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Highlights • FGF9 induced cell proliferation, EMT, migration, and invasion of mouse Lewis lung cancer (LLC) cells, in vitro. • FGF9 interacted with FGFR1 and activated FAK, AKT, and ERK/MAPK signal pathways, induced the expression of EMT key proteins (N-cadherin, vimentin, snail, MMP2, MMP3 and MMP13) and reduced the expression of E-cadherin. • FGF9 promoted liver metastasis of subcutaneous inoculated LLC tumor with tumor growth, angiogenesis, EMT and M2-macrophage infiltration in the tumor microenvironment. • The FGF9/LLC syngeneic animal model provides a useful tool for the mechanism studies of liver metastasis which is the worst prognostic factor for lung cancer patients with distant organ metastasis., Fibroblast growth factors 9 (FGF9) modulates cell proliferation, differentiation and motility for development and repair in normal cells. Abnormal activation of FGF9 signaling is associated with tumor progression in many cancers. Also, FGF9 may be an unfavorable prognostic indicator for non-small cell lung cancer patients. However, the effects and mechanisms of FGF9 in lung cancer remain elusive. In this study, we investigated the FGF9-induced effects and signal activation profiles in mouse Lewis lung carcinoma (LLC) in vitro and in vivo. Our results demonstrated that FGF9 significantly induced cell proliferation and epithelial-to-mesenchymal transition (EMT) phenomena (migration and invasion) in LLC cells. Mechanism-wise, FGF9 interacted with FGFR1 and activated FAK, AKT, and ERK/MAPK signal pathways, induced the expression of EMT key proteins (N-cadherin, vimentin, snail, MMP2, MMP3 and MMP13), and reduced the expression of E-cadherin. Moreover, in the allograft mouse model, intratumor injection of FGF9 to LLC-tumor bearing C57BL/6 mice enhanced LLC tumor growth which were the results of increased Ki67 expression and decreased cleaved caspase-3 expression compared to control groups. Furthermore, we have a novel finding that FGF9 promoted liver metastasis of subcutaneous inoculated LLC tumor with angiogenesis, EMT and M2-macrophage infiltration in the tumor microenvironment. In conclusion, FGF9 activated FAK, AKT, and ERK signaling through FGFR1 with induction of EMT to stimulate LLC tumorigenesis and hepatic metastasis. This novel FGF9/LLC allograft animal model may therefore be useful to study the mechanism of liver metastasis which is the worst prognostic factor for lung cancer patients with distant organ metastasis.

Cordycepin, a bioactive constituent from the fungus Cordyceps sinensis, could inhibit cancer cell proliferation and promote cell death via induction of cell cycle arrest, apoptosis and autophagy. Our novel finding from microarray analysis of cordycepin-treated MA-10 mouse Leydig tumor cells is that cordycepin down-regulated the mRNA levels of FGF9, FGF18, FGFR2 and FGFR3 genes in MA-10 cells. Meanwhile, the IPA-MAP pathway prediction result showed that cordycepin inhibited MA-10 cell proliferation by suppressing FGFs/FGFRs pathways. The in vitro study further revealed that cordycepin decreased FGF9-induced MA-10 cell proliferation by inhibiting the expressions of p-ERK1/2, p-Rb and E2F1, and subsequently reducing the expressions of cyclins and CDKs. In addition, a mouse allograft model was performed by intratumoral injection of FGF9 and/or intraperitoneal injection of cordycepin to MA-10-tumor bearing C57BL/6J mice. Results showed that FGF9-induced tumor growth in cordycepin-treated mice was significantly smaller than that in a PBS-treated control group. Furthermore, cordycepin decreased FGF9-induced FGFR1-4 protein expressions in vitro and in vivo. In summary, cordycepin inhibited FGF9-induced testicular tumor growth by suppressing the ERK1/2, Rb/E2F1, cell cycle pathways, and the expressions of FGFR1-4 proteins, suggesting that cordycepin can be used as a novel anticancer drug for testicular cancers.

