Exploring the Potential Therapeutic Efficacy of Diclofenac on Lymphoblast Cells

Abstract

Nonsteroidal anti-inflammatory drugs are the wide group and they show upon their action on the diverse forms of cyclooxygenase (COX). Diclofenac is one of the NSAIDs which is prescribed for the pain treatment of varied pathophysiological conditions. However, various studies have revealed caution about the safety of the drug diclofenac when it is considered for the treatment and while determining the necessary dose required for the patients concerned as overall NSAIDs show around 20-25 % of adverse drug reactions (ADR) after oral administration. Moreover, diclofenac is a choice of clinicians for varied context in the field of oncology. NSAIDs have a central role in chemoprevention and therefore might have a profound application in the settings of therapeutic regime. This particular drug has a pre tested activity for the pain therapy related to cancer and demonstrated diminished growth with reduced level of vascularisation in in vivo model study. Past study findings highlighted that drug diclofenac demonstrated the most potent activity via inhibition of cell growth and apoptosis. The mechanistic pathway of enhanced level of ROS results in a chain of events such as membrane depolarization of the mitochondria, discharge of the cytochrome c, activation of the caspase pathways and followed by the fragmentation of the nuclear materials. The purpose of this investigation was to determine the efficacy of the drug diclofenac for its action upon the lymphoblast cells, i.e., to observe the death rate of the cells with the drug application. The rationale of choosing this topic is to investigate the impact of NSAIDs specifically diclofenac upon the proliferation and redox status of lymphoblasts which would help to comprehend better the role of lymphoblasts in cancer. Therefore, to determine, the membrane redox potential alterations of T lymphoblastoid cell line named CCRF-CEM cells with and without the drug diclofenac the Reazurin assay was conducted which also determined the LC50 dosage in vitro and trypan blue assay was conducted to measure the cytotoxicity. The study findings highlighted that the drug diclofenac at the concentration of 5.00E – 02 showed the highest value of the Mean fluorescent intensity via the Reazurin assay and the lowest number of dead cells at the same concentration via the trypan blue assay. Therefore, diclofenac may be considered as a control therapy of the patients suffering from any kind of malignancies.

Introduction:

Nonsteroidal anti-inflammatory drugs (NSAIDs) are the wide group that is considered to be pharmacodynamically and pharmacokinetically miscellaneous and they show upon their action on the diverse forms of cyclooxygenase (COX) (Kołodziejska, 2018). However, this group of drugs varies in their half life, action pathways, and their dissemination at the site of inflammation within the body. The commercially available products also show variations at their dosage specifications and varied forms (Kołodziejska, 2018). Scientific evidences have also revealed that researchers are persistently trying to formulate a safer form of the drug with enhanced efficacy as the group of drug is effective for different inflammatory conditions such as osteoarthritis, rheumatoid arthritis, psoriatic arthritis, lupus erythematosus, ankylosing spondylitis, rheumatic fever and gout (Pereira-Leite, 2013). Diclofenac is one of the NSAIDs which is prescribed at a dosage of 150mg each day for the pain treatment of the above mentioned conditions. This particular drug is considered to be more efficacious in comparison to the other commercially available NSAIDs such as naproxen, ibuprofen, and celecoxib even at their highest prescribed doses (Pereira-Leite, 2013). As reported by varied investigation the drug diclofenac is considered as the reference drug or choice of clinicians for the treatment of osteoarthritis (Farmakologia, 2015). However, various studies have revealed caution about the safety of the drug diclofenac when it is considered for the treatment and while determining the necessary dose required for the patients concerned (da Costa, B.R., 2017; Farmakologia, 2015; Pereira-Leite, 2013). It is also evident that overall NSAIDs show around 20-25 % of adverse drug reactions (ADR) after oral administration (Pavelka, 2012). The adverse reactions occur when the medication is continued for prolonged period and the reactions are malfunctioning of gastrointestinal tract, central nervous and cardiovascular system, and renal system. However, there are also variations observed in terms of contraindications, ADR and therefore faces restriction on their usage (Pavelka, 2012).

