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Hypoxia, the most commonly observed characteristic in cancers, is implicated in the establishment of an immunosuppressive niche. Recent studies have indicated that extracellular vesicle (EV)-mediated cancer-stroma interactions are considered to play a critical role in the regulation of various cellular biological functions, with phenotypic consequences in recipient cells. However, the mechanisms underlying the relationship between EVs and hypoxia during cancer progression remain largely unknown. In this study, we found that EVs derived from hypoxic lung cancers increased M2-type polarization by miR-103a transfer. Decreased PTEN levels caused by hypoxic cancer-cell-derived EV miR-103a increased activation of AKT and STAT3 as well as expression of several immunosuppressive and pro-angiogeneic factors. In contrast, inhibition of miR-103a by an miRNA inhibitor effectively decreased hypoxic cancer-mediated M2-type polarization, improving the cytokine prolife of tumor infiltration macrophages. Macrophages received cancer-cell-derived EV miR-103a feedback to further enhance cancer progression and tumor angiogenesis. Finally, circulating EV miR-103a levels were higher in patients with lung cancer and closely associated with the M2 polarization. In conclusion, our results delineate a novel mechanism by which lung cancer cells induce immunosuppressive and pro-tumoral macrophages through EVs and inspire further research into the clinical application of EV inhibition or PTEN restoration for immunotherapy., Graphical Abstract, Extracellular vesicle (EV) miR-103 can be transferred from hypoxic cancer cells to macrophages, resulting in the enhancement of M2 polarization by the downregulation of miR-103a’s direct target PTEN. EV miR-103a increases the stimulatory effects of macrophages on cancer progression and angiogenesis.

Background and objectives: Bladder urothelial carcinoma is the most common type of genitourinary cancer. Patients with bladder cancer may have limited treatment efficacy related to drug toxicity, resistance or adverse effects, and novel therapeutic strategies to enhance treatment efficacy or increase sensitivity to drugs are of high clinical importance. Epigallocatechin gallate (EGCG) is a polyphenolic compound found in green tea leaves, and a potential anti-cancer agent in various cancer types through modulating and regulating multiple signaling pathways. The current study aimed to explore the role and novel therapeutic targets of EGCG on bladder urothelial carcinoma. Materials and Methods: The BFTC-905 cells, human urinary bladder transitional cell carcinoma (TCC) cell line, were treated with EGCG or water for 24 hours, and the expression profiles of mRNAs and microRNAs were analyzed using next generation sequencing (NGS). The enriched biological functions were determined using different bioinformatics databases. Results: A total of 108 differentially expressed genes in EGCG-treated bladder TCC cells were identified, which were mainly involved in nicotinamide adenine dinucleotide (NAD) biogenesis, inflammatory response and oxidation-reduction metabolism. Moreover, several microRNA-mRNA interactions that potentially participated in the response of bladder TCC to EGCG treatment, including miR-185-3p- ARRB1 (arrestin beta 1), miR-3116- MGAT5B (alpha-1,6-mannosylglycoprotein 6-beta-N-acetylglucosaminyltransferase B), miR-31-5p-TNS1 (tensin 1), miR-642a-5p-TNS1, miR-1226-3p- DLG2 (discs large homolog 2), miR-484-DLG2, and miR-22-3p- PPM1K (protein phosphatase 1K). Conclusions: The current findings provide insights into novel therapeutic targets and underlying mechanisms of action of EGCG treatment in bladder cancer.

// Wei-An Chang 1, 2 , Jen-Yu Hung 2, 3 , Shu-Fang Jian 1 , Yi-Shiuan Lin 1 , Cheng-Ying Wu 1 , Ya-Ling Hsu 4 , Po-Lin Kuo 1, 4, 5 1 Graduate Institute of Clinical Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan 2 Division of Pulmonary and Critical Care Medicine, Kaohsiung Medical University Hospital, Kaohsiung, Taiwan 3 School of Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan 4 Graduate Institute of Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan 5 Institute of Medical Science and Technology, National Sun Yat-Sen University, Kaohsiung, Taiwan Correspondence to: Po-Lin Kuo, email: kuopolin@seed.net.tw Ya-Ling Hsu, email: hsuyl326@gmail.com Keywords: laricitrin, dendritic cell, lung cancer, IL-10, STAT3 Received: August 20, 2016 Accepted: October 24, 2016 Published: November 09, 2016 ABSTRACT Natural polyphenolic compounds of grapes and their seeds are thought to be therapeutic adjuvants in a variety of diseases, including cancer prevention. This study was carried out to investigate the effect of grape phenolic compounds on the regulation of cancer-mediated immune suppression. Laricitrin exhibits the greatest potential to ameliorate the suppressive effects of lung cancer on dendritic cells’ (DCs’) differentiation, maturation and function. Human lung cancer A549 and CL1-5 cells change the phenotype of DCs that express to high levels of IL-10 and prime T cells towards an immune suppression type-2 response (Th2). Laricitrin treatment stimulated DC differentiation and maturation in the condition media of cancer cells, a finding supported by monocyte marker CD14’s disappearance and DC marker CD1a’s upregulation. Laricitrin decreases expression of IL-10 in cancer-conditioned DCs, and subsequently switches CD4 + T cell response from Th2 to Th1 in vitro and in vivo . Reversal of laricitrin on lung cancer-induced DCs’ paralysis was via inhibiting the phosphorylation of signal transducer and activator of transcription 3 (STAT3). Laricitrin also potentiated the anticancer activity of cisplatin in mouse models. Thus, laricitrin could be an efficacious immunoadjuvant and have a synergistic effect when combined with chemotherapy.

// Leong-Perng Chan 1, 2 , Ling-Feng Wang 2 , Feng-Yu Chiang 2 , Ka-Wo Lee 2 , Po-Lin Kuo 1, 3 , Chia-Hua Liang 4 1 Graduate Institute of Clinical Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan 2 Department of Otolaryngology-Head and Neck Surgery, Kaohsiung Medical University Hospital, Kaohsiung Medical University, Kaohsiung, Taiwan 3 Institute of Medical Science and Technology, National Sun Yat-Sen University, Kaohsiung, Taiwan 4 Department of Cosmetic Science and Institute of Cosmetic Science, Chia Nan University of Pharmacy and Science, Tainan, Taiwan Correspondence to: Po-Lin Kuo, email: kuopolin@seed.net.tw Chia-Hua Liang, email: tinna_ling@mail.cnu.edu.tw Keywords: HNSCC, IL-8, NOD1, cancer progression Received: May 15, 2016 Accepted: August 13, 2016 Published: August 20, 2016 ABSTRACT NOD1 (nucleotide-binding oligomerization domain 1) is overexpressed in head and neck squamous cell carcinoma (HNSCC) cells, as is IL-8 in cancer cells. However, the mechanism of the IL-8-mediated overexpression of NOD in HNSCC not been identified. This study determines whether IL-8 promotes tumor progression via the NOD signaling pathway in HNSCC. Higher IL-8, NOD1 and receptor-interacting protein kinase (RIP2) expressions were observed in HNSCC tissue than in non-cancerous matched tissue (NCMT), whereas NOD2 was weakly expressed. Furthermore, IL-8 stimulated the proliferation of HNSCC cells (SCC4, SCC9 and SCC25) but not dysplastic oral mucosa DOK cells. Exposure to IL-8 increased the clonogenicity of HNSCC cells. IL-8 siRNA inhibited cell proliferation and cell colony formation, suggesting that IL-8 is involved in HNSCC cancer progression. The expressions of CXCR1 and CXCR2 were higher in HNSCC tissue than in NCMT. HNSCC cells that were exposed to IL-8 exhibited higher expression of CXCR1/2 than did controls. The blocking of IL-8 by siRNA reduced CXCR1/2 expression in HNSCC cells, suggesting that the cancer progression of HNSCC cells that is induced by IL-8 depends on CXCR1/2. Additionally, IL-8 is associated with increased NOD1 and RIP2 expression and reduced NOD2 expression in three types of HNSCC cells. The blocking of IL-8 by siRNA reduces IL-8, NOD1 and RIP2 expressions in HNSCC cells, but not the level of NOD2. These results suggest that IL-8 has an important role in HNSCC progression via a CXCR1/2-meidated NOD1/RIP2 signaling pathway.

