HS-1793 inhibits cell proliferation in lung cancer by interfering with the interaction between p53 and MDM2

Abstract

The transcription factor or tumor suppressor protein p53 regulates numerous cellular functions, including cell proliferation, invasion, migration, senescence and apoptosis, in various types of cancer. HS-1793 is an analog of resveratrol, which exhibits anti-cancer effects on various types of cancer, including breast, prostate, colon and renal cancer, and multiple myeloma. However, to the best of our knowledge, the role of HS-1793 in lung cancer remains to be examined. The present study aimed to investigate the anti-cancer effect of HS-1793 on lung cancer and to determine its association with p53. The results revealed that HS-1793 reduced cell proliferation in lung cancer and increased p53 stability, thereby elevating the expression levels of the target genes p21 and mouse double minute 2 homolog (MDM2). When the levels of MDM2, a negative regulator of p53, are increased under normal conditions, MDM2 binds and degrades p53; however, HS-1793 inhibited this binding, confirming that p53 protein stability was increased. In conclusion, the findings of the present study provide new evidence that HS-1793 may inhibit lung cancer proliferation by disrupting the p53-MDM2 interaction.

Introduction

Lung cancer is one of the leading causes of cancer-related death worldwide. Despite great advances in novel treatments over the past few decades, the survival rate is still low compared to that of other cancers (1,2). Non-small cell lung cancer (NSCLC) is the most common form, accounting for 85% of new diagnoses of lung cancer (3,4).

p53 is closely linked with a variety of transcriptional and non-transcriptional activities that regulate cell proliferation, DNA repair, cell senescence, and cell death (5,6). Mouse double minute 2 homolog (MDM2) is an E3 ligase that negatively regulates p53. When TP53 is abnormally activated, MDM2 promotes poly-ubiquitination and causes proteasomal degradation to downregulate p53, thereby maintaining p53 at a normal level (7,8). Therefore, interference in the interaction between MDM2 and p53 is an important part of the anti-cancer treatment method targeting p53 activation (9).

HS-1793 (4-(6-hydroxy-2-naphthyl)-1.3-benzenediol), a synthetic resveratrol analog, substituted the unstable double bond present in resveratrol with a naphthalene ring (10,11). HS-1793 improved photosensitivity and metabolic instability and is more potent than resveratrol (12,13). It is a well-known drug that causes cell death by inducing apoptosis in several types of cancer, including breast, prostate, colon, and renal cancers and multiple myeloma (14–17). HS-1793 causes mitochondrial-mediated apoptosis in breast cancer and also induces cell cycle arrest by reducing Akt phosphorylation in colon cancer and multiple myeloma (14,17,18). In prostate cancer cells, it inhibits migration by inhibiting the expression of hypoxia-inducible factor-1α and vascular endothelial growth factor (19). However, the effect of HS-1793 on lung cancer has not yet been elucidated.

In the present study, we report the effect of HS-1793 on A549 and H460 cell lines harboring p53 wild type. HS-1793 inhibits the binding of p53 and MDM2, resulting in increased p53 protein stability and expression of target genes and subsequently inhibiting the proliferation of NSCLC cells. Therefore, HS-1793 acts as a binding inhibitor of p53 and MDM2 and is a potential drug target for lung cancer.

Materials and methods

Chemicals and antibodies

4-(6-Hydroxy-2-naphthyl)-1,3-benzenediol (HS-1793) was purchased from AOBIOUS Inc. Antibodies against p53 (cat. no. sc-126), GAPDH (cat. no. sc-47724), p21 waf1/Cip1 (cat. no. sc-6246), and ubiquitin (cat. no. sc-8017) were purchased from Santa Cruz Biotechnology. MDM2 (cat. no. op46) was purchased from Calbiochem^®. Anti-mouse IgG and anti-rabbit IgG were purchased from Sigma-Aldrich; Merck KGaA.

Cell culture

A549 and H460 cell lines were obtained from American Type Culture Collection (ATCC). The cells were cultured in Dulbecco's modified Eagle medium (DMEM; HyClone, GE Healthcare) or RPMI-1640 medium (HyClone) supplemented with 10% fetal bovine serum (FBS; Corning Inc.) and 1% penicillin-streptomycin (Gibco; Invitrogen; Thermo Fisher Scientific, Inc) and then incubated in a 5% CO₂ incubator at 37°C.

