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In this study, Cd-induced apoptosis in human HEK-293T human embryonic kidney 293 cells was tested Surface membrane alteration was measured as a characteristic feature of apoptotic cells. For this purpose two staining were employed at the same time, Annexin V-FITC, and propidium iodide (PI). The concentration of Cd (5 μM) was found to induce apotosis in HEK-293T cell lines, using dose dependent method after 24 hour treatment. The percentage of apoptotic cells were 3.36, 5.5, 34.63 for non-treated cells, Cd (2.5μM), and Cd (5 μM) respectively.
Cell death happens through necrosis which is an uncontrolled and passive process, or apoptosis which is controlled and energy-dependent process. Generally, two parameters are important in apoptosis: morphological changes and physiological outcomes. In necrosis swelling process, cytoplasmic vacuoles, cytoplasmic blebs were formed. Also, endoplasmic reticulum becomes expanded, swollen or ruptured mitochondria were observed, and ribosomes become disaggregated (Elmore, 2007). As a result, the contents of cells are released; adjacent cells are damaged and initiate an inflammatory response. The term apoptosis is a process by which a programmed sequence of event leads to cell death in the regulation of tissue homeostasis. Morphological and biochemical changes are two distinct parameters that this process can be characterized with. Translocation of the membrane phospholipid phosphatidylserine (PS) is one of the primary morphological changes that occurred in apoptosis. There are other morphological changes such as: margination of chromatin in the membrane of nuclear, condensation of nuclear, budding, karyorrhexis, shrinkage of the cells, and fragmentation of cell. There are two pathways that apoptosis can be induced with including, intrinsic pathway and extrinsic pathway. Intrinsic pathway contains: oxidative stress, free radicals, absence of growth factor. Extrinsic pathways mediated by activation of caspases (Karp, 2002). Mechanistically, extrinsic pathway is such a receptor mediated and it can be activated through the external messenger TNF by binding to the TNF receptor (TNFR1). In the case of intrinsic pathways caspase activation is started through the activation of proapoptotic family member Bcl-2 (Bad or Bax) and cytochrome c release from the mitochondria (Kar, 2002). Furthermore, an endoplasmic reticulum (ER)-dependent pathway has also been reported in literature (Kondoh 2001).
Morphological and physical changes in apoptosis can be monitored by flow cytometry using various procedures. Basically, flow cytometry is a technique to count and examine the particles in the micron (micrometer) or microscopic range. Cells and chromosoms are the examples of used samples.The mechanism of work in flow cytometry including two major steps: cells are suspended in a stream of fluid and passed them through detector systems. Detection is based on an optical-to-electronic coupling system. In the optical system, particles are illuminated and the resulting light directed into detectors. Scattered and emitted light signals are then converted into electronic signals and processed by a computer (BD Biosciences, 2000). The main advantage of this method is its ability to multi-parametric analysis of both physical and/or chemical features of thousands of particles per second. As a result, flow cytometry is a technique provides simultaneous separation and characterization of particles. This process occurs as they go through a beam of flight, based on specific physical characteristics (relative granularity or internal complexity, and relative fluorescence intensity). Apoptotic effects of many environmental pollutants have been reported. In this study, the apoptotic effects of cadmium on HEK-293T cells were investigated using flow cytometry methods with annexinV/PI staining. Camptothecin, a known cytotoxic drug, was used as a positive control. In this study Camptothecin used as positive control. Camptothecin is a cytotoxic plant alkaloid isolated from the plant, Camptotheca acuminate. Camptothecin is a known apoptosis inducer (Hentze, et al (2004).
Methods and Materials
Cell Culture and Treatment
A day prior to treatment, HEK-293T cells were plated in a 6-well plate in DMEM media to reach ~80 % confluence. The medium was replaced with 2 mL of the following solutions: fresh media containing no treatment, 3 μM camptothecin (positive control), 2.5 μM Cd, 5 μM Cd. The plate was then incubated at 37 oC (95% O2, 5 % CO2) for 24 hours.
Cell Harvest and Stain for Apoptosis Analysis
First, morphological changes using inverted microscope and it showed that apoptosis was occurred. Six well plate was centrifuged for 5 min at 1000 rpm. The media was removed from wells and transferred to corresponding 12x75 mm FACS sample tubes. Wells were washed with 3 mL of warm PBS buffer containing no calcium and magnesium and gently shaking for 10 seconds. The plate was then centrifuged for 5 minutes at 1000 rpm. PBS buffer was removed, 0.4 mL of trypsin-versene was added to each well and incubated at 37oC (95% O2, 5 % CO2) for 4 min to detach the cells from the surface. Original media returned to each well to stop trypsin activity. Cells were unclogged by gently pipeting for 20 seconds. The content of each well transferred back to the respective test tube.
