pyrazolo(1-5-a)pyrimidine and anthranilamide

It has previously been shown that anthranilamide-pyrazolo[1,5-a]pyrimidine conjugates activate p53 and cause apoptosis in cervical cancer cells such as HeLa and SiHa. Here we establish the role of these conjugates in activating p53 pathway by phosphorylation at Ser15, 20 and 46 residues and downregulate key oncogenic proteins such as MYCN and Mdm2 in IMR-32 neuroblastoma cells. Compounds decreased the proliferation rate of neuroblastoma cells such as IMR-32, Neuro-2a, SK-N-SH. Compound treatment resulted in G2/M cell cycle arrest. The expression of p53 dependent genes such as p21, Bax, caspases was increased with concomitant decrease of the survival proteins as well as anti-apoptotic proteins such as Akt1, E2F1 and Bcl2. In addition the expression of important microRNAs such as miR-34a, c, miR-200b, miR-107, miR-542-5p and miR-605 were significantly increased that eventually lead to the activation of apoptotic pathway. Our data revealed that conjugates of this nature cause cell cycle arrest and apoptosis in IMR-32 cells [MYCN (+) with intact wild-type p53] by activating p53 signalling and provides a lead for the development of anti-cancer therapeutics.

Topics: Antineoplastic Agents; Apoptosis; bcl-2-Associated X Protein; Cell Line, Tumor; Cyclin-Dependent Kinase Inhibitor p21; G2 Phase Cell Cycle Checkpoints; Gene Expression Regulation, Neoplastic; HeLa Cells; Humans; M Phase Cell Cycle Checkpoints; MicroRNAs; N-Myc Proto-Oncogene Protein; Neuroblastoma; Nuclear Proteins; Oncogene Proteins; ortho-Aminobenzoates; Proto-Oncogene Proteins c-mdm2; Purines; Pyrazoles; Pyrimidines; Roscovitine; Tumor Suppressor Protein p53

A library of new anthranilamide-pyrazolo[1,5-a]pyrimidine conjugates were designed, synthesized, and evaluated for their anticancer activity in cervical cancer cells such as HeLa and SiHa that possess low levels of p53. All 24 conjugates showed antiproliferative activity, while some of them exhibit significant cytotoxicity. In assays related to cell-cycle distribution, these conjugates induced G(2) /M arrest in HeLa cells and G(1) cell-cycle arrest in SiHa cells. Immunocytochemistry assays revealed that these compounds cause nuclear translocation of p53, thereby indicating the activation of p53. In cervical cancer cells, the p53 protein is degraded by E6 oncoprotein. Immunoblot and RT-PCR analyses proved the presence of mitochondria-mediated apoptosis with involvement p53 target genes such as BAX, Bcl2, and p21 (CDKI). Moreover, these compounds increased the phosphorylated forms of p53 and provide signals for apoptosis induction. Interestingly, one of the conjugates, (2-phenyl-7-(3,4,5-trimethoxyphenyl)pyrazolo[1,5-a]pyrimidin-5-yl)(4-(2-(thiophen-2-ylmethylamino)benzoyl)piperazin-1-yl)methanone, is the most promising candidate in this series and has the potential to be taken up for further detailed studies.

Topics: Antineoplastic Agents; Apoptosis; bcl-2-Associated X Protein; Caspase 3; Cell Cycle Checkpoints; Cell Line, Tumor; Cyclin-Dependent Kinase Inhibitor p21; HeLa Cells; Humans; K562 Cells; MCF-7 Cells; ortho-Aminobenzoates; Proto-Oncogene Proteins c-bcl-2; Pyrazoles; Pyrimidines; Tumor Suppressor Protein p53