KR102026142B1 - Anti-Cancer and Anti-Metastasis Composition Comprising CRIF1 Antagonist - Google Patents

Abstract

The present invention relates to a composition effective for inhibiting anticancer and cancer metastasis by inhibiting cell migration, and more particularly, comprising an inhibitor of expression of CRIF1 (CR6 interacting factor 1) gene or an inhibitor of activity of CRIF1 protein as an active ingredient. It relates to a pharmaceutical composition for anticancer and cancer metastasis inhibition.

Description

Anti-Cancer and Anti-Metastasis Composition Comprising CRIF1 Antagonist

The present invention relates to an anticancer and cancer metastasis inhibiting composition excellent in the proliferation of cancer cells and the inhibition of cell migration.

Cancer has the highest mortality rate worldwide and is the second most common cause of death in Western society after cardiovascular disease. By inducing angiogenesis, cancer cells receive nutrients and oxygen to grow indefinitely at a specific site, and move away from the existing growth site and move to a new site for growth by metastasis (metastasis). It is called. Cancer is divided into a benign tumor and a malignant tumor. A benign tumor has a relatively slow growth rate and no metastasis, whereas a malignant tumor invades other tissues and grows rapidly. It is a life threatening and very important cause of death. If the cancer leaves the site of origin and progresses to metastasis, it is usually considered to be at serious stage, so preventing metastasis is very important in the treatment of cancer. In particular, cancer metastasis, which is difficult to treat when cancer recurs, has a poor prognosis and decreases the survival rate of patients, needs to be screened early in patients with a history of cancer, and cancer metastasis can be prevented in advance. The social demand for the method is very high. On the other hand, some tumors are known to prevent recurrence and metastasis by using adjuvant chemotherapy and radiation therapy after surgical treatment, but these therapies also affect normal cells and cause serious side effects.

Cancer cells metastasize to other organs through several stages, such as the formation of neovascularization, infiltration of extracellular matrix, adhesion of tumor cells to extracellular matrix, degradation of extracellular matrix, migration of tumor cells, seeding and homing of tumor cells. do.

Cell migration is an important process essential for animal growth and physiological activity, and occurs mainly in the development of neural and vasculature or in wound healing of epithelial tissue. However, cell migration induces angiogenesis and contributes to tumor metastasis. Korean Patent No. 10-1532307 discloses a composition for inhibiting cancer metastasis containing Quescia Undulata extract that inhibits cell migration and neovascularization.

In relation to cancer metastasis, Akt is one of the key enzymes, Akt acts as an intracellular switch and is activated by phosphorylation of Akt. In particular, Akt phosphorylation is much higher in relapsed and metastasized cancer cells than in primary cancer cells, and inhibition of Akt phosphorylation has been a major target in the study of cancer recurrence and metastasis. Korean Patent No. 10-2012-0112196 reports that Hades protein, which degrades Akt and inhibits Akt activity, inhibits the proliferation and migration of cancer cells. Patent No. 10-1547297 discloses that Akt inhibition causes cell migration. Have been reported to be effective against cancer metastasis.

CRIF1 (CR6 interacting factor 1) is a protein present in mitochondria, and the lack of CRIF1 is known to induce mitochondrial dysfunction and stimulate ROS production. Korean Patent Laid-Open Publication No. 10-2016-0022628 reported that the deficiency of CRIF1 can be effectively used for diagnosis and treatment of vascular diseases related to endothelial dysfunction and vascular inflammation by causing endothelial dysfunction and vascular inflammation. Korean Patent Publication No. 10-2009-0103763 reports that CRIF1 protein has a therapeutic effect on cancer by controlling abnormal replication of DNA through the regulation of geminin through cc-domains present at the C-terminus.

However, it is not known that CRIF1 affects cell migration or that cancer metastasis affects mechanisms involved in the mechanism.

Patent Registration No. 10-1532307 Patent Publication No. 10-2012-0112196 Patent Registration No. 10-1547297 Patent Publication No. 10-2016-0022628 Patent Publication No. 10-2009-0103763

It is an object of the present invention to provide an anticancer and cancer metastasis inhibiting composition capable of inhibiting cancer cell proliferation and inhibiting cancer cell recurrence and metastasis by inhibiting cancer cell migration.

The present invention for achieving the above object relates to a pharmaceutical composition for inhibiting cancer and cancer metastasis comprising an inhibitor of the expression of CRIF1 (CR6 interacting factor 1) gene or an inhibitor of activity of CRIF1 protein as an active ingredient.

