PLAC1-specific TCR-engineered T cells mediate antigen-specific antitumor effects in breast cancer.

Placenta-specific 1 (PLAC1), a novel cancer-testis antigen (CTA), is expressed in a number of different human malignancies. It is frequently produced in breast cancer, serving a function in tumorigenesis. Adoptive immunotherapy using T cell receptor (TCR)-engineered T cells against CTA mediates objective tumor regression; however, to the best of our knowledge, targeting PLAC1 using engineered T cells has not yet been attempted. In the present study, the cDNAs encoding TCRα- and ß-chains specific for human leukocyte antigen (HLA)-A*0201-restricted PLAC1 were cloned from a cytotoxic T-lymphocyte, generated by in vitro by the stimulation of CD8+ T cells using autologous HLA-A2+ dendritic cells loaded with a PLAC1-specific peptide (p28-36, VLCSIDWFM). The TCRα/ß-chains were linked by a 2A peptide linker (TCRα-Thosea asigna virus-TCRß), and the constructs were cloned into the lentiviral vector, followed by transduction into human cytotoxic (CD8+) T cells. The efficiency of transduction was up to 25.16%, as detected by PLAC1 multimers. TCR-transduced CD8+ T cells, co-cultured with human non-metastatic breast cancer MCF-7 cells (PLAC1+, HLA-A2+) and triple-negative breast cancer MDAMB-231 cells (PLAC1+, HLA-A2+), produced interferon γ and tumor necrosis factor α, suggesting TCR activation. Furthermore, the PLAC1 TCR-transduced CD8+ T cells efficiently and specifically identified and annihilated the HLA-A2+/PLAC1+ breast cancer cell lines in a lactate dehydrogenase activity assay. Western blot analysis demonstrated that TCR transduction stimulated the production of mitogen-activated protein kinase signaling molecules, extracellular signal-regulated kinases 1/2 and nuclear factor-κB, through phosphoinositide 3-kinase γ-mediated phosphorylation of protein kinase B in CD8+ T cells. Xenograft mouse assays revealed that PLAC1 TCR-transduced CD8+T cells significantly delayed the tumor progression in mice-bearing breast cancer compared with normal saline or negative control-transduced groups. In conclusion, a novel HLA-A2-restricted and PLAC1-specific TCR was identified. The present study demonstrated PLAC1 to be a potential target for breast cancer treatment; and the usage of PLAC1-specific TCR-engineered T cells may be a novel strategy for PLAC1-positive breast cancer treatment.

Therapeutic Potential of HGF-Expressing Human Umbilical Cord Mesenchymal Stem Cells in Mice with Acute Liver Failure.

Human umbilical cord-derived mesenchymal stem cells (UCMSCs) are particularly attractive cells for cellular and gene therapy in acute liver failure (ALF). However, the efficacy of this cell therapy in animal studies needs to be significantly improved before it can be translated into clinics. In this study, we investigated the therapeutic potential of UCMSCs that overexpress hepatocyte growth factor (HGF) in an acetaminophen-induced acute liver failure mouse model. We found that the HGF-UCMSC cell therapy protected animals from acute liver failure by reducing liver damage and prolonging animal survival. The therapeutic effect of HGF-UCMSCs was associated with the increment in serum glutathione (GSH) and hepatic enzymes that maintain redox homeostasis, including γ-glutamylcysteine synthetase (γ-GCS), superoxide dismutase (SOD), and catalase (CAT). Immunohistochemical staining confirmed that HGF-UCMSCs were mobilized to the injured areas of the liver. Additionally, HGF-UCMSCs modulated apoptosis by upregulating the antiapoptotic Bcl2 and downregulating proapoptotic genes, including Bax and TNFα. Taken together, these data suggest that ectopic expression of HGF in UCMSCs protects animals from acetaminophen-induced acute liver failure through antiapoptosis and antioxidation mechanisms.

Cytotoxicity of various types of gold-mesoporous silica nanoparticles in human breast cancer cells.

