Protein Regulator of Cytokinesis 1 (PRC1) Upregulation Promotes Immune Suppression in Liver Hepatocellular Carcinoma.

Liver hepatocellular carcinoma (LIHC) is a malignant cancer with widespread prevalence. The suppressive immune environment causes largely refractory to current treatment. The protein regulator of cytokinesis 1 (PRC1) is an essential gene for cytokinesis and is involved in cancer pathogenesis. However, the functions of PRC1 have been barely clarified, especially in LIHC. Here, we investigated the expression, prognostic value, and functions of PRC1 in LIHC. Pan-cancer analysis revealed the overexpression of PRC1 in the Cancer Genome Atlas (TCGA) database. Four LIHC datasets from the Gene Expression Omnibus (GEO) database confirmed the PRC1 overexpression in LIHC. The mRNA and protein levels of PRC1 in LIHC cells were higher than in normal liver cells. The overexpression of PRC1 predicted progressed clinical stage and poor prognosis of LIHC. We further investigated the functions of PRC1 by performing the Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses, and Gene Set Enrichment Analysis (GSEA) of its coexpressing genes. High PRC1 expression was associated with increased genome instability of LIHC. Moreover, PRC1 was positively correlated with the infiltration of suppressive immune cells like T regulatory cells (Tregs) and polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs) and was negatively correlated with the effector immune cells' infiltration, including B cells and CD8+ T cells. In addition, PRC1 was positively correlated with the expression of tumor immune checkpoint molecules. Taken together, PRC1 overexpression contributes to the genome instability and the suppressive immune microenvironment of LIHC. Thus, PRC1 has the potential to be a prognostic marker and therapeutic target of LIHC.

Icaritin inhibits PLK1 to activate DNA damage response in NK/T cell lymphoma and increases sensitivity to GELOX regime.

Natural killer/T cell lymphoma (NKTCL) is a highly aggressive subtype of non-Hodgkin lymphoma. Gemcitabine, oxaliplatin, and L-asparaginase (GELOX) is one of the first-line chemotherapy regimens of NKTCL. Yet, the prognosis of NKTCL is poor. Icaritin is an herb-derived monomer from icariin with antitumor effects. We found that icaritin induced proliferation inhibition and apoptosis of NKTCL both in vitro and in vivo. Moreover, icaritin inhibited the dissemination of NKTCL in vivo. RNA sequencing revealed the Polo-like kinase 1 (PLK1) gene and DNA damage response (DDR) as the targets of icaritin. Mechanistically, icaritin inhibited PLK1 to promote checkpoint kinase 2 (Chk2) homodimerization and its T387 phosphorylation, which further activated p53, leading to the activation of the DDR pathway. Moreover, inhibiting PLK1 increased Forkhead box O3a nuclear localization, the latter of which activated ataxia telangiectasia mutated (ATM), an early sensor of DNA damage. Then ATM phosphorylated Chk2 T68 and initiated Chk2 activation. Remarkably, the combined treatment of icaritin and GELOX achieved better antitumor efficacy than single treatment in vivo. In summary, our results proved the efficacy of icaritin treating NKTCL, provided insights into its antitumor molecular mechanism, and revealed the application value of icaritin in facilitating clinical NKTCL treatment.

Resveratrol activates DNA damage response through inhibition of polo-like kinase 1 (PLK1) in natural killer/T cell lymphoma.

