Polysaccharides derived from Ganoderma lucidum fungus mycelia ameliorate indomethacin-induced small intestinal injury via induction of GM-CSF from macrophages.

Non-steroidal anti-inflammatory drugs often cause ulcers in the human small intestine, but few effective agents exist to treat such injury. Ganoderma lucidum Karst, also known as "Reishi" or "Lingzhi", is a mushroom. We previously reported that a water-soluble extract from G. lucidum fungus mycelia (MAK) has anti-inflammatory effects in murine colitis induced by trinitrobenzene sulfonic acid, and induction of granulocyte macrophage colony-stimulating factor (GM-CSF) by MAK may provide anti-inflammatory effects. However, its effects on indomethacin-induced small intestinal injuries are unknown. The present study investigated the preventative effects of MAK via immunological function and the polysaccharides from MAK on indomethacin-induced ileitis in mice. Peritoneal macrophages (PMs) were stimulated in vitro with MAK and adoptively transferred to C57BL/6 mice intraperitoneally, which were then given indomethacin. Intestinal inflammation was evaluated after 24h. We performed in vivo antibody blockade to investigate the preventive role of GM-CSF, which derived from PMs stimulated with MAK. We then used PMs stimulated with MAK pre-treated by pectinase in an adoptive transfer assay to determine the preventive role of polysaccharides. Indomethacin-induced small intestinal injury was inhibited by adoptive transfer of PMs stimulated in vitro with MAK. In this transfer model, pre-treatment with anti-GM-CSF antibody but not with control antibody reversed the improvement of small intestinal inflammation by indomethacin. Pectinase pretreatment impaired the anti-inflammatory effect of MAK. PMs stimulated by MAK appear to contribute to the anti-inflammatory response through GM-CSF in small intestinal injury induced by indomethacin. The polysaccharides may be the components that elicit the anti-inflammatory effect.

Multikinase inhibitor regorafenib inhibits the growth and metastasis of colon cancer with abundant stroma.

Interaction between tumor cells and stromal cells plays an important role in the growth and metastasis of colon cancer. We previously found that carcinoma-associated fibroblasts (CAFs) expressed platelet-derived growth factor receptor-ß (PDGFR-ß) and that PDGFR targeted therapy using imatinib or nilotinib inhibited stromal reaction. Bone marrow-derived mesenchymal stem cells (MSCs) migrate to tumor stroma and differentiate into CAFs. A novel oral multikinase inhibitor regorafenib inhibits receptor tyrosine kinases expressed on stromal cells (vascular endothelial growth factor receptor 1-3, TIE2, PDGFR-ß, and fibroblast growth factors) and tumor cells (c-KIT, RET, and BRAF). These molecules are involved in tumor growth, angiogenesis, lymphangiogenesis, and stromal activation. Therefore, we examined whether regorafenib impaired the tumor-promoting effect of CAFs/MSCs. KM12SM human colon cancer cells alone or KM12SM cells with MSCs were transplanted into the cecal wall of nude mice. Co-implantation of KM12SM cells with MSCs into the cecal wall of nude mice produced tumors with abundant stromal component and promoted tumor growth and lymph node metastasis. Single treatment with regorafenib inhibited tumor growth and metastasis by inhibiting both tumor cells and stromal reaction. This tumor-inhibitory effect of regorafenib was more obvious in tumors developed by co-implanting KM12SM cells with MSCs. Our data suggested that targeting of the tumor microenvironment with regorafenib affected tumor cell-MSC interaction, which in turn inhibited the growth and metastasis of colon cancer.

mTOR and PDGF pathway blockade inhibits liver metastasis of colorectal cancer by modulating the tumor microenvironment.

