JP7510144B2 - Anticancer drugs and their uses - Google Patents

Description

The present invention relates to an anticancer agent and its use. More specifically, the present invention relates to an anticancer agent, a kit for determining whether administration of an anticancer agent to a cancer patient is effective, and a method for screening an anticancer agent.

Homozygous deletion of tumor suppressor genes is typically observed in the genomes of cancer cells. Furthermore, it is known that deletion of tumor suppressor genes is often accompanied by deletion of adjacent genes (see, for example, Non-Patent Document 1).

For example, the RB Transcriptional Corepressor 1 (RB1) gene is a tumor suppressor gene located on the long arm of chromosome 13 (13q14), and is located near the Succinate-CoA Ligase ADP-Forming Beta Subunit (SUCLA2) gene, which is also located on the long arm of chromosome 13 (13q14).

The inventors have found that prostate cancer frequently exhibits both deletions of the RB1 gene and the SUCLA2 gene. Prostate cancer is difficult to treat due to the high frequency of the emergence of treatment-resistant clones, and there is a need to develop more effective treatment methods.

Muller F. L., et al., Passenger deletions generate therapeutic vulnerabilities in cancer, Nature, 488 (7411), 337-342, 2012.

The present invention aims to provide a new technology for treating cancer associated with deletion of the SUCLA2 gene.

［２］前記癌が、ＲＢ Ｔｒａｎｓｃｒｉｐｔｉｏｎａｌ Ｃｏｒｅｐｒｅｓｓｏｒ １（ＲＢ１）遺伝子を更に欠失している、［１］に記載の抗癌剤。
［３］前記癌が、前立腺癌、膀胱癌、浸潤性乳癌、漿液性卵巣嚢腫、膠芽腫、肉腫又は肺腺癌である、［１］又は［２］に記載の抗癌剤。
［４］ＳＵＣＬＡ２遺伝子のゲノムＤＮＡ増幅用プライマーセット、ＳＵＣＬＡ２遺伝子のゲノムＤＮＡに対するプローブ、ＳＵＣＬＡ２遺伝子のｃＤＮＡ増幅用プライマーセット、ＳＵＣＬＡ２遺伝子のｍＲＮＡに対するプローブ、又は、ＳＵＣＬＡ２タンパク質に対する特異的結合物質、を含む、癌患者に下記式（１）で表される化合物を有効成分とする抗癌剤の投与が有効か否かを判定するためのキット。

［５］癌組織試料において、ＳＵＣＬＡ２遺伝子のゲノムＤＮＡを検出する工程を含み、ＳＵＣＬＡ２遺伝子のゲノムＤＮＡの欠失が検出されたことが、前記癌組織試料が由来する癌の治療に下記式（１）で表される化合物を有効成分とする抗癌剤の投与が有効であることを示すか、ＳＵＣＬＡ２遺伝子のｍＲＮＡの発現を検出する工程を含み、ＳＵＣＬＡ２遺伝子のｍＲＮＡの発現の低下が検出されたことが、前記癌組織試料が由来する癌の治療に下記式（１）で表される化合物を有効成分とする抗癌剤の投与が有効であることを示すか、又は、ＳＵＣＬＡ２タンパク質の発現を検出する工程を含み、ＳＵＣＬＡ２タンパク質の発現の低下が検出されたことが、前記癌組織試料が由来する癌の治療に下記式（１）で表される化合物を有効成分とする抗癌剤の投与が有効であることを示す、前記癌組織試料が由来する癌の治療に下記式（１）で表される化合物を有効成分とする抗癌剤の投与が有効であるか否かを判定する方法。

［６］癌患者由来の生体試料中の、メチルマロン酸、スクシニルカルニチン、メチルマロニルカルニチン、プロピオニルカルニチン、メチルクエン酸又は３－ヒドロキシプロピオン酸の含有量を定量する工程を含み、メチルマロン酸、スクシニルカルニチン、メチルマロニルカルニチン、プロピオニルカルニチン、メチルクエン酸又は３－ヒドロキシプロピオン酸の含有量が対照と比較して有意に高いことが、前記癌患者の癌の治療に下記式（１）で表される化合物を有効成分とする抗癌剤の投与が有効であることを示す、前記癌患者の癌の治療に下記式（１）で表される化合物を有効成分とする抗癌剤の投与が有効であるか否かを判定する方法。

［７］被験物質の存在下で、癌抑制遺伝子の近傍に位置する代謝遺伝子を欠失した細胞株を培養する工程と、前記細胞株の生存率を測定する工程と、を含み、前記生存率が、対照と比較して有意に低下することが、前記被験物質が、前記代謝遺伝子の欠失を伴う癌用抗癌剤の候補であることを示す、前記代謝遺伝子の欠失を伴う癌用抗癌剤のスクリーニング方法。
［８］前記細胞株が前記癌抑制遺伝子を更に欠失している、［７］に記載のスクリーニング方法。
［９］前記癌抑制遺伝子がＲＢ１遺伝子であり、前記代謝遺伝子がＳＵＣＬＡ２遺伝子である、［８］に記載のスクリーニング方法。 The present invention includes the following aspects.
[1] An anticancer agent for a cancer associated with a deletion of the Succinate-CoA Ligase ADP-Forming Beta Subunit (SUCLA2) gene, comprising as an active ingredient a compound represented by the following formula (1):

[2] The anticancer agent according to [1], wherein the cancer further lacks RB Transcriptional Corepressor 1 (RB1) gene.
[3] The anticancer agent according to [1] or [2], wherein the cancer is prostate cancer, bladder cancer, invasive breast cancer, serous ovarian cyst, glioblastoma, sarcoma or lung adenocarcinoma.
[4] A kit for determining whether administration of an anticancer agent containing a compound represented by the following formula (1) as an active ingredient to a cancer patient is effective, the kit comprising a primer set for amplifying genomic DNA of the SUCLA2 gene, a probe for genomic DNA of the SUCLA2 gene, a primer set for amplifying cDNA of the SUCLA2 gene, a probe for mRNA of the SUCLA2 gene, or a substance that specifically binds to SUCLA2 protein.

[5] A method for determining whether administration of an anticancer agent containing a compound represented by the following formula (1) as an active ingredient is effective in treating the cancer from which the cancer tissue sample is derived, the method comprising: a step of detecting genomic DNA of SUCLA2 gene in a cancer tissue sample, wherein detection of deletion of genomic DNA of SUCLA2 gene indicates that administration of an anticancer agent containing a compound represented by the following formula (1) as an active ingredient is effective in treating the cancer from which the cancer tissue sample is derived; a step of detecting expression of mRNA of SUCLA2 gene, wherein detection of reduced expression of mRNA of SUCLA2 gene indicates that administration of an anticancer agent containing a compound represented by the following formula (1) as an active ingredient is effective in treating the cancer from which the cancer tissue sample is derived; or a step of detecting expression of SUCLA2 protein, wherein detection of reduced expression of SUCLA2 protein indicates that administration of an anticancer agent containing a compound represented by the following formula (1) as an active ingredient is effective in treating the cancer from which the cancer tissue sample is derived.

[6] A method for determining whether or not administration of an anticancer agent containing a compound represented by the following formula (1) as an active ingredient is effective in treating cancer in a cancer patient, the method comprising a step of quantifying the content of methylmalonic acid, succinylcarnitine, methylmalonylcarnitine, propionylcarnitine, methylcitric acid or 3-hydroxypropionic acid in a biological sample derived from a cancer patient, wherein a significantly higher content of methylmalonic acid, succinylcarnitine, methylmalonylcarnitine, propionylcarnitine, methylcitric acid or 3-hydroxypropionic acid compared to a control indicates that administration of an anticancer agent containing a compound represented by the following formula (1) as an active ingredient is effective in treating cancer in the cancer patient.

