Skip to main content
Download PDF

Anti-tumour activity of Panobinostat in oesophageal adenocarcinoma and squamous cell carcinoma cell lines

Clinical Epigenetics volume 16, Article number: 102 (2024) Cite this article

Abstract

Background

Oesophageal cancer remains a challenging disease with high mortality rates and few therapeutic options. In view of these difficulties, epigenetic drugs have emerged as potential alternatives for patient care. The goal of this study was to evaluate the effect and biological consequences of Panobinostat treatment, an HDAC (histone deacetylase) inhibitor already approved for treatment of patients with multiple myeloma, in oesophageal cell lines of normal and malignant origin, with the latter being representative of the two main histological subtypes: adenocarcinoma and squamous cell carcinoma.

Results

Panobinostat treatment inhibited growth and hindered proliferation, colony formation and invasion of oesophageal cancer cells. Considering HDAC tissue expression, HDAC1 was significantly upregulated in normal oesophageal epithelium in comparison with tumour tissue, whereas HDAC3 was overexpressed in oesophageal cancer compared to non-malignant mucosa. No differences between normal and tumour tissue were observed for HDAC2 and HDAC8 expression.

Conclusions

Panobinostat exposure effectively impaired malignant features of oesophageal cancer cells. Because HDAC3 was shown to be overexpressed in oesophageal tumour samples, this epigenetic drug may represent an alternative therapeutic option for oesophageal cancer patients.

Background

Oesophageal cancer is a major health burden worldwide, with high mortality rates [1]. This is a heterogenous disease, composed of two main histological subtypes: squamous cell carcinoma (SCC) and adenocarcinoma (AC). Although these tumours represent different biological entities with distinct molecular features, they share poor survival rates [1,2,3]. This is presumably a consequence of the disease being diagnosed at advanced stages due to difficulties in screening and the lack of specific targeted therapies [4]. Therefore, considering the high mortality rates of oesophageal cancer, research is now focused on new therapeutic options and also biomarkers allowing for early diagnosis.

In this regard, epigenetic alterations have recently drawn attention, because of their potential use as diagnostic and therapeutic tools. Indeed, the reversible nature of this type of alterations has made them attractive alternatives for clinical patient management. Histone deacetylation is a frequent event in cancer [5] that reduces chromatin accessibility and is catalysed by histone deacetylases (HDAC), which comprise four classes of proteins, grouped according to their homology [6]. Histone deacetylation is involved in multiple biological processes, such as autophagy, apoptosis, cell cycle control, angiogenesis and metastasis, among others, and, thus, HDAC inhibitors became interesting clinical options and have been extensively studied as anti-cancer agents [7, 8].

Panobinostat is one of such HDAC inhibitors, displaying activity at low concentrations (nanomolar range) [9], and it has already been approved in 2015 for the treatment of multiple myeloma [10, 11]. Additionally, this compound has demonstrated anti-tumour effects in pre-clinical studies in various cancer models, alone or in combination with other drugs [12,13,14,15,16,17] and several clinical trials are ongoing evaluating its efficacy in the medical setting.

Here, we evaluated the effect of Panobinostat treatment and its biological consequences in oesophageal cell lines of normal and malignant origin. Oesophageal cells were treated with Panobinostat, and its functional effects were assessed through in vitro assays. Furthermore, the expression of HDAC class I members (HDAC1, HDAC2, HDAC3 and HDAC8) was evaluated in a series of oesophageal cancer (comprising SCC and AC) and normal epithelium tissue samples.

Material and methods

Cell culture and treatments

Normal oesophageal cell line HET1A was a kind gift by Professor Raquel Almeida (i3S, University of Porto, Portugal), the AC cell line OE-19 was a kind gift of Professor Filomena Botelho (CIMAGO, University of Coimbra, Portugal), and the SCC cell line KYSE-30 was kindly gifted by Professor Fátima Baltazar (ICVS, University of Minho, Portugal). All oesophageal cells were maintained at 37ºC and 5% CO2 at low passages (maximum passage was 20). HET1A was cultured in DMEM culture medium (PAN-Biotech, Aidenbach, Germany), whereas the other cell lines were grown in RPMI culture medium (PAN-Biotech) and both media were supplemented with 10% foetal bovine serum (Biochrom, Berlin, Germany) and 1% penicillin/streptomycin (GRiSP, Porto, Portugal).

Panobinostat (Selleckchem, Houston, TX, USA) was dissolved in PBS, which was used as treatment control. For three consecutive days (72 h) cells have undergone treatment with Panobinostat: the culture medium was discarded daily and fresh medium containing the drug was added. In every assay, HET1A, OE-19 and KYSE-30 were treated with 28 nM, 24 nM and 38 nM of Panobinostat, respectively.

Cell viability assay

Oesophageal cells were seeded (1 × 104) in 96-well plates and on the following day were treated with various Panobinostat concentrations (5–100 nM). Treatments were repeated for 3 consecutive days. Cell viability was assessed using the MTT (3-(4, 5dimethylthiazol-2-yl)-2, 5-diphenyl-tetrazolium bromide) (Sigma-Aldrich/Merck, Darmstadt, Germany) assay. Evaluation was performed before the first treatment and 24 h after the last treatment. Cells were incubated with 0.5 mg/mL of MTT dissolved in culture medium and placed at 37ºC and 5%CO2 in the dark for 3 h. The resulting formazan crystals were dissolved using DMSO (dimethyl sulphoxide), and absorbance was measured in a microplate reader (FLUOstar® Omega, BMG Labtech, Ortenberg, Germany) at 540 nm.

Western blotting

Protein lysates were extracted from oesophageal cells and separated in a 10% SDS-PAGE gel. Proteins were transferred to a nitrocellulose membrane and blocked with 5% (w/v) non-fat milk in TBS containing 0.5% (v/v) Tween 20 for 1 h. Membranes were incubated with cyclin D1 (1:200 ON, clone DCS-6, Santa Cruz, Dallas, TX, USA), p21 (1:125 ON, clone SX118, Pharmingen/BD Biosciences, Franklin Lakes, NJ, USA) and with β-actin antibodies (clone A1978, Sigma, Germany) 1:10,000 ON. Following washes with TBS-T, they were incubated with horseradish secondary antibodies (Cell Signaling, USA) 1:1000 1 h and after washes with TBS-T, proteins were detected using ECL (Bio-Rad, USA). Images were analysed using the ImageJ software.

