Pharmaceutical composition for treating tumors

Abstract

The invention relates to a pharmaceutical composition for treating tumors. Specifically, through tumor inhibition pathway research, it is found that the growth of in-vivo and in-vitro cancer cells can be synergistically inhibited by combining APG-115 or Nutlin-3 with Elesclomol for use, and a new combined treatment method is provided for treatment of cancers carrying wild type p53.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and more specifically to a pharmaceutical composition for treating tumors. Background Art

[0002] The tumor suppressor p53 maintains genome integrity and prevents tumorigenesis by transcriptionally regulating the expression of genes involved in the cell cycle and DNA damage repair. However, when cells suffer irreversible damage, p53 can trigger various forms of regulated cell death (RCD) to eliminate these cells. The pro-apoptotic function of p53 was first described in 1991 and subsequently validated in mouse embryonic fibroblasts (MEFs). It has been well documented that p53 activates the transcription of pro-apoptotic genes of the BCL-2 family, such as PUMA, NOXA, and BAX, while inhibiting the expression of anti-apoptotic genes of the BCL-2 family, such as BCL-2, BCL-XL, and MCL15. p53 also physically interacts with some mitochondrial proteins to enhance apoptosis. Recently, p53 has been found to be a key regulator of ferroptosis. On the one hand, p53 promotes ferroptosis by reducing the biosynthesis of glutathione (GSH) or regulating multiple metabolic pathways related to polyunsaturated fatty acid peroxidation, glutamine catabolism, or vitamin K metabolism. On the other hand, p53 protects cells from ferroptosis by increasing GSH levels through enhancing the expression of p2116 and TIGAR17 or detoxifying lipid peroxidation through activating iPLA2β expression. In addition, p53 plays a role in regulating many other types of RCD, including necroptosis, PARP-1-dependent cell death, and autophagic cell death.

[0003] Copper death is a form of cell death that depends on copper and occurs primarily in cells that rely on mitochondrial metabolism for energy production. Several hypotheses have been proposed that p53 may play a role in the regulation of copper death. First, p53 is an important regulator of glycolysis that inhibits glycolysis and promotes the tricarboxylic acid (TCA) cycle and oxidative phosphorylation. For example, p53 inhibits the activity of the glycolysis rate-limiting enzymes hexokinase HK220 and phosphofructokinase PFKM17 or PFKP21, thereby inhibiting the glycolysis process in cancer cells. At the same time, p53 also promotes the production of acetyl-CoA22 or glutamate to provide energy for the TCA cycle. Second, p53 may promote the biosynthesis of iron-sulfur cluster proteins, which are degraded during copper death. Finally, p53 controls the biosynthesis of glutathione (GSH), which is not only a potent antioxidant but also a natural copper chelator. Although the above studies suggest that there may be a link between p53 and copper death, how p53 controls copper death and the factors involved in this regulation remain uncertain.

[0004] Therefore, technicians in this field are committed to studying and confirming new tumor inhibition mechanisms, and on this basis, developing more effective anti-tumor drugs. Summary of the invention

[0005] The present invention unexpectedly discovered through tumor suppression pathway research that the combination of APG-115 or Nutlin-3 and Elesclomol can synergistically inhibit the growth of cancer cells in vivo and in vitro, which provides a new combined treatment method for the treatment of cancers carrying wild-type p53.

[0006] A first aspect of the present invention provides a pharmaceutical composition, comprising a first component and a second component, wherein the first component is Elesclomol, and the second component is Nutlin-3 and / or APG-115.

[0007] In another preferred embodiment, the second component is Nutlin-3.

[0008] In another preferred embodiment, the second component is APG-115.

[0009] In another preferred embodiment, the molar ratio of the first component to the second component is 1:5000-100:1.

[0010] In another preferred embodiment, the molar ratio of the first component to the second component is 1:500-2000.

[0011] In another preferred embodiment, the molar ratio of the first component to the second component is 1:1000-1600.

[0012] In another preferred embodiment, the pharmaceutical composition is used to treat cancer.

[0013] In another preferred embodiment, the cancer is p53 wild-type cancer.

[0014] The second aspect of the present invention provides a method for preparing the pharmaceutical composition according to the first aspect of the present invention, wherein the method comprises mixing the first component and the second component with a pharmaceutically acceptable excipient.

[0015] The third aspect of the present invention provides a medicine kit, comprising a first component and a second component; wherein the first component is Elesclomol, and the second component is Nutlin-3 and / or APG-115.

