{'fraction_non_alphanumeric': 0.043459436055636735, 'fraction_numerical': 0.045949017697678195, 'mean_word_length': 4.81801007556675, 'pattern_counts': {'":': 0, '<': 1, '<?xml version=': 0, '>': 1, 'https://': 0, 'lorem ipsum': 0, 'www.': 0, 'xml': 0}, 'pii_count': 0, 'substrings_counts': 0, 'word_list_counts': {'cursed_substrings.json': 0, 'profanity_word_list.json': 0, 'sexual_word_list.json': 0, 'zh_pornsignals.json': 0}}
CDS-1548 induces apoptosis in HeLa cells by activating caspase 3

Cervical cancer continues to be a threat to female health globally. In the present study, the potential anticancer activity of 2-[2-hydroxyl-1-(4-methoxy phenyl) ethyl]-3-(4-benzyloxy phenyl) isoindolin-1-one (CDS-1548), was evaluated in HeLa cells. CDS-1548 is an organic small-molecule compound characterized by two chiral centers, with the nuclear parent 1H-isoindolin-1-one. CDS-1548 administration significantly elevated the transcriptional activity of p53 and its downstream target genes in a dose-dependent manner. Additionally, CDS-1548 treatment increased the expression levels of p53 and mouse double minute 2 homolog, as well as inducing apoptosis in HeLa cells. Furthermore, CDS-1548 treatment downregulated the expression of B-cell lymphoma 2, upregulated Bcl-2 homologous antagonist killer, promoted the release of cytochrome c from mitochondria to cytoplasm, and activated the production of caspase 3 and 9. Collectively, these results suggested that CDS-1548 inhibited HeLa cell proliferation by promoting G2/M cell cycle phase arrest and inducting of mitochondria-mediated apoptosis.