Metastatic tumors have been shown to establish a supportive pre-metastatic niche (PMN) in distant organs, which in turn determines disseminated tumor cells' targeting of such organs. PMN is formed through the recruitment of bone-marrow-derived cells (BMDCs); however, the role of BMDCs in PMN formation is not fully understood. On the basis of RNA-seq data and bioinformatic analysis, secretion of extracellular vesicle (EV) miR-92a by BMDCs of lung cancer-bearing mice contributes to the establishment of liver PMN. Both BMDC-derived EVs and miR-92a mimics potentiate the activation of hepatic stellate cells (HSCs), subsequently increasing extracellular matrix (ECM) deposition in mice. Consequently, remodeling of the liver microenvironment enhanced immunosuppressive cell accumulation and cancer cell attachment. EVs miR-92a directly suppressed its target SMAD7, leading to the enhancement of transforming growth factor-β signaling in HSC. Elevated levels of circulating miR-92a are found in the sera of lung cancer patients, and EVs isolated from these patients have a similar ability to increase HSCs activation and ECM protein expression. Our study reveals the sequential steps of liver PMN formation in lung cancer, providing critical mediators that prepare PMN in the liver, and identifies new targets that offer valuable options for diagnosis and therapeutic intervention.

Fatty acid metabolism is important in the regulation of breast cancer progression. Some of the proteins involved in fatty acid transport have been demonstrated to promote the proliferation, migration, and invasion in breast cancer cells. Solute carrier family 27 member 4 (SLC27A4) is a fatty acid transporter protein and is related to very long chain acyl-CoA synthetase activity. In the present study, bioinformatic analysis revealed that relatively high SLC27A4 expression was observed in all subtypes of breast tumor tissues when compared to normal breast tissues. Silencing SLC27A4 expression significantly reduced uptake of free fatty acids in two breast cancer cell lines, Hs578T and MDA-MB-231. Cell growth inhibition was observed in SLC27A4-silenced Hs578T and cell cycle was arrested at G2/M. In addition, the capacity of migration and invasion decreased in both cell lines after knockdown of SLC27A4. The epithelial&ndash, mesenchymal transition signaling pathway was inhibited because protein expression of Slug, vimentin, &alpha, smooth muscle actin, and other regulators was lower than that in control cells. Taken together, our results confirm that high SLC27A4 is associated with tumor progression in breast cancer cells. It is worth investigating whether SLC27A4 serves a diagnostic marker and therapeutic target in further studies.

Long-chain fatty acids are the most abundant fatty acids and are essential for various physiological processes. Translocation of long-chain fatty acids across cell membrane is dependent on transport proteins. Solute carrier family 27 member 6 (SLC27A6) is a transport protein which mediates long-chain fatty acid uptake. The bioinformatic analysis revealed that the expression of SLC27A6 in non-tumoral breast tissue was higher than that in tumoral breast cancer in clinic samples. When SLC27A6 expression in non-tumorigenic cell H184B5F5/M10 was repressed, the fatty acids uptake capacity and cell proliferation was inhibited, and cell cycle was delayed. The protein expression of cell cycle regulators including cell division protein kinase 4 (CDK4), CDK6, and cyclin D1 was significantly decreased in SLC27A6-silenced H184B5F5/M10. By contrast, relatively low SLC27A6 expression in tumorigenic breast cancer cell Hs578T when compared to H184B5F5/M10. Repressing SLC27A6 expression did not affect these phenotypes in Hs578T. The interaction network of SLC27A6 was further investigated via STRING database. The function of these SLC27A6-associated proteins mainly involved in lipid biosynthesis, fatty acid metabolic process, and fatty acid transport. In conclusion, this study reveals inverse correlation between SLC27A6 expression and tumoral tissues and provides a new insight into SLC27A6-mediated cell growth and cell cycle regulation in non-tumorigenic breast cells.

The endoplasmic reticulum (ER) is an organelle involved in various physiological processes such as lipid metabolism, protein synthesis and folding, and cellular calcium storage. In a physiological tumor microenvironment, hypoxia, nutrient deprivation, and calcium dysregulation cause accumulation of unfolded and misfolded proteins. Such accumulation induces ER stress and unfolded protein responses (UPRs). Increased UPR signaling pathways are associated with multiple types of cancer. The influence of ER stress on acyl‑CoA metabolic enzymes is not well understood. Evaluation of PRECOG and Kaplan‑Meier plotter databases in the present study suggested that high expression of acyl‑CoA thioesterase (ACOT)7, ACOT11, ACOT13, soluble carrier family 27 member A4 (SLC27A4) and SLC27A5 was associated with poor clinical outcomes. In addition, expression levels of ACOT7, ACOT11, SLC27A4 and SLC27A5 were not altered after induction of ER stress. By contrast, expression of some enzymes was decreased, such as those of long‑chain acyl‑CoA synthetase (ACSL)3, ACSL4 and SLC27A2. Fatty acid uptake capacity was suppressed in lung cancer cell lines A549 and CL1‑0 after thapsigargin treatment but intracellular reactive oxygen species levels were not suppressed. Gene enrichment and regulatory element analysis were performed; the results provided potential targets for further investigation. On the whole, our findings demonstrate the potential regulatory mechanism of high‑expression of acyl‑CoA metabolic enzymes, the biological effects of decreased enzyme expression levels, possible regulatory elements, and the interaction network involved in responses to ER stress in lung cancer.

Background Metastasis is the major cause of death from breast cancer. Colonization and adaption of metastatic cells in distant organs is a rate-limiting step of the cancer spreading. The underlying mechanisms responsible for the colonization of breast cancer to lung metastatic niches are not fully understood. Methods Specific gene contributions to lung metastasis were identified by comparing gene profiles of 4T1 tumors metastasizing to various organs via microarray. The oncogenic properties CXCL17 were examined by in vivo spontaneous metastasis mouse model. The chemotactic activity of CXCL17 on CD11b+Gr-1+ myeloid-derived suppressor cells (MDSCs) was examined by both in vitro and in vivo models. The therapeutic effects of MDSC depletion and platelet-derived growth factor-BB (PDGF-BB) inhibition were examined by orthotic models. Results Here, we demonstrate that breast cancer cells secrete CXCL17, which increases the accumulation of CD11b+Gr-1+ MDSCs in the lungs. Metastatic lung-infiltrating CD11b+Gr-1+ MDSCs induce angiogenesis in the lungs and facilitate cancer extravasation and survival that ultimately promote lung metastases. CXCL17 increases CD11b+Gr-1+ MDSCs to express PDGF-BB, which not only contributes to CD11b+Gr-1+ MDSC-mediated angiogenesis in the lung metastatic niche, but is also involved in the colonization of breast cancer. Consequently, both CD11b+Gr-1+ MDSC depletion and PDGF receptor inhibitor effectively prevents CXCL17-driven lung metastasis in breast cancer. More importantly, patients with high levels of CXCL17 have shorter distant metastasis-free and overall survival rates, indicators of poor prognosis. Conclusion Our study reveals that MDSCs derived by CXCL17 contribute to the establishment of a lung metastatic niche by PDGF-BB secretion and provide a rationale for development of CXCL17 or PDGF-BB antagonists to inhibit or prevent lung metastasis in cases of breast cancer. Electronic supplementary material The online version of this article (10.1186/s13058-019-1114-3) contains supplementary material, which is available to authorized users.

Clear cell renal cell carcinoma (ccRCC) is the most common type of renal cancer. In the present study, bioinformatics tools were systematically used to investigate the potential upstream effector involved in the progression of ccRCC. Using the Gene Expression Omnibus database and Library of Integrated Network-based Cellular Signatures L1000 platform, it was identified that GA-binding protein subunit β1 (GABPB1) was a potential effector gene. GABPB1 is a transcription factor subunit and its function in ccRCC is unclear. Elevated expression of GABPB1 mRNA in ccRCC was also observed in other clinical datasets from the Oncomine database. Following reverse transcription-quantitative polymerase chain reaction and western blot analysis, the ccRCC 786-O and A498 cell lines showed higher expression levels of GABPB1 than HK-2, a normal kidney cell line. Knockdown of GABPB1 in the 786-O and A498 cells significantly decreased the ability to form colonies by inducing the expression of p21Waf/Cip1. SurvExpress database analysis indicated that a higher expression of GABPB1 was associated with poor survival outcome in patients with renal cancer. These findings imply that GABPB1 serves an important role in the progression of ccRCC.

Phthalates are used as plasticizers in the manufacture of flexible vinyl, which is used in food contact applications. Phthalates have been demonstrated to have an adverse impact on human health, particularly in terms of cancer development. In the present study, we showed for the first time that benzyl butyl phthalate (BBP) potentiates the effect of tumor‑associated dendritic cells (TADCs) on the chemoresistance of breast cancer. Specific knockdown analysis revealed that S100A9 is the major factor responsible for the chemoresistance of doxorubicin/cyclophosphamide induced by BBP-stimulated TADCs in breast cancer. BBP exposure also increased tumor infiltrating myeloid-derived suppressor cell (MDSC) secretion of S100A8/A9, thereby exacerbating the resistance of breast cancer to doxorubicin with cyclophosphamide. In addition, BBP also stimulated the production of CXCL1/GROα by TADCs, which increased the angiogenesis of breast cancer in a mouse model. Inhibition of CXCL1/GROα by a neutralizing antibody, decreased the BBP-induced angiogenesis induced by BBP after chemotherapy in the mouse model. These results, for the first time, provide evidence that BBP influences the efficacy of chemotherapy by remodeling the tumor microenvironment of breast cancer.

