Increased P53 Protein Expression Research Articles

Dual-targeting class I HDAC inhibitor and ATM activator, SP-1-303, preferentially inhibits estrogen receptor positive breast cancer cell growth.

Dual-targeting chromatin regulation and DNA damage repair signaling presents a promising avenue for cancer therapy. Applying rational drug design, we synthesized a potent dual-targeting small molecule, SP-1-303. Here, we report SP-1-303 as a class I isoform selective histone deacetylase (HDAC) inhibitor and an activator of the ataxia-telangiectasia mutated protein (ATM). In vitro enzymatic assays demonstrated selective inhibition of HDAC1 and HDAC3. Cellular growth inhibition studies show that SP-1-303 differentially inhibits growth of estrogen receptor positive breast cancer (ER+ BC) cells with effective growth inhibition concentrations (EC50) for MCF-7 and T47D cells ranging from 0.32 to 0.34 μM, compared to 1.2-2.5 μM for triple negative breast cancer cells, and ~12 μM for normal breast epithelial cells. Western analysis reveals that SP-1-303 decreases estrogen receptor alpha (ER-α) expression and increases p53 protein expression, while inducing the phosphorylation of ATM and its substrates, BRCA1 and p53, in a time-dependent manner in ER+ BC cells. Pharmacokinetic evaluation demonstrates an area under the curve (AUC) of 5227.55 ng/ml × h with an elimination half-life of 1.26 h following intravenous administration in a rat model. Collectively, SP-1-303 emerges as a novel second generation class I (HDAC1 and HDAC3) selective HDAC inhibitor, and ATM activator, capable of modulating ER expression, and inhibiting growth of ER+ BC cells. Combined targeting of class I HDACs and ATM by SP-1-303 offers a promising therapeutic approach for treating ER+ breast cancers and supports further preclinical evaluation.

Degradation of estrogen receptor α induced by ionizing radiation contributes to senescence of ER+ breast cancer cells.

e12544 Background: According to the clinical and pathological characteristics, breast cancer is usually classified into estrogen receptor positive (ER+), human epidermal growth factor receptor 2 positive and triple negative. Among them, ER+ breast cancer accounts for more than two-thirds of total breast cancer. Nowadays, both ionizing radiation and endocrine therapy are used for the treatment of ER+ breast cancer. In our previous studies, it was found that significant cellular senescence and reduction of endogenous estradiol (E2) were induced by carbon ion or X-ray exposure in ER+ breast cancer cells. However, the underlying mechanism remains unclear. Methods: Two human ER+ breast cancer cell lines, MCF7 and T47D, were chosen to investigate the senescence-associated indications after 1-10 Gy of X-ray exposure. Since the elevated estrogen receptor α (ERα) is the most significant characteristic of ER+ breast cancer, the levels of ERα were detected by qRT-PCR and West Blotting analysis. In addition, MCF7 tumors were established in BALB/C nude mice and discerned using immunohistochemistry after local exposure of tumors to 2 Gy of X-rays. Results: It was confirmed that the cell survival decreased in a dose-dependent manner after 1-10 Gy of X-ray exposure in both cell lines. Increased p21 protein expression which was considered as a senescence biomarker and enhanced senescence-associated β-galactosidase activity were also observed after X-ray exposure. Meanwhile, distinct cell cycle arrest was induced by 2 Gy of X-rays in T47D cells or by 8 Gy of X-rays in MCF7 cells, implying that T47D cells were prone to enter senescence. Furthermore, both the mRNA levels and protein levels of ERα were significantly decreased at 24 hours in the two cell lines when the exposure dose was up to 4 Gy of X-rays. Reduction of glucocorticoid-regulated kinase 3 (SGK3), a transcriptional target for ERα, was also observed in both cell lines, in according with the reported SGK3-dependent survival mediated by ERα/E2 dimer in ER+ breast cancer cells. In vitro experiments, the results verified the ERα degradation and the proliferating inhibition indicated by suppression of Ki67 protein in MCF7 tumor after 2 Gy exposure. Conclusions: These findings reveal that the degradation of estrogen receptor α induced by ionizing radiation contributes to the senescence of ER+ breast cancer cells and thereby inhibits the tumor growth. The results provide a potential therapeutic strategy to enhance the radiosensitivity of ER+ breast cancer cells through regulations of ERα or ERα-regulated kinase SGK3 in radiotherapy.

