interleukin-8 and Osteosarcoma

Osteosarcoma has been reported with treatment failure in up to 40% of cases. Our laboratory had identified genes involved in the PPARγ pathway to be associated with doxorubicin (DOX) resistance. We hence used PPARγ agonist pioglitazone (PIO) to modulate DOX resistance. DOX-resistant cell line (143B-DOX) was developed by gradient exposure to DOX. The cytotoxicity to PIO and in combination with DOX was assayed in vitro, followed by HPLC to estimate the metabolites of PIO in the presence of microsomes (HLMs). Gene expression studies revealed the mechanism behind the cytotoxicity of PIO. Further, the effects were evaluated in mice bearing 143B-DOX tumors treated either with PIO (20 mg/kg/p.o or 40 mg/kg/p.o Q1D) alone or in combination with DOX (0.5 mg/kg/i.p Q2W). 143B-DOX was 50-fold resistant over parental cells. While PIO did not show any activity on its own, the addition of HLMs to the cells in culture showed over 80% cell kill within 24 h, possibly due to the metabolites of PIO as determined by HPLC. In combination with DOX, PIO had shown synergistic activity. Additionally, cytotoxicity assay in the presence of HLMs revealed that PIO on its own showed promising activity compared to its metabolites-hydroxy pioglitazone and keto pioglitazone. In vivo studies demonstrated that treatment with 40 mg/kg/p.o PIO alone showed significant activity, followed by a combination with DOX. Gene expression studies revealed that PIO could modulate drug resistance by downregulating MDR1 and IL8. Our study suggests that PIO can modulate DOX resistance in osteosarcoma cells.

Topics: Animals; Antibiotics, Antineoplastic; ATP Binding Cassette Transporter, Subfamily B, Member 1; Bone Neoplasms; Cell Line, Tumor; Disease Models, Animal; Doxorubicin; Drug Resistance, Neoplasm; Humans; Hypoglycemic Agents; Interleukin-8; Male; Mice, Nude; Osteosarcoma; Pioglitazone; Xenograft Model Antitumor Assays

Systemic immune responses in cancer patients are of tremendous importance, both to advance understanding of disease mechanisms and for development of new diagnostic testing. Minimal published information is available on the systemic cytokine response in canine osteosarcoma (OS) patients. The goal of this study was to investigate serum cytokine alterations present in OS patients at the time of diagnosis. Serum samples from 22 canine OS patients at the time of diagnosis and 18 healthy control dogs were evaluated via multiplex immunoassay for 14 analytes. Significant increases in serum interleukin-8 (IL-8) and interleukin-12p40 (IL-12p40) concentrations were found in OS patients when compared to healthy controls. The results correlate with several published studies on serum cytokine alterations in human OS patients. These data add to the growing body of knowledge on immunologic alterations in OS, including potential immunomodulatory therapy of canine patients, and support future studies on serum cytokine testing to investigate diagnostic and prognostic utility.

Topics: Animals; Dog Diseases; Dogs; Female; Interleukin-12 Subunit p40; Interleukin-8; Male; Osteosarcoma

Most circulating tumor cells (CTCs) die during the process of metastasis, but self-seeding CTCs can invade the primary tumor or form clinically meaningful metastases. This study aimed to evaluate the capacity of self-seeding CTCs to promote osteosarcoma growth and lung metastasis and to clarify the specific role of interleukin (IL)-8 in CTC self-seeding. We successfully isolated and cultured self-seeding CTCs through a self-seeding nude mouse model established using green fluorescent protein (GFP)-labeled F5M2 cells and found that self-seeding CTCs exhibit increased cellular proliferation, migration, and invasion in vitro, increased tumor growth and lung metastasis in mice, and increased IL-8 expression. Furthermore, suppressing IL-8 inhibited tumor growth and metastasis and reduced CTC seeding in primary tumors in vitro and in vivo. In osteosarcoma patients, IL-8 levels significantly correlated with the Enneking stage and metastasis. These findings demonstrate that self-seeding osteosarcoma CTCs can promote tumor growth and lung metastasis through IL-8. Their increased metastatic potential and elevated IL-8 expression suggest a novel strategy for future therapeutic interventions to prevent osteosarcoma progression and metastasis.

Topics: Animals; Cell Movement; Cell Proliferation; Gene Expression Regulation, Neoplastic; Heterografts; Humans; Interleukin-8; Lung Neoplasms; Mice; Neoplasm Invasiveness; Neoplasm Metastasis; Neoplastic Cells, Circulating; Osteosarcoma

Osteosarcoma (OS) is the representative primary malignant bone tumor with the highest incidence. It is known that malignant phenotypes of OS, such as proliferation, invasion, and metastasis, are significantly influenced not only by characteristics of the tumor itself, but also by the surrounding microenvironment. In other words, OS is considered to utilize cells in the vicinity of the tumor by changing the characteristics of these cells. Direct intercellular contact is believed to be important for this phenomenon. In the present study, we hypothesized that an interaction mediated by a humoral factor, requiring no cellular contact, might play a significant role in the progression of OS.. We developed a new co-culture model, using OS cells and mesenchymal stem cells (MSCs) without cellular contact, and found that both cell types expressed IL-8 at a high level, and FAK in OS cells was phosphorylated leading to an increase in the metastatic potential of the tumor in the co-culture condition.. It was revealed that OS cells formed a loop of signal cross-talk in which they released IL-8 as a paracrine factor, stimulating MSCs to express IL-8, and received IL-8 released by MSCs to accelerate IL-8 expression in OS cells. Administration of anti-IL-8 antibody resulted in the inhibition of FAK expression, its downstream signaling, and the invasive potential of the OS cells, resulting in decrease in metastatic lesions.. The present study might lead not only to the clarification of a new molecular mechanism of invasion and metastasis of OS, but also to the development of a new therapeutic strategy of blocking IL-8 in OS.

