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. 2021 Jan 20:7:620776.
doi: 10.3389/fvets.2020.620776. eCollection 2020.

The Small Molecule BIBR1532 Exerts Potential Anti-cancer Activities in Preclinical Models of Feline Oral Squamous Cell Carcinoma Through Inhibition of Telomerase Activity and Down-Regulation of TERT

Gennaro Altamura  1 , Barbara Degli Uberti  2 , Giorgio Galiero  2 , Giovanna De Luca  2 , Karen Power  1 , Luca Licenziato  1 , Paola Maiolino  1 , Giuseppe Borzacchiello  1
Affiliations

The Small Molecule BIBR1532 Exerts Potential Anti-cancer Activities in Preclinical Models of Feline Oral Squamous Cell Carcinoma Through Inhibition of Telomerase Activity and Down-Regulation of TERT

Gennaro Altamura et al. Front Vet Sci. .

Abstract

Expression of telomerase reverse transcriptase (TERT) and telomerase activity (TA) is a main feature of cancer, contributing to cell immortalization by causing telomeres dysfunction. BIBR1532 is a potent telomerase inhibitor that showed potential anti-tumor activities in several types of cancer, by triggering replicative senescence and apoptosis. In a previous work, we detected, for the first time, TERT expression and TA in preclinical models of feline oral squamous cell carcinoma (FOSCC); therefore, we aimed at extending our investigation by testing the effects of treatment with BIBR1532, in order to explore the role of telomerase in this tumor and foreshadow the possibility of it being considered as a future therapeutic target. In the present study, treatment of FOSCC cell lines SCCF1, SCCF2, and SCCF3 with BIBR1532 resulted in successful inhibition of TA, with subsequent cell growth stoppage and decrease in cell viability. Molecular data showed that up-regulation of cell cycle inhibitor p21, unbalancing of Bax/Bcl-2 ratio, and down-regulation of survival gene Survivin were mostly involved in the observed cellular events. Moreover, BIBR1532 diminished the expression of TERT and its transcriptional activator cMyc, resulting in the down-regulation of epidermal growth factor receptor (EGFR), phospho-ERK/ERK ratio, and matrix metalloproteinases (MMPs)-1/-2 and-9, likely as a consequence of an impairment of TERT extra-telomeric functions. Taken together, our data suggest that BIBR1532 exerts multiple anti-cancer activities in FOSCC by inhibiting telomerase pathway and interfering with signaling routes involved in cell proliferation, cell survival, and invasion, paving the way for future translational studies aimed at evaluating its possible employment in the treatment of this severe tumor of cats.

