Inhibition Of MDM2 Expression Research Articles

Icaritin activates p53 and inhibits aerobic glycolysis in liver cancer cells

Metabolic reprogramming enables cancer cells to generate energy mainly through aerobic glycolysis, which is achieved by increasing the expression levels of glycolysis-related enzymes. Therefore, the development of drugs targeting aerobic glycolysis could be an effective strategy for cancer treatment. Icaritin (ICT) is an active ingredient from the Chinese herbal plant Epimedium with several biological activities, but its anti-cancer mechanism remains inconclusive. Using normal hepatocytes and hepatoma cells, our results showed that ICT suppressed cell proliferation and clonal formation and decreased glucose consumption and lactate production in liver cancer cells. In consistent, the mRNA and protein levels of several aerobic glycolysis-related genes were decreased upon ICT treatment. Furthermore, our results demonstrated that the expression levels of the aerobic glycolysis-related proteins were correlated with the p53 status in hepatoma cells. Using PFT-α or siRNA-p53, our results confirmed that ICT regulated aerobic glycolysis in a p53-dependent manner. In addition, ICT was found to stabilize p53 at the post-translational level which might be mediated by inhibiting MDM2 expression and affecting its interaction with p53. Finally, our results demonstrated that ICT increased the levels of ROS that activated p53 via the p38 MAPK pathway. In conclusion, ICT increased intracellular ROS levels in liver cancer cells, which promoted the stabilization and activation of p53, inhibiting the expression of aerobic glycolysis-related genes and glycolysis, and ultimately leading to the suppression of liver cancer development.

Indole-3-Carbinol Stabilizes p53 to Induce miR-34a, Which Targets LDHA to Block Aerobic Glycolysis in Liver Cancer Cells.

Certain cancer cells prefer aerobic glycolysis rather than oxidative phosphorylation for energy supply. Lactate dehydrogenase A (LDHA) catalyzes the reduction of pyruvate to lactate and regains NAD+ so that glycolysis is continued. As a pivotal enzyme to promote smooth glycolysis, LDHA plays an important role in carcinogenesis. Indole-3-carbinol (I3C) has displayed antitumor activity, but the exact mechanism remains to be identified. In this study, we treated liver cancer cells with I3C, performed colony formation and cell migration, measured the expression of glycolysis-related proteins, and predicted and validated LDHA-targeting miRNA from the databases. In addition, the mRNA and protein levels of p53, glycolysis-related genes and miRNAs that regulate glycolysis were detected after I3C and siRNA-p53 treatment alone or in combination. Next, the expression and colocalization of p53 and MDM2 in liver cancer cells were evaluated after I3C treatment, and the effect of I3C on p53 protein stability was examined. The results showed that I3C inhibited cell proliferation, migration, and the expression levels of glycolysis-related gene LDHAs. MiR-34a was predicted to target LDHA, and I3C downregulated its expression. Furthermore, the combined I3C and siRNA-p53 treatment demonstrated that I3C regulated the expression of LDHA via miR-34a in a p53-dependent manner. Finally, I3C inhibited MDM2 expression and its colocalization with p53 and stabilized p53 expression. In summary, I3C inhibited the degradation of p53 by MDM2 in liver cancer cells; stable p53 induced miR-34a, which targeted LDHA, a key enzyme for aerobic glycolysis, suggesting cancer metabolism is an important target for I3C in liver cancer cells.

MiR-221-3p-mediated downregulation of MDM2 reverses the paclitaxel resistance of non-small cell lung cancer in vitro and in vivo

