Delivery of Superoxide Dismutase 3 Gene with Baculoviruses Inhibits TNF-α Triggers Vascular Smooth Muscle Cell Proliferation and Inflammation.

BACKGROUND: Superoxide dismutase 3 (SOD3), recognized as a potent free radical scavenger, exhibits antioxidant, anti-inflammatory, and anti-angiogenic properties. However, the molecular mechanisms underlying the protective effects of SOD3 on the vascular smooth muscle cell during atherosclerosis remain unclear. OBJECTIVES: This study aimed to investigate the efficacy of the baculovirus expressing SOD3 gene delivery to vascular smooth muscle cells (VSMCs) and investigate whether the overexpression of SOD3 mitigates cell proliferation and migration induced by tumor necrosis factor-α (TNF-α). METHODS: A baculoviral vector containing SOD3 cDNA (vAcMBac-CMV-IE-SOD3) was constructed and utilized to deliver the SOD3 gene into primary rat VSMCs. Cells were stimulated with recombinant TNF-α, and then cell proliferation and migration were evaluated using the bromodeoxyuridine and wound healing assay. Western blot was used to verify the expression of cell cycle regulators, cellular mediators, and proliferative biomarkers. Zymography, immunofluorescence staining, and ELISA assay were conducted to assess the expression levels of matrix metalloproteinases. RESULTS: The results demonstrated efficient and non-cytotoxic transduction of vAcMBac- CMV-IE-SOD3 in VSMCs. SOD3 overexpression significantly suppressed cell proliferation and motility by inhibiting cell cycle regulators in TNF-α-induced cells. TNF-α elevated protein levels of phospho-ERK and phospho-Akt were reduced markedly by SOD3-overexpressing. Additionally, SOD3 overexpression attenuated the elevation of MMP-2 and MMP-9, the pro-inflammatory and proliferative biomarkers. Overall, the SOD3 gene delivery exhibited potent anti-proliferation and anti-inflammation effects on TNF-α-induced VSMCs. CONCLUSION: An effective SOD3 gene delivery using a recombinant baculoviral vector has been successfully established and is useful for overexpression of the SOD gene family. This approach provides new therapeutic strategies in gene therapy against atherosclerosis.

Inhibition of Neointima Hyperplasia, Inflammation, and Reactive Oxygen Species in Balloon-Injured Arteries by HVJ Envelope Vector-Mediated Delivery of Superoxide Dismutase Gene.

Extracellular superoxide dismutase (EC-SOD) has been implicated in regulation of vascular function but its underlying molecular mechanism is largely unknown. These two-step experiments investigate whether hemagglutinating virus of Japan envelope (HVJ-E) vector-mediated EC-SOD gene delivery might protect against neointima formation, vascular inflammation, and reactive oxygen species (ROS) generation, and also explore cell growth signaling pathways. The first in-vitro experiment was performed to assess the transfection efficacy and safety of HVJ-E compared to lipofectamine®. Results revealed that HVJ-E has higher transfection efficiency and lower cytotoxicity than those of lipofectamine®. Another in-vivo study initially used balloon denudation to rat carotid artery, then delivered EC-SOD cDNA through the vector of HVJ-E. Arterial section with H&E staining from the animals 14 days after balloon injury showed a significant reduction of intima-to-media area ratio in EC-SOD transfected arteries when compared with control (empty vector-transfected arteries) (p < 0.05). Arterial tissue with EC-SOD gene delivery also exhibited lower levels of ROS, as assessed by fluorescent microphotography with dihydroethidium staining. Quantitative RT-PCR revealed that EC-SOD gene delivery significantly diminished mRNA expression of tumor necrosis factor (TNF)-α and interleukin (IL)-1ß (p < 0.05 in all comparisons). An immunoblotting assay from vascular smooth muscle cell (VSMC) cultures showed that the EC-SOD transfected group attenuated the activation of MEK1/2, ERK1/2, and Akt signaling significantly. In conclusion, EC-SOD overexpression by HVJ-E vector inhibits neointima hyperplasia, inflammation, and ROS level triggered by balloon injury. The modulation of cell growth-signaling pathways by EC-SOD in VSMCs might play an important role in these inhibitory effects.

