PDGF-BB/PDGFRß promotes epithelial-mesenchymal transition by affecting PI3K/AKT/mTOR-driven aerobic glycolysis in Wilms' tumor G401 cells.

Wilms' tumor (WT) is the most common pediatric renal malignancy. PDGFRß belongs to the type III receptor tyrosine kinase family and is known to be involved in tumor metastasis and angiogenesis. Here, we studied the effect and underlying mechanism of PDGFRß on WT G401 cells. Transwell assay and wound-healing assay were used to detect the effect of PDGFRß on G401 cells invasion and migration. Western blot and immunofluorescence were used to detect the expression of EMT-related genes. The expression of PI3K/AKT/mTOR pathway proteins was detected by Western blot. The relationship between PDGFRß and aerobic glycolysis was studied by assessing the expression of glycolysis-related enzymes detected by qRT-PCR and Western blot. The activity of HK, PK, and LDH was detected by corresponding enzyme activity kits. The concentration of lactic acid and glucose was detected by Lactic Acid Assay Kit and Glucose Assay Kit-glucose oxidase method separately. To investigate the mechanism of PDGFRß in the development of WT, the changes of glucose and lactic acid were analyzed after blocking PI3K pathway, aerobic glycolysis, or PDGFRß. The key enzyme was screened by Western blot and glucose metabolism experiment after HK2, PKM2, and PDK1 were inhibited. The results showed that PDGFRß promoted the EMT process by modulating aerobic glycolysis through PI3K/AKT/mTOR pathway in which PKM2 plays a key role. Therefore, our study of the mechanism of PDGFRß in G401 cells provides a new target for the treatment of WT.

Influence of Dichloroacetate on Wilms'Tumor in vitro.

OBJECTIVE: To investigate the effect of dichloroacetate (DCA) on Wilms' tumor (WT) G401 cells. METHODS: CCK-8 assay was used to detect the influence of DCA on G401 cells viability and 10 mmol/L DCA was selected for subsequent experiments. The expression of glycolysis-related enzymes, such as hexokinase 2 (HK2), pyruvate kinase M2 (PKM2), lactic acid dehydrogenase A (LDHA), pyruvate dehydrogenase kinase 1 (PDK1), and pyruvate dehydrogenase (PDH), were detected by qRT-PCR and western blot. The extracellular lactic acid and glucose concentrations were measured by the lactic acid assay kit and glucose oxidase method kit respectively. Flow cytometry was used to detect the effect of DCA on G401 cells apoptosis. The invasion and migration ability of G401 cells were detected by Transwell assay and wound-healing assay. RESULTS: The results showed that DCA reduced glycolysis-related enzymes expression, inhibited lactic acid production, and glucose consumption. DCA also suppressed cells growth, induced cells apoptosis and inhibited cells invasion and migration. CONCLUSION: Inhibition of aerobic glycolysis by DCA can reduce the viability of G401 cells, promote cells apoptosis and inhibit cells invasion and migration. Therefore, aerobic glycolysis may be a potential therapeutic target for Wilms' tumor.

Influence of glucose transporter 1 activity inhibition on neuroblastoma in vitro.

Most cancer cells predominantly produce their energy through a high rate of glycolysis in the presence of abundant oxygen. Glycolysis has become a target of anticancer strategies. Previous researches showed that glucose transporter 1 (GLUT1) inhibitor is effective as anticancer agents. This study assessed the effects of the selective GLUT1 inhibitor WZB117 on regulation of neuroblastoma (NB) cell line SH-SY5Y viability, cell cycle and glycolysis in vitro. SH-SY5Y cells were grown and treated with WZB117 for up to 72 h and then subjected to cell viability, qRT-PCR, Western blot and flow cytometry analysis. Level of ATP and LDH was also analyzed. The result showed that WZB117 treatment reduced tumor cells viability, downregulated level of GLUT1 protein. Moreover, WZB117 treatment arrested tumor cells at the G0-G1 phase of the cell cycle, induced tumor cells to undergo necrosis instead of apoptosis. In addition, WZB117 treatment downregulated the levels of intracellular ATP, LDH and glycolytic enzymes. Thus, WZB117-induced GLUT1 inhibition suppressed tumor cell growth, induced cell cycle arrest and reduced glycolysis metabolites in NB cells in vitro. This study suggested that GLUT1 can be used as a potential therapeutic target for NB.

