naphthoquinones and Osteosarcoma

Reportedly, the elevated expression of survivin has been observed in several tumor types, strictly involved in tumor development. In the present study, we detected elevated survivin expression in tumor tissues derived from patients with chemoresistant osteosarcoma when compared with those from chemosensitive patients. Importantly, knockdown of survivin in osteosarcoma cells significantly suppressed cell proliferation and chemoresistance both in vitro and in vivo. Simultaneously, chemotherapy mediates the upregulation of survivin in osteosarcoma cells through a survivin-based selective killing effect, resulting in the development of multidrug resistance. The utilization of tumor-derived microparticles to coencapsulate the survivin inhibitor YM155 and chemotherapeutic agents could effectively reverse multidrug resistance, leading to improved anticancer effects, as well as reduced systemic toxicity. In summary, the expression of survivin contributes to resistance toward osteosarcoma drugs, whereas employing survivin inhibiting combination therapy, based on a microparticle codelivery system, could efficiently reverse resistance and avoid potential systemic toxicity.

Topics: Animals; Cell Line, Tumor; Cell Survival; Drug Resistance, Multiple; Drug Resistance, Neoplasm; Humans; Imidazoles; Mice; Mice, Inbred BALB C; Naphthoquinones; Osteosarcoma; Survivin

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

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

Epithelial-to-mesenchymal transition is a cellular process associated with cancer invasion and metastasis. However, the antimetastatic effects of chimaphilin remain elusive. In this study, we attempted to investigate the potential use of chimaphilin as an inhibitor of TGF-β1-induced epithelial-to-mesenchymal transition in U2OS cells. We found that TGF-β1 induced epithelial-to-mesenchymal transition to promote U2OS cell invasion and metastasis. Western blotting demonstrated that chimaphilin inhibited U2OS cell invasion and migration, increased the expression of the epithelial phenotype marker E-cadherin, repressed the expression of the mesenchymal phenotype marker vimentin, as well as decreased the level of epithelial-to-mesenchymal-inducing transcription factors Snail1 and Slug during the initiation of TGF-β1-induced epithelial-to-mesenchymal transition. In this study, we revealed that chimaphilin up-regulated the E-cadherin expression level and inhibited the production of vimentin, Snail1, and Slug in TGF-β1-induced U2OS cells by blocking PI-3K/Akt and ERK 1/2 signaling pathway. Additionally, the TGF-β1-mediated phosphorylated levels of Smad2/3 were inhibited by chimaphilin pretreatment. Above all, we conclude that chimaphilin represents an effective inhibitor of the metastatic potential of U2OS cells through suppression of TGF-β1-induced epithelial-to-mesenchymal transition.

Topics: Biomarkers, Tumor; Cadherins; Cell Death; Cell Line, Tumor; Cell Movement; Epithelial-Mesenchymal Transition; Extracellular Signal-Regulated MAP Kinases; Gene Expression Regulation, Neoplastic; Humans; Naphthoquinones; Neoplasm Invasiveness; Neoplasm Metastasis; Osteosarcoma; Phosphatidylinositol 3-Kinases; Phosphorylation; Proto-Oncogene Proteins c-akt; Signal Transduction; Smad Proteins; Transcription Factors; Transforming Growth Factor beta1; Vimentin

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

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

Survivin is an important oncogenic protein expressed highly in osteosarcoma. Here, we have shown that small molecule inhibitor YM155 potently suppressed survivin expression, inhibited cell growth, and induced apoptosis in osteosarcoma cells. Furthermore, we also showed that knock down of survivin by small interfering RNA strongly inhibited cell viability in two osteosarcoma cell lines, suggesting that suppression of survivin essentially contributes to YM155-mediated anticancer activity in osteosarcoma cells. Collectively, our study suggests that YM155 holds promise for patients with osteosarcoma.