S100P, a Ca2+ binding protein, has been shown to be overexpressed in various cancers. However, its functional character in lung cancer remains largely unknown. In this study, we show that S100P increases cancer migration, invasion and metastasis in lung cancer cells. Ectopic expression of S100P increases migration, invasion and EMT in less invasive CL1-0 lung cancer cells. Conversely, knockdown of S100P suppressed migration and invasion, and caused a reversion of EMT in highly invasive lung cancer cells. These effects were transduced by increasing the interaction of S100P with integrin α7, which activated focal adhesion kinase (FAK) and AKT. Blocking FAK significantly decreased S100P-induced migration by decreasing Src and AKT activation, whereas inhibiting AKT reduced S100P upregulation on ZEB1 expression. Further study has indicated that S100P knockdown prevents the spread of highly metastatic human lung cancer in animal models. This study therefore suggests that S100P represents a critical activator of lung cancer metastasis. Detection and targeted treatment of S100P-expressing cancer is an attractive therapeutic strategy in treating lung cancer.

Bone metastasis in lung cancer results in an unfavorable outcome for patients by not only impairing the quality of life, yet also increasing the cancer-related death rates. In the present study, we discuss a novel treatment strategy that may benefit these patients. Human CD14+ monocytes treated with macrophage-colony stimulating factor (M-CSF)/receptor activator of nuclear factor κB ligand (RANKL) differentiated into osteoclasts, whereas syringetin (SGN), a flavonoid derivative found in both grapes and wine, suppressed the osteoclastogenesis in vitro in a dose-dependent manner. In addition, SGN inhibited osteoclast formation induced by human lung adenocarcinoma A549 and CL1-5 cells. The associated signaling transduction pathway in osteoclastogenesis and SGN inhibition was found to be via the AKT/mammalian target of rapamycin (mTOR) signaling pathway. Blocking AKT and mTOR by respective inhibitors significantly decreased lung adenocarcinoma-mediated osteoclastogenesis. Moreover, SGN regulated the lung adenocarcinoma-mediated interaction between osteoblasts and osteoclasts by suppressing the stimulatory effect of lung adenocarcinoma on M-CSF and RANKL production in osteoblasts, and reversing the inhibitory effect of the lung adenocarcinoma on OPG production in osteoblasts. The present study has two novel findings. It is the first to illustrate lung adenocarcinoma-mediated interaction between osteoblasts and osteoclasts, leading to osteolytic bone metastasis. It also reveals that SGN, a flavonoid derivative, directly inhibits osteoclastogenesis and reverses lung adenocarcinoma-mediated osteoclastogenesis. In conclusion, the present study suggests that SGN, a natural compound, prevents and treats bone metastasis in patients with lung cancer.

In the tumor microenvironment, soluble molecules play important role in the establishment of a pre-metastatic niche. The S100 calcium-binding protein family are inflammatory molecules that contribute to the development of a pro-inflammatory tumor microenvironment. S100B belongs to the S100 family and serum S100B (also known as S100beta) serves as a marker for metastasis in lung cancer, ovarian cancer and melanoma. However, the association between S100B and the metastasis of breast cancer is not yet well understood. In the present study, a relatively low S100B expression was observed in the tumor samples compared to normal breast tissue among online microarray datasets. When the estrogen receptor (ER)-negative breast cancer cell lines, MDA-MB-231 and Hs578T, were treated with recombinant human S100B, cell migration was significantly inhibited and epithelial cadherin expression was increased. Our results revealed that a high S100B expression predicted a good overall survival in patients with ER-negative breast cancer, and good distant metastases-free survival in all patients with breast cancer via the analysis of the KM plotter and SurvExpress databases. Although previous studies have indicated that the interaction of S100B with wild-type p53 inhibits p53 function, a high S100B expression is associated with a good prognosis in patients with p53 mutant and p53 wild-type breast cancers. On the whole, our findings demonstrate that S100B treatment suppresses the migratory capacity of ER-negative breast cancer and that S100B expression may serve a predictive marker for metastasis in breast cancer.

Several of the soluble inflammatory molecules such as cytokines and chemokines are involved in the regulation of cancer behaviors. Tumor necrosis factor (TNF)‑like weak inducer of apoptosis (TWEAK) is a member of the TNF superfamily and is a ligand of fibroblast growth factor inducible 14 (Fn14). TWEAK/Fn14 signaling pathways promote tumor progression in several types of human cancer. In the present study, we investigated the role of TWEAK through bioinformatic assay, in vitro experiments, and serum levels in patients with non‑small cell lung cancer (NSCLC). Our results indicated that TWEAK expression in normal tissues was higher than that in lung cancer tissues. In contrast, relatively higher Fn14 expression was detected in lung cancer tissues compared to normal tissues. Recombinant TWEAK treatment did not enhance and inhibit the proliferation and migration of human NSCLC cell lines including A549, H1299, CL1‑0 and CL1‑5. In addition, the serum concentration of TWEAK in normal controls was significantly higher than that in NSCLC patients. However, the TWEAK levels did not show significant difference in regards to TNM stage, cell type and metastasis status in the sera of NSCLC patients. In summary, the present study suggests that a low serum level of TWEAK may be a feature of NSCLC, and the role of TWEAK‑mediated pathways warrant further investigation.

Patients with clear cell renal cell carcinoma (ccRCC) are often diagnosed with both von Hippel-Lindau (VHL) mutations and the constitutive activation of hypoxia-inducible factor-dependent signaling. In this study, we investigated the effects of long-term hypoxia in 786-O, a VHL-defective renal cell carcinoma cell line, to identify potential genes and microRNAs associated with tumor malignancy. The transcriptomic profiles of 786-O under normoxia, short-term hypoxia and long-term hypoxia were analyzed using next-generation sequencing. The results showed that long-term hypoxia promoted the ability of colony formation and transwell migration compared to normoxia. In addition, the differentially expressed genes induced by long-term hypoxia were involved in various biological processes including cell proliferation, the tumor necrosis factor signaling pathway, basal cell carcinoma and cancer pathways. The upregulated (L1CAM and FBN1) and downregulated (AUTS2, MAPT, AGT and USH1C) genes in 786-O under long-term hypoxia were also observed in clinical ccRCC samples along with malignant grade. The expressions of these genes were significantly correlated with survival outcomes in patients with renal cancer. We also found that long-term hypoxia in 786-O resulted in decreased expressions of hsa-mir-100 and hsa-mir-378 and this effect was also observed in samples of metastatic ccRCC compared to samples of non-metastatic ccRCC. These findings may provide a new direction for the study of potential molecular mechanisms associated with the progression of ccRCC.

The bone is the most common metastatic site of breast cancer. Bone metastasis causes pain, pathologic frac- tures, and severely reduces the quality of life. Breast cancer causes osteolytic bone metastasis, which is dependent on osteo - clast-mediated bone resorption. While current treatments rely on palliative anti-resorptive agents, there is a need to develop a drug based on potential alternative therapies. This study is the first to determine that wedelolactone (WDL), a natural coumarin isolated from plants, can inhibit breast cancer- mediated osteoclastogenesis. Osteoclasts were generated from human CD14 + monocytes cultured with M-CSF/RANKL and WDL suppressed human osteoclast differentiation and activity in vitro in a dose-dependent manner. Moreover, WDL inhibited the upregulation of osteoclasts stimulated by MDA-MB-231 breast cancer cells. The activity of WDL on osteoclasts and breast cancer-mediated osteoclastogenesis was associated with the inhibition of Akt/mammalian target of the rapamycin signaling pathway (mTOR). Blocking Akt and mTOR by specific inhibitors significantly decreased osteoclast differenti - ation and bone resorption. Furthermore, WDL regulated breast cancer-enhanced interaction of osteoblasts and osteoclasts by decreasing M-CSF expression in MDA-MB-231-stimulated osteoblasts. Thus, this study suggests that WDL may be a poten - tial natural agent for preventing and treating bone destruction in patients with bone metastasis due to breast cancer.

