Hormonal, targeted and chemotherapeutic strategies largely depend on the expression of their cognate receptors and are often accompanied by intolerable toxicities. Effective and less toxic therapies for estrogen receptor negative (ER-) breast cancers are urgently needed. Here, we present the potential molecular mechanisms mediating the selective pro-apoptotic effect induced by BN107 and its principle terpene, oleanolic acid (OA), on ER- breast cancer cells. A panel of breast cancer cell lines was examined and the most significant cytotoxic effect was observed in ER- breast lines. Apoptosis was the major cellular pathway mediating the cytotoxicity of BN107. We demonstrated that sensitivity to BN107 was correlated to the status of ERalpha. Specifically, the presence of functional ERalpha protected cells from BN107-induced apoptosis and absence of ERalpha increased the sensitivity. BN107, an extract rich in OA derivatives, caused rapid alterations in cholesterol homeostasis, presumably by depleting cholesterol in lipid rafts (LRs), which subsequently interfered with signaling mediated by LRs. We showed that BN107 or OA treatment in ER- breast cancer cells resulted in rapid and specific inhibition of LR-mediated survival signaling, namely mTORC1 and mTORC2 activities, by decreasing the levels of the mTOR/FRAP1, RAPTOR and RICTOR. Cotreatment with cholesterol abolished the proapoptotic effect and restored the disrupted mTOR activities. This is the first report demonstrating possible concomitant inhibition of both mTORC1 and mTORC2 activities by modulating the levels of protein constituents present in these signaling complexes, and thus provides a basis for future development of OA-based mTOR inhibitors.

Abstract Abstract #3128 While advances in early detection and adjuvant therapy for breast cancer have had a favorable impact on survival, patients who develop metastatic breast cancer generally succumb to death. Hormonal, targeted or chemotherapeutic strategies largely depend on the expression of their cognate receptors and are often accompanied by toxicities and intolerable side effects. Effective and less toxic therapies against the more aggressive and hormonal therapy-resistant estrogen receptor negative (ER-) breast cancer are urgently needed. Botanical medicine is one of the most popular complementary and alternative medical approaches, and Chinese herbal therapies are frequently sought and used by breast cancer patients. However, the molecular mechanisms through which certain herbal extracts exert anti-breast cancer activities remain largely unknown. Bionovo Inc. has a pipeline of anti-breast cancer products (BN#) based on herbal medicine in development. Here, we present preclinical data on the potential mechanisms of the pro-apoptotic effect of BN107 on breast cancer cells. A panel of breast cancer cell lines was examined and the most significant cytotoxic effect was observed in the less-differentiated, more aggressive, ER- breast cancer lines. Apoptosis appeared to be the major cellular pathway mediating the cytotoxicity of BN107 as evident from Annexin V binding, dissipation of mitochondrial potential, activation of caspases, and DNA fragmentation. Transcriptomic analysis comparing sensitive (ER+) versus resistant (ER-) cell lines revealed distinct patterns of gene expression in response to BN107. ER- breast cancer cells responded to BN107 by upregulating genes involved in apoptotic responses and cholesterol synthesis pathways; while ER+ breast cancer cells did so by regulating genes involved in cell growth and IGF-1 receptor signaling pathways. Further molecular analysis showed that BN107 induced death preferentially in ER- cells via rapid inactivation of AKT/ mTOR pathways. In addition, the sensitivity to BN107 was greatly reduced when ER expression was introduced in MDA-MB-231, an ER- cell line highly sensitive to BN107. BN107, an extract rich in triterpenoids, caused rapid alterations in cholesterol metabolism, presumably by interfering with cell membrane permeability/integrity. Co-administration of BN107 and cholesterol abolished the pro-apoptotic effect of BN107. We found that ER- breast cancer cells rich in caveolae/lipid rafts were highly sensitive to BN107. We showed that BN107 treatment in these cells resulted in subcellular redistribution of proteins that are associated with these specialized membrane/cellular structures. In conclusion, BN107 exerts pro-apoptotic activity preferentially on the more aggressive, ER- breast cancer cells. The cytotoxic activity of BN107 may be attributed to its ability to modify membrane permeability/integrity. Activity-guided isolation and further mechanistic studies are underway to identify potential chemical constituents responsible for the selective cytotoxic activities against ER- breast cancer cells. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 3128.

