ERO1α promotes the proliferation and inhibits apoptosis of colorectal cancer cells by regulating the PI3K/AKT pathway.

Endoplasmic reticulum oxidoreductin 1α (ERO1α) is an oxidase that exists in the endoplasmic reticulum and plays an important role in regulating oxidized protein folding and tumor malignant progression. However, the specific role and mechanism of ERO1α in the progression of colorectal cancer (CRC) have not yet been fully elucidated. In this study, 280 specimens of CRC tissues and adjacent noncancerous tissues were collected to detect the expression of ERO1α and analyze the clinical significance. ERO1α was stably knocked-down in RKO and HT29 CRC cells to investigate its function and mechanism in vitro and in vivo. We found that ERO1α was remarkably upregulated in CRC tissues and high ERO1α expression is associated with N stage and poor prognosis of CRC patients. ERO1α knockdown in CRC cells significantly inhibited the proliferation and induced apoptosis while inactivating the PI3K/AKT pathway. Rescue assays revealed that AKT activator 740Y-P could reverse the effects on proliferation and apoptosis of ERO1α knockdown in CRC cells. In vivo tumorigenicity assay also confirmed that ERO1α knockdown suppressed tumor growth. Taken together, our findings demonstrated ERO1α promotes the proliferation and inhibits apoptosis of CRC cells by regulating the PI3K/AKT pathway. High expression of ERO1α is associated with poor prognosis in CRC patients, and ERO1α could be a potential therapeutic target for CRC.

Dioscin reduced chemoresistance for colon cancer and analysis of sensitizing targets.

Chemotherapy resistance is the primary cause of high mortality in patients with advanced colon cancer. The combination of small molecule compound dioscin (DIO) and traditional medicine may have a chemosensitizing effect. In this study, we reported that DIO, in combination with Oxaliplatin (L-OHP) and 5-fluorouracil (5-Fu), can effectively inhibit colon cancer cell proliferation, and co-treatment was positively related to the DIO concentration. HCT116 co-treatment with 6.4 µM L-OHP and 0.8 µM DIO significantly reduced colony formation and migration, increased apoptosis, and cell-cycle arrest in the G0/G1 and G2/M phase. DIO-assisted L-OHP significantly inhibited the xenograft model growth and exhibited low toxicity.The mRNA-sequencing combined with network pharmacological analysis suggested that the DIO sensitivity may be related to the active targets FAS, CDKN1A, ABCA1, and PPARA, which are primarily involved in regulating the cell cycle and apoptosis. Finally, our experiments suggest that DIO may enhance the L-OHP sensitivity by regulating the cell cycle through the Notch pathway.

Knockdown of mitochondrial threonyl-tRNA synthetase 2 inhibits lung adenocarcinoma cell proliferation and induces apoptosis.

Lung cancer is a significant global burden. Aminoacyl-tRNA synthetases (aaRSs) can be reliably identified by the occurrence and improvement of tumors. Threonyl-tRNA synthetase (TARS) and mitochondrial threonyl-tRNA synthetase 2 (TARS2) are both aaRSs. Many studies have shown that TARS are involved in tumor angiogenesis and metastasis. However, TARS2 has not yet been reported in tumors. This study explored the role of TARS2 in the proliferation and apoptosis of lung adenocarcinoma (LUAD). TARS2 expression in lung adenocarcinoma and non-cancerous lung tissues was detected via immunohistochemistry. Cell proliferation was detected using MTS, clone formation, and EdU staining assays. Flow cytometry was used to detect cell cycle, mitochondria reactive oxygen species (mROS) production, and apoptosis. Mitochondrial membrane potential (MMP ΔΨm) was detected using JC-1 fluorescent probes. Cell cycle, apoptosis-related pathway, and mitochondrial DNA (mtDNA) -encoded protein expression was detected via Western blotting. Finally, the effect of TARS2 on tumor growth was examined using a xenotransplanted tumor model in nude mice. We found that TARS2 was highly expressed in lung adenocarcinoma tissues and associated with poor overall survival (OS). Mechanistic analysis showed that knockdown of TARS2 inhibited proliferation through the retinoblastoma protein (RB) pathway and promoted mROS-induced apoptosis. Knockdown of TARS2 inhibits tumor growth in a xenotransplanted tumor model. TARS2 plays an important role in LUAD cell proliferation and apoptosis and may be a new therapeutic target.