MicroRNAs (miRNAs) are involved in the initiation and development of cancer and participate in drug resistance. Paclitaxel (PTX) is a first-line chemotherapy drug for advanced non-small cell lung cancer (NSCLC). The abnormal miRNA expression in NSCLC and its association with chemotherapy drug resistance remains largely unknown. The study aimed to investigate the aberrant expression of miR-221–3p in NSCLC and to elucidate its molecular mechanisms in relation to PTX resistance. PTX increased miR-221–3p expression and regulated MDM2/P53 expression in the PTX-sensitive NSCLC strain (A549). Meanwhile, miR-221–3p was rarely expressed and not interfered by PTX in PTX-resistant A549 cells (A549/Taxol). Dual-luciferase reporter assay confirmed that miR-221–3p specifically binds to MDM2 messenger RNA and inhibited MDM2 expression. The expression of MDM2 and P53 showed a negative correlation in NSCLC cell lines. MiR-221–3p down-regulation reduced the sensitivity of A549 cells to PTX, whereas its up-regulation partially reversed the A549/Taxol cells resistance to PTX and increased the chemosensitivity of A549/Taxol cells to PTX in xenograft models. Quantitative polymerase chain reaction analysis revealed that miR-221–3p expression increased, whereas the MDM2 level decreased in human NSCLC tumor tissues. Moreover, Western bolt analysis showed that P53 was lowly expressed in tumor tissues with MDM2 overexpression. Low expression of miR-221–3p in NSCLC tissues might indicate a poor T staging. In conclusion, miR-221–3p overexpression could regulate MDM2/p53 signaling pathway to reverse the PTX resistance of NSCLC and induce apoptosis in vitro and vivo.

MDM2 promotes the proliferation and inhibits the apoptosis of pituitary adenoma cells by directly interacting with p53.

Pituitary adenomas constitute one of the most common intracranial tumours. The mouse double minute 2 homologue (MDM2) is considered as an important oncogene in many tumours, but it has been little studied in pituitary adenomas and the mechanism is not well understood. The purpose of this study was to investigate the function of MDM2 and its primary mechanism of action in pituitary adenoma cells. The expression of MDM2 in pituitary adenoma cell lines and normal cells was determined by real-time polymerase chain reaction (RT-PCR). The proliferation and apoptosis of pituitary adenoma cells after inhibition of MDM2 expression were detected by MTS and flow cytometry, respectively. The protein expressions of MDM2 and p53 were detected by western blot. Co-IP was used to detect the direct binding between MDM2 and p53. The results of RT-PCR showed that MDM2 was significantly up-regulated in pituitary adenoma cell lines. Inhibition of MDM2 suppressed the proliferation and promoted apoptosis of pituitary adenoma cells. However, inhibiting the expression of MDM2 can promotethe protein expression of p53. The results of co-IP showed that MDM2 interacted with p53 by direct combination. Then, we inhibited the expressions of p53 and MDM2 simultaneously in the pituitary adenoma cells by co-transfecting siRNAs, and the results showed that, compared with the group that inhibited MDM2 alone, cell proliferation of the co-transfected group increased and apoptosis of the cotransfected group decreased, which was similar to the NC group. Taken together, these results suggest that MDM2 promoted the proliferation and inhibited the apoptosis of pituitary adenoma cells by directly interacting with p53 in pituitary adenoma cells. Therefore, MDM2-p53 may serve as a novel marker and therapeutic target for pituitary adenomas.

Long noncoding RNA TUG1 facilitates cell ovarian cancer progression through targeting MiR-29b-3p/MDM2 axis.

Ovarian cancer (OC) is one of the most aggressive female cancers in the world. OC trends to be diagnosed at an advanced stage with abdominal metastasis. Our study explored the biological function and underlying mechanism of lncRNA on OC cell proliferation and migration. The expression of turine up-regulated gene 1 (TUG1) in human OC tissues and cell lines was measured by qRT-PCR. OC cell proliferation, viability, migration, and invasion were measured by MTT assays, colony formation assays, and transwell assays in vitro. Furthermore, the nude mice xenograft model was established to determine the effects of TUG1 in vivo. The relationship between TUG1 and miR-29b-3p, as well as miR-29b-3p and MDM2 were identified using the luciferase reporter assays. We showed that the expression of TUG1 and MDM2 were significantly increased, but the expression of miR-29b-3p was remarkably decreased in OC tissues and cell lines. Knockdown of TUG1 strongly inhibited the ability of cell proliferation, colony formation, migration, and invasion in vitro. The relationship between TUG1 and miR-29b-3p, or miR-29b-3p and MDM2 were predicted by StarBase and miRanda online software. Besides, miR-29b-3p reversed the positive effect of TUG1 on the OC cell proliferation, migration, and invasion through inhibiting MDM2 expression and increasing p53 phosphorylation level. Moreover, knockdown of TUG1 suppressed tumor growth in vivo. Taken all together, this study shows that TUG1 plays a crucial oncogenic role and facilitates cell proliferation, migration, and invasion in OC through regulating miR-29b-3p/MDM2 axis.

