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Hypoxic preconditioning (HP) is a stem cell preconditioning modality designed to augment the therapeutic effects of mesenchymal stem cells (MSCs). Although autophagy is expected to play a role in HP, very little is known regarding the relationship between HP and autophagy.The adipose-derived stem cell (ASC)-secretome obtained under normoxia (NCM) and ASC-secretome obtained under HP (HCM) were obtained by culturing ASCs for 24 h under normoxic (21% partial pressure of OHP appears to induce ASCs to release a secretome with enhanced anti-apoptotic effects by promoting the autophagic process of ASCs.

Apoptotic cells are rapidly engulfed and removed by phagocytes after displaying cell surface eat-me signals. Among many phospholipids, only phosphatidylserine (PS) is known to act as an eat-me signal on apoptotic cells. Using unbiased proteomics, we identified externalized phosphatidylinositides (PIPs) as apoptotic eat-me signals recognized by CD14+phagocytes. Exofacial PIPs on the surfaces of early and late-apoptotic cells were observed in patches and blebs using anti-PI(3,4,5)P3antibody, AKT- and PLCδ PH-domains, and CD14 protein. Phagocytosis of apoptotic cells was blocked either by masking exofacial PIPs or by CD14 knockout in phagocytes. We further confirmed that exofacial PIP+thymocytes increased dramatically after in vivo irradiation and that exofacial PIP+cells represented more significant populations in tissues ofCd14−/−than WT mice, especially after induction of apoptosis. Our findings reveal exofacial PIPs to be previously unknown cell death signals recognized by CD14+phagocytes.

Tranilast, an anti-allergic drug used in the treatment of bronchial asthma, was identified as an inhibitor of the transcription factor Forkhead box O-1 (FoxO-1) by high throughput chemical library screening in the present study. Based on FoxO-1’s role in apoptotic cell death and differentiation, we examined the effect of tranilast on palmitic acid (PA)-induced cell damage in INS-1 cells. Tranilast substantially inhibited lipoapoptosis and restored glucose-stimulated insulin secretion under high PA exposure. Moreover, PA-mediated downregulation of PDX-1, MafA, and insulin expression was attenuated by tranilast. PA-induced oxidative and ER stress were also reduced in the presence of tranilast. These protective effects were accompanied by increased phosphorylation and decreased nuclear translocation of FoxO-1. Conversely, the effects of tranilast were diminished when treated in transfected cells with FoxO-1 phosphorylation mutant (S256A), suggesting that the tranilast-mediated effects are associated with inactivation of FoxO-1. Examination of the in vivo effects of tranilast using wild type and diabetic db/db mice showed improved glucose tolerance along with FoxO-1 inactivation in the pancreas of the tranilast-treated groups. Thus, we report here that tranilast has protective effects against PA-induced lipotoxic stress in INS-1 cells, at least partly, via FoxO-1 inactivation, which results in improved glucose tolerance in vivo.

To explore extracellular vesicle microRNAs (EV miRNAs) and their target mRNAs in relation to diabetic kidney disease (DKD), we performed paired plasma and urinary EV small RNA sequencing (n = 18) in patients with type 2 diabetes and DKD (n = 5) and healthy subjects (n = 4) and metabolic network analyses using our own miRNA and public mRNA datasets. We found 13 common differentially expressed EV miRNAs in both fluids and 17 target mRNAs, including RRM2, NT5E, and UGDH. Because succinate dehydrogenase B was suggested to interact with proteins encoded by these three genes, we measured urinary succinate and adenosine in a validation study (n = 194). These two urinary metabolite concentrations were associated with DKD progression. In addition, renal expressions of NT5E and UGDH proteins were increased in db/db mice with DKD compared to control mice. In conclusion, we profiled DKD-related EV miRNAs in plasma and urine samples and found their relevant target pathways.