[0016] In another preferred embodiment, the first component and the second component are independently packaged in the medicine box.

[0017] The fourth aspect of the present invention provides use of the pharmaceutical composition according to the first aspect of the present invention in the preparation of a drug for treating cancer.

[0018] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 .p53 activates FRMD4A transcription.

[0020] (AE) RT-qPCR analysis showed that HCT116 (A), CAL51 (B), HCT116 p53- / - (C), SW620 p53-R273H (D) and MDA-MB-231 p53-R280K (E) Expression of FRMD4A mRNA in cells after 48 h of treatment with the indicated drugs. (FI) RT-qPCR analysis showed that HCT116 (F), CAL51 (G), SW620 p53-R273H (H) and MDA-MB-231 p53-R280K (I) Expression of FRMD4A mRNA after cells were treated with the indicated drugs and siRNAs for 48 h. (J) RT-qPCR analysis showed the expression of FRMD4A mRNA in p53-deficient cancer cells transfected with the indicated plasmids. (K) Schematic representation of potential p53 response elements on the FRMD4A promoter. (L, M) ChIP assays combined with qPCR (L) or agarose gel electrophoresis (M) confirmed that p53 binds to p53-RE 3. (N) Schematic representation of plasmids used in luciferase reporter assay. (O) Luciferase reporter assay validated the activation of FRMD4A promoter by p53. *p<0.05, **p<0.01, ***p<0.001.

[0021] Figure 2 .p53 activates FRMD4A transcription.

[0022] (AF) RT-qPCR analysis of FRMD4A mRNA expression in wild-type p53-bearing cancer cells (AC) and p53-deficient cancer cells (DF) after 48 h of treatment with DMSO, cisplatin, 5-FU, or Nutlin-3. (G) RT-qPCR analysis of FRMD4A mRNA expression in wild-type p53-bearing A549 cells after 48 h of treatment with the indicated drugs and siRNA. (H, I) RT-qPCR analysis of FRMD4A mRNA expression in mutant p53-bearing cancer cells after 48 h of treatment with the indicated drugs and siRNA. *p<0.05, **p<0.01, ***p<0.001.

[0023] Figure 3 p53 enhances Elesclomol-induced copper death via circFRMD4A in colorectal and breast cancer cells.

[0024] (A, B) HCT116 and CAL51 cells were exposed to DMSO or Nutlin-3 and then treated with different doses of Elesclomol for 48 hours, followed by cell viability assay. (C, D) HCT116 and CAL51 cells were transfected with the indicated siRNAs and treated with different doses of Elesclomol for 48 hours, followed by cell viability assay. *p<0.05, **p<0.01, ***p<0.001.

[0025] Figure 4 The combination of Elesclomol with cisplatin or 5-FU had no synergistic inhibitory effect on colorectal cancer cells and breast cancer cells.

[0026] (A, B) HCT116 and CAL51 cells were treated with a combination of cisplatin and Elesclomol, followed by cell viability assay. The affected fraction (FA) values ​​and combination index (CI) were analyzed by Compusyn software using the Chou-Talalay method. (C, D) HCT116 and CAL51 cells were treated with a combination of 5-FU and Elesclomol, followed by cell viability assay. FA and CI value analysis methods are as described in (A, B).

[0027] Figure 5 p53 agonists and Elesclomol synergistically inhibit tumor growth.

[0028] (A) Cell viability assay of HCT116 cells treated with Nutlin-3 and Elesclomol. Affected fraction (FA) values ​​and combined index (CI) analysis were performed using the Chou-Talalay method using Compusyn software. (B, C) Cell viability assay of HCT116 and CAL51 cells treated with APG-115 and Elesclomol. FA and CI values ​​were analyzed in the same manner as described in (A). (D) Schematic diagram of xenograft experimental design and drug combinations. (EG) Growth rate (E), weight (F), and size (G) of xenograft tumors treated with Elesclomol, APG-115, or both. Data (E) and (F) are presented as mean ± SD, n = 6. *p < 0.05, **p < 0.01, ***p < 0.001.

[0029] Figure 6Body weight of nude mice treated with APG115 and / or Elesclomol. DETAILED DESCRIPTION

[0030] In order to more easily understand the present disclosure, some terms are first defined. As used in this application, unless otherwise expressly provided herein, each of the following terms should have the meaning given below. Other definitions are set forth throughout the application.

