br Materials and methods br
Materials and methods
Discussion CDK and their regulatory cyclins form a family of heterodimeric kinases that are particularly important for regulation of cathepsin g synthesis progression and transcription in tumor cells. These kinases therefore constitute biomarkers of proliferation and attractive pharmacological targets for development of anticancer therapeutics . Several classes of inhibitors have been developed to target ATP pockets, protein/protein interfaces or allosteric sites of CDK family members . CDK inhibitors have been recently investigated in preclinical and clinical settings for the treatment of several advanced or refractory blood malignancies and solid cancers . AT7519 is a potent inhibitor of several CDK family members and has favourable tolerability and efficacy in animal models compared with other CDK inhibitors in clinical development . The anti-cancer activity of AT7519 in a variety of cancers has been reported, including leukemia, multiple myeloma and cancers derived from colon, lung, breast, ovary and cervix [, , ,17]. However, whether AT7519 is effective in overcoming chemoresistance has not been revealed. To the best of our knowledge, we are the first to demonstrate the in vitro and in vivo efficacy of AT7519 in chemoresistant cancer cells. We firstly demonstrate that AT7519 is active against a panel of chemosensitive colon and cervical cancer cell lines regardless of their cellular origin and genetic background (Fig. 1). The IC50 of AT7519 on these chemosensitive cancer cell lines are 100–1000 nM, which is consistent with the previous studies on the efficacy of AT7519 in cancer [10,17]. AT7519 has been reported to inhibit proliferation of leukemia, lymphoma, and carcinomas of colon, ovarian, lung and breast cells with IC50 range from 82 to 940 nM . It is interesting that AT7519 inhibits growth of proliferating and nonproliferating fibroblast cells with IC50 at 980 nM and >10,000 nM, respectively . This suggests that AT7519 is minimal toxic to nonproliferating normal cells. The anti-cancer activity of AT7519 on chemoresistant cancer cells were evaluated using chemoresistant cancer cell lines with IC50 at least 10–20 fold higher compared with parental cell lines . We show that AT7519 potently inhibits proliferation and induces apoptosis of all tested chemoresistant cancer cell lines with nanomolar IC50 (Fig. 2A and B), suggesting the potent activity of AT7519 in chemoresistant cancer cells. This is further supported by our findings that AT7519 at sublethal concentration remarkably augments the efficacy of 5-FU and paclitaxel in colon and cervical cancer cells (Fig. 2C and D). The two chemotherapeutic agents (eg, 5-FU and paclitaxel) and two diseases (colon and cervical cancer) used in our study suggest that AT7519 is likely to be effective across a panel of chemoresistant cancers regardless of their mechanism of resistance. Importantly, AT7519 significantly inhibits growth of chemoresistant cancer cells in vivo at the dose that does not cause toxicity in mice (Fig. 4A and B). Our findings further support the previous work on the tolerability and efficacy of AT7519 in animal xenograft tumor models [8,10]. A study on the interactions of AT-7519 with ATP-binding cassette (ABC) transporter proteins shows that AT7519 does not overcome ABC transporter-mediated multidrug resistance . Using both cell culture system and xenograft mouse model, our work demonstrates that AT7519 overcome chemoresistance via inhibiting phosphorylation of CDK substrates and RNA polymerase II in chemoresistant cancer cells, which is in agreement with its ability in inhibiting CDK [7,10,11]. AT7519 selectively inhibits CDK1, 2, 4, 5 and 9. We show that AT7519 decreases phosphorylation of CDK1 substrate and CDK2 substrate in chemoresistant colon and cervical cancer cells in vitro and in vivo (Fig. 3, Fig. 4C). A significant reduction of the phosphorylation of RNA polymerase II is observed. We speculate that this might be due to the ability of AT7519 in inhibiting CDK9, which is associated with the regulation of transcriptional activity . Studies revealed that AT7519 inhibits other kinase such as glycogen synthase kinase 3β (GSK-3β) . GSK-3β inhibition has been shown to overcome chemoresistance in breast cancer . It would be interesting to investigate whether GSK-3β is involved in the action of AT7519 in chemoresistant cancer cells.