U2OS Cells Research Articles

Abstract 6416: Statins inhibit onco-dimerization of 4Ig-B7-H3

Abstract B7-H3 (CD276) is a member of the B7 family of immune regulators, but unlike the immune checkpoint targets that have revolutionized cancer therapy, blockade has resulted in mixed outcomes. B7-H3 has 2Ig and 4Ig isoforms, has no known direct ligand, and many of the physiological functions remain elusive. While protein expression of B7-H3 is differentially expressed on many solid tumors and correlates with poor survival, mechanisms of how B7-H3 elicits a pro-tumorigenic phenotype are still poorly understood, significantly hampering therapeutic efforts for targeting. Using a facile, recombinase-enhanced split-luciferase reporter, we visualized 4Ig-B7-H3 homodimerization in live U2OS cells by bioluminescence imaging and confirmed 4Ig-B7-H3 dimerization using a second non-invasive imaging technique: fluorescence lifetime imaging microscopy (FLIM) where 4Ig-B7-H3 was exogenously re-expressed following knockout of CD276 in HeLa cervical cancer cells. Homodimerization of 4Ig-B7-H3 correlated with enhanced clonogenic cell growth in HeLa, SKOv3, MDA-MB-231, and U2OS cells and tumor growth in vivo for several models. This phenotype was reversed following knockout of B7-H3 and rescued upon lentiviral re-expression. By RPPA and phospho-kinase array analysis, increases in PI3K/AKT signaling, Jak/Stat pathway, and modulators of HIF1α and NF-κβ pathways were observed with B7-H3 expression and homodimerization (p<0.05), all which are known to support increased oncogenic functions, such as increased glycolysis, increased survival and proliferation, as well as cytokine modulation and immune evasion. Next, capitalizing on our bioluminescence-based reporter of 4Ig-B7-H3 homodimerization, we performed a high-throughput small molecule screen with a 5,362 Bioactive compound library to identify modulators of 4Ig-B7-H3 dimerization. Notably, our HTS identified several HMG-CoA reductase inhibitors (statins) as significant inhibitors of B7-H3 dimerization (p<0.01). Treatment with 1 μM mevastatin or atorvastatin calcium inhibited clonogenic growth of HeLa cervical, SKOv3 ovarian and MDA-MB-231 breast cancer cells, which all endogenously express B7-H3. In vivo, treatment with 10 mg/kg of atorvastatin calcium reduced tumor growth and increased mouse survival (p=0.035) of SKOv3-ip tumors and produced functional cures (15-30%) of HeLa or MDA-MB-231 xenografts in nude mice in a B7-H3 tumor expression-dependent manner, proven through knockout and rescue experiments in live cells and animals. Thus, in the context of studies to dissect the tumor-intrinsic mechanism(s) of B7-H3 biochemical functions and activation, we identified a novel, dimerization-dependent oncogenic role for 4Ig-B7-H3 that increased oncogenic intracellular signaling activation and was modulated by statin treatment. This may provide a molecular mechanism for the statin-mediated immune modulation and enhanced cancer survival clinically reported for several tumor types. Citation Format: Margie Nicole Sutton, Sarah E. Glazer, Riccardo Muzzioli, Ping Yang, Seth T. Gammon, David Piwnica-Worms. Statins inhibit onco-dimerization of 4Ig-B7-H3 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6416.

Abstract 6203: CTC1-STN1-TEN1 (CST) complex-independent roles for STN1 in DNA double-strand break repair in pancreatic ductal adenocarcinoma

