High SNRPA1 expression leads to poor prognosis in patients with lung adenocarcinoma.

OBJECTIVE: SNRPA1, a subunit of spliceosome complex, has been implicated in diverse cancers, while its biological effect in LUAD remains elusive. Therefore, we sought to decipher the relationship between SNRPA1 expression and the prognosis of patients with LUAD and reveal the underlying molecular mechanism. MATERIALS AND METHODS: Based on the clinical data from TCGA databases, the multivariate Cox model was constructed to screen the prognostic value of SNRPA1. qRT-PCR and immunohistochemical staining were used to examine SNRPA1 mRNA and protein expression in LUAD. The effect of SNRPA1 on LUAD cell proliferation, migration, and epithelial mesenchymal transformation were examined using colony formation assays, wound healing, and western blot assays, respectively. Finally, the influence of SNRPA1 on LUAD immune microenvironment were validated from the Tumor Immune Estimation Resource database. RESULTS: SNRPA1 was significantly upregulated in both LUAD tissues and cell lines, and highly expressed SNRPA1 contributed to poor prognosis of LUAD patients. In vitro, SNRPA1 knockdown inhibited the proliferation and migration, as well as delayed the EMT differentiation of LUAD cells. Lastly, SNRPA1 was found to be positively associated with immune infiltration and some immune-check-point markers. CONCLUSIONS: Our findings indicate that SNRPA1 may be a new biomarker for prognostic prediction and a potential therapeutic target in the treatment of LUAD.

Epsin1-mediated exosomal sorting of Dll4 modulates the tubular-macrophage crosstalk in diabetic nephropathy.

Tubular epithelial cells (TECs) play critical roles in the development of diabetic nephropathy (DN), and can activate macrophages through the secretion of exosomes. However, the mechanism(s) of TEC-exosomes in macrophage activation under DN remains unknown. By mass spectrometry, 1,644 differentially expressed proteins, especially Dll4, were detected in the urine exosomes of DN patients compared with controls, which was confirmed by western blot assay. Elevated Epsin1 and Dll4/N1ICD expression was observed in kidney tissues in both DN patients and db/db mice and was positively associated with tubulointerstitial damage. Exosomes from high glucose (HG)-treated tubular cells (HK-2) with Epsin1 knockdown (KD) ameliorated macrophage activation, TNF-α, and IL-6 expression, and tubulointerstitial damage in C57BL/6 mice in vivo. In an in vitro study, enriched Dll4 was confirmed in HK-2 cells stimulated with HG, which was captured by THP-1 cells and promoted M1 macrophage activation. In addition, Epsin1 modulated the content of Dll4 in TEC-exosomes stimulated with HG. TEC-exosomes with Epsin1-KD significantly inhibited N1ICD activation and iNOS expression in THP-1 cells compared with incubation with HG alone. These findings suggested that Epsin1 could modulate tubular-macrophage crosstalk in DN by mediating exosomal sorting of Dll4 and Notch1 activation.

Construction of a fatty acid metabolism-related gene signature for predicting prognosis and immune response in breast cancer.

Background: Breast cancer has the highest incidence among malignant tumors in women, and its prevalence ranks first in global cancer morbidity. Aim: This study aimed to explore the feasibility of a prognostic model for patients with breast cancer based on the differential expression of genes related to fatty acid metabolism. Methods: The mRNA expression matrix of breast cancer and paracancer tissues was downloaded from The Cancer Genome Atlas database. The differentially expressed genes related to fatty acid metabolism were screened in R language. The TRRUST database was used to predict transcriptional regulators related to hub genes and construct an mRNA-transcription factor interaction network. A consensus clustering approach was used to identify different fatty acid regulatory patterns. In combination with patient survival data, Lasso and multivariate Cox proportional risk regression models were used to establish polygenic prognostic models based on fatty acid metabolism. The median risk score was used to categorize patients into high- and low-risk groups. Kaplan-Meier survival curves were used to analyze the survival differences between both groups. The Cox regression analysis included risk score and clinicopathological factors to determine whether risk score was an independent risk factor. Models based on genes associated with fatty acid metabolism were evaluated using receiver operating characteristic curves. A comparison was made between risk score levels and the fatty acid metabolism-associated genes in different subtypes of breast cancer. The differential gene sets of the Kyoto Encyclopedia of Genes and Genomes for screening high- and low-risk populations were compared using a gene set enrichment analysis. Furthermore, we utilized CIBERSORT to examine the abundance of immune cells in breast cancer in different clustering models. Results: High expression levels of ALDH1A1 and UBE2L6 prevented breast cancer, whereas high RDH16 expression levels increased its risk. Our comprehensive assessment of the association between prognostic risk scoring models and tumor microenvironment characteristics showed significant differences in the abundance of various immune cells between high- and low-risk breast cancer patients. Conclusions: By assessing fatty acid metabolism patterns, we gained a better understanding of the infiltration characteristics of the tumor microenvironment. Our findings are valuable for prognosis prediction and treatment of patients with breast cancer based on their clinicopathological characteristics.

