Vol. 47, No. 5, May 1999
BIOCHEMISTRY andMOLECULAR BIOLOGY INTERNATIONAL pages 765-771
MITOCHONDRIAL CHANGES DURING THE APOPTOTIC PROCESS OF HeLa CELLS EXPOSED TO CISPLATIN Jorge Melelldez-Zajgla l, Elizabeth Cruz t, Vihna Maldonado i, and Aim Maria Espinoza 2
1Laboratorio de Biologla Molecular and elnmunologid, Divisidn de Investigacidn Bdsica, lnstituto Nacional de Cancerologia, Av. San Fernando 22 Tlalpan 14000, MExico, D. E E-mail:
[email protected]
HeLa cells undergo apoptosis after exposure to cisplatin. Since mitochondria have recently been proposed as a probable effector of this type of cell death, we performed an analysis using the fluorescent cation rhodamine 123, which is transported actively by this organelle. Cisplatin induces a decrease in the mitochondrial staining, as assessed by cytofluorometric analysis. Microscopic analysis demonstrated that this effect was accompanied by damage of the mitochondria. These features were not exclusive of cisplatin, as other antineoplasic agents (taxol, etoposide) elicited similar effects. These results point toward the notion of a general effect of antineoplasic drugs over the mitochondria during induction of apoptotic cell death. SUMMARY:
Keywords: Apoptosis, Cisplatin, Etoposide, Taxol, Mitochondria, Rhodamine 123.
INTRODUCTION
Apoptosis is an active mechanism of cell death with distinctive morphological features that can be elicited by a variety of stimuli, which includes commonly used antineoplasic drugs. Several reports [1,2] have implicated p53 in the induction of cell death by apoptosis, although examples of p53-independent mechanisms are atso present [3,4]. Recently, it has been proposed that p53-induced apoptosis could depend on three steps: 1) the transcriptional induction of redox-related genes, 2) the formation of active oxygen
species and 3) the oxidative degradation
of mitochondrial
components, culminating in cell death [5]. Cisplatin (Cis-diamminedichloroplatinum II)induces HeLa cells to undergo cell death, producing DNA fragmentation and morphological changes typical of apoptosis, eighteen hours after exposure to the drug [6,7]. This drug is a chemotherapeutic agent used in the treatment of a variety of human cancers. Its clinical efficacy involves the formation of DNA lesions, generating intra-and inter strand DNA crosslinks, DNAglutathione crosslinks and monoadducts that significantly distort the structure of the 765
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double helix [8,9]. There are also evidences suggesting that the generation of active oxygen species could be participating in cisplatin toxicity. Masuda and coworkers reported that, at least in vitro, CDDP generates active oxygen species such as superoxide anion and hydroxyl radical by its interaction with DNA [10]. Also, there are reports which demonstrate that CDDP induces mitochondrial damage, depletes GHS content and protein-SH groups, inhibits Ca2+ uptake, collapses the mitochondria potential and increases lipid peroxidation of mitochondria in rat renal cortical slides [11]. Indeed, it has been shown that low doses of cisplatin, in conjunction with valinomycin, can cause extensive mitochondrial DNA damage without nuclear DNA alterations, which could be the reason for the synergistic activity of this drug combination [12]. There also has been shown a preferential binding of CDDP to mtDNA as compared to nDNA [13]. It has been proposed that altered mitochondrial potential could be a cause for resistance to cisplatin [14,15]. Recently, we showed that the transcriptional factor NF-KB, implicated in the oxidative stress response [16], was modulated when HeLa cells were exposed to cisplatin [17]. This activation was accompanied mitochondrial
antiapoptotic
protein
Bcl-2.
by a downregulation
Interestingly,
remanent
p53
of the protein,
specifically degraded in this cervical cancer cell line by its association with the papillomavirus protein E6, relocalize to the nucleus, suggesting a possible role for this protein in the apoptosis induced by this drug, even in the presence of active papillomavirus proteins [17]. For these reasons, the aim of this study is to examine the effect of cisplatin on mitochondria during the apoptotic process of HeLa cells. MATERIALS
AND
METHODS.
Cell culture: Human HeLa cells were maintained as a monolayer in Dulbecco's modified Eagle's medium containing 10 % (v/v) fetal bovine serum and incubated at 37~ in a humidified atmosphere with 5 % (v/v) CO 2 in air. Cells at 70% confluence were used in all experiments. DMEM and fetal bovine serum were obtained from GIBCO; cisplatin and other chemicals were obtained from Sigma, St. Louis. MO. Cellular viability: Cells were seeded in 24-wells chamber dishes and treated with the different drugs. At the times described, cells were fixed in -70% ethanol at -20~ washed in PBS and stained in crystal violet (1% in water). After washing, the stain was solubilized in 33% acetic acid and the absorbance determined in an ELISA reader at 570 nm. The analysis was performed by triplicate in five independent experiments. 766
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Fluorescence labeling of mitochondria: Control cells and those exposed to cisplatin (40 HM), paclitaxel of etoposide for several times were incubated with rhodamine 123 (10 ~tg/ml) for ten minutes, and washed three times with sterile PBS. Rhodamine 123 is a fluorescent cation that is specifically taken up by active mitochondria and can be visualized by fluorescence microscopy [18]. The cells were then visualized with a Zeiss microscope, and photographed in a Kodak Plus X-Pan film.. Control coverslips without rhodamine 123 did not show any staining. Parallel cultures were stained with ethidium bromide as described in [6], and low molecular weight DNA analysis performed to verify the presence of apoptosis, at optimal times after drug exposure (results not shown).
