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Plant material and extraction
Plectranthus ciliatus leaves were harvested in the Manie van der Schijff botanical garden,Pretoria, South Africa. The leaves were authenticated and a voucher specimen deposited in the Schweickerdt Herbarium, Pretoria, South Africa. The crude extract of the leaves was prepared and kindly donated by Dr. T.P. Kapewangolo, Department of Biochemistry, University of Pretoria. The plant extraction procedure was done as previously described [82]. The crude extract was stored at 4°C until use. Stock solutions were freshly prepared before each experiment by dissolving the plant extract in cell culture tested dimethyl sulfoxide (DMSO) (Sigma, Germany).
Cell culture
Human cervix adenocarcinoma (HeLa) cells were purchased from Highveld Biological (Pty) Ltd. (Johannesburg, South Africa) and grown in standard culture flasks in a humidified incubator at 37°C and 5% CO2. The growth media was Minimum Essential Medium (MEM), supplemented with 5% heat-inactivated foetal bovine serum (FBS) (Hyclone, Separations, Johannesburg, South Africa) and antimicrobial cocktail (100 U/mL penicillin, 100 μg/mL streptomycin and 250 μg/L fungizone) (Hyclone, Separations, Johannesburg, South Africa). The cells were maintained by sub-culturing the cells when 80-90% confluence was reached.
Real time cell analysis of plant extract treated HeLa cells
An RT-CES device (Roche Diagnostics, Mannheim, Germany) in a humidified incubator was used to observe the proliferation of the cells in the absence and presence of the selected plant extract. Cell titrations (5 000 – 60 000 cells / well) were done to determine the optimal cell number for the experiments. The specific concentration (1 x 105 cells/well) where the optimal exponential growth curve was found, was used for further studies as recommended by Fonteh et al. (2011) [80]. Three different concentrations (0.5 x cytotoxic concentration of a treatment affecting 50% of the cell population tested (CC50), CC50 and 2 x CC50 – determined by the tetrazolium dye XTT, Table 4.1) of the treatments were tested alongside the positive control (actinomycin D). The plant extract was dissolved in DMSO (ensure that all components were solubilized) to a stock solution of 20 mg/mL, while actinomycin D was completely dissolved in only sterile distilled water to a stock solution of 1 mg/mL. Further dilutions were made in complete media before the assays were conducted. Controls; a background (media only, no cells), untreated cells (cells and media) and a vehicle control (DMSO at 0.09%) were included every time. Electronic impedance increases as cells attach
to the electrodes and is known as the Cell Index (CI). The cells were incubated in a humidified incubator for approximately 18 hours where the unit less CI 1, after which the treatments were added to the E-plates, final volume 250 μL. Cells were monitored for a total of 90 hours. Data sets were normalized at the point where cells were treated. The Cell Index was normalized using a build in function of the Roche xCelligence Software 1.2.1. The normalized CI is calculated automatically for each well as the CI at a given time point, divided by the CI at the normalized time point (Normalized CItime = CItime / CInormalized_time). Therefore, the Normalized Cell Index for all wells must be equal one (1) at the normalization time point. Cells exhibited varying response patterns represented by CI changes that either indicated the cells as growing (increasing CI), dying (decreasing CI) or experiencing proliferation arrest (unchanging CI). Three independent experiments were conducted.
Cytotoxic concentrations of the treatment affecting 50% of the cell population tested (CC50) based on RT-CES data were calculated using Roche xCelligence Software 1.2.1. The CC50 values were expressed as the mean of three independent assays and the variability between repeats as the standard error of the mean (SEM). RT-CES graphs were used to determine whether the responses to treatment were cytotoxic or cytostatic by using guidelines published by Kustermann et al. (2013) [140]. The line at CI = 1 divided the graphs into viable / proliferating cells (CI > 1) and nonproliferating / dying cells (CI < 1) from each other (Fig. 4.1). Three typical graphs were described in Kustermann et al. (2013), where the response of cells in the absence or presence of treatments was described. Where the CI was more than 1, the cells were viable / proliferated indicative of cells under no stress. Where the CI < 1 the cells experienced toxicity. Lastly, when the growth curve moved from CI > 1 to a CI < 1 during the observation time the cells could be defined as experiencing cytostatic responses [140].
For the FTIR investigation, methods published by Zelig et al. (2009) and Machana et al. (2012) were used as a guideline during the optimization of the experiments [13,108]. Exponentially growing HeLa cells were seeded (1 x 105 cells) and incubated in 12-well plates. After 24 hours incubation, the cells were exposed to two different concentrations (CC50 and 2 x CC50) of the plant extract and actinomycin D respectively (Table 1). Vehicle (0.09% DMSO) and negative control (untreated) cells were included in the analyses. Cells were incubated for 72 hours, after which all the cells were collected from each well. Cells were centrifuged for 10 minutes, the supernatant discarded and the pellets washed twice with 1 mL phosphate buffered saline (PBS) to remove all traces of the culture media. The cells were incubated at room temperature in 1 mL 10% formalin (in PBS) for 10 minutes to fixate the cells. The cells were centrifuged for five minutes and the supernatant discarded. The cells were washed twice with 1 mL of sterile distilled water and centrifuged for three minutes to remove all traces of formalin and PBS, which could influence the spectra. The supernatant was removed and the cells suspended in 10 μL sterile distilled water. The cell suspensions (3 μL) were dried in the biosafety flow hood for one hour on sterile CaF2 discs (Crystran, UK).
Chapter 1: Introduction
Chapter 2: Literature Review
2.1 Types of cell death
2.2 How cancer cells bypass cell death mechanisms
2.3 The prevalence and treatment options for cervical cancer
2.4 Continuing chemotherapeutic research
2.5 Cytotoxic or cytostatic drugs
2.6 Background on cell lines important to this study
2.7 Conventional biochemical methods for detecting cell status
2.8 Advantages and disadvantages of conventional assays
2.9 New methodologies for monitoring cell death
2.10 Statistical analysis of vibrational spectroscopy data
2.11 Hypothesis
2.12 Primary objective
2.13 Aims
2.14 Workflow
Chapter 3: Cytotoxicity of selected cell death inducers
Abstract
3.1 Introduction
3.2 Methodology
3.3 Results and discussion
Chapter 4: Cellular injury evidenced by impedance technology and infrared microspectroscopy
Abstract
4.1 Introduction
4.2 Methods
4.3 Results and discussion
Chapter 5: Metallodrug induced apoptotic cell death and survival attempts are characterizable by Raman spectroscopy
Abstract
5.1 Introduction
5.2 Methods
5.3 Results and discussion
Chapter 6: Fourier Transform Infrared spectroscopy discloses different types of cell death in flow cytometrically sorted cells
Abstract
6.1 Introduction
6.2 Methodology
6.3 Results and discussion
Chapter 7: Fluorescent activated sorting of dead cells to determine spectral characteristics by Raman spectroscopy
Abstract
7.1 Introduction
7.2 Methodology
7.3 Results and discussion
Chapter 8: Overall conclusion
8.1 Cytotoxicity of cell death inducers (Chapter 3)
8.2 Plectranthus ciliatus – Impedance technology and FTIR spectroscopy (Chapter 4)
8.3 Metallodrug apoptosis and Raman spectroscopy (Chapter 5)
8.4 FTIR spectroscopy of sorted cells (Chapter 6)
8.5 Raman spectroscopy of sorted cells (Chapter 7)
8.6 Novel aspects of this research
8.7 Revisiting the hypothesis
8.8 Future perspectives
Chapter 9: References