Comparative assessment of direct and indirect cold atmospheric plasma effects, based on helium and argon, on human glioblastoma: an in vitro and in vivo study

Recent research has highlighted the promising potential of cold atmospheric plasma (CAP) in cancer therapy. However, variations in study outcomes are attributed to differences in CAP devices and plasma parameters, which lead to diverse compositions of plasma products, including electrons, charged particles, reactive species, UV light, and heat. This study aimed to evaluate and compare the optimal exposure time, duration, and direction-dependent cellular effects of two CAPs, based on argon and helium gases, on glioblastoma U-87 MG cancer cells and an animal model of GBM. Two plasma jets were used as low-temperature plasma sources in which helium or argon gas was ionized by high voltage (4.5 kV) and frequency (20 kHz). In vitro assessments on human GBM and normal astrocyte cell lines, using MTT assays, flow cytometry analysis, wound healing assays, and immunocytochemistry for Caspase3 and P53 proteins, demonstrated that all studied plasma jets, especially indirect argon CAP, selectively induced apoptosis, hindered tumor cell growth, and inhibited migration. These effects occurred concurrently with increased intracellular levels of reactive oxygen species and decreased total antioxidant capacity in the cells. In vivo results further supported these findings, indicating that single indirect argon and direct helium CAP therapy, equal to high dose Temozolomide treatment, induced tumor cell death in a rat model of GBM. This was concurrent with a reduction in tumor size observed through PET-CT scan imaging and a significant increase in the survival rate. Additionally, there was a decrease in GFAP protein levels, a significant GBM tumor marker, and an increase in P53 protein expression based on immunohistochemical analyses. Furthermore, Ledge beam test analysis revealed general motor function improvement after indirect argon CAP therapy, similar to Temozolomide treatment. Taken together, these results suggest that CAP therapy, using indirect argon and direct helium jets, holds great promise for clinical applications in GBM treatment.

Saw Palmetto may fight prostate cancer.

Saw Palmetto may fight prostate cancer.

Developmental Stage-Specific Hematopoietic Failure In Vivo in a Novel Model of Diamond Blackfan Anemia

Diamond Blackfan anemia (DBA) is an inherited bone marrow failure syndrome, characterized by failure of erythropoiesis at the progenitor stages, congenital anomalies, and predisposition to cancer. DBA is most frequently due to mutations in genes encoding a ribosomal protein, the most common one being mutations in RPS19. Despite the well-established role of RPS19 as well as other ribosomal proteins in the erythropoietic failure in in vitro culture systems, there is a paucity of in vivo murine models of DBA due to differences between humans and mice. Indeed, stress erythropoiesis occurs in the spleen of mouse, compensating for the erythropoietic defects observed in the bone marrow. Using CRISPR/Cas9 technologies, we generated a conditional knockout of Rps19 by flanking exon 2 with 2 loxP sites and studied hematopoiesis in this model. While Tie2-cre+;Rps19+/- animals die in utero, Vav-iCre+; Rps19+/- mice are born at expected Mendelian ratios with severe macrocytic anemia. Surprisingly, and unlike any other model generated to date, these mice present with reticulocytopenia, a critical characteristic of patients with DBA and die by post-natal day 10 (P10), due to bone marrow failure and decreased splenic stress erythropoiesis. To understand the role of ribosomal proteins during hematopoietic development, we analyzed fetal livers of Vav-iCre+; Rps19+/- embryos and control littermates at E13.5 and E17.5. Using flow cytometry, we observed defective erythropoiesis and gradual hematopoietic stem cell exhaustion at the fetal to neonatal transition. Mechanistically, and consistent with previous studies using in vitro systems, Rps19mutant cells had increased p53 protein expression and reduced mTORC1 activity. Unexpectedly, hematopoietic progenitors had enhanced global protein synthesis. Using BrDU and EdU stainings, we observed a decrease in the cell cycle duration of erythroblasts, consistent with the macrocytosis and reticulocytopenia seen in neonatal Vav-iCre+; Rps19+/- mice. Since p53 is reported to have a central role in the pathophysiology of DBA, we asked whether deletion of one allele of p53 could rescue the defects observed in Vav-iCre+; Rps19+/-. The Vav-iCre+; Rps19+/-, trp53+/-presented with improved blood counts, increased spleen size and cellularity but died by four weeks after birth. Analyses of the hematopoietic compartment in these animals at P21 revealed bone marrow failure and HSC depletion with skewed progenitor populations. The increase in global protein synthesis observed in Vav-iCre+; Rps19+/- remained unchanged in Vav-iCre+; Rps19+/-, trp53+/-, suggesting mechanisms independent of p53. Taken together, we have developed a novel in vivo model for the study of DBA. Our findings could potentially explain why DBA typically manifests after birth and highlight a critical role for Rps19 during hematopoietic differentiation both in utero and after birth.

Glycemic and nonglycemic mechanisms of congenital malformations in hyperglycemic pregnancies: a narrative review.

