KR20120106286A - Pharmaceutical composition for treating aging-related diseases comprising inhibitor of progerin and screening method thereof - Google Patents

Abstract

PURPOSE: A pharmaceutical composition containing a progerin expression inhibitor is provided to effectively treat or prevent disease caused by progerin overexpression. CONSTITUTION: A pharmaceutical composition for treating aging-related diseases contains a progerin expression inhibitor as an active ingredient. The inhibitor is pVHP(Hippel-Lindau tumor suppressor protein)-progerin binding promoter; or RNA interfering-mediated RNA of antisense RNA, RNAi, shRNA, or siRNA. A method for screening a therapeutic agent for treating aging-related diseases comprises: a step of cultuirng pVHP, progerin, and candidate drugs; and a step of selecting analyzing progerin expression level.

Description

Pharmaceutical composition for the treatment of aging-related diseases comprising a progerin expression inhibitor as an active ingredient and screening method of the progerin expression inhibitor comprising progerin and screening method

The present invention relates to a pharmaceutical composition for the treatment of aging-related diseases containing a progerin expression inhibitor, and a method for screening the progerin expression inhibitor.

Renal cell carcinoma (RCC) is a malignant tumor commonly occurring in the genitourinary system and exhibits an irregular nucleus shape, which is used as an indicator of RCC grade. In addition, p53 inactivation and radiation-resistance without genetic mutations are known features of RCC. On the other hand, pVHP (Hippel-Lindau tumor suppressor protein) frequently occurs in the early stages of RCC. However, the molecular mechanisms for such polymorphic nuclei, p53 inactivation and radiation resistance are not yet known.

Cancer is well known as an age related disease and also a genetic disease. There have been studies of why cancers are more susceptible to cancer in aged individuals, and have generally been described as being caused by multistage tumorigenesis. To reach a malignant tumor, normal cells must accumulate many genetic variations. Thus, cancer formation requires a long time. However, considering familial cancer syndromes such as Li-Fraumeni syndrome, Von Hippel Lindau syndrome and familial adenomatosis colis, tumor formation is long It does not require periods and proceeds quickly under certain conditions. In this regard, there has recently been an interesting report that the physiological function of the strong tumor suppressor, p53, is reduced during aging. Since p53 is a gatekeeper tumor suppressor, its reduced function can promote the development of cancer. Thus, aging-related changes in cellular inclusions or gene expression profiles that can inhibit p53 function will be an important clue to understanding cancer development in aging individuals.

RCC is a well-known age-related cancer, the incidence of which is apparently increased during the aging process, although not high in young individuals. RCC also exhibits nuclear membrane irregularity and resistance to IR-treatment. However, the genetic variation of p53, which has been proposed as a cause for IR-resistance, is very low. This feature means that there is a new mechanism that inhibits p53 function in RCC.

And meaningful genetic events of RCC are common mutations in pVHL. Although pVHL has been cloned from human cancer syndrome Von Hippel Lindau syndrome, its genetic variation is known to reach 70% in renal cell carcinoma. As an E3 ligase, pVHL degrades HIF-1a and blocks transcriptional activity. Since HIF-1a induces VEGF, EPO and other pro-angiogenic factors in response to hypoxia, activation of HIF-1a will be important for cancer progression, particularly tumor-angiogenesis progression. However, in many types of solid cancers, HIF-1a is stabilized and activated through the formation of hypoxic conditions that can form in the internal cell mass of tumors, and pVHL deficiency or loss of function does not appear to be essential for early cancer development. In addition, angiogenesis is necessary in the late stages of cancer and the histological features of the kidney have many well-formed blood vessels. Thus, pVHL loss is not critical for tumor formation in the early stages of RCC to achieve angiogenesis. pVHL deficiency is not detected in other types of invasive cancers. Thus, this feature suggests that a new tumor suppressor role of pVHL exists and is associated with RCC-specific function.

The incidence of RCC increases dramatically during the aging process, p53 function is reduced without genetic mutation, and pVHL is frequently mutated at an early stage, and we consider that loss of pVHL is associated with aging-related gene expression. Related to this, starting from the hypothesis that it can inhibit p53 function. To test this hypothesis, we focused on the nuclear membrane irregularities of RCC, similar to nuclear degeneration in the Hutchinson-Gilford Syndrome (HGPS), which promotes aging and death at a young age. The gene, progerin, was found to be expressed in senescent cells.

