Lidocaine induces epithelial­mesenchymal transition and aggravates cancer behaviors in non­small cell lung cancer A549 cells.

The effects of clinically relevant concentrations of lidocaine on epithelial-mesenchymal transition (EMT) and associated lung cancer behaviors have rarely been investigated. The aim of the present study was to assess the impact of lidocaine on EMT and its related phenomena, including chemoresistance. Lung cancer cell lines (A549 and LLC.LG) were incubated with various concentrations of lidocaine, 5-fluorouracil (5-FU) or both to test their effects on cell viability. Subsequently, the effects of lidocaine on various cell behaviors were assessed in vitro and in vivo using Transwell migration, colony-formation and anoikis-resistant cell aggregation assays, and human tumor cell metastasis in a chorioallantoic membrane (CAM) model quantitated by PCR analysis. Prototypical EMT markers and their molecular switch were analyzed using western blotting. In addition, a conditioned metastasis pathway was generated through Ingenuity Pathway Analysis. Based on these measured proteins (slug, vimentin and E-cadherin), the molecules involved and the alteration of genes associated with metastasis were predicted. Of note, clinically relevant concentrations of lidocaine did not affect lung cancer cell viability or alter the effects of 5-FU on cell survival; however, at this dose range, lidocaine attenuated the 5-FU-induced inhibitory effect on cell migration and promoted EMT. The expression levels of vimentin and Slug were upregulated, whereas the expression of E-cadherin was downregulated. EMT-associated anoikis resistance was also induced by lidocaine administration. In addition, portions of the lower CAM with a dense distribution of blood vessels exhibited markedly increased Alu expression 24 h following the inoculation of lidocaine-treated A549 cells on the upper CAM. Thus, at clinically relevant concentrations, lidocaine has the potential to aggravate cancer behaviors in non-small cell lung cancer cells. The phenomena accompanying lidocaine-aggravated migration and metastasis included altered prototypical EMT markers, anoikis-resistant cell aggregation and attenuation of the 5-FU-induced inhibitory effect on cell migration.

Monascin accelerates anoikis in circulating tumor cells and prevents breast cancer metastasis.

Anoikis resistance has been observed in various types of cancers in which anchorage-independent growth is a crucial step for cancer metastasis. Therefore, agents interfering with this specific cancer cell behavior may be integrated into novel antimetastatic strategies. Monascin (MS), a secondary metabolite found in Monascus species, is a known potent chemopreventive compound used for treating metabolic complications; however, the effect of MS on anoikis resistance has not been investigated. In this study, 4T1 breast cells were treated with MS under either suspension or adhesion conditions. The higher cytotoxicity of MS was more potent against suspended cells than against adherent cells. This selective cytotoxicity was due to the induction of anoikis, which was evidenced by changes in cell aggregation, caspase activity, and Annexin V/propidium iodide binding as well as the results of systemic metastasis in an animal model. Furthermore, MS inhibited E-cadherin and ß-catenin expression in the cells; the treated cells formed spherical aggregates, which suggested that anchorage-independent growth was prevented by MS. These results provide new insights into the mechanisms underlying the growth-preventing effect of MS on cancer cells and indicate the potential ability of MS to suppress metastasis.

Bidirect effects from cisplatin combine with rosmarinic acid (RA) or hot water extracts of Glechoma hederacea (HWG) on renal cancer cells.