Congenital malformations are more frequently found among children born to mothers with diabetes than in the background population. There are several complex mechanisms involved in the development of congenital malformations in the offspring of mothers with hyperglycemia, such as the overexpression of glucose transporters (GLUTs) 1 and 2, the increased activity of the hexosamine biosynthetic pathway and the reduced expression of the PAX3 gene with a consequent increase in p53 protein expression. These alterations can lead to increased glucose and free radical concentrations in the embryo, thus promoting the process of apoptosis and causing malformation. The most frequent malformations found in the offspring of mothers with diabetes are heart and neural tube defects, urinary tract and kidney malformations, and cleft lip with or without cleft palate. Strict glycemic control should be obtained before and during pregnancy, aiming to avoid or minimize the risk of congenital malformations in the offspring. Beyond hyperglycemia, several factors may also be associated with increased risks of malformations in the offspring of these women, such as obesity, multiple pregnancies, advanced maternal age, folic acid deficiency, use of angiotensin converting enzyme inhibitors and angiotensin receptor blockers, assisted reproduction techniques, and exposure to different types of environmental pollutants.

HOXA5 inhibits the proliferation of extrahepatic cholangiocarcinoma cells by enhancing MXD1 expression and activating the p53 pathway

Homeobox A5 (HOXA5) is a transcription factor in mammalian and can regulate cell differentiation, proliferation, and apoptosis as well as tumorigenesis. However, little is known on whether and how HOXA5 can regulate the malignant behaviors of cholangiocarcinoma. The methylation levels of HOXA5 were evaluated by methylation microarray and bisulfite sequencing PCR. HOXA5 expression in tissue samples was examined by immunohistochemistry and Western blot. The proliferation of tumor cells was assessed by CCK-8, EdU, and nude mouse tumorigenicity assays. The invasion, apoptosis and cell cycling of tumor cells were evaluated by Wound healing assay and flow cytometry. The interaction between HOXA5 and the MXD1 promoter was examined by CUT & Tag assay, luciferase reporter assay and chromatin immunoprecipitation. Hypermethylation in the HOXA5 promoter down-regulated HOXA5 expression in extrahepatic cholangiocarcinoma (ECCA) tissues, which was correlated with worse overall survival. HOXA5 overexpression significantly inhibited the proliferation and tumor growth. HOXA5 overexpression enhanced MXD1 expression by directly binding to the MXD1 promoter in ECCA cells. MXD1 overexpression inhibited the proliferation and tumor growth while MXD1 silencing abrogated the HOXA5-mediated proliferation inhibition. HOXA5 overexpression increased p53 protein expression in an MXD1-dependent manner. HOXA5 and MXD1 acted as tumor suppressors to inhibit the mitosis of ECCA cells by enhancing the p53 signaling. Our findings may uncover molecular mechanisms by which the HOXA5/MXD1 axis regulates the progression of ECCA, suggesting that the HOXA5/MXD1 may be therapeutic targets for ECCA.

Downregulation of krüppel-like factor 6 expression modulates extravillous trophoblast cell behavior by increasing reactive oxygen species

IntroductionPlacental extravillous trophoblasts play a crucial role in the establishment of a healthy pregnancy. Reactive oxygen species (ROS) may contribute to their differentiation and function as mediators in signaling processes or might cause oxidative stress resulting in trophoblast dysfunction. The krüppel-like transcription factor 6 (KLF6) regulates many genes involved in essential cell processes where ROS are also involved. However, whether KLF6 regulates ROS levels has not been previously investigated. Materials and MethodsKLF6 was silenced by siRNAs in HTR8-SV/neo cells, an extravillous trophoblast model. Total and mitochondrial ROS levels, as well as mitochondrial membrane potential and apoptosis were analyzed by flow cytometry. The expression of genes and proteins of interest were analyzed by qRT-PCR and Western blot, respectively. Cell response to oxidative stress, proliferation, viability, morphology, and migration were evaluated. ResultsKLF6 downregulation led to an increase in ROS and NOX4 mRNA levels, accompanied by reduced cell proliferation and increased p21 protein expression. Catalase activity, 2-Cys peroxiredoxin protein levels, Nrf2 cytoplasmic localization and hemoxygenase 1 expression, as well as mitochondrial membrane potential and cell apoptosis were not altered suggesting that ROS increase is not associated with cellular damage. Instead, KLF6 silencing induced cytoskeleton modifications and increased cell migration in a ROS-dependent manner. DiscussionPresent data reveal a novel role of KLF6 on ROS balance and signaling demonstrating that KLF6 downregulation induces an increase in ROS levels that contribute to extravillous trophoblast cell migration.

