Hirotaka Hoshi

Tumor-associated MUC5AC stimulates in vivo tumorigenicity of human pancreatic cancer

MUC5AC, a high molecular weight glycoprotein, is overexpressed in the ductal region of human pancreatic cancer but is not detectable in the normal pancreas, suggesting its association with disease development. In the present study, we investigated the in vitro and in vivo effects of MUC5AC knockdown by short interfering RNA (siRNA) in the MUC5AC-overexpressing SW1990 and BxPC3 human pancreatic cancer cell lines in order to clarify its function. Significant decreases in the expression levels of MUC5AC mRNA and protein were observed in SW1990 and BxPC3 cells that had been stably transfected with a MUC5AC siRNA expression vector (SW1990/si-MUC5AC and BxPC3/si-MUC5AC cells) compared to those in cells transfected with an si-mock vector (SW1990/si-mock and BxPC3/si-mock cells). In in vitro studies, neither type of MUC5AC-knockdown cell showed any difference in cell survival, proliferation, or morphology from the si-mock cells or parental cells. However, in vivo xenograft studies demonstrated that MUC5AC knockdown significantly reduced the tumorigenicity and suppressed the tumor growth of si-MUC5AC cells compared to those of the si-mock cells. Immunohistochemical analysis revealed that CD45R/B220+ and Gr-1+ cells had infiltrated into the tumor tissue of the SW1990/si-MUC5AC cells. Furthermore, cancer-associated antigen specific antibodies were detected at high levels in the sera from the SW1990/si-MUC5AC cell-bearing mice. These results suggest that tumor-associated MUC5AC expressed on the surface of pancreatic cancer cells supports the escape of pancreatic cancer cells from immunosurveillance. The present findings highlight a new dimension of MUC5AC as a functional immunosuppressive agent and its important role in pancreatic cancer progression.

Absorption and Tissue Distribution of an Immunomodulatory α-D-Glucan after Oral Administration of Tricholoma matsutake

Alpha-D-glucan (MPG-1) separated from Tricholoma matsutake (CM6271) has been reported to show immunomodulatory activities. In this study, the plasma concentration and tissue distribution of MPG-1 after CM6271 oral administration were investigated as part of the action mechanism analysis. When CM6271 was orally administered in a single dose to mice, MPG-1 was absorbed via the intestinal tract, appeared in plasma after 16 h, was gradually excreted from the blood, and fell to background level after 48 h. The time course analysis of MPG-1 in plasma showed the following pharmacokinetic parameters of MPG-1: tmax = 24 h; Cmax = 161.1 ng/mL; AUC(0-infinity) = 2559.7 ng x h/mL. Moreover, MPG-1 was confirmed to localize in Peyers patches, mesenteric lymph nodes (MLN), and the spleen and to promote IL-12 p70 production and NK cell activity. These results suggest that MPG-1 stimulated the intestinal immune system through Peyers patches; moreover, it was taken into the blood and stimulated the systemic immune system.

Inhibition of Decrease in Natural Killer Cell Activity in Repeatedly Restraint-Stressed Mice by a Biological Response Modifier Derived from Cultured Mycelia of the Basidiomycete Tricholoma matsutake

Objective: To develop a method to cope with stress-induced reduction in immunocompetence, we evaluated the immunomodulatory activities of a biological response modifier derived from the mycelia of the basidiomycete Tricholoma matsutake (CM6271) in mice under repeated restraint stress. Methods: C57BL/6 mice were inserted, one per tube, into 50-ml polypropylene tubes into which more than 30 ventilation holes had been drilled, and were restrained everyday for 20 days in this fashion for set periods of time. Natural killer (NK) cell activity and NK1.1-positive cell counts in the spleen, ACTH and corticosterone levels in the blood were determined. CM6271 was orally administered daily during the restraint stress period. Results: (1) When the mice were restrained in a confined space for 6 h per day for 20 days, the NK cell activity and the NK1.1-positive cell counts in the spleen significantly decreased after day 5 with an increase in the blood ACTH and corticosterone levels. (2) Oral administration of CM6271 during the restraint stress period significantly prevented the stress-induced decrease in NK cell activity. The effect was dependent on the timing, duration, and doses administered. (3) CM6271 did not significantly affect the splenic NK1.1-positive cell counts or the levels of blood ACTH and corticosterone in restraint-stressed mice. Conclusion: The above findings suggest that CM6271 inhibits the restraint stress-induced decrease of NK cell activity in a timing of administration and dose-dependent manner.

MUC5AC protects pancreatic cancer cells from TRAIL-induced death pathways

We have previously reported that a specific siRNA transfected MUC5AC could knockdown MUC5AC expression and suppress in vivo tumor growth and metastasis, although it had no effects on in vitro cell growth, cell survival, proliferation and morphology. In the present study, we investigated which host immune cells induced these effects and how the effects were induced using immunocyte-depleted animal models. The tumor growth of SW1990/si-MUC5AC cells, which show no tumor growth when implanted subcutaneously into a nude mouse, was recovered when neutrophils were removed by anti-Gr-1 mAb administration. This result suggests that MUC5AC may suppress the antitumor effects of neutrophils by allowing tumor cells to escape the host immune system. Subsequently, we investigated the effects of MUC5AC on apoptosis induction mediated by TNF-related apoptosis-inducing ligand (TRAIL), one of the antitumor mechanisms of neutrophils. SW1990/si-MUC5AC cells showed significantly increased active caspase 3 expression after the addition of TRAIL. On the other hand, SW1990/si-mock cells showed no such changes. Our results indicate that MUC5AC inhibits TRAIL‑induced apoptosis in human pancreatic cancer and may serve as an important indicator in diagnosis and prognosis.

