Knockdown of POLQ interferes the development and progression of hepatocellular carcinoma through regulating cell proliferation, apoptosis and migration.

BACKGROUND: DNA Polymerase Theta (POLQ) is a DNA polymerase involved in error-prone translesion DNA synthesis (TLS) and error-prone repair of DNA double-strand breaks (DSBs), whose function in hepatocellular carcinoma has not been investigated. METHODS: In the present study, both the data collected from the Cancer Genome Atlas (TCGA) and our group's results showed higher POLQ expression in HCC tissues than the para-cancerous tissues, which was associated with higher malignancy and poor prognosis. POLQ knockdown HCC cell model (shPOLQ) was constructed along with the corresponding negative control (shCtrl) through lentivirus infection for loss-of-function study. RESULTS: We found that, upon knockdown of POLQ, the proliferation and migration of HCC cells decreased and apoptosis percentage increased. Moreover, the percentage of cells in G2 phase significantly increased in shPOLQ group compared with shCtrl group. Xenografts in mice grafted with shPOLQ cells grew much slower than that transplanted with shCtrl cells, and expressed lower Ki67 level. Furthermore, an apoptosis-related signaling array was used to explore the involvement of downstream signaling pathways, suggesting the enhanced phosphorylation of HSP27 and JNK, and the de-activation of mTOR, PRAS40, ERK1/2 and STAT3 pathways. CONCLUSIONS: Collectively, our study revealed that POLQ may participate in the development of HCC, depletion of which may be a promising treatment strategy for HCC.

TCF21 inhibits tumor-associated angiogenesis and suppresses the growth of cholangiocarcinoma by targeting PI3K/Akt and ERK signaling.

Tumor-associated angiogenesis plays a critical role in the pathogenesis of cholangiocarcinoma (CCA). In this study, we examined the biological effects and molecular mechanisms of transcription factor 21 (TCF21) on CCA-associated angiogenesis. TCF21 expression was compared between 15 pairs of peritumor normal tissues and CCA tissues and also between normal bile duct epithelial cells and two CCA cell lines (QBC-939 and TFK-1) using real-time PCR and Western blot. With the use of both CCA cell lines as the model system, we stably expressed TCF21 by lentiviral transduction (Lv-TCF21). In vivo, we monitored xenograft growth from different CCA cells, measured tumor-associated angiogenesis by histological analysis, and determined the expressions and circulatory levels of VEGFA and PDGF-BB by immunohistochemistry and ELISA, respectively. In vitro, we assessed the effects of conditioned medium collected from different CCA cells on the viability, migration, and tube formation of endothelial cells and explored the significance of phosphatidylinositol 3-kinase/protein kinase B (PI3K/Akt), as well as ERK1/2 signaling in this process. TCF21 was significantly downregulated in CCA tissues or cell lines. Ectopic expression of TCF21 in CCA cells inhibited xenograft growth or tumor-associated angiogenesis in vivo and targeted the expression and secretion of proangiogenic factors, VEGFA and PDGF-BB. In vitro, the conditioned medium collected from Lv-TCF21 CCA cells significantly reduced the viability, migration, and tube formation of endothelial cells. On the molecular level, the targeting of PI3K/Akt and ERK1/2 signaling mediated the anti-angiogenic activity of TCF21. TCF21 presents growth-inhibitory and anti-angiogenic activities, and thus the elevation of TCF21 expression may provide therapeutic benefits for CCA. NEW & NOTEWORTHY Transcription factor 21 (TCF21) is downregulated in cholangiocarcinoma (CCA) tissues or cells. TCF21 inhibits the growth of xenografts derived from CCA cells. TCF21 suppresses in vivo tumor-associated angiogenesis. TCF21 targets expression and production of proangiogenic factors from CCA cells. The targeting of phosphatidylinositol 3-kinase/protein kinase B and ERK1/2 signaling mediates the anti-angiogenesis of TCF21.

Efficacy and safety of chemotherapy with or without targeted therapy in biliary tract cancer: A meta-analysis of 7 randomized controlled trials.

