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Cellular senescence is the specific phenotype in which cells lose the ability to proliferate in response to various mitogens or cellular stresses such as DNA damage, telomere shortening and oxidative stress.1 Cells undergoing senescence exhibit characteristics, including irreversible proliferative arrest, resistance to mitogenic and oncogenic stimuli, acquisition of a typical flat and enlarged shape, the increased expression of biomarkers of senescence, such as positive staining of senescence-associated β-galactosidase (SA-β-gal) activity, accumulation of lysosomes and chromatin remodeling accompanied by the formation of senescence-associated heterochromatin foci (SAHF).2 At the molecular level, the senescence response can be triggered by several genetic effectors, converging on the activation of p53 and Rb.2 Recent studies indicated that therapy-induced senescence can be achieved at far lower chemotherapeutic doses than those required to induce apoptosis, thus reducing the side effects of anticancer therapy.3 Moreover, as cancer cells often develop resistance to apoptosis induced by such therapies, pro-senescence therapy has recently emerged as a novel approach to treat cancers.4

Potential intervention targets for the pro-senescence approach are telomerase inhibition, p53 activation, modulation of the cell cycle and the activation of phosphatase and tensin homolog.5 Telomerase is an enzyme that is responsible for the maintenance of telomeres, essential structures that cap chromosome ends and protect chromosome stability.6 Human telomeres are composed of tandem copies of TTAGGG in DNA repeat sequences and associated proteins, which together form a protective cap** complex. Following each cellular division, telomeres become progressively shortened, leading to telomere uncap** triggering the DNA damage response (DDR), which is recognized by the MRE11-RAD50-NBS1 (MRN) complex.7, 8 The MRN complex then activates ataxia-telangiectasia mutated (ATM)/ATR and Chk1/Chk2, which in turn phosphorylate and stabilize p53.4 Activation of p53 then drives the expression of cyclin-dependent kinase (Cdk) inhibitors, such as p21, which has a direct inhibitory effect on cell cycle progression.9 In parallel with p21 expression, other Cdk inhibitors, such as p16, p15 and p27, also induce senescence, as previous described.10

Human telomerase is a ribonucleoprotein complex that consists of two essential subunits: the human telomerase reverse transcriptase (hTERT) protein and the small nuclear human telomerase RNA.6 The former provides the catalytic activity of telomerase, and the latter provides a template for telomeric repeats.11 Although telomerase stabilizes telomeres in human stem cells, cancer cells and reproductive cells, its expression remains in a repressed state in normal human somatic cells.12 In addition, telomerase has a pivotal anti-apoptotic role in cancer cells by suppressing apoptotic signaling, thereby circumventing senescence.12 Recent studies indicated that telomerase is also expressed in lung cancer and overexpressed in late dysplastic lesions.13, 14 Lung cancer is one of the most common cancer types and is responsible for the majority of cancer deaths worldwide. The poor prognosis highlights the urgent need for the development of novel therapeutic strategies for the prevention and treatment of this deadly disease.3 Recent studies showed that hTERT polymorphisms are specifically associated with several subtypes of lung cancer.15 In addition, strong evidence indicates that hTERT and the epidermal growth factor receptor interact in the etiology of lung cancer.15 Consequently, telomerase inhibition-based therapy provides a therapeutic opportunity for lung cancer.14

