Doxycycline vs Placebo at 12 Weeks in Patients With Mild Thyroid-Associated Ophthalmopathy: A Randomized Clinical Trial.

Importance: Mild thyroid-associated ophthalmopathy (TAO) negatively impacts quality of life, yet no clinical guidelines for its treatment are available. Existing evidence supports the use of doxycycline in treating mild TAO. Objective: To evaluate the short-term (12 weeks) efficacy of doxycycline in treating mild TAO. Design, Setting, and Participants: In this placebo-controlled multicenter randomized double-masked trial, 148 patients were assessed for eligibility. After exclusions (patients who were pregnant or lactating, had an allergy to tetracyclines, or had uncontrolled systematic diseases), 100 patients with mild TAO (orbital soft tissue affected mildly) at 5 centers in China were enrolled from July 2013 to December 2019 and monitored for 12 weeks. Interventions: Participants were randomly assigned 1:1 to receive doxycycline (50 mg) or placebo once daily for 12 weeks. Main Outcomes and Measures: The primary outcome was the rate of improvement at 12 weeks compared with baseline assessed by a composite indicator of eyelid aperture (reduction ≥2 mm), proptosis (reduction ≥2 mm), ocular motility (increase ≥8°), and Graves ophthalmopathy-specific quality-of-life (GO-QOL) scale score (increase ≥6 points). Adverse events were recorded. Results: A total of 50 participants were assigned to doxycycline and 50 to placebo. The mean (SD) age was 36.7 (9.1) years; 75 participants (75.0%) were female and 100 (100.0%) were Asian. Medication compliance was checked during participant interviews and by counting excess tablets. At week 12, the improvement rate was 38.0% (19 of 50) in the doxycycline group and 16.0% (8 of 50) in the placebo group (difference, 22.0%; 95% CI, 5.0-39.0; P = .01) in the intention-to-treat population. The per-protocol sensitivity analysis showed similar results (39.6% [19 of 48] vs 16.0% [8 of 50]; difference, 23.6%; 95% CI, 6.4-40.8; P = .009). No adverse events other than 1 case of mild gastric acid regurgitation was recorded in either group. Conclusions and Relevance: The results of this study indicate that oral doxycycline, 50 mg daily, resulted in greater improvement of TAO-related symptoms at 12 weeks compared with placebo in patients with mild TAO. These findings support the consideration of doxycycline for mild TAO but should be tempered by recognizing the relatively short follow-up and the size of the cohort. Trial Registration: ClinicalTrials.gov Identifier: NCT02203682.

Phylogenetic analysis of porcine circovirus 4 in Henan Province of China: A retrospective study from 2011 to 2021.

Porcine circovirus 4 (PCV4), a novel circovirus, was first discovered in April 2019 in Hunan Province of China. At present, PCV4 infection has been detected in China and South Korea. However, until 2019, there was little information about its circulating status and genetic characteristics. To further clarify the origin and prevalence of PCV4, a total of 152 clinical samples collected from 49 different swine farms of 15 cities in Henan Province of China from 2011 to 2021 were tested for the presence of PCV4 by qPCR, and the complete genome of PCV4 strains was amplified from the positive samples and sequenced. Among these samples, 45.39% (69/152) were positive for PCV4 and 86.67% (13/15) of the cities and 67.35% (33/49) of the swine farms were positive for PCV4. The genome sequences of 15 PCV4 strains were obtained, of which two PCV4 strains (HN-ZMD-201212 and HN-XX-201212) were achieved from archival samples in 2012, indicating that PCV4 has been circulating for at least 10 years in Henan Province of China. The phylogenetic analysis showed that 15 PCV4 strains in our study together with PCV4 strain HNU-AHG1-2019 were clustered into an identical but separate evolutionary branch, with genomic identity ranging from 98.2% to 98.8%. Our research further provides significant epidemiological information on PCV4 in China, which will help understand the origin and genetic characteristics of this new virus.

Simultaneous detection and differentiation of porcine circovirus 3 and 4 using a SYBR Green І-based duplex quantitative PCR assay.

