ABSTRACT
Organic acid metabolites exhibit acidic properties. These metabolites serve as intermediates in major carbon metabolic pathways and are involved in several biochemical pathways, including the tricarboxylic acid (TCA) cycle and glycolysis. They also regulate cellular activity and play crucial roles in epigenetics, tumorigenesis, and cellular signal transduction. Knowledge of the binding proteins of organic acid metabolites is crucial for understanding their biological functions. However, identifying the binding proteins of these metabolites has long been a challenging task owing to the transient and weak nature of their interactions. Moreover, traditional methods are unsuitable for the structural modification of the ligands of organic acid metabolites because these metabolites have simple and similar structures. Even minor structural modifications can significantly affect protein interactions. Thermal proteome profiling (TPP) provides a promising avenue for identifying binding proteins without the need for structural modifications. This approach has been successfully applied to the identification of the binding proteins of several metabolites. In this study, we investigated the binding proteins of two TCA cycle intermediates, i.e., succinate and fumarate, and lactate, an end-product of glycolysis, using the matrix thermal shift assay (mTSA) technique. This technique involves combining single-temperature (52 â) TPP and dose-response curve analysis to identify ligand-binding proteins with high levels of confidence and determine the binding affinity between ligands and proteins. To this end, HeLa cells were lysed, followed by protein desalting to remove endogenous metabolites from the cell lysates. The desalted cell lysates were treated with fumarate or succinate at final concentrations of 0.004, 0.04, 0.4, and 2 mmol/L in the experimental groups or 2 mmol/L sodium chloride in the control group. Considering that the cellular concentration of lactate can be as high as 2-30 mmol/L, we then applied lactate at final concentrations of 0.2, 1, 5, 10, and 25 mmol/L in the experimental groups or 25 mmol/L sodium chloride in the control group. Using high-sensitivity mass spectrometry coupled with data-independent acquisition (DIA) quantification, we quantified 5870, 5744, and 5816 proteins in succinate, fumarate, and lactate mTSA experiments, respectively. By setting stringent cut-off values (i.e., significance of changes in protein thermal stability (p-value)<0.001 and quality of the dose-response curve fitting (square of Pearson's correlation coefficient, R2)>0.95), multiple binding proteins for these organic acid metabolites from background proteins were confidently determined. Several known binding proteins were identified, notably fumarate hydratase (FH) as a binding protein for fumarate, and α-ketoglutarate-dependent dioxygenase (FTO) as a binding protein for both fumarate and succinate. Additionally, the affinity data for the interactions between these metabolites and their binding proteins were obtained, which closely matched those reported in the literature. Interestingly, ornithine aminotransferase (OAT), which is involved in amino acid biosynthesis, and 3-mercaptopyruvate sulfurtransferase (MPST), which acts as an antioxidant in cells, were identified as lactate-binding proteins. Subsequently, an orthogonal assay technique developed in our laboratory, the solvent-induced precipitation (SIP) technique, was used to validate the mTSA results. SIP identified OAT as the top target candidate, validating the mTSA-based finding that OAT is a novel lactate-binding protein. Although MPST was not identified as a lactate-binding protein by SIP, statistical analysis of MPST in the mTSA experiments with 10 or 25 mmol/L lactate revealed that MPST is a lactate-binding protein with a high level of confidence. Peptide-level empirical Bayes t-tests combined with Fisher's exact test also supported the conclusion that MPST is a lactate-binding protein. Lactate is structurally similar to pyruvate, the known binding protein of MPST. Therefore, assuming that lactate could potentially occupy the binding site of pyruvate on MPST. Overall, the novel binding proteins identified for lactate suggest their potential involvement in amino acid synthesis and redox balance regulation.
