The TGF-ß-induced up-regulation of NKG2DLs requires AKT/GSK-3ß-mediated stabilization of SP1.

Natural killer (NK) cells play an important role in preventing cancer development. NK group 2 member D (NKG2D) is an activating receptor expressed in the membrane of NK cells. Tumour cells expressing NKG2DL become susceptible to an immune-dependent rejection mainly mediated by NK cells. The paradoxical roles of transforming growth factor beta (TGF-ß) in regulation of NKG2DL are presented in many studies, but the mechanism is unclear. In this study, we showed that TGF-ß up-regulated the expression of NKG2DLs in both PC3 and HepG2 cells. The up-regulation of NKG2DLs was characterized by increasing the expression of UL16-binding proteins (ULBPs) 1 and 2. TGF-ß treatment also increased the expression of transcription factor SP1. Knockdown of SP1 significantly attenuated TGF-ß-induced up-regulation of NKG2DLs in PC3 and HepG2 cells, suggesting that SP1 plays a key role in TGF-ß-induced up-regulation of NKG2DLs. TGF-ß treatment rapidly increased SP1 protein expression while not mRNA level. It might be due to that TGF-ß can elevate SP1 stability by activating PI3K/AKT signalling pathway, subsequently inhibiting GSK-3ß activity and decreasing the association between SP1 and GSK-3ß. Knockdown of GSK-3ß further verified our findings. Taken together, these results revealed that AKT/GSK-3ß-mediated stabilization of SP1 is required for TGF-ß induced up-regulation of NKG2DLs. Our study provided valuable evidence for exploring the tumour immune modulation function of TGF-ß.

Histone deacetylase inhibitors deplete myeloid-derived suppressor cells induced by 4T1 mammary tumors in vivo and in vitro.

Myeloid-derived suppressor cells (MDSC) have been identified as a population of immature myeloid cells that suppress anti-tumor immunity. MDSC are increased in tumor-bearing hosts; thus, depletion of MDSC may enhance anti-tumor immunity. Histone deacetylase inhibitors (HDACi) are chemical agents that are primarily used against hematologic malignancies. The ability of these agents to modulate anticancer immunity has recently been extensively studied. However, the effect of HDACi on MDSC has remained largely unexplored. In the present study, we provide the first demonstration that HDACi treatment decreases MDSC accumulation in the spleen, blood and tumor bed but increases the proportion of T cells (particularly the frequency of IFN-γ- or perforin-producing CD8+ T cells) in BALB/C mice with 4T1 mammary tumors. In addition, HDACi exposure of bone marrow (BM) cells significantly eliminated the MDSC population induced by GM-CSF or the tumor burden in vitro, which was further demonstrated as functionally important to relieve the inhibitory effect of MDSC-enriched BM cells on T cell proliferation. Mechanistically, HDACi increased the apoptosis of Gr-1+ cells (almost MDSC) compared with that of Gr-1- cells, which was abrogated by the ROS scavenger N-acetylcysteine, suggesting that the HDACi-induced increase in MDSC apoptosis due to increased intracellular ROS might partially account for the observed depletion of MDSC. These findings suggest that the elimination of MDSC using an HDACi may contribute to the overall anti-tumor properties of these agents, highlighting a novel property of HDACi as potent MDSC-targeting agents, which may be used to enhance the efficacy of immunotherapeutic regimens.

Calotropin from Asclepias curasavica induces cell cycle arrest and apoptosis in cisplatin-resistant lung cancer cells.

Calotropin (M11), an active compound isolated from Asclepias curasavica L., was found to exert strong inhibitory and pro-apoptotic activity specifically against cisplatin-induced resistant non-small cell lung cancer (NSCLC) cells (A549/CDDP). Molecular mechanism study revealed that M11 induced cell cycle arrest at the G2/M phase through down-regulating cyclins, CDK1, CDK2 and up-regulating p53 and p21. Furthermore, M11 accelerated apoptosis through the mitochondrial apoptotic pathway which was accompanied by increase Bax/Bcl-2 ratio, decrease in mitochondrial membrane potential, increase in reactive oxygen species production, activations of caspases 3 and 9 as well as cleavage of poly ADP-ribose polymerase (PARP). The activation and phosphorylation of JNK was also found to be involved in M11-induced apoptosis, and SP610025 (specific JNK inhibitor) partially prevented apoptosis induced by M11. In contrast, all of the effects that M11 induce cell cycle arrest and apoptosis in A549/CDDP cells were not significant in A549 cells. Drugs with higher sensitivity against resistant tumor cells than the parent cells are rather rare. Results of this study supported the potential application of M11 on the non-small lung cancer (NSCLC) with cisplatin resistance.

