Human Osteosarcoma Cell Line U2OS Research Articles

OR26-01 A Novel Small Molecule Thyrotropin Receptor Antagonist

Abstract Disclosure: G.J. Kahaly: None. A. George: None. L. Steiner: None. T. Weirauch: None. J. Henseling: None. L. Frommer: None. J. Wolf: None. Background & aims: Treatment of both Graves’ hyperthyroidism (GH) and Graves’ orbitopathy (GO) is still far from adequate, hence new therapeutic modalities, which specifically target the autoantigens in GH/GO, are warranted. This study determined the preclinical in vitro efficacy of SYD5115, a novel thyrotropin receptor (TSH-R) antagonist. Methods: SYD5115 is a nanomolar potent low molecular weight TSH-R antagonist with in vivo activity in rats after oral dosing. The compound consists of an N-acetylated dihydropyrrole pyrimidine core containing several substituents such as a difluorinated-spirocyclohexyl ring and a chiral carbamate group on the pyrimidine ring. The TSH-R inhibiting capability of SYD5115 was tested through stimulation of wild type and chimeric TSH-R expressed in Chinese hamster ovary (CHO) cells using-two functional (stimulatory and blocking) cell-based TSH-R-Ab bioassays. TSH-R expressing human orbital fibroblasts, collected from GH+GO patients (GOF), were stimulated with the monoclonal antibody M22 or with stimulatory TSH-R-Ab (TSAb)-positive sera with cyclic adenosine monophosphate (cAMP) or hyaluronic acid (HA) release as read-outs. The effect of SYD5115 on the viability of GOF was tested in both 4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide and the “scratch” cell growth assays. Results: The new small molecule SYD5115 significantly and dose-dependently inhibited the TSH-R activation through M22 or TSAb-positive sera in all performed bioassays. Inhibition showed similar levels in the TSAb reporter bioassay and in the cAMP assay with GOF. The % inhibition and compound concentration showed a sigmoidal relationship, with all seven TSAb positive sera markedly inhibited by SYD5115. A SYD5115 dose-dependent inhibition of M22 (10ng/ml, 6 hours)-stimulated HA and/or cAMP-release from GOF was observed. Strong SYD5115-induced inhibitions of M22-stimulated cAMP production in GOF were registered with SYD5115 concentrations of one (p=0.0029), 10 (p<0.0001), 100 (p<0.0001), 1,000 (p<0.0001), and 10,000 (p<0.0001) nM/L, respectively. SYD5115-induced inhibition of M22-stimulated HA production was noted with SYD5115 concentrations of 100 (p=0.0392), 1,000 (p=0.0431), and 10,000 (p=0.0245) nM/L, respectively. The inhibitory activity of SYD5115 was confirmed in a human osteosarcoma U2OS cell line stably expressing human TSH-R with cAMP as read-out. SYD5115 induced 100% inhibition of the M22-induced cAMP levels with a potency of 193 nM. Compared to control, SYD5115 affected neither the growth nor the migration of cultivated GOF. In addition, SYD5115 did not alter the viability of GOF. Conclusions: SYD5115 blocked M22- and TSAb-induced TSH-R activity with a nanomolar potency in TSH-R overexpressed CHO cells as well as primary GOF, which demonstrates the ability of this novel small molecule to inhibit TSH-R overactivity. Presentation: Saturday, June 17, 2023

Exploring the Interaction of New Pyridoquinazoline Derivatives with G-Quadruplex in the c-MYC Promoter Region.

Novel amino-substituted pyridoquinazolinone derivatives have been designed and synthesized as potential c-MYC G-quadruplex (G4) ligands, employing an efficient methodology. All the new compounds exhibited moderate to good antiproliferative activity against the human osteosarcoma U2OS cell line. NMR and docking experiments revealed that the recently synthesized compounds interact with the Pu22 G-quadruplex in the c-MYC promoter region, establishing a 2:1 complex, with each molecule positioned over the tetrads at the 3'- and 5'-ends.

Thyrotropin Receptor Antagonism by a Novel Small Molecule: Preclinical In Vitro Observations.

