INTRODUCTION:

Cell death is categorized based on its appearance, biochemical processes, and functions, all of which are understood through detailed molecular studies¹. Apoptosis is thought to be the foremost and significant type of regulated cell death which is a completely controlled, self-destructive process of cells that is responsible in maintaining the overall homeostatic balance of organisms, triggered by signals from both the internal and external cellular environment². However, cancer cells often develop strategies to evade apoptosis by altering their genetic pathways, silencing tumor suppressor genes, and overexpressing survival proteins or have imperfections in the apoptosis pathway³.

Oxidative stress induced apoptosis has revealed as a causative factor for a wide range of kidney diseases and plays a crucial role⁴. Oxidative damage may be one of the manifestations of cellular damage in the toxicity of ADR⁵.

ADR has been demonstrated to raise intracellular levels of ROS by activating NADPH oxidase that produces superoxide radical using NADPH as a cofactor. NADPH transfers an electron to ADR, resulting in semiquinone-ADR, which then donates the electron to an oxygen molecule and leads to the formation of superoxide radical⁶. The superoxide molecule can subsequently be transformed into hydrogen peroxide by the action of superoxide dismutase, which can then be further converted into hydroxyl radicals via the Fenton reaction⁷. This alteration in ROS production can arise from direct interference with the complexes of the electron transport chain⁸. Numerous reports suggest that intracellular oxidants are produced in response to cancer chemotherapy drugs that can alter redox homeostasis in cancer cells⁹. Oxidants are best known for their ability to chemically modify susceptible macromolecules such as DNA, lipids and proteins all of which may be a target of ROS¹⁰. Consequently, ROS are vital in mediating cell injury and programmed cell death¹¹.

Chemotherapeutic agents play a dual role by effectively killing both cancer and normal cells ultimately leading to countless side effects. These agents adversely impact healthy cells that necessitates adjustments to treatment, potentially compromising its efficacy, reflecting the demand for a variety of possible therapeutic strategies¹². Their mechanisms include, inducing DNA damage, arresting cell division, and disrupting metabolic functions critical to cancer cell survival⁷. ADR is a widely used anthracycline antibiotic in cancer chemotherapy that is well-known for its multifaceted mechanisms of action. It intercalates into DNA, disrupting base pair stacking and inhibiting DNA replication and RNA transcription, which prevents the synthesis essential for cancer cell survival. ADR also obstructs topoisomerase II, resulting in the buildup of double-strand breaks in DNA and initiating apoptosis in cancerous cells¹³. Additionally, when combined with iron, ADR generates free radicals through a redox reaction, causing oxidative damage to DNA enhancing the drug's cytotoxic effects¹⁴. Despite being a powerful anticancer drug, the application of ADR is constrained by its harmful side effects^15-16 including, myelosuppression in cytopenic cells, cognitive impairment and neurodegeneration^17-19.

LA is pharmacologically relevant, readily bioavailable, and clinically used as a dietary supplement. It is also a powerful antioxidant due to its distinctive ability to operate in both aqueous and fatty environment making it an excellent candidate to protect cells from oxidative stress. Moreover, its ability to regenerate other antioxidants makes it a drug of choice to combat free radical mediated diseases. Its potential benefits have been studied against various toxicities²⁰. Its unique properties make LA an important drug of choice in health and disease prevention. LA is recognized for its strong inhibitory effects on histone deacetylase (HDAC) activity, which is crucial for the management of several conditions such as diabetes²¹. A novel, straightforward, accurate, precise, and reproducible RP-HPLC technique has been established for the quantification of LA in both bulk and pharmaceutical formulations, owing to the various advantageous effects of LA²².

