Design of Bionanomaterial of Chitosan Carbohydrate Polymer Composited with Broccoli Extract and Zinc Oxide Nanoparticles: Anticancer Activity in Human Osteosarcoma.

In the current research, a chitosan/broccoli extract/ZnO nanoparticle (CH/BE/ZnO) bionanocomposite was created. The physicochemical properties of CH/BE/ZnO bionanocomposite were investigated using a variety of methods, including field emission scanning electron microscopy (FESEM), elemental analysis (CHN-O), X-ray diffraction (XRD), Fourier transform infrared spectrum (FTIR), Brunauer-Emmett-Teller (BET), and transmission electron microscopy (TEM). The CH/BE/ZnO bionanocomposite's biological activity was assessed by examining its cytotoxicity capabilities against a bone cancer cell line (MG63). The total pore volume and specific surface area of CH/BE/ZnO are 0.134 cm3/g and 16.99 m2/g, respectively. The IC50 results for CH/BE/ZnO bionanocomposite in bone cancer investigations using the MTT test against the MG63 cell line was 115 µg/mL. The results indicate that the CH/BE/ZnO bionanocomposite is an effective chemotherapeutic agent against human osteosarcoma. The CH/BE/ZnO bionanocomposite showed high performance and structure, which means innovating nanomaterial agents for biological applications in the future.

Mild Hyperthermia-Induced Thermogenesis in the Endoplasmic Reticulum Defines Stress Response Mechanisms.

Previous studies reported that a mild, non-protein-denaturing, fever-like temperature increase induced the unfolded protein response (UPR) in mammalian cells. Our dSTORM super-resolution microscopy experiments revealed that the master regulator of the UPR, the IRE1 (inositol-requiring enzyme 1) protein, is clustered as a result of UPR activation in a human osteosarcoma cell line (U2OS) upon mild heat stress. Using ER thermo yellow, a temperature-sensitive fluorescent probe targeted to the endoplasmic reticulum (ER), we detected significant intracellular thermogenesis in mouse embryonic fibroblast (MEF) cells. Temperatures reached at least 8 °C higher than the external environment (40 °C), resulting in exceptionally high ER temperatures similar to those previously described for mitochondria. Mild heat-induced thermogenesis in the ER of MEF cells was likely due to the uncoupling of the Ca2+/ATPase (SERCA) pump. The high ER temperatures initiated a pronounced cytosolic heat-shock response in MEF cells, which was significantly lower in U2OS cells in which both the ER thermogenesis and SERCA pump uncoupling were absent. Our results suggest that depending on intrinsic cellular properties, mild hyperthermia-induced intracellular thermogenesis defines the cellular response mechanism and determines the outcome of hyperthermic stress.

Low-energy shock waves promote the cisplatin chemosensitivity of human osteosarcoma MNNG/HOS cells via the P2X7/Akt/mTOR pathway.

Background: The current dilemma of osteosarcoma treatment is the resistance of chemotherapeutic drugs after long-term usage, which also introduces life-threatening side effects. Methods and results: To minimize chemoresistance in osteosarcoma patients, the authors applied shock waves (SWs) to human osteosarcoma MNNG/HOS cells, then evaluated the cell viability and extracellular ATP levels, and further investigated the effect of SWs on cisplatin (DDP) cytotoxicity in MNNG/HOS cells. The authors' results showed that 400 SW pulses at 0.21 mJ/mm2 exhibited little influence on the MNNG/HOS cell viability. In addition, this SW condition significantly promoted the extracellular ATP release in MNNG/HOS cells. Importantly, low-energy SWs obviously increased Akt and mammalian target of rapamycin (mTOR) phosphorylation and activation in MNNG/HOS cells, which could be partially reversed in the presence of P2X7 siRNA. The authors also found that low-energy SWs strongly increased the DDP sensitivity of MNNG/HOS cells in the absence of P2X7. Conclusions: For the first time, the authors found that SW therapy reduced the DDP resistance of MNNG/HOS osteosarcoma cells when the ATP receptor P2X7 was downregulated. SW therapy may provide a novel treatment strategy for chemoresistant human osteosarcoma.

Targeting PERK-ATF4-P21 axis enhances the sensitivity of osteosarcoma HOS cells to Mppα-PDT.