Topics: Antineoplastic Agents; Apoptosis; Cell Line, Tumor; Cell Survival; Gene Expression Regulation, Neoplastic; Gene Knockdown Techniques; Humans; Imidazoles; Inhibitor of Apoptosis Proteins; Naphthoquinones; Osteosarcoma; Survivin; Tumor Stem Cell Assay

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

Osteosarcoma (OS) is the most common primary malignant bone tumor, notorious for its metastasis. We have recently shown that shikonin, an effective constituent extracted from Chinese medicinal herb, induces necroptosis in OS cells. Nevertheless, the effects of low-dose shikonin on the invasiveness of OS cells are unknown. Here, we showed that shikonin dose-dependently decreased OS cell invasiveness in both scratch wound healing assay and transwell cell migration assay. Moreover, the direct target of shikonin on cell invasiveness was found to be matrix metalloproteinase (MMP)-13. Further, the inhibitory effects of shikonin on cell invasiveness were completely abolished in MMP13-overexpressing OS cells. Together, these data suggest that shikonin may suppress OS invasiveness through MMP13 suppression. Thus, our data highlight a previous unappreciated role for shikonin in suppressing OS cell metastasis.

Topics: Apoptosis; Cell Line, Tumor; Gene Expression Regulation, Neoplastic; Humans; Matrix Metalloproteinase 13; Naphthoquinones; Neoplasm Invasiveness; Osteosarcoma

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

Osteosarcoma (OS) is a primary malignant bone tumor in humans, and is notorious mainly for its distal metastases. We have recently shown that Shikonin, an effective constituent extracted from Chinese medicinal herb, inhibits OS cell invasion through suppression of matrix metalloproteinase 13 (MMP13). However, the underlying mechanisms remain unknown.. Here, we studied the levels of tumor necrosis factor (TNF)-alpha-induced protein 8-like 2 (TIPE2) in OS cells upon Shikonin treatment. TIPE2 levels were adapted in OS cell lines through transfection with plasmids carrying transgene or short-hairpin interference RNA (shRNA), and the effects of TIPE2 adaptation on MMP13 and cell invasiveness were evaluated by RT-qPCR, Western blot, ELISA and transwell cell migration assay, respectively. TIPE2 levels in OS specimens from patients were examined and correlated with cancer metastases and patient survival.. We found that Shikonin dose-dependently decreased MMP13 levels, and increased TIPE2 levels in two OS cell lines, U2OS and SaOS-2. Overexpression of TIPE2 in U2OS significantly suppressed MMP13 levels and cell invasiveness. Depletion of TIPE2 in SaOS-2 cells significantly increased MMP13 levels and cell invasiveness. Moreover, TIPE2 levels in OS specimens were significantly decreased, compared to adjacent non-cancer bone tissue. Lower TIPE2 levels correlated with higher incidence of metastases and worse 5-year survival.. TIPE2 mediates the suppressive effects of Shikonin on MMP13 in osteosarcoma cells, and TIPE2 may be a novel therapeutic target for OS.

Topics: Adolescent; Cell Line, Tumor; Child; Female; Humans; Intracellular Signaling Peptides and Proteins; Matrix Metalloproteinase 13; Naphthoquinones; Osteosarcoma

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

Osteosarcoma, which is the most common primary bone tumor, occurs most frequently in adolescents. A number of studies have indicated that plumbagin (PL) (5-hydroxy-2-methyl-1, 4-naphthoquinone), a compound found in the plants of the Plumbaginaceae and Droseraceae families, possesses anticancer activity. However, its anticancer effects and mechanisms against osteosarcoma have not been explored. To determine the anticancer effect of PL on osteosarcoma cell lines MG-63 and U2OS, cell viability, apoptosis, cell cycle distribution, caspase-3 and caspase-9 activity and intracellular reactive oxygen species (ROS) generation were measured, and Western blot analyses were performed. PL significantly inhibited the growth of osteosarcoma cells, particularly U2OS cells. PL up-regulated the expression of p53 in U2OS cells and p21 in the two osteosarcoma cell lines causing cell cycle arrest by decreasing the expression of murine double minute 2 (MDM2)/cyclin B1 and cyclin D1. Furthermore, PL altered the ratio of Bax/Bcl-2, and may have triggered the mitochondrial apoptotic pathway, resulting in caspase-3 and caspase-9 activation. We also found that PL induced the generation of ROS in osteosarcoma cell lines. To conclude, PL exerted anticancer activity on osteosarcoma cells by inducing pro-apoptotic signaling and modulating the intracellular ROS that causes induction of apoptosis. These effects may relate to the p53 status.