Introduction
Cervical cancer is a gynecological disease that continues to threaten the health of women globally. By integrating viral DNA into human chromosomal DNA and activating proto-oncogenes (or deactivating tumor suppressor genes), the Human papilloma virus (HPV) is one of the primary causes of cervical cancer (80-90%) (1). Currently, >100 HPV variants have been identified, though few are carcinogenic (2). HPV types 16 and 18, classified as high-risk HPVs, are able to promote the progression of premalignant lesions, which unless treated, ultimately results in the development of cancer (3). Of all cervical cancer cases, ~70% have been associated with HPV 16 and 18 (4); furthermore, high-risk HPVs secrete early protein 6 that forms a complex with p53, resulting in its ubiquitination and subsequent degradation (5).
p53 is a powerful transcription factor that prevents the malignant transformation of cells, and as such is frequently inactivated following viral infection (6,7). Various stress signals, including oncogene activation, DNA damage and hypoxia, improve the stability of wild-type p53. Alterations in p53 expression level are associated with the transcription of specific p53-responsive gene, including p21, Bcl-2-associated X protein (Bax), p53 upregulated modulator of apoptosis (PUMA) and growth arrest and DNA damage-inducible 45, which are involved in p53-induced cell cycle arrest, metabolism, DNA repair, apoptosis and senescence (8,9). Previous investigations have revealed that mutations in p53 are present in ~50% human cancers (10). Under normal conditions, p53 is tightly regulated by the mouse double minute 2 homolog (Mdm2) protein via a self-regulating feedback loop (11,12). p53 is able to increase the expression level of Mdm2, which conversely inhibits p53 in three ways: i) Mdm2 binds to the transactivation domain of p53 and subsequently inhibits transcriptional activity; ii) Mdm2 blocks the nuclear export of p53; or iii) Mdm2 may function as an E3 ubiquitin protein ligase, targeting p53 for ubiquitination and degradation (13). Furthermore, the overexpression of the Mdm2 gene may result in loss of p53 function in numerous types of malignant tumor (14). Therefore, interrupting the p53-Mdm2 interaction (or Mdm2 itself) with small-molecule inhibitors may reactivate p53 and inhibit tumor growth (15,16); the development of nutlin-3 provides important proof-of-concept for the design of small-molecule inhibitors of Mdm2 (17).
The present study introduced the organic small-molecule compound 2-[2-hydroxyl-1-(4-methoxy phenyl) ethyl]-3-(4-benzyloxy phenyl) isoindolin-1-one (CDS-1548), which altered the activity of cervical cancer cell lines by promoting the accumulation of p53 and inducing apoptosis. In the present study, the method by which CDS-1548 activates p53 and inhibits tumor cell viability (by eliciting apoptosis and cell cycle arrest) was investigated.
Cell viability assay. An MTT assay was conducted to evaluate the effects of CDS-1548 on cell viability. Briefly, 5x10 3 cells/well were seeded into 96-well plates and incubated in serum-free DMEM for 24 h at 37˚C. The cells were washed with phosphate-buffered saline (PBS, pH 7.0) and treated at 37˚C with various concentrations of CDS-1548 (0.14, 0.37, 1.1, 3.3, 11, 33 and 100 µM, to a final volume of 100 µl/well) for 12, 24 and 48 h. Following treatment, 20 µl MTT solution was added and the cells were incubated for an additional 3 h at 37˚C. The culture medium was subsequently removed and 150 µl dimethyl sulfoxide (DMSO) was added to dissolve the formazan crystals. The absorbance of was assessed at 495 nm using a microplate reader and equated to the number of viable cells.
Nuclear staining. To examine the condensation and fragmentation of cellular nuclei, 5x10 4 cells/well were seeded in to 24-well plates and incubated for 24 h; the medium was then replaced with equal quantities of DMEM or DMEM + 2 µM CDS-1548. Following a further 24-h incubation at 37˚C, the cells were washed with PBS and stained with DAPI (2.5 µg/ml) for 5 min at room temperature. The morphology of the nuclei was determined by fluorescence microscopy (magnification, x10).
Cell cycle status and apoptosis assay. Early apoptotic cells were characterized by the translocation of phosphatidylserine to the outer surface of the cell membrane (22). Annexin V/PI staining was used to detect the number of apoptotic cells, according to the manufacturer's protocol. Briefly, 4x10 5 cells/well were incubated overnight in 6-well plates, and treated with 2, 5, or 10 µM CDS-1548 prior to a further 24, 48 or 72-h incubation at 37˚C. Following treatment, the cells were harvested in 15 ml tubes by trypsinization, and the medium was removed by centrifugation at 3,000 x g for 5 min at 4˚C. For the apoptotic assay, cells were washed twice in ice-cold PBS, aspirated and resuspended in binding buffer with FITC-annexin V and PI at room temperature for 15 min (in the dark). Subsequently, cells were resuspended in binding buffer and flow cytometricaly analyzed using the Beckman Flow Cytometry Analyzer (Beckman CytoFLEX; Beckman Coulter, Inc.,). For cell cycle analysis, following the aforementioned CDS-1548 treatment, cells were rinsed in PBS, resuspended in 5 ml PI solution (25 µg/ml RNase A, 50 µg/ml PI) and incubated at 37˚C for 30 min in the dark. The cell cycle distribution was determined using the Beckman Flow Cytometry Analyzer and the data were statistically analyzed using SPSS 19.0 software (IBM Corp.).
Western blotting. Following treatment with CDS-1548, the cells were lysed at 4˚C using ice-cold lysis buffer [50 mmol/l Tris (pH 8.0), 150 mmol/l NaCl, 0.1% SDS, 1% NP40 and 0.5% sodium deoxycholate] supplemented with protease/phosphatase inhibitors (1% Cocktail and 1 mmol/l phenylmethylsulfonyl fluoride). The lysates were centrifuged at 10,000 x g for 5 min at 4˚C, and the supernatant was collected. Protein quantification was performed using a Bradford assay kit and 40 µg protein/lane was separated by SDS-PAGE (10% gel). The proteins were transferred to polyvinylidene fluoride membranes (EMD Millipore) and blocked using TBS with 5% fat-free milk and 0.1% tween-20 for 1 h at room temperature. The membranes were washed twice in TBST and incubated with primary antibodies (1:200) overnight at 4˚C. The membranes were rinsed three times with ice-cold PBS and then incubated with peroxidase-conjugated secondary antibodies (1:2,000) for 2 h at room temperature. The protein bands were visualized using the BeyoECL Star kit (Beyotime Institute of Biotechnology).
Mitochondria and cytosol extraction. Briefly, cells (8x10 6 ) were seeded in a T75 flask and then incubated overnight at 37˚C. After 24-h treatment with CDS-1548 (2, 5 and 10 µM), cells were scraped from the plate in PBS and harvested by centrifugation at 500 x g for 5 min at 4˚C. The cell pellets were resuspended in extraction buffer from the Cell Mitochondria Isolation kit [20 mmol/l HEPES (pH 7.5), 1.5 mmol/l MgCl 2 , 10 mmol/l KCl, 1 mmol/l EDTA, 1 mmol/l EGTA, 1 mmol/l DTT, 0.1 mmol/l phenylmethylsulfonyl fluoride, and 250 mmol/l sucrose], and homogenized using a microhomogenizer. The homogenates were subsequently centrifuged at 750 x g for 10 min at 4˚C. To isolate mitochondrial extracts, the supernatants were re-centrifuged at 10,000 x g for 15 min at 4˚C, and the precipitate (mitochondria protein extract) and the supernatant (cytoplasmic fractions.) were retrieved.
Determination of caspase activity. Caspase-3, 8 and9 activity were determined using caspase-3/8/9 activity kits according to the manufacturer's protocols. Briefly, following treatment with CDS-1548, the cells were scraped from the culture plates in PBS (0.01 M, pH 7.4) and centrifuged at 10,000 x g at 4˚C. The cell pellets were lysed in 100 µl lysis buffer and the resulting suspension was centrifuged at 10,000 x g for 10 min at 4˚C. An equal amount of supernatant was incubated with the corresponding substrates in reaction buffer containing dithiothreitol, and the absorbance at 405 nm was determined using a microplate reader.
Statistical analysis. All quantitative data are expressed as the mean ± standard deviation of three independent experiments. Statistical differences were evaluated using analysis of variance and the Least Significant Difference post hoc test. SPSS 19.0 (SPSS, Inc., IL, USA) was used for statistical analysis and P<0.05 was considered to indicate a statistically significant difference.

