Histone Deacetylase Inhibitors Downregulate Checkpoint Kinase 1 Expression to Induce Cell Death in Non-Small Cell Lung Cancer Cells

Background: Histone deacetylase inhibitors (HDACis) are promising anticancer drugs; however, the molecular mechanisms leading to HDACi-induced cell death have not been well understood and no clear mechanism of resistance has been elucidated to explain limited efficacy of HDACis in clinical trials. Methods and Findings: Here, we show that protein levels of checkpoint kinase 1 (Chk1), which has a major role in G2 cell cycle checkpoint regulation, was markedly reduced at the protein and transcriptional levels in lung cancer cells treated with pan-and selective HDACis LBH589, scriptaid, valproic acid, apicidin, and MS-275. In HDACi treated cells Chk1 function was impaired as determined by decreased inhibitory phosphorylation of cdc25c and its downstream target cdc2 and increased expression of cdc25A and phosphorylated histone H3, a marker of mitotic entry. In time course experiments, Chk1 downregulation occurred after HDACi treatment, preceding apoptosis. Ectopic expression of Chk1 overcame HDACiinduced cell death, and pretreating cells with the cdc2 inhibitor purvalanol A blocked entry into mitosis and prevented cell death by HDACis. Finally, pharmacological inhibition of Chk1 showed strong synergistic effect with LBH589 in lung cancer cells. Conclusions: These results define a pathway through which Chk1 inhibition can mediate HDACi-induced mitotic entry and cell death and suggest that Chk1 could be an early pharmacodynamic marker to assess HDACi efficacy in clinical samples

<i>A</i>, HDACi-mediated decrease in CHK1 precedes apoptosis.

<p>A549 cells were untreated or treated with LBH589 (40 nM) for various time points. Cell lysates were prepared, and protein expression levels of cPARP, CHK1, Tyr15 phosphorylation of pCDC2 (pCDC2 ^Y15), Ser216 phosphorylation of CDC25C (pCDC25 ^S216), acetyl-H4, and cyclin B1 were determined. <i>B</i>, Quantitative Chk1 mRNA expression analysis. Total RNA was prepared from A549 cells after 24 hours of treatment with 40 nM LBH589 or vehicle. mRNA expression levels were quantified using Real-time PCR analysis. All results were normalized to GAPDH mRNA levels, and the mean and standard deviations values from four independent experiments are shown. <i>C</i>, Ectopic expression of Chk1 reverses HDACi-induced apoptosis but not histone acetylation. A549 cells were transiently transfected with an empty vector (control) or GFP- or FLAG-tagged (GFP-CHK1 or FLAG-CHK1) Chk1 expression plasmid. Forty-eight hours after transfection, cells were cultured without (control, C) or with LBH589 (L) (40 nM) for an additional 24 hour before harvesting for Western blot analysis. Treatment-induced changes in cPARP, acetyl-H4, phospho-CDC2 ^Y15, and ectopically expressed GFP-CHK1 or FLAG-CHK1 proteins were determined by Western blot analysis. β-actin expression was used as loading control. Experiments were repeated 3 times, and a representative experiment is shown. The arrows show the position of the GFP-CHK1 and FLAG-CHK1 proteins. <i>D</i>, in primary NSCLC patient samples, Chk1 protein downregulation correlates with increased cPARP <i>ex vivo</i>. Tumor samples were collected with a 23-gauge needle from patient-derived tumors, and cells were treated in duplicate with vehicle (control) or LBH589 (40 nM) for 18 hours. Following treatment, adherent and non-adherent cells were pooled, cell extracts were prepared, and expression levels of cPARP, Chk1, and acetyl-H3 were analyzed by Western blot. β-actin expression was used as loading control. SCC: squamous cell carcinoma; AC: adenocarcinoma.</p

HDACi treatment specifically inhibits CHK1 expression and upregulates its downstream signaling proteins CDC25A, CDC25C, and CDC2, involved in G₂ cell cycle checkpoint control.