BACKGROUND: Extranodal natural killer/T cell lymphoma (NKTCL) is a highly aggressive non-Hodgkin lymphoma with a poor prognosis. Resveratrol (REV), a natural nontoxic pleiotropic agent, has antitumor effects, yet not being studied in NKTCL. METHODS: We performed immunohistochemical (IHC) staining with NKTCL tumor tissues. Apoptosis and cell cycle of NKTCL cell line NK-92 were detected by using flow cytometry. Then we detected the cellular expression level of polo-like kinase 1 (PLK1) and key molecules in DNA damage response (DDR) pathway by using RNA sequencing (RNA-seq) technology, real-time PCR, and Western blot. RESULTS: In this study, we found distinguishingly expressed phosphorylated ataxia telangiectasia mutated (ATM) in human NKTCL tumor tissues compared to normal lymph nodes samples. But low levels of phosphorylated checkpoint kinase 2 (Chk2) and phosphorylated p53 were shown, suggesting that DDR pathway is blocked midway in NKTCL. REV inhibited the proliferation of NK-92 cells in a time- and dose-dependent manner, arrested cell cycle at G1 phase, and induced mitochondrial apoptosis. PLK1 was inhibited in both mRNA and protein levels by REV in NK-92 cells. At the same time, phosphorylation levels of Chk2 and p53 were upregulated. CONCLUSIONS: DDR pathway plays an important role in the pathogenesis of NKTCL. REV shows anti-NKTCL activity. The inhibition of PLK1 and the activation of DDR are vital for REV induced tumor cell apoptosis.

Antitumor effects of icaritin and the molecular mechanisms.

Traditional Chinese Medicine (TCM) has been increasingly studied for antitumor activities. Icaritin, a hydrolytic product of icariin, is an effective ingredient of the traditional Chinese herb epimedium with multiple pharmacological activities. Among them, the antitumor activity of icaritin has been widely studied and reported in tumors both in vitro and in vivo. While its exact antitumor mechanisms await revelation, icaritin has been found to regulate several key molecules and pathways concerning cell fate, including CDK-dependent pathways, mitogen-activated protein kinases (MAPKs), the serine-threonine kinase AKT, signal transducer and activator of transcription 3 (STAT3), and p53. The ability to induce cellular oxidative stress also contributes to its antitumor activity. This review outlines the results of key investigations focusing on the antitumor effects and mechanisms of icaritin.

Glutathione S-transferase A1 suppresses tumor progression and indicates better prognosis of human primary hepatocellular carcinoma.

Glutathione S-transferase (GST) family members play an important role in detoxification, metabolism and carcinogenesis. The aim of this study is to investigate the effect of Glutathione S-transferase A1 (GSTA1) on the prognosis of HCC and to understand its role in tumor progression and the possible mechanism. GSTA1 in HCC was assessed using immunohistochemical staining, and it was found that HCC patients with better pathological differentiation had higher GSTA1 abundance. Further, high GSTA1 expression was correlated with low AFP, absent PVTT, and early stage TNM for HCC patients. Higher GSTA1 indicated longer overall survival and disease-free survival, while lower GSTA1 indicated poorer prognosis. Subsequently, lentiviral vector carrying GSTA1 gene was successfully constructed and maintained high expression in 97H and SNU449 liver cancer cells. We found that high GSTA1 restrained liver cancer cell proliferation, migration and invasion in vitro. Western blot showed that LKB1 and p-AMPK were upregulated while p-mTOR, p-p70 S6 Kinase and MMP-9 were downregulated in high GSTA1 groups. Taken together, high GSTA1 correlated with satisfactory prognosis of HCC. Additionally, GSTA1 may act as a protective factor through suppression of tumorigenesis by targeting AMPK/mTOR in HCC.

Histone deacetylase inhibitor chidamide induces growth inhibition and apoptosis in NK/T lymphoma cells through ATM-Chk2-p53-p21 signalling pathway.

