1. The Role of CPT1A Enzyme in Prostate Cancer Viability
Kimberly R. Turner1
, Colton T. Pac2
, Isabel R. Schlaepher,PhD3
1. Saint Louis University, St. Louis, USA
2. University of Colorado Denver, AMC, Department of Pharmacology, Denver USA
3. University of Colorado Denver, AMC, School of Medicine, Division of Medical Oncology, Denver USA
Prostate cancer is one of the most prominate cancers in men and the second leading cause of death.
There are 3 well-established risk factors for prostate cancer: Family history (does cancer run in your
family?) race (specifically, African American men) and age (the older one is, the worse it is).
Unfortunately, we cannot change our race or our family members, nor can we stop the progress of time.
Considering the large number of men who die annually of prostate cancer (258,100 worldwide and
32,600 in USA in 2008) and the high prevalence of obesity, (35% in the United States in 2012) the
association means that tens of thousands of men die annually of obesity-related prostate cancer. While
the exact associations between obesity and prostate cancer are complex, diet and lifestyle always play a
role (Holmberg, et. al., 2013).
More recently, this uncertainty between fat, diet and prostate cancer has been fueled by new
epidemiological studies that bring attention to the role of the types of fat consumed and the risk of dying
of prostate cancer (Richman, 2013). Furthermore, the once good role of omega-3 fatty acids in guarding
off prostate cancer is now put into question as recent reports suggest that these beneficial fats may be
fueling the growth of prostate cancer tumors (Barsky, et. al., 2013). How these observations translate at
the molecular level, inside prostate cancer cells, remains unknown.
In order to shed light into the possible mechanisms of fats favoring the growth and viability of prostate
cancer cells, we studied the role of a specific enzyme that resides in the outer membrane of the
mitochondria and it is involved in bringing in the fat molecules for oxidation, just like the muscles of
our bodies do to generate energy. This enzyme is called CPT1 or Carnitoyl-palmitoyl-transferase. The
human role of this enzyme in cancer has already been proposed by others but its role in prostate cancer
cell viability remains unexplored (Linher-Melville, et. al., 2011).
The hypothesis we started with is that the cancer cells have strong avidity for fat molecules to fuel their
survival. Therefore we decided to knock down the CPT1 enzyme of the mitochondria of prostate cancer
cells and study its effects on cell viability. To explore the possible effects of this knocked down enzyme,
four different avenues were used and compared with control, non-knocked down cells. First to check
cell viability, Trypan blue cell counts were done along followed by visual comparisons to track changes
in morphological characteristics. Next to check for changes in the production of mRNA, qPCR(RT-
PCR) was performed on not only the knocked down enzyme but also another control enzyme,SCD1, to
normalize results. Finally, to test protein content, western blots were performed also with a control
enzyme,β-actin, for normalization of data.
Each test retuned significant data that points toward the great importance that CPT1 enzyme has on am
prostate cancer cell. With respect to changes in morphological characteristics, the knocked down cells
grew at a much slower rate than that of the control non-knocked down cells. They also exhibited greater
buoyance and grew in very thick clumped patterns compared to the lean control cells. The Trypan blue
cell counts revealed a significant decrease in cell growth especially when knocked down cells were

2. treated with a fat inhibitor Etomoxir (Figure1). qPCR returned data that showed a 90% decrease in the
relative gene expression of CPT1A enzyme compared to the control.Western blot analysis showed a
clear decrease in the protein content of CPT1A enzyme in the knocked down cells (Figure2).
The recent epidemiological studies have encouraged the idea that death due to prostate cancer or any
cancer for that matter is greatly increased by whether or not a person is overweight. They all translate
that cancer cells like fat and the more of it a person has, the faster and stronger PCa grows. In
conjunction with our findings, this tells us that while we were unable to completely knock down the
CPT1A gene, we were able to reduce significantly the expression of the gene. We can now clearly
associate the change in viability of the treated cells to the fact that there amount of lipids being
transported to the cell through the CPT1A enzyme was diminished.
Fig. 2: Cell viability of control and CPT1A-KD cells by Trypan blue assay. The viability of all cells with
deficient CPT1A expression drops considerably. In The CPT1A-KD cells (clone 79), the viability drops
close to zero when we use the specific CPT1 inhibitor Etomoxir.
Fig. 2: Western Blot of lysates of CPT1A KD cells (KD) and control cells (C)
References
[1] Brasky, T. B, Darke, A.K., Song, X. et. al. (2013) Plasma Phospholipid Fatty acids and prostate cancer risk in
the SELECT Trial. JNCI. 10 (in Press)
[2]Calle, E.E., Rodriguez, C., Walker-Thurmond, K. and Thun, M. J. (2003) Overweight obesity and mortality
from caner ain aprospectibelu Stvudied cohort of U.S Adults. N Engl. J. Med. 348, 1625-1638.
[3]Holomberg, L. (2013) Obesity Nutrition and prostate cancer. Insights and issue. E.A.U. 63, 8221-822
[4] Linher- Meiville, K., Zantinge, S., Sanli, T., Gerstein, H., Tsakiridis, T., Singh, G. ( 2011) Establishing a
relationship between prolactin and altered fatty acid β-Oxidation via Carnitine palmitoyl transferase in breast
cancer cells. BMC Cancer 56, 1471- 2407
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control 30 uM 50 uM
Relativecellviability
Cell viability 60 hours
Clone 3
clone
79 KD
C KD C KD KD C C
CPT1A
B-Actin