e n v i r o n m e n t a l t o x i c o l o g y a n d p h a r m a c o l o g y 3 6 ( 2 0 1 3 ) 600–610
Available online at www.sciencedirect.com
j o ur nal ho me pa ge: www.elsev ier .com/ locate /e tap
Activation of apoptotic pathway in normal, cancerovarian cells by epothilone B
Aneta Rogalska ∗, Ewa Szula, Arkadiusz Gajek, Agnieszka Marczak,Zofia JózwiakDepartment of Thermobiology, Institute of Biophysics, Faculty of Biology and Environmental Protection, University ofLodz, Pomorska 141/143, 90-236 Lodz, Poland
a r t i c l e i n f o
Article history:
Received 16 April 2013
Received in revised form
10 June 2013
Accepted 11 June 2013
Available online 19 June 2013
Keywords:
a b s t r a c t
The epothilones, a new class of microtubule-targeting agents, seem to be a very promising
alternative to the current strategy of cancer treatment. We have analyzed the aspects of
epothilone B (Epo B) on cellular metabolism of tumor (OV-90) and normal (MM 14) ovarian
cells. The observed effects were compared with those of paclitaxel (PTX), which is now a
standard for the treatment of ovarian cancer.
The results provide direct evidence that Epo B is considerably more cytotoxic to human
OV-90 ovarian cancer cells than PTX. We have found, that antitumor efficacy of this new
drug is related to its apoptosis-inducing ability, which was confirmed during measurements
Epothilone B
Human ovarian cell lines
Paclitaxel
Apoptosis
typical markers of the process. Epo B induced changes in morphology of cells, mitochondrial
membrane potential and cytochrome c release. Also a slight increase of the intracellular
calcium level was observed. Moreover, we have found that ROS production, stimulated by
Epo B, is directly involved in the induction of apoptosis via mitochondrial pathway.
on tubulin � as paclitaxel. They share one polar place at
1. Introduction
Ovarian cancer is the leading cause of death among gyne-cologic cancers in the world. Standard first line therapy inthe treatment of this kind of cancer includes microtubule-stabilizing agents (MSAs) like PTX or docetaxel (Pellicciottaet al., 2011). PTX has been established as one of the mostactive antineoplastic agents against a wide spectrum ofmalignancies: lung, ovarian and breast cancer. A macrocyclic
polyketide class of compounds has generated substantialinterest over the last few years in the areas of chem-istry, biology and medicine due to the strong inhibitory
effect on the growth of numerous cancer cells (Wang et al.,2009). Recently, several novel natural cytotoxic microtubule-stabilizing compounds have been described, including theepothilones produced by the myxobacteria Sorangium cellu-losum (Lee and Swain, 2008). Epo B and PTX stabilize themicrotubules leading to mitotic arrest and subsequent celldeath. Biological studies have shown that the epothilones sta-bilize microtubules in the same way as paclitaxel, but with aslightly higher efficiency. Epothilones bind to the same site
C7-OH, whereas the thiazole side chain is associated with adifferent region tubulin � than the taxanes (Goodin, 2008).For this reasons Epo B competitively inhibits the binding of
on 96-well plates. After 24 h the drugs were added and thecells were incubated for 72 h. At the end of incubation, themedium was removed. After two washes with PBS, 50 �l ofMTT (at a final concentration of 0.5 mg/ml) was added to the
e n v i r o n m e n t a l t o x i c o l o g y a n d
TX to the microtubules (Fojo and Menefee, 2007). Moreover,pothilones and taxanes have non-overlapping mechanismsf resistance; in particular, while overexpression of class III �-ubulin plays a major role in taxane resistance, epothilonesisplay their highest efficacy in class III �-tubulin overex-ressing malignancies (Ferrandina et al., 2012; Magnani et al.,006).
However, the biochemical mechanisms that underlinepothilone-induced cancer cell growth inhibition are notell understood. Successful treatment with chemotherapeu-
ic agents is largely dependent on their ability to trigger celleath in tumor cells and activation of apoptosis (Fernandezt al., 2010). The apoptotic cascade can be initiated by theelease from the mitochondria and cytosolic accumulationf cytochrome c. Caspases, the final executioners of apo-tosis, are activated causing degradation of cellular proteinsnd disassembly of the cell, leading to typical morpho-ogical changes such as chromatin condensation, nuclearhrinkage and the formation of apoptotic bodies (Rogalskat al., 2008). Epo B has been described as triggering apopto-is in several cell lines, as established by changes in cellembrane integrity, DNA fragmentation and morphologi-
al abnormalities, but the molecular pathways underlyinghis process are poorly described (Pellicciotta et al., 2013). Itas demonstrated that ROS and Ca2+ may act as common
ignals to regulate apoptosis in many cell types (Hamm-lvarez and Cadenas, 2008; Kania et al., 2007; Wang et al.,003). The outcome of the cellular response appears toe dependent on the type and dose of chemotherapeutictress within the cellular context. Despite the activationf apoptotic pathways by many anticancer drugs, recentvidence suggests that there are forms of chemotherapy-nduced cell death that cannot be readily classified aspoptosis or necrosis (Elmore, 2007; Igney and Krammer,002).
The aim of our study is to explore the molecular mech-nisms of the cell death induced by Epo B, the promisingicrotubule-interacting agent. Results were compared with
he well-known, widely used compound, PTX. Althoughpothilones have been described as inducing several mark-rs of apoptosis their essential mechanism of action is notlear (Bystricky and Chau, 2011; Winsel et al., 2011). In ouresearch, two models of cell lines (mouse normal and humanvarian cell line) were used. There are differences in sensi-ivity to drugs between the species. Experiments with animalell lines do not always faithfully predict or model what isbserved in human cells. However, in the literature available,here are no data on the effect of Epo B on normal cells of thevary and many studies about the mechanism of taxanes orpothilones action in vivo and in vitro are usually done on thenimal like mice, rats, dogs and monkeys (Chen et al., 2013;itzgerald et al., 2012; Hammer et al., 2010; Hennenfent andovindan, 2006; Horio et al., 2012; Jeong et al., 2013; O’Reillyt al., 2011).
