Molybdenum disulfide nanosheets based non-oxygen-dependent and heat-initiated free radical nanogenerator with antimicrobial peptides for antimicrobial, biofilm ablation and wound healing.

Chronic refractory wounds caused by multidrug-resistant (MDR) bacterial and biofilm infections are a substantial threat to human health, which presents a persistent challenge in managing clinical wound care. We here synthesized a composite nanosheet AIPH/AMP/MoS2, which can potentially be used for combined therapy because of the photothermal effect induced by MoS2, its ability to deliver antimicrobial peptides, and its ability to generate alkyl free radicals independent of oxygen. The synthesized nanosheets exhibited 61 % near-infrared (NIR) photothermal conversion efficiency, marked photothermal stability and free radical generating ability. The minimal inhibitory concentrations (MICs) of the composite nanosheets against MDR Escherichia coli (MDR E. coli) and MDR Staphylococcus aureus (MDR S. aureus) were approximately 38 µg/mL and 30 µg/mL, respectively. The composite nanosheets (150 µg/mL) effectively ablated >85 % of the bacterial biofilm under 808-nm NIR irradiation for 6 min. In the wound model experiment, approximately 90 % of the wound healed after the 4-day treatment with the composite nanosheets. The hemolysis experiment, mouse embryonic fibroblast (MEFs) cytotoxicity experiment, and mouse wound healing experiment all unveiled the excellent biocompatibility of the composite nanosheets. According to the transcriptome analysis, the composite nanosheets primarily exerted a synergistic therapeutic effect by disrupting the cellular membrane function of S. aureus and inhibiting quorum sensing mediated by the two-component system. Thus, the synthesized composite nanosheets exhibit remarkable antibacterial and biofilm ablation properties and therefore can be used to improve wound healing in chronic biofilm infections.

EuHD1 protects against inflammatory injury driven by NLRP3 inflammasome.

Non-steroidal anti-inflammatory drugs (NSAIDs) possessing anti-inflammatory, analgesic and antipyretic activities, are widely used in the treatment of osteoarthritis, rheumatism and rheumatoid arthritis. However, its long-term or large use will cause serious gastrointestinal injury or cardiovascular adverse reactions, which limits its clinical application. We have synthesized a new class of NSAIDs, EuHD1, which can release hydrogen sulfide and have better gastrointestinal safety. However, the anti-inflammatory molecular mechanism of the drug is still unclear. In this paper, we explored the mechanism of EuHD1 on NLRP3 inflammasome and its effects on acute lung injury and acute liver injury in mice. In vitro results demonstrated that EuHD1 inhibited macrophage pyroptosis and LDH release induced by LPS combined with ATP. In addition, EuHD1 blocked NLRP3 inflammasome activation and suppressed following Caspase-1 activation and secretion of mature IL-1ß. EuHD1 restrained intracellular ROS production and the formation of ASC oligomers, which inhibited the assembly and activation of NLRP3 inflammasome. In vivo results further showed that EuHD1 alleviated LPS-induced acute lung injury in mice, and inhibited the production of mature IL-1ß and Caspase-1 (p20). Besides, EuHD1 improved D-GalN/LPS-induced acute liver injury, and inhibited SOD/MDA levels and oxidative stress injury, and blocked the activation of NLRP3 inflammasome. In summary, we found that EuHD1 inhibits the assembly and activation of NLRP3 inflammasome through restraining the production of ROS and the formation of ASC oligomers, and has therapeutic effects on acute lung injury and liver injury in mice, indicating that EuHD1 has the potential to treat NLRP3 inflammasome-related diseases.

Bortezomib inhibits hepatocellular carcinoma via the Hippo-Yes-associated protein signalling pathway.