MicroRNAs derived from the insect virus HzNV-1 promote lytic infection by suppressing histone methylation.

Heliothis zea nudivirus-1 (HzNV-1) is an insect virus that can induce both lytic and latent infections in various insect cell lines. During latent infection, several microRNAs (miRNAs) are produced from persistency-associated gene 1 (pag1) as the only detectable HzNV-1 transcript. Previous studies have shown that the pag1 gene suppresses the immediate-early gene hhi1 and promotes host switching into a latent infection via miRNAs derived from pag1. Although other functions of the miRNAs derived from pag1 have not yet been elucidated, several studies have suggested that miRNAs encoded from latency-associated genes can regulate histone-associated enzymes. Because pag1 is a noncoding transcript, it potentially regulates host chromatin structure through miRNAs upon infection. Nevertheless, the exact mechanism by which pag1 alters viral infections remains unknown. In this study, we found that the pag1-encoded miRNA miR-420 suppresses expression of the histone modification-associated enzyme su(var)3-9. Therefore, this miRNA causes histone modification to promote HzNV-1 infection. These results suggest that HzNV-1 may directly influence epigenetic regulation in host cells through interactions with pag1 miRNAs to promote lytic infection. This study provides us with a better understanding of both the HzNV-1 infection pathway and the relationship between viral miRNAs and epigenetic regulation.

Deciphering the Molecular Mechanism Underlying the Inhibitory Efficacy of Taiwanese Local Pomegranate Peels against Urinary Bladder Urothelial Carcinoma.

Pomegranate (Punica granatum L.) fruit has been demonstrated to have the inhibitory activities to various tumors. In this study, we try to uncover the molecular mechanism underlying the inhibitory capability of Taiwanese local pomegranate fruit to urinary bladder urothelial carcinoma. The results collected from the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay indicated that the ethanol extract of pomegranate peel exhibited better inhibitory activity to human urinary bladder urothelial carcinoma T24 and J82 cells than that of pulp. Furthermore, the ethylacetate layer of peel ethanol extract was observed to have the best inhibitory activity against urinary bladder urothelial carcinoma cells. One of the eight fractions (PEPE2 fraction) collected from the ethylacetate layer with Diaion HP-20 column chromatography demonstrated the highest inhibitory activity in urinary bladder urothelial carcinoma cells. The results of the flow cytometry and apoptotic pathway studies suggested that the inhibitory activity of PEPE2 fraction were attributed to the UBUC cell apoptosis. To confirm the above results, our results of xenograft-induced bladder tumor in nude mice showed that the oral consumption of the ethylacetate layer (2, 5, 10 and 100 mg/kg) could decrease the volume and weight of T24 tumors and caused the apoptosis in the xenografted tumors, which was observed by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labeling assay. This study provided the likelihood that the traditionally non-edible pomegranate peel waste is re-utilized to make an affordable and promising chemopreventive product to prevent UBUC incidence or recurrence.

Risk of secondary cancers in women with breast cancer and the influence of radiotherapy: A national cohort study in Taiwan.

Breast cancer is the most common cancer in women worldwide; thus, the prolongation of survival, and the incidence and risk factors, including radiotherapy, for developing secondary malignancies are important. We compared the incidence of secondary and new primary cancers in women with breast cancer (CA) and well-matched for age, geographic region, and monthly income cancer-free controls (CA). The risk for secondary cancers with and without radiotherapy was also compared in CA women. We enrolled 2422 CA patients and CA 12,110 controls. In a 4-year follow-up, the secondary cancers risk was significant in the CA group (adjusted hazard ratio [AHR]: 1.59; 95% confidence interval [CI]: 1.17-2.18). Only the risk of uterine cancer was significant compared with the controls (AHR: 6.30; 95% CI: 2.28-17.38). CA patients and <50 years old had a higher risk for secondary cancers. Developing secondary cancers was significant in the first follow-up year (AHR: 1.51; 95% CI: 1.11-2.06). Radiotherapy had no significant effect on the CA group, but it was significant (P = 0.0298) in women ≥60 years old (elderly). We recommend monitoring secondary cancers in CA women, especially those <50 years old, and during the first year of follow-up. Radiotherapy should be used more carefully in elderly CA women.

