METHOD FOR MAINTAINING INCREASED INTRACELLULAR p53 LEVEL, INDUCED BY PLATINUM-BASED ANTICANCER DRUG, AND APPLICATION THEREOF

Abstract

The present invention relates to a method for maintaining the increased intracellular p53 level, induced by a platinum-based anticancer drug, and an application thereof and, more specifically, to a method for maintaining the increased intracellular p53 level in cells by administering a platinum-based anticancer drug and siRNA to ubiquitin ligase for p53 to a subject in need thereof in combination and sequentially, and a composition for promoting cancer cell apoptosis using the same.

According to the method of the present invention, the increased intracellular p53 expression level can be maintained for a long period of time in spite of the treatment with a low-concentration platinum-based anticancer drug, thereby effectively inducing the apoptosis of cancer cells and minimizing the drug side effect caused by the administration of the platinum-based anticancer drug, and thus the present invention can be favorably used in the prevention of cancer or the development of cancer medicines.

Claims

1. A method for potentiating a therapeutic effect of an anticancer drug in a cancer cell of a subject with a cancer, the method comprising the steps of: (a) selecting a subject who is unresponsive or poorly responsive to a platinum-based anticancer drug; (b) transducing a siRNA against ubiquitin ligase to tumor protein 53 (p53) into the cancer cell of the subject, wherein the siRNA is at least one selected from the group consisting of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10 defined by Table 1: TABLE-US-00005 TABLE1 Name SeqNo. Sequences HPVtype18 SeqNo.1 5-CAACCmGAmGCACmGACAmGmGAA-3 siRNA426 (Forward) SeqNo.2 5-UUCCUGUCGUGCUCGGUUG-3 (Reverse) HPVtype18 SeqNo.3 5-CCAACmGACmGCAmGAmGAAACA-3 siRNA450 (Forward) SeqNo.4 5-UGUmUUCUCmUGCGmUCGmUUGG-3 (Reverse) HPVtype16 SeqNo.5 5-GCAAAGACAUCmUmGmGACAAA-3 siRNA366 (Forward) SeqNo.6 5-UUUGUCCAGAUGUCUUUGC-3 (Reverse) HPVtype16 SeqNo.7 5-UCAAmGAACACmGUAmGAmGAAA-3 siRNA488 (Forward) SeqNo.8 5-UUUCUCUACGUGUUCUUGA-3 (Reverse) HPVtype16 SeqNo.9 5-GACCGGUCGAUGUAUGUCUUG-3 siRNA497 (Forward) SeqNo.10 5-AGACAmUACAmUCGACCGGmUCCA-3 (Reverse) wherein m represents a base substituted with a 2-O-Me modified nucleotide; (c) administering a platinum-based anticancer drug to the transduced cancer cell of the subject; (d) monitoring the expression level of intracellular p53 in the cancer cell of the subject; and (e) maintaining a prolonged increase in the expression level of intracellular p53 in the cancer cell of the subject above a threshold level at which the dead of the cancer cell is induced, thereby potentiating the therapeutic effect of the anticancer drug, wherein the steps of (b) and (c) are conducted simultaneously or sequentially.

2. The method according to claim 1, wherein the platinum-based anticancer drug is at least one drug selected from the group consisting of cisplatin (cis-diamminedichloroplatinum [II]), carboplatin, oxaliplatin, nedaplatin, picoplatin, triplatin tetranitrate, satraplatin, and mixtures thereof.

3. The method according to claim 1, wherein the ubiquitin ligase is at least one ligase selected from the group consisting of E6/E6-AP complex of Human Papillomavirus (HPV), E6, E6-AP, human HDM2, Pirh2 and COP1.

4. The method of claim 1, wherein the death of the cancer cell is induced via apoptosis pathway.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0066] FIGS. 1A-1F are graphs showing the intracellular TP53 kinetics when cervical cancer cells were treated with HPV E6/E7 siRNA in combination with cisplatin (426, 450, 366, 448, 497: siRNA code name, SP: siRNA pool, CDDP: cisplatin, NC: no treatment control group).