Background 8-Hydroxyquinoline derivatives have highly sensitive fluorescent chemosensors for metal ions, which are associated with anti-oxidant, anti-tumor and anti-HIV-1 properties. Head and neck squamous cell carcinoma (HNSCC) is associated with a high rate of mortality and novel anti-HNSCC drugs must be developed. Therefore, effective chemotherapy agents are required to address this public health issue. Hypothesis/Purpose The aim of this study was to investigate the inhibitory effect of tris(8-hydroxyquinoline)iron (Feq3) on the HNSCC and the underlying mechanism. Study design/Methods A novel 8-hydroxyquinoline derivative, Feq3, was synthesized. The cell viabilities were analyzed using MTT reagent. Apoptosis and the cell cycle distributions were determined by flow cytometer. Reverse transcription-polymerase chain reaction (RT-PCR), immunofluorescence, western blot, MitoSOX and CellROX stain assay were used to study the mechanism of Feq3. Feq3 combined with antioxidants NAC (N-acetylcysteine) and BSO (buthionine sulfoximine) measured the cell viability and intracellular ROS. Results Feq3 induced the death of HNSCC cells and caused them to exhibit the morphological features of apoptosis. Feq3 also induced apoptosis of SCC9 cells by cell cycle arrest during the G2/M phase and the induced arrest of SCC25 cells in the G0/G1 and G2/M phases, which was associated with decreased cyclin B1/cdc2 and cyclin D/cdk4 expressions. Feq3 increases reactive oxygen species (ROS) and reduces glutathione (GSH) levels, and responds to increased p53 and p21 expressions. Feq3 induced apoptosis by mitochondria-mediated Bax and cytochrome c up-expression and down-expression Bcl-2. Feq3 also up-regulated tBid, which interacts with the mitochondrial pathway and tumor necrosis factor-α (TNF-α)/TNF-Rs, FasL/Fas, and TNF-related apoptosis inducing ligand receptors (TRAIL-Rs)/TRAIL-dependent caspases apoptotic signaling pathway in HNSCC cells. However, Feq3 activates Fas but not FasL in SCC25 cells. Feq3 arrests the growth of HNSCC cells and is involved in the mitochondria- and death receptor (DR)-mediated caspases apoptotic pathway. Conclusion This study is the first to suggest that apoptosis mediates the anti-HNSCC of Feq3. Feq3 has potential as a cancer therapeutic agent against HNSCC.

2-Amino-1-methyl-6-phenylimidazo [4,5-b]pyridine (PhIP) which can be detected in processed meats and red meats, is a potential carcinogen for renal cell carcinoma (RCC). Approximately 30% of patients with metastatic RCC have bone metastases, and the prognosis of RCC with bone metastases is poor. Thus, the aim of the present study was to investigate whether PhIP induced bone metastases and to develop novel therapeutic agents. Our data revealed that PhIP pre-treatment increased the production of parathyroid hormone-related protein (PTHrP) in human 786-O renal cell carcinoma cells. Subsequently, the cultures of human osteoblasts with PhIP-stimulated condition medium of 786-O increased the expression of the macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor kappa-B ligand (RANKL), and decreased the expression of osteoprotegerin (OPG). In addition, PhIP-mediated PTHrP up-regulated as well as increased IL-8 secretion in 786-O cells, and then contributed to 786-O-mediated bone resorption. Furthermore, 6-shogaol, which is an active ingredient in ginger, showed suppressive effects on PhIP-mediated bone resorption. In summary, this is the first study to demonstrate that PhIP pre-treatment increases the stimulatory effect of human renal cell carcinoma 786-O on osteoclastogenesis activity directly by PTHrP. In addition, 6-shogaol treatment reverses PhIP-mediated bone resorption. It suggests that 6-shogaol treatment results in bone resorption activity in the RCC model in vitro.

This study investigates the anticancer effect of arctigenin (ATG), a natural lignan product of Arctium lappa L., in human breast cancer MDA-MB-231 cells. Results indicate that ATG inhibits MDA-MB-231 cell growth by inducing apoptosis in vitro and in vivo. ATG triggers the mitochondrial caspase-independent pathways, as indicated by changes in Bax/Bcl-2 ratio, resulting in AIF and EndoG nuclear translocation. ATG increased cellular reactive oxygen species (ROS) production by increasing p22(phox)/NADPH oxidase 1 interaction and decreasing glutathione level. ATG clearly increases the activation of p38 MAPK, but not JNK and ERK1/2. Antioxidant EUK-8, a synthetic catalytic superoxide and hydrogen peroxide scavenger, significantly decreases ATG-mediated p38 activation and apoptosis. Blocking p38 with a specific inhibitor suppresses ATG-mediated Bcl-2 downregulation and apoptosis. Moreover, ATG activates ATF-2, a transcription factor activated by p38, and then upregulates histone H3K9 trimethylation in the Bcl-2 gene promoter region, resulting in Bcl-2 downregulation. Taken together, the results demonstrate that ATG induces apoptosis of MDA-MB-231 cells via the ROS/p38 MAPK pathway and epigenetic regulation of Bcl-2 by upregulation of histone H3K9 trimethylation.

Background Bone is a common site of metastasis for lung cancer, and is associated with significant morbidity and a dismal prognosis. MicroRNAs (miRNAs) are increasingly implicated in regulating the progression of malignancies. Methods The efficacy of miR-33a or anti-miR-33a plasmid was assessed by Real-time PCR. Luciferase assays were using One-Glo Luciferase Assay System. Measurement of secreted factors was determined by ELISA kit. Results We have found that miR-33a, which is downregulated in lung cancer cells, directly targets PTHrP (parathyroid hormone-related protein), a potent stimulator of osteoclastic bone resorption, leading to decreased osteolytic bone metastasis. We also found that miR-33a levels are inversely correlated with PTHrP expression between human normal bronchial cell line and lung cancer cell lines. The reintroduction of miR-33a reduces the stimulatory effect of A549 on the production of osteoclastogenesis activator RANKL (receptor activator of nuclear factor kappa-B ligand) and M-CSF (macrophage colony-stimulating factor) on osteoblasts, while the expression of PTHrP is decreased in A549 cells. miR-33a overexpression also reduces the inhibitory activity of A549 on the production of OPG (osteoprotegerin), an osteoclastogenesis inhibitor. In addition, miR-33a-mediated PTHrP downregulation results in decreased IL-8 secretion in A549, which contributes to decreased lung cancer-mediated osteoclast differentiation and bone resorption. Conclusions These findings have led us to conclude that miR-33a may be a potent tumor suppressor, which inhibits direct and indirect osteoclastogenesis through repression of PTHrP. General significance miR-33a may even predict a poor prognosis for lung cancer patients.

Gemifloxacin (GMF) is an orally administered broad-spectrum fluoroquinolone antimicrobial agent used to treat acute bacterial exacerbation of pneumonia and bronchitis. Although fluoroquinolone antibiotics have also been found to have anti-inflammatory and anticancer effects, studies on the effect of GMF on treating colon cancer have been relatively rare. To the best of our knowledge, this is the first report to describe the antimetastasis activities of GMF in colon cancer and the possible mechanisms involved. Results have shown that GMF inhibits the migration and invasion of colon cancer SW620 and LoVo cells and causes epithelial mesenchymal transition (EMT). In addition, GMF suppresses the activation of NF-κB and cell migration and invasion induced by TNF-αand inhibits the TAK1/TAB2 interaction, resulting in decreased IκB phosphorylation and NF-κB nuclear translocation in SW620 cells. Furthermore, Snail, a critical transcriptional factor of EMT, was downregulated after GMF treatment. Overexpression of Snail by cDNA transfection significantly decreases the inhibitory effect of GMF on EMT and cell migration and invasion. In conclusion, GMF may be a novel anticancer agent for the treatment of metastasis in colon cancer.

Hypoxia plays a critical role during the evolution of malignant cells and tumour microenvironment (TME).Tumour-derived exosomes contain informative microRNAs involved in the interaction of cancer and stromal cells, thus contributing to tissue remodelling of tumour microenvironment. This study aims to clarify how hypoxia affects tumour angiogenesis through exosomes shed from lung cancer cells. Lung cancer cells produce more exosomes under hypoxic conditions than do parental cells under normoxic conditions. miR-23a was significantly upregulated in exosomes from lung cancer under hypoxic conditions. Exosomal miR-23a directly suppressed its target prolyl hydroxylase 1 and 2 (PHD1 and 2), leading to the accumulation of hypoxia-inducible factor-1 α (HIF-1 α) in endothelial cells. Consequently, hypoxic lung cancer cells enhanced angiogenesis by exosomes derived from hypoxic cancer under both normoxic and hypoxic conditions. In addition, exosomal miR-23a also inhibits tight junction protein ZO-1, thereby increasing vascular permeability and cancer transendothelial migration. Inhibition of miR-23a by inhibitor administration decreased angiogenesis and tumour growth in a mouse model. Furthermore, elevated levels of circulating miR-23a are found in the sera of lung cancer patients, and miR-23a levels are positively correlated with proangiogenic activities. Taken together, our study reveals the clinical relevance and prognostic value of cancer-derived exosomal miR-23a under hypoxic conditions, and investigates a unique intercellular communication, mediated by cancer-derived exosomes, which modulates tumour vasculature.

