WO2016127789A1 - 绿原酸在制备通过p53、pi3k-akt和mapk通路预防和治疗肺母细胞瘤药物中的用途 - Google Patents

绿原酸在制备通过p53、pi3k-akt和mapk通路预防和治疗肺母细胞瘤药物中的用途 Download PDF

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WO2016127789A1
WO2016127789A1 PCT/CN2016/072126 CN2016072126W WO2016127789A1 WO 2016127789 A1 WO2016127789 A1 WO 2016127789A1 CN 2016072126 W CN2016072126 W CN 2016072126W WO 2016127789 A1 WO2016127789 A1 WO 2016127789A1
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chlorogenic acid
cells
pulmonary blastoma
pi3k
akt
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English (en)
French (fr)
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张洁
张金兰
李梦林
黄骏
黄望
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四川九章生物科技有限公司
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    • 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
    • A61K31/21—Esters, e.g. nitroglycerine, selenocyanates
    • A61K31/215—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids
    • A61K31/216—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids of acids having aromatic rings, e.g. benactizyne, clofibrate

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  • the invention relates to the use of chlorogenic acid in the preparation of a medicament for treating pulmonary blastoma, in particular to the use of chlorogenic acid for the preparation of a medicament for preventing and treating pulmonary blastoma through the P53, PI3K-Akt and MAPK pathways, belonging to the field of medicine .
  • Chlorogenic acid also known as coffee tannin, chemically known as 3-0-caffeoylquinic acid, is derived from caffeic acid and quinic acid.
  • Quinic acid consists of a carboxylic acid.
  • Chlorogenic acid is a product of aerobic respiration metabolism in plants. It is the main active ingredient in many Chinese herbal medicines and fruits and vegetables. It has many biological activities, such as cardiovascular protection, anti-oxidation, anti-ultraviolet and anti-radiation effects. Anti-mutagenic and anti-cancer effects, antibacterial effects, antiviral effects, lipid-lowering and hypoglycemic effects, immunomodulatory effects, etc. It has a wide range of applications in the fields of pharmaceutical, chemical and food.
  • Pulmonary blastoma is a rare primary malignant tumor of the lung, divided into adult pulmonary blastoma and childhood pleural pulmonary blastoma.
  • the clinical symptoms and imaging examination of pulmonary blastoma are not specific, easy to be misdiagnosed, and difficult to diagnose before surgery.
  • Postoperative pathology should be considered in combination with morphology and immunohistochemistry.
  • Surgical treatment is preferred for this disease.
  • the surgical approach is mainly lobectomy and mediastinal lymph node dissection. Prognosis is related to pathological type, stage and tumor location, and the overall prognosis is poor.
  • the disease advocates surgery-based comprehensive treatment, the surgical plan is similar to lung cancer, with lung or whole lung resection combined with mediastinal, hilar lymph node dissection, postoperative adjuvant chemotherapy, radiotherapy is not accurate, and advocates postoperative radiotherapy is rare.
  • the disease is not sensitive to chemotherapy, and what chemotherapy drugs and chemotherapy regimens are currently in use are still inconclusive.
  • an object of the present invention is to provide a new use of chlorogenic acid.
  • the above object of the present invention is achieved by the following technical solution: a use of chlorogenic acid for the preparation of a medicament for preventing and treating pulmonary blastoma.
  • chlorogenic acid is capable of inhibiting the formation of tumor blood vessels, thereby treating pulmonary blastoma and controlling the metastasis of pulmonary blastoma.
  • Chlorogenic acid is used for the prevention and treatment of pulmonary blastoma through the P53, PI3K-Akt and MAPK pathways. Specifically, chlorogenic acid activates the P53 pathway and promotes apoptosis of tumor cells to achieve the goal of treating pulmonary blastoma.
  • chlorogenic acid acts as a target inhibitor of PI3K and AKT, inhibits PI3K-Akt activity, specifically inhibits tumor angiogenesis, thereby treating pulmonary blastoma and controlling the metastasis of pulmonary blastoma.
  • chlorogenic acid acts as a MAPK kinase A target inhibitor of kinase that inhibits MAPK kinase kinase activity, thereby inhibiting MAPK kinase activity, thereby inhibiting MAPK activity, specifically inhibiting tumor vascular endothelial cells and tumor angiogenesis, thereby treating pulmonary blastoma and controlling pulmonary blastoma Transfer.
  • P53 is a tumor suppressor protein that plays an important role in preventing the occurrence and development of tumors, such as apoptosis of tumor cells and inhibition of tumor blood vessel formation.
  • P53 protein can inhibit the cell reproductive cycle to stay at the G 1 /S rhythm point, to achieve DNA repair identification, to initiate DNA repair; if DNA is not repairable, P53 initiates apoptosis process to avoid the continued division and growth of tumor cells .
  • the mutated p53 protein will likely lose the ability to form an effective binding to DNA to signal the cessation of cell division. Therefore, the damaged cells will undergo uncontrolled cell division, and eventually the formation will lead to the development of tumors.
  • chlorogenic acid acts on the target gene of P53 protein in the p53 signaling pathway, activates its target gene, expresses P53 protein, and makes it biologically active to inhibit tumor development and antitumor. effect.
  • PI3K-Akt signaling pathway plays a key role in promoting cell proliferation and inhibiting apoptosis in vivo by affecting the activation state of various effector molecules in the downstream.