[0067] FIG. 1A shows that Hela cells (left panel) and CaSki cells (right panel) were transduced with 20 nM of HPV E6/E7 siRNA and exposed to 10 M of cisplatin. After 24 hours, whole cell lysates were collected for induction of TP53 and immunoblot analysis of E6, E7 efficiency. -actin was used as a control.

[0068] FIG. 1B shows that cell viability was confirmed by WST analysis. The error bars mean the meanSD of independent experiments.

[0069] FIG. 1C shows that the degree of induction of TP53 and hyperphosphorylated RB in tumor cells treated with HPV E6/E7 siRNA was evaluated using TP53 and E2F luciferase reporter activity.

[0070] FIG. 1D shows that Hela cells (upper panel) and CaSki cells (lower panel) were treated with E6/E7 specific siRNA alone and/or with cisplatin (CDDP). Silent restoration of endogenous TP53, RB and 18E7, 16E7, 16E6 and 18E6 was analyzed with other sub-target genes at designated time points. 8-actin was used as a control.

[0071] FIG. 1E shows the ratio of the relative intensities of TP53, E6, E2F, and E7 obtained from corresponding western blots.

[0072] FIG. 1F shows that after treatment with Hela cells (left panel) and CaSki cells (right panel) with E6/E7 specific siRNA alone and/or CDDP, CDKN1A transcript levels in each cell were analyzed by real-time quantitative PCR (qPCR). The error bars mean the meanSD of independent experiments.

[0073] FIGS. 2A-2B show the results of real-time qPCR analysis of the effect of HPV E6/E7 siRNA alone or in combination with CDDP on the expression of a gene well known as a target gene of TP53 (A: Hela cells, B: CaSki cells).

[0074] FIGS. 3A-3D show the results of analysis of the effect of E6/E7 deletion on TP53 kinetics and cell survival.

[0075] FIG. 3A shows that a schematic representation of the GFP-TP53 reporter construct was used to form stable cell lines. The characterized TP53-RE contains the TP53 consensus binding site (green box) and the arrows represent the minimal TATA box with the GFP-reporter gene. IncuCyte was used as 9 images of all wells every 30 minutes. Time-lapse microscopic images of live Hela cells were performed with 20 nM of SP, 10 M of CDDP monotherapy, and mixed treatment of SP and CDDP, respectively. The selection of representative images after post-transduction was expressed by a fluorescence and phase-contrast image merge with a control siRNA-treated cell as a control group (Representative IncuCyte images of live cell video recordings made for 5 days. scale bar=40 m. GFP-TP53 Reporter stable Hela cells).

[0076] FIG. 3B shows that growth rate (upper left), GFP count (upper right), GFP intensity (lower right), and the normalization of GFP counts (lower left) indicate CDDP alone and/or combination therapies in response to HPV E6/E7 siRNA (Scale bar: 20 m. Arrows indicate induction of TP53-responsive element (RE)-driven GFP reporter gene expression and HPV E6/E7 siRNA treatment time).

[0077] FIG. 3C shows that growth rate (upper left), GFP count (upper right), GFP intensity (lower left), and GFP count normalization (lower right) of GFP-TP53 reporter stable CaSki cells in response to HPV E6/E7 siRNA alone and/or combination treatment (Arrows indicate induction of TP53-responsive element (RE) driven GFP reporter gene expression and HPV E6/E7 siRNA treatment time).

[0078] FIG. 3D shows a simulation of TP53 activity by HPV E6/E7 siRNA alone, CDDP alone or a combination thereof.

[0079] FIGS. 4A-4B show the results of observing the expression of GFP-TP53 and RFP-E2F for HPV E6/E7 siRNA alone and/or in combination with CDDP.

[0080] FIG. 4A shows that stable Hela cells transformed with cyanene RFP-E2F and GFP-TP53 were cultured and time-dependent changes of RFP-E2F in GFP-TP53 were photographed by confocal microscopy to demonstrate the silencing effect of the HPV E6/E7 oncogene in the Hela cells (red indicates Ex/Em=565 nm/650 nm and green indicates Ex/Em=495 nm/545 nm). The fluorescence image of the cell overlaps the phase difference image).

