Cytoprotective activity of amifostine on HeLa cell cultures treated with daunorubicin and evaluation of gene expression by RT-PCR

Abstract

There are reports that amifostine protects normal tissues against chemotherapy and radiotherapy in cancer treatment. In this study, daunorubicin effects on cell kinetics parameters and apoptosis were examined both singly and in combination on HeLa cell culture. Optimum doses of daunorubicin and amifostine (0.1, 1 μg/mL, respectively) were applied for 24 and 48 h. Cell kinetic parameters such as growth rate (WST-1 colorimetric assay), mitotic and apoptotic index were applied to identify cytotoxicity that was induced by daunorubicin. In addition, to evaluate the molecular mechanism of cytoprotectivity we studied DNA degradation in agarose gel electrophoresis and expression of two genes, bcl-2 and bax, known to be important for the regulation of apoptosis. The cytotoxic effect correlated with a decline of growth rate and mitotic index, and a significant increase of apoptotic index, when HeLa cells were treated with daunorubicin alone or in combination with amifostine treatment (p < 0.05-0.001). The enhancement of the cytotoxicity by daunorubicin was closely associated with DNA degradation. Investigation of bcl-2/bax expression level by RT-PCR showed that amifostine did not protect tumor cell lines against daunorubicin cytotoxicity.

Keywords::

• HeLa
• DNA
• bcl-2/bax
• cytoprotectivity

Introduction

Chemotherapy plays a critical role in virtually every phase of cancer treatment (Zeng et al., 2000). The role of chemotherapeutic pharmacodynamic and pharmacokinetic properties has increasingly been investigated during the last few years (Plosker & Faulds, 1993; Zeng et al., 2000; Özcan Arıcan et al., 2007). One of the major limitations of cancer chemotherapy is the indiscriminate injury of normal tissue, leading to organ toxicity and consequent dose limitation/treatment failure. Several antioxidants have been tested and shown to protect normal tissues from chemotherapeutic toxicity (Das et al., 2003). Among these agents, amifostine (AMI) has cytoprotective action. AMI, previously known as WR (Walter-Reed) 2721, was originally developed as a radioprotective reagent during the Cold War in the 1950s. Nowadays, it is considered as a broad-spectrum radioprotective and chemoprotective drug, and it has been clinically used to reduce the toxic side-effects induced by anticancer agents (Grochova & Ŝmardova, 2007).

Chemotherapeutic agents including daunorubicin (DAU) are known to induce apoptosis (Skladanowski & Konopa, 1993; Hueber et al., 2003). Apoptosis, as a distinct type of cell death, is governed by a number of regulating genes mediated by apoptotic signals. Bcl-2 family proteins were demonstrated to be important apoptosis regulators after drug treatment (Periman et al., 1999; Bruckheimer et al., 2000). Bcl-2 and bax are two discrete members of the gene family involved in the regulation of cellular apoptosis (Hanada et al., 1995). Among the variety of proteins that are produced in response to apoptotic signals are those mediating DNA fragmentation, such as bax, bcl-xs and bak, as well as the anti-apoptotic ones, such as bcl-2, bcl-xl (Werner et al., 2002; Oliver et al., 2005).

In recent years, the in vitro cytotoxic activity and cytoprotective effect of antineoplastic agents has been investigated. In vitro, AMI’s cytoprotectivity and DAU sensivity was related to different mechanisms (Viera et al., 2002; Das et al., 2003. 2008; Dai et al., 2005; Roˇźalski et al., 2005). Several factors might explain the discrepancies regarding the influence of bcl-2 and bax on apoptosis (Jäckel et al., 1999). Therefore, we have investigated the expression and possible role of bcl-2 and bax on cytoprotectivity in vitro.

In this study we examined the cytotoxicity of DAU with growth inhibition and mitotic index (MI) as the cell kinetic parameters. Also, DNA profiles of treatment groups were screened by agarose gel electrophoresis and bcl-2/bax gene expression by RT-PCR. We examined the apoptotic index (AI) parameter to investigate cytotoxicity, induced by DAU and AMI+DAU.