Proliferation assay

Oesophageal cell lines were seeded (5 × 103) in 96-well plates and treated daily with Panobinostat for the following 3 days. Assays were performed at the 0 h time point and 24 h after the final treatment using the Cell Proliferation ELISA, BrdU (colorimetric) kit (Roche/Sigma–Aldrich/Merck, Darmstadt, Germany) following manufacturer’s guidelines. Absorbance was evaluated using a microplate reader (FLUOstar® Omega, BMG Labtech) at 450 nm with background subtraction at 690 nm; all values were normalised for the first time point (0 h) and the vehicle condition.

Apoptosis assay

Oesophageal cell lines were seeded (2 × 105) in flasks and treated daily with Panobinostat for the following 3 days. The FITC Annexin V Apoptosis Detection Kit with 7-AAD (Biolegend, San Diego, CA, USA) was used according to manufacturer’s protocol to identify the apoptotic oesophageal cells. Data were acquired by flow cytometry using a FACS Canto™ II Cell Analyzer (BD Biosciences) and analysed using the FlowJo™ software (BD Biosciences).

Colony formation assay

Oesophageal cells were seeded (5 × 103 cells) in 6-well plates and treated daily with Panobinostat for the following 3 days. Cells were incubated for an additional 10 days post-treatment for OE-19 and 3 days for KYSE-30 to allow colonies to grow. Upon colony formation, cells were washed in PBS (phosphate-buffered saline), followed by fixation with methanol (Supelco/Sigma–Aldrich/Merck, Darmstadt, Germany) for 10 min and another wash with PBS. Subsequently, colonies were stained with Hemacolor solution 2 (Sigma–Aldrich/Merck) for 5 min, washed with PBS, incubated with Hemacolor solution 3 (Sigma–Aldrich / Merck) for 5 min and washed with PBS. Finally, colonies were washed with running water for 2 min and allowed to dry overnight. The survival fraction (SF) was calculated considering the plating efficiency (PE) of the control, using the following equations: PE = number of colonies counted on control ÷ number of cells plated × 100 and SF = number of colonies counted ÷ (cells plated × [PE ÷ 100]).

Cell invasion assay

Oesophageal cell lines were seeded (2 × 105) in flasks and treated with Panobinostat for 72 h. 24 h after the final treatment, cells were harvested and seeded (1 × 105 for OE-19 and 5 × 104 for KYSE-30) in Matrigel® invasion chambers (Corning, Corning, NY, USA) in serum-free medium. After a defined time period (72 h for OE-19 and 24 h for KYSE-30), the chambers were washed with PBS, the cells in the lower part of the chamber were incubated with paraformaldehyde 4% (ChemCruz/Santa Cruz Biotechnology) for 2 min, while the cells in the upper part of the chamber were removed. After washing with PBS, cells were fixed using methanol (Supelco/Sigma-Aldrich/Merck) for 20 min, followed by another wash with PBS. Finally, cell staining was performed with Crystal Violet (Active Motif, Carlsbad, CA, USA) for 10 min and cells were washed with PBS. The whole membrane was photographed under a stereomicroscope (model S2X16, Olympus) and the invasive cells were counted using the ImageJ software Cell Counter Plugin.

Patients’ selection and tissue microarray (TMA) construction

Oesophageal tissue samples (n = 161) were obtained from the archives of the Department of Pathology of IPO Porto, Porto, Portugal. The series comprised 112 cases of oesophageal cancer, including 70 SCC and 42 AC, as well as 49 normal mucosae (derived from the oesophageal margin of gastrectomies performed for gastric cancer treatment). The cases included in this series were collected between 2007 and 2018.

Representative areas in all oesophageal tissue samples were selected by a pathologist using hematoxylin and eosin-stained sections and marked on the corresponding paraffin blocks. Three tissue cores of 2 mm in diameter were extracted from each selected donor block and deposited in a recipient paraffin block, using a TMA workstation (Abcam, Cambridge, UK). Twenty-four TMAs were constructed (10 for SCC, 7 for AC and 7 for normal tissue), each containing 24 samples, arranged in a 6 × 4 sector. Each TMA included 3 cores used for orientation purposes only: in the cancer TMAs, liver and normal oesophagus were used, whereas liver and colon mucosa were used in the normal tissue TMAs. In order to homogenise the paraffin of the recipient blocks and the paraffins of the cores extracted from the donor blocks, the TMAs were kept overnight at 37ºC. They were subsequently placed for 1 h at room temperature, followed by two cycles of 1 h at 37ºC plus 1 h at room temperature.

This study was approved by the Institutional Review Board (Comissão de Ética para a Saúde) of IPO Porto (CES 202/017). All the procedures involving the use of human samples were performed in accordance with the national ethical standards and following the 1964 Helsinki declaration and its later amendments or comparable ethical standards.

Immunohistochemistry

Immunohistochemistry was performed on 3-µm-thick sections, using the Novolink Max Polymer Detection System (Leica Biosystems, Newcastle, UK). Briefly, after deparaffinisation, epitope retrieval was performed in the microwave for 20 min, using a 10 mM citrate buffer solution (pH = 6) (Vector Laboratories, Newark, CA, USA) for HDAC1 and HDAC2 and a 1 mM EDTA buffer solution (pH = 8) (Sigma-Aldrich/Merck) for HDAC3 and HDAC8. Endogenous peroxidase activity was blocked in a 0.6% H2O2 solution for 20 min, and non-specific binding was hindered in a 1:50 solution of horse serum in antibody diluent for 20 min. Primary antibody incubation was performed as follows: HDAC1 (1:1000, clone 5C11, Sigma-Aldrich/Merck), HDAC2 (1:50, clone C-8, Santa Cruz Biotechnology, Dallas, TX, USA), HDAC3 (1:1000, clone Y415, Abcam) and HDAC8 (1:200, clone 48, Sigma-Aldrich/Merck) all for 1 h at room temperature. Afterwards, tissue slides were incubated with post-primary block for 30 min, followed by the polymer solution for 30 min and stained with 3,3′-diaminobenzidine (Sigma-Aldrich). Finally, slides were counterstained with haematoxylin, dehydrated and cover-slipped using Entellan (Sigma-Aldrich/Merck). Gastric mucosa, prostate (normal and malignant), endometrial carcinoma and colon carcinoma were used as positive controls for HDAC1, HDAC2, HDAC3, and HDAC8, respectively.

