Telmisartan inhibits cell proliferation by blocking nuclear translocation of ProHB-EGF C-terminal fragment in colon cancer cells

Abstract

Background aims: Current treatment target toward advanced colorectal cancers is mainly focused on the epidermal growth factor receptor (EGFR) signaling, but its additive effects with chemotherapy are still limited. A disintegrin and metalloproteinase (ADAM) cleaves the proheparin-binding epidermal growth factor like growth factor (proHB-EGF). And soluble HB-EGF activates EGFR. In parallel, the carboxy-terminal fragment of proHB-EGF (HB-EGF-CTF) translocates into the inner nuclear membrane, and subsequently exerts on the regulation of cell proliferation by binding nuclear promyelocytic leukemia zinc finger (PLZF) protein, a transcriptional repressor, thereby causing its nuclear export. We hypothesized that the inhibition of HB-EGF-CTF nuclear translocation may be a new strategy in preventing cell proliferation.

Methods: 12-O-tetradecanoylphorbor-13-acetate (TPA) was treated to activate ADAM. Nine-thousand chemical compounds were screened for their efficacies in blocking the binding of HB-EGF-CTF to promyelocytic leukemia zinc finger (PLZF) with Alphascreen system. The obtained candidates were then used to block the binding of HB-EGF-CTF to PLZF in colon cancer cells, HT29 and HCT116. Cell proliferation was investigated with a growth curve assay. The intracellular localization, and association between HB-EGF-CTF and PLZF, was assessed with immunofluorescent staining, and immunoprecipitation and Western blotting, respectively. The effects of obtained candidates on EGFR phosphorylation and on nuclear translocation of HB-EGF-CTF and export of PLZF during the angiotensin II type1 receptor (AT1R) knockdown were also investigated.

Results: Telmisartan and candesartan were found to be potential candidates. Telmisartan inhibited TPA-induced cell proliferation stronger than candesartan. Telmisartan, but not candesartan blocked the nuclear translocation of HB-EGF-CTF, and binding of HB-EGF-CTF to PLZF, during TPA stimulation. Both telmisartan and candesartan did not inhibit TPA-induced EGFR phosphorylation, and telmisartan, but not candesartan, inhibited TPA-induced nuclear translocation of HB-EGF-CTF after knockdown of AT1R.

Conclusions: The inhibition of HB-EGF-CTF nuclear translocation with telmisartan may be a novel strategy in preventing cell proliferation.

Figure 1. Dual signaling pathways of EGFR phosphorylation and HB-EGF C-terminal fragment nuclear translocation during cell proliferation.

Quoted from and modified. TPA induces an ADAM-mediated cleavage of proHB-EGF, and results in the ectodomain shedding of its N-terminal fragment and generation of an intracellular C-terminal fragment (CTF). The soluble HB-EGF binds to the EGFR and induces a rapid transient phosphorylation of EGFR. This phosphorylation results in the transcription of various genes. Meanwhile the HB-EGF-CTF is translocated into the nucleus, where it subsequently induces the nuclear export of PLZF. This results in the progression of cell cycle. The potent inhibitor blocks the nuclear translocation of HB-EGF-CTF. P indicates phosphorylation. Abbreviations: EGFR; epidermal growth factor receptor, TPA; 12-O-tetradecanoylphorbol-13-acetate, PKCδ; protein kinase Cδ, ADAM; a disintegrin and metalloproteinase, HB-EGF; heparin-binding EGF-like growth factor, CTF; C-terminal fragment, MAPK; mitogen-activated protein kinase, PLZF; promyelocytic leukemia zinc finger.

Figure 2. Binding of EGFR ligand-CTFs to ZnF5-8 region of PLZF.

