naphthoquinones and Bone-Neoplasms

Although chondrosarcoma is the second most common primary malignant bone tumor, treatment options are limited due to its extensive resistance to a chemo- and radiation therapy. Since shikonin has shown potent anticancer activity in various types of cancer cells, it represents a promising compound for the development of a new therapeutic approach.. The dose-relationships of shikonin and its derivatives acetylshikonin and cyclopropylshikonin on two human chondrosarcoma cell lines were measured using the CellTiter-Glo®. The changes in the cell cycle were presented by flow cytometry. Protein phosphorylation and expression apoptotic markers, MAPKs and their downstream targets were analyzed using western blotting and gene expression were evaluated using RT-qPCR.. These data demonstrated the significant anti-tumorigenic effect of shikonin derivatives in chondrosarcoma and encourage further research.

Topics: Apoptosis; Bone Neoplasms; Cell Cycle Checkpoints; Cell Line, Tumor; Chondrosarcoma; Humans; Mitogen-Activated Protein Kinases; Naphthoquinones; Receptors, Death Domain

Prostate cancer (PC) metastasizes to the skeleton forming predominantly sclerotic lesions, and there is currently no cure for bone metastatic disease. The transcription factor signal transducer and activator of transcription 3 (STAT3) is implicated as a metastatic driver, but its potential as therapeutic target in bone metastasis has not been investigated. In this study, we evaluated for the first time a STAT3 inhibitor, Napabucasin, as a therapeutic option for bone metastatic PC.. Effects of STAT3 inhibitors, Stattic and Napabucasin, on metastatic potential in PC cells were studied in vitro by assessment of migration capacity, self-renewal potential, and tumorsphere formation. For evaluation of the role of STAT3 in initial skeletal establishment of PC cells as well as in progressed castration-resistant PC (CRPC) in bone, human VCaP prostate cancer cells were inoculated in the tibia of mice which subsequently were treated with the STAT3 inhibitor Napabucasin. Bone specimens were analyzed using computed tomography (CT), immunohistochemistry, and quantitative polymerase chain reaction.. The small molecule STAT3 inhibitors Stattic and Napabucasin both effectively impaired metastatic potential of PC cells in vitro. Furthermore, treatment with Napabucasin prevented metastatic establishment in tibial bones in vivo and thereby also the tumor-induced sclerotic bone response seen in vehicle-treated VCaP xenografts. In addition, treatment with Napabucasin of established bone CRPC significantly decreased both tumor burden and tumor-induced trabecular bone volume compared with effects seen in vehicle-treated animals. Anti-mitotic effects were confirmed by decreased Ki67 staining in Napabucasin-treated xenografts compared with vehicle-treated xenografts. Alterations of gene expression in the femoral bone marrow (BM) niche toward the maintenance of hematopoietic stem cells and the myeloid lineage were demonstrated by quantitative real-time polymerase chain reaction and were further reflected by a substantial increase in the number of erythrocytes in BM of Napabucasin-treated mice. Furthermore, a unique pattern of STAT3 phosphorylation in osteoblasts/stromal cells surrounding the areas of tumor cells was demonstrated immunohistochemically in bone xenograft models using several different PC cell lines.. Inhibition of STAT3 activity disrupts the bone metastatic niche and targets both the skeletal establishment of PC and advanced bone metastatic CRPC in mice, suggesting STAT3 as a candidate for molecular targeted therapies of skeletal metastatic disease.