Nodal signaling modulates the expression of Oct-4 via nuclear translocation of ß-catenin in lung and prostate cancer cells.

Nodal is a member of transforming growth factor beta (TGF-ß) superfamily. Nodal promotes the self-renewal of human cancer stem cells (CSCs) and triggers carcinogenesis of human cancers via an autocrine manner through Smad2/3 pathway. In our study, generation of Nodal-overexpressed cancer cells was constructed, and the effect of Nodal on the stem cell marker Oct-4 was evaluated by overexpression or blocked Nodal/ALKs signaling pathway in non-small cell lung cancer cells A549 and prostate cancer cells PC3. Functionally, Nodal also increased the proliferation via the ß-catenin nuclear translocation. This increase was attributed to GSK-3ß dephosphorylating, and activin receptor-like kinase 4/7 (ALK4/7) played a major role in human cancer cells. Our study provides a positive understanding of Nodal function in cancer cells and suggests a potential novel target for clinical therapeutic research.

AKT/GSK-3ß regulates stability and transcription of snail which is crucial for bFGF-induced epithelial-mesenchymal transition of prostate cancer cells.

BACKGROUND: Epithelial-mesenchymal transition (EMT) plays a pivotal role in the development of metastatic cancers. Basic fibroblast growth factor (bFGF) is significantly elevated in metastatic prostate cancers, which has been mentioned mainly to induce EMT in normal cells. However, there is no description about bFGF induced EMT and its underlying mechanism in prostate cancer cells. METHODS: Western blotting, immunofluorescence and qRT-PCR assays were used to study protein or mRNA expression profiles of the EMT. Wound healing scratch, migration and invasion assays were used to test the motility of cells undergoing EMT. More methods were used to explore the underlying mechanisms. RESULTS: We demonstrated that bFGF promoted EMT and motility of human prostate cancer PC-3 cells. Both protein and mRNA expression of Snail were rapidly increased after bFGF treatment. Ectopic expression of Snail triggered EMT and enhanced cell motility in PC-3 cells, and knockdown of Snail almost abolished bFGF induced EMT, suggesting the critical role of Snail. Mechanistic study demonstrated that bFGF promoted the stability, nuclear localization and transcription of Snail by inhibiting the activity of glycogen synthase kinase 3 beta (GSK-3ß) through phosphatidylinositide 3 kinases (PI3K)/protein kinase B (AKT) signaling pathway. CONCLUSIONS: It is concluded that bFGF can promote EMT and motility of PC-3 cells, and AKT/GSK-3ß signaling pathway controls the stability, localization and transcription of Snail which is crucial for this bFGF induced EMT. GENERAL SIGNIFICANCE: To our knowledge, this is the first study to demonstrate that bFGF can induce EMT via AKT/GSK-3ß/Snail signaling pathway in prostate cancer cells.

Nodal promotes the generation of M2-like macrophages and downregulates the expression of IL-12.

Nodal, a member of the TGF-ß superfamily, is an embryonic morphogen that is upregulated in different types of tumors. Nodal increases the tumorigenesis by inducing angiogenesis and promoting metastasis. Importantly, Nodal inhibition suppresses the growth and invasion of tumor. Since tumor-associated macrophages (TAMs) are the major infiltrating leukocytes in most cancers, we investigated whether Nodal is involved in the differentiation of TAMs. Our results revealed that Nodal inhibition in tumor microenvironment upregulated the production of IL-12 in macrophages and reversed TAMs to classically activated macrophage phenotype. In contrast, treatment with recombinant Nodal (rNodal) decreased the expression of IL-12 in murine macrophages. Furthermore, rNodal promoted macrophage polarization to an alternatively activated macrophage-like/TAM phenotype and modulated its function. These results suggest that Nodal may play an important role in macrophage polarization and downregulation of IL-12. The rescued antitumor function of TAMs via the inhibition of Nodal expression could be a new therapeutic strategy for cancer treatment.

