Anti-cancer Drug that Targeting AFR Receptor
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Figure 1 Cell viability data based on the use of the XTT assay
Two different lung tumor cell lines were tested: one expressing the mutated AFR receptor (blue line) and one that expresses wild-type AFR (orange line). Figure 1a represented AFR lung cancer cell line with Drug X and figure1b represented AFR lung cancer cell line with drug Y. Cells were seeded in wells of a 96 well plate at 3 x 10*4 cells/well before treatment with the drugs at the indicated concentrations for 72 hours (triplicate samples at each concentration). XTT assay reagent was then added to the wells and the absorbance measured at 450 nm. Control wells ("no cells") contained medium but no cells.
The Cell Proliferation XTT assay is a colorimetric assay for the nonradioactive analysis of cellular proliferation, viability, quantification of cytotoxic and cytostatic compounds like anticancer drugs and pharmaceutical compounds, evaluation of growth-inhibitory antibodies and physiological mediators that able to inhibit cell growth (Sigma-Aldrich, 2016). Sample is adherent or suspension which cultured in 96-well microplates. A fictional receptor (AFR) was targeted by anti-cancer drug to inhibit the mutated AFR function, causing an anti-proliferative effect and even cell death. XTT ((2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide)) assay was used to measure cell viability in the two different lung tumor cell lines, one expressing the mutated AFR receptor (AFR mut) and one that expresses wild-type AFR (AFR wt). Drug Y was observed to hinder cancer cell growth dose dependently (Figure 1). The amount of observed absorbance increases with cell number and incubation time for 72hours when a maximum absorbance level is reached. The plot of the XTT assay data should create a curve with a linear part. This area exhibits the highest sensitivity to changes produced by the experimental parameters.
Establishing the XTT assay data for treatment of AFR mutated and AFR wild-type lung cancer cell line with drug X from 0nm to 1600nm (Figure 1a), It was illustrated that the cell viability of mutated AFR increased considerably from 1.1 to 1.3 between 0nm and 800nm and then became steady at approximately 1.4 from 800nm to 1600nm. Compared with the wild type of AFR (Figure1a), the trend of the line was similar to the mutated one but the cell viability was first rised from 0.9 to 1.2 between 0nm and 800nm and finally exhibited a loss of linearity when greater than 800nm. The lung cancer cell line with drug X represented a cell proliferation assay because the absorbance values of the mutated one were greater than control conditions, representing an increase in cell proliferation and viability. On the other hand, investigating the XTT assay data for treatment of AFR mutated and AFR wild-type lung cancer cell line with drug Y from 0nm to 1600nm, It was indicated that the cell viability of mutated AFR started off at about 1.1 from 0nm to 50nm and then occurred a dramatic drop from 1.1 to 0.1 between 50nm to 400nm and finally lied on 0.03 at 1600nm (Figure1b). Compared with the wild type of AFR (Figure1b), the line was also decreasing but showed a completely different trend. The cell viability decreased from 0.89 to 0.88 between 0nm and 800nm stably and then exhibited a significant drop when greater than 800nm and finally lied on 0.8 of 1600nm. The lung cancer cell line with drug Y represented a cell apoptosis assay as the absorbance values were lower than control conditions, showing a decrease in cell proliferation and resulted from cellular necrosis or apoptosis. From the data analysis, drug Y was successfully tested to inhibit the function of mutated AFR which have an anti-proliferative effect to cause cell death and drug Y could be put forward for further testing.
Figure 2 Flow cytometry apoptosis data: Annexin V -FITC (x axis) and propidium iodide (Y axis)
The proapoptotic effect of drug X and Y. The AFRmut cell line was treated with each drug at a concentration of 800 nM for 24 hours. The cells were harvested, resuspended in buffer and then treated with Annexin V-FITC and propidium iodide. Cytograms of annexin-V-FITC binding against PI uptake show three populations: (i) viable cells (low FITC and low PI signal) in gate Lower Left; (ii) early apoptotic cells (high FITC and low PI signal) in gate Lower Right and (iii) cells that lost membrane integrity which results in late apoptosis (high FITC and high PI signal) in gate Upper Right. The percentage of cell death increases in the tumor cancer cell line of drug Y, as demonstrated by incorporation of Annexin V.
The inhibition of cell proliferation caused by AFRmut was related to the apoptosis induction. Apoptosis is programmed as cell death which is important in development, in normal function of certain tissues and in the response to the damage. Apoptosis involves events which results in the destruction of cell. Annexin V -FITC is a protein that binds to phosphotidylserine which can be labelled and used to detect apoptotic cells. The flow cytometry apoptosis data was plotted on a dot plot and the lower left was represented the viable cells while the upper right and the lower right were represented the dead cells and the apoptotic cells respectively.