Ovarian cancer patients frequently relapse after first-ine treatment based on platinum–taxane doublets. Hence,pothilones might represent a therapeutic alternative in thisetting. Epo B has undergone parallel clinical development butts future role in ovarian cancer therapy remains to be definedDiaz-Padilla and Oza, 2011).
m a c o l o g y 3 6 ( 2 0 1 3 ) 600–610 601
2. Materials and methods
2.1. Chemicals
PTX was obtained from Sequoia Research Products(Pangbourne, United Kingdom). Epo B, 2′,7′-dichlorodihydrofluorescein diacetate (DCFH2-DA),5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethyl-benzimidazol-carbocyanine iodide (JC-1), and trypsin-EDTA were purchasedfrom Sigma (St. Louis, USA). Fluo-4 NW Calcium AssayKits was obtained from Molecular Probes (Eugene, USA).Cytochrome c kit was purchased from Invitrogen (Camarillo,USA). Medium (RPMI 1640) and fetal bovine serum (FBS) weresupplied by Cambrex (Basel, Switzerland). All other chemicalsand solvents were of high analytical grade and were obtainedfrom Sigma (St. Louis, USA).
2.2. Ovarian cells culture
MM14 (mouse normal ovarian cell line) and OV-90 (human ovar-ian papillary serous adenocarcinoma cell line) were obtained fromAmerican Type Culture Collection (Rockville, MD, USA). MM14cells were grown as a monolayer with Dulbecco’s ModifiedEagle’s Medium (DMEM), supplemented with 10% fetal bovineserum, penicillin (10 U/ml) and streptomycin (50 �g/ml) instandard conditions: 37 ◦C, 100% humidity, the atmospherebeing 5% CO2 and 95% normal air. OV-90 cells were also grownas a monolayer with a special medium, which consisted of199 and MCDB medium, supplemented with 15% fetal bovineserum, penicillin (10 U/ml) and streptomycin (50 �g/ml). Thecells were routinely screened for Mycoplasma contamination.
2.3. Cells culture
Cells were plated into 96-well microplates at a density of25 × 103 cells per well for MTT, ROS, calcium assay and mito-chondrial potential measurements. The Petri dishes were usedfor double staining Hoechst 33258/PI (3 × 105 cells/dish) andfor cytochrome c assay (3 × 106 cells/dish). After 24 h (timenecessary to ensure that the cells were in the exponentialgrowth phase) different concentrations of drugs (Epo B or PTX)(depending on the assessment method) were added and thecells were incubated with the drugs in a CO2 incubator for dif-ferent periods of time (2–48 h). In all experiments, some of thesamples were preincubated with an antioxidant (3 mM NAC-concentration evaluated experimentally) for 1 h, and then thedrugs were added at appropriate concentrations and incuba-tion was continued for the required period of time under thesame conditions.
2.4. Cytotoxicity assay
The cytotoxicity of the drugs to ovarian cells was estimatedby the standard microplate MTT colorimetric method. Log-arithmically growing cells (25 × 103 cells/well) were seeded
602 e n v i r o n m e n t a l t o x i c o l o g y a n
cells. Subsequently, the microplates were incubated for 4 hat 37 ◦C. The medium in each well was aspirated. Then theviolet formazan crystals that formed as a result of MTT reduc-tion within metabolically viable cells were dissolved with100 �l of DMSO per well. Absorbance was measured at 570 nmwith a microplate reader (Awareness Technology Inc., USA)(Carmichael et al., 1987a,b). On the basis of the MTT assay, theIC50 parameter was determined by dividing gained absorbancevalues of the drug-treated samples by those of control cells.
2.5. ROS production
The level of ROS was measured by the DCFH2-DA assay.The probe is a membrane permeable molecule that is enzy-matically hydrolyzed by intracellular esterases to DCFH2.Upon oxidation, it yields highly fluorescent dichlorofluores-cein (DCF) (Halliwell and Whiteman, 2004; Hempel et al.,1999). Intracellular ROS production was determined directly incell monolayers in black 96-well flat-bottom microtiter platesusing a Fluoroskan Ascent FL microplate reader (Labsystems,Sweden). Cells in complete medium were incubated with IC50
concentrations of Epo B or PTX (designated respectively byusing MTT assay) for 2–48 h in the presence or absence of anantioxidant. To determine the production of ROS, cells weretreated with 5 �M of DCFH2-DA at 37 ◦C for 30 min, and thefluorescence of DCF was measured at 530 nm after excitationat 485 nm. Assays were performed in Hank’s buffered salt solu-tion (HBSS).
2.6. Mitochondrial membrane potential
Ovarian cells were seeded into black 96-well titrationmicroplates. After 24 h with the drugs at IC50 concentrationsor chlorophenylhydrazone (CCCP), an uncoupling mitochon-drial agent at various concentrations (0.01–10 �M) was added.The cells were incubated with the drugs or CCCP for 2, 4,24, and 48 h. At the end of treatment, the medium wasremoved and the cells were incubated in total darknesswith 5 �M JC-1 in HBSS for 30 min at 37 ◦C. JC-1 has theproperty of spontaneously forming red-fluorescent dimersunder high mitochondrial potential, whereas its monomericform, prevalent in cells with low ��m, shows green fluo-rescence. Thus, changes in the red/green fluorescence ratioreflect the variation of ��m. The fluorescence of both JC-1monomers and dimers was measured on a Fluoroskan AscentFL microplate reader (Labsystems, Sweden) using filter pairsof �ex530 nm/�em590 nm (dimers) and �ex485 nm/�em538 nm(monomers). The results given in the figures are shown as aratio of dimer to monomer fluorescence in relation to the con-trol fluorescence ratio, taken as 100%. The cells presented inthe images were incubated with IC50 concentrations of Epo Bor PTX for 24 h. JC-1 fluorescence was photographed imme-diately after drug treatment with an inverted Olympus IX70fluorescence microscope.
2.7. Monitoring of apoptosis and necrosis
The appearance of apoptotic and necrotic cells was moni-tored by double staining with Hoechst 33258 and PI usinga fluorescence microscopy Olympus IX70, Japan. Cells were
a r m a c o l o g y 3 6 ( 2 0 1 3 ) 600–610
cultured with IC50 concentrations of the drugs for 2–48 h. Atcertain time points, cells were removed from culture dishesby trypsinization, centrifuged and suspended in PBS at afinal concentration of 1 × 106 cells/ml. 1 �l of Hoechst 33258(0.13 mM) and 1 �l of PI (0.23 mM) were added to 100 �l of cellsuspension. The cells were then incubated at room temper-ature for 10 min in total darkness (Gasiorowski et al., 2001)then dropped onto microscopic slides and examined. The cellswere classified on the basis of their morphological and stain-ing characteristics as: live (blue fluorescence), early apoptoticcells (intensive bright blue fluorescence), late apoptotic cells(blue-violet fluorescence) and necrotic (red fluorescence). Rep-resentative areas of cells stained at 48 h of incubation werechosen for photographic documentation.
