Loss of PDK4 expression promotes proliferation, tumorigenicity, motility and invasion of hepatocellular carcinoma cells.

Although the roles and underlying mechanisms of other PDK family members (i.e., PDK1, PDK2 and PDK3) in tumor progression have been extensively investigated and are well understood, the functions and underlying molecular mechanisms of pyruvate dehydrogenase kinase 4 (PDK4) in the tumorigenesis and progression of various cancers [including hepatocellular carcinoma (HCC)] remain largely unknown. In this study, we examined the expression profile of PDK4 in HCC clinical tissue specimens and the roles of PDK4 in the proliferation, tumorigenicity, motility and invasion of HCC cells. The immunohistochemistry (IHC) and quantitative real-time PCR (qRT-PCR) results revealed that PDK4 was significantly downregulated in the cohort of HCC clinical specimens. Additionally, PDK4 protein was found in both the nucleus and cytoplasm of HCC cells based on an immunofluorescence (ICC) assay, and PDK4 protein was also found in the nucleus and cytoplasm of cancer cells contained in HCC clinical specimens based on IHC. The CCK-8 assay and cell colony formation assay demonstrated that stable depletion of endogenous PDK4 by lentivirus-mediated RNA interference (RNAi) markedly promoted the proliferation of HCC cell lines (i.e., BEL-7402 and BEL-7404 cells) in vitro, while PDK4 silencing significantly enhanced the tumorigenic ability of BEL-7404 cells in vivo. In addition to enhance proliferation and tumorigenesis induced by PDK4 silencing, additional studies demonstrated that knockdown of PDK4 led to increase migration and invasion of BEL-7402 and BEL-7404 cells in vitro. Taken together, these findings suggest that the loss of PDK4 expression contributes to HCC malignant progression.

Recognition and killing of cancer stem-like cell population in hepatocellular carcinoma cells by cytokine-induced killer cells via NKG2d-ligands recognition.

There is an urgent need for more potent and safer approaches to eradicate cancer stem cells (CSCs) for curing cancer. In this study, we investigate cancer-killing activity (CKA) of cytokine-induced killer (CIK) cells against CSCs of hepatocellular carcinoma (HCC). To visualize CSCs in vitro by fluorescence imaging, and image and quantify CSCs in tumor xenograft-bearing mice by bioluminescence imaging, HCC cells were engineered with CSC detector vector encoding GFP and luciferase controlled by Nanog promoter. We found that CIK cells have a strong CKA in vitro against putative CSCs of HCC, as shown by tumorsphere formation and time-lapse imaging. Additionally, time-lapse recording firstly revealed that putative CSCs were attacked simultaneously by many CIK cells and finally eradicated by CIK cells, indicating the necessity of achieving sufficient effector-to-target ratios. We firstly illustrated that anti-NKG2D antibody blocking partially but significantly inhibited CKA of CIK cells against putative CSCs. More importantly, intravenous infusion of CIK cells remarkably delayed tumor growth in mice with a significant decrease in putative CSC number monitored by bioluminescence imaging. Taken together, these findings demonstrate CKA of CIK cells against putative CSCs of HCC, at least in part, by NKG2D-ligands recognition.

Hes1 triggers epithelial-mesenchymal transition (EMT)-like cellular marker alterations and promotes invasion and metastasis of nasopharyngeal carcinoma by activating the PTEN/AKT pathway.

