WO2020248192A1 - 一种具有抗肿瘤作用的组合物及其应用 - Google Patents

一种具有抗肿瘤作用的组合物及其应用 Download PDF

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WO2020248192A1
WO2020248192A1 PCT/CN2019/091116 CN2019091116W WO2020248192A1 WO 2020248192 A1 WO2020248192 A1 WO 2020248192A1 CN 2019091116 W CN2019091116 W CN 2019091116W WO 2020248192 A1 WO2020248192 A1 WO 2020248192A1
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metformin
composition
tumor
mitochondrial
cells
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万晓春
章桂忠
刘曌
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Shenzhen Institute of Advanced Technology of CAS
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/13—Amines
    • A61K31/155—Amidines (), e.g. guanidine (H2N—C(=NH)—NH2), isourea (N=C(OH)—NH2), isothiourea (—N=C(SH)—NH2)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/47—Quinolines; Isoquinolines
    • A61K31/4706—4-Aminoquinolines; 8-Aminoquinolines, e.g. chloroquine, primaquine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/517—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with carbocyclic ring systems, e.g. quinazoline, perimidine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/12—Cyclic peptides, e.g. bacitracins; Polymyxins; Gramicidins S, C; Tyrocidins A, B or C
    • A61K38/13—Cyclosporins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • the invention belongs to the field of medicine, and particularly relates to a composition with anti-tumor effect and its application.
  • Tumor is a non-hereditary genetic disease. Under the action of tumor-causing factors, normal cells have changed their genes, losing normal regulation of their growth, leading to abnormal proliferation. Tumor cells have three significant basic characteristics, namely: immortality, migration and loss of contact inhibition. Tumor therapy, whether it is traditional surgery, radiotherapy and chemotherapy, or new molecular targeted therapy, immune cell therapy, the core idea is to kill and remove tumor cells. Despite the rapid development of biomedical technology in recent years, the current level of tumor treatment is still not high. Tumor cells are immortal. How to break this immortality to kill and eliminate cancer cells is the core of various treatment methods. At present, one of the mechanisms of commonly used clinical tumor treatments such as chemotherapy, radiotherapy, hormone therapy and some biological therapies is to induce tumor cell apoptosis.
  • Metformin is the first-line treatment for type 2 diabetes.
  • metformin is directly related to the reduction of tumor incidence in patients with type 2 diabetes, and further discovered that it has an inhibitory effect on the growth of a variety of tumors and is a potential anti-tumor drug.
  • the researchers also found that the dose of metformin used was relatively large, and the dose could not be reached in the blood circulation or tumor site through oral administration. Therefore, the single use of metformin cannot effectively play an anti-tumor effect, which limits the application potential of metformin.
  • the embodiment of the present invention provides a composition with anti-tumor effect and its application to solve the problem that when metformin is used as an anti-tumor active ingredient in the prior art, metformin enhances mitochondrial autophagy in tumor cells and causes damage in tumor cells. Mitochondria are removed, and tumor cells continue to survive.
  • composition with anti-tumor effect includes at least one of mitochondrial autophagy inhibitors and lysosomal inhibitors. Species, and metformin.
  • an anti-tumor drug is provided, and the anti-tumor drug includes the above-mentioned composition with anti-tumor effect.
  • metformin when metformin is used as the anti-tumor active ingredient, metformin can cause mitochondrial damage in tumor cells and inhibit tumor growth by inhibiting mitochondrial respiration, but at the same time, tumor cells treated with metformin can promote intracellular mitochondrial autophagy , Leading to the removal of damaged mitochondria in tumor cells and the continued survival of tumor cells.
  • the composition with anti-tumor effects provided by the present invention includes at least one of mitochondrial autophagy inhibitors and lysosomal inhibitors, And metformin.
  • mitochondrial autophagy inhibitors can block mitochondrial autophagy, make the damaged mitochondria in the cell not cleared, affect the stability of the cell mitochondrial network and cell survival, and induce apoptosis; another On the other hand, since mitochondrial autophagy needs to fuse with lysosomes to degrade damaged mitochondria, adding lysosomal inhibitors can block the degradation of damaged mitochondria, inhibit the removal of damaged mitochondria, and make A large number of damaged mitochondria accumulate in the cell and further induce cell apoptosis.
  • the composition with anti-tumor effect includes at least one of mitochondrial autophagy inhibitors, lysosomal inhibitors, and metformin. The two act synergistically to significantly promote the death of tumor cells induced by metformin, and effectively play an anti-tumor effect. The effect of tumors.
  • the present invention also provides an anti-tumor drug.
  • the anti-tumor drug includes the above-mentioned composition with anti-tumor effect.
  • the prepared anti-tumor drug has significant drug effect and good anti-tumor effect.
  • Figure 1 is an analysis of the mitochondrial membrane potential of lymphoma cells stimulated by metformin according to the first embodiment of the present invention
  • Figure 2 is an analysis of mitochondrial autophagy in cervical cancer cell lines stimulated by metformin according to the second embodiment of the present invention.
  • Figure 3 is an analysis of the death of cervical cancer cell line Hela by the metformin (Met) and metformin/Cyclosporin A (Met/Cyclosporin A) composition provided by the third embodiment of the present invention;
  • Figure 4 is an analysis of the death of cervical cancer cell line Hela by the metformin (Met) and metformin/Mdivi-1 (Met/Mdivi-1) composition provided by the third embodiment of the present invention
  • Fig. 5 is an analysis of the death of lymphoma cells (Raji) by the metformin (Met) and metformin/Cyclosporin A (Met/Cyclosporin A) composition provided by the third embodiment of the present invention;
  • Fig. 6 is an analysis of the death of lymphoma cells (Raji) by the metformin (Met) and metformin/Mdivi-1 (Met/Mdivi-1) composition provided by the third embodiment of the present invention;
  • Fig. 7 is an analysis of the death of cervical cancer cell line Hela by the metformin (Met) and metformin/chloroquine (Met/Chloroquine) composition provided by the third embodiment of the present invention.
  • the receiving device obtains the identification information of the wearable device from the RFID electronic tag of the wearable device, and sends the identification information of the wearable device to a forwarding node, and the wearable device converts the acquired vital sign data into Chirp data, the wearable device sends the chirp data to the forwarding node.
  • Metformin solution 350 mg metformin (sigma, D150959) was dissolved in 10 mL PBS to make a 200 mM stock solution.
