KR102597832B1 - A Composition for Enhancing Anti-Cancer Effect of Sorafenib - Google Patents

Abstract

The present invention relates to a composition for preventing or treating cancer, specifically hepatocellular carcinoma, and a composition for preventing or treating diseases related to SIRT3 (Sirtuin 3) deficiency. The present invention can be usefully used as an efficient combination treatment method that can significantly extend the survival period of cancer patients by suppressing resistance to poorly differentiated compounds, especially sorafenib, and improving treatment sensitivity. In addition, the screening method for SIRT3 activators of the present invention is based on the negative correlation between SIRT3 and CDK4/6 newly identified in the present invention, and is a fundamental therapeutic candidate for various diseases caused by reduced expression or dysfunction of SIRT3. Materials can be searched accurately and quickly.

Description

Composition for enhancing the anti-cancer effect of sorafenib {A Composition for Enhancing Anti-Cancer Effect of Sorafenib}

The present invention relates to a composition for enhancing the therapeutic sensitivity of poorly differentiated anticancer agents for solid tumors, and specifically, to a composition for enhancing the responsiveness of hepatocellular carcinoma to sorafenib.

Hepatocellular carcinoma (HCC) is one of the leading causes of cancer-related death worldwide [1]. Patients with early-stage HCC have no noticeable symptoms, and most HCC is discovered after it has progressed to a considerable extent, making it difficult for patients to receive appropriate treatment such as surgical resection or liver transplantation [2].

Although surgical treatment improves the course of the disease, there is a risk of recurrence even in early HCC. Sorafenib (Nexavar), an oral multi-kinase inhibitor, has been used as a first-line chemotherapy for patients with advanced HCC [3]. Although sorafenib extends the median survival time of patients by 3-5 months, its use is limited due to its high resistance rate and serious side effects [4-6]. Accordingly, the demand for strategies to enhance the anticancer activity of sorafenib is gradually increasing.

Sirtuins (SIRT1-7) are key regulators of tumorigenic processes such as cell cycle progression, cell survival, metabolism, and angiogenesis [7-9]. SIRT3, a mitochondrial sirtuin, deacetylates and activates several enzymes involved in cellular redox balance and defense against oxidative damage [10-12]. Several studies have suggested that SIRT3 plays a dual function in cancer [13-15]. SIRT3 functions as an oncogene in oral cancer and melanoma by maintaining ROS levels below a certain threshold to prevent apoptosis and promote cell proliferation [16, 17]. On the other hand, SIRT3 has been identified as a tumor suppressor in HCC [18, 19], breast cancer [20], ovarian cancer [21], and leukemia [22]. Furthermore, SIRT3 has been reported to be involved in metabolic reprogramming (Warburg) and triggering cell death under stress conditions [23, 24]. In fact, high expression of SIRT3 is associated with a favorable prognosis and increased overall survival in HCC patients [25]. Therefore, regulation of SIRT3 expression could be a new strategy for personalized treatment of tumors.

Numerous papers and patent documents are referenced and citations are indicated throughout this specification. The disclosures of the cited papers and patent documents are incorporated herein by reference in their entirety to more clearly explain the content of the present invention and the level of technical field to which the present invention pertains.

Patent Document 1. U.S. Patent Publication No. 2020-0054635

The present inventors have made extensive research efforts to develop a method to improve the prognosis and survival rate of solid cancer patients by overcoming resistance to chemotherapy drugs and improving treatment responsiveness. As a result, when the expression level or activity of SIRT3 is increased, the treatment sensitivity to poorly differentiated anticancer drugs represented by the following formula (1), specifically sorafenib, is significantly improved, thereby improving the migration, cell cycle progression, and proliferation of cancer cells. The present invention was completed by discovering that a synergistic anti-cancer effect was significantly inhibited.

Therefore, an object of the present invention is to provide a composition for preventing or treating cancer, specifically hepatocellular carcinoma.

Another object of the present invention is to provide a composition for preventing or treating diseases related to SIRT3 (Sirtuin 3) deficiency.

Another object of the present invention is to provide a screening method for Sirtuin 3 (SIRT3) activator.

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

According to one aspect of the present invention, the present invention

(a) Activator of Sirtuin 3 (SIRT3); and

(b) Provided is a composition for preventing or treating cancer comprising a compound represented by the following formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient:

Formula 1

In Formula 1, R ₁ is C ₁ -C ₃ alkyl, R ₂ is halogen, and R ₃ to R ₅ are each independently hydrogen or halogen.

The present inventors have made extensive research efforts to develop a method to improve the prognosis and survival rate of solid cancer patients by overcoming resistance to chemotherapy drugs and improving treatment responsiveness. As a result, when the expression level or activity of SIRT3 is increased, the treatment sensitivity to the poorly differentiated anticancer agent represented by Formula 1, specifically sorafenib, is significantly improved, thereby increasing the migration, cell cycle progression, and proliferation of cancer cells. It was found that a synergistic anti-cancer effect was observed that was significantly inhibited.

As used herein, the term “activator of SIRT3” refers to an active ingredient that enhances the expression level or activity of SIRT3 protein, for example, the expression of SIRT3, a protein whose sequence and structure is already known in the art, to a gene or protein. It includes, but is not limited to, nucleic acid molecules, peptides, proteins, compounds and natural products that enhance the level or enhance intrinsic biological activity. Accordingly, the activator of SIRT3 that can be used in the present invention includes means that can directly increase the level of SIRT3 protein in the subject, such as the SIRT3 protein itself or a gene carrier loaded with the gene encoding it, as well as its expression level. All active ingredients that affect activity are included. Therefore, the term “activator of SIRT3 protein” is used interchangeably with “SIRT3 protein agonist.”

As used herein, the term “alkyl” refers to a straight-chain or branched saturated hydrocarbon group and includes, for example, methyl, ethyl, propyl, isopropyl, etc. C ₁ -C ₃ alkyl refers to an alkyl group having an alkyl unit having 1 to 3 carbon atoms, and when C ₁ -C ₃ alkyl is substituted, the carbon number of the substituent is not included.

As used herein, the term “halogen” refers to the halogen group elements and includes, for example, fluoro, chloro, bromo, and iodo.

According to a specific embodiment of the present invention, in Formula 1, R ₁ is C ₁ alkyl, and R ₂ is Cl. More specifically, in Formula 1, R ₃ to R ₅ are F.