Impaired Notch Signaling Leads to a Decrease in p53 Activity and Mitotic Catastrophe in Aged Muscle Stem Cells

The decline of tissue regenerative potential with age correlates with impaired stem cell function. However, limited strategies are available for therapeutic modulation of stem cell function during aging. Using skeletal muscle stem cells (MuSCs) as a model system, we identify cell death by mitotic catastrophe as a cause of impaired stem cell proliferative expansion in aged animals. The mitotic cell death is caused by a deficiency in Notch activators in the microenvironment. We discover that ligand-dependent stimulation of Notch activates p53 in MuSCs via inhibition of Mdm2 expression through Hey transcription factors during normal muscle regeneration and that this pathway is impaired in aged animals. Pharmacologic activation of p53 promotes the expansion of aged MuSCs invivo. Altogether, these findings illuminate a Notch-p53 signaling axis that plays an important role in MuSC survival during activation and is dysregulated during aging, contributing to the age-related decline in muscle regenerative potential.

Abstract 4867: Treating hepatocellular carcinoma metastasis and overcoming chemoresistance through inhibiting the MDM2 oncogene

Abstract Hepatocellular carcinoma (HCC) poses a major health problem worldwide and novel, effective therapeutic approaches are urgently needed. The MDM2 oncogene is amplified and overexpressed in HCC and associated with its initiation, progression, metastasis, and chemoresistence. MDM2 has been demonstrated to be as a promising therapeutic target for the treatment of HCC. However, most of the current MDM2 inhibitors have been designed to block the binding between MDM2 and p53, and have limited efficacy against tumors with mutant or deficient p53. We recently performed a high-throughput virtual and cell-based screening, yielding a novel MDM2 inhibitor, termed SP141. The present study was designed to evaluate the anti-HCC activity of SP141, its potential to chemosensitize HCC cells to other anticancer agents, and to determine the underlying mechanism(s) of action of SP141 in various in vitro and in vivo models with different backgrounds of p53 (wildtype, mutant or null). Our results demonstrated that SP141 inhibited cell growth and prevented cell migration and invasion, independent of p53. Mechanistically, SP141 directly bound to the MDM2 protein and promoted MDM2 degradation. The inhibition of MDM2 by SP141 also increased the sensitivity of HCC cells to sorafenib. In addition, in orthotopic and patient-derived xenograft (PDX) models, SP141 inhibited MDM2 expression and suppressed tumor growth and metastasis, without any host toxicity. Furthermore, mechanistic study demonstrated that the inhibition of MDM2 by SP141 was essential for its anti-HCC activities. In conclusion, MDM2 inhibition by SP141 resulted in the inhibition of HCC tumor growth and metastasis and sensitization of HCC cells to chemotherapy, regardless of their p53 status. These results provide support for the further development of SP141 as a lead candidate for the treatment of HCC. (Supported by NIH R01 CA186662 and R01CA214019 and ACS RSG-15-009-01-CDD.) Citation Format: Wei Wang, Bo Hu, Jiang-Jiang Qin, Jianwen Cheng, Xin Li, Ming Hu, John Buolamwini, Xin-Rong Yang, Jia Fan, Ruiwen Zhang. Treating hepatocellular carcinoma metastasis and overcoming chemoresistance through inhibiting the MDM2 oncogene [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4867.

2-Methoxy-5((3,4,5-trimethosyphenyl)seleninyl) phenol inhibits MDM2 and induces apoptosis in breast cancer cells through a p53-independent pathway

2-Methoxy-5((3,4,5-trimethosyphenyl)seleninyl) phenol (SQ) is a novel synthesized combretastatin A-4 (CA-4) analog that can be classified as a microtubule inhibitor. Our previous study demonstrated that SQ induced G2/M phase arrest and promoted apoptosis progression in breast cancer cells. In the present study, we found that SQ dissociated the MDM2-p53 complex and directly induced MDM2 degradation through the ubiquitin-dependent proteasome pathway in MCF-7 and MDA-MB-231 cells. Further, p53 was activated by SQ through regulation of its transcription, translation, and post-translation modification. More specifically, we demonstrated that SQ induced caspase-dependent but p53-independent apoptosis, and this apoptosis is associated with the inhibition of MDM2. We also showed that SQ exhibited superior in vivo efficacy and low toxicity than CA-4. The immunofluorescence histochemistry study indicated that SQ also inhibited MDM2 expression in vivo. In summary, we report for the first time that SQ shows excellent anti-breast cancer activity in vivo and in vitro and induces p53-independent apoptosis, which is associated with MDM2 inhibition. Therefore, the novel compound SQ has potential for therapeutic treatment of both wild-type and mutant p53 breast cancer.

