| Objectives:ATRA is applied in clinical use to induce the differentiation of tumor cells, especially leukemia cells. However, the RA resistance restricts its further application in clinical treatment. Nutlins are the inhibitor of the interaction between p53 and its E3 ligase-MDM2. This research will focus on whether Nutlin-1 could enhance ATRA's ability to induce the differentiation of leukemia cells and the mechanisms involved will be studied. Also we will make some primary research on the role of Nulin-1's target-MDM2 in ATRA - induced differentiation. Here we hope to find some new ways to conquer the RA resistance and also elucidate some novel function of MDM2.Methods:Three leukemia cell lines:HL60 (p53 null), NB4 (p53 mutant), U937 (p53 wild type) were used to test whether Nutlin-1 could strengthen the differentiation-inducing activity of ATRA. (1) MTT assay was used to detect the cytotoxicity of Nutlin-1 and ATRA at high concentrations. (2) PI staining followed by flow cytometry determined the apoptosis rate. (3) The level of cell differentiation after the treatment with ATRA alone or in combination with Nutlin-1 was tested through detection of CD11b expression and NBT reduction assay. (4) p53 was silenced in U937 cells through siRNA technique. (5) RT-PCR and Western Blot were used to detect the mRNA level of MDR1, c-myc, CEBP/βand the protein levels of p-gp, RARa. (6) The efflux of the p-gp substrates JC1 and Rhodamine123 by cells was observed through flow cytometry. (7) p-gp ATPase activity of Nutlin-1 was determined using the p-gp-GloTM assay systems. Molecular docking model showed the interaction between p-gp and Nutlin-1.Studying the role of MDM2 in ATRA-induced differentiation of solid tumors: neuroblastoma CHP126 cells and osteosarcoma U2OS cells. (1) Following Indicators were used to detect the differentiation of CHP126 cells:the morphological changes and formation of neurite were photographed by microscopy; the relative length of the neuritis was calculated by Image Pro 5.0 software; Western Blot was used to detect the expression levels of molecular marker MAP2 andβ3-tubulin. (2) Following indicators were used to detect differentiation of U2OS cells:the inhibition of cell growth by ATRA was determined by counting cells; RT-PCR and Western Blot were used to detect the expression of molecular marker ALP, RUNX2 and OPN. (3) p53 and MDM2 were silenced through shRNA and siRNA techniques respectively. (4) Cells were transfected with the plasmid PCMV-myc3-MDM2 to have MDM2 over-expressed. Then the plasmid stably-expressed CHP126 cells and U20S cells were built up through screening the monoclonal population by the treatment with G418. (5) RT-PCR and Western Blot were used to detect the mRNA and protein levels of MDM2.Results:Only in U937 cells which had wild-type p53, Nutlin-1 exhibited cytotoxicity shown by MTT assay. Also Nutlin-1 could induce apoptosis of U937 cells, but not in HL60 or NB4 cells. The result of detecting CD11b expression showed that when HL60 cells were treated with ATRA alone, the differentiation rate was only 15.57%, but when ATRA was combined with 5μM,10μM,20μM Nutlin-1, the differentiation rates increased to 19.04%,21.83%,30.16%. In NB4 cells, this combination effect was much more obvious. The result of NBT reduction assay demonstrated that in HL60, NB4, U937 cells, ATRA could respectively induce 12.68%,46.20%,31.00% reduction rates, but when ATRA was combined with Nutlin-1, the rates changed to 20.08%,83.06%, 31.94%. Meanwhile, after we silenced p53 in U937 cells, Nutlin-1 was also unable to strengthen the differentiation-inducing activity of ATRA. When HL60 and NB4 cells were treated with ATRA for 1 to 3 days, both the protein level of p-gp and the mRNA level of MDR1 increased, which could not be observed in U937 cells. Then we found that pretreatment with ATRA for 3 days could result in increased efflux of JC1 and Rhodaminel23, which were the substrates of p-gp. At the same time, we tested the cytotoxicity of ATRA (0-50μM) in NB4 cells through MTT assay. The result showed that pretreatment with 0.01μM ATRA could reduce the sensitivity of NB4 cells to ATRA at high-concentrations, but when ATRA was combined with Verapamil, the reduction of ATRA's cytotoxicity could be reversed. In the p-gp ATPase activity assay, Nutlin-1 induced this activity in a concentration-dependent manner, demonstrating that Nutlin-1 was the possible substrate of p-gp. Also the molecular docking model exhibited the interaction between Nultin-la or Nutlin-1 b with p-gp. The results of RT-PCR showed that compared with ATRA alone, ATRA combined with Nutlin-1 could inhibit the mRNA expression of c-myc, or induce the expression of CEBP/βmuch more greatly. Also the decrease of the protein level of RARa induced by ATRA was more intense when the HL60 cells and NB4 cells were treated with ATRA and Nutlin-1.When CHP126 and U2OS cells were treated with ATRA for 1 to 3 days, the protein level of MDM2 was greatly reduced while its mRNA level remained almost the same. When MDM2 was silenced in CHP126 cells, treatment with ATRA for 5 days could induce more neurite formation and the expression level of molecular differentiation marker MAP2 was much higher. Also when U2OS cells were treated with ATRA for 7 days, silencing MDM2 could strengthen the expression of differentiation marker ALP, RUNX2 and OPN induced by ATRA while the inhibition of cell growth was more intense. In MDM2 over-expressed cells, the differentiation-inducing activity of ATRA was inhibited shown by various differentiation indicators. In the process of ATRA treatment, p53 protein levels didn't change greatly in both of the two cell lines. After p53 was silenced in U2OS cells, ATRA could still induce more expression of RUNX2 and OPN when MDM2 was silenced subsequently, which indicated that maybe p53 was not involved in MDM2's role in ATRA-induced differentiation.Conclusions:The small molecule inhibitor of MDM2, Nutlin-1 could strengthen differentiation-inducing activity of ATRA. However, this combination effect was less related with p53 than p-gp which was involved in ATRA resistance. As the target of Nutlin-1, MDM2 played a negative role in ATRA-induced CHP126 and U2OS cells differentiation, and this role was probably independent of p53. Here we showed for the first time that Nutlin-1 was the substrate of p-gp, interacting with the protein and inhibiting the efflux of ATRA by p-gp. |
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