iNOS In Cells Research Articles

Hierarchically Structured Hydroxyapatite Particles Facilitate the Enhanced Integration and Selective Anti-Tumor Effects of Amphiphilic Prodrug for Osteosarcoma Therapy.

Efficient delivery of cargo into target cells is a formidable challenge in modern medicine. Despite the great promise of biomimetic hydroxyapatite (HA) particles in tissue engineering, their potential applications in bone tumor therapy, particularly their structure-function relationships in cargo delivery to target cells, have not yet been well explored. In this study, biomimetic multifunctional composite microparticles (Bm-cMPs) are developed by integrating an amphiphilic prodrug of curcumin with hierarchically structured HA microspheres (Hs-hMPs). Then, the effects of the hierarchical structure of vehicles on the integration and delivery of cargo as well as the anti-osteosarcoma (OS) effect of the composite are determined. Different hierarchical structures of the vehicles strongly influence the self-assembly behavior of the prodrug. The flake-like crystals of Hs-hMPs enable the highest loading capacity and enhance the stability of the cargo. Compared to the normal cells, OS cells exhibit 3.56-times better uptake of flake-like Hs-hMPs, facilitating the selective anti-tumor effect of the prodrug. Moreover, Bm-cMPs suppress tumor growth and metastasis by promoting apoptosis and inhibiting cell proliferation and tumor vascularization. The findings shed light on the potential application of Bm-cMPs and suggest a feasible strategy for developing an effective targeted therapy platform using hierarchically structured minerals for OS treatment.

Attenuation of Endoplasmic Reticulum Stress Enhances Carvacrol-Induced Apoptosis in Osteosarcoma Cell Lines.

Carvacrol is a monoterpenoid phenol that has excellent antimicrobial, antiviral, and anti-inflammatory activities. It can also improve wound healing. However, few studies have explored its antitumor effect on osteosarcoma. In this report, we tried to determine the potential efficacy of carvacrol against osteosarcoma cell lines. Our data revealed that carvacrol exposure inhibited the proliferation of osteosarcoma HOS and U-2 OS cells. In addition, carvacrol exposure enhanced the levels of cleaved PARP and caspase 3 and increased annexin V-positive cells, indicating that carvacrol exposure triggers apoptosis in osteosarcoma cell lines. Furthermore, the levels of reactive oxygen species (ROS) were enhanced after carvacrol exposure and cotreatment with NAC, the ROS scavenger, decreased the levels of cleaved PARP and caspase 3, suggesting the involvement of ROS in carvacrol-induced apoptosis. Importantly, we found that carvacrol exposure triggered several protein expressions related to endoplasmic reticulum (ER) stress, including GRP78/Bip, IRE1a, PERK, and CHOP, in HOS and U-2 OS cells, indicating that carvacrol exposure could result in ER stress in these cell lines. Cotreatment with the ER stress inhibitor 4-PBA increased the levels of cleaved PARP and caspase 3 and further suppressed cellular proliferation in carvacrol-exposed osteosarcoma cell lines. Overall, the results indicate that induced ER stress can protect cells from apoptosis, but increased ROS contributes to apoptosis in carvacrol-treated cells. In this report, we first demonstrate the role of ER stress in carvacrol-induced apoptosis and suggest that ER stress could be targeted to enhance the antitumor activity of carvacrol in osteosarcoma cell lines.

Dysregulation of circRNA-0076906 and circRNA-0134944 is Correlated with Susceptibility to Osteoporosis and Osteoporotic Fracture in Postmenopausal Females from the Chinese Han Population.

