Results

Evaluating the possible mechanism of action of a novel anti-fungal drug on cdc20gene expression in AML cell lines
Authors
Departmental and Contact Address
Abstract
Acute myeloid leukemia (AML) is fast progressing cancer of the blood which originates in the bone marrow..Increased expression of Cdc20 has been implicated in various cancerous cells and is considered to be a potential target for anti-cancer drugs.
The present study was conducted to evaluate the mechanism of action, of a novel drug used to treat AML. The drug is an established antifungal agent and has shown to cause cytotoxicity in AML cell lines. The evaluation was based on RNA levels and cDNA expressions, derived from AML cell lines compared to non-cancerous cell lines.
Reverse transcription was used to get the cDNA imprints. The qRT-PCR approach was implemented to evaluate the changes in expression of RNA samples (based on cDNA expressions).
The Cycle Threshold (CT) signals were detected as the positive reaction in the qRT-PCR assay. The average CT values were highest for the sample that was treated by drug for the cdc20 gene. This indicated the novel drug must have reduced the overall expression of cdc20 gene compared to the samples that were untreated with the novel drug.
The time bound evaluation the 2^-ddCT values were evaluated. These values indicated the change of expression of cDNA in the same sample with respect to an endogenous control over a period of time. It was indicated that the novel drug reduced the expression of cdc20 by 63%, compared to calibrators. Moreover, the single time dCT values for test samples and calibrators complied with the results of relative expression of cdc20 gene
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Introduction
Acute myeloid leukemia (AML) is fast progressing cancer of the blood which originates in the bone marrow. The maturity of the bone marrow cells is prevented in AML. Instead, immature blast cells with increased capacity of mitosis are released into the blood. Around 11 billion blast cells are formed and released from the bone marrow. Thus, AML is a process whereby the hematopoietic precursor cells are transformed in such a way leading to limitless cell cycles and cell divisions. These cells do not undergo apoptosis or programmed cell death, which leads to the progression of cancer. AML is also designated by other classifications like myelocytic, granulocytic, myelogenous and non-lymphocytic leukemia. In this type of leukemia the affected cells are RBCs, WBCs and Platelets but do not include lymphocytes. AML can affect various organs like the lymph nodes, liver, spleen and testicles (1).
The risk factors for AML includes smoking, chronic exposure to benzene (as it damages the DNA of bone marrow cells), chemotherapeutic treatment with alkylating agents and topoisomerase II inhibitors and presence of Down’s syndrome or Fanconianemia.

AML may also be potentiated by polycythemia or myelofibrosis or other forms of blood cancer. AML is the most common form of acute leukemia that affects older people. It is more prevalent in individuals >60 years of age and predominantly occurs in males. However, 15% to 25% cases of childhood acute leukemia are due to AML. The risk of AML increases to around 10 fold from the age range of 30-34 years to the age range in 65 to 69 years. During the former age range there is a prevalence of AML in 1 out of 100,000 while in the later age range the prevalence of AML is 10 out of 100,000. Beyond the age of 70 years till the age of 85 years the prevalence is far higher(1).
Cdc20 is a specific cell division checkpoint protein that helps in the activation of anaphase-promoting complex (APC). It is product of the cdc20 gene. For, a cell to undergo mitosis (cell division), anaphase is an important step. In this step the sister chromatids are separated and start to move towards different poles from the equatorial plate (metaphase). This arrangement is important for cell division. Hence, cdc20 helps in normal cell division and progression of cancer cells (metastasis). Cdc20 binds to phosphorylated APC and activates it. Increased activation of APC/cyclin complex leads to inhibition of a protein called, Securin. Securin inhibits a cell from entering the Anaphase because it prevents the separation of chromatids to the opposite poles. Activation of APC thus helps a cell to enter the anaphase by overcoming the inhibition of Securin. Cdc20 also inhibits the cyclin-dependent kinase that was used to phosphorylate APC, after the mitotic phase is over. Hence, cdc20 acts as cell cycle checkpoints and regulates both mitosis and also inhibits premature mitosis (2).
