Effects of Combination of Estradiol with Selective Progesterone Receptor Modulators (SPRMs) on Human Breast Cancer Cells In Vitro and In Vivo.

Use of estrogen or estrogen/progestin combination was an approved regimen for menopausal hormonal therapy (MHT). However, more recent patient-centered studies revealed an increase in the incidence of breast cancer in women receiving menopausal hormone therapy with estrogen plus progestin rather than estrogen alone. Tissue selective estrogen complex (TSEC) has been proposed to eliminate the progesterone component of MHT with supporting evidences. Based on our previous studies it is evident that SPRMs have a safer profile on endometrium in preventing unopposed estrogenicity. We hypothesized that a combination of estradiol (E2) with selective progesterone receptor modulator (SPRM) to exert a safer profile on endometrium will also reduce mammary gland proliferation and could be used to prevent breast cancer when used in MHT. In order to test our hypothesis, we compared the estradiol alone or in combination with our novel SPRMs, EC312 and EC313. The compounds were effectively controlled E2 mediated cell proliferation and induced apoptosis in T47D breast cancer cells. The observed effects were found comparable that of BZD in vitro. The effects of SPRMs were confirmed by receptor binding studies as well as gene and protein expression studies. Proliferation markers were found downregulated with EC312/313 treatment in vitro and reduced E2 induced mammary gland proliferation, evidenced as reduced ductal branching and terminal end bud growth in vivo. These data supporting our hypothesis that E2+EC312/EC313 blocked the estrogen action may provide basic rationale to further test the clinical efficacy of SPRMs to prevent breast cancer incidence in postmenopausal women undergoing MHT.

Gossypin as a novel selective dual inhibitor of V-RAF murine sarcoma viral oncogene homolog B1 and cyclin-dependent kinase 4 for melanoma.

Mutation in the BRAF gene (BRAFV600E) exists in nearly 70% of human melanomas. Targeted therapy against BRAFV600E kinase using a recently identified RAF-selective inhibitor, PLX4032, has been successful in early clinical trials. However, in patients with the normal BRAF allele (wild-type), PLX4032 is protumorigenic. This conundrum identifies the unmet need for novel therapeutic agents to target BRAFV600E kinase that are not counterproductive. We have identified gossypin, a pentahydroxy flavone, as a potent antimelanoma agent. Gossypin inhibited human melanoma cell proliferation, in vitro, in melanoma cell lines that harbor both BRAFV600E kinase and cyclin-dependent kinase 4 (CDK4) as well as in cells with BRAF wild-type allele. Gossypin inhibited kinase activities of BRAFV600E and CDK4, in vitro, possibly through direct binding of gossypin with these kinases, as confirmed by molecular docking studies. For cells harboring the BRAFV600E, gossypin inhibited cell proliferation through abrogation of the MEK-ERK-cyclin D1 pathway and in cells with BRAF wild-type allele, through attenuation of the retinoblastoma-cyclin D1 pathway. Furthermore, gossypin significantly inhibited melanoma growth in an organotypic three-dimensional skin culture mimicking human skin. Gossypin (10 and 100 mg/kg) treatment for 10 days in human melanoma (A375) cell xenograft tumors harboring BRAFV600E significantly reduced tumor volume through induction of apoptosis and increased survival rate in mice, and the effect was significantly superior to that of PLX4032 (10 mg/kg) or roscovitine 10 mg/kg. In summary, this study identified gossypin as a novel agent with dual inhibitory effects for BRAFV600E kinase and CDK4 for treatment of melanoma.