Norcantharidin inhibits IL-6-induced epithelial­mesenchymal transition via the JAK2/STAT3/TWIST signaling pathway in hepatocellular carcinoma cells.

Epithelial-mesenchymal transition (EMT), plays a vital role in hepatocellular carcinoma (HCC) development and metastasis. Norcantharidin (NCTD; 7-oxabicyclo (2.2.1) heptane-2,3-dicarboxylic anhydride) plays anticancer roles in the regulation of tumor cell proliferation, apoptosis and migration. However, the molecular mechanism of HCC EMT and the effects of NCTD in the HCC EMT process have been either poorly elucidated or not studied. In this study, HCC EMT was induced by the treatment of IL-6 and various concentrations of NCTD (0, 30, 60 and 120 µM) were treated with HCC cell lines, HCCLM3 and SMMC-7721. We investigated the effect of NCTD on the invasion of HCC cells by using Transwell assay. Immunofluorescence staining, western blot analysis and quantitative RT-PCR were performed to evaluate the protein and mRNA expression levels of HCC cells. Here, using cell line models, our data demonstrated that interleukin 6 (IL-6) induced EMT through the JAK/STAT3/TWIST pathway in HCC. Moreover, our studies revealed that NCTD markedly inhibited IL-6-induced EMT and cell invasiveness. Signaling studies revealed that NCTD sufficiently suppressed JAK/STAT3/TWIST signaling to reverse the IL-6-promoting effects. Collectively, these data provide evidence for the use of NCTD as a potential anticancer drug in HCC metastatic patients.

Chinese patent medicine Fei-Liu-Ping ointment as an adjunctive treatment for non-small cell lung cancer: protocol for a systematic review.

INTRODUCTION: Fei-Liu-Ping ointment has been widely applied as adjunctive drug in the treatment of non-small cell lung cancer (NSCLC). However, there has been no systematic review of research findings regarding the efficacy of this treatment. Here, we provide a protocol for assessing the effectiveness and safety of Fei-Liu-Ping ointment in the treatment of NSCLC. METHODS AND ANALYSIS: The electronic databases to be searched will include MEDLINE (PubMed), Cochrane Central Register of Controlled Trials (CENTRAL) in the Cochrane Library, Excerpt Medica Database (EMBASE), China National Knowledge Infrastructure (CNKI), China Scientific Journal Database (VIP), Wanfang Database and Chinese Biomedical Literature Database (CBM). Papers in English or Chinese published from inception to 2016 will be included without any restrictions. We will conduct a meta-analysis of randomised controlled trial if possible. The therapeutic effects according to the standard for treatment of solid tumours by the WHO and the quality of life as evaluated by Karnofsky score and weight will be applied as the primary outcomes. We will also evaluate the data synthesis and risk of bias using Review Manager 5.3 software. DISSEMINATION: The results of this review will offer implications for the use of Fei-Liu-Ping ointment as an adjunctive treatment for NSCLC. This knowledge will inform recommendations by surgeons and researchers who are interested in the treatment of NSCLC. The results of this systematic review will be disseminated through presentation at a conference and publication of the data in a peer-reviewed journal. TRIAL REGISTRATION NUMBER: PROSPERO CRD42016036911.

Association between Tumor Vasculogenic Mimicry and the Poor Prognosis of Gastric Cancer in China: An Updated Systematic Review and Meta-Analysis.

Background. Vasculogenic mimicry can promote tumor growth and metastasis. This article is aimed at conducting a systematic meta-analysis to explore the clinicopathological and prognostic significance of vasculogenic mimicry and gastric cancer. Methods. We searched Pubmed, EMBASE, Cochrane Library, China National Knowledge Infrastructure, and the VIP and Wanfang Database for eligible studies. We manually searched for printed journals and relevant textbooks. Subgroups analyses were performed based on the region, manuscript quality, methods of vasculogenic mimicry identification, pathology, and number of patients. Results. Nine studies with 997 patients were included in this meta-analysis. A significant association was observed between vasculogenic mimicry-positive patients and those with gastric cancer with poor overall survival (hazard ratio = 2.24, 95% confidence interval: 1.45-3.47), poor pathological grading, high tumor node metastasis clinical stage, lymph node metastasis, deep tumor invasion, and distant metastasis. Conclusions. Vasculogenic mimicry is associated with a poor prognosis in patients with gastric cancer in China. Clinical studies with large samples are needed worldwide and standardized protocols should be adopted in the future to achieve a better understanding of the relationship between gastric cancer and vasculogenic mimicry.