Abstract KRAS mutation occurs in 90-95% of pancreatic ductal adenocarcinoma (PDAC) and is a driver of PDAC; however, the underlying molecular mechanisms by which KRAS mutation maintains survival and promotes progression of PDAC need further refinement. We herein report that KRAS mutations increase STN1 (also named OBFC1) expression leading to enhanced repair of DNA double strand breaks (DSBs) in PDAC. Analysis of The Cancer Genome Atlas databases reveal that STN1 is significantly upregulated in PDAC tissue and, is associated with KRAS mutation and poor survival. Silencing KRAS or pharmacologic inhibition of KRAS signaling decreased STN1 expression in PDAC cells. Canonically, STN1 forms a CST (CTC1-STN1-TEN1) complex, which is important for telomere duplication and maintenance. Intriguingly, siRNA-mediated depletion of STN1 (but not CTC1 or TEN1) sensitized PDAC cells to ionizing radiation and chemotherapy as assessed by radiation clonogenic assay and alamarBlue assay, respectively. Consistently, immunoblotting and immunofluorescence analyses of γH2AX (marker of DNA damage) demonstrated that depletion of STN1 (but not CTC1 or TEN1) resulted in DNA damage. To explore the underlying molecular mechanisms, we performed proteomic analysis and found that STN1 physically interacts with many proteins important for DNA repair, replication and cell cycle progression. Of note, we revealed unreported interactions of STN1 with ATM, ATR and DNA-PKcs, key components of DNA damage response signaling. Furthermore, homologous recombination and non-homologous end joining repair reporter assays demonstrated that STN1 silencing reduced both homologous recombination and non-homologous end joining repair of DSBs in U2OS and PDAC cells. Moreover, flow cytometry and immunofluorescence analyses showed that knockdown of STN1 by siRNA impaired cell cycle arrest at S and G2/M phases which was accompanied with mitotic catastrophe in response to ionizing radiation. Our findings have revealed novel CST complex-independent roles for STN1 in DSB repair and suggest STN1 is a novel biomarker for PDAC as well as potentially other cancers. Citation Format: Changxian Shen, Linlin Yang, Sergio Corrales-Guerrero, Sindhu Nair, Terence Williams. CTC1-STN1-TEN1 (CST) complex-independent roles for STN1 in DNA double-strand break repair in pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6203.

Vrk2 sensitizes u2os cell to chemotherapeutic agents by negatively regulating g3bp1-mediated stress granule formation.

Vrk2 sensitizes u2os cell to chemotherapeutic agents by negatively regulating g3bp1-mediated stress granule formation.

Cinobufacini injection suppresses the proliferation of human osteosarcoma cells by inhibiting PIN1-YAP/TAZ signaling pathway.

Cinobufacini injection (CI), an aqueous extract of Cutis Bufonis, is clinically used for cancer therapy in China, but its molecular mechanism for the treatment of osteosarcoma (OS) remains unclear. We constructed U2OS ectopic subcutaneous tumor model to verify the anti-OS effect of CI in vivo. Meanwhile, cell proliferation of U2OS and MG63 cells was monitored in vitro using the CCK-8 assay, colony formation and morphological changes. Cell cycle arrest and apoptosis were detected by flow cytometry and western blot, which showed that CI significantly inhibited proliferation, induced cell cycle arrest and apoptosis in human OS cells. The further RNA-seq results identified that the Hippo signaling pathway was involved in the anti-OS effect of CI. YAP/TAZ are two major components of the Hippo pathway in breast cancer and are positively regulated by prolyl isomerase PIN1, we assessed their role in OS using both clinicopathological sections and western blots. CI also inhibited PIN1 enzyme activity in a dose-dependent manner, which resulted in impaired PIN1, YAP, and TAZ expression in vitro and in vivo. Additionally, 15 potential compounds of CI were found to occupy the PIN1 kinase domain and inhibit its activity. In summary, CI plays an anti-OS role by down-regulating the PIN1-YAP/TAZ pathway.

Cuproptosis signature and PLCD3 predicts immune infiltration and drug responses in osteosarcoma.

Osteosarcoma (OS) is a cancer that is frequently found in children and adolescents and has made little improvement in terms of prognosis in recent years. A recently discovered type of programmed cell death called cuproptosis is mediated by copper ions and the tricarboxylic acid (TCA) cycle. The expression patterns, roles, and prognostic and predictive capabilities of the cuproptosis regulating genes were investigated in this work. TARGET and GEO provided transcriptional profiling of OS. To find different cuproptosis gene expression patterns, consensus clustering was used. To identify hub genes linked to cuproptosis, differential expression (DE) and weighted gene co-expression network analysis (WGCNA) were used. Cox regression and Random Survival Forest were used to build an evaluation model for prognosis. For various clusters/subgroups, GSVA, mRNAsi, and other immune infiltration experiments were carried out. The drug-responsive study was carried out by the Oncopredict algorithm. Cuproptosis genes displayed two unique patterns of expression, and high expression of FDX1 was associated with a poor outcome in OS patients. The TCA cycle and other tumor-promoting pathways were validated by the functional study, and activation of the cuproptosis genes may also be connected with immunosuppressive state. The robust survival prediction ability of a five-gene prognostic model was verified. This rating method also took stemness and immunosuppressive characteristics into account. Additionally, it can be associated with a higher sensitivity to medications that block PI3K/AKT/mTOR signaling as well as numerous chemoresistances. U2OS cell migration and proliferation may be encouraged by PLCD3. The relevance of PLCD3 in immunotherapy prediction was verified. The prognostic significance, expressing patterns, and functions of cuproptosis in OS were revealed in this work on a preliminary basis. The cuproptosis-related scoring model worked well for predicting prognosis and chemoresistance.

