Detection method for platinum-based drug-resistant ovarian cancer and screening method for candidate substances for tumor treatment or prevention

Abstract

To provide a detection method for platinum-based drug-resistant ovarian cancer.SOLUTION: The present invention provides a detection method for platinum-based drug-resistant ovarian cancer, based on the expression level of the Fdx1 gene in ovarian cancer cells taken from a subject as an indicator.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present disclosure relates to a method for detecting platinum-resistant ovarian cancer, and a method for screening candidate substances for treating or preventing tumors. [Background technology]

[0002] Ovarian cancer is known to have the highest mortality rate among gynecological malignant tumors, with approximately 13,000 cases and approximately 5,000 deaths per year in Japan. Since complete removal of ovarian cancer by surgical therapy alone is difficult, sensitivity to chemotherapy is important.

[0003] As a chemotherapy, TC therapy, which combines paclitaxel with a platinum agent such as carboplatin, is known. However, the acquisition of resistance to platinum agents is a problem. Specifically, during initial treatment with a platinum agent, approximately 80% of patients show sensitivity to the agent, with a response rate of approximately 40-80%. However, if recurrence occurs within 6 months after treatment, the cancer is classified as platinum-resistant ovarian cancer, with a response rate of approximately 10-20%, and a very poor prognosis. For this reason, there is a need to establish an early diagnosis method and develop a treatment method for platinum-resistant ovarian cancer. [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Oncogene. 2003 Oct 20;22(47):7265-79.doi:10.1038 / sj.onc.1206933. [Non-Patent Document 2] Nat Rev Drug Discov.2005 Apr;4(4):307-20.doi:10.1038 / nrd1691. [Non-Patent Document 3] Cancer Res Treat.2018 Apr;50(2):445-460.doi:10.4143 / crt.2016.572.Epub 2017 May 10. Summary of the Invention [Problem to be solved by the invention]

[0005] The present disclosure aims to provide a method for detecting platinum-resistant ovarian cancer. Another aim of the present disclosure is to provide a method for screening candidate substances for treating or preventing tumors, more specifically, candidate substances for treating or preventing ovarian cancer. [Means for solving the problem]

[0006] The inventors discovered that the expression level of the Fdx1 gene, which encodes the iron-sulfur cluster protein Fdx1, which is mainly localized in mitochondria, increases with the acquisition of cisplatin resistance. They also discovered that the Fdx1 protein, the expression level of which increases, contributes to the acquisition of cisplatin resistance by suppressing cisplatin-induced ferroptosis (iron-dependent cell death), and have made further improvements.

[0007] The present disclosure includes, for example, the subject matter described in the following sections: Section 1. A method for detecting platinum-resistant ovarian cancer using the expression level of the Fdx1 gene in ovarian cancer cells collected from a subject as an indicator. Section 2. (I) contacting a test substance with an Fdx1 gene-expressing cell; and (II) A method for screening a candidate substance for treating or preventing a tumor, comprising a step of measuring the expression level of the Fdx1 gene in the Fdx1 gene-expressing cells. Section 3. Item 3. The method according to item 2, wherein the tumor is ovarian cancer. Section 4. The method of claim 3, wherein the ovarian cancer is platinum-resistant ovarian cancer. Section 5. Item 4. The method according to Item 2 or 3, wherein the Fdx1 gene-expressing cells are ovarian cancer cells. Effect of the Invention

[0008] These results indicate that Fdx1 is useful as a diagnostic marker for platinum-resistant ovarian cancer, and that Fdx1 contributes to ferroptosis resistance in platinum-resistant ovarian cancer. These results demonstrate that Fdx1 is a novel therapeutic target for platinum-resistant ovarian cancer, and are useful for establishing novel therapeutic methods that target the novel inhibitory mechanism of ferroptosis by platinum agents. Methods for detecting platinum-resistant ovarian cancer are provided. Also provided are methods for screening for candidate agents for the treatment or prevention of tumors. [Brief description of the drawings]

