Theranostic System With Combination Of Plumbagin And Zoledronic Acid To Attenuate Breast Cancer Bone Metastasis

ObjectiveBone metastasis is the major complication of malignant breast cancer,contributing to the degeneration of life quality and the decrease of life span significantly in breast cancer patients.Within the three metastatic microenvironments,namely primary tumor microenvironment(PTM),circulation microenvironment(CM)and bone microenvironment(BM),the biotic cellular candidates(like osteocytes and osteoclasts)in BM create a vivid crosstalk with breast cancer cells,resulting in the vicious cycle that facilitates the localization of breast cancer cells in BM and the subsequent cancer-induced osteolysis.These are presented as skeletal related events(SREs),showing intractable bone pain,pathological fracture,hypercalcium and vertebrate compression,etc.Zoledronic acid is the first-line reagent in treating SREs by targeting osteoclasts to attenuate osteoclastogenesis.However,its anti-tumoriogenesis ability remains largely debatable.Therefore,it is important to target cancer cells simultaneously and realize high-cencentration drug release pattern in localized osteolysis lesions.Herein,we intended to deploy the combination of one natural small-molecule compound,plumbagin(PL),with zoledronic acid(ZA)in breast cancer bone metastasis treatment and analyze underlying synergistic mechanisms.Also,we established ZA-anchored PL-loaded pH-responsive Gadolinium(Gd)3+-doped upconversion nanoparticles for bimodal upconversion luminescence(UCL)/magnetic resonance(MR)imaging,leading to the effective theranostic effects in early breast cancer bone metastasis.We aimed to provide promising theranostic strategy for breast cancer bone metastasis at early stage.Methods1.In vitro proliferation,apoptosis and migration ability of breast cancer cells were evaluated with combined treatment of PL with ZA for analyzing potential synergistic effects and underlying anti-tumor machanisms.On the basis of intratibial breast cancer cells injection nude mice model,the combined anti-tumoriogenesis ability of PL with ZA was assessed with non-invasive in vivo imaging system(IVIS)and histologic analyses in vivo.2.In vitro proliferation and differentiation of osteoclast precursors were assessed with combined treatment of PL with ZA for analyzing the inhibition of breast cancer-induced osteoclastogenesis and underlying anti-osteoclast mechanisms.On the basis of intratibial breast cancer cells injection nude mice model,the combined anti-osteoclastogenesis ability of PL with ZA was assessed with X-ray,?CT,ELISA and histologic analyses in vivo.3.ZA-anchored PL-loaded pH-responsive Gd3+-doped bimodal upconversion nanoparticle was established and characterized for exploring its effect on osteocyte-induced osteoclastogenesis inhibition with clarified mechanisms.On the basis of intracardiac breast cancer cells injection bone metastasis nude mice model,the theranostic and osteocyte-targeting effects of nanosystem were assessed with X-ray/IVIS/UCL/MR/?CT scanning,Scanning electron microscope(SEM),Energy disperse X-ray spectroscopy(EDX)and histologic analyses in vivo.Results1.The combination of PL with ZA could inhibit breast cancer proliferation and migration while promote cancer cells apoptosis synergistically in vitro.Combined treatment reduced the expressions of PARP and Bcl-2 whereas increased the expressions of ID 1?p21 and cleaved caspase 3 synergistically,as further illustrated by the regulation of Notch-1-Bcl-2 signaling to promote breast cancer apoptosis.In intratibial breast cancer model,dual drugs decreased breast cancer growth and stimulated apoptosis synergistically in vivo bone microenvironment.2.The combination of PL with ZA could attenuate osteoclast precursors proliferation synergistically in vitro.Dual drugs treatment inhibited RANKL and cancer condition medium induced osteoclast formation synergistically,mainly through spatial simulation of adenosine phosphate(ANP)for competitively inhibiting its binding and phosphorylation to Erk/Jnk,thereby down-regulating activation of NF-?B/MAPK signalings and downstream c-Fos?c-Jun and NFATcl molecules productions.Combined treatment also inhibited cancer-associated osteoclast activation and osteolysis synergistically in vivo.3.Bone-targeting upconversion nanoparticles could decrease the expressions of RANKL and Sclerostin in osteocytes for inhibiting osteocyte-induced osteoclast formation in vitro.In intracardiac injection breast cancer bone metastasis model,bimodal nanoparticles could discover tiny bone lesions earlier than convertional X-ray imaging while impeding breast cancer cells colonization and related osteolysis in bone microenvironment by pH-responsive PL release simultaneously in vivo.Conclusions1.Combined treatment of PL with ZA inhibited breast cancer growth and promoted cancer cells apoptosis synergistically through Notch-1-Bcl-2 signaling both in vivo and in vitro.2.Combined treatment of PL with ZA inhibited breast cancer induced osteoclast activation and osteolysis through spatial simulation of ANP to competitively attenuate its binding and phosphorylation towards Erk/Jnk,thereby inhibiting osteoclast formation both in vivo and in vitro.3.ZA-anchored PL-loaded bone-targeting upconversion nanoparticles could detect bone lesions in early stage and target osteocyte-osteoclast-cancer cells,thereby inhibiting breast cancer bone metastasis progression and realizing theranostic effects in vivo.