Upregulation Of Fas Expression Research Articles

Impact of dietary fat levels and fatty acid composition on milk fat synthesis in sows at peak lactation

BackgroundDietary fat is important for energy provision and immune function of lactating sows and their progeny. However, knowledge on the impact of fat on mammary transcription of lipogenic genes, de novo fat synthesis, and milk fatty acid (FA) output is sparse in sows. This study aimed to evaluate impacts of dietary fat levels and FA composition on these traits in sows. Forty second-parity sows (Danish Landrace × Yorkshire) were assigned to 1 of 5 dietary treatments from d 108 of gestation until weaning (d 28 of lactation): low-fat control diet (3% added animal fat); or 1 of 4 high-fat diets with 8% added fat: coconut oil (CO), fish oil (FO), sunflower oil (SO), or 4% octanoic acid plus 4% FO (OFO). Three approaches were taken to estimate de novo milk fat synthesis from glucose and body fat.ResultsDaily intake of FA was lowest in low-fat sows within fat levels (P < 0.01) and in OFO and FO sows within high-fat diets (P < 0.01). Daily milk outputs of fat, FA, energy, and FA-derived carbon reflected to a large extent the intake of those. On average, estimates for de novo fat synthesis were 82 or 194 g/d from glucose according to method 1 or 2 and 255 g de novo + mobilized FA/d according to method 3. The low-fat diet increased mammary FAS expression (P < 0.05) and de novo fat synthesis (method 1; P = 0.13) within fat levels. The OFO diet increased de novo fat synthesis (method 1; P < 0.05) and numerically upregulated mammary FAS expression compared to the other high-fat diets. Across diets, a daily intake of 440 g digestible FA minimized milk fat originating from glucose and mobilized body fat.ConclusionsSows fed diets with low-fat or octanoic acid, through upregulating FAS expression, increased mammary de novo fat synthesis whereas the milk FA output remained low in sows fed the low-fat diet or high-fat OFO or FO diets, indicating that dietary FA intake, dietary fat level, and body fat mobilization in concert determine de novo fat synthesis, amount and profiles of FA in milk.

Receptor Activator of Nuclear Factor (Nf)-kb Ligand Promotes T Helper 17 Cell Differentiation through Fas

ABSTRACT T helper 17 (Th17) cells play important role in the defense against pathogens and autoimmune diseases. Many cytokines can induce Th17 cell differentiation. However, the mechanism of Th17 cell differentiation is not well clarified. RankL, a member of the TNF superfamily, binds with Rank and then participates in the proliferation and differentiation of many kinds of cells. Recent studies showed that RankL-Rank signaling is closely related to Th17 differentiation and function. The detail of the Rank-RankL pathway in Th17 cell differentiation is still unclear. To illustrate the role of Rank-RankL in Th17 differentiation, naive CD4 + T cells were differentiated into Th17 cells with or without RankL stimulation. During Th17 differentiation, the expression of Rank obviously increased. The RankL stimulation significantly increased Th17 cell differentiation indicated by increased IL-17-positive cell number, highly expressed IL-17 and IL-22 and elevated IL-17 secretion. These effects were canceled by Rank-Fc addition. In further study, RankL treatment during Th17 differentiation up-regulated Fas expression. Fas knockdown inhibited the Th17 differentiation promoted by RankL. In this study, it was confirmed that Rank-RankL signaling could promote Th17 cell differentiation through Fas induction.

Copper(II) and iron(III) complexes of chiral dehydroabietic acid derived from natural rosin: metal effect on structure and cytotoxicity.