BACKGROUND: Cisplatin (CDDP) is a chemotherapeutic drug which also causes adverse side effects. Glechoma hederacea is a traditional Chinese herb belonging to the Labiatae family and has many biological activities. Our previous study indicated that rosmarinic acid (RA) was the most abundant phytochemical in G. hederacea. However, the antioxidant or anti-inflammatory effects of the combined treatment of G. hederacea, RA and CDDP on human renal cell carcinoma (RCC) 786-O cells have not been clearly demonstrated. We aimed to investigate the bioefficacy of hot water extracts of G. hederacea (HWG) and RA in inhibiting RCC 786-O cell activity and its synergism with CDDP against metastatic renal cancer cell. METHODS: Bioactivities of the combination treatment of HWG, RA, HWG/CDDP and RA/CDDP were assessed using the MTT assay and transwell migration, and the crude extract/compound efficacy was evaluated using wound healing migration assays, flow cytometry and western blotting. RESULTS: Our study indicates that CDDP inhibits 786-O cell proliferation and migration and HWG and RA protect against these effects. On the other hand, HWG and RA demonstrate a low cytotoxic effect in human renal proximal tubular epithelial cell line -2 (HK-2 cells). Cell cycle analysis found that HWG/CDDP and RA/CDDP combined treatment exerted cytotoxicity by inducing G2/M arrest and apoptosis. RA in combined with CDDP significantly inhibiting the expression of p-FAK (Tyr 925) in RCC 786-O cells in vitro. CONCLUSION: We propose that the inhibition of RA on RCC 786-O cell invasion and migration may partly occur through the downregulation of FAK phosphorylation. The HWG/CDDP and RA/CDDP combined treatments may be effective strategies for intervention of RCC 786-O cell activity.

Infraclavicular brachial plexus block in adults: a comprehensive review based on a unified nomenclature system.

Over the last decade, considerable progress has been made regarding infraclavicular brachial plexus block (ICB) in adults, especially since the introduction of ultrasound guidance. The advancements in ICB have been attributed to the development of various approaches to improve the success rate and reduce complications. This has also necessitated a unified nomenclature system to facilitate comparison among different approaches. This review aimed to propose an anatomical nomenclature system by classifying ICB approaches into proximal and distal ones to aid future research and provide practice advisories according to recent updates. We also comprehensively discuss various aspects of this nomenclature system. Our review suggests that ultrasound-guided ICB should be categorized as an advanced technique that should be performed under supervision and dual guidance. For one-shot block, the conventional distal approach is still preferred but should be modified to follow ergonomic practice, with the arm in the proper position. For continuous ICB, the proximal approach is promising for reducing local anesthetic volume and increasing efficacy. Nevertheless, further studies are warranted in this direction. We provide practice advisories to maximize safety and minimize adverse events, and recommend designing future studies on ICB according to these findings based on the unified nomenclature system.

Prospective comparison of (4S)-4-(3-18F-fluoropropyl)-L-glutamate versus 18F-fluorodeoxyglucose PET/CT for detecting metastases from pancreatic ductal adenocarcinoma: a proof-of-concept study.

PURPOSE: (4S)-4-(3-18F-Fluoropropyl)-L-glutamate (FSPG) positron emission tomography (PET) reflects system xC- transporter (xCT) expression. FSPG PET has been used to detect brain, lung, breast and liver cancer with only modest success. There is no report on the use of FSPG PET in pancreatic ductal adenocarcinoma (PDAC), presumably because of normal xCT expression in the pancreas. Nonetheless, the tissue-specific expression of xCT in the pancreas suggests that FSPG PET may be ideal for identifying metastasized PDAC. METHODS: The performance of FSPG in detecting PDAC metastases was compared with that of 18F-fluorodeoxyglucose (FDG) in small-animal PET studies in seven PDAC tumour-bearing mice and in prospective PET/computed tomography (CT) studies in 23 patients with tissue-confirmed PDAC of stage III or stage IV. All PET/CT results were correlated with the results of histopathology or contrast-enhanced CT (ceCT) performed 3 and 6 months later. RESULTS: In the rodent model, FSPG PET consistently found more PDAC metastases earlier than FDG PET. FSPG PET showed a trend for a higher sensitivity, specificity and diagnostic accuracy than FDG PET in detecting PDAC metastases in a patient-based analysis: 95.0%, 100.0% and 95.7%, and 90.0%, 66.7% and 90.0%, respectively. In a lesion-based analysis, FSPG PET identified significantly more PDAC metastases, especially in the liver, than FDG PET (109 vs. 95; P = 0.0001, 95% CI 4.9-14.6). The tumour-to-background ratios for FSPG and FDG uptake on positive scans were similar (FSPG 4.2 ± 4.3, FDG 3.6 ± 3.0; P = 0.44, 95% CI -1.11 to 0.48), despite a lower tumour maximum standardized uptake value in FSPG-avid lesions (FSPG 4.2 + 2.3, FDG 7.7 + 5.7; P = 0.002, 95% CI 0.70-4.10). Because of the lower physiological activity of FSPG in the liver, FSPG PET images of the liver are more easy to interpret than FDG PET images, and therefore the use of FSPG improves the detection of liver metastasis. CONCLUSION: FSPG PET is superior to FDG PET in detecting metastasized PDAC, especially in the liver.