Hepatocellular carcinoma (HCC) is one of the principle causes of cancer-associated death throughout the world. However, the patients with HCC are insensitive to traditional drugs and lack effective therapeutic drugs. Dysregulation of Hippo-Yes-associated protein (YAP) signalling is closely associated with HCC. Bortezomib (BTZ) is mainly used in clinical multiple myeloma. It has recently been confirmed that BTZ could suppress cell proliferation in many different types of cancer. Nevertheless, the precise effects of BTZ on HCC and its possible interactions with the Hippo-YAP signalling pathway in HCC cells remain largely unknown. In this study, HCC cell lines (HepG2 and Huh7) and nude mice with xenograft tumours were used to evaluate the influences of BTZ. Furthermore, we focused on exploring whether BTZ exerts its anti-HCC effect through the Hippo-YAP signalling pathway and aimed to lay a theoretical foundation for BTZ as a potential therapeutic drug for HCC. Herein, our results disclose a new mechanism of BTZ in controlling the cell growth of HCC. BTZ downregulates the level of YAP by promoting LATS1 expression to inhibit the growth of HCC cells, which leads to the phosphorylation of YAP and limits YAP nuclear translocation. In sum, our data confirmed that the Hippo-YAP signalling pathway mediates the anti-HCC effects of BTZ.

M378 exhibits anti-inflammatory activities through NLRP3 signaling pathway.

Nonsteroidal anti-inflammatory drugs (NSAIDs) are frequently used drugs due to their values in attenuating pain, fever and inflammation. Unfortunately, conspicuous adverse effects, such as gastrointestinal (GI) damage and/or cardiovascular events have impeded their application in clinic. M378 is a novel hydrogen sulfide-releasing NSAIDs with uncompromised potency and negligible toxicity compared to the existing NSAIDs. However, its anti-inflammatory activity and mechanism are still an enigma. Here we investigated the effect of M378 on the NLRP3 inflammasome signaling pathway and addressed the underlying molecular mechanism. Our data in vitro showed that M378 dose-dependently inhibited the cleavage of Caspase-1 and the secretion of active IL-1ß and blocked NLRP3-dependent pyroptosis in LPS-primed J774A.1 macrophages. Furthermore, M378 remarkably inhibited upstream ASC oligomerization and ROS production regarding the process of NLRP3 inflammasome assembly. Our data in vivo demonstrated that M378 protected mice from acute liver injury, reducing the levels of ALT/AST and IL-1ß and improving hepatic pathological damages. Immunoblot analysis revealed that M378 inhibited the expressions of Caspase-1 and IL-1ß in liver tissues of ALI mice. We also showed that M378 alleviated IL-1ß secretion and peritoneal neutrophils infiltration in MSU-elicited acute peritonitis mice. In conclusion, M378 exerted its anti-inflammatory effect both in vitro and in vivo and its mechanisms are at least connected to its inhibitory performance on the generation of ASC oligomers and ROS production. These findings give an insight. into the molecular mechanism of hydrogen sulfide-releasing NSAIDs and support a potent therapeutic role of M378 in the treatment of NLRP3-driven inflammatory diseases.

Study on the Purity of Gold Leaf in a SO2 Atmosphere at Ambient Temperature.

Gold leaf samples of different purities were corroded in a SO2 atmosphere at three different relative humidities (30%, 60%, 90%) at ambient temperature, and the effects on color, gloss, and morphology were studied. Results showed that a corrosion rate of 0.0898 g/cm2 could be attained after 6 weeks at high humidity. Color changes also occurred during the gold leaf corrosion process, and many thin pits formed on the surfaces, as shown by SEM. EDX results showed that these pits contained C, O, and S compounds. By comparing the results of different gold purity samples and different relative humidity conditions, it could be concluded that both gold content and humidity play an important role in SO2 atmosphere corrosion. These conclusions are helpful for the conservation of gold leaf decorative cultural relics.

Evaluation of nine active components of Lu-Jiao Fang in normal and hypertrophic rat myocardia via LC-MS/MS.

Lu-Jiao Fang (LJF), a traditional Chinese medicine prescription, can improve the cardiac function of chronic heart failure (CHF) patients; however, knowledge about the cardiac distribution of LJF, especially in CHF animal models, is rather limited. This work aimed to explore the cardiac distribution of LJF in pressure overload-induced CHF rats at the last gavage administration of LJF after 30 weeks of treatment. LC-MS/MS methods for analyzing nine active components (i.e. loganin, hesperidin, epimedin C, icariin, psoralen, isopsoralen, baohuoside I, morroniside and specnuezhenide) of LJF in cardiac tissue samples were established, and the components were then analyzed in left ventricular wall (LVW) and right ventricular wall (RVW) in parallel at same time point postdose for three dose groups. The results showed that most analytical component levels in LVW (hypertrophic myocardium) were only 39-74% of those in RVW (normal myocardium); however, psoralen and isopsoralen levels in LVW were equal to or even greater than the levels in RVW, suggesting that the hypertrophic myocardium tissue affinity of psoralen and isopsoralen might overcome the negative effect of decreased blood flow on distribution. This study indicated that the pathological state may influence drug distribution, and the efficacy of psoralen and isopsoralen for improving CHF deserves further investigation.