Many circRNAs, such as circRNA-0076906 and circRNA-0134944, have been reported to participate in the pathogenesis of osteoporosis via sponging miRNAs in postmenopausal female patients. In this study, we aimed to study potential signaling pathways underlying the role of certain circRNAs, miRNAs and their target genes in the pathogenesis of osteoporotic fracture in postmenopausal females. Quantitative real-time PCR was performed to analyze the expression of circRNAs, miRNAs and their targets genes. Luciferase assays were carried out to explore the regulatory relationship between circ_0076906/miR-548i/OGN and circ_0134944/miR-630/TLR4. Osteoporosis and fracture were positively correlated to the expression of circ_0134944, miR-548i and TLR4, but negatively correlated to the expression of circ_0076906, miR-630 and OGN in the peripheral blood and bone tissue samples of postmenopausal women. Luciferase activities of wild-type circ_0076906 and OGN were inhibited by miR-548i, and the luciferase activities of wild-type circ_0134944 and TLR4 were suppressed by miR-630 in MG-63 and U-2 OS cells. Inhibition of circ_0076906 expression in MG-63 and U-2 OS cells activated the expression of miR-548i and inhibited the expression of OGN. Moreover, the overexpression of circ_0134944 in MG-63 and U-2 OS cells suppressed the expression of miR-630 and enhanced the expression of TLR4. This study implied that the dysregulation of circRNA-0076906 and circRNA-0134944 modulated their specific signaling and thus contributed to the severity of osteoporosis, increasing the risk of osteoporotic fracture.

The synergistic anticancer effect of CBD andDOX inosteosarcoma.

Osteosarcoma is a malignant tumor that can present with pain in the bones, joints, and local masses. The incidence is highest in adolescents, and the most common sites are the distal femur, proximal tibia and proximal humerus metaphyseal. Doxorubicin is the first-line chemotherapeutic agent for the treatment of osteosarcoma, but it has many side effects. Cannabidiol is a non-psychoactive plant cannabinoid cannabinol (CBD) that has been shown to be effective against osteosarcoma; however, the molecular targets and mechanisms of CBD action in osteosarcoma remain unclear. Cell proliferation, migration, invasion and colony formation were analyzed using two drugs alone or in combination to evaluate their inhibitory effects on the malignant characteristics of OS cells. Apoptosis and the cell cycle were detected by flow cytometry. The synergistic inhibitory effect of doxorubicin/cannabidiol on tumors was also detected in nude mouse xenotransplantation models. Through analysis of two osteosarcoma cell lines, MG63 and U2R, it was found that the cannabidiol/doxorubicin combination treatment synergistically inhibited growth, migration and invasion and induced apoptosis, blocking G2 stagnation in OS cells. Further mechanistic exploration suggests that the PI3K-AKT-mTOR pathway and MAPK pathway play an important role in the synergistic inhibitory effect of the two drugs in osteosarcoma. Finally, in vivo experimental results showed that the cannabidiol/doxorubicin combination treatment significantly reduced the number of tumor xenografts compared to cannabidiol alone or doxorubicin alone. Our findings in this study suggest that cannabidiol and doxorubicin have a synergistic anticancer effect on OS cells, and their combined application may be a promising treatment strategy for 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.

MiR-141-3p Promotes the Cisplatin Sensitivity of Osteosarcoma Cell through Targeting the Glutaminase [GLS]-Mediated Glutamine Metabolism.

This study aimed to evaluate the roles and molecular targets of miRNA-141-3p in the cisplatin sensitivity of osteosarcoma. Osteosarcoma is one of the most common-type bone tumors, occurring mainly in children and adolescents. Cancer cells display dysregulated cellular metabolism, such as the abnormally elevated glutamine metabolism. Non-coding RNA miRNA-141-3p has been reported to act as a tumor suppressor in osteosarcoma. Currently, the precise molecular mechanisms for the miR- 141-3p-mediated chemosensitivity through regulating glutamine metabolism remain unclear. We collected thirty paired OS tumors and their adjacent normal tissues. The osteosarcoma cell lines [Saos-2] and normal osteoblast cells, hFOB1.19, were used for in vitro experiments. RT-qPCR and Western blot were applied for gene expression detections. Targets of miR-141-3p were predicted from starBase. The MTT and flow cytometric assays were performed to determine cell growth and apoptosis rates. The cellular glutamine metabolism was monitored by glutamine uptake assay and the glutaminase [GLS] activity assay. We reported that miR-141-3p were significantly downregulated in osteosarcoma tissues and cells. Overexpression of miR-141-3p suppressed OS cell growth and sensitized OS cells to cisplatin. In addition, glutamine metabolism was significantly increased in osteosarcoma. We characterized that GLS played oncogenic roles in osteosarcoma and validated GLS was a direct target of miR-141-3p in OS cells. Rescue experiments consistently demonstrated that miR-141-3p-promoted cisplatin sensitivity was achieved by targeting GLS directly. Overall, our findings revealed new molecular mechanisms of the miR-141- 3p-modulated cisplatin sensitization through targeting the GLS-glutamine metabolism pathway. This study will contribute to developing new therapeutic approaches for the treatments of chemoresistant osteosarcoma.

