tretinoin and Bone-Neoplasms

The evaluation of new therapies in prostate cancer requires unique end points for agents with diverse mechanisms of action. Because retinoic acid may have a confounding effect on prostate-specific antigen, we incorporated a pathological end point into the outcome assessment of two sequential clinical trials using all-trans-retinoic acid (ATRA) and the combination of 13-cis-retinoic acid and IFN-2a (cRA¿IFN). Pre- and posttherapy tumor biopsy specimens were studied for histological changes, apoptosis (terminal deoxynucleotidyl transferase-mediated nick end labeling assay), and proliferation index (Ki67). Prostate-specific membrane antigen (PSMA) expression was also evaluated using two different monoclonal antibodies to its intracellular domain (Cytogen 7E11 and Hybritech PM2). Fourteen patients with androgen-independent disease were treated with ATRA (50 mg/m2 p.o. every 8 h daily) and 16 androgen-independent and 4 androgen-dependent patients were treated with cRA¿IFN (10 mg/kg/day cRA plus 3, 6, or 9 million units daily IFN). Both therapies were well tolerated, with fatigue and cheilitis being the most common adverse events. Clinical activity, assessed by radiographs and serum prostate-specific antigen, was minimal, and the majority of patients progressed within 3 months. One patient with androgen-dependent disease had prolonged stabilization for >1 year. The majority of cases (95%) showed no gross histological changes and no difference in apoptotic or proliferative indices. Increased PSMA immunoreactivity was seen in seven of nine (78%) cases using PM2 antibody and in two of nine (22%) cases using the 7E11 antibody. Although antitumor effects were modest, the results suggest a role for retinoids in modulating the expression of PSMA on prostate cancer cells.

Topics: Aged; Antigens, Surface; Antineoplastic Agents; Antineoplastic Combined Chemotherapy Protocols; Biopsy; Bone Neoplasms; Carboxypeptidases; Cheilitis; Dyspnea; Exanthema; Fatigue; Glutamate Carboxypeptidase II; Hematologic Diseases; Humans; Interferon alpha-2; Interferon-alpha; Ki-67 Antigen; Liver; Male; Middle Aged; Prostate; Prostate-Specific Antigen; Prostatic Neoplasms; Recombinant Proteins; Transaminases; Treatment Outcome; Tretinoin

The United States Environmental Protection Agency has proposed a tiered testing strategy for chemical hazard evaluation based on new approach methods (NAMs). The first tier includes in vitro profiling assays applicable to many (human) cell types, such as high-throughput transcriptomics (HTTr) and high-throughput phenotypic profiling (HTPP). The goals of this study were to: (1) harmonize the seeding density of U-2 OS human osteosarcoma cells for use in both assays; (2) compare HTTr- versus HTPP-derived potency estimates for 11 mechanistically diverse chemicals; (3) identify candidate reference chemicals for monitoring assay performance in future screens; and (4) characterize the transcriptional and phenotypic changes in detail for all-trans retinoic acid (ATRA) as a model compound known for its adverse effects on osteoblast differentiation. The results of this evaluation showed that (1) HTPP conducted at low (400 cells/well) and high (3000 cells/well) seeding densities yielded comparable potency estimates and similar phenotypic profiles for the tested chemicals; (2) HTPP and HTTr resulted in comparable potency estimates for changes in cellular morphology and gene expression, respectively; (3) three test chemicals (etoposide, ATRA, dexamethasone) produced concentration-dependent effects on cellular morphology and gene expression that were consistent with known modes-of-action, demonstrating their suitability for use as reference chemicals for monitoring assay performance; and (4) ATRA produced phenotypic changes that were highly similar to other retinoic acid receptor activators (AM580, arotinoid acid) and some retinoid X receptor activators (bexarotene, methoprene acid). This phenotype was observed concurrently with autoregulation of the RARB gene. Both effects were prevented by pre-treating U-2 OS cells with pharmacological antagonists of their respective receptors. Thus, the observed phenotype could be considered characteristic of retinoic acid pathway activation in U-2 OS cells. These findings lay the groundwork for combinatorial screening of chemicals using HTTr and HTPP to generate complementary information for the first tier of a NAM-based chemical hazard evaluation strategy.

Topics: Bone Neoplasms; Humans; Phenotype; Receptors, Retinoic Acid; RNA-Seq; Tretinoin; United States

