Modulation of Doxorubicin-Induced Oxidative Stress by a Grape ( Vitis vinifera L.) Seed Extract in Normal and Tumor Cells

Abstract

The major limitation of Doxorubicin (Dox) clinical use is the development of chronic and acute toxic side effects induced through the generation of reactive oxygen species. The present work was designated to investigate in vitro effects of a red grape-seed hydroethanolic extract Burgund Mare (BM), in associated administration with Dox (30 min before drug administration) in normal (Hfl-1) and tumor cell lines (HepG2 and Mls). The BM concentrations administered were below the level of the extract cytotoxiciy threshold (40 μg gallic acid [GA] Eq/mL; 37.5, 25.0, and 12.5 μg GA Eq/mL). The antioxidant capacity of the BM extract was assessed by measuring the acute toxicity at 24 h, lipid peroxides (LP), and protein oxidation. In normal cells, the product statistically decreased cytotoxicity and markedly inhibited LP and protein carbonyl (PC) formation, in a dose-dependent relationship. On contrary, in tumor cells, such treatment resulted in a reversed effect, cell death, malondialdehyde, and PC contents increasing with BM dose enhancement. BM extract treatment prior to subsequent administration of Dox afforded a differential protection against Dox-negative toxic side effects in normal cells without weakening (even enhancing) Dox's antitumor activity.

Introduction

D oxorubicin (Dox) is a highly used chemotherapeutic compound applied in the treatment of a wide variety of malignancies such as breast cancer, childhood solid tumors, soft tissue sarcomas, Hodgkin and non-Hodgkin lymphomas, and acute leukemia.^1
–3 Nevertheless, the major limitation of Dox's clinical use is the development of chronic and acute toxic side effects it produces. Now, it is well documented that the mechanism of Dox-induced toxicity involves the generation of reactive oxygen species (ROS), and cardiomyopathy is the most prevalent consequence^2

–5 because heart has a great sensibility to damage induced by free radicals.⁶ When the antioxidant capacity of cells is overwhelmed by an excessive generation of these free radicals and reactive molecules, it will result in the oxidative stress. An uncontrolled oxidative stress initiates a series of harmful biochemical events that further could be associated with diverse pathological processes. The main cellular damages caused by these species include lipid oxidation^7,8 and protein oxidation (PO)^9,10 and DNA damage lesions.^11,12

Lipid peroxidation is a complex process whereby polyunsaturated fatty acids (PUFAs) in the cellular membrane undergo reactions with oxygen to yield lipid peroxides,¹³ and this breakage of PUFAs chains compromises the membrane integrity functioning.^14,15 In addition, the end product of this process, malondialdehyde (MDA), is itself a highly cytotoxic molecule.^16

–19

Protein oxidation produces the cleavage of polypeptide chain and oxidation of side chains in aminoacid residues.²⁰ This way, the protein structural and functions could be severely impaired,²¹ leading to cellular tissue alterations responsible for development of various diseases, such as cancer, Alzheimer's disease, muscular dystrophy,^1,13 and aging.²²

The free radical-mediated damages seem to give a perfect rationale for cellular miocardic protection against Dox's undesirable side effects by using antioxidants. The naturally occurring antioxidants have been already assessed as potential candidates for the antioxidant therapies.²³ The effects of various plant-origin extracts, containing phenolic compounds, in alleviating ROS negative activity have been proved both in vitro on cardiac myoblasts²⁴ and in vivo.^23,25
–27 Therefore, an immediate approach against Dox prooxidative effects might be the combination of Dox delivery with an antioxidant product, a modality that allows reducing the Dox toxic side effects, if possible, without diminishing the drug cytostatic activity.

