Systematic analysis of the role of LDHs subtype in pan-cancer demonstrates the importance of LDHD in the prognosis of hepatocellular carcinoma patients.

BACKGROUND: Lactate dehydrogenase (LDHs) is an enzyme involved in anaerobic glycolysis, including LDHA, LDHB, LDHC and LDHD. Given the regulatory role in the biological progression of certain tumors, we analyzed the role of LDHs in pan-cancers. METHODS: Cox regression, Kaplan-Meier curves, Receiver Operating Characteristic (ROC) curves, and correlation of clinical indicators in tumor patients were used to assess the prognostic significance of LDHs in pan-cancer. The TCGA, HPA, TIMER, UALCAN, TISIDB, and Cellminer databases were used to investigate the correlation between the expression of LDHs and immune subtypes, immune checkpoint genes, methylation levels, tumor mutational load, microsatellite instability, tumor-infiltrating immune cells and drug sensitivity. The cBioPortal database was also used to identify genomic abnormalities of LDHs in pan-cancer. A comprehensive assessment of the biological functions of LDHs was performed using GSEA. In vitro, HepG2 and Huh7 cells were transfected with LDHD siRNA and GFP-LDHD, the proliferation capacity of cells was examined using CCK-8, EdU, and colony formation assays; the migration and invasion of cells was detected by wound healing and transwell assays; western blotting was used to detect the levels of MMP-2, MMP-9, E-cadherin, N-cadherin and Akt phosphorylation. RESULTS: LDHs were differentially expressed in a variety of human tumor tissues. LDHs subtypes can act as pro-oncogenes or anti-oncogenes in different types of cancer and have an impact on the prognosis of patients with tumors by influencing their clinicopathological characteristics. LDHs were differentially expressed in tumor immune subtypes and molecular subtypes. In addition, LDHs expression correlated with immune checkpoint genes, tumor mutational load, and microsatellite instability. LDHD was identified to play an important role in the prognosis of HCC patients, according to a comprehensive analysis of LDHs in pan-cancer. In HepG2 and Huh7 cells, knockdown of LDHD promoted cell proliferation, migration, and invasion, promoted the protein expression levels of MMP-2, MMP-9, N-cadherin, and Akt phosphorylation, but inhibited the protein expression level of E-cadherin. In addition, LDHD overexpression showed the opposite changes. CONCLUSION: LDHs subtypes can be used as potential prognostic markers for certain cancers. Prognostic and immunotherapeutic analysis indicated that LDHD plays an important role in the prognosis of HCC patients. In vitro experiments revealed that LDHD can affect HCC proliferation, migration, and invasion by regulating MMPs expression and EMT via Akt signaling pathway, which provides a new perspective on the anti-cancer molecular mechanism of LDHD in HCC.

Calunduloside E inhibits HepG2 cell proliferation and migration via p38/JNK-HMGB1 signalling axis.

High-mobility group box 1 (HMGB1), a highly conserved chromosome protein, is considered as a potential therapeutic target and novel biomarker because of its regulation in the proliferation and metastasis of Hepatocellular carcinoma (HCC). Calenduloside E (CE), a natural active product, has been reported to anti-cancer effect. However, the role and underlying molecular mechanism of CE in HCC is still unclear. The purpose of this study is to investigate the effects of CE on the proliferation and migration of HCC, and then explore the possible underlying molecular mechanism. HepG2 cells were treated with CE or transfected with HMGB1 shRNA plasmids, EdU and colony formation assays were used to detect cell proliferation ability. Wound healing and transwell assays were used to determine the role of CE in cell migration. The expression of Cyclins, PCNA, MMPs, HMGB1, N-cadherin, E-cadherin and phosphorylation of p38, ERK and JNK were all detected using Western blotting. Our results showed that CE inhibited HepG2 cells proliferation and migration in a dose dependent manner; reduced the expression levels of Cycins, PCNA, HMGB1, MMPs and N-cadherin; up-regulated E-cadherin expression; enhanced the phosphorylation of p38 and JNK signalling pathways. Blocking the activation of p38 and JNK obviously reversed CE-mediated inhibitory effects on HepG2 cell proliferation and migration; reversed CE-induced down-regulation of Cyclins, PCNA, MMPs, N-cadherin and HMGB1, as well as E-cadherin up-regulation. In conclusion, our study suggested that CE reduces the expression levels of Cyclins, MMPs and epithelial-mesenchymal transformation (EMT) through p38/JNK-HMGB1 signaling axis and then inhibits HepG2 cells proliferation and migration in HepG2 cells. This study provides a new perspective for the anti-tumour molecular mechanism of CE in HCC.

miR-24-3p promotes cell migration and proliferation in lung cancer by targeting SOX7.

