The phosphoinositide 3-kinase inhibitor ZSTK474 increases the susceptibility of osteosarcoma cells to oncolytic vesicular stomatitis virus VSVΔ51 via aggravating endoplasmic reticulum stress.

Blockage of phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt) signal pathway is effective to increase the cytotoxic effects of oncolytic virus on cancer cells, but the detailed mechanisms are still largely unknown. Based on this, the present study managed to investigate the anti-tumor effects of PI3K inhibitor ZSTK474 and oncolytic vesicular stomatitis virus VSVΔ51 combination treatments on osteosarcoma (OS) in vitro and in vivo. Specifically, ZSTK474 aggravated the inhibiting effects of VSVΔ51 on osteosarcoma development by triggering endoplasmic reticulum (ER)-stress mediated apoptotic cell death. Mechanistically, either ZSTK474 or VSVΔ51 alone had limited effects on cell viability in osteosarcoma cells, while ZSTK474 and VSVΔ51 combination treatments significantly induced osteosarcoma cell apoptosis. Interestingly, VSVΔ51 increased the expression levels of IRE1α and p-PERK to initiate ER stress in osteosarcoma cells, which were aggravated by co-treating cells with ZSTK474. Next, the promoting effects of ZSTK474-VSVΔ51 combined treatment on osteosarcoma cell death were abrogated by the ER-stress inhibitor 4-phenyl butyric acid (4-PBA), indicating that ZSTK474 enhanced the cytotoxic effects of VSVΔ51 on osteosarcoma cells in an ER-stress dependent manner. Finally, the xenograft tumor-bearing mice models were established, and the results showed that ZSTK474-VSVΔ51 combined treatment synergistically hindered tumorigenesis of osteosarcoma cells in vivo. Taken together, our data suggested that ZSTK474 was a novel agent to enhance the cytotoxic effects of VSVΔ51 on osteosarcoma by aggravating ER-stress, and the present study might provide alternative therapy treatments for osteosarcoma in clinic.

Bavachin Induces Ferroptosis through the STAT3/P53/SLC7A11 Axis in Osteosarcoma Cells.

Ferroptosis is a new form of regulated cell death, which is mediated by intracellular iron. Although it is reported that bavachin has antitumour effects on several tumour cells and prompts the reactive oxygen species (ROS) generation, it is unclear whether ferroptosis can be induced by bavachin in osteosarcoma (OS) cells. In this study, we found that bavachin inhibits the viability of MG63 and HOS OS cell lines along with an increase in the ferrous iron level, ROS accumulation, malondialdehyde overexpression, and glutathione depletion. Moreover, iron chelators (deferoxamine), antioxidants (Vit E), and ferroptosis inhibitors (ferrostatin-1 and liproxstatin-1) reverse bavachin-induced cell death. Bavachin also altered the mitochondrial morphology of OS cells, leading to smaller mitochondria, higher density of the mitochondrial membrane, and reduced mitochondrial cristae. Further investigation showed that bavachin upregulated the expression of transferrin receptor, divalent metal transporter-1, and P53, along with downregulating the expression of ferritin light chain, ferritin heavy chain, p-STAT3 (705), SLC7A11, and glutathione peroxidase-4 in OS cells. More importantly, STAT3 overexpression, SLC7A11 overexpression, and pretreatment with pifithrin-α (P53 inhibitor) rescued OS cell ferroptosis induced by bavachin. The results show that bavachin induces ferroptosis via the STAT3/P53/SLC7A11 axis in OS cells.

FOCAL3D: A 3-dimensional clustering package for single-molecule localization microscopy.