Proliferation assay and cell cycle

Cell viability was determined using a cell count kit (CCK)-8 assay kit (Dojindo), according to the manufacturer's instructions. Briefly, lung cancer cells were seeded into a 96-well microplate, and CCK-8 solution (10/100 µl medium) was added to each well of the plate. After incubation of the plate for 1–4 h in a CO₂ incubator at 37°C, the absorbance of each well was measured at 450 nm using a microplate reader (Molecular Devices) with a reference wavelength of 650 nm. Lung cancer cells were treated with HS-1793 at the dose indicated in the figures. Cell cycle distribution was determined by DNA staining with PI. Cells were harvested and fixed in 70% ethanol. Cell pellets were suspended in PI and simultaneously treated with RNase at 37°C for 30 min. The percentage of cells in different phases of the cell cycle was measured using a FACScanto II flow cytometer (BD biosciences).

Clonogenic assay

Cells were plated at equal densities in 6-well plates (1×10³ cells/well) for 24 h and then treated with HS-1793 at the doses and times indicated in Fig. 1. After treatment, the cells were trypsinized, serially diluted, and re-plated. The cells were grown at 37°C with 5% CO₂ for 7 days, and the colonies were fixed and stained with 0.01% crystal violet. Subsequently, the stained dyes were extracted, and the optical density (OD) at 560 nm was measured using a 96-well plate reader (Molecular Devices).

Figure 1.

HS-1793 diminishes the viability of A549 and H460 cells. (A) A549 and H460 cells were treated with HS-1793 at different concentrations (0, 1, 2.5, 5, 7.5, and 10 µM) for 24 h, and absorbance was measured at 450 nm after 1 h incubation, using a CCK-8 reagent to confirm cell viability. Results are expressed as fold change. Data indicate the mean ± standard deviation (SD) of three independent experiments. *P<0.05, **P<0.01 vs. control. (B) The clonogenic assay was performed using the same cells as above. After treatment with HS-1793 in a dose-dependent manner, the media was replaced after 24 h. The media was then changed once every 2–3 days; the cells were fixed after 7 days of observation and counted by staining with crystal violet. Results are expressed as fold change. Data indicate the mean ± SD of three independent experiments. *P<0.05, **P<0.01 vs. control. CCK-8, cell counting kit-8.

Protein isolation and western blot analysis

Cells were lysed in lysis buffer A [20 mM HEPES (pH 7.5), 150 mM NaCl, 1 mM EDTA, 2 mM EGTA, 1% Triton X-100, 10% glycerol, and protease inhibitor cocktail Set II (Sigma-Aldrich; Merck KGaA)], and the cellular debris was removed by centrifugation at 10,000 × g for 10 min. Proteins were separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred onto nitrocellulose membranes, blocked with 5% skim milk in 0.01 M TBS (pH 7.5) containing 0.5% Tween-20, and labelled with the appropriate primary antibodies. Antigen-antibody complexes were detected based on chemiluminescence detection (Abclone). Protein band intensity was measured using the ImageJ software.

Cycloheximide chase assay

Cycloheximide (CHX) chase assay was performed to evaluate the regulation of p53 protein stability by HS-1793. Cells were pre-incubated with or without 5 µM HS-1793 for 2 h. Subsequently, 20 µg/ml CHX was added to inhibit protein synthesis. The cells were collected at 0, 1, 3, 6, and 9 h following treatment with CHX. Total cell lysates were analyzed using western blot with anti-p53 and anti-GAPDH. The results from western blot analysis were quantified using ImageJ, and the stable p53 protein level was quantified.

RNA isolation and quantitative reverse transcription-quantitative polymerase chain reaction (RT-qPCR)

RNA was extracted from A549 and H460 cells using an Accuprep^® Universal RNA extraction kit (Bioneer). Briefly, 1 µg RNA was reverse-transcribed with oligo (dT) 12–18 primers using the First-Strand cDNA Synthesis Kit (Fermentas; Thermo Fisher Scientific, Inc.). All reactions were performed in triplicate, and the β-2-microglobulin (B2M) gene was used as control. Using the comparative threshold cycle (Ct) or standard method, relative quantification of p53 gene expression was calculated after normalization against B2M for each sample. The primers for qPCR were designed as follows: p53 primer, forward, 5′-CCTCACCATCATCACACTGG-3′ and reverse, 5′-CCTCATTCAGCTCTCGGAAC-3′; B2M primer, forward, 5′-CTCGCTCCGTGGCCTTAG-3′ and reverse, 5′-CAAATGCGGCATCTTCAA-3′.

