KR20110085318A - Suppression method for doxorubicin resistance in ovarian carcinoma using interference shrna inhibiting nrf2 transcription - Google Patents

Abstract

PURPOSE: A method for suppressing ovarian cancer resistance of doxorubicin through Nrf2 transcription inhibition using interference shRNA is provided. CONSTITUTION: A method for suppressing ovarian cancer resistance of doxorubicin is performed using an interference shRNA(sequence number 26) which suppresses Nfr2(NF-E2-relating faction 2). The ovarian cancer resistance suppression of doxorubicin causes significant doxorubicin sensitivity in human ovarian cancer SKOV3 cell line, OV90 cell line, and A2780DR cell line. A lentivirus contains interference Nrf2-targeting shRNA(sequence number 26) expression plasmid which suppress Nrf2 transcription.

Description

Suppression method for doxorubicin resistance in ovarian carcinoma using interference shRNA inhibiting Nrf2 transcription}

The present invention relates to a method for inhibiting ovarian cancer resistance of doxorubicin through the inhibition of Nrf2 transcription using interference shRNA. More specifically, the ovary of the anticancer drug doxorubicin through inhibition of Nrf2 transcription factor through interference shRNA that inhibits transcription of NF-E2-related factor 2 (Nrf2), which protects cells from reactive oxygen species stress. A method for inhibiting cancer resistance.

Increased free radical species (ROS) is observed in the cancer microenvironment. Metabolic activity and mitochondrial dysfunction in cancer cells are known to increase ROS production. Carcinogenic signals, such as Ras activation, can increase ROS production, which in turn stimulates mitogen pathways in cancer cells. Therefore, it is estimated that the antioxidant defense system may be an important factor for the survival of cancer cells in the further oxidative conditions that can be induced by the exogenous anticancer chemicals. Indeed, gene expression associated with the antioxidant defense system in a wide range of cancer cells is greatly increased compared to normal cells. Increased cellular glutathione (GSH) content has been observed in many types of resistant tumors. Increased heme-oxygenase-1 (HO-1) expression confers tumor cell survival and inhibits apoptosis. Increased levels of detoxifying enzymes that are members of the antioxidant defense system and drug transport proteins such as multidrug-resistant protein (MRP) are known to contribute to cancer cell survival by promoting the rapid removal of anticancer agents from cells.

Transcription factor NF-E2-related factor-2 (Nrf2) plays an important role in maintaining cellular redox status and protecting cells from various oxidative damages. Inducible expression of a broad spectrum of genes encoding various cytoprotective proteins is regulated primarily by Nrf2 by binding to an antioxidant-responsive element (ARE) in its promoter. Low levels of Nrf2 are maintained through KEAP1-dependent proteosome degradation under normal homeostatic conditions. KEAP1, originally isolated from the cytoplasm as an Nrf2-binding protein, functions as a backbone protein for Nrf2 and Cullin-3 E3 ubiquitin ligase and enhances the sustained degradation of Nrf2 by 26S proteosomes.

The binding of Nrf2 to KEAP1 (Kelch-like ECH-associated protein 1) under oxidative conditions or in the presence of an inducer can be affected through the modification of several cysteine residues in KEAP1, which alters Nrf2 from proteolysis and accumulates in the cell nucleus. It triggers the metastatic activity of antioxidant response element (ARE) -containing genes. After successful development of Nrf2-deficient mice, a variety of significant studies have been conducted to investigate the phenotypic differences of these mutant mice in response to oxidative damage and the results obtained confirm the importance of Nrf2 as a cytoprotective gene. Developmental body evidence, however, indicates that aberrant activation of Nrf2 in cancer cells enhances the antioxidant defense system, providing growth benefits and chemotherapeutic resistance. Several studies have shown that the structural activation of Nrf2 has been associated with somatic mutations in KEAP1 in some lung, gallbladder and breast cancer examples.

Doxorubicin (Adriamycin) is an effective anthracycline chemotherapeutic agent and is widely used for the treatment of hematopoietic solid tumors, including ovarian cancer. However, its anticancer mechanism has not yet been fully understood, and several mechanisms of action can be proposed as models of doxorubicin-mediated cytotoxic apoptosis: (i) DNA damage via doxorubicin-induced DNA insertion, (ii) quinone structure. Inhibition of DNA synthesis by ROS production by redox cycle and (i) stabilization of topoisomerase II-DNA complex. Clinical use of doxorubicin is mainly limited by dose-dependent toxicity in normal organs, including heart cells. Dose-limiting side effects of doxorubicin can often be exacerbated by increased resistance causing treatment failure. Several endogenous factors mediating doxorubicin resistance have been identified and cellular defects such as p53 loss and dysfunction of death receptors have been linked to insufficient reactivity of cancer cells to anthracyclines. In particular, an increase in intracellular GSH pool has been shown to correlate with reduced doxorubicin sensitivity of tumor cells. Since ROS production is one of the doxorubicin anticancer mechanisms, the levels of the antioxidant defense system in cancer cells determine the susceptibility to doxorubicin chemotherapy. Moreover, when tumor cells exert adaptive activation of the antioxidant defense system in response to doxorubicin, cancer cell selection results in the acquisition of chemical resistance.

In the present invention, we investigated the role of Nrf2-antioxidant system in doxorubicin resistance of ovarian cancer cells. Four types of ovarian cancer cell lines were used to determine whether doxorubicin susceptibility and Nrf2 activity were correlated and doxorubicin-resistant A2780DR cells were established.

According to the experiments of the present invention, high doxorubicin-sensitive cell lines were measured to have lower Nrf2 activity than doxorubicin-resistant cell lines. Moreover, established A2780DR cells selected for doxorubicin resistance showed GSH-synthetic enzyme expression with increased levels of Nrf2 activity and accompanying increase in total GSH pool. In contrast, stable inhibition of Nrf2 expression in A2780DR cells by the use of a lentiviral shRNA delivery system was able to restore doxorubicin sensitivity with inhibition of antioxidant response element (ARE) -acting gene expression.

Therefore, the present inventors confirmed that doxorubicin-mediated anticancer activity was increased in the absence of the Nrf2 protein system, and the present invention was completed by confirming that Nrf2 transcription factor inhibition by interference shRNA application reduced ovarian cancer resistance of doxorubicin.

The problem to be solved by the present invention is to confirm that doxorubicin-mediated anticancer activity is increased in the absence of the Nrf2 protein system, to determine that inhibition of Nrf2 transcription factor by the application of interference shRNA reduces ovarian cancer resistance of doxorubicin.

An object of the present invention to provide a method for inhibiting ovarian cancer resistance of the anticancer drug doxorubicin using an interference shRNA (SEQ ID NO: 26) that inhibits the transcription of Nrf2 (NF-E2-related factor 2) that protects cells from reactive oxygen species stress. will be.

At this time, the inhibition of ovarian cancer resistance of the doxorubicin is characterized by imparting doxorubicin sensitivity significantly to the human ovarian cancer SKOV3 cell line, ovarian cancer OV90 cell line and ovarian cancer A2780DR cell line.

In addition, inhibition of doxorubicin resistance using the interfering shRNA may be inhibited by glutamate-cysteine ligase catalytic subunit (GCLC), heme-oxygenase-1 (HO-1) and glutathione (GSH) through inhibition of Nrf2 transcription in resistant ovarian cancer cell lines. Characterized in that it is induced through inhibition of expression.

Meanwhile, another object of the present invention is a lentivirus comprising an interfering Nrf2-targeting shRNA (SEQ ID NO: 26) expression plasmid that inhibits transcription of Nrf2 (NF-E2-related factor 2) that protects cells from reactive oxygen species stress. To provide.

