The use of fluid-phase 3D printing to pattern alginate-gelatin hydrogel properties to guide cell growth and behaviourin vitro.

Three-dimensional (3D) (bio)printing technology has boosted the advancement of the biomedical field. However, tissue engineering is an evolving field and (bio)printing biomimetic constructions for tissue formation is still a challenge. As a new methodology to facilitate the construction of more complex structures, we suggest the use of the fluid-phase 3D printing to pattern the scaffold's properties. The methodology consists of an exchangeable fluid-phase printing medium in which the constructions are fabricated and patterned during the printing process. Using the fluid-phase methodology, the biological and mechanical properties can be tailored promoting cell behaviour guidance and compartmentalization. In this study, we first assessed different formulations of alginate/gelatin to create a stable substrate capable to promote massive cell colonizationin vitroover time. Overall, formulations with lower gelatin content and 2-(N-morpholino)ethanesulfonic acid (MES) buffer as a solvent showed better stability under cell culture conditions and enhanced U2OS cell growth. Next, the fluid-phase showed better printing fidelity and resolution in comparison to air printing as it diminished the collapsing and the spread of the hydrogel strand. In sequence, the fluid-phase methodology was used to create functionalized alginate-gelatin-arginylglycylaspartic acid peptide (RGD) hydrogels via carbodiimides chemistry. The alginate-gelatin-RGD hydrogels showed an increase of 2.97-fold in cell growth and more spread substrate colonization in comparison to alginate-gelatin hydrogel. Moreover, the fluid-phase methodology was used to add RGD molecules to pre-determined parts of the alginate-gelatin substrate during the printing process promoting U2OS cell compartmentalization. In addition, different substrate stiffnesses were also created via fluid-phase by crosslinking the hydrogel with different concentrations of CaCl2during the printing process. As a result, the U2OS cells were also compartmentalized on the stiffer parts of the printings. Finally, our results showed that by combining stiffer hydrogel with RGD increasing concentrations we can create a synergetic effect and boost cell metabolism by up to 3.17-fold. This work presents an idea of a new printing process for tailoring multiple parameters in hydrogel substrates by using fluid-phase to generate more faithful replication of thein vivoenvironment.

Overexpression of miR-506-3p reversed doxorubicin resistance in drug-resistant osteosarcoma cells.

Background and objective: Osteosarcoma is a common primary malignant tumor of bone, and doxorubicin is one of the most widely used therapeutic drugs. While the problem of doxorubicin resistance limits the long-term treatment benefits in osteosarcoma patients. The role of miRNAs and their target genes in osteosarcoma have become increasingly prominent. Currently, there is no report on miR-506-3p reversing doxorubicin resistance by targeting STAT3 in osteosarcoma. The purpose of this study was to investigate the molecular mechanism that overexpression of miR-506-3p reverses doxorubicin resistance in drug-resistant osteosarcoma cells. Methods: Doxorubicin-resistant osteosarcoma cells (U-2OS/Dox) were constructed by intermittent stepwise increasing stoichiometry. The target genes of miR-506-3p were predicted by bioinformatics approach and the targeting relationship between miR-506-3p and STAT3 was detected using dual luciferase reporter assay. U-2OS/Dox cells were treated with miR-506-3p overexpression and STAT3 silencing respectively. Then Western blot and RT-qPCR were used to detect the protein and mRNA expression levels of JAK2/STAT3 signaling pathway, drug-resistant and apoptotic associated molecules. The migration and invasion were assessed by cell scratch assay and transwell assay. The cell proliferative viability and apoptosis were investigated by CCK8 assay and flow cytometry assay. Results: U-2OS/Dox cells were successfully constructed with a 14.4-fold resistance. MiR-506-3p is directly bound to the 3'-UTR of STAT3 mRNA. Compared with U-2OS cells, the mRNA expression of miR-506-3p was reduced in U-2OS/Dox cells. Overexpression of miR-506-3p decreased the mRNA expression levels of JAK2, STAT3, MDR1/ABCB1, MRP1/ABCC1, Survivin and Bcl-2, and decreased the protein expression levels of p-JAK2, STAT3, MDR1/ABCB1, MRP1/ABCC1, Survivin and Bcl-2, and conversely increased Bax expression. It also inhibited the proliferation, migration and invasion of U-2OS/Dox cells and promoted cells apoptosis. The results of STAT3 silencing experiments in the above indicators were consistent with that of miR-506-3p overexpression. Conclusion: Overexpression of miR-506-3p could inhibit the JAK2/STAT3 pathway and the malignant biological behaviors, then further reverse doxorubicin resistance in drug-resistant osteosarcoma cells. The study reported a new molecular mechanism for reversing the resistance of osteosarcoma to doxorubicin chemotherapy and provided theoretical support for solving the clinical problems of doxorubicin resistance in osteosarcoma.