Topics: Animals; Antibodies; Cell Line, Tumor; Cell Movement; Cell Proliferation; Coculture Techniques; Focal Adhesion Kinase 1; Humans; Interleukin-8; Mesenchymal Stem Cells; Mice; Mice, Nude; Neoplasm Metastasis; Osteosarcoma; Phosphorylation; Recombinant Proteins; Signal Transduction; Transplantation, Heterologous; Tumor Microenvironment; Up-Regulation

The loss of appropriate cell adhesion normally induces apoptosis via a process termed anoikis. The aim of this study was to investigate the effects of mesenchymal stem cells (MSCs) in the cancer microenvironment on the anoikis resistance and pulmonary metastasis of osteosarcoma (OS) cells, and to evaluate the critical role of the interleukin (IL)-8/C-X-C chemokine receptor (CXCR) 1/Akt-signaling pathway in these processes. Metastatic OS subtype cells, which did or did not interact with MSC-conditioned medium (MSC-CM) in vitro, were isolated from the pulmonary site and named Saos2-lung-M. Both MSC-CM and IL-8 treatment increased the anoikis resistance of Saos2 cells in vitro. Moreover, exogenous MSC-CM promoted the survival and metastasis of Saos2 cells in nude mice. Saos2-lung-M cells were more malignant and resistant to anoikis than parental cells. MSCs secreted IL-8, thereby protecting OS cells from anoikis. Blocking the IL-8/CXCR1/Akt pathway via CXCR1 knockdown inhibited the pulmonary metastasis of Saos2-lung-MSCs and prolonged the survival of tumor-bearing mice. In conclusion, MSCs enhanced OS cell resistance to anoikis and pulmonary metastasis via regulation of the IL-8/CXCR1/Akt pathway. These findings suggest that MSCs can "select for" OS cells with high metastatic potential in vivo, and highlight CXCR1 as a key target in the regulation of pulmonary metastasis of OS cells.

Topics: Animals; Anoikis; Bone Neoplasms; Cells, Cultured; Culture Media, Conditioned; Disease Progression; Gene Expression Regulation, Neoplastic; Humans; Interleukin-8; Lung Neoplasms; Male; Mesenchymal Stem Cells; Mice; Mice, Inbred BALB C; Mice, Nude; Osteosarcoma; Proto-Oncogene Proteins c-akt; Receptors, Interleukin-8A; Signal Transduction; Tumor Microenvironment

Osteosarcoma (OS), a malignant tumor of bone, kills through aggressive metastatic spread almost exclusively to the lung. Mechanisms driving this tropism for lung tissue remain unknown, though likely invoke specific interactions between tumor cells and other cells within the lung metastatic niche. Aberrant overexpression of ΔNp63 in OS cells directly drives production of IL-6 and CXCL8. All these factors were expressed at higher levels in OS lung metastases than in matched primary tumors from the same patients. Expression in cell lines correlated strongly with lung colonization efficiency in murine xenograft models. Lentivirus-mediated expression endowed poorly metastatic OS cells with increased metastatic capacity. Disruption of IL-6 and CXCL8 signaling using genetic or pharmaceutical inhibitors had minimal effects on tumor cell proliferation in vitro or in vivo, but combination treatment inhibited metastasis across multiple models of metastatic OS. Strong interactions occurred between OS cells and both primary bronchial epithelial cells and bronchial smooth muscle cells that drove feed-forward amplification of IL-6 and CXCL8 production. These results identify IL-6 and CXCL8 as primary mediators of OS lung tropism and suggest pleiotropic, redundant mechanisms by which they might effect metastasis. Combination therapy studies demonstrate proof of concept for targeting these tumor-lung interactions to affect metastatic disease.

Topics: Adolescent; Adult; Animals; Antineoplastic Combined Chemotherapy Protocols; Bone and Bones; Bone Neoplasms; Cell Line, Tumor; Cell Proliferation; Child; Cytokine Receptor gp130; Drug Evaluation, Preclinical; Follow-Up Studies; Humans; Hydrazines; Interleukin-6; Interleukin-8; Lung; Lung Neoplasms; Male; Mice; Osteosarcoma; Primary Cell Culture; Quinoxalines; Receptors, Interleukin-8A; Sulfonamides; Xenograft Model Antitumor Assays; Young Adult

Emerging evidence shows that cytokines such as interleukins (ILs) are involved in the progression and chemoresistance of multiple tumors, including osteosarcoma (OS). Our present study established the doxorubicin (Dox) resistant human OS MG-63 and HOS cells and named them MG-63/Dox and HOS/Dox, respectively. The expression of IL-8, while not VEGFA, IL-32, or IL-34, was significantly increased in OS/Dox cells as compared with that in the parental cells. IL-8 neutralization antibody can significantly increase the Dox sensitivity of OS/Dox cells. Further, IL-8 can up regulate ABCB1, which encodes one important ATP-binding cassette (ABC) transporter /P-glycoprotein (P-gp). Mechanically, IL-8 increased the transcription of ABCB1 via up regulating its promoter activity, while had no effect on its protein or mRNA stability. Targeted inhibition of p65 can attenuate IL-8 induced transcription of ABCB1 in OS cells. Treatment OS cells with 5-aza-dC, the inhibitor of DNMT, had no effect on expression of IL-8. Expression of HDAC6 in MG-63/Dox and HOS/Dox cells was significantly greater than that in their parental cells. Knockdown of HDAC6 can suppress the expression of IL-8 in OS cells. Collectively, our data showed that HDAC6 mediated upregulation of IL-8 can regulate the Dox sensitivity of OS cells via transcriptionally regulating the expression of ABCB1. Targeted inhibition of IL-8 might be a potent potential approach for overcome the Dox resistance of OS cells and helpful for clinical therapy of OS patients.

