Expression of p53 synergistically augments caspases-mediated apoptosis induced by replication-competent adenoviruses in pancreatic carcinoma cells.

We examined cytotoxicity of replication-competent type 5 adenoviruses (Ad5) in human pancreatic carcinoma cells with a p53-defective genotype. The replication-competent Ad5 of which E1A gene was activated by exogenous transcriptional regulatory sequences, derived from the midkine and survivin genes, achieved cytotoxicity to the pancreatic carcinoma. These cells were susceptible to replication-incompetent Ad5 expressing the wild-type p53 gene. We also produced the replication-competent Ad5 bearing the same exogenous regulatory sequences and the type 35 Ad-derived fiber-knob region, and showed that the cytotoxicity was comparable to that of the replication-competent Ad5 prototype. We then investigated possible combinatory effects of the fiber-modified replication-competent Ad and Ad5 expressing the wild-type p53 gene, both of which did not interfere respective infections. The combination produced synergistic cytotoxic effects with enhanced cleavages of caspase-3 and PARP molecules, and with increased sub-G1 fractions and annexin V-positive populations although the viral production of the replication-competent Ad was rather suppressed by expressed p53. Pancreatic cells infected with both Ad showed increase of p53 and decrease of MDM2 and p21 levels, compared with those infected with Ad expressing the p53 gene. These data collectively indicated that replication-competent Ad augmented susceptibility of pancreatic cells to apoptosis through upregulated p53 expression.

Administration of DNA Encoding the Interleukin-27 Gene Augments Antitumour Responses through Non-adaptive Immunity.

DNA-mediated immunization of a tumour antigen is a possible immunotherapy for cancer, and interleukin (IL)-27 has diverse functions in adaptive immunity. In this study, we examined whether IL-27 DNA administration enhanced antitumour effects in mice vaccinated with DNA encoding a putative tumour antigen, ß-galactosidase (ß-gal). An intramuscular injection of cardiotoxin before DNA administration facilitated the exogenous gene expression. In mice received ß-gal and IL-27 DNA, growth of ß-gal-positive P815 tumours was retarded and survival of the mice was prolonged. Development of ß-gal-positive Colon 26 tumours was suppressed by vaccination of ß-gal DNA and further inhibited by additional IL-27 DNA administration or IL-12 family cytokines. Nevertheless, a population of ß-gal-specific CD8(+) T cells did not increase, and production of anti-ß-gal antibody was not enhanced by IL-27 DNA administration. Spleen cells from mice bearing IL-27-expressing Colon 26 tumours showed greater YAC-1-targeted cytotoxicity although CD3(-)/DX5(+) natural killer (NK) cell numbers remained unchanged. Recombinant IL-27 enhanced YAC-1-targeted cytotoxicity of IL-2-primed splenic NK cells and augmented a phosphorylation of signal transducer and activator of transcription 3 and an expression of perforin. These data collectively indicate that IL-27 DNA administration activates NK cells and augments vaccination effects of DNA encoding a tumour antigen through non-adaptive immune responses.

Zoledronic acid induces apoptosis and S-phase arrest in mesothelioma through inhibiting Rab family proteins and topoisomerase II actions.

Zoledronic acid (ZOL), a nitrogen-containing bisphosphonate, produced anti-tumor effects through apoptosis induction or S-phase arrest depending on human mesothelioma cells tested. An addition of isoprenoid, geranylgeraniol but not farnesol, negated these ZOL-induced effects, indicating that the ZOL-mediated effects were attributable to depletion of geranylgeranyl pyrophosphates which were substrates for prenylation processes of small guanine-nucleotide-binding regulatory proteins (small G proteins). ZOL-treated cells decreased a ratio of membrane to cytoplasmic fractions in RhoA, Cdc42 and Rab6 but less significantly Rac1 proteins, indicating that these proteins were possible targets for ZOL-induced actions. We further analyzed which small G proteins were responsible for the three ZOL-induced effects, caspase-mediated apoptosis, S-phase arrest and morphological changes, using inhibitors for respective small G proteins and siRNA for Cdc42. ZOL-induced apoptosis is due to insufficient prenylation of Rab proteins because an inhibitor of geranlygeranyl transferase II that was specific for Rab family proteins prenylation, but not others inhibitors, activated the same apoptotic pathways that ZOL did. ZOL suppressed an endogenous topoisomerase II activity, which was associated with apoptosis and S-phase arrest in respective cells because we detected the same cell cycle changes in etoposide-treated cells. Inhibitors for geranlygeranyl transferase I and for RhoA produced morphological changes and disrupted actin fiber structures, both of which were similar to those by ZOL treatments. These data demonstrated that anti-tumor effects by ZOL were attributable to inhibited functions of respective small G proteins and topoisomerase II activity, and suggested that cellular factors were involved in the differential cell cycle changes.

Combination of adenoviruses expressing melanoma differentiation-associated gene-7 and chemotherapeutic agents produces enhanced cytotoxicity on esophageal carcinoma.