Single-cell genomic variation induced by mutational processes in cancer.

How cell-to-cell copy number alterations that underpin genomic instability1 in human cancers drive genomic and phenotypic variation, and consequently the evolution of cancer2, remains understudied. Here, by applying scaled single-cell whole-genome sequencing3 to wild-type, TP53-deficient and TP53-deficient;BRCA1-deficient or TP53-deficient;BRCA2-deficient mammary epithelial cells (13,818 genomes), and to primary triple-negative breast cancer (TNBC) and high-grade serous ovarian cancer (HGSC) cells (22,057 genomes), we identify three distinct 'foreground' mutational patterns that are defined by cell-to-cell structural variation. Cell- and clone-specific high-level amplifications, parallel haplotype-specific copy number alterations and copy number segment length variation (serrate structural variations) had measurable phenotypic and evolutionary consequences. In TNBC and HGSC, clone-specific high-level amplifications in known oncogenes were highly prevalent in tumours bearing fold-back inversions, relative to tumours with homologous recombination deficiency, and were associated with increased clone-to-clone phenotypic variation. Parallel haplotype-specific alterations were also commonly observed, leading to phylogenetic evolutionary diversity and clone-specific mono-allelic expression. Serrate variants were increased in tumours with fold-back inversions and were highly correlated with increased genomic diversity of cellular populations. Together, our findings show that cell-to-cell structural variation contributes to the origins of phenotypic and evolutionary diversity in TNBC and HGSC, and provide insight into the genomic and mutational states of individual cancer cells.

Paeoniflorin ameliorates ischemic injury in rat brain via inhibiting cytochrome c/caspase3/HDAC4 pathway.

Paeoniflorin (PF), a bioactive monoterpene glucoside, has shown a variety of pharmacological effects such as anti-inflammation and autophagy modulation etc. In this study, we investigated whether and how PF exerted a protective effect against ischemic brain injury in vivo and in vitro. Primary rat cortical neurons underwent oxygen/glucose deprivation/reperfusion (OGD/R) for 90 min. We showed that after OGD/R, a short fragment of histone deacetylase 4 (HDAC4) produced by caspase3-mediated degradation was markedly accumulated in the nucleus and the activity of caspase3 was increased. Treatment with PF (100 nM, 1 µM) significantly improved the viability of cortical neurons after OGD/R. Furthermore, PF treatment could maintain HDAC4 intrinsic subcellular localization and reduce the caspase3 activity without changing the HDAC4 at the transcriptional level. PF treatment significantly reduced OGD/R-caused inhibition of transcriptional factor MEF2 expression and increased the expression of downstream proteins such as GDNF, BDNF, and Bcl-xl, thus exerting a great anti-apoptosis effect as revealed by TUNEL staining. The beneficial effects of PF were almost canceled in HDAC4 (D289E)-transfected PC12 cells after OGD/R. In addition, PF treatment reduced the caspase9 activity, rescued the release of cytochrome c from mitochondria, and maintained the integrity of mitochondria membrane. We conducted in vivo experiments in 90-min-middle cerebral artery occlusion (MCAO) rat model. The rats were administered PF (20, 40 mg/kg, ip, 3 times at the reperfusion, 24 h and 48 h after the surgery). We showed that PF administration dose-dependently reduced infarction area, improved neurological symptoms, and maintained HDAC4 localization in rats after MCAO. These results demonstrate that PF is effective in protecting against ischemic brain injury and inhibit apoptosis through inhibiting the cytochrome c/caspase3/HDAC4 pathway.