Subject(s)
Citric Acid Cycle , Humans , HeLa Cells , Succinic Acid/metabolism , Succinic Acid/chemistry , Fumarates/metabolism , Fumarates/chemistryABSTRACT
OBJECTIVE: The primary objective of this study was to explore the clinical characteristics of apoplectic intratumoral hemorrhage in gliomas and offer insights for improving the diagnosis and treatment of this disease. METHODS: We analyzed the clinical data of 35 patients with glioma and hemorrhage. There were eight cases of multiple cerebral lobe involvement, and 22 cases involved a single lobe. Twenty-one patients had a preoperative Glasgow Coma Scale (GCS) score of ≥ 9 and had a craniotomy with tumor resection and hematoma evacuation after undergoing preoperative preparation. A total of 14 patients with GCS < 9, including one with thalamic hemorrhage breaking into the ventricles and acute obstructive hydrocephalus, underwent craniotomy for tumor resection after external ventricular drainage (EVD). One patient had combined thrombocytopenia, which was surgically treated after platelet levels were normalized through transfusion. The remaining 12 patients received immediate intervention in the form of craniotomy hematoma evacuation and tumor resection. RESULTS: We performed subtotal resection on three tumors of thalamic origin and two tumors of corpus callosum origin, but we were able to successfully resect all the tumors in other locations that were gross total resection Pathology results showed that 71.43% of cases accounted for WHO-grade 4 tumors. Among the 21 patients with a GCS score of ≥ 9, two died perioperatively. Fourteen patients had a GCS score < 9, of which eight patients died perioperatively. CONCLUSIONS: Patients with a preoperative GCS score ≥ 9 who underwent subemergency surgery and received aggressive treatment showed a reasonable prognosis. We found their long-term outcomes to be correlated with the pathology findings. On the other hand, patients with a preoperative GCS score < 9 required emergency treatment and had a high perioperative mortality rate.
Subject(s)
Brain Neoplasms , Glioma , Humans , Glioma/complications , Glioma/surgery , Male , Female , Brain Neoplasms/surgery , Brain Neoplasms/complications , Middle Aged , Adult , Aged , Young Adult , Adolescent , Cerebral Hemorrhage/surgery , Cerebral Hemorrhage/diagnosis , Cerebral Hemorrhage/complications , Child , Craniotomy/methods , Glasgow Coma Scale , Retrospective Studies , Treatment OutcomeABSTRACT
Endothelial cells (ECs) present in the tumor microenvironment (TME) exhibit significant diversity that may impact the efficacy of anti-tumor treatments. Thus, our study sought to elucidate the various clusters of ECs present in pancreatic ductal adenocarcinoma (PDAC) and explore their possible interactions and influence on clinical outcomes. We obtained single-cell transcriptome data from 24 PDAC tumors and 11 normal pancreases, minimizing any batch effects between samples. Next, we compared the relative abundance of various ECs clusters across distinct sample types. Pseudo-time analysis was employed to investigate the differentiation origin of ECs. A variety of bioinformatics methods were used to investigate potential communication between ECs and malignant cells, as well as assess metabolic changes, pathway alterations, and immune-related markers expression within distinct EC clusters. Lastly, we investigated the impact of particular ECs clusters on patient prognosis in bulk transcriptome data. Our study identified seven distinct clusters of ECs, denoted as CA4+ ECs, MMP2+ ECs, SPP1+ ECs, MT1F+ ECs, CCL5+ ECs, RGS5+ ECs, and TYROBP+ ECs. Pseudo-time analysis suggested that the loss of CA4+ ECs and MT1F+ ECs may promote malignant progression. Cell communication elucidated that MT1F+ ECs exhibited the strongest outgoing interaction strength, whereas RGS5+ ECs displayed the strongest incoming interaction strength. Furthermore, TYROBP+ ECs exhibited greater metabolic activity, and notably, CCL5+ ECs displayed increased expression of immune-related molecules. Lastly, across cohorts of bulk transcriptome levels, CA4+ ECs, MT1F+ ECs, and RGS5+ ECs consistently demonstrated prognostic indicative effects. PDAC patients exhibit the presence of seven distinct EC clusters, each demonstrating significant metabolic and immunological heterogeneity. Targeted therapeutic approaches directed toward CA4+ ECs and MT1F+ ECs may prove advantageous in addressing challenges associated with PDAC treatment. Additionally, variations in the relative abundance of CA4+ ECs, MT1F+ ECs, and RGS5+ ECs were indicated as predictive of patient prognosis.