However, this particular drug is a choice of clinicians for varied context in the field of oncology (Pavelka, 2012). There is a recent upsurge that NSAIDs have a central role in chemoprevention and therefore might have a profound application in the settings of therapeutic regime (Kowalski, 2011). The drug diclofenac shows varied reactions within our body such as immunological reactions, in the aspect of radiation and chemo sensitivity, and metabolic pathway of tumour. This particular drug has a pre tested activity for the pain therapy related to cancer, and is utilised in the topical form for the treatment of a pre-cancerous lesion known as actinic keratosis (Kowalski, 2011). Preclinical evidence with regard to cancer therapy in rat models have revealed that when chemically stimulated forms of tumours are treated with diclofenac demonstrated diminished growth with reduced level of vascularisation (Michałowska, 2011). However, further in vivo model study revealed that this particular drug enhanced the flow of blood within the tumour which might involve any particular mechanism of prostaglandins for determining blood vessels permeability (Michałowska, 2011). Diclofenac was also tested within the neuroblastoma cell lines and xenograft models for determining its activity (Wełnicki, 2012). The study of Johnsen et al revealed that there was an enhanced expression of COX-2 among 27 tissue samples extracted from 28 paediatric patients suffering from neuroblastoma (Johnsen, 2004). In vitro study have revealed that diclofenac inhibited the growth of the neuroblastoma cell line with an IC50 value ranging in between 100 to 600 μM and had revealed enhanced level of apoptosis with diclofenac (Johnsen, 2004). Similarly another research group investigated the combined effect of NSAIDs on the cell lines of ovarian cancer namely CAOV-3, SKOV-3, 36M2 and SW626 (Pantziarka, 2016). The study findings highlighted that drug diclofenac in the concentration range of 20–200 μM along with other drug such as sulindac sulphide demonstrated the most potent activity via inhibition of cell growth and apoptosis among all the four cell lines (Pantziarka, 2016). Other study on cell viability showed that diclofenac application revealed diminished viability of cells at concentration of 50 μM among the serous ovarian adenocarcinoma cell lines HEY and OVCAR5 cell line and at a concentration of 250 μM for the UCl-101 line (Sriuttha, 2018). Scientific investigation has also revealed that diclofenac could potentially induce high level of apoptosis and diminished level of proliferation of the Cutaneous T-cell lymphomas (CTCL) cells which is responsible for mixed assortment form of non-Hodgkin's lymphomas of the skin (Fischer, 2011). The drug also demonstrated further sensitization of the TRAIL-provoked apoptosis. It is already established that with treatment of NSAIDs the caspase cascade gets activated which involves caspase-3, -8, -9 whereas there is reduced potential observed for the membrane of mitochondria (Johnsen, 2005). The study investigation also highlighted that cytochrome c was observed to be released only with the application of diclofenac. Therefore these studies provided a scientific base for the utilisation of NSAIDs specifically diclofenac for the therapy of the patients suffering from Cutaneous T-cell lymphomas (Fischer, 2011; Johnsen, 2005).

Therefore, for the purpose of this assignment, the investigation will be carried out to determine the efficacy of the drug diclofenac for its action upon the lymphoblast cells, i.e., to observe the death rate of the cells with the drug application. However, there will be one concern to study the effect of drug also upon the healthy normal cells of the body. The rationale of choosing this topic is to investigate the impact of NSAIDs specifically diclofenac upon the proliferation and redox status of lymphoblasts which would help to comprehend better the role of lymphoblasts in cancer which is one of the leading cause of mortality throughout the globe. As it is also evident that the causal factors of cancer is variable in most of the cases, therefore instead of conducting any pinpoint research investigation we can study upon the control therapy for the cure of the disease which will be contribute to the existing scientific knowledge and will never be a waste. Moreover, the topic chosen for this research is a major concern in the present state so any research showing any positive impact in this field can contribute to take a step forward for the treatment of the disease for those individuals who are suffering from cancer. Thus, the aim of the project is to determine the efficacy of the drug diclofenac for its action upon the lymphoblast cells versus the normal healthy cells via the cell viability and death assay.

Materials and Methodologies:

Chemicals used for the assay:

Diclofenac (provide Company name with product code), Hydrogen peroxide (provide Company name with product code), Absolute ethanol (provide Company name with product code), Phosphate Buffer saline (PBS) (provide Company name with product code), Trypan Blue dye (provide Company name with product code), Resazurin dye (provide Company name with product code), RPMI 1640, Foetal Bovine Serum, L-glutamine, antibiotics namely penicillin, ampicillin and streptomycin (provide Company name with product code for all).

Cell Line used:

T lymphoblastoid cell line named CCRF-CEM cells were procured from European Collection of Authenticated Cell Cultures (ECACC, Catalogue No. 85112105).

Media used for cell maintenance and growth:

RPMI 1640 with 10% foetal bovine serum supplemented with 2mM L-glutamine and 10% antibiotics namely penicillin, ampicillin and streptomycin. Provide details with Company name and product code, Country details.

Instruments used:

Provide details of the spectrophotometer scanner, company details, country, and instrument code.

Inverted and Light Microscope details

Methodologies used:

Cell Culture and Maintenance:

T lymphoblastoid cell line named CCRF-CEM cells were cultured using the complete growth medium RPMI 1640 with 10% foetal bovine serum supplemented with 2mM L-glutamine and (10%) antibiotics namely penicillin, ampicillin and streptomycin. 5x10^5 cells/ml were cultured in T25 (containing 5ml media) flasks via suspension method under complete aseptic condition with sterile complete growth medium and allowed to grow within 5% carbon dioxide incubator at 37° C for the next 48 hours. The cell media and concentration was observed every day and fresh media was recharged every 48 hours (Yssel, 2005). When the cells reached around 80% confluency, observed under inverted microscope the splitting of cells were done to maintain the concentration of cells around 5x10^5 cells. All the steps were conducted with biosafety cabinet level 2 to maintain the aseptic condition with sterile media solutions and chemicals (Yssel, 2005).

