Phytosterols for Cancer Treatment

1. Phytosterols for
Cancer Treatment
Josh Nooner, BS, CSCS
Phytochemicals
11/19/15

2. Objectives
 Provide a brief overview of phytosterols
 List food sources and bioavailability
 Examine dosing and contraindications
 Provide a literature review of human, animal, and in vitro studies
 Discuss major research findings and applicability to human health and disease

3. Introduction
 Phytosterols - Plant-derived compounds that are similar in structure and
function to cholesterol
 Includes both sterols and stanols
o Sterols - Have a double bond in the sterol ring (90%)
o Stanols – Lack a double bond in the sterol ring (10%)
 The most abundant sterols in plants and the human diet
are sitosterol and campesterol
 Shown to reduce LDL cholesterol, improve lipid profiles,
and prevent cardiovascular disease
 But what are the effects on cancer?

4. Food Sources Food Source Amount of
Beta-
Sitosterol in
3.5oz
portion
Amount of
Beta-
Sitosterol in
200 calories
Avocado 260mg 95 mg
Vegetable oil 240mg 93mg
Margarine 215mg 77mg
Pistachios 200mg 71mg
Hazelnuts 90mg 34mg
Dark Chocolate 90mg 34mg
Macadamias 85mg 30mg
Pecans 75mg 27mg
Walnuts 57mg 20mg
1 Egg 42mg 15mg
o Avocados
o Vegetable oils
o Fortified margarine
o Nuts
o Seeds
o Dark Chocolate

5. Bioavailability
 Studies show a range of absorption from 0.5-8%
 Very low bioavailability, caused by 3 mechanisms
o Low absorption into the enterocyte
o Poor esterification once inside the enterocyte
o High biliary excretion rate once in the liver

6. Major Research Findings
 Most anticancer effects of phytosterols have been demonstrated in animal
and in vitro studies
 Need to be confirmed in carefully controlled human clinical trials
 Epidemiological studies - Only studies that have been done in humans
 I will review 8 research articles
o 4 animal studies
o 3 in vitro studies
o 1 human epidemiological study

7. Animal Studies

8. Animal Study 1: Phytosterols inhibit the tumor growth and lipoprotein
oxidizability induced by a high-fat diet in mice with inherited breast cancer
 Methods: 4-week-old female PyMT Tg mice were randomized into 2 groups. Those
consuming and those not consuming a 2% phytosterol supplement added to the
powdered food on either an
1. LFLC diet (6.2% fat, no cholesterol, energy density 3.1 kcal/g, calories from
protein, fat and carbohydrate, 24%, 18% and 58%, respectively
2. HFHC diet (21.2% fat, 0.2% cholesterol, energy density 4.5 kcal/g, calories from
protein, fat and carbohydrate, 15.2%, 42% and 42.7%, respectively
o Phytosterols were composed of 20% campesterol, 22% stigmasterol and 41% β-
sitosterol
o Mice were maintained on the diets until euthanized (at 4, 8 or 13 weeks of age)

9. Animal Study 1: Phytosterols inhibit the tumor growth and lipoprotein
oxidizability induced by a high-fat diet in mice with inherited breast cancer
 Dietary phytosterol supplementation delayed tumor onset and progression in the setting of a typical Western diet
 The protection against cancer provided by phytosterols occurred at the same dose that reduced blood cholesterol and
cardiovascular risk
 Phytosterols decreased levels of Cyclin D1, a gene that leads to breast cancer when activated

10. Animal Study 2: In vitro and in vivo (SCID mice) effects of phytosterols on
the growth and dissemination of human prostate cancer PC-3 cells
 Methods:
 PC-3 cells: supplemented with either cholesterol, campesterol, or beta-sitosterol.
o Tested tumor invasiveness, cell migration,
 In vivo: Mice were fed for two weeks ad libitum either
1. Cholesterol supplemented diet
2. Phytosterol supplemented diet
o Tumor cells were then injected and their growth was monitored for 8 weeks

11. Animal Study 2: In vitro and in vivo (SCID mice) effects of phytosterols on
the growth and dissemination of human prostate cancer PC-3 cells
 Results:
 Cell density and cell migration were significantly reduced in the phytosterol supplemented cells
 Total tumor area at 8 wks was significantly lower in the phytosterol fed mice

12. Animal Study 3: Protective effect of plant sterols against chemically
induced colon tumors in rats
 Methods: 4 experimental groups
1. Control chow and intracolonic0.9% NaCIsolution N=10
2. Control chow plus B-sitosterol (0.2%) and intracolonic 0.9% NaCI solution N=10
3. Control chow and intracolonic MNU3 N=71
4. Control chow plus B-sitosterol (0.2%) and intracolonic MNU N=48
 28 week intervention period
 At Week 28, the colon was opened, and the number of tumors was recorded

