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black cohosh research poster

  1. 1. Black cohosh (Cimicifuga racemosa): A potential remedy for breast cancer Black cohosh-A gift from Native Americans Black cohosh(Cinicifuga racemosa) is a herbaceous perennial plant. Native to North America, the root and rhizomes of black cohoshwere used by Native American women for menstrual cramps, difficult childbirth and complicated menopause, as well as other conditions likedysmeorrhea, colic and rheumatism. Native Americans subsequently introduced the herb to the American colonists, who used it for women’s complaints, as well as illness like bronchiitis, nervous disorders and inflammation. Today, black cohoshis widely used in various pharmaceutical preparations. It is widely used in the U.S. and Europe against menopausal symptoms, suchas hot flashes, sweats, irritability and vaginal dryness. The action of black cohoshis attributed to synergy of its active components. The main active compounds ofthe herb’s root are believed to be the triterpene glycosides (TTGs), including actein and cimifungoside. Other possible biologically active compounds include the isoflavone formononetin, the TTGs 27-deoxyactein and racemoside, as well as the aromatic (CAEs), sugars and long-chain fatty acids. One aim of this research was to examine the effects of the various black cohoshcompounds on the growth of human breast cancer cells, including inhibition effect, cell cycle progression and expression proteins involved in cell cycle control. Another aim of the research was to identify new approaches to breast cancer prevention and therapy, leading to following studies concerning the use of herbology in cancer treatment. Cimicifuga racemosa structure of the triterpene glycoside actein:
  2. 2. Basic structure of cinnamic acid esters: Methodology: This research was library-based, and took place at the library of the University of East London (on Stratford campus). It included the collection and analysis of data from 29 different scientific articles concerning the anti-breast-cancer activity and properties of black cohosh(Cimicifuga racemosa) and its different compounds. Thearticles for the research were obtained through PubMed and Google Scholar. Results: Ethanolic and isopropanolic extracts of black cohoshrhizomes inhibited the growth of both estrogen-dependent MCF-7 and estrogen-independent MDA-MB human breast cancer cells. Black cohoshalso showed no estrogenic activity in MCF-7, but antagonised these activities. In addition, estrogen-receptor-negative (ER-) were significantly more sensitive than ER+ cells. There was dose-dependentanti-proliferative action of black cohoshin breast cancer cells, probably evoked by genomic (ER and non-ER mediated) mechanisms, due to the various properties of different components of the herb. Also, it was found that black cohosh doesn’tbind to ER-alpha or beta. However, there were no differences in sensitivity of MCF- 7 and MDA-MB cell lines to either TTGs or CAEs, according to IC50 values. In both cell types, the herb had no significant effect on the conversion of androstenedione to estradiol at any dose, and only the highest doses inhibited the conversion of estrone to estradiol. Black cohoshwas also found to initiate cytotoxicity through induction of apoptosis. An ethanolic extract of the plant was found to inhibit the activity of cyclin D1 promoter and increase the activity of P21cip1 promoter in ER- human breast cancer cells. Ethyl acetate fraction of black cohoshinhibited growth of MCF-7 (ER+, Her2 low) cells and induced cell cycle arrest at G1 after treatment with 30 micrograms/mL, and at G2/M after treatment with 60 micrograms/mL. This suggests that the fraction contains a mixture of components, with the more active/abundant component causing G1 arrest, and the less active causing G2/M arrest. Therefore, it is possible that at high concentrations the fraction affects proteins regulating later phases in the cell cycle. CAEs turned out to be the more potent inhibitors of proliferation and apoptosis inducers in MCF-7 cells. Black cohoshhas as anti-proliferative effect, therefore genes involved in proliferation control are significantly over-expressed. Transcripts related to cell cycle regulation and DNA replication are regulated in a manner supporting cell cycle arrest. Genes, whose products are involved in the transition from G1 to S-phase, appears to be down-regulated, such as cyclins (CCNA2, CCNE2, CCNF), cdk2 and transcription regulators (E2F2, PCNA, SKP2), whereas
  3. 3. transcription of inhibitory genes cyclin G2 (CCNG2), GADD45A (growth arrest and DNA- damage-inducible) and P21cip1 was increased. Since actein was found to alter the expression of genes involved in calcium metabolism and the Na-K-ATPase affects calcium metabolism, the ability of actein to inhibit the activity of the ATPase and activate related down-stream pathways is possible. Na-K-ATPase mediates many stress responses and proliferation pathways that are affected by actein. Actein may act through interaction with the Na-K-ATPase promoters in the cell membrane and induction of clustering of ATPasewith neighbouring proteins in micro-domains. Some CAEs are also potential inhibitors of EGFR and Her2 proteins, which have a proven connection with breast cancer. It had also been shown that CAEs have selective anti- proliferative activity towards cancer cells. Among the compounds isolated from black cohoshwith anti-oxidant behaviour, methyl caffeate has the highest potency in protecting DNA against single-strand cleavage. Also, both caffeic and ferulic acids protectDNA by reducing reactive oxygen species. Functional categoriesof genesregulatedinMCF-7cellsafter24 h incubationwithblackcohoshextract.Geneswere groupedin5 large groups(Apoptosis,Proliferation,General Growth,Signaling& Transport,Metabolism),some consistingof subgroups.Genesthatare notclearlyassociatedwiththese groupsare summarizedinthe category others.The categorystressresponse containsgenesalsogroupedintoone of the 6 mainclasses.Each bar represents the numberof genesthat were up- (dark) ordownregulated(white) inthe respective group.