[0031] Copper death is a type of cell death induced by copper that primarily affects cells that rely on mitochondrial metabolism. Although p53 regulates glycolytic metabolism, it is unclear whether this tumor suppressor plays a role in copper death. The results of the present invention show that specific p53 agonists (Nutlin-3 and APG-115) and Elesclomol can synergistically inhibit cancer growth. This study provides a new combination therapy for the treatment of cancers in which the p53 gene is wild-type.

[0032] Elesclomol

[0033] Elesclomol is called Yilisimo or Yilisimo in Chinese (CAS No.: 488832-69-5), and its chemical structure is as follows:

[0034]

[0035] Nutlin-3

[0036] The chemical name of Nutlin-3 is: (+ / -)-4-(4,5-bis(4-chlorophenyl)-2-(2-isopropoxy-4-methoxyphenyl)-4,5-dihydro-1H-imidazole-1-carbonyl)piperazine-2-one (CAS No.: 890090-75-2), and the chemical structure is as follows:

[0037]

[0038] APG-115

[0039] The CAS number of APG-115 is 1818393-16-6, and its chemical structure is as follows:

[0040]

[0041] Pharmaceutical composition

[0042] The term "pharmaceutical composition" refers to a mixture containing a therapeutically effective amount of one or more of the compounds and their pharmaceutically acceptable tautomers, solvates, hydrates or salts, and other pharmaceutically acceptable carriers. The purpose of preparing the compounds into a pharmaceutical composition is to more conveniently administer them to the subject.

[0043] According to one aspect of the present invention, the present invention provides a pharmaceutical composition, comprising a first component and a second component, wherein the first component is Elesclomol, and the second component is Nutlin-3 and / or APG-115.

[0044] According to some embodiments of the present invention, the molar concentration ratio of the first component to the second component is about 1:5000-100:1, for example, 1:4000-10:1, 1:3000-10:1, 1:2000-10:1, 1:1000-10:1, 1:1000-10:1, 1:100-10:1, 1:10-10:1.

[0045] According to some embodiments of the present invention, the pharmaceutical composition is used to treat cancer.

[0046] According to some embodiments of the present invention, the cancer includes prostate cancer, colorectal cancer, lung cancer and breast cancer.

[0047] According to some embodiments of the present invention, the total content of the first component and the second component accounts for 1-100% of the pharmaceutical composition, for example, 1-99.5%, 1-99%, 1-90%, 1-80%, 1-70%, 1-60%, 1-50%, 1-40%, 1-30%, 1-20%, 1-10%, 10-100%, 10-99.5%, 10-99%, 10-90%, 10~80%、10~70%、10~60%、10~50%、10~40%、10~30%、10~20%、20~100%、20~99.5%、20~99%、20~90%、20~80%、20~70%、20~60%、20~50%、20~40%、20~30%、30~100%、30~99.5%、30~99%、 30-90%, 30-80%, 30-70%, 30-60%, 30-50%, 30-40%, 40-100%, 40-99.5%, 40-99%, 40-90%, 40-80%, 40-70%, 40-60%, 40-50%, 50-100%, 50-99.5%, 50-99%, 50-90%, 50-80%, 50-70%, 50-60%, 60-100%, 60-99.5%, 60-99%, 60-90%, 60-80%, 60-70%, 70-100%, 70-99.5%, 70-99%, 70-90%, 70-80%, 80-100%, 80-99.5%, 80-99%, 80-90%, 90-100%, 90-99.5%, or 90-99%.

[0048] According to certain embodiments of the present application, the total content of the first component and the second component accounts for about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, %, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 100%.

[0049] Drug synergy: When two or more drugs are used together, if their directions of action are consistent, the effect of mutual enhancement is called synergy, and the total effect exceeds the sum of the effects of each drug when used alone. In other words, the effect of the combined use of two drugs is greater than the efficacy of any one drug used alone, and greater than the additive effect of the two drugs.

[0050] The term "about" may refer to a value or composition within an acceptable error range for a particular value or composition determined by one of ordinary skill in the art, which will depend in part on how the value or composition is measured or determined. In the present application, when "about" is used to modify a numerical value, it means that the numerical value may fluctuate up or down within the range of ±10%, ±9%, ±8%, ±7%, ±6%, ±5%, ±4%, ±3%, ±2% or ±1%.

[0051] The term "modulate" includes treating, preventing or interfering.

