Triptolide-induced suppression of phospholipase D expression inhibits proliferation of MDA-MB-231 breast cancer cells

In spite of the importance of phospholipase D (PLD) in cell proliferation and tumorigenesis, little is known about the molecules regulating PLD expression. Thus, identification of small molecules inhibiting PLD expression would be an important advance for PLD-mediated physiology. We examined one such here, denoted "Triptolide", which was identified in a chemical screen for inhibitors of PLD expression using cell assay system based on measurement of PLD promoter activity. Triptolide significantly suppressed the expression of both PLD1 and PLD2 with sub-µM potency in MDA-MB-231 breast cancer cells as analyzed by promoter assay and RT-PCR. Moreover, triptolide abolished the protein level of PLD in a time and dose-dependent manner. Triptolide-induced PLD1 downregulation was also observed in all the cancer cells examined, suggesting a general phenomenon detected in various cancer cells. Decrease of PLD expression by triptolide suppressed both basal and PMA-induced PLD activity. In addition, triptolide inhibited activation of NFκB which increased PLD1 expression. Ultimately, downregulation of PLD by triptolide inhibited proliferation of breast cancer cells. Taken together, we demonstrate that triptolide suppresses the expression of PLD via inhibition of NFκB activation and then decreases cell proliferation.     

Phospholipase D inhibitor enhances radiosensitivity of breast cancer cells

Radiation and drug resistance remain the major challenges and causes of mortality in the treatment of locally advanced, recurrent and metastatic breast cancer. Dysregulation of phospholipase D (PLD) has been found in several human cancers and is associated with resistance to anticancer drugs. In the present study, we evaluated the effects of PLD inhibition on cell survival, cell death and DNA damage after exposure to ionizing radiation (IR). Combined IR treatment and PLD inhibition led to an increase in the radiation-induced apoptosis of MDA-MB-231 metastatic breast cancer cells. The selective inhibition of PLD1 and PLD2 led to a significant decrease in the IR-induced colony formation of breast cancer cells. Moreover, PLD inhibition suppressed the radiation-induced activation of extracellular signal-regulated kinase and enhanced the radiation-stimulated phosphorylation of the mitogen-activated protein kinases p38 and c-Jun N-terminal kinase. Furthermore, PLD inhibition, in combination with radiation, was very effective at inducing DNA damage, when compared with radiation alone. Taken together, these results suggest that PLD may be a useful target molecule for the enhancement of the radiotherapy effect.     

The hydrophobic amino acids involved in the interdomain association of phospholipase D1 regulate the shuttling of phospholipase D1 from vesicular organelles into the nucleus

Phospholipase D (PLD) catalyzes the hydrolysis of phosphatidylcholine to generate the lipid second messenger, phosphatidic acid. PLD is localized in most cellular organelles, where it is likely to play different roles in signal transduction. PLD1 is primarily localized in vesicular structures such as endosomes, lysosomes and autophagosomes. However, the factors defining its localization are less clear. In this study, we found that four hydrophobic residues present in the N-terminal HKD catalytic motif of PLD1, which is involved in intramolecular association, are responsible for vesicular localization. Site-directed mutagenesis of the residues dramatically disrupted vesicular localization of PLD1. Interestingly, the hydrophobic residues of PLD1 are also involved in the interruption of its nuclear localization. Mutation of the residues increased the association of PLD1 with importin-β, which is known to mediate nuclear importation, and induced the localization of PLD1 from vesicles into the nucleus. Taken together, these data suggest that the hydrophobic amino acids involved in the interdomain association of PLD1 are required for vesicular localization and disturbance of its nuclear localization.     

Substrate requirements of phospholipase D from Streptomyces netropsis in the transphosphatidylation synthesis of phospolipids

The effect of the structure of the phospholipid substrate on the yield of phosphatidyl-5′-thymidine by transphosphatidylation catalyzed by phospholipase D from Streptomyces netropsis was studied. The reaction and product yield depended on the structures of the polar and nonpolar parts, the hydrophobic-hydrophilic balance, and the degree of unsaturation of the fatty-acids in the phospholipid substrate. __________ Translated from Khimiya Prirodnykh Soedinenii, No. 1, pp. 26–29, January–February, 2006.     