Accordingly, the present inventors have found a correlation between the characteristics of RCC and elevated expression of progerin. As a result, nuclear membrane irregularity of RCC was improved by elimination of progerin, and p53 reactivity to DNA damage was restored by progerin elimination. Elevated expression of progerin and inactivation of p53 were due to pVHL dysfunction. In addition, pVHL interacts with progerin and blocks progerin-induced p14 / ARF inactivation, while progerin removes p14, resulting in inactivation of p53 and nuclear membrane irregularity, progerin expression in human leukemia Since it was detected in samples and supercultured cell lines derived therefrom, it was found that progerin expression plays an important role in the progression of cancer, especially in aged individuals.

An object of the present invention to provide a pharmaceutical composition for the treatment of aging-related diseases containing a progerin expression inhibitor as an active ingredient.

Another object of the present invention is to provide a method for screening a drug for treating aging-related diseases, comprising selecting a candidate drug that inhibits progerin expression.

In order to achieve the above object, the present invention provides a pharmaceutical composition for the treatment of aging-related diseases containing a progerin expression inhibitor as an active ingredient.

The progerin expression inhibitor may include a binding promoter between Hippel-Lindau tumor suppressor protein (pVHP) and progerin; Or RNA that mediates RNA interference for progerin expression selected from the group consisting of antisense RNA, interfering RNA, short hairpin RNA (shRNA) and short interfering RNA (siRNA).

Preferably, the progerin expression inhibitor may block the inactivation of p53 by inhibiting binding between progerin and p14 by promoting binding between Hippel-Lindau tumor suppressor protein (pVHP) and progerin.

The aging-related disease may be a disease selected from the group consisting of cancer disease and premature ejaculation, the cancer disease may be a cancer disease selected from renal cancer, leukemia or prostate cancer, and the premature ejaculation is selected from Warner syndrome or Hutchinson Gilford syndrome. It may be mania.

The present inventors have found that the expression of progerin is increased in aging-related diseases such as RCC, and reducing the increased expression of progerin improves nuclear membrane irregularity, which is a characteristic of RCC, and the reactivity of p53 due to DNA damage to progerin removal. It was confirmed that it is restored again by. pVHL interacted with progerin to block progerin-induced p14 / AFR inactivation, thereby activating p53 to enhance tumor suppression. Progerin expression was also detected in human leukemia samples and primary cultured cell lines derived therefrom. Thus, it has been found that expression of progerin plays an important role in cancer progression, particularly in cancer progression in aged individuals.

Therefore, substances that inhibit the expression of progerin, particularly those that promote the binding of pVHL and progerin, may be used to treat diseases caused by progerin overexpression, such as aging-related diseases, such as RCC, leukemia, prostate cancer, premature ejaculation, etc. It can be a promising medicinal target for.

The pharmaceutical composition according to the present invention may further comprise a suitable carrier, excipient or diluent commonly used in the preparation of the pharmaceutical composition.

Carriers, excipients or diluents usable in the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, Methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, mineral oil, and the like.

The pharmaceutical composition can be used in the form of powder, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols and the like, oral formulations, suppositories, and sterile injectable solutions, respectively, according to conventional methods.

In the case of formulation, a diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, or a surfactant is usually used. Solid form preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and the solid form preparations include at least one excipient such as starch, calcium carbonate, sucrose ( Prepare by mixing sucrose or lactose, gelatin, etc.

In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Oral liquid preparations include suspensions, solvents, emulsions, and syrups, and may include various excipients, such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin. .

Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Examples of the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used.

The amount of active ingredient in the pharmaceutical composition may vary depending on the age, sex, weight, route of administration, degree of disease, type of disease, etc. of the patient, and may be administered once to several times daily. Thus, such dosages do not limit the scope of the invention in any aspect.

The pharmaceutical composition may be administered to various mammals such as mice, mice, livestock, humans, and the like. All modes of administration can be expected, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intrauterine dural or intracerebroventricular injection.

The present invention also provides a method for screening a drug for treating aging-related diseases, comprising selecting a candidate drug that inhibits progerin expression.

More preferably, the screening method comprises the steps of culturing a Hippel-Lindau tumor suppressor protein (pVHP), progerin and candidate drugs; And analyzing candidate progerin expression levels in the culture to promote binding between pVHP (Hippel-Lindau tumor suppressor protein) -progerin to select candidate drugs that inhibit the binding between progerin and p14. .

The aging-related diseases include cancer diseases selected from the group consisting of kidney cancer, leukemia and prostate cancer; Or premature ejaculation selected from Warner syndrome or Hutchinson Gilford syndrome.