High-content image screening to identify chemical modulators for peroxisome and ferroptosis.

BACKGROUND: The peroxisome is a dynamic organelle with variety in number, size, shape, and activity in different cell types and physiological states. Recent studies have implicated peroxisomal homeostasis in ferroptosis susceptibility. Here, we developed a U-2OS cell line with a fluorescent peroxisomal tag and screened a target-selective chemical library through high-content imaging analysis. METHODS: U-2OS cells stably expressing the mOrange2-Peroxisomes2 tag were generated to screen a target-selective inhibitor library. The nuclear DNA was counterstained with Hoechst 33342 for cell cycle analysis. Cellular images were recorded and quantitatively analyzed through a high-content imaging platform. The effect of selected compounds on ferroptosis induction was analyzed in combination with ferroptosis inducers (RSL3 and erastin). Flow cytometry analysis was conducted to assess the level of reactive oxygen species (ROS) and cell death events. RESULTS: Through the quantification of DNA content and peroxisomal signals in single cells, we demonstrated that peroxisomal abundance was closely linked with cell cycle progression and that peroxisomal biogenesis mainly occurred in the G1/S phase. We further identified compounds that positively and negatively regulated peroxisomal abundance without significantly affecting the cell cycle distribution. Some compounds promoted peroxisomal signals by inducing oxidative stress, while others regulated peroxisomal abundance independent of redox status. Importantly, compounds with peroxisome-enhancing activity potentiated ferroptosis induction. CONCLUSIONS: Our findings pinpoint novel cellular targets that might be involved in peroxisome homeostasis and indicate that compounds promoting peroxisomal abundance could be jointly applied with ferroptosis inducers to potentiate anticancer effect.

Functional Characterization of Circadian Nuclear Receptors REV-ERBα and REV-ERBß in Human Osteosarcoma Cell Cultures.

REV-ERBα and its paralog, REV-ERBß, encoded by NR1D1 and NR1D2 genes, are key nuclear receptors that link the circadian timing system and metabolic homeostasis. Since heme is an endogenous ligand, REV-ERBs have been considered key components of the circadian molecular clock and can be pharmacologically targeted to treat various circadian rhythm-related diseases, such as cardiometabolic, inflammatory, and neuropsychiatric diseases, as well as cancer. REV-ERBs are believed to be functionally redundant and compensatory, although they often affect the expression of gene subsets in an isoform-specific manner. Therefore, this study aimed to identify the redundant and distinct roles of each isoform in controlling its target genes by comparing the transcriptome profiles of a panel of mutant U2OS human osteosarcoma cells in which either NR1D1 or NR1D2 was ablated. Indeed, our transcriptomic analyses revealed that most REV-ERB-regulated genes are controlled by redundant or even additive actions. However, the RNA expression profiles of each single mutant cell line also provide strong evidence for isoform-dependent actions. For example, REV-ERBα is more responsible for regulating the NF-κΒ signaling pathway, whereas a group of extracellular matrix components requires REV-ERBß to maintain their expression. We found that REV-ERBs have isoform-selective functions in the regulation of certain circadian output pathways despite their overlapping roles in the circadian molecular clock. Thus, the development of isoform-selective REV-ERB modulators can help treat metabolic disturbances and certain types of cancer.