Despite the fact that various characteristics of apoptotic cells have been studied using contemporary techniques, chromatin condensation and nuclear fragmentation remain significant markers of apoptosis²³. Dual AO/EB fluorescent staining, visualized under a fluorescent microscope, can be used to identify apoptosis-associated changes of cell membranes during the process of apoptosis²⁴. This method can also accurately distinguish cells in different stages of apoptosis²⁵. The classification of cell death within a particular model should always include a morphological evaluation in conjunction with at least one other test. Fluorescence light microscopy uses fluorescent dyes that bind to DNA and have different absorption properties, like AO/EB staining. This method is preferred due to its ease of use, speed, and precision, allowing for the simultaneous evaluation of the apoptotic index and the status of the cell membrane.AO penetrates every cell, resulting in nuclei that appear green. EB can only enter cells when the cytoplasmic membrane is compromised, leading to a red-stained nucleus that obscures the AO effect. Consequently, live cells show a clearly defined green nucleus; early apoptotic cells have a vibrant greenish-yellow nucleus with condensed or fragmented chromatin; whereas late apoptotic cells display condensed and fragmented orange chromatin. Cells undergoing necrosis show an undamaged orange nucleus²⁶.

Vero cells are well established kidney epithelial cell line with high reproducibility, ease of maintenance, and a stable genetic background compared to many human-derived lines. They also provide a reliable in vitro system to study nephrotoxic insults since they retain several renal epithelial characteristics, including brush-border enzyme activity and sensitivity to oxidative stress. Therefore, the goal of this work was to determine whether the antineoplastic drug, ADR causes apoptosis in Vero cells by inducing formation of intracellular oxidants, which are widely thought to cause contended damage to critical organs as a side effect of cancer treatment, and weather the same be ameliorated with the use of a universal antioxidant LA.

MATERIALS AND METHOD:

Reagents:

LA, ADR, AO, EB, and Ethylenediaminetetraacetic acid (EDTA) along with Trypsin were obtained from Sigma Aldrich Co, located in St. Louis, USA. Antibiotics, Minimum Essential Medium (MEM), and Fetal Bovine Serum (FBS) were sourced from GIBCO-BRL, based in Grand Island, NY, USA. All other utilized chemicals and reagents were of analytical quality.

Propagation of Vero cell culture:

This study employed cell lines sourced from the kidney epithelial cells of the African green monkey (Vero). Vero cells cultured in MEM were supplemented with 10% FBS, streptomycin (0.02mg/ml) and penicillin (1 x 10² IU/ml) while maintaining the pH at 7.4 and temperature at 37 degree Celsius in a 5% CO₂ and 95% air environment. The medium was changed a few times weekly, and the cells were cultured to confluence in MEM in 24 well plates (1 x 10⁵ cells/well).

Trypsinization and valuation of cell density:

Cell viability was assessed utilizing the conventional trypan blue exclusion method. Prior to their use, cell cultures were visually inspected and harvested promptly upon reaching confluence, demonstrating over 99% viability in all experiments. Cells underwent treatment with a trypsin EDTA solution for 2 minutes and were then washed twice with phosphate buffered saline (PBS). They were re-suspended in MEM for further sub culturing and viability assessments. A sample of the re-suspended pellet was combined with 0.05% trypan blue. The cells were then examined directly under a light microscope, which allowed for the observation of the relative quantity of stained dead cells.

Experimental set up:

To study the effect of ADR on Vero cells, sub confluent cultures of Vero cells (1 x 10⁵ cells/ml) were exposed for different length of time duration (6 and 12hours) to 1μM of ADR either alone or with LA (100μg/ml of media) added 60min prior to the addition of ADR. LA was added to the culture medium on the day of plating. The ED ₅₀ for LA for cytotoxicity was found to be 6μg/ml and the effect increased with increasing concentration reaching a maximum at 100μg/ml. Hence a dosage of 100μg of LA /ml of media was adopted in this study.

AO and EB staining:

Apoptotic cells were enumerated by AO and EB, which stains the cell nuclei²⁷. Sterile coverslips were placed in the wells before the start of the experiment. The media was removed after a 12-hour incubation time in the presence of ADR and LA. Cells in each well were stained with 50μl of a combination of AO and EB in PBS (50μg/ml + 50μg/ml). Coverslips were carefully removed to visualize the cells under fluorescence light microscopy (450 - 490nm). Only the late apoptotic cells were counted in blinded, random, nonbiased manner. For each sample, 100 cells per well and 6 wells per condition were calculated. The percentage of apoptotic cells was determined using the formula: total number of apoptotic cells/total number of cells counted) x 100

Lipid peroxidation:

Cells in wells were sonicated along with media and used for the measure of lipid peroxides. The method of Devasagayam et al²⁸ was employed to assay malondialdehyde (MDA) released.

