Measuring Cytotoxicity in HepG2 Cells

Measuring Cytotoxicity in HepG2 Cells Measuring cytotoxicity in HepG2 cells treated with cisplatin or 5-fluorouracil, via flow cytometry as a measure of effectiveness of the drugs at treating cancer Introduction Cytotoxicity is the quality of being toxic to cells, resulting in cell death (Rang et al. 2012). In the majority of therapeutics cytotoxicity is not a desirable characteristic. However, in the treatment of cancer many chemotherapy drugs, through cytotoxicity are designed to selectively induce apoptosis, a form of cell death (Rang et al. 2012). There are three main forms of cell death, these are: apoptosis, autophagy and necrosis. Apoptosis is the physiological process by which cells are destroyed in a controlled manner (Elmore 2007). There are four basic steps involved; the first is cell shrinkage, blebs begin to form and condensation of chromatin occurs. Next, membrane blebbing occurs and enzymes begin the process of nuclear fragmentation. In the late stages of apoptosis, the cell is broken down into apoptotic bodies which contain the cells components and nuclear fragments. The final stage is the phagocytosis of apoptotic bodies (Ulukaya et al. 2011). One of the main ways in which phagocytes recognise apoptotic cells is the presentation of phosphatidylserine (PS). Ordinarily, this phospholipid is found on the inner leaflet of the membrane bilayer, during apoptosis it translocates to the outer leaflet and binds to a PS receptor present on the phagocyte (Wu et al. 2006). In autophagy, targeted components such as organelles are isolated in an autophagosome; a form of vesicle with a lipid bilayer membrane. These autophagosomes are degraded by lysosomes (Kroemer, G et al. 2009). Necrosis is often thought of as uncontrolled cell death. However, there is an increasing amount of evidence that suggests it is regulated by transduction pathways and other mechanisms (Kroemer, G et al. 2009). It is caused by factors such as trauma, infection and toxins. It is characterised by the swelling of cells which causes the cell membrane to rupture, resulting in the release of intracellular components. These intracellular components can trigger a local inflammatory response. In contrast to apoptosis, the phagocytic stage does not occur in necrosis (Golstein, Pierre and Kroemer, Guido 2007). As a result, dead cells and their intracellular components remain at the site of necrosis, this build-up of debris may lead to conditions such as gangrene. Due to its detrimental effects, it’s preferential that necrosis is not produced by therapeutics. Cisplatin and 5-fluorouracil are chemotherapy agents, which both cause apoptosis by interfering with DNA via its replication and repair. Cisplatin crosslinks with purine bases in DNA which interferes with DNA repair mechanisms, this means that DNA damage is not fixed triggering apoptosis (Dasari and Bernard Tchounwou 2014). 5-flurouracil is an antimetabolite, it is metabolised into 5-fluorodeoxyuridine monophosphate (5-FdUMP) which is then incorporated into both DNA and RNA and also inhibits thymidilate synthase (Parker and Stivers 2011). HepG2 cells are derived from the hepatocellular carcinoma cells of a 15 year old, American, Caucasian, male and are epithelial in nature. They are commonly used as an in vitro model of human hepatocellular carcinoma (Costantini et al. 2013). Flow cytometry is a powerful tool that allows for the rapid differentiation and characterisation of particles including cells. A flow cytometer is made up of three main components: fluidics, optics and electronics. When a sample is injected into a flow cytometer, the fluidics system is responsible for producing a stream of single particles, this relies on the principle of laminar flow. This allows the particles to be interrogated by the laser beam individually. When a particle passes through the laser beam it scatters light. The amount of forward scatter is proportional to the size of the cell. The side scatter is proportional to the complexity of the cell. If a fluorophore is present, it can be excited by a specific wavelength of light. It will then undergo a Stokes shift and a different wavelength of light will be emitted which can be measured by the electronics of the flow cytometer (Macey 2007, Maxwell and Hannon-Fletcher 2009). The fluorophore can be conjugated with proteins suc h as annexin V which have a known high affinity to specific molecules present on the cell surface. Annexin V is a 35.7kDa, calcium sensitive, phospholipid binding protein. It has a high affinity to phosphatidylserine. The binding of fluorophore labelled annexin V is used as a common measure of apoptotic cells by flow cytometry (van Genderen et al. 2008). Aims To measure the effectiveness of two anti-cancer drugs by measuring the level of apoptosis in a population of HepG2 cells after being treated with