Black cohosh research

Black cohosh: A potential remedy for breast cancer

Black cohosh- background:

Black cohosh (Cimicifugaracemosa) is an herbaceous perennial plant. It’s a coarse woodland herb with large compound leaves and a thick, knotted rhizome system (F. Firenzuoli, 2007). Native to North America, the root and rhizomes of black cohosh were used by Native American women throughout their life for menstrual cramps, difficult childbirth and complicated menopause, as well as other conditions such as dysmenorrhea, colic and rheumatism. Native Americans subsequently introduced the herb to the American colonists, who used it for women’s complaints, as well as illness such as bronchitis, nervous disorders, inflammation and uterine disorders (J.L. Mayo, 1998).

Today, black cohosh is widely used in various pharmaceutical industrial preparations, often mixed with other medicinal plants (F. Firenzuoli, 2007). It is widely used in the U.S. and Europe to help alleviate menopausal symptoms, such as hot flashes, sweats, irritability and vaginal dryness.

The action of black cohosh is attributed to the synergy of the entire profile of is active components (J.L. Mayo, 1998). The primary active constituent of the herb’s root is believed to be the triterpene glycoside fraction, including actein and cimicifungoside (B. Klinger, 2003). Other potentially biologically active substances of the rhizome include isoflavone (phytoestrogen) formononetin, the triterpene glycosides 27- deoxyactein and racemoside, as well as the aromatic acids ferulic acids and isoferulic acid. In addition, cimicifungoside appears to affect the hypothalamus- pituitary axis, resulting in reproductive and nervous system effects, while the aromatic acids are believed to be anti-

inflammatory (J.L. Mayo, 1998). Other components of the herb are cinnamic acid esters, sugars and long- chain fatty acids (L.S. Einbond, 2004).

Breast cancer mechanisms- background:

The growth promoting effects on breast cancer cells are attributed to estrogenic actions on estrogen receptor (ER) alpha. Most breast cancers express estrogen receptors. In these cases, estrogen is required for continued growth, and in post- menopausal women the major source of estradiol is through local conversion of circulating androgens and estrone sulphate. Breast tissue can convert circulating androgens through aromatase and 17- beta- hydroxysteroid dehydrogenases and estrone sulphate to estradiol through steroid sulphatase and 17- beta- HSD. All these enzymes are highly expressed in breast cancer tissue, such that the concentration of estradiol in breast tumours is about 20- fold higher than in the circulation. The reductive activity of 17- beta- HSD1 that drives estrone to estradiol is dominant in breast cancers, while oxidative 17- beta- HSD2 activity that catalyses the reverse reaction predominates in normal breast tissue. Steroid sulphatase mRNA expression is correlated with breast cancer, and it has been proposed that the sulphatase pathway is more important than aromatase (S. Rice, 2007). Therefore, 17- beta- HSDs act as molecular switches. Patients with tumours with high 17- beta- HSD1 expression have significantly shortened survival rates (J.M. Day, 2008).

In addition, the growth factor receptor kinase epidermal growth factor receptor (EGFR) plays important roles in the development, progression, aggressiveness and metastasis of many tumours. EGFR (or Her1) and the related human epidermal growth factor receptor Her2

have been proved to be relevant for cancer too (Y. Qian, 2010).

Another factor affecting breast cancer potential are free radicals and other reactive oxygen species (ROS) that are constantly generated and cause damage to biomolecules. This process is regulated by existence of antioxidants, DNA repair systems and replacement of damaged lipids and proteins. Scavenging of ROS can protect against cancer. DNA is a significant target of oxidative stress, because continuous oxidative damage contributes to development of cancer. This may be due to effects on cellular proliferation, prevention of apoptosis, damage to DNA repair enzymes and damage to DNA polymerases that can decrease fidelity of replication. Accordingly, if direct damage to DNA bases caused by ROS contributes to development of cancer, agents that reduce such damage should decrease the risk of cancer development (J.E. Burdette, 2002).

Another relevant factor is apoptosis, which is an essential regulatory mechanism. An imbalance between mitosis and apoptosis has pathologic implications and has been associated with many autoimmune disorders, tumours and viral infections. Caspases are the central executioners of apoptosis, and one of their actions is the cleavage of the intermediate filament cytokeratin 18 in epithelial cells. There are increasing levels of caspases during apoptotic cell deaths (K. Hostanska, 2004).