Single-molecule localization microscopy (SMLM) is a powerful tool for studying intracellular structure and macromolecular organization at the nanoscale. The increasingly massive pointillistic data sets generated by SMLM require the development of new and highly efficient quantification tools. Here we present FOCAL3D, an accurate, flexible and exceedingly fast (scaling linearly with the number of localizations) density-based algorithm for quantifying spatial clustering in large 3D SMLM data sets. Unlike DBSCAN, which is perhaps the most commonly employed density-based clustering algorithm, an optimum set of parameters for FOCAL3D may be objectively determined. We initially validate the performance of FOCAL3D on simulated datasets at varying noise levels and for a range of cluster sizes. These simulated datasets are used to illustrate the parametric insensitivity of the algorithm, in contrast to DBSCAN, and clustering metrics such as the F1 and Silhouette score indicate that FOCAL3D is highly accurate, even in the presence of significant background noise and mixed populations of variable sized clusters, once optimized. We then apply FOCAL3D to 3D astigmatic dSTORM images of the nuclear pore complex (NPC) in human osteosaracoma cells, illustrating both the validity of the parameter optimization and the ability of the algorithm to accurately cluster complex, heterogeneous 3D clusters in a biological dataset. FOCAL3D is provided as an open source software package written in Python.

Scaffold-based 3D cellular models mimicking the heterogeneity of osteosarcoma stem cell niche.

The failure of the osteosarcoma conventional therapies leads to the growing need for novel therapeutic strategies. The lack of specificity for the Cancer Stem Cells (CSCs) population has been recently identified as the main limitation in the current therapies. Moreover, the traditional two-dimensional (2D) in vitro models, employed in the drug testing and screening as well as in the study of cell and molecular biology, are affected by a poor in vitro-in vivo translation ability. To overcome these limitations, this work provides two tumour engineering approaches as new tools to address osteosarcoma and improve therapy outcomes. In detail, two different hydroxyapatite-based bone-mimicking scaffolds were used to recapitulate aspects of the in vivo tumour microenvironment, focusing on CSCs niche. The biological performance of human osteosarcoma cell lines (MG63 and SAOS-2) and enriched-CSCs were deeply analysed in these complex cell culture models. The results highlight the fundamental role of the tumour microenvironment proving the mimicry of osteosarcoma stem cell niche by the use of CSCs together with the biomimetic scaffolds, compared to conventional 2D culture systems. These advanced 3D cell culture in vitro tumour models could improve the predictivity of preclinical studies and strongly enhance the clinical translation.

The Role of Pre-Clinical 3-Dimensional Models of Osteosarcoma.

Treatment for osteosarcoma (OS) has been largely unchanged for several decades, with typical therapies being a mixture of chemotherapy and surgery. Although therapeutic targets and products against cancer are being continually developed, only a limited number have proved therapeutically active in OS. Thus, the understanding of the OS microenvironment and its interactions are becoming more important in developing new therapies. Three-dimensional (3D) models are important tools in increasing our understanding of complex mechanisms and interactions, such as in OS. In this review, in vivo animal models, in vitro 3D models and in ovo chorioallantoic membrane (CAM) models, are evaluated and discussed as to their contribution in understanding the progressive nature of OS, and cancer research. We aim to provide insight and prospective future directions into the potential translation of 3D models in OS.

Co-delivery of doxorubicin and paclitaxel by reduction/pH dual responsive nanocarriers for osteosarcoma therapy.

Nanoparticle-based drug delivery system offers a promising platform for combination cancer therapy. However, the inefficient drug release in cells reduces the therapeutic efficacy of cancer nanomedicines. Herein, a PEGylated poly(α-lipoic acid) copolymer (mPEG-PαLA) was prepared and used as a reduction/pH dual responsive nanocarrier to simultaneously deliver paclitaxel (PTX) and doxorubicin (DOX) for osteosarcoma therapy. The amphiphilic mPEG-PαLA could efficiently encapsulate both PTX and DOX during its self-assembly into micelles in aqueous solution to generate PTX and DOX co-loaded nanoparticles (NP-PTX-DOX). The as-prepared NP-PTX-DOX showed enhanced PTX and DOX release in response to reductive and acidic stimuli. Moreover, the dual-drug loaded nanoparticles were efficiently internalized by K7 osteosarcoma cells and released drugs intracellularly, as confirmed by flow cytometry analysis and confocal laser scanning microscopy. Consequently, NP-PTX-DOX exhibited synergistic therapeutic effects and induced enhanced cell apoptosis in K7 cells. Furthermore, NP-PTX-DOX presented improved biodistribution and higher tumor growth inhibition efficacy compared to the control groups in a murine osteosarcoma model. Altogether, the results of this work indicate that the proposed strategy is promising for osteosarcoma therapy using mPEG-PαLA copolymer as a dual-responsive nanocarrier to co-deliver anticancer drugs.