cisplatin (15 µg/mL) or 5-fluorouracil (25 µg/mL) for 48 hours and compare those to a population of untreated cells via flow cytometry. Objectives To count and fix a population of HepG2 cells before staining them with fluorescently labelled Annexin V in preparation for analysis using a flow cytometer. To measure the level of fluorescence in 5000 treated and untreated cells using a flow cytometer as well as measuring the minimal and maximal fluorescence in a negative and positive control. Based on the fact the fluorescence is directly proportional to apoptosis determine which treatment is significantly better and provide evidence from the literature as to why this is the case. Methods Please refer to laboratory schedule (Hatch 2014). Following the collection of data descriptive statistics and a one-way ANOVA test were carried out the results of which can be found in the appendix. Results The negative control was used to identify if there was any background fluorescence. In table 1 (found in the appendix) the mean fluorescence for the negative control was 144. Therefore, fluorescence that is significantly higher than this level can be attributed to the excitation of the fluorophore (Alexa 488). Figure 1 Cytotoxicity of HepG2 cells measured as fluorescence using flow cytometry. The bars are means of triplicates, the standard error has been used for the error bars. *statistically significant compared to control (blue for negative, red for positive), p In figure 1, a greater level of fluorescence was seen for the samples treated with cisplatin and 5-fluorouracil compared to that of the untreated sample. Cisplatin produced the greatest level of fluorescence of the two treated samples. Discussion The data used was a class set, some of the data provided was produced by those running the laboratory session as some samples did not contain a pellet which was needed to run the flow cytometry analysis. The five sets of data were compared with one another, it was found that the differences were statistically significant (p As mentioned in results, cisplatin showed a greater mean fluorescence compared to 5-fluorouracil, Results were 726 and 540 respectively. Seeing as fluorescence is directly proportional to phosphotidylserine expression, which in turn is a direct measure of apoptosis. It can be assumed that cisplatin produced the highest level of apoptosis and is most cytotoxic to HepG2 cells. Although a higher concentration of the drugs were used (150 µg/mL) this result has also been seen in the literature (Brenes et al. 2007). Although it was not stained with annexin V, the negative control showed a mean fluorescence of 144; this is due to auto fluorescence of the cells. The fluorophore which was conjugated with annexin V was Alexa 488. It fluoresces at a wavelength of 488nm, this is within the green part of the spectrum. It is well documented that molecules found within all cells, including hepatocytes also fluoresce at a green wavelength, the most notable of these are flavins which fluoresce at around 560nm (Croce, Anna Cleta et al. 2007, Croce, Anna C et al. 2014). FAD (flavin adenine dinucleotide) is an example of a flavin and is a redox cofactor used in metabolism. HepG2 cells are a liver carcinoma cell line. Due to their cancerous nature there would be a high level of metabolism within these cells and hence, a high level of FAD. The low level of fluorescence seen in the untreated sample is expected as there should be no apoptosis induced by cytotoxicity. Therefore, it is not surprising that the difference between the fluorescence of the untreated sample and the negative control and the treated samples is less statistically significant. In this experiment, only one stain was used in this experiment (Annexin V), this allows for the identification of cells that are cytotoxic. To evaluate the true effectiveness of a chemotherapy drug, the cell viability and necrosis is also needed. Necrosis and late stage apoptosis can be measured using propidium iodide (PI). PI binds to nucleic acids and is impermeable to intact membranes, it is often used in combination with annexin V as it fluoresces red and there is little overlap between the wavelengths of red and green light. Therefore, compensation is not needed after flow cytometry has been carried out. It has been observed in the literature that the cytotoxicity of 5-fluorouracil is dependent on both dose and exposure time (Okamura et al. 2008). We only looked at one dose and exposure time for both drugs. It has also been observed that the use of cisplatin in combination with 5-fluorouracil or other chemotherapy drugs has a synergistic and additive effect in hepatoma cell lines (Kogure et al. 2004), both of these could be investigated further is the experiment were carried out again. Conclusion In conclusion, cisplatin was shown to produce a higher level of apoptosis in HepG2 cells