Epac activation ameliorates tubulointerstitial inflammation in diabetic nephropathy.

Tubulointerstitial inflammation plays an important role in the progression of diabetic nephropathy (DN), and tubular epithelial cells (TECs) are crucial promoters of the inflammatory cascade. Exchange protein activated by cAMP (Epac) has been shown to suppress the angiotensin II (Ang-II)-induced release of inflammatory cytokines in tubular cells. However, the role of Epac in TEC-mediated tubulointerstitial inflammation in DN remains unknown. We found that administering the Epac agonist 8-pCPT-2'-O-Me-cAMP (8-O-cAMP) to db/db mice inhibited tubulointerstitial inflammation characterized by macrophage infiltration and increased inflammatory cytokine release and consequently alleviated tubulointerstitial fibrosis in the kidney. Furthermore, 8-O-cAMP administration restored CCAAT/enhancer binding protein ß (C/EBP-ß) expression and further upregulated the expression of Suppressor of cytokine signaling 3 (SOCS3), while inhibiting p-STAT3, MCP-1, IL-6, and TNF-α expression in the kidney cortex in db/db mice. And in vitro study showed that macrophage migration and MCP-1 expression induced by high glucose (HG, 30 mM) were notably reduced by 8-O-cAMP in human renal proximal tubule epithelial (HK-2) cells. In addition, 8-O-cAMP treatment restored C/EBP-ß expression in HK-2 cells and promoted C/EBP-ß translocation to the nucleus, where it transcriptionally upregulated SOCS3 expression, subsequently inhibiting STAT3 phosphorylation. Under HG conditions, siRNA-mediated knockdown of C/EBP-ß or SOCS3 in HK-2 cells partially blocked the inhibitory effect of Epac activation on the release of MCP-1. In contrast, SOCS3 overexpression inhibited HG-induced activation of STAT3 and MCP-1 expression in HK-2 cells. These findings indicate that Epac activation via 8-O-cAMP ameliorates tubulointerstitial inflammation in DN through the C/EBP-ß/SOCS3/STAT3 pathway.

Characteristics and clinical significance of polyploid giant cancer cells in laryngeal carcinoma.

OBJECTIVES: We aimed to construct an induction system for polyploid giant cancer cells (PGCCs), as well as to investigate PGCC features and clinical significance. METHODS: A laryngeal neoplasm-PGCC induction system was constructed using paclitaxel liposomes (PTX). We used western blots to compare expression of epithelial-mesenchymal transition-related proteins, stem cell interrelated proteins, and cyclin-associated proteins. We then measured PGCC count in tissue samples of patients with laryngeal neoplasms and analyzed its relationship with prognosis. Statistical significance was determined using t-tests. RESULTS: PTX successfully induced PGCCs. Western blotting showed that CyclinB1, CDC25C, CDK1, E-cadherin, and EIF-4A expression decreased in PGCCs compared with normal cancer cells, whereas vimentin and CD133 expression increased. Number of PGCCs in laryngeal cancer tissues and overall survival time were inversely correlated (P < .05). CONCLUSIONS: PTX successfully induces PGCC formation in laryngeal carcinoma, which may be the cause of poor prognosis in patients with laryngeal cancer.Level of Evidence: 4.

Molecular and biochemical mechanisms underlying boron-induced alleviation of cadmium toxicity in rice seedlings.

Both cadmium (Cd) contamination and boron (B) deficiency in farmland soils pose a threat to the yield and quality of crops in Southern China. The present study investigated the mechanisms by which B reduces Cd accumulation in rice (Oryza sativa) seedlings. Boron supplementation partially restored the decline in shoot and root biomass caused by Cd treatment (26% and 33%, respectively), with no significant difference between the B+Cd and control groups. We also found that B significantly reduced shoot and root Cd concentrations (by 64% and 25%, respectively) but increased Cd concentration (by 43%) and proportion (from 38% to 55%) in root cell walls. Transcriptome analysis and biochemical tests suggested that B supplementation enhanced lignin and pectin biosynthesis, pectin demethylation, and sulfur and glutathione metabolism. Moreover, B decreased the expression of some Cd-induced transporter-related genes (i.e., HMA2, Nramp1, and several ABC genes). These results indicate that B relieved Cd toxicity and reduced Cd accumulation in rice seedlings by restraining Cd uptake and translocation from root to shoot by improving Cd tolerance and chelation ability. These novel findings would benefit further investigations into how B influences Cd uptake, translocation, detoxification, and accumulation in crops.