Arctigenin protects against steatosis in WRL68 hepatocytes through activation of phosphoinositide 3-kinase/protein kinase B and AMP-activated protein kinase pathways.

Arctigenin (ATG), a lignin extracted from Arctium lappa (L.), exerts antioxidant and anti-inflammatory effects. We hypothesized that ATG exerts a protective effect on hepatocytes by preventing nonalcoholic fatty liver disease (NAFLD) progression associated with lipid oxidation-associated lipotoxicity and inflammation. We established an in vitro NAFLD cell model by using normal WRL68 hepatocytes to investigate oleic acid (OA) accumulation and the potential bioactive role of ATG. The results revealed that ATG inhibited OA-induced lipid accumulation, lipid peroxidation, and inflammation in WRL68 hepatocytes, as determined using Oil Red O staining, thiobarbituric acid reactive substance assay, and inflammation antibody array assays. Quantitative RT-PCR analysis demonstrated that ATG significantly mitigated the expression of acetylcoenzyme A carboxylase 1 and sterol regulatory element-binding protein-1 and significantly increased the expression of carnitine palmitoyltransferase 1 and peroxisome proliferator-activated receptor alpha. The 40 targets of the Human Inflammation Antibody Array indicated that ATG significantly inhibited the elevation of the U937 lymphocyte chemoattractant, ICAM-1, IL-1ß, IL-6, IL-6sR, IL-7, and IL-8. ATG could activate the phosphoinositide 3-kinase/protein kinase B (PI3K/AKT) and AMP-activated protein kinase (AMPK) pathways and could increase the phosphorylation levels of Akt and AMPK to mediate cell survival, lipid metabolism, oxidation stress, and inflammation. Thus, we demonstrated that ATG could inhibit NAFLD progression associated with lipid oxidation-associated lipotoxicity and inflammation, and we provided insights into the underlying mechanisms and revealed potential targets to enable a thorough understanding of NAFLD progression.

Novel effect and the mechanistic insights of fruiting body extract of medicinal fungus Antrodia cinnamomea against T47D breast cancer.

INTRODUCTION: Tamoxifen, an anti-oestrogenic drug for estrogen receptor positive (ER+) breast cancer, was observed to stimulate tumor growth or drug resistance in patients. Antrodia cinnamomea (AC), a precious medicinal fungus has been traditionally used as a folk remedy for cancers in Asian countries. The objective of this study was to investigate the bioefficacy and the underlying molecular mechanisms of the AC fruiting bodies extracts (AC-3E) against human ER+ T47D breast cancer cells, and compare the effect with that of tamoxifen. METHODS: Cell proliferation, migration, TUNEL assay, western blotting, time-lapse confocal microscopy analyses, chorioallantoic membrane assay, and a xenograft BALB/c nude mouse system were used in this study. Chemical fingerprinting of AC-3E was established using LC-MS. RESULTS: AC-3E attenuated T47D breast cancer cell activity by deregulating the PI3K/Akt/mTOR signaling pathway and key cell-cycle mediators, and inducing apoptosis. AC-3E also effectively inhibited tube-like structures of endothelial cells, blood vessel branching and microvessel formation ex vivo and in vivo. Significant preventive and therapeutic effects against T47D mammary tumor growth of AC-3E was observed comparable or superior to tamoxifen treatment in xenograft BALB/c nude mice. Dehydroeburicoic acid (2) was characterized as the main chemical constituent in AC-3E against breast cancer. CONCLUSION: This study suggests that AC-3E extracts can be employed as a double-barreled approach to treat human ER+ breast cancer by attacking both cancer cells and tumor-associated blood vessel cells.