Lung cancer (LC) is one of the leading causes of cancer-related death in the world. miR-24-3p plays critical roles in many cancer types, including LC. In this study, we first investigated whether miR-24-3p promoted LC cell migration and proliferation in vitro. We used three bioinformatics algorithms to predict the miR-24-3p target gene to study the molecular mechanism by which miR-24-3p contributes to LC progression. Then, we used the luciferase reporter assay to identify whether SOX7 was a direct target of miR-24-3p. Moreover, Western blotting and a quantitative real time-polymerase chain reaction analysis showed that miR-24-3p downregulated SOX7 protein expression by a post-transcriptional mechanism. Finally, we determined that SOX7 had opposing effects to those of miR-24-3p on LC cell proliferation and migration, suggesting that miR-24-3p promotes cell proliferation and migration by directly targeting SOX7. Furthermore, miR-24-3p accelerated tumor growth in xenograft mice by targeting SOX7. These results provide the first clue that miR-24-3p could play a role as an oncomiR in LC by regulating SOX7.

Crosstalk between JNK and NF-κB signaling pathways via HSP27 phosphorylation in HepG2 cells.

The crosstalk of intracellular signaling pathways is extremely complex. Previous studies have shown that there is a potential crosstalk between MAPKs and NF-κB signaling pathways. It has been reported that JNK regulates cell survival under some conditions. But the molecular mechanism through which JNK regulates cell survival is still unclear. In the present study, we hypothesized that there was a crosstalk between JNK and NF-κB signaling pathway regulating cell survival and HSP27 phosphorylation mediates such a crosstalk. Our data showed that in HepG2 cells, suppression of JNK activation by a specific inhibitor or overexpression of JNK inactive mutant enhanced TNF-α-induced apoptosis. In addition, reduction of JNK activation attenuated HSP27 phosphorylation envoked by TNF-α, especially the phosphorylation of HSP27 at serine 78 residue. Our results also showed that suppression of JNK activation reduced the degradation of IκB-α, but did not affect IKK phosphorylation upon TNF-α stimulation. Co-immunoprecipitation experiments demonstrated that JNK regulated the degradation of IκB-α through promoting the formation of HSP27/IKK/IκB-α ternary complex in response to TNF-α. Suppression of JNK activation hindered HSP27 phosphorylation at Ser78 residue and subsequently reduced the interaction between IKK and IκB-α. Taken together, our study suggests that through modulation the phosphorylation of HSP27, JNK plays an important roles in cell survival via regulating NF-κB signaling pathway.

[Effect of sodium aescinate in inducing human breast cancer MCF-7 cells apoptosis by inhibiting AKT, ERK and upstream signal SRC activity].

To study the effect of sodium aescinate in inducing human breast cancer MCF-7 cells apoptosis and its possible mechanism. MTT assay was used to detect the inhibitory effect of sodium aescinate on the proliferation of MCF-7 cells. The morphological changes were observed under inverted microscope. DAPI nuclear staining was used to detect the changes in cell nucleus. Annexin V-FITC/PI flow cytometry was adopted to test the apoptosis rate. Changes in apoptosis-related proteins (PARP, cleaved caspase-8 and pro-caspase-3), cell survival-associated signal molecules (AKT and ERK) and their common upstream kinase SRC was detected by Western blotting. The result showed that after different concentrations of sodium aescinate were used to treat breast cancer MCF-7 cells, they inhibited the proliferation of MCF-7 cells in a dose-dependent manner, induced cell apoptosis (typical morphological changes in nucleus, significant increase in cell apoptosis rate). The expressions of cleaved PARP and caspase-8 increased, while the expression of pro-caspase-3 decreased, which further verified sodium aescinate's effect in inducing cell apoptosis. Sodium aescinate significantly inhibited the phosphorylation of cell survival-related signal molecules (AKT, ERK) and down-regulate the activation of their common up-stream kinase SRC. The findings indicated that sodium aescinate can block signals transiting to downstream molecules AKT, ERK, inhibit the proliferation of breast cancer cell MCF-7 cell apoptosis and induced cell apoptosis by suppressing the activation of SRC.

Analysis of m6A-related lncRNAs for prognostic and immunotherapeutic response in hepatocellular carcinoma.

