We next examined the effect of gene knockdown about the G2/M DNA injury checkpoint in these cells by monitoring the percentage of mitotic cells eight, twelve, 16, twenty, and 24 h right after siRNA transfection. In contrast with SN 38 treated cells transfected with handle siRNA, cells transfected with siRNA particular for Chk1 or Wee1 showed a progressive increase in mitotic index. The kinetics of mitotic entry were considerably more rapidly in cells transfected with the two Chk1 and Wee1 siRNA than in individuals transfected with every single personal oligonucleotide.
On the other hand, the extent of checkpoint escape seen in cells mGluR transfected using the pooled oligonucleotides was reduced than what one particular would have anticipated in the event the combined influence of down regulating just about every kinase was additive, suggesting that Chk1 and Wee1 may possibly function along precisely the same signaling pathway in controlling the G2/M checkpoint. Together, gene knockdown of Chk1 and Wee1 recapitulated in component the pharmacological effects of 17AAG in triggering abrogation on the G2/M checkpoint. Ultimately, we explored the therapeutic prospective of combining SN 38 and 17AAG to target p53 defective cells. Apoptosis was measured in parental and p53 null HCT116 immediately after combined treatment with SN 38 and 17AAG in numerous schedules. As proven in Fig. 6A, single agent therapy with 20 nM SN 38 or 500 nM 17AAG resulted in minimal apoptosis in each cell lines.
The mixture of SN 38 and 17AAG was ineffective in leading to apoptosis during the parental cells, no matter the sequence of drug treatment. This end result is in agreement with all the flow cytometry information, which showed no abrogation on the G2/M checkpoint by 17AAG within this cell line. Within the other hand, in p53 null cells, concurrent treatment method with SN 38 and 17AAG for 24 h resulted VEGFR inhibition inside a marked increase in apoptosis. Sequential treatment with SN 38 followed by 17AAG also induced an increase in apoptosis, which seemed to become a delayed phenomenon as the incidence of apoptosis improved additional when sequential therapy was followed by an supplemental 24 h of drug washout.
Pretreatment with 17AAG followed by SN 38 didn’t outcome in apoptosis in each cell lines, yet again reliable with the final results from cell cycle analysis demonstrating no abrogation from the G2/M checkpoint when the two agents were offered on this sequence. Examination of the nuclear morphology of cells in mitosis after concurrent or sequential SN 38 and 17AAG therapy exposed the presence of GSK-3 inhibition condensed but disorganized chromatin without the need of discernible metaphases or anaphases. We corroborated our apoptosis research by using a viability assay and formally evaluated the nature of the interaction among SN 38 and 17AAG in both parental and p53 null cells. The IC50 values of SN 38 were comparable in the two cell lines and p53 cells, respectively. p53 null cells have been extra delicate than their parental counterpart to 17AAG on this assay.