AKT1 induces Nanog promoter in a SUMOylation-dependent manner in different pluripotent contexts.

AKT/PKB is a kinase crucial for pluripotency maintenance in pluripotent stem cells. Multiple post-translational modifications modulate its activity. We have previously demonstrated that AKT1 induces the expression of the pluripotency transcription factor Nanog in a SUMOylation-dependent manner in mouse embryonic stem cells. Here, we studied different cellular contexts and main candidates that could mediate this induction. Our results strongly suggest the pluripotency transcription factors OCT4 and SOX2 are not essential mediators. Additionally, we concluded that this induction takes place in different pluripotent contexts but not in terminally differentiated cells. Finally, the cross-matching analysis of ESCs, iPSCs and MEFs transcriptomes and AKT1 phosphorylation targets provided new clues about possible factors that could be involved in the SUMOylation-dependent Nanog induction by AKT.

Role of pH and Crosslinking Ions on Cell Viability and Metabolic Activity in Alginate-Gelatin 3D Prints.

Alginate-gelatin hydrogels are extensively used in bioengineering. However, despite different formulations being used to grow different cell types in vitro, their pH and its effect, together with the crosslinking ions of these formulations, are still infrequently assessed. In this work, we study how these elements can affect hydrogel stability and printability and influence cell viability and metabolism on the resulting 3D prints. Our results show that both the buffer pH and crosslinking ion (Ca2+ or Ba2+) influence the swelling and degradation rates of prints. Moreover, buffer pH influenced the printability of hydrogel in the air but did not when printed directly in a fluid-phase CaCl2 or BaCl2 crosslinking bath. In addition, both U2OS and NIH/3T3 cells showed greater cell metabolic activity on one-layer prints crosslinked with Ca2+. In addition, Ba2+ increased the cell death of NIH/3T3 cells while having no effect on U2OS cell viability. The pH of the buffer also had an important impact on the cell behavior. U2OS cells showed a 2.25-fold cell metabolism increase on one-layer prints prepared at pH 8.0 in comparison to those prepared at pH 5.5, whereas NIH/3T3 cells showed greater metabolism on one-layer prints with pH 7.0. Finally, we observed a difference in the cell arrangement of U2OS cells growing on prints prepared from hydrogels with an acidic buffer in comparison to cells growing on those prepared using a neutral or basic buffer. These results show that both pH and the crosslinking ion influence hydrogel strength and cell behavior.

Single-Molecule Telomere Assay via Optical Mapping (SMTA-OM) Can Potentially Define the ALT Positivity of Cancer.

Telomeres play an essential role in protecting the ends of linear chromosomes and maintaining the integrity of the human genome. One of the key hallmarks of cancers is their replicative immortality. As many as 85-90% of cancers activate the expression of telomerase (TEL+) as the telomere maintenance mechanism (TMM), and 10-15% of cancers utilize the homology-dependent repair (HDR)-based Alternative Lengthening of Telomere (ALT+) pathway. Here, we performed statistical analysis of our previously reported telomere profiling results from Single Molecule Telomere Assay via Optical Mapping (SMTA-OM), which is capable of quantifying individual telomeres from single molecules across all chromosomes. By comparing the telomeric features from SMTA-OM in TEL+ and ALT+ cancer cells, we demonstrated that ALT+ cancer cells display certain unique telomeric profiles, including increased fusions/internal telomere-like sequence (ITS+), fusions/internal telomere-like sequence loss (ITS-), telomere-free ends (TFE), super-long telomeres, and telomere length heterogeneity, compared to TEL+ cancer cells. Therefore, we propose that ALT+ cancer cells can be differentiated from TEL+ cancer cells using the SMTA-OM readouts as biomarkers. In addition, we observed variations in SMTA-OM readouts between different ALT+ cell lines that may potentially be used as biomarkers for discerning subtypes of ALT+ cancer and monitoring the response to cancer therapy.