  • Development is closely related, chlorogenic acid acts on the key point p110 ⁇ subunit and pAkt in the PI3K-Akt signaling pathway or target genes acting on these targets, causing these targets to lose their original activity or to express these target proteins.
  • the mitogen-activated protein kinase (MAPK) cascade is a superfamily of silk/threonine protein kinases widely present in cells, and is an important substance that transmits cytoplasmic signals to the nucleus and causes changes in the nucleus.
  • MAPK can be divided into 4 subfamilies: ERK, p38, JNK and ERK5.
  • Activation of p38MAPK and ERK causes morphological changes in cells, from cuboid to long fusiform, accompanied by down-regulation of E-cadherin, promoting cell growth, migration and invasion.
  • JNK plays an important role in various physiological and pathological processes such as cell cycle, production, apoptosis and cell stress.
  • Chlorogenic acid acts on ERK and p38MAPK in the MAPK signaling pathway, inhibiting the activity of ERK and p38MAPK, thereby inhibiting the biology, migration and invasion of tumor cells.
  • Chlorogenic acid acts on JNK, activates JNK signaling pathway, and promotes apoptosis of tumor cells.
  • the drug is a preparation prepared by adding chlorogenic acid as an active ingredient, adding a pharmaceutically acceptable adjuvant or an auxiliary component.
  • Another object of the present invention is to provide a formulation for treating a pulmonary blastoma comprising chlorogenic acid and a pharmaceutically acceptable excipient or adjuvant.
  • the excipients or excipients include, but are not limited to, mannitol, sodium bisulfite, starch, dextrin powder, absolute ethanol, water for injection, powdered sugar, lactose, hypromellose, magnesium stearate, sucrose, Povidone K30.
  • the preparation of the present invention is an oral preparation or an injection preparation.
  • the preparation contains 1-10 mg of chlorogenic acid per unit preparation, and the clinical use dose is 1-100 mg/kg.
  • the drug has a relatively obvious therapeutic effect on pulmonary blastoma and has a good prognosis.
  • Figure 1 is a graph showing the effect of chlorogenic acid on the proliferation of pulmonary blastoma cells.
  • Figure 2 is a schematic diagram showing chlorogenic acid-induced apoptosis of pulmonary blastoma cells.
  • Figure 3 is a schematic diagram showing the expression of caspase 3 protein by Western blotting.
  • Figure 4 is a schematic diagram of cell cycle state detection at different dose concentrations.
  • Figure 5 is a schematic diagram of serine phosphorylation at position 15 of p53 protein.
  • Figure 6 is a schematic diagram showing apoptosis of lung blastoma cells induced by chlorogenic acid (20 mg/ml).
  • Figure 7 shows the expression of key proteins of the PI3K-Akt pathway in lungblastoma cells and normal cells (Western Blot results).
  • Figure 8 is a dose-effect relationship of chlorogenic acid on the proliferation of lungblastoma cells (p ⁇ 0.05).
  • Figure 9 is a graphical representation of the effect of chlorogenic acid on the cell cycle of pulmonary blastoma.
  • Figure 10 is a graphical representation of the effect of chlorogenic acid on the cell cycle of pulmonary blastoma.
  • Figure 11 is a flow cytometry apoptosis map of lung blastoma cells affected by chlorogenic acid.
  • Figure 12 is a graph showing the effect of chlorogenic acid on the expression of pAkt in pulmonary blastoma cells.
  • Figure 13 is a graph showing the effect of chlorogenic acid on the expression of pAkt in pulmonary blastoma cells.
  • Figure 14 is a graph showing the effect of MTT detection of chlorogenic acid on proliferation of pulmonary blastoma cells.
  • Figure 15 is a schematic diagram showing the effect of green number on the proliferation of pulmonary blastoma cells by cell number method.
  • Figure 16 is a graph showing the effect of chlorogenic acid (20 mg/ml) on apoptosis of pulmonary blastoma cells.
  • Figure 17 is a graph showing the effect of chlorogenic acid (20 mg/ml) on MAPK signaling pathway in pulmonary blastoma cells.
  • Figure 18 is a schematic diagram showing the effect of chlorogenic acid on apoptosis of pulmonary blastoma cells through MAPK signaling pathway.
  • the p53 tumor suppressor gene is a transcription factor that plays a key role in regulating cell death and various cellular processes.
  • Cell events involved in regulation of p53 include cell cycle, apoptosis, DNA damage repair, and senescence.
  • chlorogenic acid can induce apoptosis in pulmonary blastoma cells.
  • Pifithrin- ⁇ is a specific p53 protein inhibitor. After treatment of pneumoblastoma cells with pifithrin- ⁇ , flow cytometry experiments showed that apoptosis of lung blastoma cells was completely inhibited.
  • Apoptosis of pulmonary blastoma cells was detected by FITC-Annexin V flow cytometry (BD apoptosis detection kit).
  • a. Collecting cells When the cells in the cell culture flask reach 60-80% confluence, add 2 ml of EDTA-pancreatin to the cell culture flask, and observe under microscope that 90% of the cells are detached from the bottle wall and 5 ml of whole culture is blown to terminate digestion and collection. The cells were centrifuged at 200 g for 10 min to obtain a cell pellet. A 5 ml medium suspension was added and the cells were counted under a microscope.