  • Cyclosporin A solution 50 mg cyclosporin A A (Selleck, S2286) dissolved in 831.5 Make a 50 mM stock solution in uL DMSO.
  • Mdivi-1 solution 20 mg Mdivi-1 dissolved in 1.13 Prepare 50 mM stock solution in mL DMSO.
  • Chloroquine solution 0.25 g chloroquine (Sigma, C6628-25G, MW515.86), add 9.7 mL opti-MEM to prepare a 50 mM stock solution.
  • Metformin an inhibitor of mitochondrial respiratory chain complex I, can inhibit mitochondrial respiration, thereby affecting the main source of intracellular reactive oxygen species.
  • mitochondria When mitochondria are attacked by intracellular reactive oxygen species, their DNA, proteins, and lipids will suffer varying degrees of damage, causing abnormalities in the electron transport chain of mitochondria, leading to further accumulation of intracellular reactive oxygen species and damage to mitochondria, destroying cell homeostasis.
  • ATP cannot be hydrolyzed, unable to provide energy for the matrix, and release death-related proteins, promote cell death, and inhibit tumor cell growth.
  • the mitochondrial membrane potential of cancer cells decreases after metformin treatment, and more mitochondria lose membrane potential in the cell. , The addition of metformin can cause mitochondrial damage in tumor cells.
  • mitochondrial autophagy is a process that selectively removes damaged and redundant mitochondria, which is critical to the stability of cell mitochondrial network and cell survival.
  • mitochondrial damage it is often Inducing mitochondrial autophagy, autophagy can prevent damaged mitochondria from releasing pro-apoptotic proteins. Therefore, inducing autophagy can inhibit cell apoptosis and eliminate damaged mitochondria to maintain cell survival.
  • metformin when metformin is used as an anti-tumor active ingredient, after metformin treatment, it will enhance mitochondrial autophagy in tumor cells, resulting in the removal of damaged mitochondria in tumor cells and the continued survival of tumor cells. Therefore,
  • the example of the present invention provides a composition having an anti-tumor effect, which includes at least one of a mitochondrial autophagy inhibitor, a lysosomal inhibitor, and metformin.
  • the composition with anti-tumor effect includes at least one of a mitochondrial autophagy inhibitor, a lysosomal inhibitor, and metformin.
  • a mitochondrial autophagy inhibitor block mitochondrial autophagy, so that the damaged mitochondria in the cell cannot be removed, which affects the stability of the cell mitochondrial network and the survival of the cell.
  • the composition with anti-tumor effect includes metformin and a mitochondrial autophagy inhibitor. In some embodiments, the composition with anti-tumor effect includes metformin and a lysosomal inhibitor. In some embodiments, the composition with anti-tumor effect includes metformin, mitochondrial autophagy inhibitor and lysosomal inhibitor.
  • the mitochondrial autophagy inhibitor is selected from any one of cyclosporin A, Mdivi-1, and 3-methyladenine.
  • the composition with anti-tumor effect comprises metformin and the mitochondrial autophagy inhibitor Cyclosporin A (Cyclosporin A, CsA).
  • Cyclosporin A Cyclosporin A
  • the addition of metformin can inhibit the growth of tumor cells by inhibiting mitochondrial respiration in tumor cells, causing tumor cell mitochondrial damage and promoting cell death.
  • Cyclosporin A is a neutral cyclic polypeptide composed of 11 amino acid residues, which affects mitochondria by inhibiting the opening of mitochondrial permeability transition pore (MTP), inhibits mitochondrial autophagy, and inhibits mitochondria from purging themselves. Therefore, the combination of metformin and cyclosporine A, an inhibitor of mitochondrial autophagy, can promote metformin to induce cell death.
  • MTP mitochondrial permeability transition pore
  • the metformin and the mitochondrial autophagy inhibitor cyclosporin A composition is a composition solution, and in the composition solution, the concentration of metformin is 50 ⁇ M-30 mM, and the concentration of cyclosporin A is 0.1 ⁇ M-20 ⁇ M. In the specific embodiment of the present invention, the concentration of cyclosporin A solution is 5 ⁇ M; the concentration of metformin solution is 10 mM. If the added concentration is too low, the combined solution of metformin and cyclosporin A does not achieve the effect of promoting metformin to induce cell death; if the added concentration is too high, it will cause other toxic and side effects to the cells.
  • the composition with anti-tumor effect comprises metformin and Mdivi-1, an inhibitor of mitochondrial autophagy.
  • Mdivi-1 is a selective inhibitor of mitochondrial DRP (Dynein-related GTPase) division and an inhibitor of mitochondrial division DRP (Dnm1). Its addition is mainly to inhibit the activity of the mitochondrial division regulator DRP and the division starter protein Dnm1, block mitochondrial division, thereby inhibiting type I mitochondrial autophagy.
  • the combination of metformin and mitochondrial autophagy inhibitor Mdivi-1 can further promote metformin-induced cell apoptosis.
  • the composition of metformin and the mitochondrial autophagy inhibitor Mdivi-1 is a composition solution, and in the composition solution, the concentration of metformin is 50 ⁇ M-30 mM, and the concentration of Mdivi-1 is 0.1 ⁇ M-50 ⁇ M.
  • the added concentration of the metformin solution is 10 mM, and the added concentration of the Mdivi-1 solution is 10 ⁇ M. If the added concentration is too low, the combined solution of metformin and Mdivi-1 will not promote the effect of metformin in inducing cell death; if the added concentration is too high, it will cause other toxic and side effects to cells.
  • the autophagy process that occurs in the cell is a highly conserved process in eukaryotes.
  • a combination of a lysosomal inhibitor and metformin is selected to prepare a composition having an anti-tumor effect. Adding a lysosomal inhibitor can block the degradation of damaged mitochondria, inhibit the removal of damaged mitochondria, accumulate a large number of damaged mitochondria in the cell, and further induce cell death. Together with metformin, the two have a synergistic effect, which can significantly promote the death of tumor cells induced by metformin and effectively play an anti-tumor effect.
  • the lysosomal inhibitor is selected from chloroquine, bafaloromycin A1, nocodazole, vinblastine, hydroxychloroquine, cysteine protease inhibitor, aspartic protease Any one of inhibitors.