Metastatic prostate cancer in the bone induces bone-forming lesions that contribute to progression and therapy resistance. Prostate cancer-induced bone formation originates from endothelial cells (EC) that have undergone endothelial-to-osteoblast (EC-to-OSB) transition in response to tumor-secreted BMP4. Current strategies targeting prostate cancer-induced bone formation are lacking. Here, we show that activation of retinoic acid receptor (RAR) inhibits EC-to-OSB transition and reduces prostate cancer-induced bone formation. Treatment with palovarotene, an RARγ agonist being tested for heterotopic ossification in fibrodysplasia ossificans progressiva, inhibited EC-to-OSB transition and osteoblast mineralization in vitro and decreased tumor-induced bone formation and tumor growth in several osteogenic prostate cancer models, and similar effects were observed with the pan-RAR agonist all-trans-retinoic acid (ATRA). Knockdown of RARα, β, or γ isoforms in ECs blocked BMP4-induced EC-to-OSB transition and osteoblast mineralization, indicating a role for all three isoforms in prostate cancer-induced bone formation. Furthermore, treatment with palovarotene or ATRA reduced plasma Tenascin C, a factor secreted from EC-OSB cells, which may be used to monitor treatment response. Mechanistically, BMP4-activated pSmad1 formed a complex with RAR in the nucleus of ECs to activate EC-to-OSB transition. RAR activation by palovarotene or ATRA caused pSmad1 degradation by recruiting the E3-ubiquitin ligase Smad ubiquitination regulatory factor1 (Smurf1) to the nuclear pSmad1/RARγ complex, thus blocking EC-to-OSB transition. Collectively, these findings suggest that palovarotene can be repurposed to target prostate cancer-induced bone formation to improve clinical outcomes for patients with bone metastasis.. This study provides mechanistic insights into how RAR agonists suppress prostate cancer-induced bone formation and offers a rationale for developing RAR agonists for prostate cancer bone metastasis therapy. See related commentary by Bhowmick and Bhowmick, p. 2975.

Topics: Bone Neoplasms; Endothelial Cells; Humans; Male; Osteoblasts; Prostatic Neoplasms; Receptors, Retinoic Acid; Tretinoin; Ubiquitin-Protein Ligases

Osteosarcoma (OS) is the most common primary malignant bone tumor in adolescents. The molecular mechanism behind OS progression and metastasis remains poorly understood, which limits the effectiveness of current therapies. RNA N. Liquid chromatography-tandem mass spectrometry (LC-MS/MS), dot blotting, and colorimetric ELISA were used to detect m. We observed the abundance of m. Our study revealed that METTL14 contributes to OS progression and ATRA resistance as an m. This work was supported by the National Natural Science Foundation of China (Grants 81972510 and 81772864).

Topics: Adolescent; Bone Neoplasms; Cell Line, Tumor; Chromatography, Liquid; Humans; Methyltransferases; Osteosarcoma; RNA; RNA-Binding Proteins; RNA, Messenger; Tandem Mass Spectrometry; Trans-Activators; Tretinoin; Tumor Suppressor Proteins

The prognosis of patients with osteosarcoma in many cases remains poor, and life expectancy with lung metastases is around 12 months. Chemotherapy and radiotherapy can only temporarily control neoplastic progression, followed by developing chemo and radioresistant tumours.. This is a retrospective observational study on 15 patients diagnosed with osteosarcoma and treated by a multitherapy approach. The multitherapy consisted of somatostatin and analogous (octreotide) all-trans-retinoic acid (ATRA), β-Carotene, axerophthol dissolved in vitamin E, vitamin D, vitamin C, melatonin (MLT), proteoglycans, glycosaminoglycans, hydroxyurea, and sodium butyrate.. This multitherapy increased the survival rate and life quality, without overt toxicity, compared to the standard treatment for osteosarcomas. The agents in this approach have several functions. They exert antiproliferative, antiangiogenic, cytostatic, antioxidant, antimetastatic, and immunomodulating features. Moreover, the inclusion of ATRA, MLT, and sodium butyrate has reinforced antitumor properties on cancer stem cells. Furthermore, the non-cytolytic and non-cytotoxic metronomic hydroxyurea dosage increased the biological therapy outcome by strengthening antitumor capability.. This multitherapy approach is effective against osteosarcoma.. The multistrategy of this multitherapy therapy are inhibiting the proliferative-invasiveness and neoplastic angiogenesis, silencing the survival system of cancer stem cells, enhancing the immunomodulatory and antioxidant activities, improving vitality and efficiency of normal cells, and depressing the efficiency and vitality of neoplastic ones.

Topics: Antioxidants; Bone Neoplasms; Butyric Acid; Humans; Hydroxyurea; Melatonin; Osteosarcoma; Tretinoin

Osteosarcoma, a common type of bone cancer in children, and represents an aggressive and fetal cancer worldwide. Osteosarcoma initiating cells are considered to be a subpopulation of cancer cells which contribute to the progression, recurrence, metastasis and multi-drug resistance of osteosarcoma. CD133 is considered to be one marker for osteosarcoma initiating cells. All-trans retinoic acid (ATRA), an active metabolite of vitamin A under the family retinoid, is an up-and-coming drug which was able to effectively treat various cancer initiating cells. Nevertheless, there have been no research that reported the activity of ATRA against osteosarcoma initiating cells. In this research, we hereby examined the potential activity of ATRA in osteosarcoma initiating cells, and developed lipid-polymer nanoparticles with CD133 aptamers for targeted ATRA delivery to osteosarcoma initiating cells. Using the cytotoxicity assay, colony formation assay, tumorsphere formation assay and flow cytometry, the therapeutic effect of ATRA and ATRA-loaded lipid-polymer nanoparticles conjugated with CD133 aptamers (ATRA-PLNP-CD133) against osteosarcoma initiating cells were investigated. The results showed that ATRA exerted potent activity towards osteosarcoma initiating cells. ATRA-PLNP-CD133, which showed a size of 129.9 nm and a sustained release of ATRA during 144 h, was demonstrated to efficiently and specifically promote the ATRA delivery to osteosarcoma initiating cells, and achieve superior therapeutic efficacy in osteosarcoma compared with ATRA and non-targeted nanoparticles. This is the first report of the therapeutic efficacy of ATRA towards osteosarcoma initiating cells, and the increased ATRA delivery by nanoparticles to osteosarcoma initiating cells using CD133 aptamers. ATRA-PLNP-CD133 represent an up-and coming approach for the therapy of osteosarcoma initiating cells.