  • Species plate blastoma cells were inoculated 1x10 6 cells in 100mm petri dishes.
  • the cell culture dish was placed in a 5% CO 2 and cultured in a 37 ° C incubator for 24 hours.
  • the cells were treated with 20 mg/ml chlorogenic acid and 100 ⁇ M pifithrin- ⁇ on the second day; the control group was treated with DMSO.
  • the cell culture dishes were placed in a 5% CO 2 and cultured in a 37 ° C cell incubator for 30 mins to 24 hours.
  • the pulmonary blastoma cells were treated with chlorogenic acid (40 mg/ml in the high dose group, 20 mg/ml in the middle dose group and 5 mg/ml in the low dose group) for 24, 48 and 72 hours, and the cycle state was observed by flow cytometry.
  • a. Collecting cells When the cells in the cell culture flask reach 60-80% confluence, add 2 ml of EDTA-pancreatin to the cell culture flask, and observe under microscope that 90% of the cells are detached from the bottle wall and 5 ml of whole culture is blown to terminate digestion and collection. The cells were centrifuged at 200 g for 10 min to obtain a cell pellet. A 5 ml medium suspension was added and the cells were counted under a microscope.
  • Species plate blastoma cells were inoculated 1x10 6 cells in 100mm petri dishes.
  • the cell culture dish was placed in a 5% CO 2 and cultured in a 37 ° C incubator for 24 hours.
  • Flow cytometry The cells were collected by EDTA-trypsin at the indicated culture time, and the cells were washed three times with PBS; the cells were resuspended in 1 ml of buffer, vortexed while adding 4 ml of 70% pre-chilled ethanol, fixed at 4 °C. More than 12 hours; wash the PBS solution three times, add 400 ⁇ L PBS buffer to resuspend the cells, add PI and RNaseA to a final concentration of 50 ⁇ g / ml, incubate at room temperature for 30 mins in the dark, and measure the cell cycle state by flow cytometry.
  • Chlorogenic acid inhibits proliferation of pulmonary blastoma cells by inducing apoptosis. Decreased proliferation of pulmonary blastoma cells is the result of chlorogenic acid directly inducing apoptosis in pulmonary blastoma cells.
  • Flow cytometry results showed that after treatment with low dose of chlorogenic acid (5mg/ml), the cell surface bound with AnnexinV and PI staining increased. After adding chlorogenic acid, FITC-AnnexinV and FITC-AnnexinV could be observed as early as 30 minutes. PI positive cells increased ( Figure 2).
  • Chlorogenic acid activates the p53 signaling pathway.
  • the p53 tumor suppressor gene is a transcription factor that plays a key role in regulating cell death and various cellular processes.
  • Cell events involved in regulation of p53 include cell cycle, apoptosis, DNA damage repair, and senescence.
  • chlorogenic acid can induce apoptosis in pulmonary blastoma cells.
  • Western blotting showed that serine phosphorylation of p53 protein at position 15 increased rapidly after chlorogenic acid (20 mg/ml) treatment of lungblastoma cells (Fig. 5), indicating that p53 was activated.
  • Pifithrin- ⁇ is a specific p53 protein inhibitor.
  • chlorogenic acid (20 mg/ml) induced complete inhibition of apoptosis in lung blastoma cells (Fig. 6).
  • the positive rate of FITC-AnnexinV staining in the control group was 11%; the positive rate of FITC-AnnexinV staining in the chlorogenic acid treatment group; the positive rate of FITC-AnnexinV staining in the pifithrin- ⁇ group was 10%.
  • Apoptosis experiments further demonstrated that chlorogenic acid induced apoptosis in lung blastoma cells by activating the p53 signaling pathway.
  • Example 2 Example of inhibition of PI3K-Akt activity by chlorogenic acid
  • PI3K-Akt is one of the most important signal transduction pathways in cell biological functions such as cell proliferation, apoptosis and cell cycle. Its abnormal activation is closely related to the occurrence and development of malignant tumors. Chlorogenic acid can effectively block the corresponding molecular targets in the PI3K-Akt signaling pathway of lungblastoma cells, inhibit the growth and proliferation of lungblastoma cells, induce cell cycle arrest, promote apoptosis, and significantly down-regulate Expression and activation of signal proteins downstream of the signaling pathway.
  • the pulmonary blastoma cells were cultured in DMEM medium containing 10% calf serum, 100 IU/L penicillin and 100 mg/L streptomycin were added, and placed in a constant temperature incubator at 37 ° C, 5% CO 2 for routine culture. After the cells are over the bottom of the bottle, discard the culture solution, rinse twice with PBS solution, digest with 0.25% trypsin and 0.02% EDTA for 5 minutes, then divide the flask for culture, and pass it once every 2 to 3 days. Several growth phase cells were used for the experiment.
  • the logoblastoma cells in logarithmic growth phase were trypsinized and inoculated into a six-well culture plate, placed in a 5% CO 2 incubator, and cultured at 37 ° C. When growing to 80% confluent state, the total cell extraction was collected. Protein, protein concentration was determined by BCA method. The expression of PI3K (p110 ⁇ subunit) and pAkt (S473, T308) in cells was detected by Western Blot.