Melanoma differentiation-associated gene-7/interleukin 24 inhibits invasion and migration of human cervical cancer cells in vitro.

OBJECTIVE: In this study, we used an adenoviral vector -melanoma differentiation-associated gene-7 (Ad-mda7) to examine the effect of the ectopic production of MDA-7/IL-24 on cell migration and invasion by human cervical cancer cells. METHODS: The study took place in the Department of Biochemistry and Molecular Biology, Chongqing Medical University, Chongqing, China, between April 2006 and November 2006. The change of metastasis of cervical cancer cells (CaSki) cells were detected by Cell Migration Assay and Cell Invasion Assay after treated with Ad-mda7. The production of proteins associated with cell migration and invasion were detected by western blot. RESULTS: Cervical cancer cells treated in vitro with Ad-mda7 migrated and invaded less than cells treated with phosphate-buffered saline (PBS) or Ad-Luc (vector control). Melanoma differentiation-associated gene-7 /IL-24 inhibited migration and invasion by down-regulating the production of matrix metalloproteinase-2 (MMP-2) and by up-regulating the production of p38 mitogen-activated protein kinase. relative to PBS and Ad-Luc. CONCLUSION: These results show that MDA-7/IL-24 inhibits invasion and migration by cervical cancer cells by down- or up- regulating proteins associated with these processes, resulting in reduced metastasis. Thus, Ad-mda7 should be considered a therapeutic agent that can inhibit primary tumor growth and prevent metastasis.

An analysis on transcriptional regulation activity of human XBP1 gene 5' upstream DNA sequences.

OBJECTIVE: To analyze the transcription activation and possible regulation mechanism of human X-box binding protein 1(XBP1)gene 5'upstream DNA sequence in different cell lines. METHODS: Six kinds of XBP1 promoter deletion mutants were cloned into pGEM-Teasy vector, which included XBP1 gene 5' upstream -1039 to 66 bp,-859 to 66 bp,-623 to 66 bp,-351 to 66 bp,-227 to 66 bp,-227 to -45 bp respectively. Every deletion mutant sequence was cut from Teasy-XBP1p by KpnI and Xho I, and subcloned into pCAT3-Basic to produce a set of constructs termed as p1-XBP1p, p2-XBP1p, p3-XBP1p, p4-XBP1p, p5-XBP1p, p6-XBP1p, respectively. The transcription activity of each construct was detected after transiently transfecting K562, HepG2,NIH-3T3 and L0(2)cell with FuGENE 6 transfection reagent. Cells transfected by pCAT3-Basic or pCAT3-Promoter were used as negative and positive controls. The activity of chloramphenicol acetyltransferase(CAT), which reflects the transcription activation of the XBP1 gene promoter, was detected by ELISA after 48 hours of transfection. RESULTS: The reporter vectors of six kinds of XBP1 promoter deletion mutants were successfully constructed, as confirmed by restriction enzyme digestion and sequencing. The activities of p4-XBP1p and p5-XBP1p were higher than the other deletion mutants in K562 and HepG2. And the activity of p5-XBP1p was the highest in HepG2. There was no activity detected from any transfected NIH-3T3. CONCLUSION: The XBP1 gene promoter can transactivate its downstream gene to transcription. The core sequence of XBP1 promoter was implied between -227 bp and 66 bp. This sequence was connected with the transcriptional activity of XBP1 promoter closely. Its transcription activity varies with different cell lines. XBP1 promoter might drive gene expression with cell-type specificity.

Dendritic cells induce specific cytotoxic T lymphocytes against prostate cancer TRAMP-C2 cells loaded with freeze- thaw antigen and PEP-3 peptide.