Tumour cell invasion is a complex process, which is characterised by alterations in cellular attachment, proteolytic and migratory activities. The invasive potential of cancer cells is linked to their capacity to degrade basement membrane and extracellular matrix to create a path for migration. Metastasis is a complex multi- step process, involving cell adhesion, invasion

and motility. Hence, interruption of one or more of these steps is one approach of anti- metastatic therapy. Migration and invasion are key functional activities in the progression of early stage breast cancer into a more aggressive state. Cellular growth independent from basement membrane, as a prerequisite for migration and invasion is one of the hallmarks of the metastatic phenotype. An invasiveness of breast cancer cells is frequently associated with absence of estrogen receptors (K. Hostanska, 2007).

Black cohosh anti- breast cancer activity:

Extracts of black cohosh rhizomes have been recognised as a rational choice for the treatment and prevention of breast cancer. Ethanolic and isopropanolic extracts of black cohosh rhizomes inhibited the growth of both estrogen- dependent MCF-7 and estrogen- independent MDA-MB-231 human breast cancer cells (K. Hostanska, 2007).

According to S. Rockwell and co, there is considerable debate about whether black cohosh has estrogenic or anti-estrogenic activities, since conflicting findings were found. However, according to another research, C. racemosa shows weak binding activity to estrogen receptor, and shows no estrogenic activity in MCF-7 cells, and no gene expression in estrogen-inducible cells, but antagonises these activities. Black cohosh extract significantly inhibits estrogen-induced proliferation of MCF-7 cells adapted to estrogen-free medium. In addition, the effect of E2 on proliferation and gene expression in estrogen-inducible cells is antagonised by whole extract of black cohosh. ER- cells are 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-mediated) and non-ER-mediated mechanisms, because of the various physic-chemical properties of individual components of black cohosh. The spontaneous apoptotic rate of MCF-7 cells in comparison to MDA-MB-231 cells was higher. This suggests that black cohosh cytotoxicity appears to be explained in part by induction of apoptosis. MCF-7 cells, despite of their caspase-3 deficiency, show a high responsiveness to black cohosh treatment. Therefore, the apoptotic action of black cohosh must be mediated through mechanisms other than its weak binding to ER (K. Hostanska, 2004).

treatment

dose (micrograms/ ml) survival (%) mcf-7 ic50 (micrograms/ml)

control 0 100 +/- 3.8

icr 38.7 97.5 +/- 2.3 128.5 +/- 4.977.4 95.2 +/- 3.4

154.8 89 +/- 4.2ttg 0.1 96.9 +/- 1.9

1 93.5 +/- 1.5 >1005 86.7 +/- 2.7

cae 0.1 94.3 +/- 1.9

1 90.9 +/- 2.4 ~25^a5 83.3 +/- 3.7

treatment

dose (micrograms/ ml)

control0 survival (%) mda-mb231 ic50 (micrograms/ml)

icr38.7 100 +/- 6.5

77.4 104.3 +/- 1.2 92.5 +/- 3.5

154.8 89 +/- 2.7

ttg0.1 90.9 +/- 4.1

1 103.9 +/- 0.8

5 102.6 +/- 1.1 96.3 +/- 5.3

cae0.1 102.5 +/- 2.6

1 104.6 +/- 1.4

5 103.1 +/- 2.3 26.2 +/- 1.4

96.7 +/- 3.9

Table 1: Effects of treatments by black cohosh extract, TTGs and CAEs on proliferation of MCF-7 and MDA-MB-231, reflected through the IC50 values obtained (L.S. Einbond, 2004).

Table 2: 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.

In addition, another research claims that extracts of black cohosh don’t bind to ER-alpha or beta (S. Rice, 2007). MDA-MB-231 cells showed a higher sensitivity to the cytotoxic effects of black cohosh than MCF-7 cells. However, there were no differences in sensitivity of both cell types to either triterpene glycosides or cinnamic acid esters, according to IC50 values. Therefore, synergistic action between the different plant compounds is likely (K. Hostanska, 2007). In both MCF-7 and MDA cells, black cohosh 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 (S. Rice, 2007).

