Novel CDK9 inhibitor oroxylin A promotes wild-type P53 stability and prevents hepatocellular carcinoma progression by disrupting both MDM2 and SIRT1 signaling.

Hepatocellular carcinoma (HCC) is one of the most lethal tumours worldwide. However, the effects of first-line sorafenib treatment in advanced HCC fail to prolong patients' survival due to the highly heterogeneous characteristics of HCC etiology. Cyclin-dependent kinase 9 (CDK9) is an important target in the continuous development of cancer therapy. Here, we demonstrate that CDK9 is closely associated with the progression of HCC and can serve as an HCC therapeutic target by modulating the recovery of wild-type p53 (wt-p53) function. We prove that mouse double minute 2 homologue (MDM2) and Sirtuin 1 (SIRT1) are phosphorylated by CDK9 at Ser166 and Ser47, respectively. Inhibition of CDK9 not only reduces the MDM2-mediated ubiquitination and degradation of wt-p53 but also increases wt-p53 stability by suppressing deacetylase activity of SIRT1. Thus, inhibition of CDK9 promotes the wt-p53 stabilization and prevents HCC progression. However, excessive inhibition by high concentrations of specific CDK9 inhibitors counteracts the promotion of p53 stability and reduces their anti-HCC activity because of extreme general transcription repression. The effects of a novel CDK9 inhibitor named oroxylin A (OA) from Scutellaria baicalensis are explored, with the results indicating that OA shows moderate and controlled inhibition of CDK9 activity and expression, and stabilizes wt-p53 by inhibiting CDK9-regulated MDM2 and SIRT1 signaling. These outcomes indicate the high therapeutic potential of OA against HCC and its low toxicity in normal tissue. This study demonstrates a novel mechanism for the regulation of wt-p53 by CDK9 and indicates that OA is a potential candidate for HCC therapy.

EGFR suppresses p53 function by promoting p53 binding to DNA-PKcs: a noncanonical regulatory axis between EGFR and wild-type p53 in glioblastoma.

BACKGROUND: Epidermal growth factor receptor (EGFR) amplification and TP53 mutation are the two most common genetic alterations in glioblastoma multiforme (GBM). A comprehensive analysis of the TCGA GBM database revealed a subgroup with near mutual exclusivity of EGFR amplification and TP53 mutations indicative of a role of EGFR in regulating wild-type-p53 (wt-p53) function. The relationship between EGFR amplification and wt-p53 function remains undefined and this study describes the biological significance of this interaction in GBM. METHODS: Mass spectrometry was used to identify EGFR-dependent p53-interacting proteins. The p53 and DNA-dependent protein kinase catalytic subunit (DNA-PKcs) interaction was detected by co-immunoprecipitation. We used CRISPR-Cas9 gene editing to knockout EGFR and DNA-PKcs and the Edit-R CRIPSR-Cas9 system for conditional knockout of EGFR. ROS activity was measured with a CM-H2DCFDA probe, and real-time PCR was used to quantify expression of p53 target genes. RESULTS: Using glioma sphere-forming cells (GSCs), we identified, DNA-PKcs as a p53 interacting protein that functionally inhibits p53 activity. We demonstrate that EGFR knockdown increased wt-p53 transcriptional activity, which was associated with decreased binding between p53 and DNA-PKcs. We further show that inhibition of DNA-PKcs either by siRNA or an inhibitor (nedisertib) increased wt-p53 transcriptional activity, which was not enhanced further by EGFR knockdown, indicating that EGFR suppressed wt-p53 activity through DNA-PKcs binding with p53. Finally, using conditional EGFR-knockout GSCs, we show that depleting EGFR increased animal survival in mice transplanted with wt-p53 GSCs. CONCLUSION: This study demonstrates that EGFR signaling inhibits wt-p53 function in GBM by promoting an interaction between p53 and DNA-PKcs.

Targeting p53 as a promising therapeutic option for cancer by re-activating the wt or mutant p53's tumor suppression.

p53 protein, a product of the TP53 tumor suppressor gene, controls the cellular genome's integrity and is an important regulator of cell cycling, proliferation, apoptosis and metabolism. Mutations of TP53 or inactivation of its gene product are among the first events initiating malignant transformation. The consequent loss of control over the cell cycle, resulting in accelerated cell proliferation and facilitating metabolic reprogramming, gives the initiated (premalignant) cells numerous advantages over healthy cells. Interestingly, p53 status is not only an important marker in cancer diagnosis; it has also become a promising target of personalized therapy. Depending on the TP53 status different therapeutic options have been developed. (Re)-activation of p53 functionality in cancer cells offers promising new alternatives to existing oncological therapies.