Preparation of working concentration of the drug diclofenac:

To assay the complete solubility of the drug diclofenac the solvent used are distilled sterile water, sterile Phosphate buffer saline (PBS) and ethanol (10%, 20% and 100%). It was observed the drug was completely soluble in absolute (98 – 100%) ethanol solvent. Therefore, 0.08g of diclofenac was dissolved in 50 mmol per Litre of absolute ethanol to conduct the cell viability assay. Then the final solution was filtered using sterile syringe 0.45um filter and all the steps were conducted with biosafety cabinet level 2 to maintain the aseptic condition. Absolute ethanol without the drug diclofenac of same dilutions was used as negative control for the cell viability assay. For positive control the same dilution of hydrogen peroxide solution was used.

Determinations of the LC50 of the drug diclofenac using the Resazurin dye Assay:

LC50 is considered to be the dosage of the drug that kills around 50% of the cells while culturing the cells i.e., the cytotoxicity. Reazurin assay is the assay in which the compound can be used to measure the metabolism level of the cell that act as an indicator for the varied physiological processes such as the cell proliferation, viability and the cyto-toxicity (Präbst, 2017). This is considered to be a colorimetric assay where the nonfluorecent dye named resazurin undergoes chemical reactions to get reduced to form the strong fluorescent compound named resorufin which is purple or pink in colour. The basic principle behind this reaction is that the live cells contain the mitochondrial reductase enzymes which could show the reduction of the chemical resazurin to resorufin. The major beneficial features with this assay are: it is of the same principle of the tetrazolium assay (MTT) and it is extremely easy to carry out and inexpensive. Then the resultant solution colour was quantified within the range of the wavelength 500 to 600 nm with the help of a multi well scanner or spectrophotometer. The intensity of the colour of the solution has a direct relation with the viability of the cells (Präbst, 2017). 1x106/ml concentration of CCRF-CEM cells (sterile condition) was used for the cell viability assay. 100 µl solutions of cells within the complete growth medium was dispensed into each well of the 2 x 96 wells microtitre plates. Then 100 µl of the drug solution was added to the first well and aspirated well for mixing. Following this, a series of 10-fold dilutions was conducted up to 6 times using the sterile diclofenac solution. Column 1 – 6 of the microtitre plates were used for the drug concentrations, column 7 – 12 were used for the negative controls of ethanol, only media containing live cells. Then the microtitre plate was allowed to incubate within 5% carbon dioxide incubator at 37° C for the next 48 hours. Then 20 µl of sterile Resazurin dye was added to all the wells and incubated for another 2 hours at 37° C within 5% carbon dioxide incubator. Then the plates were scanned using spectrophotometer set at the excitation wavelength 540 nm and emission wavelength 570 nm to read the fluorescence (Präbst, 2017).

Pattern of experimentation in a 96 well micro-titre plate:

From this experiment we have to determine the LC50 value and the redox potential with and without the drug diclofenac (Präbst, 2017).

Trypan blue assay:

The trypan blue assay helps to determine the viable status of cells within a suspension of cells. Therefore it is also being referred as the dye exclusion method. The assay is considered because of the basic principle that it used to determine to distinguish the status of cells are that the live cells possess exact cell membrane that do not allow the entry of certain dyes like trypan blue, eosin and propidium in comparison to the dead cells (Strober, 2015).

The optimization process of this particular assay was carried out in a 96 well microtitre plate which will include both the positive and the negative controls. The cell suspension will contain the number of cells and this will correlate with the Reazurin assay. To each of the wells of the plate, 100 µl volumes of cells will be added which will be followed by the same volume of 100 µl of saponin or hydrogen peroxide. For every group of the experiment four wells were dedicated and at the end of the experiment the contents of the wells were pooled using the Gilson pipette and the cells were stained by the trypan blue assay procedure. The microtitre plate was allowed to incubate at the at 37° C within 5% carbon dioxide incubator for the next 48 hours following which the trypan blue assay procedure was carried out (Strober, 2015). 20 µl of the cell suspension of the concentration 5x10^5 cells/ml that was used to seed the T25 flask for culturing the cells was taken in a separate 1.5mL of eppendorf or centrifuge tube and mixed with 0.4% of trypan blue dye diluted with phosphate buffer saline solution. The cell suspension and the dye solution was mixed with a ratio of 1:1 and was kept for strictly 3 minutes (maximum) for mixing and staining of the cells. Both the solutions were aspirated well for proper mixing and after 3 minutes and within 5 minutes a drop of the mixture was counted using the haemocytometer for the number of viable cells with the aid of light microscope. Then the unstained (viable) cells were counted along with the stained (dead) cells separately using the haemocytometer chamber. The calculation of the total number of viable cells observed and present within 1 mL of the aliquot of the mixture of cell suspension and trypan blue dye was the multiplication of the total number of viable cells with two which is the dilution factor of the cell suspension with the dye trypan blue. Moreover, the total number of cells present within the aliquot was calculated by the summation of total number of viable and unviable cells observed under the microscope and multiplying the number with two. The percentage of the viable cells was counted by using the below mentioned formula as: Viable cells (%) = Total number of unstained cells present within 1 mL of the aliquot * 100 Total number of cells present within 1 mL of the aliquot For this assay the positive control used is the hydrogen peroxide for measurement of cell death. 1% H2O2 has been used as positive control for both the Reazurin assay and the trypan blue assay (Strober, 2015).