Background: Treatment of Graves' hyperthyroidism (GH) and Graves' orbitopathy (GO) is far from adequate, and hence, new substances that specifically target the autoantigens in GH/GO are warranted. This study determined the preclinical in vitro efficacy of SYD5115, a novel low-molecular-weight compound that inhibits the thyrotropin receptor (TSH-R). Methods: The TSH-R inhibiting capability of SYD5115 was tested through stimulation of wild-type and chimeric TSH-R expressed in Chinese hamster ovary (CHO) cells using two functional (stimulatory and blocking) cell-based TSH-R-Ab bioassays. TSH-R expressing human orbital fibroblasts, collected from GH+GO patients (GOF), were stimulated with the monoclonal antibody M22 or with stimulatory TSH-R-Ab (TSAb)-positive sera with cyclic adenosine monophosphate (cAMP) or hyaluronic acid (HA) release as readouts. The effect of SYD5115 on the viability of GOF was tested in 4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide and scratch cell growth assays. Results: SYD5115 significantly and dose dependently inhibited the TSH-R activation through M22 or TSAb-positive sera in all performed bioassays. Inhibition showed similar levels in the TSAb reporter bioassay and in the cAMP assay with GOF. The % inhibition and compound concentration showed a sigmoidal relationship, with all seven TSAb-positive sera markedly inhibited by SYD5115. An SYD5115 dose-dependent inhibition of M22 (10 ng/mL, 6 hours)-stimulated HA and/or cAMP-release from GOF was observed. Strong SYD5115-induced inhibitions of M22-stimulated cAMP production in GOF were registered with SYD5115 concentrations of 1 (p = 0.0029), 10 (p < 0.0001), 100 (p < 0.0001), 1,000 (p < 0.0001), and 10,000 (p < 0.0001) nM, respectively. SYD5115-induced inhibition of M22-stimulated HA production was noted with SYD5115 concentrations of 100 (p = 0.0392), 1000 (p = 0.0431), and 10,000 (p = 0.0245) nM, respectively. The inhibitory activity of SYD5115 was confirmed in a human osteosarcoma U2OS cell line stably expressing human TSH-R with cAMP as readout. SYD5115 induced 100% inhibition of the M22-induced cAMP levels with a potency of 193 nM. Compared with control, SYD5115 did neither impact the growth nor the migration of cultivated GOF. In addition, SYD5115 did not alter the viability of GOF. Conclusions: SYD5115 blocked M22- and TSAb-induced TSH-R activity with a nanomolar potency in TSH-R-overexpressed CHO cells as well as primary GOF, which demonstrates the ability of this small molecule to block TSH-R overactivity.

COPS6 promotes tumor progression and reduces CD8+ T cell infiltration by repressing IL-6 production to facilitate tumor immune evasion in breast cancer.

Due to poor T cell infiltration, tumors evade immune surveillance. Increased CD8+ T cell infiltration in breast cancer suggests a satisfactory response to immunotherapy. COPS6 has been identified as an oncogene, but its role in regulating antitumor immune responses has not been defined. In this study, we investigated the impact of COPS6 on tumor immune evasion in vivo. Tumor transplantation models were established in C57BL/6 J mice and BALB/c nude mice. Flow cytometry was conducted to identify the role of COPS6 on tumor-infiltrating CD8+ T cells. By analyzing the TCGA and GTEx cohort, we found that COPS6 expression was significantly up-regulated in a variety of cancers. In human osteosarcoma cell line U2OS and non-small cell lung cancer cell line H1299, we showed that p53 negatively regulated COPS6 promoter activity. In human breast cancer MCF-7 cells, COPS6 overexpression stimulated p-AKT expression as well as the proliferation and malignant transformation of tumor cells, whereas knockdown of COPS6 caused opposite effects. Knockdown of COPS6 also significantly suppressed the growth of mouse mammary cancer EMT6 xenografts in BALB/c nude mice. Bioinformatics analysis suggested that COPS6 was a mediator of IL-6 production in the tumor microenvironment and a negative regulator of CD8+ T cell tumor infiltration in breast cancer. In C57BL6 mice bearing EMT6 xenografts, COPS6 knockdown in the EMT6 cells increased the number of tumor-infiltrating CD8+ T cells, while knockdown of IL-6 in COPS6KD EMT6 cells diminished tumor infiltrating CD8+ T cells. We conclude that COPS6 promotes breast cancer progression by reducing CD8+ T cell infiltration and function via the regulation of IL-6 secretion. This study clarifies the role of p53/COPS6/IL-6/CD8+ tumor infiltrating lymphocytes signaling in breast cancer progression and immune evasion, opening a new path for development of COPS6-targeting therapies to enhance tumor immunogenicity and treat immunologically "cold" breast cancer.