[0009] [Figure 1] The results of analyzing the cell viability of A2780, A2780cis, OVK18, and OVK18cis at each cisplatin concentration using WST8 are shown. [Diagram 2] The results of RNA sequencing and gene expression analysis for A2780, A2780cis, OVK18, and OVK18cis are shown. [Diagram 3] 1 shows the results of tissue staining for an ovarian cancer patient. [Figure 4] The results of analyzing cell viability when cisplatin or deferoxamine was added to OVK18 cells, the results of staining mitochondrial membrane potential when cisplatin was added to OVK18 and OVK18cis, and the results of staining lipid peroxides when cisplatin was added to OVK18 and OVK18cis are shown. [Diagram 5] The results of staining for mitochondrial membrane potential, staining for lipid peroxide, and measurement of cell viability when Fdx1 expression is suppressed are shown. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0010] Each embodiment included in the present disclosure will be described in further detail below. The present disclosure includes a method for detecting platinum-resistant ovarian cancer, which uses the expression level of Fdx1 gene in ovarian cancer cells collected from a subject as an index. In this specification, the method may be referred to as "the detection method of the present disclosure" or the like.

[0011] Platinum preparations (also called platinum drugs) are preparations that exhibit antitumor effects by binding to the DNA of cancer cells, inhibiting DNA replication and inducing apoptosis of cancer cells. Examples of platinum preparations include carboplatin, cisplatin, nedaplatin, etc. These may be used alone or in combination of two or more. Among these, cisplatin is preferred.

[0012] As used herein, "platinum-resistant ovarian cancer" refers to ovarian cancer that is resistant to chemotherapy containing a platinum agent, or ovarian cancer that has recurred or may recur within six months after completion of chemotherapy containing a platinum agent.

[0013] As used herein, "platinum-sensitive ovarian cancer" refers to ovarian cancer that responds to chemotherapy containing a platinum agent as initial treatment, or ovarian cancer that has recurred or may recur more than six months after completion of chemotherapy containing a platinum agent.

[0014] As used herein, the term "platinum-sensitive ovarian cancer cells" refers to ovarian cancer cells derived from a patient with platinum-sensitive ovarian cancer. The cells may be primary cultured cells, passaged cells, or established cells.

[0015] In this specification, the term "platinum-resistant ovarian cancer cells" refers to ovarian cancer cells derived from a patient with platinum-resistant ovarian cancer, or ovarian cancer cells that have a significantly improved survival rate in the presence of a specific concentration of platinum (e.g., 4 μM cisplatin, etc.) compared to platinum-sensitive ovarian cancer cells. The cells may be primary cultured cells, passaged cells, or established cells.

[0016] Examples of ovarian cancers targeted by the detection method of the present disclosure include serous cancer, endometrioid cancer, clear cell cancer, mucinous cancer, etc. These may be used alone or in combination of two or more. Among these, endometrioid cancer is preferred.

[0017] The subject is preferably a mammal. Examples of mammals include humans, monkeys, dogs, cats, mice, rats, sheep, cows, horses, etc. Examples of human subjects include ovarian cancer patients, humans suspected of having ovarian cancer, etc.

[0018] The method for collecting ovarian cancer cells is not particularly limited. For example, a known method can be appropriately selected depending on the type of sample containing ovarian cancer cells collected from a subject. Examples of samples containing ovarian cancer cells include body fluids (e.g., ascites, pleural effusion, etc.), tissues (e.g., primary lesions (ovaries, etc.), disseminated lesions (peritoneum, omentum, etc.), metastatic lesions (lymph nodes, etc.)), etc. For example, they may be biopsy specimens or pathological tissue sections. These may be used alone or in combination of two or more.

[0019] Fdx1 (Ferredoxin 1) is an iron-sulfur cluster protein that is mainly localized in mitochondria. For example, the accession number of the mRNA sequence of the human Fdx1 gene is NM_004109.

[0020] The expression level of the Fdx1 gene can be measured by measuring the expression level of Fdx1 mRNA or protein. The method for measuring the expression level of Fdx1 gene is not particularly limited. For example, the method for measuring the expression level of m-RNA includes quantitative RT-PCR, in situ hybridization, FISH, etc. For example, the method for measuring the expression level of protein includes Western blotting, ELISA, immunostaining, etc.

