Induction of p27 contributes to inhibitory effect of isorhapontigenin (ISO) on malignant transformation of human urothelial cells

ABSTRACT

Bladder cancer (BC) is the most expensive cancer to manage on a per-patient basis, costing about $4 billion in total healthcare expenditure per annum in America alone. Therefore, identifying a natural compound for prevention of BC is of tremendous importance for managing this disease. Previous studies have identified isorhapontigenin (ISO) as having an 85% preventive effect against invasive BC formation induced by N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN). The results showed here that ISO treatment inhibited EGF-induced cell transformation of human urothelial cells through induction of tumor suppressor p27 transcription secondary to activation of an E2F1-dependentpathway.ISOtreatmentrenderedcellsresistanttoEGF-induced anchorage-independent growth concurrent with p27 protein induction in both UROtsa and SV-HUC-1 cells. ISO inhibition of EGF-induced cell transformation could be completely reversed by knockdown of p27, indicating that this protein was essential for the noted ISO inhibitory action. Mechanistic studies revealed that ISO treatment resulted in increased expression of E2F1, which in turn bound to its binding site in p27 promoter and initiated p27 transcription. The E2F1 induction was due to the elevation of its translation caused by ISO-induced miR-205 downregulation. Consistently, miR-205 was found to be overexpressed in human BCs, and ectopic expression of miR-205 mitigated ISO inhibitory effects against EGF-induced outcomes. Collectively, the results here demonstrate that ISO exhibits its preventive effect on EGF-induced human urothelial cell transformation by induction of p27 through a miR-205/E2F1 axis. This is distinct from what has been described for the therapeutic effects of ISO on human BC cells.

Introduction

Isorhapontigenin (ISO) is a stilbene compound originally isolated from the Chinese herb Gnetum cleistostachyum and is now available commercially [1]. A number of studies have depicted therapeutic effects of ISO on human BCs both in vitro and in vivo [2]. For instance, ISO treatment affects cell cycle G₀/G₁ arrest and inhibition of cancer cell anchorage-independent growth through down-regulating miR-137/Sp1/cyclin D1 axis in human BC cells [2,3]. Other studies also show that ISO is capable of inhibiting patient-derived glioblastoma sphere formation via regulation of the miR-145/SOX2/cyclin D1 axis [4]. Thus, it is clear that ISO holds a potential therapeutic against BC. Given that there are differential mechanisms involved in therapeutic and preventive actions in many previous studies and Jiang et al. have already identified that ISO shows the extremely strong preventive effect on mouse MIBC formation in vivo [5], current studies explored the effect and molecular mechanisms underlying ISO prevention of EGF-induced human urothelial cell transformation.

As a key regulator of the cell cycle, p27 has a canonical function in the inhibition of cyclin-dependent kinase (CDK) activity. Apart from this, p27 holds many CDK-independent functions, including cytoskeletal dynamics and cellular plasticity, cell migration, and stem-cell proliferation and differentiation [6–8]. Other studies suggest that an additional function for p27 is as a putative tumor suppressor in various human cancers [9,10]. Unlike many tumor suppressors that are mutated in cancer, p27 has a low mutation rate in many cancers, and a low abundance of p27 is closely related to the tumor initiation and progression in cancer patients [10–13]. Previous studies have shown that upregulating p27 expression inhibits the anchorage-independent growth of human BC cells [14–16]. Thus, p27 protein inhibition is associated with bladder cancer development.

Given that there are differential mechanisms involved in therapeutic and preventive actions and previous study has already shown that application of ISO results in an extremely strong preventive effect against mouse MIBC formation in vivo [2]. The current studies reported here explored the molecular mechanisms underlying ISO preventive effects against EGF-induced human urothelial cell transformation. Just as one of the recent studies demonstrates that ISO application elevated p27 expression in BBN-induced mouse bladder tissues [14], the study here showed that ISO treatment down-regulated miR-205, and this, in turn, reduced miR-205 inhibition of E2F1 protein translation. As a consequence, the now-elevated levels of E2F1 specifically promoted p27 transcription, leading to cell growth arrest and inhibition of EGF-induced transformation of human urothelial cells by a known BC carcinogen.

Materials and methods

Plasmids, antibodies, and other reagents

The plasmids of shRNA targeting p27 (shp27), E2F1 (shE2F1), the human full-size p27 promoter-luciferase construct (p27 PF), and the deletion mutants (p27 KPN Ι and p27 Sac II) were mentioned in previous publications [14,17]. The 3’-untranslated region (3’-UTR) fragment of human E2F1 was cloned into pMIR luciferase reporter vector by targeting 5’-CCG CTC GAG CAG GGCTTG GAG GGA CC-3’ and 5’-CGA CGC GTG TTA GGA CCA AAA CAG CGA GG-3’ at the XhoI and MluI sits. A mutation of the miR-205 binding site in the E2F1 3’-UTR was created using site-directed mutagenesis by the overlap extension PCR. The miR-205 expression construct pcDNA3.2/V5 hsa-miR-205 (Plasmid #26312) was purchased from Addgene (Cambridge, MA, USA).