Osteosarcoma (OS) is the most prevalent type of malignant bone tumor in adolescents. The overall survival of OS patients has reached a plateau recently. Thus, there is an urgent need to develop approaches to improve the sensitivity of OS to therapies. Pyropheophorbide-α methyl ester-mediated photodynamic therapy (MPPα-PDT) is a new type of tumor therapy, and elucidating its mechanism is helpful to improve its anti-tumor efficacy. Here, we investigated how PERK signaling promotes the human OS (HOS) cell survival induced by MPPα-PDT, as overcoming this may enhance sensitivity to MPPα-PDT. We found that MPPα-PDT combined with PERK inhibitor GSK2656157 enhanced HOS cell apoptosis by suppressing autophagy and p21. Autophagy inhibition and p21 depletion enhanced cell death, indicating pro-survival effects in MPPα-PDT. Notably, p21 was found to be an effector of the PERK-Atf4 pathway, which could positively regulate autophagy mediated by MPPα-PDT. In conclusion, we found that the combination of MPPα-PDT and GSK2656157 enhanced apoptosis in HOS cells by inhibiting autophagy. Mechanistically, this autophagy is p21-dependent and can be suppressed by GSK2656157, thereby enhancing sensitivity to MPPα-PDT.

Impact of Conventional and Potential New Metal-Based Drugs on Lipid Metabolism in Osteosarcoma MG-63 Cells.

This work investigated the mechanisms of action of conventional drugs, cisplatin and oxaliplatin, and the potentially less deleterious drug Pd2Spermine (Spm) and its Pt(II) analog, against osteosarcoma MG-63 cells, using nuclear-magnetic-resonance metabolomics of the cellular lipidome. The Pt(II) chelates induced different responses, namely regarding polyunsaturated-fatty-acids (increased upon cisplatin), suggesting that cisplatin-treated cells have higher membrane fluidity/permeability, thus facilitating cell entry and justifying higher cytotoxicity. Both conventional drugs significantly increased triglyceride levels, while Pt2Spm maintained control levels; this may reflect enhanced apoptotic behavior for conventional drugs, but not for Pt2Spm. Compared to Pt2Spm, the more cytotoxic Pd2Spm (IC50 comparable to cisplatin) induced a distinct phospholipids profile, possibly reflecting enhanced de novo biosynthesis to modulate membrane fluidity and drug-accessibility to cells, similarly to cisplatin. However, Pd2Spm differed from cisplatin in that cells had equivalent (low) levels of triglycerides as Pt2Spm, suggesting the absence/low extent of apoptosis. Our results suggest that Pd2Spm acts on MG-63 cells mainly through adaptation of cell membrane fluidity, whereas cisplatin seems to couple a similar effect with typical signs of apoptosis. These results were discussed in articulation with reported polar metabolome adaptations, building on the insight of these drugs' mechanisms, and particularly of Pd2Spm as a possible cisplatin substitute.

Genistein Induces Antiproliferative Activity and Apoptosis in Human Osteosarcoma Saos-2 Cells.

BACKGROUND/AIM: Genistein (4', 5, 7-trihydroxyisoflavone) and daidzein (4', 7-dihydroxyisoflavone) are isoflavones derived from soybean and have anti-cancer effects in various cells. However, the effects of genistein and daidzein on the human osteosarcoma cell line Saos-2 has not been investigated before. MATERIALS AND METHODS: Human osteosarcoma Saos-2 cells were treated with genistein for 24 and 48 hours. Cytotoxicity and apoptosis were measured. RESULTS: Genistein significantly inhibited proliferation of Saos-2 cells stronger than daidzein in a dose-dependent manner (0 to 80 µM). Genistein also significantly suppressed Saos-2 cell viability in a dose-dependent manner (0 to 100 µM). In contrast, daidzein did not affect Saos-2 cell viability. Real-time PCR revealed that genistein caused G1-arrest by increasing the expression of p21 and p27 mRNAs in Saos-2 cells. In addition, genistein induced apoptosis through the up-regulation of effector caspase-3/7 activity in Saos-2 cells. Genistein also enhanced initiator caspase-9 and TNF-α mRNA expression in cells. CONCLUSION: Genistein may inhibit proliferation through the up-regulation of p21 and p27 and viability by inducing apoptosis in Saos-2 cells.