Molecular evidence for better efficacy of hypocrellin A and oleanolic acid combination in suppression of HCC growth.

Hepatocellular carcinoma (HCC) is one of the most frequently occurring cancer worldwide and the fifth most common malignancy. The Hippo pathway has been found to play a critical role in cancer development. YAP, a transcriptional coactivator, is the major downstream effector of the Hippo signaling pathway. Hypocrellin A (HA), a natural perylenequinone light-sensitive compound, is usually used for the therapy of eukoplakia of the vulva and keloids in China. Oleanolic acid (OA), a pentacyclic triterpene compound, is prevalent in the treatment of human liver diseases such as viral hepatitis. In this study, we aimed to explore the mechanism by which HA modulates the Hippo/YAP signaling pathway in HCC cells and the anticancer effect of HA combined with OA. Treatment with HA resulted in a decrease in cell viability and migration in HCC cells. Furthermore, we found that HA decreased the YAP and TEAD protein levels of the Hippo pathway. Next we demonstrated that HA could potentiate OA's effect on HCC cells. Our results indicated that HA could inhibit the growth of HCC cells in darkness through Hippo-YAP signaling and HA combined with OA had a better effect than HA or OA alone.Thus, our results provide an alternative combinational inhibitor to combat hepatocellular carcinoma diseases.

COX-2 Forms Regulatory Loop with YAP to Promote Proliferation and Tumorigenesis of Hepatocellular Carcinoma Cells.

COX-2 and YAP are shown to be highly associated with hepatocellular carcinoma (HCC) and frequently upregulated during tumor formation. However, despite their importance, whether there is a mutual interaction between COX-2 and YAP and how they regulate each other are not clear. In this paper, we showed that COX-2 overexpression in HCC cell lines resulted in increased levels of YAP mRNA, protein, and its target genes. COX-2 promoted proliferation of HCC cell lines, and knockdown of YAP antagonized this effect. In addition, our results indicated that EP2 and Wnt/ß-Catenin mediate the transcriptional induction of YAP by COX-2. On the other hand, YAP increased COX-2 expression at the level of transcription requiring intact TEAD binding sites in the COX-2 promoter. Collectively, these findings indicated that COX-2 is not only a stimulus of YAP but also a target of Hippo-YAP pathway, thus forming a positive feedback circuit, COX-2-PGE2-EP2-Gαs-ß-catenin-YAP-COX-2. In a further study, we showed that inhibition of YAP and COX-2 acted synergistically and more efficiently reduced the growth of HCC cells and tumor formation than either of them alone, suggesting that dual governing of YAP and COX-2 may lead to the discovery of promising therapeutic strategies for HCC patients via blocking this positive feedback loop.

Robust iterative closest point algorithm based on global reference point for rotation invariant registration.

The iterative closest point (ICP) algorithm is efficient and accurate for rigid registration but it needs the good initial parameters. It is easily failed when the rotation angle between two point sets is large. To deal with this problem, a new objective function is proposed by introducing a rotation invariant feature based on the Euclidean distance between each point and a global reference point, where the global reference point is a rotation invariant. After that, this optimization problem is solved by a variant of ICP algorithm, which is an iterative method. Firstly, the accurate correspondence is established by using the weighted rotation invariant feature distance and position distance together. Secondly, the rigid transformation is solved by the singular value decomposition method. Thirdly, the weight is adjusted to control the relative contribution of the positions and features. Finally this new algorithm accomplishes the registration by a coarse-to-fine way whatever the initial rotation angle is, which is demonstrated to converge monotonically. The experimental results validate that the proposed algorithm is more accurate and robust compared with the original ICP algorithm.