Piperine improves the sensitivity of osteosarcoma cells to doxorubicin by inducing apoptosis and inhibiting the PI3K/AKT/GSK-3β pathway

BackgroundOsteosarcoma is a primary bone malignancy associated with the highest incidence rate. Chemotherapy for osteosarcoma has not substantially changed, and survival of patients with metastatic tumours has reached a plateau. Doxorubicin (DOX) is a broad-spectrum anti-osteosarcoma drug; however, its application is limited due to its high cardiotoxicity. Piperine (PIP) has been verified to drive certain cancer cell death and increases chemosensitivity of DOX. However, the effects of PIP in promoting the chemosensitivity of osteosarcoma to DOX have not been studied.MethodsWe examined the combined effect of PIP and DOX on U2OS and 143B osteosarcoma cells. CCK-8 assays, scratch assays, flow cytometry analysis, and western blotting were performed. Furthermore, the effect of PIP combined with DOX on osteosarcoma tumours was observed in vivo using nude mice.ResultsPIP can increase the chemosensitivity of U2OS and 143B cells to DOX. Both in vitro and in vivo results showed the dramatic inhibition of cell proliferation and tumour growth by the combined therapy group compared to monotherapy groups. Apoptosis analysis revealed that PIP augments DOX-induced cell apoptosis by upregulating BAX and P53 expression, as well as reducing Bcl-2 expression. Furthermore, PIP also attenuated the initiation of the PI3K/AKT/GSK-3β signaling pathway in osteosarcoma cells by altering the expression levels of P-AKT, P-PI3K and P-GSK3β.ConclusionsThis study revealed for the first time that PIP can potentiate the sensitivity and cytotoxicity of DOX during osteosarcoma therapy in vitro and in vivo, which probably achieved by inhibiting the PI3K/AKT/GSK-3β signalling pathway.

Differential contributions of phosphotransferases CEPT1 and CHPT1 to phosphatidylcholine homeostasis and lipid droplet biogenesis

The CDP-choline (Kennedy) pathway culminates with the synthesis of phosphatidylcholine (PC) and phosphatidylethanolamine (PE) by choline/ethanolamine phosphotransferase 1 (CEPT1) in the endoplasmic reticulum (ER), and PC synthesis by choline phosphotransferase 1 (CHPT1) in the Golgi apparatus. Whether the PC and PE synthesized by CEPT1 and CHPT1 in the ER and Golgi apparatus has different cellular functions has not been formally addressed. Here we used CRISPR editing to generate CEPT1-and CHPT1-knockout (KO) U2OS cells to assess the differential contribution of the enzymes to feed-back regulation of nuclear CTP:phosphocholine cytidylyltransferase (CCT)α, the rate-limiting enzyme in PC synthesis, and lipid droplet (LD) biogenesis. We found that CEPT1-KO cells had a 50% and 80% reduction in PC and PE synthesis, respectively, while PC synthesis in CHPT1-KO cells was also reduced by 50%. CEPT1 knockout caused the post-transcriptional induction of CCTα protein expression as well as its dephosphorylation and constitutive localization on the inner nuclear membrane and nucleoplasmic reticulum. This activated CCTα phenotype was prevented by incubating CEPT1-KO cells with PC liposomes to restore end-product inhibition. Additionally, we determined that CEPT1 was in close proximity to cytoplasmic LDs, and CEPT1 knockout resulted in the accumulation of small cytoplasmic LDs, as well as increased nuclear LDs enriched in CCTα. In contrast, CHPT1 knockout had no effect on CCTα regulation or LD biogenesis. Thus, CEPT1 and CHPT1 contribute equally to PC synthesis; however, only PC synthesized by CEPT1 in the ER regulates CCTα and the biogenesis of cytoplasmic and nuclear LDs.