Topics: Animals; Benzofurans; Bone Neoplasms; Cell Line, Tumor; Cell Proliferation; Cyclic S-Oxides; Male; Mice; Naphthoquinones; Prostatic Neoplasms; STAT3 Transcription Factor; Tibia

Osteosarcoma (OS) is a malignant bone neoplasm, mostly occurring in pediatric patients. OS is characterized by a highly aggressive and metastatically active tumor. Chemotherapy followed by surgical excision is the treatment of choice but is often associated with both chemoresistance and relapse. Hence, it is important to develop further understanding of OS pathogenesis and identify potential therapeutic targets. Both the signal transducer and activator of transcription 3 (STAT3) and mammalian target of rapamycin (mTOR) have been implicated in OS pathogenesis. Crosstalk between mTOR and STAT3 signaling has been shown to regulate hypoxia-induced angiogenesis in other diseases. In this study, we determined using OS cell lines if there is a crosstalk between these two pathways and how that impacts sensitivity to treatment with Rapamycin. OS cell lines exhibited differential sensitivity to mTOR inhibitor Rapamycin. Evaluation of phosphorylated STAT3 showed that in Rapamycin-sensitive 143B cells, the inhibitor decreased phosphorylation of STAT3 at Y705, but not at S727 whereas, in Rapamycin-resistant U2OS cells, the inhibitor decreased S727 phosphorylation but not Y705. However, knockdown of STAT3 in U2OS cells made them sensitive to Rapamycin. Immunofluorescence (IF) analysis showed that mTOR is constitutively activated in the 143B cells but is suppressed in the U2OS cells, indicating that this might be their reason for being resistant to Rapamycin. Both cell lines were sensitive to treatment with the STAT3 inhibitor Napabucasin (NP). Treatment with NP inhibited STAT3 activation at Y705 and additionally inhibited mTOR activation, indicating crosstalk between STAT3 and mTOR signaling pathways. Rapamycin could effectively prevent lung metastasis in an orthotropic OS mice model using 143B cells. However, Rapamycin could not inhibit lung metastasis in mice injected with U2OS cells. The STAT3 inhibitor NP attenuated lung metastasis with the U2OS cells. Our results thus established yet undefined crosstalk of STAT3 and mTOR signaling pathways in OS and highlight the possibility of using mTOR inhibitors for treatment in patients with OS.

Topics: Animals; Antibiotics, Antineoplastic; Benzofurans; Bone Neoplasms; Cell Line, Tumor; Drug Resistance, Neoplasm; Female; Humans; Mice, Inbred BALB C; Naphthoquinones; Osteosarcoma; Phosphorylation; Signal Transduction; Sirolimus; STAT3 Transcription Factor; TOR Serine-Threonine Kinases; Xenograft Model Antitumor Assays

Canine osteosarcoma (OSA) is an aggressive and highly malignant primary bone tumor. Its poor survival outcome remains problematic despite recent advances in anti-cancer therapy, therefore highlighting the need for alternative treatment options or drug repositioning. The aim of this study was to determine if YM155, a small-molecule survivin inhibitor, potentiates the chemotherapeutic efficacy of etoposide against canine OSA in vitro and in vivo. In cell culture, YM155 enhanced the cytotoxic effect of etoposide against canine OSA cell lines; however, the molecular mechanism behind this effect was heterogeneous, as only one cell line had an elevated apoptotic level. In addition, this effect was not associated with survivin suppression in two of the cell lines. These results suggest that the molecular target of YM155 is not restricted to survivin alone. When tested on a murine xenograft model, the average tumor volume of the combination treatment group (YM155, 5 mg/kg, intraperitoneally, 5 consecutive days/week; and etoposide, 20 mg/kg, intraperitoneally, every 5 days) was 66% smaller than the control group, although this difference was not statistically significant (P=0.17). Further studies to improve the treatment protocol are necessary to confirm the findings of this study.

Topics: Animals; Antineoplastic Agents; Apoptosis; Bone Neoplasms; Cell Proliferation; Dog Diseases; Dogs; Drug Synergism; Etoposide; Humans; Imidazoles; Mice; Naphthoquinones; Osteosarcoma; Survivin; Xenograft Model Antitumor Assays

