Recombinant Newcastle disease virus expressing human IFN-λ1 (rL-hIFN-λ1)-induced apoptosis of A549 cells is connected to endoplasmic reticulum stress pathways.

BACKGROUND: IFN-λs are a kind of cytokine with anti-tumor, immunomodulatory, and anti-proliferative activity. Recent studies have shown that the recombinant Newcastle disease virus expresses human IFN-λ1 (rL-hIFN-λ1), which plays a role in gastric cancer cell apoptosis. Endoplasmic reticulum stress (ERS) induces autophagy and apoptosis in tumor cells. In this study, we explored the relationship between ERS and rL-hIFN-λ1-induced apoptosis of lung adenocarcinoma A549 cells and its underlying mechanism. METHODS: First, we investigated the effect of rL-hIFN-λ1 on cellular proliferation, migration, and proteins associated with ERS, autophagy, and apoptosis of A549. Second, after administration of the ERS inhibitor, the associated proteins induced by rL-hIFN-λ1 were detected. Finally, a subcutaneous mouse model was used to examine the effect of rL-hIFN-λ1 on tumor growth and the ERS and apoptosis associated proteins in tumor tissues. RESULTS: The results showed that the proliferation and migration of A549 cells, and tumor tissue growth were significantly inhibited and the ERS, autophagy, and apoptosis associated proteins were upregulated in the experimental group. Additionally, both 4-PBA and knockdown of PERK or CHOP reduced the levels of rL-hIFN-λ1-induced autophagy and apoptosis-associated proteins. BCL-2 knockdown caused autophagy and apoptosis associated protein upregulation. CONCLUSIONS: In summary, rL-hIFN-λ1 inhibited cell proliferation and activated ERS, autophagy, and apoptosis in A549 cells and tissues, and when ERS pathways were blocked, the inhibiting effect was even more pronounced. Therefore, the recombinant Newcastle disease virus rL-hIFN-λ1-induced apoptosis of A549 cells is connected to ER stress and could be a promising therapeutic agent for lung adenocarcinoma.

Knockdown of TWIST enhances the cytotoxicity of chemotherapeutic drugs in doxorubicin-resistant HepG2 cells by suppressing MDR1 and EMT.

The transcription factor twist family bHLH transcription factor 1 (TWIST), which is a member of the basic helix-loop-helix class of proteins, is known to induce epithelial-mesenchymal transition (EMT) and promote cancer metastasis. TWIST has previously been reported to be associated with multidrug resistance (MDR), since its depletion increases drug sensitivity. Although these previous studies have established a strong association between EMT and MDR, the molecular mechanism remains obscure. The present study demonstrated that TWIST protein expression was elevated in liver cancer, and was positively correlated with multidrug resistance protein 1 (MDR1) expression. Conversely, MDR1 was negatively correlated with E­cadherin expression in liver cancer samples. In addition, the present study indicated that doxorubicin-resistant HepG2 (R­HepG2) cells acquired an EMT phenotype. TWIST was also more highly expressed in R­HepG2 cells compared with in parental HepG2 cells. Knockdown of TWIST increased the sensitivity of R­HepG2 cells to 5-fluroracil, cisplatin and doxorubicin through a reduction in MDR1 expression and drug efflux ability. Furthermore, knockdown of TWIST in R­HepG2 cells inhibited the migratory ability of cells and suppressed the EMT phenotype. These findings demonstrated that targeting TWIST may be considered a novel strategy to overcome drug resistance in liver cancer.

SPOCK1 promotes the invasion and metastasis of gastric cancer through Slug-induced epithelial-mesenchymal transition.

Metastasis is a crucial impediment to the successful treatment for gastric cancer. SPOCK1 has been demonstrated to facilitate cancer metastasis in certain types of cancers; however, the role of SPOCK1 in the invasion and metastasis of gastric cancer remains elusive. SPOCK1 and epithelial-mesenchymal transition (EMT)-related biomarkers were detected by immunohistochemistry and Western blot in gastric cancer specimens. Other methods including stably transfected against SPOCK1 into gastric cancer cells, Western blot, migration and invasion assays in vitro and metastasis assay in vivo were also performed. The elevated expression of SPOCK1 correlates with EMT-related markers in human gastric cancer tissue, clinical metastasis and a poor prognosis in patients with gastric cancer. In addition, knockdown of SPOCK1 expression significantly inhibits the invasion and metastasis of gastric cancer cells in vitro and in vivo, inversely, SPOCK1 overexpression results in the opposite effect. Interestingly, SPOCK1 expression has no effect on cell proliferation in vitro and in vivo. Regarding the mechanism(s) of SPOCK1-induced cells invasion and metastasis, we prove that Slug-induced EMT is involved in SPOCK1-facilitating gastric cancer cells invasion and metastasis. The elevated SPOCK1 expression is closely correlated with cancer metastasis and patient survival, and SPOCK1 promotes the invasion and metastasis of gastric cancer through Slug-mediated EMT, thereby possibly providing a novel therapeutic target for gastric cancer.