[7] A method for screening an anticancer drug for a cancer involving deletion of a metabolic gene, comprising the steps of culturing a cell line lacking a metabolic gene located near a cancer suppressor gene in the presence of a test substance and measuring the viability of the cell line, wherein a significant decrease in the viability compared to a control indicates that the test substance is a candidate for an anticancer drug for a cancer involving deletion of the metabolic gene.
[8] The screening method described in [7], wherein the cell line further lacks the tumor suppressor gene.
[9] The screening method described in [8], wherein the tumor suppressor gene is the RB1 gene and the metabolic gene is the SUCLA2 gene.

The present invention aims to provide a new technology for treating cancer associated with deletion of the SUCLA2 gene.

6A and 6B are graphs showing the results of Experimental Example 1. 13 is a graph showing the results of screening in Experimental Example 6. 13 is a graph showing the results of measuring cell viability in Experimental Example 7. 6( a ) to 6 ( d ) are graphs showing the results of flow cytometry in Experimental Example 8. Graphs (a) and (b) are graphs created based on the results of FIGS. 4(a) to (d). 13 is a graph showing the results of measuring tumor volume in Experimental Example 9. 13(a) and (b) are representative photographs of the cancer-bearing model mice of the control group and the thymoquinone-administered group in Experimental Example 9. 1 shows photographs of tumor masses excised from cancer-bearing model mice of the control group and the thymoquinone-administered group in Experimental Example 9. 9 is a graph showing the results of measuring the tumor weight of the tumor mass shown in FIG. 8. 13 is a graph showing the results of measuring the number of cleaved caspase 3-positive cells in Experimental Example 10. 1 is a photograph showing the results of Western blotting in Experimental Example 12. 1 is a graph showing the results of flow cytometry analysis in Experimental Example 13.

[Anti-cancer drugs]
In one embodiment, the present invention provides an anticancer agent for cancer with deletion of the SUCLA2 gene, which contains as an active ingredient a compound represented by the following formula (1). The compound represented by the following formula (1) is thymoquinone (CAS number: 490-91-5). As described later in the Examples, the anticancer agent of this embodiment has high cytotoxicity against cancer cells with deletion of the SUCLA2 gene.

Cancers against which the anticancer agent of this embodiment is effective often lack the RB1 gene along with the SUCLA2 gene. This is thought to be because when a tumor suppressor gene is deleted during the malignant progression of cancer, nearby genes are also deleted. Examples of cancers that lack the RB1 gene along with the SUCLA2 gene include prostate cancer, bladder cancer, invasive breast cancer, serous ovarian cyst, glioblastoma, sarcoma, and lung adenocarcinoma.

The SUCLA2 protein is a protein that constitutes the β subunit of the Succinate-CoA Ligase heterodimer, an enzyme that converts succinyl-CoA to succinic acid in the TCA cycle. When the SUCLA2 protein is deleted or its activity is reduced due to mutation, methylmalonyl-CoA mutase is inhibited. As a result, it is known that the concentrations of methylmalonic acid, succinylcarnitine, methylmalonylcarnitine, and propionylcarnitine in the blood increase. It is also known that the concentrations of methylmalonic acid, methylcitric acid, and 3-hydroxypropionic acid in the urine increase.

The anticancer agent of this embodiment is effective against cancers in which the above-mentioned activity of the SUCLA2 protein, i.e., the activity of converting succinyl-CoA to succinic acid, is reduced. In this specification, "cancer associated with deletion of the SUCLA2 gene" may refer to cancers in which a part or all of the SUCLA2 locus is deleted homozygously or heterozygously, cancers in which a mutation that reduces the expression of the SUCLA2 gene has occurred homozygously or heterozygously in the SUCLA2 locus, cancers in which a mutation that reduces the above-mentioned activity of the SUCLA2 protein has occurred homozygously or heterozygously in the SUCLA2 locus, etc.

The anticancer agent of this embodiment is preferably formulated as a pharmaceutical composition mixed with a pharma- ceutical acceptable carrier. The pharmaceutical composition may be in a dosage form for oral use or a dosage form for parenteral use. Dosage forms for oral use include, for example, tablets, capsules, elixirs, microcapsules, etc. Dosage forms for parenteral use include, for example, injections, inhalants, suppositories, patches, etc.

Examples of pharma- ceutically acceptable carriers include solvents such as sterile water and saline; binders such as gelatin, corn starch, gum tragacanth, and gum arabic; excipients such as crystalline cellulose; and leavening agents such as alginic acid.

The pharmaceutical composition may further contain additives. Examples of additives include lubricants such as magnesium stearate; sweeteners such as sucrose, lactose, and saccharin; flavorings such as peppermint and saffron oil; stabilizers such as benzyl alcohol and phenol; buffers such as phosphates and sodium acetate; solubilizers such as benzyl benzoate and benzyl alcohol; antioxidants; and preservatives.

Pharmaceutical compositions can be formulated by combining the above-mentioned carriers and additives appropriately and mixing them in a unit dosage form required for generally accepted pharmaceutical practice.

Administration to a patient can be by methods known to those skilled in the art, such as intra-arterial injection, intravenous injection, intranasal, transbronchial, intramuscular, transdermal, or oral administration. The dosage varies depending on the patient's weight and age, the patient's symptoms, the method of administration, etc., but a person skilled in the art can select an appropriate dosage.

The dosage of the anticancer agent of this embodiment varies depending on the patient's symptoms, etc., but in the case of oral administration, it is generally considered appropriate to administer approximately 300 to 5000 mg, for example approximately 1000 to 4000 mg, for example approximately 200 to 2000 mg, per day to an adult (body weight 60 kg) once a day or in divided doses several times a day.

When administered parenterally, the dosage will vary depending on the patient's symptoms, the target organ, the method of administration, etc., but when administered systemically, it is generally considered appropriate for an adult (body weight 60 kg) to administer approximately 1-10 g, for example approximately 1-5 g, for example approximately 1-2 g, once a day or in several divided doses. When administered locally, it is generally considered appropriate for an adult (body weight 60 kg) to administer approximately 300-1200 mg, for example approximately 300-800 mg, for example approximately 100-600 mg, once a day or in several divided doses.

[kit]
In one embodiment, the present invention provides a kit for determining whether administration of an anticancer agent containing thymoquinone as an active ingredient to a cancer patient is effective or not, the kit comprising a primer set for amplifying genomic DNA of SUCLA2 gene, a probe for genomic DNA of SUCLA2 gene, a primer set for amplifying cDNA of SUCLA2 gene, a probe for mRNA of SUCLA2 gene, or a substance that specifically binds to SUCLA2 protein. The kit of this embodiment makes it possible to determine whether administration of an anticancer agent containing thymoquinone as an active ingredient to a cancer patient is effective or not.

(Primer set for amplifying genomic DNA)
The kit of this embodiment may include a primer set for amplifying genomic DNA of the SUCLA2 gene. The base sequence of the primer set is not particularly limited as long as it can amplify the SUCLA2 gene on the genome.

When genomic DNA in a cancer tissue sample from a cancer patient is amplified with a primer set for amplifying genomic DNA of the SUCLA2 gene, amplification of the SUCLA2 gene is observed if the cancer cells in the cancer tissue sample retain the SUCLA2 gene on their genomic DNA.