(A) Schema of FLAG-tagged full length PLZF consisting of FLAG, BTB, Center, and nine ZnFs. (B) HT1080 cells stably expressing pro-HB-EGF, pro-TGF-α, pro-AR and pro-EPR were transiently transfected with an expression vector encoding FLAG-tagged PLZF. PLZF protein expression of cell lysates (lane1), as well as, cells treated with 100 nM TPA for 1 h, and probed with anti-FLAG antibodies following immunoprecipitation with anti-EGFR ligands-CTF antibodies (lane2) and an anti-normal rabbit IgG (lane3). (C) GST pull-down assay. Cell lysates containing FLAG-tagged PLZF derivatives were incubated with GST (lane 2), GST-HB-EGF-CTF (lane 3), GST-TGF-α-CTF (lane4), GST-AR-CTF (lane 5), and GST-EPR-CTF (lane 6) beads for 2 h, and bound proteins were detected by immunoblotting with an anti-FLAG antibody. (D) Schema of FLAG-tagged PLZF derivatives. The binding properties of PLZF derivatives to GST-fused- HB-EGF-CTF, TGF-α-CTF, AR-CTF and EPR-CTF GST in a pull-down assay, as summarized in the right lanes of each structure. Binding properties are based on the estimation of band intensity with are relative to the control band and indicated by++(>50%),+(50–10%), and − (<10%).

Figure 3. The interaction of EGFR-ligand-CTFs and PLZF, and high-throughput screening for inhibitors blocking the interaction.

(A) Direct interaction between EGFR-ligand-CTF and PLZF (GST-tagged-ZnF5-8) with the SPR system. The recombinant biotin-EGFR- ligand-CTFs were individually immobilized, by SA sensor chips. GST-Zn5-8 with concentrations ranging from 0.03 to 1.0 µM was then injected with running buffer at 25°C and a flow rate of 30 µL/min for 2 min. (B) The expression vector encoding CFP-PLZF was co-transfected into wild-type HT1080 cells and HT1080 cells stably expressing either proHB-EGF, proTGF-α, proAR or proEPR. The cells were cultured for 24 h and then pretreated in serum-free medium with KB-R7785. Cells were then treated in the serum-free conditioned medium with TPA, and the subcellular localization of the CFP fusion protein was observed. To determine the percentage of cells (mean±SD) demonstrating nuclear localization of CFP-PLZF, cells were counted in at least two transfections, and at least 200 cells expressing CFP-PLZF were examined in each experiment. *P<0.05 for the stimulus effect during TPA treatment vs. no treatment, and **P<0.05 for the inhibitory effect during KB-R7785 treatment vs. TPA treatment. (C) A schematic of the high-throughput Alphascreen system. Upon excitation at 680 nm, ambient oxygen is converted to singlet oxygen (¹O₂) by a photosensitizer present in the donor beads. If the acceptor beads are in close proximity (<200 nm), ¹O₂ transfers its energy to thioxene derivatives present in the acceptor beads leading to emission of light at 520–620 nm. One complex consists of streptavidin-coated donor beads and biotinylated EGFR ligands-CTF. Another consists of anti-GST antibody-conjugated acceptor beads and GST-ZF5-8. If the association between EGFR ligand-CTF and ZnF5-8 occurs, the beads are close enough to allow detection of a signal. Any inhibitors of this interaction would increase the distance between the beads, and the signal would be lost. (D) Alphascreen signals of GST or GST-Zn5-8 incubated with various concentrations of biotin-HB-EGF-CTF. (E) Alphascreen signals of biotin-HB-EGF-CTF incubated with various concentrations of GST or GST-Zn5-8. (E) The binding abilities of HB-EGF, TGF-α, amphiregulin (AR), and epiregulin (EPR), to PLZF with Alphascreen system.

Figure 4. The potent candidates as inhibitors and their inhibitory effects on cell proliferation.

