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.     

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‐β (PDGFR‐β) 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‐β, 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.     

Expression of activated platelet-derived growth factor receptor in stromal cells of human colon carcinomas is associated with metastatic potential

Platelet-derived growth factor receptor (PDGF-R) expression has been reported in a variety of cancers, including colorectal, breast, lung, ovarian and pancreatic cancers, but the role of PDGF-R expression in the development and progression of colon carcinoma has not yet been elucidated. The purpose of this study was to examine the expression of PDGF and PDGF-R in human colon carcinomas. The expression of PDGF, PDGF-R and phosphorylated PDGF-R (p-PDGF-R) was examined by immunofluorescence in 12 surgical specimens of colon carcinoma and in human colon carcinoma cells growing in the subcutis (ectopic site) and the cecal wall (orthotopic site) of nude mice. In most surgical specimens, tumor cells expressed PDGF-A and -B subunits, without corresponding levels of PDGF-Ralpha and PDGF-Rbeta. PDGF-Rbeta was predominantly expressed by tumor-associated stromal cells and pericytes of tumor vasculature. The expression of PDGF-Rbeta in the stroma was associated with advanced stage disease. Under culture conditions, human colon carcinoma cell lines expressed PDGF-A and -B, but not PDGF-R. In orthotopic tumors, the KM12 cells (Duke's stage B) expressed PDGF-A and -B, but PDGF-Rbeta was expressed only by stromal cells and pericytes in the tumor vasculature. This expression of PDGF-Rbeta by stromal cells and pericytes was higher in tumors growing at the orthotopic site than in those at the ectopic site. The expression of PDGF-Rbeta in the stroma was higher in highly metastatic KM12SM tumors than in low metastatic KM12C tumors. In conclusion, the expression of PDGF-Rbeta in stromal cells is influenced by the organ-specific microenvironment and is associated with metastatic potential.     

Early tumor growth in metastatic organs influenced by the microenvironment is an important factor which provides organ specificity of colon cancer metastasis

We have previously demonstrated that liver metastases in nude mice and lung metastases in nude rats occurred specifically, when KM12SM human colon carcinoma cells were inoculated orthotopically into the cecal wall of nude mice and rats. To clarify the relationship between the tumor growth potential in the metastatic organs and the metastatic organ preference in these two metastatic models, we have evaluated the in vitro cell growth activities affected by the organ conditioned medium (CM) from the liver and lung, and the in vivo growth activities of the ectopic implanted tumors in the liver and lung. The tumorigenicity of the ectopic implanted tumors was 100% in mouse liver, 33% in rat liver, 50% in mouse lung, and 75% in rat lung. The crude liver CM of the animals showed inhibitory activities for KM12SM cell growth in a dosage-dependent manner, and the crude lung CM stimulated KM12SM cell growth. The liver CM of nude mice inhibited the KM12SM cell growth more strongly compared with the CM of nude rats, and the lung CM of nude rats was more strongly stimulated compared with the CM of nude mice. The liver CM of nude mice had non-heparin binding factors, which stimulated or inhibited KM12SM cell growth, in a molecular weight range of 50 to 100 kDa. By contrast, the liver CM of nude rats showed no growth stimulating activity for KM12SM cells. These results suggest that the metastatic organ specificity of KM12SM cells may depend on the early tumor growth influenced by the microenvironment in metastatic organs.     

In vivo tracking of the tropism of mesenchymal stem cells to malignant gliomas using reporter gene‐based MR imaging

Mesenchymal stem cells (MSCs) have emerged as a promising cellular vehicle for gene therapy of malignant gliomas due to their property of tumor tropism. However, MSCs may show bidirectional and divergent effects on tumor growth. Therefore, a robust surveillance system with a capacity for noninvasive monitoring of the homing, distribution and fate of stem cells in vivo is highly desired for developing stem cell‐based gene therapies for tumors. In this study, we used ferritin gene‐based magnetic resonance imaging (MRI) to track the tumor tropism of MSCs in a rat orthotopic xenograft model of malignant glioma. MSCs were transduced with lentiviral vectors expressing ferritin heavy chain (FTH) and enhanced green fluorescent protein (eGFP). Intra‐arterial, intravenous and intertumoral injections of these FTH transgenic MSCs (FTH‐MSCs) were performed in rats bearing intracranial orthotopic C6 gliomas. The FTH‐MSCs were detected as hypointense signals on T2‐ and T2*‐weighted images on a 3.0 T clinical MRI. After intra‐arterial injection, 17% of FTH‐MSCs migrated toward the tumor and gradually diffused throughout the orthotopic glioma. This dynamic process could be tracked in vivo by MRI up to 10 days of follow‐up, as confirmed by histology. Moreover, the tumor tropism of MSCs showed no appreciable impact on the progression of the tumor. These results suggest that FTH reporter gene‐based MRI can be used to reliably track the tropism and fate of MSCs after their systemic transplantation in orthotopic gliomas. This real‐time in vivo tracking system will facilitate the future development of stem cell‐based therapies for malignant gliomas.     