[Expression of Ras-Associated Protein 1 in the Vascular Endothelium of Bone Tissue with Non-Traumatic Osteonecrosis of Femoral Head].

Objective: To investigate the difference in the expression of Ras-associated protein 1 (Rap1) in necrotic and healthy areas of non-traumatic osteonecrosis of femoral head (NONFH) patients. Methods: Femoral head tissue samples from 30 cases of NONFH and 30 cases of traumatic osteonecrosis of the femoral head (TONFH) were collected after hip replacement surgery, respectively. No significant difference of Association Research Circulation Osseous (ARCO) staging was found between the NONFH and the TONFH groups ( Z=-0.769, P=0.442). In the NONFH group, 8 patients were ARCO stage IIIb, 10 were stage IV, and 12 were stage V, while in the TONFH ground, 11 patients were ARCO stage IIIb, 9 were stage IV, and 10 were stage V. There were 19 males and 11 females in the NONFH group, with an average age of 49.6 yr. (26-69 yr.), and 16 males and 14 females in the TONFH group, with an average age of 54.2 yr. (37-68 yr.). There was no significant difference in gender or age between the two groups ( P>0.05). Specimens were collected from different bone areas, including those from the necrotic areas (area A) and the healthy areas (area B) of the NONFH group, and those from the healthy areas (area B') of the TONFH group, i.e., the control group. Western blot and quantitative real-time reverse transcription PCR (qRT-PCR) were used to analyze the different expression of Rap1, vascular endothelial growth factor (VEGF) protein, phosphoinositide 3-kinase (PI3K), and Akt protein and their corresponding mRNA in the three areas of bone tissue. HE staining and immunohistochemisty staining were done in order to observe the morphological changes of each area. Results: Western blot results indicated that there was no statistical difference in the relative expression of Rap1, VEGF, PI3K, and Akt proteins ( P>0.05). The relative expressions of Rap1, VEGF, PI3K, and Akt proteins in the area A were lower than those in the area B and the difference was statistically significant ( P<0.05). qRT-PCR results showed that the relative expressions of Rap1, VEGF, PI3 K and Akt mRNA in area A were lower than those of area B, and a statistical difference was found ( P<0.05). The relative expression of the mRNA of Rap1, VEGF , PI3 K and Akt in area B and area B' were not significantly different ( P>0.05). HE staining and immunohistochemisty staining showed that chondrocytes decreased in the necrotic area (area A) of NONFH, chondrocytes nucleus disappeared, subchondral bone trabeculae were broken, bone trabeculae thickened, and empty bone lacunae appeared. Granulation tissues composed of new capillaries and fibrous cells have proliferated and crawled around the necrotic area. Positive expressions of the Rap1, VEGF, PI3K and Akt proteins in area A were weaker than those of the normal area. In addition, there were positive expressions of Rap1, PI3K and Akt on the trabecular bone of both area A and area B at similar intensity of expression. There were strong positive expressions of Rap1, VEGF, PI3K and Akt on the intima of arterioles and venules, and on the peripheral stromal cell membrane, but the positive expression in area A was significantly lower than that in area B. However, the positive expression positions and intensity of all indicators were similar in area B and area B'. Conclusion: The necrosis in NONFH may be related to vascular endothelial damages caused by the inhibition of the Rap1-PI3K/Akt signaling pathways and the subsequent decline in the protein expression.

Clinical and prognostic significance of CC chemokine receptor type 8 protein expression in gastrointestinal stromal tumors.