Chordoma, slow growing bone tumours originating from remnants of the notochord, leave affected patients with a median survival of six years. The high recurrence rate of chordoma, together with limited treatment options and bad overall prognosis, make the development of new treatment options urgently necessary.. In this study, the potential of two natural products, silibinin and β-β-dimethylacrylshikonin (DMAS), was tested on clival (MUG-CC1 and UM-Chor1) as well as sacral (MUG-Chor1 and U-CH2) chordoma cell lines. The treatment was administered both as single- and combined therapy.. For investigation of cell viability, the Cell Titer 96 Aqueous Non-Radioactive Cell Proliferation Assay Kit was used. Apoptosis induction was studied by flow cytometry, (Annexin V/SYTOX Green, caspase-3) and RT-qPCR. Pathway analyses were performed by western blot.. Both drugs were found to reduce cell viability alone as well as in combination in a dose dependent manner, with DMAS being more efficient than silibinin. The mode of cell death was mainly apoptosis in DMAS treated samples, while the combination therapy led to apoptosis as well as late-apoptosis/necrosis. Silibinin therapy alone, although reducing cell viability, did not lead to significant apoptotic effects in the performed assays. Focussing on the molecular mechanism of DMAS induced apoptosis, it was found that major genes of the mitochondrial apoptosis pathway, like NOXA and PUMA were overexpressed. Additionally, western blot experiments showed a decrease of ERK/pERK, STAT3/pSTAT3 (Tyr705) and AKT/pAKT expression/activation levels under DMAS treatment.. DMAS is a promising new candidate for chordoma therapy, while silibinin or a combination of both is less favourable.

Topics: Antineoplastic Agents, Phytogenic; Apoptosis; Bone Neoplasms; Boraginaceae; Caspase 3; Cell Line, Tumor; Cell Survival; Chordoma; Humans; Mitochondria; Naphthoquinones; Plant Roots; Signal Transduction; Silybin; Silymarin

Osteosarcoma is the most common bone cancer. Despite advances, molecular mechanisms associated with osteosarcoma have not been fully understood. Hence, an effective treatment for osteosarcoma has yet to be developed. Even though signal transducer and activator of transcription3 (STAT3) has been implicated, its role in pathogenesis of osteosarcoma is not fully determined. In this study, we investigated the antitumor effect of napabucasin (NP) (BBI608), an inhibitor of STAT3 on osteosarcoma in vitro and in vivo and studied the underlying molecular mechanism.. Cell viability, colony formation, apoptosis, tumor growth and metastasis assays were performed to examine the effect of NP on osteosarcoma in vitro and in vivo. Real-time RT-PCR, western analysis, immunofluorescence and reporter assays were used to monitor the expression and activity of proteins and underlying molecular pathways. Protein synthesis, co-immunoprecipitation and CAP binding assays were carried out to understand NP-mediated mechanism of actions in osteosarcoma cells.. Our results show that NP treatment decreases cell viability and induces apoptosis in several osteosarcoma cell lines. NP treatment suppresses both expression and phosphorylation of STAT3 in addition to blocking STAT3-mediated transcription and downstream target proteins in osteosarcoma cells. Furthermore, NP inhibits protein synthesis through regulation of the eukaryotic initiation factor 4E (eIF4E) and eIF4E-binding protein 1 (4E-BP1). NP also inhibits the progression of osteosarcoma tumors and metastasis in vivo in an orthotopic tibial model of osteosarcoma.. Taken together, our investigation reveals that NP acts through a novel mechanism and inhibits osteosarcoma growth and metastasis, and could be investigated clinically for treating osteosarcoma patients alone or in combination with other drugs.

Topics: Animals; Apoptosis; Benzofurans; Bone Neoplasms; Cell Line, Tumor; Female; Humans; Mice; Mice, Inbred BALB C; Mice, Nude; Naphthoquinones; Neoplasm Metastasis; Osteosarcoma; Protein Synthesis Inhibitors; Random Allocation; STAT3 Transcription Factor; Xenograft Model Antitumor Assays

