naphthoquinones and Osteolysis

The treatment of breast cancer-induced osteolysis remains a challenge in clinical settings. Here, we explored the effect and mechanism of combined treatment with zoledronic acid (ZA) and plumbagin (PL), a widely investigated component derived from Plumbago zeylanica, against breast cancer-induced osteoclastogenesis. We found that the combined treatment with PL and ZA suppressed cell viability of precursor osteoclasts and synergistically inhibited MDA-MB-231-induced osteoclast formation (combination index=0.28) with the abrogation of recombinant mouse receptor activator of nuclear factor-κB ligand (RANKL)-induced activation of NF-κB/MAPK (nuclear factor-κB/mitogen-activated protein kinase) pathways. Molecular docking suggested a putative binding area within c-Jun N-terminal kinase/extracellular signal-regulated kinase (JNK/Erk) protease active sites through the structural mimicking of adenosine phosphate (ANP) by the spatial combination of PL with ZA. A homogeneous time-resolved fluorescence assay further illustrated the direct competitiveness of the dual drugs against ANP docking to phosphorylated JNK/Erk, contributing to the inhibited downstream expression of c-Jun/c-Fos/NFATc-1 (nuclear factor of activated T cells, cytoplasmic, calcineurin-dependent 1). Then, in vivo testing demonstrated that the combined administration of PL and ZA attenuated breast cancer growth in the bone microenvironment. Additionally, these molecules prevented the destruction of proximal tibia, with significant reduction of tartrate-resistant acid phosphatase (TRAcP)-positive osteoclast cells and potentiation of apoptotic cancer cells, to a greater extent when combined than when the drugs were applied independently. Altogether, the combination treatment with PL and ZA could significantly and synergistically suppress osteoclastogenesis and inhibit tumorigenesis both in vitro and in vivo by simulating the spatial structure of ANP to inhibit competitively phosphorylation of c-Jun N-terminal kinase/extracellular signal-regulated kinase (JNK/Erk).

Topics: Adenine Nucleotides; Animals; Antineoplastic Combined Chemotherapy Protocols; Breast Neoplasms; Cell Line, Tumor; Diphosphonates; Disease Models, Animal; Drug Synergism; Female; Humans; Imidazoles; JNK Mitogen-Activated Protein Kinases; MAP Kinase Signaling System; Mice; Mice, Inbred BALB C; Mice, Nude; Naphthoquinones; Osteoclasts; Osteolysis; Phosphorylation; Random Allocation; Signal Transduction; Zoledronic Acid

Bone loss is one of the major complications of advanced cancers such as breast cancer, prostate cancer, and multiple myeloma; agents that can suppress this bone loss have therapeutic potential. Extensive research within the last decade has revealed that RANKL, a member of the tumor necrosis factor superfamily, plays a major role in cancer-associated bone resorption and thus is a therapeutic target. We investigated the potential of vitamin K3 analogue plumbagin (derived from Chitrak, an Ayurvedic medicinal plant) to modulate RANKL signaling, osteoclastogenesis, and breast cancer-induced osteolysis. Plumbagin suppressed RANKL-induced NF-κB activation in mouse monocytes, an osteoclast precursor cell, through sequential inhibition of activation of IκBα kinase, IκBα phosphorylation, and IκBα degradation. Plumbagin also suppressed differentiation of these cells into osteoclasts induced either by RANKL or by human breast cancer or human multiple myeloma cells. When examined for its ability to prevent human breast cancer-induced bone loss in animals, plumbagin (2 mg/kg body weight) administered via the intraperitoneal route significantly decreased osteolytic lesions, resulting in preservation of bone volume in nude mice bearing human breast tumors. Overall, our results indicate that plumbagin, a vitamin K analogue, is a potent inhibitor of osteoclastogenesis induced by tumor cells and of breast cancer-induced osteolytic metastasis through suppression of RANKL signaling.

Topics: Animals; Antineoplastic Agents, Phytogenic; Blotting, Western; Bone Neoplasms; Bone Resorption; Breast Neoplasms; Cell Line; Cell Line, Tumor; Female; Humans; I-kappa B Proteins; Macrophages; Mice; Mice, Inbred BALB C; Mice, Nude; Naphthoquinones; NF-kappa B; NF-KappaB Inhibitor alpha; Osteoclasts; Osteogenesis; Osteolysis; RANK Ligand; Signal Transduction; Xenograft Model Antitumor Assays

Bone metastasis is a common and serious consequence of breast cancer. Bidirectional interaction between tumor cells and the bone marrow microenvironment drives a so-called 'vicious cycle' that promotes tumor cell malignancy and stimulates osteolysis. Targeting these interactions and pathways in the tumor-bone microenvironment has been an encouraging strategy for bone metastasis therapy. In the present study, we examined the effects of plumbagin on breast cancer bone metastasis. Our data indicated that plumbagin inhibited cancer cell migration and invasion, suppressed the expression of osteoclast-activating factors, altered the cancer cell induced RANKL/OPG ratio in osteoblasts, and blocked both cancer cell- and RANKL-stimulated osteoclastogenesis. In mouse model of bone metastasis, we further demonstrated that plumbagin significantly repressed breast cancer cell metastasis and osteolysis, inhibited cancer cell induced-osteoclastogenesis and the secretion of osteoclast-activating factors in vivo. At the molecular level, we found that plumbagin abrogated RANKL-induced NF-κB and MAPK pathways by blocking RANK association with TRAF6 in osteoclastogenesis, and by inhibiting the expression of osteoclast-activating factors through the suppression of NF-κB activity in breast cancer cells. Taken together, our data demonstrate that plumbagin inhibits breast tumor bone metastasis and osteolysis by modulating the tumor-bone microenvironment and that plumbagin may serve as a novel agent in the treatment of tumor bone metastasis.

Topics: Actins; Animals; Antigens, Differentiation; Antineoplastic Agents; Bone and Bones; Bone Density Conservation Agents; Bone Neoplasms; Breast Neoplasms; Cell Differentiation; Cell Line, Tumor; Cell Survival; Cytokines; Enzyme Activation; Female; Gene Expression; Humans; Inhibitory Concentration 50; Macrophages; Mice; Mice, Inbred BALB C; Mice, Nude; Mitogen-Activated Protein Kinases; Naphthoquinones; Neoplasm Invasiveness; NF-kappa B; Osteoclasts; Osteolysis; Protein Binding; RANK Ligand; Receptor Activator of Nuclear Factor-kappa B; Signal Transduction; TNF Receptor-Associated Factor 6; Tumor Microenvironment; Xenograft Model Antitumor Assays