Merging targeted systemic gene delivery and systemic chemotherapy against cancer, chemovirotherapy, has the potential to improve chemotherapy and gene therapy treatments and overcome cancer resistance. We introduced a bacteriophage (phage) vector, named human adeno-associated virus (AAV)/phage or AAVP, for the systemic targeting of therapeutic genes to cancer. The vector was designed as a hybrid between a recombinant adeno-associated virus genome (rAAV) and a filamentous phage capsid. To achieve tumor targeting, we displayed on the phage capsid the double-cyclic CDCRGDCFC (RGD4C) ligand that binds the alpha-V/beta-3 (α_vβ₃) integrin receptor. Here, we investigated a combination of doxorubicin chemotherapeutic drug and targeted gene delivery by the RGD4C/AAVP vector. Firstly, we showed that doxorubicin boosts transgene expression from the RGD4C/AAVP in two-dimensional (2D) cell cultures and three-dimensional (3D) tumor spheres established from human and murine cancer cells, while preserving selective gene delivery by RGD4C/AAVP. Next, we confirmed that doxorubicin does not increase vector attachment to cancer cells nor vector cell entry. In contrast, doxorubicin may alter the intracellular trafficking of the vector by facilitating nuclear accumulation of the RGD4C/AAVP genome through destabilization of the nuclear membrane. Finally, a combination of doxorubicin and RGD4C/AAVP-targeted suicide gene therapy exerts a synergistic effect to destroy human and murine tumor cells in 2D and 3D tumor sphere settings.

Non-alcoholic fatty liver disease (NAFLD) is caused by excessive accumulation of fat within the liver, leading to further severe conditions such as non-alcoholic steatohepatitis (NASH). Progression of healthy liver to steatosis and NASH is not yet fully understood in terms of process and response. Hepatic oxidative stress is believed to be one of the factors driving steatosis to NASH. Oxidative protein modification is the major cause of protein functional impairment in which alteration of key hepatic enzymes is likely to be a crucial factor for NAFLD biology. In the present study, we aimed to discover carbonylated protein profiles involving in NAFLD biology in vitro. Hepatocyte cell line was used to induce steatosis with fatty acids (FA) in the presence and absence of menadione (oxidative stress inducer). Two-dimensional gel electrophoresis-based proteomics and dinitrophenyl hydrazine derivatization technique were used to identify carbonylated proteins. Sequentially, in order to view changes in protein carbonylation pathway, enrichment using Funrich algorithm was performed. The selected carbonylated proteins were validated with western blot and carbonylated sites were further identified by high-resolution LC-MS/MS. Proteomic results and pathway analysis revealed that carbonylated proteins are involved in NASH pathogenesis pathways in which most of them play important roles in energy metabolisms. Particularly, carbonylation level of ATP synthase subunit α (ATP5A), a key protein in cellular respiration, was reduced after FA and FA with oxidative stress treatment, whereas its expression was not altered. Carbonylated sites on this protein were identified and it was revealed that these sites are located in nucleotide binding region. Modification of these sites may, therefore, disturb ATP5A activity. As a consequence, the lower carbonylation level on ATP5A after FA treatment solely or with oxidative stress can increase ATP production. The reduction in carbonylated level of ATP5A might occur to generate more energy in response to pathological conditions, in our case, fat accumulation and oxidative stress in hepatocytes. This would imply the association between protein carbonylation and molecular response to development of steatosis and NASH.

Background: Human papillomavirus (HPV) infection causes around 90% of cervical cancer cases, and cervical cancer is a leading cause of female mortality worldwide. HPV-derived oncoprotein E7 participates in cervical carcinogenesis by inducing aberrant host DNA methylation. However, the targeting specificity of E7 methylation of host genes is not fully understood but is important in the down-regulation of crucial proteins of the hallmark cancer pathways. In this study, we aim to link E7-driven aberrations in the host proteome to corresponding gene promoter hypermethylation events in the hope of providing novel therapeutic targets and biomarkers to indicate the progression of cervical cancer. Methods: HEK293 cells were transfected with pcDNA3.1-E7 plasmid and empty vector and subjected to mass spectrometry-based proteomic analysis. Down-regulated proteins (where relative abundance was determined significant by paired T-test) relevant to cancer pathways were selected as gene candidates for mRNA transcript abundance measurement by qPCR and expression compared with that in SiHa cells (HPV type 16 positive). Methylation Specific PCR was used to determine promoter hypermethylation in genes downregulated in both SiHa and transfected HEK293 cell lines. The FunRich and STRING databases were used for identification of potential regulatory transcription factors and the proteins interacting with transcription factor gene candidates, respectively. Results: Approximately 400 proteins totally were identified in proteomics analysis. The transcripts of six genes involved in the host immune response and cell proliferation (PTMS, C1QBP, BCAP31, CDKN2A, ZMYM6 and HIST1H1D) were down-regulated, corresponding to proteomic results. Methylation assays showed four gene promoters (PTMS, C1QBP, BCAP31 and CDKN2A) were hypermethylated with 61, 55.5, 70 and 78% increased methylation, respectively. Those four genes can be regulated by the GA-binding protein alpha chain, specificity protein 1 and ETS-like protein-1 transcription factors, as identified from FunRich database predictions. Conclusions: HPV E7 altered the HEK293 proteome, particularly with respect to proteins involved in cell proliferation and host immunity. Down-regulation of these proteins appears to be partly mediated via host DNA methylation. E7 possibly complexes with the transcription factors of its targeting genes and DNMT1, allowing methylation of specific target gene promoters.