Asthma and many autoimmune diseases, such as systemic lupus erythematosus, have been reported to associate with vitamin D deficiency recently. Growth-related oncogene-α (GRO-α)/CXCL1, a neutrophil-related chemokine, have an important influence on the chronic inflammation of these diseases. It is unknown whether vitamin D has regulatory effects on GRO-α expression in human monocytes. To this end, the human monocytic leukemia cell line, THP-1, and human primary monocytes were pretreated with 1α, 25-(OH)(2)D(3), and was stimulated with lipopolysaccharide (LPS). Supernatants were collected to determine GRO-α level by ELISA. The intracellular signaling was investigated by nuclear factor (NF)-κB inhibitor, the mitogen-activated protein kinase (MAPK) inhibitors, and Western blot. In our studies, LPS-induced GRO-α was significantly enhanced in THP-1 cells, but suppressed in human primary monocytes by 1α, 25-(OH)(2)D(3). Western blotting revealed that 1α, 25-(OH)(2)D(3) increased LPS-stimulated pp38 expression in THP-1 cells, but suppressed LPS-stimulated pMEK1/2-pERK and pJNK in human primary monocytes. In conclusion, the opposite effects of 1α, 25-(OH)(2)D(3) on GRO-α expression in THP-1 cells and human primary monocytes indicated that the data from THP-1 cells should be further confirmed by human primary monocytes. Moreover, vitamin D3 may have potentiality in treating GRO-α-related chronic inflammatory diseases, like asthma and autoimmune diseases.

The interaction of cancer within a microenvironment is an important factor determining cancer development. This study analyzed the soluble factors secreted by tumor-associated dendritic cells (TADCs), which are responsible for increasing lung cancer growth, migration, invasion, and epithelial-to-mesenchymal transition. Addition of amphiregulin, present in large amounts in TADC-conditioned medium (CM), mimicked the inductive effect of TADC-CM on lung cancer progression, supported by the enhancement of cell proliferation, migration, and invasion as well as osteolytic bone metastases phenotypes. In contrast, neutralization of amphiregulin from TADC-CM decreased the advanced malignancy-inductive properties of TADC-CM. Significant upregulation of amphiregulin has been seen in tumor-infiltrating CD11c+ DCs in human lung cancer samples and patients’ sera. The enhancement of amphiregulin in TADCs has also been noted in mice transplanted with lung cancer cells. Induction of lung cancer progression by TADC-derived amphiregulin is associated with increased STAT3 and AKT activation, which subsequently increases the expression of cyclin D, Twist, and Snail. Blocking AKT significantly decreases TADC-CM and amphiregulin-mediated migration by decreasing the upregulation of Snail, whereas inhibition of STAT3 reduced the modulation of TADC-derived amphiregulin on Twist and cyclin D expression, suggesting that cooperation of STAT3 and AKT plays a critical role in TADC-mediated cancer progression. Moreover, mice treated with anti-amphiregulin Abs showed decreased incidence of cancer development and increased survival rates. Our study suggests that inhibition of amphiregulin or amphiregulin-related signaling is an attractive therapeutic target in lung cancer patients.

This study is the first to investigate the anticancer effects of α-mangostin in human glioblastoma cells. α-Mangostin decreases cell viability by inducing autophagic cell death but not apoptosis. Pretreatment of cells with the autophagy inhibitors 3-methyladenine (3-MA) and bafilomycin or knockdown beclin-1, resulted in the suppression of α-mangostin-mediated cell death. We also found that liver kinase B1 (LKB1)/AMP-activated protein kinase (AMPK) signaling is a critical mediator of α-mangostin-induced inhibition of cell growth. Activation of AMPK induces α-mangostin-mediated phosphorylation of raptor, which subsequently associates with 14-3-3γ and results in the loss of mTORC1 activity. The phosphorylation of both downstream targets of mTORC1, p70 ribosomal protein S6 kinase (p70S6 kinase) and 4E-BP1, is also diminished by activation of AMPK. Furthermore, the inhibition of AMPK expression with shRNAs or an inhibitor of AMPK reduced α-mangostin-induced autophagy and raptor phosphorylation, supporting the theory that activation of AMPK is beneficial to autophagy. A further investigation revealed that α-mangostin also induced autophagic cell death in transplanted glioblastoma in nude mice. Together, these results suggest a critical role for AMPK activation in the α-mangostin-induced autophagy of human glioblastoma cells.

Lung cancer, one of the leading causes of death worldwide, is often associated with a state of immune suppression, but the molecular and functional basis remains enigmatic. Evidence is provided in this paper supporting the role of lung cancer-derived soluble lectin, galectin-1, as a culprit in dendritic cell (DC) anergy. We have shown that galectin-1 is highly expressed in lung cancer cell lines, together with the serum and surgical samples from lung cancer patients. Functionally, lung cancer-derived galectin-1 has been shown to alter the phenotypes of monocyte-derived DCs (MdDCs) and impair alloreactive T cell response, concomitant with the increase of CD4+CD25+FOXP3+ regulatory T cells. The regulatory effect of galectin-1 is mediated, in part, through its ability to induce, in an Id3 (inhibitor of DNA binding 3)-dependent manner, the expression of IL-10 in monocytes and MdDCs. This effect is inhibited by the addition of lactose, which normalizes the phenotypic and functional alterations seen in MdDCs. Of note, significant upregulation of IL-10 was seen in tumor-infiltrating CD11c+ DCs in human lung cancer samples. This was also noted in mice transplanted with lung cancer cells, but not in those receiving tumor cells with galectin-1 knockdown. Furthermore, a significant reduction was noted in lung cancer incidence and in the levels of IL-10–expressing, tumor-infiltrating DCs, in mice receiving galectin-1–silenced tumor cells. These results thus suggest that the galectin-1/IL-10 functional axis may be crucial in lung cancer-mediated immune suppression, and that galectin-1 may serve as a target in the development of lung cancer immunotherapy.

This study is the first to investigate the anticancer effect of tricetin (TCN) in two human liver cancer cell lines, Hep G2 and PLC/PRF/5. TCN induced cancer cell death treatment by triggering mitochondrial and death receptor 5 (DR5) apoptotic pathways. Exposure of Hep G2 and PLC/PRF/5 cells to TCN resulted in cellular glutathione reduction and ROS generation, accompanied by JNK activation and apoptosis. Both of the antioxidants vitamin C and catalase significantly decreased apoptosis by inhibiting the phosphorylation of JNK and subsequently triggering DR5 cell death pathways. The reduction of JNK expression by siRNA decreased TCN-mediated Bim cleavage, DR5 up-regulation, and apoptosis. Furthermore, daily TCN intraperitoneal injections in nude mice with PLC/PRF/5 subcutaneous tumors resulted in an approximately 60% decrease of mean tumor volume, compared with vehicle-treated controls. Taken together, the results of the present study indicate that TCN-induced cell death in liver cancer cells is initiated by ROS generation and that both intrinsic and extrinsic apoptotic pathways contribute to the cell death caused by this highly promising cancer chemopreventive agent.

This study is the first to investigate the anticancer effect of 6-dehydrogingerdione (DGE), an active constituent of dietary ginger, in human breast cancer MDA-MB-231 and MCF-7 cells. DGE exhibited effective cell growth inhibition by inducing cancer cells to undergo G2/M phase arrest and apoptosis. Blockade of cell cycle was associated with increased levels of p21, and reduced amounts of cyclin B1, cyclin A, Cdc2 and Cdc25C. DGE also enhanced the levels of inactivated phosphorylated Cdc2 and Cdc25C. DGE triggered the mitochondrial apoptotic pathway indicated by a change in Bax/Bcl-2 ratios, resulting in caspase-9 activation. We also found the generation of reactive oxygen species is a critical mediator in DGE-induced cell growth inhibition. DGE clearly increased the activation of apoptosis signal-regulating kinase 1 and c-Jun N-terminal kinase (JNK), but not extracellular signal-regulated kinase 1/2 (ERK1/2) and p38. In addition, antioxidants vitamin C and catalase significantly decreased DGE-mediated JNK activation and apoptosis. Moreover, blocking JNK by specific inhibitors suppressed DGE-triggered mitochondrial apoptotic pathway. Taken together, these findings suggest that a critical role for reactive oxygen species and JNK in DGE-mediated apoptosis of human breast cancer.

