KR20060066610A - Method of enhancing the effect of anti-cancer drug by inhibition of the activity of ampk - Google Patents

Abstract

The present invention provides a method of enhancing the efficacy of the anticancer agent by inhibiting the activity of AMPK in the use of an anticancer agent for inhibiting the proliferation and / or metastasis of cancer. According to the method of the present invention, it is possible to significantly enhance the efficacy of anticancer agents by inhibiting the activity of AMPK, which is very useful for inhibiting the proliferation and metastasis of cancer using anticancer agents. Moreover, the use of such anticancer agents may be enabled for cancer cells that are resistant to existing anticancer agents.

Description

Method of enhancing the effect of anti-cancer drug by inhibition of the activity of AMPK}

Figure 1 shows that after treating cisplatin in AGS gastric cancer cells in Example 1, the phosphorylation of AMPK Thr172 ("p-AMPK"), the amount of AMPK protein ("AMPK"), and the degree of phosphorylation of ACC Ser79 ("p-ACC"). ) And the amount of ACC protein ("ACC") is measured by Westen blot analysis.

Figure 2a to 2d is a graph showing the measurement of cell viability by time and concentration when treated with cisplatin after AGS cancer cells infected with AMPK-WT and AMPK-DN adenosine virus in Example 2, respectively, PI and Hoechst dye Figures and graphs showing the extent of apoptosis, respectively, using;

3A and 3B show the phosphorylation of p53 Ser 15 residue and the amount of p53 protein when Western Alot was infected with AMPK-WT and AMPK-DN adenosine virus in Example 3 and treated with cisplatin and Western blot analysis. Figures showing the results confirmed by the RT-PCR synthesis of p53 and the lower target substrate;

3c shows cell viability of HCT116p53 − / − and HCT116p53 + / + and wild type p53 transfected with HAMP116 p53 − / − cancer cells transfected with AMPK-WT and AMPK-DN adenosine virus after treatment with cisplatin. Is a graph measured by;

 FIG. 4 shows that AGS cells were infected with AMPK-WT and AMPK-DN in Example 4, respectively, and treated with 30 μM cisplatin or 30 μM cisplatin and caffeine, an ATR inhibitor, after 6 hours. p-p53 "), the amount of p53 protein (" p53 ") and the amount of p21 protein (" p21 ") is a diagram showing the results measured by Westen blot analysis;

5a and 5b are micrographs showing the appearance of cells when treated with various anticancer agents after infection with various cancer cells in Example 3 with AMPK-WT and AMPK-DN adenosine virus, respectively.

The present invention provides a method of enhancing the efficacy of the anticancer agent by inhibiting the activity of AMP-activated protein kinase ("AMPK") when using a medicament ("anticancer agent") for inhibiting the proliferation and / or metastasis of cancer. .

AMPK is a serine / threonine protein kinase that is activated under various stress conditions that deplete the energy of the cell and plays an important role in maintaining energy homeostasis and is essential for cell survival. More specifically, AMPK is central to activating enzymes involved in pathways that produce ATP by inhibiting the activity of enzymes involved in pathways that consume ATP by controlling their activity by the AMP / ATP ratio caused by stress in cells. It is known as protein. In other words, AMPK is activated by metabolic stress to promote glucose uptake and fatty acid oxidation, thereby enhancing cellular energy status, thereby ultimately increasing the survival rate of cells.

AMPK is a heterotrimer consisting of three subunits, and catalytic activity is possessed by such subunits. Recently, cDNA sequences of these subunits have been identified and isomers for each subunit have also been found.

Looking at the activation mechanism of AMPK, AMPK combined with AMP is phosphorylated by AMPK kinase, an upstream kinase, and the site is known as Threonine 172 of the subunit. Recently, the present inventors have reported that AMPK plays a very important role in metabolism of cancer cells, presumably it will be an important target protein in the treatment of cancer.

