Engagement of Components of DNA-Break Repair Complex and NFκB in Hsp70A1A Transcription Upregulation by Heat Shock.

An involvement of components of DNA-break repair (DBR) complex including DNA-dependent protein kinase (DNA-PK) and poly-ADP-ribose polymerase 1 (PARP-1) in transcription regulation in response to distinct cellular signalling has been revealed by different laboratories. Here, we explored the involvement of DNA-PK and PARP-1 in the heat shock induced transcription of Hsp70A1A. We find that inhibition of both the catalytic subunit of DNA-PK (DNA-PKc), and Ku70, a regulatory subunit of DNA-PK holo-enzyme compromises transcription of Hsp70A1A under heat shock treatment. In immunoprecipitation based experiments we find that Ku70 or DNA-PK holoenzyme associates with NFκB. This NFκB associated complex also carries PARP-1. Downregulation of both NFκB and PARP-1 compromises Hsp70A1A transcription induced by heat shock treatment. Alteration of three bases by site directed mutagenesis within the consensus κB sequence motif identified on the promoter affected inducibility of Hsp70A1A transcription by heat shock treatment. These results suggest that NFκB engaged with the κB motif on the promoter cooperates in Hsp70A1A activation under heat shock in human cells as part of a DBR complex including DNA-PK and PARP-1

NFκB modulates Hsp70A1A promoter activity under heat shock.

<p>A) Schematic showing the regions of Hsp70A1A promoter fragments cloned upstream of renila luciferase reporter (Rluc). The locations of heat shock elements (dHSE, distal heat shock element; pHSE, proximal heat shock element), NF-Y and κB consensus sequence (κBc) and a less conserved κB (?) site are indicated with respect to transcription start site (+1, an arrow). The direction of the arrow indicates the direction of transcription. B) RT-PCR assay with transcripts prepared from HEK293 cells 48 h post transfection with the constructs shown in panel ‘A’. Representative ethidium bromide stained agarose gels showing relative levels of indicated transcripts. C) Estimation of the band intensities of luciferase cDNA vs those of GFP and GAPDH as the internal control. D) RT-PCR assay with transcripts prepared from HEK293 cells 48 h posttransfection with the constructs indicated (also shown in panel A, constructs #3 and #3(M)). Representative ethidium bromide stained agarose gels showing relative levels of transcripts as indicated. E) Estimation of the band intensities of lucifearse cDNA vs those of GFP and GAPDH as the internal control. HS, heat shock.</p

Interaction of p65/RelA with Ku70, DNA-PKc and PARP-1 determined by immunoprecipitation coupled immunoblot experiments.

<p>Whole cell lysates (WCL) of HeLa cells stably expressing FLAG-Ku70 or transiently expressing FLAG-p65/RelA proteins pretreated with heat shock (HS) or not were used. A) Detection of p65/RelA using anti-p65 antibody by immunoblot in immunoprecipitate (IP) obtained from WCL of cells stably expressing FLAG-ku70 using anti-FLAG (M2) antibody. Asterisk denotes a non-specific band. The same membrane was stripped to detect the presence of FLAG-Ku70 protein (lower panel) using anti-FLAG antibody. B) Immunoblot to detect Ku70 or DNA-PKc in immunoprecipitate isolated with anti-FLAG antibody from WCL of cells expressing FLAG-p65/RelA pretreated as indicated. FLAG-p65/RelA level was detected by reblotting the same membrane with the anti-FLAG antibody (FLAG-Ab). The amounts of WCL analyzed as an internal control to identify the target protein band were indicated on the right. C) Immunoblot showing PARP-1 in immunoprecipitate isolated from WCL used in panel B. D) RT-PCR analysis of cDNAs prepared from cells treated with PARP-1 specific siRNA (siPARP-1) or mock to show the effects on Hsp70A1A transcription as indicated by representative agarose gels stained with ethidium bromide. E) The abundance of the indicated transcripts/cDNAs (indicated in panel D) were estimated by RT-qPCR normalized with GAPDH level as internal control. One-way ANOVA with Turkey’s post-test was used to analyse the data where *p < 0.05, **p < 0.01 and ***p < 0.001 respectively. F) Immunoblot showing downregulation of PARP-1 protein in WCL isolated from HeLa cells pre-treated with si-PARP-1 or mock treated. Fold change shown was estimated by densitometric scanning of PARP-1 vs β-Actin bands.</p

The requirement of p65/RelA in Hsp70A1A transcription under heat shock.