[0052] The term "treatment" refers to administering the drug of the present invention to a subject in need of treatment for the purpose of curing, alleviating, improving, alleviating, affecting the disease, symptoms, or predisposition of the subject. The subjects of the present invention include mice, rabbits, monkeys, humans, and other mammals.

[0053] The term "therapeutically effective amount" refers to the amount of a drug that can achieve the therapeutic purpose in the body of the treated subject. It should be understood by those skilled in the art that the "therapeutically effective amount" may vary depending on the route of administration of the drug, the pharmaceutical excipients used, and the combination of the drug with other drugs.

[0054] The pharmaceutical composition of the present invention comprises the drug (active ingredient) of the present invention within a safe and effective amount and a pharmacologically acceptable excipient or carrier. The "safe and effective amount" means that the amount of the active ingredient is sufficient to significantly improve the condition without causing serious side effects. Generally, the pharmaceutical composition contains 0.001-1000 mg of active ingredient / dose, preferably 0.05-300 mg of active ingredient / dose, and more preferably, 0.5-200 mg of active ingredient / dose.

[0055] The active ingredient of the present invention and its pharmacologically acceptable salt can be made into various preparations, which contain the active ingredient of the present invention or its pharmacologically acceptable salt within the safe and effective amount range and a pharmacologically acceptable excipient or carrier. The "safe and effective amount" means that the amount of the active ingredient is sufficient to significantly improve the condition without causing serious side effects. The safe and effective amount of the active ingredient is determined according to the specific circumstances such as the age, condition, and course of treatment of the subject.

[0056] "Pharmacologically acceptable excipients or carriers" refer to: one or more compatible solid or liquid fillers or gel substances, which are suitable for human use and must have sufficient purity and sufficiently low toxicity. "Compatibility" here means that the components in the composition can be mixed with the compounds of the present invention and with each other without significantly reducing the efficacy of the compounds. Some examples of pharmacologically acceptable excipients or carriers include cellulose and its derivatives (such as sodium carboxymethyl cellulose, sodium ethyl cellulose, cellulose acetate, etc.), gelatin, talc, solid lubricants (such as stearic acid, magnesium stearate), calcium sulfate, vegetable oils (such as soybean oil, sesame oil, peanut oil, olive oil, etc.), polyols (such as propylene glycol, glycerol, mannitol, sorbitol, etc.), emulsifiers (such as, wetting agents (such as sodium lauryl sulfate), colorants, flavoring agents, stabilizers, antioxidants, preservatives, pyrogen-free water, etc.

[0057] When the composition of the present invention is applied, it can be administered orally, rectally, parenterally (intravenously, intramuscularly or subcutaneously), or topically. It can be prepared into any pharmaceutically acceptable dosage form, including but not limited to tablets, oral agents, granules, injections, liposomes, targeted drug delivery injections, pills, capsules, granules, powders, suppositories, powders, ointments, patches, injections, solutions, suspensions, sprays, lotions, drops, liniments, etc. The pharmaceutical composition can be prepared in the form of dry powder and mixed with sterile water or buffer before administration to prepare a solution form. The pH of the buffer is usually 3-11, preferably 5-9, and more preferably 7-8.

[0058] The composition of the present invention can be administered alone or in combination with other pharmaceutically acceptable compounds.

[0059] Microcapsules containing the composition of the present invention can be used for sustained-release administration of the active ingredient of the present invention. The sustained-release preparation of the active ingredient of the present invention can be prepared with a lactic acid glycolic acid polymer (PLGA) having good biocompatibility and broad biodegradability. The degradation products of PLGA, lactic acid and glycolic acid, can be quickly cleared by the human body. Moreover, the degradation capacity of the polymer can be extended from several months to several years depending on its molecular weight and composition (Lewis, "Controlled release of bioactive agents form lactide / glycolide polymer," in: M.Chasin and R.Langer (Eds.), Biodegradable Polymers as Drug Delivery Systems (Marcel Dekker: New York, 1990), pp.1-41)).

[0060] When using the pharmaceutical composition, a safe and effective amount of the active ingredient of the present invention is applied to a mammal (such as a human) in need of treatment, wherein the dosage during administration is a pharmaceutically effective dosage, and for a person weighing 60 kg, the dosage per administration is usually 0.01 to 300 mg, preferably 0.5 to 100 mg. Of course, the specific dosage should also take into account factors such as the route of administration and the health status of the patient, which are all within the skill of a skilled physician.

