Estrogen Induced Metastatic Modulators MMP-2 and MMP-9 Are Targets of 3,3′-Diindolylmethane in Thyroid Cancer

Thyroid cancer is the most common endocrine related cancer with increasing incidences during the past five years. Current treatments for thyroid cancer, such as surgery or radioactive iodine therapy, often require patients to be on lifelong thyroid hormone replacement therapy and given the significant recurrence rates of thyroid cancer, new preventive modalities are needed. The present study investigates the property of a natural dietary compound found in cruciferous vegetables, 3,3'-diindolylmethane (DIM), to target the metastatic phenotype of thyroid cancer cells through a functional estrogen receptor.Thyroid cancer cell lines were treated with estrogen and/or DIM and subjected to in vitro adhesion, migration and invasion assays to investigate the anti-metastatic and anti-estrogenic effects of DIM. We observed that DIM inhibits estrogen mediated increase in thyroid cell migration, adhesion and invasion, which is also supported by ER-α downregulation (siRNA) studies. Western blot and zymography analyses provided direct evidence for this DIM mediated inhibition of E(2) enhanced metastasis associated events by virtue of targeting essential proteolytic enzymes, namely MMP-2 and MMP-9.Our data reports for the first time that DIM displays anti-estrogenic like activity by inhibiting estradiol enhanced thyroid cancer cell proliferation and in vitro metastasis associated events, namely adhesion, migration and invasion. Most significantly, MMP-2 and MMP-9, which are known to promote and enhance metastasis, were determined to be targets of DIM. This anti-estrogen like property of DIM may lead to the development of a novel preventive and/or therapeutic dietary supplement for thyroid cancer patients by targeting progression of the disease

Participation of HSP27 in the antiapoptotic action of 17β-estradiol in skeletal muscle cells

Exposure to 17β-estradiol prior to induction of apoptosis protects skeletal muscle cells against damage. The mechanism involved in this protective action of the hormone is poorly understood. In the present study, using the murine muscle cell line C2C12, evidence was obtained that inhibition of H2O2-induced apoptosis by the estrogen requires the participation of heat shock protein 27 (HSP27). Reverse transcriptase polymerase chain reaction, Western blot, and immunocytochemistry assays showed that 17β-estradiol induces a time-dependent (5–60 min) increase in the expression of HSP27. In addition, in presence of quercetin, an inhibitor of HSPs, the antiapoptotic effect of the hormone was diminished. More specifically, blockage experiments with short interference RNA targeting HSP27 confirmed the role of this chaperone in the protective effect of the steroid. 17β-Estradiol abolished caspase-3 cleavage elicited by H2O2. Coimmunoprecipitation assays suggested physical interaction of HSP27 with caspase-3 in presence of estradiol. Furthermore, we observed that this chaperone interacts with estrogen receptors (ER) β in mitochondria. Then, this study suggests that HSP27 plays a new role in the antiapoptotic action triggered by 17β-estradiol by modulating caspase-3 activity and stabilizing ERβ in skeletal muscle cells

Involvement of G‐proteins in chitosan‐induced Anthraquinone synthesis in Rubia tinctorum

Elicitation with chitosan (200 mg/l) significantly stimulates (100%) anthraquinone (Aq) synthesis in Rubia tinctorum L. cultures, but the mechanism of elicitation is largely unknown.We recently showed that the effects of the elicitor involve phospholipase C (PLC), protein kinase C (PKC), phosphoinositide 3-kinase (PI3K) and mitogen activated protein kinase (MAPK) cascades. Here, we show that the elicitor action on Aq production can be blocked with the intracellular Ca2+ chelator BAPTA-AM but not in a Ca2+-free medium (+EGTA) or in the presence of the voltage-dependent Ca2+ channel antagonists verapamil and nifedipine. In agreement with these observations, spectrofluorimetric measurements in Fura 2-loaded R. tinctorum cells show that chitosan increases intracellular Ca2+ concentration in a medium devoid of calcium. Short treatment intervals (1?5 min) of cells with the elicitor significantly increased DAG and IP3 formation. Moreover, the PI-PLC inhibitors neomycin and U73122 diminished to a great extent chitosan-induced Aq synthesis. Blockers of Ca2+ release from inner stores such as 2-APB, TMB-8, caffeine, ruthenium red and dantrolene inhibited elicitation. Chitosan rapidly stimulated (phosphorylated) MAPK. This effect was significantly decreased by the calcium modulators used above. However, EGTA did not prevent activation of MAPK. Compound LY24009, a blocker of PI3K, inhibited MAPK phosphorylation by chitosan. Accordingly, an increase in PI3K activity was observed in parallel. The results of this study show that chitosan induction of anthraquinone synthesis in R. tinctorum involves the stimulation of PLC, intracellular Ca2+ mobilization and PI3K, which mediate MAPK activation.Fil: Vasconsuelo, Andrea Anahi. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Ciencias Biológicas y Biomédicas del Sur. Universidad Nacional del Sur. Departamento de Biología, Bioquímica y Farmacia. Instituto de Ciencias Biológicas y Biomédicas del Sur; ArgentinaFil: Picotto, Gabriela. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigaciones en Ciencias de la Salud. Universidad Nacional de Córdoba. Instituto de Investigaciones en Ciencias de la Salud; ArgentinaFil: Giuletti, Ana M.. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica; ArgentinaFil: Boland, Ricardo Leopoldo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Ciencias Biológicas y Biomédicas del Sur. Universidad Nacional del Sur. Departamento de Biología, Bioquímica y Farmacia. Instituto de Ciencias Biológicas y Biomédicas del Sur; Argentin