International Journal of
Molecular Sciences Article
Lomefloxacin Induces Oxidative Stress and Apoptosis in COLO829 Melanoma Cells Artur Beberok *, Dorota Wrze´sniok, Martyna Szlachta, Jakub Rok, Zuzanna Rzepka, Michalina Respondek and Ewa Buszman Department of Pharmaceutical Chemistry, School of Pharmacy with the Division of Laboratory Medicine, Medical University of Silesia, Jagiellonska ´ 4, 41-200 Sosnowiec, Poland;
[email protected] (D.W.);
[email protected] (M.S.);
[email protected] (J.R.);
[email protected] (Z.R.);
[email protected] (M.R.);
[email protected] (E.B.) * Correspondence:
[email protected]; Tel./Fax: +48-32-364-1611 Received: 27 August 2017; Accepted: 16 October 2017; Published: 20 October 2017
Abstract: Although some fluoroquinolones have been found to exert anti-tumor activity, studies on the effect of these drugs on melanoma cells are relatively rare. The aim of this study was to examine the effect of lomefloxacin on cell viability, reactive oxygen species production, redox balance, cell cycle distribution, DNA fragmentation, and apoptosis in COLO829 melanoma cells. Lomefloxacin decreases the cell viability in a dose- and time-dependent manner. For COLO829 cells treated with the drug for 24, 48, and 72 h, the values of IC50 were found to be 0.51, 0.33, and 0.25 mmol/L, respectively. The analyzed drug also altered the redox signaling pathways, as shown by intracellular reactive oxygen species overproduction and endogeneous glutathione depletion. After lomefloxacin treatment, the cells were arrested in S- and G2/M-phase, suggesting a mechanism related to topoisomerase II inhibition. DNA fragmentation was observed when the cells were exposed to increasing lomefloxacin concentrations and a prolongation of incubation time. Moreover, it was demonstrated that the drug induced mitochondrial membrane breakdown as an early hallmark of apoptosis. The obtained results provide a strong molecular basis for the pharmacologic effect underlying the potential use of lomefloxacin as a valuable agent for the treatment of melanoma in vivo. Keywords: lomefloxacin; melanoma; oxidative stress; DNA fragmentation; apoptosis
1. Introduction Melanoma, the most deadly and aggressive form of skin cancer, derives from pigment-producing cells, namely, melanocytes [1]. These cells are characterized by pro-survival mechanisms that counteract damage-causing factors, including UV radiation [2]. The complex mechanisms of resisting cell death, already active in melanocytes, are further extended in melanoma cells, contributing to the melanoma pro-survival phenotype [1]. Throughout the years, the incidence of melanoma has continued to increase worldwide. According to the predictions of the American Cancer Society, about 90,000 new cases of melanoma will be diagnosed in 2017 [3]. Moreover, the statistics estimate a doubling of melanoma incidence every 10 to 20 years [4]. The prognosis for patients with advanced stages of cutaneous melanoma is poor, with a median survival time of less than one year [5] and a five year survival rate from 5 to 19% [4]. Until 2011, a chemotherapeutic alkylating agent, dacarbazine, was the standard drug for patients with metastatic melanoma, despite its modest efficacy and lack of documented survival benefit [6,7]. Recently, significant progress has been made towards melanoma treatment by approving monoclonal antibodies like ipilimumab, nivolumab, and prembrolizumab, as well as tyrosine kinases inhibitors like vemurafenib, dabrafenib, and trametinib [7–9]. However the persisting high toll of deaths resulting