  • the pulmonary blastoma cells in the logarithmic growth phase were seeded in a six-well plate at a density of 1 to 5 x 10 4 cells/well. After 24 hours of culture, chlorogenic acid (5, 20 and 40 mg/ml) was added for 24 hours, the cell culture solution was decanted, and the cells were washed once with ice PBS. The cells were harvested by trypsinization, and cells were collected by PI staining (apoptosis detection using FITC-AnnexinV and PI double staining), and cell cycle and apoptosis were detected by flow cytometry. Each of the same experiments was repeated 3 times.
  • the pulmonary blastoma cells in the logarithmic growth phase were seeded in a six-well plate at a density of 1 to 5 x 10 4 cells/well. After 24 hours of culture, chlorogenic acids (5, 10, 15 and 20 mg/ml) were added for 48 hours, and the cells were collected to extract total protein. The expression of PI3K (p110 ⁇ subunit) and pAkt (S473, T308) in cells was detected by Western Blot.
  • Control group 10.2 ⁇ 2.7 Chlorogenic acid (5mg/ml) 20.3 ⁇ 3.5** Chlorogenic acid (10mg/ml) 30.1 ⁇ 8.5** Chlorogenic acid (15mg/ml) 38.9 ⁇ 9.2** Chlorogenic acid (20mg/ml) 49.6 ⁇ 12.6**
  • Pulmonaryblastoma cells were treated with chlorogenic acid at five dose concentrations of 5, 10, 15 and 20 mg/ml for different times to detect cell proliferation (Fig. 14, Fig. 15).
  • Example 4 Example of chlorogenic acid prevention and treatment of pulmonary blastoma
  • Mouse lung blastoma model C57BL/6 mice, male, 18-20 g.
  • the tumor tissues with good growth were taken and diluted with a 1:3 ratio of sterile physiological saline to prepare a tumor cell suspension, and 0.2 ml of tumor fluid was inoculated into the back of each mouse.
  • Animals were randomized the next day after inoculation, weighed, and dosing.
  • the solvent control group was administered by intraperitoneal injection of 0.2 ml per 10 g of mice once a day.
  • the chlorogenic acid injection was administered in an amount of 0.2 ml per 10 g of mice per day, once a day for 13 days.