<p>A) Representative ethidium bromide stained agarose gels showing relative transcripts levels prepared from HeLa cells as indicated determined by RT-PCR. B) Estimation of the effect of p65 knockdown on Hsp70A1A transcription shown in panel A by RT-qPCR using GAPDH as an internal control. One-way ANOVA with Turkey’s post test was used to analyse the data where ***p < 0.001. C) Immunoblot showing downregulation of p65/RelA protein in WCL isolated from HeLa cells pre-treated with sip65 or scramble siRNA. Fold change shown was estimated by densitometric scanning of intensities of bands of p65/RelA vs β-actin. D) Representative ethidium bromide stained agarose gels showing the effects on Hsp70A1A following the treatment of cells as indicated. PMA treated cells were included as positive control. Andro, andrographolide. E) Estimation of intensities of the bands in panel D. GAPDH level was measured as an internal control. One-way ANOVA with Turkey’s post test was used to analyse the data where ***p < 0.001. F) p65/RelA presence in the nucleus following heat shock (plus 45 min recovery at 37°C) or PMA treatment detected by indirect immunofluorescence technique visualised by a confocal microscope. Antibodies against the epitopes were used to detect transiently expressed the FLAG-p65 or his₆-HSF1 protein, respectively. The images were taken in 63x magnification with a 3x optical zoom. HS, heat shock; NHS, non heat shock. AF, alexa flour. The bar graph on the right show the estimates of cells (%) with nuclear p65. Estimation was carried out using student’s <i>t</i> test, *p<0.05. G) Translocation of FLAG-p65 and his₆-HSF1 in isolated cytoplasmic (C) and nuclear (N) protein fractions by immunoblot using anti-FLAG and anti-his₆ antibody, respectively. The α-tubulin protein level was estimated by immunoblot as a cytoplasmic protein marker. Net nuclear translocation of p65/RelA and his₆HSF1 was estimated by subtracting residual cytoplasm contamination in the nuclear fraction equivalent to α-tubulin level.</p

DNA-PK holoenzyme is required for induction of Hsp70A1A transcription in response to heat shock (HS).

<p>cDNAs prepared from cells pre-treated with Ku70 specific (siKu70), or scrambled siRNA were subjected to PCR to determine expression levels of indicated genes. A) Representative ethidium bromide stained agarose gels indicating the relative levels of Ku70 and Hsp70A1A transcripts. B) The abundance of the indicated transcripts/cDNAs (as indicated in panel A) were estimated by RT-qPCR normalized with normalized with β-actin level. One-way ANOVA with Turkey’s post-test was used to analyse the data where ***p < 0.001. C) Immunoblot showing downregulation of Ku70 protein in whole cell lysate isolated from HeLa cells pre-treated with siKu70 or scramble siRNA. Fold change shown was estimated by densitometric scanning of intensities of bands of Ku70 vs β-actin. D) Representative ethidium bromide stained agarose gels showing the levels of transcripts isolated from cells pre-treated with siKu70, or shDNA-PKc or both siKu70 and shDNA-PKc simultaneously determined by RT-PCR assay. E) Estimation of the intensities of bands shown in panel D through densitometric scanning. The β-actin level was used as the loading control (bottom panel). One-way ANOVA with Turkey’s post-test was used to analyse the data where ***p < 0.001.</p

DNA-PKc is required for heat shock mediated transcription induction of Hsp70A1A gene in human cells.

