Oleanolic acid inhibits hypoxic tumor-derived exosomes-induced premetastatic niche formation in hepatocellular carcinoma by targeting ERK1/2-NFκB signaling.

BACKGROUND: Pulmonary premetastatic niche (PMN) formation plays a key role in the lung metastasis of hepatocellular carcinoma (HCC). Hypoxia promotes the secretion of tumor-derived exosomes (TDEs) and facilitates the formation of PMN. However, the mechanisms remain unexplored. METHODS: TDEs from normoxic (N-TDEs) or hypoxic (H-TDEs) HCC cells were used to induce fibroblast activation in vitro and PMN formation in vivo. Oleanolic acid (OA) was intragastrically administered to TDEs-preconditioned mice. Bioinformatics analysis and drug affinity responsive target stability (DARTS) assays were performed to identify targets of OA in fibroblasts. RESULTS: H-TDEs induced activation of pulmonary fibroblasts, promoted formation of pulmonary PMN and subsequently facilitated lung metastasis of HCC. OA inhibited TDEs-induced PMN formation and lung metastasis and suppressed TDEs-mediated fibroblast activation. MAPK1 and MAPK3 (ERK1/2) were the potential targets of OA. Furthermore, H-TDEs enhanced ERK1/2 phosphorylation in fibroblasts in vitro and in vivo, which was suppressed by OA treatment. Blocking ERK1/2 signaling with its inhibitor abated H-TDEs-induced activation of fibroblasts and PMN formation. H-TDEs-induced phosphorylation of ERK1/2 in fibroblasts touched off the activation NF-κB p65, which was mitigated by OA. In addition, the ERK activator C16-PAF recovered the activation of ERK1/2 and NF-κB p65 in H-TDEs-stimulated MRC5 cells upon OA treatment. CONCLUSION: The present study offers insights into the prevention of TDEs-induced PMN, which has been insufficiently investigated. OA suppresses the activation of inflammatory fibroblasts and the development of pulmonary PMN by targeting ERK1/2 and thereby has therapeutic potential in the prevention of lung metastasis of HCC.

Hypoxia-induced exosomes facilitate lung pre-metastatic niche formation in hepatocellular carcinoma through the miR-4508-RFX1-IL17A-p38 MAPK-NF-κB pathway.

Background: Hypoxia plays an important role in the lung metastasis of hepatocellular carcinoma (HCC). However, the process by which hypoxia promotes the formation of a pre-metastatic niche (PMN) and its underlying mechanism remain unclear. Methods: Exosomes derived from normoxic and hypoxic HCC cells were collected to induce fibroblast activation in vitro and PMN formation in vivo. The micro RNA (miR) profiles of the exosomes were sequenced to identify differentially expressed miRNAs. Gain- and loss-of-function analyses were performed to investigate miR-4508 function. Dual-luciferase, western blotting, and real-time reverse transcription-PCR analyses were used to identify the direct targets of miR-4508 and its downstream signaling pathways. To demonstrate the roles of hypoxic tumor-derived exosomes (H-TDEs) and miR-4508 in the lung metastasis of liver cancer, H22 tumor cells were injected through the tail vein of mice. Blood plasma-derived exosomes from patients with HCC who underwent transarterial chemoembolization (TACE) were applied to determine clinical correlations. Results: We demonstrated that H-TDEs activated lung fibroblasts and facilitated PMN formation, thereby promoting lung metastasis in mice. Screening for upregulated exosomal miRNAs revealed that miR-4508 and its target, regulatory factor X1 (RFX1), were involved in H-TDE-induced lung PMN formation. Moreover, miR-4508 was significantly upregulated in plasma exosomes derived from patients with HCC after TACE. We confirmed that the p38 MAPK-NF-κB signaling pathway is involved in RFX1 knockdown-induced fibroblast activation and PMN formation. In addition, IL17A, a downstream target of RFX1, was identified as a link between RFX1 knockdown and p38 MAPK activation in fibroblasts. Conclusion: Hypoxia enhances the release of TDEs enriched with miR-4508, thereby promoting lung PMN formation by targeting the RFX1-IL17A-p38 MAPK-NF-κB pathway. These findings highlight a novel mechanism underlying hypoxia-induced pulmonary metastasis of HCC.

