Bcl-2 Inhibited Induction of Apoptosis by Camptothecin in HeLa Cells
November 26, 2019
Cellular Biology Lab
The Bcl-2 protein suppresses the apoptosis pathway in cells, which can lead to cancer growth. Bcl-2 can be inhibited by chemotherapeutic drugs such as camptothecin, this removes the cancerous cell through apoptosis. The purpose of this experiment was to evaluate 5% FBS, 10% FBS, and 10% FBS + camptothecin to determine which is the best for growing HeLa cells, and the Bcl-2 protein was targeted to quantify Bcl-2 expression. The hypothesize stated that 10% FBS would be the best media and have the highest expression of Bcl-2, 5% FBS would be the second-best media with intermediate expression, and 10% FBS + camptothecin would be the worst with the lowest Bcl-2 expression. The best media would have the highest confluency while the worst media would have the lowest confluency. The thickness and visibility of protein bands in the western blot are proportional to the concentration of the protein, therefore higher expression of Bcl-2 would appear as thicker and bolder bands. This was achieved through the Western Blotting technique which involves three main steps: 1) separation of proteins by molecular mass, 2) the transfer of proteins from a gel to a blotting paper, and 3) probing for the target protein with primary and secondary antibodies. The hypothesize was accepted, meaning that 10% FBS is the best media and has the highest Bcl-2 expression while 10% FBS + camptothecin is the worst and has the lowest Bcl-2 expression. This means that camptothecin has the potential to be an effective anti-cancer treatment by inducing apoptosis in cells.
Cell death can occur in one of two forms: apoptosis or necrosis (Cook et al. 1999). Necrosis occurs in response to tissue injury, for example, a severe burn. Apoptosis on the other hand is a programmed cell death that eliminates damaged cells. This programmed cell death occurs in the presence of a cell death signal or in the absence of factors necessary to the cell’s survival. This process is regulated by a family of proteins called the Bcl-2 family, this family includes Bcl-2 and Bcl-xL proteins which are antiapoptotic as well as the proapoptotic Bax and Bad proteins (Cook et al. 1999). This family of proteins is typically found on the mitochondrial membrane because apoptosis is partially initiated by the loss of cytochrome c from the intermembrane space of the mitochondria in the absence of survival factors (Cook et al. 1999). When cytochrome c is released into the cytosol it forms a complex with caspase-9, this complex initiates a cascade that eventually activates caspase-3, at which point apoptosis begins (Cook et al. 1999). Bcl-2 and Bcl-xL prevent apoptosis by holding cytochrome c in the intermembrane space of the mitochondria, meaning that high expression of these proteins prevents cell death (Cook et al. 1999). On the other hand, high expression of the proapoptotic Bad and Bax proteins promotes the loss of cytochrome c from the mitochondrial intermembrane and initiates apoptosis (Cook et al. 1999). Several studies have shown estrogen and other sex hormones stimulate an increase in Bcl-2 expression (Leung and Wang, 1999).
The natural regulation of Bcl-2 is disrupted by chemotherapeutic drugs including the cytotoxic camptothecin which is used in breast cancer treatment (Leung and Wang, 1999). Camptothecin works by inhibiting Bcl-2, this allows cytochrome c to leave the mitochondrial intermembrane and apoptosis to take place (Leung and Wang, 1999). This would be harmful to a healthy cell but drugs like camptothecin can be helpful in destroying cancer cells (Leung and Wang, 1999). Camptothecin only kills cells that are actively dividing, so non-dividing cells like neurons or infrequently dividing muscle cells are generally not affected by camptothecin treatment; this is because it inhibits DNA topoisomerase-I, the enzyme that makes single-stranded DNA cuts during DNA replication and transcription to relieve supercoiling (Morris and Heller, 1996). This prevents the formation of a phosphodiester bond between the DNA fragments (Venditto and Simanek, 2013), in HeLa cells this damage occurs mostly during the synthesis phase of interphase and results in cell-death (Papazisis et al. 2004). Camptothecin also has been shown to inhibit the estrogen-stimulated increased expression of Bcl-2, the consequence of this is that cancer cells treated with camptothecin have lower levels of Bcl-2 mRNA, which is the amino acid sequence that codes for the Bcl-2 protein during DNA translation (Leung and Wang, 1999).