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from melanoma, as well as a rapid increase in resistance to targeted drugs, is observed [6,7]. Therefore the search for novel chemotherapeutic agents for the treatment of melanoma is still needed. Lomefloxacin belongs to fluoroquinolones, a class of synthetic antibiotics that are widely used for the treatment of various infections [10]. Their mechanism of bactericidal action involves the inhibition of type II isomerases, namely, DNA gyrase (topoisomerase II) and topoisomerase IV, which areprocaryotic enzymes that are functional analogs of eukaryotic topoisomerase II [11]. By selectively cleaving, rearranging, and religating double helixes, type II topoisomerases are able to relax supercoiled DNA and catalyze the decatenation of interlinked DNA molecules [12]. Fluoroquinolones form stable topoisomerase-drug-DNA complexes and inhibit helix religation. The occurrence of double-stranded DNA breaks disturbs replication and transcription processes, leading to cell death [13,14]. Human topoisomerase II is well validated as a target for some classes of anti-cancer chemotherapeutic drugs, including anthracyclines (doxorubicin, epirubicin, daunorubicin, idarubicin, aclarubicin), mitoxantrone, epipodophyllotoxins (etoposide, teniposide), and amsacrine [15]. These agents act primarily by inhibiting the topoisomerase II-mediated religation of double-strand DNA breaks and thereby inducing apoptosis of cancer cells [15,16]. The clinical use of these antineoplastic drugs is limited by their sensitivity to P-glycoprotein-mediated efflux, as well as to high toxicity [11]. Therefore the search for novel human topoisomerase II inhibitors that avoid resistance due to P-glycoprotein expression is still reasonable. Cancer cells exhibit higher levels of oxidative stress compared to normal cells. Disproportional increases in intracellular reactive oxygen species (ROS) may selectively induce cancer cell death either through random damaging functions of ROS or by the specific induction of apoptosis via the mitochondrial pathway [17,18]. Several in vitro [19,20] and clinical [21] studies have revealed that fluoroquinolones may cause the depletion of antioxidant enzyme activity and therefore induce significant disruption of the cellular antioxidant status. Simultaneous topoisomerase II inhibition, apoptosis induction, and oxidative stress generation might be an effective strategy for inducing melanoma cell death. Therefore, we investigated, for the first time, the pro-apoptotic and pro-oxidant activity of lomefloxacin towards the melanotic COLO829 melanoma cell line. The aim of this study was to examine the effect of lomefloxacin on cell viability, reactive oxygen species production, redox balance, cell cycle distribution, DNA fragmentation, and apoptosis in COLO829 melanoma cells. In order to provide strong experimental evidence for the use of lomefloxacin as a potential drug for the treatment of melanoma, the obtained results were compared with previously received data for normal human melanocytes [22]. 2. Results 2.1. Lomefloxacin Decreases the Viability of COLO829 Cells To evaluate the effect of lomefloxacin on the viability of human COLO829 melanoma cells, a WST-1 (4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulphonate) assay was performed. As shown in Figure 1, the treatment of cells with lomefloxacin concentrations from 0.1 to 1.0 mmol/L for 24 h resulted in the decrease of cell viability by 12 to 68%. The cytotoxic response was more marked after a prolongation of incubation time up to 48 h or 72 h, wherein the viability of COLO829 cells treated with 0.1, 0.5, and 1.0 mmol/L lomefloxacin solutions decreased to 86%, 37%, and 12% or 81%, 28%, and 5%, respectively. After the incubation of the cells with lower drug concentrations (from 0.0001 to 0.05 mmol/L), the loss in cell viability was not statistically significant. For COLO829 cells treated with lomefloxacin for 24, 48, and 72 h, the values of IC50 (the concentration of a drug that produces a loss in cell viability of 50%) were found to be 0.51, 0.33, and 0.25 mmol/L, respectively.