R ₁ is C ₁ alkyl (methyl), R ₂ is Cl, and R ₃ to R ₅ are F. The compound of Formula 1 is sorafenib (sorafenib), 4-[4-({[4-chloro-3-(tri fluoromethyl)phenyl]carbamoyl}amino)phenoxy]-N-methylpyridine-2-carboxamide)

As used herein, the term “responsiveness” refers to the degree to which symptoms of a disease are alleviated, suppressed, eliminated, or alleviated by an individual suffering from a specific disease by a therapeutic composition for the disease, and has the same meaning as “treatment sensitivity.” It is used. Individuals with low responsiveness may be considered resistant or resistant to the therapeutic agent.

As used herein, the term “pharmaceutically acceptable salt” includes salts derived from pharmaceutically acceptable inorganic acids, organic acids, or bases. Examples of suitable acids include hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, perchloric acid, fumaric acid, maleic acid, phosphoric acid, glycolic acid, lactic acid, salicylic acid, succinic acid, toluene-p-sulfonic acid, tartaric acid, acetic acid, trifluoroacetic acid, citric acid, methane. Examples include sulfonic acid, formic acid, benzoic acid, malonic acid, naphthalene-2-sulfonic acid, and benzenesulfonic acid. Salts derived from suitable bases may include alkali metals such as sodium, alkaline earth metals such as magnesium, ammonium, and the like.

As used herein, the term “prevention” refers to suppressing the occurrence of a disease or disease in a subject who has not been diagnosed as having the disease or disease but is likely to develop the disease or disease.

As used herein, the term “treatment” refers to (a) inhibiting the development of a disease, condition or symptom; (b) alleviation of a disease, condition or symptom; or (c) means eliminating a disease, condition or symptom. When the composition of the present invention is administered to a subject, it binds directly to TUFM, thereby activating the influx of autophagy and inducing lipolysis, suppressing the development of metabolic disease symptoms due to excessive accumulation of fat, eliminating or alleviating them. Do it. Accordingly, the composition of the present invention may itself be a composition for treating these diseases, or may be administered together with other pharmacological ingredients and applied as a treatment adjuvant for these diseases. Accordingly, in this specification, the term “treatment” or “therapeutic agent” includes the meaning of “therapeutic aid” or “therapeutic aid.”

As used herein, the term “administration” or “administer” refers to directly administering a therapeutically effective amount of the composition of the present invention to a subject so that the same amount is formed in the subject's body.

In the present invention, the term “therapeutically effective amount” refers to the content of the composition in which the pharmacological ingredients in the composition are contained in a sufficient amount to provide a therapeutic or preventive effect to the individual to whom the pharmaceutical composition of the present invention is to be administered. It is meant to include a “prophylactic effective amount.”

As used herein, the term “subject” includes, without limitation, humans, mice, rats, guinea pigs, dogs, cats, horses, cattle, pigs, monkeys, chimpanzees, baboons, or rhesus monkeys. Specifically, the subject of the present invention is a human.

According to a specific embodiment of the present invention, the activator of Sirtuin 3 (SIRT3) is selected from the group consisting of SIRT3 protein, nucleic acid molecules encoding it, and cyclin-dependent kinase (CDK) 4/6 inhibitors.

As shown in the Examples described later, the present inventors observed a negative correlation between the expression level of SIRT3 and phosphorylation of retinoblastoma protein (Rb), and were the first to identify that CDK4/6 and SIRT3 expression also have a negative correlation. It was discovered that the expression of SIRT3 could be increased through inhibition of CDK4/6.

As used herein, the term “CDK 4/6 inhibitor” refers to a substance that causes a decrease in the activity or expression of CDK 4 and/or CDK6 enzymes, thereby making the activity or expression of CDK 4 and/or CDK6 undetectable or It refers to a substance that reduces the activity or expression of CDK 4 and/or CDK6 not only when it exists at an insignificant level, but also to the extent that SIRT3 expression can be significantly increased.

As used herein, the term “reduced expression” refers to a state in which the expression level of CDK 4/6 is reduced by more than 20%, more specifically, more than 30%, and more specifically, more than 40% compared to the control group. It can mean.

As used herein, the term “reduction in activity” refers to a measurable significant decrease in the in vivo intrinsic kinase activity of CDK 4/6 compared to the control group, for example, a decrease of more than 20% compared to the control group, more specifically. may mean a decrease of more than 30%, more specifically, a decrease of more than 40%. A decrease in activity includes not only a simple decrease in function but also an ultimate inhibition of activity due to a decrease in stability.

Inhibitors of CDK 4/6 include, for example, shRNA, siRNA, miRNA, ribozyme, and PNA that inhibit the expression of CDK 4/6 enzyme at the gene level, the amino acid sequence and encoding nucleotide sequence of which are already known in the art. The CRISPR system includes (peptide nucleic acids), antisense oligonucleotides, and guide RNA that recognizes target genes, as well as antibodies or aptamers that inhibit at the protein level, as well as small molecule compounds, peptides, and natural products that inhibit their activity. However, without being limited thereto, all possible inhibition methods at the gene and protein levels may be used.

According to a specific embodiment of the present invention, the CDK 4/6 inhibitor of the present invention may be an antibody or antigen-binding fragment thereof that inhibits the expression or activity of CDK 4/6 at the protein level. Antibodies that specifically recognize CDK 4/6 are polyclonal or monoclonal antibodies, and are preferably monoclonal antibodies.

The antibody of the present invention can be prepared using methods commonly practiced in the art, such as the fusion method (Kohler and Milstein, European Journal of Immunology , 6:511-519 (1976)), the recombinant DNA method (US Pat. No. 4,816,567) ) or phage antibody library method (Clackson et al, Nature , 352:624-628 (1991) and Marks et al, J. Mol. Biol. , 222:58, 1-597 (1991)). . For general procedures for antibody preparation, see Harlow, E. and Lane, D., Using Antibodies: A Laboratory Manual , Cold Spring Harbor Press, New York, 1999; and Zola, H., Monoclonal Antibodies: A Manual of Techniques , CRC Press, Inc., Boca Raton, Florida, 1984.

As used herein, the term “antigen binding fragment” refers to a portion of a polypeptide to which an antigen can bind among the overall structure of an immunoglobulin, such as F(ab')2, Fab', Fab, Fv, and Including, but not limited to scFv.