DEPDC1 and KIF4A synergistically inhibit the malignant biological behavior of osteosarcoma cells through Hippo signaling pathway

The treatment of osteosarcoma (OS) is still mainly surgery combined with systematic chemotherapy, and gene therapy is expected to improve the survival rate of patients. This study aimed to explore the effect of DEP domain 1 protein (DEPDC1) and kinesin super-family protein 4A (KIF4A) in OS and understand its mechanism. Th expression of DEPDC1 and KIF4A in OS cells was detected by RT-PCR and western blot. The viability, proliferation, invasion and migration of OS cells and tube formation of human umbilical vein endothelial cells (HUVECs) after indicated treatment were in turn detected by CCK-8 assay, EdU staining, wound healing assay, transwell assay and tube formation assay. The interaction between DEPDC1 and KIF4A was predicted by STRING and confirmed by co-immunoprecipitation. The expression of epithelial-mesenchymal transition (EMT)-related proteins, tube formation-related proteins and Hippo signaling pathway proteins was detected by western blot. As a result, the expression of DEPDC1 and KIF4A was all increased in U2OS cells. Down-regulation of DEPDC1 suppressed the viability, proliferation, invasion and migration of U2OS cells and tube formation of HUVECs, accompanied by the increased expression of E-cadherin and decreased expression of N-cadherin, Vimentin and VEGF. DEPDC1 was confirmed to be interacted with KIF4A. Upregulation of KIF4A partially reversed the effect of DEPDC1 interference on the above biological behaviors of U2OS cells. Down-regulation of DEPDC1 promoted the expression of p-LATS1 and p-YAP in Hippo signaling pathway, which was reversed by upregulation of KIF4A. In conclusion, down-regulation of DEPDC1 inhibited the malignant biological behavior of OS cells through the activation of Hippo signaling pathway, which could be reversed by upregulation of KIF4A.

Ursonic acid inhibits migration and invasion of human osteosarcoma cells through the suppression of mitogen-activated protein kinases and matrix metalloproteinases.

Osteosarcoma (OS) is the most common form of bone malignancy. Although contemporary chemotherapy and surgery have improved the prognosis of those with OS, developing new OS therapies has proven difficult for some time. The activation of the matrix metalloproteinase (MMP) and mitogen-activated protein kinase (MAPK) signaling pathways can induce metastasis, which is an obstacle to OS treatment. Ursonic acid (UNA) is a phytochemical with the potential to cure a variety of human ailments, including cancer. In this study, we investigated the anti-tumor properties of UNA in MG63 cells. We conducted colony formation assay, wound healing assay, and Boyden chamber assays to investigate the anti-OS effects of UNA. UNA was found to significantly inhibit the proliferative, migratory, and invasive abilities of MG63 cells. This bioactivity of UNA was mediated by the inhibition of extracellular signal-regulated kinase (ERK) and p38 and reduction of MMP-2 transcriptional expression as observed in western blot analysis, gelatin zymography and RT-PCR. Anti-OS activities of UNA were also observed in Saos2 and U2OS cells, indicating that its anti-cancer properties are not specific to cell types. Our findings suggest that UNA has the potential for use in anti-metastatic drugs in the treatment of OS.

PINK1 regulates apoptosis of osteosarcoma as the target gene of cisplatin

BackgroundOsteosarcoma is a common primary bone malignancy prevalent among adolescents and young adults. PTEN-induced kinase 1 (PINK1) regulates Parkinson's disease, but its role in cancers is unknown.ObjectiveThis study was designed to analyze the mechanism by which PINK1 affects osteosarcoma using bioinformatics and cell experiments.Materials and methodsThe gene expression profiles were downloaded from the TARGET database. Several online databases were used to analyze the expression and protein‒protein interaction networks. CCK-8 cell viability assays and cisplatin treatment were used to assess cell activity with or without cisplatin treatment. Acridine orange/ethidium bromide (AO/EB) fluorescence staining was used to calculate the percentage of apoptotic cells.ResultsThrough bioinformatics analysis, we found that high expression of PINK1 was associated with poor prognosis in patients with osteosarcoma, and PINK1 inhibited apoptosis and promoted proliferation pathways. Next, we found that both PINK1 mRNA and protein levels were upregulated in osteosarcoma tissues. Additionally, we found that PTEN was reduced, while FOXO3a was markedly increased in osteosarcoma, suggesting that FOXO3a and not PTEN induced the overexpression of PINK1. CCK-8 and clonogenic assays showed that the knockdown of PINK1 decreased the growth of U2OS osteosarcoma cells. Ki67 immunofluorescence staining revealed that reduced cell proliferation in U2OS cells resulted in the depletion of PINK1. In addition, our AO/EB staining results indicated that the knockdown of PINK1 resulted in an increase in apoptotic cells and increased the levels of cleaved caspase-3. Furthermore, our experiments revealed that cisplatin promotes OS cell apoptosis by downregulating PINK1.ConclusionCollectively, our findings demonstrate that PINK1 is crucially involved in osteosarcoma and suggests that it can promote the apoptosis of OS cells as the downstream target gene of cisplatin.