Background: RNA methylation modifications are important post-translational modifications that are regulated in an epigenetic manner. Recently, N6-methyladenosine (m6A) RNA modifications have emerged as potential epigenetic markers in tumor biology. Methods: Gene expression and clinicopathological data of LIHC were obtained from the cancer genome atlas (TCGA) database. The relationship between long non-coding RNAs (lncRNAs) and m6A-related genes was determined by gene expression analysis using Perl and R software. Co-expression network of m6A-lncRNA was constructed, and the relevant lncRNAs associated with prognosis were identified using univariate Cox regression analysis. These lncRNAs were then divided into two clusters (cluster 1 and cluster 2) to determine the differences in survival, pathoclinical parameters, and immune cell infiltration between the different lncRNA subtypes. The least absolute shrinkage and selection operator (LASSO) was carried out for regression analysis and prognostic model. The HCC patients were randomly divided into a train group and a test group. According to the median risk score of the model, HCC patients were divided into high-risk and low-risk groups. We built models using the train group and confirmed them through the test group. The m6A-lncRNAs derived from the models were analyzed for the tumor mutational burden (TMB), immune evasion and immune function using R software. AL355574.1 was identified as an important m6A-associated lncRNA and selected for further investigation. Finally, in vitro experiments were conducted to confirm the effect of AL355574.1 on the biological function of HCC and the possible biological mechanisms. Huh7 and HepG2 cells were transfected with AL355574.1 siRNA and cell proliferation ability was measured by CCK-8, EdU and colony formation assays. Wound healing and transwell assays were used to determine the cell migration capacity. The expression levels of MMP-2, MMP-9, E-cadherin, N-cadherin and Akt/mTOR phosphorylation were all determined by Western blotting. Results: The lncRNAs with significant prognostic value were classified into two subtypes by a consistent clustering analysis. We found that the clinical features, immune cell infiltration and tumor microenvironment (TME) were significantly different between the lncRNA subtypes. Our analysis revealed significant correlations between these different lncRNA subtypes and immune infiltrating and stromal cells. We created the final risk profile using LASSO regression, which notably included three lncRNAs (AL355574.1, AL158166.1, TMCC1-AS1). A prognostic signature consisting of the three lncRNAs was constructed, and the model showed excellent prognostic predictive ability. The overall survival (OS) of the low-risk cohort was significantly higher than that of the high-risk cohort in both the train and test group. Both risk score [hazard ratio (HR)=1.062; P<0.001] and stage (HR=1.647; P< 0.001) were considered independent indicators of HCC prognosis by univariate and multivariate Cox regression analysis. In Huh7 and HepG2 cells, AL355574.1 knockdown inhibited cell proliferation and migration, suppressed the protein expression levels of MMP-2, MMP-9, N-cadherin and Akt/mTOR phosphorylation, but promoted the protein expression levels of E-cadherin. Conclusions: This study established a predictive model for the OS of HCC patients, and these OS-related m6A-lncRNAs, especially AL355574.1 may play a potential role in the progression of HCC. In vitro experiments also showed that AL355574.1 could enhance the expression of MMPs and EMT through the Akt/mTOR signaling pathway, thereby affected the proliferation and migration of HCC. This provides a new perspective on the anticancer molecular mechanism of AL355574.1 in HCC.

Methane-Water Interfacial Tension in Nanopores: A Dissipative Particle Dynamics Study.

Imbibition of fracturing fluid in deep shale nanopores has a significant effect on shale gas production. One of the key parameters affecting imbibition is the interfacial tension of the methane-water system. However, studies on the methane-water interfacial tension in nanopores are very limited, and obtaining the accurate value of the methane-water interfacial tension at the nanoscale is difficult and time-consuming. In this work, a dissipative particle dynamics simulation model was built to study the methane-water interfacial tension in nanopores. This model provides reliable access to methane-water interfacial tension for deep shales under high-temperature, high-pressure conditions at low computation cost. It can be easily used to compute the methane-water interfacial tension in nanopores or the confined space in wide application scenarios. A sensitivity study of methane-water interfacial tension on a variety of factors was conducted. Results demonstrate that under high-pressure conditions, the increase in pressure leads to the rise of interfacial tension. When pressure increases from 20 to 120 MPa, interfacial tension rises from 0.0275 to 0.12 N/m, which contributes to the severe imbibition of fracturing fluid in deep shales. The confinement effect was observed by investigating the influence of pore size. Interfacial tension almost remains unchanged in pores smaller than 7 nm because most of the confined space is occupied by interface layer molecules in these pores. When pore size increases from 7 to 15 nm, the confinement effect is reduced. The interfacial tension experiences a growth from 0.1155 to 0.27 mN/m. Compared with pressure and pore size, the effect of temperature on interfacial tension can be neglected during deep shale gas production.

Baicalein reduces lipopolysaccharide-induced inflammation via suppressing JAK/STATs activation and ROS production.