The ability to quantify Bcl-2 expression is useful when examining the effectiveness of chemotherapeutic drugs or any other cancer treatment that is associated with the Bcl-2 pathway or for determining if the cancer cells are becoming resistant to a drug-treatment. One of the most common ways to identify and quantify Bcl-2 protein expression is through a technique called Western Blotting. This technique has three main steps: separating proteins by molecular mass, transferring proteins from the SDS gel to a transfer membrane, and then probing for the target protein with primary and secondary antibodies so the protein can be visualized.
In order to separate the proteins by size they must first be liberated from the cell, to accomplish this the adhesive cells must be scraped from their culture in cold PBS (Mahmood and Yang, 2012). Then a lysis buffer such as RIPA or MPER is added to the dislodged cells, this inhibits proteases and phosphatases and makes the cells hypotonic which encourages the cell to burst (Mahmood and Yang, 2012). The lysed cells are spun in a centrifuge to purify the protein in the form of supernatant. In order to determine the exact amount of protein in each sample the concentration of the samples is determined through spectrophotometry (Mahmood and Yang, 2012), the samples are compared to a standard such as a Bicinchoninic acid Protein Assay and a standard curve is generated and used to calculate the concentration of each sample. A gel is placed into an SDS-PAGE electrophoresis chamber with buffer and then the marker and samples are added to the wells (Mahmood and Yang, 2012). The gel can be run at a higher voltage for a shorter amount of time for quicker results or on a lower voltage for a longer amount of time for more reliable results (Mahmood and Yang, 2012). This process separates the proteins by molecular mass, larger proteins will be at the top of the gel and the smaller proteins will appear at the bottom of the gel because they traveled further through the gel.
After the proteins are separated by size, they can be transferred to a membrane with a transfer apparatus because otherwise they would not be visualized, the proteins become fixed on the membrane/blot (Mahmood and Yang, 2012). The apparatus is assembled with the negatively charged electrodes below the gel and the positively charged electrodes above the transfer membrane, so the negatively charged proteins move towards the positive charge and transfer from the gel to the membrane (Mahmood and Yang, 2012). The proteins are negatively charged because they are treated with SDS.
Finally, the target protein is probed for using antibodies. The transfer membrane binds to all proteins and antibodies in an indiscriminate manner; therefore, a blocking solution is required to prevent this and only bind specific antibodies (Mahmood and Yang, 2012). Usually, milk is used in the blocking process because it contains the casein which binds to proteins and prevents antigens from non-specifically binding (Mahmood and Yang, 2012). After incubating with the blocking solution, it is time to add the antibodies. The primary antibody should bind to the antigen on the target protein (Mahmood and Yang, 2012). For example, a Bcl-2 antibody is selected as the primary antibody when probing for Bcl-2. The second antibody is chemiluminescent and binds to the primary antibody on the antigen-antibody complex (Mahmood and Yang, 2012). A chemiluminescence imager can be used to view the secondary antibody, which is also known as the label antibody because this corresponds to the position of the protein in respect to the molecular ladder on the blot, this allows for the visualization of the protein size and relative concentration between samples (Mahmood and Yang, 2012).
This experiment aims to discover how variable concentrations of growth factors and the presence of a chemotherapeutic drug growth in HeLa cells and resistant to apoptosis. Specifically, this experiment will culture HeLa cells in three different solutions; 5% FBS (Fetal Bovine Serum), 10% FBS, and 10% FBS + camptothecin. The Fetal Bovine Serum contains growth factors and γ‐globulins that activate DNA transcription and translation (Asayama and Yao, 2017). The Bcl-2 proteins in each of these cultures will be isolated and analyzed with Western Blotting (DeCicco-Skinner, 2019). After the proteins are isolated, equally concentrated protein samples from each will be separated by mass by SDS-PAGE electrophoresis (DeCicco-Skinner, 2019). The proteins will be transferred to the PVDF transfer membrane, afterward, the membrane will be probed for the Bcl-2 protein with the primary antibody (Bcl-2) and the secondary antibody so that the banding pattern of the proteins can be visualized using a chemiluminescence imager (DeCicco-Skinner, 2019). The target protein is Bcl-2 because it is an anti-apoptotic protein, so its concentration is proportional to the health and growth of the cells. Cells with low expression of Bcl-2 would be more likely to undergo apoptosis, while cells with high expression of Bcl-2 are more likely to be growing rapidly.