When the expression of CRIF1 is inhibited, it inhibits Akt activity by inhibiting Akt phosphorylation, which is known as one of the key enzymes in conventional cancer metastasis. In addition, inhibition of expression of CRIF1 has been shown to inhibit cell migration, which is one of the important stages of cancer metastasis, and inhibition of cell migration is achieved by regulating the expression of Rho GDI2, which is known to regulate cell migration. Such inhibition of Akt activity or inhibition of cell migration is essential for inhibition of cancer metastasis, meaning that an inhibitor of expression of CRIF1 gene or an inhibitor of activity of CRIF1 protein is useful for inhibition of cancer metastasis.

In fact, induction of CRIF1 deficiency by knocking down CRIF1 in breast cancer cell lines, unlike the conventional report that inhibiting the expression of CRIF1 suppresses the proliferation of cancer cells, CRIF1 deficiency inhibits the proliferation of cancer cells I could confirm that. The effect of inhibiting proliferation was more pronounced at lower glucose concentrations, which is a mitochondrial protein that regulates transcription of the OXPHOS complex. Sufficient glucose seems to reduce the effect of inhibiting growth by CRIF1 deficiency.

Deficiency of CRIF1 has also been shown to reduce cell migration in breast cancer cell lines, effectively inhibiting cancer cell metastasis.

The expression inhibitor of the CRIF1 gene may be an antisense oligonucleotide or siRNA that specifically binds to the CRIF1 gene. As used herein, an "antisense nucleotide that specifically binds to the CRIF1 gene" refers to an oligonucleotide that specifically binds to the mRNA of CRIF1 by having a sequence complementary to a specific region of the mRNA of CRIF1 to inhibit the translation of the mRNA into a protein. Means. The antisense nutotide may be composed of DNA, RNA, PNA or derivatives thereof. As used herein, "siRNA" refers to double-stranded RNA that inhibits the production of proteins by cleaving the mRNA of CRIF1. Antisense nucleotides and siRNAs that specifically bind to the CRIF1 gene may be readily designed by those skilled in the art with reference to the mRNA sequences of human CRIF1 known in the art. In the following examples, double-stranded RNA consisting of SEQ ID NO: 1 and SEQ ID NO: 2 was used as siRNA, but is not limited thereto.

Sense: 5'-UGGAGGCCGAAGAACGCGAAUGGUA-3 '(SEQ ID NO: 1)

Antisense: 5'-UACCAUUCGCGUUCUUCGGCCUCCA-3 '(SEQ ID NO: 2)

The activity inhibitor of the protein may be an antibody specific for the CRIF1 protein. The antibody specifically binds to the CRIF1 protein to inhibit the activity of the CRIF1 protein, and may be a polyclonal antibody or a monoclonal antibody. Antibodies to the CRIF1 protein can be prepared by methods conventional in the art.

The pharmaceutical composition may be mixed with itself or with a pharmaceutically acceptable carrier, forming agent, diluent, etc. by a method known in the pharmaceutical field to form a powder, granule, tablet, capsule or injection. Can be prepared and used. The pharmaceutical composition according to the invention may be administered in a pharmaceutically effective amount. In the present invention, the 'pharmaceutically effective amount' means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment. Effective dose levels are well known in the patient's disease type, severity, age, gender, drug activity, drug sensitivity, time of administration, route of administration and rate of release, duration of treatment, factors including concurrent medications, and other medical fields. It can be determined according to the factor. The compositions of the present invention may be administered alone or in combination with other therapeutic agents and may be administered sequentially or simultaneously with conventional therapeutic agents. Considering all of the above factors, it is important to administer an amount that can obtain the maximum effect in a minimum amount without side effects, which can be easily determined by those skilled in the art.

As described above, according to the composition of the present invention, inhibiting metastasis of cancer by inhibiting Akt phosphorylation and cell migration, and inhibiting cancer cell proliferation and cancer cell migration, it may be usefully used as a composition for inhibiting cancer and cancer metastasis. .

1 is an electrophoretic image and quantitative graph showing Akt phosphorylation inhibition by CRIF1 knockdown.
2 is an electrophoretic image and quantitative graph showing increased Rho GDI2 expression by CRIF1 knockdown.
Figure 3 is an image and quantitative graph showing the effect of inhibiting cell migration by CRIF1 knockdown.
4 is a graph showing reduction of CRIF1 expression by CRIF1 knockdown in breast cancer cell lines.
Figure 5 is a graph showing the cell proliferation inhibitory effect by CRIF1 knockdown in breast cancer cell line.
Figure 6 is an image and quantitative graph showing the cell migration inhibitory effect by CRIF1 knockdown in breast cancer cell line.

