Studies On Mechanism And Effect Of Genistein On Bone Metastasis Of Breast Cancer

BackgroundBone metastasis of breast cancer usually activates osteoclasts, resulting in osteolytic lesions, which could cause many complications including intractable bone pain, pathological fractures, hypercalcemia and nerve compression syndromes, and seriously decreases the quality of life, but the precise mechanisms underlying bone metastasis of breast cancer have not been clear. Now, the studies mainly focus on the increase of osteoclasts activity induced by breast cancer cells. However, to inhibit solely osteoclasts activity could not completely succeed to block the progress of osteolytic lesion. And in recent years, studies have found that breast cancer cells may also affect other cells in bone microenvironment, especially the biological function of osteoblasts. Thus, promoting osteoblasts activity may be important in clinical treatment of breast cancer with bone metastasis as well as inhibiting osteoclasts activity. The clinical treatment of breast cancer and its bone metastasis is quite difficult, and because of side effects and drug resistance in endocrine therapy, there has been heightened interest to find selective estrogen receptor modulator and alternative natural estrogen in treatment and prevention of breast cancer and bone metastasis. Genistein is a natural isoflavone compound and could bind with estrogen receptor to exert weak estrogen-like effects without inducing adverse effects. It also exerts anticancer and antioxidant effects, promotes bone formation and inhibits bone resorption in normal condition of bone metabolism. But in pathological condition of bone metastasis, whether genistein could regulate bone metabolism and reverse bone loss needs further studies.ObjectiveEstablishing nude mouse model of breast cancer bone metastasis and administering genistein prophylactically and therapeutically were to observe the effect of genistein on the formation and development of osteolytic bone metastases, and bone mass, bone mircostructure and biological functions of osteblasts and osteoclasts in pathological condition of bone metastasis. And using the osteoblasts harvested from calvaria of Sprague Dawley rats cultured with conditioned medium collected from two types of human breast cancer cells with different metastatic abilities was to study the effect of human breast cancer cells on biological function of osteoblasts, and the effect of genistein on osteoblasts coultured with conditioned medium which was collected from breast cancer cells. The objective of this study was to investigate the effect of genistein on bone metastasis of breast cancer and the possible mechanisms underlying it from animal and cell experiments, to change the clinical treatment of simply inhibiting osteoclasts activity and provide a theoretical basis and new idea of intervention for preventing or reversing bone loss.MethodsIn animal study, female BALB/c-nu/nu mice were injected with human breast cancer cells, MDA-MB-231,into left cardiac ventricle to form osteolytic bone metastases, and administered genistein prophylactically and therapeutically, that is, administered genistein subcutaneously after radiologically small but defined osteolytic metastases had been observed (protocol 1),simultaneously with cancer cells inoculation (protocol 2) and prophylactically 7 days before inoculation of cancer cells (protocol 3).At the end of the experiment, all animals were examined by radiography for assessing the number and volume of osteolytic bone metastases. Blood samples were immediately collected, and then nude mice were sacrificed by cervical dislocation. Hindlimbs were collected to make decalcified and undecalcified bone specimens. Then the number of osteoclasts in the left hindlimb was counted by tartrate-resistant acid phosphatase (TRAP) staining, bone histomorphometry including bone mass and microstructure parameters in the right tibia was analyzed by Goldner's Trichrome staining, and an automatic biochemical analyzer was used for measurements of serum calcium, phosphorus and alkaline phosphatase levels. These analyses were to investigate the effect of genistein on the formation and development of osteolytic bone metastasis, and bone mass, bone mircostructure and biological functions of osteblasts and osteoclasts in pathological condition of bone metastasis.Meanwhile, the osteoblasts harvested from calvaria of Sprague Dawley rats were treated with conditioned medium collected from MCF-7 estrogen-receptor positive, or MDA-MB-231 estrogen-receptor negative, breast cancer cells. Then proliferation was measured by MTT method, alkaline phosphatase (ALP) activity was assessed by the p-nitrophenyl phosphate (PNPP) method, and mineralizd nodules were confirmed by