ABSTRACT
BACKGROUND & AIMS: Aberrant DNA methylation is frequent in colorectal cancer (CRC), but underlying mechanisms and pathologic consequences are poorly understood. METHODS: We disrupted active DNA demethylation genes Tet1 and/or Tdg from ApcMin mice and characterized the methylome and transcriptome of colonic adenomas. Data were compared to human colonic adenocarcinomas (COAD) in The Cancer Genome Atlas. RESULTS: There were increased numbers of small intestinal adenomas in ApcMin mice expressing the TdgN151A allele, whereas Tet1-deficient and Tet1/TdgN151A-double heterozygous ApcMin colonic adenomas were larger with features of erosion and invasion. We detected reduction in global DNA hypomethylation in colonic adenomas from Tet1- and Tdg-mutant ApcMin mice and hypermethylation of CpG islands in Tet1-mutant ApcMin adenomas. Up-regulation of inflammatory, immune, and interferon response genes was present in Tet1- and Tdg-mutant colonic adenomas compared to control ApcMin adenomas. This up-regulation was also seen in murine colonic organoids and human CRC lines infected with lentiviruses expressing TET1 or TDG short hairpin RNA. A 127-gene inflammatory signature separated colonic adenocarcinomas into 4 groups, closely aligned with their microsatellite or chromosomal instability and characterized by different levels of DNA methylation and DNMT1 expression that anticorrelated with TET1 expression. Tumors with the CpG island methylator phenotype (CIMP) had concerted high DNMT1/low TET1 expression. TET1 or TDG knockdown in CRC lines enhanced killing by natural killer cells. CONCLUSIONS: Our findings reveal a novel epigenetic regulation, linked to the type of genomic instability, by which TET1/TDG-mediated DNA demethylation decreases methylation levels and inflammatory/interferon/immune responses. CIMP in CRC is triggered by an imbalance of methylating activities over demethylating activities. These mice represent a model of CIMP CRC.
Subject(s)
Adenocarcinoma , Adenoma , Colonic Neoplasms , Colorectal Neoplasms , Animals , Humans , Mice , Adenocarcinoma/genetics , Adenocarcinoma/pathology , Adenoma/genetics , Adenoma/pathology , Carcinogenesis/genetics , Cell Transformation, Neoplastic/genetics , Colonic Neoplasms/genetics , Colorectal Neoplasms/genetics , Colorectal Neoplasms/pathology , CpG Islands/genetics , DNA Methylation , DNA-Binding Proteins/genetics , Epigenesis, Genetic , Mixed Function Oxygenases/genetics , Phenotype , Proto-Oncogene Proteins/geneticsABSTRACT
Prostate cell lines from diverse backgrounds are important to addressing disparities in prostate cancer (PCa) incidence and mortality rates among Black men. ACRJ-PC28 was developed from a transrectal needle biopsy and established via inactivation of the CDKN2A locus and simultaneous expression of human telomerase. Characterization assays included growth curve analysis, immunoblots, IHC, 3D cultures, immunofluorescence imaging, confocal microscopy, flow cytometry, WGS, and RNA-Seq. ACRJ-PC28 has been passaged more than 40 times in vitro over 10 months with a doubling time of 45 hours. STR profiling confirmed the novelty and human origin of the cell line. RNA-Seq confirmed the expression of prostate specific genes alpha-methylacyl-CoA racemase (AMACR) and NKX3.1 and Neuroendocrine specific markers synaptophysin (SYP) and enolase 2 (ENO2) and IHC confirmed the presence of AMACR. Immunoblots indicated the cell line is of basal-luminal type; expresses p53 and pRB and is AR negative. WGS confirmed the absence of exonic mutations and the presence of intronic variants that appear to not affect function of AR, p53, and pRB. RNA-Seq data revealed numerous TP53 and RB1 mRNA splice variants and the lack of AR mRNA expression. This is consistent with retention of p53 function in response to DNA damage and pRB function in response to contact inhibition. Soft agar anchorage-independent analysis indicated that the cells are transformed, confirmed by principal component analysis (PCA) where ACRJ-PC28 cells cluster alongside other PCa tumor tissues, yet was distinct. The novel methodology described should advance prostate cell line development, addressing the disparity in PCa among Black men.