BACKGROUND: Gastrointestinal stromal tumors (GISTs) are the most common mesenchymal neoplasms of the gastrointestinal tract. Surgical resection and tyrosine kinase inhibitors are defined as the main treatments but cannot cure patients with advanced GIST, which eventually develops into recurrence and acquired drug resistance. Therefore, it is necessary to identify prognostic biomarkers and new therapeutic targets for GISTs. CC chemokine receptor type 8 (CCR8) protein participates in regulation of immune responses. Recent studies on CCR8 in non-small cell lung cancer and colorectal cancer showed that it was highly expressed in tumor-infiltrating regulatory T cells and correlated with a poor prognosis. AIM: To detect CCR8 expression in GIST tissues and analyze its relationships with clinicopathological features and prognosis in patients with GISTs. METHODS: Tissue samples were used for the tissue microarrays construction. The microarrays were then subjected to immunohistochemical analyses to detect CCR8 expression. Next, Kaplan-Meier analysis was utilized to calculate the survival rate of patients with complete follow-up data, and the potential prognostic value of CCR8 was evaluated by Cox regression analysis. Finally, a Gene Ontology/Kyoto Encyclopedia of Genes and Genomes single-gene enrichment chart of CCR8 was constructed using the STRING database. RESULTS: CCR8-positive signals were detected as brown or brown-yellow particles by immunohistochemistry located in the cytoplasm. Among 125 tissue samples, 74 had CCR8 high expression and 51 had low or negative expression. Statistical analyses suggested CCR8 was significantly correlated with tumor size, mitotic index, AFIP-Miettinen risk classification and tumor location. Kaplan-Meier and multivariate analyses showed that patients with low or negative CCR8 expression, mitotic index < 5/high-power fields (HPF) and tumor diameter < 5 cm had a better prognosis. Based on the STRING database, CCR8 was significantly enriched in biological processes such as tumor immunity, T lymphocyte chemotaxis, migration and pathways like the nuclear factor-κB and tumor necrosis factor pathways as well as intestinal immune regulation networks. CONCLUSION: CCR8 is a prognostic biomarker for malignant potential of GISTs, with high expression correlated with malignancy and poor prognosis.

Diagnostic and prognostic value of the peripheral natural killer cell levels in gastric cancer.

Peripheral blood lymphocyte subsets have been reported to be useful as prognostic and/or diagnostic markers for patients with cancer. However, the clinical value of peripheral blood lymphocyte subsets in gastric cancer (GC) has remained elusive. In the present study, peripheral CD3+, CD4+ and CD8+ T lymphocytes, B cells (CD19+), regulatory T cells (Tregs; CD4+CD25+CD127-) and natural killer (NK) cells (CD3-CDl6+CD56+) were detected by flow cytometry in 122 patients with GC, 80 healthy donors (HDs) and 80 patients with gastric ulcer (GU). NK cells (CD56+) were detected by immunohistochemical (IHC) analysis in 20 GC and three GU tissue samples. A receiver-operating characteristic (ROC) curve was used to determine the threshold of the peripheral NK cell level and survival analysis was performed to assess its prognostic value in patients with GC. The results indicated that the peripheral NK cell proportion in patients with GC (18.77%) was significantly higher than that in the HD (12.19%) and GU (12.74%) groups. IHC analysis suggested that the NK level in GC tumor samples was correlated with that in paired serum samples. ROC curve analysis indicated that the peripheral NK cell level (15.16%) was able to effectively identify patients with GC, a diagnostic sensitivity of 75.41% and a specificity of 77.45% were determined. Multivariate logistic regression analysis revealed that the peripheral NK cell level was independently associated with the T stage and survival analysis demonstrated that high levels of peripheral NK cells were associated with poor prognosis of patients with GC. In conclusion, the peripheral NK cell level may be a diagnostic and prognostic marker for patients with GC.

High expression of G protein subunit gamma 13 is associated with poor prognosis of gastrointestinal stromal tumor.

The G protein subunit gamma 13 (GNG13) plays an important role in olfaction, vision, and biological behavior. However, our knowledge of the relationship between GNG13 expression and the clinicopathological features of gastrointestinal tumors is insufficient. Therefore, we used the Oncomine database to evaluate the expression of GNG13 mRNA in gastric cancer, the result showed that there was no significant difference in the expression of GNG13 between gastric cancer and adjacent normal tissues, and GNG13 mRNA expression was assessed in 32 matched pairs of Gastrointestinal adenocarcinoma tissues and adjacent normal tissues as well as 32 matched pairs of gastrointestinal stromal tumor (GIST) and adjacent normal tissues by quantitative reverse transcription-polymerase chain reaction analysis. The results suggested that GNG13 is upregulated in gastrointestinal stromal tumors. Immunohistochemical analysis was used to detect the GNG13 in the tissues of 123 patients with GIST. High cytoplasmic expression of GNG13, which was observed in 65.85 % of GIST patients, significantly correlated with mitotic index(P = 0.036) and tumor size(P = 0.024). Multiple logistic regression analysis showed that the expression of GNG13 was significantly associated with tumor size. Kaplan-Meier analysis indicated that high GNG13 expression was associated with poor prognosis of GIST. Multivariate Cox regression analysis indicated that the expression of GNG13, mitotic index and tumor size were independent adverse prognostic factors of GIST. These findings suggest that GNG13 is associated with the malignant phenotype of GIST and may serve as a marker of poor prognosis.

