Apoptosis DOI 10.1007/s10495-014-1010-3
ORIGINAL PAPER
The checkpoint 1 kinase inhibitor LY2603618 induces cell cycle arrest, DNA damage response and autophagy in cancer cells Feng-Ze Wang • Hong-rong Fei • Ying-Jie Cui • Ying-Kun Sun • Zhao-Mei Li • Xue-Ying Wang • Xiao-Yi Yang • Ji-Guo Zhang • Bao-Liang Sun
Ó Springer Science+Business Media New York 2014
Abstract Chemotherapy- or radiotherapy-induced DNA damage activates the Chk1-dependent DNA damage response (DDR) and cell cycle checkpoints to facilitate cell survival. Numerous attempts have been made to identify specific Chk1 inhibitors to enhance the efficiency of chemotherapy or radiotherapy. In this study, we investigated the molecular mechanisms underlying the antitumor activity of LY2603618, a potent and selective small molecule inhibitor of Chk1 protein kinase, in human lung cancer cells. Treatment of cancer cells with LY2603618 caused cell cycle arrest in the G2/M phase. A marked induction of DDR, including the phosphorylation of ATM,
Feng-Ze Wang and Hong-rong Fei have contributed equally to this work. F.-Z. Wang Z.-M. Li X.-Y. Wang School of Biological Science, Taishan Medical University, Taian 271016, People’s Republic of China F.-Z. Wang X.-Y. Yang B.-L. Sun (&) Key Lab of cerebral microcirculation in Universities of Shandong (Taishan Medical University), Taishan Medical University, Taian 271000, People’s Republic of China e-mail: [email protected] H. Fei J.-G. Zhang (&) School of Pharmacology, Taishan Medical University, Taian 271016, People’s Republic of China e-mail: [email protected] Y.-J. Cui Institute of Atherosclerosis, Taishan Medical University, Taian 271000, People’s Republic of China Y.-K. Sun College of Landscape Architecture and Forestry, Qingdao Agricultural University, Qingdao 266109, People’s Republic of China
Chk2, p53 and histone H2AX, was observed after LY2603618 treatment. LY2603618 inhibited Chk1 autophosphorylation (S296 Chk1) and increased DNA damagemediated Chk1 phosphorylation (S345 Chk1). In addition, LY2603618-treated lung cancer cells transitioned from LC3-I to LC3-II, a hallmark of autophagy. Blocking autophagy with chloroquine (CQ) further enhanced LY26036180 s inhibitory effect on cell viability/proliferation. LY2603618 also significantly increased p38 and c-Jun N-terminal kinase (JNK) phosphorylation. Pretreatment with the JNK inhibitor reduced cleavage of caspase-3 and PARP levels in LY2603618-treated cells. These results suggest the following: (i) the biological consequences of LY2603618 in lung cancer cells is associated with both inhibition of Chk1 phosphorylation on S296 and activation of the DNA damage response network; and (ii) the anticancer property of LY2603618 might be increased by inhibiting autophagy. Keywords Chk1 inhibitor LY2603618 G2/M phase DNA damage Autophagy
Introduction Genomic integrity maintenance is critical for cell survival and cell growth. DNA damage from normal metabolic processes and environmental factors induces cell cycle arrest in eukaryotic cells via checkpoint activation or DNA repair machineries to repair the damaged DNA [1, 2]. However, when DNA damage is irreparable, the damaged cells undergo apoptosis to avoid passing on the potentially lethal errors in DNA to their daughter cells. The cellular responses induced by DNA damage, including cell cycle checkpoint, DNA repair and DNA damage-induced
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programmed cell death, are collectively called the DNA damage response (DDR) [3], a versatile response to genomic DNA damages. Cells commonly respond to DDR by activating G1/S checkpoint and/or G2/M checkpoint, and the latter prevents the cells that have suffered DNA damage during G2 phase from initiating mitosis or from progressing into G2 phase despite unrepaired damage inflicted during previous S or G1 phases [4]. Key proteins involved in DNA damage and DNA strand break repair signaling are ATM (Ataxia telangiectasiamutated) and ATR (ATM and Rad3-related), two serine/ threonine kinase members of the phosphoinositide 3-kinase-like family. In response to DNA damage, ATM and/or ATR initiate cell cycle arrest via Chk1 and Chk2, and facilitate DNA repair complex activation to preserve DNA integrity [5, 6]. ATM is usually considered a response to DNA damage caused by ionizing radiation or oxidative stress, and it phosphorylates Chk2. In contrast, ATR primarily senses UV damage or replication stress and relays its effect via Chk1 phosphorylation. ATM/ATRChk1/Chk2 pathway activation leads to Cdc2 (Cdk1) inhibition through phosphorylation-mediated Cdc25 inactivation, thereby triggering G2/M phase arrest [7]. There are three isoforms of Cdc25: Cdc25A, Cdc25B, and Cdc25C. Cdc25B is thought to function at the G2/M cell cycle transition, and Cdc25C acts on M phase to sustain cyclinB1/Cdc2 activity [8]. Because DDR facilitates cancer cell survival after DNA damage, there is strong clinical interest in combining checkpoint inhibitors with DNA damage agents to increase cancer sensitivity [9, 10]. The G1/S checkpoint is frequently inactivated or lost in cancer due to p53 mutations, and the G2/M checkpoint may be important for cancer cells to respond to DNA-damaging chemotherapeutic agents [11]. The Chk1-mediated signaling pathway is required for the G2/M arrest in response to DNA-damaging agents. Chk1 phosphorylates and inhibits Cdc25, consequently blocking cyclinB1/Cdc2 activation and mitotic entry when DNA synthesis is inhibited [12]. Chk1 inhibitors may selectively enhance genotoxic agent activity in tumors by inhibiting the S and G2 checkpoints, while normal cells are rescued by their competent DDR, and many preclinical and clinical studies have focused on Chk1 inhibition to enhance tumor cell susceptibility to chemotherapy [13, 14]. A small molecule inhibitor of Chk1, LY2603618, was recently found to impair DNA synthesis and induce H2AX phosphorylation in cancer cells [15, 16]. However, the underlying mechanisms of its anticancer properties still need to be elucidated. In this study, we investigated the effects of LY2603618 on cell cycle progression, DNA damage responses, cell apoptosis and autophagy in nonsmall cell lung cancer cells. We found that LY2603618
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treatment of lung cancer cells resulted in accumulation of cells in the G2/M phase. LY2603618 also induced apoptosis and autophagy. Inhibition of autophagy enhanced LY2603618-mediated cleavage of caspase-3 and PARP. Treatment with a JNK inhibitor partially alleviated LY2603618-induced cell death.