Subject(s)
Neuroendocrine Cells , Prostatic Neoplasms , Male , Humans , Tumor Suppressor Protein p53/genetics , Neuroendocrine Cells/metabolism , Prostatic Neoplasms/genetics , Cell Line , RNA, Messenger , Caribbean RegionABSTRACT
Background: Supply chain disruptions during the COVID-19 pandemic have affected the availability of components for specimen collection kits to detect SARS-CoV-2. Plastic injection molding offers a rapid and cheap method for mass production of swabs for upper respiratory tract sampling. Local production of virus transport medium increases flexibility to assemble sample collection kits if the medium provides appropriate stability for SARS-CoV-2 detection. Methods: A locally produced virus transport medium and a novel injection molded plastic swab were validated for SARS-CoV-2 detection by reverse-transcription quantitative polymerase chain reaction. Both components were compared to standard counterparts using viral reference material and representative patient samples. Results: Clinical testing showed no significant differences between molded and flocked swabs. Commercial and in-house virus transport media provided stable test results for over 40 days of specimen storage and showed no differences in test results using patient samples. Conclusions: This collection kit provides new supply chain options for SARS-CoV-2 testing.
Subject(s)
COVID-19 , Neoplasms , Humans , SARS-CoV-2 , COVID-19 Testing , Pandemics , Nasopharynx/chemistry , Specimen Handling/methods , Culture Media , RNA, ViralABSTRACT
Single-molecule localization microscopy is a powerful tool for visualizing subcellular structures, interactions and protein functions in biological research. However, inhomogeneous refractive indices inside cells and tissues distort the fluorescent signal emitted from single-molecule probes, which rapidly degrades resolution with increasing depth. We propose a method that enables the construction of an in situ 3D response of single emitters directly from single-molecule blinking datasets, and therefore allows their locations to be pinpointed with precision that achieves the Cramér-Rao lower bound and uncompromised fidelity. We demonstrate this method, named in situ PSF retrieval (INSPR), across a range of cellular and tissue architectures, from mitochondrial networks and nuclear pores in mammalian cells to amyloid-ß plaques and dendrites in brain tissues and elastic fibers in developing cartilage of mice. This advancement expands the routine applicability of super-resolution microscopy from selected cellular targets near coverslips to intra- and extracellular targets deep inside tissues.
Subject(s)
Brain/metabolism , Cartilage/metabolism , Imaging, Three-Dimensional/methods , Microscopy, Fluorescence/methods , Nanotechnology/methods , Plaque, Amyloid/metabolism , Single Molecule Imaging/methods , Animals , Brain/pathology , Cartilage/pathology , Cell Nucleus/metabolism , Cells, Cultured , Image Interpretation, Computer-Assisted/methods , Male , Mice , Mitochondria/metabolism , Molecular Imaging/methods , Nuclear Pore/metabolism , Plaque, Amyloid/pathologyABSTRACT
Fluorescence nanoscopy has become an indispensable tool for studying organelle structures, protein dynamics, and interactions in biological sciences. Single-molecule localization microscopy can now routinely achieve 10-50 nm resolution through fluorescently labeled specimens in lateral optical sections. However, visualizing structures organized along the axial direction demands scanning and imaging each of the lateral imaging planes with fine intervals throughout the whole cell. This iterative process suffers from photobleaching of tagged probes, is susceptible to alignment artifacts and also limits the imaging speed. Here, we focused on the axial plane super-resolution imaging which integrated the single-objective light-sheet illumination and axial plane optical imaging with single-molecule localization technique to resolve nanoscale cellular architectures along the axial (or depth) dimension without scanning. We demonstrated that this method is compatible with DNA points accumulation for imaging in nanoscale topography (DNA-PAINT) and exchange-PAINT by virtue of its light-sheet illumination, allowing multiplexed super-resolution imaging throughout the depth of whole cells. We further demonstrated this proposed system by resolving the axial distributions of intracellular organelles such as microtubules, mitochondria, and nuclear pore complexes in both COS-7 cells and glioblastoma patient-derived tumor cells.
ABSTRACT
Three-dimensional cell culture has the potential to revolutionize toxicology studies by allowing human-based reproduction of essential elements of organs. Beyond the study of toxicants on the most susceptible organs such as liver, kidney, skin, lung, gastrointestinal tract, testis, heart and brain, carcinogenesis research will also greatly benefit from 3D cell culture models representing any normal tissue. No tissue function can be suitably reproduced without the appropriate tissue architecture whether mimicking acini, ducts or tubes, sheets of cells or more complex cellular organizations like hepatic cords. In this review, we illustrate the fundamental characteristics of polarity that is an essential architectural feature of organs for which different 3D cell culture models are available for toxicology studies in vitro. The value of tissue polarity for the development of more accurate carcinogenesis studies is also exemplified, and the concept of using extracellular gradients of gaseous or chemical substances produced with microfluidics in 3D cell culture is discussed. Indeed such gradients-on-a-chip might bring unprecedented information to better determine permissible exposure levels. Finally, the impact of tissue architecture, established via cell-matrix interactions, on the cell nucleus is emphasized in light of the importance in toxicology of morphological and epigenetic alterations of this organelle.