Background Clinically, liver fibrosis and cholestasis are two major disease entities, ultimately leading to hepatic failure. Although autophagy plays a substantial role in the pathogenesis of these diseases, its precise mechanism has not been determined yet. Materials and methods Mouse models of liver fibrosis or cholestasis were obtained after the serial administration of thioacetamide (TAA) or surgical bile duct ligation (BDL), respectively. Then, after obtaining liver specimens at specific time points, we compared the expression of makers related to apoptosis (cleaved caspases), inflammation (CD68), necrosis (high-mobility group box 1), phospho-c-Jun N-terminal kinase (p-JNK), and autophagy (microtubule-associated protein light chain 3B and p62) in the fibrotic or cholestatic mouse livers, by polymerase chain reaction, Western blot analysis, immunohistochemistry, and immunofluorescence. Results Although cholestatic livers exhibited the tendency of progressively increasing the expression of most apoptosis-related markers (cleaved caspases), it was not prominent when it was compared with the tendency found in the livers of TAA-treated mice. Contrastingly, the necrosis-related factor (high-mobility group box 1) was significantly increased in the livers of BDL mice over time, reaching their peak values on day 7 after BDL. In addition, the inflammation-related factor (CD68) was highly expressed in BDL mice compared with TAA-treated mice over time. Autophagy marker studies indicated that autophagy was upregulated in fibrotic livers, whereas it was downregulated in cholestatic livers. We also observed mild to moderate activation of p-JNK in the livers of TAA-treated mice, whereas significantly higher p-JNK activation was detected in the livers of BDL mice. Conclusions Unlike TAA-treated mice, BDL mice exhibited higher expression of the markers related with inflammation and necrosis, especially including p-JNK, while maintaining low levels of autophagic process. Therefore, obstructive cholestasis is characterized by higher p-JNK activation, which could be related with marked necrotic cell death resulting from extensive inflammation and little chance of compensatory autophagy.

Purpose mTORC1 and mTORC2 inhibition by Ku-0063794 could confer profound anticancer effects against cancer cells because it eliminates feedback activation of Akt. Herein, we aimed to determine anticancer effects of docetaxel and Ku-0063794, individually or in combination, against breast cancer cells, especially triple-negative breast cancer (TNBC) cells.Materials and Methods MCF-7 breast cancer and MDA-MB-231 TNBC cell lines for in vitro studies and mouse xenograft model for in vivo studies were used to investigate the effect of docetaxel, Ku-0063794, or their combination.Results In the in vitro experiments, combination therapy synergistically reduced cell viability and induced higher apoptotic cell death in breast cancer cells than the individual monotherapies (p < 0.05). Western blot analysis and flow cytometric analysis showed that the combination therapy induced higher apoptotic cell death than the individual monotherapies (p < 0.05). In the in vivo experiment, docetaxel and Ku-0063794 combination therapy reduced the growth of MDA-MB-231 cells xenografted in the nude mice better than in the individual monotherapies (p < 0.05). Immunohistochemistry showed that the combination therapy induced the highest expression of cleaved caspase-3 and the lowest expression of Bcl-xL in the MDA-MB-231 cells xenografted in the nude mice (p < 0.05). Western blot analysis and immunofluorescence, incorporating both in vitro and in vivo experiments, consistently validated that unlike individual monotherapies, docetaxel and Ku-0063794 combination therapy significantly inhibited epithelial-mesenchymal transition (EMT) and autophagy (p < 0.05).Conclusion These data suggest that docetaxel and Ku-0063794 combination therapy has higher anticancer activities over individual monotherapies against MDA-MB-231 TNBC cells through a greater inhibition of autophagy and EMT.

Here, we provide the possibility of a novel chemotherapeutic agent against gastric cancer cells, comprising the combination of 5-fluorouracil (5-FU) and a mitochondria-targeting self-assembly peptide, which is a phenylalanine dipeptide with triphenyl phosphonium (Mito-FF). The anticancer effects and mechanisms of 5-FU and Mito-FF, individually or in combination, were compared through both in vitro and in vivo models of gastric cancer. Our experiments consistently demonstrated that the 5-FU and Mito-FF combination therapy was superior to monotherapy with either, as manifested by both higher reduction of proliferation as well as an induction of apoptotic cell death. Interestingly, we found that combining 5-FU with Mito-FF leads to a significant increase of reactive oxygen species (ROS) and reduction of antioxidant enzymes in gastric cancer cells. Moreover, the inhibition of ROS abrogated the pro-apoptotic effects of combination therapy, suggesting that enhanced oxidative stress could be the principal mechanism of the action of combination therapy. We conclude that the combination of 5-FU and Mito-FF exerts potent antineoplastic activity against gastric cancer cells, primarily by promoting ROS generation and suppressing the activities of antioxidant enzymes.