Molecular Modes of Action of an Aqueous Nerium oleander Extract in Cancer Cells In Vitro and In Vivo

Cancer drug resistance remains a major obstacle in clinical oncology. As most anticancer drugs are of natural origin, we investigated the anticancer potential of a standardized cold-water leaf extract from Nerium oleander L., termed Breastin. The phytochemical characterization by nuclear magnetic resonance spectroscopy (NMR) and low- and high-resolution mass spectrometry revealed several monoglycosidic cardenolides as major constituents (adynerin, neritaloside, odoroside A, odoroside H, oleandrin, and vanderoside). Breastin inhibited the growth of 14 cell lines from hematopoietic tumors and 5 of 6 carcinomas. Remarkably, the cellular responsiveness of odoroside H and neritaloside was not correlated with all other classical drug resistance mechanisms, i.e., ATP-binding cassette transporters (ABCB1, ABCB5, ABCC1, ABCG2), oncogenes (EGFR, RAS), tumor suppressors (TP53, WT1), and others (GSTP1, HSP90, proliferation rate), in 59 tumor cell lines of the National Cancer Institute (NCI, USA), indicating that Breastin may indeed bypass drug resistance. COMPARE analyses with 153 anticancer agents in 74 tumor cell lines of the Oncotest panel revealed frequent correlations of Breastin with mitosis-inhibiting drugs. Using tubulin-GFP-transfected U2OS cells and confocal microscopy, it was found that the microtubule-disturbing effect of Breastin was comparable to that of the tubulin-depolymerizing drug paclitaxel. This result was verified by a tubulin polymerization assay in vitro and molecular docking in silico. Proteome profiling of 3171 proteins in the NCI panel revealed protein subsets whose expression significantly correlated with cellular responsiveness to odoroside H and neritaloside, indicating that protein expression profiles can be identified to predict the sensitivity or resistance of tumor cells to Breastin constituents. Breastin moderately inhibited breast cancer xenograft tumors in vivo. Remarkably, in contrast to what was observed with paclitaxel monotherapy, the combination of paclitaxel and Breastin prevented tumor relapse, indicating Breastin's potential for drug combination regimens.

Expansion microscopy for imaging the cell-material interface.

Surface topography on the scale of tens of nanometers to several micrometers substantially affects cell adhesion, migration, and differentiation. Recent studies using electron microscopy and super-resolution microscopy provide insight into how cells interact with surface nanotopography; however, the complex sample preparation and expensive imaging equipment required for these methods makes them not easily accessible. Expansion microscopy (ExM) is an affordable approach to image beyond the diffraction limit, but ExM cannot be readily applied to image the cell-material interface as most materials do not expand. Here, we develop a protocol that allows the use of ExM to resolve the cell-material interface with high resolution. We apply the technique to image the interface between U2OS cells and nanostructured substrates as well as the interface between primary osteoblasts with titanium dental implants. The high spatial resolution enabled by ExM reveals that although AP2 and F-actin both accumulate at curved membranes induced by vertical nanostructures, they are spatially segregated. Using ExM, we also reliably image how osteoblasts interact with roughened titanium implant surfaces below the diffraction limit; this is of great interest to understand osseointegration of the implants but has up to now been a significant technical challenge due to the irregular shape, the large volume, and the opacity of the titanium implants that have rendered them incompatible with other super-resolution techniques. We believe that our protocol will enable the use of ExM as a powerful tool for cell-material interface studies.

Dissecting diffusion and oligomerization dynamics of GPCRs in live cells using single-particle tracking.

