Pharmaceutical composition for inhibition of cancer cell metastasis including siRNA for ribosomal protein S3

Abstract

The present invention relates to a pharmaceutical composition for inhibiting cancer cell metastasis, the pharmaceutical composition including at least one of: (1) si-rpS3/203 binding to a 203.sup.rd base sequence of rpS3 mRNA; (2) si-rpS3/635 binding to a base sequence corresponding to a position 635 of the rpS3 mRNA; (3) si-rpS3/747 binding to a base sequence corresponding to a position 747 of the rpS3 mRNA; and (4) si-rpS3/766 binding to a base sequence corresponding to a position 766 of the rpS3 mRNA.

Claims

1. A pharmaceutical composition for inhibiting cancer cell metastasis, the pharmaceutical composition comprising at least one of: si-rpS3/203 binding to a 203.sup.rd base sequence of rpS3 mRNA, wherein the si-rpS3/203 is represented by a base sequence of SEQ ID NO: 1.

2. A pharmaceutical composition for inhibiting cancer cell metastasis, the pharmaceutical composition comprising si-rpS3/635 binding to a 635.sup.th base sequence of rpS3 mRNA, wherein the si-rpS3/635 is represented by a base sequence of SEQ ID NO: 2.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 is an image showing the result in which the protein expression is specifically inhibited in the rpS3 by prepared si-rpS3s in a human fibrosarcoma cell line and a human skin melanoma cell line.

(2) FIGS. 2A to 2C show images representing changing aspects of a malignancy pattern of a cancer cell line by prepared si-rpS3s.

DETAILED DESCRIPTION OF THE INVENTION

(3) Hereinafter, the present invention will be described in more detail with reference to examples and drawings. It will be obvious that the purpose of the following examples is just to specifically describe the present invention in detail and the examples are not to be construed to limit the scope of the present invention.

Example 1: Protein Modification Through Glycosylation of 165th Glycine Amino Acid Related to Secretion of rpS3

(4) An LC-MS/MS scheme was performed by concentrating only the rpS3 protein secreted by an immunoprecipitation scheme through an antibody against the rpS3 protein from a culture medium of a cancer cell line cultured by the research team of the present invention.

(5) It was confirmed that the rpS3 protein secreted through the performed scheme is subject to the protein modification through a glycosylation process occurred at the 165.sup.th asparagine amino acid.

Example 2: Preparation of Si-rpS3/203, 635, 747, and 766 (SEQ ID Nos: 1-4, Respectively) which are Si-RNAs for Reducing the Expression of rpS3 Protein Expressed in Cancer Cell Line

(6) In order to regulate the expression of the rpS3 protein in the cancer cell line at the mRNA level, si-RNAs (Si-rpS3/203 and si-rpS3/635) each constituting 21 nucleotides starting from the 203.sup.th and 635.sup.th nucleotides of the rpS3 mRNA were prepared, respectively, through a sequence analysis.

(7) Particularly, the prepared si-RNAs are open reading frames (ORFs) which are portions included in the translation into proteins of the rpS3 mRNA, and Si-rpS3/747 and si-rpS3/766 starting from the 747.sup.th and 766.sup.th nucleotides are portions included in an untranslated region (UTR).

(8) Each sequence is shown in Table 1.

(9) TABLE-US-00001 Sequence Identifier si-RNA sequence Started matching site si-rpS3/203 SEQ ID NO: 1 aac tga ctg ctg tag ttc aga 203.sup.rd nucleotide of the ORF si-rpS3/635 SEQ ID NO: 2 aac cca aag atg aga tac tgc 635.sup.th nucleotide of the ORF si-rpS3/747 SEQ ID NO: 3 cag ctg tat tct gga gtc t 747.sup.th nucleotide of the UTR si-rpS3/766 SEQ ID NO: 4 gga tgt tgc tct cta aag a 766.sup.th nucleotide of the UTR

Experimental Example 1: Implementation of Regulating the Protein Expression by Using Si-rpS3/203, 635, 747, and 766 which are Si-RNAs of the rpS3 Protein

(10) In order to confirm whether the si-rpS3/203, 635, 747 and 766, SEQ ID NO. 1, 2, 3, and 4, respectively, which are the si-RNAs obtained in Example 2 can regulate the expression of the rpS3 protein, 50 pmol of the si-RNA was injected into the human fibrosarcoma cell line (HT1080) and the human skin melanoma cell line (WM115) and then cultured for 48 hours.