Abstract 5289: Activating p53 through MDM2 and WIP1 inhibition effectively inhibits tumors in liver adenocarcinoma

Abstract Intrahepatic cholangiocarcinoma (iCCA) is an adenocarcinoma arising from the intrahepatic bile duct and accounts for the second most cases of primary liver cancers after hepatocellular carcinoma. Lacking effective treatment leads to a poor prognosis for advanced iCCA so new targeted therapy is warranted. Impaired wild-type (WT) p53 tumor suppressor function by its negative regulators frequently occurs in iCCA. Therefore, restoration of WT p53 function by inhibiting its negative regulators is a therapeutic strategy being explored for cancer treatment. Both MDM2 inhibitor (MDM2i, RG7388) stabilizing p53 and WIP1 inhibitor (WIP1i, GSK2830371) increasing p53 phosphorylation lead to maximize p53 function. The combination of MDM2i/WIP1i has been reported in severe cancer types but no in vivo study has been done. In the current study, both liver adenocarcinoma cell lines, RBE and SK-Hep-1, were treated with the RG7388 alone and in combination with GSK2830371. Cell proliferation, clonogenicity, protein and mRNA expressions, and cell cycle distribution were performed to investigate the effect and mechanism of growth suppression. To evaluate the antitumor efficacy of RG7388 and GSK2830371 in vivo, xenografts with SK-Hep-1 cells were treated with combination therapy for two weeks in NOD-SCID mice. The combination of MDM2i and WIP1i significantly increased the growth inhibition, cytotoxic activity, increased p53 protein expression, and phosphorylation (Ser 15), leading to transactivation of downstream targets (p21 and MDM2). The in vivo test demonstrated that the combination treatment can significantly inhibit tumor growth. In this study, liver adenocarcinoma cell lines were inhibited by the combination via reactivation of p53 function evidenced by increased p53 expression, phosphorylation and its downstream targets. This efficacy was also validated by in vivo study. The current research provides a novel strategy for targeting the p53 pathway in liver adenocarcinoma. Citation Format: Chiao-Ping Chen, Chun-Nan Yeh, Yi-Ru Pan, Yu-Tien Hsiao, Chih-Hong Lo, Cai-Jhen Jhuang, Chiao-En Wu. Activating p53 through MDM2 and WIP1 inhibition effectively inhibits tumors in liver adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5289.

MEDB-08. Inhibition of different mitotic targets demonstrated distinct DNA damage and cell death response in p53-mutant medulloblastoma

BACKGROUND: In normal cells, cell cycle is tightly regulated by mitotic proteins to ensure smooth transition through each phase of cell division. Here, we examine two proteins – KIF11, a mitotic kinesin, responsible for assembly and maintenance of mitotic spindle during mitosis; and MELK, a serine/threonine kinase, essential for mitotic progression. Cancer cells can upregulate MELK and KIF11 to promote uncontrolled cell division and protect the cells from apoptotic cell death, leading to tumorigenesis. AIMS: We investigated the response of p53-mutant medulloblastoma (MB) by inhibiting KIF11 and MELK separately to study the effects on cell cycle progression and cell death mechanisms. RESULTS: Cell proliferation was suppressed by inhibition of either KIF11 or MELK in MB, independent of p53-mutant status. Regardless of p53-mutant status, inhibiting KIF11 induced cell cycle arrest at G2/M. In contrast, inhibiting MELK (IC50 dose) induced more prominent G2/M arrest in p53-mutant cells compared to p53-wildtype cells. In p53-mutant MB, arrested cells during MELK inhibition subsequently underwent apoptotic cell death at 24h and 48h. With KIF11 inhibition, p53-mutant cells at 24h were already in necrotic stage. p53-mutant cells reached necrotic stage in a shorter time with KIF11 inhibition than MELK inhibition. On immunoblotting, independent of p53-mutant status, KIF11 inhibition produces more significant increase in DNA damage marker and c-PARP indicative of apoptosis, compared to MELK inhibition. Treatment with KIF11 or MELK inhibitor increased p53 protein expression in p53-wildtype (normal stress response). However, in p53-mutant cells, p53 protein expression decreased post-KIF11-inhibition, but remained unchanged post-MELK inhibition. In-vivo, inhibiting KIF11 was less tolerable in a patient-derived orthotopic xenograft model with p53-mutation. CONCLUSION: Inhibition of either mitotic target KIF11 or MELK, can induce anti-proliferative effects in MB. In p53-mutant MB, DNA damage and cell death response with KIF11-inhibition are more marked.

Resistance to mutant KRASV12-induced senescence in an hTERT/Cdk4-immortalized normal human bronchial epithelial cell line