Topics: ATP Binding Cassette Transporter, Subfamily B, Member 1; Cell Line, Tumor; Doxorubicin; Drug Resistance, Neoplasm; Gene Expression Regulation, Neoplastic; Histone Deacetylase 6; Humans; Interleukin-8; Osteosarcoma; Transcription, Genetic; Up-Regulation

Estrogenic signals can regulate the progression of osteosarcoma (OS) via classic estrogen receptor α/β (ERα/β). G protein-coupled estrogen receptor (GPER) can mediate the non-genomic effects of estrogen and regulate the progression of various cancers. Our present study revealed that the expression of GPER in OS cells and tissues was lower than that in their corresponding controls. Activation of GPER via its specific agonist G-1 can decrease the proliferation, migration, and invasion of OS cells. By screening the expression of cytokines involved in the progression of OS, we found that activation of GPER can inhibit the expression of interleukin-6 (IL-6) and IL-8 in OS cells. Recombinant IL-6 (rIL-6) or rIL-8 can attenuate G-1 suppressed migration of OS cells. Mechanically, activation of GPER can rapidly decease the phosphorylation and nuclear translocation of NF-κB in OS cells. While over expression of p65 significantly attenuated G-1 induced down regulation of IL-6/IL-8. Further, G-1 can decrease the activation of p38-MAPK, which can further shorten the half-life of IL-8 mRNA. Collectively, we revealed that GPER can suppress the migration and invasion of OS cells via inhibition of IL-6 and IL-8. It suggested that GPER might be a potential therapy target for OS treatment.

Topics: Cell Line, Tumor; Cell Movement; Down-Regulation; Humans; Interleukin-6; Interleukin-8; Osteosarcoma; p38 Mitogen-Activated Protein Kinases; Receptors, G-Protein-Coupled; RNA, Messenger

Chemokine cysteine-X-cysteine motif ligand 8 (CXCL8) is up-regulated in many malignancies, indicating that CXCL8 takes part in tumor progression. However, the expression and function of CXCL8 in osteosarcoma remained not fully elucidated. In this study, expressions of 12 cytokines and chemokines were measured in the serum from 12 of normal controls (NCs) and 25 of osteosarcoma patients. The human osteosarcoma cell line MG-63 was stimulated by recombinant CXCL8 to further analyze invasion, proliferation, apoptosis, cell cycles, cytokine secretions, and signaling pathways. We found that serum concentrations of CXCL8 and vascular endothelial growth factor were elevated in osteosarcoma patients in comparison with those in NCs. CXCL8 stimulation led to enhancement of invasion and suppression of late stage apoptosis in MG-63 cells. Moreover, secretions of MMPs by MG-63 cells were also increased upon stimulation. However, early stage apoptosis, proliferation, and cell cycles were not affected by CXCL8 treatment. Furthermore, CXCL8 stimulation induced elevations of phosphorylated PI3K and Akt, but not PKC or FAK. In conclusion, our findings suggested that CXCL8 enhanced the invasion and suppressed late stage apoptosis of osteosarcoma cells probably via influencing PI3K/Akt signaling pathway and elevating the expression of MMPs. CXCL8 may promote disease progression of osteosarcoma as a protumorigenic molecule, and may be served as a new therapeutic target for osteosarcoma.

Topics: Adult; Apoptosis; Bone Neoplasms; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Female; Humans; Interleukin-8; Male; Neoplasm Invasiveness; Osteosarcoma; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; Signal Transduction; Vascular Endothelial Growth Factor A

Interleukin-8 (IL-8) is an angiogenic chemokine that plays a potent role in both development and progression of many human malignancies. However, there are no data about the role of IL-8 polymorphism in development of osteosarcoma. A hospital-based case-control study was conducted among 190 patients with osteosarcoma and 190 healthy controls to investigate the possible association between the IL-8 -251 A/T and +781 C/T polymorphisms, respectively, and the risk of osteosarcoma. Significant differences of genotype distribution were observed between osteosarcoma cases and controls at the IL-8 -251T/A genotypes. Compared with the IL-8 -251T/A homozygote TT, the heterozygous TA genotype was associated with significantly increased risk for osteosarcoma (odds ratio (OR) = 2.16, 95 % confidence interval (CI) = (1.38-4.52), P = 0.021); the AA genotype was associated with increased risk for osteosarcoma (OR = 1.94, 95 % CI = 1.31-3.83, P = 0.018). TA and AA combined variants were associated with increased risk for osteosarcoma compared with the TT genotype (OR = 1.72, 95 % CI = 1.45-4.41, P = 0.023). Moreover, the genotype AA of IL-8 -251T/A carried a higher risk of osteosarcoma metastasis and later Enneking stages, compared with the TT genotype. However, the genotype and allele frequencies of IL-8 +781 C/T polymorphisms in osteosarcoma patients were not significantly different from controls. Our results showed that the IL-8 -251 A/T genotype was associated with increased risk for development and metastasis of osteosarcoma in Chinese Han population.