Phenolic compounds of grapes (particularly red) have attracted much interest due to their antioxidant properties and herewith their potential benefits for human health. For this reason, grape seed extracts have became popular in recent years even as nutritional supplements as antioxidants.^23,26 The present work was designated to investigate the in vitro effects of a red grape-seed hydroethanolic extract (BM), in associated administration with Dox in normal cells Hfl-1 (normal lung fibroblasts) and tumor cell lines Mls (ovarian carcinoma) and HepG2 (hepatocarcinoma), respectively.

For this purpose, we measured the Dox cytotoxicity, lipid peroxides, and protein carbonyl levels in treatments prior to subsequent administration of Dox.

Materials and Methods

General materials

Seeds of red grapes (Vitis vinifera L.), variety Burgund Mare (BM), were obtained from Recas vineyard, Romania. The hydroethanolic extract BM (50/50, v/v), after total polyphenols (TPs) content determination, was standardized at 3 mg gallic acid (GA) Eq/mL.

Doxorubicin hydrochloride (Dox) was from Zhejizang Hisun Pharmaceuticals Co. Ltd. All other chemicals and reagents were of analytical purity. BM extracts and Dox solutions were freshly prepared and kept cool (4°C–6°C).

Cell culture materials

Human fetal lung fibroblasts (Hfl-1) and human hepatocarcinoma (Hep G2) were from ECACC. Human ovary carcinoma (Mls) was a gift from Dr. Y. Schiffenbeuer, Medical Technology, Izrael. Culture media (from Sigma-Aldrich) were F-12 Nutrient mixture for Hfl-1, RPMI-1040 for HepG2, and Dulbecco's modified Eagle's medium for Mls, respectively. All media contained 10% fetal bovine serum.

Grape seed extract (BM) obtaining and characterization

One part (w) of finely grounded seeds was treated with 20 parts (v) solvent (water/ethanol, 50/50; v/v) and refluxed on a water bath for 30 min. After cooling, the product was filtered and centrifuged²⁸ and standardized at 3 mg GA Eq/mL.

1. Determination of total polyphenolic content (TP) was performed according to the Folin-Ciocalteu colorimetric method.²⁹ Samples were correspondingly diluted as a function of their expected phenolic content. To 1 mL of diluted sample, 5 mL Folin-Ciocalteu reagent (Merck) was added. After 5 min of stirring, the mixture was treated with 4 mL 7.5% Na₂CO₃ solution. After standing for 2 h at room temperature in dark, the samples' absorbance was measured at 740 nm (Spectrophotometer UV-Vis, Jasco, V-530) versus blank. The absorbance values were multiplied by the dilution factor in order to obtain the polyphenolic concentration. This was expressed as GA mg Eq/mL solution, used as standard for calibration.

2. Procyanidins (PA) measurement was done by the butanol-HCl assay.^30,31 A reaction mixture consisting of 2 mL BM extract and 50 mL 2 N HCl was heated under reflux for 50 min. After cooling to room temperature, the mixture was twice extracted with 25 mL n-butanol each, and absorbance at 550 nm was read. The content in PA was expressed as leucocyanidine as reference substance.³¹

3. Anthocyanidins quantification was done by the pH-differential method.³² The method is based on the reversible structural transformation that anthocyanin pigments undergo at pH 1.0 (colored oxonium form) and at pH 4.5 (colorless hemiketal form).

Two dilutions of extract, one with sodium phosphate buffer (pH 1.0) and the other with sodium acetate buffer (pH 4.5), were prepared. Both solutions were left to equilibrate for 15 min and the absorbance A=(A₅₂₀−A₇₀₀)_pH _1.0−(A₅₂₀−A₇₀₀)_pH _4.5 was used in computing the total amount of monomeric anthocyans and expressed as cyanidine-3-glycoside (mg/mL).

4. Cell-proliferation assay. The cell viability was measured by the MTT cleavage test described by Mosmann.³³ The cells seeded in 96-well plates were treated, at subconfluent cell density, with cumulative doses of Dox (0.2–200 μM), alone or supplemented with three concentrations of BM extract (37.5, 25.0, and 12.5 μg GA Eq/mL), 30 min before the addition of Dox. The cells were then incubated for 24 h and the colorimetric determinations were done in the presence of MTT dye [3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium bromide] (Sigma-Aldrich), at 492 nm with a plate reader (Tecan Sunrise).