Materials and methods Reagent LY2603618 was a gift from Lanmu Chemistry (Shanghai, China), and it was dissolved in DMSO. 3-[4, 5-dimethylthiazol-2-y-l]-2, 5-diphenyltetrazolium bromide (MTT) and chloroquine (CQ) were purchased from Sigma-Aldrich (St. Louis, MO). SP600125 was purchased from Calbiochem (La Jolla, CA, USA), SB203580 was purchased from Santa Cruz Technology (CA, USA). ECL Western blot detection reagents were purchased from PerkinElmer (Rockford, USA). Antibodies specific to b-actin, Atg-5 and LC3-II were obtained from Sigma-Aldrich. Rabbit anti-caspase-9, caspase-3, PARP, JNK, ATM, p-ATM, ATR, p-ATR, p-Chk2, p-Chk1(S345), p-Chk1(S296), c-H2AX, p-Cdc25C, p-p53, Cdc2, survivin and p-p38 antibodies were purchased from Cell Signaling Technology (Shanghai, China). Mouse antiChk2, p21, p27 and p-JNK antibodies were purchased from BD Biosciences. Anti-cyclinD1, Cdc25A, Cdc25B, cyclinB1, p53, ERK, p-ERK and p38 antibodies were purchased from Santa Cruz Technology. Anti-Chk1 and Cdc25C antibodies were purchased from Epitomics (Hangzhou, China). The antiH2AX antibody was obtained from Sangon Biotech (Shanghai, China). Cells and cell culture A549 and H1299 cell lines were obtained from the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China).Cells were cultured in RPMI medium 1640 (Gibco) plus 10 % fetal bovine serum (Gibco) in a humidified atmosphere (37 °C; 5 % CO2). Cell viability/proliferation assay A549 or H1299 cells were plated in 96-well plates for 24 h. The medium was removed, and the cells were treated with various concentrations of the test compound (LY2603618). A total volume of 20 lL MTT (5 mg/mL) was added for 4 h. After removal of the medium and MTT, 150 lL DMSO was added to each well, and the optical density was detected at 490 nm using a microplate reader.
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Cell cycle distribution analysis by flow cytometry Cells were treated with LY2603618 and DMSO as a control. After trypsinization, cells were fixed in 70 % ethanol at 4 °C overnight. The cells were washed twice with PBS and incubated for 30 min in the dark in PBS containing propidium iodide (PI) and RNase A. Stained cells were analyzed by a FACScan flow cytometry and CellQuest analysis software (Becton–Dickinson, San Jose, CA).
at 37 °C, and DAPI was used to stain the nuclei. The signals were detected by a fluorescence confocal microscope with the appropriate filter sets. Statistical analysis All results were expressed as mean ± SD. Each value is the mean of at least 3 independent experiments. Statistical analysis was performed by the student’s t test. P \ 0.05 was considered significant.
Apoptosis assay To evaluate apoptosis induction, TUNEL staining was performed according to the manufacturer’s instructions. Briefly, after exposure to LY2603618, cells were washed with PBS and fixed in 4 % (w/v) paraformaldehyde in PBS for 1 h at room temperature. Cells were then permeabilized with 0.1 % Triton X-100 for 2 min on ice. TUNEL staining was carried out using the In Situ Cell Death Detection Kit, Fluorescein (Roche, IN, USA). The stained cells were visualized with a fluorescence microscope (Olympus, Tokyo, Japan). GFP-LC3 localization by fluorescence microscopy Cells were seeded into 12-well plates and transfected with 0.5 lg GFP-LC3 plasmid using Lipofectamine 2000 (Invitrogen Life Technologies, CA, USA) according to the manufacturer’s instructions. After 24 h, cells were incubated with LY2603618 for another 24 h. Fluorescent signals were detected by a fluorescence microscope with the appropriate filter sets. Western blot analysis Cells were lysed with ice-cold RIPA lysis buffer containing PMSF and protease inhibitor cocktail for 20 min on ice, and the lysates were centrifuged at 13,000 g for 20 min at 4 °C. The supernatants were used as total cell lysates. The proteins were separated by SDS-PAGE and transferred onto nitrocellulose filter membranes. Membranes were blocked with 5 % milk powder and incubated with specific antibodies. Detection of the target proteins on the membranes was performed using the ECL Western Blotting Detection Reagents. Immunoflourescence assays Cultured cells were fixed with cold 100 % methanol at -20 °C for 10 min and permeabilized with PBS-0.1 %Triton X-100 for 5 min. Coverslips were blocked with 3 % BSA, and the cells were subsequently incubated with an anti-c-H2AX antibody (Abcam, Hong Kong) for 2 h. After washing, cells were incubated with a FITC-conjugated secondary antibody (Santa Cruz, 1:100 dilution) for 30 min
Results LY2603618 induces cell cycle arrest at the G2/M phase in wild-type and p53-deficient lung cancer cells To determine the antitumor activities of LY2603618, we first assessed the effect of LY2603618 on cell cycle distribution by flow cytometry. As shown in Fig. 1a, A549 control cell populations in the G1, S and G2/M phases were 54.88, 31.81 and 13.31 %, respectively. After 24 h of incubation with 5 or 10 lM LY2603618, the percentage of G2/M phase cells increased to 32.31 and 38.95 %, respectively. We observed similar changes in cell cycle distribution after LY2603618 treatment in H1299 cells, indicating that LY2603618 induces cell cycle arrest in G2/M phase in human lung cancer cells. In mammalian cells, cyclinB1 and Cdc2 form a complex and cooperate to promote the G2/M phase transition. Therefore, we next investigated whether LY2603618 regulated cyclinB1 and Cdc2 expression in lung cancer cells. LY2603618 treatment considerably inhibited cyclinB1 and Cdc2 expression in both A549 and H1299 cells. The Cdc25C phosphatase is thought to regulate Cdc2 phosphorylation, and thus we also examined Cdc25C phosphorylation. As shown in Fig. 1b, p-Cdc25C (S216) levels decreased in LY2603618-treated cells, and Cdc25B expression was also inhibited. However, p21 and p27 expression increased upon LY2603618 treatment. We did not observe any significant changes in Cdc25A, Cdc25C and cyclinD1 levels. Phosphorylation of histone H3 on Ser10 plays an important role in mammalian mitotic chromatin condensation [17]. Thus, we examined whether LY2603618 had an effect on histone H3 phosphorylation. We found that 24 h LY2603618 treatment induced increased phosphorylation of histone H3 (Fig. 1b). LY2603618 activates DNA damage sensor kinases in A549 and H1299 cells To investigate the mechanisms of G2/M cell cycle arrest triggered by LY2603618, we investigated the genotoxicity
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Fig. 1 LY2603618 induces G2/M arrest in non-small cell lung cancer cells. a A549 and H1299 cells were exposed to different concentrations of LY2603618 for 24 h, and cell cycle distributions were measured by flow cytometry. b The effect of LY2603618 on cell
cycle regulatory proteins was examined by western blot analysis. Cells were treated with LY2603618 for 24 h, and total cell lysates were analyzed for cell cycle regulatory proteins with specific antibodies. b-actin was used as an internal control
of this compound using immunofluorescence staining to monitor the formation of c-H2AX foci in A549 lung cancer cells. As shown in Fig. 2a, LY2603618 treatment increased the formation of c-H2AX foci in lung cancer cells. We then examined phosphorylation of the nuclear histone H2AX by western blot assay and found that LY2603618 induced total H2AX expression and H2AX phosphorylation (Fig. 2b). We also observed an increase in H2AX phosphorylation levels in LY2603618-treated H1299 cells. To confirm that
LY2603618 inhibited Chk1 activities in lung cancer cells, we analyzed Chk1 signaling by western blot analysis. As shown in Fig. 2c, increasing concentrations of LY2603618 induced the downregulation of Chk1 autophosphorylation (S296), suggesting that LY2603618 inhibits Chk1. We also found that DNA damage-mediated phosphorylation of Chk1 (S345) and Chk2 (T68) were increased in response to LY2603618 treatment (Fig. 2c). To further elucidate the mechanisms leading to LY2603618-mediated DNA
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Fig. 2 Analysis of LY2603618-induced DNA damage in human lung cancer cells. a DNA double-strand breaks, as measured by c-H2AX, were induced by LY2603618 treatment. A549 cells were exposed to different concentrations of LY2603618 for 24 h. Cells were then fixed and immunostained with anti-c-H2AX antibody and DAPI. b LY2603618 induced H2AX phosphorylation in lung cancer cells. A549 and H1299 cells were treated with LY2603618 for 24 h.