[0061] Method for preparing pharmaceutical composition

[0062] According to one aspect of the present invention, the present invention provides a method for preparing the pharmaceutical composition, wherein the method comprises mixing the first component and the second component with a pharmaceutically acceptable excipient.

[0063] The term "excipient" means a pharmaceutically acceptable ingredient that does not have any pharmacological effect and is generally used in the pharmaceutical technology of preparing granules and / or solid oral dosage forms and / or liquid injection formulations. Excipients can act as carriers, diluents, or dissolution modifiers, absorption enhancers, stabilizers, or auxiliary agents for preparation and other effects. Excipients useful in preparing pharmaceutical compositions are generally safe, nontoxic, and acceptable for medical use and pharmaceutical use. "Excipients" or "pharmaceutically acceptable excipients" used in this specification include one or more such excipients.

[0064] Pill Box

[0065] The terms "kit" or "test kit" are used interchangeably in this application. The present application discloses a kit comprising a therapeutically effective amount of the therapeutic agent or pharmaceutical composition. According to certain embodiments of the present application, the kit further comprises one or more other therapeutic agents. According to certain embodiments of the present application, the kit further comprises instructions for use. According to certain embodiments of the present application, the kit further comprises a device for a corresponding administration method, such as but not limited to a needle.

[0066] According to one aspect of the present invention, the present invention provides a medicine kit, comprising the first component and the second component.

[0067] Treatment

[0068] According to one aspect of the present invention, the present invention provides a method for treating cancer using the pharmaceutical composition.

[0069] According to some embodiments of the present invention, the pharmaceutical composition comprises the first component and the second component.

[0070] Unless otherwise specified in this application or clearly contradicted by the context, the terms "a", "an", "said", "the", "at least one" and similar references used in the context of describing this application (including the context of the claims) are interpreted to cover the singular and the plural. Unless otherwise specified in this application or clearly contradicted by the context, the terms "comprising", "having", "including" and "containing" used in this application are interpreted as open terms (i.e., "including but not limited to"). Unless otherwise specified in this application or clearly contradicted by the context, all methods described in this application can be performed in any suitable order according to the understanding of those skilled in the art.

[0071] Materials and Methods

[0072] Cell culture and transient transfection

[0073] Cancer cell lines HCT116, RKO, CAL51, MCF-7, and A549 carrying wild-type p53, cancer cell lines SW620, MDA-MB-231, H1975, and ES-2 carrying mutant p53, and cancer cell lines HCT116 p53- / - and H1299 lacking p53 were cultured in DMEM medium containing 10% fetal bovine serum, penicillin (100 U / ml), and streptomycin (0.1 mg / ml). All cells were cultured in a 5% CO 2 The cells were incubated at 37°C in a humidified environment. All cells were confirmed to be free of mycoplasma contamination by PCR analysis. Plasmids and siRNAs were transiently transfected using Hieff Trans liposome transfection reagent according to the manufacturer's protocol (Shanghai Yisheng Biotechnology Co., Ltd.).

[0074] Clinical samples of colorectal cancer and breast cancer

[0075] In this study, a total of 100 paraffin-embedded tissue sections from the First Affiliated Hospital of Nanchang University were collected and analyzed by in situ hybridization (ISH). Among them, 20 pairs of colorectal cancer tissues and paired normal tissues were used to analyze the expression of circFRMD4A, while 80 colorectal cancer tissues were used to analyze overall survival based on ISH scores. In addition, seven pairs of breast cancer and adjacent normal tissues were used for RT-qPCR analysis. This study has been approved by the Human Research Ethics Committee of the First Affiliated Hospital of Nanchang University.

[0076] Reverse transcription and real-time quantitative PCR

[0077] According to the manufacturer's protocol, total RNA was extracted from cells and tissues using RNAiso Plus (Takara, Japan); genomic DNA (gDNA) was extracted from cells using the TIANamp genomic DNA kit (TIANGEN); cDNA synthesis was performed using RT SuperMix for qPCR (+gDNA wiper) (Vazyme); RT-qPCR analysis was performed using SYBR qPCR Master Mix. -ΔΔCt Methods The relative expression was calculated, with GAPDH as the internal reference gene.

[0078] RNA interference and construction of stable cell lines

[0079] All siRNAs and shRNAs were synthesized and purified by Shanghai GenePharma Co., Ltd. (GenePharma). Cells were seeded in 6-well plates at appropriate density. siRNA transfection was performed using Hieff Trans liposomal transfection reagent according to the manufacturer's protocol (Yeasen). After 24–48 hours of culture after transfection, cells were collected for RT-qPCR or immunoblot (IB) analysis.