Role of placenta growth factor in cancer and inflammation

Accumulating evidences have documented that angiogenesis is closely linked to inflammation and regulators of angiogenesis play key roles in various inflammatory conditions. PlGF is an angiogenic protein belonging to the VEGF family and is upregulated mainly in pathologic conditions. Recently, PlGF was discovered having a proinflammatory role in inflammatory arthritis and its serum level drew attention not only as a useful surrogate biomarker but also a potential therapeutic target in atherosclerosis and various cancers. Particularly, PlGF has attractive clinical values because endogenous PlGF is redundant for vascular development and physiological vessel maintenance in healthy adults. However, there have been conflicting results about the efficacy of PlGF inhibition depending on the experimental and clinical settings. Further close investigations for resolving the puzzle of PlGF biology are required.     

The presence of high level soluble herpes virus entry mediator in sera of gastric cancer patients

The development of gastric cancer (GC) is closely related to chronic inflammation caused by Helicobacter pylori infection, and herpes virus entry mediator (HVEM) is a receptor expressed on the surface of leukocytes that mediates potent inflammatory responses in animal models. However, the role of HVEM in human GC has not been studied. Previously, we showed that the interaction of HVEM on human leukocytes with its ligand LIGHT induces intracellular calcium mobilization, which results in inflammatory responses including induction of proinflammatory cytokine production and anti-bacterial activities. In this study, we report that leukocytes from GC patients express lower levels of membrane HVEM (mHVEM) and have lower LIGHT-induced bactericidal activities than those from healthy controls (HC). In contrast, levels of soluble HVEM (sHVEM) in the sera of GC patients were significantly higher than in those of HC. We found that monocyte membrane-bound HVEM is released into the medium when cells are activated by proinflammatory cytokines such as TNF-α and IL-8, which are elevated in the sera of GC patients. mHVEM level dropped in parallel with the release of sHVEM, and release was completely blocked by the metalloprotease inhibitor, GM6001. We also found that the low level of mHVEM on GC patient leukocytes was correlated with low LIGHT-induced bactericidal activities against H. pylori and S. aureus and production of reactive oxygen species. Our results indicate that mHVEM on leukocytes and sHVEM in sera may contribute to the development and/or progression of GC.     

Knockdown of GGCT inhibits cell proliferation and induces late apoptosis in human gastric cancer

Background

Gamma glutamylcyclotransferase (GGCT) has been proved to be involved in various cancers, but the biological function of GGCT in gastric cancer is still largely unknown.

Methods

The expression level of GGCT was evaluated by informatics analyses based on the Oncomine database. GGCT gene was then effectively knocked down via lentivirus mediated short hairpin RNA (shRNA) system. Then a series of functional assays, including MTT, colony formation and flow cytometry analysis were conducted on gastric cancer cells following GGCT knockdown.

Results

We found GGCT is commonly up-regulated in gastric cancer tissues. Furthermore, MTT analysis showed that GGCT depletion significantly inhibited cell proliferation in MGC80-3 and AGS cells. Colony formation assay revealed that depletion of GGCT reduced the colony formation ability in gastric cancer cells. What’s more, cell cycle analysis showed that depletion of GGCT induced gastric cancer cell cycle arrested G2/M phase. More importantly, cell apoptosis analysis further revealed that GGCT inhibition induced early and late cell apoptosis in gastric cancer.

Conclusion

This study suggests GGCT is essential for gastric cancer proliferation and its downregulation may provide a potential anticancer therapy for gastric cancer.