MiR-125b-5p inhibits proliferation and migration of osteosarcoma cells by negatively regulating RAB3D expression

To investigate the inhibitory effect of miR-125b-5p on proliferation and migration of osteosarcoma and the role of RAB3D in mediating this effect. The expression level of miR-125b-5p was detected by qRT-PCR in a normal bone cell line (hFOB1.19) and in two osteosarcoma OS cell lines (MG63 and HOS). A miR-125b-5p mimic or inhibitor was transfected in the osteosarcoma cell lines via liposome and the changes in cell proliferation and migration were detected with EDU and Transwell experiments. Bioinformatic analysis was conducted for predicting the target gene of miR-125b-5p, and the expression level of RAB3D in hFOB1.19, MG63, and HOS cells was detected by Western blotting. In the two osteosarcoma cell lines transfected with miR-125b-5p mimic or inhibitor, the expression levels of RAB3D mRNA and protein in osteosarcoma cells were examined with qRT-PCR and Western blotting. The effects of RAB3D overexpression, RAB3D knockdown, or overexpression of both miR-125b-5p and RAB3D on the proliferation and migration of cells were assessed using EDU and Transwell experiments. The two osteosarcoma cell lines had significantly lower expression levels of miR-125b-5p (P < 0.05). Bioinformatic analysis predicted that RAB3D was a possible target gene regulated by miR-125b-5p. In osteosarcoma cells, overexpression of miR-125b-5p significantly lowered the expression of RAB3D protein (P < 0.05); inhibiting miR-125b-5p expression significantly decreased RAB3D expression only at the protein level (P < 0.05) without obviously affecting its mRNA level. Modulation of miR-125b-5p and RAB3D levels produced opposite effects on proliferation and migration of osteosarcoma cells, and in cells with overexpression of both miR-125b-5p and RAB3D, the effect of RAB3D on cell proliferation and migration was blocked by miR-125b-5p overexpression (P < 0.05). Overexpression of miR-125b-5p inhibits the proliferation and migration of osteosarcoma cells by regulating the expression of RAB3D at the post-transcriptional level.

Abstract B036: Platelets promote in vitro osteosarcoma cell migration and in vivo metastatic lung seeding

