Assessment of Apoptotic Effect of Carbamazepine on Tongue Squamous Cell Carcinoma Cell Line (in vitro Study)

doi:10.21203/rs.3.rs-5047741/v1

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Assessment of Apoptotic Effect of Carbamazepine on Tongue Squamous Cell Carcinoma Cell Line (in vitro Study)

https://doi.org/10.21203/rs.3.rs-5047741/v1

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Background

This study investigates the apoptotic and anti-cancerous effects of carbamazepine on the tongue squamous cell carcinoma (HNO-97) cell line.

Methods

The research compares carbamazepine-treated HNO-97 cells with untreated HNO-97 cells as a control. The Hematoxylin and Eosin staining method was used to observe cellular morphological changes. MTT assay was performed to assess cell viability. ELISA technique was done to measure reactive oxygen species and p53 upregulated modulator for investigating the drug apoptotic and potential anticancer effect.

Results

Thestudy findings indicate that carbamazepine reduces cell viability in a concentration-dependent manner via induction of apoptosis by increase production of reactive oxygen species and p53 upregulated modulator as they led finally to induction of apoptosis in oral cancer cells, which were confirmed by ELISA. Prominent cellular apoptotic signs were detected microscopically for the oral cancer cells in both early and late stages of apoptosis.

Conclusion

The study findings suggested the potent apoptotic and anti-cancerous effect of carbamazepine on oral squamous cell carcinoma cells through the activation of the caspase apoptotic pathway. This drug may serve as a promising adjuvant therapy for oral cancers.

Carbamazepine (CBZ)

HNO-97 cell line

PUMA

MTT assay

ELISA

ROS

H&E

Oral squamous cell carcinoma (OSCC) is one of the most common human cancers, affecting the quality of life of more than 400,000 people each year, and may lead to their death [1]. OSCC causes difficulties in many aspects such as chewing and swallowing, also speech and esthetic problems that can badly impact patients’ quality of life [2].

In the past decades, despite the significant progress and development in analysis and treatments of OSCC including radiotherapy and chemotherapy, the five-year survival rate does not exceed 50% of counted cases [2]. Therefore acquiring additional information of the mechanisms underlying the incidence and improvement of OSCC would constantly facilitate the development of better novel treatment options.

Carbamazepine (CBZ) is an FDA approved antiepileptic drug that belongs to the category of dibenzazepine [3]. It causes stabilization of the inactivated state of sodium channels in the brain. This leads to less excitation of the brain cells and decreasing seizures. CBZ has also been shown to potentiate GABA receptors made up of α1, β2 and γ2 subunits. It is also known to have antimanic and other neuroprotective effects. Thus this drug is usually employed in the treatment of trigeminal neuralgia [3, 4]. CBZ has been shown to inhibit histone deacetylase (HDAC) activity, which may contribute to its neuroprotective effects. This inhibition is documented to modulate gene expression, potentially offering therapeutic benefits in neurological conditions beyond its antiepileptic properties [4].

HDAC usually contributes to the process of regulation of gene expression, thus it is known for its oncogenic manner in many types of neoplasms. HDAC inhibitors were known for their potent effect on cell cycle arrest (cell death) and also anti-angiogenic effect in some neoplasms. For illustration, vorinostat, romidepsin, and belinostat, administrated as HDAC inhibitors have been approved for some T cell lymphoma and also panobinostat for treatment of multiple myeloma [5]. HDAC inhibitors were reported to have promising antitumor effects together with standard chemotherapy and/or radiotherapy [6]. CBZ was previously found to inhibit HDAC in an in-vitro study oncolon adenocarcinoma cell line [7].

Reactive oxygen species (ROS) are known as molecules produced during cellular metabolism, which are playing dual roles as signaling molecules and oxidative stress inducers when their cellular levels are elevated [8]. Its imbalance can damage cellular components; therefore it is obviously linked to many diseases like cancer and neurodegenerative disorders. Cells are equipped with complex antioxidant defense mechanisms to counteract ROS effects. Understanding ROS interactions is vital for explaining disease mechanisms and developing therapies aimed to restore cellular balance and mitigating oxidative stress-related diseases [8].