Osteosarcoma is the most common primary malignant bone tumor. Cancer cells employ a host of mechanisms to develop resistance to adriamycin (ADM) or other chemotherapeutic drugs. Shikonin (SK), an active constituent extracted from a Chinese medicinal herb, has been shown to cooperate with ADM in the treatment of osteosarcoma and certain other types of cancer by contributing to the response rate of chemotherapy and the side effects. The aim of the present study was to investigate the role and underlying mechanism of SK in chemotherapy for osteosarcoma. In the present study, a CCK-8 assay was performed to assess cell survival rate in vitro. Western blot analysis was performed to determine the expression levels of B‑cell lymphoma 2‑associated X protein (Bax), caspase‑3, caspase‑8, and poly (ADP‑ribose) polymerase (PARP). Flow cytometry was used to analyze cell cycle and cell death. The survival rate of cells decreased significantly in a dose‑ and time‑dependent manner when treated with a combination of SK and ADM. Western blot analysis revealed increased expression levels of Bax, caspase‑3, caspase‑8 and PARP in U2OS and MG63 cells 48 h following treatment with SK and ADM. Flow cytometric analysis showed that the combined treatment of SK and ADM significantly induced apoptosis in the osteosarcoma cells. Taken together SK cooperated with ADM to promote apoptosis, possibly by inducing caspase‑3‑ and caspase‑8‑dependent apoptosis. SK may be a potential enhancer in the treatment of drug‑resistant primary osteosarcoma.

Topics: Antibiotics, Antineoplastic; Apoptosis; Bone Neoplasms; Caspase 3; Caspase 8; Cell Line, Tumor; Cell Proliferation; Cell Survival; Doxorubicin; Drug Synergism; Humans; Naphthoquinones; Osteosarcoma

The early detection and thus treatment of breast cancer bone metastasis remain a big challenge clinically. As the most abundant cells within bone tissue, osteocytes have been found to manipulate the activity of early cancer bone metastasis by its crosstalk with cancer cells and osteoclasts. However, conventional bone-targeting nanomedicine has limited bone-lesion specificity and ignores the vital role of osteocytes during breast cancer bone metastasis. Also, it lacks detailed insight into the therapeutic mechanisms, which hinders the following translational practice. Previously, we have shown that a combination of zoledronic acid (ZA) and plumbagin (PL) synergistically alleviates cancer-induced bone destruction. Herein, we further develop a pH-responsive bone-targeting drug delivery system, i.e., the ZA-anchored bimodal mesoporous slica covered gadolinium(III) upconversion nanoparticles loaded with PL, to detect and treat bone metastasis sensitively and specifically at an early stage. This multifunctional nanosystem can target osteocytes to release PL as controlled by pH, decreasing osteocytic RANKL expression synergistically through the structural simulation of adenosine phosphate, which competitively inhibits the phosphorylation of osteocytic protein kinase-a, cAMP-response element binding protein, extracellular regulated protein kinase, and c-Jun N-terminal kinase. More importantly, by establishing a breast cancer bone metastasis mice model via intracardiac injection, we show that tumoriogenesis and osteoclastogenesis can both be attenuated significantly. We thereby realize the effective theranostics of tiny bone metastasis in breast cancer bone metastasis. Our work highlights the significance of theranostic nanomedicine and osteocyte-targeting therapy in the treatment of early bone metastasis, which could be applied in achieving efficient theranostic effects for other bone diseases.

Topics: Animals; Antineoplastic Agents, Phytogenic; Bone Density Conservation Agents; Bone Neoplasms; Breast Neoplasms; Cell Line, Tumor; Delayed-Action Preparations; Drug Delivery Systems; Female; Gadolinium; Humans; Luminescent Agents; Mice; Mice, Nude; Nanoparticles; Naphthoquinones; Optical Imaging; Osteocytes; Silicon Dioxide; Theranostic Nanomedicine; Zoledronic Acid