BDH2 triggers ROS-induced cell death and autophagy by promoting Nrf2 ubiquitination in gastric cancer.

BACKGROUND: 3-Hydroxy butyrate dehydrogenase 2 (BDH2) is a short-chain dehydrogenase/reductase family member that plays a key role in the development and pathogenesis of human cancers. However, the role of BDH2 in gastric cancer (GC) remains largely unclear. Our study aimed to ascertain the regulatory mechanisms of BDH2 in GC, which could be used to develop new therapeutic strategies. METHODS: Western blotting, immunohistochemistry, and RT-PCR were used to investigate the expression of BDH2 in GC specimens and cell lines. Its correlation with the clinicopathological characteristics and prognosis of GC patients was analysed. Functional assays, such as CCK-8 and TUNEL assays, transmission electron microscopy, and an in vivo tumour growth assay, were performed to examine the proliferation, apoptosis, and autophagy of GC cells. Related molecular mechanisms were clarified by luciferase reporter, coimmunoprecipitation, and ubiquitination assays. RESULTS: BDH2 was markedly downregulated in GC tissues and cells, and the low expression of BDH2 was associated with poor survival of GC patients. Functionally, BDH2 overexpression significantly induced apoptosis and autophagy in vitro and in vivo. Mechanistically, BDH2 promoted Keap1 interaction with Nrf2 to increase the ubiquitination level of Nrf2. Ubiquitination/degradation of Nrf2 inhibited the activity of ARE to increase accumulation of reactive oxygen species (ROS), thereby inhibiting the phosphorylation levels of AktSer473 and mTORSer2448. CONCLUSIONS: Our study indicates that BDH2 is an important tumour suppressor in GC. BDH2 regulates intracellular ROS levels to mediate the PI3K/Akt/mTOR pathway through Keap1/Nrf2/ARE signalling, thereby inhibiting the growth of GC.

Annexin A2 promotion of hepatocellular carcinoma tumorigenesis via the immune microenvironment.

Hepatocellular carcinoma (HCC) is the most common primary liver cancer with a dismal prognosis, especially when diagnosed at advanced stages. Annexin A2 (ANXA2), is found to promote cancer progression and therapeutic resistance. However, the underlining mechanisms of ANXA2 in immune escape of HCC remain poorly understood up to now. Herein, we summarized the molecular function of ANXA2 in HCC and its relationship with prognosis. Furthermore, we tentatively elucidated the underlying mechanism of ANXA2 immune escape of HCC by upregulating the proportion of regulatory T cells and the expression of several inhibitory molecules, and by downregulating the proportion of natural killer cells and dendritic cells and the expression of several inhibitory molecules or effector molecules. We expect a lot of in-depth studies to further reveal the underlying mechanism of ANXA2 in immune escape of HCC in the future.

Rafoxanide promotes apoptosis and autophagy of gastric cancer cells by suppressing PI3K /Akt/mTOR pathway.

Rafoxanide is commonly used as anti-helminthic medicine in veterinary medicine, a main compound of salicylanilide. Previous studies have reported that rafoxanide, as an inhibitor of BRAF V600E mutant protein, inhibits the growth of colorectal cancer, multiple myeloma, and skin cancer. However, its therapeutic effect on gastric cancer (GC) and the potential mechanism has not been investigated. Here, we have found that rafoxanide inhibited the proliferation of GC cells in vitro, arrested the cell cycle in the G0/G1 phase, and promoted apoptosis and autophagy in GC cells. Treatment with specific autophagy inhibitor 3-methyladenine drastically inhibited the apoptotic cell death effect by suppressing the switch from autophagy to apoptosis. Mechanistically, we found that rafoxanide inhibited the growth of GC cells in vitro by inhibiting the activity of the PI3K/Akt/mTOR signaling pathway. This process induced autophagy, which essentially resulted in the apoptosis of GC cells. Results from subcutaneous implanted tumor models in nude mice also indicated that rafoxanide inhibited the growth of GC cells in vivo. Taken together, our findings revealed that rafoxanide inhibited the growth of GC cells both in vitro and vivo, indicating a potential drug candidate for the treatment of GC.