Porcine circovirus 4 (PCV4) was a novel circovirus identified from diseased pigs in 2019 in Hunan Province, China, and PCV3 and PCV4 co-infection has been reported. In order to detect and differentiate PCV3 and PCV4 simultaneously, the SYBR Green І-based duplex quantitative PCR (qPCR) assay was established in the present study. The two viruses could be easily distinguished by different Tm values: 86.5°C for PCV3 and 79°C for PCV4, while other porcine pathogens did not shown specific melting peaks. The detection limits of this duplex qPCR assay were 51.7 copies/µL for PCV3 and 67.7 copies/µL for PCV4, and both of the intra-assay and inter-assay of the CV analysis of this assay were less than 2.0 %. Sixty-four clinical samples from 22 different swine farms were screened by the duplex qPCR assay. The results showed that the positive detection rate of PCV3 was 37.5 % (24/64) and PCV4 was 34.38 % (22/64), and PCV3 and PCV4 co-infection rate was 17.19 % (11/64). The detection rate of the duplex qPCR assay was higher than that of the conventional PCR assay. The duplex qPCR was of high sensitivity and specificity, being able to provide technical support for clinical detection, differential diagnosis and control of PCV3 and PCV4.

Detection and genetic characteristics of porcine bocavirus in central China.

To investigate the epidemic profile and genetic diversity of porcine bocavirus (PBoV), 281 clinical samples, including 236 intestinal tissue samples and 45 fecal samples were collected from diarrheic piglets on 37 different pig farms in central China, and two SYBR Green I-based quantitative PCR assays were developed to detect PBoV1/2 and PBoV3/4/5, respectively. One hundred forty-eight (52.67%) of the 281 clinical samples were positive for PBoV1/2, 117 (41.63%) were positive for PBoV3/4/5, 55 (19.57%) were positive for both PBoV1/2 and PBoV3/4/5, and 86.49% (32/37) of the pig farms were positive for PBoV. Overall, the prevalence of PBoV was 74.73% (210/281) in central China. Subsequently, nearly full-length genomic sequences of two PBoV strains (designated CH/HNZM and PBoV-TY) from two different farms were determined. Phylogenetic analysis demonstrated that the two PBoV strains obtained in this study belonged to the PBoV G2 group and had a close relationship to 10 other PBoV G2 strains but differed genetically from PBoV G1, PBoV G3, and seven other bocaviruses. CH/HNZM and PBoV-TY were closely related to the PBoV strain GD18 (KJ755666), which may be derived from the PBoV strains 0912/2012 (MH558677) and 57AT-HU (KF206160) through recombination. Compared with reference strain ZJD (HM053694)-China, more amino acid variation was found in the NS1 proteins of CH/HNZM and PBoV-TY. These data extend our understanding of the molecular epidemiology and evolution of PBoV.

Molecular detection and phylogenetic analysis of Porcine circovirus 4 in Henan and Shanxi Provinces of China.

Porcine circovirus 4 (PCV4), a new circovirus with a distinct relationship to other circoviruses, was identified in 2019 in several pigs with severe clinical disease in Hunan Province, China. To investigate the epidemic profile and genetic diversity of the virus, 63 clinical samples were collected from 24 different pig farms in 14 cities in Henan and Shanxi Provinces, China, between February 2018 and December 2019, and the partial Cap gene of PCV4 was amplified by PCR. Among the 63 samples, 16 (25.40%) were positive for PCV4, and 50% (12/24) of the pig farms were positive for PCV4. PCV4 was detected in samples from pigs with different clinical presentations. One PCV4 strain (Henan-LY1-2019) was sequenced in this study, and shared 98.4% genomic nucleotide identity with PCV4 strain HNU-AHG1-2019 (accession no. MK986820) detected on a pig farm in Hunan Province in 2019. A phylogenetic analysis based on the genomes of Henan-LY1-2019 and 31 reference strains showed that the Henan-LY1-2019 strain together with PCV4 strain HNU-AHG1-2019 was grouped in a relatively independent sub-branch, and separated from other viruses in the genus Circovirus. The results of this study extend our understanding of the molecular epidemiology of PCV4.

Characteristics of the spike and ORF3 genes of porcine epidemic diarrhea virus in Henan and Shanxi provinces of China.