Osteosarcoma is the most common primary bone tumor that occurs in children and adolescents. Osteosarcoma has a poor prognosis and is often unresponsive to chemotherapy. Therefore, it remains a challenge to identify a novel strategy to effectively treat osteosarcoma. The present study demonstrated a novel opportunity in osteosarcoma treatment using the natural compound plumbagin. Plumbagin reduced cell viability in osteosarcoma cells but not normal bone cells, as determined by MTT assay and colony formation assay. Plumbagin induced cell apoptosis by mitochondrial dysfunction, which in turn promoted Ca2+ release and endoplasmic reticulum (ER)‑stress, as determined by DAPI staining assay, DNA fragmentation assay, flow cytometry and western blotting analysis. In addition, plumbagin improved reactive oxygen species (ROS) generation, as determined by flow cytometry. Finally, these apoptotic cascades activated caspase‑3 and caspase‑9 to elicit apoptosis response. Our results demonstrated the anticancer effect of plumbagin by inducing cell apoptosis in osteosarcoma cells. In conclusion, plumbagin activated the apoptosis signaling pathway through eliciting ROS, ER stress, mitochondria dysfunction, and finally causing caspase activation. These results indicated that plumbagin may serve as potential antitumor drug by its multifunctional effects in osteosarcoma.

Topics: Antineoplastic Agents, Phytogenic; Apoptosis; Bone Neoplasms; Calcium; Caspases; Cell Cycle; Cell Line, Tumor; Cell Survival; Endoplasmic Reticulum Stress; Humans; Mitochondria; Models, Biological; Naphthoquinones; Osteosarcoma; Reactive Oxygen Species; Signal Transduction

Chimaphilin, an active compound separated from pyrola, possesses the highly efficient antitumor activities. Insulin-like growth factor-I receptor (IGF-IR) plays an important role in tumor cell survival. To look for effective strategies for interrupting IGF-IR signaling pathway, we found that chimaphilin can inhibit the receptor tyrosine kinase activity of IGF-IR. Chimaphilin inhibited the growth of both drug-sensitive and drug-resistant osteosarcoma cell lines in a time and dose-dependent manner; however, it showed relatively little toxicity in normal osteoblast cell lines. Chimaphilin can increase the sensitivity of doxorubicin in doxorubicin-resistant osteosarcoma cell lines. Additionally, small interfering RNA downregulation of IGF-IR expression in drug-resistant cell lines also caused resensitization to doxorubicin. Above all, we conclude that chimaphilin represents a valuable natural source and may potentially be applicable for reversing the drug-resistant phenotype in osteosarcoma therapy.

Topics: Apoptosis; Bone Neoplasms; Cell Line, Tumor; Cell Proliferation; Drug Resistance, Neoplasm; Humans; Naphthoquinones; Osteosarcoma; Receptor, IGF Type 1

YM155, a novel small-molecule inhibitor of survivin, is known to exert antitumor effects on various cancers, including breast, prostate and lung cancer. However, there are few studies describing the inhibitory effect of YM155 on human osteosarcoma (OS) which highly expresses survivin. Here, we tested the effects of YM155 on OS cells by several in vitro experiments. It was found that YM155 inhibited cell proliferation, colony formation, migration and invasion, induced cell apoptosis, as well as increased caspase-3, -8 and -9 activity in the OS cell lines in a dose-dependent manner. We also found that YM155 suppressed Mcl-1 and survivin expression without affecting the expression of anti-apoptotic proteins X-linked inhibitor of apoptosis (XIAP) and Bcl-2. In addition, YM155 decreased phosphoinositide 3-kinase (PI3K) and AKT expression without effecting total PI3K and AKT in the OS cell lines, which contributed to suppression of OS tumor growth at least in part. In addition, YM155 also suppressed tumor growth in vivo, reducing the size of OS MG63 cell xenografts. Taken together, the findings revealed that YM155 suppresses the tumor growth of OS in vitro and in vivo, suggesting that YM155 has potential as a therapeutic agent for the treatment of OS.