Keywords: BIBR1532; EGFR; MMP; TERT; cancer; feline oral squamous cell carcinoma; telomerase activity.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
BIBR1532 down-regulates telomerase activity (TA) in a dose-dependent manner in FOSCC cell lines. (A) SCCF1, SCCF2, and SCCF3 were treated with BIBR1532 at 25, 50, and 100 μM vs DMSO and analyzed by telomeric repeat amplification protocol (TRAP) assay. Representative gels out of at least three independent experiments are illustrated (C–: negative control, sample with no lysate; L100bp: 100 bp DNA ladder, the first band from the bottom is 100 bp; L50bp: 50 bp DNA ladder, the first band from the bottom is 50 bp). (B) Quantification of TA by densitometric analysis of gel scans. Data were calculated as the ratio between TERT products ladder and 36 bp internal standard and represent the mean ± standard deviations of at least three independent experiments. Changes in TA were calculated and expressed as % for each BIBR1532 dose compared with its respective DMSO control set as 100% (statistically significant, *P < 0.05; **P < 0.01).
Figure 2
Figure 2
BIBR1532 inhibits cell growth and decreases cell viability in FOSCC cell lines. (A) SCCF1, SCCF2, and SCCF3 were treated with BIBR1532 at 25, 50, and 100 μM vs DMSO and analyzed by cell counting. The inhibitory effect on cell growth of each dose of BIBR1532 was calculated and expressed as % of decrease compared with its respective DMSO control set as 100%. Mean ± standard deviations (SD) from at least four independent experiments are shown. (B) Cell viability assessed by trypan blue exclusion assay. The effect of each dose of was calculated and expressed as % of decrease compared with its respective DMSO control set as 100%; the plots represent mean ± SD from at least four independent experiments (statistically significant, *P < 0.05; **P < 0.01).
Figure 3
Figure 3
Gene expression changes in p21, Bcl-2, Bax, and Survivin in FOSCC cell lines treated with BIBR1532. SCCF1, SCCF2, and SCCF3 were treated with BIBR1532 at 25, 50, and 100 μM vs DMSO and analyzed by qPCR for p21 (A), Bcl-2 (B), and Bax (C). Bax/Bcl-2 transcriptional ratio (D) and Survivin gene expression (E) were also calculated as a measure of activated apoptosis. Data were normalized for β2-microglobulin and expressed as relative quantization by the 2−ΔΔCt method. Changes in relative mRNA levels for each dose of BIBR1532 were calculated and compared with their respective DMSO control set as 1. Plots represent the mean ± standard deviations of at least three repeated, independent experiments performed in technical triplicate (statistically significant, *P < 0.05; **P < 0.01).
Figure 4
Figure 4
BIBR1532 down-regulates cMyc and TERT in FOSCC cell lines. (A) SCCF1, SCCF2, and SCCF3 were treated with BIBR1532 at 50 μM vs DMSO and analyzed by qPCR for TERT and cMyc gene expression. Data were normalized for β2-microglobulin as housekeeping gene and expressed as relative quantization by the 2−ΔΔCt method. Changes in relative mRNA levels were calculated for SCCF1, SCCF2, and SCCF3 treated with BIBR1532 compared with their respective DMSO control set as 1. Plots represent the mean ± standard deviations (SD) of at least three repeated, independent experiments performed in technical triplicate. (B) A representative Western blotting (WB) gel for TERT protein expression in BIBR1532- vs. DMSO-treated cells. The blot was stripped and reprobed for β-actin to ensure comparable protein loading and allow normalization. For each cell line, boxes are cut from the same gel at the same exposure time and properly aligned according to molecular standards loaded onto the gel. Full scans from original gels are shown in Supplementary Figure 3. (C) Mean densitometric values normalized for β-actin expression ± SD from at least three repeated, independent WB experiments. Changes in protein levels were calculated for SCCF1, SCCF2, and SCCF3 treated with BIBR1532 compared with their respective DMSO control set as 1 (statistically significant, *P < 0.05 and **P < 0.01).
Figure 5
Figure 5
BIBR1532 down-regulates EGFR pathway in FOSCC cell lines. (A) SCCF1, SCCF2, and SCCF3 were treated with BIBR1532 50 μM vs. DMSO and analyzed by qPCR for EGFR gene expression. Data were normalized for β2-microglobulin and expressed as relative quantization by the 2−ΔΔCt method. Changes in relative mRNA levels were calculated for SCCF1, SCCF2, and SCCF3 treated with BIBR1532 compared with their respective DMSO control set as 1. Plots represent the mean ± standard deviations (SD) of at least three repeated, independent experiments performed in technical triplicate. (B) Representative WB for EGFR, ERK, and pERK in BIBR1532- vs. DMSO-treated cells. HeLa whole cell lysate run along with feline samples confirmed the identity of the bands. The blot was stripped and reprobed for β-actin to ensure comparable protein loading and allow normalization. (C) Densitometric values of EGFR normalized for β-actin and densitometric ratio pERK/ERK; data are expressed as mean ± SD from at least three repeated, independent experiments. Changes in protein levels were calculated for SCCF1, SCCF2, and SCCF3 treated with BIBR1532 compared with their respective DMSO control set as 1 (statistically significant, *P < 0.05 and **P < 0.01).
Figure 6
Figure 6
BIBR1532 down-regulates MMP-1/-2/-9 in FOSCC cell lines. (A) SCCF1, SCCF2, and SCCF3 were treated with BIBR1532 50 μM vs. DMSO and analyzed by Western blotting (WB) for MMP-1/-2/-9. Representative WB experiments showing lower MMP-1/-2/-9 expression at protein level in BIBR1532- vs. DMSO-treated cells are shown. Blots were stripped and reprobed for β-actin to ensure comparable protein loading and allow normalization. For each cell line, boxes are cut from the same gel at the same exposure time and properly aligned according to molecular standards loaded onto the gel. Full scans from original gels are shown in Supplementary Figure 4. (B) Mean densitometric values normalized for β-actin expression ± SD from at least two repeated, independent experiments. Changes in MMPs protein levels were calculated for SCCF1, SCCF2, and SCCF3 treated with BIBR1532 compared with their respective DMSO control set as 1 (statistically significant, *P < 0.05 and **P < 0.01).
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References

    1. Deng Y, Chan SS, Chang S. Telomere dysfunction and tumour suppression: the senescence connection. Nat Rev Cancer. (2008) 8:450–8. 10.1038/nrc2393 - DOI - PMC - PubMed
    1. Low KC, Tergaonkar V. Telomerase: central regulator of all of the hallmarks of cancer. Trends Biochem Sci. (2013) 38:426–34. 10.1016/j.tibs.2013.07.001 - DOI - PubMed
    1. Cerni C. Telomeres, telomerase, and myc. An update. Mutat Res. (2000) 462:31–47. 10.1016/s1383-5742(99)00091-5 - DOI - PubMed
    1. McMurray HR, McCance DJ. Human papillomavirus type 16 E6 activates TERT gene transcription through induction of c-Myc and release of USF-mediated repression. J Virol. (2003) 77:9852–61. 10.1128/jvi.77.18.9852-9861.2003 - DOI - PMC - PubMed
    1. Smith LL, Coller HA, Roberts JM. Telomerase modulates expression of growth-controlling genes and enhances cell proliferation. Nat Cell Biol. (2003) 5:474–9. 10.1038/ncb985 - DOI - PubMed