Abstract 6382: Activation of transcription factors, p65 and NFATc1, mediated by WNT5a, induces up-regulation of novel immune checkpoint CNTN4 expression

Abstract We previously confirmed that contactin 4 (CNTN4) is highly expressed in the various human tumor tissues and inhibits mouse and human T cell activity by suppressing a cascade of signaling processes associated with T cell activation through binding with amyloid precursor protein (APP) of T cells. However, the molecular mechanism for the expression of CNTN4 in tumors has yet to be elucidated. So, we investigated whether oncogenic ligands regulate the expression of CNTN4 through CNTN4 promoter (- 2,000 bp ~ + 100 bp)-based reporter gene assays in HEK293 cells using oncogenic ligands. Here, we identified that WNT5a significantly increased the expression of CNTN4 in a dose-dependent manner. Furthermore, the treatment of the WNT5a ligand in the human colorectal cancer cell line SW480, which expresses CNTN4 at a low level, increased the expression of CNTN4. In addition, CNTN4 expression was decreased when the WNT5a antagonist (Box5, a WNT5a-derived N-butyloxycarbonyl hexapeptide) was treated in the human osteosarcoma cell line U2OS expressing CNTN4 endogenously. The changes in the expression of CNTN4 were observed by RT-PCR and FACS. Treatment of the WNT5a ligand or WNT5a antagonist changed the transcriptional expression of CNTN4 in each cell line and affected the cell surface expression of CNTN4. Since WNT5a ultimately increases the activity of transcription factors such as NFATc1 or p65 to increase the transcriptional expression of the target gene, we further investigated the effect of NFATc1 or p65 on the expression of CNTN4. We found that NFATc1 or p65 binds to the promoter of CNTN4 and increases the expression of transcription. Moreover, it was recently reported that anti-PD-1-refractory melanoma exhibits elevated WNT ligand signaling activity, especially upregulated WNT7b and WNT5a in the WNT ligands. Following this background, the expression level of CNTN4 can be increased in the refractory environment to immunotherapy induced by elevated WNT signaling. Therefore, we constructed B16F10 murine melanoma cells overexpressing WNT5a (B16F10/WNT5a) to assess the anti-tumor efficacy by administering GENA-104A16, the anti-CNTN4 humanized monoclonal antibody, in the syngeneic mice model with increased expression of CNTN4 induced by WNT5a, and in vivo efficacy by treatment of GENA-104A16 is under assessment in the B16F10/WNT5a syngeneic mice model. Through the investigation, we expect to support the rationale and strategy for a combination therapy approach of GENA-104A16 for non-responding patients to immunotherapy. Citation Format: Mi Young Cha, Bu-Nam Jeon, Hyeokgu Kang, Kyung Mi Park, Hansoo Park. Activation of transcription factors, p65 and NFATc1, mediated by WNT5a, induces up-regulation of novel immune checkpoint CNTN4 expression [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 6382.

An Exploration of Osteosarcoma Metastasis Diagnostic Markers Based on Tumor-Associated Neutrophils.

The high rate of the recurrence and metastasis of osteosarcoma (OS) is the major cause of its poor prognosis. There is a strong correlation between tumor-associated neutrophils (TANs) and tumor progression, progression, and metastasis. This study aimed to identify potential markers that could predict OS metastasis based on analysis of TANs in the tissues of OS patients. A single-cell sequencing dataset (GSE152048), containing seven primary OS lesions, two recurrent OS lesions, and two lung metastatic OS lesions was used for TANs subset identification using the R software (version 4.1.0, R Project for Statistical Computing, Vienna, Austria; https://www.r-project.org/). Immune cell infiltration and immune score were analyzed using CIBERSORT algorithm and ESTIMATE database, respectively. The differentially expressed genes (DEGs) of TANs were used for weighted gene co-expression network analysis (WGCNA) to screen key genes associated with OS metastasis. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) functional enrichment analysis were used to analyze the functions and signaling pathways involved in key genes. The mRNA levels of protein phosphatase 2 regulatory subunit B'gamma (PPP2R5C) were validated in human osteosarcoma cell lines U2-OS and MG63, human normal cervical endometrial cell line HUCEC, and human foreskin fibroblast (HFF-1) cell line by real-time qPCR (RT-qPCR). PPP2R5C-siRNA991 was transfected into U2-OS and MG63 for 48 h, then the expression levels of PPP2R5C, AKT serine/threonine kinase (AKT), and phospho-AKT (p-AKT) were determined by RT-qPCR and Western blotting. Cell proliferation, migration, and apoptosis were measured by cell counting kit-8 (CCK-8), Transwell, and flow cytometry, respectively. We identified TANs subsets in primary, metastatic, and recurrent OS. Immune infiltration analysis showed that TANs were expressed in OS. Compared with non-metastatic OS, metastatic OS had lower stromal score, immune score, ESTIMATE score, and higher tumor purity. WGCNA classified DEGs into five clusters, according to their function and identified PPP2R5C, protein phosphatase 2 regulatory subunit B'epsilon (PPP2R5E), tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein gamma (YWHAG) and CREB binding protein (CREBBP), as potential markers that may affect TANs-induced OS metastasis via hypoxia inducible factor 1 (HIF-1), phosphatidylinositol 3-kinases (PI3K)-AKT and Janus kinase (JAK)-signal transducers and activators of transcription (STAT) signaling pathways. In vitro experiments demonstrated that the mRNA and protein expressions of PPP2R5C, PPP2R5E, YWHAG, and CREBBP were highly expressed in U2-OS and MG63 cells (p < 0.01). Furthermore, PPP2R5C reduced proliferation and migration (p < 0.01) and increased apoptosis and p-AKT protein levels in U2-OS and MG6 cells (p < 0.01). PPP2R5C affects OS metastasis via PI3K/AKT pathway, which may be a potential marker for OS metastasis and recurrence.