Enzyme release:

The spent media from each well was recuperated and subjected to centrifugation to remove crystals and cellular debris, and the resulting supernatant was utilized for enzyme analysis. LDH, a cytoplasmic marker; NAG, a lysosomal marker and γ-GT, a brush-border membrane marker was assayed as per the protocols of King²⁹, Maruhn³⁰, Orlowski and Meister³¹ respectively. The secretion of these enzymes in the spent medium, can therefore be considered as indicators of the release of all components, including those, that cause tissue damage, found within the same cellular compartment.

Statistical report:

The data analysis was conducted using SPSS software, employing one-way analysis of variance. Post hoc testing was conducted for comparisons between groups using least significant difference method. The results of the tests were presented as mean±standard deviation (SD). The level of statistical significance was set at p<0.05.

RESULT AND DISCUSSION:

ADR is a highly active antineoplastic agent that induces cell killing as well as a number of non-specific side effects³². ADR exhibits its effect both at the cell surface and at the nuclear level, but it is tough to describe the pathway that ultimately leads to cell death¹². Considerable evidence supports the concept that ADR-induced oxygen free radicals causes a variety of biochemical alterations including DNA damage, that is of clinical importance in mediating the toxicity associated with its clinical usage³³. Generation of ROS contributes to the mechanism of cytotoxicity in cells treated with ADR³⁴. Therefore, ROS involvement has been deduced from established markers: (i) characteristic apoptotic features under AO/EB staining, (ii) significant lipid peroxidation (MDA formation), (iii) enzyme leakage (LDH, NAG, γ-GT), which strongly support a ROS-mediated pathway.

Apoptosis:

Staining of Vero cells revealed multiple apoptotic cells upon ADR administration which were seen increasing with time. Viable cells were seen having green fluorescent nuclei whereas the early apoptotic cells displayed yellow chromatin and late apoptotic cells showed orange chromatin (Figure 1) which is in consonance with previous research finding²⁶. Figure 2 represents the apoptotic cell counting of our study. The mean±SD values of apoptotic cell count were 7±2 (control) and 42±09 in Vero cells administered ADR (12-hour exposure). Apoptotic score in 6-hour treatment was 16±05 (ADR). When compared with control, the mean apoptotic cell count in ADR treated rats was much higher and found to be statistically significant.

Researchers have indicated that, exposure to ADR leads to the heightened activity of NADPH oxidase that has been demonstrated to cause apoptosis in H9c2 cells³⁵. Evidence from H9c2 and hepatocyte studies help reinforce the general role of ROS in doxorubicin toxicity. Some antioxidants have been indicated to defend DNA damage consequently inhibiting apoptosis³⁶. Our study extends this knowledge specifically to Vero cells, confirming that kidney epithelial cells undergo similar oxidative and apoptotic injury, which can be alleviated by LA. LA pretreatment reduced the apoptotic score to nearly that of control cells. The mechanism by which LA abrogate ADR-induced apoptosis in Vero cells might be a consequence of its antioxidant property and its efficacy in chelating iron and copper, needed to catalyze the formation of ROS by ADR. Cells in culture are capable of taking up LA, reducing it to DHLA finally releasing the reduced molecule³⁷.

Pierce et al³⁸ has reported the safety extended by LA, preventing apoptosis caused by tumor necrosis factor and actinomycin D in differentiated murine hepatocytes cell lines. LA, a redox-active medication and essential nutrient, is reduced within cells to form the powerful reductant DHLA. This conversion notably boosts Fas-mediated apoptosis in leukemic Jurkat cells, yet does not influence peripheral blood lymphocytes from healthy subjects³⁹. Our findings advocate that one of the factors of ADR-induced apoptosis in Vero cell may be ROS.