To date, numerous studies focused on either identifying and testing natural agents or synthesizing compounds that inhibit telomerase activity in cancer cells, resulting in the loss of telomere maintenance and induction of senescence.10 Pterostilbene (trans-3,5-dimethoxy-4’-hydroxystilbene, PT), a dimethyl ether analog of resveratrol, has similar pharmacologic properties but better pharmacokinetic characteristics (more lipophilic, higher potential for cellular uptake, higher oral absorption and longer half-life) than resveratrol.16 The anticancer effects of PT include the inducing of cell cycle arrest, apoptosis, necrosis and autophagy in a few cancer cell lines.17, 18, 19, 20 A current molecular docking study performed on PT with the crystal structure of telomerase in cancer cells indicated a good interaction between PT and the active site of telomerase.21 However, to the best of our knowledge, no previous study has examined the direct effects of telomerase inhibition via PT treatment in cancer cells. Therefore, it is worthwhile to determine whether low-dose PT suppresses the growth of lung cancer cells via the induction of senescence through inhibition of telomerase activity. In addition, telomere erosion was linked p53 to senescence or apoptosis.1 Induction of p53 is pivotal for the initiation and maintenance of senescence mainly through DDR signaling.1 Abrogation of DDR or loss of p53 was reported to impair senescence.1 Disruption of the normal function of p53 may disrupt the cellular response leading to a reduced therapeutic response or increased overall resistance.22 Moreover, p53 mutation was reported significantly correlated with a poor prognosis in lung cancer patients.22 Therefore, our current study also investigated the potency of PT-induced senescence by targeting telomerase in lung cancer cells with different p53 phenotypes.

Results

PT inhibits cell growth and induces S phase arrest in lung cancer cell lines

H460 (p53 wild type) and H1299 (p53 null) lung cancer cells were treated with different doses (50, 75 and 100 μM) of PT for 24–96 h. The results indicated that 100 μM PT induced rapid cell death (Supplementary Figures 1A and 1B). PT at a lower dose (50 μM) slowed cell proliferation after 96 h of treatment (Supplementary Figures 1A and 1B), suggesting that a lower concentration of PT is sufficient to inhibit the growth of lung cancer cells. Then, we tested the growth inhibition effects of lower concentrations of PT (12.5, 25 and 50 μM) for 24–96 h. The results showed that lower concentrations of PT significantly decreased cell growth (Figure 1a). Moreover, H460 and H1299 cells became heterogeneous and contained a number of flattened cells with enlarged nuclei compared with normal cells after PT treatment (Supplementary Figure 1C). Accordingly, we hypothesized that a lower concentration of PT (50 μM) may trigger senescence in lung cancer cells. To further elucidate senescence-associated growth inhibition, we analyzed the cell cycle profile of both cells in the presence of PT (50 μM) using fluorescence-activated cell sorting. As shown in Figures 1b and c, lung cancer cells treated with PT showed a slightly increased sub-G0/G1 phase, a decreased G0/G1 phase and gradually accumulated in the S phase over time, compared with mock-treated cells. The results indicated that lung cancer cells entered ‘prolonged arrest’ through the loss of proliferative potential after PT treatment. We then examined the protein expression patterns of cell cycle related regulators in PT-treated cells. Figure 1d shows increased accumulation of cyclin E and cyclin A accompanied by decreases in cyclin B expression, confirming S phase arrest after treatment with 50 μM PT for 12, 24 and 48 h. The repeated experiments showed a subtle increase in expression of cyclin A at 12 h after PT treatment, which was followed by its gradual accumulation in a time-dependent manner (Supplementary Figure 1D). In addition, the expression levels of p-Cdk2 (Tyr15) (an inactivated form of Cdk2) and p53 in H460 cells were also time-dependently upregulated in response to PT treatment (Supplementary Figure 1D). Moreover, the results showed a significant increase of p21 and p27 expression in both cell types (Figure 1d). These data suggest that the p53/p21 axis signaling is likely involved in S phase arrest in PT-treated H460 cells, whereas in H1299 cells, PT-induced p21 expression may be independent of p53 activation (Figure 1d).

