Fatty Acids Induce Tumor Growth (links lead to published papers in .pdf format)

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                   Due to the switch from saturated fats to the poly-unsaturated fats over the past 3 decades, the Western diet has increased in essential fatty acids by 20 fold. Some of these unsaturated fats can promote growth of prostate and colorectal  tumors. Our newly published studies have helped elucidate the role of prostaglandin synthase 2 (PGS2), also known as cyclo-oxygenase-2 (COX-2) up-regulated in prostate cancer. This up-regulation is associated with growth stimulation and carcinogenesis. Our goal is to identify and evaluate the molecular link between a high fat diet, LDLr and prostate cancer. Preliminary data suggest that a significant percentage of prostate tumor cells respond to essential fatty acids LA and AA by up-regulating the immediate early genes c-fos and cox-2 thereby accelerating tumor growth. In addition, our studies suggest that delivery of AA by an over-expressed low-density lipoprotein receptor (LDLr) may be one factor in prostate tumor growth. Studies in this laboratory have shown that fatty acids induce prostate tumors to express cyclo-oxygenase 2 (COX-2) a immediate early gene which is found in growing tissues. Recent evidence has demonstrated that the action of the essential fatty acids is working through the prostaglandin E2 receptor EP-4. If the findings indicate that reductions in dietary essential fatty acids, COX-2 inhibitors or blocking the fatty acid action with prostaglandin receptor (EP-4) antagonist  would reduce growth of prostate cancers.
Fatty acids induce immediate early gene expression in human PC-3 cells  Induction of c-fos is a primary event in growth induction by sera or growth factors and is one of the earliest known effects on gene expression by mitogens. AA induction of c-fos occurs within minutes, this is followed 2 days later by growth stimulation. Taken together these data implicate AA as a growth mitogen. The effect is FA dose dependent. In this figure, we see that very small amounts of arachidonic acid added to PC-3 cells induce the immediate early gene, c-fos, within 30 minutes; this induction occurs in a dose responsive manner. Using specific eicosanoid inhibitors, kinase and EP blockers, we will study the signal transduction pathways responsible for upregulation of immediate early growth genes.

               Arachidonic acid upregulation of cox-2

                                  Figure 2                                                      Figure 3
Text Box: Figure 2: AA upregulation of COX-2 mRNA: Cells were grown in 2% FCS media for 24 hours before addition of AA. at varying concentrations (0.1, 0.5, 5 or 10 ug/ml). RNA was collected at 3 hours after addition of treatment and analyzed by RTPCR as described. Figure 3 AA upregulation of COX-2 enzyme activity. Cells were grown in 2% FCS media for 24 hours before addition of AA. AA was added at varying concentrations (0.1, 0.5, 5 or 10 ug/ml) for 2.5 hours. Media with 1% FCS was added to the cells with 1 uM AA for 30 more minutes before media was collected at the end of the 3-hour time point and analyzed for synthesis of PGE2.


In addition to upregulation of c-fos, AA induces cox-2 mRNA expression within 30 minutes, reaching its peak effect at 3 hours. There was no induction of COX-1 mRNA by AA. Appearance of the message (Figure 2) preceded the enzyme by approximately 1 hour. Controls are treated with carrier albumin at the same time as treatments. Growth followed approximately 24 hours later. In PC-3 cells from 2-3 hours after AA treatment, there is upregulation of another immediate early gene, cox-2.  Treatment with AA increased COX-2 enzyme activity in a dose dependent manner.

 LA and AA and their product, PGE2 accelerates Human Prostate Cancer Growth.

 

 

 

 

The PC-3 cells are incubated with AA ± flurbiprofen (a NSAID) for 24 hours before cell counts are taken. There is a significant reduction in prostate tumor cell growth when cyclooxygenase is inhibited with 5uM NSAID flurbiprofen.  There was no induction of COX-1 with AA; therefore these data would suggest that the essential fatty acids are inducing growth through actions of COX-2 and production of PGE2.

Essential fatty acids (found in corn oil) increase growth of 2 human prostate tumor lines, OA (olive oil) or EPA (fish oil) do not . Cell number was determined by Hoechst staining and/or CyQuant analysis. We found that LA, AA and their metabolic product, PGE2 stimulate proliferation of cancer cells suggesting a role of the essential fatty acids in stimulation of cell growth. In addition, there were striking increases in PGE2 synthesis in the AA and LA treated cells, no increase was seen in PGE2 production with OA or EPA.  Fig.6 shows results in the DU-145 tumor line.

Text Box: Figure 5: Regulation of growth by fatty acids: comparison of PC-3 growth induction by FA. PC-3 cells were grown in 2% FCS media for 24 hours before addition of supplement. After 7.5 ug/ml of AA, OA, EPA, PGE2 or LA were added to depleted media, cells were grown for 24 hours before cell counts. Data is presented as percent of control ± S.D. N =4

 

 


Prostate tumor lines have un-regulated uptake of LDL, which allows unlimited essential fatty acid delivery to the cell.
 Loss of LDLr feedback regulation in prostate cancer:   In other studies  shown on the next page, we have demonstrated that the lack of LDL feedback both at the mRNA and functional level is lost in many prostate cancers.  In many cell types expression of LDLr is feedback regulated by cholesterol, which is delivered by LDL.  In this figure we investigated how LDLr was regulated by LDL in human prostate cells.  Presence of LDL or whole serum reduced LDLr mRNA level (measured by RT-PCR) in the PreC (normal prostate cells), compared to those grown in LPDS (Fig. 7A).  The PC-3 cancer cells, however, expressed similar amount of LDLr in the presence or absence of exogenous LDL or serum lipoprotein (Fig. 7B).  This suggests that some prostate cancer cells lack normal LDLr feedback regulation at the mRNA level. In the figure on the following page, we show that feedback regulation of the receptor is also lost at the functional/protein level. (Fig. 7C) Regulation of LDLr expression was further studied at the functional level measured by uptake of fluorescent-labeled LDL.

Control prostate cells, PreC cells grown in LPDS had strong fluorescent signals showing active uptake of the labeled LDL at the cell surface as well as internalization.  When cells were grown in presence of LDL or whole serum, the signals were much weaker.  In the PC-3 cancer cells, fluorescent signal uptake remained unchanged in most cells regardless of the presence of lipoproteins.  Human fibroblasts derived from a familial hypercholesterolemia patient, which lacks functional LDLr expression, was used as negative control in this experiment.

 

Impact of Research findings: Prostate carcinoma is one of the most common cancers in the U.S. The incidence of prostate tumor in Japan is low in comparison to the U.S. However, when Japanese immigrate to the US, the incidence rate is the same. Since almost a third of the male population harbors small microscopic prostate cancers, it is important to define and minimize dietary factors that accelerate growth of these tumors. growth of prostate tumors. Although multiple human and animal studies have suggested that fatty acids are associated with increased occurrence of prostate cancer, there are no reports on mechanism of action by which fatty acids accelerate tumor growth.  If the findings indicate that reductions in essential fatty acids or EP4 receptor antagonists reduce growth of prostate cancers, the information could be immediately implemented in the clinic. This area of study has potential value both in terms of cancer biology and therapy.