Corrigendum to "The Chinese Medicine, Jiedu Recipe, Inhibits the Epithelial Mesenchymal Transition of Hepatocellular Carcinoma via the Regulation of Smad2/3 Dependent and Independent Pathways".

[This corrects the article DOI: 10.1155/2018/5629304.].

Effect and Molecular Mechanisms of Jiedu Recipe on Hypoxia-Induced Angiogenesis after Transcatheter Arterial Chemoembolization in Hepatocellular Carcinoma.

Transcatheter arterial chemoembolization (TACE) is one of the effective treatment methods for hepatocellular carcinoma (HCC) in middle and late phases. However, TACE-induced hypoxia may promote the angiogenesis and section of some cytokines, such as IL-8, and, thereby, lead to tumor metastasis. Therefore, we investigated the effect of Jiedu Recipe (JR), which has been demonstrated as an effective Traditional Chinese Medicine (TCM) recipe on HCC, on TACE-induced cytokines upregulation and hypoxia-induced angiogenesis. A total of 88 hepatocellular carcinoma (HCC) patients treated with TACE were enrolled and divided into a JR group or control group. TACE induced significant increases of neutrophil lymphocyte ratio (NLR), platelet lymphocyte ratio (PLR), IL-1ß, IL-2R, IL-6, and IL-8. JR treatment significantly inhibited the elevation of IL-8 compared with control. In vitro, JR significantly inhibited the hypoxia-induced overexpression of IL-8, HIF-1α, and VEGF mRNA in Huh 7 cells. ELISA assay demonstrated the effect of JR on IL-8 expression. Both hypoxia and IL-8 may promote angiogenesis which was suppressed by JR. Western blot showed that IL-8 upregulated the expression of phosphorylation of AKT, ERK, NF-κB, and VEGFR, which were inhibited by JR. On the other hand, effects of IL-8 on the increase of p-AKT and p-ERK were also blocked by LY294002 and U0126, respectively. In conclusion, our results indicated that JR may inhibit hypoxia-induced angiogenesis through suppressing IL-8/HIF-1α/PI3K and MAPK/ERK pathways after TACE in HCC patients.

UDCA Inhibits Hypoxic Hepatocellular Carcinoma Cell-Induced Angiogenesis Through Suppressing HIF-1α/VEGF/IL-8 Intercellular Signaling.

Background: A hypoxic microenvironment may induce angiogenesis and promote the development of hepatocellular carcinoma (HCC). The aim of this study was to evaluate whether ursodeoxycholic acid (UDCA) may inhibit hypoxic HCC cell-induced angiogenesis and the possible mechanisms. Methods: Tube formation and matrigel plug angiogenesis assays were used to evaluate angiogenesis in vitro and in vivo, respectively. Real-time PCR, enzyme-linked immunosorbent assay, and Western blot were used to evaluate the mRNA and protein expressions of hypoxia-inducible factor-1α (HIF-1α), vascular endothelial growth factor (VEGF), and IL-8, respectively. Dual-luciferase reporter assay was applied to assess the reporter gene expression of hypoxia-response element (HRE). Results: UDCA antagonized hypoxic Huh 7 cell-induced tube formation of EA.hy 926 cells. In HCC cells, UDCA inhibited hypoxia-induced upregulation of VEGF and IL-8 both in mRNA and protein levels. UDCA also inhibited IL-8-induced angiogenesis in vitro and in vivo through suppressing IL-8-induced phosphorylation of ERK. The levels of HIF-1α mRNA and protein and HRE-driven luciferase activity in HCC cells were upregulated by hypoxia and were all inhibited by UDCA. The proteasome inhibitor MG132 antagonized the effect of UDCA on HIF-1α degradation. In hypoxic condition, the phosphorylation of ERK and AKT was obviously increased in HCC cells, which was suppressed by UDCA. Transfection of the HIF-1α overexpression plasmid reversed the effects of UDCA on hypoxic HCC cell-induced angiogenesis, HRE activity, and expressions of IL-8 and VEGF. Conclusions: Our results demonstrated that UDCA could inhibit hypoxic HCC cell-induced angiogenesis through suppressing HIF-1α/VEGF/IL-8-mediated intercellular signaling between HCC cells and endothelial cells.