Figure 1
figure 1

Effects of PT on the growth inhibition and cell cycle arrest in lung cancer cells. (a) H460 and H11299 lung cancer cells were plated in six-well plates for 24 h and then treated with different concentrations of PT (0, 12.5, 25 and 50 μM) for 24, 48, 72 and 96 h. Cell numbers were counted daily by Trypan blue exclusion assay. Data represented the mean±S.E.M. of three independent experiments. *P<0.05 compared with the control group (0 μM). (b) H460 and H1299 were seeded in six-well plates and treated with 50 μM PT for the indicated times (24, 48, 72 and 96 h). Cells were collected and incubated with 40 μg/ml of PI for 15 min and subjected to flow cytometry analysis to examine the cell distribution at each phase of the cell cycle. Data are presented as representative graphs from three independent experiments. sub-G0/G1, G0/G1, S and G2/M phases are indicated as 1, 2, 3 and 4, respectively. (c) The percentage of cells in cell cycle phase was determined using FlowJo 7.6.1 software. Data represent the mean±S.E.M. (n=3, *P<0.05 compared with the control (d) H460 (left panel) and H1299 cells (right panel) were treated with 50 μM PT for indicated times then cell lysates were isolated and immunoblotted with anti-cyclin E, cyclin A, cyclin B, p53, p21 and p27 antibodies. Membranes were probed with an anti-GAPDH antibody to confirm equal loading of proteins. Representative data from one of three independent experiments are shown

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p53 wild-type H460 cells are more sensitive to PT-induced senescence than p53 null H1299 cells

Based on the proliferation arrest and morphological changes, we propose that the lung cancer cells underwent senescence in response to PT. We then assayed several markers for senescence. The first marker was SA-β-gal activity, which is the most ubiquitous cellular senescence marker.23 The enlarged cells exhibited an increase in SA-β-gal activity (Figure 2a), with up to 40% of H460 cells staining positive, compared with 20% of H1299 cells under the same treatment for 48 h (Supplementary Figures 2A and 2B). Then, we investigated the replicative and regenerative potential in PT-treated cells using a colony formation assay because colony formation ability is lost in senescent cells even in the presence of a mitogen.24 Lung cancer cells were treated with PT for 72 h, and PT was washed out with fresh medium (Figure 2b). Indeed, the results showed a marked retardation of replicative potential after an additional 9-day incubation in lung cancer cells in the absence of PT (Figure 2c). PT significantly suppressed colony-forming activity in a dose- and time-dependent manner compared with the different p53 status of lung cancer cells, such that, H1299 cells displayed higher colony-forming efficacy than H460 cells under the same PT treatment conditions (Figures 2d and e). Another hallmark of senescence is the formation of SAHF, which is known as areas of condensed and transcriptionally silenced DNA that can be detected by co-staining of DAPI and tri-methylation of histone H3 on lysine 9 (H3K9me3).25 Figure 2f shows the positive staining for H3K9me3 cells is also significantly increased in H460 cells compared with a slight increase in H1299 cells under the same treatment conditions. These results clearly showed that a lower concentration of PT-induced cellular senescence in lung cancer cells and that p53 wild-type H460 cells were more sensitive to PT-induced senescence than p53 null H1299 cells.

Figure 2
figure 2

PT-induced senescence in lung cancer cells. Senescence morphology of H460 and H1299 cells treated with 50 μM PT for 48 h. Cells were stained for β-gal. The representative images are shown with arrows indicating senescent morphology (a), bar: 100 μm. The experiment scheme for measuring loss of replication and regenerative potential (RP) was presented in (b). Cells were seeding overnight and then treated with or without 50 μM PT for 72 h. PT was then removed and cells could recover for additional 9 days (total period of 12 days). Then, colonies were stained with crystal violet (c). (d) Lung cancer cells were treated with 25, 50 or 75 μM PT for 72 h then PT was removed and the cells were allowed to recover for additional 9 days. (e) The colony-forming efficiency was also examined in lung cancer cells treated with 50 μM PT for 24 and 48 h and then cultivated in drug-free medium for additional 9 days. Data represent the mean±S.E.M. (n=3, *P<0.05 compared with the control). Y axis represents % decreases in the number of colonies relative to control. (f) Immunofluorescence analysis of the senescent heterochromatin foci stained with H3K9me3 (green) and with DAPI (blue) to visualize DNA in H460 and H1299 cells treated with PT (50 μM) for 72 h (Bar: 20 μm)