Apoptosis is a normal controlled cell death process, which is known to be crucial for maintaining tissue health and regulating development, immunity, and response to cellular stress. Apoptosis can be triggered by various intrinsic and extrinsic signals, including DNA damage and cellular stress. Dysregulation of apoptosis can lead to various pathological conditions including cancer [9]. The p53-upregulated modulator of apoptosis (PUMA) is one of the previously known apoptotic proteins involved in the intrinsic mitochondrial pathway of the apoptotic process. It is activated/ stimulated by p53 in response to stress signals and elevated ROS production. The known and documented function of PUMA is to neutralize anti-apoptotic proteins, leading to mitochondrial outer membrane permeabilization and also the initiation of the apoptotic cascade [9]. PUMA is usually detected in regulation of apoptosis, which is known to be implicated in numerous physiological and pathological processes. Therefore PUMA is a potential target for therapeutic intervention in many pathological conditions like cancer [10].

This study aims to assess the apoptotic and anti-cancerous effects of CBZ on the OSCC cell line by performing MTT assays and measuring ROS and PUMA cellular expression via enzyme-linked immunosorbent assay (ELISA) technique. The novelty lies in exploring CBZ’s potential as an anticancer effect by various methods that was not tested previously and to determine its clinical apoptotic and anti-cancerous effect alongside of its antiepileptic role.

Chemicals and Materials

The human SCC cell line (HNO-97) from the tongue was acquired from Nawah Scientific Center (Almokattam, Cairo, Egypt) and it was stored in liquid nitrogen containers at -196 C°. CBZ was obtained from Novartis Pharmaceutical Company (Cairo, Egypt). Fetal Bovine Serum (FBS) was sourced from GIBCO (USA). Phosphate-Buffered Saline (PBS) was obtained from ADWIA Company (Egypt), and was procured from Sigma Aldrich Company (USA). Sodium Bicarbonate (2%) and penicillin–streptomycin antibiotics (1%) were purchased from Invitrogen Company (USA) RPMI 1640 medium with L-Glutamine was acquired from Sigma Aldrich Company (USA).

For the MTT viability assay, Dimethyl Sulfoxide (DMSO) and isopropanol reagents were obtained from Sigma Aldrich Chemicals Company (USA). Regarding the agents used in morphological analysis by H&E staining, EOSIN Y (free acid) and Hematoxylin (Hydroxybrazilin) were both purchased from MyBioSource (San Diego, CA, USA). In addition to Ethyl Alcohol used in this technique was obtained from Elgomhorrya Company (Egypt) which was also used in this technique.

The Human ROS ELISA Kit (catalog no. AMS.E01R002) and also the Human PUMA ELISA Kit (catalog no. MBS2500852) were both purchased from MyBioSource (San Diego, USA).

Preparation of CBZ Drug

The CBZ drug solution was prepared at a concentration of 10 mg/mL and dissolved in dimethyl sulfoxide (DMSO). It was then serially diluted in serum-free medium. The dissolved CBZ in DMSO used in experiments at different concentrations (0.01ug/ml- 10000 ug/ml) to calculate the IC50% The diluted drug was added to the intervention group only pre-culture HNO-97 cells (t/HNO-97) in a 96-well plate (2.19×104 cells/cm2). The plate was microscopically assessed post treatment after 24 hours.

Culturing of Cell Lines

The procedures were conducted at the International Centre for Advanced Research (ICTAR) in Egypt. The laminar flow hood was sanitized with 70% alcohol and exposed to UV light before use. RPMI 1640 medium supplemented with 10% FBS and 1% penicillin-streptomycin was used for cell culture. Cells were incubated at 37°C with a 5% CO2 incubator. Cell viability was monitored using an inverted phase contrast microscope. Cells were maintained by three washing with PBS, treating with trypsin, and then resuspended in growth media. The cells were cultured in 96-well plates and incubated until confluence [11].

Cell cultures were inspected under an inverted microscope, and upon reaching 70–80% confluence, cells were subcultured. The medium was removed, cells were washed with PBS, and trypsin was added. A trypsin Solution (0.25%) was prepared by dissolving 0.25 grams of powdered trypsin in 100 mL PBS (pH 7.4). After detachment, fresh RPMI 1640 medium with 10% FBS was added to inhibit trypsin. Cells were dispersed, resuspended, and plated in new culture flasks or 96-well plates as needed [12]. Finally we have two main study groups, the intervention group composed of HNO-97 cells treated with CBZ drug (t/HNO-97) and the control group, composed of untreated HNO-07 cells (C/HNO-97)

Measuring Cell Viability Using MTT Assay:

The MTT assay quantifies viable cells based on mitochondrial activity. Cells were treated with CBZ in the intervention group (t/HNO-97) and absorbance was measured at 570 nm. The half-maximal inhibitory concentration (IC50) represented the concentration of the tested CBZ drug required to inhibit 50% of a specific activity. The IC₅₀ dose was determined using MASTER PLEX 2010 software, for cytotoxicity and cell viability calculation [13].