2.8. Intracellular calcium assay
Intracellular calcium level was determined using the fluores-cent probe Fluo-4-NW, which crosses the plasma membrane ofliving cells. After entering the cell, Fluo-4-NW is converted bycytosolic hydrolases to the active form, having the possibilityof binding calcium ions. As a result of joining, the calcium ionsprobe emits fluorescence (�em = 538 nm) after excitation witha light of wavelength (�ex = 485 nm). The cells were plated into96-well black plates (25 × 104 cells/well) and then treated withEpo B and PTX at IC50 concentration. Incubations were car-ried out with drugs for 2–48 h. Then the medium was removedand the cells were washed with PBS. Finally the dye loadingsolution (Fluo-4-NW, probenecid, HBSS buffer, 20 mM HEPES)was added in a volume of 100 �l per well. Measurements weremade using a fluorescence plate reader (Fluoroskan Ascent FL.,Labsystem).
2.9. Measurements the level of intracellularcytochrome c
The generation of cytochrome c was measured according tothe manufacturer’s protocol. The cells were plated (3 × 106) in5 ml of culture medium into 100 mm Petri dishes. After 24 h,drugs were added at IC50 concentration and the cells wereincubated for 4 and 48 h. After drug treatment, the cells werewashed with PBS and re-suspended in ice cold cytosol extrac-tion buffer containing PMSF (phenylmethanesulfonylfluoride)and protease inhibitor coctail. Cell lysate was centrifugedat 10,000 × g for 30 min at 4 ◦C. The supernatant (cytosolicfraction) was collected and stored at −80 ◦C. The direct mea-surement of cytochrome c release from mitochondria wasevaluated with the Human cytochrome c Immunoassay. Thisquantitative sandwich assay technique measures the levelof cytochrome c in cytoplasm by using monoclonal antibodyspecific for cytochrome c, which has been pre-coated ontomicroplate. Clarified cytoplasm extracts were added to trip-licate wells for determining the cellular level of cytochrome c.The reactions were continued for 2 h at room temperature. Theplate was washed four times with washing buffer and thenincubated for 2 h with cytochrome conjugate (100 �l/well).
Next, when the plate was aspirated and washed 4 times, a100 �l substrate solution per well was added and the sam-ples were incubated in total darkness, at room temperaturefor 30 min. The reaction was stopped by the addition of 2 M
e n v i r o n m e n t a l t o x i c o l o g y a n d p h a r m a c o l o g y 3 6 ( 2 0 1 3 ) 600–610 603
Fig. 1 – The comparison of the cytotoxicity of PTX and Epo B for MM14 and OV-90 cells. Values of IC50 parameter wasdetermined by the MTT test after 72 h incubation with the drugs. The values are the mean ± SD of five independentexperiments in 6 repeats each. (*) Statistically significant differences in comparison to the control cells taken as 100%,P < 0.005.
s4
3
3
Ts7(r–ds
3
CFgeoi
ulfuric acid generating a yellow color which was recorded at50 nm with a PowerWave, BioTek microplate reader.
. Results
.1. Drugs cytotoxicity
he studied cell lines exhibited a significantly different sen-itivity to both compounds. Generally, cancer cells were.26-fold more sensitive to Epo B (IC50 – 27.4 nM) than to PTXIC50 – 199 nM). The normal MM14 cells were five times moreesistant to PTX (IC50 = 1000 nM) and about 3-fold to Epo B (IC50
70.46 nM) than cancer OV-90 cell line, P < 0.005 (Fig. 1). Theifferences between the IC50 values of Epo B and PTX weretatistically significant (P < 0.0001) for both cell lines.
.2. ROS detection
hanges in ROS level after drug treatment are shown inig. 2. Our results clearly demonstrate that Epo B is able to
enerate ROS in ovarian cancer cells and also, to a lesserxtent, in MM14 cells. In OV-90 cell line, a gradual increasef DCF fluorescence intensity with the increasing time of
ncubation with Epo B and PTX was observed. The maximal
increase in ROS level occurred after 24 h incubation with drugs(Epo B – 33%; PTX – 25%; P < 0.05). Extending the time of incu-bation (48 h) resulted in a slow decrease in the level of ROS.In the case of normal cells, a statistically significant rise inROS level was found only after 2 h of treatment both withEpo B and PTX (about 20%) (P < 0.05). Antioxidant (NAC) hasensured notable protection of the cells against reactive oxygenspecies.
3.3. Mitochondrial membrane potential
Fig. 3 shows the accumulation of JC-1 within the activemitochondria of the investigated cells after exposure to IC50
concentration of Epo B and PTX. Changes in the ratio ofred/green fluorescence of the fluorogenic probe reflect thevariation of ��m. In drug treated cells, a remarkable increasein green fluorescence of JC-1 monomers is visible, indicating areduction in mitochondrial membrane potential, in contrast tored fluorescence of JC-1 dimers expressed mainly in untreated(control) cells with high mitochondrial potential (Denk et al.,
2012). Changes in ��m were dependent on the type of drugand the period of treatment time (Fig. 4B) (P < 0.05). Apprecia-ble depolarization of the mitochondrial membrane in normalovarian cell line was visible after 24 h of incubation which led
604 e n v i r o n m e n t a l t o x i c o l o g y a n d p h
Fig. 2 – The effect of Epo B and PTX on the ROS productionin MM14 and OV-90 cells. Each point represents themean ± SD of four independent experiments in 6 repeatseach. (*) Statistically significant changes in comparison tocontrol cells, taken as 100%, P < 0.05; (+) Statisticallysignificant changes in comparison to the samplespreincubated 1 h with 3 mM NAC and then incubated with
PTX-19%) (P < 0.05). The level of cytochrome c, at 48 h treatment
Epo B or PTX, P < 0.05.