A novel optically pure dinuclear copper(II) complex of a rosin derivative dehydroabietic acid (DHA, HL) was synthesized and fully characterized. The in vitro antitumor activities of the copper(II) complex Cu2(µ2-O)(L)4(DMF)2 (1) were explored and compared with those of a trinuclear iron(III) complex [Fe3(µ3-O)(L)6(CH3OH)2(CH3O)]·H2O (2). 1 was more cytotoxic than 2, and the in vitro cytotoxicity of 1 was comparable to that of cisplatin and oxaliplatin. The metal coordination improved the cytotoxicity of DHA. 1 could arrest cycle in G1 phase and induce apoptosis in MCF-7 cell. 1 increased reactive oxygen species level, GSSG/GSH ratio, and Ca2+ production, and caused the loss of mitochondrial membrane potential (Δψm) in MCF-7 cells. The up-regulated Bax and down-regulated Bcl-2 expression levels, caspase-9/caspase-3 activation, and the release of Cyt c demonstrate that 1 triggered mitochondria-mediated intrinsic apoptosis in MCF-7 cells. Caspase-8/caspase-4 activation and up-regulated Fas expression indicate that death receptor-mediated extrinsic apoptosis was included. Comet assay and up-regulated γ-H2AX and p53 expressions confirmed that 1 caused DNA damage in MCF-7 cells. Moreover, 1 led to enhancement of the biomarker of lipid peroxidation and the indicator of protein carbonylation in MCF-7 cells. All the results suggest that 1 could kill MCF-7 cells by generating oxidative stress, impairing DNA, promoting lipid peroxidation and protein carbonylation, and inducing apoptosis and autophagy. Furthermore, 1 also displayed antimetastatic activities with inhibition of cell invasion and migration, together with antiangiogenesis properties. On the whole, copper complex based on rosin derivatives is worth developing as metal-based antitumor drugs.

Fas Ligand Enhances Apoptosis of Human Lung Cancer Cells Cotreated with RIG-I-like Receptor Agonist and Radiation.

Retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs) play key roles in the antiviral response, but recent works show that RLR activation elicits anticancer activity as well, including apoptosis. Previously, we demonstrated that the anticancer activity of the RLR agonist Poly(I:C)-HMW/LyoVec™ [Poly(I:C)-HMW] against human lung cancer cells was enhanced by cotreatment with ionizing radiation (IR). In addition, cotreatment with Poly(I:C)-HMW and IR induced apoptosis in a Fas-independent manner, and increased Fas expression on the cell surface. The current study investigated the resultant hypothesis that Fas ligand (FasL) may enhance apoptosis in lung cancer cells cotreated with Poly(I:C)-HMW+IR. FasL was added into culture medium at 24 h following cotreatment with Poly(I:C)- HMW+IR, after upregulation of cell surface Fas expression on human lung cancer cells A549 and H1299 have already been discussed. FasL enhanced the apoptosis of A549 and H1299 cells treated with Poly(I:C)-HMW+IR. Similarly, IR alone - and not Poly(I:C)-HMW - resulted in the upregulation of cell surface Fas expression followed by a high response to FasL-induced apoptosis, thus suggesting that the high sensitivity of cells treated with Poly(I:C)-HMW+IR to FasL-induced apoptosis resulted from the cellular response to IR. Finally, knockdown of Fas by siRNA confirmed that the high response of treated cells to FasL-induced apoptosis is dependent on Fas expression. In summary, the present study indicates that upregulated Fas expression following cotreatment with Poly(I:C)-HMW and IR is responsive to FasL-induced apoptosis, and a combination of RLR agonist, IR, and FasL could be a potential promising cancer therapy.

The potential target of double negative T cells in cancer immunotherapy.

e15180 Background: Double negative T cell (DNT) is defined by the expression of CD3 (or TCRαβ) with the absence of CD4, CD8 and iNKT cell markers. DNTs account for 1-3% of the total peripheral lymphocytes. Although healthy donor cells derived allogeneic DNTs were reported to have anti-tumor effects in several cancer models, the function of DNTs in tumor microenvironment is not well understood. In this study, we aim to characterize DNTs in cancer tissues and lymph nodes of colorectal cancer patients including their immunological functions. Methods: A total of 14 colorectal cancer tissues and regional lymph nodes were collected and examined for the proportion and the immunological functions of DNTs by FACS analysis. Results: DNTs, which were defined in this study as TCRαβ positive with CD4-, CD8- and CD56- T cells, were detected in colorectal cancer tissues. DNT cells account for more than 5% of TCRαβ-positive populations in 12 cases and out of which 7 cases were more than 20% observed in this study. In all of these 7 cases, DNT cells were also found to be higher in the tumor tissues than in non-metastatic lymph nodes. In addition to colorectal cancer, DNTs were found to be more than 5% of TCRαβ-positive populations in cancer tissues in 3 out of the 6 cases of ovarian and uterine cancer cases.To further evaluate the potential immunological functions of DNTs, expression of cell membrane protein markers were evaluated. CD3ε expression was down regulated in almost all of the DNTs. The CD107a expression of DNTs were higher than that of CD8+ T cells in tumor tissues. When co-culturing DNTs with tumor cells, CD107a expression of DNTs was significantly upregulated. Notably, the intracellular production of perforin and granzyme B were lower than CD8+ T cells. Fas-L expression were found to be higher on DNT cells compared to other T cell populations, suggesting DNT might possess cytotoxic activity in the tumor microenvironment. It is known that upregulation of Fas expression is caused by T cell activation and subsequently induces T cell apoptosis. The upregulation of Fas expression was also observed in DNT cells from non-metastatic lymph nodes. In contrast, Fas expression of DNT cells in tumor tissues was observed to be lower than CD4+ or CD8+ T cells, suggesting that DNT could escape from apoptosis. This downregulation of Fas expression was speculated to be one of the reasons for the accumulation of DNTs in tumor tissues. Conclusions: Although the function of DNTs was still not well elucidated, the accumulation of DNT cells in tumor microenvironment suggests DNTs could be a potential target for cancer immunotherapy.