Interstitial serum albumin empowers osteosarcoma cells with FAIM2 transcription to obtain viability via dedifferentiation.

During hematogenous metastasis, cancer cells escape from primary lesions and enter into the circulatory system, and only a few can colonize distant organs. However, the mechanism of cell survival and metastasis in the hematopoietic environment remains unclear. Angiorrhea is the character of pathological neovascularization in malignant tumors and commonly detected in osteosarcoma (OS), a bone tumor that prefers circulatory metastasis. In the present study, we focused on the notable role of serum albumin, the highest content in blood plasma, on OS progression. Our results indicated that serum albumin might act as a barrier against exogenous cancer cells during hematogenous metastasis. OS cells with high metastatic potential could gradually obtain strong viability through dedifferentiation under the effect of serum albumin in the angiorrhea region. Further exploration showed that serum albumin could increase the intracellular calcium concentration by activating the voltage-dependent calcium channel Cav2.1 in OS cells to affect the cytoskeleton, sequentially leading to dedifferentiation. Dedifferentiated OS cells with increased FAS apoptosis inhibitory molecule 2 (FAIM2) expression would gradually acquire survival ability, whereas knockdown of FAIM2 caused apoptosis in serum albumin. Moreover, FAIM2 overexpression rescued the viability of OS cells with low metastatic potential in serum albumin. In clinical specimens, OS cells showed markedly stronger positive staining of FAIM2 in the angiorrhea area. Taken together, our findings indicate that serum albumin in the angiorrhea region is a critical substance during pulmonary metastasis of OS cells. Angiorrhea is a nonnegligible prognostic element and FAIM2 might serve as a promising therapeutic target.

High-resolution large-area imaging of nanoscale structure and mineralization of a sclerosing osteosarcoma in human bone.

Osteosarcoma is the most common primary bone cancer type in humans. It is predominantly found in young individuals, with a second peak later in life. The tumour is formed by malignant osteoblasts and consists of collagenous, sometimes also mineralized, bone matrix. While the morphology of osteosarcoma has been well studied, there is virtually no information about the nanostructure of the tumour and changes in mineralization on the nanoscale level. In the present paper, human bone tissue inside, next to and remote from a sclerosing osteosarcoma was studied with small angle x-ray scattering, x-ray diffraction and electron microscopy. Quantitative evaluation of nanostructure parameters was combined with high resolution, large area mapping to obtain microscopic images with nanostructure parameter contrast. It was found that the tumour regions were characterized by a notable reduction in mineral particle size, while the mineral content was even higher than that in normal bone. Furthermore, the normal preferential orientation of mineral particles along the longitudinal direction of corticalis or trabeculae was largely suppressed. Also the bone mineral crystal structure was affected: severe crystal lattice distortions were detected in mineralized tumour tissue pointing to a different ion substitution of hydroxyl apatite in tumorous tissue than in healthy tissue.

Roles of microRNA-22 in Suppressing Proliferation and Promoting Sensitivity of Osteosarcoma Cells via Metadherin-mediated Autophagy.