The cells were analyzed by western blot analysis using anti-c-H2AX or anti-b-actin antibodies. c LY2603618 treatment resulted in increased phosphorylation of ATM, Chk2 and p53. Cells were treated with the indicated concentrations of LY2603618 (1–20 lM) for 24 h. ATM/ATR-Chk1/Chk2 signaling pathway proteins were measured by western blot with specific antibodies
damage, we examined ATM and ATR phosphorylation following LY2603618 exposure. Western blot results showed that LY2603618 increased ATM phosphorylation at S1981, whereas LY2603618 had no effect on ATR phosphorylation at residue S428. Total p53 levels and phosphorylated p53 (S15) levels were also increased after LY2603618 treatment (Fig. 2c).
fashion. We also examined whether LY2603618 affected survivin expression because survivin was reported to play a crucial role in regulating apoptosis [18]. We found that LY2603618 dose-dependently suppressed survivin expression in both A549 and H1299 cells.
LY2603618-induced apoptosis is associated with caspase activation and survivin inhibition Apoptosis is another important mechanism involved in cancer therapy. Therefore, we examined apoptosis in LY2603618-treated cells by identifying TUNEL-positive cells. As shown in Fig. 3a, LY2603618 treatment induced apoptosis of non-small cell lung carcinoma cells. To elucidate whether LY2603618-treated cancer cells express canonical apoptotic markers, we examined the effects of LY2603618 on apoptosis-related protein levels. As shown in Fig. 3b, treatment of cancer cells with LY2603618 for 24 h increased the cleavage of the initiator caspase-9, caspase-3 and PARP in a dose-dependent
Blocking autophagy enhances LY2603618 cytotoxic activity on human lung cancer cells The conversion of soluble LC3-I to lipid bound LC3-II via proteolytic cleavage and lipidation shows autophagy induction [19]. To study whether LY2603618 induced autophagy in lung adenocarcinoma cells, we examined the formation of LC3 dots and conversion of LC3-I to LC3-II. pEGFP-LC3-transfected cells were treated with or without LY2603618 for 24 h and subjected to immunofluorescence for visualization of GFP-LC3 expression. We found that LY2603618 induced the redistribution of GFP-LC3 from a diffuse distribution (control cells) to punctate structures (LY2603618-treated cells) (Fig. 4a), which indicates that LY2603618 induced autophagy in lung cancer cells. Next, we examined LC3-II protein levels, a promising
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LY2603618 and CQ markedly decreased A549 cell viability/proliferation compared to the control group, indicating that the suppression of autophagy enhanced LY2603618induced cell viability/proliferation inhibition. In agreement, the combination of LY2603618 and CQ was more potent than LY2603618 alone in inducing apoptosis, as evidenced by the increased cleavage of caspase-3 and PARP (Fig. 4d). These data indicate that LY2603618-induced autophagy represents a pro-survival mechanism, inhibition of which drives lung cancer cells to death. We also detected changes in LY2603618-induced H2AX expression in the presence of CQ. As shown in Fig. 4e, inhibition of autophagy significantly enhanced LY2603618-mediated H2AX phosphorylation, suggesting that inhibition of autophagy with CQ potentiates the LY2603618-induced DNA damage response. LY2603618 increases JNK and p38 MAPK phosphorylation in lung cancer cells
Fig. 3 The apoptotic effect of LY2603618 on human lung cancer cells. a Cells were treated with different concentrations of LY2603618 for 24 h. Apoptosis was analyzed by TUNEL staining. Results showed the presence of fragmented DNA in LY2603618treated cells. b LY2603618 treatment induced the cleavage of PARP, caspase-9, caspase-3, and decreased survivin expression. After cells were treated with LY2603618 for 24 h, total cell lysates were harvested and analyzed by western blot with specific antibodies against PARP, caspase-9, caspase-3 and survivin. b-actin was used as a loading control
autophagosomal marker. Western blot results showed significantly increased conversion of LC3-I to LC3-II with increasing concentrations of LY2603618 (Fig. 4b). We also measured the expression of autophagy-related proteins Atg5 in LY2603618- treated cells and found that LY2603618 increased Atg5 levels (Fig. 4b). These results indicate that LY2603618 stimulates the conversion of LC3I to LC3-II and induces autophagy. We further determined the role of LY2603618-induced autophagy in cell survival or cell death in lung cancer cells by blocking autophagy with CQ, a lysosomal protease inhibitor. As shown in Fig. 4c, the combination of
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It has been previously shown that MAPK signaling is associated with cell proliferation, survival, DNA damage and autophagy [20, 21]. To determine whether the MAPK pathway was affected by LY2603618 in lung cancer cells, we detected the phosphorylation of JNK and p38 MAPK by western blot analysis. The results showed that LY2603618 increased JNK and p38 MAPK phosphorylation in a dosedependent manner after 24 h (Fig. 5a). To determine whether activated JNK and/or p38 MAPK play a role in LY2603618-induced caspase cleavage, A549 cells were incubated with the JNK inhibitor SP600125 or p38 MAPK inhibitor SB203580. SP600125 partially prevented LY2603618-induced cleavage of caspase-3 and PARP (Fig. 5b). Moreover, SP600125 significantly blocked LY26036180 s inhibition of cell viability/proliferation (Fig. 5c). These results suggest that LY2603618induced apoptosis in non-small cell lung cancer cells is associated with JNK activation.