All statistical analyses were done with a GraphPad Prism software program, version 5.0 (GraphPad).

Dose–response curves, IC₅₀, were calculated by fitting the data with a four-parametric nonlinear regression equation. The results are expressed as the mean±standard error of the mean (SEM) in triplicate measurements from two separate experiments (n=6). Statistical comparison between groups was made by one-way analysis of variance (ANOVA) followed by Tukey's multiple comparison test (P<.05 statistic significant values).

5. Cell culture and treatment protocol in lipid peroxides (LP) and protein carbonyl (PC) measurements: cells were grown to confluence in 75-cm² Cole flasks. Dox alone (10 μM) or in pretreatment with three doses of BM (37.5, 25.0, and 12.5 μg GA Eq/mL) was given to the cells, and the system was incubated for 24 h. Cell samples were homogenized in a lysis buffer containing Tris 100 mM (pH 7.4), 0.2% Triton X-100, ethylenediaminetetraacetic acid 1 mM, and 1% protease inhibitor.

Cell homogenates were then centrifuged at 2000 RCF for 10 min (Hettich Universal 32 R centrifuge). The supernatant was collected for protein determination using the Bradford method³⁴ and estimated by reading at 280 nm using a standard curve obtained with bovine serum albumin (BSA). The supernatants of cellular lysates were further used in the determination of lipid oxidation and protein oxidation parameters.

Lipid peroxidation. MDA is the most commonly used marker of lipid peroxides (LP).^35,36 MDA level was measured using the fluorimetric method with 2-thiobarbituric acid as described.³⁵ The samples (50 μL) were heated in a boiling water bath for 1 h with a solution of 10 mM 2-thiobarbituric acid in 75 mM K₂HPO₄ (pH 3) solution. After cooling, the reaction product was extracted in n-butanol. The MDA was measured in the organic phase using a synchronous technique with excitation at 534 nm and emission at 548 nm. The MDA values are expressed as nmol/mg protein.

Protein carbonyl. The quantification analysis of protein oxidation in terms of the formation of protein carbonyl was done using 2,4-dinitrophenyl hydrazine (DNPH).³⁷ The samples (0.5 mL) were treated with 3 mL solution of 10 mM DNPH in 2.5 N HCl, and the mixture was kept for 1 h at room temperature in the dark. After protein precipitation with 20% trichloroacetic acid and centrifugation, the washed protein pellet was dissolved in 1.2 mL 6 M guanidine hydrochloride. The absorbance was read at 355 nm, and the protein carbonyl content was determined using the absorption coefficient for aliphatic hydrazones (22.000 M⁻¹·cm⁻¹). The final concentration of proteins in each sample was estimated by reading at 280 nm using a standard curve obtained with BSA standards. PC values were expressed as nmol carbonyl/mg protein. To confirm BM antioxidative capacity, the effect of a powerful antioxidant catechin, epigallocatechin-galate (EGCG, 25 μM), was evaluated on the same type of cell lines.

The results are expressed as the mean±SEM in four separate experiments (n=6). Statistical comparison between groups was made by one-way ANOVA and the Dunnett's multiple comparison test (P<.05 statistic significant values).

In all experiments linearity was assessed by linear regression analysis of three IC₅₀ versus BM doses and linear trend post-test.

Results

Physicochemical (partial) characterization of BM extract

Total phenolic substance, total procyanidinic, and anthocyanic contents in the BM hydroethanolic extract as well as the main antioxidant parameters reported earlier by DPPH· assay: EC₅₀ _fs, final state efficient concentration, the time required to reach the final state, redox reaction stoichiometry (n),³⁸ and 2,2-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) assays³⁹ are illustrated in Table 1.