[0021] In the detection method of the present disclosure, for example, when the expression level of the Fdx1 gene in ovarian cancer cells collected from a subject is higher than that in platinum-sensitive ovarian cancer cells, the ovarian cancer cells collected from the subject may be determined to be platinum-resistant ovarian cancer cells. For example, the expression level of the Fdx1 gene in ovarian cancer cells collected from a subject is preferably 1.2 times or more higher than that in platinum-sensitive ovarian cancer cells. The upper or lower limit of the range may be, for example, about 1.3 times, 1.4 times, 1.5 times, 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, or 10 times. Specifically, it may be about 1.2 to 10 times. In addition, in the detection method of the present disclosure, for example, the expression level of the Fdx1 gene can also be compared between the same specimens. For example, when the expression level of the Fdx1 gene in ovarian cancer cells collected from a specimen previously diagnosed as a platinum-sensitive ovarian cancer patient is higher than the expression level of the Fdx1 gene in platinum-sensitive ovarian cancer cells previously collected from the specimen, the ovarian cancer cells collected from the specimen can be determined to be platinum-resistant ovarian cancer cells. For example, the expression level of the Fdx1 gene in ovarian cancer cells collected from a specimen previously diagnosed as a platinum-sensitive ovarian cancer patient is preferably 1.2 times or more higher than the expression level of the Fdx1 gene in platinum-sensitive ovarian cancer cells previously collected from the specimen. The upper or lower limit of the range may be, for example, about 1.3 times, 1.4 times, 1.5 times, 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, or 10 times. Specifically, it may be about 1.2 to 10 times.

[0022] According to the detection method of the present disclosure, platinum-resistant ovarian cancer can be detected. In other words, according to the detection method of the present disclosure, platinum-sensitive ovarian cancer can be distinguished from platinum-resistant ovarian cancer.

[0023] The present disclosure also encompasses a method for screening a candidate substance for treating or preventing a tumor, comprising the steps of (I) contacting a test substance with a cell expressing the Fdx1 gene, and (II) measuring the expression level of the Fdx1 gene in the cell expressing the Fdx1 gene. In the present specification, the method may be referred to as the "screening method of the present disclosure" or the like. In addition, in the present specification, each step may be referred to as step (I), step (II), or the like.

[0024] The Fdx1 gene-expressing cells are not particularly limited as long as they can express the Fdx1 gene. For example, cancer cells and the like can be mentioned. Examples of cancer cells include ovarian cancer cells and the like. In addition, the cancer cells may be cancer cells in which the Fdx1 gene is overexpressed by artificial manipulation. The overexpression of the Fdx1 gene can be carried out by a known method.

[0025] In addition, the ovarian cancer cells may be platinum-sensitive ovarian cancer cells or platinum-resistant ovarian cancer cells, of which platinum-resistant ovarian cancer cells are preferred.

[0026] Examples of platinum-sensitive ovarian cancer cells include ovarian cancer cells isolated from patients with platinum-sensitive ovarian cancer, ovarian cancer cells present in non-human mammals with platinum-sensitive ovarian cancer, and commercially available platinum-sensitive ovarian cancer cells. A non-human mammal having platinum-sensitive ovarian cancer can be obtained, for example, by transplanting platinum-sensitive ovarian cancer cells into the non-human mammal. Non-human mammals include, for example, monkeys, dogs, cats, mice, rats, sheep, cows, horses, and the like. Commercially available platinum-sensitive ovarian cancer cells include, for example, A2780 cells, OVK18 cells, and the like.

[0027] Examples of platinum-resistant ovarian cancer cells include ovarian cancer cells isolated from patients with platinum-resistant ovarian cancer, ovarian cancer cells present in non-human mammals with platinum-resistant ovarian cancer, and commercially available platinum-resistant ovarian cancer cells. A non-human mammal having platinum-resistant ovarian cancer can be obtained, for example, by transplanting platinum-resistant ovarian cancer cells into the non-human mammal. Non-human mammals include, for example, monkeys, dogs, cats, mice, rats, sheep, cows, horses, and the like. Commercially available platinum-resistant ovarian cancer cells include, for example, A2780cis cells. Platinum-resistant ovarian cancer cells can also be obtained by known methods, such as exposing platinum-sensitive ovarian cancer cells to a platinum agent and selecting cells that can survive in the presence of a platinum agent. Examples of platinum preparations include carboplatin, cisplatin, nedaplatin, etc. These may be used alone or in combination of two or more. Among these, cisplatin is preferred.

