Lupeol, the major compound of the dichloromethane extract of Millettia macrophylla Benth (Fabaceae), displays estrogenic effects in ovariectomized rats.

The present work deals with the assessment of the in vitro and in vivo estrogenic effects of the triterpenoids (lupenone, lupeol, and stigmastenone) isolated from Millettia macrophylla extract. The in vitro estrogenicity was performed by a reporter gene assay and estrogen receptor-α (ERα) target gene expression, whereas the in vivo estrogenicity was evaluated by a 3-day uterotrophic assay in ovariectomized rats. As results, lupenone and lupeol did not transactivate ERα as well as ERß of human embryonic kidney 293T (HEK293T) cells. However, lupeol seems to be antagonistic to estrogen (E2) only in HEK293T-ERα (10-9 and 10-8  µM). Furthermore, lupeol slightly upregulated GREB1 gene expression at the concentration of 1 µM, suggesting a weak activation of endogenous ERα. In vivo, only lupeol at a dose of 1 mg/kg significantly increased the uterine wet weight (p < 0.05), uterine (p < 0.05), and vaginal (p < 0.01) epithelial heights. The concomitant administration of lupeol (1 mg/kg) with a pure antiestrogen fulvestrant abrogated its effects only in the vagina, whereas in combination with E2, lupeol exhibited a significant antiestrogen-like effect in uterine wet weight and synergistic effects on endometrium. Lupeol has estrogenic effects that is partly through ERs transcriptional activity and does involve alternative mechanisms that are still to be uncovered.

Timeless Is a Novel Estrogen Receptor Co-activator Involved in Multiple Signaling Pathways in MCF-7 Cells.

Activation of estrogen receptor α (ERα) stimulates cell division and tumor growth by modulating the expression of ERα target genes. This activation involves the recruitment of specific proteins with activities that are still not fully understood. Timeless, the human homolog of the Drosophila gene involved in circadian rhythm, was previously shown to be a strong predictor of tamoxifen relapse, and is involved in genomic stability and cell cycle control. In this study, we investigated the interplay between Timeless and ERα, and showed that human Timeless is an ERα coactivator. Timeless binds to ERα and enhances its transcriptional activity. Overexpressing Timeless increases PARP1 expression and enhances ERα-induced gene regulation through the proximal LXXLL motif on Timeless protein and ERα PARylation. Finally, Timeless is recruited with ERα on the GREB1 and cMyc promoters. These data, the first to link Timeless to steroid hormone function, provide a mechanistic basis for its clinical association with tamoxifen resistance. Thus, our results identify Timeless as another key regulator of ERα in controlling ERα transactivation.

Crateva adansonii DC, an African ethnomedicinal plant, exerts cytotoxicity in vitro and prevents experimental mammary tumorigenesis in vivo.

ETHNOPHARMACOLOGICAL RELEVANCE: Crateva adansonii DC is a plant traditionally used in Cameroon to treat constipation, asthma, snakebites, postmenopausal complaints and cancers. AIM: The anticancer potential of the dichloromethane/methanol extract of C. adansonii stem barks was investigated using human breast cancer cell and 7,12 dimethylbenz(a)anththracene (DMBA)-induced mammary tumorigenesis model in rats. MATERIAL AND METHODS: The cytotoxicity of C. adansonii extract was assessed in vitro towards breast carcinoma (MCF-7 and MDA-MB-231) and non-tumoral cell lines (NIH/3T3 and HUVEC) by Alamar Blue assay. Furthermore, in vivo studies were performed on female Wistar rats treated either with C. adansonii extract at a dose of 75 or 300mg/kg body weight or with tamoxifen (3.3mg/kg body weight), starting 1 week prior DMBA treatment and lasted 12 weeks. The investigation focused on tumour burden, tumour DNA fingerprint, morphological, histological, hematological, and biochemical parameters. RESULTS: CC50 values for the in vitro assays were 289µg/mL against MCF-7 cells and >500µg/mL in others cells, leading to a selectivity index ≥1.73. C. adansonii extract significantly (p<0.001) revealed in vivo the reduction of the cumulative tumour yield (87.23%), total tumour burden (88.64%), average tumour weight (71.11%) and tumour volume (78.07%) at the dose of 75mg/kg as compared to DMBA control group. A weak effect was also observed at 300mg/kg. This extract showed a moderate hyperplasia at the dose of 75mg/kg while at 300mg/kg no significant change was noted as compared to DMBA group. It protected rats from the DNA alteration induced by DMBA and increased antioxydant enzymes activities in mammary gland tissue homogenates. In addition, Ultra-High Performance Liquid Chromatography/ESI-QTOF-Mass Spectrometry analysis of C. adansonii extract detected structure-related of many well-known anticancer agents such as flavane gallate, flavonol, phenylpropanoïds, sesquiterpene derivatives, gallotannins and lignans. The LD50 of C. adansonii was estimated to be greater than 5000mg/kg. CONCLUSIONS: These aforementioned results suggest that the C. adansonii extract may possess antitumor constituents, which could combat breast cancer and prevent chemically-induced breast cancer in rats.