to an approximately 39% loss of mitochondrial potential afterPTX treatment while smaller changes were observed for Epo B(28%) (P < 0.05). The above changes between drugs action werestatistically significant. The effect of drugs on mitochondrialmembrane depolarization was different in cancer cell line.Epo B induced a considerable drop in mitochondrial mem-brane potential by 23% after 2 h, 36% after 4 h and 25% after24 h incubation. At the same time, PTX caused a consider-able lower decrease of ��m in ovarian cancer cells (about20%) (P < 0.05). As a positive control, prior to JC-1 labeling, cellswere preincubated with CCCP, a protonophoric uncoupler ofoxidative phosphorylation, for the same period of time as thatused for drug treatment. CCCP causes loss of mitochondrialmembrane potential and increases respiration (Wang et al.,2012). Upon incubation with CCCP, a profound and expectedfall in membrane potential was observed depending on the
time of treatment and concentration of uncoupler. The high-est changes were observed after 2 h of OV-90 cell line treatment(65% drop) with 10 �M CCCP. In normal cell line, we detected a
a r m a c o l o g y 3 6 ( 2 0 1 3 ) 600–610
decrease of ��m to 45% of control value (P < 0.05). At longertimes of incubation we showed a gradual increase of themitochondrial potential to the level of control (Fig. 4A). NACpretreatment effectively inhibited Epo B and PTX-induced lossof mitochondrial membrane potential in both cell lines. Theseresults confirm the considerably higher cytotoxic activity ofEpo B as compared to PTX and imply that ROS induced by Epo Bare responsible for the dissipation of mitochondrial potential.
3.4. Morphological changes in Epo B and PTX treatedcell lines
To directly assess the effect of tested drugs at the level ofthe single cell, we used fluorescence microscopy. We exam-ined permeating and intercalating of the fluorescene probes:Hoechst 33258 and propidium iodide. Numerous changes incell morphology, typical either for apoptosis and necrosis weredetected (Fig. 5). Alterations in the structure, size and shapeof the cell nucleus were detected after the treatment with EpoB. Cells exhibited blue fluorescing chromatin of condensedappearance without any red fluorescence. After a prolongedincubation time (48 h), profound chromatin condensation, cellshrinkage and nuclear fragmentation as well as a formationof apoptotic bodies, impairment of the plasma membrane andcell disintegration were also observed. In both tested cell linessome cells with condensed chromatin were present in the pop-ulation exposed to drugs and also showed red fluorescence,indicating loss of membrane integrity. The increase in theamount of apoptotic cells was noted at 48 h after treatmentwith Epo B. In summary, under these experimental conditionswe have shown that both drugs can induce apoptosis andnecrosis in cancer cells. Epo B, compared to PTX, generatedmore intensive apoptotic changes and in a shorter time whilea considerably higher number of cells with necrotic morphol-ogy were observed after PTX treatment. Pretreatment of testedcells with the antioxidant, partially reduced both apoptoticand necrotic cell population.
3.5. Intracellular distribution of calcium
Significant changes in the level of intracellular Ca2+ werenoted at 2 h and 4 h after incubation of both types of cell lineswith PTX (Fig. 6) (P < 0.05). At these times, the content of Ca2+
increased in the normal cells by about 20% and 30%, and incancer OV-90 cells by 12% and 27%, respectively for 2 and 4 h(P < 0.05). Under the same conditions, Epo B did not induceevident changes in the level of Ca2+ in normal or cancer cells.
3.6. Activation of mitochondrial pathway of apoptosis
Normal cells treated with Epo B and PTX showed an increase inthe level of cytosolic cytochrome (about 30% and 100%, respec-tively) only after 48 h of incubation (Fig. 7) (P < 0.05). Treatmentof cancer cell line with drugs leads to an increase in the cytoso-lic level of cytochrome c after only 4 h of incubation (Epo B-83%,
with Epo B, decreased to the value of control. The oppositeeffect was observed in the case of PTX. The level of cytochromec increased even more.
e n v i r o n m e n t a l t o x i c o l o g y a n d p h a r m a c o l o g y 3 6 ( 2 0 1 3 ) 600–610 605
Fig. 3 – Fluorescent microscopy images of control cells and cells treated with IC50 concentration of Epo B and PTX for 24 h,(A) MM14 cell line, (B) OV-90 cell line, in the absence or presence of antioxidant N-acetylcysteine. The cells were stainedwith JC-1 probe. Yellow-orange fluorescence of JC-1 dimers is present in the cell areas with high mitochondrial membranepotential, while green fluorescence of JC-monomers is prevalent in the cell areas with low mitochondrial membranepotential. The cells were viewed under an inverted fluorescence microscope (Olympus IX70, Japan), bar 50 �M. � enlarge theselected cells, bar 10 �M. (For interpretation of the references to color in this figure legend, the reader is referred to the webv
4
AdfawdtBtucflo
ersion of this article).
. Discussion
poptosis is required for the elimination of unwanted andamaged cells in physiological conditions. So it is importantor normal tissue development and homeostasis. However,poptotic cell death occurs also in response to cellular stresshich participates in many forms of pathological cell loss andisorders. In the present study, we investigated the contribu-ion of the apoptotic changes to the cytotoxic effects of Epo
in comparison to PTX. In both cell lines the apoptotic fea-ures appeared as early as 2 h after incubation with Epo B. The
se of specific fluorochrome allowed the confirmation of thehromatin condensation which occurred in the form of blueuorescent crescents near the cell membrane. The formationf apoptotic bodies was also observed.
The determination of the effect of Epo B and PTX on cellviability of tested cell lines showed that ovarian cancer cellline is more sensitive to Epo B and PTX than normal cells. Theresults of Tanaka et al. proved that mouse cells are relativelyresistant to PTX. As for nude mouse tumor cells, sensitivity to12 anticancer drugs was significantly lower than that of theprimary tumor cells, including PTX (Tanaka et al., 2008).