As used herein, the term “specifically binding” has the same meaning as “specifically recognizing,” meaning that an antigen and an antibody (or a fragment thereof) specifically interact through an immunological reaction. It means to do.

The present invention can also inhibit CDK 4/6 activity at the protein level by using an aptamer that specifically binds to CDK 4/6 instead of an antibody. As used herein, the term “aptamer” refers to a single-stranded nucleic acid (RNA or DNA) molecule or peptide molecule that binds to a specific target substance with high affinity and specificity. For a general discussion of aptamers, see Hoppe-Seyler F, Butz K "Peptide aptamers: powerful new tools for molecular medicine". J Mol Med. 78(8):426-30(2000); Cohen BA, Colas P, Brent R. “An artificial cell-cycle inhibitor isolated from a combinatorial library”. Proc Natl Acad Sci USA. 95(24):14272-7 (1998).

According to a specific embodiment of the present invention, the CDK 4/6 inhibitor may be a nucleic acid molecule that inhibits the expression of a nucleotide encoding the CDK 4/6 enzyme.

As used herein, the term “nucleic acid molecule” is meant to comprehensively include DNA (gDNA and cDNA) and RNA molecules, and nucleotides, which are the basic structural units in nucleic acid molecules, include not only natural nucleotides but also analogs with modified sugar or base sites. (analogue) is also included (Scheit, Nucleotide Analogs , John Wiley, New York (1980); Uhlman and Peyman, Chemical Reviews , 90:543-584 (1990)).

As used herein, the term “nucleic acid molecule that inhibits expression” specifically recognizes the target gene by including a complementary nucleic acid sequence that can hybridize with the target gene and can cause modifications in the nucleotide structure that cause a decrease in its function. refers to a nucleic acid molecule, and includes, for example, the above-described shRNA, siRNA, miRNA, ribozyme, PNA, antisense oligonucleotide, and gRNA included in the CRISPR system.

As used herein, the term “complementary” means that the expression-inhibiting nucleic acid molecule is sufficiently complementary to selectively hybridize to the target nucleic acid sequence under predetermined annealing or hybridization conditions, and is substantially complementary and completely complementary. It has a meaning that encompasses everything that is (perfectly complementary), and preferably means that it is completely complementary. As used herein, the term “substantially complementary sequence” includes not only completely identical sequences, but also sequences that are partially mismatched with the sequence being compared, to the extent that sequence-specific hybridization can occur by annealing to a specific sequence. am.

As used herein, the term “shRNA (small hairpin RNA)” is a single strand consisting of 50-70 nucleotides forming a stem-loop structure in vivo , and is used to suppress the expression of a target gene through RNA interference. It refers to an RNA sequence that creates a tight hairpin structure. Typically, long RNAs of 19 to 29 nucleotides complement each other on both sides of the loop region of 5 to 10 nucleotides to form a double-stranded stem. In order to ensure constant expression, they are introduced into cells through a vector containing the U6 promoter. It is transduced and is usually passed on to daughter cells, allowing the inhibition of expression of the target gene to be inherited.

As used herein, the term “siRNA” refers to a short double-stranded RNA that can induce RNA interference (RNAi) through cleavage of a specific mRNA. It consists of a sense RNA strand with a sequence homologous to the mRNA of the target gene and an antisense RNA strand with a complementary sequence. The total length is 10 to 100 bases, preferably 15 to 80 bases, and most preferably 20 to 70 bases, and has blunt ends or cohesive ends if the expression of the target gene can be suppressed by the RNAi effect. All ends are possible. The sticky end structure can be either a structure where the 3rd end protrudes or a structure where the 5th end protrudes.

As used herein, the term “miRNA (microRNA)” refers to an oligonucleotide that is not expressed in cells and has a short stem-loop structure and refers to a single-stranded RNA molecule that inhibits target gene expression through complementary binding to the mRNA of the target gene. do.

As used herein, the term “ribozyme” is a type of RNA and refers to an RNA molecule that has the same function as an enzyme that recognizes the base sequence of a specific RNA and cleaves it on its own. The ribozyme consists of a region that specifically binds to the complementary base sequence of the target mRNA strand and a region that cleaves the target RNA.

As used herein, the term “Peptide nucleic acid (PNA)” refers to a molecule that has the properties of both nucleic acid and protein and is capable of complementary binding to DNA or RNA. PNA is not found in nature but is artificially synthesized through chemical methods. It forms a double strand through hybridization with natural nucleic acids of complementary base sequences and regulates the expression of target genes.

As used herein, the term “antisense oligonucleotide” refers to a nucleotide sequence complementary to the sequence of a specific mRNA, which binds to the complementary sequence in the target mRNA and carries out its translation into a protein, translocation into the cytoplasm, maturation, or any other activity. It refers to a nucleic acid molecule that inhibits essential activities for overall biological function. Antisense oligonucleotides may be modified at one or more base, sugar, or backbone positions to enhance efficacy (De Mesmaeker et al., Curr Opin Struct Biol. , 5(3):343-55, 1995) . The oligonucleotide backbone can be modified with phosphorothioate, phosphotriester, methyl phosphonate, short-chain alkyl, cycloalkyl, short-chain heteroatomic, heterocyclic sugars, etc.

As used herein, the term “gRNA (guideRNA)” refers to an RNA molecule used in a gene editing system that recognizes a target gene and specifically cuts the recognized region by inducing a nuclease. A representative example of such a gene editing system is the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) system.

The nucleic acid molecule of the present invention described above can inhibit the expression of CDK 4/6 at the gene level by expressing it in a subject.

As used herein, the term “express” means causing a subject to express an exogenous gene or artificially introducing it using a gene delivery system to increase the natural expression level of an endogenous gene, so that the gene is expressed in the subject's cells. This means that replication becomes possible as an extrachromosomal factor or through completion of chromosomal integration. Accordingly, the term “expression” has the same meaning as “transformation,” “transfection,” or “transduction.”

As used herein, the term “gene delivery system” refers to all means for transporting genes into cells, and gene delivery has the same meaning as transduction of genes into cells. At the tissue level, the term gene transfer has the same meaning as spread of genes. Accordingly, the gene delivery system of the present invention can be described as a gene penetration system and a gene diffusion system.