OBJECTIVE: To investigate the precise molecular mechanisms by which baicalein exerts beneficial biochemical activities in RAW264.7 macrophages treated with LPS. MATERIALS AND METHODS: RAW264.7 cells were cultured in the absence or presence of baicalein together with or without LPS. iNOS and COX-2 expression were measured by western blot and RT-PCR analyses. TNF-α, IL-1ß, and IL-6 were determined by using double-antibody sandwich ELISA. Phosphorylations of JAK1 and JAK2, and of STAT1 and STAT3 were detected by western blotting. Nuclear translocation of STAT1 and STAT3 was visualized by confocal microscopy. ROS production was detected by ROS assay. RESULTS: Baicalein significantly reduced the phosphorylation of STAT1 and STAT3 and the phosphorylation of JAK1 and JAK2, but without affecting MAPKs phosphorylation in LPS-stimulated RAW264.7 cells. Baicalein suppressed the nuclear translocation of STAT1 and STAT3 and inhibited production of iNOS upon LPS-stimulation, resulting in the inhibition of releases of NO and pro-inflammatory cytokines such as IL-1ß, IL-6, and TNF-α, in a dose-dependent manner. In addition, we found that baicalein reduced the LPS-induced accumulation of ROS, confirming that baicalein serves as an antioxidant. CONCLUSIONS: Our results suggested that suppressing JAK/STATs activation and interfering with ROS production might contribute to the anti-inflammatory action of baicalein in macrophages.

CLEC5A regulates the proliferation and migration of colon cancer via the AKT/mTOR signaling pathway.

Background: The purpose of this study is to understand the CLEC5A mechanism in colon cancer's proliferation and migration. Methods: The CLEC5A expression levels in colon cancer tissues were analyzed using bioinformatics method based on Oncomine and The Cancer Genome Atlas (TCGA) databases, which were further tested by immunohistochemistry (IHC) and quantitative real-time polymerase chain reaction (qRT-PCR). The CLEC5A expression levels in 4 types of colon cancer cell lines (HCT116, SW620, HT29, and SW480) were also examined by qRT-PCR. We constructed CLEC5A knockdown cell lines and used colony formation, Cell Counting Kit-8 (CCK-8), 5-Ethynyl-2'-deoxyuridine (EdU), wound healing, and transwell assays for investigating the CLEC5A function in colon cancer's proliferation and migration. A CLEC5A silencing nude mice model was established to measure the scale, weight, and growth rate of tumor xenograft. In CLEC5A knockdown cell lines and xenograft tissues, the levels of cell cycle and epithelial-mesenchymal transition (EMT)-related proteins were detected using Western blot (WB), and the phosphorylation levels of AKT/mTOR pathway key proteins were also detected by WB. On the basis of gene expression data retrieved from TCGA database, a relevance between CLEC5A and AKT/mTOR pathway in colon cancer was examined by gene set enrichment analysis (GSEA), and correlation analysis of CLEC5A and COL1A1 was employed to confirm their interaction. Results: Bioinformatics analysis, IHC staining, and qRT-PCR assay results all showed the significant high levels of CLEC5A expression in colon cancer tissues and cells, and positive links between CLEC5A levels and lymph node metastasis, vascular metastasis, and tumor-node-metastasis (TNM) stages of colon cancer patients. The suppressive effects of CLEC5A knockdown on colon cancer's proliferation and migration were verified in cell function and nude mice tumorigenesis assays. WB analysis further indicated that CLEC5A knockdown could inhibit cell cycle, and EMT processes, as well as AKT/mTOR pathway phosphorylation in colon cancer. On the basis of TCGA data, CLEC5A's activation effect on AKT/mTOR pathway had been confirmed by GSEA analysis, and the interaction between CLEC5A and COL1A1 was also revealed through correlation analysis in colon cancer. Conclusions: CLEC5A may promote the development and migration of colon cancer by triggering the AKT/mTOR signaling pathway. Furthermore, COL1A1 could serve as the target gene of CLEC5A.

Effects of photodynamic therapy on Streptococcus mutans and enamel remineralization of multifunctional TiO2-HAP composite nanomaterials.