Our results indicate that Epo B may display cancer-specificcytotoxic properties, which are desired in the search for novelchemotherapeutics. The therapeutic index (IC50 of normalcells/IC50 of cancer cells) was 2.6. Moreover, normal cell lineexhibited a delayed response to the collapse of mitochondrial
membrane potential (at 24 h time point) and recovered at 48 hwhereas cancer cell line exhibited faster (start at 2 h) and sus-tained response (from 2 h to 48 h), suggesting that OV-90 cellsare more sensitive to Epo B in comparison to PTX. Increased
606 e n v i r o n m e n t a l t o x i c o l o g y a n d p h a r m a c o l o g y 3 6 ( 2 0 1 3 ) 600–610
Fig. 4 – Changes in mitochondrial membrane potential (��m) of MM14 and OV-90 cells incubated with CCCP (A) and withEpo B or PTX in the absence and presence of NAC (B). Fluorescence ratio of JC-1 dimers/JC-1 monomers of control wasassumed as 100%. Results are presented as means ± SD of four experiments in 5 repeats each. (*) Statistically significantdifferences between drugs or CCCP-treated and untreated control cells (taken as 100%), P < 0.05; (+) statistically significantdifferences between the probes incubated with drugs in the presence and absence of antioxidant, P < 0.05; (#) Statisticallysignificant differences observed between the samples incubated with Epo B in comparison to the effect after treatment withPTX, P < 0.05.
resistance of tumor cells to drugs like PTX, is probably relatedto the ability to produce defense mechanisms like overexpres-sion of ABC transporters. Cancer cells are characterized bytheir limitless replication potential and the mechanisms oftissue invasion (Jakobisiak et al., 2003). They have abnormalredox systems. It has been reported that many types of cancercells have increased levels of ROS compared with their normalcounterparts. Changes in redox status may facilitate cancerprogression. In using ROS as a signal transducer, cancer cellsmay acquire proliferative, metastatic and angiogenic proper-ties as well (Kobayashi and Suda, 2012). Several reports suggestthat cancer cells have the ability to reduce ROS includingthe activation of ROS-scavenging systems such as glutathione(GSH), as well as the inhibition of apoptosis (Jakobisiak et al.,2003; Trachootham et al., 2009). Oxidative stress is a keycomponent in linking toxicity to the multistage carcino-genic process. A primary mechanism of the action of manychemotherapy drugs against cancer cells is the formation of
ROS or other free radicals. A new generation of reactive oxygenspecies may induce cancer cell death through non-specificoxidation of cellular components and could be an early sig-nal that mediates apoptosis (Fuchs-Tarlovsky, 2012). For this
reason we measured the level of ROS in the context of apo-ptosis induction. Based on the performed tests, it was notedthat changes in the level of ROS occur more rapidly in normalcells. The ability of Epo B to generate ROS was also confirmedin our earlier study on SKOV-3 cell line, where we noted thatNAC increased cell viability by about 20% in comparison to theprobes incubated with Epo B (Rogalska et al., 2013). In neurob-lastoma cells, as in our results, an early ROS production frommitochondria might play an important role in Epo B inducedapoptosis through modifications in mitochondrial membranepermeability. The kinetic analysis of O2
− generation revealeda significant increase within the first 2 h of treatment (33%increase for IC90), which was maintained up to 24 h. However,the enhancement H2O2 formation decrease from 51% at 2 h to32% after 6 h of Epo B incubation (Khawaja et al., 2008). In ourstudies we also observed that the greatest level of ROS in can-cer cells was found at 24 h while at 48 h we observed decreasedvalue in this parameter. H2O2 accumulation was an early and
crucial step in paclitaxel cytotoxicity and was probably relatedto the overproduction of O2
•− and to its rapid conversion intoH2O2. DCFH2-DA was used to indirectly measure mainly cellu-lar generation of hydrogen peroxide (H2O2) before it reacted to
e n v i r o n m e n t a l t o x i c o l o g y a n d p h a r m a c o l o g y 3 6 ( 2 0 1 3 ) 600–610 607
Fig. 5 – The MM14 cells (A) and OV-90 cells (B) stained with Hoechst 33258 and propidium iodide visualized by fluorescencemicroscopy observed after 48 h incubation of the cells with Epo B and PTX, bar 50 �M. Representative cells with typicalmorphological features of apoptosis and necrosis (A′) and (B′), respectively: viable; early apoptotic; late apoptotic; necroticc
fts
iem1PmPbagogbfci
ells, bar 10 �M.
orm stable organic peroxides (Alexandre et al., 2006). Perhapshis explains the decrease in the level of ROS observed in ourtudies.
It has been well-established that a moderate rise inntracellular ionized calcium triggers apoptosis in manyxperimental models. The loss of calcium homeostasis is aore determining factor in apoptotic cell death (Kluck et al.,
994). We observed an increase of intracellular calcium in bothTX treated cell lines and a lack of significant changes of [Ca2+]ediated by Epo B. Moreover, [Ca2+] decreased at 48 h after
TX treatment of both cell lines. Literature data indicate thatesides Ca2+, other pro-apoptotic condition must be met forpoptosis to occur. In accordance with Giorgi et al. we sug-est that mitochondria acts as “coincidence detectors” wherenly contemporary application of both signals, Ca2+ and ROS,enerate apoptosis mediated by PTX (Giorgi et al., 2012). It has
een reported that ROS generation releases free calcium ionsrom intracellular store sites. In addition, oxidative stress alsoauses a rise in [Ca2+] in cytoplasm, which induces calciumnflux into mitochondria and nuclei to control apoptosis in
chondrocytes (Asada et al., 2001). In our studies we observeda similar temporal relationship between the increase in ROSlevel and Ca2+ concentrations. It should be emphasized espe-cially a decline in both ROS and [Ca2+] after a long incubationperiod. This may indicate that there is a link between thesetwo parameters and that they together are involved in theinduction of apoptosis.
The studies of other researchers have reported that whenmitochondria are sensitized by oxidative stress, even smallrises in mitochondrial [Ca2+] (not detectable by tested method)could promote opening of the mitochondrial permeabilitytransition pore (mPTP) and induce apoptosis (Giorgi et al.,2008, 2012). In our experiment, the reactive oxygen speciesproduced after treatment with Epo B may disrupt the mito-chondria. On the other hand, Kluck et al. (1994) noted (usingcalcium chelators in cultured cells) that raised level of intra-
cellular ionized calcium is not universally present duringthe induction of apoptosis. Similar results were obtained inmelanoma cell line MM200, where the increase in the level ofcalcium was observed after treatment with docetaxel (DTX).