Sinomenine inhibits A549 human lung cancer cell invasion by mediating the STAT3 signaling pathway.

Increasing evidence suggests that the failure of lung cancer treatment may occur as a result of tumor invasion and metastasis. Signal transducer and activator of transcription 3 (STAT3), an epithelial-mesenchymal transition-inducing transcription factor, is a key signaling molecule involved in the proliferation, apoptosis, invasion and metastasis of tumor cells. Sinomenine is an alkaloid compound with an antineoplastic potential against a variety of cancer cells. The aim of the present study was to assess the antitumor mechanisms of sinomenine in the A549 human lung cancer cell line. The results demonstrated that sinomenine manifested dose-dependent cytotoxicity and induced apoptosis in A549 cells. The protein expression of Janus kinase 2, STAT3, phosphorylated-STAT3, Snail, N-cadherin and vimentin decreased in sinomenine-treated cells, while E-cadherin protein expression increased. The regulation of STAT3, N-cadherin and E-cadherin by sinomenine was further confirmed by reverse transcription-quantitative polymerase chain reaction and immunofluorescent staining. It was demonstrated that sinomenine exerts inhibitory effects on A549 human lung cancer cell invasion, possibly through the inhibition of STAT3 signaling. These results provide a novel insight into the role of sinomenine in the treatment of non-small cell lung cancer.

Anti-tumor enhancement of Fei-Liu-Ping ointment in combination with celecoxib via cyclooxygenase-2-mediated lung metastatic inflammatory microenvironment in Lewis lung carcinoma xenograft mouse model.

BACKGROUND: Fei-Liu-Ping (FLP) ointment is an oral prescription medication that has been widely applied to treat lung cancer patients in China. Regulation of the metastatic microenvironment is an important therapeutic approach for prevention and treatment of tumor recurrence and metastasis. The advantage of Traditional Chinese Medicine management of lung cancer lies in the prevention of recurrence and metastasis. Our previous study has demonstrated that FLP ointment could regulate lung inflammatory microenvironment in vitro. However, the effects of FLP on the tumor microenvironment in vivo are still poorly understood. The objective of this study is to investigate the effect of FLP alone or in combination with celecoxib in the prevention of lung cancer progression by Cyclooxygenase (Cox)-2 mediated tumor inflammatory microenvironment in vivo. METHODS: 120 Lewis lung carcinoma xenograft mice were divided equally into four groups: vehicle, FLP, celecoxib, and FLP plus celecoxib. The dynamic growth of the xenografted tumors was observed using an in vivo fluorescence imaging system. Mice were sacrificed on day 14, day 21, and day 28, and tumor specimens and lung tissues were harvested to detect the metastasis-associated protein expression. RESULTS: Tumor inhibition rate was 15.4, 44.2, 47.4 % at day 14, 37.3, 34.7, 61.5 % at day 21, and 15.5, 10.3, 32.5 % at day 28 after treatment of FLP, celecoxib, and FLP plus celecoxib, respectively. Upon treatment of FLP and celecoxib together, lung metastasis rate was 30 % (8 metastatic nodules) lower than other groups. FLP inhibited Cox-2 expression in a time-dependent manner. Moreover, FLP inhibited N-cadherin, matrix metalloproteinases (MMP)-9, and Vimentin expression. Treatment of FLP in combination with celecoxib was more effective than FLP or celecoxib alone in inhibiting vascular endothelial growth factor, platelet-derived growth factor receptors ß, microsomal Prostaglandin E synthase-1, MMP-2, MMP-9, N-cadherin, and Vimentin expression, but increased E-cadherin expression. CONCLUSIONS: FLP inhibited tumor growth and metastasis in a Lewis lung xenograft mice model through the Cox-2 pathway. FLP in combination with celecoxib enhanced the antitumor growth and anti-metastasis effects. Traditional Chinese herbs combined with anti-inflammatory drugs might offer a promising strategy to prevent tumor metastasis.