BACKGROUND: As photosensitizer and photocatalyst, titanium dioxide (TiO2) can produce a photodynamic reaction for antibacterial treatment. This study aims to explore a Titanium dioxide/nano-hydroxyapatite (TiO2-HAP) composite combined with the dental curing lamp (385-515 nm) in clinical which could inhibit the dental plaque biofilm formed by Streptococcus mutans (S. mutans) and promote the enamel surface remineralization simultaneously. METHODS: X-ray Diffraction (XRD) and high resolution transmission electron microscope (HRTEM) were used to detect the characterization of TiO2-HAP composite nanomaterials. Photodynamic properties of TiO2-HAP were detected by Diffuse reflectance spectrum (DRS) and fluorescence spectroscopy. Bacterial growth was measured by reading the absorbance of bacterial cultures and confocal microscope was used to observe the biofilm removal ability of nanomaterials. The ability of TiO2-HAP to promote enamel remineralization was measured by Scanning electron microscope (SEM). RESULTS: The OD 600 of S. mutans was 0.76 in the control group and 0.13 in group of TiO2-HAP with exposure to light-emitting diode (LED) (150 mW/cm2) for 5 min, suggesting its sustained antibacterial potency and inhibition of the metabolic activity of dental plaque microcosm biofilm. Also, the release of calcium and phosphorus ions in TiO2-HAP can promote enamel mineralization simultaneously. After 15 days of remineralization, the Ca/P ratio of demineralized enamel surface increased from 1.28 to 1.67, which was similar to that of normal enamel. CONCLUSIONS: The TiO2-HAP exhibit a promising anti-bacterial activity and remineralization capacity which can prevent the occurrence of caries to the greatest extent and promote the biomimetic mineralization of dental tissues.

[Dihydromyricetin inhibits proliferation and migration of gastric cancer cells through regulating Akt/STAT3 signaling pathways and HMGB1 expression].

OBJECTIVE: To investigate the inhibitory effects of dihydromyricetin on the proliferation and migration of gastric cancer BGC-823 cells and explore the molecular mechanisms. METHODS: BGC-823 cells in routine culture were treated with different concentrations of dihydromyricetin (0, 40, 60, 80, 100, and 120 µg/mL) for 24 h, and the changes in cell viability were detected using CCK-8 assay; colony forming assay and Transwell assay were performed to assess the changes in colonyforming and migration abilities of the cells, respectively. The levels of MMP-2 and MMP-9 in the treated cells were determined using ELISA, and Western blotting was used to detect the expressions of E-cadherin, N-cadherin, cyclin D1, cyclin E1, HSP70 and HMGB1 and the phosphorylation levels of Akt and Stat3. RESULTS: CCK-8 assay showed that dihydromyricetin treatment dose-dependently inhibited the viability of BGC-823 cells (P < 0.05). Treatment with dihydromyricetin obviously suppressed the proliferation and migration of BGC-823 cells, significantly reduced the expression levels of cyclin D1, cyclin E1 and Ncadherin, enhanced E-cadherin expression, inhibited the phosphorylation of Akt and stat3, and downregulated HMGB1 expression in the cells. The results of ELISA demonstrated significantly lowered levels of MMP-2 and MMP-9 in dihydromyricetin-treated cells. CONCLUSIONS: Dihydromyricetin inhibits the proliferation and migration of BGC-823 cells through suppressing the activation of Akt/stat3 signaling pathways and HMGB1 expression.

High mobility group box 1 regulates gastric cancer cell proliferation and migration via RAGE-mTOR/ERK feedback loop.

Gastric cancer (GC) is a common malignancy tumour in China. Despite various therapeutic approaches to improve the survival rate of GC patients, the effectiveness of currently available treatments remains unsatisfactory. High mobility group box 1 (HMGB1) is reported to play a role in tumour development. However, the molecular mechanisms involved in HMGB1-mediated regulation of proliferation and migration of GC cells remain unclear. In the present study, we demonstrated that HMGB1 is highly expressed in GC cells and tissue. In HGC-27 GC cells, HMGB1 overexpression or HMGB1 RNA interference both demonstrated that HMGB1 could promote GC cell proliferation and migration. Investigation of the underlying molecular mechanisms revealed that HMGB1 enhanced cyclins expression, induced epithelial-to-mesenchymal transition and matrix metalloproteinase (MMPs) expression and promoted RAGE expression as well as RAGE-mediated activation of Akt/mTOR/P70S6K and ERK/P90RSK/CREB signalling pathways. We also found that inhibition of ERK and mTOR using specific inhibitors reduced recombinant human HMGB1-induced RAGE expression, suggesting that the RAGE-mTOR/ERK positive feedback loop is involved in HMGB1-induced GC cell proliferation and migration. Our study highlights a novel mechanism by which HMGB1 promotes GC cell proliferation and migration via RAGE-mediated Akt-mTOR and ERK-CREB signalling pathways which also involves the RAGE-mTOR/ERK feedback loop. These findings indicate that HMGB1 is a potential therapeutic target for GC.

Swelling of Shales by Supercritical Carbon Dioxide and Its Relationship to Sorption.