[0028] The test substance is not particularly limited. For example, it may be a compound or a composition. The compound may be, for example, a low molecular weight compound, a nucleic acid (e.g., DNA, RNA, etc.), a protein (e.g., an antibody or a part thereof, etc.), or a polymer compound such as a polymer. The composition may also be an extract obtained from an organism (e.g., an animal, a plant, a microorganism, etc.), or may be a combination of two or more compounds.

[0029] In step (I), the method of contacting the test substance with the Fdx1 gene-expressing cells is not particularly limited. For example, the Fdx1 gene-expressing cells and the test substance may be added to an appropriate solvent, or the test substance may be added to the Fdx1 gene-expressing cells. The suitable solvent is not particularly limited, and examples thereof include water, liquid medium for cell culture, and the like. The concentration of the test substance can be appropriately set depending on the type of the test substance. The temperature during contact is not particularly limited and can be, for example, about 25 to 40°C, or about 30 to 40°C, etc. The contact time is not particularly limited and can be, for example, about 1 minute to 24 hours, or about 3 minutes to 12 hours. During the contact, for example, stirring or shaking can be performed as necessary. Furthermore, when the Fdx1 gene-expressing cells are present in a non-human mammal, methods for contacting the test substance with the Fdx1 gene-expressing cells in step (I) include a method of administering the test substance to the non-human mammal.

[0030] In step (II), the expression level of the Fdx1 gene can be measured by measuring the expression level of Fdx1 mRNA or protein. The method for measuring the expression level of Fdx1 gene is not particularly limited. For example, the method for measuring the expression level of m-RNA includes quantitative RT-PCR, in situ hybridization, FISH, etc. For example, the method for measuring the expression level of protein includes Western blotting, ELISA, immunostaining, etc.

[0031] According to the screening method of the present disclosure, for example, a substance that reduces the expression level of the Fdx1 gene when the test substance is added compared to when the test substance is not added can be screened as a candidate substance for treating or preventing a tumor.

[0032] For example, the expression level of the Fdx1 gene may be decreased by 10% or more when the test substance is added, compared to when the test substance is not added. The upper or lower limit of the range may be, for example, about 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%. Specifically, it may be about 10 to 99%.

[0033] Tumors targeted by the screening method of the present disclosure include, for example, ovarian cancer. Examples of ovarian cancer include serous cancer, endometrioid cancer, clear cell cancer, mucinous cancer, etc. These may be used alone or in combination of two or more. Among these, endometrioid cancer is preferred. In addition, the ovarian cancer may be platinum-sensitive ovarian cancer or platinum-resistant ovarian cancer, of which platinum-resistant ovarian cancer is preferred.

[0034] As shown in the examples below, platinum drugs are thought to induce ferroptosis (iron-dependent cell death) in ovarian cancer by increasing mitochondrial membrane potential. Also, as shown in the examples below, Fdx1 is thought to contribute to the acquisition of platinum drug resistance by suppressing ferroptosis mediated by an increase in mitochondrial membrane potential caused by platinum drugs. Without wishing to be bound by theory, it is speculated that the tumor treatment or prevention candidate substance obtained by the screening method of the present disclosure can exert a tumor treatment or prevention effect by suppressing the expression of Fdx1 gene, thereby releasing the suppression of ferroptosis in platinum-resistant ovarian cancer, or promoting ferroptosis by platinum drugs, etc. In other words, the tumor treatment or prevention candidate substance obtained by the screening method of the present disclosure can also be said to be a candidate substance for enhancing the antitumor effect of platinum drugs.

[0035] In addition, in this specification, the term "comprising" includes "consisting essentially of" and "consisting of." In addition, the present disclosure includes any combination of the constituent elements described in this specification.

[0036] In addition, the various characteristics (properties, structures, functions, etc.) described in each embodiment of the present disclosure above may be combined in any way to specify the subject matter included in the present disclosure. In other words, the present disclosure includes all subject matter consisting of all combinations of each combinable characteristic described in this specification. EXAMPLES

[0037] The contents of the present disclosure will be specifically explained using the following experimental examples. However, the present disclosure is not limited to these in any way. In the following, unless otherwise specified, the experiments are performed under atmospheric pressure and room temperature conditions. Furthermore, unless otherwise specified, "%" means "volume %".