Discussion In the past several years, various specific small molecule Chk1 inhibitors have been developed [22]. LY2603618 has entered into phase I clinical trials and showed acceptable safety and pharmacokinetic profiles. The present study was performed to determine the anticancer effect of LY2603618 in non-small cell lung cancer cells and to investigate the underlying cellular mechanism. We have demonstrated that targeting non-small cell lung cancer with LY2603618 induces cell cycle arrest in G2/M, accompanied by a decrease in cyclinB1, Cdc2, Cdc25B and p-Cdc25C expression, and increased levels of p21 and p27. LY2603618 inhibits phosphorylation of Chk1 at Ser296 but induces
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Fig. 4 Induction of autophagy in LY2603618-treated lung cancer cells. a Formation of LC3-positive vesicles in LY2603618-treated GFP-LC3 transduced cells. Lung cancer cells were transfected with GFP-LC3 plasmids and treated with 0, 5 or 10 lM LY2603618 for 24 h, followed by observation of GFP-LC3 by fluorescence microscopy. b A549 and H1299 cells were treated with the indicated concentrations of LY2603618 for 24 h and subjected to western blot to detect the levels of LC3-I, LC3-II and Atg-5 with b-actin as an
internal control. c The effects of the autophagic inhibitor CQ on LY2603618-induced cell viability/proliferation inhibition. Cells were treated with 20 lM CQ for 2 h before the addition of 10 lM LY2603618. After 24 h, cell viability/proliferation was determined by MTT. d The activation of caspase-3 and PARP was measure by western blot analysis in A549 cells. e Cells were treated with 20 lM LY2603618 and/or 20 lM CQ for 24 h. The levels of LC3-II and c-H2AX were determined by western blot analysis
phosphorylation of Chk1 at Ser345. LY2603618 treatment provoked the DNA damage response and apoptosis. Inhibition of autophagy by CQ increased LY26036180 s effect on cell viability/proliferation. LY2603618 may act via the MAPK signaling pathway to elicit its anti-cancer effect. Cell cycle arrest during the DNA damage checkpoint is carried out by the sequential action of ATM/ATR-Chk1/
Chk2-Cdc25A/Cdc25C, and it eventually results in the inhibitory phosphorylation of Cdc2 and activation of the cyclinB1/Cdc2 complex [23, 24]. As an essential kinase involved in cell cycle checkpoints, Chk1 was phosphorylated at Ser345, Ser317, and Ser296 in response to DNA damage [25]. Ser317 and Ser345 phosphorylation is predominantly
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Apoptosis Fig. 5 LY2603618 activates JNK1/2 and p38 MAPK in human lung cancer cell lines. a Cells were treated with the indicated concentrations of LY2603618 for 24 h, and the levels of JNK1/2 and p38 phosphorylation were determined by western blot analysis. b LY2603618-induced JNK activation is involved in apoptosis. A549 cells were treated with 20 lM SP600125 or 20 lM SB203580 1 h before the addition of LY2603618 (20 lM) for 24 h. Caspase-3 and PARP levels were detected by western blot analysis. c The LY2603618-mediated reduction of lung cancer cell viability/ proliferation was significantly inhibited by SP600125
catalyzed by ATR, while Ser296 phosphorylation occurs through autophosphorylation [26]. Our studies showed, for the first time, that LY2603618 inhibited Ser296 Chk1 phosphorylation in human lung cancer cells. However, phosphorylated Ser-345 Chk1 (pS345 Chk1) levels significantly increased in response to low doses of LY2603618, suggesting that LY2603618 induces the DNA damage response and further amplifies ATR/ATM-mediated Chk1 phosphorylation (S345) in human lung cancer cells. This is consistent with a report by Parsels LA and Riesterer O in which the two Chk1 inhibitors AZD7762 and XL-844 also induced Chk1 (S345) phosphorylation [27, 28]. We found that LY2603618 strongly induces the DNA damage response in lung cancer cells. When cells are exposed to DNA-damaging chemotherapeutic agents, double-stranded breaks (DSBs) are generated, rapidly resulting in H2AX phosphorylation at Ser139 (c-H2AX) by ATM, ATR or DNA-PK. Because this process is robust, fast and correlates well with each DSB, H2AX phosphorylation is considered as a sensitive marker of DNA damage [29]. Our data presented here demonstrate that LY2603618 induced the formation of c-H2AX foci and increased cH2AX levels in human cancer cells. We observed increased phosphorylation of ATM, Chk2 and p53 after LY2603618 treatment. Therefore, LY2603618 induced the whole spectrum of the DNA damage response pathway.
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Although 1 lM LY2603618 robustly activated DNA damage response factors, including c-H2AX and p-ATM, phosphorylation of Chk1 at Ser296 was only slightly inhibited at this concentration of LY2603618, suggesting that LY2603618 may activate the DSB response pathway independently of Chk1 inhibition. DNA-damaging agents trigger complex responses in human cells, including the activation of cell cycle checkpoints to facilitate repair, and the induction of apoptotic cell death or growth arrest via accelerated cellular senescence, depending on the genetic background of the cells and the extent of DNA damage. We found that sublethal concentrations of LY2603618 induced DNA damage and inhibited cell proliferation, and at high concentrations (5–20 lM), it induced cell cycle arrest apoptosis and necrosis. However, all of our measurements were performed 24 h after LY2603618 treatment. The time frame we used may only allow us to examine the early DNA damage response. Because DNA-damaging agents induce a dynamic range of DNA damage, responses may continue for a few more days after the initial insults. Whether LY2603618-inhibited tumor cell growth involves other mechanisms such as DNA damage-induced senescence requires further investigation [30, 31]. If the extent of DNA damage exceeds repair capacity, additional signaling pathways are activated to eliminate the
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autophagy in immunofluorescence studies to observe autophagic vesicle formation and by western blot to measure the ratio of LC3-II to LC3-I. Crosstalk between apoptosis and autophagy in cancer or radio-therapy treatment is very complex. In certain cancers, the cells can induce autophagy to resist the effect of chemotherapy. On the contrary, autophagy and apoptosis are often induced by the same stimuli and share similar effectors and regulators [33]. Here, we showed that LY2603618 induced autophagy in lung cancer cells, as demonstrated by an increase in the number of GFP-LC3 puncta, and promoted LC3 degradation. Inhibition of autophagy with CQ strongly enhanced LY2603618-induced apoptosis in A549 cells. In summary, we have demonstrated that the biological effects of LY2603618 in lung cancer cells are associated with not only inhibition of Chk-1 phosphorylation on S296, but also with the activation of the DNA damage response network (Fig. 6). Our results suggest that the anticancer property of LY2603618 might be enhanced when used in combination with an autophagy inhibitor. Fig. 6 Schematic depiction of LY26036180 s effects on lung cancer cells. LY2603618 inhibits Chk1 autophosphorylation (Ser296), which is required for maintaining basal Chk1 activity. As a result of LY2603618 treatment, DNA damage accumulates, which in turn amplifies ATM signaling, leading to an increase in phosphorylation of Chk1 (S345), Chk2 (T68) and H2AX, and activates the recruitment of DNA repair machinery. Chk1/Chk2-mediated inactivation of Cdc25 plays a major role in the dissociation of the cyclinB1/Cdc2 complex, leading to G2/M phase arrest by LY2603618. In addition, LY2603618 also induces autophagy, which inhibits LY2603618-indcued lung cancer cell apoptosis
damaged cells. Therefore, we investigated LY26036180 s effect on cell apoptosis, because apoptosis is a major mechanism used to eliminate cancer cells, and targeting apoptotic signaling intermediates could be an effective strategy for cancer prevention and treatment. The LY2603618-induced apoptosis was caused by the cleavage of caspase-3, caspase-9 and PARP. Furthermore, we observed a decrease in survivin expression, an anti-apoptosis protein, after LY2603618 treatment. Pretreatment of A549 cells with a JNK inhibitor attenuated LY2603618induced cleavage of caspase-3, PARP and inhibition of cell viability (predicated by the MTT assay), suggesting that LY2603618 may act via JNK activation to exert its apoptosis-inducing effects. Autophagy is a dynamic process in which a portion of the cytoplasm is sequestered in an autophagosome and subsequently degraded upon fusion with a lysosome [32]. It is usually considered that the conversion of soluble LC3-I to lipid-bound LC3-II is associated with autophagosome formation, with its characteristic double-membrane cytosolic vesicle. Therefore, LC3 has been used as a marker of
Acknowledgments This work was supported by grants from the National Natural Science Foundation of China (No. 81272683 to Feng-Ze Wang, No. 81271275 to Bao-Liang Sun). Conflict of interest of interest.