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To further confirm the pro-senescence potency of PT, A549 lung cancer cells and MCF7 breast cancer cells were treated with 50 μM PT for 24 h and the SA-β-gal activity was quantified by C12FDG staining (Supplementary Figures 2C and 2D). The results showed that PT induced approximately 35% senescence in both cells, which clearly demonstrates that PT is a promising senescence-promoting agent in a variety of cancer cell lines.

p53 contributes to PT-mediated telomerase inhibition and senescence induction

We therefore examined whether the p53 status in lung cancer cells can affect senescence induction by PT. To this end, H1299 cells with stable ectopic expression of p53 (H1299-p53+) were used. After treatment with PT for 24 h, the expression of p53 was significantly increased in H460 and H1299-p53+ cells (Figure 3a). As expected, PT induced more senescent characteristics in H1299-p53+ cells than in p53 null H1299 cells, determined by SA-β-gal activity (Figure 3b and Supplementary Figure 3) and C12FDG staining (Figure 3c). Quantification of C12FDG staining showed that 50 μM PT induced 30% and 40% senescence at 24 and 48 h, respectively, in H1299-p53+ cells, whereas no >20% senescence was observed in H1299 cells (Figure 3d). In addition, we established stable p53 knocked down H460 cell lines (H460-p53-/1 and H460-p53-/2) using two different p53 short hairpin RNA (shRNA) constructs (Figure 3e). As shown in Figure 3f, knockdown of p53 resulted in a significant decrease in SA-β-gal activity by a maximum of approximately 20% in H460-p53-/2 cells after treatment with 50 μM PT for 24 h. The results showed that the p53-mediated pathway enhances PT-induced senescence.

Figure 3
figure 3

PT-induced senescence in lung cancer cells mediated by p53. (a) Protein expression of p53 in H460, H1299 and H1299-p53+ lung cancer cells after treated with 50 μM PT for 24 h. (b) Senescence morphology of H460 and H1299 cells treated with 50 μM PT for 48 h. Bar: 100 μm. (c) H460, H1299, and H1299-p53+ cells treated with 50 and 75 μM PT for 24 and 48 h and then incubated with C12FDG to detect SA-β gal activities by flow cytometry. X axis: FSC-H, Y axis: FL1-H. (d) The percentage of SA-β gal-positive cells detected by C12FDG staining is shown. Data represent the mean±S.E.M. of three independent experiments. *P<0.05, significantly higher than control group in different time course category. #P<0.05, significantly higher than H1299 groups. (e) Western blot analysis showed the expression of p53 in H460 cells and p53 stable knockdown cell lines (H460-p53-/1 and H460-p53-/2). (f) The percentage of SA-β gal-positive cells detected by C12FDG staining was shown in H460, H460-p53-/1 and H460-p53-/2 cells treated with 50 μM PT for 24 h. Data represent the mean±S.E.M. of three independent experiments. *P<0.05, significantly higher than control groups, #P<0.05, significantly lower than H460 PT-treated groups. X axis: FSC-H, Y axis: FL1-H

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As the S phase is tightly regulated to ensure genome duplication and stability, alteration of the replication process by replicative stress may induce S phase checkpoint activation. Replicative stress induced by telomerase inactivation was implicated in the onset of cellular senescence.26 We next examined the telomerase inhibitory effects of PT in H460 and H1299 cells. As shown in Figure 4, following PT treatment for 6–48 h, both hTERT activity and protein expression in H460 cells were significantly decreased compared with H1299 cells (Figures 4a and b). We further confirmed whether the inhibition of hTERT activity and expression is mediated by p53, and the results revealed that hTERT expression and activity were reduced in H1299-p53+ cells similar to H460 cells treated with PT (Figures 4c and d). Next, we analyzed hTERT and cyclin A expression in H460, H460-p53-/1 and H460-p53-/2 cells. We observed that the expression of hTERT was decreased in H460 cells treated with PT, whereas the expression of hTERT was increased in p53 knockdown cells after PT treatment compared with H460 PT-treated groups (Figure 4e). Importantly, p53 knockdown reduced cyclin A accumulation after PT treatment. These results provide evidence that supports the requirement of p53 for hTERT inhibition and, may explain the mechanism underlying PT-induced senescence.