Histone deacetylase inhibitor induction of epithelial-mesenchymal transitions via up-regulation of Snail facilitates cancer progression.

Histone deacetylase inhibitors (HDACIs) are now emerging as a new class of anticancer drugs. Some of them have been used in clinical treatment for tumors, most impressively in the hematological tumors. But their single-agent activities in epithelial-derived tumors are limited. The mechanisms of these actions of HDACIs are not yet well understood. In this study, it was found for the first time that HDACIs were able to induce epithelial-mesenchymal transitions (EMT) which is believed to trigger tumor cell invasion and metastasis. We show that HDACIs induce fibroblast-like morphology, up-regulate Snail and Vimentin and down-regulate E-cadherin in epithelial cell-derived tumor cell lines. It demonstrates that HDACI treatment enhances further Snail acetylation and reduces its ubiquitylation, and induces Snail transcription as well as Snail nuclear translocation in CNE2 cells. Snail knockdown by siRNAs prevents the change in cell morphology and Vimentin up-regulation in response to HDACIs. The results suggested that Snail plays an important role in the HDACI-induced EMT. It is very crucial for a better understanding of clinical therapeutical failure of HDACIs in the patients with epithelial cell-derived cancers. Therefore, our results indicate that more attention should be paid to the cancer treatment using HDACIs due to the fact that it will enhance the spread risks of cancer cells to facilitate cancer progression and it is very important to select appropriate drugs for different tumors.

The role of indoleamine 2,3-dioxygenase (IDO) in immune tolerance: focus on macrophage polarization of THP-1 cells.

Macrophages can be divided into two groups as M1 and M2 phenotype. Our results and other groups revealed that IFN-γ can up-regulate the IDO expression and differentiate THP-1 cells to M1 phenotype. Therefore we hypothesized that IDO may play potential roles in macrophage differentiation. Interesting, our results indicated that the ectopic IDO increases the expression of M2 markers such as IL-10 and CXCR4 while decreases the M1 markers such as CCR7 and IL-12p35. In contrast, the knockdown of IDO expression in THP-1 cells resulted in increased M1 markers and lower M2 markers. Our results suggested that the expression intensity of IDO modulates macrophages differentiation. These finding support the counter-regulatory role for IDO with regarding to the polarization of macrophages to restrain excessive or inappropriate immune activation in inflammatory or tumor microenvironment. It throws new light on the mechanisms about the immunosuppressive effect of IDO in tumor or inflammatory diseases.

Snail regulated by PKC/GSK-3ß pathway is crucial for EGF-induced epithelial-mesenchymal transition (EMT) of cancer cells.

Cancer metastasis is considered a major challenge in cancer therapy. Recently, epidermal growth factor (EGF)/epidermal growth factor receptor (EGFR) signaling has been shown to induce epithelial-mesenchymal transition (EMT) and thereby to promote cancer metastasis. However, the underlying mechanism has not been fully elucidated. We demonstrate that EGF can induce EMT in human prostate and lung cancer cells and thus promote invasion and migration. EGF-induced EMT has been characterized by the cells acquiring mesenchymal spindle-like morphology and increasing their expression of N-cadherin and fibronectin, with a concomitant decrease of E-cadherin. Both protein and mRNA expression of transcription factor Snail rapidly increases after EGF treatment. The knockdown of Snail significantly attenuates EGF-induced EMT, suggesting that Snail is crucial for this process. To determine the way that Snail is accumulated, we demonstrate (1) that EGF promotes the stability of Snail via inhibiting the activity of glycogen synthase kinase 3 beta (GSK-3ß), (2) that protein kinase C (PKC) rather than the phosphatidylinositide 3-kinase (PI3K)/protein kinase B (AKT) signaling pathway is responsible for GSK-3ß inhibition and (3) that GSK-3ß inhibition promotes the transcription of Snail. Taken together, these results reveal that the PKC/GSK-3ß signaling pathway controls both the stability and transcription of Snail, which is crucial for EMT induced by EGF in PC-3 and A549 cells. Our study suggests a novel signaling pathway for Snail regulation and provides a better understanding of growth-factor-induced tumor EMT and metastasis.