[0038] Cell culture and transfection The endometrioid ovarian cancer cell lines A2780 (parental) and cisplatin-resistant A2780 (A2780cis) were obtained from ECACC (Salisbury, United Kingdom). Another endometrioid ovarian cancer cell line, OVK18 (parental), was obtained from the Riken BRC Cell Bank (Ibaraki, Japan). Cisplatin-resistant OVK18 (OVK18cis) cells were prepared by exposing parental OVK18 cells to cisplatin at a final concentration of 0.1 μM, and then increasing the concentration of cisplatin in the surviving cells. Finally, OVK18cis cells were defined as cells that could survive in the presence of 6 μM cisplatin. A2780cis and OVK18cis cells were maintained in the presence of cisplatin (1.5 μM). All cells were cultured in RPMI-1640 (Nacalai Tesque, Tokyo, Japan) containing 10% (v / v) FBS at 37°C and 5% CO 2 The short tandem repeat profiles of these cells were analyzed (BEX Co., Ltd., Tokyo) to confirm that these cells were free of contamination.

[0039] siRNA transfection was performed as previously reported (Okamoto D., et al., Genes Cells., 2022, 27, 5, 368-375). A2780, A2780cis, OVK18, and OVK18cis cells were transfected with each siRNA using Lipofectamine RNAiMAX reagent (Thermo Fisher Scientific, Waltham, MA) according to the manufacturer's instructions. Specifically, 30 nmol / L siRNA was mixed with RNAiMAX reagent diluted with Opti-MEM (Thermo Fisher Scientific), incubated at room temperature (20-25 °C) for 20 minutes, and added to the cultured cells. The following target sequences were used as siRNAs: siFdx1#1: 5'-CGAUCUGGCAUAUGGACUA-3' (SEQ ID NO: 1), and 5'-UAGUCCAUAUGCCAGAUCG-3' (SEQ ID NO: 2) siFdx1#2: 5'-GCCAAAUCUGUUUGACAAA-3' (SEQ ID NO: 3), and 5'-UUUGUCAAACAGAUUGGC-3' (SEQ ID NO: 4)

[0040] Cell viability assay Cell viability was assessed by WST8 assay using Cell Counting Kit 8 (CCK8; Dojindo, Kumamoto, Japan) according to the manufacturer's instructions. Specifically, 1,000–5,000 cells were seeded into each well of a 96-well plate with 100 μl of medium / well. After the cells were treated with each drug (cisplatin: 0.1–64 μM; deferoxamine: 100 μM; penicillamine: 1 μM) for 48–96 h, the cells were incubated in fresh medium containing 10% (v / v) CCK8 reagent for 2 h, and the absorbance of the culture medium in each well was measured at a wavelength of 450 nm.

[0041] RNA extraction and quantitative RT-PCR RNA extraction and quantitative RT-PCR were performed as previously reported (Avincsal MO., et al., Oncol. Rep., 2021, 46, 148). Total RNA was extracted from cultured cells using Sepasol-RNA I SuperG (Nacalai Tesque), and cDNA was synthesized from these RNAs using PrimeScript RT Reagent (Takara Bio, Shiga). Expression of each target gene was quantified using the LightCycler 480 II system (Roche, Basel, Switzerland). The amount of target mRNA was normalized to the amount of 18S ribosomal RNA. The following primers were used: Fdx1: 5'-AACAGACAGATCACGGTTGGG-3' (SEQ ID NO: 5), and 5'-GGTCTTGCCCACATCAATGG-3' (SEQ ID NO: 6) Timp1: 5'-ACTACCTGCAGTTGTGGCT-3' (SEQ ID NO: 7), and 5'-CTGTCCGTCCAAGCAA-3' (SEQ ID NO: 8) Usp17l11: 5'-CAGCTCAGTGTCCAGCAA-3' (SEQ ID NO: 9), and 5'-AGTTAACGTCTTGGAGGCCG-3' (SEQ ID NO: 10)