Topics: AC133 Antigen; Animals; Antineoplastic Agents; Aptamers, Nucleotide; Bone Neoplasms; Dose-Response Relationship, Drug; Drug Delivery Systems; Humans; Lipids; Male; Mice; Mice, Inbred BALB C; Mice, Nude; Nanoparticles; Osteosarcoma; Polymers; Tretinoin; Xenograft Model Antitumor Assays

Retinoic acid receptor alpha (RARα) has a critical role in the differentiation process of osteosarcoma cells induced by all-trans retinoic acid (ATRA). However, degradation of RARα through ubiquitin proteasome pathway weakens the differentiation efficiency of osteosarcoma cells. In this study, we discover that murine double minute-2 (MDM2) acts as an E3 ubiquitin ligase to target RARα for degradation. We observe that MDM2 is required for RARα polyubiquitination and proteasomal degradation because downregulation of MDM2 by short hairpin RNA results in the accumulation of RARα, and MDM2 overexpression promotes the degradation of RARα. We also demonstrate that the N-terminal domain of MDM2 (amino acids 1-109) is the major RARα-binding site. Importantly, endogenous MDM2 levels are not only upregulated in human primary osteosarcoma blasts but are also inversely correlated with the level of osteopontin, which is a marker of bone differentiation. Moreover, MDM2 impairs the ATRA-induced osteoblastic differentiation of osteosarcoma cells, whereas an inhibitor of the MDM2 ubiquitin ligase synergizes with ATRA to enhance the differentiation of osteosarcoma cells and primary osteosarcoma blasts. Therefore, our study indicates that MDM2 serves as an E3 ubiquitin ligase to regulate the degradation of RARα and suggests that MDM2 is a novel therapeutic target for ATRA-based differentiation therapeutic approaches in osteosarcoma.

Topics: Antineoplastic Agents; Bone Neoplasms; Cell Differentiation; Cell Line, Tumor; Humans; Osteoblasts; Osteosarcoma; Proto-Oncogene Proteins c-mdm2; Retinoic Acid Receptor alpha; Tretinoin

The induced differentiation of tumor cells into mature phenotypes is a promising strategy in cancer therapy. In this study, the effects of combined treatment with all-trans retinoic acid (ATRA) and lipoxygenase/cyclooxygenase inhibitors were examined in two osteosarcoma cell lines, Saos-2 and OSA-01. Caffeic acid and celecoxib were used as inhibitors of 5-lipoxygenase and of cyclooxygenase-2, respectively. Changes in the cell proliferation, matrix mineralization, and occurrence of differentiation markers were evaluated in treated cell populations at intervals. The results confirmed the capability of caffeic acid to enhance the antiproliferative effect of ATRA in both cell lines. In contrast, celecoxib showed the same effect in Saos-2 cells only. Furthermore, the extension of matrix mineralization was observed after combined treatment with ATRA and celecoxib or caffeic acid. The increased expression of osteogenic differentiation markers was observed in both cell lines after the combined application of ATRA and inhibitors. The obtained results clearly demonstrate the capability of lipoxygenase/cyclooxygenase inhibitors to enhance the antiproliferative and differentiating effect of ATRA in osteosarcoma cells, although some of these effects are specific and depend on the biological features of the respective tumor or cell line.

Topics: Antineoplastic Agents; Bone Neoplasms; Caffeic Acids; Celecoxib; Cell Differentiation; Cell Line, Tumor; Cell Proliferation; Cyclooxygenase Inhibitors; Humans; Lipoxygenase Inhibitors; Osteosarcoma; Pyrazoles; Sulfonamides; Tretinoin

Topics: Adult; Aminoglycosides; Antibodies, Monoclonal; Antibodies, Monoclonal, Humanized; Antineoplastic Agents; Antineoplastic Combined Chemotherapy Protocols; Arsenic Trioxide; Arsenicals; Benzoates; Bone Marrow; Bone Neoplasms; Combined Modality Therapy; Doxorubicin; Drug Resistance, Neoplasm; Gemtuzumab; Hematopoietic Stem Cell Transplantation; Humans; Idarubicin; Leukemia, Promyelocytic, Acute; Leukemic Infiltration; Male; Mercaptopurine; Methotrexate; Oxides; Radionuclide Imaging; Remission Induction; Salvage Therapy; Sarcoma, Myeloid; Soft Tissue Neoplasms; Tetrahydronaphthalenes; Tretinoin