Mutant KRAS, the most frequently occurring (∼30%) driver oncogene in lung adenocarcinoma, induces normal epithelial cells to undergo senescence. This phenomenon, called “oncogene-induced senescence (OIS)”, prevents mutant KRAS-induced malignant transformation. We have previously reported that mutant KRASV12 induces OIS in a subset of normal human bronchial epithelial cell line immortalized with hTERT and Cdk4. Understanding the mechanism and efficacy of this important cancer prevention mechanism is a key knowledge gap. Therefore, this study investigates mutant KRASV12-induced OIS in upregulated telomerase combined with the p16/RB pathway inactivation in normal bronchial epithelial cells. The normal (non-transformed and non-tumorigenic) human bronchial epithelial cell line HBEC3 (also called “HBEC3KT”), immortalized with hTERT (“T”) and Cdk4 (“K”), was used in this study. HBEC3 that expressed mutant KRASV12 in a doxycycline-regulated manner was established (designated as HBEC3-RIN2). Controlled induction of mutant KRASV12 expression induced partial epithelial-to-mesenchymal transition in HBEC3-RIN2 cells, which was associated with upregulated expression of ZEB1 and SNAIL. Mutant KRASV12 caused the majority of HBEC3-RIN2 to undergo morphological changes; suggestive of senescence, which was associated with enhanced autophagic flux. Upon mutant KRASV12 expression, only a small HBEC3-RIN2 cell subset underwent senescence, as assessed by a senescence-associated β-galactosidase staining (SA-βG) method. Furthermore, mutant KRASV12 enhanced cell growth, evaluated by colorimetric proliferation assay, and liquid and soft agar colony formation assays, partially through increased phosphorylated AKT and ERK expression but did not affect cell division, or cell cycle status. Intriguingly, mutant KRASV12 reduced p53 protein expression but increased p21 protein expression by prolonging its half-life. These results indicate that an hTERT/Cdk4 -immortalized normal bronchial epithelial cell line is partially resistant to mutant KRASV12-induced senescence. This suggests that OIS does not efficiently suppress KRASV12-induced transformation in the context of the simultaneous occurrence of telomerase upregulation and inactivation of the p16/Rb pathway.

Salviolone from Salvia miltiorrhiza Roots Impairs Cell Cycle Progression, Colony Formation, and Metalloproteinase-2 Activity in A375 Melanoma Cells: Involvement of P21(Cip1/Waf1) Expression and STAT3 Phosphorylation.

Melanoma is a highly malignant solid tumor characterized by an elevated growth and propagation rate. Since, often, melanoma treatment cannot prevent recurrences and the appearance of metastasis, new anti-melanoma agents need to be discovered. Salvia miltiorrhiza roots are a source of diterpenoid derivatives, natural compounds with several biological activities, including antiproliferative and anticancer effects. Seven diterpenoid derivatives were purified from S. miltiorrhiza roots and identified by NMR and MS analysis. Tanshinone IIA and cryptotanshinone were detected as the main components of S. miltiorrhiza root ethanol extract. Although their antitumor activity is already known, they have been confirmed to induce a reduction in A375 and MeWo melanoma cell growth. Likewise, salviolone has been shown to impair the viability of melanoma cells without affecting the growth of normal melanocytes. The underlying anticancer activity of salviolone has been investigated and compared to that of cryptotanshinone in A375 cells, showing an increased P21 protein expression in a P53-dependent manner. In that way, salviolone, even more than cryptotanshinone, displays a multitarget effect on cell-cycle-related proteins. Besides, it modulates the phosphorylation level of the signal transducer and activator of transcription (STAT)3. Unexpectedly, salviolone and cryptotanshinone induce sustained activation of the extracellular signal-regulated kinases (ERK)1/2 and the protein kinase B (Akt). However, the blockage of ERK1/2 or Akt activities suggests that kinase activation does not hinder their ability to inhibit A375 cell growth. Finally, salviolone and cryptotanshinone inhibit to a comparable extent some crucial malignancy features of A375 melanoma cells, such as colony formation in soft agar and metalloproteinase-2 activity. In conclusion, it has been shown for the first time that salviolone, harboring a different molecular structure than tanshinone IIA and cryptotanshinone, exhibits a pleiotropic effect against melanoma by hampering cell cycle progression, STAT3 signaling, and malignant phenotype of A375 melanoma cells.

Suppression of CDCA3 inhibits prostate cancer progression via NF‑κB/cyclin D1 signaling inactivation and p21 accumulation.

Dysregulation of the cell cycle contributes to tumor progression. Cell division cycle-associated 3 (CDCA3) is a known trigger of mitotic entry and has been demonstrated to be constitutively upregulated in tumors. It is therefore associated with carcinogenic properties reported in various cancers. However, the role of CDCA3 in prostate cancer is unclear. In the present study, western blotting and analysis of gene expression profiling datasets determined that CDCA3 expression was upregulated in prostate cancer and was associated with a poor prognosis. CDCA3 knockdown in DU145 and PC-3 cells led to decreased cell proliferation and increased apoptosis, with increased protein expression levels of cleaved-caspase3. Further experiments demonstrated that downregulated CDCA3 expression levels induced G0/G1 phase arrest, which was attributed to increased p21 protein expression levels and decreased cyclin D1 expression levels via the regulation of NF-κB signaling proteins (NFκB-p105/p50, IKKα/β, and pho-NFκB-p65). In conclusion, these results indicated that CDCA3 may serve a crucial role in prostate cancer and consequently, CDCA3 knockdown may be used as a potential therapeutic target.