YAP transcriptionally regulates COX-2 expression and GCCSysm-4 (G-4), a dual YAP/COX-2 inhibitor, overcomes drug resistance in colorectal cancer.

BACKGROUND: Chemotherapy resistance remains a major challenge in cancer treatment. COX-2 (cyclooxygenase 2) is involved in drug resistance and poor prognosis of many neoplastic diseases or cancers. However, investigations identifying new modulators of COX-2 pathway and searching for new chemicals targeting these valid resistant biomarkers are still greatly needed. METHODS: HCT15, HCT-116, HT-29, COLO205, FHC, IMCE, SW480 cell lines were used to detect the expression of YAP and COX-2. Site-directed mutagenesis, luciferase reporter analysis and ChIP assay were used to test whether YAP activated COX-2 transcription through interaction with TEAD binding sites in the promoter of COX-2. Cell line models exhibiting overexpression or knockdown of some genes were generated using transfection agents. Coimmunoprecipitation was used to detect protein mutual interaction. mRNA and protein levels were measured by qRT-PCR and western blot respectively. RESULTS: Here, we reported that both YAP and COX-2 were overexpressed in colorectal cancer cells. YAP increased COX-2 expression at the level of transcription requiring intact TEAD binding sites in the COX-2 promoter. YAP conferred drug resistance through COX-2 and its related effectors such as MCL, MDR, Survivin. GCCSysm-4 (G-4), a YAP and COX-2 inhibitor, effectively inhibited both YAP and COX-2 activation, induced apoptosis and decreased viability in Taxol-resistant cells. Inhibition of YAP and COX-2 acted synergistically and more efficiently reduced the resistance of CRC cells than either of them alone. CONCLUSIONS: Our data provide new mechanisms that YAP is a new upstream regulator of COX-2 pathway and plays an important role in conferring resistance in CRC cells. G-4, targeting YAP-COX-2, may be a novel valuable strategy to combat resistance in CRC.

YAP and 14-3-3γ are involved in HS-OA-induced growth inhibition of hepatocellular carcinoma cells: A novel mechanism for hydrogen sulfide releasing oleanolic acid.

Hydrogen sulfide-releasing oleanolic acid (HS-OA) is an emerging novel class of compounds and consists of an oleanolic acid (OA) and a H2S-releasing moiety. Although it exhibits improved anti-inflammatory activity, its potency in human cancers has not been understood yet. In this study, we examined the effects of HS-OA on the growth of liver cancer cell lines and the underlying mechanisms.HS-OA inhibited the growth of all four cancer cell lines studied, with potencies of 10- to 30-fold greater than that of its counterpart (OA). HS-OA induced significant apoptosis and decreased viability, clonogenic activity and migration of Hep G2 cells. Further studies showed that HS-OA resulted in the reduction of YAP expression and its downstream targets, CTGF and CYR 61, thus promoting cell apoptosis. In addition, HS-OA caused a decrease of 14-3-3γ expression, which led to Bad translocation to the mitochondria, ΔΨm loss, cytochrome c release, caspase activation and a recovery of 14-3-3γ reversed these effects induced by HS-OA.These findings indicate that YAP and 14-3-3γ are involved in HS-OA's effects on liver cancer cells and identifying HS-OA as a potential new drug candidate for cancer therapy.

Verteporfin can Reverse the Paclitaxel Resistance Induced by YAP Over-Expression in HCT-8/T Cells without Photoactivation through Inhibiting YAP Expression.

BACKGROUND/AIMS: Paclitaxel (PTX) is one of the most effective anti-cancer drugs. However, multiple drug resistance is still the main factor that hinders the effective treatment of cancer with PTX. Several factors including YAP over-expression can cause PTX resistance. In this study, we aimed to verify the role YAP plays in PTX resistance, explore the reversal of PTX resistance by verteporfin (VP) and investigate the effect of combination therapy of PTX and VP on the PTX resistant colon cancer cells (HCT-8/T). METHODS: To study the relationship between YAP and PTX resistance, a stable YAP-over-expression or YAP silencing cell line was generated by transfected with YAP-plasmids or siYAP-RNA. WST-1 assay was performed to detect the cytotoxicity of PTX on HCT-8 and HCT-8/T cells. Clone formation assay and Transwell assay was preformed to determine the cell proliferation and invasion ability respectively. Immunofluorescence and Western blot analysis was performed for protein detection. RESULTS: YAP was stronger expressed in HCT-8/T than in HCT-8, and PTX resistance was positively correlated with the level of YAP expression. VP, a strongly YAP inhibitor, could reduce the PTX resistance on HCT-8/T cells without light activation by inhibiting YAP. Beside, VP and PTX combination therapy showed synergism on inhibition of YAP and cytotoxicity to HCT-8/T. Moreover, verteporfin and PTX combination therapy affect the invasion and colony formation ability and induce apoptosis of HCT-8/T cells. CONCLUSIONS: VP can reverse the PTX resistance induced by YAP over-expression in HCT-8/T cells without photoactivation through inhibiting YAP expression.