Recombinant Newcastle disease virus rL-RVG enhances the apoptosis and inhibits the migration of A549 lung adenocarcinoma cells via regulating alpha 7 nicotinic acetylcholine receptors in vitro.

BACKGROUND: The aim of this study were to investigate the possible pro-apoptotic mechanisms of the recombinant Newcastle disease virus (NDV) strain rL-RVG, which expresses the rabies virus glycoprotein, in A549 lung adenocarcinoma cells via the regulation of alpha 7 nicotinic acetylcholine receptors (α7 nAChRs) and to analyze the relationships between α7 nAChR expression in lung cancer and the clinical pathological features. METHODS: α7 nAChR expression in A549, LΑ795, and small-cell lung carcinoma (SCLC) cells, among others, was detected using reverse transcription polymerase chain reaction (RT-PCR). The optimal α7 nAChR antagonist and agonist concentrations for affecting A549 lung adenocarcinoma cells were detected using MTT assays. The α7 nAChR expression in A549 cells after various treatments was assessed by Western blot, immunofluorescence and RT-PCR analyses. Apoptosis in the various groups was also monitored by Western blot and TUNEL assays, followed by the detection of cell migration via transwell and scratch tests. Furthermore, α7 nAChR expression was examined by immunohistochemistry in lung cancer tissue samples from 130 patients and 40 pericancerous tissue samples, and the apoptotis in lung adenocarcinoma tissue was detected by Tunel assay, Then, the expression levels and clinicopathological characteristics were analyzed. RESULTS: Of the A549, LΑ795, SCLC and U251 cell lines, the A549 cells exhibited the highest α7 nAChR expression. The cells infected with rL-RVG exhibited high RVG gene and protein expression. The rL-RVG group exhibited weaker α7 nAChR expression compared with the methyllycaconitine citrate hydrate (MLA, an α7 nAChR antagonist) and NDV groups. At the same time, the MLA and rL-RVG treatments significantly inhibited proliferation and migration and promoted apoptosis in the lung cancer cells (P < 0.05). The expression of α7 nAChR was upregulated in lung cancer tissue compared with pericancerous tissue (P = 0.000) and was significantly related to smoking, clinical tumor-node-metastases stage, and histological differentiation (P < 0.05). The AI in lung adenocarcinoma tissue in high-medium differentiation group was lower than that in low differentiation group (p < 0.01). CONCLUSIONS: An antagonist of α7 nAChR may be used as a molecular target for lung adenocarcinoma therapy. Recombinant NDV rL-RVG enhances the apoptosis and inhibits the migration of A549 lung adenocarcinoma cells by regulating α7 nAChR signaling pathways.

Genetically engineered Newcastle disease virus expressing human interferon-λ1 induces apoptosis in gastric adenocarcinoma cells and modulates the Th1/Th2 immune response.

Interferon-λ1 (IFN-λ1), a recently discovered cytokine of the type III IFN family, was found to be a therapeutic alternative to type I IFN in terms of tumors. Using reverse genetics technique, we generated a recombinant Newcastle disease virus (NDV) LaSota strains named as human IFN­λ1 recombinant adenovirus (rL-hIFN-λ1) containing human IFN-λ1 gene and further evaluated the expressing of IFN-λ1 in human gastric adenocarcinoma cell line SGC-7901 after infected with rL-hIFN-λ1 by using western blot analysis, RT-PCR and immunofluorescence analyses. IFN-λl specific receptor IFNLR1 was detected on several gastric tumor cell lines including SGC-7901 and AGS and on PBMCs.The expression of the IFN-λ1 proteins reached a high level detected in the supernatant harvested 24 h after the infection of tumor cells. The proliferation changes of SGC infected with rL-hIFN-λ1 was significantly inhibited compared with NDV-infected group. Apoptosis was significantly induced by rL-hIFN-λ1 in gastric cancer cells compared with NDV virus tested by TUNEL assay, western blot analysis and Annexin V flow cytometry. Due to the high dose of IFN-λ1 expressed by the rL-hIFN-λ1-infected tumor cells, the immune study showed that rL-hIFN-λ1 increased IFN-γ production [the T helper cell subtype 1 (Th1) response] and inhibited interleukin (IL)-13 production [the T helper cell subtype 2 (Th2) response] to change the Th1/Th2 response of tumor microenvironment which inhibited tumor growth. This study aims at building recombinant NDV rL-hIFN-λ1 as an efficient antitumor agent.