Tumor growth and metastasis are not determined by cancer cells alone but also by a variety of stromal cells, and platelet-derived growth factor receptors (PDGF-Rs) are overexpressed by various stromal cell populations. Activation of PI3K-AKT-mTOR signaling is frequently observed in many cancer types. We investigated whether the mTOR inhibitor everolimus, alone or in combination with the PDGF-R tyrosine kinase inhibitor nilotinib, can inhibit growth and metastasis of human colon cancer. The effects of nilotinib and everolimus on tumor growth and metastasis were examined in an orthotopic mouse model of human colon cancer and a model of liver metastasis. After treatment with nilotinib (versus distilled water), the stromal reaction of xenografts growing in the cecal wall and liver was significantly decreased. After treatment with everolimus, the stromal reaction did not decrease, but tumor cell proliferation and microvessel density decreased. With the two drugs in combination, both stromal reaction and tumor cell proliferation decreased and apoptosis of tumor cells increased, resulting in remarkable inhibition of tumor growth at both the orthotopic and the metastatic site. Concurrent inhibition of tumor cells and activated stromal cells by a PDGF-R tyrosine kinase inhibitor and an mTOR inhibitor used in combination may represent a novel therapeutic approach for colorectal cancer.

Crucial involvement of the CCL3-CCR5 axis-mediated fibroblast accumulation in colitis-associated carcinogenesis in mice.

Patients with inflammatory bowel diseases often develop colon carcinoma. Combined treatment of azoxymethane (AOM) and dextran sulfate sodium (DSS) recapitulates colitis-associated cancer in mice. AOM/DSS-induced tumor formation was reduced in CCL3- or its specific receptor, CCR5-deficient mice despite the presence of a massive infiltration of inflammatory cells. However, AOM/DSS-induced type I collagen-positive fibroblast accumulation in the colon was reduced in CCL3- or CCR5-deficient mice. This was associated with depressed expression of heparin-binding epidermal growth factor-like growth factor (HB-EGF), which is expressed mainly by fibroblasts. Moreover in vitro, CCL3 induced fibroblasts to proliferate and to enhance HB-EGF expression. Furthermore, CCR5 blockade reduced tumor formation together with reduced fibroblast accumulation and HB-EGF expression, even when administered after the development of multiple colon tumors. Thus, CCL3-CCR5-mediated fibroblast accumulation may be required, in addition to leukocyte infiltration, to induce full-blown colitis-associated carcinogenesis. Our studies shed light on a therapeutic potential of CCR5 antagonist for patients with colitis-associated cancer.

Stroma-directed imatinib therapy impairs the tumor-promoting effect of bone marrow-derived mesenchymal stem cells in an orthotopic transplantation model of colon cancer.

Bone marrow-derived mesenchymal stem cells (MSCs) are reported to contribute to formation of tumor-promoting stromal cells. We reported recently that, in an orthotopic nude mice model of colon cancer, MSCs traveled to tumor stroma, where they differentiated into carcinoma-associated fibroblast (CAF)-like cells. We also found that CAFs express platelet-derived growth factor receptor (PDGFR) at a high level and that imatinib therapy targeting PDGFR in CAFs inhibits growth and metastasis of human colon cancer. These findings led us to examine whether the tumor-promoting effect of MSCs is impaired by blockade of PDGFR signaling achieved with imatinib. Orthotopic transplantation and splenic injection of human MSCs along with KM12SM human colon cancer cells, in comparison with transplantation of KM12SM cells alone, resulted in significantly greater promotion of tumor growth and liver metastasis. The KM12SM + MSC xenograft enhanced cell proliferation and angiogenesis and inhibited tumor cell apoptosis. When tumor-bearing animals were treated with imatinib, there was no significant increase in primary tumor volume or total volume of liver metastases, despite the KM12SM+MSC xenograft, and survival in the mixed-cell group was prolonged by imatinib treatment. Moreover, the ability of MSCs to migrate to tumor stroma was impaired, and the number of MSCs surviving in the tumor microenvironment was significantly decreased. In in vitro experiments, treatment with imatinib inhibited migration of MSCs. Our data suggest that blockade of PDGF signaling pathways influences the interaction between bone marrow-derived MSCs and tumor cells in the tumor microenvironment and, hence, inhibits the progressive growth of colon cancer.