It is hypothesized that 10% FBS will be the best media for growing HeLa cells due to the high nutrient content, 5% FBS is hypothesized to be the second-best media for growing HeLa cells because it will not has as many nutrients for the HeLa cells to replicate, and 10% FBS + camptothecin is hypothesized to be the worst media for growing Hela cells because it will kill them through apoptosis. Additionally, it is hypothesized that the HeLa cells grown in the 10% FBS + camptothecin solution will have the lowest expression of Bcl-2 because camptothecin is a cytotoxic drug and has been shown to induce apoptosis and inhibit expression of Bcl-2 proteins (Venditto and Simanek, 2013). On the other hand, HeLa cells grown in the 10% FBS solution are hypothesized to have the highest expression of Bcl-2 because this sample has the greatest availability of growth factors and nutrients. Finally, HeLa cells grown in the 5% FBS solution are hypothesized to have an intermediate expression of Bcl-2 with respect to the two other samples, because while this sample has the lowest availability of growth factors and nutrients it does not contain any cytotoxic drugs.
If the hypothesis is correct then HeLa cells grown in 10% FBS will be the most confluent and have the least number of floating cells, 5% FBS will be the second most confluent, and 10% FBS + camptothecin will be the least confluent with the greatest number of floating cells. The floating cells indicate that the HeLa cell has detached from the substrate and died. Furthermore, observations of the blotted membrane with a chemiluminescence imager will reveal that the protein band in the 10% FBS lane will be the thickest, indicating the highest expression of Bcl-2. Conversely, the protein band in the 10% FBS + camptothecin lane will be either non-existent or the thinnest and most faint, meaning it has the lowest expression of Bcl-2. Finally, the protein band in the 5% FBS lane will appear intermediate to the two other samples, it will be thinner and fainter than the 10% FBS sample but thicker and more visible than the 10% FBS + camptothecin, this would be intermediate Bcl-2 expression.
Methods and Materials
First, the media was removed from cell culture Petri dishes containing 5% FBS, 10% FBS (A and B), and 10% FBS + camptothecin, then the Petri dishes were washed twice with cold PBS. Next, RIPA with protease and phosphate inhibitors were added to each petri dish. The cells were scraped, and the contents transferred to a tube, the contents were mixed together, and then the samples were incubated on ice for 35 minutes. After the cells ruptured and released their proteins into the solution the samples were spun at 4 degrees Celcius at 12,000 rpm for 5 minutes. The supernatant was transferred to new tubes. The protein samples were added to wells on a well-plate along with RIPA buffer in a 1:5 dilution ratio. Protein standards ranging from 2mg/mL to 25 µg/mL were added to separate wells so that the protein concentration of the samples could be measured by the absorbance at 562 nm. A working agent was added to each of the wells. After this, the well-plate was covered and incubated at 37 degrees Celcius for 30 minutes and then a plate reader was used to measure the absorbance of all of the samples at 562 nm so that a standard curve of protein concentrations could be calculated (DeCicco-Skinner, 2019).
Gel and Gel Box Preparation
To begin the 4-12% Tris-Glycine gel with Tris-Glycine buffer was rinsed with water and placed in the gel box creating a tight seal. Next, the inner and outer chambers of the gel box were filled with1x MOPS running buffer (DeCicco-Skinner, 2019).
Sample Preparation and SDS-Page
While the gel equilibrated with the buffer for 10 minutes, samples containing 20 µg of protein were prepared from the original protein samples, RIPA lysis buffer, and loading dye. These samples were heated for 10 minutes at 70 degrees Celcius and then placed on ice. The protein marker was loaded into a gel lane and then the samples containing 5% FBS, 10% FBS, and 10% FBS + camptothecin were loaded into the next lanes. Then the gel ran for 35 minutes at 200 volts. Afterward, the gel was removed from the cassette plates with a gel knife, and the gel was cut to leave only the central region so that it could fit in a petri dish filled with deionized water (DeCicco-Skinner, 2019).
