Cytotoxic Property of Grias neuberthii Extract on Human Colon Cancer Cells: A Crucial Role of Autophagy.
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ABSTRACT:
SUBMITTER: Guaman-Ortiz LM
PROVIDER: S-EPMC7152961 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Despite significant progress in the diagnosis and treatment of colorectal cancer, drug resistance continues to be a major limitation of therapy. In this regard, studies aimed at creating combination therapy are gaining popularity. One of the most promising adjuvants are inhibitors of the proteostasis system, chaperone machinery, and autophagy. The main HSP regulator, HSF1, is overactivated in cancer cells and autophagy sustains the survival of malignant cells. In this work, we focused on the selection of combination therapy for the treatment of rectal cancer cells obtained from patients after tumor biopsy without prior treatment. We characterized the migration, proliferation, and chaperone status in the resulting lines and also found them to be resistant to a number of drugs widely used in the clinic. However, these cells were sensitive to the autophagy inhibitor, chloroquine. For combination therapy, we used an HSF1 activity inhibitor discovered earlier in our laboratory, the cardenolide CL-43, which has already been proven as an auxiliary component of combined therapy in established cell lines. CL-43 effectively suppressed HSF1 activity and Hsp70 expression in all investigated cells. We tested the autophagy inhibitor, chloroquine, in combination with CL-43. Our results indicate that the use of an inhibitor of HSF1 activity in combination with an autophagy inhibitor results in effective cancer cell death, therefore, this therapeutic approach may be a promising treatment regimen for certain patients.
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Project description:BackgroundCancer cells maintain high rates of glycolysis. Pyruvate dehydrogenase kinases (PDK) contribute to this phenomenon, which favours apoptosis resistance and cellular transformation. We previously reported upregulation of PDK4 in normal mucosa of colorectal cancer (CRC) patients compared with controls and in preneoplastic intestine of our mouse model. Decreased methylation of four consecutive PDK4 CpGs was observed in normal mucosa of patients. Although other members of the PDK family have been investigated for transformation potential, PDK4 has not been extensively studied.MethodsPDK4 methylation in blood of CRC patients and controls was evaluated by pyrosequencing. PDK4 expression in human colon carcinoma cells was down-regulated by RNAi. Cellular migration and invasion, apoptosis and qRT-PCR of key genes were assessed.ResultsPyrosequencing revealed decreased methylation of the same four consecutive CpGs in the blood of patients compared with controls. Cellular migration and invasion were reduced and apoptosis was increased following transient or stable inhibition of PDK4. Expression of vimentin, HIF-1 and VEGFA was reduced.ConclusionsThese studies demonstrate the involvement of PDK4 in transformation. Methylation assessment of PDK4 in the blood may be useful for non-invasive CRC detection. PDK4 should be considered as a target for development of anticancer strategies and therapies.
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Project description:The research for innovative treatments against colon adenocarcinomas is still a great challenge. Acacia catechu Willd. heartwood extract (AC) has health-promoting qualities, especially at the gastrointestinal level. This study characterized AC for its catechins content and investigated the apoptosis-enhancing effect in human colorectal adenocarcinoma HT-29 cells, along with its ability to spare healthy tissue. MTT assay was used to describe the time course, concentration dependence and reversibility of AC-mediated cytotoxicity. Cell cycle analysis and AV-PI and DAPI-staining were performed to evaluate apoptosis, together with ROS formation, mitochondrial membrane potential (MMP) changes and caspase activities. Rat ileum and colon rings were tested for their viability and functionality to explore AC effects on healthy tissue. Quantitative analysis highlighted that AC was rich in (±)-catechin (31.5 ± 0.82 mg/g) and (-)-epicatechin (12.5 ± 0.42 mg/g). AC irreversibly decreased cell viability in a concentration-dependent, but not time-dependent fashion. Cytotoxicity was accompanied by increases in apoptotic cells and ROS, a reduction in MMP and increases in caspase-9 and 3 activities. AC did not affect rat ileum and colon rings' viability and functionality, suggesting a safe profile toward healthy tissue. The present findings outline the potential of AC for colon cancer treatment.
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Project description:Autophagy modulation is now recognized as a potential therapeutic approach for cancer (including colorectal cancer), yet the molecular mechanisms regulating autophagy in response to cellular stress are still not well understood. MicroRNAs (miRNAs) have been found to play important roles in controlling many cellular functions, including growth, metabolism and stress response. The physiological importance of the miRNA-autophagy interconnection is only beginning to be elucidated. MiRNA microarray technology facilitates analysis of global miRNA expression in certain situations. In this study, we explored the expression profile of miRNAs during the response of human colon cancer cells (HT29s) to 5-FU treatment and nutrient starvation using miRNA microarray analysis. The alteration of miRNA expression showed the same pattern under both conditions was further testified by qRT-PCR in three human colon cancer cell lines. In addition, bioinformatic prediction of target genes, pathway analysis and gene network analysis were performed to better understand the roles of these miRNAs in the regulation of autophagy. We identified and selected four downregulated miRNAs including hsa-miR-302a-3p and 27 upregulated miRNAs under these two conditions as having the potential to target genes involved in the regulation of autophagy in human colon cancer cells. They have the potential to modulate autophagy in 5-FU-based chemotherapy in colorectal cancer.