Overexpression of the transcriptional factor Hes1 (hairy and enhancer of split-1) has been observed in numerous cancers, but the precise roles of Hes1 in epithelial-mesenchymal transition (EMT), cancer invasion and metastasis remain unknown. Our current study firstly revealed that Hes1 upregulation in a cohort of human nasopharyngeal carcinoma (NPC) biopsies is significantly associated with the EMT, invasive and metastatic phenotypes of cancer. In the present study, we found that Hes1 overexpression triggered EMT-like cellular marker alterations of NPC cells, whereas knockdown of Hes1 through shRNA reversed the EMT-like phenotypes, as strongly supported by Hes1-mediated EMT in NPC clinical specimens described above. Gain-of-function and loss-of-function experiments demonstrated that Hes1 promoted the migration and invasion of NPC cells in vitro. In addition, exogenous expression of Hes1 significantly enhanced the metastatic ability of NPC cells in vivo. Chromatin immunoprecipitation (ChIP) assays showed that Hes1 inhibited PTEN expression in NPC cells through binding to PTEN promoter region. Increased Hes1 expression and decreased PTEN expression were also observed in a cohort of NPC biopsies. Additional studies demonstrated that Hes1-induced EMT-like molecular changes and increased motility and invasion of NPC cells were mediated by PTEN. Taken together, our results suggest, for what we believe is the first time, that Hes1 plays an important role in the invasion and metastasis of NPC through inhibiting PTEN expression to trigger EMT-like phenotypes.

Cytokine-induced killer cells efficiently kill stem-like cancer cells of nasopharyngeal carcinoma via the NKG2D-ligands recognition.

Cancer stem cells (CSCs) are considered to be the root cause for cancer treatment failure. Thus, there remains an urgent need for more potent and safer therapies against CSCs for curing cancer. In this study, the antitumor activity of cytokine-induced killer (CIK) cells against putative CSCs of nasopharyngeal carcinoma (NPC) was fully evaluated in vitro and in vivo. To visualize putative CSCs in vitro by fluorescence imaging, and image and quantify putative CSCs in tumor xenograft-bearing mice by in vivo bioluminescence imaging, NPC cells were engineered with CSC detector vector encoding GFP and luciferase (Luc) under control of Nanog promoter. Our study reported in vitro intense tumor-killing activity of CIK cells against putative CSCs of NPC, as revealed by percentage analysis of side population cells, tumorsphere formation assay and Nanog-promoter-GFP-Luc reporter gene strategy plus time-lapse recording. Additionally, time-lapse imaging firstly illustrated that GFP-labeled or PKH26-labeled putative CSCs or tumorspheres were usually attacked simultaneously by many CIK cells and finally killed by CIK cells, suggesting the necessity of achieving sufficient effector-to-target ratios. We firstly confirmed that NKG2D blockade by anti-NKG2D antibody significantly but partially abrogated CIK cell-mediated cytolysis against putative CSCs. More importantly, intravenous infusion of CIK cells significantly delayed tumor growth in NOD/SCID mice, accompanied by a remarkable reduction in putative CSC number monitored by whole-body bioluminescence imaging. Taken together, our findings suggest that CIK cells demonstrate the intense tumor-killing activity against putative CSCs of NPC, at least in part, by NKG2D-ligands recognition. These results indicate that CIK cell-based therapeutic strategy against CSCs presents a promising and safe approach for cancer treatment.

MicroRNA-19 triggers epithelial-mesenchymal transition of lung cancer cells accompanied by growth inhibition.

The miR-19 family (miR-19a and miR-19b-1) are key oncogenic components of the miR-17-92 cluster. Overexpression of miR-19 is strongly associated with cancer invasion and metastasis, and poor prognosis of cancer patients. However, the underlying mechanisms remain largely unknown. In the present study, we found that enforced expression of miR-19 including miR-19a and miR-19b-1 triggered epithelial-mesenchymal transition (EMT) of lung cancer cells A549 and HCC827 as shown by mesenchymal-like morphological conversion, downregulation of epithelial proteins (e.g., E-cadherin, ZO-1 (zona occludens 1), and α-catenin), upregulation of mesenchymal proteins (e.g., vimentin, fibronectin 1, N-cadherin, and snail1), formation of stress fibers, and reduced cell adhesion. In addition, enhanced migration and invasion were observed in the cancer cells A549 and HCC827 undergoing EMT. In contrast, silencing of endogenous miR-19 reversed EMT and reduced the migration and invasion abilities of A549 and HCC827 cells. DNA microarray results revealed significant changes of the expression of genes related to EMT, migration, and metastasis of miR-19-expressing A549 cells. Moreover, siRNA-mediated knockdown of PTEN, a target of miR-19, also resulted in EMT, migration, and invasion of A549 and HCC827 cells, suggesting that PTEN is involved in miR-19-induced EMT, migration and invasion of lung cancer cells. Furthermore, lung cancer cells undergoing EMT induced by miR-19 demonstrated reduced proliferation in vitro and in vivo, and enhanced resistance to apoptosis caused by TNF-α. Taken together, these findings suggest that miR-19 triggers EMT, which has an important role in the invasion and migration of lung cancer cells, accompanied by the reduced proliferation of cells.