Shale gas is a promising energy source offering additional energy security over concerns of fossil fuel depletion. Injecting CO2 into depleted shale gas reservoirs might provide a feasible solution for CO2 storage and enhanced gas recovery. However, shale strain caused by the CO2 injection as well as CO2 sequestration in the reservoir needs to be considered during shale gas production. For this purpose, this paper examines the adsorption capacities, CO2-induced swelling, and He-induced strain of shales at 0-16 MPa and 35-75 °C. The maximum excess adsorption at different temperatures correlated with the bulk phase density: as the CO2 temperature increased, the maximum excess adsorption density decreased. The density of the adsorbed phase, obtained using the Dubinin-Radushkevich model, was used to fit the excess adsorption data. At low pressure, the CO2-induced strain on shale was caused by the gas adsorption, whereas at high pressure, it was caused by gas pressure. The absolute adsorption linearly correlated with the adsorption-induced strain.

Aloin Inhibits the Proliferation and Migration of Gastric Cancer Cells by Regulating NOX2-ROS-Mediated Pro-Survival Signal Pathways.

BACKGROUND: Aloin has been reported to have many pharmacological effects including anti-inflammatory, anti-oxidant and anti-tumour activities. However, the precise molecular mechanisms underlying the anti-tumour properties of aloin are yet to be elucidated. METHODS: HGC-27 and BGC-823 gastric cancer cells were treated with aloin. EdU and colony formation assays were used to detect the proliferation ability of cells. The migration of cells was detected using wound healing and transwell assays. Western blotting was used to detect the levels of cyclinD1, cyclin E1, MMPs, N-cadherin, E-cadherin and NOX2. The phosphorylation of Akt, mTOR, P70S6K, S6, Src, stat3 and IκBα were also detected by Western blotting. Flow cytometry was used to detect the cell cycle distribution.The location of p65 in cells was determined by using a confocal microscopy assay. The total amounts of ROS present in cells were measured using an ROS assay kit. RESULTS: Here, we found that aloin inhibited the proliferation and migration of HGC-27 and BGC-823 gastric cancer cells using a combination of EdU, colony formation, wound healing and transwell assays. Further investigations revealed that aloin decreased the protein expression levels of cyclin D1, N-cadherin, and the matrix metalloproteinases (MMP)-2 and MMP-9; increased E-cadherin expression in a dose-dependent manner; inhibited reactive oxygen species (ROS) generation; and mediated the activation of Akt-mTOR, signal transducer and activator of transcription-3 (Stat3), and NF-κB signalling pathways. Our results also indicated that aloin is able to attenuate the expression levels of the two regulatory proteins of nicotinamide adenine dinucleotide phosphate oxidase 2 (NOX2), p47phox and p22phox, but had no effect on the level of gp91phox. N-acetylcysteine treatment of gastric cancer cells inhibited ROS production and Akt-mTOR, Stat3, and IκBα phosphorylation. Taken together, our data suggest that aloin inhibits the proliferation and migration of gastric cancer cells by downregulating NOX2-ROS-mediated activation of the Akt-mTOR, Stat3, and NF-κB signalling pathways. CONCLUSION: Our findings suggest a potential role for aloin in the prevention of gastric cancer cell proliferation and migration and provide novel insights into the anti-cancer properties of aloin.

miR-593-5p inhibit cell proliferation by targeting PLK1 in non small cell lung cancer cells.

Worldwide, lung cancer has the highest rates of mortality and morbidity, with the majority of its pathology attributable to non-small cell lung cancer (NSCLC). MicroRNAs are pivotal in the occurrence and development of cancer. However, the role of miRNA-593-5p in the progression of NSCLC is not clear. In this study, we investigate, in vitro, whether miRNA-593-5p inhibits NSCLC cell proliferation. To clarify its specific mechanism of inhibition, we used bioinformatics to predict its target genes and identified PLK1. Luciferase reporter assay confirmed the binding of miR-593-5p to the PLK1 3'-UTR in a sequence-specific manner in NSCLC cells. Additionally, we also found through Western blot and quantitative RT-PCR that miR-593-5p down-regulates the expression of PLK1 protein. Finally, PLK1 overexpression was shown to disinhibit NSCLC cell proliferation. Taken together, this evidence suggests that miR-593-5p inhibits NSCLC cell proliferation by inhibiting PLK1 expression.

[Calenduloside E inhibits lipopolysaccharide-induced inflammatory response by inhibiting activation of ROS-mediated JAK1-stat3 signaling pathway in RAW264.7 cells].