Abstract Osteosarcoma (OS) is the most common primary bone cancer worldwide where it mainly affects adolescents. Metastatic OS has a 20% survival rate and treatment options remain poor, thus understanding mechanisms that contribute to OS metastasis is critical for improved patient outcomes. Platelets, commonly recognized for their role in hemostasis, have been shown to promote metastasis in several cancer models. However, most previous studies investigating platelet contributions to metastasis have focused on carcinomas while their role in osteosarcoma remains poorly understood. Previous studies by our lab have shown that platelet interactions with breast cancer cells resulted in increased migration in vitro and increased lung metastases in vivo. Thus, we hypothesized that OS cell migration will be enhanced in the presence of platelets, and that platelet depletion in vivo will inhibit OS lung seeding. Cell suspensions of SaOs2 and MG63.3 cells in serum free media were fluorescently stained and seeded, in triplicates, in the top chamber of an 8um pore Fluoroblock migration assay wells. In the bottom chamber, media and either Tyrode’s buffer or 1.5x106 isolated human platelets were added. The presence of platelets resulted in a 3.5- and 4.6-fold increase of migrated MG63.3 and SaOs2 cells respectively. For in vivo analysis, cohorts of eight-week-old, male Nod Scid-Gamma (NSG), Nod Scid, and Nude mice were randomly assigned to either the control or platelet depletion group. Mice were depleted of platelets by administration of Anti-Platelet glycoprotein Ib alpha (GPIbα) antibody and, 5x105 GFP transduced MG63.3 cells were injected via tail vein. 24 hrs later, the mice were euthanized, and lungs were collected and microscopically imaged. Platelet depletion in NSG mice resulted in a 48% reduction in MG63.3 lung seeding compared to the isotype control recipient mice. This result was more dramatic in the less immunocompromised Nude and Nod Scid mice with a 61% and 87% reduction of MG63.3 lung seeding in the platelet depleted mice respectively. Finally, RNAseq and was performed on seven OS cell lines co-cultured with isolated mouse platelets, and alterations in gene expression were compared to control cells. Gene Enrichment Set Analysis (GSEA) of the data indicated that platelet co-incubation significantly upregulated pathways related to inflammatory responses, Tumor Necrosis Factor (TNF)-a signaling, and Interleukin (IL)-6 signaling. These results suggest that platelet secreted factors facilitate OS cell migration in vitro while an immune dependent mechanism is involved in the role of platelets in the in vivo lung seeding of OS cells. Citation Format: Olivia K. Travis, Ruishan Wang, Myriam Labelle. Platelets promote in vitro osteosarcoma cell migration and in vivo metastatic lung seeding [abstract]. In: Proceedings of the AACR Special Conference: Cancer Metastasis; 2022 Nov 14-17; Portland, OR. Philadelphia (PA): AACR; Cancer Res 2022;83(2 Suppl_2):Abstract nr B036.

Aa-Z2 triggers ROS-induced apoptosis of osteosarcoma by targeting PDK-1

BackgroundOsteosarcoma (OS) is the most frequent cancer derived from bone, and the prognosis of OS is poor. Metabolic alterations have been previously reported to contribute to the development of OS, and arsenic compounds have been suggested to exhibit strong anti-OS effects. However, few studies have described the therapeutic efficiency of arsenic compounds by targeting metabolism in OS.MethodsHere, we presented a novel organo-arsenic compound, Aa-Z2, and its antitumour efficacy against OS both in vitro and in vivo.ResultsAa-Z2 induced OS cell apoptosis, G2/M phase arrest, and autophagy through the accumulation of reactive oxygen species (ROS). Elevated ROS functioned by promoting the mitochondrial-dependent caspase cascade and attenuating the PI3K/Akt/mTOR signalling pathway. N-acetylcysteine (NAC), a kind of ROS scavenger, could reverse the effects of Aa-Z2 treatment on 143B and HOS cells. Specifically, by targeting pyruvate dehydrogenase kinase 1 (PDK-1), Aa-Z2 induced changes in mitochondrial membrane potential and alterations in glucose metabolism to accumulate ROS. Overexpression of PDK-1 could partially desensitize OS cells to Aa-Z2 treatment. Importantly, Aa-Z2 suppressed tumour growth in our xenograft osteosarcoma model.ConclusionThe study provides new insights into the mechanism of Aa-Z2-related metabolic alterations in OS inhibition, as well as pharmacologic evidence supporting the development of metabolism-targeting therapeutics.

Selenium-incorporated mesoporous silica nanoparticles for osteosarcoma therapy.