Topics: Adolescent; Adult; Alleles; Asian People; Case-Control Studies; Child; China; Female; Genetic Association Studies; Genetic Predisposition to Disease; Genotype; Humans; Interleukin-8; Male; Neoplasm Metastasis; Neoplasm Staging; Odds Ratio; Osteosarcoma; Polymorphism, Single Nucleotide; Population Surveillance; Risk; Young Adult

p63 is a structural homolog within the 53 family encoding two isoforms, ΔNp63 and TAp63. The oncogenic activity of ΔNp63 has been demonstrated in multiple cancers, however the underlying mechanisms that contribute to tumorigenesis are poorly characterized. Osteosarcoma (OSA) is the most common primary bone tumor in dogs, exhibiting clinical behavior and molecular biology essentially identical to its human counterpart. The purpose of this study was to evaluate the potential contribution of ΔNp63 to the biology of canine OSA. As demonstrated by qRT-PCR, nearly all canine OSA cell lines and tissues overexpressed ΔNp63 relative to normal control osteoblasts. Inhibition of ΔNp63 by RNAi selectively induced apoptosis in the OSA cell lines overexpressing ΔNp63. Knockdown of ΔNp63 upregulated expression of the proapoptotic Bcl-2 family members Puma and Noxa independent of p53. However the effects of ΔNp63 required transactivating isoforms of p73, suggesting that ΔNp63 promotes survival in OSA by repressing p73-dependent apoptosis. In addition, ΔNp63 modulated angiogenesis and invasion through its effects on VEGF-A and IL-8 expression, and STAT3 phosphorylation. Lastly, the capacity of canine OSA cell lines to form pulmonary metastasis was directly related to expression levels of ΔNp63 in a murine model of metastatic OSA. Together, these data demonstrate that ΔNp63 inhibits apoptosis and promotes metastasis, supporting continued evaluation of this oncogene as a therapeutic target in both human and canine OSA.

Topics: Animals; Apoptosis; Bone Neoplasms; Cell Line, Tumor; Cell Survival; Dogs; Female; Gene Expression Regulation, Neoplastic; Gene Knockdown Techniques; Interleukin-8; Lung Neoplasms; Mice; Mice, SCID; Neoplasm Invasiveness; Neovascularization, Pathologic; Osteoblasts; Osteosarcoma; Phosphorylation; Protein Isoforms; Proto-Oncogene Proteins c-bcl-2; RNA Interference; RNA, Small Interfering; Sarcoma, Experimental; STAT3 Transcription Factor; Transcription Factors; Tumor Protein p73; Tumor Suppressor Protein p53; Tumor Suppressor Proteins; Vascular Endothelial Growth Factor A

Chemotherapy resistance is a major cause of poor prognoses for osteosarcoma patients. This study aimed to determine whether CXCR1 gene knockdown improves the sensitivity of osteosarcomas to chemotherapy. Both CXCR1 expression and cisplatin sensitivity were investigated and compared in two osteosarcoma cell lines. Sensitivity to the chemotherapy drug cisplatin and apoptosis were investigated with or without stimulation via Interleukin-8 (IL-8), which is a ligand of CXCR1. Furthermore, activation of the Akt signaling pathway was determined. Finally, luciferase-labeled CXCR1-knockdown Saos2-lung cells were injected into the tibiae of nude mice that were treated with cisplatin thereafter. We found that CXCR1 expression and cisplatin sensitivity were negatively correlated in osteosarcoma cell lines. IL-8-induced reduction in sensitivity could be blocked by silencing CXCR1, and CXCR1 knockdown suppressed the Akt signaling pathway. Moreover, CXCR1-knockdown tumors were significantly smaller than control tumors, which was consistent with the luciferase intensity results. The expression levels of IL-8, CXCR1 and p-Akt were suppressed in CXCR1-knockdown cells. Taken together, these data indicate that CXCR1 gene knockdown in osteosarcoma cells improved the sensitivity to chemotherapy and that this process might be regulated in part by the IL-8/CXCR1/Akt signaling pathway.

Topics: Animals; Apoptosis; Cell Line, Tumor; Cisplatin; Humans; Interleukin-8; Mice; Mice, Nude; Osteosarcoma; Promoter Regions, Genetic; Proto-Oncogene Proteins c-akt; Receptors, Interleukin-8A; Transfection

Osteosarcoma (OS) is the most common malignant bone tumor in patients under 20 years old. Studies have shown that cytokines play important roles in regulating immune responses in OS. In the current study, we investigated the effect of cytokines on OS by assessing serum cytokine profiles. Serum levels of 11 cytokines were measured by multiplex protein arrays in 58 patients with OS and 72 healthy controls. Results showed that serum levels of interleukin 1 receptor antagonist (IL-1Ra), IL-6, IL-8, and tumor necrosis factor-alpha (TNF-α) were significantly increased in patients than in controls (2.5-fold, 2.4-fold, 2.7-fold, and 2.1-fold, respectively). When comparing the expression of cytokines in OS patients with different clinical parameters, cases with osteoblastic subtype revealed increased level of IL-6 than patients with other subtypes (p < 0.05); cases with metastasis demonstrated significantly higher level of TNF-α than those without metastasis (p < 0.05), whereas OS patients whose tumor size were bigger than 8 cm presented elevated levels of IL-8 and TNF-α than those with small tumor size (p < 0.05 and p < 0.05, respectively). These data indicated that IL-1Ra, IL-6, IL-8, and TNF-α were associated with increased risk of OS, in which IL-8 and TNF-α may be further correlated with the progression of this disease.