Materials and methods

Cell culture

The tumor cell line used in our experiments was HeLa, initiated from a human cervical carcinoma in 1951 and continuously grown in cell culture since that date. These cells were grown regularly by passage twice a week in our laboratory. Cells were cultured in minimum essential medium (MEM) (Sigma) containing 10% fetal bovine serum (FBS) (Gibco) at 37°C in a humidified atmosphere of 5% CO₂ in air. Cells were washed with Hank’s balanced salt solution (HBSS) and harvested using 0.25% trypsin (Gibco) for 3 min. Then cells were centrifuged at 1500 rpm for 3 min. The supernatant was discarded and the pellet was diluted with MEM. Cells were seeded 30.000 cells/well in 96-well plates. After incubation at 37°C for 24 h, experiments were done.

Drug treatments

AMI (Ethyol^®, 50 mg) (Bioscience) and DAU (Daun orubicin, 20 mg) (Carlo Erba) were dissolved in MEM as a 1 mg/mL stock solution supplemented with 10% FBS. The pH of the drug solution was adjusted to 7.4 with NaOH. All assays testing AMI and DAU were protected from light. The inhibitory concentration 50% (IC₅₀) dose of DAU was determined in HeLa cell culture (özcan Arıcan et al., 2005). The experiments (using doses of 0.1 and 1 μg/mL, respectively) were carried out in 4 groups: Control, AMI, DAU and AMI+DAU combinations. HeLa cells were tested for 24 and 48 h.

Cytotoxicity assay

The effect of chemotherapeutic agents on the growth rate of HeLa cells was evaluated with the WST-1 assay kit (Premix WST-1, Takara, cat no: MK400). The WST-1 assay was applied to identify cytotoxicity that was formed by drugs for 24 and 48 h. Therefore, WST-1 reagent was added, and 4 h later, the 96 well plates were read on a Mquant Microelisa reader, using a test wavelength of 570 nm, and a reference wavelength of 630 nm.

Mitotic index analysis

MI was studied by the method of Feulgen. Before the cells were treated with Feulgen, they were prepared with 1 N HCl at room temperature for 1 min and then hydrolyzed with 1 N HCl for 10.5 min at 60°C. After the slides were treated with Feulgen, they were rinsed for a few min in distilled water and stained with 10% Giemsa stain solution pH 6.8, for 3 min and washed twice in phosphate buffer. After staining, the slides were rinsed in distilled water and air-dried. Finally, MI was calculated by counting metaphases, anaphases and telophases for each tested drug concentration and control. At least 3,000 cells were examined from each slide for MI.

Apoptosis assay

For the determination of the AI, cells were fixed under methanol and stained with 49, 6-diamidine-2 phenylindole (DAPI). Following extensive washing in phosphate-buffered saline (PBS), slides were scored (double-blind) under the fluorescence microscope. AI represents the percentage of fragmented nuclei and was determined on a microscopic field of at least 100 area/each experimental point by the same scorer.

DNA extraction

Genomic DNA was isolated from HeLa cells as described in the protocol of manufacturer (Apopladder Ex™ Kit, Takara, Cat. No. MK600). The DNA concentration of samples was at A₂₆₀ and electrophoresed in 2% (w/v) agarose containing 1 μg/mL ethidium bromide.

Total RNA extraction

Total RNA was isolated from HeLa cells via Invitrogen, Purelink Micro-to-Midi Total RNA Purification System (Cat no. 12183-018). Total RNA content was measured with visible spectrophotometer (GBC Cintra 20).

RT-PCR analysis

HeLa cells (2.5 10⁵) were seeded on plastic culture dishes ( 60 mm in diameter) in 5 mL of MEM. Cells were precultured for 24 and 48 h, and then total RNA from the cells was extracted using a total RNA purification system (Invitrogen, Cat no. 12183-018). RT-PCR was carried out using SuperScript III one-step RT-PCR system with Platinum Taq DNA polymerase described by the manufacturer (Invitrogen, Cat no. 12574-026) with the PCR primers from Takara Apoprimer set (Takara Bcl-2 family, Cat no. 6623). Single strand cDNA was synthesized by incubation at 55°C for 30 min. The reaction mixture was then held at 94°C for 2 min to denature the RNA/cDNA hybrid and inactivate the AMV-RT.