To investigate the epidemic characteristics of porcine epidemic diarrhea virus (PEDV), 135 clinical samples (including intestinal tissues and feces) were collected from diseased piglets during outbreaks of diarrhea from 2015 to 2019 on farms in Henan and Shanxi provinces of China where swine had been immunized with attenuated PEDV (CV777). A total of 86 clinical samples (86/135, 63.7%) were positive for PEDV by RT-PCR, and subsequently, the complete spike (S) and ORF3 genes of 32 PEDV samples were sequenced. Phylogenetic analysis showed that the 32 PEDV strains obtained in this study belonged to group 2 (pandemic variant strains) and had a close relationship to 17 Chinese strains after 2010, two South Korean strains (KNU-1305 and KNU-1807), three American strains (PC22A-P140.BI, USA/Colorado/2013, and USA/OK10240-6/2017) and a Mexican strain (PEDV/MEX/QRO/02/2017), but differed genetically from a South Korean strain (SM98), a European strain (Br1/87), a Chinese strain (LZC), and a vaccine strain (CV777). G2-a subgroup strains were the dominant pandemic variant strains circulating in Henan and Shanxi provinces of China. Furthermore, a cross-recombination event was identified in the S region of the SX/TY2/2017 strain, and the putative parental strains were the epidemic strains CH/GDGZ/2012 and CH/YZ1/2015, identified in China in 2012 and 2015, respectively. These results provide further information about PEDV evolution, which could improve our understanding of the circulation of PEDV in Henan and Shanxi provinces. This information will also be helpful for developing new strategies for prevention and control of variant strains.

Development of a duplex SYBR Greenâ based real-time PCR assay for detection of porcine epidemic diarrhea virus and porcine bocavirus3/4/5.

The duplex real-time PCR assay based on SYBR Green І was developed for detection of porcine epidemic diarrhea virus (PEDV) and porcine bocavirus (PBoV) 3/4/5 genotypes simultaneously. Two pairs of specific primers were designed targeting the N gene sequence of PEDV and VP1 gene sequence of PBoV3/4/5. PEDV and PBoV3/4/5 could be distinguished by their different melting temperatures (Tm) in one sample. The Tm value of PEDV was 83.5 °C, and the Tm value of PBoV3/4/5 was 78.5 °C, while other swine pathogens showed no specific melting peaks. The detection limits of this assay were 10 copies/µL for both PEDV and PBoV3/4/5. A total of sixty-three intestinal tissue samples were collected from piglets suffering from diarrhea, and the viral nucleic acids detected and identified by the real-time PCR assay and conventional PCR assay. The duplex real-time PCR detection results showed that the prevalence of PEDV and PBoV3/4/5 was 85.7% and 46%, respectively, and the co-infection rate of the two viruses was 28.6%. These results indicated that this duplex real-time PCR assay was a sensitive, specific and reproducible method for differentiating PEDV and PBoV3/4/5 or their co-infection.

Simultaneous detection of classical swine fever virus and porcine circovirus 3 by SYBR green I-based duplex real-time fluorescence quantitative PCR.

In the present study, the SYBR green I-based duplex quantitative polymerase chain reaction (qPCR) was developed for simultaneous detection of classical swine fever virus (CSFV) and porcine circovirus 3 (PCV3). The assay was used to detect both CSFV and PCV3 in one sample by their distinct melting temperatures (melting peaks at 87°C for CSFV and 81.5 °C for PCV3), and no specific fluorescence signals were detected for other non-targeted porcine pathogens. The assay had a high degree of linearity (R2 > 0.998) with the detection limits of 23 copies/µL for CSFV and 36 copies/µL for PCV3, and exhibited high repeatability and reproducibility with a low coefficient of variation below 2.0% in both intra- and inter-assay. In this study, 130 clinical samples collected from sick pigs in the field were tested by this assay with the positive rates of 9.23% (12/130) for CSFV and 21.54% (28/130) for PCV3 respectively, and the positive rate of CSFV and PCV3 co-infection was 6.92% (9/130). Our results showed that the developed method was a reliable diagnostic tool to monitor and survey CSFV, PCV3 and CSFV/PCV3 co-infection in the field.

Development of a TB green II-based duplex real-time fluorescence quantitative PCR assay for the simultaneous detection of porcine circovirus 2 and 3.