The authors declare that there are no conflicts
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ORIGINAL PAPER
The checkpoint 1 kinase inhibitor LY2603618 induces cell cycle arrest, DNA damage response and autophagy in cancer cells Feng-Ze Wang • Hong-rong Fei • Ying-Jie Cui • Ying-Kun Sun • Zhao-Mei Li • Xue-Ying Wang • Xiao-Yi Yang • Ji-Guo Zhang • Bao-Liang Sun
Ó Springer Science+Business Media New York 2014
Abstract Chemotherapy- or radiotherapy-induced DNA damage activates the Chk1-dependent DNA damage response (DDR) and cell cycle checkpoints to facilitate cell survival. Numerous attempts have been made to identify specific Chk1 inhibitors to enhance the efficiency of chemotherapy or radiotherapy. In this study, we investigated the molecular mechanisms underlying the antitumor activity of LY2603618, a potent and selective small molecule inhibitor of Chk1 protein kinase, in human lung cancer cells. Treatment of cancer cells with LY2603618 caused cell cycle arrest in the G2/M phase. A marked induction of DDR, including the phosphorylation of ATM,
Feng-Ze Wang and Hong-rong Fei have contributed equally to this work. F.-Z. Wang Z.-M. Li X.-Y. Wang School of Biological Science, Taishan Medical University, Taian 271016, People’s Republic of China F.-Z. Wang X.-Y. Yang B.-L. Sun (&) Key Lab of cerebral microcirculation in Universities of Shandong (Taishan Medical University), Taishan Medical University, Taian 271000, People’s Republic of China e-mail: [email protected] H. Fei J.-G. Zhang (&) School of Pharmacology, Taishan Medical University, Taian 271016, People’s Republic of China e-mail: [email protected] Y.-J. Cui Institute of Atherosclerosis, Taishan Medical University, Taian 271000, People’s Republic of China Y.-K. Sun College of Landscape Architecture and Forestry, Qingdao Agricultural University, Qingdao 266109, People’s Republic of China
Chk2, p53 and histone H2AX, was observed after LY2603618 treatment. LY2603618 inhibited Chk1 autophosphorylation (S296 Chk1) and increased DNA damagemediated Chk1 phosphorylation (S345 Chk1). In addition, LY2603618-treated lung cancer cells transitioned from LC3-I to LC3-II, a hallmark of autophagy. Blocking autophagy with chloroquine (CQ) further enhanced LY26036180 s inhibitory effect on cell viability/proliferation. LY2603618 also significantly increased p38 and c-Jun N-terminal kinase (JNK) phosphorylation. Pretreatment with the JNK inhibitor reduced cleavage of caspase-3 and PARP levels in LY2603618-treated cells. These results suggest the following: (i) the biological consequences of LY2603618 in lung cancer cells is associated with both inhibition of Chk1 phosphorylation on S296 and activation of the DNA damage response network; and (ii) the anticancer property of LY2603618 might be increased by inhibiting autophagy. Keywords Chk1 inhibitor LY2603618 G2/M phase DNA damage Autophagy
Introduction Genomic integrity maintenance is critical for cell survival and cell growth. DNA damage from normal metabolic processes and environmental factors induces cell cycle arrest in eukaryotic cells via checkpoint activation or DNA repair machineries to repair the damaged DNA [1, 2]. However, when DNA damage is irreparable, the damaged cells undergo apoptosis to avoid passing on the potentially lethal errors in DNA to their daughter cells. The cellular responses induced by DNA damage, including cell cycle checkpoint, DNA repair and DNA damage-induced
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programmed cell death, are collectively called the DNA damage response (DDR) [3], a versatile response to genomic DNA damages. Cells commonly respond to DDR by activating G1/S checkpoint and/or G2/M checkpoint, and the latter prevents the cells that have suffered DNA damage during G2 phase from initiating mitosis or from progressing into G2 phase despite unrepaired damage inflicted during previous S or G1 phases [4]. Key proteins involved in DNA damage and DNA strand break repair signaling are ATM (Ataxia telangiectasiamutated) and ATR (ATM and Rad3-related), two serine/ threonine kinase members of the phosphoinositide 3-kinase-like family. In response to DNA damage, ATM and/or ATR initiate cell cycle arrest via Chk1 and Chk2, and facilitate DNA repair complex activation to preserve DNA integrity [5, 6]. ATM is usually considered a response to DNA damage caused by ionizing radiation or oxidative stress, and it phosphorylates Chk2. In contrast, ATR primarily senses UV damage or replication stress and relays its effect via Chk1 phosphorylation. ATM/ATRChk1/Chk2 pathway activation leads to Cdc2 (Cdk1) inhibition through phosphorylation-mediated Cdc25 inactivation, thereby triggering G2/M phase arrest [7]. There are three isoforms of Cdc25: Cdc25A, Cdc25B, and Cdc25C. Cdc25B is thought to function at the G2/M cell cycle transition, and Cdc25C acts on M phase to sustain cyclinB1/Cdc2 activity [8]. Because DDR facilitates cancer cell survival after DNA damage, there is strong clinical interest in combining checkpoint inhibitors with DNA damage agents to increase cancer sensitivity [9, 10]. The G1/S checkpoint is frequently inactivated or lost in cancer due to p53 mutations, and the G2/M checkpoint may be important for cancer cells to respond to DNA-damaging chemotherapeutic agents [11]. The Chk1-mediated signaling pathway is required for the G2/M arrest in response to DNA-damaging agents. Chk1 phosphorylates and inhibits Cdc25, consequently blocking cyclinB1/Cdc2 activation and mitotic entry when DNA synthesis is inhibited [12]. Chk1 inhibitors may selectively enhance genotoxic agent activity in tumors by inhibiting the S and G2 checkpoints, while normal cells are rescued by their competent DDR, and many preclinical and clinical studies have focused on Chk1 inhibition to enhance tumor cell susceptibility to chemotherapy [13, 14]. A small molecule inhibitor of Chk1, LY2603618, was recently found to impair DNA synthesis and induce H2AX phosphorylation in cancer cells [15, 16]. However, the underlying mechanisms of its anticancer properties still need to be elucidated. In this study, we investigated the effects of LY2603618 on cell cycle progression, DNA damage responses, cell apoptosis and autophagy in nonsmall cell lung cancer cells. We found that LY2603618
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treatment of lung cancer cells resulted in accumulation of cells in the G2/M phase. LY2603618 also induced apoptosis and autophagy. Inhibition of autophagy enhanced LY2603618-mediated cleavage of caspase-3 and PARP. Treatment with a JNK inhibitor partially alleviated LY2603618-induced cell death.