Efficacy of complementary and integrative medicine on health-related quality of life in cancer patients: a systematic review and meta-analysis.

Complementary and integrative medicine (CIM) has been used for improving health-related quality of life (HRQOL) in patients with cancer. The objective of this review is to evaluate the effects of CIMs on the HRQOL of cancer patients. We identified randomized controlled trials (RCTs) involving patients with cancer at any stage by retrieving electronic databases from the inception to February 14, 2018 (Systematic Review Registration: PROSPERO CRD42018091609). The main outcomes were HRQOL scores and related domains such as physical well-being scores. The standardized mean difference was used for the analysis and heterogeneity was assessed with the I 2 statistic. A Bayesian framework was used to estimate the ranking order of efficacy in HRQOL change. Finally, 34 RCTs with 3,010 patients were included. As a whole, the results showed clearly superior efficacy of CIM in improving HRQOL. For different domains of HRQOL, different CIM interventions may play different roles. The ranking order of efficacy in change HRQOL was qigong plus mindfulness, Chinese herbal medicine, multimodal complementary medicine, qigong, nutritional supplement, mindfulness, acupuncture, yoga, and massage, and it was different among different domains. There was no evidence of publication bias. In conclusion, CIM may improve the HRQOL of cancer patients. More studies, especially focusing on male cancer patients, are needed to increase the confidence level of our findings.

Melittin Inhibits Hypoxia-Induced Vasculogenic Mimicry Formation and Epithelial-Mesenchymal Transition through Suppression of HIF-1α/Akt Pathway in Liver Cancer.

In this study, we investigated whether melittin could suppress hypoxia-induced vasculogenic mimicry (VM) formation in liver cancer and explored the underlying mechanisms. Melittin significantly inhibited the proliferation of liver cancer cells with or without CoCl2 presence. Melittin also significantly inhibited CoCl2-induced migration, invasion, and VM formation of liver cancer cells. CoCl2 treatment suppressed the expression of E-cadherin and elevated the expression of N-cadherin and Vimentin. Melittin reversed the changes in the protein and mRNA levels of these epithelial-mesenchymal transition (EMT) markers. CoCl2-induced accumulation of HIF-1α increased the level of phosphorylated Akt and upregulated the expression of VEGF and MMP-2/9. Melittin decreased the HIF-1α level and thereby suppressed the levels of p-Akt, VEGF, and MMP-2/9. In addition, the inhibitor of PI3K/Akt also suppressed CoCl2-induced EMT and liver cancer cells migration, and the activator of Akt, SC-79, partly blocked the effect of melittin on CoCl2-induced EMT and liver cancer cells migration. In the xenograft tumor model in nude mice, melittin treatment significantly suppressed the tumor growth, VM formation, and HIF-1α expression in the tumor. In conclusion, this study indicates melittin may inhibit hypoxia-induced VM formation and EMT in liver cancer through inhibiting HIF-1α/Akt pathway.

Identification of Traditional Chinese Medicine Constitutions and Physiological Indexes Risk Factors in Metabolic Syndrome: A Data Mining Approach.