Porcine circovirus 3 (PCV3), as a newly emerged circovirus, is widely distributed in pig populations worldwide. Co-infection of PCV2 and PCV3 has been reported frequently in clinical samples. In the present study, a TB Green II-based duplex real-time polymerase chain reaction (qPCR) was developed to rapidly and differentially detect PCV2 and PCV3. The assay specifically detected PCV2 and PCV3, with no fluorescence signals being detected for other non-targeted pig pathogens. The duplex qPCR showed a high degree of linearity (R2 > 0.998), and its limits of detection were 10 and 78 copies/µL for PCV2 and PCV3, respectively. The duplex qPCR could detect and differentiate PCV2 (melting peaks at 85.5 °C) and PCV3 (melting peaks at 82.5 °C), and showed high repeatability and reproducibility, with intra- and inter-assay coefficients of variation of less than 2.0%. Fifty-six tissue samples from 18 pig farms were used to evaluate the duplex qPCR method. The results revealed infection rates of 66.07% (37/56) and 39.28% (22/56) for PCV2 and PCV3, respectively. The PCV2 + PCV3 co-infection rate was 39.28% (22/56). The developed method could be used as an efficient molecular biology tool for epidemiological investigations of PCV2 and PCV3.

STAT3 signaling drives EZH2 transcriptional activation and mediates poor prognosis in gastric cancer.

BACKGROUND: STAT3 signaling plays the pivotal role in tumorigenesis through EZH2 epigenetic modification, which enhanced STAT3 activity by increased tyrosine phosphorylation of STAT3. Here, another possible feedback mechanism and clinical significance of EZH2 and STAT3 were investigated in gastric cancer (GC). METHODS: STAT3, p-STAT3 (Tyr 705) and EZH2 expression were examined in 63 GC specimens with matched normal tissues by IHC staining. EZH2 and STAT3 were also identified in five GC cell lines using RT-PCR and western blot analyses. p-STAT3 protein was detected by western blotting. In order to investigate whether EZH2 expression was directly regulated by STAT3, EZH2 expression was further detected using siRNA for STAT3 or IL-6 stimulation, with dual luciferase reporter analyses, electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP) assays. The clinical significance of STAT3, p-STAT3 and EZH2 expression was evaluated by multi-factor COX regression and Kaplan-Meier analyses. RESULTS: Hyper-activation of STAT3, p-STAT3 and EZH2 expression were observed in GC cells and tissues. STAT3 signaling was correlated with EZH2 expression in GC (R = 0.373, P = 0.003), which was consistent with our data showing that STAT3 as the transcriptional factor enhanced EZH2 transcriptional activity by binding the relative promoter region (-214 ~ -206). STAT3 was an independent signature for poor survival (P = 0.002). Patients with STAT3+/EZH2+ or p-STAT3+/EZH2+ had a worse outcome than others (P < 0.001); Besides, high levels of STAT3 and EZH2 was associated with advanced TNM staging (P = 0.017). Moreover, treatment with a combination of siSTAT3 and EZH2-specific inhibitor, 3-deazaneplanocin A (DZNEP), increased the apoptotic ratio of cells. It is benefit for targeting STAT3-EZH2 interplay in GC treatment. CONCLUSIONS: Our results indicate that STAT3 status mediated EZH2 upregulation, associated with advanced TNM stage and poor prognosis, suggesting that combination with knockdown of STAT3 and EZH2 inhibitor might be a novel therapy in GC treatment. Collectively, STAT3, p-STAT3 and EZH2 expression were provided for the precision medicine in GC patients.

GKN1 inhibits cell invasion in gastric cancer by inactivating the NF-kappaB pathway.

Metastasis is a relatively early event and a major cause of death in gastric cancer (GC) patients. Gastrokine 1 (GKN1) is a stomach-specific protein that is normally expressed in gastric mucosa but not in primary tumors or cell lines. We and others have demonstrated that GKN1 inhibits cell growth; however, its role in metastasis is not clear. In this study, we explored the role of GKN1 in cell invasion. Immunohistochemistry (IHC) was used to measure the expression of GKN1 in precancerous lesions and in GCs. The cell invasion assay was employed to examine the effect of GKN1 on cell invasion. The molecular mechanism of GKN1 in inhibiting GC cell invasion in vitro was explored by western blotting. We noted a gradual decrease in GKN1 expression from normal mucosa to dysplastic gastric tissue to GC, and that low GKN1 expression was associated with metastasis (P=0.003). We showed that GKN1 inhibits cell invasion by downregulating MMP2 expression through the NF-κB pathway. These results provide molecular evidence that GKN1 inhibits metastasis in GC cells, and indicate that GKN1 is a potential novel therapeutic target for gastric cancer.

The effect of anti-TGF-ß2 antibody functionalized intraocular lens on lens epithelial cell migration and epithelial-mesenchymal transition.