Tryptanthrin reduces mast cell proliferation promoted by TSLP through modulation of MDM2 and p53.

Atopic dermatitis (AD) results from complex interactions between mast cells and inflammatory mediators. An inflammatory mediator, thymic stromal lymphopoietin (TSLP) is known to promote mast cell proliferation through up-regulation of mouse double minute 2 (MDM2, a negative regulator of p53) and aggravate AD. In this study, we investigated whether tryptanthrin (TR, an anti-inflammatory agent) would regulate TSLP-induced mast cell proliferation and TSLP-induced a pro-inflammatory cytokine, tumor necrosis factor (TNF)-α production from mast cells. Human mast cell line (HMC-1) cells were treated with TR and stimulated with TSLP. Proliferation was measured with a bromodeoxyuridine incorporation assay. And pro- and anti-apoptotic factors were analyzed with quantitative real-time PCR, Western blot analysis, and ELISA. The mRNA expression and production of TNF-α were analyzed with quantitative real-time PCR and ELISA. TR significantly inhibited the proliferation of HMC-1 cells promoted by TSLP. TR inhibited MDM2 expression, whereas TR increased the expression of p53, poly ADP-ribose polymerase, and caspase-3 in the TSLP-stimulated HMC-1 cells. TR significantly inhibited Ki67 mRNA expression as well as mRNA expression and production of interleukin (IL)-13 in the TSLP-stimulated HMC-1 cells. Moreover, TR significantly suppressed mRNA expression and production of TNF-α in the TSLP-stimulated HMC-1 cells. Finally, the mRNA expression of IL-7 receptor α chain and TSLP receptor was inhibited by TR in the TSLP-stimulated HMC-1 cells. Our results suggest that TR determined with new concept has intensive potential for the treatment of mast cell-mediated allergic diseases, such as AD.

Inhibition of MDM2 expression by rosmarinic acid in TSLP-stimulated mast cell

Rosmarinic acid (RA) has an anti-inflammatory property while thymic stromal lymphopoietin (TSLP) has an important role in mast cell-mediated inflammatory responses. Thus, the aim of this study was to determine the regulatory effect of RA in TSLP-stimulated human mast cell line, HMC-1 cells, and short ragweed pollen-induced allergic conjunctivitis mouse model. As a result, we found that RA significantly decreased the TSLP-induced mast cell proliferation and murine double minute (MDM) 2 expression. RA significantly decreased the levels of interleukin (IL)-13 and phosphorylated the signal transducer and activation of transcription 6 in the TSLP-stimulated HMC-1 cells. RA induced the increment of p53 levels, caspase-3 activation, and poly-ADP-ribose polymerase cleavage and the reduction of the procaspase-3 and Bcl2. RA significantly reduced the production of tumor necrosis factor-α, IL-1β, and IL-6 on the TSLP-stimulated HMC-1 cells. In addition, RA significantly reduced the levels of IgE, IL-4, and TSLP in the short ragweed pollen-induced allergic conjunctivitis mouse model. In conclusion, the results of the study suggest that RA has a significant anti-inflammatory effect on TSLP-induced inflammatory reactions. These effects of RA are likely to be mediated through inhibiting the MDM2 increased by TSLP.

Overcoming Akt Induced Therapeutic Resistance in Breast Cancer through siRNA and Thymoquinone Encapsulated Multilamellar Gold Niosomes.

Akt overexpression in cancer causes resistance to traditional chemotherapeutics. Silencing Akt through siRNA provides new therapeutic options; however, poor in vivo siRNA pharmacokinetics impede translation. We demonstrate that acidic milieu-sensitive multilamellar gold niosomes (Nio-Au) permit targeted delivery of both Akt-siRNA and thymoquinone (TQ) in tamoxifen-resistant and Akt-overexpressing MCF7 breast cancer cells. Octadecylamine groups of functionalized gold nanoparticles impart cationic attribute to niosomes, stabilized through polyethylene glycol. TQ's aqueous insolubility renders its encapsulation within hydrophobic core, and negatively charged siRNA binds in hydrophilic region of cationic niosomes. These niosomes were exploited to effectively knockdown Akt, thereby sensitizing cells to TQ. Immunoblot studies revealed enhanced apoptosis by inducing p53 and inhibiting MDM2 expression, which was consistent with in vivo xenograft studies. This innovative strategy, using Nio-Au to simultaneously deliver siRNA (devoid of any chemical modification) and therapeutic drug, provides an efficacious approach for treating therapy-resistant cancers with significant translational potential.