On the other hand, artificial manipulation in cell signal transduction pathways induced by chemotherapeutic agents in cancer cells increases the effectiveness of chemotherapeutic agents. In many cases, chemotherapy has been studied in combination with chemotherapy and target protein to treat cancer, and it is of great importance in clinical practice. Depending on the nature of the therapeutic agent, anticancer chemotherapy treatments may be based on the nature of the therapeutic agent: 1) alkylating agents (cyclophosphamide, cisplatin, nitrosourea), 2) antimetabolic agents (Fluouracil, methotrexate). 3) Anticancer antibiotics (Actinomycin-D, Doxorubicin, Adriamycin, Mitomycin-C, Bleomycin), 4) Alkaloids (Vinblastine) (Vinblastine), Vincristine (Vincristine), Bindesine (Vindesine), etoposide (Etoposide), etc. Among them, cisplatin (cisplatin (cis-diamminedichloroplatinum; CDDP)), an alkylating agent anticancer agent, is the most effective cancer treatment Known. Cisplatin is one of the most useful drugs among 30 kinds of anticancer drugs currently used in the clinic, and is effective for cancers such as testicular cancer, ovarian cancer, lung cancer, head and neck cancer, bladder cancer, gastric cancer, cervical cancer, and prostate cancer. Cisplatin is a DNA adduct that cross-links to an intrastrand or interstrand, thereby interfering with DNA replication and transcription. It also interferes with the growth of cancer cells and induces apoptosis. Apoptosis by cisplatin is achieved by the expression of signal transduction proteins by DNA damage, and their signal transduction pathways play an important role in the death of cancer cells.

Among them, p53, a cancer suppressor protein, is an important protein expressed by DNA damage. When a stimulant that causes DNA damage enters the cell, the expression level of p53 increases to regulate proteins such as p21, a representative downstream protein, resulting in cell cycle arrest and DNA repair. It also plays a role in killing cells by expressing Bax protein, a pro-apoptotic protein in the stream. The activity of p53 is regulated by several mechanisms, upstream of which ATM / ATR kinase is located. p53 normally binds to a protein called MDM2 in normal cells, a proteasome protein that ubiquitinylates and continues to inhibit the expression of p53, resulting in DNA damage from the outside. This triggers phosphorylation of p53, which breaks its binding to MDM2, which in turn stabilizes p53 and increases its expression. It enters the nucleus and binds to DNA, increasing the transcription of target genes. Therefore, the cancer treatment method through the regulation of the signal transduction pathway of p53 has also been reported as an important cancer therapy.

Recently, many cisplatin-resistant cancers have been reported, although the mechanism is not clear, but some studies have revealed mechanisms such as overexpression of mismatch repair proteins in cancer cells or overexpression of anti-apoptotic proteins. have. Currently, since cancer cells with such resistance are problematic and have a risk of recurrence after cancer treatment, studies on treatment methods by cisplatin and other chemicals or by modulating cisplatin and intracellular expression proteins are actively conducted. Is in progress. In addition, advances in the molecular and biological understanding of cancer have led to the discovery of many molecular biologicals that play a decisive role in cancer development, and new therapeutic methods targeting this have been developed and used in combination with existing anticancer therapies. It is expected to improve.

Accordingly, the present inventors have determined that AMPK, a protein that is central to energy metabolism by intracellular stress, will be an important new target material for anticancer treatment, thereby confirming the role of AMPK in the process of cancer cell death of various anticancer drugs currently used in the clinic. Attempts, and suggest the possibility of combined chemotherapy through AMPK regulation.

The inventors, through various experiments in the Examples described below, have found that the inhibition of AMPK activity surprisingly significantly increases the efficacy of existing anticancer agents, including cisplatin, and has led to the completion of the present invention.

Accordingly, the present invention provides a method of enhancing the pharmacological effect by the anticancer agent by inhibiting the activity of AMPK in the use of a medicament ("anticancer agent") for inhibiting the proliferation and / or metastasis of cancer.

In the present invention, the in vivo mechanism for the enhancement of the anticancer drug efficacy according to the inhibition of the activity of AMPK cannot be accurately explained, but the activity of p53, a tumor suppressor protein whose expression is increased by the anticancer agent, and its expression are further increased to increase cancer cells. Is thought to include a process that is more sensitive to anticancer drugs and promotes cell death.

The subject cancer to which the method of the present invention can be applied is not particularly limited, for example, testicular cancer, ovarian cancer, lung cancer, head and neck cancer, bladder cancer, gastric cancer, cervical cancer, small cell lung cancer, acute leukemia, malignant melanoma, breast cancer. , Liver cancer, lymphoma, multiple myeloma, colon cancer, pancreatic cancer, sarcoma, thyroid cancer, prostate cancer, various blood tumors, skin cancer and the like. In the embodiment of the present invention, according to the suppression of AMPK activity in cancer cells, such as gastric cancer GAS, cervical cancer HeLa, liver cancer Hep3B, breast cancer MCF-7, prostate cancer DU145 The anti-cancer drug's potentiating effect was confirmed.

The anticancer agent that can be used in the method of the present invention is also not particularly limited as long as it is a drug that inhibits the proliferation and / or metastasis of the cancer. In an embodiment of the present invention, cisplatin, an alkylating agent, doxorubicin, an anticancer antibiotic, and The use of alkaloid etoposide (trade name) has been shown to enhance drug efficacy. Among them, it was found to be particularly preferable in the use of cisplatin. The method of the invention makes use of anticancer agents particularly difficult to exert due to the emergence of resistant cancer cells.