Impact of Manganese and Chromate on Specific DNA Double-Strand Break Repair Pathways.

Manganese is an essential trace element; nevertheless, on conditions of overload, it becomes toxic, with neurotoxicity being the main concern. Chromate is a well-known human carcinogen. The underlying mechanisms seem to be oxidative stress as well as direct DNA damage in the case of chromate, but also interactions with DNA repair systems in both cases. However, the impact of manganese and chromate on DNA double-strand break (DSB) repair pathways is largely unknown. In the present study, we examined the induction of DSB as well as the effect on specific DNA DSB repair mechanisms, namely homologous recombination (HR), non-homologous end joining (NHEJ), single strand annealing (SSA), and microhomology-mediated end joining (MMEJ). We applied DSB repair pathway-specific reporter cell lines, pulsed field gel electrophoresis as well as gene expression analysis, and investigated the binding of specific DNA repair proteins via immunoflourescence. While manganese did not seem to induce DNA DSB and had no impact on NHEJ and MMEJ, HR and SSA were inhibited. In the case of chromate, the induction of DSB was further supported. Regarding DSB repair, no inhibition was seen in the case of NHEJ and SSA, but HR was diminished and MMEJ was activated in a pronounced manner. The results indicate a specific inhibition of error-free HR by manganese and chromate, with a shift towards error-prone DSB repair mechanisms in both cases. These observations suggest the induction of genomic instability and may explain the microsatellite instability involved in chromate-induced carcinogenicity.

The MMP-2 histone H3 N-terminal tail protease is selectively targeted to the transcription start sites of active genes.

BACKGROUND: Proteolysis of the histone H3 N-terminal tail (H3NT) is an evolutionarily conserved epigenomic feature of nearly all eukaryotes, generating a cleaved H3 product that is retained in ~ 5-10% of the genome. Although H3NT proteolysis within chromatin was first reported over 60 years ago, the genomic sites targeted for H3NT proteolysis and the impact of this histone modification on chromatin structure and function remain largely unknown. The goal of this study was to identify the specific regions targeted for H3NT proteolysis and investigate the consequence of H3NT "clipping" on local histone post-translational modification (PTM) dynamics. RESULTS: Leveraging recent findings that matrix metalloproteinase 2 (MMP-2) functions as the principal nuclear H3NT protease in the human U2OS osteosarcoma cell line, a ChIP-Seq approach was used to map MMP-2 localization genome wide. The results indicate that MMP-2 is selectively targeted to the transcription start sites (TSSs) of protein coding genes, primarily at the + 1 nucleosome. MMP-2 localization was exclusive to highly expressed genes, further supporting a functional role for H3NT proteolysis in transcriptional regulation. MMP-2 dependent H3NT proteolysis at the TSSs of these genes resulted in a > twofold reduction of activation-associated histone H3 PTMs, including H3K4me3, H3K9ac and H3K18ac. One of genes requiring MMP-2 mediated H3NT proteolysis for proficient expression was the lysosomal cathepsin B protease (CTSB), which we discovered functions as a secondary nuclear H3NT protease in U2OS cells. CONCLUSIONS: This study revealed that the MMP-2 H3NT protease is selectively targeted to the TSSs of active protein coding genes in U2OS cells. The resulting H3NT proteolysis directly alters local histone H3 PTM patterns at TSSs, which likely functions to regulate transcription. MMP-2 mediated H3NT proteolysis directly activates CTSB, a secondary H3NT protease that generates additional cleaved H3 products within chromatin.

Acetylshikonin induces apoptosis of human osteosarcoma U2OS cells by triggering ROS-dependent multiple signal pathways.

