Tumor suppressor p53 is a multifunctional transcription factor that regulates diverse cell fates, including apoptosis and autophagy in tumor biology. p53 overexpression enhances the antitumor activity of oncolytic adenoviruses; however, the molecular mechanism of this occurrence remains unclear. We previously developed a tumor-specific replication-competent oncolytic adenovirus, OBP-301, that kills human osteosarcoma cells, but some human osteosarcoma cells were OBP-301-resistant. In this study, we investigated the antitumor activity of a p53-expressing oncolytic adenovirus, OBP-702, and the molecular mechanism of the p53-mediated cell death pathway in OBP-301-resistant human osteosarcoma cells. The cytopathic activity of OBP-702 was examined in OBP-301-sensitive (U2OS and HOS) and OBP-301-resistant (SaOS-2 and MNNG/ HOS) human osteosarcoma cells. The molecular mechanism in the OBP-702-mediated induction of two cell death pathways, apoptosis and autophagy, was investigated in OBP-301-resistant osteosarcoma cells. The antitumor effect of OBP-702 was further assessed using an orthotopic OBP-301-resistant MNNG/HOS osteosarcoma xenograft tumor model. OBP-702 suppressed the viability of OBP-301-sensitive and -resistant osteosarcoma cells more efficiently than OBP-301 or a replication-deficient p53-expressing adenovirus (Adp53). OBP-702 induced more profound apoptosis and autophagy when compared with OBP-301 or Ad-p53. E1A-mediated miR-93/106b upregulation induced p21 suppression, leading to p53-mediated apoptosis and autophagy in OBP-702-infected cells. p53 overexpression enhanced adenovirus-mediated autophagy through activation of damage-regulated autophagy modulator (DRAM). Moreover, OBP-702 suppressed tumor growth in an orthotopic OBP-301-resistant MNNG/HOS xenograft tumor model. These results suggest that OBP-702-mediated p53 transactivation is a promising antitumor strategy to induce dual apoptotic and autophagic cell death pathways via regulation of miRNA and DRAM in human osteosarcoma cells.

Autophagy is known to have a cytoprotective role under various cellular stresses; however, it also results in robust cell death as an important safeguard mechanism that protects the organism against invading pathogens and unwanted cancer cells. Autophagy is regulated by cell signalling including microRNA (miRNA), a post-transcriptional regulator of gene expression. Here, we show that genetically engineered telomerase-specific oncolytic adenovirus induced miR-7 expression, which is significantly associated with its cytopathic activity in human cancer cells. Virus-mediated miR-7 upregulation depended on enhanced expression of the E2F1 protein. Ectopic expression of miR-7 suppressed cell viability and induced autophagy by inhibiting epidermal growth factor receptor (EGFR) expression. Our results suggest that oncolytic adenovirus induces autophagic cell death through an E2F1-miR-7-EGFR pathway in human cancer cells, providing a novel insight into the molecular mechanism of an anticancer virotherapy.

PurposeThe combination of hepatitis B immunoglobulin (HBIg) and nucleos(t)ide analogues has been accepted as the best treatment to control hepatitis B recurrence after orthotopic liver transplantation (OLT). However, the optimal dose of HBIg remains unclear. We have previously reported that high-dose HBIg in the early period followed by low-dose HBIg with nucleos(t)ide analogues offers reliable and cost-effective control of hepatitis B recurrence. The aim of this study was to investigate intrahepatic hepatitis B virus (HBV) reinfection status with our clinically successful protocol.MethodsWe quantified levels of intrahepatic HBV covalently closed circular (ccc) deoxyribonucleic acid (DNA) and serum hepatitis B core-related antigen (HBcrAg), a new serological marker that can estimate intrahepatic cccDNA levels. Nucleos(t)ide analogues were administered in all cases.ResultsNo patients showed recurrence of hepatitis B surface antigen (HBsAg) or HBV-DNA. However, HBV, cccDNA, and HBcrAg were positive in 57% and 48% of patients after OLT, respectively. Pre-OLT serum HBV-DNA and HBcrAg levels correlated linearly with post-OLT cccDNA levels (r = 0.534, P < 0.05, and r = 0.634, P < 0.05, respectively). High serum HBV-DNA and HBcrAg levels, particularly with >3 log10 copies/mL and >4 log10 IU/mL, respectively, at the time of OLT, were associated with high levels of post-OLT cccDNA. Even with our successful protocol, nearly half of patients showed HBV reinfection.ConclusionsPatients with high serum HBV-DNA and HBcrAg levels before OLT (particularly >3 log10 copies/mL and >4 log10 IU/mL, respectively) should be followed with care for HBV recurrence.

Trastuzumab, a humanized antibody targeting HER2, exhibits remarkable therapeutic efficacy against HER2-positive breast and gastric cancers; however, acquired resistance presents a formidable obstacle to long-term tumor responses in the majority of patients. Here, we show the mechanism of resistance to trastuzumab in HER2-positive human cancer cells and explore the molecular sensitization by exogenous expression of HER2-extracellular domain (ECD) in HER2-negative or trastuzumab-resistant human cancer cells. We found that long-term exposure to trastuzumab induced resistance in HER2-positive cancer cells; HER2 expression was downregulated, and antibody-dependent cellular cytotoxicity (ADCC) activity was impaired. We next examined the hypothesis that trastuzumab-resistant cells could be re-sensitized by the transfer of non-functional HER2-ECD. Exogenous HER2-ECD expression induced by the stable transfection of a plasmid vector or infection with a replication-deficient adenovirus vector had no apparent effect on the signaling pathway, but strongly enhanced ADCC activity in low HER2-expressing or trastuzumab-resistant human cancer cells. Our data indicate that restoration of HER2-ECD expression sensitizes HER2-negative or HER2-downregulated human cancer cells to trastuzumab-mediated ADCC, an outcome that has important implications for the treatment of human cancers.

“Cell-in-cell” denotes an invasive phenotype in which one cell actively internalizes in another. The novel human T-cell line HOZOT, established from human umbilical cord blood, was shown to penetrate a variety of human cancer cells but not normal cells. Oncolytic viruses are emerging as biological therapies for human cancers; however, efficient viral delivery is limited by a lack of tumor-specific homing and presence of pre-existing or therapy-induced neutralizing antibodies. Here, we report a new, intriguing approach using HOZOT cells to transmit biologics such as oncolytic viruses into human cancer cells by cell-in-cell invasion. HOZOT cells were successfully loaded via human CD46 antigen with an attenuated adenovirus containing the fiber protein of adenovirus serotype 35 (OBP-401/F35), in which the telomerase promoter regulates viral replication. OBP-401/F35–loaded HOZOT cells were efficiently internalized into human cancer cells and exhibited tumor-specific killing by release of viruses, even in the presence of anti-viral neutralizing antibodies. Moreover, intraperitoneal administration of HOZOT cells loaded with OBP-401/F35 significantly suppressed peritoneally disseminated tumor growth in mice. This unique cell-in-cell property provides a platform for selective delivery of biologics into human cancer cells, which has important implications for the treatment of human cancers.