[0081] FIG. 4B shows that mean intensity data were obtained as a time-lapse confocal image of Dual reporter stable cells. Cells were taken every 20 minutes from 12 hours to 24 hours. In particular, the metastatic pattern was observed in HPV E6/E7 siRNA pool transformed cells at 19 to 21 hours

[0082] FIGS. 5A-5D show the results that the effect of triple combination treatment (cisplatin, paclitaxel and siRNA) on HPV positive cervical cancer cells was confirmed in vitro or in vivo (CDDP: cisplatin, PTX: paclitaxel).

[0083] FIG. 5A shows that Hela cells were treated with E6/E7 specific siRNA, CDDP +paclitaxel, or a mixture thereof. The induction of endogenous TP53, hypophosphorylated RB and silencing 18E7, 16E7, 16E6 and 18E6 were analyzed with other sub-target genes at the indicated time points. -actin was used as a control.

[0084] FIG. 5B shows that CDKN1A transcript levels were analyzed by real-time quantitative (q) PCR. The error bars represent the meanSD of independent experiments.

[0085] FIG. 5C shows that the effects of the three mixtures on the activity of GFP-TP53 were analyzed and exhibited as cell growth rate (upper left), GFP count (upper right), and GFP intensity (lower right).

[0086] FIG. 5D shows that Hela cells were treated with HPV E6/E7 siRNA pools, CDDP and CDDP +paclitaxel, or mixtures thereof. Cell cycle analysis and percentage of cells in each step (G0-G1, S and G2/M) were measured.

[0087] FIG. 6 shows a graphical representation of the role of HPV E6/E7 oncogene and HPV E6/E7 siRNA combination therapy in HPV-infected cervical cancer cells.

[0088] FIG. 7 shows the results of checking the expression level of GFP-TP53 over time in HeLa cells when cisplatin alone, siRNA pool alone and/or cisplatin were treated.

DETAILED DESCRIPTION OF THE INVENTION

[0089] Hereinafter, the present invention will be described in detail.

[0090] However, the following examples are illustrative of the present invention, and the present invention is not limited to the following examples.

EXAMPLE 1

Effect of Cisplatin and HPV E6/E7 siRNA on TP53 in Cervical Cancer Cells

Example 1-1

[0091] Effect of HPV E6/E7 siRNA co-administered with Cisplatin in Cervical Cancer Cells

[0092] Cervical cancer cell and HeLa cervical cancer cell lines (HeLa; ATCC CCL-2) infected with HPV type 18 virus and SiHa cervical cancer cell lines (SiHa; ATCC HTB-35) infected with HPV type 16 virus were plated on a 6-well plate with 510.sup.4 or 110.sup.5 cells and cultured in RPMI 1640 or DMEM medium for 24 hours at 37 C. and 5% carbon dioxide, respectively. siRNA intracellular transfection was performed with DharmaFect (Dharmacon, Lafayette, Colo., USA) according to manufacturer's instructions.

[0093] HeLa cervical carcinoma cells infected with HPV type 18 virus were transformed by pooling with each of siRNAs of 426 and 450 sequences, or respectively. The HPV type 16 virus-infected siHa cell line was transformed by pooling siRNAs of 366, 448 and 497 sequences, or respectively.

[0094] In the Western blot, whole-cell lysate was extracted with RIPA buffer (10 mM Tris (pH.4), 0.15 M NaCl, 5 mM EDTA, 1% Triton X-100, 0.5% deoxycholic acid sodium salt and 0.1% SDS). After centrifugation, the protein concentration of the supernatant was measured using a BCA protein assay kit (Thermo Fisher Scientific Inc). Protein samples were placed in sample buffer and boiled for 5 minutes for complete denaturation. The samples were then applied to 6%, 10%, or 15% polyacrylamide gels and transferred to a 0.4 m PVDF membrane (Milipore, Bilerica). After transfer, the membranes were blocked with 5% skim milk, incubated with the appropriate concentration of primary antibody and horseradish peroxidase-conjugated IgG. Finally, the expression level of the protein was detected using ECL solution. The antibody used in the Western blotting experiment was TP53 [DO7], HPV 18-E6 [G7], HPV16-E6 [C1P5], HPV 18-E7 [F7], HPV16-E7 [ED17], while antibodies from Santa Cruz Biotechnology (St. Louis, Calif.) were used for -Actin [C4].