According to L.S. Einbond and co, the proliferation of ER+ and ER- human breast cancer cells was inhibited via induction of apoptosis through activation of caspases. Further, fractions of black cohosh enriched in triterpene glycosides or cinnamic acid esters, inhibited cell growth and induced apoptosis. An ethanolic extract inhibited the activity of the cyclin D1 promoter and increased the activity of the P21cipi1 promoter in the 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 microgram/ml, and the G2/M after treatment with 60 microgram/ml. This suggests that the fraction contains a mixture of components with the more active or abundant component causing G1 arrest, and the less active causing G2/M arrest, or individual components in

the same fraction exert different effects at different concentrations. Therefore, it is possible that at high concentrations the fraction affects proteins that regulate later phases in the cell cycle (L.S. Einbond, 2007).

Table 3: The effects of black cohosh extract and isolated actein on proliferation of MCF-7 breast cancer cells (F.Gaube, 2007).

Apoptosis, the essential regulatory mechanism of programmed cell death, is responsible for the observed inhibition of the proliferation of breast cancer cells by isopropanolic extract (iCR). Apoptosis induced by iCR involved cleavage of cytokeratin 18 and caspase activation. However, in MCF-7 cells, black cohosh extract also induced cell cycle arrest at G1 and G2/M, which proposes as an additional pathway. Cinnamic acid esters were the more potent inhibitor of proliferation and apoptosis inducer in MCF-7 cells (K. Hostanska, 2007).

Black cohosh has an anti-proliferative effect, therefore genes involved in proliferation control are significantly over-represented. 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, appear to be down regulated, such as cyclins (CCNA2, CCNE2, CCNF), cyclin-dependent kinase 2 (cdk2) and transcription regulators (E2F2, PCNA, SKP2), whereas transcription of inhibitory genes cyclin G2 (CCNG2), GADD45A (growt arrest and DNA-damage-inducible, alpha) and P21cip1 was increased. Elevated levels of CCNG2, CCNB1TP1, FOXO3A, GADD45A and P21cip1 genes, as well as down-regulation of cyclin A2 (CCNA2) and cdk2 provided evidence that cell cycle progression might be additionally arrested at the G2/M-

checkpoint. The level of various DNA replication related genes (CDC6, CDT1, FEN1, MCM2, MCM3, MCM4, pfs2, RFC3) was also reduced, thereby suggesting a reduction in the replication rate. In addition to regulation of genes involved in proliferation control, there is also regulation of apoptosis-linked genes in a pro-apoptotic manner. An increase in apoptosis events also contributes to decrease in cellular proliferation. In cells treated with black cohosh, the transcript of apoptosis inhibitor surviving was down-regulated, whereas genes coding for apoptosis-inducing and supporting products were increased. FOXO3A, GADD45A, GDF15 and P21cip1, whose mRNA levels increased, are also connected with apoptosis in addition to their role in cell cycle control. Transcript of tyrosyl-tRNA-synthetase (YARS), whose secretion is liked to apoptotic events, was up-regulated. Down-regulation of lymphoid-specific helicase (HELLS), is associated with apoptosis. The up-regulation of JNK1 and DDIT3 is related to stress induced apoptosis. P8, IER3 and DDIT4, whose transcript is strongly up-regulated, are expressed under cellular stress, and are associated with both pro and anti-apoptotic events. Over-expression of transcripts involved with cellular stress is statistically significant after treatment with black cohosh. 40 transcripts associated with metabolic stress response, such as hypoxia, unfolded protein response in the endoplasmic reticulum or starvation for amino acids or glucose, have been identified. Transcript of HIF1-alpha/ARNT (HIF1) binds to hypoxia-responsive elements, thereby regulating the expression of hypoxia-response genes. VEGF, HMOX1, BHLHB2, P21cip1 and DDIT4, whose transcript was also up-regulated, are known to be direct target genes. A hypoxia response pathway via mTOR including inactivation of EIF4EBP1, and finally resulting in increased mRNA translation is