Topics: Adolescent; Child; Disease Progression; Female; Gene Expression Regulation, Neoplastic; Humans; Immunity, Innate; Interleukin 1 Receptor Antagonist Protein; Interleukin-6; Interleukin-8; Male; Neoplasm Metastasis; Osteosarcoma; Tumor Necrosis Factor-alpha

Serum levels of sRANKL, RANK, OPG, IL-8, IL-6, IL-16, MMP-2, and calcitonin were measured by ELISA in patients with malignant, borderline, and benign bone tumors and in healthy individuals (control). Serum levels of RANK, OPG, IL-8, IL-6, and the OPG/sRANKL ratio were significantly higher, while the level of MMP-2 was significantly lower in patients with bone tumors than in controls. Serum concentration of IL-16 in osteosarcoma patients was significantly lower than in chondrosarcoma patients. No significant differences between bone sarcomas of different differentiation were detected for any of the studied markers. Calcitonin level depended on the tumor location and type.

Topics: Adolescent; Adult; Aged; Bone Neoplasms; Calcitonin; Case-Control Studies; Chondrosarcoma; Chordoma; Female; Gene Expression; Humans; Interleukin-16; Interleukin-6; Interleukin-8; Male; Matrix Metalloproteinase 2; Middle Aged; Neoplasms; Osteoprotegerin; Osteosarcoma; RANK Ligand; Receptor Activator of Nuclear Factor-kappa B

Chronic inflammation represents one of the hallmarks of cancer. Of special relevance to the malignant process is the pro-inflammatory cytokine IL-1 playing a crucial role in cancer-related inflammation. Recent observations indicate increased IL-1 levels in an animal model of human osteosarcoma, the most frequent primary malignant bone tumor in man. In patients with bone sarcomas, increased serum levels of tumor-promoting cytokines, including IL-6, IL-8 and VEGF can be found, correlating with poor overall survival. The link between cancer and inflammation makes it clear that there is a need to reduce the external factors inducing inflammation as a preventive or therapeutical measure. Therefore, in the present study the effects of anti-inflammatory IL-1 receptor antagonist (IL-1Ra) was tested alone and in combination with (-)-epigallocatechin-3-gallate (EGCG), an anti-inflammatory chemopreventive agent from green tea, on the production of IL-1-induced tumorigenic factors in U-2 OS human osteosarcoma cells. We found that IL-1Ra and EGCG downregulated IL-1-induced IL-6 and IL-8 release from U-2 OS cells by 65-85%. IL-1Ra and EGCG also reduced secretion of invasiveness-promoting MMP-2 and pro-angiogenic VEGF to 62-75% without affecting the metabolic response and caspase-3 activity. In conclusion, downregulation of IL-1-induced tumorigenic factors (IL-6, IL-8, VEGF, MMP-2) in U-2 OS by IL-1Ra and EGCG may positively affect tumor-associated inflammation and, as a consequence, lead to reduction in angiogenesis and invasiveness. This renders a combined administration of EGCG and IL-1Ra a promising approach as an adjuvant therapy in patients with osteosarcoma.

Topics: Antineoplastic Agents; Caspase 3; Catechin; Cell Line, Tumor; Cell Survival; Cell Transformation, Neoplastic; Drug Synergism; Enzyme Activation; Humans; Interleukin 1 Receptor Antagonist Protein; Interleukin-1; Interleukin-6; Interleukin-8; Matrix Metalloproteinase 2; Osteosarcoma; Receptors, Interleukin-1; Vascular Endothelial Growth Factor A

The lack of good preclinical models has hampered anticancer drug discovery. Standard preclinical protocols require the growth of cells in high throughput two-dimensional (2D) culture systems. However, such in vitro drug testing methods yield drug efficacy results that differ greatly from animal models. Conversely, it is much more difficult and expensive to use animal models for large-scale molecular biology research. It is conceivable that three-dimensional (3D) growth may be responsible for some of these changes. Porous silk sponges were fabricated through freeze drying and seeded with 143.98.2 osteosarcoma cells. Molecular profiles were obtained by carrying out real-time polymerase chain reaction for angiogenic growth factors and proliferation markers for osteosarcoma cells grown under 2D, 3D, and SCID mouse xenograft conditions. The angiogenic factor expression profiles for cells grown in 2D differed greatly from the 3D silk scaffold model (P < 0.05 for bFGF, HIF-1α, IL-8, and VEGF-A), whereas 3D tumor model profiles were found to be able to approximate that for the in vivo tumor better with no statistically different expression of HIF-1α and VEGF-A between the two. Immunohistochemistry staining for HIF-1α, VEGF-A, and VEGF receptor on osteosarcoma cells grown on the scaffolds validated the results obtained with the gene expression profiles. The results suggest that 3D tumor models could be used to bridge the gap between in vitro and in vivo tumor studies, and aid in the study of mechanisms activated during tumorigenesis for the development of novel targeted chemotherapy.

