P2 Script (Jian Wei) Preparation Flashcards
Slide 15: Materials & Methods (M&M)
Let us move on to the materials and methods we employed in our study.
Slide 16: M&M Overview
HEK293 cells were cultured in DMEM
- Supplemented with 10% foetal bovine serum and 1% penicillin/streptomycin
- Optimisation studies were performed with MTT assay
- More specifically explored the IC50 of cisplatin and pre-treatment study
- To understand the effects of NAOs against CIC
- Following optimisation studies, we performed quantification assays to explore the various mechanisms of action as mentioned in the research objectives
Slide 17: Cell Viability Studies
Incubation & Seeding:
- All incubation conditions were at 37 degrees Celsius in 5% carbon dioxide.
- For all studies in this slide, we seeded 10,000 cells per well in 96-well plates
- Incubated them overnight for cells to adhere to wells
(A) Determining IC50 of cisplatin
- Replace media with 100 uL of varying concentrations of cisplatin
- Incubated them for 24h before conducting the MTT assay
- In which we replace the media with 100 uL of diluted MTT solution and incubate the plates for 3 hours
- Then replace the MTT solutions with 150 uL of DMSO and gently shake the plates for 15 minutes at 200 RPM
- Absorbance was then read at 570nm with Magellen 6 reader
In the case of determining the baseline cytotoxicity of NAOs and pre-treatment studies:
- Instead of replacing media with cisplatin, we replaced the media with varying concentrations of alpha-mangostin, curcumin and lycopene
- Then incubated for 24h
(B) To determine the baseline cytotoxicity of NAOs, we performed the MTT assay after 24h
(C) Whereas for the pre-treatment studies, we replaced the media with 70uM of cisplatin (IC50 determined in part A) and incubated for 24h. Subsequently, we performed the MTT assay as well.
Slide 18: Quantification Assays
- A round of NAO pre-treatment assay was first performed, where varying seeding densities were employed.
- Following this, the cells were incubated overnight to adhere to the plates.
- Subsequently, cells were subjected to pre-treatment with 1 uM of respective NAOs, followed by a 24-hour incubation
- Then, NAOs were replaced with 70uM cisplatin and a final round of 24-hour incubation
- Samples were processed accordingly for the quantification assays, namely:
1) DNA staining by propidium iodide
2) Caspase-3 fluorometric assay, and
3) TNF-alpha ELISA
Slide 19: Results
Let us now move on to our results.
Slide 20: Cisplatin IC50
- After running three biological replicates of IC50 assay
- Determined 24-hour IC50 of cisplatin of HEK293 cells
- Average value of 70 uM
- Subsequently used as our treatment concentration
Slide 21: NAO Pre-Treatment Studies
- Statistically significant protection exerted against CIC at concentrations of:
1) 0.1 uM and 1 uM of alpha-mangostin,
2) 1 uM of curcumin and
3) 1 uM of lycopene, - Mean percentage cell viability boxed out in red were significantly higher than their respective cisplatin-only positive controls.
- Concentration-dependent protective effect observed in all three NAO pre-treatment groups
- Postulate that below threshold value of 1 uM, CIN is increasingly ameliorated as NAO pre-treatment concentration increase
- However, beyond 1 uM, the beneficial protective effect is subsequently outweighed by its cytotoxic effect
- Resulting in a lower mean percentage cell viability displayed at higher concentrations past the threshold
For an equal basis of comparisons, 1 uM of each NAO was used in the subsequent quantification assays.
Slide 22: Cell Cycle Analysis
- Identified cisplatin-only positive control had a significant percentage increase of cells at sub-G0/G1 phase as compared to DMEM-only negative control
- Coupled with a significant percentage decrease of cells in G2/M phase
- Suggest that cell cycle arrest occurred at sub-G0/G1 phase upon treating HEK293 cells with 70uM cisplatin
- Samples which have undergone 1 uM of NAO pre-treatment, followed by 70uM cisplatin treatment displayed a significant percentage decrease of cells at sub-G0/G1 phase as compared to cisplatin-only positive control
- Cells at sub-G0/G1 phase can be considered apoptotic
- Postulate that pre-treatment with 1 uM of NAO may be able to reduce apoptosis caused by 70 uM cisplatin treatment
Slide 23: Caspase-3 Fluorometric Assay
Link from Slide 22:
- Since all three pre-treatment samples with NAOs displayed a significant reduction in cell cycle arrest at sub-G0/G1 phase as compared to cisplatin-only positive control
- By quantifying caspase-3 levels, which is the main executioner protein involved in apoptosis
- Understand the extent of apoptosis mitigated by NAOs via the caspase-dependent pathway
As shown in our graph:
- Samples pre-treated with 1 uM of alpha-mangostin and curcumin may be able to reduce caspase-3 production via the caspase-dependent pathway, when compared to the cisplatin-only positive control
- However, pre-treatment with 1 uM of lycopene showed an increase in caspase-3 levels instead.
- Expln: Mechanism of lycopene in attenuating cisplatin-induced apoptosis involves the caspase-independent pathway instead
- Further studies needed to confirm this postulation
Slide 24: TNF-alpha ELISA
- By quantifying TNF-alpha production in our samples, we can deduce if pre-treatment with NAO can mediate the inflammatory response included by cisplatin damage.
- However, the optical densities measured for all eight samples were similar to the optical densities measured for the TNF-alpha protein Standard 6, Standard 7 and the zero standard
Expln:
- Equipment used to measure was not adequately sensitive to detect the levels of TNF-alpha produced
- Another could be there was insufficient production of TNF-alpha by HEK293 cells released in the cell culture supernatant to be detected
- Propose that even if all three NAOs can reduce TNF-alpha levels, it is not likely that this mechanism plays a major role in reducing CIN