Extracellular matrix modulates expression of growth factors and growth-factor receptors in liver-colonizing colon-cancer cell lines

Site-specific metastasis is determined by the extracellular matrix (ECM) of the colonized organ. We have shown that hepatocyte-derived ECM stimulated proliferation of colon-cancer cells via induction of autocrine growth factors and their receptors. The ECM component responsible was heparin proteoglycan. We therefore investigated the effect of exogenously added heparin on colon cell lines of varying liver-colonizing potential. The cells were grown on typical liver matrix components, such as fibronectin and collagens type I and IV. We assessed the effect of these matrix components on clonal growth, proliferation and expression of autocrine growth factors and their receptors. The clonal growth of the KM12 cells was not affected by heparin, while the other cell lines were inhibited by heparin. Cell proliferation in weakly metastatic KM12, but not in strongly metastatic KM12SM, was inhibited by heparin on plastic. Weakly metastatic LS174T, but not strongly metastatic LiM6, was inhibited by heparin on fibronectin. Expression of erb-B2 was also differently modulated by heparin in weakly metastatic vs. highly metastatic cells. In weakly metastatic cells, heparin reduced erb-B2 levels when cells were on plastic and fibronectin, while in strongly metastatic cells, erb-B2 was induced by heparin. In all 4 cell lines, mRNA for cripto was induced by heparin when the cells were grown on fibronectin. In KM12SM cells, amphiregulin was induced by heparin in cells on fibronectin and collagen IV. We show that soluble heparin, similar in its carbohydrate chemistry to liver heparin proteoglycan, regulates the growth of colon-cancer cells. This effect depends on other matrix components found in the liver and is mediated in part by EGF family members. Int. J. Cancer 77:295–301, 1998.© 1998 Wiley-Liss, Inc.     

Inhibition of experimental colon cancer metastasis by the GABA-receptor agonist nembutal

Gamma-aminobutyric acid (GABA) is an inhibitory neurotransmitter, and GABA receptors have been recently detected on epithelial colon cancer cells. Nembutal (pentobarbital) is a barbiturate with GABA-agonistic effects. We demonstrate that GABA receptors are present on colon cancer cell lines (KM12SM, HT29, RKO). Nembutal (0.1-500 microg/ml) continuous exposure resulted in an IC50 level of 58 microg/ml for the KM12SM cells and 120 microg/ml for the HT29 cells. Nembutal reduced cellular cAMP concentration in colon cancer cells and resulted in a dose and time dependent decrease in MMP-2 and MMP-9 levels. In the KM12SM intracecal injected mice, 9 of 10 mice in the metaphane group developed a primary tumor (mean weight = 2.16 g +/- 0.76) compared to 7 of 10 mice in the nembutal group (mean weight = 0.41 g +/- 0.21, p = 0.03). In the KM12SM intrasplenic injected mice, the tumor weight in the spleen was 85% smaller in the nembutal group compared to the metaphane group (p = 0.008). In the HT29 injected mice, the metaphane group and nembutal group had similar tumor incidence, but combined tumor weight (primary tumor and liver metastases) was significantly higher in the metaphane group (1.61+/- 0.45 g) versus 0.07 +/- 0.05 g; p = 0.008. The incidence of liver metastases in the nembutal group was zero compared to eight out of nine in the metaphane group. To the best of our knowledge, this is the first evidence that nembutal is a potent inhibitor of primary colon cancer and metastasis. These findings may have therapeutic implications for the treatment of colon and other cancers.     