Microbeads mediated oral plasmid DNA delivery using polymethacrylate vectors: an effectual groundwork for colorectal cancer.

This study was aimed to develop and evaluate p53 polyplex-loaded enteric-coated calcium pectinate microbeads for oral gene delivery as an effective novel alternative for colorectal cancer therapy. Mutation in p53 is the key event in colorectal cancer (CRC) and an important target for the treatment of CRC through gene therapy. Polymethacrylates-based non-viral vectors were evaluated for their ability to complex, protect and transfect p53 (wt) into colon cancer cell line. Polyplexes were formulated by complexation of cationic polymer with anionic pDNA at different N/P ratios. p53 polyplex-loaded calcium pectinate (CP) microbeads were prepared by ionotropic gelation of pectin with calcium chloride and coated with Eudragit® S100. In vitro release studies showed that enteric-coated CP microbeads protected the release of p53 polyplex in upper GIT with less than 10% release. In-vitro cell line studies and in vivo studies in rat showed that polymethacrylate carrier could transfect the pDNA effectively. Results of in vivo gene expression study further confirmed the ability of enteric-coated calcium pectinate microbeads to deliver pDNA specifically to rat colon. Conclusively, enteric-coated calcium pectinate microbeads released p53 polyplex specifically in colon and could serve as an effective alternative for CRC therapy.

EFFECT OF wt-P53 PROTEIN ON TELOMERASE ACTIVITY IN KELOID FIBROBLASTS / 中国修复重建外科杂志

Objective To evaluated the role of wt-P53 protein in telomerase regulation in keloid fibroblasts(KFBs). Methods The fibroblasts were derived from human keloid tissue which was proved by pathological diagnosis. KFBs were divided into 2 groups, the transfection group and the untransfection group. wt-p53 gene was transfected into the fibroblasts by adenovirus vectors in the transfection group. The KFBs untransfected with wt-p53 gene served as control (untransfection group). After 48 hours of transfection, the expression of wt-P53 protein was analyzed by both Western blotting and immunofluorescence method, respectively. The telomerase activity was evaluated by TRAP-ELISA after 1-7 days of transfection.Results All the KFBs from 2 groups expressed wt-P53 protein. But the expression level of wt-P53 protein in the transfection group was significantly higher than that in the untransfection group. At the same time of high expression of wt-P53 protein, the telomerase activity of KFBs in transfection group was significantly lower than that in the untransfection group( P＜0.05). Conclusion High level expression of wt-P53 protein can transiently inhibit the telomerase activity of KFBs.

Influence of Wt-p53 gene by adenovirus vector on proliferation of keloid fibroblasts / 中华医学美学美容杂志

Objective To study wt-p53 gene's influence on the proliferation of keloid fibroblasts in vitro. Methods wt-p53 gene was transfected into keloid fibroblasts by adenovirus vector. wt-p53 mRNA was analyzed by RT-PCR; wt-p53 protein was evaluated by indirectiy immunofluorescence; The ability of proliferation of keloid fibroblasts was analyzed by cell growing curves; The cell cycle of KFB was checked by FCAS. Results The expression of wt-p53 mRNA and protein was obviously higher in the fibroblasts of the experimental group than those of control group; the rate of G_0~G_1 in cell cycle was higher in the fibroblasts of the experimental group than those of control group; at the same time, the rate of G_2~M was lower in fibroblasts of the experimental group than those of control group (P

The research of p33~(ING1),wt-p53 growth suppressing and collapsing effect toward stomach cancer cell strain / 肿瘤研究与临床

Objective To discuss the growth suppressing, apoptosing effect of new type tumor-supressor gene-p33ING1 in stomach cancer cell strain, and to explore new strategies and methods in tumor therapy. Methods The PCDNA3/p33ING1 nuclear expressing microsome was constructed, p33ING1 and wt-p53 were implanted to human stomach cancer cell both and to evaluate the effect of p33ING1 and p53 toward stomach cancer cell and synergism between them. Results The PCDNA3/p33ING1 nuclear expressing microsome was successfully constructed. The human stomach cancer cell strain SSCG-7901 under implantation of p33ING1 and wt-p53 showed a significant decrease in cell growth, the coupling time was delayed, DNA synthetic phase was shortened and G0/G1 phase prolonged. The cell collapse increased. Conclusions Despite of the tumor-inhibiting effect and biochemical activation of p33ING1, it also plays a role with p53 gene in controling growth of stomach cancer cell, inducing cell collapse and hampering cell proliferation cycle. P33ING1 and p53 are synergistic to each other.