Fibroblast growth factor 9 (FGF9) promotes cancer progression; however, its role in cell proliferation related to tumorigenesis remains elusive. We investigated how FGF9 affected MA‐10 mouse Leydig tumor cell proliferation and found that FGF9 significantly induced cell proliferation by activating ERK1/2 and retinoblastoma (Rb) phosphorylations within 15 minutes. Subsequently, the expressions of E2F1 and the cell cycle regulators: cyclin D1, cyclin E1 and cyclin‐dependent kinase 4 (CDK4) in G1 phase and cyclin A1, CDK2 and CDK1 in S‐G2/M phases were increased at 12 hours after FGF9 treatment; and cyclin B1 in G2/M phases were induced at 24 hours after FGF9 stimulation, whereas the phosphorylations of p53, p21 and p27 were not affected by FGF9. Moreover, FGF9‐induced effects were inhibited by MEK inhibitor PD98059, indicating FGF9 activated the Rb/E2F pathway to accelerate MA‐10 cell proliferation by activating ERK1/2. Immunoprecipitation assay and ChIP‐quantitative PCR results showed that FGF9‐induced Rb phosphorylation led to the dissociation of Rb‐E2F1 complexes and thereby enhanced the transactivations of E2F1 target genes, Cyclin D1, Cyclin E1 and Cyclin A1. Silencing of FGF receptor 2 (FGFR2) using lentiviral shRNA inhibited FGF9‐induced ERK1/2 phosphorylation and cell proliferation, indicating that FGFR2 is the obligate receptor for FGF9 to bind and activate the signaling pathway in MA‐10 cells. Furthermore, in a severe combined immunodeficiency mouse xenograft model, FGF9 significantly promoted MA‐10 tumor growth, a consequence of increased cell proliferation and decreased apoptosis. Conclusively, FGF9 interacts with FGFR2 to activate ERK1/2, Rb/E2F1 and cell cycle pathways to induce MA‐10 cell proliferation in vitro and tumor growth in vivo.

Glycine N-methyltransferase is a protein with many functions. In addition to catalyzing the production of sarcosine in the one carbon metabolism pathway, it plays a role in the detoxification of environmental carcinogens such as benzo[a]pyrene, aflatoxin B1, and aristocholic acid. There is also increasing evidence suggesting a role of GNMT deficiency in liver carcinogenesis. In this review, we discuss the role of GNMT in the detoxification of xenobiotics and the mechanism of GNMT suppression during liver tumorigenesis. The protective role of GNMT in the liver allows GNMT to not only serve as a marker of liver disease, but also potentially be applied in the treatment of liver disorders and hepatocellular carcinoma. We describe the potential use of GNMT in gene therapy and we introduce the development of a GNMT promoter reporter assay that can be used to screen medicinal drugs and herbal libraries for natural compounds with anti-cancer properties.

Cordyceps sinensis has various biological and pharmacological functions, and it has been claimed as a tonic supplement for sexual and reproductive dysfunctions for a long time in oriental society. In this article, the in vitro and in vivo effects of C. sinensis and cordycepin on mouse Leydig cell steroidogenesis are briefly described, the stimulatory mechanisms are summarized, and the recent findings related to the alternative substances regulating male reproductive functions are also discussed.

Fibronectin (FN) expressed by tumor cells has been known to be tumor suppressive but the pericellular FN (periFN) assembled on circulating tumor cells appears to evidently promote distant metastasis. Whereas the regulation of periFN assembly in suspended cells has currently been under investigation, how it is regulated in adherent tumor cells and the role of periFN in primary tumor growth remain elusive. Techniques of RNAi, plasmid transfections, immunoblotting, fluorescence/immunohistochemistry staining, cell proliferation assays, and primary tumor growth in C57BL6 mice and Fischer 344 rats were employed in this study. We found that endogenously synthesized FN in adherent tumor cells was required for periFN assembly which was aligned by RhoA-organized actin stress fiber (SF). Depleting periFN on adherent tumor cells congruently promoted in vivo tumor growth but surprisingly did not autonomously impact on in vitro tumor cell proliferation and apoptosis, suggestive of a non-autonomous role of periFN in in vivo tumor growth. We showed that the proliferative ability of shFN-expressing tumor cells was higher than shScramble cells did in the presence of fibroblasts. Altogether, these results suggested that depriving RhoA/SF-regulated periFN matrices non-autonomously promotes fibroblast-mediated tumor cell growth.