Osteosarcoma is the most common primary malignancy of bone. The long-term survival of osteosarcoma patients hinges on our ability to prevent and/or treat recurrent and metastatic lesions. Here, we investigated the activation of peroxisome proliferator-activated receptor gamma (PPARgamma) and retinoid receptors as a means of differentiation therapy for human osteosarcoma.. We examined the endogenous expression of PPARgamma and retinoid receptors in a panel of osteosarcoma cells. Ligands or adenovirus-mediated overexpression of these receptors were tested to inhibit proliferation and induce apoptosis of osteosarcoma cells. Osteosarcoma cells overexpressing the receptors were introduced into an orthotopic tumor model. The effect of these ligands on osteoblastic differentiation was further investigated.. Endogenous expression of PPARgamma and isotypes of retinoic acid receptor (RAR) and retinoid X receptor (RXR) is detected in most osteosarcoma cells. Troglitazone, 9-cis retinoic acid (RA), and all-trans RA, as well as overexpression of PPARgamma, RARalpha, and RXRalpha, inhibit osteosarcoma cell proliferation and induce apoptosis. A synergistic inhibitory effect on osteosarcoma cell proliferation is observed between troglitazone and retinoids, as well as with the overexpression pairs of PPARgamma/RARalpha, or PPARgamma/RXRalpha. Overexpression of PPARgamma, RARalpha, RXRalpha, or in combinations inhibits osteosarcoma tumor growth and cell proliferation in vivo. Retinoids (and to a lesser extent, troglitazone) are shown to promote osteogenic differentiation of osteosarcoma cells and mesenchymal stem cells.. Activation of PPARgamma, RARalpha, and RXRalpha may act synergistically on inhibiting osteosarcoma cell proliferation and tumor growth, which is at least partially mediated by promoting osteoblastic differentiation of osteosarcoma cells.

Topics: Adenoviridae; Animals; Antineoplastic Agents; Apoptosis; Blotting, Western; Bone Neoplasms; Cell Differentiation; Cell Line, Tumor; Cell Proliferation; Drug Synergism; Humans; Luciferases; Mesenchymal Stem Cells; Mice; Osteosarcoma; PPAR gamma; Receptors, Retinoic Acid; Retinoic Acid Receptor alpha; Retinoid X Receptors; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Tretinoin

To identify potential biomarkers of therapy response, we have previously done a large-scale gain-of-function genetic screen to identify genes whose expression confers resistance to histone deacetylase inhibitors (HDACI). This genetic screen identified two genes with a role in retinoic acid signaling, suggesting that HDACIs target retinoic acid signaling as part of their anticancer effect. We study here a third gene identified in this genetic screen, UNC45A, and assess its role in retinoic acid signaling and responses to HDACIs using cell-based proliferation and differentiation assays and transcriptional reporter gene assays. The vertebrate Unc45 genes are known for their roles in muscle development and the assembly and cochaperoning of the muscle motor protein myosin. Here, we report that human UNC45A (GCUNC45) can render transformed cells resistant to treatment with HDACIs. We show that UNC45A also inhibits signaling through the retinoic acid receptor alpha. Expression of UNC45A inhibits retinoic acid-induced proliferation arrest and differentiation of human neuroblastoma cells and inhibits the induction of endogenous retinoic acid receptor target genes. These data establish an unexpected role for UNC45A in causing resistance to both HDACI drugs and retinoic acid. Moreover, our data lend further support to the notion that HDACIs exert their anticancer effect, at least in part, through an effect on retinoic acid signaling.

Topics: Amino Acid Sequence; Animals; Bone Neoplasms; Cell Differentiation; Cell Growth Processes; Cell Line, Tumor; Drug Resistance, Neoplasm; Histone Deacetylase Inhibitors; Histone Deacetylases; Humans; Intracellular Signaling Peptides and Proteins; Mice; Molecular Sequence Data; Neuroblastoma; Osteosarcoma; Receptors, Retinoic Acid; Retinoic Acid Receptor alpha; Signal Transduction; Tretinoin

While tissue-nonspecific alkaline phosphatase (TNSALP) is a well-known indicator of bone formation and all-trans-retinoic acid a key regulator of that process, the relationship between TNSALP and retinoic acid has not yet been clearly described. The aim of the present study was therefore to clarify the mechanism by which retinoic acid modulates expression of TNSALP. After culturing SaOS-2 human osteoblastic osteosarcoma cells in the presence or absence of 10(-6) M all-trans-retinoic acid, real-time RT-PCR confirmed that retinoic acid up-regulates expression of TNSALP mRNA. Notably, this time-dependent induction of TNSALP expression was accompanied by a corresponding increase in detected catalytic activity of the enzyme. When we then isolated the 5'-upstream region of the human TNSALP gene and carried out luciferase assays with a set of deletion mutants, we found that the promoter region contains a retinoic acid response element-like motif. Moreover, electrophoretic mobility shift assays showed that the nuclear extract bound to the motif. It thus appears that retinoic acid regulates the expression of human TNSALP via a retinoic acid response element in the genes promoter region.

