Kunitaka Hirose

A Candidate for Cancer Gene Therapy: MIP-lα Gene Transfer to an Adenocarcinoma Cell Line Reduced T\imorigenicity and Induced Protective Immunity in Immunocompetent Mice

AbstractPurpose. To evaluate the possibility of cancer gene therapy by the gene delivery of chemokine, the effects of human macrophage inflammatory protein lα (hu-MIP-lα), murine-macrophage inflammatory protein lα (mu-MIP-lα), and human-interleukin 8 (hu-IL-8) on tumor progression and immunization were studied. Methods. Cachexia-inducing and highly tumorigenic adenocarcinoma cells (cell line colon 26, clone 20) were transfected with either a control plasmid, hu-MIP-lα, mu-MIP-lα, or hu-IL-8 expression vector. The production of hu-MIP-1α reached >1.5 ng/ml in vitro when transfectant cells were cultured at a cell density of 2 × 105 cells in 7 ml for 3 days. Immunocompetent BALB/c mice were inoculated into the footpad with the tumor cells, and then primary tumor growth, morphological analyses, and tumor immunogenicity were studied. Results. The secretion of hu-MIP-lα, mu-MIP-lα, and hu-IL-8 did not affect the growth rate in vitro. Reduced tumorigenicities in vivo were observed in transfected cells with hu-MIP-lα and mu-MIP-lα. Morphologic observation of the site of inoculation of cells transfected with hu-MIP-lα showed infiltration of macrophages and neutrophils on the 5th day after the inoculation. Mice that had rejected cells transfected with hu-MIP-lα gene were immune to a subsequent challenge with the parental cells. Conclusions. The rejection of the cells depends on cytolysis and generates potent and long lasting antitumor immunity. These data suggest that tumor cells transfected with the MIP-lα gene might be useful as an effective therapy for the treatment of certain tumors.

Induction of gene expression for immunomodulating cytokines in peripheral blood mononuclear cells in response to orally administered PSK, an immunomodulating protein-bound polysaccharide

The protein-bound polysaccharide extracted from a fungus, PSK, has been used as a biological response modifier in the treatment of cancer patients in Japan for over 16 years. The administration of PSK to tumor-bearing rodents inhibited tumor growth and modulated immune responses. Recently, an in vitro study has revealed that PSK is a strong inducer of cytokine gene expression and production in human peripheral blood mononuclear cells (PBMC). To establish whether PSK has cytokine-inducing activities in vivo, we have orally administered PSK (1 g, the clinical dose) to 12 healthy volunteers and 9 gastric cancer patients who had undergone gastrectomy, and assessed the gene expression for cytokines in PBMC of each subject. As determined by the reverse-transcribed polymerase chain reaction method, the induction of gene expression for both tumor necrosis factor α and interleukin-8 (IL-8) was detected in PBMC from 5 of the 12 healthy volunteers (42%) and 4 of the 9 patients (44%). Furthermore, the concentration of serum IL-8 was elevated in 5 healthy volunteers given PSK orally, who had shown induction of IL-8 gene expression, as detected by enzyme-linked immunosorbent assay. These findings indicate that responsiveness of PBMC to PSK, in terms of gene expression and production of cytokines, varies among individuals. Thus, when using PSK to treat cancer patients, it seems advisable to select patients on the basis of their responsiveness to PSK. We speculate that the cytokines induced by PSK might mediate the immunoenhancing action of this agent in vivo.

Role of Interleukin-6 in Uncoupling of Bone In Vivo in a Human Squamous Carcinoma Coproducing Parathyroid Hormone-Related Peptide and Interleukin-6

OCC tumor has been established from a human squamous carcinoma associated with humoral hypercalcemia of malignancy (HHM) and shown to overproduce parathyroid hormone‐related peptide (PTHrP) and cause aggressive hypercalcemia when implanted into nude rats. In the present study, we have demonstrated by reverse transcription‐polymerase chain reaction and Northern blot analysis that OCC tumor also overexpressed interleukin 6 (IL‐6) mRNA and that tumor‐bearing animals exhibited a marked increase in plasma IL‐6 as well as PTHrP concentrations. When a monoclonal antibody against human IL‐6 was injected to block the activities of tumor‐derived IL‐6, bone loss in tumor‐bearing animals was significantly prevented. Quantitative bone histomorphometric analysis revealed that treatment with anti–IL‐6 antibody caused a substantial decrease in both osteoclast number and eroded surface (as parameters of bone resorption) and also a significant increase in the mineral apposition rate, but little effect on the osteoblastic surface. These results provide in vivo evidence suggesting that in tumors coproducing IL‐6 and PTHrP, IL‐6 is involved not only in the acceleration of osteoclastic bone resorption but also, at least in part, in the suppression of osteoblastic functions in HHM syndrome.