  • the composition with anti-tumor effect includes metformin and chloroquine (Chloroquine, CQ). Among them, the addition of metformin can inhibit the growth of tumor cells by inhibiting mitochondrial respiration or promote tumor cell death through mitochondrial damage. . After tumor cells feel the mitochondrial damage induced by metformin, they feedback up-regulate mitochondrial autophagy to maintain cell survival.
  • Chloroquine itself is weakly alkaline and has lysosomal properties. Once it selectively enters the lysosome, it will destroy the acidic environment of the lysosome and inhibit the activities of monoacylglycerol lipase and phospholipase A2. , Thereby inhibiting lysosomal function. Therefore, when combined with metformin, chloroquine can inhibit autophagy by destroying the function of lysosomes and improve the killing effect of metformin on cancer cells.
  • the metformin and chloroquine (Chloroquine, CQ) composition is a composition solution, and in the composition solution, the concentration of metformin is 50 ⁇ M-30 mM, and the concentration of chloroquine is 5-100 ⁇ M.
  • the added concentration of the metformin solution is 10 mM, and the added concentration of the chloroquine solution is 50 ⁇ M. If the added concentration is too low, the combined solution of metformin and chloroquine does not promote the effect of metformin in inducing cell death; if the added concentration is too high, it will cause other toxic and side effects to cells.
  • tumor cells from different sources are selected to detect the effect of metformin and its composition on tumor cell apoptosis.
  • the tumor cells are selected from any one of cervical cancer cell lines and hematoma cell lines, wherein the hematoma cell lines may be lymphoma cells.
  • the lymphoma cells (Raji) are transferred at a density of 1 ⁇ 106 cells/well, and the cervical cancer cell line (Hela) is transferred at a density of 2 ⁇ 105 cells/well, and the cells can be cultured overnight.
  • the solution of the composition with anti-tumor effect is added to the tumor cells cultured overnight and cultured for 36-48 hours.
  • the culture time is determined according to the sensitivity of different cells. Cervical cancer cell line (Hela) cells can be cultured for 36 hours, and other hematoma cells need 48 hours. If the culture time is too short, the synergistic effect of anti-tumor drugs will not be obvious.
  • flow cytometry is used to detect the effect of metformin and its composition on tumor cell apoptosis.
  • metformin When metformin is used as an anti-tumor active ingredient, metformin can cause mitochondrial damage in tumor cells and inhibit tumor growth by inhibiting mitochondrial respiration, but at the same time, metformin-treated tumor cells can promote intracellular mitochondrial autophagy, resulting in tumor cells. Damaged mitochondria are removed and tumor cells continue to survive.
  • the composition with anti-tumor effect provided by the present invention includes at least one of mitochondrial autophagy inhibitors, lysosomal inhibitors, and metformin.
  • mitochondrial autophagy inhibitors can block mitochondrial autophagy, make the damaged mitochondria in the cell not cleared, affect the stability of the cell mitochondrial network and cell survival, and induce apoptosis; another On the other hand, since mitochondrial autophagy needs to fuse with lysosomes to degrade damaged mitochondria, adding lysosomal inhibitors can block the degradation of damaged mitochondria, inhibit the removal of damaged mitochondria, and make A large number of damaged mitochondria accumulate in the cell and further induce cell apoptosis.
  • the composition with anti-tumor effect includes at least one of mitochondrial autophagy inhibitors, lysosomal inhibitors, and metformin. The two act synergistically to significantly promote the death of tumor cells induced by metformin, and effectively play an anti-tumor effect. The effect of tumors.
  • the present invention also provides an anti-tumor drug.
  • the anti-tumor drug includes the above-mentioned composition with anti-tumor effect.
  • the prepared anti-tumor drug has significant drug effect and good anti-tumor effect.
  • Lymphoma cancer cells (Raji) were seeded in a 12-well plate at a density of 1 ⁇ 106 cells/well. After overnight culture, the experimental group was stimulated with 10 mM metformin solution for 18 h. The cells were collected and stained with JC-1 (Biyuntian, C2006), and flow cytometric detection of mitochondrial membrane potential.
  • the results of the experiment are shown in Figure 1.
  • the flow cytometer JC-1 is used to detect mitochondrial membrane potential.
  • JC-1 is an ideal fluorescent probe for detecting mitochondrial membrane potential, which can detect cells, tissues or purification.
  • the mitochondrial membrane potential When the mitochondrial membrane potential is high, JC-1 aggregates in the mitochondrial matrix to form a polymer (JC-1 red (aggregates)), which appears to produce red fluorescence; when the mitochondrial membrane potential is low, JC-1 cannot accumulate in In the mitochondrial matrix, JC-1 is a monomer (JC-1 green (monomers)) at this time, which exhibits green fluorescence.
  • JC-1 green When not treated with metformin solution, the content of JC-1 green (ie JC-1 monomer) is 2.68%, and when treated with metformin solution, the content of JC-1 green (ie JC-1 monomer) is 9.33%; therefore After treatment with metformin solution, the JC-1 red/green ratio (JC-1 red/green ratio) decreases, which means that the mitochondrial membrane potential of cancer cells is reduced.
  • JC-1 is a monomer and cannot be aggregated in the mitochondrial matrix. Polymer, the red fluorescence intensity is reduced; and the membrane potential loss of mitochondria in cells treated with metformin solution is more, indicating that metformin can cause mitochondrial damage in tumor cells, which may be caused by mitochondrial respiratory disorders.
  • Cell processing Inoculate the cervical cancer cell line (Hela cells) stably expressing mt-mKeima protein in a 12-well plate at 2 ⁇ 105 cells/well. After overnight culture, add 10 mM metformin solution to continue processing for 18 hours, collect the cells, and flow cytometry Cytometry detects the red and green fluorescence intensity of mt-mKemia protein.
  • mt-mKeima is a mitochondrial-localized protein, which is sensitive to pH.
  • the mt-mKemia protein is in the neutral environment of mitochondria (mt-mKeima). At neutral pH, it emits green fluorescence, and at the end of mitochondrial autophagy, mt-mKemia protein enters the lysosome and is in an acidic environment (mt-mKemia at acidic pH), and the protein turns to emit red fluorescence. Therefore, by detecting the red and green fluorescence changes of mt-mKeima protein, it can reflect the situation of cell mitochondrial autophagy.