Migration and epithelial-mesenchymal transition (EMT) of lens epithelial cells (LECs) are main causes of central posterior capsule opacification after cataract extraction combined with intraocular lens (IOL) implantation. In this study, commercially available hydrophobic acrylic IOLs were first pretreated with atmospheric pressure glow discharge plasma to produce plenty of negatively charged chemical groups onto IOL surface, then polyethylenimine was deposited onto IOL surfaces as a precursor monolayer, and then anti-TGF-ß2 (anti-T) antibody and poly-l-lysine were sequentially deposited onto IOL surface for four cycles followed by another upmost monolayer of anti-T antibody via layer-by-layer self-assembly technique. After the fabrication of anti-T antibody multilayers on IOL surface, the surface characteristics of the anti-T antibody functionalized IOL, as well as its effect on LECs adhesion, proliferation, migration and EMT were then tested in this study. Our results revealed that anti-T antibody multilayers could be successfully immobilized onto IOL surfaces by plasma pretreatment and layer-by-layer self-assembly technique, and could keep stable for at least 3 months on IOL surface. The anti-T antibody immobilized in the multilayers on IOL surfaces showed good immunological activity by its specific antigen-antibody interaction with exogenous TGF-ß2. Anti-T antibody functionalized IOL surface was as smooth and flat as the untreated IOL surface. No difference in optical or physical properties was found between the anti-T antibody functionalized IOLs and the untreated IOLs. Compared with the untreated IOLs, the anti-T antibody functionalized IOL greatly inhibited LECs from migration and EMT, yet showed only transient inhibition to LECs adhesion and no inhibition to LECs proliferation. With these data, we demonstrate a simple, inexpensive, and feasible method to fabricate surface functionalized IOL for in situ capture and neutralization of TGF-ß2 in the capsular bag, which might be a possible solution to preventing posterior capsule opacification after cataract surgery.

Clinicopathological significance of altered metallothionein 2A expression in gastric cancer according to Lauren's classification.

BACKGROUND: Dysregulated metallothionein 2A (MT2A) has been implicated in carcinogenesis. The purpose of this study was to investigate the expression of MT2A in gastric cancer (GC) and its correlation with prognosis. METHODS: Reverse transcription-polymerase chain reaction and real-time polymerase chain reaction were used to detect the mRNA expression of MT2A in 12 GC cell lines, normal gastric epithelial GES-1 cells, and 36 GC and adjacent normal tissues. MT2A protein expression was determined in 258 GC tissues and 171 adjacent normal tissues by immunohistochemistry. RESULTS: MT2A mRNA expression was lower in GC cells and primary tumors than in GES-1 cells and adjacent normal tissues, respectively. High protein expression of MT2A was present in 130 of 171 normal tissues (76.0%) and in 56 of 258 GC tissues (21.7%; P < 0.001). MT2A protein expression was higher in well/moderately differentiated GC (22/54; 40.7%) than in poorly differentiated GC (34/204; 16.7%; P < 0.001). Moreover, the protein expression of MT2A was lower in diffuse-type GC (6/82; 7.3%) than in intestinal-type GC (50/176; 28.4%; P = 0.0001). Importantly, MT2A expression was an independent prognostic factor for GC, and decreased MT2A expression was associated with poor clinical outcome (P < 0.001). The expression status of MT2A could predict prognosis in intestinal and diffuse-type GCs. CONCLUSION: Expression status of MT2A might be a useful prognostic biomarker for GC, especially when used in combination with Lauren's classification.

MAWBP and MAWD inhibit proliferation and invasion in gastric cancer.

