KR101859641B1 - Anti-cancer composition comprising artemisin derivative and Nrf inhibitor - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for preventing or treating cancer, comprising: artemisin or derivatives thereof; and a nuclear factor erythroid 2-related factor 2 (Nrf2) inhibitor. In addition, the present invention relates to an anticancer adjuvant and an anticancer adjuvant screening method. Artemisin or derivatives thereof; and a Nrf2 inhibitor according to the present invention cause ferroptosis in cancer cells to induce selective apoptosis of cancer cells; and shows anticancer effects on cancers resistant to cisplatin which is a conventional anticancer agent, and thus may be beneficially used as a novel anticancer agent. In addition, since resistance to artemisin or derivatives thereof can be improved through the newly screened Nrf3 inhibitor, artemisin or derivatives thereof according to the present invention can be utilized as a novel composite anticancer preparation that has overcome the limitations of conventional anticancer agents with respect to resistance.

Description

An anticancer composition comprising an artemisin derivative and an Nrf2 inhibitor,

The present invention relates to artemisin or a derivative thereof; And a nuclear factor erythroid 2-related factor 2 (Nrf2) inhibitor. The present invention also relates to a method for screening anticancer agents and anticancer adjuvants.

In today's rapidly developing medical technology, cancer is still one of the most threatening diseases in the world, and many people are diagnosed with cancer and die of cancer or its associated complications.

Head and neck cancer is a cancer that occurs in tissues such as nasal cavity, pharynx, larynx, salivary gland, thyroid gland, and the incidence is about 5% of all malignant tumors worldwide. Although the incidence of head and neck cancer is increasing worldwide, cisplatin, which is the most potent anticancer drug currently used in clinical practice, has been used for the treatment of head and neck cancer. However, resistance to cisplatin is continuously induced, . Therefore, there is a great need for a new anticancer drug that can replace this.

Cisplatin is known to be very effective in treating various types of cancer such as lung cancer, breast cancer, bladder cancer, stomach cancer, cervical cancer, myeloma and the like, as well as head and neck cancer. Cisplatin is a heavy metal compound containing platinum and has two chlorine atoms and two ammonia molecules bonded in a cis-form centering on a platinum atom. The cisplatin bonds to two adjacent guanines on the DNA strand, Forms an interstrand crosslink to inhibit DNA synthesis. Namely, it binds to DNA double helix structure existing in the nucleus of cancer cells, inhibits DNA replication, inhibits growth and proliferation of cancer cells, and removes cancer cells to exhibit anticancer effects. However, since cisplatin has increased clinical problems due to its resistance, there is a need for a new anticancer drug and a therapeutic method that can replace the cisplatin.

Artemisin-based drugs are sesquiterpene lactone endoperoxides extracted and isolated from the leaves of Artemisia annua and are known as anti-malarial drugs. Artemisinin and its derivatives are described in WHO articles on the Good Agricultural Products and Collection System (GACP) for Artemisia annua L. (WHO article 2006). Artemisin is known as an anti-malarial drug, but recently, research on new medicinal uses such as tuberculosis, cancer, and brain tumor is actively under way. However, there have been no reports of established studies and mechanisms for the new medicinal uses of artemisin drugs. In particular, there is a lack of research on methods to overcome resistance and induced resistance .

The inventors of the present invention have investigated the anticancer effect of artemisin or a derivative thereof. When artemisin or its derivative induces ferroptosis in cancer cells and shows resistance to it, And the present invention has been completed.

Accordingly, an object of the present invention is to provide artemisin or a derivative thereof; And a Nrf2 (nuclear factor erythroid 2-related factor 2) inhibitor. The present invention also provides a pharmaceutical composition for prevention or treatment of cancer comprising at least one of Nrf2 (nuclear factor erythroid 2-related factor 2) And to screen for new chemotherapeutic adjuvants for overcoming the resistance.

In order to accomplish the above object, the present invention relates to artemisin or a derivative thereof; And a nuclear factor erythroid 2-related factor 2 (Nrf2) inhibitor.

The present invention also provides an anticancer therapy adjuvant composition for overcoming resistance to artemisin or a derivative thereof, which comprises a nuclear factor erythroid 2-related factor 2 (Nrf2) inhibitor.

The present invention also provides a method for treating cancer, comprising the steps of: treating a candidate substance against cancer cells resistant to artemisin or a derivative thereof; And selecting a substance inducing a decrease in expression of Nrf2, HO-1 and NQO1 in the cancer cell; And a method for screening anticancer adjuvants for overcoming resistance to artemisin or derivatives thereof.

Artemisin or a derivative thereof according to the present invention; And Nrf2 inhibitor not only induces selective killing of cancer cells by inducing ferrotocyte in cancer cells but also can be used as a new anticancer agent because it can exhibit anticancer effect against the existing anticancer drug cisplatin resistant cancer. In addition, resistance to artemisin or its derivatives can be improved through a novel screening Nrf2 inhibitor, which can be used as a new combination chemotherapeutic agent that overcomes the limitations of existing anticancer drugs against resistance.