Subject(s)
Carcinoma, Pancreatic Ductal , Pancreatic Neoplasms , Humans , Transcriptome , Endothelial Cells/metabolism , Endothelial Cells/pathology , Tumor Microenvironment/genetics , Carcinoma, Pancreatic Ductal/genetics , Carcinoma, Pancreatic Ductal/drug therapy , Carcinoma, Pancreatic Ductal/metabolism , Pancreatic Neoplasms/genetics , Pancreatic Neoplasms/drug therapy , Gene Expression Profiling/methods , BiomarkersABSTRACT
BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal cancer with limited treatment options. The PI3K/AKT/mTOR pathway is commonly activated in PDAC and plays a critical role in its progression. METHODS AND RESULTS: In this study, the effect of taselisib (a selective PI3K inhibitor) on PDAC cell proliferation was investigated, and a significant decrease in viability was observed with increasing concentrations of taselisib. Differential analysis on samples from the Genotype-Tissue Expression and The Cancer Genome Atlas databases revealed 24 dysregulated PI3K/AKT/mTOR pathway-related genes (PRGs). Unsupervised clustering-based analysis of transcriptome cohorts revealed two clusters with high consistency between RNA-seq and microarray cohorts. Cluster B had higher enrichment of immune cells, particularly CD8+ T cells, and lower levels of immunosuppressive Treg cells. Moreover, we investigated the relationship between drug sensitivity and different clusters and found that cluster A had a better response to PI3K/AKT/mTOR pathway-related inhibitors and chemotherapy. Finally, cluster A exhibited significant activation of PI3K/AKT/mTOR and related oncogenic pathways, contributing to poor prognosis. The study also developed a risk score based on the expression profiles of PRGs and machine learning, which showed a significant increase in overall survival time among patients in the low-risk group. Importantly, the PI3K/AKT/mTOR pathway could be used to better predict individual risk scores, as evidenced by stratified survival analysis. CONCLUSIONS: These findings suggest that targeting the PI3K/AKT/mTOR pathway may have therapeutic potential in PDAC, and distinct pathway states, immune modulation and tumor microenvironments have prognostic value.
Subject(s)
Carcinoma, Pancreatic Ductal , Pancreatic Neoplasms , Humans , Proto-Oncogene Proteins c-akt/genetics , Proto-Oncogene Proteins c-akt/metabolism , Phosphatidylinositol 3-Kinases/genetics , Phosphatidylinositol 3-Kinases/metabolism , Transcriptome , CD8-Positive T-Lymphocytes/metabolism , Carcinoma, Pancreatic Ductal/genetics , Pancreatic Neoplasms/metabolism , TOR Serine-Threonine Kinases/genetics , TOR Serine-Threonine Kinases/metabolism , Cell Proliferation/genetics , Tumor MicroenvironmentABSTRACT
Metastasis is the major cause of death in patients with pancreatic ductal adenocarcinoma (PDAC), and circulating tumor cells (CTCs) play an important role in the development of metastasis. However, few studies have uncovered the metastasis mechanism of PDAC based on CTCs. In this study, the existing bulk RNA-sequencing (bulk RNA-seq) and single-cell sequencing (scRNA-seq) data for CTCs in pancreatic cancer were obtained from the Gene Expression Omnibus (GEO) database. Analysis of tumor-infiltrating immune cells (TIICs) by CIBERSORT showed that the CTCs enriched from the peripheral blood of metastatic PDAC were found to contain a high proportion of T cell regulators (Tregs) and macrophages, while the proportion of dendritic cells (DCs) was lower than that enriched from localized PDAC. Through weighted gene co-expression network analysis (WGCNA) and the result of scRNA-seq, we identified the hub module (265 genes) and 87 marker genes, respectively, which were highly associated with metastasis. The results of functional enrichment analysis indicated that the two gene sets mentioned above are mainly involved in cell adhesion and cytoskeleton and epithelial-mesenchymal transition (EMT). Finally, we found that HMGB3 was the hub gene according to the Venn diagram. The expression of HMGB3 in PDAC was significantly higher than that in normal tissues (protein and mRNA levels). HMGB3 expression was significantly positively correlated with both EMT-related molecules and CTC cluster-related markers. Furthermore, it was also found that HMGB3 mutations were favorably related to tumor-associated immune cells through the TIMER2.0 online tool. We further demonstrated that PDAC patients with higher HMGB3 expression had significantly worse overall survival (OS) in multiple datasets. In summary, our study suggests that HMGB3 is a hub gene associated with EMT in CTCs, the formation of CTC clusters, and infiltration patterns of immune cells favorable for tumor progression and metastasis to distant organs.
