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 cohosh were 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 cohosh is widely used in various pharmaceutical preparations. It is widely used in the U.S. and Europe against menopausal symptoms, such as hot flashes, sweats, irritability and vaginal dryness.

The action of black cohosh is attributed to synergy of its active components. The main active compounds of the 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 cohosh compounds 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:

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. The articles for the research were obtained through PubMed and Google Scholar.

Results:

Ethanolic and isopropanolic extracts of black cohosh rhizomes inhibited the growth of both estrogen-dependent MCF-7 and estrogen-independent MDA-MB human breast cancer cells.

Black cohosh also 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-dependent anti-proliferative action of black cohosh in 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’t bind 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 cohosh was 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 cohosh inhibited 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 cohosh has 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

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 ATPase with 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 cohosh with anti-oxidant behaviour, methyl caffeate has the highest potency in protecting DNA against single-strand cleavage. Also, both caffeic and ferulic acids protect DNA by reducing reactive oxygen species.

Functional categories of genes regulated in MCF-7 cells after 24 h incubation with black cohosh extract. Genes were grouped in 5 large groups (Apoptosis, Proliferation, General Growth, Signaling & Transport, Metabolism), some consisting of subgroups. Genes that are not clearly associated with these groups are summarized in the category others. The category stress response contains genes also grouped into one of the 6 main classes. Each bar represents the number of genes that were up- (dark) or downregulated (white) in the respective group.

. Effect of iCR, TTG and CAE on tumor cell growth. MCF-7 and MDA-MB 231 cells were treated for 24 h with or without substances at the indicated concentrations. Attached cells were stained with crystal violet and the absorbance of the cell lysate was measured at 540 nm. Data are expressed as mean±SD of triplicate wells from two independent experiments. *p<0.05 vs. untreated control.

Discussion:

From the aspect of 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 cohosh than MCF-7 cells.

It also seems, for this reason, that the activity of black cohosh on 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 cohosh compounds 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.

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