FIG. 1 is a graph showing the results of confirming selective cancer cell killing effect upon treatment with artesanate. FIG. 1 (A) shows the cytotoxic effect of cisplatin-resistant cancer and susceptible cancer according to the treatment concentration of arthesinate, FIG. 1 (B) shows the cytotoxic effect of arthisinate treatment in head and neck cancer cells and normal cells, C is a colony forming ability according to treatment with arthesinate in head and neck cancer cells and normal cells, and D in FIG. 1 shows the results of comparing anti-cancer effects of arthesinate in parent cells and cisplatin-resistant cells (Arts: ).
FIG. 2 is a graph showing the cell numbers (A), aresinate and HTL (holo-transferrin), DFO and trolox in combination treatment with arthesinate and DFO (deferoxamine), antioxidant trolox (B) and cell viability (C) in cisplatin-resistant cancer cells according to the present invention (* indicates P <0.01 for aresinate alone treatment).
Figure 3 shows the total ROS and lipid ROS (ROS) activity of aresinate alone or necro-statin-1 (Nec-1, 20 [mu] M), ferrostatin (* Indicates P <0.01 for treatment with artesonate alone).
FIG. 4 is a graph showing changes in protein expression of xCT, CD44, RAD51, p53, Nrf2 and Keap1 according to artesinate treatment in cisplatin sensitive or resistant cancer (A) and Nrf2-ARE according to artesinate treatment in cisplatin- (B) showing the results of confirming the expression changes of the mechanism involved proteins (Nrf2, Keap1, Ho-1, NQO1). FIG. 4C is a graph showing changes in the expression of Nrf2, Keap1 and HO-1 according to treatment with erastin or sulfasalazine (SAS), an artesinate and ferrotic inducer (* P < 0.01 relative to NT control).
FIG. 5 is a graph showing changes in NFE2L2 (Nrf2) mRNA according to treatment with arestinate and erastin in cisplatin resistant cancer cells (* P <0.01 relative to NT control).
FIG. 6 shows (C) the change in NRf2 expression (A), the change in NFE2L2 (Nrf2) transcription activity (B), and the change in expression of HMOX1 and NQO1 mRNA in the cisplatin resistant cancer cells according to the treatment with aresinate or erastin (* P < 0.01 relative to NT control).
FIG. 7 is a graph showing inhibition of KEAP mRNA expression by siKEAP1 in head and neck cancer cells (A) and inhibition of protein expression (B) and cell number change (C) of Nrf2 and HO- 0.05 relative to control). (siCtr: siControl, Lipo: lipofectamine).
8 is a diagram showing a result of confirming the cell survival rate of the Artemisia can carbonate and siKEAP1, siNFE2L2 (Nrf2) and teurigo nelrin (trig) combined treatment (* P <0.05 relative to control ; * P <0.01 between the groups.) .
FIG. 9 is a graph showing the results of measuring cellular GSH and lipid ROS levels in the cisplatin-resistant cells, alone or in combination with treatment with arthesinate and trigonelline (* P <0.05 relative to control; * P <0.01 between the groups.
FIG. 10 is a graph showing the results of overcoming aresinate resistance by an Nrf2 inhibitor. FIG. 10A shows the results of confirming reduction of mRNA level of NFE2L2 (Nrf2) by siNFE2L2 treatment and siHMOX1 treatment and reduction of mRNA level of HMOX1. FIG. 10B is a graph showing the cell survival rate of the change in susceptibility to artesonate according to the treatment with siRNA or siHMOC and Trolox alone or in combination. (* P <0.01 relative to siCtr, ** P <0.01 relative to siNFE2L2 transfection. Fig. 10C shows the results of confirming resistance to overcome of the combination treatment of aresinate with siRNAr2, an Nrf2 inhibitor. 10 and 11 show changes in cellular GSH and cellular ROS levels due to treatment with siNFE2L2 and artesinate, respectively, and the result of blocking by trolox treatment (* P < 0.05 and ** P < 0.01 compared between groups).
Figure 11 shows the results of genetic inhibition (shNrf2) and pharmacological inhibition (trigonelline) of aresinate and Nrf2 in mice to affect glutathione deficiency and γH2AX formation leading to iron-dependent anti-cancer effects Fig. FIG. 11A is a graph showing changes in tumor weight and Nfr2 expression by treatment with KEAP1 shRNA and artesonate (* P < 0.05 versus the shRNA control). 11B and 11C show the result of confirming the change in tumor volume (B) and cellular GSH (C) by shKeap1, artesanate, trigonelline alone or in combination (* P <0.05 compared between two groups). FIG. 11D shows the result of confirming the change in tumor volume according to the test group of Trigonelin, Artesonate, shNrf2 alone or in combination. FIGS. 11E and 11F are graphs showing changes in cellular GSH level and γH2AX formation in cells after shNfr2, artesinate, and trigonellin alone or in combination in cisplatin resistant cancer. FIG. (* P <0.05 compared between two groups; ** P <0.01 relative to control or between two groups).
12 is a schematic diagram showing the ferrotocyte-inducing mechanism according to the present invention.

The present invention relates to artemisin or a derivative thereof; And a nuclear factor erythroid 2-related factor 2 (Nrf2) inhibitor.

Artemisin or a derivative thereof according to the present invention; And Nrf2 (nuclear factor erythroid 2-related factor 2) inhibitors induce pertro- sis in cancer cells to induce selective killing of cancer cells as well as anticancer effects against cisplatin resistant cancer, which is an existing cancer drug.

In the present invention, the derivatives of artemisin may include all derivatives of artemisin known in the art as far as they can induce the selective death of cancer cells through the activity of ferrotocyte in cancer cells, Are artemisinin, hydroartemisinin, dihydroartemisinin, artemether, arteether, arteflene, artesunate, and the like. , At least one member selected from the group consisting of aresinate salt, propylcarbonate dihydroartemisinin, bis-ether artelinic acid, and dihydroxydihydroartemisinin. have. In the present invention, artesunate is used as a preferred example, and artesanate is referred to as dihydroartemisinine-12-alpha-succinate or succinyl dihydroartemisinin or sodium artesunate or Quinghaosu reduced succinate ester or butanedioic acid, (3R, 5aS, 6R, 8aS, 9R, 10S, 12R, 12aR) -decahydro-3,6,9-trimethyl-3,12-epoxy- (white powder or crystal) at room temperature and has a chemical structure of C ₁₉ H ₂₈ O ₈ and a molecular weight of 384.42082 g / cm &lt; 3 &gt; mole.

Artesinate, which is one example of artemisin or derivative of the present invention, exhibits excellent anticancer activity in both platinum-resistant and cancer-resistant platinum-based anticancer drugs, which are one of typical anticancer drugs, and is a safe drug that does not induce apoptosis in normal cells. In particular, the arthesinate of the present invention exhibits an activity of killing cancer cells by inducing iron-dependent ROS-mediated iron ferrooptic cell death, that is, ferrotocyte. In addition, aresinate treatment induces anticancer activity through a mechanism of increasing total ROS and lipid ROS levels in the cancer cells treated therewith.

The Nrf2 inhibitor of the present invention inhibits the activity of Nrf2, and may include, without limitation, a substance that genetically inhibits Nrf2, or a small molecule that inhibits the activity of an Nrf2 inhibitor. Nrf2 plays a major role in the transcriptional activation of genes regulated by the antioxidant response element (ARE), and genes regulated by ARE mainly encode antioxidant enzymes.

The Nrf inhibitor of the present invention may preferably be at least one selected from the group consisting of antisense of Nrf2, dsRNA, ribozyme, aptamer, fragment of Nrf2 binding protein, antibody or fragment of antibody.

Said "antisense" refers to a nucleic acid comprising a sequence complementary to an mRNA encoding NRF2. Antisense can consist of DNA, RNA or both. The antisense need not be 100% complementary to the mRNA of the target NRF2. Antisense may include non-complementary bases as long as they can specifically hybridize under stringent conditions (Sambrook et al. 1989). When an antisense is introduced into a cell, it binds to the target polynucleotide and inhibits transcription, RNA processing, translation or stability. In addition to the antisense polynucleotides, the antisense includes polynucleotide mimetics comprising modified verbone, and 3 ' and 5 ' terminal regions. Such antisense can be prepared by appropriately designing from the sequence information of NRF2 and using methods well known to those skilled in the art (e.g., chemical synthesis).

