CN113855805A - 一种含cdk4/6抑制剂的抗癌组合物及其应用 - Google Patents
一种含cdk4/6抑制剂的抗癌组合物及其应用 Download PDFInfo
- Publication number
- CN113855805A CN113855805A CN202111137231.3A CN202111137231A CN113855805A CN 113855805 A CN113855805 A CN 113855805A CN 202111137231 A CN202111137231 A CN 202111137231A CN 113855805 A CN113855805 A CN 113855805A
- Authority
- CN
- China
- Prior art keywords
- inhibitor
- abemaciclib
- cdk4
- cells
- napabucasin
- 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.)
- Granted
Links
- 229940124297 CDK 4/6 inhibitor Drugs 0.000 title claims abstract description 18
- 230000001093 anti-cancer Effects 0.000 title claims abstract description 17
- 239000000203 mixture Substances 0.000 title claims abstract description 17
- 238000011282 treatment Methods 0.000 claims abstract description 17
- 108010017324 STAT3 Transcription Factor Proteins 0.000 claims abstract description 12
- 102000004495 STAT3 Transcription Factor Human genes 0.000 claims abstract description 12
- 239000003112 inhibitor Substances 0.000 claims abstract description 10
- 229950001573 abemaciclib Drugs 0.000 claims description 32
- UZWDCWONPYILKI-UHFFFAOYSA-N n-[5-[(4-ethylpiperazin-1-yl)methyl]pyridin-2-yl]-5-fluoro-4-(7-fluoro-2-methyl-3-propan-2-ylbenzimidazol-5-yl)pyrimidin-2-amine Chemical compound C1CN(CC)CCN1CC(C=N1)=CC=C1NC1=NC=C(F)C(C=2C=C3N(C(C)C)C(C)=NC3=C(F)C=2)=N1 UZWDCWONPYILKI-UHFFFAOYSA-N 0.000 claims description 32
- DPHUWDIXHNQOSY-UHFFFAOYSA-N napabucasin Chemical group O=C1C2=CC=CC=C2C(=O)C2=C1OC(C(=O)C)=C2 DPHUWDIXHNQOSY-UHFFFAOYSA-N 0.000 claims description 20
- 229950011456 napabucasin Drugs 0.000 claims description 20
- 206010058467 Lung neoplasm malignant Diseases 0.000 claims description 12
- 201000005202 lung cancer Diseases 0.000 claims description 12
- 208000020816 lung neoplasm Diseases 0.000 claims description 12
- 208000002154 non-small cell lung carcinoma Diseases 0.000 claims description 12
- 208000029729 tumor suppressor gene on chromosome 11 Diseases 0.000 claims description 12
- 239000002246 antineoplastic agent Substances 0.000 claims description 9
- 230000005012 migration Effects 0.000 claims description 7
- 238000013508 migration Methods 0.000 claims description 7
- 229940041181 antineoplastic drug Drugs 0.000 claims description 6
- 230000002401 inhibitory effect Effects 0.000 claims description 5
- 108010025464 Cyclin-Dependent Kinase 4 Proteins 0.000 claims description 4
- 102000013698 Cyclin-Dependent Kinase 6 Human genes 0.000 claims description 4
- 108010025468 Cyclin-Dependent Kinase 6 Proteins 0.000 claims description 4
- 102100036252 Cyclin-dependent kinase 4 Human genes 0.000 claims description 4
- PDGKHKMBHVFCMG-UHFFFAOYSA-N 2-[[5-(4-methylpiperazin-1-yl)pyridin-2-yl]amino]spiro[7,8-dihydropyrazino[5,6]pyrrolo[1,2-d]pyrimidine-9,1'-cyclohexane]-6-one Chemical compound C1CN(C)CCN1C(C=N1)=CC=C1NC1=NC=C(C=C2N3C4(CCCCC4)CNC2=O)C3=N1 PDGKHKMBHVFCMG-UHFFFAOYSA-N 0.000 claims description 3
