The Association of Propranolol with 2-Deoxy-D-Glucose Reduces the Metabolism and the Proliferation of Cutaneous Squamous Carcinoma A431 Cell Line

Research Article

Austin J Dermatolog. 2023; 10(1): 1107.

The Association of Propranolol with 2-Deoxy-D-Glucose Reduces the Metabolism and the Proliferation of Cutaneous Squamous Carcinoma A431 Cell Line

Carolina V De Almeida1; Marianna Buscemi1, Aida Cavallo1, Matteo Lulli2, Ilenia Foffa1, Tamer Al Kayal1, Giorgio Soldani1, Paola Losi1*

¹Institute of Clinical Physiology, CNR, Massa, Italy

²Department of Experimental and Clinical Biomedical Sciences “Mario Serio”, Università degli Studi di Firenze, Italy

*Corresponding author: Paola Losi Institute of Clinical Physiology, CNR, Via Aurelia sud, 54100, Massa, Italy. Email: paola.losi@ifc.cnr.it

Received: June 12, 2023 Accepted: July 12, 2023 Published: July 19, 2023

Abstract

Non-selective β-blocking (±)-Propranolol Hydrochloride was demonstrated to improve the progression-free survival of oncological patients. Since the expression of β-adrenoceptors in the epidermal squamous cell carcinoma was described, we hypothesized that the topical application of a β- adrenoceptors-blockers over the tumor lesion may decrease its extension before the surgical excision, becoming an adjuvant therapy against cutaneous squamous cell carcinoma. However, it is known that β-AR-blocker anti-cancer activity as a single agent is limited. Hence, we suggested that the combination of propranolol with the glucose analog 2-Deoxy-D-glucose could improve its antiproliferative effect through the induction of metabolic stress.

Propranolol and 2-Deoxy-D-glucose effect on A431 squamous cell carcinoma and normal keratinocytes evaluating metabolic activity, proliferation and apoptosis through MTT, immunofluorescence Ki-67 and AnnexinV assays, cell cycle analysis and migration assay.

Our results demonstrated that the addition of 2-Deoxy-D-glucose low dose to propranolol improve its effect on reduction of A431 cells metabolism and proliferation, similar effect was observed on HaCaT viability and mobility. However, the HaCaT migration ability is not completely compromised.

The combination of propranolol with a low dose of 2-Deoxy-D-glucose could be a promising treatment to be topically applied avoiding systemic adverse effects in patients with cutaneous squamous cell carcinoma.

Keywords: Propranolol hydrochloride; 2-Deoxy-d-glucose; Cutaneous squamous cell carcinoma; A431 cell line

Introduction

Cutaneous Squamous Cell Carcinoma (cSCC) is the second most frequent skin cancer in white ethnic populations worldwide [1,2] and even if most of the cases are easily cured by surgical removal, this cancer remains the cause of the majority of Non-Melanoma Skin Cancer (NMSC) deaths. This is due to “high-risk SCCs”, which are associated with significant metastasis, morbidity, and death [3,4]. Among the main cause of cSCC, DNA damage by Ultraviolet (UV) radiation exposure is the most common [5], since it causes the deregulation of important signaling pathways that are involved in the cell cycle, apoptosis, DNA repair and cell differentiation [6,7]. Other risk factors for cSCC promotion are immunosuppression [8], Human Papilloma Virus (HPV) infection [9,10], genetic disorders [11] and smoking [12]. Sporadically, cSCC can be also associated with non-healing wounds/scarring, or chronic lesions preceded by chronic inflammatory processes [13, 14].

Usually, in situ cSCC may be controlled by different interventions, including electrodessication and curettage, topical therapy, cryotherapy, and photodynamic therapy; however, since these treatments are not appropriate for invasive cSCC [15], surgical excision is usually indicated. The surgical procedure creates wounds that could be small, superficial, and amenable to primary closure, but often they can be large, deep, and extensive needing more complex closure and covering. Specifically scalp injuries, due to low elasticity, can be devastating and can require significantly more extensive surgeries, concerning both the number and complexity [16]. Consequently, we believe that an effective therapeutic strategy could be to control the extension of the lesion before its excision aspiring for less invasive surgery with less devastating wounds.

Multiple intracellular signal transduction pathways, involved in events such as cellular replication, inflammation, angiogenesis, apoptosis, cell motility and trafficking, activation of tumor-associated viruses, DNA damage repair, cellular immune response and epithelial–mesenchymal transition (reviewed by Coelho M et al) [17], are regulated through interactions of a- and β-Adrenoceptors (AR) and Catecholamine (CA) neurotransmitters [18]. Tumor cells may express β-AR, and the involvement of β-adrenergic signaling in the progression of malignant diseases has been increasingly [1] recognized [19-21]. The use of beta-blocker therapy can reduce the incidence of prostate cancer [22] and improve the prognosis of patients with breast [23] and hepatocellular [24] cancer. The expression of β-AR in the A431 cSCC human cells was described in 1987 by Kashles and Levitzki [25], which leads us to believe that cSCC proliferation may be controlled by using β-AR-blockers. Thus, we hypothesized that the topical application of a β-AR-blocker over the tumor lesion may decrease/restrain its extension before the surgical excision, becoming an adjuvant\therapy against cSCC. The topical application of β-AR-blocker, timolol [26,27] and propranolol [28], was already described on infantile hemangioma, with no collateral effects.