Inhibitory effects of CDS-1548 on HeLa cells.
To assess the inhibitory effects of CDS-1548 on HeLa cells (Fig. 1A), a concentration-escalation experiment was performed (Fig. 1B). CDS-1548 reduced cell viability in a dose-and time-dependent manner. The MTT assay revealed that the half maximal inhibitory concentration (IC 50 ) of CDS-1548 in HeLa cells was markedly reduced following increasing treatment durations. The IC 50 at 12, 24 and 48 h were 100.0, 4.3 and 4.0 µM, respectively. Notably, at 11 µM, CDS-1548 significantly enhanced cell cytotoxicity following 24-and 48-h of treatment, suggesting that this was the optimum concentration in HeLa cells. However, significant differences were not observed between the 24-and 48-treatments due to the cycling time of the HeLa cell line.

CDS-1548 induces G 2 /M arrest in HeLa cells.
To determine whether CDS-1548 caused cell-cycle arrest, a flow cytometric cell cycle assay was performed using HeLa cells following 24 h of treatment with 0, 2, 5, or 10 µM CDS-1548. A significantly increased percentage of G 2 /M-phase cells was observed from 25.115% (0 µM) to 38.65% (2 µM), 73.03% (5 µM) or 89.62% (10 µM CDS-1548; Fig. 2A). To further elucidate the potential molecular mechanism underlying CDS-1548-induced cell-cycle arrest, the expression levels of proteins associated with G 2 /M phase progression were determined. The results indicated that CDS-1548 downregulated the expression level of cyclin B1 in a dose-dependent manner, upregulated the expression levels of p-CHK1 and decreased the levels of CDC25C compared with the control-treated cells. However, CDS-1548 did not alter the expression levels of CHK2 or its phosphorylation (Fig. 2B). Alterations in the expression levels of p-ATR and the histone γ-H2AX, DNA-damage markers associated with CHK1, were also determined. CDS-1548 treatment increased the level of γ-H2AX expression and ATR phosphorylation. These data suggested that CDS-1548 induced cell cycle arrest at the G 2 /M-phase via DNA-damage checkpoint pathways.

CDS-1548 induces p53-dependent apoptosis in human cervical cancer cells.
The present study investigated whether the inhibitory effect of CDS-1548 on HeLa cells also induced cell death. As illustrated in Fig. 3A, chromatin condensation and apoptotic bodies were observed in the nuclei subsequent to treatment with CDS-1548. Furthermore, flow cytometric analysis indicated that CDS-1548 induced the accumulation of early apoptotic cells (annexin V+/PI-), suggesting that apoptosis in HeLa cells was dose-and time-dependent (Fig. 3B). After 24-h treatment with CDS-1548 (2, 5 and 10 µM) the percentages of early apoptotic cells were 2.82, 9.21 and 14.7%, and the percentages of necrotic cells (annexin V+/PI+) were 3.69, 2.95 and 4.16% respectively. Compared with 24-h CDS-1548 treatment, the proportions of early apoptotic cells in the 48-h treatment group was significantly increased, to 3.33, 35.51 and 51.32%; the percentages of necrotic cells were 0.89, 3.05 and 6.41% for 2, 5, and 10 µM CDS-1548, respectively. These results indicated that the inhibitory effects observed in response to CDS-1548 were primarily associated with the induction of apoptosis, and not necrosis in HeLa cells.
To confirm the effect of CDS-1548 on p53 activation, the expression levels of p53, p-p53 and the downstream target genes of p53 were determined. Fig. 3C and D illustrate that the mRNA and protein expression levels of p53, p21 and PUMA were increased in a dose-dependent manner in HeLa cells, compared with control treated cells. Furthermore, the upregulation of Mdm2 protein expression was observed. These results demonstrated that p53 accumulated in HeLa cells following CDS-1548 treatment, resulting in an increase in the expression levels of p21, Mdm2 and PUMA in a p53-associated manner.