- RHXHGRAEPCAFML-UHFFFAOYSA-N 7-cyclopentyl-n,n-dimethyl-2-[(5-piperazin-1-ylpyridin-2-yl)amino]pyrrolo[2,3-d]pyrimidine-6-carboxamide Chemical compound N1=C2N(C3CCCC3)C(C(=O)N(C)C)=CC2=CN=C1NC(N=C1)=CC=C1N1CCNCC1 RHXHGRAEPCAFML-UHFFFAOYSA-N 0.000 claims description 3
- 108050002653 Retinoblastoma protein Proteins 0.000 claims description 3
- 230000009545 invasion Effects 0.000 claims description 3
- 229960004390 palbociclib Drugs 0.000 claims description 3
- AHJRHEGDXFFMBM-UHFFFAOYSA-N palbociclib Chemical compound N1=C2N(C3CCCC3)C(=O)C(C(=O)C)=C(C)C2=CN=C1NC(N=C1)=CC=C1N1CCNCC1 AHJRHEGDXFFMBM-UHFFFAOYSA-N 0.000 claims description 3
- 230000035755 proliferation Effects 0.000 claims description 3
- 229950003687 ribociclib Drugs 0.000 claims description 3
- 229950007127 trilaciclib Drugs 0.000 claims description 3
- 239000004480 active ingredient Substances 0.000 claims description 2
- 230000026731 phosphorylation Effects 0.000 claims 1
- 238000006366 phosphorylation reaction Methods 0.000 claims 1
- 206010059866 Drug resistance Diseases 0.000 abstract description 7
- 230000000694 effects Effects 0.000 abstract description 7
- 230000002195 synergetic effect Effects 0.000 abstract description 7
- 206010028980 Neoplasm Diseases 0.000 abstract description 4
- 210000004027 cell Anatomy 0.000 description 37
- 238000002474 experimental method Methods 0.000 description 13
- 230000004663 cell proliferation Effects 0.000 description 7
- 239000003814 drug Substances 0.000 description 6
- XACKNLSZYYIACO-DJLDLDEBSA-N edoxudine Chemical compound O=C1NC(=O)C(CC)=CN1[C@@H]1O[C@H](CO)[C@@H](O)C1 XACKNLSZYYIACO-DJLDLDEBSA-N 0.000 description 5
- 210000004881 tumor cell Anatomy 0.000 description 5
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 4
- 239000012091 fetal bovine serum Substances 0.000 description 4
- 102000003910 Cyclin D Human genes 0.000 description 3
- 108090000259 Cyclin D Proteins 0.000 description 3
- 201000011510 cancer Diseases 0.000 description 3
- 230000012292 cell migration Effects 0.000 description 3
- 239000013078 crystal Substances 0.000 description 3
- 229940079593 drug Drugs 0.000 description 3
- 239000000890 drug combination Substances 0.000 description 3
- 230000035876 healing Effects 0.000 description 3
- 230000005764 inhibitory process Effects 0.000 description 3
- 238000001262 western blot Methods 0.000 description 3
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 2
- 102100031181 Glyceraldehyde-3-phosphate dehydrogenase Human genes 0.000 description 2
- 101001056180 Homo sapiens Induced myeloid leukemia cell differentiation protein Mcl-1 Proteins 0.000 description 2
- 102100026539 Induced myeloid leukemia cell differentiation protein Mcl-1 Human genes 0.000 description 2
- 239000002033 PVDF binder Substances 0.000 description 2
- 229930040373 Paraformaldehyde Natural products 0.000 description 2