Immunohistochemical staining was evaluated considering the percentage of positive cells (extension) and intensity of staining. The score for extension was defined from 0 to 10 (10 corresponding to 100% of positive cells), and intensity was set from 1 to 3 (corresponding to weak, moderate and strong expression, respectively). The final score was obtained by multiplying the extension and intensity scores.

Statistical analysis

GraphPad Prism software version 7 was used to perform statistical analyses. Differences between conditions were assessed using Mann–Whitney or Kruskal–Wallis nonparametric tests, with Dunn’s correction. In all analyses, statistical significance was considered when p values were lower than 0.05: *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. All results are presented as mean ± standard deviation for each group and are representative of at least three independent experiments.

Results

Panobinostat inhibits oesophageal cell viability

The cytotoxic effect of Panobinostat was evaluated in oesophageal cell lines HET1A, OE-19 and KYSE-30 after 3 days of treatment, and the corresponding EC50 (half maximal effective concentration) values were calculated. The EC50 values for Panobinostat in HET1A, OE-19 and KYSE-30 were 28.3 nM, 24.4 nM and 37.9 nM, respectively (Fig. 1). The normal oesophageal cell line HET1A and the AC cell line OE-19 displayed comparable EC50 values, being more sensitive than the SCC cells (KYSE-30) to the treatment with Panobinostat.

Fig. 1
figure 1

Dose–response curves of oesophageal cell lines upon Panobinostat treatment. EC50 values after 3 days of treatment are indicated in each graph

Full size image

Panobinostat hinders cell proliferation and induces apoptosis in oesophageal cell lines

In vitro assays were performed to evaluate the functional effects of Panobinostat treatment. All oesophageal cell lines exposed to Panobinostat exhibited lower proliferation rates than vehicle-treated cells and differences were statistically significant (Fig. 2).

Fig. 2
figure 2

Effect of Panobinostat treatment in the proliferation rates of oesophageal cell lines. Values were normalised to the vehicle condition. (V—vehicle, P—Panobinostat; drug concentration: HET1A—28 nM, OE-19—24 nM and KYSE-30—38 nM)

Full size image

Treatment with Panobinostat induced an increase in the percentage of apoptotic cells in HET1A and OE-19 oesophageal cells (Fig. 3). In contrast, KYSE-30 cells only displayed a small decline in the apoptotic response after drug exposure when compared to vehicle-treated cells. In SCC, it was demonstrated that cell cycle arrest was a consequence of p21 expression increase as well as the reduction in cyclin D1 expression (18). Similarly, upon Panobinostat treatment, HET1A and OE-19 exhibited p21 expression upregulation, whereas a marginal decrease was observed in KYSE-30 cells (Fig. 4 and Additional file 1: Fig. S1). Accordingly, Cyclin D1 expression levels were also increased following drug administration in all oesophageal cell lines. Furthermore, the effect of Panobinostat treatment on class I HDAC members protein levels was evaluated (Additional file 2: Fig. S2, Additional file 3: Fig. S3, Additional file 4: Fig. S4, Additional file 5: Fig. S5). Collectively, the expression levels of HDAC1, 2, 3 and 8 were decreased following drug treatment and this effect was stronger in oesophageal cancer cell lines (OE-19 and KYSE-30) than in the normal oesophageal cell line HET1A.

Fig. 3
figure 3

Effect of Panobinostat treatment in the apoptosis of oesophageal cell lines. (V—vehicle, P—Panobinostat; drug concentration: HET1A—28 nM, OE-19—24 nM and KYSE-30—38 nM)

Full size image
Fig. 4
figure 4

– Effect of Panobinostat treatment in the expression of p21 and Cyclin D1 in oesophageal cell lines. Images were analysed using the ImageJ software. (V—vehicle, P—Panobinostat; drug concentration: HET1A—28 nM, OE-19—24 nM and KYSE-30—38 nM)

Full size image

Panobinostat reduces colony formation and inhibits cell invasion in oesophageal cancer cells

The clonogenic and invasion assays were only performed in OE-19 and KYSE-30 cell lines, since they assess features that are typical of tumour cells. Regarding colony formation, the treatment with Panobinostat significantly decreased the number of colonies in both oesophageal cancer cell lines when compared to control cells (Fig. 5).

Fig. 5
figure 5

Effect of Panobinostat treatment in the colony formation ability of oesophageal cancer cells. Values were normalised to the vehicle condition. (V—vehicle, P—Panobinostat; drug concentration: HET1A—28 nM, OE-19—24 nM and KYSE-30—38 nM)

Full size image

OE-19 and KYSE-30 treated with Panobinostat exhibited a statistically significant lower percentage of invasive cells than vehicle-treated cells (Fig. 6).

Fig. 6
figure 6

Effect of Panobinostat treatment in the invasive capacity of oesophageal cancer cells. Values were normalised to the vehicle condition. (V—vehicle, P—Panobinostat; drug concentration: HET1A—28 nM, OE-19—24 nM and KYSE-30—38 nM)

Full size image

Expression of HDAC class I members in normal and cancerous oesophageal tissue samples

The expression of HDAC class I members (HDAC1, HDAC2, HDAC3 and HDAC8) was evaluated by immunohistochemistry in oesophageal samples arranged in TMAs comprising both normal mucosa and cancer tissue. All proteins were expressed in the cell nucleus (Fig. 7). In normal tissue, expression was observed predominantly in the basal and parabasal cells with decreasing levels of expression towards the surface of the epithelium.