The effects of the present invention confirm that doxorubicin-mediated anticancer activity is increased in the absence of Nrf2 protein system and that Nrf2 transcription factor inhibition by interference shRNA application reduces ovarian cancer resistance of doxorubicin by ovarian cancer of doxorubicin using Nrf2 interfering shRNA. It is to provide a method of inhibiting resistance.

1 shows the chemical sensitivity of human ovarian carcinoma cell lines. A2780, SKOV3 (SKOV), TOV21G (TOV) and OV90 cells were plated on 96-well plates at a density of 5000 cells per well and incubated with the carrier. For 24 hours (A) Cisplatin (1-32 μg / ml), (B) Doxorubicin (0.1-80 μM), (C) Cyclophosphamide (4-64 mM) or (D) 5-FU (4- 64 μM). Cell viability was monitored by MTT assay. Values are mean ± SE of 8 measurements.
2 shows Nrf2 target gene expression levels of ovarian cancer cell lines. 2 (A) shows Nrf2 transcription levels and their target genes in OV90, SKOV3, TOV21G and A2780 cells. Total RNA was used for RT-PCR analysis to measure the levels of subunits of GCL (GCLC and GCLM), GR, NQO1, Nrf2, UGT1A6 and β-actin. Relative intensities of GCLC, GCLM and GR were obtained after normalization using β-actin levels. Similar results were obtained in three independent experiments. 2 (B) shows the total GSH content in four ovarian cancer cell lines and the transcriptional levels of xCT, a cystine transporter in resistant SKVO3 and sensitive A2780 cells. Total GSH content is expressed as the amount of GSH per mg of protein. Values are the mean ± SE of 4 experiments. 2 (C) shows the transcriptional activity of Nrf2 and AP-1 in SKOV3 and A2780 cells. Luciferase receptor plasmids containing three copies of human NQQ1 ARE or a single copy of mouse GSTA1 ARE were transfected into cells and luciferase activity was monitored using the Dual Luciferase System. Luciferase receptor plasmids with TRE were transfected into cells for measuring AP-1 transcriptional activity. Values are mean ± SE of 4 experiments. 2 (D) shows EMSA for measuring Nrf2-ARE binding activity. 6 μg of nucleus protein was incubated with biotin-labeled ARE for 20 minutes. More than 100-fold unlabeled ARE or Nrf2-specific antibody (1 μg) was added to the incubation mixture for competitive binding. The whole binding mixture was run on 4% polyacrylamide gels and transferred to the nylon membrane. Labeled ARE was detected by incubating the membrane with streptavidin-carrot peroxidase conjugate and chemiluminescent substrate. Similar results were obtained in three independent analyzes. 2 (E) shows the nuclear Nrf2 protein levels in SKOV3 and A2780 cells. Nucleic acid protein extracts from SKOV3 and A2780 cells were used for immunoblot analysis of Nrf2. 2 (F) shows transcript levels of NRF1 and BACH1 in SKOV3 and A2780 cell lines.
3 shows that doxorubicin increases Nrf2 target gene expression in A2780 cells. In FIG. 3 (A) A2780 cells were incubated with vehicle (Veh) or doxorubicin (0.75 or 1.5 μM) for 24 hours and total RNA extracted. Transcript levels of GCLC, GR, NQO1, HO-1, and AKR1C1 / 2 were measured using RT-PCR analysis. Relative strength was obtained after normalization with 28S values. Similar results were obtained in three independent measurements. 3 (B) shows transcript levels of GCLC and AKR1C1 / 2 after 24 hours incubation with 2.5 μM SFN in A2780 cells. Relative strength was obtained after normalization with 28S values.
4 shows the establishment of doxorubicin-resistant A2780DR cells. In FIG. 4 (A) A2780DR cells were established by exposing A2780 cells to progressively increasing concentrations of doxorubicin (0.02-0.12 μM). A2780 and A2780DR cells were incubated with doxorubicin (0-3.2 μM) for 72 hours and cell viability was monitored by MTT assay. Values are the mean ± SE of 8 measurements. 4 (B) shows stable resistance of A2780DR cells to doxorubicin treatment. Established A2780 cells were maintained for 4 weeks in medium without doxorubicin (A2780DRND) and cell viability was monitored after 72 hours of incubation with vehicle (v) or doxorubicin (0.2 or 1.6 μM). Values are the mean ± SE of 8 measurements. 4 (C) shows doxorubicin resistance of A2780DR cells. Cells were incubated with doxorubicin (0-4 μM) for 24 hours and cell viability was measured by MTT assay. 4 (D and E) show cisplatin and cyclophoside sensitivity of A2780DR cells. Cells were incubated with cisplatin (D; 0-32 μg / ml) or cyclophosphamide (E; 0-32 mM) for 24 hours and viable cell numbers were monitored by MTT assay. Values are the mean ± SE of 8 measurements. ^A P <0.05 compared to parent A2780.
5 shows a decrease in doxorubicin-induced apoptosis response in A2780DR cells. 5 (A) shows TUNEL analysis after doxorubicin treatment. A2780 and A2780DR cells were incubated with the vehicle (Veh) or 1.5 μ doxorubicin for 24 hours. Apoptosis TUNEL staining was performed by labeling DNA nicks with fluorescein-12-dUTP and chromatin counterstaining was performed by staining with PI. Apoptosis green staining and nucleus red staining were observed using fluorescence microscopy. Similar results were obtained in three independent measurements. 5 (B) shows inhibition of doxorubicin-induced caspase-3 activation in A2780DR cells. Cells were incubated with Veh or doxorubicin (2 or 4 μM) for 24 hours and activated caspase-3 cleavage form (17 kDa) and β-tubulin levels were measured with specific antibodies.
6 shows an increased Nrf2-GSH system in doxorubicin-resistant A2780DR cells. 6 (A) shows the transcript levels of Nrf2 target genes in A2780 and A2780DR cells. Levels of GCLC, GCLM, GR, HO-1, xCT, MRP1 and β-actin were measured using RT-PCR analysis. Bar graphs show the relative intensities of Nrf2 target gene levels normalized to the levels of β-actin. Values are mean ± SE of 3 experiments. ^A P <0.05 compared to parent A2780. 6 (C) shows Nrf2 transcriptional activity in A2780 and A2780DR cells. Cells were transfected with human NQQ1 ARE receptor plasmids and ARE-acting luciferase activity was measured. Values are mean ± SE of 4 experiments. ^A P <0.05 compared to parent A2780. 6 (D) shows EMSA of Nrf2-ARE binding activity. A2780 cells were treated with vehicle or SFN for 6 hours and nucleated protein was extracted. Biotin-labeled AREs were incubated with nucleus proteins from carrier-treated A2780 (Veh) or SFN-treated A2780 (SFN), and more than 100-fold unlabeled AREs were added to the incubation mixture with carrier and A2780 for competitive binding. It became. Nuclear protein was prepared from untreated cells to compare ARE binding activity in A2780 and A2780DR (right column). The whole binding mixture was run on 4% polyacrylamide gels and transferred to the nylon membrane. Labeled ARE was detected by incubating the membrane with streptavidin-carrot peroxidase conjugate and chemiluminescent substrate. Similar results were obtained in three independent analyzes. 6 (E) shows the levels of nucleus Nrf2 and total KEAP1 in A2780 and A2780DR cells. Cell nucleus proteins and total lysates were used to detect Nrf2 and KEAP1, respectively, using immunoblot analysis. Similar results were obtained in three independent analyzes.
Figure 7 shows NF-κB activity in A2780 cells. 7 (A) shows increased NF-κB activity in A2780DR cells. Luciferase receptor plasmids containing NF-κB response elements were transfected into cells and luciferase activity was monitored using the Dual Luciferase System. 7 (B) shows GCLC promoter activity in A2780 and A2780DR cells. The p1088-GCLC luciferase plasmid containing the NF-κB binding site but without functional ARE was transfected into cells and a double luciferase assay was performed. Values are mean ± SE of 4 or 5 measurements. ^A P <0.05 compared to parent A2780.
8 shows the establishment and recovery of chemical sensitivity of Nrf2-inhibited A2780 cells. 8 (A) shows inhibition of Nrf2 and target gene expression by lentiviral delivery of Nrf2 shRNA. A2780DR cells were transduced with lentiviral particles comprising scrambled RNA-expressing virus plasmids or Nrf2-specific shRNA-expressing virus plasmids. After puromycin selection, stable cell lines expressing scrambled RNA (A2780DR-scRNA) or Nrf2-targeting shRNA (A2780DR-shRNA) were established. Transcript levels of Nrf2, GCLC, HO-1 and HPRT were monitored by RT-PCR analysis. Similar results were obtained in three independent experiments. 8 (B) shows cell viability after incubation with doxorubicin. A2780DR, A2780DRscRNA and A2780DR-shRNA cells were incubated with carrier or doxorubicin (1 and 2 μM) for 48 hours. Cell viability was monitored by MTT assay. Values are the mean ± SE of 8 measurements. ^A P <0.05 compared to A2780DR-scRNA control. 8 (C) shows flow cytometric analysis of PI stained apoptosis cells. A2780DR, A2780DR-scRNA and A2780DR-shRNA were incubated with vehicle (Veh) or 1.5 μ doxorubicin for 24 hours and stained with PI after ethanol fixation. PI FACS analysis was performed and apoptosis cells were measured by measuring sub-G ₁ phase fractions. The figure is the cell fraction of sub-G ₁ phase out of 20,000 instrument cells. 8 (D) shows doxorubicin susceptibility of Nrf2 shRNA expressing A2780 parent cells. Lentivirus particles comprising nonspecific scrambled RNA expression plasmids or Nrf2-targeting shRNA expression plasmids were transfected into parent A2780 cells and stable cell lines were established after puromycin selection and colony isolation. A2780 cells with scrambled RNA (A2780-scRNA) or Nrf2 shRNA (A2780-shRNA) were incubated with doxorubicin (0.2 or 0.4 μM) for 24 hours and cell viability was monitored by MTT assay. Values are the mean ± SE of 8 measurements.

