PKR-NF-κB Pathway Upstream of IFN-ß Induction Is Dysregulated in Oncolytic Sindbis Virus-infected HeLa Cells.

BACKGROUND/AIM: Sindbis virus (SINV) is a naturally occurring oncolytic virus that kills cancer cells and is less harmful to normal cells. In this study, a recombinant SINV, which expressed green and blue fluorescent proteins, was used to precisely analyze SINV infection and replication. MATERIALS AND METHODS: Antiviral responses, including IFN-ß mRNA, protein kinase R (PKR), NF-B, and caspase 3/7, were analyzed in SINV-infected cancerous HeLa cells and normal human fibroblast TIG-1-20 cells. RESULTS: SINV could infect, replicate, and proliferate both in HeLa and TIG-1-20 cells, causing lytic infection only in HeLa cells. SINV grew preferentially in HeLa cells causing remarkable apoptosis. IFN-ß mRNA expression was suppressed in SINV-infected HeLa cells compared to that in TIG-1-20 cells. Further analyses of PKR and NF-B upstream of IFN-ß induction revealed that the compromised response in the PKR-NF-B pathway during early infection coincided with IFN induction suppression in HeLa cells. CONCLUSION: Dysregulation of PKR in HeLa cells is the determinant of SINV oncolysis.

CCL299, a Benzimidazole Derivative Induces G1 Phase Arrest and Apoptosis in Cancer Cells.

BACKGROUND/AIM: Benzimidazoles are considered potential anticancer candidates. We herein studied the anticancer activity of CCL299, 4-(1H-1,3-benzodiazol-1-yl) benzonitrile. MATERIALS AND METHODS: In this in vitro study, we used ATP assays, flow cytometry, western blotting, and caspase-3/7 assays to evaluate the effects of CCL299 on cell proliferation, cell-cycle progression and apoptosis. RESULTS: ATP assays showed that CCL299 inhibited cell growth in the hepatoblastoma cell line HepG2 and the cervical cancer cell line HEp-2, without exhibiting cytotoxic effects on non-cancer cells and TIG-1-20 fibroblasts. Flow cytometry, western blotting, and caspase-3/7 assays revealed that CCL299 induced G1-phase cell-cycle arrest followed by apoptosis that was associated with up-regulation of p-p53 (Ser15) and p21 expression and the down-regulation of p-CDK2 (Thr160) expression. CONCLUSION: CCL299 exhibits cytotoxic activity via apoptosis in a subset of cancer cells, and should be considered as a promising anticancer candidate agent.

CCL113, a novel sulfonamide, induces selective mitotic arrest and apoptosis in HeLa and HepG2 cells.

Targeting cell­cycle regulation to hinder cancer cell proliferation is a promising anticancer strategy. The present study investigated the effects of a novel sulfonamide, CCL113, on cell cycle progression in cancer cell lines (HeLa and HepG2), a noncancerous cell line (Vero) and a normal human fibroblast cell line (TIG­1­20). The present results showed that treatment with CCL113 significantly decreased the viability of the cancer cells. FACS analyses showed that CCL113 treatment increased the proportion of cancerous and noncancerous cells in the G2/M phase. Analyses of cell cycle regulatory proteins showed that CCL113 treatment inhibited the activity of CDK1 in HeLa cells, possibly due to the decrease in the level of Cdc25B/C proteins and arrest in the M phase. Using time­lapse imaging­assisted analyses of HeLa and Vero cells expressing fluorescent ubiquitination­based cell cycle indicator (FUCCI), it was observed that CCL113 treatment led to a prolonged G2 phase at the G2/M checkpoint and arrest in the M phase in both cell lines. This possibly activated the DNA damage response in noncancerous cells, while inducing mitotic arrest leading to apoptosis in the cancer cells. The results of molecular docking studies suggested that CCL113 might have the potential to bind to the taxol­binding site on ß­tubulin. In conclusion, CCL113 holds potential as a reliable anticancer drug due to its ability to induce mitotic arrest followed by apoptosis of cancer cells and to activate the DNA damage response in noncancerous cells, thereby facilitating exit from the cell cycle.

Immunohistochemical detection of an AGE, a ligand for macrophage receptor, in peritoneum of CAPD patients.

BACKGROUND: In patients on long-term continuous ambulatory peritoneal dialysis (CAPD), ultrafiltration (UF) capacity of peritoneal membrane may be impaired due to accumulation of advanced glycation end products (AGEs). This study aimed to elucidate the characteristics of a novel anti-AGE antibody, ODI-GLC19, and to demonstrate AGE accumulation in the peritoneum of CAPD patients using the antibody. METHODS: A monoclonal anti-AGE antibody (ODI-GLC19) was prepared by immunizing female balb/c mice using D-glucose-modified keyhole limpet hemocyanin. The characteristics of ODI-GLC19 were determined by enzyme-linked immunosorbent assay and receptor binding inhibition assay. Immunohistochemistry using ODI-GLC19 was performed to detect AGE in peritoneal tissues obtained from patients with nonrenal disease, and CAPD patients with normal and low UF. RESULTS: ODI-GLC19 reacted with glycolaldehyde-modified BSA (GA-BSA) and glucose-modified BSA (GLC-BSA), but not with imidazolone or N epsilon-(carboxymethyl)lysine. GA-BSA and GLC-BSA strongly bound to cultured macrophages. Time-dependent recognition of newly formed GA-BSA by ODI-GLC19 was similar to that by macrophages. The binding of GA-BSA to macrophages was inhibited by ODI-GLC19 in a dose-dependent manner. Immunohistochemical studies revealed that ODI-GLC19-positive AGE was exclusively detected in peritoneal cells including macrophages, and its staining intensity was more prominent in the peritoneum of CAPD patients, especially with low UF, than in patients with nonrenal disease. CONCLUSIONS: A novel monoclonal anti-AGE antibody, ODI-GLC19, recognizes a ligand for an AGE receptor on macrophages. Incorporation of AGE into peritoneal cells including macrophages may be involved in progressive peritoneal dysfunction in CAPD patients.