Protein Transfer with the iBlot2 Dry Blotting System
For this procedure an iBlot2 transfer stack was obtained, the stack contained a PVDF transfer membrane, buffer, copper electrodes, filter paper, and an absorbent pad. The iBlot was separated into separate Top and Bottom Stacks so that the transfer membrane was the uppermost surface in the Bottom Stack. The plastic tray containing the bottom stack was placed onto the blotting surface of the Gel Transfer Device so that the electrical contacts of the two elements would be unobstructed and aligned. The electrophoresed gel was placed in the Bottom Stack and covered by wet filter paper. Then a blotting roller was used to remove air bubbles trapped in the membrane. The plastic separator was removed from the Top Stack, and then the Top Stack with the transfer gel layer exposed was placed on top of the filter paper. An absorbent pad was placed on top of the Top Stack, again this was arranged so that the electrical contacts aligned with the Gel Transfer Device. The process of protein transfer was initiated using the P0 method on the Gel Transfer Device, this ran for 7 minutes. After the protein transfer was complete, the absorbent pad and components of the Top Stack were discarded, the gel was also discarded because the protein markers had moved onto the membrane. A permanent marker was used to label the positions of the protein marker on the membrane and then the membrane was transferred to a petri dish filled with water (DeCicco-Skinner, 2019).
A blocking solution of 5% non-fat milk in TBST was added to the petri dish with the membrane and incubated at 4 degrees Celcius until 24 hours before the antibody visualization procedure. At this point, the primary antibody (Bcl-2 antibody) was added to the solution at a 1:500 dilution and incubated again at 4 degrees Celcius for 24 hours (DeCicco-Skinner, 2019).
To wash away the primary antibody (Bcl-2) the membrane was washed three times with TBST, each time rocking the solution for 10 minutes. After the membrane was washed the secondary antibody was added in a 1:2000 dilution. The membrane with the secondary antibody was rocked for 45 minutes to facilitate its binding to the Fc region of the Bcl-2 antibody. To wash away the secondary antibody the membrane was washed three times with TBST, each time rocking the solution for 5 minutes. Developing solution was prepared in a 1:1 ratio with chemical A and chemical B. While the solution was prepared the protein blot was allowed to slightly air dry. The developing solution was added to the petri dish containing the protein blot so that it covered the entire surface. After 5 minutes the blot membrane was removed from the solution and placed onto a Kimwipe to remove any excess developing solution, then the membrane was placed between two sheet protectors. Finally, the banding pattern was visualized using the chemiluminescence imager (DeCicco-Skinner, 2019).
Table 1: Confluency and Health Observations for Cell Culture Treatments
Table 1: Displaying observations regarding the overall appearance and confluency of the four cell culture treatments: sample 1 (5% FBS), sample 2A (10% FBS), sample 2B (10% FBS), and sample 3 (10% FBS + camptothecin). Cell cultures treated with 10% FBS had the greatest confluency and appeared the healthiest, treatments of 5% FBS produced cells that appeared healthy but had lower confluency, the cell culture treated with 10% FBS + camptothecin had the lowest confluency and contained many dead floating cells.
The appearance and confluency of the four cell cultures were examined prior to protein isolation. Sample 1 (5% FBS) contained cells that appeared healthy because there were very few or no floating cells, meaning that most of the cells were alive and adhered to the petri dish. The cells were growing on top of each other forming giant masses of cells. Confluency varied in the culture but overall there was 55% confluency. Sample 2A (10% FBS) contained the greatest number of cells and they all appeared very healthy, meaning that very few or no floaters were seen because most adhered to the petri dish. These cells also were growing on top of each other forming layers and giant masses. Confluency was approximately 95% throughout this culture. Sample 2B (10% FBS) contained cells that were adhered to the petri dish and appeared healthy for the most part, but there was a small quantity of floating dead cells. Confluency was approximately 65% throughout the culture. Sample 3 (10% FBS + camptothecin) contained many sickly or dead cells; most of the cells were floating around and not adhered to the petri dish. The cells in this culture gathered into giant masses. Confluency varied in the culture, in most places there was 3% confluency, but when the cells formed masses the confluency increased to 13%.
Table 2: Preparation of 20 µg Protein Samples for each Cell Treatment using RIPA Buffer and Loading Dye
Table 2: Displaying the volume of reagents used to prepare samples containing 20 µg protein for the four treatment groups. The second column (protein conc.) displays the concentration of protein from each of the original isolated protein samples in µg/uL. The third column (volume of protein for 20 µg) displays the volume of protein in µL used to prepare the samples. The fourth column (volume of RIPA Buffer) displays the volume of buffer in µL used to prepare the samples. The fifth column (volume of loading dye) displays the volume of loading dye in µL used to prepare the samples.