  • the experimental animals were divided into 7 groups, negative control group, solvent control group, chlorogenic acid 5 mg/kg, 10 mg/kg, and 20 mg/kg three dose groups. 15 animals per group. After the chlorogenic acid was stopped, the animals were sacrificed the next day, weighing, weighing and weighing. The tumor inhibition rate (%) was calculated from the tumor weight. Body weight and tumor weight were expressed as mean ⁇ standard deviation (x ⁇ SD) and between each administration group and the negative control group.

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Abstract

本发明公开了一种绿原酸在制备治疗肺母细胞瘤的药物中的用途，具体涉及绿原酸制备通过P53、PI3K-Akt和MAPK通路预防和治疗肺母细胞瘤的药物中的用途。本发明还公开了一种治疗肺母细胞瘤的制剂，所述制剂包含绿原酸和药物上可接受的赋形剂或辅料。

Description

绿原酸在制备通过P53、PI3K-Akt和MAPK通路预防和治疗肺母细胞瘤药物中的用途 技术领域
本发明涉及绿原酸在制备治疗肺母细胞瘤的药物中的用途，具体涉及绿原酸制备通过P53、PI3K-Akt和MAPK通路预防和治疗肺母细胞瘤的药物中的用途，属于药物领域。
背景技术
绿原酸(Chlorogenic acid,CGA)，又名咖啡单宁酸，化学名为3-0-咖啡酰奎尼酸(3-0-caffeoylquinic acid)，是由咖啡酸(Caffeic acid)与奎尼酸(Quinic acid)组成的羧酚酸。
绿原酸是植物体有氧呼吸代谢的产物，是许多中药材以及水果蔬菜中的主要有效成分，具有多种生物活性，如:心血管保护作用、抗氧化作用、抗紫外及抗辐射作用、抗诱变及抗癌作用、抗菌作用、抗病毒作用、降脂降糖作用、免疫调节作用等。在医药化工和食品等领域都具有广泛的应用。
肺母细胞瘤是一种罕见肺部原发恶性肿瘤，分为成人型肺母细胞瘤和儿童胸膜肺母细胞瘤。肺母细胞瘤的临床症状及影像学检查无特异性，容易误诊，术前诊断困难，术后病理需结合形态学和免疫组化综合考虑。本病首选手术治疗，手术方式以肺叶切除加纵隔淋巴结清扫术为主。预后与病理类型、分期及肿瘤部位等有关，总体预后差。
目前，临床上用于治疗肺母细胞瘤的唯一手段是手术。本病主张以手术为主的综合治疗，手术方案与肺癌相似，以肺叶或者全肺切除配合纵隔、肺门淋巴结清扫，术后辅助化疗，放疗的疗效不确切，主张术后放疗者甚少。该病对化疗不敏感，目前应用何种化疗药物以及何种化疗方案还没有定论。
发明内容
针对上述技术问题，本发明的目的在于提供一种绿原酸的新用途。
本发明的上述目的是通过如下技术方案实现的：一种绿原酸在制备预防和治疗肺母细胞瘤的药物中的应用。
在本发明中，绿原酸能够抑制肿瘤血管的生成，从而治疗肺母细胞瘤和控制肺母细胞瘤的转移。绿原酸是通过P53、PI3K-Akt和MAPK通路达到预防和治疗肺母细胞瘤的目的。具体而言，绿原酸是激活P53通路，促进肿瘤细胞的凋亡而达到治疗肺母细胞瘤的目的。此外，绿原酸是作为PI3K和AKT的靶点抑制剂，抑制PI3K-Akt活性，具体是抑制肿瘤血管的生成，从而治疗肺母细胞瘤和控制肺母细胞瘤的转移。此外，绿原酸作为MAPK激酶 激酶的靶点抑制剂，抑制MAPK激酶激酶活性，从而抑制MAPK激酶活性，从而再抑制MAPK活性，具体是抑制肿瘤血管内皮细胞和肿瘤血管的生成，从而治疗肺母细胞瘤和控制肺母细胞瘤的转移。
P53是一个肿瘤抑制蛋白，在避免肿瘤的发生和发展中起着重要的作用，例如，肿瘤细胞的凋亡和抑制肿瘤新血管的生成等。P53蛋白能抑制细胞生殖周期停留于G1/S的节律点上，达成DNA修复的辨识，以启动DNA的修复；若DNA不可修复，则P53启动细胞凋亡程序，避免肿瘤细胞的继续分裂生长。突变后的p53蛋白将可能丧失与DNA形成有效结合的能力，以发出停止细胞分裂的信号。因此，受损细胞将不受控制的进行细胞分裂，最终形成导致肿瘤的发生发展。
发明人研究发现，绿原酸作用于细胞内p53信号通路中的P53蛋白的靶基因上，激活其靶基因，表达P53蛋白，使其具有生物活性以抑制肿瘤的发生发展，起到抗肿瘤的作用。PI3K-Akt信号通路作为细胞内重要信号转导通路之一，通过影响下游多种效应分子的活化状态，在体内发挥着促进细胞增殖和抑制凋亡的关键作用，它与多种肿瘤的发生和发展密切相关，绿原酸作用于PI3K-Akt信号通路中的关键点p110α亚基和pAkt或者作用于这些靶点的靶基因，使这些靶点失去原有的活性或者使这些靶点蛋白的表达受到抑制，从而抑制肿瘤的发生发展、促进肿瘤细胞的凋亡和抑制肿瘤细胞的浸袭和转移。丝裂原活化蛋白激酶(mitogen-activated protein kinase，MAPK)级联是细胞内广泛存在的丝/苏氨酸蛋白激酶超家族，是将细胞质的信号传递至细胞核并引起细胞核发生变化的重要物质，MAPK可分为4个亚族：ERK、p38、JNK和ERK5。p38MAPK和ERK的激活，使细胞发生形态学改变，由立方形转变为长梭形，并伴随着E-cadherin下调，促进细胞生长、迁移及侵袭。JNK在细胞周期、生产、凋亡和细胞应激等多种生理和病理过程中起重要作用。绿原酸作用于MAPK信号通路中的ERK和p38MAPK，抑制ERK和p38MAPK的活，从而抑制肿瘤细胞的生物、迁移和侵袭。绿原酸作用于JNK，激活JNK信号通路，促进肿瘤细胞的凋亡。