Topics: Alkaline Phosphatase; Base Sequence; Bone Neoplasms; Cell Line, Tumor; Cloning, Molecular; DNA, Neoplasm; Electrophoretic Mobility Shift Assay; Gene Expression Regulation, Enzymologic; Humans; Molecular Sequence Data; Osteosarcoma; Plasmids; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Transcription, Genetic; Transfection; Tretinoin

In this report, we studied the relationship between telomerase activity and in vitro differentiation of osteosarcoma cells. Human osteosarcoma cells (HOS-8603) were treated with all-trans-retinoic acid (RA) and dexamethasone (DEX). Cell cycle phase, alkaline phosphatase (AP) activity, telomerase activity, and human telomerase RNA (hTR) in treated cells were detected. The results showed that the treated cells underwent morphologic differentiation. AP activity of the cells increased significantly. The proportion of the cells in S and G2/M phases was increased. A pronounced decline in telomerase activity was observed, but no significant difference in the amount of hTR expressed, when compared with the control. This study demonstrates that: (1) both RA and DEX can inhibit cell growth and induce morphologic and functional differentiation of HOS-8603 cells; (2) telomerase is an enzyme system regulated during induced differentiation of HOS-8603 cells; (3) significantly decreased telomerase activity may be an indicator of differentiation but does not parallel the expression level of hTR; and (4) the regulation of telomerase is directly linked to cell differentiation not cell cycle.

Topics: Alkaline Phosphatase; Blotting, Northern; Bone Neoplasms; Cell Cycle; Cell Differentiation; Cell Division; Dexamethasone; Dose-Response Relationship, Drug; Down-Regulation; Humans; Osteosarcoma; RNA, Messenger; Telomerase; Tretinoin; Tumor Cells, Cultured

To determine differentiation and growth inhibition effects of retinoids on canine osteosarcoma cells.. 3 osteosarcoma cell lines established from osteosarcomas in dogs.. Osteosarcoma cells were incubated with various concentrations of all-trans-retinoic acid and 9-cis-retinoic acid or control medium, counted daily for 10 days, and evaluated for morphologic changes. Synthesis of DNA was measured by use of a cell proliferation ELISA. To analyze effect of retinoids on colony formation on plastic dishes, cells were cultured for 14 days, fixed, and stained; number of colonies was counted.. In a dose-dependent manner, both retinoids induced morphologic differentiation and growth inhibition in the 3 osteosarcoma cell lines and inhibited each cell's ability to form anchorage-dependent colonies.. Retinoids induced differentiation of osteosarcoma cells of dogs, resulting in altered expression of their malignant phenotype. Induction of differentiation by retinoids may have potential as an adjunctive treatment for osteosarcoma in dogs.

Topics: Alitretinoin; Animals; Antimetabolites; Antineoplastic Agents; Bone Neoplasms; Bromodeoxyuridine; Cell Division; DNA; Dog Diseases; Dogs; Dose-Response Relationship, Drug; Enzyme-Linked Immunosorbent Assay; Microscopy, Phase-Contrast; Osteosarcoma; Tretinoin; Tumor Cells, Cultured

The effect of two retinoids, all- trans and 9- cis retinoic acid, on the differentiation of three canine osteosarcoma cells (OOS, HOS, and POS) was examined using markers specifically expressed by phenotypic osteoblasts. Both retinoids induced morphologic differentiation in all the canine osteosarcoma cells. Retinoids enhanced cell flattening and spreading, as well as reduction in cell overlapping. Alkaline phosphatase (ALP) activity and ALP staining was enhanced in OOS, and HOS cells, but decreased in POS cells. These results may suggest that OOS and HOS cells have immature osteoblastic properties and POS cells have mature osteoblastic properties. Retinoids decreased osteocalcin production in all the osteosarcoma cells. They induced an increase in production of type I collagen in HOS and POS cells, but a decrease in OOS cells. These results indicate that retinoids induce differentiation of canine osteosarcoma cells, resulting in an altered expression of their malignant phenotype.

Topics: Alitretinoin; Alkaline Phosphatase; Animals; Antineoplastic Agents; Bone Neoplasms; Cell Differentiation; Collagen; Dog Diseases; Dogs; Osteocalcin; Osteosarcoma; Tretinoin; Tumor Cells, Cultured

Pulmonary metastasis is a major cause of death and a major obstacle to the successful treatment of canine osteosarcoma. However, the residual capacity of the neoplasia for differentiation and its susceptibility to undergo apoptosis may be used to suppress its growth and metastatic properties. The highly metastasizing POS (HMPOS) canine osteosarcoma cell line which preferentially metastasize to the lungs was used to test the possible efficacy of 22-oxa-calcitriol (OCT) and all- trans retinoic acid (ATRA) to inhibit growth and pulmonary metastasis of the subcutaneously grown osteosarcoma in nude mice. Treatments in vitro, morphologically elongated and increased alkaline phosphatase activity and staining of cells. Tumour growth in vivo was inhibited significantly and the combination treatment of OCT and ATRA (OCT + ATRA) exerted a synergistic and stronger suppression at concentration of 1.0 microg kg(-1)body weight when given subcutaneously three times a week for 5 weeks. The subcutaneous tumours of the control mice consisted of osteoblast-like cells and isolated chondroblast-like cells, but formed several areas of osteoid and increased amount of collagen tissue in all treated mice. Pinpoint macrometastatic nodules developed only in all control mice. Micrometastatic nodule developed only in two of six mice treated with ATRA. However, nodule size and number, and lung wet weight were all reduced significantly. Metastasis were not seen in the mice treated with OCT or OCT + ATRA. This study demonstrated that inhibition of growth and pulmonary metastasis was induced by subcutaneous treatment with these drugs and suggest that both its differentiating and apoptotic inducing activities may be responsible for the antitumour effects. These drugs may be useful in the clinic as an adjunct for the treatment of canine osteosarcoma.