  4. 4. . Effectof iCR, TTG and CAE on tumor cell growth.MCF-7 and MDA-MB 231 cellswere treatedfor24 h withor withoutsubstancesatthe indicatedconcentrations.Attachedcellswerestainedwithcrystal violetandthe absorbance of the cell lysate wasmeasuredat540 nm.Data are expressedasmean±SDof triplicate wellsfromtwo independentexperiments.*p<0.05 vs.untreatedcontrol.
  5. 5. Discussion: From the aspectof general function of black cohosh, the herb seems to have anti- cancer properties, since both ethanolic and isopropanolic extracts of black cohosh rhizomes inhibit growth of both estrogen-dependent MCF-7 and estrogen- independent MDA-MB cell lines. In addition, MDA-MB-231 cells showed a higher sensitivity to the cytotoxic effects of black cohoshthan MCF-7 cells. It also seems, for this reason, that the activity of black cohoshon breast cancer cells is mainly non- estrogenic. This is also supported by the fact that there was no significant effect on the conversion of estrone to estradiol. It is far more likely that black cohosh, especially its actein fraction, causes cytotoxic effects on breast cancer cells through apoptosis induction through the Her2 receptor, as well as the Na-K-ATPase enzyme. Action via binding to Her2 is likely, since the Her2+ MDA-MB cell lines are more sensitive to the cytotoxic effect than MCF-7 (Her2 low) cells. Also, MCF-7 cells transfected for Her2 are more sensitive than normal MCF-7 cells to actein. Actein also seems to act via activation of Na-K-ATPase, due to actein’s effect on calcium metabolism related genes, which are also related to ATPase activation. This makes sense also due to actein’s role in alteration of actin filaments’ distribution, through which apoptosis is induces. Inhibition of ATPase has also been shown to be related to a compound’s lipid-affinity, and actein has steroid-like sterol composition, that makes it lipophilic enough for this role. Unlike TTGs, CAEs do seem to inhibit EGFR, in addition to Her2. It most likely acts through reduction of DNA damage by protection from reactive oxygen species. Synergy between different black cohoshcompounds is likely, since it has been shown to inhibit breast cancer through several pathways. Probably the binding of actein to ATPase makes the cell membrane more penetrable through structure alteration, which allows CAE access. Then, the CAEs can act against the reactive oxygen species, and also inhibit EGFRs. By binding to ATPase and Her2, actein also has anti-cancer activity in its own right, but also increases the effectiveness of CAEs. References: 1. B. Klinger. 2003. Black cohosh.American family physician, volume 68, no. 1, p.114-116. 2. J.L. Mayo. 1998. Black cohosh and chaste berry: Herbs valued by women for centuries. Clinical nutrition insights,vol. 6, no. 15, p.1-4. 3. F. Firenzuoli. 2011. Black cohosh hepatic safety: Follow-up of 107 patients consuming a special cimicifuga racemosa rhizome herbal extract and review of literature. Evidence- based complementary and alternative medicine, vol. 2011, p.1-7. 4. A. Huntley. 2004. The safety of black cohosh (cimicifugaracemosa). Expert opinion, vol. 3, no. 6, p. 615-623. 5. P.W. Whiting. 2002. Black cohosh and other herbal remedies associated with acute hepatitis. MJA, vol. 177, p. 432-435. 6. D. Joy. 2008. Black cohosh:A cause of abnormal post-menopausalliver function tests. Climacteric, vol.11, p.84-88. 7. R. Teschke. 2008. Suspected hepatotoxicity by cimicifugaracemosa rhizome (black cohosh root): Critical analysis and structured causality assessment.Phytomedicine, vol.16, p.72-84.