[0042] RNA-Seq analysis Total RNA was extracted from A2780, A2780cis, OVK18, and OVK18cis cells using Sepasol-RNA I SuperG (Nacalai Tesque). AmpliSeq libraries were generated using the Ion AmpliSeq Transcriptome Human Gene Expression Kit (Thermo Fisher Scientific) according to the manufacturer's instructions. Libraries were amplified using the Ion OneTouch 2 System (Thermo Fisher Scientific). Libraries were then sequenced using Ion Torrent PGM (Thermo Fisher Scientific) or Ion S5 (Thermo Fisher Scientific). RNA-Seq reads were analyzed using CLC bio Genomics Workbench Version 12.0 (CLC Bio, Aarhus, Denmark). Genes with q-values ​​<0.001 and fold change >0 between A2780 and A2780cis cells, or between OVK18 and OVK18cis cells, were extracted.

[0043] Flow cytometry analysis 5×10 5 Cells were seeded in 6 cm dishes and then treated with each drug (cisplatin: 2.0 μM; deferoxamine: 100 μM; rotenone: 0.5 μM) or their combination. To monitor lipid peroxidation or mitochondrial membrane potential, Liperfluo (Dojindo Laboratories) or MT-1 (Dojindo Laboratories) were added to each dish according to the manufacturer's instructions and incubated for 30 min. Flow cytometry analysis was then performed using a BD LSRFortessa™ X-20 (BD Biosciences, Franklin Lakes, NJ, USA).

[0044] Immunohistochemical analysis Ovarian cancer tissue specimens resected from 45 patients at Kobe University Hospital were fixed and embedded in paraffin. Then, cylindrical tissues were cut from each paraffin block and re-embedded in one paraffin block to prepare tissue microarrays (TMA) for dissection. Endometrioid ovarian cancer cases were selected from the TMA and classified into platinum-sensitive and platinum-resistant groups according to their clinical course. The obtained TMA sections were incubated with an antibody against Fdx1 (12592-1-AP, 1:200, Thermo Fisher Scientific) overnight at 4°C, and then incubated with anti-rabbit IgG antibody conjugated to HRP-labeled polymer (ImmPRESS Reagent kit Peroxidase; Vector Laboratories, Burlingame, CA, USA) for 30 min at room temperature. Secondary antibodies were visualized using DAB Chromogen (Dako Cytomation, Glostrup, Denmark), and nuclei were stained with hematoxylin. Specimens were observed under a BZ-X700 microscope (Keyence, Osaka, Japan). Clinical tissue specimens were obtained and analyzed in accordance with procedures approved by the Institutional Review Board of Kobe University Hospital (Nos. B200076 and B220122).

[0045] statistical analysis Data were analyzed using BellCurve for Excel (Social Survey Research Information Co., Ltd., Tokyo). Student's t-test was used for comparisons between two groups, and one-way ANOVA followed by Tukey's honest significant difference test was used for comparisons between three or more groups. Statistical significance was determined as *P<0.05, **P<0.01, ***P<0.001.

[0046] The cell viability of A2780, A2780cis, OVK18, and OVK18cis at each cisplatin concentration was analyzed using WST8.

[0047] As shown in FIG. 1, it was confirmed that OVK18cis exhibits cisplatin resistance compared to OVK18, and A2780cis exhibits cisplatin resistance compared to A2780.

[0048] We used RNA sequencing to analyze genes whose expression levels were increased with cisplatin resistance between A2780cis and A2780, and between OVK18cis and OVK18.

[0049] As shown in Figure 2, the results of RNA sequencing analysis showed that the expression of 137 genes was increased in A2780 upon acquisition of cisplatin resistance, and the expression of 9 genes was increased in OVK18 upon acquisition of cisplatin resistance. Three genes, Fdx1, Timp1, and Usp17l11, were identified as common to both cells. The three genes were analyzed using quantitative RT-PCR, and as shown in FIG. 2, only Fdx1 was significantly increased in expression in the cisplatin-resistant lines.

[0050] Using the method described above, tissue staining was performed on three tissue samples derived from patients with platinum-sensitive ovarian cancer and three tissue samples derived from patients with platinum-resistant ovarian cancer.