Flavonoids, Breast Cancer Chemopreventive and/or Chemotherapeutic Agents.

Flavonoids are secondary metabolites abundantly present in commonly consumed fruits and vegetables. They possess diverse properties such as anti-inflammatory, anti-oxidant and anti-cancer. Epidemiologic studies suggest that an enrich flavonoids diet is linked to a decreased risk of breast cancer. These protective properties are due to the alteration of numerous signalling pathways involved in cancer-related phenomena such as inflammation and proliferation. Human clinical trials examining the effect of supplementation of some flavonoids on disease prevention have been conducted. There is no natural flavonoid that has been approved for the treatment of breast cancer. However, natural flavonoids served as lead compounds in the synthesis of cancer chemopreventive and/or therapeutic agents.

Flavonoids, Breast Cancer Chemopreventive and/or Chemotherapeutic Agents

Flavonoids are secondary metabolites abundantly present in commonly consumed fruits and vegetables. They possess diverse properties such as anti-inflammatory, anti-oxidant and anti-cancer. Epidemiologic studies suggest that an enrich flavonoids diet is linked to a decreased risk of breast cancer. These protective properties are due to the alteration of numerous signalling pathways involved in cancer-related phenomena such as inflammation and proliferation. Human clinical trials examining the effect of supplementation of some flavonoids on disease prevention have been conducted. There is no natural flavonoid that has been approved for the treatment of breast cancer. However, natural flavonoids served as lead compounds in the synthesis of cancer chemopreventive and/or therapeutic agents.

Preventive effects of the methanol soluble fraction of Millettia macrophylla Benth (Fabaceae) on an osteoporosis-like model of ovariectomized Wistar rats.

BACKGROUND: Millettia macrophylla Benth is a Cameroonian medicinal plant traditionally used to alleviate menopause-related problems. The methanol soluble fraction of this plant was shown to exhibit estrogenic effects in vitro in Human Embryonic kidney cells, and in vivo on ovariectomized rat following the classical uterotrophic assay. Since estrogens have been involved in bone remodeling process, the present study then aimed at evaluating bone loss preventive effects of the methanol soluble fraction of Millettia macrophylla (MM-met) in ovariectomized rat model. METHODS: Twenty-five healthy Wistar female rats (3-month-old) were randomly assigned to a sham-operated group and to four treated ovariectomized (OVX) groups. Treatments lasted 8 weeks and animals were sacrificed. The uterus, the femoral and the tibia bones of each animal were collected, weighed and fixed in 10% formalin for histological analysis. RESULTS: Results showed that ovariectomy decreased uterine wet weight (p<0.01), induced body weight gain (p<0.01), decreased both femoral and tibia bone density and mineral content and increased alkaline phosphatase activity (p<0.05). E2V and MM-met treatments in general prevented bone mass loss and/or bone density loss. At all tested doses, MM-met induced a significant decrease of alkaline phosphatase activity (p<0.05). As observed with E2V, MM-met also induced a significant protective effect on bone, and this was indicated by an abundance of bone marrow in an almost intact trabecular network. CONCLUSIONS: The overall results show that the methanol soluble fraction of Millettia macrophylla may prevent ovariectomy-induced bone mass loss and deterioration of the trabecular microarchitecture.

Elucidation of Underlying Mechanisms by Which Millettia macrophylla Benth Induces Its Estrogenic Activity.

Millettia macrophylla is used traditionally to treat menopause related symptoms. This plant was shown to exhibit estrogenic effects in vitro on human embryonic kidney cells and in vivo on ovariectomized rats. The present study aimed at elucidating underlying mechanisms by which M. macrophylla induced its estrogenic effects. To accomplish our goal, kidney Hek293T cells transiently transfected with estrogen alpha or beta receptor expression plasmids were cotreated with a pure antiestrogen ICI 182,780 and the dichloromethane or methanol soluble fractions of M. macrophylla. To follow up, we cotreated ovariectomized rats with both extracts and ICI 182,780 for 3 days in the classical uterotrophic assay. Animals were then sacrificed and the uterine wet weight, total protein levels in uteri, uterine, and vaginal epithelial heights, and mammary gland were assessed. In vitro, the results suggested that the induction of the estrogenic activity by M. macrophylla is due to the binding of its secondary metabolites to ERα and ERß. In vivo, the cotreatment of extracts and ICI 182,780 significantly abrogated the biological responses induced by the extracts alone. Taken together, these results indicate that the active principles of M. macrophylla induce their beneficial effects on menopausal symptoms by activating the ERs.