Selenium (Se) compounds are promising chemotherapeutics due to their ability to inhibit cancer cell activity via the generation of reactive oxygen species (ROS). However, to circumvent adverse effects on bone healthy cells, new methods are needed to allow intracellular Se delivery. Mesoporous silica nanoparticles (MSNs) are promising carriers for therapeutic ion delivery due to their biocompability, rapid uptake via endocytosis, and ability to efficiently incorporate ions within their tunable structure. With the aim of selectively inhibiting cancer cells, here we developed three types of MSNs and investigated their ability to deliver Se. Specifically, MSNs containing SeO32- loaded on the surface and in the pores (MSN-SeL), SeO32- doped in the silica matrix (Se-MSNs) and Se nanoparticles (SeNP) coated with mesoporous silica (SeNP-MSNs), were successfully synthesized. All synthesized nanoparticles were stable in neutral conditions but showed rapid Se release in the presence of glutathione (GSH) and nicotinamide adenine dinucleotide phosphate (NADPH). Furthermore, all nanoparticles were cytotoxic towards SaoS-2 cells and showed significantly lower toxicity towards healthy osteoblasts, where Se doped MSNs showed lowest toxicity towards osteoblasts. We further show that the nanoparticles could induce ROS and cell apoptosis. Here we demonstrate MSNs as promising Se delivery carriers for osteosarcoma (OS) therapy.

TYMSOS-miR-101-3p-NETO2 axis promotes osteosarcoma progression

BackgroundOsteosarcoma (OS) is a type of bone cancer most often affects pre-teens and teens, but it is still a rare disorder. Neuropilin and tolloid-like 2 (NETO2) has been reported to promote OS progression, but its upstream mechanism in OS cells remains obscure. MethodsQuantitative real-time PCR (RT-qPCR) and Western blot were conducted to examine RNA and protein levels, separately. Functional assays were performed to assess the impact of NETO2 on OS cell malignancy. Moreover, bioinformatics analyses and mechanism experiments were performed to identify the upstream mechanism of NETO2 in OS cells. ResultsFunctionally, NETO2 depletion repressed cell proliferation, migration and invasion as well as epithelial-mesenchymal transition (EMT) but triggered the apoptosis of OS cells. NETO2 is directly targeted and negatively regulated by microRNA-101-3p (miR-101-3p). Mechanically, miR-101-3p could combine with long noncoding RNA (lncRNA) TYMS opposite strand RNA (TYMSOS) in OS cells. In addition, our study proved that TYMSOS promotes the malignancy of OS via elevating NETO2 expression as miR-101-3p sponge. ConclusionTYMSOS-miR-101-3p-NETO2 axis promotes the malignant behaviors of OS cells, which might offer a novel sight for OS treatment.

Pan-Cancer analysis of TERT and Validation in Osteosarcoma Cell Lines

PurposeTo investigate the possible functions of TERT in pan-cancer and OS. MethodsFirst, differential TERT gene expression analysis was conducted using multi-omics data integrative analyses, including differential expression, prognosis, the correlation between infiltrating inflammatory immune cells, and mutation in pan-cancer. Furthermore, differential TERT gene expression analysis was conducted using mRNA expression profiles related to OS based on the GEO Datasets. Various differentially expressed genes were chosen based on a fitness threshold for further investigations. Finally, the function of the TERT gene was assessed in OS cells, including cellular proliferation, migration, and metastasis. ResultsPan-cancer research demonstrated that variable expression of TERT was not only associated with numerous types of human cancer but was also intimately linked to DNA methylation. Bioinformatic investigation revealed a link between the differential expression of TERT with immune cell infiltration in the tumor microenvironment (TME). In vitro studies indicated that inhibition of TERT decreased OS cell proliferation, motility, and metastasis. ConclusionTERT may serve as a useful genomic biomarker for the diagnosis and prediction of pan-cancer and as a prospective therapeutic target for the treatment of OS.

Rapamycin-induced autophagy in osteosarcoma cells is mediated via the biglycan/Wnt/β-catenin signaling axis.