1. Norsolorinic acid, isolated from Aspergillus nidulans, has been shown to have antiproliferative activity in T24 human bladder cancer cells by arresting the cell cycle at the G(0)/G(1) phase and inducing apoptosis. The aim of the present study was to investigate the antiproliferative activity of norsolorinic acid in T24 human bladder cancer cells. 2. The effects of norsolorinic acid (1, 5, 10 and 20 micromol/L) on the proliferation of T24 cells and on the distribution of cells within different phases of the cell cycle were investigated indirectly using an XTT assay and a flow cytometer, respectively. Factors affecting the cell cycle and apoptosis, including p53, p21, Fas receptor, Fas ligand (FasL) and caspase 8 activity, were examined using ELISA. 3. The results showed that norsolorinic acid inhibited proliferation of T24 cells in a dose-dependent manner, with an IC(50) of 10.5 micromol/L. The effect involved the induction of cell cycle arrest at the G(0)/G(1) phase and apoptosis. 4. These results demonstrate that G(0)/G(1) phase arrest is due to increased expression of p21 in cells treated with norsolorinic acid (10 and 20 micromol/L) for 24 h. Moreover, enhanced Fas and membrane-bound Fas ligand (mFasL) may be responsible for the apoptotic effect of norsolorinic acid. Thus, the present study reports, for the first time, that induction of p21 and the Fas/mFas ligand apoptotic system may participate in the antiproliferative action of norsolorinic acid in T24 human bladder cancer cells.

Background and aim Huang-lian-jie-du-tang (HLJDT; Japanese name, oren-gedoku-to) is a traditional Chinese medicine prescription known to possess anti-inflammatory activity. Our study reports here for the first time the anticancer effect of HLJDT in two human liver cancer cell lines, Hep G2 and PLC/PRF/5. Methods Inhibition of cell proliferation by HLJDT was measured by sodium 3'-(1-(phenylamino-carbonyl)-3,4-tetrazolium)-bis(4-methoxy-6-nitro) benzene-sulfonic acid hydrate (XTT) assay. Clonogenic assay was used to elucidate the possible differences in long-term effects of HLJDT on human liver cancer cells. Cell cycle distribution was determined by flow cytometry. Apoptosis was detected using electrophoresis and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick endlabeling (TUNEL) assay. Protein expressions were determined by immunoblot assay. The activity of nuclear factor-kappa B (NF-kappaB) was determined by Trans-AM ELISA kit. In vivo tumor activity was assessed by xenograft study. Results HLJDT significantly increased the expression of inactivated phospho-Cdc2 and phospho-Cdc25C, and decreased the levels of cyclin A, cyclin B1, Cdc2, and Cdc25C, thereby contributing to cell-cycle arrest. HLJDT increased the expression of Bax and Bak, but decreased the level of Bcl-2 and Bcl-XL, and subsequently triggered the mitochondrial apoptotic pathway. In addition, HLJDT also inhibited cell-survival signaling by enhancing the amount of IkappaBalpha in the cytoplasm, reducing the level and activity of NF-kappaB in the nucleus, and subsequently attenuating the expression of Bcl-XL in Hep G2 and PLC/PRF/5 cells(.) The inhibitory effect mediated by HLJDT on cell growth was also demonstrated in a nude mouse model, in which the liver cancer cells induced tumor xenograft shrank considerably following treatment with HLJDT. Conclusions Taken together, these results suggest a potential anticancer effect of HLJDT against human liver cancer cells.

This study is the first to investigate the anticancer effect of plumbagin in human melanoma A375.S2 cells. Plumbagin exhibited effective cell growth inhibition by inducing cancer cells to undergo S-G2/M phase arrest and apoptosis. Further investigation revealed that plumbagin’s inhibition of cell growth was also evident in a nude mice model. Blockade of cell cycle was associated with increased levels of p21, and reduced amounts of cyclin B1, cyclin A, Cdc2, and Cdc25C. Plumbagin also enhanced the levels of inactivated phosphorylated Cdc2 and Cdc25C. Plumbagin triggered the mitochondrial apoptotic pathway indicated by a change in Bax/Bcl-2 ratios, resulting in caspase-9 activation. We also found the generation of ROS is a critical mediator in plumbagin-induced cell growth inhibition. Plumbagin increased the activation of apoptosis signal-regulating kinase 1, JNK and extracellular signal-regulated kinase 1/2 (ERK1/2), but not p38. In addition, antioxidants vitamin C and catalase significantly decreased plumbagin-mediated c-Jun N-terminal kinase (JNK) activation and apoptosis. Moreover, blocking ERK and JNK by specific inhibitors suppressed plumbagin-triggered mitochondrial apoptotic pathway. Taken together, these results imply a critical role for ROS and JNK in the plumbagin’s anticancer activity.

The inhibition of β-catenin/LEF-1 signaling is an emerging strategy in cancer therapy. However, clinical targeted treatment of the β-catenin/LEF-1 complex remains relatively ineffective. Therefore, development of specific molecular targets is a key approach for identifying new cancer therapeutics. Thus, we attempted to synthesize a peptide (TAT-NLS-BLBD-6) that could interfere with the interaction of β-catenin and LEF-1 at nuclei in human breast cancer cells. TAT-NLS-BLBD-6 directly interacted with β-catenin and inhibited breast cancer cell growth, invasion, migration and colony formation as well as increased arrest of sub-G1 phase and apoptosis; it also suppressed breast tumor growth in nude mouse and zebrafish xenotransplantation models, showed no signs of toxicity and did not affect body weight. Furthermore, the human global gene expression profiles and Ingenuity Pathway Analysis software showed that the TAT-NLS-BLBD-6 downstream target genes were associated with the HER-2 and IL-9 signaling pathways. TAT-NLS-BLBD-6 commonly down-regulated 27 candidate genes in MCF-7 and MDA-MB-231 cells, which are concurrent with Wnt downstream target genes in human breast cancer. Our study suggests that TAT-NLS-BLBD-6 is a promising drug candidate for the development of effective therapeutics specific for Wnt/β-catenin signaling inhibition.

This study is the first to investigate the anticancer effect of isoobtusilactone A (IOA) in two human breast cancer cell lines, MCF-7 and MDA-MB-231. IOA exhibited effective cell growth inhibition by inducing cancer cells to undergo G2-M phase arrest and apoptosis. Further investigation revealed that IOA's inhibition of cell growth was also evident in a nude mice model. Cell cycle blockade was associated with increased levels of p21 and reduced amounts of cyclin B1, cyclin A, cdc2, and cdc25C. IOA also enhanced the levels of inactivated phosphorylated cdc2 and cdc25C. IOA triggered the mitochondrial apoptotic pathway, as indicated by a change in Bax/Bcl-2 ratios, resulting in mitochondrial membrane potential loss, cytochrome c release, and caspase-9 activation. We also found that the generation of reactive oxygen species (ROS) is a critical mediator in IOA-induced cell growth inhibition. Enhancement of ROS by IOA activated apoptosis signal-regulating kinase 1 (ASK1) resulted in the increased activation of c-Jun NH2-terminal kinase and p38. Antioxidants EUK8 and N-acetyl cystenine significantly decreased apoptosis by inhibiting the ASK1 dephosphorylation at Ser967 and subsequently increased the interaction of ASK1 with thioredoxin or 14-3-3 proteins. Moreover, blocking ASK1 by small interfering RNA inhibition completely suppressed IOA-induced apoptosis. Taken together, these results imply a critical role for ROS and ASK1 in IOA's anticancer activity. [Cancer Res 2007;67(15):7406–20]

In this study, we first report the anti-invasive effect of ethylacetate extract from Antrodia cinnamomea (EAC) fruiting bodies in the human liver cancer cell line PLC/PRF/5. Treatment with EAC decreased the cancer invasion of PLC/PRF/5 cells in a dose-dependent manner. This effect was strongly associated with a concomitant decrease in either the level or activity of VEGF, MMP-2, MMP-9 and MT1-MMP, and an increase in the expression of TIMP-1 and TIMP-2. EAC inhibited constitutively activated and inducible NF-kappaB in both its DNA-binding activity and transcriptional activity. Furthermore, EAC also inhibited the TNF-alpha-activated NF-kappaB-dependent reporter gene expression of MMP-9 and VEGF, and the invasion of cancer cells. EAC also exhibited an inhibitory effect on angiogenesis in a Matrigel Plug Angiogenesis Assay. Further investigation revealed that EAC's inhibition of cancer cell growth and invasion was also evident in a nude mice model. Our results indicate that EAC inhibits the activation of NF-kappaB, and may provide a molecular basis for drug development using EAC as an anti-invasive agent in the prevention and treatment of cancer.

