39 research outputs found
Determination of maximum tolerated dose and toxicity of Inauhzin in mice
AbstractReactivating the tumor suppressor p53 offers an attractive strategy for developing cancer therapy. We recently identified Inauhzin (INZ) as a novel non-genotoxic p53-activating compound. To develop INZ into a clinically applicable anticancer drug, we have initiated preclinical toxicity studies. Here, we report our study on determining the maximum tolerated dose (MTD) of INZ analog, Inauhzin-C (INZ (C)), following intraperitoneal (i.p.) administration (Phase A) and its toxicity following i.p. administration over a period of 5-day dosing plus 2-day recovery (Phase B) in CD-1 mice. The Phase A study showed that the MTD of INZ (C) is 200mg/kg for female and 250mg/kg for male, respectively. The Phase B study showed that the administration of INZ (C) via 5-day consecutive i.p. injection is tolerated by female CD-1 mice at all dose levels tested from 50mg/kg to 120mg/kg without significant changes in biochemical and pathological parameters in the animals. Together, these results indicate that our previously determined effective dose of INZ at 30–60mg/kg via i.p. is quite safe to mice, and imply that this compound have the features worthy for further development into a clinically applicable drug
The role of IMP dehydrogenase 2 in Inauhzin-induced ribosomal stress
The ‘ribosomal stress (RS)-p53 pathway’ is triggered by any stressor or genetic alteration that disrupts ribosomal biogenesis, and mediated by several ribosomal proteins (RPs), such as RPL11 and RPL5, which inhibit MDM2 and activate p53. Inosine monophosphate (IMP) dehydrogenase 2 (IMPDH2) is a rate-limiting enzyme in de novo guanine nucleotide biosynthesis and crucial for maintaining cellular guanine deoxy- and ribonucleotide pools needed for DNA and RNA synthesis. It is highly expressed in many malignancies. We previously showed that inhibition of IMPDH2 leads to p53 activation by causing RS. Surprisingly, our current study reveals that Inauzhin (INZ), a novel non-genotoxic p53 activator by inhibiting SIRT1, can also inhibit cellular IMPDH2 activity, and reduce the levels of cellular GTP and GTP-binding nucleostemin that is essential for rRNA processing. Consequently, INZ induces RS and the RPL11/RPL5-MDM2 interaction, activating p53. These results support the new notion that INZ suppresses cancer cell growth by dually targeting SIRT1 and IMPDH2
Mutant p53 Gains Its Function via c-Myc Activation upon CDK4 Phosphorylation at Serine 249 and Consequent PIN1 Binding
TP53 missense mutations significantly influence the development and progression of various human cancers via their gain of new functions (GOF) through different mechanisms. Here we report a unique mechanism underlying the GOF of p53-R249S (p53-RS), a p53 mutant frequently detected in human hepatocellular carcinoma (HCC) that is highly related to hepatitis B infection and aflatoxin B1. A CDK inhibitor blocks p53-RS\u2019s nuclear translocation in HCC, whereas CDK4 interacts with p53-RS in the G1/S phase of the cells, phosphorylates it, and enhances its nuclear localization. This is coupled with binding of a peptidyl-prolyl cis-trans isomerase NIMA-interacting 1 (PIN1) to p53-RS, but not the p53 form with mutations of four serines/threonines previously shown to be crucial for PIN1 binding. As a result, p53-RS interacts with c-Myc and enhances c-Myc-dependent rDNA transcription key for ribosomal biogenesis. These results unveil a CDK4-PIN1-p53-RS-c-Myc pathway as a novel mechanism for the GOF of p53-RS in HCC
Global effect of inauhzin on human p53-responsive transcriptome.