Evaluating the Potential Anticancer Properties of Salvia triloba in Human-Osteosarcoma U2OS Cell Line and Ovarian Adenocarcinoma SKOV3 Cell Line

Salvia triloba (S. triloba) is an herb inherently linked to traditional medicine systems in the Eastern Mediterranean region. There is minimal experimental evidence however, regarding the anticancer effects of S. triloba in both osteosarcoma and ovarian cancer. In this study, we investigated the effects of crude (macerated) S. triloba ethanol and acetone leaf extracts on viability, migratory ability, and the expression of genes regulating these activities in U2OS and SKOV3 cells using MTT assay, scratch-wound healing/trans-well migration assay, and RT-qPCR respectively. MTT assay results indicated that the acetone extract significantly reduced both U2OS and SKOV3 cell viability with half-maximal inhibitory concentrations (IC50) of 54.51 ± 1.10 µg/mL and 75.96 ± 1.0237 µg/mL respectively; these concentrations further displayed negligible hemolytic activity. The combination of acetone extract (19 µg/mL) and paclitaxel (0.787 µg/mL) displayed synergy and reduced SKOV3 cell viability by over 90%. Additionally, the trans-well migration assay illustrated that the acetone extract (IC50) inhibited both U2OS and SKOV3 cell migration by more than 50%. Moreover, S. triloba acetone extract significantly downregulated the steady-state mRNA expression of key genes involved in driving select cancer hallmarks. Four fractions were generated from the acetone extract by thin layer chromatography (TLC), and the obtained retention factors (Rf) (ranging from 0.2 to 0.8) suggested a mixture of high and moderately polar compounds whose bioactivities require further investigation. In addition, FTIR measurements of the extract revealed peaks corresponding to OH, aliphatic CH, and ester groups suggesting the presence of phenolic compounds, terpenes, and polysaccharides. Altogether, these results suggest that S. triloba possesses potential therapeutic compounds that inhibit cell proliferation and migration, and modulate several genes involved in osteosarcoma and ovarian carcinoma progression.

Synthesis and Antitumor Activity of a GSH Inert Bisphosphonate Platinum (II) Complex

Osteosarcoma is a highly aggressive neoplasm. Traditional platinum chemotherapeutic agents for osteosarcoma inevitably have acquired drug resistance and serious side effects, which have limited their utility. To slow down the reaction of platinum drugs with glutathione (GSH) is a strategy to overcome the resistance of platinum chemotherapeutic agents. Herein, the unique design of a GSH inert bisphosphonate platinum complex cis-{di(amino)platinum[tetraethyl 2,2-bis(2-pyridinylmethyl)methylidene-1,1-bisphosphonate]} (DBPP) is reported. MTT assay demonstrates that DBPP showed moderate inhibition towards human osteosarcoma cell line U2OS cells. The cytostatic action of DBPP is related to conformational conversion from B-DNA to A-DNA and the unwinding of pUC19 DNA. DBPP could also destroy the tertiary structure of human serum albumin (HSA). Notably, 31P NMR and 1H NMR indicate that DBPP can hardly chelate with GSH, which could overcome the GSH-induced side effects. We envision that this unique design of the platinum complex would open up new ways to overcome GSH-induced resistance.

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.