Biglycan is a class I secreted small leucine-rich proteoglycan (SLRP), which regulates signaling pathways connected to bone pathologies. Autophagy is a vital catabolic process with a dual role in cancer progression. Here, we show that biglycan inhibits autophagy in two osteosarcoma cell lines (P ≤ 0.001), while rapamycin-induced autophagy decreases biglycan expression in MG63 osteosarcoma cells and abrogates the biglycan-induced cell growth increase (P ≤ 0.001). Rapamycin also inhibits β-catenin translocation to the nucleus, inhibiting the Wnt pathway (P ≤ 0.001) and reducing biglycan's colocalization with the Wnt coreceptor LRP6 (P ≤ 0.05). Furthermore, biglycan exhibits protective effects against the chemotherapeutic drug doxorubicin in MG63 OS cells through an autophagy-dependent manner (P ≤ 0.05). Cotreatment of these cells with rapamycin and doxorubicin enhances cells response to doxorubicin by decreasing biglycan (P ≤ 0.001) and β-catenin (P ≤ 0.05) expression. Biglycan deficiency leads to increased caspase-3 activation (P ≤ 0.05), suggesting increased apoptosis of biglycan-deficient cells treated with doxorubicin. Computational models of LRP6 and biglycan complexes suggest that biglycan changes the receptor's ability to interact with other signaling molecules by affecting the interdomain bending angles in the receptor structure. Biglycan binding to LRP6 activates the Wnt pathway and β-catenin nuclear translocation by disrupting β-catenin degradation complex (P ≤ 0.01 and P ≤ 0.05). Interestingly, this mechanism is not followed in moderately differentiated, biglycan-nonexpressing U-2OS OS cells. To sum up, biglycan exhibits protective effects against the doxorubicin in MG63 OS cells by activating the Wnt signaling pathway and inhibiting autophagy.

LncRNA CASC9 promotes cell proliferation and invasion in osteosarcoma through targeting miR-874-3p/SOX12 axis

BackgroundOsteosarcoma (OS) is a common primary malignant bone tumor. This study aimed to explore the biological role of long on-coding RNA (lncRNA) CASC9 and its regulatory mechanism in OC.MethodsThe CASC9 expressions in OS cells and tissues were measured using qRT-PCR. The functional role of CASC9 in OC was studied using MTT assay, colony formation assay, transwell invasion assay, and xenograft tumor assay. In addition, the mechanism of CASC9 function was determined using luciferase reporter assay. Western blot was used to analyze protein expressions in our paper.ResultsLncRNA CASC9 was found to be up-regulated in OS. Knockdown of CASC9 inhibited the proliferation and invasion of OS cells. Besides, miR-874-3p was identified as the target of CASC9, and SOX12 acted as a potential target of miR-874-3p. The down-regulation of miR-874-3p recovered the reduction in cell invasion and proliferation in vitro which were induced by CASC9 knockdown and delayed the tumor progression in vivo.ConclusionLncRNA CASC9 promotes cell proliferation and invasion in OS via miR-874-3p/SOX12 axis. Our study might provide novel biomarkers and potential therapeutic targets for OS treatment.

Characterization of drug effects on cell cultures from phase-contrast microscopy images

In this work, we classify chemotherapeutic agents (topoisomerase inhibitors) based on their effect on U-2 OS cells. We use phase-contrast microscopy images, which are faster and easier to obtain than fluorescence images and support live cell imaging. We use a convolutional neural network (CNN) trained end-to-end directly on the input images without requiring for manual segmentations or any other auxiliary data. Our method can distinguish between tested cytotoxic drugs with an accuracy of 98%, provided that their mechanism of action differs, outperforming previous work. The results are even better when substance-specific concentrations are used. We show the benefit of sharing the extracted features over all classes (drugs). Finally, a 2D visualization of these features reveals clusters, which correspond well to known class labels, suggesting the possible use of our methodology for drug discovery application in analyzing new, unseen drugs.