Synthesis and biological evaluation of novel hydrogen sulfide releasing glycyrrhetic acid derivatives.

A series of hybrids, which are composed of glycyrrhetic acid (GA) and slowly hydrogen sulfide-releasing donor ADT-OH, were designed and synthesized to develop anticancer and anti-inflammatory agents. Most of the compounds, whose inhibitory rates were comparable to or higher than those of GA and aspirin, respectively, significantly inhibited xylene-induced ear edema in mice. Especially, compound V4 exhibited the most potent inhibitory rate of 60.7%. Furthermore, preliminary structure-activity relationship studies demonstrated that 3-substituted GA derivatives had stronger anti-inflammatory activities than the corresponding 3-unsubstituted GA derivatives. In addition, anti-proliferative activities of compounds V1-9 were evaluated in three different human cancer cell lines. Compound V4 showed the most high potency against all three tumor cell lines with IC50 values ranging from 10.01 µM in Hep G2 cells to 17.8 µM in MDA-MB-231 cells, which were superior to positive GA.

A LC-MS/MS method for the determination of stachyose in rat plasma and its application to a pharmacokinetic study.

A sensitive, simple and rapid analytical method based on a liquid chromatography-tandem mass spetrometry (LC-MS/MS) has been established and validated for the determination of stachyose in rat plasma. Plasma samples were prepared by protein precipitation with acetonitrile. Separation of stachyose and nystose (internal standard, IS) was achieved using acetonitrile-water (55:45, v/v) as the mobile phase at a flow rate of 1 ml/min for 6 min on an Asahipak NH2P-50 4E column with an Asahipak NH2P-50G 4A guard column. Detection and quantification were conducted by LC-MS/MS method in the negative ion mode using multiple reaction monitoring (MRM) transitions at m/z [M-H](-) 665.4→383.1 for stachyose and 665.5→485.0 for IS, respectively. The method was linear over the concentration ranges of 100-30000 ng/ml with a lower limit of quantification (LLOQ) of 100 ng/ml. The intra- and inter- day precision were all within 8.7% and the accuracy ranged from 97.2-108.4% and 98.3-102.4%, respectively. Stability studies indicated that stachyose was stable under short-term, long-term and three freeze-thaw storage conditions. The method was successfully applied to a pharmacokinetic study involving pulmonary administration of micronized Rehmannia glutinosa oligosaccharides (RGOS) to rats.

The hepatobiliary disposition of timosaponin b2 is highly dependent on influx/efflux transporters but not metabolism.

The purpose of this study was to characterize the hepatobiliary disposition of timosaponin B2 (TB-2), a natural saponin. Although TB-2 has multiple pharmacologic activities, the mechanism of its hepatobiliary disposition has not been explored. Because the metabolism of TB-2 is limited and the accumulation of TB-2 in primary hepatocytes is highly temperature dependent (93% of its accumulation is due to active uptake), the contribution of hepatic transporters was investigated. Organic anion-transporting polypeptide (OATP) 1B1- and OATP1B3-transfected human embryonic kidney 293 cells were employed. TB-2 serves as a substrate for OATP1B1 and OATP1B3, with the former playing a predominant role in the hepatic uptake of TB-2. An inhibition study in sandwich-cultured rat hepatocytes suggested that TB-2 is a substrate for both breast cancer resistance protein (Bcrp) and multidrug resistance-associated protein 2 (Mrp2), consistent with its high biliary excretion index (43.1-44.9%). This hypothesis was further verified in BCRP and MRP2 membrane vesicles. The cooperation of uptake and efflux transporters in TB-2 hepatic disposition could partially explain the double-peak phenomenon observed in rat plasma and liver and biliary clearance, which accounted for 70% of the total TB-2 clearance. Moreover, TB-2 significantly increased the rosuvastatin concentration in rat plasma in a concentration-dependent manner and decreased its biliary excretion, which corresponded to reductions in rosuvastatin accumulation in hepatocytes and the biliary excretion index in sandwich-cultured rat hepatocytes, representing a perfect example of a potential saponin-statin drug-drug interaction. These studies demonstrate that transporters (Oatp, Bcrp/Mrp2), but not metabolism, contribute significantly to rat TB-2 hepatobiliary disposition.