Impact of high glucose on metastasis of colon cancer cells.

AIM: To investigate the possible mechanism of how glucose promotes invasion and metastasis of colon cancer cells. METHODS: CT-26 rat colorectal cancer cells were cultured in different concentrations of glucose environments (10, 20, and 30 mmol/L). Wound healing assay and transwell chamber invasion assay were utilized to test the migration and invasion, respectively. In order to understand the role of signal transducer and activator of transcription 3 (STAT3) in the process, STAT3 inhibitors, including Stattic (an STAT3 specific inhibitor) and small interfering RNA targeting STAT3, were used to block STAT3 function to evaluate their impact on CT-26 cell motion. To verify whether STAT3 and matrix metalloproteinase-9 (MMP-9) protein expression is associated with glucose-induced cell movement, Western blot was used to compare the differences in the expression of MMP-9 and STAT3 in cells incubated with and without STAT3 inhibitors in high glucose condition. RESULTS: In both wound healing and invasion assays, the migration and invasion of CT-26 cells increased gradually with the increase in glucose concentration. However, the glucose-induced migration and invasion were obviously inhibited by STAT3 inhibitors (P<0.05). Similarly, in Western blot assessment, both MMP-9 and STAT3 expression increased under a high glucose environment and the highest expression was achieved when 30 mmol/L glucose was used. However, in cells treated with 30 mmol/L mannitol, either MMP-9 or STAT3 expression did not increase (P>0.05). When STAT3 inhibitors were added in the 30 mM glucose group, not only STAT3 but also MMP-9 expression decreased significantly (P<0.05). CONCLUSION: Our study provides evidence that glucose can promote both migration and invasion of CT-26 cells, and that the STAT3-induced MMP-9 signal pathway is involved in this process.

Suppression of urinary bladder urothelial carcinoma cell by the ethanol extract of pomegranate fruit through cell cycle arrest and apoptosis.

BACKGROUND: Pomegranate possesses many medicinal properties such as antioxidant, anti-inflammation and antitumor. It has been extensively used as a folk medicine by many cultures. Pomegranate fruit has been shown to have the inhibitory efficacy against prostate cancer and lung cancer in vitro and in vivo. It can be exploited in chemoprevention and chemotherapy of prostate cancer. In this study we examined the anti-cancer efficacy of pomegranate fruit grown in Taiwan against urinary bladder urothelial carcinoma (UBUC) and its mechanism of action. METHODS: Edible portion of Taiwanese pomegranate was extracted using ethanol and the anti-cancer effectiveness of ethanol extract was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Flow cytometry and western immunoblotting were exploited to uncover the molecular pathways underlying anti-UBUC activity of Taiwanese pomegranate ethanol extract. RESULTS: This study demonstrated that Taiwanese pomegranate fruit ethanol extract (PEE) could effectively restrict the proliferation of UBUC T24 and J82 cells. Cell cycle analyses indicated that the S phase arrest induced by PEE treatment might be caused by an increase in cyclin A protein level and a decrease in the expression of cyclin-dependent kinase 1. The results of western immunoblotting demonstrated that PEE treatment could not only evoke the activation of pro-caspase-3, -8,-9 but also increase Bax/Bcl-2 ratio in T24 cells. The above observations implicated that PEE administration might trigger the apoptosis in T24 cells through death receptor signaling and mitochondrial damage pathway. Besides we found that PEE exposure to T24 cells could provoke intensive activation of procaspase-12 and enhance the expressions of CHOP and Bip, endoplasmic reticulum (ER) stress marker, suggesting that ER stress might be the cardinal apoptotic mechanism of PEE-induced inhibition of bladder cancer cell. CONCLUSIONS: The analytical results of this study help to provide insight into the molecular mechanism of induced bladder cancer cell apoptosis by pomegranate and to develop novel mechanism-based chemopreventive strategy for bladder cancer.