Pattern of experimentation in a 96 well microtitre plate:

Statistical Analysis:

All the experiments were conducted thrice and the mean ± SEM of the data were plotted and a paired T – test was conducted to carry out the statistical significance of the data. T test was considered for each comparative variable and if the p value is less than 0.05 then it is considered to be significant.

Results:

The redox potential with and without the drug diclofenac

Figure 1: Measuring redox FIU using Resazurin assay of CCRF-CEM cells exposed to different concentrations with and without diclofenac. Graph represents Mean ± SEM, N=8 of live and dead cells . For statistical analysis a paired t-test was performed (*): p≤0.05.

The value obtained for the control solvent 1% H2O2 is 900 FIU.

The determination of LC50 value of the drug diclofenac on the cell line CCRF-CEM cells using the Resazurin assay

Figure 2: Determination of LC50 value of the drug diclofenac on the cell line CCRF-CEM cells using the Resazurin assay

The determination of cytotoxicity using the Trypan blue assay

Measuring cytotoxicity using Trypan Blue assay. Typran blue assay of dead CCRF cells exposed to different concentrations without diclofenac. Graph represents Mean ± SEM, N=8 of dead cells. Graph represents Mean ± SEM, N=8.

Discussion:

Reazurin assay is the assay in which the compound can be used to measure the metabolism level of the cell that act as an indicator for the varied physiological processes within the cells (Präbst, 2017). The trade name of the compound is alamer blue which is generally considered to be non toxic in nature only for the shorter span of incubation period as suggested within the manufacturer’s protocol (Präbst, 2017). The basic principle behind this reaction is that the live cells contain the mitochondrial reductase enzymes which could show the reduction of the chemical resazurin to resorufin (Erikstein, 2010). Moreover, it is also mentioned that the intensity of the colour of the solution has a direct relation with the viability of the cells (Präbst, 2017; Erikstein, 2010). Live cells produces a high intensity of fluorescence in comparison to dead cells that do not produce any fluorescence as their internal reduction oxidation potential has been altered due to the presence of the drug diclofenac (Präbst, 2017). This is a colorimetric assay where the nonfluorecent dye named resazurin, blue in colour, undergoes chemical reactions to get reduced to form the strong fluorescent compound named resorufin which is purple or pink in colour (Präbst, 2017). It was obtained from the first figure that the mean concentration of the drug diclofenac that showed the highest value of Mean fluorescent intensity (FIU) is 5.00E – 02 (Refer Figure: 1). As mentioned above that LC50 is considered to be the dosage of the drug that kills around 50% of the cells while culturing the cells i.e., the cytotoxicity (Erikstein, 2010). From the second figure the drug concentration that showed 50% response is 0.005 μg / mL (please check the unit, Refer Figure: 2). Thus with the utilisation of this assay the impact of the varied concentration of the drug on the cells could be studied. One thing that has to be taken with caution regarding the Reazurin assay is that strict maintenance of the incubation period as prolonged incubation again changes the compound resorufin to hydroresorfin which is a colourless non fluorescent second state reduced compound and this will totally hamper the colorimetric reaction (Präbst, 2017; Erikstein, 2010). To determine the cytotoxicity, trypan blue assay was used which was initially named by the scientist Paul Erlich in the year, 1904 (Avelar-Freitas, 2014). As discussed earlier, the basic principle of the assay procedure involves determining the viable status of cells within a suspension of cells. Therefore it is also being referred as the dye exclusion method (Avelar-Freitas, 2014). The assay is considered because of the basic principle that it used to determine to distinguish the status of cells are that the live cells possess exact cell membrane that do not allow the entry of certain dyes like trypan blue, eosin and propidium in comparison to the dead cells (Kim, 2011). The figure 3 showed the number of dead cells resulted due to the different concentration of the drug diclofenac. At higher concentration of the drug the numbers of dead cells observed are higher in number in comparison to lower concentration of the drug. However at the concentration of the drug diclofenac 5.00E – 02 the number of dead cells observed was found to be lowest which also showed correlation with the redox potential findings of the cells with the drug (Refer Figure: 1 and 3). All the graphs represent a Mean ± SEM of the findings and the findings were compared via paired T test to obtain the statistical significance and all the values showed validity with resulting p value of ≤0.05. The statistical significance was measured using the Microsoft Excel version 2007 for the assay findings. The trypan blue assay was chosen as because the scientific evidence have reported that the dye exclusion test is the simple method that determines the viable status of cells based on the integrity of the cell membrane (Kim, 2011). In this regard a more sophisticated method is suggested to score the viability of the cells by tagging a fluorescent dye with the dye exclusion method and observing the cellular membrane integrity using a fluorescent microscope. This is recommended because of the problem that though the membrane integrity of the cells may be abnormal but the cells will become absolutely viable by repairing itself (Kim, 2011). Another potential problem is that a little bit of dye is up taken by the cells sometimes due to certain injury which might remain undiagnosed. With the emerging technologies in the field of scientific investigation, more sophisticated technologies are coming up such as the dye exclusion method with the aid of flow cytometry (Avelar-Freitas, 2014). This assay also can be done with the trypan blue dye as this makes bonding with the proteins and the emission of the fluorescence signal is measured using the flow cytometer at wavelength of 660 nm when the bovine serum albumin is used for the assay procedure (Avelar-Freitas, 2014). As per scientific evidences there is various assay procedure where the dye utilised binds or intercalate with the nucleic acids or DNA material via the probable hydrogen bonding mechanisms between the dye and the polymer for the localization of the dyes namely phenanthridinium analogues or acridine which are generally used for the anticancer and antiviral therapies (Nygren, 1998). Therefore, with the utilization of the simple yet useful assay procedure the viable cells could be recognized with the aid of only light microscopy which helped to determine the cell cytotoxicity of the drug diclofenac (Nygren, 1998).