Since the transcription factor Nrf2 up-regulates a broad array of antioxidant genes by binding to the antioxidant-responsive element (ARE), it is firmly known that Nrf2 is important for healthy cell survival in response to oxidative stress. Therefore, the hypothesis of adaptive activation of the Nrf2 system after exposure of cancer cells to chemotherapeutic agents can be established, which means that chemo resistance is obtained by tumors.

The present invention measured the potential role of Nrf2 signaling in enhancing acquisition resistance to doxorubicin. The human ovarian carcinoma cell line A2780, which is highly susceptible to doxorubicin, exhibited low ARE binding and ARE-acting luciferase activity levels as well as suppressed expression of its target genes as compared to resistant ovarian carcinoma SKOV3 and OV90 cells. Doxorubicin-resistant A2780DR cells established after exposure to increasing concentrations of doxorubicin showed refractory to doxorubicin-induced apoptosis. Acquisition of doxorubicin resistance in A2780 cells was accompanied by increased Nrf2 activity and subsequent expression of catalytic subunits of γ-glutamylcysteine ligase and increased total GSH content.

An important role of Nrf2 in the acquisition chemical resistance of A2780DR cells can be confirmed by the recovery of doxorubicin susceptibility after stable expression of Nrf2-specific shRNA (SEQ ID NO: 26) in A2780DR cells, whereas inhibition of Nrf2 is doxorubicin in parental A2780 cells The sensitivity could not be further enhanced. These results indicate that Nrf2 activity levels are a determinant for doxorubicin sensitivity in ovarian carcinoma cell lines and that adaptive activation of the Nrf2 system may be involved in the increased resistance obtained for anthracycline treatment.

Therefore, the present invention confirms that doxorubicin-mediated anticancer activity is increased in the absence of Nrf2 protein system, and that inhibition of Nrf2 protein expression by interference shRNA decreases ovarian cancer resistance of doxorubicin, thereby ovarian cancer resistance of doxorubicin using Nrf2 interfering shRNA. It is to provide a suppression method.

Hereinafter, the present invention will be described in more detail.

A2780 Cells Susceptible to Anticancer Drug Doxorubicin

Platinum-based anticancer agents are used in combination with doxorubicin, alkylating agents and paclitaxel for the treatment of ovarian cancer. However, endogenous and acquired resistance to these cytotoxic drugs is very common. To investigate the potential relationship of Nrf2 to chemical resistance, the present invention used four types of human ovarian cancer cell lines, including OV90, SKOV3, TOV21G and A2780, and evaluated their susceptibility to various anticancer agents.

Platinum-based cisplatin, anthracycline doxorubicin, alkylated cyclophosphamide and anti-metabolite 5-fluorouracil (5-FU) as anticancer agents were incubated in cells for 24 hours and cell viability was monitored using MTT assay. . SKOV3 and OV90 cells showed high resistance to these anticancer agents; Treatment with cisplatin, doxorubicin and 5-FU did not significantly reduce cell viability and 80 μM doxorubicin resulted in 70-100% survival (FIGS. 1A, B and D). A2780 was most sensitive to these chemicals, and after 72 hours of incubation, the IC ₅₀ for doxorubicin was 0.033 μM, a significant difference from OV90 and SKOV3. No significant difference in cell sensitivity to cyclophosphamide was observed for the four cell lines (FIG. 1C). However, TOV21G cells were very sensitive to doxorubicin and cisplatin compared to SKOV3 and OV90. These results indicate that the A2780 cell line is very sensitive to doxorubicin and cisplatin while SKOV3 and OV90 are resistant cell lines.

Chemotherapy-sensitive A2780 with low levels of Nrf2 activity, antioxidant gene expression and cellular GSH content compared to resistant cell lines

The present invention was then examined for Nrf2 target gene expression levels in these cell lines. Nrf2 is known to play an important role in regulating both basal and inducible expression of GCLC and GCLM, GR, GSTs, NQOl and UDP glucuronosyl transferase (UGT). Doxorubicin resistant OV90 and SKOV3 cells showed higher levels of GCLC, GR, and UGT1A6 than those of susceptible A2780 (FIG. 2A) when the transcript levels for these genes were assessed using RT-PCR analysis (FIG. 2A). There was no significant difference in the GCLM and NQO16 expression levels. Although resistant OV90 expressed high levels of Nrf2 mRNA, transcript levels for Nrf2 itself did not show a significant correlation with doxorubicin sensitivity.

Increased glutamate-cysteine ligase catalytic subunit (GCLC) expression can lead to increased GSH biosynthesis in resistant cell lines, and measurement of total GSH content could confirm this association, which means that SKOV3 and OV90 are more than A2780 and TOV21G. Has a high level of cellular GSH pools; The total GSH content of SKOV3 was 4.5-fold of A2780 (FIG. 2B). In addition to increased GSH-synthesizing enzyme expression, SKOV3 cells exhibited significantly higher levels of cystine transporter xCT, one of the Nrf2 target genes compared to A2780 cells, as well as increased GSH biosynthesis with increased GSH pool in SKOV3 cells. Mediated by increased availability of cysteine. These results therefore indicate a positive association between the expression levels of several Nrf2 target genes in ovarian cancer cells and the total GSH levels and chemical resistance.