Proteomic exploration of the impacts of pomegranate fruit juice on the global gene expression of prostate cancer cell.

Prostate cancer has been known to be the second highest cause of death in cancer among men. Pomegranate is rich in polyphenols with the potent antioxidant activity and inhibits cell proliferation, invasion, and promotes apoptosis in various cancer cells. This study demonstrated that pomegranate fruit juice could effectively hinder the proliferation of human prostate cancer DU145 cell. The results of apoptotic analyses implicated that fruit juice might trigger the apoptosis in DU145 cells via death receptor signaling and mitochondrial damage pathway. In this study, we exploited 2DE-based proteomics to compare nine pairs of the proteome maps collected from untreated and treated DU145 cells to identify the differentially expressed proteins. Comparative proteomics indicated that 11 proteins were deregulated in affected DU145 cells with three upregulated and eight downregulated proteins. These dys-regulated proteins participated in cytoskeletal functions, antiapoptosis, proteasome activity, NF-κB signaling, cancer cell proliferation, invasion, and angiogenesis. Western immunoblotting were implemented to confirm the deregulated proteins and the downstream signaling proteins. The analytical results of this study help to provide insight into the molecular mechanism of inducing prostate cancer cell apoptosis by pomegranate fruit juice and to develop a novel mechanism-based chemopreventive strategy for prostate cancer.

Heliothis zea nudivirus 1 gene hhi1 induces apoptosis which is blocked by the Hz-iap2 gene and a noncoding gene, pag1.

Heliothis zea nudivirus 1 (HzNV-1 or Hz-1 virus), previously regarded as a nonoccluded baculovirus, recently has been placed in the Nudivirus genus. This virus generates HzNV-1 HindIII-I 1 (hhi1) and many other transcripts during productive viral infection; during latent viral infection, however, persistency-associated gene 1 (pag1) is the only gene expressed. In this report, we used transient expression assays to show that hhi1 can trigger strong apoptosis in transfected cells, which can be blocked, at least partially, by the inhibitor of apoptosis genes Autographa californica iap2 (Ac-iap2) and H. zea iap2 (Hz-iap2). In addition to these two genes, unexpectedly, pag1, which encodes a noncoding RNA with no detectable protein product, was found to efficiently suppress hhi1-induced apoptosis. The assay of pro-Sf-caspase-1 processing by hhi1 transfection did not detect the small P12 subunit at any of the time intervals tested, suggesting that hhi1 of HzNV-1 induces apoptosis through alternative caspase pathways.

The early gene hhi1 reactivates Heliothis zea nudivirus 1 in latently infected cells.

Heliothis zea nudivirus 1 (HzNV-1), previously known as Hz-1 virus, is an insect virus able to establish both productive and latent infections in several lepidopteran insect cells. Here, we have cloned and characterized one of the HzNV-1 early genes, hhi1, which maps to the HindIII-I fragment of the viral genome. During the productive viral infection, a 6.2-kb hhi1 transcript was detectable as early as 0.5 h postinfection (hpi). The level of transcript reached a maximum at 2 hpi and gradually decreased after 4 hpi. The transcript was not detectable during the latent phase of viral infection. Upon cycloheximide treatment, much higher levels of hhi1 transcript were detected throughout the productive viral infection cycle, suggesting that newly synthesized proteins are not needed for the expression of hhi1. Nevertheless, viral coinfection can further stimulate the expression of transfected hhi1 promoter in a plasmid. Transient hhi1 expression in latently infected cells resulted in a significant increase in virus titer and viral DNA propagation, suggesting that hhi1 plays a critical role in viral reactivation. Additional experiments showed that six early genes, which possibly function in transcription or DNA replication, were activated in the latent cells upon hhi1 transfection. Among these six genes, orf90 and orf121 expression could be induced by hhi1 alone without the need for other viral genes. Our discovery should be useful for future mechanistic study of the switches of latent/productive HzNV-1 viral infections.

Genomic and host range studies of Maruca vitrata nucleopolyhedrovirus.