Purpose Everolimus only inhibits mammalian target of rapamycin complex 1 (mTORC1), whereas Ku0063794 inhibits both mTORC1 and mTORC2. Although they have similar anticancer effects, their combination has a synergistic effect against hepatocellular carcinoma (HCC) cells. We aimed to determine the mechanism underlying the synergistic effects of everolimus and Ku0063794 associated with autophagy in HCC cells. Materials and methods We compared the effects of everolimus and Ku0063794, individually or in combination, on both the in vitro and in vivo models of HCCs. Results HepG2 cells treated with both agents had significantly lower rates of cell proliferation and higher apoptosis than the individual monotherapies (p l 0.05). Autophagic studies consistently indicated that, unlike the monotherapies, the combination therapy significantly reduced autophagy (p l 0.05). Autophagic blockage directly promoted the pro-apoptotic effects of combination therapy, suggesting autophagy as the survival mechanism of HCC cells. Unlike the monotherapies, combination therapy showed the potential to inhibit sirtuin 1 (SIRT1), the positive regulator of autophagy. SIRT1 overexpression abrogated the autophagy-inhibiting and pro-apoptotic effects of combination therapy. In a nude mouse xenograft model, the shrinkage of tumors was more prominent in mice treated with combination therapy than in mice treated with the respective monotherapies (p l 0.05). The immunohistochemical and immunofluorescence stains of the tumor obtained from the xenograft model showed that combination therapy had the potential of reducing autophagy and promoting apoptosis. Conclusion The combination of everolimus and Ku0063794 potentiates anticancer effects on HCCs through a decrease in autophagy, which is prompted by SIRT1 downregulation.

Accumulation of tissue macrophages is a significant characteristic of disease-associated chronic inflammation, and facilitates the progression of disease pathology. However, the functional roles of these bone marrow-derived macrophages (BMDMs) in aging are unclear. Here, we identified agedependent macrophage accumulation in the bone marrow, showing that aging significantly increases the number of M1 macrophages and impairs polarization of BMDMs. We found that age-related dysregulation of BMDMs is associated with abnormal overexpression of the anti-inflammatory interleukin-10. BMDM dysregulation in aging impairs the expression levels of pro-inflammatory cytokines and genes involved in B-cell maturation and activation. Phagocytosis of apoptotic Jurkat cells by BMDMs was reduced because of low expression of phagocytic receptor CD14, indicating that increased apoptotic cells may result from defective phagocytosis of apoptotic cells in the BM of aged mice. Therefore, CD14 may represent a promising target for preventing BMDM dysregulation, and macrophage accumulation may provide diagnostic and therapeutic clues. [BMB Reports 2017; 50(1): 43-48].

There is lots of evidence to support the critical involvement of mTOR signaling in the carcinogenesis of hepatocellular carcinoma (HCC). However, it has not been determined how the roles of individual mTORC1 and mTORC2 inhibitors played in the HCC therapeutics. We thus compared the effects of everolimus, Ku0063794, and a combination of the two therapies on HCC cells, using various in vitro studies (HepG2, Hep3B, and Huh7 cells), ex vivo culturing of HCC tissues obtained from patients, and the in vivo mouse xenograft model of HCC cells. Our in vitro, ex vivo, and in vivo experiments consistently demonstrated that everolimus and Ku0063794 combination therapy was superior to individual monotherapies, as manifested by higher reduction of proliferation, migration, and invasion of HCC cells, and the higher inhibition of EMT process as well. Although individual monotherapies could not inhibit SIRT1 (positive regulator of EMT) expression, the combination therapy significantly inhibited SIRT1 expression. However, overexpression of SIRT1 mitigated the EMT-inhibiting effect of the combination therapy, suggesting that the combination therapy inhibits the EMT by way of suppressing SIRT1 expression. Therefore, when considering everolimus as an anti-HCC agent, the improved anticancer effects provided by combining it with an inhibitor of both mTORC1 and mTORC2 should be recognized.

