KR20190115812A - Pharmaceutical composition for treating xCT inhibitor- resistant cancer - Google Patents

Abstract

The present invention relates to a composition for enhancing sensitivity to xCT inhibitors comprising an inhibitor or activity inhibitor of CISD2 protein as an active ingredient. The present invention also relates to a method of providing information on determining whether to administer an xCT inhibitor or a method of screening a substance for enhancing sensitivity of an xCT inhibitor. The composition of the present invention can be effectively used as an anticancer agent or anticancer adjuvant by effectively overcoming the resistance to pereptosis apoptosis caused by xCT inhibitors.

Description

Pharmaceutical composition for treating xCT inhibitor-resistant cancer

The present invention relates to a composition for enhancing sensitivity to an xCT (system x _c ^- cystine / glutamate antiporter) inhibitor including a CISD2 (CDGSH iron sulfur domain 2) inhibitor.

Sulfasalazine (Sulfasalazine, SAS) is widely used to treat inflammatory arthritis and intestinal diseases, including rheumatoid arthritis, ulcerative colitis and Crohn's disease (Pouillon L et al., 2017). The drug has been reused to induce the death of therapeutic resistant cancer cells in combination with monotherapy or other chemotherapy drugs or radiation therapy (Sleire L et al., 2015; Lo M et al., 2010; Shitara K et al. ., 2017). SAS inhibits xCT (system x _c ^-- cystine / glutamate antiporter) to show anti-inflammatory and anti-cancer effects (Guan J et al., 2009). xCT exchanges extracellular cystine for intracellular glutamate as a source of glutathione (GSH), a major cellular antioxidant (Dixon SJ et al., 2014). SAS-induced cystine deficiency significantly inhibits significant reduction of intracellular GSH and tumor growth in vivo without severe toxicity (Doxsee DW et al., 2007). Inhibition of SAS-induced xCT sensitizes cancer cells to ferroptosis, a recently known form of iron-dependent, non-apototic cell death (Dixon SJ et al., 2012).

Ferroptosis is distinguished from apoptosis, necroptosis and autophagic cell death and is a new form of cell death through iron accumulation and lipid peroxidation. Major molecules related to ferroptosis include glutathione peroxidase (GPX4), an essential regulator of xCT and ferroptosis that inhibits lipid peroxidation (Yang WS et al., 2014). Inhibition of xCT and GPX4 can eradicate cancer cells resistant to conventional chemotherapy or radiation therapy (Xie Y et al., 2016). Inhibition of xCT induces GSH deficiency by blocking cystine uptake and makes cancer cells sensitive to chemotherapy agents (Yoshikawa M et al., 2013; Liu DS et al., 2017). GPX4 inhibition is also mesenchymal treatment resistant, making sensitive cancer cells susceptible to ferroptotic cancer cell death (Hangauer MJ et al., 2017).

Nutrient-deprivation autophagy factor-1 (NAF-1) is one of the iron-sulfur (FeS) protein families, encoded by CDSDSH iron sulfur domain 2 (CISD2) (Tamir S et al., 2013). This protein transfers 2Fe-2S clusters into apo-acceptor proteins and iron into mitochondria, which plays an important role in neurodevelopment, skeletal muscle maintenance and longevity (Chen YF et al., 2010). Overexpression of NAF-1 or mitoNEET is associated with aggressive phenotypes and clinical outcomes of various human cancers, the silence of which suppresses tumor proliferation (Sohn YS et al., 2013; Wang L et al., 2016; Chen B et al., 2015; Yang L et al., 2016). mitoNEET has recently been reported as a negative regulator of ferroptotic cancer cell death that prevents mitochondrial lipid peroxidation (Yuan H et al., 2016).

Resistance to xCT inhibition allows treatment resistant cancer cells to avoid cell death, including ferroptosis. NAF-1 is a protein that interacts with mitoNEET and is likely to be involved in the mechanism of resistance to ferroptotic cancer cell death. A deeper understanding of the mechanism of resistance to ferroptosis will facilitate the implementation of new approaches to overcome cancer resistance.

The matters described as the background art are only for the purpose of improving the understanding of the background of the present invention and should not be taken as acknowledging that they correspond to the related art already known to those skilled in the art.