Regulatory loop between lncRNA FAS-AS1 and DNMT3b controls FAS expression in hydroquinone-treated TK6 cells and benzene-exposed workers

Hydroquinone (HQ), one of the main metabolites of benzene, is a well-known human leukemogen. However, the specific mechanism of how benzene or HQ contributes to the development of leukemia is unknown. In a previous study, we demonstrated the upregulation of DNA methyltransferase (DNMT) expression in HQ-induced malignant transformed TK6 (HQ-TK6) cells. Here, we investigated whether a regulatory loop between the long noncoding RNA FAS-AS1 and DNMT3b exists in HQ-TK6 cells and benzene-exposed workers. We found that the expression of FAS-AS1 was downregulated in HQ-TK6 cells and workers exposed to benzene longer than 1.5 years via histone acetylation, and FAS-AS1 expression was negatively correlated with the time of benzene exposure. Restoration of FAS-AS1 in HQ-TK6 cells promoted apoptosis and inhibited tumorigenicity in female nude mice. Interestingly, treatment with a DNMT inhibitor (5-aza-2-deoxycytidine), histone deacetylase inhibitor (trichostatin A), or DNMT3b knockout led to increased FAS-AS1 through increased H3K27ac protein expression in HQ-TK6 cells, and DNMT3b knockout decreased H3K27ac and DNMT3b enrichment to the FAS-AS1 promoter region, which suggested that DNMT3b and/or histone acetylation involve FAS-AS1 expression. Importantly, restoration of FAS-AS1 resulted in reduced expression of DNMT3b and SIRT1 and increased expression of FAS in both HQ-TK6 cells and xenograft tissues. Moreover, the average DNMT3b expression in 17 paired workers exposed to benzene within 1.5 years was decreased, but that of the remaining 103 paired workers with longer exposure times was increased. Conversely, DNMT3b was negatively correlated with FAS-AS1 expression. Both FAS-AS1 and DNMT3b influenced the enrichment of H3K27ac in the FAS promoter region by regulating the expression of SIRT1, consequently upregulating FAS expression. Taken together, these observations demonstrate crosstalk between FAS-AS1 and DNMT3b via a mutual inhibition loop and indicate a new mechanism by which FAS-AS1 regulates the expression of FAS in benzene-related carcinogenesis.

Abstract 4966: The SUV39H1-H3K9me3 axis mediates colon carcinoma cell intrinsic apoptosis and immune evasion