Spindle assembly checkpoint (SAC) acts as a monitor to detect the proper alignment of chromosomes to the mitotic spindles before anaphase or cell division ensues. Thus, SAC acts as an inhibitor of APC-cdc20 and controls accurate mitosis. However, when chromosomal misalignment occurs, mitotic checkpoint complex (MCC) consisting of three proteins Mad2, Bub3 and BubR1 attach to APC-cdc20 and inactivate it. Thus, premature mitosis is prevented. In cases of malignancy this mechanism of inhibition is weakened which causes the cancerous cells to gain or lose genetic material. In AML, there is an abnormal expression of a protein called AML-1ETO which causes the chromosomal aberrations (3).
Truncated AML-1ETO has been demonstrated to interfere with the integrity of SAC. Hence, these cells produced repressed amounts of BubR1. Decreased BubR1 resulted in ineffective inhibition of APC-cdc20, which causes premature mitosis and progression of cancer in AML(3).Kasumi-1 cells (low BubR1 expressed, myeloblastic cells) and HL-60 cell line (AML cells showing normal BubR1 expression) were transfected with retrovirus-delivered BubR1. When induced by doxycycline the expression of Bub1 expression was increased these cells (4). Thus, it may be predicted that BUbR1 expressions will lead to inhibition of APc-cdc20 and inhibit premature mitosis. Targeting cdc-20 holds a promise for alleviating AML as inhibition of cdc20 and thus APC will prevent premature mitosis and progression of immature blasts into the blood.
The present study evaluated the possibilities of a novel drug in the treatment of AML. The drug has been already established as an effective antifungal and recent studies have indicated its preferential cytotoxicity in AML and leukemic stem cells, compared to non-cancerous hematopoietic stem cells. However, the mechanism of its cytotoxicity is unknown. Increased expression of Cdc20 has been implicated in various cancerous cells and is considered to be a potential target for anti-cancer drugs. Hence, the present study evaluated the impact of the novel drug on Cdc20 expression. The evaluation was based on RNA levels and cDNA expressions, derived from AML cell lines compared to non-cancerous cell lines.
Methodology
Principle
Collection and treatment of samples
RNA was extracted from a cell line that was isolated from a patient suffering from AML. Some of these cell lines were treated with the novel drug and some cell lines were kept untreated. RT-qPCR was done to evaluate the gene expression of Cdc20 (5).
Selection of samples for analysis
The final selection of RNA samples was based on the highest amount of RNA concentration and purity of the RNA extracted based on spectrophotometer readings at 260nm and 280nm. 3 pairs of Eppendorf tubes were taken(5).
Each pair contained RNA from treated and untreated cell lines. 3 pairs were used for replication and extract the most viable RNA content. Two samples, including one each from the replications were taken. The two samples respectively taken were sample “a” (derived from untreated cell line with 242.4ng/ul RNA concentration with OD260/OD280 = 1.87) and sample “d” (derived from drug-treated cell line with 298.2ng/ul RNA concentration with OD260/OD280 = 1.85)
Reverse transcription for deriving cDNA from extracted and selected RNA samples
The samples “a” and “d” were subjected to RT-PCRfor generating cDNA of the respective RNAs. A master reaction mix was prepared for each of the samples in a 0.5ml Eppendorf tube (with appropriate amounts of reaction buffer and enzyme mix). The final reaction volumes were destined to be 20ul in PCR tubes. The tubes were filled with requisite volume of sterile nuclease-free water to make up the volume along with RNA samples. 3 such tubes for “a”, “d” and control (which did not have any transcripts), were finally evaluated for getting the cDNA. Hence PCR tubes with “a” consisted of 4.13ul of RNA sample (242.4ng/ul RNA concentration) + 4.87ul sterile water + 10ul 2*RT Buffer +1ul 20*Enzyme Mix), “d” consisted of 3.35ul of RNA sample (298.2ng/ul RNA concentration) + 5.65ul sterile water + 10ul 2*RT Buffer +1ul 20*Enzyme Mix) and finally the tube with “no transcript” consisted of 9ul sterile water + 10ul 2*RT Buffer +1ul 20*Enzyme Mix. The PCR tubes were then subjected to thermocycler. Poly-T primers were used to align with the RNA sequences for obtaining cDNA. cDNA was used for obtaining the exact gene (target gene) portions to study the impact of the novel drug on Cdc20 gene(5).