Anti-stromal therapy with imatinib inhibits growth and metastasis of gastric carcinoma in an orthotopic nude mouse model.

Recent studies have revealed that platelet-derived growth factor (PDGF) plays a role in promoting progressive tumor growth in several organs; however, whether PDGF plays such a role in gastric carcinoma is undetermined. We examined whether inhibition of PDGF receptor (PDGF-R) tyrosine kinase signaling by imatinib affects tumor growth and metastasis in an orthotopic nude mouse model of human gastric carcinoma. TMK-1 human gastric carcinoma cells were injected into the gastric wall of nude mice. Groups of mice (n = 10 each) received sterile water (control), low-dose imatinib (50 mg/kg/day), high-dose imatinib (200 mg/kg/day), cancer chemotherapeutic agent irinotecan (5 mg/kg/week), or imatinib (50 mg/kg/day or 200 mg/kg/day) and irinotecan (5 mg/kg/week) in combination for 28 days. Tumor growth and metastasis were assessed. Resected tumors were analyzed immunohistochemically. Carcinoma-associated fibroblasts, pericytes and lymphatic endothelial cells in stroma expressed high levels of PDGF-R; carcinoma cells did not. Treatment with imatinib alone did not inhibit tumor growth and metastasis; however, treatment with irinotecan alone or combined with imatinib significantly inhibited tumor growth. Only treatment with high-dose imatinib and irinotecan in combination inhibited lymph node and peritoneal metastases. Immunohistochemically, only imatinib alone or in combination with irinotecan was shown to significantly decrease the stromal reaction, microvessel area and pericyte coverage of tumor microvessels. These effects were marked with high-dose imatinib. In conclusion, administration of PDGF-R tyrosine kinase inhibitor in combination with irinotecan appears to impair the progressive growth of gastric carcinoma by blockade of PDGF-R signaling pathways in stromal cells.

Mesenchymal stem cells enhance growth and metastasis of colon cancer.

Recently, mesenchymal stem cells (MSCs) were reported to migrate to tumor stroma as well as injured tissue. We examined the role of human MSCs in tumor stroma using an orthotopic nude mice model of KM12SM colon cancer. In in vivo experiments, systemically injected MSCs migrated to the stroma of orthotopic colon tumors and metastatic liver tumors. Orthotopic transplantation of KM12SM cells mixed with MSCs resulted in greater tumor weight than did transplantation of KM12SM cells alone. The survival rate was significantly lower in the mixed-cell group, and liver metastasis was seen only in this group. Moreover, tumors resulting from transplantation of mixed cells had a significantly higher proliferating cell nuclear antigen labeling index, significantly greater microvessel area and significantly lower apoptotic index. Splenic injection of KM12SM cells mixed with MSCs, in comparison to splenic injection of KM12SM cells alone, resulted in a significantly greater number of liver metastases. MSCs incorporated into the stroma of primary and metastatic tumors expressed α-smooth muscle actin and platelet-derived growth factor receptor-ß as carcinoma-associated fibroblast (CAF) markers. In in vitro experiments, KM12SM cells recruited MSCs, and MSCs stimulated migration and invasion of tumor cells through the release of soluble factors. Collectively, MSCs migrate and differentiate into CAFs in tumor stroma, and they promote growth and metastasis of colon cancer by enhancing angiogenesis, migration and invasion and by inhibiting apoptosis of tumor cells.

Potential role for vascular endothelial growth factor-D as an autocrine factor for human gastric carcinoma cells.