"dsRNA" is a double-stranded RNA structure that inhibits gene expression by RNA interference (RNAi), including siRNA (short interfering RNA) and shRNA (short hairpin RNA). The dsRNA does not need to have 100% homology with the sequence of the target gene as long as it inhibits the expression of the target gene. A portion of the dsRNA can be replaced with DNA for stabilization or for another purpose (s). Usually siRNA is a double stranded RNA consisting of 21-23 bases. siRNA can be produced by methods well known to those skilled in the art, for example, by chemical synthesis, or as analogs of native RNA. shRNA is a RNA short chain having a hairpin turn structure. The shRNA can be produced by a method well known to those skilled in the art, for example, by chemical synthesis, or by introducing DNA encoding shRNA into a cell to express the DNA. SiRNA sequences as Nrf inhibitors illustratively used in the present invention may be an siRNA pair represented by SEQ ID NO: 1 and 2, an siRNA pair represented by SEQ ID NO: 3 and 4, an siRNA pair represented by SEQ ID NO: 15 and 16, The sequence may be an shRNA pair represented by SEQ ID NOs: 19 and 20, an shRNA pair represented by SEQ ID NOs: 21 and 22, an shRNA pair represented by SEQ ID NOs: 23 and 24, and 90 to 99%, preferably 95 to 99% Or &lt; / RTI &gt; homologous sequences.

"Ribozyme" has RNA processing catalytic activity and can perform RNA cleavage, pasting, insertion and transfer. The structure of the ribozyme may include a hemmer head, a hairpin, and the like.

"Aptamer" is a nucleic acid that binds to a protein-like substance. The form of the nucleic acid may be double stranded or single stranded. The length of the aptamer is not limited as long as it can specifically bind to the target molecule, and is, for example, 10 to 200 nucleotides, preferably 10 to 100 nucleotides, more preferably 15 to 80 nucleotides, Lt; RTI ID = 0.0 &gt; 15 &lt; / RTI &gt; to 50 nucleotides. The aptamers may be selected using methods well known to those skilled in the art. For example, SELEX (Systematic Evolution of Ligands by Exponential Enrichment) (Tuerk, C. and Gold, L., 1990, Science, 249, 505-510) can be applied.

"Fragment of NRF2-binding protein" is a protein fragment that binds to NRF2 and inhibits NRF2 from performing its original function. For example, fragments of NRF2-binding proteins can be obtained by preparing partial peptides of such proteins and selecting peptides that bind to NRF2. In addition, to improve its stability or enhance its inhibitory activity, the peptide can be modified appropriately and an amino acid mutation can be introduced into a portion of the peptide.

As used herein, a "fragment of an antibody" is a peptide comprising a portion (partial fragment) of an antibody or a portion of an antibody, which retains the activity of the antibody against the antigen. The fragments of the antibody may be selected from the group consisting of F (ab ') ₂ , Fab', Fab, short chain Fv (abbreviated as "scFv" hereinafter), disulfide bonded Fv (hereinafter abbreviated as "dsFv" (Hereinafter abbreviated as "diabodies &quot;), or a peptide comprising a CDR.

In addition, the NRf2 inhibitor of the present invention may be a small molecule substance capable of inhibiting the activity of Nrf2, preferably trigonelline, ascorbic acid, luteolin, ochratoxin A, chrysin, brusatol, and all- trans retinoic acid (ATRA).

As described above, the Nrf2 inhibitor of the present invention plays an auxiliary role in increasing the sensitivity to artemisin or its derivative in cancer cells which exhibit resistance to artemisin or its derivatives or decrease in sensitivity, and it is an artemisin or derivative thereof When treated together with cancer cells, cellular GSH levels are markedly reduced, γH2AX formation can be markedly increased, and lipid ROS accumulation can be promoted, thus helping iron-dependent anticancer activity of aresinate, . &Lt; / RTI &gt;

The cancer of the present invention may include all kinds of cancer cells in which artemisinin or a derivative thereof can induce selective cancer cell death through ferrothisis and the cancer cell resistance is improved due to the combined administration of the Nrf2 inhibitor, It may be cervical cancer. In the present invention, head and neck cancer is generically referred to as cancer except for the thyroid gland among the carcinomas that occur on the upper part of the clavicle. That is, it includes squamous cancer, pharyngeal cancer, tongue cancer, laryngeal cancer, and the like.

Also, artemisin or a derivative thereof of the present invention; And Nrf2 (nuclear factor erythroid 2-related factor 2) inhibitor is a novel anticancer agent which can be used as a more effective anticancer agent in cancerous carcinomas resistant or resistant to existing anticancer agents. Examples of the anticancer agent which is widely used but has a problem of resistance or resistance include a platinum-based anticancer agent and may be one or more anticancer agents selected from the group consisting of cisplatin, carboplatin and nedaplatin. Therefore, the present invention can be effectively used for carcinomas resistant to conventional anticancer agents.

The artemisin or a derivative thereof of the present invention is characterized by causing ferrotocis in cancer cells. The term &quot; ferroptosis &quot; refers to iron cell necrosis, which means a new cancer cell death mechanism that reacts with iron ions in the molecule to produce reactive oxygen species, thereby killing cancer cells. That is, artemisin or its derivatives induce the generation of active oxygen species and accumulation of lipid peroxidation, which are iron-dependent, thereby inducing selective death of cancer cells.

Accordingly, the artemisin or a derivative thereof of the present invention can be characterized in that it reduces glutathione or increases active oxygen species in cancer cells.

The cancer of the present invention, on the other hand, is now, but not limited to, a ferrotocyte resistant cancer. This is because the present invention includes an Nrf2 inhibitor as an ingredient for overcoming resistance to ferrotocyte, and it is difficult for artemisin or its derivative, which induces ferrotocyte to induce anticancer activity, to act smoothly This is because administration of Nrf2 inhibitor in combination with artemisin or its derivative in ferrotocyte-resistant cancer has specific superiority in increasing the susceptibility or sensitivity of the ferrotocyte-resistant cancer and improving the anti-cancer effect.

Accordingly, the present invention also relates to an anticancer therapeutic composition for overcoming resistance to artemisin or a derivative thereof, which comprises a nuclear factor erythroid 2-related factor 2 (Nrf2) inhibitor.

As used herein, the term " composition for assisting in the chemotherapy "means a composition that synergistically increases the effect of chemotherapy by increasing the sensitivity of cancer cells to an anticancer drug when applied in combination with treatment with an anticancer drug.

Therefore, the anticancer therapy adjuvant composition can be used as an adjunct to cancer treatment with resistance and can be administered simultaneously or sequentially with anticancer drugs as anticancer adjuvants.

The composition of the present invention comprises a pharmaceutically acceptable carrier. The carrier which is included in the pharmaceutical composition of the present invention and which is usually used at the time of preparation is lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, calcium silicate, (S), microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, mineral oil, saline, PBS And the like, but are not limited thereto.