Isorhapontigenin (ISO, purity >99.9%) was obtained from Higher Biotech (Shanghai, China) and dissolved by dimethyl sulfoxide (DMSO, Sigma, St Louis, MO) to make a 20 mM stock solution, stored at −80°C freezer. Protein synthesis inhibitor cycloheximide (CHX, sc-3508) was bought from Santa Cruz (Dallas, TX).

Cell culture and transfection

The SV40 immortalized human urothelial cell line (SV-HUC-1) was purchased from the Chinese Academy of Sciences Cell Bank (Shanghai, China). The cells were cultured in F-12 K medium supplemented with 10% FBS in a 95% air/5% CO₂ incubator under 37°C. UROtsa cell line is a normal human urothelial cell, which was a kind gift from Dr. Scott H. Garrent (University of North Dakota, Grand Forks, ND), and was cultured in RPMI1640 with 10% FBS [18]. The cell line is authenticated 6 months before starting current studies by testing STR loci and gender using the PowerPlex® 16 HS System provided by Genetica DNA Laboratories (Burlington, NC 27215 USA).

The shRNA constructs against E2F1 and p27 were purchased from Open Biosystems (Thermo Fisher Scientific) and were transfected into UROtsa cells. Moreover, the human miR-205 constitutively expressed plasmid or negative control plasmid were transfected into cells using Lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. For stable transfection, cell cultures were subjected to puromycin selection (0.2–0.3 μg/ml), and cells surviving from the antibiotics selection were pooled as stable mass transfectants.

Western blot

The presentation of the whole-cell extracts preparation and protein concentrations determination was in previous publications [19,20]. Briefly, cells were plated in six-well plates and then cultured in 0.1% FBS-containing medium for 12 hours, followed by treatment with different doses of ISO in the presence of EGF for varying lengths of time as indicated. Subsequently, whole cells were lysed in lysis buffer(50 mM Tris pH 7.5, 0.15 M NaCl, 1% NP-40, 0.1% SDS,0.5% sodium deoxycholate, 50 mM NaF, 1 mM EDTA, 1 mM DTT, 1 mM Na3VO4) with protease inhibitors and phosphatase inhibitors. After incubation on ice for 30 min, the supernatant was harvested by spinning at 13,600 rpm for 25 min at 4°C, followed by the measurement of protein concentration using the BCA protein assay kit (23,225; Thermo), and then equal aliquots of cell extracts were separated on SDS-polyacrylamide gels, and the gel concentration is from 8% to 13%. The proteins were then transferred to PVDF membranes (Bio-Rad, Hercules, CA, USA) at 250 mA for 75 min, blocked, and probed with a specific primary antibody, followed by incubation with AP-conjugated secondary antibody and then treatment with ECL Western blot substrate. The images were acquired using the PhosphorImager Typhoon FLA 7000 imager (GE Healthcare, Pittsburgh, PA).

Antibodies are followed: E2F1 (3742; Cell Signaling Technology; 1:1000), p27 (sc-1641; Santa Cruz; 1:1000), Cyclin D1 (sc-8396; Santa Cruz; 1:1000), Cyclin E (sc-377,100; Santa Cruz; 1:1000), CDK4 (sc-23,896; Santa Cruz; 1:1000), CDK6 (sc-7961; Santa Cruz; 1:1000), β-Actin (66,009-1-1 g; Proteintech; 1:10,000), GAPDH (60,004-1-1 g; Proteintech; 1:2000), FOXO1 (2880; Cell Signaling Technology; 1:1000), Sp1 (sc-420; Santa Cruz; 1:1000), p65 (3033; Cell Signaling Technology; 1:1000).

Flow cytometry assay

Cells (3 × 10⁵) were cultured in each well of 6-well plates till 60–70% confluence. The cell culture medium was then replaced with 0.1% FBS medium and cultured for 12 h. After ISO treatment for 24 h in medium containing 2% FBS with or without EGF (20 ng/ml), the cells were collected and fixed with pre-cold 70% ethanol overnight kept in the −20°C. The fixed cells were then stained in Propidium Iodide staining solution (Propidium Iodide 50 mg/ml, RNase A 10 mg/ml, and 0.1% Triton X-100) for about 30 minutes on ice. The DNA content was evaluated using Flow Cytometry with the Epics XL FACS (Beckman Coulter, Miami, FL), and the results were analyzed FlowJo software.

Luciferase assay

Dual-luciferase reporter assay was subjected to the luciferase assay system (Promega Corp., Madison, WI), as described previously [2]. In brief, UROtsa cells were transfected with each of the indicated luciferase reporters construct in combination with the pRL-TK vector (Promega, Madison/WI) as an internal control. The transfectants were seeded into dedicated 96-well plates at a density of

10,000 cells per well and then treated with different doses of ISO in the presence of EGF for 24 hours.

Whole-cell extracts were prepared using cell lysis buffer (25 mM Tris-phosphate [pH 7.8] 2 mmol/L EDTA, 1% Triton X-100, and 10% glycerol), and the luciferase activity in the extract was then detected in a Microplate Luminometer LB96V microplate luminometer (Berthold GmbH & Co. KG, Bad Wildbad, Germany).