  6. 6. 8. E.T. Enbom. 2014. Mechanismof hepatotoxicity due to black cohosh (cimicifugaracemosa): Histological, immunohistochemical and electron microscopy analysis of 2 liver biopsies with clinical correlation. Experimental and molecular pathology,vol.96, p.279-283. 9. L.S. Einbond. 2008. Growth inhibitory activity of extracts and compounds from cimicifugaracemosa on human breast cancer cells. Phytomedicine, vol.15, p.504-511. 10. H. Tamaki. 2009. Inhibitory effects of herbal extracts on breast cancer resistance protein (BCRP) and structure-inhibitory potency relationship of isoflavonoids. Drug metabolism pharmacokinetics, vol.25, no.2, p.170-179. 11. J.E. Burdette. 2002. Black cohosh protects against menadione-induced DNA damage through scavenging of reactive oxygen species:Bioassay-directed isolation and characterization of active principles. Journal of agricultural and food chemistry, vol.50, p.7022-7028. 12. L.S. Einbond. 2004. Growth inhibitory activity of extracts and purifies components of black cohosh on human breast cancer cells. Breast cancer research and treatment, vol.83, p.221-231. 13. S. Rice. 2006. Ethanolic extracts of black cohosh inhibit growth and estradiol synthesis from estrone sulphate in breast cancer cells. Maturitas, vol.56, p.359-367. 14. R.L. Ruhlen. 2008. Black cohosh:Insights into its mechanism(s) of action. Integrative medicine insights,vol.3, p.21-32. 15. F.Gaube. 2007. Gene expression profiling reveals effects of cimicifugaracemosa (black cohosh)on the estrogen receptorpositive breast cancer cell line MCF-7. BMC pharmacology, vol.7, no.11, p.1-19. 16. K. Hostanska. 2007. Inhibitory effect if an isopropanolic extract of black cohosh on the invasiveness ofMDA-MB-231 human breast cancer cells. In vivo, vol.21, p.349-356. 17. L.S. Einbond. 2007. Gene expression analysis of the mechanisms whereby black cohosh inhibits human breast cancer cell growth. Anti-cancer research, vol.27, p.697-712. 18. S.N. Kovalchuk. 2006. Estrogenic activity of triterpene glycosides in yeast two-hybrid assay.The journal of steroid biochemistry and molecular biology, vol.101, p.226-231. 19. Y. Huang. 2010. Fukinolic acid derivatives and triterpene glycosides from black cohosh inhibit CYP isoenzymes, but not cytotoxic to Hep-G2. Curr drug saf, vol.5, no.2, p.118- 124. 20. L.S. Einbond, 2008. Actein inhibits the Na-K-ATPase and enhances the growth inhibitory effect of digitoxin on human breast cancer cells. Biochemical and biophysical research communications, vol.375, p.608-613. 21. L.S. Einbond. 2007. The growth inhibitory effect of actein on human breast cancer cells is associated with activation of stress response pathways.Internationaljournal of cancer, vol.121, p.2073-2083. 22. S. Rockwell. 2005. Alteration of the effects of cancer therapy agents on breast cancer cells by the herbal medicine black cohosh.Breast cancer research and treatment, vol.90, p.233-239. 23. L. Sun. 2007. Cimicifoetisides A and B, 2 cytotoxic cycloartanetriterpenoid glycosides from the rhizomes of cimicifugafoetida, inhibit proliferation of cancer cells. Beilstein journal of organic chemistry, vol.3 no.3, p.1-6. 24. K. Hostanska.2004. Cimicifugaracemosa extract inhibits proliferation of estrogen receptor-positive and negative human breast cancer cell lines by induction of apoptosis. Breast cancer research and treatment, vol.84, p.151-160. 25. Y. Qian. 2010. Synthesis,molecular modelling and biological evaluation of cinnamic acid metronidazole esterderivatives as novel anti-cancer agents.Bio-organic and medicinal chemistry, vol.18, p.4991-4996. 26. K. Kristan. 2006. Cinnamic and cinnamamids inhibit fungal 17-beta-hydroxysteroid dehydrogenase.Molecular and cellular endocrinology,vol.243, p.239-241. 27. J.M. Day. 2008. Design and validation of specific inhibitors of 17-beta-hydroxysteroid dehydrogenases fortherapeutic application in breast and prostate cancer, and in endometriosis. Endocrine-related cancer, vol.15, p.665-692. 28. M. Cardenas. 2006. Anti-tumour activity of some natural flavonoids and synthetic derivatives on various human and murine cancer cell lines. Bio-organic and medicinal chemistry, vol.14, p.2966-2971. 29. S. Gobec. 2004. Cinnamic acid esters as potent inhibitors of fungal 17-beta- hydroxysteroid dehydrogenase-a model enzyme of the short-chain dehydrogenase/ reductase superfamily. Bio-organic and medicinal chemistry, vol.14, p.3933-3936.