(A) Twelve candidate compound’s structural formula based on efficacy for blocking binding of HB-EGF-CTF or AR-CTF to Zn5-8 of PLZF with Alphascreen system. (B) Inhibitory effects of twelve candidate compounds on TPA-induced cell proliferation in keratinocytes with a cell proliferation assay. 2×10⁴ cells were treated in the conditioned media with or without the twelve candidate compounds during TPA stimulation, and absorbance at 450 nm was determined every 12 h till 60 h. **P<0.05 for the inhibitory effects during 12 candidates treatment vs. TPA treatment. (C, D) The inhibitory effects of the candidates, specifically compound no.8016 and telmisartan, on the binding of AR-CTF to PLZF. Plots with the percent inhibition against various concentrations of compound no.8016 and telmisartan presented with the IC₅₀ values observed by Alphascreen system. (E) Inhibitory effects of three ARB candidates on the binding of AR-CTF to PLZF. Plots with the percent inhibition against various concentrations of each inhibitor and inhibition presented with IC₅₀ values.

Figure 5. Inhibitory effects of no.8016 and four ARBs on cell proliferation.

(A-D) Inhibitory effects of compound no.8016 (A) and four ARBs (B) on TPA-induced cell proliferation in keratinocytes with a cell proliferation assay. 2×10⁴ cells were treated in conditioned media with or without the no.8016 or four ARBs during TPA stimulation, and absorbance at 450 nm was determined every 12 h till 60 h. (A, C) Inhibition of cell proliferation with no.8016 and telmisartan was verified following a wash at 12 h. (D) The cells were also observed with microscopy. **P<0.05 for the inhibitory effects during 12 candidates and four ARBs treatment vs. TPA treatment.

Figure 6. Cell proliferation through EGFR pathway and nuclear translocation of HB-EGF-CTF during TPA stimulation.

TPA-induced cell proliferation through EGFR and nuclear translocation of HB-EGF-CTF signaling. (A) Growth curve assay. HT29 cell numbers which were counted daily, 24 h (i.e.day1) after cells were seeded in three dependent colonies that were cultured in conditioned media. The values are means of three independent experiments. (B) Cell numbers of colonies cultured in 5% FBS conditioned media with or without TPA, KB-R7785, AG1478, and recombinant HB-EGF on day6. The cells were also observed with microscopy (×200) *P <0.05 for the stimulus effect, and **P<0.05 for the inhibitory effect. (C) Effects of KB-R7785 and AG1478 on TPA-induced nuclear translocation of HB-EGF-CTF and nuclear export of PLZF. Cells were treated with TPA following preincubation with or without KB-R7785 and AG1478. Immunofluorescent staining with anti-HB-EGF-CTF antibodies (red signals), anti-PLZF antibodies (green) and DAPI (blue), which stains for nuclei was performed. Images were obtained on a fluorescence microscope (×200). The white bar indicated 10 µm. (D) Effects of KB-R7785 on the association between HB-EGF-CTF and PLZF after TPA stimulation. Cells were treated with TPA at various times following preincubation with or without KB-R7785. Blotted samples were probed with antibodies against PLZF after immunoprecipitation with anti-HB-EGF-CTF antibody (upper panel). The total amount of HB-EGF-CTF in the immunoprecipitates was determined by reprobing the same blot with an anti-HB-EGF antibody (lower panel).

Figure 7. The inhibitory effects of telmisartan and candesartan on TPA-induced cell proliferation in colon cancer cells.