Cell transformation assay (soft agar assay)

Urothelial cell transformation was evaluated by determining anchorage-independent growth ability as described in previous studies [15]. In brief, cells (5 × 10⁴) in 10% FBS basal medium Eagle (BME) with the indicated concentration of ISO and/or EGF containing 0.33% soft agar were seeded over the 0.5% soft agar basal layer of modified Eagle’s medium supplemented with 10% FBS, indicated concentration ISO and/or EGF in each well of 6-well plates. Two weeks late, 1 ml of 0.33% soft agar containing the indicated concentration of ISO and/or EGF was added into each well of the 6-well plate. Colonies were observed under a microscope, and only colonies with over 32 cells were scored after another 2 ~ 3 weeks (UROtsa) or 3 ~ 4 weeks (SV-HUC-1) of incubation.

Quantitative real-time PCR

Total RNA was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA), and 0.5 μg total RNA was used for reverse transcription to synthesize cDNA with Super-Script First-Strand Synthesis system (Invitrogen, Carlsbad, CA). PCR amplification was carried out using PowerUp SYBR Green Master Mix (Invitrogen, Grand Island, NY, USA) with Specific primers as below. Primers for human p27 (forward, 5’-ACC CGC CCG AGG AGG AAG ATG T-3’; reverse, 5’-GCG CGG GGG CCT GTA GTA GAA C-3’); the primers for human E2F1 (forward, 5’-AGT TCA TCA GCC TTT CCC CAC C-3’; reverse, 5’-GAC AAG GTG AGC ATC TCT GGA AAC-3’); and The primers for human GAPDH (forward: 5’-AGA AGG CTG GGG CTC ATT TG-3’, reverse: 5’-AGG GGC CAT CCACAG TCT TC-3’) as used in previous studies [21].

The miRNA was extracted using miRNeasy Mini Kit (QIAGEN, Valencia, CA, USA); 1 μg total

RNA was used for reverse transcription. Analysis of miRNAs was carried out using the miScript

PCR system (QIAGEN, Valencia, CA, USA) by QuantStudio Real-time PCR system (Applied Biosystems, Beverly, MA). The primers were obtained from Invitrogen (Grand Island, NY, USA), and the inner control was U6. The initial activation was performed as previously described at 95°C for 15 min and followed by 40 cycles, denaturation at 95°C for 15 s, annealing at 55°C for 30 s, and extension at 70°C for 30 s.

ATP cell viability assay

Cell viability was determined utilizing the CellTiter-Glo Luminescent Cell Viability Assay Kit following the manufacturer’s instructions as described in previous studies [5]. Results were read on a microplate luminometer LB 96 V (Berthold GmbH & Co. KG).

Chromatin immunoprecipitation assay

The ChIP assay was carried out by utilizing EZ-ChIP kit (Millipore, MA). Briefly, the indicated proteins and chromatin DNA fragments were isolated as described [14,15]. Furthermore, the E2F1 antibody (sc-251) was bought from Santa Cruz (Dallas, TX). In order to amplify the specific region containing the putative responsive elements of the human p27 promoter, PCR was used with the following pair of primers, p27 promoter (−324 site): 5’-CCT CTG AAA GCA TTC ACT AG-3’ and 5’-AGT TAA GGC CAT GGA CAT T-3’; p27 promoter (−1254 site):5’-CAT CAA GCT CAT CTG TAG CA-3’ and 5’-GGT GGA AGG TAA AGT AGG AC-3’.

Immunofluorescence

Cells were fixed with 4% paraformaldehyde for 10 min, permeabilized in 0.5% Triton X-100 for 15 min, and incubated in PBS and 5% goat serum blocking solution for 45 min. Then, the cells were incubated with anti-E2F1 and anti-p27 antibodies in 5% goat serum for 1.5 h and stained with Alexa

Fluor 488 and Alexa Fluor 568 conjugated secondary antibody for another 1 h. Afterward, cells were washed twice in blocking solution and a third time in 1 × PBS, and nuclei were counterstained using DAPI. Ultimately, cells were viewed under a fluorescence microscope.

Fluorescence in Situ Hybridization (FISH)

Cy3-labeled miR-205 probes were designed and synthesized by Shanghai GenePharma Co., Ltd.

Nuclei were counterstained with DAPI. The signals of the probes were determined with a Fluorescent In Situ Hybridization Kit (Gene Pharma: F12101/50) according to the manufacturer’s instructions. Images were acquired using an inverted fluorescence microscope (Nikon Eclipse Tisr, Tokyo, Japan). The probes

[³⁵S]-labeled methionine-cysteine pulse assay

UROtsa(vector) and UROtsa(miR-205) were incubated with methionine–cysteine-free DMEM (GibcoBRL, Grand Island, NY) containing 2% dialyzed fetal calf serum in the presence of 10 μM MG132 for 30 min. The cells were then treated with 20 μM of ISO diluted in 2% FBS methionine/cysteine-free DMEM containing ³⁵S-label methionine/cysteine (250 mCi per dish, Trans ³⁵S-label, ICN) for the indicated time periods. The cells were extracted with 1× lysis buffer (Cell Signaling Technology) containing complete proteinase inhibitor mixture (Roche) [22]. Total lysate of 500 ug was incubated with anti-E2F1 antibody and protein G-agarose (Santa Cruz Biotechnology, C1014) for 16 h at 4°C. The immunoprecipitated samples were washed for five times, heated at 100°C for 5 min, and then subjected to SDS-PAGE analysis. The ³⁵S-labeled E2F1 protein was explored with the PhosphorImager Typhoon FLA 7000 (GE, Pittsburgh, PA).