Effect of thermal annealing on a bilayer polyvinyl alcohol/polyacrylic acid electrospun hydrogel nanofibres loaded with doxorubicin and clarithromycin for a synergism effect against osteosarcoma cells

Polyvinyl alcohol/polyacrylic acid (PVA/PAA) bilayer hydrogel nanofibres were successfully fabricated by electrospinning and physically crosslinked via heat treatment. The effects of the thermal annealing process on the structure, morphology, swelling, thermal properties and hydrophilicity of electrospun nanofibres were investigated. In addition, these membranes were also used to incorporate doxorubicin and clarithromycin for osteosarcoma treatment, one in each layer. These drugs were used because it is hypothesized in this work that a synergism occurs between both drugs. So, these membranes were analyzed towards their dual-drug release and potential cytotoxicity towards the U2OS human osteosarcoma cell line. Moreover, the water contact angle, disintegration, swelling and weight loss studies confirmed the rapid swelling and improved water stability of the annealed PVA/PAA bilayer nanofibres. The annealed bilayer nanofibres exhibited an increase in the average diameter and degree of crystallinity. In addition, the results revealed that a variation occurred in the degree of hydrophilicity of annealed PVA/PAA bilayer nanofibres. The PAA nanofibres surface exhibited higher hydrophilicity than the PVA nanofibres surface. Drug delivery presented to be as fast rate release for clarithromycin and slow-rate release for doxorubicin, which may be advantageous because both drugs exhibited to be synergetic for certain dosages presenting the combination of the drugs higher than 50% of cell inhibition, while these membranes had higher inhibition values (up to 90%), which was attributed to the PAA but also the drugs. These unique properties are of potential interest in drug delivery applications for dual drug delivery where the tunability of surfaces is desirable.

Development, Characterization and Cell Viability Inhibition of PVA Spheres Loaded with Doxorubicin and 4'-Amino-1-Naphthyl-Chalcone (D14) for Osteosarcoma.

Chalcones (1,3-diaryl-2-propen-1-ones) are naturally occurring polyphenols with known anticancer activity against a variety of tumor cell lines, including osteosarcoma (OS). In this paper, we present the preparation and characterization of spheres (~2 mm) from polyvinyl alcohol (PVA) containing a combination of 4′-Amino-1-Naphthyl-Chalcone (D14) and doxorubicin, to act as a new polymeric dual-drug anticancer delivery. D14 is a potent inhibitor of osteosarcoma progression and, when combined with doxorubicin, presents a synergetic effect; hence, physically crosslinked PVA spheres loaded with D14 and doxorubicin were prepared using liquid nitrogen and six freeze–thawing cycles. Physical-chemical characterization using a scanning electron microscope (SEM), differential scanning calorimetry (DSC), Fourier transform infrared spectroscopy (FTIR) and X-ray diffraction (XRD) presented that the drugs were incorporated into the spheres via weak interactions between the drugs and the polymeric chains, resulting in overall good drug stability. The cytotoxicity activity of the PVA spheres co-encapsulating both drugs was tested against the U2OS human osteosarcoma cell line by 3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide (MTT) assay, and compared to the spheres carrying either D14 or doxorubicin alone. The co-delivery showed a cytotoxic effect 2.6-fold greater than doxorubicin alone, revealing a significant synergistic effect with a coefficient of drug interaction (CDI) of 0.49. The obtained results suggest this developed PVA sphere as a potential dual-drug delivery system that could be used for the prominent synergistic anticancer activity of co-delivering D14 and doxorubicin, providing a new potential strategy for improved osteosarcoma treatment.

Abstract PO-082: Radiation therapy enhances the potential of the oncolytic virus in the treatment of osteosarcoma