The pharmaceutical composition according to the present invention is effective for the treatment or prevention of diseases caused by progerin overexpression, especially diseases caused in aged individuals such as kidney cancer, leukemia, prostate cancer, schizophrenia, etc. For example, it is possible to specifically select a drug that promotes the binding between pVHL and progerin, thereby effectively developing a therapeutic agent for diseases caused by aging individuals such as kidney cancer, leukemia, prostate cancer, and premature ejaculation.

Figure 1 shows the effect of progerin on the nuclear membrane irregularity of RCC, A is nuclear membrane irregularity in human RCC cell line C2, B is the shape of nuclear degeneration of progerin in RCC, C is transcriptional transcription of progerin in RCC Expression, D is progerin expression at the level of transcription, E is the effect of si-progerin on nuclear membrane irregularity of RCC, F is the effect of si-progerin on nuclear shape of HGPS, respectively.
Figure 2 shows the effect of pVHL on progerin expression, A is the effect of pVHL overexpression on progerin expression, B is the effect of si-VHL on progerin expression, C is in pVHL-transfected C2V cells Progerin analysis, D is the effect of pVHLdml on progerin half-life, E is the effect of pVHL mutation on progerin expression, F is the effect of pVHL on nuclear modification of HGPS cells,
Figure 3 shows the effect of progerin on p53, where A is the effect of si-progerin on p53 expression in caki-2 cells, B is the effect of si-progerin on p53 expression in C2 cells, C FHIT or RKIP effect on p53 in C2 cells, D is si-progerin transfection p53 immunostaining assay in C2 cells, E is si-progerin effect on DNA damaging sensitivity in C2 cells, F is shows the effect of si-progerin on the transcriptional activity of p53, respectively
Figure 4 shows a progerin correlation for p53 induction by pVHL, where A is the effect of pVHL transfection on p53 expression in C2 cells, pVHL on p53 induction in caki-2 cells, C is Effect of pVHL on p53 induction in A549, D on pVHL on p53 induction in HCT116, E on pVHL on p53 induction following si-progerin treatment in caki-2 cells, F on HGPS Si-progerin's effect on p53 induction in, G indicates the effect of pVHL on p53 induction in HGPS, respectively
5 shows the effect of progerin on p14-mediated p53 activation, where A is the sensitivity of p53 in human mania and B is the effect of Neutlin-3 on p53 induction in HGPS cells, C is C2 cells Si-progerin's effect on DNA damage response in, D shows the effect of p41 overexpression on progerin-induced p53 inhibition, E indicates si-p14's effect on p53 induction by si-progerin, respectively ,
Figure 6 shows the effect of pVHL on the direct binding of p14-progerin, A is the effect of pVHL on the interaction between p14 and progerin, B is the interaction between p14-progerin, C is GST-p14 GST-pulldown analysis, D is the effect of progerin on the interaction between p53-p14, E is the effect of progerin on p14 half-life, F is the effect of pVHL on p14 half-life,
7 shows progerin expression in human leukemia samples, where A is progerin expression in leukemia samples, B is progerin expression in established cell lines, C is cell survival rate by si-progerin, and D is si-progerin's effect on p53 expression and DNA damage response, E represents the mechanism of cancer progression with aging, respectively.
Figure 8 is the analysis of the nucleation of RCC, A is DAPI staining analysis in caki-2 (left) and C2 cells, B is the effect of si-progerin on progerin expression, C is si on nuclear shape Shows the effects of progerin,
Figure 9 shows the effect of HIF-1a on the regulation of progerin expression of pVHL, A is nuclear shape in C2 and pVHL-transfected C2V, B is the effect of si-HIF1a on progerin expression, C is pro Effect of HIF-1a overexpression on guerin induction, D represents the effect of pVHL on progerin transcripts, respectively,
10 shows a direct correlation between pVHL and progerin, A represents the interaction between progerin and pVHL, B represents the GST-pulldown assay,
Figure 11 shows the effect of si-progerin on p53 induction, A is immunostaining analysis of p53 in C2 cells, B is the effect of si-progerin on p53 induction in HGPS, C is in C2V cells Shows the si-progerin effect on p53 induction of
Figure 12 shows the effect of progerin on p14 regulation, A is synergistic effect of p14 and si-progerin, B is the effect of p14 on the nucleus shape, respectively.

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, these examples are merely to illustrate the invention, the content of the present invention is not limited by these examples.