OBJECTIVE: To analyze the effect of microRNA-22 on autophagy and proliferation and to investigate the underlying molecular mechanism of osteosarcoma cell chemotherapy sensitivity. METHODS: MG-63 cells were divided into four groups, including a control group, a negative control (NC) group, a cisplatin group, and a cisplatin + miR-22 group. Proliferation of MG-63 cells that had been treated with cisplatin and transfected with miR-22 mimics was determined using MTT assay and colony formation assay. We assessed the degree of autophagy using flow cytometry through cellular staining of the autophagy lysosomal marker monodansylcadaverine (MDC). The effect of microRNA-22 on autophagy was observed along with the expression levels of Beclin1, LC3, metadherin (MTDH) and ATG5 by western blot and quantitative reverse transcription polymerase chain reaction (qRT-PCR). Luciferase reporter assay revealed the targeted binding site between miR-22 and the 3'-untranslated region (3'-UTR) of MTDH mRNA. Western blot and qRT-PCR were used to explore the level of MTDH in the control group, the NC group, the cisplatin group, and the miR-22 group for 6, 12, and 24 h. RESULTS: In the in vitro study, the MTT results indicated that the MG-63 cells with overexpression of miR-22 exhibited a significant decline in the proliferation capacity compared with the control group (0.513 ± 0.001, P < 0.0005). Similar to the MTT results, MG-63 cells that were transfected with miR-22 mimic (101.0 ± 10.58) formed fewer colonies compared with the cisplatin group (129.7 ± 4.163). MDC staining revealed that miR-22-overexpressing osteosarcoma (OS) cells treated with cisplatin showed a significant decrease in the expression of autophagy (7.747 ± 0.117, P < 0.0001). Our data revealed that miR-22 could regulate not only autophagy but also proliferation in the chemosensitivity of osteosarcoma cells. We found that miR-22 sensitized osteosarcoma cells to cisplatin treatment by regulating autophagy-related genes. In addition, Luciferase Reporter Assay revealed that miR-22 negatively regulated autophagy through direct targeting of MTDH. We performed western blot analysis to detect the MTDH expression level. The results revealed that the overexpression of miR-22 obviously decreased the expression of MTDH (1.081 ± 0.023, P < 0.001). CONCLUSION: Inhibition of miR-22 ameliorated the anticancer drug-induced cell proliferation decrease in osteosarcoma cells. MTDH was identified as the miR-22 target in OS cells and MTDH-triggered autophagy played a key function in the miR-22-associated chemotherapy sensitivity.

Inhibition of LCMR1 and ATG12 by demethylation-activated miR-570-3p is involved in the anti-metastasis effects of metformin on human osteosarcoma.

Epidemiological studies have demonstrated that metformin could mitigate the progression of several tumors. Although it has been proved that metformin could cause demethylation of DNA and lead to up-regulation of some encoding genes and non-coding RNAs, there is little data about the effects of metformin on metastasis, and the interaction between metastasis and autophagy in human osteosarcoma cells. Here, we found miR-570-3p was significantly down-regulated in human metastatic osteosarcoma tissues but not in non-metastatic osteosarcoma tissues. Metformin attenuates the metastasis and autophagy in osteosarcoma. Interestingly, this autophagy favors osteosarcoma cells invasion. Moreover, reduction of metformin-induced inhibition of autophagy could reverse the invasion suppression in osteosarcoma. Mechanistically, metformin increases miR-570-3p by the demethylation of DNA, and the upregulation of miR-570-3p repressed the translation of its target, LCMR1 and ATG12. Our results, for the first time, presents evidence that the miR-570-3p-mediated suppression of LCMR1 and ATG12 is involved in the metformin-induced inhibition of metastasis in osteosarcoma cells.

Paeoniflorin induces G2/M cell cycle arrest and caspase-dependent apoptosis through the upregulation of Bcl-2 X-associated protein and downregulation of B-cell lymphoma 2 in human osteosarcoma cells.

Paeoniflorin (PF), extracted from the peony root, has been proved to possess antineoplastic activity in different cancer cell lines. However, it remains unclear whether PF has an antineoplastic effect against osteosarcoma cells. The present study investigated the effects and the specific mechanism of PF on various human osteosarcoma cell lines. Using the multiple methods to detect the activity of PF on HOS and Saos­2 human osteosarcoma cell lines, including an MTS assay, flow cytometry, transmission electron microscopy and western blotting, it was demonstrated that PF induces inhibition of proliferation, G2/M phase cell cycle arrest and apoptosis in the osteosarcoma cell lines in vitro, and activation of cleaved­caspase­3 and cleaved­poly (ADPribose) polymerase in a dose­dependent manner. Furthermore, the pro­apoptotic factors Bcl­2 X­associated protein and BH3 interacting domain death agonist were uregulated, while the anti­apoptotic factors B­cell lymphoma 2 (Bcl­2) and Bcl­2­extra large were downregulated. In conclusion, these results demonstrated that PF has a promising therapeutic potential in for osteosarcoma.