608 e n v i r o n m e n t a l t o x i c o l o g y a n d p h a r m a c o l o g y 3 6 ( 2 0 1 3 ) 600–610
Fig. 6 – Influence of Epo B and PTX on intracellular level ofCa2+ in MM14 and OV-90 cells. The cells were treated withIC50 concentrations of drugs for 2, 4, 24 or 48 h. Inexperiments with antioxidant, cells were preincubatedwith 3 mM NAC, then drugs were added and incubationwas continued for another 2–48 h. The results representmean ± SD of four independent experiments in 4 repeatseach. *P < 0.05 in comparison to respective control cellstaken as 100%, #P < 0.05 indicates significant differencesbetween drugs-treated cells and samples preincubated
Fig. 7 – Changes in intracellular cytochrome c level of MM14and OV90 cells incubated with Epo B or PTX in IC50 for 4and 48 h. The results represent the mean ± SD of fourindependent experiments in 3 repeats each. Absorbance ofcontrol was assumed as 100%. (*) Statistically significant
with NAC.
In cells pretreated with calcium chelator (BAPTA-AM) theincrease in calcium level induced by DTX was inhibited but ithad no effect on apoptosis induced by docetaxel. The authorspostulated that, docetaxel-induced apoptosis is mediated byendoplasmic reticulum (ER) stress and the main road lead-ing to cell death were JNK pathway and endonuclease 1�
(IRE1�) which is a part of the stress-response signaling path-ways (Mhaidat et al., 2008). One of the elements challengingthe function of endoplasmatic reticulum (ER) – Golgi networkis disruption of calcium homeostasis resulting in ER stress.Moreover the authors reveal that resistance of colorectal can-cer cells to paclitaxel induced apoptosis is also mediated bythe activation of MEK/ERK signaling pathway especially byGRP78 (chaperon glucose regulated protein 78) (Mhaidat et al.,2009).
The release of cytochrome c into the cytosol is a key phe-
nomenon for the activation of caspase 9 and the internal pathof apoptosis. Microtubule-Stabilizing Agents (MSAs) cause anefflux of mitochondrial cytochrome c and apoptotic cell death
changes in comparison to control cells, P < 0.05.
by inhibiting the movement of microtubules, resulting inimpairment of their functioning. Partial confirmation of thishypothesis was performed using PTX, which, after 2–3 h ofincubation, contributed to the impairment of mitochondrialmovement (Shprung and Gozes, 2009). In our experiments,the greatest changes were observed after treating the cancercells with Epo B. The level of cytochrome c in the cytosolincreased by 80% (compared to control), after 4 h of incuba-tion with Epo B and only by 19% after treatment with PTX.Mitochondria in MM14 cell line disintegrated at the longertimes after exposure to the Epo B (48 h), which is important forpotential therapy. In addition, at 48 h the level of cytochromec in the cytosol of normal cells was about 70% higher in thepresence of PTX than the Epo B. Formation of apoptosomelead to the activation of executive caspase-3 which can cleavea number of cellular proteins and made the morphologicfeatures. The caspase-3 activity was assessed in the medul-loblastoma cell lines after Epo B treatment. Enzyme activitywas increased over time in the D425Med and the DAOY cell
lines (Oehler et al., 2011). Moreover, caspases-3 activationincreased in non small cell lung cancer cells (Gan et al., 2011)and SW620 human colon cancer cells (Lee et al., 2007). Epo
e n v i r o n m e n t a l t o x i c o l o g y a n d
derivative, BMS 247550, induces the mitochondrial releasend cytosolic accumulation of Smac/DIABLO, along withytochrome c, in human acute leukemia Jurkat T cells andnduces PARP cleavage activity of caspase-3 higher in theresence of PTX than the Epo B (Guo et al., 2002). After Flude-
one (26-trifluoro-(E)-9,10-dehydro-12,13-desoxyepothilone B)reatment, caspase-3, -8, and -9 were activated in multiple
yeloma (MM) cells. Immunoblots of whole-cell lysateshowed typical 17-kDa products of caspase-3 (Wu et al., 2005).
The molecular link between mitochondrial ROS andicrotubule targeted agent (MTAs) activity remains to be
stablished. Here, we showed that the early increase in theevel of ROS induced by Epo B preceded mitochondrial mem-rane alterations and cytochrome c release in the normal cells.he side effects of PTX and emerging multidrug resistance,timulate the research to look for a novel class of natural com-ounds, like epothilones which have better water solubilitynd simpler synthesis (Fauzee et al., 2012). Our results showhat Epo B is more cytotoxic than PTX to OV-90 ovarian can-er cells. In our earlier studies we examined the same in theKOV-3 ovarian cancer cells. These two cell lines differ in the
evel of p-glycoprotein and, consequently, drug resistance. Theomparison of our previous and current research leads to aery valuable observation. In OV-90 cells the IC50 of PTX wasigher than in SKOV-3 cells, but the Epo B was equally effec-
ive in both cell lines. The potency of Epo B we can confirm byhe calculation of the ratio of the IC50 PTX/IC50 Epo B, whichor SKOV-3 cells is 4.54, and for OV-90 cells is 7.23. This con-rms that Epo B can be a very promising compound for thereatment of patients with multidrug resistance. The secondovelty in this article is the demonstration for the first time,hat after treatment with Epo B, an increase in the level ofCa2+] and a high outflow of cytochrome c from mitochondriaere observed. However, further studies to explain the signal-
ng pathways triggered by Epo B, are being carried out in ouraboratory. Only a detailed explanation of pathways leading tohe death of normal and neoplastic cells will allow the creationf the correct model of epothilones treatment.
onflict of interest statement
one declared.
cknowledgements
he authors wish to thank Mrs. Marzena Pacholska for valu-ble technical assistance.
This work was supported by Grant N N405 100939 of theinistry of Science and Higher Education, Poland.
e f e r e n c e s
lexandre, J., Batteux, F., Nicco, C., Chereau, C., Laurent, A.,
Guillevin, L., Weill, B., Goldwasser, F., 2006. Accumulation ofhydrogen peroxide is an early and crucial step forpaclitaxel-induced cancer cell death both in vitro and in vivo.Int. J. Cancer 119, 41–48.
m a c o l o g y 3 6 ( 2 0 1 3 ) 600–610 609
Asada, S., Fukuda, K., Nishisaka, F., Matsukawa, M., Hamanisi, C.,2001. Hydrogen peroxide induces apoptosis of chondrocytes;involvement of calcium ion and extracellular signal-regulatedprotein kinase. Inflamm. Res. 50, 19–23.
Bystricky, B., Chau, I., 2011. Patupilone in cancer treatment.Expert Opin. Invest. Drugs 20, 107–117.