  • the mt-mKeima protein When the metformin (Met) solution is not used for treatment, the mt-mKeima protein is in an acidic environment (mt-mKemia at acidic pH) of 16.2%. After treatment with the metformin solution, the detection of mt-mKeima protein emits red fluorescence, mt-mKeima The protein in an acidic environment (mt-mKemia at acidic pH) is 28.7%. It shows that the protein is in an acidic environment as the mitochondria enter the lysosome, that is, the intensity of mt-mKemia at acidic pH increases, indicating that the cell mitochondrial autophagy (mitophagy) is enhanced.
  • the composition with anti-tumor effect of the present invention includes at least one of mitochondrial autophagy inhibitors, lysosomal inhibitors, and metformin.
  • Lymphoma cells Raji or cervical cancer cell line Hela were seeded in 12-well plates at a density of 1 ⁇ 106 cells/well and 2 ⁇ 105 cells/well respectively;
  • metformin solution Metal, 50 ⁇ m-30 mM
  • composition metalformin/cyclosporin A solution, 10 mM/5 ⁇ M; metformin/Mdivi-1 solution, 10 mM/10 ⁇ M; or metformin/chloroquine solution, 10 mM/50 ⁇ M;
  • the concentrations of the two were 10 mM and 5 ⁇ M, respectively; in the metformin/Mdivi-1 composition solution, the concentrations of the two were 10 mM and 10 ⁇ M, respectively; metformin In the chloroquine/chloroquine composition solution, the concentrations of the two are 10 mM and 50 ⁇ M respectively.
  • PI-positive cells are dead cells.
  • Metformin (Met) and Metformin/Cyclosporin A The effect of the composition on the death of cervical cancer cell line Hela: It can be seen from Figure 3 that the higher the PI staining solution I is, the higher the cell death rate. Among them, the cell death rate (Cell death) of the blank control group was 7.02%, and the cell death rate (Cell death) of adding metformin (Met) was 24.1%; when cyclosporin A (CsA) was added, the blank control group was added The cell death rate obtained after cyclosporin A treatment was 11.6%, and the cell death rate obtained after treatment with metformin and cyclosporin A group was 46.3%. Therefore, the mitochondrial autophagy inhibitor Cyclosporin A (CsA) significantly promotes the death of Hela cells induced by Metformin (Met).
  • CsA mitochondrial autophagy inhibitor Cyclosporin A
  • Metformin (Met) and Metformin/Cyclosporin A The effect of the composition on the death of lymphoma cells (Raji): It can be seen from Figure 5 that the higher the PI staining solution I is, the higher the death rate of the cells. Among them, the cell death rate of the blank control group was 4.41%, and the cell death rate of the metformin group was 17.3%; when cyclosporin A was added, the blank control group was treated with cyclosporin A The cell death rate (Cell death) obtained afterwards was 6.59%, and the cell death rate (Cell death) obtained after the treatment with metformin and cyclosporin A was 45.4%. Therefore, the mitochondrial autophagy inhibitor cyclosporin A ( CsA) significantly promotes the death of Raji cells induced by metformin (Met).