The aqueous extract of Anemarrhena asphodeloides (BN108) induces apoptosis in various cancer cell lines but is significantly less cytotoxic in non-transformed cells. Chemical fractionation of BN108 showed that its cytotoxicity is associated with timosaponins, steroidal saponins of coprostane type. Timosaponin BII (TBII) is a major saponin in BN108, but it shows little cytotoxicity. A much less abundant TAIII induces cell death in tumor cells but not in normal cells, reproducing the selectivity of the total extract BN108. Glycosidase treatment, by removing the extra sugar moiety in TBII, converts it to TAIII and confers cytotoxic activity. Analysis of the mechanisms of death induced by TAIII revealed activation of two distinct pro-apoptotic pathways: first, inhibition of mTORC1 manifested in much reduced phosphorylation of mTORC1 targets; second, induction of endoplasmic reticulum stress culminating in phosphorylation of eIF2 alpha and activation of caspase 4. These pro-apoptotic pathways are activated by TAIII selectively in tumor cells but not in normal cells. Both pathways play a causative role in TAIII cytotoxicity, as restoration of either mTOR activity or relief of ER stress alone offer only partial protection from TAIII. Inhibition of mTORC1 and induction of ER stress apparently contribute to the induction of the previously reported autophagic response in TAIII-treated cells. TAIII induced autophagy plays a protective role in TAIII induced death signaling, and failure to mount autophagic response is associated with heightened sensitivity to TAIII induced apoptosis. The multiple death-promoting and apparently tumor-selective responses to TAIII, its ability to inhibit mTORC1, and the possibility of further enhancing its cytotoxicity by pharmacological inhibition of autophagy, make TAIII an attractive candidate for development as a cancer therapeutic agent.

Abstract Abstract #3018 We present preclinical data on the potential mechanisms of the cytotoxic effect of the herbal extract BN108 towards cancer cells. Botanical medicine is one of the most popular complementary medical approaches, and herbal therapies are frequently sought and used by breast cancer patients. However, the molecular mechanisms through which certain herbal extracts exert growth inhibitory activity on breast cancer cells remain largely unknown. Treatment of a panel of breast cancer cell lines and normal mammary cells with BN108 induced cell death selectively in breast cancer lines. Normal mammary epithelial cells and fibroblasts were resistant to the cytotoxic effects of BN108. Breast cancer cells sensitive to BN108 underwent apoptotic death confirmed by Annexin V staining, caspase activation, cleavage of PARP and DNA fragmentation. In particular, caspases 4 and 9, whose activation is observed during apoptosis induced by endoplasmic reticulum stress, were proteolytically activated. Inhibition of caspase 4 partially protected breast cancer cells from cell death induced by BN108. Expression array analysis of cells treated with BN108 showed induction of expression of several known pro-apoptotic and anti-proliferative genes such as REDD1, p21CIP, cyclin G2, stratifin and more. BN108 induced rapid inactivation of AKT and mTOR kinases in breast cancer but not in non-transformed cells. Moreover, the well-defined targets of mTORC1, S6kinase and 4eBP1 were inactivated in BN108 treated cells. The expression array analysis also showed the induction by BN108 of numerous genes whose products code for enzymes within the cholesterol synthesis pathway. Interestingly, a transient increase in cholesterol synthesis was seen in breast cancer cells but not in normal cells following BN108 treatment. In general, cholesterol levels are higher in breast cancer cells compared to normal cells. Steroidal saponins are major components of BN108 extract, and they are known to deplete cell membrane cholesterol. Similar to BN108 extract, treatment with purified timosaponin AIII (a component of BN108 extract) also induced apoptosis and changes in cholesterol production in breast cancer cells but not in normal cells. It is possible that steroidal saponins induce a very transient depletion of cholesterol, followed by inactivation of AKT and mTOR, and negative feedback inhibition of the cholesterol synthesis pathway that is, in the end, is futile. None of these events are observed in normal cells. In conclusion, BN108 extract contains cytotoxic activity selective for transformed versus normal cells. These selective cytotoxic properties of BN108 could be related to its differential effects on cholesterol synthesis in breast cancer cells versus normal cells, as well as inhibition of major oncogenic pathways. Future studies will be aimed at understanding the molecular relationship between the BN1008 effect on cholesterol synthesis and induction of apoptosis, which may give rise to a unique pathway for targeting tumor cells. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 3018.