Down-regulation of c-Met inhibits growth in the liver of human colorectal carcinoma cells

Overexpression of c-Met, the protein tyrosine kinase receptor for the hepatocyte growth factor/scatter factor, has been implicated in the progression and metastasis of human colorectal carcinoma. To examine the role of c-Met on in vitro and in vivo growth of human colon tumor cell lines, stable subclones of the high metastatic human colorectal carcinoma cell line, KM20, isolated from a Dukes' D patient, with reduced c-Met expression were obtained after transfection with a c-Met-specific targeting ribozyme. The subclones were only modestly reduced in c-Met expression because of c-Met playing an important role in cellular survival. However, a 60-90% reduction in basal c-Met autophosphorylation and kinase activity were observed. Correlating with the reduction in c-Met kinase activity, subclones with reduced c-Met expression had significantly reduced in vitro growth rates and soft-agar colony-forming abilities. The in vivo growth of these cells was examined at both the ectopic SQ site and the orthotopic site of metastatic growth, the liver. SQ growth was delayed significantly in the c-Met down-regulated clones compared with controls, with tumors growing on loss of the ribozyme construct. In contrast, tumor incidence was significantly reduced when the c-Met down-regulated cells were grown in the orthotopic liver site. Thus, c-Met activation may be important in metastatic growth of colon tumor cells in the liver. Collectively these data demonstrate that a small reduction in c-Met protein levels leads to profound biological effects, and potential c-Met inhibitors may be of therapeutic value in treatment of colon cancer.     

Modulation of Doxorubicin sensitivity and level of p-glycoprotein expression in human colon-carcinoma cells by ectopic and orthotopic environments in nude-mice

The purpose of the study was to determine whether the organ environment can influence the response of colon cancer cells to chemotherapy. The highly metastatic human colon cancer cell line KM12L4, previously selected for production of liver metastases in nude mice, was injected into the cecal wall and into the spleen to produce liver metastases, and into the subcutis of nude mice. Doxorubicin (DOX) at 10 mg/kg or saline (control) was injected intravenously on days 7 and 16 after tumor cell injection. The in vivo response of tumors growing in the cecum, liver, and subcutaneous (s.c.) sites as well as the DOX sensitivity of cell lines established from liver and s.c. tumors were compared. Colon cancers growing s.c. were more sensitive to DOX than tumors growing in the cecal wall or liver of nude mice. The difference in response to DOX between s.c. tumors (sensitive) and liver tumors (resistant) was not due to selection of cell populations with different sensitivity to DOX, or differences in DOX distribution. PKC activity was lower in tumors of the liver and the cecum than in s.c. tumors. The expression of P-glycoprotein as determined by flow cytometric analysis of tumor cells harvested from lesions in different organs correlated inversely with their sensitivity to DOX. Increased levels of P-glycoprotein correlated with mdr-1, mdr-3 mRNA expression as determined by Northern analysis. Collectively, the data show that the organ environment influences the response of human colon carcinoma cells to DOX and recommend that animal models of this disease for experimental therapeutic studies employ orthotopic implantation of tumor cells.     

Repression of MLH1 and MGMT genes in colon mucosa adjacent to implanted cancer in athymic mouse

Hyperplastic mucosa adjacent to colon cancer, being a reactive change, accelerates cancer progression and its metastasis through expression of angiogenic factors. We investigated promoter methylation in hyperplastic mucosa adjacent to orthotopic KM12SM colon cancer in mice. In the hyperplastic mucosa adjacent to KM12SM tumors in the cecum of athymic mice, reductions in the levels of the mutL homologue 1 (MLH1) and O6-methylguanine-DNA methyltransferase (MGMT) proteins were detected by immunohistochemistry and immunoblotting. To examine the effects of growth factors and cytokines on promoter methylation and repressed expression of the MLH1 and MGMT genes, a rat intestinal epithelial cell line, IEC6, was treated with epidermal growth factor (EGF) and interleukin (IL)-15 for 35 days. Protein levels of MLH1 and MGMT were reduced in EGF- and IL-15-treated IEC6 cells. A methylation-sensitive restriction enzyme assay revealed that CpG methylation was present in the promoter regions of the MLH1 and MGMT genes in DNAs extracted from hyperplastic mucosa adjacent to KM12SM tumors. These findings suggest that promoter CpG methylation affects expression of MLH1 MGMT genes in hyperplastic mucosa adjacent to colon cancer.     

