Guoxiong Xu

Pyridoxine 5′-phosphate oxidase is a novel therapeutic target and regulated by the TGF-β signalling pathway in epithelial ovarian cancer

Pyridoxine 5′-phosphate oxidase (PNPO) is an enzyme that converts pyridoxine 5′-phosphate into pyridoxal 5′-phosphate (PLP), an active form of vitamin B6 implicated in several types of cancer. However, the role of PNPO and its regulatory mechanism in epithelial ovarian cancer (EOC) are unknown. In the present study, PNPO expression in human ovarian tumour tissue and its association with the clinicopathological features of patients with EOC were examined. Further, the biological function of PNPO in EOC cells and in xenograft was evaluated. We demonstrated for the first time that PNPO was overexpressed in human EOC. Knockdown of PNPO induced EOC cell apoptosis, arrested cell cycle at G2/M phase, decreased cell proliferation, migration and invasion. Xenografts of PNPO-shRNA-expressing cells into the nude mouse attenuated tumour growth. PNPO at mRNA and protein levels in EOC cells was decreased after transforming growth factor-β1 (TGF-β1) treatment. The inhibitory effect of TGF-β1 on PNPO expression was abolished in the presence of SB-431542, a TGF-β type I receptor kinase inhibitor. Moreover, we found that TGF-β1-mediated PNPO expression was at least in part through the upregulation of miR-143-3p. These data indicate a mechanism underlying PNPO regulation by the TGF-β signalling pathway. Furthermore, PLP administration reduced PNPO expression and decreased EOC cell proliferation, suggesting a feedback loop between PLP and PNPO. Thus, our findings reveal that PNPO can serve as a novel tissue biomarker of EOC and may be a potential target for therapeutic intervention.

Biomarker triplet NAMPT/VEGF/HER2 as a de novo detection panel for the diagnosis and prognosis of human breast cancer

The early detection of breast cancer, the most common malignant tumor disease in women worldwide, relies on mammography and self breast examination. Here we evaluated the concentration of nicotinamide phosphoribosyltransferase (NAMPT), vascular endothelial growth factor (VEGF) and human epidermal growth factor receptor-2 (HER2) in serum and their expression in breast tissues associated with the clinicopathological features of patients with benign and malignant breast tumors. The immunohistochemical analysis showed that NAMPT, VEGF and HER2 proteins were overexpressed in breast tumors. The highest expression was observed in malignant tumors, low in benign tumors and negative in the adjacent normal tissue, indicating that the triplets may be progression markers and correlated with each other. The detection rate of NAMPT, VEGF and HER2 alone in tissue was 54.17, 64.58 and 60.42%, respectively, and was increased to about 79% in double combination and to 90% in triple combination. The basal levels of serum NAMPT, VEGF and HER2 in healthy controls were 94.90±4.24 pg/ml, 87.02±2.41 pg/ml and 1.12±0.04 ng/ml, respectively, measured by ELISA and found to be increased by 6.64-, 1.76- and 2.52-fold, respectively, in patients with malignant breast tumor. These elevated serum levels of NAMPT, VEGF and HER2 in patients were decreased after tumor removal, suggesting that these molecules are the indicators of treatment efficacy. The combined measurement of these triplets together may improve the sensitivity of breast cancer diagnosis and may potentially be used as a testing panel for the detection of malignant tumors, the assessment of treatment effectiveness and the monitoring of the disease progression in patients with breast cancer. Thus, we propose that the biomarker triplet NAMPT/VEGF/HER2 can be used as a de novo detection panel for the diagnosis and prognosis of human breast cancer.