Time to Local Recurrence as a Predictor of Survival in Adult Head and Neck Soft Tissue Sarcoma.

OBJECTIVES: We sought to evaluate the impact of the time interval from surgical resection to local recurrence (TTLR) on clinical outcomes in head and neck soft tissue sarcoma (HNSTS). METHODS: A total of 401 patients who underwent R0 resection for primary HNSTS were included in this study. Patients with local recurrence as the first event after their initial resection were divided into early local recurrence (ELR) or late local recurrence (LLR) groups according to TTLR. Multiple survival analyses were performed to identify the independent prognostic predictors of overall survival (OS) and survival after local recurrence (SAR). RESULTS: Two hundred and nine of the 401 patients (52.1%) developed local recurrence during a median follow-up period of 134.6 months. Patients in the ELR group had a shorter median OS time (35.0 vs. 120.6, p < 0.001) and lower 5-year OS rate (47.7% vs. 80.9%, p < 0.001) than those in the LLR group. Moreover, the ELR group exhibited worse SAR (p = 0.001) than the LLR group, and multivariate analyses demonstrated TTLR as an independent prognostic factor for SAR (p = 0.048) and OS (p = 0.004). Additionally, re-resection significantly prolonged SAR than other salvage interventions or no treatment (p < 0.001). CONCLUSION: In patients with HNSTS, ELR after R0 resection presents adverse effects on OS and SAR than those with LLR, and TTLR could serve as a promising predictor for survival. Salvage therapies, especially the re-resection could improve SAR and should be recommended when there are surgical indications after recurrence. LEVEL OF EVIDENCE: 3 Laryngoscope, 133:2174-2182, 2023.

Expression of indoleamine 2,3-dioxygenase in nasopharyngeal carcinoma impairs the cytolytic function of peripheral blood lymphocytes.

BACKGROUND: Tumor-specific cytotoxic T cells and infiltrating lymphocytes are frequently found in tumor tissues in patients with nasopharyngeal carcinoma (NPC). Most patients with NPC, however, especially those with advanced stages, have a poor clinical prognosis despite conventional immunotherapy. The aim of this work was to examine the effect of indoleamine 2,3-dioxygenase (IDO), an immunosuppressive enzyme, on the lymphocyte function in NPC. METHODS: The NPC cell line CNE2 was treated by interferon-gamma (IFNgamma) and the levels of IDO expression was analyzed by Western blotting and reverse phase high-performance liquid chromatography (HPLC). Lymphocytes from health human exposed to the milieu created by IDO-positive CNE2 cells and the lymphocyte cytotoxicity to target tumor cells was analyzed by standard lactate dehydrogenase (LDH) release assay. Additionally, expression of IDO was determined by Immunohistochemical assay in the tumor tissues form clinically evaluated NPC. RESULTS: IDO expression was acutely induced in the NPC cell line CNE2 by low dose interferon-gamma (IFNgamma) or by co-incubation with activated lymphocytes. Exposure to the milieu created by IDO-positive CNE2 cells did not promote lymphocyte death, but lymphocyte cytotoxicity against target tumor cells was impaired. The suppression of lymphocyte cytotoxic function was fully restored when the conditioned medium was replaced by fresh medium for 24 h. In additionally, the IDO-positive cells were found scattered in the tumor tissues from patients with NPC. CONCLUSION: Altogether, these findings suggest that IDO-mediated immunosuppression may be involved in the tumor immune evasion, and that blocking IDO activity in tumor cells may help to re-establish an effective anti-tumor T cell response in NPC.

[Preliminary study on molecular mechanism of curcumine anti-mouse melanoma].