Vascular endothelial growth factor (VEGF)-D induces lymphangiogenesis by activating VEGF receptor (VEGFR)-3, which is expressed mainly by lymphatic endothelial cells. VEGFR-3 has also been detected in several types of malignant cells, but the significance of VEGFR-3 expression by malignant cells remains unclear. We examined the expression and function of VEGF-D/VEGFR-3 in human gastric carcinoma cells. Expression of VEGF-D and VEGFR-3 was analyzed in three human gastric carcinoma cell lines and 29 surgical specimens. cDNA microarray analysis was used to examine the effect of VEGF-D on the expression of genes associated with disease progression in VEGFR-3-expressing KKLS cells. VEGF-D-transfected cells and control cells were transplanted into the gastric wall of nude mice. In 10 of the 29 (34%) gastric carcinoma specimens and two of the three cell lines, cancer cells expressed both VEGF-D and VEGFR-3. In vitro treatment of KKLS cells with exogenous VEGF-D increased expression of cyclin D1 and Bcl-2 and stimulated cell proliferation. VEGF-D transfection into KKLS cells resulted in stimulation of angiogenesis, lymphangiogenesis, and cell proliferation, and in inhibition of apoptosis. VEGF-D may participate in the progression of human gastric carcinoma by acting via autocrine and paracrine mechanisms.

Expression of platelet-derived growth factor (PDGF)-B and PDGF-receptor ß is associated with lymphatic metastasis in human gastric carcinoma.

Recent study of murine fibrosarcoma has revealed that platelet-derived growth factor (PDGF) plays a direct role in promoting lymphangiogenesis and metastatic spread to lymph nodes. Thus, we investigated the relation between PDGF and PDGF receptor (PDGF-R) expression and lymphatic metastasis in human gastric carcinoma. We examined PDGF-B and PDGF-Rß expression in four human gastric carcinoma cell lines (TMK-1, MKN-1, MKN-45, and KKLS) and in 38 surgical specimens of gastric carcinoma. PDGF-B and PDGF-Rß expression was examined by immunofluorescence in surgical specimens and in human gastric carcinoma cells (TMK-1) implanted orthotopically in nude mice. Groups of mice (n = 10, each) received saline (control) or PDGF-R tyrosine kinase inhibitor imatinib. PDGF-B and PDGF-Rß mRNA expression was significantly higher in patients with lymph node metastasis than in those without and was also significantly higher in diffuse-type carcinoma than in intestinal-type carcinoma. In surgical specimens, tumor cells expressed PDGF-B, but PDGF-Rß was expressed predominantly by stromal cells. Under culture conditions, expression of PDGF-B mRNA was found in all of the gastric cell lines, albeit at different levels. In orthotopic TMK-1 tumors, cancer cells expressed PDGF-B but not PDGF-Rß. PDGF-Rß was expressed by stromal cells, including lymphatic endothelial cells. Four weeks of treatment with imatinib significantly decreased the area of lymphatic vessels. Our data indicate that secretion of PDGF-B by gastric carcinoma cells and expression of PDGF-Rß by tumor-associated stromal cells are associated with lymphatic metastasis. Blockade of PDGF-R signaling pathways may inhibit lymph node metastasis of gastric carcinoma.

Antibodies to Helicobacter pylori and CagA protein are associated with the response to antibacterial therapy in patients with H. pylori-positive API2-MALT1-negative gastric MALT lymphoma.

The aim of this study was to clarify predictive factors for response to eradication therapy in cases of Helicobacter pylori (H. pylori)-positive API2-MALT1-negative gastric mucosa-associated lymphoid tissue (MALT) lymphoma. Sixty-six patients who were examined for H. pylori infection and the presence of the API2-MALT1 chimeric transcript and who underwent H. pylori eradication therapy as first-line therapy, were enrolled in this study. Immunohistochemical markers (p53, Ki-67, and BCL10), microsatellite instability, loss of heterozygosity, serum levels of antibodies (anti-H. pylori and anti-CagA), and markers for gastritis (gastrin and pepsinogens) were examined, and the results were compared between patients whose tumors regressed completely after eradication therapy (responders) and patients whose tumors did not regress (non-responders). Of the 66 patients with localized gastric MALT lymphoma, 47 (71.2%) showed complete remission after eradication therapy. None of the H. pylori-negative (n = 9) and/or API2-MALT1-positive (n = 7) patients responded to antibacterial treatment. Of 44 patients with H. pylori-positive API2-MALT1-negative gastric MALT lymphoma, 38 (86.4%) showed complete remission after eradication therapy. Titers of antibodies against H. pylori and CagA protein were significantly higher in the responders than in the non-responders (P = 0.0235 and 0.0089, respectively). No significant difference between the groups was observed for the other factors. In conclusion, measurement of titers of serum antibodies to H. pylori and CagA protein may be useful for predicting the response to eradication therapy in patients with H. pylori-positive API2-MALT1-negative gastric MALT lymphoma.