A Novel Prognostic Model Based on Ferroptosis-Related Gene Signature for Bladder Cancer.

BACKGROUND: Bladder cancer (BC) is a molecular heterogeneous malignant tumor; the treatment strategies for advanced-stage patients were limited. Therefore, it is vital for improving the clinical outcome of BC patients to identify key biomarkers affecting prognosis. Ferroptosis is a newly discovered programmed cell death and plays a crucial role in the occurrence and progression of tumors. Ferroptosis-related genes (FRGs) can be promising candidate biomarkers in BC. The objective of our study was to construct a prognostic model to improve the prognosis prediction of BC. METHODS: The mRNA expression profiles and corresponding clinical data of bladder urothelial carcinoma (BLCA) patients were downloaded from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) databases. FRGs were identified by downloading data from FerrDb. Differential analysis was performed to identify differentially expressed genes (DEGs) related to ferroptosis. Univariate and multivariate Cox regression analyses were conducted to establish a prognostic model in the TCGA cohort. BLCA patients from the GEO cohort were used for validation. Gene ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and single-sample gene set enrichment analysis (ssGSEA) were used to explore underlying mechanisms. RESULTS: Nine genes (ALB, BID, FADS2, FANCD2, IFNG, MIOX, PLIN4, SCD, and SLC2A3) were identified to construct a prognostic model. Patients were classified into high-risk and low-risk groups according to the signature-based risk score. Receiver operating characteristic (ROC) and Kaplan-Meier (K-M) survival analysis confirmed the superior predictive performance of the novel survival model based on the nine-FRG signature. Multivariate Cox regression analyses showed that risk score was an independent risk factor associated with overall survival (OS). GO and KEGG enrichment analysis indicated that apart from ferroptosis-related pathways, immune-related pathways were significantly enriched. ssGSEA analysis indicated that the immune status was different between the two risk groups. CONCLUSION: The results of our study indicated that a novel prognostic model based on the nine-FRG signature can be used for prognostic prediction in BC patients. FRGs are potential prognostic biomarkers and therapeutic targets.

High-resolution metabolomic biomarkers for lung cancer diagnosis and prognosis.

Lung cancer is the leading cause of human cancer mortality due to the lack of early diagnosis technology. The low-dose computed tomography scan (LDCT) is one of the main techniques to screen cancers. However, LDCT still has a risk of radiation exposure and it is not suitable for the general public. In this study, plasma metabolic profiles of lung cancer were performed using a comprehensive metabolomic method with different liquid chromatography methods coupled with a Q-Exactive high-resolution mass spectrometer. Metabolites with different polarities (amino acids, fatty acids, and acylcarnitines) can be detected and identified as differential metabolites of lung cancer in small volumes of plasma. Logistic regression models were further developed to identify cancer stages and types using those significant biomarkers. Using the Variable Importance in Projection (VIP) and the area under the curve (AUC) scores, we have successfully identified the top 5, 10, and 20 metabolites that can be used to differentiate lung cancer stages and types. The discrimination accuracy and AUC score can be as high as 0.829 and 0.869 using the five most significant metabolites. This study demonstrated that using 5 + metabolites (Palmitic acid, Heptadecanoic acid, 4-Oxoproline, Tridecanoic acid, Ornithine, and etc.) has the potential for early lung cancer screening. This finding is useful for transferring the diagnostic technology onto a point-of-care device for lung cancer diagnosis and prognosis.

ERO1α mediates endoplasmic reticulum stress-induced apoptosis via microRNA-101/EZH2 axis in colon cancer RKO and HT-29 cells.