Materials and methods Reagent LY2603618 was a gift from Lanmu Chemistry (Shanghai, China), and it was dissolved in DMSO. 3-[4, 5-dimethylthiazol-2-y-l]-2, 5-diphenyltetrazolium bromide (MTT) and chloroquine (CQ) were purchased from Sigma-Aldrich (St. Louis, MO). SP600125 was purchased from Calbiochem (La Jolla, CA, USA), SB203580 was purchased from Santa Cruz Technology (CA, USA). ECL Western blot detection reagents were purchased from PerkinElmer (Rockford, USA). Antibodies specific to b-actin, Atg-5 and LC3-II were obtained from Sigma-Aldrich. Rabbit anti-caspase-9, caspase-3, PARP, JNK, ATM, p-ATM, ATR, p-ATR, p-Chk2, p-Chk1(S345), p-Chk1(S296), c-H2AX, p-Cdc25C, p-p53, Cdc2, survivin and p-p38 antibodies were purchased from Cell Signaling Technology (Shanghai, China). Mouse antiChk2, p21, p27 and p-JNK antibodies were purchased from BD Biosciences. Anti-cyclinD1, Cdc25A, Cdc25B, cyclinB1, p53, ERK, p-ERK and p38 antibodies were purchased from Santa Cruz Technology. Anti-Chk1 and Cdc25C antibodies were purchased from Epitomics (Hangzhou, China). The antiH2AX antibody was obtained from Sangon Biotech (Shanghai, China). Cells and cell culture A549 and H1299 cell lines were obtained from the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China).Cells were cultured in RPMI medium 1640 (Gibco) plus 10 % fetal bovine serum (Gibco) in a humidified atmosphere (37 °C; 5 % CO2). Cell viability/proliferation assay A549 or H1299 cells were plated in 96-well plates for 24 h. The medium was removed, and the cells were treated with various concentrations of the test compound (LY2603618). A total volume of 20 lL MTT (5 mg/mL) was added for 4 h. After removal of the medium and MTT, 150 lL DMSO was added to each well, and the optical density was detected at 490 nm using a microplate reader.
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Cell cycle distribution analysis by flow cytometry Cells were treated with LY2603618 and DMSO as a control. After trypsinization, cells were fixed in 70 % ethanol at 4 °C overnight. The cells were washed twice with PBS and incubated for 30 min in the dark in PBS containing propidium iodide (PI) and RNase A. Stained cells were analyzed by a FACScan flow cytometry and CellQuest analysis software (Becton–Dickinson, San Jose, CA).
at 37 °C, and DAPI was used to stain the nuclei. The signals were detected by a fluorescence confocal microscope with the appropriate filter sets. Statistical analysis All results were expressed as mean ± SD. Each value is the mean of at least 3 independent experiments. Statistical analysis was performed by the student’s t test. P \ 0.05 was considered significant.
Apoptosis assay To evaluate apoptosis induction, TUNEL staining was performed according to the manufacturer’s instructions. Briefly, after exposure to LY2603618, cells were washed with PBS and fixed in 4 % (w/v) paraformaldehyde in PBS for 1 h at room temperature. Cells were then permeabilized with 0.1 % Triton X-100 for 2 min on ice. TUNEL staining was carried out using the In Situ Cell Death Detection Kit, Fluorescein (Roche, IN, USA). The stained cells were visualized with a fluorescence microscope (Olympus, Tokyo, Japan). GFP-LC3 localization by fluorescence microscopy Cells were seeded into 12-well plates and transfected with 0.5 lg GFP-LC3 plasmid using Lipofectamine 2000 (Invitrogen Life Technologies, CA, USA) according to the manufacturer’s instructions. After 24 h, cells were incubated with LY2603618 for another 24 h. Fluorescent signals were detected by a fluorescence microscope with the appropriate filter sets. Western blot analysis Cells were lysed with ice-cold RIPA lysis buffer containing PMSF and protease inhibitor cocktail for 20 min on ice, and the lysates were centrifuged at 13,000 g for 20 min at 4 °C. The supernatants were used as total cell lysates. The proteins were separated by SDS-PAGE and transferred onto nitrocellulose filter membranes. Membranes were blocked with 5 % milk powder and incubated with specific antibodies. Detection of the target proteins on the membranes was performed using the ECL Western Blotting Detection Reagents. Immunoflourescence assays Cultured cells were fixed with cold 100 % methanol at -20 °C for 10 min and permeabilized with PBS-0.1 %Triton X-100 for 5 min. Coverslips were blocked with 3 % BSA, and the cells were subsequently incubated with an anti-c-H2AX antibody (Abcam, Hong Kong) for 2 h. After washing, cells were incubated with a FITC-conjugated secondary antibody (Santa Cruz, 1:100 dilution) for 30 min
Results LY2603618 induces cell cycle arrest at the G2/M phase in wild-type and p53-deficient lung cancer cells To determine the antitumor activities of LY2603618, we first assessed the effect of LY2603618 on cell cycle distribution by flow cytometry. As shown in Fig. 1a, A549 control cell populations in the G1, S and G2/M phases were 54.88, 31.81 and 13.31 %, respectively. After 24 h of incubation with 5 or 10 lM LY2603618, the percentage of G2/M phase cells increased to 32.31 and 38.95 %, respectively. We observed similar changes in cell cycle distribution after LY2603618 treatment in H1299 cells, indicating that LY2603618 induces cell cycle arrest in G2/M phase in human lung cancer cells. In mammalian cells, cyclinB1 and Cdc2 form a complex and cooperate to promote the G2/M phase transition. Therefore, we next investigated whether LY2603618 regulated cyclinB1 and Cdc2 expression in lung cancer cells. LY2603618 treatment considerably inhibited cyclinB1 and Cdc2 expression in both A549 and H1299 cells. The Cdc25C phosphatase is thought to regulate Cdc2 phosphorylation, and thus we also examined Cdc25C phosphorylation. As shown in Fig. 1b, p-Cdc25C (S216) levels decreased in LY2603618-treated cells, and Cdc25B expression was also inhibited. However, p21 and p27 expression increased upon LY2603618 treatment. We did not observe any significant changes in Cdc25A, Cdc25C and cyclinD1 levels. Phosphorylation of histone H3 on Ser10 plays an important role in mammalian mitotic chromatin condensation [17]. Thus, we examined whether LY2603618 had an effect on histone H3 phosphorylation. We found that 24 h LY2603618 treatment induced increased phosphorylation of histone H3 (Fig. 1b). LY2603618 activates DNA damage sensor kinases in A549 and H1299 cells To investigate the mechanisms of G2/M cell cycle arrest triggered by LY2603618, we investigated the genotoxicity
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Fig. 1 LY2603618 induces G2/M arrest in non-small cell lung cancer cells. a A549 and H1299 cells were exposed to different concentrations of LY2603618 for 24 h, and cell cycle distributions were measured by flow cytometry. b The effect of LY2603618 on cell
cycle regulatory proteins was examined by western blot analysis. Cells were treated with LY2603618 for 24 h, and total cell lysates were analyzed for cell cycle regulatory proteins with specific antibodies. b-actin was used as an internal control
of this compound using immunofluorescence staining to monitor the formation of c-H2AX foci in A549 lung cancer cells. As shown in Fig. 2a, LY2603618 treatment increased the formation of c-H2AX foci in lung cancer cells. We then examined phosphorylation of the nuclear histone H2AX by western blot assay and found that LY2603618 induced total H2AX expression and H2AX phosphorylation (Fig. 2b). We also observed an increase in H2AX phosphorylation levels in LY2603618-treated H1299 cells. To confirm that
LY2603618 inhibited Chk1 activities in lung cancer cells, we analyzed Chk1 signaling by western blot analysis. As shown in Fig. 2c, increasing concentrations of LY2603618 induced the downregulation of Chk1 autophosphorylation (S296), suggesting that LY2603618 inhibits Chk1. We also found that DNA damage-mediated phosphorylation of Chk1 (S345) and Chk2 (T68) were increased in response to LY2603618 treatment (Fig. 2c). To further elucidate the mechanisms leading to LY2603618-mediated DNA
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Fig. 2 Analysis of LY2603618-induced DNA damage in human lung cancer cells. a DNA double-strand breaks, as measured by c-H2AX, were induced by LY2603618 treatment. A549 cells were exposed to different concentrations of LY2603618 for 24 h. Cells were then fixed and immunostained with anti-c-H2AX antibody and DAPI. b LY2603618 induced H2AX phosphorylation in lung cancer cells. A549 and H1299 cells were treated with LY2603618 for 24 h.