Human bladder cancer tissues

Human BC tissues and their adjacent non-tumor bladder tissues were obtained from Union Hospital of Tongji Medical College (Wuhan, China). And the comprehensive information of tissues were described in previous studies [23].

Statistical methods

Statistical analysis was performed using Prism5.01 software (GraphPad). The student’s t-test was utilized to determine the significance between various groups. All data were presented as means ± SD. P < 0.05 was considered significant.

Results

ISO repressed EGF-induced cell cycle transition and cell transformation in human urothelial cells

Although ISO’s therapeutic effects have been reported in both in vitro and in vivo [2,5,14], its chemopreventive potential has not been explored yet. To explore the chemopreventive potential of ISO, we first selected nontoxic doses of ISO on cell viability in normal urothelial cell lines. As shown in Figure 1(a), ISO at doses over 30 μM did show cytotoxicity to UROtsa cells, whereas it at doses of 20 μM and less did not show observable cytotoxic effect on UROtsa cells. The IC₅₀ of ISO on the UROtsa cell line was 37.69 ± 1 μM (n = 3) and the SV-HUC-1 cell line was 36.6 ± 0.93 μM (n = 3) (Fig. S1a) in this ATPase activity assays. Thus, 5–20 μM were employed for current studies. It has been reported that the epidermal growth factor receptor (EGFR) was overexpressed in BCs [16], and previous studies have identified that EGF induces UROtsa cell transformation [18]. To examine the effects of ISO on anchorage-independent growth of EGF-induced cell transformation, UROtsa cells were employed to evaluate the inhibition of ISO on EGF-induced UROtsa cell transformation in soft agar assay. Results showed that co-incubation of cells with ISO exhibited a dramatic inhibition of EGF-induced cell transformation in a dose-dependent manner in UROtsa cells (Figure 1(b-d)). Similarly, the considerable inhibition of ISO treatment on EGF-induced cell transformation was also observed in SV-HUC-1 cells (Figures 1(c, e)). Consistently, the result obtained from cell cycle analysis via flow cytometry indicated that ISO treatment-induced cell cycle G₀-G₁ growth arrest in both cell lines in a concentration-dependent manner (Figures 1(f, g)). Our results revealed that ISO treatment inhibits EGF-induced cell transformation concurrently with induction of G₀-G₁ cell growth arrest in UROtsa and SV-HUC-1 cells.

Figure 1.

ISO inhibited EGF-induced cell transformation and induced G₀/G₁ cell-cycle arrest in human urothelial cells (a) UROtsa cells were treated with different doses of ISO for 48 hrs, and the cell extracts were subjected to ATPase activity assay. (b-e) Representative images of UROtsa cells (b & d) and SV-HUC-1 cells (c & e) were treated with EGF (20 ng/ml) and various concentrations of ISO in soft agar assay for determination of cell transformation. The results were obtained from three independent experiments. The symbol (*) indicates a significant difference (P < 0.01). (f & g) UROtsa cells and SV-HUC-1 cells were pre-treated ISO at indicated doses for 3 hrs and then co-tread with EGF, and flow cytometry was performed to evaluate the cell cycles. The results presented are one of 3 independent experiments.

ISO inhibition of EGF-induced cell transformation and G₀-G₁ growth arrest was mediated by its induction of p27 in human urothelial cells

Previous studies revealed that ISO downregulated Cyclin D1 protein expression and induced cell cycle G₀-G₁ growth arrest in Glioblastoma (GBM) and bladder cancer [3,4,14]. To explore the mechanisms of ISO treatment in inhibition of EGF-induced cell transformation and induction of G₀/G₁ growth arrest, Western blotting was performed to screen for potential ISO downstream targets in both UROtsa and SV-HUC-1 cells. The results indicated that ISO treatment explicitly induced protein expression of p27 and Cyclin D1 in both cells, while it only showed a positive effect on Cyclin E expression in UROtsa, but not in SV-HUC-1 cells. In contrast, ISO treatment did not show any consistent effect on CDK4 and CDK6 (Figures 2(a, b)). Given the consistent effect on both cells and their biological role in cell cycle modulation, we anticipated that p27 might be an ISO-targeted downstream effector for induction of G₀/G₁ growth arrest and inhibition of EGF-induced cell transformation. To test this notion, we stably transfected shRNA specific targeting p27 (shp27#1 and shp27#2) into UROtsa cells. As shown in Figures 2(c, d), knockdown of p27 in UROtsa (shp27#1 and shp27#2) cells (Figure 2(c)) abolished ISO induction of p27 and G₀-G₁ growth arrest (Figures 2(d, e)), as well as reversed inhibition of EGF-induced anchorage-independent growth by ISO in UROtsa cells (Figures 2(f, g)). These results approved that elevated p27 mediates ISO induction of G₀/G₁ growth arrest and inhibition of EGF-induced anchorage-independent growth in UROtsa cells.