Although colon cancer is a leading and typical gastrointestinal tumor, there is little published data on the underlying molecular mechanisms of endoplasmic reticulum (ER) stress. Here, we investigated the role of ERO1α and its impact on microRNA (miR)-101 expression and ER stress in colon cancer cells. Cell ER stress was established by treating RKO or HT-29 cells with 1 µM thapsigargin (THG). Cell biological behaviors were detected using CCK-8, bromodeoxyuridine assay, flow cytometry and western blot. We also investigated the expression of ERO1α and miR-101 after THG treatment using RT-qPCR. Moreover, effects of ERO1α and miR-101 on ER stress of colon cancer cells were detected. Additionally, miR-101 impact on EZH2 expression and relevance of this regulation was confirmed by RT-qPCR and luciferase reporter. The regulation of miR-101/EZH2 axis and Wnt/ß-catenin pathway in ER stress were investigated. Our results demonstrated that THG induced ER stress in colon cancer cells. Silencing ERO1α further promoted ER stress-induced cell apoptosis. ERO1α knockdown up-regulated miR-101 expression and promoted colon cancer cell apoptosis via regulating miR-101. Surprisingly, miR-101 negatively regulated EZH2 expression via miRNA-mRNA targeting. Moreover, ER stress promoted colon cancer cell apoptosis via regulating miR-101/EZH2 axis. Wnt/ß-catenin pathway was also involved in the regulation of ERO1α/miR-101/EZH2 in ER stress of colon cancer cells. These findings illustrated that silencing ERO1α regulated ER stress-induced apoptosis via miR-101/EZH2 axis in RKO and HT-29 cells.

LncRNA MEG3 promotes endoplasmic reticulum stress and suppresses proliferation and invasion of colorectal carcinoma cells through the MEG3/miR-103a-3p/PDHB ceRNA pathway.

LncRNA maternally expressed gene 3 (MEG3) is a potential prognostic and diagnostic biomarker in colorectal carcinoma (CC). However, its cellular functions and mechanism remain not fully uncovered. Relative expression of MEG3, miRNA (miR)-103a-3p, and pyruvate dehydrogenase E1 subunit beta (PDHB) was detected by RT-qPCR and western blotting. Cell proliferation was measured by CCK-8 assay, colony formation assay, and flow cytometry, as well as xenograft tumor assay. Transwell assay examined cell invasion. Endoplasmic reticulum (ER) stress was evaluated by western blotting. Dual-luciferase reporter assay and RNA immunoprecipitation determined the relationship between miR-103a-3p and MEG3 or PDHB. Expression of MEG3 was downregulated in human CC tumor tissues and cells (SW620 and HCT116), accompanied by higher miR-103a-3p and lower PDHB. Restoring MEG3 suppressed cell viability, colony formation ability, and invasion, arrested cell cycle, and induced apoptosis rate in SW620 and HCT116 cells, as well as promoted expression of ER stress-related proteins (GRP78, ATF6, CHOP, caspase-3, and caspase-9). Furthermore, MEG3 overexpression hindered tumor growth and facilitated ER stress in vivo. Molecularly, miR-103a-3p was a target of MEG3, and further targeted PDHB. Similarly, in function, blocking miR-103a-3p suppressed CC in vitro by affecting proliferation, invasion, and ER stress; in addition, restoring miR-103a-3p partially counteracted the suppressive role of MEG3 in CC cells. MEG3 sponged miR-103a-3p to suppress CC malignancy by inducing ER stress and inhibiting cell proliferation and invasion via upregulating PDHB, suggesting a novel MEG3/miR-103a-3p/PDHB ceRNA pathway.

Knockdown of microsomal glutathione S-transferase 1 inhibits lung adenocarcinoma cell proliferation and induces apoptosis.

BACKGROUND: Lung cancer has been the most common cancer worldwide. Microsomal glutathione S-transferase 1 (MGST1) has been reported to play vital roles in oxidative stress, tumor occurrence and drug resistance. However, the biological function and molecular mechanism of MGST1 in lung adenocarcinoma (LUAD) has not yet been elucidated. METHODS: The expression of MGST1 in LUAD tissues and cell lines was evaluated by immunohistochemistry and western blotting, respectively. MGST1 was knocked down by shRNA lentivirus. Cell proliferation was evaluated by MTS, colony formation and EdU assays. Apoptosis was detected by flow cytometry. The potential molecules involved in cell proliferation and apoptosis were examined by western blotting. Finally, the effect of MGST1 on tumor growth in vivo was evaluated in a nude mouse xenograft model. RESULTS: TCGA database analysis and immunohistochemistry demonstrated that MGST1 was highly expressed in LUAD tissues. MGST1 expression in LUAD was correlated with AJCC stage and poor overall survival of patients. MGST1 knockdown significantly inhibited LUAD cell proliferation and induced apoptosis. Mechanistic analyses revealed that MGST1 knockdown might inhibit cell proliferation by inactivating the AKT/GSK-3ß pathway signaling and promote cell apoptosis by regulating the mitochondrial apoptosis pathway related proteins. Moreover, knockdown of MGST1 suppressed tumor growth in vivo. CONCLUSIONS: MGST1 plays an important role in LUAD tumorigenesis and might serve as a potential prognostic factor and therapeutic target in LUAD.

