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Cytotoxic and proapoptotic effect of doxycycline – An in vitro study on the human skin melanoma cells
Toxicology in Vitro 65 (2020) 104790
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Cytotoxic and proapoptotic effect of doxycycline – An in vitro study on the human skin melanoma cells
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Jakub Rok , Marta Karkoszka, Zuzanna Rzepka, Michalina Respondek, Klaudia Banach, Artur Beberok, Dorota Wrześniok Department of Pharmaceutical Chemistry, Faculty of Pharmaceutical Sciences in Sosnowiec, Medical University of Silesia, Katowice, Poland, Jagiellońska 4, 41-200 Sosnowiec, Poland
A R T I C LE I N FO
A B S T R A C T
Keywords: Doxycycline Melanoma Apoptosis Cell cycle DNA fragmentation Mitochondrial membrane potential
Doxycycline is a semisynthetic, second generation tetracycline. Currently, it is used, among others, in the treatment of acne and skin infections. Moreover, doxycycline has many valuable nonantibiotic properties, including anti-inflammatory, immunosuppressive and anticancer effects. Recent studies showed that the drug had the ability to inhibit the adhesion and migration of cancer cells, as well as affected their growth and proliferation and induced apoptosis. The purpose of this study was to examine the antimelanoma effect of doxycycline. The obtained results demonstrated that doxycycline decreased the viability and inhibited the proliferation of human melanoma cells, proportionally to the drug concentration and the treatment time. It was stated that doxycycline disturbed the homeostasis of the cells by lowering intracellular level of reduced thiols. In addition, the treatment changed the cell cycle profile and triggered the DNA fragmentation. Mitochondria of melanoma cells exposed to the drug had lowered membrane potential, which indicated cells apoptosis. Finally, doxycycline induced the externalization phosphatidylserine – a well-known hallmark of apoptosis, confirmed by results of annexin V test. The presented study contributes to the increase of knowledge about nonantibacterial action of doxycycline, including the influence on human cancer cells and indicates new potential possibility of effective treatment of malignant melanoma.
1. Introduction Malignant melanoma is considered one of the most aggressive human cancers with constantly rising incidence worldwide, especially in Celtic and Caucasian races (Rastrelli et al., 2014a). The growing public health problem is caused by high morbidity, poor prognosis and lack of effective current therapeutic methods (Liu and Sheikh, 2014). It is estimated that approximately 350,000 melanomas and 13 million non-melanoma skin cancers occur every year and lead to 81,000 deaths (Silva et al., 2018). In 2014 there were 76,100 new cases of melanoma diagnosed, which constituted about 4.6% of new cancer cases (Holmes, 2014). Although melanoma accounts for a little over 2% of skin neoplasms, it is the cause of about 80% of deaths due to cancerous skin lesions (Liu and Sheikh, 2014; Miller and Mihm, 2006). The incidence of melanoma cases increases in most white populations worldwide. The problem concerns mainly Australasian, North American, and European populations (von Schuckmann et al., 2019). Melanoma is a tumor that derives from pigment-producing cells – melanocytes and their neural crest cells precursors (Lo and Fisher, ⁎
2014). Both genetic predispositions and environmental factors are significant in the development and progression of this tumor (Rastrelli et al., 2014b). More than 90% of this skin cancers are located within the skin. In 1 out of 10 diagnosed melanomas, the primary tumor lesions are located in other places where melanocytes occur, such as the eyeball or oral mucosa (Schadendorf et al., 2015; Sulaimon and Kitchell, 2003). Surgical removal of melanoma with a margin of healthy tissue and assessment of the surrounding lymph nodes is the basic method of therapy for this type of cancer. An appropriate treatment for primary focus enables effective treatment of patients who have not metastasized (Garbe et al., 2016). Dacarbazine and temozolomide are major drugs used in melanoma chemotherapy. Approximately 15% of patients respond to treatment with these agents, with a 5-year survival rate of less than 2%. In more advanced stages of the disease, radiotherapy and immunotherapy are applied (Bahtia et al., 2009). Interleukin-2 and interferon alpha are used in immunotherapy, allowing for remission in about 16% of patients (Kee and McArthur, 2017). More modern immunotherapy methods are based on the use of monoclonal antibodies
Corresponding author. E-mail address: [email protected] (J. Rok).
https://doi.org/10.1016/j.tiv.2020.104790 Received 26 November 2019; Received in revised form 4 February 2020; Accepted 6 February 2020 Available online 08 February 2020 0887-2333/ © 2020 Elsevier Ltd. All rights reserved.
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propidium iodide, 1.2 μg/ml acridine orange in DMSO), Solution 7 (200 μg/ml JC-1), Solution 8 (1 μg/ml DAPI in PBS), Solution 15 (500 μg/ml Hoechst 33342), Solution 16 (500 μg/ml propidium iodide), NC-Slide A8 and A2 as well as Via-1-Cassette (acridine orange and DAPI fluorophores) were obtained from ChemoMetec (Denmark). Annexin V-CF488A conjugate as well as Annexin V binding buffer were purchased from Biotium (UK).
against the CTLA-4 protein (ipilimumab) and the PD-1 receptor (pembrolizumab, nivolumab). Treatment effects are visible in approximately 15% of patients when ipilimumab is used and 50% during therapy directed against the PD-1 receptor (Homet and Ribas, 2014). The advancement of knowledge about molecular mechanisms for the development of this type of cancer has allowed the invention of drugs that include vemurafenib and trametinib (Ho and Tsao, 2015). Despite the fact that surgical treatment of early melanoma leads to high percentage of cures, advanced and metastatic melanoma presents tendency to rapidly metastasize and an innate resistance to chemotherapy (Homet and Ribas, 2014). The above information indicates that melanoma still remains, a potentially fatal malignancy (Rastrelli et al., 2014b). Tetracyclines are a group of drugs, constantly and widely used since their invention in 1948 (Griffin et al., 2010). These antibiotics owe their popularity to a range of beneficial properties, such as a broad spectrum of action against both Gram (+) and Gram (−) bacteria, good tolerance and favorable pharmacokinetic properties (Broghi and Palma, 2014). Tetracycylines have a bacteriostatic effect associated with binding the drug with the 16S rRNA in 30S subunit of the bacterial ribosome. This prevents the attachment of aminoacyl tRNA to A site and disrupts protein synthesis (Chukwudi, 2016). Doxycycline is a semisynthetic drug that belongs to the second generation of tetracyclines. Currently, doxycycline is used in the treatment of acne and skin infections of various origins (Cuhna et al., 2018). In addition to well-known antibiotic effects, an extensive research on doxycycline has revealed a range of highly valuable nonantibiotic properties. Anti-inflammatory, immunosuppressive and anticancer effects of doxycycline are the reasons for conducting numerous researches on alternative usage of this drug (Bahrami et al., 2012). The nonantibiotic effect of doxycycline is mainly the result of its ability to inhibit metalloproteinases activity (MMP). Doxycycline is currently the only inhibitor of MMP enzymes that has been approved for usage by the FDA in the treatment of periodontitis at sub-antibiotic dose (SDD) is (Caton and Ryan, 2011). Recent reports of antitumor activity of doxycycline have raised the greatest interest. It has been found that the drug has the ability to inhibit the adhesion and migration of cancer cells. In addition, doxycycline may affect their growth and proliferation and induce apoptosis (Sun et al., 2009). Molecular mechanisms of the antitumor action of doxycycline have not been fully understood, but until now it has been shown that it may be associated among others with: i) inhibition of the MMP-2 and MMP-9 metalloproteinases activity, ii) activation of apoptosis signal-regulated kinase 1, c-Jun N-terminal kinase and caspases which inducing apoptosis, iii) reduction of mitochondria membrane potential, iv) induction of oxidative stress in cancer cells (Shieh et al., 2010). The purpose of this study was to examine the antimelanoma effect of doxycycline by the analysis of cells viability, morphology, cell cycle, vitality, DNA fragmentation and mitochondrial membrane potential as well as the results of annexin V assay.
2.2. Cell culture Human skin melanotic melanoma cell line, COLO 829, was purchased from ATCC (CRL-1974™, USA). The cells were cultured in RPMI 1640 medium (with L-glutamine) enriched by inactivated fetal bovine serum to a final concentration of 10%, penicillin (100 U/ml), neomycin (10 μg/ml) and amphotericin B (0.25 μg/ml). Cells were grown in a 5% CO2 incubator CB 160 (BINDER, Germany) at 37 °C with 5% relative humidity. 2.3. Cytotoxicity assessment The viability of melanotic melanoma cells COLO 829 was determined by the WST-1 colorimetric assay. WST-1 (4-[3-(4-iodophenyl)2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzenedisulphonate) is a water-soluble tetrazolium salt, which is cleaved to formazan by cells mitochondrial dehydrogenases. The amount of produced formazan correlates with the number of viable cells. In brief, 2.5 × 103 cells per well were placed in a 96-well microplate in a supplemented RPMI 1640 growth medium and incubated at 37 °C and 5% CO2 for 24 h. After the incubation, the medium was removed and cells were treated with doxycycline solutions in a concentration ranging from 1 × 10−7 M to 2,5 × 10−4 M. After 21 h, 45 h and 69 h of incubation, 10 μl of WST-1 solution was added to each well, and the incubation was continued for 3 h. The absorbance of the samples was measured at 440 nm using a reference wavelength at 650 nm. The measurement was made using microplate reader Infinite 200 PRO (TECAN, Switzerland). The controls were normalized to 100% for each assay and the treatments were expressed as the percentage of the controls. 2.4. Cell morphology assessment Photographic documentation of COLO 829 cells abundance and morphology under the influence of doxycycline was carried out during the research. Cells were seeded in T-75 flasks (2 × 106 cells/flask) and cultured in RPMI supplemented medium, as described above. The treatment with doxycycline in concentration 100 μM began 24 h after seeding. The cells culture was examined under light inverted microscope NIKON TS100F (Japan) after 24 h, 48 h and a 72 h-long incubation with the drug. 2.5. Cell cycle assay The cell cycle analysis of COLO 829 cells were performed using a NucleoCounter® NC-3000™ fluorescent imaging cytometer (Denmark). The analysis is based on measurements of the DNA content within investigated cells population. The COLO 829 cells were seeded in T-75 flasks at a density of 2 × 106 cells per flask. The treatment by doxycycline in a concentration 50 μM and 100 μM was evaluated after 24 h, 48 h and 72 h of incubation. The cells were harvested by trypsinization, loaded into Via-1-Casette and counted using NucleoCounter NC-3000 image cytometer. Afterwards, 1 × 106 cells were suspended in 0.5 ml PBS and fixed with 4.5 ml of 70% cold ethanol for at least 12 h at 0-4 °C. Then the cells were centrifuged, the ethanol was removed and the cells pellets were resuspended in PBS and centrifuged again for 5 min at 500 g. Each of the obtained cells pellets was resuspended in 0.5 ml of Solution 3 and incubated for 5 min at 37 °C. The stained cells were loaded into NC-Slide A8 and analyzed using „Fixed Cell Cycle-DAPI
2. Materials and methods 2.1. Chemicals and reagents Doxycycline hyclate, C22H24N2O8 x HCl x 0,5H2O x 0,5C2H6O, penicillin and amphotericin B were purchased from Sigma-Aldrich Inc. (USA). Neomycin was obtained from Amara (Poland). Fetal bovine serum was purchased from Gibco (Poland). The growth medium for melanoma cells, RPMI 1640, as well as trypsin inhibitor were acquired from Cascade Biologics (UK). Trypsin/EDTA was obtained from Cytogen (Poland). Cell Proliferation Reagent WST-1 was purchased from Roche GmbH (Germany). Solution 3 (1 μg/ml DAPI, 0.1% triton X100 in PBS), Solution 5 (400 μg/ml VitaBright-48™, 500 μg/ml 2
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2.9. Annexin V assay
Assay” protocol by NC-3000 image cytometer. The obtained DNA content histograms were used to demarcate different phases of cell cycle in tested samples.
