230 research outputs found
Enhancement of autophagy is a potential modality for tumors refractory to radiotherapy
Radiotherapy is a well-established treatment for cancer. However, the existence of radioresistant cells is one of the major obstacles in radiotherapy. In order to understand the mechanism of cellular radioresistance and develop more effective radiotherapy, we have established clinically relevant radioresistant (CRR) cell lines, which continue to proliferate under daily exposure to 2 Gray (Gy) of X-rays for >30 days. X-ray irradiation significantly induced autophagic cells in parental cells, which was exiguous in CRR cells, suggesting that autophagic cell death is involved in cellular radiosensitivity. An autophagy inducer, rapamycin sensitized CRR cells to the level of parental cells and suppressed cell growth. An autophagy inhibitor, 3-methyladenine induced radioresistance of parental cells. Furthermore, inhibition of autophagy by knockdown of Beclin-1 made parental cells radioresistant to acute radiation. These suggest that the suppression of autophagic cell death but not apoptosis is mainly involved in cellular radioresistance. Therefore, the enhancement of autophagy may have a considerable impact on the treatment of radioresistant tumor
1,1-Bis(3'-indolyl)-1-(p-substituted phenyl)methanes induce autophagic cell death in estrogen receptor negative breast cancer
Signal transducer and activator of transcription 3 is involved in cell growth and survival of human rhabdomyosarcoma and osteosarcoma cells
The role of RelA (p65) threonine 505 phosphorylation in the regulation of cell growth, survival, and migration
The NF-ΞΊB family of transcription factors is a well-established regulator of the immune and inflammatory responses and also plays a key role in other cellular processes, including cell death, proliferation, and migration. Conserved residues in the trans-activation domain of RelA, which can be posttranslationally modified, regulate divergent NF-ΞΊB functions in response to different cellular stimuli. Using rela(β/β) mouse embryonic fibroblasts reconstituted with RelA, we find that mutation of the threonine 505 (T505) phospho site to alanine has wide-ranging effects on NF-ΞΊB function. These include previously described effects on chemotherapeutic drug-induced apoptosis, as well as new roles for this modification in autophagy, cell proliferation, and migration. This last effect was associated with alterations in the actin cytoskeleton and expression of cellular migrationβassociated genes such as WAVE3 and Ξ±-actinin 4. We also define a new component of cisplatin-induced, RelA T505βdependent apoptosis, involving induction of NOXA gene expression, an effect explained at least in part through induction of the p53 homologue, p73. Therefore, in contrast to other RelA phosphorylation events, which positively regulate NF-ΞΊB function, we identified RelA T505 phosphorylation as a negative regulator of its ability to induce diverse cellular processes such as apoptosis, autophagy, proliferation, and migration
DOR/Tp53inp2 and Tp53inp1 Constitute a Metazoan Gene Family Encoding Dual Regulators of Autophagy and Transcription
Human DOR/TP53INP2 displays a unique bifunctional role as a modulator of autophagy and gene transcription. However, the domains or regions of DOR that participate in those functions have not been identified. Here we have performed structure/function analyses of DOR guided by identification of conserved regions in the DOR gene family by phylogenetic reconstructions. We show that DOR is present in metazoan species. Invertebrates harbor only one gene, DOR/Tp53inp2, and in the common ancestor of vertebrates Tp53inp1 may have arisen by gene duplication. In keeping with these data, we show that human TP53INP1 regulates autophagy and that different DOR/TP53INP2 and TP53INP1 proteins display transcriptional activity. The use of molecular evolutionary information has been instrumental to determine the regions that participate in DOR functions. DOR and TP53INP1 proteins share two highly conserved regions (region 1, aa residues 28β42; region 2, 66β112 in human DOR). Mutation of conserved hydrophobic residues in region 1 of DOR (that are part of a nuclear export signal, NES) reduces transcriptional activity, and blocks nuclear exit and autophagic activity under autophagy-activated conditions. We also identify a functional and conserved LC3-interacting motif (LIR) in region 1 of DOR and TP53INP1 proteins. Mutation of conserved acidic residues in region 2 of DOR reduces transcriptional activity, impairs nuclear exit in response to autophagy activation, and disrupts autophagy. Taken together, our data reveal DOR and TP53INP1 as dual regulators of transcription and autophagy, and identify two conserved regions in the DOR family that concentrate multiple functions crucial for autophagy and transcription
Combination of adenoviral virotherapy and temozolomide chemotherapy eradicates malignant glioma through autophagic and apoptotic cell death in vivo