Trombosis en paciente pediátrico con osteosarcoma / (Thrombosis in pediatric patient with osteosarcoma)

ResumenPaciente masculino de 12 años, con cuadro de 2 meses de evolución de dolor en cadera derecha y claudicación, con circulación colateral abdominal y edema de miembro inferior derecho al examen físico, con radiografías de pelvis que muestran masa en cresta iliaca derecha; ultrasonido de abdomen y miembro inferior derecho (con doppler ) con masa en cresta iliaca derecha asociada a trombo oclusivo total de vena iliaca común derecha, hasta vena cava inferior en su desembocadura en aurícula derecha; además, tomografía de tórax con múltiples vasos de circulación colateral con extenso trombo en la vena cava inferior, que se extiende a la aurícula derecha, cruzando la válvula tricúspide e insinuándose en el ventrículo derecho, con múltiples nódulos pulmonares bilaterales, que por sus características, son altamente sugestivos de lesiones metastásicas, y tomografía de pelvis que muestra extensa lesión infiltrante del hueso iliaco derecho, la cual compromete la totalidad del hueso hasta la altura del techo acetabular. Además, se documenta masa de tejido blando que desplaza músculos adyacentes, estructuras vasculares y vísceras de la pelvis hacia la izquierda, con patrón blástico, con áreas líticas que comprometen el aspecto lateral derecho de S3 y S4, encontrándose también infiltradas por la neoplasia.Debido al patrón de destrucción ósea y aspecto morfológico del trombo, se desaconseja trombectomía, y se realiza biopsia tumoral, con reporte de osteosarcoma; se inicia quimioterapia.

AbstractA 12-year-old male patient, with a 2-month history of pain in the right hip and claudication, with collateral abdominal circulation and lower right limb edema on physical examination, with pelvic radiographs showing a right iliac crest mass; ultrasound of abdomen and lower right limb (with Doppler) with right iliac crest mass associated with total occlusive thrombus of right common iliac vein, to inferior vena cava at its right atrial outlet; also chest tomography with multiple collateral circulation vessels with extensive thrombus in the inferior vena cava, extending to the right atrium, crossing the tricuspid valve and insinuating itself in the right ventricle, with multiple bilateral pulmonary nodules that, because of their characteristics are highly suggestive of metastatic lesions, and pelvic tomography which shows extensive infiltrating lesion of the right iliac bone, which compromises the entire bone to the height of the acetabular roof; in addition, a mass of soft tissue is documented that displaces adjacent muscles, vascular structures and viscera of the pelvis to the left, with blastic pattern, with lithic areas that compromise the right lateral aspect of S3 and S4, being also infiltrated by the neoplasia.Due to the bone destruction pattern and the morphological aspect of the thrombus, thrombectomy is advised against and tumor biopsy is performed with osteosarcoma report and chemotherapy is initiated.

The tumor microenvironment promotes cancer progression and cell migration.

The tumor microenvironment contributes to cancer progression, in part through interactions between tumor and normal stromal cells. This study analyzed morphological and molecular changes induced in co-cultured human fibroblasts (HFs) and the MG-63 osteosarcoma cell line. Co-cultured cell monolayers were morphologically analyzed using high resolution scanning electron microscopy (HR-SEM), and trans-well assays were performed to assess cell migration and invasion. Proteins involved in inflammatory responses, cancer cell invasion, and angiogenesis were assessed using western blotting. HR-SEM showed progressive spatial orientation loss by fibroblasts in contact with MG-63s, while MG-63s proliferated rapidly and invaded HF space. Trans-well assays showed enhanced MG-63 migration in the presence of HFs. IL-6 expression was increased in co-cultured HFs, possibly stimulated by TNF-α. HFs do not normally express YKL-40 but did so in co-culture. Band densitometric analyses showed that increasing YKL-40 expression was followed by VEGF overexpression, especially in MG-63s. Finally, our results confirmed fibroblasts as the main matrix metalloproteinase source in this tumor microenvironment. Our study sheds new light on how tumor-stroma interactions promote tumor development and progression, and may support identification of novel anti-cancer therapeutics.