The pharmaceutical composition of the present invention may further contain a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifying agent, a suspending agent, a preservative, etc., in addition to the above components. Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington ' s Pharmaceutical Sciences (19th ed., 1995).

The pharmaceutical compositions of the present invention can be administered orally or parenterally, preferably orally or subcutaneously, and can be used together, simultaneously, and sequentially, including additional components for chemotherapy.

The appropriate dosage of the pharmaceutical composition of the present invention may vary depending on such factors as formulation method, administration method, age, body weight, sex, pathological condition, food, administration time, route of administration, excretion rate, .

The present invention also relates to artemisin or a derivative thereof; And Nrf2 (nuclear factor erythroid 2-related factor 2) inhibitor to a subject.

The subject is preferably a mammal, including a human, and may include, without limitation, any potential patient population suffering from, suffering from, or being sick with cancer. The artemisin or a derivative thereof; And Nrf2 (nuclear factor erythroid 2-related factor 2) inhibitors can be delivered to a subject in a pharmaceutically effective amount.

The present invention also relates to a method for the treatment and / or prophylaxis of cancer, comprising administering an Nrf2 (nuclear factor erythroid 2-related factor 2) inhibitor together with artemisin or a derivative thereof; A method for overcoming resistance to or resistance to artemisin or a derivative thereof; Or a method for enhancing sensitivity or sensitivity. Detailed descriptions related to the present treatment method are omitted in order to avoid duplication with the above-described description.

The present invention also provides a method for treating cancer, comprising the steps of: treating a candidate substance against cancer cells resistant to artemisin or a derivative thereof; And selecting a substance inducing a decrease in expression of Nrf2, HO-1 and NQO1 in the cancer cell; To a method of screening for anticancer adjuvant for overcoming resistance to artemisin or a derivative thereof.

In the present invention, artemicin or a derivative thereof, preferably artesinate, may exhibit anticancer activity by ferrotocys in cisplatin-resistant or sensitive cancer, but the anticancer activity of Nrf2- ARE pathway activation. Thus confirming that inhibition or blocking of the Nrf2-ARE pathway plays an important role in overcoming the resistance caused by treatment with artemisin or its derivatives. Therefore, when a substance capable of inhibiting or blocking the Nrf2-ARE pathway is identified in cancer cells resistant to artemisin or its derivatives, it can be used as an anti-cancer adjuvant for resistant cancer cells against artemisin or its derivatives. Accordingly, the present invention provides a method for overcoming resistance to artemisin or a derivative thereof by selecting a substance that causes a decrease in the expression of Nrf2, HO-1 (Heme oxygenase-1) and NQO1 (NAD (P) H quinone dehydrogenase 1) A method for screening a cancer-preventing adjuvant is provided.

Such reduction of expression includes both reduction of transcription activity or reduction of protein expression, and such reduction can be carried out using any method known in the art for measuring the amount of a transcript or a protein encoded therefrom. More specifically, by immunoprecipitation, radioimmunoassay (RIA), enzyme immunoassay (ELISA), immunohistochemistry, RT-PCR, Western blotting and flow cytometry (FACS) But it is not limited thereto. In addition, the degree of activity can be measured by any one selected from the group consisting of SDS-PAGE, immunofluorescence, enzyme immunoassay (ELISA), mass spectrometry and protein chip, but is not limited thereto.

Hereinafter, the present invention will be described in detail by way of examples. However, the following examples are illustrative of the present invention, and the contents of the present invention are not limited by the following examples.

Materials and Methods

Cell line

HNC cell line (HN2-10) and SNU cell line were purchased from Korean Cell Line Bank and confirmed by STR (short tandem repeat) based DNA fingerprinting and multiplex PCR. The cells were cultured in RPMI 1640 medium containing EMEM medium or 10% fetal bovine serum at 37 ° C under a 5% carbon dioxide atmosphere. Normal oral keratinocytes or fibroblasts were obtained from patients who underwent oral surgery and were used for in vitro experiments. Three types of cisplatin resistant HNC cell lines (HN3-cisR, HN4-cisR, and HN9-cisR) were obtained through treatment of parental HN3, HN4, and HN9 cells, respectively, with increasing cisplatin concentrations. The IC50 of cisplatin (Sigma-Aldrich, St. Louis, MO, USA) was 2.2 to 3.5 uM in parental cells and 25.5 to 38.9 uM in cisplatin-resistant HNC cells.

 cell Survival rate  Experiment

Changes in cell viability by combination with 1 to 100 uM exposure to arthesinate (Sigma-Aldrich), 100 uM trigonelline (Sigma-Aldrich), Nrf2 genetic inhibitors and activators were assessed by MTT, rypan blue exclusion, clonogenic assay. For the MTT assay, cells were incubated with MTT (3- [4,5-dimethyl-2-thiazolyl] -2,5-diphenyl-2H-tetrazolium bromide (Sigma-Aldrich) The incubation was carried out in solubilization buffer for a period of time. The absorbance was then measured using a SpectraMax M2 microplate reader (Molecular Devices, Sunnyvale, Calif., USA). The trypan blue exclusion assay was performed with 0.4% trypan blue staining and cells were counted with a hemocytometer.

Clonogenic assays were performed using 0.5% crystal violet solution and colonies (> 50 cells) were quantified after 14 days of culture. Cell death assays were performed using Annexin V and Pi staining. Positive stained cells were counted using a flow cytometer and Cell Quest Pro software (BD Biosciences, Franklin Lakes, NJ, USA), and all assays were repeated three times. In drug interactions, the combination index (CI) was calculated using Chou-Talalay (ComboSyn, Inc., Paramus, NJ, USA) using CI <1 defined as synergistic interaction.

Glutathione  Synthetic and ROS  Measure

Cellular GSH levels of cell lysates exposed to other drugs were measured using the GSH colorimetric detection kit (BioVision Inc., Milpitas, CA, USA). Cellular ROS production in supernatants of HNC cell eluates was measured using 10 μM 2 ', 7'-dichlorofluorescein diacetate (DCF-DA) (cytosolic ROS; Enzo Life Sciences, Farmingdale, NY, USA) or 2 μM C11-BODIPY C11 peroxidation; Thermo Fisher Scientific) for 30 minutes at 37 ° C. The ROS levels were analyzed using a FACSCalibur flow cytometer equipped with CellQuest Pro (BD Biosciences).