Abstract 1064: RASSF10 suppresses colorectal cancer growth by activating p53 signaling and sensitizes colorectal cancer cell to docetaxel

Abstract RASSF10 has previously been reported to be frequently methylated in a number of malignancies. To understand the importance of RASSF10 inactivation in colorectal carcinogenesis, eight colorectal cancer cell lines, 89 cases of primary colorectal cancer and 5 cases of normal colorectal mucosa were examined. Methylation specific PCR, western blot, siRNA, gene expression array and xenograft mice were employed. The expression of RASSF10 was was regulated by promoter regional methylation in colorectal cancer cells. RASSF10 was methylated in 60.7% (54/89) of primary colorectal cancers and was positively associated with tumor stage (p<0.05) and metastasis (p<0.05). Restoration of RASSF10 led to inhibition of colorectal cancer cell proliferation in vitro and in vivo and increased apoptosis. Gene expression arrays discovered RASSF10 inhibition of MDM2 expression as a mediator of these effects, which was confirmed with RT- PCR and western blot. RASSF10 was shown to activate P53 signaling in RKO and HCT116 cells after UV exposure, and sensitized these cells to docetaxel. In conclusion, our study demonstrates RASSF10 is frequently methylated in human colorectal cancer leading to loss of expression. RASSF10 normally suppresses human colorectal cancer growth by activating P53 signaling in colorectal cancer, and restored expression sensitizes colorectal cancer to docetaxel. Citation Format: Jing Guo, Yage Yang, Yunsheng Yang, Enqiang Linghu, Qimin Zhan, Malcolm V. Brock, James G. Herman, Bingyong Zhang, Mingzhou Guo. RASSF10 suppresses colorectal cancer growth by activating p53 signaling and sensitizes colorectal cancer cell to docetaxel. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1064. doi:10.1158/1538-7445.AM2015-1064