Anticancer effect of the traditional Chinese medicine herb Maytenus compound via the EGFR/PI3K/AKT/GSK3ß pathway.

BACKGROUND: Cancer is a leading cause of death worldwide; folk anticancer medicinal plants have applied for cancer treatment. The Maytenus compound tablet as traditional Chinese compound medicine has been approved for alleviating hyperplasia of mammary glands, whether it can inhibit cancer still unknown. The study was to evaluate the anticancer activity of the Maytenus compound tablet. METHODS: MTS assay evaluated the anti-proliferation effect of the Maytenus compound on H226, A2058, 786O and HeLa cancer cells and two normal epithelial cell lines, namely, 16HBE and Hecate. Nude mouse xenograft tumor model using H226 and HeLa cells examined the drug's anticancer effect in vivo. Western blot assay studied the possible mechanism. RESULTS: The Maytenus compound indicated obvious ability to against proliferation in four strains of cancer cells, particularly against H226 cells by an IC50 of 85.47±10.06 µg/mL and against HeLa cells by an IC50 of 128.74±17.46 µg/mL. However, it had a low cytotoxicity in human normal epithelial cell lines 16HBE with an IC50 of 4,555.86±25.21 µg/mL and Hecate with an IC50 of 833.56±181.88 µg/mL. The Maytenus compound at the 2.45 g/kg oral dosages inhibited the proliferation of H226 cells and HeLa cells in nude mouse with inhibitory rates of 36.06% and 26.45%, respectively, and no organ toxicity. The Maytenus compound could significantly downregulate the expression of pEGFR, pPI3K, pAKT, pGSK3ß, ß-catenin, and c-MYC and upregulate the protein expression of GSK3ß. CONCLUSIONS: The Maytenus compound has significant anticancer activities against human cancer H226 and HeLa cells both in vitro and in vivo, highlighting it may be an anticancer medicine.

Prevalence and Molecular Study of G6PD Deficiency in the Dai and Jingpo Ethnic Groups in the Dehong Prefecture of the Yunnan Province.

OBJECTIVES: To estimate the prevalence and mutation types of G6PD deficiency and evaluate the relationship between G6PD genotypes and erythrocyte phenotypes in the Dai and Jingpo ethnic groups in the Dehong prefecture of the Yunnan province, China. METHODS: G6PD deficiency was screened in Dai (1,530 individuals) and Jingpo (372 individuals) populations using a modified G6PD/6PGD ratio assay. Red blood cell traits were analyzed using the Sysmex XE2100 fully automated blood analyzer. PCR-direct sequencing for G6PD genotyping analysis was performed, and then the linkage disequilibrium blocks of the target SNPs were constructed with Haploview 4.2 software. RESULTS: The prevalence of G6PD deficiency was higher in the Dai ethnic group (8.63%) than in the Jingpo ethnic group (5.91%). The major mutations in descending order were rs137852314 G>A, rs72554664 G>A, rs72554665 G>T, and rs137852341 G>T. Hemoglobin concentration was significantly lower in the rs137852314 G>A group than in the normal group (p = 0.021). Mean corpuscular volume and mean corpuscular hemoglobin were substantially higher in the rs137852341 G>T group compared to the normal group (p = 0.049, p = 0.042). A linkage disequilibrium block of 13 SNPs was constructed for the G6PD deficiency group from the Dai sample. CONCLUSIONS: The Dai and Jingpo ethnic groups have distinctive incidence rates and gene frequencies of G6PD deficiency, and the genotypes of G6PD deficiency are associated with erythrocyte phenotypes.

Up-regulation of microRNA-340 promotes osteosarcoma cell apoptosis while suppressing proliferation, migration, and invasion by inactivating the CTNNB1-mediated Notch signaling pathway.