The systematic treatment based on gemcitabine plus cisplatin is recommended as the current standard chemotherapy for unresectable or metastatic biliary tract cancers. However, the exact benefits from the recognized regime are still dismal. We thus elicit this study in an attempt to analyze whether targeted therapy coupled with various chemotherapy could produce improvement of survival benefits. The clinical trials were searched electronically from databases till July 2016 published in English and Chinese. Nine hundred and sixty-four patients from 7 trials were identified in our analysis. The overall analysis achieved a significantly higher overall response rate (ORR) among the patients treated with targeted drugs plus chemotherapy than chemotherapy alone (OR=1.87; 95% CI: 1.37-2.57; P=0.000), but failed in the overall progression-free survival (PFS) [mean difference (MD)=0.63; 95% CI:-0.45-1.72; P=0.26] and overall survival (OS) (MD=-0.67; 95% CI:-2.54-1.20; P=0.49). In the sub analysis, better ORR was obtained with the addition of EGFR (OR=1.75; 95% CI: 1.20-2.56; P=0.004) and VEGFR (OR=2.5; 95% CI: 1.28-4.87; P=0.007) targeted therapy. Furthermore, the sub analysis of EGFR target showed an significant improvement on PFS (MD=1.36; 95% CI: 0.29-2.43; P=0.01). No significant differences were observed in the incidences of neutropenia (OR=1.37; 95% CI: 0.89-2.12), thrombocytopenia (OR=1.40; 95% CI: 0.83-2.39), anemia (OR=1.21; 95% CI: 0.62-2.38), peripheral neuropathy (OR=1.52; 95% CI: 0.81-2.88), increased AST/ALT (OR=1.40; 95% CI: 0.82-2.39) as well as fatigue (OR=1.65; 95% CI: 0.96-2.84) in either of the treatment groups. In conclusion, better ORR associated with chemotherapy combined with targeted therapy (both targeting EGFR and VEGF) is found in the present meta-analysis without the cost of increased unacceptable toxicities, but regretfully not for the OS. The sub-analysis of targeting EGFR instead of VEGF obtains a superior PFS. Otherwise, there is no statistically significant difference in the overall PFS between the combination regime and chemotherapy alone. Given the paucity of favorable data, we need further studies to characterize optimal targeted agents to confirm the potential value to biliary tract cancer.

A low serum Tat-interacting protein 30 level is a diagnostic and prognostic biomarker for hepatocellular carcinoma.

The present study aimed to evaluate the diagnostic and prognostic value of Tat-interacting protein 30 (HTATIP2/TIP30) levels alone and in combination with α-fetoprotein (AFP) for the evaluation of hepatocellular carcinoma (HCC) patients. ELISA and immunohistochemical measurements on the serum and tissue of HTATIP2/TIP30 protein from HCC patients and normal controls were made. Receiver operating characteristic (ROC) curve analyses of AFP and HTATIP2/TIP30 were performed, as well as logistic regression analysis of APF combined with HTATIP2/TIP30. Log-rank analysis was used to correlate the prognosis with various levels of HTATIP2/TIP30. HTATIP2/TIP30 levels were significantly lower in the HCC group compared with the control group (4.50±2.63 vs. 9.50±2.04 ng/ml, P<0.001). ROC analysis revealed an optimal cut-off point at 7.27 ng/ml HTATIP2/TIP30 for separating the HCC from the control groups. The sensitivity and specificity were 84.6 and 93.7% (P<0.001), respectively. ROC areas of HTATIP2/TIP30 (0.928, P<0.001) were significantly higher than those for AFP (P<0.001). The area under the curve of the HTATIP2/TIP30 and AFP combination was 0.950 (P<0.001). Log-rank tests revealed that the recurrence-free survival time of the group with HTATIP2/TIP30>5.71 ng/ml was significantly higher than that of the control group (P<0.001). This is the first study to demonstrate that HTATIP2/TIP30 levels in serum may be an effective biomarker for the diagnosis and prognosis of HCC.

Peptidoglycans promotes human leukemic THP-1 cell apoptosis and differentiation.