alizarin red S (ARS) staining and the area of bone nodules was measured to observe the mineralization capacity. The mRNA levels of BGP,OPG and RANKL in osteoblasts were determined by RT-PCR. These analyses were to investigate the effect of two types of human breast cancer cells with different metastatic abilities on biological function of osteoblasts. Meanwhile, adding different concentrations of genistein into conditioned medium investigates the effect of genistein on osteoblasts in pathological condition of bone metastasis and the possible mechanisms underlying it.ResultsThe animal study showed that:at day 21,nude mice in protocol 1 given injections with MDA-MB-231 breast cancer cells into the left ventricle of the heart showed small but distinct osteolytic bone metastases at multiple areas, expecially at limbs by radiological examination. Genistein administered prophylactically (protocol 2:simultaneously with cancer cells inoculation or protocol 3:prophylactically 7 days before inoculation of cancer cells) reduced the number and volume of osteolytic bone metastases assessed by radiography and the number of TRAP-positive osteoclasts in the different extents. Furthermore, histomorphometrical analysis revealed that genistein administered prophylactically markedly increased trabecular area (Tb.Ar%), trabecular number (Tb.N) and trabecular thickness (Tb.Th), and decreased trabecular separation (Tb.Sp). That is, genistein could exert prophylactic effect on the formation of osteolytic bone metastases and bone loss and bone microstructure damage induced by bone metastasis. Even if administered after osteolytic bone metastases formation (protocol 1:therapeutic treatment), genistein also markedly reduced the number and volume of osteolytic bone metastases, and slowed bone loss and bone microstructure damage induced by bone metastases. Meanwhile, genistein partly reversed the high level of serum ALP in bone metastases of breast caner, and had certain repair in metabolic imbalance of serum calcium and phosphorus.The cell study showed that:when osteoblasts were treated with conditioned medium from MDA-MB-231 or MCF-7, the cells became long and spindle-like, assumed a fibroblastic morphology. Conditioned medium from breast cancer cells significantly inhibited proliferation of osteoblasts:compared with those cultured with vehicle control medium, the proliferation inhibition rates of cultured with 50% conditioned medium from MDA-MB-231 or MCF-7 cells on osteoblasts were 18.1%,13.0%,19.2%,19.3% and 15.8%,20.8%,33.9%,28.7%on day 1,day 3,day 5,and day 7, respectively (P<0.01).Moreover, conditioned medium significantly inhibited ALP activity of osteoblasts in a dose-dependent manner, especially addition of 50% conditioned medium inhibited ALP activity by 31.9% and 47.5%(P<0.01). Conditioned medium also significantly inhibited bone nodule formation of osteoblasts in a dose-dependent manner, especially at 50% conditioned medium group, the bone nodule formation was almost completely inhibited and the areas of bone nodules diminished by 89% and 74% compared with the vehicle control medium group (P<0.01).Meanwhile, conditioned medium significantly decreased the mRNA levels of BGP and OPG, and increased the mRNA level of RANKL.While, the proliferation and ALP activity were increased in the different extents compared with condition medium group when osteoblasts were treated with different concentrations of genistein:510-7mol/L,5×10-8mol/L and 5×10-9mol/L. The ALP activity were increased by 22.7%,32.4%,63.5%and 27.7%,32.0%,58.3%,respectively (P<0.05). Genistcin also increased the area of bone nodule formation, improved osteoblastic mineralization ability inhibited by conditioned medium, and increased the mRNA levels of BGP and OPG, and decreased the mRNA level of RANKL.ConclusionsGenistein could inhibit the formation and development of osteolytic bone metastases and partly reverse excessive bone resorption, bone loss and bone microstructure damage induced by bone metastases of breast cancer, exert prophylactic and therapeutic effect on bone health in bone metastasis of breast cancer. Meanwhile, breast cancer cells with different metastatic abilities could inhibit proliferation, decrease ALP activity and disrupt the mineralization process of osteoblasts, that is, may decrease bone formation through regulating biological function of osteoblasts. While, genistein could improve biological function of osteoblasts suppressed by breast cancer cells through promoting directly proliferation, differentiation and mineralization of osteoblasts, up-regulating the mRNA levels of BGP and OPG and down-regulating the mRNA level of RANKL.This may be one of the mechanisms underlying beneficial effect of genistein on bone health in bone metastasis of breast cancer.