Morphological analysis using H&E staining:

H&E staining was performed using EOSIN Y (free acid) and Hematoxylin (Hydroxybrazilin). After application of CBZ and preparation of the study groups, cells were collected and centrifuged, then processed with ethanol to form a pellet. Cell pellets were gently removed from the tube and placed into a cassette lined with biopsy filter paper. Subsequently the pellet cells were resuspended in PBS, and 50µL was applied to a sterilized glass slide. Cells were fixed by ethanol on slides, rehydrated, and stained with H&E staining. The slides were then dehydrated using ascending concentrations (50%, 70%, and 100%) of ethyl alcohol. The prepared H&E slides were mounted, and examined by Leica light Microscope (Germany) for morphological examination [14].

Sample Preparation for ELISA:

Cells were spun down, washed with PBS, and resuspended in a 1.0 mL of cell lysis buffer containing phenylmethanesulfonyl fluoride (1.0 mM PMSF, Cell Signaling Technologies) to the adhered cell layer in each well. The supernatant was diluted for the ELISA assay [15, 16]. We followed guidelines documented in previous study Afifi et al., (2022) regarding ELISA technique, as for ROS samples were incubated with ROS HRP conjugate, washed, and the reaction was developed with substrate solution. Absorbance (the optical density) was precisely identified and quantified at 450 nm using a microplate reader [17]. For PUMA ELISA, samples were added to pre-coated wells, incubated with detection antibodies, and developed with substrate solution. Absorbance (the optical density) was precisely identified and quantified at 450 nm using a microplate reader [17].

Statistical Analysis

Analysis was performed using IBM SPSS version 25 (Statistical Package for the Social Sciences; IBM Corp, Armonk, NY, USA) and Microsoft Excel for data handling and graphical presentation. The normality of the data was assessed with the Kolmogorov-Smirnov Test. Data were normally distributed and comparison was done between the two study groups using an independent t-test. Results were considered significant when p < 0.05.

Cell viability and cytotoxicity MTT assay:

The cell viability percentage in the intervention group (t/HNO-97) showed gradual decrease with increasing concentrations of the CBZ. In a 96 well plate, the living cells were detected by forming purple formazan crystals. In contrast, the non-living cells were clinically observed in yellow color (Fig. 1). The lowest value of cell viability was recorded as (9.31%) by applying the highest concentration of the CBZ drug (10000 µ/ml). While the highest cell viability (101.04%) was recorded at the lowest concentration of CBZ (4.5 µ / ml). The IC₅₀ value of the drug in the intervention group was calculated to be 220 µg/ml. The values were listed in the following table (table 1) and plotted in (Fig. 2).

Figure (1): A photograph showing the color changes in the MTT in the intervention group (t/HNO-97). The living cells were detected by purple formazan crystals, while the dead cells appeared yellow in color.

Table (1): Variable concentrations of CBZ with corresponding cell viability % of the intervention group.

t/HNO-97
Drug Concentration (ug/ml)	Viability%
10000	9.31
5000	13.13
2500	20.69
1250	24.72
625	41.88
312.5	70.97
156	99.10
78	100.21
39	100.56
19.5	101.94
9.5	99.24
4.5	101.04
IC50	220.1

Figure (2): A linear graph showing viability % of cells at different concentrations of CBZ.

Morphological Analysis by H&E Staining:

a. The Control Group (c/HNO-97)

The untreated malignant OSCC cells in this group showed cellular and nuclear pleomorphism, nuclear hyperchromatism and an increase in the nuclear/cytoplasmic ratio (Fig. 3A).

b. The Intervention Group (t/ HNO-97)

Malignant squamous treated with CBZ drug revealed apoptotic cells at early and late stages of apoptosis. The cells showed cellular and nuclear shrinkage, peripheral condensation of chromatin, nuclear fragmentation and apoptotic bodies. Swollen necrotic cells with ruptured cell membrane were also detected (Fig. 3B, 3C, 3D).