1.Pouillon L, Bossuyt P, Vanderstukken J, Moulin D, Netter P, Danese S, et al. Management of patients with inflammatory bowel disease and spondyloarthritis. Expert review of clinical pharmacology 2017; 10: 1363-74 Sleire L, Skeie BS, Netland IA, Forde HE, Dodoo E, Selheim F, et al. Drug repurposing: sulfasalazine sensitizes gliomas to gamma knife radiosurgery by blocking cystine uptake through system Xc-, leading to glutathione depletion. Oncogene 2015; 34: 5951-9 Lo M, Ling V, Low C, Wang YZ, Gout PW. Potential use of the anti-inflammatory drug, sulfasalazine, for targeted therapy of pancreatic cancer. Current oncology (Toronto, Ont) 2010; 17: 9-16 Shitara K, Doi T, Nagano O, Fukutani M, Hasegawa H, Nomura S, et al. Phase 1 study of sulfasalazine and cisplatin for patients with CD44v-positive gastric cancer refractory to cisplatin (EPOC1407). Gastric cancer: official journal of the International Gastric Cancer Association and the Japanese Gastric Cancer Association 2017; 20: 1004-9 Guan J, Lo M, Dockery P, Mahon S, Karp CM, Buckley AR, et al. The xc-cystine / glutamate antiporter as a potential therapeutic target for small-cell lung cancer: use of sulfasalazine. Cancer chemotherapy and pharmacology 2009; 64: 463-72 Dixon SJ, Patel DN, Welsch M, Skouta R, Lee ED, Hayano M, et al. Pharmacological inhibition of cystine-glutamate exchange induces endoplasmic reticulum stress and ferroptosis. eLife 2014; 3: e02523 Doxsee DW, Gout PW, Kurita T, Lo M, Buckley AR, Wang Y, et al. Sulfasalazine-induced cystine starvation: potential use for prostate cancer therapy. The Prostate 2007; 67: 162-71 Dixon SJ, Lemberg KM, Lamprecht MR, Skouta R, Zaitsev EM, Gleason CE, et al. Ferroptosis: an iron-dependent form of nonapoptotic cell death. Cell 2012; 149: 1060-72 Yang WS, Sri Ramaratnam R, Welsch ME, Shimada K, Skouta R, Viswanathan VS, et al. Regulation of ferroptotic cancer cell death by GPX4. Cell 2014; 156: 317-31 Xie Y, Hou W, Song X, Yu Y, Huang J, Sun X, et al. Ferroptosis: process and function. Cell death and differentiation 2016; 23: 369-79 Yoshikawa M, Tsuchihashi K, Ishimoto T, Yae T, Motohara T, Sugihara E, et al. X CT inhibition depletes CD44v-expressing tumor cells that are resistant to EGFR-targeted therapy in head and neck squamous cell carcinoma. Cancer research 2013; 73: 1855-66 Liu DS, Duong CP, Haupt S, Montgomery KG, House CM, Azar WJ, et al. Inhibiting the system xC (-) / glutathione axis selectively targets cancers with mutant-p53 accumulation. Nature communications 2017; 8: 14844 Hangauer MJ, Viswanathan VS, Ryan MJ, Bole D, Eaton JK, Matov A, et al. Drug-tolerant persister cancer cells are vulnerable to GPX4 inhibition. Nature 2017; 551: 247-50 Tamir S, Zuris JA, Agranat L, Lipper CH, Conlan AR, Michaeli D, et al. Nutrient-deprivation autophagy factor-1 (NAF-1): biochemical properties of a novel cellular target for anti-diabetic drugs. PloS one 2013; 8: e61202 Chen YF, Wu CY, Kirby R, Kao CH, Tsai TF. A role for the CISD2 gene in lifespan control and human disease. Annals of the New York Academy of Sciences 2010; 1201: 58-64 Sohn YS, Tamir S, Song L, Michaeli D, Matouk I, Conlan AR, et al. NAF-1 and mitoNEET are central to human breast cancer proliferation by maintaining mitochondrial homeostasis and promoting tumor growth. Proceedings of the National Academy of Sciences of the United States of America 2013; 110: 14676-81 Wang L, Ouyang F, Liu X, Wu S, Wu HM, Xu Y, et al. Overexpressed CISD2 has prognostic value in human gastric cancer and promotes gastric cancer cell proliferation and tumorigenesis via AKT signaling pathway. Oncotarget 2016; 7: 3791-805 Chen B, Shen S, Wu J, Hua Y, Kuang M, Li S, et al. CISD2 associated with proliferation indicates negative prognosis in patients with hepatocellular carcinoma. International journal of clinical and experimental pathology 2015; 8: 13725-38 Yang L, Hong S, Wang Y, He Z, Liang S, Chen H, et al. A novel prognostic score model incorporating CDGSH iron sulfur domain2 (CISD2) predicts risk of disease progression in laryngeal squamous cell carcinoma. Oncotarget 2016; 7: 22 720-32 Yuan H, Li X, Zhang X, Kang R, Tang D. CISD1 inhibits ferroptosis by protection against mitochondrial lipid peroxidation. Biochemical and biophysical research communications 2016; 478: 838-44

The present inventors made an effort to elucidate the mechanism by which xCT inhibitors useful as existing cancer therapeutic agents acquire resistance in connection with cell death including pereptosis. As a result, the overexpression of CISD2 confers resistance to pereptosis of the xCT inhibitor, and the present invention was completed by revealing that inhibition of CISD2 can overcome the pereptosis resistance induced by the xCT inhibitor.

Accordingly, it is an object of the present invention to provide a composition for enhancing sensitivity to xCT inhibitors comprising an inhibitor or activity inhibitor of CISD2 protein as an active ingredient.

Another object of the present invention is to provide a pharmaceutical composition for preventing or treating cancer, comprising an activity inhibitor or expression inhibitor of CISD2 protein, and an xCT inhibitor as an active ingredient.

Another object of the present invention to provide an anti-cancer adjuvant for xCT inhibitor resistant cancer comprising an active inhibitor or expression inhibitor of CISD2 protein as an active ingredient.

It is another object of the present invention to provide a method of providing information on determining whether to administer an xCT inhibitor.

It is another object of the present invention to provide a method for screening a substance for enhancing sensitivity of an xCT inhibitor.

Other objects and advantages of the present invention will become apparent from the following detailed description, claims and drawings.

According to an aspect of the present invention, the present invention provides a composition for enhancing sensitivity to xCT (system x _c ^- cystine / glutamate antiporter) inhibitors containing an inhibitor or activity inhibitor of CISD2 (CDGSH iron sulfur domain 2) protein as an active ingredient To provide.

The inventors have discovered the mechanism by which xCT inhibitors acquire resistance in relation to cell death, including pereptosis, and overexpression of CISD2 confers resistance to pereptosis of xCT inhibitors, and when the xSD inhibitor inhibits CISD2, It was confirmed that it is possible to overcome the resistance to pereptosis caused by.

The xCT inhibitor is not limited, but is preferably sulfasalazine (Sulfasalazine, SAS), carboxyphenylglycine (CPG), elastin, monosodium glutamate, aminoadipate, aminopimel Aminopimelate, homocysteate, L-serine-O-sulphate, ibotenate, bromomoibotenate and quisqualate ( quisqualate).

The activity inhibitor or expression inhibitor of the CISD2 protein is characterized by enhancing the sensitivity of the xCT inhibitor.

The expression inhibitor of the CISD2 protein is a group consisting of antisense nucleotides, short interfering RNAs (siRNAs), short hairpin RNAs (shRNAs), and miRNAs, which complementarily bind to mRNAs of genes encoding CISD2 proteins. It is preferably any one selected from.

The activity inhibitor of the CISD2 protein is preferably any one selected from the group consisting of compounds, peptides, peptide mimetics, aptamers, antibodies, and natural products that specifically bind to CISD2 protein.

The compound specifically binding to the CISD2 protein as an activity inhibitor of the CISD2 protein is preferably thiazolidinedione, more preferably pioglitazone (PGZ) or rosiglitazone (RGZ).

According to a preferred embodiment of the present invention, the composition of the present invention is to be used when treating a cancer disease using an xCT inhibitor.