Acetylshikonin a natural compound isolated from the root of Lithospermum erythrorhizon and one of the shikonin derivatives which possess promising anticarcinogenic ability. In this study, we attempted to investigate the anti-cancer potential of acetylshikonin towards osteosarcoma U2OS cells. The effects of acetylshikonin towards the treatment of U2OS cells showed that decreased cell proliferation and inhibited migration ability of cells which are experimentally assessed via wide range of assays including MTT, WST-1, cell counting, colony formation assays, wound healing assay and gelatin zymography assay. We also observed that early apoptosis and late apoptosis were increased through fluorescence-activated cell sorter (FACS) analysis. Terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labeling (TUNEL) assay showed that acetylshikonin induced DNA fragmentation. Western blot analysis revealed the apoptotic effect of acetylshikonin by measuring of proteins such as cleaved caspase -9, -8, -3, -6, -7, and Bcl-2 family. We observed that ROS level and DNA damage were increased via DCF-DA assay and comet assay. In terms of the presence of ROS, induction of apoptosis was detected by measuring proteins such as cleaved caspase 3, PARP, Bcl-2 and Bax. We suggested that the reactions were related to the nuclear translocation of FOXO3 through western blot of cytoplasmic/nuclear protein fractionation. We finally demonstrated that the knockdown of the FOXO3 induced the decrease of the apoptosis-associated proteins via western blot of FOXO3 siRNA transfection. Taken together, these results suggested that acetylshikonin might induce ROS-mediated apoptosis in a FOXO3-dependent manner against osteosarcoma cells. Therefore, acetylshikonin may be elucidated as an effective candidate for the treatment of osteosarcoma.

Radiometric Assay of ABHD2 Activity.

The α,ß-hydrolase fold-containing protein 2 (ABHD2) is a serine hydrolase, responsible for the cleavage of endogenous 2-arachidonoylglycerol (2-AG). ABHD2 is activated by progesterone, thus, it is considered a nonnuclear receptor of this steroid hormone that terminates its biological effects. The products of ABHD2-catalyzed cleavage by the natural substrate 2-AG are glycerol and arachidonic acid; here, instead of 2-AG, the radioactive substrate 2-oleoyl-[3H]glycerol has been used as already done in various acylglycerol lipase activity assays. The amount of [3H]glycerol released allows to measure ABHD2 enzymatic activity.

Systemic ASBT inactivation protects against liver damage in obstructive cholestasis in mice.

Background & Aims: Non-absorbable inhibitors of the apical sodium-dependent bile acid transporter (ASBT; also called ileal bile acid transporter [IBAT]) are recently approved or in clinical development for multiple cholestatic liver disorders and lead to a reduction in pruritus and (markers for) liver injury. Unfortunately, non-absorbable ASBT inhibitors (ASBTi) can induce diarrhoea or may be ineffective if cholestasis is extensive and largely precludes intestinal excretion of bile acids. Systemically acting ASBTi that divert bile salts towards renal excretion may alleviate these issues. Methods: Bile duct ligation (BDL) was performed in ASBT-deficient (ASBT knockout [KO]) mice as a model for chronic systemic ASBT inhibition in obstructive cholestasis. Co-infusion of radiolabelled taurocholate and inulin was used to quantify renal bile salt excretion after BDL. In a second (wild-type) mouse model, a combination of obeticholic acid (OCA) and intestine-restricted ASBT inhibition was used to lower the bile salt pool size before BDL. Results: After BDL, ASBT KO mice had reduced plasma bilirubin and alkaline phosphatase compared with wild-type mice with BDL and showed a marked reduction in liver necrotic areas at histopathological analysis, suggesting decreased BDL-induced liver damage. Furthermore, ASBT KO mice had reduced bile salt pool size, lower plasma taurine-conjugated polyhydroxylated bile salt, and increased urinary bile salt excretion. Pretreatment with OCA + ASBTi in wild-type mice reduced the pool size and greatly improved liver injury markers and liver histology. Conclusions: A reduced bile salt pool at the onset of cholestasis effectively lowers cholestatic liver injury in mice. Systemic ASBT inhibition may be valuable as treatment for cholestatic liver disease by lowering the pool size and increasing renal bile salt output even under conditions of minimal faecal bile salt secretion. Lay summary: Novel treatment approaches against cholestatic liver disease (resulting in reduced or blocked flow of bile) involve non-absorbable inhibitors of the bile acid transport protein ASBT, but these are not always effective and/or can cause unwanted side effects. In this study, we demonstrate that systemic inhibition/inactivation of ASBT protects mice against developing severe cholestatic liver injury after bile duct ligation, by reducing bile salt pool size and increasing renal bile salt excretion.