On the other hand, if the cancer cells in the cancer tissue sample have a homozygous deletion of the SUCLA2 gene on the genomic DNA, no amplification of the SUCLA2 gene is observed. Also, if the cancer cells in the cancer tissue sample have a heterozygous deletion of the SUCLA2 gene on the genomic DNA, reduced amplification of the SUCLA2 gene is observed.

If the cancer cells in the cancer tissue sample have a homozygous or heterozygous deletion of the SUCLA2 gene, it can be determined that administration of an anticancer drug containing thymoquinone as an active ingredient is effective in treating the cancer of the cancer patient from whom the cancer tissue sample was derived.

(Probes for Genomic DNA)
The kit of this embodiment may include a probe for the genomic DNA of the SUCLA2 gene. The base sequence of the probe is not particularly limited as long as it can detect the SUCLA2 gene on the genome. The probe may form a nucleic acid array.

When a probe for the genomic DNA of the SUCLA2 gene is hybridized to genomic DNA from a cancer tissue sample from a cancer patient, if the cancer cells in the cancer tissue sample have the SUCLA2 gene on their genomic DNA, the probe hybridizes and the presence of the SUCLA2 gene can be detected.

On the other hand, if the cancer cells in the cancer tissue sample have a homozygous deletion of the SUCLA2 gene on the genomic DNA, the probe cannot hybridize and the presence of the SUCLA2 gene is not observed. Also, if the cancer cells in the cancer tissue sample have a heterozygous deletion of the SUCLA2 gene on the genomic DNA, a decrease in the amount of probe hybridizing to the SUCLA2 gene is observed.

If the cancer cells in the cancer tissue sample have a homozygous or heterozygous deletion of the SUCLA2 gene, it can be determined that administration of an anticancer drug containing thymoquinone as an active ingredient is effective in treating the cancer of the cancer patient from whom the cancer tissue sample was derived.

(Primer set for cDNA amplification)
The kit of this embodiment may include a primer set for amplifying cDNA of the SUCLA2 gene. The base sequences of the primer set are not particularly limited as long as they can amplify cDNA of the SUCLA2 gene.

When RNA is extracted from a cancer tissue sample from a cancer patient and amplified by RT-PCR using a primer set for amplifying cDNA of the SUCLA2 gene, amplification of cDNA of the SUCLA2 gene is observed if the cancer cells in the cancer tissue sample express mRNA of the SUCLA2 gene.

On the other hand, when the cancer cells in the cancer tissue sample do not express the mRNA of the SUCLA2 gene, amplification of the cDNA of the SUCLA2 gene is not observed. Examples of cases in which the cancer cells do not express the mRNA of the SUCLA2 gene include cases where the SUCLA2 gene in the genomic DNA is homozygously deleted, cases where the mRNA of the SUCLA2 gene cannot be expressed due to a genetic mutation, etc.

In addition, cancer cells in a cancer tissue sample may have reduced mRNA expression of the SUCLA2 gene compared to control cells. Examples of such cases include cases where the SUCLA2 gene in genomic DNA is heterozygously deleted, and cases where the expression level of mRNA of the SUCLA2 gene is reduced due to genetic mutations, etc. In such cases, the amount of cDNA of the SUCLA2 gene amplified by RT-PCR of RNA derived from cancer cells in a cancer tissue sample is reduced compared to control cells. Here, an example of the control cell is a wild-type cell.

If the cancer cells in the cancer tissue sample do not express SUCLA2 gene mRNA or have reduced expression of SUCLA2 gene mRNA, it can be determined that administration of an anticancer agent containing thymoquinone as an active ingredient is effective in treating the cancer of the cancer patient from whom the cancer tissue sample was derived.

(Probes for mRNA)
The kit of this embodiment may include a probe for the mRNA of the SUCLA2 gene. The base sequence of the probe is not particularly limited as long as it can detect the mRNA of the SUCLA2 gene. The probe may form a nucleic acid array.

When RNA is extracted from a cancer tissue sample from a cancer patient and a probe for the mRNA of the SUCLA2 gene is hybridized, if the cancer cells in the cancer tissue sample express the mRNA of the SUCLA2 gene, the probe will hybridize and the presence of the mRNA of the SUCLA2 gene can be detected.

On the other hand, if the cancer cells in the cancer tissue sample do not express the SUCLA2 gene mRNA, the probe cannot hybridize and the presence of the SUCLA2 gene mRNA is not detected. Examples of cases in which the cancer cells do not express the SUCLA2 gene mRNA include cases where the SUCLA2 gene in the genomic DNA is homozygously deleted, and cases where the SUCLA2 gene mRNA cannot be expressed due to a genetic mutation, etc.

In addition, cancer cells in a cancer tissue sample may have reduced mRNA expression of the SUCLA2 gene compared to control cells. Examples of such cases include cases where the SUCLA2 gene in genomic DNA is heterozygously deleted, and cases where the expression level of mRNA of the SUCLA2 gene is reduced due to genetic mutations, etc. In such cases, the amount of hybridization of the probe to RNA extracted from cancer cells in a cancer tissue sample is reduced compared to control cells. Here, an example of the control cell is a wild-type cell.

If the cancer cells in the cancer tissue sample do not express SUCLA2 gene mRNA or have reduced expression of SUCLA2 gene mRNA, it can be determined that administration of an anticancer agent containing thymoquinone as an active ingredient is effective in treating the cancer of the cancer patient from whom the cancer tissue sample was derived.

(Specific binding substance)
The kit of this embodiment may contain a substance that specifically binds to SUCLA2 protein. Examples of the specific binding substance include antibodies, antibody fragments, aptamers, etc. Examples of the antibody fragments include F(ab') ₂ , Fab', Fab, Fv, scFv, etc. The specific binding substance is not particularly limited as long as it can detect SUCLA2 protein. The specific binding substance may be bound to a solid phase to constitute, for example, a flow strip, an array, etc.

When proteins are extracted from a cancer tissue sample from a cancer patient and contacted with a substance that specifically binds to SUCLA2 protein, if the cancer cells in the cancer tissue sample express SUCLA2 protein, the specific binding substance will bind to the protein, making it possible to detect the presence of SUCLA2 protein.

On the other hand, if the cancer cells in the cancer tissue sample do not express SUCLA2 protein, the specific binding substance cannot bind, and the presence of SUCLA2 protein is not observed. Examples of cases in which cancer cells do not express SUCLA2 protein include cases where the SUCLA2 gene on genomic DNA is homozygously deleted, cases where gene mutations or the like have made it impossible to express SUCLA2 gene mRNA, and cases where gene mutations or the like have made it impossible to translate SUCLA2 gene mRNA.

In addition, the cancer cells in the cancer tissue sample may have reduced expression of SUCLA2 protein compared to the control cells. Examples of such cases include cases where the SUCLA2 gene in the genomic DNA is heterozygously deleted, cases where the expression level of the mRNA of the SUCLA2 gene is reduced due to genetic mutations, and cases where the translation efficiency of the mRNA of the SUCLA2 gene is reduced due to genetic mutations. In such cases, the binding amount of the specific binding substance to the protein extracted from the cancer cells in the cancer tissue sample is reduced compared to the control cells. Here, an example of the control cell is a wild-type cell.

If the cancer cells in the cancer tissue sample do not express SUCLA2 protein or have a reduced expression level of SUCLA2 protein, it can be determined that administration of an anticancer agent containing thymoquinone as an active ingredient is effective in treating the cancer of the cancer patient from whom the cancer tissue sample was derived.