Topics: Animals; Cell Line, Tumor; Cell Proliferation; Female; Fibroblast Growth Factor 2; Hydrogen-Ion Concentration; Hypoxia-Inducible Factor 1, alpha Subunit; Immunohistochemistry; Interleukin-8; Mice; Mice, SCID; Microscopy, Phase-Contrast; Osteosarcoma; Oxygen; Silk; Tissue Scaffolds; Vascular Endothelial Growth Factor A

Cigarette smoking is a major risk factor in the development and further progression of periodontal diseases. However, little is known about how nicotine influences the expression of osteolytic mediators in cigarette smoking-associated periodontal diseases. The aim of this study was to investigate the expression of interleukin-1, interleukin-8, receptor activator of nuclear factor-kappaB ligand (RANKL), gelatinases and tissue-type plasminogen activator in U2OS cells (from the human osteosarcoma cell line) stimulated with nicotine.. Differences in the expression of interleukin-1, interleukin-8 and RANKL mRNAs, in response to exposure to various concentrations of nicotine (0, 0.125, 0.25, 0.5 and 1 mm) were evaluated in U2OS cells using the reverse transcription-polymerase chain reaction.In addition, the levels of interleukin-1, interleukin-8 and RANKL proteins were determined using enzyme-linked immunosorbent assays. The gelatinolytic and caseinolytic activities in nicotine treated-U2OS cells were demonstrated using gelatin and casein zymography, respectively.. Nicotine was found to increase the expression of interleukin-1, interleukin-8 and RANKL mRNA and protein in U2OS cells (p < 0.05). The gelatin zymograms revealed that matrix metalloproteinase (MMP)-2 and MMP-9 were secreted by U2OS cells. The secretion of MMP-2 and MMP-9 occurred in a dose-dependent manner that was dependent on the concentration of nicotine (p < 0.05). Casein zymography exhibited a caseinolytic band with a molecular weight of 70 kDa, indicative of the presence of tissue-type plasminogen activator. Tissue-type plasminogen activator was also found to be up-regulated by nicotine in a dose-dependent manner (p < 0.05).. Taken together, the results of the present study indicated that smoking modulation of bone destruction in periodontal disease may involve various osteolytic mediators, such as interleukin-1, interleukin-8, RANKL, MMP-2, MMP-9, and tissue-type plasminogen activator.

Topics: Caseins; Cell Line, Tumor; Dose-Response Relationship, Drug; Gelatinases; Humans; Interleukin-1; Interleukin-8; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Molecular Weight; Nicotine; Nicotinic Agonists; Osteolysis; Osteosarcoma; RANK Ligand; Time Factors; Tissue Plasminogen Activator; Up-Regulation

Pulmonary metastasis continues to be the most common cause of death in osteosarcoma. Indeed, the 5-year survival for newly diagnosed osteosarcoma patients has not significantly changed in over 20 years. Further understanding of the mechanisms of metastasis and resistance for this aggressive pediatric cancer is necessary. Pet dogs naturally develop osteosarcoma providing a novel opportunity to model metastasis development and progression. Given the accelerated biology of canine osteosarcoma, we hypothesized that a direct comparison of canine and pediatric osteosarcoma expression profiles may help identify novel metastasis-associated tumor targets that have been missed through the study of the human cancer alone.. Using parallel oligonucleotide array platforms, shared orthologues between species were identified and normalized. The osteosarcoma expression signatures could not distinguish the canine and human diseases by hierarchical clustering. Cross-species target mining identified two genes, interleukin-8 (IL-8) and solute carrier family 1 (glial high affinity glutamate transporter), member 3 (SLC1A3), which were uniformly expressed in dog but not in all pediatric osteosarcoma patient samples. Expression of these genes in an independent population of pediatric osteosarcoma patients was associated with poor outcome (p = 0.020 and p = 0.026, respectively). Validation of IL-8 and SLC1A3 protein expression in pediatric osteosarcoma tissues further supported the potential value of these novel targets. Ongoing evaluation will validate the biological significance of these targets and their associated pathways.. Collectively, these data support the strong similarities between human and canine osteosarcoma and underline the opportunities provided by a comparative oncology approach as a means to improve our understanding of cancer biology and therapies.

Topics: Animals; Cell Line, Tumor; Child; Cluster Analysis; Comparative Genomic Hybridization; Dogs; Excitatory Amino Acid Transporter 1; Gene Expression Profiling; Genomics; Humans; Interleukin-8; Oligonucleotide Array Sequence Analysis; Osteosarcoma; RNA, Neoplasm

Drug resistance continues to be a stumbling block in achieving a better cure rate in several cancers, including osteosarcoma. To understand this, we developed a doxorubicin drug-resistant osteosarcoma cell line (143B-DR-DOX). This cell line had an IC50 of 75 micromol/l compared with the parental 143B cell line's IC50 of 0.4 micromol/l. Using a 22000 70-mer oligomicroarray, gene expression studies were performed in four replicates. Data analysis was done using the TIGR Microarray suite. Seventy-four genes were found to be either upregulated (21) or downregulated (53). Real time quantitative-PCR was done on 21 genes, which confirmed the gene expression data for 11 genes. Choosing the significant fold change criteria of greater than 2-fold upregulation or downregulation, four genes including multidrug resistance 1, interleukin-8, Krüppel-like factor 2 and MGC4175 were found to be upregulated and seven genes including epidermal growth factor receptor-coamplified and overexpressed protein, uridine phosphorylase 1, a disintegrin and metalloproteinase domain 19, cytochrome C1, SEC, S-adenosyl homocysteine hydrolase and p53 were found to be downregulated. The data suggest that apart from the known gene alterations in doxorubicin resistance (multidrug resistance 1, topoisomerase IIbeta), others can also contribute to the drug-resistance phenotype. The involvement of interleukin-8 and Krüppel-like factor 2 suggests that the peroxisome proliferator-activated receptors gamma pathway may also be involved in doxorubicin drug resistance in the 143B-DR-DOX cell line.