Figure 4
figure 4

PT inhibited telomerase enzyme activity and protein expression in lung cancer cells. (a) H460 and H1299 cells were treated with 50 μM PT for the indicated times (6, 24 and 48 h) and telomerase enzyme activity were measured using a TRAPeze RT Telomerase Detection Kit. (b) Cell lysates extracted from H460 and H1299 cells treated with 50 μM PT for 6, 24 and 48 h were submitted to western blot analysis to detect the protein expression of hTERT. Equal loading was confirmed by GAPDH staining. (c) Western blot analysis for hTERT expression and (d) telomerase enzyme activity was measured by TRAPeze RT Telomerase Detection Kit in H460, H1299 and H1299-p53+ cells treated with 50 μM PT for 24 h. Data represented the mean±S.E.M. of three independent experiments. *P<0.05, compared with control groups. #P<0.05, significantly lower than H1299 groups. (e) Protein expression in H460, H460-p53-/1 and H460-p530-/2 cells treated with 50 μM PT for 24 h. The membrane was probed with anti-GAPDH to confirm equal loading of proteins. The number below each line indicates the relative intensity of protein expression compared with H460 control groups (p53) or each group without PT treatment (hTERT and cyclin A) (defined as 1)

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hTERT overexpression confers resistance to senescence induced by PT

The inhibition of telomerase can lead to induction of the DDR, replication fork stalling, activation of the intra-S-phase checkpoint and senescence.25 As shown in Figures 5a and b, we provide the evidence of DNA damage in cells following hTERT inhibition using a comet assay. A marked increased in DNA strand breaks (DSBs) in PT-treated H460 cells compared with H1299 cells under the same treatment conditions was observed. Other marker for DSBs including activation of DNA damage checkpoint factor NBS1 and γH2AX (the phosphorylated H2AX) were detected (Figure 5c). In addition, DDR activated ATM and its downstream kinase Chk2, and the subsequent inactivation of cdc25A, which is Chk2 substrate27 (Figure 5d).

Figure 5
figure 5

PT-induced DNA damage in lung cancer cells. (a) Photomicrography of H460 and H1299 cells treated with 50 μM PT for 24 h and analyzed using comet assay. (b) DNA damage level expressed as tail length (μm) calculated using Komet 5.5 software (Kinetic Imaging Ltd., London, UK) after 24 h of 50 μM PT treatment in H460 and H1299 cells (mean±S.E.M., n=3, *P<0.05 compared with control groups). (c) H460 and H1299 cells were treated with 50 μM PT for 12, 24 and 48 h. Total-cell lysates were subjected to western blot analysis for (c) DNA damage proteins and (d) DNA sensor kinase (ATM), cell cycle checkpoint kinase (Chk2), and cell cycle regulatory protein (cdc25A). Membranes were probed with an anti-GAPDH antibody to confirm equal loading of proteins. Results are representative of three independent experiments

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To gain further insight into whether hTERT inhibition contribute to PT-induced senescence, we established an hTERT-overexpressing H460 cell line (Figure 6a). hTERT protein expression and activity were significantly increased in H460-hTERT+ cells compared with the H460 vector control cells (Figures 6a and b). Enforcement of hTERT expression rescued telomerase activity (Figure 6c) and prevented PT-induced senescence as determined by C12FDG staining and SA-β-gal staining compared with H460 vector control cells (Figure 6d). In addition, we found that the activation of ATM and induction of γH2AX were decreased in H460-hTERT+ cells after PT treatment (Figure 6e), indicating that hTERT overexpression rescues PT-induced DNA damage and senescence. These findings highlight the impact of telomerase inhibition by PT, which may initiate senescence by enforcing permanent cell cycle arrest when the ATM/Chk2 DNA damage checkpoint pathway is activated.