The samples were prepared so that each contained 20 µg protein. Sample 1 (5% FBS) contained 2.8 µL of a protein with a 7.1 µg/µL concentration, 17.2 µL of RIPA buffer, and 6 µL of loading dye. Sample 2A (10% FBS) contained 6.1 µL of a protein with a 3.3 µg/µL concentration, 13.9 µL of RIPA buffer, and 6 µL of loading dye. Sample 2B (10% FBS) contained 6.9 µL of a protein with a 2.9 µg/µL concentration, 13.1 µL of RIPA buffer, and 6 µL of loading dye. Sample 3 (10% FBS + camptothecin) contained 6.1 µL of a protein with a 3.3 µg/µL concentration, 13.9 µL of RIPA buffer, and 6 µL of loading dye.
Figure 1: Reverse Contrast Image from a Chemiluminescence Imager allowing for the Antibody Visualization
Figure 1: Displaying the results from the Western Blot of three protein samples. To the left in the “M” lane is the Novex Sharp Pre-Stained Protein Standard, it shows molecular weights measured in kDa. The second lane “5%” which contained the Sample 1 of 5% FBS shows some expression of the Bcl-2 protein. The third lane “10%” which contained Sample 2A of 10% FBS shows the greatest expression of the Bcl-2 protein. The fourth lane “10% + C” which contained Sample 3 of 10% FBS + camptothecin shows the expression of the Bcl-2 protein. The Bcl-2 Protein appeared between the 20 and 30 kDa molecular markers for all three samples.
Using a Chemiluminescence Imager allowing for the visualization of Bcl-2 expression, this is because the secondary antibody is chemiluminescent, and it binds to the primary antibody (Bcl-2 antibody) which binds to antigens on the Bcl-2 protein. Thus, the banding pattern that appeared in Figure 1 allows for the visualization of Bcl-2 expression.
Sample 1 in lane 2 which contained 5% FBS displayed expression of the Bcl-2 protein, in comparison to the other two protein samples, the western blot results for this sample appeared intermediately bold and thick (Figure 1). Sample 2A in lane 3 which contained 10% FBS displayed expression of the Bcl-2 protein, in comparison to the other two protein samples, the western blot results for this sample appeared the boldest and thickest (Figure 1). Sample 3 in lane 4 which contained 10% FBS + camptothecin also displayed expression of the Bcl-2 protein, in comparison to the other two protein samples, the western blot results for this sample appeared the least bold and thick (Figure 1).
For all three samples, Bcl-2 expression appeared between the 20 and 30 kDa markers of lane 1 (Figure 1).
This purpose of this experiment was to explore how the concentration of fetal bovine serum (5% or 10% FBS) and presence of the cytotoxic camptothecin would affect the growth of HeLa cells and their resistance to apoptosis To accomplish this the expression of the Bcl-2 protein was quantified from each sample through Western Blotting. It was important to keep the protein ice cold to inhibit the proteases that would denature the protein (Mahmood and Yang, 2012). Additionally, while it was important to culture the HeLa cells in the sterile environment of flow hood to prevent contamination this is not necessary for protein isolation. HeLa cells are anchorage-dependent meaning that they will only grow if they have adhered to the media. During the protein isolation step, the cells were scraped from the media, which halted DNA replication since they were no longer attached to the media. This protein was targeted because it is an anti-apoptotic protein, so its concentration provides information about the health of the cell. Cells with low expression of Bcl-2 would be more likely to undergo apoptosis, while cells with high expression of Bcl-2 are more likely to be growing rapidly. This is an important technique with applications to treating cancer because cancer cells can be killed by inhibiting Bcl-2. Thus, the effectiveness and drug-resistance of Bcl-2 associated chemotherapeutic drugs can be investigated in this manner.
It was hypothesized that the 10% FBS would be the best medium for growing HeLa cells because of the high nutrient content and that HeLa cells grown in 10% FBS would have the highest expression of Bcl-2. On the other hand, a 10% FBS + camptothecin media was hypothesized to be the worst for growing HeLa cells and that the HeLa cells grown in the 10% FBS + camptothecin media would have the lowest expression of Bcl-2 because camptothecin is a cytotoxic drug and is known to induce apoptosis and inhibit expression of Bcl-2 proteins (Venditto and Simanek, 2013). Finally, it was hypothesized that 5% FBS would be the second-best media for growing HeLa cells because it does not has as many nutrients as the 10% FBS media for the HeLa cells to replicate and that it would have an intermediate expression of Bcl-2 in respect to the two other samples because this sample did not contain a cytotoxic drug but had less available growth factors and nutrients than the 10% FBS sample.