Generation and characterization of human U-2 OS cell lines with the CRISPR/Cas9-edited protoporphyrinogen oxidase IX gene

In humans, disruptions in the heme biosynthetic pathway are associated with various types of porphyrias, including variegate porphyria that results from the decreased activity of protoporphyrinogen oxidase IX (PPO; E.C.1.3.3.4), the enzyme catalyzing the penultimate step of the heme biosynthesis. Here we report the generation and characterization of human cell lines, in which PPO was inactivated using the CRISPR/Cas9 system. The PPO knock-out (PPO-KO) cell lines are viable with the normal proliferation rate and show massive accumulation of protoporphyrinogen IX, the PPO substrate. Observed low heme levels trigger a decrease in the amount of functional heme containing respiratory complexes III and IV and overall reduced oxygen consumption rates. Untargeted proteomics further revealed dysregulation of 22 cellular proteins, including strong upregulation of 5-aminolevulinic acid synthase, the major regulatory protein of the heme biosynthesis, as well as additional ten targets with unknown association to heme metabolism. Importantly, knock-in of PPO into PPO-KO cells rescued their wild-type phenotype, confirming the specificity of our model. Overall, our model system exploiting a non-erythroid human U-2 OS cell line reveals physiological consequences of the PPO ablation at the cellular level and can serve as a tool to study various aspects of dysregulated heme metabolism associated with variegate porphyria.

PLOD2 high expression associates with immune infiltration and facilitates cancer progression in osteosarcoma.

BackgroundOsteosarcoma (OS) is the most common primary malignant bone tumors in children and adolescents. Procollagen-lysine, 2-oxoglutarate 5-dioxygenase 2 (PLOD2) is a key gene in mediating the formation of the stabilized collagen cross-link, playing an important role in the progression of cancer. However, the interaction between OS and PLOD2 has not been clarified so far.MethodsThe target gene PLOD2 was screened through our own RNA-seq results and other two RNA-seq results from GEO database. The expression of PLOD2 in OS was detected by RT-qPCR, Western blot and immunohistochemistry. Functional experiments were performed to investigate the role of PLOD2 in OS cell invasion, migration and angiogenesis in vitro. An OS lung metastasis model was established to investigate the function of PLOD2 in OS metastasis and angiogenesis in vivo. The role of PLOD2 in immune infiltration in OS was explored by KEGG/GO analysis and immune infiltration analysis with TARGET, TCGA and TIMER.ResultsPLOD2 was high-expressed in OS, which was related to poor prognosis of OS patients. PLOD2 promoted OS cell migration, invasion and angiogenesis in vitro and aggravated OS metastasis and angiogenesis in vivo. Bioinformatic analysis showed that PLOD2 played an important role in immune cell infiltration in OS, including CD8 positive T cells, macrophages M0 cells, DC cells, endothelial cells, iDC cells, ly endothelial cells, MEP cells, mv endothelial cells, native B cells, smooth muscle cells and Th1 cells. Immunohistochemical results showed that the expression of CD4 and CD8A was negatively correlated with the expression of PLOD2 in OS.ConclusionPLOD2 was high-expressed in OS and promoted OS migration, invasion and angiogenesis in vitro and facilitated OS metastasis and angiogenesis in vivo. PLOD2 was associated with immune cell infiltration in OS, which could be a promising target to treat OS patients with metastasis and utilized to guide clinical immunotherapy in the future.

Induced resistance to ifosfamide in osteosarcoma cells suggests a more aggressive tumor profile

AimsOsteosarcoma (OS) is the most common primary malignant bone sarcoma among children and adolescents. Treatment is based on neo-adjuvant and adjuvant chemotherapy, using the standard drugs cisplatin, methotrexate, doxorubicin, and ifosfamide (IFO). Due to the high capacity of tumor resistance, the current work aimed to analyze genes related to cycle control and cell differentiation in OS cells sensitive to and with induced resistance to IFO. This was to assess whether the differentiated expression of these genes may affect resistance to the drug IFO used in OS treatment, and thus establish possible biomarkers of disease progression. Materials and methodsIn this work, the treatment-sensitive OS U2OS lineage was used, and the same lineage was submitted to the process of induction of IFO resistance. These cells were evaluated by MTT, migration and proliferation assays and submitted to gene expression analysis. Key findingsThe results demonstrate that after induction of resistance to IFO, resistant U2OS cells show a more aggressive tumor behavior, with greater capacity for cell migration, proliferation, and invasion compared to sensitive cells. Gene analysis indicates that resistance-induced cells have differentiated expression of the genes EPB41L3, GADD45A, IER3, OXCT1, UBE2L6, UBE2A ALPL, and EFNB2. Our results suggest new perspectives on possible resistance biomarkers, especially the genes EFNB2 and EPB41L3, given that these genes have rarely been studied their expression linked to osteosarcoma. They show how the resistance induction model can be useful for studies on tumor cell behavior.