Real-Time Quantitative RT-PCR (qRT-PCR) for evaluating expression of target gene
The qRT-PCR approach was implemented to evaluate the change in expression of RNA samples that were treated with experimental drug compared to samples that were untreated(5). Hence, this method helped to quantify the expression of RNA over certain periods of time. A housekeeping gene GAPDH was incorporated as an endogenous control measure, to standardize for the error (variations) in the input of nucleic acid(5).
The cDNAs were collected from thermocycler. 5ng/ulcDNA were used as because the cDNA volumes were made to 40uL. This volume was made to use 20uL for the qRT-PCR. The RNA obtained earlier was extrapolated with cDNA final volume of 40ul. For each sample of cDNA that were subjected to qRT-PCR two reaction sets were used(5). Each reaction was examined for the expression of housekeeping gene and the gene of interest (cdc20) was examined. 2 replicates were used for the cDNA samples (treated and untreated with novel drug). The two replications were done to obtain the average expression of both the genes. The RNA samples (treated and untreated with novel drug) and the control sample was assayed once. The RNA sample was also used as a control to confirm that the RNA sample was not contaminated with genomic DNA. The control sample did not contain nucleic acids and it contained the reagents and the rest of volume was made up with nuclease free sterile water.
The cDNA samples were probed with cdc20 and GAPDH primers separately. The RNA samples were not probed with cdc20 gene. However, it was probed with GAPDH. Thus, each pair will be assayed for 4 reactions and hence there will be a total of 12 assays. For the qRT-PCR, 14ul of Sybrgreen PCR master mix was taken in the PCR tubes along with 5ul of cDNA (at a concentration of 5ng/ul) or RNA (5ul) or sterile nuclease-free (5ul) water. 1ul primers were added to each to make the total volume to 20ul. All the samples were run on ABI One plus Real-time Machine.
Assay Evaluation
The qRT-PCR assay was evaluated through detection of positive reaction from the accumulation of fluorescent signal from the cdc20 and GAPDH probes (6).
Results
The qRT-PCR assay was evaluated through detection of positive reaction from the accumulation of fluorescent signal. The accumulation of such signal is denoted by their CT values. CT values indicate the number of cycles that are required for the fluorescent signal to cross the threshold level (when it crosses the background level) to get detected. The value is inversely proportional to the amount of targeted nucleic acid in the specific sample.
In this experiment, the CT values were calculated and expressed in different ways. Firstly, CT value of cdc20 (target gene) was compared to the CT of the GAPDH (reference gene) for both the samples (treated with the drug and untreated with the novel drug). The sample (treated with drug) was considered the test sample while the sample (untreated with drug) was considered as the calibrator (Ref. Table 1). Next, the Δct of both the samples were calculated from the average CT values obtained from the cdc20gene and the GAPDH gene. It was done to find out exclusively the CT value generated from cdc20 component with respect to nucleic acid background fluorescent variations (as indicated by GAPDH).
Table 1: The average CT value for cdc20 and GAPDH in calibrator and test sample
Sample Gene
Average of CT for cdc20 Average CT value for GAPDH
Samples that was not treated with X drug (calibrator) 21.354094505
13.6109089851
Samples that was treated with X drug (test) 22.633182526
13.459040165
In Table 1 the average CT value of cdc20 in calibrator and test samples are indicated. The CT value for cdc20 by itself was not enough to interpret the data because there might have been error in handling the sample. For example if the volume pipette in one sample was more than the volume pipette for the other sample, chances of error would be higher. Therefore, the CT value of cdc20 in both sample types was normalized for the CT value of GAPDH (reference gene). Thus, the CT value for the GAPDH was subtracted from the CT value for the cdc20 for the parameter Δct (Table2). However, the above two data represented the average expressions of cdc20 in both the samples.
