Fumikazu Koyama

The Membrane-Anchored Matrix Metalloproteinase (MMP) Regulator RECK in Combination with MMP-9 Serves as an Informative Prognostic Indicator for Colorectal Cancer

Purpose: RECK, a membrane-anchored regulator of matrix metalloproteinases (MMPs), is widely expressed in healthy tissue, whereas it is expressed at lower levels in many tumor-derived cell lines. Studies in mice and cultured cells have shown that restoration of RECK expression inhibits tumor invasion, metastasis, and angiogenesis. However, the clinical relevance of these findings remains to be fully documented. Here we examined the expression of RECK and one of its targets, MMP-9, in colorectal cancer tissue. Experimental Design: The RECK and MMP-9 expression levels in colorectal cancer samples from 53 patients were determined by immunohistochemical techniques. The expression level of each protein was scored, and the patients were divided into two groups based on these scores. In 33 cases, we performed gelatin zymography to estimate the degree of MMP-2 and MMP-9 activation. Microvessel density and vascular endothelial growth factor (VEGF) expression were also evaluated histologically. Results: RECK protein was detected in 30 of 53 (56.6%) specimens. Importantly, patients with tumors expressing relatively high levels of RECK (high-RECK group) had a significantly lower risk of recurrence than did patients with tumors expressing relatively low levels of RECK (low-RECK group; P = 0.011). Moreover, RECK-dominant (RECK score ≥ MMP-9 score) patients showed a significantly lower incidence of recurrence than did MMP-9-dominant patients (P = 0.0003). Multivariate analysis revealed that the RECK/MMP-9 balance was an independent prognostic factor (P = 0.0122). The expression of VEGF and microvessel density were inversely correlated with the level of RECK expression. Conclusions: RECK/MMP-9-balance is an informative prognostic indicator for colorectal cancer. Our data also suggest that RECK suppresses tumor angiogenesis, probably by limiting the availability of VEGF in tumor tissues.

Combined suicide gene therapy for human colon cancer cells using adenovirus-mediated transfer of escherichia coli cytosine deaminase gene and Escherichia coli uracil phosphoribosyltransferase gene with 5-fluorocytosine.

The virus-directed enzyme/prodrug system using the Escherichia coli cytosine deaminase (CD) gene and 5-fluorocytosine (5-FC) suffers from a sensitivity limitation in many tumor cells. The E. coli uracil phosphoribosyltransferase (UPRT), which is a pyrimidine salvage enzyme, directly converts 5-fluorouracil (5-FU) to 5-fluorouridine monophosphate at the first step of its activating pathway. To improve the antitumoral effect of the CD/5-FC system, we investigated a combined suicide gene transduction therapy for human colon cancer cells using two separate adenovirus vectors expressing the E. coli CD and E. coli UPRT genes and systemic 5-FC administration (the CD, UPRT/5-FC system). The present study demonstrates that the CD, UPRT/5-FC system generates a co-operative effect of CD and UPRT, resulting in dramatic increases in both RNA- and DNA-directed active forms, including 5-fluorouridine triphosphate incorporated into RNA, 5-fluorodeoxyuridine monophosphate, and the thymidylate synthase inhibition rate, compared with the CD/5-FC system. Furthermore a significant increase in the 5-FC sensitivity of colon cancer cells was demonstrated in the CD, UPRT/5-FC system compared with the CD/5-FC system in vitro and in vivo. These results suggest that the CD, UPRT/5-FC system is a powerful approach in gene therapy for colorectal cancer.