The double-stranded cDNA was produced and amplified with the following PCR conditions: 40 cycles of template denaturation at 94°C for  15 s primer annealing at 60°C for  30 s and primer extension at 68°C for 1 min. A final elongation step at 68°C for 5 min was performed. Reaction was stopped at 4°C. PCR products were separated on Tris-acetate-EDTA 1.5% agarose gels containing 100 ng/mL ethidium bromide.

Statistical analysis

The data were analyzed statistically using ANOVA. Dunnett’s test (between control and treatment groups) and Student Newman-Keuls tests (between treatment groups) were used for multiple comparisons. Apoptotic index analysis was evaluated by Student’s t-test. Data analyzed were from a minimum of three independent experiments.

Results

Growth rate effects of AMI and DAU

The absorbance value of AMI, DAU and AMI+DAU treatment groups of HeLa cells are shown in Table 1. The absorbance value for drugs was higher for 24 h than for 48 h in HeLa cells. When the HeLa cells were treated for 24 and 48 h with DAU, their growth and viability were significantly decreased in time-dependent manners relative to control (p <0.001) (Figure 1).

Table 1.  Effect of AMI and DAU either alone or in combination for 24 and 48 h in HeLa cells, given in ± SD.

Treatment groups	Absorbance value (570 nm)
	24 h	48 h
Control	10 ± 1.68 × 10^−2	11 ± 1.53 × 10^−2
AMI	9 ± 0.54 × 10^−2	10 ± 0.88 × 10^−2
DAU	4.2 ± 0.84 × 10^−2*	2.4 ± 0.33 × 10^−2*
AMI + DAU	5.0 ± 0.66 × 10^−2*	2.0 ± 0.43 × 10^−2*

*p <0.001.

Figure 1.  Cell growth inhibition of HeLa cell lines by AMI, DAU and AMI+DAU treatment for 24 and 48 h.

Mitotic index of treated cells

The values of MI for 24 and 48 h decreased in HeLa cells when treated with DAU and AMI+DAU, as seen in Table 2. These decreases were statistically significant when compared to control and single AMI treatment (p <0.001). Another part of the cytotoxic effect of these drugs is inhibition of MI in HeLa cells.

Table 2.  Mitotic index values of treated cells, given in ± SD.

Treatment groups	24 h	48 h
Control	3.75 ± 0.66	4.36 ± 0.56
AMI	3.99 ± 0.09	4.03 ± 0.77
DAU	2.12* ± 0.01	1.24* ± 0.37
AMI + DAU	2.44* ± 0.22	1.87* ± 0.02

*p <0.05 (according to control).

Apoptosis assay of treated cells

Apoptotic cell number of HeLa cell culture treated with AMI, DAU and AMI+DAU, as shown in Table 3, revealed that drugs express part of their cytotoxic effects by induction apoptosis. The values of AI also increased with increasing treatment time (Figure 2). The apoptotic cell number and AI values in DAU and AMI+DAU treatment was higher than control and single AMI treatment for both treatment times.

Table 3.  Apoptotic cell number of drugs in HeLa cell lines.

Treatment groups	24 h	48 h
Control	4 ± 0.88	6 ± 1.44
AMI	3 ± 1.03	7 ± 1.97
DAU	28* ± 2.06	46* ± 2.48
AMI + DAU	30* ± 3.99	44* ± 4.01

*p <0.05 (according to control).

Figure 2.  Apoptotic index of HeLa cell lines treated with drugs for 24 and 48 h (respectively).

DNA profiles of treated cells

Agarose gel electrophoresis of DNA from HeLa cells treated with AMI, DAU and AMI+DAU is shown in Figure 3 for 24 and 48 h, respectively. As a result, high DNA degradation was determined in single DAU treatment for 24 and 48 h. When the DNA levels of HeLa cells were compared in treatment groups, the most effective result was obtained in single DAU treatment for 48 h.

Figure 3.  DNA profiles of AMI, DAU and AMI+DAU treatment groups in HeLa cells for 24 and 48 h (M, Marker; C, Control; 1, AMI; 2, DAU (↓) and 3, AMI+DAU for 24 h; C, Control; 4, AMI; 5, DAU (↓) and 6, AMI+DAU for 48 h).