The cancer is preferably head and neck cancer, lung cancer, ovarian cancer, colon cancer, colon cancer, pancreatic cancer, liver cancer, cervical cancer, kidney cancer, gastric cancer, prostate cancer, breast cancer, brain tumor, uterine cancer, bladder cancer and blood cancer It is selected from the group consisting of, more preferably is selected from the group consisting of head and neck cancer, lung cancer, ovarian cancer and colorectal cancer.

Antisense  Nucleotide

Antisense nucleotides, as defined in Watson-click base pairs, bind (hybridize) the complementary sequences of DNA, immature-mRNA or mature mRNA to disrupt the flow of genetic information as a protein in DNA. The nature of antisense nucleotides specific to the target sequence makes them exceptionally multifunctional. Since antisense nucleotides are long chains of monomeric units they can be easily synthesized for the target RNA sequence. Many recent studies have demonstrated the utility of antisense nucleotides as biochemical means for studying target proteins (Rothenberg et al., J. Natl. Cancer Inst., 81: 1539-1544, 1999). The use of antisense nucleotides can be considered as a novel form of inhibitor because of recent advances in nucleotide synthesis and in the field of nucleotide synthesis exhibiting improved cell line uptake, target binding affinity, and nuclease resistance.

Peptide Mimetics (Peptide Minetics )

Peptide Minetics are peptides or nonpeptides that inhibit the binding domain of CISD2 protein leading to CISD2 activity. Major residues of the non-hydrolyzable peptide analogs include β-turn dipeptide cores (Nagai et al. Tetrahedron Lett 26: 647, 1985), keto-methylene pseudopeptides (Ewenson et al. J Med chem 29: 295, 1986; And Ewenson et al. In Peptides: Structure and Function (Proceedings of the 9th AmeriCan Peptide Symposium) Pierce chemi Cal co. Rockland, IL, 1985), Huffman et al. In Peptides: chemistry and Biology, GR Marshall ed., EScOM Publisher: Leiden, Netherlands, 1988), benzodiazepines (Freidinger et al. In Peptides; chemistry and Biology, GR Marshall ed., EScOM Publisher: Leiden, Netherlands, 1988), β-aminoalcohol (Gordon et al. Biochem Biophys Res commun 126: 419 1985) and substituted gamma lactam ring (Garvey et al. in Peptides: chemistry and Biology, GR Marshell ed., EScOM Publisher: Leiden, Netherlands, 1988).

siRNA  molecule

The sense RNA and the antisense RNA form a double stranded RNA molecule, wherein the sense RNA is preferably an siRNA molecule comprising a nucleic acid sequence identical to the target sequence of a contiguous nucleotide of some of the CISD2 mRNA. The siRNA molecule is preferably composed of a sense sequence consisting of 10 to 30 bases selected from the base sequence of the CISD2 gene and an antisense sequence complementarily binding to the sense sequence, but is not limited thereto. Any double-stranded RNA molecule having a sense sequence capable of complementarily binding to a nucleotide sequence can be used. Most preferably, the antisense sequence has a sequence complementary to the sense sequence.

Antibodies

CISD2 antibodies can be used both prepared by CISD2 injection or purchased commercially. In addition, the antibodies include polyclonal antibodies, monoclonal antibodies, fragments capable of binding epitopes, and the like.

Polyclonal antibodies can be produced by conventional methods of injecting CISD2 into an animal and collecting blood from the animal to obtain a serum comprising the antibody. Such polyclonal antibodies can be purified by any method known in the art and can be made from any animal species host, such as goats, rabbits, sheep, monkeys, horses, pigs, cattle, dogs, chickens, and the like.

Monoclonal antibodies can be prepared using any technique that provides for the production of antibody molecules through the culture of continuous cell lines. Such techniques include, but are not limited to, hybridoma technology, human B-cell line hybridoma technology, and EBV-hybridoma technology (Kohler G et al., Nature 256: 495-497, 1975; Kozbor). D et al., J Immunol Methods 81: 31-42, 1985; cote RJ et al., Proc Natl ACad Sci 80: 2026-2030, 1983; and cole SP et al., Mol cell Biol 62: 109-120, 1984).

In addition, antibody fragments containing specific binding sites for the CISD2 can be prepared. For example, but not limited to, F (ab ') 2 fragments can be prepared by digesting antibody molecules with pepsin, and Fab fragments can be prepared by reducing the disulfide bridges of F (ab') 2 fragments. Alternatively, the Fab expression library can be made small to quickly and simply identify monoclonal Fab fragments with the desired specificity (Huse WD et al., Science 254: 1275-1281, 1989).

The antibody can be bound to a solid substrate to facilitate subsequent steps such as washing or separation of the complex. Solid substrates include synthetic resins, nitrocellulose, glass substrates, metal substrates, glass fibers, microspheres and microbeads. In addition, the synthetic resins include polyester, polyvinyl chloride, polystyrene, polypropylene, PVDF and nylon.

Aptamers ( Aptamer )

Aptamers are single-stranded nucleic acids (DNA, RNA or modified nucleic acids) that have a stable tertiary structure and are capable of binding to target molecules with high affinity and specificity. Aptamers have been developed since the first development of an aptamer excavation technology called Systematic Evolution of Ligands by EXponential enrichment (Ellington, AD and Szostak, JW., Nature 346: 818-822, 1990). Many aptamers have been unearthed that can bind to various target molecules. Aptamers are compared to single antibodies because of their inherent high affinity (usually pM levels) and their specificity to bind to target molecules, and thus have high potential as alternative antibodies, particularly as "chemical antibodies."

In one embodiment of the present invention, it was confirmed that apoptosis was induced when CISD2 siRNA was treated to colorectal cancer cell lines resistant to xCT inhibitors. Moreover, tumor growth in vivo was significantly inhibited when CISD2 inhibitors and xCT inhibitors were injected in tumor-grafted mice. Therefore, CISD2 protein expression or activity inhibitor may be usefully used as an active ingredient of a composition for enhancing sensitivity to xCT inhibitors.

The composition may contain one or more active ingredients exhibiting the same or similar function in addition to the CISD2 expression or activity inhibitor.