≪ Example 1 >

1. Cell Culture and Reagent Preparation

Cell lines used in the present invention were purchased from ATCC and maintained in a growth chamber at 37 ° C. in RPMI-1640 (A549, HCT116) or DMEM (293) containing 10% FBS and 1% antibiotics. Other cell lines It was provided by Jung YJ (Pusan University) and maintained in a growth chamber at 37 ° C. in C2 (UMRC2), C2V (UMRC2V) or DMEM (CAKI2) containing 10% FBS and 1% antibiotic.

Human fibroblasts (AG03141; female 30 years old) from Warner syndrome patients, human fibroblasts (AG01972; female 14 years old) from HGPS patients and human fibroblasts from GM (GM 00038; female 9 years old) from Coriell Cell Repositories Was purchased and maintained in EMEM medium containing 15% FBS, 2 mM Glu (no antibiotics).

Common chemical inhibitors including nocodazole and colsamide were purchased from Calbiochem and p14 / ARF (MS-850-P0) emerin was purchased from Novocastra. In addition, the antibodies used in the present invention, that is, the antibody of progerin (sc-81611), the antibody of Lamin A (sc-20680), the antibody of GST (sc-138), the antibody of GFP (sc-9996), and the VHL Antibody (sc-17780), p53 (DO-1) (sc-126), actin antibody (sc-1616), MDM2 antibody (sc-965) and His antibody (sc8-036) were obtained from Santa Cruz, α-FLAG antibody (F3165) was purchased from sigma, p21 antibody (# 2946) and p-chk2 antibody (# 2661) from Cell Signaling.

2. Vector Preparation and Transfection

GFP-fusion progerin and GFP-fusion lamin A expression vectors were provided from Scaffidi and Misteli (NCI). pVHL mammalian expression vector It was provided by Jung YJ (Pusan University). Myc-ARF vector and p21-luciferase vector were purchased from Addgene. si-progerin and si-p14 were constructed according to the previously known literature (Scaffidi and Misteli, 2005; Voorhoeve and Agami, 2005). For mammalian expression of these vectors, transfection was performed using Jetpei (polyplus). 1.5 μg of vector was mixed with 1.5 μl of Jetpei reagent dissolved in 150 nM NaCl solution. After the reaction mixture was incubated for 15 minutes at room temperature, the reaction mixture was added to the cells. After 3 hours, the medium containing 10% FBS was replaced with serum free medium.

3. Recombinant Protein Preparation and GST-Pulldown Assay

To investigate the protein-protein interactions, 100 amino acids were cloned from the upstream of the terminal codon via PCR to produce recombinant lamin A-C-terminal regions (L-C), progerin-C-terminal regions (Prog). Full length p14 and VHL were produced by PCR and cloned in pGEX vector. Each clone was identified by DNA sequencing. The recombinant protein was purified using a nickel column.

For binding assays, GST-bead-fusion lamin A-C or progerin-C were reacted with 293 cell lysates obtained from p14 / ARF transfected cells for 2 hours at room temperature. GST-p14 or pVHL was then reacted with GFP-Lamine A or progerin-transformed 293 cell lysate for 2 hours at room temperature. After washing twice with PBS and once with RIPA, the precipitates were collected, transferred to SDS-PAGE, and Western blot analysis was performed using anti-p14 and GST.

4. Immunoprecipitation (IP) and Western Blot (WB) Analysis

Whole cell lysates were prepared in RIPA buffer and the lysates were centrifuged at 14,000 rpm for 30 minutes. 20 μg of cell extracts were separated via SDS-polyacrylamide gel electrophoresis and transferred onto PVDF membrane. The membranes were incubated at 4 ° C. for 1 hour to overnight and reacted with the appropriate primary antibody for 1 hour at room temperature followed by reaction with the secondary antibody. Peroxidase activity was detected by chemiluminescence using the ECL kit (Intron) according to the manufacturer's instructions.

To investigate the interaction between pVHL and Lamin A, an antibody against pVHL or Lamin A / C (2 g / sample) was added to the protein extract. After incubation with stirring at 4 ° C. for 2 hours, Protein A and Protein G were added. After washing twice with PBS, the precipitate was dissolved in RIPA buffer and SDS sample buffer.

