CN116531382B - Apy29在制备治疗食管鳞癌的药物中的应用 - Google Patents
Apy29在制备治疗食管鳞癌的药物中的应用 Download PDFInfo
- Publication number
- CN116531382B CN116531382B CN202310726061.5A CN202310726061A CN116531382B CN 116531382 B CN116531382 B CN 116531382B CN 202310726061 A CN202310726061 A CN 202310726061A CN 116531382 B CN116531382 B CN 116531382B
- Authority
- CN
- China
- Prior art keywords
- esophageal squamous
- apy29
- squamous carcinoma
- cells
- application
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- WJNBSTLIALIIEW-UHFFFAOYSA-N 2-n-(3h-benzimidazol-5-yl)-4-n-(5-cyclopropyl-1h-pyrazol-3-yl)pyrimidine-2,4-diamine Chemical compound C1CC1C1=NNC(NC=2N=C(NC=3C=C4N=CNC4=CC=3)N=CC=2)=C1 WJNBSTLIALIIEW-UHFFFAOYSA-N 0.000 title claims abstract description 48
- 206010041823 squamous cell carcinoma Diseases 0.000 title claims abstract description 39
- 239000003814 drug Substances 0.000 title claims abstract description 15
- 238000002360 preparation method Methods 0.000 title abstract description 5
- 238000001959 radiotherapy Methods 0.000 claims abstract description 15
- 238000013508 migration Methods 0.000 claims abstract description 8
- 230000006907 apoptotic process Effects 0.000 claims abstract description 6
- 230000004709 cell invasion Effects 0.000 claims description 3
- 230000012292 cell migration Effects 0.000 claims description 3
- 230000004663 cell proliferation Effects 0.000 claims description 2
- 101001051723 Homo sapiens Ribosomal protein S6 kinase alpha-6 Proteins 0.000 abstract description 13
- 102100024897 Ribosomal protein S6 kinase alpha-6 Human genes 0.000 abstract description 13
- 238000011282 treatment Methods 0.000 abstract description 8
- 230000005012 migration Effects 0.000 abstract description 6
- 230000009545 invasion Effects 0.000 abstract description 5
- 230000035755 proliferation Effects 0.000 abstract description 5
- 229940079593 drug Drugs 0.000 abstract description 4
- 238000004393 prognosis Methods 0.000 abstract description 3
- 238000011160 research Methods 0.000 abstract description 3
- 210000004027 cell Anatomy 0.000 description 39
- 208000036765 Squamous cell carcinoma of the esophagus Diseases 0.000 description 13
- 208000007276 esophageal squamous cell carcinoma Diseases 0.000 description 13
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 9
- 239000002609 medium Substances 0.000 description 9
- 239000000758 substrate Substances 0.000 description 8
- 238000001514 detection method Methods 0.000 description 7
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 6
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 6
- 238000002474 experimental method Methods 0.000 description 6
- 239000003153 chemical reaction reagent Substances 0.000 description 5