Topics: Animals; Antineoplastic Agents; Bone Neoplasms; Cell Movement; Cell Proliferation; Gene Expression Regulation, Neoplastic; Humans; Imidazoles; Male; Mice; Naphthoquinones; Osteosarcoma; Xenograft Model Antitumor Assays

Chemoresistance is the principal reason for poor survival and disease recurrence in osteosarcoma patients. Survivin, a family member of the inhibitor of apoptosis proteins, plays an important role in inhibition of apoptosis. Survivin is expressed in a vast majority of human cancers, which is often correlated with poor prognosis in a wide variety of cancer patients. Furthermore, survivin expression is often related with chemoresistance in cancer cells, including osteosarcoma (OS). Here, we evaluated the therapeutic potential of YM155, a selective survivin suppressant alone and in combination with cisplatin using human OS models.. U-2 OS, SW1353, MG-63 cells were treated with YM155, and/or cisplatin, and cell viability, apoptosis, survivin protein expression levels were then evaluated. Furthermore, the efficacy of YM155 combined with cisplatin was further examined in established xenograft models.. YM155 was sufficient to induce spontaneous apoptosis of OS cells. Combination with YM155 significantly augmented the cytotoxicity of cisplatin in OS cells. Combination treatment of YM155 and cisplatin showed antiproliferative effects and induced a greater rate of apoptosis than the sum of the single-treatment rates and promoted tumor regression in established OS xenograft models.. Our findings provide evidence that YM155 could act as a survivin inhibitor on OS cells. Chemotherapeutic approaches using YM155 might enhance the benefit of the cisplatin in the treatment of OS cells. YM155 could be further developed as a potential therapeutic agent for the treatment of OS.

Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Bone Neoplasms; Cell Line, Tumor; Cell Survival; Cisplatin; Drug Resistance, Neoplasm; Humans; Imidazoles; Inhibitor of Apoptosis Proteins; Mice; Mice, Inbred ICR; Models, Animal; Naphthoquinones; Neoplasm Recurrence, Local; Osteosarcoma; Survivin; Xenograft Model Antitumor Assays

To investigate the effects of plumbagin, a naphthoquinone derived from the medicinal plant Plumbago zeylanica, on human breast cancer cell growth and the cancer cell-induced osteolysis in the bone microenvironment of mice.. Human breast cancer cell subline MDA-MB-231SA with the ability to spread and grow in the bone was tested. The cell proliferation was determined using the CCK-8 assay. Apoptosis was detected with Annexin V/PI double-labeled flow cytometry. Red fluorescent protein-labeled MDA-MB-231SArfp cells were injected into the right tibia of female BALB/c-nu/nu mice. Three days after the inoculation, the mice were injected with plumbagin (2, 4, or 6 mg/kg, ip) 5 times per week for 7 weeks. The growth of the tumor cells was monitored using an in vivo imaging system. After the mice were sacrificed, the hind limbs were removed for radiographic and histological analyses.. Plumbagin (2.5-20 μmol/L) concentration-dependently inhibited the cell viability and induced apoptosis of MDA-MB-231SA cells in vitro (the IC50 value of inhibition of cell viability was 14.7 μmol/L). Administration of plumbagin to breast cancer bearing mice delayed the tumor growth by 2-3 weeks and reduced the tumor volume by 44%-74%. The in vivo imaging study showed that plumbagin dose-dependently inhibited MDA-MB-231SArfp cell growth in bone microenvironment. Furthermore, X-ray images and micro-CT study demonstrated that plumbagin reduced bone erosion area and prevented a decrease in bone tissue volume. Histological studies showed that plumbagin dose-dependently inhibited the breast cancer cell growth, enhanced the cell apoptosis and reduced the number of TRAcP-positive osteoclasts.. Plumbagin inhibits the cell growth and induces apoptosis in human breast cancer cells in mice bone microenvironment, leading to significant reduction in osteolytic lesions caused by the tumor cells.