Abstract Human colon cancer, except for the small subset of microsatellite instable (MSI) colon cancer, does not respond to anti-PD-L1/PD-1 immune checkpoint inhibitor (ICI) immunotherapy. A well-established notion is that MSI colon cancer harbors various mutations that serve as neoantigens to generate tumor-specific cytotoxic T lymphocytes (CTLs). Because CTLs suppress tumor through inducing tumor cell apoptosis, we hypothesized that colon cancer cell intrinsic apoptosis resistance is potentially another mechanism underlying microsatellite stable (MSS) colon cancer nonresponse to ICI immunotherapy. If tumor cells are not sensitive to apoptosis induction, then tumor cells cannot be killed by CTLs regardless of how potent the CTLs are. To test this hypothesis, we analyzed MSI and MSS human colon carcinoma specimens and developed a small molecule that targets H3K9me3-mediated cell death pathways as a sensitizer for colon cancer ICI immunotherapy. As expected, we observed high level of CTL infiltration in all eight MSI colon carcinoma specimens examined. However, five of the nine MSS colon carcinoma specimens also exhibited high level of CTL infiltration, suggesting that CTL level is not the sole mechanism underlying MSS colon cancer non-response to ICI immunotherapy. Analysis of the TCGA database revealed that SUV39H1, a histone methyltransferase that catalyzes H3K9me3, is significantly elevated in human colon carcinoma specimens compared to normal human colon tissues. A SUV39H1 structure-based virtual chemical library screening in combination with functional assays identified a small-molecule SUV39H1 inhibitor. Further analysis of the top forty-three hits identified a potent SUV39H1 inhibitor. Structure modifications were then performed and a chemical synthesis procedure was developed to synthesize a novel SUV39H1 inhibitor, termed F5446. F5446 has an EC50 of 0.496 µM against SUV39H1 enzymatic activity and suppresses human colon carcinoma cell growth in a dose-dependent manner in vitro. F5446 suppresses colon carcinoma growth through upregulating key cell cycle regulators to induce cell cycle arrest at the S phase. Furthermore, F5446 also decreased H3K9me3 at the FAS promoter to upregulate Fas expression. Consequently, a sublethal dose of F5446 effectively overcame human colon carcinoma cell resistance to apoptosis induced by FasL, a ligand on activated CTLs in vitro. Treatment of colon carcinoma-bearing mice with F5446 and anti-PD-1 both significantly suppress tumor growth in vivo. Our data determined that F5446 is a novel SUV39H1 inhibitor that has the potential to be further developed as a sensitizer to overcome colon carcinoma resistance to ICI immunotherapy through activating the Fas-FasL pathway. Citation Format: Chunwan Lu, Dafeng Yang, John D. Klement, Aaron H. Colby, Mark W. Grinstaff, Cedric Pearce, Nicholas H. Oberlies, Thomas Albers, Iryna Lebedyeva, Kebin Liu. The SUV39H1-H3K9me3 axis mediates colon carcinoma cell intrinsic apoptosis and immune evasion [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4966.

A novel biphenyl urea compound, TPD7, stimulates apoptosis through modulating Fas signaling and Bcl-2 family proteins in cervical cancer.

We recently reported that TPD7 suppressed tumor cell proliferation, and inhibited invasion, through the suppression of C-X-C chemokine receptor type4 (CXCR4). In the present study, we investigated the anticancer effect of TPD7 on apoptosis and invasion of cervical cancer HeLa cells. Cell cycle analysis revealed that TPD7 decreased cyclin-dependent kinase (CDK)1 and cyclinD1 expression, and increased cyclin A expression, following Sphase blockade. TPD7 induced chromatin condensation and significantly elevated the number of apoptotic cells, suggesting that its inhibitory effect on HeLa cells was due to the induction of cell cycle blockade and apoptosis. Mechanistically, TPD7 altered the extrinsic apoptosis pathway by upregulating Fas expression, and the intrinsic pathway by modulating Bcl-2 family proteins, p53, and NF-κB p65, leading to enhanced apoptosis. TPD7 inhibited HeLa cell invasion by downregulating the expression of matrix metalloproteinase (MMP)-9 and CXCR4 proteins. Invivo experiments revealed that TPD7 inhibited tumor growth in HeLa cell xenografted mice. These findings indicated that TPD7 may be a potential chemoprevention agent for the management of cervical carcinoma.

P38: MicroRNA‐184 promotes apoptosis of trophoblast cells by up‐regulating Fas expression and inhibits differentiation of decidual regulatory T cells at the maternal‐fetal interface

P38: MicroRNA‐184 promotes apoptosis of trophoblast cells by up‐regulating Fas expression and inhibits differentiation of decidual regulatory T cells at the maternal‐fetal interface

Evaluate the response of Apoptosis, Angiogenesis and Cancer Therapies

Angiogenesis, the growth of new blood vessels from the existing vasculature, and is maintained in adult tissues by the balanced presence of both angiogenic inducers and inhibitors in the tissue milieu. When inducers predominate, vascular endothelial cells (VECs) become activated and in this activated VECs, distinct cell signaling pathways are initiated providing the specificity of anti-angiogenic therapies to the tumor vasculature. VEC apoptosis has been well documented in regressing vessels, and it has been shown that, in addition to activating the VECs, some inducers such as vascular endothelial growth factor also up-regulate Fas expression, thus sensitizing the cell to apoptotic stimuli. Endogenous angiogenesis inhibitors, such as thrombospondin-1(TSP-1) and pigment epithelium-derived factor (PEDF), stimulate signaling cascades within the VECs and also induce the expression of Fas ligand in activated VECs. Therefore, when inhibitors predominate, the apoptotic cascade is initiated ,thus anti-angiogenic therapies can target the inducer supply or directly target the VECs. Although clinical studies suggest that anti-angiogenic therapies may prove to be most effective when used in combination with traditional therapies