Hereinafter, the present invention will be described in more detail with reference to the accompanying examples. However, such an embodiment is only an example for easily describing the content and scope of the technical idea of the present invention, whereby the technical scope of the present invention is not limited or changed. It will be apparent to those skilled in the art that various modifications and variations are possible within the scope of the present invention based on these examples.

EXAMPLE

Example 1 Evaluation of the Effect of CRIF1 on the Cell Migration of Vascular Endothelial Cells

1) Knockdown of CRIF1 Gene by Cell Culture and siRNA Infection

Human umbilical vein endothelial cells (HUVECs) were purchased from Clonetics (USA) and incubated in Endothelial Growth Medium-2 (Lonza, USA) at 37 ° C., 5% CO ₂ conditions according to the manufacturer's instructions. Sub-confluent proliferative HUVECs were used at 2-8 passages. HUVECs were infected with siRNA against CRIF1 of SEQ ID NOs 1 and 2 as described above using Lipofectamine 2000 (Invitrogen, USA) according to the manufacturer's manual. Infected cells were incubated for 48 hours at 37 ° C., 5% CO ₂ for knockdown of the CRIF1 gene. The control group was infected with Stealth RNAi Negative Control Duplexes and Medium GC Duplex (Invitrogen) instead of siRNAs of SEQ ID NOs: 1 and 2.

2) Confirmation of Akt Phosphorylation Inhibition by CRIF1 Knockdown

In 1), siRNA-infected cells were cultured with RIPA (10 mM Tris-HCl at pH 8.0, 1 mM EDTA, 140 mM NaCl, 0.1% SDS, 0.1% 200 sodium deoxycholate, and 1% Triton X-100) and protease inhibitor cocktail ( Lysis using Roche). 30 μg of the lysate was dissolved in SDS-PAGE and then transferred to the PVDF membrane to quantify CRIF1, Akt and p-Akt proteins by conventional Western blot methods known in the art. As an antibody for Western blot, Anti-CRIF1, anti-phospho Akt antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA), and Akt antibody was purchased from Cell Signaling Technology (Beverly, MA, USA). Perxosidase conjugated secondary antibody (Santa Cruz) was treated for visualization of the separated proteins, and chemiluminescent signals were developed using Super Fisher West Pico or Femto Substrate from Thermo Fisher Scientific. The density of developed blots and bands was quantified with Gel Doc 2000 Chemi Doc system using Quantity One software (Bio-Rad, Hercules, CA).

Figure 1 is an electrophoretic image and quantitative graph showing the Western blot results show that Akt phosphorylation process is also inhibited as the expression of CRIF1 is reduced by the infection of siRNA.

3) Evaluation of expression level of Rho GDI2 by CRIF1 knockdown

Rho GDI2 is known to regulate cell migration. This was confirmed by Western blot whether the knockdown of CRIF1 influences the expression of Rho GDI2. Western blot was performed in the same manner as 2), and an anti-Rho GDI2 antibody (Santa Cruz Biotechnology) was used as the antibody.

2 is an electrophoretic photograph and a quantitative graph showing the Wessett blot results. 2 shows that CRIF1 can regulate cell mobility by regulating the expression of Rho GDI2.

4) Wound healing assasy

HUVECs were cultured according to the method described in 1) in 6-well plates and infected with siRNA for CRIF1. The cells were then further incubated for 24 hours, wound to a constant width using a 200 μl pipette tip sterilized in the fused cell monolayer, and the debris was washed using PBS. Thereafter, 48 hours of incubation was performed, and wound recovery ability was evaluated by measuring the moving distance of the cells at the wound site using Image J software from the photos before and after the culture.

Figure 3 is a graph quantifying the wound image and the microscope image of the wound recovery evaluation experiment. 3 shows that the wound recovery ability is greatly reduced when CRIF1 is knocked down compared to the control group. From this, when the expression of CRIF1 is suppressed, the mobility of cells is reduced. This is consistent with the increased expression of Rho GDI2, which inhibits cell mobility of 3), suggesting that CRIF1 affects cell mobility by regulating the expression of Rho GDI2.