Recombinant Newcastle disease virus (rL-RVG) triggers autophagy and apoptosis in gastric carcinoma cells by inducing ER stress.

We have reported that the recombinant avirulent Newcastle disease virus (NDV) LaSota strain expressing the rabies virus glycoprotein (rL-RVG) could induce autophagy and apoptosis in gastric carcinoma cells. In the present study, we explored the upstream regulators, endoplasmic reticulum (ER) stress that induce autophagy and apoptosis and the relationships among them. For this purpose, SGC-7901 and HGC cells were infected with rL-RVG. NDV LaSota strain and phosphate-buffered saline (PBS) were treated as the control groups. Western blotting and immunofluorescence microscopy were used to detect the expression of the ER stress-related proteins glucose-regulated protein 78 (GRP78) and the transcription factor GADD153 (CHOP), among others. The expression of beclin-1 and the conversion of light chain (LC) 3-I were used to determine the occurrence of autophagy, and flow cytometry (FCM) and western blotting were used to examine apoptosis-related protein expression. Transmission electron microscopy was also performed to monitor the ultrastructure of the cells. Moreover, small interfering (si) RNA was used to knock down CHOP expression. rL-RVG treatment increased the expression of ER stress-related proteins, such as GRP78, CHOP, activating transcriptional factor 6 (ATF6), X-box-binding protein 1 (XBP-1), and phosphorylated eukaryotic initiation factor 2 (p-eIF2α), in a time- and concentration-dependent manner, and knockdown of CHOP reduced LC3-II conversion and beclin-1 expression. When ER stress was inhibited with 4-PBA, the expression of both autophagy-related proteins and apoptosis-related proteins markedly decreased. Interestingly, inhibition of autophagy with 3-methyladenine (3MA) decreased not only apoptosis-related protein expression but also ER stress-related protein expression. Moreover, we found that downregulation of the c-Jun N-terminal kinase (JNK) pathway by SP600125 reduced LC3-II conversion, beclin-1 expression and caspase-3 activation. Collectively, the results suggest that rL-RVG increased ER stress in three branch pathways (ATF6, inositol-requiring enzyme 1 (IRE1), and PKR-like ER protein kinase (PERK)) that are upstream regulators of autophagy and apoptosis. Moreover, the IRE1-JNK pathway played an important role in switching ER stress to autophagy. These findings will provide molecular bases for developing rL-RVG into a drug candidate for the treatment of gastric carcinoma.

GALC gene is downregulated by promoter hypermethylation in Epstein-Barr virus-associated nasopharyngeal carcinoma.

The aim of the study was to investigate the tumor-suppressor effect of GALC in Epstein-Barr virus (EBV)-associated nasopharyngeal carcinoma (NPC) and determine whether GALC was downregulated by promoter hypermethy-lation in the NPC cell line, CNE-2Z. Forty-one archival NPC biopsy specimens were compared with 15 chronic nasopharyngitis specimens. EBV-encoded RNA (EBER) was verified by in situ hybridization and GALC protein expression was analyzed by immunohistochemistry. Promoter methylation in CNE-2Z cells was analyzed by bisulfite sequencing polymerase chain reaction. The functional role of GALC in NPC was investigated by restoring GALC expression in CNE-2Z cells via treatment with the DNA-deme-thylating agent 5-Aza-2'-deoxycytidine (5-Aza­dC). EBER was expressed in 92.68% NPC specimens but no chronic nasopharyngitis specimens (P<0.01). GALC protein was present in 60% of chronic nasopharyngitis specimens and 24.39% NPC specimens (P<0.05). GALC protein expression was present significantly more frequently in tumors without lymph node metastasis than in those with metastasis (P<0.05). Logistic regression showed that GALC protein expression protected against lymph node metastasis (P<0.05). GALC protein expression was not correlated with age, gender and TNM stage (P>0.05). Treatment of GALC-negative CNE-2Z cells with 5-Aza-dC reduced GALC promoter methylation and restored GALC expression in a dose-dependent manner (P<0.05). The re-expression of GALC in CNE-2Z cells reduced cell proliferation and migration compared to the controls (P<0.05). GALC was downregulated by promoter hypermethylation and contributed to the pathogenesis of EBV-associated NPC. The findings showed the putative tumor-suppressor effect of GALC in NPC.

