Q&A Bank Flashcards
Why did you use MTT assay to determine cell viability?
- Relatively easy screening tool to indicate cell viability and cytotoxicity
- Living cells metabolise water-soluble MTT solution in a stoichiometric manner into insoluble purple formazan crystals
- Proportion of formazan crystals detected by colorimetric absorbance upon addition of DMSO determine extent of cells remained alive after cisplatin treatment.
- Limitation include the accuracy in ensuring the formazan crystals are not withdrawn in the process is quoted in literature, but its ease of access remains to be one of its selling point as a convenient screening tool
Why did you mention that there was a statistical significant decrease in the G2/M phase between treatment groups against cisplatin-only positive control?
- G2/M phase is the interphase leading to the mitosis of cells
- Generally have higher DNA content in G2/M phase in preparation for mitosis
- Reduction in % cells in G2/M phase due to significant % increase in G0/G1 phase
- With greater amount of cell cycle arrest in the growth phase, cells are unable to proceed into the subsequent phases of cell division
What do you mean when you mentioned that lycopene could be undergoing caspase-INdependent apoptosis?
- Slide 22: All three NAOs significantly reduced % of cells in sub-G0/G1 phase compared to cisplatin-only positive control at 1 uM
- Slide 23: Only 1 uM of AMG & CCM pre-treatment were able to reduce caspase-3 levels
- Caspase-3 is the main executioner protein for caspase-dependent apoptosis
- Indicates that lycopene reduces apoptosis via caspase-INdependent apoptosis that translate to the results of decreasing % of cells in sub-G0/G1 phase
You mentioned that antioxidants have a greater potential to reduce CIN, as compared to the other two in vivo strategies you have brought up in Slide 9. Can you elaborate why so?
1st: Cimetidine (OCT2 inhibitor)
- MOA: Reduce cellular uptake of cisplatin via OCT2 primarily
- However, in the study with rats between WT & OCT2 gene knockout groups, copper transporter 1 (Ctr1) gene is not knocked out.
- Signs of nephrotoxicity apparent still since cisplatin can be uptaked via Ctr1 instead
- Authors cautioned that their results are not conclusive, and speculative at best.
- Even when clinical trials were conducted, it was a case-control study with two groups of 9 patients each and not adequately powered. Intervention group involved a combination of verapamil (non-DHP CCB) and cimetidine.
- Reduction in CIN (i.e. no sig. diff. in GFR, filtration fraction & effective renal plasma flow) could not be attributed singly to cimetidine as a result
- Hence, its potential as CIN prophylaxis is limited at present.
2nd: Downregulation of TNF-alpha expression
- Primarily produced in macrophages through induction of toll-like receptors
- Managed to find HEK293 cells did produce TNF-alpha, but the production was amplified with HEK293 cells transfected with TNF-alpha gene.
- Literature review: Discovered that NAOs have anti-inflammatory properties, thus we tried to investigate if NAOs can “kill two birds with one stone” by observing if they can mitigate CIN through TNF-alpha downregulation, thus having a greater potential to be used as CIN prophylaxis
Why did you NOT perform tests to determine the effects of ROS quenching, i.e. the presumed primary MOA of NAOs?
- Performed ROS quantification via H2DCFDA (2’,7’ –dichlorofluorescin diacetate) assay in our experiments
- However, results were inconclusive due to potential degradation of H2DCFDA dye and inability to receive fresh batches of H2DCFDA dye in time to perform adequate number of replicates for reproducibility of results
- Mulitple studies have been conducted in literature with ample evidence that NAOs can reduce ROS generation.
- Thus, we decided to not present our inconclusive findings.
Why did you specifically choose this range of concentrations to use your reagents (i.e. 1 uM NAOs & 70 uM cisplatin)?
- Generally used a large range of concentrations for reagents used e.g. 1 to 200 uM for cisplatin, 0.1 to 25 uM for alpha-mangostin and 0.01 to 10 uM for curcumin and lycopene
- To determine the whole spectrum of activities caused by reagents & increased the odds of detecting extreme effects brought about by their use
- Generated the list of concentrations used based on serial dilution from respective stock solutions that are reasonably convenient to generate
Since you mentioned that NAOs are derived from fruits and food substances, such as mangosteen, turmeric and tomatoes, how will this affect its administration in the future?
- Poor aqueous solubility of lipophilic compounds
- Circumvented the solubility issue with dissolution of alpha-mangostin and curcumin in DMSO, and lycopene in THF for our in vitro study
- Use of liposomal formulations can be explored in subsequent experiments to determine if there are any differences in the protective effect conferred against CIN when compared to free formulation
- Direct intake as fruits may not be the most optimal due to lack in potency in small concentrations naturally found in food substances
- Quantification of NAOs via natural intake may be difficult as well
You mentioned that you have performed cytotoxicity tests for NAOs. How were the results?
