In vitro Effects on Cancer Cells

Researchers at King’s College London examined Artemis’ standardized extracts—Bilberry, Chokeberry, and Elderberry on malignant human brain tumor cell lines in vitro. Using time-lapse video recording and flow cytometry they found increased cell death with chokeberry and bilberry extracts and down-regulation of MMP-9 secretion with the chokeberry extract. They proposed that chokeberry flavonoids might be promising candidates as novel treatments for brain tumor therapy.

Christidou, M., Rooprai, H.K., Rucklidge, G., Davis, D., Clayton, P.R., Zicha, D. Pilkington, G.J. 2001. The effect of three berry flavonoids on malignant human brain tumour cell lines in vitro. Presented at 14th International Conference on Brain Tumor Research and Therapy, May 27-30. Asheville, NC.

Researchers at the University of Maryland investigated the effect of Artemis’ standardized extracts – Bilberry and Chokeberry, on the growth of the HT-29 colon carcinoma cell line in vitro. Both extracts were found to inhibit the growth of HT-29 cells (50μg monomeric anthocyanin/mL), but the inhibition levels varied despite the fact that the extracts were standardized for equal anthocyanin content. This suggests that the anthocyanin profile may impact biological activity. Growth inhibition and cell morphological changes demonstrated a dose-dependent effect and maximum growth inhibition (up to 50% of control) was observed with Artemis’ standardized chokeberry extract.

The researchers also found that the normal human colon epithelial cell line (NCM460) grew normally in the presence of Artemis’ standardized chokeberry and bilberry extracts with little to no inhibition. However, HT-29 carcinoma cells were susceptible to growth inhibition by Artemis’ standardized chokeberry and bilberry extracts and it appeared to be resulting from a cytostatic effect rather than a cytotoxic effect. The berries’ chemical composition and combination of both anthocyanins and other polyphenols play a role in HT-29 colonic cell growth inhibition. The researchers concluded that anthocyanins and/or other compounds in the chokeberry and bilberry extracts may be potential chemopreventive agents for colon cancer.

Further flow cytometric analyses of Artemis’ standardized chokeberry extract indicated an altered progression of the chokeberry-treated HT-29 cells through the cell cycle as compared to untreated controls. Overall, Artemis’ standardized chokeberry extract down-regulated COX2 mRNA levels but not COX1 mRNA levels in HT-29 cells. It was also reported that cyclooxygenase-2 gene expressions were down-regulated in chokeberry-treated HT-29 cells. Artemis’ standardized chokeberry extract arrested the cell cycle at two phases: G1/G0 and G2/M. When the cell cycle was blocked, p21WAF1 and p27 KIP1 gene expressions increased concomitantly and there was decreased expression of cyclin A and B genes. The aberrant colonic cells were viable; thus, this finding elucidates the proposed cytostatic paradigm.

Zhao, C., Malik, M., Magnuson, B.A., and Giusti, M.M. 2002. The inhibitory effect of different anthocyanin-rich extracts on cancer cell growth. Presented at the Institute of Food Technologists Annual Meeting, June 16- 19, Anaheim, CA.

Malik, M., Zhao, C., Schoene, N.W., Giusti, M.M., Moyer, M.P., and Magnuson, B.A. 2002. Inhibition of colon cancer cell growth by anthocyanin-rich extracts from fruits. Presented at Colon Cancer: Genetics to Prevention Conference, March 7-10, Philadelphia, PA.

Malik, M., Zhao, C., Schoene, N., Guisti, M.M., Moyer. M.P., Magnuson, B.A.. 2003. Anthocyanin-Rich extract from Aronia melanocarpa E. induces a cell cycle block in colon cancer but not normal colonic cells. Nutrition and Cancer. 46(2) 186-196.

Zhao, C., Giusti, M.M., Malik, M., Moyer, M.P., Magnuson, B. 2004. Effects of commercial anthocyanin-rich extracts on colonic cancer and nontumorigenic colonic cell growth. J.Agric.Food Chem. 52: 6122-6128.

This team then collaborated with Ohio State University to take their investigations a step further and look into structure-function relationships of anthocyanin-rich extracts exhibiting chemoprotective qualities. In an assay investigating human colon cancer growth inhibition, purple corn was shown to be the most potent, followed by Artemis’ chokeberry and bilberry extracts. They determined that anthocyanins, as opposed to other flavonoids, played a major role in the chemoprotection and exerted an additive interaction with other phenolics present. Nonacylated monoglycosylated anthocyanins had the greatest inhibitory effect on HT-29 cell proliferation, suggesting the specific anthocyanin chemical structure does affect chemoprotection. These findings help to shape the scope of future research toward discovering which specific chemical structures (or combinations thereof) contribute to desired health effects.