IMP-3 promotes migration and invasion of melanoma cells by modulating the expression of HMGA2 and predicts poor prognosis in melanoma.

IGF II mRNA-binding protein 3 (IMP-3) has been reported to be a marker of melanoma progression. However, the mechanisms by which it impacts melanoma are incompletely understood. In this study, we investigate the clinical significance of IMP-3 in melanoma progression and also its underlying mechanisms. We found that IMP-3 expression was much higher in advanced-stage/metastatic melanomas and that it was associated with a poor prognosis (P=0.001). Univariate analysis showed that IMP-3 expression was associated with stage III/IV melanomas (odds ratio=5.40, P=0.031) and the acral lentiginous subtype (odds ratio=3.93, P=0.0034). MeWo cells with overexpression of IMP-3 showed enhanced proliferation and migration and significantly increased tumorigenesis and metastatic ability in nude mice. We further demonstrated that IMP-3 could bind and enhance the stability of the mRNA of high mobility group AT-hook 2 (HMGA2). It was also confirmed that IMP-3 had an important role in melanoma invasion and metastasis through regulating HMGA2 mRNA expression. IMP-3 expression was positively correlated with HMGA2 expression in melanoma cells and also in melanoma tissues. Our results show that IMP-3 expression is a strong prognostic factor for melanoma, especially acral lentiginous melanoma.

Silibinin and Paclitaxel Cotreatment Significantly Suppress the Activity and Lung Metastasis of Triple Negative 4T1 Mammary Tumor Cell in Mice.

The in vitro and in vivo bioactivities of silibinin (SB), paclitaxel (PTX) and SB and PTX in combination (SB+PTX) against murine metastatic mammary 4T1 cancer cell line were investigated. Isobologram and combination index (CI) analyses showed that SB and PTX can function synergistically in the inhibition of 4T1 cell proliferation with a CI value < 1. Both SB and PTX alone or SB+PTX treatment inhibited 4T1 cell migration and motility possibly through downregulation of the serpin protease nexin-1 (PN-1) and N-cadherin expression, inhibition of matrix metalloprotease (MMP)-9 activity, and upregulation of E-cadherin. Flow cytometry and Western blot analyses demonstrated that both drugs deregulated cell-cycle mediators and induced apoptosis in 4T1 cells. A real-time in vivo bioluminescence imaging system to monitor the breast cancer cell metastasis in syngeneic BALB/c mice was established using a stable 4T1(pGL-COX-2/Luc) cell clone carrying a COX-2 promoter driven-luciferase reporter gene. In vivo study using the allograft 4T1(pGL-COX-2/Luc) metastatic mouse model indicated that SB co-treated with PTX can significantly suppress lung metastasis of 4T1 cells likely through inhibiting cell proliferation and angiogenesis. Together, this study demonstrates that SB could act synergistically with PTX in 4T1 cells, providing a therapeutic option for highly metastatic triple negative breast cancer.

Dimerumic acid inhibits SW620 cell invasion by attenuating H2O2-mediated MMP-7 expression via JNK/C-Jun and ERK/C-Fos activation in an AP-1-dependent manner.