Conditionally replicative adenoviruses (CRAds) represent a novel treatment strategy for malignant glioma. Recent studies suggest that the cytopathic effect elicited by these vectors is mediated through autophagy, a form of programmed cell death. Likewise, temozolomide (TMZ), a chemotherapeutic agent used for the treatment of malignant gliomas, also triggers autophagic cell death. In this study, we examined the potential to combine the two treatments in the setting of experimental glioma. In vitro, pretreatment with TMZ followed by CRAd-Surivin-pk7 enhanced cytotoxicity against a panel of glioma cell lines. Western blot analysis showed increased expression of BAX and p53, decreased expression of BCL2 and elevated level of APG5. Treatment with TMZ followed by CRAd-Survivin-pk7 (CRAd-S-pk7) led to a significant over-expression of autophagy markers, acidic vesicular organelles and light-chain 3 (LC3). These results were further evaluated in vivo, in which 90% of the mice with intracranial tumours were long-term survivors (>100 days) after treatment with TMZ and CRAd-S-pk7 (P<0.01). Analysis of tumours ex vivo showed expression of both LC3 and cleaved Caspase-3, proving that both autophagy and apoptosis are responsible for cell death in vivo. These results suggest that combination of chemovirotherapy offers a powerful tool against malignant glioma and should be further explored in the clinical setting
A potent betulinic acid analogue ascertains an antagonistic mechanism between autophagy and proteasomal degradation pathway in HT-29 cells
Betulinic acid (BA), a member of pentacyclic triterpenes has shown important biological activities like
anti-bacterial, anti-malarial, anti-inflammatory and most interestingly anticancer property. To overcome its poor
aqueous solubility and low bioavailability, structural modifications of its functional groups are made to generate
novel lead(s) having better efficacy and less toxicity than the parent compound. BA analogue, 2c was found most
potent inhibitor of colon cancer cell line, HT-29 cells with IC50 value 14.9 ΞΌM which is significantly lower than
standard drug 5-fluorouracil as well as parent compound, Betulinic acid. We have studied another mode of PCD,
autophagy which is one of the important constituent of cellular catabolic system as well as we also studied
proteasomal degradation pathway to investigate whole catabolic pathway after exploration of 2c on HT-29 cells.
Mechanism of autophagic cell death was studied using fluorescent dye like acridine orange (AO) and
monodansylcadaverin (MDC) staining by using fluorescence microscopy. Various autophagic protein expression
levels were determined by Western Blotting, qRT-PCR and Immunostaining. Confocal Laser Scanning Microscopy
(CLSM) was used to study the colocalization of various autophagic proteins. These were accompanied by formation
of autophagic vacuoles as revealed by FACS and transmission electron microscopy (TEM). Proteasomal degradation
pathway was studied by proteasome-Gloβ’ assay systems using luminometer.The formation of autophagic vacuoles in HT-29 cells after 2c treatment was determined by fluorescence
staining β confirming the occurrence of autophagy. In addition, 2c was found to alter expression levels of different autophagic proteins like Beclin-1, Atg 5, Atg 7, Atg 5-Atg 12, LC3B and autophagic adapter protein, p62. Furthermore we found the formation of autophagolysosome by colocalization of LAMP-1 with LC3B, LC3B with Lysosome, p62 with lysosome. Finally, as proteasomal degradation pathway downregulated after 2c treatment colocalization of ubiquitin
with lysosome and LC3B with p62 was studied to confirm that protein degradation in autophagy induced HT-29 cells
follows autolysosomal pathway. In summary, betulinic acid analogue, 2c was able to induce autophagy in HT-29 cells and as proteasomal degradation pathway downregulated after 2c treatment so protein degradation in autophagy induced HT-29 cell
Inhibition of platelet-derived growth factor signalling induces autophagy in malignant glioma cells
Galectin-3 and Beclin1/Atg6 Genes In Human Cancers: Using cDNA Tissue Panel, qRT-PCR, and Logistic Regression Model to Identify Cancer Cell Biomarkers
Cancer biomarkers are sought to support cancer diagnosis, predict cancer patient response to treatment and survival. Identifying reliable biomarkers for predicting cancer treatment response needs understanding of all aspects of cancer cell death and survival. Galectin-3 and Beclin1 are involved in two coordinated pathways of programmed cell death, apoptosis and autophagy and are linked to necroptosis/necrosis. The aim of the study was to quantify galectin-3 and Beclin1 mRNA in human cancer tissue cDNA panels and determine their utility as biomarkers of cancer cell survival.A panel of 96 cDNAs from eight (8) different normal and cancer tissue types were used for quantitative real-time polymerase chain reaction (qRT-PCR) using ABI7900HT. Miner2.0, a web-based 4- and 3-parameter logistic regression software was used to derive individual well polymerase chain reaction efficiencies (E) and cycle threshold (Ct) values. Miner software derived formula was used to calculate mRNA levels and then fold changes. The ratios of cancer to normal tissue levels of galectin-3 and Beclin1 were calculated (using the mean for each tissue type). Relative mRNA expressions for galectin-3 were higher than for Beclin1 in all tissue (normal and cancer) types. In cancer tissues, breast, kidney, thyroid and prostate had the highest galectin-3 mRNA levels compared to normal tissues. High levels of Beclin1 mRNA levels were in liver and prostate cancers when compared to normal tissues. Breast, kidney and thyroid cancers had high galectin-3 levels and low Beclin1 levels.Galectin-3 expression patterns in normal and cancer tissues support its reported roles in human cancer. Beclin1 expression pattern supports its roles in cancer cell survival and in treatment response. qRT-PCR analysis method used may enable high throughput studies to generate molecular biomarker sets for diagnosis and predicting cancer treatment response
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