known to be inhibited by DDIT4. This could explain the increase of EIF4EBP1 mRNA observed. The increase of CCAAT, EPAS1, EGR1 and SESN2 mRNA are also related to hypoxia. There is also regulation of genes related to endoplasmic reticulum stress response (UPR), which involves activation of 3 different pathways: transcription of JNK1 was regulated, which is a target of one UPR-pathway and its activation may lead to apoptosis. Phosphorylation of eIF2-alpha is involved not only in UPR, but also in response to hypoxia and other cellular stresses. PERK, whose mRof the mRNA-level was increased by black cohosh, is a kinase linking hypoxia stress response and UPR to eIF2-alpha-phosphorylation, whereas amino acid and glucose starvation response acts via GCN2 kinase. As a result of eIF2-alpha-phosphorylation, the translation of most mRNAs is inhibited, but the translation of ATF4 is increased. There is an up-regulation of the ATF4 gene, as well as various ATF4-induced downstream target genes, like ASNS, ATF3, CHOP, GADD45A, HERPUD1 and HSPA5. Gene products of these transcripts are involved in cell survival and tumour-genesis, as well as apoptotic events. Some processes of protein turnover are affected by black cohosh extract. The expression levels of various ubiquitin cycle-related genes were influenced by black cohosh. Some of these transcripts code for products involved with cell cycle progression, and are regulated in a cell cycle arresting manner. Inhibitory CCNB1IP1 is up regulated, while SKP2 and UHRF1 are down regulated (F. Gaube, 2007).

Table 4: 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 (K. Hostanska, 2007).

Motility is another property of malignant cells needed for them to migrate from the primary site to a secondary organ. Any alteration of this property would interrupt the metastatic cascade. Also, iCR treated cells had a moderately reduced motility. Low doses of triterpene glycosides and cinnamic acid esters didn’t affect cell migration. The mechanisms by which iCR (triterpene glycosides and cinnamic acid esters) inhibit cell invasion is not clear and needs further investigation. Cyclo-oxygenase (cox)-2 enzyme plays a role in the metastatic process of cancer. Hence, the inhibition mechanism of iCR could be cox-2 related (K. Hostanska, 2007).

Triterpene glycosides and breast cancer:

Figure 1: Structure of actein (L.S. Einbond, 2008).

The growth inhibition activity of black cohosh extracts appears to be related, in part, to their triterpene glycoside composition (L.S. Einbond, 2008). Triterpene glycosides are well known as a group of secondary metabolites, typically found in plants. They possess a wide spectrum of biological activities, including

cytotoxic, haemolytic, anti-fungal and antibacterial properties. This results from their ability to form a complex with unsaturated sterols of cellular membranes. The physiological effects of glycosides are probably related to the certain structural conformity between the glycosides and steroidal hormones like glucocorticoids and their formation of a complex with steroidal receptors (S.N. Kovalchuk, 2006). The most potent cimicifuga component tested is actein, which has an acetyl group at position c-25. Thus, the acetyl group at this position enhances growth inhibitory activity. MCF-7 cells transfected for Her2 are more sensitive than the parental MCF-7 cells to growth inhibitory effects of actein, indicating that that Her2 plays a role in the action of actein. Treatment with actein alters the distribution of actin filaments and induces apoptosis in these cells. Treatment with actein induces a stress response in human breast cancer cells, and the growth inhibitory effect of black cohosh on these cells is thought to be mainly due to the triterpene glycoside fraction, rather than the isoferulic (cinnamic acid ester) content. Since the colonies were smaller on average in the actein-treated cells, actein appears to decrease the rate of cell proliferation. Therefore, actein appears to have cytostatic as well as cytotoxic activity. MDA-MB-453 cells, which are ER- and Her2+, were the most sensitive of the cells to actein. Treatment of MCF-7 or MDA-MB-453 cells with actein altered their cell structure, since the actin filaments around the cell nuclei and the nuclei itself appeared doughnut-shaped. Aggregation of actin around the nucleus occurs in response to cell stress (L.S. Einbond, 2008). Actein induces cell cycle arrest at G1. Cyclin D1 plays a critical role in mediating the transition from G1 to S and is over-expressed in 50-60% of human breast carcinomas, and also over-expressed in several human breast