OBJECTIVE: To investigate the effects of curcumine on mouse B16 melanoma growth and possible mechanism of Bcl-2, P53 and glutathione in tumor cells. METHODS: The inhibitory effect on growth of melanoma in vivo were examined by mice melanoma models transplanted B16 cells to C57BL/6J mice. MTT method was used to assay the contribution of curcumine to B16 cells in vitro. The apoptosis and expression of Bcl-2, P53 gene of B16 cells were analyzed by flow cytometry, and HPLC assay was used to detect the change of GSH in B16 melanom tissues of C57BL/6J mouse caused by curcumine. RESULTS: Curcumine had obvious inhibitory effect on the growth of mouse B16 melanoma in time and dose dependent manner and the gene expression of bcl-2 in B16 cells decreased after 24 hours supplied with curcumine, whereas P53 protein expression increased; Curcumine depressed the GSH quantity in melanoma tissues. CONCLUSION: The growth inhibitory effect of curcumine on mouse melanom is proved in vivo and in vitro respectively. Curcumine can induce some cells to apoptosis which may be relevant to downregulation of bcl-2 expression and upregulation of P53 expression as well as exhaustion of GSH in tumor organization.

AP-1 confers resistance to anti-cancer therapy by activating XIAP.

The underlying cause of treatment failure in many cancer patients is intrinsic and acquired resistance to chemotherapy. Recently, histone deacetylase (HDAC) inhibitors have developed into a promising cancer treatment. However, resistance mechanism induced by HDAC inhibitors remains largely unknown. Here we report that a HDAC inhibitor, JNJ-2648158 induced transcription of XIAP by activating AP-1 expression, which conferring resistance to chemotherapeutics. Our results showed that high expression of c-Fos caused by HDAC inhibitor promoted AP-1 formation during acquired resistance towards chemo-drugs, indicating an extremely poor clinical outcome in breast cancers and liver cancers. Our study reveals a novel regulatory mechanism towards chemo-drug resistance, and suggests that XIAP may serve as a potential therapeutic target in those chemo-resistant cancer cells.

Identification of TBK1 and IKKε, the non-canonical IκB kinases, as crucial pro-survival factors in HTLV-1-transformed T lymphocytes.

Persistent activation of NF-κB is a prerequisite for development of adult T cell leukemia-lymphoma (ATL) caused by human T cell leukemia virus type 1 (HTLV-1). HTLV-1 genome encodes a viral transforming protein named Tax, which constitutively activates the canonical IκB kinases (IKK), the central regulator of NF-κB signaling. However, the role of the non-canonical IκB kinases, TBK1 and IKKε, in the pathogenesis of HTLV-1-associated leukemia has not been evaluated. We here show that TBK1/IKKε are crucial pro-survival molecules by maintaining persistent activity of Stat3. Consistent with this finding, silencing Stat3 by the specific shRNA or by the chemical inhibitor ruxolitinib results in drastic impediment of leukemia cell growth. We further find that in HTLV-1-transformed T cells expressing Tax, TBK1 co-localizes with the canonical IκB kinases and Tax in the lipid raft microdomains. The wild type Tax, but not the Tax mutant defective in activating the canonical IKK, promotes the lipid raft translocation of TBK1. This phenomenon correlates with Tax activation of both NF-κB and Stat3. Tax does not interact directly with TBK1/IKKε, and it rather engages a molecular crosstalk between the canonical IKKs and TBK1/IKKε. Our data, therefore, demonstrate a key role of TBK1/IKKε in the survival and proliferation of HTLV-1-transformed T cells and implicate a potential therapy targeting TBK1/IKKε and Stat3 in controlling HTLV-1-mediated oncogenesis.

CCL21 Facilitates Chemoresistance and Cancer Stem Cell-Like Properties of Colorectal Cancer Cells through AKT/GSK-3ß/Snail Signals.

Some evidence indicated that chemoresistance associates with the acquisition of cancer stem-like properties. Recent studies suggested that chemokines can promote the chemoresistance and stem cell properties in various cancer cells, while the underling mechanism is still not completely illustrated. In our study, we found that CCL21 can upregulate the expression of P-glycoprotein (P-gp) and stem cell property markers such as Bmi-1, Nanog, and OCT-4 in colorectal cancer (CRC) HCT116 cells and then improve the cell survival rate and mammosphere formation. Our results suggested that Snail was crucial for CCL21-mediated chemoresistance and cancer stem cell property in CRC cells. Further, we observed that CCL21 treatment increased the protein but not mRNA levels of Snail, which suggested that CCL21 upregulates Snail via posttranscriptional ways. The downstream signals AKT/GSK-3ß mediated CCL21 induced the upregulation of Snail due to the fact that CCL21 treatment can obviously phosphorylate both AKT and GSK-3ß. The inhibitor of PI3K/Akt, LY294002 significantly abolished CCL21 induced chemoresistance and mammosphere formation of HCT116 cells. Collectively, our results in the present study revealed that CCL21 can facilitate chemoresistance and stem cell property of CRC cells via the upregulation of P-gp, Bmi-1, Nanog, and OCT-4 through AKT/GSK-3ß/Snail signals, which suggested a potential therapeutic approach to CRC patients.

