase, has been demonstrated to exhibit its antitumor activity by the blockade of EGF receptor, the role of signaling pathways downstream of EGFR in gefitinib sensitivity remains unknown. In this study, we investigated the mechanistic role of Src and Ras, major oncogene products implicated in the pathogenesis of many human cancers in gefitinib sensitivity. Methods: Using parental and v-src- or c-H-ras-transfected HAG-1 human gallbladder adenocarcinoma cell lines, effects of gefitinib on cytotoxicity, cell cycle purtubation and apoptosis, and tyrosine phosphorylation of EGFR, Akt, and Erk were determined by WST-1 assay, flow cytometry, and Western blots, respectively. Results: Activated Ras and Src conferred a strong resistance to gefitinib by nearly 30-fold and 200-fold, respectively. Gefitinib induced accumulation of cells in the G0/G1 phase of the cell cycle at 24-h, with progressive expansion of apoptotic cell population in parental HAG-1 cells, but these effects were completely abolished in v-src- or c-H-ras-transfected cell line. Upon gefitinib treatment, EGFR activation and subsequent downstream activation through Erk and Akt were significantly inhibited in HAG-1 cells. By contrast, gefinitib failed to inhibit the activation of both Akt and Erk in v-src-transfected cells and Erk, but not Akt in c-H-ras-transfected cells, despite the blockade of EGFR activation in these respective cell lines. Treatment of v-src-transfected cells with herbimycin A, a Src tyrosine kinase inhibitor, partially reversed the gefitinib resistance, with concomitant inhibition of Akt and Erk. Conclusion: Our results suggest that activated Ras and Src could induce gefitinib resistance by activating either or both of Akt and Erk signaling pathways, thus providing a strategic rationale for assessment of these specific signaling molecules downstream of EGFR to customize treatment.

CCK2R identifies and regulates gastric antral stem cell states and carcinogenesis

Objective Progastrin is the incompletely cleaved precursor of gastrin that is secreted by G-cells in the gastric antrum. Both gastrin and progastrin bind to the CCK2 receptor (Cckbr or CCK2R) expressed on a subset of gastric epithelial cells. Little is known about how gastrin peptides and CCK2R regulate gastric stem cells and carcinogenesis. Interconversion among progenitors in the intestine is documented, but the mechanisms by which this occurs are poorly defined. Design We generated CCK2R-CreERT mice and performed inducible lineage tracing experiments. CCK2R+ antral cells and Lgr5+ antral stem cells were cultured in a three-dimensional in vitro system. We crossed progastrin-overexpressing mice with Lgr5-GFP-CreERT mice and examined the role of progastrin and CCK2R in Lgr5+ stem cells during MNU-induced carcinogenesis. Results Through lineage tracing experiments, we found that CCK2R defines antral stem cells at position +4, which overlapped with an Lgr5neg or low cell population but was distinct from typical antral Lgr5high stem cells. Treatment with progastrin interconverts Lgr5neg or low CCK2R+ cells into Lgr5high cells, increases CCK2R+ cell numbers and promotes gland fission and carcinogenesis in response to the chemical carcinogen MNU. Pharmacological inhibition or genetic ablation of CCK2R attenuated progastrin-dependent stem cell expansion and carcinogenesis. Conclusions CCK2R labels +4 antral stem cells that can be activated and expanded by progastrin, thus identifying one hormonal trigger for gastric stem cell interconversion and a potential target for gastric cancer chemoprevention and therapy.

Intracellular signal transduction of interferon on the suppression of haematopoietic progenitor cell growth