Table 2
Sample Δct
Samples that was not treated with X drug (calibrator) 7.74
Samples that was treated with X drug (test) 9.185
To evaluate the fold expression of cdc20 in both the samples at any given time or over a period of time 2^ (- ΔΔCT) method was used. The next step was to calibrate ΔCT of the test sample to the ΔCT of the calibrator. In other words, the ΔCT of the calibrator was subtracted from the ΔCT of the test sample (Table 3). The ΔΔCT obtained was1.445 which represented the change in expression of the cdc20 between the test and calibrator conditions, normalized for any differences in loading between the test and calibrator samples. 2^-ΔCT of the respective samples indicated the relative expression of cdc20 over a period of time. More the value of 2^-ΔCT, more is the presence of the particular nucleic acid segment at any given time. Similarly, ΔΔCT denoted difference in ΔCT samples over two-time durations. 2^-ΔΔCT indicated that lesser is its value, lesser is the presence of that particular nucleic acid compared to the earlier period, which means the expression of that portion of nucleic acid has decreased. 2^-ΔΔCT was calculated from the formula(6):
2^-ΔΔCT = 2^-((CT_target – CT_endo)_time2 – (CT_target – CT_endo)_time1)
In this formula, as required for the present analysis ΔΔCT denoted difference in ΔCT test sample over ΔCT of the calibrator. While, 2^-ΔΔCT indicated the fold difference in cdc20 expression between test and calibrator.
Table 3
2^-(ΔCT) ΔΔCT 2^(- ΔΔCT)
Calibrator 4.677651 0 1
Test 1.718065 1.445 0.367292158
The fold difference in expression in cdc20 between the calibrator and test samples was obtained by calculating2^ (- ΔΔCT) (Table3). It was observed that the drug has down regulated the expression of cdc20 by 0.633 fold (Fig1).

Fig1: Comparative Quantitation of cdc20 expression in test and calibrator samples using Livak’s method(6). The fold expression of cdc20 in the test sample was significantly reduced(p<0.05).

Fig 2: Exponential relationship of cdc20 between test and calibrator sample. ΔCT method generated essentially the same results as ΔΔCT, however it tells the difference between the two conditions in absolute terms rather than the fold change. The absolute change in cdc20 expressions was also lowered significantly for the test sample (p<0.05).
2^- (ΔCT) for the test sample was 4.677 whereas 2^- (ΔCT) for the calibrator was 1.718. (Table3) (Fig2). This was another way for normalization against reference gene. The graph shows reduce 2^- (ΔCT) value in test sample when compared to the calibrator. This indicated that the drug down regulated cdc20 expression in AML cells.
Discussion
Acute Myeloid Leukemia is a life-threatening condition manifested by rapid progression of immature hematopoietic stem cells in the blood. This leads to the fast progression of cancer due to uncontrolled cell division. Left untreated AML can affect various organs like the lymph nodes, liver, spleen and testicles through metastasis. The risk factors for AML include smoking, chronic exposure to benzene, treatment with alkylating agents and topoisomerase II inhibitors. AML is the most common form of acute leukemia that affects older people. It is more prevalent in individuals >60 years of age and predominantly occurs in males.
Cdc20, a specific cell division checkpoint protein has been associated with AML and other forms of cancer. Cdc20 helps in activation of anaphase-promoting complex (APC). This leads to mitosis and progression of cancer cells. Hence, targeting the cdc20 gene and controlling its expression holds the promise of prevention of premature mitosis. The decrease in premature mitosis would lead t deceased progression of immature hematopoietic stem cells in the blood and alleviate the progression of AML. Drugs are aimed to target the expression of cdc20 gene either directly. Doxycycline increased the uptake of BubRI which lead to inhibition of APC-cdc20 complex. The direct actions are based on site-directed actions on the cdc20 genome.