More specifically, the CDK 4/6 inhibitor is a compound represented by the following formula (2) or a pharmaceutically acceptable salt thereof:

Formula 2

According to a specific embodiment of the present invention, the cancer prevented or treated with the composition of the present invention is hepatocellular carcinoma.

According to another aspect of the present invention, the present invention provides a composition for preventing or treating diseases related to SIRT3 (Sirtuin 3) deficiency, comprising a CDK (cyclin-dependent kinase) 4/6 inhibitor as an active ingredient.

Since the CDK 4/6 inhibitor used in the present invention has already been described in detail, its description is omitted to avoid duplication.

As used herein, the term “SIRT3 deficiency-related disease” refers to a disease caused by reduced expression or dysfunction of SIRT3. SIRT3, the primary NAD-dependent deacetylating enzyme present in mitochondria, plays a key role in regulating oxidative stress, mitochondrial function, and metabolism by removing the toxicity of free radicals and catalyzing the removal of fatty acids through beta-oxidation. , its deficiency is known to accelerate neurodegeneration in the brain. Accordingly, SIRT3 deficiency-related diseases that can be prevented or treated with the composition of the present invention are diseases related to mitochondrial dysfunction, specifically neurodegenerative diseases, and more specifically, Alzheimer's disease and Parkinson's disease. , amyotrophic lateral sclerosis, Huntington's disease, Creutzfeldt-Jakob disease, Lewy body dementia, cerebral amyloid angiopathy, Pick's disease ( Pick's disease) and multiple sclerosis, but are not limited thereto.

According to another aspect of the present invention, the present invention provides a method for screening a Sirtuin 3 (SIRT3) activator comprising the following steps:

(a) contacting a candidate material with a biological sample containing CDK (cyclin-dependent kinase) 4/6 or cells expressing it;

(b) measuring the activity or expression level of CDK 4/6 in the sample,

When the activity or expression level of CDK 4/6 is decreased, the candidate substance is determined to be a SIRT3 activator.

In the present invention, the term “biological sample” refers to any sample obtained from mammals, including humans, containing CDK 4/6 or cells expressing it, including, but not limited to, tissues, organs, cells, or cell culture fluid. .

The term “candidate” used while referring to the screening method of the present invention refers to a substance added to a sample containing CDK 4/6 or cells expressing it to test whether it affects the activity or expression level of CDK 4/6. Refers to an unknown substance used in screening. The test substances include, but are not limited to, compounds, nucleotides, peptides, and natural extracts. The step of measuring the expression level or activity of CDK 4/6 in a biological sample treated with a test substance can be performed by various expression level and activity measurement methods known in the art. As a result of the measurement, if the expression level or activity of CDK 4/6 is decreased, the test substance can be determined to be an activator of SIRT3.

According to the present invention, the expression level of CDK 4/6 enzyme, which is the screening target of the present invention, can be measured according to an immunoassay method using an antigen-antibody reaction. This immunoassay can be performed according to various immunoassay or immunostaining protocols developed conventionally.

The expression level of the CDK 4/6 enzyme can also be measured at the gene level using an agent that measures the expression level of the gene encoding the CDK 4/6 enzyme. An agent for measuring the expression level of the gene is, for example, a primer or probe that specifically binds to the nucleic acid sequence of the CDK 4/6 gene.

As used herein, the term “primer” refers to conditions that induce the synthesis of a primer extension product complementary to a nucleic acid chain (template), i.e., the presence of nucleotides and a polymerizing agent such as DNA polymerase, and synthesis under conditions of appropriate temperature and pH. refers to an oligonucleotide that acts as the starting point. Specifically, the primer is a single strand of deoxyribonucleotide. Primers used in the present invention may include naturally occurring dNMP (i.e., dAMP, dGMP, dCMP, and dTMP), modified nucleotides, or non-natural nucleotides, and may also include ribonucleotides.

In the present invention, the term “complementary” means that a primer or probe is sufficiently complementary to selectively hybridize to a target nucleic acid sequence under predetermined annealing or hybridization conditions, and is substantially complementary and completely complementary. It means encompassing all cases of (perfectly complementary), and specifically means completely complementary cases. As used herein, the term “substantially complementary sequence” refers not only to completely identical sequences, but also to sequences that are partially mismatched with the sequence being compared, to the extent that they can anneal to a specific sequence and serve as a primer.

As used herein, the term “probe” refers to a linear oligomer having a natural or modified monomer or linkage containing deoxyribonucleotides and ribonucleotides that can hybridize to a specific nucleotide sequence. Specifically, the probes are single-stranded, more specifically deoxyribonucleotides, for maximum efficiency in hybridization.

According to a specific embodiment of the present invention, the cells expressing CDK 4/6 are liver tissue-derived cells.

According to a specific embodiment of the present invention, the SIRT3 activator is a composition for enhancing the therapeutic responsiveness of sorafenib in hepatocellular carcinoma.

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

(a) The present invention provides a composition for preventing or treating cancer, specifically hepatocellular carcinoma, and a composition for preventing or treating diseases related to SIRT3 (Sirtuin 3) deficiency.

(b) The present invention can be usefully used as an efficient combination treatment method that can significantly extend the survival period of cancer patients by suppressing resistance to poorly differentiated compounds, especially sorafenib, and improving treatment sensitivity.

(c) In addition, the screening method for SIRT3 activators of the present invention is based on the negative correlation between SIRT3 and CDK4/6 newly identified by the present inventors for various diseases caused by reduced expression or dysfunction of SIRT3. Fundamental therapeutic candidate substances can be searched accurately and quickly.