The study findings highlighted that the drug diclofenac at the concentration of 5.00E – 02 showed the highest value of the Mean fluorescent intensity via the Reazurin assay and the lowest number of dead cells at the same concentration via the trypan blue assay on the selected T lymphoblastoid cell line named CCRF-CEM cells. Thus the impact of NSAIDs specifically diclofenac upon the proliferation and redox status of lymphoblasts which had been experimentally comprehended to evaluate better the role of lymphoblasts in malignancies and justified the objective of the research up taken. The efficacy of the drug diclofenac for its action upon the lymphoblast cells versus the normal healthy cells via the cell viability and death assay had enabled to develop a control strategy for the cure of the disease which had contributed to the existing scientific knowledge. It was reported that the drug diclofenac previously reported apoptosis on the cell line of neuroblastoma SH-SY5Y and it was observed to be time and dosage dependent (Cecere, 2010). The mechanism involved the early initiation of reactive oxygen species (ROS). The enzyme named mitochondrial superoxide dismutase (SOD2) plays a very critical function as a defensive mechanism against the ROS which in turn provides protection against the several stimulus initiation of apoptosis (Gómez-Lechón, 2003). It is evident that the drug diclofenac diminishes the level of proteins and the activity of the enzymes SOD2 without resulting any potential impairments of the level of mRNA of the neuroblastoma SH-SY5Y cell line (Cecere, 2010). However, the drug diclofenac induced changes within the membrane of the mitochondria organelle thus resulting in the discharge of the chemical cytochrome c (Tsutsumi, 2004). The drug alters the membrane potential of the mitochondria and this involves a possible mechanism of apoptosis induced by the drug and makes use of the significant role of SOD2 in the whole process (Lindskog, 2006). Though diclofenac is a widely used NSAID in the clinical field but it resulted in cytotoxicity in many cell lines in vitro experiments (Kokoszka, 2001). However, this physiological behaviour is common among the widely used NSAIDs and also the anticancer agents. Therefore varied investigations on NSAIDs showing higher specificity for the inhibitors of cyclooxigenase-2 recommended that they can be used as anticancer agents (Peterson, 1985). Moreover it is also reported that the joint action of the NSAIDs and the anticancer drugs shows more potent action and in this regard diclofenac can serve as an example which stimulated the chemotherapeutic effect of some anticancer drugs on the cell lines of neuroblastoma (Zerbini, 2011). The individuals who are treated with NSAID diclofenac goes through oxidative injury resulting in the alteration of the permeability impacting upon the redox potential of the mitochondria of the hepatocytes (Kokoszka, 2001). The drug inducing apoptosis is The study findings highlighted that the drug diclofenac at the concentration of 5.00E – 02 showed the highest value of the Mean fluorescent intensity via the Reazurin assay and the lowest number of dead cells at the same concentration via the trypan blue assay on the selected T lymphoblastoid cell line named CCRF-CEM cells. Thus the impact of NSAIDs specifically diclofenac upon the proliferation and redox status of lymphoblasts which had been experimentally comprehended to evaluate better the role of lymphoblasts in malignancies and justified the objective of the research up taken. The efficacy of the drug diclofenac for its action upon the lymphoblast cells versus the normal healthy cells via the cell viability and death assay had enabled to develop a control strategy for the cure of the disease which had contributed to the existing scientific knowledge. It was reported that the drug diclofenac previously reported apoptosis on the cell line of neuroblastoma SH-SY5Y and it was observed to be time and dosage dependent (Cecere, 2010). The mechanism involved the early initiation of reactive oxygen species (ROS). The enzyme named mitochondrial superoxide