Cell Counting And MTT Essay For Etoposide Induced Cellular Toxicity On HeLa Cells.

Abstract

Describe about the Cell counting and MTT Assay?
 

We report here about the etoposide induced cellular toxicity on HeLa cells as etoposide is a chemotherapeutic agent and has been characterised as Topoisomerase II inhibitor and DNA damaging agent. To analyse etoposite induced cellular toxicity MTT assasy was performed. Analysing the results upon considering group S3 of HeLa cells as control with 100% survival, it was observed that S1 and S2 group alone show 50.4% and 67.4% survival respectively which is in agreement to the number of cells seeded for the experiment. However, treatment with Etoposide shows a decrease in survival percentage as compared to cells alone. Upon comparison of S1+Et group with S1 alone, only 48.9% survival was observed. Similarly, S2+Et and S3+Et group showed 55.3 and 47.3% survival only in comparison to S2 and S3 groups respectively. This indicates that maximum inhibitory effect of etoposide was observed in treatment with S3 group and minimum anticancerous potential was observed in treatment with S2 group. Etoposide treatment to S1 group however, showed  significant variation in percentage survival of HeLa cells.

Effects on etoposide result in damage to DNA in various forms such as DNA fragmentation, DSB, SSB, damage to repair mechanisms, all of which can cumulatively result in deleterious side-effects like cancer and eventually cell death. The mode of action followed by etoposide induced DNA damage and consequent apoptosis, mainly involves radiolytic splitting of water followed by production of various ROS/RNS like hydroxyl radicals (HO•), Superoxide radicals etc. The deleterious biological effects of etoposide are manifested in form of altered cellular metabolic and proliferative activity, cell cycle arrest, induction of apoptosis and eventually cell death (Kausch et al., 2005).. Several indigenous cellular mechanisms play an important role in protecting living organisms against cellular toxicities, e.g., shielding cells from lipid peroxidation, scavenging of secondary free radicals, suppression of protracted oxidative stress, decrease in O₂ concentration and enhancement of DNA repair (Sypniewski et al., 2013). Etoside is known to produce enzymatic and morphological changes, like increases in lysosomal enzyme content, H₂O₂ production, membrane ruffling, etc. It has also been documented that phagocytic activity and the production of ROS such as H₂O₂ and O₂^– are increased. Etoposide exhibits an important role in therapy for many kinds of cancer. However, it can also result in inflammation and accompanying injury (Ibuki and Goto, 2004).

HeLa cell line is an immortal human cell line which is very traditionally used in clinical research. This cell line was established in 1951 from a lady name Henrietta Lacks, who died because of cervical cancer and this cell line, was said to remarkably durable at that time. Later this cell line was extensively used in various researches like analysing radiation effects, polio AIIDS research and notably in cancer research. The main speciality of this cell line is that it abnormally grows rapidly more rapidly than any other cancerous cell line as during division it has rapid and active telomere region (Bottomley et al., 1969).

To observe the etoposide induced cellular toxicity MTT cell viability assay was performed with observe test sets of cell. MTT cell viability assay is basically works on the concept of reduction of MTT and formation of formazan crystals. It is a unique assay that is designed in 96 well or 8 well plate formats and could analyse cell toxicities in rapid duration of time. MTT is basically 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and assay was first of its kind to observe cell in homogeneous suspension. MTT tetrazolium assay has been widely accepted and is been routinely used to validate chemotherapeutic compounds, drugs and natural product for their cellular toxicity against cell of specific origin. According to concept the live cell which is metabolically converts the MTT to formazan crystal but when cell die it loses its ability to convert the MTT to formazan hence it serves as a marker for viable cells. While assay several precaution must be taken as sensitivity of assay depends upon concentration of MTT ( 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide), duration of incubation, metabolic positions and number of cells. In the present study toxic effect of etopoide was done to established the cell viability and percentage survival against incubation with etoposide. Prior to experiment it is assume that etoposide forms a ternary structure that inhibits the topoisomerase and leads to DNA strand breaks and toxicity and expected to reduce the cell number compared to untreated control samples.

Material

Hela cell line, Etoposide, High glucose Dulbecco Modified Eagle Medium (HG-DMEM), Trypan Blue, Fetal Bovine Serum (FBS), and RNase were obtained from Sigma-Aldrich, St Louis, MO, USA. Phosphate buffered saline (PBS) and 3-(4,5-dimethyl-2-yl)-2,5-diphenyl-2II-tetrazolium bromide (MTT) were procured from certified reagent supplier.