本发明中，作为优选，所述的药物是由绿原酸为有效成分，加入药学上可接受的辅料或辅助性成分制备而成的制剂。
本发明的另一目的在于，提供一种治疗肺母细胞瘤的制剂，所述制剂包含绿原酸和药物上可接受的赋形剂或辅料。所述辅料或赋形剂包括但不限于甘露醇、亚硫酸氢钠、淀粉、糊精粉、无水乙醇、注射用水、糖粉、乳糖、羟丙甲纤维素、硬脂酸镁、蔗糖、聚维酮K30。
具体而言，本发明所述的制剂是口服制剂或注射制剂。
其中，所述的制剂每单位制剂含绿原酸1-1000mg，临床使用剂量为：1-100mg/kg。
本发明的有益效果在于提供一种可以用于治疗肺母细胞瘤的绿原酸和药学上可接受的赋形剂或辅料组成的制剂。所述药物对肺母细胞瘤有较为明显的治疗效果且预后性较好。
附图说明
图1为绿原酸抑制肺母细胞瘤细胞增生的效果图。
图2为绿原酸诱导肺母细胞瘤细胞凋亡的示意图。
图3为蛋白印迹实验检测caspase 3蛋白表达示意图。
图4为不同剂量浓度下细胞周期状态检测示意图。
图5为p53蛋白15位丝氨酸磷酸化示意图。
图6为绿原酸(20mg/ml)诱导肺母细胞瘤细胞凋亡示意图。
图7为PI3K-Akt通路关键蛋白在肺母细胞瘤细胞和正常细胞中的表达(Western Blot结果)。
图8为绿原酸对肺母细胞瘤细胞增殖能力影响的量效关系(p<0.05)。
图9为绿原酸对肺母细胞瘤细胞周期的影响示意图。
图10为绿原酸对肺母细胞瘤细胞周期的影响示意图。
图11为肺母细胞瘤细胞受绿原酸影响的流式细胞仪凋亡图谱。
图12为绿原酸对肺母细胞瘤细胞pAkt表达的影响示意图。
图13为绿原酸对肺母细胞瘤细胞pAkt表达的影响示意图。
图14为MTT检测绿原酸对肺母细胞瘤细胞增殖的影响示意图。
图15为细胞数法检测绿原对肺母细胞瘤细胞增殖的影响示意图。
图16为绿原酸(20mg/ml)对肺母细胞瘤细胞凋亡的影响示意图。
图17为绿原酸(20mg/ml)对肺母细胞瘤细胞中MAPK信号通路的影响示意图。
图18为绿原酸通过MAPK信号通路影响肺母细胞瘤细胞的凋亡示意图。
具体实施方式
实施例1绿原酸激活P53实施例
p53肿瘤抑癌基因是一个转录因子，在调控细胞死亡和各种细胞程序中起关键作用。p53参与调控的细胞事件包括细胞周期、细胞凋亡、DNA损伤修复以及衰老等。实验显示绿原酸能够诱导肺母细胞瘤细胞凋亡。pifithrin-α是一种特异性p53蛋白抑制剂，用pifithrin-α处理肺母细胞瘤细胞后，流式细胞实验显示肺母细胞瘤细胞凋亡完全被抑制。
(1)细胞凋亡实验
用FITC-Annexin V流式细胞实验(BD公司凋亡检测试剂盒)检测肺母细胞瘤细胞凋亡。
a.收集细胞待细胞培养瓶中细胞达到60-80％汇合度时，向细胞培养瓶中加入2ml EDTA-胰酶，显微镜下观察待90％细胞脱离瓶壁时加入5ml全培养吹打终止消化收集细胞，200g离心10mins得到细胞沉淀。加入5ml培养基重悬液，显微镜下计数细胞。
b.种板将肺母细胞瘤细胞1x106个接种于100mm细胞培养皿。将细胞培养皿置于5％CO2，37℃培养箱中培养24小时。
c.细胞贴壁后，第二日用20mg/ml绿原酸、100μM pifithrin-α处理细胞；对照组用DMSO处理细胞。将细胞培养皿置于5％CO2，37℃细胞培养箱中培养30mins到24小时。
d.流式细胞实验第三日用EDTA-胰酶消化收集细胞，200g离心10mins，细胞用PBS液洗2次；用1xBinding缓冲液重悬制成1x106/ml细胞悬液；将100μL细胞悬液转移至5ml流式管中，加入5μLAnnexin V及5μL Propidium iodide(PI)，混匀并室温避光孵育15mins；向各流式管中加入4005μL 1xBinding缓冲液，涡旋震荡后上流式细胞仪进行检测。
e.实验结果用FlowJo软件进行分析。
(2)细胞周期实验
用绿原酸(大剂量组40mg/ml、中剂量组20mg/ml和低剂量组5mg/ml)处理肺母细胞瘤细胞24、48和72小时后，流式细胞实验观察周期状态。
a.收集细胞待细胞培养瓶中细胞达到60-80％汇合度时，向细胞培养瓶中加入2ml EDTA-胰酶，显微镜下观察待90％细胞脱离瓶壁时加入5ml全培养吹打终止消化收集细胞，200g离心10mins得到细胞沉淀。加入5ml培养基重悬液，显微镜下计数细胞。
b.种板将肺母细胞瘤细胞1x106个接种于100mm细胞培养皿。将细胞培养皿置于5％CO2，37℃培养箱中培养24小时。
c.细胞贴壁后，第二日用40mg/ml、20mg/ml和5mg/ml绿原酸；对照组用DMSO处理细胞。将细胞培养皿置于5％CO2，37℃细胞培养箱中培养24、48和72小时。