ABSTRACT
Recent evidence indicates that the long noncoding RNA (lncRNA) cancer susceptibility candidate 2 (CASC2) is involved in tumorigenesis of several types of cancer through targeting microRNAs (miRs); however, the molecular mechanism of CASC2 in pancreatic cancer remains elusive. In the present study, the expression levels of CASC2, miR24 and mucin 6 (MUC6) were measured in pancreatic cancer specimens and cell lines by reverse transcriptionquantitative PCR. Western blotting was used to determine the protein expression levels of MUC6, Integrin ß4 (ITGB4), phosphorylated (p)focal adhesion kinase (FAK) and several epithelialtomesenchymal transition markers in pancreatic cancer cells. MTT, colony formation, wound healing, Transwell and flow cytometry assays were performed to detect cell proliferation, colony formation, migration, invasion and apoptosis, respectively, in vitro. Morphological changes of pancreatic cancer cells were assessed by light microscopy. The interactions between CASC2, miR24 and MUC6 were assessed by the dualluciferase reporter assay. A tumor xenograft model was generated to investigate tumor growth in vivo. CASC2 and MUC6 were downregulated, and miR24 was upregulated in pancreatic cancer specimens and cell lines. Functionally, CASC2 overexpression or miR24 knockdown suppressed pancreatic cancer cell proliferation, colony formation, migration and invasion, and promoted apoptosis. Additionally, they altered cellcell adhesion as demonstrated by the attenuated ITGB4, pFAK and Ncadherin protein levels, as well as morphological changes. Mechanistically, CASC2 sponged miR24 and activated its downstream target MUC6 to suppress pancreatic cancer growth and progression. CASC2 exerted tumorsuppressive functions in pancreatic cancer through the miR24/MUC6 axis, which may be a promising target for pancreatic cancer therapy.
Subject(s)
Gene Expression Regulation, Neoplastic , MicroRNAs/metabolism , Mucin-6/genetics , Pancreatic Neoplasms/genetics , Tumor Suppressor Proteins/metabolism , Aged , Animals , Apoptosis/genetics , Cell Line, Tumor , Cell Movement/genetics , Cell Proliferation/genetics , Disease Progression , Female , Humans , Male , Mice , Middle Aged , Neoplasm Invasiveness/genetics , Pancreas/pathology , Pancreas/surgery , Pancreatectomy , Pancreatic Neoplasms/pathology , Pancreatic Neoplasms/surgery , Up-Regulation , Xenograft Model Antitumor AssaysABSTRACT
Preclinical Research A novel series of ten 5-hydroxy, 5-substituted benzene sulfonamide pyrimidine-2,4,6-triones were synthesized and their structures ascertained using (1) H-NMR, (13) C-NMR, mass and elemental analysis. These compounds were subsequently tested for inhibition of MMP-2 and MMP-9 where most exhibited activity with compound 5i being the most potent against MMP-2 and MMP-9 with IC50 values of 2.35 nM and 8.24 nM, respectively. Compound 5i was further analyzed in a mouse LPS-induced acute lung injury model where it had protective activity. Histochemical studies indicated that 5i improved the vascular integrity of the lung. Drug Dev Res 77 : 251-257, 2016. © 2016 Wiley Periodicals, Inc.
Subject(s)
Acute Lung Injury/drug therapy , Pyrimidines/pharmacology , Sulfonamides/pharmacology , Acute Lung Injury/pathology , Animals , Disease Models, Animal , Gelatinases/antagonists & inhibitors , Inhibitory Concentration 50 , Lipopolysaccharides/toxicity , Magnetic Resonance Spectroscopy , Male , Matrix Metalloproteinase 2/metabolism , Matrix Metalloproteinase 9/metabolism , Matrix Metalloproteinase Inhibitors/chemical synthesis , Matrix Metalloproteinase Inhibitors/chemistry , Matrix Metalloproteinase Inhibitors/pharmacology , Mice , Mice, Inbred BALB C , Pyrimidines/chemical synthesis , Pyrimidines/chemistry , Structure-Activity Relationship , Sulfonamides/chemical synthesis , Sulfonamides/chemistry , BenzenesulfonamidesABSTRACT
Hepatocellular carcinoma (HCC) is a highly aggressive form of carcinoma with poor prognosis, and HCC-associated mortality primarily occurs due to migration and invasion of HCC cells. The manipulation of epigenetic proteins, such as BRD4, has recently emerged as an alternative therapeutic strategy. The present study aimed to investigate the novel mechanism of BRD4 involvement in the migration and invasion of HCC cells. Reverse transcription-quantitative polymerase chain reaction was used to assess BRD4 mRNA expression levels in HCC cell lines. This analysis demonstrated that BRD4 was significantly overexpressed in HCC cell lines compared with a human immortalized normal liver cell line. A short hairpin RNA (shRNA) was then used to suppress BRD4 expression in HCC cells, and resulted in impaired HCC cell proliferation, migration and invasion. When the HepG2 HCC cell line was treated with recombinant human sonic hedgehog (SHH) peptide, the migration and invasion capabilities of HepG2 cells that were inhibited by BRD4 silencing were restored. BRD4 induced cell migration and invasion in HepG2 cells through the activation of matrix metalloproteinase (MMP)-2 and MMP-9, mediated by the SHH signaling pathway. Taken together, the results of the present study demonstrated the importance of BRD4 in HCC cell proliferation and metastasis. Thus, BRD4 is a potential novel target for the development of therapeutic approaches against HCC.
