Cancer has long established
Cancer has long established it's presence on the medical podium as one of the deadliest disease in the world. In the United Kingdom alone, the incidence of cancer has been increasing at alarming rate as we noticed a 25% increase in incidence rate between the year 1977 and 2006.1 With a staggering figure of approximately 293,601 cases reported in the year 2006, 155,484 cancer-related death were reported the following year.1 This generally illustrates the importance of developing new novel anticancer drugs to tackle this problem.
Cancer or also known malignant tumour develops owing to the uncontrolled proliferation of cells and the development of the ability to metastasize and to promote angiogenesis. All of which favours cell growth and takes place preferentially due to the mutation of various proteins and pathway. A classic example would be the RAS signaling pathway. RAS, an active G-protein when bound to GTP and inactive when GDP bound, was documented to be relatively active in cancerous cell and hence promotes the abnormal proliferation of cells.2 Asides from that, p53 which presents as a tumour suppressor protein too undergoes mutation in which causing it to lose its apoptotic and antiangiogenic activity that is vitally important in preventing cancer. Besides that, it has also been documented that RAF kinases which are serine/threonine phosphotransferase involved in the mitogenic cycle also undergoes similar mutation (increase in activity), causing abnormal cell proliferation.2 Interestingly, NF-κB that undergoes extensive activation through RAF signaling would therefore benefit off RAF kinases mutation as we observed an increase in the properties of cancer namely its proliferative, metastatic, proangiogenic and antiapoptotic nature.2
Therefore, targeting specific constituent of the signaling cascade for cellular proliferation would deem reasonable in terms of treating cancer. Among the best widely used anticancer drug would be cisplatin. Known also as as cis-diamminedichloroplatinum(II) or cis-DDP, cisplatin was documented to exert its cytotoxicity through means of binding to the cell nucleus of both DNA and non-DNA target (forming Platinum-DNA adducts) in which would promotes cell death by apoptosis and necrosis.3 Taxol on the other hand, is use to treat cancer as it was found promote polymerization of which would lead to the formation of very stable yet nonfunctional microtubules.4 This of which would ultimately damnpen the mitotic and metastatic nature of cancerous cells. Aside from that, anthracycline antibiotics have also been used many years now for the treatment of cancer. These groups of drug mainly act by inhibiting DNA synthesis through means of intercalative binding to DNA.5 A splendid example for this group of drug would be epidoxorubicin which portrays not only an immunogenic but also a cytotoxic profile against cancer cells. As we are aware, cancer also normally results from the inflammatory response induced by the excessive signaling from both the intracellular tyrosine kinases and tyrosine kinases receptors.6 Therefore, by inhibiting tyrosine kinase, the phosphorylation of epidermal growth factor receptor(EGFR) involved in promoting tumour mitogenesis, metastasis and angiogenesis would be suppressed.6 The group of drugs bringing such profound effects is famously known as tyrphostins and a famous example would be AG1478.
Therefore, this experiment was carry out with the aim is to study and compare the effect of the four anticancer drugs on cell proliferation and to determine which drug is more potent.
(add in the effect at different concentration- cell death effect amplified ?) one aim?
The aim of the analysis is to quantify the degree of inhibition of cell growth produced by each of the 4 drugs
tested (cisplatin, taxol, epidoxorubicin, AG1728) and to determine which drug is the most potent (i.e. requires
the least concentration of drug to effect inhibition).
2.0 Materials and Methods
Cell Lines, Culture Medium and Incubation condition. A431 cells which is derived from a cancerous human epidermal squamous carcinoma cell were incubated at 37°C for 24hours in Dulbecco's Modified Eagle's Medium (DMEM) [Purchased from: Invitrogen. Product No.: 10938-0325] under atmospheric condition of 5% CO2 and air (ratio: 1:19). The culture medium was also supplemented with 10% (v/v) Fetal Calf Serum (FCS) [Purchased from: Invitrogen. Product No.: 16010-0169], 250IUml-1 penicillin/250microgml-1 streptomycin (Pen:strep) [Purchased from: Invitrogen. Product No.: 15140-122] and 2mM L-glutamine [Purchased from: Invitrogen. Product No.: 25030-024]. Approximately 1 million (106) cultured cells were then seeded in the wells of a 96 well plate.