5. Immunofluorescence (IF) Analysis

To examine the nuclear shape, cells transfected with the defined vector were fixed with 100% methanol at 4 ° C. for 10 minutes. After washing with PBS, the cells were incubated for 1 hour with blocking buffer (PBS + 1% BSA + normal Gout Ab). After washing twice with PBS, incubated with anti-lamin A / C antibody (1: 200) in blocking buffer for 2 hours, followed by anti-rabbit Ab-FITC or anti-rabbit Ab-loader in blocking buffer for 2 hours. Incubated with Min (1: 1000) and mounted. Nuclei were stained with DAPI, and immunofluorescence signals were analyzed by fluorescence microscopy (Zeizz).

6. RNA Isolation and RT-PCR

For RT-PCR, total cellular RNA was extracted using the Qiagen RNA Extraction Kit. After measuring RNA concentration, 1 μg total RNA was reverse transcribed into cDNA using MMLV RT (invitrogen) and random hexamers. RT-PCR was performed using the following specific primers:

Lamin A / C-forward primer: SEQ ID NO: 1 (5'-AAGGAGATGACCTGCTCCATC-3 '), reverse primer: SEQ ID NO: 2 (5'-TTTCTTTGGCTTCAAGCCCCC-3')

GAPDH-forward primer: SEQ ID NO: 3 (5'-ATCTTCCAGGAGCGAGATCCC-3 '), reverse primer: SEQ ID NO: 4 (5'-AGTGAGCTTCCCGTTCAGCTC-3')

7. MTT Analysis

To measure cell viability, cells were transfected with defined vectors or si-RNA for 24 hours. After washing, cells were treated with adriamycin and camptothecin for 2 hours. For MTT analysis, cells were incubated with 0.5 mg / ml MTT solution for 4 hours at 37 ° C. After the excess solution was removed, the precipitate was quantified by dissolving with 200 μl DMSO and measuring absorbance at 540 nm.

8. Luciferase Analysis

For analysis of p21 activity, p21-luc vector was transfected into 293 cells for 24 hours as siRNA. After washing, cells were treated with adriamycin for 2 hours. After washing with promega buffer, cells were lysed with Lysis buffer. Luciferase activity was measured by Luminometer.

9. Human Leukemia Sample Preparation

Blood samples from leukemia patients and normal people were provided by the Pusan National University Medical Center. After collecting the WBCs, the cells were stored at −70 ° C. until use. Diagnosis was carried out in a general procedure. In order to establish a cell line, WBCs were cultured in DMEM (containing 15% FBS) to obtain three kinds of stably grown cells.

10. Experimental Results

1) The nuclear membrane irregularity of RCC is due to increased expression of progerin.

UMRC2 (C2), a human RCC cell line, showed similar nuclear membrane irregularities with HGPS cells (FIGS. 1A and 1B). Similar nuclear membrane irregularities were also observed in the Caki-2 cell line (FIG. 8A).

Since nuclear modification in HGPS cells affects progerin expression, progerin transcripts were examined by RT-PCR in RCC cell line C2 and found that C2 increased progerin expression compared to breast cancer cell line MCF-7. (FIG. 1C) and this progerin expression was observed by Western blot (FIG. 1D).

In order to determine whether progerin is involved in nuclear membrane irregularity of RCC, si-RNA for progerin was constructed and transfected into C2 and Caki2 cells, and nuclear membrane irregularity of RCC cells was improved by removal of progerin (FIG. 1E and 8C). In addition, removal of progerin increased lamin A / C expression (FIGS. 1D and 1E).

In addition, as a result of observing the inhibitory effect of progerin on lamin A / C expression, removal of progerin improved nuclear modification in HGPS cells (FIG. 1F).

From these results, it was confirmed that nuclear modification of HGPS and nuclear membrane irregularity of RCC were caused by increased expression of progerin.

2) pVHL inhibits progerin expression.

Since pVHL is frequently modified in RCC, the correlation between pVHL and nuclear modification was examined. That is, comparing the nuclear shape of C2 with the pVHL-stabilized transfected cell line UMRC2V (C2V), the re-expression of pVHL in C2 (C2V) improved the nuclear shape (FIG. 9A).

To determine how pVHL modulates progerin expression, examining the effects of pVHL on progerin expression, the overexpression of pVHL inhibits progerin expression (FIG. 2A), whereas the overexpression of si-pVHL is pro Guerin expression was increased (FIG. 2B). However, lamin A expression was not affected by pVHL (FIGS. 2A and 2B).

In addition, since pVHL inhibits HIF-1a expression, examining the effect of HIF-1a on progerin expression, HIF-1a overexpression or knockdown did not explicitly change progerin expression (FIG. 9B and FIG. 9C). These results confirmed that pVHL-induced progerin inhibition was due to a pathway independent of HIF-1a.