Pterocarnin A, isolated from the bark of Pterocarya stenoptera (Juylandaceae), was investigated for its antiproliferative activity in human breast adenocarcinoma MCF-7 cells. To identify the anticancer mechanism of pterocarnin A, we assayed its effects on apoptosis, cell cycle distribution, and levels of p53, p21/WAF1, Fas/APO-1 receptor and Fas ligand. The results showed that pterocarnin A induced apoptosis of MCF-7 cells without mediation of p53 and p21/WAF1. We suggest that the Fas/Fas ligand apoptotic system is the main pathway of pterocarnin A-mediated apoptosis of MCF-7 cells. Our study reports here for the first time that the activity of the Fas/Fas ligand apoptotic system may participate in the antiproliferative activity of pterocarnin A in MCF-7 cells.

Myricetin (3,3',4',5,5',7-hexahydroxyflavone), a flavonoid compound, is present in vegetables and fruits. By means of alkaline phosphatase (ALP) activity, osteocalcin, and type I collagen enzyme-linked immunosorbent assay (ELISA), we have shown that myricetin exhibits a significant induction of differentiation in MG-63 and hFOB human osteoblasts. Alkaline phosphatase and osteocalcin are phenotypic markers for early-stage differentiated osteoblasts and terminally differentiated osteoblasts, respectively. Our results indicate that myricetin stimulates osteoblast differentiation at various stages, from maturation to terminally differentiated osteoblasts. Induction of differentiation by myricetin is associated with increased bone morphogenetic protein-2 (BMP-2) production. The BMP-2 antagonist noggin blocked myricetin-mediated ALP activity and osteocalcin secretion enhancement, indicating that BMP-2 production is required in myricetin-mediated osteoblast maturation and differentiation. Induction of differentiation by myricetin is associated with increased activation of SMAD1/5/8 and p38 mitogen-activated protein kinases. Cotreatment of p38 inhibitor SB203580 inhibited myricetin-mediated ALP upregulation and osteocalcin production. In conclusion, myricetin increased BMP-2 synthesis, and subsequently activated SMAD1/5/8 and p38 MAPK, and this effect may contribute to its action on the induction of osteoblast maturation and differentiation, followed by an increase of bone mass.

Recent studies indicate that a high level of nesfatin-1/Nucleobindin-2 (NUCB-2) is associated with poor outcome and promotes cell migration in breast cancer and prostate cancer. However, the role of NUCB2 is not well known in colon cancer. In this study, NUCB-2 level in colon cancer tissue was higher than that in non-tumor tissue. Suppression of NUCB-2 in a colon cancer cell line SW620 inhibited migration and invasion. The microarray analysis showed that low expression level of transcription factor ZEB1 in NUCB-2 knockdowned SW620 cells. In addition, expression level of epithelial-mesenchymal transition (EMT)-related molecules including N-cadherin, E-cadherin, β-catenin, Slug and Twist was affected by NUCB-2 suppression and ZEB1-denepdent pathway. The signaling pathway liver kinase B1(LKB1)/AMP-dependent protein kinase (AMPK)/target of rapamycin complex (TORC) 1 was involved in regulation of NUCB-2-mediated metastasis and EMT properties. Suppression of NUCB-2 inhibited tumor nodules formation in a murine colon tumor model as well. In summary, nesfatin-1/NUCB-2 enhanced migration, invasion and EMT in colon cancer cells through LKB1/AMPK/TORC1/ZEB1 pathways in vitro and in vivo.

Antrodia cinnamomea is well known in Taiwan as a traditional medicine for treating cancer and inflammation. The purpose of this study was to evaluate the apoptotic effects of ethylacetate extract from A. cinnamomea (EAC) fruiting bodies in Hep 3B, a liver cancer cell line. EAC decreased cell proliferation of Hep 3B cells by inducing apoptotic cell death. EAC treatment increased the level of calcium (Ca2+) in the cytoplasm and triggered the subsequent activation of calpain and caspase-12. EAC also initiated the mitochondrial apoptotic pathway through regulation of Bcl-2 family proteins expression, release of cytochrome c, and activation of caspase-9 in Hep 3B cells. Furthermore, the mitochondrial apoptotic pathway amplified the calpain pathway by Bid and Bax interaction and Ca2+ translocation. We have therefore concluded that the molecular mechanisms during EAC-mediated proliferation inhibition in Hep 3B cells were due to: (1) apoptosis induction, (2) triggering of Ca2+/calpain pathway, (3) disruption of mitochondrial function, and (4) apoptotic signaling being amplified by cross-talk between the calpain/Bid/Bax and Ca2+/mitochondrial apoptotic pathways.

Apigenin, a common dietary flavonoid abundantly present in fruits and vegetables, is believed to possess preventive and therapeutic potential against cancers. In this study, the anti-hepatoma property of apigenin was evaluated on three different human hapatoma cells, namely Hep G2, Hep 3B, and PLC/PRF/5 cells. Results showed that apigenin exhibited a significant growth inhibition against the three selected hepatoma cell lines but not the normal murine liver BNL CL.2 cells. Interestingly, it was shown to possess a similar potency as a commercial anti-hepatoma agent 5-flurouracil (5-FU: positive control) against Hep G2 cells, with IC 50 value of 8.02±1.30 μg/ml. Therefore, we conducted our study further to investigate the cellular mechanism of apigenin effect on Hep G2 cell death. Using DNA ladder and flow cytometric analysis, apigenin was found to induce apoptosis in Hep G2 cells. It also increased the accumulation of p53 and further enhanced the level of p21/WAF1. Together, it was shown that the apoptosis induced by apigenin in Hep G2 cells was possibly mediated through the p53-dependent pathway and the induction of p21 expression, which was probably associated with the cell cycle arrest in G 2 /M phase. The present study concludes that the anti-hepatoma activity of apigenin is as effective as 5-FU and its apoptotic mechanism might be mediated through the p53-dependent pathway and the induction of p21 expression.

Ellipticine, a cytotoxic plant alkaloid, is known to inhibit topoisomerase II. Here we report the mechanism of apoptosis induction and cell cycle arrest by ellipticine in human breast MDA-MB-231 cancer cells. Ellipticine treatment arrested MDA-MB-231 cells at the G 2 /M phase after 6 h of treatment. This effect was strongly associated with a concomitant decrease in the level of cyclin B 1 , Cdc25 and Cdc2, and increase in phospho-Cdc2 (Tyr15). In addition, ellipticine also induced apoptosis in MDA-MB-231 cells, as determined by using both DNA fragmentation and Annexin-V staining assay. Ellipticine increased the expression of Bax, but decreased the level of Bcl-2, Bcl-X L and X-linked inhibitor of apoptosis protein (XIAP), and subsequently triggered the mitochondrial apoptotic pathway (release of cytochrome c, and activation of caspase-9 and -3). In addition, pre-treatment of cells with caspase-9 inhibitor inhibited ellipticine-induced cell proliferation and apoptosis, indicating that caspase-9 activation was involved in MDA-MB-231 cell apoptosis induced by ellipticine. Taken together, our study suggests that the inhibition of cell cycle progression signaling and initiation of the mitochondrial apoptotic system may participate in the anti-proliferative activity of ellipticine in MDA-MB-231 cells.

Isoliquiritigenin (ISL) is a natural pigment with the simple chalcone structure 4,2′,4′-trihydroxychalcone. In this study, we report ISL induced inhibition in the proliferation of human hepatoma cells (Hep G2) for the first time. The cell proliferation inhibition achieved by ISL treatment resulted in a G2/M-phase arrest and programmed cell death. ISL treatment was found to result in the upregulation of p53, p21/WAF1, Fas/APO-1 receptor, Fas ligand, Bax and NOXA, but not in Bad. To elevate the role of p53 in these functions, we generated Hep G2 cells that express the dominant negative p53, which blocks the transcriptional activity of p53. The enhancement of p21/WAF1, Fas/APO-1, Bax and NOXA were decreased in Hep G2 cells that lack functional p53. Furthermore, Hep G2 cells were significantly more resistant to ISL when the activity of p53 was blocked. These results demonstrated that ISL-inducible p53 plays a key apoptotic role, and may do so by regulating the expression of specific target molecules that promotes efficient apoptotic cell death following G2/M-cell cycle arrest.