Recently, gold nanoparticles (AuNPs) have shown promising biological applications due to their unique electronic and optical properties. However, the potential toxicity of AuNPs remains a major hurdle that impedes their use in clinical settings. Mesoporous silica is very suitable for the use as a coating material for AuNPs and might not only reduce the cytotoxicity of cetyltrimethylammonium bromide-coated AuNPs but might also facilitate the loading and delivery of drugs. Herein, three types of rod-like gold-mesoporous silica nanoparticles (termed bare AuNPs, core-shell Au@mSiO2NPs, and Janus Au@mSiO2NPs) were specially designed, and the effects of these AuNPs on cellular uptake, toxic behavior, and mechanism were then systematically studied. Our results indicate that bare AuNPs exerted higher toxicity than the Au@mSiO2NPs and that Janus Au@mSiO2NPs exhibited the lowest toxicity in human breast cancer MCF-7 cells, consistent with the endocytosis capacity of the nanoparticles, which followed the order, bare AuNPs > core-shell Au@mSiO2NPs > Janus Au@mSiO2NPs. More importantly, the AuNPs-induced apoptosis of MCF-7 cells exhibited features that were characteristic of intracellular reactive oxygen species (ROS) generation, activation of c-Jun-N-terminal kinase (JNK) phosphorylation, an enhanced Bax-to-Bcl-2 ratio, and loss of the mitochondrial membrane potential. Simultaneously, cytochrome c was released from mitochondria, and the caspase-3/9 cascade was activated. Moreover, both ROS scavenger (N-acetylcysteine) and JNK inhibitor (SP600125) partly blocked the induction of apoptosis in all AuNPs-treated cells. Taken together, these findings suggest that all AuNPs induce apoptosis through the ROS-/JNK-mediated mitochondrial pathway. Thus, Janus Au@mSiO2NPs exhibit the potential for applications in biomedicine, thus aiding the clinical translation of AuNPs.

Mucin 1 gene silencing inhibits the growth of SMMC-7721 human hepatoma cells through Bax-mediated mitochondrial and caspase-8-mediated death receptor apoptotic pathways.

Mucin 1 (MUC1) is an oncogene that has a crucial role in the pathogenesis and progression of the majority of epithelial malignant tumors. Our previous study demonstrated that MUC1 gene silencing inhibited the growth of SMMC­7721 cells in vitro and in vivo, however, whether this growth inhibition is associated with apoptotic cell death remains to be elucidated. In the present study, it was found that MUC1 gene silencing not only resulted in the inhibition of SMMC­7721 cell growth, determined using a clone formation assay in vitro and a tumor xenograft mouse model with an in vivo imaging system, but also induced apoptotic alterations in SMMC­7721 cells, determined using Hoechst 33342 staining, flow cytometry with an Annexin V-PE staining and a DNA ladder assay. Further investigation using western blotting revealed that cytochrome c was released from the mitochondria into the cytoplasm, and caspase­8 and caspase­9 were activated in MUC1 gene­silenced SMMC­7721 cells. The pro­apoptotic protein Bcl­2­associated X protein (Bax) and the tumor suppressor p53 were increased, while the anti­apoptotic protein B­cell lymphoma 2 was decreased in MUC1 gene­silenced cells. In addition, results from the co­immunoprecipitation experiments demonstrated that the MUC1 cytoplasmic tail can bind directly to Bax or caspase­8 and these interactions were reduced upon MUC1 gene silencing in SMMC­7721 cells. The above results indicate that MUC1 gene silencing induces growth inhibition in SMMC­7721 cells through Bax­mediated mitochondrial and caspase-8-mediated death receptor apoptotic pathways.

Mucin1 promotes the migration and invasion of hepatocellular carcinoma cells via JNK-mediated phosphorylation of Smad2 at the C-terminal and linker regions.