Topics: Antineoplastic Agents, Phytogenic; Apoptosis; Caspase 3; Caspase 9; Cell Cycle Checkpoints; Cell Line, Tumor; Cyclin B1; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p21; Humans; Naphthoquinones; Osteosarcoma; Proto-Oncogene Proteins c-mdm2; Reactive Oxygen Species; Tumor Suppressor Protein p53

The anti-cancer effect of β-lapachone (β-lap) is positively related to the cellular activity of NAD(P)H:quinone oxidoreductase (NQO1). Heat shock has been reported to elevate cellular NQO1. The effect of heating on the NQO1 expression in human osteosarcoma cells (HOS) and the response of the cells to the combined treatment with β-lap and hyperthermia was investigated.. The effects of β-lap alone, hyperthermia alone and in combination to cause clonogenic death and apoptosis in HOS cells were elucidated. The effect of heating on the NQO1 expression was evaluated with western blot analysis. The effect of β-lap on the cell cycle distribution was elucidated with flow cytometry and to cause DNA damage was determined by assessing the γH2AX foci formation.. Treatment of HOS cells with β-lap at 42°C was markedly more effective than that at 37°C in causing clonogenic cell death. Heating caused a long-lasting up-regulation of NQO1 in the cells, and sensitised the cells to β-lap. The γH2AX foci formation was increased immediately after β-lap treatment and preheating increased the β-lap-induced γH2AX foci formation.. The sensitivity of HOS cells to β-lap was increased not only during heating but also after heating as demonstrated by the increase in the clonogenic cell death and γH2AX foci formation. The increase in β-lap sensitivity after heating appeared to be due to the heat-induced elevation of NQO1 activity.

Topics: Cell Line, Tumor; Cell Survival; Combined Modality Therapy; Histones; Humans; Hyperthermia, Induced; NAD(P)H Dehydrogenase (Quinone); Naphthoquinones; Osteosarcoma

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

The effect of the novel anticancer 2-methylnaphtho[2,3-b]furan-4,9-dione (FNQ3) on human osteosarcoma cell lines (HuO9 and HuO9N2) was investigated. The IC50 values of FNQ3 were 5.95 microM for HuO9 and 3.86 microM for HuO9N2, while that for normal fibroblasts (WI-38 cell line) was 35.8 microM. The selectivity in antitumour activity which was estimated from the IC50 ratio of normal fibroblasts to tumour cells was 6.0 and 9.3 fold for HuO9 and HuO9N2, respectively. FNQ3 at 23.6 microM selectively injured mitochondria of HuO9 cells starting at 36 h and HuO9N2 cells at 24 h, whereas WI-38 cells were unaffected even after 72 h. These results demonstrated that FNQ3 was selectively toxic to the mitochondria of osteosarcoma cells similar to carcinoma cells (Pan et al. (1997) J. Electron Microsc. 46: 181), in comparison to normal cells.

Topics: Antineoplastic Agents, Phytogenic; Cell Division; Cell Line; Fibroblasts; Humans; Mitochondria; Naphthoquinones; Osteosarcoma; Tumor Cells, Cultured

Some 2,3-bis(substituted methyl)naphthazarins and related compounds were synthesized by the Diels-Alder reaction of benzoquinone and 2,3-dimethylbutadiene followed by oxidation and substitution reactions. These compounds were prepared as potential biological alkylating agents. Screening results indicated that 1,4-diacetyl-6,7-dimethyl-4a,5,8,8a-tetrahydronaphthalene and 5,8-bis(benzoyloxy)-2,3-dimethyl-1,4-naphthoquinone possessed borderline activity against leukemia P388 and that naphthazarin diacetate possessed confirmed cytotoxicity against the cell culture of human epidermoid carcinoma of the nasopharynx.

Topics: Animals; Antineoplastic Agents; Carcinoma 256, Walker; Carcinoma, Ehrlich Tumor; Leukemia L1210; Leukemia, Experimental; Mice; Naphthoquinones; Osteosarcoma; Sarcoma 180; Sarcoma, Experimental