AIM: To investigate role of putative mitogen-activated protein kinase activator with WD40 repeats (MAWD)/MAWD binding protein (MAWBP) in gastric cancer (GC). METHODS: MAWBP and MAWD mRNA expression level was examined by real-time reverse transcriptase-polymerase chain reaction and semi-quantitative polymerase chain reaction in six GC cell lines. Western blotting was used to examine the protein expression levels. We developed GC cells that stably overexpressed MAWBP and MAWD, and downregulated expression by RNA interference assay. Proliferation and migration of these GC cells were analyzed by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl tetrazolium bromide (MTT), soft agar, tumorigenicity, migration and transwell assays. The effect of expression of MAWBP and MAWD on transforming growth factor (TGF)-ß1-induced epithelial-mesenchymal transition (EMT) was examined by transfection of MAWBP and MAWD into GC cells. We detected the levels of EMT markers E-cadherin, N-cadherin and Snail in GC cells overexpressing MAWBP and MAWD by Western blotting. The effect of MAWBP and MAWD on TGF-ß signal was detected by analysis of phosphorylation level and nuclear translocation of Smad3 using Western blotting and immunofluorescence. RESULTS: Among the GC cell lines, expression of endogenous MAWBP and MAWD was lowest in SGC7901 cells and highest in BGC823 cells. MAWBP and MAWD were stably overexpressed in SGC7901 cells and knocked down in BGC823 cells. MAWBP and MAWD inhibited GC cell proliferation in vitro and in vivo. MTT assay showed that overexpression of MAWBP and MAWD suppressed growth of SGC7901 cells (P < 0.001), while knockdown of these genes promoted growth of BGC823 cells (P < 0.001). Soft agar colony formation experiments showed that overexpression of MAWBP and MAWD alone or together reduced colony formation compared with vector group in SGC7901 (86.25 ± 8.43, 12.75 ± 4.49, 30 ± 6.41 vs 336.75 ± 22.55, P < 0.001), and knocked-down MAWBP and MAWD demonstrated opposite effects (131.25 ± 16.54, 88.75 ± 11.12, 341.75 ± 22.23 vs 30.25 ± 8.07, P < 0.001). Tumorigenicity experiments revealed that overexpressed MAWBP and MAWD inhibited GC cell proliferation in vivo (P < 0.001). MAWBP and MAWD also inhibited GC cell invasion. Transwell assay showed that the number of traverse cells of MAWBP, MAWD and coexpression group were more than that in vector group (84 ± 16.57, 98.33 ± 9.8, 29 ± 16.39 vs 298 ± 11.86, P < 0.001). Coexpression of MAWBP and MAWD significantly decreased the cells traversing the matrix membrane. Conversely, knocked-down MAWBP and MAWD correspondingly promoted invasion of GC cells (100.67 ± 14.57, 72.66 ± 8.51, 330.67 ± 20.55 vs 27 ± 11.53, P < 0.001). More importantly, coexpression of MAWBP and MAWD promoted EMT. Cells that coexpressed MAWBP and MAWD displayed a pebble-like shape and tight cell-cell adhesion, while vector cells showed a classical mesenchymal phenotype. Western blotting showed that expression of E-cadherin was increased, and expression of N-cadherin and Snail was decreased when cells coexpressed MAWBP and MAWD and were treated with TGF-ß1. Nuclear translocation of p-Smad3 was reduced by attenuating its phosphorylation. CONCLUSION: Coexpression of MAWBP and MAWD inhibited EMT, and EMT-aided malignant cell progression was suppressed.

Overexpression of p42.3 promotes cell growth and tumorigenicity in hepatocellular carcinoma.

AIM: To investigate the association of p42.3 expression with clinicopathological characteristics and the biological function of p42.3 in human hepatocellular carcinoma (HCC). METHODS: We used reverse transcription-polymerase chain reaction (RT-PCR), quantitative real-time RT-PCR and western blotting to detect p42.3 mRNA and protein expression in hepatic cell lines. We examined primary HCC samples and matched adjacent normal tissue by immunohistochemistry to investigate the correlation between p42.3 expression and clinicopathological features. HepG2 cells were transfected with a pIRES2-EGFP-p42.3 expression vector to examine the function of the p42.3 gene. Transfected cells were analyzed for their viability and malignant transformation abilities by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, colony formation assay, and tumorigenicity assay in nude mice. RESULTS: p42.3 is differentially expressed in primary HCC tumors and cell lines. Approximately 69.6% (96/138) of cells were p42.3-positive in hepatic tumor tissues, while 30.7% (35/114) were p42.3-positive in tumor-adjacent normal tissues. Clinicopathological characteristics of the HCC specimens revealed a significant correlation between p42.3 expression and tumor differentiation (P = 0.031). However, p42.3 positivity was not related to tumor tumor-node-metastasis classification, hepatitis B virus status, or hepatoma type. Regarding p42.3 overexpression in stably transfected HepG2 cells, we discovered significant enhancement of cancer cell growth and colony formation in vitro, and significantly enhanced tumorigenicity in nude mice. Western blot analysis of cell cycle proteins revealed that enhanced p42.3 levels promote upregulation of proliferating cell nuclear antigen, cyclin B1 and mitotic arrest deficient 2. CONCLUSION: p42.3 promotes tumorigenicity and tumor growth in HCC and may be a potential target for future clinical cancer therapeutics.