Comparative Study of Ozonated Glycerol and Macrogol Ointment on Bone Matrix Production by Human Osteosarcoma Cell Line Saos-2.

Ozonated glycerol is glycerol containing ozone, has no unpleasant odor, and has a long half-life. To apply ozonated glycerol for clinical use, ozonated macrogol ointment has been developed by adding macrogol ointment to ozonated glycerol to increase the retention in the affected area. However, the effects of ozone on this macrogol ointment were unclear. The viscosity of the ozonated macrogol ointment was approximately two times higher than that of ozonated glycerol. The effect of the ozonated macrogol ointment on the human osteosarcoma cell line Saos-2 (Saos-2 cells) proliferation, type 1 collagen production, and alkaline phosphatase (ALP) activity were studied. The proliferation of Saos-2 cells was assessed using MTT and DNA synthesis assays. Type 1 collagen production and ALP activity were studied using ELISA and ALP assays. Cells were treated for 24 h with or without 0.05, 0.5, or 5 ppm ozonated macrogol ointment. The 0.5 ppm ozonated macrogol ointment significantly elevated Saos-2 cell proliferation, type 1 collagen production, and ALP activity. These results also showed almost the same trend as for ozonated glycerol.

Relative Biological Effectiveness Values of Spot-scanning Proton Beam Therapy at Shonan Kamakura General Hospital.

BACKGROUND/AIM: This study aimed to confirm the relative biological effectiveness (RBE) values of the proton beam therapy (PBT) system installed in Shonan Kamakura General Hospital. MATERIALS AND METHODS: Clonogenic cell-survival assays were performed with a human salivary gland (HSG) cell line, a human tongue squamous-cell carcinoma cell line (SAS), and a human osteosarcoma cell line (MG-63). Cells were irradiated with proton beams and X-rays with different doses (1.8, 3.6, 5.5, and 7.3 Gy for proton beams, and 2, 4, 6, and 8 Gy for X-rays). Proton beam irradiation used spot-scanning methods and three different depths (at the proximal, center, and distal sides of the spread-out Bragg peak). RBE values were obtained from a comparison of the dose that resulted in a surviving fraction of 10% (D10). RESULTS: D10 of proton beams at the proximal, center, and distal sides and X-rays in HSG were 4.71, 4.71, 4.51, and 5.25 Gy, respectively; those in SAS were 5.08, 5.04, 5.01, and 5.59 Gy, respectively; and those in MG-63 were 5.36, 5.42, 5.12, and 6.06 Gy, respectively. The RBE10 values at the proximal, center, and distal sides in HSG were 1.11, 1.11, and 1.16 respectively; those in SAS were 1.10, 1.11, and 1.12, respectively; and those in MG-63 were 1.13, 1.12, and 1.18, respectively. CONCLUSION: RBE10 values of 1.10-1.18 were confirmed by in vitro experiments using the PBT system. These results are considered acceptable for clinical use in terms of therapeutic efficacy and safety.

Curcumin and its Analogs and Carriers: Potential Therapeutic Strategies for Human Osteosarcoma.

Curcumin is a natural polyphenol phytochemical derived from turmeric with antioxidant, anti-inflammatory, and anticancer properties but is concerned about poor solubility in water, absorption, and metabolic stability. Potent metastatic osteosarcoma is the most common primary bone cancer in children, adolescents, and young adults. It is responsible for low survival rates because of its high rate of metastasis to the lungs. To improve poor bioavailability, numerous curcumin analogs were developed to possess anticancer characteristics through a variety of biological pathways involved in cytotoxicity, proliferation, autophagy, sensitizing chemotherapy, and metastases. This review provides an overview of their various pharmacological functions, molecular mechanisms, and therapeutic potential as a remedy for human osteosarcoma. To enhance therapeutic efficacy, several liposomal nanoparticles, nanocarriers, multifunctional micelles, and three-dimensional printed scaffolds have also been developed for the controlled delivery of curcumin targeting human osteosarcoma cells. Consequently, curcumin and several potential analogs and delivery formulations are optimistic candidates to improve the currently available strategy for human osteosarcoma. However, further insight into the mechanism of action of promising curcumin analogs and the development of carriers in clinical trials of osteosarcoma needs to be investigated to improve their overall potency and clinical utility, in particular the anti-metastatic effect.