Osteosarcoma (OS) is the most common histological form of primary bone cancer. It is most prevalent in teenagers and young adults. The present study aims at exploring the regulatory effect of microRNA-340 (miR-340) on OS cell proliferation, invasion, migration, and apoptosis via regulating the Notch signaling pathway by targeting ß-catenin (cadherin-associated protein) 1 (CTNNB1). OS tissues belonging to 45 patients and normal femoral head tissues of 45 amputees were selected. Cells were allocated to different groups. In situ hybridization was performed to determine the positive rate of miR-340 expression while immunohistochemistry was used to determine that of CTNNB1 and B-cell lymphoma 2 (Bcl-2). We used a series of experiments to measure the expressions of related factors and assess rates of cell proliferation, migration, invasion, cycle, and apoptosis respectively. Our results show that miR-340 was expressed a higher level in normal tissue than OS tissue. Expression of Notch, CTNNB1, hairy and enhancer of split 1 (Hes1), Bcl-2, Runt-related transcription factor 2 (Runx2), and osteocalcin increased and that of miR-340, Bcl-2 interacting mediator of cell death (BIM), and Bcl-2 associated protein X (Bax) decreased in OS tissues. U-2OS cell line had the highest miR-340 expression. We also found that the up-regulation of miR-340 had increased expression of miR-340, BIM, and Bax but decreased expression of Notch, CTNNB1, Hes1, Bcl-2, Runx2, and osteocalcin. Up-regulation of miR-340p lead to increased cell apoptosis, suppressed cell proliferation, migration, and invasion. Our study demonstrates that overexpression of miR-340 could suppress OS cell proliferation, migration, and invasion as well as promoting OS cell apoptosis by inactivating the Notch signaling pathway via down-regulating CTNNB1. Functional miR-340 overexpression might be a future therapeutic strategy for OS.

Effects of MicroRNA-206 on Osteosarcoma Cell Proliferation, Apoptosis, Migration and Invasion by Targeting ANXA2 Through the AKT Signaling Pathway.

BACKGROUND/AIMS: This study aimed to investigate the mechanism by which microRNA-206 (miR-206) affects the proliferation, apoptosis, migration and invasion of osteosarcoma (OS) cells by targeting ANXA2 via the AKT signaling pathway. METHODS: A total of 132 OS tissues and 120 osteochondroma tissues were examined in this study. The targeting relationship between miR-206 and ANXA2 was verified with a dual-luciferase reporter assay. The miR-206 expression and ANXA2, AKT, PARP, FASN, Survivin, Bax, Mcl-1 and Bcl-1 mRNA and protein expression in the above two groups were examined by qRT-PCR and western blotting. The cultured OS cells were divided into 6 groups: a blank group, negative control (NC) group, miR-206 mimic group, miR-206 inhibitor group, si-ANXA2 group and miR-206 inhibitor + si-ANXA2 group. Cell cycle and apoptosis were assessed by flow cytometry, cell migration was examined with a wound-healing assay, and cell invasion was assessed with a Transwell assay. Pearson correlation analysis was used to determine the correlation between ANXA2 mRNA expression and miR-206 expression in OS. RESULTS: OS tissues exhibited increased mRNA and protein expression of ANXA2, AKT, PARP, FASN, Survivin, Mcl-1 and Bcl-2; decreased miR-206 expression; and decreased Bax mRNA and protein expression. ANXA2 mRNA expression was strongly negatively correlated with miR-206 expression in OS. ANXA2 was found to be a miR-206 target gene. In the miR-206 mimic group and the si-ANXA2 group, the mRNA and protein expression of ANXA2, AKT, PARP, FASN, Survivin, Mcl-1 and Bcl-1 decreased markedly, cell proliferation was inhibited, apoptosis was promoted, higher cell growth in G1 phase and decreased growth in S phase was detected, and decreased cell migration and invasion were observed compared with those in the blank group. CONCLUSION: The current results demonstrate that miR-206 overexpression inhibits OS cell proliferation, migration and invasion and promotes apoptosis through targeting ANXA2 by blocking the AKT signaling pathway.

MicroRNA-370 inhibits the growth and metastasis of lung cancer by down-regulating epidermal growth factor receptor expression.