Topical treatment with Xiaozheng Zhitong Paste alleviates bone cancer pain by inhibiting proteinase-activated receptor 2 signaling pathway.

Herbal analgesic Xiaozheng Zhitong Paste (XZP) and related modifications are often used in traditional Chinese medicine to manage cancer pain. However, its underlying mechanism remains unknown. To investigate the effects and mechanism of XZP on bone cancer pain in a rat model of breast cancer-induced bone pain, a bone cancer pain model was established by inoculating Walker 256 cells into Wistar rats. Bone cancer-bearing rats were topically treated with different doses of XZP or injected with 5 mg/kg of osteoprotegerin (OPG) as positive control. Bone destruction, bone mineral content (BMC) and bone mineral density (BMD) were analyzed by radiology. Paw withdrawal threshold (PWT) and paw withdrawal latency (PWL) were examined to determine pain levels. Trypsin, TNF-α and IL-1ß serum levels were determined using enzyme-linked immunosorbent assay (ELISA). Central sensitization markers such as c-Fos, GFAP, IBA1 and CGRP, as well as proteinase-activated receptor 2 (PAR2) signaling pathway mediators such as PAR2, PKC-γ, PKA and TRPV1, were determined by quantitative RT-PCR and western blotting assay. XZP treatment significantly mitigated bone cancer-related nociceptive behavior, bone damage, BMC and BMD; and decreased radiological scores in rats. XZP treatment significantly inhibited IBA1, GFAP, c-Fos and CGRP expressions in the spinal cord; and significantly mitigated trypsin, TNF-α and IL-1ß serum levels. Furthermore, PAR2, PKC-γ, PKA and TRPV1 relative mRNA levels and protein expression in bone lesions were significantly reduced in rats treated with XZP. XZP significantly alleviates breast cancer-induced bone pain by inhibiting the PAR2 signaling pathway.

The mechanism of µ-opioid receptor (MOR)-TRPV1 crosstalk in TRPV1 activation involves morphine anti-nociception, tolerance and dependence.

Initiated by the activation of various nociceptors, pain is a reaction to specific stimulus modalities. The µ-opioid receptor (MOR) agonists, including morphine, remain the most potent analgesics to treat patients with moderate to severe pain. However, the utility of MOR agonists is limited by the adverse effects associated with the use of these drugs, including analgesic tolerance and physical dependence. A strong connection has been suggested between the expression of the transient receptor potential vanilloid type 1 (TRPV1) ion channel and the development of inflammatory hyperalgesia. TRPV1 is important for thermal nociception induction, and is mainly expressed on sensory neurons. Recent reports suggest that opioid or TRPV1 receptor agonist exposure has contrasting consequences for anti-nociception, tolerance and dependence. Chronic morphine exposure modulates TRPV1 activation and induces the anti-nociception effects of morphine. The regulation of many downstream targets of TRPV1 plays a critical role in this process, including calcitonin gene-related peptide (CGRP) and substance P (SP). Additional factors also include capsaicin treatment blocking the anti-nociception effects of morphine in rats, as well as opioid modulation of TRPV1 responses through the cAMP-dependent PKA pathway and MAPK signaling pathways. Here, we review new insights concerning the mechanism underlying MOR-TRPV1 crosstalk and signaling pathways and discuss the potential mechanisms of morphine-induced anti-nociception, tolerance and dependence associated with the TRPV1 signaling pathway and highlight how understanding these mechanisms might help find therapeutic targets for the treatment of morphine induced antinociception, tolerance and dependence.

Review on the Applications and Molecular Mechanisms of Xihuang Pill in Tumor Treatment.

Xihuang pill (XH) is a complementary and alternative medicine that has been used in traditional Chinese medicine (TCM) for the treatment of tumors since the 18th century. XH has clinical effects on non-Hodgkin lymphoma, breast cancer, gastric cancer, liver cancer, and bone metastasis. XH can also inhibit the growth of tumor cells and cancer stem cells, prevent tumor invasion and angiogenesis, and regulate the tumor microenvironment. XH is composed of Ru Xiang (olibanum), Mo Yao (Commiphora myrrha), She Xiang (Moschus), and Niu Huang (Calculus bovis). Some of the compounds found in these ingredients exert multiple antitumor effects and may synergize with the other ingredients. We aimed to summarize the clinical applications and molecular mechanisms of XH and its chemical composition. This review will provide potential new strategies and alternative perspectives for tumor treatments and basic research into complementary and alternative medicine.