Topics: Alkaline Phosphatase; Animals; Antineoplastic Agents; Biomarkers; Bone Neoplasms; Calcitriol; Cell Division; Dog Diseases; Dogs; Female; Lung Neoplasms; Mice; Mice, Nude; Osteosarcoma; Transplantation, Heterologous; Tretinoin; Tumor Cells, Cultured

To determine effects of all-trans and 9-cis retinoic acid (RA) on tumor growth and metastatic ability of canine osteosarcoma cells transplanted into athymic (nude) mice.. Forty-five 5-week-old female BALB/c nude mice.. 1 X 10(7) POS osteosarcoma cells were transplanted subcutaneously into the intrascapular region of mice. All-trans RA (3 or 30 microg/kg of body weight in 0.1 ml of sesame oil), 9-cis RA (3 or 30 mg/kg in 0.1 ml of sesame oil), or sesame oil (0.1 ml; control treatment) were administered intragastrically 5 d/wk for 4 weeks beginning 3 days after transplantation (n = 4 mice/group) or after formation of a palpable tumor (5 mice/group). Tumor weight was estimated weekly by measuring tumor length and width, and retinoid toxic effects were evaluated daily. Two weeks after the final treatment, mice were euthanatized, and number of mice with pulmonary metastases was determined.. Adverse treatment effects were not detected. Tumor weight was less in mice treated with either dose of 9-cis RA than in control mice, although this difference was not significant. Treatment with 30 mg of 9-cis RA/kg initiated after tumor formation significantly reduced the incidence of pulmonary metastasis, compared with the control group.. 9-cis RA decreased the incidence of pulmonary metastasis in nude mice transplanted with canine osteosarcoma cells and may be a potential adjunct therapy for treatment of osteosarcoma in dogs.

Topics: Alitretinoin; Animals; Antineoplastic Agents; Bone Neoplasms; Dog Diseases; Dogs; Female; Lung Neoplasms; Mice; Mice, Inbred BALB C; Mice, Nude; Neoplasm Metastasis; Neoplasm Transplantation; Osteosarcoma; Transplantation, Heterologous; Tretinoin; Tumor Cells, Cultured

The effects of 22-oxacalcitriol (OCT), calcitriol and all-trans retinoic acid (ATRA) on the induction of functional differentiation and growth inhibition of the canine osteosarcoma cell line POS were investigated in vitro via bone differentiation markers and proliferation assays, respectively. The intracellular alkaline phosphatase (ALP) activity and the gamma-carboxyglutamic acid osteocalcin (GLA-OC) and procollagen type I C peptide (PIP) production were used as markers of differentiation. Treatment with 10(-8) M concentrations of all drugs for 72 h significantly inhibited growth (P < 0.0001) and increased ALP activity and GLA-OC and PIP production in POS. OCT, calcitriol and ATRA significantly increased the: ALP activity from 1.58 +/- 0.14 mumol/min/mg protein (mean +/- SD; control) to 2.50 +/- 0.09 (P < 0.0001), 2.30 +/- 0.14 (P < 0.0001) and 2.00 +/- 0.14 (P = 0.0008), respectively; GLA-OC production from 0.71 +/- 0.01 ng/ml (control) to 2.87 +/- 0.01 (P < 0.0001), 2.87 +/- 0.11 (P < 0.0001) and 1.36 +/- 0.06 (P < 0.0001), respectively; and PIP production from 433.91 +/- 23.29 ng/ml (control) to 536.54 +/- 15.46 (P = 0.0002), 497.06 +/- 1.99 (P = 0.0028) and 481.66 +/- 0.01 (P = 0.0104), respectively. This study demonstrated that treatment with these drugs induced a phenotypic maturation of POS cells into cells that exhibit the properties of functionally mature bone cells with parallel growth inhibition. The effects of these drugs on functional differentiation may be useful to complement the progression of a normal osteogenic differentiation process in the sarcoma.

Topics: Alkaline Phosphatase; Animals; Antineoplastic Agents; Bone Neoplasms; Calcitriol; Dog Diseases; Dogs; Osteocalcin; Osteosarcoma; Peptide Fragments; Procollagen; Tretinoin; Tumor Cells, Cultured

The enhanced expression of the regulatory subunit of cyclic AMP (cAMP)-dependent protein kinase type I, RIalpha, has been correlated with cancer cell growth. Retinoic acid (RA) has been shown to play an important role in the regulation of proliferation and differentiation in neoplastic cells. In the present study, the effects of cAMP analogue 8-chlorocyclic-AMP (8-Cl-cAMP) and RA (both singly and combined) on growth inhibition and apoptosis in Ewing's sarcoma CHP-100 cells were evaluated. The inhibitory effects of 8-Cl-cAMP and RA (9-cis-RA, 13-cis-RA, and all-trans-RA) on cell viability were time and dose related. The degree of growth inhibition induced by 9-cis-RA was the greatest among all of the RA analogues (13-cis-RA and all-trans-RA) examined. The combined effects of 8-Cl-cAMP and RA on the induction of growth arrest at the G0-G1 stage of the cell cycle, apoptosis, down-regulation of RIalpha, and cleavage of poly(ADP-ribose) polymerase were synergistic. In conclusion, it is clear that RA and 8-Cl-cAMP act in a synergistic fashion and have potential for combination chemotherapy for the treatment of malignant disease.