In the western world, there is an increasing trend of occurrence in testicular cancer. Treatment of malignant testicular cancer is primarily combined surgery with various chemical drugs. Propofol has been frequently used as an anesthetic and sedative induction agent, which could modulate different γ‑aminobutyric acid receptors in the central nervous system. Studies demonstrated that propofol activates endoplasmic reticulum stress to induce apoptosis in lung cancer. However, it remains elusive whether propofol regulates caspase and/or mitogen‑activated protein kinase (MAPK) pathways to induce apoptosis in Leydig tumor cells. In the present study, MA‑10 mouse Leydig tumor cells were treated with propofol, and possible signal pathways associated with apoptosis were investigated. Results demonstrated that increasing dosage of propofol (300‑600 µM) for 24 h significantly decreased cell viability in MA‑10 cells (P

The incidence of testicular cancer is increasing worldwide. Leydig cell tumors represent one type of sex cord-stromal testis malignancy, which tend to respond unfavorably to chemotherapies. Identifying more efficient treatment strategies is therefore crucial for patients. The present study aimed to investigate the apoptotic effects of arsenic compounds and their underlying mechanisms. The results indicated that sodium arsenite and dimethylarsenic acid induced apoptosis of the murine Leydig tumor cell line, MA-10. These apoptotic effects were characterized morphologically by membrane blebbing and cell detachment assays, biochemically using a cell viability assay, and cytologically by flow cytometry analysis. Western blotting demonstrated that caspases-3, -8 and -9, and poly(ADP-ribose) polymerase protein levels were increased compared with untreated MA-10 cells; however, the caspase inhibitor, Z-VAD-fmk, reversed these effects. In conclusion, the present study has shown that sodium arsenite and dimethylarsenic acid may activate the intrinsic and extrinsic caspase pathways, and induce MA-10 cell apoptosis. These results suggest that sodium arsenite and dimethylarsenic acid may represent novel approaches to treat clinically unmanageable forms of testicular cancer.

Fu-Chi Kang,1,* Shu-Chun Wang,2,* Ming-Min Chang,2 Bo-Syong Pan,3 Kar-Lok Wong,4 Ka-Shun Cheng,4,5 Edmund Cheung So,4,6 Bu-Miin Huang2,7 1Department of Anesthesia, Chi Mei Medical Center, Chiali, Tainan, Taiwan, Republic of China; 2Department of Cell Biology and Anatomy, College of Medicine, National Cheng Kung University, Tainan, Taiwan, Republic of China; 3Department of Cancer Biology, Wake Forest University School of Medicine, Winston Salem, NC, USA; 4Department of Anesthesia, China Medical University, Taichung, Taiwan, Republic of China; 5Department of Anesthesiology, The Qingdao University Yuhuangding Hospital, Yantai, Shandong, China; 6Department of Anesthesia, An Nan Hospital, China Medical University, Tainan, Taiwan, Republic of China; 7Department of Medical Research, China Medical University Hospital, China Medical University, Taichung, Taiwan, Republic of China *These authors contributed equally to this work Background: Midazolam (MDZ) has powerful hypnosis, amnesia, anti-anxiety and anticonvulsant effects. Studies have shown that prenatally developmental toxicity of diazepam can be observed in many organs/tissues. However, it remains elusive in male reproductive system.Materials and methods: TM3 mouse Leydig progenitor cell line was used to determine whether MDZ has any unfavorable effects.Results: Midazolam significantly decreased cell viability in dose- and time-dependent manners in TM3 cells. In flow cytometry analysis, midazolam significantly increased subG1 phase cell numbers, and annexin V/PI double staining assay further confirmed that MDZ induced apoptosis in TM3 cells. Moreover, MDZ significantly induced the expression of caspase-8 and -3 proteins and the phosphorylation of JNK, ERK1/2 and p38. Besides, MDZ didn’t activate Akt pathway in TM3 cells. Furthermore, the expressions of p-EIF2α, ATF4, ATF3 and CHOP were induced by midazolam, suggesting that midazolam could induce apoptosis through endoplasmic reticulum (ER) stress in TM3 cells. Additionally, the expressions of cyclin A, cyclin B and CDK1 were inhibited by midazolam through the regulation of p53 in TM3 cells, indicating that midazolam could regulate cell cycle to induce apoptosis.Conclusion: Midazolam could activate caspase, MAPKs and ER stress pathways and impede Akt pathway and cell cycle to induce apoptosis in TM3 mouse Leydig progenitor cells. Keywords: midazolam, TM3, Leydig progenitor cells, apoptosis, caspase, MAPKs, ER stress, cell cycle&nbsp