13. Animal Study 3: Protective effect of plant sterols against chemically
induced colon tumors in rats
 Results:
 Decrease in colonic tumor formation when the plant sterol B-sitosterol was
added to the diet

14. Animal Study 4: β-sitosterol, β-sitosterol glucoside, and a mixture of β-
sitosterol and β-sitosterol glucoside modulate the growth of estrogen-
responsive breast cancer cells in vitro and in ovariectomized athymic
mice
 Methods: Estrogen pellet and MCF-7 cells injected into ovariectomized
female mice.
 4 experimental groups:
1. Negative control
2. BSS (Beta – Sitosterol)
3. BSSG (Beta – Sitosterol glucoside)
4. MC (BSS:BSSG = 99:1)
 Estrogenic and antiestrogenic effects of dietary phytosterols on tumor growth
measured
 18 week intervention period

15. Animal Study 4: β-sitosterol, β-sitosterol glucoside, and a mixture of β-
sitosterol and β-sitosterol glucoside modulate the growth of estrogen-
responsive breast cancer cells in vitro and in ovariectomized athymic
mice
 Results:
 Dietary BSS and MC reduced E2-induced MCF-7 tumor growth
 Dietary phytosterols lowered the plasma E2 level by 35.3%

16. In Vitro Studies

17. In Vitro Study 1: Chemopreventive potential of β-sitosterol in
experimental colon cancer model-an in vitro and in vivo study
 Methods:
o β-sitosterol was isolated from A. curassavica leaves.
o The ability to induce apoptosis was determined by its in vitro antiradical activity and cytotoxic
studies using human colon adenocarcinoma cell lines.
 1,2-dimethylhydrazine (DMH, 20 mg/kg b.w.) was injected into male Wistar rats.
 Rats were supplemented with β-sitosterol in 3 different concentrations
1. 5 mg/kg bw
2. 10 mg/kg bw
3. 20 mg/kg bw
 16 week experimental period

18. In Vitro Study 1: Chemopreventive potential of β-sitosterol in
experimental colon cancer model-an in vitro and in vivo study
• Significant induction of apoptotic cells by B-sitosterol

19. In Vitro Study 1: Chemopreventive potential of β-sitosterol in
experimental colon cancer model-an in vitro and in vivo study
• Significant reduction in ACF lesions with increased B-sitosterol
• Dose dependent decrease in B-Catenin (cell adhesion)
and PCNA (DNA Replication) genes

20. In Vitro Study 2: Cholesterol and phytosterols differentially regulate the
expression of caveolin 1 and a downstream prostate cell growth-
suppressor gene
 Methods:
 PC-3 and DU145 cells were treated with either cholesterol or phytosterols for 72h
o Necrosis and cell growth were measured
o Induction of cell growth-suppressor gene expression was evaluated
o Apoptosis was evaluated

21. In Vitro Study 2: Cholesterol and phytosterols differentially regulate the
expression of caveolin 1 and a downstream prostate cell growth-
suppressor gene
 NDRG1 gene (tumor suppressor gene) increased with campsiterol
 Caveolin 1 gene (tumor suppressor gene) increased with campsiterol

22. In Vitro Study 2: Cholesterol and phytosterols differentially regulate the
expression of caveolin 1 and a downstream prostate cell growth-
suppressor gene
• Decrease in anti-apoptotic genes and increase in pro-apoptotic and tumor suppressor genes in phytosterol group
• Opposite effect in cholesterol group

23. In Vitro Study 3: β-Sitosterol inhibits cell growth and induces apoptosis in
SGC-7901 human stomach cancer cells
 Methods: SGC-7901 human stomach cancer cells
 Proliferation, cytotoxicity, and apoptosis were examined using various assays and western blotting
 Cells treated with different concentrations of β-sitosterol

24. In Vitro Study 3: β-Sitosterol inhibits cell growth and induces apoptosis in
SGC-7901 human stomach cancer cells
• Dose dependent decrease in proliferation of stomach cancer cells

25. In Vitro Study 3: β-Sitosterol inhibits cell growth and induces apoptosis in
SGC-7901 human stomach cancer cells
 Results:
• B-sitosterol increases expression of pro-apoptotic genes, decreases anti-apoptotic genes
• B-sitosterol decreases size of stomach cancer cells

