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Drug for inhibiting proliferation of tumor cells and application thereof

A tumor cell proliferation and tumor cell technology, which is used in anti-tumor drugs, drug combinations, pharmaceutical formulations, etc.

Inactive Publication Date: 2012-07-04
INST OF RADIATION MEDICINE ACAD OF MILITARY MEDICAL SCI OF THE PLA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] So far, there is no report that RPL26 directly regulates MDM2 and inhibits tumor cell proliferation by affecting the activity of the tumor suppressor molecule p53

Method used

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  • Drug for inhibiting proliferation of tumor cells and application thereof
  • Drug for inhibiting proliferation of tumor cells and application thereof
  • Drug for inhibiting proliferation of tumor cells and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Embodiment 1, the construction of RPL26 expression vector

[0027] According to the cDNA sequence of RPL26 (GenBank Accession No.M_000987), design the forward primer ML26-1: 5'-gtacGTCGACCatgaagtttaatccctttg-3' (the capital letter is the Sal I restriction site) and the reverse primer ML26-6: 5'- tcagGCGGCCGCttcctgcatcttctcaatg-3' (the uppercase letter is the Not I restriction site), and the adult liver cDNA library (purchased from Invitrogen) was used as a template to amplify the coding gene of RPL26 by PCR. The PCR amplified product and the pCMV-Myc (purchased from Clontech Company) vector were digested with Sal I and Not I respectively and ligated, and the ligated product was transferred into E.coli JM109 competent cells, and ampicillin resistance was used to screen positive Cloning, extracting plasmids for enzyme digestion identification, sequencing the clones with correct enzyme digestion identification results, and naming the obtained recombinant expression vector ...

Embodiment 2

[0032] Embodiment 2, the regulation of RPL26 to MDM2-p53 pathway

[0033] 1. The effect of RPL26 on the transcriptional activation activity of wild-type p53

[0034] The plasmid pGL13-Luc (Yu J, Zhang L, Hwang P M, Rago C, Kinzler K W and Vogelstein B. (1999). Identification and classification of p53-regulated genes. Proc NatlAcad Sci US A 96: 14517-14522.), pRL- CMV (purchased from Promega), pCMV-MDM2 (Zhang Y, Wolf G W, Bhat K, Jin A, Allio T, Burkhart W A et al. (2003). Ribosomalprotein L 11 negatively regulates oncoprotein MDM2 and mediates a p53-dependent ribosomal- stress checkpoint pathway.Mol Cell Biol 23:8902-8912.) and the plasmid pMyc-RPL26 constructed in Example 1 above, according to figure 1 The indicated mixes were transfected with osteosarcoma cells (U2-OS cells) containing wild-type p53 (purchased from American Standard Biological Collection). figure 1 Among them, + indicates that the substance is added, - indicates that the substance is not added, 1× indicat...

Embodiment 3

[0050] Example 3, the mechanism of RPL26 affecting the MDM2-p53 pathway

[0051] 1. The effect of RPL26 on the ubiquitination of MDM2 and p53

[0052] The above plasmids pFlag-p53, pCMV-MDM2 and pHA-Ub (Zhang Y, Wolf G W, Bhat K, Jin A, Allio T, Burkhart W A et al. (2003). Ribosomal protein L 11 negatively regulates on coprotein MDM2 and mediates a p53 -dependent ribosomal-stress checkpoint pathway. Mol Cell Biol 23: 8902-8912.) and the plasmid pMyc-RPL26 constructed in Example 1 above, according to Figure 5 Mix as indicated and transfect H1299 cells. Figure 5 In , + means to add the substance, - means not to add the substance. In 25cm by Lipofectamine2000 transfection method 2 / 50ml culture flask for transfection, and each group of plasmids transfected 3 flasks.

[0053] After 24 hours of transfection, replace with fresh medium containing 20 μM proteasome inhibitor MG132 (purchased from Sigma Company) and continue to culture for 8 hours, aspirate the medium, wash the ce...

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Abstract

The invention discloses a polypeptide for inhibiting proliferation of tumor cells, which is of a fragment with maximum length of 145 amino acid residues at 46-100 position from N-terminal, comprising ribosomal protein RPL26. The reporter gene experiments, Western blotting, co-immunoprecipitation and cell proliferation detection experiments confirm that RPL26 as well as MDM2 and p53 form a complex, and the combination of RPL26 and MDM2 can obviously reduce the ubiquitin ligase activity of MDM2, thereby improving the stability and transcriptional activation of p53. The overexpression of RPL26 can obviously inhibit the proliferation of the tumor cells, and the function is related to the activity of p53. RPL26 can be assisted for the clinical treatment of cancer and has great significance.

Description

technical field [0001] The invention relates to a medicine for inhibiting tumor cell proliferation and its application. Background technique [0002] In normal mammalian cells, the tumor suppressor protein p53 is mainly maintained at a low level by ubiquitin ligase E3 and 26S proteasomal degradation pathway. When cells face stress signals such as oncogene activation and DNA damage, the degradation of the tumor suppressor protein p53 will be inhibited, the amount of p53 will increase rapidly, the transcriptional activation function will be activated, and the activation of downstream target genes will be induced, thereby causing cell cycle arrest. Stress responses such as stagnation, apoptosis, or DNA damage repair. Studies have shown that the change of p53 protein level has a great relationship with its function. Therefore, the p53 ubiquitination degradation pathway is very important for the regulation of p53 function. [0003] MDM2 is an important ubiquitin ligase E3 resp...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/47A61K38/17A61P35/00
Inventor 贺福初杨晓明张莹王建
Owner INST OF RADIATION MEDICINE ACAD OF MILITARY MEDICAL SCI OF THE PLA
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