LRIG2 regulates cell proliferation, migration and apoptosis of osteosarcoma

BackgroundOsteosarcoma (OS) is one of the malignant bone tumors with strong aggressiveness and poor prognosis. Leucine-rich repeats and immunoglobulin-like domains2 (LRIG2) is closely associated with the poor prognosis of a variety of tumors, but the role of LRIG2 in osteosarcoma and the underlying molecular mechanism remains unclear.ObjectiveThe aim of this study was to determine the function of LRIG2 in OS and the related molecular mechanism on cell proliferation, apoptosis and migration of OS.MethodsThe mRNA and protein expression of LRIG2 in OS tissues and cells was detected by qRT-PCR, western blot (WB) assay and immunohistochemistry (IHC). The cell counting Kit-8 (CCK-8), clone formation, transwell, TdT-mediated dUTP Nick-End Labeling (TUNEL) and WB assay were applied to determine the proliferation, migration and apoptosis abilities of OS cells and its molecular mechanisms. Spontaneous metastasis xenografts were established to confirm the role of LRIG2 in vivo.ResultsLRIG2 exhibited high expression in OS tissues and OS cell lines and the expression of which was significantly correlated with Enneking stage of patients, knockdown LRIG2 expression significantly inhibited OS cell proliferation, migration and enhanced apoptosis. Silencing LRIG2 also suppressed the growth of subcutaneous transplanted tumor in nude mice. Further, the mechanism investigation revealed that the protein level of cell proapoptotic proteins (Bax, caspase9 and caspase3) all increased attributed to LRIG2 deficiency, whereas expression of anti-apoptotic protein BCL2 decreased. LRIG2 silencing led to the decrease phosphorylation of AKT signaling, a decrease expression of vimentin and N-cadherin. Additionally, silencing LRIG2 significantly decreased the rate of tumor growth and tumor size.ConclusionsLRIG2 acts as an oncogene in osteosarcoma, and it might become a novel target in the treatment of human OS.

Knock-out of 5-lipoxygenase in overexpressing tumor cells—consequences on gene expression and cellular function

5-Lipoxygenase (5-LO), the central enzyme in the biosynthesis of leukotrienes, is frequently expressed in human solid malignancies even though the enzyme is not present in the corresponding healthy tissues. There is little knowledge on the consequences of this expression for the tumor cells regarding gene expression and cellular function. We established a knockout (KO) of 5-LO in different cancer cell lines (HCT-116, HT-29, U-2 OS) and studied the consequences on global gene expression using next generation sequencing. Furthermore, cell viability, proliferation, migration and multicellular tumor spheroid (MCTS) formation were studied in these cells. Our results show that 5-LO influences the gene expression and cancer cell function in a cell type-dependent manner. The enzyme affected genes involved in cell adhesion, extracellular matrix formation, G protein signaling and cytoskeleton organization. Furthermore, absence of 5-LO elevated TGFβ2 expression in HCT-116 cells while MCP-1, fractalkine and platelet-derived growth factor expression was attenuated in U-2 OS cells suggesting that tumor cell-derived 5-LO shapes the tumor microenvironment. In line with the gene expression data, KO of 5-LO had an impact on cell proliferation, motility and MCTS formation. Interestingly, pharmacological inhibition of 5-LO only partly mimicked the KO suggesting that also noncanonical functions are involved.