Figure 2.

ISO induced p27 expression in human urothelial cells. (a & b) UROtsa cells and SV-HUC-1 cells were pretreated with indicated doses of ISO for 3 hrs and then followed by co-incubated with ISO and EGF (20 ng/ml) for 24 hrs. The cell extracts were subjected to Western blotting for determination of expression of p27, cyclinD1, cyclin E, CDK6, CDK4 proteins. (c) UROtsa cells stably transfected with p27 knockdown constructs, and Western blot was employed to evaluate the knockdown efficiency of p27 protein. (d) UROtsa(Nonsense), UROtsa(shp27#1), UROtsa(shp27#2) were treated with EGF (20 ng/ml) alone or ISO+EGF for 24 hrs, and the cell extracts were then subjected to Western blot for determination of p27 protein alteration. (e) Cell cycle analyses of UROtsa(Nonsense, shp27#1, shp27#2) were carried out using flow cytometry as described in Materials and Methods. Data represent one of 3 independent experiments. (f & g) UROtsa (Nonsense, shp27#1, shp27#2) were treated with medium or EGF (20 ng/ml) with or without ISO (20 μM) for three weeks. Images of colonies in soft agar were captured and colonies were counted and presented. The results were shown as colonies per 50,000 cells from three different experiments. The symbol (*) displays a significant difference (P < 0.01).

ISO treatment promoted p27 transcription by specific upregulation of E2F1 in human urothelial cells

To investigate the mechanisms underlying the ISO induction of p27 protein, we examined the effect of ISO on p27 mRNA level. The results evidenced that ISO treatment increased p27 mRNA expression in BC cells (Figure 3(a)). Therefore, we transfected UROtsa cells with three different-length fragments of human p27 promoter luciferase reporters, p27 PF, KPNI, and SACII, as indicated in Figure 3(b)). The transfectants were then exposed to ISO for determination of luciferase reporters’ activity. The results showed that ISO-induced promoter transactivation was observed in p27 PF and KPNI promoter-driven reporters but not in p27 SACII promoter in UROtsa cells (Figure 3(c)), revealing that transcription factor binding site(s) responsible for ISO increasing p27 promoter transcription should be in the promoter region of −1324 to +162. The results obtained from analysis of this region showed that the transcription factor binding sites are E2F1, SP1, FOXO1, and p65 (Figure 3(d)). Thus, we determined the alteration of relevant transcription factor expression in the UROtsa cells and SV-HUC-1 cells that were treated with ISO (Figsure 3(e, f)). The results showed that E2F1 exhibited a consistent induction by ISO in both cell lines, whereas others showed no remarkable alteration in both cells. These results suggest that E2F1 might be the effective transcription factor being involved in the regulation of p27 by ISO. To test this, shRNAs particularly targeting E2F1 (shE2F1#1 and shE2F1#2) were stably transfected into UROtsa cells. As shown in Figure 4(a, b), the western blot and immunofluorescence results showed that P27 protein abundance was also significantly down-regulated after E2F1 was knockdown. Then, the transfectants were treated with ISO and (or) EGF to determine the potential effect of E2F1 in the regulation of p27 expression and cell cycle. As expected, ISO treatment up-regulated E2F1, and P27 protein levels and knockdown of E2F1 attenuated p27 protein expression, p27 promoter activity, and p27 mRNA expression as compared with those in UROtsa(nonsense) transfectants (Figure 4(c-e)). Further, knockdown of E2F1 also abolished the effect of ISO on induction of G₀-G₁ growth arrest in UROtsa cells (Figure 4(f)). In addition, the chromatin immunoprecipitation (ChIP) assay was performed using an anti-E2F1 antibody, and the results showed that E2F1 specifically binds to the p27 promoter only at −324 site, not at −1254 site (Figure 4(g)). Collectively, our results strongly demonstrate that E2F1 is the critical transcriptional factor mediating ISO inhibition of malignant transformation of human urothelial cells by directly binding to the p27 promoter, thereby increasing p27 expression.

Figure 3.

ISO treatment promoted p27 mRNA Transcription in human urothelial cells. (a)The expression levels of p27 mRNA in UROtsa cells were determined by real-time PCR after cells treated with EGF (20 ng/ml) and/or ISO for the indicated dose for 12 hrs. (b) Schematic diagrams of the p27 promoter and its deletion fragment (p27 PF, p27KPNI, and SACII). (c) Indicated promoter-driven luciferase reporters (p27 PF, p27 KPN Ι, and p27 SAC ΙΙ) were transfected into UROtsa cells, respectively, and the transfectants were then treated with ISO and/or EGF (20 ng/ml) for 24 hrs. The cell extracts were subjected to Dual-Luciferase Reporter Assay to determine p27 promoter transcriptional activity. The symbol (*) indicates a significant increase in ISO treatment (P < 0.01). (d) Schematic diagrams of potential transcriptional factor binding sites in the human p27 promoter region between – 1324 and +162. (e & f) Cell extracts as indicated were subjected to Western blot to examine the expression of the related transcription factors.

