• BMB Reports
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• v.47 no.4
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• pp.221-226
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• 2014

Drug-resistance and imbalance of apoptotic regulation limit chemotherapy clinical application for the human hepatocellular carcinoma (HCC) treatment. The reactivation of p53 is an attractive therapeutic strategy in cancer with disrupted-p53 function. Nutlin-3, a MDM2 antagonist, has antitumor activity in various cancers. The post-translational modifications of p53 are a hot topic, but there are some controversy ideas about the function of phospho-$Ser^{392}$-p53 protein in cancer cell lines in response to Nutlin-3. Therefore, we investigated the relationship between Nutlin-3 and phospho-$Ser^{392}$-p53 protein expression levels in SMMC-7721 (wild-type TP53) and HuH-7 cells (mutant TP53). We demonstrated that Nutlin-3 induced apoptosis through down-regulation phospho-$Ser^{392}$-p53 in two HCC cells. The result suggests that inhibition of p53 phosphorylation on $Ser^{392}$ presents an alternative for HCC chemotherapy.

• Asian Pacific Journal of Cancer Prevention
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• v.16 no.9
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• pp.3759-3765
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• 2015

Background: Approaches in disruption of MDM2-p53 interactions have now emerged as an important therapeutic strategy in resurrecting wild type p53 functional status. The present study highlights virtual screening strategies in identification of high affinity small molecule non-peptidic inhibitors. Nutlin3A and RG7112 belonging to compound class of Cis-imidazoline, MI219 of Spiro-oxindole class and Benzodiazepine derived TDP 665759 served as query small molecules for similarity search with a threshold of 95%. The query molecules and the similar molecules corresponding to each query were docked at the transactivation binding cleft of MDM2 protein. Aided by MolDock algorithm, high affinity compound against MDM2 was retrieved. Patch Dock supervised Protein-Protein interactions were established between MDM2 and ligand (query and similar) bound and free states of p53. Compounds with PubCid 68870345, 77819398, 71132874, and 11952782 respectively structurally similar to Nutlin3A, RG7112, Mi219 and TDP 665759 demonstrated higher affinity to MDM2 in comparison to their parent compounds. Evident from the protein-protein interaction studies, all the similar compounds except for 77819398 (similar to RG 7112) showed appreciable inhibitory potential. Of particular relevance, compound 68870345 akin to Nutlin 3A had highest inhibitory potential that respectively showed 1.3, 1.2, 1.16 and 1.26 folds higher inhibitory potential than Nutilin 3A, MI 219, RG 7112 and TDP 1665759. Compound 68870345 was further mapped for structure based pharamacophoric features. In the study, we report Cis-imidazoline derivative compound; Pubcid: 68870345 to have highest inhibitory potential in blocking MDM2-p53 interactions hitherto discovered.

• Oncology Letters
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• v.41 no.2
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• pp.1075-1082
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• 2019

In a previous study, we reported that α-smooth muscle actin (α-SMA), one of the mesenchymal marker proteins, is highly expressed in tamoxifen-resistant breast cancer (TamR) cells. However, the exact mechanism of α-SMA expression in TamR cells is not fully understood. Here, we investigated the effect of TP53 on α-SMA expression in breast cancer cells. The levels of α-SMA mRNA and protein expression were analyzed by real-time PCR and western blotting, respectively. In estrogen receptor-positive [ER(+)] breast cancer patients, aberrant α-SMA expression was found to be associated with a poor prognosis. The level of α-SMA expression was significantly increased in established TamR cells compared to tamoxifen-sensitive (TamS) cells. To verify the regulatory mechanism of α-SMA expression, we analyzed diverse kinase activities between TamS and TamR cells. The activity of TP53 was markedly increased in the TamR cells. When TamS cells were treated with TP53 activator, Nutlin3 (Nut3), α-SMA expression was increased in the TamS cells. In addition, α-SMA expression was significantly increased by TP53 overexpression in breast cancer cells. On the contrary, the basal level of α-SMA expression was decreased by the TP53 inhibitor, pifithrin-α (PFT-α). Taken together, we demonstrated that α-SMA expression is regulated by TP53 activity in TamR cells.