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Project description:BackgroundAutophagy plays important roles in odontogenic differentiation of dental pulp cells (DPCs) in the developmental stage of tooth bud. Few studies have reported the role of autophagy during reparative dentin formation process. The objective of this study was to discover gene expression pattern correlated to autophagy and their role during odontogenic differentiation process in DPCs.MethodsAfter tooth cavities were prepared on the mesial surface of lower first molar crown of rats. Odontogenic differentiation and reparative dentin formation were assessed based on detection of morphology change with hematoxylin and eosin staining.ResultsAfter tooth cavities were prepared on the mesial surface of lower first molar crown of rats, odontogenic differentiation and reparative dentin formation were assessed based on detection of morphology change with hematoxylin and eosin staining and dentin sialophosphoprotein (DSPP), whereas autophagy inhibitor 3-methyladenine (3MA) reversed. Results of quantitative polymerized chain reaction array of autophagosome formation related genes revealed that GABARAPL2 was prominently upregulated while expression of other ATG8 family members were moderately increased after tooth cavity preparation. In addition, human DPCs incubated in differentiation medium predominantly upregulated MAP1LC3C, which selectively decreased by 3MA but not by autophagy enhancer trehalose. Knock-down of MAP1LC3C using shRNA resulted in strong downregulation of dentin matrix protein 1 and DSPP as well-known odontogenic marker compared to knock-down of MAP1LC3B during odontogenic differentiation process of human DPCs.ConclusionOur results suggest that MAP1LC3C plays a crucial role in odontogenic differentiation of human DPCs via regulating autophagic flux.
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Project description:Silver nanoparticles (AgNPs) have gained attention for use in cancer therapy. In this study, AgNPs were biosynthesized using naringenin. We investigated the anti-colon cancer activities of biogenic AgNPs through transcriptome analysis using RNA sequencing, and the mechanisms of AgNPs in regulating colon cancer cell growth. The synthesized AgNPs were characterized using UV⁻visible spectroscopy (UV⁻vis), X-ray diffraction (XRD), Fourier-transform infrared spectroscopy (FTIR), dynamic light scattering (DLS), and transmission electron microscopy (TEM). The AgNPs were spherical with sizes of 2⁻10 nm. Cytotoxicity assays indicated that the AgNPs in HCT116 colorectal cancer cells were very effective at low concentrations. The viability and proliferation of colon cancer cells treated with 5 µg/mL biogenic AgNPs were reduced by 50%. Increased lactate dehydrogenase leakage (LDH), reactive oxygen species (ROS) generation, malondialdehyde (MDA), and decreased dead-cell protease activity and ATP generation were observed. This impaired mitochondrial function and DNA damage led to cell death. The AgNPs upregulated and downregulated the most highly ranked biological processes of oxidation⁻reduction and cell-cycle regulation, respectively. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed that AgNPs upregulated GADD45G in the p53 pathway. Thus, the AgNP tumor suppressive effects were mediated by cell apoptosis following DNA damage, as well as by mitochondrial dysfunction and cell-cycle arrest following aberrant regulation of p53 effector proteins. It is of interest to mention that, to the best of our knowledge, this study is the first report demonstrating cellular responses and molecular pathways analysis of AgNPs in HCT116 colorectal cancer cells.
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Project description:The nucleolus is the site of ribosome biogenesis and has been recently described as important sensor for a variety of cellular stressors. In the last two decades, it has been largely demonstrated that many chemotherapeutics act by inhibiting early or late rRNA processing steps with consequent alteration of ribosome biogenesis and activation of nucleolar stress response. The overall result is cell cycle arrest and/or apoptotic cell death of cancer cells. Our previously data demonstrated that ribosomal protein uL3 is a key sensor of nucleolar stress activated by common chemotherapeutic agents in cancer cells lacking p53. We have also demonstrated that uL3 status is associated to chemoresistance; down-regulation of uL3 makes some chemotherapeutic drugs ineffective. Here, we demonstrate that in colon cancer cells, the uL3 status affects rRNA synthesis and processing with consequent activation of uL3-mediated nucleolar stress pathway. Transcriptome analysis of HCT 116p53-/- cells expressing uL3 and of a cell sub line stably depleted of uL3 treated with Actinomycin D suggests a new extra-ribosomal role of uL3 in the regulation of autophagic process. By using confocal microscopy and Western blotting experiments, we demonstrated that uL3 acts as inhibitory factor of autophagic process; the absence of uL3 is associated to increase of autophagic flux and to chemoresistance. Furthermore, experiments conducted in presence of chloroquine, a known inhibitor of autophagy, indicate a role of uL3 in chloroquine-mediated inhibition of autophagy. On the basis of these results and our previous findings, we hypothesize that the absence of uL3 in cancer cells might inhibit cancer cell response to drug treatment through the activation of cytoprotective autophagy. The restoration of uL3 could enhance the activity of many drugs thanks to its pro-apoptotic and anti-autophagic activity.