The annexin V assay is commonly used to detect cells apoptosis. The test is based on high affinity of a cellular protein annexin V to phosphatidylserine whose translocation to the outer membrane layer is already observed in early apoptosis. Dyes using in the assay, except FITC labeled annexin V (Annexin V-CF488A), involve Hoechst 33342 – Solution 15 (stains total population) and propidium iodide – solution 16 (stains late apoptotic and necrotic cells). The test and its analysis were performed using the fluorescence image cytometer NucleoCounter NC3000 (Denmark). Tested cells were seeded in T-75 flasks at a density of 2 × 106 cells per flask and after 24 h they were treated with doxycycline in a concentration 100 μM for 24 h and 48 h. After the treatment, the cells were detached by trypsinization and counted using the NC-3000 image cytometer. In the next step, 3.0 × 105 cells were suspended in 100 μl of Annexin V binding buffer and fixed with 2 μl of Annexin V-CF488A conjugate and 2 μl of Hoechst33342. The cells were then incubated for 15 min at 37 °C. Afterwards, the stained cells were subsequently centrifuged at 400 g for 5 min and the supernatant was removed. The cell pellets were resuspended in 300 μl of Annexin V binding buffer. The suspension was centrifuged twice under the conditions described above. Finally, the cell pellets were resuspended in 100 μl of Annexin V binding buffer and 2 μl of Solution 16 was added. The samples were analyzed immediately using NCSlideA8 and „Annexin V Assay” protocol. The obtained scatter-plots were used to demarcate the percentage of heathy cells, early apoptotic as well as late apoptotic cells, respectively.
2.6. Vitality assay: the analysis of cellular thiols level The estimation of cells vitality was performed by the use of the fluorescence imaging cytometer NucleoCounter® NC-3000™ (Denmark). The analysis is based on the assessment of cellular thiols level using a specific dye – VitaBright-48™. The treatment with doxycycline in concentration 50 μM and 100 μM began 24 h after reaching the logarithmic growth phase and was conducted for 24 h, 48 h or 72 h. Before the test, the cells were harvested by trypsinization, loaded into Via-1-Casette, and counted using the NC-3000 image cytometer. Subsequently, according to the producer, 1 × 106 cells were suspended in 0,5 ml of PBS, and then 10 μl of Solution 5 was added into 190 μl of the cell suspension. The stained cells were loaded into the NC-Slide A8 and measured using the „Vitality (VB-48) Assay” protocol in the NC-3000 image cytometer. High fluorescence intensity of a particular cell indicates that the cell has a high level of reduced thiols, such as GSH, which is an indicator of overall health status (vitality). The obtained VB-48™ intensity histograms were used to demarcate healthy and low vitality subpopulation of the tested samples.
2.7. DNA fragmentation assay The assessment of DNA fragmentation in COLO 829 cells were performed using a fluorescent imaging cytometer NucleoCounter® NC3000™ (Denmark). The analysis is based on the detection of cells with high molecular weight fragmented DNA, stained with DAPI. The investigated cells were tested after 24 h, 48 h and 72 h incubation with doxycycline in a concentration 50 μM and 100 μM. After the treatment, the cells were detached by trypsinization, counted, suspended in PBS (2 × 106 cells/ml) and fixed with 70% cold ethanol for at least 12 h at 0-4 °C. Afterwards, the cells were centrifuged. Obtained cells pellets were resuspended in PBS, centrifuged again for 5 min at 500 g and next resuspended in 0.5 ml of Solution 3. Stained cells were incubated for 5 min at 37 °C. and loaded into NC-Slide A8 to perform the analysis. The obtained histograms were used to demarcate subpopulation of the tested cells culture with fragmented DNA.
2.10. Statistical analysis Statistical analysis of the results was performed using GraphPad Prism 6.01 Software. In all experiments mean values of at least three separate experiments performed in triplicate (n = 9) ± standard deviation of the mean (SD) were calculated. The results were analyzed statistically of Student's t-test, one-way ANOVA as well as Dunnett's comparison test. The Kolomogorov-Smirnov test checked the compliance of the distribution results and the Brown-Forsythe test checked the variances of the compared groups meet the homogeneity assumption. In all cases the statistical significance was found for p-value to be lower than 0.05. 3. Results
2.8. Mitochondrial potential assay
3.1. Cytotoxic effect of doxycycline on melanoma cells
The mitochondrial transmembrane potential (ΔΨm) was measured by the use of the fluorescence image cytometer NucleoCounter NC-3000 (Denmark). Decrease of the potential usually precedes cells apoptosis. The method is based on fluorescent cationic dye JC-1, which accumulates in the mitochondria in the potential depended manner. In healthy cells, it achieves a high concentration inside the mitochondria and aggregates to red fluorescent form. In turn, in apoptotic cells it localizes in the cells cytoplasm emitting green fluorescence. COLO 829 cells were seeded in T-75 flasks at a density of 2 × 106 cells per flask and after 24 h they were treated with doxycycline in a concentration 100 μM for 24 h and 48 h. After the treatment, the cells were harvested by trypsinization and counted using the NC-3000 image cytometer. Afterwards, 12.5 μl of Solution 7 was added to 1.0 × 106 cells, which were next incubated for 10 min at 37 °C. The stained cells were then centrifuged at 400 g for 5 min and washed twice with PBS. The obtained cell pellets were resuspended in 0.25 ml Solution 8 and analyzed immediately using NC-Slide A8 and „Mitochondrial Potential Assay” protocol. The results presented in the form of scatter-plots were used to demarcate the percentage of polarized/healthy cells and depolarized/apoptotic cells.
The cytotoxicity of doxycycline was tested for the concentrations ranging from 0.1 to 250 μM (Fig. 1A). The time of cells exposure to investigated drug was 24 h, 48 h and 72 h. The obtained results showed the melanoma cell viability decreased proportionally to the drug concentration as well as the time of the treatment. The significant reduction of viable cells, below 50% of control, was observed in the concentrations from 50 μM after 24 h (ca. 42% of control) and in the concentrations from 25 μM after 48 h (ca. 44% of control) as well as 72 h (ca. 30% of control). The viability of melanoma cells for the highest tested concentration was 28.5%, 9.5% and 3.7%, for 24 h, 48 h and 72 h-long incubation, respectively. On the basis of the results, the EC50 values were calculated to be 74.35 μM, 32.28 μM and 16.25 μM for following incubation time. Excepting the cytotoxicity, proliferative potential of melanoma cells was evaluated (Fig. 1B). It was stated that doxycycline in concentrations ranging from 0.1 μM to 10 μM did not influence cells divisions during 24 h and 48 h-long treatment. A gentle but statistically significant decrease of the proliferation rate was observed after a 72 h-long treatment for the concentrations of 5 μM, 7.5 μM and 10 μM. However, doxycycline caused notable inhibition of the cell proliferation in the 3
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Fig. 1. The effect of doxycycline on melanoma cells COLO 829 viability and proliferation. Cells were treated with doxycycline concentrations ranging from 0.1 μM to 250 μM for 24 h, 48 h, and 72 h. Mean values ± SD from three independent experiments are presented. ⁎p < .05, ⁎⁎p < .005 vs control. (A). Cell viability examined by the WST-1 assay. Data are expressed as % of the controls. (B). Anti-proliferative effect of doxycycline on human melanoma cells COLO 829 determined by the WST-1 assay. (C). Photographs of human melanoma cells COLO 829 exposed to 100 μM of doxycycline for 24 h, 48 h and 72 h. Scale bar 250 μm.
concentration of 25 μM after 48 h and 72 h. The divisions of the cells treated with 50 μM, 75 μM and 100 μM of doxycycline were on the same low level, independently on the treatment time. The effect of doxycycline on a culture of melanoma cells can be observed in Fig. 1C. It is noticed that untreated cells proliferate all the time, creating characteristic clusters. Control cells retain their typical morphology, independently of a confluence stage. In turn, doxycycline inhibits cells proliferation and causes noticeable changes of the cell morphology. Melanoma cells exposed to the drug are round and located separately. Moreover, spherical, detached cells appear in the treated cell culture.
of treatment. The changes involve ca. 5% increase in the percentage of cells in S and G2/M phase as well as ca. 12% decrease in the percentage of cells in G1/G0 phase. Moreover, there was 9% and 5% reduction observed in the amount of the cells in G1/G0 phase after 48 h and 72 h, respectively, as well as 8% increase of the amount of cells in G2/M phase. In turn, 100 μM of doxycycline induces a growth of the percentage of melanoma cells in sub-G1 phase by about 7%, 9% and 14% after 24 h, 48 h and 72 h, respectively. Furthermore, an amount of the cells in G2/ M phase was reduced almost twice to ca. 12%. An increase of cells number in S phase was also observed when compared to the control. The results were 19% and 18% for 24 h and 48 h treatment, respectively.
3.2. The effect of doxycycline on melanoma cells cycle The effect of doxycycline on melanoma cell cycle was evaluated in the concentrations of 50 μM and 100 μM, after 24 h, 48 h and 72 h treatment. The results presented in Fig. 2 show that the drug at the concentration of 50 μM changes the cell cycle profile mostly after 24 h
3.3. Doxycycline depreciates melanoma cells vitality by decrease a level of reduced thiols The analysis of cells vitality in the form of intracellular thiols level 4
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Fig. 2. The effect of doxycycline on the cell cycle profile of human melanoma cells COLO 829. The cells were incubated with doxycycline at the concentrations of 50 μM and 100 μM for 24 h, 48 h and 72 h. Representative histograms demonstrate distribution of cells in the different phases of the cycle for each tested group. Markers in the histograms respond to different stages of the cells cycle, as follow: M1 – sub-G1, M2 – G0/G1, M3 – S, M4 – G2/M. Mean values of the percentage of a particular subpopulations and responding standard deviations are presented in the bar graph.
was performed for 50 μM and 100 μM of doxycycline using a specific reagent - VitaBright-48™. The obtained results presented in Fig. 3 show the drug depreciates the level of reduced thiols leading to low vitality of the melanoma cells. The observed effect is proportional to the drug concentration and the time of incubation. The highest level of reduced thiols – 86%, was stated in no-treatment control cells. In turn, the highest percentage of low vitality cells with a small amount of reduced thiols – 48%, was remarked for the drug concentration of 100 μM and 72 h-long incubation. It is worth noticing, the most significant distinctions between the effects of tested concentrations occur after the treatment for 24 h and 72 h. The level of melanoma cells vitality after 24 h incubation was 74% and 62% for 50 μM and 100 μM concentration, respectively. In the case of 72 h-long treatment, the responding
results were stated to be 62% and 52%.