The composition can be administered orally or parenterally during clinical administration and intraperitoneal injection, rectal injection, subcutaneous injection, intravenous injection, intramuscular injection, intrauterine dural injection, cerebrovascular injection or intrathoracic injection during parenteral administration. And can be used in the form of general pharmaceutical formulations.

The composition can be used alone or in combination with methods using surgery, radiation therapy, hormone therapy, chemotherapy and biological response modifiers.

The daily dosage of the composition is about 0.0001 to 100 mg / kg, preferably 0.001 to 10 mg / kg, preferably administered once or several times a day, but the weight, age, sex, health, diet of the patient The range varies depending on the time of administration, the method of administration, the rate of excretion and the severity of the disease.

The composition of the present invention may be administered in various parenteral formulations during actual clinical administration, when formulated using a diluent or excipient such as fillers, extenders, binders, wetting agents, disintegrating agents, surfactants, etc., which are commonly used. do. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories. As the non-aqueous solvent and the suspension solvent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like can be used. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used.

According to another aspect of the present invention, the present invention provides a prophylactic or therapeutic agent comprising an active inhibitor or expression inhibitor of CISD2 (CDGSH iron sulfur domain 2) protein and an xCT (system x _c ^- cystine / glutamate antiporter) inhibitor as an active ingredient. It provides a pharmaceutical composition.

According to another embodiment of the present invention, the present invention provides an anticancer agent for xCT (system x _c ^- cystine / glutamate antiporter) inhibitor resistant cancer comprising an active inhibitor or expression inhibitor of CISD2 (CDGSH iron sulfur domain 2) protein as an active ingredient. Provide supplements.

Since activity inhibitors or expression inhibitors of the CISD2 protein and xCT inhibitors used in the present invention have already been described above, the description is omitted to avoid excessive duplication.

The anticancer adjuvant may be administered orally or parenterally during clinical administration and intraperitoneal injection, rectal injection, subcutaneous injection, intravenous injection, intramuscular injection, intrauterine dural injection, cerebrovascular injection or intrathoracic injection during parenteral administration. It can be administered by injection and can be used in the form of a general pharmaceutical formulation.

The anticancer adjuvant may be used alone or in combination with methods using surgery, radiation therapy, hormone therapy, chemotherapy and biological response modifiers.

The daily dosage of the anticancer adjuvant is about 0.0001 to 100 mg / kg, preferably 0.001 to 10 mg / kg, preferably administered once or several times a day, but the weight, age, sex, health status, The range varies depending on the diet, the time of administration, the method of administration, the rate of excretion and the severity of the disease.

The anticancer adjuvant of the present invention may be administered in various parenteral dosage forms in actual clinical administration, and when formulated, diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, surfactants, etc., which are commonly used, may be used. It is prepared. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories. As the non-aqueous solvent and the suspension solvent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like can be used. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used.

The present invention also provides a composition for treating or preventing cancer comprising the anticancer adjuvant.

In one embodiment of the present invention, when the CISD2 expression or activity inhibitor according to the present invention is treated with an xCT inhibitor, cancer-specific apoptosis of head and neck cancer cell lines resistant to the xCT inhibitor is increased. In addition, in vivo effects of cancer specific cell death of CISD2 expression or activity inhibitors according to the present invention were observed in mouse models transplanted with tumors.

Therefore, the anticancer adjuvant containing the CISD2 expression or activity inhibitor according to the present invention can be usefully used as an active ingredient of a composition for treating or preventing cancer.

The cancer treatment or prevention composition may contain one or more active ingredients that exhibit the same or similar function in addition to the CISD2 expression or activity inhibitor.

According to another aspect of the present invention, the present invention provides a method of providing information on determining whether to administer an xCT (system x _c ^- cystine / glutamate antiporter) inhibitor comprising the following steps:

(a) measuring the activity or expression level of the CDGSH iron sulfur domain 2 (CISD2) protein present in a sample isolated from a cancer patient; And

(b) determining that the xCT inhibitor can be administered if the activity or expression level of CISD2 protein present in the sample isolated from the patient is not significantly higher than that of normal or non-xCT inhibitor non-resistant patients.

The inventors confirmed that the levels of CISD2 mRNA and protein were significantly higher in cancer cell lines resistant to xCT inhibitors compared to cell lines that did not. Thus, comparing the activity or expression level of the CISD2 protein may provide useful information in determining whether to administer the xCT inhibitor.

The subject (patient or normal) in the present invention is not limited and is preferably selected from the group consisting of mammals, more preferably humans, rats, mice, monkeys, dogs, cats, cattle, horses, pigs, sheep and goats. Mammals, most preferably humans.

Samples included in the method of the present invention are not limited as long as they contain the genetic information related to the CISD2 of the subject naturally or artificially separated from the subject, preferably, feces, cells, blood, plasma, serum, hair or Urine and the like.

According to another aspect of the present invention, the present invention provides a method for screening a substance for enhancing sensitivity of an xCT (system x _c ^- cystine / glutamate antiporter) inhibitor comprising the following steps:

(a) treating a candidate material with a sample containing a CDGSH iron sulfur domain 2 (CISD2) protein;

(b) measuring the activity or expression level of the CISD2 protein treated with the candidate; And

(c) determining that the candidate substance can increase the sensitivity of the xCT inhibitor when the activity of the CISD2 protein is decreased or the expression of the CISD2 protein is reduced.

The candidate may inhibit the activity or inhibit the expression of the CISD2 protein, thereby enhancing the sensitivity of the xCT inhibitor.

Candidates capable of inhibiting the expression of the CISD2 protein include antisense nucleotides, short interfering RNAs (siRNAs), and short hairpin RNAs (shRNAs), which complementarily bind to mRNAs of genes encoding CISD2 proteins. ) And miRNA is preferably any one selected from the group consisting of.

Candidates that can inhibit the activity of the CISD2 protein is preferably any one selected from the group consisting of compounds, peptides, peptide mimetics, aptamers, antibodies, and natural products that specifically bind to CISD2 protein.

The features and advantages of the present invention are summarized as follows:

(Iii) The present invention provides a composition for enhancing sensitivity to xCT inhibitors comprising an inhibitor or activity inhibitor of CISD2 protein as an active ingredient.