The innate immune system coordinates the inflammatory response to pathogens. To do so, its cells must discriminate self from non-self utilizing receptors that identify molecules synthesized exclusively by microbes. Toll- like receptors have a crucial role in the detection of microbial infection in mammals and insects. In mammals, they have evolved to recognize conserved products unique to microbial metabolism. These include lipopolysaccharide (LPS), lipotechoic acids, and peptidoglycans (PGN). We show here that TLRs, including TLR2, are expressed on the THP-1 human leukemia cell line. Activation of TLR2 signaling in THP-1 by PGN induces the synthesis of various soluble factors and proteins including interleukin-1ß, interleukin-8 and TNF-α and apoptosis of THP-1 with PGN dose and time dependence. Moreover , in this study we show that PGN induces apoptosis of THP-1 cells in a TNF-α-dependent manner. These findings indicate that TLR2 signaling results in a cascade leading to tumor apoptosis and differentiation, which may suggest new clinical prospects using TLR2 agonists as cytotoxic agents in certain cancers.

[DNA damage during umbilical cord blood expansion ex vivo].

The aim of this study was to detect DNA damage during expansion ex vivo of umbilical cord blood (UCB) hematopoietic cells and explore the optimal harvest time for culture of CB hematopoietic cells. Mononuclear cells (MNCs) separated from UCB were cultured in a serum-free system supplemented with cytokines and colony forming units were assessed by semisolid culture at the same time. On day 0, 7, 14 and 21 cells were collected for single cell gel electrophoresis (SCGE) analysis and CFUs were also assayed by SCGE, CD34+ cells and CD133+ cells were quantitated by fluorescence-activated cell sorting (FACS). The results showed that the percentage of CD34+ and CD133+ cells was found to be highest after short-term culture (<14 days) and the cord blood DNA damage rate was observed to be less than 5.0% at earlier time points, but at day 21 the DNA damage rate was 28.2%, which was higher than that at day 0 (p=0.000), the tail length of the DNA comet was longer than that at day 0 (p=0.000). The tail lengths of DNA damage on other time points were not significantly different from that at day 0. It is concluded that the DNA damage rate is less than 5.0% after short-term (<14 days) culture of UCB cells ex vivo by using this method. After 14 days DNA damage rate increases significantly. The optimal harvest time of cord blood cells after culture ex vivo would be within 14 days.

[Isolation, culture and identification of two types of endothelial progenitor cells from human umbilical cord blood].

The aim of this study was to establish the method of isolating and culturing endothelial progenitor cells (EPCs) from human umbilical cord blood. Mononuclear cells (MNCs) from human umbilical cord blood were cultured by using culture system supplemented with endothelial cell-conditioned medium. The obtained two types of cells were purified by picking up colonies, identified by uptake of acetylated low-density lipoprotein (Ac-LDL) and binding to lectin [Ulex European Agglatinin (UEA-1)], and were analyzed for the expression of markers by flow cytometry. The results showed that there were significant differences between two types of cells in proliferation, so they were referred as circulating angiogenic cells (CACs) and high proliferative potential endothelial progenitor cells (HPP-EPCs), respectively. They were in accordance with the standards of EPCs, could uptake DiI-Ac-LDL and bind to UEA-1, and expressed the markers of endothelial cells, such as CD31, CD144 and vWF detected by immunocytochemistry. The transcription of CD31, KDR, CD144 and ENOS in both of them could be detected by RT-PCR, but FACS analysis showed significant differences of surface marker expression between them. In conclusion, two types of EPCs are successfully obtained by culturing MNCs isolated from human umbilical cord blood using endothelial cell-conditioned medium.

[LFA-1 and VLA-4 involved in vasoendothelial adhesion and transendothelial migration of human high proliferative potential endothelial progenitor cells].

To investigate whether lymphocyte function-associated antigen 1 (LFA-1) and very late antigen 4 (VLA-4) are involved in vasoendothelial adhesion and transendothelial migration of high proliferative potential endothelial progenitor cells (HPP-EPCs), flow cytometry was used to analyze the expression of integrin beta1 and beta2, the expression of intercellular adhesion molecule (ICAM-1, 2) and vascular cell adhesion molecule (VCAM-1) in mouse bone marrow endothelial cells (mBMECs). The adhesion and transmigration through endothelial cells of the HPP-EPCs blocked by functional grade neutralizing antibodies of VLA-4 and LFA-1 were studied in vitro. The results revealed that HPP-EPCs were positive for CD11a and CD49d in HPP-EPCs. The expression of ICAM-1and VCAM-1 of mBMECs increased after activated by IL-1beta and TNF-alpha. The results of adhesion in vitro revealed that the numbers of the adhered and migrated cells in the CD11a antibody group, in the CD49d antibody group and in the combinational antibody group were less than those in the isotype control antibody group. Furthermore, the number of adhered and migrated cells in the combinational antibody group was less than that in the CD11a or the CD49d antibody group (p < 0.05). It is concluded that both LFA-1 and VLA-4 are involved in vasoendothelial adhesion and transendothelial migration of HPP-EPCs.