OBJECTIVE: In order to find the predictive indexes for metabolic syndrome (MS), a data mining method was used to identify significant physiological indexes and traditional Chinese medicine (TCM) constitutions. METHODS: The annual health check-up data including physical examination data; biochemical tests and Constitution in Chinese Medicine Questionnaire (CCMQ) measurement data from 2014 to 2016 were screened according to the inclusion and exclusion criteria. A predictive matrix was established by the longitudinal data of three consecutive years. TreeNet machine learning algorithm was applied to build prediction model to uncover the dependence relationship between physiological indexes, TCM constitutions, and MS. RESULTS: By model testing, the overall accuracy rate for prediction model by TreeNet was 73.23%. Top 12.31% individuals in test group (n=325) that have higher probability of having MS covered 23.68% MS patients, showing 0.92 times more risk of having MS than the general population. Importance of ranked top 15 was listed in descending order . The top 5 variables of great importance in MS prediction were TBIL difference between 2014 and 2015 (D_TBIL), TBIL in 2014 (TBIL 2014), LDL-C difference between 2014 and 2015 (D_LDL-C), CCMQ scores for balanced constitution in 2015 (balanced constitution 2015), and TCH in 2015 (TCH 2015). When D_TBIL was between 0 and 2, TBIL 2014 was between 10 and 15, D_LDL-C was above 19, balanced constitution 2015 was below 60, or TCH 2015 was above 5.7, the incidence of MS was higher. Furthermore, there were interactions between balanced constitution 2015 score and TBIL 2014 or D_LDL-C in MS prediction. CONCLUSION: Balanced constitution, TBIL, LDL-C, and TCH level can act as predictors for MS. The combination of TCM constitution and physiological indexes can give early warning to MS.

The Chinese Medicine, Jiedu Recipe, Inhibits the Epithelial Mesenchymal Transition of Hepatocellular Carcinoma via the Regulation of Smad2/3 Dependent and Independent Pathways.

Hepatocellular carcinoma (HCC) is one of the most common malignant tumors worldwide. In China, traditional Chinese herb medicine has been widely used in the treatment of HCC. Jiedu Recipe (JR) is a common used prescription which has shown good results against HCC. However, the exact mechanisms of JR are still unknown. Therefore, we investigated the efficacy of JR on HCC in the current study. JR inhibited the cell viability of both SMMC-7721 and Huh7 cells in both time- and dose-dependent manners. Transwell assay revealed that JR decreased the number of migrated cells of SMMC-7721 cells. JR treatment increased the E-cadherin expression level and decreased the levels of p-Smad2/3 and Smad2/3. Further study showed that JR reversed the effect of TGFß1 on the expression of E-cadherin, vimentin, N-cadherin, and MMP2/9. JR also significantly inhibited TGFß1-induced migration and invasion of SMMC-7721 and Huh7 cells determined by wound healing assay and transwell assay. TGFß1 treatment increased the phosphorylation of Smad2/3, p38 MAPK, JNK, ERK1/2, and Akt in SMMC-7721 cells and pretreatment with JR blocked TGFß1-induced activation of Smad2/3 and Akt and MAPKs. In conclusion, JR inhibits liver cancer cells migration and invasion through epithelial mesenchymal transition (EMT) inhibition via Smad2/3 dependent and independent pathways, suggesting it is an effective therapeutic strategy against HCC metastasis.

Inhibition of cFLIP overcomes acquired resistance to sorafenib via reducing ER stress­related autophagy in hepatocellular carcinoma.

Treatment with sorafenib remains the first­line therapy for patients with advanced stage hepatocellular carcinoma (HCC), however, it has limited effect due to the acquired resistance of HCC. Elucidating the potential mechanism can assist in developing promising strategies to overcome this resistance. In the present study, a sorafenib­refractory HCC cell was established from the Huh7 parental cell line, which was resistant to sorafenib mediated­cytotoxicity in vitro. The cell inhibition rate and apoptosis of cells were determined by MTT assay and flow cytometry, respectively. Electronic microscopy was used to detect autophagy in cells. The expression levels of endoplasmic reticulum stress (ERS)­related protein, apoptosis­related protein and cFLIP were examined by western blot analysis. Co­immunoprecipitation was used to examine the ubiquitination of cFLIP. It was found that sustained exposure to sorafenib activated ERS in the HCC cells. The ERS inhibitor partly increased sorafenib­induced cell death in these cells. In addition, ERS­induced autophagy was important in resistance to sorafenib, as inhibiting autophagy led to the resistant HCC cells becoming more sensitive to sorafenib. However, ERS­induced apoptosis did not differ between sorafenib­sensitive HCC cells and sorafenib­refractory HCC cells. The knockdown of cFLIP reversed the acquired sorafenib resistance by activating caspase­8 and inhibiting activated ERS in the sorafenib­resistant HCC cells. Mechanistically, a sustained increased in cFLIP was found to be dependent on USP2­induced deubiquitination. In conclusion, cFLIP was identified as a potential target for overcoming the acquired sorafenib resistance in HCC. These effects occurred partially through reducing ERS­related autophagy in HCC.