(A-D) Inhibitory effects of telmisartan, but not candesartan, on TPA-induced cell proliferation in HT29 cells (A), HCT116 (B), SW480 (C), and CaCo2 (D) with CCK-8 kit assay. After 24 h of plating, 2×10⁵ cells were incubated for 72 h in conditioned media with or without telmisartan, candesartan and/or TPA. Each bar represents the means of six independent experiments. *P<0.05 for the stimulus effect, and **P<0.05 for the inhibitory effect. (E and G) In growth curve assay, HT29 and HCT116 cell numbers were counted daily in three dependent colonies cultured in conditioned media. The values are means of three independent experiments. ×; Control (5%FBS), ▪; TPA (100 nM), ○; telmisartan (30 µM), •; TPA+telmisartan (30 µM), △; candesartan (30 µM), ▴; TPA+candesartan (30 µM). (F and H) Cell numbers in colonies cultured in conditioned media with or without TPA, telmisartan and candesartan on day 5 or 6. *P<0.05 for the stimulus effect, and **P <0.05 for the inhibitory effect. Lane 1; Control (5% FBS white box), 2; TPA (100 nM), 3; TPA+telmisartan (30 µM), 4; TPA+candesartan (30 µM), 5; telmisartan (30 µM), 6; candesartan (30 µM). (I) Knockdown of ADAM12 with siRNA. Probing with an anti-ADAM12 antibody (upper panel) and anti- β-actin antibody (lower panel). (J) Inhibitory effects of telmisartan on TPA-induced cell proliferation in HT29 cells with CCK-8 kit assay following knockdown of ADAM12 with siRNA. *P<0.05 for the stimulus effect, and **P <0.05 for the inhibitory effect. (K) Inhibitory effects of telmisartan and GW9662 on TPA-induced cell proliferation in HT29 cells with CCK-8 kit assay. *P<0.05 for the stimulus effect, and **P<0.05 for the inhibitory effect.

Figure 8. The effects of telmisartan or candesartan on localization and binding of HB-EGF-CTF and PLZF.

(A and B) Immunofluorescent stainings with anti-HB-EGF-CTF antibodies (red), anti-PLZF antibodies (green), and DAPI (blue), in the presence or absence of TPA after pretreatment with telmisartan or candesartan. Images were obtained on a fluorescence microscope (×200). The white bar indicated 10 µm. (C) Effects of telmisartan or candesartan on the TPA-induced association between HB-EGF-CTF and PLZF. HT29 cells were preincubated with or without telmisartan or candesartan. The cells were then treated with TPA for 0, 30, 60 and 120 min. Blotted samples were probed with an anti-PLZF antibody after immunoprecipitation with the anti-HB-EGF-CTF antibody (upper panel). The total amount of HB-EGF-CTF in the immunoprecipitates was determined by reprobing the same blot with an anti-HB-EGF-CTF antibody (lower panel). (D)Three staining regions were randomly selected under a ×200 field and the total for three regions was calculated as the positive rate of cells displaying nuclear staining. The percentage of cells showing nuclear staining is shown for 100 nM of TPA, and 30 µM of telmisartan and candesartan. Images were obtained on a fluorescence microscope (×200). The white bar indicated 10 µm. *P<0.05 for the stimulus effect, and **P<0.05 for the inhibitory effect.

Figure 9. The effects of telmisartan and candesartan on HB-EGF-CTF nuclear translocation and cell proliferation during AT1R depletion.

(A) Effects of telmisartan or candesartan on TPA-induced EGFR phosphorylation. Cells were preincubated with or without telmisartan or candesartan, and then treated with TPA for 0, 15, 60 and 120 min. Blotted samples were probed with an anti-phosphotyrosine antibody after immunoprecipitation with an anti-EGFR antibody (upper panel). The total amount of EGFR in the immunoprecipitates was determined by reprobing the same blot with an anti-EGFR antibody (lower panel). (B) Effects of telmisartan or candesartan on TPA-induced nuclear translocation of HB-EGF-CTF and nuclear export of PLZF following knockdown of AT1R with siRNA. Probing with an anti-AT1R antibody (upper panel) and anti-β-actin antibody (lower panel). (C) Cells were then treated with TPA following preincubation with or without telmisartan or candesartan. Immunofluorescent stainings with anti-HB-EGF-CTF antibodies (red), anti-PLZF antibodies (green) and DAPI (blue) were performed following knockdown of AT1R with siRNA. Images were obtained on a fluorescence microscope (×400). The white bar indicated 10 µm. (D)The inhibitory effects of telmisartan and candesartan on TPA-induced cell proliferation in HT29 cells with CCK-8 kit assay following knockdown of AT1R with siRNA. *P<0.05 for the stimulus effect, and **P<0.05 for the inhibitory effect.