- 230000000694 effects Effects 0.000 description 5
- 230000005764 inhibitory process Effects 0.000 description 5
- 239000000243 solution Substances 0.000 description 5
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 5
- 108091000080 Phosphotransferase Proteins 0.000 description 4
- 238000011534 incubation Methods 0.000 description 4
- 108010082117 matrigel Proteins 0.000 description 4
- 239000012528 membrane Substances 0.000 description 4
- 230000004048 modification Effects 0.000 description 4
- 238000012986 modification Methods 0.000 description 4
- 102000020233 phosphotransferase Human genes 0.000 description 4
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 3
- 238000002738 Giemsa staining Methods 0.000 description 3
- 206010061534 Oesophageal squamous cell carcinoma Diseases 0.000 description 3
- 239000006285 cell suspension Substances 0.000 description 3
- 150000001875 compounds Chemical class 0.000 description 3
- 238000012258 culturing Methods 0.000 description 3
- 238000010790 dilution Methods 0.000 description 3
- 239000012895 dilution Substances 0.000 description 3
- 239000012091 fetal bovine serum Substances 0.000 description 3
- 239000011148 porous material Substances 0.000 description 3
- 229920000742 Cotton Polymers 0.000 description 2
- 102000001301 EGF receptor Human genes 0.000 description 2
- 108060006698 EGF receptor Proteins 0.000 description 2
- 102100030013 Endoribonuclease Human genes 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- 208000000461 Esophageal Neoplasms Diseases 0.000 description 2
- 101001010783 Homo sapiens Endoribonuclease Proteins 0.000 description 2
- 206010030155 Oesophageal carcinoma Diseases 0.000 description 2
- 230000018199 S phase Effects 0.000 description 2
- 108010087230 Sincalide Proteins 0.000 description 2
- 238000002835 absorbance Methods 0.000 description 2
- 239000004480 active ingredient Substances 0.000 description 2
- 230000004075 alteration Effects 0.000 description 2
- 239000000872 buffer Substances 0.000 description 2
- 238000010609 cell counting kit-8 assay Methods 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 238000009826 distribution Methods 0.000 description 2
- 201000004101 esophageal cancer Diseases 0.000 description 2
- 239000000834 fixative Substances 0.000 description 2
- 238000000684 flow cytometry Methods 0.000 description 2
- 230000012010 growth Effects 0.000 description 2
- 239000001963 growth medium Substances 0.000 description 2
- 239000003112 inhibitor Substances 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 238000004020 luminiscence type Methods 0.000 description 2
- 239000011259 mixed solution Substances 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 230000000737 periodic effect Effects 0.000 description 2