The method of inhibiting the activity of AMPK in the method of the present invention is not limited to the followings, but a method of administering a drug ("AMPK activity inhibitor") that exhibits inhibition of AMPK activity is representatively mentioned. Such inhibitors of AMPK activity include both substances already known in the art but subsequently identified or synthesized. In particular, AMPK inhibitors known in the art for use in other applications may be newly used for enhancing anticancer drug efficacy by the methods of the present invention.

Such AMPK activity inhibitors may be used before, concurrently with or after administration of the anticancer agent, preferably before administration of the anticancer agent.

The present invention also provides a pharmaceutical composition for preventing and treating cancer, comprising a pharmacologically effective amount of an AMPK activity inhibitor and an anticancer agent, and a pharmaceutically acceptable carrier, diluent, or excipient, or a combination thereof.

The present invention also provides a method of inhibiting the activity of AMPK to inhibit the proliferation and / or metastasis of cancer.

AMPK is an essential protein for cell survival because it is activated under various stress conditions that deplete the energy of the cell and plays an important role in maintaining energy homeostasis. Thus, inhibition of such AMPK activity can be very fatal for cancer cells, which have a very rapid proliferation rate compared to normal cells.

Especially preferably, anticancer agents can be administered with the inhibition of such AMPK activity.

The present invention also provides a method of preventing and treating cancer by inhibiting the activity of AMPK.

The prevention and treatment of such cancers can be more preferably achieved by combined administration of an anticancer agent. In addition, according to the method of the present invention has an effect that can effectively remove the cancer cells recurring after surgery.

Hereinafter, the content of the present invention will be described in more detail with reference to Examples, but the scope of the present invention is not limited thereto.

EXAMPLE

The conditions, procedures, measurement methods, and the like used in the experiment will be described below.

One. Cell Culture and Materials Used

AGS, HCT116, DU145, MCF-7, Hep3B, and HeLa cells were cultured by adding 10% fetal bovine serum to RPMI1640 medium and feeding 5% CO ₂ at 37 ° C. Cisplatin was purchased from Sigma and Anti-phospho Acetyl-CoA carboxylase and phospho p53 and p53 antibodies were purchased from Cell Signal.

2. Western Blot Analysis

Cells were digested with phosphatase inhibitors in lysis buffer containing 1% NP-40 and 30 ㎍ protein was isolated with 10% SDS-polyacrylamide gel. The activity of AMPK was determined by measuring the phosphorylation level of Acetyl-CoA Carboxylase Ser79, which is an intracellular substrate. Phosphorylation or expression of p53 was measured using phospho-p53 Ser15 or p53 antibody. Infected AMPK construct expression was measured using C-myc 9E10 antibody.

3. Cell Viability and Apoptosis Detection

Live cells were measured using trypan blue dye, and also by 3- (4,5-dimethylthiazolyl) -2,5-diphenyltetrazolium bromide (MTT) assay. Apoptosis was determined by FACS and confocal microscopy after propidium iodide (PI) dye and Hoechast dye were added directly to the cells.

4. Adenovirus preparation and Infection

Wild type and dominant negative form cDNA of AMPK subunit was obtained by PCR. Dominant negative form is a form in which Asp157 of the AMPK subunit is substituted with Ala, and does not function as a kinase. The cDNA was cloned into pAV1, an adenovirus shuttle vector, and transformed into human embryonic kidney 293 cells with E1-deleted adenovirus serotype 5 genome to purify recombinant adenovirus. Purified adenovirus was mixed in a growth medium without serum and left in the cells for 2 hours, and then the experiment was performed 24 hours after the complete medium was added.

5. RT- PCR

Total RNA was isolated and separated using TRIzol (Life Technologies, Glasgow, United Kingdom), and synthesized cDNA using cDNA synthesis Kit (Amersham Pharmacia Biotech, Piscataway, NewJersey) and PCR amplification using specific primer of each substrate It was.