cancer cell lines. Treatment of MCF-7 cells with 40 microgram/ml of actein for 3 or 10 hours resulted in a partial decrease, and treatment for 24 hours caused a marked decrease in cellular levels of cyclin D1, when compared to untreated cells. After treatment with 40 microgram/ml for 24 hours, there was a complete loss of the protein. Normal mammary epithelial cells do not express cyclin D1. Cyclin D1 binds to and activates the cyclin-dependent kinases cdk4 and cdk6. The resulting complexes phosphorylate and inactivate pRb, thereby preventing pRb from inhibiting the transcription factor E2F, thus allowing the cells to progress from G1 to S. The ability of actein to arrest cells in G1 may be due to the decreased expression of cyclin D1 and cdk4 and the increased expression of P21cip1, which results in a decrease in the level of the hyper phosphorylated form of pRb. The level of epidermal growth factor (EGFR), which is over-expressed in various cancers, was not significantly affected by actein treatment. Nor was there a consistent effect of actein on the phosphorylated and activated form of the EGFR (p-EGFR), but there was a significant decrease with the 40 micrograms/ml dose at 24 hours. The EtoAc fraction exhibited the greatest growth inhibitory activity. This fraction inhibited growth of both the ER+ MCF-7 and ER-/Her2+ MDA-MB-453 human breast cancer cell lines, with IC50 values of 18 micrograms/ml and 10 micrograms/ml, respectively. Actein turned out to be the most potent compound. It decreased the level of cyclin D1 mRNA within 3 hours of treatment, and significantly reduced the level at 24 hour, suggesting an effect at the level of transcription. The level of the EGFR was not altered after treatment with actein, nor was there a consistent effect on the level of the phosphorylated form of EGFR (p-EGFR), which reflected its state of activation. Thus, the EGFR does not appear

to be a direct target for actein (L.S. Einbond, 2004). There are several potential mediators of the growth inhibitory effect of actein, including increased expression of ATF3 and DDIT3, and decreased expression of the cell cycle related genes cyclin E2 and cell division cycle 25A. The increased expression of the transcription factor ATF3 may be of particular interest, since it can repress the transcription of several survival genes. The number of genes affected increases with the dose of actein and the duration of exposure. Actein alters the expression of 5 genes: DDIT4, WIPI49, HERPUD1, SLC7A11 and LETMD1, which are involved in cellular responses to diverse stresses, including DNA damage. The effect of actein on expression of genes related to the integrated stress response are not limited to the MDA-MB-453 cell line, since treatment of the ER+ MCF-7 cell line with actein also induced increased expression of genes involved in calcium metabolism and the Na-K-ATPase effects calcium metabolism. Hence, the ability of actein to inhibit the activity of the Na-K-ATPase and activate related down-stream pathways is possible. Na-K-ATPase mediates many stress responses and proliferation pathways that are affected by actein. Therefore, actein subsequently phosphorylates downstream proteins. Inhibition of the enzyme has been shown to be related to a compound’s ability to interact with the enzyme’s lipid-rich environment. 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. GRP78, which is expressed on the cell surface and is involved in endocytosis of Na-K-ATPase in cells, is also activated by actein and may therefore be instrumental in actein-mediated ATPase inhibition (L.S. Einbond, 2008).

Other triterpene glycosides potentially affecting proliferation of breast cancer cells are 23-epi-deoxyactein, cimifungoside and cimiracemoside A, that inhibit MCF-7 cell proliferation, and cimiracemoside G which is cytotoxic to human oral squamous cell carcinoma, but not to normal human fibroblasts (R.L. Ruhlen, 2008). Also, cimiracemoside A and B both reduce in dose-dependent manner the single-stranded DNA breaks induced by menandione in breast cancer cells (J.E. Burdette, 2002).

Cinnamic acid esters and breast cancer:

Figure 2: Basic structure of a cinnamic acid ester (L.S. Einbond, 2008).