Antisense epidermal growth factor receptor delivered by adenoviral vector blocks tumor growth in human gastric cancer

Epidermal growth factor receptor (EGFR) protein overexpression is commonly found in human gastric cancer, and its gene amplification is known to correlate with poor prognosis in gastric cancer patients. With regard to therapy trials targeting EGFR, it has been reported that stable transfection of EGFR antisense or treatment with antibody against EGFR results in growth suppression of human cancer cells that express high levels of EGFR. We have designed an adenovirus-expressing antisense EGFR and have investigated its effect on the growth of gastric cancer in vitro and in vivo. Following infection with EGFR antisense RNA-expressing adenovirus (Ad-EAS), the cell surface EGFR protein levels of infected cancer cells were markedly reduced, and the in vitro growth of Ad-EAS-infected cells was significantly inhibited relative to control-infected cells in all three gastric cancer cell lines (AGS, KKLS, and MKN28) studied here (P < .0002). In a nude mouse subcutaneous tumor system, in vivo tumor growth of MKN28 was significantly inhibited after Ad-EAS treatment, and inhibition on day 48 was 93% by volume compared with that of untreated controls. These results suggest that an adenoviral vector system targeting the down-regulation of EGFR could be a good candidate for the therapy of gastric cancers that overexpress EGFR.

Adenovirus-mediated gene transduction of truncated IκBα enhances radiosensitivity in human colon cancer cells

Nuclear factor kappa B (NF‐κB) is a transcription factor that is known to regulate apoptosis when cells are exposed to DNA‐damaging agents such as ionizing radiation and cytotoxic drugs. We sought to determine if inhibition of NF‐κB could enhance radiosensitivity in human colon cancer cells in vitro and in vivo. To inhibit NF‐κB activation specifically, we constructed a recombinant adenovirus vector expressing a truncated form of the inhibitor protein lκβα (lκBαΔN) that lacks the phosphorylation sites essential for activation of NF‐κB, and transfected two human colon cancer cell lines (HT29 and HCT15) with this vector. In vitro colony‐forming assays revealed that the overexpression of the stable IκBα by AxIκBαΔN infection significantly suppressed cell growth after irradiation in both cell lines as compared to infection with a control vector, AxLacZ. Treatment with AxIκBαΔN and irradiation successfully inhibited the growth of HT29 xenografted subcutaneous tumors in nude mice with an 83.8% volume reduction on day 38 as compared to the untreated tumors. Furthermore, it was demonstrated that apoptosis was increased by adenovirus‐mediated gene transduction of IκBaLΔN in vitro and in vivo. These results indicated that inhibition of NF‐κB could enhance radiosensitivity through an increase in radiation‐induced apoptosis. We believe that radio‐gene therapy using adenovirusmediated gene transduction of IκBαΔN could be an attractive candidate as a treatment strategy for colorectal cancer.

Tumor suppression effect using NK4, a molecule acting as an antagonist of HGF, on human gastric carcinomas

Hepatocyte growth factor (HGF) is involved in malignant behavior of cancers as a mediator of tumor-stromal interactions, facilitating tumor invasion and metastasis. We have investigated whether a blockade of HGF using recombinant NK4, an HGF antagonist, would lead to growth inhibition of the human gastric carcinoma cell line, TMK1. To evaluate the function of endogenous NK4 and investigate its potential inhibitory effect, TMK1 cells were transfected with NK4 plasmid. After selection, NK4-expressing cells (T11) were obtained, and cell growth was evaluated. Significant growth inhibition was observed in the T11-group compared to the control both in vitro and in vivo. Moreover, we investigated the effect of exogenous NK4 transferred by an adenovirus vector (AdCMV.NK4). Cell proliferation of AdCMV.NK4 infected TMK1 cells was significantly inhibited compared with the control group. We also assessed the in vivo tumor suppression effect of AdCMV.NK4. The tumor volume following treatment with AdCMV.NK4 was significantly inhibited compared to that of the control group. These findings indicate that NK4 gene expression has a potential role in controlling proliferation of cancer cells. In conclusion, NK4 is a promising therapeutic agent and its gene delivery may be a new approach to treating patients with advanced gastric cancer.