dismutase (SOD2) plays a very critical function as a defensive mechanism against the ROS which in turn provides protection against the several stimulus initiation of apoptosis (Gómez-Lechón, 2003). It is evident that the drug diclofenac diminishes the level of proteins and the activity of the enzymes SOD2 without resulting any potential impairments of the level of mRNA of the neuroblastoma SH-SY5Y cell line (Cecere, 2010). However, the drug diclofenac induced changes within the membrane of the mitochondria organelle thus resulting in the discharge of the chemical cytochrome c (Tsutsumi, 2004). The drug alters the membrane potential of the mitochondria and this involves a possible mechanism of apoptosis induced by the drug and makes use of the significant role of SOD2 in the whole process (Lindskog, 2006). Though diclofenac is a widely used NSAID in the clinical field but it resulted in cytotoxicity in many cell lines in vitro experiments (Kokoszka, 2001). However, this physiological behaviour is common among the widely used NSAIDs and also the anticancer agents. Therefore varied investigations on NSAIDs showing higher specificity for the inhibitors of cyclooxigenase-2 recommended that they can be used as anticancer agents (Peterson, 1985). Moreover it is also reported that the joint action of the NSAIDs and the anticancer drugs shows more potent action and in this regard diclofenac can serve as an example which stimulated the chemotherapeutic effect of some anticancer drugs on the cell lines of neuroblastoma (Zerbini, 2011). The individuals who are treated with NSAID diclofenac goes through oxidative injury resulting in the alteration of the permeability impacting upon the redox potential of the mitochondria of the hepatocytes (Kokoszka, 2001). The drug inducing apoptosis is considered to be the essential step behind the mitochondrial involving pathway for the cytotoxic nature and past scientific evidence had demonstrated its effect upon the nerve cells also (Zerbini, 2011). On the basis of the therapeutic dosage of the diclofenac for patients in between the dosage value 50–150 mg/day, the peak level of the drug that can be achieved within the plasma of the patients are within the value of 10 and 30 μM. However, in certain situations the level of the drug can become high due to prolonged therapy, over dosage and also due to the problem of limited clearance from the body (Lindskog, 2006). Past studies have also reported that diclofenac with a concentration level of 150 μM at the highest level do not show any cytotoxicity. Moreover, in case of clinical applications where a large therapeutic dosage is required caution must be taken because the drug diclofenac has the ability to cross the blood brain barrier (Lindskog, 2006). The mechanistic pathway of enhanced level of ROS results in a chain of events such as membrane depolarization of the mitochondria, discharge of the cytochrome c, activation of the caspase pathways and followed by the fragmentation of the nuclear materials (Cecere, 2010). Moreover, this mechanism was also demonstrated by other studies by the drug diclofenac for the apoptotic mechanism among the gastric and renal cells (Sriuttha, 2018). Therefore, in our study also we have shown that there was a membrane redox potential alteration in presence and absence of the drug diclofenac and the cytotoxicity shown by the drug which might be due to the following membrane potential alterations of the mitochondria and this involves a possible mechanism of apoptosis induced by the drug and makes use of the significant role of SOD2 in the whole process (Cecere, 2010). Therefore this study provided a scientific base for the utilisation of NSAIDs specifically diclofenac for the therapy of the patients suffering from any kind of malignancies. For students who are seeking biomedical science dissertation help, get comprehensive support and guidance that is tailored to individual research needs. The assistance is given with literature review, methodology development, data analysis, and other elements. Our experienced professionals are there to help you achieve academic success.