If the hypothesis was correct then it was predicted that the HeLa cells grown in 10% FBS would be the most confluent and have the least amount of floating cells, 5% FBS would be the second most confluent, and that 10% FBS + camptothecin would be the least confluent with the greatest number of floating cells. Additionally, the protein band in the 10% FBS lane on the blot membrane would be the thickest of the three samples, which is an indicator that it has the highest expression of Bcl-2. Conversely, the protein band in the 10% FBS + camptothecin lane was predicted to be either non-existent or the thinnest and most faint, which would have indicated the lowest expression of Bcl-2. Finally, the protein band in the 5% FBS lane was predicted to appear as an intermediate to the two other samples, it will be thinner and fainter than the 10% FBS sample but thicker and more visible than the 10% FBS + camptothecin, of this would mean that it has intermediate Bcl-2 expression.
Before the protein isolation step in the Western Blotting technique, the confluency of each of the samples was examined. As predicted, the HeLa cells grown in 10% FBS were the most confluent; Sample 2A was 95% confluent and Sample 2B was 65% confluent (Table 1). There were very little or no floating dead cells in this media meaning that the HeLa cells grown in 10% FBS were healthy and alive. Furthermore, the HeLa cells grown in the 10% FBS + camptothecin media were the least confluent as was predicted; Sample 3 varied from 3% to 13% confluent (Table 1). There were many dead and sickly cells in this media, in fact, most of the cells were floating meaning that the HeLa cells grown in 10% FBS + camptothecin were mostly dead or dying. Finally, as was predicted the HeLa cells grown in 5% FBS were the second most confluent; Sample 1 was 55% confluent (Table 1). Most of these cells were healthy and adhered to the flask. Since the results of this part of the experiment match the prediction the first part of the hypothesis can be accepted: 10% FBS is the best medium for growing HeLa cells because those cultures were the most confluent (95% and 65%), 10% FBS + camptothecin is the worst medium for growing HeLa cells because that culture was least confluent (3% to 12%) and there were many dead cells, and 5% FBS is the second-best media for growing HeLa cells because it was the second most confluent sample (55%). Therefore, it can be concluded that the rate of HeLa cell growth increases when the concentration of Fetal Bovine Serum is increased and that camptothecin inhibits the growth of HeLa cells and induces apoptosis. These findings correspond to other research, an experiment published in the National Academy of Sciences found that human fibroblasts grew better in 20% FBS than 10% FBS. (Peehl and Stanbridge, 1981).
Analysis through spectrophotometry revealed that the 5% FBS culture (Sample 1) had a protein concentration of 7.1 µg/µL concentration, the 10% FBS cultures had protein concentrations of 3.3 µg/µL and 2.9 µg/µL (Sample 2A and Sample 2B respectively), and the 10% FBS + camptothecin culture had a protein concentration of 0 µg/µL concentration so it was replaced with a new sample (Sample 3) containing 3.3 µg/µL concentration (Table 2). It is no surprise that the 10% FBS + camptothecin culture originally had such a low protein concentration because it also had such low confluency and so many floating dead cells, in such condition’s DNA translation would not occur. On the other hand, it is surprising that the 5% FBS culture (Sample 1) had the highest concentration of protein because it was less confluent than the 10% FBS cultures.
A Chemiluminescence Imager was used to view the chemiluminescent secondary antibodies on the blot membrane. This works because the secondary antibody binds to the primary antibody (Bcl-2 antibody) which binds to antigens on the Bcl-2 protein. This produced the banding pattern on the blot membrane that allowed for the visualization of protein bands revealing protein bands composed of the molecular ladder and Bcl-2 proteins (Figure 1). As was predicted the 10% FBS sample displayed the highest expression of the Bcl-2 protein, this was concluded because it had the boldest and thickest protein bands out of the three samples (Figure 1 – Lane 3 “10”). Also corresponding to the predication, 10% FBS + camptothecin displayed the lowest expression of the Bcl-2 protein, this was concluded because it has the thinnest and most faint protein band out of the three samples (Figure 1- Lane 4 “10 + C”). Finally, the 5% FBS sample also appeared as expected, displaying an intermediate expression of the Bcl-2 protein, this was concluded because the protein band was thicker and more visible than the “10% + C” sample in Lane 4 but not nearly as thick or as bold as the “10%” sample in Lane 3 (Figure 4 – Lane 2 “5”). For all three samples, Bcl-2 expression appeared between the 20 and 30 kDa markers of lane 1 (Figure 1). There were no abnormalities on the blot but some of the bands for the molecular ladder were very faint, so it was difficult to determine the size in kDa that each band represented. This may have resulted in the molecular size of the Bcl-2 protein bands being misread.