  • CsA mitochondrial autophagy inhibitor cyclosporin A

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Abstract

一种具有抗肿瘤作用的组合物及其应用，所述具有抗肿瘤作用的组合物包括线粒体自噬抑制剂、溶酶体抑制剂中的至少一种，以及二甲双胍。通过上述组合物，添加二甲双胍，其能够通过抑制线粒体呼吸抑制肿瘤生长，同时细胞内线粒体自噬能力增强；加入线粒体自噬抑制剂或者溶酶体抑制剂阻断线粒体自噬，能够显著促进二甲双胍诱导的肿瘤细胞死亡。

Description

一种具有抗肿瘤作用的组合物及其应用 技术领域
本发明属于药物领域，尤其涉及一种具有抗肿瘤作用的组合物及其应用。
 背景技术
肿瘤是一种非遗传的基因病。正常细胞在致瘤因素作用下，基因发生了改变，失去对其生长的正常调控，导致异常增生。肿瘤细胞有三个显著的基本特征，即：不死性，迁移性和失去接触抑制。肿瘤治疗，不管是传统的手术、放化疗，还是新生的分子靶向治疗、免疫细胞治疗，其核心思想都是杀死、清除肿瘤细胞。尽管近年来生物医药技术发展突发猛进，但目前的肿瘤治疗水平依然不高。肿瘤细胞具有不死性，如何打破这种不死性以杀死、清除癌细胞是各种治疗方法的核心。目前临床上常用的肿瘤治疗手段如化疗、放疗、激素治疗及一些生物疗法的作用机制之一便是诱导肿瘤细胞发生凋亡。不管是传统的放化疗还是新兴的生物疗法，都无法彻底灭杀肿瘤，耐药和复发案例时有发生。因此，临床上亟需一些新的肿瘤治疗方案/药物。然而，受限于肿瘤细胞异质性，单一治疗方法/药物很难彻底杀死所有肿瘤细胞。更糟糕的是，这种不完全的治疗一方面会助长那些对该疗法不敏感的肿瘤细胞爆发性增殖，导致治疗失败或复发；另一方面肿瘤细胞基因组原本就不稳定，治疗过程中产生的生存压力往往促使原本对药物敏感的肿瘤细胞发生基因组以及表达谱的改变，进化出抵抗治疗的能力，即发生耐药。此外，尽管抗肿瘤策略越来越多，也日趋先进，但目前仍有很大一部分患者无法从现有的治疗策略中获益，或者初期获益后出现耐药。因此，目前的肿瘤治疗方法都无法彻底战胜肿瘤，总体治疗水平不高。
二甲双胍是治疗二型糖尿病的一线药物。除了降血糖作用，近年来的研究发现二甲双胍与二型糖尿病患者的肿瘤发病率降低直接相关，并进一步发现其对多种肿瘤生长具有抑制作用，是一种有潜力的抗肿瘤药物。但在这些研究中，研究人员也发现，二甲双胍的使用剂量较大，通过口服的方式无法在血液循环或者肿瘤部位达到该剂量。因此，单一使用二甲双胍，无法有效地起到抗肿瘤的作用效果，限制了二甲双胍的应用潜力。
 技术问题
本发明实施例提供了一种具有抗肿瘤作用的组合物及其应用，以解决现有技术中使用二甲双胍作为抗肿瘤活性成分时，二甲双胍增强肿瘤细胞内线粒体自噬，导致肿瘤细胞中的受损线粒体得以清除，肿瘤细胞继续维持生存的问题。
 技术解决方案
本发明实施例是这样实现的，第一方面，提供了一种具有抗肿瘤作用的组合物，所述具有抗肿瘤作用的组合物包括线粒体自噬抑制剂、溶酶体抑制剂中的至少一种，以及二甲双胍。
第二方面，提供了一种抗肿瘤药物，所述抗肿瘤药物包括上述的具有抗肿瘤作用的组合物。
 有益效果
本发明实施例中，以二甲双胍作为抗肿瘤活性成分时，二甲双胍能够通过抑制线粒体呼吸，导致肿瘤细胞中线粒体损伤、抑制肿瘤生长，但同时，二甲双胍处理过的肿瘤细胞，能促进细胞内线粒体自噬，导致肿瘤细胞中的受损线粒体得以清除，肿瘤细胞继续维持生存，本发明提供的的一种具有抗肿瘤作用的组合物包括线粒体自噬抑制剂、溶酶体抑制剂中的至少一种，以及二甲双胍。添加线粒体自噬抑制剂，线粒体自噬抑制剂能够阻断线粒体自噬，使细胞内受损的线粒体得不到清除，影响细胞线粒体网络的稳定性以及细胞的生存，诱导细胞凋亡；另一方面，由于线粒体自噬发生后需要与溶酶体融合才能将受损线粒体降解，添加溶酶体抑制剂，溶酶体抑制剂能够阻断受损线粒体的降解，抑制受损线粒体的清除，使细胞内积累大量受损的线粒体，进一步诱导细胞凋亡。所述具有抗肿瘤作用的组合物包括线粒体自噬抑制剂、溶酶体抑制剂中的至少一种，以及二甲双胍，二者协同作用，能够显著促进二甲双胍诱导的肿瘤细胞死亡，有效地起到抗肿瘤的作用效果。
本发明还提供了一种抗肿瘤药物，所述抗肿瘤药物包括上述的具有抗肿瘤作用的组合物，所制备的抗肿瘤药物药效显著，抗肿瘤效果好。
 附图说明
图1是本发明第一实施例提供的经二甲双胍刺激的淋巴瘤细胞线粒体膜电位的分析；
图2是本发明第二实施例提供的经二甲双胍刺激的宫颈癌细胞系线粒体自噬的分析。
图3是本发明第三实施例提供的二甲双胍（Met）及二甲双胍/环孢素A（Met/Cyclosporin A）组合物对宫颈癌细胞系Hela死亡的分析；
图4是本发明第三实施例提供的二甲双胍（Met）及二甲双胍/Mdivi-1（Met/Mdivi-1）组合物对宫颈癌细胞系Hela死亡的分析；
图5是本发明第三实施例提供的二甲双胍（Met）及二甲双胍/环孢素A（Met/Cyclosporin A）组合物对淋巴瘤细胞（Raji）死亡的分析；
图6是本发明第三实施例提供的二甲双胍（Met）及二甲双胍/Mdivi-1（Met/Mdivi-1）组合物对淋巴瘤细胞（Raji）死亡的分析；
图7是本发明第三实施例提供的二甲双胍（Met）及二甲双胍/氯喹（Met/Chloroquine）组合物对宫颈癌细胞系Hela死亡的分析。
 本发明的实施方式