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Figure 1. The effect of lomefloxacin on the viability of COLO829 melanoma cells. The cells were
Figure 1. The effect of lomefloxacin on the viability of COLO829 melanoma cells. The cells were treated treated with various lomefloxacin concentrations (0.0001–1.0 mmol/L) for 24, 48, and 72 h and with various lomefloxacin (0.0001–1.0 mmol/L) for 24, 48, and 72 h and examined by examined by the concentrations WST-1 (4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene the WST-1 (4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulphonate) assay. are expressed as percentages the controls. Mean values ±disulphonate) standard error Figure 1. The effect ofData lomefloxacin on the viability ofofCOLO829 melanoma cells. The cells wereassay. thewith meanvarious (SEM) from three independent experiments (n = 3) performed in triplicate are presented. Data treated areofexpressed as percentages of the controls. Mean values ± standard error of the mean (SEM) lomefloxacin concentrations (0.0001–1.0 mmol/L) for 24, 48, and 72 h and ** p < 0.005 versus control samples. from examined three independent (n = 3) performed in triplicate are presented. ** p < 0.005 versus by the experiments WST-1 (4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulphonate) control samples. assay. Data are expressed as percentages of the controls. Mean values ± standard error
2.2. Lomefloxacin Induces Morphological Changes in COLO829 Cells of the mean (SEM) from three independent experiments (n = 3) performed in triplicate are presented. ** pThe < 0.005 versus control samples. cells was estimated by the use of a light inverted microscope at morphology of COLO829 2.2. Lomefloxacin Induces Morphological Changes in COLO829 Cells 40× magnification. Figure 2 shows the morphological changes observed in COLO829 cells after 2.2.incubation Lomefloxacin Induces Morphological Changes in COLO829 Cells with lomefloxacin at cells a concentration of 1.0 mmol/L for 24, and 72 h. Whilemicroscope the The morphology of COLO829 was estimated by the use of 48, a light inverted untreated cells (Figure 2A,C,E) grew adherently in culture flasks and had regular shapes and sizes, at 40× magnification. Figure 2 showscells thewas morphological changes in COLO829 cells The morphology of COLO829 estimated by the use ofobserved a light inverted microscope at after the cells treated with lomefloxacin at a concentration of 1.0 mmol/L for 24, 48, and 72 h (Figure 2B,D,F) incubation with lomefloxacin at a concentration of 1.0 mmol/L for 24, 48, and 72 h. While the untreated 40× magnification. Figure 2 shows the morphological changes observed in COLO829 cells after became rounded and lost their regular shape and size. Moreover, a loss of cell to cell contact and a cells incubation (Figure 2A,C,E) grew adherently in culture flasks had for regular shapes and with lomefloxacin at a concentration of 1.0and mmol/L 24, 48, and 72 h. sizes, While the the cells decrease in cell number was observed. After 48 and 72 h of incubation with lomefloxacin (Figure untreated cells (Figure 2A,C,E) grew adherently in culture flasks and had regular shapes and sizes, treated with lomefloxacin at a concentration of 1.0 mmol/L for 24, 48, and 72 h (Figure 2B,D,F) 2D,F), most of the COLO829 melanoma cells were detached from their substratum, displaying thebecame thetypical cells withregular lomefloxacin at and a concentration mmol/L forcell 24, 48, and contact 72 h (Figure rounded andtreated lost their shape size. Moreover, a loss of to cell and2B,D,F) a decrease morphological changes observed during the of cell1.0 death process.
became rounded lost theirAfter regular Moreover, a with loss oflomefloxacin cell to cell contact and a2D,F), in cell number was and observed. 48shape and and 72 hsize. of incubation (Figure decrease in cell number was observed. After 48 and 72 h of incubation with lomefloxacin (Figure most of the COLO829 melanoma cells were detached from their substratum, displaying the typical 2D,F), most of the COLO829 melanoma cells were detached from their substratum, displaying the morphological changes observed during the cell death process. typical morphological changes observed during the cell death process.
Figure 2. Cont.