[Large scale expansion of hematopoietic progenitor cells from umbilical cord blood by magnet stirred culture system].

The aim of this study was to expand hematopoietic progenitor cells at large scale by magnet stirred culture system. Mononuclear cell from umbilical cord blood were cultured in serum-free medium with stem cell factor, FIT-3 ligand and thrombopoietin. Firstly, the role of magnet on cell growth and colony-forming was studied by static culture on 0, 25 and 50 mT. Then the expansion multiple of cells, colony-forming and expression of surface markers were studied in magnet stirred culture by cell counting, colony-forming assay and flow cytometry. The results indicated that there was no difference in multiple of total cell expansion and numbers of hematopoietic colonies between 0, 25 and 50 mT groups and spinner groups (all p > 0.05). After 7 day cultures, the multiple of total cell expansion in magnet stirred culture was higher than that in static culture (p < 0.01). The numbers of CFU-GM (colony-forming unit-granulocyte/macrophage) and CFU-E (erythroid colony forming unit) in magnet stirred culture were higher than those in static culture, (p < 0.05). The primitive cells (CD34(+), CD34(+)/CD38(-) or CD133(+)) of the expanded cells in magnet stirred culture were less than those in static culture (p < 0.05). However, the CD184(+) or CD62L(+) expanded cells were more than that in static culture (p < 0.05). It is concluded that magnet stirred culture favors the expansion of hematopoietic progenitor cells. The results will be finally confirmed in further in vivo experiments and clinical applications.

Angiogenic potential difference between two types of endothelial progenitor cells from human umbilical cord blood.

The hierarchy of endothelial progenitor cells (EPCs) in human umbilical cord blood has been disclosed. In this study we compare, for the first time, the angiogenic potential difference between two types of EPCs. We cultured mononuclear cells (MNCs) isolated from human umbilical cord blood using endothelial cell-conditioned medium and obtained two types of EPCs, referred to as circulating angiogenic cells (CACs) and high proliferative potential endothelial progenitor cells (HPP-EPCs). Both types of cells possess characteristics of EPCs, including expressing CD31, VE-cadherin, KDR and von Willebrand factor, uptake of Ac-LDL and binding to lectin. However, unlike CACs, which express CD14 but not CD133, HPP-EPCs express CD133 but not CD14. Also, unlike CACs, HPP-EPCs display stronger proliferation and clonogenic potential in vitro and show stronger ability to promote vascular growth in the hind-limb model of ischemia in mice (BALB/C-nu) in vivo.

[99mTc-MIBI myocardial perfusion tomography for monitoring the anthracycline cardiotoxicity].

OBJECTIVE: To investigate the value of 99mTc-MIBI myocardial perfusion tomography monitoring the cardiotoxicity induced by anthracycline. METHODS: Twenty-three patients with anthracycline chemotherapy were examined by electrocardiogram (ECG), myocardial enzyme (CK-MB), nuclear angiography for detecting left ventricular ejection fraction (LVEF) and 99mTc-MIBI myocardial perfusion tomography for detecting myocardial relative quantity (MRQ). These examinations were repeated after every chemotherapy. RESULTS: The MRQ after one period of anthracycline chemotherapy was significantly lower than the pretherapy in 23 patients (P < 0.01). The MRQ significantly decreased after one period of chemotherapy in 11 patients treated by pirarubicin, in 6 by epirubicin, and 6 by mitoxantrone (P < 0.05). There was not significant change in the mean value of ECG and CK-MB after one period of chemotherapy (P > 0.05). After multiple-period anthracycline chemotherapy in 10 patients, a decrease was observed in MRQ (P < 0.01). There was not significant difference in MRQ between multiple periods and one period therapy (P > 0.05) and in LVEF in the period before and after multiple-period chemotherapy (P > 0.05). CONCLUSION: 99mTc-MIBI myocardial perfusion tomography can monitor the anthracycline cardiotoxicity and its changes are earlier than LVEF's. 99mTC-MIBI myocardial perfusion tomography may be helpful to the clinical treatment for anthracycline.