Colchicine as a novel drug for the treatment of osteosarcoma through drug repositioning based on an FDA drug library.

Background: Colchicine is a traditional medication that is currently approved to treat gout and familial Mediterranean fever (FMF). However, colchicine has a wide range of anti-inflammatory activities, and several studies have indicated that it may be useful in a variety of other conditions, such as rheumatic disease, cardiac disease, and cancer. Osteosarcoma, the most common type of bone sarcoma, is derived from primitive bone-forming mesenchymal cells. In this study, we investigated whether colchicine could be used to treat osteosarcoma through the regulation of cell cycle signaling. Methods: Two human osteosarcoma cell lines, U2OS and Saos-2, were used. A clonogenic assay was used to determine the antiproliferative effects of colchicine on osteosarcoma cells. Reactive oxygen species (ROS) production and apoptosis were measured by flow cytometry. Migration and invasion assays were performed to investigate the inhibitory effects of colchicine. The signaling pathways related to colchicine treatment were verified by GO biological process (GOBP) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses. Results: Colchicine was selected as the lead compound based on the results of initial screening and cell viability assays conducted in Saos-2 and U2Os cells. Colchicine reduced the viability of Saos-2 and U2OS cells in a concentration-dependent manner. It also significantly inhibited colony-forming ability and induced ROS production and apoptosis. It also inhibited the migration and invasion of both Saos-2 and U2OS cells. GOBP and KEGG enrichment analyses indicated the involvement of microtubule-based processes and cancer-related pathways. Conclusions: These findings suggest that colchicine has therapeutic potential in osteosarcoma.

Recovering mixtures of fast-diffusing states from short single-particle trajectories.

Single-particle tracking (SPT) directly measures the dynamics of proteins in living cells and is a powerful tool to dissect molecular mechanisms of cellular regulation. Interpretation of SPT with fast-diffusing proteins in mammalian cells, however, is complicated by technical limitations imposed by fast image acquisition. These limitations include short trajectory length due to photobleaching and shallow depth of field, high localization error due to the low photon budget imposed by short integration times, and cell-to-cell variability. To address these issues, we investigated methods inspired by Bayesian nonparametrics to infer distributions of state parameters from SPT data with short trajectories, variable localization precision, and absence of prior knowledge about the number of underlying states. We discuss the advantages and disadvantages of these approaches relative to other frameworks for SPT analysis.

Differential impact of lipid raft depletion on platelet-derived growth factor (PDGF)-induced ERK1/2 MAP-kinase, SRC and AKT signaling.

It has become clear that lipid rafts functions as signaling hotspots connecting cell surface receptors to intracellular signaling pathways. However, the exact involvement of lipid rafts in receptor tyrosine kinase signaling is still poorly understood. In this study, we have analyzed platelet-derived growth factor (PDGF) receptor ß (PDGFR-ß) signaling in two different cell lines depleted of cholesterol, and as a consequence, disruption of lipid rafts. Cholesterol depletion of BJ-hTERT fibroblasts using methyl-ß-cyclodextrin (MßCD) did not affect PDGFR-ß activation as measured by its tyrosine phosphorylation. However, we did observe a small reduction in AKT phosphorylation and a more robust decrease of ERK1/2 activation. In contrast, in the osteosarcoma cell line U2OS, we noticed a deficient receptor activation. Interestingly, in U2OS cells, the ERK1/2 pathway was unaffected, but instead AKT and SRC signaling was reduced. These results suggest that cell type specific wiring of signaling pathways can lead to differential sensitivity to cholesterol depletion. Furthermore, MßCD treatment had a much more pronounced morphological effect on U2OS compared to BJ-hTERT cells. This is consistent with a previous report claiming that cancer cells are more sensitive to cholesterol depletion than normal cells. Our data supports the possibility that cholesterol lowering drugs may impede tumor growth.