There is a high likelihood that this band that appears between 20 kDa and 30 kDa is Bcl-2 because the blocking with milk should have blocked binding of any antibodies besides the Bcl-2 antibody and chemiluminescent secondary antibody. Furthermore, experimental results published in the journal of Circulation Research report Bcl-2 to show Bcl-2 protein bands at 26 kDa and 32 kDa for cardiac myocytes (Cook et al. 1999). Similar findings were published in the scientific journal Blood, which discovered through Western Blotting that myeloid leukemias and lymphocytes appear at 26 kDa (Delia et al. 1992). Furthermore, the Western Blot technique revealed that the Bcl-2 protein appeared at 22.5 kDa in mice, these findings write published in The Journal of Biological Chemistry (Vance et al. 1996). Similarly, findings in the journal Stroke detected Bcl-2 proteins at 26 kDa in the neurons of mice (Wu et al. 2003). The most commonly reported molecular size of Bcl-2 is 26 kDa so the band that fell between 20 kDa and 30kDa is most likely a Bcl-2 protein with a molecular size of 26 kDa. However, it is possible that this band could be a different protein from the Bcl-2 family such as Bcl-xL which has a molecular size of 28 kDa or Bax which has a molecular weight of 21 kDa (Wu et al. 2003). To be sure that the observed is actually Bcl-2 then a positive and negative control should be included in the experiment. The positive control would be something that the primary antibody will definitely bind to, this positive control could be pure Bcl-2 protein. The positive control produces a protein band next to the other samples. The negative control would be something that the primary antibody would not bind to, such as beta-actin (Mahmood and Yang, 2012). If the banding appeared in the same location for the negative control, then this would indicate that non-specific antigen bonding occurred. Including duplicates of each sample would have also served as a positive control, because if the two duplicates did not produce identical results then it would indicate that there was some experimental error in preparing the equal protein concentrations or if the bands appeared in different locations it would mean that other antibodies are non-specifically binding to the membrane.
The hypothesis can be accepted because the results match the prediction and the Bcl-2 band size correspond to those from scientific literature: the 10% FBS sample has the highest expression of Bcl-2, the 5% FBS has an intermediate expression of Bcl-2, and the 10% FBS + camptothecin sample has the lowest expression of Bcl-2. The significance of this is that the sample with the lowest expression of Bcl-2 will be best at killing cancer cells. It was important to include camptothecin in this experiment because if it was excluded then it might be incorrectly concluded that cancer cells cannot undergo apoptosis when higher concentrations of nutrients and growth factors are present. Of course, this is not true because when camptothecin is added to 10% FBS it inhibits expression of the Bcl-2 protein. The importance of these findings is that camptothecin can still be an effective cancer drug even in conditions that would otherwise promote cell growth and typically suppress apoptosis (a high concentration of growth factors for example).
It was important that equal concentrations of protein samples were used in Western Blot rather than equal concentrations of protein volume because the samples had different concentrations of protein per unit of volume. If the samples had been loaded to the wells so that they all used the same volume of protein, then some of the wells would end up with higher or lower concentrations of protein. This could result in one of the samples being falsely represented as having a higher expression of the targeted Bcl-2 protein, or conversely, a sample can be misrepresented as having a lower expression of Bcl-2 than in reality. For example, if a higher concentration of 10% FBS + camptothecin protein was injected into a well then it would produce a Bcl-2 protein band that was thicker and bolder than the other samples. This is why the concentration of protein in each sample was calculated through spectrophotometry and then diluted in the loading dye and RIPA buffer (Table 2).