According to the differential expression level of the Nrf2 target gene, ARE activity was associated with resistance; Luciferase activity from three copies of human NQQ1 ARE was 40-fold higher in SKOV3 than A2780 (FIG. 2C). The human NQQ1 gene contains ARE (5'-TGACTCAGC-3 ') with a recessed TPA-responsive element (TRE; 5'-TGAG / CTCA-3'), and high levels of NQQ1 ARE activity seen in SKOV3 cells Reflects AP-1 activity. However, increased ARE activity in SKOV3 cells is considered primarily due to increased Nrf2 activity; Luciferase activity from three copies of TRE was 2.7-fold higher in SKOV3 cells than in A2780 cells, but without mouse APSTA binding site (5'-AAATGACATTGCTAATGGTGACAAAGCAAC-3 '(SEQ ID NO: 1). A single copy of)) showed 3.3-fold higher activity in SKOV3 cells than A2780.

Similarly, EMSA showed that the NQQ1 ARE-binding activity of SKOV3-derived nucleus proteins was even higher than A2780, indicating that increased Nrf2 activity in SKOV3 was associated with increased DNA binding activity (FIG. 2D). The binding activity of SKOV3 cell nucleus protein to NQQ1 ARE can be initiated by the addition of Nrf2 antibody as well as unlabeled ARE, which confirmed the specific binding of Nrf2 and NQQ1 ARE. Recently, branched somatic mutations in the KEAP1 gene have been associated with impaired degradation of Nrf2, which leads to the accumulation of Nrf2 in several tumors.

However, the present invention could not indicate that resistant SKOV3 cells accumulate high levels of Nrf2 protein when compared to susceptible A2780. In immunoblot analysis, measuring the nuclear protein levels of Nrf2 protein, no significant difference was observed between resistant SKOV3 and sensitive A2780 (FIG. 2E). Several studies have demonstrated that ARE can be recognized by other transcription factors, including several inhibitors such as NRF1 and BACH1. In particular, BACH1 is known to inhibit ARE activity compared to Nrf2. Thus, low levels of ARE activity in A2780 cell lines not correlated with very high levels of Nrf2 protein are mediated by differential expression of Nrf1 or BACH1. However, the measurement of transcript levels of Nrf1 and BACH1 of the present invention showed no significant difference between the two cell lines. The present results therefore suggest that doxorubicin-sensitive A2780 cells exhibit low structural activity of Nrf2 with low levels of GSH pools.

Nrf2-antioxidant system that can be activated by acute treatment with A2780 intracellular doxorubicin

We then questioned whether the response to doxorubicin treatment of A2780 cells involves altered expression of the Nrf2 target gene. Some anticancer agents are known to affect ARE-acting gene expression through activation of Nrf2. In particular, increased ROS after doxorubicin treatment can be expected to activate the Nrf2 system. When cancer cells use the Nrf2 defense system to adapt to the anticancer environment, nonresponsive resistant tumor cells will proliferate rapidly. To investigate this possibility, A2780 cells were first incubated with 0.75 and 1.5 μM doxorubicin for 24 hours and RT-PCR analysis was performed to estimate the expression levels of Nrf2 target antioxidant genes. Doxorubicin treatment increased transcripts of Nrf2 target genes including GCLC, NQO1 and HO-1 in a dose-dependent manner; GCLC levels increased 2.8-fold after treatment for 24 hours with 1.5 μM doxorubicin (FIG. 3A). Moreover, the expression of AKR1C1 / 2, known to be regulated by Nrf2 in human cells, was significantly increased by doxorubicin treatment in these cells.

The levels of Nrf2 target genes induced by doxorubicin treatment were similar to sulfoaphane, a well-characterized Nrf2 activator in many types of cell lines: GCLC induction in 1.5 μM doxorubicin and 2.5 μM SFN-treated cells, respectively, was 2.8. -And 2.3-fold and AKR1C1 / 2 induction was 7.8- and 4.6-fold (Figures 3A and B). These results indicate that short-term treatment of A2780 cells with doxorubicin can increase the Nrf2 pathway and this adaptation is involved in obtaining resistance.

Establishment of doxorubicin-resistant A2780DR cells

Given the activation of Nrf2 by transient exposure to doxorubicin, it can be speculated that long-term exposure to doxorubicin would enable the establishment of resistant cell lines with increased defense systems. Based on this hypothesis, the present invention established a doxorubicin-resistant A2780DR cell line by exposing A2780 cells to doxorubicin at progressively increasing concentrations (0.015-0.12 μM) over three months. The resistant cell lines obtained were maintained in 0.12 μM doxorubicin for at least 1 month and used for further characterization. When cells were incubated with doxorubicin for 72 hours and the number of viable cells was measured using MTT assay, A2780DR showed increased resistance to doxorubicin: IC ₅₀ of doxorubicin in A2780DR was 1.27 μM, which is 38 times higher than A2780. High (FIG. 4A). The stability of the established resistance was confirmed by incubating A2780DR in 1 week of doxorubicin free medium, confirming similar reactivity to doxorubicin (FIG. 4B).

The present invention then questioned whether A2780DR cells were cross-resistant to other forms of anticancer agents, including daunorubicin, cisplatin and cyclophosphamide. A2780DR was found to have increased resistance to anthracycline anticancer drug daunorubicin with IC ₅₀ increased by 7.5-fold compared to A2780 (FIG. 4C). However, sensitivity to cisplatin and cyclophosphamide was not altered in A2780DR cells compared to A2780 (FIGS. 4D and E).

A2780DR Cells Showing Insufficient Response to Doxorubicin-Induced Apoptosis

Doxorubicin-induced apoptosis was measured in sensitive and resistant A2780 cells to confirm the results obtained in the MTT assay. Cells were incubated with doxorubicin for 24 hours and doxorubicin-induced DNA cleavage was assessed using TUNEL analysis. At 1.5 μM doxorubicin incubation, A2780 showed dUTP-labeled foci, which reflected the final time of apoptosis while A2780DR did not show any labeled focal. As a result of the TUNEL analysis, doxorubicin incubation increased cleavage caspase-3 protein levels at 4 μM doxorubicin only within A2780 but not A2780DR (FIG. 5B). Further analysis of p53 activity showed that A2780DR cells did not exhibit inhibited transcriptional activity of p53 activity compared to parent A2780, indicating that doxorubicin resistance of A2780DR is not associated with p53 pathway alteration. These results confirm the resistance of A2780DR cells to doxorubicin-induced apoptosis.

Increased Nrf2 activity and its target gene expression in doxorubicin-resistant A2780DR cells

Next, the present invention was intended to investigate the involvement of Nrf2 in the increase in Nrf2 activity in A2780DR as well as its target gene expression and acquisition resistance. First, when evaluating GCLC, GCLM, GR and HO-1 expression using RT-PCR analysis, the present invention shows that the transcript levels for GCLC and HO-1, which are genes increased by doxorubicin treatment for 24 hours, are higher in A2780DR than in A2780. Found high (FIG. 6A). Moreover, there was no statistical significance, but MRP1 expression, known as one of the Nrf2 target genes, in various types of cells was very high in A2780DR cells compared to parent A2780. The figures for GCLM, GR and xCT did not show significant changes. Moreover, AKR1C1 / 2 expression increased by acute treatment of doxorubicin was not increased in A2780DR. Secondly increased GCLC was associated with cellular GSH content: total cellular GSH pool in A2780DR was 2.1-fold higher than A2780 (FIG. 6B). Since the expression level of xCT in A2780DR was not significantly increased (FIG. 6A), increased GCLC expression in these resistant cells may be mainly due to increased GSH content. Third, NQQ1 ARE-activated luciferase activity in A2780DR was 2.7-fold higher than A2780. Fourth, the ARE-binding activity of the nuclear protein derived from A2780DR was 2-fold higher than that of A2780 (Figure 6D, lanes 5 and 4). Specificity of ARE binding in the EMSA of the present invention is a measure of competitive binding to the addition of unlabeled ARE and detection of increased nucleus protein binding to ARE in cells treated with Nrf2 activator SFN (FIG. 6D, lanes 2 and 1) (FIG. 6D, lanes 2 and 3). Fifth, the Nrf2 protein levels measured in total cell lysates derived from A2780DR were somewhat higher than A2780 in a similar pattern (FIG. 6E). However, the present invention could not detect significant alterations in KEAP1 protein levels. These results thus indicate that doxorubicin resistance obtained in A2780DR cells is accompanied by increased Nrf2 basal activity, which can lead to an increase in GSH.