Differential expression of Rab27A/B correlates with clinical outcome in hepatocellular carcinoma.

AIM: To investigate the association of Rab27A and Rab27B expression with clinicopathological characteristics and prognosis of hepatocellular carcinoma (HCC). METHODS: We used reverse transcription polymerase chain reaction (RT-PCR), real-time PCR, and Western blotting to detect Rab27A and Rab27B mRNA and protein expression in 5 human HCC lines and the immortalized hepatic HL-7702 cell line. We further examined 148 primary HCC samples matched with adjacent normal tissue and 80 non-HCC specimens by immunohistochemistry to evaluate the correlation of Rab27A and Rab27B expression with clinicopathological features and prognosis. RESULTS: Our data showed that Rab27A and Rab27B were differentially expressed in cell lines and primary HCC tumors. Rab27A mRNA and protein were detected in 67% (4/6) of human cell lines and 80% (4/5) of HCC cell lines, while Rab27B was found in 50% (3/6) of human lines and 40% (2/5) of HCC lines. Rab27A expression was higher in primary HCC (46.2%, 66/143) than in matched adjacent tissue (24.3%, 33/136, P < 0.001), whereas immunopositivity for Rab27B was lower in primary HCC (57.4%, 81/141) than in matched adjacent tissue (87.5%, 119/136, P < 0.001). Analysis of clinicopathological characteristics of 148 HCC specimens revealed significant correlations between Rab27A and Rab27B expression and tumor tumor-node-metastasis (TNM) classification (P = 0.046 and P = 0.027, respectively), and between strong Rab27A expression and tumor differentiation grade (P = 0.008). Survival analyses revealed that patients with Rab27A(+) or Rab27B(+) tumors had significantly reduced overall survival compared with that of patients with Rab27A(-) or Rab27B(-) tumors (P = 0.015 and P = 0.005, respectively). Risk analyses revealed that Rab27B(+) and TNM III-IV were independent poor prognosis factors associated with a 3.36- and 3.37-fold higher relative risk of death, respectively. CONCLUSION: Rab27A and Rab27B expression were closely correlated with tumor progression and can be valuable prognostic indicators for HCC patients.

Tumor suppressor XIAP-Associated factor 1 (XAF1) cooperates with tumor necrosis factor-related apoptosis-inducing ligand to suppress colon cancer growth and trigger tumor regression.

BACKGROUND: XIAP-associated factor 1 (XAF1) antagonizes the anticaspase activity of XIAP (X-linked inhibitor of apoptosis) and functions as a tumor suppressor in colon cancer. The tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is known as a potential anticancer agent. In this study, the synergistic effect of XAF1 and TRAIL on colon cancer growth was investigated. METHODS: Adeno-XAF1 virus was generated and purified. Cell apoptosis was detected by flow-cytometry and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assay. Protein expression of the different genes was determined by Western blot analysis. Tumorigenesis and tumor growth were assessed in subcutaneous nude mouse xenograft experiments. RESULTS: Stable overexpression of XAF1-sensitized colon cancer cells to TRAIL-induced apoptosis significantly increased the activity of caspase 3, 7, 8, and 9; released cytochrome c; and down-regulated XIAP, survivin, and c-IAP-2. The restoration of XAF1 expression mediated by adenovirus (adeno-XAF1) directly induced apoptosis, and synergized TRAIL-induced apoptosis in colon cancer cells. Ex vivo transduction of adeno-XAF1 suppressed colon cancer formation in vivo. Furthermore, adeno-XAF1 treatment of mice significantly inhibited tumor growth, strongly enhanced TRAIL-induced apoptosis and antitumor activity in colon cancer xenograft models in vivo, and markedly prolonged the survival. Notably, the combined treatment with adeno-XAF1 and TRAIL completely eradicated the established tumors without detectable toxicity in normal tissue. CONCLUSIONS: The combined restoration of XAF1 expression and TRAIL treatment may be a potent strategy for colon cancer therapy.

Expression status of S100A14 and S100A4 correlates with metastatic potential and clinical outcome in colorectal cancer after surgery.