Stomatin-like protein 2 is associated with the clinicopathological features of human papillary thyroid cancer and is regulated by TGF-β in thyroid cancer cells

Papillary thyroid cancer (PTC) accounts for 80-90% of all cases of thyroid malignancies. Stomatin-like protein 2 (SLP-2) is a novel member of the stomatin superfamily and is found in several types of human tumors. However, whether it is expressed in human PTC is unknown. In the present study, we aimed to explore the diagnostic value of SLP-2 in patients with PTC and to investigate whether SLP-2 expression is regulated by transforming growth factor-β (TGF-β), a cytokine which plays an important role in PTC tumorigenesis. A total of 107 patients consisting of 99 cases of classical and 8 cases of follicular variant PTC was examined. The expression of SLP-2 mRNA and protein was examined by immunohistochemistry (IHC) and qPCR, respectively. We found that SLP-2 was overexpressed in human PTC. The expression of SLP-2 was significantly associated with clinicopathological features of the PTC cases. Particularly, increased SLP-2 expression was mainly correlated with primary tumors >1 cm in size, with late stage tumors and with metastatic lymph nodes. The expression of SLP-2 was correlated with the expression of Ki-67, a cell proliferation marker, in PTC tissues as detected by IHC. SLP-2 was upregulated by TGF-β1 in PTC cells as evaluated by western blotting. The present data revealed for the first time that patients with PTC exhibited SLP-2 overexpression that was associated with clinicopathological features. The correlation between SLP-2 expression and proliferation marker Ki-67 may be characteristic of PTC and may reflect PTC progression. SLP-2 was upregulated by TGF-β1, indicating a possible role of SLP-2 in PTC tumorigenesis. Our data suggest that SLP-2 may be considered as a useful diagnostic marker and therapeutic target for PTC.

High-dose irradiation in combination with toll-like receptor 9 agonist CpG oligodeoxynucleotide 7909 downregulates PD-L1 expression via the NF-κB signaling pathway in non-small cell lung cancer cells.

Objectives Irradiation resistance appears as local recurrence and distant metastasis in advanced stages of non-small cell lung cancer (NSCLC). High-dose irradiation combined with immunotherapy improved overall survival and local control of NSCLC. This study explored the underlying molecular mechanism by which the effect of high-dose irradiation plus toll-like receptor 9 (TLR9) agonist CpG oligodeoxynucleotide (CpG ODN) 7909 on NSCLC. Materials and methods NSCLC H460 cells were exposed to constant high-dose irradiation (6.37 Gy) in irradiation (IR) group and the irradiation plus CpG group. Gene expression was assessed using quantitative reverse transcriptase-polymerase chain reaction and Western blot. Knockdown of nuclear factor kappa B (NF-κB) p65 expression was conducted using p65 siRNA. Results Expression of programmed death-ligand 1 (PD-L1) mRNA was significantly decreased in IR combined with CpG ODN 7909 group compared with the control or IR-alone groups (P<0.05). TLR9 expression was also obviously increased in the combination group compared with the control (P<0.05). Moreover, expression of NF-κB p65 was apparently reduced in the combination group compared with the control (P<0.05). However, expression of PD-L1 was significantly decreased after knockdown of p65 in IR group (P<0.05), but increased in the combination group (P<0.05) and slightly increased in CpG ODN-alone group (P<0.05), which was opposite to that without p65 knockdown group. Conclusion This study demonstrated that radiotherapy combined with CpG ODN 7909 was able to downregulate PD-L1 expression through inhibition via the NF-κB signaling pathway.

Cytidine monophosphate kinase is inhibited by the TGF-β signalling pathway through the upregulation of miR-130b-3p in human epithelial ovarian cancer