Bcl-2 and bax expression of HeLa cells

In our study, single DAU treatment modified the expression of bcl-2 and changed the ratio of bcl-2 over bax, thereby inducing more apoptosis than other experimental treatment, especially for 24 h (Figures 4 and 5). Bcl-2/bax gene expression levels showed that both bcl-2 and bax expression levels increase on only DAU experiment groups. There was no expression difference between control and AMI+DAU combined group.

Figure 4.  Bcl-2 expression of HeLa cells (M, Marker; C, Control; 1, AMI; 2, DAU and 3, AMI+DAU for 24 h; C, Control; 4, AMI; 5, DAU and 6, AMI+DAU for 48 h).

Figure 5.  Bax expression of HeLa cells (M, Marker; C, Control; 1, AMI; 2, DAU and 3, AMI+DAU for 24 h; C, Control; 4, AMI; 5, DAU and 6, AMI+DAU for 48 h).

Discussion

In our experiment, assessed parameters showed that DAU has cytotoxic effects both at 24 and 48 h, thus it has been observed that it causes significant (p < 0.001) time-dependent decrease on the proliferation rate of HeLa cells. It also has been shown using proliferation rate, MI, AI, agarose gel electrophoresis and RT-PCR techniques that AMI does not moderate cytoprotective effects over the cytotoxic effects of DAU. As a result, we agree the cytotoxic effect of AMI does not occur on tumor cell lines (Özcan Arıcan, 2005).

DNA synthesis inhibition, free radical formation and lipid peroxidation, DNA binding and alkylation, DNA cross-linking, interference with DNA strand separation and helicase activity, direct membrane effects, and the initiation of DNA damage via the inhibition of topoisomerase II are mechanisms responsible for the antiproliferative and cytotoxic effects of daunorubicin (Meriwether & Bachur, 1972; Keprtova et al., 1993; Gewirtz, 1999). The anthracyclines have been shown to induce apoptotic cell death (Ling et al., 1993; Skladanowski & Konopa, 1993; Jaffrezou et al., 1996), although this is likely to be the final cellular response to upstream events such as inhibition of topoisomerase II (Gewirtz, 1999). Grazide et al. (2004) studied the effect of blocking GSC in their highly drug-sensitive leukemic models. They demonstrated that by using the classic GCS inhibitors, daunorubicin and Ara-C induced apoptosis was blocked. In recent years, several studies have reported the cytoprotectivity and apoptotic activities of anticancer drugs of with cell culture (Mirjolet et al., 2000; Nitsu et al., 2000; Lucianot et al., 2006; Takara et al., 2006).

Criteria such as proliferation rate and DNA fragmentation which were assessed in our experiments are limited to explain the mechanism of cytotoxic and cytoprotective effects of drugs on the cellular level. However, previous studies are consistent with our findings as cytotoxicity mediated by DAU increases depending on time, and the cytoprotective effect of AMI does not occur with tumor cells.

The effect of AMI, DAU, and AMI+DAU combinations on HeLa cell DNA profiles are shown in Figure 3. The largest DNA sample degradation was caused by DAU; there is no cytoprotective effect of combined AMI application in tumor cells.

In summary, consistent with the literature, our findings indicate a lack of cytoprotective effects of AMI on tumor cells. As shown by AI values, DAU and AMI+DAU treatment groups have high values relative to control and single AMI experimental groups (p <0.05). DNA profiles and RT-PCR results support AI and MI results in single and combined DAU treatments. In addition, we looked at bcl-2/bax gene expression levels, we could see both bcl-2 and bax expression levels increase on only DAU experiment groups. There was no expression difference between control and AMI+DAU groups. For future studies, the cytoprotective effect of AMI mediated by bcl-2 and bax regulation can be investigated. The results of this study show the extent of spontaneous apoptosis seems to be independent of the expression of bcl-2 and bax, but correlates with the mitotic activity and apoptotic index in HeLa cell lines. Our results also suggest the mechanisms underlying this process are complex and largely unknown; it will be difficult to assess the contribution of individual genes to apoptosis in vitro and in vivo.

Acknowledgements

This work was supported by the Research Fund of the University of Istanbul. Project number BYP-749/21072005.

Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.