P21-Driven Multifusion Gene System for Evaluating the Efficacy of Histone Deacetylase Inhibitors by In Vivo Molecular Imaging and for Transcription Targeting Therapy of Cancer Mediated by Histone Deacetylase Inhibitor

Abstract

Overexpressed histone deacetylase (HDAC) activity has been linked with tumor initiation and progression that prompt the development of histone deacetylase inhibitors (HDACIs) as anticancer agents. HDACI was reported to be able to activate p21 promoter through the SP1 binding sites in the proximal region of p21 WAF1/CIP1 promoter. In this study, we established a p21 WAF1/CIP1 promoter-driven triple-fused reporter gene system (p21-3H) to evaluate the efficacy of HDACI and the ganciclovir (GCV)-mediated anticancer effect contributed by HDACI-induced and p21-driven truncated herpes simplex virus-1 thymidine kinase sr39 mutant (ttksr39) in vitro and in vivo. Methods: The p21-3H construct was generated and stably or transiently transfected into H1299 cell lines. These cells were treated with trichostatin A or vorinostat (suberoylanilide hydroxamic acid [SAHA]) to evaluate the activation of p21 promoter-driven reporter gene expression by in vitro confocal fluorescence microscopy, luciferase assay, 2′-fluoro-2′-deoxyarabinofuranosyl-5-ethyluracil ( 3 H-FEAU) cellular uptake, in vivo bioluminescence imaging, and 9-(4-18 F-fluoro-3-hydroxymethylbutyl) guanine ( 18 F-FHBG) small-animal PET imaging. The therapeutic efficacy on p21-3H-expressing tumor xenografts was assessed by daily administration with SAHA (100 mg/kg intraperitoneally) or GCV (20 mg/kg) for 9 d, followed by tumor volume measurement. Results: On treatment with trichostatin A or SAHA, H1299 cells carrying p21-3H showed a significant increase of luciferase activity, cellular uptake of 3 H-FEAU (Moravek), and DsRed expression. In vivo tumor xenografts carrying p21-3H also showed increased luciferase activity by luminescent imaging and enhanced accumulation of 18 F-FHBG by small-animal PET imaging. Furthermore, when cells transfected with p21-3H or p21/PstI-3H (which lacks p53-binding sites) were treated, the increase of luciferase activity was similar in both groups, indicating that HDACI-induced p21 promoter activation is independent of p53. Both in vitro and in vivo results showed improved therapeutic effect by combined treatment of GCV and HDACI. Conclusion: We have established an HDACI-inducible, p21-driven reporter system that has the potential for evaluating the anticancer effect of HDACIs on cancer cells by multiple molecular imaging modalities. Furthermore, ttksr39 in a p21-3H reporter construct provides a potential combination with thymidine kinase-mediated gene therapy to optimize the therapeutic benefit of HDACI.

Histone Deacetylase Inhibitors Romidepsin and Vorinostat Promote Hepatitis B Virus Replication by Inducing Cell Cycle Arrest

“…31,32 Increased expression of p21/p27 and, subsequently, cell cycle arrest are common responses to HDACi treatment. 33 Consistent with a previous study, our results showed that the expression of cyclin-dependent kinase inhibitors, such as p21 and p27, were significantly increased, while cyclin D1/E and CDK2, which are required for the G1/S checkpoint complex, were significantly decreased after FK228/SAHA treatment. In fact, several studies have indicated that HBV replication is cell cycle-dependent and highly associated with the growth status of hepatocytes.…”

STAT3/NF-κB-Regulated Lentiviral TK/GCV Suicide Gene Therapy for Cisplatin-Resistant Triple-Negative Breast Cancer

“…Quantitative evaluation of the uptake of radiotracer in vitro of TK in cells transient transfected with Lenti-CMV-DsRedm-tk and Lenti-STAT3-NF-κB-DsRedm-tk was measured by 3 H-FEAU uptake assay as previously described 24 . In brief, MDA-MB-231 cells transfected with equal amount of Lenti-CMV-DsRedm-tk and Lenti-STAT3-NF-κB-DsRedm-tk plasmid with untransfected cells (control) were seeded in 12-well plates (1×10 5 per well) in triplicate and incubated overnight, followed by 10 ng/mL TNF-α (R&D systems, cat.210-TA-005, Minneapolis, MN, USA) or DMSO treatment for 24 h. Culture medium was then replaced by 0.5 mL of medium containing 29-fluoro-29-deoxyarabinofuranosyl-5-ethyluracil ( 3 H-FEAU) (7.4 kBq [0.2 μCi]), and incubated for 120 minutes at 37˚C with 5% CO 2 .…”

Molecular Mechanism of the Cell Death Induced by the Histone Deacetylase Pan Inhibitor LBH589 (Panobinostat) in Wilms Tumor Cells

“…2–10% of important biological processes genes were changed when inhibition of HDACs [ 12 ]. In addition to modifying histones, HDACs also target many other non-histone protein substrates to regulate gene expression [ 13 , 14 ]. Recently, HDACs have received increasing attention because HDAC-inhibiting compounds are being developed as promising anti cancer therapeutics.…”