Subject(s)
Cell Culture Techniques , Toxicology/trends , Animals , Cell Culture Techniques/instrumentation , Cell Polarity , Humans , Microfluidics , Toxicity TestsABSTRACT
Oncogenic protein E6 of human papillomavirus type 16 (HPV-16) is believed to involve in the aberrant methylation in cervical cancer as it upregulates DNA methyltransferase 1 (DNMT1) through tumor suppressor p53. In addition, DNA demethylating agent induces the expression of one of the HPV-16 E6 regulated microRNAs (miRs), miR-23b, in human cervical carcinoma SiHa cells. Thus, the importance of DNA methylation and miR-23b in HPV-16 E6 associated cervical cancer development is investigated. In the present study, however, it is found that miR-23b is not embedded in any typical CpG island. Nevertheless, a functional CpG island is predicted in the promoter region of C9orf3, the host gene of miR-23b, and is validated by methylation-specific PCR and bisulfite genomic sequencing analyses. Besides, c-MET is confirmed to be a target gene of miR-23b. Silencing of HPV-16 E6 is found to increase the expression of miR-23b, decrease the expression of c-MET and thus induce the apoptosis of SiHa cells through the c-MET downstream signaling pathway. Taken together, the tumor suppressive miR-23b is epigenetically inactivated through its host gene C9orf3 and this is probably a critical pathway during HPV-16 E6 associated cervical cancer development.
Subject(s)
Aminopeptidases/genetics , DNA Methylation , Gene Expression Regulation, Neoplastic , MicroRNAs/genetics , Oncogene Proteins, Viral/genetics , Repressor Proteins/genetics , Apoptosis/drug effects , Apoptosis/genetics , Azacitidine/analogs & derivatives , Azacitidine/pharmacology , Blotting, Western , Cell Line, Tumor , DNA (Cytosine-5-)-Methyltransferase 1 , DNA (Cytosine-5-)-Methyltransferases/antagonists & inhibitors , DNA (Cytosine-5-)-Methyltransferases/genetics , DNA (Cytosine-5-)-Methyltransferases/metabolism , Decitabine , Enzyme Inhibitors/pharmacology , Female , HCT116 Cells , Human papillomavirus 16/genetics , Human papillomavirus 16/metabolism , Human papillomavirus 16/physiology , Humans , Promoter Regions, Genetic/genetics , Proto-Oncogene Proteins c-met/genetics , Proto-Oncogene Proteins c-met/metabolism , RNA Interference , Reverse Transcriptase Polymerase Chain Reaction , Uterine Cervical Neoplasms/genetics , Uterine Cervical Neoplasms/pathology , Uterine Cervical Neoplasms/virologyABSTRACT
Multidrug resistance (MDR) is one of the major reasons for failure in cancer chemotherapy and its suppression may increase the efficacy of therapy. The human multidrug resistance 1 (MDR1) gene encodes the plasma membrane P-glycoprotein (P-gp) that pumps various anti-cancer agents out of the cancer cell. R-HepG2 and MES-SA/Dx5 cells are doxorubicin induced P-gp over-expressed MDR sublines of human hepatocellular carcinoma HepG2 cells and human uterine carcinoma MES-SA cells respectively. Herein, we observed that clitocine, a natural compound extracted from Leucopaxillus giganteus, presented similar cytotoxicity in multidrug resistant cell lines compared with their parental cell lines and significantly suppressed the expression of P-gp in R-HepG2 and MES-SA/Dx5 cells. Further study showed that the clitocine increased the sensitivity and intracellular accumulation of doxorubicin in R-HepG2 cells accompanying down-regulated MDR1 mRNA level and promoter activity, indicating the reversal effect of MDR by clitocine. A 5'-serial truncation analysis of the MDR1 promoter defined a region from position -450 to -193 to be critical for clitocine suppression of MDR1. Mutation of a consensus NF-κB binding site in the defined region and overexpression of NF-κB p65 could offset the suppression effect of clitocine on MDR1 promoter. By immunohistochemistry, clitocine was confirmed to suppress the protein levels of both P-gp and NF-κB p65 in R-HepG2 cells and tumors. Clitocine also inhibited the expression of NF-κB p65 in MES-SA/Dx5. More importantly, clitocine could suppress the NF-κB activation even in presence of doxorubicin. Taken together; our results suggested that clitocine could reverse P-gp associated MDR via down-regulation of NF-κB.
