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Molecular Weight Characterization of an Almond Component Cytotoxic to Gastrointestinal Cancer Cell Lines (2020-US-EPO-612)

Steve Figard, Director of Cancer Research Lab, Bob Jones University
Evan Becker, student, Bob Jones University
Luke Brown, student, Bob Jones University
Emily Swager, student, Bob Jones University
Rachel Westphal, student, Bob Jones University

 

Colorectal cancer is both the third most common and the third leading cause of deaths associated with cancer.  Previous studies in this lab have demonstrated the in vitro cytotoxic effect of almonds on human GI cancers.  An almond extract was prepared and processed using a pseudo-digestion procedure in order to mimic the effects the extract would have in a physiological system.  This extract demonstrated a dose response in vitro cytotoxicity to human gastric adenocarcinoma and was cytotoxic to a human colorectal adenocarcinoma, but had no effect on a healthy human colon epithelial cell line. 

The extract was processed through filters with molecular weight cutoffs of 100,000 Da and 5,000 Da to estimate the size of any anticancer molecules, and it was found that the responsible molecules were less than 5,000 Da in molecular weight.  In addition, a polyphenol extract of the almonds was prepared and shown to have similar effects as the whole almond extract. It was concluded that the anticancer agents are likely polyphenols. Finally, four flavonoids commonly found in almonds were compared to the polyphenol extract, and they showed similar cytotoxicity.

 

 

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Transcript

Evan Becker Hello, this is the
2019 BJU cancer research team.
My name is Evan Becker and along with me today are Luke Brown,
Emily Swagger, Rachel Westfall and we are all under the direction of Dr. Stephen Figard at the Department of Biology and Bob Jones University.
And our paper, our presentation is on the molecular weight characterization of an almond component cytotoxic to gastrointestinal cancer cell lines.
All right, for the introduction.
Colorectal cancer is currently of great concern in the medical community as it is the third leading cause of cancer deaths for both men and women nationwide.
Previous research from the BJU cancer lab has shown promise by demonstrating that almonds have a cytotoxic effect on LoVo colorectal cancer in vitro.
A pseudo digestion procedure for an almond extract was also used as a way to mimic how the extract would work in a physiological system.
The same almond extract was shown to induce a dose response and the human gastric adenocarcinoma cancer cell line AGS.
Also this extract causes no negative effects in the human colon epithelial cell line CCD, indicating that the cytotoxic effects of almonds do not affect normal healthy cells.
Passing our almond extract through molecular weight cut off filters of 100,000 Daltons and 5000 Daltons respectively,
we were able to determine that the molecules present in the almonds inducing the set of toxic effect must be smaller than 5000 Daltons.
As a result, polyphenols were determined to be a possible cause of the cytotoxic effect and the polyphenol extract was conducted on the almonds with this treatment showing very similar cytotoxic effects on the cancer cell lines.
I think we're ready.
Rachel Westphal To the cell lines that we use included AGS, which is stomach cancer; LoVo, which is colon cancer; and CCD, which was our normal cell line.
We used 5-fluorouracil as our positive control for cell death. And we used an in vitro pseudo digestion of the almonds to mimic physiological digest...physiological digestion.
WST cell proliferation assay, we utilize that to determine absorbance with a plate reader and use that to calculate percent viability.
And then JMP was used for statistical analysis, we used ANOVA analysis, the Tukey-Kramer HSD test and the Wilcox and non parametric comparison. p values less than 0.05 were considered statistically significant.
Can go to the next slide. Yeah.
Luke Brown All right. The first set of tests I'd like to introduce you to are tests regarding the AGS human cancer cell line and this is a human gastric adino carcinoma.
Now JMP was an important tool for us because it helped us to, first of all, determine the standard deviation in a few pilot studies we conducted.
This then allowed us to use the software to run a power analysis to determine our sample size. Now looking at some of the data we got here,
first of all, you'll see among both Figure 1 and Figure 2 what it has in common is PBS phosphate buffered saline and 5-FU or 5-fluorouracil,
which is well documented and established cancer treatment for cancer, such as gastric and colorectal cancers. Now I'd like to direct your attention to Figure 1 here.
Previous studies in our lab had already established that it seems almond extract does have some sort of cytotoxic effect that's selective to these cancer lines.
Well, we hope to establish in Figure 1 here is, first of all, the dosage effect. And second of all, we wanted to narrow down
what molecular weight, we'd be looking at to establish what compound or compounds are responsible for this effect.
As you see, moving up from 12% almond extract all the way to 100% almond extract, we do see a dosage response. And we were able to establish this using the Tukey-Kramer HSD test inside JMP to be able to establish that these groups are statistically significantly different.
Now you see that both the 100,000 molecular weight cut off filter and less than 5000 molecular weight cut off filter
are statistically the same as the 100% almond extract. This led us to believe that whatever compound or compounds is responsible for this effect is going to be relatively small at less than 5,000 Daltons.
Given this, we moved on to Figure 2 and we looked at some research showing that flavonoids have been shown to have a similar effect in walnuts.
cyanidin, delphinidin, malvidin and petunidin.
As you can see both the extract and all these flavonoids were actually more effective than the 5-FU in this treatment.
So given this information, I'm going to pass the next slide to Emily here to give you some more information about another set of data.
Emily So to make sure that we weren't just looking at results particular to AGS, we also ran another cancer cell line called LoVo. LoVo is a colon cancer cell line.
As you can see, we didn't do all of the extensive dosage treatments on this particular one, because we'd kind of shown that with AGS. We were just particularly looking at
is this particular to a certain line? So for LoVo, you can see that we don't have as low a value for 5-FU.