<p>cDNAs prepared from HT1080 cells pretreated with shRNA against catalytic subunit of DNA-PK (shDNA-PKc) were subjected to PCR for testing its effects on the transcription of Hsp70A1A gene using the procedures described in the materials and methods. A) Representative ethidium bromide stained agarose gels showing relative transcript levels of DNA-PKc, Hsp70A1A and β-actin (as loading control). B) Estimation of DNA-PKc and the Hsp70A1A mRNA levels in indicated samples (indicated in panel A) by the RTq-PCR normalized with β-actin level. One-way ANOVA with Turkey’s post-test was used to analyse the data where **p < 0.01. C) Immunoblot showing downregulation of DNA-PKc protein in whole cell lysate isolated from HeLa cells pre-treated with shDNA-PKc or mock treated. Fold change shown was estimated by densitometric scanning of intensities of bands of DNA-PKc vs β-actin. D) Representative agarose gels stained with ethidium bromide showing the effect on Hsp70A1A transcription upon chemical inhibition (100 μM for 24 h) of DNA-PK determined by RT-PCR. E) Estimation of the effect of chemical inhibition DNA-PK by RT-qPCR assay on transcription of Hsp70A1A normalized with β-actin level. One-way ANOVA with Turkey’s post-test was used to analyze the data where **p < 0.001.</p

Actin–Curcumin Interaction: Insights into the Mechanism of Actin Polymerization Inhibition

Curcumin, derived from rhizomes of the <i>Curcuma longa</i> plant, is known to possess a wide range of medicinal properties. We have examined the interaction of curcumin with actin and determined their binding and thermodynamic parameters using isothermal titration calorimetry. Curcumin is weakly fluorescent in aqueous solution, and binding to actin enhances fluorescence several fold with a large blue shift in the emission maximum. Curcumin inhibits microfilament formation, which is similar to its role in inhibiting microtubule formation. We synthesized a series of stable curcumin analogues to examine their affinity for actin and their ability to inhibit actin self-assembly. Results show that curcumin is a ligand with two symmetrical halves, each of which possesses no activity individually. Oxazole, pyrazole, and acetyl derivatives are less effective than curcumin at inhibiting actin self-assembly, whereas a benzylidiene derivative is more effective. Cell biology studies suggest that disorganization of the actin network leads to destabilization of filaments in the presence of curcumin. Molecular docking reveals that curcumin binds close to the cytochalasin binding site of actin. Further molecular dynamics studies reveal a possible allosteric effect in which curcumin binding at the “barbed end” of actin is transmitted to the “pointed end”, where conformational changes disrupt interactions with the adjacent actin monomer to interrupt filament formation. Finally, the recognition and binding of actin by curcumin is yet another example of its unique ability to target multiple receptors

Actin–Curcumin Interaction: Insights into the Mechanism of Actin Polymerization Inhibition

Curcumin, derived from rhizomes of the <i>Curcuma longa</i> plant, is known to possess a wide range of medicinal properties. We have examined the interaction of curcumin with actin and determined their binding and thermodynamic parameters using isothermal titration calorimetry. Curcumin is weakly fluorescent in aqueous solution, and binding to actin enhances fluorescence several fold with a large blue shift in the emission maximum. Curcumin inhibits microfilament formation, which is similar to its role in inhibiting microtubule formation. We synthesized a series of stable curcumin analogues to examine their affinity for actin and their ability to inhibit actin self-assembly. Results show that curcumin is a ligand with two symmetrical halves, each of which possesses no activity individually. Oxazole, pyrazole, and acetyl derivatives are less effective than curcumin at inhibiting actin self-assembly, whereas a benzylidiene derivative is more effective. Cell biology studies suggest that disorganization of the actin network leads to destabilization of filaments in the presence of curcumin. Molecular docking reveals that curcumin binds close to the cytochalasin binding site of actin. Further molecular dynamics studies reveal a possible allosteric effect in which curcumin binding at the “barbed end” of actin is transmitted to the “pointed end”, where conformational changes disrupt interactions with the adjacent actin monomer to interrupt filament formation. Finally, the recognition and binding of actin by curcumin is yet another example of its unique ability to target multiple receptors