We investigated the anti-tumour effects and the underlying molecular mechanisms of a new oral histone deacetylase inhibitor (HDACi), chidamide, in NK/T cell lymphoma (NKTCL), a rare and highly aggressive non-Hodgkin lymphoma with poor outcomes. SNT-8 and SNK-10 NKTCL cell lines were exposed to different concentrations of chidamide for the indicated time. The treated cells were analysed for cell proliferation, cell cycle progression, and cell apoptosis. Proteins in the AKT/mTOR and MAPK signalling pathways and the DNA damage response (DDR) cell cycle checkpoint pathway were measured by Western blotting. Chidamide inhibited cell proliferation in a dose- and time-dependent manner, arrested cell cycle progression at the G0/G1 phase, and induced apoptosis in the NKTCL cell lines. In addition, we found that chidamide suppressed the phosphorylation levels of proteins in the AKT/mTOR and MAPK signalling pathways and activated the DDR cell cycle checkpoint pathway, that is, the ATM-Chk2-p53-p21 pathway. Expression of EBV genes was also assessed by Real-Time PCR. Chidamide induced EBV lytic-phase gene expression in EBV-positive NKTCL. Our results provide evidence that chidamide shows antitumour effects by inhibiting the AKT/mTOR and MAPK signalling pathways and activating the ATM-Chk2-p53-p21 signalling pathway in vitro.

Resveratrol inhibits Extranodal NK/T cell lymphoma through activation of DNA damage response pathway.

BACKGROUND: Extranodal NK/T cell lymphoma (NKTCL) is a highly aggressive non-Hodgkin lymphoma with poor prognosis. Resveratrol (RSV, 3,5,4'-trihydroxystilbene), a natural nontoxic phenolic compound found in the skin of grapes and some other spermatophytes, performs multiple bioactivities, such as antioxidant activity, anti-aging activity, reduction of cardiovascular disease risk and anticarcinogenic effect. Here we report the anti-tumor effect of RSV in NKTCL cell lines SNT-8, SNK-10 and SNT-16. RESULTS: RSV inhibited NKTCL cell proliferation in a dose- and time-dependent manner and arrested cell cycle at S phase. It induced NKTCL cells apoptosis through mitochondrial pathway, shown as down-regulation of MCl-1 and survivin, up-regulation of Bax and Bad, and activation of caspase-9 and caspase-3. In addition, we found that RSV suppressed the phosphorylation level of AKT and Stat3, and activated DNA damage response (DDR) pathway directly or through up-regulation of Zta of Epstein-Barr virus (EBV). Furthermore, using KU55933 as the inhibitor of pATM, we verified that DDR played an important role in RSV inducing NKTCL apoptosis. RSV also showed synergistic effect on activating DDR pathway in combination with etoposide or ionizing radiation, which resulted in cell proliferation inhibition and apoptosis. CONCLUSIONS: Our results provide in vitro evidence that RSV produces anti-tumor effect by activating DDR pathway in an ATM/Chk2/p53 dependent manner. So we suggest that RSV may be worthy for further study as an anti-tumor drug for NKTCL treatment.

Steroidogenic Acute Regulatory Protein (StAR) Overexpression Reduces Inflammation and Insulin Resistance in Obese Mice.

Steroidogenic acute regulatory protein (StAR), a mitochondrial cholesterol delivery protein, plays a beneficial role in hyperlipidemia, NAFLD, and endothelial inflammation. Elevated circulating fatty acids and low grade inflammation are known as key risk factors of insulin resistance and type 2 diabetes. In the present study, C57BL/6J mice were fed with HFD and infected with recombinant adenovirus expressing StAR by tail-vein injection. Intraperitoneal glucose/insulin tolerance test was performed to assess the insulin sensitivity. Morphological analysis and intramuscular lipid determination were used to illustrate the adipose hypertrophy and ectopic fat accumulation in skeletal muscle. The levels of inflammatory factor and nitric oxide were determined by ELISA and classic Griess reagent methods, respectively. The fatty acids composition was analysis using gas chromatography-mass spectrometry (GC-MS). The expression of genes associated with inflammation and insulin resistance were determined by Western blotting and qPCR to elucidate the underlying mechanism. We demonstrated that StAR overexpression ameliorated insulin resistance and systemic inflammatory response with the reduction of adipose hypertrophy and intramuscular lipid in HFD-fed mice. In addition, StAR overexpression increased serum unsaturated fatty acids (UFAs) and PPARγ expression in muscle and adipose tissue of obese mice. In conclusion, StAR may activate PPARγ by increasing UFAs, which leads to a protective role in systemic inflammation and insulin resistance in obese mice. J. Cell. Biochem. 118: 3932-3942, 2017. © 2017 Wiley Periodicals, Inc.