Some cinnamic acid derivatives are also potential inhibitors of EGFR and Her2. The IC50 value for inhibition of Her2 kinase is higher than that observed for EGFR kinase. However, they have the same trends of inhibitory effect. It is evident that there is also a reasonable correlation between the EGFR and Her2 inhibitory activities. Thus, this isn’t surprising considering the high sequence homology of the catalytic domains of these 2 kinases (Y. Qian, 2010). A study of the cytotoxic activity of cinnamic acid derivatives also showed that they have an anti-

proliferative activity without toxicity towards non-cancer cells, in general (M. Cardenas, 2006). Methyl caffeate, caffeic acid, ferulic acid, fukinolic acid, cimicifugic acid A and cimiracemate B are all cinnamic acid derivatives that contain a phenol group para to the conjugated ethylene side chain. It is thought that the hydrogen on the phenol can be abstracted easily by a radical, resulting in formation of phenoxy radical. The unpaired electron can delocalize across the entire molecule, hence stability. Among the compounds found to have antioxidant activity, those with the greatest number of phenolic groups tended to be the most effective free radical scavengers. The presence of a second phenolic group allows formation of a catechol, which is important for antioxidant behaviour. Methyl caffeate has the highest potency in protecting DNA against single-strand cleavage with breast cancer cells. The conversion of the acid group to the methoxy group decreases the polarity and might facilitate the transport of this compound across the cell membrane, where it can scavenge free radicals. Ferulic acid and caffeic acid also have a dose-dependent reduction in DNA damage. The caffeic and ferulic acids protect DNA by reducing reactive oxygen species (ROS) (J.E. Burdette, 2002).

Phytoestrogens and breast cancer:

Black cohosh also contains phytoestrogens (flavonoids) that were also shown to inhibit growth of breast cancer cells through breast cancer resistance protein (BCRP) in some types of cancer cells. They can also inhibit both aromatase and 17-beta-HSD1, although their IC50s are generally low (S. Rice, 2007). 17-beta-HSD inhibitors consisting of a non-steroid core and devoid of a residual steroidogenic activity, like phytoestrogens, have potential for inhibiting cancer proliferation. The

biosynthesis of phytoestrogens proceeds via cinnamic acid or related phenolic acids, like caffeic or ferulic acid that are also present in black cohosh. Many flavonoids have been shown to be potent inhibitors of type 1, 3 and 5 17-beta-HSD. Flavonoids can inhibit both the oxidative and reductive reactions catalysed by 17-beta-HSD at low micro-molar concentrations (K. Kristan, 2006). Flavonoids with a hydroxyl group in position 7 of the A-ring, which mimics the D-ring of steroids, inhibit 17-beta-HSD1. Unfortunately, they are not useful as therapeutic inhibitors, as they are often estrogenic, or aren’t specific for 17-beta-HSD1, having inhibitory effects on other steroidal enzymes and receptors (J.M. Day, 2003). Some flavonoids also produce moderately active compounds in MCF-7 cell lines, but only ones containing a chlorine or bromine atom (M. Cardenas, 2006).

Black cohosh and hepatotoxicity:

Recently, the European medicines agency has proposed a causal relationship for acute liver disease, observed under treatment with black cohosh (R. Teschke, 2008). To identify herbal remedy as being responsible for hepatotoxicity, there should be a clear demonstration of a temporal relationship between consumption of the product and development of the illness (D. Joy, 2008). According to one research, this was not the case for black cohosh in general (R. Teschke, 2008). Another research by F. Firenzuoli and co also claims that black cohosh has a very good safety profile, as confirmed in a review of more than 3800 women. However, according to that same research, there were also 42 cases of suspected hepatotoxic reactions in patients consuming the herb, which gives it a potential connection with hepatotoxicity. Also, according to a research by D. Joy

and co, there have been several case reports of hepatotoxic effects of varying severity associated with black cohosh. 2 reviews have found it to be well tolerated when taken up to 6 months. According to E.T. Enbom and co, the safety of black cohosh has been evaluated during several clinical trials, resulting in the conclusion the adverse effects secondary to black cohosh consumption are relatively common, including liver damage. The extent of hepatotoxic effects of black cohosh extract varies and depends on gender, age, duration of treatment and underlying liver pathology (E.T. Enbom, 2014).

Also, genetic polymorphisms have a strong influence on drug metabolism and may increase the risk of hepatotoxicity (F. Firenzuoli, 2007). It is also important to remember that since the liver is central to metabolic disposition of all drugs, drug-induced hepatic injury is a potential complication of nearly every medication. However, the mechanisms of liver injury by black cohosh remain unclear.