MicroRNA-122 triggers mesenchymal-epithelial transition and suppresses hepatocellular carcinoma cell motility and invasion by targeting RhoA.

The loss of microRNA-122 (miR-122) expression is strongly associated with increased invasion and metastasis, and poor prognosis of hepatocellular carcinoma (HCC), however, the underlying mechanisms remain poorly understood. In the present study, we observed that miR-122 over-expression in HCC cell lines Sk-hep-1 and Bel-7402 triggered the mesenchymal-epithelial transition (MET), as demonstrated by epithelial-like morphological changes, up-regulated epithelial proteins (E-cadherin, ZO-1, α-catenin, occludin, BVES, and MST4), and down-regulated mesenchymal proteins (vimentin and fibronectin). The over-expression of miRNA-122 also caused cytoskeleton disruption, RhoA/Rock pathway inactivation, enhanced cell adhesion, and suppression of migration and invasion of Sk-hep-1 and Bel-7402 cells, whereas, these effects could be reversed through miR-122 inhibition. Additional studies demonstrated that the inhibition of wild-type RhoA function induced MET and inhibited cell migration and invasion, while RhoA over-expression reversed miR-122-induced MET and inhibition of migration and invasion of HCC cells, suggesting that miR-122 induced MET and suppressed the migration and invasion of HCC cells by targeting RhoA. Moreover, our results demonstrated that HNF4α up-regulated its target gene miR-122 that subsequently induced MET and inhibited cell migration and invasion, whereas miR-122 inhibition reversed these HNF4α-induced phenotypes. These results revealed functional and mechanistic links among the tumor suppressors HNF4α, miR-122, and RhoA in EMT and invasive and metastatic phenotypes of HCC. Taken together, our study provides the first evidence that the HNF4α/miR-122/RhoA axis negatively regulates EMT and the migration and invasion of HCC cells.

Hydrolysis of timosaponin BII by the crude enzyme from Aspergillus niger AS 3.0739.

Timosaponin BII (1), a steroidal saponin showing potential anti-dementia activity, was regioselectively hydrolyzed into its deglycosyl derivatives by the crude enzyme from Aspergillus niger AS 3.0739. Three biotransformation products, timosaponin BII-a (2), timosaponin BII-b (3), and timosaponin BII-c (4), were purified and their structures were elucidated on the basis of 1D NMR, 2D NMR, FAB-MS, and HR-ESI-MS spectral data. Compounds 2 and 3 are new compounds.

Enzymatic synthesis of alpha-glucosyl-timosaponin BII catalyzed by the extremely thermophilic enzyme: Toruzyme 3.0L.

Timosaponin BII (BII), a steroidal saponin showing potential anti-dementia activity, was converted into its glucosylation derivatives by Toruzyme 3.0L. Nine products with different degrees of glucosylation were purified and their structures were elucidated on the basis of (13)C NMR, HR-ESI-MS, and FAB-MS spectra data. The active enzyme in Toruzyme 3.0L was purified to electrophoretic homogeneity by tracking BII-glycosylase activity and was identified as Cyclodextrin-glycosyltransferase (CGTase, EC 2.4.1.19) by ESI-Q-TOF MS/MS. In this work, we found that the active enzyme catalyzed the synthesis of alpha-(1-->4)-linked glucosyl-BII when dextrin instead of an expensive activated sugar was used as the donor and showed a high thermal tolerance with the most favorable enzymatic activity at 100 degrees C. In addition, we also found that the alpha-amylases and CGTase, that is, GH13 family enzymes, all exhibited similar activities, which were able to catalyze glucosylation in steroidal saponins. But other kinds of amylases, such as gamma-amylase (GH15 family), had no such activity under the same reaction conditions.