Luteolin is a common constituent of many kinds of fruits and vegetables. It possesses the anti-neoplastic activities against several human cancers, but its activity against hepatocellular carcinoma (HCC) is seldom mentioned. To evaluate the activity against HCC and to provide information about the mechanism, we tested luteolin against five human hepatoma cell lines, namely HepG2, SK-Hep-1, PLC/PRF/5, Hep3B, and HA22T/VGH, with XTT assay and flow cytometry. The results showed that luteolin inhibited PLC/PRF/5, Hep3B and HA22T/VGH at a concentration of 1 microg/ml, but it needed 5 microg/ml to inhibit HepG2 and 10 microg/ml for SK-Hep1 (P

Isoliquiritigenin (4,2',4'-trihydroxychalcone, ISL) is a natural pigment with a simple chalcone structure. In this study, we report the ISL-induced inhibition on the growth of human hepatoma cells (Hep G2) for the first time. The cell growth inhibition achieved by ISL treatment resulted in programmed cell death in a caspase activation-dependent manner, with an IC50 of 10.51 microg/mL. Outcomes of ISL treatment included the up-regulation of IkappaBalpha expression in the cytoplasm, and the decrease of NF-kappaB level as well as its activity in the nucleus. In addition, ISL also suppressed the expression of Bcl-XL and c-IAP1/2 protein, the downstream target molecule of NF-kappaB. These results demonstrated that ISL treatment inhibited the NF-kappaB cell survival-signaling pathway and induced apoptotic cell death in Hep G2 cells.

This study first investigates the anticancer effect of asiatic acid in two human breast cancer cell lines, MCF-7 and MDA-MB-231. Asiatic acid exhibited effective cell growth inhibition by inducing cancer cells to undergo S-G2/M phase arrest and apoptosis. Blockade of cell cycle was associated with increased p21/WAF1 levels and reduced amounts of cyclinB1, cyclinA, Cdc2, and Cdc25C in a p53-independent manner. Asiatic acid also reduced Cdc2 function by increasing the association of p21/WAF1/Cdc2 complex and the level of inactivated phospho-Cdc2 and phospho-Cdc25C. Asiatic acid treatment triggered the mitochondrial apoptotic pathway indicated by changing Bax/Bcl-2 ratios, cytochrome c release, and caspase-9 activation, but it did not act on Fas/Fas ligand pathways and the activation of caspase-8. We also found that mitogen-activated protein kinases (MAPKs), extracellular signal-regulated kinase (ERK1/2), and p38, but not c-Jun NH2-terminal kinase (JNK), are critical mediators in asiatic acid-induced cell growth inhibition. U0126 [1,4-diamino-2,3-dicyano-1,4-bis(2-aminophenylthio)butadiene] or SB203580 [4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)-1H-imidazole], specific inhibitors of mitogen-activated protein kinase kinase and p38 kinase activities, significantly decreased or delayed apoptosis. Asiatic acid was likely to confine the breast cancer cells in the S-G2/M phase mainly through the p38 pathway, because both SB203580 and p38 small interfering RNA (siRNA) inhibition significantly attenuated the accumulation of inactive phospho-Cdc2 and phospho-Cdc25C proteins and the cell numbers of S-G2/M phase. Moreover, U0126 and ERK siRNA inhibition completely suppressed asiatic acid-induced Bcl-2 phosphorylation and Bax up-regulation, and caspase-9 activation. Together, these results imply a critical role for ERK1/2 and p38 but not JNK, p53, and Fas/Fas ligand in asiatic acid-induced S-G2/M arrest and apoptosis of human breast cancer cells.

Ursolic acid (UA) is a pentacyclic triterpene compound isolated from many types of medicinal plants and is present in human diet. It has been reported to possess a wide range of pharmacological properties, and is one of the most promising chemopreventive agents for cancer. Here, we report that UA inhibits the cell proliferation of human lung cancer cell line A549 and provide a molecular understanding of this effect. The results showed that UA blocked cell cycle progression in the G1 phase that was associated with a marked decrease in the protein expression of cyclin D1, D2, and E and their activating partner cdk2, 4, and 6 with concomitant induction of p21/WAF1. This accumulation of p21/WAF1 might be through a p53-dependent manner. Further, UA treatment also resulted in the triggering of apoptosis as determined by DNA fragmentation assay. This effect was found to correlate with the up-regulation of Fas/APO-1, Fas ligand, and Bax, and down-regulation of NF-kappaB, Bcl-2, and Bcl-XL. Taken together, our study indicated that UA might be a potential chemopreventive agent for lung cancer.

1. Isoliquiritigenin (ISL) is a natural pigment with the simple chalcone structure 4,2',4'-trihydroxychalcone. In the present study, we report, for the first time, ISL-induced inhibition of the proliferation of the human non-small cell lung cancer A549 cell line. 2. The results showed that ISL not only inhibited A549 cell proliferation, but also induced apoptosis and blocked cell cycle progression in the G1 phase. An ELISA assay demonstrated that ISL significantly increased the expression of p53 and p21/WAF1 protein, which caused cell cycle arrest. 3. An enhancement in Fas and its two ligands, namely membrane-bound Fas ligand (mFasL) and soluble Fas ligand (sFasL), may be responsible for the apoptotic effect induced by ISL. 4. Taken together, the results indicate that the p53 and Fas/FasL apoptotic system may participate in the antiproliferative activity of ISL in A549 cells.

This study has two novel findings: it is not only the first to demonstrate that tumor-associated dendritic cells (TADCs) facilitate lung and breast cancer metastasis in vitro and in vivo by secreting inflammatory mediator CC-chemokine ligand 2 (CCL2), but it is also the first to reveal that 6-shogaol can decrease cancer development and progression by inhibiting the production of TADC-derived CCL2. Human lung cancer A549 and breast cancer MDA-MB-231 cells increase TADCs to express high levels of CCL2, which increase cancer stem cell features, migration, and invasion, as well as immunosuppressive tumor-associated macrophage infiltration. 6-Shogaol decreases cancer-induced up-regulation of CCL2 in TADCs, preventing the enhancing effects of TADCs on tumorigenesis and metastatic properties in A549 and MDA-MB-231 cells. A549 and MDA-MB-231 cells enhance CCL2 expression by increasing the phosphorylation of signal transducer and activator of transcription 3 (STAT3), and the activation of STAT3 induced by A549 and MDA-MB-231 is completely inhibited by 6-shogaol. 6-Shogaol also decreases the metastasis of lung and breast cancers in mice. 6-Shogaol exerts significant anticancer effects on lung and breast cells in vitro and in vivo by targeting the CCL2 secreted by TADCs. Thus, 6-shogaol may have the potential of being an efficacious immunotherapeutic agent for cancers.

Developing novel chemo-prevention techniques and advancing treatment are key elements to beating lung cancer, the most common cause of cancer mortality worldwide. Our previous cohort study showed that cysteinyl leukotriene receptor antagonists, mainly montelukast, decreased the lung cancer risk in asthma patients. In the current study, we conducted in vivo and in vitro experiments to demonstrate the inhibiting effect of montelukast on lung cancer and to investigate the underlying mechanisms. Using Lewis lung carcinoma-bearing mice, we showed that feeding montelukast significantly delayed the tumor growth in mice (p < 0.0001). Montelukast inhibited cell proliferation and colony formation and induced the cell death of lung cancer cells. Further investigation showed the down-regulation of B-cell lymphoma 2 (Bcl-2), up-regulation of Bcl-2 homologous antagonist/killer (Bak), and nuclear translocation of apoptosis-inducing factor (AIF) in montelukast-treated lung cancer cells. Montelukast also markedly decreased the phosphorylation of several proteins, such as with no lysine 1 (WNK1), protein kinase B (Akt), extracellular signal-regulated kinase 1/2 (Erk1/2), MAPK/Erk kinase (MEK), and proline-rich Akt substrate of 40-kDa (PRAS40), which might contribute to cell death. In conclusion, montelukast induced lung cancer cell death via the nuclear translocation of AIF. This study confirmed the chemo-preventive effect of montelukast shown in our previous cohort study. The utility of montelukast in cancer prevention and treatment thus deserves further studies.

Lung cancer is the leading cause of cancer death worldwide, with metastasis underlying majority of related deaths. Angiomotin (AMOT), a scaffold protein, has been shown to interact with oncogenic Yes-associated protein/transcriptional co-activator with a PDZ-binding motif (YAP/TAZ) proteins, suggesting a potential role in tumor progression. However, the functional role of AMOT in lung cancer remains unknown. This study aimed to identify the patho-physiological characteristics of AMOT in lung cancer progression. Results revealed that AMOT expression was significantly decreased in clinical lung cancer specimens. Knockdown of AMOT in a low metastatic CL1-0 lung cancer cell line initiated cancer proliferation, migration, invasion and epithelial-mesenchymal transition. The trigger of cancer progression caused by AMOT loss was transduced by decreased cytoplasmic sequestration and increased nuclear translocation of oncogenic co-activators YAP/TAZ, leading to increased expression of the growth factor, Cyr61. Tumor promotion by AMOT knockdown was reversed when YAP/TAZ or Cyr61 was absent. Further, AMOT knockdown increased the growth and spread of Lewis lung carcinoma in vivo. These findings suggest that AMOT is a crucial suppressor of lung cancer metastasis and highlight its critical role as a tumor suppressor and its potential as a prognostic biomarker and therapeutic target for lung cancer.