In order to confirm the effect of pVHL on progerin, progerin expression in C2V compared to C2 was analyzed by Western blot, but the expression of progerin was reduced in pVHL expressing C2V cells (FIG. 2C). Transcripts of were not significantly inhibited by pVHL transfection (FIG. 2D). Thus, pVHL was found to regulate progerin at the post-transcriptional stage.

In addition, since pVHL is known as an E3 ligase, the investigation of the pVHL dependent half-life of progerin through pulse-chase analysis revealed that pVHL explicitly inhibited progerin expression while inhibiting lamin A. Not (FIG. 2D). At this time, the wave tracking analysis was performed by monitoring the rate at which the amount of protein decreases for a certain time after preventing the start of protein using cyclohexamide (CHX).

In order to confirm the role of pVHL on progerin expression, after transfection with mutant pVHL and measuring progerin expression, unlike wild-type pVHL, mutant pVHL with impaired E3-ligase activity suppressed progerin expression. Failed (Figure 2E).

Investigating the effect of pVHL on nuclear modification of HGPS cells via IF staining with Western A / C and Western blot, similar to RCC cells, pVHL overexpression improved nuclear shape (FIG. 2F).

From these results, it can be seen that pVHL inhibits progerin expression through protein turnover enhancement.

3) Progerin inhibits p53 through direct binding between progerin-pVHL.

Investigation of the interaction between pVHL and progerin by IP analysis revealed that the two proteins precipitated with the pVHL (Flag) antibody (FIG. 10A). Although lamin A moved with pVHL, the binding affinity between pVHL-progerin was stronger than the binding affinity between pVHL and lamin A, and GST-pulldown analysis confirmed direct binding between progerin-pVHL. (FIG. 3B).

In addition, one of the important features of RCC is that it is known to inactivate p53 without genetic variation, so we examined the effect on well-known p53 inhibitors of p53 inhibitors, including MDM2, COPi and Parc-1, in C2 cells. Knockdown of this p53 inhibitor did not induce p53 expression (FIG. 10A).

And the correlation between p53 inactivation and progerin expression was examined as follows. First, as a result of investigating p53 expression in HGPS cells, p53 expression was the lowest in HGPS compared to normal fibroblasts (FIG. 10B), but the removal of progerin was able to induce p53 expression (FIG. 10B). In addition, the effects on p53 expression and DNA damage in Caki-2 were confirmed after transfection with si-progerin. Very low p53 expression and low sensitivity to Adr were recovered by si-progerin (FIG. 4A). Similar results could be obtained from C2 cells (FIG. 4B).

FHIT and RKIP are known to be defective in RCC. Investigations of the relevance of p53 regulation in RCC cell lines revealed that although RKIP partially induces p53, the increase in p53 expression is entirely dependent on si-progerin. (Figure 4C). Immunofluorescence staining gave similar results that si-progerin can induce p53 expression (FIG. 4D).

In order to confirm reactivation of p53 in response to progerin knockdown, the transcriptional activity of p53 was monitored in the p21-luc system, indicating that Adr treatment did not increase p21-luc expression in control C2 cells (FIG. 3D). In contrast, si-progerin was able to restore Adr-induced p21-luc activation (FIG. 3E).

As a result of monitoring cell viability through MTT analysis, DNA damage-induced cell death was restored by si-progerin (FIG. 3F).

These results show that elevated expression of progerin may block p53-induced cell death and tumor suppression.

4) pVHL activates p53 progenerally.

Since pVHL blocks progerin expression (FIG. 2), investigating the effect of pVHL on p53 expression in RCC shows that forced expression of pVHL results in not only MDM2 inhibitor Neutlin-3 in C2 and Caki-2 cell lines, The reactivity of p53 to DNA damaging agents was restored (FIGS. 4A and 4B). P53 expression in C2V was higher than C2 cell lines under non-irritating conditions.

Since pVHL is reported to be able to activate p53 through direct binding, the effects of pVHL on the progerin-negative cell lines HCT116 and A549 were examined. No significant induction of p53 or synergistic effects by DNA damaging agents was observed in these cell lines (FIGS. 4C and 4D). These results confirmed that pVHL-mediated p53 induction was obtained by progerin inhibition.

In addition, transfection of si-progerin and pVHL synergistically enhanced p53 expression (FIG. 4E). However, no enhancement of p53 reactivity or p53 induction against DNA damage in C2V was observed by si-progerin (FIG. 11C). These results confirm that p53 inactivation in RCC is due to elevated expression of progerin, which is obtained by pVHL defects.