[0095] To investigate the effect of HPV E6/E7 siRNA conjugated with cisplatin (CDDP) on TP53 in cervical cancer cells, the following experiment was conducted.

[0096] Hela cells and CaSki cells, which are cervical cancer cells, were transformed with HPV E6/E7 siRNA and treated with 10 M of cisplatin for 24 hours. Cell lysates were collected and subjected to western blotting by methods known in the art.

[0097] As a result, as shown in FIG. 1A, the amount of HPV E6/E7 protein expression was not observed in the group treated with cisplatin, and the amount of TP53 expression was increased.

Example 1-2

Cell Apoptotic Effects of Cisplatin and HPV E6/E7 siRNA

[0098] To investigate the effect of cisplatin (CDDP) and HPV E6/E7 siRNA on cell viability in cervical cancer cells, the following experiment was conducted.

[0099] Cervical cancer cells, Hela and CaSki cells were transformed with HPV E6/E7 siRNA and cultured for 24 hours with 10 M of cisplatin. WST analysis was performed to confirm cell viability.

[0100] Cell counts were determined by water soluble tetrazolium salts (WST) (EZ-Cytox kit; Daeil Lab Service, Seoul, Korea). EZ-Cytox solution (50 l) added to each well of a 12-well plate and incubated for 2 to 3 hours. Live cells were measured (485 nm) using a GENios Pro microplate reader (Tecan Trading AG, Mannedorf, Switzerland) in a 96-well plate.

[0101] As a result, as shown in FIG. 1B, cell viability was decreased in the treated group compared to the group not treated with cisplatin

Example 1-3

Measurement of Enzyme Activity of TP53 by Cisplatin and HPV E6/E7 siRNA

[0102] To investigate the effects of cisplatin (CDDP) and HPV E6/E7 siRNA on enzyme activity in cervical cancer cells, the following experiment was conducted.

[0103] Hela cells and CaSki cells, treated with cisplatin (CDDP) and HPV E6/E7 siRNA, were double-transformed with pTA-TP53-Luc (TP53 reporter) and pTA-E2F-Luc (E2F reporter) vectors. The above luciferase reporter vector system was purchased from Clontech pathway profiling system (Mountain View, Calif., USA). Luciferase assay was performed using Dual-Luciferase Reporter Assay System (Promega) after 24 hours of transfection, and luminescence activity was measured using a GENios Pro microplate reader (Tecan Trading AG, Mannedorf, Switzerland).

[0104] As a result, as shown in FIG. 1C, the cisplatin treatment of Hela cells and CaSki cells showed an increase in the activity of TP53, but a further decrease in the activity of E2F.

Example 1-4

Effect of Cisplatin and HPV E6/E7 siRNA on the Expression of TP53

[0105] To investigate the effects of cisplatin (CDDP) and HPV E6/E7 siRNA on enzyme activity in cervical cancer cells, the following experiment was conducted.

[0106] Hela cells and CaSki cells were cultured and transformed in the same manner as described in Example 1-1, then treated with cisplatin. Cell lysates were then obtained and subjected to western blotting.

[0107] In western blotting, antibodies of Santa Cruz Biotechnology (St. Louis, Calif.) were used for TP53 [DO7], HPV 18-E6 [G7], HPV 16-E6 [C1P5], HPV 18-E7 [F7], HPV 16-E7 [ED17], E2F-1 [KH195], Cyclin-E [M20], -Actin [C4] and antibody of BD Pharmingen was used for RB [554164], antibody of cell signaling was used for phospho-TP53 (ser-15) [9284].

[0108] As a result, as shown in FIGS. 1D and 1E, when cisplatin was treated, the expression level of TP53 was increased and the expression level of pRB was increased. In addition, the expression levels of 18-E6 and 16-E6, 18-E7 and 16-E7 were decreased (FIG. 1D, FIG. 1E).