     

Tunicamycin enhances TRAIL-induced apoptosis by inhibition of cyclin D1 and the subsequent downregulation of survivin

TNF-related apoptosis-inducing ligand (TRAIL) has been proposed as a promising cancer therapy that preferentially induces apoptosis in cancer cells, but not most normal tissues. However, many cancers are resistant to TRAIL by mechanisms that are poorly understood. In this study, we showed that tunicamycin, a naturally occurring antibiotic, was a potent enhancer of TRAIL-induced apoptosis through downregulation of survivin. The tunicamycin-mediated sensitization to TRAIL was efficiently reduced by forced expression of survivin, suggesting that the sensitization was mediated at least in part through inhibition of survivin expression. Tunicamycin also repressed expression of cyclin D1, a cell cycle regulator commonly overexpressed in thyroid carcinoma. Furthermore, silencing cyclin D1 by RNA interference reduced survivin expression and sensitized thyroid cancer cells to TRAIL; in contrast, forced expression of cyclin D1 attenuated tunicamycin-potentiated TRAIL-induced apoptosis via over-riding downregulation of survivin. Collectively, our results demonstrated that tunicamycin promoted TRAIL-induced apoptosis, at least in part, by inhibiting the expression of cyclin D1 and subsequent survivin. Of note, tunicamycin did not sensitize the differentiated thyroid epithelial cells to TRAIL-induced apoptosis. Thus, combined treatment with tunicamycin and TRAIL may offer an attractive strategy for safely treating resistant thyroid cancers.     

Inhibition of cell proliferation by a resveratrol analog in human pancreatic and breast cancer cells

Resveratrol has been reported to possess cancer preventive properties. In this study, we analyzed anti-tumor activity of a newly synthesized resveratrol analog, cis-3,4'',5-trimethoxy-3''-hydroxystilbene (hereafter called 11b) towards breast and pancreatic cancer cell lines. 11b treatments reduced the proliferation of human pancreatic and breast cancer cells, arrested cells in the G2/M phase, and increased the percentage of cells in the subG1/G0 fraction. The 11b treatments also increased the total levels of mitotic checkpoint proteins such as BubR1, Aurora B, Cyclin B, and phosphorylated histone H3. Mechanistically, 11b blocks microtubule polymerization in vitro and it disturbed microtubule networks in both pancreatic and breast cancer cell lines. Computational modeling of the 11b-tubulin interaction indicates that the dimethoxyphenyl group of 11b can bind to the colchicine binding site of tubulin. Our studies show that the 11b treatment effects occur at lower concentrations than similar effects associated with resveratrol treatments and that microtubules may be the primary target for the observed effects of 11b. These studies suggest that 11b should be further examined as a potentially potent clinical chemotherapeutic agent for treating pancreatic and breast cancer patients.     

Implication of leucyl-tRNA synthetase 1 (LARS1) over-expression in growth and migration of lung cancer cells detected by siRNA targeted knock-down analysis

Molecular mechanism of lung carcinogenesis and its aggressive nature is still largely elusive. To uncover the biomarkers related with tumorigenesis and behavior of lung cancer, we screened novel differentially expressed genes (DEG) in A549 lung cancer cell line by comparison with CCD-25Lu, normal pulmonary epithelial cell line, using annealing control primer(ACP)-based GeneFishing system. Of the DEGs, over-expression of leucyl-tRNA synthetase 1 (LARS1) was prominent and this up-regulation was confirmed by immunoblotting and real-time quantitative RT-PCR analysis. In addition to A549 cell line, primary lung cancer tissues also expressed higher level of LARS1 mRNA than their normal counter tissues. To explore the oncogenic potential of LARS1 over-expression in lung cancer, we knocked-down LARS1 by treating siRNA and observed the tumor behavior. LARS1 knock-down cells showed reduced ability to migrate through transwell membrane and to form colonies in both soft agar and culture plate. Taken together, these findings suggest that LARS1 may play roles in migration and growth of lung cancer cells, which suggest its potential implication in lung tumorigenesis.     