Figure 6
figure 6

Exogenous telomerase expression rescued telomerase activity and decreased senescence induced by PT. (a) H460 cells were transiently transfected with either pcDNA-3.1 vector or pcDNA-3.1 hTERT-3HA plasmid for 24 and 48 h. Cell lysates were analyzed for the expression of hTERT by western blot analysis and (b) telomerase enzyme activity. (c) The telomerase enzyme activity of the vector or hTERT transfected H460 cells treated with 50 μM PT for 24 h were measured using a TRAPeze RT Telomerase Detection Kit. (mean±S.E.M., n=3, *P<0.05, significantly lower than control groups. #P<0.05, significantly higher than vector groups). (d) The SA β-gal activity of cells treated as in (b) was stained with C12FDG and analyzed by flow cytometry. X axis: FSC-H, Y axis: FL1-H. Data represented the mean±S.E.M. of three independent experiments. *P<0.05, compared with control groups. #P<0.05, significantly lower than vector groups. (e) Cells were treated as described in (b) and immunoblotting was performed with anti-hTERT, p-ATM, ATM, γH2AX, and GAPDH antibodies. Results are representative of three independent experiments. (f) Proposed model summarizing PT-induced senescence in lung cancer cells. In p53 wild-type H460 cells, PT inhibits hTERT enzyme activity and protein expression resulting in the subsequent induction of DNA damage, activation of ATM/Chk2 and p53, and S phase arrest. Activation of p53 positive feedback provokes hTERT downregulation, resulting in senescence in H460 cells. Interestingly, PT slightly inhibited hTERT enzyme activity resulting in less senescence in H1299 cells, suggesting that PT-induced senescence in lung cancer cells partially through p53-mediated hTERT inhibition

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Discussion

Cellular senescence inhibits tumor progression in vivo, thus making it an attractive therapeutic target for cancer.10 The drug concentration of anticancer agents or the dose of radiation required to induce senescence is lower than that necessary to kill cells; therefore, senescence-inducing treatments provide the advantage of enhancing treatment efficacy and reducing side effects in anticancer therapy.9 In this study, we demonstrated a novel anticancer effect of PT through senescence induction, which was preferentially observed in p53+ lung cancer cells. The possible underlying mechanism is as follows: PT→inhibition of telomerase activity and protein expression→DNA damage→ATM/Chk2/p53 activation→p21 induction→prolonged S phase arrest→senescence (Figure 6f). In addition, ATM/Chk2-activated p53 may act as a feedback regulation to inhibit hTERT expression to increase DNA damage and senescence in H460 cells (Figure 6f). To the best of our knowledge, this is the first report investigating the induction of senescence in lung cancer cells by primarily targeting hTERT using low-dose PT.

With regard to the role of hTERT in lung cancer biology, ectopic expression of hTERT in primary lung epithelial cells immortalizes cells, indicating that increased hTERT activity may increase the cell proliferation capacity of normal cells.15 Multiple studies verified the hTERT expression level using qRT-PCR and showed that hTERT expression is significantly higher in tumor tissues of non-small cell lung cancer than in normal tissues.15 Mechanistic studies indicated that hTERT promotes epithelial proliferation through transcriptional pathways, including the Myc and Wnt pathways.15 Therefore, downregulation of hTERT subsequently reduced telomerase activity and led to lung adenocarcinoma apoptosis and reduced tumor size, indicating that telomerase is an attractive target for lung cancer therapy.Statistical analyses

Results are expressed as the mean±S.E.M. Experimental data were analyzed using Student’s t-test. Differences were statistically significant when the P-value was <0.05.