RNA interference and gene transfection

To induce silencing of the KEAP1 gene, cisplatin-sensitive HN9 cells were dispensed. To induce silencing of the NFE2L2 and HMOX1 genes, cisplatin-resistant HN3- and HN9-cisR cells were dispensed. Cells were transfected 24 hours later with 10 nM siRNA or scrambled control siRNA (Integrated DNA Technologies, Coralville, IA, USA). For stable KEAP1 or NFE2L2 knockdown, HNC-cisR cells were transfected with a lentiviral vector containing KEAP1 or NFE2L2 or control shRNA (Transomic, Huntsville, AL, USA). Cells with stable transfection were selected by treatment with 2 ug / ml of puromycin. siRNA or shRNA induces gene silencing, and the effect of gene silencing is confirmed by Western blotting and RT-PCR.

The siRNA and shRNA sequences used in the present invention are as follows.

siRNA sequence information siRNA  Kinds company number Forward Reverse NFE2L2
(Nrf2) IDTDNA One 5'-GUUACAACUAGAUGAAGAGACAGGT -3 '(SEQ ID NO: 1) 5'-ACCUGUCUCUUCAUCUAGUUGUAACUG -3 '(SEQ ID NO: 2) 2 5'-GCAAGAUUUAGAUCAUUUGAAAGAT -3 '(SEQ ID NO: 3) 5'-AUCUUUCAAAUGAUCUAAAUCUUGCUC -3 '(SEQ ID NO: 4) 3 5'-UCAACGAAAUGAUGUCCAAAGAGCA -3 '(SEQ ID NO: 5) 5'-UGCUCUUUGGACAUCAUUUCGUUGAAG -3 '(SEQ ID NO: 6) KEAP1
IDTDNA
One 5'-AGAACAGACAAACUAGUGUCUUUCA -3 '(SEQ ID NO: 7) 5'-UGAAAGACACUAGUUAGUCUGUUCUUU -3 '(SEQ ID NO: 8) 2 5'-AGCGCUACGAUGUGGAAACAGAGAC-3 '(SEQ ID NO: 9) 5'-GUCUCUGUUUCCACAUCGUAGCGCUCC-3 '(SEQ ID NO: 10) 3 5'-AGAGGAACGAGUGGCGAAUGAUCAC-3 '(SEQ ID NO: 11) 5'-GUGAUCAUUCGCCACUCGUUCCUCUCU -3 '(SEQ ID NO: 12) HO1 IDTDNA One 5'-AACAUUGUCUGAUAGUAGCUUGAAA -3 '(SEQ ID NO: 13) 5'-UUUCAAGCUACUAUCAGACAAUGUUGU -3 '(SEQ ID NO: 14) 2 5'-UAAACAACAUUGUCUGAUAGUAGCT -3 '(SEQ ID NO: 15) 5'-AGCUACUAUCAGACAAUGUUGUUUAUU -3 '(SEQ ID NO: 16) 3 5'-GGUCCUUACACUCAGCUUUCUGGTG -3 '(SEQ ID NO: 17) 5'-CACCAGAAAGCUGAGUGUAAGGACCCA-3 '(SEQ ID NO: 18)

* IDT DNA = Integrated DNA Technologies, Coralville, IA, USA; HO1 = heme oxygenase 1.

shRNA sequence information shRNA  Kinds company number Forward Reverse NFE2L2
(Nrf2) Tranomic One 5'-TAGATCAGAAACATCAATGGGC-3 '(SEQ ID NO: 19) 5'-ACCCATTGATGTTTCTGATCTA-3 '(SEQ ID NO: 20) 2 5'-TTTCGTTGAAGTCAACAACAGG-3 '(SEQ ID NO: 21) 5'-ACTGTTGTTGACTTCAACGAAA-3 '(SEQ ID NO: 22) 3 5'-TTGAGCTTCATTGAACTGCTCT-3 '(SEQ ID NO: 23) 5'-CGAGCAGTTCAATGAAGCTCAA-3 '(SEQ ID NO: 24)

Western Blasting

 Cells were plated, grown to 70% confluence, and then drug treated. Cells were lysed in RIPA lysis buffer (Thermo Fisher Scientific), 4 ° C. A total of 50 ug of protein was eluted by 10-12% gel SDS-PAGE and transferred to nitrocellulose or polyvinylidene dipride membranes and labeled with primary and secondary antibodies. The following primary antibodies were used for Western blotting: Nrf2, Keap1, NQO1, HO-1 (Abcam, Cambridge, UK), xCT, p53, RAD51, and CD44 (Cell Signaling Technology, Danvers, MA). β-actin (Sigma-Aldrich) was used as a loading control and all antibodies were diluted 1: 500 to 1: 5000.

Nrf2  Transcription activation analysis

The transcriptional activity of Nrf2 was analyzed using the Cignal Antioxidant Response Reporter kit (Qiagen, Valencia, CA, USA) according to the manufacturer's manual.

Preclinical  exam

All animal tests were carried out with the approval of Asan's Animal Care and Use Committee. Six-week-old athymic BALB / c male nude mice (nu / nu) were purchased from the central animal and HN9 cells (5x10 ⁶ ) with or without KEAP1 shRNA transfection were subcutaneously injected into each side . Mice with apparent nodules after cell injection were treated with vehicle, aresinate, or triethoteline. After euthanasia, the tumors were extracted and compared in each group. In other pre-clinical experiments, it was subcutaneously injected or control has the NFE2L2 shRNA transfection or without HN9-cisR cells (5x10 ⁶⁾ on the side of a nude mouse. Six different treatments were performed on the mice from the day that the nodules were found after tumor transplantation: shCtr + vehicle, shCtr + trigonellin, shCtr + arthesinate, shCtr + arthesinate + trigonelline, shNrf2 + vehicle, shNrf2 + arthesinate. The aresinate was orally administered at 50 mg / kg daily and the trigonelline administered orally at 50 mg / kg daily. After euthanasia, tumors were isolated and immunofluorescence staining for cellular GSH measurement and γH2AX formation was performed. The two-tailed Mann-Whitney U test was used to compare statistical significance among the other treatment groups.

Example  One. Artesanate  Determination of selective cancer killing effect

To determine whether aresinate could exhibit selective killing effects on cisplatin-sensitive cancer (HN3, HN4, HN9) and cisplatin resistant cancer (HN3-cisR, HN4-cisR, HN9-cisR) cells, The cell viability was analyzed by treatment with 1 to 100 uM of Nate, and the results are shown in FIG.