HeLa cells is cervical cancer cell line and were procured and maintained in the institutional cell culture facilty. Stock of  cells were cultured in routine passages in DMEM containing 25 mM glucose, 43 mM bicarbonate, 25mM HEPES buffer, 5000U penicillin, and 5mg/ml streptomycin at 37°C under 5% CO₂ atmosphere supplemented with 10% heat-inactivated FBS. Cells were passaged 2-3 times each week by scraping to maintain them in healthy log phase. For the experiments, cells were seeded at low density (0.5 × 10⁶ cells/ml) in 25cm² diameter Petridishes calculation was done with the help of hemocytometer.

After cell seeding three set of cultured cells petridishes were selected and named as S1, S2 and S2. Calculating the cell through haemocytometer where group S1, group S2 and group S3 having approximately 20x10⁴ cells /ml. Fixed concentration of etoposide i.e 40µM was added to every petridish and incubated for 24h for analysis.

Method

1:1 dilution of cell suspension of trypan blue was prepared by adding 20µl of trypan blue with 20µ of cell suspension and gently vortexing for few minutes and then 10µl of cell suspension was taken for cell counting at haemocytometer and in other chamber alone trypan blue was taken. Microscope was adjusted to focus to observe crosshatch grid at 10X magnification. Cell viability assay was performed according to standard protocol designed by assay guideline manual. Cytotoxicity and metabolic activity was determined using the microtiter MTT assay (Berridge et al. 1996).  MTT is a yellow water-soluble tetrazolium dye that is reduced by living cells to a water-insoluble purple formazan. HeLa cells were seeded at a density of 5000 cells/well in 96 well ELISA plates and left overnight for adherence. Different sets of cells were selected and 40µM of etoposide was added and their toxicity was assessed at 24h. Effect of etoposide was analysed by incubating cells. MTT (final concentration 1mg/ml) was added to the media incubated at 37á´¼C for 2hrs. After completion of incubation period, growth media and MTT were discarded and the purple formazan crystals formed were solubilized by adding DMSO to each well. The plates were shaken at room temperature for 15 min to aid in dissolving crystals. Absorbance of the coloured compound was taken using spectrophotometer at dual wavelength 540nm and 630nm. The relative metabolic activity was expressed as a percentage of the non-treated control.

For calculation of viable cell T25 was placed under microscope and calculated using the following formula

Viable Cells/ml = Average viable cell count per square x Dilution Factor x 10⁴

Experiment was performed in triplicate and at least thrice to achieve randomised data is expressing mean ± standard deviation. The variation between control and test groups was calculated. Significant variation between these groups was analysed by one-way-anova (one way analysis of variance) which was followed by bon ferroni test. pp<0.05.

Cell Culture

After 24h of incubation with etoposide the cell viability of HeLa cells were calculated to indentify the etoposide induced cell cytotoxicity. As analysed at the time of drug treatment and endpoint of three subsets of HeLa cell groups were prepared having three flasks for each subgroup having particular cell number and denoted by S1, S2 and S3. Etoposide is a compound that acts as an Topoisomerase-II inhibitor in the cellular environment and induces cellular toxicities. Result of the present study supports the earlier evidence that denotes that etoposide induces the cellular toxicities and as treatment of etoposide clearly inhibits the cell cycle and reduces the cell no in every set of cells. At the time drug treatment the cells number was calculated and in S1, S2 and S3 set the cell number was 20x10⁴ cells /ml as calculated in haemocytometer.  

Figure1. Analysis of etopide induced cellular and metabolic toxicity in HeLa cell line though MTT cell viability assay.

Where etoposide significantly inhibits the cell survival in S2 and S3 HeLa cells group while moderately reduces cell viability in S1 group. Where S1,S2 and S3 set having 20X10⁴ cells/ml, with incubation of 40µM Etoposide. *;where p 

Table1. Analysis of etopoise induced cellular toxicity and viable cell number after treatment.

Etoposide significantly induces the cell viability in all the cell subset of HeLa cell line    

In the treatment regimen the 40 µM of etoposide reduces the percentage cell survival as in S1 subset after etoposide treatment percentage cell survival was 48.86% (S1+Et), in S2 subset at etoposide treatment percentage cell survival decrease to 55.25% (S2+Et) and in S3 it was 47.25% after etoposide treatment (S3+Et) (Figure 1., Table1). Calculating the inhibition in the cell viability etoposide significantly inhibits the viable cell number as shown in Table 1 and figure 2.
 