ABSTRACT
Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive carcinoma with a poor prognosis. To date, there is no effective treatment for this fatal disease. The manipulation of epigenetic proteins, such as BRD4, has recently emerged as an alternative therapeutic strategy. Our objective was to analyze the effect of BRD4 on the cell progression and chemoresistance of PDAC and the novel mechanisms involved. In the present study, we firstly revealed that the expression of BRD4 was significantly upregulated in PDAC cell lines, compared to that in human pancreatic duct epithelial cells. An in vitro assay showed that the suppression of BRD4 impaired PDAC cell viability and proliferation. Similarly, the tumor growth rate was also decreased in vivo after silencing of BRD4. Furthermore, we showed that the expression of BRD4 was increased after treatment with gemcitabine (GEM). Combination treatment of GEM and BRD4 silencing had a synergistic effect on the chemotherapeutic efficacy in the PANC-1 and MIAPaCa-2 cell lines, and significantly promoted apoptosis. In particular, we demonstrated that BRD4 activated the Sonic hedgehog (Shh) signaling pathway members in a ligand-independent manner in the PDAC cells. Together, our results indicate the important role of BRD4 in PDAC cell proliferation and chemoresistance and suggests that BRD4 is a promising target directed against the transcriptional program of PDAC.
Subject(s)
Carcinoma, Pancreatic Ductal/pathology , Deoxycytidine/analogs & derivatives , Drug Resistance, Neoplasm/drug effects , Neoplasm Proteins/physiology , Nuclear Proteins/physiology , Pancreatic Neoplasms/pathology , Transcription Factors/physiology , Animals , Apoptosis/drug effects , Carcinoma, Pancreatic Ductal/drug therapy , Cell Cycle Proteins , Cell Division/drug effects , Cell Line, Tumor , Cell Survival/drug effects , Deoxycytidine/pharmacology , Deoxycytidine/therapeutic use , Disease Progression , Epithelial Cells/drug effects , Gene Expression Regulation, Neoplastic/drug effects , Hedgehog Proteins/biosynthesis , Hedgehog Proteins/genetics , Hedgehog Proteins/physiology , Humans , Male , Mice , Mice, Nude , Neoplasm Proteins/antagonists & inhibitors , Neoplasm Proteins/biosynthesis , Neoplasm Proteins/genetics , Nuclear Proteins/antagonists & inhibitors , Nuclear Proteins/biosynthesis , Nuclear Proteins/genetics , Pancreatic Neoplasms/drug therapy , RNA Interference , RNA, Small Interfering/genetics , Signal Transduction/physiology , Transcription Factors/antagonists & inhibitors , Transcription Factors/biosynthesis , Transcription Factors/genetics , Transfection , Tumor Stem Cell Assay , Xenograft Model Antitumor Assays , GemcitabineABSTRACT
BACKGROUND: This study aimed to evaluate the feasibility, safety, and cosmetic results of a novel technique, transumbilical single-incision laparoscopic appendectomy (TSILA), using a single working channel with conventional instruments. PATIENTS AND METHODS: The study enrolled 84 consecutive patients undergoing laparoscopic appendectomy for acute appendicitis. To test the advantages of TSILA on the management of patients with acute appendicitis, a prospective randomized clinical trial was conducted. Surgical outcomes such as operation time, complication, and hospital stay of 42 patients undergoing TSILA were analyzed and compared with those of 42 patients undergoing classic 3-port appendectomy. All patients received a follow-up visit for 3 to 12 months. RESULTS: The study consisted of 42 patients undergoing TSILA and 42 patients undergoing classic 3-port laparoscopic appendectomy with an average age of 34.1 and 34.9 years, respectively. The mean operative time of TSILA did not show any difference when compared with the classic procedure (84.8 vs. 77.9 min, P=0.271). No operative complications occurred in patients undergoing TSILA, whereas 2 patients undergoing the classic procedure showed incisional infection. The average postoperative hospital stay was 2.7 days in the TSILA group and 2.9 days in the classic procedure group with no difference (P=0.316). At the follow-up visit, no patient showed any evidence of incisional hernia. The transumbilical incisions were visible minimally, and the cosmetic scores given by patients undergoing TSILA was higher than that given by patients undergoing the classic procedure (4.5 vs. 3.9, P<0.001). CONCLUSIONS: The results of the study demonstrate that laparoscopic appendectomy can be achieved through a single umbilical incision and a single working channel using conventional instruments and that this approach is successful, safe, economic, and esthetic.