Mitophagy Effects of Protodioscin on Human Osteosarcoma Cells by Inhibition of p38MAPK Targeting NIX/LC3 Axis.

Protodioscin (PD) is a steroidal saponin with various pharmacological activities, including neuro-protective, anti-inflammatory, and anti-tumor activities. However, the effect of PD on human osteosarcoma (OS) cells is unclear. In this study, we found that PD significantly inhibits the growth of human HOS and 143B OS cells through the upregulation of apoptotic-related proteins (cleaved caspase-3, cleaved caspase-9, and cleaved PARP) and mitophagy-related proteins (LC3B and NIX), which contribute to the induction of apoptosis, and MMP (mitochondrial membrane potential) dysfunction and mitophagy. The inhibition of LC3 or NIX was shown to decrease apoptosis and mitophagy in PD-treated OS cells. The knockdown of p38MAPK by siRNA decreased mitochondrial dysfunction, autophagy, mitophagy, and the NIX/LC3B expression in the PD-treated OS cells. A binding affinity analysis revealed that the smaller the KD value (-7.6 Kcal/mol and -8.9 Kcal/mol, respectively), the greater the binding affinity in the PD-NIX and PD-LC3 complexes. These findings show the inhibitory effects of PD-induced mitophagy in human OS cells and may represent a novel therapeutic strategy for human OS, by targeting the NIX/LC3 pathways.

Identification of optimal reference genes for gene expression normalization in human osteosarcoma cell lines under proliferative conditions.

The molecular pathogenesis and therapeutic target research studies on osteosarcoma (OS) have developed well during the last few years using various OS cell lines with reverse transcription quantitative polymerase chain reaction (RT-qPCR). However, the identification of suitable reference genes of RT-qPCR for OS cell lines has not been reported. Here, we conducted the normalization research of 12 reference genes (GAPDH, ACTB, 18S, B2M, ALAS1, GUSB, HPRT1, HMBS, PPIA, PUM1, RPL29, and TBP) for gene expression analysis in four kinds of human OS cell lines (U2OS, Saos-2, HOS, and MG-63) to improve the investigation of molecular mechanisms and the accuracy of diagnosis and prognostic molecular targets of OS. The gene expression stability and applicability of the 12 reference gene candidates were determined using geNorm, NormFinder, and BestKeeper software. The results indicated that PUM1 and the combination of PPIA + ALAS1 were recommended as the optimal reference gene in these four different sources of human OS cell lines under proliferative conditions. The present study identified the most suitable reference genes and reference gene combinations for OS cell lines under proliferative conditions in order to use in gene expression profile analysis. A reliable standardized method has the potential to improve the understanding of the biological mechanisms underlying OS in the future.

Bisdemethoxycurcumin suppresses human osteosarcoma U­2 OS cell migration and invasion via affecting the PI3K/Akt/NF­κB, PI3K/Akt/GSK3ß and MAPK signaling pathways in vitro.

The metastasis of human osteosarcoma (OS) shows a difficult­to­treat clinical scenario and results in decreased quality of life and diminished survival rates. Finding or developing novel treatments to improve the life quality of patients is urgent. Bisdemethoxycurcumin (BDMC), a natural product, was obtained from the rhizome of turmeric (Curcuma longa) and exerts antitumor activities in numerous human cancer cell lines. At present, there is no study showing BDMC effects on OS cell migration and invasion. In the present study, the effects of BDMC on cell migration and invasion of OS U­2 OS cells were investigated in vitro. Cell viability and proliferation were measured by flow cytometric and MTT assays, respectively. Cell motility, MMP­2 and ­9 activity, and cell migration and invasion were assayed by scratch wound healing, gelatin zymography, and Transwell chamber assays, respectively. The protein expression levels were measured by western blotting. BDMC at 20 and 40 µM significantly reduced total cell viability, and BDMC at 5 and 10 µM significantly inhibited cell motility in U­2 OS cells. BDMC significantly suppressed the activities of MMP­2 and MMP­9 in U­2 OS cells. BDMC suppressed cell invasion and migration after 24 h treatment in U­2 OS cells, and these effects were in a dose­dependently manner. Results from western blotting indicated that BDMC significantly decreased the protein expression levels of PI3K/Akt/NF­κB, PI3K/Akt/GSK3ß, and MAPK pathway in U­2 OS cells. Furthermore, BDMC inhibited uPA, MMP­2, MMP­9, MMP­13, N­cadherin, VE­cadherin, and vimentin but increased E­cadherin in U­2 OS cells. Based on these observations, it was suggested that BDMC may be a potential candidate against migration and invasion of human OS cells in the future.