Topics: 8-Bromo Cyclic Adenosine Monophosphate; Antineoplastic Agents; Apoptosis; Bone Neoplasms; Cell Cycle; Cell Division; Cell Survival; Drug Synergism; Enzyme-Linked Immunosorbent Assay; Humans; Nucleosomes; Sarcoma, Ewing; Tretinoin; Tumor Cells, Cultured

Tumors are heterogeneous in terms of morphology and susceptibility to drugs or radiation. Among primary and metastatic cells of a human renal carcinoma, a population (type II) of larger cells with prominent nucleoli, eosinophilic globules of intermediate filaments in paranuclear bundles, margination of subcellular organelles and peripheral pools of glycogen was evident. Paranuclear structures were recognized by monoclonal antibodies specific for cytokeratin 8, 18 and 19, but not by vimentin specific antibodies. We propagated a cell line in vitro (referred to as BKR cells), and observed culture in vitro, the almost complete disappearance of the type II cells. Pharmacological agents that influence cell differentiation, such as retinoic acid, rescued the expression of type II cells in vitro. Long-term treatments with insulin or alpha-interferon, but not with the epithelial growth factor (EGF), similarly differentiated BKR cells and abated their susceptibility to spontaneous and actinomycin-D induced apoptosis. These data support the contention that differentiation of tumor cells is actively maintained in vivo and further strengthen the caveat on tumor lines stabilized in vitro, that poorly represent the morphologic and antigenic heterogeneity of neoplasms in vivo.

Topics: Apoptosis; Blotting, Western; Bone Neoplasms; Carcinoma, Renal Cell; Cell Size; Fas Ligand Protein; Humans; Immunohistochemistry; Insulin; Kidney Neoplasms; Membrane Glycoproteins; Microscopy, Electron; Phenotype; Sacrum; Tretinoin; Tumor Cells, Cultured

The effects of steroid and thyroid hormones are mediated by intracellular hormone receptors. An important mechanism modulating target tissue responsiveness to hormones is homologous and heterologous regulation of the receptors. We have characterized the expression of steroid hormone receptors in human MG-63 osteosarcoma cells. The MG-63 cells express receptor mRNAs for glucocorticoids, estrogen, retinoic acid, and 1,25(OH)2D3. We found that only the vitamin D receptor (VDR) mRNA concentration was influenced by the hormones. The stability of the VDR message was identical in control, dexamethasone- and estradiol-treated cells. On the other hand, both 1,25(OH)2D3 and retinoic acid separately stabilized the VDR mRNA levels increasing the apparent half-life by 11 h and 6 h, respectively. The VDR protein levels, however, as measured by immunoprecipitation, increased only after the 1,25(OH)2D3 treatment.

Topics: Blotting, Northern; Bone Neoplasms; Calcitriol; Cell Line; Dexamethasone; Estradiol; Gene Expression; Humans; Kinetics; Osteosarcoma; Receptors, Calcitriol; Receptors, Estrogen; Receptors, Glucocorticoid; Receptors, Retinoic Acid; Receptors, Steroid; RNA, Messenger; Steroids; Time Factors; Tretinoin; Tumor Cells, Cultured

Treatment of human MG-63 osteosarcoma cells with human recombinant transforming growth factor beta 1 (TGF-beta 1) was found to inhibit cell proliferation. In addition, 1,25-dihydroxyvitamin D3 [1,25(OH)2D3]-induced osteocalcin synthesis was greatly influenced by TGF-beta 1. Dose- and time-dependent inhibition was seen both in medium osteocalcin and the corresponding mRNA concentrations. Furthermore, TGF-beta 1 decreased osteocalcin synthesis modulated negatively by dexamethasone or positively by retinoic acid. The stability of osteocalcin mRNA was not decreased by the TGF-beta 1 treatment, but in vitro transcription assays demonstrated diminished osteocalcin gene transcription caused by the TGF-beta 1 treatment. Binding of vitamin D receptor (VDR) to an oligonucleotide probe containing the osteocalcin vitamin D response element (VDRE) was not influenced by TGF-beta 1, however. Incubation of the cells with the serine/threonine kinase inhibitor H-7 did not block the ability of TGF-beta 1 to decrease osteocalcin synthesis but caused a further inhibition. Also, the 1,25(OH)2D3-induced osteocalcin synthesis was decreased by H-7 treatment, suggesting that phosphorylation as such is involved in the transcriptional activation mechanism of VDR. These results demonstrate that TGF-beta 1 is a strong inhibitor of the synthesis of osteocalcin, a calcium binding protein participating in bone mineralization, by counteracting the stimulatory effects of other hormones on its synthesis. We further suggest that TGF-beta 1 affects the synthesis of osteocalcin at the level of transcription through mechanism(s) different from the serine/threonine kinase pathway.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Base Sequence; Bone Neoplasms; Calcitriol; Cell Division; Dexamethasone; Dose-Response Relationship, Drug; Drug Interactions; Gene Expression Regulation, Neoplastic; Humans; Isoquinolines; Molecular Sequence Data; Oligonucleotide Probes; Osteocalcin; Osteosarcoma; Phosphorylation; Piperazines; Protein Kinase C; Receptors, Calcitriol; RNA, Messenger; Transcription, Genetic; Transforming Growth Factor beta; Tretinoin; Tumor Cells, Cultured; Vitamin D-Binding Protein