Fig. 7
figure 7

Immunohistochemical staining of HDAC1, HDAC2, HDAC3 and HDAC8 in the different types of oesophageal tissue. (N—normal, AC—adenocarcinoma, SCC—squamous cell carcinoma, magnification: 200x)

Full size image

HDAC1 expression levels were significantly lower in oesophageal cancer compared with normal tissue (Fig. 8). Considering HDAC2, the expression levels were similar in normal and tumour tissue. HDAC3 was upregulated in oesophageal carcinomas in comparison with non-malignant mucosa and this difference was statistically significant (p < 0.01). The same trend was observed for HDAC8 expression (p = 0.58).

Fig. 8
figure 8

HDAC1, HDAC2, HDAC3 and HDAC8 expression levels in normal oesophagus and tumour tissue. (N—normal, T—tumour)

Full size image

Concerning histological subtypes, differences were disclosed between normal oesophagus and SCC, as well as between AC and SCC for HDAC1 expression (Fig. 9). Regarding HDAC2, AC samples presented significantly higher levels of expression than SCC. HDAC3 was upregulated in AC comparatively to normal tissue and SCC. Considering HDAC8, AC samples exhibited significantly higher expression levels than SCC.

Fig. 9
figure 9

HDAC1, HDAC2, HDAC3 and HDAC8 expression levels in normal oesophageal tissue, AC and SCC. (AC—adenocarcinoma, SCC—squamous cell carcinoma)

Full size image

Discussion

Oesophageal cancer is a challenging disease, with poor survival rates and lacking effective targeted therapies [1, 4]. With the only treatment modalities being surgical resection and radiochemotherapy [19, 20], alternative options are urgently needed and have been the focus of intensive research.

Epigenetic alterations, due to their plastic nature, are good candidates for therapeutic targets. Here, we evaluated the functional effects of Panobinostat, a pan-HDAC inhibitor already approved for the treatment of multiple myeloma [11], in oesophageal cell lines. Additionally, the expression of HDAC class I members (HDAC1, HDAC2, HDAC3 and HDAC8) was assessed in oesophageal SCC, AC and non-malignant mucosa to evaluate their potential therapeutic actionability.

Using oesophageal cell lines of both tumour and non-malignant origin, phenotypic assays have been performed to evaluate the effect of Panobinostat treatment in various cell features. After drug exposure, a reduction in cell viability was detected in all the tested oesophageal cell lines. Although they display comparable EC50 values, OE-19 tumour cells were slightly more sensitive to Panobinostat treatment than the normal HET1A cells, an effect that has been observed in prostate cells in a previous study by our group [21]. We believe that some degree of toxicity upon Panobinostat treatment of normal oesophageal cells would be expected, since these cells also present epigenetic machinery, namely HDAC proteins, and are therefore susceptible of being affected by this drug. This effect may be reflected in decreased cell viability and proliferation, as well as in apoptosis induction. Our data also confirmed other reports which described an inhibitory effect on cell viability upon drug exposure [21,22,23,24,25,26,27,28]. In agreement with this, Panobinostat significantly suppressed proliferation of non-malignant and tumour oesophageal cells, further validating the growth inhibitory effects of this drug. Importantly, similar results on cell proliferation have been observed and reported in other cancer types, such as prostate and testicular tumours, as well as melanomas [21, 29, 30].

Interestingly, the exposure of oesophageal cell lines to Panobinostat produced discrepant results in apoptosis, with a dual outcome: it promoted cell death in HET1A and OE-19, whereas the opposite effect was observed in KYSE-30 cells. This result is probably a consequence of the different sensitivity to this drug exhibited by the cell lines, which is reflected in their EC50 values. Numerous studies have reported apoptosis induction after Panobinostat treatment [22,23,24,25,26, 28, 31,32,33,34], thus lending support to the results we have obtained in HET1A and OE-19 cell lines. Furthermore, in SCC, cell cycle arrest was accomplished by simultaneous upregulation of p21 and cyclin D1 expression decline [18]. A similar result was observed in thyroid cancer [28]. Here, we demonstrated an increment in p21 expression following Panobinostat treatment in HET1A (normal) and OE-19 (AC) cells, making ours the first study to evaluate the molecular mechanisms of action of Panobinostat in these types of oesophageal samples. Also in SCC cells, MS-275 (entinostat), a specific inhibitor of class I HDACs [35], induced apoptosis with a concomitant decrease of cyclin D1 and cyclin A expression (36). Upon Panobinostat exposure, KYSE-30 cells (SCC) showed a reduction in the percentage of apoptotic cells, which is in line with the slight increase in Cyclin D1 expression levels observed, as well as with the minimal p21 expression decrease. The other tested cell lines also exhibited Cyclin D1 expression upregulation, although to a lesser extent compared to the pronounced increase observed for p21 expression.

Upon Panobinostat treatment, there was also a significant decline in the invasive capacity of oesophageal cancer cell lines. Panobinostat has been previously shown by our group to decrease the invasive capacity of prostate cancer cells [21] and similar results were obtained in other tumour models, such as breast, thyroid and liver cancer [22, 32, 37,38,39]. Concomitantly, Panobinostat exposure or HDAC-silencing has reduced migration of malignant cells, namely in gastric, thyroid, breast and bladder cancer [33, 37,38,39,40]. Moreover, a reduction of the number of colonies of oesophageal cancer cell lines upon drug exposure was observed, confirming previous results in SCC cells [18] and, to the best of our knowledge, describing the same effect in AC cells for the first time. Furthermore, the effect of Panobinostat treatment in the downregulation of the clonogenic potential of cancer cells is well described in prostate, gastric, renal, colorectal, glioblastoma, ovary and thyroid tumours [21, 23, 24, 26,27,28, 33, 34]. Thus, Panobinostat demonstrated an effective ability to counteract these important malignant traits, foreseeing relevant anti-tumour activity in vivo.