Expression of human GCLC is known to be primarily controlled by ARE4 located ˜3 kb upstream in the 5 ′ flanking region of the GCLC promoter; However, putative binding sites for NF-κB of ˜1052 bp have also been reported to control the expression of GCLC in response to pro-inflammatory cytokines. Since the nuclear transcription factor NF-κB is increased in many types of tumors and contributes to overexpression of antioxidant genes such as GCLC and SOD, the present invention questions whether the increased NF-κB activity in A2780DR is associated with increased GCLC expression. Filed. As shown in FIG. 7A, NF-κB-activated luciferase activity in A2780DR cells was significantly higher than A2780 normal cells. These results indicate that increased NF-κB activity in A2780DR is a causative factor for increased GCLC expression. However, when the p1088-GCLC-luciferase plasmid containing a non-ARE NF-κB site was transfected into A2780 and A2780DR cells, GCLC promoter activity showed similar values in these two cell lines (FIG. 7B). These results support the notion that increased GCLC expression and obtained resistance in A2780DR cells are not associated with increased NF-κB signaling and that Nrf2 signaling activation is a central mechanism for increased GCLC expression.

Nrf2 knockdown by lentiviral delivery of shRNA capable of restoring doxorubicin sensitivity in A2780DR cells

The results of the present invention indicate that increased Nrf2 survival signaling is a direct cause of acquired resistance to doxorubicin in ovarian cancer A2780 cells, and we therefore question whether doxorubicin resistance may be reversed by inhibition. Nrf2 inhibition was attempted in A2780DR cells. For this purpose, expression plasmids for Nrf2-specific shRNA or nonspecific scrambled RNA were transferred into A2780DR cells using a lentiviral system. Lentiviral particles comprising Nrf2 targeting shRNA plasmids or scrambled RNA plasmids were generated in HEK 293T cells and transduced into A2780DR for 24 hours. Puromycin selection was then performed for 3-4 weeks to isolate stable cell lines. Individual colonies were isolated and Nrf2 inhibition was verified in each colony.

The present invention then measured the reactivity to doxorubicin in one of the stable cell lines established in isolated colonies. Transcript levels for Nrf2 were inhibited by 80% in Nrf2-inhibited A2780DR-shRNA cells compared to scrambled control A2780DR-scRNA cells (FIG. 8A). In a similar pattern, GCLC and HO-1 mRNA levels were reduced to 70 and 40%, respectively, indicating that increased GCLC and HO-1 levels in A2780DR are due to activation of Nrf2 signaling. When these cells were incubated with doxorubicin for 24 hours and MTT assay was performed, cell viability after doxorubicin treatment was somewhat affected by administration of scRNA compared to normal A2780DR (FIG. 8B). This reduction in viability is explained by the observation of the present invention that cells with stable scRNA expression have 15-20% less total GSH pool than untransduced A2780DR cells. Stable expression of Nrf2 shRNA significantly increased apoptosis when cell viability was compared to scRNA control cells: A2780DR-shRNA group while 71 and 51% cell viability was observed after 1 and 2 μM doxorubicin treatment in A2780DR-scRNA Only 35 and 28% cells survived. The present invention then determined the amount of apoptotic cells by measuring the cell fraction of sub-G ₁ phase using PI-FACS analysis. Cells were treated with vehicle or 1.5 μM doxorubicin for 24 hours, fixed and stained with PI. The proportion of sub-G ₁ st cells was measured to estimate the cells in the apoptotic response. As shown in FIG. 8C, the cell ratio of sub-G ₁ phase was not affected by doxorubicin treatment in A2780DR cells. Incubation with doxorubicin increased the proportion of sub-G ₁ cells in Nrf2-inhibited A2780DR (A2780DR-shRNA): the ratio increased from 4.7 to 32.5% in A2780DR-shRNA while 6.5 to 12 in the A2780DR-scRNA control group. Increased by%. However, delivery of Nrf2 shRNA with low levels of Nrf2 activity and GSH pool into parental A2780 cells did not further increase doxorubicin sensitivity (FIG. 8D). Therefore, these results confirmed that the acquired doxorubicin resistance in A2780DR cells was mainly mediated by Nrf2 pathway activation.

Since the transcription factor Nrf2 has been identified as a major regulator of GSH-synthetic enzyme and expression of many antioxidant proteins such as HO-1, a possible link to Nrf2's cancer resistance can be inferred. Moreover, since more than 50% of anticancer agents can induce oxidative stress, it is highly possible that tumor cells from patients undergoing chemotherapy have acquired resistance with an increased Nrf2-antioxidant system. In the present invention we show that the activity of Nrf2 and its target gene expression such as GCLC are significantly higher in doxorubicin-resistant cell lines SKOV3 and OV90 than doxorubicin sensitive cell line A2780. Similarly established doxorubicin-resistant A2780DR cells showed increased Nrf2 activity with increased GCLC expression. The direct association of Nrf2 with doxorubicin resistance may be supported by the results indicating partial recovery of sensitivity after stable inhibition of Nrf2 in A2780DR cells. However, inhibition of Nrf2 in parental A2780 cells did not affect doxorubicin sensitivity, indicating that increased Nrf2 activity in A2780DR is clearly associated with doxorubicin resistance.

The involvement of Nrf2 in cancer cell survival can be supported by several recent findings confirming the constitutive activation of Nrf2 in various types of tumors. In most cases, activation of Nrf2 in tumors causes mutations in KEAP1. Padmanabhan, B. et al., Mol. Cell 21: 689-700, 2006 identified mutations in the DGR domain of KEAP1 protein that induces glycine to cysteine substitution in tumors and cell lines from lung cancer patients. These mutations resulted in a defect in the Nrf2 response function of KEAP1 and the resulting accumulation of Nrf2 in the cell. Similarly, Singh, A. et al., PLoS Med. 3: e420, 2006 reported that human lung tumors have a high frequency of mutations in the Kelch or interstitial domains of KEAP1. A recent study by Singh, A. et al. Found that Nrf2 somatic mutations were found in 11 of 103 patients with early lung cancer, with most mutations located in the KEAP1 binding domain causing damage to binding of Nrf2 to KEAP1. Reported. These recent findings support the view that cells with aberrant activation of Nrf2 favor survival and rapid growth in high oxidative tumor conditions and that these cells can be readily selected during the carcinogenic process. In the present invention, SKOV3 possesses higher levels of Nrf2 activity than A2780 as well as its target gene expression such as GCLC, UGT1A6 and xCT, but no significant difference was found in the protein levels of Nrf2 and KEAP1 between these two cell lines. Further studies will be needed to elucidate the molecular mechanism of the high transcriptional activity of Nrf2 in SKOV3 compared to A2780; However, the involvement of Nrf2 in SKOV3 resistance is considered important in that the endogenous resistance of SKOV3 to cisplatin can be weakened by siRNA-based inhibition of Nrf2.