Abnormal microRNA-370 (miR-370) expression has been frequently reported in several types of cancers, including lung cancer. However, the role and molecular mechanisms of miR-370 in regulating the growth and metastasis of lung cancer have not been clarified. Here, we show higher levels of epidermal growth factor receptor (EGFR), but lower levels of miR-370 expression in most human lung cancer cells and non-tumor cells. Induction of miR-370 over-expression significantly reduced the levels of EGFR expression and the EGFR 3'untranslated region (UTR)-regulated luciferase activity in XWLC-05 and H157 cells, suggesting that miR-370 may bind to the 3'UTR of EGFR mRNA. Compared with the control cells, induction of miR370 overexpression significantly inhibited the proliferation, clone formation capacity, migration and invasion of XWLC-05 and H157 cells while miR-370 inhibitor over-expression enhanced their tumor behaviors in vitro. Furthermore, miR-370 over-expression down-regulated the EGFR and hypoxia-inducible factor (HIF)-1α expression, and attenuated the extracellular single-regulated kinase (ERK)1/2 and AKT phosphorylation in XWLC-05 and H157 cells. In contrast, miR370 inhibitor over-expression increased the EGFR and HIF-1α expression as well as the ERK1/2 and AKT phosphorylation in XWLC-05 and H157 cells. Moreover, miR-370 over-expression significantly reduced the levels of EGFR and CD31 expression and inhibited the growth and lung metastasis of xenograft NSCLC tumors in mice. Our study indicates that miR-370 may bind to the 3'UTR of EGFR to inhibit EGFR expression and the growth, angiogenesis and metastasis of non-small cell lung cancer by down-regulating the ERK1/2 and AKT signaling.

Acquired resistance to HSP90 inhibitor 17-AAG and increased metastatic potential are associated with MUC1 expression in colon carcinoma cells.

Heat shock protein 90 (HSP90) is a molecular chaperone required for the stability and function of many proteins. The chaperoning of oncoproteins by HSP90 enhances the survival, growth, and invasive potential of cancer cells. HSP90 inhibitors are promising new anticancer agents, in which the benzoquinone ansamycin 17-allylamino-17-demethoxygeldanamycin (17-AAG) is currently in clinical evaluation. However, the implications of acquired resistance to this class of drug remain largely unexplored. In the present study, we have generated isogenic human colon cancer cell lines that are resistant to 17-AAG by continued culturing in the compound. Cross-resistance was found with another HSP90 inhibitor 17-dimethylaminoethylamino-17-demethoxygeldanamycin. The resistant cells showed obvious morphology changes with a metastatic phenotype and significant increases in migration and adhesion to collagens. Western blotting analysis of epithelial-mesenchymal transition molecular markers found that expression of E-cadherin downregulated, whereas expression of N-cadherin and ß-catenin upregulated in the resistant cells. Mucin 1 (MUC1) has been reported to mediate metastasis as well as chemical resistance in many cancers. Here, we found that MUC1 expression was significantly elevated in the acquired drug resistance cells. 17-AAG treatment could decrease MUC1 more in parental cells than in acquired 17-AAG-resistant cells. Further study found that knockdown of MUC1 expression by small interfering RNA could obviously re-sensitize the resistant cells to 17-AAG treatment, and decrease the cell migration and adhesion. These were coupled with a downregulation in N-cadherin and ß-catenin. The results indicate that HSP90 inhibitor therapies in colon carcinomas could generate resistance and increase metastatic potential that might mediated by upregulation of MUC1 expression. Findings from this study further our understanding of the potential clinical effects of HSP90-directed therapies in colon carcinomas.

Down-regulated SOX4 expression suppresses cell proliferation, metastasis and induces apoptosis in Xuanwei female lung cancer patients.

The transcription factor SOX4 has functional importance in foetal lung maturation and tumorigenesis in a number of cancers. However, its biological functions in the progression of lung tumorigenesis remain unclear. In this study, we found that the expression levels of SOX4 mRNA and protein were significantly higher in Xuanwei female lung cancer tissues than in benign lung lesions. The patients with high expression of the SOX4 protein had a higher pathological grade, lymph node (LN) metastasis, poor tumor differentiation and worse prognosis than those patients with low expression of SOX4. Knockdown of the SOX4 gene in the Xuanwei female lung cancer cell line XWLC-05 resulted in apoptotic morphological changes, decreased cell proliferation, invasion and migration. Furthermore, knockdown of the SOX4 gene resulted in obvious sub-G1 peaks and induction of apoptosis through upregulation of caspase-3 expression, while in cells treated with a caspase-3 inhibitor, apoptosis induced by silencing SOX4 expression was inhibited. In vivo analysis in nude mice further confirmed that knockdown of SOX4 suppressed tumor growth. In conclusion, SOX4 appears to be an important tumor suppressor gene in the regulation of Xuanwei female lung cancer cell proliferation, apoptosis and metastases, and it may be a potential target for effective lung cancer therapy.