We examined the combinatory antitumor effects of adenoviruses expressing human mda-7/IL-24 gene (Ad-mda-7) and chemotherapeutic agents on nine kinds of human esophageal carcinoma cells. All the carcinoma cells expressed the melanoma differentiation-associated gene-7/interleukin-24 (MDA-7/IL-24) receptor complexes, IL-20R2 and either IL-20R1 or IL-22R1, and were susceptible to Ad-mda-7, whereas fibroblasts were positive only for IL-20R2 gene and resistant to Ad-mda-7-mediated cytotoxicity. Sensitivity of these esophageal carcinoma cells to Ad-mda-7 was however lower than that to Ad expressing the wild-type p53 gene. We thereby investigated a possible combination of Ad-mda-7 and anticancer agents and found that Ad-mda-7 with 5-fluorouracil (5-FU), cisplatin, mitomycin C or etoposide produced greater cytotoxic effects than those by Ad-mda-7 or the agent alone. Half-maximal inhibitory concentration values of the agents in respective cells were decreased by the combination with Ad-mda-7. Cell cycle analyses showed that Ad-mda-7 and 5-FU increased G2/M-phase and S-phase populations, respectively, and the combination augmented sub-G1 populations. Ad-mda-7-treated cells showed cleavages of caspase-8, -9 and -3 and poly (ADP-ribose) polymerase, but the cleavage levels were not different from those of the combination-treated cells. Ad-mda-7 treatments upregulated Akt phosphorylation but suppressed IκB-α levels, whereas 5-FU treatments induced phosphorylation of p53 and extracellular signal-regulated protein kinases 1 and 2. Molecular changes caused by the combination were similar to those by Ad-mda-7 treatments, but the Ad-mda-7-mediated upregulation of Akt phosphorylation decreased with the combination. These data collectively suggest that Ad-mda-7 induced apoptosis despite Akt activation and that the combinatory antitumor effects with 5-FU were produced partly by downregulating the Ad-mda-7-induced Akt activation.

Heat-shock protein 90 inhibitors synergistically enhance melanoma differentiation-associated gene-7-mediated cell killing of human pancreatic carcinoma.

Pancreatic cancer is one of the intractable diseases and an effective therapeutic strategy is required to improve the prognosis. We examined possible antitumor effects of adenoviruses expressing melanoma differentiation-associated gene-7/interleukin-24 (Ad-mda-7) and a heat-shock protein 90 (Hsp90) inhibitor to human pancreatic carcinoma cells. Ad-mda-7 and an Hsp90 inhibitor, geldanamycin (GA), produced cytotoxic effects, and a combinatory use of Ad-mda-7 and GA further achieved synergistic effects. Administration of N-acetyl-L-cysteine, an inhibitor of reactive oxygen species, eliminated Ad-mda-7- and GA-mediated cytotoxicity. Ad-mda-7 augmented phosphorylated AKT levels but GA did not influence the phosphorylation. GA-treated cells showed cleavage of poly-(ADP-ribose) polymerase but not caspase-3, and upregulated Hsp70 and LC3A/B II levels, whereas Ad-mda-7-treated cells did not. GA treatments augmented ubiquitination and markedly increased melanoma differentiation-associated gene-7 (MDA-7) expression levels. These findings suggest that Ad-mda-7-mediated cytotoxicity is dependent on reactive oxygen species but independent of apoptosis or autophagy, and that GA-mediated cytotoxicity was linked with caspase-independent apoptosis and/or autophagy. A mechanism underlying the combinatory effects of Ad-mda-7 and GA remained complex and the synergism is attributable to multiple factors including increased MDA-7 protein stability by GA.

Phase II trial of gefitinib alone without radiation therapy for Japanese patients with brain metastases from EGFR-mutant lung adenocarcinoma.

BACKGROUND: Brain metastases (BM) are a common in patients with lung cancer. Although whole-brain radiation therapy (WBRT) is the standard therapy, it may have a risk of decline in cognitive function of patients. In this study, we evaluated the efficacy of gefitinib alone without radiation therapy for the treatment of patients with BM from lung adenocarcinoma. MATERIALS AND METHODS: Eligible patients had BM from lung adenocarcinoma with epidermal growth factor receptor (EGFR) mutations. Gefitinib was given at 250 mg orally once a day until tumor progression or unacceptable toxicity. RESULTS: Forty-one patients were enrolled. The response rate was 87.8%. No patient experienced grade ≥4 toxicity. The median progression-free survival time was 14.5 months (95% CI, 10.2-18.3 months), and the median overall survival time was 21.9 months (95% CI, 18.5-30.3 months). In compared with L858R, exon 19 deletion was associated with better outcome of patients after treatment with gefitinib in both progression-free (p = 0.003) and overall survival (p = 0.025). CONCLUSION: Favorable response of BM to gefitinib even without irradiation was demonstrated. Exon 19 deletion was both a predictive and prognostic marker of patients with BM treated by gefitinib.

High CC chemokine receptor 7 expression improves postoperative prognosis of lung adenocarcinoma patients.

BACKGROUND: Chemokines and chemokine receptors not only have significant roles in cancer metastasis and tumorigenesis but also act as antitumour agents. The interaction between the Crk-like adaptor protein (CrkL), which is encoded by the CRKL gene, and non-receptor tyrosine kinase c-ABL is reported to transform many cells into malignant cells. We examined the effects of CC chemokine receptor 7 (CCR7), CCR7 ligands and CrkL and c-ABL in lung adenocarcinoma. METHODS: One hundred and twenty patients with lung adenocarcinoma were included in this historical cohort analysis. We examined CCR7 and CCR7 ligands and CrkL and c-ABL mRNA expressions in surgically resected lung adenocarcinoma specimens and evaluated their contribution to prognosis, and the relationship with epidermal growth factor receptor (EGFR) and TP53 mutations. RESULTS: High CCR7 mRNA expressions indicated better prognoses than those of the groups with low CCR7 mRNA expressions (P=0.007, HR=2.00, 95% CI of ratio: 1.22 -3.31). In lung adenocarcinoma, CrkL and c-ABL mRNAs were related to CCR7 mRNA expression (P<0.0001). CrkL and c-ABL mRNA expressions were influenced by EGFR mutations. A high expression of CCL19 was a good prognostic factor of lung adenocarcinoma. CONCLUSION: We propose that CCR7 and CCL19 are clinically good prognostic factors and that CCR7 is strongly related to CrkL and c-ABL kinase mRNA expression in lung adenocarcinoma.