The cells were analyzed by western blot analysis using anti-c-H2AX or anti-b-actin antibodies. c LY2603618 treatment resulted in increased phosphorylation of ATM, Chk2 and p53. Cells were treated with the indicated concentrations of LY2603618 (1–20 lM) for 24 h. ATM/ATR-Chk1/Chk2 signaling pathway proteins were measured by western blot with specific antibodies
damage, we examined ATM and ATR phosphorylation following LY2603618 exposure. Western blot results showed that LY2603618 increased ATM phosphorylation at S1981, whereas LY2603618 had no effect on ATR phosphorylation at residue S428. Total p53 levels and phosphorylated p53 (S15) levels were also increased after LY2603618 treatment (Fig. 2c).
fashion. We also examined whether LY2603618 affected survivin expression because survivin was reported to play a crucial role in regulating apoptosis [18]. We found that LY2603618 dose-dependently suppressed survivin expression in both A549 and H1299 cells.
LY2603618-induced apoptosis is associated with caspase activation and survivin inhibition Apoptosis is another important mechanism involved in cancer therapy. Therefore, we examined apoptosis in LY2603618-treated cells by identifying TUNEL-positive cells. As shown in Fig. 3a, LY2603618 treatment induced apoptosis of non-small cell lung carcinoma cells. To elucidate whether LY2603618-treated cancer cells express canonical apoptotic markers, we examined the effects of LY2603618 on apoptosis-related protein levels. As shown in Fig. 3b, treatment of cancer cells with LY2603618 for 24 h increased the cleavage of the initiator caspase-9, caspase-3 and PARP in a dose-dependent
Blocking autophagy enhances LY2603618 cytotoxic activity on human lung cancer cells The conversion of soluble LC3-I to lipid bound LC3-II via proteolytic cleavage and lipidation shows autophagy induction [19]. To study whether LY2603618 induced autophagy in lung adenocarcinoma cells, we examined the formation of LC3 dots and conversion of LC3-I to LC3-II. pEGFP-LC3-transfected cells were treated with or without LY2603618 for 24 h and subjected to immunofluorescence for visualization of GFP-LC3 expression. We found that LY2603618 induced the redistribution of GFP-LC3 from a diffuse distribution (control cells) to punctate structures (LY2603618-treated cells) (Fig. 4a), which indicates that LY2603618 induced autophagy in lung cancer cells. Next, we examined LC3-II protein levels, a promising
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LY2603618 and CQ markedly decreased A549 cell viability/proliferation compared to the control group, indicating that the suppression of autophagy enhanced LY2603618induced cell viability/proliferation inhibition. In agreement, the combination of LY2603618 and CQ was more potent than LY2603618 alone in inducing apoptosis, as evidenced by the increased cleavage of caspase-3 and PARP (Fig. 4d). These data indicate that LY2603618-induced autophagy represents a pro-survival mechanism, inhibition of which drives lung cancer cells to death. We also detected changes in LY2603618-induced H2AX expression in the presence of CQ. As shown in Fig. 4e, inhibition of autophagy significantly enhanced LY2603618-mediated H2AX phosphorylation, suggesting that inhibition of autophagy with CQ potentiates the LY2603618-induced DNA damage response. LY2603618 increases JNK and p38 MAPK phosphorylation in lung cancer cells
Fig. 3 The apoptotic effect of LY2603618 on human lung cancer cells. a Cells were treated with different concentrations of LY2603618 for 24 h. Apoptosis was analyzed by TUNEL staining. Results showed the presence of fragmented DNA in LY2603618treated cells. b LY2603618 treatment induced the cleavage of PARP, caspase-9, caspase-3, and decreased survivin expression. After cells were treated with LY2603618 for 24 h, total cell lysates were harvested and analyzed by western blot with specific antibodies against PARP, caspase-9, caspase-3 and survivin. b-actin was used as a loading control
autophagosomal marker. Western blot results showed significantly increased conversion of LC3-I to LC3-II with increasing concentrations of LY2603618 (Fig. 4b). We also measured the expression of autophagy-related proteins Atg5 in LY2603618- treated cells and found that LY2603618 increased Atg5 levels (Fig. 4b). These results indicate that LY2603618 stimulates the conversion of LC3I to LC3-II and induces autophagy. We further determined the role of LY2603618-induced autophagy in cell survival or cell death in lung cancer cells by blocking autophagy with CQ, a lysosomal protease inhibitor. As shown in Fig. 4c, the combination of
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It has been previously shown that MAPK signaling is associated with cell proliferation, survival, DNA damage and autophagy [20, 21]. To determine whether the MAPK pathway was affected by LY2603618 in lung cancer cells, we detected the phosphorylation of JNK and p38 MAPK by western blot analysis. The results showed that LY2603618 increased JNK and p38 MAPK phosphorylation in a dosedependent manner after 24 h (Fig. 5a). To determine whether activated JNK and/or p38 MAPK play a role in LY2603618-induced caspase cleavage, A549 cells were incubated with the JNK inhibitor SP600125 or p38 MAPK inhibitor SB203580. SP600125 partially prevented LY2603618-induced cleavage of caspase-3 and PARP (Fig. 5b). Moreover, SP600125 significantly blocked LY26036180 s inhibition of cell viability/proliferation (Fig. 5c). These results suggest that LY2603618induced apoptosis in non-small cell lung cancer cells is associated with JNK activation.