3.4. Doxycycline induces DNA fragmentation in melanoma cells The evaluation of DNA fragmentation process in melanoma cells was performed after 24 h, 48 h and 72 h-long treatment with doxycycline in the concentrations of 50 μM and 100 μM. The results presented in Fig. 4 indicate that 50 μM of doxycycline increases the percentage of cells with fragmented DNA only by 3% for 24 h and 48 h incubation and 4% for 72 h-long treatment, when compared to the control. Much more noticeable changes were remarked in cell culture exposed to 100 μM of tested drug. In this case, the cells population with fragmented DNA was risen to 26%, 35% and 36% for following treatment time. 5
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Fig. 3. The influence of doxycycline on vitality of human melanoma cells COLO 829. Histograms demonstrate representative distribution of the cells population for each tested group. Marker M1 represents unhealthy cells with decreased level of reduced thiols. Marker M2 responds the subpopulation of healthy cells with high level of reduced thiols. Mean values of the percentage of cells with high level of reduced thiols and responding standard deviations are presented in the bar graph. ⁎⁎p < .005 vs control.
percentage of this population to 56% and to 34% for 24 h and 48 h-long incubation, respectively. It means that, in order, 44% and 66% of cells treated with doxycycline have depolarized mitochondrial membrane. This effect indicates a high possibility of cells apoptosis.
Taking into consideration described alterations in melanoma cells caused by doxycycline, the evaluation of mitochondrial potential and annexin V test was conducted using 100 μM concentration.
3.5. Doxycycline decreases mitochondrial membrane potential of melanoma cells
3.6. Doxycycline triggers externalization of phosphatidylserine and activates melanoma cells apoptosis
Disruption of mitochondrial membrane potential is considered as a state preceding cell apoptosis. The assay was performed by the use of a high specific dye JC-1, which is able to accumulate in the mitochondrial matrix. The obtained results (Fig. 5) show that 92% of untreated melanoma cells have polarized mitochondrial membrane. In turn, the treatment with doxycycline in the concentration of 100 μM decrease the
The annexin V assay allows to evaluate translocation of phosphatidylserine to the outer layer of the cell membrane, which is an indicator of the cell apoptosis. In order to confirm the process, the tested cell culture was treated with doxycycline in the concentration of 100 μM for 24 h and 48 h. The obtained results (Fig. 6) show the drug 6
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Fig. 4. The effect of doxycycline on DNA fragmentation in human melanoma cells COLO 829. The cells were treated with doxycycline at the concentrations of 50 μM and 100 μM for 24 h, 48 h and 72 h. Representative results of the experiments were displayed in the histograms. Marker M1 represents the percentage of cells with fragmented DNA. Mean values of the percentage of cells with fragmented DNA and responding standard deviations are presented in the bar graph. ⁎p < .05, ⁎⁎p < .005 vs control.
cure are not satisfying, mainly due to the lack of efficacy. All these problems generate the need of searching for new drugs and new methods of therapy. Doxycycline is the second generation tetracycline antibiotic possessing various nonantibacterial activities. The anti-cancer effect belongs to the most frequent described and tested actions. So far the effect has been confirmed in many in vitro studies, among other on leukemia, prostate and breast cancer cell lines. Not only does doxycycline influence the growth, proliferation and apoptosis of cancer cells, but it also inhibits metastasis and acts as a radio-sensitizer (Ali et al., 2017). Moreover, the drug has many other positive features, including almost complete absorption after oral administration, good tolerance, low cost and universal availability (Holmes and Charles, 2009). It is also worth emphasizing the possibility of doxycycline to form complexes with
after 24 h rises the percentage of early apoptotic cells by ca. 52% when compared to the control. The population of late apoptotic cells for this treatment time was determined to ca. 36%. In turn, after another 24 h of incubation, the percentage of early and late apoptotic cells in the melanoma culture was estimated to be ca. 23% and ca. 81%. 4. Discussion Melanoma is a cancer that occurs as a result of the malignant transformation of skin pigment cells – melanocytes. It is characterized by the worst prognosis and the highest mortality among the typical epidermal cancers. The rapid increase of melanoma incidence is currently observed, especially in Caucasian populations (Potrony et al., 2015). Despite various available ways of treatment, the results of the 7
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Fig. 5. The influence of doxycycline on mitochondrial membrane potential in human melanoma cells COLO 829. Representative scatter plots demonstrate a tested cells population divided by a gate to polarized cells (Q1ur - upper right quadrant) and cells with depolarized mitochondrial membrane (Q1lr - lower right quadrant). Mean values of the percentage of cells with decreased mitochondrial membrane potential and responding standard deviations are presented in the bar graph. ⁎⁎p < .005 vs control.
The first step of the study was to examine the cytotoxicity of doxycycline on melanoma cells. The obtained results showed that doxycycline decreased cells viability and inhibited cells proliferation in the exposure time and concentration-depended manner. The noticeable
melanin polymers which can lead, in a similar way to other tetracyclines, to accumulation of the drug in pigmented tissue (Banning and Heard, 2002; Rok et al., 2019). Having referred to the above issues, it seems reasonable to investigate antimelanoma effect of doxycycline.
Fig. 6. The annexin V assay of human melanoma cells COLO 829 treated with doxycycline for 24 h and 48 h. Representative scatter plots demonstrate a tested cells population divided by a gate to three subpopulations: healthy cells (Q1ll - lower left quadrant), early apoptotic cells (Q1lr - lower right quadrant) and late apoptotic cells (Q1ur - uper right quadrant). Mean values of the percentage of apoptotic cells and responding standard deviations are presented in the bar graph. ⁎⁎p < .005 vs control. 8
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(Foroodi et al., 2009), small cell lung cancer – NCI-H446 and lung carcinoma – A549 (Qin et al., 2015). These findings suggest that doxycycline changes the cell cycle profile depending on a kind of cancer and probably on a molecular disturbance of cell cycle regulation in particular cell line. One of biochemical factors influencing the cell cycle are reactive oxygen species. It was stated that oxidative stress reduced cell proliferation, among others by inhibiting the passage of cells from the G0 to the G1 phase, slowing the S phase by inhibiting DNA synthesis or by cell cycle checkpoints arrest. Moreover, ROS interact with cellular macromolecules (nucleic acids, proteins, lipids), damage their structure and biological functions leading to cell death (Conklin, 2004). In general, cancer cells have the basal level of ROS higher than normal cells. For this reason, their tolerance of the increase ROS level is smaller than in normal cells (Tang et al., 2019). Among anticancer agents there are many drugs which act through ROS-dependent pathway. It was confirmed that some of cytotoxic antibiotics (doxorubicin, mitomycin C, mitoxantrone, actinomycin D), alkylating agents (nimustine), antimetabolites (gemcitabine, mercaptopurine) and antimitotic agents (paclitaxel, vinblastine, vinorelbine) were able to induce oxidative stress (Yokoyama et al., 2017). The most abundant and well-known non-enzymatic antioxidant is glutathione (GSH). The molecule is considered to be essential for redox homeostasis and cell survival (Gorrini et al., 2013). GSH represents reduced form of thiols which can be oxidized to its corresponding disulfide (GSSG), reducing simultaneously reactive oxygen species (Baba and Bhatnagar, 2018). The results obtained in the study show that doxycycline significantly decreases the level of reduced thiols. The observed changes are dependent on the drug concentration as well as the treatment time. The drug depreciated the percentage of melanoma cells with high level of reduced thiol close to 50%. The finding suggests that one of mechanisms of doxycycline antimelanoma effect is generation of oxidative stress and redox imbalance. Moreover, in the earlier study we demonstrated that doxycycline unexposed to UVA radiation did not influence the activity of antioxidant enzymes (SOD, CAT, GPx) in normal melanocytes, even at the toxic concentration (Rok et al., 2015). It lets us presume that doxycycline does not induce oxidative stress in normal melanocytes, thus its influence on melanoma cells redox balance can be considered as selective. Glutathione, besides its antioxidant activity, influences other molecular and cellular processes including apoptotic signaling pathways (Circu and Aw, 2012). Moreover, it is worth noticing that (GSH) depletion is a common feature of cell apoptosis triggered by death receptors-dependent mechanisms, oxidative stress or and cytotoxic drugs (Franco and Cidlowski, 2009). In reference to the obtained results, the influence of doxycycline on apoptosis process in melanoma cells was examined. The analysis of apoptosis involved DNA fragmentation as well as the measurements of mitochondrial potential and annexin V. One of admitted biochemical hallmarks of apoptosis is DNA fragmentation – a process of DNA cleavage into oligonucleosome-sized fragments. The process is controlled by activation of the apoptosisspecific endonucleases and provides the removal of DNA and the timely completion of apoptosis (Zhang and Xu, 2002). Signal pathways leading to DNA fragmentation, are mediated mainly by the family of cysteinyl aspartate-specific protease caspases (Kitazumi and Tsukahara, 2011). Beyond caspases, DNA fragmentation may occur in a caspase-independent manner. In this case, the process is caused by proapoptotic proteins, released from the mitochondria: apoptosis inducing factor AIF and endonuclease G (Elmore, 2007). Several earlier conducted studies indicated that cytotoxicity of doxycycline was connected to the induction of DNA fragmentation. The phenomenon was confirmed during experiments on leukemic cells HL60 and K562 as well as pancreatic cancer cells PANC-1and MDAPanc-28 (Song et al., 2014; Fares et al., 2015; Son et al., 2009; Fujioka et al., 2004). The results obtained in our study show that doxycycline induces DNA fragmentation in the melanoma cells COLO 829. The largest