Enhanced Expression of Manganese Superoxide Dismutase mRNA and Increased TNFα mRNA Expression by Gastric Mucosa in Gastric Cancer

Abstract. Manganese superoxide dismutase (Mn-SOD), a mitochondrial enzyme, is a cytokine-regulated acute-phase protein that protects cells from free radicals. The current investigations examined the in vivo regulation of the expression of Mn-SOD mRNA and tumor necrosis factor α (TNFα) mRNA in gastric carcinoma tissue. The expression of these transcripts in breast carcinoma tissue also was examined because breast cancer is a much more TNF-sensitive tumor than gastric cancer. TNFα mRNA was markedly increased in gastric carcinoma tissue (p < 0.005). There were significantly higher levels of Mn-SOD mRNA in gastric carcinoma tissue than in noncancerous tissue (p < 0.0001). The level of Mn-SOD mRNA in gastric carcinoma tissue was higher than that in breast carcinoma tissue (p < 0.005). Up-regulation of Mn-SOD mRNA in gastric carcinoma tissue most likely serves as a protective mechanism against superoxide radicals and TNF cytotoxicity.

The nm23-H1 gene as a predictor of sensitivity to chemotherapeutic agents in oesophageal squamous cell carcinoma

SummaryRecently, nm23-H1, an anti-metastasis gene, has been reported to correlate with sensitivity to chemotherapeutic agents including cisplatin in human breast and ovarian carcinoma cells. The aim of this study was to evaluate a role for nm23-H1 in responsiveness to cisplatin-based chemotherapy in patients with oesophageal squamous cell carcinoma (OSCC). The expression of nm23-H1 protein was examined immunohistochemically in 32 eligible patients with OSCC who underwent adjuvant chemotherapy with cisplatin, etoposide, and 5-fluorouracil after tumour resection. Fifteen (46.9%) of 32 patients were positive for nm23-H1 staining and 17 (53.1%) were negative. Both disease-free survival and overall survival rates of nm23-H1-negative patients were significantly shorter than in nm23-H1-positive patients (P < 0.01 for both). There was no significant difference in clinicopathologic characteristics between nm23-H1-positive and nm23-H1-negative groups. Multivariate analysis also showed that nm23-H1 expression was the most significant factor for overall survival of OSCC patients included in this study (P = 0.0007). To further study the role of nm23-H1, a human OSCC cell line (YES-2) was transfected with a plasmid containing a fragment of the nm23-H1 cDNA in an antisense orientation. Reduced expression of nm23-H1 protein in the antisense-transfected (AS) clones was found by Western blot analysis as compared to wild-type YES-2 and YES-2/Neo (clone transfected with the neomycin resistance gene alone). MTT (3-(4,5-dimethyl-2-thiazol)-2,5-diphenyl-2H tetrazolium bromide) assay showed that reduced expression of the nm23-H1 protein in AS clones was consistent with the degree of increased resistance to cisplatin but not etoposide or 5-fluorouracil. These data support the conclusion that reduced expression of nm23-H1 may be associated with resistance to cisplatin, suggesting the value of nm23-H1 expression as a prognostic marker for OSCC patients who are to undergo cisplatin-based chemotherapy.

Tumour cells engineered to secrete interleukin-15 augment anti-tumour immune responses in vivo.

SummaryWe examined the effect of interleukin-15 (IL-15) gene transfer into tumour cells on the host’s anti-tumour response. In BALB/c mice IL-15 producing Meth-A cells (Meth-A/IL-15) underwent complete rejection, in a response characterized by massive infiltration of CD4+ T-cells and neutrophils. In contrast, Meth-A cells transfected with vector alone (Meth-A/Neo) grew rapidly. Moreover, rechallenged parental cells also were rejected in association with CD8+ T-cell infiltration. However, in nude mice there was no drastic difference between Meth-A/IL-15 and Meth-A/Neo cells. These results demonstrate that IL-15-secreting tumour cells can stimulate local and systemic T-cell-dependent immunity and therefore may have a potential role in cancer therapy.

Interleukin-1 receptor antagonist mRNA expression and the progression of gastric carcinoma.