- 230000009471 action Effects 0.000 description 2
- 238000001516 cell proliferation assay Methods 0.000 description 2
- 230000005757 colony formation Effects 0.000 description 2
- 238000011284 combination treatment Methods 0.000 description 2
- 239000007850 fluorescent dye Substances 0.000 description 2
- 108020004445 glyceraldehyde-3-phosphate dehydrogenase Proteins 0.000 description 2
- 239000001963 growth medium Substances 0.000 description 2
- 238000010348 incorporation Methods 0.000 description 2
- 239000012528 membrane Substances 0.000 description 2
- 238000010232 migration assay Methods 0.000 description 2
- 230000035772 mutation Effects 0.000 description 2
- 229920002866 paraformaldehyde Polymers 0.000 description 2
- 229920002981 polyvinylidene fluoride Polymers 0.000 description 2
- 239000003531 protein hydrolysate Substances 0.000 description 2
- 230000009822 protein phosphorylation Effects 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 108090000623 proteins and genes Proteins 0.000 description 2
- 230000019491 signal transduction Effects 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 238000002626 targeted therapy Methods 0.000 description 2
- RWQNBRDOKXIBIV-UHFFFAOYSA-N thymine Chemical compound CC1=CNC(=O)NC1=O RWQNBRDOKXIBIV-UHFFFAOYSA-N 0.000 description 2
- 206010065553 Bone marrow failure Diseases 0.000 description 1
- 206010006187 Breast cancer Diseases 0.000 description 1
- 108020004414 DNA Proteins 0.000 description 1
- 102000053602 DNA Human genes 0.000 description 1
- 108091000080 Phosphotransferase Proteins 0.000 description 1
- 206010041067 Small cell lung cancer Diseases 0.000 description 1
- IQFYYKKMVGJFEH-XLPZGREQSA-N Thymidine Chemical class O=C1NC(=O)C(C)=CN1[C@@H]1O[C@H](CO)[C@@H](O)C1 IQFYYKKMVGJFEH-XLPZGREQSA-N 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 230000008235 cell cycle pathway Effects 0.000 description 1
- 210000003855 cell nucleus Anatomy 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 239000005482 chemotactic factor Substances 0.000 description 1
- 238000002512 chemotherapy Methods 0.000 description 1
- 230000003021 clonogenic effect Effects 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 239000002875 cyclin dependent kinase inhibitor Substances 0.000 description 1
- 229940043378 cyclin-dependent kinase inhibitor Drugs 0.000 description 1
- 238000012217 deletion Methods 0.000 description 1
- 230000037430 deletion Effects 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 208000035475 disorder Diseases 0.000 description 1
- 238000003384 imaging method Methods 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 238000002372 labelling Methods 0.000 description 1
- 230000036210 malignancy Effects 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 238000000034 method Methods 0.000 description 1