OBJECTIVE: To investigate the effect of calenduloside E on lipopolysaccharide (LPS)-induced inflammatory response in RAW264.7 cells and explore the underlying molecular mechanism. METHODS: CCK-8 assay was used to examine the effect of different concentrations of calenduloside E (0-30 µg/mL) on the viability of RAW264.7 cells. The release of the pro-inflammatory cytokines tumor necrosis factor-α (TNF-α) and interleukin-1ß (IL-1ß) in RAW264.7 cells in response to pretreatment with 6, 8, and 10 µg/mL calenduloside E for 2 h followed by stimulation with 100 ng/mL LPS was detected using enzyme-linked immunosorbent assay (ELISA). The expression levels of iNOS and COX-2 and the activation of JAK-stats, MAPKs and NF-кB signaling pathways in the treated cells were determined using Western blotting. A reactive oxygen species (ROS) detection kit was used to detect ROS production in the cells, and the nuclear translocation of the transcription factor stat3 was observed by laser confocal microscopy. RESULTS: Calenduloside E below 20 µg/mL did not significantly affect the viability of RAW264.7 cells. Calenduloside E dose-dependently decreased the expression levels of iNOS and COX-2 induced by LPS, inhibited LPS-induced release of TNF-α and IL-1ß, and suppressed LPS-induced JAK1-stat3 signaling pathway activation and stat3 nuclear translocation. Calenduloside E also significantly reduced ROS production induced by LPS in RAW264.7 cells. CONCLUSIONS: Calenduloside E inhibits LPS-induced inflammatory response by blocking ROS-mediated activation of JAK1-stat3 signaling pathway in RAW264.7 cells.

The molecular mechanisms of Aloin induce gastric cancer cells apoptosis by targeting High Mobility Group Box 1.

Purpose: Aloin (ALO), a bioactive ingredient extracted from aloe vera, has anti-tumor effects. High Mobility Group Box 1 (HMGB1), a highly conserved nuclear DNA-binding protein, has been implicated in various cancer types. Highly expressed HMGB1 is closely associated with tumor cells apoptosis, proliferation and migration. We investigated the specific molecular mechanisms by which ALO-induced apoptosis by targeting HMGB1 in gastric cancer cells. Materials and methods: Human gastric cancer HGC-27 cells were treated with different doses of ALO (100, 200 and 400 µg/ml) for 24 h, after which DAPI staining was used to observe the nuclear morphology, Annexin V/PI double staining assay was used to determine the rate of apoptosis; Western blotting was used to detect the levels of PARP, pro-caspase3, HMGB1 and RAGE; nuclear translocation of HMGB1 was determined by conducting a nucleoplasm separation experiment. The Enzyme linked immunosorbent assay (ELISA) assay was used to detect release of HMGB1. The HGC-27 cells, transfected with HMGB1 shRNA plasmids, were stimulated with ALO for 24 h, after which a flow cytometry assay was used to detect the rate of apoptosis. HGC-27 cells were pre-treated with or without ALO and then stimulated with rhHMGB1, the phosphorylation of Akt, mTOR, P70S6K, S6, 4EBP1, ERK, P90RSK, cAMP regulatory element binding (CREB) were detected by Western blotting. Results: After different doses of ALO treatment, the nuclei showed morphological changes characteristic of apoptosis. Apoptotic rates were enhanced in a dose dependent manner. The level of cleaved PARP was enhanced and pro-caspase3, HMGB1 and RAGE levels were reduced, HMGB1 nuclear translocation and release were inhibited. The activation of rhHMGB1-induced Akt-mTOR-P70S6K and ERK-CREB signalling pathways was inhibited by ALO. Blocking these signalling pathways by special inhibitors and HMGB1 knockdown could enhance ALO-induced HGC-27 cell apoptosis. Conclusion: ALO- induced HGC-27 cell apoptosis by down-regulating expressions of HMGB1 and RAGE, inhibiting HMGB1 release and then suppressing rhHMGB1-induced activation of Akt-mTOR-P70S6K and ERK-P90RSK-CREB signalling pathways.

Inhibition of ROS-mediated activation Src-MAPK/AKT signaling by orientin alleviates H2O2-induced apoptosis in PC12 cells.