Acknowledgement:

Conclusion:

The paper detailed about the working mechanism of Nonsteroidal anti-inflammatory drugs (NSAIDs) which are a broad group having varied half life, action pathways, and their dissemination at the site of inflammation within the body. They show action upon the on the diverse forms of cyclooxygenase (COX). Diclofenac is one of the NSAIDs which is prescribed at a dosage of 150mg each day for the pain treatment. It is considered as the reference drug or choice of clinicians for the treatment of osteoarthritis. However, various studies have revealed caution about the safety of the drug diclofenac when it is considered for the treatment as overall NSAIDs show around 20-25 % of adverse drug reactions after oral administration. This particular drug is a choice of clinicians for varied context in the field of oncology as NSAIDs have a central role in chemoprevention and therefore might have a profound application in the settings of therapeutic regime. Therefore, the investigation was carried out to determine the efficacy of the drug diclofenac for its action upon the lymphoblast cells, i.e., to observe the death rate of the cells with the drug application. The study findings highlighted that the drug diclofenac at the concentration of 5.00E – 02 showed the highest value of the Mean fluorescent intensity via the Reazurin assay and the lowest number of dead cells at the same concentration via the trypan blue assay on the selected T lymphoblastoid cell line named CCRF-CEM cells. There was a membrane redox potential alteration in presence and absence of the drug diclofenac and the cytotoxicity shown by the drug which might be due to the following membrane potential alterations of the mitochondria and this involves a possible mechanism of apoptosis induced by the drug and makes use of the significant role of SOD2 in the whole process. Therefore, diclofenac can be considered as a control therapy of the patients suffering from any kind of malignancies.

References:

Avelar-Freitas, B.A., Almeida, V.G., Pinto, M.C.X., Mourão, F.A.G., Massensini, A.R., Martins-Filho, O.A., Rocha-Vieira, E. and Brito-Melo, G.E.A., 2014. Trypan blue exclusion assay by flow cytometry. Brazilian Journal of Medical and Biological Research, 47(4), pp.307-315.

Braun, F.K., Al-Yacoub, N., Plötz, M., Möbs, M., Sterry, W. and Eberle, J., 2012. Nonsteroidal anti-inflammatory drugs induce apoptosis in cutaneous T-cell lymphoma cells and enhance their sensitivity for TNF-related apoptosis-inducing ligand. Journal of investigative dermatology, 132(2), pp.429-439.

Cecere, F., Iuliano, A., Albano, F., Zappelli, C., Castellano, I., Grimaldi, P., Masullo, M., De Vendittis, E. and Ruocco, M.R., 2010. Diclofenac-induced apoptosis in the neuroblastoma cell line SH-SY5Y: possible involvement of the mitochondrial superoxide dismutase. Journal of Biomedicine and Biotechnology, 2010.

da Costa, B.R., Reichenbach, S., Keller, N., Nartey, L., Wandel, S., Jüni, P. and Trelle, S., 2017. Effectiveness of non-steroidal anti-inflammatory drugs for the treatment of pain in knee and hip osteoarthritis: a network meta-analysis. The Lancet, 390(10090), pp.e21-e33.

Erikstein, B.S., Hagland, H.R., Nikolaisen, J., Kulawiec, M., Singh, K.K., Gjertsen, B.T. and Tronstad, K.J., 2010. Cellular stress induced by resazurin leads to autophagy and cell death via production of reactive oxygen species and mitochondrial impairment. Journal of cellular biochemistry, 111(3), pp.574-584.

Farmakologia, R.C.G., 2015. Czelej.

Fischer, S.M., Hawk, E.T. and Lubet, R.A., 2011. Coxibs and other nonsteroidal anti-inflammatory drugs in animal models of cancer chemoprevention. Cancer prevention research, 4(11), pp.1728-1735.

Gómez-Lechón, M.J., Ponsoda, X., O’Connor, E., Donato, T., Castell, J.V. and Jover, R., 2003. Diclofenac induces apoptosis in hepatocytes by alteration of mitochondrial function and generation of ROS. Biochemical pharmacology, 66(11), pp.2155-2167.

Gurpinar, E., Grizzle, W.E. and Piazza, G.A., 2013. COX-independent mechanisms of cancer chemoprevention by anti-inflammatory drugs. Frontiers in oncology, 3, p.181.

Johnsen, J.I., Lindskog, M., Ponthan, F., Pettersen, I., Elfman, L., Orrego, A., Sveinbjörnsson, B. and Kogner, P., 2004. Cyclooxygenase-2 is expressed in neuroblastoma, and nonsteroidal anti-inflammatory drugs induce apoptosis and inhibit tumor growth in vivo. Cancer research, 64(20), pp.7210-7215.

Johnsen, J.I., Lindskog, M., Ponthan, F., Pettersen, I., Elfman, L., Orrego, A., Sveinbjörnsson, B. and Kogner, P., 2005. NSAIDs in neuroblastoma therapy. Cancer letters, 228(1-2), pp.195-201.

Kim, J.S., Nam, M.H., An, S.S.A., Lim, C.S., Hur, D.S., Chung, C. and Chang, J.K., 2011. Comparison of the automated fluorescence microscopic viability test with the conventional and flow cytometry methods. Journal of clinical laboratory analysis, 25(2), pp.90-94.

Kokoszka, J.E., Coskun, P., Esposito, L.A. and Wallace, D.C., 2001. Increased mitochondrial oxidative stress in the Sod2 (+/−) mouse results in the age-related decline of mitochondrial function culminating in increased apoptosis. Proceedings of the National Academy of Sciences, 98(5), pp.2278-2283.