96 well plate arrangement. Each drug (Cisplatin, Taxol, Epidoxorubicin and AG1478) were allocated 3 columns each on the 12 column 96 well plate. The first row (Row A) for each drug was assigned as the positive control in which consisted of 100µL of growth media. The last row (Row H) for each drug was on the other hand allocated as the negative control, consisting of only 100µL of sterile water. Alternatively, the second row (Row B) for each drug was assigned as the vehicle control in which consisted of 90µL of growth media and 10µL of suitable vehicle corresponding to that particular drug. Conversely, 90µL of growth media was added into all the remaining wells.
Drug Treatment.Cisplatin [Purchased from: Sigma Chemical Co. Product No.: P4391], Paclitaxel (Taxol) [Purchased from: Merck Bioscience Ltd. Product No.: 580555]. Epidoxorubicin [Purchased from: Merck Bioscience Ltd. Product No.: 324905] and AG1478 [Purchased from: Merck Bioscience Ltd. Product No.: 658552] were prepared from their stock solution through serial dilution to provide a series of drug concentration ranging from 0.1µM to 1000µM in which would provide a final bath drug concentration of 0.01µM to 100µM with the addition of 10µL of drugs of varying concentration to their corresponding concentration. The cells were then incubated for another 48 hours.
MTT assay. After 48 hours of incubation and the media subsequently aspirated, 100µL of media and 10µL of yellow tetrazolium MTT (3-(4, 5dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide) were then added into each of the well of the plate. The plate was then left to incubate for an additional 2 hours. The media was then carefully aspirated again and 100µL of DMSO (Dimethyl Sulfoxide) was then added in each well to solubilise the formed crystals, allowing it to be spectrophotometrically quantified by a molecular device known as SpectraMax 190 plate reader.
Data Analysis.The readings obtained from the SpectraMax 190 plate reader were given in terms of absorbance units. Using Bio-BarChart, these absorbance values were used to construct a histogram in relations to the drug concentration used. In addition to that, with the assumption that the mean value of the triplicate date obtained for the vehicle control represents 100% cell growth, the fraction of inhibition for each drug at their specific concentration were measured as follow:
The fractional inhibition determined was then used to construct a concentration-response curve which allowed us to determine the IC50 (The concentration that is required to achieve 50% of the maximum inhibition response) for each drug. BioGraph was the programme used.
Anova test F test tukey wise comparison test
All data were analysed by one way ANOVA: significant was defined as p<0.05
Suitable vehicle- be specific?
Figure 1. The mean absorbance value against epidoxorubicin concentration. A431 cells that were grown on the 96 well plate were subjected to increasing concentration of epidoxorubicin ranging from 0.01µM to 100µM and left to be incubated for 48hours. MTT assay was then carried out as described in the methods section to evaluate the properties of epidoxorubicin in inhibiting cell proliferation. The absorbance value were taken as the mean of the triplicate sampling ± standard error and plotted against epidoxorubicin concentration. The absorbance value for the positive control, vehicle control and negative controls were also used for means of comparison.
The histogram clearly illustrates the effectiveness of epidoxorubicin in inhibiting the proliferation of A431 cells. Even though epidoxorubicin portrays a concentration-response pattern in inhibiting cell proliferation, it is evident to us that the only within the effective concentration of 100µM to 1µM that epidoxorubicin has a more profound effect. This is proven as within those concentrations, epidoxorubicin illustrates more than 50% inhibition properties on A431 cells growth (with the assumption that the positive controls portray 100% cell growth). It is also reasonable for use to conclude that the inhibition effects portrayed by 100µM epidoxorubicin is approximately twice as effective of those 10µM, 5 times as effective than 1µM, 17 times as effective than 0.01µM and 18 times as effective of those of 0.01µM.