Protease-activated receptor 2 antagonist potentiates analgesic effects of systemic morphine in a rat model of bone cancer pain.

BACKGROUND AND OBJECTIVES: Bone cancer pain affects the quality of life of cancer patients. This study was aimed at investigating the analgesic effects of combined therapies with an antagonist of proteinase-activated receptor (PAR) 2 and morphine on pain-related behaviors in a rat model of bone cancer pain. METHODS: Female Wistar rats were inoculated intramedullarily with Walker 256 cells into their tibias. The analgesic effects of intraperitoneal treatment with morphine and/or intrathecal with the PAR2 antagonist, FSLLRY-NH2, on bone cancer pain-related behaviors in rats were examined. RESULTS: Treatment with morphine at 3 or 10 mg/kg significantly improved limb-use and weight-bearing scores and reduced the number of spontaneous flinches in rats. Treatment with FSLLRY-NH2 at 10 mmol/L also significantly improved limb use and weight bearing scores, and reduced the number of spontaneous flinches in rats. Combination a sub-analgesic dose of FSLLRY-NH2 (0.1 mmol/L) and morphine further elevated limb-use and weight-bearing scores and reduced the number of flinches compared with the effects of morphine alone in rats. CONCLUSIONS: Our data indicate that the combination of morphine and FSLLRY-NH2 has potent analgesic effects on bone cancer pain and our findings may aid in design of new strategies for the treatment of bone cancer pain.

Topical Treatment with Xiaozheng Zhitong Paste (XZP) Alleviates Bone Destruction and Bone Cancer Pain in a Rat Model of Prostate Cancer-Induced Bone Pain by Modulating the RANKL/RANK/OPG Signaling.

To explore the effects and mechanisms of Xiaozheng Zhitong Paste (XZP) on bone cancer pain, Wistar rats were inoculated with vehicle or prostate cancer PC-3 into the tibia bone and treated topically with inert paste, XZP at 15.75, 31.5, or 63 g/kg twice per day for 21 days. Their bone structural damage, nociceptive behaviors, bone osteoclast and osteoblast activity, and the levels of OPG, RANL, RNAK, PTHrP, IGF-1, M-CSF, IL-8, and TNF-α were examined. In comparison with that in the placebo group, significantly reduced numbers of invaded cancer cells, decreased levels of bone damage and mechanical threshold and paw withdrawal latency, lower levels of serum TRACP5b, ICTP, PINP, and BAP, and less levels of bone osteoblast and osteoclast activity were detected in the XZP-treated rats (P<0.05). Moreover, significantly increased levels of bone OPG but significantly decreased levels of RANL, RNAK, PTHrP, IGF-1, M-CSF, IL-8, and TNF-α were detected in the XZP-treated rats (P<0.05 for all). Together, XZP treatment significantly mitigated the cancer-induced bone damage and bone osteoclast and osteoblast activity and alleviated prostate cancer-induced bone pain by modulating the RANKL/RANK/OPG pathway and bone cancer-related inflammation in rats.

Engagement of signaling pathways of protease-activated receptor 2 and µ-opioid receptor in bone cancer pain and morphine tolerance.