The oncolytic peptide LTX-315 triggers necrotic cell death.

The oncolytic peptide LTX-315 has been designed for killing human cancer cells and turned out to stimulate anti-cancer immune responses when locally injected into tumors established in immunocompetent mice. Here, we investigated the question whether LTX-315 induces apoptosis or necrosis. Transmission electron microscopy or morphometric analysis of chromatin-stained tumor cells revealed that LTX-315 failed to induce apoptotic nuclear condensation and rather induced a necrotic phenotype. Accordingly, LTX-315 failed to stimulate the activation of caspase-3, and inhibition of caspases by means of Z-VAD-fmk was unable to reduce cell killing by LTX-315. In addition, 2 prominent inhibitors of regulated necrosis (necroptosis), namely, necrostatin-1 and cycosporin A, failed to reduce LTX-315-induced cell death. In conclusion, it appears that LTX-315 triggers unregulated necrosis, which may contribute to its pro-inflammatory and pro-immune effects.

High-performance thin-layer chromatography for the evaluation of voacamine intracellular concentration related to its cytotoxic effect.

Previous investigations demonstrated that pretreatment with non-cytotoxic concentrations of voacamine had a chemosensitizing effect on cultured multidrug resistant osteosarcoma cells exposed to doxorubicin; whereas when used alone at high concentrations voacamine induced apoptosis-independent cell death on both sensitive and resistant cells. To gain insight into the mechanism of action of voacamine at the subcellular level, we developed an analytical high-performance thin-layer chromatography technique to assess the intracellular content of voacamine that could be correlated with the induction of cell death and consequent morphological and ultrastructural changes. The results of the quantitative analysis not only did allow us to measure both the amount of unmodified voacamine molecules (determined by the method) and the amount of molecules which reacted with cellular components (undetectable), but also to confirm the findings of our previous studies and support the validity of this method.

5-Aminolevulinic Acid-Based Sonodynamic Therapy Induces the Apoptosis of Osteosarcoma in Mice.

OBJECTIVE: Sonodynamic therapy (SDT) is promising for treatment of cancer, but its effect on osteosarcoma is unclear. This study examined the effect of 5-Aminolevulinic Acid (5-ALA)-based SDT on the growth of implanted osteosarcoma and their potential mechanisms in vivo and in vitro. METHODS: The dose and metabolism of 5-ALA and ultrasound periods were optimized in a mouse model of induced osteosarcoma and in UMR-106 cells. The effects of ALA-SDT on the proliferation and apoptosis of UMR-106 cells and the growth of implanted osteosarcoma were examined. The levels of mitochondrial membrane potential (ΔψM), ROS production, BcL-2, Bax, p53 and caspase 3 expression in UMR-106 cells were determined. RESULTS: Treatment with 5-ALA for eight hours was optimal for ALA-SDT in the mouse tumor model and treatment with 2 mM 5-ALA for 6 hours and ultrasound (1.0 MHz 2.0 W/cm2) for 7 min were optimal for UMR-106 cells. SDT, but not 5-ALA, alone inhibited the growth of implanted osteosarcoma in mice (P<0.01) and reduced the viability of UMR-106 cells (p<0.05). ALA-SDT further reduced the tumor volumes and viability of UMR-106 cells (p<0.01 for both). Pre-treatment with 5-ALA significantly enhanced the SDT-mediated apoptosis (p<0.01) and morphological changes. Furthermore, ALA-SDT significantly reduced the levels of ΔψM, but increased levels of ROS in UMR-106 cells (p<0.05 or p<0.01 vs. the Control or the Ultrasound). Moreover, ALA-SDT inhibited the proliferation of osteosarcoma cells and BcL-2 expression, but increased levels of Bax, p53 and caspase 3 expression in the implanted osteosarcoma tissues (p<0.05 or p<0.01 vs. the Control or the Ultrasound). CONCLUSIONS: The ALA-SDT significantly inhibited osteosarcoma growth in vivo and reduced UMR-106 cell survival by inducing osteosarcoma cell apoptosis through the ROS-related mitochondrial pathway.