(Ii) The present invention also provides a method of providing information on determining whether to administer an xCT inhibitor or a method of screening a substance for enhancing sensitivity of an xCT inhibitor.

(Iii) The composition of the present invention can be effectively used as an anticancer agent or an anticancer adjuvant by effectively overcoming the resistance to pereptosis apoptosis induced by the xCT inhibitor.

1 shows the results of SAS inducing various levels of ferroptotic cell death in HNC. (( A , B ) Viability of HNC cells exposed to different concentrations of sulfasalazine (SAS) for 72 hours. ( C ) Staining PI positive cells exposed to 0.5 and 1 mM SAS for 72 hours and counting with fluorescence microscopy Analysis by flow cytometry NT is a dimethyl sulfoxide control group not treated with SAS ( D ) Cell glutathione (GSH) in HN10 and HN11 cells exposed to SAS for 12 hours with or without 0.5 mM trolox pretreatment ( E ) Changes in cellular lipid ROS levels of HNC cells exposed to 1 mM SAS for 12 hours Cells were also deferoxamine (DFO, 100 mM), ferrostatin-1 (Fer-1, 20 μM) or alpha tocopherol (αTP). , 1 mM) Error bars indicate standard error of three replicates *: P <0.01 compared to control or other treatment groups)
Figure 2 shows that CISD2 expression is associated with resistance to ferroptotic cancer cell death. (( A , B ) Level of CISD2 mRNA and protein in HN10 and HN11 cells. ( C ) Rhodamine B-[(1,10-phenanthroline-5-yl) -aminocarbonyl] benzyl ester (0.5 and Accumulation of mitochondrial iron measured in cells treated with 1 mM SAS for 24 hours) ( D , E ) Mitochondrial MDA and iron ion concentrations were measured in HNC cells exposed to SAS for 24 hours. F ) Measurement of ΔΨM using TMRE (tetramethylrhodamine ethyl ester) after exposure to SAS for 24 hours *: P <0.01 compared to group
Figure 3 shows that CISD2 overexpression induces resistance to ferroptotic cancer cell death. (( A , B ) Effect of CISD2 Overexpression Induced by CISD2 Gene Transfection on Cell Growth Changes of SAS-Sensitive HN11 Cells ( CE ) Cytoplasmic Lipid ROS in HN11 Cells Transfected with Vector Control (vtr) or CISD2 Overexpression Vector , Mitochondrial MDA and RPA were measured and then exposed to 0.5 and 1 mM SAS for 24 hours. *: P <0.01 compared between groups. ( F ) 0.5 or 1 mM SAS, 10 μM in HN11 cells transfected with gene ( F ). Determination of PI positive fractions when exposed to PGZ or transfected with siCISD2 *: P <0.01 compared to control or two groups, **: P <0.001 compared to control or two groups)
Figure 4 shows the results of the inhibition of CISD2 reverse the resistance to ferroptotic cancer cell death. (( AC ) SAS resistant HN10 cells were transfected with siRNA control (siCtr) or siCISD2 mRNA amount ( A ), cell number change ( B ) and PI positive cell number ( C ) after transfection in the absence or presence of SAS ( DE ) Results of mitochondrial MDA, RPA, iron ions, and ΔΨM in HN10 cells transfected with siCtr or siCISD2 for 24 hours followed by exposure to 0.5 and 1 mM SAS. <0.01)
Figure 5 shows the results of PGZ inhibit the growth of HNC cells resistant to ferroptotic cancer cell death. (( A ) Growth of HN10 cells exposed to 1 mM SAS with or without 10 μm PGZ. ( B-F ) PI after exposure to HN10 cells alone or in combination with 0.5 and 1 mM SAS. Positive, lipid ROS, RPA, and mitosox levels measured *: P <0.01 compared between groups)
6 shows the results of PGZ sensitizing HNC cells to SAS treatment in vivo . (( AC ) shows tumor size, weight and weight changes in nude mice transplanted with HN10 cells. These mice received different treatments for vehicle (NT), SAS, PGZ, or a combination thereof. ( D , E ) Lipid ROS And measurement of RPA (F) Immunofluorescence staining of γH2AX formation (red) in transplanted tumors AU comparison between differently treated tumors Error bars indicate standard error *: P <0.05 compared to control or other treatment groups , **: P <0.01 compared to control or other treatment groups.)
FIG. 7 shows whether PGZ or RGZ, which is a thiazolidinedione-based drug as a CISD2 inhibitor, improves SAS-induced ferroptosis in various cancer cells.
8 is a result confirming whether PGZ or RGZ, a typical thiazolidinedione-based drug as a CISD2 inhibitor, improves irastin-induced ferroptosis in various cancer cells.
9 shows the mechanism by which CISD2 inhibition reverses resistance to SAS-induced ferroptosis in HNC cells. SAS induces ferroptosis in cancer cells through inhibition of xCT and depletion of GSH. However, increased CISD2 expression contributes to ferroptosis resistance through reduced levels of mitochondrial iron ions. Genetic inhibition of CISD2 or the use of PGZ reverses ferroptosis resistance of cancer cells to SAS by increasing iron ion levels in mitochondria.

Hereinafter, the present invention will be described in more detail with reference to the following examples. These examples are only for illustrating the present invention in more detail, it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples in accordance with the gist of the present invention. .

Experimental Materials and Methods

1. Cell Culture and Reagents

This study included ANC hospital HNC cell line (AMC-HN2-11) (Kim et al., Establishment and characterization of nine new head and neck cancer cell lines, Acta oto-laryngologica 1997; 117: 775-84) and SNU cell line ( SNU-1041, -1066, and -1076) were used and the cell lines were purchased from the Korea Cell Line Bank (Seoul, Korea). Also, A549, HCT116 and SKOV3 cell lines were purchased from ATCC (American Type Culture Collection, Manassas, VA, USA). The cell lines were verified using short tandem repeat (STR) -based DNA fingerprinting and multiplex PCR. Cells were prepared at 37 ° C., 5% CO ₂ using either Eagle's minimum essential medium (EMEM) or RPMI 1640 Medium (Roswell Park Memorial Institute 1640 Medium) (Thermo Fisher Scientific, Waltham, Mass., USA) with 10% FBS. Incubated. Representative drugs of xCT inhibitors such as SAS (sulfasalazine, sc-204312, Santa Cruz Biotechnology, Dallas, TX, USA) or Erastin (Sellechem; Houston, TX, USA) and thiazolidinedione series according to the designated time and dose Representative drugs of PGZ (pioglitazone; E6910, Sigma-Aldrich, St. Louis, MO, USA) or RGZ (rosiglitazone; Sigma-Aldrich; St. Louise, MO, USA) were treated with the cells.

