Analysis of Isoliquiritigenin effect on mitoNEET expression
Kayle J Marsh, Rachel M Hemmerlin, Ashley M Loe
Department of Chemistry, Slippery Rock University
Overview of Melanoma
Melanoma represents the most aggressive and deadliest form
of skin cancer. It is the fifth most common cancer among men
and women with an estimated 196,000 new cases in the United
States in 2021.1 It occurs from genetic mutations in
melanocytes, cells that produce pigments.2 Melanocytes are
located in the basal layer of the epidermis. Their growth is
regulated by the surrounding keratinocytes in a variety of
pathways.3 Treatments for this cancer are limited. Most
common
practice
includes
surgery,
chemotherapy,
immunotherapies, and other targeted therapies none of which
provide a cure.4 Alternative and effective therapies are greatly
needed to potentially cure this aggressive cancer.

Preliminary Studies
Initial plating of the A375 cells were done using 10,000, 25,000,
50,000, 75,000, and 100,000 cells per 35 mm dish. After
examination, it was concluded approximately 50,000 cells per dish
was optimal. The amount of lipofectamine 2000 for transfection was
tested with 3, 6, 9, 12, 15, and 18 μL per dish. Only the 3 μL and 6
μL survived the transfection. The results after imaging showed
using 3 μL was most ideal for the experiment. Figure 2 shows
representative images from the transfection optimization
experiment.

Figure 5: (A) Control, A375 cells expressing mitoNEET-GFP. (B) A375 cells expressing mitoNEET-GFP after exposure to 0.5 μM ISL. (C) A375 cells
expressing mitoNEET-GFP after exposure to 5 μM ISL. (D) A375 cells expressing mitoNEET-GFP after exposure to 50 μM ISL.

Figure 2: A375 cells expressing mitoNEET-GFP after transfection with 3 μL lipofectamine 2000 (A) or 6 μL lipofectamine 2000 (B).

MitoNEET
MitoNEET is a newly discovered mitochondrial protein. It is an
integral protein localized in the outer mitochondrial membrane
(OMM). Its name is based on its subcellular localization and
the presence of the amino acid sequence Asn-Glu-Glu-Thr
(NEET). MitoNEET is also identified as part of the unique
39 amino acid sequence,
CDGSH, a domain in residues
55-93 that act similarly, to a
zinc finger and is likely involved
with iron binding. The protein
also contains a N-terminal
α-helix with a redox active
iron-sulfur domain, [2Fe-2S].
These components have shown
that mitoNEET is able to play a
role in the regulation of energy
metabolism in the mitochondria.6

Figure 3. Structure and possible functional implications of mitoNEET. Paddock,
Mark L.; Wiley, Sandra E.; Axelrod, Herbert L.; Cohen, Aina E.; Roy, Melinda;
Abresch, Edward C.; Capraro, Dominique; Murphy, Anne N.; Nechushtai, Rachel;
Dixon, Jack E.; Jennings, Patricia A. MitoNEET is a uniquely folded 2Fe​2S outer mitochondrial membrane protein stabilized by pioglitazone. PNAS. 2007.
104(36). 14342-14347.

MitoNEET in the presence of ISL

The concentration of mitoNEET-GFP was varied using four
concentrations including 100, 200, 500, and 1000 ng/μL. It was
determined that the ideal concentration was 150 ng/μL for each
dish of cells. Figure 3 shows each concentration of mitoNEET-GFP
after imaging.

The mean fluorescence intensity (a.u) was examined for
each trial which is shown in Figure 6. In the experiment using
0.1, 1, 10, and 100 μM the results were 318 ±14, 286 ±15,
274 ±17, and 236 ±15, respectively with the control being 337
±10. For the trial using 0.5, 5, 50 μM the fluorescence
intensities were 167 ±7, 165 ±14, and 142 ±9, respectively
with the control being 235 ±10. The raw integrated densities
were measured and are shown in Figure 7.

Figure 6: (A) Mean fluorescence intensity of A375 cells expressing mitoNEET-GFP for control, 0.1 μM ISL, 1 μM ISL, 10 μM ISL, and 100 μM ISL. (B) Mean
fluorescence intensity of A375 cells expressing mitoNEET-GFP for control, 0.5 μM ISL, 5 μM ISL, and 50 μM ISL. (n= 50,000 ), (Data are mean values ± S.D.)

Figure 3: (A) A375 cells with 100 ng/μL mitoNEET-GFP. (B) A375 cells with 200 ng/μL mitoNEET-GFP. (C) A375 cells with 500 ng/μL mitoNEET-GFP. (D) A375 cells with
1000 ng/μL mitoNEET-GFP.

MitoNEET in the presence of ISL
Trials of 0.1, 1, 10, and 100 μM and 0.5, 5, 50, and 500 μM were
added to the A375 cells tagged with the mitoNEET-GFP. Results are
shown in Figure 4 and Figure 5.

Figure 7: (A) Mean raw integrated density of A375 cells expressing mitoNEET-GFP for control, 0.1 μM ISL, 1 μM ISL, 10 μM ISL, and 100 μM ISL. (B) Mean
raw integrated density of A375 cells expressing mitoNEET-GFP for control, 0.5 μM ISL, 5 μM ISL, and 50 μM ISL. (n= 50,000), (Data are mean values ± S.D.)

Isoliquiritigenin

Conclusion

Isoliquiritigenin (ISL), is a bioreactive compound that is derived
from licorice root. It is commonly found in foods and Chinese
herbal medicines.7 This root is a member of the flavonoids,
which promote health and disease prevention. Many studies
have revealed that they are affective against cardiovascular
disease and cancer.8 More specifically they are known to have
anti-tumor effects in vitro and in vivo.9 ISL has a promising
outlook when considering cancer treatment options.

ACKNOWLEDGMENTS
This work was supported, in whole or in part, by Slippery Rock University, and the
SRU Chemistry Department

Overall, treatment of A375 cells with ISL downregulated the
expression of mitoNEET making it a promising target for future
cancer treatments. Additional studies will measure the effect of
mitoNEET on cell viability and production of ROS in the
presence of additional treatments in combination with ISL.

References
1 Skin

Figure 4: (A) Control, A375 cells expressing mitoNEET-GFP. (B) A375 cells expressing mitoNEET-GFP after exposure to 0.1 μM ISL. (C) A375 cells
expressing mitoNEET-GFP after exposure to 1 μM ISL. (D) A375 cells expressing mitoNEET-GFP after exposure to 10 μM ISL. (E) A375 cells expressing
mitoNEET-GFP after exposure to 100 μM ISL.

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