Reactive oxygen species (ROS) such as hydrogen peroxide (H2O2) in the tumor microenvironment play important roles in tumor invasion and metastasis. Recently, ROS have been reported to cause a significant increase in the production and expression of matrix metalloproteinase (MMP)-7, which is closely correlated with metastatic colorectal cancer. The present study was undertaken to evaluate the scavenging activity of dimerumic acid (DMA) for H2O2 isolated from Monascus-fermented rice to investigate the inhibitory effects of DMA on the invasive potential of SW620 human colon cancer cells, and to explore the mechanisms underlying both these phenomena. Our results showed that increased MMP-7 expression due to H2O2 exposure was mediated by activation of mitogen-activated protein kinases (MAPKs) such as Jun N-terminal kinase (JNK), extracellular-regulated kinase (ERK), and p38 kinase. DMA pretreatment suppressed activation of H2O2-mediated MAPK pathways and cell invasion. Moreover, H2O2-triggered MMP-7 production was demonstrated via JNK/c-Jun and ERK/c-Fos activation in an activating protein 1 (AP-1)-dependent manner. Taken together, these results suggest that DMA suppresses H2O2-induced cell invasion by inhibiting AP-1-mediated MMP-7 gene transcription via the JNK/c-Jun and ERK/c-Fos signaling pathways in SW620 human colon cancer cells. Our data suggest that DMA may be useful in minimizing the development of colorectal metastasis. In the future, DMA supplementation may be a beneficial antioxidant to enhance surgical outcomes.

Red Mold Rice Mitigates Oral Carcinogenesis in 7,12-Dimethyl-1,2-Benz[a]anthracene-Induced Oral Carcinogenesis in Hamster.

The prevalence of oral tumor has exponentially increased in recent years; however, the effective therapies or prevention strategies are not sufficient. Red mold rice is a traditional Chinese food, and several reports have demonstrated that red mold rice had an anti-tumor effect. However, the possible anti-tumor mechanisms of the red mold rice are unclear. In this study, we examined the anti-tumor effect of red mold rice on 7,12-dimethyl-1,2-benz[a]anthracene (DMBA)-induced oral tumor in hamster. The ethanol extract of red mold rice (RMRE) treatment significantly decreases the levels of DMBA-induced reactive oxygen species, nitro oxide and prostaglandin E(2) than those of the lovastatin-treated group (P < .001). Moreover, RMRE decreases the formation of oral tumor induced by DMBA. Monacolin K, monascin, ankaflavin or other red mold rice metabolites had been reported to decrease inflammation and oxidative stress and exerted anti-tumor effects. Therefore, we evaluated the anti-inflammation and anti-oxidative stress effects of monacolin K, monascin, ankaflavin and citrinin in lipopolysaccharide-treated RAW264.7 cells. We found that RMRE reduced the LPS-induced nitrite levels in RAW264.7 cells better than monacolin K, monascin, ankaflavin or citrinin (P < .05).

The effect of Monascus secondary polyketide metabolites, monascin and ankaflavin, on adipogenesis and lipolysis activity in 3T3-L1.

The aim of the present work is to investigate the effects of Monascus secondary metabolites, monascin (MS) and ankaflavin (AK), on cell proliferation, adipogenesis, lipolysis and heparin-releasable lipoprotein lipase (HR-LPL) in 3T3-L1 preadipocyte. MS and AK inhibit the proliferation of 3T3-L1 cells in a dose-dependent fashion. At 8 µg/mL concentration MS inhibits proliferation for 80.5% after 48 h, whereas the value for AK is 69.2%. Adipogenesis is inhibited by MS and AK without dose-dependency. Triglyceride is decreased 37.1% and 41.1% respectively by treating 0.125 µg/mL MS and AK. Adipocyte-specific transcription factors peroxisome proliferator-activated receptor γ (PPARγ), CCAAT/enhancer binding protein ß (C/EBPß), C/EBPδ and C/EBPα mRNA levels are measured by real-time polymerase chain reaction. The expression of the four transcriptional factors analyzed (PPARγ, C/EBPß, C/EBPδ and C/EBPα) is reduced at the initial and the middle period. At the later period, there is no effect on the expression of PPARγ and C/EBPα by treating MS and AK. Furthermore, both MS and AK increase basal lipolysis of mature adipocytes by 113.2% and 278.3% upregulation, respectively. And both MS and AK reduce the activity of HR-LPL, by 45.3% and 58.1% reduction, respectively. This study reveals for the first time that Monascus secondary metabolites, MS and AK, can prevent the differentiation of preadipocyte and stimulate basal lipolysis of mature adipocytes, avoiding the accumulation of lipid.