The limitations of stem cells have led researchers to investigate the secretome, which is the secretory materials in stem cells, since the principal mechanism of action of stem cells is mediated by the secretome. In this study, we determined the antifibrotic potential of the secretome released from miR-150-transfected adipose-derived stromal cells (ASCs). The secretome released from ASCs that were transfected with antifibrotic miR-150 was obtained (referred to as the miR-150 secretome). To validate the antifibrotic effects of the miR-150 secretome, we generated in vitro and in vivo models of liver fibrosis by treating human hepatic stellate cells (LX2 cells) with thioacetamide (TAA) and subcutaneous injection of TAA into mice, respectively. In the in vitro model, more significant reductions in the expression of fibrosis-related markers, such as TGFβ, Col1A1, and α-SMA, were observed by using the miR-150 secretome than the control secretome, specifically in TAA-treated LX2 cells. In the in vivo model, infusion of the miR-150 secretome into mice with liver fibrosis abrogated the increase in serum levels of systemic inflammatory cytokines, such as IL-6 and TNF-α, and induced increased expression of antifibrotic, proliferation, and antioxidant activity markers in the liver. Our in vitro and in vivo experiments indicate that the miR-150 secretome is superior to the naive secretome in terms of ameliorating liver fibrosis, minimizing systemic inflammatory responses, and promoting antioxidant enzyme expression. Therefore, we conclude that miR-150 transfection into ASCs has the potential to induce the release of secretory materials with enhanced antifibrotic, proliferative, and antioxidant properties., Regenerative medicine: Training stem cells to fix liver fibrosis A mixture of molecules produced by genetically modified stem cells could help repair the damage associated with liver fibrosis. Fat-derived adipose stem cells (ASCs) secrete proteins and nucleic acids that can facilitate tissue regeneration, but the natural mixture of molecules secreted (the ‘secretome’) is insufficient to reverse advanced fibrosis. Researchers led by Say-June Kim of the Catholic University of Korea, Seoul, South Korea, have boosted the potency of this cell-derived treatment by engineering ASCs to produce an RNA called miR-150. This RNA inhibits biological processes that drive fibrosis. Experiments in cultured cells and a mouse model of fibrosis confirmed that miR-150 consistently improved the ASC secretome’s capacity to control liver fibrosis and minimize systemic inflammatory responses. This approach could thus offer a safe strategy for promoting tissue regeneration and preventing liver failure.

Background Although methyl-tertiary butyl ether (MTBE) is the only clinical topical agent for gallstone dissolution, its use is limited by its side effects mostly arising from a relatively low boiling point (55 °C). In this study, we developed the gallstone-dissolving compound containing an aromatic moiety, named 2-methoxy-6-methylpyridine (MMP) with higher boiling point (156 °C), and compared its effectiveness and toxicities with MTBE. Methods The dissolubility of MTBE and MMP in vitro was determined by placing human gallstones in glass containers with either solvent and, then, measuring their dry weights. Their dissolubility in vivo was determined by comparing the weights of solvent-treated gallstones and control (dimethyl sulfoxide)-treated gallstones, after directly injecting each solvent into the gallbladder in hamster models with cholesterol and pigmented gallstones. Results In the in vitro dissolution test, MMP demonstrated statistically higher dissolubility than did MTBE for cholesterol and pigmented gallstones (88.2% vs. 65.7%, 50.8% vs. 29.0%, respectively; P

Background Secretome refers to the total set of molecules secreted or surface-shed by stem cells. The limitations of stem cell research have led numerous investigators to turn their attention to the use of secretome instead of stem cells. In this study, we intended to reinforce antifibrotic properties of the secretome released from adipose-derived stem cells (ASCs) transfected with miR-214. Methods We generated miR-214-transfected ASCs, and extracted the secretome (miR214-secretome) from conditioned media of the transfected ASCs through a series of ultrafiltrations. Subsequently, we intravenously injected the miR-214-secretome into mice with liver fibrosis, and determined the effects of miR-214-secretome on liver fibrosis. Results Compared with that by naïve secretome, liver fibrosis was ameliorated by intravenous infusion of miR-214-secretome into mice with liver fibrosis, which was demonstrated by significantly lower expression of fibrosis-related markers (alpha-smooth muscle actin, transforming growth factor-β, and metalloproteinases-2) in the livers as well as lower fibrotic scores in the special stained livers compared with naïve secretome. The infusion of miR-214-secretome also led to lesser local and systemic inflammation, higher expression of an antioxidant enzyme (superoxide dismutase), and higher liver proliferative and synthetic function. Conclusion MicroRNA-214 transfection stimulates ASCs to release the secretome with higher antifibrotic and anti-inflammatory properties. miR-214-secretome is thus expected to be one of the prominent ways of overcoming liver fibrosis, if further studies consistently validate its safety and efficiency., Graphical Abstract