Figure 4.

Knockdown of E2F1 reversed ISO inhibition of EGF-induced Anchorage-independent growth and the G₀/G₁ growth in UROtsa cells. E2F1 knockdown constructs were transfected into UROtsa cells. (a) The whole-cell lysis of UROtsa stable transfectants was subjected to Western blot for determination of E2F1 and p27 expression. (b) Immunofluorescence and DAPI staining of the indicated transfectants were performed to determine the expression of E2F1 and p27. (c) The cell extracts were subjected to Western Blot for determination of E2F1 and p27 expression; (d) Luciferase activity was evaluated by the dual-luciferase reporter assay system for the indicated transfectants upon ISO and/or EGF treatments. (e) The indicated transfectants were treated with EGF and/or ISO (0, 5, 10, 20 μM) for 12 hrs and the cell extracts were subjected to real-time PCR for determination of p27 mRNA expression. The error bars represent the SD from three independent experiments and Symbol “*” indicates a significant difference (P < 0.01). (f) Cell cycle analyses of UROtsa (Nonsense, shE2F1#1, shE2F1#2) were subjected to flow cytometry for determination of cell cycles. Data represent one of 3 independent experiments. (g) A ChIP assay was carried out using anti-E2F1 antibody to detect the interaction of E2F1 with p27 promoter.

ISO treatment attenuated miR-205, and in turn reduced its inhibition of E2F1 protein translation and further promoted p27 induction, and consequently inhibiting cell cycle progression and transformation in human urothelial cells

To clarify the mechanism underlying ISO upregulation of E2F1 expression, we examined p27 mRNA level in UROtsa cells treated with ISO. The results indicated that ISO treatment did not affect E2F1 mRNA expression (Figure 5(a)). Our results also showed that E2F1 increment by ISO did not occur at protein degradation level (Figure 5(b)). Based on the results above, we proposed that ISO regulated E2F1 at the level of a protein translation. Given that ISO could regulate protein translation via regulating protein mRNA 3′-UTR and miRNA in bladder cancer [2], we transfected E2F1 mRNA-3′-UTR luciferase reporter into UROtsa cells. The results indicated that ISO treatment resulted in the increment of EGF-induced its 3′-UTR activity in a dose-dependent manner (Figure 5(c)). The results from analyzing miRNA binding sites in E2F1 mRNA 3′-UTR luciferase reporter region with the Targetscan database revealed that there were multiple putative binding sites for miRNA, including miR-205, miR-17-5P, miR-136-5P, miR-372-3P, as shown in Figure 5(d). Therefore, real-time PCR was employed to evaluate the expression levels of those potential miRNAs in UROtsa cells followed ISO treatment. As shown in Figure 5(e), only miR-205 was significantly downregulated due to ISO treatment, while other miRNAs were not affected by ISO (Figure 5(e)). Furthermore, fluorescence in situ hybridization experiments also confirmed that the abundance of miR-205 in UROtsa cells gradually decreased with the increasing of ISO treatment concentration. Meanwhile, miR-205 was mainly located in the nucleoli and cytoplasm, and ISO treatment did not change the subcellular localization of miR-205 (Figure 5(f)). To test whether miR-205 could bind directly to the 3′-UTR of E2F1 mRNA for inhibition of E2F1 translation, we constructed the point mutation of the miR-205 binding site in the E2F1 3′-UTR of mRNA luciferase reporter as displayed in Figure 5(g). The point mutation of the miR-205 binding site in E2F1 mRNA 3′-UTR-luciferase reporter resulted in a completely no response of E2F1 mRNA 3′-UTR luciferase reporter following ISO treatment (Figure 5(h)), suggesting that miR-205 binding site is crucial for ISO induction of E2F1 3’-UTR of mRNA luciferase activity.

Figure 5.

ISO treatment inhibited miR-205 and miR-205 specific targeted 3’-UTR of E2F1 mRNA in UROtsa cells. (a) Real-time PCR was used to determine E2F1 mRNA expression levels in UROtsa cells afterward cells were treated with EGF (20 ng/ml) and/or ISO for 12 hrs. (b) The cells were treated with ISO (20 μM) and/or EGF (20 ng/ml) for 3 hrs, and the cells were then co-incubated with cycloheximide CHX (50 μg/ml) for the indicated time period. The cell extracts were subjected to Western blot for determination of E2F1 protein expression. GAPDH was used as the protein loading control. (c) E2F1-3’-UTR luciferase reporter together with pRL-TK reporter were transiently transfected into UROtsa cells. The transfectants were treated with EGF (20 ng/ml) and/or 20 μM of ISO for 24 hrs. The cell extracts were subjected to determination of luciferase activity with normalization to internal TK activity. The results was presented as relative E2F1-3’-UTR activity. (d) The potential miRNA binding sites in human 3’-UTR of E2F1 mRNA that were analyzed using TargetScan database. (e) UROtsa cells were treated with ISO and/or EGF (20 ng/ml) for 24 hrs, and the real-time PCR was performed to determine alteration of microRNA expression levels. (f) Fluorescence in Situ Hybridization was performed for miR-205 in UROtsa cells treated with ISO and/or EGF (20 ng/ml) for 24 hrs. (g) Schematic diagrams of the construction of miR-205 binding site mutant of E2F1-3’-UTR luciferase reporter. (h) The wild-type and mutant E2F1-3’-UTR luciferase reporters were transiently transfected with pRL-TK into UROtsa cells, respectively, and the transfectants were treated with ISO and/or EGF (20 ng/ml) for 24 hrs and the cell extracts were subjected to determine luciferase activity. The asterisks (*) indicate a significant difference (P < 0.01).