Phenolic Phytochemicals for Prevention and Treatment of Colorectal Cancer: A Critical Evaluation of In Vivo Studies

Colorectal cancer (CRC) is the third most diagnosed and second leading cause of cancer-related death worldwide. Limitations with existing treatment regimens have demanded the search for better treatment options. Different phytochemicals with promising anti-CRC activities have been reported, with the molecular mechanism of actions still emerging. This review aims to summarize recent progress on the study of natural phenolic compounds in ameliorating CRC using in vivo models. This review followed the guidelines of the Preferred Reporting Items for Systematic Reporting and Meta-Analysis. Information on the relevant topic was gathered by searching the PubMed, Scopus, ScienceDirect, and Web of Science databases using keywords, such as &ldquo;colorectal cancer&rdquo; AND &ldquo;phenolic compounds&rdquo;, &ldquo;colorectal cancer&rdquo; AND &ldquo;polyphenol&rdquo;, &ldquo;colorectal cancer&rdquo; AND &ldquo;phenolic acids&rdquo;, &ldquo;colorectal cancer&rdquo; AND &ldquo;flavonoids&rdquo;, &ldquo;colorectal cancer&rdquo; AND &ldquo;stilbene&rdquo;, and &ldquo;colorectal cancer&rdquo; AND &ldquo;lignan&rdquo; from the reputed peer-reviewed journals published over the last 20 years. Publications that incorporated in vivo experimental designs and produced statistically significant results were considered for this review. Many of these polyphenols demonstrate anti-CRC activities by inhibiting key cellular factors. This inhibition has been demonstrated by antiapoptotic effects, antiproliferative effects, or by upregulating factors responsible for cell cycle arrest or cell death in various in vivo CRC models. Numerous studies from independent laboratories have highlighted different plant phenolic compounds for their anti-CRC activities. While promising anti-CRC activity in many of these agents has created interest in this area, in-depth mechanistic and well-designed clinical studies are needed to support the therapeutic use of these compounds for the prevention and treatment of CRC

Phenolic Phytochemicals for Prevention and Treatment of Colorectal Cancer: A Critical Evaluation of In Vivo Studies

Colorectal cancer (CRC) is the third most diagnosed and second leading cause of cancer-related death worldwide. Limitations with existing treatment regimens have demanded the search for better treatment options. Different phytochemicals with promising anti-CRC activities have been reported, with the molecular mechanism of actions still emerging. This review aims to summarize recent progress on the study of natural phenolic compounds in ameliorating CRC using in vivo models. This review followed the guidelines of the Preferred Reporting Items for Systematic Reporting and Meta-Analysis. Information on the relevant topic was gathered by searching the PubMed, Scopus, ScienceDirect, and Web of Science databases using keywords, such as “colorectal cancer” AND “phenolic compounds”, “colorectal cancer” AND “polyphenol”, “colorectal cancer” AND “phenolic acids”, “colorectal cancer” AND “flavonoids”, “colorectal cancer” AND “stilbene”, and “colorectal cancer” AND “lignan” from the reputed peer-reviewed journals published over the last 20 years. Publications that incorporated in vivo experimental designs and produced statistically significant results were considered for this review. Many of these polyphenols demonstrate anti-CRC activities by inhibiting key cellular factors. This inhibition has been demonstrated by antiapoptotic effects, antiproliferative effects, or by upregulating factors responsible for cell cycle arrest or cell death in various in vivo CRC models. Numerous studies from independent laboratories have highlighted different plant phenolic compounds for their anti-CRC activities. While promising anti-CRC activity in many of these agents has created interest in this area, in-depth mechanistic and well-designed clinical studies are needed to support the therapeutic use of these compounds for the prevention and treatment of CRC