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

This paper aims to validate if intrapancreatic injection of penicillin G can enhance hardness and suture holding capacity (SHC) of the pancreas through prompting the fibrosis process. Soft pancreatic texture is constantly mentioned as one of the most contributory predictors of postoperative pancreatic fistula (POPF). Soft pancreas has poor SHC and higher incidence of parenchymal tearing, frequently leading to POPF. From a library of 114 antibiotic compounds, we identified that penicillin G substantially enhanced pancreatic hardness and SHC in experimental mice. Specifically, we injected penicillin G directly into the pancreas. On determined dates, we measured the pancreatic hardness and SHC, respectively, and performed molecular and histological examinations for estimation of the degree of fibrosis. The intrapancreatic injection of penicillin G activated human pancreatic stellate cells (HPSCs) to produce various fibrotic materials such as transforming growth factor-&beta, 1 (TGF-&beta, 1) and metalloproteinases-2. The pancreatic hardness and SHC were increased to the maximum at the second day after injection and then it gradually subsided demonstrating its reversibility. Pretreatment of mice with SB431542, an inhibitor of the TGF-&beta, 1 receptor, before injecting penicillin G intrapancreatically, significantly abrogated the increase of both pancreatic hardness and SHC caused by penicillin G. This suggested that penicillin G promotes pancreatic fibrosis through the TGF-&beta, 1 signaling pathway. Intrapancreatic injection of penicillin G promotes pancreatic hardness and SHC by enhancing pancreatic fibrosis. We thus think that penicillin G could be utilized to prevent and minimize POPF, after validating its actual effectiveness and safety by further studies.

Background A hypoxic-preconditioned secretome from stem cells reportedly promotes the functional and regenerative capacity of the liver more effectively than a control secretome. However, the optimum oxygen partial pressure (pO2) in the cell culture system that maximizes the therapeutic potential of the secretome has not yet been determined. Methods We first determined the cellular alterations in adipose tissue-derived stem cells (ASCs) cultured under different pO2 (21%, 10%, 5%, and 1%). Subsequently, partially hepatectomized mice were injected with the secretome of ASCs cultured under different pO2, and then sera and liver specimens were obtained for analyses. Results Of all AML12 cells cultured under different pO2, the AML12 cells cultured under 1% pO2 showed the highest mRNA expression of proliferation-associated markers (IL-6, HGF, and VEGF). In the cell proliferation assay, the AML12 cells cultured with the secretome of 1% pO2 showed the highest cell proliferation, followed by the cells cultured with the secretome of 21%, 10%, and 5% pO2, in that order. When injected into the partially hepatectomized mice, the 1% pO2 secretome most significantly increased the number of Ki67-positive cells, reduced serum levels of proinflammatory mediators (IL-6 and TNF-α), and reduced serum levels of liver transaminases. In addition, analysis of the liver specimens indicated that injection with the 1% pO2 secretome maximized the expression of the intermediate molecules of the PIP3/Akt and IL-6/STAT3 signaling pathways, all of which are known to promote liver regeneration. Conclusions The data of this study suggest that the secretome of ASCs cultured under 1% pO2 has the highest liver reparative and regenerative potential of all the secretomes tested here. Electronic supplementary material The online version of this article (doi:10.1186/s13287-017-0635-x) contains supplementary material, which is available to authorized users.

Varieties of transforming growth factor-β (TGF-β) antagonists have been developed to intervene with excessive TGF-β signalling activity in cancer. Activin receptor-like kinase5 (ALK5) inhibitors antagonize TGF-β signalling by blocking TGF-β receptor-activated Smad (R-Smad) phosphorylation. Here we report the novel mechanisms how ALK5 inhibitors exert a therapeutic effect on a mouse B16 melanoma model. Oral treatment with a novel ALK5 inhibitor, EW-7197 (2.5 mg/kg daily) or a representative ALK5 inhibitor, LY-2157299 (75 mg/kg bid) suppressed the progression of melanoma with enhanced cytotoxic T-lymphocyte (CTL) responses. Notably, ALK5 inhibitors not only blocked R-Smad phosphorylation, but also induced ubiquitin-mediated degradation of the common Smad, Smad4 mainly in CD8(+) T cells in melanoma-bearing mice. Accordingly, T-cell-specific deletion of Smad4 was sufficient to suppress the progression of melanoma. We further identified eomesodermin (Eomes), the T-box transcription factor regulating CTL functions, as a specific target repressed by TGF-β via Smad4 and Smad3 in CD8(+) T cells. Thus, ALK5 inhibition enhances anti-melanoma CTL responses through ubiquitin-mediated degradation of Smad4 in addition to the direct inhibitory effect on R-Smad phosphorylation.