Steroidogenic acute regulatory protein (StAR) overexpression attenuates HFD-induced hepatic steatosis and insulin resistance.

Non-alcoholic fatty liver disease (NAFLD) covers a wide spectrum of liver pathology. Intracellular lipid accumulation is the first step in the development and progression of NAFLD. Steroidogenic acute regulatory protein (StAR) plays an important role in the synthesis of bile acid and intracellular lipid homeostasis and cholesterol metabolism. We hypothesize that StAR is involved in non-alcoholic fatty liver disease (NAFLD) pathogenesis. The hypothesis was identified using free fatty acid (FFA)-overloaded NAFLD in vitro model and high-fat diet (HFD)-induced NAFLD mouse model transfected by recombinant adenovirus encoding StAR (StAR). StAR expression was also examined in pathology samples of patients with fatty liver by immunohistochemical staining. We found that the expression level of StAR was reduced in the livers obtained from fatty liver patients and NAFLD mice. Additionally, StAR overexpression decreased the levels of hepatic lipids and maintained the hepatic glucose homeostasis due to the activation of farnesoid x receptor (FXR). StAR overexpression attenuated the impairment of insulin signaling in fatty liver. This protective role of StAR was owing to a reduction of intracellular diacylglycerol levels and the phosphorylation of PKCε. Furthermore, FXR inactivation reversed the observed beneficial effects of StAR. The present study revealed that StAR overexpression can reduce hepatic lipid accumulation, regulate glucose metabolism and attenuate insulin resistance through a mechanism involving the activation of FXR. Our study suggests that StAR may be a potential therapeutic target for NAFLD.

Therapeutic effect of low molecular weight chitosan containing sepia ink on ethanol-induced gastric ulcer in rats

ABSTRACT PURPOSE: To evaluate the role of low molecular chitosan containing sepia ink (LMCS) in ethanol-induced (5 ml/kg) gastric ulcer in rats. METHODS: Animals were divided into four groups (n = 12): normal group (Normal), negative control group (Con), experiment group (LMCS) and positive control Omeprazole group (OMZ). Gastric empty rate was detected in the first 7 days. Rats were sacrificed at 7, 14 and 21 day for histology and ELISA detections. RESULTS: Gastric empty was no significant differences among the groups (P > 0.05). Histological observation showed gastric mucosal LMCS treated had better healing effect. Hydroxyproline (Hyp) was significantly increased from 7 day (P < 0.05). LMCS significantly inhibited malondialdehyde (MDA) generation for lipid peroxidation from 7 day (P < 0.05). LMCS significantly promoted the activity of superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) at the earlier stage (P < 0.05). OMZ had the similar effects above. As for myeloperoxidase (MPO), LMCS significantly decreased and restored it to normal levels from 7 day (P < 0.05), it is earlier than OMZ which is from 14 day. CONCLUSION: LMCS can improve gastric mucosa tissue repair, exert significant influences on oxidative and antioxidant enzyme activities and neutrophil infiltration.

Effects of chitin and sepia ink hybrid sponge on the healing of burning wound rats and its impact on macrophages in vitro.

PURPOSE: To detect whether chitin and sepia ink sponge (CS) can promote wound healing and elevate impact of CS on phagocytosis ability of macrophages. METHODS: Forty-eight rats were assigned to four groups: Normal group (Normal), negative control group (Con), chitin and sepia ink sponge group (CS) and positive control Surgicel Gauze(r) group (SG). Deep second-degree burn model was created in rats. Wound area was recorded by digital imaging and determined using Image J software. Samples were collected and kept at -80oC on 3d, 7d, 14d and 21d for cytokines detecting. Transforming growth factor (TGF)-ß1, interleukin (IL)-6, matrix metalloproteinase (MMP)-1, hydroxyproline (Hyp) and macrophage activity reflected by tumor necrosis factor (TNF)-α were determined by enzyme-linked immunosorbent assay (ELISA). RESULTS: Comparing to Con and SG, scabs in CS group fell off and basically healed on 21 day. TGF-ß1, IL-6, MMP-1 and Hyp were significantly increased by CS and SG comparing to Con (p < 0.05), CS had more apparently adjustment on TGF-ß1 and MMP-1 compared to SG; results in vitro indicated CS significantly promoted phagocytosis ability of macrophages reflected in TNF-α (p < 0.05). CONCLUSION: CS improved wound healing through exerting significant influences on secretion of kinds of cytokines and activating macrophages.