Subject(s)
Appendectomy/methods , Appendicitis/surgery , Laparoscopes , Laparoscopy/methods , Umbilicus/surgery , Adolescent , Adult , Aged , Appendicitis/diagnosis , Equipment Design , Female , Follow-Up Studies , Humans , Laparoscopy/adverse effects , Length of Stay , Male , Middle Aged , Minimally Invasive Surgical Procedures/adverse effects , Minimally Invasive Surgical Procedures/methods , Pain, Postoperative/physiopathology , Prospective Studies , Risk Assessment , Time Factors , Treatment Outcome , Young AdultABSTRACT
The PNP/6-methylpurine 2'-deoxyriboside (6MePdR) system is an efficient gene-directed enzyme prodrug therapy system with significant antitumor activities. In this system, Escherichia coli purine nucleoside phosphorylase (ePNP) activates nontoxic 6MePdR into potent antitumor drug 6-methylpurine (6MeP). The Salmonella typhimurium PNP (sPNP) gene has a 96-% sequence homology in comparison with ePNP and also has the ability to convert 6MePdR to 6MeP. In this study, we used tumor-targeting S. typhimurium VNP20009 expressing endogenous PNP gene constitutively to activate 6MePdR and a combination treatment of bacteria and prodrug in B16F10 melanoma model. The conversion of 6MePdR to 6MeP by S. typhimurium was analyzed by HPLC and the enzyme activity of sPNP was confirmed by in vitro (tetrazolium-based colorimetric assay) MTT cytotoxicity assay. After systemic administration of VNP20009 to mice, the bacteria largely accumulated and specifically delivered endogenous sPNP in the tumor. In comparison with VNP20009 or 6MePdR treatment alone, combined administration of VNP20009 followed by 6MePdR treatment significantly delayed the growth of B16F10 tumor and increased the CD8(+) T-cell infiltration. In summary, our results demonstrated that the combination therapy of S. typhimurium and prodrug 6MePdR is a promising strategy for cancer therapy.
Subject(s)
Antineoplastic Agents/therapeutic use , Melanoma, Experimental/therapy , Prodrugs/therapeutic use , Purine Nucleosides/therapeutic use , Salmonella typhimurium/physiology , Amino Acid Sequence , Animals , Apoptosis , Chromatography, High Pressure Liquid , Female , In Situ Nick-End Labeling , Melanoma, Experimental/drug therapy , Melanoma, Experimental/pathology , Mice , Mice, Inbred C57BL , Molecular Sequence Data , Purine-Nucleoside Phosphorylase/chemistry , Sequence Homology, Amino AcidABSTRACT
BACKGROUND: Pancreatic cancer is one of the most lethal human cancers with a very low survival rate of 5 years. Conventional cancer treatments including surgery, radiation, chemotherapy or combinations of these show little effect on this disease. Several proteins have been proved critical to the development and the progression of pancreatic cancer. The aim of this study was to investigate the effect of resveratrol on apoptosis in pancreatic cancer cells. METHODS: Several pancreatic cancer cell lines were screened by resveratrol, and its toxicity was tested by normal pancreatic cells. Western blotting was then performed to analyze the molecular mechanism of resveratrol induced apoptosis of pancreatic cancer cell lines. RESULTS: In the screened pancreatic cancer cell lines, capan-2 and colo357 showed high sensitivity to resveratrol induced apoptosis. Resveratrol exhibited insignificant toxicity to normal pancreatic cells. In resveratrol sensitive cells, capan-2 and colo357, the activation of caspase-3 was detected and showed significant caspase-3 activation upon resveratrol treatment; p53 and p21 were also detected up-regulated upon resveratrol treatment. CONCLUSION: Resveratrol provides a promising anti-tumor strategy to fight against pancreatic cancer.