Table 1.

Polyphenolic Content and Antioxidant Activity of Burgund Mare Variety Extract

	Units	Values	References
Total polyphenols (TPs)	mg/mL	3	Postescu et al. ²⁸
Procyanidins	mg/mL	1.073±0.03	Present study
Cyanidins	mg/mL	3.17×10⁻³	Present study
Antioxidant activity
DPPH•
EC₅₀ fs	mmol GA Eq/mmol DPPH	0.072±0.002	Dicu et al. ³⁸
T_EC50 fs	Min	7.5
n	—	6.9
ABTS	nmol Trolox Eq/mL	59.89±0.02	Filip et al. ³⁹

BM influence on cells toxicity in associated treatments with Dox

The BM concentrations to be administered were selected below the level of the extract cytotoxiciy threshold (40 μg GA Eq/mL), as previously determined,⁴⁰ this check-point being used as a guide in BM extract pretreatments with Dox. The cell viability was counted following a Dox administration alone or in pretreatment regimens with three variable doses of BM (37.5, 25.0, and 12.5 μg GA Eq/mL). The results, expressed as the ratio IC₅₀ Dox+BM/IC₅₀ Dox showed (Fig. 1) that Dox combined treatments of cells with BM doses, synergistically protected normal cells (IC₅₀ values increased), at 24 h of incubation. The protection declined with dose decrease, from +70.1% to +32.8% and 0.0%, respectively (Fig. 1a), as compared with control (Dox alone; P=.028, ANOVA). The dose–effect dependence was linear.

FIG. 1.

Cells survival ratio: IC₅₀ Dox+BM/IC₅₀ Dox, in associated treatments (BM+Dox), in (a) normal Hfl-1, and tumor cells (b) Mls, (c) HepG2. Burgund Mare variety concentrations used: 37.5, 25.0, and 12.5 μg GA Eq/mL and were selected within the nontoxic range of BM concentration (>95% cell survival). IC₅₀ values were calculated by nonlinear regression four-parameters sigmoidal curve fit; statistical analysis was done by one-way analysis of variance (ANOVA) followed by Tukey's multiple comparations post-test; each column represents the mean±standard error of the mean (SEM) of three measurements each in two separate experiments; *P<.05, **P<.01 (Tukey's test).

On contrary, in tumor cells (Mls and HepG2) Dox's antiproliferative effect enhanced with BM concentrations and ranged between −51.5% to −27.7% and −21.4% cell survival (P=.005) for Mls and −15.5%, −5.8%, and 0.00% (ns) for HepG2 cells, respectively (Fig. 1b, c). Tukey's test revealed significant differences between normal and tumor cells at the dose of 37.5 μg GA Eq/mL (P=.0001), as well as between doze size in normal cells (P=.001).

Lipid peroxidation

Incubation of both normal and tumor cells with Dox (10 μM) resulted in significant induction of LP production (measured in terms of MDA equivalents) compared with the untreated cells (data not shown). Therefore we assessed the BM extract treatments influence as of the ratio of LP content in treated/untreated cells_.

As is depicted in Figure 2, BM pretreatments given 30 min before Dox administration, with variable concentrations of 37.5, 25.0, and 12.5 μg GA Eq/mL depleted MDA levels, in normal Hfl 1 cells, in a linearly dose-dependent manner −44.53%, −33.08%, and −27.36%, respectively (EGCG, 25 μM: −30.19%) as compared with Dox (P=.0057, ANOVA). The decline caused by all three doses was statistically significant (.001<P<.05, Dunnett's test) (Fig. 2a).

FIG. 2.

Effects of BM extract on Dox-induced lipid peroxidation (LP) in (a) Hfl-1 cells, (b) Mls, and (c) HepG2 cells. LP levels were calculated from the ratio treated/control cells in Dox alone (10 μM), Dox+BM ( 37.5, 25.0, 12.5 μg GA Eq/mL), and epigallocatechin-galate (EGCG), all versus untreated control; statistical analysis was done by one-way ANOVA followed by Dunnett's multiple comparations post–test, and each column represents the mean±SEM in four repeated experiments; *P<.05, **P<.01, ***P<.001 (Dunnett's test).