Anticancer Activity of Pasak Bumi Root Extract (Eurycoma longifolia Jack) on Raji Cells.

<b>Background and Objective:</b> The use of the roots of the pasak bumi (<i>E. longifolia</i> Jack) to treat cancer has been studied widely, however, the scientific basis of these plants used as an anticancer drug is widely unknown. The purpose of this study was to examine the anticancer activity of ethyl acetate and non-ethyl acetate fractions of pasak bumi roots in Raji cells. <b>Materials and Methods:</b> The cytotoxicity test is using the direct cell count method with trypan blue staining. The growth inhibition is using doubling time analysis of Raji cells. Observation of the apoptotic events of Raji cells used ethidium bromide staining, while observing the expression of p53 protein in Raji cells was done by immunohistochemical staining. <b>Results:</b> The results of the cytotoxicity and doubling time test showed that the activity of the non-ethyl acetate fraction was greater than that of the roots of pasak bumi. The lower concentration of non-ethyl acetate fraction of pasak bumi roots was able to delay the multiplication time of Raji cells which was greater than that of ethyl acetate. The results of the cytotoxicity and doubling time test showed that the activity of the non-ethyl acetate fraction was greater than that of the roots of pasak bumi. <b>Conclusion:</b> It can be concluded that the ethyl acetate and non-ethyl acetate fractions of the roots of pasak bumi have cytotoxic and antiproliferative activity on Raji cells, however they cannot induce apoptosis in Raji cells. The death of Raji cells is through the mechanism of inhibiting Raji cell proliferation as evidenced by an increase in p53 protein expression.

WIP1 Inhibition by GSK2830371 Potentiates HDM201 through Enhanced p53 Phosphorylation and Activation in Liver Adenocarcinoma Cells.

Simple SummaryPatients with advanced intrahepatic cholangiocarcinoma (iCCA) have a very poor prognosis, and no targeted therapy is approved for advanced iCCA. A therapeutic strategy for wild-type p53 cancers is the reactivation of p53 by inhibition of its the negative regulators, MDM2, and WIP1. In the present study, we used HDM201 (an MDM2-p53 binding antagonist) to increase p53 stabilization and upregulate the expression of downstream targets (p21 and MDM2) in RBE and SK-Hep-1 liver adenocarcinoma cell lines. The survival rate and clonogenicity decreased after HDM201 treatment in a dose-dependent manner. Combined treatment with HDM201 and GSK2830371 (WIP1 inhibitor) increased p53 phosphorylation, leading to sustained p53 activation. This combination treatment resulted in G2/M phase arrest and promoted cytotoxicity compared with MDM2 inhibitor monotherapy. Furthermore, increased expression of p53 signaling pathway target genes were identified following combination treatment with HDM201 and GSK2830371, suggesting potential roles for this combination strategy in iCCA therapy.Background: Intrahepatic cholangiocarcinoma (iCCA) is an adenocarcinoma arising from the intrahepatic bile duct. It is the second most common primary liver cancer and has a poor prognosis. Activation of p53 by targeting its negative regulators, MDM2 and WIP1, is a potential therapy for wild-type p53 cancers, but few reports for iCCA or liver adenocarcinoma exist. Methods: Both RBE and SK-Hep-1 liver adenocarcinoma cell lines were treated with the HDM201 (Siremadlin) MDM2-p53 binding antagonist alone or in combination with the GSK2830371 WIP1 phosphatase inhibitor. Cell proliferation, clonogenicity, protein and mRNA expression, cell cycle distribution, and RNA sequencing were performed to investigate the effect and mechanism of this combination. Results: GSK2830371 alone demonstrated minimal activity on proliferation and colony formation, but potentiated growth inhibition (two-fold decrease in GI50) and cytotoxicity (four-fold decrease in IC50) by HDM201 on RBE and SK-Hep-1 cells. HDM201 increased p53 protein expression, leading to transactivation of downstream targets (p21 and MDM2). Combination with GSK2830371 increased p53 phosphorylation, resulting in an increase in both p53 accumulation and p53-dependent trans-activation. G2/M arrest was observed by flow cytometry after this treatment combination. RNA sequencing identified 21 significantly up-regulated genes and five downregulated genes following p53 reactivation by HDM201 in combination with GSK2830371 at 6 h and 24 h time points compared with untreated controls. These genes were predominantly known transcriptional targets regulated by the p53 signaling pathway, indicating enhanced p53 activation as the predominant effect of this combination. Conclusion: The current study demonstrated that GSK2830371 enhanced the p53-dependent antiproliferative and cytotoxic effect of HDM201 on RBE and SK-Hep-1 cells, providing a novel strategy for potentiating the efficacy of targeting the p53 pathway in iCCA.