Apoptosis gene reprograming of human peripheral blood mononuclear cells induced by radioiodine-131 (131I) irradiation.

Background & objectives: The nature of adaptable change of B-cell lymphoma-2 (BCL-2) and/or Bcl2-associated X protein (BAX) gene expression in the human peripheral blood mononuclear cells (PBMCs) irradiated by radioiodine in thyroid diseases therapy is not fully understood. In this study, the alternation of apoptotic gene expression was evaluated while the PBMCs collected from healthy volunteers were irradiated by the radioiodine-131 (131I). Methods: Fasting blood samples were obtained from healthy volunteers. PBMCs from group 0 to 6 were incubated and exposed to different doses of 131I in cell suspension for 6, 12, 24 and 48 h. The apoptosis rates and expression of BCL-2 and BAX genes of PBMCs were examined. Results: The apoptosis rate in the human PBMCs was gradually enhanced after six hour irradiation. The values of BCL-2 and BAX gene expression in groups 1-6 were higher than in group 0 within 6 h of irradiation, and then, these were decreased gradually from 6 to 12 h. BCL: -2 gene expression increased in groups 1-3 after 12 h irradiation, but there was no difference in groups 4-6. The ratio of BCL-2/BAX gene expression among groups 4-6 gradually decreased during the period from 6 to 12 h, and it was significantly lower than in the group 0 at 12, 24 and 48 h. Interpretation & conclusions: The expression of BCL-2 and BAX genes was initially upregulated following irradiation. Later, the balance of BCL-2/BAX genes expression was adjusted, and then, PBMCs underwent apoptosis at higher doses of radiation.

High-level expression of PRSS3 correlates with metastasis and poor prognosis in patients with gastric cancer.

BACKGROUND AND OBJECTIVES: Serine protease-3 (PRSS3) is a known contributor to the genesis and development of malignant tumors, although its role in gastric cancer (GC) is still unclear. METHODS: PRSS3 expression in GC tissue samples and its relationship with clinicopathological features were analyzed. Effects of GC cellular responses to the introduction of small interfering RNA (siRNA)-mediated and short hairpin RNA (shRNA)-mediated interference with tumor PRSS3 expression were also assessed. RESULTS: PRSS3 was significantly upregulated in GC tissues, and PRSS3 protein levels were higher in tumors that developed metastases soon after the surgery compared with those that remained metastasis-free. High expression of PRSS3 was associated with tumor N staging and independently predictive of postoperative prognosis in patients with GC. The V1 variant of PRSS3 was primarily detected in GC tissue and cell lines, the others (V2-V4) being scarcely detectable. Methylation and demethylation drugs had no impact on expression levels of any PRSS3 transcriptional variant. The downregulated PRSS3 expression suppressed GC cell growth, migration, and invasion in vitro and in vivo. CONCLUSIONS: PRSS3 appears to act as an oncogene of GC. High PRSS3 expression portends postoperative metastasis, serving as an effective biomarker of poor therapeutic outcomes.

Tra2ß silencing suppresses cell proliferation in laryngeal squamous cell carcinoma via inhibiting PI3K/AKT signaling.

