Johnson Faye M, Saigal Babita, Tran Hai, Donato Nicholas J
Department of Thoracic/Head and Neck Medical Oncology, The University of Texas M. D. Anderson Cancer Center, Houston, TX 77030-4009, USA.
Clin Cancer Res. 2007 Jul 15;13(14):4233-44. doi: 10.1158/1078-0432.CCR-06-2981.
The Src family of kinases (SFKs) regulate multiple signal transduction cascades and influence proliferation, motility, survival, and angiogenesis. Dasatinib inhibits SFKs, which leads to cytotoxicity, cell cycle arrest, apoptosis, and decreased invasion of cancer cells. Signal transducer and activator of transcription 3 (STAT3) is a latent transcription factor that regulates survival and proliferation. Dasatinib results in rapid and durable inhibition of c-Src, whereas STAT3 undergoes only transient inactivation. We hypothesized that the reactivation of STAT3 after dasatinib treatment represents the engagement of a compensatory signal for cell survival that blocks the antitumor effects of SFK inhibition.
The effects of upstream inhibitors on STAT3 activation were assessed with western blotting and a quantitative bioplex phosphoprotein assay. We used the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay to determine the cytotoxicity and propidium iodine/annexin V staining with fluorescence-activated cell sorting (FACS) analysis to evaluate cell cycle change and apoptosis. The combination index was calculated by the Chou-Talalay equation. Cytokines were quantitated using a multiplexed, particle-based FACS analysis.
C-Src and several downstream molecules were rapidly and durably inhibited by dasatinib. However, STAT3 was reactivated by 24 h. The addition of JAK inhibitors during dasatinib incubation resulted in sustained inhibition of STAT3, although JAK activation by dasatinib was not shown. Combined SFK and JAK inhibition resulted in synergistic cytotoxicity due to increased apoptosis.
The reactivation of STAT3 during dasatinib treatment is caused by the engagement of a compensatory pathway that suppresses the antitumor effects of SFK inhibition and allows cancer cell survival. Abrogation of this pathway resulted in synergistic cytotoxicity. Given that STAT3 reactivation occurred in 14 of 15 solid tumor cell lines, dasatinib combined with Janus-activated kinase inhibitors may have widespread application in cancer treatment.
Src激酶家族（SFKs）调节多种信号转导级联反应，并影响细胞增殖、运动、存活及血管生成。达沙替尼可抑制SFKs，进而导致细胞毒性、细胞周期停滞、细胞凋亡以及癌细胞侵袭能力下降。信号转导及转录激活因子3（STAT3）是一种潜在的转录因子，可调节细胞存活和增殖。达沙替尼可迅速且持久地抑制c-Src，而STAT3仅经历短暂失活。我们推测，达沙替尼治疗后STAT3的重新激活代表了一种细胞存活补偿信号的参与，该信号阻断了SFK抑制的抗肿瘤作用。
采用蛋白质免疫印迹法和定量生物复合体磷酸化蛋白分析评估上游抑制剂对STAT3激活的影响。我们使用3-(4,5-二甲基噻唑-2-基)-2,5-二苯基四氮唑溴盐（MTT）法测定细胞毒性，并通过荧光激活细胞分选（FACS）分析进行碘化丙啶/膜联蛋白V染色，以评估细胞周期变化和细胞凋亡。采用Chou-Talalay方程计算联合指数。使用基于微粒的多重FACS分析对细胞因子进行定量。
达沙替尼可迅速且持久地抑制c-Src及多个下游分子。然而，STAT3在24小时后重新激活。在达沙替尼孵育期间添加JAK抑制剂可导致STAT3持续抑制，尽管未显示达沙替尼对JAK的激活作用。联合抑制SFK和JAK由于细胞凋亡增加而导致协同细胞毒性。
达沙替尼治疗期间STAT3的重新激活是由一种补偿途径的参与引起的，该途径抑制了SFK抑制的抗肿瘤作用并使癌细胞存活。消除该途径可导致协同细胞毒性。鉴于15种实体瘤细胞系中有14种出现STAT3重新激活，达沙替尼联合Janus激活激酶抑制剂可能在癌症治疗中有广泛应用。