[Method of Determination]
First Embodiment
The determination method of the first embodiment is a method for determining whether administration of an anticancer agent containing thymoquinone as an active ingredient is effective in treating the cancer from which a cancer tissue sample is derived, and is any one of the following methods (i) to (iii):
(i) A method comprising a step of detecting genomic DNA of the SUCLA2 gene in a cancer tissue sample, wherein detection of deletion of the genomic DNA of the SUCLA2 gene indicates that administration of an anticancer agent containing thymoquinone as an active ingredient is effective in treating the cancer from which the cancer tissue sample is derived.
(ii) A method comprising a step of detecting the expression of SUCLA2 gene mRNA in a cancer tissue sample, wherein detection of a decrease in the expression of SUCLA2 gene mRNA indicates that administration of an anticancer agent containing thymoquinone as an active ingredient is effective in treating the cancer from which the cancer tissue sample is derived.
(iii) A method comprising a step of detecting expression of SUCLA2 protein in a cancer tissue sample, wherein detection of a decrease in expression of SUCLA2 protein indicates that administration of an anticancer agent containing thymoquinone as an active ingredient is effective in treating the cancer from which the cancer tissue sample is derived.

Methods including a step of detecting genomic DNA
The determination method of this embodiment may be a method that includes (i) a step of detecting genomic DNA of the SUCLA2 gene in a cancer tissue sample, and detection of deletion of the genomic DNA of the SUCLA2 gene indicates that administration of an anticancer agent containing thymoquinone as an active ingredient is effective in treating the cancer from which the cancer tissue sample is derived.

Detection of the genomic DNA of the SUCLA2 gene can be carried out as described above using the above-mentioned primer set for amplifying the genomic DNA of the SUCLA2 gene, a probe for the genomic DNA of the SUCLA2 gene, etc.

If the cancer cells in the cancer tissue sample have a homozygous or heterozygous deletion of the SUCLA2 gene on the genomic DNA, it can be determined that administration of an anticancer drug containing thymoquinone as an active ingredient is effective in treating the cancer from which the cancer tissue sample is derived.

Methods Including a Step of Detecting Expression at the Gene Level
The determination method of this embodiment may be a method that includes (ii) a step of detecting the expression of mRNA of the SUCLA2 gene in a cancer tissue sample, and detection of a decrease in the expression of mRNA of the SUCLA2 gene indicates that administration of an anticancer agent containing thymoquinone as an active ingredient is effective in treating the cancer from which the cancer tissue sample is derived.

Detection of gene-level expression of the SUCLA2 gene can be carried out as described above using the above-mentioned primer set for amplifying cDNA of the SUCLA2 gene, a probe for the mRNA of the SUCLA2 gene, etc.

When the cancer cells in the cancer tissue sample do not express the mRNA of the SUCLA2 gene, or the expression level of the mRNA of the SUCLA2 gene is reduced compared to the control cells, it can be determined that administration of an anticancer agent containing thymoquinone as an active ingredient is effective in treating the cancer from which the cancer tissue sample is derived. Here, an example of the control cell is a wild-type cell.

Methods Including a Step of Detecting Expression at the Protein Level
The determination method of this embodiment may be a method that includes (iii) a step of detecting expression of SUCLA2 protein in a cancer tissue sample, and detection of a decrease in expression of SUCLA2 protein indicates that administration of an anticancer agent containing thymoquinone as an active ingredient is effective in treating the cancer from which the cancer tissue sample is derived.

Detection of SUCLA2 protein can be carried out as described above using a substance that specifically binds to the SUCLA2 protein.

If the cancer cells in the cancer tissue sample do not express SUCLA2 protein or have a reduced expression level of SUCLA2 protein compared to the control cells, it can be determined that administration of an anticancer agent containing thymoquinone as an active ingredient is effective in treating the cancer from which the cancer tissue sample is derived. Here, an example of the control cell is a wild-type cell.

Second Embodiment
The determination method of the second embodiment is a method for determining whether or not administration of an anticancer drug containing thymoquinone as an active ingredient is effective in treating cancer in a cancer patient, and includes a step of quantifying the content of methylmalonic acid, succinylcarnitine, methylmalonylcarnitine, propionylcarnitine, methylcitric acid or 3-hydroxypropionic acid in a biological sample derived from a cancer patient, and a significantly higher content of methylmalonic acid, succinylcarnitine, methylmalonylcarnitine, propionylcarnitine, methylcitric acid or 3-hydroxypropionic acid compared to a control indicates that administration of an anticancer drug containing thymoquinone as an active ingredient is effective in treating cancer in the cancer patient.

The determination method of the second embodiment is mainly different from the determination method of the first embodiment in that the content of methylmalonic acid, succinylcarnitine, methylmalonylcarnitine, propionylcarnitine, methylcitric acid, or 3-hydroxypropionic acid in a biological sample is used as an index. As described above, SUCLA2 protein is a subunit of an enzyme that converts succinyl-CoA to succinic acid in the TCA cycle. When SUCLA2 protein is deleted or its activity is reduced, methylmalonyl-CoA mutase is inhibited. As a result, it is known that the concentrations of methylmalonic acid, succinylcarnitine, methylmalonylcarnitine, and propionylcarnitine in the blood increase. It is also known that the concentrations of methylmalonic acid, methylcitric acid, and 3-hydroxypropionic acid in the urine increase.

In the determination method of the second embodiment, urine, blood, etc. can be used as the biological sample. The content of methylmalonic acid, succinylcarnitine, methylmalonylcarnitine, propionylcarnitine, methylcitric acid, or 3-hydroxypropionic acid in the biological sample can be quantified by, for example, liquid chromatography (LC)-mass spectrometry (MS), LC-MS/MS, gas chromatography (GC)-MS, GC-MS/MS, etc.

When the content of methylmalonic acid, succinylcarnitine, methylmalonylcarnitine, propionylcarnitine, methylcitric acid or 3-hydroxypropionic acid in a biological sample derived from a cancer patient is significantly higher than that of a control, it can be determined that administration of an anticancer agent containing thymoquinone as an active ingredient is effective in treating the cancer of the cancer patient. Here, for example, a biological sample derived from a healthy individual can be used as the control.

[Screening method]
In one embodiment, the present invention provides a screening method for anticancer agents for cancers involving deletion of a metabolic gene, comprising the steps of culturing a cell line lacking a metabolic gene located near a cancer suppressor gene in the presence of a test substance and measuring the viability of the cell line, wherein a significant decrease in the viability compared to a control indicates that the test substance is a candidate anticancer agent for cancers involving deletion of the metabolic gene.

The screening method of this embodiment makes it possible to screen for anticancer drugs that are effective against cancers involving metabolic gene deletions. There are no particular limitations on the test substance, and for example, natural compound libraries, synthetic compound libraries, existing drug libraries, etc. can be used.

As used herein, metabolic genes refer to genes that code for enzymes that regulate metabolic pathways in energy homeostasis in vivo.

In this specification, "a metabolic gene located near a cancer suppressor gene" refers to a metabolic gene that may be deleted together with a cancer suppressor gene when the cancer suppressor gene is deleted. More specifically, examples of such metabolic genes include those that are located on the same chromosome as the cancer suppressor gene and are located within 500 kb, for example, within 300 kb, for example, within 200 kb, from the cancer suppressor gene.