[Targeted Sox4 gene-knock-down suppresses xenograft growth of Xuanwei female lung cancer cell line XWLC-05 cells in nude mice].

OBJECTIVE: To study the effect of targeted Sox4 gene-knock-down on the growth of xenografts of Xuanwei female lung cancer cell line XWLC-05 cells in nude mice. METHODS: Recombinant plasmid pGFP-V-RS-Sox4 shRNA was constructed and transfected into XWLC-05 cells. Real-time quantitative PCR and Western blot were applied to confirm the effect of Sox4 gene-knock-down. XWLC-05 cells stably transfected with the plasmids were inoculated into nude mice to establish the xenograft model. The nude mouse status, tumor formation and tumor growth were observed, and the tumor inhibition rate was calculated. CT scan was performed to assess the metastasis of xenografts. Immunohistochemical staining was applied to detect Sox4 and ki-67 protein expression. RESULTS: Recombinant plasmid pGFP-V-RS-A-Sox4 shRNA which can effectively knocking-down Sox4 gene was successfully constructed and the stable transfected cells were selected by puromycin-screening. The success rate of tumor cell inoculation was 100% in the mice of all groups except those inoculated with saline. The body weight of all mice inoculated with parental XWLC-05 cells (blank control), pGFP-V-RS-scram shRNA trsfected XWLC-05 cells (negative control), and pGFP-V-RS-Sox4 shRNA transfected XWLC-05 cells was increased to a varying degree, but there was no significant difference among the groups (P > 0.05 ). The growth of xenografts was significantly inhibited after silencing the Sox4 gene expression when compared with that of the blank and negative controls (P < 0.05) . The volume of removed tumors of the Sox4 gene-inhibited mice was (2.30 ± 0.34) cm(3) , significantly smaller than that of the negative control (3.99 ± 0.45) cm(3) and the blank control (4.03 ± 0.42) cm(3) (P < 0.05) . The weight of removed tumors of Sox4 gene-inhibited mice was (0.86 ± 0.14) g, significantly lower than that of the negative control (1.84 ± 0.27) g and blank control (1.86 ± 0.22) g, (P < 0.05). Immunohistochemical staining showed that Sox4 and ki-67 proteins mainly expressed in cell nuclei. The staining was significantly decreased in xenografts of Sox4-inhibited mice when compared with the negative and blank controls (P < 0.05). No distant metastasis was found in any mouse by CT imaging and pathological examination during the observation period. CONCLUSIONS: The xenograft model of Xuanwei female lung cancer cell line XWLC-05 cells in nude mice is successfully established. Knocking-down of Sox4 gene can suppress the xenograft tumor growth.

Aberrant expression of trefoil factor 3 is associated with colorectal carcinoma metastasis.

BACKGROUND: Recent evidence has indicated that the trefoil factor family possesses pivotal roles in the progression of human cancer. Aberrant expression of trefoil factor 3 (TFF3) has been reported to correlate with an aggressive tumor phenotype. However, the clinical importance of TFF3 expression in colorectal carcinomas (CRCs) has rarely been addressed. PURPOSE: To investigate the putative role of TFF3 in colorectal carcinogenesis and progress, and to clarify whether TFF3 could be a serum marker for CRCs. MATERIALS AND METHODS: Fifty-six CRCs were sequenced for TFF3 mutations; subsets of the primary tumors were subjected to real-time quantitative reverse transcription polymerase chain reaction (RT-PCR) and immunohistochemistry analyses and serum TFF3 was detected by enzyme-linked immunosorbent assay (ELISA) for patients with CRCs. RESULTS: No variants were detected in the code area of TFF3; TFF3 mRNA is increased in CRCs but not up to statistic significance when compared with paired normal colonic mucosa; TFF3 staining by immunohistochemistry in primary CRCs showed that increased expression of TFF3 is associated with lymph node metastases(LNM), and no significant differences were found with respect to patient's sex, cancer cell differentiation and stage. Serum TFF3 is significantly elevated in patients with CRCs, especially CRCs with LNM. CONCLUSION: The results indicate that TFF3 point mutations seem to be a rare event in colorectal carcinogenesis; TFF3 expression may play a role in promoting lymph node metastases of CRCs and serum TFF3 may be a potential useful marker for patients with CRCs and their metastases.

Trefoil factor 1 gene alternations and expression in colorectal carcinomas.