- IZTQOLKUZKXIRV-YRVFCXMDSA-N sincalide Chemical compound C([C@@H](C(=O)N[C@@H](CCSC)C(=O)NCC(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC=1C=CC=CC=1)C(N)=O)NC(=O)[C@@H](N)CC(O)=O)C1=CC=C(OS(O)(=O)=O)C=C1 IZTQOLKUZKXIRV-YRVFCXMDSA-N 0.000 description 2
- 239000012192 staining solution Substances 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 230000001225 therapeutic effect Effects 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- 102400000888 Cholecystokinin-8 Human genes 0.000 description 1
- 101800005151 Cholecystokinin-8 Proteins 0.000 description 1
- 206010059866 Drug resistance Diseases 0.000 description 1
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 1
- 101001012157 Homo sapiens Receptor tyrosine-protein kinase erbB-2 Proteins 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 102100030086 Receptor tyrosine-protein kinase erbB-2 Human genes 0.000 description 1
- 108010083644 Ribonucleases Proteins 0.000 description 1
- 102000006382 Ribonucleases Human genes 0.000 description 1
- 102000005789 Vascular Endothelial Growth Factors Human genes 0.000 description 1
- 108010019530 Vascular Endothelial Growth Factors Proteins 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 238000007605 air drying Methods 0.000 description 1
- 239000002246 antineoplastic agent Substances 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 239000012131 assay buffer Substances 0.000 description 1
- 230000035578 autophosphorylation Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 230000010261 cell growth Effects 0.000 description 1
- 238000002737 cell proliferation kit Methods 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 238000010367 cloning Methods 0.000 description 1
- 230000005757 colony formation Effects 0.000 description 1
- 229940127089 cytotoxic agent Drugs 0.000 description 1
- 239000008367 deionised water Substances 0.000 description 1
- 229910021641 deionized water Inorganic materials 0.000 description 1
- 238000004043 dyeing Methods 0.000 description 1
- 102000052116 epidermal growth factor receptor activity proteins Human genes 0.000 description 1
- 108700015053 epidermal growth factor receptor activity proteins Proteins 0.000 description 1
- 239000003292 glue Substances 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 238000000021 kinase assay Methods 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 238000011242 molecular targeted therapy Methods 0.000 description 1
- YOHYSYJDKVYCJI-UHFFFAOYSA-N n-[3-[[6-[3-(trifluoromethyl)anilino]pyrimidin-4-yl]amino]phenyl]cyclopropanecarboxamide Chemical compound FC(F)(F)C1=CC=CC(NC=2N=CN=C(NC=3C=C(NC(=O)C4CC4)C=CC=3)C=2)=C1 YOHYSYJDKVYCJI-UHFFFAOYSA-N 0.000 description 1
- 230000001575 pathological effect Effects 0.000 description 1