Effects of antineoplaston AS2-1 against post-operative lung metastasis in orthotopically implanted colon cancer in nude rat

We have investigated the efficacy and mechanisms of antineoplaston AS2-1 against post-operative lung metastasis following removal of implanted human colon cancer in nude rat. The influence of AS2-1 on in vitro KM12SM human colon carcinoma cell activities (growth, cell cycle, and apoptosis) was evaluated. AS2-1 was administered perorally after removal of the implanted KM12SM cecal cancer in nude rat. AS2-1 inhibited KM12SM cell proliferation through G1 cell arrest and, at a higher concentration, induction of apoptosis. AS2-1 showed significant reduction in lung metastasis at 5 weeks after cecal removal. The survival rate in the AS2-1 group was significantly higher than that in the control. TUNEL staining on the lung metastatic tumors revealed that the apoptosis index (AI) in the AS2-1 group was significantly higher. Antineoplaston AS2-1 showed an antimetastatic effect against post-operative lung metastases from colon cancer through G1 cell arrest and the subsequent induction of apoptosis.     

Targeting tumor‐infiltrating Ly6G+ myeloid cells improves sorafenib efficacy in mouse orthotopic hepatocellular carcinoma

Sorafenib, a multikinase inhibitor with antiangiogenic activity, is an approved therapy for hepatocellular carcinoma (HCC). It is unclear whether the proinflammatory and immunosuppressive mechanisms may limit the therapeutic efficacy of sorafenib in HCC. We used a syngeneic mouse liver cancer cell line to establish orthotopic liver or subcutaneous tumors to study how proinflammatory and immunosuppressive mechanisms impact on the efficacy of sorafenib. We found sorafenib exhibited a potent therapeutic effect in subcutaneous tumors, but a less potent effect in orthotopic liver tumors. The protein levels of interleukin‐6 (IL‐6) and vascular endothelial growth factor A (VEGF‐A) were persistently elevated in orthotopic liver tumors, but not in subcutaneous tumors, treated with sorafenib. Likewise, the tumor‐infiltrating Ly6G⁺ myeloid‐derived suppressor cells (MDSCs) and immune suppressors were increased in orthotopic liver tumors, not in subcutaneous tumors, treated with sorafenib. The tumor‐infiltrating Ly6G⁺ MDSCs of sorafenib‐treated orthotopic liver tumors significantly induced IL‐10 and TGF‐β expressing CD4⁺ T cells, and downregulated the cytotoxic activity of CD8⁺ T cells. IL‐6, but not VEGF‐A, protected Ly6G⁺ MDSCs from sorafenib‐induced cell death in vitro. The combination of anti‐Ly6G antibody or anti‐IL‐6 antibody with sorafenib significantly reduced the cell proportion of Ly6G⁺ MDSCs in orthotopic liver tumors, enhanced the T cells proliferation and improved the therapeutic effect of sorafenib synergistically. Modulating tumor microenvironment through targeting tumor‐infiltrating Ly6G⁺MDSCs represents a potential strategy to improve the anti‐HCC efficacy of sorafenib.     

Ectopic expression of neutrophil gelatinase-associated lipocalin suppresses the invasion and liver metastasis of colon cancer cells

Neutrophil gelatinase-associated lipocalin (NGAL), also known as lipocalin 2, is a 25-kDa lipocalin initially purified from neutrophil granules. It is thought to play a role in regulating cellular growth since its expression is highly upregulated in a variety of proliferative cells such as cancer cells. However, experimental evidence showing a clear causal relationship between NGAL expression and the proliferation of tumor cells is lacking. Here, we found NGAL expression in highly and poorly metastatic colon cancer cell lines of the same genetic origin correlated inversely with the metastatic potential of these cells, which suggests NGAL participates in the metastatic process. To explore the role NGAL plays in tumor growth and metastasis, the KM12SM human colon cancer cell line, which is highly metastatic while showing decreased NGAL expression, was genetically manipulated to overexpress NGAL. The effects of this on tumor growth and liver metastasis were then analyzed using experimental animal models established by injecting BALB/c nude mice with tumor cells subcutaneously or intrasplenically. Ectopic expression of NGAL in the colon cancer cells had little effect on the growth and viability of the tumor cells both in vitro and in vivo. However, NGAL expression not only suppressed the ability of the colon carcinoma cells to invade Matrigel in vitro, it also substantially inhibited liver metastasis in an experimental animal model. Collectively, these results indicate that NGAL may be a candidate metastasis suppressor in colon cancer cells.     

Production of interleukin 15 by human colon cancer cells is associated with induction of mucosal hyperplasia, angiogenesis, and metastasis.