Tumor Antigen ROR1 Targeted Delivery of MiR-29b in Chronic Lymphocytic Leukemia Induces Epigenetic Reprograming Resulting in Cell Cycle Arrest

Aggressive Chronic lymphocytic leukemia (CLL) exhibits reduced miR-29b with poor outcome. Strategically increasing miR-29b to reprogram CLL cells provides therapeutic potential. However, miRs are sensitive to degradation in circulation and encapsulation by cationic lipocomplex leads non-specific delivery, thus causing limited uptake in CLL cells. To overcome these limitations, we designed miR29b-encapsuled immunoliposome (2A2-miR-29b-ILP) targeting ROR1, a receptor tyrosine kinase expressed in leukemic but not normal B cells. The 2A2-ILP enhanced miR-29b uptake by >20-fold compared to free miR29b and 3-fold to IgG-ILP control. Interestingly, 2A2-miR-29b-ILP treatment did not directly affect cellular viability nor BCL2 pathways, but significantly decreased the global DNA methylation through downregulation of DNMTs and SP1 in primary CLL cells and a human CLL cell line OSU-CLL. (n=3 CLL, DNMT1: p= 0.0115; DNMT3a: p=0.0231, SP1; p=0.0031). Consistent with the downregulation of DNMTs, decreased global DNA methylation was observed in OSU-CLL cell line one week after treatment with 2A2-ILP-miR-29b (n=3, p=0.0003). Furthermore, using a double transgenic Eμ-TCL-1-ROR1 mouse model expressing human ROR1 (hROR1) on leukemic B cells, we further demonstrated the therapeutic benefit of enhanced survival with 2A2-miR-29b-ILP via cellular reprograming by downregulation of DNMT1 and DNMT3a in-vivo. Using the hROR1+CD19+CD5+ leukemic cell adoptive transfer model, we observed significant in-vivo efficacy of 2A2-miR-29b-ILP (also referred as 2A2-29b) formulation, compared to scramble control (referred as 2A2-SC), associated with a) decreased number of circulating leukemic B220+CD5+ cells (58±3% decrease in 2A2-29b treatment, p=0.0184; 2A2-29b: n=9; 2A2-SC: n=7) b) reduced splenomegaly (53±2% decrease in 2A2-29b treatment, p=0.0002, n=4) c) with extended survival (p=0.0075, 2A2-29b: n=9; 2A2-SC: n=7). Moreover, our RNAseq profiling of hROR1xTCL1 splenocyte implied reprogramming of cell cycle by miR-29b treatment relieving the leukemic progression. Gene expression of miR-29b treated leukemic splenocytes revealed 128 of 233 differentially expressed genes to be associated with cell growth and proliferation. The in-vivo miR29b-treated splenocytes exhibited compromised cell cycle, associated with decreased SP1 and increased p21 protein expression in our immunoblotting analysis. Importantly, SP1-knockdown resulted in significant decrease in miR29b mediated cell cycle arrest implicating SP1 is involved in miR-29b cell cycle arrest/suppression. In conclusion, 2A2-ILP effectively delivered functional miR-29b, resulting in DNMTs downregulation, reduction of DNA methylation and anti-leukemic activity. These studies form a basis of leukemic cell targeted delivery of miR29b as a promising therapeutic approach for CLL without compromising the ROR1-ve normal B cell counterparts.[Chi-Ling Chiang is a recipient of Pelotonia Graduate Student Fellowship. This work was supported by NIH Grants] DisclosuresByrd:Acerta Pharma: Research Funding; Janssen: Research Funding; Genentech: Research Funding; Pharmacyclics: Research Funding; The Ohio State University: Patents & Royalties: OSU-2S.

Calcium-binding and coiled-coil domain 2 promotes the proliferation and suppresses apoptosis of prostate cancer cells.

Prostate cancer (PCa) is considered to be one of the most common tumors in men. Calcium-binding and coiled-coil domain 2 (CALCOCO2) is a known important xenophagy receptor, which mediates intracellular bacterial degradation. To the best of the authors' knowledge, the present study is the first to demonstrate that CALCOCO2 functions as an oncogene in PCa. The results of the current study indicated that CALCOCO2 knockdown suppressed cell proliferation and colony formation, whereas it promoted apoptosis of PCa cells. In addition, knockdown of CALCOCO2 in PCa cells reduced cyclin-E1 and increased p53 protein expression. Bioinformatics analysis revealed that CALCOCO2 was associated with 'autophagosome assembly', 'nucleophagy' and 'nucleic acid metabolic process' biological processes and interacted with sequestosome-1, microtubule-associated proteins 1A/1B light chain 3 (MAP1LC3)B, γ-aminobutyric acid receptor-associated protein, IκB kinase subunit γ and MAP1LC3C. Moreover, CALCOCO2 protein levels were indicated to be significantly increased in PCa samples compared with normal prostate tissues. These results suggested that CALCOCO2 may be of value as a diagnostic and therapeutic target in PCa.