Slide 41:
- Baseline cytotoxicity results showed a concentration-dependent protective effect similar to our pre-treatment studies
- 1 uM is the maximal non-lethal concentration before cytotoxicity is observed in HEK293 cells, where %CV is reduced past this threshold concentration
- Given that HEK293 cells are acceptable kidney models to be extrapolated to human kidneys, we are unable to determine if at 1 uM, it is cytotoxic to other organs in humans.
- NAO pre-treatment and cisplatin treatment lasted for 24h only, thus we are unable to determine any time-dependent repercussions caused by NAOs past 24h.
Slide 42:
- Examine if there are any solvent effects in cytotoxicity
- Highest concentrations of DMSO and THF present for the dissolution of NAOs have also been explored
- Conclusively ruled out that there is no significant difference in %CV resulting from the use of DMSO and THF
Literature review:
- Alpha-mangostin can cause teratogenicity & mortality in zebrafish embryos at concentrations above 6 uM
- Low (rats: 0.1 g/kg of body weight; hamsters: 0.2 g/kg of body weight) to medium doses (rats: 1.1 g/kg of body weight; hamsters: 2.1 g/kg of body weight) of curcumin did not induce any side effects in acute and chronic toxicity tests in either rats or hamsters
- Lycopene has no significant toxicity has been observed in rats treated with lycopene beadlet formulations in the diet at doses of up to 500 mg/kg body weight/day for 14 weeks or 1000 mg/kg bw/day for 4 weeks
Since your experiments involved a pre-incubation of NAOs prior to cisplatin treatment, did you also explore co-incubation and post-incubation of NAOs?
No.
- Prioritise pre-incubation studies of NAOs to ensure greatest possible concentration of NAOs retained in HEK293 cells to enable investigation into its MOA in attenuating CIC in HEK293 cells
Co-incubation:
- Unsure whether the entry of cisplatin and NAOs into HEK293 will be impeded with co-administration and the actual amounts entering the cells
- Focus of study is investigation of MOA of NAOs in reducing CIN, thus our priority is ensuring that NAOs are present intracellularly in HEK293 cells before cisplatin treatment for more conclusive results.
- Co-incubation may result in HEK293 cells being killed first before NAOs can step in to protect them, which may result in variable results obtained to inform us of a postulation.
Post-incubation:
- Narrower window to observe any protective effects from NAOs against CIC
- MOA of cisplatin is the irreversible alkylation of DNA at N7 atom of guanine primarily
- Apoptosis may be triggered prior to NAO treatment post-cisplatin administration, which defeats the intended purpose to investigate any protective MOA exerted by NAOs
What are the strengths and limitations of your study?
(+) Human embryonic kidney 293 cells used to simulate the human kidney in in vitro setting, thus improving generalizability of our results to extrapolate to clinical settings
(+) Methods used in quantification assays are well-established in literature, and followed closely to manufacturer’s instructions backed by literature; thus results obtained are accurate and reliable as far as possible
(-) Lack of time to explore time-dependent effects past 24h for more comprehensive review of NAO pre-treatment effects
(-) Caspase-8 & caspase-9 signalling pathways, amongst others, require more time to explore and investigate, especially if there is indeed a delay in effect observed past 24h NAO pre-treatment
Why is the currently employed strategy of using aggressive pre-hydration limited in its effectiveness in mitigating CIN?
Conflicting evidence suggesting diuretics either aggravate or reduce CIN
- Neither platinum content in plasma and kidney, nor degree of cisplatin-induced necrosis is positively influenced by diuretics
- Randomised study by Al-Sarraf et al. observed protection in kidney function with hydration + mannitol + cisplatin ONLY after first cycle, as compared to hydration + cisplatin
- No convincing effect in subsequent cycles of cisplatin
- Likely electrolyte losses when using diuretics -> require electrolyte supplementation
Pre-hydration 12h prior to cisplatin administration will induce diuresis of at least 100mL/h urine output
- Minimise electrolyte losses
- Unable to attenuate long-term renal toxicity from developing, given irreversible DNA damage incurred thru MOA of cisplatin
- Thus a growing need for more effective nephroprotective strategies to be implemented
Why did you use two different post-hoc analyses (i.e. Dunnett’s & Tukey’s) in your statistical analyses?
Pre-treatment studies: Dunnett’s multiple comparison test
- Single point of comparison made against i.e. cisplatin-only positive control
- Compares %CV of respective NAO pre-treatment groups against cisplatin-only positive
Cell cycle analysis: Tukey’s multiple comparison test
- Multiple points of comparison are made instead:
(1) Comparing % of cells in sub-G0/G1 phase between cisplatin-only positive control AGAINST DMEM-only negative control,
(2) comparing % of cells in sub-G0/G1 phase of NAO pre-treatment groups AGAINST cisplatin-only positive control, and
(3) comparing % of cells in G2/M phase of NAO pre-treatment groups and cisplatin-only positive control AGAINST DMEM-only negative control.