Capsaicin (8-methyl-N-vanillyl-6-nonenamide), a pungent ingredient of hot chili peppers, has been reported to possess substantial anticarcinogenic and antimutagenic activities. In the present study, we investigated the effect of capsaicin on induction of apoptosis in highly metastatic B16-F10 murine melanoma cells. Capsaicin inhibited growth of B16-F10 cells in a concentration-dependent manner. Proapoptotic effect of capsaicin was evidenced by nuclear condensation, internucleosomal DNA fragmentation, in situ terminal nick-end labeling of fragmented DNA (TUNEL), and an increased sub G1 fraction. Treatment of B16-F10 cells with capsaicin caused release of mitochondrial cytochrome c, activation of caspase-3, and cleavage of poly (ADP-ribose) polymerase in a dose-dependent manner. Furthermore, Bcl-2 expression in the B16-F10 cells was slightly down-regulated by capsaicin treatment. In contrast, there were no alterations in the levels of Bax in capsaicin-treated cells. Collectively, these findings indicate that capsaicin-induces apoptosis of B16-F10 melanoma cells via down-regulation the Bcl-2.

As the criteria for liver donation have been extended to include marginal donors, liver grafts are becoming particularly vulnerable to hepatic ischemia-reperfusion injury (IRI). However, no specific measures have been validated to ameliorate hepatic IRI. In this article, we explored whether everolimus has protective effects against hepatic IRI in relation with autophagy. The effects of everolimus were investigated in both in vitro and in vivo hepatic IRI models. Mouse hepatocyte AML12 cells and BALB/c mice were utilized for the establishment of each model. In the IRI-induced AML12 cells, everolimus treatment increased the expressions of autophagic markers (microtubule-associated protein 1 light chain 3 and p62) and decreased pro-apoptotic proteins (cleaved caspase 3 and cleaved poly-ADP ribose polymerase). The blockage of autophagy, using either bafilomycin A1 or si-autophagy-related protein 5, abrogated these anti-apoptosis effects of everolimus. Subsequently, everolimus administration to the hepatic IRI-induced mice provided hepatoprotective effects in terms of (1) decreasing the expressions of pro-apoptotic proteins, (2) inhibiting the release of pro-inflammatory cytokines (IL-6 and tumor necrosis factor-α), (3) reducing elevated liver enzymes (aspartate transaminase, alanine transaminase, and ammonia), and (4) restoring liver histopathology. These findings suggest that everolimus protects the liver against hepatic IRI by way of activating autophagy, and thus could be a potential therapeutic agent for hepatic IRI.

Rosiglitazone is a well-known anti-diabetic drug that increases insulin sensitivity via peroxisome proliferator-activated receptor γ (PPARγ) activation, but unfortunately it causes bone loss in animals and humans. A previous study showed that prolyl hydroxylase domain protein (PHD) plays a role in rosiglitazone-induced adipocyte differentiation. Based on the inverse relationship between adipocyte and osteoblast differentiation, we investigated whether PHD is involved in the effects of rosiglitazone on osteoblast differentiation. Rosiglitazone inhibited osteoblast differentiation in a concentration-dependent manner, and in parallel induced three PHD isoforms (PHD1, 2, and 3). PHD inhibitors and knockdown of each isoform prevented the inhibitory effects of rosiglitazone on osteoblast differentiation and increased the expression of Runx2, a transcription factor essential for osteoblastogenesis. MG-132, a proteasomal inhibitor also prevented the rosiglitazone-induced degradation of Runx2. Furthermore, both increased PHD isoform expressions and reduced osteoblast differentiation by rosiglitazone were prevented by PPARγ antagonists, indicating these effects were mediated via PPARγ activation. In vivo oral administration of rosiglitazone to female ICR mice for 8 weeks reduced bone mineral densities and plasma alkaline phosphatase (ALP) activity, and increased PHD expression in femoral primary bone marrow cells and the ubiquitination of Runx2. Together, this suggests that the rosiglitazone-induced suppression of osteoblast differentiation is at least partly induced via PPARγ-mediated PHD induction and subsequent promotion of the ubiquitination and degradation of Runx2.