Doxorubicin-Loaded Lipid Nanoparticles Coated with Calcium Phosphate as a Potential Tool in Human and Canine Osteosarcoma Therapy.

Osteosarcoma (OSA) is the most frequently diagnosed primary malignant bone tumor in humans and dogs. In both species, standard chemotherapy can be limited by multidrug resistance of neoplastic cells, which prevents intracellular accumulation of cytotoxic drugs, resulting in chemotherapy failure. In this study, a lipophilic ester of doxorubicin (C12DOXO) was loaded into nanoparticles (NPs) using the "cold microemulsion dilution" method. The resulting NPs were then coated with calcium phosphate (CaP) in two different ways to have calcium or phosphate ions externally exposed on the surface. These systems were characterized by determining mean diameter, zeta potential, and drug entrapment efficiency; afterward, they were tested on human and canine OSA cells to study the role that the coating might play in increasing both drug uptake into tumor cells and cytotoxicity. Mean diameter of the developed NPs was in the 200-300 nm range, zeta potential depended on the coating type, and C12DOXO entrapment efficiency was in the 60-75% range. Results of studies on human and canine OSA cells were very similar and showed an increase in drug uptake and cytotoxicity for CaP-coated NPs, especially when calcium ions were externally exposed. Therefore, applications in both human and veterinary medicine can be planned in the near future.

Synergistic Antitumor Interaction of Risedronate Sodium and Standard Anticancer Agents in Canine (D-17) and Human Osteosarcoma (U-2 OS) Cell Lines.

The study discusses in vitro cytotoxicity of a combination of cytostatic drugs (doxorubicin, cisplatin, carboplatin, etoposide) and risedronate sodium against canine and human osteosarcoma (D-17 and U-2 OS). Standard protocols were used for the preparation of cell cultures and evaluation of their viability and apoptosis. MTT assay assessed the culture viability and EC50, while the apoptotic effect of the drugs was checked with a TUNEL assay. Doxorubicin alone showed the strongest cytotoxicity against D-17 (0.056 ± 0.019 µg/mL) and U-2 OS (0.051 ± 0.003 µg/mL), while the lowest cytotoxicity was observed for carboplatin (D-17, 6.45 ± 0.2 µg/mL and U2-OS, 27.5 ± 2.3 µg/mL). Risedronate sodium at 100, 10 and 1 µg/mL lowered viability in OS cell lines by 53.38 ± 1.46 and 49.56 ± 0.7%, 97.08 ± 3.32 and 74.92 ± 4.01%, and 102.67 ± 3.56 and 94.56 ± 3.52%, respectively. In all analyzed drug combinations, risedronate sodium significantly (* p < 0.05) increased the cytotoxicity against tested osteosarcoma cell lines. The decrease in cell viability caused by the studied compound combinations was weaker in canine than in human cell cultures. A combination of doxorubicin (all concentrations), cisplatin (1 µg/mL) and etoposide (1 µg/mL) with 100 µg/mL of risedronate sodium significantly improved the cytotoxicity of the drugs against canine and human osteosarcoma. Administration of carboplatin (1 µg/mL) and risedronate sodium (100 µg/mL), compared to carboplatin per se, produced no significant differences in cytotoxicity against the D-17 cell culture but significantly enhanced cytotoxicity in the U-2 OS line. The strongest apoptosis in both lines was detected for 0.01 µg/mL doxorubicin combined with 100 µg/mL risedronate sodium or 1 µg/mL cisplatin and 100 µg/mL risedronate sodium. In all combinations, the tested compounds revealed a synergistic mechanism of action.

Establishment and application of a human osteosarcoma U-2OS cell line that can stably express Cas9 protein.