Example 1-5

Effect of Cisplatin and HPV E6/E7 siRNA on the expression of CDKN1A

[0109] To investigate the effects of cisplatin (CDDP) and HPV E6/E7 siRNA on enzyme activity in cervical cancer cells, the following experiment was conducted.

[0110] Hela cells and CaSki cells were cultured and transformed in the same manner as described in the above example, then treated with cisplatin. RNA was then extracted from the cells and subjected to real-time qPCR.

[0111] The sequences of primers and probes used in the PCR were as follows and all were made in TIB MOLBIOL (Berlin, Germany).

TABLE-US-00002 CDKN1Aforward 5-CGAAGTCAGTTCCTTGTGGAG-3 CDKN1Areverse 5-CATGGGTTCTGACGGACAT-3 TaqManprobe 5-FAM-CAGAGGAG-Darkquencher-3

[0112] mRNA levels were determined at 530 and 705 nm wavelengths using the LightCycler Real-time PCR Detection System (Roche Diagnostics, Basel, Switzerland).

[0113] As a result, as shown in FIG. 1F, the amount of CDKN1A mRNA expression was increased when Hela cells (left) and CaSki cells (right) were treated with cisplatin and E6/E7 siRNA.

Example 2

Effect of Cisplatin and HPV E6/E7 siRNA on the Expression of TP53-Related Genes in Cervical Cancer Cells

[0114] To investigate the effects of cisplatin (CDDP) and HPV E6/E7 siRNA on the expression of TP53 target genes in cervical cancer cells, the following experiment was conducted.

[0115] The well-known TP53 target genes were classified according to function by cell cycle arrest and DNA recovery, TP53 expression control, apoptosis and regulation of senescence. Hela cells and CaSki cells were cultured and transformed in the same manner as described in the above example, and treated with cisplatin. RNA was then extracted from the cells and subjected to real-time qPCR.

[0116] The sequences of primers and probes used in the PCR were as shown in below Table 2 and were all produced in TIB MOLBIOL (Berlin, Germany). mRNA levels were determined at 530 and 705 nm wavelengths using the LightCycler Real-time PCR Detection System (Roche Diagnostics, Basel, Switzerland).

TABLE-US-00003 TABLE2 SequencesofprimersandprobesforqRT-PCR Name Sequence CDKN1Aforward(SeqNo.11) 5-CGAAGTCAGTTCCTTGTGGAG-3 CDKN1Areverse(SeqNo.12) 5-CATGGGTTCTGACGGACAT-3 TaqManprobe(SeqNo.13) 5-FAM-CAGAGGAG-Darkquencher-3 APAF1forward(SeqNo.14) 5-CCTGTTGTCTCTTCTTCCAGTGT-3 APAF1reverse(SeqNo.15) 5-AAAACAACTGGCCTCTGTGG-3 TaqManprobe(SeqNo.16) 5-FAM-AGGTGGAG-Darkquencher-3 BAXforward(SeqNo.17) 5-GAACCATCATGGGCTGGA-3 BAXreverse(SeqNo.18) 5-CGTCCCAAAGTAGGAGAGGA-3 TaqManprobe(SeqNo.19) 5-FAM-CTTCCTCC-Darkquencher-3 PMLforward(SeqNo.20) 5-GAGCCCCGTCATAGGAAGT-3 PMLreverse(SeqNo.21) 5-CACAACGCGTTCCTCTCC-3 TaqManprobe(SeqNo.22) 5-FAM-GCAGGAAG-Darkquencher-3 YPEL3forward(SeqNo.23) 5-AACCACGACGACCTCATCTC-3 YPEL3reverse(SeqNo.24) 5-AGCCCACGTTCACCACTG-3 TaqManprobe(SeqNo.25) 5-FAM-CCAGGGCA-Darkquencher-3 GADD45Aforward(SeqNo.26) 5-CCCCGATAACGTGGTGTT-3 GADD45Areverse(SeqNo.27) 5-GCCACATCTCTGTCGTCGT-3 TaqManprobe(SeqNo.28) 5-FAM-GCCTGCTG-Darkquencher-3 XPCforward(SeqNo.29) 5-AGACCATACCAGAGCCCATTT-3 XPCreverse(SeqNo.30) 5-AGGCTGGTCCATGTGTTTTG-3 TaqManprobe(SeqNo.31) 5-FAM-GGGAGAAG-Darkquencher-3 PPM1D(Wip1)forward(SeqNo.32) 5-CCCATGTTCTACACCACCAGT-3 PPM1D(Wip1)reverse(SeqNo.33) 5-TGGTCCTTAGAATTCACCCTTG-3 TaqManprobe(SeqNo.34) 5-FAM-TGGAGGAG-Darkquencher-3 MDM2forward(SeqNo.35) 5-CCATGATCTACAGGAACTTGGTAGTA-3 MDM2reverse(SeqNo.36) 5-TCACTCACAGATGTACCTGAGTCC-3 TaqManprobe(SeqNo.37) 5-FAM-TCCTGCTG-Darkquencher-3 HPRT1forward(SeqNo.38) 5-TGACCTTGATTTATTTTGCATACC-3 HPRT1reverse(SeqNo.39) 5-CGAGCAAGACGTTCAGTCCT-3 TaqManprobe(SeqNo.40) 5-FAM-GCTGAGGA-Darkquencher-3