Figure (3): photomicrographs of the study groups, (a) cell lines in the control group showing pleomorphic malignant cells with nuclear hyperchromatism (red arrow) and an increase in the nuclear/cytoplasmic ratio (black arrow). (b) Showing cells in the intervention group with shrunken apoptotic cells & nuclei (red arrows), peripheral condensation of chromatin (blue arrows), and nuclear fragmentation (green arrow) and apoptotic bodies (yellow arrows). (c) Showing cells in the intervention group with shrunken apoptotic cells & nuclei and irregular cell membranes (red arrows), apoptotic bodies (yellow arrows). (d) Showing cells in the intervention group with shrunken apoptotic cells & shrunken nuclei and irregular cell membranes (red arrows), apoptotic bodies (yellow arrows) and swollen necrotic cells with ruptured cell membrane (gray arrows) (H&E, x1000, Oil).

Expression of ROS Using ELISA:

Regarding the reported values for the ROS, The intervention group (t/HNO-97) yielded higher values of ROS expression (477.9 ± 12.3) than the control group (c/HNO-97) (126.6 ± 9.9). The independent t-test revealed a significant difference between both study groups as (p = 0.001), (table 2).

Table (2): ROS expression levels among the experimental groups

Sample		Results	t-value	p-value
Groups	Cells	ROS g/ml (Mean ± SD)
Intervention group	t /HNO-97	477.9 ± 12.3	31.460	0.001
Control group	c/HNO-97	126.6 ± 9.9
Significant at p < 0.05*

Expression of Apoptotic Marker (PUMA) Using ELISA

Regarding the reported values for the PUMA expression, statistical analysis revealed that the level of expression of PUMA was higher in the intervention group (t/HNO97) (2.5 ± 0.07) than the control group (c/HNO-97) (0.99 ± 0.06).The independent t- test revealed a significant difference between both study groups as p = 0.002 (table 3).

Table (3): PUMA expression levels among the experimental groups

Sample		Results	t-value	P-value
Study groups	Cells	PUMA ug/ml (Mean ± SD)
Intervention group	t /HNO97	2.5 ± 0.07	22.3	0.002*
Control group	c/HNO-97	0.99 ± 0.06

*significant at p < 0.05

The pragmatic sharp rise in the incidence of OSCC as more than 400,000 people worldwide, beside the documented side effects related to the administration of most of the chemotherapeutics agents were potent essentials for constant searching about other effective alternatives for treatment of OSCC [1]. Many reported issues regarding mastication, swallowing, communication and aesthetics are reported as a complication of such OSCC, which is considered as one of the most prevalent cancers nowadays [1]. These documented complications usually have a bad impact on the patient’s quality of life. Thus, there are continuous notable advancements in cancer research regarding development of new therapies especially the anticancer target therapy for such critical disease with high rate of mortality[2].

CBZ is typically used to treat clinical trigeminal neuralgia cases as it possesses antimanic and potent neuro prophylactic properties [3, 4]. CBZ drug was chosen (as an antiepileptic drug) in the current study, as it was reported previously that combination of CBZ with conventional chemotherapy and/or radiation therapy enhances their antitumor benefits [16]. In a previously performed in-vitro study on cancer cell line, CBZ was discovered and reported with its ability to inhibit HDAC [5–7].

The anticancer effect of HDAC inhibitors was earlier confirmed through a documented case-control study by Salminen et al. (2016), who concluded that the use of antiseizure drugs such as CBZ (HDAC inhibitor) could potentially reduce the clinical risk of developing malignancy. They compared the use of antiepileptic drugs among individuals with prostate cancer (cases) to those without prostate cancer (controls) [18].

Angiogenesis-related genes including vascular endothelial growth factor and Hypoxia-inducible factor were found to be down-regulated in response to the anticancer and antiangiogenic effects of HDAC inhibitors [5, 6]. To date, according to our knowledge, Akbarzadeh et al. (2016) has reported only the cytotoxic potentials of CBZ against human colon cancer (SW480) cell line [16]. This study was followed by Sohaiband Ezhilarasan, 2020, that suggested CBZ drug to be a promising anticancer agent in treating colon adenocarcinoma cell line (HT-29 cells); this was attributed to its robust HDAC inhibitory property [7].