An approximation solution to refinery crude oil scheduling problem with demand uncertainty using joint constrained programming.

This paper is devoted to develop an approximation method for scheduling refinery crude oil operations by taking into consideration the demand uncertainty. In the stochastic model the demand uncertainty is modeled as random variables which follow a joint multivariate distribution with a specific correlation structure. Compared to deterministic models in existing works, the stochastic model can be more practical for optimizing crude oil operations. Using joint chance constraints, the demand uncertainty is treated by specifying proximity level on the satisfaction of product demands. However, the joint chance constraints usually hold strong nonlinearity and consequently, it is still hard to handle it directly. In this paper, an approximation method combines a relax-and-tight technique to approximately transform the joint chance constraints to a serial of parameterized linear constraints so that the complicated problem can be attacked iteratively. The basic idea behind this approach is to approximate, as much as possible, nonlinear constraints by a lot of easily handled linear constraints which will lead to a well balance between the problem complexity and tractability. Case studies are conducted to demonstrate the proposed methods. Results show that the operation cost can be reduced effectively compared with the case without considering the demand correlation.

Growth inhibitory effect of KYKZL-1 on Hep G2 cells via inhibition of AA metabolites and caspase-3 pathway and cell cycle arrest.

KYKZL-1, a newly synthesized compound with COX/5-LOX dual inhibition, was subjected to the inhibitory activity test on Hep G2 growth. We found that KYKZL-1 inhibited the growth of Hep G2 cells via inducing apoptosis. Further studies showed that KYKZL-1 activated caspase-3 through cytochrome c release from mitochondria and down regulation of Bcl-2/Bax ratio and reduced the high level of COX-2 and 5-LOX. As shown in its anti-inflammatory effect, KYKZL-1 also exhibited inhibitory effect on the PGE2 and LTB4 production in Hep G2 cells. Accordingly, exogenous addition of PGE2 or LTB4 reversed the decreases in cell viability. In addition, KYKZL-1 caused cell cycle arrest at the S-G2 checkpoint via the activation of p21(CIP1) protein and down-regulation of cyclin A expression. These data indicate that the growth inhibitory effect of KYKZL-1 is associated with inhibition of AA metabolites and caspase-3 pathway and cell cycle arrest. Combined with our previous findings, KYKZL-1 exhibiting COX/5-LOX inhibition may be a promising potential agent not only for inflammation control but also for cancer prevention/therapy with an enhanced gastric safety profile.

Inhibition of inflammatory mediators contributes to the anti-inflammatory activity of KYKZL-1 via MAPK and NF-κB pathway.

KYKZL-1, a newly synthesized compound with COX/5-LOX dual inhibition, was subjected to the anti-inflammatory activity test focusing on its modulation of inflammatory mediators as well as intracellular MAPK and NF-κB signaling pathways. In acute ear edema model, pretreatment with KYKZL-1 (p.o.) dose-dependently inhibited the xylene-induced ear edema in mice with a higher inhibition than diclofenac. In a three-day TPA-induced inflammation, KYKZL-1 also showed significant anti-inflammatory activity with inhibition ranging between 20% and 64%. In gastric lesion test, KYKZL-1 elicited markedly fewer stomach lesions with a low index of ulcer as compared to diclofenac in rats. In further studies, KYKZL-1 was found to significantly inhibit the production of NO, PGE2, LTB4 in LPS challenged RAW264.7, which is parallel to its attenuation of the expression of iNOS, COX-2, 5-LOX mRNAs or proteins and inhibition of phosphorylation of p38 and ERK MAPKs and activation of NF-κB. Taken together, our data indicate that KYKZL-1 comprises dual inhibition of COX and 5-LOX and exerts an obvious anti-inflammatory activity with an enhanced gastric safety profile via simultaneous inhibition of phosphorylation of p38 and ERK MAPKs and activation of NF-κB.