为了使本发明的目的、技术方案及优点更加清楚明白，以下结合附图及实施例，对本发明进行进一步详细说明。应当理解，此处所描述的具体实施例仅仅用以解释本发明，并不用于限定本发明。
本发明实施例中，接收设备从穿戴设备的RFID电子标签中获取所述穿戴设备的标识信息，并将所述穿戴设备的标识信息发送至转发节点，穿戴设备将获取的生命体征数据转换为啁啾数据，穿戴设备将所述啁啾数据发送至转发节点。
为了说明本发明所述的技术方案，下面通过具体实施例来进行说明。
本发明中，各溶液配制方法如下：
二甲双胍溶液：350 mg二甲双胍（sigma, D150959）溶于10 mL PBS中，配成200 mM贮存液。
环孢素A溶液：50 mg环孢素A A (Selleck, S2286) 溶于831.5 uL DMSO中，配成50 mM贮存液。
Mdivi-1溶液：20 mg Mdivi-1溶于1.13 mL DMSO中，配成50 mM贮存液。
氯喹溶液：0.25 g氯喹 (Sigma, C6628-25G, MW515.86)，加入9.7 mL opti-MEM，配制成50 mM贮存液。
二甲双胍，是一种线粒体呼吸链复合物I的抑制剂，能够通过抑制线粒体呼吸，进而影响细胞内活性氧的主要来源。当线粒体受到胞内活性氧的攻击时，其DNA、蛋白质、脂质会遭受不同程度的损伤，使线粒体的电子传递链发生异常，导致胞内活性氧进一步积累及线粒体损伤，破坏细胞稳态，导致ATP无法水解，无法为基体提供能量，并释放死亡相关蛋白，促进细胞死亡，抑制肿瘤细胞生长；同时，二甲双胍处理后癌细胞线粒体膜电位降低，并且细胞中膜电位丧失的线粒体更多，因此，添加二甲双胍会导致肿瘤细胞线粒体损伤。但是，同时，经二甲双胍处理后，细胞线粒体自噬增强，而线粒体自噬一种选择性清除受损、冗余线粒体的过程，对细胞线粒体网络稳定和细胞生存至关重要，线粒体损伤后，往往诱导线粒体自噬，自噬能够防止损伤的线粒体释放促进凋亡蛋白，因此诱导自噬，可以抑制细胞凋亡，用以清除受损线粒体以维持细胞生存。
即：使用二甲双胍作为抗肿瘤活性成分时，经二甲双胍处理后，会增强肿瘤细胞内线粒体自噬，导致肿瘤细胞中的受损线粒体得以清除，肿瘤细胞继续维持生存。鉴于此，
本发明实例提供一种具有抗肿瘤作用的组合物，所述具有抗肿瘤作用的组合物包括线粒体自噬抑制剂、溶酶体抑制剂中的至少一种，以及二甲双胍。
具体的，为了提高二甲双胍的抗肿瘤的效果，所述具有抗肿瘤作用的组合物包括线粒体自噬抑制剂、溶酶体抑制剂中的至少一种，以及二甲双胍。其中，添加线粒体自噬抑制剂，阻断线粒体自噬，使细胞内受损的线粒体得不到清除，影响细胞线粒体网络的稳定性以及细胞的生存。
在一些实施例中，所述具有抗肿瘤作用的组合物包含二甲双胍与线粒体自噬抑制剂。在一些实施例中，所述具有抗肿瘤作用的组合物包含二甲双胍与溶酶体抑制剂。在一些实施例中，所述具有抗肿瘤作用的组合物包含二甲双胍、线粒体自噬抑制剂及溶酶体抑制剂。
优选的，所述线粒体自噬抑制剂选自环孢素A、Mdivi-1、3-甲基腺嘌呤的任意一种。
在一个具体实施例中，所述的具有抗肿瘤作用的组合物包含二甲双胍与线粒体自噬抑制剂环孢素A（Cyclosporin A, CsA）。其中，添加二甲双胍能够通过抑制肿瘤细胞中线粒体呼吸，导致肿瘤细胞线粒体损伤，促进细胞死亡，以抑制肿瘤细胞的生长。环孢素A由11个氨基酸残基组成的中性环状多肽，通过抑制线粒体通透性转换孔（MTP）的开放而影响线粒体，抑制线粒体自噬，抑制线粒体进行自身清除。因此，二甲双胍组合线粒体自噬抑制剂环孢素A进行作用，可以促进二甲双胍诱导细胞死亡。
具体的，所述二甲双胍与线粒体自噬抑制剂环孢素A组合物为组合物溶液，且在所述组合物溶液中，二甲双胍的浓度为50 μM-30 mM，环孢素A的浓度为0.1 μM-20 μM。在本发明具体实施例中，环孢素A溶液的添加浓度为5 μM；二甲双胍溶液的添加浓度为10 mM。若添加的浓度太低，二甲双胍与环孢素A的组合物溶液没有达到促进二甲双胍诱导细胞死亡的效果；若添加的浓度太高，则会对细胞造成其他毒副作用。
在另一个具体实施例中，所述的具有抗肿瘤作用的组合物包含二甲双胍与线粒体自噬抑制剂Mdivi-1。其中，Mdivi-1是线粒体DRP（发动蛋白相关GTP酶）分裂选择性抑制剂以及线粒体分裂发动蛋白（Dnm1）抑制剂。其添加主要是抑制线粒体分裂调控因子DRP及分裂发动蛋白Dnm1的活性，阻断线粒体分裂，从而抑制I型线粒体自噬。二甲双胍组合线粒体自噬抑制剂Mdivi-1进行作用，可进一步促进二甲双胍诱导细胞凋亡。
具体的，所述二甲双胍与线粒体自噬抑制剂Mdivi-1组合物为组合物溶液，且所述组合物溶液中，二甲双胍的浓度为50 μM-30 mM，Mdivi-1的浓度为0.1 μM-50 μM。在本发明具体实施例中，所述二甲双胍溶液的添加浓度为10 mM，Mdivi-1溶液的添加浓度为10 μM。若添加的浓度太低，二甲双胍与Mdivi-1的组合物溶液没有促进二甲双胍诱导细胞死亡的效果；若添加的浓度太高，则会对细胞造成其他毒副作用。细胞中发生的自噬过程，属于真核生物中高度保守的过程，均发生在细胞质中，自噬发生后均在溶酶体中实现蛋白的降解，因此，当细胞中线粒体受到损伤发生自噬过程之后，均需要与溶酶体融合方能实现将受损线粒体进行降解。具体的，本发明优选实施例中，选择溶酶体抑制剂与二甲双胍进行组合制备具有抗肿瘤作用的组合物。添加溶酶体抑制剂，能够阻断受损线粒体的降解，抑制受损线粒体的清除，使细胞内积累大量受损的线粒体，进一步诱导细胞死亡。再协同二甲双胍，二者协同作用，能够显著促进二甲双胍诱导的肿瘤细胞死亡，有效地起到抗肿瘤的作用效果。
在本发明优选实施例中，所述溶酶体抑制剂选自氯喹、巴法络霉素A1、诺考达唑、长春碱、羟氯喹、半胱氨酸蛋白酶抑制剂、天冬氨酸蛋白酶抑制剂的任意一种。在本发明具体实施例中，所述具有抗肿瘤作用的组合物包含二甲双胍和氯喹（Chloroquine，CQ）。其中，二甲双胍的添加能够通过抑制线粒体呼吸，以抑制肿瘤细胞的生长或通过线粒体损伤促进肿瘤细胞死亡。。肿瘤细胞感受到二甲双胍诱导的线粒体损伤后，反馈性上调线粒体自噬以维持细胞生存。氯喹本身具有弱碱性，同时具有趋溶酶体的特性，一旦其选择性地进入溶酶体内，就会破坏溶酶体的酸性环境，抑制单酰基甘油脂肪酶、磷脂酶A2等酶的活性，从而抑制溶酶体功能。因此，与二甲双胍联合应用，氯喹可以通过破坏溶酶体的功能抑制自噬，提高二甲双胍对癌细胞的杀伤作用。具体的，所述二甲双胍和氯喹（Chloroquine，CQ）组合物为组合物溶液，且所述组合物溶液中，二甲双胍的浓度为50 μM-30 mM，氯喹的浓度为5-100 μM。在本发明具体实施例中，所述二甲双胍溶液的添加浓度为10 mM，氯喹溶液的添加浓度为50 μM。若添加的浓度太低，二甲双胍与氯喹的组合物溶液没有促进二甲双胍诱导细胞死亡的效果；若添加的浓度太高，则会对细胞造成其他毒副作用。