We studied the mechanism of the cytotoxic activity of BZL101, an aqueous extract from the herb Scutellaria barbata D. Don, which is currently in phase II clinical trial in patients with advanced breast cancer. The phase I trial showed favorable toxicity profile and promising efficacy. We report here that BZL101 induces cell death in breast cancer cells but not in non-transformed mammary epithelial cells. This selective cytotoxicity is based on strong induction by BZL101 of reactive oxygen species (ROS) in tumor cells. As a consequence, BZL101 treated cancer cells develop extensive oxidative DNA damage and succumb to necrotic death. Data from the expression profiling of cells treated with BZL101 are strongly supportive of a death pathway that involves oxidative stress, DNA damage and activation of death-promoting genes. In breast cancer cells oxidative damage induced by BZL101 leads to the hyperactivation of poly (ADP-ribose) polymerase (PARP), followed by a sustained decrease in levels of NAD and depletion of ATP, neither of which are observed in non-transformed cells. The hyperactivation of PARP is instrumental in the necrotic death program induced by BZL101, because inhibition of PARP results in suppression of necrosis and activation of the apoptotic death program. BZL101 treatment leads to the inhibition of glycolysis selectively in tumor cells, evident from the decrease in the enzymatic activities within the glycolytic pathway and the inhibition of lactate production. Because tumor cells frequently rely on glycolysis for energy production, the observed inhibition of glycolysis is likely a key factor in the energetic collapse and necrotic death that occurs selectively in breast cancer cells. The promising selectivity of BZL101 towards cancer cells is based on metabolic differences between highly glycolytic tumor cells and normal cells.

BACKGROUND:Botanical therapies are often used by breast cancer patients yet few clinical trials have evaluated their safety and efficacy. We studied mechanisms of activity and performed a phase I clinical trial in patients with advanced breast cancer to evaluate BZL101, an aqueous extract from Scutellaria barbata.METHODS:Preclinical studies were conducted in vitro to characterize cell death induced by BZL101. In a phase I trial, eligible patients had histologically confirmed, measurable metastatic breast cancer. Treatment consisted of 350 ml per day of oral BZL101, administered as sole cancer therapy until disease progression, toxicity or personal preference to discontinue. Primary endpoints were safety, toxicity and tumor response.RESULTS:BZL101 extract induced strong growth inhibition and apoptosis of breast cancer cell lines. In the phase I trial, 21 patients received BZL101. Mean age was 54 years (30-77) and mean number of prior treatments for metastatic disease was 3.9 (0-10). There were no grade III or IV adverse events (AEs). The most frequently reported BZL101-related grade I and II AEs included: nausea (38%), diarrhea (24%), headache (19%) flatulence (14%), vomiting (10%), constipation (10%), and fatigue (10%). Sixteen patients were evaluable for response. Four patients had stable disease (SD) for >90 days (25%) and 3/16 had SD for >180 days (19%). Five patients had objective tumor regression, one of which was 1 mm short of a PR based on RECIST criteria.CONCLUSIONS:BZL 101 inhibits breast cancer cell lines by inducing apoptosis. In a phase I clinical trial, BZL101 was safe and had a favorable toxicity profile. BZL101 demonstrated encouraging clinical activity in this heavily pretreated population.

Statins are cholesterol-lowering drugs with pleiotropic activities including inhibition of isoprenylation reactions and reduction of signals driving cell proliferation and survival responses. The objectives of this study were to examine the effects of statins on breast cancer cells, both in vitro and in vivo, and to begin to determine their mechanism of action. We evaluated the effects of statins on breast cancer cell growth, phosphoprotein signaling intermediates, survival/apoptosis regulators, cell cycle regulators, and activated transcription factors. We also examined the in vivo effect of statin administration in a mouse ErbB(2+) breast cancer model. Only lipophilic statins had direct anticancer activity in vitro. Breast cancer cells with activated Ras or ErbB2 pathways seemed to be more sensitive than those overexpressing estrogen receptor, and this correlated with endogenous levels of activated nuclear factor kappa B (NF-kappa B). Key intermediates regulating cell survival by NF-kappa B activation, as well as cell proliferation by the mitogen activated protein kinase cascade, were among the earliest phosphoproteins influenced by statin treatment. These early effects were followed by declines in activator protein-1 and NF-kappa B activation and concordant changes in other mediators of proliferation and apoptosis. In vivo results showed that oral dosing of statins significantly inhibited the growth of a mouse mammary carcinoma. Lipophilic statins can exert direct anticancer activity in vitro by reducing proliferation and survival signals in susceptible breast cancer phenotypes. Tumor growth inhibition in vivo using a clinically relevant statin dose also seems to be associated with reduced tumor cell proliferation and survival. These findings provide supporting rationale for future statin trials in breast cancer patients.