Specific examples of metabolic genes located near tumor suppressor genes include the SUCLA2 gene located near the tumor suppressor gene RB1 gene, the Methylthioadenosine Phosphorylase (MTAP) gene located near the tumor suppressor genes Cyclin Dependent Kinase Inhibitor 2A (CDKN2A) gene and Cyclin Dependent Kinase Inhibitor 2B (CDKN2B) gene, the Malic Enzyme 2 (ME2) gene located near the tumor suppressor gene SMAD Family Member 4 (SMAD4) gene, and the Acetyl-CoA gene located near the tumor suppressor gene ATM Serine/Threonine Kinase (ATM) gene. These include the Acetyltransferase 1 (ACAT1) gene, the Sterol-C5-Desaturase (SC5D) gene located near the tumor suppressor gene Rho Guanine Nucleotide Exchange Factor 12 (ARHGER12) gene, and the Glutamate Dehydrogenase 1 (GLUD1) gene located near the tumor suppressor gene Phosphatase And Tensin Homolog (PTEN) gene.

In the screening method of this embodiment, the cell line (test subject cell) used for screening may be deleted of a metabolic gene and may retain a nearby cancer suppressor gene, or may be deleted of both a metabolic gene and a nearby cancer suppressor gene. The cell line that has deleted a metabolic gene located near a cancer suppressor gene may be a cell artificially created by genome editing or the like, or may be an established cell line.

In the screening method of this embodiment, the method for measuring the viability of the cell line is not particularly limited, and any commonly used method can be used as appropriate. For example, dead cells may be selectively stained with trypan blue, propidium iodide (PI), or the like, and the ratio of dead cells to the total number of cells may be used as the viability, or the number of live cells may be measured using a method using a tetrazolium compound such as 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), and the viability may be calculated.

When the test cells are cultured in the presence of a test substance and the survival rate of the test cells is significantly reduced compared to the control, the test substance can be determined to be a candidate for an anticancer drug for cancer involving the deletion of a metabolic gene. Here, examples of the control that can be used include wild-type cells, cells in which a metabolic gene has been deleted and a metabolic gene has been further reintroduced, etc.

Whether or not the survival rate of the test cells has decreased compared to that of the control cells may be determined, for example, by calculating the killing ratio according to the following formula (F1).
Killing rate = Viability of test cells / Viability of control cells ... (F1)

When the viability of the test cells is equal to that of the control cells, the kill ratio is 1. A kill ratio value lower than 1 indicates that the viability of the test cells is lower than that of the control cells. A lower kill ratio value indicates that the test substance has specific cytotoxicity to the test cells.

[Other embodiments]
In one embodiment, the present invention provides a method for treating cancer associated with deletion of the SUCLA2 gene, comprising administering to a patient in need of treatment an effective amount of thymoquinone, the cancer associated with deletion of the SUCLA2 gene being as described above.

In one embodiment, the present invention provides a method for treating cancer, comprising the steps of: determining whether or not administration of thymoquinone is effective for treating cancer in a cancer patient by any of the following methods (i) to (iv); and administering an effective amount of thymoquinone to the cancer patient when it is determined that administration of thymoquinone is effective for treating cancer in the cancer patient:
(i) A method comprising a step of detecting genomic DNA of the SUCLA2 gene in a cancer tissue sample derived from a cancer patient, and determining that administration of thymoquinone is effective in treating the cancer in the cancer patient when deletion of the genomic DNA of the SUCLA2 gene is detected.
(ii) A method comprising a step of detecting expression of SUCLA2 gene mRNA in a cancer tissue sample derived from a cancer patient, and determining that administration of thymoquinone is effective in treating the cancer in the cancer patient when a decrease in expression of SUCLA2 gene mRNA is detected.
(iii) A method comprising a step of detecting expression of SUCLA2 protein in a cancer tissue sample derived from a cancer patient, and determining that administration of thymoquinone is effective in treating the cancer in the cancer patient when a decrease in expression of SUCLA2 protein is detected.
(iv) A method for determining that administration of thymoquinone is effective in treating cancer in a cancer patient, comprising a step of quantifying the content of methylmalonic acid, succinylcarnitine, methylmalonylcarnitine, propionylcarnitine, methylcitric acid or 3-hydroxypropionic acid in a biological sample derived from a cancer patient, when the content of methylmalonic acid, succinylcarnitine, methylmalonylcarnitine, propionylcarnitine, methylcitric acid or 3-hydroxypropionic acid is significantly higher than that of a control.

In (iv) above, the biological sample is the same as that described above, and urine, blood, etc. can be used. The content of methylmalonic acid, succinylcarnitine, methylmalonylcarnitine, propionylcarnitine, methylcitric acid, or 3-hydroxypropionic acid in the biological sample can be quantified, for example, by LC-MS, LC-MS/MS, GC-MS, GC-MS/MS, etc. As a control, for example, a biological sample derived from a healthy individual can be used.

In one embodiment, the present invention provides thymoquinone for the treatment of cancers associated with deletion of the SUCLA2 gene, as described above.

In one embodiment, the present invention provides the use of thymoquinone to manufacture a medicament for treating a cancer associated with a deletion of the SUCLA2 gene. The cancer associated with a deletion of the SUCLA2 gene is as described above.

The present invention will now be described in more detail with reference to examples, but the present invention is not limited to the following examples.

[Experimental Example 1]
(RB1 and SUCLA2 gene deletions in various cancers 1)
Using the public cancer genome analysis database (cBioportal), deletions of the RB1 gene and the SUCLA2 gene in various types of cancer were examined.

Figures 1(a) and (b) are graphs showing the analysis results. Figure 1(a) shows the results for the RB1 gene, and Figure 1(b) shows the results for the SUCLA2 gene.

As a result, it was found that deletion of the RB1 gene occurs in various carcinomas, including prostate cancer, serous ovarian cyst, bladder cancer, sarcoma, and invasive breast cancer. Furthermore, it was found that deletion of the SUCLA2 gene occurs in various carcinomas, including prostate cancer, bladder cancer, invasive breast cancer, serous ovarian cyst, glioblastoma, sarcoma, and lung adenocarcinoma.

[Experimental Example 2]
(RB1 and SUCLA2 gene deletions in various cancers 2)
The expression of RB1 protein and SUCLA2 protein in cancer tissue sections from prostate cancer patients was examined by immunostaining. As the cancer tissue sections, a commercially available tissue microarray (product name: "Tissue Microarray Human Tumor Tissue (Prostate Tumor, matched Prostatic Intraepithelial Neoplasia (PIN) and Normal Tissue)", catalog number: "TM0016", Provitro) was used. In this tissue microarray, the patients were blinded to their information.

Table 1 below shows the results of an investigation into the expression of RB1 protein and SUCLA2 protein in cancer tissues derived from prostate cancer patients. As a result, it was revealed that in 6 out of 7 cases of prostate cancer in which RB1 protein expression was lost, SUCLA2 protein expression was also lost. This result indicates that, at least in prostate cancer, both the RB1 gene and the SUCLA2 gene are frequently lost.

[Experimental Example 3]
(Establishment of SUCLA2 knockout cell line)
The SUCLA2 gene of the human prostate cancer cell line DU145 was knocked out by genome editing to produce SUCLA2 knockout cells. Specifically, a DNA fragment in which a restriction enzyme site (BpiI) was added to the complementary strand of the 20-base base sequence of the SUCLA2 gene (CCGAGGCCGGTGGTTCCGAA, SEQ ID NO: 1) was inserted into a PX458 vector (Addgene) treated with the restriction enzyme BpiI to obtain a PX458-sgSUCLA2 vector.

Subsequently, 8 μg of PX458-sgSUCLA2 vector and 2 μg of pcDNA3.1 vector (Thermo Fisher Scientific) were simultaneously introduced into DU145 cells at a concentration of 5 × 10 ⁶ cells/mL. Gene introduction was performed using a NeonTransfection System (Thermo Fisher Scientific) according to the protocol under the conditions of a pulse voltage of 1,250 (V), a pulse width of 2 (ms), a pulse number of 2, and a 100 μL tip.