Effect of heparin-derived oligosaccharide on vascular smooth muscle cell proliferation through inhibition of PKC-alpha expression.

In this study, the effect of heparin-derived oligosaccharide (HDO) on bovine vascular smooth muscle cell (VSMC) proliferation and signal transduction mechanism involved were investigated. The levels of PKC-alpha protein and mRNA were determined by cell-based ELISA, RT-PCR, Western blotting and immunocytochemical methods. Meanwhile, mRNA levels of c-jun, c-myc and c-fos were assayed by RT-PCR method. The results showed that HDO inhibited newborn calf serum (NCS)-induced expression of PKC-alpha and proto-oncogenes, which may be one of the mechanisms for the inhibition of VSMC proliferation by HDO. Flow cytometry analysis indicated that HDO blocked NCS-induced cell cycle progression by arresting cells at G0/G1 phase. The results imply that HDO inhibits VSMC proliferation by moderating the gene level of PKC-alpha, eventually inhibiting proto-oncogene mRNA expression and blocking G1/S transition.

Effect of heparin-derived oligosaccharide on vascular smooth muscle cell proliferation and the signal transduction mechanisms involved.

PURPOSE: In this study, the effect of heparin-derived oligosaccharide (HDO) on vascular endothelial growth factor (VEGF) induced vascular smooth muscle cell (VSMC) proliferation and the signal transduction mechanisms involved were investigated. METHODS: MTT assays were used to measure VSMC proliferation, flow cytometry to analyze cell cycle distribution, RT-PCR for detection of gene transcript levels, and cell-based ELISA, Western blotting and immunocytochemical methods to detect the expression of PKC-α, ERK 1/2, p-ERK 1/2, Akt, p-Akt, p-PDK1 and p-GSK-3ß. RESULTS: HDO at concentrations of 0.01, 0.1 and 1 µmol·L(-1) dose-dependently inhibited VEGF-induced VSMC proliferation with inhibition indices of 6.8 %, 13.1 % and 28.9 %, respectively. Similar concentrations of HDO dose-dependently decreased the percentage of VEGF-induced cells in S phase to 3.6 %, 3.4 %, and 5.4 %, while increasing that of cells arrested in the G0/G1 phase to 80 %, 82 % and 83.6 %. HDO at 0.01, 0.1 or 1 µmol·L(-1) inhibited VEGF-induced PKC-α mRNA expression, with inhibition indices of 9.2 %, 16.1 % and 54.0 %. HDO at 0.1 or 1 µmol·L(-1) inhibited VEGF-induced proto-oncogene mRNA expression, with inhibition indices of 5.2 % and 6.6 % for c-jun, 8.8 % and 11.6 % for c-myc, and 6.5 % and 11.9 % for c-fos, respectively. Additionally, treatment with 0.01, 0.1 or 1 µmol·L(-1) HDO, inhibited VEGF-induced expression of some proliferation related proteins with inhibition indices of 33.2 %, 56.3 % and 77.0 % for PKC-α, 33.7 %, 38.7 % and 53.2 % for p-Akt, 3.5 %, 24.2 % and 49.3 % for p-ERK 1/2, 39.2 %, 71.8 % and 80.7 % for p-PDK 1 and 41.4 %, 89.4 % and 92.4 % for p-GSK-3ß, respectively. The results showed that HDO inhibited PKC-α, c-jun, c-fos and c-myc mRNA transcription, and also down-regulated phosphorylation levels of ERK 1/2 and Akt. CONCLUSION: Our study demonstrates that HDO inhibits transcription of proliferation-related proto-oncogenes and arrests G1/S transition through inhibition of the PKC, MAPK and Akt/PI3K pathways in association with inhibition of VSMC proliferation. This altered molecular signature may explain one mechanism of HDO-mediated inhibition of VSMC proliferation.