To evaluate the safety of adenovirus-mediated gene transfer, we investigated differential protein expression after transducing adenoviral vector containing the p16(INK4a) tumor suppressor gene (Ad5CMV-p16) into Balb/c nude mice. We found that adenovirus-mediated p16(INK4a) gene transfer inhibited experimental lung metastasis, and that the intratumoral injection of Ad5CMV-p16 resulted in regression of A549 cell xenografted tumors in Balb/c nude mice. We investigated changes in protein expression after intratumoral injection of Ad5CMV-p16 or Ad5CMV (10(10) plaque-forming units) into A549 cell xenografted Balb/c nude mice by two-dimensional gel electrophoresis /matrix-assisted laser desorption/ionization-time of flight mass spectrometry. Compared with the control (serum-free medium treated tumor cells) Ad5CMV-p16 gene transfer changed the expression of 29 proteins including heterogeneous nuclear ribonucleoprotein, protein phosphatase 2, 14-3-3 zeta protein, alpha-tubulin, and glutathione-S-transferase P1. Moreover, both Ad5CMV-p16 and Ad5CMV up-regulated the expression of glutathione-S-transferase P1. In addition, Ad5CMV-p16 gene transfer did not seem to increase the expression of tumorigenicity-related protein in Balb/c nude mice. Further studies will be needed to investigate the effect of Ad5CMV-p16 on normal human cells and tissues for safety evaluation. These results suggest that the p16 gene seems to have an important role in apoptosis as well as in cell cycle arrest in non-small cell lung cancer.

Tumor-associated macrophages (TAMs) accumulate in various cancers and promote tumor angiogenesis and metastasis, and thus may be ideal targets for the clinical diagnosis of tumor metastasis with high specificity. However, there are few specific markers to distinguish between TAMs and normal or inflammatory macrophages. Here, we show that TAMs localize in green fluorescent protein-labeled tumors of metastatic lymph nodes (MLNs) from B16F1 melanoma cells but not in necrotic tumor regions, suggesting that TAMs may promote the growth of tumor cells and the progression of tumor metastasis. Furthermore, we isolated pure populations of TAMs from MLNs and characterized their gene expression signatures compared to peritoneal macrophages (PMs), and found that TAMs significantly overexpress immunosuppressive cytokines such as IL-4, IL-10, and TGF-β as well as proangiogenic factors such as VEGF, TIE2, and CD31. Notably, immunological analysis revealed that TIE2(+)/CD31(+) macrophages constitute the predominant population of TAMs that infiltrate MLNs, distinct from tissue or inflammatory macrophages. Importantly, these TIE2(+)/CD31(+) macrophages also heavily infiltrated MLNs from human breast cancer biopsies but not reactive hyperplastic LNs. Thus, TIE2(+)/ CD31(+) macrophages may be a unique histopathological biomarker for detecting metastasis in clinical diagnosis, and a novel and promising target for TAM-specific cancer therapy.

Capsaicin (trans-8-methyl-N-vanillyl-6-nonenamide), the major pungent ingredient of red pepper, has been reported to possess anti-carcinogenic and anti-mutagenic activities. In this study, the anti-migration activity of capsaicin on highly metastatic B16-F10 melanoma cells was investigated. Capsaicin significantly inhibited the migration of melanoma cells without showing obvious cellular cytotoxicity at low doses. This effect correlated with the down-regulation of phosphatidylinositol 3-kinase (PI3-K) and its downstream target, Akt. Although B16-F10 cell migration was increased by the PI3-K activator through the activation of Akt, these PI3-K activator-induced phenomena were attenuated by capsaicin. Moreover, capsaicin was found to significantly inhibit Rac1 activity in a pull-down assay. These results demonstrate that capsaicin inhibits the migration of B16-F10 cells through the inhibition of the PI3-K/Akt/Rac1 signal pathway. The present investigation suggests that capsaicin targets PI3-K/Akt/ Rac1-mediated cellular events in B16-F10 melanoma cells. Consequently, capsaicin administration should be considered an effective approach for the suppression of invasion and metastasis in malignant melanoma chemotherapy.