- 230000026731 phosphorylation Effects 0.000 description 1
- 238000006366 phosphorylation reaction Methods 0.000 description 1
- 239000013641 positive control Substances 0.000 description 1
- 230000005855 radiation Effects 0.000 description 1
- 238000005201 scrubbing Methods 0.000 description 1
- 239000012679 serum free medium Substances 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- 239000008399 tap water Substances 0.000 description 1
- 235000020679 tap water Nutrition 0.000 description 1
- 238000002626 targeted therapy Methods 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 238000010257 thawing Methods 0.000 description 1
- 229940126585 therapeutic drug Drugs 0.000 description 1
- 238000002560 therapeutic procedure Methods 0.000 description 1
- 238000011277 treatment modality Methods 0.000 description 1
- 210000004881 tumor cell Anatomy 0.000 description 1
Classifications
-
- 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
- A61K31/506—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim not condensed and containing further heterocyclic rings
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K41/00—Medicinal preparations obtained by treating materials with wave energy or particle radiation ; Therapies using these preparations
- A61K41/0038—Radiosensitizing, i.e. administration of pharmaceutical agents that enhance the effect of radiotherapy
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
- A61P35/04—Antineoplastic agents specific for metastasis
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
本发明属于医药领域，具体涉及APY29在制备治疗食管鳞癌的药物中的应用。本发明的研究结果表明，APY29能够高效抑制RSK4酶的表达，且能够高效的促进食管鳞癌细胞凋亡，抑制食管鳞癌细胞的增殖、侵袭和迁移，还可减轻食管鳞癌细胞的放疗抵抗，具有作为食管鳞癌放疗增敏剂的潜力，该结果为食管鳞癌患者的治疗和预后提供了一种有效的技术手段。
Description
技术领域
本发明属于医药领域，具体涉及APY29在制备治疗食管鳞癌的药物中的应用。
背景技术
食管鳞癌(esophageal squamous cell carcinoma，ESCC)是食管癌最常见的组织学类型。尽管临床上有针对ESCC的多种治疗方法，但晚期ESCC的5年总生存率仅为15％。放射治疗是ESCC一种重要的治疗方式，特别是对那些手术不可切除的晚期食管癌患者。遗憾的是，肿瘤细胞的放疗抵抗会导致ESCC的复发和治疗失败，大多数患者在病理缓解后仍有复发，放疗还会引起多种副作用。由于放疗的局限性，研制开发针对ESCC的靶向治疗药物十分必要。
ESCC分子靶向治疗主要的靶点有表皮生长因子受体(epidermal growth factorreceptor，EGFR)、人表皮生长因子受体2(human epidermal factor，VEGF)等，但目前关于靶向治疗的研究仍处于起始阶段，ESCC精准的治疗靶点还不十分明确，耐药性的出现使正常剂量的靶向药物不再发挥应有的抑癌作用。因此，开发新的化疗药物对ESCC患者的治疗和预后具有重要意义。
APY29是一种I类IRE1α抑制剂，与IRE1α的ATP结合位点相结合，抑制其自我磷酸化(IC50＝280nM)、增强其RNase作用(EC50＝460nM)，目前尚无APY29在食管鳞癌中的报道。
发明内容
本发明的目的在于提供APY29的新用途。
第一方面，本发明提供APY29在制备治疗食管鳞癌的药物中的应用。
进一步的，所述APY29促进食管鳞癌细胞凋亡。
进一步的，所述APY29抑制食管鳞癌细胞增殖。
进一步的，所述APY29抑制食管鳞癌细胞侵袭和迁移。
第二方面，本发明提供一种治疗食管鳞癌的药物，所述药物包括有效剂量的APY29。
进一步的，APY29为所述药物的唯一有效成分或有效成分之一。
更进一步的，所述药物促进食管鳞癌细胞凋亡，抑制食管鳞癌细胞增殖、侵袭和迁移。
第三方面，本发明提供APY29在制备食管鳞癌放疗增敏剂中的应用。
第四方面，本发明提供一种食管鳞癌放疗增敏剂，所述食管鳞癌放疗增敏剂包括有效剂量的APY29。
进一步的，所述APY29抑制食管鳞癌细胞的放疗抵抗。
与现有技术相比，本发明具有如下有益效果：