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Figure 2. 2. Lomefloxacin induces morphological changes inchanges COLO829 in melanoma cells: melanoma control COLO829 Figure Lomefloxacin induces morphological control COLO829 cells incubated for (A) 24 h, (C) 48 h, and (E) 72 h; cells COLO829 exposed to lomefloxacin at cells: cells incubated for (A) 24 h, (C) 48 h, and (E) 72 h; cells exposed to lomefloxacin at concentration control COLO829 cells incubated for(B) (A)2424 (C) 72The h; cells toalomefloxacin at a concentration of 1.0 mmol/L for h, h, (D) 4848 h, h, (F)and and (E) 72 h. cells exposed were observed under a of 1.0 mmol/L for (B) 24 h, (D) 48 h, (F) and 72 h. The cells were observed under a light a concentration 1.0 mmol/L formagnification (B) 24 h, (D)(scale 48 h,bar (F)250 and 72 h. The cells were observedinverted under a light invertedofmicroscope at 40× µm). microscope at 40 × magnification (scale bar 250 µm). light inverted microscope at 40× magnification (scale bar 250 µm). 2.3. Lomefloxacin Induces ROS Generation in COLO829 Cells
2.3. Induces ROS Generation in COLO829 Cells 2.3. Lomefloxacin Lomefloxacin Induces ROSwas Generation COLO829 H2DCFDA staining used toin detect ROSCells generation in COLO829 cells exposed to lomefloxacin treatment. As shown in Figure 3, the exposure COLO829 cells lomefloxacin leads H usedused to detect ROS generation inofCOLO829 cells to exposed to lomefloxacin H22DCFDA DCFDA staining stainingwas was to detect ROS generation in COLO829 cells exposed to to ROS overproduction in a concentration-dependent manner. The treatment of cells with treatment. As shown in Figure 3, the exposure of COLO829 cells to lomefloxacin leads to ROS lomefloxacin treatment. As shown in Figure 3, the exposure of COLO829 cells to lomefloxacin leads lomefloxacin at concenrations 0.1, 0.5, and 1.0 mmol/L for 24 h enhanced ROS production by 38%, overproduction in a concentration-dependent manner. Themanner. treatmentThe of cells with lomefloxacin at to ROS overproduction in a concentration-dependent treatment of cells with 93%, and 137%, respectively, in comparison to the untreated cells (controls). concenrations 0.1, 0.5, and 1.0 mmol/L for 24 enhanced by 38%, 93%, and lomefloxacin at concenrations 0.1, 0.5, and 1.0hmmol/L forROS 24 hproduction enhanced ROS production by137%, 38%, respectively, in comparison to in thecomparison untreated cells (controls). 93%, and 137%, respectively, to the untreated cells (controls).
Figure 3. Lomefloxacin induces reactive oxygen species (ROS) production in COLO829 melanoma cells. The cells were exposed to the drug in concentrations of 0.1, 0.5, and 1.0 mmol/L for 24 h. The data are expressed as percentages of the controls normalized to a number of living cells. Mean values ± SEM from three independent experiments (n = 3) performed in triplicate are presented. ** p < 0.005 Figure Lomefloxacin induces reactive oxygen species (ROS) production in COLO829 melanoma versus control samples. Figure 3.3.Lomefloxacin induces reactive oxygen species (ROS) production in COLO829 melanoma cells.
cells. Thewere cellsexposed were exposed to the in concentrations of and 0.1, 1.0 0.5,mmol/L and 1.0 mmol/L h. The The cells to the drug in drug concentrations of 0.1, 0.5, for 24 h. for The24 data are 2.4. Lomefloxacin Decreases the Level of Cellular Reduced Glutathione (GSH) data are expressed as percentages of the controls normalized to a number of living cells. Mean values expressed as percentages of the controls normalized to a number of living cells. Mean values ± SEM ± SEM from three experiments (nearly = 3) performed triplicate are presented. p versus < 0.005 A decrease inindependent the cellular GSH level is an sign thein progression of cell death in**response from three independent experiments (n = 3) performed inof triplicate are presented. ** p < 0.005 versus control samples. to different pro-apoptotic agents. There is a strong correlation between cellular GSH depletion and control samples. the progression of apoptosis [23]. This phenomenon seems to be attributed mainly by direct GSH 2.4. Lomefloxacin Lomefloxacin Decreases the Level ofCellular Cellular Reduced Glutathione(GSH) (GSH) oxidation promoted bythe ROS. As of shown in Figure 4, Glutathione lomefloxacin caused a cellular decrease in the 2.4. Decreases