Black cohosh contains a mixture of alkaloids, tannins and terpenoids, and diterpenoids that have been shown in animal models to result in liver injury, either by reactive metabolites, or by an autoimmune mechanism (P.W. Whiting, 2002). The mechanism of hepatotoxicity due to black cohosh is probably idiosyncratic, and has identical presentation to troxis necrosis that occurs during autoimmune hepatitis (F. Firenzuoli, 2007). Another study demonstrated formation of immunologic synapses between hepatocytes and lymphocytes, and by accumulation of lipid peroxidation products. Clinical data showed that discontinuation of black cohosh ingestion has stopped this process and led to recovery (E.T. Enbom, 2014). Also, it has been hypothesizes that the hepatotoxic effect could be due to inhibition of

CYP3A4, a cytochrome p450 enzyme responsible for metabolism of several drugs in the liver. This could have particular implications regarding drug efficacy and toxicity. Serious adverse interactions may result from ingestion of drugs that are CYP3A4 substrates (D.Joy, 2008).

However, natural and synthetic estrogens can alter liver physiology, and it has been reported that black cohosh extracts and isolated compounds do not possess estrogenic activity. But black cohosh also contains several catechols, like caffeic acid, fukinolic acid and cimicifugic acid A and B. That could be of significant concern in toxicology, because they could be activated metabolically or chemically, to electrophilic quinones. This could lead to formation of reactive oxygen species. However, black cohosh did not show any effect on lipids, glucose, insulin and fibrinogen (F. Firenzuoli, 2007).

Factors that could contribute to toxicity also include mistaken identity of the plant, wrong part of the plant being used, incorrect storage, contamination during preparation, pesticides and heavy metals, and inconsistency in nomenclature and labelling of the final product (D. Joy, 2008).

Discussion:

First of all, it is important to mention that there wasn’t enough consistent data regarding the IC50 values of each component of black cohosh, or the whole herb extract, to draw statistical analysis through 2- way ANOVA or X^2 test from a few sets of information collected from different researches, neither was there enough data regarding, for instance, the number of

genes expressed while treatment with different concentrations of black cohosh components, or quantitative sets of data regarding the same type of cell line (for each line). So, for this reason, ANOVA or X^2 could not be performed as desired, since the sets of data obtained from all the articles analysed were too few and different from one another.

From the aspect of the function of black cohosh extract in general, this herb seems to have anti-carcinogenic properties, since both ethanolic and isopropanolic extracts of black cohosh rhizomes inhibit growth of both estrogen-dependent MCF-7 and various estrogen-independent MDA-MB human breast cancer cell lines. There is also evidence that MDA-MB-231 showed a higher sensitivity to the cytotoxic effects of black cohosh than MCF-7 cells. However, there were no differences in sensitivity of both cell types to either triterpene glycosides or cinnamic acid esters, according to IC50 values.

It also seems most likely that the activity of black cohosh on breast cancer cells is largely non-estrogenic, except for a weak binding of flavonoid phytoestrogens to estrogen receptors. This assumption is supported by the fact that black cohosh showed no estrogenic activity in MCF-7 cells, or on gene expression in those cells, but rather antagonising activity. Also, in both MCF-7 and MDA cells, black cohosh 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.

It is far more likely that black cohosh, especially its actein part of the triterpene glycoside fraction, causes cytotoxic effects on breast cancer cells through mediated apoptosis induced through the Her2 receptor, and also the Na-K-ATPase membrane-bound enzyme.

Activity through binding to Her2 is likely, since the Her2+ MDA-MB cell lines were far more sensitive to the cytotoxic effect than their parental MCF-7 cells to growth inhibitory effects of actein, indicating that Her2 plays a role in the action of actein.

Actein seems to act through activation of Na-K-ATPase, since actein alters genes involved in calcium metabolism and the Na-K-ATPase affects calcium metabolism. Therefore, the ability of actein to inhibit Na-K-ATPase activity and activate related down-stream pathways is possible. This makes sense, also because actein alters the distribution of actin filaments and induces apoptosis through that mechanism, and calcium is a known factor in the function of actin as a cell-skeleton compound. Inhibition of Na-K-ATPase has been also shown to be related to a compound’s ability to interact with the enzyme’s lipid-rich environment, and actein, as a tritepene glycoside, does possess some steroid-like lipid conformation. Actein’s inhibition of ATPase is relatively weak, so a promoter-dependent mode of action is suggested. Actein may act through interaction with the Na-K-ATPase promoters in the cell membrane and induction of ATPase clustering with neighbouring proteins in micro-domains. Also, GRP78, which is expressed on the cell surface and is involved in endocytosis of ATPase in cells, is also activated by actein and may therefore be instrumental in actein-mediated ATPase inhibition.