Recent evidence has shown that non-selective β-blocking (±)-Propranolol Hydrochloride, improved the progression-free survival of breast cancer patients [29], and reduced the risk of developing head and neck, prostate, esophagus, stomach, and colon cancers [30] It is known that the (±)-Propranolol Hydrochloride anti-cancer activity is due to its ability to inhibit the mitochondrial metabolism, which can increase the cell glycolytic activity resulting in elevated metabolism and switch towards aerobic glycolysis, which could stimulate the tumor progression and drug resistance. However, its anti-cancer activity as a single agent was demonstrated to be limited [31].

Hence, based on the well-known warburg effect, cancer cells boost glucose uptake and conversion into lactate in the presence of high oxygen tension, exploiting the aerobic glycolysis, we suggested the combination of propranolol with the glucose analog 2-Deoxy-D-glucose (2-DG) aiming to improve its antiproliferative effect. 2DG is a well-known antidiabetic drug, which by competition can inhibit glucose uptake, blocking the first critical step of glucose metabolism and mitochondrial respiration, inducing metabolic stress [32]. 2DG increases autophagy, a ubiquitous cellular catabolic process that under conditions of protracted stresses suppresses tumorigenesis [33]. 2DG treatment alone does not significantly induce cancer cell death, but it may use with specific agents or to exert a synergistic therapeutic action.

To confirm these hypotheses, we performed in vitro assays using the human A431 cSCC cell line and the human keratinocytes HaCaT cells. We demonstrated that the addition of 2DG to (±)-Propranolol Hydrochloride therapy can improve its effect on A431 cells metabolism and proliferation.

Materials and Methods

Propranolol Solution

(±)-Propranolol Hydrochloride (Sigma St. Louis, MO, USA) dilutions were made using 1:1 (v:v) Dulbecco's Modified Eagle's Medium: Nutrient Mixture F-12 Ham (SF-DMEM: F12) (Sigma St. Louis) in a stock solution of 400μM, filtered with a 0.22μm (Millipore, Burlington, MA, USA). The solution was diluted freshly before each experiment to different concentrations as indicated in each assay. Corroborating with the results of Bustamante et al. 2019 [34] whom demonstrated that on melanocytes 200μM of propranolol has a cytotoxic effect, but not with 50μM, we started to observe effects only with concentrations over 100μM (data not shown), thus, we performed the experiments using concentrations from 100 to 300μM (100, 150, 200 and 300μM).

2DG Solution

2-Deoxy-D-glucose (Calbiochem, San Diego, CA, USA) dilutions were made using 1:1 (v:v) Dulbecco's Modified Eagle's Medium: Nutrient Mixture F-12 Ham (SF-DMEM:F12) (Sigma St. Louis) in a stock solution of 5mM, filtered with a 0.22μm (Millipore). The solution was diluted freshly before each experiment to different concentrations as indicated in each assay. Based on the literature, we investigated the cytotoxic effect of 2DG in five different concentrations: 1, 2, 3, 4 and 5mM.

Cell Culture

The epidermal squamous cell carcinoma A431 (ATCC® CRL-1555™) (Cell Applications, San Diego, CA, USA) was cultivated on DMEM/Ham's F12 medium (Sigma St. Louis), added with 1x nonessential amino acids (Lonza™ BioWhittaker™. Basel, Switzerland), L-glutamine (2.5mM), gentamicin (1μL/mL) and FBS 10%. The medium was routinely changed every 3 days and at confluence, cells were subcultured (split ratio 1:5) by trypsinization (0.5% trypsin/0.02% EDTA). Human keratinocytes HaCat (Istituto Zooprofilattico Sperimentale della Lombardia e dell'Emilia Romagna "Bruno Ubertini") were cultured in RPMI 1640 supplemented with 10% FBS, 2mM L-Glutamine, 100μg/mL streptomycin and 100U/mL penicillin.

Metabolic Viability – MTT Assay

To evaluate the effect of different doses of propranolol on cellular metabolic activity, we used the colorimetric assay with MTT [3-(4,5 dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide]. Briefly, cells (5x103 cells/well) were seeded into 96-well plates. After 24h of incubation, the medium was added with the specific doses of propranolol diluted in complete cell culture medium (150–350μM), or complete medium which was used as a reference. After 48h, MTT phosphate buffered solution (final concentration of 0.1mg/mL) was added to each well and cultures were incubated at 37°C for 3h. The supernatant was removed from the wells by slow aspiration and replaced with DMSO (100μl per plate) to solubilize the MTT tetrazolium dye. At the end of incubation time, the Optical Density (OD) was measured at 550nm wavelength using a microplate reader (Spectrafluor Plus; TECAN Austria GmbH, Grödig, Austria). Three replications were used for each analysis. The percentage of cell viability was calculated vs. the complete medium (assumed as 100%).