[0051] As shown in Figure 3, Fdx1 expression was observed in some ovarian cancer cells in samples from platinum-sensitive ovarian cancer patients. In contrast, samples from platinum-resistant ovarian cancer patients tended to have higher Fdx1 expression than samples from platinum-sensitive ovarian cancer patients. These results suggest that Fdx1 expression may increase in ovarian cancer tissues as they acquire cisplatin resistance.

[0052] Cisplatin is known to be cytotoxic by inducing DNA damage (Non-Patent Documents 1 and 2). In recent years, it has been reported that cisplatin may be cytotoxic by inducing ferroptosis, which is iron-dependent cell death (Non-Patent Document 3). Therefore, to examine whether cisplatin induces ovarian cancer cell death in an iron-dependent manner, an experiment was carried out in which deferoxamine (100 μM), an iron chelator, was added.

[0053] As shown in Figure 4, cisplatin-induced cell death of OVK18 was suppressed in the presence of deferoxamine. Similar results were also observed in A2780 (data not shown). This suggests that cisplatin may induce ferroptosis in ovarian cancer.

[0054] The inventors also clarified that most of Fdx1 is localized in mitochondria (data not shown), and then investigated the relationship between cisplatin and mitochondrial function. Specifically, the mitochondrial membrane potential when cisplatin was added to OVK18 and OVK18cis was stained with a mitochondrial membrane potential measuring reagent called MT1, and then analyzed by flow cytometry.

[0055] As shown in Figure 4, cisplatin treatment increased the mitochondrial membrane potential in OVK18, but cisplatin did not increase the mitochondrial membrane potential in OVK18cis. This suggests that cisplatin may induce ferroptosis by increasing the mitochondrial membrane potential.

[0056] Since ferroptosis is characterized by an increase in lipid peroxides, we confirmed whether lipid peroxides increased upon administration of cisplatin.

[0057] As shown in Figure 4, the addition of cisplatin to OVK18 increased lipid peroxides, which may induce ferroptosis. On the other hand, it was shown that the increase in lipid peroxides caused by cisplatin administration was suppressed in OVK18cis, and it was revealed that cisplatin-resistant ovarian cancer cells are resistant to ferroptosis caused by cisplatin. Similar results were also observed in A2780 (data not shown).

[0058] After transfecting OVK18cis with siRNA against Fdx1 or control siRNA, the mitochondrial membrane potential was analyzed by flow cytometry after staining with MT1 when cisplatin was added. After transfecting OVK18cis with siRNA against Fdx1 or control siRNA, lipid peroxidation upon addition of cisplatin was analyzed by flow cytometry after staining with Liperfluo. In addition, siRNA against Fdx1 or control siRNA was introduced into OVK18cis, and the cell viability when cisplatin was added was analyzed using WST8.

[0059] As shown in FIG. 5, suppression of Fdx1 expression in OVK18cis increased the mitochondrial membrane potential upon administration of cisplatin. It was revealed that suppressing Fdx1 expression in OVK18cis increased lipid peroxidation after the addition of cisplatin. Furthermore, it was found that suppression of Fdx1 expression promoted cisplatin-induced cell death in OVK18cis. These findings suggest that in OVK18cis, Fdx1 inhibits ferroptosis by suppressing the increase in mitochondrial membrane potential caused by cisplatin, possibly contributing to the acquisition of cisplatin resistance. [Industrial Applicability]

[0060] This can be used for early diagnosis of platinum-resistant ovarian cancer and the development of new treatment methods.

Claims

1. A method for detecting platinum-resistant ovarian cancer, which uses the expression level of the Fdx1 gene in ovarian cancer cells collected from a subject as an indicator.

2. (I) contacting a test substance with an Fdx1 gene-expressing cell; and (II) A method for screening a candidate substance for treating or preventing a tumor, comprising a step of measuring the expression level of the Fdx1 gene in the Fdx1 gene-expressing cells.

3. The method of claim 2 , wherein the tumor is ovarian cancer.

4. 4. The method of claim 3, wherein the ovarian cancer is platinum-resistant ovarian cancer.

5. The method according to claim 2 or 3, wherein the Fdx1 gene expressing cells are ovarian cancer cells.

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