Celastrol induces apoptosis and autophagy via the ROS/JNK signaling pathway in human osteosarcoma cells: an in vitro and in vivo study.

Osteosarcoma is the most common primary malignant tumor of bone, the long-term survival of which has stagnated in the past several decades. Celastrol, a triterpene from traditional Chinese medicine, has been proved to possess potent anti-tumor effect on various cancers. However, the effect of celastrol on human osteosarcoma and the underlying mechanisms remains to be elucidated. We reported here that celastrol could inhibit cell proliferation by causing G2/M phase arrest. Exposure to celastrol resulted in the activation of caspase-3, -8, and -9, indicating that celastrol induced apoptosis through both extrinsic and intrinsic pathways. Autophagy occurred in celastrol-treated cells as evidenced by formation of autophagosome and accumulation of LC3B-II. The celastrol-induced cell death was remarkably restored by the combination of autophagy and apoptosis inhibitors. Furthermore, inhibition of apoptosis enhanced autophagy while suppression of autophagy diminished apoptosis. Celastrol also induced JNK activation and ROS generation. The JNK inhibitor significantly attenuated celastrol-triggered apoptosis and autophagy while ROS scavenger could completely reverse them. The ROS scavenger also prevented G2/M phase arrest and phosphorylation of JNK. Importantly, we found that celastrol had the similar effects on primary osteosarcoma cells. Finally, in vivo, celastrol suppressed tumor growth in the mouse xenograft model. Taken together, our results revealed that celastrol caused G2/M phase arrest, induced apoptosis and autophagy via the ROS/JNK signaling pathway in human osteosarcoma cells. Celastrol is therefore a promising candidate for development of antitumor drugs targeting osteosarcoma.

The oncolytic adenovirus Δ24-RGD in combination with cisplatin exerts a potent anti-osteosarcoma activity.

Osteosarcoma is the most common malignant bone tumor in children and adolescents. The presence of metastases and the lack of response to conventional treatment are the major adverse prognostic factors. Therefore, there is an urgent need for new treatment strategies that overcome both of these problems. Our purpose was to elucidate whether the use of the oncolytic adenovirus Δ24-RGD alone or in combination with standard chemotherapy would be effective, in vitro and in vivo, against osteosarcoma. Our results showed that Δ24-RGD exerted a potent antitumor effect against osteosarcoma cell lines that was increased by the addition of cisplatin. Δ24-RGD osteosarcoma treatment resulted in autophagy in vitro that was further enhanced when combined with cisplatin. Of importance, administration of Δ24-RGD and/or cisplatin, in novel orthotopic and two lung metastatic models in vivo resulted in a significant reduction of tumor burden meanwhile maintaining a safe toxicity profile. Together, our data underscore the potential of Δ24-RGD to become a realistic therapeutic option for primary and metastatic pediatric osteosarcoma. Moreover, this study warrants a future clinical trial to evaluate the safety and efficacy of Δ24-RGD for this devastating disease.

Mitochondrial dysfunction and permeability transition in osteosarcoma cells showing the Warburg effect.

Metabolic reprogramming in cancer is manifested by persistent aerobic glycolysis and suppression of mitochondrial function and is known as the Warburg effect. The Warburg effect contributes to cancer progression and is considered to be a promising therapeutic target. Understanding the mechanisms used by cancer cells to suppress their mitochondria may lead to development of new approaches to reverse metabolic reprogramming. We have evaluated mitochondrial function and morphology in poorly respiring LM7 and 143B osteosarcoma (OS) cell lines showing the Warburg effect in comparison with actively respiring Saos2 and HOS OS cells and noncancerous osteoblastic hFOB cells. In LM7 and 143B cells, we detected markers of the mitochondrial permeability transition (MPT), such as mitochondrial swelling, depolarization, and membrane permeabilization. In addition, we detected mitochondrial swelling in human OS xenografts in mice and archival human OS specimens using electron microscopy. The MPT inhibitor sanglifehrin A reversed MPT markers and increased respiration in LM7 and 143B cells. Our data suggest that the MPT may play a role in suppression of mitochondrial function, contributing to the Warburg effect in cancer.