2. Cell Viability and Apoptosis Analysis

The cells were exposed to SAS or PGZ, or a combination thereof, followed by MTT (3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide, Sigma-Aldrich), trypan Cell viability and extent of cell death were analyzed for 72 hours using trypan blue exclusion and propidium iodide staining. Control cells were exposed to the same amount of dimethyl sulfoxide (DMSO). Cells were incubated with MTT for 4 hours and then incubated with solubilization buffer for 2 hours. Absorbance was measured at 570 nm using a SpectraMax M2 microplate reader (Molecular Devices, Sunnyvale, Calif., USA). The trypan blue exclusion test included 0.4% trypan blue staining and counting through hemocytometer. For PI staining, samples were washed twice with PBS and stained with 2.5 μg / ml propidium iodide (Sigma-Aldrich) in PBS (each plate for 30 minutes). Stained cells were analyzed using a FACSCalibur flow cytometer equipped with CellQuest Pro (BD Biosciences, San Jose, Calif., USA) and observed under ZEISS fluorescence microscopy (Oberkochen Germany). The mean fluorescence intensity of each group was normalized to the mean fluorescence intensity of the control group.

3. GSH and ROS Levels and Lipid Peroxidation Analysis

Cellular GSH levels of HNC cell lysates were measured using a GSH colorimetric detection kit (BioVision Inc., Milpitas, Calif., USA). The generation of cellular reactive oxygen species (ROS) in the supernatant of HNC cell lysates was 2 μM of 2 ', 7'-dichlorofluorescein diacetate (DCF-DA) (cytosolic ROS, Enzo Life Sciences, Farmingdale, -BODIPY C11 (lipid peroxidation; Thermo Fisher Scientific) was evaluated by treatment for 30 minutes at 37 ° C. ROS levels were analyzed with a FACSCalibur flow cytometer equipped with CellQuest Pro (BD Biosciences, Franklin Lakes, NJ, USA). Cellular lipid peroxidation was evaluated by measuring the concentration of malondialdehyde (MDA), the final product of lipid peroxidation, using a lipid peroxidation assay kit (Sigma-Aldrich).

4. Mitochondrial Separation, Iron, Superoxide Generation and Membrane Potential Analysis

Mitochondria were isolated using the Mitochondrial Separation Kit (Thermo Fisher Scientific). Ferrous levels in cells or mitochondria were measured using an iron assay kit (Sigma-Aldrich). Accumulation levels of mitochondrial iron include rhodamine B-[(1,10-phenanthroline-5-yl) -aminocarbonyl] benzyl ester (RPA), fluorescent non-toxic iron sensor (Enzo Biochem, Inc., Farmingdale, NY, USA). Mitochondrial superoxide production was assessed by mitoSOX (Thermo Fisher Scientific) in viable cancer cells. Arbitrary fluorescence units (AU) were compared between different treatment groups. Mitochondrial membrane potential (ΔΨm) was measured by staining with 200 nM tetramethyl rhodamine ethyl ester (TMRE, Thermo Fisher Scientific) for 20 minutes. The mean fluorescence intensity of each group was normalized to the mean fluorescence intensity of the control group.

5. RNA Interference and Gene Transfection

In order to silence CISD2 , SAS-resistant HN10 cells were seeded. 24 hours later, it was transfected to human CISD2 or scrambled control siRNA cells with small interfering RNA (siRNA) of 10 nmol / L for the (RNAi TriFECTa ^® Kit, Integrated DNA Technologies, Coralville, IA , USA) as a target. SensiFAST ^TM SYBR ^® No-ROX Kit confirmed by (Bioline International, Toronto, Canada) 1-2 μg total RNA ( each sample) RT-qPCR (reverse transcription- quantitative PCR) for gene silencing with siRNA are derived from using the It was. This was done after synthesizing cDNA with SensiFAST ^™ cDNA Synthesis Kit (Bioline International) and Western blotting with anti-NAF-1 antibody. To produce a cell which stably over-expressing the CISD2, HN11 control plasmid or CISD2 the cells were transfected stably infected with the expression plasmid (Transomic, Huntsville, AL, USA ). CISD2 overexpression was confirmed using RT-qPCR and western blotting.

6. Immunoblotting

Cells were plated, grown to 70% confluent, and then treated with the indicated drugs. Cells were lysed at 4 ° C. in cell lysis buffer (Cell Signaling Technology, Danvers, MA, USA) containing a protease / phosphatase inhibitor cocktail (Cell Signaling Technology). A total of 5-15 μg of protein was analyzed by SDS-PAGE on a 10% -15% gel, transferred to nitrocellulose or polyvinylidene difluoride membrane and probed with primary and secondary antibodies. Primary antibodies were used: NAF-1 (60758S, Cell Signaling Technology), GPX4 (ab125066, Abcam, Cambridge, MA, USA), Bcl-2 (B cell lymphoma 2, ab117115, Abcam), NCOA4 ( nuclear receptor coactivator 4; A302-272A, Bethyl Laboratories, Montgomery, TX, USA), beclin-1 (4122s, Cell Signaling Technology), FTH1 (ferritin heavy chain 1; 4393, Cell Signaling Technology). β-actin (BS6007M, BioWorld, Atlanta, GA, USA) was used as total loading control. All antibodies were diluted 1: 500 to 1: 10000.

7. Tumor Genograph

All animal study procedures were performed according to a protocol approved by the Institutional Animal Care and Use Committee (IACUC) at Asan Hospital. Six week sterile BALB / c male nude mice (nu / nu) were purchased from Central Animals Co., Ltd. (Seoul, South Korea). SAS-resistant HN10 cells were injected subcutaneously on the sides of nude mice. After nodules were found in tumor implants, the mice were subjected to the following other treatments: vehicle, SAS (250 mg / kg / day intraperitoneal administration), PGZ (20 mg / kg / day oral administration), or SAS PGZ simultaneous administration. Each group contained 10 mice. Tumor size and weight of each mouse were measured twice a week and volume was calculated as (length × width ² ) / 2. Mice were sacrificed and tumors were isolated and analyzed by cytoplasmic lipid ROS and mitochondrial iron levels and immunofluorescence staining of γH2AX. Arbitrary fluorescence units (AU) were compared between differently treated tumors.