In the present invention a correlation between doxorubicin resistance and increased cellular GSH pool could be found. Increased levels of GSH in doxorubicin-resistant SKOV3 and A2780DR cells contributed to the up-regulation of enzyme biosynthesis. In particular, acquisition resistance of A2780DR cells was not accompanied by an increase in cystine transporter xCT overexpressed in resistant SKOV3 cells, indicating a major role of GCLC in the increased GSH pool in A2780DR. There has long been a premise that increased cellular GSH pools exhibit refractory responses to various forms of anticancer agents. Bracht, K. et al., Anticancer Drugs 17: 41-51, 2006 demonstrated a significant inverse correlation between intracellular GSH concentration and doxorubicin sensitivity in a panel of 14 human cancer cell lines. In many tumors, GSH-synthetic enzymes are overexpressed. GSH may play an important role in determining the chemosensitivity of cancer cells in two ways: (i) GSH neutralizes and promotes the removal of reactive anticancer agents, and (ii) GSH modulates apoptosis sensitivity. For example, when the GSH concentration is increased, activation of the apoptosis pathway by Fas ligand is greatly attenuated. The results of the present invention support the new concept that increased GCLC and increased GSH pools present in many types of refractory tumors are mediated by adaptive activation of the Nrf2 system. Kim, SK et al., Free Radic. Biol. Med. A recent study by 45: 537-546, 2008 demonstrated that breast cancer cells with acquired resistance to tamoxifen accumulate Nrf2 protein and increase GCLC expression.

It is not currently clear which mechanism by which doxorubicin resistant cells obtains high levels of Nrf2 activity and that selected cells increase the expression of only a limited amount of Nrf2 target gene. Protein levels for Nrf2 in A2780DR cells were slightly increased compared to parental A2780, after which ARE binding activity was significantly increased along with ARE transcriptional activity, while KEAP1 levels did not show any significant change. As shown in other types of tumor cells, alteration of KEAP1 and resulting stabilization of Nrf2 occur, but the detailed molecular mechanism of Nrf2 activation in A2780DR should be elucidated.

Another question is why the expression of many other known Nrf2 target genes, including GCLM, GR, NQO1, xCT and AKR1C, is not significantly increased in A2780DR cells. These observations raised questions about whether transcription factors other than Nrf2 mediate increased expression of GCLC and HO-1 in A2780DR. For inducible expression of human GCLC and HO-1, Nrf2 and NF-κB play an important role. NF-κB has been shown to be increased in A2780DR cells, and the involvement of NF-κB in increased GCLC expression can be inferred.

However, the results of the present invention indicate that Nrf2 is an important factor mediating increased GCLC expression and resulting doxorubicin resistance. First, the human GCLC promoter, including the NF-κB response element but lacking functional ARE, showed similar levels of response in both A2780 and A2780DR, indicating that NF-κB was not involved in the differential expression of GCLC in A2780DR cells. Indicates. Secondly, increased expression levels of GCLC and HO-1 in A2780DR were significantly decreased by stable inhibition of Nrf2. Third, the doxorubicin resistance of A2780DR was effectively weakened by Nrf2 knockdown. Doxorubicin-related signaling pathways are very complex and resistance mechanisms are not fully understood. Therefore, it is possible for A2780DR cells to develop resistance through alteration of many signaling compounds and transcription factors other than Nrf2. Modulation of a wide array of transcription factor activity can be obtained during adaptation and these complex changes lead to a final increase in a limited number of Nrf2-regulated genes in A2780DR.

In conclusion, the present invention shows that increased adaptability of Nrf2 activity may be related to obtaining anthracycline resistance in ovarian carcinoma cells. Thus, proper regulation of Nrf2 activity may be a possible strategy to prevent the development of acquisition resistance to anticancer agents.

The present invention will be described in more detail with reference to the following examples. However, these examples do not limit the scope of the present invention.

material

Doxorubicin, daunorubicin, cyclophosphamide, cisplatin and 5-fluorouracil (5-FU) were purchased from Sigma (Saint Louis, MO, USA). Antibodies that recognize Nrf2 and β-actin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Caspase-3 antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA). Receptor plasmids containing human NQO1 ARE are described in Dr. The mouse GSTA1 ARE-containing luciferase plasmid was donated to Nobunao Wakabayashi (Johns Hopkins University, Baltimore, MD, USA) and was described in Manandhar, S. et al., Eur. J. Pharmacol. Prepared as described in 577: 17-27, 2007. The p1088-GCLC promoter-luciferase plasmid Provided by JC Fernandez-Checa (University of Barcelona, Spain). NF-κB and AP-1 receptor plasmids were purchased from Panomics (Redwood City, CA, USA). Lentivirus plasmids and virus packing mixes with Nrf2 shRNA were purchased from Sigma.

Example 1 Cell Culture

Ovarian cancer cell lines OV90 and TOV21G were obtained from the American Type Culture Collection (ATCC; Rockville, MD, USA), 10% heat-inactivated fetal bovine serum (FBS; Hyclone, Logan, UT, USA) and 1% penicillin / streptomycin Cultured in growth medium containing 1: 1 MCDB 105 (Sigma) and M199 (Sigma) supplemented with (Hyclone). A2780 cell line was purchased from European Collection of Cell Cultures (Salisbury, Wiltshire, UK) and SKOV3 was obtained from Korean Cell Line Bank (Kwanak-gu, Seoul, South Korea). SKOV3 and A2780 were maintained in RPMI 1640 (Cambrex Bio Science, Walkersville, MD, USA) supplemented with 10% FBS (Hyclone) and 1% penicillin / streptomycin (Hyclone). HEK 293T cells were obtained from ATCC and maintained in DMEM (Hyclone) supplemented with 10% FBS and 1% penicillin / streptomycin. All cells were grown at 37 ° C. in a humidified 5% CO ₂ atmosphere.

Example 2 Establishment of doxorubicin-resistant A2780DR cells

Doxorubicin-resistant A2780 cells (A2780DR) were established by selecting cells that survived after long-term incubation with low concentrations of doxorubicin. 0.015 μM doxorubicin was applied as starting concentration and the concentration gradually increased to 0.12 μM over a 3 month period. The recovered cell growth rate and IC50 of doxorubicin measured in A2780DR cells after 1 month of exposure to 0.12 μM doxorubicin were not significantly altered after incubation in normal medium without doxorubicin for 1 week, which established resistance stability of established cells. Indicates.

Example 3 MTT Analysis

Cells were plated at a density of 5 × 10 ³ cell counts / well in 96-well plates. 24 hours post cell incubation were treated with various concentrations of doxorubicin, daunorubicin, cyclophosphamide, cisplatin or 5-FU. MTT solution (2 mg / ml) was then added to each well and cells were further incubated for 4 hours. After removal of the MTT solution 100 μl of dimethyl sulfoxide was added to each well and the absorbance was measured at 540 nm using a Versamax microplate reader (Sunnyvale, CA, USA).