[0080] Cell viability assay

[0081] Cells were transfected with the indicated siRNA or plasmid and seeded in 96-well plates at a density of 3000 cells per well. After the cells were cultured for the indicated time, Cell Counting Kit-8 (CCK-8) reagent (Yeasen) was added to the culture and incubated for 2 hours. Cell viability was measured at 450 nm using a microplate reader. For the cell viability assay associated with copper death, cells were seeded in 96-well plates at a density of 9000 cells per well 24 hours after transfection and treated with the indicated reagents or DMSO for 48 hours. Cell viability was then determined using CCK-8 as described above.

[0082] Colony formation assay

[0083] The cells were seeded in 6-well plates at a density of 1000 cells per well, and the culture medium was changed every two days for 14 days. The colonies were washed with PBS, fixed with methanol, and stained with a mixture of methanol and crystal violet for 30 minutes at room temperature. They were then rinsed with running water, the plates were dried, and photographed.

[0084] Flow cytometry detection of apoptosis

[0085] Apoptosis was detected by flow cytometry using the Annexin-V-PE / 7-AAD kit (Vazyme) according to the manufacturer's protocol. That is, cells were collected 48 hours after transfection with the specified siRNA or plasmid and stained with Annexin-V-PE and 7-AAD for 20 minutes at room temperature in the dark. The percentage of apoptotic cells was then measured using a flow cytometer (Beckman Coulter).

[0086] Western Blotting

[0087] Proteins were extracted using RIPA buffer and separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and then transferred to a PVDF membrane. The membrane was blocked with 5% skim milk and incubated with primary antibodies overnight. The membrane was washed three times and incubated with secondary antibodies at a dilution ratio of 1:10000. Proteins were visualized using ECL chemiluminescent reagents (Yeasen).

[0088] Chromatin Immunoprecipitation (ChIP)

[0089] Cells were seeded in 100 mm dishes and cultured for 24 hours. Cells were cross-linked with 37% formaldehyde for 10 minutes at room temperature, then neutralized with 800 μl 10× glycine for 5 minutes and washed three times with cold PBS. Subsequently, cells were scraped and suspended in cell lysis buffer (50 mM Tris-HCl pH 7.5, 140 mM NaCl, 1 mM EDTA, 10% glycerol, 0.5% NP-40, 0.25% TritonX-100, and protease inhibitor cocktail) and incubated on ice for 20 minutes, vortexing every 5 minutes. Cell nuclei were collected and resuspended in 400 μl nuclear lysis buffer (50 mM Tris-HCl pH 8.0, 10 mM EDTA, 1% SDS, and protease inhibitor cocktail). After sonication (200 W, 60 cycles, 30 s on, 30 s off each time), the lysates were centrifuged at 12,000 g for 5 min, and the supernatants were incubated with anti-p53 antibody or IgG at 4°C overnight and then with protein A / G magnetic beads at 4°C for 2 h. The beads were washed sequentially with low salt wash buffer (50 mM Tris-HCl pH 8.0, 0.1% SDS, 0.5% sodium deoxycholate, 1 mM EDTA, 1% NP-40, and 150 mM NaCl), high salt wash buffer (50 mM Tris-HCl pH 8.0, 0.1% SDS, 0.5% sodium deoxycholate, 1 mM EDTA, 1% NP-40, and 500 mM NaCl), LiCl wash buffer (50 mM Tris-HCl pH 8.0, 250 mM LiCl, 0.1% SDS, 0.5% sodium deoxycholate, 1 mM EDTA, and 1% NP-40), and TE buffer (10 mM Tris HCl pH 8.0, and 1 mM EDTA). The protein-DNA complex was eluted with ChIP elution buffer (1% SDS and 0.1 M NaHCO 3 After de-crosslinking at 62°C for 2 h, DNA was extracted and analyzed by qPCR and agarose gel electrophoresis.

[0090] Luciferase Reporter Assay

[0091] Cells were seeded in 24-well plates and transfected with pGL3-basic, pGL3-p53-RE1 / 2 or pGL3-p53-RE1 / 2 / 3 plasmids, as well as plasmids encoding Renilla luciferase and p53. After 48 h of culture, cells were harvested and luciferase activity was measured using the Dual-Luciferase Reporter Gene Assay System (Promega, Madison, WI, USA) according to the manufacturer's protocol.