Flow cytometry: For the measure of mitochondrial membrane potential, cells without treatment and cells exposed to cisplatin (40 t/M) for 6 and 18 hours were incubated for 10 min with rhodamine (10 ~g/ml) washed three times with PBS, detached and centrifuged 800 X g for 5 rain. The pellet was then dissolved with sterile PBS-glucose (4 g/L) and analyzed on a Becton-Dickinson FACStar Plus cytometer with excitation at 488 nm and emission at 585 nm and 640 nm. At least 10,000 events were acquired for each sample. Cells were maintained in complete dark and in aseptic condition all the time.
RESULTS
Since the death of HeLa cells induced by cisplatin shows DNA fragmentation and apoptotic features eighteen hours after exposure to the drug [6], we analyzed the mitochondrial function and morphology within this time. Control cells and cells exposed to drug were incubated with rhodamine
123, and visualized
with
epifluorescence. During the first hours after treatment, we did not found changes in the intensity of the staining nor in the morphology of the mitochondria. Nevertheless, nine hours after exposure, the intensity of cellular fluorescence decreases, accompanied by a diffuse mitochondrial
staining pattern in which shortened organelles prevailed.
These results became more evident prolonging the exposure to the drug. Fifteen hours after exposure (panel C), a sharp difference was observed between control cells and cells exposed to cisplatin (figure 1). These changes were present before the onset of morphological changes (panel E) or DNA breakage (not shown). To further characterize this changes, we extended our studies a cytometric analysis, which revealed a progressive increase in the number of cells with less intracellular rhodamine (figure 2). Finally, to determine if this mitochondrial changes were exclusive of cisplatin exposure, we induced apoptosis in HeLa cells with two other antineoplastic agents 767
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known to induce apoptosis in this cell line [19,20]. As shown in figure 3, twenty four hours after exposure of HeLa cells to 500 nM of taxol, mitochondria also present signs of damage (figure 3B). Exposure to 50 IJ.M of etoposide resulted in similar changes (figure 3). This changes were also present before the onset of apoptotic morphology (panel C and D) and DNA breakage (not shown).
E
~D
C
~,~lb e a e ql
i,
Figure 1
C
A
E=
Rhodamine 123 staining
Figure 2
768
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Figure 3
DISCUSSION
In this report, we found that cisplatin induces important mitochondrial changes in HeLa cells. By cytofluorometric analysis we found that cell~ exposed to cisplatin retained less rhodamine 123, a change compatible with the notion of a collapsed mitochondrial potential as a feature of the apoptotic process. These changes were present predominantly at later time points, just previous to the apoptotic morphological appearance previously reported [6], although earlier changes were found, which support the hypothesis of the mitochondria as a later effector of cellular death. The loss of mitochondrial potential reported here disagree with an earlier report of Shinomiya, et al [21]. These authors report an increase in rhodamine 123 uptake in EL-4 lymphoma cells exposed to a 10 #g/ml concentration of cisplatin. Although the authors did not specifically address the presence or timing of apoptosis, the cisplatin concentrations used are similar, so it could be expected that these cells undergo apoptosis at later time points. The conflicting results could be due to the presence of an early increase of rhodamine uptake with a later loss in membrane potential, although this is not the case in our model, in which we did not found any increase in the time points analyzed (one to 18 hours). The difference may a!so be due to a cell-specific response to this antineoplasic drug. 769
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It has been suggested that apoptosis-inducing
agents can trigger the
uncoupling of electron transport from ATP production, leading to a decrease in mitochondrial potential and a corresponding production of reactive oxygen species [22,23]. In this regard, it has been proposed that p53-induced apoptosis could depend on the transcription of redox related genes that, finally, produces active oxygen species and degradation of mit0chondrial components [5]. Although not directly tested, the nuclear translocation of p53 in HeLa cells after cisplatin exposure, avoiding E6induced degradation [17], could suggest a conserved p53 function, which could be supporting the latter hypothesis. Nevertheless, HeLa cells do not depend on RNA or protein synthesis to elicit apoptosis after exposure to cisplatin [24]. This could be due to an inefficient
p53 transactivation
function,
and, therefore, a p53-independent
mechanism; the participation of short-lived p53 down-regulated genes as negative apoptosis inducers; the participation of other molecules signaling to the mitochondria, or the direct participation of the mitochondria without nuclear signaling. The mitochondrial changes associated with the exposure of antineoplasic drugs were not restricted
to cisplatin,
as paclitaxel,
a microtubule
poison and etoposide,
a
topoisomerase inhibitor, produced similar damage. The present study supports the hypothesis of the mitochondria as an organelle that participates in the apoptosis induced by antineoplasic drugs. REFERENCES
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