Prostate cancer is the most common cancer in men. In this study, we investigated immune responses of cytotoxic T lymphocytes (CTLs) against TRAMP-C2 prostate cancer cells after activation by dendritic cells (DCs) loaded with TRAMP-C2 freeze-thaw antigen and/or PEP-3 peptide in vitro. Bone marrow-derived DC from the bone marrow of the C57BL/6 were induced to mature by using the cytokine of rhGM-CSF and rhIL-4, and loaded with either the freeze-thaw antigen or PEP-3 peptide or both of them. Maturation of DCs was detected by flow cytometry. The killing efficiency of the CTLs on TRAMP-C2 cells were detected by flow cytometry, CCK8, colony formation, transwell migration, and wound-healing assay. The levels of the IFN-γ, TNF-ß and IL-12 were measured by enzyme-linked immunosorbent assay (ELISA). Compared with the unloaded DCs, the loaded DCs had significantly increased expression of several phenotypes related to DC maturation. CTLs activated by DCs loaded with freeze-thaw antigen and PEP-3 peptide had more evident cytotoxicity against TRAMP-C2 cells in vitro. The secretion levels of IFN-γ, TNF-ß and IL-12, secreted by DCs loaded with antigen and PEP-3 and interaction with T cells, were higher than in the other groups. Our results suggest that the CTLs activated by DCs loaded with TRAMP-C2 freeze-thaw antigen and PEP-3 peptide exert a remarkable killing efficiency against TRAMP-C2 cells in vitro.

Cytotoxic T lymphocytes elicited by dendritic cell-targeted delivery of human papillomavirus type-16 E6/E7 fusion gene exert lethal effects on CaSki cells.

Human papillomavirus (HPV) is the primary etiologic agent of cervical cancer. Consideration of safety and non human leukocyte antigen restriction, protein vaccine has become the most likely form of HPV therapeutic vaccine, although none have so far been reported as effective. Since tumor cells consistently express the two proteins E6 and E7, most therapeutic vaccines target one or both of them. In this study, we fabricated DC vaccines by transducing replication-defective recombinant adenoviruses expressing E6/E7 fusion gene of HPV-16, to investigate the lethal effects of specific cytotoxic T lymphocytes (CTL) against CaSki cells in vitro. Mouse immature dendritic cells (DC) were generated from bone marrow, and transfected with pAd-E6/E7 to prepare a DC vaccine and to induce specific CTL. The surface expression of CD40, CD68, MHC II and CD11c was assessed by flow cytometry (FCM), and the lethal effects of CTL against CaSki cells were determined by DAPI, FCM and CCK-8 methods. Immature mouse DC was successfully transfected by pAd-E6/E7 in vitro, and the transfecting efficiency was 40%-50%. A DC vaccine was successfully prepared and was used to induce specific CTL. Experimental results showed that the percentage of apoptosis and killing rate of CaSki cells were significantly increased by coculturing with the specific CTL (p <0.05). These results illustrated that a DC vaccine modified by HPV-16 E6/E7 gene can induce apoptosis of CaSki cells by inducing CTL, which may be used as a new strategy for biological treatment of cervical cancer.

DAXX silencing suppresses mouse ovarian surface epithelial cell growth by inducing senescence and DNA damage.

Mouse ovarian surface epithelium (OSE) is a single layer of cubodial epithelial cells that covers the ovary surface and is involved in regulating the secretion and transport of 17ß-hydroxysteroid dehydrogenase. Recently, OSE cells have attracted particular interest as a major source of ovarian cancer. Death-associated protein DAXX along with PML (promyelocytic leukemia protein) nuclear bodies (PML-NBs) reportedly play roles in transcriptional regulation and apoptosis. However, little is known regarding a role for DAXX in mOSE cells. In this study, we both over-expressed DAXX and depleted DAXX in primary mOSE cells. We found that Daxx deletion accelerated senescence in a p53/p21-dependent manner and promoted DNA damage by interacting with PML bodies without affecting cell cycle progression. These results suggest that DAXX may transform mOSE cells to an ovarian oncogenic phenotype and may be an anti-cancer target.

[HPV caused pathological changes in genital system of mice].