Based on your results, which NAO will you recommend pursuing further studies in mitigating CIN? Why so?
α-mangostin.
- Larger safety margin to show therapeutic effect since significant increase in %CV in pre-treatment studies at both 0.1 μM and 1 μM of AMG, but only 1 μM for curcumin & lycopene
- Even baseline cytotoxicity showed that at 10 μM of AMG, %CV did not drastically decline, highlighting its already larger therapeutic window available for AMG to exert its protective effect.
- Comparing against 1 μM of curcumin and lycopene pre-treatment, in our caspase-3 assay, α-mangostin continues to show a decreasing trend of caspase-3 activation.
- Curcumin alone, however, did trigger caspase-3 activation, despite showing a decline in caspase-3 activation, thus it is less optimal to recommend curcumin over α-mangostin.
- Lycopene did not show a decline in capsase-3 levels, and since our results are not conclusive to indicate that lycopene attenuates caspase-INdependent apoptosis leading to CIN, AMG stands a higher chance instead.
What optimisation will you suggest if you want to have more conclusive results on whether NAO pre-treatment can mitigate CIN via TNF-α downregulation?
Include a positive control known to trigger TNF-α production in our TNF-α ELISA, to show that HEK293 cells can secrete TNF-α (Look for possible positive controls)
However, it is possible that HEK293 cell line does not produce TNF-α in large amounts, as the study that showed HEK293 cells secreting TNF-α were using HEK293 cells transfected with TNF-α gene to amplify its production, and NOT wild type HEK293 cell line. This leads to questionable generalisability of results to actual physiological conditions.
Possible optimisation can also include testing TNF-α production with primary renal tubular cells, instead of HEK293 cell lines, to see if a similar trend (i.e. TNF-α downregulation by NAO) is observed.
Why do you use these specific cell counts for the respective quantification assays?
Dependent on the culture plates or wells used to subculture, and in accordance with manufacturers’ recommendation.
- If too much, cells overgrowth, and die, due to lack of culture media & excessive waste
- If too little, cells may not be comfortable multiplying and quantification of endpoint markers may remain in the undetectable range
What are the other MOA of CIN that future studies can be explored to see if NAO can mitigate CIN thru those pathways?
Adapted from Manohar 2018:
1) DNA damage via intercalation / alkylation
2) Cytoplasmic organelle dysfunction with endoplasmic reticulum stress & mitochondrial dysfunction, leading to caspase-dependent apoptosis
3) Death receptor-mediated / Caspase-INdepndent apoptosis, inclusive of autophagy
4) Oxidative stress
5) Inflammation - mediated by TNF-α and other cytokines
What are the primary PK parameters of cisplatin?
- ROA: IV
- Platinum is extensively bound to plasma protein (fu = 0.1); but unclear for cisplatin
- Free cisplatin is freely filtered at the glomerulus, by virtue of its low MW = 301.1 g/mol < 500 g/mol & uncharged character; largely excreted unchanged in urine.
- Rat & human studies suggest possible active secretion of cisplatin
- T1/2 = 30 min, but platinum from cisplatin is slowly eliminated with min. T1/2 = 5 days
What are the primary PK parameters of α-mangostin?
- Poor aqueous solubility → Low F due to intensive first-pass metabolism with PO ROA
- Via IV ROA, postulate it is likely to hit 1 μM at physiological condition since able to hit C0 = 17.88mg/mL = 43,557 μM in rats (MW = 410.5 g/mol)
- Only one PK study thus far using rat models
- Large V = 6.82 L/kg
- Biphasic distribution, including a rapid distribution phase and a slow elimination phase
- M: Phase II conjugation via glucuronidation & sulfation
- CL = 1.54 L/h/kg
- T1/2= 3.46 h
What are the primary PK parameters of curcumin?
- Poor aqueous solubility → Low F
- Cmax with single PO dose of 10 g and 12 g respectively in healthy human volunteers = 2.30 ± 0.26 μg/mL and 1.73 ± 0.19 μg/mL = 6.24 μM and 4.70 μM physiologically
- M: Proposed biotransformation of curcumin (MW = 368.39 g/mol) into dihydrocurcumin, tetrahydrocurcumin & hexahydrocurcumin (CYP metabolism), curcumin sulfate & curcumin glucoronide (Phase II metabolism)
- T1/2 = 6.77 ± 0.83 h
What are the primary PK parameters of lycopene?
- Poor aqueous solubility → Low F
- 28-day sampling period of increasing conc. of single PO doses of 10mg to 120 mg lycopene as tomato paste formulation
- Cmax = 0.075–0.120 μM
- T1/2 = 28.1 - 61.6h
- CL/F = 98.6 - 286.4 mL/min
- Vß/F = 2.12 - 18.54 L/kg