CDS-1548 triggers apoptosis by activating the mitochondriamediated pathway.
Previous investigations have demonstrated that p53-mediated apoptotic cell death is associated with the intrinsic mitochondrial pathway (23,24). The present study investigated the expression of PARP and pro-caspase-3 (an effector caspase) to determine whether cell death was apoptotic in nature. As presented in Fig. 4A, CDS-1548 resulted in the cleavage of PARP and decreased the expression levels of pro-caspase 9 and 3 in a dose-dependent manner; the expression level of pro-caspase 8 was not altered. These results suggested that CDS-1548 induced apoptotic cell death. Furthermore, CDS-1548 treatment downregulated Bcl-2 expression levels and upregulated those of BAK, but not BAX, resulting in the release of cytochrome c into the cytosol   of HeLa cells. The caspase activity assay further indicated an increase in caspase 3 and 9 activity following CDS-1548 treatment, while caspase 8 activity remained unaltered (Fig. 4B). These results implied that CDS-1548 treatment inhibited Bcl-2 expression, but promoted BAK expression and cytochrome c release, inducing apoptosis via the mitochondria-mediated signaling pathway.

Discussion
Cervical cancer is an important threat to female health globally (25). Currently, regular screening and vaccination are effective means of preventing cervical cancer (26)(27)(28)(29), though these methods are expensive and may not be affordable in developing countries (30). Chemotherapeutic treatments, although effective in a proportion of patients, are not completely curative and are accompanied by severe side effects (31). As an alternative approach, small molecule inhibitors that destroy cancerous cells with less toxic effects on normal cells may be beneficial (32). In previous years, potent, selective and efficacious small-molecule inhibitors have been successfully developed, and a number of these compounds have been advanced into clinical trials for the treatment of human cancers (33).
In the present study, the anticancer effect of CDS-1548 on HeLa cells was evaluated. CDS-1548 is characterized by two chiral centers, with 1H-isoindolin-1-one as a nuclear parent. The results of the present study revealed that CDS-1548 treatment induced the activation of p53 and elevated the mRNA and protein expression levels of p53-targeted genes. Additionally, p21 may suppress CDK1 and cyclin B1 expression by inhibiting either CDK activity or impeding the formation of the CDK-cyclin B1 complex, thereby resulting in cell cycle arrest at the G 2 /M phase (34). Furthermore, the upregulated expression level of PUMA, an important regulatory factor of p53-mediated apoptosis (35), was observed. It was hypothesized that CDS-1548-induced p53 activation and elevated expression levels of p21 and PUMA served important roles in the promotion of G 2/ M cell cycle arrest and apoptosis. The inhibition of Bcl-2 expression, the upregulation of BAK, the release of cytochrome c and the activation of caspase 3 and caspase 9 are considered to be markers of early-stage apoptosis; however, the potential function of CDS-1548 as an inhibitor of p53 requires further investigation by experimentation with alternative cell lines and in vivo studies.
The proliferation of cancer cells depends on a defect or dysfunction at the G 1 checkpoint, and subsequent entry into the S and G 2 phases, where DNA damage repair is initiated (36). Therefore, cancer cells in the G 2 /M phase are sensitive to the cytotoxic effects of chemotherapeutic drugs. More importantly, the activation of p53 promotes apoptosis upon G 2 /M phase arrest in response to DNA damage (37). The results of the present study indicated that CDS-1548 induced cell cycle arrest at the G 2 /M phase, which was accompanied by the downregulation of cyclin B1, CDK1 and CDC25C. Additionally, the phosphorylation of CHK1 and the upregulation of ATR expression (involved in the DNA-damage response) were observed. Of note, there were no observed changes in CHK2 protein expression level in HeLa cells. Subsequently, it was confirmed that CDS-1548 treatment significantly induced the phosphorylation of ATR and γ-H2AX in HeLa cells, collectively suggesting that CDS-1548 induced p53-dependent apoptosis by promoting G 2 /M phase arrest.
Further investigation is required to address the potential molecular mechanisms involved in CDS-1548-mediated p53 activation. Specifically, the regulatory action of CDS-1548 on Mdm2 and p53 is an important feedback loop that requires further clarification through in vitro and in vivo anti-tumor activity assays.
In conclusion, the present study demonstrated that the novel small-molecule inhibitor CDS-1548 possessed cytotoxicity against cancer cells. Furthermore, CDS-1548 triggered apoptosis via p53 accumulation and cell cycle arrest at the G 2 /M phase, indicating that the use of small-molecule inhibitors that target p53 may be a potential strategy for the treatment of cervical cancer.