Figure 1.
Panobinostat suppresses BRCA1, CHK1, and RAD51 protein and transcript expression and induces apoptosis in AML cell lines.
THP-1 cells were treated with variable concentrations of panobinostat for 48 h. Whole cell lysates were subjected to Western blotting (Panels A&D). Total RNAs were isolated and mRNA levels were determined by Real-time RT-PCR (Panel B). Apoptotic events were determined by annexin V/PI staining and flow cytometry analysis (Panel C). Protein and mRNA levels for BRCA1, CHK1, and RAD51 genes were determined by Western blotting (Panel E) and Real-time PCR (Panel F), respectively, in THP-1 cells treated with 10 nM panobinostat for up to 48 h. CTS, OCI-AML3 or U937 AML cells were treated with variable concentrations of panobinostat for 48 h. Whole cell lysates were subjected to Western blotting to measure protein levels for BRCA1, CHK1, and RAD51 in the cells (Panel G). The levels of apoptosis induced by panobinostat were determined by flow cytometry analysis with annexin V/PI staining (Panel H). Transcript levels for BRCA1, CHK1, and RAD51 genes were determined by Real-time RT-PCR (Panels I–K).
Table 1.
Effects of panobinostat treatment on the transcript levels for BRCA1, CHK1, and RAD51 genes in diagnostic AML blasts.
Figure 2.
Panobinostat cooperates with cytarabine or DNR in inducing DNA DSBs and apoptosis, and abrogates S and/or G2/M cell cycle checkpoint activation induced by cytarabine or DNR in THP-1 and OCI-AML3 AML cells.
THP-1 or OCI-AML3 cells were treated with cytarabine or DNR, alone or in combination with panobinostat for 48 h. Early and late apoptosis events in the cells were determined by annexin V/PI staining and flow cytometry analyses (Panels A&B). Whole cell lysates were subjected to Western blotting (Panels C&D). Cell cycle distribution was determined by PI staining and flow cytometry analysis (Panels E&F). **indicates p<0.005.
Figure 3.
The roles of BRCA1, CHK1, and RAD51 in cytarabine- or DNR-induced DNA DSBs and apoptosis in THP-1 cells.
THP-1 cells were infected with BRCA1, CHK1, RAD51, or non-target control (NTC) shRNA lentivirus overnight, washed and then treated with 4 µM cytarabine or 25 nM DNR for 48 h. shRNA knockdown of BRCA1, CHK1, or RAD51, and induction of γH2AX by cytarabine or DNR were determined by Western blotting (Panels A–C). The lane headings indicate the treatment conditions ‘Control’ ‘Ara-C’ or ‘DNR’ and the+or – indicate the shRNA-treated cells from which the sample was derived. Apoptotic events in the cells were determined by annexin V/PI staining and flow cytometry analyses (Panel D). THP-1 cells were infected with BRCA1, CHK1, or RAD51 shRNA lentivirus overnight. The cells were washed three times with complete medium and cultured in virus-free complete medium for up to 72 h. The cells were then treated with 50 µM cytarabine or 2 µM DNR for 3 h and the drugs were washed out, and the cells were cultured in drug-free complete medium for up to 8 h. DNA damage was assessed by COMET assay. Representative images at the 8 h time point are shown (Panel E). The median percent DNA in the tail from at least three replicate gels are shown plus or minus the standard error of the mean (Panel F). *indicates p<0.05.
Figure 4.
Antileukemic activity of panobinostat alone, cytarabine alone, and panobinostat plus cytarabine in a U937 xenograft model.
NOD-SCID-IL2Rγnull (NSG) mice were injected with luciferase-labeled U937 cells and treated 3 days later with panobinostat (5 mg/kg once daily for 3 weeks), ara-C (6.25 mg/kg once daily for 4 weeks), or combination (panobinostat 5 mg/kg once daily for 3 weeks plus ara-C 6.25 mg/kg once daily for 4 weeks). Serial bioluminescence images of representative mice receiving panobinostat alone (n = 8), ara-C alone (n = 10), or panobinostat plus ara-C (n = 8) (Panel A). When bioluminescence reached 1.5×107 p/s/cm2/sr, day 17 after injection of U937 cells, mice received panobinostat (5 mg/kg once daily×2). Four hours after the second dose mice were sacrificed and Bone Marrows were harvested. Pellets were lysed and subjected to Western Blot (Panel B). Tumor progression monitored by quantitative biophotonic imaging analysis of control and treatment groups (Panel C). A plot of overall survival probability, estimated with the Kaplan–Meier method (Panel D).
Figure 5.
E2F1 is a critical mediator of the suppression of BRAC1, CHK1, and RAD51 by panobinostat in THP-1 cells.
THP-1 cells were treated with cytarabine or DNR alone or in combination with panobinostat for 48 h. Transcript levels for BRCA1, CHK1, RAD51, and E2F1 genes were determined by Real-time RT-PCR (Panels A&C). Whole cell lysates were subjected to Western blotting and probed with E2F1 antibody (Panel B). In vivo binding of E2F1 to the putative binding sites located in the BRCA1, CHK1, or RAD51 promoter was determined by ChIP assays with or without 10 nM panobinostat treatment for 48 h with the use of real-time PCR as described in “Material and Methods” (Panel D). THP-1 cells were infected with E2F1 or non-target control (NTC) shRNA lentivirus overnight, washed and cultured for another 72 h. shRNA knockdown of E2F1 was determined by Western blotting (Panel E) and real-time RT-PCR (Panel F). Effects of E2F1 knockdown on the transcript levels for BRCA1, CHK1, or RAD51 were determined by real-time RT-PCR (Panel F). *indicates p<0.05, **indicates p<0.005, ***indicates p<0.0005.
Figure 6.
A proposed model of molecular mechanisms underlying the cooperative induction of apoptosis by HDACIs and DNA damaging agents in AML cells.
HDACIs suppress the transcript and protein levels of the transcription factor gene E2F1, leading to decreased expression of BRCA1, CHK1, and RAD51. Decreased expression of BRCA1, CHK1, and RAD51 would result in weakened repair of DNA DSBs induced by DNA damaging chemotherapeutic agents (e.g., cytarabine or DNR), thus enhancing apoptosis. At the same time, downregulation of BRCA1 and CHK1 by HDACIs would result in abrogation of cell cycle checkpoint activation (S and/or G2/M) induced by DNA damaging agents, thus forcing cells carrying DNA lesions to progress in the cell cycle and undergo apoptosis.