The complete genome of the Maruca vitrata nucleopolyhedrovirus (MaviNPV) isolated from the legume pod borer, Maruca vitrata (Lepidoptera: Pyralidae), was sequenced. It was found to be 111 953 bp in length, with an overall 39 % G+C content, and contained 126 open reading frames (ORFs) encoding predicted proteins of over 50 aa. The gene content and gene order of MaviNPV have the highest similarity to those of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) and their shared homologous genes are 100 % collinear. In fact, MaviNPV seems to be a mini-AcMNPV that is native to Taiwan and possesses a smaller genome with fewer auxiliary genes than the AcMNPV type species. Except for one ORF (Mv74), all of the MaviNPV ORFs have homologues in the AcMNPV genome. MaviNPV is the first lepidopteran-specific baculovirus to lack homologues of vfgf and odv-e66. In addition, MaviNPV lacks the baculovirus repeat ORF (bro) gene that corresponds to AcMNPV ORF2. Five homologous regions (hrs) were located within the MaviNPV genome, and these contained a total of 44 imperfect palindromes. Phylogenetic analysis of the whole genome revealed that MaviNPV was separated from the common ancestor of AcMNPV and Bombyx mori nucleopolyhedrovirus before these two viral species diverged from each other. Moreover, replication of MaviNPV in several cell lines and an egfp-MaviNPV infection assay revealed that IPLB-LD-652Y cells are only partially permissive to MaviNPV, which supports our conclusion that MaviNPV is a distinct species of the group I lepidopteran NPVs.

Cooperation of ie1 and p35 genes in the activation of baculovirus AcMNPV and HzNV-1 promoters.

HzNV-1 is a non-occluded virus belongs to the family of the baculovirus. One of the first detectable transcripts expressed by HzNV-1 virus infection is a 6.2 kb gene, hhi1, located in the HindIII-I fragment of the viral genome. Here we show that infection of baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV) could activate the expression of the hhi1 promoter. By using constructs containing progressive deletions of the upstream regulatory regions of the hhi1 gene, we demonstrated that the most highly activated area was located between nucleotides -62 to +277 of the hhi1 promoter. We subsequently searched the entire 130 kb AcMNPV genome and identified two baculovirus genes, ie1 and p35, that their cooperation is required for the activation of the hhi1 promoter. Further, by taking advantages of a baculovirus DNA chip and low background baculovirus gene expressions in the mammalian cells, we went on to identify a specific set of baculoviral genes, including orf21 and orf25, that could be specifically activated by the combination of ie1 and p35 genes. We conclude that a unique cooperative mechanism of ie1 and p35 exists in the genome of AcMNPV, which can activate the expression of a specific set of AcMNPV and HzNV-1 promoters.

A cell line (NTU-MV) established from Maruca vitrata (Lepidoptera: Pyralidae): Characterization, viral susceptibility, and polyhedra production.

Here we describe the establishment of a new cell line, NTU-MV, derived from pupal tissues of an economically important pest, the legume pod borer Maruca vitrata. This cell line contained four major cell types: polymorphic cells, round cells, spindle-shaped cells, and comma cells. The doubling time of MV cells in TNM-FH medium supplemented with 8% FBS at 28 degrees C was 27h. The chromosome numbers of MV cells varied widely from 16 to 268. Compared to other insect cell lines, the MV cell line produced distinct isozyme patterns with esterase, malate dehydrogenase (MDH), and lactate dehydrogenase (LDH). Confirmation that NTU-MV was derived from M. vitrata was demonstrated by showing that the sequence of the internal transcribed spacer regions (ITS) of the MV cells was 98% identical to that of M. vitrata larvae. Two NTU-MV cell strains, NTU-MV1 and NTU-MV56, were selected based on susceptibility to MaviMNPV (M. vitrata multiple nucleopolyhedrovirus). NTU-MV, MV1, and MV56 cells showed a high susceptibility to MaviMNPV and produced high yields of polyhedra (47-50OBs/cell, 4x10(7)-5.96x10(7)OBs/ml) after 2 weeks of MaviMNPV infection. We conclude that the NTU-MV cell line will be a useful tool for studying MaviMNPV as well as for the mass production of MaviMNPV polyhedra for the biocontrol of M. vitrata.