cytotoxic effect was observed from the concentration of 50 μM. Doxycycline in this concentration stopped cells divisions and reduced cells viability by ca. 58% already after 24 h. The calculated EC50 values were 74.35 μM, 32.28 μM and 16.25 μM for 24 h, 48 h and 72 h-long incubation, respectively. Previously conducted studies on the melanoma cell line COLO 829 showed that dacarbazine – one of the antimelanoma reference drug and sulindac in the concentration of 50 μM decreased cells viability after a 24-h incubation by 42.3% and 46.3%, respectively. Interestingly, even doubling of the tested drugs concentration had no significant effect. The observed reduction of cells viability at concentration 100 μM was 47.3% and 46.3%, respectively (Miliński et al., 2017). It proves doxycycline is more cytotoxic and shows better antimelanoma effect then dacarbazine and sulindac. Additionally, the EC50 values for doxycycline are lower than obtained in other studies for fluoroquinolone derivatives: lomefloxacin (EC50 24h = 510 μM, EC50 48h = 330 μM, EC50 72h = 250 μM), ciprofloxacin (EC50 24h = 740 μM, EC50 48h = 170 μM, EC50 72h = 100 μM) and moxifloxacin (EC50 24h = 400 μM, EC50 48h = 220 μM, EC50 72h = 150 μM) (Bebrok et al., 2017; Bebrok et al., 2018; Beberok et al., 2019). The studies of anticancer activity of doxycycline were also conducted using another cancer cell lines. In case of breast and lung cancer cells, the obtained values of EC50 are lower than calculated for COLO 829. It was stated that doxycycline decreased viability of breast cancer cells MCF7 and MDA-MB-468 by 50% after 72-h incubation at concentration of 11.39 μM and 7.13 μM, respectively (Zhang et al., 2017). In turn, the analysis of lung cancer cells showed the EC50 value after 48 h incubation is 1.70 μM and 1.06 μM for NCI-H446 and A549, respectively (Qin et al., 2015). Presented results indicate different sensitivity of particular call line to the cytotoxic effect of doxycycline. Due to promising results obtained in the first part of the study, the evaluation of cell cycle, thiols level, DNA fragmentation as well as the analysis of mitochondrial potential and annexin V was made. Doxycycline was used in subsequent studies in concentrations of 50 μM and 100 μM. The cell cycle is the series of following events which leads to doubling of cellular components and to segregating each cell into daughter cells. The individual phases of cell cycle are marked with symbols connected with their roles: the resting state (quiescence) – G0, DNA replication (DNA synthesis) – S-phase; two gap phases (the growth of cells) separate S phase and mitosis are known as G1 and G2; chromosomes segregation and cell division (mitosis) – M-phase (Casimiro et al., 2012; Barnum and O'Connell, 2014). Normal cells undergo the cycle in response to mitogenic signals, whereas cancer cells proliferate without growth stimuli in an unregulated way. That is why the cancer is often considered a disease of the cell cycle (Diaz-Moralli et al., 2013). Deregulation of the cell cycle and cell division underlies malignant transformation of normal cells into tumor cells. Molecular mechanisms, including a system of cyclin-dependent kinases (CDKs), which regulate these processes, have become a target for potential anticancer drugs. In the case of human melanoma, disruptions of CDK1, CDK2 and CDK4 were stated (Otto and Sicinski, 2017; Bai et al., 2017). Basing on the obtained results, it can be stated that doxycycline doesn't work in phase-specific manner on melanoma cells. The observed changes differ depending both on a concentration, and on the time of incubation. It seems that the most significant variations are the decrease the percentage of cells in the G2/M phase, and the increase the number of cells in the sub-G1 phase at the concentration of 100 μM. The influence of doxycycline on a cell cycle profile was also examined on another cancer cell lines. The analysis of prostate adenocarcinoma cells PC-3 showed that doxycycline alone as well as in combination with dacarbazine significantly increased the percentage of cells in G2/M phase after 72 h treatment (Zhu et al., 2017). On the other hand, the arrest of the cell cycle in the G1/G0 phase was noticed in the case of following cancer cell lines: epithelioid carcinoma of the pancreas – PANC-1 (Son et al., 2009), breast adenocarcinoma – MDA-MB-231 9
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tested concentrations (50 μM ̴ 22 μg/ml; 100 μM ̴ 44 μg/ml) are higher than serum concentrations observed during a standard antibacterial therapy. Nowadays, the maximum recommended dose of doxycycline in serious infections is 300 mg daily and the usual daily dosage is 200 mg. (Holmes and Charles, 2009). Pharmacokinetic data for doxycycline indicates that a single dose of 200 mg achieves peak serum concentrations of approximately 2.6–5.9 μg/ml after oral administration and approximately 9.3 μg/ml for intravenous route. It was also measured that serum Cmax for a dose of 500 mg p.o. is 15.3 μg/ml (Agwuh and MacGowan, 2006). However, it is worth emphasizing that the concentrations used in our study reflect efficacy of short-time and single-dose therapy. Moreover, doxycycline has some properties which can be preferred in the melanoma treatment. Among others, the drug is lipophilic and widely distributed, including the skin, where it is accumulated (Valentin et al., 2009). The clinical trial shows the accumulation ratio in the skin after 2 weeks therapy with a relatively low dosage - 40 mg/day of doxycycline ranges from 1.8 to 2.7, depends on dietary conditions (Pal et al., 2018). The possibility of doxycycline to form complexes with melanin allows to draw conclusions that the accumulation ratio in highly pigmented cells, like melanoma, would be much higher. In conclusion, it was stated for the first time that doxycycline decreased the viability and inhibited the proliferation of human melanoma cells, proportionally to the drug concentration and the treatment time. The obtained results indicated that doxycycline disturbed the homeostasis of the tested cells. The drug lowered cells vitality by decreasing the intracellular level of reduced thiols as well as changed the cell cycle profile. In addition, we demonstrated that doxycycline triggered the DNA fragmentation during the treatment. The melanoma cells exposed to the drug were characterized also by the lowered mitochondrial membrane potential, which indicated cells apoptosis. Finally, doxycycline appeared to be the inductor of phosphatidylserine externalization – well known hallmark of apoptosis, which was confirmed by results of annexin V test. The conducted study contributes to the increase of knowledge about nonantibacterial action of doxycycline, including the influence on human cancer cells. It is worth emphasizing there is a need of successive studies on the antimelanoma effect of doxycycline taking into account a long-time therapy, the process of accumulation in the skin or a possibility of topical application. However, in relation to not satisfied results of malignant melanoma treatment, it brings new advice on searching for a more effective therapy.
increase was noticed for doxycycline in the concentration of 100 μM after 48 h and 72 h treatment. In this case, the percentage of cells with fragmented DNA was about 35%. In turn, the tested drug in half of the concentration elevated the cells population with fragmented DNA only up to 15%. For this reason, the next analyses were made only for the higher concentration of doxycycline. Mitochondria are one of the possible target for doxycycline in relation to its anticancer activity. The mechanism of the action involves, among others, an inhibition of mitochondrial protein synthesis and a decrease of the mitochondrial energy-generating capacity. The study with cultures of the human cancer cells A549, COLO357 and HT29 showed that doxycycline, more than a chemically modified tetracycline COL-3, specifically affects mitochondrially encoded proteins (Protasoni et al., 2018). Mitochondrial dysfunction through decreasing mitochondrial membrane potential (MMP), mitochondrial respiration and altering the energy production, was also stated in human glioblastoma cell lines A172 and U87, treated with doxycycline (Tan et al., 2017). In the study on the melanoma cell line COLO 829 doxycycline augmented the percentage of cells with decreased mitochondrial membrane potential by about 36% and 58% after 24 h and 48 h treatment, respectively. The finding is another evidence to suggest that doxycycline triggers melanoma cells apoptosis. Decreased MMP is not only one of the apoptosis hallmarks, but is also required to release from the mitochondria to the cytoplasm apoptogenic factors: cytochrome c and AIF (Gottlieb et al., 2003; Ly et al., 2003). Another characteristic molecular feature of apoptosis is the change of phospholipids localization in the plasma membrane. The distribution of phospholipids is asymmetric in healthy cells. The outer leaflet mostly contains electrically neutral phosphatidylcholine and sphyngomielin, whereas phosphatidylethanolamine as well as anionic phosphatidylserine and phosphatidic acid are located in the cytosolic layer (Demchenko, 2012). In an apoptotic cell, the enzyme γ-scramblase is activated resulting in phosphatidylserine flipping to the outer leaflet of the plasma membrane (Yang et al., 2012). In general, the loss of the asymmetric distribution makes apoptotic cells more detectable for removing that cells macrophages (Wlodkowic et al., 2012). Moreover, the appearance of phosphatidylserine in the outer membrane layer, considered as a near-universal event in apoptosis, has become the way for detection of this kind of cell death. The fluorochrome-conjugated annexin V, a natural human anticoagulant protein, is currently the most frequently used a marker of apoptosis. The method of apoptosis detection is based on relatively high affinity (Kd ̴ 10−7–10−8 M) of annexin V to negatively charged phosphatidylserine (Demchenko, 2012; Wlodkowic et al., 2012). Earlier conducted studies showed that doxycycline increased the number of annexin V-positive cells after 72-h treatment in population of breast cancer cells MCF7 ( ̴ 10% of population) and MDA-MB-468 (̴ 45% of population) as well as prostate cancer cells PC3 ( ̴ 12% of population) (Zhang et al., 2017; Zhu et al., 2017). According to the study on melanoma cells COLO 829, doxycycline induced apoptosis in most treated cells. The percentage of early and late apoptotic cells was about 60% and 35% after 24 h and 22% and 60% after 48 h, respectively. The results show and confirm relatively quickly and clearly noticeable induction of apoptosis in investigated melanoma cells by the tested drug. They also suggest that melanoma cells can be more sensitive to doxycycline then other cancer cells. The performed in vitro studies show doxycycline anticancer activity in treatment of human melanoma cells. The influence of drug on COLO 829 cells depends on its concentration. The action of doxycycline in the concentration of 50 μM includes generally cytotoxic, antiproliferative and oxidative stress-inducing effects. In turn, the concentration 100 μM causes DNA fragmentation, disruption of mitochondrial membrane potential and triggers cells apoptosis, additionally. The observed effects rise the question about possibility of doxycycline usage in the melanoma treatment in vivo. The problem of directly transferring conditions in vitro to in vivo is complex and requires consideration of various factors. Taking into account literature data, it can be stated that the
Declaration of Competing Interest The authors: Jakub Rok, Marta Karkoszka, Zuzanna Rzepka, Michalina Respondek, Klaudia Banach, Artur Beberok and Dorota Wrześniok declare that there are no conflicts of interest. The manuscript entitled: “Cytotoxic and proapoptotic effect of doxycycline – an in vitro study on the human skin melanoma cells”, which all the listed authors have read and approved of, has not been submitted elsewhere for publication, in whole or in part, and is permitted to be published in this journal. Acknowledgment This work was financially supported by the Medical University of Silesia in Katowice (Grants No. KNW-2-O26/N/9/K and KNW-1-037/K/ 9/O). References Agwuh, K.N., MacGowan, A., 2006. Pharmacokinetics and pharmacodynamics of the tetracyclines including glycylcyclines. J. Antimicrob. Chemother. 58, 256–265. Ali, I., Alfarouk, K.O., Reshkin, S.J., Ibrahim, M.E., 2017. Anticancer agents med. Chem. 17, 1617–1623.