Immunoprecipitation (IP)

For co-IP, A549 and H460 cells were treated with HS-1793 (5 µM) or DMSO for 9 h or 18 h. Cells for endogenous IP were lysed in lysis buffer A. The lysate was immunoprecipitated with antibodies to detect p53, MDM2, or normal immunoglobulin G. Protein A/G sepharose beads (Santa Cruz Biotechnology) were then applied, and the final precipitated proteins were quantified using western blot analysis with the corresponding antibodies.

Statistical analysis

All data are obtained from three replicate experiments and expressed as mean ± standard deviation of three independent experiments. Statistical difference between multiple groups were analyzed using one-way analysis of variance (ANOVA) followed by Tukey's post hoc test, using SigmaPlot 12.0 software (2013; Systat Software Inc.). P<0.05 was considered to indicate a statistically significant difference.

Results

HS-1793 diminishes cell viability in A549 and H460 cells

HS-1793 affects cell viability in various carcinomas (14–17). The anti-cancer effect was extensively examined in H1299, A549 and H460 lung cancer cells by CCK-8 assay. Results showed that HS-1793 significantly inhibited cell viability in A549 and H460 cells (high dose; 7.5 and 10 µM) but not in H1299 cells, suggesting that A549 and H460 cells were HS-1793 sensitive (Fig. S1). Therefore, we decided to proceed with our studies in A549 and H460 cells. As shown in Fig. 1A, cell viability was determined after treatment of A549 and H460 cells with 0 to 10 µM HS-1793 for 24 h. HS-1793 has a slightly weaker effect on cell viability at concentrations up to 5 µM at the 24 h time point. However, at concentrations of 7.5 and 10 µM, HS-1793 induced a significant loss of viability (Fig. 1A). We made similar observation at the different time points (Fig. S2) We also determined G0-G1 arrest after exposure to low doses of HS-1793 (<7.5 µM); however, the proportion of cells in the G2-M was significantly increased after exposure to 10 µM (S3). In addition, the clonogenic assay demonstrated that HS-1793 diminished colony production in a dose-dependent manner (Fig. 1B). Based on these results, we focused on the effects of HS-1793 at a concentration of 5 µM and a 24 h treatment in subsequent experiments.

HS-1793 increases p53 protein expression and its target genes

Based on the findings of previous breast cancer studies, to confirm the relationship between HS-1793 and p53 in lung cancer, p53 protein level was confirmed using western blotting. We first compared the basal level of p53 protein between A549 and H460 cells. As shown in Fig. S3, the basal level of p53 protein in A549 cells is approximately 50% higher than that in H460 cells (Fig. S4). Next, to evaluate whether HS-1793 regulated p53 protein level, we treated A549 and H460 cells with concentration-dependent manner. HS-1793 increased the p53 protein level in a concentration-dependent manner (Fig. 2A and B). As shown in Fig. 2C and D, HS-1793 also increased the p53 protein level in a time-dependent manner. Next, we confirmed whether p53 regulates its target genes when upregulated by HS-1793. A549 and H460 cells were treated with HS-1793 at different doses and time durations, cell lysates were prepared, and western blot analysis was performed. As shown in Fig. 3A and B, HS-1793 significantly increased the protein expression of p21 and MDM2 in A549 and H460 cells in a dose- and time-dependent manner. We also evaluated the mRNA levels of p53 target gene (p21) in a time-dependent manner and made a similar observation in A549 and H460 cells in response to HS-1793 treatment (Fig. S5).

Figure 2.

HS-1793 increases p53 protein expression. The protein expression of p53 in (A) A549 and (B) H460 cells was measured by western blot analysis. GAPDH was used as a loading control at each concentration (0, 2.5, 5, 7.5, and 10 µM) for 24 h. The experiment was independently performed twice, quantified through the ImageJ program, normalized with GAPDH, and displayed as a graph. The expression of p53 in (C) A549 and (D) H460 cells was confirmed in a time-dependent manner (0, 3, 6, 9, 12, and 24 h) by selecting 5 µM concentration of HS-1793, which showed a significant increase in the data above. Results are expressed as fold change. Data indicate the mean ± standard deviation of two independent experiments. *P<0.05, **P<0.01 vs. control. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

Figure 3.