Osteosarcoma is the most common primary malignant bone tumor, and U-2OS is a common osteosarcoma cell model. The study obtained a human osteosarcoma U-2OS tool cell line which could stably express Cas9 protein, and we reported its production method and application. Firstly, we introduced a Cas9 protein expression gene and an antibiotic screening marker gene through CRISPR/Cas9 system to construct a human osteosarcoma U-2OS tool cell line which could stably express Cas9 protein. Secondly, as the cell line could stably express Cas9 protein, it was only transfected alone a small sgRNA fragment for related gene editing, we then transfected, respectively, a small ETV4 and MALAT1 sgRNA fragment to U-2OS tool cell line for gene editing. Lastly, the Q-PCR results showed that the transcription levels of ETV4 and MALAT1 were significantly decreased, and western blotting result showed that the translation level of ETV4 was significantly decreased, these results indicated that the constructed U-2OS tool cell line could effectively edit protein-coding gene (ETV4) and long non-coding RNA gene (MALAT1). The results of this study also indicated that the constructed U-2OS tool cell line could greatly improve the efficiency of gene editing. Therefore, the genetic engineering cell line provided by the study is of great significance for studying the pathogenesis and regulatory network of osteosarcoma, and for preventing and treating bone tumor as soon as possible.

Fabrication, microstructure characterization, and degradation performance of electrospun mats based on poly(3-hydroxybutyrate-co-3 hydroxyvalerate)/polyethylene glycol blend for potential tissue engineering.

There have been strong demands for nanofibrous scaffolds fabricated by electrospinning for various fields due to their various advantages. Electrospun poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) fibre mats were prepared. The effects of processing variables as well as the inclusion of poly(ethylene glycol) (PEG) on the morphologies of generated fibres were investigated using Fourier-transform infrared spectroscopy and scanning electron microscopy. The average fibrous diameter was monitored in the range 400-3000 nm relying on the total content of PEG. The fluorescence cell imaging of electrospun mats was also explored. The results of cell viability demonstrated that skin fibroblast BJ-1 cells showed different adhesions and growth rates for the three kinds of PHBV fibres. Electrospun PHBV mats with low amount of PEG offer a high-quality medium for cell growth. Therefore, those mats exhibited high potential for soft tissue engineering, in particular wound healing.

Antihyperglycemic effect of Vernonia amygdalina and in vitro evaluation of its antiproliferative activity on human osteosarcoma MG-63.

Introduction: the leaves of Vernonia amygdalina (V. amygdalina) are consumed as food in sub-Saharan Africa (SSA). In traditional medicine, this plant is widely used in the treatment of cancer and diabetes mellitus. In the present study, we evaluated the antihyperglycemic and the antiproliferative activities of the hydroalcoholic extract of V. amygdalina leaves (HAEVa). Methods: we conducted an experimental descriptive and analytical study with a prospective data collection from May 2019 to July 2020. For the in vivo study, the experiments were carried out on albino male rats of Wistar strain (Rattus norvegicus). Antihyperglycemic activity was performed in vivo in dexamethasone-induced insulin-resistant rats using the oral glucose tolerance test (OGTT). The biocompatibility and the antiproliferative activity of extract were performed in vitro respectively on rabbit primary dermal fibroblasts (RPDF) and human osteosarcoma MG-63 cells using the 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The data were analyzed with the GraphPad Prism software version 5.0.3. The statistical analyses were obtained by the analysis of variance (ANOVA), followed by Bonferroni´s post-test. P<0.05 was considered as the minimal level of statistical significance. Results: regarding to the antiproliferative investigation, extract at 125, 250 µg/mL exhibited a significant cytotoxic effect on human osteosarcoma MG-63 compared to the vehicle (p<0.001) in a dose-response manner after 24h, 48h of exposure to HAEVa. Interestingly, HAEVa in concentrations of 125 and 250µg/ml showed no cytotoxicity (p>0.05) on RPDF after the different times of exposure. However, HAEVa in a high concentration of 500 µg/mL wasn´t biocompatible with RPDF. HAEVa also prevented postprandial blood glucose level in dexamethasone-induced insulin-resistant rats at both doses tested (p>0.05 and p<0.01 at doses of 50 and 100 mg/kg respectively). Conclusion: the results of this study suggest that HAEVa has antiproliferative properties on MG-63 osteosarcoma in vitro and also inhibits in vivo the postprandial blood glucose level in dexamethasone-induced insulin-resistant rats.

Calcium Phosphate-Coated Lipid Nanoparticles as a Potential Tool in Bone Diseases Therapy.