Abstract Background: Combination of neoadjuvant chemotherapy and surgery is the standard treatment approach for osteosarcoma. Despite aggressive treatment protocol, ~40% of patients do not respond and relapse, the survival rate is poor for these patients. Conditionally replicative adenovirus (CRAds) destroying cancer cell while sparing normal cells is a new treatment approach for cancers. We developed a new oncolytic adenovirus which grows using mRNA stabilization system found in many cancer cells. Our recent report showed that this virus replicated in cancer cells where AU-rich element (ARE) containing mRNA is stabilized. ARE-mRNA is stabilized in the majority types of cancers mediate by binding with HuR (Human antigen R), which exports target ARE-mRNA to the cytoplasm. Radiotherapy (RT) is not a first-line treatment option for osteosarcoma as osteosarcoma cells are not killed by radiation easily. But in certain conditions radiotherapy can be used where surgical resection is not technically possible without undue morbidity or when surgery is refused. In response to ionizing radiation and UV radiation DNA damage response (DDR) can influence HuR action either by the increasing cytoplasmic HuR abundance or affecting the binding affinity to target ARE-mRNA. The purpose of the present study was to analyze the efficacy of the combination treatment of RT and oncolytic adenovirus for osteosarcoma. Materials and Methods: E4rof6-deleted adenovirus (dl355) and human osteosarcoma cell lines U2OS and Saos2 were used in this study. Cell viability assays were carried out to evaluate the cytotoxicity and synergism of dl355 and RT combination. Chou and Talalay’s median-effect method was used to calculate the combination index. CPE assay was used to assess the cytopathic effect of the combination. Viral replication with or without radiation combination was investigated by hexon staining. Western blot analysis was performed to examine viral protein expression. Results: We found that RT treatment enhanced the cytoplasmic relocalization of HuR; thus irradiation up-regulated the propagations of dl355. Also, radiation-induced upregulation of viral protein expression was observed by western blotting. The RT and dl355 combination treatment increased the cytolysis of U2OS, and Saos2 cells in a synergistic manner compared to RT or virus only. Conclusion: Radiotherapy up-regulated of oncolytic adenovirus, which grows using the ARE-mRNA stabilization system, and this cytolytic effect provides a rationale for this combination. This combination approach has the possibility of a promising strategy for osteosarcoma. Citation Format: Elora Hossain, Fumihiro Higashino. Radiation therapy enhances the potential of the oncolytic virus in the treatment of osteosarcoma [abstract]. In: Proceedings of the AACR Virtual Special Conference on Radiation Science and Medicine; 2021 Mar 2-3. Philadelphia (PA): AACR; Clin Cancer Res 2021;27(8_Suppl):Abstract nr PO-082.

LncRNA FOXD2-AS1 knockdown inhibits the resistance of human osteosarcoma cells to cisplatin by inhibiting miR-143 expression.

The aim of this study was to explore the effect of long non-coding ribonucleic acid (lncRNA) FOXD2-adjacent opposite strand RNA 1 (FOXD2-AS1) on the sensitivity of osteosarcoma cells to cisplatin and its possible underlying mechanism. Our findings might help to provide a certain reference for clinically preventing the drug resistance of osteosarcoma cells. Cisplatin with a certain concentration gradient was used to induce the stable acquired resistance of human osteosarcoma U2-OS cell line. Subsequently, the expression level of lncRNA FOXD2-AS1 was determined in osteosarcoma cells in non-resistance group (Control group) and Cisplatin-resistance group (Cisplatin-RES group), respectively. Next, the cell line with stable lncRNA FOXD2-AS1 knockdown was constructed in Cisplatin-RES group using small interfering RNA (siRNA). The effects of stable knockdown of lncRNA FOXD2-AS1 on the proliferation of human osteosarcoma cells and the half-maximal inhibitory concentration (IC50) of cisplatin were detected by Cell Counting Kit-8 (CCK-8) assay. 5-ethynyl-2'-deoxyuridine (EdU) staining was performed to measure deoxyribonucleic acid (DNA) replication level in each group of cells. The protein expression levels of apoptosis-associated genes B-cell lymphoma 2 (Bcl-2) and Bcl-2 associated X protein (Bax) in each group of cells were measured via Western blotting. The migration and invasion abilities of cells in each group were determined using wound-healing assay and transwell assay. In addition, the expression of micro RNA (miR)-143 in each group of cells was detected via Reverse Transcription-Polymerase Chain Reaction (RT-PCR). Compared with Control group, the expression level of lncRNA FOXD2-AS1 rose significantly in cells in Cisplatin-RES group (p<0.05). Knockdown of FOXD2-AS1 evidently decreased the IC50 of cisplatin in human osteosarcoma cells (p<0.05). According to EdU staining results, the knockdown of FOXD2-AS1 distinctly inhibited the proliferation of osteosarcoma cells (p<0.05). Western blotting results demonstrated that the knockdown of FOXD2-AS1 remarkably upregulated the expression of pro-apoptotic protein Bax and repressed that of anti-apoptotic protein Bcl-2 in drug-resistant human osteosarcoma cells (p<0.05). Moreover, the knockdown of FOXD2-AS1 significantly weakened the migration and invasion abilities of drug-resistant human osteosarcoma cells (p<0.05). Finally, it was found that the expression level of miR-143 was distinctly elevated in drug-resistant human osteosarcoma cells after knockdown of FOXD2-AS1 (p<0.05). LncRNA FOXD2-AS1 knockdown inhibits the resistance of human osteosarcoma cells to cisplatin, promotes their apoptosis and weakens their invasion and migration abilities. The possible underlying mechanism may be related to the inhibition of miR-143 expression by lncRNA FOXD2-AS1 in drug-resistant cell lines.

Multimodal assessment of autophagy in mammalian cells with a novel, LC3-based tandem reporter.