Topics: Antibiotics, Antineoplastic; ATP Binding Cassette Transporter, Subfamily B, Member 1; Cell Line, Tumor; DNA Topoisomerases, Type II; DNA-Binding Proteins; Doxorubicin; Drug Resistance, Neoplasm; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Humans; Inhibitory Concentration 50; Interleukin-8; Kruppel-Like Transcription Factors; Oligonucleotide Array Sequence Analysis; Osteosarcoma; Polymerase Chain Reaction; PPAR gamma

Interactions between acute myelogenous leukemia (AML) blasts and non-leukemic cells in the bone marrow seem to be important for both disease development and susceptibility to chemotherapy. Recent studies have focused on the endothelial cells, but other non-leukemic cells may also be involved. In the present study we investigated how osteoblasts affect native human AML blasts.. AML cells were derived from a large group of consecutive patients. The AML blasts and osteoblastic sarcoma cell lines (Cal72, SJSA-1) were incubated together in different chambers separated by a semipermeable membrane. We investigated effects of co-culture on proliferation, apoptosis and cytokine release.. The cross-talk between these two cell populations, achieved via release of soluble mediators, resulted in increased AML blast proliferation, including increased proliferation of clonogenic progenitors, but did not affect spontaneous in vitro apoptosis. Both interleukin (IL) 1-b and granulocyte-macrophage colony-stimulating factor were involved in this growth-enhancing cross-talk, and normal osteoblasts could also increase the AML blast proliferation. Furthermore, co-culture of AML blasts with osteoblastic sarcoma cells as well as normal osteoblasts increased the levels of the pro-angiogenic mediator IL8.. Our in vitro results suggest that the release of soluble mediators by osteoblasts supports leukemic hematopoiesis through two major mechanisms: (i) direct enhancement of AML blast proliferation; and (ii) enhanced angiogenesis caused by increased IL8 levels.

Topics: Apoptosis; Cell Communication; Cell Proliferation; Coculture Techniques; Cytokines; Humans; Interleukin-8; Leukemia, Myeloid, Acute; Neovascularization, Pathologic; Osteoblasts; Osteosarcoma; Tumor Cells, Cultured

The cytokines IL-6, initially recognized as a regulator of immune and inflammatory response and IL-8, a potential regulator of angiogenesis, also regulate the growth of many tumor cells. Human cancer cells selected for multidrug resistance to common chemotherapeutic agents demonstrate increased expression of IL-6 and IL-8. To determine whether IL-6 or IL-8 overexpression contributes directly to the drug resistant phenotype, IL-6 or IL-8 cDNA were introduced into the paclitaxel sensitive human osteosarcoma cell line U-2OS using the pIRESneo bicistronic expression vector. Interleukin-6 and IL-8 transfectants were selected for either high IL-6 or IL-8 secretion and evaluated in drug resistance assays. Two IL-6 and two IL-8 secreting clones express IL-6 or IL-8 levels of 10 ng/ml and 1 ng/ml in culture, while parental U-2OS and pIRESneo vector transfected control cells express IL-6 and IL-8 levels of 0.005 ng/ml and 0.1 ng/ml, respectively. MTT cytotoxicity with IL-6 transfected cells demonstrates a five-fold increase in resistance to paclitaxel and a four-fold increase in resistance to doxorubicin as compared to U-2OS. There are no changes in mitoxantrone or topotecan resistance in the IL-6 transfectants as compared to parental U-2OS. Northern analysis of IL-6 transfectants demonstrates that the resistant phenotype is not related to increased levels of MDR-1, MRP-1, or LRP. Western analysis also confirms that P-glycoprotein levels are not altered in IL-6 transfectants. Further supporting an MDR-1 independent mechanism of drug resistance, verapamil cannot reverse paclitaxel resistance in transfected cells, findings further supported by rhodamine 123 exclusion data. Treatment of IL-6 transfected cells with paclitaxel, compared with drug-sensitive parental U-2OS, shows U-2OS(IL-6) are significantly more resistant to apoptosis induced by paclitaxel and exhibit decreased proteolytic activation of caspase-3. In contrast U-2OS(IL-8) transfectants demonstrate no appreciable increase in paclitaxel resistance when compared with parental cells. In summary, while both IL-6 and IL-8 are overexpressed in paclitaxel resistant cell lines, only IL-6 has the potential to contribute directly to paclitaxel and doxorubicin resistance in U-2OS. This resistance is through a non-MDR-1 pathway.