P53: sense 5`-CCTTCCCAGAAAACCTACCA-3`,

      anti-sense 5`-TCATAGGGCACCACCACACT-3`

Bax: sense 5`-GCGAATTCCATGGACGGGTCCGGGGAG-3`

      anti-sense 5`-CGCTCGAGTCAGCCCATGTTCTTCCAG-3`

P21: sense 5`-GCGACTGTGATGCGCTAAT-3`,

      anti-sense 5`-GGCGTTTGGAGTGGTAGAAA-3`

β-actin: sense 5'-GTGGGGGCGCCCAGGCACCA-3 ',

           anti-sense 5'-CTCCTTAATGTCACGCACCATTTC-3 '

Example 1

Cisplatin confirmed that the DNA damage agent activates AMPK. Specifically, AGS gastric cancer cells were treated with cisplatin at 0, 7.5, 15, 30, 60 and 90 μM, respectively, and after 1 hour, the degree of phosphorylation of AMPK Thr172 ("p-AMPK") and the amount of AMPK protein ("AMPK") The degree of phosphorylation of ACC Ser79 ("p-ACC") and the amount of ACC protein ("ACC") were measured by Westen blot analysis.

As a result is shown in Figure 1, it was confirmed that the activity of AMPK concentration-dependently increased by cisplatin.

Example 2

After AGS gastric cancer cells were infected with recombinant adenovirus capable of expressing dominant negative cDNA of AMPK, the effect of cisplatin, an anticancer agent, on the viability of the cells was measured by MTT assay method compared to the control group. Specifically, the group not administered cisplatin ("None"); Group administered 30 μM cisplatin (“Cisplatin”); Cancer cells were infected with AMPK-WT (wild type) adenovirus and treated with 30 μM cisplatin 24 hours later (“WT + Cisplatin”); And 24 hours after the cancer cells were infected with AMPK-DN (dominant-negative) adenovirus was administered to 30 μM cisplatin ("DN + Cisplatin") and the group treated with Cisplatin by concentration.

The results are disclosed in FIGS. 2A to 2D. First, referring to FIG. 2A, the apoptosis effect of cisplatin in AGS gastric cancer cells was very insignificant, but had little effect on the viability of the cells, but the activity of AMPK was suppressed. Showed a very pronounced anticancer effect, and about 60% of the cells were killed within 24 hours. The nature of this cell death can be confirmed in Figure 2c and 2d that apoptosis. Specifically, when cisplatin was treated to cells in which AMPK activity was suppressed, the nucleus of the cells was condensed by Hoechast staining, and when PI staining, subG0 cell populations were increased. Shows that they are dying of apoptosis. Therefore, even cancer cells exhibiting cisplatin resistance are expected to show anticancer improvement effects by combination therapy through inhibition of AMPK activity.

Example 3

Cisplatin is known to block cancer cell proliferation or induce cell death by increasing the expression and activity of p53 protein with tumor suppressor function as a DNA damage agent. p53 protein is activated by various post-translational modifications, and phosphorylation is also known as an important regulatory mechanism. p53 has a lot of phosphorylation sites, of which Serine15 phosphorylation plays the most important role. When this site is phosphorylated, the activity of p53 protein is increased and stabilized in the cytoplasm.

Therefore, this experiment confirmed the correlation between AMPK activity inhibition and p53. Specifically, AGS cells were infected with AMPK-WT and AMPK-DN, respectively, and after 24 hours of treatment with 30 μM cisplatin for 12 hours, the degree of phosphorylation of p53 Ser15 residues (“p-p53”) and the amount of p53 protein (“p53”). ") Was measured by Westen blot analysis. Since AMPK expressed in infected cells contains c-myc epitopte, its expression level was confirmed using c-myc antibody. In addition, the expression levels of p53, p21, and Bax were confirmed using RT-PCR. In addition, HCT16 ^{p53 + / +} , HCT116 ^{p53 -/-} and HCT116 ^{p53 -/-} cells with wild-type p53 were used to determine the role of p53 in cell death.

3A to 3C, the results show that the degree of phosphorylation of p53 ser15 was increased by cisplatin and the amount of p53 protein was increased at the same time. In addition, when cisplatin was treated after inhibiting the activity of intracellular AMPK, it can be seen that this degree was further increased. Therefore, it usually means that AMPK inhibits the activity of p53. And, p53 did not change the mRNA level of itself, it was confirmed that increases the synthesis of the target molecules p21 and Bax of the lower. Experiments using HCT16 ^{p53 + / +} , HCT116 ^{p53 -/-} and HCT116 ^{p53 -/-} cells with wild-type p53 inhibited the induction of cisplatin and AMPK in HCT16 ^{p53 -/-} and suppressed wild- The degree of apoptosis was restored by HCT116 ^{p53 -/-} cells injected with type p53, indicating that p53 plays an important role in cell death. Thus, AMPK inhibition is believed to increase sensitivity to anticancer agents through the induction of p53.