Interleukin-1 receptor antagonist (IL-1ra), an endogeneous inhibitor of IL-1, plays an immunosuppressive role in vivo by blocking the proinflammatory effects of IL-1. In the present study, we examined whether IL-1ra expression in human gastric carcinoma correlates with tumor progression and/or metastatic potential. The reverse transcription-polymerase chain reaction was used to compare the expression of the secreted form of IL-1ra (sIL-1ra) and the intracellular form of IL-1ra (icIL-1ra) mRNA in tumor and corresponding benign tissue obtained from 38 patients with gastric carcinoma. The incidence of sIL-1ra mRNA expression was significantly higher in tumor (52%) than in corresponding benign tissue (18%) (P = 0.002). On the contrary, icIL-1ra mRNA was detected in all tumors and benign tissues. The expression of sIL-1ra mRNA by malignant tissue correlated positively with both lymph node metastasis (P = 0.008) and liver metastasis (P = 0.015). There was no association between tumor sIL-lra mRNA expression and other clinicopathologic factors. The degree of regional lymph node reaction, such as sinus histiocytosis, in tumors expressing sI-1ra mRNA was significantly weaker than that in tumors without sIL-1ra mRNA expression (5/20 vs. 12/18, P = 0.010). These results demonstrate that the altered expression of sIL-1ra by malignant tissue may be related to the progression of gastric carcinoma via modulating host immune response.

Downregulation of intracellular nm23-H1 prevents cisplatin-induced DNA damage in oesophageal cancer cells: possible association with Na(+), K(+)-ATPase.

Previously, we showed that expression of nm23-H1 is associated inversely with sensitivity to cisplatin in human oesophageal squamous cell carcinoma (OSCC). The present study was undertaken to investigate the association of nm23-H1 expression with cisplatin-induced DNA damage in OSCC using antisense nm23-H1 transfectants. YES-2/AS-12, an antisense nm23-H1-transfected OSCC cell line, showed significantly reduced expression of intracellular nm23-H1 protein compared with that in parental YES-2 cells and YES-2/Neo transfectants. Surface expression of nm23-H1 protein was not observed in any of the three cell lines. PCR analysis for DNA damage demonstrated that YES-2/AS-12 cells were more resistant to nuclear and mitochondrial DNA damage by cisplatin than were YES-2/Neo cells. In addition, mitochondrial membrane potentials and DNA fragmentation assays confirmed that YES-2/AS-12 was more resistant than YES-2/Neo to apoptosis induced by cisplatin. In contrast, YES-2/AS-12 was more sensitive to ouabain, a selective inhibitor of Na+, K+-ATPase, than YES-2 and YES-2/Neo. Pre-treatment with ouabain resulted in no differences in cisplatin sensitivity between the three cell lines examined. Intracellular platinum level in YES-2/AS-12 was significantly lower than that in YES-2 and YES-2/Neo following incubation with cisplatin, whereas ouabain pre-treatment resulted in no differences in intracellular platinum accumulations between the three cell lines. Our data support the conclusion that reduced expression of intracellular nm23-H1 in OSCC cells is associated with cisplatin resistance via the prevention of both nuclear and mitochondrial DNA damage and suggest that it may be related to Na+, K+-ATPase activity, which is responsible for intracellular cisplatin accumulation.

A biological response modifier, PSK, inhibits human immunodeficiency virus infection in vitro

PSK, a biological response modifier (BRM), was studied to determine its anti-viral activity on human immunodeficiency virus (HIV) in vitro. Either a novel infection system using human T-cell lymphotropic virus type I (HTLV-I)-carrying MT-4 cells or a coculture system using MOLT-4 cells and its virus-producing cells MOLT-4/HIVHTLV-IIIB which induces multinucleated giant cells very efficiently was used. PSK almost completely blocked the cytopathic effect such as giant cell formation and HIV-specific antigen expression both in MT-4 cells and MOLT-4 cells at a concentration of 0.4 and 0.8 mg/ml, respectively. Pretreatment of the virus with PSK may specifically interfere with early stages of HIV infection by modifying the viral receptor.

A biological response modifier, PSK, inhibits reverse transcriptase in vitro

We found that PSK has an antiviral effect on human immunodeficiency virus (HIV) in vitro. One of the mechanisms of this effect is attributable to the inhibition of binding of HIV with lymphocytes. Here, we found that PSK inhibits reverse transcriptase in a non-competitive way in vitro. Such inhibition may be important in its anti-HIV effect as well as its inhibitory effect on the binding of HIV with lymphocytes.