Figure 1 is a diagram showing the SIRT3 expression pattern according to ¹⁸ F-FDG absorption in hepatocellular carcinoma (HCC) patients. Figure 1a shows the results of RT-PCR using a SIRT3-specific probe on RNA extracted from frozen HCC samples obtained after transsphenoidal surgery. Expression of target genes was normalized to B2M (housekeeping gene) using the 2-ΔΔCt method. The boundary of the box closest to 0 represents 25%, the line within the box represents the median, and the boundary of the box furthest from 0 represents 75%. Figure 1B shows immunofluorescence and DAPI counterstaining results for formalin-fixed, paraffin-embedded human HCC samples. Scale bar: 50 μm. Statistical analysis was performed using GraphPad Prism. Results were expressed as mean ± standard deviation. Comparison between groups was performed using the Mann-Whitney test. *P<0.05
Figure 2 This figure shows SIRT3 expression in hepatocellular carcinoma (HCC) cell lines and HCC transplant models. Figure 2a shows the results of measuring SIRT3 expression in four different HCC cell lines by quantitative RT-PCR. The expression levels of target genes were normalized to the housekeeping gene B2M. Data are expressed as mean ± standard deviation for three independent experiments. Figure 2b shows the results of Western blotting using antibodies against SIRT3 and actin in HCC cell lines. Figure 2c shows the results of immunohistochemical analysis using SIRT3, GLUT1, and Ki67 antibodies on formalin-fixed, paraffin-embedded liver tissue obtained from an HCC transplant model. Scale bar: 20μm. Figure 2d shows the results of transducing Huh7 cells with the MOCK vector and pcDNA-SIRT3, culturing them for 48 hours, extracting the proteins, and measuring the expression of SIRT3, Ki67, and actin by Western blotting. Figure 2e shows the results of measuring glucose absorption using the Glucose-Glo assay. Data are expressed as mean ± standard deviation for three independent experiments. Statistical analysis was performed using GraphPad Prism, and comparison between groups was performed using the Mann-Whitney test. *P < 0.01.
Figure 3 is a diagram showing the relationship between SIRT3 expression level and sorafenib treatment. Figure 3a shows that SIRT3 expression is reduced when hepatocellular carcinoma (HCC) cells are treated with sorafenib. HCC cells were cultured with DMSO, 1 μM sorafenib, or 10 μM sorafenib. After 48 hours, SIRT3 and actin levels were measured by Western blotting. Figure 3b shows the results of treating Huh7 cells with 10 μM sorafenib 24 hours after transduction with MOCK or pcDNA-SIRT3 and measuring SIRT3 and β-actin expression by Western blotting after 24 hours of culture. Figure 3c shows the results of measuring cell proliferation using the WST-1 assay. Data are expressed as mean ± standard deviation for three independent experiments. Figure 3d is a picture showing the results of measuring cell proliferation by WST-1 assay 48 hours after culturing SIRT3 KD or control cell lines with sorafenib at the indicated concentration. Statistical analysis was performed using GraphPad Prism. Results were expressed as mean ± standard deviation. Comparison between groups was performed using the Mann-Whitney test. *P <0.05; **P < 0.01
Figure 4 is a diagram showing the negative correlation between SIRT3 and retinoblastoma protein (Rb). Figure 4a shows the results of immunohistochemical analysis performed on formalin-fixed, paraffin-embedded (FFPE) liver tissue of an HCC transplant model with an antibody against phosphor-Rb (pRb) and counterstaining with hematoxylin. Scale bar: 50 μm. Figure 4b is a diagram showing the results of RNA extraction from a frozen HCC sample after transsphenoidal surgery and RT-PCR using a probe specific for Rb. Expression of target genes was normalized to B2M (housekeeping gene). The boundary of the box closest to 0 represents 25%, the line within the box represents the median, and the boundary of the box furthest from 0 represents 75%. *P<0.05. Figure 4c shows the results of immunohistochemical analysis performed on FFPE liver tissue from a human HCC patient using SIRT3 (left) and pRb (right) antibodies and counterstaining with hematoxylin. Scale bar: 50μm
Figure 5 This figure shows that SIRT3 is induced by PD0332991 treatment. Figure 5a shows the results of measuring SIRT3 and actin levels by Western blotting 48 hours after culturing HepG2, Hep3B, SK-Hep1, and Huh7 cells with DMSO, 1 μM PD0332991, or 10 μM PD0332991. Western blotting was performed 48 hours after transduction of HepG2 cells, Huh7 cells, and SK-Hep1 cells with scramble siRNA or siRNA against CDK4/6 (Figure 5b). Figure 5c is a diagram showing gene fold change derived through gene expression profiling after transduction of HepG2 cells with scramble siRNA or siRNA for CDK4/6. All data were uploaded to the GEO database under accession number GSE145389.
Figure 6 This figure shows that sensitivity to sorafenib increased due to combined administration of sorafenib and PD0332991. HepG2 cells were cultured with DMSO or 10 μM PD0332991 in the presence or absence of 1 μM sorafenib, and SIRT3 levels were analyzed by qRT-PCR 48 h later (Figure 6a). The expression level of the target gene was normalized to the housekeeping gene B2M. Data are expressed as the mean value ± standard deviation for three independent experiments. Cells were treated in the same way, proteins were extracted, and the expression of SIRT3 and actin was measured by Western blotting (Figure 6b). Figure 6c shows the results of analyzing proliferation after 48 hours using the WST-1 assay. Huh7 cells were cultured with DMSO or 10 μM PD0332991 in the presence or absence of 1 μM sorafenib, and after 48 h, proteins were extracted and the expression of SIRT3 and actin was measured by Western blotting ( Fig. 6D ). Figure 6e shows the results of analyzing proliferation after 48 hours using the WST-1 assay. The results were expressed as the mean ± standard deviation for data from six replicate experiments.
Figure 7 This figure shows that the combined treatment of sorafenib and PD0332991 in HepG2 cells and Huh7 cells enhanced growth inhibition. Figure 7a is a diagram showing the results of the mobility assay. 24 hours after treatment with the indicated drugs, HepG2 cells and Huh7 cells were fixed and visualized by crystal violet staining. Figures 7b and 7c are pictures showing the results of quantitative analysis of cell movement on Matrigel-coated filters. Counts were made in five random fields at ×200 magnification. Figures 7d and 7e show the results of apoptosis analysis using Annexin V-APC/propidium iodide (PI) double staining on HepG2 and Huh7 cells 24 hours after treatment with the indicated drugs. Viable cells negative for both Annexin V and PI by two-color flow cytometry dot plot; Early apoptotic cells positive for Annexin V and negative for PI; and the respective percentages of double-positive terminal apoptotic/necrotic cells. Results are expressed as mean ± standard deviation (n = 3). Statistical analysis was performed using GraphPad Prism. Results are expressed as mean ± standard error (range). Comparison between groups was performed using the Mann-Whitney test. *, P <0.05;**, P <0.01;;***, P < 0.001
Figure 8 is a diagram showing the results of measuring SIRT3 expression according to ¹⁸ F-FDG absorption in HCC patients through Western blotting (Figure 8a) and band quantification using Image J (Figure 8b). Statistical analysis was performed using GraphPad Prism. Results are expressed as mean ± standard deviation. Comparison between groups was performed using the Mann-Whitney test. *P < 0.05.
Figure 9 is a picture showing the synergistic antitumor effect of sorafenib and PD0332991. Figure 9a shows the results of cell cycle analysis after co-administration of sorafenib and PD0332991 in HepG2 and Huh7 cells. Figure 9b shows the results of co-administration of sorafenib and PD0332991 to HepG2 and Huh7 cells, followed by immunofluorescence staining with Ki67 and counterstaining with DAPI. Figure 9c shows the results of counting Ki67 positive cells, and Figure 9d shows the results of measuring apoptosis of HepG2 and Huh7 cells through Annexin V-APC/propidium iodide (PI) double staining.