Autophagy is involved in recombinant Newcastle disease virus (rL-RVG)-induced cell death of stomach adenocarcinoma cells in vitro.

Oncolytic viruses can kill malignant cells while sparing normal cells. Multiple pathways are involved in this action. The antitumor effects of viral infection on SGC-7901 and AGS cells were investigated. We measured endoplasmic reticulum stress and autophagy caused by the recombinant avirulent Newcastle disease virus (NDV) LaSota strain expressing the rabies virus glycoprotein (rL-RVG) and the NDV wild-type strain. The dose-response curves were analyzed using the MTT assay. The expression of RVG was detected by western blotting, RT-PCR and immunofluorescence analyses. Cell death and autophagy were observed using transmission electron microscopy, TUNEL and western blotting. Endoplasmic reticulum stress and the mitochondrial transmembrane potential were detected by western blotting and immunofluorescence, respectively. Immunofluorescence, western blot and RT-PCR analyses indicated that RVG gene and protein were expressed in SGC-7901 and AGS cells infected by rL-RVG. MTT and TUNEL analyses showed that the growth of SGC-7901 and AGS cells in the rL-RVG-infected group was significantly inhibited compared with the wild-type NDV-infected group (p<0.05). Western blot analysis indicated that rL-RVG and NDV induced increases in apoptosis, endoplasmic reticulum stress, and autophagy in the SGC-7901 and AGS cells. However, apoptosis and autophagy decreased in these cells after the application of the autophagy pathway inhibitor 3-MA or ATG-5-specific siRNA. Immunofluorescence analysis showed that the mitochondrial membrane potential collapsed. Taken together, these results indicate that the rL-RVG virus group is much more powerful compared with the NDV-infected group (p<0.05). rL-RVG and NDV are potent antitumor agents that induce autophagy.

Expression of miRNA-146a in nasopharyngeal carcinoma is upregulated by Epstein-Barr virus latent membrane protein 1.

We aimed to investigate the relationship between miRNA-146a and latent membrane protein 1 (LMP1) of Epstein-Barr virus (EBV) in nasopharyngeal carcinoma (NPC). The expression levels of LMP1 in 40 cases of NPC, 28 cases of chronic nasopharyngitis and NPC cell lines CNE1 and CNE1-GL (in which LMP1 was stably transfected) were detected by immunohistochemical staining. The expression of miRNA-146a in 16 cases of NPC, 13 cases of chronic nasopharyngitis and cell lines was determined by quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis. A plasmid containing the luciferase gene under the control of miRNA-146a promoter (pri-miRNA-146a) was constructed and transfected into NPC cells, and the luciferase activity was detected. LMP1 was positive in 17.9% (5/28) of chronic nasopharyngitis cases and 62.5% (25/40) of NPC cases (p<0.01). The miRNA-146a levels in NPC were significantly higher than that in chronic nasopharyngitis (p<0.01), and were higher in CNE1-GL cells than those in CNE1 cells (p<0.01). The expression of miRNA-146a in human NPC was elevated by EBV-associated antigen LMP1, probably through the activation of the miRNA-146a promoter.

[Pathology of myocardial fatty infiltration: an autopsy report from 117 cases].

OBJECTIVE: To explore the risk factors related to the formation of myocardial fatty infiltration and possible pathological consequences. METHODS: The macroscopic and microscopic findings in 117 autopsy cases with myocardial fatty infiltration were examined during October, 2001 to June, 2009. RESULTS: There was a significant positive correlation between the macroscopic grading of subepicardial adipose tissue and the microscopic myocardial fatty infiltrative degree(r(s) = 0.57, P < 0.01) but there was no correlations between the myocardial fatty infiltrative degree and age as well as coronary arteriosclerosis (all P > 0.05). The percent of myocardial atrophy was 39.32% (46/117), and the rate of myocardial atrophy in mild myocardial fatty infiltration group (13/63, 20.63%) was significantly lower than that in moderate myocardial fatty infiltration group (22/39, 34.92%; chi(2) = 12.14, P < 0.01) and in severe myocardial fatty infiltration group (11/15, 73.33%; chi(2) = 13.42, P < 0.01). There were 28 sudden cardiac deaths among the 117 cases including 6 deaths due to myocardial fatty infiltration. CONCLUSIONS: Myocardial fatty infiltration is often associated with myocardial atrophy, even with sudden cardiac death but is not an accompanying pathologic changes of aging and coronary arteriosclerosis.

[Pathology of accidental electrocution: an autopsy study of 16 cases].