Example 2 Evaluation of the Effect of CRIF1 on Cancer Cell Proliferation and Cell Migration in Cancer Cells

1) Knockdown of CRIF1 Gene by Cell Culture and siRNA Infection

MCF-7 (ER +, PR +, Her2-) cell lines were purchased from ATCC, and DMEM medium containing 4 mM L-glutamine, 100 ug / ml streptomycin, 100 U / ml penicillin, 10% FBS, 0.1 mM non-essential amino acid was used. Culture at 37 ° C., 5% CO ₂ .

The BT-549 (tri-negative breast cancer) cell line was also purchased from ATCC, using RPMI medium containing 4 mM L-glutamine, 100 ug / ml streptomycin, 100 U / ml penicillin, 10% FBS, and 0.1 mM non-essential amino acid. It was cultured under the condition of ℃, 5% CO ₂ .

Breast cancer cell lines cultured by this method were infected with siRNA against CRIF1 of SEQ ID NOS: 1 and 2 described above using Lipofectamine 2000 (Invitrogen, USA) according to the manufacturer's manual. Infected cells were incubated for 48 hours at 37 ° C., 5% CO ₂ for knockdown of the CRIF1 gene. The control group was infected with Stealth RNAi Negative Control Duplexes and Medium GC Duplex (Invitrogen) instead of siRNAs of SEQ ID NOs: 1 and 2.

Expression of CRIF1 was quantified by Western blot using the cells infected with siRNA according to the method of Example 1 2). 4 shows that the results showed that the infection of siRNA effectively knocked down the CRIF1 gene in both MCF-7 and BT-549 cell lines.

2) Confirmation of cancer cell proliferation inhibitory effect by knockdown of CRIF1 gene

MCF-7 and BT-549 cells were cultured in 6-well plates, respectively, as described in 1) above, followed by knocking down the CRIF1 gene in the same manner. After 48 hours of incubation, 50 μl of the reagent of CCK-8 kit (Dojindo, Japan) was treated, and after 1 hour, the cell proliferation rate was measured by measuring the OD value at 450 nm. Relative proliferation was obtained by setting the cell number of the control group to 100 and the results are shown in FIG. 5. It can be seen from FIG. 5 that the expression of CRIF1 is suppressed in both MCF-7 and BT-549 cell lines, thereby inhibiting the proliferation of cancer cells.

In particular, the low concentration of glucose was more significant effect of inhibiting cancer cell proliferation, because it is considered that the high glucose concentration does not require energy metabolism separately because of sufficient nutrients necessary for cancer cell proliferation.

3) Confirmation of cancer cell migration inhibitory effect by knockdown of CRIF1 gene

The effects of the CRIF1 gene on the migration of cancer cells were evaluated by MCF-7 and BT-549 cell lines in the same manner as in Example 4).

Figure 6 is a graph quantifying the wound recovery capacity and the microscopic image of the wound recovery capacity evaluation experiment showing the results, (A) is MCF-7 cell line, (B) is the result for BT-549 cell line. In FIG. 6, when knocking down CRIF1, the wound recovery ability is greatly reduced compared to the control group. From this, it can be seen that the mobility of cancer cells is also reduced when the expression of CRIF1 is suppressed. Therefore, inhibitors of expression of the CRIF1 gene or inhibitors of the activity of the CRIF1 protein may be usefully used for the inhibition of cancer metastasis.

<110> The Industry & Academic Cooperation in Chungnam National University (IAC) <120> Anti-Cancer and Anti-Metastasis Composition Comprising CRIF1          Antagonist <130> P0318-172 <150> KR 10-2017-0035167 <151> 2017-03-21 <160> 2 <170> KoPatentIn 3.0 <210> 1 <211> 25 <212> RNA <213> Artificial Sequence <220> <223> sense siRNA for CRIF1 <400> 1 uggaggccga agaacgcgaa uggua 25 <210> 2 <211> 25 <212> RNA <213> Artificial Sequence <220> <223> antisense siRNA for CRIF1 <400> 2 uaccauucgc guucuucggc cucca 25

Claims (5)

delete A pharmaceutical composition for inhibiting cancer and metastasis to breast cancer, characterized in that it comprises an antisense nucleotide or siRNA that specifically binds to CRIF1 (CR6 interacting factor 1) gene.
The method of claim 2,
Said siRNA is a pharmaceutical composition for inhibiting cancer and cancer metastasis, characterized in that having the sequence of SEQ ID NO: 1 and 2.
A pharmaceutical composition for inhibiting cancer and metastasis to breast cancer, characterized in that it comprises an antibody specific for CRIF1 (CR6 interacting factor 1) protein. delete

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