Subject(s)
ATP Binding Cassette Transporter, Subfamily B, Member 1/metabolism , Antineoplastic Agents/pharmacology , Down-Regulation/drug effects , Drug Resistance, Multiple/drug effects , Pyrimidine Nucleosides/pharmacology , Transcription Factor RelA/genetics , ATP Binding Cassette Transporter, Subfamily B, Member 1/genetics , Cell Proliferation/drug effects , Drug Resistance, Multiple/genetics , Gene Expression Regulation, Neoplastic/drug effects , Hep G2 Cells , Humans , Promoter Regions, Genetic/drug effects , Promoter Regions, Genetic/genetics , X-Linked Inhibitor of Apoptosis Protein/geneticsABSTRACT
It was found that psoralen derivative could perform a Friedel-Crafts acylation smoothly with acetic anhydride to give 5'-acetylpsoralen in a 73% yield. In the presence of boron trifluoride etherate, 5'-acetylpsoralen reacted with both aromatic amines and aliphatic amine smoothly to afford 5'-Schiff-base group substituted psoralen derivatives in 72%-92% yields. The novel synthetic method has the advantages of cheap materials, mild reaction conditions, good yields and high regioselectivity in the Friedel-Crafts acylation. Cell viability assay by MTT demonstrates that some of the psoralen derivatives 6 have antiproliferative activities.
Subject(s)
Cell Proliferation/drug effects , Furocoumarins/chemical synthesis , Furocoumarins/pharmacology , Acylation , Boranes/chemistry , Cell Line, Tumor , Furocoumarins/chemistry , Humans , Molecular Structure , Schiff Bases/chemistryABSTRACT
BACKGROUND/AIMS: Coumarins are natural compounds found in many plants that possess medical value by itself and its modified derivatives. METHOD: Six novel coumarin derivatives were synthesized and examined for their potential anticancer cytotoxicity. RESULT: Among the 6 derivatives, 3,5-dimethyl-(7)H-furo[3,2-g]chromen-7-one (DMFC) presented the strongest cytotoxicity against human hepatoma HepG2 cells in vitro with an IC(50) value of 8.46 ± 0.28 µM in a 48-hour treatment. Further experiments revealed that DMFC induced apoptosis in HepG2 cells through both extrinsic and intrinsic apoptotic pathways in a p53-dependent manner. Mechanistically, DMFC activated caspases 3, 8 and 9, depolarized mitochondrial membrane potential and induced cytochrome c and apoptosis-inducing factor release. DMFC-induced apoptosis was also characterized by DNA fragmentation, phosphatidylserine externalization and sub-G1 peak in DNA histograms. Moreover, both caspase 8 and 9 inhibitors suppressed the apoptosis induced by DMFC. Western blot analyses revealed that DMFC also significantly increased the expression levels of p53, Fas death receptor, Fas-associated death domain protein and proapoptotic Bcl-2 family members such as Bax, Bad and tBid, as well as decreased the levels of pro-survival members such as Bcl-2 and Bcl-xl. CONCLUSION: DMFC is potentially an effective therapeutic agent in liver cancer therapy.
Subject(s)
Antineoplastic Agents/pharmacology , Apoptosis/drug effects , Benzofurans/pharmacology , Carcinoma, Hepatocellular/drug therapy , Coumarins/pharmacology , Liver Neoplasms/drug therapy , Tumor Suppressor Protein p53/metabolism , Apoptosis Inducing Factor/metabolism , Carcinoma, Hepatocellular/genetics , Carcinoma, Hepatocellular/metabolism , Carcinoma, Hepatocellular/pathology , Caspases/metabolism , Cytochromes c/metabolism , DNA Fragmentation/drug effects , Hep G2 Cells , Humans , Liver Neoplasms/genetics , Liver Neoplasms/metabolism , Liver Neoplasms/pathology , Membrane Potential, Mitochondrial/drug effects , Phosphatidylserines/metabolism , Proto-Oncogene Proteins c-bcl-2/biosynthesis , Proto-Oncogene Proteins c-bcl-2/genetics , Tumor Suppressor Protein p53/biosynthesis , Tumor Suppressor Protein p53/genetics , bcl-X Protein/biosynthesis , bcl-X Protein/genetics , fas Receptor/biosynthesis , fas Receptor/geneticsABSTRACT
P-glycoprotein (Pgp), an efflux pump, was confirmed the first time to regulate the expressions of miR/gene in cells. Pgp is known to be associated with multidrug resistance. RHepG2 cells, the multidrug resistant subline of human hepatocellular carcinoma HepG2 cells, expressed higher levels of Pgp as well as miR-16, and lower level of Bcl-2 than the parental cells. In addition, RHepG2 cells were more radiation sensitive and showed more pronounced radiation-induced apoptotic cell death than the parental cells. Mechanistic analysis revealed that transfection with mdr1 specific antisense oligos suppressed radiation-induced apoptosis in HepG2 cells. On the other hand, ectopic mdr1 expression enhanced radiation-induced apoptosis in HepG2 cells, SK-HEP-1 cells, MiHa cells, and furthermore, induced miR-16 and suppressed its target gene Bcl-2 in HepG2 cells. Moreover, the enhancement effects of Pgp and miR-16 on radiation-induced apoptosis were counteracted by overexpression of Bcl-2. The Pgp effect on miR-16/Bcl-2 was suppressed by Pgp blocker verapamil indicating the importance of the efflux of Pgp substrates. The present study is the first to reveal the role of Pgp in regulation of miRNA/gene expressions. The findings may provide new perspective in understanding the biological function of Pgp.