Kołodziejska, J. and Kołodziejczyk, M., 2018. Diclofenac in the treatment of pain in patients with rheumatic diseases. Reumatologia, 56(3), p.174.

Kołodziejska, J. and Kołodziejczyk, M., 2018. Diclofenac in the treatment of pain in patients with rheumatic diseases. Reumatologia, 56(3), p.174.

Kowalski, M.L., Makowska, J.S., Blanca, M., Bavbek, S., Bochenek, G., Bousquet, J., Bousquet, P., Celik, G., Demoly, P., Gomes, E.R. and Niżankowska‐Mogilnicka, E., 2011. Hypersensitivity to nonsteroidal anti‐inflammatory drugs (NSAIDs)–classification, diagnosis and management: review of the EAACI/ENDA# and GA2LEN/HANNA. Allergy, 66(7), pp.818-829.

Lindskog, M., Gleissman, H., Ponthan, F., Castro, J., Kogner, P. and Johnsen, J.I., 2006. Neuroblastoma cell death in response to docosahexaenoic acid: Sensitization to chemotherapy and arsenic‐induced oxidative stress. International Journal of Cancer, 118(10), pp.2584-2593.

Michałowska, M., Safety of use of non-steroidal anti-inflammatory drugs with particular emphasis on OTC-over-the-counter drugs.

Nygren, J., Svanvik, N. and Kubista, M., 1998. The interactions between the fluorescent dye thiazole orange and DNA. Biopolymers: Original Research on Biomolecules, 46(1), pp.39-51.

Pantziarka, P., Sukhatme, V., Bouche, G., Meheus, L. and Sukhatme, V.P., 2016. Repurposing Drugs in Oncology (ReDO)—diclofenac as an anti-cancer agent. Ecancermedicalscience, 10.

Pavelka, K., 2012. A comparison of the therapeutic efficacy of diclofenac in osteoarthritis: a systematic review of randomised controlled trials. Current medical research and opinion, 28(1), pp.163-178.

Pereira-Leite, C., Nunes, C. and Reis, S., 2013. Interaction of nonsteroidal anti-inflammatory drugs with membranes: in vitro assessment and relevance for their biological actions. Progress in lipid research, 52(4), pp.571-584.

Peterson, H.I., Alpsten, M., Skolnik, G. and Karlsson, L., 1985. Influence of a prostaglandin synthesis inhibitor and of thrombocytopenia on tumor blood flow and tumor vascular permeability. Experimental studies in the rat. Anticancer research, 5(3), pp.253-257.

Präbst, K., Engelhardt, H., Ringgeler, S. and Hübner, H., 2017. Basic colorimetric proliferation assays: MTT, WST, and resazurin. In Cell viability assays (pp. 1-17). Humana Press, New York, NY.

Sriuttha, P., Sirichanchuen, B. and Permsuwan, U., 2018. Hepatotoxicity of nonsteroidal anti-inflammatory drugs: a systematic review of randomized controlled trials. International journal of hepatology, 2018.

Strober, W., 2015. Trypan blue exclusion test of cell viability. Current protocols in immunology, 111(1), pp.A3-B.

Tsutsumi, S., Gotoh, T., Tomisato, W., Mima, S., Hoshino, T., Hwang, H.J., Takenaka, H., Tsuchiya, T., Mori, M. and Mizushima, T., 2004. Endoplasmic reticulum stress response is involved in nonsteroidal anti-inflammatory drug-induced apoptosis. Cell Death & Differentiation, 11(9), pp.1009-1016.

Wełnicki, M., 2012. Mamcarz A. Stosowanie niesterydowych leków przeciwzapalnych u pacjentów kardiologicznych–aktualny stan wiedzy. Kardiol Prakt, 6, pp.33-37.

Yssel, H. and Spits, H., 2005. Generation and maintenance of cloned human T cell lines. Current protocols in immunology, 65(1), pp.7-19.

Zerbini, L.F., Tamura, R.E., Correa, R.G., Czibere, A., Cordeiro, J., Bhasin, M., Simabuco, F.M., Wang, Y., Gu, X., Li, L. and Sarkar, D., 2011. Combinatorial effect of non-steroidal anti-inflammatory drugs and NF-κB inhibitors in ovarian cancer therapy. PLoS One, 6(9), p.e24285.

Zerbini, L.F., Czibere, A., Wang, Y., Correa, R.G., Otu, H., Joseph, M., Takayasu, Y., Silver, M., Gu, X., Ruchusatsawat, K. and Li, L., 2006. A Novel Pathway Involving Melanoma Differentiation Associated Gene-7/Interleukin-24 Mediates Nonsteroidal Anti-inflammatory Drug–Induced Apoptosis and Growth Arrest of Cancer Cells. Cancer Research, 66(24), pp.11922-11931.

Continue your exploration of Effect of Arc Length and Intraocular Pressure on Corneal Radius of Curvature with our related content.