Carmichael, J., Degraff, W.G., Gazdar, A.F., Minna, J.D., Mitchell,J.B., 1987a. Evaluation of a tetrazolium-based semiautomatedcolorimetric assay – assessment of chemosensitivity testing.Cancer Res. 47, 936–942.
Carmichael, J., Degraff, W.G., Gazdar, A.F., Minna, J.D., Mitchell,J.B., 1987b. Evaluation of a tetrazolium-based semiautomatedcolorimetric assay – assessment of radiosensitivity. CancerRes. 47, 943–946.
Chen, J., Chen, Y., Chen, Z., 2013. MiR-125a/b regulates theactivation of cancer stem cells in paclitaxel-resistant coloncancer. Cancer Invest. 31, 17–23.
Diaz-Padilla, I., Oza, A.M., 2011. Epothilones in the treatment ofovarian cancer. Future Oncol. 7, 559–568.
Elmore, S., 2007. Apoptosis: a review of programmed cell death.Toxicol. Pathol. 35, 495–516.
Fauzee, N.J., Wang, Y.L., Dong, Z., Li, Q.G., Wang, T., Mandarry,M.T., Xu, L., Pan, J., 2012. Novel hydrophilic docetaxel(CQMU-0519) analogue inhibits proliferation and inducesapoptosis in human A549 lung, SKVO3 ovarian and MCF7breast carcinoma cell lines. Cell Proliferat. 45, 352–364.
Fernandez, Y., Cueva, J., Palomo, A.G., Ramos, M., de Juan, A.,Calvo, L., Garcia-Mata, J., Garcia-Teijido, P., Pelaez, I.,Garcia-Estevez, L., 2010. Novel therapeutic approaches to thetreatment of metastatic breast cancer. Cancer Treat. Rev. 36,33–42.
Ferrandina, G., Mariani, M., Andreoli, M., Shahabi, S., Scambia, G.,Ferlini, C., 2012. Novel drugs targeting microtubules: the roleof epothilones. Curr. Pharm. Des. 18, 2793–2803.
Fitzgerald, D.P., Emerson, D.L., Qian, Y., Anwar, T., Liewehr, D.J.,Steinberg, S.M., Silberman, S., Palmieri, D., Steeg, P.S., 2012.TPI-287, a new taxane family member, reduces the brainmetastatic colonization of breast cancer cells. Mol. CancerTher. 11, 1959–1967.
Fojo, T., Menefee, M., 2007. Mechanisms of multidrug resistance:the potential role of microtubule-stabilizing agents. Ann.Oncol. 18 (Suppl. 5), v3–v8.
Fuchs-Tarlovsky, V., 2012. Role of antioxidants in cancer therapy.Nutrition.
Gan, P.P., McCarroll, J.A., Byrne, F.L., Garner, J., Kavallaris, M., 2011.Specific beta-tubulin isotypes can functionally enhance ordiminish epothilone B sensitivity in non-small cell lungcancer cells. PLoS ONE 6, e21717.
Gasiorowski, K., Brokos, B., Kulma, A., Ogorzalek, A., Skorkowska,K., 2001. A comparison of the methods applied to detectapoptosis in genotoxically-damaged lymphocytes cultured inthe presence of four antimutagens. Cell. Mol. Biol. Lett. 6,141–159.
Giorgi, C., Baldassari, F., Bononi, A., Bonora, M., De Marchi, E.,Marchi, S., Missiroli, S., Patergnani, S., Rimessi, A., Suski, J.M.,Wieckowski, M.R., Pinton, P., 2012. Mitochondrial Ca(2+) andapoptosis. Cell Calcium 52, 36–43.
Giorgi, C., Romagnoli, A., Pinton, P., Rizzuto, R., 2008. Ca2+
signaling, mitochondria and cell death. Curr. Mol. Med. 8,119–130.
Goodin, S., 2008. Novel cytotoxic agents: epothilones. Am. J.
Health Syst. Pharm. 65, S10–S15.
Guo, F., Nimmanapalli, R., Paranawithana, S., Wittman, S., Griffin,D., Bali, P., O’Bryan, E., Fumero, C., Wang, H.G., Bhalla, K., 2002.
610 e n v i r o n m e n t a l t o x i c o l o g y a n
Ectopic overexpression of second mitochondria-derivedactivator of caspases (Smac/DIABLO) or cotreatment withN-terminus of Smac/DIABLO peptide potentiates epothilone Bderivative-(BMS 247550) and Apo-2L/TRAIL-inducedapoptosis. Blood 99, 3419–3426.
Halliwell, B., Whiteman, M., 2004. Measuring reactive species andoxidative damage in vivo and in cell culture: how should youdo it and what do the results mean? Br. J. Pharmacol. 142,231–255.
Hamm-Alvarez, S., Cadenas, E., 2008. Mitochondrial medicineand mitochondrion-based therapeutics. Adv. Drug Deliv. Rev.60, 1437–1438.
Hammer, S., Sommer, A., Fichtner, I., Becker, M., Rolff, J., Merk, J.,Klar, U., Hoffmann, J., 2010. Comparative profiling of the novelepothilone, sagopilone, in xenografts derived from primarynon-small cell lung cancer. Clin. Cancer Res. 16, 1452–1465.
Hennenfent, K.L., Govindan, R., 2006. Novel formulations oftaxanes: a review. Old wine in a new bottle? Ann. Oncol. 17,735–749.
Horio, M., Kato, T., Mii, S., Enomoto, A., Asai, M., Asai, N.,Murakumo, Y., Shibata, K., Kikkawa, F., Takahashi, M., 2012.Expression of RET finger protein predicts chemoresistance inepithelial ovarian cancer. Cancer Med. 1, 218–229.
Igney, F.H., Krammer, P.H., 2002. Death and anti-death: tumourresistance to apoptosis. Nat. Rev. Cancer 2, 277–288.
Jakobisiak, M., Lasek, W., Golab, J., 2003. Natural mechanismsprotecting against cancer. Immunol. Lett. 90, 103–122.
Jeong, J.Y., Kim, K.S., Moon, J.S., Song, J.A., Choi, S.H., Kim, K.I.,Kim, T.H., An, H.J., 2013. Targeted inhibition of phosphatidylinositol-3-kinase p110beta, but not p110alpha, enhancesapoptosis and sensitivity to paclitaxel in chemoresistantovarian cancers. Apoptosis.