Subject(s)
ATP Binding Cassette Transporter, Subfamily B, Member 1/physiology , Apoptosis/genetics , Gene Expression Regulation, Neoplastic , MicroRNAs/genetics , Proto-Oncogene Proteins c-bcl-2/genetics , Radiation Tolerance/genetics , ATP Binding Cassette Transporter, Subfamily B , ATP Binding Cassette Transporter, Subfamily B, Member 1/antagonists & inhibitors , ATP Binding Cassette Transporter, Subfamily B, Member 1/genetics , Carcinoma, Hepatocellular/genetics , Carcinoma, Hepatocellular/metabolism , Cell Line, Tumor , Drug Resistance, Multiple/genetics , Drug Resistance, Neoplasm/genetics , Gamma Rays , Humans , Verapamil/pharmacologyABSTRACT
The HPV-16 early proteins E6 and E7 are considered to function as oncoproteins in cervical cancer. DNA methyltransferase 1 (DNMT1) is one of the enzymes involved in epigenetic silencing of tumor suppressor genes. In the present study, the functional role and regulation of DNMT1 in HPV-16 E6 associated cervical cancer development were examined. Knockdown of E6 in HPV-16 positive human cervical carcinoma SiHa and CaSki cells led to the increase in p53, repression of DNMT1 protein and promoter activity. Moreover, p53 knockdown increased the DNMT1 protein as well as promoter activity, indicating that p53 may mediate E6 upregulation of DNMT1. In addition, E6 knockdown induced growth retardation in SiHa cells, and the effect was partially reverted by DNMT1 overexpression. The results suggest that HPV-16 E6 may act through p53/ DNMT1 to regulate the development of cervical cancer.
Subject(s)
DNA (Cytosine-5-)-Methyltransferases/genetics , Oncogene Proteins, Viral/physiology , Repressor Proteins/physiology , Tumor Suppressor Protein p53/antagonists & inhibitors , Tumor Suppressor Protein p53/genetics , Carcinoma/etiology , Carcinoma/genetics , Carcinoma/metabolism , Cell Line, Tumor , Cell Proliferation/drug effects , DNA (Cytosine-5-)-Methyltransferase 1 , DNA (Cytosine-5-)-Methyltransferases/metabolism , Down-Regulation/drug effects , Down-Regulation/genetics , Female , Gene Expression Regulation, Neoplastic/drug effects , Gene Knockdown Techniques , Human papillomavirus 16/metabolism , Human papillomavirus 16/physiology , Humans , Oncogene Proteins, Viral/genetics , Oncogene Proteins, Viral/metabolism , Papillomavirus Infections/complications , Papillomavirus Infections/genetics , RNA, Small Interfering/pharmacology , Repressor Proteins/genetics , Repressor Proteins/metabolism , Tumor Suppressor Protein p53/metabolism , Up-Regulation/drug effects , Up-Regulation/physiology , Uterine Cervical Neoplasms/etiology , Uterine Cervical Neoplasms/genetics , Uterine Cervical Neoplasms/metabolismABSTRACT
BAD (BCL-2 antagonist of cell death) is a pro-apoptotic BCL-2 family protein that plays a critical role in the regulation of apoptotic response. This study presents direct evidence that AF1q increased the radiation-induced apoptosis through up-regulation of BAD in human squamous carcinoma A431 cells and the key transcription factor involved is NF-kappaB. The minimal promoter sequence of BAD was identified; the activity was increased in AF1q stable transfectants and decreased upon AF1q siRNA transfection. The NF-kappaB consensus binding sequence is detected on BAD promoter. Inactivation of NF-kappaB by NF-kappaB inhibitor Bay 11-7082 or NF-kappaB p65 siRNA suppressed the expression and promoter activity of BAD; the suppression is more obvious in AF1q stable transfectants which also have an elevated NF-kappaB level. Mutation of putative NF-kappaB motif decreased the BAD promoter activity. The binding of NF-kappaB to the BAD promoter was confirmed by chromatin-immunoprecipitation. These findings indicate that AF1q up-regulation of BAD is through its effect on NF-kappaB and this may hint of its oncogenic mechanism in cancer.