26. Human Studies

27. Human Study 1: Phytosterols and risk of lung cancer: a case-control study
in Uruguay
 Methods: All patients with newly diagnosed primary lung cancer diagnosed in the four major
hospitals of Montevideo included.
 463 cases with lung cancer and 465 hospitalized controls
 Detailed medical, lifestyle, and diet questionnaire
 96% response rate

28. Human Study 1: Phytosterols and risk of lung cancer: a case-control study
in Uruguay
 Significantly lower risk of developing lung cancer in the highest quintiles of phytosterol
consumption

29. Mechanisms

30. Dosing
 Studies recommend 200-400mg/day of phytosterols
o This will keep the serum level at the necessary concentration to see positive effects
o Must keep the serum concentration relatively constant
o Eat a variety of phytosterol containing foods daily
 Most Americans get 80mg/day

31. Contraindications
 Two cases where people should avoid phytosterols
1. Those with Sitosterolemia – A very rare hereditary disease that results from
a mutation in both copies of the ABCG5 or ABCG8 gene
o Rare autosomal recessive inherited lipid metabolic disorder
o ABC transport proteins ineffective
o Leads to elevated blood sterols, atherosclerosis, and xanthomas
2. Those pregnant and lactating – Lack of research in this area

32. Application to Human Health and Disease
 Phytosterols shown to:
1. Increase apoptosis of cancerous cells
2. Reduce ROS levels and prevent oxidative damage
3. Increase antioxidant enzymes
4. Reduce blood cholesterol
5. Decrease inflammatory cytokines
6. Decrease angiogenesis
 Phytosterols are a promising treatment for many types of cancer
 Will be used as a dietary intervention in a clinical setting in the future

33. Future Studies
 Short and long term effects
 Interactions with prescription drugs
 Mechanisms of prevention

34. Research Summary
Research Article Area Investigated Results
Llaverias, G., Escolà-Gil, J. C., Lerma, E., Julve, J.,
Pons, C., Cabré, A., ... & Blanco-Vaca, F. (2013).
Examined the effects of a dietary phytosterol
supplement on tumor onset and progression in breast
cancer.
Dietary phytosterol supplementation delayed tumor
onset and progression in the setting of a typical Western
diet and suggest that phytosterols may exert these
anticancer effects by preventing oxidative damage.
Awad, A. B., Fink, C. S., Williams, H., & Kim, U The dietary effect of phytosterols vs. cholesterol on the
growth and metastasis of the PC-3 human prostate
cancer cells in SCID mice.
Phytosterols both indirectly and directly inhibited the
growth and metastasis of PC-3 cells.
Raicht, R. F., Cohen, B. I., Fazzini, E. P., Sarwal, A. N.,
& Takahashi, M
The effect of B-sitosterol on colon tumor formation in
rats treated with a carcinogen.
There is a decrease in colonic tumor formation when the
plant sterol B-sitosterol was added to the diet.
Ju, Y. H., Clausen, L. M., Allred, K. F., Almada, A. L., &
Helferich, W. G.
Evaluated the estrogenic and antiestrogenic effects of
BSS, BSSG, and MC (0.001 to 150 mol/L) on the
proliferation of Michigan Cancer Foundation 7 (MCF-7)
cells in vitro.
BSS and MC stimulated MCF-7 cell growth in vitro.
Although BSSG comprises only 1% of MC, BSSG made MC
less estrogenic than BSS alone in vitro. However, dietary
BSS and MC protected against E2-stimulated MCF-7
tumor growth and lowered circulating E2 levels.
Baskar, A. A., Ignacimuthu, S., Paulraj, G. M., & Al
Numair, K. S.
To broaden the understanding of the anticancer
potential of β-sitosterol in in vitro cancer model and
DMH-induced experimental colon carcinogenesis model.
β-sitosterol induced significant dose-dependent growth
inhibition of colon cells, induced apoptosis by
scavenging reactive oxygen species, and suppressed the
expression of β-catenin and PCNA antigens in human
colon cancer cells.
Ifere, G. O., Equan, A., Gordon, K., Nagappan, P.,
Igietseme, J. U., & Ananaba, G. A
To show the distinction between the apoptotic and anti-
proliferative signaling of phytosterols and cholesterol-
enrichment in prostate cancer cell lines, mediated by
the differential transcription of caveolin-1, and N-myc
downstream-regulated gene 1 (NDRG1), a pro-apoptotic
androgen-regulated tumor suppressor.
Cholesterol-enrichment promoted cell growth (P<0.05),
while phytosterols significantly induced growth-
suppression (P<0.05) and apoptosis. Cell cycle analysis
showed that contrary to cholesterol, phytosterols
decreased mitotic subpopulations
Zhao, Y., Chang, S. K., Qu, G., Li, T., & Cui, H. Investigate the effect of β-sitosterol on proliferation and
apoptosis in SGC-7901 stomach cancer cells in vitro and
to study the possible mechanisms of action involved.
B-sitosterol suppresses the proliferation and induces the
cell cytotoxicity of SGC-7901 stomach cancer cells in a
time and dose-dependent manner.
Mendilaharsu, M., De Stefani, E., Deneo-Pellegrini, H.,
Carzoglio, J., & Ronco, A.
To establish a possible protective role of plant sterols in
lung carcinogenesis.
Total plant sterol intake was associated with a reduction
in risk of 50% when contrasting the upper exposure
quartile with the lower.