[0092] Analysis of combined medication

[0093] The combined effect of p53 inducers (5-FU, cisplatin, Nutlin-3 and APG-115) with Elesclomol was evaluated by cell viability assay. The affected fraction (FA) values ​​and the combination index (CI) were analyzed by the Chou-Talalay method using Compusyn software. The CI value indicates the effect of the drug combination: synergistic (CI<1), additive (CI=1) or antagonistic (CI>1).

[0094] Mouse xenograft tumor model

[0095] Mouse xenograft experiments followed ethical guidelines and were approved by the Animal Welfare Committee of Fudan University Shanghai Cancer Center. Six-week-old BALB / c nude mice were purchased and maintained from the Department of Laboratory Animal Science, Fudan University Shanghai Cancer Center. To evaluate the in vivo function of circFRMD4A, 8 × 10 mice stably expressing empty vector or circFRMD4A plasmid were placed in the 6 HCT116 cells stably expressing shNC or shcircFRMD4A; 5×10 6 HCT116 cells were implanted subcutaneously in nude mice. Tumor growth was monitored twice a week using a vernier caliper. For drug treatment, 8×10 6 HCT116 cells were subcutaneously implanted into nude mice. Seven days later, the mice were randomly divided into four groups: control group, Elesclomol group, APG-115 group and Elesclomol+APG-115 group. Elesclomol (5 mg / kg) was administered daily by intraperitoneal injection (five days a week for two weeks). APG-115 (50 mg / kg) was administered intragastrically (five days a week for two weeks). The control group was given an equal amount of normal saline. The weight of the mice was monitored and recorded daily. The formula "volume = length × width was used. 2 × 0.5” to measure and calculate tumor volume.

[0096] Statistical analysis

[0097] All in vitro data were obtained from three independent replicates. Data are presented as mean ± standard deviation (SD). Differences between two or more groups were compared using Student's t test or one-way analysis of variance (ANOVA). The overall survival of patients with colorectal cancer was analyzed using the Kaplan-Meier method. p values ​​< 0.05 were considered statistically significant. ns indicates no statistically significant difference, *p < 0.05, **p < 0.01, ***p < 0.001.

[0098] The present invention is further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples that do not specify specific conditions are usually performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are calculated by weight.

[0099] Example 1p53 activates transcription of FRMD4A

[0100] The results of the present invention showed that in cancer cells carrying wild-type p53, including HCT116, RKO, CAL51, MCF7 and A549, the transcription level of FRMD4A (expression of circFRMD4A) increased after treatment with 5-FU, cisplatin or Nutlin-3 ( Figure 1 A, B, Figure 2 AC).

[0101] However, in cancer cells lacking or harboring mutant p53, these treatments had little effect on FRMD4A transcript levels ( Figure 1 CE Figure 2 DF).

[0102] In addition, the expression of p53 was knocked down to determine whether the upregulation of FRMD4A transcription levels was dependent on p53. The results showed that in cancer cells carrying wild-type p53, p53 knockout significantly reversed the increase in FRMD4A transcription levels induced by 5-FU, cisplatin or Nutlin-3 ( Figure 1 F, G and Figure 2 G), while knockdown of mutant p53 had no effect on the transcription level of FRMD4A ( Figure 1 H, I, Figure 2 of H and I).

[0103] The results also showed that the expression of the maternal gene FRMD4A increased after treatment with 5-FU, cisplatin or Nutlin-3 ( Figure 1 A, B, Figure 2 AC), and this increase was dependent on the presence of wild-type p53 ( Figure 1 CJ Figure 2 This suggests that FRMD4A may be a target gene of p53.

[0104] To test this hypothesis, we used the p53MH algorithm26 to analyze the genomic sequence upstream of the transcription start site (TIS) of FRMD4A and identified three possible p53 response elements (REs) ( Figure 1 By ChIP-qPCR and agarose gel electrophoresis analysis, the present invention verifies that p53 specifically binds to RE3 located at -1121 to -1099 upstream of TIS, but does not bind to RE1 or RE2 ( Figure 1 of L and M).

[0105] To determine whether p53 activates the transcription of FRMD4A through these REs, the present invention performed a luciferase reporter gene assay and found that RE3 is indispensable for the p53-induced increase in luciferase activity ( Figure 1 N and O). These findings suggest that p53 activates FRMD4A expression at the transcriptional level.