Crucial insights about the signaling and trafficking mechanisms of G Protein Coupled Receptors (GPCRs) can be derived from their membrane diffusion properties. Previous studies in our lab (Shivnaraine et al., JACS 2016, Li at al., Biophys. J. 2018) have shown that the M2 muscarinic receptor (M2R) can be purified as oligomers, yet the oligomerization dynamics and the implications for signalling in live cells remain elusive. Here we used single-molecule fluorescence techniques, such as single-particle tracking (SPT) and single-molecule photobleaching (smPB), to characterize the dynamic distribution of M2R in HEK293 cells. To that end, receptors have been co-expressed at their N-terminus either with a green fluorescent protein (eGFP), or with a HaloTag for labelling with HaloTag ligand (HTL) dyes, such as JF549 HTL. For comparison, we applied the same techniques to μ-opioid receptors (MORs) in live U2OS cells. For both cases, measured diffusion maps are spatially and temporally heterogeneous, with receptors transitioning between normal and anomalous diffusion regimes. The analysis of experimental data was reinforced by simulations and by control measurements on monomeric (CD86) and dimeric (CD28) membrane proteins. Intensity traces of immobile, single receptor complexes and of monomeric/dimeric controls in the membrane of fixed cells were analyzed using an in-house smPB code based on a Bayesian algorithm (Garry et al., J. Chem. Phys. 2020). The results confirm the presence, correlated with expression levels, of higher order M2R oligomers in cells, and lay out the foundation for subsequent studies of their functional role.

Single-spot spiral phase plate for tracking 3D particle motion.

We have imaged and tracked submicron fluorescent particles in three dimensions by inserting a spiral phase plate (SPP) of our design into a conventional wide-field optical microscope. Our novel SPP consists of concentric annular zones that each impose a unique quantized orbital angular momentum (OAM) on the ring of fluorescent light it intercepts. Each ring of the SPP consists of a vortex surface that produces one additional unit of OAM than the smaller ring just inside it, with the seven successive rings each contributing n = 1 to 7 quantum units of OAM respectively. The light rings interfere to produce a single-spot rotating point spread function (SS-RPSF). This image ‘spot’ rotates with the axial depth of a particle, permitting 3D mapping of the particle position. The SPP is easily incorporated into existing microscopes. We used the DIC slider here for the SPP. 3D positions of fluorescent beads were retrieved using template matching. Bead positions could be inferred over a 6 µm depth range. Best results were obtained over a depth range of 2.3 μm, with a mean absolute error of 20 nm in this range. As proof-of-concept for live-cell imaging we tracked DNA loci and determined their diffusion coefficients in the 3D environment of the nucleus of U2OS cells. We are pursuing chromatin tracking experiments to better understand how DNA damage affects chromatin motions and, reciprocally, how chromatin dynamics impacts the DNA damage response. Beyond this specific application, our SPP-based imaging approach should enable researchers to track dynamic fluorescent objects in samples with high temporal resolution because x, y, and z are determined at exactly the same time.

TMBIM4 Deficiency Facilitates NLRP3 Inflammasome Activation-Induced Pyroptosis of Trophoblasts: A Potential Pathogenesis of Preeclampsia

Impaired invasion of EVTs results in inadequate remodelling of arteries and poor placentation, leading to PE. TMBIM4 was found to promote the migration and invasion of human osteosarcoma U2-OS and breast cancer MCF7 cell lines. However, the effect of TMBIM4 on trophoblast biological behaviour and its relevance to PE pathophysiology remain unclear. In this study, we confirmed that TMBIM4 was highly expressed in cytotrophoblasts, syncytiotrophoblasts, and EVTs of the human placenta during early pregnancy. By comparing the expression levels of TMBIM4 in the placenta of women with normal-term pregnancy and PE, TMBIM4 was found to be significantly decreased in PE. Thereafter, we determined the expression of TMBIM4 in the LPS-treated first-trimester human trophoblast cell line HTR-8/SVneo (mimicking a PE-like cell model), and determined the effect of TMBIM4 on trophoblast function and its underlying mechanism. LPS treatment reduced the expression of TMBIM4 and induced NLRP3 inflammasome activity in HTR-8/SVneo cells. KO of TMBIM4 in the HTR-8/SVneo cell line impaired cell viability, migration, and invasion, which was more severe in the LPS/ATP-treated TMBIM4-KO cell line. Moreover, TMBIM4 deficiency enhanced NLRP3 inflammasome activity and promoted subsequent pyroptosis, with or without LPS/ATP treatment. The negative relationship between TMBIM4 expression and NLRP3 inflammatory activity was verified in PE placentas. Inhibiting the NLRP3 inflammasome with MCC950 in HTR-8/SVneo cells alleviated LPS/ATP-induced pyroptosis and damaged cell function in the TMBIM4-KO cell line. Overall, this study revealed a new PE-associated protein, TMBIM4, and its biological significance in trophoblast pyroptosis mediated by the NLRP3 inflammasome. TMBIM4 may serve as a potential target for the treatment of placental inflammation-associated PE.