Discussion In the past several years, various specific small molecule Chk1 inhibitors have been developed [22]. LY2603618 has entered into phase I clinical trials and showed acceptable safety and pharmacokinetic profiles. The present study was performed to determine the anticancer effect of LY2603618 in non-small cell lung cancer cells and to investigate the underlying cellular mechanism. We have demonstrated that targeting non-small cell lung cancer with LY2603618 induces cell cycle arrest in G2/M, accompanied by a decrease in cyclinB1, Cdc2, Cdc25B and p-Cdc25C expression, and increased levels of p21 and p27. LY2603618 inhibits phosphorylation of Chk1 at Ser296 but induces
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Fig. 4 Induction of autophagy in LY2603618-treated lung cancer cells. a Formation of LC3-positive vesicles in LY2603618-treated GFP-LC3 transduced cells. Lung cancer cells were transfected with GFP-LC3 plasmids and treated with 0, 5 or 10 lM LY2603618 for 24 h, followed by observation of GFP-LC3 by fluorescence microscopy. b A549 and H1299 cells were treated with the indicated concentrations of LY2603618 for 24 h and subjected to western blot to detect the levels of LC3-I, LC3-II and Atg-5 with b-actin as an
internal control. c The effects of the autophagic inhibitor CQ on LY2603618-induced cell viability/proliferation inhibition. Cells were treated with 20 lM CQ for 2 h before the addition of 10 lM LY2603618. After 24 h, cell viability/proliferation was determined by MTT. d The activation of caspase-3 and PARP was measure by western blot analysis in A549 cells. e Cells were treated with 20 lM LY2603618 and/or 20 lM CQ for 24 h. The levels of LC3-II and c-H2AX were determined by western blot analysis
phosphorylation of Chk1 at Ser345. LY2603618 treatment provoked the DNA damage response and apoptosis. Inhibition of autophagy by CQ increased LY26036180 s effect on cell viability/proliferation. LY2603618 may act via the MAPK signaling pathway to elicit its anti-cancer effect. Cell cycle arrest during the DNA damage checkpoint is carried out by the sequential action of ATM/ATR-Chk1/
Chk2-Cdc25A/Cdc25C, and it eventually results in the inhibitory phosphorylation of Cdc2 and activation of the cyclinB1/Cdc2 complex [23, 24]. As an essential kinase involved in cell cycle checkpoints, Chk1 was phosphorylated at Ser345, Ser317, and Ser296 in response to DNA damage [25]. Ser317 and Ser345 phosphorylation is predominantly
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Apoptosis Fig. 5 LY2603618 activates JNK1/2 and p38 MAPK in human lung cancer cell lines. a Cells were treated with the indicated concentrations of LY2603618 for 24 h, and the levels of JNK1/2 and p38 phosphorylation were determined by western blot analysis. b LY2603618-induced JNK activation is involved in apoptosis. A549 cells were treated with 20 lM SP600125 or 20 lM SB203580 1 h before the addition of LY2603618 (20 lM) for 24 h. Caspase-3 and PARP levels were detected by western blot analysis. c The LY2603618-mediated reduction of lung cancer cell viability/ proliferation was significantly inhibited by SP600125
catalyzed by ATR, while Ser296 phosphorylation occurs through autophosphorylation [26]. Our studies showed, for the first time, that LY2603618 inhibited Ser296 Chk1 phosphorylation in human lung cancer cells. However, phosphorylated Ser-345 Chk1 (pS345 Chk1) levels significantly increased in response to low doses of LY2603618, suggesting that LY2603618 induces the DNA damage response and further amplifies ATR/ATM-mediated Chk1 phosphorylation (S345) in human lung cancer cells. This is consistent with a report by Parsels LA and Riesterer O in which the two Chk1 inhibitors AZD7762 and XL-844 also induced Chk1 (S345) phosphorylation [27, 28]. We found that LY2603618 strongly induces the DNA damage response in lung cancer cells. When cells are exposed to DNA-damaging chemotherapeutic agents, double-stranded breaks (DSBs) are generated, rapidly resulting in H2AX phosphorylation at Ser139 (c-H2AX) by ATM, ATR or DNA-PK. Because this process is robust, fast and correlates well with each DSB, H2AX phosphorylation is considered as a sensitive marker of DNA damage [29]. Our data presented here demonstrate that LY2603618 induced the formation of c-H2AX foci and increased cH2AX levels in human cancer cells. We observed increased phosphorylation of ATM, Chk2 and p53 after LY2603618 treatment. Therefore, LY2603618 induced the whole spectrum of the DNA damage response pathway.
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Although 1 lM LY2603618 robustly activated DNA damage response factors, including c-H2AX and p-ATM, phosphorylation of Chk1 at Ser296 was only slightly inhibited at this concentration of LY2603618, suggesting that LY2603618 may activate the DSB response pathway independently of Chk1 inhibition. DNA-damaging agents trigger complex responses in human cells, including the activation of cell cycle checkpoints to facilitate repair, and the induction of apoptotic cell death or growth arrest via accelerated cellular senescence, depending on the genetic background of the cells and the extent of DNA damage. We found that sublethal concentrations of LY2603618 induced DNA damage and inhibited cell proliferation, and at high concentrations (5–20 lM), it induced cell cycle arrest apoptosis and necrosis. However, all of our measurements were performed 24 h after LY2603618 treatment. The time frame we used may only allow us to examine the early DNA damage response. Because DNA-damaging agents induce a dynamic range of DNA damage, responses may continue for a few more days after the initial insults. Whether LY2603618-inhibited tumor cell growth involves other mechanisms such as DNA damage-induced senescence requires further investigation [30, 31]. If the extent of DNA damage exceeds repair capacity, additional signaling pathways are activated to eliminate the
Apoptosis
autophagy in immunofluorescence studies to observe autophagic vesicle formation and by western blot to measure the ratio of LC3-II to LC3-I. Crosstalk between apoptosis and autophagy in cancer or radio-therapy treatment is very complex. In certain cancers, the cells can induce autophagy to resist the effect of chemotherapy. On the contrary, autophagy and apoptosis are often induced by the same stimuli and share similar effectors and regulators [33]. Here, we showed that LY2603618 induced autophagy in lung cancer cells, as demonstrated by an increase in the number of GFP-LC3 puncta, and promoted LC3 degradation. Inhibition of autophagy with CQ strongly enhanced LY2603618-induced apoptosis in A549 cells. In summary, we have demonstrated that the biological effects of LY2603618 in lung cancer cells are associated with not only inhibition of Chk-1 phosphorylation on S296, but also with the activation of the DNA damage response network (Fig. 6). Our results suggest that the anticancer property of LY2603618 might be enhanced when used in combination with an autophagy inhibitor. Fig. 6 Schematic depiction of LY26036180 s effects on lung cancer cells. LY2603618 inhibits Chk1 autophosphorylation (Ser296), which is required for maintaining basal Chk1 activity. As a result of LY2603618 treatment, DNA damage accumulates, which in turn amplifies ATM signaling, leading to an increase in phosphorylation of Chk1 (S345), Chk2 (T68) and H2AX, and activates the recruitment of DNA repair machinery. Chk1/Chk2-mediated inactivation of Cdc25 plays a major role in the dissociation of the cyclinB1/Cdc2 complex, leading to G2/M phase arrest by LY2603618. In addition, LY2603618 also induces autophagy, which inhibits LY2603618-indcued lung cancer cell apoptosis
damaged cells. Therefore, we investigated LY26036180 s effect on cell apoptosis, because apoptosis is a major mechanism used to eliminate cancer cells, and targeting apoptotic signaling intermediates could be an effective strategy for cancer prevention and treatment. The LY2603618-induced apoptosis was caused by the cleavage of caspase-3, caspase-9 and PARP. Furthermore, we observed a decrease in survivin expression, an anti-apoptosis protein, after LY2603618 treatment. Pretreatment of A549 cells with a JNK inhibitor attenuated LY2603618induced cleavage of caspase-3, PARP and inhibition of cell viability (predicated by the MTT assay), suggesting that LY2603618 may act via JNK activation to exert its apoptosis-inducing effects. Autophagy is a dynamic process in which a portion of the cytoplasm is sequestered in an autophagosome and subsequently degraded upon fusion with a lysosome [32]. It is usually considered that the conversion of soluble LC3-I to lipid-bound LC3-II is associated with autophagosome formation, with its characteristic double-membrane cytosolic vesicle. Therefore, LC3 has been used as a marker of
Acknowledgments This work was supported by grants from the National Natural Science Foundation of China (No. 81272683 to Feng-Ze Wang, No. 81271275 to Bao-Liang Sun). Conflict of interest of interest.
The authors declare that there are no conflicts
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