After gene transfer, the cells were cultured for 72 hours at 5% CO ₂ and 37° C., and then the medium was replaced with a medium containing 400 μg/mL G418 and cultured for 2 weeks to select cells into which both PX458-sgSUCLA2 and pcDNA3.1 had been introduced.

Next, the cells after drug selection were seeded in a 96-well plate at 0.3 cells/well and cultured for 1.5 months to obtain clones. Among the obtained clones, those in which SUCLA2 expression was not confirmed by Western blotting and a frameshift was confirmed in the base sequence of the SUCLA2 gene by Sanger sequencing were used as SUCLA2 knockout cells in the following experiments.

[Experimental Example 4]
(Preparation of recombinant lentiviral vector and virus solution for expressing SUCLA2 gene)
A recombinant lentivirus vector for expressing the SUCLA2 gene was prepared, and a virus solution was prepared. First, total RNA was prepared from DU145 cells. Then, cDNA was synthesized from total RNA using PrimeScript II 1st strand cDNA Synthesis Kit (Takara Bio) according to the protocol.

Next, using the synthesized cDNA as a template, a PCR reaction was performed using KOD plus (Toyobo) to amplify a SUCLA2 gene fragment with the attB1 sequence added to the 5' end, the attB2 sequence added to the 3' end, and the stop codon deleted.

The resulting PCR product was then cloned into the pDONR223 vector using Gateway BP Clonase II Enzyme mix (Thermo Fisher Scientific). The base sequence of the vector was then sequenced using a DNA sequencer 3130 (Thermo Fisher Scientific) to confirm that the desired genetic recombination had occurred.

Next, the SUCLA2 gene fragment cloned into the pDONR223 vector was subcloned into the pLenti6.3/V5-DEST vector (Thermo Fisher Scientific) using Gateway LR Clonase Enzyme mix (Thermo Fisher Scientific). Hereinafter, this vector may be referred to as the "SUCLA2 expression plasmid."

In addition, the LacZ gene fragment was subcloned into the pLenti6.3/V5-DEST vector using a similar method. Hereinafter, this vector may be referred to as the "LacZ expression plasmid."

Next, recombinant lentivirus was produced by the following method: First, 293FT cells (Thermo Fisher Scientific) were seeded in DMEM containing 10% fetal bovine serum (FBS) at 6 x ¹⁰⁶ cells/10 cm dish, and cultured at 37°C under 5% _CO2 for 24 hours.

Next, the packaging plasmid and 1 μg of SUCLA2 expression plasmid were introduced into 293FT cells. As a control, a LacZ expression plasmid was also introduced. Gene introduction was performed by adding 3 μg of DNA and 9 μL of 1 mg/mL polyethyleneimine (molecular weight 15,000) to 1 mL of OPTI-MEM, leaving it at room temperature for 15 minutes, and then adding the mixture dropwise to the medium.

The medium was replaced 18 hours after gene transfer, and collected 48 hours later. The collected medium was filtered through a 0.45 μm PVDF membrane (Sartorius), concentrated using polyethylene glycol, and 8 μg/mL polybrene (Nacalai Tesque) was added to prepare a virus solution.

[Experimental Example 5]
(Establishment of SUCLA2-reconstituted cell line)
The SUCLA2 gene was introduced into the SUCLA2 knockout cells prepared in Experimental Example 3 using the virus solution prepared in Experimental Example 4 to prepare a SUCLA2 reconstituted cell line.

Specifically, first, SUCLA2 knockout cells were seeded in a 6-well plate at 3 × ¹⁰ cells/well. Then, the virus solution prepared in Experimental Example 4 was added to the medium, and the SUCLA2 knockout cells were contacted with the virus for 24 hours to infect the SUCLA2 knockout cells with the virus.

Then, the virus-infected cells were cultured in regular DMEM medium (Fujifilm Wako Pure Chemical Industries, Ltd.) for 24 hours. Then, 2 μg/mL puromycin (Invivogen) was added to the medium and cultured for 48 hours to select virus-infected cells. The obtained cell line was used as SUCLA2-reconstituted cells in the following experiments.

In addition, the virus solution prepared using the LacZ expression plasmid was infected into SUCLA2 knockout cells in the same manner as above, and virus-infected cells were selected. The resulting cell line was used in the following experiment as SUCLA2 knockout cells (control).

[Experimental Example 6]
(Screening of compound libraries)
Using the SUCLA2 knockout cells prepared in Experimental Example 3 and the SUCLA2 reconstituted cells prepared in Experimental Example 5, a compound exhibiting cytotoxicity specific to SUCLA2 knockout cells was screened.

Specifically, each compound contained in the compound library (Enzo Life Sciences) was added to the medium of SUCLA2 knockout cells and SUCLA2 reconstituted cells at a final concentration of 5 μg/mL and cultured for 48 hours. Then, the viability of each cell was measured using a commercially available kit (product name "Cell count reagent SF", Nacalai Tesque). Then, the killing ratio was calculated according to the following formula (F2).
Killing ratio = viability of SUCLA2-reconstituted cells / viability of SUCLA2 knockout cells ... (F2)

Figure 2 is a graph showing the results of the screening. The vertical axis of the graph shows the killing rate, and the horizontal axis shows the compound number. A higher killing rate value indicates more cytotoxicity specific to SUCLA2 knockout cells. As a result, it was revealed that thymoquinone has cytotoxicity specific to SUCLA2 knockout cells.

[Experimental Example 7]
(Verification of SUCLA2 deletion dependency of thymoquinone cytotoxicity)
We investigated whether the cytotoxicity of thymoquinone is SUCLA2 deletion-dependent. Specifically, the SUCLA2 knockout cells prepared in Experimental Example 3 and the SUCLA2 reconstituted cells prepared in Experimental Example 5 were seeded at 3 x 105 cells/6 cm dish. At the same time as seeding, 0, 5, ¹⁰ , 15, 20, and 25 μg/mL of thymoquinone was added to the medium, and the cells were cultured at 37°C under 5% _CO2 for 48 hours.

Then, the culture supernatant and remaining cells were collected, and an equal volume of 0.5% trypan blue (Nacalai Tesque) was added to the cell suspension and mixed. The total cell count and the number of trypan blue-positive cells (dead cells) were then measured using a fully automated cell counter TC20 (Bio-Rad). The ratio of the number of trypan blue-negative cells to the total cell count was then calculated as the cell viability.

In some cases, cell viability was calculated by the following method: First, the SUCLA2 knockout cells prepared in Experimental Example 3 and the SUCLA2 reconstituted cells prepared in Experimental Example 5 were seeded on a 96-well plate at 1 x ¹⁰⁴ cells/well. Simultaneously with the seeding, 0, 5, 10, 15, 20, and 25 μg/mL thymoquinone was added to the medium, and the cells were cultured at 5% _CO2 and 37°C for 48 hours.

Next, 10 μL of the viable cell count measurement reagent SF (Nacalai Tesque) was added to each well and allowed to react for 2 hours. The absorbance at 460 nm was then measured using a microplate reader (product name "iMark", Bio-Rad). The cell viability was calculated by subtracting the blank absorbance from the absorbance of cells to which thymoquinone had not been added, and taking this value as 100%.

Figure 3 is a graph of a dose-response curve created based on cell viability. In Figure 3, "-R" indicates the results for SUCLA2-reconstituted cells, and "KO" indicates the results for SUCLA2 knockout cells. The horizontal axis of Figure 3 indicates the concentration of thymoquinone, and the vertical axis indicates the response rate (%).