From the apoptosis data (Figure2), it was observed that the untreated condition showed 77% viable cells and 21% of apoptotic effect. When AFRmut was treated with drug X, the viable cells were increased by 19% and the apoptotic effect was largely decreased to 3%. In contrast, the incorporation with drug Y lead to considerable anti-proliferative effect where 58% of apoptotic cells were observed and viable cells only remained as 40%. Related to the induction of programmed cell death, the majority of cells from the two tumor cell lines were alive upon carrier treatment, the use of AFRmut lead to the entrance of these cells into apoptosis with incorporation of Annexin V. The treatment of drug Y showed a significant apoptotic effect than the drug X, which has a potential to inhibit the function of mutated AFR and results in anti-proliferative effect.
Figure 3 Flow cytometry cell cycle data: x-axis shows propidium iodide fluorescence and y-axis shows the number of cells
The AFRmut cell line was treated with drug X and Y at a concentration of 800 nM for 24 hours and then fixed with ethanol and stained with propidium iodide before analysis in a flow cytometer. Treating with drug Y showed no cells in G2/M phase.
The inhibition of cell proliferation induced by AFRmut was correlated with cell cycle which was evaluated with propidium iodide by flow cytometry. Cell cycle demonstrated the cell progression through a division cycle which result in cell growth and separation into two daughter cells. Live cell observation of cellular DNA and distribution of cell cycle are valuable to regulate apoptosis, and also the tumor behavior and suppressor gene systems. Cells would be distributed into three phases of cell cycle: G0 /G1 phase (2N), S phase (DNA synthesis with various DNA), and G2 /M phase (4N) to identify apoptotic cells with partial DNA content desirablyÂ (Krishnakumar R, Kraus W ,2010). Propidium iodide fluorescence was needed in these actions to bind DNA.
From the flow cytometry results by histogram (figure 3), the untreated condition showed 42% of cells in G0/G1 phase while 31% in S phase and 27% in G2/M phase. When the AFRmut was treated with drug X, the cell number in G1 phase dropped by 7% while there was increase of 6% in S Phase. DNA replication occurs more effectively during S phase. Tumor cells with a higher proportion of cells in S phase as tumors were growed faster and more aggressive in that phase so drug X induced proliferation of mutated AFR. However, the addition of drug Y caused effective anti-proliferation effect by increasing the cell numbers in G1 phase from 42% to 86% since the cell has left the cycle and has stopped dividing. There was increase in the cell numbers and grow in size. The decrease from 31% to 14% for S phase indicated the ineffective DNA replication. Moreover, there was no cells observed in G2/M phase when treated with drug Y which represented the increasing numbers of apoptotic cells and the G2/M phase arrest, which failed the mitosis progression.
Drug Y can be an effective anticancer drug which targets mutated AFR and inhibit its function to lead an anti-proliferative effect. In the present study, two lung tumor cell lines, mutated AFR receptor (AFRmut) and wild-type AFR (AFRwt), Drug Y was found that it could inhibit the growth of the AFR in vitro. The IC50 values of AFRmut and AFRwt were with the same range of 450nm. Additionally, the cell cycle revealed that Drug Y arrested mutated AFR at S phase and G2/M phase. Furthermore, the data demonstrated that Drug Y induced apoptosis in the mechanism assessed by 3 different methods: cell viability assay, apoptosis assay and the cell cycle of flow cytometry. Taken together, these results suggest that the inhibitory effect of Drug Y on cell proliferation in mutated AFR is mediated through induction of cell cycle arrest and apoptotic cell death.
The limitation is the flow cytometry are only authorize the relative abundances of cell cycle phases compared to another (Dick FA, Dyson NJ, 2002). These actions can be examined by evaluating a coordinate sample of cells with mitotic inhibitor like nocodazole or G1/S inhibitor like aphidicolin. As these drugs establish a prevalent arrest in M-phase or early S-phase accordingly, moderately proliferating cells will gather at the drug inferred arrest location. For instance, cells arrested in G1 BY pRB expression will stay in G1 phase regardless of nocodazole treatment even regulating cells will accumulate in M-phase
Overall, the data demonstrated drug Y is able to develop an anti-cancer drug that targets a receptor called AFR (A Fictional Receptor) to block its activity. Drug Y successfully inhibits the function of mutated AFR by evaluating the cell viability assay, apoptosis assay and the cell cycle of flow cytometry. The assays showed an anti-proliferative effect and even cause cell death, which explains its significant antitumor activity in the various experimental investigations that have assessed. Thus, further studies about the way of administration, dose limiting toxicities and recommended doses are needed for the preclinical development of drug Y to evaluate the potential of the compound as an anticancer drug.
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