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Project description:BackgroundMutations that activate the PI3K/AKT/mTOR pathway are relatively common in urothelial (bladder) cancers, but how these pathway mutations affect AKT dependency is not known. We characterized the relationship between AKT pathway mutational status and sensitivity to the effects of the selective AKT kinase inhibitor AZ7328 using a panel of 12 well-characterized human bladder cancer cell lines.MethodsSequenome DNA sequencing was performed to identify mutations in a panel of 12 urothelial cancer cell lines. Drug-induced proliferative inhibition and apoptosis were quantified using MTT assays and propidium iodide staining with FACS analyses. Protein activation via phosphorylation was measured by immunoblotting. Autophagy was measured by LC3 immunofluorescence and immunoblotting.ResultsAZ7328 inhibited proliferation and AKT substrate phosphorylation in a concentration-dependent manner but had minimal effects on apoptosis. Proliferative inhibition correlated loosely with the presence of activating PIK3CA mutations and was strengthened in combination with the mTOR inhibitor rapamycin. AZ7328 induced autophagy in some of the lines, and in the cells exposed to a combination of AZ7328 and chemical autophagy inhibitors apoptosis was induced.ConclusionsThe cytostatic effects of AZ7328 correlate with PIK3CA mutations and are greatly enhanced by dual pathway inhibition using an mTOR inhibitor. Furthermore, AZ7328 can interact with autophagy inhibitors to induce apoptosis in some cell lines. Overall, our results support the further evaluation of combinations of PI3K/AKT/mTOR pathway and autophagy inhibitors in pre-clinical in vivo models and ultimately in patients with PIK3CA mutant bladder cancers.
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Project description:Tooth enamel is generated by ameloblasts. Any failure in amelogenesis results in defects in the enamel, a condition known as amelogenesis imperfecta. Here, we report that mice with deficient autophagy in epithelial-derived tissues (K14-Cre;Atg7F/F and K14-Cre;Atg3F/F conditional knockout mice) exhibit amelogenesis imperfecta. Micro-computed tomography imaging confirmed that enamel density and thickness were significantly reduced in the teeth of these mice. At the molecular level, ameloblast differentiation was compromised through ectopic accumulation and activation of NRF2, a specific substrate of autophagy. Through bioinformatic analyses, we identified Bcl11b, Dlx3, Klk4, Ltbp3, Nectin1, and Pax9 as candidate genes related to amelogenesis imperfecta and the NRF2-mediated pathway. To investigate the effects of the ectopic NRF2 pathway activation caused by the autophagy deficiency, we analyzed target gene expression and NRF2 binding to the promoter region of candidate target genes and found suppressed gene expression of Bcl11b, Dlx3, Klk4, and Nectin1 but not of Ltbp3 and Pax9. Taken together, our findings indicate that autophagy plays a crucial role in ameloblast differentiation and that its failure results in amelogenesis imperfecta through ectopic NRF2 activation.
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Project description:Hypoxia occurs in a wide variety of physiological and pathological conditions, including tumorigenesis. Tumor cells have to adapt to hypoxia by altering their gene expression and protein synthesis. Here, we showed that hypoxia inhibits translation through activation of PERK and inactivation of mTOR in human colon cancer HCT116 cells. Prolonged hypoxia (1% O2, 16 h) dramatically inhibits general translation in HCT116 cells, yet selected mRNAs remain efficiently translated under such a condition. Using microarray analysis of polysome- associated mRNAs, we identified a large number of hypoxia-regulated genes at the translational level. Efficiently translated mRNAs during hypoxia were validated by polysome profiling and quantitative real-time RT-PCR. Pathway enrichment analysis showed that many of the up-regulated genes are involved in lysosome, glycan and lipid metabolism, antigen presentation, cell adhesion, and remodeling of the extracellular matrix and cytoskeleton. The majority of down-regulated genes are involved in apoptosis, ubiquitin-mediated proteolysis, and oxidative phosphorylation. Further investigation showed that hypoxia induces lysosomal autophagy and mitochondrial dysfunction through translational regulation in HCT116 cells. The abundance of several translation factors and the mTOR kinase activity are involved in hypoxia-induced mitochondrial autophagy in HCT116 cells. Our studies highlight the importance of translational regulation for tumor cell adaptation to hypoxia.
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