Prognostic Role of Platelet-to-Lymphocyte Ratio in Hepatocellular Carcinoma with Different BCLC Stages: A Systematic Review and Meta-Analysis.

The role of platelet-to-lymphocyte ratio (PLR) in the prognosis of hepatocellular carcinoma (HCC) patients with different Barcelona Clinic Liver Cancer (BCLC) stages remains controversial. This systematic review and meta-analysis aimed to determine the efficacy of PLR on HCC prognosis. Five electronic databases were searched for clinical trials focusing on the role of PLR in the prognosis of HCC. A total of 297 potential studies were initially identified, and 9 studies comprising 2449 patients were finally enrolled to evaluate the association between the pretreatment PLR and clinical outcomes of overall survival (OS), disease-free survival (DFS), and event occurrence in patients with HCC in different BCLC stages. An elevated pretreatment PLR indicated unfavorable worse OS (HR = 1.73; 95% CI: (1.46, 2.04); P < 0.00001) and DFS (HR = 1.30; 95% CI: (1.06, 1.60); P = 0.01). Subgroup analysis indicated that high PLR indicated poor OS among BCLC-B/C patients without heterogeneity, while PLR in BCLC-A patients indicated high statistical heterogeneity with I2 value of 78%. As for the correlation between PLR and event occurrence, high PLR was related to poor clinical event occurrence only among BCLC-C patients, though obvious heterogeneity was observed in all different BCLC stages. In conclusion, PLR may be a significant biomarker in the prognosis of HCC in different BCLC stages.

Effectiveness of Traditional Chinese Medicineas as an Adjunct Therapy for Refractory Schizophrenia: A Systematic Review and Meta Analysis.

Although recent studies focused on traditional Chinese medicine (TCM) for the treatment of refractory schizophrenia have reported that it may be beneficial, there is still lack of convincing evidence and critical meta-analytic work regarding its effectiveness as an adjunctive therapy. Therefore, we performed a meta-analysis to investigate the effectiveness of TCM in combination with antipsychotics for refractory schizophrenia. Fourteen articles involving 1725 patients published as of December 2016 were included which compared antipsychotic therapies to either TCM alone, or TCM as an adjunctive therapy. TCM was observed to have beneficial effects on aspects of the Positive and Negative Syndrome Scale (PANSS) including total score changes and negative score changes, as well as clinical effects estimated with PANSS or the Brief Psychiatric Rating Scale (BPRS). The changes in extrapyramidal side effects (RSESE) scores from baseline to the end of the treatment period were similar in two groups of related trials. TCM was also reported to mitigate some anti-psychotic related side-effects and overall, TCM adjuvant therapy was generally safe and well tolerated. While, the results indicated the potential utility of TCM as an alternative adjunctive therapeutic for refractory schizophrenia treatment, there remains a need for further high-quality studies.

Regulatory effects of four ginsenoside monomers in humoral immunity of systemic lupus erythematosus.

Ginsenosides Rb1, Rh1, Rg1 and Rg3 are known as the main active components extracted from the roots of the Panax ginseng C.A. Meyer, and were reported to have immunoregulatory effects. Disruption of B-cell immune regulation during the pathogenesis of systemic lupus erythematosus (SLE) may lead to the production of large amounts of antibodies. The present study investigated the effects of the four ginsenoside monomers on B-cell immune regulation and observed that they inhibited the proliferation and secretion of B cells induced by LPS, caused an upregulation of the expression of apoptosis-associated proteins Fas/Fas ligand and caspase-3, the expression of FcγRIIB (CD32) as well as the proportion of inactive B cells (CD19+CD27-). These results indicate that Rb1, Rh1, Rg1 and Rg3 inhibit the humoral immunity of SLE, among which Rh1 exhibited the most obvious inhibitory effect.