Anti-protein-bound polysaccharide-K monoclonal antibody binds the active structure and neutralizes direct antitumor action of the compound

Protein-bound polysaccharide K (PSK) is extracted and purified from Coriolus versicolor (CM101), and is used as an anti-cancer agent. In this study, focusing on the direct actions of PSK, we investigated whether PSK reaches tumor and immune tissues with its active structure remaining intact, and the direct action of PSK was evaluated by its antitumor effects against MethA fibrosarcomas implanted in immunodeficient NOD/SCID mice. The results obtained suggest that PSK reaches the tumor tissue in its active form and exhibits antitumor effects against MethA cells.

Construction of a novel cell-based assay for the evaluation of anti-EGFR drug efficacy against EGFR mutation

Epidermal growth factor receptor (EGFR) overexpression and EGFR-mediated signaling pathway dysregulation have been observed in tumors from patients with various cancers, especially non-small cell lung cancer. Thus, several anti-EGFR drugs have been developed for cancer therapy. For patients with known EGFR activating mutations (EGFR exon 19 in-frame deletions and exon 21 L858R substitution), treatment with an EGFR tyrosine kinase inhibitor (EGFR TKI; gefitinib, erlotinib or afatinib) represents standard first-line therapy. However, the clinical efficacy of these TKIs is ultimately limited by the development of acquired drug resistance such as by mutation of the gatekeeper T790 residue (T790M). To overcome this acquired drug resistance and develop novel anti-EGFR drugs, a cell-based assay system for EGFR TKI resistance mutant-selective inhibitors is required. We constructed a novel cell-based assay for the evaluation of EGFR TKI efficacy against EGFR mutation. To this end, we established non-tumorigenic immortalized breast epithelial cells that proliferate dependent on EGF (MCF 10A cells), which stably overexpress mutant EGFR. We found that the cells expressing EGFR containing the T790M mutation showed higher resistance against gefitinib, erlotinib and afatinib compared with cells expressing wild-type EGFR. In contrast, L858R mutant-expressing cells exhibited higher TKI sensitivity. The effect of T790M-selective inhibitors (osimertinib and rociletinib) on T790M mutant-expressing cells was significantly higher than gefitinib and erlotinib. Finally, when compared with commercially available isogenic MCF 10A cell lines carrying introduced mutations in EGFR, our EGFR mutant-overexpressing cells exhibited obviously higher responsiveness to EGFR TKIs depending on the underlying mutations because of the higher levels of EGFR phosphorylation in the EGFR mutant-overexpressing cells than in the isogenic cell lines. In conclusion, we successfully developed a novel cell-based assay for evaluating the efficacy of anti-EGFR drugs against EGFR mutation.

Evaluation of anticancer agents using patient-derived tumor organoids characteristically similar to source tissues

Patient-derived tumor xenograft models represent a promising preclinical cancer model that better replicates disease, compared with traditional cell culture; however, their use is low-throughput and costly. To overcome this limitation, patient-derived tumor organoids (PDOs) were established from human lung, ovarian and uterine tumor tissues, among others, to accurately and efficiently recapitulate the tissue architecture and function. PDOs were able to be cultured for >6 months, and formed cell clusters with similar morphologies to their source tumors. Comparative histological and comprehensive gene expression analyses proved that the characteristics of PDOs were similar to those of their source tumors, even following long-term expansion in culture. At present, 53 PDOs have been established by the Fukushima Translational Research Project, and were designated as Fukushima PDOs (F-PDOs). In addition, the in vivo tumorigenesis of certain F-PDOs was confirmed using a xenograft model. The present study represents a detailed analysis of three F-PDOs (termed REME9, 11 and 16) established from endometrial cancer tissues. These were used for cell growth inhibition experiments using anticancer agents. A suitable high-throughput assay system, with 96- or 384-well plates, was designed for each F-PDO, and the efficacy of the anticancer agents was subsequently evaluated. REME9 and 11 exhibited distinct responses and increased resistance to the drugs, as compared with conventional cancer cell lines (AN3 CA and RL95-2). REME9 and 11, which were established from tumors that originated in patients who did not respond to paclitaxel and carboplatin (the standard chemotherapy for endometrial cancer), exhibited high resistance (half-maximal inhibitory concentration >10 µM) to the two agents. Therefore, assay systems using F-PDOs may be utilized to evaluate anticancer agents using conditions that better reflect clinical conditions, compared with conventional methods using cancer cell lines, and to discover markers that identify the pharmacological effects of anticancer agents.

Evaluation system for arrhythmogenic potential of drugs using human-induced pluripotent stem cell-derived cardiomyocytes and gene expression analysis

In recent years, human-induced pluripotent stem cell-derived cardiomyocytes (hiPS-CMs) have been widely used to develop evaluation systems for drug cardiotoxicity, including the arrhythmia caused by QT prolongation. To accurately assess the arrhythmogenic potential of drugs, associated with QT prolongation, we developed an evaluation system using hiPS-CMs and gene expression analysis. hiPS-CMs were treated with 8 arrhythmogenic and 17 non-arrhythmogenic drugs at several concentrations for 24 hr to comprehensively analyze gene expression. The results showed that 19 genes were upregulated in the arrhythmogenic drug-treated cells compared with their expression levels in the non-treated and non-arrhythmogenic drug-treated cells. The arrhythmogenic risks of the drugs were evaluated by scoring gene expression levels. The results indicated that arrhythmogenic risks could be inferred when cells were treated at a concentration 100 times higher than the maximum blood concentration of the drug. Thus, we succeeded in developing a system for evaluation of the arrhythmogenic potential of drugs using gene expression analysis.