35. References
 Awad, A. B., Fink, C. S., Williams, H., & Kim, U. (2001). In vitro and in vivo (SCID mice) effects of phytosterols
on the growth and dissemination of human prostate cancer PC-3 cells. European Journal of Cancer
Prevention, 10(6), 507-513.
 Baskar, A. A., Ignacimuthu, S., Paulraj, G. M., & Al Numair, K. S. (2010). Chemopreventive potential of β-
sitosterol in experimental colon cancer model-an in vitro and in vivo study. BMC complementary and
alternative medicine,10(1), 24.
 Bradford, P. G., & Awad, A. B. (2007). Phytosterols as anticancer compounds.Molecular nutrition & food
research, 51(2), 161-170.
 Bradford, P. G., & Awad, A. B. (2010). Modulation of signal transduction in cancer cells by
phytosterols. Biofactors, 36(4), 241-247.
 Ding, H., Chin, Y. W., Kinghorn, A. D., & D’Ambrosio, S. M. (2007, October). Chemopreventive characteristics
of avocado fruit. In Seminars in cancer biology (Vol. 17, No. 5, pp. 386-394). Academic Press.
 Higdon, J. (2005). Phytosterols. Retrieved from http://lpi.oregonstate.edu/mic/dietary-
factors/phytochemicals/phytosterols
 Ifere, G. O., Equan, A., Gordon, K., Nagappan, P., Igietseme, J. U., & Ananaba, G. A. (2010). Cholesterol and
phytosterols differentially regulate the expression of caveolin 1 and a downstream prostate cell growth-
suppressor gene. Cancer epidemiology, 34(4), 461-471.
 Ju, Y. H., Clausen, L. M., Allred, K. F., Almada, A. L., & Helferich, W. G. (2004). β-sitosterol, β-sitosterol
glucoside, and a mixture of β-sitosterol and β-sitosterol glucoside modulate the growth of estrogen-responsive
breast cancer cells in vitro and in ovariectomized athymic mice. The journal of nutrition,134(5), 1145-1151.

36. References Cont.
 Landauer, E. (2008). Healthy Male Prostate Diet. Retrieved from http://www.peak-health-now.com/male-prostate-
diet.html
 Llaverias, G., Escolà-Gil, J. C., Lerma, E., Julve, J., Pons, C., Cabré, A., ... & Blanco-Vaca, F. (2013). Phytosterols
inhibit the tumor growth and lipoprotein oxidizability induced by a high-fat diet in mice with inherited breast
cancer. The Journal of nutritional biochemistry, 24(1), 39-48.
 Mendilaharsu, M., De Stefani, E., Deneo-Pellegrini, H., Carzoglio, J., & Ronco, A. (1998). Phytosterols and risk of lung
cancer: a case-control study in Uruguay. Lung Cancer, 21(1), 37-45.
 Raicht, R. F., Cohen, B. I., Fazzini, E. P., Sarwal, A. N., & Takahashi, M. (1980). Protective effect of plant sterols
against chemically induced colon tumors in rats. Cancer Research, 40(2), 403-405.
 Ramprasath, V. R., & Awad, A. B. (2015). Role of Phytosterols in Cancer Prevention and Treatment. Journal of AOAC
International.
 Woyengo, T. A., Ramprasath, V. R., & Jones, P. J. H. (2009). Anticancer effects of phytosterols. European Journal of
Clinical Nutrition, 63(7), 813-820.
 Zhao, Y., Chang, S. K., Qu, G., Li, T., & Cui, H. (2009). β-Sitosterol inhibits cell growth and induces apoptosis in SGC-
7901 human stomach cancer cells.Journal of agricultural and food chemistry, 57(12), 5211-5218.

37. Questions?