在本发明优选实施例中，选用不同来源的肿瘤细胞检测二甲双胍及其组合物对肿瘤细胞凋亡的影响。优选的，所述肿瘤细胞选自宫颈癌细胞系及血液瘤细胞系的任意一种，其中，血液瘤细胞系可选淋巴瘤细胞。优选的，所述淋巴瘤细胞（Raji）以1×106细胞/孔的密度进行转接，宫颈癌细胞系（Hela）以2×105细胞/孔的密度进行转接，过夜培养即可。
优选的，在所述过夜培养的肿瘤细胞中，添加所述具有抗肿瘤作用的组合物的溶液，培养36-48 h。优选的，所述培养时间是根据不用细胞的敏感性不同进行确定的，宫颈癌细胞系（Hela）细胞培养36小时即可，其他血液瘤细胞需要48小时。若培养时间太短会导致抗肿瘤药物协同效果不明显。
优选的，利用流式细胞术检测二甲双胍及其组合物对肿瘤细胞凋亡的影响。
以二甲双胍作为抗肿瘤活性成分时，二甲双胍能够通过抑制线粒体呼吸，导致肿瘤细胞中线粒体损伤、抑制肿瘤生长，但同时，二甲双胍处理过的肿瘤细胞，能促进细胞内线粒体自噬，导致肿瘤细胞中的受损线粒体得以清除，肿瘤细胞继续维持生存，本发明提供的的一种具有抗肿瘤作用的组合物包括线粒体自噬抑制剂、溶酶体抑制剂中的至少一种，以及二甲双胍。添加线粒体自噬抑制剂，线粒体自噬抑制剂能够阻断线粒体自噬，使细胞内受损的线粒体得不到清除，影响细胞线粒体网络的稳定性以及细胞的生存，诱导细胞凋亡；另一方面，由于线粒体自噬发生后需要与溶酶体融合才能将受损线粒体降解，添加溶酶体抑制剂，溶酶体抑制剂能够阻断受损线粒体的降解，抑制受损线粒体的清除，使细胞内积累大量受损的线粒体，进一步诱导细胞凋亡。所述具有抗肿瘤作用的组合物包括线粒体自噬抑制剂、溶酶体抑制剂中的至少一种，以及二甲双胍，二者协同作用，能够显著促进二甲双胍诱导的肿瘤细胞死亡，有效地起到抗肿瘤的作用效果。
相应的，本发明还提供了一种抗肿瘤药物，所述抗肿瘤药物包括上述的具有抗肿瘤作用的组合物，所制备的抗肿瘤药物药效显著，抗肿瘤效果好。
下面结合具体实施例的内容进一步进行说明。
实施例一：
线粒体膜电位的测定
细胞处理：将淋巴癌细胞（Raji）以1×106细胞/孔的密度接种12孔板中，过夜培养后，实验组加10 mM 二甲双胍溶液刺激18 h。收集细胞并进行JC-1(碧云天，C2006)染色，流式检测线粒体膜电位。
实验结果如图1分析可知，利用流式细胞仪JC-1进行线粒体膜电位检测，其中，JC-1是一种理想的用于检测线粒体膜电位的荧光探针，可以检测细胞、组织或纯化的线粒体膜电位。当线粒体膜电位较高时，JC-1聚集在线粒体基质中，形成聚合物（JC-1 red (aggregates)），表现为产生红色荧光；当线粒体膜电位较低时，JC-1不能聚集在线粒体基质中，此时JC-1为单体（JC-1 green(monomers)），表现为产生绿色荧光。
当没有用二甲双胍溶液处理时，JC-1 green（即JC-1单体）含量为2.68%，当利用二甲双胍溶液处理后，JC-1 green（即JC-1单体）含量为9.33%；因此，利用二甲双胍溶液处理后，JC-1 red/green比值（JC-1 red/green ratio）降低，即表示癌细胞线粒体膜电位降低，JC-1为单体，不能聚集在线粒体基质中，没有形成聚合物，红色荧光强度降低；且利用二甲双胍溶液处理得到的细胞中膜电位丧失的线粒体更多，表明二甲双胍会导致肿瘤细胞线粒体损伤，可能是线粒体呼吸紊乱导致的。
实施例二：
线粒体自噬的测定
细胞处理：将稳定表达mt-mKeima蛋白的宫颈癌细胞系（Hela细胞）以2×105细胞/孔接种于12孔板，过夜培养后，加10 mM二甲双胍溶液继续处理18h, 收集细胞，流式细胞术检测mt-mKemia蛋白的红绿荧光强度。
实验结果如图2，其中，mt-mKeima是线粒体定位的蛋白，对酸碱度敏感，mt-mKemia蛋白处于线粒体的中性环境（mt-mKeima at neutral pH）中时发出绿色荧光，而当线粒体自噬末期，mt-mKemia蛋白随着线粒体进入溶酶体而处于酸性环境（mt-mKemia at acidic pH），蛋白转而发出红色荧光。因此，通过检测mt-mKeima蛋白的红绿荧光改变，可以反应细胞线粒体自噬的情况。
当没有用二甲双胍（Met）溶液进行处理时，mt-mKeima蛋白处于酸性环境（mt-mKemia at acidic pH）为16.2%，而利用二甲双胍溶液处理后，检测mt-mKeima蛋白发出红色荧光，mt-mKeima蛋白处于酸性环境（mt-mKemia at acidic pH）为28.7%。说明蛋白随着线粒体进入溶酶体而处于酸性环境，即mt-mKemia at acidic pH的强度增强，说明细胞线粒体自噬（mitophagy）增强。
上述实施例1、实施例2的结果表明二甲双胍溶液对癌细胞的处理，会导致癌细胞中线粒体膜电位降低，且细胞中膜电位丧失的线粒体会增加；同时，经过二甲双胍溶液处理后，细胞中线粒体的自噬能力也会增强。因此，本发明的具有抗肿瘤作用的组合物，所述具有抗肿瘤作用的组合物包括线粒体自噬抑制剂、溶酶体抑制剂中的至少一种，以及二甲双胍。
实施例三：
流式细胞术检测二甲双胍（Metformin, Met）及其组合物对肿瘤细胞凋亡的影响
1．细胞处理
1）淋巴瘤细胞（Raji）或宫颈癌细胞系Hela分别以1×106细胞/孔和2×105细胞/孔的密度接种12孔板中；
2）过夜培养后，加二甲双胍溶液（Met, 50 μm-30 mM）或组合物（二甲双胍/环孢素A溶液, 10 mM/5 μM; 二甲双胍/Mdivi-1溶液, 10 mM/10 μM; 或二甲双胍/氯喹溶液, 10 mM/50 μM;）进行培养36-48 h后，检测细胞死亡情况。
其中，二甲双胍/环孢素A组合物溶液中，二者的添加浓度分别为10 mM、5 μM；二甲双胍/Mdivi-1组合物溶液中，二者的添加浓度分别为 10 mM、10 μM；二甲双胍/氯喹组合物溶液中，二者的添加浓度分别为10 mM、50 μM。