Example 4 Total RNA Extraction and RT-PCR Analysis

After cell treatment with doxorubicin, total RNA was isolated from the cells using Trizol reagent (Invitrogen, Carlsbad, Calif., USA). For cDNA synthesis, 200 μg of total RNA was raised to 0.5 μg / μl [4] _12-18 , 200 U / μl Moloney mouse leukemia virus reverse transcriptases (Invitrogen) and Fail Safe master buffer (Epicentre, Madison, WI, USA) The reverse transcriptase reaction was carried out by incubating with the reaction mixture containing. PCR amplification of each gene was performed in a thermal cycler (Bio-Rad, Hercules, CA, USA) and the amplification conditions were 27-30 cycles of 40 seconds at 95 ° C., 30 seconds at 56 ° C. and 30 seconds at 72 ° C. . Primers were synthesized in Bioneer (Daejeon, South Korea) and primer sequences for human genes were NQOl, 5'-GATATTGTGGCTGAACAA-3 '(SEQ ID NO: 2) and 5'-TGCTATATGTCAGTTGAG-3' (SEQ ID NO: 3); Nrf2, 5'-ATAGCTGAGCCCAGTATC-3 '(SEQ ID NO: 4) and 5'-CATGCACGTGAGTGCTCT-3' (SEQ ID NO: 5); NRF1, 5'-TGGCTGATGCTTCAGAATTG-3 '(SEQ ID NO: 6) and 5'-ACTGTAGCTCCCTGCTGCAT-3' (SEQ ID NO: 7); BACH1, 5'-CAAGGAAATGCAAAAGCCTC-3 '(SEQ ID NO: 8) and 5'-CATTTGGCTGAACAGAAGCA-3' (SEQ ID NO: 9); Catalytic subunits of GCL (GCLC), 5'-AGACATTGATTGTCGCTG-3 '(SEQ ID NO: 10) and 5'-TGGTCAGACTCATTAGCA-3' (SEQ ID NO: 11); Regulatory subunits of GCL (GCLM), 5'-CAGATGTCTTGGAATGCACT-3 '(SEQ ID NO: 12) and 5'-GCTGTTCCAACTGTGTTTTG-3' (SEQ ID NO: 13); Glutathione reductase (GR), 5'-TCTAAGACATCACTGATG-3 '(SEQ ID NO: 14) and 5'-GAATTCGTCTACGATGAT-3' (SEQ ID NO: 15); HO-1, 5'-CAGAAGAGCTGCACCGCAAG-3 '(SEQ ID NO: 16) and 5'-GGTAGAGCTGCTTGAACTTG-3' (SEQ ID NO: 17); Cystine vehicle (xCT), 5'-GCACTTTGAACAAAGGTCAGTGGGG-3 '(SEQ ID NO: 18) and 5'-CCACCATGCCCGGCTCACAG-3' (SEQ ID NO: 19); Multi-drug resistance-related protein-1 (MRP1), 5'-TGACCATTGACGAGAACAAC-3 '(SEQ ID NO: 20) and 5'-TGGGAGAGAAAGATCCTCAG-3' (SEQ ID NO: 21); Aldoketo-reductase 1C1 / 2 (AKR1C1 / 2), 5'-GCCTAAACAGAAATGTGCGA-3 '(SEQ ID NO: 22) and 5'-TCACCAAGCAGGAGAGATTT-3' (SEQ ID NO: 23); And β-actin, 5'-CCATGTACGTTGCTATCCAG-3 '(SEQ ID NO: 24) and 5'-GGCCATCTCTTGCTCGAAGT-3' (SEQ ID NO: 25). PCR products were analyzed on a 1.2% agarose gel and images were taken using the Visi Doc-It imaging system (UVP, Upland, CA, USA). The intensity of each blot was quantified using ImageJ software (National Institutes of Health, Bethesda, MD, USA).

Example 5 Preparation of Cell Nucleus Extract

Nucleic acid proteins from A2780 and A2780DR cells are described in Manandhar, S. et al., Eur. J. Pharmacol. Extracted as described in 577: 17-27, 2007. Briefly, the natural nucleus fraction is used to homogenize the cells in homogenization buffer (2 M sucrose, 1 M Hepes, 2 M MgCl ₂ , 2 M KCl, 30% glycerol, 0.5 M EDTA, 1 M dithiothreitol (DTT), protease inhibitors). Digestion with cocktail (Sigma) and 10% NP-40) was followed by centrifugation at 12,000 g for 15 minutes. The nucleus protein was obtained from the natural nucleus fraction obtained by incubating with extraction buffer containing 20 mM Hepes (pH 7.9), 1.5 mM MgCl ₂ , 420 mM NaCl, 10% glycerol, 0.2 mM EDTA and protease inhibitor cocktail on ice for 30 minutes. Extracted. Protein concentration was measured by RC DC Protein Assay Kit (Bio-Rad).

Example 6 Immunoblot Analysis

Cell lysates were added dropwise on 6 or 12% SDS-polyacrylamide gels and separated by electrophoresis. The protein on the gel is transferred to a nitrocellulose membrane (Whatman, Dassel, Germany) and the membrane is TPBS buffer (8 g / L NaCl, 0.2 g / L KCl, 1.44 g / L Na ₂ HPO ₄ , 0.24 g / L KH ₂ PO Blocked for 1 hour with 0 5% Scheme Milk in ₄ and 2 ml / L Tween 20). This was followed by incubation with a secondary antibody (Bio-Rad) conjugated to carrot peroxidase for 1 hour after the primary antibody incubation was performed overnight. Detection was performed with Enhanced Chemiluminescence Reagent (Amersham Biosciences, Buckinghamshire, UK). As Nrf2 standard, nucleus protein obtained from Nrf2-transfected mouse papilloma (PE) cells was used.

Example 7 Receptor Plasmid Transfection and Luciferase Activity Measurement

Cells were transfected with plasmids at 50% fusion using Welfect-Ex Plus transfection reagent (WelGene, Daegu, South Korea). Briefly, cells were treated with 0.5 μg ARE-luciferase plasmid, 0.05 μg pRLtk control plasmid and transfection reagent (Welfect-Ex 2 μg and Enhancer-Q 1.5 μg) in serum- and antibiotic-free OptiMEM (Invitrogen). It was incubated with the containing transfection complex. Transfection lasted 18 hours and then recovered in complete medium for the next 24 hours. The cells were then lysed and firefly and Renilla lucifera in total cell lysate using a double-luciferase assay kit (Promega) with 20/20 ⁿ luminometer (Turner Biosystems, Sunnyvale, CA, USA) First activity was measured.

Example 8 Electrophoretic Movement Change Analysis (EMSA)

EMSA was performed using a Lightshift chemiluminescence kit (Pierce, Rockford, IL, USA) according to the manufacturer's protocol for detection of Nrf2-ARE binding activity. Briefly, 50 ng of sense and antisense DNA for human NQQ1 ARE (5'-GCAGTCACAGTGACTCAGCAGAATCT-3 ') was obtained with 5 μM biotin-11-dUTP and 10 U terminal deoxynucleotidine in TdT buffer for 30 minutes at 37 ° C. Biotin was labeled by incubation with transferase (TdT). The reaction was stopped by the addition of 0.2 M EDTA. Labeled DNA was extracted by simple centrifugation at 13,000 g after addition of 50 μg of chloroform: isoamyl alcohol (24: 1). The top aqueous phase containing labeled DNA was stored for annealing and further binding reactions of the labeled sense and antisense DNA. Binding reaction mixture (1X binding buffer (100 mM Tris, 500 mM KCl, 10 mM DTT, pH 7.5), 7.5% glycerol, 2 mM MgCl ₂ , 50 ng / μl poly (dI-) for binding of nucleus proteins to ARE. dC)) was incubated with 6 μg of nuclear protein for 20 minutes at room temperature and 5 μl of 5 × dropping buffer was added. More than 100-fold unlabeled ARE or Nrf2 antibody (1 μg) was added to the incubation mixture for binding competition. 20 μl of the combined mixture was then added dropwise onto 4% natural polyacrylamide gel for electrophoretic separation and then transferred to the nylon membrane. After completion of the transfer DNA was cross-linked to the membrane at 120 mJ / cm 2 with a 254 nm tube mounted CL-1000 ultraviolet cross-linker (UVP, Upland, CA, USA) for 60 seconds exposure. Chemiluminescent detection of biotin-labeled DNA was performed by incubation of the membrane with streptavidin-carrot peroxidase conjugates and chemiluminescent substrates.