Kania, K., Zych, A., Jozwiak, Z., 2007. Involvement of reactiveoxygen species in aclarubicin-induced death of humantrisomic and diabetic fibroblasts. Toxicol. In Vitro 21,1010–1019.
Khawaja, N.R., Carre, M., Kovacic, H., Esteve, M.A., Braguer, D.,2008. Patupilone-induced apoptosis is mediated bymitochondrial reactive oxygen species through Bimrelocalization to mitochondria. Mol. Pharmacol. 74,1072–1083.
Kluck, R.M., McDougall, C.A., Harmon, B.V., Halliday, J.W., 1994.Calcium chelators induce apoptosis—evidence that raisedintracellular ionised calcium is not essential for apoptosis.Biochim. Biophys. Acta 1223, 247–254.
Kobayashi, C.I., Suda, T., 2012. Regulation of reactive oxygenspecies in stem cells and cancer stem cells. J. Cell. Physiol.227, 421–430.
Lee, J.J., Swain, S.M., 2008. The epothilones: translating from thelaboratory to the clinic. Clin. Cancer Res. 14, 1618–1624.
Lee, S.H., Son, S.M., Son, D.J., Kim, S.M., Kim, T.J., Song, S., Moon,D.C., Lee, H.W., Ryu, J.C., Yoon, D.Y., Hong, J.T., 2007.Epothilones induce human colon cancer SW620 cell apoptosisvia the tubulin polymerization independent activation of thenuclear factor-kappaB/IkappaB kinase signal pathway. Mol.Cancer Ther. 6, 2786–2797.
Magnani, M., Ortuso, F., Soro, S., Alcaro, S., Tramontano, A., Botta,M., 2006. The betaI/betaIII-tubulin isoforms and theircomplexes with antimitotic agents. Docking and moleculardynamics studies. FEBS J. 273, 3301–3310.
a r m a c o l o g y 3 6 ( 2 0 1 3 ) 600–610
Mhaidat, N.M., Alali, F.Q., Matalqah, S.M., Matalka, I.I., Jaradat,S.A., Al-Sawalha, N.A., Thorne, R.F., 2009. Inhibition of MEKsensitizes paclitaxel-induced apoptosis of human colorectalcancer cells by downregulation of GRP78. Anticancer Drugs 20,601–606.
Mhaidat, N.M., Thorne, R., Zhang, X.D., Hersey, P., 2008.Involvement of endoplasmic reticulum stress indocetaxel-induced JNK-dependent apoptosis of humanmelanoma. Apoptosis 13, 1505–1512.
O’Reilly, T., McSheehy, P.M., Wartmann, M., Lassota, P., Brandt, R.,Lane, H.A., 2011. Evaluation of the mTOR inhibitor,everolimus, in combination with cytotoxic antitumor agentsusing human tumor models in vitro and in vivo. AnticancerDrugs 22, 58–78.
Oehler, C., von Bueren, A.O., Furmanova, P., Broggini-Tenzer, A.,Orlowski, K., Rutkowski, S., Frei, K., Grotzer, M.A., Pruschy, M.,2011. The microtubule stabilizer patupilone (epothilone B) is apotent radiosensitizer in medulloblastoma cells. Neuro Oncol.13, 1000–1010.
Pellicciotta, I., Yang, C.P., Goldberg, G.L., Shahabi, S., 2011.Epothilone B enhances Class I HLA and HLA-A2 surfacemolecule expression in ovarian cancer cells. Gynecol. Oncol.122, 625–631.
Pellicciotta, I., Yang, C.P., Venditti, C.A., Goldberg, G.L., Shahabi,S., 2013. Response to microtubule-interacting agents inprimary epithelial ovarian cancer cells. Cancer Cell Int. 13, 33.
Rogalska, A., Koceva-Chyla, A., Jozwiak, Z., 2008.Aclarubicin-induced ROS generation and collapse ofmitochondrial membrane potential in human cancer celllines. Chem. Biol. Interact. 176, 58–70.
Rogalska, A., Marczak, A., Gajek, A., Szwed, M., Sliwinska, A.,Drzewoski, J., Jozwiak, Z., 2013. Induction of apoptosis inhuman ovarian cancer cells by new anticancer compounds,epothilone A and B. Toxicol. In Vitro 27, 239–249.
Shprung, T., Gozes, I., 2009. A novel method for analyzingmitochondrial movement: inhibition by paclitaxel in apheochromocytoma cell model. J. Mol. Neurosci. 37, 254–262.
Tanaka, T., Toujima, S., Utsunomiya, T., Yukawa, K., Umesaki, N.,2008. Experimental characterization of recurrent ovarianimmature teratoma cells after optimal surgery. Oncol. Rep. 20,13–23.
Trachootham, D., Alexandre, J., Huang, P., 2009. Targeting cancercells by ROS-mediated mechanisms: a radical therapeuticapproach? Nat. Rev. Drug Discov. 8, 579–591.
Wang, H., Yang, X., Zhang, Z., Xu, H., 2003. Both calcium and ROSas common signals mediate Na(2)SeO(3)-induced apoptosis inSW480 human colonic carcinoma cells. J. Inorg. Biochem. 97,221–230.
Wang, J., Zhang, H., Ying, L., Wang, C., Jiang, N., Zhou, Y., Wang,H., Bai, H., 2009. Five new epothilone metabolites fromSorangium cellulosum strain So0157-2. J. Antibiot. (Tokyo) 62,483–487.
Wang, S., Xiao, W., Shan, S., Jiang, C., Chen, M., Zhang, Y., Lu, S.,Chen, J., Zhang, C., Chen, Q., Long, M., 2012. Multi-patterneddynamics of mitochondrial fission and fusion in a living cell.PLoS ONE 7, e19879.
Winsel, S., Sommer, A., Eschenbrenner, J., Mittelstaedt, K., Klar,U., Hammer, S., Hoffmann, J., 2011. Molecular mode of actionand role of TP53 in the sensitivity to the novel epothilonesagopilone (ZK-EPO) in A549 non-small cell lung cancer cells.PLoS ONE 6, e19273.
Danishefsky, S.J., Moore, M.A., 2005. Investigation ofantitumor effects of synthetic epothilone analogs in humanmyeloma models in vitro and in vivo. Proc. Natl. Acad. Sci.USA 102, 10640–10645.