- 230000001617 migratory effect Effects 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 235000021049 nutrient content Nutrition 0.000 description 1
- 230000037361 pathway Effects 0.000 description 1
- 230000000149 penetrating effect Effects 0.000 description 1
- 239000008363 phosphate buffer Substances 0.000 description 1
- 102000020233 phosphotransferase Human genes 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 230000010076 replication Effects 0.000 description 1
- 230000003362 replicative effect Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 230000008261 resistance mechanism Effects 0.000 description 1
- 238000007789 sealing Methods 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 239000002356 single layer Substances 0.000 description 1
- 235000020183 skimmed milk Nutrition 0.000 description 1
- 208000000587 small cell lung carcinoma Diseases 0.000 description 1
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 229940113082 thymine Drugs 0.000 description 1
- 230000035899 viability Effects 0.000 description 1
- 239000012130 whole-cell lysate Substances 0.000 description 1
Images
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
- A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
-
- 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
- A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
- A61K31/34—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having five-membered rings with one oxygen as the only ring hetero atom, e.g. isosorbide
- A61K31/343—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having five-membered rings with one oxygen as the only ring hetero atom, e.g. isosorbide condensed with a carbocyclic ring, e.g. coumaran, bufuralol, befunolol, clobenfurol, amiodarone
-
- 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
- A61K31/496—Non-condensed piperazines containing further heterocyclic rings, e.g. rifampin, thiothixene or sparfloxacin
-
- 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
- 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
- A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
-
- 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
Landscapes
- Health & Medical Sciences (AREA)
- Veterinary Medicine (AREA)
- Chemical & Material Sciences (AREA)
- Medicinal Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Life Sciences & Earth Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- General Health & Medical Sciences (AREA)
- Public Health (AREA)
- Epidemiology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Organic Chemistry (AREA)
- Oncology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
本发明公开了一种含CDK4/6抑制剂抗癌组合物及其应用，该抗癌组合物由CDK4/6抑制剂和STAT3抑制剂组成，采用本发明的组合物，两者可产生协同作用，从而克服靶向抑制剂的耐药并提高临床疗效，为肿瘤精准治疗开启新窗口。
Description
技术领域
本发明属于生物医药领域，具体涉及一种含CDK4/6抑制剂的抗癌组合物及其应用。
背景技术
肺癌是是全球发病率和癌症相关死亡率最高的恶性肿瘤，其中非小细胞肺癌(Non-Small Cell Lung Cancer,NSCLC)约占85％。近年来肺癌治疗取得重要进展，特别是靶向治疗带来革命性突破，但原发或继发性耐药问题已成为限制其临床疗效的瓶颈。因此探究肺癌靶向治疗耐药机制并寻求克服靶向治疗耐药的新策略已成为现阶段研究领域的热点。