PURPOSE: The purpose of this study was to identify a mediator produced by human colon cancer cells that is responsible for the induction of hyperplasia in the adjacent mucosa. EXPERIMENTAL DESIGN: Seventy human colon cancer surgical specimens were immunostained to determine the presence of cytokines that can induce hyperplasia in the adjacent mucosal. Human colon cancer cells with low and high metastatic potential were implanted into the cecal wall of nude mice. The resulting lesions were studied by immunohistochemistry to detect possible mediators of mucosal hyperplasia. RESULTS: Immunostaining of 70 colon cancer specimens from 70 patients suggested that mucosal hyperplasia and distant metastasis were associated with the expression of interleukin (IL)-15 and, to a lesser extent, transforming growth factor alpha. The production of IL-15 by colon cancer cells was not associated with the infiltration of natural killer cells into the tumors. Cecal tumors produced in nude mice by human colon cancer cells with low and high metastatic potential (KM12C and KM12SM cells, respectively) expressed similar levels of transforming growth factor alpha, and expression of IL-15 was detected only in the metastatic KM12SM cells and was associated with hyperplasia of the surrounding mucosa. The expression of the IL-15 receptor in rat intestinal epithelial cells (IEC6 cells) was confirmed by immunoblotting with antibodies against IL-15 receptor alpha and IL-2 receptor beta and gamma subunits and by a binding assay using (125)I-labeled IL-15 (K(d) = 0.011 nM). IL-15 stimulated proliferation of the IEC6 cells, even under serum starvation. Treatment of IEC6 cells with IL-15 decreased doxorubicin-mediated cytotoxicity. In IEC6 cells treated with either IL-15- or KM12SM-conditioned medium, immunoblotting revealed a decrease in the production of p21Waf1, Bax, and Bak and an increase in the production of cyclin E, proliferating cell nuclear antigen, the phosphorylated active form of AKT, basic fibroblast growth factor, and vascular endothelial growth factor, changes associated with cell growth, survival, and induction of angiogenesis. CONCLUSIONS: These data indicate that IL-15 produced by metastatic colon carcinoma cells can induce hyperplasia in the mucosa adjacent to colon cancer, thus contributing to angiogenesis and progression of the disease.     

Correlation between the presence of microvilli and the growth or metastatic potential of tumor cells

We used an electron microscope to examine microvilli which appear on the surfaces of various tumor cells with high or low growth potential and/or metastatic ability. The results show that a greater number of microvilli appeared on the surfaces of tumor cells (QRpP and ERpP) which possess high growth potential than on tumor cells (QR and ER) with low growth potential. We also observed that microvilli were more abundant on the surface of highly metastatic clone cells, i.e. c-SST-2 (cl-2), mouse B16 melanoma (F-10) and human colon carcinoma (KM12SM) than on weakly metastatic clone cells, c-SST-2 (cl-4-2), B16 (F-1) and (KM12C). At the same time, more microvilli were observed on the surface of B16 BL6 cells, which were obtained from the metastatic site of the B16 F10 cells, than on the surface of the parent B16 F10 cells. Immunoelectron microscopy revealed that the c-neu oncogene product, which is closely related to an epidermal growth factor receptor, was positively stained in the microvilli of tumor cells (ERpP) with high growth potential and high metastatic ability, whereas the tumor cells (ER) with low growth potential and weak metastatic ability were not stained. These findings suggest that the increased presence of microvilli correlates closely with the growth potential and metastatic ability of tumor cells.     

Correlation between the Presence of Microvilli and the Growth or Metastatic Potential of Tumor Cells

We used an electron microscope to examine microvilli which appear on the surfaces of various tumor cells with high or low growth potential and/or metastatic ability. The results show that a greater number of microvilli appeared on the surfaces of tumor cells (QRpP and ERpP) which possess high growth potential than on tumor cells (QR and ER) with low growth potential. We also observed that microvilli were more abundant on the surface of highly metastatic clone cells, i.e. c-SST-2 (cl-2), mouse B16 melanoma (F-10) and human colon carcinoma (KM12SM) than on weakly metastatic clone cells, c-SST-2 (cl-4-2), B16 (F-1) and (KM12C). At the same time, more microvilli were observed on the surface of B16 BL6 cells, which were obtained from the metastatic site of the B16 F10 cells, than on the surface of the parent B16 F10 cells. Immunoelectron microscopy revealed that the c- neu oncogene product, which is closely related to an epidermal growth factor receptor, was positively stained in the microvilli of tumor cells (ERpP) with high growth potential and high metastatic ability, whereas the tumor cells (ER) with low growth potential and weak metastatic ability were not stained. These findings suggest that the increased presence of microvilli correlates closely with the growth potential and metastatic ability of tumor cells.     