In addition, examination of p53 expression in si-progerin or pVHL-transfected HGPS cells showed that under both conditions, progerin was commonly decreased and p53 was increased (FIGS. 4F and 4G).

Considering these results, it can be seen that pVHL-mediated p53 activation is obtained through progerin inhibition.

5) Progerin blocks p14-mediated p53 activation.

In order to elucidate the detailed molecular mechanism by which progerin blocks p53 function, the responsiveness of p53 to DNA damage was examined in human progeria cell lines (HGPS and WS). While clearly increased, HGPS and Warner syndrome cells (WS) showed no induction of p53 (FIG. 5A). Warner syndrome cells are known to not respond to DNA damage-induced p53 activation, and it has been observed that WS cells can express progerin. From these results, it can be seen that progerin can block the DNA damage-induced p53 activation pathway.

Since p53 is regulated by the MDM2 pathway, progerin's involvement in MDM2-mediated p53 inhibition is investigated by treatment of Neutlin-3 which better blocks binding between MDM2 and p53 and induces p53 expression. P53 was clearly induced in normal cells (FIG. 4B), but HGPS partially responded (FIG. 5B). In addition, Neutlin-3 clearly did not induce p53 in C2 and Caki-2 cell lines (FIG. 4B). Taken together, these results suggest that elevated expression of progerin is associated with DNA damage-induced p53 activation and MDM2-mediated p53 inhibition.

In addition, since p14 / ARF is an MDM2 inhibitor and is activated by ATM / ATR-dependent DNA damage signals, p14 / ARF is considered to be a strong candidate for missing links in progerin-mediated p53 inhibition and to investigate this, As a result of Western blot analysis of p53 expression after transfection with ARF, DNA damage did not induce p53 expression in C2 cells. However, si-progerin restored this reactivity (FIG. 5C). In addition, si-progerin increased the expression of p14 / ARF (FIG. 5C). In order to confirm this, after expression of p53 in HGPS after transfection with si-progerin and p14, the removal of progerin and overexpression of p14 in these cells induced synergistically p53 expression (FIG. 12A).

In addition, examining the effect of p14 on progerin-induced p53 inactivation, p14 / ARF enhanced the reactivity of p53 in HCT116 and progerin blocked p53 activation (FIG. 5D). However, forced expression of p14 overcomes progerin-induced p53 inhibition (FIG. 5D). In addition, si-progerin-induced p53 activation in Caki-2 was blocked by si-RNA for p14 (FIG. 5E). These results indicate that p14 acts downstream of progerin and progerin blocks p53 activation through inhibition of p14.

In addition, because progerin can induce nuclear transformation, investigating the effect of p14 on nuclear degeneration of HGPS, p14 alone could improve nuclear degeneration even though pVHL and si-progerin reduced nuclear irregularity and degeneration. (FIG. 11B). From these results, it can be seen that the binding between progerin-p14 in p53 regulation is limited, and nuclear degeneration or irregularity results from elevated expression of progerin.

6) pVHL blocks the interaction between progerin and p14.

To determine the mechanism by which progerin blocks p14's function, the GST pulldown assay monitors the interaction between progerin and p14, suggesting that p14 specifically interacts with progerin rather than lamin A. Found (FIG. 6A). To confirm specific interactions between p14 and progerin, NBS-transfected cell lysates were added. However, NBS protein did not interact with prominin as well as lamin A (FIG. 6A). In contrast, addition of pVHL-cell lysates blocked the binding of p14-progerin (FIG. 6B).

In addition, as a result of examining the interaction using GST-p14 in order to avoid the artifact (artifact), GST-p14 showed a specific binding to progerin as in the previous results (Fig. 6C). In addition, pVHL blocked the interaction between GST-p14-progerin. However, pVHL was not associated with the p14-GST protein (FIG. 6C).

These results support the previous results that pVHL binds to progerin and blocks p14, and it can be seen that p14-progerin interaction is interrupted by pVHL. Thus, in pVHL-deficient cells such as RCC, progerin may bind to p14 and cause inactivation of p53. To confirm this, progerin's effect on p14-p53 relevance was examined and the interaction between p53-p14 was inhibited by progerin (FIG. 6D). Considering these results, elevated progerin under pVHL-deficient conditions blocked p14 resulting in inactivation of p53. In addition, protein stability of p14 was measured because the removal of progerin induced p14 expression (FIG. 5C). Wave follow-up analysis confirmed that progerin reduced the half-life of p14 while si-progerin extended the half-life of p14 (FIG. 6E). In addition, prolongation of p14 half-life was observed in pVHL-transfected cells (FIG. 6F). These results indicate that pVHL prolongs the half-life of p14 through progerin inhibition.