Effects of chitin and sepia ink hybrid sponge on the healing of burning wound rats and its impact on macrophages in vitro

PURPOSE: To detect whether chitin and sepia ink sponge (CS) can promote wound healing and elevate impact of CS on phagocytosis ability of macrophages. METHODS: Forty-eight rats were assigned to four groups: Normal group (Normal), negative control group (Con), chitin and sepia ink sponge group (CS) and positive control Surgicel Gauze(r) group (SG). Deep second-degree burn model was created in rats. Wound area was recorded by digital imaging and determined using Image J software. Samples were collected and kept at -80oC on 3d, 7d, 14d and 21d for cytokines detecting. Transforming growth factor (TGF)-β1, interleukin (IL)-6, matrix metalloproteinase (MMP)-1, hydroxyproline (Hyp) and macrophage activity reflected by tumor necrosis factor (TNF)-α were determined by enzyme-linked immunosorbent assay (ELISA). RESULTS: Comparing to Con and SG, scabs in CS group fell off and basically healed on 21 day. TGF-β1, IL-6, MMP-1 and Hyp were significantly increased by CS and SG comparing to Con (p < 0.05), CS had more apparently adjustment on TGF-β1 and MMP-1 compared to SG; results in vitro indicated CS significantly promoted phagocytosis ability of macrophages reflected in TNF-α (p < 0.05). CONCLUSION: CS improved wound healing through exerting significant influences on secretion of kinds of cytokines and activating macrophages.

Icaritin induces lytic cytotoxicity in extranodal NK/T-cell lymphoma.

BACKGROUND: Extranodal NK/T-cell lymphoma (ENKL) is an aggressive hematological malignancy associated with Epstein-Barr virus (EBV) infection. It is often resistant to conventional chemotherapy and has a poor prognosis. Icaritin, a compound derived from Chinese herbal medicine, Herba Epimedii, has been reported to exert antitumor effects on a variety of cancer cell lines. In the present study, we investigated the cytotoxic effects of Icaritin on the two EBV-positive ENKL cell lines SNK-10 and SNT-8, along with the underlying molecular mechanisms. METHODS: ENKL cell lines SNK-10 and SNT-8 were exposed to different concentrations of Icaritin for the indicated time. Treated cells were analyzed for cell proliferation, cell cycle, and cell apoptosis. Phosphorylation of Stat3 and Akt proteins in signaling pathways and the EBV-encoded LMP1 proteins were measured by Western blot. Expression of EBV genes was assessed by Real-Time PCR. RESULTS: Our results showed that Icaritin dose-dependently inhibits ENKL cell proliferation and induces apoptosis and cell cycle arrest at G2/M phase. Additionally, Icaritin upregulates Bax, downregulates Bcl-2 and pBad, and activates caspase-3 and caspase-9. The anti-proliferative and pro-apoptotic effects of Icaritin are likely mediated by inhibition of Stat3 and Akt pathways through LMP1 downregulation. Importantly, Icaritin induces EBV lytic gene expression in ENKL cells, and the combination of Icaritin and the antiviral drug ganciclovir (GCV) is more effective in inducing ENKL cells apoptosis than Icaritin or GCV alone. CONCLUSIONS: These findings indicate that EBV-targeted approaches may have significant therapeutic potential for ENKL treatment.