As a result, the _IC50 (50% inhibitory concentration) of thymoquinone against SUCLA2 knockout cells was 5.77 μg/mL, and the _IC50 of thymoquinone against SUCLA2-reconstituted cells was 18 μg/mL, indicating that the cytotoxicity of thymoquinone is SUCLA2 deletion-dependent.

[Experimental Example 8]
(Study of cytotoxicity of thymoquinone)
We investigated whether cytotoxicity caused by thymoquinone was accompanied by apoptosis. Specifically, the SUCLA2 knockout cells prepared in Experimental Example 3 and the SUCLA2 reconstituted cells prepared in Experimental Example 5 were seeded at 6 x ¹⁰⁵ cells/6 cm dish. At the same time as seeding, 10 μg/mL thymoquinone was added to the medium, and the cells were cultured at 5% _CO2 and 37°C for 48 hours. As a control, cells were also prepared to which only the solvent (DMSO) was added instead of thymoquinone.

Next, the floating and adherent cells in the medium were collected and washed twice with phosphate-buffered saline (PBS). Annexin V-positive cells were then stained using the eBioscience Annexin V-FITC Apoptosis Detection Kit (Thermo Fisher Scientific). Dead cells were stained with propidium iodide (PI).

Then, each cell was analyzed using FACS Canto II (BD Biosciences). Cells with high Annexin V-FITC fluorescence values and low PI fluorescence values were early apoptotic cells, and cells with high Annexin V-FITC and PI fluorescence values were late apoptotic cells.

Figure 4(a) is a graph showing the results of adding DMSO to SUCLA2 knockout cells. Figure 4(b) is a graph showing the results of adding thymoquinone to SUCLA2 knockout cells. Figure 4(c) is a graph showing the results of adding DMSO to SUCLA2 reconstituted cells. Figure 4(d) is a graph showing the results of adding thymoquinone to SUCLA2 reconstituted cells. In Figures 4(a) to (d), the vertical axis shows the fluorescence intensity of PI, and the horizontal axis shows the fluorescence intensity of Annexin V-FITC.

Figures 5(a) and (b) are graphs prepared based on the results of Figures 4(a) to (d). Figure 5(a) is a graph showing the percentage of cells that underwent apoptosis, and Figure 5(b) is a graph showing the percentage of live cells. In Figures 5(a) and (b), "LacZ" indicates the results for SUCLA2 knockout cells, "A2R" indicates the results for SUCLA2 reconstituted cells, "DMSO" indicates the results after addition of dimethyl sulfoxide, and "TQ" indicates thymoquinone. As a result, it was revealed that the cytotoxicity of SUCLA2-deficient cells by thymoquinone is accompanied by apoptosis.

[Experimental Example 9]
(In vivo efficacy evaluation of thymoquinone by repeated administration)
The SUCLA2 knockout cells prepared in Experimental Example 3 and the SUCLA2 reconstituted cells prepared in Experimental Example 5 were each transplanted at a cell number of 5 x ¹⁰⁶ /mouse together with Matrigel (BD Biosciences) into the dorsal subcutaneous tissue of KSN/slc mice (8 weeks old, male) to prepare a tumor-bearing mouse model. The tumor-bearing mouse model was divided into a TQ administration group in which thymoquinone was administered frequently, and a control group in which only a solvent was administered instead of thymoquinone, with 5 mice in each group.

After the average tumor diameter of each individual reached 7 mm, the TQ-treated group was intraperitoneally administered thymoquinone at 20 mg/kg body weight five times every 48 hours. Meanwhile, the control group was intraperitoneally administered the solvent alone five times at the same timing as the TQ-treated group. Corn oil for biochemistry (Fujifilm Wako Pure Chemical Industries) was used as the solvent.

For mice in each group, tumor volume was measured 0, 3, 6, 9, and 11 days after the start of thymoquinone administration. Tumor volume was measured as follows. The long and short diameters of the tumors formed subcutaneously on the back were measured using digital calipers, and the tumor volume was calculated as (long diameter mm × short diameter mm × short diameter mm)/2.

24 hours after the final administration, tumor masses formed subcutaneously on the back were excised, and tumor weight and individual body weight were measured. GraphPad Prism was used to compare tumor weight and tumor volume between groups. Tukey-Kramer tests were performed between the control group and the TQ-administered group for each cell type for the values of tumor weight and tumor volume on each day, and significant differences were determined at the P<0.01 level in a two-sided test.

Fig. 6 is a graph showing the results of measuring tumor volume. In Fig. 6, the horizontal axis indicates the time (days) from the start of administration of thymoquinone, and the vertical axis indicates the average tumor volume (mm ³ ). In addition, "KO Cont." indicates the results in the control group of tumors formed by transplanting SUCLA2 knockout cells, "-R Cont." indicates the results in the control group of tumors formed by transplanting SUCLA2-reconstituted cells, "KO+TQ" indicates the results in the TQ-administered group of tumors formed by transplanting SUCLA2 knockout cells, and "-R+TQ" indicates the results in the TQ-administered group of tumors formed by transplanting SUCLA2-reconstituted cells.

Figures 7(a) and (b) are representative photographs of control and TQ-treated mice 11 days after the start of thymoquinone administration. Figure 7(a) shows a control mouse, and Figure 7(b) shows a TQ-treated mouse. In Figures 7(a) and (b), "SUCLA2 KO" indicates a tumor formed by transplanting SUCLA2 knockout cells, and "SUCLA2 R" indicates a tumor formed by transplanting SUCLA2-reconstituted cells.

Figure 8 shows photographs of tumor masses excised from mice in the control and TQ-administered groups 11 days after the start of thymoquinone administration. In Figure 8, "Control" shows a photograph of a tumor mass excised from a mouse in the control group, and "TQ" shows a photograph of a tumor mass excised from a mouse in the TQ-administered group. In Figure 8, "SUCLA2-KO" shows a tumor formed by transplanting SUCLA2 knockout cells, and "SUCLA2-R" shows a tumor formed by transplanting SUCLA2-reconstituted cells.

Figure 9 is a graph showing the results of measuring the tumor weight of the tumor mass shown in Figure 8. In Figure 9, the vertical axis of the graph indicates tumor weight, "A2 KO" indicates a tumor formed by transplanting SUCLA2 knockout cells, "A2 R" indicates a tumor formed by transplanting SUCLA2 reconstituted cells, "Cont" indicates the control group, and "TQ" indicates the TQ administration group. In addition, "*" indicates a significant difference at p<0.05, "**" indicates a significant difference at p<0.01, and "ns" indicates no significant difference.

As a result, it was found that administration of thymoquinone significantly reduced the volume of tumors formed by transplantation of SUCLA2 knockout cells. This result indicates that thymoquinone has high cytotoxicity against cancers with SUCLA2 gene deletion in vivo.

[Experimental Example 10]
(Immunohistological analysis of cancer tissue in a tumor-bearing mouse model administered thymoquinone repeatedly)
In Experimental Example 10, tumor masses excised from the TQ-administered group and the control group were fixed overnight at 4°C in 4% paraformaldehyde and embedded in paraffin according to a standard method. Then, 4 μm-thick slices were prepared from the paraffin-embedded tumor samples. Then, the samples were deparaffinized using Histo-Clear (Cosmo Bio), and the Histo-Clear (Cosmo Bio) was removed with ethanol. Then, the samples were washed with running tap water for 10 minutes, followed by washing three times with PBS for 5 minutes. Then, hematoxylin-eosin staining and immunohistochemical staining were performed according to a standard protocol.