Figure 2. Analysis of number of viable cells after etoposide treatment. Where S1, S2 and S3 sets were incubated with etoposide that induces cellular toxicity to HeLa cell that leads to significantly inhibited cell number. Where *; p<0.001.

The number of viable section the S1 group was found to be 9.6X10⁴ cells/ml which was significantly less that control (P<0.001). in set S2 and S3 the cell no was 8.8X10⁴ cells /ml and 9.2X10⁴cells/ml respectively (P<0.001)indication the cytotoxic effect of etoposide.

Cell viability assay is used identify the compounds and other moilcules or drug for their cellular toxicities that could easily lead to the cell death by inhibiting the cell differentiation ad proliferation. Basic concept of the cell based assay is used to analyse the binding of receptor and management and monitoring of cell organelles. In this assay the the cells which are viable and have healthy metabolism have an ability to convert MTT to blue formazan having absorbance near 570nm (Berridge et al., 1996). Only live cell have the ability for conversion of MTT to formazan which involves NADH having role to provide electrons to MTT (He et al., 2015). The formazan is insoluble crystal that is deposited at the surface of cells and it is very essential to dissolve these crystals to avoid hindrance during observation. So DMSO is used to stabilise the crystal. Etoposide is chemotoxic agent and it is achemotherapeutic drug that belongs to topoisomerase-II inhibitor class and it is known to induced cellular and metabolic toxicity, it is also known to induce apoptosis that leads to cell death. 

Treatment

The metabolic viability of cells is a prime indicator of its health, well-being, surviving potential and clonogenicity (Huyck et al., 2012). Viability was assessed using colorimetric assay using MTT against etoposide treatment alone. However, at etoposide treatment cells began to steadily lose their metabolic viability which could be attributed to the cumulative toxic nature. HeLa cells were treated with etoposide and cell viability was measured and there was significant loss in viability at all the tested groups (P<0.001). This postulates a hypothesis that presumably pretreatment with etoposide inhibits the cell activity and decreases their metabolic activity substantially. Usually cells are known to have intrinsic superoxide ions as a mechanism for respiratory burst and to kill pathogens. Despite of this fact, increased presence of ROS within the cells due to etoposide treatment could prove lethal. Upon stimulation with etoposide it could be assume that there will be significant increase in intracellular ROS levels that may be fatal. It could be concluded that etoposide could be toxic and can cause an upsurge in ROS levels that execute in apotosis and loss of cellular viability. There is several evidences that denotes that treatment with etoposide may lead to loss of mitochondrial potential as well as loss of mitochondrial mass to the cellular moieties (Hande, 1998). Mitochondrial metabolism plays a pivotal role in the survival and growth at cellular level and even slight permeability transition provides a preliminary signal for apoptosis initiation. Changes in MMP are critically decisive towards cell life-death transition and the maintenance of MMP homeostasis is measured by a change in the membrane potential (Huang, 2002). Hence, analysis of modulations pertaining to effect of etoposide and its manifestations in the cellular metabolism and cell differentiation can be easily correlated.

Loss of Mitochondrial membrane potential may lead to loss of mitochondrial mass and may leads to signalling cascade that induces apoptosis and cell cycle arrest. Proceeding with etoposide incubation and loss in cell viability indicates that cells failed to continue to progress ahead from G₁ phase further in the cell cycle.Cells usually remain in S phase for the period sufficiently required for the cells to synthesize and replicate DNA for proceeding towards mitosis. Therefore, it can be deciphered that more damaged cells will remain in S phase for a longer duration in order to detect DNA abnormalities and for rectification of the irregularities in order to have an uninterrupted replication (Abraham, 2001). In the case of cellular toxicity their is blockage in S phase in a all the groups. It has been validated that regulation of G₂/M checkpoint is imperative as it ensures that cells initiate mitosis only after restoring impaired DNA after replication (Wang et al., 2000). Cells having a defective G₂/M checkpoint enter into mitosis prior to DNA repair, hence leading to cell death by undergoing apoptosis and efforts to enhance this effect may increase the cytotoxicity induced by etoposide (DiPaola, 2002). p53 is known to regulate G₂/M transition either through induction of p21 and 14-3-3σ (Stratifin), a protein proficient in sequestering cyclin B1-Cdc2 complexes in the cytoplasm or via activation of transcription of apoptosis-linked genes. Stratifin, p21 and Gadd45 are amongst the major transcriptional objectives of p53 which inhibit Cdc2 crucial for entry into mitosis (Luk et al., 2005).all these prosurvival factor have been inhibited by etoposide so it could be easily correlated regarding cell death by induction of apoptosis and DNA defect.  Present study indicates that pre-treatment with etopoide to HeLa cells results in a steady decrease in cell number thus it could be assume that this might be because of cell cycle arrest and providing no time prerequisite for DNA repair resulting in further cell cycle termination.