Pain is one of the most common and distressing symptoms suffered by patients with progression of cancer. Using a rat model of bone cancer, recent findings suggest that proteinase-activated receptor 2 (PAR2) signaling pathways contribute to neuropathic pain and blocking PAR2 amplifies antinociceptive effects of systemic morphine. The purpose of our study was to examine the underlying mechanisms responsible for the role of PAR2 in regulating bone cancer-evoked pain and the tolerance of systemic morphine. Breast sarcocarcinoma Walker 256 cells were implanted into the tibia bone cavity of rats and this evoked significant mechanical and thermal hyperalgesia. Our results showed that the protein expression of PAR2 and its downstream pathways (protein kinases namely, PKCε and PKA) and transient receptor potential vanilloid 1 (TRPV1) were amplified in the dorsal horn of the spinal cord of bone cancer rats compared to control rats. Blocking spinal PAR2 by using FSLLRY-NH2 significantly attenuated the activities of PKCε/PKA signaling pathways and TRPV1 expression as well as mechanical and thermal hyperalgesia. Also, inhibition of PKCε/PKA and TRPV1 significantly diminished the hyperalgesia observed in bone cancer rats. Additionally, blocking PAR2 enhanced the attenuations of PKCε/PKA and cyclic adenosine monophosphate induced by morphine and further extended analgesia of morphine via µ-opioid receptor (MOR). Our data revealed specific signaling pathways, leading to bone cancer pain, including the activation of PAR2, downstream PKCε/PKA, TRPV1 and resultant sensitization of MOR. Targeting one or more of these signaling molecules may present new opportunities for treatment and management of bone cancer pain often observed in clinics.

New insights into protease-activated receptor 4 signaling pathways in the pathogenesis of inflammation and neuropathic pain: a literature review.

Pain is an unpleasant sensory and emotional experience that is commonly associated with actual or potential tissue damage. Despite decades of pain research, many patients continue to suffer from chronic pain that is refractory to current treatments. Accumulating evidence has indicated an important role of protease-activated receptor 4 (PAR4) in the pathogenesis of inflammation and neuropathic pain. Here we reviewed PAR4 expression and activation via intracellular signaling pathways and the role of PAR4 signaling pathways in the development and maintenance of pain. Understanding PAR4 and its corresponding signaling pathways will provide insight to further explore the molecular basis of pain, which will also help to identify new targets for pharmacological intervention for pain relief.

Protease-activated receptor 4: a critical participator in inflammatory response.

Protease-activated receptors (PARs) are G protein-coupled receptors of which four members PAR1, PAR2, PAR3, and PAR4 have been identified, characterized by a typical mechanism of activation involving various related proteases. The amino-terminal sequence of PARs is cleaved by a broad array of proteases, leading to specific proteolytic cleavage which forms endogenous tethered ligands to induce agonist-biased PAR activation. The biological effect of PARs activated by coagulation proteases to regulate hemostasis and thrombosis plays an enormous role in the cardiovascular system, while PAR4 can also be activated by trypsin, cathepsin G, the activated factor X of the coagulation cascade, and trypsin IV. Irrespective of its role in thrombin-induced platelet aggregation, PAR4 activation is believed to be involved in inflammatory lesions, as show by investigations that have unmasked the effects of PAR4 on neutrophil recruitment, the regulation of edema, and plasma extravasation. This review summarizes the roles of PAR4 in coagulation and other extracellular protease pathways, which activate PAR4 to participate in normal regulation and disease.

Increased expression of protease-activated receptor 2 and 4 within dorsal root ganglia in a rat model of bone cancer pain.

In an effort to understand the underlying mechanisms of cancer-induced bone pain, we investigated the presence of two protease-activated receptors, protease-activated receptor 2 (PAR2), and protease-activated receptor 4 (PAR4), in dorsal root ganglia (DRGs) neurons in an animal model of bone cancer pain. Female Wistar rats were randomized into three groups: tumor-bearing animals killed after 14 days (D14) and tumor-bearing animals killed after 21 days (D21) group and a sham operation group. After establishment of the Walker 256 carcinoma bone cancer pain model, behavioral tests were carried out to determine both the spontaneous nocifensive behavior and the paw withdrawal threshold (PWT) of mechanical and thermal hyperalgesia in these rats. Subsequently, real-time RT-PCR, Western bolt, and immunofluorescence were used to determine the messenger RNA (mRNA) and protein expression of PAR2 and PAR4 in the ipsilateral lumbar 4-5 DRG neurons. Rats in the D21 treatment group displayed a significant increase in spontaneous nocifensive behavior scores compared with the sham group as well as a considerably decreased withdrawal threshold in mechanical allodynia and thermal stimulation. Compared to sham group, the relative mRNA and protein expression of PAR2 and PAR4 was significantly upregulated in the D14 group and D21 groups, concurrent with tumor growth and proliferation. In addition, we identified the co-expression of PAR2 and PAR4 in the DRG neurons. The upregulation of mRNA and protein levels as well as the co-localization of PAR2 and PAR4 in DRG neurons suggests their novel involvement in the development and maintenance of bone cancer pain.