Level Reduced level of glutathione in its reduced state. Following image cytometric analyses after the exposure of A decrease decrease in in thecellular cellular GSH GSH level level is is an an early early sign sign of of the the progression progressionof of cell cell death death in in response response A COLO829 cells the to lomefloxacin in concentrations of 0.1 and 1.0 mmol/L for 24 h, the percentage of PI to different different pro-apoptotic pro-apoptotic agents. agents. There There is is aa strong strongcorrelation correlation between between cellular cellular GSH GSH depletion depletion and and to (propidium iodide) negative cells with low vitality (with reduced GSH levels) increased from 5 to 11 the progression of apoptosis [23]. This phenomenon seems to be attributed mainly by direct GSH and 13%, respectively. The [23]. response was more markedseems after the of mainly the incubation timeGSH the progression of apoptosis This phenomenon toprolongation be attributed by direct oxidation promoted ROS. in Figure 4, mmol/L, lomefloxacin caused aofcellular decrease the up topromoted 48 h; for lomefloxacin at ashown concentration of 0.1 the percentage cells with reduced oxidation bybyROS. AsAs shown in Figure 4, lomefloxacin caused a cellular decrease in thein level level of glutathione in its reduced image the cytometric analyses after the exposure of GSH levels increased from tostate. 42%.Following Simultaneously, treatment of the COLO829 cells with of glutathione in its reduced state.7 Following image cytometric analyses after exposure of COLO829
COLO829 cells to lomefloxacin in concentrations 0.1 and 1.0 24 h, theof percentage of PI cells to lomefloxacin in concentrations of 0.1 and 1.0ofmmol/L for mmol/L 24 h, the for percentage PI (propidium (propidium iodide) negative cells with low vitality (with reduced GSH levels) increased from 5 to 11 iodide) negative cells with low vitality (with reduced GSH levels) increased from 5 to 11 and 13%, and 13%, respectively. The response more after marked the prolongation of the incubation time respectively. The response was morewas marked theafter prolongation of the incubation time up to up to 48 h; for lomefloxacin at a concentration of 0.1 mmol/L, the percentage of cells with reduced GSH levels increased from 7 to 42%. Simultaneously, the treatment of COLO829 cells with
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at a concentration of 0.1 mmol/L, the percentage of cells with reduced5GSH of 17 levels increased from 7 to 42%. Simultaneously, the treatment of COLO829 cells with lomefloxacin lomefloxacin in concentrations 0.1 and for 1.0 24 mmol/L 24 and 48the h increased theofpercentage PI in concentrations of 0.1 and 1.0of mmol/L and 48for h increased percentage PI positiveofcells positive cellsfrom (dead cells) to 31% from 3 to 28%, respectively. (dead cells) 6 to 31%from and 6from 3 toand 28%, respectively.
Figure 4. Analysis of the cellular Cellular Reduced Glutathione (GSH) levels in COLO829 cells after Figure 4. Analysis of the cellular Cellular Reduced Glutathione (GSH) levels in COLO829 cells after exposure to lomefloxacin treatment. (A) Histograms presenting the changes of GSH levels in cells exposure to lomefloxacin treatment. (A) Histograms presenting the changes of GSH levels in cells exposed to lomefloxacin in concentrations of 0.1 and 1.0 mmol/L. The presented histograms are exposed to lomefloxacin in concentrations of 0.1 and 1.0 mmol/L. The presented histograms are representative of three independent experiments with similar results. Q1ur are dead cells; Q1lr are representative of three independent experiments with similar results. Q1ur are dead cells; Q1lr are cells with low GSH levels (with low vitality). (B) The effect of lomefloxacin on cellular GSH levels in cells with low GSH levels (with low vitality). (B) The effect of lomefloxacin on cellular GSH levels in COLO829 COLO829cells. cells.The Thecells cellswere weretreated treated with with lomefloxacin lomefloxacin in in concentrations concentrations of of 0.1 0.1 and and 1.0 1.0 mmol/L mmol/Lfor for 24 and 48 h. Mean values ± SEM from three independent experiments (n = 3) performed in triplicate 24 and 48 h. Mean values ± SEM from three independent experiments (n = 3) performed in triplicate are arepresented. presented.****pp