OBJECTIVES/HYPOTHESIS: Transformer-2 protein homolog beta (Tra2ß) generally plays an important role in various human cancers, but its role and the underlying mechanisms in laryngeal squamous cell carcinoma (LSCC) remained unknown. So this study aimed to assess the clinical significance and regulatory mechanisms of Tra2ß in LSCC. STUDY DESIGN: Laboratory analysis. METHODS: Expression of Tra2ß was compared in human LSCC tissue samples and paired adjacent normal tissue samples. The in vitro effects of Tra2ß expression in Hep-2 cells on their proliferation, invasion, and migration were assessed by CCK-8 assays, Matrigel invasion, and transwell migration assays. In addition, the effects of downregulation of Tra2ß on the activation of PI3K/AKT signaling pathway were measured using Western blot analysis. The effect of Tra2ß on the growth of tumors was detected in the Hep-2-injected xenograft models in vivo. RESULTS: Reverse-transcription quantitative polymerase chain reaction analysis and immunochemistry analysis indicated that the increased expression of Tra2ß in LSCC was significantly associated with poor differentiation, lymph node metastasis, and advanced clinical stage. In vitro knockdown of Tra2ß caused a significant decrease in the proliferation, invasion, and migration of Hep-2 cells. Tra2ß silencing decreased the expression of Bcl-2 but increased Bax and Caspase-3 both in mRNA and protein levels. Furthermore, knockdown of Tra2ß eliminated the suppressive effects of activation of PI3K/AKT signaling. In vivo knockdown of Tra2ß significantly inhibited the tumor growth of Hep-2-injected xenograft mice. CONCLUSIONS: The results of the present study demonstrated that knockdown of Tra2ß inhibits the proliferation and invasion of LSCC cells, at least partly via inhibiting PI3K/AKT signaling. LEVEL OF EVIDENCE: NA Laryngoscope, 129:E318-E328, 2019.

Downregulation of Lgr6 inhibits proliferation and invasion and increases apoptosis in human colorectal cancer.

The aim of the present study was to analyze the role of leucine­rich repeat­containing G­protein coupled receptor 6 (Lgr6) in the proliferation and invasion of colorectal cancer (CRC) cells, and to investigate its possible mechanisms. The expression of Lgr6 in CRC tissues was observed by real time­quantitative polymerase chain reaction and western blotting. Then cell viability, apoptosis and cell invasion was measured by MTT, flow cytometry or Matrigel­Transwell system, respectively in CRC cells after transfected with Lgr6 siRNA or Lgr6 vector. Furthermore, the expression of apoptosis­associated protein and PI3K/AKT signaling (phosphorylated­PI3K, phosphorylated­AKT, t­PI3K, t­AKT) were measured by real­time PCR/or western blot analysis. The results demonstrated that the level of Lgr6 was higher in CRC tissues than that in adjacent tissues, and Lgr6 overexpression increased CRC proliferation, and invasion of CRC cells in vitro. Notably, suppressing the expression of Lgr6 in CRC cells increased the expression of B­cell lymphoma-2 (Bcl­2)­associated X protein and caspase­3, but decreased the expression of Bcl­2 at the mRNA and protein levels. Lgr6 also had the ability to regulate the phosphoinositide 3­kinase/AKT signaling pathway. It was concluded that Lgr6 has a tumor­promoting role in the development of CRC, and may serve as a potential diagnostic and prognostic biomarker for the disease.

Low expression of RASSF10 is associated with poor survival in patients with colorectal cancer.

The RASSF10 has been identified as a tumor suppressor in human colorectal cancer (CRC). However, the expression of RASSF10 in patients with CRC has not been evaluated for its potential use as a biomarker in the diagnosis and prognosis assessment of CRC. We analyzed the expression of RASSF10 mRNA (n=30) and protein (n=205) in CRC and matched noncancerous colon tissue samples to explore the relationships among RASSF10 expression, clinicopathological factors, and prognosis in patients with CRC. Our results showed that the expression of RASSF10 mRNA and protein in CRC-adjacent tissues was higher than that in CRC tissues. Low RASSF10 expression was associated with the T stage (P=.037, odds ratio, 0.664; 95% confidence interval, 0.452-0.975) and the N stage (P<.001, odds ratio, 0.318; 95% confidence interval, 0.184-0.549) of the tumors. In addition, univariate analysis revealed that patients with CRC with lower RASSF10 expression had poorer overall survival (OS; P<.001) and disease-free survival (DFS; P<.001). The 5-year OS and DFS rates were 48.2% and 28.3%, respectively, in patients with low RASSF10 expression and 82.2% and 62.6%, respectively, in patients with high RASSF10 expression. Multivariate Cox regression analysis revealed that the strongest predictors of OS and DFS were RASSF10 expression (P<.001 and P<.001, respectively), T stage (P=.003 and P=.009, respectively), and N stage (P=.005 and P=.026, respectively). These results demonstrate that low expression of RASSF10 in CRC tissues is significantly correlated with poor survival after curative resection and may serve as a useful biomarker predictive of CRC prognosis.