As shown in Fig. 1 (A), artesanate showed apoptosis effects on cisplatin-sensitive cancer (HN3, HN4, HN9) and cisplatin resistant cancer (HN3-cisR, HN4-cisR, HN9-cisR) cells in a dose- . HN9 was more susceptible to artesanate than HN3 or HN4 and all cells died at 100 uM concentration. In addition, as shown in FIG. 1B, the HNC cell line and the normal cell line were susceptible to artesonate, and the cell survival rate of HNC cell line was significantly decreased. However, most normal human oral keratinocytes (HOK) and oral fibroblast HOF) did not show apoptotic effect despite 50 uM aresinate treatment. That is, it was confirmed that artesanate does not induce apoptosis in normal cells and induces selective apoptosis of cancer cells. In addition, as shown in Fig. 1C, the colony forming ability (CFA) was markedly decreased in cisplatin-sensitive HNC cells and decreased in cisplatin-resistant HNC cells. On the other hand, colony forming ability was preserved in normal cells. Also, as shown in Figure 1D, cells were observed with microphotocytes after 72 hours of treatment with vehicle or 50 uM of artesinate, indicating that the artesinate treatment inhibited the growth of cells in cisplatin resistant or susceptible cells Respectively.

Example  2. Of head and neck cancer Artesanate Iron castle  Cell death ferrotoptic  cell death

 2.1 Cell number  And Colony  Confirm formation inhibition effect

 It was confirmed that aresinate induced the death of cancer cells, and it was confirmed that HNC cells induced iron-dependent ROS-mediated iron cell death, that is, ferrotocyte. More specifically, 50 uM of aresinate was treated with cisplatin-sensitive cells for 1 to 4 days, and the change in cell number was confirmed. The cells were either pretreated with HTF (holo-transferrin), 100 mM iron chelator deferoxamine (DFO), or 0.5 mM antioxidant trolox. The results of confirming the change in cell number are shown in Fig. 2A.

In addition, cisplatin resistant HNC cells were treated with arthesinate for 72 hours to confirm the effect of colony formation, and the effects of artesonate treatment, HTF, DFO, and Trolox treatment were compared to compare colony formation effects. The results are shown in Fig. 2B. As shown in Figures 2A and B, arthesinate significantly reduced the number of cells and colonies, and the effect of reducing arthesinate by colony was reversed by treatment with DFO and antioxidant trolox.

2.2 Identification of effects on cell death

The effects of annexin V and PI staining and flow cytometry analysis on cell death were analyzed. Cell viability was determined by measuring cellular ATP levels in HN9 cells exposed to 50 uM for 72 hours. HTF, DFO, and Trolox pretreated cell lines were placed for confirmation of the mechanism, and the effect of apoptosis was confirmed for the combination of artesonate alone, HFT or DFO or trolox, and the results are shown in FIG. 2C. As shown in Figure 2C, the cell viability as determined by Annexin V-PI staining and ATP measurements was significantly reduced in the HNC cells treated with artesonate. Pretreatment of DFO and trolox, on the other hand, restored ATP levels and decreased apoptosis.

Cell viability was further reduced by the addition of holo-transferrin (20 ug / ml), but this effect was markedly blocked by pretreatment with iron chelator dexammin (100 mM) or antioxidant trolox (0.5 mM).

2.3 Total ROS And lipids To ROS  Check the effect on

In HNC cells treated with 50 uM artesinate with or without necro-statin-1 (Nec-1, 20 uM), ferrostatin (Fer-1, 20 uM), and trolox (0.5 mM) The lipid ROS levels (DCF-DA and BODIPY C11 measurements) were confirmed and the results are shown in FIG.

As shown in FIG. 3, total ROS and lipid ROS levels following artesonate treatment were significantly increased in artesinate-treated HNC cells, and this effect was observed in ferrostatin-1 (20 uM) Or co-culture with trolox (0.5 mM). However, it was not blocked by necro-statin-1 (20 uM) treatment.

These results are summarized to confirm that aresinate induces cell death through iron-dependent ROS-mediated iron ferrooptic cell death in HNC cells.

Example  3. Artesanate  Depending on the treatment Nrf2 - Activation of antioxidant factor pathway

Through the above Example 2, it was confirmed that the artesanate showed the cancer cell killing effect through the induction of iron blast cell death in HNC. Based on these confirmations, we further investigated the mechanism of HNC cell resistance to artesonate. More specifically, HN9 and HN9-cisR cells and cisplatin-resistant HNC cells were treated with 10 to 25 uM of artesinate for 24 hours, and changes in expression of the related proteins were confirmed by Western blotting. The results are shown in FIG.

As shown in Figs. 4A and 4B, artesanate increased the expression of Nrf2 during iron cell death of HNC cells. Treatment of aresinate reduced levels of xCT, RAD51, and Keap1 proteins, but increased levels of p53 and Nrf2 protein in HN9 and HN9-cis9 cells. Basal Nrf2 levels were higher in cisplatin-resistant HNC cells compared to cisplatin-sensitive cells, and Nrf2, HO-1, and NQO1 were activated in HNC cells after arthesinate exposure.

HN9-cisR cells were treated with 20uM erastin or 2mM sulfasalazine (SAS), a representative ferrotocyte inducing substance, and Nrf2, Keap1, and HO -1 protein levels were compared with the aresinate treatment group. As a control group, the dimethyl sulfoxide treatment group (NT) was used, and the results are shown in Fig.

As shown in Fig. 4C, the aresinate treatment reduced Keapl and increased Nrf2 and HO-1, as shown by ferrotic inducers erastin and sulfasalazine. In other words, arthesinate proved to be a cause of ferrotosis-inducing substances, but surprisingly, resistance to ferrotosis could be induced by activating the pathway of Nrf2-antioxidant response element.

In addition, the level of NFE2L2 (Nrf2) mRNA after treatment with dimethyl sulfoxide, artesinate (50uM), and erastin (20uM) for 24 hours in three cisplatin resistant HNC cells was measured and the results are shown in FIG.

As shown in Fig. 5, the level of Nrf2 mRNA was not changed in all three cisplatin-resistant HNC cells even when treated with artesinate or erastin.

In order to determine whether Nrf2 is regulated at the level of transcription activity according to the above results, the expression of Nrf2 in the HNC cell nuclear extract was changed for 24 hours with 50 uM aresinate or 20 uM Erastin And confirmed after treatment. The results are shown in Fig.

As shown in FIG. 6, the expression of Nrf2 in the nuclear extract of HNC cells treated with artesonate or erastin was markedly increased, confirming that the transcriptional activity was increased (A and B). Also, it was confirmed that HO-1 and NQO1 mRNA levels were changed in cisplatin-resistant cells (C).

These results indicate that the treatment with aresinate is a substance capable of increasing Nrf2 expression at the level of transcriptional activity in cancer cells and activates the Nrf2-antioxidant response element pathway to increase the resistance to arthesinate .