Example 9 GSH Content Measurement

Cells were grown for 24 hours in 6-well plates and lysed with 5% metaphosphate solution. Optical density was monitored for 4 minutes after incubation with 30 μl of cell lysate and 30 μl 5 ′, 5-dithiobis (2-nitrobenzene acid), GR and β-NADPH for determination of total GSH content. Protein concentration was measured using the BCA Protein Assay Kit (Pierce).

Example 10 TUNEL Analysis

A2780 and A2780DR cells were seeded and grown overnight on 8-well chamber slides at a density of 5 × 10 ⁴ cells per well. Carrier or doxorubicin (1.5 μM) treatment was then performed for an additional 24 hours. Cells were then fixed with 4% paraformaldehyde solution at 4 ° C. for 30 minutes and permeated with 0.2% Triton X-100 solution for 5 minutes at room temperature. Terminal deoxynucleotidyl transferase dUTP gap-terminal labeling (TUNEL) was performed according to the manufacturer's protocol (Promega). Cells were counterstained for 15 minutes at room temperature with propidium iodide (PI; 1 μg / ml) and observation was performed using a fluorescence microscope (Eclipse TE 2000U; Nikon, Melville, NY, USA).

Example 11 Preparation of Lentiviral Particles with Nrf2 shRNA Expression Vectors

Lentivirus particles with shRNA were generated in HEK 293T cells after transfection with Nrf2 shRNA expression plasmid (Sigma) using Mission lentiviral packing mix (Sigma). Briefly HEK 293T cells were dispensed on 60-mm plates at a density of 7 × 10 ⁵ cells per well. The next day medium was replaced with OptiMEM (Invitrogen) in the absence of homeostasis followed by 1.5 μg pLKO.1-Nrf2 shRNA and packing containing human Nrf2-specific shRNA (5'-CCGGGCTCCTACTGTGATGTGAAATCTCGAGATTTCACATCACAGTAGGA-3 ') (SEQ ID NO: 26) The mix was transfected into cells using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. pLKO.1-scrambled RNA plasmid was used as nonspecific control RNA. On the second day medium with the transfection complex was removed and complete medium was placed in each well. Medium containing lentiviral particles on the third and fourth days was incubated and the harvested virus particles were stored at −70 ° C. after low speed centrifugation to remove cellular debris.

Example 12 Establishment of Nrf2-Knockdown A2780DR Cells

A2780DR cells were dispensed on 6-well plates at a density of 2 × 10 ⁵ cells per well and comprise scrambled nonspecific RNA expression plasmids or Nrf2 shRNA expression plasmids in the presence of 8 μg / ml hedimethrin bromide (Sigma). Transduced with lentiviral particles. Transduction lasted for 24 hours and then recovered for 48 hours in complete medium. For selection of cells with target plasmids, cells were transferred to 96-well plates at a density of 100 cells per well and grown in medium with 1 μg / ml puromycin (Sigma). Each colony isolated was retained for 3 weeks and Nrf2 inhibition was verified within each colony using RT-PCR analysis of Nrf2 and its target gene levels. Nrf2-inhibited parental A2780 cells were also established after transduction of Nrf2 shRNA expressing plasmid-containing lentivirus particles and selection of puromycin (1 μg / ml).

Example 13 Apoptosis Analysis

Cells were plated on 6-well plates at a density of 2 × 10 ⁵ cells per well and grown overnight. After 24-hour incubation with 1.5 μM doxorubicin, cells were harvested and fixed with ice-cooled 70% ethanol at 4 ° C. for at least 4 hours. The immobilized cells were then resuspended in 250 μl of PI solution (100 μg / ml; Sigma) and incubated at 37 ° C. for 20 minutes. Apoptotic cells were measured by measuring the cell population of sub-G ₁ phase using Becton-Dickinson FACSCalibur (San Jose, Calif., USA) and analysis software CellQuest (Becton-Dickinson).

Statistical analysis

Statistical significance was measured by Student-Newman-Keuls comparison (SigmaStat analysis software; SPSS, Chicago, IL, USA) after Studuent correspondence t test or one-way ANOVA. IC ₅₀ numerical measurements were performed using GraphPad Prism (San Diego, CA, USA).

<110> Industry-Academic Cooperation Foundation, Yeungnam University <120> Suppression method for doxorubicin resistance in ovarian          carcinoma using interference shRNA inhibiting Nrf2 transcription <160> 26 <170> KopatentIn 1.71 <210> 1 <211> 30 <212> DNA <213> a single copy of the murine GSTA2 ARE <400> 1 aaatgacatt gctaatggtg acaaagcaac 30 <210> 2 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 2 gatattgtgg ctgaacaa 18 <210> 3 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 3 tgctatatgt cagttgag 18 <210> 4 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 4 atagctgagc ccagtatc 18 <210> 5 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 5 catgcacgtg agtgctct 18 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 6 tggctgatgc ttcagaattg 20 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 7 actgtagctc cctgctgcat 20 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 8 caaggaaatg caaaagcctc 20 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 9 catttggctg aacagaagca 20 <210> 10 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 10 agacattgat tgtcgctg 18 <210> 11 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 11 tggtcagact cattagca 18 <210> 12 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 12 cagatgtctt ggaatgcact 20 <210> 13 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 13 gctgttccaa ctgtgttttg 20 <210> 14 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 14 tctaagacat cactgatg 18 <210> 15 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 15 gaattcgtct acgatgat 18 <210> 16 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 16 cagaagagct gcaccgcaag 20 <210> 17 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 17 ggtagagctg cttgaacttg 20 <210> 18 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 18 gcactttgaa caaaggtcag tgggg 25 <210> 19 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 19 ccaccatgcc cggctcacag 20 <210> 20 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 20 tgaccattga cgagaacaac 20 <210> 21 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 21 tgggagagaa agatcctcag 20 <210> 22 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 22 gcctaaacag aaatgtgcga 20 <210> 23 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 23 tcaccaagca ggagagattt 20 <210> 24 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 24 ccatgtacgt tgctatccag 20 <210> 25 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 25 ggccatctct tgctcgaagt 20 <210> 26 <211> 50 <212> DNA <213> human NRF2-specific shRNA <400> 26 ccgggctcct actgtgatgt gaaatctcga gatttcacat cacagtagga 50

Claims (4)

Method for inhibiting ovarian cancer resistance of anticancer drug doxorubicin using interference shRNA (SEQ ID NO: 26) that inhibits transcription of Nrf2 (NF-E2-related factor 2) that protects cells from reactive oxygen species stress
The method of claim 1, wherein the inhibition of ovarian cancer resistance of the doxorubicin is a ovary of the anticancer drug doxorubicin using an interference shRNA, characterized in that it imparts significant doxorubicin sensitivity to human ovarian cancer SKOV3 cell line, ovarian cancer OV90 cell line and ovarian cancer A2780DR cell line How to inhibit cancer resistance
The method of claim 1, wherein the inhibition of doxorubicin resistance using the interfering shRNA is characterized in that glutamate-cysteine ligase catalytic subunit (GCLC), heme-oxygenase-1 (HO-1), through inhibition of Nrf2 transcription in resistant ovarian cancer cell lines. And method for inhibiting ovarian cancer resistance of the anticancer drug doxorubicin using interference shRNA, which is induced by inhibiting the expression of glutathione (GSH)
Lentiviruses comprising interfering Nrf2-targeting shRNA (SEQ ID NO: 26) expression plasmids that inhibit transcription of Nrf2 (NF-E2-related factor 2) that protects cells from reactive oxygen species stress

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