8. Measurement of ferroptosis effect in various cancer cell lines other than head and neck cancer

Under the same conditions as the experiment using the SAS or SAS + PGZ, various cancers including the head and neck cancer cell line in combination with PGZ or RGZ, which is a representative drug of the thiazolidinedione family, using SAS or Erastin, which is a representative drug of xCT inhibitors Cell viability in cell lines (lung cancer (A459), colon cancer (HCT 116), ovarian cancer (SKOV3)) was tested.

9. Statistical Analysis

Data are expressed as mean ± standard error. Statistical significance for differences between treatment groups was assessed using ANOVA analysis with the Mann-Whitney U-test or Bonferroni post-hoc test. The IBM ^® SPSS ^® Statistics version 24.0 statistical software for Windows (IBM, Armonk, NY, USA) was used. Statistical significance was defined as two-sided P value <0.05.

Experiment result

1. SAS Induces Multiple Levels of Ferroptotic Cell Death in HNC

SAS reduced the viability of HNC cells in a concentration dependent *? **? * Manner, with HN9 and HN11 cells having the highest sensitivity and HN10 cells showing the lowest (P <0.01) sensitivity (FIGS. 1A and 1B). ). IC ₅₀ of SAS was significantly different between HN10 and HN11 cell lines selected for the next experiment (0.95 vs 0.17 mM, P <0.01). Increased PI staining was observed in HN11 cells but no significant changes were observed in HN10 cells when treated with 0.5 mM SAS (P <0.01) (FIG. 1C). However, GSH levels decreased significantly in both HN10 and HN11 cells after SAS treatment, but recovered when pretreated with the antioxidant trolox (P <0.01) (FIG. 1D). The level of cellular lipid ROS was significantly increased in SAS sensitive HN11 cells treated with 1 mM SAS for 12 hours (P <0.01) (FIG. 1E). Cell death and lipid ROS induced by SAS treatment in HNC cells were reversed by pretreatment with iron chelator deferoxamine, ferroptosis inhibitor ferrostatin-1, or α-tocopherol, representing a typical pattern of ferroptotic cell death.

2. CISD2 expression is associated with resistance to ferroptotic cancer cell death

The levels of CISD2 mRNA and protein were significantly higher in SAS-resistant HN10 cells than in SAS-sensitive HN11 cells (P <0.01) (FIGS. 2A-2B). Mitochondrial iron levels measured by RPA assay after 24 hours of exposure to SAS were significantly increased in HN11 cells but not significantly increased in HN10 cells (P <0.01) (FIG. 2C). The levels of mitochondrial MDA and iron ions were also significantly increased in HN11 cells, while ΔΨM was significantly decreased in HN11 cells but not significantly in HN10 cells (P <0.01) (FIGS. 2D-2F). All changes were reversed by pretreatment with iron chelator deferoxamine.

We next examined the effect of CISD2 overexpression on the growth and survival of SAS-sensitive HN11 cells. CISD2 overexpression can be induced by CISD2 gene transfer in cancer cells. CISD2 overexpression showed that CISD2-transfected HN11 cells induce resistance to ferroptotic cell death and restore reduced *? **? * Cancer cell growth by SAS treatment (FIGS. 3A-3B). In HN11 cells transfected with CISD2, the levels of cytoplasmic lipids ROS, mitochondrial MDA and mitochondrial iron were significantly reduced (P <0.01) (FIGS. 3C-3E). Cell death was also significantly reduced (P <0.01), but reversed by silencing of the CISD2 gene or in combination with 10 μM pioglitazone (FIG. 3F).

3. CISD2 Inhibition Reverses Resistance to Ferroptotic Cancer Cell Death

Silence of the CISD2 gene significantly reduced the growth of SAS-resistant, siCISD2-type infected HN10 cells (P <0.01) (FIGS. 4A-4B). SAS treatment induced killing of siCISD2-transfected HN10 cells compared to the vector control, as revealed by increased positive PI staining (FIG. 4C). Mitochondrial levels of MDA, RPA and iron ions were significantly increased in HN10 cells transfected with siCISD2 (P <0.01) (FIGS. 4D-4F). ΔΨM was significantly decreased (P <0.01) in siCISD2-silenced cells (FIG. 4E).

NAF-1 is located on both ER (endoplasmic reticulum) and mitochondrial outer membrane, and plays a role in cellular autophagy as a mediator of Bcl-2-induced antagonism of Beclin-1 dependent autophagy in ER. In addition, NCOA4 is abundantly present in the double membrane of autophagosomes and acts as a selective cargo receptor for the autophagy turnover of ferritin (ferritinophagy). Therefore, we investigated the expression of ferritinophagy-related proteins in siCISD2 or si control-type infected HNC cells. Western blot analysis showed that dose-dependently reduced GPX4 expression in siCISD2 and si control-form infected HN10 cells. However, although the expression of NCOA4, beclin-1, Bcl-2 and FTH1 proteins was significantly decreased in siCISD2-type infected HN10 cells, there was no significant change in the vector control. NCOA4 was highly localized in the cytoplasm by treating siCISD2-form infected cells with SAS.

Pioglitazone (PGZ) is a thiazolidinedione (TZD) drug that is a peroxisome proliferator-activated receptor gamma (PPAR-γ) ligand drug that enhances insulin sensitivity, induces fat cell differentiation and inhibits cancer cell proliferation (Kole) L et al., 2016). PGZ is also known to inhibit the ability of NAF-1 to deliver 2Fe-2S clusters as apo-acceptor proteins and iron to mitochondria (Tamir S et al., 2013). Combination treatment with PGZ and SAS significantly reduced the growth and survival of SAS resistant HN10 cells and increased cell death (P <0.01) (FIGS. 5A-5B). Lipid ROS, RPA and mitoSOX expression levels were significantly increased in a SAS dose dependent manner (FIGS. 5D-5F). Taken together, PGZ overcomes resistance to SAS-induced ferroptotic cancer cell death through the accumulation of iron ions in mitochondria.