than 5-fluorouracil. However, to fully evaluate the effectiveness of an anti-cancer agent, measures of viability and necrosis are also needed. The differences between the two drugs may be due to the different mechanisms by which the drugs work. The introduction of 5-dUMP into DNA and RNA is a more subtle mechanism compared to the cross-linking of DNA via cis-platin. This cross-linking may be detected by cells more readily resulting in higher levels of apoptosis. As HepG2 cells were used, these results may only be the case for hepatocellular carcinomas. References Brenes, O., Arce, F., Gà ¤tjens-Boniche, O., and Dà ­az, C. (2007) â€Å"Characterization of Cell Death Events Induced by Anti-Neoplastic Drugs Cisplatin, Paclitaxel and 5-Fluorouracil on Human Hepatoma Cell Lines: Possible Mechanisms of Cell Resistance.† Biomedicine pharmacotherapy = Biomà ©decine pharmacothà ©rapie 61 (6), 347–55 Costantini, S., Di Bernardo, G., Cammarota, M., Castello, G., and Colonna, G. 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Error 95% Confidence Interval for Mean Minimum Maximum Lower Bound Upper Bound 1.00 3 144.3333 11.93035 6.88799 114.6967 173.9700 136.00 158.00 2.00 3 1866.6667 32.65476 18.85323 1785.5477 1947.7856 1836.00 1901.00 3.00 3 214.3333 10.59874 6.11919 188.0046 240.6621 203.00 224.00 4.00 3 864.0000 20.22375 11.67619 813.7614 914.2386 849.00 887.00 5.00 3 540.6667 51.93586 29.98518 411.6508 669.6825 484.00 586.00 Total 15 726.0000 647.67772 167.22967 367.3280 1084.6720 136.00 1901.00 Table 2 Descriptive statistics where 1.00 is the negative control, 2.00 is the positive control, 3.00 is the untreated sample, 4.00 is the sample treated with cisplatin and 5.00 is the sample treated with 5-fluorouracil. N Mean Std. Deviation Std. Error 95% Confidence Interval for Mean Minimum Maximum Lower Bound Upper Bound 1.00 3 144.3333 11.93035 6.88799 114.6967 173.9700 136.00 158.00 2.00 3 1866.6667 32.65476 18.85323 1785.5477 1947.7856 1836.00 1901.00 3.00 3 214.3333 10.59874 6.11919 188.0046 240.6621 203.00 224.00 4.00 3 864.0000 20.22375 11.67619 813.7614 914.2386 849.00 887.00 5.00 3 540.6667 51.93586 29.98518 411.6508 669.6825 484.00 586.00 Total 15 726.0000 647.67772 167.22967 367.3280 1084.6720 136.00 1901.00 Table 3 ANOVA analysis carried out on the data Sum of Squares df Mean Square F Sig. Between Groups 5863955.333 4 1465988.833 1655.612 .000 Within Groups 8854.667 10 885.467 Total 5872810.000 14 Table 4 Ad-hoc test Tukeys range tests and LSD showing statistical significance between groups. All differences between groups are statistically significant (p Multiple Comparisons Dependent Variable: Fluorescence (I) Treatment (J) Treatment Mean Difference (I-J) Std. Error Sig. 95% Confidence Interval Lower Bound Upper Bound Tukey HSD 1.00 2.00 -1722.33333* 24.29632 .000 -1802.2945 -1642.3722 3.00 -70.00000 24.29632 .094 -149.9612 9.9612 4.00 -719.66667* 24.29632 .000 -799.6278 -639.7055 5.00 -396.33333* 24.29632 .000 -476.2945 -316.3722 2.00 1.00 1722.33333* 24.29632 .000 1642.3722 1802.2945 3.00 1652.33333* 24.29632 .000 1572.3722 1732.2945 4.00 1002.66667* 24.29632 .000 922.7055 1082.6278 5.00 1326.00000* 24.29632 .000 1246.0388 1405.9612 3.00 1.00 70.00000 24.29632 .094 -9.9612 149.9612 2.00 -1652.33333* 24.29632 .000 -1732.2945 -1572.3722 4.00 -649.66667* 24.29632 .000 -729.6278 -569.7055 5.00 -326.33333* 24.29632 .000 -406.2945 -246.3722 4.00 1.00 719.66667* 24.29632 .000 639.7055 799.6278 2.00 -1002.66667* 24.29632 .000 -1082.6278 -922.7055 3.00 649.66667* 24.29632 .000 569.7055 729.6278 5.00 323.33333* 24.29632 .000 243.3722 403.2945 5.00 1.00 396.33333* 24.29632 .000 316.3722 476.2945 2.00 -1326.00000* 24.29632 .000 -1405.9612 -1246.0388 3.00 326.33333* 24.29632 .000 246.3722 406.2945 4.00 -323.33333* 24.29632 .000 -403.2945 -243.3722 LSD 1.00 2.00 -1722.33333* 24.29632 .000 -1776.4689 -1668.1978 3.00 -70.00000* 24.29632 .016 -124.1356 -15.8644 4.00 -719.66667* 24.29632 .000 -773.8022 -665.5311 5.00 -396.33333* 24.29632 .000 -450.4689 -342.1978 2.00 1.00 1722.33333* 24.29632 .000 1668.1978 1776.4689 3.00 1652.33333* 24.29632 .000 1598.1978 1706.4689 4.00 1002.66667* 24.29632 .000 948.5311 1056.8022 5.00 1326.00000* 24.29632 .000 1271.8644 1380.1356 3.00 1.00 70.00000* 24.29632 .016 15.8644 124.1356 2.00 -1652.33333* 24.29632 .000 -1706.4689 -1598.1978 4.00 -649.66667* 24.29632 .000 -703.8022 -595.5311 5.00 -326.33333* 24.29632 .000 -380.4689 -272.1978 4.00 1.00 719.66667* 24.29632 .000 665.5311 773.8022 2.00 -1002.66667* 24.29632 .000 -1056.8022 -948.5311 3.00 649.66667* 24.29632 .000 595.5311 703.8022 5.00 323.33333* 24.29632 .000 269.1978 377.4689 5.00 1.00 396.33333* 24.29632 .000 342.1978 450.4689 2.00 -1326.00000* 24.29632 .000 -1380.1356 -1271.8644 3.00 326.33333* 24.29632 .000 272.1978 380.4689 4.00 -323.33333* 24.29632 .000 -377.4689 -269.1978 *. The mean difference is significant at the 0.05 level.