Conclusion

We conclude from our studies that etoposide can lead to loss of cellular metabolic viability in challenged HeLa cells. It also escalates the generation of intracellular ROS levels and it could be assume that it might crefurbishes fluctuations in mitochondrial membrane potential (MMP).  Role in etoposide in abrogating deleterious manifestations of damages by means of causing cell cycle arrest. Additionally, following elucidation of precise mechanism of action, it has prospective applicability in developing a targeted approach aimed at alleviating bystander effect during chemotherapy is yet to done in future.

References:

Bottomley, R. H., Trainer, A. L., Griffin, M. J. (1969) ‘Enzymatic and chromosomal characterization of HeLa variants’. Journal of Cell Biololgy. Vol 41 no, 3, pp 806–815.

Sypniewski, D., Bednarek, I., GaÅ‚ka, S., Loch, T., BÅ‚aszczyk, D., SoÅ‚tysik, D. (2013) ‘Cytotoxicity of etoposide in cancer cell lines in vitro after BCL-2 and C-RAF gene silencing with antisense oligonucleotides.’ Acta Poloniae Pharmaceutica. Vol.70, no.1, pp.87-97.

Ibuki, Y., and Goto, R. (2004). ‘Ionizing radiation-induced macrophage activiation: Augmentation of nitric oxide production and its significance.’ Cellular and Molecular Biology, Vol. 50, Online: OL617-OL626.

Kausch, I., Jiang, H., Thode, B., Doehn, C., Kruger, S., Jocham, D. (2005). ‘Inhibition of bcl-2 enhances the efficacy of chemotherapy in renal cell carcinoma. European Urology.’ Vol.47, no.5, pp.703-709.

He, Z., Yuan, J., Qi, P., Zhang, L., Wang, Z. (2015). ‘Atorvastatin induces autophagic cell death in prostate cancer cells in vitro.’ Molecular Medicine Report.. doi: 10.3892

Hande, K., R. (1998). ‘Etoposide: four decades of development of a topoisomerase II inhibitor’. European Journal of Cancer, Vol. 34, no.10, pp.1514–1521.

Huang, S. (2002) ‘Development of a High Throughput Screening Assay for Mitochondrial Membrane Potential in Living Cells.’ Journal of Biomolecular Screening. Vol. 7, no.4, pp 383-389.

Abraham, R,.T,. (2001) ‘Cell cycle checkpoint signaling through the ATM and ATR kinases.’ Genes and Development. Vol. 15, no.17, pp 2177-2196.

Wang, X., McGowan, C., H., Zhao, M., He, L., Downey, J., S., Fearns, C., Wang, Y., Huang, S., Han, J. (2000) ‘Involvement of the MKK6-p38g Cascade in γ-Radiation-Induced Cell Cycle Arrest.’ Molecular and Cellular Biology. Vol. 20, no.13 pp. 4543-4552.

Berridge, M.,V., Tan, A.,S., McCoy, K., D., Wang, R. (1996). The biochemical and cellular basis of cell proliferation assays that use tetrazolium salts. Biochemica. Vol.4, : 14– 19

Huyck, L., Ampe, C., Van Troys, M. (2012). ‘The XTT Cell Proliferation Assay Applied to Cell Layers Embedded in Three-Dimensional Matrix.’ Assay and Drug Development Technologies Vol.10, no.4, pp.382-392.

DiPaola, R.,S. (2002). ‘To arrest or not to G2-M cell cycle arrest.’ Clinical and Cancer Research. Vol.8, pp.3311–3314.

Luk, S.,C.,W., Siu, S.,W., F.,, Lai, C., K., Wu, Y., J., Pang, S., F. (2005) ‘Cell Cycle Arrest by a Natural Product via G2/M Checkpoint.’ International journal of Medical Science. Vol.2, no.2, pp 64-69.

Brazina, J., Svadlenka, J., Macurek, L., Andera, L., Hodny, Z., Bartek, J., Hanzlikova, H. (2015) DNA damage-induced regulatory interplay between DAXX, p53, ATM kinase and Wip1 phosphatase. Cell Cycle. Vol. 4, no.3, pp.375-387