Topics: Animals; Antineoplastic Agents, Phytogenic; Bone Neoplasms; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Cell Survival; Dose-Response Relationship, Drug; Female; Humans; Mice; Mice, Inbred BALB C; Mice, Nude; Naphthoquinones; Osteoclasts; Tumor Microenvironment

Osteosarcoma is the most frequent primary malignant bone tumor, notorious for its lung metastasis. Shikonin, an effective constituent extracted from Chinese medicinal herb, was demonstrated to induce necroptosis in some cancers.. MTT assay was performed to detect cell survival rate in vitro. Flow cytometry was used to analyze cell cycle and cell death. Western blot was performed to determine the expression levels of RIP1, RIP3, caspase-3, caspase-6 and PARP. The tibial primary and lung metastatic osteosarcoma models were used to evaluate the anti-tumor effect of shikonin in vivo.. The cell survival rate was decreased in a dose and time dependent manner when treated with shikonin. No major change in cell cycle was observed after shikonin treatment. The cell death induced by shikonin could be mostly rescued by specific necroptosis inhibitor necrostatin-1, but not by general caspase inhibitor Z-VAD-FMK. The number of necrotic cells caused by shikonin was decreased after being pretreated with Nec-1 detected by flow cytometry in K7 cells. After 8-hour treatment of shikonin, the expression levels of RIP1 and RIP3 were increased while caspase-3, caspase-6 and PARP were not activated in K7 and U2OS cells determined by Western blot. Size of primary tumor and lung metastasis in shikonin treated group were significantly reduced. The protein levels of RIP1 and RIP3 in primary tumor tissues were increased by shikonin. The overall survival of lung metastatic models was longer compared with control group (p < 0.001).. Shikonin had prompt but profound anti-tumor effect on both primary and metastatic osteosarcoma, probably by inducing RIP1 and RIP3 dependent necroptosis. Shikonin would be a potential anti-tumor agent on the treatment of primary and metastatic osteosarcoma.

Topics: Animals; Antineoplastic Agents; Apoptosis; Bone Neoplasms; Cell Line, Tumor; Cell Survival; Drug Screening Assays, Antitumor; Drugs, Chinese Herbal; Female; Humans; Lung Neoplasms; Mice; Mice, Inbred BALB C; Naphthoquinones; Necrosis; Neoplasm Transplantation; Nuclear Pore Complex Proteins; Osteosarcoma; Receptor-Interacting Protein Serine-Threonine Kinases; RNA-Binding Proteins; Up-Regulation

BIRC5 (Survivin), an inhibitor of apoptosis protein (IAP), is over-expressed in several human cancers and increased expression is associated with poor prognosis. The goal of the current study was to evaluate the role of BIRC5 in Ewing sarcoma (ES), the second most common pediatric bone sarcoma.. BIRC5 protein expression was determined in ES cell lines using Western Blot analysis. Functional role of survivin on growth and viability of ES cells was assessed by siRNA knockdown of BIRC5 and by using a small molecule inhibitor YM155. Immunohistochemical analysis for BIRC5 protein was performed on patient tumor samples using an anti-survivin antibody. The degree of BIRC5 protein expression was correlated with clinical parameters and patient outcome.. BIRC5 is over-expressed in a panel of ES cell lines. Gene silencing of BIRC5 in the ES cell line TC-71 decreases cell growth by more than 50% for each BIRC5 siRNA construct compared to non-silencing siRNA control constructs. YM155 also reduces ES cell growth and viability with an EC(50) ranging from 2.8 to 6.2 nM. BIRC5 protein is expressed in majority of the ES tumor samples with minimal expression in normal tissue (P < 0.005). Tumors with more than 50% expression are associated with worse overall survival than tumors with less than 50% expression (Hazard Ratio: 6.05; CI: 1.7-21.4; P = 0.04).. BIRC5 is over-expressed in ES cell lines and tumor samples. Further, it plays an important role in cell growth and viability in vitro. Higher degree of expression in patients is an independent poor prognostic factor.