Asclepiasterol, a novel C21 steroidal glycoside derived from Asclepias curassavica, reverses tumor multidrug resistance by down-regulating P-glycoprotein expression.

Multidrug resistance (MDR) mediated by P-glycoprotein (P-gp) is a major cause of cancer therapy failure. In this study, we identified a novel C21 steroidal glycoside, asclepiasterol, capable of reversing P-gp-mediated MDR. Asclepiasterol (2.5 and 5.0µM) enhanced the cytotoxity of P-gp substrate anticancer drugs in MCF-7/ADR and HepG-2/ADM cells. MDR cells were more responsive to paclitaxel in the presence of asclepiasterol, and colony formation of MDR cells was only reduced upon treatment with a combination of asclepiasterol and doxorubicin. Consistent with these findings, asclepiasterol treatment increased the intracellular accumulation of doxorubicin and rhodamine 123 (Rh123) in MDR cells. Asclepiasterol decreased expression of P-gp protein without stimulating or suppressing MDR1 mRNA levels. Asclepiasterol-mediated P-gp suppression caused inhibition of ERK1/2 phosphorylation in two MDR cell types, and EGF, an activator of the MAPK/ERK pathway, reversed the P-gp down-regulation, implicating the MAPK/ERK pathway in asclepiasterol-mediated P-gp down-regulation. These results suggest that asclepiasterol could be developed as a modulator for reversing P-gp-mediated MDR in P-gp-overexpressing cancer variants.

Autophagy is involved in TGF-ß1-induced protective mechanisms and formation of cancer-associated fibroblasts phenotype in tumor microenvironment.

Transforming growth factor-ß1 (TGF-ß1) present in tumor microenvironment acts in a coordinated fashion to either suppress or promote tumor development. However, the molecular mechanisms underlying the effects of TGF-ß1 on tumor microenvironment are not well understood. Our clinical data showed a positive association between TGF-ß1 expression and cancer-associated fibroblasts (CAFs) in tumor microenvironment of breast cancer patients. Thus we employed starved NIH3T3 fibroblasts in vitro and 4T1 cells mixed with NIH3T3 fibroblasts xenograft model in vivo to simulate nutritional deprivation of tumor microenvironment to explore the effects of TGF-ß1. We demonstrated that TGF-ß1 protected NIH3T3 fibroblasts from Star-induced growth inhibition, mitochondrial damage and cell apoptosis. Interestingly, TGF-ß1 induced the formation of CAFs phenotype in starvation (Star)-treated NIH3T3 fibroblasts and xenografted Balb/c mice, which promoted breast cancer tumor growth. In both models, autophagy agonist rapamycin increased TGF-ß1-induced protective effects and formation of CAFs phenotypes, while autophagy inhibitor 3-methyladenine, Atg5 knockdown or TGF-ß type I receptor kinase inhibitor LY-2157299 blocked TGF-ß1 induced these effects. Taken together, our results indicated that TGF-ß/Smad autophagy was involved in TGF-ß1-induced protective effects and formation of CAFs phenotype in tumor microenvironment, which may be used as therapy targets in breast cancer.

A SIRT3/AMPK/autophagy network orchestrates the protective effects of trans-resveratrol in stressed peritoneal macrophages and RAW 264.7 macrophages.