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Cytotoxic and proapoptotic effect of doxycycline – An in vitro study on the human skin melanoma cells
T
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Jakub Rok , Marta Karkoszka, Zuzanna Rzepka, Michalina Respondek, Klaudia Banach, Artur Beberok, Dorota Wrześniok Department of Pharmaceutical Chemistry, Faculty of Pharmaceutical Sciences in Sosnowiec, Medical University of Silesia, Katowice, Poland, Jagiellońska 4, 41-200 Sosnowiec, Poland
A R T I C LE I N FO
A B S T R A C T
Keywords: Doxycycline Melanoma Apoptosis Cell cycle DNA fragmentation Mitochondrial membrane potential
Doxycycline is a semisynthetic, second generation tetracycline. Currently, it is used, among others, in the treatment of acne and skin infections. Moreover, doxycycline has many valuable nonantibiotic properties, including anti-inflammatory, immunosuppressive and anticancer effects. Recent studies showed that the drug had the ability to inhibit the adhesion and migration of cancer cells, as well as affected their growth and proliferation and induced apoptosis. The purpose of this study was to examine the antimelanoma effect of doxycycline. The obtained results demonstrated that doxycycline decreased the viability and inhibited the proliferation of human melanoma cells, proportionally to the drug concentration and the treatment time. It was stated that doxycycline disturbed the homeostasis of the cells by lowering intracellular level of reduced thiols. In addition, the treatment changed the cell cycle profile and triggered the DNA fragmentation. Mitochondria of melanoma cells exposed to the drug had lowered membrane potential, which indicated cells apoptosis. Finally, doxycycline induced the externalization phosphatidylserine – a well-known hallmark of apoptosis, confirmed by results of annexin V test. The presented study contributes to the increase of knowledge about nonantibacterial action of doxycycline, including the influence on human cancer cells and indicates new potential possibility of effective treatment of malignant melanoma.
1. Introduction Malignant melanoma is considered one of the most aggressive human cancers with constantly rising incidence worldwide, especially in Celtic and Caucasian races (Rastrelli et al., 2014a). The growing public health problem is caused by high morbidity, poor prognosis and lack of effective current therapeutic methods (Liu and Sheikh, 2014). It is estimated that approximately 350,000 melanomas and 13 million non-melanoma skin cancers occur every year and lead to 81,000 deaths (Silva et al., 2018). In 2014 there were 76,100 new cases of melanoma diagnosed, which constituted about 4.6% of new cancer cases (Holmes, 2014). Although melanoma accounts for a little over 2% of skin neoplasms, it is the cause of about 80% of deaths due to cancerous skin lesions (Liu and Sheikh, 2014; Miller and Mihm, 2006). The incidence of melanoma cases increases in most white populations worldwide. The problem concerns mainly Australasian, North American, and European populations (von Schuckmann et al., 2019). Melanoma is a tumor that derives from pigment-producing cells – melanocytes and their neural crest cells precursors (Lo and Fisher, ⁎
2014). Both genetic predispositions and environmental factors are significant in the development and progression of this tumor (Rastrelli et al., 2014b). More than 90% of this skin cancers are located within the skin. In 1 out of 10 diagnosed melanomas, the primary tumor lesions are located in other places where melanocytes occur, such as the eyeball or oral mucosa (Schadendorf et al., 2015; Sulaimon and Kitchell, 2003). Surgical removal of melanoma with a margin of healthy tissue and assessment of the surrounding lymph nodes is the basic method of therapy for this type of cancer. An appropriate treatment for primary focus enables effective treatment of patients who have not metastasized (Garbe et al., 2016). Dacarbazine and temozolomide are major drugs used in melanoma chemotherapy. Approximately 15% of patients respond to treatment with these agents, with a 5-year survival rate of less than 2%. In more advanced stages of the disease, radiotherapy and immunotherapy are applied (Bahtia et al., 2009). Interleukin-2 and interferon alpha are used in immunotherapy, allowing for remission in about 16% of patients (Kee and McArthur, 2017). More modern immunotherapy methods are based on the use of monoclonal antibodies
Corresponding author. E-mail address: [email protected] (J. Rok).
https://doi.org/10.1016/j.tiv.2020.104790 Received 26 November 2019; Received in revised form 4 February 2020; Accepted 6 February 2020 Available online 08 February 2020 0887-2333/ © 2020 Elsevier Ltd. All rights reserved.
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propidium iodide, 1.2 μg/ml acridine orange in DMSO), Solution 7 (200 μg/ml JC-1), Solution 8 (1 μg/ml DAPI in PBS), Solution 15 (500 μg/ml Hoechst 33342), Solution 16 (500 μg/ml propidium iodide), NC-Slide A8 and A2 as well as Via-1-Cassette (acridine orange and DAPI fluorophores) were obtained from ChemoMetec (Denmark). Annexin V-CF488A conjugate as well as Annexin V binding buffer were purchased from Biotium (UK).
against the CTLA-4 protein (ipilimumab) and the PD-1 receptor (pembrolizumab, nivolumab). Treatment effects are visible in approximately 15% of patients when ipilimumab is used and 50% during therapy directed against the PD-1 receptor (Homet and Ribas, 2014). The advancement of knowledge about molecular mechanisms for the development of this type of cancer has allowed the invention of drugs that include vemurafenib and trametinib (Ho and Tsao, 2015). Despite the fact that surgical treatment of early melanoma leads to high percentage of cures, advanced and metastatic melanoma presents tendency to rapidly metastasize and an innate resistance to chemotherapy (Homet and Ribas, 2014). The above information indicates that melanoma still remains, a potentially fatal malignancy (Rastrelli et al., 2014b). Tetracyclines are a group of drugs, constantly and widely used since their invention in 1948 (Griffin et al., 2010). These antibiotics owe their popularity to a range of beneficial properties, such as a broad spectrum of action against both Gram (+) and Gram (−) bacteria, good tolerance and favorable pharmacokinetic properties (Broghi and Palma, 2014). Tetracycylines have a bacteriostatic effect associated with binding the drug with the 16S rRNA in 30S subunit of the bacterial ribosome. This prevents the attachment of aminoacyl tRNA to A site and disrupts protein synthesis (Chukwudi, 2016). Doxycycline is a semisynthetic drug that belongs to the second generation of tetracyclines. Currently, doxycycline is used in the treatment of acne and skin infections of various origins (Cuhna et al., 2018). In addition to well-known antibiotic effects, an extensive research on doxycycline has revealed a range of highly valuable nonantibiotic properties. Anti-inflammatory, immunosuppressive and anticancer effects of doxycycline are the reasons for conducting numerous researches on alternative usage of this drug (Bahrami et al., 2012). The nonantibiotic effect of doxycycline is mainly the result of its ability to inhibit metalloproteinases activity (MMP). Doxycycline is currently the only inhibitor of MMP enzymes that has been approved for usage by the FDA in the treatment of periodontitis at sub-antibiotic dose (SDD) is (Caton and Ryan, 2011). Recent reports of antitumor activity of doxycycline have raised the greatest interest. It has been found that the drug has the ability to inhibit the adhesion and migration of cancer cells. In addition, doxycycline may affect their growth and proliferation and induce apoptosis (Sun et al., 2009). Molecular mechanisms of the antitumor action of doxycycline have not been fully understood, but until now it has been shown that it may be associated among others with: i) inhibition of the MMP-2 and MMP-9 metalloproteinases activity, ii) activation of apoptosis signal-regulated kinase 1, c-Jun N-terminal kinase and caspases which inducing apoptosis, iii) reduction of mitochondria membrane potential, iv) induction of oxidative stress in cancer cells (Shieh et al., 2010). The purpose of this study was to examine the antimelanoma effect of doxycycline by the analysis of cells viability, morphology, cell cycle, vitality, DNA fragmentation and mitochondrial membrane potential as well as the results of annexin V assay.
2.2. Cell culture Human skin melanotic melanoma cell line, COLO 829, was purchased from ATCC (CRL-1974™, USA). The cells were cultured in RPMI 1640 medium (with L-glutamine) enriched by inactivated fetal bovine serum to a final concentration of 10%, penicillin (100 U/ml), neomycin (10 μg/ml) and amphotericin B (0.25 μg/ml). Cells were grown in a 5% CO2 incubator CB 160 (BINDER, Germany) at 37 °C with 5% relative humidity. 2.3. Cytotoxicity assessment The viability of melanotic melanoma cells COLO 829 was determined by the WST-1 colorimetric assay. WST-1 (4-[3-(4-iodophenyl)2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzenedisulphonate) is a water-soluble tetrazolium salt, which is cleaved to formazan by cells mitochondrial dehydrogenases. The amount of produced formazan correlates with the number of viable cells. In brief, 2.5 × 103 cells per well were placed in a 96-well microplate in a supplemented RPMI 1640 growth medium and incubated at 37 °C and 5% CO2 for 24 h. After the incubation, the medium was removed and cells were treated with doxycycline solutions in a concentration ranging from 1 × 10−7 M to 2,5 × 10−4 M. After 21 h, 45 h and 69 h of incubation, 10 μl of WST-1 solution was added to each well, and the incubation was continued for 3 h. The absorbance of the samples was measured at 440 nm using a reference wavelength at 650 nm. The measurement was made using microplate reader Infinite 200 PRO (TECAN, Switzerland). The controls were normalized to 100% for each assay and the treatments were expressed as the percentage of the controls. 2.4. Cell morphology assessment Photographic documentation of COLO 829 cells abundance and morphology under the influence of doxycycline was carried out during the research. Cells were seeded in T-75 flasks (2 × 106 cells/flask) and cultured in RPMI supplemented medium, as described above. The treatment with doxycycline in concentration 100 μM began 24 h after seeding. The cells culture was examined under light inverted microscope NIKON TS100F (Japan) after 24 h, 48 h and a 72 h-long incubation with the drug. 2.5. Cell cycle assay The cell cycle analysis of COLO 829 cells were performed using a NucleoCounter® NC-3000™ fluorescent imaging cytometer (Denmark). The analysis is based on measurements of the DNA content within investigated cells population. The COLO 829 cells were seeded in T-75 flasks at a density of 2 × 106 cells per flask. The treatment by doxycycline in a concentration 50 μM and 100 μM was evaluated after 24 h, 48 h and 72 h of incubation. The cells were harvested by trypsinization, loaded into Via-1-Casette and counted using NucleoCounter NC-3000 image cytometer. Afterwards, 1 × 106 cells were suspended in 0.5 ml PBS and fixed with 4.5 ml of 70% cold ethanol for at least 12 h at 0-4 °C. Then the cells were centrifuged, the ethanol was removed and the cells pellets were resuspended in PBS and centrifuged again for 5 min at 500 g. Each of the obtained cells pellets was resuspended in 0.5 ml of Solution 3 and incubated for 5 min at 37 °C. The stained cells were loaded into NC-Slide A8 and analyzed using „Fixed Cell Cycle-DAPI
2. Materials and methods 2.1. Chemicals and reagents Doxycycline hyclate, C22H24N2O8 x HCl x 0,5H2O x 0,5C2H6O, penicillin and amphotericin B were purchased from Sigma-Aldrich Inc. (USA). Neomycin was obtained from Amara (Poland). Fetal bovine serum was purchased from Gibco (Poland). The growth medium for melanoma cells, RPMI 1640, as well as trypsin inhibitor were acquired from Cascade Biologics (UK). Trypsin/EDTA was obtained from Cytogen (Poland). Cell Proliferation Reagent WST-1 was purchased from Roche GmbH (Germany). Solution 3 (1 μg/ml DAPI, 0.1% triton X100 in PBS), Solution 5 (400 μg/ml VitaBright-48™, 500 μg/ml 2
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2.9. Annexin V assay
Assay” protocol by NC-3000 image cytometer. The obtained DNA content histograms were used to demarcate different phases of cell cycle in tested samples.
