University of Nairobi Effects of Lupeol and Resveratrol Reflection Paper i upload a poster i want you to write a reflection about i, try to finish it as so

University of Nairobi Effects of Lupeol and Resveratrol Reflection Paper i upload a poster i want you to write a reflection about i, try to finish it as soon as possble The Effects of Lupeol and Resveratrol, Novel Anti-Cancer
Agents, on Chk1 Mutant Saccharomyces cerevisiae
Bradley Joseph Lagemann and María Fernanda Zúñiga
Dept. of Biology, Univ. of the Incarnate Word, San Antonio, TX 78209
Introduction
Methods
Lupeol and Resveratrol are found in various medicinal plants worldwide and they have been
shown to have antineoplastic characteristics in several human cancer cell lines. In this
study we illustrate the effects of these compounds on cell cycle progression in mutant and
wild type Saccharomyces cerevisiae. To further elucidate their ability to control unregulated
cellular growth, we tested them on mutant checkpoint 1 (CHK1) S. cerevisiae. CHK1 is a
gene that controls the production of checkpoint 1 kinase, a key enzyme in the activation of
the DNA damage response (Fig 1). We hypothesize that Lupeol and Resveratrol will arrest
mutant CHK1 S. cerevisiae in S phase of cell cycle, leading to apoptosis through an
inhibitory mechanism on DNA replication machinery. It is our goal to utilize S. cerevisiae as
a model system to identify if these compounds can inhibit unregulated cellular growth.
Through this study we hope to continue the conversation about naturopathic compounds
and encourage further exploration of their antineoplastic effects.
Cell Maintenance & Growth
S. Cerevisiae were grown in YPD broth and incubated at 37oC.
To measure cell growth and viability, parent and mutant cells
were counted utilizing a hemocytometer and trypan blue.
Fig. 2: Parental and Mutant Yeast Cultures & Cell Viability
Parental and Mutant CHK1 yeast cultures in YPD (left).
Viable Cell plating of parental and mutant cultures (right).
Fig. 1: The Cell Cycle
Pharmacology
RNA Extraction
25mg of 94% Lupeol (Fig. 3) was purchased from Sigma Aldrich. Research in
human cervical cancer cell lines suggests an optimal Lupeol concentration of
100µM to induce S-phase arrest and apoptosis. Resveratrol was obtained from
Whole Foods in the form of a suplement. In research, Resveratrol has been seen
to cause apoptosis or cell cycle arrest in the G1-phase. During our experiments
this concentration of Lupeol was utilized in a 1:1 DMSO/Ethanol solution and
Resveratrol in a 20% ethanol solution. After treatment, 48 hours was allowed to
pass before conducting any experiments.
RNA was extracted both treated and control strains using YeaStar RNA kit. 1.5 mL of each inoculum was centrifuged
and the supernatant was discarded. Then 80 µL of YR digestion buffer and 5 µL of Zymolase was added to each pellet
and incubated for 1 hour at 37 oC. After Centrifuging 160 µL of YR lysis buffer was added to each tube and vortexed.
The supernatant was added to filter cups from the YeaStar kit and centrifuged. Then 200 µL of RNA wash buffer was
added to each filter cup and centrifuged. Wash and centrifuge was repeated twice. Filter cups were then moved to a
new Eppendorf tube and washed with and 60 µL of RNAse/DNAse free water. RNA concentration was determined
using Nanodrop. RNA was stored at -80oC.
cDNA Synthesis
cDNA was synthesized by using 10 µL of extracted RNA, 4 µL
of Trans Amp buffer, 5 µL of nuclease free water, 1 µL of
reverse transcriptase (Sigma), and a thermocycler set to the
following parameters: 25oC for 10 mins, 42 oC for 15 mins, 85
oC for 5 mins, and 4 oC (hold). cDNA was stored at -20 oC.
PCR & Gel Electrophoresis
Fig. 3: Lupeol (left) and Resveretrol (right) Structures
Hemocytometer Counts
To determine the effect Lupeol and Resveratrol had on cell
growth and viability, hemocytometer counts and viable cell
plating were performed. Significant differences were noted
between average cell counts of the MC/MT and PC/PT
inoculums.
Fig. 4: Viable Cell Plating after
(Fig. 4 & 5)
treatment with 100µM
PCR was conducted on CHK1 and Cdc25 genes. 18 µL of ultrapure water was added to 5 GE
Healthcare PCR reaction beads (dNTPs & DNA Polymerase). 5µL of PC, PT, MC, and MT cDNA
was added to 4 PCR tubes. Then1 µL of forward and reverse primers for the gene of interest was
added, and the tubes were placed in the thermocycler. The heat cycle was 95 oC for 10 mins, 95
oC for 15 sec, 60 o C for 15 sec, 72 oC for 15 sec, repeat steps 2-4 thirty times, and 72 oC (final
extension) for 2 min. After completion of the PCR, gel electrophoresis of the amplified DNA was
run on an Invitrogen 1.2 % Agarose E-gel, 20 µL of a 1kB molecular weight ladder, 2 µL of 5x
orange dye in each PCR tube and 20 µL cDNA. The gel was run on the 2% setting for 20 minutes
then imaged on a BIO RAD Gel Doc molecular imager.