本发明提供了APY29的新用途，本发明的研究结果表明，APY29能够高效抑制RSK4酶的表达，且能够高效的促进食管鳞癌细胞凋亡，抑制食管鳞癌细胞的增殖、侵袭和迁移，还可减轻食管鳞癌细胞的放疗抵抗，具有作为食管鳞癌放疗增敏剂的潜力，该结果为ESCC患者的治疗和预后提供了一种有效的技术手段。
附图说明
图1为APY29的化学结构式。
图2为APY29对RSK4酶的半抑制曲线。
图3为经APY29刺激的食管鳞癌细胞(TE-10)的周期分布。
图4为3μm APY29处理下的食管鳞癌细胞(TE-10)的生长曲线。
图5为APY29对食管鳞癌细胞(TE-10)侵袭和迁移的影响，其中，A为细胞吉姆萨染色图，B为A的定量统计结果。
图6为APY29对食管鳞癌细胞(TE-10)的放疗抵抗的作用。
具体实施方式
下面结合附图和具体实施例对本发明进行详细说明，但不应理解为本发明的限制。如未特殊说明，下述实施例中所用的技术手段为本领域技术人员所熟知的常规手段，下述实施例中所用的材料、试剂等，如无特殊说明，均可从商业途径得到。
本发明披露了化合物APY29的新用途，所述APY29的分子式为C17H16N8，CAS No.:1216665-49-4，化学结构式如图1所示。
实施例1：APY29对RSK4酶的抑制
1实验方法
本次实验利用ADP-Glo方法检测APY29在RSK4酶上的作APY29起始浓度为10μM，5倍梯度稀释，2个重复，6个浓度。
(1)冰上解冻RSK4酶，RSK Substrate，kinase assay buffer III(5×缓冲液)，DTT(0.1M)和ATP(10mM)，并且以上试剂在整个实验过程中需要一直放置在冰上。
(2)将5×的缓冲液用去离子水配置成1×的缓冲液，并在其中加入DTT，DTT在1×缓冲液中的浓度为50μM；
(3)将1μl/孔5×待测化合物加入到白色微孔板中，微孔板在离心机上1000转离心1分钟；
阳性对照孔(Pos.Ctrl)：1μl/孔化合物稀释溶剂；
空白对照孔(Blank)：1μl/孔1×缓冲液。
(4)RSK4酶完全解冻后，使用1×的缓冲液将RSK4酶稀释到1ng/μl，取2μl/孔加入到白色微孔板中，此时每孔中RSK4的酶量为2ng；空白对照孔加入2μl/孔1×缓冲液；此步骤请在冰上进行，加完后，微孔板在离心机上1000转离心1分钟；
(5)配制RSK Substrate/ATP混合液：
RSK Substrate/ATP混合液：取130μl的RSK Substrate(1mg/ml)加入3.25μl 5mMATP和127μl的2×缓冲液(注意此处是按比例稀释)，此时ATP浓度为62.5μM，RSK Substrate浓度为0.5mg/ml；此步骤请在冰上进行；
(6)取2μl/孔RSK Substrate/ATP的混合溶液到白色微孔板中，此时RSKSubstrate浓度为0.2mg/ml，ATP浓度为25μM，加完后微孔板1000转离心1分钟；
(7)离心完毕后，给微孔板贴上膜，压紧贴膜，在25℃中孵育1小时；
(8)将Promega的试剂盒中需要用的ADP-GloTM reagent和Kinase Detection相关试剂平衡到室温，并按照说明书将Kinase Detection buffer和Kinase DetectionSubstrate混合备用。
(9)结束孵育后，取5μl/孔ADP-GloTM reagent加入到白色微孔板中，微孔板1000转离心1分钟，25℃中孵育40分钟；
(10)结束孵育后，取Kinase Detection混合液10μl/孔加入到微孔板中，微孔板1000转离心1分钟，25℃中孵育30分钟；
(11)结束孵育后在读板器上进行化学发光(Luminescence)检测，读取发光值(RLU)；
(12)酶抑制率计算：
％Inhibition＝100-(RLU(Sample)-RLU(Blank))/(RLU(Pos.Ctrl)-RLU(Blank))×100％。
2实验结果
如图2所示，随着APY29浓度的升高，APY29对RSK4激酶活性的抑制作用也增强。采用GraphPad软件进行曲线拟合，得出IC50值为55.10nM。结果表明APY29能够有效抑制RSK4的磷酸化，抑制RSK4的激活，是有效的RSK4抑制剂。
实施例2：APY29对食管鳞癌细胞生长的调控
1实验方法
本次实验采用APY29刺激食管鳞癌细胞系(TE10)，CCK8检测APY29对食管鳞癌细胞增殖的影响，流式细胞术检测食管鳞癌细胞周期分布，Transwell检测APY29对食管鳞癌细胞迁移的影响，同时以不加药物作为空白对照(Black或Vector)。
1.1流式细胞术检测步骤：
(1)在TE10细胞状态良好的对数生长期进行试验，保证瓶细胞数量少为1x106个/ml。配制固定液比例为1×PBS:无水乙醇＝1:2，每瓶细胞至少1ml固定液。
(2)用4℃预冷的1×PBS洗两次，尽量吸净1×PBS后加入固定液重悬，上流式细胞仪进行检测。
(3)将结果数据整理并进行统计。
1.2CCK8检测步骤：
使用上海陶术生物科技有限公司的CCK-8细胞增殖试剂盒完成。
(1)96孔板接种细胞悬液，每孔100μL，每孔2,000个细胞。
(2)按照实验需要，进行培养或给与药物，处理适当时间。
(3)每孔加入CCK-8溶液10μL，37℃孵育。
(4)酶标仪选择450nm波长测定吸光度值。以时间和吸光度值绘制生长曲线。
1.3细胞迁移：
(1)将培养好的TE10细胞消化制备成单细胞悬液，用PBS洗一遍。
(2)对照组使用加有DMSO的DMEM培养基重悬至5×105个/ml，实验组使用3μMAPY29的DMEM培养基重悬至5×105个/ml。
(3)24孔板小室的下室加入500μl含10％胎牛血清的DMEM培养基，上室分别加入200μl实验组和对照组的细胞重悬液。
(4)常规培养24h。
(5)吸出小室内培养基，用无水甲醇固定小室细胞15min，PBS漂洗。
(6)24孔板中加入吉姆萨染色液，染色20min，使用PBS清洗。
(7)用棉签小心擦去膜内层细胞，将膜风干。
(8)显微镜下拍照，取5个高倍视野计数，取平均值。
1.4细胞侵袭：
(1)将冻存于-80℃冰箱中的BD Matrigel胶置于4℃过夜，解冻为液态。
(2)使用无血清培养基按照1：8稀释浓度稀释为50μg/ml Matrigel，充分混匀后，将60μl Matrigel胶加入到Transwell小室的上室。
(3)将培养好的TE10细胞消化制备成单细胞悬液，用PBS洗一遍。