Mucin1 (MUC1), as an oncogene, plays a key role in the progression and tumorigenesis of many human adenocarcinomas. In this study, wound-healing, transwell migration and matrigel invasion assays showed that MUC1 promotes human hepatocellular carcinoma (HCC) cell migration and invasion by MUC1 gene silencing and overexpressing. Treatment with exogenous transforming growth factor beta (TGF-ß)1, TGF-ß type I receptor (TßRI) inhibitor, TGF-ß1 siRNAs, or activator protein 1 (AP-1) inhibitor to MUC1-overexpressing HCC cells revealed that MUC1-induced autocrine TGF-ß via JNK/AP-1 pathway promotes the cell migration and invasion. In addition, the migration and invasion of HCC cells were more significantly inhibited by JNK inhibitor compared with that by TßRI inhibitor or TGF-ß1 siRNAs. Further studies demonstrated that MUC1-mediated JNK activation not only enhances the phosphorylation of Smad2 C-terminal at Ser-465/467 site (Smad2C) through TGF-ß/TßRI, but also directly enhances the phosphorylation of Smad2 linker region at Ser-245/250/255 site (Smad2L), and then both of them collaborate to upregulate matrix metalloproteinase (MMP)-9-mediated cell migration and invasion of HCC. These results indicate that MUC1 is an attractive target in liver cancer therapy.

Mucin1 shifts Smad3 signaling from the tumor-suppressive pSmad3C/p21(WAF1) pathway to the oncogenic pSmad3L/c-Myc pathway by activating JNK in human hepatocellular carcinoma cells.

Mucin1 (MUC1) is a transmembrane glycoprotein that acts as an oncogene in human hepatic tumorigenesis. Hepatocellular carcinoma (HCC) cells often gain advantage by reducing the tumor-suppressive activity of transforming growth factor beta (TGF-ß) together with stimulation of its oncogenic activity as in MUC1 expressing HCC cells; however, molecular mechanisms remain largely unknown. Type I TGF-ß receptor (TßRI) and c-Jun NH2-terminal kinase (JNK) differentially phosphorylate Smad3 mediator to create 2 phosphorylated forms: COOH-terminally phosphorylated Smad3 (pSmad3C) and linker-phosphorylated Smad3 (pSmad3L). Here, we report that MUC1 overexpression in HCC cell lines suppresses TßRI-mediated pSmad3C signaling which involves growth inhibition by up-regulating p21(WAF1). Instead, MUC1 directly activates JNK to stimulate oncogenic pSmad3L signaling, which fosters cell proliferation by up-regulating c-Myc. Conversely, MUC1 gene silencing in MUC1 expressing HCC cells results in preserved tumor-suppressive function via pSmad3C, while eliminating pSmad3L-mediated oncogenic activity both in vitro and in vivo. In addition, high correlation between MUC1 and pSmad3L/c-Myc but not pSmad3C/p21(WAF1) expression was observed in HCC tissues from patients. Collectively, these results indicate that MUC1 shifts Smad3 signaling from a tumor-suppressive pSmad3C/p21(WAF1) to an oncogenic pSmad3L/c-Myc pathway by directly activating JNK in HCC cells, suggesting that MUC1 is an important target for HCC therapy.

Escherichia coli maltose-binding protein (MBP) directly induces mouse Th1 activation through upregulating TLR2 and downregulating TLR4 expressions.

Maltose-binding protein (MBP), a component of the maltose transport system of Escherichia coli, has been commonly thought to have minimal bioactivity. Our previous studies demonstrated that MBP could significantly enhance Bacillus Calmette-Guerin (BCG)-induced T helper 1 (Th1) cell activation in mice. In the present study, we analyzed the effect of MBP on mouse T cells and found that MBP promoted the proliferation and IFN-γ production of CD4(+) T cells, suggesting that MBP directly induces Th1 activation. To explore the mechanism of Th1 activation, the expression of Toll-like receptor 2/4 (TLR2/4) on purified mouse CD4(+) T cells was detected. The results showed that MBP up-regulated TLR2 while down-regulated TLR4 expression, accompanied by a clear increase in MyD88 expression and IκB phosphorylation. Notably, the addition of anti-TLR2 antibody abrogated the MBP-induced CD4(+) T cells proliferation, IFN-γ secretion and MyD88 expression, whereas the addition of anti-TLR4 antibody exhibited a contradictive effect. Besides, the block of either TLR2 or TLR4 both reduced IκB phosphorylation. These results above suggest that TLR2-mediated MyD88-dependent pathway contributes to MBP-induced Th1 activation, while TLR4 appears to counteract this effect via MyD88-independent pathway.