d.流式细胞实验在指定的培养时间用EDTA-胰酶消化收集细胞，用PBS液洗细胞3次；用1ml缓冲液重悬细胞，旋涡震荡同时加入4ml 70％预冷乙醇，4℃固定12小时以上；PBS液洗3次，加入400μLPBS缓冲液重悬细胞，加入PI和RNaseA至终浓度50μg/ml，室温避光孵育30mins后用流式细胞仪测定细胞周期状态。
e.实验结果用FlowJo软件进行分析。
(3)实验结果
a.首先检测绿原酸对肺母细胞瘤细胞增生的影响。MTT实验发现，绿原酸低剂量组5mg/ml时，肺母细胞瘤细胞即受抑制；绿原酸中剂量组20mg/ml时，肺母细胞瘤细胞受到显著的抑 制(p<0.05)(图1)。
图1中实验结果是3次独立MTT实验数据统计的累计，在实验中绿原酸每个剂量高有16个重复孔，实验结果可重复性高。
b.绿原酸通过诱导凋亡抑制肺母细胞瘤细胞增生。肺母细胞瘤细胞增生降低是绿原酸直接诱导肺母细胞瘤细胞凋亡的结果。流式细胞实验结果显示，用低剂量绿原酸(5mg/ml)处理细胞后，细胞表面结合AnnexinV和PI染色增加，在加绿原酸后，最早在30分钟即能观察到FITC-AnnexinV和PI阳性细胞增加(图2)。蛋白印迹实验证实，用20mg/ml的绿原酸处理肺母细胞瘤细胞24，48和72小时后，裂解caspase3蛋白表达增加(图3)。用不同剂量浓度的绿原处理肺母细胞瘤细胞24、48和72小时后，用PI对细胞进行染色，再用流式细胞仪检测细胞周期状态改变(图4)。
c.绿原酸激活p53信号通路。p53肿瘤抑癌基因是一个转录因子，在调控细胞死亡和各种细胞程序中起关键的作用。p53参与调控的细胞事件包括细胞周期、细胞凋亡、DNA损伤修复以及衰老等。实验显示，绿原酸能够诱导肺母细胞瘤细胞凋亡。同时，蛋白印迹实验显示，绿原酸(20mg/ml)处理肺母细胞瘤细胞后，p53蛋白15位丝氨酸磷酸化迅速增加(图5)，说明p53被激活。
pifithrin-α是一种特异性p53蛋白质抑制剂。用pifithrin-α预处理肺母细胞瘤细胞后，流式细胞实验显示绿原酸(20mg/ml)诱导肺母细胞瘤细胞凋亡完全被抑制(图6)。对照组中细胞FITC-AnnexinV染色阳性率为11％；绿原酸处理组细胞FITC-AnnexinV染色阳性率为；pifithrin-α组中细胞FITC-AnnexinV染色阳性率为10％。细胞凋亡实验进一步证明，绿原酸诱导肺母细胞瘤细胞凋亡是通过激活p53信号通路。
实施例2绿原酸抑制PI3K-Akt活性实施例
PI3K-Akt是细胞增殖、细胞凋亡及细胞周期等细胞生物学功能最重要的信号转导通路之一，其异常激活与恶性肿瘤的发生发展密切相关。绿原酸能有效的阻断肺母细胞瘤细胞PI3K-Akt信号通路上的相应的分子靶点，抑制肺母细胞瘤细胞的生长增殖，诱导细胞周期阻滞，促进细胞凋亡，并显著下调信号通路下游信号蛋白质的表达及活化。
(1)实验方法
肺母细胞瘤细胞培养于含10％小牛血清的DMEM培养液中，添加100IU/L的青霉素和100mg/L的链霉素，置于37℃，5％CO2的恒温培养箱中常规培养，待细胞长满瓶底后，弃掉培养液，PBS液冲洗两次，再用0.25％的胰酶和0.02％的EDTA消化5分钟，再分瓶培养，2至3天传代一次，取对数生长期细胞用于实验。
a.PI3K-Akt通路活性检测
取对数生长期的肺母细胞瘤细胞经胰酶消化后接种于六孔培养板中，置于5％CO2培养箱，37℃培养，待生长至80％融合状态时，收集细胞提取总蛋白，用BCA法测定蛋白浓度。用Western Blot法检测细胞中PI3K(p110α亚单位)和pAkt(S473，T308)的表达。
b.细胞周期及凋亡分析
将处于对数生长期的肺母细胞瘤细胞，按1～5x104细胞/孔密度接种于六孔板中。培养24小时后，分别加绿原酸(5、20和40mg/ml)作用24小时，倒去细胞培养液，用冰PBS洗细胞一次。加入胰蛋白酶消化，收集细胞，采用PI单染(细胞凋亡检测采用FITC-AnnexinV和PI双染法)，用流式细胞仪分别进行细胞周期和细胞凋亡检测。每一相同实验重复3次。
c.绿原酸对肺母细胞瘤细胞PI3K-Akt通路活性影响
将处于对数生长期的肺母细胞瘤细胞，按1～5x104细胞/孔密度接种于六孔板中。培养24小时后，分别加绿原酸(5、10、15和20mg/ml)作用48小时，收集细胞，提取总蛋白。用Western Blot方法检测细胞中PI3K(p110α亚单位)和pAkt(S473，T308)的表达。
d.统计分析
采用SPSS11.5软件进行数据统计分析，所有数据表示为平均值±标准误差(Means±SEM)。
(2)结果
a.PI3K-Akt通路关键蛋白在正常细胞和肺母细胞瘤细胞中的表达Western Blot分析发现，PI3K-Akt通路中的关键蛋白PI3K(p110α亚单位)在肺母细胞瘤细胞和正常细胞中的表达无显著差异；总pAkt蛋白变化表达水平无明显差异；pAkt(S473，T308)在肺母细胞瘤中的表达较正常细胞显著升高(p<0.05)(图7)。
绿原酸对肺母细胞瘤细胞增殖抑制作用MTT比色法检测结果显示，量效关系上绿原酸(5、10、15和20mg/ml)呈剂量依赖的抑制肺母细胞瘤细胞的生长(p<0.05)(图8)。
c.绿原酸对肺母细胞瘤细胞周期变化及凋亡率的影响流式细胞仪细胞周期检测结果显示，绿原酸(20mg/ml)作用24小时后，肺母细胞瘤细胞出现明显的G0/G1期阻滞现象，相应的处于S期的细胞比例与对照组相比较显著减少(p<0.05)(图9、图10)。