Anticancer effect of SN-38 on colon cancer cell lines with different metastatic potential

SN-38 is an active metabolite of the topoisomerase I inhibitor irinotecan. The mechanism behind its antitumor effect in colorectal cancer is not fully understood. In this study, we examined the response of colon cancer cell lines with different metastatic potential to SN-38. The parental human colon cancer cell line KM12C and its two highly metastatic derivatives KM12SM and KM12L4a were cultivated in 5% CO2 at 37 degrees C for 24 h and then exposed to SN-38 (2.5 microg/ml) at 37 degrees C for 4, 24 and 48 h, respectively. The cell cycle was measured by flow cytometry, apoptotic activity was determined by flow cytometry and immunocytochemistry and the expression of topoisomerase I, Bax and survivin proteins were examined by Western blot. The exposure of the cells to SN-38 induced S-phase and G2 arrest (P<0.0001) and the KM12L4a cells had the highest response in a time-dependent manner (P<0.0001). The rates of apoptosis in the KM12SM (P=0.001) and KM12L4a cell lines (P=0.01) were increased time-dependently, though there was no such change in the KM12C cells. The expression of topoisomerase I protein was decreased in each cell line tested and the expression of Bax protein was increased, especially in KM12L4a. In conclusion, the effect of SN-38 on the colon cancer cell lines was mediated via conducting S-phase and G2 arrest and apoptosis. This effect was found in the cell lines with higher metastatic potentials, indicating that SN-38 can be used to treat advanced colon cancers.     

Differential effector mechanisms induced by vaccination with MUC1 DNA in the rejection of colon carcinoma growth at orthotopic sites and metastases

The effects of MUC1 DNA vaccination on the orthotopic growth and liver metastasis of colon carcinoma cells were investigated in mice. Vaccination with MUC1 DNA resulted in immune responses that were effective in suppressing mouse colon carcinoma cells transfected with MUC1 cDNA. CD4⁺ T cells but not CD8⁺ T cells mediated this antitumor response as shown by the in vivo depletion of lymphocyte subpopulations with the use of anti‐CD4 or anti‐CD8 antibody. The effects of neutralizing antibodies in vivo revealed that the predominant effector molecule in preventing orthotopic tumor growth was FasL, whereas the effector molecule effective in preventing liver metastasis was tumor necrosis factor‐α. Colon carcinoma cells isolated from tumors growing in the ceca, spleens, and livers were shown to be equally sensitive to FasL and tumor necrosis factor‐α. The results strongly suggest that elimination of tumor cells initiated by DNA vaccination in the present protocol is mediated by antigen‐specific CD4⁺ T cells and the effector mechanisms in the cecum and in the liver are distinct due to a unique organ microenvironment. (Cancer Sci 2008; 99: 2477–2484)     

Relationship between the anti-metastatic effect of UFT and in vitro chemosensitivity to 5-FU in metastatic tumors from orthotopic implanted colon cancer in nude rats

We have investigated the correlation between the in vitro chemosensitivity to 5-FU, measured using the collagen gel droplet embedded culture drug sensitivity test (CD-DST), and the anti-tumor effect of UFT, a prodrug of 5-FU, in metastatic tumors from orthotopic implanted colon cancer in nude rats. Human colon cancer cells (KM12SM) were injected into the cecal wall of the nude rats. Five weeks later, the implanted cecal tumors were removed. Oral UFT (a daily dose of 30 mg/kg) was administered postoperatively for four weeks. After the UFT administration period, the lung and lymph nodes were analyzed macroscopically and microscopically. In vitro chemosensitivity to 5-FU in the lung and lymph node metastases was tested using CD-DST, and the enzymatic activities of thymidine synthetase (TS) and dihydropyrimidine dehydrogenase (DPD) in the lung and lymph node metastases were measured. A daily administration of UFT produced an inhibitory effect on lung metastasis compared with the control group. However, there was no difference in the frequency of lymph node metastasis. The inhibition rate produced by 5-FU in CD-DST was significantly higher for lung metastases than for lymph node metastases. There was no difference in the TS and DPD activities between the metastatic tumoral tissues. These results suggest that the organ specificity of the anti-tumor effects of UFT on colon metastases may be determined by CD-DST of 5-FU for individual tumors. The TS and DPD activity in the tumoral tissues may not affect the organ specificity of the anti-tumor effect of UFT on colon metastases.