Finally, the expression of HDAC class I members was assessed in normal and malignant (both SCC and AC) oesophageal tissue. Lower HDAC1 expression levels have been detected in oesophageal cancer in comparison with non-malignant tissue. Published data concerning HDAC1 expression in oesophageal cancer, however, are conflicting. Unlike our findings, it has been reported that HDAC1 is more highly expressed in oesophageal tumours than normal mucosa [36, 41, 42], but only SCC samples have been evaluated, whereas our data include both AC and SCC histological subtypes. Nonetheless, among our samples (normal, AC and SCC), the lowest HDAC1 levels were detected in SCC. Another study compared the expression in SCC and the corresponding normal epithelium and found a marginal increase in HDAC1 expression in the non-malignant mucosa in comparison with the tumour tissue (100% versus 95% of cases) [43]. However, only 20 samples were evaluated and the tumour-adjacent epithelium was considered as normal tissue, whereas we have used tissue from the oesophageal margin of gastrectomy specimens as normal mucosa, thus excluding potential carcinogenic field effects. A similar amount of HDAC1 expression in SCC and the corresponding non-malignant mucosa has also been reported [44], whereas, in AC, one report found that the majority of tumour cases presented low or negative expression (45).

Concerning HDAC2, no differences were detected between normal and tumour tissue, although AC samples presented higher levels of expression than SCC. In contrast to our data, HDAC2 expression was shown to be upregulated in SCC compared with non-cancerous tissue [36, 43], whereas in AC 68% of cases exhibited moderate or high expression (45).

As for HDAC3 expression, significant differences were observed, with non-malignant oesophageal mucosa displaying significantly lower levels than tumour tissue. Among tumour subtypes, HDAC3 expression was higher in AC than in SCC. HDAC3 has been described as more frequently expressed in SCC than in normal tissue (95% versus 80% of cases) [43], but, in that study, blocks that contained both normal and tumour tissue were used. Another study also reported higher HDAC3 expression in SCC in comparison with adjacent epithelium along with lower patients’ overall survival [46]. Ahrens and colleagues [47] observed lower HDAC1, HDAC2 and HDAC3 expression in 10–20% of SCC and AC compared with normal mucosa, but they have only assessed 10 samples of each histological subtype and the respective case-matched oesophageal non-malignant epithelium. HDAC3 deregulation has been implicated in various types of diseases, with its inhibitors being suggested as therapeutic options [48, 49]. In SCC, HDAC3 silencing promoted apoptosis and inhibited proliferation, migration and invasion [46].

Considering HDAC8, malignant tissue displayed higher expression levels than normal oesophagus, but this difference failed to reach statistical significance. AC samples exhibited higher expression levels than normal oesophageal tissue and SCC. Only one study reported on HDAC8 expression, showing that it was more frequently expressed in SCC than in non-malignant mucosa (43), but, to the best of our knowledge, no report has been published concerning oesophageal AC. As far as we know, this is the first study reporting on the expression of all class I HDACs in a comprehensive series of oesophageal samples comprising SCC, AC and normal epithelium. Variations in results of immunohistochemical analysis may derive from differences in populations under study, technical protocols (e.g. antibody clones) and origin of the control (normal) tissue. Nonetheless, it should be emphasised that most previously published studies used small tissue series, which may also impact the results as it might not capture the full range of expression across oesophageal tumours and even normal mucosa.

Conclusions

Altogether, our results demonstrate that Panobinostat treatment exerts anti-tumour effects in oesophageal cell lines, endorsing HDACs as potential therapeutic targets in oesophageal cancer. To confirm this, further studies will be required to identify which members of the HDAC family are responsible for the observed outcomes, thus enabling the use of more specific targeted therapies. Nonetheless, immunoexpression analysis suggests HDAC3 as the most likely candidate for therapeutic actionability, considering its overexpression in cancerous tissues. Further validation in an in vivo oesophageal cancer context is now required to further test the efficacy of Panobinostat and eventually consider testing it in a clinical trial.

Availability of data and materials

All data generated or analysed during this study are included in this article.

Abbreviations

AC:

Adenocarcinoma

DMSO:

Dimethyl sulphoxide

EC50 :

Half maximal effective concentration

HDAC:

Histone deacetylase

MTT:

3-(4, 5Dimethylthiazol-2-yl)-2, 5-diphenyl-tetrazolium bromide

ON:

Overnight

PBS:

Phosphate-buffered saline

PE:

Plating efficiency

SCC:

Squamous cell carcinoma

SDS-PAGE:

Sodium dodecyl-sulphate polyacrylamide gel electrophoresis

SF:

Survival fraction

TBS:

Tris-buffered saline

TBS-T:

Tris-buffered saline-tween

TMA:

Tissue microarray

References

  1. Morgan E, Soerjomataram I, Rumgay H, Coleman HG, Thrift AP, Vignat J, et al. The global landscape of esophageal squamous cell carcinoma and esophageal adenocarcinoma incidence and mortality in 2020 and projections to 2040: new estimates from GLOBOCAN 2020. Gastroenterology. 2022;163(3):649-58.e2.

    Article  PubMed  Google Scholar 

  2. Ferlay J, Colombet M, Soerjomataram I, Mathers C, Parkin DM, Pineros M, et al. Estimating the global cancer incidence and mortality in 2018: GLOBOCAN sources and methods. Int J Cancer. 2019;144(8):1941–53.

    Article  PubMed  CAS  Google Scholar 

  3. Cancer Genome Atlas Research Network. Integrated genomic characterization of oesophageal carcinoma. Nature. 2017;541(7636):169-75

  4. Rustgi AK, El-Serag HB. Esophageal carcinoma. N Engl J Med. 2014;371(26):2499–509.

    Article  PubMed  Google Scholar 

  5. Neganova ME, Klochkov SG, Aleksandrova YR, Aliev G. Histone modifications in epigenetic regulation of cancer: Perspectives and achieved progress. Semin Cancer Biol. 2022;83:452–71.

