In the rapidly expanding era of cancer target therapy, regulators of apoptosis are emerging as attractive therapeutic targets. X-linked inhibitor of apoptosis (XIAP) is of specific interest owing to its characteristic overexpression in a wide variety of neoplasms, with a resultant survival advantage for tumor cells and treatment resistance. In this study, we examined three pyrazolo [3,4-d] pyridazine derivatives (PPDs) through molecular modeling and studied their modes of interaction with XIAP-BIR3 domain. PPD-1, which possessed the highest binding affinity with XIAP, was tested on A549 (lung cancer cell line); HCT-116 (colorectal carcinoma cell line); HEPG2 (liver carcinoma cell line), HFB4 (normal human skin melanocyte cell line) and WI-38 (human embryonic lung fibroblasts). In comparison to cisplatin as a positive control, PPD-1 yielded remarkable cytotoxicity on all cancer cell lines, with the highest anti-tumor activity on A549 and a favorable therapeutic ratio. Flow cytometry studies concluded that PPD-1 treatment induces Sub G1 and G2/M cell cycle arrest and apoptosis. The percentage of apoptotic cells in PPD-1 treated A549 cells was considerably higher than that in untreated cells (10.06% vs 0.57%, respectively). To further investigate the mechanism of induction of apoptosis by PPD-1, Real time-PCR was used to quantify the expression levels of key apoptotic regulators. Significant overexpression of the effector capsase-3, pro-apoptotic bax and tumor suppressor gene p53 were noted as compared to untreated cells (7.19 folds, 7.28 folds, and 5.08 folds, respectively). Moreover, PPD-1 inhibited the expression of the anti-apoptotic bcl-2 gene to 0.22 folds. These findings demonstrate that PPD-1 treatment disrupts the Bcl-2/BAX balance in lung cancer cell lines, leading to apoptosis induction possibly through intrinsic mitochondria-dependent pathway. These novel insights elucidate the mechanism of PPD-1 cytotoxicity in lung cancer cell lines and offer a promising therapeutic approach that needs further study.

在快速发展的癌症靶标治疗时代，细胞凋亡的调节剂正逐渐成为有吸引力的治疗靶标。X连锁的凋亡抑制剂（XIAP）由于其在多种肿瘤中的特征性过表达而具有特殊的意义，因此具有肿瘤细胞的生存优势和治疗耐药性。在这项研究中，我们研究了三种吡唑并[3,4- d哒嗪衍生物（PPDs）通过分子建模并研究了它们与XIAP-BIR3结构域的相互作用方式。在A549（肺癌细胞系）上测试了与XIAP具有最高结合亲和力的PPD-1；HCT-116（结肠直肠癌细胞系）；HEPG2（肝癌细胞系），HFB4（正常人皮肤黑色素细胞系）和WI-38（人胚胎肺成纤维细胞）。与顺铂作为阳性对照相比，PPD-1在所有癌细胞系上均产生了显着的细胞毒性，在A549上具有最高的抗肿瘤活性，并具有良好的治疗率。流式细胞术研究得出结论，PPD-1处理可诱导Sub G1和G2 / M细胞周期停滞和凋亡。PPD-1处理的A549细胞中凋亡细胞的百分比明显高于未处理的细胞（分别为10.06％对0.57％）。为了进一步研究PPD-1诱导凋亡的机制，使用实时荧光定量PCR来量化关键凋亡调节因子的表达水平。效应子的明显过表达与未处理的细胞相比，注意到了capsase-3，促凋亡的bax和肿瘤抑制基因p53（分别为7.19倍，7.28倍和5.08倍）。此外，PPD-1将抗凋亡bcl-2基因的表达抑制了0.22倍。这些发现表明，PPD-1处理破坏了肺癌细胞系中的Bcl-2 / BAX平衡，可能通过内在的线粒体依赖性途径导致凋亡的诱导。这些新颖的见解阐明了PPD-1在肺癌细胞系中的细胞毒性机制，并提供了有希望的治疗方法，需要进一步研究。