This western blot experiment only probed for one target protein, but usually, multiple proteins are detected in a single western blot. If this experiment also included a Bax antibody, then the confidence in the Bcl-2 results would increase. While Bcl-2 is an antiapoptotic protein, Bax has the opposite effect because it is a proapoptotic protein. Bax proteins promote the loss of cytochrome c from the mitochondrial intermembrane and initiate apoptosis (Cook et al. 1999). Multiple experiments have shown that Bcl-2 and Bax have an inverse relationship, meaning that when concentrations of Bcl-2 are low then concentrations of Bax are high, and vice versus (Venditto and Simanek, 2013). Thus, probing for Bax should produce the opposite effect of Bcl-2: the 10% FBS sample should have the lowest expression of Bax indicated by the faintest Bax protein band, the 5% FBS has an intermediate expression of Bax, and the 10% FBS + camptothecin sample has the highest expression of Bax indicated by the boldest Bax protein band. The Bax protein is 21 kDa (Wu et al. 2003), so this would produce a second row of protein bands below the Bcl-2 bands.
Finally, this experiment could be improved by running the electrophoresis at a lower voltage for a longer amount of time to better separate the proteins. An interesting future experiment would be to explore how the length of incubation time for 5% FBS, 10% FBS, and 10% FBS + camptothecin effects expression of Bcl-2 proteins; research suggests that longer incubation would result in lower concentrations of Bcl-2 in the 5% and 10% FBS samples (Papazisis et al. 2004). This experiment could also be replicated but instead of probing for Bcl-2, Bcl-xL primary antibodies could be used instead to probe for Bcl-xL. This should produce similar results to the Bcl-2 experiment because it is also an antiapoptotic protein, however, it should produce protein bands that are 28 kDa instead of 26 kDa (Wu et al. 2003).
Cook, S. A., P. H. Sugden, and A. Clerk. (1999) Regulation of Bcl-2 Family Proteins During Development and in Response to Oxidative Stress in Cardiac Myocytes. Circulation Research. 85: 940-949.
DeCicco-Skinner, K. 2019. Weeks 9-11: Protein Isolation for Western Blot (part 1). Bio-320 Cell Biology lab American University: Washington, D.C.
DeCicco-Skinner, K. 2019. Weeks 9-11: SDS-PAGE and Transfer for Western Blot (parts 2 and 3). Bio-320 Cell Biology lab American University: Washington, D.C.
Delia, D., A. Aiello, D. Soligo, E. Fontanella, C. Melani, F. Pezzella, M. A. Pierotti, G. Della-Porta. (1992) Bcl-2 proto-oncogene expression in normal and neoplastic human myeloid cells. Blood. 79(5): 1291-8.
Leung, L. K., and T. T. Y. Wang. (1999) Differential effects of chemotherapeutic agents on the Bcl‐2/Bax apoptosis pathway in human breast cancer cell line MCF‐7. Breast Cancer Research and Treatment. 55(1): 73-83.
Mahmood, T., and P. Yang. (2012). Western Blot: Technique, Theory, and Trouble Shooting. North American Journal of Medical Sciences. 4(9): 429-434.
Morris, E. J., and H. M. Geller. (1996) Induction of neuronal apoptosis by camptothecin, an inhibitor of DNA topoisomerase-I: evidence for cell cycle-independent toxicity. J Cell Biol. 134(3): 757-70.
Papazisis, K. T., T. G. Kalemi, D. Zambouli, G. D. Geromichalos, A. F. Lambropoulos, A. Kotsis, L. L. Boutis, and A. H. Kortsaris. (2004) Synergistic effects of protein tyrosine kinase inhibitor genistein with camptothecins against three cell lines in vitro. Cancer Letters. 233(2): 255-256.
Peehl, D. M., and E. J. Stanbridge. (1981) Anchorage-independent growth of normal human fibroblasts. National Academy of Sciences. 78(5): 3053-3057.
Vance, B. A., C. M. Zacharchuk, and D. M. Segal. (1996) Recombinant Mouse Bcl-2(1-203). The Journal of Biological Chemistry. 271: 30811-30815.
Venditto, V. J., and E. E. Simanek. (2013) Cancer Therapies Utilizing the Camptothecins: A Review of in Vivo Literature. Mol Pharm. 7(2): 307-349.
Wu, C., H. Fujihara, J. Yao, S. Qi, H. Li, K. Shimoji, and H. Baba. (2003) Different Expression Patterns of Bcl-2, Bcl-xl, and Bax Proteins After Sublethal Forebrain Ischemia in C57Black/Crj6 Mouse Striatum. Stroke. 34(7):1803-8.
Yao, T., and Y. Asayama. (2017) Animal‐cell culture media: History, characteristics, and current issues. Reproductive Medicine and Biology. 16(2): 99-117.