Progress in research on the effects of traditional Chinese medicine on the tumor microenvironment.

Tumor microenvironment (TME) has received more and more attention as modern medical research has begun to understand its importance in tumorigenesis. The occurrence, development, metastasis and drug resistance of tumors are closely related to TME. TME is a complicated system, including nontumor cells, their secreted cytokines, extracellular matrix, among other components. The concepts of wholism and multitarget regulation in traditional Chinese medicine (TCM) make it well suited to the regulation of TME. In this paper, the authors reviewed the progress of TME research and the effect of TCM on TME, providing some views of Chinese medicine in antitumor research.

Emodin inhibits migration and invasion of MHCC-97H human hepatocellular carcinoma cells.

Emodin, an anthraquinone derivative from the root and rhizome of Rheum palmatum L., was found to have antitumor effects in different types of cancer by regulating multi-molecular targets. The aim of the present study was to explore the effect of emodin on the migration and invasion of MHCC-97H human hepatocellular carcinoma cells and the underlying molecular mechanisms. Firstly, it was demonstrated that emodin can inhibit cell proliferation and induce apoptosis of cells in a time- and dose-dependent manner, using a MTT assay and flow cytometry, respectively. However, when emodin concentration was <50 µmol/l, it had little effect on the inhibition of proliferation or the induction of apoptosis. Then, it was observed that emodin can significantly suppress cell migration and invasion with a treatment dose <50 µmol/l compared with the control (P<0.05), which was not attributed to a decrease in cell number. Further study demonstrated that emodin significantly suppressed the expression levels of matrix metalloproteinase (MMP)-2 and MMP-9 compared with the control, which may be mediated by the activation of the p38 mitogen-activated protein kinases (MAPK) signaling pathway and suppression of extracellular signal regulated kinase (ERK)/MAPK and phosphatidylinositol 3-kinase/Akt signaling pathways. Therefore, the present study, for the first time, used MHCC-97H cells, which have the high potential of malignant invasion, to demonstrate that emodin may inhibit cell migration and invasion.

Emodin induces hepatocellular carcinoma cell apoptosis through MAPK and PI3K/AKT signaling pathways in vitro and in vivo.

Emodin is an active ingredient derived from root and rhizome of Rheum palmatum L and many studies have reported that it exhibits anticancer effects in a number of human tumors. However, there is little information demonstrating the possible effects of emodin on the proliferation and apoptosis of hepatocellular carcinoma (HCC). In the present study, we show that emodin may inhibit the proliferation of SMMC-7721 cells in a dose- and time-dependent manner and induced apoptosis of cells in a concentration-dependent manner after treatment for 24 h. Moreover, we further discovered that the possible molecular mechanisms involved may relate to the mitogen-activated protein kinase (MAPK) and phosphoinositide 3-kinase (PI3K)/AKT signaling pathways. Emodin may induce the phosphorylation of extracellular-signal-regulated kinase (ERK) and p38 while mildly suppressed the expression of p-c-Jun-N-terminal kinase (p-JNK). However, emodin did not affect the expression of the total (t)-ERK, t-p38 or t-JNK. Furthermore, emodin also suppressed the activation of p-AKT, but not the t-AKT. In vivo, we found that emodin suppressed tumor growth in experimental mice without an obvious change in body weight, which may work through the antiproliferation and apoptosis inducing effects. Moreover, emodin improves the liver and kidney function in mice, revealing that emodin may improve the life quality of the mice with implanted tumors. In conclusion, the above findings indicate that emodin may be a potentially effective and safe drug to induce apoptosis of HCC.

[Research progress in anti-tumor effect of emodin].

Emodin is one of the main active ingredient of Rheum palmatum, and has anti-inflammatory, anti-bacterial, anti-viral and other effects. In recent years, it arouse concern since it has a significant anti-tumor effect with low toxicity. In this paper we mainly report the anti-cancer effects of emodin according to the studies of the past five years, including four parts such as inhibit tumor growth, inhibit migration and invasion, enhance the efficacy of combination therapy, increase chemosensitivity and attenuated side effects. We hope that our work may provide a reference for further study.