Subject(s)
Apoptosis/genetics , Apoptosis/radiation effects , Blood Proteins/physiology , Carcinoma, Squamous Cell/pathology , NF-kappa B/physiology , Neoplasm Proteins/physiology , bcl-Associated Death Protein/genetics , Apoptosis/drug effects , Base Sequence , Blood Proteins/antagonists & inhibitors , Blood Proteins/genetics , Carcinoma, Squamous Cell/genetics , Carcinoma, Squamous Cell/metabolism , Cell Line, Tumor , Gamma Rays , Gene Expression Regulation, Neoplastic/drug effects , Gene Expression Regulation, Neoplastic/radiation effects , Humans , Molecular Sequence Data , NF-kappa B/antagonists & inhibitors , NF-kappa B/genetics , NF-kappa B/metabolism , Neoplasm Proteins/antagonists & inhibitors , Neoplasm Proteins/genetics , Promoter Regions, Genetic , Protein Binding , Proto-Oncogene Proteins , RNA, Small Interfering/pharmacology , Transcription, Genetic/drug effects , Transcription, Genetic/genetics , Up-Regulation/drug effects , Up-Regulation/genetics , bcl-Associated Death Protein/metabolismABSTRACT
Epigallocatechin gallate (EGCG) is a major type of green tea polyphenols and is known to have cancer prevention effect. MicroRNAs (miRNAs) are 19 to 25 nucleotides and are believed to be important in gene regulation. In the present study, the influence of EGCG on the expressions of miRNAs in human cancer cells was investigated as this has not yet been reported. By miRNA microarray analysis, EGCG treatment was found to modify the expressions of some of the miRNAs in human hepatocellular carcinoma HepG2 cells, 13 were up-regulated and 48 were down-regulated. miR-16 is one of the miRNAs up-regulated by EGCG and one of its target genes is confirmed to be the anti-apoptotic protein Bcl-2. EGCG treatment induced apoptosis and down-regulated Bcl-2 in HepG2 cells. Transfection with anti-miR-16 inhibitor suppressed miR-16 expression and counteracted the EGCG effects on Bcl-2 down-regulation and also induction of apoptosis in cells. Results from the present study confirm the role of miR-16 in mediating the apoptotic effect of EGCG and also support the importance of miRNAs in the regulation of the biological activity of EGCG.
Subject(s)
Anticarcinogenic Agents/pharmacology , Apoptosis/drug effects , Carcinoma, Hepatocellular/metabolism , Catechin/analogs & derivatives , Liver Neoplasms/metabolism , MicroRNAs/metabolism , Up-Regulation/drug effects , Catechin/pharmacology , DNA Fragmentation/drug effects , Gene Expression Profiling , Gene Expression Regulation, Neoplastic/drug effects , Genes, Reporter , Genes, bcl-2/drug effects , Hep G2 Cells , Humans , MicroRNAs/antagonists & inhibitors , MicroRNAs/genetics , Oligonucleotide Array Sequence Analysis , Polymerase Chain Reaction , Proto-Oncogene Proteins c-bcl-2/genetics , Proto-Oncogene Proteins c-bcl-2/metabolism , Tea/chemistryABSTRACT
H19 is an imprinted oncofetal non-coding RNA recently shown to be the precursor of miR-675. The pathophysiological roles of H19 and its mature product miR-675 to carcinogenesis have, however, not been defined. By quantitative reverse transcription-polymerase chain reaction, both H19 and miR-675 were found to be upregulated in human colon cancer cell lines and primary human colorectal cancer (CRC) tissues compared with adjacent non-cancerous tissues. Subsequently, the tumor suppressor retinoblastoma (RB) was confirmed to be a direct target of miR-675 as the microRNA suppressed the activity of the luciferase reporter carrying the 3'-untranslated region of RB messenger RNA that contains the miR-675-binding site. Suppression of miR-675 by transfection with anti-miR-675 increased RB expression and at the same time, decreased cell growth and soft agar colony formation in human colon cancer cells. Reciprocally, enhanced miR-675 expression by transfection with miR-675 precursor decreased RB expression, increased tumor cell growth and soft agar colony formation. Moreover, the inverse relationship between the expressions of RB and H19/miR-675 was also revealed in human CRC tissues and colon cancer cell lines. Our findings demonstrate that H19-derived miR-675, through downregulation of its target RB, regulates the CRC development and thus may serve as a potential target for CRC therapy.