In Mls cells, LP production increased after administration of three doses of BM with +49.90%, 26.16%, and 6.55%, respectively (P=.002) and +1.78% in EGCG. In HepG2 cells, the stimulatory effect occurred only at the dose of 37.5 μg GA Eq/mL (+33.4%) and not at lower doses (25.0 and 12.5 μg GA Eq/mL) when −6.80% and −20.66% declines were found (P=.0012) (EGCG decreased by −25.04%).

Protein carbonyls

Determination of protein carbonyls is the most often employed index of protein oxidation.^22,41 When supplemented with BM extract, normal fibroblasts (Hfl1) followed a similar behavior in PC levels as LP profile, namely, a dose–effect decrease of −30.71%, −14.44%, and −10.01% (P=.0035) and −24.14% for EGCG. Tumor cells (Mls and HepG2) responded in a dualistic manner, displaying an increase in PC content of +11.49% and +7.87%, respectively, only when the most concentrated BM solution was administered. It was followed by an inhibition at lower doses of extract (25.0 and 12.5 μg GA Eq/mL), even for EGCG (Fig. 3). Though PC levels slightly decreased below those in untreated cells (Dox alone), the dose–effect dependence remained significant (P=.0026 for Mls and P=.0033) for HepG2 cells, and they strictly obey the linear dose–effect shape. EGCC behaved similarly exhibiting lower PC values than Dox alone. Generally, PC formation is less responsive to BM treatments as seen from the lack of statistic significance in this measurement.

FIG. 3.

Effects of BM extract on Dox-induced protein carbonyl (PC) production in (a) Hfl-1 cells, (b) Mls, and (c) HepG2 cells. PC levels were calculated from the ratio treated/control cells in Dox alone (10 μM), Dox+BM ( 37.5, 25.0, 12.5 μg GA Eq/mL), and EGCG, all versus untreated control; statistical analysis was done by one-way ANOVA followed by Dunnett's multiple comparations post-test and each column represents the mean±SEM in four repeated experiments; *P<.05, **P<.01 (Dunnett's test).

In normal cells, regardless the dose size, BM supplementation inhibited both lipid oxidation and protein oxidation compared with that of Dox given alone. In tumor cells, the influence was rather indefinite. Thus, the highest BM dose (37.5 μg GA Eq/mL) evidently produced an enhanced production of LP and PC, while the doses 25.0 and 12.5 μg GA Eq/mL lowered both oxidation parameter levels below their basal values, but preserved a dose–effect relationship. With a single exception, the PL level in Hfl-1 cells, the linear-trend post-test indicated a significant dose-dependant variation, .001<P<.05, in all experiments.

Discussion

Long-term administration of the antracycline derivative Dox causes a cumulative dose-dependant cardiotoxicity.^1,3
–5 The mechanism of this Dox-induced damaging side effect is attributed to free radical generation, leading to oxidative stress and subsequent stimulation of lipid oxidation and protein oxidation.^6,7

It is well documented that the oxidative stress commonly stimulates the accumulation of the key cytotoxic lipid peroxides, ultimately the final product MDA, which is the principal and most studied product of PUFAs' peroxidation. This aldehyde is a highly toxic molecule and should be considered as a marker of lipid peroxidation. It interacts with DNA and proteins being potentially mutagenic and atherogenic.¹⁷

Protein oxidation, as well, is highly detrimental to cell membrane structure and function, and its elevated levels have been linked to damaging effects, such as loss of fluidity, inactivation of membrane enzymes, increased membrane permeability, and, ultimately, the rupture of cell membrane.^14,15