Topics: Antineoplastic Agents; Antineoplastic Agents, Phytogenic; ATP Binding Cassette Transporter, Subfamily B, Member 1; Blotting, Northern; Blotting, Western; Caspase 3; Caspases; Cell Division; DNA, Complementary; Dose-Response Relationship, Drug; Drug Resistance, Neoplasm; Enzyme Activation; Enzyme-Linked Immunosorbent Assay; Genetic Vectors; Humans; Interleukin-6; Interleukin-8; Mitoxantrone; Osteosarcoma; Paclitaxel; Phenotype; RNA; Tetrazolium Salts; Thiazoles; Time Factors; Topotecan; Transfection; Tumor Cells, Cultured

We have investigated both constitutive- and cytokine-induced secretion of interleukin-8 (IL-8) and its regulation by dexamethasone and 17 beta-estradiol in normal human bone marrow stromal (HBMS), osteoblast-like cells (hOB), and osteosarcoma MG-63 cells. Although HBMS cells secrete low levels of IL-8 constitutively, treatment with IL-1 beta and tumor necrosis factor-alpha (TNF-alpha) induced IL-8 secretion. Their effects were synergistic but IL-8 production was not affected by 17 beta-estradiol. Human osteosarcoma MG-63 cells also secreted low levels of IL-8 constitutively; the production was induced by IL-1 beta and TNF-alpha and was also not affected by 17 beta-estradiol. The magnitude of the response to cytokine stimulation of IL-8 in MG-63 cells was much lower than that of HBMS and hOB cells, indicating differences in response in normal and osteoblastic osteosarcoma cells. Dexamethasone (10(-7) M) significantly inhibited IL-1 beta plus TNF-alpha stimulated IL-8 production in HBMS, MG-63, and hOB cells. The accumulated results demonstrate that IL-8 is secreted by HBMS, MG-63, and hOB cells, suggesting that IL-8 may play a role in the regulation of bone cell function. These data also emphasize the importance of glucocorticoids in controlling cytokine secretion in HBMS, hOB, and MG-63 cells.

Topics: Bone Marrow; Bone Marrow Cells; Bone Neoplasms; Cells, Cultured; Culture Media, Conditioned; Dexamethasone; Drug Synergism; Enzyme-Linked Immunosorbent Assay; Estradiol; Humans; Interleukin-1; Interleukin-8; Osteoblasts; Osteosarcoma; Ribs; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha

Tumor cells are capable of simultaneously producing a number of related inflammatory peptides, now classified as chemokines. We have isolated a new human granulocyte chemotactic protein (GCP-2), coproduced with interleukin-8 (GCP-1/IL-8) by osteosarcoma cells. Furthermore, the bovine homologue of human GCP-2 was purified from kidney tumor cells using the same isolation procedure. Both chemokines occur in at least four NH2-terminally truncated forms. These 5-6 kDa proteins do not differ in potency and efficacy as granulocyte chemotactic factors using a standard in vitro migration assay. The complete primary structures of human and bovine GCP-2 were disclosed by sequencing peptide fragments derived from the natural proteins. On the basis of the conservation of four cysteine residues, the two molecules are to be classified within the C-X-C chemokine family, including IL-8. Human and bovine GCP-2 are 67% similar at the amino acid level. Their sequences show only weak similarity with that of IL-8, and human GCP-2 does not cross-react in a radioimmunoassay for IL-8. Human and bovine GCP-2 are specific granulocyte chemotactic factors in that they do not attract human monocytes. Bovine GCP-2 is not species specific since it is at least as active as human GCP-2 on human granulocytes. Both chemokines can also activate postreceptor mechanisms leading to release of gelatinase B by granulocytes. This is indicative for a possible role in inflammation and tumor cell invasion.

Topics: Amino Acid Sequence; Animals; Cattle; Cell Line; Chemokine CXCL6; Chemokines, CXC; Chemotaxis, Leukocyte; Chromatography, High Pressure Liquid; Cytokines; Granulocytes; Humans; In Vitro Techniques; Interleukin-8; Molecular Sequence Data; Monocytes; Osteosarcoma; Sequence Homology, Amino Acid; Tumor Cells, Cultured

Stimulated human osteosarcoma cells (MG-63) were used as a source of granulocyte chemotactic protein (GCP). In addition to the previously isolated GCP-1/IL-8, natural forms of GRO alpha, GRO gamma, and IP-10 were purified and identified by amino acid sequence analysis. Further, a novel GCP, GCP-2, was isolated in its natural form (6 kDa) and was found to be structurally related to the other members of the IL-8 family. GRO alpha, IP-10, and GCP-2 showed heterogeneity, in that several forms of each protein were recovered. These differed in truncation at the amino terminus. Reverse phase HPLC allowed us to separate four such different forms of GCP-2. These tumor-derived factors were compared in granulocyte activation and chemotaxis assays. IL-8 induced neutrophil gelatinase B release at 2 nM, but GRO alpha and GCP-2 showed a 5- to 10-fold lower specific activity. When the migration of granulocytes through polycarbonate micropore membranes was measured, GCP-2 and GRO alpha had a maximal chemotactic index comparable to that of IL-8. The minimal effective dose for GCP-2 and GRO alpha was 3 to 10 nM, whereas the specific activity of IL-8 was at least 10-fold higher. IP-10 was not active in this assay at doses up to 100 nM. Finally, in vivo chemotaxis was measured by using granulocyte recruitment in the rabbit skin model. After intradermal injection of 200 ng/site, GCP-2 provoked a significant granulocyte infiltration, albeit to a lesser extent than did IL-8 and GRO alpha. GCP-2 did not attract monocytes in vivo nor did it induce the cells in vitro to migrate or to produce enzyme. In conclusion, this study reveals a new member of the IL-8 family and shows that these related inflammatory mediators possess different potencies and efficacies towards granulocytes.

Topics: Amino Acid Sequence; Animals; Chemokine CXCL10; Chemokines, CXC; Chemotactic Factors; Chemotaxis, Leukocyte; Cytokines; Granulocytes; Humans; Interleukin-8; Molecular Sequence Data; Osteosarcoma; Rabbits; Tumor Cells, Cultured