Regulation of Mdm2 mRNA Stability by RNA-binding Protein RNPC1

The murine double minute-2 (Mdm2) oncoprotein is an E3 ligase and a key regulator of a variety of fundamental cellular processes [1]. Mdm2 was originally identified as being amplified on double-minute chromosomes in transformed mouse fibroblasts [2]. Soon after its discovery, Mdm2 was found to be a negative regulator of tumor suppressor p53. The importance of Mdm2 in controlling the p53 activity is demonstrated in Mdm2 knockout mice. Mdm2-null embryos die very early during gestation, but additional deletion of p53 rescues them from death. Nevertheless, Mdm2 has p53-independent activities in promoting tumorigenesis. Due to its critical role in tumor development, understanding the mechanism by which Mdm2 expression is controlled will lay the foundation for therapeutic strategies by targeting Mdm2 for cancer management. The RNPC1 gene, also called RBM38, encodes an RNA-binding protein and is expressed as two isoforms, RNPC1a and RNPC1b. Both RNPC1a and RNPC1b contain a putative RNA recognition motif, which shares a high homology with the ones in HuR and Musashi. Recently, we showed that RNPC1 is a target of p53 and can in turn translationally repress p53 expression [3,4]. Thus, RNPC1 forms a feedback-regulatory loop with p53. In a recent publication, we described a novel regulation of Mdm2 by RNPC1 via mRNA stability [5] (Fig. ​(Fig.1A).1A). In particular, we generated several stable cell lines that can inducibly express ectopic RNPC1 under the control of tetracycline-responsive promoter. We found that upon induction, ectopic RNPC1 is able to inhibit Mdm2 expression at the level of transcript and protein regardless of p53. Consistent with this, knockdown or knockout of endogenous RNPC1 led to an increased level of Mdm2 transcript and protein independent of p53. Mechanistically, we found that RNPC1 is able to bind multiple AU-/U-rich elements in Mdm2 3'untranslated region (3'UTR) and subsequently, destabilizes Mdm2 transcripts. We also found that the AU-/U-rich elements in Mdm2 3'UTR and the RNA-binding domain in RNPC1 are required for RNPC1 to inhibit Mdm2 expression. Together, these data suggest that RNPC1 is a critical regulator of Mdm2 and further studies are needed to address that the biological significance of these regulation under a physiological setting. The finding that RNPC1 destabilizes Mdm2 transcripts may add an additional level of control to p53-Mdm2 autoregulatory feedback loop, which dictates cellular p53 levels and activity (Fig. ​(Fig.1B).1B). Briefly, RNPC1 is a target of the p53 and a negative regulator of p53 and Mdm2. Thus, it is likely that under non-stress conditions, RNPC1 and Mdm2 restrain p53 expression via translational repression and protein degradation, respectively. In response to various stimuli such as DNA damage, p53 is accumulated and transactivates both RNPC1 and Mdm2. Consequently, RNPC1 induction leads to decreased Mdm2 expression, which would release p53 from Mdm2-mediated inhibition. We would like to mention that although RNPC1 inhibits p53 translation, this regulation may be less effective under stress conditions owing to a global inhibition of protein synthesis. In addition to RNPC1, Mdm2 can be regulated by several other RNA-binding proteins at posttranscriptional level. For example, Mdm2 is regulated by HuR via mRNA stability [6]. In addition, Mdm2 is found be regulated by La protein via protein translation, which may play a critical role in leukemogenesis [7]. Furthermore, a number of ribosomal proteins, such as RPL5, RPL7, RPL11, RPL23, and RPS27L, are found to interact with Mdm2 and inhibit its E3 ligase activity [1]. Since altered expression of RBPs has been found in many types of human cancer, understanding how Mdm2 is regulated by RBPs and whether this regulation contributes to tumorigenesis might provide novel therapeutic strategies for cancer treatment. In summary, our data suggest that RNPC1 is a critical regulator of Mdm2. As both RNPC1 and Mdm2 expression is altered in several types of human cancers, this regulation may represent an important mechanism for cancer development and RNPC1 may be used for targeting tumors with high Mdm2 expression. Figure 1 (A) A proposed model for RNPC1-mediated Mdm2 repression. (B) A proposed model for the interplay among RNPC1, Mdm2, and p53.

Triptolide Inhibits MDM2 and Induces Apoptosis in Acute Lymphoblastic Leukemia Cells through a p53-Independent Pathway

Triptolide, a natural product derived from the Chinese plant Tripterygium wilfordii, is reported to exhibit antitumor effects in a broad range of cancers. The antitumor activity of triptolide is associated with its biologic activities, as it inhibits various proproliferative or antiapoptotic factors that are dominantly expressed in given types of cancer cells. Herein, we show that triptolide induced apoptosis in a subgroup of acute lymphoblastic leukemia (ALL) cells overexpressing the MDM2 oncoprotein by inhibiting MDM2 expression. More specifically, we found that triptolide inhibited MDM2 at the transcriptional level by suppressing its mRNA synthesis. This MDM2 inhibition led in turn to increased levels of p53 protein; however, p53 functionality was not activated due to the fact that triptolide-treated cells lacked induction of p21 and PUMA as well as in G(1) cell-cycle arrest. Triptolide-mediated downregulation of MDM2 increased inhibition of X-linked inhibitor of apoptosis protein (XIAP), its translational target, in a manner distinct from reactions to cellular stress and DNA-damaging agent ionizing radiation that induce XIAP due to p53-activated MDM2. These results suggest that increased inhibition of XIAP due to downregulation of MDM2 may play a critical role in triptolide-induced apoptosis in MDM2-overexpressing cancers.