7) Expression of progerin is elevated in human leukemia.

Since progerin is able to inhibit p53 through p14 activation, it is estimated that progerin expression will be increased in other types of cancer. To confirm this, the expression of progerin was expressed in 16 leukemia blood samples and 3 normal blood samples. Investigate. According to the literature, leukemia is known to exhibit resistance to chemotherapy and radiotherapy and polymorphic nuclei, with half of the 19 samples expressing progerin (FIG. 7A). However, the normal sample did not show progerin expression and no difference was observed between acute myeloid leukemia and acute lymphocytic leukemia (Table 1).

From these samples, three types of leukemia cell lines (KDJ62, SJW2.2 and SJW 92) were established, and progerin expression was examined, and SJW2.2 and SJW 92 showed progerin expression (FIG. 7B). . Cell viability was examined after progerin removal, and SJW 92 was found to be sensitive to si-progerin. However, KDJ62 did not show a response to si-progerin (FIG. 7C). In addition, p53 activation and p-chk2 DNA damage response were examined using these cell lines. In KDJ 62, si-progerin inhibited p53 activation and p-chk2 in response to adriamycin and Neutlin-3. Did not induce. In contrast, SJW2.2 and SWJ92 showed reactivity to si-progerin (FIG. 7D).

From these results, SJW 92 and SJW2.2 showed progerin-dependent p53 inactivation like RCC. Thus, several types of cancer, including RCC, leukemia, and prostate cancer, block p53-induced tumor suppressor effect through p14-induced p53 activation and progerin overexpression that blocks DNA damage signals (FIG. 7E).

<110> Pusan National University Industry-University Cooperation Foundation <120> Pharmaceutical composition for treating aging-related diseases          comprising inhibitor of progerin and screening method <130> DP-2010-0744 <160> 4 <170> Kopatentin 1.71 <210> 1 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for lamin A / C <400> 1 aaggagatga cctgctccat c 21 <210> 2 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for lamin A / C <400> 2 tttctttggc ttcaagcccc c 21 <210> 3 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for GAPDH <400> 3 atcttccagg agcgagatcc c 21 <210> 4 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for GAPDH <400> 4 agtgagcttc ccgttcagct c 21

Claims (9)

A pharmaceutical composition for the treatment of aging-related diseases containing a progerin expression inhibitor as an active ingredient. The method according to claim 1, wherein the progerin expression inhibitor is a pVHP (Hippel-Lindau tumor suppressor protein) -progerin binding promoter; Or antisense RNA, interfering RNA, short hairpin RNA (shRNA) and short interfering RNA (siRNA) for the treatment of aging-related diseases, characterized in that any one selected from the RNA mediating RNA interference for progerin expression selected from the group consisting of. Pharmaceutic composition. The method according to claim 1 or 2, wherein the progerin expression inhibitor is characterized by blocking the inactivation of p53 by promoting the binding between the ppel HP (Lipu-Lindau tumor suppressor protein) and progerin to inhibit the binding between progerin and p14 Pharmaceutical composition for the treatment of aging-related diseases. The pharmaceutical composition for treatment of an aging-related disease according to claim 1 or 2, wherein the aging-related disease is a disease selected from cancer disease or premature ejaculation. The pharmaceutical composition of claim 4, wherein the cancer disease is selected from the group consisting of kidney cancer, leukemia and prostate cancer. The pharmaceutical composition of claim 4, wherein the premature ejaculation is selected from Warner syndrome or Hutchinson Gilford syndrome. A method for screening a therapeutic agent for aging-related diseases, comprising selecting a candidate drug that inhibits progerin expression. The method of claim 7, wherein the screening method comprises the steps of culturing a Hippel-Lindau tumor suppressor protein (pVHP), progerin and candidate drugs; And analyzing candidate progerin expression levels in the culture to promote binding between pVHP (Hippel-Lindau tumor suppressor protein) -progerin to select candidate drugs that inhibit the binding between progerin and p14. Screening method for treating aging-related diseases. The method of claim 7 or 8, wherein the aging-related disease is a cancer disease selected from the group consisting of kidney cancer, leukemia and prostate cancer; And any disease selected from the group consisting of premature ejaculation selected from Warner syndrome or Hutchinson Gilford syndrome.

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