Subject(s)
Adenocarcinoma/genetics , Cell Transformation, Neoplastic/genetics , Colorectal Neoplasms/genetics , MicroRNAs/physiology , Neoplasm Proteins/physiology , RNA, Neoplasm/physiology , RNA, Untranslated/genetics , Retinoblastoma Protein/physiology , 3' Untranslated Regions/genetics , Adenocarcinoma/pathology , Binding Sites , Cell Division , Colorectal Neoplasms/pathology , Down-Regulation , Fibroblasts/cytology , Gene Expression Profiling , Humans , MicroRNAs/genetics , RNA Interference , RNA, Long Noncoding , RNA, Neoplasm/genetics , RNA, Small Interfering/pharmacology , Transfection , Tumor Cells, Cultured/pathology , Tumor Stem Cell AssayABSTRACT
Hepatocellular carcinoma HepG2 cells (G cells) were subjected to selection first with gamma-radiation and then doxorubicin (Dox). The radiation treatment consisted of 2 Gy for 10 days (G2) or 10 Gy for 2 days (G10) and the Dox treatment was continuous exposure for up to 10 microM. Compared with respective parental G, G2, G10 cells, the Dox-selected cells showed mdr1 amplification/P-glycoprotein overexpression, Dox resistance and also less intracellular Dox accumulation. Verapamil reversed the drug resistance and increased the Dox accumulation in all cells. Decay in drug resistance and reduction in mdr1 amplification/P-glycoprotein overexpression were observed in the Dox-selected cells culturing in Dox-free condition. Among the Dox-selected cells, G2R cells showed the highest levels of drug resistance, mdr1 amplification, but the least resistance decay. Results from the study indicate the possible influence of radiation treatment on the development of drug resistance in cancer cells and it may even lead to a highly resistant phenotype.
Subject(s)
Drug Resistance, Neoplasm/radiation effects , Gamma Rays/adverse effects , Gene Expression/radiation effects , ATP Binding Cassette Transporter, Subfamily B, Member 1/biosynthesis , ATP Binding Cassette Transporter, Subfamily B, Member 1/genetics , Antineoplastic Agents/pharmacology , Blotting, Southern , Blotting, Western , Cell Line, Tumor , Combined Modality Therapy/adverse effects , Doxorubicin/pharmacology , Drug Resistance, Neoplasm/drug effects , Drug Resistance, Neoplasm/genetics , Humans , Radiotherapy/adverse effects , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
Hepatoma-derived growth factor (HDGF) is frequently overexpressed in human cancer. The growth factor was previously demonstrated to be a survival factor as knock-down of HDGF suppresses the growth and induces apoptosis in human cancer cells through the Bad-mediated intrinsic apoptotic pathway. However, inactivation of Bad cannot completely repress the apoptosis induced upon HDGF knock-down, indicating the presence of other unidentified pathways. In the present study, HDGF knock-down was shown to trigger the Fas-mediated extrinsic apoptotic pathway in human hepatocellular carcinoma HepG2 cells through NF-kappaB signaling pathway. Increases in Fas expression and fas promoter activity were detected upon HDGF knock-down by Western blot analysis and luciferase reporter assay. Knock-down of fas inhibited HDGF knock-down effect on apoptosis induction and growth suppression as revealed by annexin V binding assay and soft agar assay. Down-regulation of IkappaBalpha was also observed upon HDGF knock-down. Overexpression of IkappaBalpha by transient transfection or inhibition of NF-kappaB by BAY11-7082 suppressed HDGF knock-down effect on fas promoter activation, Fas up-regulation, apoptosis induction and growth suppression. Furthermore, the interaction of Fas-mediated extrinsic and Bad-mediated intrinsic apoptotic pathways was demonstrated as a stronger inhibition on apoptosis induction and growth suppression upon HDGF knock-down was observed when both pathways were inactivated. The results therefore suggested that, through both intrinsic and extrinsic apoptotic pathways, HDGF may function as a survival factor and be a potential target for cancer therapy.