To investigate whether S100A14 and S100A4 expression correlates with metastatic potential and prognosis in colorectal cancer (CRC), we firstly used RT-PCR analysis to detect mRNA expression of S100A14 and S100A4 in 40 pairs of fresh tumor samples matched with adjacent normal tissues. We then evaluated the clinical significance of our findings with immunohistochemistry on 115 samples of formalin-fixed and paraffin-embedded tumors on tissue microarrays. Typically, we identified decreased S100A14 mRNA levels (52.5%, 21/40), and increased S100A4 mRNA levels (70.0%, 28/40) in primary CRC samples. In addition, down-regulated or absent S100A14 expression was detected in 56.5% of samples (65/115) and was correlated with poor differentiation (P=0.010). In contrast, overexpressed S100A4 was detected in 57.4% of samples (66/115) and was associated with lymph node metastasis (P=0.001). Simultaneous S100A14 low-expression and S100A4 high-expression was correlated with high CRC metastatic potential (P<0.001). Taken together, the signature derived from the combined expression status of S100A14 and S100A4 could be a valuable prognostic indicator in CRC.

Restoration of XAF1 expression induces apoptosis and inhibits tumor growth in gastric cancer.

XAF1 (XIAP-associated factor 1) is a novel XIAP binding protein that can antagonize XIAP and sensitize cells to other cell death triggers. Our previous results have shown that aberrant hypermethylation of the CpG sites in XAF1 promoter is strongly associated with lower expression of XAF1 in gastric cancers. In our study, we investigated the effect of restoration of XAF1 expression on growth of gastric cancers. We found that the restoration of XAF1 expression suppressed anchorage-dependent and -independent growth and increased sensitivity to TRAIL and drug-induced apoptosis. Stable cell clones expressing XAF1 exhibited delayed tumor initiation in nude mice. Restoration of XAF1 expression mediated by adenovirus vector greatly increased apoptosis in gastric cancer cell lines in a time- and dose-dependent manner and sensitized cancer cells to TRAIL and drugs-induced apoptosis. Adeno-XAF1 transduction induced cell cycle G2/M arrest and upregulated the expression of p21 and downregulated the expression of cyclin B1 and cdc2. Notably, adeno-XAF1 treatment significantly inhibited tumor growth, strongly enhanced the antitumor activity of TRAIL in a gastric cancer xenograft model in vivo, and significantly prolonged the survival time of animals bearing tumor xenografts. Complete eradication of established tumors was achieved on combined treatment with adeno-XAF1 and TRAIL. Our results document that the restoration of XAF1 inhibits gastric tumorigenesis and tumor growth and that XAF1 is a promising candidate for cancer gene therapy.

Gene therapy for colon cancer by adeno-associated viral vector-mediated transfer of survivin Cys84Ala mutant.

BACKGROUND AND AIMS: Reactivation of survivin expression is involved in carcinogenesis and angiogenesis in colon cancer. Previous in vitro studies showed that mutation of the cysteine residue at position 84 (Cys84Ala) of survivin generates a dominant-negative mutant that triggers mitotic catastrophe and apoptosis. We investigated the therapeutic effect of the adeno-associated virus (AAV)-mediated survivin mutant (Cys84Ala) on colon cancer. METHODS: Survivin mutant (Cys84Ala) (Sur-Mut(Cys84Ala)) was cloned into the AAV expression vector pAM/CAG-WPRE.poly(A) to generate recombinant AAV-Sur-Mut(Cys84Ala) virus. Cell proliferation, apoptosis, mitotic catastrophe, and tumor growth were measured in vitro and in vivo. RESULTS: Transduction of colon cancer cells with rAAV-Sur-Mut(Cys84Ala) inhibited cell proliferation and induced apoptosis and mitotic catastrophe in vitro. rAAV-Sur-Mut(Cys84Ala) sensitized colon cancer cells to chemotherapeutic drugs. Furthermore, expression of survivin mutant mediated by AAV inhibited tumorigenesis in colon cancer cells. Intratumoral injection of rAAV-Sur-Mut(Cys84Ala) significantly induced apoptosis and mitotic catastrophe and inhibited angiogenesis and tumor growth in a colon cancer xenograft model in vivo. No obvious cytotoxicity to other tissues was observed. More importantly, rAAV-Sur-Mut(Cys84Ala) expression strongly enhanced the antitumor activity of 5-Fluorouracil (5-FU), resulting in regression of established tumors. CONCLUSIONS: Our results showed that rAAV-Sur-Mut(Cys84Ala) induced apoptosis and mitotic catastrophe and inhibited tumor angiogenesis and tumor growth. Thus, use of AAV-mediated survivin mutant (Cys84Ala) is a promising strategy in colon cancer gene therapy.