Suitable reference genes for relative quantification of miRNA expression in prostate cancer

Real time quantitative PCR (qPCR) is the method of choice for miRNA expression studies. For relative quantification of miRNAs, normalization to proper reference genes is mandatory. Currently, no validated reference genes for miRNA qPCR in prostate cancer are available. In this study, the expression of four putative reference genes (hsa-miR-16, hsa-miR-130b, RNU6-2, SNORD7) was examined with regard to their use as normalizer. After SNORD7 was already shown an inappropriate reference gene in preliminary experiments using total RNA pools, we studied the expression of the putative reference genes in tissue and normal adjacent tissue sample pairs from 76 men with untreated prostate carcinoma collected after radical prostatectomy. hsa-miR-130b and RNU6-2 showed no significantly different expression between the matched malignant and non-malignant tissue samples, whereas hsa-miR-16 was significantly underexpressed in malignant tissue. Softwares geNorm and Normfinder predicted hsa- miR-130b and the geometric mean of hsa-miR-130b and RNU6-2 as the most stable reference genes. Normalization of the four miRNAs hsa-miR-96, hsa- miR-125b, hsa-miR-205, and hsa-miR-375, which were previously shown to be regulated, shows that normalization to hsa-mir-16 can lead to biased results. We recommend using hsa-miR-130b or the geometric mean of hsa-miR-130b and small RNA RNU6-2 for normalization in miRNA expression studies of prostate cancer.     

Chitinase inhibits growth of human breast and colorectal cancer cells in vitro and Ehrlich ascites carcinoma cells in vivo

Trichosanthes dioica seed extract was loaded on a QA-cellulose column and the unbound fraction with the chitinase activity was run on SDS-PAGE. Multiple bands were observed and were separated by a Sephadex G-50 column. The combination of the 6 and 33 kDa masses supported the degradation of chitinase as purified earlier. Only the 33 kDa fraction contained sugar and showed chitinase activity. The chitinase was also isolated by using a chitin column. At 200 µg/ml protein concentration, the chitinase inhibited 49.1 %, 48.8 % and 38.12 % of Ehrlich ascites carcinoma, HCT-116 and MCF-7 cells growth, respectively, in a dose-dependent manner. Exactly, 46 % and 82 % EAC cell growth inhibition were observed after treating the EAC cells bearing Swiss albino mice with the chitinase at the doses of 1.0 and 2.0 mg/Kg/day respectively. EAC, HCT-116 and MCF-7 cells growth inhibitions were due to the induction of apoptosis. ROS was accumulated in HCT-116 and MCF-7 cells. After treatment of HCT-116 cells, the expression level of p53 and TNFα genes increased and PARP gene decreased. On the other hand, elevated expression was observed for PARP, MAPK, NFκB, FAS, FADD, and Caspase-8 genes in MCF-7 cells. The induction of apoptosis in HCT-116 was further confirmed by caspase protein expression. The chitinase causes ‘S’ cell cycle arrest in MCF-7 and HCT-116 cells. T. dioica seed chitinase inhibited EAC, HCT-116 and MCF-7 cells by inducing apoptosis in vitro and EAC in vivo in mice. These promising results indicated that T. dioica seed chitinase can be an anticancer agent.     

Chemomechanical and morphological properties with proliferation of keratinocyte cells of electrospun poyhydroxyalkanoate fibers incorporated with essential oil

Delivery systems based on electrospun polymeric nanofibers have shown potential for delivery of bioactive and plant extract formulations. This research focused on the fabrication of polyhydroxyalkanoate (PHA) copolymer nanofibers as a vehicle for loading of the Thai traditional herbal extract of Plai oil (Zingiber cassumunar Roxb). Nanofibers were formed by dissolving PHA and Plai oil together in dichloromethane solvent, with the PHA concentration being varied (5, 8, and 10%), followed by electrospinning for 4 hours. Based on the submicron diameters of the nanofibers, 8% PHA proved to be the optimal concentration. The concentration of Plai oil (10, 20, and 30%) was used, and hence, the solution viscosity influenced the nanofiber synthesis and physical properties of the nanofibers were obtained. Scanning electron microscope results indicated that the average diameters of cylindrical PHA nanofibers loaded with Plai oil (10%, 20%, and 30%) were 1.10, 1.01, and 1.11 μm, respectively, highlighting that fibers composed of 20% Plai oil were classifiable as nanofibers. Tensile testing of 20% Plai oil‐loaded nanofibers indicated that stiffness and elongation at break were within the acceptable range. Fourier transform infrared and differential scanning calorimetry measurements highlighted the presence of terpenen‐4‐ol, a component found in Plai oil, in the nanofiber film samples of PHA/Plai oil, confirming its inclusion in the systems. In addition, cell proliferation was set up to confirm the morphology and toxicity of skin keratinocyte cell line, and the results show that the HaCaT cells were attached on the PHA nanofibers which the nanofibers containing 20% Plai oil may affect cell behavior in spite the fact that it is not toxic to the cells.     