Resveratrol gains a great interest for its strong antioxidant properties, while the molecular mechanisms underlie the beneficial effects on psychosocial stress remain controversial. In this study, we demonstrated that resveratrol protected peritoneal macrophages and RAW 264.7 cells from stress-induced decrease in the total cell count, phagocytic capability, reactive oxygen species generation, monodansylcadaverine and mitochondrial membrane potential in stressed mice. Resveratrol promoted stress-induced autophagy in both models. Modulation of autophagy by rapamycin or 3-methyladenine regulated the protective effect of resveratrol, suggesting a role of autophagy in the protective mechanisms of resveratrol. The comparison studies revealed that distinct mechanisms were implicated in the protective effect of resveratrol and other antioxidants (vitamin C and edaravone). Resveratrol promoted autophagy via upregulating SIRT3 expression and phosphorylation of AMP-activated protein kinase (AMPK). Knockdown of SIRT3 resulted in decreased autophagy and abolished protective effect of resveratrol. SIRT1 was also involved in the protective mechanism of resveratrol, although its effect on autophagy was unnoticeable. Pharmacological manipulation of autophagy modulated the effects of resveratrol on SIRT3 and AMPK, revealing the engagement of a positive feedback loop. In sharp contrast, vitamin C and edaravone effectively protected macrophages from stress-induced cytotoxicity, accompanied by downregulated SIRT3 expression and AMPK phosphorylation, and decreased level of autophagy response. Taken together, we conclude that a SIRT3/AMPK/autophagy network orchestrates in the protective effect of resveratrol in macrophages.

Endogenous Nodal promotes melanoma undergoing epithelial-mesenchymal transition via Snail and Slug in vitro and in vivo.

Nodal, an important embryonic morphogen, has been reported to modulate tumorigenesis. Epithelial-mesenchymal transition (EMT) plays an important role in cancer metastasis. We have previously reported that recombinant Nodal treatment can promote melanoma undergoing EMT, but the effects of endogenous Nodal on EMT are still unknown. Here we generated both Nodal-overexpression and -knockdown stable cell lines to investigate the in vitro and in vivo characteristics of Nodal-induced EMT in murine melanoma cells. Nodal-overexpression cells displayed increased migration ability, accompanied by typical phenotype changes of EMT. In contrast, Nodal-knockdown stable cells repressed the EMT phenotype as well as reduced cell motility. Results of animal experiments confirmed that overexpression of Nodal can promote the metastasis of melanoma tumor in vivo. Mechanistically, we found that Nodal-induced expression of Snail and Slug involves its activation of ALK/Smads and PI3k/AKT pathways, which is an important process in the Nodal-induced EMT. However, we also found that the EMT phenotype was not completely inhibited by blocking the paracrine activity of Nodal in Nodal overexpression cell line suggesting the presence of additional mechanism(s) in the Nodal-induced EMT. This study provides a better understanding of Nodal function in melanoma, and suggests targeting Nodal as a potential strategy for melanoma therapey.

TGF-ß and EGF induced HLA-I downregulation is associated with epithelial-mesenchymal transition (EMT) through upregulation of snail in prostate cancer cells.

Human leukocyte antigen class I antigens (HLA-I) is essential in immune response by presenting antigenic peptides to cytotoxic T lymphocytes. Downregulation of HLA-I is observed in primary and metastatic prostate cancers, which facilitates them escape from immune surveillance, thereby promotes prostate cancer progression. In addition, elevated level of growth factors like TGF-ß or EGF in microenvironment is related to the prostate cancer deterioration. Thus, we wondered whether TGF-ß or EGF was involved in the regulation of HLA-I during the development of prostate cancer cells. In this study, we demonstrated that TGF-ß and EGF both downregulated the expression of HLA-I, thereby attenuated the cytotoxic T cell mediated lysis of prostate cancer cells. Next, we revealed that TGF-ß and EGF induced downregulation of HLA-I is associated with classical epithelial-mesenchymal transition (EMT) morphological changes and expression profiles. We further illustrated that overexpression of Snail is crucial for HLA-I downregulation and its association with EMT. At last, we discussed that NF-κB/p65 is the plausible target for Snail to induce HLA-I downregulation. Taken together, this is the first evidence to reveal that both TGF-ß and EGF can induce HLA-I downregulation which is then proven to be associated with EMT in prostate cancer cells. These discoveries provide a deeper understanding of growth factors induced immune escape and introduce potential therapeutic targets for prostate cancers.