[0117] As a result, as shown in FIGS. 2A-2B, the expression levels of GADD45A, XPC, MDM2, PPM1 D, BAX, APAF1, PML and YPEL3 were increased when cisplatin and HPV E6/E7 siRNA were treated together.

Example 3

Effect of Cisplatin and HPV E6/E7 siRNA on Apoptosis in Cervical Cancer Cells

[0118] To investigate the effects of cisplatin (CDDP) and HPV E6/E7 siRNA on the activity of TP53 and apoptosis in cervical cancer cells, the following experiment was conducted.

[0119] HeLa and CaSki cells were cultured on a 6-well plate and transformed with a GFP-TP53 vector having a fluorescent substance to construct a stable cell line. The GFP-TP53 vector utilized a lentivirus system with puormycin (Sigma-Aldrich, St. Louis, Mo.) resistance marker.

[0120] Cisplatin (CDDP) and HPV E6/E7 siRNA were treated in the above prepared stable cell line. The surviving cancer cells in each well were then photographed for 5 days using an IncuCyte HD system (Essen Instruments, Ann Arbor, Michigan) using the time-lapse technique. In addition, the cell proliferation rate and the number and intensity of GFP, which signify TP53, for each HPV E6/E7 siRNA were analyzed using Incucyte ZOOM software (Essen Bioscience).

[0121] As a result, as shown in FIGS. 3A-3D, when the siRNA and cisplatin were treated together in Hela cells, the cell proliferation rate was decreased and the number of GFP-bound TP53 was increased with time (FIG. 3A, 3B). Also, in CaSki cells treated with siRNA and cisplatin, the cell proliferation rate decreased, and the number of GFP-bound TP53 was further increased with time (FIG. 3C).

Example 4

TP53 and E2F in Response to the Binding of Cisplatin to HPV E6/E7 siRNA in Cervical Cancer Cells

[0122] To investigate the changes of TP53 and E2F in the binding of cisplatin (CDDP) and HPV E6/E7 siRNA in cervical cancer cells, the following experiment was conducted.

[0123] Hela cells were cultured in 96-well plates to transform GFP- conjugated TP53 and RFP-conjugated E2F and then treated with HPV E6/E7 siRNA or cisplatin. Subsequently, changes in GFP-TP53 and RFP-E2F over time in Hela cells with silencing effects of HPV E6/E7 siRNA were recorded using confocal microscopy. Red was taken at Ex/Em=565 nm/650 nm and green was taken at Ex/Em=495 nm/545 nm. The mean intensity of each signal was observed using time-lapse confocal images of stable cells photographed every 20 minutes from 12 hours to 24 hours.