Summary. Interferon (IFN)‐α and IFN‐γ suppress the growth of haematopoietic progenitor cells. IFN‐α activates Janus kinase‐1 (Jak1) and Tyrosine kinase‐2 (Tyk2), followed by the phosphorylation of the signal transducers and activators of transcription, Stat1 and Stat2. IFN‐γ activates Jak1 and Jak2, followed by the activation of Stat1. Activated Stats bind the promoter regions of IFN‐inducible genes. We evaluated the role of Tyk2 and Stat1 in the IFN‐mediated inhibition of haematopoietic progenitor cell growth. While IFN‐α (1000 U/ml) suppressed the number of granulocyte‐macrophage colony‐forming units (CFU‐GM) or erythroid burst‐forming units (BFU‐E) from wild‐type mouse bone marrow cells, this suppression was partially inhibited by a deficiency in Tyk2 and completely inhibited by a deficiency in Stat1. High levels of IFN‐α (10 000 U/ml) suppressed the CFU‐GM or BFU‐E obtained from Stat1‐deficient mice, but did not suppress this growth in cells from Tyk2‐deficient mice. Stat1 was phosphorylated by IFN‐α in Tyk2‐deficient cells, although the level of phosphorylation was weaker than that observed in wild type mice. Thus, the inhibitory signal on haematopoietic progenitor cells mediated by IFN‐α may be transduced by two signalling pathways, one regulated by Tyk2 and the other dependent on Stat1. IFN‐γ also suppressed the number of CFU‐GM or BFU‐E, and this pathway was mediated by IFN‐γ in a Stat1‐dependent manner, independently of Tyk2.

Burden on Oncologists When Communicating the Discontinuation of Anticancer Treatment

Objective Communicating the discontinuation of anticancer treatment to patients is a difficult task. The primary aim of this study was to clarify the level of oncologist-reported burden when communicating about discontinuation of an anticancer treatment. The secondary aims were (i) to identify the sources of burden contributing to their levels and (ii) to explore the useful strategies to alleviate their burden. Methods A multicenter nationwide questionnaire survey was conducted on 620 oncologists across Japan (response rate, 67%). Results High levels of perceived burden were reported by 47% of respondents, and 17% reported that they sometimes, often or always wanted to stop oncology work because of this burden. There was a significant association between high levels of burden and: a feeling that breaking bad news would deprive the patient of hope; concern that the patients family would blame the oncologist; concern that the patient may lose self-control; and a feeling that there was not enough time to break the bad news. Strategies perceived to be useful by oncologists included training in how to effectively communicate to patients discontinuation of anticancer treatment, a reduction in total workload to allow sufficient time to break bad news, and development of a multidisciplinary model to facilitate cooperation with other professionals and facilities. Conclusions Many oncologists reported high levels of burden relating to communication of discontinuation of anticancer treatment. A specific communication skills training program, sufficient time for communication and development of a multidisciplinary model could help alleviate the burden on oncologists.

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.

Phase I Study to Assess the Safety, Tolerability and Pharmacokinetics of AZD4877 in Japanese Patients with Solid Tumors

Introduction AZD4877 is a potent Eg5 inhibitor that has been shown to have an acceptable tolerability profile in a Phase I study of Western patients with solid tumors. This study was conducted to evaluate the safety, pharmacokinetic (PK) profile, maximum tolerated dose (MTD) and efficacy of AZD4877 in a Japanese population with solid tumors. Methods In this Phase I, open-label, dose-escalation study, AZD4877 (10, 15, 20 or 25 mg) was administered as a 1-hour intravenous infusion on days 1, 8 and 15 of repeated 28-day cycles to Japanese patients with advanced solid tumors. Adverse events (AEs) were evaluated according to Common Terminology Criteria for Adverse Events (CTCAE) version 3.0. PK variables were assessed pre- and post dosing. The MTD of AZD4877 was determined by evaluating dose-limiting toxicities (DLTs). Efficacy was evaluated by assessing best response according to Response Evaluation Criteria In Solid Tumors version 1.0. Results Of the 21 patients enrolled, 18 received at least one dose of AZD4877 (N = 3 in both the 10 and 15 mg cohorts, N = 6 in both the 20 and 25 mg cohorts). The most commonly reported AEs were fatigue and nausea (39% of patients each). One patient in each of the 20 and 25 mg cohorts experienced a DLT (neutropenia and febrile neutropenia). Dose escalation was halted at 25 mg and the MTD was not defined in this population. CTCAE grade ≥3 abnormal laboratory findings/vital signs were reported in 12 patients, with neutropenia (56%) and leukopenia (44%) being the most commonly reported. Exposure to AZD4877 was not fully dose proportional and AZD4877 clearance and elimination half-life appeared independent of dose. The best response to AZD4877 was stable disease in five of 16 evaluable patients. Conclusion AZD4877 up to doses of 25 mg was well tolerated in Japanese patients. There was little evidence of clinical efficacy.