Example 4

Cisplatin-induced DNA damage agents are known to phosphorylate p53 ser-15 via the ATR pathway. Therefore, in this experiment, we confirmed whether ser53 phosphorylation of p53 by AMPK inhibition was related to ATR pathway. Specifically, AGS cells were infected with AMPK-WT and AMPK-DN, respectively, and after 24 hours of treatment with 30 μM cisplatin or 30 μM cisplatin and caffeine, an ATR inhibitor, the degree of phosphorylation of p53 Ser15 residue after 6 hours (“p-p53 "), amount of p53 protein (" p53 ") and amount of p21 protein (" p21 ") were measured by Westen blot analysis. Since AMPK expressed in infected cells contains c-myc epitopte, its expression level was confirmed using c-myc antibody.

As a result, as shown in Figure 4, the increase in the degree of phosphorylation of p53 ser15 by cisplatin was completely inhibited by caffeine, but the phosphorylation of p53 ser15 further increased by AMPK inhibition was only partially inhibited by caffeine. Therefore, phosphorylation of p53 ser15 by AMPK inhibition is thought to exist other than ATR pathway.

Example 5

Since doxorubicin, etoposide, cisplatin, etc. are representative among the various anticancer drugs currently used in cancer patients in the clinical trials, these experiments were used to observe the apoptosis effect of AMPK inhibition in various cancer cells. Specifically, HeLa (cervical cancer cells), Hep3B (liver cancer cells), MCF-7 (bladder cancer cells) and DU145 (prostate cancer cells) were infected with adenoviruses of AMPK-WT and AMPK-DN, respectively, and after 24 hours, doxorubicin ( 10 M), etoposide (50 M) and cisplatin (30 M) were treated for 24 hours, and cell death was observed under a microscope. The results are shown in Figures 5a and 5b, it can be seen that each anti-cancer drugs can kill the cells more effectively when artificially inhibited the activity of AMPK in all cancer cells and anti-cancer drug treatment conditions.

As described above, according to the method of the present invention, it is possible to significantly enhance the efficacy of anticancer agents by inhibiting the activity of AMPK, which is very useful for inhibiting cancer proliferation and activity using anticancer agents. In addition, it is expected to enable the use of such anticancer agents for cancer cells resistant to the existing anticancer agents, and to obtain an effect of effectively removing cancer cells that recur after surgery. In addition, the type of cancer to which the method of the present invention can be applied and the type of anticancer agent are not particularly limited.

Those skilled in the art to which the present invention pertains will be able to make various applications and modifications within the scope of the present invention based on the above contents.

Claims (13)

A method of enhancing the pharmacological effect of an anticancer agent by inhibiting the activity of AMP-activated protein kinase ("AMPK") in the use of a medicament ("anticancer agent") to inhibit cancer proliferation and / or metastasis. According to claim 1, Inhibition of the activity of the AMPK, the activity of p53, a tumor suppressor protein that is increased by the anticancer agent and its expression is further increased, the cancer cells are more sensitive to the anticancer agent and promotes cell death Characterized in that the method. According to claim 1, wherein the cancer is testicular cancer, ovarian cancer, lung cancer, head and neck cancer, bladder cancer, gastric cancer, cervical cancer, small cell lung cancer, acute leukemia, malignant melanoma, breast cancer, liver cancer, lymphoma, multiple myeloma, colon cancer, pancreatic cancer , Sarcoma, thyroid cancer, prostate cancer, various blood tumors, skin cancer, and the like. 4. The method of claim 3, wherein the cancer is gastric cancer induced by GAS, cervical cancer induced by HeLa, liver cancer induced by Hep3B, breast cancer induced by MCF-7, or prostate cancer caused by DU145. How to feature. The method of claim 1, wherein the anticancer agent is selected from cisplatin, doxorubicin, and etoposide. The method of claim 1, wherein the anticancer agent is an anticancer agent which is difficult to exert an anticancer effect by itself due to the appearance of resistant cancer cells. The method of claim 1, wherein the inhibition of AMPK activity is obtained by administering a drug that exhibits inhibition of AMPK activity. 8. The method of claim 7, wherein said AMPK activity inhibitor is used before, simultaneously with, or after administration of said anticancer agent. A pharmaceutical composition for the prevention and treatment of cancer, comprising a pharmacologically effective amount of an AMPK activity inhibitor and an anticancer agent, and a pharmaceutically acceptable carrier, diluent, or excipient, or a combination thereof. A method for inhibiting the proliferation and / or metastasis of cancer by inhibiting the activity of AMPK. The method of claim 10, wherein the anticancer agent is administered with the inhibition of the activity of the AMPK. Method of preventing and treating cancer by inhibiting the activity of AMPK. 13. The method of claim 12, wherein the prevention and treatment of cancer is achieved by combined administration of an anticancer agent.

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