This study novelty was to assess the anticancer effect of CBZ drug on OSCC cell line which was not previously detected on this type of malignant tumor cells, and ensure if CBZ can be clinically introduced as adjuvant anticancer agent. The tongue SCC cell line was chosen in this study owing to its simplicity in methods of extraction and cultivation[17].

The MTT assay was used to assess the cytotoxicity of CBZ in HNO-9 cells, as it is one of the most commonly utilized cytotoxicity assays according to the current literature. This is attributed to its availability, simplicity, inexpensive and affordable type of techniques, (considered as a gold standard assay) to assess cytotoxicity [17, 19].

Regarding our findings in the MTT Assay, the cell viability percentage in the intervention group (t/HNO-97) showed gradual decrease with increasing concentrations of the CBZ drug. Similar results were documented through an in vitro study by Sohaib and Ezhilarasan (2020), who reported the cytotoxicity of the CBZ drug after giving various doses to adenocarcinoma cell line (HT-29 cells) for 24 hours. CBZ administration caused a significant (P < 0.001) and concentration-dependent cytotoxicity in HT-29 cells at concentration ranges of 5, 10, 20, 40, and 80 µg/ml They previously suggested that the cytotoxic potentials of CBZ drug was attributed to the accumulation of ROS intracellular molecules in HT-29 cells. Which finally will elevate the induction of the oxidative stress, which subsequently damages the cell membrane thus; it will trigger the process of cellular apoptosis [7].

For establishment of the proper CBZ drug dose, IC50 value on (t/HNO-97) cell line was determined in our study. This is a very informative test, which is widely used in laboratories to measure drug efficacy [20].In our study the IC50 of CBZ drug in (t/HNO-97) group was estimated to be 220 µg/ ml by the MTT assay, ensuring that this dose of CBZ is required to inhibit 50% of TSCC cell viability and growth.

In a similar and previously published study done by Akbarzadeh et al. (2016) who also determined the IC50 value for CBZ and reported it to be lower than the IC50 value recorded in this study, as they were assessing anticancer effect of Valproic acid (VPA) and CBZ on colon cancer cell lines. The difference in the previously mentioned values is attributed to the difference in the type of cell lines used in the experiments [16].

Examination of cellular morphological changes under ordinary transmitted light microscopy after H&E staining of cell lines in both study groups was done in this study. H&E staining is considered as a gold standard diagnostic staining for examination of histological and cytological changes in morphology [21, 22]. According to the scientific literature the process of apoptosis was microscopically distinguished by some characteristic morphological changes in the cell as (cellular shrinkage, dense chromatin condensation and formation of apoptotic bodies). All these features could be figured out easily from our documented photomicrographs of H&E-stained slides in the intervention group (t/HNO-97). Our reported apoptotic cellular signs in SCC cell lines stained by H&E staining are similar to the findings documented previously by Mohamed Aziz et al. (2021) as they investigated apoptotic effect of graviola on HNO-97 cell line [23]. In addition to another study done by Oh et al. (2020) who investigated the apoptotic effect of Formononetin, (a phytoestrogen extracted from herbal plant) on pharyngeal SCC cell line [24].

The ELISA technique was used as a method of quantification of ROS activity in our study as it is a well-known available and affordable test. We used to quantify ROS cellular expression in this study as previous studies have suggested that HDAC inhibitors can cause cytotoxicity in a range of cancer cells by generating ROS [17].

Results have shown that the (t/ HNO-97) group has statistically significantly higher measured values of ROS expression by ELISA than the (c/ HNO-97) group. The reported findings regarding ROS expression were on the same line with a study by Eghbal et al. (2013). It was found that the treatment of CBZ drug in freshly isolated rat hepatocytes induced oxidative stress, which cumulated the generation of ROS and products of lipid peroxidation. Also, another study previously tested the effect of Syzygiumcumini on OSCC, documented those treatments caused cell death and increased the presence of ROS within the cells, leading to cellular damage and apoptosis. The OSCC cells in those studies showed elevated levels of apoptosis and morphological changes in membrane composition [25, 26].