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

Example

Experiment method

Human HCC samples

The study of the present invention was conducted under the approval of the Clinical Research Ethics Committee of Yonsei University Severance Hospital, and the experiment was conducted in compliance with the current research ethics guidelines (Yonsei IRB number: 4-2015-0904). Patients were selected using a previously reported method [26].

compound

PD0332991 was purchased from TOCRIS Bioscience (Bristol, UK), and sorafenib was purchased from Santa Cruz (Dallas, TX, USA). PD0332991 and sorafenib were dissolved in DMSO (Sigma Aldrich, St. Louis, MO, USA) at a concentration of 10 mM. All reagents were stored at -80°C.

Cell lines and cell culture

Human HCC cell lines HepG2, Hep3B, skHep1, and Huh7 were purchased from the Korean Cell Line Bank. HepG2 was cultured in RPMI, and Hep3B, skHep1, and Huh7 were cultured in DMEM (Dulbecco's modified Eagle's medium). 10% fetal bovine serum (FBS; Hyclone) and 1% penicillin streptomycin were added to all culture media. Cells were maintained at 37°C and 5% CO ₂ in a humidified incubator. For three-dimensional spheroid formation, Costar® Ultra-Low attachment multi-well plates (MerkKGaA→Corning, Darmstadt, Germany) were used. HCC cells were plated at 5000 cells/well and centrifuged at 179 xg for 1 minute. Spheroids were observed 1-2 days after plating. Hep3B, skHep1, and Huh7 cell lines were plated and cultured for 24 hours before transduction. siRNA transduction was performed using Lipofectamine or RNAi-MAX reagents (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions. The hSIRT3 plasmid (sc-61, 555-SH) or scramble shRNA plasmid (sc-108,060) was co-transfected using Lipofectamine 2000 (Invitrogen, 12,566,014). After 72 hours of culture, the cells were treated with puromycin (2 μg/mL) to form stable cell line clones.

Cell proliferation assays and glucose measurements

The WST-1 colorimetric assay (Roche, Mannheim, Germany) to measure cell viability was performed 48 hours later according to the manufacturer's instructions. Huh7 cells were placed in a 96-well plate and transfected with MOCK or pcDNA-SIRT3 plasmid. After 48 hours of treatment, glucose uptake was measured using a glucose assay (Promega, Germany) according to the manufacturer's instructions. Absorbance was measured at 440 nm and 640 nm using a microplate reader (Molecular Devices, CA, USA).

RNA isolation and sequencing

Total RNA was isolated using TRIzol reagent (Invitrogen). RNA quality was evaluated with an Agilent 2100 bioanalyzer using the RNA 6000 Nano Chip (Agilent Technologies, Amstelveen, The Netherlands), and RNA was quantified with an ND-2000 spectrophotometer (Thermo Inc., DE, USA). For control and test RNA samples, libraries were constructed using the QuantSeq 3′mRNA-Seq Library Prep Kit (Lexogen, Inc., Austria) according to the manufacturer's instructions. In summary, 500ng of total RNA was prepared for each sample, an oligo-dT primer containing an Illumina compatible sequence at the 5' end was hybridized to the RNA, and reverse transcription was performed. After RNA template digestion, second-strand synthesis was initiated with random primers containing an Illumina compatible-linker sequence at the 5′ end. The double-stranded library was purified with magnetic beads to remove all reaction composition. The library was amplified and the complete adapter sequence required for cluster formation was added. The amplified library was purified and high-throughput sequencing (HTS) was performed using NextSeq 500 (Illumina, Inc., USA).

Real-time PCR

Total RNA was extracted using TRIzol (Invitrogen), and cDNA was synthesized from 500 ng of total RNA using ReverTra Ace qPCR RT Master Mix and gDNA Remover (Toyobo, Osaka, Japan). Quantitative RT-PCR was performed on a C1000 a → a C1000 Thermal Cycler (Bio-Rad) using SYBR Green Real Time PCR Master Mix (Toyobo, Osaka, Japan). Gene expression levels were normalized to the beta-2 microglobulin (B2M) mRNA expression level of the corresponding cDNA sample. All PCR primers were purchased from Bioneer (Daejeon, Korea). The following primers were used: SIRT3 (forward"??* 5′-GAAACTACAAGCCCAACGTCA-3′, reverse 5′-AAGGTTTCCATGAGCTTCAACC-3′), RB1 (forward"??* 5′-GAAGCAACCCTCCTAAACCAC-3′, reverse 5′) -CTGCTTTTGCATTCGTGTTCG-3′), and B2M (forward"??* 5′-TTACTCACGTCATCCAGCAGA-3′, reverse 5′-AGAAAGACCAGTCCTTGCTGA-3′).