The annexin V assay is commonly used to detect cells apoptosis. The test is based on high affinity of a cellular protein annexin V to phosphatidylserine whose translocation to the outer membrane layer is already observed in early apoptosis. Dyes using in the assay, except FITC labeled annexin V (Annexin V-CF488A), involve Hoechst 33342 – Solution 15 (stains total population) and propidium iodide – solution 16 (stains late apoptotic and necrotic cells). The test and its analysis were performed using the fluorescence image cytometer NucleoCounter NC3000 (Denmark). Tested cells were seeded in T-75 flasks at a density of 2 × 106 cells per flask and after 24 h they were treated with doxycycline in a concentration 100 μM for 24 h and 48 h. After the treatment, the cells were detached by trypsinization and counted using the NC-3000 image cytometer. In the next step, 3.0 × 105 cells were suspended in 100 μl of Annexin V binding buffer and fixed with 2 μl of Annexin V-CF488A conjugate and 2 μl of Hoechst33342. The cells were then incubated for 15 min at 37 °C. Afterwards, the stained cells were subsequently centrifuged at 400 g for 5 min and the supernatant was removed. The cell pellets were resuspended in 300 μl of Annexin V binding buffer. The suspension was centrifuged twice under the conditions described above. Finally, the cell pellets were resuspended in 100 μl of Annexin V binding buffer and 2 μl of Solution 16 was added. The samples were analyzed immediately using NCSlideA8 and „Annexin V Assay” protocol. The obtained scatter-plots were used to demarcate the percentage of heathy cells, early apoptotic as well as late apoptotic cells, respectively.
2.6. Vitality assay: the analysis of cellular thiols level The estimation of cells vitality was performed by the use of the fluorescence imaging cytometer NucleoCounter® NC-3000™ (Denmark). The analysis is based on the assessment of cellular thiols level using a specific dye – VitaBright-48™. The treatment with doxycycline in concentration 50 μM and 100 μM began 24 h after reaching the logarithmic growth phase and was conducted for 24 h, 48 h or 72 h. Before the test, the cells were harvested by trypsinization, loaded into Via-1-Casette, and counted using the NC-3000 image cytometer. Subsequently, according to the producer, 1 × 106 cells were suspended in 0,5 ml of PBS, and then 10 μl of Solution 5 was added into 190 μl of the cell suspension. The stained cells were loaded into the NC-Slide A8 and measured using the „Vitality (VB-48) Assay” protocol in the NC-3000 image cytometer. High fluorescence intensity of a particular cell indicates that the cell has a high level of reduced thiols, such as GSH, which is an indicator of overall health status (vitality). The obtained VB-48™ intensity histograms were used to demarcate healthy and low vitality subpopulation of the tested samples.
2.7. DNA fragmentation assay The assessment of DNA fragmentation in COLO 829 cells were performed using a fluorescent imaging cytometer NucleoCounter® NC3000™ (Denmark). The analysis is based on the detection of cells with high molecular weight fragmented DNA, stained with DAPI. The investigated cells were tested after 24 h, 48 h and 72 h incubation with doxycycline in a concentration 50 μM and 100 μM. After the treatment, the cells were detached by trypsinization, counted, suspended in PBS (2 × 106 cells/ml) and fixed with 70% cold ethanol for at least 12 h at 0-4 °C. Afterwards, the cells were centrifuged. Obtained cells pellets were resuspended in PBS, centrifuged again for 5 min at 500 g and next resuspended in 0.5 ml of Solution 3. Stained cells were incubated for 5 min at 37 °C. and loaded into NC-Slide A8 to perform the analysis. The obtained histograms were used to demarcate subpopulation of the tested cells culture with fragmented DNA.
2.10. Statistical analysis Statistical analysis of the results was performed using GraphPad Prism 6.01 Software. In all experiments mean values of at least three separate experiments performed in triplicate (n = 9) ± standard deviation of the mean (SD) were calculated. The results were analyzed statistically of Student's t-test, one-way ANOVA as well as Dunnett's comparison test. The Kolomogorov-Smirnov test checked the compliance of the distribution results and the Brown-Forsythe test checked the variances of the compared groups meet the homogeneity assumption. In all cases the statistical significance was found for p-value to be lower than 0.05. 3. Results
2.8. Mitochondrial potential assay
3.1. Cytotoxic effect of doxycycline on melanoma cells
The mitochondrial transmembrane potential (ΔΨm) was measured by the use of the fluorescence image cytometer NucleoCounter NC-3000 (Denmark). Decrease of the potential usually precedes cells apoptosis. The method is based on fluorescent cationic dye JC-1, which accumulates in the mitochondria in the potential depended manner. In healthy cells, it achieves a high concentration inside the mitochondria and aggregates to red fluorescent form. In turn, in apoptotic cells it localizes in the cells cytoplasm emitting green fluorescence. COLO 829 cells were seeded in T-75 flasks at a density of 2 × 106 cells per flask and after 24 h they were treated with doxycycline in a concentration 100 μM for 24 h and 48 h. After the treatment, the cells were harvested by trypsinization and counted using the NC-3000 image cytometer. Afterwards, 12.5 μl of Solution 7 was added to 1.0 × 106 cells, which were next incubated for 10 min at 37 °C. The stained cells were then centrifuged at 400 g for 5 min and washed twice with PBS. The obtained cell pellets were resuspended in 0.25 ml Solution 8 and analyzed immediately using NC-Slide A8 and „Mitochondrial Potential Assay” protocol. The results presented in the form of scatter-plots were used to demarcate the percentage of polarized/healthy cells and depolarized/apoptotic cells.
The cytotoxicity of doxycycline was tested for the concentrations ranging from 0.1 to 250 μM (Fig. 1A). The time of cells exposure to investigated drug was 24 h, 48 h and 72 h. The obtained results showed the melanoma cell viability decreased proportionally to the drug concentration as well as the time of the treatment. The significant reduction of viable cells, below 50% of control, was observed in the concentrations from 50 μM after 24 h (ca. 42% of control) and in the concentrations from 25 μM after 48 h (ca. 44% of control) as well as 72 h (ca. 30% of control). The viability of melanoma cells for the highest tested concentration was 28.5%, 9.5% and 3.7%, for 24 h, 48 h and 72 h-long incubation, respectively. On the basis of the results, the EC50 values were calculated to be 74.35 μM, 32.28 μM and 16.25 μM for following incubation time. Excepting the cytotoxicity, proliferative potential of melanoma cells was evaluated (Fig. 1B). It was stated that doxycycline in concentrations ranging from 0.1 μM to 10 μM did not influence cells divisions during 24 h and 48 h-long treatment. A gentle but statistically significant decrease of the proliferation rate was observed after a 72 h-long treatment for the concentrations of 5 μM, 7.5 μM and 10 μM. However, doxycycline caused notable inhibition of the cell proliferation in the 3
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Fig. 1. The effect of doxycycline on melanoma cells COLO 829 viability and proliferation. Cells were treated with doxycycline concentrations ranging from 0.1 μM to 250 μM for 24 h, 48 h, and 72 h. Mean values ± SD from three independent experiments are presented. ⁎p < .05, ⁎⁎p < .005 vs control. (A). Cell viability examined by the WST-1 assay. Data are expressed as % of the controls. (B). Anti-proliferative effect of doxycycline on human melanoma cells COLO 829 determined by the WST-1 assay. (C). Photographs of human melanoma cells COLO 829 exposed to 100 μM of doxycycline for 24 h, 48 h and 72 h. Scale bar 250 μm.
concentration of 25 μM after 48 h and 72 h. The divisions of the cells treated with 50 μM, 75 μM and 100 μM of doxycycline were on the same low level, independently on the treatment time. The effect of doxycycline on a culture of melanoma cells can be observed in Fig. 1C. It is noticed that untreated cells proliferate all the time, creating characteristic clusters. Control cells retain their typical morphology, independently of a confluence stage. In turn, doxycycline inhibits cells proliferation and causes noticeable changes of the cell morphology. Melanoma cells exposed to the drug are round and located separately. Moreover, spherical, detached cells appear in the treated cell culture.
of treatment. The changes involve ca. 5% increase in the percentage of cells in S and G2/M phase as well as ca. 12% decrease in the percentage of cells in G1/G0 phase. Moreover, there was 9% and 5% reduction observed in the amount of the cells in G1/G0 phase after 48 h and 72 h, respectively, as well as 8% increase of the amount of cells in G2/M phase. In turn, 100 μM of doxycycline induces a growth of the percentage of melanoma cells in sub-G1 phase by about 7%, 9% and 14% after 24 h, 48 h and 72 h, respectively. Furthermore, an amount of the cells in G2/ M phase was reduced almost twice to ca. 12%. An increase of cells number in S phase was also observed when compared to the control. The results were 19% and 18% for 24 h and 48 h treatment, respectively.
3.2. The effect of doxycycline on melanoma cells cycle The effect of doxycycline on melanoma cell cycle was evaluated in the concentrations of 50 μM and 100 μM, after 24 h, 48 h and 72 h treatment. The results presented in Fig. 2 show that the drug at the concentration of 50 μM changes the cell cycle profile mostly after 24 h
3.3. Doxycycline depreciates melanoma cells vitality by decrease a level of reduced thiols The analysis of cells vitality in the form of intracellular thiols level 4
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Fig. 2. The effect of doxycycline on the cell cycle profile of human melanoma cells COLO 829. The cells were incubated with doxycycline at the concentrations of 50 μM and 100 μM for 24 h, 48 h and 72 h. Representative histograms demonstrate distribution of cells in the different phases of the cycle for each tested group. Markers in the histograms respond to different stages of the cells cycle, as follow: M1 – sub-G1, M2 – G0/G1, M3 – S, M4 – G2/M. Mean values of the percentage of a particular subpopulations and responding standard deviations are presented in the bar graph.
was performed for 50 μM and 100 μM of doxycycline using a specific reagent - VitaBright-48™. The obtained results presented in Fig. 3 show the drug depreciates the level of reduced thiols leading to low vitality of the melanoma cells. The observed effect is proportional to the drug concentration and the time of incubation. The highest level of reduced thiols – 86%, was stated in no-treatment control cells. In turn, the highest percentage of low vitality cells with a small amount of reduced thiols – 48%, was remarked for the drug concentration of 100 μM and 72 h-long incubation. It is worth noticing, the most significant distinctions between the effects of tested concentrations occur after the treatment for 24 h and 72 h. The level of melanoma cells vitality after 24 h incubation was 74% and 62% for 50 μM and 100 μM concentration, respectively. In the case of 72 h-long treatment, the responding
results were stated to be 62% and 52%.