Fig 4: SDS Page Gel
Protein Extraction & SDS-Page
First we grew 106 inoculums of our 4 experimental groups. Then we added 3 mL of the inoculums to 4 canonical tubes and centrifuged them. The supernatant was discarded and 500 µL of ultrapure
water and 100 µL of 0.2M NaOH was added to the tubes. The tubes were incubated at room temp for 10 minutes, then centrifuged. The supernatant was removed and placed into an Eppendorf tube
containing 50 µL of Lammeli Buffer. The pellet from each canonical tube was resuspended in 500 µL of ultrapure water and 50 µL of Lammeli Buffer, then placed in an Eppendorf tube. Tubes were stored
at -20oC. Extracted protein was run on an SDS Page gel. To perform this, we boiled our protein samples for 5 mins then add 20 µL of each sample to the SDS Page gel. 20 µL of a protein kaleidoscope
ladder was used as a reference marker. Gels were run between 98-108 V for 45min – 1hr. Once complete, the gels were stained with Coomassie dye, rocked on a gel rocker for 24 hours, and imaged
using a BIO RAD Gel Doc molecular imager.
Results:
Figure 6: PCR Gel (Left) & SDS-Page Gel (Right)
Figure 4: Viable Cell Plating Post Treatment
Figure 4: Shows the colony forming units for each of the dilution series in each experimental group. Using number of colony
f orming units and the lev el of dilution, we are able to calculate the av erage number of cells in each of the respective yeast
inoculums.
Figure 6A: The PCR gel shows three distinct bands in lanes 2, 4, and 6 at the 100 kB mark. The bands in lane 4 &
6 indicate that the f orward and rev erse primers have dimerized meaning the PCR failed to amplify our gene of
interest. Lane, the negative control (NC) has no cDNA present and shows the dimerized primers. The SDS-Page gel
shows the v arious sizes of proteins present in each experimental group’s pellet and supernatant.
Figure 5: Hemocytometer Data Analysis
Figure 5: (A) Mutant control hemocy tometer data showed a signif icant increase in the av erage number of cells compared to the
Figure 6B:
parental control. (B) Parental treated hemocytometer data showed a significant increase in the average number of cells compared to
the parental control. (C) Mutant treated hemocytometer data showed a significant decrease in the average number of cells
compared to the mutant control, and (D) Mutant treated hemocytometer data showed a signif icant decrease in the av erage number
of cells compared to the parental treated.
Conclusions
Discussion/Future Directions
•
•
•
The hemocytometer data suggests that Lupeol causes a significant decrease in the average number of
cells in CHK1 mutants (Fig. 5).
PCR data inconclusive, due to limited [RNA]. (Fig. 6).
Thinking like a scientist….
•
•
This experiment has really highlighted the time commitment it takes to conduct complex research.
2. Although I thoroughly enjoyed this process, I do not think a research career is for me…well maybe as an
MD/PhD candidate.
•
Through this experience we learned how to apply the scientific method and
critically/statistically analyze collected data.
In future research we would like to utilize S. cerevisiae as a model system to identify specific
mechanisms of how these compounds can act to inhibit unregulated cellular growth
Acknowledgements
Funding by the UIW Department of Biology. Dr. Veronica Acosta and the biology laboratory staff for growing
our cell cultures, and my lab partner Fernanda Zuniga for keeping me on track.
Results:
Figure #?:
Figure #?:
Figure #? Title?
Figure 4: Figure Legend….
Figure 4: Figure Legend….
Figure #?: Figure Legend…..
Conclusions
• Bullet points are good here
• Reference Fig#
• Only Conclusions
Discussion/Future Directions
• Here you can talk about your
experience.
• What did you learn?
• What would you do if you had all
the grant money in the world next
time you did these experiments?
Thinking like a scientist….
Acknowledgements
•
•
Funding by the UIW Department of Biology. The X antibodies were obtained from the Developmental
Studies Hybridoma Bank, created by the NICHD of the NIH and maintained at The University of Iowa,
Department of Biology, Iowa City, IA 52242. You can thank your lab partner or anyone else here as
well.
•
Answer these questions…
1. How did this experience change the way you
think of the work that scientists do?
2. Would you consider going into a research
career?
Lupeol is one of many triterpenoids found in various medicinal plants w orldwide and has been show n to have
antineoplastic characteristics in several human cancer cell lines. In this study w e illustrate the effects of Lupeol on
cell cycle progression in mutant and w ild type Saccharomyces cerevisiae. To further elucidate Lupeol’s ability to
control unregulated cellular grow th, we tested Lupeol on mutant checkpoint 1 (CHK1) S. cerevisiae. CHK1 is a
gene that controls the production of checkpoint 1 kinase, a key enzyme in the activation of the DNA damage
response (DDR). We hypothesize that Lupeol w ill arrest mutant CHK1 S. cerevisiae in S phase of cell cycle,
eventually leading to apoptosis through an inhibitory mechanism on the DNA replication machinery. It is our goal
to utilize S. cerevisiae as a model system to identify any potential mechanisms of how this triterpenoid can act to
inhibit unregulated cellular grow th. Through this study w e hope to continue the conversation about Lupeol and
encourage further exploration of the antineoplastic effects of all triterpenoids.

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