Effects of Millettia macrophylla (Fabaceae) extracts on estrogen target organs of female wistar rat.

The present study aims to determine the estrogenicity of Millettia macrophylla, a Cameroonian medicinal plant, in ovariectomized rats and to investigate the underlying mechanisms, in order to justify scientifically its traditional use. To accomplish this objective, we used dichloromethane (DCM) and methanol (MeOH) extracts of the stem bark of M. macrophylla. In the cell culture based assay, the MeOH extract significantly transactivated estrogen receptor α (ERα) and estrogen receptor ß (ERß); in addition, the estrogen-like effects of both, DCM and MeOH extracts, could be inhibited in vitro by the pure ER antagonist ICI 182,780, indicating that these effects were primarily mediated through ERs. In animal experiments, both DCM and MeOH extracts significantly increased the uterine and vaginal epithelial heights in the 3-day treatment assay, while only the MeOH extract exhibited such effects in the sub-chronic treatment regimen. Furthermore, the MeOH extract significantly decreased fasting serum triglycerides, total cholesterol levels and artherogenic risk in the sub-chronic treatment. These results indicate that M. macrophylla extracts have estrogen-like effects supporting their traditional use in Cameroon to alleviate some menopausal problems (See graphical abstract in Supplementary Fig. 1, available in the online version only).

Phytotherapy and women's reproductive health: the Cameroonian perspective.

Approximately 80 % of the population in Africa use traditional medicinal plants to improve their state of health. The reason of such a wide use of medicinal plants has been mainly attributed to their accessibility and affordability. Expectation of little if any side effects, of a "natural" and therefore safe treatment regimen, as well as traditional beliefs additionally contribute to their popularity. Several of these plants are used by women to relieve problems related to their reproductive health, during or after their reproductive life, during pregnancy, or following parturition. The African pharmacopoeia thus provides plants used for preventing and/or treating gynecological infections, dysmenorrhea, irregular menstruations, oligomenorrhea or protracted menstruation, and infertility. Such plants may then be used as antimicrobians, emmenagogues, or as suppressors of uterine flow. African medicinal plants are also used during pregnancy for prenatal care, against fetal malposition or malpresentation, retained dead fetus, and against threatened abortion. Some others are used as anti-fertilizing drugs for birth control. Such plants may exert various activities, namely, anti-implantation or early abortifacient, anti-zygotic, blastocytotoxic, and anti-ovulatory effects. Some herbs could also act as sexual drive suppressors or as a post-coital contraceptive by reducing the fertility index. A number of these plants have already been subject to scientific investigations and many of their properties have been assessed as estrogenic, oxytocic, or anti-implantation. Taking into account the diversity of the African pharmacopoeia, we are still at an early stage in the phytochemical and pharmacological characterization of these medicinal plants that affect the female reproductive system, in order to determine, through in vitro and in vivo studies, their pharmacological properties and their active principles.

In vitro estrogenic activity of two major compounds from the stem bark of Erythrina lysistemon (Fabaceae).

Plant-derived estrogen-like compounds, so called phytoestrogens, are given much attention due to their potential therapeutic use. In our previous work the ethylacetate extract of Erythrina lysistemon stem bark showed estrogenic effects on cell culture systems and ovariectomized Wistar rats. Using classical chromatographic methods, two constituents of Erythrina lysistemon have been isolated, referred to here as compounds 1 (alpinumisoflavone) and 2 (abyssinone V-4'-methyl-ether), and their structures successfully determined using spectroscopic techniques. To test their binding affinity, the ligand binding assay has been used on estrogen α receptor, and estrogen ß receptor. Furthermore, transactivation assay in stably or transiently transfected human osteosarcoma (U2OS-estrogen α receptor and estrogen ß receptor) cells were used to examine their estrogenic activity. The regulations of some estrogen receptor target genes were also investigated. Both compounds bind to estrogen α and ß receptors. They significantly increased luciferase activity in a dose-dependent manner and induced the endogenous estrogen receptor-estrogen response element (ERE) interaction in U2OS-estrogen α receptor and estrogen ß receptor cells. In contrast, when co-treated with E2, compound 2 did not antagonize E2 activity in both systems whereas, 1 significantly suppressed E2 activity despite its low binding affinity to estrogen ß receptor. This result suggests a non-competitive mechanism. Both compounds also altered the expression of estrogen receptor target genes such as growth regulation by estrogen in breast cancer 1 (GREB1) and Cyclin D1 in breast cells. These results suggest that compounds 1 and 2 endow estrogenic activity and may be the active principles of Erythrina lysistemon.