Antibacterial piperazine‐derived quaternized copolyesters with controlled degradability

AbstractThe slow degradability of aliphatic polyesters can be attributed to their poor hydrophilicity. In this article, we report on a series of biodegradable and biocompatible pH‐responsive piperazine‐based copolyesters labeled P2‐P7 synthesized by melt polycondensation approach that exhibit tunable hydrophilicity with antibacterial properties. X‐ray diffraction analysis showed the copolymers to be highly crystalline, with their crystallinity in the range of 41%–55%. Contact angle measurements showed moderate hydrophilicity, with a water contact angle of 59°–71° for P2‐P7 suggesting that the incorporation of piperazine units in the copolymer backbone moderately improves their hydrophilicity. N‐alkylation with methyl iodide further enhanced their hydrophilicity with their measured contact angles changing from 62° in unalkylated P2 to 27° in the fully alkylated analogue P12. The effect of crystallinity and hydrophilicity on polyester degradation was systematically investigated by hydrolytic degradation studies. The quaternized copolymer P10 showed ~39% reduction in molecular weight, whereas only ~19% of P2 was degraded in 8 weeks, thus exhibiting faster degradation in the quaternized polyester. The biocompatibility with U2OS cells and the antibacterial properties established their importance as potential biomedical materials.

P53 inhibits CTR1-mediated cisplatin absorption by suppressing SP1 nuclear translocation in osteosarcoma.

Osteosarcoma (OS) is a malignant bone tumor mainly affecting children and young adolescents. Cisplatin is a first-line chemotherapy drug for OS, however, drug resistance severely limits the survival of OS. Nevertheless, cellular factors in cisplatin resistance for OS remain obscure. In this study, the function and potential mechanism of p53 in cisplatin absorption were explored in OS cells. The CRISPR-Cas9 gene editing technology was performed to obtain p53 gene knock-out U2OS cells. The p53 over-expression 143B cell line was established by lentivirus-mediated virus infection. Moreover, the functions of p53 and CTR1 in cisplatin absorption were assessed by inductively coupled plasma mass spectrometry (ICP-MS) through CTR1 over-expression and knock-down. Further, the DNA binding activity of SP1 on CTR1 gene promoter was determined by dual-luciferase assay and chromatin immunoprecipitation (ChIP) assay. The functional regulation of p53 on SP1 was studied by nucleocytoplasmic separation assay and electrophoretic mobility shift assay (EMSA). The interaction between p53 and SP1 was verified by Co-Immunoprecipitation assay. Under cisplatin treatment, p53 knock-out promoted CTR1 expression and cisplatin uptake, while p53 overexpression inhibited CTR1 expression and cisplatin uptake. Moreover, p53 regulated CTR1 level not by binding to CTR1 promoter directly but by suppressing the nuclear translocation of transcription factor specificity protein 1 (SP1). It was verified that SP1 is directly bound with CTR1 promoter. SP1 overexpression stimulated CTR1 expression, and SP1 knock-down attenuated CTR1 expression. The p53 might function as a negative regulator in CTR1 mediated cisplatin absorption, and the p53-SP1-CTR1 axis is a target for cisplatin resistance.

Chitosan targets PI3K/Akt/FoxO3a axis to up-regulate FAM172A and suppress MAPK/ERK pathway to exert anti-tumor effect in osteosarcoma