Autophagy is an important intracellular pathway for the degradation of superfluous or harmful subcellular materials, thereby playing a critical role in the maintenance of cell health under normal and stress-related conditions. Researchers interrogating autophagic activity in mammalian cell lines often leverage complementary assay technologies to confirm observations. The Autophagy LC3 HiBiT Reporter assay system utilizes a tandem reporter module, HiBiT-HaloTag, fused to a key marker of autophagic activity, LC3B protein, to enable multiple, cell-based assay modalities. This novel autophagy reporter expressed in a single cell line supports (a) a bioluminescent, homogeneous, plate-reader assay for rapid and quantitative assessment of changes in the level of the LC3-based reporter, (b) a fluorescence-based imaging approach to monitor reporter subcellular distribution in live cells, and (c) an antibody-free, protein blotting method to detect the relative amounts of the LC3-I and LC-II forms of the reporter associated with modulation of autophagic flux. Here we detail protocols for all three assay modalities applied to a U2OS human osteosarcoma cell line stably expressing the novel autophagy reporter, enabling the identification of modulators of autophagic activity and subsequent confirmation of mechanism of action.

Serum factor(s) from lung adenocarcinoma patients regulates the molecular clock expression.

Lung cancer is a leading cause of cancer-associated deaths worldwide. Lung cancer may lead to circadian disruption, which could contribute to the development of lung cancer. Recently, several studies using animal models indicated that tumors influence systemic circadian homeostasis in remote tissues. However, it is unclear whether carcinoma of the lungs influences remote circadian rhythm, whether this effect exists in humans, and whether signals from the tumor travel through the blood. In this study, we used a cell-based assay to determine whether serum from patients with lung adenocarcinoma could modulate the molecular clock. We found that the daily oscillation period of Bmal1 was significantly lengthened following treatment with serum from untreated lung adenocarcinoma patients. In addition, heat inactivation of this serum abolished the effect, suggesting that a heat-sensitive circulating factor(s) is present in the serum of untreated lung adenocarcinoma patients. Using real-time PCR, we also examined the mRNA abundance of Bmal1, Cry1, and Per1 in human osteosarcoma u2os cell line, HUVECs and A549 cell lines. The expression of Bmal1 was changed in A549 cells in the presence of sera from lung adenocarcinoma patients. Our study revealed a direct effect of serum from lung adenocarcinoma patients on the molecular clock.

Spritz: A Proteogenomic Database Engine.

Proteoforms are the workhorses of the cell, and subtle differences between their amino acid sequences or post-translational modifications (PTMs) can change their biological function. To most effectively identify and quantify proteoforms in genetically diverse samples by mass spectrometry (MS), it is advantageous to search the MS data against a sample-specific protein database that is tailored to the sample being analyzed, in that it contains the correct amino acid sequences and relevant PTMs for that sample. To this end, we have developed Spritz (https://smith-chem-wisc.github.io/Spritz/), an open-source software tool for generating protein databases annotated with sequence variations and PTMs. We provide a simple graphical user interface for Windows and scripts that can be run on any operating system. Spritz automatically sets up and executes approximately 20 tools, which enable the construction of a proteogenomic database from only raw RNA sequencing data. Sequence variations that are discovered in RNA sequencing data upon comparison to the Ensembl reference genome are annotated on proteins in these databases, and PTM annotations are transferred from UniProt. Modifications can also be discovered and added to the database using bottom-up mass spectrometry data and global PTM discovery in MetaMorpheus. We demonstrate that such sample-specific databases allow the identification of variant peptides, modified variant peptides, and variant proteoforms by searching bottom-up and top-down proteomic data from the Jurkat human T lymphocyte cell line and demonstrate the identification of phosphorylated variant sites with phosphoproteomic data from the U2OS human osteosarcoma cell line.

Encapsulation of Nedaplatin in Novel PEGylated Liposomes Increases Its Cytotoxicity and Genotoxicity against A549 and U2OS Human Cancer Cells.