Mucin1 mediates autocrine transforming growth factor beta signaling through activating the c-Jun N-terminal kinase/activator protein 1 pathway in human hepatocellular carcinoma cells.

In a previous study, we observed by global gene expression analysis that oncogene mucin1 (MUC1) silencing decreased transforming growth factor beta (TGF-ß) signaling in the human hepatocellular carcinoma (HCC) cell line SMMC-7721. In this study, we report that MUC1 overexpression enhanced the levels of phosphorylated Smad3 linker region (p-Smad3L) (Ser-213) and its target gene MMP-9 in HCC cells, suggesting that MUC1 mediates TGF-ß signaling. To investigate the effect of MUC1 on TGF-ß signaling, we determined TGF-ß secretion in MUC1 gene silencing and overexpressing cell lines. MUC1 expression enhanced not only TGF-ß1 expression at the mRNA and protein levels but also luciferase activity driven by a TGF-ß promoter, as well as elevated the activation of c-Jun N-terminal kinase (JNK) and c-Jun, a member of the activation protein 1 (AP-1) transcription factor family. Furthermore, pharmacological reduction of TGF-ß receptor (TßR), JNK and c-Jun activity inhibited MUC1-induced autocrine TGF-ß signaling. Moreover, a co-immunoprecipitation assay showed that MUC1 directly bound and activated JNK. In addition, both MUC1-induced TGF-ß secretion and exogenous TGF-ß1 significantly increased Smad signaling and cell migration, which were markedly inhibited by either TßR inhibitor or small interfering RNA silencing of TGF-ß1 gene in HCC cells. The high correlation between MUC1 and TGF-ß1 or p-Smad3L (Ser-213) expression was shown in tumor tissues from HCC patients by immunohistochemical staining analysis. Collectively, these results indicate that MUC1 mediates autocrine TGF-ß signaling by activating the JNK/AP-1 pathway in HCC cells. Therefore, MUC1 plays a key role in HCC progression and could serve as an attractive target for HCC therapy.

MUC1 and maltose­binding protein recombinant fusion protein combined with Bacillus Calmette­Guerin induces MUC1­specific and nonspecific anti­tumor immunity in mice.

Human mucin 1 (MUC1) is a target for immunotherapy. The major problem associated with MUC1­based cancer vaccines is the weakness of the immunogenicity of MUC1. The present study aimed to develop an efficient cancer vaccine through generating a recombinant fusion protein consisting of MUC1 and maltose­binding protein (MBP) by inserting seven tandem repeats encoding the human MUC1 gene into the pMAL­c2 expression vector. Bacillus Calmette­Guerin (BCG) was used as an adjuvant. MUC1 was found to predominantly induce T helper type 2 (Th2) cell responses. MUC1/BCG and MUC1­MBP were found to generate T helper (Th) type 1 and 2 responses, while MUC1­MBP/BCG induced a Th1 immune profile and stimulated MUC1­specific cytotoxic T lymphocyte killing activity. MUC1­MBP, as well as MBP and BCG alone were found to induce natural killer (NK) cell activity, with MUC1­MBP/BCG observed to synergistically induce NK cell activity. Furthermore, MUC1­MBP/BCG significantly inhibited MUC1+ B16 cell growth in mice. These findings show that MBP augments the immunogenicity of MUC1 and that BCG enhances the efficacy of the MUC1­MBP vaccine. Thus, MUC1­MBP/BCG may have potential as a cancer vaccine for clinical application.

Escherichia coli maltose-binding protein activates mouse peritoneal macrophages and induces M1 polarization via TLR2/4 in vivo and in vitro.