Western blotting

Western blotting was performed according to a previously reported method [26]. Primary antibodies used were: SIRT3 (Cell Signaling Technology, Danvers MA, USA; clone C73E3; diluted 1:1000), CDK4 (DCS156, 1:1000), CDK6 (DCS83, 1:1000), Phospho- Rb(Ser807/811) (D20B12, 1:1000), Rb(4H1, 1:2000), PCNA(D3H8P, 1:2000), GLUT1(1:2000) (purchased from Abcam (Cambridge, UK)) and Ki67 (Santa Cruz, Dallas TX, USA; MIB-1, 1:500). Western blotting experiments on biological replicates showed similar expression data, supporting the reproducibility of the results. ChemiDoc For band quantification, images were analyzed with Image Lab software (Bio-Rad, Hercules, California, USA).

flow cytometry

To quantify apoptosis, double staining was performed using Annexin V-FITC Apoptosis Detection Kit (BD Pharmingen™, NJ, USA) and propidium iodide (PI) according to the manufacturer's instructions. After incubation with the indicated drugs, HepG2 and Huh7 cells were collected, washed twice with ice-cold PBS, and resuspended in 200 μL of binding buffer. Annexin V-FITC was added to the cells and incubated in dark conditions at 25°C for 15 minutes. PI (10 mL) was added to the tube and incubated in dark conditions at 4°C for 5 minutes. Afterwards, flow cytometry was performed on the samples using CELL Quest software (BD). Cell fractions in different quadrants were analyzed using quadrant statistical analysis. Cells in the lower right quadrant (annexin-V+/PI-) show early apoptosis and those in the upper right quadrant (annexin-V+/PI+) show late apoptosis. For cell cycle analysis, HepG2 and Huh7 cells were collected after incubation with the indicated drugs and incubated at 4°C in 70% ethanol for 1 hour. After washing with PBS, cells were incubated with PI at a concentration of 5 μg/mL and RNase A at a concentration of 10 mg/mL and incubated at 37°C for 30 min-hours. DNA content was analyzed with FlowJo software (Tree StarInc., Ashland, OR, USA).

movement analysis

Chemomigration assays were performed using 24-well plates and uncoated polycarbonate membranes (BioCoat; BD Biosciences, Heidelberg, Germany). PD0332991 or a combination of sorafenib and PD033291 was added to a total of 50,000 cells in medium containing 0.1% FBS and sorafenib. The lower wells were filled with medium supplemented with 10% FBS. 24 hours later, the migrated cells were fixed with 100% methanol and stained with 1.5% (w/v) toluidine blue dissolved in water. Images were recorded on an Olympus BX53 microscope using Olympus Cell Sens software (Carl Zeiss Microscopy, GmbH, Jena, Germany).

Immunostaining

Immunohistochemistry (IHC) and immunofluorescence (IF) were performed according to previously reported methods [27]. Immunostaining was performed using the following primary antibodies: SIRT3 (Cell Signaling Technology, Danvers MA, USA; clone C73E3; dilution 1:500); Ki67 (Dako, Glostrup, Denmark; MIB-1;1:500); GLUT1 (1:500) and Ki67 (SP6, 1:500, Abcam, Cambridge, UK). Images were recorded on an Olympus BX53 microscope using Olympus Cell Sens software (Carl Zeiss Microscopy, GmbH, Jena, Germany). The percentage of Ki67-positive cells and phosphorylated retinoblastoma protein (pRb) was calculated by counting cells with DAPI-stained nuclei.

Cancer gene atlas (TCGA) data analysis

mRNA levels of TCGA liver HCC data were obtained from the OncoLnc TCGA data portal ( www.oncolnc .org). A correlation graph between two different genes was derived using 360 HCC samples with high- and low-expression gene groups (50-50 percentile). GraphPad Prism 5 (GraphPad Software, San Diego, CA, USA) was used for mapping.

statistical analysis

Statistical analysis was performed using GraphPad Prism software (GraphPad Software, Inc., San Diego, CA). Results were expressed as mean ± standard error (range), and a P value <0.05 was considered to have statistical significance. Comparison between groups was performed using the Mann-Whitney test.

Experiment result

SIRT3 expression in HCC patients

¹⁸ Glucose metabolism was assessed using F-fluorodeoxyglucose (FDG) PET/CT. To investigate the relationship between glucose uptake and SIRT3 expression, 21 HCC patients were divided into two groups according to ¹⁸ F-FDG uptake: 9 HCC patients with high glycolytic activity and ¹⁸ F-FDG uptake and 18 patients with high glycolytic activity and ^{18 F} -FDG uptake. Twelve HCC patients with low F-FDG uptake. The mRNA expression of SIRT3 was higher in the low-glycolytic group (Figure 1a). To confirm these observations, IHC analysis was performed on HCC tissues from two groups (n=6 for each group). Patients with low FDG uptake had low GLUT1 and Ki67 expressions and high SIRT3 expression in the tumor area (Figure 1b). In the group with high FDG uptake, high Ki67 and GLUT1 expressions and low SIRT3 expression were observed. Additionally, the expression of SIRT3 in HCC patients was observed by Western blotting (Figure 8). A decrease in SIRT3 was observed in HCC patients with high ¹⁸ F-FDG uptake. Taken together, SIRT3 expression appeared to be associated with glycolytic metabolism and cell proliferation in HCC patients.

SIRT3 expression differences in HCC cells

The expression of SIRT3 was measured in three HCC cell lines (HepG2, Hep3B and Huh7) and their transplantation models showing different proliferation rates and glycolytic activities. HepG2 cells showed the highest SIRT3 expression at both mRNA and protein levels (Figures 2A and 2B). Similar to human HCC patients, a negative correlation between GLUT1 and SIRT3 was also observed in the HCC transplant model (Figure 2c). Additionally, there was a significant negative correlation between SIRT3 and Ki67. HepG2 and Hep3B cells showed low Ki67 expression and high SIRT3 expression, whereas Huh7 and SK-HEP1 cells showed high Ki67 expression and low SIRT3 expression. These results are also consistent with the results of the TCGA database analysis of human HCC samples. Spearman correlation also showed a significant negative correlation between SIRT3 and Ki67 (Spearman coefficient r= -0.3093, P<0.0001) and between SIRT3 and HK2 (Spearman coefficient r= -0.239, P<0.0001). showed (Spearman coefficient r= *?*0.3093, P<0.0001). To further confirm these findings, SIRT3 was overexpressed in Huh 7 cells, which have low basal expression of SIRT3. SIRT3 overexpression reduced Ki67 and GLUT1 expression and also significantly inhibited glucose uptake (Figures 2d and 2e). Taken together, it can be seen that SIRT3 expression has a negative correlation with glycolytic metabolism and cell proliferation in HCC cells and their transplantation models.

Relationship between SIRT3 expression level and sorafenib treatment

Since the experimental results of the present invention showed that SIRT3 plays an important role in HCC, HCC cell lines were cultured with sorafenib for 48 hours to determine whether the expression of SIRT3 changes by sorafenib treatment. As a result, it was confirmed that SIRT3 expression in HCC cells decreased after treatment with 10 μM sorafenib (Figure 3a). Furthermore, Huh7 cell proliferation was significantly reduced when SIRT3 was transfected (Figures 3b and 3c). In addition, as a result of producing SIRT3 KD cell line using HepG2 cells, it was observed that sensitivity to sorafenib was significantly reduced compared to control cells, confirming that SIRT3 expression enhances the responsiveness of HCC cells to sorafenib ( Figure 3d).