New insights of nociceptor sensitization in bone cancer pain.

INTRODUCTION: Numerous studies have shown that an intact CNS is required for the conscious perception of cancer-induced bone pain (CIBP) and that changes in the CNS are clearly evident. Accordingly, the blockage of nociceptive stimulus into the CNS can effectively relieve or markedly attenuate CIBP, revealing the clinical implication of the blockage of ongoing peripheral inputs for the control of CIBP. AREAS COVERED: In this review, the heterogeneity and excitability of nociceptors in bone are covered. Furthermore, their role in initiating and maintaining CIBP is also described. EXPERT OPINION: Developing mechanistic therapies to treat CIBP is a challenge, but they have the potential to fundamentally change our ability to effectively block/relieve CIBP and increase the functional status and quality of life of patients with bone metastasis. Further studies are desperately needed at both the preclinical and clinical levels to determine whether the targets as mentioned in this review are viable and feasible for patient populations.

PAR2-mediated upregulation of BDNF contributes to central sensitization in bone cancer pain.

BACKGROUND: Bone cancer pain is currently a major clinical challenge for the management of cancer patients, and the cellular and molecular mechanisms underlying the spinal sensitization remain unclear. While several studies demonstrated the critical role of proteinase-activated receptor (PAR2) in the pathogenesis of several types of inflammatory or neuropathic pain, the involvement of spinal PAR2 and the pertinent signaling in the central sensitization is not determined yet in the rodent model of bone cancer pain. FINDINGS: Implantation of tumor cells into the tibias induced significant thermal hyperalgesia and mechanical allodynia, and enhanced glutamatergic strength in the ipsilateral dorsal horn. Significantly increased brain-derived neurotrophic factor (BDNF) expression was detected in the dorsal horn, and blockade of spinal BDNF signaling attenuated the enhancement of glutamatergic strength, thermal hyperalgesia and mechanical allodynia in the rats with bone cancer pain. Significantly increased spinal PAR2 expression was also observed, and inhibition of PAR2 signaling ameliorated BDNF upsurge, enhanced glutamatergic strength, and thermal hyperalgesia and mechanical allodynia. Inhibition of NF-κB pathway, the downstream of PAR2 signaling, also significantly decreased the spinal BDNF expression, glutamatergic strength of dorsal horn neurons, and thermal hyperalgesia and mechanical allodynia. CONCLUSION: The present study demonstrated that activation of PAR2 triggered NF-κB signaling and significantly upregulated the BDNF function, which critically contributed to the enhancement of glutamatergic transmission in spinal dorsal horn and thermal and mechanical hypersensitivity in the rats with bone cancer. This indicated that PAR2 - NF-κB signaling might become a novel target for the treatment of pain in patients with bone cancer.

[Prognostic analysis of 42 patients with gastric neuroendocrine carcinoma].

OBJECTIVE: To investigate the clinicopathological characteristics and prognosis of gastric neuroendocrine carcinoma (NEC). METHODS: Clinical data of 42 patients with gastric neuroendocrine carcinoma admitted in the Cancer Hospital of Zhengzhou University between May 2006 and July 2011 were analyzed retrospectively. The prognostic factors were determined by Log-rank test. RESULTS: Gastric NEC was found in 42 (0.83%) of 5046 patients with gastric cancer during the same period, including 37 males and 5 females. The average age of the patients was 63 years old at the diagnosis. Forty patients underwent R0 resection and 2 patients R1 resection. Forty patients received routine adjuvant chemotherapy with fluorouracil plus oxaliplatin. The median follow-up duration was 26.0 months (range 4-70 months). The median survival was 25.0 months, and the overall 1-, 3-, 5-year survival rates were 71.4%, 26.2% and 11.9%, respectively. Univariate analysis revealed that maximum tumor diameter, tumor invasion depth, lymph node metastasis, lymphatic invasion, stage, and curability were associated with survival (all P<0.05). CONCLUSIONS: Gastric NEC is rare. Curative operation is essential for improving the prognosis, while the choice of comprehensive treatment after surgery should be optimized.