Subject(s)
Apoptosis/drug effects , Stilbenes/pharmacology , Blotting, Western , Caspase 3/metabolism , Cell Survival/drug effects , Humans , Mitogen-Activated Protein Kinases/metabolism , Pancreatic Neoplasms/metabolism , Resveratrol , Tumor Cells, CulturedABSTRACT
OBJECTIVE: To establish a stable high red fluorescent protein (RFP)-expressing orthotopic transplantation nude mice spontaneous metastasis model of pancreatic cancer. METHODS: Stable high RFP-expressing cells SW1990-RFP were injected subcutaneously into mice to establish subcutaneous implantation model. Fluorescent tumor piece from subcutaneous was transplanted into the body of the pancreas to establish surgical orthotopic implantation model. The growth of primary tumor, metastasis and micrometastasis were assessed by whole-body fluorescence imaging system. RESULTS: Twelve RFP orthotopic transplantation nude mice metastasis models of pancreatic cancer were established successfully, the percentage of success rate was 100%. RFP-labeled pancreatic cancer growth could be monitored in real time way. The micrometastasis of primary lesions were detected in early stage with whole-body fluorescence imaging system. CONCLUSIONS: The RFP orthotopic transplantation nude mice metastasis model of pancreatic cancer is stable and reliable, and can be observed dynamically in vitro in a noninvasive way, with much higher sensitivity and specificity.
Subject(s)
Luminescent Proteins , Pancreatic Neoplasms/pathology , Xenograft Model Antitumor Assays , Animals , Disease Models, Animal , Female , Male , Mice , Mice, Nude , Neoplasm Metastasis , Pancreas/pathology , Red Fluorescent ProteinABSTRACT
BACKGROUND: Pancreatic cancer is one of the most common tumors and has a 5-year survival for all stages of less than 5%. Most patients with pancreatic cancer are diagnosed at an advanced stage and therefore are not candidates for surgical resection. In recent years, investigation into alternative treatment strategies for this aggressive disease has led to advances in the field of gene therapy for pancreatic cancer. E. coli purine nucleoside phosphorylase/6-methylpurine deoxyribose (ePNP/MePdR) is a suicide gene/prodrug system where PNP enzyme cleaves nontoxic MePdR into cytotoxic membrane-permeable compounds 6-methylpurine (MeP) with high bystander activity. hTERT is expressed in cell lines and tissues for telomerase activity. In this study we examined the efficacy of ePNP under the control of hTERT promoter sequences and assessed the selective killing effects of the ePNP/prodrug MePdR system on pancreatic tumors. METHODS: Recombinant pET-PNP was established. The protein of E. coli PNPase was expressed and an antibody to E. coli PNPase was prepared. Transcriptional activities of hTERT promoter sequences were analyzed using a luciferase reporter gene. A recombinant phTERT-ePNP vector was constructed. The ePNP/MePdR system affects SW1990 human pancreatic cancer cell lines in vitro. RESULTS: The hTERT promoter had high transcriptional activity and conferred specificity on cancer cell lines. The antibody to E. coli PNPase was demonstrated to be specific for the ePNP protein. The MePdR treatment induced a high in vitro cytotoxicity on the sole hTERT-ePNP-producing cell lines and affected SW1990 cells in a dose-dependent manner. CONCLUSIONS: The hTERT promoter control of the ePNP/MePdR system can provide a beneficial anti-tumor treatment in pancreatic cancer cell lines including a good bystander killing effect.
Subject(s)
Escherichia coli/enzymology , Genetic Therapy , Pancreatic Neoplasms/therapy , Promoter Regions, Genetic , Purine Nucleosides/therapeutic use , Purine-Nucleoside Phosphorylase/genetics , Telomerase/genetics , Cell Line, Tumor , HumansABSTRACT
OBJECTIVE: To investigate the expression of NF-kappaB in peripheral blood polymorphonuclear leukocyte (PMN) of acute pancreatitis (AP) and to assess the preventive effectiveness of pyrrolidine dithiocarbamate (PDTC) on NF-kappaB in vitro. METHODS: Nineteen patients and 16 healthy individuals as control were enrolled in this study. The expression of NF-kappaB in PMNs was determined by gel electrophoretic mobility shift assay (EMSA). Routine clinical examination results and computed tomography findings of AP were recorded in all patients. RESULTS: The PMNs from the patients with AP showed higher levels of NF-kappaB activities than those from control subjects (P < 0.01), severe acute pancreatitis (SAP) group showed much higher than mild acute pancreatitis (MAP) group (P < 0.05). In vitro, PDTC could reduce the NF-kappaB activity in PMNs of patients with AP, and its effectiveness at 2 mmol/L was stronger than at 1 mmol/L (P < 0.05). The PMNs from control subjects pretreated with 2 mmol/L PDTC before stimulation with the plasma from patients with SAP showed lower levels of NF-kappaB activities than did those untreated (P < 0.05). CONCLUSION: The NF-kappaB activation in peripheral blood PMNs participate in the course of acute pancreatitis and can be inhibited by PDTC in vitro.