3.4. Doxycycline induces DNA fragmentation in melanoma cells The evaluation of DNA fragmentation process in melanoma cells was performed after 24 h, 48 h and 72 h-long treatment with doxycycline in the concentrations of 50 μM and 100 μM. The results presented in Fig. 4 indicate that 50 μM of doxycycline increases the percentage of cells with fragmented DNA only by 3% for 24 h and 48 h incubation and 4% for 72 h-long treatment, when compared to the control. Much more noticeable changes were remarked in cell culture exposed to 100 μM of tested drug. In this case, the cells population with fragmented DNA was risen to 26%, 35% and 36% for following treatment time. 5
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Fig. 3. The influence of doxycycline on vitality of human melanoma cells COLO 829. Histograms demonstrate representative distribution of the cells population for each tested group. Marker M1 represents unhealthy cells with decreased level of reduced thiols. Marker M2 responds the subpopulation of healthy cells with high level of reduced thiols. Mean values of the percentage of cells with high level of reduced thiols and responding standard deviations are presented in the bar graph. ⁎⁎p < .005 vs control.
percentage of this population to 56% and to 34% for 24 h and 48 h-long incubation, respectively. It means that, in order, 44% and 66% of cells treated with doxycycline have depolarized mitochondrial membrane. This effect indicates a high possibility of cells apoptosis.
Taking into consideration described alterations in melanoma cells caused by doxycycline, the evaluation of mitochondrial potential and annexin V test was conducted using 100 μM concentration.
3.5. Doxycycline decreases mitochondrial membrane potential of melanoma cells
3.6. Doxycycline triggers externalization of phosphatidylserine and activates melanoma cells apoptosis
Disruption of mitochondrial membrane potential is considered as a state preceding cell apoptosis. The assay was performed by the use of a high specific dye JC-1, which is able to accumulate in the mitochondrial matrix. The obtained results (Fig. 5) show that 92% of untreated melanoma cells have polarized mitochondrial membrane. In turn, the treatment with doxycycline in the concentration of 100 μM decrease the
The annexin V assay allows to evaluate translocation of phosphatidylserine to the outer layer of the cell membrane, which is an indicator of the cell apoptosis. In order to confirm the process, the tested cell culture was treated with doxycycline in the concentration of 100 μM for 24 h and 48 h. The obtained results (Fig. 6) show the drug 6
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Fig. 4. The effect of doxycycline on DNA fragmentation in human melanoma cells COLO 829. The cells were treated with doxycycline at the concentrations of 50 μM and 100 μM for 24 h, 48 h and 72 h. Representative results of the experiments were displayed in the histograms. Marker M1 represents the percentage of cells with fragmented DNA. Mean values of the percentage of cells with fragmented DNA and responding standard deviations are presented in the bar graph. ⁎p < .05, ⁎⁎p < .005 vs control.
cure are not satisfying, mainly due to the lack of efficacy. All these problems generate the need of searching for new drugs and new methods of therapy. Doxycycline is the second generation tetracycline antibiotic possessing various nonantibacterial activities. The anti-cancer effect belongs to the most frequent described and tested actions. So far the effect has been confirmed in many in vitro studies, among other on leukemia, prostate and breast cancer cell lines. Not only does doxycycline influence the growth, proliferation and apoptosis of cancer cells, but it also inhibits metastasis and acts as a radio-sensitizer (Ali et al., 2017). Moreover, the drug has many other positive features, including almost complete absorption after oral administration, good tolerance, low cost and universal availability (Holmes and Charles, 2009). It is also worth emphasizing the possibility of doxycycline to form complexes with
after 24 h rises the percentage of early apoptotic cells by ca. 52% when compared to the control. The population of late apoptotic cells for this treatment time was determined to ca. 36%. In turn, after another 24 h of incubation, the percentage of early and late apoptotic cells in the melanoma culture was estimated to be ca. 23% and ca. 81%. 4. Discussion Melanoma is a cancer that occurs as a result of the malignant transformation of skin pigment cells – melanocytes. It is characterized by the worst prognosis and the highest mortality among the typical epidermal cancers. The rapid increase of melanoma incidence is currently observed, especially in Caucasian populations (Potrony et al., 2015). Despite various available ways of treatment, the results of the 7
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Fig. 5. The influence of doxycycline on mitochondrial membrane potential in human melanoma cells COLO 829. Representative scatter plots demonstrate a tested cells population divided by a gate to polarized cells (Q1ur - upper right quadrant) and cells with depolarized mitochondrial membrane (Q1lr - lower right quadrant). Mean values of the percentage of cells with decreased mitochondrial membrane potential and responding standard deviations are presented in the bar graph. ⁎⁎p < .005 vs control.
The first step of the study was to examine the cytotoxicity of doxycycline on melanoma cells. The obtained results showed that doxycycline decreased cells viability and inhibited cells proliferation in the exposure time and concentration-depended manner. The noticeable
melanin polymers which can lead, in a similar way to other tetracyclines, to accumulation of the drug in pigmented tissue (Banning and Heard, 2002; Rok et al., 2019). Having referred to the above issues, it seems reasonable to investigate antimelanoma effect of doxycycline.
Fig. 6. The annexin V assay of human melanoma cells COLO 829 treated with doxycycline for 24 h and 48 h. Representative scatter plots demonstrate a tested cells population divided by a gate to three subpopulations: healthy cells (Q1ll - lower left quadrant), early apoptotic cells (Q1lr - lower right quadrant) and late apoptotic cells (Q1ur - uper right quadrant). Mean values of the percentage of apoptotic cells and responding standard deviations are presented in the bar graph. ⁎⁎p < .005 vs control. 8
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(Foroodi et al., 2009), small cell lung cancer – NCI-H446 and lung carcinoma – A549 (Qin et al., 2015). These findings suggest that doxycycline changes the cell cycle profile depending on a kind of cancer and probably on a molecular disturbance of cell cycle regulation in particular cell line. One of biochemical factors influencing the cell cycle are reactive oxygen species. It was stated that oxidative stress reduced cell proliferation, among others by inhibiting the passage of cells from the G0 to the G1 phase, slowing the S phase by inhibiting DNA synthesis or by cell cycle checkpoints arrest. Moreover, ROS interact with cellular macromolecules (nucleic acids, proteins, lipids), damage their structure and biological functions leading to cell death (Conklin, 2004). In general, cancer cells have the basal level of ROS higher than normal cells. For this reason, their tolerance of the increase ROS level is smaller than in normal cells (Tang et al., 2019). Among anticancer agents there are many drugs which act through ROS-dependent pathway. It was confirmed that some of cytotoxic antibiotics (doxorubicin, mitomycin C, mitoxantrone, actinomycin D), alkylating agents (nimustine), antimetabolites (gemcitabine, mercaptopurine) and antimitotic agents (paclitaxel, vinblastine, vinorelbine) were able to induce oxidative stress (Yokoyama et al., 2017). The most abundant and well-known non-enzymatic antioxidant is glutathione (GSH). The molecule is considered to be essential for redox homeostasis and cell survival (Gorrini et al., 2013). GSH represents reduced form of thiols which can be oxidized to its corresponding disulfide (GSSG), reducing simultaneously reactive oxygen species (Baba and Bhatnagar, 2018). The results obtained in the study show that doxycycline significantly decreases the level of reduced thiols. The observed changes are dependent on the drug concentration as well as the treatment time. The drug depreciated the percentage of melanoma cells with high level of reduced thiol close to 50%. The finding suggests that one of mechanisms of doxycycline antimelanoma effect is generation of oxidative stress and redox imbalance. Moreover, in the earlier study we demonstrated that doxycycline unexposed to UVA radiation did not influence the activity of antioxidant enzymes (SOD, CAT, GPx) in normal melanocytes, even at the toxic concentration (Rok et al., 2015). It lets us presume that doxycycline does not induce oxidative stress in normal melanocytes, thus its influence on melanoma cells redox balance can be considered as selective. Glutathione, besides its antioxidant activity, influences other molecular and cellular processes including apoptotic signaling pathways (Circu and Aw, 2012). Moreover, it is worth noticing that (GSH) depletion is a common feature of cell apoptosis triggered by death receptors-dependent mechanisms, oxidative stress or and cytotoxic drugs (Franco and Cidlowski, 2009). In reference to the obtained results, the influence of doxycycline on apoptosis process in melanoma cells was examined. The analysis of apoptosis involved DNA fragmentation as well as the measurements of mitochondrial potential and annexin V. One of admitted biochemical hallmarks of apoptosis is DNA fragmentation – a process of DNA cleavage into oligonucleosome-sized fragments. The process is controlled by activation of the apoptosisspecific endonucleases and provides the removal of DNA and the timely completion of apoptosis (Zhang and Xu, 2002). Signal pathways leading to DNA fragmentation, are mediated mainly by the family of cysteinyl aspartate-specific protease caspases (Kitazumi and Tsukahara, 2011). Beyond caspases, DNA fragmentation may occur in a caspase-independent manner. In this case, the process is caused by proapoptotic proteins, released from the mitochondria: apoptosis inducing factor AIF and endonuclease G (Elmore, 2007). Several earlier conducted studies indicated that cytotoxicity of doxycycline was connected to the induction of DNA fragmentation. The phenomenon was confirmed during experiments on leukemic cells HL60 and K562 as well as pancreatic cancer cells PANC-1and MDAPanc-28 (Song et al., 2014; Fares et al., 2015; Son et al., 2009; Fujioka et al., 2004). The results obtained in our study show that doxycycline induces DNA fragmentation in the melanoma cells COLO 829. The largest