The present study was conducted to evaluate the mechanism of action, of a novel drug used for the management of AML. The drug is an established antifungal and has shown the potential to alleviate AML. Increased expression of Cdc20 has been implicated in various cancerous cells and is considered to be a potential target for anti-cancer drugs. Hence, the present study evaluated the impact of the novel drug on Cdc20 expression.
The qRT-PCR approach was implemented to evaluate the changes in expression of RNA samples (based on cDNA expressions) that were treated with experimental drug compared to samples that were untreated. This method was used to quantify the expression of RNA over certain periods of time. A housekeeping gene GAPDH was incorporated as an endogenous control. Equal volumes of cDNA, RNA and control were subjected qRT-PCR.
RNA sample was used in the qPCR as another control. This was done to make sure that RNA sample was not contaminated with genomic DNA. The presence of genomic DNA will produce a false result about gene expression. This is because RNA will not be amplified by qPCR. The average CT values were highest for the sample that was treated by drug for the cdc20 gene. This indicated the novel drug must have reduced the overall expression of cdc20 gene compared to the samples that were untreated with the novel drug.
Hence, the novel drug may have acted and directly or indirectly on the cdc20 gene to reduce its overall expression. The conclusion was robust after controlling for the background fluorescence emitted by non-cdc20 genomes. The estimations for each sample were performed in duplicate. In the calibrator (untreated samples) the CT values were 22.1 and 20.5 for cdc20. This variation in CT value led to high coefficient of variation around 0.051. This variation is slightly unacceptable; although the CT value of cdc20 in both samples was normalized for the CT value of GAPDH. To increase the validity of the results the estimations must be carried out with a few replications in future studies.
The study provided strong evidence that this drug has down regulated cdc20. Therefore drug might be applicable at very early stage of AML. The study might be a simplification of establishing the role of Cdc20 in the genesis of or progression of AML. Cancer is caused by interaction of various genes and Cdc20 protein might just be an important factor in regulating the concerted pathways that lead to AML (4). Hence, the effect of the novel drug must be evaluated holistically and its effect on the final cancer pathway should be established.
The limitations of this study may be attributed to the standardization of the sample or background noise while detecting the probes. However, adequate measures were taken for standardization and purity of the RNA samples extracted from the AML cell lines. Moreover, the use of GAPDH as the housekeeping gene and normalizing the CT values with respect to cdc20 gene provided adequate reliability and sensitivity to the study.
Since, the study elucidated the level gene targeting of the novel drug, future studies may be oriented towards finding the target of the drug on the expressed and translated cdc20 protein. This is because the study reflected that cdc20 gene expression is reduced in the target cells but it is not completely eliminated. Hence, there is every chance that some cdc20 functional protein will be available and may form the APC-cdc20 complex that may result in premature mitosis and lead to the progression of cancer. Therefore, some post-translational modifications of cdc20 protein with regard to the action of novel drug may be studied. This would help in specifying the dose f the drug according to the stages of cancer (AML) and provide a better prognosis.
The results concluded that the drug has down regulated the expression of cdc20, however the exact mechanism remains unrevealed. There is a possibility that the novel drug might have over expressed the gene Mad2. Mad2 is a gene that encodes for Mad2 protein. This protein recruits two additional checkpoint proteins, BubR1 and Bub3, to form the so-called mitotic checkpoint complex (MCC) (7). MCC inhibits the formation of active APC-Cdc20. Thus, premature mitosis is prevented as revealed from review of literature (4).
One approach that could help to understand the mechanism of action of the novel drug is to use the microarray technique. The 2 RNA samples should be extracted from the 2 AML cell lines. One cell line must be treated by the drug and the other one is not. RNA will be converted to cDNA (by RT) which should be used as a template for microarray assay. This assay will enable to evaluate the differences in gene expression in the whole genome. There might be possibilities of an operon concept in expression of cdc20 gene, which may also be revealed from such studies.
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