Osteosarcoma (OS) is a serve and the most frequent primary malignant tumor of bone. Chitosan was reported to have anti-tumor effect on human cancers including OS. However, the molecular mechanism by which chitosan suppresses tumor growth is not fully illustrated. In this study, human OS cell lines, including both Saos-2 and U2OS cells, were used to dissect the underlying mechanisms. RNA sequencing results show that a candidate biomarker family with sequence similarity 172 member A (FAM172A) was up-regulated in both of the two cell lines treated with chitosan. We observed that the mitogen-activated protein kinase (MAPK) signaling pathway could be inactivated by chitosan, and the MAPK inhibition caused by chitosan was reversed by FAM172A knockdown. Moreover, we uncovered a direct interaction between C-terminal domain of FAM172A (311–415) and mitogen-activated protein kinase kinase 1 (MEK1) (270–307) by immunoprecipitation assay. Finally, we also found that chitosan could bind with subunit p85 of PI3K to further inactivate the PI3K/Akt pathway. Taken together, our study demonstrates that chitosan binds with PI3K p85 subunit to suppress the activity of PI3K/Akt pathway to up-regulate the expression of FAM172A, and which exerts its function by suppressing phosphorylation of MEK1/2 and blocking the activity of MAPK/ERK signaling pathway. Taken together, our study deepens the understanding of the molecular mechanism of MAPK/ERK pathway inhibition induced by chitosan, and provides insights into the development of new targets to enhance the pharmacological effect of chitosan against OS.

Nuclear receptor modulators inhibit osteosarcoma cell proliferation and tumour growth by regulating the mTOR signaling pathway

Osteosarcoma is the most common primary malignant bone tumour in children and adolescents. Chemoresistance leads to poor responses to conventional therapy in patients with osteosarcoma. The discovery of novel effective therapeutic targets and drugs is still the main focus of osteosarcoma research. Nuclear receptors (NRs) have shown substantial promise as novel therapeutic targets for various cancers. In the present study, we performed a drug screen using 29 chemicals that specifically target 17 NRs in several different human osteosarcoma and osteoblast cell lines. The retinoic acid receptor beta (RARb) antagonist LE135, peroxisome proliferator activated receptor gamma (PPARg) antagonist T0070907, liver X receptor (LXR) agonist T0901317 and Rev-Erba agonist SR9011 significantly inhibited the proliferation of malignant osteosarcoma cells (U2OS, HOS-MNNG and Saos-2 cells) but did not inhibit the growth of normal osteoblasts. The effects of these NR modulators on osteosarcoma cells occurred in a dose-dependent manner and were not observed in NR-knockout osteosarcoma cells. These NR modulators also significantly inhibited osteosarcoma growth in vivo and enhanced the antitumour effect of doxorubicin (DOX). Transcriptomic and immunoblotting results showed that these NR modulators may inhibit the growth of osteosarcoma cells by regulating the PI3K/AKT/mTOR and ERK/mTOR pathways. DDIT4, which blocks mTOR activation, was identified as one of the common downstream target genes of these NRs. DDIT4 knockout significantly attenuated the inhibitory effects of these NR modulators on osteosarcoma cell growth. Together, our results revealed that modulators of RARb, PPARg, LXRs and Rev-Erba inhibit osteosarcoma growth both in vitro and in vivo through the mTOR signaling pathway, suggesting that treatment with these NR modulators is a novel potential therapeutic strategy.

FGF23 promotes proliferation, migration and invasion by regulating miR-340-5p in osteosarcoma

BackgroundIncreasing evidences have been indicated that FGF23 is associated with the biological behavior of malignant tumors, but its role in osteosarcoma and the specific mechanism need to be elucidated. The purpose of this study is to investigate the effects of FGF23 on the proliferation, migration and invasion of osteosarcoma cells, and the possible molecular mechanisms.MethodsWestern blot was used to detect differences in FGF23 expression in osteosarcoma cells MG-63 and U2-OS and osteoblasts hFOB1.19. FGF23-overexpressing adenoviruses and FGF-silencing plasmids were transfected into osteosarcoma cells, and transfection efficiency was verified using Western blot. MTT and colony formation assays were performed to detect osteosarcoma cell proliferation. Cell cycle was measured by flow cytometry. Scratch assay, holographic imaging cell analyzer Holomonitor ® M4 and transwell were applied to detect cell migration and invasion. Dual-luciferase reporter assay was performed to validate the interaction between FGF23 and miR-340-5p. Changes in miR-340-5p mRNA levels were measured by QRT-PCR.ResultsFGF23 is highly expressed in osteosarcoma cells compared to hFOB1.19. Overexpression of FGF23 significantly promoted the proliferation, migration and invasion of MG-63 and U2-OS cells. MiR-340-5p is a target of FGF23. Transfection of miR-340-5p mimics reversed the promoting effects of FGF23 on proliferation, migration and invasion of MG-63 and U2-OS cells.ConclusionFGF23 promotes osteosarcoma cell proliferation, migration and invasion by targeting miR-340-5p gene expression.