Negative correlation between SIRT3 and pRb

PD0332991, a selective inhibitor for CDK4/CDK6 kinases, can block the phosphorylation activity of Rb [28]. By measuring pRb expression in HCC patients, we sought to determine whether PD0332991 could be a candidate drug that increases the sensitivity of sorafenib through regulating SIRT3 expression. IHC using pRb-specific antibody was performed on the HCC transplant model. Huh7 and skHep1-transplantation models showing low SIRT3 expression had high expression levels of pRb, confirming that the expression of pRb and SIRT3 had a negative correlation in the HCC transplantation model (Figure 4a). In human HCC, Rb mRNA levels were significantly higher and SIRT3 mRNA was relatively higher in the high ¹⁸ F-FDG group compared to the low ¹⁸ F-FDG group (Figure 4b). Analysis of TCGA data showed that Spearman correlation also showed a significant negative correlation between SIRT3 and Rb1 expression levels in human HCC patients (Spearman coefficient r= -0.3408, P<0.0001). Furthermore, a negative correlation was also observed between pRb and SIRT3 expression in human HCC patients (Figure 4c).

SIRT3 expression was increased by CDK4/6 inhibitor treatment

Since SIRT3 and pRb were found to be related, we investigated whether PD0332991 could improve the sensitivity of HCC cells to sorafenib by regulating the expression of SIRT3. Upon treatment with PD0332991, SIRT3 expression increased in HepG2. In SK-HEP1 and Huh7 cells, SIRT3 expression slightly increased compared to the control group (Figure 5a), but no significant changes were observed in Hep3B cells. It is possible that Rb mutations exist in Hep3B cells that cause relative resistance to CDK4/6 inhibition [29, 30]. Therefore, Hep3B cells were excluded from further testing. In addition to PD0332991 treatment, changes in SIRT3 expression by CDK4/6 knockdown in HCC cells were investigated. Similar to PD0332991 treatment, knocking down CDK4/6 in HepG2 cells increased SIRT3 expression, while expression of PCNA, a proliferation marker, decreased (Figures 5b-5d). As our data showed that SIRT3 expression was negatively correlated with glucose metabolism (Figures 1A and 1B), microarray analysis revealed changes in the expression of glycolytic- and TCA-related genes after CDK4/6 inhibition in HepG2 cells. was investigated. As a result, the expression of glycolysis-related genes including SLC2A1 (fold change: 0.12), PFKP (fold change: 0.341), PKM (fold change: 0.457) and HK2 (fold change: 0.693) in CDK4/6 KD HepG2 cells. A decrease was observed (Figure 5e). Furthermore, the most dysregulated genes between the two groups (Scramble vs. CDK4/6 KD) belong to the following categories: DNA replication, meiotic cell cycle progression, chromosome segregation, regulation of fatty acid oxidation, and regulation of lipid catabolic processes. The rate of glycolysis-related gene dysregulation after PD0332991 treatment was lower than that seen after CDK4/6 KD (Figure 5E). Accordingly, the present inventors discovered a new mechanism for suppressing glycolysis and cell proliferation by regulating SIRT3 expression through CDK4/6 inhibition.

Enhancement of sorafenib anticancer effect through combined administration with PD0332991

Next, we sought to investigate whether increasing the expression level of SIRT3 through PD0332991, a CDK4/6 inhibitor, would enhance the anticancer effect of sorafenib on HCC cells. As a result of combined treatment of sorafenib and PD0332991 in HepG2, both mRNA and protein expression of SIRT3 increased (Figures 6a and 6b). In addition, cell survival rate decreased more clearly compared to the single treatment group (Figure 6c). For further confirmation, three-dimensional spheroids of HepG2 cells were generated, and as a result, the size of the spheroids decreased after the combined administration of the two drugs compared to the single administration group. Through Western blotting analysis, it was confirmed that SIRT3 expression increased compared to the control and sorafenib treated groups after PD0332991 and the combined administration of the two drugs. Huh7 cells also showed the same results as HepG2 cells except for SIRT3 mRNA expression, which had a very low basal level (Figures 6d and 6e). To confirm the increase in anticancer effect by combined administration, cell migration was measured after treatment with sorafenib, PD0332991, or a combination thereof. The migration of HepG2 and Huh7 cells was further reduced in the combined treatment group compared to the single treatment group (Figures 7a-7c). Cell cycle analysis, ki67 immunostaining, and apoptosis assay were performed on HepG2 cells and Huh7 cells. As a result of cell cycle analysis, it was confirmed that the G2/M phase was reduced in HepG2 cells compared to the single administration group. In Huh7 cells, G2/M and S phases were reduced by co-administration (Figure 9a). In addition, immunostaining for Ki67 in HepG2 and huh7 cells showed that Ki67-positive cells were dramatically reduced when administered in combination (Figures 9b and 9c). Furthermore, the Annexin V-PI staining results showed that combined administration of sorafenib and PD033291 increased early and late apoptosis (FIGS. 7D and 9D). Taken together, it can be seen that increasing the expression of SIRT3 through CDK4/6 inhibition increases the sensitivity of HCC cells to sorafenib.

As the specific parts of the present invention have been described in detail above, it is clear to those skilled in the art that these specific techniques are merely preferred embodiments and do not limit the scope of the present invention. Accordingly, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

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Claims (12)

delete delete delete delete delete delete delete delete delete A screening method for Sirtuin 3 (SIRT3) activator comprising the following steps:
(a) contacting a candidate material with a biological sample containing CDK (cyclin-dependent kinase) 4/6 or cells expressing it;
(b) measuring the activity or expression level of CDK 4/6 in the sample,
When the activity or expression level of CDK 4/6 is decreased, the candidate substance is determined to be a SIRT3 activator.
The method of claim 10, wherein the cells expressing CDK 4/6 are cells derived from liver tissue.
The method of claim 10, wherein the SIRT3 activator is a composition for enhancing treatment responsiveness to sorafenib in hepatocellular carcinoma.