Therefore, a logic approach to fight Dox's prooxidative effects could be the combinant delivery of the cytostatic with an antioxidant, whereby the Dox's undesirable side effects might be alleviated without diminishing its cytostatic activity. The cytoprotective effects of antioxidants against Dox were usually associated with the inhibition of lipid oxidation and protein oxidation, regardless the type and structure of antioxidants.^42

–45 Crude extracts are typical complex mixtures containing a multitude of components. The synergy of action of these components is superior when compared with single purified active ingredients.⁴⁵

We assessed the antioxidant capacity of BM extract employing three characteristic in vitro models, acute toxicity, lipid and protein oxidation in combined treatments with Dox. Thus, addition of BM extract to normal cells exerted a cytoprotective effect following the oxidative treatment with Dox. The product markedly inhibited LP and PC formation, both products being suitable markers for oxidative stress. The antioxidant potency of extract was dose dependent because cell survival increased, whereas MDA and protein carbonyl levels decreased by increasing the extract polyphenolic content.

On contrary, in tumor cells, such treatment resulted in a reversed effect, cell death, MDA, and protein carbonyl contents increasing with BM dose extract enhancement. Therefore, the antioxidant activity of BM extract was asserted by its differential capacity to prevent Dox toxicity, lipid and protein oxidative damage in normal cells (Hfl-1) but not in tumoral systems (Mls and HepG2 cells). A synergistic efficacy of a grape seed extract in combination with Dox has been reported in the treatment of human breast carcinoma cells.⁴⁶

The cytoprotective activity of BM might be assigned to its antioxidative properties so the attenuated toxicity in normal cells, respectively, the enhanced toxicity in tumor cells could be related with an inhibition, respectively, a stimulation of lipid oxidation and protein oxidation products formation. In a similar experiment⁴⁷ previously carried out with a palm oil enriched in tocotrienols, we revealed a different pattern, namely, this antioxidant product indiscriminately protected both normal and tumor cells in treatments with Dox.

Concerning the BM extract activity, we recently reported that topical application significantly reduced glutathione formation, glutathione peroxidase, and caspase activities in skin of mice irradiated with ultraviolet type B (UVB).⁴⁸ Similarly, BM addition inhibited UVB-induced sunburn cells, cyclobutane pyrimidine dimers (CPDs), and reduced levels of interleukin 6 and tumor necrosis factor α compared to UVB alone in a single dose regimen.³⁹ Additionally, after multiple doses of UVB, the product restored manganese superoxide dismutase, respectively, increased catalase and glutathione peroxidase activities, and reduced the percentage of CPD positive cells of the skin.⁴⁹ All these findings confirmed BM extract's antioxidative potential and its effectiveness in reducing the oxidative stress effects.

EGCG, a component of green tea, is known as an efficient agent in protecting the organisms against the oxidative stress. This strong free-radical scavenger was found as a potent suppressor of LP in human hepatoma cells and colorectal adenoma intestinal cells.^42,50 This was the rationale we have chosen this compound as reference substance in our experiments dealing with modulation of LP and PC production. The results revealed that BM extract exhibited/demonstrated antioxidant effects generally better than EGCG (25 μM), which in HepG2 cells, significantly favored LP and PC production.

The role of cytostatics is to destroy as much tumor cells, and to not affect or to have less effect on normal cells, although the production of free radicals by Dox is involved in its action, regarding both antitumor and toxic effects.⁶ BM extract treatment prior to subsequent administration of Dox afforded a differential protection against Dox negative toxic side effects in normal cells without weakening (even enhancing) Dox antitumor activity. BM extract might be used as a supplement in Dox treatments, its administration resulting in the reduction of risk factors associated with Dox administration in normal but not in tumor cells.

The outcome of this study holds promise for further in vitro studies targeting to elucidate the mechanisms of this process, the benefits of polyphenols from fruits and vegetables being increasingly accepted.

Footnotes

Author Disclosure Statement

No competing financial interests exist.

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