    Article  PubMed  CAS  Google Scholar 

  6. Glozak MA, Seto E. Histone deacetylases and cancer. Oncogene. 2007;26(37):5420–32.

    Article  PubMed  CAS  Google Scholar 

  7. Li Z, Zhu WG. Targeting histone deacetylases for cancer therapy: from molecular mechanisms to clinical implications. Int J Biol Sci. 2014;10(7):757–70.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  8. Hai R, Yang D, Zheng F, Wang W, Han X, Bode AM, et al. The emerging roles of HDACs and their therapeutic implications in cancer. Eur J Pharmacol. 2022;931:175216.

    Article  PubMed  CAS  Google Scholar 

  9. Miller TA, Witter DJ, Belvedere S. Histone deacetylase inhibitors. J Med Chem. 2003;46(24):5097–116.

    Article  PubMed  CAS  Google Scholar 

  10. San-Miguel JF, Hungria VT, Yoon SS, Beksac M, Dimopoulos MA, Elghandour A, et al. Panobinostat plus bortezomib and dexamethasone versus placebo plus bortezomib and dexamethasone in patients with relapsed or relapsed and refractory multiple myeloma: a multicentre, randomised, double-blind phase 3 trial. Lancet Oncol. 2014;15(11):1195–206.

    Article  PubMed  CAS  Google Scholar 

  11. Raedler LA. Farydak (Panobinostat): First HDAC Inhibitor Approved for Patients with Relapsed Multiple Myeloma. Am Health Drug Benefits. 2016;9((Spec Feature)):84–7.

    PubMed  PubMed Central  Google Scholar 

  12. Harttrampf AC, da Costa MEM, Renoult A, Daudigeos-Dubus E, Geoerger B. Histone deacetylase inhibitor panobinostat induces antitumor activity in epithelioid sarcoma and rhabdoid tumor by growth factor receptor modulation. BMC Cancer. 2021;21(1):833.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  13. Hazama Y, Tsujioka T, Kitanaka A, Tohyama K, Shimoya K. Histone deacetylase inhibitor, panobinostat, exerts anti-proliferative effect with partial normalization from aberrant epigenetic states on granulosa cell tumor cell lines. PLoS ONE. 2022;17(7):e0271245.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  14. Lu X, Liu M, Yang J, Que Y, Zhang X. Panobinostat enhances NK cell cytotoxicity in soft tissue sarcoma. Clin Exp Immunol. 2022;209(2):127–39.

    Article  PubMed  PubMed Central  Google Scholar 

  15. Ovejero-Sánchez M, González-Sarmiento R, Herrero AB. Synergistic effect of Chloroquine and Panobinostat in ovarian cancer through induction of DNA damage and inhibition of DNA repair. Neoplasia. 2021;23(5):515–28.

    Article  PubMed  PubMed Central  Google Scholar 

  16. Wu K, Zhang H, Zhou L, Chen L, Mo C, Xu S, et al. Histone deacetylase inhibitor panobinostat in combination with rapamycin confers enhanced efficacy against triple-negative breast cancer. Exp Cell Res. 2022. 113362.

  17. Xiao L, Somers K, Murray J, Pandher R, Karsa M, Ronca E, et al. Dual targeting of chromatin stability by the curaxin CBL0137 and histone deacetylase inhibitor panobinostat shows significant preclinical efficacy in neuroblastoma. Clin Cancer Res. 2021;27(15):4338–52.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  18. Cheng YW, Liao LD, Yang Q, Chen Y, Nie PJ, Zhang XJ, et al. The histone deacetylase inhibitor panobinostat exerts anticancer effects on esophageal squamous cell carcinoma cells by inducing cell cycle arrest. Cell Biochem Funct. 2018;36(8):398–407.

    Article  PubMed  CAS  Google Scholar 

  19. Puhr HC, Prager GW, Ilhan-Mutlu A. How we treat esophageal squamous cell carcinoma. ESMO Open. 2023;8(1):100789.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  20. Sheikh M, Roshandel G, McCormack V, Malekzadeh R. Current Status and Future Prospects for Esophageal Cancer. Cancers (Basel). 2023;15(3).

  21. Pacheco MB, Camilo V, Lopes N, Moreira-Silva F, Correia MP, Henrique R, et al. Hydralazine and Panobinostat Attenuate Malignant Properties of Prostate Cancer Cell Lines. Pharmaceuticals (Basel). 2021;14(7).

  22. Qin G, Li Y, Xu X, Wang X, Zhang K, Tang Y, et al. Panobinostat (LBH589) inhibits Wnt/β-catenin signaling pathway via upregulating APCL expression in breast cancer. Cell Signal. 2019;59:62–75.

    Article  PubMed  CAS  Google Scholar 

  23. Sato A, Asano T, Isono M, Ito K, Asano T. Ritonavir acts synergistically with panobinostat to enhance histone acetylation and inhibit renal cancer growth. Mol Clin Oncol. 2014;2(6):1016–22.

    Article  PubMed  PubMed Central  Google Scholar 

  24. Helland Ø, Popa M, Bischof K, Gjertsen BT, McCormack E, Bjørge L. The HDACi panobinostat shows growth inhibition both in vitro and in a bioluminescent orthotopic surgical xenograft model of ovarian cancer. PLoS ONE. 2016;11(6):e0158208.

    Article  PubMed  PubMed Central  Google Scholar 

  25. Tate CR, Rhodes LV, Segar HC, Driver JL, Pounder FN, Burow ME, et al. Targeting triple-negative breast cancer cells with the histone deacetylase inhibitor panobinostat. Breast Cancer Res. 2012;14(3):R79.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  26. LaBonte MJ, Wilson PM, Fazzone W, Russell J, Louie SG, El-Khoueiry A, et al. The dual EGFR/HER2 inhibitor lapatinib synergistically enhances the antitumor activity of the histone deacetylase inhibitor panobinostat in colorectal cancer models. Cancer Res. 2011;71(10):3635–48.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  27. Wilson PM, Labonte MJ, Martin SC, Kuwahara ST, El-Khoueiry A, Lenz HJ, et al. Sustained inhibition of deacetylases is required for the antitumor activity of the histone deactylase inhibitors panobinostat and vorinostat in models of colorectal cancer. Invest New Drugs. 2013;31(4):845–57.