Example  4. Nrf2  Due to inhibition Artesanate  Overcome resistance - in vitro

To further confirm whether iron-induced apoptosis of arthesinate in HNC cells is associated with activation of Nrf2, activation of the Nrf2-ARE pathway through silencing of the KEAP1 gene, confirmation of susceptibility to arthesinate, Nrf inhibitor trigonellin treatment And confirmation of changes in susceptibility by

4.1 KEAP1  Due to inhibition Artesanate  Confirm reduction of susceptibility to

First, in order to inhibit the expression of KEAP1 gene, cisplatin-sensitive HN9 cells were transfected with siKEAP1 or siNrf2 inhibitor shown in Table 1, siRNA control (siCtr), and then expression of Keap1 mRNA, expression of proteins involved in Nrf2-ARE pathway The change in cell number was confirmed, and the results are shown in Fig.

As shown in FIG. 7, siRNA transfection for KEAP1 reduced mRNA expression of KEAP1 (A), resulting in an increase in protein expression of Nrf2 and increased expression of HO-1 protein such as Nrf2-ARE pathway Respectively. Treatment with aresinate for the siKEAP-1 transfected group further increased the protein expression of Nrf2 (B). In addition, it was confirmed that in Keap1 silenced HN9 cells, even after aresinate treatment, the number of HN9 cells increased, resulting in increased resistance to artesonate and decreased anticancer effect. (C) These results showed that inhibition of KEAP1 expression Activates the Nrf2-ARE pathway and consequently resistance to artesonate is induced through activation of the Nrf2-ARE pathway.

  4.2 Nrf2  Restoration of susceptibility and mechanism of inhibitor treatment

Since the reduced susceptibility of Keap1 silenced HN9 cells to aresinate was found to be associated with the Nrf2-ARE pathway, reduced susceptibility was restored by complex treatment with the Nrf2 inhibitor trigonelline (100 uM) And the results are shown in FIG.

As shown in FIG. 8, the resistance of the HN9 cells induced by KEAP1 silencing by siKEAP1 to artesonate was reversed by the inhibition of Nrf2 by treatment with siNFE2L2. In addition, the cell survival rate was also decreased in the treatment group treated with siKEAP1 and trigonelline, and it was confirmed that the suppression of the susceptibility to aresinate by KEAP1 silencing can be reversed through the treatment with Nrf2 inhibitor. That is, it is expected that the problem of resistance to artesonate can be solved by simultaneously suppressing Nrf2.

In order to confirm the above-described sensitivity-restoring mechanism, the levels of cellular GSH and lipid ROS following treatment with artesonate and Nrf2 inhibitor trigonelline were determined, and the results are shown in FIG.

As shown in Fig. 9, in the cisplatin resistant cell line, aresinate treatment significantly reduced cellular glutathione (GSH) levels and induced lipid ROS accumulation, and this effect was markedly increased by co-culture with trigonellin . This suggests that co-cultivation with trigonellin helps reduce GSH levels and promotes lipid ROS accumulation, thereby increasing the iron-dependent anticancer activity of aresinate and overcoming the resistance to arthesinate-induced ferrotocyte This is the result.

Example  5. Nrf2 Depending on the suppression of the -ARE path Artesanate  Confirm resistance overcome

It was confirmed in Example 4 that the Nrf2-ARE pathway is an important mechanism for acquiring resistance to the iron-dependent anticancer activity of the artesinate. Thus, when the pathway is inhibited, whether or not the resistance of the cell to the artesinate can be improved Was more specifically confirmed through silencing of genes using siRNAs of Nrf2 and HO-1 shown in Table 1, and the results are shown in Fig.

As shown in FIG. 10, the silencing of the Nrf2 and / or HO-1 genes reduces the cell survival rate of cisplatin-resistant HNC cells after aresinate exposure compared to siRNA control cells (FIGS. 10A and B). These results clearly show that Nrf2 inhibition can be an important factor to overcome the resistance to arthesinate. That is, in HN3-cisR cells transfected with resistant HNC siNrf2, the cell viability and cellular GSH levels were significantly decreased, and cellular ROS levels were significantly increased compared to the siRNA control. This effect was also blocked by pretreatment of the antioxidant trolox (Figs. 10C-E).

Example  6. Nrf2  Due to inhibition Artesanate  Confirmation of overcoming resistance - in vivo

The effect of Nrf2 genetic or pharmacological silencing in HNC cells was tested in vivo. Whether the aresinate-induced tumor growth inhibition effect was reduced by KEAP1 inhibition was confirmed by tumor weight and volume measurements, and whether the resistance was overcome by co-treatment of the Nrf2 inhibitor trigonelline was also confirmed. For HN3-cisR in vivo experiments, 50 mg / kg aresinate or trigonelline was orally administered daily.

In addition, whether or not the genetic suppression (shNrf2) and pharmacological inhibition (trigonelline) of aresinate and Nrf2 in mice can affect the increase of glutathione deficiency and γH2AX formation leading to iron-dependent anti-cancer effects is shown in the shRNA sequence of Table 2 And the result was shown in Fig.

As shown in Fig. 11, tumor weight reduction effect was reduced in nude mice treated with Keap1 shRNA transfected HN9 cells as compared to shRNA control mice (A). These results are the same as those of the previous cell experiments in which the iron-dependent anti-cancer effect is reduced through the activation of Nrf2 when Keap1 is inhibited. In addition, co-treatment of the Nrf2 inhibitor trigonelline significantly reduced in vivo tumor growth and cellular GSH levels in mice bearing Keap1-silenced, Nrf2 activated HN9 cells (B and C).

The single treatment of the Nrf2 inhibitor trigonelline did not significantly reduce the tumor volume, but the joint treatment of artesinate and trigonelline significantly reduced the tumor volume in HNC cells. Similarly, the treatment of shNrf2 for genetic Nrf2 inhibition did not significantly reduce the tumor volume, but the tumor volume significantly decreased in the experimental group treated with shNrf2 and artesinate (D). These results indicate that Nrf2 inhibitor acts as an adjuvant to assist in the anticancer activity of aresinate.

In order to confirm the mechanism induced by the co-administration with the Nrf2 inhibitor, a comparison of the cellular GSH level and the formation of [gamma] H2AX in HCN-9 cisR cells revealed that in the tumors of mice in which artesinate and shNRf2 transfection or trigonellin were co- Cellular GSH levels were markedly decreased and γH2AX formation was markedly increased, and it was confirmed that the combination of the combination therapy improved the resistance to ferrotocyte and induce apoptosis (E, F).

These results indicate that artesanate induces the susceptibility of artesonate in resistant HNC cells through γH2AX formation and GSH deficiency, and Nrf2 inhibitor plays a role in supporting such action, It can be confirmed that the resistance can be overcome.

  The genetic suppression of Nrf2 or the treatment of Nrf-inhibitory small molecule materials such as trigonelline for blocking the Nrf2-ARE pathway leading to resistance to ferrotocyte, can be induced by treatment with cisplatin sensitive or resistant cancer cells It is possible to overcome the ferrotocyse resistance of the present invention.