For immunohistochemical staining, antigen activation was performed by heating with 10 mM citrate buffer (pH 6) for 10 minutes, and after cooling at room temperature, washing was performed twice with distilled water for 5 minutes. Subsequently, endogenous _peroxidase activation was performed with 3% _H2O2 for 10 minutes, followed by washing with running water and PBS.

Next, blocking was performed using PBS containing 1% bovine serum albumin (BSA), 5% normal goat serum, and 0.1% Triton-X100. The primary antibody was then reacted. Anti-cleaved caspase 3 antibody (Cell Signaling Technology) was used as the primary antibody. Each antibody was diluted 400-fold and incubated overnight at 4°C.

Next, EnVision+Dual Link System-HRP (a type for both mouse and rabbit primary antibodies, Dako) was dropped onto the sections as a secondary antibody, and the HRP-conjugated polymer was allowed to react. Then, the sections were washed three times with PBS for 5 minutes each, and a color reaction was carried out using Liquid DAB+ Substrate Chromogen System (Dako).

The color reaction time was 5 minutes. After the color reaction, the sections were washed with PBS for 5 minutes three times, counterstained with hematoxylin solution (Sakura Finetech Japan), dehydrated, and cleared. Then, the sections were mounted using VectaMount Permanent Mounting Medium (Vector Laboratories).

Subsequently, microscopic observation was performed to measure the number of cleaved caspase 3-positive cells. Figure 10 is a graph showing the results of measuring the number of cleaved caspase 3-positive cells in one field of view. In Figure 10, "Cont." indicates the result of tumor masses excised from the control group, "TQ" indicates the result of tumor masses excised from the TQ-administered group, "KO" indicates the result of tumors formed by transplanting SUCLA2 knockout cells, and "-R" indicates the result of tumors formed by transplanting SUCLA2-reconstituted cells.

The results showed that administration of thymoquinone to tumors formed by transplanting SUCLA2 knockout cells significantly increased the number of cleaved caspase 3-positive cells.

Incidentally, Geminin protein is a protein whose expression is known to decrease specifically in the G1 phase of the cell cycle. Further investigation revealed that administration of thymoquinone to tumors formed by transplanting SUCLA2 knockout cells significantly reduced the number of Geminin-positive cells.

[Experimental Example 11]
(Expression analysis of cell death-inducing genes using Cap analysis gene expression (CAGE) sequencing method)
The SUCLA2 reconstituted cells prepared in Experimental Example 5 were seeded at 1.2 x ¹⁰⁶ cells/10 cm dish. After culturing for 24 hours, thymoquinone was added to a final concentration of 10 μg/mL, and the cells were cultured at 5% _CO2 and 37°C for 6 hours. Total RNA was then recovered from the cells and purified. RNA purification was performed using RNeasy Mini Kit (Qiagen) according to the protocol.

RNA with a purity of 7.0 or higher, and A260/280 and 260/230 of 1.7 or higher, as measured using a Bioanalyzer 2100 (Agilent Technologies), was used to create the CAGE library.

First, cDNA was synthesized from the 5'-end cap of RNA using reverse transcriptase, and an arbitrary CAGE barcode tag was added at the same time. Next, the CAGE-tagged cDNA was sequenced using NextSeq 550 (Illumina), and RNA containing ribosomes and bases other than A/C/G/T was removed from the obtained sequence, after which it was mapped to the human hg19 genome using Burrows-Wheeler Aligner software (v0.5.9).

Gene expression was analyzed by clustering the mapped tags using the REproducible CLUSTERING method. However, genes that did not meet the maximum irreproducible discovery rate of 0.1 and the minimum tags per million of 0.1 were excluded.

The detection of differentially expressed genes and gene ontology analysis were performed using the Weighted Average Differentiation method. Furthermore, the dataset analysis of differentially expressed genes was performed using Gene Set Enrichment Analysis. The results of the gene ontology analysis are shown in Table 2 below. In Table 2, "GO Term" indicates biological process, and "FDR" indicates false discovery rate. As a result, it was revealed that thymoquinone treatment causes changes in the expression of genes involved in the execution of apoptosis.

[Experimental Example 12]
(Study of the apoptosis-inducing effect of thymoquinone)
The SUCLA2 knockout cells prepared in Experimental Example 3 and the SUCLA2 reconstituted cells prepared in Experimental Example 5 were seeded at 6 x ¹⁰⁵ cells/6 cm dish. Simultaneously with the seeding, thymoquinone was added to the medium to a final concentration of 10 µg/mL, and the cells were cultured for 6 hours.

Next, proteins were extracted using a RIPA modified cell lysis buffer, and the protein concentration was measured using the TaKaRa BCA Protein Assay Kit (Takara Bio Inc.), followed by SDS-polyacrylamide gel electrophoresis (PAGE) and transfer onto a PVDF membrane. Next, cleaved caspase 3 and cleaved Poly(ADP-Ribose) Polymerase (PARP) were detected by Western blotting to evaluate the apoptosis-inducing effect.

Figure 11 is a photograph showing the results of Western blotting. In Figure 11, "KO" indicates the results for SUCLA2 knockout cells, and "-R" indicates the results for SUCLA2 reconstituted cells. "DM" indicates the results when DMSO was added as a control, and "TQ" indicates the results when thymoquinone was added. "Cont." indicates the results for the parent cell line. As a result, it was revealed that the protein amount of cleaved caspase 3, which is the molecule that executes apoptosis, increased in the thymoquinone-treated group.

[Experimental Example 13]
(Z-VAD-FMK inhibits the apoptosis-inducing effect of thymoquinone)
The SUCLA2 knockout cells prepared in Experimental Example 3 and the SUCLA2 reconstituted cells prepared in Experimental Example 5 were seeded at 6 x ¹⁰⁵ cells/6 cm dish. Simultaneously with the seeding, thymoquinone was added to the medium at a final concentration of 10 μg/mL either alone or together with a cell-permeable pan-caspase inhibitor, Z-VAD-FMK (Promega), at a final concentration of 20 μM, and the cells were cultured at 5% _CO2 and 37°C for 48 hours.

Next, the floating and adherent cells in the medium were collected and washed twice with PBS. Next, Annexin V-positive cells were stained using eBioscience Annexin V-FITC Apoptosis Detection Kit (Thermo Fisher Scientific). Dead cells were stained with propidium iodide (PI). Next, each cell was analyzed using FACS Canto II (BD Biosciences).

Figure 12 is a graph showing the results of flow cytometry analysis. In Figure 12, the vertical axis indicates the percentage of annexin V positive cells (cells undergoing apoptosis), "SUCLA2-KO" indicates the results for SUCLA2 knockout cells, and "SUCLA2-R" indicates the results for SUCLA2 reconstituted cells. "DMSO" indicates the results when DMSO was added as a control, "TQ" indicates the results when thymoquinone was added, and "TQ+Z-VAD-FMK" indicates the results when thymoquinone and Z-VAD-FMK were added. "N.S." indicates that no significant difference was observed.

As a result, it was found that administration of thymoquinone induced apoptosis in SUCLA2 knockout cells, and that addition of Z-VAD-FMK together with thymoquinone inhibited apoptosis.

The present invention aims to provide a new technology for treating cancer associated with deletion of the SUCLA2 gene.

Claims (2)

An anticancer agent for prostate cancer associated with deletion of the Succinate-CoA Ligase ADP-Forming Beta Subunit (SUCLA2) gene, comprising as an active ingredient a compound represented by the following formula (1):

The anticancer agent according to claim 1, wherein the prostate cancer further lacks the RB Transcriptional Corepressor 1 (RB1) gene.

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