Effects of Monascus-fermented rice extract on malignant cell-associated neovascularization and intravasation determined using the chicken embryo chorioallantoic membrane model.

The traditional Chinese medicine, Hong-Qu, also called red mold rice in the United States and Europe, is used for treating blood stasis, a disorder related to hyperlipidemia and atherosclerosis. In addition to improving metabolic syndrome, extracts from Monascus-fermented rice inhibit the proliferation of various cancer cells in vitro and in vivo. The objective was to examine the effect of red mold rice ethanol extract (RMRE) on angiogenesis, invasion, and metastasis during tumor progression. RMRE significantly inhibited the proliferation of SW480 and SW620 human colorectal carcinoma cells in a dose- and time-dependent manner by using the MTT assay. A capillary-like network morphology was observed after the addition of 20 ng/mL vascular endothelial growth factor or SW620 culture-conditional medium, which was not seen after RMRE treatment. Moreover, spontaneous intravasation into Matrigel grafts of SW620 cells from the upper to the lower layers in the chick embryo chorioallantoic membrane (CAM) model was detected by the polymerase chain reaction (PCR) amplification of human Alu genomic DNA from the lower CAMs in the RMRE-untreated group. Neovascularization increased to 75.3% +/- 11.6% by SW620 cells onplant with Matrigel grafts in the CAM model. However, RMRE significantly reduced CAM neovascularization in a dose-dependent manner. Finally, RMRE effectively decreased the activity of matrix metalloproteinase (MMP)-7 as determined by reverse transcription PCR (RT-PCR), Western blotting, and casein zymography assays. In summary, Monascus-fermented products exert a potent effect on tumor growth and activation, suggesting that they may serve as supplementary agents in adjuvant cancer therapy.

Red mold rice promoted antioxidase activity against oxidative injury and improved the memory ability of zinc-deficient rats.

Zn deficiency is a common disease leading to memory impairment with increasing age. This study evaluated the protection effects of red mold rice (RMR) administration and Zn supplementation against memory and learning ability impairments from oxidative stress caused by Zn deficiency. Rats (4 weeks old) were induced to be Zn deficiency by a Zn-deficient diet for 12 weeks. After that, rats were administered Zn, 1xRMR, 5xRMR, and various dosages of RMR plus Zn, respectively. Decreases of antioxidant enzyme activities in the hippocampus and cortex were observed, and the levels of Ca, Fe, and Mg were increased in the hippocampus and cortex of Zn-deficient rats, leading to memory and learning ability injury. However, the administration of RMR (1- or 5-fold dosage) and with or without Zn significantly improved the antioxidase and neural activity to maintain cortex and hippocampus functions. This study demonstrates that RMR is a possible functional food for the prevention or cure of neural injury associated with Zn deficiency.

The Monascus metabolite monacolin K reduces tumor progression and metastasis of Lewis lung carcinoma cells.

Monascus -fermented red mold rice extract (RMRE) offers valuable therapeutic benefits and has been extensively used for centuries in East Asia. Monascus secondary polyketide metabolites, including monacolin K (MK) and ankaflavin (AK), have been reported to have antitumor-initiating effects on cancer progression. This paper reports that the oral administration of RMRE dramatically inhibited the metastatic ability of murine Lewis lung carcinoma (LLC) cells in syngeneic C57BL/6 mice caused by the decline of serum vascular endothelial growth factor (VEGF) levels compared with untreated metastatic groups. MK is a key antimetastatic and antiangiogenesis compound in RMRE, as shown by down-regulation of VEGF-stimulated invasive activity in LLC cells by Matrigel-coating transwell and tube-forming assays and reverse transcription-Polymerase Chain Reaction (RT-PCR). Therefore, application of RMRE may serve as a nontoxic natural chemopreventive or antineoplastic agent in the development of cancer adjuvant chemotherapy.