OBJECTIVE: To study the pathologic findings seen in lethal cases due to accidental electrocution. METHODS: The macroscopic and microscopic findings in 16 autopsy cases died of electrocution encountered during the period from January, 2001 to July, 2008 were retrospectively reviewed. RESULTS: Typical electric marks were found on gross examination in 5 of the 16 cases studied. Histologically, 11 of the 16 cases showed evidence of electric burn. The morphologic features of atypical electric marks varied. Simple epidermal exfoliation and color changes were relatively common. Pathologic changes in internal viscera included disarray of myocardial fibers. Rupture of myocardial fibers was readily identified than in non-electrocution death. Sometimes, focal interstitial hemorrhage and polarization of endothelial cells were seen. CONCLUSIONS: The electric marks on the skin, as confirmed by histologic examination, remain important sequelae of electrocution. The pathologic changes seen in myocardium provide additional clues to the diagnosis.

[Histopathological characterization of drug-related death].

OBJECTIVE: To study the histopathological changes in drug-related death cases in order to provide valuable information for its diagnosis. METHODS: Thirty cases of drug-related death were collected for systemic autopsy and histopathology examination. Ante mortem history and other informations of each case were also reviewed and analyzed. RESULTS: Injection marks, emaciation, asphyxia and histopathological changes in critical organs and tissues correlated with addiction behavior. In the 30 cases, 20% died of diseases, 33.3% acute drug intoxication, 26.7% quitting drug, 10% sudden death, and 10% outside violence. CONCLUSION: Systemic autopsy and histopathology examination in drug-related death are useful for determination of the cause of death in these cases.

[Construction and screening of a ribozyme targeting telomerase RNA and effects of telomerase ribozyme on proliferation and apoptosis of CNE-2Z cells].

BACKGROUND & OBJECTIVE: It was proved that telomerase is an important determinant in tumor progression and cell immortalization. Ribozyme is a special kind of trans-acting RNA with endonuclease activity and sequence-specific catalytic RNA molecules, which can cleave target RNA. It was reported that telomerase activity is present in human poorly-differentiated nasopharyngeal carcinoma (NPC) CNE-2Z cells. This study was designed to construct eukaryotic expression plasmids containing telomerase ribozyme (teloRZ)gene targeting the template region of human telomerase RNA (hTR) and then to transfect the plasmids into CNE-2Z cells by electroporation to investigate the effect of teloRZ on proliferation and apoptosis of those transfected CNE-2Z cells. METHODS: Hammer ribozyme gene teloRZ directed against telomerase RNA templet was designed and synthesized to serve as a telomerase inhibitor. Three different eukaryotic expression plasmids carried with the green fluorescent protein (GFP) reporter gene and puromycin-resistance gene and containing teloRZ gene were constructed. They were referred to as pGFPuro-teloRZ2.1, pGFPuro-teloRZ7.1, and pGFPuro-teloRZ7.7 and differed in the relative orientation of the genes for telomerase-ribozyme and puromycin-resistance. The CNE-2Z cells were transfected with three expression plasmids and control plasmid pPAT-GFP by electroporation. The expression of GFP was detected by fluorescent microscope; cellular proliferation index (PI) and apoptosis were investigated by flow cytometry analysis and fluorescence staining. RESULTS: PI of CNE-2ZGTR7.1 cells transfected by plasmid pGFPuro-teloRZ7.1 (25.100%+/-0.141%)was significantly lower than those of CNE-2Z cells untransfected by any plasmid (53.663%+/-16.981%),CNE-2ZG cells transfected by control plasmid pPAT-GFP (61.575%+/-5.166%),CNE-2ZGTR2.1 cells transfected by plasmid pGFPuro-teloRZ2.1 (61.500%+/-20.082%), and CNE-2ZGTR7.7 cells transfected by plasmid pGFPuro-teloRZ7.7 (59.400%+/-13.933%) (P< 0.01). GFP was detected in CNE-2ZG cells,CNE-2ZGTR7.1 cells, and CNE-2ZGTR7.7 cells;while there was no GFP expression in CNE-2Z cells and CNE-2ZGTR2.1 cells. The plasmid pGFPuro-teloRZ7.1 was selected from 3 plasmids for further experiments. Apoptosis could be observed in CNE-2ZGTR7.1 cells after 12 generations. There was no apoptosis occurring in CNE-2Z and CNE-2ZG cells. CONCLUSION: The teloRZ7.1 gene was electroporated successfully into CNE-2Z cells. TeloRZ7.1 can inhibit the proliferation and induce apoptosis of CNE-2Z cells. These findings suggest the potential application of ribozyme teloRZ7.1 as telomerase inhibitor.