In order to stain the cells, test tubes containing cell suspension were centrifuged for 5 min at 1000 rpm. The supernatant was removed (small amount of supernatant was left to prevent any damage to cells). Cells were washed twice with 2 mL of cold PBS mixed and centrifuged after each wash. Supernatant was removed and 100 μL of 1X annexin V binding buffer added to each tube. After mix, 5 uL of fluorescein isothiocyanate (FITC)-conjugated annexin V and 5 uL of fluorescent dye propidium iodide (PI) were added. They mixed and incubated in the dark place for 15 min at room temperature. The volume of 400 uL of annexin binding buffer was added to each well and samples were loaded into a FACS instrument (Beckton Dickinson). Flow cytometer equipped with a FACS loader rack for analysis. Data acquisition and analyze was performed using Cell-Quest Pro software.
Results and Discussion
The cytotoxicity effect of different concentrations of cadmium solutions on HEK-293T cells were studied using flow cytometry technique. The staining step was performed using annexin V and PI. This step is critical to differentiate between intact cells, early apoptotic cells and necrotic cells. Basically, Annexin V has a good affinity for the translocated phosphatidylserine of the apoptotic cells membrane. On the other hand, it can also bind necrotic cells due to internal access to the leakiness of the cell membrane. Thus, more staining by PI, which is a dye that can only enter necrotic cells through their damaged plasma membrane is important. Because the presence of PI staining distinguishe necrotic cells from viable and apoptotic cells. In this study camptotecin used as positive control. Populations of different areas were gated using variable gating (appendix 1). They are displayed on a forward scatter dot fluorescence plot of FL2-PI versus FL3-Anexin V dot plot. Each sample was performed in triplicate (Table1). The setting parameters for FACS instrument are presented in the appendix 2. Instrument settings are vary for different types of assay and types of the cell lines.
2.5 μM Cd
5.0 μM Cd
Viable Cells (R2)
Apoptotic Cells (R4)
Necrotic Cells (R3)
Table1. A. Dot plot result from flow cytometry analysis of histogram to show apoptotic effect of Cd on HEK-293T cells. Cells were treated with various concentrations of Cd, treatment control media or camptothecin for 24 hours before they were harvested for analysis using flow cytometry with annexin/PI staining as described in the methods section.
Average amount for above table is presented in Table2. Each sample was run for three time and numbers are the average for triplicate measurement.
2.5 μM Cd
5.0 μM Cd
Viable Cells (R2)
Apoptotic Cells (R4)
Necrotic Cells (R3)
Table2. Average of data in Table 1in triplicate
Viable cells (double negative) are presented in gate R2, apoptotic cells (PI-/annexin+) in gate R4 and necrotic cells in gate R3 (appendix1). As it is clear in Table 2, a significant difference was observed in apoptotic cells in the 2.5 μM Cd treated cells and 5 μM Cd treated cells.
Based on the data obtained by FACS corresponding graph was presented in Figure 1.
Figure1. Summary of apoptosic, viable, and necrotic cell percentage in HEK-293T cells in various conditions including, negative control, positive control, 2.5μM Cd concentration, and 5.0 Cd concentration.
As it is clear in the Figure 1, Cadmium induced apoptosis at 5 μM but not at 2.5 μM. Apoptotic cell percentages in the positive control (campthotecine 3 μM) and Cd 2.5 μM -treated cells were not significantly different. Kidney cells are very sensitive to toxicity and they are primary targets for toxicity of either cadmium or camptothecin .The reaon could be that Camptothecin lost its efficiency and potency. Morphological changes of Cd treatment were observed using the microscope. In the case of 2.5 μM Cd, the shrinkage was observed for the cells. For 5 μM Cd treated cells blebs and apoptotic bodies were observed. This microscope observation is a helpful tool to correlate the data of visible tests to FACS data. Different stain procedure can be employed to evaluate apoptosis at several stages. One of them is depsipher staining which is useful to assess mitochondrial membrane potential. Another one is LDS-751 staining that is used to study the conformational changes of DNA. Also, annexin/PI staining which is helpful to study surface membrane alterations and SUB-G1 DNA staining for study fragmentation of DNA (Davies). Thus, that would be helpful to combine various detection methods (satining) to increase the precision of the data. Moreover, progress of apoptosis occurred at different rate in different cell lines and this might be necessary to study the apoptosis within longer incubation period of time. This should be mentioned that during this experiment after 24 hours treatment of cells with Camptothecin (3 μM) no considerable apoptosis was observed. Also, cell apoptosis was limited to less than 6.5% for this treatment. Apparently, Camptothecin as a positive control for this experiment was not efficient to induce apoptotic effect to the cells. One of the reasons could be that Camptothecin lost its efficiency due to the expiration date. Another reason might be that the storage procedure was not proper. Higher concentration of Camptothecin could be employed to induce the apoptosis to cells.