Metastasis is a major cause of cancer deaths. Seeking alternative prognostic biomarkers may enable oncologists to make accurate therapeutic decisions to benefit cancer patients. Cumulated evidence reveals that fibronectin (FN) is highly correlated with cancer metastasis but has not been deemed as a prognostic biomarker due to its broad tissue distribution patterns and complicated physiological and pathological functionalities that significantly interfere with the judgmental accuracy. Combining other FN-related factors may make FN possible as a useful prognostic biomarker. Clathrin, a highly protease-susceptible cytoplasmic molecule, is known to affect pericellular FN (periFN) assembly via regulating cell surface FN receptors or FN matrix turnover by coating the endocytic vesicles. Researching our previously published proteomics database of 660 differential secretome proteins expressed in human lung adenocarcinoma cell lines and performing double immunofluorescent staining for periFN and clathrin, we recognized an inverse relationship between them. However, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) data contradicted this relationship, which could be corrected by the addition of a mixture of protease inhibitors into nonmetastatic cancer cell lysates. These results suggested that nonmetastatic cells express either higher levels of cellular proteases or less amounts of protease inhibitors. By examining our proteomic database and reviewing the literature, we conclude that clathrin expression and assembly is inversely correlated with metastatic potential of FNhigh cancer cells mainly related to the expression of protease inhibitors, instead of proteases. It is worth investigating whether such an inverse relationship between FN/protease inhibitors and clathrin in human cancers could clinically be incorporated into the prognostic strategy for various cancer types.

Figure S1. The reduced lung metastasis of FN-silenced suspended LLC cells is not due to inhibited cell proliferation and to the lowered mouse body weights. Figure S2. PS is among other stilbenoids the best inhibitor against periFN assembly of suspended mouse lung cancer cell line LLC, human lung cancer cell line CL1-5, and rat glioblastoma cell line CNS-1 TR50. Figure S3. Viabilities of PS-treated suspended LLC cells. Suspended LLC cells were similarly treated with DMSO or PS for up to 48 h, stained, and analyzed as described in methodology. Figure S4. Viabilities of PS-treated adherent LLC cells. Figure S5. PS-pretreatment for LLC cells does not impair their growth and migratory activities upon reseeding onto dishes. Figure S6. PS inhibits periFN assembly and metastasis of LLC cells. Figure S7. Migration and invasion activities of the reseeded LLC cells that were pretreated with various concentrations of PS in suspension. Figure S8. PS has no effect on activities of JNK and p38. Figure S9. Effects of PI3K inhibitor and shAKT on polyFN assemblies and expression levels of AKT and ERK. Figure S10. LW:BWs from each mouse bearing intravenously inoculated suspended LLC cells that were treated with DMSO, PS, PS+LY, or PS+LY+U0126 as described in Fig. 8a. (PDF 1380Â kb)

The purposes of this study were to separate the effect of iron status from the effect of acute iron intake on tissue retention of aluminum and Ga-67 and to evaluate Ga-67 as a marker for aluminum. Anemic and control rats were dosed by gavage with a citrate solution containing 20 microCi Ga-67 with no added aluminum and iron (Gavage Ga-67), with 0.8 mmol aluminum (Gavage Al), with 0.8 mmol iron (Gavage Fe), or with both 0.8 mmol aluminum and 0.8 mmol iron (Gavage Fe and Al). After 24 h, anemic rats in the Gavage Al treatment had lower concentrations of aluminum in their tibias, kidneys, and spleens than control rats in that treatment. In contrast, anemic rats dosed with only Ga-67 (Gavage Ga-67 treatment) had lower concentrations of Ga-67 in their tibias and kidneys, but greater concentrations of Ga-67 in their livers and spleens than control rats in that treatment. The single dose of iron had no effect on tissue aluminum concentrations but depressed tissue Ga-67 concentrations. All rats accumulated aluminum predominantly in bone and control rats accumulated Ga-67 predominantly in bone, but anemic rats accumulated Ga-67 predominantly in liver. A major limitation of Ga-67 as a marker for aluminum is its greater sensitivity than aluminum to iron intake and status.