Small Interfering RNA–Mediated Suppression of Fas Modulate Apoptosis and Proliferation in Rat Intervertebral Disc Cells

Study DesignIn vitro cell culture model.PurposeTo investigate the effect of small interfering RNA (siRNA) on Fas expression, apoptosis, and proliferation in serum-deprived rat disc cells.Overview of LiteratureSynthetic siRNA can trigger an RNA interference (RNAi) response in mammalian cells and precipitate the inhibition of specific gene expression. However, the potential utility of siRNA technology in downregulation of specific genes associated with disc cell apoptosis remains unclear.MethodsRat disc cells were isolated and cultured in the presence of either 10% fetal bovine serum (FBS) (normal control) or 0% FBS (serum deprivation to induce apoptosis) for 48 hours. Fas expression, apoptosis, and proliferation were determined. Additionally, siRNA oligonucleotides against Fas (Fas siRNA) were transfected into rat disc cells to suppress Fas expression. Changes in Fas expression were assessed by reverse transcription-polymerase chain reaction and semiquantitatively analyzed using densitometry. The effect of Fas siRNA on apoptosis and proliferation of rat disc cells were also determined. Negative siRNA and transfection agent alone (Mock) were used as controls.ResultsSerum deprivation increased apoptosis by 40.3% (p<0.001), decreased proliferation by 45.3% (p<0.001), and upregulated Fas expression. Additionally, Fas siRNA suppressed Fas expression in serum-deprived cultures, with 68.5% reduction at the mRNA level compared to the control cultures (p<0.001). Finally, Fas siRNA–mediated suppression of Fas expression significantly inhibited apoptosis by 9.3% and increased proliferation by 21% in serum-deprived cultures (p<0.05 for both).ConclusionsThe observed dual positive effect of Fas siRNA might be a powerful therapeutic approach for disc degeneration by suppression of harmful gene expression.

The cross talk between cervical carcinoma cells and vascular endothelial cells mediated by IL-27 restrains angiogenesis.

To explore whether cervical carcinoma cell-derived interleukin-27 (IL-27) modulates the angiogenesis of vascular endothelial cells. The expression of IL-27 in cervical cancer tissues and cervical cell lines was analyzed by immunohistochemistry, ELISA and flow cytometry. Then, the effects of IL-27 on the proliferation and apoptosis-related molecules and angiogenesis in vitro of human umbilical vein endothelial cells (HUVECs) were investigated. Finally, in vivo experiment was performed to further confirm the effects of IL-27. Compared with cervicitis, the cervical cancer tissues highly expressed IL-27. Both HeLa and CaSki cells secreted IL-27, and HUVECs expressed low levels of IL-27 receptors (IL-27R). However, the co-culture of cervical cell lines and HUVECs led to a significant elevation of IL-27R on HUVECs. Co-culturing with IL-27-overexpressed HeLa cells downregulated Ki-67 and Bcl-2 and upregulated Fas expression in HUVECs. In addition, overexpression of IL-27 in HeLa cells and CasKi cells secreted less IL-8 and could further restrict angiogenesis compared with control cells in vitro. In the subcutaneous tumorous model of C57/BL6 mouse, there were decreased vessel density and tumor volume when inoculation with IL-27-overexpressed TC-1 cells. This study indicates that IL-27 secreted by cervical carcinoma cells restricts the angiogenesis in a paracrine manner in the pathogenesis of cervical cancer.

Hydroquinone induces TK6 cell growth arrest and apoptosis through PARP-1/p53 regulatory pathway.