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.

Tanshinol suppresses osteosarcoma by specifically inducing apoptosis of U2-OS cells through p53-mediated mechanism.

ETHNOPHARMACOLOGICAL RELEVANCE: Radix Salviae miltiorrhizae (also called Danshen in traditional Chinese medicine) is a famous herbal medicine, which has been frequently used to treat blood stasis syndrome including osteosarcoma (OS) in traditional Chinese medicine. Main components of Danshen have been assumed to exhibit anti-OS capacity. Nevertheless, tanshinol (TS, main component of Danshen)'s efficacy and mechanism in OS hasn't been clearly described ever since. This drew our attention, since OS is the most frequent primary bone carcinomas in children and adolescents, with a high incidence and fatality rate. Unfortunately, chemotherapy for OS has faced many clinical challenges due to the increasing chemoresistance and recurrence. This study was then designed to deeply explore TS's role in OS therapy. AIM OF THE STUDY: To explore the anti-OS efficacy and mechanism of TS, we conducted in vivo and in vitro experiments by using a zebrafish xenograft model and U2-OS cells. MATERIALS AND METHODS: CCK-8 assay, DAPI and γ-H2A.X immunofluorescence staining, and flow cytometry (apoptosis verification) were employed to determine the anti-proliferative and pro-apoptotic effects of TS. qPCR and Western blot were used to examine TS's molecular actions and mechanism on apoptosis of U2-OS cells. RESULTS: The in vivo data showed that TS significantly inhibited U2-OS tumor growth in larval zebrafish from 2 to 20 ng/mL. In vitro data indicated that TS exerted significant anti-proliferative and pro-apoptotic effects on U2-OS cells in a dose-dependent manner. Moreover, TS has no inhibitory effect on bMSCs, suggesting its safety on normal bone-forming cells. Molecular data illustrated that TS obviously activated the p53 signaling-related proteins (p-p53, Bax, CASP3, CASP9) and its upstream JNK (p-JNK, p-c-JUN) and ATM (p-ATM) signaling molecules through phosphorylation and cleavage, followed by up-regulation of the pro-apoptotic genes, NOXA, PUMA, TP53, BAX, and BIM, and down-regulation of Bcl-2 protein. CONCLUSION: In sum, TS specifically induced apoptosis of U2-OS cells by activating p53 signaling pathways, indicating TS as a promising candidate for OS treatment.

TRIP13 knockdown inhibits the proliferation, migration, invasion, and promotes apoptosis by suppressing PI3K/AKT signaling pathway in U2OS cells.

BACKGROUND: Although osteosarcoma (OS) is the most common malignant bone tumor, the biological mechanism underlying its incidence and improvement remains unclear. This study investigated early diagnosis and treatment objectives using bioinformatics strategies and performed experimental verification. METHODS AND RESULTS: The top 10 OS hub genes-CCNA2, CCNB1, AURKA, TRIP13, RFC4, DLGAP5, NDC80, CDC20, CDK1, and KIF20A-were screened using bioinformatics methods. TRIP13 was chosen for validation after reviewing literature. TRIP13 was shown to be substantially expressed in OS tissues and cells, according to Western blotting (WB) and quantitative real-time polymerase chain reaction data. Subsequently, TRIP13 knockdown enhanced apoptosis and decreased proliferation, migration, and invasion in U2OS cells, as validated by the cell counting kit-8 test, Hoechst 33,258 staining, wound healing assay, and WB. In addition, the levels of p-PI3K/PI3K and p-AKT/AKT in U2OS cells markedly decreased after TRIP13 knockdown. Culturing U2OS cells, in which TRIP13 expression was downregulated, in a medium supplemented with a PI3K/AKT inhibitor further reduced their proliferation, migration, and invasion and increased their apoptosis. CONCLUSIONS: TRIP13 knockdown reduced U2OS cell proliferation, migration, and invasion via a possible mechanism involving the PI3K/AKT signaling pathway.