Cell proliferation Assay

Cells were seeded (5x103 cells/well) in a 96-wells plate and incubated for 24h. Then, we treated the cells with 200μM of propranolol (Prop), 0.5mM 2DG (2DG) or 200μM of propranolol and 0.5mM of 2DG (Prop+2DG). Control was made by adding fresh complete medium. Three replications were used for each analysis. Cells were detached and alive cells were manually counted in three times: before treatments, 24 and 48h after the treatments.

Immunofluorescence Ki-67

Cells were grown overnight on glass coverslips and then treated with 200μM of propranolol during 24h. Cells were washed twice with 1mL of cold Phosphate Buffered Saline (PBS), fixed for 20 min in 3.7% paraformaldehyde in PBS and permeabilized with 0.3% Triton X-100 in PBS for 5 min. Cells were incubated in the blocking buffer (5% FBS and 0.3% Triton X-100 in PBS) for 1 h at room temperature. Then, the cells were incubated overnight at 4°C with primary antibody Ki-67 (sc-23900) (Santa Cruz Biotechnology, Dallas, TX, USA) and successively for 1h with the anti-mouse Alexa Fluor 488 (Cat #A21121) (ThermoFisher, Waltham, MA, USA) at room temperature. After staining of the nuclei with Hoechst 33242 dye (Sigma St. Louis), the cells were dried, mounted onto glass slides with DPX Mountant for histology (Sigma St. Louis), and examined with confocal microscopy using a Nikon Eclipse TE2000-U (Nikon, Tokyo, Japan). A single composite image was obtained by the superimposition of 6 optical sections for each sample observed. The collected images were analyzed by Image J software. All the experiments were repeated three times.

Apoptosis Assay

Apoptosis was detected by flow cytometry by using (BV421)-Annexin-V and the nonvital dye 7-amino-actinomycin D (7AAD) double staining (BD Biosciences, Franklin Lakes, NJ, USA). A431 cells were inoculated into six-well plates with 5x105 cells/well and cultured for 24h. The growth of cells converged to approximately 70%. Cells were then treated with Prop, 2DG or Prop+2DG. After 24h of treatment, floating and adherent cells were collected and resuspended in binding buffer (BD Biosciences). (BV421)-Annexin V and 7AAD were added, the samples were incubated for 20 min in the dark at 4°C and analyzed by FACSCanto II and FlowJo software (BD Biosciences). Experiments were performed three times.

Cell Cycle Analysis

Flow cytometry analysis of DNA content was performed to assess the cell cycle phase distribution in control conditions (not treated) or after the treatments were added to logarithmically growing A431 cells. After 48h exposure, A431 cells were harvested by trypsinization, and a solution containing 50μg/mL propidium iodide (Sigma Aldrich), 0.1% w/v trisodium citrate and 0.1% NP40 was added. Samples were then incubated for 30min at 4°C in the dark and nuclei analysed with a FACSCanto II flow cytometer and FlowJo software (BD Biosciences) Experiments were performed three times.

Cell Migration Assay

The cells (20x104) were seeded into 24-well plates, cultured to confluence, and scratched by scraping with a 10μL pipette tip. Following PBS washes, cultures were treated with Prop, 2DG or Prop+2DG. Control wells received a serum-free medium or complete culture medium. At 20, 40 and 60h after scratching, digital images of cells were captured by a phase contrast microscope (Axiovert 25, Zeiss, Milan, Italy; O.M. 50X) equipped with a digital camera (EOS 1000D, Canon, Milano, Italy). Scratch closure was qualitatively analyzed with respect to time 0, by measuring the wound width/area change. Such method uses several metrics to quantify migration, including the percentage difference in the wound width at each time point.

Statistical Analysis

Differences in the experimental groups were assessed using analysis of variance (ANOVA). To avoid bias due to the variability between the experiments, the factor defining the different experimental groups was crossed with a second factor defining the different experiments (two-way ANOVA). P-values lower than 0.05 were considered statistically significant. Figures are representative of all experiments that were realized during the study.

Results

Combination of (±)-Propranolol Hydrochloride and Low Dose of 2DG Decreases the Proliferation of Human A431 Cells

To determine the sensitivity of A431 cells to different concentrations of (±)-Propranolol Hydrochloride and 2DG, we performed the MTT test that establishes cell metabolism by measuring the functionality of mitochondrial dehydrogenases. The effects of (±)-Propranolol Hydrochloride were shown only after 48 h of treatment in all concentrations with a significant effect observed from 200μM. Results revealed that propranolol reduced cell metabolism in a dose-dependent manner (Figure 1A).