(4)对照组使用加有DMSO的DMEM培养基重悬至5×105个/ml，实验组使用3μMAPY29的DMEM培养基重悬至5×105个/ml。
(5)在Transwell小室的下室加入500μl含10％胎牛血清的DMEM培养基，上室分别加入200μl实验组和对照组的细胞重悬液。
(6)将Transwell小室置于恒温细胞培养箱中培养24h。
(7)培养结束后，取出Transwell小室，使用蘸有PBS的棉签擦洗上室，去除Matrigel胶；
(8)将小室放入到甲醇中，固定20min；
(9)24孔板中加入吉姆萨染色液，染色20min，使用PBS清洗。
(10)显微镜下拍照，取5个高倍视野计数，取平均值。
2实验结果
如图3-5所示，与对照组相比，经APY29刺激的S期TE10细胞显著增多，提示APY29使TE10细胞发生S期阻滞，促进细胞凋亡，且TE10细胞的增殖、侵袭和迁移能力均受到显著的抑制作用。
实施例3：APY29对食管鳞癌细胞的放疗抵抗的作用
1实验方法
辐射克隆形成实验
(1)将TE10细胞置于含10％胎牛血清的1640培养基，实验组加3μMAPY29，对照组不加，将不同组细胞进行0Gy或8Gy剂量的X线照射后，正常培养24h。
(2)调整细胞浓度至1×103个/ml，取200μl接种至6cm培养皿，加10ml培养基。
(3)常规培养2周，出现肉眼可见集落形成时停止培养。
(4)弃去培养基，PBS洗涤2次。
(5)无水甲醇5ml固定15min，弃固定液。
(6)加入吉姆萨染液染色20min，自来水冲洗，空气干燥。
(7)拍照，并计数肉眼可见的克隆数，计算克隆形成率＝克隆数/接种细胞数。
2实验结果
如图6所示，APY29处理可减轻TE10细胞的放疗抵抗，该结果为食管鳞癌的治疗提供新的思路。
尽管已描述了本发明的优选实施例，但本领域内的技术人员一旦得知了基本创造性概念，则可对这些实施例作出另外的变更和修改。所以，所附权利要求意欲解释为包括优选实施例以及落入本发明范围的所有变更和修改。
显然，本领域的技术人员可以对本发明进行各种改动和变型而不脱离本发明的精神和范围。这样，倘若本发明的这些修改和变型属于本发明权利要求及其等同技术的范围之内，则本发明也意图包含这些改动和变型在内。
Claims (5)
1.APY29在制备治疗食管鳞癌的药物中的应用,其特征在于，所述APY29的分子式为C17H16N8，CAS号为1216665-49-4。
2.根据权利要求1所述的应用，其特征在于，所述APY29促进食管鳞癌细胞凋亡。
3.根据权利要求1所述的应用，其特征在于，所述APY29抑制食管鳞癌细胞增殖。
4.根据权利要求1所述的应用，其特征在于，所述APY29抑制食管鳞癌细胞侵袭和迁移。
5.APY29在制备食管鳞癌放疗增敏剂中的应用，其特征在于，所述APY29的分子式为C17H16N8，CAS号为1216665-49-4。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310726061.5A CN116531382B (zh) | 2023-06-19 | 2023-06-19 | Apy29在制备治疗食管鳞癌的药物中的应用 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310726061.5A CN116531382B (zh) | 2023-06-19 | 2023-06-19 | Apy29在制备治疗食管鳞癌的药物中的应用 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN116531382A CN116531382A (zh) | 2023-08-04 |
| CN116531382B true CN116531382B (zh) | 2023-10-10 |
Family
ID=87443787
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202310726061.5A Active CN116531382B (zh) | 2023-06-19 | 2023-06-19 | Apy29在制备治疗食管鳞癌的药物中的应用 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN116531382B (zh) |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106822905A (zh) * | 2017-03-08 | 2017-06-13 | 暨南大学 | 含Survivin抑制剂和IRE1抑制剂的药物及用途 |
| CN115969855A (zh) * | 2022-12-15 | 2023-04-18 | 中国人民解放军空军军医大学 | Apy29在制备rsk4激酶抑制剂中的用途 |
-
2023
- 2023-06-19 CN CN202310726061.5A patent/CN116531382B/zh active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106822905A (zh) * | 2017-03-08 | 2017-06-13 | 暨南大学 | 含Survivin抑制剂和IRE1抑制剂的药物及用途 |
| CN115969855A (zh) * | 2022-12-15 | 2023-04-18 | 中国人民解放军空军军医大学 | Apy29在制备rsk4激酶抑制剂中的用途 |
Also Published As
| Publication number | Publication date |
|---|---|
| CN116531382A (zh) | 2023-08-04 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Guo et al. | Breast cancer cell‐derived exosomal miR‐20a‐5p promotes the proliferation and differentiation of osteoclasts by targeting SRCIN1 | |