Abstract 14425: Coronary Microvascular Disease in Type 2 Diabetes is Linked to the Upregulation of P53

Diabetes mellitus is a prominent risk factor of coronary microvascular disease (CMD) and the comorbidity of CMD in diabetic patients leads to an increasing occurrence of cardiac mortality. Increased protein O -linked β-N-acetylglucosaminylation ( O -GlcNAcylation), a post-translational modification of proteins, is implicated in the development of vascular complications in diabetes. Therefore, we investigated the molecular mechanisms in which excess protein O -GlcNAcylation advances the progression of CMD in diabetes through coronary endothelial cell dysfunction. We hypothesize that excess protein O -GlcNAcylation will up-regulate p53 which will induce the development of CMD. We had conducted in vivo and in vitro experiments in control mice, TALLYHO/Jng (TH) mice, a polygenic type 2 diabetic mouse model, and endothelial cell-specific O -GlcNAcase (OGA, a regulatory enzyme that catalyzes the removal of O- GlcNAc from proteins)-overexpressing TH mice. We found that compared to control mice, TH mice exhibited a decrease in coronary flow velocity reserve (CFVR, an indicator of coronary microvascular function), reduced capillary density, and increased endothelial cell apoptosis in the left ventricle. In addition, TH mice showed a significant increase of p53 protein expression in mouse coronary endothelial cells (MCECs) compared to control mice. Moreover, overexpression of OGA lowered protein O -GlcNAcylation and decreased p53 levels in MCECs. p53 inhibition with pifithrin-α restoratively increased CFVR and cardiac contractility in TH mice. From this data, we conclude OGA overexpression lowers p53 expression levels and thus attenuates endothelial cell apoptosis and restores CFVR and cardiac function in diabetes. The downregulation of p53 through OGA overexpression could be a potential therapeutic target for CMD in diabetic patients.

Blockade of TRIM59 enhances esophageal cancer cell chemosensitivity to cisplatin by upregulating p53.

Human esophageal cancer (hESC) cell motility adopts various modes, resulting in hESC progression and poor survival. However, how tripartite motif 59 (TRIM59), as the ubiquitination machinery, participates in hESC metastasis is not completely understood. The results indicated that TRIM59 was aberrantly upregulated in hESC tissues compared with adjacent healthy esophageal tissues, which was associated with poor survival and advanced TNM state among patients with hESC. Moreover, patients with hESC with higher TRIM59 expression displayed undetectable p53 expression, which contributed to enhanced progression and motility of hESC. At the molecular level, TRIM59 was indicated to be an E3 putative ubiquitin ligase that targeted the p53 protein, leading to increased degradation of p53, which resulted in decreased chemosensitivity to cisplatin. TRIM59 knockdown reduced TRIM59 expression, increased p53 protein expression, and decreased hESC cell viability, clone formation and migration compared with the small interfering RNA negative control (siNC) group. Furthermore, hESC cell lines were more sensitive to cisplatin in the TRIM59-knockdown group compared with the siNC group. The results indicated a relationship between TRIM59, p53 and the chemosensitivity of cisplatin. The present study suggested that TRIM59 may serve as a promising prognostic indicator for patients with hESC.

Renal Programming by Transient Postnatal Overfeeding: The Role of Senescence Pathways.

BackgroundEarly nutrition influences the risk of chronic kidney diseases (CKDs) development in adulthood. Mechanisms underlying the early programming of altered renal function remain incompletely understood. This study aims at characterizing the role of cell senescence pathways in early programming of CKD after transient postnatal overfeeding.Materials and MethodsReduced litters of 3 mice pups and standard litters of 9 mice pups were obtained to induce overfed animals during lactation and control animals, respectively. Animals were sacrificed at 24 days (weaning) or at 7 months of life (adulthood). Body weight, blood pressure, kidney weight, and glomerular count were assessed in both groups. Senescence pathways were investigated using β-Galactosidase staining and Western blotting of P16, P21, P53, P-Rb/Rb, and Sirtuin 1 (Sirt1) proteins.ResultsEarly overfed animals had a higher body weight, a higher blood pressure at adulthood, and a higher glomerular number endowment compared to the control group. A higher β-Galactosidase activity, a significant increase in P53 protein expression (p = 0.0045) and a significant decrease in P-Rb/Rb ratio (p = 0.02), were observed at weaning in animals who underwent early postnatal overfeeding. Protein expression of Sirt1, a protective factor against accelerated stress-induced senescence, was significantly decreased (p = 0.03) at weaning in early overfed animals.ConclusionEarly postnatal overfeeding by litter size reduction is associated with increased expression of factors involved in cellular senescence pathways, and decreased expression of Sirt 1 in the mouse kidney at weaning. These alterations may contribute to CKD programming after early postnatal overfeeding.