HOS-8603 is a newly established human osteosarcoma cell line with phenotypic characteristics of osteoblasts. When these cells were grown in monolayer culture in the presence of dexamethasone (Dex) or retinoic acid (RA), there was a significant inhibition of proliferation in a concentration-dependent manner. The combined effects of Dex and RA depended upon the concentrations: at low concentrations (< 10 nM) the effects of Dex and RA were additive, whereas at high concentrations the effects were antagonistic. Anchorage-independent growth studies performed in methylcellulose culture indicated that Dex or RA inhibited colony formation by HOS-8603 cells. Treatment of HOS-8603 cells with 100 nM Dex induced alkaline phosphatase activity in a time-dependent manner, reaching a maximum of about 6.5-fold over basal levels. All these effects of Dex on HOS-8603 cells could be reversed by RU 486, a potent antiglucocorticoid. Based upon saturation of specific binding and Scatchard plot analysis, we demonstrated that a saturable, high-affinity glucocorticoid receptor (GR) existed in HOS-8603 cells, suggesting that the effects of glucocorticoids on HOS-8603 cells are mediated by the specific GR. Finally, we further investigated the homologous and heterologous regulation of GR in HOS-8603 cells. Treatment of these cells with Dex led to a time-dependent decrease in GR concentrations. This homologous GR downregulation occurred not only at the level of hormone binding but also at the level of GR mRNA. In contrast, RA was capable of increasing GR concentrations in a concentration- and time-dependent manner.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Alkaline Phosphatase; Bone Neoplasms; Cell Adhesion; Cell Differentiation; Cell Division; Cell Line; Dexamethasone; Dose-Response Relationship, Drug; Drug Interactions; Humans; Kinetics; Osteosarcoma; Receptors, Glucocorticoid; Tretinoin; Tumor Cells, Cultured

The inducibility of 1 alpha,25-dihydroxyvitamin D3 [1,25-(OH)2D3] binding by all-trans-retinoic acid (RA) was examined in tumor-derived clonal bone cell lines, in established clonal cell lines derived from normal embryonic bone, and in cultured bone cell populations freshly isolated from 18- and 21-day fetal and 5-day-old neonatal rat calvaria. Levels of 1,25-(OH)2D3 binding were determined using a single saturating dose (84 pM) of 3H-labeled 1,25-(OH)2D3. Bone-derived tumor cell lines (ROS 17/2.8, ROS 17/2, RCJ 3.2T.1, RCJ 3.2.4.1CAM, RCJ 3.2CE2.1) possessed high basal levels of binding and showed increases in 1,25-(OH)2D3 binding after culture for 24 hours in the presence of 10(-5) M RA. The non-tumor-derived established bone cell lines (RCB 2.2A, RCB 2.2B, RCB 2.2C, RCB 2.2D) showed low basal 1,25-(OH)2D3 binding levels and no change in response to RA, while first subcultures of bone cell populations derived from fetal and neonatal rat calvaria showed decreased 1,25-(OH)2D3 binding, following similar treatment with RA. In representative cell populations, the dose dependency of the RA effect was established. The observed differences in response to RA in the cell lines tested seem to be dependent on whether the cells originated from normal or tumor tissue.

Topics: Animals; Bone and Bones; Bone Neoplasms; Cell Line; Dose-Response Relationship, Drug; Rats; Receptors, Calcitriol; Receptors, Steroid; Tretinoin

The histogenesis of Ewing's sarcoma remains unknown. Recent studies have suggested a relationship to an unusual form of childhood neural tumor, often termed peripheral neuroepithelioma or primitive neuroectodermal tumor. Five Ewing's sarcoma tumor cell lines were studied for evidence of a neural phenotype. Under normal culture conditions, no morphologic evidence of neural differentiation was detected. Treatment with retinoic acid, an agent known to induce marked neural differentiation in neuroblastoma, had no demonstrable effect. Treatment with either cyclic AMP or TPA, in contrast, induced pronounced morphologic evidence of neural differentiation. Cells developed elongate processes with varicosities by phase-contrast microscopy; filaments, microtubules, and uraniffin-positive dense core granules were present by electron microscopy. Three neural markers (NSE, NFTP, and cholinesterase) were absent or barely detectable in untreated cells, but became abundant after treatment. These results provide convincing evidence for a neural histogenesis of Ewing's sarcoma. They also suggest a close relationship between Ewing's sarcoma and peripheral neural tumors, including the chest wall tumor described by Askin, but only a distant relationship to neuroblastoma.

Topics: Adolescent; Adult; Animals; Bone Neoplasms; Cell Differentiation; Cells, Cultured; Cholinesterases; Cyclic AMP; Cytoskeletal Proteins; Female; Humans; Male; Mice; Mice, Nude; Microscopy, Electron; Neoplasms, Nerve Tissue; Nerve Growth Factors; Phosphopyruvate Hydratase; Sarcoma, Ewing; Tretinoin