The purposes of this study were to evaluate the effect of nutritional status in regard to iron on aluminum distribution and turnover and to evaluate Ga-67 as a marker for aluminum. Anemic (n = 27) and normal (n = 30) rats were dosed by gavage with 0.8 mmoles of aluminum and 20 microCi Ga-67 in a 0.75 mol/l citrate solution and sacrificed 1, 3, 6, 9, 15, and 21 days later. Anemic rats generally retained more aluminum in their livers but less in tibias and spleens than normal rats. The half-lives of aluminum in liver (56 vs 17 days), muscle (33 vs 16 days), and serum (12 vs 8 days) were significantly greater in anemic than normal rats, respectively. Total body retention of Ga-67 could be described on the basis of a two-compartment model. The turnover of Ga-67 from the first compartment was rapid (half-life = 0.8 and 0.6 days) in anemic and normal rats, respectively, and was similar to the turnover of Ga-67 from the Gl tract (half-life = 0.7 and 0.6 days in anemic and normal rats, respectively). The turnover of Ga-67 from the second compartment was also rapid (2.8 vs 4.0 days in anemic and normal rats, respectively). Anemia affected the retention of Ga-67 more than the retention of aluminum; anemic rats retained more Ga-67 in their livers, spleens, kidneys, hearts, and muscles but less in their tibias than normal rats. In general, Ga-67 was not a satisfactory marker for aluminum distribution and turnover in normal and anemic rats.

Tumor cells chronically progress, involving a series of aberrant activities within distinct organs, to accomplish final metastasis. The role of fibronectin (FN) in tumor progression has been controversial, being either tumor-suppressive or metastatic-promoting. Nevertheless, little effort has been made to reconcile these discrepancies in a systemic manner. Here, we deprived metastatic tumor cells of pericellular FN (periFN) in the spontaneous metastasis assay and demonstrated a dual role of FN. We found that, while knocking down FN expression of Lewis lung carcinoma cells (FNKD-LLC) increased the tumor sizes and migratory activities, it significantly decreased the tumor metastasis in the lungs. Histological examination revealed that subcutaneous tumors of mice bearing FNKD LLC invaded the surrounding tissues to a higher extent than those of mice bearing control scramble-LLC. Immunohistochemical staining with antibodies against CD31, FN, and casepase 3, revealed that FNKD-LLC rendered more angiogenesis, tumor intravasation, and fibrous FN deposited in the surrounding inflammatory tissues, without altering the tumor apoptosis, suggesting that the FN expression in tumor cells indeed plays a role in suppressing the tumor progression in the primary tumor tissues. Conversely, the metastatic tumor nodule numbers in the lungs were significantly reduced for the FNKD-LLC cells, clearly indicative of a metastatic-promoting role of periFN. We reasoned these seemingly conflicting phenomena by hypothesizing that the FN expression of neoplastic lung carcinoma cells is initially downregulated to trigger fibrotic microenvironments (FN deposition in extracellular matrices) to which pro-inflammatory leukocytes are recruited, leading to the enhanced angiogenesis and tumor intravasation. However, the early circulating lung cancer cells fail to colonize distant organs (including lungs) until periFN is re-assembled in the later progression stages, thus facilitating the tumor cell adhesion to CD26 on endothelial cells and consequently extravasation and metastasis. This hypothesis of temporal-spatial regulations was clinically supported by comparing and statistically analyzing the FN expression levels in lung cancer tissues collected from patients hospitalized by the NCKU hospital for surgery within 2003 through 2006 with low and high survivals. This study was supported by Grant # MOST 102-2320-B-006 -031- and MOST 103-2325-B-006 -009 -. Citation Format: Hung-Chi Cheng, Ming-Min Chang, Yau-Lin Tseng. The dual role of fibronectin in lung cancer progression. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2274. doi:10.1158/1538-7445.AM2015-2274