2．流式细胞术分析细胞死亡情况
1）收集细胞悬液，将细胞悬液3200 rpm，4℃离心5 min，弃掉上清，收集沉淀中的细胞；
3）用预冷的PBS洗涤细胞1次，然后用400 μL PI染色液（碘化丙啶，0.05 mg/mL）重悬细胞；
4）4 ℃避光染色15 min；
5）流式细胞仪检测凋亡情况：PI阳性的细胞为死亡细胞。
二甲双胍（Met）及二甲双胍/环孢素A（Met/Cyclosporin A）组合物对宫颈癌细胞系Hela死亡的影响：由图3可知，当 PI染色液I阳性越高，表示细胞的死亡率越高。其中，空白对照组的细胞死亡率（Cell death）为7.02%，加了二甲双胍（Met）的细胞死亡率（Cell death）为24.1%；当加入了环孢素A（CsA），空白对照组加入环孢素A处理后得到的细胞死亡率（Cell death）为11.6%，加了二甲双胍和环孢素A组处理后得到的细胞死亡率（Cell death）为46.3%，因此，线粒体自噬抑制剂环孢素A（CsA）显著促进二甲双胍（Met）诱导的Hela细胞死亡。
二甲双胍（Met）及二甲双胍/Mdivi-1（Met/Mdivi-1）组合物对宫颈癌细胞系Hela死亡的影响：由图4可知，当 PI染色液I阳性越高，表示细胞的死亡率越高。其中，空白对照组的细胞死亡率（Cell death）为9.68%，加了二甲双胍组的细胞死亡率（Cell death）为26.2%；当加入了Mdivi-1，空白对照组加入Mdivi-1处理后得到的细胞死亡率（Cell death）为12.7%，加了二甲双胍和Mdivi-1组处理后得到的细胞死亡率（Cell death）为42.9%，因此，线粒体自噬抑制剂Mdivi-1显著促进二甲双胍（Met）诱导的Hela细胞死亡。
二甲双胍（Met）及二甲双胍/环孢素A（Met/Cyclosporin A）组合物对淋巴瘤细胞（Raji）死亡的影响：由图5可知，当 PI染色液I阳性越高，表示细胞的死亡率越高。其中，空白对照组的细胞死亡率（Cell death）为4.41%，加了二甲双胍组的细胞死亡率（Cell death）为17.3%；当加入了环孢素A，空白对照组加入环孢素A处理后得到的细胞死亡率（Cell death）为6.59%，加了二甲双胍和环孢素A组处理后得到的细胞死亡率（Cell death）为45.4%，因此，线粒体自噬抑制剂环孢素A（CsA）显著促进二甲双胍（Met）诱导的Raji细胞死亡。
二甲双胍（Met）及二甲双胍/Mdivi-1（Met/Mdivi-1）组合物对淋巴瘤细胞（Raji）死亡的影响：由图6可知，当 PI染色液I阳性越高，表示细胞的死亡率越高。其中，空白对照组的细胞死亡率（Cell death）为3.63%，加了二甲双胍组的细胞死亡率（Cell death）为14.4%；当加入了Mdivi-1，空白对照组加入Mdivi-1处理后得到的细胞死亡率（Cell death）为4.79%，加了二甲双胍和Mdivi-1组处理后得到的细胞死亡率（Cell death）为28.3%，因此，线粒体自噬抑制剂Mdivi-1显著促进二甲双胍（Met）诱导的Raji细胞死亡。
二甲双胍（Met）及二甲双胍/氯喹二甲双胍/氯喹（Met/Chloroquine）组合物对宫颈癌细胞系Hela死亡的影响：由图7可知，当PI染色液I阳性越高，表示细胞的死亡率越高。其中，空白对照组的细胞死亡率（Cell death）为4.28%，加了二甲双胍组的细胞死亡率（Cell death）为10.8%；当加入了氯喹（CQ），空白对照组加入氯喹处理后得到的细胞死亡率（Cell death）为7.64%，加了二甲双胍和氯喹组处理后得到的细胞死亡率（Cell death）为22.4%，因此，溶酶体抑制剂氯喹（CQ）显著促进二甲双胍（Met）诱导的Hela细胞死亡。
以上所述实施例仅用以说明本发明的技术方案，而非对其限制；尽管参照前述实施例对本发明进行了详细的说明，本领域的普通技术人员应当理解：其依然可以对前述各实施例所记载的技术方案进行修改，或者对其中部分技术特征进行等同替换；而这些修改或者替换，并不使相应技术方案的本质脱离本发明各实施例技术方案的精神和范围，均应包含在本发明的保护范围之内。

Claims (10)

  1. 一种具有抗肿瘤作用的组合物，其特征在于，所述具有抗肿瘤作用的组合物包括线粒体自噬抑制剂、溶酶体抑制剂中的至少一种，以及二甲双胍。
  2. 根据权利要求1所述的具有抗肿瘤作用的组合物，其特征在于，所述线粒体自噬抑制剂选自环孢素A、Mdivi-1、3-甲基腺嘌呤的任意一种。
  3. 根据权利要求1所述的具有抗肿瘤作用的组合物，其特征在于，所述溶酶体抑制剂选自氯喹、巴法络霉素A1、诺考达唑、长春碱、羟氯喹、半胱氨酸蛋白酶抑制剂、天冬氨酸蛋白酶抑制剂的任意一种。
  4. 根据权利要求1-3任一所述的具有抗肿瘤作用的组合物，其特征在于，所述抗肿瘤作用的组合物包括环孢素A及二甲双胍。
  5. 根据权利要求4所述的具有抗肿瘤作用的组合物，其特征在于，所述组合物为组合物溶液，且所述组合物溶液中，二甲双胍的浓度为50 μM-30 mM，环孢素A的浓度为0.1 μM-20μM。
  6. 根据权利要求1-3任一所述的具有抗肿瘤作用的组合物，其特征在于，所述抗肿瘤作用的组合物包括Mdivi-1及二甲双胍。
  7. 根据权利要求6所述的具有抗肿瘤作用的组合物，其特征在于，所述组合物为组合物溶液，且所述组合物溶液中，二甲双胍的浓度为50 μM-30 mM，Mdivi-1的浓度为0.1 μM-50μM。
  8. 根据权利要求1-3任一所述的具有抗肿瘤作用的组合物，其特征在于，所述抗肿瘤作用的组合物包括氯喹及二甲双胍。
  9. 根据权利要求8所述的具有抗肿瘤作用的组合物，其特征在于，所述组合物为组合物溶液，且所述组合物溶液中，二甲双胍的浓度为50 μM-30 mM，氯喹的浓度为5-100μM。
  10. 一种抗肿瘤药物，其特征在于，所述抗肿瘤药物包括如权利要求1至9任一项所述的具有抗肿瘤作用的组合物。
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