Cytidine monophosphate kinase (CMPK), a member of the nucleoside monophosphate kinase family, plays an important role in the biosynthesis of nucleoside metabolism, DNA repair and tumour development. In this study, we demonstrated for the first time that CMPK was overexpressed in human ovarian epithelial borderline and malignant tumours using tissue microarray. Knockdown of CMPK significantly inhibited epithelial ovarian cancer (EOC) cell proliferation, migration and invasion. Furthermore, CMPK-shRNA inhibited PCNA, MMP-2, MMP-9 and vimentin expression, increased E-cadherin expression and arrested cell cycle at the G2/M phase. Suppression of CMPK resulted in a decrease of EOC cell microtissue formation and colony formation in vitro. Overexpression of miR-130b-3p decreased CMPK expression, whereas anti-miR-130b-3p increased CMPK expression. Moreover, TGF-β1 inhibited the expression of CMPK, which was blocked in the presence of a TGF-β type I receptor, SB431542, and was abolished by the inhibitor of miR-130b-3p, indicating that CMPK is regulated by the TGF-β signalling pathway through the upregulation of miR-130b-3p. Thus, our data identify that overexpression of CMPK occurs in EOC and reveal a mechanism underlying the regulation of CMPK by the TGF-β signalling pathway. We could consider CMPK as an EOC biomarker and targeting CMPK by decreasing its expression may be beneficial in patients with EOC.

Regulative Effect of Nampt on Tumor Progression and Cell Viability in Human Colorectal Cancer.

Colorectal cancer (CRC) is the third most common cancer disease. Here we examined Nampt expression in patients with CRC and the effect of Nampt on cell viability in CRC cells. Nampt protein was overexpressed in colorectal adenoma as well as colorectal carcinoma. The immunoreactive staining of Nampt was negative in the adjacent normal colorectal tissue, weak in colorectal adenoma, and strong in colorectal carcinoma, which may represent tumor progression. Further evaluation of clinical data showed that Nampt expression was not correlated with the clinicopathological characteristics of CRC. Additionally, our in vitro studies demonstrated that Nampt promotes CRC cell viability, whereas the Nampt inhibitor FK866 suppressed CRC cell viability, which was in concordance with the previous studies in other cancer cells. Treatment with Nampt-siRNA reduced the Nampt protein expression resulting in the inhibition of the cell viability of HCT116 and Caco2. Thus, the involvement of Nampt in cell growth indicates that Nampt may play an important role in colorectal tumorigenesis. As a consequence, our results suggest that Nampt may be considered as a progression marker of colorectal tumor and a potentially therapeutic target for the treatment of CRC.

CpG oligodeoxyribonucleotide 7909 enhances radiosensitivity via downregulating Oct-4 expression in radioresistant lung cancer cells.

Radiotherapy is a powerful cure for local advanced non-small cell lung cancer. However, radioresistance and tumor relapse still occur in a high proportion of patients. Octamer-4 (Oct-4), a transcription factor of the POU family, plays a key role in maintaining chemoradioresistant properties and regulating cancer progression. In this study, we demonstrated that Oct-4 expression was significantly increased in radioresistant H460 (H460R) cell line. CpG oligodeoxyribonucleotide (CpG-ODN) 7909 sensitized H460R cells when combined with irradiation treatment. The clonogenic capacity was significantly decreased, and the values of D0 and Dq were lower than those of irradiation alone group. The sensitive enhancement ratio (SER) of D0 was 1.224. This combined treatment led to a dramatic reduction in Oct-4 expression in a dose-dependent manner and also showed increased percentage of cells in the radiosensitive G2/M phase relative to either treatment alone. These results identified that Oct-4 was involved in radioresistance. CpG-ODN 7909 could enhance radiosensitivity partly through downregulating Oct-4 expression in radioresistant lung cancer cells.