流式细胞仪凋亡实验检测结果显示，绿原酸作用肺母细胞瘤细胞24小时后，肺母细胞瘤细胞凋亡率与对照组相比较显著升高(p<0.05)(图11、表1)。
表1.各剂量组处理肺母细胞瘤细胞凋亡率
Figure PCTCN2016072126-appb-000001
分组 肺母细胞瘤细胞(％)
对照组 10.2±2.7
绿原酸(5mg/ml) 20.3±3.5**
绿原酸(10mg/ml) 30.1±8.5**
绿原酸(15mg/ml) 38.9±9.2**
绿原酸(20mg/ml) 49.6±12.6**
**：与对照组相比，p<0.05。
d.绿原酸对肺母细胞瘤细胞Akt活性的影响Western Blot结果显示，肺母细胞瘤细胞经过绿原酸(20mg/ml)孵育48小时后，pAkt(T308和S473)的表达水平与对照组相比显著降低(p<0.05)(图12、图13)。
实施例3绿原酸抑制MAPK信号通路活性实施例
(1)以5、10、15和20mg/ml四种剂量浓度的绿原酸分别处理肺母细胞瘤细胞不同时间，检测细胞的增殖情况(图14，图15)。
流式细胞仪检测发现，20mg/mg的绿原酸作用于肺母细胞瘤细胞24小时后，与正常的control组相比，在实相图上出现典型性细胞亚二倍体凋亡峰，细胞凋亡率为39.59％，明显高于对照组的凋亡率1.89％(表2，图16)。
表2.绿原酸对肺母细胞瘤细胞凋亡的影响(n＝15，means±SEM)
Figure PCTCN2016072126-appb-000002
注：与正常对照组比较，*p<0.05。
(2)为证明绿原诱导肺母细胞瘤细胞凋亡的信号通路，用绿原酸处理培养的肺母细胞瘤细胞，观察信号通路中的三个主要成员，即ERK1/2、JNK和p38MAPK的变化。用Western Blot检测三个信号转导蛋白的磷酸化水平变化。结果显示，当20mg/ml绿原酸作用于肺母细胞瘤细胞24小时后，开始升高，48小时明显升高。ERK和p38MAPK48小时后明显降低，见图17。表明绿原是通过激活肺母细胞瘤细胞中MAPK通路中的JNK，抑制ERK和p38MAPK来控制肺母细胞瘤细胞的增殖和凋亡。
为证明绿原酸激活JNK、抑制ERK和p38MAPK信号通路与肺母细胞瘤细胞凋亡有关，用信号通路的抑制剂PD98059处理细胞后用流式细胞仪检测细胞的凋亡情况。如图18所示，阻断ERK和p38MAPK信号通路后，与单纯绿原酸诱导条件下相比凋亡率进一步上升(图18C)。以上结果表明JNK、ERK和p38MAPK参与了绿原酸诱导肺母细胞瘤细胞凋亡。
实施例4绿原酸预防和治疗肺母细胞瘤实施例
小鼠肺母细胞瘤模型，选用C57BL/6小鼠，雄性，18-20g。实验时，取生长良好的肿瘤组织，用无菌生理盐水按1：3比例稀释后制成肿瘤细胞悬液，每只小鼠腋背部接种0.2ml瘤液。接种后次日动物随机分组，称重，并开始给药。溶剂对照组按每10g小鼠腹腔注射0.2ml给药，每日1次。绿原酸注射液给药体积为每10g小鼠腹腔注射0.2ml，每日1次，连续给药13天。
实验动物共分7组，阴性对照组、溶剂对照组、绿原酸5mg/kg，10mg/kg，20mg/kg三个剂量组。每组15只动物。绿原酸停药后次日处死动物，称体重，剥瘤并称瘤重。根据肿瘤重量计算肿瘤抑制率(％)。体重及瘤重用均值±标准差(x±SD)表示，并进行各给药组与阴性对照组之间。
注射给予注射用绿原酸对小鼠肺母细胞瘤呈明显的剂量依赖性的生长抑制作用。(表3)。
表3.绿原酸对小鼠肺母细胞瘤的抗肿瘤作用
Figure PCTCN2016072126-appb-000003
注：***P<0.001，与阴性对照组比较。

Claims (10)

  1. 一种绿原酸在制备预防和治疗肺母细胞瘤的药物中的应用。
  2. 如权利要求1所述的应用，所述药物是绿原酸通过P53、PI3K-Akt和MAPK通路预防和治疗肺母细胞瘤。
  3. 如权利要求2所述的应用，所述药物是绿原酸激活P53通路，促进肿瘤细胞的凋亡而达到治疗肺母细胞瘤。
  4. 如权利要求2或3所述的应用，所述药物是绿原酸作为PI3K和AKT的靶点抑制剂，抑制PI3K-Akt活性，优选所述药物是绿原酸抑制肿瘤血管的生成，从而治疗肺母细胞瘤和控制肺母细胞瘤的转移。
  5. 如权利要求2-4任一项所述的应用，所述药物是绿原酸作为MAPK激酶激酶的靶点抑制剂，抑制MAPK激酶激酶活性，从而抑制MAPK激酶活性，从而再抑制MAPK活性。
  6. 如权利要求5所述的应用，所述药物是绿原酸抑制肿瘤血管内皮细胞和肿瘤血管的生成，从而治疗肺母细胞瘤和控制肺母细胞瘤的转移。
  7. 如权利要求1-6任一项所述的应用，所述的药物包括以绿原酸和药学上可接受的辅料或辅助性成分。
  8. 一种用于治疗治疗肺母细胞瘤的药物，包括绿原酸和药学上可接受的辅料和/或赋形剂。
  9. 如权利要求8所述的药物，所述药物为注射制剂或口服剂。
  10. 如权利要求8或9所述的药物，所述的药物使用剂量为1-100mg/kg。
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