Aqueous extracts of 12 Chinese medicinal herbs, Anemarrhena asphodeloides, Artemisia argyi, Commiphora myrrha, Duchesnea indica, Gleditsia sinensis, Ligustrum lucidum, Rheum palmatum, Rubia cordifolia, Salvia chinensis, Scutellaria barbata, Uncaria rhychophylla and Vaccaria segetalis were evaluated for their antiproliferative activity on eight cancer cell lines as well as on normal human mammary epithelial cells. Five human and three murine cancer cell lines representing different tissues (breast, lung, pancreas and prostate) were used. All the crude aqueous extracts demonstrated growth inhibitory activity on some or all of the cancer cell lines, but only two showed activity against the normal mammary epithelial cells. Overall, the murine cell lines tended to be more sensitive to most of the extracts compared with the human cell lines. Among the human cell lines, cell type specificity was observed for two extracts. These results indicate the potential use of traditional Chinese medicinal herbs as antineoplastic agents and suggest that further studies evaluating their mechanism(s) of action and the isolation of active antitumor compounds are warranted.

The tetrazolium salt 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) is used to determine cell viability in assays of cell proliferation and cytotoxicity. MTT is reduced in metabolically active cells to yield an insoluble purple formazon product. However, in the present study, we demonstrate that some botanical extracts can reduce MTT in the absence of living cells. Treatment of these extracts with iodoacetic acid (IAA) to alkylate free thiol groups inhibited their ability to reduce MTT, indicating that free thiols in the botanical extracts are responsible, at least in part, for the reduction of MTT to the formazon product. These results suggest that caution must be taken when interpreting the results of experiments using the MTT assay to measure the effects of botanical extracts on cell growth.

1003 Background: Statins are potent cholesterol-lowering drugs with pleiotropic activities including inhibition of Ras, Rac and Rho isoprenylation, altered PI3K/AKT activity and eNOS production, and reduced ubiquinone synthesis. These activities are independent of cholesterol reduction and may explain the prevention of breast cancer and other malignancies seen in a number of studies. Methods: We compared hydrophilic (pravastatin, P) vs. hydrophobic (lovastatin, L; simvastatin, S; fluvastatin, F) statins for their in vitro growth inhibitory effects on a panel of human breast cancer cell lines: MDA-MB-231, SKBR3, BT-474, and MCF-7. To evaluate statin potential to silence expression of specific oncogenes, stable MCF-7 sublines expressing ErbB2 or cyclinD1 promoter-driven luciferase reporters were assayed for growth and promoter activity after statin treatment. Finally, we evaluated the in vivo impact of statin administration in a murine ErbB2 breast cancer model. Results: In vitro results indicate that only hydrophobic statins have direct anticancer activity. There is a >2 log IC50 range in statin sensitivity (e.g. IC50 for S and F: <1um in MDA-231 vs. 91 and 85 for MCF-7, respectively). Cells with activated Ras or ErbB2 pathways appeared more sensitive than those overexpressing ER or cyclin D1. The NCI/DTP screen of L and S against >50 non-breast human cancer cell lines produced mean cytotoxic values (LC50, 48h) of 29 uM and 25 uM, respectively; with no apparent correlation between L and S on these cell lines (COMPARE analysis). Only S and F produced promoter-specific oncogene inhibition, with ErbB2 showing greater promoter sensitivity than cyclinD1. Finally, oral dosing of S (1–2mg/kg/day) in mice implanted with MCNeuA mammary cancer cells produced 50% inhibition in mean tumor size by day 27 (518 vs. 1019 mm3; p<0.001). Conclusions: Hydrophobic statins exert direct anticancer activity in vitro, especially in cell lines with activated Ras or ErbB2 pathways, and inhibit ErbB2-dependent breast tumorigenic growth in vivo at a daily dose comparable to that approved for cholesterol reduction in humans. Among the statins tested, F demonstrated the most potent antiproliferative and oncogene-specific inhibitory activity. No significant financial relationships to disclose.

Chinese medicinal herbs are traditionally used to prevent and treat a variety of diseases, including cancer. These herbal preparations are purported to have many biological effects including direct antiproliferative effects on cancer cells, anti-mutagenic activity, and stimulatory or suppressive effects on immune responses. The present study investigates the effects of aqueous extracts from seventy-one Chinese medicinal herbs on the growth of five breast cancer cell lines (SK-BR-3, MCF7, MDA-MB-231, BT-474 and MCNeuA). Twenty-one percent (15 out of 71) of the extracts demonstrated greater than 50% growth inhibition on at least 4 of the 5 cell lines. Dose-response curves were obtained for several of the most potent crude extracts and demonstrated IC50 values ranging from < 10 micrograms/ml to > 1 mg/ml. Six of seven herbs tested induced high molecular weight DNA fragmentation, an early marker of apoptosis, while one of these also induced low molecular weight DNA fragmentation. Flow cytometric analysis of breast cancer cells exposed to one of these herbs (Rheum palmatum) suggested that it arrests cells in the G2/M phase of the cell cycle. These results indicate that many of the herbs used in traditional Chinese medicine for the treatment of cancer have significant growth inhibitory effects on breast cancer cells in vitro.