Subject(s)
NF-kappa B/blood , Neutrophils/metabolism , Pancreatitis/metabolism , Pyrrolidines/pharmacology , Thiocarbamates/pharmacology , Acute Disease , Adult , Aged , Aged, 80 and over , Female , Humans , Male , Middle Aged , NF-kappa B/biosynthesis , Neutrophils/drug effectsABSTRACT
AIM: To directly investigate the relationship between telomerase activity and its subunit expression and the inhibitory effect of antisense hTR on pancreatic carcinogenesis. METHODS: We examined the telomerase activity and its subunit expression by cell culture, polymerase chain reaction (PCR), PCR-silver staining, PCR-ELISA, DNA sequencing, MTT and flow cytometry methods. RESULTS: PCR-silver staining and PCR-ELISA methods had the same specificity and sensitivity as the TRAP method. Telomerase activity was detected in the extract of the 10(th),20(th) and 30(th) passages of P3 cells,while it was absent in fibroblasts. Furthermore, after the 30th generation, the proliferation period of fibroblast cells was significantly prolonged. Telomerase activity and hTERTmRNA were detected in two pancreatic carcinoma cell lines, but were found to be negative in human fibroblast cells. Telomerase activity and hTERTmRNA were tested in pancreatic carcinoma specimens of 24 cases. The telomerase activity was positive in 21 of the 24 cases (87.5 %), and the hTERTmRNA in 20 cases (83.3 %). In adjacent normal tissues positive rates were both 12.5 %. There was a significant difference between the two groups. This indicated a significant correlation between the expression level of telomerase activity and histologic differentiation, metastasis and advanced clinical stage of pancreatic carcinoma. Our findings showed that the expressions of hTR and TP1mRNA were not correlated with the activity of telomerase but the expression of hTERTmRNA was. After treatment with PS-ODNs, telomerase activity in P(3) cells weakened and the inhibiting effect became stronger with an increase in PS-ODNs concentration. There was a significant difference between different PS-ODN groups (P<0.05). Inhibition of telomerase activity occurred most significant with PS-ODN1. The results of the FCM test of pancreatic cancer P(3) cells showed an increase in the apoptotic rate with increasing PS-ODN1 and PS-ODN2 concentrations. CONCLUSION: The expression of telomerase activity has a significant relationship to carcinogenesis. A strong correlation exists between telomerase activity and hTERTmRNA expression. The up-regulation of hTERTmRNA expression may play a critical role in human carcinogenesis. The expression of telomerase activity and its subunit level in pancreatic carcinoma significantly correlate with the clinical stage of pancreatic carcinoma and hence, may be helpful in its diagnosis and prognosis. The anti-hTR complementary to the template region of hTR is sufficient to inhibit P3 cell telomerase activity and cell proliferation in vitro, and can lead to a profound induction of programmed cell death.
Subject(s)
Carcinoma/enzymology , Carcinoma/pathology , Oligonucleotides, Antisense/pharmacology , Pancreatic Neoplasms/enzymology , Pancreatic Neoplasms/pathology , RNA/genetics , Telomerase/genetics , Telomerase/metabolism , Carrier Proteins/metabolism , Cell Division/drug effects , DNA-Binding Proteins , Female , Humans , Male , Middle Aged , Neoplasm Staging , RNA/metabolism , RNA, Messenger/metabolism , RNA-Binding Proteins , Telomerase/adverse effects , Tumor Cells, CulturedABSTRACT
This paper is to investigate PS-ODN's (antisense-PS-ODN of hTR,sense-PS-ODN of hTR and random sequence) effects on telomerase activity and proliferation of P3 pancreatic cancer cells,and to find a novel method for gene therapy of pancreatic cancer. The results indicate that the anti-hTR complementary to the template region of hTR is sufficient to inhibit P3 cell telomerase activity and cell proliferation in vitro,and as a result, they can lead to a profound induction of programmed cell death. Telomerase represents an interesting and promising anticancer drug target and anti-telomerase technology may have potential significance in tumor therapy.