Figure 2.The effects of 4 individual anti-cancer drugs on A431 cell growth. A431 cells that were grown on the 96 well plate were subjected to increasing concentration of Cisplatin, Taxol, Epidoxorubicin and AG1478 ranging from 0.01µM to 100µM and left to be incubated for 48hours. MTT assay was then carried out as described in the methods section to evaluate the properties of these drugs in inhibiting cell proliferation. The mean values of the factional inhibition obtained were then used to plot against the corresponding concentration of individual drugs. The error bar indicates the standard error(s.e) of triplicate sampling.
It is evident to us that all four anticancer drugs tested portrayed a concentration-dependent inhibition on A431 cells proliferation, with Epidoxorubicin proving most potent with an IC50 of 4.701E-007 +/- 2.275E-007 (s.e.). The next most potent drug would be Cisplatin with an IC50 of 6.573E-006 +/- 6.59E-007 (s.e.) followed by AG1478 (IC50 = 6.267E-005 +/- 8.065E-006 (s.e.)) and Taxol (IC50 = 7.288E-005 +/- 0.06076 (s.e.)). It would be also worthy to note the result obtained for Taxol which derived a highly varied standard error range may not truly justify its true potency in inhibiting cell growth.
Figure 3.Concentration-response curve of Epidoxorubicin. The growth inhibition effects of Epidoxorubicin on A431 cells were conducted using the MTT assay and were measured spectrophotometrically as described in the methods section. As this experiment was conducted under a triplicate manner, the mean values of the factional inhibition were used to plot against the corresponding concentration of Epidoxorubicin that were used. The concentrations used were ranged from 0.01µM to 100µM. The error bar indicates the standard error of the triplicate samples.
The graph plotted clearly indicates that Epidoxorubicin illustrates a strong positive inhibiting characteristic towards A431 cells. With an IC50 of 4.701E-007 +/- 2.275E-007 (s.e.), Epidoxorubicin was concluded to be a highly potent anticancer drug. Since Epidoxorubicin portrays a concentration-response property, this explains the low fractional inhibition that is exerted by 100µM (Lower the fractional inhibition, higher the effectiveness of the drug in inhibiting cell proliferation).
Figure 4.The Mean pIC50 value for individual drugs. The mean pIC50 value for individual drugs were obtained from the class sample (n= 136). The error bar in this case represents the standard deviation of the whole sample.
It is strongly apparent to us that the mean pIC50 value varies for individual drugs. However, to further support this finding, an F test in accordance to the ANOVA test was carried out in order to determine whether the differences in the mean pIC50 value between these drugs were significant (The ANOVA F test result datasheet generated off Minitab is included at the end of this report). With a P value of 0.000 and an F statistic value of 32.85, the differences in the mean pIC50 value between these drugs were concluded to be not only significant but also large respectively. As the drugs have been proven to have significant differences in their mean pIC50 value, a Tukey pair-wise comparison test was then carried out in order to study and investigate the significance of those differences for every pair of drugs. The Tukey results obtained indicates that the mean pIC50 values for AG1478 and Cisplatin are not significantly different whilst the mean pIC50 value for Epidoxorubicin and Taxol respectively are deemed significantly different from the other two. With larger mean pIC50 values, Epidoxorubicin and Taxol were concluded to be the two most potent anticancer drugs among the four with Epidoxorubicin being more superior to Taxol. On the other hand, AG1478 and Cisplatin are deemed to be equipotent.
A431 cells express high levels of EGF receptors and in contrast to normal cells with much lower numbers of EGF receptors are growth inhibited by EGF Cell line used - brief introduction Taxol - talk bout resistance Therefore incorporate a humanized anitibody ? Limitations use other cell lines A431 is a squamous carcinoma cell line that over expresses the EGF receptor (13) and due to this fact features a constitutive activation of receptor dependent signalling processes. HeLa is derived from a cervical carcinoma, which is infected with HSV and expresses E6 and E7, viral proteins that neutralize the Rb and p53 tumour suppressor proteins (14,15). MCF7 is an estrogen responsive mammary carcinoma cell line that over expresses the anti-apoptotic Bcl-2 protein (16). These cell lines are widely used to study drug responses, because they represent common human cancers and feature different transforming principles important to the aetiology of cancer.