Topics: Adolescent; Adult; Apoptosis; Biomarkers; Bone Neoplasms; Cell Line, Tumor; Cell Proliferation; Child; Child, Preschool; Female; Humans; Imidazoles; Inhibitor of Apoptosis Proteins; Male; Naphthoquinones; Prognosis; Sarcoma, Ewing; Survivin

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

Shikonin, a major ingredient in the Chinese traditional herb Lithospermum erythrorhixon, exhibits multiple biological functions including antimicrobial, anti-inflammatory, and antitumor effects. In this study, we delineated the molecular mechanisms of shikonin in the apoptosis of 143B osteosarcoma cells. Shikonin reduced the cell viability of 143B cells in a dose- and time-dependent manner. The IC(50) at 24 h and 48 h for 143B cells was 4.55 and 2.01microM, respectively. A significantly elicited hypodiploid cell population was found in cells treated with 2, 4, and 8microM shikonin for 24 h. Moreover, treatment with shikonin induced reactive oxygen species (ROS) generation, increased extracellular signal-regulated kinase (ERK) phosphorylation, decreased B-cell lymphoma-2 (Bcl2) expression, and was accompanied by poly(ADP-ribose) polymerase (PARP) cleavage. Pretreatment with the antioxidant agent N-acetyl cysteine (NAC) not only reversed shikonin-induced ROS generation but also significantly attenuated the cytotoxic effects of shikonin in 143B cells. Furthermore, NAC attenuated shikonin-induced ERK phosphorylation. Taken together, our results reveal that shikonin increased ROS generation and ERK activation, and reduced Bcl2, which consequently caused the cells to undergo apoptosis. Therefore, shikonin may be a promising chemotherapeutic agent for osteosarcoma treatment.

Topics: Acetylcysteine; Anti-Inflammatory Agents, Non-Steroidal; Antineoplastic Agents, Phytogenic; Antioxidants; Apoptosis; Bone Neoplasms; Cell Line, Tumor; Dose-Response Relationship, Drug; Drugs, Chinese Herbal; Extracellular Signal-Regulated MAP Kinases; Humans; Inhibitory Concentration 50; Naphthoquinones; Osteosarcoma; Phosphorylation; Phytotherapy; Poly(ADP-ribose) Polymerases; Proto-Oncogene Proteins c-bcl-2; Reactive Oxygen Species

beta-Lapachone, a novel anticancer drug, induces various human carcinoma cells to undergo apoptotic cell death. However, we report here that, in human osteocarcinoma (U2-OS) cells, beta-lapachone induces necrosis rather than apoptosis. beta-Lapachone-induced necrotic cell death in U2-OS cells was characterized by propidium iodide uptake, cytochrome c release, a decreased mitochondrial membrane potential, and ATP depletion. The mitochondrial potential transition (MPT), including the reduction of the mitochondrial transmembrane potential and the release of mitochondrial cytochrome c, occurred in beta-lapachone-treated cells; cotreatment of these cells with cyclosporin A, an inhibitor of MPT pore, failed to prevent necrotic cell death. This indicates that the MPT transition does not play a crucial role in this process. Furthermore, beta-lapachone-induced necrosis was independent of oxidative stress and caspase activation. However, excessive poly(ADP-ribose) polymerase (PARP) activation and subsequent depletion of intracellular NAD(+) and ATP were seen in beta-lapachone-treated U2-OS cells. Cotreatment with a PARP inhibitor, 3-aminobenzamide, decreased beta-lapachone-induced PARP activation and provided significant protection from necrosis by preventing depletion of intracellular NAD(+) and ATP. Taken together, our results suggest that PARP plays an important role in the signaling pathway for beta-lapachone-induced necrosis in U2-OS cells.

Topics: Adenosine Triphosphate; Antineoplastic Agents, Phytogenic; Apoptosis; Blotting, Western; Bone Neoplasms; Cell Cycle; Cytochrome c Group; DNA Damage; DNA Fragmentation; Enzyme Activation; Flow Cytometry; Genes, p53; Humans; In Situ Nick-End Labeling; Membrane Potentials; NAD; Naphthoquinones; Osteosarcoma; Poly(ADP-ribose) Polymerase Inhibitors; Poly(ADP-ribose) Polymerases; Reactive Oxygen Species; Signal Transduction; Tumor Cells, Cultured