Hydroquinone (HQ), one of the most important metabolites derived from benzene, induces cell cycle arrest and apoptosis. Poly(ADP-ribose) polymerase-1 (PARP-1) participates in various biological processes, including DNA repair and cell cycle regulation. To explore whether PARP-1 regulatory pathway mediated HQ-induced cell cycle arrest and apoptosis, we assessed the effect of PARP-1 suppression on induction of apoptosis analyzed by FACSCalibur flow cytometer in PARP-1 deficientTK6 cells (TK6-shPARP-1). We observed an increase in the fraction of cells in G1 phase by 7.6% and increased apoptosis by 4.5% in PARP-1-deficient TK6 cells (TK6-shPARP-1) compared to those negative control cells (TK6-shNC cells) in response to HQ treatment. Furthermore, HQ might activate the extrinsic pathways of apoptosis via up-regulation of Fas expression, followed by caspase-3 activation, apoptotic body, and sub G1 accumulation. Enhanced p53 expression was observed in TK6-shPARP-1 cells than in TK6-shNC cells after HQ treatment. In contrast, Fas expression was lower in TK6-shPARP-1 cells than in TK6-shNC cells. Therefore, we conclude that HQ may activate apoptotic signals via Fas up-regulation and p53-mediated apoptosis in TK6-shNC cells. The reduction of PARP-1 expression further intensified up-regulation of p53 in TK6-shPARP-1 cells, resulting in an increased G1→S phase cell arrest and apoptosis in TK6-shPARP-1 cells compared to TK6-shNC cells.

Induction of apoptosis in human cervical carcinoma Hela cells with active components of Menispermum dauricum

Menispermum dauricum DC possesses a wide range of pharmacological effects. In this study, the mechanism of apoptosis induced by active components of M. dauricum was investigated in the human cervical carcinoma HeLa cell line. HeLa cells were treated with different M. dauricum concentrations over different time periods. The proliferation-inhibitory rate and cytotoxic effect of HeLa cells were measured by using the methyl thiazolyl tetrazolium (MTT) assay, and the apoptotic rate was detected by flow cytometry. Expressions of caspase-9, caspase-8, caspase-3, Bcl-2, and Fas proteins, in the apoptotic pathway, and the expression of nuclear factor-kappa B (NF-κB) were detected by SP immunocytochemistry. The MTT assay showed that active components of M. dauricum could significantly inhibit the growth of HeLa cells in a dose- and time-dependent manner (P<0.01). The Sub-Gl peak was found by flow cytometry, and the maximal apoptosis rate was 24.93%. Immunocytochemistry showed that after treatment with M. dauricum, the expressions of caspase-8, caspase-9, caspase-3, Fas protein, and NF-κB all increased, and the expression of the Bcl-2 protein decreased, with significant differences relative to the control group (P<0.01). Apoptosis in HeLa cells could be induced by active components of M. dauricum through the NF-κB signal transduction pathway and the caspase pathway, which was related to the downregulation of Bcl-2 expression and the upregulation of Fas expression.

Krüppel-Like Factor 6 Rendered Rat Schwann Cell More Sensitive to Apoptosis via Upregulating FAS Expression

Krüppel-like factor 6 (KLF6) is a tumor suppressor gene and play a role in the regulation of cell proliferation and apoptosis. After the peripheral nerve injury (PNI), the microenvironment created by surrounding Schwann cells (SCs) is a critical determinant of its regenerative potential. In this study, we examined the effects of KLF6 on SCs responses during PNI. Both KLF6 mRNA and protein expression levels were upregulated in the injured sciatic nerve, and immunofluorescence results showed that many KLF6-positive cells simultaneously expressed the SC markers S-100 and p75NTR. The apoptosis inducers TNFα and cisplatin upregulated KLF6 expression in primary cultured SCs and the SC line RSC96. Although KLF6 overexpression exacerbated cisplatin- and TNFα-induced apoptosis, expression levels of the apoptosis regulators Bcl2 and Bax were not significantly affected in either KLF6-overexpressing or KLF6-depleted RSC96 cells. Realtime PCR arrays and qRT-PCR demonstrated that KLF6 overexpression upregulated four pro-apoptotic genes, FAS, TNF, TNFSF12, and PYCARD, and inhibited expression of the anti-apoptotic IL10 gene expression. Further analysis revealed that FAS protein expression was positively correlated with KLF6 expression in SCs. These data suggest that KLF6 upregulation may render SCs more vulnerable to apoptosis after injury via upregulating FAS expression.