Aciculatin Induces p53-Dependent Apoptosis via MDM2 Depletion in Human Cancer Cells In Vitro and In Vivo

Aciculatin, a natural compound extracted from the medicinal herb Chrysopogon aciculatus, shows potent anti-cancer potency. This study is the first to prove that aciculatin induces cell death in human cancer cells and HCT116 mouse xenografts due to G1 arrest and subsequent apoptosis. The primary reason for cell cycle arrest and cell death was p53 accumulation followed by increased p21 level, dephosphorylation of Rb protein, PUMA expression, and induction of apoptotic signals such as cleavage of caspase-9, caspase-3, and PARP. We demonstrated that p53 allele-null (−/−) (p53-KO) HCT116 cells were more resistant to aciculatin than cells with wild-type p53 (+/+). The same result was achieved by knocking down p53 with siRNA in p53 wild-type cells, indicating that p53 plays a crucial role in aciculatin-induced apoptosis. Although DNA damage is the most common event leading to p53 activation, we found only weak evidence of DNA damage after aciculatin treatment. Interestingly, the aciculatin-induced downregulation of MDM2, an important negative regulator of p53, contributed to p53 accumulation. The anti-cancer activity and importance of p53 after aciculatin treatment were also confirmed in the HCT116 xenograft models. Collectively, these results indicate that aciculatin treatment induces cell cycle arrest and apoptosis via inhibition of MDM2 expression, thereby inducing p53 accumulation without significant DNA damage and genome toxicity.

Natural Product Ginsenoside 25-OCH3-PPD Inhibits Breast Cancer Growth and Metastasis through Down-Regulating MDM2

Although ginseng and related herbs have a long history of utility for various health benefits, their application in cancer therapy and underlying mechanisms of action are not fully understood. Our recent work has shown that 20(S)-25-methoxyl-dammarane-3β, 12β, 20-triol (25-OCH3-PPD), a newly identified ginsenoside from Panax notoginseng, exerts activities against a variety of cancer cells in vitro and in vivo. This study was designed to investigate its anti-breast cancer activity and the underlying mechanisms of action. We observed that 25-OCH3-PPD decreased the survival of breast cancer cells by induction of apoptosis and G1 phase arrest and inhibited the growth of breast cancer xenografts in vivo. We further demonstrated that, in a dose- and time-dependent manner, 25-OCH3-PPD inhibited MDM2 expression at both transcriptional and post-translational levels in human breast cancer cells with various p53 statuses (wild type and mutant). Moreover, 25-OCH3-PPD inhibited in vitro cell migration, reduced the expression of epithelial-to-mesenchymal transition (EMT) markers, and prevented in vivo metastasis of breast cancer. In summary, 25-OCH3-PPD is a potential therapeutic and anti-metastatic agent for human breast cancer through down-regulating MDM2. Further preclinical and clinical development of this agent is warranted.

Nandrolone, an Anabolic Steroid, Stabilizes Numb Protein Through Inhibition of mdm2 in C2C12 Myoblasts

Nandrolone, an anabolic steroid, slows denervation atrophy of rat muscle, prevents denervation-induced nuclear accumulation of intracellular domain of the Notch receptor, and elevates expression of Numb. Numb acts as an inhibitor of Notch signaling and promotes myogenic differentiation of satellite cells. Turnover of Numb is regulated by mdm2, an E3 ubiquitin ligase. With these considerations in mind, we investigated the effects of nandrolone on the expression of Numb and mdm2 proteins and determined the effect of mdm2 on nandrolone-induced alterations in Numb protein in C2C12 myoblasts. When C2C12 cells were cultured in a medium favoring differentiation (Dulbecco modified Eagle medium containing 2% horse serum), nandrolone up-regulated Numb protein levels in a time-dependent manner and prolonged Numb protein half-life from 10 to 18 hours. In contrast, nandrolone reduced the expression of mdm2 protein. To determine whether the decreased mdm2 expression induced by nandrolone was responsible for the increased levels and prolonged half-life of Numb protein in this cell line, mdm2-small interfering RNA (siRNA) was employed to inhibit mdm2 expression. Compared to cells transfected with scrambled siRNA (negative control), transfection with mdm2-siRNA increased basal Numb protein expression but abolished the further increase in Numb protein levels by nandrolone. In addition, transfection of mdm2-siRNA mimicked the effect of nandrolone to prolong the half-life of Numb protein. Moreover, when C2C12 cells were forced to overexpress mdm2, there was a significant decline in the expression of both basal and inducible Numb protein. Our data suggest that nandrolone, by a novel mechanism for this agent in a muscle cell type, increases Numb protein levels in C2C12 myoblasts by stabilizing Numb protein against degradation, at least in part, via suppression of mdm2 expression.