Increased p53 expression by HS-1793 upregulates its target genes p21 and MDM2. (A and B) A549 and H460 cells were treated with HS-1793 (A) in a dose-dependent manner for 24 h and (B) in a time-dependent manner (5 µM). The cells were subjected to western blotting using the indicated antibodies. This experiment was independently performed twice, protein expression was quantified through the ImageJ program, normalized to tubulin expression, and displayed as a graph. Results are expressed as fold change. Data indicate the mean ± standard deviation of two independent experiments. *P<0.05, **P<0.01 vs. control. MDM2, mouse double minute 2 homolog.

HS-1793-induced p53 protein expression is mediated by a post-translational mechanism

A CHX chase assay was performed to determine whether the upregulation of p53 expression by HS-1793 was due to transcription upregulation or increased protein stability. A549 (Fig. 4A) and H460 cells (Fig. 4B) were first treated with HS-1793 and then with CHX for the indicated durations. As shown in Fig. 4A and B, p53 half-life increased in the presence of HS-1793. In contrast, the mRNA expression of p53 was not significantly changed in the presence of HS-1793 (Fig. 4C and D).

Figure 4.

HS-1793 increases protein stability but not p53 transcription. (A) A549 and (B) H460 cells were pre-treated with HS-1793 for 6 h; incubated with CHX (20 µg/ml) for 0, 1, 3, 6, and 9 h; harvested; and analyzed using western blot. Band intensity was quantified by normalizing p53 with GAPDH as a control using the ImageJ program. (C and D) A549 and H460 cells were treated with HS-1793 (C) in a dose-dependent manner for 24 h and (D) in a time-dependent manner (5 µM). mRNA expression of the cells was measured using a quantitative polymerase chain reaction. B2M was used as a control and normalized to obtain a relative quantification graph. Results are expressed as fold change. Data indicate the mean ± standard deviation of independent experiments performed twice. CHX, cycloheximide; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; B2M, beta-2-microglobulin.

HS-1793 blocks the interaction between p53 and MDM2

To identify the molecular mechanism underlying p53 stability, we performed an IP assay using antibodies specific for p53 and MDM2. As shown in Fig. 5A and B, p53 and MDM2 were strongly bound to each other when antibodies for p53 or MDM2 were used. In contrast, HS-1793-treated cells decreased to precipitate either protein, emphasizing that HS-1793 regulates the interaction between p53 and MDM2.

Figure 5.

HS-1793 blocks the interaction between p53 and MDM2. The interaction between p53 and MDM2 was confirmed by immunoprecipitation with p53 and MDM2 antibodies. (A) After 9 h treatment with HS-1793 in A549, cells were harvested, and the cell are lysed in lysis buffer A. The lysates (1 mg) was incubated with primary antibodies (2 µg; anti-p53 or 1 µg anti-MDM2) overnight at 4°C. Subsequently, the supernatant was incubated with 50 µl protein A/G sepharose beads for 4 h at 4°C, and the beads were washed with lysis buffer A. A western blot experiment was then performed with the lysate to confirm the interaction. (B) In H460, HS-1793 was treated for 18 h, and the experiment was conducted under the same conditions as above (A). MDM2, mouse double minute 2 homolog.

Discussion

Lung cancer is one of the most common carcinomas worldwide and is the primary cause of all cancer-related deaths in both male and female cancer patients. There are four major histological types of lung cancer, including small cell lung cancer (SCLC) and three types of NSCLC (20,21). As NSCLC accounts for over 85% of new lung cancer diagnoses, its treatment is of great importance (22–24). Early detection and multimodal care have selectively shown high patient survival rates (25,26). However, the overall treatment and survival rates for NSCLC are low, and further studies are needed.

Resveratrol has demonstrated potential anti-cancer activity against several types of human cancer, including prostate (27,28), breast (29,30), colorectal (31,32), and skin cancers (33), and has also been used as a chemotherapeutic drug to treat various lung cancer cells (34–36). Its anti-lung cancer effect has been widely studied, with results suggesting that it could exert potent anti-tumor effects by upregulating microtubule-associated protein 1 light chain 3 (37) and p53 (38), downregulating smad activator 2 and 4 (39), and inhibiting the phosphatidylinositol-3-kinase pathway (40). Although resveratrol has potential therapeutic properties, it has poor pharmacokinetic properties, such as increased photosensitivity and rapid metabolization (41,42). We, therefore, tested the anti-cancer effect of resveratrol in lung cancer using its synthetic analog HS-1793.