<p><i>A</i>, A549, PC9, H1299, H292, H358, H441 and HCC827 cells were cultured in the presence of vehicle (C), or LBH589 (LBH) 40 nM for 24 hours and expression levels of cPARP, phosphorylation of CDC2 (pCDC2 ^Y15), CHK1, and acetylated histone H4 (acetyl-H4) were determined by Western blot and quantitated using AlphaEase software. β-actin was used as loading control. <i>B</i>, PC9 and A549 cells were cultured in the presence of vehicle (C), or LBH589 (LBH) 40 nM, or scriptaid (S) 1 µM for 24 hours and expression levels of cPARP, tyrosine-15 phosphorylation of CDC2 (pCDC2 ^Y15), serine-216 phosphorylation of CDC25C (pCDC25c ^S216), CDC25A (T-CDC25A), CDC25C (T-CDC25C) and CDC2 (T-CDC2), acetylated histone H4 (acetyl-H4), and cyclin B1 were determined by Western blot analysis. β-actin was used as loading control. <i>C</i>, drug-mediated changes in the expression of CHK1, CHK2, AKT, and c-RAF were determined by Western blot analysis. β-actin was used as loading control. <i>D</i>, PC9 or A549 cells were treated with or without 40 nM LBH589 and analyzed for annexin positive cells using the BD Annexin V-FITC/7-AAD Flow Cytometry kit. <i>E</i>, A549 cells were treated with MS-275 (MS), (500 nM), valproic acid (VA) (1 Mm), or apicidin (Api) (400 nM) for 24 h. Expression levels of cPARP, CHK1, pCDC2 ^Y15, and β-actin were determined by Western blot analysis. All experiments were repeated at least three times.</p

LBH589 treatment leads to mitotic abnormalities and cytokinesis failures.

<p>A549 cells were treated with vehicle (control) or 40 nM LBH589 for 24 hours. <i>A</i>, arrows show bi- or multinucleated cells with impaired cytokinesis in LBH589-treated NSCLC cells. <i>B</i>, cells were fixed and stained with DAPI or cleaved poly (ADP-ribose) polymerase (cPARP) antibody (×100 or ×400 magnification). <i>C</i>, Western blot analysis demonstrating that HDAC inhibition by LBH589 causes histone H3 phosphorylation (H3-P10), histone H4 acetylation (Acety-H4), and PARP cleavage (cPARP) in A549 cells treated with LBH589 for 24 hours. β-actin was used as loading control.</p

LBH589 and Chk1 inhibitor treatment shows a synergistic effect in NSCLC cells.

<p><i>A</i>, A549 cells were treated with LBH589 40 nM and/or a UCN-01 (250 nM) for 24 hours, cell extracts were prepared, and Western blot analysis was performed with PARP (t: total, c: cleaved). Experiments were repeated at least 3 times, and a representative experiment is shown. <i>B</i>, A549 cells were treated with LBH589 and UCN-01 either alone or in combination at a constant ratio (1∶40) for 72 hours. Drug concentrations are indicated on the horizontal axis and plotted against cell viability of control wells, which was arbitrarily set at 100% viability for each experiment. Error bars represent ± SD of 4 replicate wells. <i>C</i>, combined effects of LBH589 and Chk1 inhibitor UCN-01 were quantified with the Chou and Talalay combination index (CI) method (40). The CI used for drug combination analyses was determined by the isobologram equation (see text). Ranking symbols (+/−) indicate average calculated Chou and Talalay combination index (CI) range (+++, strong synergism).</p

Purvalanol A (Pur A) pretreatment diminishes the cytotoxic effect of LBH589 in A549 cells.

<p>Cells treated with LBH589 40 nM with or without Pur A (10 µM) pretreatment were analyzed by flow cytometry for cell cycle distribution or by Western blot to determine drug-mediated changes in cPARP. β-actin was used as a loading control.</p