Mebendazole on Apoptosis mechanism in Cancer / Şahin and Kara _________________________________________________________________
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INTRODUCTION
The fact that drugs used in the treatment of cancer in humans and
animals have serious side effects, and the desired treatment success
is still not achieved in many types of cancer, compels scientists to
search for new strategies and treatment methods in the battle against
cancer. One of these emerging strategies is the investigation of
non–cancer drugs that may exhibit anti–cancer activity when used
in cancer treatments [1, 2].
In numerous studies examining its anti–cancer activity,
Mebendazole has been compared with conventional antineoplastic
drugs. The literature reports that the apoptosis–inducing effects of
Mebendazole, which contribute to its anti–cancer effects, primarily
stem from the destabilization of the tubulin protein an important
component of the cytoskeleton [3, 4, 5].
Paclitaxel acts by stabilizing the microtubulin structure formed
through the combination of tubulin dimers, while vincristine functions
as a microtubulin destabilizer by depolymerizing the microtubulin
protein formed through tubulin dimer polymerization [6, 7]. Both drugs
induce cell death by disrupting the mitotic division in cells undergoing
mitosis [8]. It has been documented in the literature that Mebendazole
exhibits anti–cancer activity by reducing the levels of depolymerized
tubulin [9, 10, 11]. However, there is a need for further information and
studies on the anti–cancer ecacy of mebendazole when combined
with a known antineoplastic drug. Thus, this study aims to evaluate
the anti–cancer activity of Mebendazole by comparing its effects in
combination with vincristine and paclitaxel.
The present study aims to assess the in vitro effects of mebendazole
on cell proliferation, cell healing, apoptosis induction, and caspase 3, 8,
and 9 levels in healthy and cancerous lung cells (MRC–5 and NCI–H209)
MATERIALS AND METHODS
Cell culture and reagents
Human lung broblast cells (MRC–5) and human small cell lung
carcinoma cells (NCI–H209) were obtained from the Cell Culture
Laboratory at HÜKÜK, Alum Institute, Turkey. Dulbecco's Minimum
Essential Medium (Thermo Fisher Scientic, DMEM, USA) was used
for cell culture. The media were supplemented with 10% FBS (Sigma–
Aldrich) and penicillin–streptomycin (100 U·mL
-1
Invitrogen). All cells
were maintained in a standard incubator (Nüve, EC160, Turkey) at 37°C
with 5% CO
2
. Mebendazole (Mbz), Paclitaxel (Pac), and Vincristine (Vin)
were purchased from Abcam in analytical purity (Cambridge, UK).
Cell viability and proliferation assays
Cell viability and proliferation were assessed using the MTT Cell
Proliferation assay (Biovision, Massachusetts, USA). The cells were
counted on a Thoma slide (2×10
4
cells/well), and then treated with
Mbz, Vin, Pac, Vin + Mbz combination, and Pac + Mbz combinations
at doses ranging from 0.5 to 250 µM in 96–well plates. To determine
the number of viable and proliferating cells, absorbance values were
measured using an Epoch Elisa reader (BioTek, Vermont, USA) at a
wavelength of 590 nm. The experiments were repeated three times.
Caspase–3, 8 and 9 experiments
Colorimetric caspase assay kits (Catalog no: K106, K113, K119,
Massachusetts, USA) were utilized to determine the levels of
caspases. The cells were counted (1×10
6
cells/well), and then treated
with Mbz, Vin, Pac, Vin + Mbz combination, and Pac + Mbz combinations
in 96–well plates at a dose range of 0.5–2 µM. After 24 hours of drug
administration, the experiments were conducted following the
experimental protocol. The absorbance values were measured at a
wavelength of 400 nm using an EPOCH ELISA reader (BioTek, Vermont,
USA). The experiment was repeated at least three times.
Apoptotic Deoxyribonucleic acid (DNA) laddern level
Apoptotic DNA levels were determined using an assay kit from
Biovision (Massachusetts, USA). The drug groups were applied to cells
in 96–well plates at a density of 1×10
6
cells/well, and the cells were
incubated for 24 hours. The experiments were conducted following
the protocol provided with the assay kit. At the end of the experiment,
DNA fragments were separated on a 1.2% agarose gel containing
0.5 µg·mL
-1
ethidium bromide, and the gel was analyzed using a UV
transilluminator device (Maestrogene, Hsinchu, Taiwan). The density
of DNA bands was determined using the image analysis program
(Image J 1.48s processing software, National Institutes of Health,
MD, USA). The experiment was repeated at least twice.
In vitro wound healing experiments
To evaluate cell proliferation and cell migration under in–vitro
conditions, a cellular wound healing experiment was conducted
[12, 13]. After the cells were counted (1×10
6
cells/well), they were
seeded into 6–well plates. Once the cells reached full conuence, a
standardized cellular wound was created in each plate by scratching
the monolayer cell layer. Following the application of control and drug
treatments at a dose range of 0.5–2 µM, the cells were incubated
(Thermo Fisher Scientic, DMEM, USA) in serum–free cell media for
24 hours. After the incubation period, the cellular wound areas were
visualized using a light microscope, and the resulting images were
analyzed using the image analysis program (Image J 1.48s processing
software, National Institutes of Health, MD, USA). In this experiment,
only MRC–5 cells were used since NCI–H209 cells were not suitable
for this particular assay due to their suspension nature
Statistical analysis
The experiments were repeated three times, and standard deviations
were calculated. Analysis of variance was performed using a one–way
ANOVA test, and within–group comparisons were conducted using
Tukey's test. A P–value of <0.05 was considered statistically signicant.
RESULTS AND DISCUSSION
Effect on cell viability and proliferation
Upon examining the obtained results, it was observed that cell
proliferation in NCI–H209 cancer cells decreased in all groups
administered with the drugs, depending on the dosage (FIG. 1).
Similar decreases in cell proliferation were also observed in healthy
MRC–5 cells across all treatment groups (FIG. 1). In the groups where
mebendazole was administered solely to healthy lung broblast
cells, cell viability was noted to be higher compared to other groups.
Additionally, the cell viability ratio in healthy lung cells treated with
mebendazole and vincristine was observed to surpass the cell viability
ratios obtained within the corresponding groups in cancer cells.
This implies that the cytotoxic effect of mebendazole, whether
administered alone or in combination with vincristine, is lower on