细胞周期通路紊乱失调常见于在肺癌等恶性肿瘤。部分肺癌患者存在细胞周期蛋白D(Cyclin D)和细胞周期依赖激酶(Cyclin D-Dependence Kinase,CDK)4/6扩增、点突变或CDK抑制因子缺失、点突变和多重改变等，为CDK4/6抑制剂的肺癌治疗提供理论依据。目前已有一种CDK4/6抑制剂Trilaciclib获批应用于降低广泛期小细胞肺癌患者在接受某些类型化疗时出现的骨髓抑制频率。Palbociclib、Ribociclib、Abemaciclib三种CDK4/6抑制剂已应用于ER+/HER2-晚期乳腺癌的临床治疗，治疗效用较好，但治疗后期不可避免地出现耐药问题。
发明内容
本发明提供了一种含CDK4/6抑制剂抗癌组合物及其应用，该抗癌组合物可有效避免现有的CDK4/6抑制剂的耐药问题。
本发明的技术方案如下：
一种抗癌组合物，由CDK4/6抑制剂和STAT3抑制剂组成。
作为优选，所述的CDK4/6抑制剂为Trilaciclib、Palbociclib、Ribociclib和Abemaciclib中的一种或者多种。
作为优选，所述的STAT3抑制剂为Napabucasin。
本发明还提供了一种所述的抗癌组合物在制备抗癌药物中的应用，所述的抗癌药物以所述的抗癌组合物作为活性成分。
作为优选，所述的抗癌药物用于治疗肺癌，作为进一步的优选，所述的抗癌药物用于治疗非小细胞肺癌。
作为优选，所述的抗癌药物用于抑制肺癌细胞的增殖，抑制肺癌细胞的侵袭迁移，降低RB蛋白磷酸化以及CYCLIND1、CDK4、CDK6蛋白的表达水平。
作为优选，所述的CDK4/6抑制剂Abemaciclib，所述的STAT3抑制剂为Napabucasin。作为进一步的优选，所述的Abemaciclib与所述的Napabucasin的摩尔比为0.25～0.75：0.05～0.2；作为更进一步的优选，所述的Abemaciclib的浓度为0.25～0.75μM，所述的Napabucasin的浓度为0.05～0.2μM，作为进一步的优选，所述的抗癌药物抑制H1975、PC9细胞的增殖。
同现有技术相比，本发明的有益效果体现在：
采用本发明的组合物，可克服靶向抑制剂的耐药并提高临床疗效，为肿瘤精准治疗开启新窗口。
附图说明
图1为实施例1中Abemaciclib处理后的NSCLC细胞STAT3信号通路激活结果；
图2为实施例2中Abemaciclib和Napabucasin联合抑制NSCLC细胞增殖结果；
图3为实施例3中Abemaciclib和Napabucasin联合抑制NSCLC细胞侵袭迁移结果；
图4为实施例4中Abemaciclib和Napabucasin联合抑制RB通路的结果。
具体实施方式
下面结合具体实施例对本发明做进一步的描述。
实施例1 Western blot实验
本实施例通过Western blot实验分析CDK4/6抑制剂Abemaciclib对肿瘤细胞蛋白表达的影响。在6孔板中铺入NSCLC细胞H1975，待细胞贴壁后加入不同浓度(0，0.5μM，1μM，2μM)的Abemaciclib；药物作用72h后，吸弃上清液，加入细胞蛋白裂解液提取全细胞蛋白裂解液，配制蛋白上样体系；将上样体系在10％SDS-PAGE凝胶中电泳分离，后转移至PVDF膜上，PVDF膜应用5％脱脂牛奶中封闭1h，在4℃与相应一抗PSTAT3、STAT3、MCL-1、GAPDH孵育过夜；使用相应二抗标记一抗后进行成像，结果如图1所示。结果显示：Abemaciclib处理72h后，H1975细胞中P-STAT3、STAT3、MCL-1蛋白表达明显升高。结果提示Abemaciclib处理反馈激活STAT3信号通路。
实施例2 Abemaciclib联合Napabucasin的细胞增殖实验
本实施例通过细胞增殖实验(MTT实验、EdU掺入实验、集落形成实验)，进一步研究CDK4/6抑制剂Abemaciclib联合STAT3抑制剂Napabucasin对NSCLC细胞增殖的影响。
MTT实验：在96孔板中铺入细胞(2000个/孔)，待细胞贴壁后分别加入不同浓度的Abemaciclib(0，0.25μM，0.5μM，0.75μM)和Napabucasin(0，0.05μM，0.1μM，0.2μM)，药物作用72h后加入5mg/mL MTT溶液(25μL/孔)。避光孵育4h后种植培养，小心吸弃孔内上清液，加入二甲基亚砜溶液(150ul/孔)溶解结晶，应用酶标仪在490nm波长上测定各孔吸光度，计算联合指数(Combination Index,CI)，结果如图2A所示。联合指数(CI)可判断两者的相互作用，当0.9≤CI≤1.1，表示两者为叠加作用；当0.8≤CI＜0.9，表示两者为低度协同作用；当0.6≤CI＜0.8，表示两者为中度协同作用；当0.4≤CI＜0.6，表示两者为高度协同作用；当0.2≤CI＜0.4，表示两者为强协同作用。结果显示，当采用不同浓度的Abemaciclib和Napabucasin进行联合处理时，表现出不同的协同作用。针对H1975细胞，Abemaciclib和Napabucasin药物联合指数为0.101-0.286，两者为强协同作用；针对PC9细胞，药物联合指数为0.52-0.61，两者为高度(中度)协同作用。由图2A可知，H1975细胞的药物联合指数如下表：
| Abemaciclib(μM) | 0.25 | 0.25 | 0.25 | 0.5 | 0.5 | 0.5 | 0.75 | 0.75 | 0.75 |
| Napabucasin(μM) | 0.05 | 0.1 | 0.2 | 0.05 | 0.1 | 0.2 | 0.05 | 0.1 | 0.2 |
| CI | 0.210 | 0.213 | 0.110 | 0.248 | 0.281 | 0.105 | 0.286 | 0.280 | 0.101 |
由图2B可知，PC9细胞的药物联合指数如下表：
| Abemaciclib(μM) | 0.25 | 0.25 | 0.25 | 0.5 | 0.5 | 0.5 | 0.75 | 0.75 | 0.75 |
| Napabucasin(μM) | 0.05 | 0.1 | 0.2 | 0.05 | 0.1 | 0.2 | 0.05 | 0.1 | 0.2 |
| CI | 0.53 | 0.54 | 0.59 | 0.52 | 0.54 | 0.58 | 0.56 | 0.6 | 0.61 |
EdU掺入实验：EdU(5-Ethynyl-2’-deoxyuridine)是一种胸腺嘧啶核苷类似物，能够在细胞增殖时期代替胸腺嘧啶(T)渗入正在复制的DNA分子中，通过观察细胞核荧光染色情况判断DNA复制活性，示踪细胞增殖情况。在24孔板中铺入细胞(2×104个/孔)，待细胞贴壁后分别加入0.5μM Abemaciclib和0.3μM Napabucasin，培养24h后，根据EdU试剂盒操作手册进行操作，并在荧光显微镜下观察细胞核荧光染色情况。EdU细胞增殖检测提示Abemaciclib联合Napabucasin可以增强对H1975、PC9细胞增殖的抑制(图2B)。
克隆形成实验：肿瘤细胞具有独立生存能力，形成细胞集落，通过集落形成实验可以了解细胞增殖情况。在6孔板中铺入H1975、PC9细胞(800-1000个/孔)，待细胞贴壁后分别加入0.25μM Abemaciclib和0.2μMNapabucasin，作用24h后撤去药物，加入新鲜培养基继续培养1周，待细胞形成肉眼可见的集落；用4％多聚甲醛固定后应用结晶紫溶液染色，比较各处理组集落的大小和数量，结果如图2C所示。结果显示，联合处理明显抑制NSCLC细胞的克隆形成数量和大小。
实施例3 Abemaciclib联合Napabucasin的细胞迁移实验
本实施例通过划痕愈合和Transwell实验进一步分析Abemaciclib和Napabucasin联合处理对NSCLC细胞迁移能力的影响。
划痕愈合实验：划痕愈合实验模拟了细胞在体内的迁移过程，可反映细胞的迁移能力。当细胞生长融合成单层状态时，人为创建“划痕”，划痕边缘的细胞会逐渐进入空白区域使“划痕”愈合，在划痕创建开始和定期捕获图像以比较空白区域的大小，细胞迁移速率。在6孔板中铺入PC9细胞(1×106个/孔)，待细胞融合至80-90％时，用200ul黄枪头划出划痕，用磷酸盐缓冲液洗去漂浮细胞后加入含2％FBS的新鲜血清以及0.25μMAbemaciclib和0.2μM Napabucasin，于药物作用0h、36h分别显微镜镜下拍摄划痕区域，结果如图3A所示。结果显示联合处理明显抑制了PC9细胞的迁移能力。
Transwell实验：在Transwell小室的上室铺入肿瘤细胞，下室加入胎牛血清(fatal bovine serum,FBS)或某些特定的趋化因子，肿瘤细胞会向营养成分高的下室移动，通过计数进入下室的细胞数量可反映肿瘤细胞的迁移能力。在Transwell小室的上室铺入细胞(2×104个/孔)，上、下室均为无血清基础培养基，待细胞饥饿24h后，在上室分别加入0.25μMAbemaciclib和0.2μM Napabucasin，下室替换为含10％FBS的新鲜培养基；药物作用24h后培养基，用4％多聚甲醛固定，并用结晶紫溶液染色，比较各处理组下室的细胞数量，结果如图3B所示。H1975细胞的Transwell实验结果进一步支持联合处理抑制细胞迁移能力。
实施例4 Abemaciclib联合Napabucasin的机制研究
本实施例通过Western blot实验进一步探究了Abemaciclib和Napabucasin联合可能的机制。在6孔板中铺入NSCLC细胞H1975、PC9，待细胞贴壁后分别加入0.5μMAbemaciclib和0.3μM Napabucasin，收集全细胞蛋白裂解液，具体操作见上述，用p-RB、RB、CYCLIND1、CDK4、CDK6、GAPDH一抗及相应二抗标记并成像，结果如图4所示。结果显示，Abemaciclib和Napabucasin联合可进一步降低RB蛋白磷酸化以及CYCLIND1、CDK4、CDK6蛋白的表达水平(图4)。
Claims (10)
1.一种抗癌组合物，其特征在于，由CDK4/6抑制剂和STAT3抑制剂组成。
2.根据权利要求1所述的抗癌组合物，其特征在于，所述的CDK4/6抑制剂为Trilaciclib、Palbociclib、Ribociclib和Abemaciclib中的一种或者多种。
3.根据权利要求1所述的抗癌组合物，其特征在于，所述的STAT3抑制剂为Napabucasin。
4.一种如权利要求1～3任一项所述的抗癌组合物在制备抗癌药物中的应用，其特征在于，所述的抗癌药物以所述的抗癌组合物作为活性成分。
5.根据权利要求4所述的应用，其特征在于，所述的抗癌药物用于治疗肺癌。
6.根据权利要求4所述的应用，其特征在于，所述的抗癌药物用于治疗非小细胞肺癌。
7.根据权利要求4所述的应用，其特征在于，所述的抗癌药物用于抑制肺癌细胞的增殖，抑制肺癌细胞的侵袭迁移，降低RB蛋白磷酸化以及CYCLIND1、CDK4、CDK6蛋白的表达水平。
8.根据权利要求4所述的应用，其特征在于，所述的CDK4/6抑制剂Abemaciclib，所述的STAT3抑制剂为Napabucasin。
9.根据权利要求8所述的应用，其特征在于，所述的Abemaciclib与所述的Napabucasin的摩尔比为0.25～0.75：0.05～0.2。
10.根据权利要求8所述的应用，其特征在于，所述的Abemaciclib的浓度为0.25～0.75μM，所述的Napabucasin的浓度为0.05～0.2μM。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111137231.3A CN113855805B (zh) | 2021-09-27 | 2021-09-27 | 一种含cdk4/6抑制剂的抗癌组合物及其应用 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111137231.3A CN113855805B (zh) | 2021-09-27 | 2021-09-27 | 一种含cdk4/6抑制剂的抗癌组合物及其应用 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN113855805A true CN113855805A (zh) | 2021-12-31 |
| CN113855805B CN113855805B (zh) | 2023-09-19 |
Family
ID=78991320
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202111137231.3A Active CN113855805B (zh) | 2021-09-27 | 2021-09-27 | 一种含cdk4/6抑制剂的抗癌组合物及其应用 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN113855805B (zh) |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2019200224A1 (en) * | 2018-04-13 | 2019-10-17 | The Broad Institute, Inc. | Synergistic drug combinations predicted from genomic features and single-agent response profiles |
-
2021
- 2021-09-27 CN CN202111137231.3A patent/CN113855805B/zh active Active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2019200224A1 (en) * | 2018-04-13 | 2019-10-17 | The Broad Institute, Inc. | Synergistic drug combinations predicted from genomic features and single-agent response profiles |
Non-Patent Citations (4)
| Title |
|---|
| KETTNER N. M. 等: ""Combined inhibition of STAT-3 & DNA repair in palbociclib resistant breast cancer"", 《CANCER RESEARCH》 * |
| LI CHUNGEN 等: ""A novel series of napabucasin derivatives as orally active inhibitors of signal transducer and activator of transcription 3 (STAT3)"", 《EUROPEAN JOURNAL OF MEDICINAL CHEMISTRY》 * |
| 刘子齐 等: ""CDK4/6 信号通路靶向治疗剂在癌症治疗中的研究进展"", 《生物工程学报》 * |
| 秦琼 等: "CDK4/6抑制剂在非小细胞肺癌治疗中的研究进展", 《中国肺癌杂志》 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN113855805B (zh) | 2023-09-19 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Ottaiano et al. | Inhibitory effects of anti-CXCR4 antibodies on human colon cancer cells | |
| Sun et al. | Resveratrol suppresses the growth and metastatic potential of cervical cancer by inhibiting STAT3Tyr705 phosphorylation | |
| Hui et al. | Radiosensitization by inhibiting STAT1 in renal cell carcinoma | |
| Deng et al. | Long non-coding RNA HOTTIP promotes tumor growth and inhibits cell apoptosis in lung cancer | |
| CN105001168A (zh) | 三烷氧基取代的苯并喹唑啉类酪氨酸激酶抑制剂及其用途 | |
| CN108913662A (zh) | 一种肺癌功能性高通量用药筛选的方法 | |
| Lee et al. | Regulatory effects of Siegesbeckia glabrescens on non-small cell lung cancer cell proliferation and invasion | |
| CN113855805A (zh) | 一种含cdk4/6抑制剂的抗癌组合物及其应用 | |
| CN115252622B (zh) | 醛糖还原酶阻化剂及其在制备用于治疗肺癌的药物中的应用 | |
| CN106265632B (zh) | 青蒿素 b抑制a549细胞迁移侵袭的作用 | |
| US20210263039A1 (en) | Application of niemann-pick c1 protein in diagnosis and treatment of cancer | |
| Haskell et al. | Comparative survival in tissue culture of normal and neoplastic human cells exposed to adriamycin | |
| CN112386587B (zh) | 石斛酚在制备早期膀胱癌治疗药物中的用途及分子标记 | |
| CN114949226A (zh) | B7-h3基因抑制剂在制备治疗非小细胞肺癌药物中的应用 | |
| CN112336865A (zh) | 吡硫锌在肺癌治疗中的应用 | |
| CN112206238A (zh) | 金诺芬在肺癌治疗中的应用 | |
| CN112438980A (zh) | 米氮平与甲氨蝶呤联合用药在制备治疗肝癌药物中的应用 | |
| CN109498618A (zh) | 一种抑制卵巢癌细胞的药物组合物 | |
| CN116270617B (zh) | Arp2/3复合物抑制剂CK-666和多西他赛组合用于癌症治疗的制药用途 | |
| CN108434133A (zh) | 一种小白菊内酯用作骨形成蛋白9抑制剂的生物医药用途 | |
| Valerlote et al. | Growth characteristics of MOPC-315 plasmacytoma and response to anticancer agents | |
| Cheng et al. | Reducing expression of dynamin-related protein 1 increases radiation sensitivity of glioblastoma cells | |
| CN116942680A (zh) | 一种治疗耐药的非小细胞肺癌的药物组合物 | |
| CN114366740B (zh) | 化合物a-6在制备广谱抗癌药物中的应用 | |
| Yang | Targeting Glutamine/Signal Transducer and Activator of Transcription 3 Axis in Pancreatic Ductal Adenocarcinoma |
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 |