Transcatheter Arterial Chemoembolization Using Cisplatin Powder Mixed with Degradable Starch Microspheres for Colorectal Liver Metastases after FOLFOX Failure: Results of a Phase I/II Study

PURPOSE To report the results of a phase I/II study of a transcatheter arterial chemoembolization protocol using cisplatin powder and degradable starch microspheres (DSM) for unresectable colorectal liver metastases after failure of FOLFOX (5-flourouracil, leucovorin plus oxaliplatin) chemotherapy conducted to determine the recommended dose of cisplatin powder and to assess the efficacy and safety of the protocol. MATERIALS AND METHODS A fine-powder formulation of cisplatin was mixed with DSM and administered via the hepatic artery every 4 weeks. In phase I, three cohorts of patients received escalating doses of cisplatin powder: 50 mg/m(2), 65 mg/m(2), and 80 mg/m(2). In phase II, tumor response, toxicity, and survival times were assessed. RESULTS The study enrolled 24 patients. Previously, FOLFOX had been administered to all patients, an irinotecan-containing regimen had been administered to 12 patients, and bevacizumab or cetuximab or both had been administered to 14 patients. In phase I, dose-limiting toxicity did not appear at any level, and the recommended dose of cisplatin powder was determined to be 80 mg/m(2). In phase II, a tumor response rate of 61.1% was achieved. The median hepatic progression-free survival and overall survival were 8.8 months (95% confidence interval [CI], 4.06-13.5 mo) and 21.1 months (95% CI, 8.37-33.8 mo). The following grade 3 toxicities were observed: thrombocytopenia (12.5%), aspartate transaminase elevation (33.3%), alanine transaminase elevation (12.5%), hyponatremia (8.3%), and cholecystitis (4.2%). CONCLUSIONS This study shows that transcatheter arterial chemoembolization with cisplatin powder at a dose of 80 mg/m(2) mixed with DSM is well tolerated and can produce a high response rate with a long survival time for patients with unresectable colorectal liver metastases after failure of FOLFOX.

Adenoviral-mediated transfer of Escherichia coli uracil phosphoribosyltransferase (UPRT) gene to modulate the sensitivity of the human colon cancer cells to 5-fluorouracil.

5-Fluorouracil (5-FU) has been used as a chemotherapeutic drug for colorectal cancer. Escherichia coli uracil phosphoribosyltransferase (UPRT), a pyrimidine salvage enzyme, converts 5-FU into 5-fluorouridine monophosphate (5-FUMP) at the initial step of 5-FU activation. We investigated the effects of adenoviral-mediated transfer of the E. coli UPRT gene into human colon cancer cells on 5-FU metabolism and 5-FU chemosensitivity. Three cell lines were used (HT29, KM12 and SW1116). The intracellular levels of 5-fluorodeoxyuridine monophosphate (5-FdUMP) and 5-FU incorporated into RNA after 5-FU treatment in cells infected with adenovirus containing the UPRT gene (AdCA-UPRT) were significantly higher than those of non-infected cells. This was accompanied by marked inhibition of thymidylate synthase (TS) in all cell lines. Furthermore, HT29, KM12 and SW1116 infected with AdCA-UPRT were, respectively, 13.1-, 30.2- and 70.5-fold more sensitive to 5-FU than non-infected cells. Most importantly, treatment with AdCA-UPRT and 5-FU effectively inhibited the growth of HT29-xenografted subcutaneous tumours in nude mice. Therefore, AdCA-UPRT/5-FU treatment had the potential to enhance the actions of 5-FU at both the DNA and RNA levels. Treatment augmented the sensitivity of human colon cancer cells to 5-FU both in vitro and in vivo. We conclude that adenoviral-mediated transfer of the E. coli UPRT gene into colon cancer cells can achieve biochemical modulation of 5-FU and this provides a new approach in the treatment of colorectal cancer.