[0106] To verify this hypothesis, the present invention used different concentrations of Elesclomol alone or in combination with low-dose Nutlin-3 to treat cells, and then performed cell viability assays. The results showed that the presence of Nutlin-3 increased the sensitivity of cancer cells to Elesclomol ( Figure 3 A and B).

[0107] Conversely, p53 knockout rendered cancer cells resistant to elesclomol-induced copper death ( Figure 3 C and D). These results suggest that p53 increases the sensitivity of colorectal cancer and breast cancer cells to copper death by activating the transcription of FRMD4A.

[0108] Example 2 Elesclomol and p53 agonists synergistically inhibit tumor growth

[0109] Since the above results indicate that p53 activation enhances the sensitivity of cancer cells to copper death induced by Elesclomol, the present invention speculates that p53 inducers and Elesclomol may synergistically inhibit tumor growth.

[0110] To this end, the present invention evaluated the effects of Elesclomol in combination with various drugs (including cisplatin, 5-FU, Nutlin-3 and APG-115) on cell viability by calculating the combination index (CI) value.

[0111] Although genotoxic drugs such as cisplatin and 5-FU can activate p53 through DNA damage stress, their combination with Elesclomol did not show synergistic effects on cancer cell growth ( Figure 4 AD).

[0112] Interestingly, the combination of Elesclomol with the p53 agonists Nutlin-3 and APG-115 synergistically inhibited the growth of colorectal and breast cancer cells, as all CI values ​​were less than 1.0 ( Figure 5 AC).

[0113] The nude mouse xenograft tumor model was used to test whether APG-115 could synergistically inhibit the growth of colorectal cancer in vivo with Elesclomol. After subcutaneous inoculation of HCT116 cells in nude mice, they were randomly divided into four groups and treated with saline, APG-115, Elesclomol, and the combination of APG-115 and Elesclomol for ten days ( Figure 5 D).

[0114] The results showed that either drug alone reduced the growth rate, weight, and size of xenograft tumors ( Figure 5 More importantly, their combination can significantly inhibit tumor growth than single treatment ( Figure 5 EG), and the side effects are acceptable ( Figure 6 ).

[0115] Together, these results suggest that the combination of the p53 agonists Nutlin-3 and APG-115 and the copper death inducer Elesclomol is a promising strategy for treating cancer.

[0116] discuss

[0117] As a transcription factor, p53 regulates the expression of many genes, including protein-coding genes and non-coding genes. The present invention found that p53 induced the transcription of FRMD4A and enhanced their sensitivity to copper death induced by Elesclomol, thereby playing a tumor suppressor function. Further studies unexpectedly found that specific p53 agonists Nutlin-3 and APG-115 (rather than cisplatin and 5-FU) that activate p53 can synergize with Elesclomol to inhibit the growth of colorectal cancer and breast cancer cells. Therefore, the findings of the present invention show that specific p53 agonists (Nutlin-3 or APG-115) and Elesclomol can synergistically inhibit cancers carrying wild-type p53.

[0118] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference individually. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

Claims

1. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises a first component and a second component, wherein the first component is Elesclomol, and the second component is Nutlin-3 and / or APG-115.

2. The pharmaceutical composition according to claim 1, characterized in that The second component is Nutlin-3.

3. The pharmaceutical composition according to claim 1, characterized in that The second component is APG-115.

4. The pharmaceutical composition according to claim 1, characterized in that The molar ratio of the first component to the second component is 1:5000-100:

1.

5. The pharmaceutical composition according to claim 1, characterized in that The molar ratio of the first component to the second component is 1:500-2000.

6. The pharmaceutical composition according to claim 1, characterized in that The pharmaceutical composition is used for treating cancer.

7. A method for preparing the pharmaceutical composition according to claim 1, characterized in that: The method comprises the steps of: mixing a first component and a second component with a pharmaceutically acceptable excipient; wherein the first component is Elesclomol, and the second component is Nutlin-3 and / or APG-115.

8. A medicine box, characterized in that: The drug kit comprises a first component and a second component; wherein the first component is Elesclomol, and the second component is Nutlin-3 and / or APG-115.

9. The medicine box according to claim 8, characterized in that The first component and the second component are independently packaged in the medicine kit.

10. Use of the pharmaceutical composition according to claim 1 in preparing a drug for treating cancer.

Images