HS-1793 has enhanced pharmacokinetic properties, such as reduced photosensitivity and metabolic instability, and is more potent than resveratrol (12,13). HS-1793 exhibits anti-cancer effects such as cell cycle arrest and cell death in various carcinomas (14–18,43), but its effects have not been demonstrated in lung cancer. We have determined the effect of HS-193 on the growth of human lung cancer cells. HS-1793 significantly inhibited the proliferation of both A549 and H460 cells harboring wild-type p53. However, H1299 cells containing p53 null were less affected (Fig. S3), suggesting that the p53 signaling pathway may play a pivotal role in HS-1793 treated cells. Therefore, we decided to proceed with our studies in A549 and H460 cells harboring wild-type p53 allele.

In a previous paper, Kim et al identified a relationship between HS-1793 and p53 in breast cancer (18), but the exact mechanism was unclear. In this study, we showed that HS-1793 induced a dose-dependent increase in p53 expression. In addition, we revealed that HS-1793 could significantly increase p53 stability in both A549 and H460 cells. Although a similar effects of HS-1793 observed for both cells, unlike A549 cells, expression of the target genes of p53, including p21 and MDM2, was delayed in H460 cells after HS-1793. While wild-type p53 expression is observed in A549 and H460 cells, the relative protein level is not equal between the cell lines. As shown in Fig. S4, the protein level in A549 cells is approximately 50% higher than that in H460 cells. The differential expression of p53 between these two cancer cell lines may be attributed to the response of the HS-1793-treated cells.

Several reports demonstrated that Akt represses p53 protein stability via Akt-mediated phosphorylation of MDM2 (44–47). Phosphorylation of MDM2 by Akt enhances the nuclear accumulation of MDM2 and consequently augments the destabilization of p53 by MDM2. Therefore, the Akt/MDM2 and MDM2/p53 signaling pathways serve an important role in the regulation of apoptosis and proliferation. We have previously shown that HS-1793 regulates p-Akt activity as an inhibitor of Akt-HSP90 binding (17). Notably, in multiple myeloma, Akt inhibition by HS-1793 results in NF-κB pathway inhibition. We also have analyzed the dose-dependent effect of HS-1793 on the p-Akt level in both A549 and H460 cells. Interestingly, western blot analysis revealed that the p-Akt level was markedly downregulated in response to HS-1793 in A549 cells, whereas a gradual reduction in the p-Akt level was observed in H460 cells (data not shown). Therefore, the subtle difference between the two cell lines for HS-1793 is probably due to p-Akt levels. However, it is also unclear how HS-1793 regulates p53 expression and target genes in lung cancer cells. Therefore, we consider the detailed mechanisms for HS-1793-mediated regulation of p53 target genes by defining a subset of lung tumors for further study.

p53 is a tumor suppressor gene that inhibits tumor development by activating transcription factors related to the cell cycle and apoptosis (48,49). Conversely, MDM2 is a tumor-promoting gene that acts as a negative regulator of p53 (50,51). In various tumors, overexpression of MDM2 inhibits p53 and promotes cell growth (9). Reactivation strategies of wild-type p53, which do not function properly, such as inhibition of E3 ligase activity of MDM2 and using drugs that bind to the p53 pockets of MDM2, are known to be effective chemotherapy methods (52). Thus, we assessed the association between p53 and MDM2 in HS-1793-treated A549 and H460 cells. Our findings revealed that HS-1793 markedly reduced p53-MDM2 interactions. However, further studies are needed to determine whether HS-1793 interferes with the binding of p53 to MDM2 directly or indirectly.

We demonstrated that HS-1793 decreased cell proliferation in lung cancer and increased p53 stability to elevate the expression of its target genes p21 and MDM2. Under normal conditions, MDM2, a negative regulator of p53, binds to and degrades p53 by acting as an E3 ligase. However, this function is blocked by HS-1793, confirming that p53 protein stability is increased. In summary, the anti-cancer effect of HS-1793 in lung cancer is due to p53 upregulation via blocking the p53-MDM2 interaction.

Funding Statement

This research was supported by the Basic Research Program through the National Research Foundation of Korea (NRF) and funded by the MSIT (grant no. NRF-2020R1A4A1016029).

Availability of data and materials

The datasets used and/or analyzed during this study are available from the corresponding author upon reasonable request.

Authors' contributions

CL and SWJ designed the research. CL, PCWL and SS performed the research. SS and SJ analyzed the data. SS and SWJ wrote the paper. CL, PCWL, SS and SWJ confirm the authenticity of all the raw data. All authors read and approved the final manuscript.

Ethics approval and consent to participate

Not applicable.

Patient consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.