cytotoxic effect was observed from the concentration of 50 μM. Doxycycline in this concentration stopped cells divisions and reduced cells viability by ca. 58% already after 24 h. The calculated EC50 values were 74.35 μM, 32.28 μM and 16.25 μM for 24 h, 48 h and 72 h-long incubation, respectively. Previously conducted studies on the melanoma cell line COLO 829 showed that dacarbazine – one of the antimelanoma reference drug and sulindac in the concentration of 50 μM decreased cells viability after a 24-h incubation by 42.3% and 46.3%, respectively. Interestingly, even doubling of the tested drugs concentration had no significant effect. The observed reduction of cells viability at concentration 100 μM was 47.3% and 46.3%, respectively (Miliński et al., 2017). It proves doxycycline is more cytotoxic and shows better antimelanoma effect then dacarbazine and sulindac. Additionally, the EC50 values for doxycycline are lower than obtained in other studies for fluoroquinolone derivatives: lomefloxacin (EC50 24h = 510 μM, EC50 48h = 330 μM, EC50 72h = 250 μM), ciprofloxacin (EC50 24h = 740 μM, EC50 48h = 170 μM, EC50 72h = 100 μM) and moxifloxacin (EC50 24h = 400 μM, EC50 48h = 220 μM, EC50 72h = 150 μM) (Bebrok et al., 2017; Bebrok et al., 2018; Beberok et al., 2019). The studies of anticancer activity of doxycycline were also conducted using another cancer cell lines. In case of breast and lung cancer cells, the obtained values of EC50 are lower than calculated for COLO 829. It was stated that doxycycline decreased viability of breast cancer cells MCF7 and MDA-MB-468 by 50% after 72-h incubation at concentration of 11.39 μM and 7.13 μM, respectively (Zhang et al., 2017). In turn, the analysis of lung cancer cells showed the EC50 value after 48 h incubation is 1.70 μM and 1.06 μM for NCI-H446 and A549, respectively (Qin et al., 2015). Presented results indicate different sensitivity of particular call line to the cytotoxic effect of doxycycline. Due to promising results obtained in the first part of the study, the evaluation of cell cycle, thiols level, DNA fragmentation as well as the analysis of mitochondrial potential and annexin V was made. Doxycycline was used in subsequent studies in concentrations of 50 μM and 100 μM. The cell cycle is the series of following events which leads to doubling of cellular components and to segregating each cell into daughter cells. The individual phases of cell cycle are marked with symbols connected with their roles: the resting state (quiescence) – G0, DNA replication (DNA synthesis) – S-phase; two gap phases (the growth of cells) separate S phase and mitosis are known as G1 and G2; chromosomes segregation and cell division (mitosis) – M-phase (Casimiro et al., 2012; Barnum and O'Connell, 2014). Normal cells undergo the cycle in response to mitogenic signals, whereas cancer cells proliferate without growth stimuli in an unregulated way. That is why the cancer is often considered a disease of the cell cycle (Diaz-Moralli et al., 2013). Deregulation of the cell cycle and cell division underlies malignant transformation of normal cells into tumor cells. Molecular mechanisms, including a system of cyclin-dependent kinases (CDKs), which regulate these processes, have become a target for potential anticancer drugs. In the case of human melanoma, disruptions of CDK1, CDK2 and CDK4 were stated (Otto and Sicinski, 2017; Bai et al., 2017). Basing on the obtained results, it can be stated that doxycycline doesn't work in phase-specific manner on melanoma cells. The observed changes differ depending both on a concentration, and on the time of incubation. It seems that the most significant variations are the decrease the percentage of cells in the G2/M phase, and the increase the number of cells in the sub-G1 phase at the concentration of 100 μM. The influence of doxycycline on a cell cycle profile was also examined on another cancer cell lines. The analysis of prostate adenocarcinoma cells PC-3 showed that doxycycline alone as well as in combination with dacarbazine significantly increased the percentage of cells in G2/M phase after 72 h treatment (Zhu et al., 2017). On the other hand, the arrest of the cell cycle in the G1/G0 phase was noticed in the case of following cancer cell lines: epithelioid carcinoma of the pancreas – PANC-1 (Son et al., 2009), breast adenocarcinoma – MDA-MB-231 9
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tested concentrations (50 μM ̴ 22 μg/ml; 100 μM ̴ 44 μg/ml) are higher than serum concentrations observed during a standard antibacterial therapy. Nowadays, the maximum recommended dose of doxycycline in serious infections is 300 mg daily and the usual daily dosage is 200 mg. (Holmes and Charles, 2009). Pharmacokinetic data for doxycycline indicates that a single dose of 200 mg achieves peak serum concentrations of approximately 2.6–5.9 μg/ml after oral administration and approximately 9.3 μg/ml for intravenous route. It was also measured that serum Cmax for a dose of 500 mg p.o. is 15.3 μg/ml (Agwuh and MacGowan, 2006). However, it is worth emphasizing that the concentrations used in our study reflect efficacy of short-time and single-dose therapy. Moreover, doxycycline has some properties which can be preferred in the melanoma treatment. Among others, the drug is lipophilic and widely distributed, including the skin, where it is accumulated (Valentin et al., 2009). The clinical trial shows the accumulation ratio in the skin after 2 weeks therapy with a relatively low dosage - 40 mg/day of doxycycline ranges from 1.8 to 2.7, depends on dietary conditions (Pal et al., 2018). The possibility of doxycycline to form complexes with melanin allows to draw conclusions that the accumulation ratio in highly pigmented cells, like melanoma, would be much higher. In conclusion, it was stated for the first time that doxycycline decreased the viability and inhibited the proliferation of human melanoma cells, proportionally to the drug concentration and the treatment time. The obtained results indicated that doxycycline disturbed the homeostasis of the tested cells. The drug lowered cells vitality by decreasing the intracellular level of reduced thiols as well as changed the cell cycle profile. In addition, we demonstrated that doxycycline triggered the DNA fragmentation during the treatment. The melanoma cells exposed to the drug were characterized also by the lowered mitochondrial membrane potential, which indicated cells apoptosis. Finally, doxycycline appeared to be the inductor of phosphatidylserine externalization – well known hallmark of apoptosis, which was confirmed by results of annexin V test. The conducted study contributes to the increase of knowledge about nonantibacterial action of doxycycline, including the influence on human cancer cells. It is worth emphasizing there is a need of successive studies on the antimelanoma effect of doxycycline taking into account a long-time therapy, the process of accumulation in the skin or a possibility of topical application. However, in relation to not satisfied results of malignant melanoma treatment, it brings new advice on searching for a more effective therapy.
increase was noticed for doxycycline in the concentration of 100 μM after 48 h and 72 h treatment. In this case, the percentage of cells with fragmented DNA was about 35%. In turn, the tested drug in half of the concentration elevated the cells population with fragmented DNA only up to 15%. For this reason, the next analyses were made only for the higher concentration of doxycycline. Mitochondria are one of the possible target for doxycycline in relation to its anticancer activity. The mechanism of the action involves, among others, an inhibition of mitochondrial protein synthesis and a decrease of the mitochondrial energy-generating capacity. The study with cultures of the human cancer cells A549, COLO357 and HT29 showed that doxycycline, more than a chemically modified tetracycline COL-3, specifically affects mitochondrially encoded proteins (Protasoni et al., 2018). Mitochondrial dysfunction through decreasing mitochondrial membrane potential (MMP), mitochondrial respiration and altering the energy production, was also stated in human glioblastoma cell lines A172 and U87, treated with doxycycline (Tan et al., 2017). In the study on the melanoma cell line COLO 829 doxycycline augmented the percentage of cells with decreased mitochondrial membrane potential by about 36% and 58% after 24 h and 48 h treatment, respectively. The finding is another evidence to suggest that doxycycline triggers melanoma cells apoptosis. Decreased MMP is not only one of the apoptosis hallmarks, but is also required to release from the mitochondria to the cytoplasm apoptogenic factors: cytochrome c and AIF (Gottlieb et al., 2003; Ly et al., 2003). Another characteristic molecular feature of apoptosis is the change of phospholipids localization in the plasma membrane. The distribution of phospholipids is asymmetric in healthy cells. The outer leaflet mostly contains electrically neutral phosphatidylcholine and sphyngomielin, whereas phosphatidylethanolamine as well as anionic phosphatidylserine and phosphatidic acid are located in the cytosolic layer (Demchenko, 2012). In an apoptotic cell, the enzyme γ-scramblase is activated resulting in phosphatidylserine flipping to the outer leaflet of the plasma membrane (Yang et al., 2012). In general, the loss of the asymmetric distribution makes apoptotic cells more detectable for removing that cells macrophages (Wlodkowic et al., 2012). Moreover, the appearance of phosphatidylserine in the outer membrane layer, considered as a near-universal event in apoptosis, has become the way for detection of this kind of cell death. The fluorochrome-conjugated annexin V, a natural human anticoagulant protein, is currently the most frequently used a marker of apoptosis. The method of apoptosis detection is based on relatively high affinity (Kd ̴ 10−7–10−8 M) of annexin V to negatively charged phosphatidylserine (Demchenko, 2012; Wlodkowic et al., 2012). Earlier conducted studies showed that doxycycline increased the number of annexin V-positive cells after 72-h treatment in population of breast cancer cells MCF7 ( ̴ 10% of population) and MDA-MB-468 (̴ 45% of population) as well as prostate cancer cells PC3 ( ̴ 12% of population) (Zhang et al., 2017; Zhu et al., 2017). According to the study on melanoma cells COLO 829, doxycycline induced apoptosis in most treated cells. The percentage of early and late apoptotic cells was about 60% and 35% after 24 h and 22% and 60% after 48 h, respectively. The results show and confirm relatively quickly and clearly noticeable induction of apoptosis in investigated melanoma cells by the tested drug. They also suggest that melanoma cells can be more sensitive to doxycycline then other cancer cells. The performed in vitro studies show doxycycline anticancer activity in treatment of human melanoma cells. The influence of drug on COLO 829 cells depends on its concentration. The action of doxycycline in the concentration of 50 μM includes generally cytotoxic, antiproliferative and oxidative stress-inducing effects. In turn, the concentration 100 μM causes DNA fragmentation, disruption of mitochondrial membrane potential and triggers cells apoptosis, additionally. The observed effects rise the question about possibility of doxycycline usage in the melanoma treatment in vivo. The problem of directly transferring conditions in vitro to in vivo is complex and requires consideration of various factors. Taking into account literature data, it can be stated that the
Declaration of Competing Interest The authors: Jakub Rok, Marta Karkoszka, Zuzanna Rzepka, Michalina Respondek, Klaudia Banach, Artur Beberok and Dorota Wrześniok declare that there are no conflicts of interest. The manuscript entitled: “Cytotoxic and proapoptotic effect of doxycycline – an in vitro study on the human skin melanoma cells”, which all the listed authors have read and approved of, has not been submitted elsewhere for publication, in whole or in part, and is permitted to be published in this journal. Acknowledgment This work was financially supported by the Medical University of Silesia in Katowice (Grants No. KNW-2-O26/N/9/K and KNW-1-037/K/ 9/O). References Agwuh, K.N., MacGowan, A., 2006. Pharmacokinetics and pharmacodynamics of the tetracyclines including glycylcyclines. J. Antimicrob. Chemother. 58, 256–265. Ali, I., Alfarouk, K.O., Reshkin, S.J., Ibrahim, M.E., 2017. Anticancer agents med. Chem. 17, 1617–1623.
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