Subject(s)
Apoptosis , Carcinoma, Hepatocellular/metabolism , Carcinoma, Hepatocellular/pathology , Intercellular Signaling Peptides and Proteins/genetics , Liver Neoplasms/metabolism , Liver Neoplasms/pathology , Annexin A5/analysis , Annexin A5/metabolism , Carcinoma, Hepatocellular/genetics , Cell Line, Tumor , DNA Fragmentation/drug effects , Gene Expression Regulation, Neoplastic/drug effects , Genes, Reporter , Humans , Liver Neoplasms/genetics , Luciferases, Renilla/metabolism , Models, Biological , NF-kappa B/antagonists & inhibitors , NF-kappa B/metabolism , Nitriles/pharmacology , Oligonucleotides, Antisense/pharmacology , RNA, Small Interfering/metabolism , Signal Transduction , Sulfones/pharmacology , Transfection , bcl-Associated Death Protein/metabolism , fas Receptor/biosynthesisABSTRACT
During the search of new anti-cancer agent from high fungi, the ethyl acetate extract of the mushroom Suillus placidus was found to exhibit a significant cytotoxic activity against human hepatoma HepG2 cells. With bioassay-guided fractionation, a cytotoxic component suillin was isolated from the extract. The anti-cancer effect of suillin was subsequently examined in 8 human cancer cell lines by using MTT assay. It is of interest to note that human liver cancer cells (HepG2 cells, Hep3B cells, and SK-Hep-1) were preferentially killed by suillin with an IC(50) of approximately 2microM in a 48h treatment. Mechanistically. suillin was found for the first time to induce apoptosis in HepG2 cells as characterized by DNA fragmentation, phosphatidyl-serine (PS) externalization, activation of caspase-3, -8, -9, depolarization of mitochondrial membrane potential, as well as release of cytochrome c into the cytosol. Moreover, the apoptosis induced by suillin was suppressed by both caspase-8 and -9 inhibitors. Western blot analysis revealed significant increases in the protein levels of Fas death receptor, adaptor FADD protein, pro-apoptotic protein Bad and a decline of Bid. These results suggest that the induction of apoptosis by suillin is through both death receptor and mitochondrial pathways. Taken together, our results suggest that suillin might be an effective agent to treat liver cancer.
Subject(s)
Agaricales/chemistry , Apoptosis/drug effects , Diterpenes/pharmacology , Phenols/pharmacology , Blotting, Western , Cell Line, Tumor , Cell Proliferation/drug effects , Humans , Phosphatidylserines/metabolismABSTRACT
Estrogen-related receptor alpha (ERRalpha) is primarily thought to regulate energy homeostasis through interacting with peroxisome proliferator-activated receptor gamma coactivator-1alpha and -1beta (PGC-1alpha and -1beta). They coordinately control the transcription of genes in the oxidative phosphorylation pathway. In addition to its role in energy metabolism, ERRalpha has also been implicated as a prognostic marker for breast, ovarian, colon and prostate cancers. In this study, we found that an ERRalpha inverse agonist XCT-790 induced cell death in HepG2 hepatocarcinoma and its multi-drug resistance (MDR) sub-line R-HepG2. Using a dye Mitotracker Green which stains mitochondrion independent of mitochondrial membrane potential (DeltaPsi(m)), we found that XCT-790 dose-dependently decreased mitochondrial mass. Intriguingly, XCT-790 increased DeltaPsi(m) upon short term treatment but decreased DeltaPsi(m) upon longer term treatment. The changes of DeltaPsi(m) in turn promoted the production of reactive oxygen species (ROS) and led to ROS-mediated caspases 3/7, 8, 9 activation and cell death. Importantly, we established that an anti-oxidative compound Mn(III) Tetra(4-benzoic acid) porphyrin chloride (MnTBAP) blocked the caspases activities and cell death increased by XCT-790 treatment. Finally, we found that XCT-790 synergized with paclitaxel to induce cell death in multi-drug resistance sub-line R-HepG2. Our results provide a conceptual framework for further developing chemotherapeutics based on suppressing ERRalpha activity.
Subject(s)
Drug Resistance, Neoplasm , Nitriles/pharmacology , Receptors, Estrogen/agonists , Thiazoles/pharmacology , Apoptosis/drug effects , Blotting, Western , Caspases/metabolism , Cell Line, Tumor , DNA, Mitochondrial/genetics , Humans , Mitochondria/drug effects , Polymerase Chain Reaction , Reactive Oxygen Species/metabolism , ERRalpha Estrogen-Related ReceptorABSTRACT
The Ras proto-oncogene mediates a wide variety of cellular events and is frequently mutated in cancer. MicroRNAs (miRNAs) may regulate the development of cancer through their effect on the target genes. In the search of miRNAs that target on Ras, miR-18a* is the first time confirmed to target on K-Ras and furthermore not on N- and H-Ras. miR-18a* repression by transfection with anti-miR-18a* inhibitor increased the K-Ras expression as well as the luciferase activity of a reporter construct containing the 3'-untranslated region of K-Ras messenger RNA. Furthermore, the miR-18a* repression also increased the cell proliferation and promoted the anchorage-independent growth in soft agar of human squamous carcinoma A431 cells, colon adenocarcinoma HT-29 cells and fetal hepatic WRL-68 cells. On the other hand, ectopic expression of miR-18a* by transfection with miR-18a* precursor suppressed K-Ras expression, cell proliferation and anchorage-independent growth of A431 cells. The increase in cell proliferation and anchorage-independent growth upon miR-18a* repression was, however, rendered by the Ras inhibitor farnesylthiosalicylic acid. In conclusion, miR-18a* may function as a tumor suppressor by targeting on K-Ras. Therefore, the miRNA may also be a potential therapeutic agent or target for cancer therapy.