The inhibitory effect of a novel phosphodiesterase 4 inhibitor ZL-n-91 on proliferation of osteosarcoma U20S cells / 中国药理学通报

Aim To explore the anti-cancer effects of ZL-n-91, a novel and highly selective phosphodiesterase 4 inhibitor, on the osteosarcoma U2OS cells.Methods CCK-8 assay was used to detect the inhibitory effect of ZL-n-91 with different concentrations(0, 20, 40, 80, 160, 240, 320, 400, 480 μmol·L-1)and different intervention time(0, 24, 48, 72, 96 h)on the proliferation of U2OS cells.Tablet clone forming experiment was used to detect the effect of ZL-n-91 on the clonality of U2OS cells.Flow cytometry was used to detect the cell apoptosis and cell cycle distribution.Western blot was employed to detect the expression of Bcl-2, CDK2, CDK4, CyclinD1, CyclinE1 protein.Results The inhibitory rate of ZL-n-91 on U2OS cells was concentration- and time-dependent(P<0.05), and its half inhibition rate IC50 was 174.1 μmol·L-1.ZL-n-91 significantly inhibited the clonality of U2OS cells(P<0.01).ZL-n-91 significantly induced cell apoptosis, and caused cell cycle arrest at G0/G1 phase in U2OS cells(P<0.01).The results of Western blot showed that ZL-n-91 significantly down-regulated the expression of Bcl-2, CDK2, CDK4, CyclinD1, CyclinE1 proteins in U2OS cells(P<0.05).Conclusions The novel selective phosphodiesterase 4 inhibitor, ZL-n-91, can significantly inhibit the proliferation of osteosarcoma U2OS cells with induction of cell cycle arrest and cell apoptosis, and may become a potential anti-cancer agent.

Early Recognition of the PCL/Fibrous Carbon Nanocomposites Interaction with Osteoblast-like Cells by Raman Spectroscopy.

Poly(ε-caprolactone) (PCL) is a biocompatible resorbable material, but its use is limited due to the fact that it is characterized by the lack of cell adhesion to its surface. Various chemical and physical methods are described in the literature, as well as modifications with various nanoparticles aimed at giving it such surface properties that would positively affect cell adhesion. Nanomaterials, in the form of membranes, were obtained by the introduction of multi-walled carbon nanotubes (MWCNTs and functionalized nanotubes, MWCNTs-f) as well as electro-spun carbon nanofibers (ESCNFs, and functionalized nanofibers, ESCNFs-f) into a PCL matrix. Their properties were compared with that of reference, unmodified PCL membrane. Human osteoblast-like cell line, U-2 OS (expressing green fluorescent protein, GFP) was seeded on the evaluated nanomaterial membranes at relatively low confluency and cultured in the standard cell culture conditions. The attachment and the growth of the cell populations on the polymer and nanocomposite samples were monitored throughout the first week of culture with fluorescence microscopy. Simultaneously, Raman microspectroscopy was also used to track the dependence of U-2 OS cell development on the type of nanomaterial, and it has proven to be the best method for the early detection of nanomaterial/cell interactions. The differentiation of interactions depending on the type of nanoadditive is indicated by the ν(COC) vibration range, which indicates the interaction with PCL membranes with carbon nanotubes, while it is irrelevant for PCL with carbon nanofibers, for which no changes are observed. The vibration range ω(CH2) indicates the interaction for PCL with carbon nanofibers with seeded cells. The crystallinity of the area ν(C=O) increases for PCL/MWCNTs and for PCL/MWCNTs-f, while it decreases for PCL/ESCNFs and for PCL/ESCNFs-f with seeded cells. The crystallinity of the membranes, which is determined by Raman microspectroscopy, allows for the assessment of polymer structure changes and their degradability caused by the secretion of cell products into the ECM and the differentiation of interactions depending on the carbon nanostructure. The obtained nanocomposite membranes are promising bioactive materials.