The above results suggest that ISO might inhibition of miR-205 might contribute to the anti-cancer activity of ISO. Thus, we next evaluated the status of miR-205 expression in human BCs. As shown in Figure 6(a), miR-205 expression was remarkably upregulated in human BCs in comparison to their adjacent non-tumor bladder tissues. This finding was greatly supported by the results obtained from the analysis of TCGA database (Fig. S1b-1c). To further evaluate the contribution of miR-205 downregulation to ISO inhibition of EGF-induced cell transformation, UROtsa cells were transfected with miR-205 overexpression construct. The results showed that overexpression miR-205, as identified in Figure 6(b) completely abolished both basal level and ISO-induced level of E2F1 and p27 protein expression in comparison to that in UROtsa(vector) transfectants (Figure 6(c)). Further, the results from [³⁵S]-labeled methionine and cysteine pulse showed that ISO treatment did result in E2F1 new protein translation in UROtsa(Vector) cells, whereas ectopic expression of miR-205 almost completely abolished E2F1 protein translation (Figure 6(d)). Consistently, over-expression miR-205 also reversed ISO-caused G₀/G₁ cell cycle arrest and inhibition of cell transformation in UROtsa cells (Figure 6(e-g)). Taken together, these results here demonstrate that ISO could inhibit miR-205 expression and that this, in turn, led to enhanced E2F1 protein translation, increases in p27 mRNA transcription, and eventually inhibition of EGF-induced G₀/G₁ phase transition and anchorage-independent growth in human urothelial cells (Figure 6(h)).

Figure 6.

Overexpression of miR-205 inhibited expression of E2F1 and P27 and reversed ISO inhibition of EGF-induced Anchorage-independent growth and induction of G₀/G₁ growth arrest in UROtsa cells. (a) The relative expression levels of miR-205 in BC tissues in comparison to normal bladder tissues were determined by real-time PCR. The asterisks (*) indicate a significant difference (P < 0.01). (b) UROtsa cells were transfected stably with human miR-205 constitutively expressed plasmid, and real-time PCR was carried out to identify the stable transfectants. (c) UROtsa(Vector) and UROtsa(miR-205) were treated with EGF (20 ng/ml) and/or 20 μM of ISO for 24 hrs, and cell extracts were subjected to Western blot for determination of E2F1 and p27 expression. (d) The transfectants as indicated were treated with ISO (20 μM) for 12 hrs. The newly synthesized E2F1 protein was monitored by pulse assay using [³⁵S]-labeled methionine-cysteine. The whole-cell lysate (WCL) was used as total new synthesized protein control and Coomassie blue staining was used as protein loading control. (e) The cell transfectants were performed with flow cytometry for analysis of cell cycle distribution after treatment of cells with EGF (20 ng/ml) and/or 20 μM of ISO for 24 hrs. (f & g) The transfectants were subjected to soft agar assay with the indicated treatment for determination of their Anchorage-independent growth abilities. (h) The proposed schematic for the cascade underlying ISO inhibition of malignant transformation of human urothelial cells through upregulation of p27 in an E2F1-dependent manner.

Discussion

BCs are the most expensive cancers to be treated in clinics [24], therefore finding means, such as prevention, to decrease the incidence of BC is of highly significant. Although ISO has recently been found to have an anti-cancer effect on BCs both in vitro and in vivo [2,5], the chemopreventive effect and mechanisms of ISO’s action on BC remains unclear. In the study reported here, it is shown for the first time that ISO inhibits EGF-induced transformation of human urothelial cells. These in vitro results are consistent with in vivo results showing that ISO inhibits BBN-induced high invasive BC formation in mice and further strongly suggest that ISO is a high promised compound for chemoprevention of human BCs [2,5].