(11) Then, the cells were inserted into Tris-NaCl-NP40 buffer (TNN buffer) containing protease inhibitors, and dissolved through a freeze-thawing scheme.

(12) The cell lysate obtained through the above process was quantified to prepare total cell protein of 20 g, an electrophoresis was performed in 12% SDS acrylamide gel, and the cell lysate was moved to a nitrocellulose membrane.

(13) The total cell protein rooted on the nitrocellulose membrane was subject to a blocking reaction for one hour using a blocking solution formed of dried skim milk, and reacted with a solution, in which a polyclonal rpS3 antibody was diluted at a concentration of 0.2 g/mL, for one hour at the room temperature.

(14) Those samples were treated with horseradish peroxidase conjugated goat anti-rabbit and anti-mouse IgG serum for one hour at the room temperature, treated with chemiluminescence substrate (Roche Diagnostics cat. #1 501 399), and sensitized to an X-ray film (FIG. 1).

(15) According to the experimental result, the si-rpS3/203, 635, 747 and 766, which are the si-RNAs for the rpS3 protein prepared in the present invention, can specifically inhibit the protein expression in the rpS3 in the human fibrosarcoma cell line (HT1080) and the human skin melanoma cell line (WM115).

Experimental Example 2: Examination of Changing Aspects of Malignancy Patterns of the Cancer Cell Lines by Using Si-rpS3/203, 635, 747, and 766 of the Present Invention

(16) As confirmed in Experimental Example 1, in order to examine the changing aspects of malignancy patterns of the cancer cell lines by using the Si-rpS3/203, 635, 747, and 766, (SEQ ID NO. 1, 2, 3, and 4 respectively) which are the si-RNAs for the rpS3 protein prepared in Example 2, the protein expression is inhibited by modulating mRNA levels, and then cell shapes of the human fibroblastoma cell line (HT1080) and the human skin melanoma cell line (WM115) were confirmed using a 3D culturing scheme.

(17) The cells were pretreated with 300 L of matrigel in a culture container having 24 culture grooves, cultured and solidified for 15 minutes at the temperature of 37 C. Then, 600 L of a culture medium containing 2% matrigel together with 0.110.sup.4 cells is added and cultured for seven days. Then, shape changes of the cells in the culture vessel were observed through a microscope.

(18) A surface of a cancer cell which can be metastasized protrudes, however, a surface of a normal cell which cannot be metastasized has a rounded shape.

(19) Referring to FIG. 2A, it was confirmed that, in both of the human fibrosarcoma cell line (HT1080) and the human skin melanoma cell line (WM115), the cells were changed into rounded shapes which are the shapes at a normal cell level by the si-rpS3/203, 635, 747, and 766 (SEQ ID NO. 1, 2, 3, and 4, respectively) where the expression of the rpS3 protein is regulated at the mRNA level.

(20) Referring to FIG. 2B, in the case of a ribosomal protein rpS6 and a RACK1 protein, any change of the cell shape the same as that of rpS3 by the si-RNA was not observed under the same experimental conditions.

(21) Referring to FIG. 2C, it was confirmed that the migration of the cell was remarkably lowered in an experimental group where mutant rpS3 proteins were expressed from the inspection of the migration of the cell (HT1080) in which a condition (rpS3-WT) of the expression of an original rpS3 is compared with a condition (RpS3-N165G) that a rpS3 plasmid DNA obtained by mutating the 165.sup.th amino acid serving as an important portion in the glycosylation process related to the secretion of the rpS3 protein is inserted and expressed.

(22) The present invention has been described in detail according to the present invention, and it will be apparent to those skilled in the art that the specific description is only for a preferred embodiment and is not limited to the scope of the present invention. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.