Jing, Pu. Bomser, J., Schwartz, S., He, J., Magnuson, B., Guisti, M. 2008. Structure-function relationships of anthocyanins from various anthocyanin-rich extracts on the inhibition of colon cancer cell growth.. J.Agric.Food Chem. 56(20): 9391-9398.

Researchers from the University of Portsmouth, England, conducted studies to evaluate the effects of Artemis’ Standardized Chokeberry extract on a malignant brain tumor cell line, NP750. The qualitative analysis by immunocytochemistry and confirmation by the quantitative analysis using flow cytometry showed the down regulation of CD44 expression by 85%. Additionally, the chokeberry extract downregulated the gene expression of MMP-2, -14, -15, -24, TIMP-1, -2, -3, and EGFr. The fact that the chokeberry extract can cross the blood-brain barrier and demonstrates anti-invasive potential is promising for its use as a therapeutic agent in malignant brain tumor management.

Rooprai, H.K., Christidou, M., Kavies, D., Nuttall, R., and Pilkington, G.J. 2003. Influence of chokeberry extract on parameters of glioma invasion. Poster.

Researchers at Indiana-Purdue University in Fort Wayne, Indiana tested Artemis’ BerryDefense™ Blend components on an LnCAP cell line (Lymph Node CAncer of the Prostate). When different concentrations of the berry extracts were added to the tumor cell line, the cells exhibited an anomalous (shriveled) form and the growth level was significantly suppressed. Western blot analysis revealed that the Artemis BerryDefense™ Blend contains an extract component that alters Protein Kinase C (PKC) levels. PKC, a regulatory enzyme, is essential in cell response, cell growth, and apoptosis. The data is consistent with decreased LnCaP cell proliferation. This finding suggests a plausible function as an anti-cancer factor.

Blumenthal, E.J., and Bush, M.C. 2006. The effects of berry extracts on immune system function and LnCap cancer cell growth. Unpublished data-manuscript in preparation.

See Appendix I for additional in vitro research supporting the protective effects of high anthocyanin berry extracts against cancer cells.

In vivo Effects on Cancer Cells

A joint animal study by researchers from both Ohio State University and University of Maryland showed that Artemis’ standardized chokeberry and bilberry extracts inhibited multiple biomarkers of colon cancer in rats, providing in vivo confirmation of the chemopreventive activity observed in previous in vitro studies. Azoxymethane (AZO)-induced rats expressed aberrant crypt foci (ACF); ACF are pre-neoplastic lesions that develop in the colon causing a cancerous state and are the preliminary biomarkers for colon cancer in both rodents and humans. After feeding the rats diets containing Artemis’ berry extracts, the researchers reported that Artemis’ standardized chokeberry and bilberry were effective in reducing the number and size of the ACF. Additionally, a decrease in anomalous colonic cell proliferation was observed in chokeberry and bilberry-fed rats. Hence, the high anthocyanin levels in chokeberry and bilberry may suggest a putative chemopreventive role.

The researchers further indicated that the urine from the rats fed with chokeberry and bilberry diets contained anthocyanin levels of 23.6 mg/l for chokeberry and 7.8 mg/l for bilberry. Fecal analyses showed anthocyanin levels of 1.7 mg/l for chokeberry and 2.0 mg/l for bilberry. The fecal moisture content and fecal amounts were higher in bilberry and chokeberry-fed rats compared to the control. Researchers found a drastic fecal bile acid reduction in chokeberry (.55μmol/g per day) and bilberry (1.26μmol/g per day) fed rats. Bioavailability studies revealed that the presence of anthocyanins reduced fecal bile. Bile acids and other tumor-inducing compounds promote colon cancer, therefore, the ability of Artemis’ standardized chokeberry and bilberry extracts to reduce fecal bile in feces is a significant finding. In conclusion, the chemical composition, metabolism, and the functional bioavailability all contribute to the chemopreventive role of anthocyanins.

Lala, G., Malik, M., Zhao, C., He, J., Kwon, Y., Giusti, M.M., Magnuson, B. 2006. Anthocyanin-rich extracts inhibit multiple biomarkers of colon cancer in rats. Nutrition and Cancer. 54(1): 84-93.

Angiogenesis is a complex vascularization process essential for the development of neoplastic tumors. Researchers from the Military Medical Academy in Poland tested Artemis’ Standardized Chokeberry extract on induced angiogenesis in mice. When the chokeberry extract was intragastrically administered, there was a clear inhibition in the development of newly formed angiogenic vessels by over 60%. This significant inhibition of vessel neoplasia suggests that the chokeberry extract possesses antiangiogenic effects.

Krzesinski P., Kura, M., Wojciech, S., and Bartosz, S. Effect of anthocyanins from Aronia melanocarpa Elliott on skin angiogenesis reaction in mice. Przegl. Wojsk.-Med, 2002, 44(2): 123-127.

See Appendix I for additional in vivo research supporting the protective effects of high anthocyanin berry extracts against cancer cells.