A Questionnaire Survey on the Sequence of Events Prior to Undergoing Colonoscopy: The Influence of the Behavioral Response after a Fecal Occult Blood Test on the Early Detection of Colorectal Cancer.

Objective The fecal occult blood (FOB) test is commonly used for colorectal cancer screening; however, it is uncertain if further diagnostic interventions, such as a colonoscopy, should be performed based on its results. Method To better understand patient behavior following the FOB test, 6,414 patients (3,807 men and 2,607 women) who underwent colonoscopy between August 2015 and March 2016 at any of the 26 medical institutions throughout Hiroshima Prefecture were invited to participate in the study. All patients provided their written consent, after which they completed a questionnaire, and their colonoscopy results were obtained. These datasets were analyzed in a blinded manner, and the unique codes linking the records were revealed at the end of the analysis. Results Of the total study population, 4,749 patients (74.0%) had previously undergone FOB testing. After classification of common behavioral responses that the patients displayed following their FOB test, the group who had undergone the test several times, who had not had positive test results in the past, and whose latest FOB test results were positive had a significantly higher diagnosis rate of both early- and advanced-stage cancer than the other groups. Furthermore, patients in whom several previous FOB test results had been negative whose previous colonoscopy was positive were associated with a higher diagnosis rate of early-stage cancer than other groups. Conclusion These results suggested that colonoscopy should be performed immediately for patients with positive FOB test results due to their association with colorectal cancer and the possible detection of cancer at an early stage.

[A case report of anaphylactoid purpura with acute abdominal pain secondary to trauma caused by traffic accident].

A 17-year-old man was admitted to our hospital with multiple fractures resulting from traffic accident. After treatment of fractures, his general status was improved. However, one month after traffic accident, he suffered severe pain in the epigastrium. Ultrasonography and computed tomography showed thickening of the intestinal wall in the duodenum, ileum, and ascending colon. Nineteen days after the onset of abdominal pain, small hemorrhagic spots appeared on both of the lower legs. Subsequently, he developed proteinuria and hematuria. Purpura nephritis was diagnosed in biopsy specimens of the kidney. Anaphylactoid purpura associated with traffic accident is very rare and it is difficult to diagnose without skin and renal symptoms.

Mesenchymal Stem Cells Induce Epithelial to Mesenchymal Transition in Colon Cancer Cells through Direct Cell-to-Cell Contact.

We previously reported that in an orthotopic nude mouse model of human colon cancer, bone marrow-derived mesenchymal stem cells (MSCs) migrated to the tumor stroma and promoted tumor growth and metastasis. Here, we evaluated the proliferation and migration ability of cancer cells cocultured with MSCs to elucidate the mechanism of interaction between cancer cells and MSCs. Proliferation and migration of cancer cells increased following direct coculture with MSCs but not following indirect coculture. Thus, we hypothesized that direct contact between cancer cells and MSCs was important. We performed a microarray analysis of gene expression in KM12SM colon cancer cells directly cocultured with MSCs. Expression of epithelial-mesenchymal transition (EMT)-related genes such as fibronectin (FN), SPARC, and galectin 1 was increased by direct coculture with MSCs. We also confirmed the upregulation of these genes with real-time polymerase chain reaction. Gene expression was not elevated in cancer cells indirectly cocultured with MSCs. Among the EMT-related genes upregulated by direct coculture with MSCs, we examined the immune localization of FN, a well-known EMT marker. In coculture assay in chamber slides, expression of FN was seen only at the edges of cancer clusters where cancer cells directly contacted MSCs. FN expression in cancer cells increased at the tumor periphery and invasive edge in orthotopic nude mouse tumors and human colon cancer tissues. These results suggest that MSCs induce EMT in colon cancer cells via direct cell-to-cell contact and may play an important role in colon cancer metastasis.