<110> University of Ulsan Foundation for Industry Cooperation          THE ASAN FOUNDATION <120> Anti-cancer composition comprising artemisin derivative and Nrf          inhibitor <130> ASAN1-10 <160> 24 <170> Kopatentin 2.0 <210> 1 <211> 25 <212> RNA <213> Artificial Sequence <220> <223> NFE2L2 siRNA forward <400> 1 guuacaacua gaugaagaga caggt 25 <210> 2 <211> 27 <212> RNA <213> Artificial Sequence <220> <223> NFE2L2 siRNA reverse <400> 2 accugucucu ucaucuaguu guaacug 27 <210> 3 <211> 25 <212> RNA <213> Artificial Sequence <220> <223> NFE2L2 siRNA forward <400> 3 gcaagauuua gaucauuuga aagat 25 <210> 4 <211> 27 <212> RNA <213> Artificial Sequence <220> <223> NFE2L2 siRNA reverse <400> 4 aucuuucaaa ugaucuaaau cuugcuc 27 <210> 5 <211> 25 <212> RNA <213> Artificial Sequence <220> <223> NFE2L2 siRNA forward <400> 5 ucaacgaaau gauguccaaa gagca 25 <210> 6 <211> 27 <212> RNA <213> Artificial Sequence <220> <223> NFE2L2 siRNA reverse <400> 6 ugcucuuugg acaucauuuc guugaag 27 <210> 7 <211> 25 <212> RNA <213> Artificial Sequence <220> <223> KEAP1 siRNA forward <400> 7 agaacagacu aacuaguguc uuuca 25 <210> 8 <211> 27 <212> RNA <213> Artificial Sequence <220> <223> KEAP1 siRNA reverse <400> 8 ugaaagacac uaguuagucu guucuuu 27 <210> 9 <211> 25 <212> RNA <213> Artificial Sequence <220> <223> KEAP1 siRNA forward <400> 9 agcgcuacga uguggaaaca gagac 25 <210> 10 <211> 27 <212> RNA <213> Artificial Sequence <220> <223> KEAP1 siRNA reverse <400> 10 gucucuguuu ccacaucgua gcgcucc 27 <210> 11 <211> 25 <212> RNA <213> Artificial Sequence <220> <223> KEAP1 siRNA forward <400> 11 agaggaacga guggcgaaug aucac 25 <210> 12 <211> 27 <212> RNA <213> Artificial Sequence <220> <223> KEAP1 siRNA reverse <400> 12 gugaucauuc gccacucguu ccucucu 27 <210> 13 <211> 25 <212> RNA <213> Artificial Sequence <220> <223> HO1 siRNA forward <400> 13 aacauugucu gauaguagcu ugaaa 25 <210> 14 <211> 27 <212> RNA <213> Artificial Sequence <220> <223> HO1 siRNA reverse <400> 14 uuucaagcua cuaucagaca auguugu 27 <210> 15 <211> 25 <212> RNA <213> Artificial Sequence <220> <223> HO1 siRNA forward <400> 15 uaaacaacau ugucugauag uagct 25 <210> 16 <211> 27 <212> RNA <213> Artificial Sequence <220> <223> HO1 siRNA reverse <400> 16 agcuacuauc agacaauguu guuuauu 27 <210> 17 <211> 25 <212> RNA <213> Artificial Sequence <220> <223> HO1 siRNA forward <400> 17 gguccuuaca cucagcuuuc uggtg 25 <210> 18 <211> 27 <212> RNA <213> Artificial Sequence <220> <223> HO1 siRNA reverse <400> 18 ugcucuuugg acaucauuuc guugaag 27 <210> 19 <211> 22 <212> RNA <213> Artificial Sequence <220> <223> NFE2L2 shRNA forward <400> 19 tagatcagaa acatcaatgg gc 22 <210> 20 <211> 22 <212> RNA <213> Artificial Sequence <220> <223> NFE2L2 shRNA reverse <400> 20 acccattgat gtttctgatc ta 22 <210> 21 <211> 22 <212> RNA <213> Artificial Sequence <220> <223> NFE2L2 shRNA forward <400> 21 tttcgttgaa gtcaacaaca gg 22 <210> 22 <211> 22 <212> RNA <213> Artificial Sequence <220> <223> NFE2L2 shRNA reverse <400> 22 actgttgttg acttcaacga aa 22 <210> 23 <211> 22 <212> RNA <213> Artificial Sequence <220> <223> NFE2L2 shRNA forward <400> 23 ttgagcttca ttgaactgct ct 22 <210> 24 <211> 22 <212> RNA <213> Artificial Sequence <220> <223> NFE2L2 shRNA reverse <400> 24 cgagcagttc aatgaagctc aa 22

Claims (10)

Artesinate or a pharmaceutically acceptable salt thereof; And Nrf2 (nuclear factor erythroid 2-related factor 2) inhibitors,
The Nrf2 inhibitor may be selected from the group consisting of antisense, dsRNA, ribozyme, aptamer, fragments of Nrf2 binding protein, fragments of antibodies or antibodies, trigonelline, ascorbic acid, luteolin, ochratoxin A, A pharmaceutical composition for preventing or treating cancer, which is at least one selected from the group consisting of chrysin, brusatol, and all- trans retinoic acid (ATRA).
delete delete delete The pharmaceutical composition for preventing or treating cancer according to claim 1, wherein the cancer is head and neck cancer.
The pharmaceutical composition according to claim 1, wherein the cancer is a resistant cancer against a platinum-based anticancer drug selected from the group consisting of cisplatin, carboplatin and nedaplatin. Gt;
The pharmaceutical composition for preventing or treating cancer according to claim 1, wherein the artesinate or a pharmaceutically acceptable salt thereof induces ferroptosis in cancer cells.
The pharmaceutical composition for preventing or treating cancer according to claim 1, wherein the cancer is a pertortisis-resistant cancer.
Nrf2 (nuclear factor erythroid 2-related factor 2) inhibitor,
The Nrf2 inhibitor may be selected from the group consisting of antisense, dsRNA, ribozyme, aptamer, fragments of Nrf2 binding protein, fragments of antibodies or antibodies, trigonelline, ascorbic acid, luteolin, ochratoxin A, Wherein the anticancer agent is at least one selected from the group consisting of chrysin, brusatol and all- trans retinoic acid. The anticancer agent for overcoming resistance to artesinate or a pharmaceutically acceptable salt thereof, A composition for therapeutic use.
Treating the candidate substance with a resistant cancer cell against an artesinate or a pharmaceutically acceptable salt thereof; And
Selecting a substance which induces a decrease in expression of Nrf2, HO-1 (Heme oxygenase-1) or NQO1 (NAD (P) H quinone dehydrogenase 1) in the cancer cell; Or a pharmaceutically acceptable salt thereof. &Lt; Desc / Clms Page number 19 &gt;

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