4. PGZ is in vivo ( in vivo Sensitively induce HNC cells during SAS treatment

Mice transplanted with HN10 cells were treated with vehicle (NT), SAS, PGZ or SAS + PGZ for 21 days. All mice survived tumor cell transplantation and treatment and were euthanized 21 days after treatment. Tumor growth in vivo was moderately inhibited by SAS or PGZ alone administration and significantly inhibited by SAS + PGZ combination (P <0.01) (FIGS. 6A and 6B). Body weight and daily intake decreased in the control group, but the total tumor size increased significantly, but there was no significant change in the drug treatment group (P <0.05). The levels of lipid ROS and RPA measured in tumors were significantly higher in the SAS + PGZ combination group than in the control or other treatment groups (P <0.05). ΓH2AX formation in tumor cells was significantly higher in the SAS + PGZ group than in the other groups (P <0.01). Histological examination of living organs showed no significant difference between these groups.

5. CISD2 Inhibitors Sensitively Induce Various Cancer Cells When Treated with xCT Inhibitors

PGZ or RGZ, a representative thiazolidinedione family of drugs as CISD2 inhibitors, enhanced SAS-induced ferroptosis (FIGS. 7A-E). 7A and B show cell viability at 72 hours exposure to head and neck cancer cells of sulfasalazine alone (SAS, 0.1-0.5 mM) and pioglitazone (PGZ, 10 μM) and rosiglitazone (RGZ, 10 μM), 7C is Lung cancer cells (A549), 7D show colorectal cancer cells (HCT 116) and 7E show results in ovarian cancer cells (SKOV3). Error bars represent standard deviation through three iterations and show synergistic effects of cell death by combination in all types of cancer cell lines.

In addition, PGZ or RGZ enhanced the ferroptosis induced by Erastin in various cancer cells (FIGS. 8A-E). 8A and B show cell viability at 72 hours of exposure to head and neck cancer cells of irastin alone (Erastin, 1-20 μM) and pioglitazone (PGZ, 10 μM) and rosiglitazone (RGZ, 10 μM), 8C Results in lung cancer cells (A549), 8D in colorectal cancer cells (HCT 116) and 8E in ovarian cancer cells (SKOV3). Error bars represent standard deviation through three iterations and show a synergistic effect of cell death by combination in all types of cancer cell lines.

Having described the specific part of the present invention in detail, it is apparent to those skilled in the art that the specific technology is merely a preferred embodiment, and the scope of the present invention is not limited thereto. Therefore, the substantial scope of the present invention will be defined by the appended claims and equivalents thereof.

Claims (13)

A composition for enhancing sensitivity to xCT (system x _c ^- cystine / glutamate antiporter) inhibitor comprising an inhibitor or activity inhibitor of CDSDSH (CDGSH iron sulfur domain 2) protein as an active ingredient.
The method of claim 1,
The expression inhibitor of the CISD2 protein is a composition, characterized in that any one selected from the group consisting of antisense nucleotides, siRNA, shRNA and miRNA complementary to the mRNA of the gene encoding the CISD2 protein.
The method of claim 1,
The activity inhibitor of the CISD2 protein is a composition, characterized in that any one selected from the group consisting of compounds, peptides, peptide mimetics, aptamers, antibodies and natural products that specifically bind to CISD2 protein.
The method of claim 3,
Said compound is thiazolidinedione.
The method of claim 4, wherein
The thiazolidinedione is pioglitazone (pioglitazone, PGZ) or rosiglitazone (rosiglitazone, RGZ), characterized in that the composition.
The method of claim 1,
The xCT inhibitors are sulfasalazine (Sulfasalazine, SAS), carboxyphenylglycine (CPG), erastin (Erastin), monosodium glutamate, aminoadipate, aminopimelate, homopimelate Characterized by being homocysteate, L-serine-O-sulphate, ibotenate, bromomoibotenate or quisqualate. Composition.
The method of claim 1,
The composition is a composition characterized in that it is used when treating cancer diseases using xCT inhibitors.
The method of claim 7, wherein
The cancer is selected from the group consisting of head and neck cancer, lung cancer, ovarian cancer, colon cancer, colon cancer, pancreatic cancer, liver cancer, cervical cancer, kidney cancer, gastric cancer, prostate cancer, breast cancer, brain tumor, uterine cancer, bladder cancer and blood cancer. Composition.
A pharmaceutical composition for preventing or treating cancer, comprising an activity inhibitor or expression inhibitor of a CDGSH iron sulfur domain 2 (CISD2) protein and an xCT (system x _c ^- cystine / glutamate antiporter) inhibitor as an active ingredient.
An anticancer adjuvant for xCT (system x _c ^- cystine / glutamate antiporter) inhibitor resistant cancer comprising an activity inhibitor or expression inhibitor of CISD2 (CDGSH iron sulfur domain 2) protein as an active ingredient.
The method of claim 10,
The activity inhibitor or expression inhibitor of the CISD2 protein is anticancer adjuvant, characterized in that to enhance the sensitivity of the xCT inhibitor.
Providing information on determining whether to administer an xCT (system x _c ^- cystine / glutamate antiporter) inhibitor comprising the following steps:
(a) measuring the activity or expression level of the CDGSH iron sulfur domain 2 (CISD2) protein present in a sample isolated from a cancer patient; And
(b) determining that the xCT inhibitor can be administered if the activity or expression level of CISD2 protein present in the sample isolated from the patient is not significantly higher than that of normal or non-xCT inhibitor non-resistant patients.
A method for screening a substance for enhancing sensitivity of an xCT (system x _c ^- cystine / glutamate antiporter) inhibitor comprising the following steps:
(a) treating a candidate material with a sample containing a CDGSH iron sulfur domain 2 (CISD2) protein;
(b) measuring the activity or expression level of the CISD2 protein treated with the candidate; And
(c) determining that the candidate substance can increase the sensitivity of the xCT inhibitor when the activity of the CISD2 protein is decreased or the expression of the CISD2 protein is reduced.