Figure 2: Percentage of apoptotic cells after exposure to ADR and LA Bars represent the mean±SD for 100cells/well and six wells/condition Comparisons were made between a-groups and b-groups and values are statistically significant at *p˂0.05

Lipid peroxidation:

ADR exercises its effect on normal cells, leading to the progression of serious side effects including nephrotoxicity. Ritov and colleagues⁴⁰ identified a method for specifically probing oxidative stress in membrane phospholipids within live cells, showing that the peroxidation of phosphatidylserine occurs prior to its externalization. Free radicals are involved in the pathogenesis of ADR-induced nephrotoxicity¹⁵ and cardiotoxicity¹⁶. LPO is thought to be one of the possible biochemical mechanisms involved in nephrotoxicity due to ADR¹⁵ that was measured as MDA coupled with TBA. The levels of MDA were found to be markedly elevated (p<0.05, figure 3) in Vero cells that were treated with ADR, with the highest levels occurring after 12 hours of exposure.

Figure 3: Time dependant increase in LPO on exposure of Vero cells to ADR and LA Bars represent the mean±SD for six wells/condition Comparisons were made between a-groups I and II and b-groups II and III Values are statistically significant at *P ˂ 0.05

The present results are consistent with that of Benchekroun and Robert⁴¹ who have reported an increase in LPO in rat glioblastoma cells incubated with ADR, indicating cytotoxicity. LA pretreatment effectively reduced the MDA levels to that of control group cells, thus mitigating free radical injury to the cells thereby proving its antioxidant property.

Enzyme Release:

Figures 4 represents the enzymes released into the spent media as a result of ADR-induced cell damage. The enzyme levels of LDH, NAG and γ-GT in the media were observed to be significantly higher following a 12-hour exposure, although elevated levels were noted at certain earlier time points. The release of the soluble cytoplasmic enzyme, LDH, into the extracellular space indicates damage to the plasma membrane, making LDH leakage a marker for cellular injury. The production of ROS and the leakage of LDH were notably increased in podocytes affected by ADR-induced damage⁴².

Figure 4: Effect of ADR and LA on enzyme activities in the spent medium Bars represent the mean±SD for six wells/condition Comparisons were made between a-groups I and II and b-groups II and III Values are statistically significant at *P˂0.05

Assessing the excretion levels of the lysosomal enzyme NAG could act as a trustworthy and accurate marker of kidney injury. A reduction in NAG activity within renal tissue might be attributed to damage to the lysosomal membrane induced by ADR, potentially leading to enzyme leakage. γ-GT is found on the luminal surface of the excretory and absorptive cells seen lining various glands and ducts in the body, with its highest activity levels occurring in the kidneys⁴³. The reduction in γ-GT activity within the kidneys may be attributed to enhanced tubule damage, as the brush borders of the proximal tubule continue to be the primary toxic targets. Our prior research indicated that LA has a protective effect against nephrotoxicity in rats⁴⁴.

CONCLUSION:

The inhibitory effect of LA on cell proliferation was substantiated using cell lines.LA pretreatment to cell cultures demonstrated a significant decrease in MDA content and restored the activities of key cellular enzymes (LDH, NAG, γ-GT) when challenged with ADR.Thus the study explicated the involvement of ROS in cellular injury and apoptosis, establishing the cytoprotective nature of LA.

ACKNOWLEDGEMENT:

The first author wishes to thank the Lady Tata Memorial Trust, Mumbai, India, for funding the research work. The authors also wish to thank Dr. P. Varalakshmi for her insights and guidance. The support rendered by Dr. Santhosh M, for providing us with Vero cell lines is gratefully acknowledged.

CONFLICTS OF INTEREST:

The authors declare that there are no conflicts of interest.

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Received on 01.02.2025 Revised on 18.07.2025

Accepted on 17.10.2025 Published on 16.03.2026

Available online from March 18, 2026

Research J. Pharmacy and Technology. 2026;19(3):1085-1090.

DOI: 10.52711/0974-360X.2026.00154

This work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License. Creative Commons License.