PURPOSE: Reactive oxygen species (ROS) are considered a direct cause of neurodegenerative diseases (NDDs). Drugs developed to target ROS are effective for the treatment of NDDs. Orientin is a pyrone glucoside extracted from Polygonum orientale, and it exhibits many pharmacological activities. In this study, we aimed to determine whether orientin could relieve hydrogen peroxide (H2O2)-induced neuronal apoptosis and to investigate the specific target of orientin. MATERIALS AND METHODS: In this study, the neuroprotective effect and its possible mechanisms of orientin in mouse pheochromocytoma cell line (PC12) cells stimulated by H2O2, establishing an oxidative stress model, were investigated. And we further tested the role of ROS in the neuroprotective effects of orientin. RESULTS: Orientin (5-100 µg/mL) did not cause toxicity in PC12 cells but significantly decreased H2O2-induced reduction in PC12 cell viability, cell apoptosis rates, and nuclear condensation. It also inhibited the activation of caspase-3 and degradation of poly(ADP-ribose) polymerase (PARP). Under the stimulation of H2O2, MAPKs (ERK, JNK, and p38), AKT, and Src signaling proteins in PC12 cells were activated in a time-dependent manner. The application of inhibitors that were specific for MAPKs, AKT, and Src effectively alleviated H2O2-induced cell apoptosis. In addition, the Src inhibitor decreased the activation of MAPKs and AKT signaling. More importantly, orientin effectively decreased H2O2-induced phosphorylation of MAPKs, AKT, and Src signaling proteins. Finally, we confirmed that orientin effectively inhibited H2O2-induced accumulation of ROS in cells. In addition, ROS inhibitors blocked the Src-MAPKs/AKT signaling pathway-dependent cell apoptosis stimulated by H2O2. CONCLUSION: These results indicate that alleviation of H2O2-induced cell apoptosis by orientin is Src-MAPKs/AKT dependent. Overall, our study confirms that orientin alleviates H2O2-induced cell apoptosis by inhibiting the ROS-mediated activation of Src-MAPKs/AKT signaling.

[Chrysin inhibits lipopolysaccharide-induced inflammatory responses of macrophages via JAK-STATs signaling pathway].

OBJECTIVE: To investigate the mechanism of chrysin in regulating lipopolysaccharide (LPS)-induced inflammation in RAW264.7 cells. METHODS: RAW264.7 cells were treated with different concentrations (0, 5, 10, 20, 40, 60, 80, 100, 150, and 200 µg/mL) of chrysin for 24 h, and the cell viability was measured using CCK-8. RAW264.7 cells were pre-treated with 10, 30, or 60 µg/mL chrysin for 2 h before stimulation with LPS for different times. The levels of TNF-α, IL-6 and MCP-1 were detected by ELISA, and Western blotting was used to detect the phosphorylation of JAK- 1, JAK-2, STAT-1 and STAT-3. The level of reactive oxygen species in RAW264.7 cells was detected by CM-H2DCFDA fluorescence probe. The effect of ROS on LPS-induced JAK-STATs signal and the inflammatory response of RAW264.7 cells was detected by ROS scavenger NAC. The transcription factors STAT-1 and STAT-3 nuclear translocation were observed by laser confocal microscopy. RESULTS: Chrysin below 60 µg/mL did not significantly affect the viability of RAW264.7 cells. At 10, 30, and 60 µg/mL, chrysin dose-dependently inhibited the expression of iNOS induced by LPS. Chrysin treatment also inhibited LPS-induced phosphorylation of JAK-STATs, nuclear translocation of STAT1 and STAT3, release of TNF-α, IL-6 and MCP-1, and the production of ROS in RAW264.7 cells; ROS acted as an upstream signal to mediate the activation of JAK-STATs signaling pathway. CONCLUSION: Chrysin blocks the activity of JAK-STATs mediated by ROS to inhibit LPS-induced inflammatory response in RAW264.7 cells.

[Aloin induces apoptosis via regulating the activation of MAPKs signaling pathway in human gastric cancer cells in vitro].

OBJECTIVE: To investigate the effect of aloin on apoptosis of human gastric cancer cells and explore the molecular mechanism. METHODS: Gastric cancer MKN-28 and HGC-27 cells were cultured routinely in 1640 medium supplemented with 10% fetal bovine serum and 10% non-essential amino acids (for HGC-27 cells) and treated with different concentrations of aloin for different durations. The cell viability, cell nuclear morphology, and apoptotic rate of the cells were detected using CCK-8 assay, DAPI staining and AnnexinV-FITC/PI, respectively; Western blotting was used to detect the expression levels of PARP, procaspase 3 and the phosphorylation of p38, ERK and JNK. The cells were treated with specific inhibitors of p38, ERK and JNK, and the inhibitory effects on these pathways were detected with Western blotting; DAPI staining was used to detect the effects of inhibitors on apoptosis of gastric cancer cells. RESULTS: Aloin dose-dependently inhibited the viability and induced apoptosis of HGC-27 and MKN-28 cells. Alion treatment obvious enhanced the phosphorylation of p38 and JNK but decreased ERK phosphorylation in the cells. Blocking ERK activation with the ERK inhibitor obviously enhanced aloin-induced cell apoptosis, where inhibiting p38 and JNK activation partly reversed alion-induced apoptosis in the cells. CONCLUSIONS: Aloin induces apoptosis of human gastric cancer cells in vitro by activating p38 and JNK signaling pathways and inhibiting ERK signaling pathway.