The Histone Deacetylase Inhibitor, MS-275 (Entinostat), Downregulates c-FLIP, Sensitizes Osteosarcoma Cells to FasL, and Induces the Regression of Osteosarcoma Lung Metastases

The purpose of this study was to determine the effects of the histone deacetylase inhibitor, MS-275, on the Fas signaling pathway and susceptibility of osteosarcoma (OS) to Fas ligand (FasL)-induced cell death. OS metastasizes almost exclusively to the lungs. We have shown that Fas expression in OS cells is inversely correlated with their metastatic potential. Fas(+) cells are rapidly eliminated when they enter the lungs via interaction with FasL, which is constitutively expressed in the lungs. Fas(-) OS cells escape this FasL-induced apoptosis and survive in the lung microenvironment. Moreover, upregulation of Fas in established OS lung metastases results in tumor regression. Therefore, agents that upregulate Fas expression or activate the Fas signaling pathway may have therapeutic potential. Treatment of Fas(-) metastatic OS cell lines with 2 µM MS-275 sensitized cells to FasL-induced cell death in vitro. We found that MS-275 did not alter the expression of Fas on the cell surface; rather it resulted in the downregulation of the anti-apoptotic protein, c-FLIP (cellular FLICE-inhibitory protein), by inhibiting c-FLIP mRNA. Downregulation of c- FLIP correlated with caspase activation and apoptosis induction. Treatment of nu/nu-mice with established OS lung metastases with oral MS-275 resulted in tumor regression, increased apoptosis and a significant inhibition of c-FLIP expression in tumors. Histopathological examination of mice showed no evidence of significant toxicity. Overall, these results suggest that the mechanism by which MS-275 sensitizes OS cells and lung metastases to FasL-induced cell death may be by a direct reduction in the expression of c-FLIP.

Correlation of hepatitis B virus X antigen expression with liver cell apoptosis

To study the roles of hepatitis B virus (HBV) X antigen (HBxAg) in development of HBV-related liver diseases and carcinogenesis. Liver tissues were collected from patients with HBV infection (HBV carriers, n = 14; chronic hepatitis B (CHB), n = 24), HBV-related liver cirrhosis (LC, n = 20), or hepatocellular carcinoma (HCC, n = 20). Immunohistochemistry was used to detect the expression of HBxAg and the host apoptosis-related genes Fas and Fas ligand (Fas-L). The correlations of HBxAg with HBV DNA level in serum, inflammation grade, and fibrosis stage were statistically analyzed. Liver inflammation grade and fibrosis stage were in accordance with Knodell standard. x2test and Fisher's exact test were adopted in count data, x2split method was adopted in pariwise comparisons between multiple samples, Rank-sum test was adopted in ranked data, Spearman rank correlation analysis was adopted in correlation analysis. The rates of HBxAg-positivity were similar between the patients with HBV infection (71.1%), LC (60.0%), and HCC (65.0%) (x2= 0.754, P = 0.686). The rates of Fas- and Fas-L-positivity in liver cells were also similar between the three groups (Fas: 28.9% vs. 20.0% vs. 5.0%, x2= 4.667, P = 0.101; Fas-L: 36.8% vs. 50.0% vs. 60.0%, x2= 2.988, P = 0.225). However, the positive rate of Fas in lymphocytes of liver tissue was significantly higher in the HCC patients than in the HBV-infected patients (90.0% vs. 68.4%, Z = -4.360, P = 0.00001). The expressions of HBxAg and Fas-L corresponded to regions of severe inflammation in tissues from LC patients and some HCC patients. Furthermore, the expression of HBxAg was positively correlated with Fas (r = 0.304, P = 0.02) and Fas-L (r = 0.368, P = 0.004) in the HBV-infected patients and LC patients, and the expression of Fas was positively correlated with that of Fas-L (r = 0.448, P = 0.0004). Patients with high and medium loads of HBV DNA showed significantly higher rates of HBxAg-positivity than those with low loads (88.9% and 69.2% vs. 26.7%, P less than 0.05). In the early stage of chronic HBV infection, HBxAg may induce liver cell apoptosis by up-regulating Fas expression, and in the later stage, HBxAg may induce immune escape by up-regulating Fas-L expression in liver cells. Together, HBxAg and high HBV DNA load may promote chronic HBV infection and progression to hepatocarcinogenesis.

