Abstract:
Colorectal cancer (CRC) is one of the leading causes of cancer death in the world. Deregulation of cyclin- dependent kinases (CDKs) is often observed in CRC making them attractive drug targets. We investigated the efficacy of three multitarget CDK inhibitors viz. riviciclib (also known as P276-00) roscovitine and UCN-01 with varied specificities for CDKs against human CRC cell lines of different genetic background viz. Colo-205, HCT116 and HCT-15. Among the three drugs, UCN-01 was found to be the most potent followed by rivicilcib. Roscovitine was the least potent across these cell lines. Nonetheless, all these drugs exerted cytotoxicity and inhibited clonogenic potential of CRC cells. Cell cycle studies using flow cytometry revealed that these drugs abrogated cell cycle progression and lead to accumulation of cells in the sub G1 phase indicating increased apoptosis. Further, apoptosis was also confirmed by annexin V/PI, Terminal deoxynucleotidyl transferase–mediated nick end labeling (TUNEL) and caspase-3/7 glo assays. Protein expression analysis demonstrated that these drugs downregulated expression of their respective target CDKs and cyclins as well as their activity and also modulated expression of caspases and pro- and anti-apoptotic proteins. Further, riviciclib, roscovitine and UCN-01 were found to inhibit RNA polymerase II carboxy- temrinal domain (CTD) phosphorylation at Ser 2/5 thus inhibiting transcription via downregulation of CDK9/cyclin T1 and CDK7/cyclin H (i.e. transcription-regulatory CDK/cyclin complexes) activity. This lead to rapid downregulation of short-lived anti-apoptotic protein Mcl-1 which is implicated in not only cell survival but also drug resistance of CRC cells. Hypoxia-inducible factor 1 alpha (HIF-1α) overexpression is typically associated with mortality and metastasis of many solid tumors including CRC. Riviciclib and roscovitine specifically inhibited hypoxia-induced HIF-1 transcriptional activity in luciferase reporter assay and also downregulated hypoxia-induced HIF-1α accumulation in metastatic CRC cell line HCT116. All the three drugs abrogated migration of HCT116 cells and also inhibited vascular endothelial growth factor (VEGF)- stimulated tube formation of human umbilical vein endothelial cells (HUVECs) indicating their anti- angiogenic potential. Moreover, these CDK inhibitors exhibited synergism when used in combination with standard chemotherapeutic drugs against CRC cells. Thus, we hereby provide proof-of-concept for the potential use of multitarget CDK inhibitors as promising therapeutic drugs against CRC.
Audience Take Away Notes:
- The underlying biology of CRC is complex and heterogeneous. There is an unmet medical need in order to treat CRC patients with surgically incurable disease. Trials with novel approaches and agents that attempt to minimize toxicity and improve efficacy are urgently needed
- Given the hyperactivation of one or more redundant CDKs in CRC, it is implicated that multitarget CDK inhibitors are promising for CRC treatment. This study provides a preclinical framework for potential clinical application of such multispectrum CDK inhibitors against CRC
- Much of the anticancer drug development phase relies on ‘in vitro’ model systems i.e. cancer cell lines since these are less expensive and less time-consuming as compared to animal tumor models. Functional assays and mechanistic studies using these cell lines assess the potential of candidate drugs that alter the target and facilitate the selection of the most promising candidate drug e.g. CDK inhibitors