Although polycyclic aromatic hydrocarbons (PAHs) have been known to suppress immune responses, few studies have addressed the immunotoxicity of benzo[k]fluoranthene (B[k]F). In this study, we investigated the immunosuppression by B[k]F, both in vivo and in vitro, in female BALB/c mice. To assess the effects of B[k]F on humoral immunity as splenic antibody response to sheep red blood cells (SRBCs), B[k]F was given a single dose or once daily for 7 consecutive days po with 30, 60, and 120 micromol/kg. B[k]F reduced the number of antibody-forming cells (AFCs) in a dose-dependent manner. Subacute treatment with B[k]F caused weight increases in liver and decreases in spleen and thymus. The number of AFCs was dramatically decreased by B[k]F in a dose-dependent manner. In a subsequent study, mice were subacutely exposed to the same doses of B[k]F without an immunization with SRBCs, followed by splenic and thymic lymphocyte phenotypings using a flow cytometry and ex vivo mitogen-stimulated proliferation. B[k]F-exposed mice exhibited reduced splenic and thymic cellularity, decreased numbers of total T cells, CD4(+) cells, and CD8(+) cells in spleen, and immature CD4(+)CD8(+) cells, CD4(+)CD8(-) cells, and CD8(+)CD4(-) cells in thymus. The number of CD4(+) IL-2(+) cells was reduced by about 11%, 31%, and 53% following exposure of mice to 30, 60, and 120 micromol/kg of B[k]F, respectively. In the ex vivo lymphocyte proliferation assay, B[k]F inhibited splenocyte proliferation by LPS and Con A. In the in vitro mitogen-stimulated proliferation by untreated splenic suspensions, B[k]F only suppressed splenocyte proliferation to LPS. These results suggested that B[k]F-induced immunosuppression might be mediated, at least in part, through the IL-2 production, and caused by mechanisms associated with metabolic processes.

Certain medicinal plants contain anti-inflammatory and antioxidative substances that can exert chemopreventive effects. Our previous studies have demonstrated that the methanol extract of Alpinia oxyphylla Miquel (Zingiberaceae) inhibits tumor promotion in mouse skin. Two major diarylheptanoids named yakuchinone A (1-[4'-hydroxy-3'-methoxyphenyl]-7-phenyl-3-heptanone) andyakuchinone B (1-[4'-hydroxy-3'-methoxyphenyl]-7-phenylhept-1-en-3-one) have been isolated from this medicinal plant. Both compounds have strong inhibitory effects on the synthesis of prostaglandins and leukotrienes in vitro. In the present work, we show that both yakuchinone A and yakuchinone B inhibit the expression of cyclooxygenase-2 (COX-2) and of inducible nitric oxide synthase (iNOS) as well as the expression of tumor necrosis factor (TNF)-alpha mRNA in mouse skin treated with the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA). Topical application on mouse skin of these diarylheptanoids also attenuated the TPA-induced DNA binding activity of the ubiquitous eukaryotic transcription factor NF-kappaB that plays a crucial role in regulating the expression of the aforementioned proinflammatory enzymes and cytokines in response to a wide variety of external stimuli. These findings suggest that diarylheptanoids contained in Alpinia oxyphylla down-regulate COX-2 and iNOS expression through suppression of NF-kappaB activation in the TPA-treated mouse skin.

In recent years, there have been considerable efforts to search for naturally occurring substances for intervention of carcinogenesis. Many components from medicinal or dietary plants have been identified to possess potential chemopreventive properties. For instance, curcumin, a yellow colouring agent from turmeric (Curcuma longa Linn., Zingiberaceae) has been shown to inhibit tumor formation in diverse animal models. Alpinia oxyphylla Miquel that also belongs to ginger family has been used in oriental herbal medicine. In the present work, we have evaluated the anti-tumor promoting potential of yakuchinone A (1-[4'-hydroxy-3'-methoxyphenyl]-7-phenyl-3-heptanone) and yakuchinone B (1-[4'-hydroxy-3'-methoxyphenyl]-7-phenylhept-1-en-3-one), major pungent ingredients of A. oxyphylla. Thus, topical application of yakuchinone A or B significantly suppressed TPA-induced epidermal ornithine decarboxylase activity. They also reduced TPA-stimulated production of tumor necrosis factor-alpha in cultured human promyelocytic leukemia (HL-60) cells. Both compounds blunted the TPA-induced superoxide generation in differentiated HL-60 cells in a concentration-related manner and also inhibited lipid peroxidation in rat brain homogenates. Furthermore, yakuchinone A and yakuchinone B nullified the activation of the activator protein-1 (AP-1) in immortalized mouse fibroblast cells in culture. These findings indicate that pungent diarylheptanoids from A. oxyphylla have anti-tumor promotional properties that can contribute to their chemopreventive potential.