The recombined adenovirus DNA was transfected into 293 cells for packing and amplification of replication-deficient Ad-CMV-E6/E7, Ad-K14 -E6/E7 virus was purified by CsCl density gradient centrifugation , recombined adenovirus Ad-CMV-E6/E7, Ad-K14 -E6/E7 were used as experimental group, while pAd-CMV and pAdtrack-K14 were used as control group. Four of them were injected through one main vein of nude mice tail respectively. These mice were then treated with 0.05 mg 17beta-estradiol over 12 weeks. Mice were anaesthesiaed with 2.5% Avertint and the vagina, mammary gland, ovaries and uterus were dissected and fixed in 3.75% paraformaldehyde overnight at 4 degrees C. Paraffin-embedded sections, HE staining and identification of P53 and Bcl-2 protein via immunohistochemistry were performed. The expression of E6/E7 was verified by RT-PCR in different tissue of nude mice. HE staining showed evident hyperplasy in cervix-uterus transformation zone of experimental group 2. The expression of mutant P53 and Bcl-2 were higher than control group via immunohistochemical S-P method in uterus stroma-cell. Western blotting also showed that E6 protein was expressed. The expression of E6/E7 was higher than control group by human cytokeratin promoter 14 and hyperlasy changes were detected in epithelial tissue of cervix-uterus transformation zone.

RNAi-mediated silencing of the Bmi-1 gene causes growth inhibition and enhances doxorubicin-induced apoptosis in MCF-7 cells.

The oncogene Bmi-1 is a member of the Polycomb group gene family. Its expression is found to be greatly increased in a number of malignant tumors including breast cancer. This could suggest Bmi-1 as a potent therapeutic target. In this study, RNAi was introduced to down-regulate the expression of Bmi-1 in a highly malignant breast adenocarcinoma cell line, MCF-7. A thorough study of the biological behavior and chemosensitivity changes of the MCF-7 cells was carried out in context to the therapeutic potential of Bmi-1. The results obtained indicated that siRNA targeting of Bmi-1 could lead to an efficient and specific inhibition of endogenous Bmi-1 activity. The mRNA and protein expression of Bmi-1 were determined by RT-PCR and Western blot, respectively. Furthermore, silencing of Bmi-1 resulted in a drastic inhibition of the growth of MCF-7 cells as well as G(1) /S phase transition. The number of target cells was found to increase in phase G (0) /G (1) and decrease in the S phase, but no increase in the basal level of apoptosis was noticed. On the other hand, a reduction in the expression of cyclin D1 and an increase in the expression of p21 were also noticed. Silencing of Bmi-1 made the MCF-7 cells more sensitive to the chemotherapeutic agent doxorubicin and induced a significantly higher percentage of apoptotic cells. Here, we report on a study regarding the RNAi-mediated silencing of the Bmi-1 gene in breast cancer.

RNAi-mediated silencing of the Bmi-1 gene causes growth inhibition and enhances doxorubicin-induced apoptosis in MCF-7 cells

The oncogene Bmi-1 is a member of the Polycomb group gene family. Its expression is found to be greatly increased in a number of malignant tumors including breast cancer. This could suggest Bmi-1 as a potent therapeutic target. In this study, RNAi was introduced to down-regulate the expression of Bmi-1 in a highly malignant breast adenocarcinoma cell line, MCF-7. A thorough study of the biological behavior and chemosensitivity changes of the MCF-7 cells was carried out in context to the therapeutic potential of Bmi-1. The results obtained indicated that siRNA targeting of Bmi-1 could lead to an efficient and specific inhibition of endogenous Bmi-1 activity. The mRNA and protein expression of Bmi-1 were determined by RT-PCR and Western blot, respectively. Furthermore, silencing of Bmi-1 resulted in a drastic inhibition of the growth of MCF-7 cells as well as G1/S phase transition. The number of target cells was found to increase in phase G0/G1 and decrease in the S phase, but no increase in the basal level of apoptosis was noticed. On the other hand, a reduction in the expression of cyclin D1 and an increase in the expression of p21 were also noticed. Silencing of Bmi-1 made the MCF-7 cells more sensitive to the chemotherapeutic agent doxorubicin and induced a significantly higher percentage of apoptotic cells. Here, we report on a study regarding the RNAi-mediated silencing of the Bmi-1 gene in breast cancer.