| Wu et al. | Retracted: effects of Glut1 gene silencing on proliferation, differentiation, and apoptosis of colorectal cancer cells by targeting the TGF‐β/PI3K‐AKT‐mTOR signaling pathway | |
| US11344601B2 (en) | Tumor microenvironment-related target TAK1 and application thereof in inhibition of tumor | |
| Wang et al. | Knockdown of TRIM47 inhibits breast cancer tumorigenesis and progression through the inactivation of PI3K/Akt pathway | |
| Wu et al. | Tumour necrosis factor‐α‐induced protein 8‐like 2 is a novel regulator of proliferation, migration, and invasion in human rectal adenocarcinoma cells | |
| Yun et al. | Hypoxia-induced PGC-1α regulates mitochondrial function and tumorigenesis of colorectal cancer cells | |
| Zhu et al. | Retracted: MicroRNA‐198 inhibition of HGF/c‐MET signaling pathway overcomes resistance to radiotherapy and induces apoptosis in human non–small‐cell lung cancer | |
| WO2024061385A1 (zh) | Selonsertib在制备治疗癌症的药物中的用途 | |
| Dong et al. | Presence and function of stress granules in atrial fibrillation | |
| Lu et al. | Downregulation of PDGF-D inhibits proliferation and invasion in breast cancer MDA-MB-231 cells | |
| Wang et al. | ERα36 gene silencing promotes tau protein phosphorylation, inhibits cell proliferation, and induces apoptosis in human neuroblastoma SH‐SY5Y cells | |
| CN116531382B (zh) | Apy29在制备治疗食管鳞癌的药物中的应用 | |
| CN112656808A (zh) | 一种肝素寡糖在制备抗肿瘤药物中的应用 | |
| Sipos et al. | Survival of HT29 Cancer Cells Is Affected by IGF1R Inhibition via Modulation of Self-DNA-Triggered TLR9 Signaling and the Autophagy Response | |
| Zhan et al. | SAR1A regulates the RhoA/YAP and autophagy signaling pathways to influence osteosarcoma invasion and metastasis | |
| Jiang et al. | Extracellular vesicles promotes liver metastasis of lung cancer by ALAHM increasing hepatocellular secretion of HGF | |
| Yu et al. | B4GALNT2 Gene Promotes Proliferation, and Invasiveness and Migration Abilities of Model Triple Negative Breast Cancer (TNBC) Cells by Interacting With HLA-B Protein | |
| CN116763792B (zh) | Hg-14-10-04在制备治疗食管鳞癌的药物中的应用 | |
| CN116898857A (zh) | RGB-286638 free base在制备治疗食管鳞癌的药物中的应用 | |
| CN116942671B (zh) | ALK inhibitor 2在制备治疗食管鳞癌的药物中的应用 | |
| CN116531385A (zh) | Czc-54252在制备治疗食管鳞癌的药物中的应用 | |
| Gungorduk et al. | Immunolocalization of ERK1/2 and p-AKT in normal endometrium, endometrial hyperplasia, and early and advanced stage endometrioid endometrial adenocancer and their prognostic significance in malignant group | |
| CN104958306B (zh) | 化合物Hu‑17单独或联合铂类药物在制备治疗卵巢癌药物中的应用 | |
| WO2020113409A1 (zh) | 酰基辅酶a∶胆固醇酰基转移酶-1在肝癌诊疗中的应用 | |
| Gendrau‐Sanclemente et al. | Ovarian cancer relies on the PDGFRβ–fibronectin axis for tumorsphere formation and metastatic spread |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |