ST1571对恶性多发性神经胶质母细胞瘤（GBM）的体外抑瘤作用

目的：研究酪氨酸激酶抑制剂(ST1571)对恶性胶质瘤细胞的肿瘤生物学调节作用，方法：用免疫组化法探查血小板源生长因子(PDGF)及其受体在T98G，U87MG，LNZ308，RG四种胶质瘤细胞的表达；MTT法用于检查ST1571／TMZ单独或联合应用对胶质瘤细胞的增殖抑制作用，ST1571诱导的细胞凋亡途径由流式细胞仪检测，免疫印迹杂交法用来探查ST1571抵抗细胞的蛋白激酶网络变化。结果：所用胶质瘤细胞均不同程度的表达PDGF及受体.ST1571与TMZ相互协同作用于胶质瘤细胞株T98G；101μmol／LST1571可部分抑制所测胶质瘤细胞增殖、可完全抑制其形成集落；高于101μmol／L浓度的ST1571可快速诱导胶质瘤细胞凋亡或坏死：胶质瘤细胞株T98G与ST1571长期共培养后，瘤细胞蛋白激酶网络有较大改变。结论：研究表明，PDGF受体酪氨酸激酶抑制剂ST1571有较强的抑制恶性胶质瘤细胞(GBM)增殖的作用并可能与其他化疗药物协同抗肿瘤。     

STI571对食管癌细胞株CE-48T和CE-81T的体外杀伤作用

背景与目的:酪氨酸激酶是细胞增殖和分化的介导者,有研究显示,其在食管癌的发生发展中起重要的促进作用。STI571为血小板衍生的生长因子受体β(platelet-derivedgrowthfactorreceptorβ,PDGFR-β)的酪氨酸激酶抑制剂;食管癌组织中可高表达PDGFR-β。本实验的目的就是检测靶向治疗新药STI571对食管癌细胞株的体外杀伤作用。方法:使用Westernblot分析检测两种细胞株CE-48T和CE-81T中PDGFR-α和PDGFR-β的表达,然后应用MTT法测出STI571对两种细胞的IC50值,以AnnexinV/PI染色检测细胞凋亡情况,再用Westernblot检测STI571处理前后p-PDGFR-β的表达。结果:CE-48T细胞表达PDGFR-β而不表达PDGFR-α;而CE-81T细胞既不表达PDGFR-β,也不表达PDGFR-α。STI571对PDGFR-β阳性的CE-48T细胞具有很强的抑制作用,其平均IC50值为(8.32±1.50)μmol/L,而对PDGFR-β阴性的CE-81T细胞的抑制作用较弱,其平均IC50值为(41.02±7.64)μmol/L。AnnexinV/PI染色检测表明以STI571以10μmol/L作用CE-48T细胞12h,细胞凋亡率为(52.43±5.30)%,增加作用时间和药物浓度不能增加凋亡率。使用STI571处理CE-48T细胞后,p-PDGFR-β的表达受到抑制,而对CE-81T细胞没有影响。结论:STI571可诱导PDGFR-β表达阳性食管癌细胞凋亡,在体外有明显的杀伤作用,p-PDGFR-β可能是STI571的主要作用靶点。     

microRNA-133b对胶质母细胞瘤细胞迁移和侵袭的影响

目的探讨microRNA(miR)-133b对胶质母细胞瘤(GBM)细胞的作用,并分析其作用的分子机制。方法应用实时逆转录聚合酶链反应(RT-PCR)检测20例人GBM标本及正常人脑胶质细胞系HEB中miR-133b的表达水平。Transwell侵袭实验评估miR-133b对GBM细胞迁移和侵袭的影响。Western blotting和荧光素酶报告实验来确定miR-133b的靶基因。结果与正常人脑胶质细胞系HEB比较,miR-133b在GBM标本中的相对表达量明显降低(1.0±0.17 vs.2.42±0.69,P<0.05);转染miR-133b mimic后GBM迁移和侵袭跨膜细胞数分别为66±17和60±14,均低于转染NC组(P<0.05);与转染NC组比较,转染miR-133b mimic后MMP-14蛋白表达水平明显下降(P<0.05)。荧光素酶报告基因分析实验进一步验证mir-133b靶向调节MMP-14。结论 miR-133b通过直接靶向调节MMP-14抑制GBM的迁移和侵袭能力,可作为GBM诊断和治疗的候选靶点。     

NT4p53(N15)Ant融合基因重组腺病毒的构建及其体外抑瘤作用

背景与目的:有研究发现P53氨基端15肽与穿膜肽(antennapedia,Ant)的融合肽能迅速穿透细胞膜,直接、快速引起乳腺癌、胰腺癌细胞坏死而非凋亡,但对正常细胞几乎没有毒性。本研究构建缺陷型腺病毒载体表达NT4p53(N15)Ant融合基因,研究其体外抑瘤作用。方法:应用Ad-EasyTM系统,在大肠杆菌同源重组,构建NT4p53(N15)Ant腺病毒表达载体,在HEK-293细胞内成功包装并鉴定后,感染肝癌细胞株HepG2,用倒置相差显微镜、透射电镜观察细胞形态学变化、四甲基偶氮唑蓝(MTT)比色实验及培养基中的乳酸脱氢酶(lactatedehydrogenase,LDH)测定研究其体外抑瘤作用。结果:Ad-NT4p53(N15)Ant对肝癌细胞株HepG2有明显抑制作用,48、72、96h细胞存活率分别为36.67%、20.47%、17.82%,与空病毒处理组比较差异有统计学意义(P<0.05);电镜观察到感染的HepG2细胞胞膜、核膜破坏,胞浆内出现囊泡状物质,染色质聚集。Ad-NT4p53(N15)Ant处理HepG2细胞24、48、72、96h时培养液中LDH释放分别为94、236、267、313U/L,较空病毒处理组明显增加,其差异有统计学意义(P<0.05)。结论:构建的腺病毒载体Ad-NT4p53(N15)Ant感染HepG2细胞后能抑制细胞增殖。     

人LI-LAK细胞和环磷酰胺对小鼠S_(180)肉瘤的体内外抗瘤作用

本文用人LI—LAK细胞作了对小鼠S_(180)肿瘤细胞的体外杀伤试验,并与环磷酰胺分别及协同对小鼠S_(180)荷瘤鼠体内抗瘤试验。结果表明:人LI—LAK细胞在体内外均有明显的抗小鼠S_(180)肿瘤的作用。经人LI—LAK细胞及环磷酰胺治疗,S_(180)荷瘤鼠存活期显著延长,部分可完全治愈。     

A Tyrosine-specific Protein Kinase Inhibitor, α-Cyano-3-ethoxy-4-hydroxy-5-phenylthiomethylcinnamamide, Blocks the Phosphorylation of Tyrosine Kinase Substrate in Intact Cells

Inhibition by α-cyano-3-ethoxy-4-hydroxy-5-phenylthiomethylcinnamamide (ST 638) of tyrosine-specific protein kinase was examined using epidermal growth factor (EGF)-treated A431 cells at the concentration of 25 to 100 μ M . ST 638 had negligible effects on the growth and morphology of A431 cells and on EGF binding to its receptor, and subsequent down-regulation of the receptor. ST 638 specifically inhibited EGF-induced phosphorylation of tyrosine residues of whole cell proteins in a dose-dependent manner without affecting the phosphorylation of serine and threonine residues. ST 638 greatly inhibited the EGF-induced phosphorylation of lipocortin I at 25 μ M , and yet had a negligible effect on the EGF-induced phosphorylation of EGF receptor. Neither the amount of [³⁵S]methionine-labeled lipocortin I nor the serine/threonine phosphorylation level of fodrin β-subunit was affected by the same concentration of ST 638. These results indicate that the phosphorylation of lipocortin I is not relevant to the transformation of A431 cells. In cell lines transformed by src or fgr oncogene encoding tyrosine kinase, ST 638 also inhibited phosphorylation of calpactin I (p36) without affecting that of the oncogene products. Two-dimensional polyacrylamide gel electrophoresis showed that ST 638 specifically inhibited the EGF-induced phosphorylation and dephosphorylation of cellular proteins in A431 cells.     

The Synergistic Inhibitory Effect of STI571 in Combination with Arsenic Trioxide (As2O3) on Multidrug-Resistant Leukemic Cells

OBJECTIVE To study the synergistic effect of STI571, an inhibitor of tyrosine kinase, in combination with arsenic trioxide As2O3 on a multidrug-resistant leukemia cell line expressing bcr-abl.METHODS The cytotoxic effect of STI571 alone or in combination with different concentrations of As2O3 on the bcr-abl and mdr1 -positive leukemia cell line, K562-n/VCR, was examined by the MTT method.RESULTS One μmol/L of STI571 alone had no significant cytotoxic effect on K562-n/VCR cells. However the cytotoxic effect increased markedly when combined with As2O3 at concentrations of 10-5, 10-6, 10-7 and 10-8 mol/L. The IC50 of K562-n/VCR cells in As2O3 group was 1.879 μmol/L, with. Upon addition of STI571, the IC50 decreased to 0.155 μmol/L resulting in a synergistic cytotoxic effect on K562-n/VCR ceils that was increased 12.1 times.CONCLUSION A combination of STI571 with As2O3 has a more powerful inhibitory effect on leukemia cells expressing positive bcr-abl and positive mdrl compared to the effect with As2O3 alone.     

Inhibition of abl Oncogene Tyrosine Kinase Induces Erythroid Differentiation of Human Myelogenous Leukemia K562 Cells

The human chronic myelogenous leukemia cell line K562 expresses a structurally altered c- abl protein with tyrosine kinase activity. Erythroid differentiation of K562 cells was induced by tyrosine kinase inhibitors, but not by other kinase inhibitors. Treatment of K562 cells with 5'd(TACTGGCCGCTG-AAGGGC)3', complementary to the second exon (codons 2 to 7) of c- abl mRNA, inhibited cell growth and induced benzidine-positive cells in a dose-dependent manner. However, exposure to the sense oligomer did not induce erythroid differentiation of the cells. These results suggest that inhibition of abl tyrosine kinase activity is closely related to induction of erythroid differentiation of K562 cells. A multidrug-resistant subline (K562R) was induced to undergo erythroid differentiation by tyrosine kinase inhibitors such as genistein or herbimycin A as effectively as the parent K562 cells were. Therefore, tyrosine kinase inhibitors might be useful as cancer chemotherapeutic agents against some multidrug-resistant leukemias having abnormally high activity of oncogene tyrosine kinase(s).     

MTT assays cannot be utilized to study the effects of STI571/Gleevec on the viability of solid tumor cell lines

Purpose  This study will determine whether MTT assays accurately assess the effect of STI571 (Gleevec; Abl kinase inhibitor) on the viability of cancer cells containing highly active Abl kinases. Methods  Growth kinetics, tritiated thymidine, fluorescent caspase, MTT, and Cell Titer Glo (CTG) assays were used to determine the effect of STI571 on growth, proliferation, apoptosis, and viability of melanoma and breast cancer cells. Results  STI571 inhibited growth and proliferation, and increased apoptosis. However, MTT assays indicated that STI571 increased cell viability. In contrast, STI571 induced a dose-dependent decrease in viability using CTG assays. Conclusions  Doses of STI571 (1–10 μM) required to inhibit endogenous Abl kinases interfere with the MTT assay, and therefore MTT cannot be used to determine the effect of STI571 on viability using these doses. Additionally, caution should be utilized when interpreting the results of MTT assays used to screen kinase inhibitors for anti-cancer activity, as drug effectiveness may be minimized.     

β-Hydroxyisovalerylshikonin Is a Novel and Potent Inhibitor of Protein Tyrosine Kinases

β-Hydroxyisovalerylshikonin (β-HIVS), a compound isolated from Lithospermium radix , most efficiently induced cell-death in two lines of lung cancer cells, namely, NCI-H522 and DMS114, whereas shikonin was effective against a wide variety of tumor cell lines. During our studies of the mechanism of action of β-HIVS on tumor cells, we found that this compound inhibited protein tyrosine kinase (PTK) activity. The tyrosine kinase activities of a receptor for EGF (EGFR) and v-Src were strongly inhibited and that of KDR/Flk–1 was weakly inhibited by β-HIVS. The inhibition by β-HIVS of the activities of EGFR and v-Src was much stronger than that by shikonin. The IC₅₀values of β-HIVS for EGFR and v-Src were approximately 0.7 μM and 1 μM , respectively. Moreover, the inhibition of v-Src by β-HIVS was non-competitive with respect to ATP. These results strongly suggest that the action of β-HIVS, as well as that of shikonin, involves the inhibition of PTK, and they also suggest the possibility of producing a novel group of PTK inhibitors based on shikonin as the parent compound.     

(Pre‐)Clinical Pharmacology and Activity of Pazopanib,a Novel Multikinase Angiogenesis Inhibitor

Pazopanib is a recently approved, novel tyrosine kinase inhibitor specifically designed to impair angiogenesis by abrogating vascular endothelial growth factor receptor 2 (VEGFR‐2) to exert its function. Pazopanib inhibits VEGF‐induced endothelial cell proliferation in vitro and angiogenesis in vivo and demonstrates antitumor activity in mouse models. Furthermore, the pazopanib concentration resulting in maximal inhibition of VEGFR‐2 phosphorylation in vivo was in line with the steady‐state concentration required to inhibit growth of tumor xenografts, suggesting that pazopanib's mechanism of action is indeed through VEGFR‐2 inhibition. In a phase I trial, a generally well‐tolerated dose was identified at which the majority of patients achieved pazopanib plasma concentrations above the concentration required for maximal in vivo inhibition of VEGFR‐2 phosphorylation in preclinical models. Administered as monotherapy, evidence of antitumor activity was observed in phase II studies in several tumor types, including soft tissue sarcoma, renal cell cancer (RCC), ovarian cancer, and non‐small cell lung cancer. Recently, the U.S. Food and Drug Administration granted approval for treatment with pazopanib in patients with RCC based on the longer progression‐free survival time observed with this agent in a placebo‐controlled, randomized trial. This review summarizes the preclinical and clinical pharmacokinetics and pharmacodynamics of pazopanib, as well as data on clinical activity, that ultimately resulted in its recent approval.     

Determination of drug synergism between the tyrosine kinase inhibitors NSC 680410 (adaphostin) and/or STI571 (imatinib mesylate,Gleevec) with cytotoxic drugs against human leukemia cell lines

The primary growth factor receptors involved in angiogenesis and lymphomagenesis can be grouped into the vascular endothelial growth factor (VEGF) receptors and related families. Inhibition of VEGF and other growth factors, including c-Abl, c-Kit, platelet-derived growth factor (PDGF), epidermal growth factor (EGF) and insulin-like growth factor (IGF), or their receptors containing tyrosine kinase domains by antiangiogenesis drugs disrupts cell survival signal transduction pathways and may contribute to the proapoptotic pathways in malignant cells. However, clinical trials suggest that signal transduction inhibitors have considerable antitumor activity when used as single agents only for a short time, most likely due to the development of drug resistance by the host or by the tumor cells. In order to prevent this problem and to augment their antitumor efficacy, these agents could be administered in combination with cytotoxic antineoplastic drugs. We hypothesized that the combination of the antiangiogenesis tyrosine kinase inhibitors with cytotoxic drugs would produce synergistic drug regimens. Two human T-lymphoblastic leukemia cell lines that express VEGF-R1, CEM/0 (wild-type, WT) and the drug-resistant clone CEM/ara-C/I/ASNase-0.5-2, were utilized in the drug combination studies. NSC 680410, a tyrosine kinase inhibitor given at 0.1 to 1 microM for 72 h, inhibited VEGF secretion and leukemic cell growth at 90% of vehicle-treated control cultures with an IC50 value of less than 1 microM. The cytotoxic drugs idarubicin (IDA), fludarabine (Fludara), and cytosine arabinoside (ara-C) were used for the various drug combinations. One-, two-, three-, and four-drug treatments were tested. Cell viability was documented by the MTT assay and photomicrographic estimation of apoptotic cells. Both the combination index (CI) and isobologram evaluations demonstrated strong synergism between these drugs and the tyrosine kinase inhibitor. NSC 680410 was highly synergistic with IDA, IDA + ara-C, and IDA + Fludara + ara-C, over the respective cytotoxic drug regimens at concentrations easily achieved in patient plasma. NSC 680410 potentiated the activity of IDA in both leukemia cell lines by 17.8- and 221.4-fold in the WT and drug-resistant line, respectively. The activity of NSC 680410 + IDA + ara-C was also potentiated by 58.8-fold in the WT line, and the activity of NSC 680410 + IDA + Fludara + ara-C by 2.4- and 6.47x10(6)-fold in the WT and drug-resistant lines, respectively. The results suggest that IDA was not needed for optimal synergistic activity in the CEM/0 cells, but IDA was a necessary component to obtain drug synergism in the drug-resistant clone. Similarly, STI571 (imatinib mesylate, Gleevec), the p210(bcr/abl) tyrosine kinase inhibitor, demonstrated synergism with Fludara + ara-C or IDA + ara-C. Most importantly STI571 showed synergism with NSC 680410, suggesting that these drugs inhibit different tyrosine kinase domains in human leukemia cells. Lastly, pretreatment of leukemic cells with NSC 680410 showed additivity with gamma radiation in comparison to either treatment modality alone. The data, taken together, suggest that by inhibiting the pro-survival signal transduction pathway (VEGF-R1) and DNA replication by cytotoxic drugs, leukemic cells undergo apoptosis in a synergistic manner. In conclusion, the combinations of antiangiogenesis and DNA-damaging cytotoxic drugs are highly synergistic regimens in both WT and drug-resistant leukemic cell lines and they should be examined further.