BACKGROUND: Previously, we reported that Inauhzin (INZ) induces p53 activity and suppresses tumor growth by inhibiting Sirt1. However, it remains unknown whether INZ may globally affect p53-dependent gene expression or not. Herein, we have conducted microarray and real-time PCR analyses of gene expression to determine the global effect of INZ on human p53-responsive transcriptome. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we conducted microarray analysis followed by PCR validation of general gene expression in HCT116(p53+/+) and HCT116(p53-/-) cells treated with or without INZ. Microarray data showed that 324 genes were up-regulated by ≥ 2.3-fold and 266 genes were down-regulated by ≥ 2-fold in response to INZ treatment in a p53-dependent manner. GO analysis for these genes further revealed that INZ affects several biological processes, including apoptosis (GO:0006915), cell cycle (GO:0007049), immune system process (GO:0002376), and cell adhesion (GO:0007155), which are in line with p53 functions in cells. Also, pathway and STRING analyses of these genes indicated that the p53-signaling pathway is the most significant pathway responsive to INZ treatment as predicted, since a number of these p53 target genes have been previously reported and some of them were validated by RT-qPCR. Finally, among the 9 tested and highly expressed genes, ACBD4, APOBEC3C, and FLJ14327 could be novel p53 target genes, for they were up-regulated by INZ in HCT116(p53+/+) cells, but not in HCT116(p53-/-) cells. CONCLUSIONS/SIGNIFICANCE: From our whole genome microarray analysis followed by validation with RT-qPCR, we found that INZ can indeed induce the expression of p53 target genes at a larger scale or globally. Our findings not only verify that INZ indeed activates the p53 signaling pathway, but also provide useful information for identifying novel INZ and/or p53 targets. The global effect of INZ on human p53-responsive transcriptome could also be instrumental to the future design of INZ clinical trials
SSRP1 functions as a co-activator of the transcriptional activator p63
The p53 homolog p63 is a transcriptional activator. Here, we describe the identification of an HMG1-like protein SSRP1 as a co-activator of p63. Over expression of wild-type, but not deletion mutant, SSRP1 remarkably enhanced p63γ-dependent luciferase activity, G(1) arrest, apoptosis and expression of endogenous PIG3, p21(Waf1/cip1) and MDM2 in human p53-deficient lung carcinoma H1299 cells and mouse embryonic fibroblasts. Also, SSRP1 interacted to p63γ in vitro and in cells, and resided with p63γ at the p53-responsive DNA element sites of the cellular endogenous MDM2 and p21(Waf1/cip1) promoters. Moreover, N-terminus-deleted p63 (ΔN-p63) bound to neither SSRP1 nor its central domain in vitro. Accordingly, SSRP1 was unable to stimulate ΔN-p63-mediated residual luciferase activity and apoptosis in cells. Finally, the ectopic expression of the central p63-binding domain of SSRP1 inhibited p63-dependent transcription in cells. Thus, these results suggest that SSRP1 stimulates p63 activity by associating with this activator at the promoter
Structure and activity analysis of Inauhzin analogs as novel antitumor compounds that induce p53 and inhibit cell growth.
Identifying effective small molecules that specifically target the p53 pathway in cancer has been an exciting, though challenging, approach for the development of anti-cancer therapy. We recently identified Inauhzin (INZ) as a novel p53 activator, selectively and efficiently suppressing tumor growth without displaying genotoxicity and with little toxicity to normal cells. In order to reveal the structural features essential for anti-cancer activity of this small molecule, we have synthesized a panel of INZ analogs and evaluated their ability to induce cellular p53 and to inhibit cell growth in cell-based assays. This study as described here leads to the discovery of INZ analog 37 that displays much better potency than INZ in both of p53 activation and cell growth inhibition in several human cancer cell lines including H460 and HCT116(+/+) cells. This INZ analog exhibited much less effect on p53-null H1299 cells and HCT116(-/-) cells, and importantly no toxicity on normal human p53-containing WI-38 cells. Hence, our results not only unveil key chemical features for INZ activity, but also identify the newly synthesized INZ analog 37 as a better small molecule for further development of anti-cancer therapy
Ribosomopathies: Mechanisms of Disease
Ribosomopathies are diseases caused by alterations in the structure or function of ribosomal components. Progress in our understanding of the role of the ribosome in translational and transcriptional regulation has clarified the mechanisms of the ribosomopathies and the relationship between ribosomal dysfunction and other diseases, especially cancer. This review aims to discuss these topics with updated information