Following the discovery of cisplatin over 50 years ago, platinum-based drugs have been a widely used and effective form of cancer therapy, primarily causing cell death by inducing DNA damage and triggering apoptosis. However, the dose-limiting toxicity of these drugs has led to the development of second and third generation platinum-based drugs that maintain the cytotoxicity of cisplatin but have a more acceptable side-effect profile. In addition to the creation of new analogs, tumor delivery systems such as liposome encapsulated platinum drugs have been developed and are currently in clinical trials. In this study, we have created the first PEGylated liposomal form of nedaplatin using thin film hydration. Nedaplatin, the main focus of this study, has been exclusively used in Japan for the treatment of non-small cell lung cancer, head and neck, esophageal, bladder, ovarian and cervical cancer. Here, we investigate the cytotoxic and genotoxic effects of free and liposomal nedaplatin on the human non-small cell lung cancer cell line A549 and human osteosarcoma cell line U2OS. We use a variety of assays including ICP MS and the highly sensitive histone H2AX assay to assess drug internalization and to quantify DNA damage induction. Strikingly, we show that by encapsulating nedaplatin in PEGylated liposomes, the platinum uptake cytotoxicity and genotoxicity of nedaplatin was significantly enhanced in both cancer cell lines. Moreover, the enhanced platinum uptake as well as the cytotoxic/antiproliferative effect of liposomal nedaplatin appears to be selective to cancer cells as it was not observed on two noncancer cell lines. This is the first study to develop PEGylated liposomal nedaplatin and to demonstrate the superior cell delivery potential of this product.

Biological effectiveness of very high gamma dose rate and its implication for radiological protection

Many experimental studies are carried out to compare biological effectiveness of high dose rate (HDR) with that of low dose rate (LDR). The rational for this is the uncertainty regarding the value of the dose rate effectiveness factor (DREF) used in radiological protection. While a LDR is defined as 0.1 mGy/min or lower, anything above that is seen as HDR. In cell and animal experiments, a dose rate around 1 Gy/min is usually used as representative for HDR. However, atomic bomb survivors, the reference cohort for radiological protection, were exposed to tens of Gy/min. The important question is whether gamma radiation delivered at very high dose rate (VHDR—several Gy/min) is more effective in inducing DNA damage than that delivered at HDR. The aim of this investigation was to compare the biological effectiveness of gamma radiation delivered at VHDR (8.25 Gy/min) with that of HDR (0.38 Gy/min or 0.79 Gy/min). Experiments were carried out with human peripheral mononuclear cells (PBMC) and the human osteosarcoma cell line U2OS. Endpoints related to DNA damage response were analysed. The results show that in PBMC, VHDR is more effective than HDR in inducing gene expression and micronuclei. In U2OS cells, the repair of 53BP1 foci was delayed after VHDR indicating a higher level of damage complexity, but no VHDR effect was observed at the level of micronuclei and clonogenic cell survival. We suggest that the DREF value may be underestimated when the biological effectiveness of HDR and LDR is compared.

MicroRNA-9-5p inhibits osteosarcoma cell promotion, metastasis and resistance to apoptosis via negatively targeting Grb2-associated binding protein 2

The study explores the inhibition effects of MicroRNAs in osteosarcoma, as a means of suggesting it as treatment for bone cancer. MicroRNAs (miRNAs) are a sort of noncoding RNA molecules that regulates gene expression by targeting mRNAs and play critical roles in tumor development. This study probed the effect of miR-9-5p on osteosarcoma development. Human osteosarcoma cell lines U2-OS, 143B, MG63 and HOS and normal human osteoblast cell line hFOB were cultivated and expression of miR-9-5p and Grb2-associated binding protein 2 (Gab2) measured. The binding of miR-9-5p and Gab2 was confirmed using a bio-information program and dual luciferase reporter gene assay. Loss-of-functions of miR-9-5p and Gab2 were performed to measure their roles in osteosarcoma cell proliferation, invasion, migration and resistance to death. Result showed high miR-9-5p expression and low Gab2 expression in osteosarcoma cells, particularly in U2-OS cells. miR-9-5p directly bound to the 3untranslated region of Gab2. Down-regulated miR-9-5p induced U2-OS cell proliferation, invasion and the resistance to death, while conversely, silenced Gab2 led to an opposite trend on U2-OS cell growth and metastasis. Moreover, co-effect of inhibited miR-9-5p and silenced Gab2 led to decreased cell proliferation but promoted cell apoptosis compared to inhibited miR-9-5p alone, while it led to enhanced cell proliferation and invasion, but reduced cell apoptosis compared to silenced Gab2 alone. To conclude, this study demonstrated that miR-9-5p could inhibit osteosarcoma cell proliferation, invasion, migration and resistance to death via negatively targeting Gab2.

Two new naphthoate derivatives from Morinda officinalis

Two new naphthoate derivatives, including a symmetrical dimer (1) and a monomer (2), were separated from the roots of Morinda officinalis var. hirsuta. Their structures were characterized on the basis of spectroscopic means especially MS and NMR methods. Biological evaluations revealed that the two compounds did not show inhibition against both cholinesterases AChE and BChE, while the dimer (1) did exhibit moderate growth inhibitory activity toward one human osteosarcoma cell line U2OS with an IC50 value of 18.5 ± 1.1 μM.