AIMS AND BACKGROUND: Aberrant expression of the trefoil factor family (TFF) has been recognized to be involved in the development and/or progression of various solid tumors. Increased trefoil factor 1 (TFF1) expression is found associated with tumor progression in some tumors, and TFF1 missense mutations have been detected in gastric cancer. The aim of the study was to analyze TFF1 alternations and expression in colorectal carcinoma and their correlation with cancer progression and pathological aspects. METHODS: TFF1 mutations were detected in colorectal carcinomas by DNA sequencing. TFF1 mRNA and protein levels in subsets of the primary tumors were determined using quantitative reverse transcription polymerase chain reaction and immunohistochemistry analyses. The serum level of TFF1 was also detected by enzyme-linked immunosorbent assay for patients with colorectal carcinoma. RESULTS: Five variants were detected in the 5'-untranslation region and intron 1 of TFF1. TFF1 expression was increased in colorectal carcinoma compared to paired distal colonic mucosa. Immunohistochemistry in primary colorectal carcinoma showed no significant differences in tumor TFF1 levels with respect to clinicopathological parameters such as the patient's sex, cancer differentiation, stage and lymph node metastasis. However, serum TFF1 levels were significantly elevated in patients with colorectal carcinoma compared to healthy individuals. CONCLUSIONS: The results indicate that TFF1 missense mutations seem to be a rare event in colorectal carcinogenesis. Serum TFF1 may be a potential useful marker for patients with colorectal carcinoma.

The frog trefoil factor Bm-TFF2 activates human platelets via Gq and G12/13 signaling pathway.

Bm-TFF2 is an amphibian trefoil factor purified from the Bombina maxima skin secretion that is highly toxic to mammals. We previously reported that Bm-TFF2 activates human platelets via protease-activated receptor 1. In this study, for a better understanding of platelet activation induced by Bm-TFF2, we used affinity chromatography and pharmacological inhibitors to investigate the downstream signaling pathway. Using Bm-TFF2-affinity chromatography, Gq was specifically eluted from the Bm-TFF2-coulped column. Pharmacological inhibitors such as U73122, Xestospongin C, BAPTA-AM and Gö6976 can significantly inhibit Bm-TFF2-induced platelet aggregation. These results suggested that Gq activation and the downstream PLCß-IP3 receptor-cytoplasmic Ca(2+)-PKC signaling pathway is crucial for Bm-TFF2 to stimulate platelet aggregation. Furthermore, Bm-TFF2 induced strong platelet shape change at the concentrations of 5nM, in which the Ca(2+) mobilization of the platelets stimulated was not detectable. The p160(ROCK) inhibitorY27632 totally inhibited the shape change, indicating that Bm-TFF2 may activate the G12/13 pathway which leads to the activation of RhoA-p160(ROCK). In conclusion, Bm-TFF2 induced platelet activation mainly via the Gq and G12/13 signaling pathway. This study on the signaling pathway of Bm-TFF2 stimulation may help us understand the toxicity of B. maxima skin secretion to the human platelets.

Ginsenoside-Rg1 from Panax notoginseng prevents hepatic fibrosis induced by thioacetamide in rats.

Panax notoginseng saponins have recently been reported to suppress liver fibrosis. Since ginsenoside-Rg1 is the most abundant component of P.notoginseng saponins, we investigated the effect of ginsenoside-Rg1 on experimental liver fibrosis in rats. Histological analysis revealed that ginsenoside-Rg1 significantly improved the extent of liver fibrosis in rats induced by thioacetamide. Ginsenoside-Rg1 markedly suppressed the serum levels of fibrotic markers and hepatic hydroxyproline content in rats treated with thioacetamide. Ginsenoside-Rg1 also reduced the serum levels of alanine transaminase, aspartate transaminase and alkaline phosphatase. Finally, ginsenoside-Rg1 attenuated the levels of thiobarbituric acid reactive substances in livers of rats treated by thioacetamide. In cultured hepatic stellate cells, ginsenoside-Rg1 markedly inhibited cell proliferation, activation and formation of reactive oxygen species stimulated by platelet-derived growth factor-BB (PDGF-BB). Additionally, ginsenoside-Rg1 down-regulated the expression of PDGF receptor-beta by reducing the nuclear factor-kappaB activity, which was required for the gene expression. These results suggest that ginsenoside-Rg1, which exhibits its antioxidant and antifibrotic properties, may be of potential therapeutic value in protecting the liver fibrosis.