Enhancement of osteosarcoma cell sensitivity to cisplatin using paclitaxel in the presence of hyperthermia

Purpose: This paper aimed to evaluate the effects of a combination of paclitaxel and cisplatin on osteosarcoma (OS) cell lines in the presence of hyperthermia and to investigate the related mechanism.Materials and methods: Two types of OS cell lines (OS732 and MG63) were treated with paclitaxel and cisplatin in the presence of hyperthermia. The survival rate was measured by MTT assay, and the clonogenic rate was measured by a clonogenic assay. The cellular changes were observed with an inverted phase contrast microscope and a fluorescence microscope. The apoptotic effect was analysed with flow cytometry (FCM). Fas expression by the OS cell lines was measured by western blot. Fas expression in OS tissue was measured by immunohistochemistry.Results: Our study indicated that 1 h after the application of a combination of 10 μg/mL paclitaxel and 5 μg/mL cisplatin to OS cells at 43 °C, the survival rate of the OS cells was 11.96%, which was significantly lower than when either 10 μg/mL paclitaxel (45.02%) or 5 μg/mL cisplatin (48.69%) was applied alone (p < 0.01). Additionally, the clonogenic assay demonstrated that the clonogenic survival rate in the OS cells of the combination group was lower than that in the individual groups. Moreover, the cellular changes and apoptosis rates indicated that apoptosis in the combined application group was much greater than when either drug was applied individually. Fas expression by OS cell lines was increased by the combination of paclitaxel and cisplatin under hyperthermic conditions. More importantly, our study revealed low Fas expression in OS, which better explained the up-regulation of Fas achieved by the combination of paclitaxel and cisplatin in the presence of hyperthermia.Conclusions: The combination of paclitaxel and cisplatin increases the effects of thermochemotherapy on OS cell lines, primarily through the induction of apoptosis by the up-regulation of Fas expression.

Triptolide-Mediated Apoptosis by Suppression of Focal Adhesion Kinase through Extrinsic and Intrinsic Pathways in Human Melanoma Cells.

Triptolide (TPL) has been shown to inhibit cell proliferation and induce apoptosis in various human cancer cells; however, the precise mechanism of apoptosis induced by TPL in human melanoma cells has not yet been elucidated. In this study, we investigated the precise mechanism underlying cytocidal effects of TPL on human melanoma cells. Treatment of human melanoma cells with TPL significantly inhibited cell growth and induced apoptosis, as evidenced by flow cytometry and annexin V-fluorescein isothiocyanate analyses. TPL increased the levels of Fas and Fas-associated death domain (FADD) and induced cleavage of Bid by activation of caspase-8 and cytochrome c release from mitochondria to the cytosol, which resulted in activation of caspase-9 and caspase-3. Moreover, TPL-induced apoptosis in SK-MEL-2 cells was mediated through dephosphorylation of focal adhesion kinase (FAK) and its cleavage by caspase-8-mediated caspase-3 activation via upregulation of Fas expression. We also found that TPL mediated the dissociation of receptor-interacting protein (RIP) from FAK and enhanced the formation of RIP/Fas complex formation initiating cell death. In conclusion, our data firstly demonstrated that TPL induces apoptosis by both extrinsic and intrinsic apoptosis pathways in human melanoma cells and identified that RIP shuttles between Fas and FAK to mediate apoptosis.

Downregulation of Fas activity rescues early onset of diabetes inc-KitWv/+mice

c-Kit and its ligand stem cell factor (SCF) are important for β-cell survival and maturation; meanwhile, interactions between the Fas receptor (Fas) and Fas ligand are capable of triggering β-cell apoptosis. Disruption of c-Kit signaling leads to severe loss of β-cell mass and function with upregulation of Fas expression in c-Kit(Wv/+) mouse islets, suggesting that there is a critical balance between c-Kit and Fas activation in β-cells. In the present study, we investigated the interrelationship between c-Kit and Fas activation that mediates β-cell survival and function. We generated double mutant, c-Kit(Wv/+);Fas(lpr/lpr) (Wv(-/-)), mice to study the physiological and functional role of Fas with respect to β-cell function in c-Kit(Wv/+) mice. Isolated islets from these mice and the INS-1 cell line were used. We observed that islets in c-Kit(Wv/+) mice showed a significant increase in β-cell apoptosis along with upregulated p53 and Fas expression. These results were verified in vitro in INS-1 cells treated with SCF or c-Kit siRNA combined with a p53 inhibitor and Fas siRNA. In vivo, Wv(-/-) mice displayed improved β-cell function, with significantly enhanced insulin secretion and increased β-cell mass and proliferation compared with Wv(+/+) mice. This improvement was associated with downregulation of the Fas-mediated caspase-dependent apoptotic pathway and upregulation of the cFlip/NF-κB pathway. These findings demonstrate that a balance between the c-Kit and Fas signaling pathways is critical in the regulation of β-cell survival and function.