Abstract 1996: Anticancer efficacy and mechanism of action of novel ginsenoside 25-OCH3-PPD, a natural product MDM2 inhibitor

Abstract Ginseng and related herbs have been used for various health benefits in over 2000 years. However, their mechanisms of action are not fully understood. In our laboratory, we have been isolating and characterizing ginsenosides as anticancer agents. 20(S)-25-methoxyl-dammarane-3α, 12α, 20-triol (25-OCH3-PPD), a newly identified natural product from Panax notoginseng, has shown activities against a variety of cancer cells in vitro and in vivo. However, the mechanisms of action for the novel anticancer agent are largely unknown. In this study, we discovered a novel mode of action for 25-OCH3-PPD: direct inhibition of the MDM2 oncoprotein. It is well established that the MDM2 oncogene plays a major role in human cancer development and progression. Its tumorigenic properties are associated with both p53-dependent and -independent pathways. We first demonstrated that, in a dose-dependent manner, 25-OCH3-PPD inhibited MDM2 expression in human breast cancer cell lines with different p53 backgrounds, including MCF-7 (p53 wild type and p53 knockout) and MDA-MB-468 (p53 mutate) cells. We further found that 25-OCH3-PPD inhibited MDM2 expression at the transcriptional level, affecting MDM2 promoter activity. We also observed that 25-OCH3-PPD decreased the survival, inhibited proliferation, induced apoptosis and led to G1 cell cycle arrest in the test breast cancer cells. The anti-cancer activities of 25-OCH3-PPD were reduced in MCF7 inducible MDM2-overexpression cells, further supporting the relevance of the MDM2 inhibitory effect to its anti-cancer activity. Moreover, 25-OCH3-PPD inhibited the growth of breast cancer xenograft tumors in vivo which was associated with its MDM2 inhibitory effects. 25-OCH3-PPD demonstrated low toxicity to non-cancer cells, and did not have any observable toxicity in animals. In summary, our preclinical data indicate that 25-OCH3-PPD is a potential therapeutic agent and that further preclinical and clinical development of this agent is warranted. These results also provide an example of a natural product/oncogene interaction as a novel mechanism of action for chemotherapeutic agents. (This work was supported by NIH/NCI grants R01 CA112029 and R01 CA121211 and a grant (BCTR070731) from Susan G Komen for the Cure.) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1996. doi:1538-7445.AM2012-1996

Gambogic acid mediates apoptosis as a p53 inducer through down-regulation of mdm2 in wild-type p53-expressing cancer cells

Gambogic acid (GA) is a natural product with potent apoptotic activity. Here, we showed that GA broadly inhibited the growth of cancer cells that expressed wild-type p53 as determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazol-iumbromide assay, (3)H-thymidine incorporation analysis, and an in vivo mouse xenograft model. GA induced massive cell apoptosis as judged by Annexin V and propidium iodide dual-staining experiments. Furthermore, we found that GA partially induced cancer cell growth inhibition in a p53-dependent manner because cell survival could be restored after endogenous p53 was attenuated by p53 transcriptional repressor pifithrin-alpha or p53 small interfering RNA. Interestingly, GA had no influence on p53 mRNA synthesis but dramatically enhanced its protein expression. This unique observation could be accounted for by the down-regulation of mdm2 at both mRNA and protein levels. It is concluded that GA enhances p53 protein level through inhibition of mdm2 expression and thereby hampers p53 harboring tumor growth.

P53 mediates the negative regulation of MDM2 by orphan receptor TR3

MDM2 is an oncoprotein whose transforming potential is activated by overexpression. The expression level of MDM2 is negatively regulated by orphan receptor TR3 that mainly acts as a transcriptional factor to regulate gene expression. However, the underlying mechanism is largely unclear. Here, we present the first evidence that inhibition of TR3 on MDM2 is mediated by p53. We found that TR3 directly interacts with p53 but not MDM2, and such interaction is critical for TR3 to inhibit MDM2 expression. TR3 downregulates p53 transcriptional activity by blocking its acetylation, leading to a decrease on the transcription level of MDM2. Furthermore, TR3 binding to p53 obstructs its ubiquitination and degradation induced by MDM2, resulting in the MDM2 ubiquitination and degradation. In addition, TR3 could enhance p53-mediated apoptosis induced by UV irradiation. Taken together, our findings demonstrate that p53 mediates the suppression of TR3 on MDM2 at both transcriptional and post-transcriptional level and suggest TR3 as a potential target to develop new anticancer agents that restrict MDM2-induced tumor progression.