[0124] As a result, as shown in FIGS. 4A-4B, it was confirmed that the expression level of GFP-conjugated TP53 was increased after treatment with HPV E6/E7 siRNA compared with before treatment, and that the green signal was stronger over time. The expression level of GFP-conjugated TP53 increased in 19 to 21 hours, and the amount of expression of RFP-bound E2F was decreased inversely (FIG. 4A).

[0125] On the other hand, in the case of the combination treatment of cisplatin (CDDP) and HPV E6/E7 siRNA, the expression level of GFP-bound TP53 increased at 12 to 14 hours, and the expression level of RFP-bound E2F decreased (FIG. 4B).

Example 5

Therapeutic Effect of Triple Mixtures on HPV Positive Cervical Cancer Cells

Example 5-1

Therapeutic Effect of Triple Mixtures on HPV Positive Cervical Cancer Cells In Vitro

[0126] To investigate the therapeutic effects of treatment with cisplatin (CDDP), HPV E6/E7 siRNA and anticancer drugs in HPV-positive cervical cancer cells, the following experiment was conducted.

[0127] Hela cells were treated with HPV E6/E7 siRNA, cisplatin, and paclitaxel (PTX), an anticancer drug. Then cell lysates were obtained and subjected to Western blotting, and RNA was extracted and subjected to RT-qPCR. Sequences of primers and probes are as follows, all of which were produced in TIB MOLBIOL (Berlin, Germany).

TABLE-US-00004 CDKN1Aforward 5-CGAAGTCAGTTCCTTGTGGAG-3, CDKN1Areverse 5-CATGGGTTCTGACGGACAT-3, TaqManprobe 5-FAM-CAGAGGAG-Darkquencher-3

[0128] mRNA levels were determined at 530 and 705 nm wavelengths using the LightCycler Real-time PCR Detection System (Roche Diagnostics, Basel, Switzerland).

[0129] In addition, the cell proliferation rate, number and intensity of GFP were measured using an IncuCyte HD system (Essen Instruments, Ann Arbor, Michigan) using GFP-conjugated TP53. In addition, Hela cells were treated with HPV E6/E7 siRNA, cisplatin and cisplatin+paclitaxel, or a mixture thereof, followed by cell cycle analysis via FACS assay and the percentages (% cells) of cells in each step (G0-G1, S and G2/M) were measured.

[0130] The results are shown in FIGS. 5A to 5D.

[0131] When HeLa cells were treated with HPV E6/E7 siRNA, cisplatin, and paclitaxel, protein expression of TP53 increased over time, but protein expression of 18-E6 and 18-E7 decreased (FIG. 5A). In addition, when Hela cells were treated with HPV E6/E7 siRNA pool (SP, 20 nM), cisplatin (5 M), and paclitaxel (10 nM) together, the expression level of CDKN1A mRNA was increased (FIG. 5B). In addition, When Hela cells, transformed with GFP-TP53, were treated with the three mixtures, cell proliferation was inhibited, the number of GFP was increased, and the intensity of GFP was increased (FIG. 5C).

[0132] On the other hand, when the Hela cells were treated with HPV E6/E7 siRNA pool (SP, 20 nM), cisplatin (5 M), and paclitaxel (10 nM), the proportion of Sub-G1 phase cells was high and the ratio of G0/G1 phase cells was low. This confirms that the mixtures of HPV E6/E7 siRNA, cisplatin, and paclitaxel inhibit the cell cycle of G1 and G2/M.

INDUSTRIAL AVAILABILITY

[0133] The present invention is to provide a method of maintaining elevated levels of p53 in cells, by administering a platinum-based anticancer drug and an siRNA against ubiquitin ligase to p53 to a subject in need thereof, in combination or sequentially. The method according to the present invention makes it possible to maintain the increased expression level of intracellular p53 for a long period of time even after treatment with a low concentration of platinum-based anticancer drug, thereby effectively inducing the death of cancer cells and minimizing the side effects of drug administration upon administration of the platinum-based anticancer drug, and can be usefully used for the development of a preventive or therapeutic agent for cancer, thus being highly industrially applicable.