In the current study, quantification of PUMA expression as potent cellular apoptotic indicator cellular was performed by ELISA for both study groups. This test is used in some reported studies as an alternative indicator for caspase − 3, which was studied previously in an invitro study [16]. PUMA was chosen for its implication in triggering intrinsic pathways of the process of cellular apoptosis. In addition, it played a new function in controlling the anticancer effects of MEK inhibitors [17, 27]. Regarding our PUMA expression findings, it was clearly observed in (t/HNO-97) with statistically significantly greater values than in (c/HNO-97). Illustrating this point form cellular/molecular aspect, the DNA damage usually activates accumulation of ROS then the ATM/ATR-p53-PUMA-Bax oligomerization then cytochrome c release and therefore leads to caspase-9 activation pathway. After the activation of initiator caspase (caspase-9), a final activation of caspase-3 will take place, thus leading to start the mechanisms of cellular apoptosis.this mechanism was clearly illustrated by Sohaib and Ezhilarasan (2020), who documented the potential apoptotic effect of CBZ drug on adenocarcinoma cell line (HT-29 cells) by measuring caspase-3 cellular levels [7].

Our reported findings were compared to another previous reported study which used VPA drug, which was found to increase the cytotoxicity of temozolomide (a drug used to treat specific types of brain cancer) by inducing apoptosis via boosting the activation of the p53 pathway and elevating the expression of its downstream target gene, PUMA [28]. Taken together with the previously mentioned studies and the current findings of decreased cell viability, increased cellular ROS and PUMA cellular levels induced by CBZ drug in OSCC cells, we strongly suggest that this CBZ drug has anticancer potentials against OSCC. Our provided data is likely to be confirmed by further in-vivo studies. However, we acknowledge the limitations of our in vitro study, including the lack of the vivo validation and the potential differences in drug effects in a living organism compared to cell cultures.

Depending on our gathered information, the potent apoptotic effect of CBZ drug, which was confirmed through MTT Assay after investigation for the first time on HNO-97 cell line. Also the significant elevation in the accumulated ROS concentration found in CBZ treated OSCC cells is responsible for the morphological changes related to apoptosis and increased PUMA expression, which will finally activate the caspase pathway for cellular apoptosis. Therefore, CBZ may be introduced in anticancer adjunctive treatment modality.

CBZ

Carbamazepine

DMSO

dimethylsulfoxide

ELISA

enzyme-linked immunosorbent assay

FBS

Fetal Bovine Serum

H&E

Hematoxylin and Eosin

HDAC

Histone deacetylase

MTT Assay

3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide) assay

OSCC

Oral squamous cell carcinoma

PBS

phosphate buffer saline

PUMA

p53-upregulated modulator of apoptosis

ROS

Reactive oxygen species

VPA

Valproic acid

Authors’ Contributions

RahafAbdelWahab: Conceptualization, Writing original draft preparation and investigation.

Mohsen Kazem: Conceptualization and reviewing final manuscript

Mai Elhalawany: reviewing and editing.

Heba Ahmed Saleh: investigation, formal analysis, reviewing and editing

Funding: this work was self-funded by the first author

Data availability: The data supporting the conclusions of this article is included within the article

Ethics approval: All experimental protocols were approved by the Ethical Committee of Faculty of Dentistry, Cairo University. This study was registered with no (11 11 22) after reviewing the protocol.

Consent to participate: “not applicable”.

Competing interests: The authors declare no competing interests.

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No competing interests reported.

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13 Sep, 2024
Submission checks completed at journal
13 Sep, 2024
First submitted to journal
07 Sep, 2024

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Assessment of Apoptotic Effect of Carbamazepine on Tongue Squamous Cell Carcinoma Cell Line (in vitro Study)

Status:

Version 1

Abstract

Background

Methods

Results

Conclusion

Figures

Introduction

Materials and Methods

Chemicals and Materials

Preparation of CBZ Drug

Culturing of Cell Lines

Measuring Cell Viability Using MTT Assay:

Morphological analysis using H&E staining:

Sample Preparation for ELISA:

Statistical Analysis

Results

Cell viability and cytotoxicity MTT assay:

a. The Control Group (c/HNO-97)

b. The Intervention Group (t/ HNO-97)

Expression of ROS Using ELISA:

Expression of Apoptotic Marker (PUMA) Using ELISA

Discussion

Conclusion

Abbreviations

Declarations

References

Additional Declarations

Status:

Version 1