    Article  PubMed  CAS  Google Scholar 

  28. Catalano MG, Pugliese M, Gargantini E, Grange C, Bussolati B, Asioli S, et al. Cytotoxic activity of the histone deacetylase inhibitor panobinostat (LBH589) in anaplastic thyroid cancer in vitro and in vivo. Int J Cancer. 2012;130(3):694–704.

    Article  PubMed  CAS  Google Scholar 

  29. Lobo J, Guimarães-Teixeira C, Barros-Silva D, Miranda-Gonçalves V, Camilo V, Guimarães R, et al. Efficacy of HDAC Inhibitors Belinostat and Panobinostat against Cisplatin-Sensitive and Cisplatin-Resistant Testicular Germ Cell Tumors. Cancers (Basel). 2020;12(10).

  30. Landreville S, Agapova OA, Matatall KA, Kneass ZT, Onken MD, Lee RS, et al. Histone deacetylase inhibitors induce growth arrest and differentiation in uveal melanoma. Clin Cancer Res. 2012;18(2):408–16.

    Article  PubMed  CAS  Google Scholar 

  31. Gao L, Gao M, Yang G, Tao Y, Kong Y, Yang R, et al. Synergistic activity of carfilzomib and panobinostat in multiple myeloma cells via modulation of ROS generation and ERK1/2. Biomed Res Int. 2015;2015:459052.

    Article  PubMed  PubMed Central  Google Scholar 

  32. Song X, Wang J, Zheng T, Song R, Liang Y, Bhatta N, et al. LBH589 Inhibits proliferation and metastasis of hepatocellular carcinoma via inhibition of gankyrin/STAT3/Akt pathway. Mol Cancer. 2013;12(1):114.

    Article  PubMed  PubMed Central  Google Scholar 

  33. Lee NR, Kim DY, Jin H, Meng R, Chae OH, Kim SH, et al. 2021 Inactivation of the Akt/FOXM1 Signaling Pathway by Panobinostat Suppresses the Proliferation and Metastasis of Gastric Cancer Cells. Int J Mol Sci 22(11).

  34. De La Rosa J, Urdiciain A, Zazpe I, Zelaya MV, Meléndez B, Rey JA, et al. The synergistic effect of DZ-NEP, panobinostat and temozolomide reduces clonogenicity and induces apoptosis in glioblastoma cells. Int J Oncol. 2020;56(1):283–300.

    PubMed  Google Scholar 

  35. Khan N, Jeffers M, Kumar S, Hackett C, Boldog F, Khramtsov N, et al. Determination of the class and isoform selectivity of small-molecule histone deacetylase inhibitors. Biochem J. 2008;409(2):581–9.

    Article  PubMed  CAS  Google Scholar 

  36. Ma S, Liu T, Xu L, Wang Y, Zhou J, Huang T, et al. Histone deacetylases inhibitor MS-275 suppresses human esophageal squamous cell carcinoma cell growth and progression via the PI3K/Akt/mTOR pathway. J Cell Physiol. 2019;234(12):22400–10.

    Article  PubMed  CAS  Google Scholar 

  37. Catalano MG, Fortunati N, Pugliese M, Marano F, Ortoleva L, Poli R, et al. Histone deacetylase inhibition modulates E-cadherin expression and suppresses migration and invasion of anaplastic thyroid cancer cells. J Clin Endocrinol Metab. 2012;97(7):E1150–9.

    Article  PubMed  CAS  Google Scholar 

  38. Fortunati N, Marano F, Bandino A, Frairia R, Catalano MG, Boccuzzi G. The pan-histone deacetylase inhibitor LBH589 (panobinostat) alters the invasive breast cancer cell phenotype. Int J Oncol. 2014;44(3):700–8.

    Article  PubMed  CAS  Google Scholar 

  39. Rhodes LV, Tate CR, Segar HC, Burks HE, Phamduy TB, Hoang V, et al. Suppression of triple-negative breast cancer metastasis by pan-DAC inhibitor panobinostat via inhibition of ZEB family of EMT master regulators. Breast Cancer Res Treat. 2014;145(3):593–604.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  40. Junqueira-Neto S, Vieira FQ, Montezuma D, Costa NR, Antunes L, Baptista T, et al. Phenotypic impact of deregulated expression of class I histone deacetylases in urothelial cell carcinoma of the bladder. Mol Carcinog. 2015;54(7):523–31.

    Article  PubMed  CAS  Google Scholar 

  41. Zhu Y, Yuan T, Zhang Y, Shi J, Bai L, Duan X, et al. AR-42: a pan-HDAC inhibitor with antitumor and antiangiogenic activities in esophageal squamous cell carcinoma. Drug Des Devel Ther. 2019;13:4321–30.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  42. Zhong L, Zhou S, Tong R, Shi J, Bai L, Zhu Y, et al. Preclinical assessment of histone deacetylase inhibitor quisinostat as a therapeutic agent against esophageal squamous cell carcinoma. Invest New Drugs. 2019;37(4):616–24.

    Article  PubMed  CAS  Google Scholar 

  43. Nakagawa M, Oda Y, Eguchi T, Aishima S, Yao T, Hosoi F, et al. Expression profile of class I histone deacetylases in human cancer tissues. Oncol Rep. 2007;18(4):769–74.

    PubMed  CAS  Google Scholar 

  44. Toh Y, Yamamoto M, Endo K, Ikeda Y, Baba H, Kohnoe S, et al. Histone H4 acetylation and histone deacetylase 1 expression in esophageal squamous cell carcinoma. Oncol Rep. 2003;10(2):333–8.

    PubMed  CAS  Google Scholar 

  45. Langer R, Mutze K, Becker K, Feith M, Ott K, Hofler H, et al. Expression of class I histone deacetylases (HDAC1 and HDAC2) in oesophageal adenocarcinomas: an immunohistochemical study. J Clin Pathol. 2010;63(11):994–8.