Maltose-binding protein (MBP) is a component of the maltose transport system of Escherichia coli. Our previous study found that MBP combined with Bacillus Calmette-Guerin (BCG) increases the percentage of activated macrophages in the spleen and the pinocytic activity of peritoneal macrophages in vivo. However, the effect of MBP alone on macrophages remains unclear. In the present study, the results showed that MBP enhanced LPS-stimulated macrophage activity in vivo. Subsequently, we investigated the regulatory effect of MBP on mouse peritoneal macrophages in vitro and the possible underlying mechanism. The results showed that MBP directly promoted macrophage phagocytic activity and increased the production of NO, IL-1ß and IL-6. Notably, macrophage phenotypic analysis showed that MBP significantly increased iNOS, IL-12p70 and CD16/32. In contrast, MBP decreased the secretion of IL-10 and slightly decreased Arg-1 mRNA and CD206 protein expression. These results suggested that MBP activated macrophages and polarized them into M1 macrophages. Further study found that MBP directly bound to macrophages and upregulated TLR2 mRNA expression. This process was accompanied by a clear increase in MyD88 expression and phosphorylation of p38 MAPK and IκB-α, but these effects were largely abrogated by pretreatment with anti-TLR2 or anti-TLR4 antibodies. The effects of MBP on macrophage NO production were also partially inhibited by anti-TLR2 and/or anti-TLR4 antibodies. Furthermore, the effect of MBP on IL-12 and IL-10 secretion was largely influenced by the NF-κB inhibitor PDTC and the p38 MAPK inhibitor SB203580. These results suggest that MBP directly activates macrophages and induces M1 polarization through a process that may involve TLR2 and TLR4.

Impact of Mucin1 knockdown on the phenotypic characteristics of the human hepatocellular carcinoma cell line SMMC-7721.

Mucin1 (MUC1) is a transmembrane glycoprotein that plays a key role as an oncogene in the tumorigenesis of many human adenocarcinomas. However, the role of MUC1 in human hepatocellular carcinoma (HCC) progression remains unclear. In the present study, we silenced MUC1 to investigate its effect on the human HCC cell line SMMC-7721 and found that knockdown of MUC1 significantly inhibited cell proliferation, enhanced cell-cell aggregation and induced apoptosis. No significant differences were found in in vitro migration or invasion. We also observed that knockdown of MUC1 decreased the translocation of ß­catenin to the nucleus, reduced the activity of T cell factor and blocked the expression of cyclin D1 and c-Myc. In addition, MUC1 knockdown enhanced the expression of E-cadherin, a molecular chaperone of ß­catenin that plays an important role in cell-cell aggregation. In vivo assays demonstrated that there was no tumor growth in mice injected with MUC1-silenced cells. Global gene expression analysis showed that a series of genes encoding molecules in the Wnt/ß­catenin, nuclear factor-κB (NF-κB), mitogen-activated protein kinase (MAPK), insulin, transforming growth factor ß (TGF-ß) and vascular endothelial growth factor (VEGF) signaling pathways were all influenced by the knockdown of MUC1, and these may contribute to the phenotypic alterations observed. Collectively, our results indicate that MUC1 plays a key role in HCC tumorigenesis.

Expression of human full-length MUC1 inhibits the proliferation and migration of a B16 mouse melanoma cell line.

Mucin 1 (MUC1) is a large transmembrane glycoprotein that is aberrantly overexpressed in most adenocarcinomas and certain hematological malignancies. MUC1 is known to function as an oncogene with roles in both tumor formation and progression, making it a potential target for immunotherapy. B16-MUC1 cells with human full-length MUC1 are frequently used to study the antitumor activities of MUC1-based vaccines. However, we found that the growth of B16-MUC1 cells was significantly reduced in vitro. Therefore, in this study, we established two MUC1-positive clones, B16-MUC1 9-12 and B16-MUC1 9-23, and one empty vector control clone, B16-neo, to investigate the effects of MUC1 on the cancer-related characteristics of B16 cells in vitro and in vivo. Our results demonstrated that, compared with MUC1-negative cells, cells expressing MUC1 exhibited decreased cell proliferation, increased cell cycle arrest and reduced cell migratory and invasive capacities. We further investigated several MUC1-related molecules of the ß-catenin pathway, and found that the expression of MUC1 decreased the translocation of ß-catenin into the nucleus, reduced the activity of T cell factor (TCF) and blocked the expression of cyclin D1 and c-Myc. Moreover, when inoculated into BALB/c nude mice, cells expressing MUC1 developed smaller tumors compared with the control cells. These results demonstrate that MUC1 expression negatively affects the malignancy of B16 cells, and suggest that the regulatory mechanisms of MUC1 as an oncoprotein are more complex than previously appreciated.