MALAT1 affects ovarian cancer cell behavior and patient survival

Epithelial ovarian cancer (EOC) is one of the most lethal malignancies of the female reproductive organs. Increasing evidence has revealed that long non-coding RNAs (lncRNAs) participate in tumorigenesis. Metastasis associated lung adenocarcinoma transcript 1 (MALAT1) is an lncRNA and plays a role in various types of tumors. However, the function of MALAT1 on cellular behavior in EOC remains unclear. The current study explored the expression of MALAT1 in ovarian cancer tissues and in EOC cell lines. Quantitative RT-PCR analysis revealed that the expression of MALAT1 was higher in human ovarian malignant tumor tissues and EOC cells than in normal ovarian tissues and non-tumorous human ovarian surface epithelial cells, respectively. By analyzing the online database Kaplan-Meier Plotter, MALAT1 was identified to be correlated with the overall survival (OS) and progression-free survival (PFS) of patients with ovarian cancer. Furthermore, knockdown of MALAT1 by small interfering RNA (siRNA) significantly decreased EOC cell viability, migration, and invasion. Finally, dual-luciferase reporter assays demonstrated that MALAT1 interacted with miR-143-3p, a miRNA that plays a role in EOC as demonstrated in our previous study. Inhibition of MALAT1 resulted in an increase of miR-143-3p expression, leading to a decrease of CMPK protein expression. In conclusion, our results indicated that MALAT1 was overexpressed in EOC. Silencing of MALAT1 decreased EOC cell viability and inhibited EOC cell migration and invasion. These data revealed that MALAT1 may serve as a new therapeutic target of human EOC.

Antitumor effects of nadroparin combined with radiotherapy in Lewis lung cancer models

Background The beneficial antitumor effects of low-molecular-weight heparins (LMWHs) have previously been investigated in basic and clinical studies. In this study, the antitumor efficacy of nadroparin combined with radiotherapy was investigated in vivo. Methods A total of 48 tumor-bearing mice were randomly divided into six groups (n=8 per group): control group, irradiation group (X), LMWH1,000 group, LMWH2,000 group, LMWH1,000+X group and LMWH2,000+X group. Following this, tumor growth, weight and inhibitory rate, as well as the survival of mice in each group, were determined. Levels of serum interleukin (IL)-6 and transforming growth factor (TGF)-β1 were determined via enzyme-linked immunosorbent assay (ELISA) analyses. The expression levels of CD34 were investigated using immunohistochemistry analyses to represent the microvascular density (MVD) values of tumor tissues. In addition, tumor cell apoptosis was investigated using TdT-mediated dUTP nick end labeling (TUNEL) analysis post treatment. The expression levels of survivin were analyzed by Western blotting. Results The volumes and weights of tumors in the treatment groups were demonstrated to be significantly decreased, which was most obvious in the LMWH2,000+X group. The tumor inhibitory rate was significantly increased in the treated mice. ELISA assays demonstrated that the concentrations of serum IL-6 and TGF-β1 were significantly decreased in the LMWH2,000+X group. In addition, the decreased CD34 expression was found in the combined treatment groups. TUNEL assays demonstrated that the apoptosis rate was increased in treated mice, and the highest apoptosis rate was exhibited by the LMWH2,000+X group. Results of Western blotting demonstrated that combinatory treatment with both nadroparin and X-ray irradiation significantly inhibited the expression of survivin. Conclusion These results demonstrated that a combinatory treatment strategy of nadroparin with fractionated irradiation had a strong synergistic antitumor effect in vivo, which may be associated with the promotion of apoptosis, inhibited secretion of TGF-β1 and IL-6 and down-regulation of CD34 and survivin expression.

Data on the association of CMPK1 with clinicopathological features and biological effect in human epithelial ovarian cancer

Human epithelial ovarian cancer (EOC) is the most lethal gynecological disease. However, the molecular mechanisms by which transforming growth factor-β (TGF-β) regulates ovarian tumor progression markers remain unclear. The present data show cytidine monophosphate kinase (CMPK) as an EOC biomarker and are related to the article entitled “Cytidine monophosphate kinase is inhibited by the TGF-β signalling pathway through the upregulation of miR-130b-3p in human epithelial ovarian cancer” [1]. CMPK, as well as cystatin B [2] and β-2-microglobulin [3], is overexpressed in human epithelial-type ovarian tumors. CMPK is an enzyme required for nucleic acid biosynthesis [4] and is regulated by the TGF-β signaling pathway in EOC cells [1]. Furthermore, the data show the effect of CMPK-shRNA on EOC cell apoptosis and TGF-β-induced Smad2 phosphorylation. CMPK expression in two EOC cell lines OVCAR-3 and SK-OV-3 is regulated by multiple miRNAs and some of these miRNAs may affect EOC chemoresistance [5].