• BMB Reports
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• v.51 no.10
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• pp.526-531
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• 2018

Protein-Protein Interactions (PPIs) play essential roles in diverse biological processes and their misregulations are associated with a wide range of diseases. Especially, the growing attention to PPIs as a new class of therapeutic target is increasing the need for an efficient method of cell-based PPI analysis. Thus, we newly developed a robust PPI assay (SeePPI) based on the co-translocation of interacting proteins to the discrete subcellular compartment 'processing body' (p-body) inside living cells, enabling a facile analysis of PPI by the enriched fluorescent signal. The feasibility and strength of SeePPI (${\underline{S}}ignal$ ${\underline{e}}nhancement$ ${\underline{e}}xclusively$ on ${\underline{P}}-body$ for ${\underline{P}}rotein-protein$ ${\underline{I}}nteraction$) assay was firmly demonstrated with FKBP12/FRB interaction induced by rapamycin within seconds in real-time analysis of living cells, indicating its recapitulation of physiological PPI dynamics. In addition, we applied p53/MDM2 interaction and its dissociation by Nutlin-3 to SeePPI assay and further confirmed that SeePPI was quantitative and well reflected the endogenous PPI. Our SeePPI assay will provide another useful tool to achieve an efficient analysis of PPIs and their modulators in cells.

• Molecules and Cells
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• v.43 no.1
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• pp.23-33
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• 2020

NF-κB signaling through both canonical and non-canonical pathways plays a central role in immune responses and inflammation. NF-κB-inducing kinase (NIK) stabilization is a key step in activation of the non-canonical pathway and its dysregulation implicated in various hematologic malignancies. The tumor suppressor, p53, is an established cellular gatekeeper of proliferation. Abnormalities of the TP53 gene have been detected in more than half of all human cancers. While the non-canonical NF-κB and p53 pathways have been explored for several decades, no studies to date have documented potential cross-talk between these two cancer-related mechanisms. Here, we demonstrate that p53 negatively regulates NIK in an miRNA-dependent manner. Overexpression of p53 decreased the levels of NIK, leading to inhibition of the non-canonical NF-κB pathway. Conversely, its knockdown led to increased levels of NIK, IKKα phosphorylation, and p100 processing. Additionally, miR-34b induced by nutlin-3 directly targeted the coding sequences (CDS) of NIK. Treatment with anti-miR-34b-5p augmented NIK levels and subsequent non-canonical NF-κB signaling. Our collective findings support a novel cross-talk mechanism between non-canonical NF-κB and p53.

• International Journal of Molecular Medicine
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• v.46 no.5
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• pp.1923-1937
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• 2020

It has been suggested that oxidative stress involving reactive oxygen species (ROS) induces granulosa cell apoptosis, leading to follicular atresia, and that T-lymphokine-activated killer cell-originated protein kinase (TOPK) suppresses cancer cell apoptosis induced by several stimuli. However, it remains to be determined whether TOPK affects oxidative stress-induced granulosa cell apoptosis. The present study demonstrates that TOPK inhibition increases human granulosa COV434 cell apoptosis induced by hydrogen peroxide (H₂O₂). Co-treatment with the TOPK inhibitor, OTS514, in combination with H₂O₂ increased p53 acetylation and its expression, whereas it decreased Sirtuin 1 (SIRT1) expression, contributing to the promotion of apoptosis. In addition, the SIRT1 activator, resveratrol, or the SIRT1 inhibitor, Ex527, reduced or elevated H₂O₂-induced COV434 cell apoptosis, respectively. Furthermore, the p53 inhibitor, Pifithrin-μ, diminished the augmentation in poly(ADP-ribose) polymerase (PARP) cleavage induced by OTS514 plus H₂O₂, while the Mdm2 antagonist, Nutlin 3, increased PARP cleavage. Moreover, OTS514 further decreased the SIRT1 transcriptional activity decreased by H₂O₂, but promoted the H₂O₂-induced p53 or p21 transcriptional activity. Notably, the expression of exogenous p53 reduced SIRT1 transcriptional activity. Taken together, the findings of the present study demonstrate that TOPK inhibition promotes p53-mediated granulosa cell apoptosis through SIRT1 downregulation in response to H₂O₂. Therefore, it can be concluded that TOPK suppresses H₂O₂-induced apoptosis through the modulation of the p53/SIRT1 axis, suggesting a potential role of TOPK in the regulation of human granulosa cell apoptosis, leading to the promotion of abnormal follicular development.