Brassinin inhibits proliferation and induces cell cycle arrest and apoptosis in nasopharyngeal cancer C666-1 cells

BackgroundNasopharyngeal cancer is a tumor that occurs in the mucous epithelium of the nasopharynx. Due to its rapid growth and early metastatic nature, the successful treatment of nasopharyngeal cancer is highly challenging.ObjectiveHere, we intended to assess the in vitro anticancer property of brassinin against the nasopharyngeal cancer C666-1 cells.MethodologyThe in vitro free radical scavenging property of the brassinin was assessed by various free radical scavenging activities such as FRAP, DPPH, chemiluminescence (CL), and ORAC assays. The cytotoxic level of the brassinin (1–50 µM) against the nasopharyngeal cancer C666-1 cells and normal Vero cells were assessed by the MTT cytotoxicity assay. The levels of TBARS, GSH, and the SOD activity was assessed using kits. The level of ROS generation, MMP, and apoptosis were investigated by the respective fluorescent staining techniques. The flow cytometry analysis was done to scrutinize the cell cycle arrest. The Bax/Bcl-2 level and caspase activities were examined using respective kits.ResultsThe brassinin treatment effectively scavenged the free radicals, which are assessed by the FRAP, DPPH, chemiluminescence (CL), and ORAC assays. The proliferation of brassinin treated C666-1 cells were decreased remarkably, while the same concentration of brassinin did not disturbed the Vero cell viability. The 30 µM of brassinin effectively increased the ROS production, depleted the MMP, and stimulated the apoptosis in the C666-1 cells. The brassinin increased the TBARS and depleted the GSH and SOD in the C666-1 cells. The flow cytometry analysis revealed that the brassinin administration improved the G0/G1 ratio and decreased the proportion of cells with ‘S’ and ‘G2/M’ phase. The Bax, caspase-3 and ?9 were elevated and Bcl-2 level was decreased in the brassinin administered C666-1 cells.ConclusionOur findings discovered that the brassinin has the capacity to prevent the proliferation and stimulate the apoptotic cell death C666‐1 cells via blocking cell cycle and increasing oxidative stress and apoptotic markers. Hence, it can be a talented therapeutic agent to treat the nasopharyngeal cancer in the future.     

Optimization of fibrin gelation for enhanced cell seeding and proliferation in regenerative medicine applications

In order to improve the cell seeding efficiency and cell compatibility inside porous tissue scaffolds, a method of fibrin gel‐mediated cell encapsulation inside the scaffold was optimized. Disc‐type poly(d ,l ‐glycolic‐co‐lactic acid) (PLGA) scaffolds without a dense surface skin layer were fabricated using an established solvent casting and particulate leaching method as a model porous scaffold, which showed high porosity ranging from 90 ± 2% to 96 ± 2%. The thrombin and fibrinogen concentration as precursors of fibrin gel was varied to control the gelation kinetics as measured by rheology analysis, and optimized conditions were developed for a uniform fibrin gel formation with the target cells inside the porous PLGA scaffold. The fibroblast cell seeding accompanied by a uniform fibrin gel formation at an optimized gelation condition inside the PLGA scaffold resulted in an increase in cell seeding efficiency, a better cell proliferation, and an increase in final cell density inside the scaffold. Scanning electron microscopy images revealed that cells were better spread and grown by fibrin gel encapsulation inside scaffold compared with the case of bare PLGA scaffold. Copyright © 2016 John Wiley & Sons, Ltd.