    Article  PubMed  CAS  Google Scholar 

  46. Yang Y, Zhang Y, Lin Z, Wu K, He Z, Zhu D, et al. Silencing of histone deacetylase 3 suppresses the development of esophageal squamous cell carcinoma through regulation of miR-494-mediated TGIF1. Cancer Cell Int. 2022;22(1):191.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  47. Ahrens TD, Timme S, Hoeppner J, Ostendorp J, Hembach S, Follo M, et al. Selective inhibition of esophageal cancer cells by combination of HDAC inhibitors and azacytidine. Epigenetics. 2015;10(5):431–45.

    Article  PubMed  PubMed Central  Google Scholar 

  48. He R, Liu B, Geng B, Li N, Geng Q. The role of HDAC3 and its inhibitors in regulation of oxidative stress and chronic diseases. Cell Death Discov. 2023;9(1):131.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  49. Ho TCS, Chan AHY, Ganesan A. Thirty years of hdac inhibitors: 2020 insight and hindsight. J Med Chem. 2020;63(21):12460–84.

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgements

Not applicable.

Funding

CJ research is supported by the Research Center of the Portuguese Institute of Porto (BF.CBEG CI-IPOP- 27-2016). NL holds a Junior Researcher position funded by FCT—Fundação para a Ciência e Tecnologia (Contract UID/DTP/0076/POCI-01-0145-FEDER-006868). SS was supported by a fellowship (SFRH/BD/143717/2019) granted by FCT. BTF was funded by CI‐IPOP/PD/Kymab. VMG holds a Junior Researcher position UIDP/00776/2020–3C funded through CI-IPOP Programmatic funding 2020–2023 (reference UIDP/00776/2020) from FCT. MPC was funded by FCT (Contract CEECINST/00091/2018).

Author information

Authors and Affiliations

  1. Cancer Biology and Epigenetics Group, Research Center of IPO Porto (CI-IPOP) – CI-IPOP@RISE (Health Research Network), Portuguese Oncology Institute of Porto (IPO Porto)/Porto Comprehensive Cancer Center Raquel Seruca (Porto.CCC), Research Center-LAB 3, F Bdg, 1st Floor, Rua Dr. António Bernardino de Almeida, 4200-072, Porto, Portugal

    Nair Lopes, Sofia Salta, Bianca Troncarelli Flores, Vera Miranda-Gonçalves, Margareta P. Correia, Rui Henrique & Carmen Jerónimo

  2. Doctoral Program in Pathology and Molecular Genetics, ICBAS – School of Medicine and Biomedical Sciences – University of Porto (ICBAS-UP), Rua de Jorge Viterbo Ferreira, 228, 4050-313, Porto, Portugal

    Sofia Salta

  3. Department of Pathology and Molecular Immunology, ICBAS-School of Medicine and Biomedical Sciences, University of Porto (ICBAS-UP), Rua de Jorge Viterbo Ferreira, 228, 4050-313, Porto, Portugal

    Vera Miranda-Gonçalves, Margareta P. Correia, Rui Henrique & Carmen Jerónimo

  4. Department of Pathology, Portuguese Oncology Institute of Porto (IPO Porto)/Porto Comprehensive Cancer Center Raquel Seruca (Porto.CCC), Rua Dr. António Bernardino de Almeida, 4200-072, Porto, Portugal

    Davide Gigliano, Rita Guimarães & Rui Henrique

Authors
  1. Nair Lopes
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Sofia Salta
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Bianca Troncarelli Flores
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Vera Miranda-Gonçalves
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Margareta P. Correia
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Davide Gigliano
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Rita Guimarães
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Rui Henrique
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Carmen Jerónimo
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

CJ conceived the study. CJ and NL designed the experiments. NL, VMG and RG performed the experiments. NL, SS, BTF, MPC and DG analysed the data. NL wrote the manuscript. RH and CJ revised the manuscript. All authors read and approved the manuscript.

Corresponding author

Correspondence to Carmen Jerónimo.

Ethics declarations

Ethics approval and consent to participate

This study has been approved by the Institutional Review Board (Comissão de Ética para a Saúde) of IPO Porto (CES 202/017). All the procedures involving the use of human samples were in accordance with the national ethical standards and following the 1964 Helsinki declaration and its later amendments or comparable ethical standards.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Additional information

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary Information

Additional file 1: Fig. S1

. Effect of Panobinostat treatment on the expression of Cyclin D1 and p21 in oesophageal cell lines HET1A, OE-19 and KYSE-30 (V – vehicle, P – Panobinostat).

Additional file 2: Fig. S2

. Effect of Panobinostat treatment on the expression of HDAC1 in oesophageal cell lines HET1A, OE-19 and KYSE-30 (V—vehicle, P—Panobinostat).

Additional file 3: Fig. S3

. Effect of Panobinostat treatment on the expression of HDAC2 in oesophageal cell lines HET1A, OE-19 and KYSE-30 (V—vehicle, P—Panobinostat).

Additional file 4: Fig. S4

. Effect of Panobinostat treatment on the expression of HDAC3 in oesophageal cell lines HET1A, OE-19 and KYSE-30 (V—vehicle, P—Panobinostat).

Additional file 5: Fig. S5

. Effect of Panobinostat treatment on the expression of HDAC8 in oesophageal cell lines HET1A, OE-19 and KYSE-30 (V—vehicle, P—Panobinostat).

Rights and permissions

Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if you modified the licensed material. You do not have permission under this licence to share adapted material derived from this article or parts of it. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by-nc-nd/4.0/.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Lopes, N., Salta, S., Flores, B.T. et al. Anti-tumour activity of Panobinostat in oesophageal adenocarcinoma and squamous cell carcinoma cell lines. Clin Epigenet 16, 102 (2024). https://doi.org/10.1186/s13148-024-01700-3

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: https://doi.org/10.1186/s13148-024-01700-3

Keywords

Clinical Epigenetics

ISSN: 1868-7083

Contact us