Combining molecular targeted drugs to inhibit both cancer cells and activated stromal cells in gastric cancer.

Recent studies have revealed that PDGF plays a role in promoting progressive tumor growth in several cancers, including gastric cancer. Cancer-associated fibroblasts, pericytes, and lymphatic endothelial cells in stroma express high levels of PDGF receptor (PDGF-R); cancer cells and vascular endothelial cells do not. Mammalian target of rapamycin (mTOR) is a serine/threonine kinase that increases the production of proteins that stimulate key cellular processes such as cell growth and proliferation, cell metabolism, and angiogenesis. In the present study, we examined the effects of PDGF-R tyrosine kinase inhibitor (nilotinib) and mTOR inhibitor (everolimus) on tumor stroma in an orthotopic nude mice model of human gastric cancer. Expression of PDGF-B and PDGF-Rß mRNAs was associated with stromal volume. Treatment with nilotinib did not suppress tumor growth but significantly decreased stromal reactivity, lymphatic invasion, lymphatic vessel area, and pericyte coverage of tumor microvessels. In contrast, treatment with everolimus decreased tumor growth and microvessel density but not stromal reactivity. Nilotinib and everolimus in combination reduced both the growth rate and stromal reaction. Target molecule-based inhibition of cancer-stromal cell interaction appears promising as an effective antitumor therapy.

Stroma-directed molecular targeted therapy in gastric cancer.

Recent studies in molecular and cellular biology have shown that tumor growth and metastasis are not determined by cancer cells alone, but also by a variety of stromal cells. Tumor stroma contains abundant extracellular matrix and several types of cells, including carcinoma-associated fibroblasts (CAFs), endothelial cells, pericytes and inflammatory cells including macrophages. In gastric cancer tissues, tumor cells express platelet-derived growth factor (PDGF)-B. Stromal cells, including CAFs, pericytes and lymphatic endothelial cells, express PDGF receptor (PDGFR)-ß. Administration of PDGFR tyrosine kinase inhibitor significantly decreases stromal reaction, lymphatic vessel area and pericyte coverage of tumor microvessels. Administration of PDGFR tyrosine kinase inhibitor in combination with cytotoxic chemotherapeutic drug(s) impairs the progressive growth and metastasis of gastric cancer. Activated stroma might serve as a novel therapeutic target in cases of gastric cancer.

Rapid progression of Epstein-Barr-virus-positive gastric diffuse large B-cell lymphoma during chemoradiotherapy: a case report.

A 61-year-old woman was referred to our hospital with epigastralgia and appetite loss. Barium examination and upper gastrointestinal endoscopy revealed uneven erythematous mucosa with multiple elevated lesions from the gastric fornix to the upper corpus. Abdominal computed tomography showed thickening of the wall of the fornix and swelling of perigastric lymph nodes, but whole-body gallium scintigraphy and bone marrow examination did not indicate further involvement. Biopsy specimens showed diffuse infiltration of large atypical lymphoid cells in which Epstein-Barr virus (EBV) was detected by in situ hybridization. Diffuse large B-cell lymphoma (DLBCL), stage II1, was diagnosed. Combination chemotherapy [cyclophosphamide, adriamycin, vincristine, and prednisone (CHOP)], was given, and this was followed by radiotherapy. Partial remission was achieved by chemotherapy, but the disease progressed rapidly during radiotherapy. Because the reported prognosis of EBV-positive DLBCL is unfavorable, the therapeutic strategy for EBV-positive gastric DLBCL should be considered carefully.