ISO is a dietary resveratrol derivative whose anti-cancer has been studied in many previous studies [25–27]. It is very interesting to note that the potency of ISO was much higher than resveratrol in some pre-clinical pharmacokinetic analyses of ISO [28]. Recent studies demonstrate that ISO exhibits an anti-proliferation effect by inducing cell-cycle G₀–G₁ arrest [2,3] and attenuates BC invasion through downregulation of MMP-2 expression [5]. While it would clearly be of great interest to further explore the anti-cancer potentials of ISO, its effects and the molecular mechanisms underlying its potential use as a preventive agent against BC still are not explored yet. Our current studies showed that ISO at sublethal doses effectively inhibits EGF-induced cell transformation and cell cycle transition in human urothelial cells. We also observed that ISO treatment transcriptionally upregulated p27 by enhancing the binding of E2F1 to p27 promoter region, while ISO-promoted E2F1 protein translation was mediated by its inhibition of miR-205, which decreased its binding to 3′-UTR of E2F1 mRNA, and consequently resulting in more E2F1 protein translation and its dependent p27 transcription, as well as growth inhibition of human urothelial cells. These results reveal that ISO holds the prevention of EGF-induced human urothelial cell transformation by attenuating miR-205 expression, thereby upregulation of its targeted E2F1 protein translation and its downstream effector p27 expression.

p27 is a non-canonical oncosuppressor that rarely have mutations or deletion in human cancers [13]. A decreased level of p27 is associated with cancer growth, progression, and poor prognosis in various of cancers, including BCs [29–31]. Hence, the understanding molecular mechanism of p27 dysregulation as well as identification of drugs that can reverse p27 downregulation at either transcriptional and post-translational are of clinical importance for management of cancer development. Forkhead box O proteins (FOXO4, FOXO3a, and FOXO1a) are transcription factors that can increase p27 transcription, by which results in cancer cell G₀/G₁ growth arrest [32,33]. Recent studies reveal that ISO treatment upregulates p27 transcription by increasing FOXO1 binding to p27 promoter region in human BC cells. Conversely, another transcription factor c-myc was identified as a negative regulatory transcription factor for p27 transcription [32,34]. Transcription factor E2F1 could also bind to the p27 promoter in vivo and activates p27 transcription [35]. The results from animal experiments show that E2F1-null mice are likely to have more tumor development [36,37], suggesting that E2F1 acts as a suppressor of tumor growth. The results from analyzing gene expression profile of 165 BC samples surgically removed tumors also reveal that E2F1 expression is strongly associated with the status of invasive BC [38]. Moreover, recent studies demonstrate that E2F1 is crucial for promotion of BC invasion via upregulating Hsp70 transcription [39]. Distinctly different from the role of E2F1 in the suppression of BC cell invasion, our experimental results from current studies intensively illuminated that E2F1 could directly bind to the p27 promoter and results in p27 induction, which in turn mediates ISO inhibition of EGF-induced anchorage-independent growth in human urothelial cells. Our study also found that overexpression of miR-205 promotes the transformation of human urothelial cells, while ISO treatment inhibits miR-205 expression, by which reduces its binding to 3’-UTR E2F1 mRNA, therefore promotes the E2F1 protein translation. The expression of miR-205 was consistently upregulated in several cancers, including, nasopharyngeal carcinoma and endometrial carcinoma [40–43]. The results from current studies showed that miR-205 was upregulated in human BCs and that ISO treatment attenuated miR-205 levels. Given current studies revealing that miR-205 directly targets 3’-UTR of E2F1, and overexpression of miR-205 completely reverses ISO-induced E2F1 expression and p27 induction, as well as inhibition of cell transformation by ISO, we anticipated that attenuation of miR-205 plays an essential role in ISO prevention of urothelial cell transformation.

In summary, we demonstrate that natural compound ISO inhibits EGF-induced human urothelial cell transformation by induction of p27 through miR-205/E2F1 axis. Given that previous studies on the anti-tumor activity of ISO, this study further refined the prophylactic value of ISO in management of BC. For the purpose of completely recognizing the chemoprophylactic effect of ISO, future studies should further explore how ISO treatment initiates miR-205 downregulation both in vitro and in vivo.

Funding Statement

This work was partially supported by grants from Natural Science Foundation of China (NSFC81773391 to C.H.); Oujiang Research Project (OJQD2022006 to C.H.); Natural Science Foundation of Ningbo (202003N4205 to J.S.); the Research Foundation of Ningbo Institute of Life and Health Industry, University of Chinese Academy of Sciences (research fund of ningbo institute of life and health industry, university of Chinese academy of sciences 2020YJY0209 to J.S.); the National Natural Science Foundation of China (82003199 to J.S.).

Disclosure statement

No potential conflict of interest was reported by the author(s).

Ethical standards

Ethical approval: All specimens were obtained with appropriate informed consent from the patients and the Medical Ethics Committee of China. And the comprehensive information of tissues were described in Materials and Methods.

Author contributions

MH, XH, JX and JS acquisition and analysis of the data, drafting the manuscript; ZT, JW, HC, XW and PS, acquisition of data, critically reviewed the article; HY and JS analysis and interpretation of the data, critically reviewed the article; and CH conception and design, analysis and interpretation of the data, drafting and revising the manuscript. All the authors have read and approved the final manuscript.

Data availability

The authors declare that all data supporting the findings of this study are available with the article or from the corresponding author upon reasonable request.

Supplementary material

Supplemental data for this article can be accessed online at https://doi.org/10.1080/15384101.2022.2074623