Phytosterols for Cancer Treatment Powerpoint

Phytosterols for Cancer Treatment

Josh Nooner, BS, CSCSPhytochemicals

11/19/15

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

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?

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

Bioavailability

Studies show a range of absorption from 0.5-8% Very low bioavailability, caused by 3 mechanisms

o Low absorption into the enterocyteo Poor esterification once inside the enterocyteo High biliary excretion rate once in the liver

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 studieso 3 in vitro studieso 1 human epidemiological study

Animal Studies

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)

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

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 diet2. Phytosterol supplemented dieto Tumor cells were then injected and their growth was monitored for 8 weeks

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

Animal Study 3: Protective effect of plant sterols against chemically induced colon tumors in rats

Methods: 4 experimental groups1. Control chow and intracolonic0.9% NaCIsolution N=102. Control chow plus B-sitosterol (0.2%) and intracolonic 0.9% NaCI solution

N=103. Control chow and intracolonic MNU3 N=714. 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

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

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 control2. 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

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%

In Vitro Studies

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 bw2. 10 mg/kg bw3. 20 mg/kg bw

16 week experimental period


• Significant induction of apoptotic cells by B-sitosterol


• Significant reduction in ACF lesions with increased B-sitosterol

• Dose dependent decrease in B-Catenin (cell adhesion) and PCNA (DNA Replication) genes

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 measuredo Induction of cell growth-suppressor gene expression was evaluatedo Apoptosis was evaluated


NDRG1 gene (tumor suppressor gene) increased with campsiterol Caveolin 1 gene (tumor suppressor gene) increased with campsiterol


• Decrease in anti-apoptotic genes and increase in pro-apoptotic and tumor suppressor genes in phytosterol group

• Opposite effect in cholesterol group

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


• Dose dependent decrease in proliferation of stomach cancer cells


Results:

• B-sitosterol increases expression of pro-apoptotic genes, decreases anti-apoptotic genes• B-sitosterol decreases size of stomach cancer cells

Human Studies

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

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

Mechanisms

Dosing

Studies recommend 200-400mg/day of phytosterolso This will keep the serum level at the necessary concentration to see positive effectso Must keep the serum concentration relatively constanto Eat a variety of phytosterol containing foods daily

Most Americans get 80mg/day

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 geneo Rare autosomal recessive inherited lipid metabolic disordero ABC transport proteins ineffectiveo Leads to elevated blood sterols, atherosclerosis, and xanthomas

2. Those pregnant and lactating – Lack of research in this area

Application to Human Health and Disease Phytosterols shown to:

1. Increase apoptosis of cancerous cells2. Reduce ROS levels and prevent oxidative damage3. Increase antioxidant enzymes4. Reduce blood cholesterol5. Decrease inflammatory cytokines6. 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

Future Studies

Short and long term effects Interactions with prescription drugs Mechanisms of prevention

Research SummaryResearch Article Area Investigated ResultsLlaverias, 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.

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.

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.

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Phytosterols for Cancer Treatment Powerpoint