However, the level of epidermal growth factor (EGFR, or Her1), which is over-expressed in various cancers, was not significantly affected by actein treatment. Nor was there a consistent effect of actein on the phosphorylated and activated form of EGFR (p-EGFR), except for a significant decrease with the 40

micrograms/ml dose for 24 hours. Therefore, the EGFR does not appear to be a direct target for actein.

It appears that the main cytotoxic mechanism through which the breast cancer cells’ proliferation decrease is apoptosis-related, via activation of caspases. Fractions of black cohosh enriched in triterpene glycosides or cinnamic acid esters inhibited cell growth and induced apoptosis. An ethanolic extract inhibite the activity of the cyclin promoter and increased the activity of the P21cip1 promoter in the ER- human breast cancer cells. Ethyl acetate fraction of black cohosh inhibited growth of MCF-7 cells and induced cell cycle arrest at G1 after treatment with 30 micrograms/ml, and 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, I tis possible that at high concentrations the fraction affects proteins that regulate later cell cycle phases. I tis possible, according to this data that the triterpene glycoside fraction works as a limiting factor, hence over a certain concentration there is no added advantage of the concentration increase, but under a certain concentration- the other fraction, cinnamic acid esters, becomes more influential and potent (especially if the receptors for triterpene glycosides are limited in number). Therefore, cinnamic acid esters also seemed to be more potent in MCF-7 cell cytotoxicity, which could also indicate an additional pathway for cinnamic acid esters function.

As expected, genes involved in proliferation control are significantly over-represented in cells treated with black cohosh. Transcripts related to cell cycle regulation and DNA replication are regulated in a manner supporting cell cycle arrest: genes whose products are involved in

transition from G1 to S-phase are down-regulated (cyclins, cdk2, transcription regulators), while transcription of inhibitory genes (cyclin G2, GADD45A and P21cpi1) are increased. The level of DNA replication genes was also reduced, hence the probability of regulation of pro-apoptotic genes (like surviving or tRNA synthetase).

Unlike triterpene glycosides, some cinnamic acid esters do seem to have inhibitory effect on EGFR, in addition to Her2, and they have a high and selective anti-proliferative activity against cancer cells, due to this fact. This is probably the alternative anti-cancer pathway demonstrated against MCF-7 cell proliferation. Cinnamic acid esters have anti-oxidative properties against formation of carcinoma also by conversion of the acid group to methoxy group. This protects DNA against single-strand cleavage by decreasing the polarity and might initiate the transport of the compound across the cell membrane, where it can scavenge free radicals. Methyl caffeate was found to have the highest potency via this pathway, although also ferulic acid and caffeic acid have a dose-dependent reduction in DNA damage, by protecting DNA through reduction of reactive oxygen species.

According to a few researches, synergistic action between the different compounds of black cohosh is likely. This makes sense, since the herb has been shown to inhibit breast cancer cell proliferation through more than one pathway. It is probably likely that by binding to the Na-K-ATPase enzyme, actein makes the cell membrane more penetrable through alteration of its structure (due to its steroid-like sterol composition), which allows more access for the cinnamic acid esters. After entering the cell, the cinnamic acid esters can perform their activity against reactive oxygen species,

and prevent DNA damage by free radicals. Entering the cell also enables the cinnamic acid esters to inhibit EGFRs, an attribute that actein and other triterpene glycosides lack. While triterpene glycosides are lipophilic, cinnamic acid esters seem to be hydrophilic, so are therefore more dependent on triterpene glycosides as agents for cell penetration. By binding to ATPase and Her2, actein also has anti-cancer activities in its own right, mainly of apoptosis induction and also cytostatic activity, but also increases the effectiveness of cinnamic acid esters and their anti-oxidative activities.

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