The treatment of bone diseases (including osteoporosis, osteoarthritis, and bone cancer) often results in reduced efficiency and/or adverse reactions due to the fact that it is not specifically targeted to the site of action. The employment of a suitable carrier should increase drug location to the site of bone disease. The purpose of this study is to prepare and characterize lipid nanoparticles (NPs) coated with calcium phosphate (CaP-NPs). A coating method, to date used only to obtain liposomes covered with CaP, is herein partially-modified to prepare CaP-coated lipid NPs. An extensive physico-chemical characterization was achieved by employing several techniques (DLS, SEM and TEM, and both combined with EDS, XRD, and FTIR) that confirmed the feasibility of the developed coating method. Preliminary uptake studies on human osteosarcoma cells (U-2OS) were performed by entrapping, as a lipid probe, Sudan Red III in NPs. The obtained data provided evidence that CaP-NPs showed higher cell accumulation than uncoated NPs. This result may have important implications for the development of drug loaded CaP-NPs to be tested in vitro with a view of planning future treatment of bone diseases, and indicate that CaP-NPs are potential vehicles for selective drug delivery to bone tissue.

Identification of Conserved Pappalysin 1-Derived Circular RNA-Mediated Competing Endogenous RNA in Osteosarcoma.

The etiology of osteosarcoma (OS) is complex and not fully understood till now. This study aimed to identify the miRNAs, circRNAs, and genes (mRNAs) that are differentially expressed in OS cell lines to investigate the mechanism of circRNA-associated competing endogenous RNAs (ceRNAs) in OS. Microarray datasets reporting mRNA (GSE70414), miRNA (GSE70367), and circRNA changes (GSE96964) in human OS cell lines were downloaded, differentially expressed (DE) RNAs were identified, and DEmRNAs were used for the annotation of Gene Ontology (GO) biological processes (BP), and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. The mechanisms of DEcircRNA-mediated ceRNAs were identified in a step-by-step process. A total of 326 DEmRNAs, 45 DEmiRNAs, and 110 DEcircRNAs were identified from 3 datasets. The DEmRNAs were associated with GO BP terms, including cholesterol biosynthetic process, angiogenesis, extracellular matrix organization and KEGG pathways, including p53 signaling pathway and biosynthesis of antibiotics. The final ceRNA network consisted of 8 DEcircRNAs, including 5 pappalysin (PAPPA) 1-derived DEcircRNAs (hsa_circ_0005456, hsa_circ_0088209, hsa_circ_0002052, hsa_circ_0088214 and has_circ_0008792, all downregulated), 3 DEmiRNAs (hsa-miR-760, hsa-miR-4665-5p and hsa-miR-4539, all upregulated), and downregulated genes (including MMP13 and HMOX1). The ceRNA regulation network of OS was built, which played important roles in the pathogenesis of OS and might be of great importance in therapy.

Response of Osteosarcoma Cell Metabolism to Platinum and Palladium Chelates as Potential New Drugs.

This paper reports the first metabolomics study of the impact of new chelates Pt2Spm and Pd2Spm (Spm = Spermine) on human osteosarcoma cellular metabolism, compared to the conventional platinum drugs cisplatin and oxaliplatin, in order to investigate the effects of different metal centers and ligands. Nuclear Magnetic Resonance metabolomics was used to identify meaningful metabolite variations in polar cell extracts collected during exposure to each of the four chelates. Cisplatin and oxaliplatin induced similar metabolic fingerprints of changing metabolite levels (affecting many amino acids, organic acids, nucleotides, choline compounds and other compounds), thus suggesting similar mechanisms of action. For these platinum drugs, a consistent uptake of amino acids is noted, along with an increase in nucleotides and derivatives, namely involved in glycosylation pathways. The Spm chelates elicit a markedly distinct metabolic signature, where inverse features are observed particularly for amino acids and nucleotides. Furthermore, Pd2Spm prompts a weaker response from osteosarcoma cells as compared to its platinum analogue, which is interesting as the palladium chelate exhibits higher cytotoxicity. Putative suggestions are discussed as to the affected cellular pathways and the origins of the distinct responses. This work demonstrates the value of untargeted metabolomics in measuring the response of cancer cells to either conventional or potential new drugs, seeking further understanding (or possible markers) of drug performance at the molecular level.