A new possibility for repairing the anal dysfunction by promoting regeneration of the reflex pathways in the enteric nervous system

Moderate rectal distension elicits recto-rectal reflex contractions and simultaneous recto-internal anal sphincter reflex relaxations that together comprise the defecation reflex. Both reflexes are controlled by 1) pelvic nerves, 2) lumbar colonic nerves, and 3) enteric nervous system. The aim of the present study was to explore a novel approach to repairing the defecation reflex dysfunction by using the plasticity of enteric nervous pathways. Experiments were performed in anesthetized guinea pigs with ethyl carbamate. The rectum 30 mm oral from the anal verge was transected without damage to extrinsic nerves, and subsequent end-to-end one-layer anastomosis was performed. Recovery of the defecation reflex and associated reflex pathways were evaluated. Eight weeks after sectioning of intrinsic reflex nerve pathways in the rectum, the defecation reflex recovered to the control level, accompanied with regeneration of reflex pathways. The 5-HT(4)-receptor agonist mosapride (0.5 and 1.0 mg/kg) significantly (P < 0.01) enhanced the recovered defecation reflex 8 wk after surgery. Two weeks after local treatment with brain-derived neurotrophic factor (BDNF: 10(-6) g/ml) at the rectal anastomotic site, the recto-internal anal sphincter reflex relaxations recovered and some bundles of fine nerve fibers were shown to interconnect the oral and anal ends of the myenteric plexus. These results suggested a possibility for repairing the anal dysfunction by promoting regeneration of the reflex pathways in the enteric nervous system with local application of BDNF.

Extent and contraindications for sacral amputation in patients with recurrent rectal cancer: a systematic literature review

BackgroundAbdominosacral amputation is a potentially curative surgical approach for patients with recurrent rectal cancer. Previous reports have described differing extents of sacral resection. Most of these reports stated that high sacral involvement of the tumor is a contraindication for surgery; however, the basis for this is unclear.MethodsIn this study, we reviewed the highest level of sacral amputation and the “contraindications” for this technique. Using a systematic literature survey, we analyzed the theoretical basis and the changes in surgical indications for recurrent rectal cancer.ResultsWe retrieved 33 articles from Medline and one study from the Cochrane Center Register of Controlled Trials. The highest level of resection was at the level of L5/S and S1 in one article, S1/2 and S2 in nine articles and S2/3 and S3 in 11 articles. Fifteen articles stated contraindications regarding sacral level, including tumor involvement of S1, the S1/2 junction, or the level above the S2/3 junction. Reasons stated for these contraindications included the risks associated with surgery, namely bladder dysfunction, anorectal dysfunction, genital dysfunction, walking disorder, and spinal fluid leak. In terms of the rationale for the contraindications, three articles referred to four previously published reviews or case series. None of these supporting publications were randomized controlled trials and they did not include any statistical evaluation.ConclusionThe consensus for contraindications for sacral amputation was formed empirically, without strong supporting evidence. The balance between curability and dysfunction should be further evaluated scientifically.

Tumor angiogenesis predicts recurrence with normal serum carcinoembryonic antigen in advanced rectal carcinoma patients.

Many studies have established the usefulness of serum carcinoembryonic antigen (CEA) oriented serial monitoring for predicting recurrence and prognosis; however, few studies have so far investigated serum CEA-negative recurrence. The aim of this study was to elucidate the nature of CEA-negative recurrence regarding tumor angiogenesis. Fifty-seven patients with T3/T4 rectal cancer were divided into the two groups according to the serum CEA status. Angiogenesis was defined as the intratumoral vessel count by immunohistochemical staining using CD31. The CD31 count was significantly higher in the recurrent patients in both groups and the ratio of nodal involvement was significantly higher in the recurrent patients of the CEA-negative group. Local recurrence mainly developed in the CEA-negative group; however, the CD31 count did not predict the sites of recurrence nor the relapse period in the both groups. A multivariate analysis showed a high CD31 count >26) to be a prognostic factor not only for recurrence but also for survival (P 5 0.001, 0.043, respectively). These results suggest that a high degree of tumor angiogenesis in sections of T3/T4 rectal cancer may therefore be an important predictor for CEAnegative recurrence.