Effect of mesenchymal stem cells on hypoxia-induced desensitization of ß2-adrenergic receptors in rat osteosarcoma cells.

The ß2-adrenergic receptor (ß2AR) mediates the effects of chronic stress in several neoplasms, however, ß2AR signaling is impaired by hypoxia in various tissues. While hypoxia is a common feature significant in the progression of solid tumors, little is known about the effect of hypoxia on ß2AR signaling in the tumor microenvironment. Previously, it has been reported that the systemic administration of mesenchymal stem cells (MSCs) increased the engraftment and metastatic colonization of rat osteosarcoma (OS) cells. In the current study, the effect of MSCs on the hypoxia-induced desensitization of the ß2AR in OS cells was investigated. Epinephrine, norepinephrine and isoproterenol increased the cellular proliferation of the rat OS cell line COS1NR and rat MSCs in a dose-dependent and ß2AR antagonist-sensitive manner. While isoproterenol had significant proliferative effects on MSCs under normoxic and hypoxic conditions, COS1NR cells did not respond under hypoxic conditions. A sensitivity assay for the ß2AR revealed that hypoxia impaired the sensitivity of COS1NR cells, whereas hypoxia did not affect MSCs. An immunoassay revealed no significant change in the expression of hypoxia-inducible factor-1α (HIF1α) in COS1NR cells, whilst an immunoassay demonstrated a 15% increase in MSCs following isoproterenol stimulation. In COS1NR cells co-cultured with MSCs under hypoxic conditions, isoproterenol caused a significant increase in proliferation and this effect was inhibited by an anti-interleukin (IL)-6 antibody. A tumor formation assay in syngeneic rats revealed that the systemic administration of MSCs enhances the growth of OS and the effect of MSCs was inhibited by IL-6 neutralization. In conclusion, MSCs are resistant to the hypoxia-induced desensitization to ß2AR. Hypoxia caused a siginificant desensitization of the ß2AR in COS1NR cells alone, whereas MSCs may support tumor progression through cellular interactions.

Low concentrations of alendronate increase the local invasive potential of osteoblastic sarcoma cell lines via connexin 43 activation.

Bisphosphonates (BPs) are agents used for treating disorders of excessive bone resorption. In addition, due to their cell-killing activity, BPs were potent candidates for adjuvant cancer therapy. On the other hand, low-concentrations of BPs have been reported to increase cellular viability in several types of tumor cells. Therefore, we focused on the effect of BPs on cellular aggressiveness of malignant bone tumors at low concentrations. MTS assay was performed using osteosarcoma cell lines MG63 and HOS, fibrosarcoma cell line HT1080, and prostate cancer cell line PC3. All the cell lines showed toxicity at high concentrations. On the other hand, at lower concentrations, the cellular viabilities of HOS and MG63 were rather higher than those of untreated controls. Since this tendency was most evident, HOS was used for further assays, including cellular motility, bone resorption activity, and cathepsin K activity. The low-concentration of alendronate enhanced cellular viability and motility, which correlated with the expression of connexin 43 at the mRNA and protein levels. Interestingly, oleamide, a potent connexin 43 inhibitor, had an inhibitory effect on the enhanced proliferation. Our data suggest that alendronate may enhance the proliferation of osteoblastic cell line through connexin 43 activation.

Sphere-forming stem-like cell populations with drug resistance in human sarcoma cell lines.

The presence of cancer stem cells in both solid and hematopoietic malignancies, has been recently linked to their pathogenesis. Sarcomas are rare, and diversely characterized by degrees of mesenchymal differentiation. The aim of the current study was to demonstrate whether the human sarcoma cell lines, osteosarcoma MG63, Ewing's sarcoma HTB166, fibrosarcoma HT1080, possess the stem-like properties which may contribute to the drug-resistance. All cell lines possessed an ability to form spherical, clonal expanding colonies (sarcospheres) in anchorage-independent, serum-starved conditions. Sarcospheres showed the stem-like properties with the ability of self-renewal, and increased expression of the stem cell-related genes such as Nanog, OCT3/4 SOX2 and DNA repair enzyme genes, MLH1 and MSH2. Sarcospheres showed strong resistance to doxorubicin and cisplatin, and caffeine, a DNA repair inhibitor, enhanced the efficacy of those drugs, suggesting that the drug resistance in sarcosphere cells was partly related to the efficient DNA repair ability. These results indicate that human sarcoma cell lines contain stem-like cell populations with strong drug resistance, and DNA repair inhibitor could enhance the efficacy of chemo-drugs against sarcomas.

Growth inhibition and induction of apoptosis by 2-methoxyestradiol in rat osteosarcoma and malignant fibrous histiocytoma cell lines.

2-Methoxyestradiol (2-ME) has been found to possess antitumor activity in vivo and in vitro. It has been suggested that 2-ME induces apoptosis resulting in G2/M arrest of tumor cells. In this study, the effect of 2-ME was evaluated in rat osteosarcoma and malignant fibrous histiocytoma (MFH) cell lines. 2-ME was used at final concentrations of 100 nM to 2 microM. The effect of 2-ME on cell growth was measured by the MTS assay. Induction of apoptosis and activation of caspase-3 were investigated along with apoptosis-related gene expression. The data showed that 2-ME significantly inhibited cell growth, inducing apoptosis. The activity of caspase-3 was increased at 20 h and 40 h in both cell lines. 2-ME induced p16 expression, which was possibly involved in the apoptotic process. These results suggested that the 2-ME-induced apoptosis of rat osteosarcoma and rat MFH cells was accompanied by caspase-3 activation through p16 induction.

Reduced expression of INK4a/ARF genes in stem-like sphere cells from rat sarcomas.

The presence of cancer stem cells, in both hematopoietic and solid malignancies, has been recently linked to their pathogenesis. We aimed to identify the characteristics and stem-like properties of sphere-colony forming cells in rat osteosarcoma and malignant fibrous histiocytoma cell lines. The results showed that both cell lines possessed an ability to form spherical, clonally expanding colonies in anchorage-independent, serum-starved conditions in N2/1% methylcellulose medium. The sphere cells showed stem-like properties with the ability to self-renew, and expressed the stem cell-related STAT3 and Bmi1 genes. Interestingly, spheres from both sarcomas remarkably decreased the expression of INK4a/ARF locus genes, p16(INK4a) and p19(ARF), which could be related to the resistance against cell senescence and apoptosis. Spheres showed strong tumorigenicity with metastatic potential in vivo via the inoculation into syngeneic rats, suggesting the presence of these populations might contribute to the tumor development such as metastasis via the resistance to apoptotic stimuli.

Growth inhibition and induction of apoptosis by flavopiridol in rat lung adenocarcinoma, osteosarcoma and malignant fibrous histiocytoma cell lines.

Flavopiridol is the potent inhibitor of cdks sharing its function with endogenous cdk inhibitors, and causes arrest at both the G1 and G2 phases of the cell cycle resulting in apoptosis in various tumor cell lines. Cyclin-dependent kinase inhibitor p16INK4a induces cell cycle arrest in G1 or G2 or both, and is inactivated in many malignant tumors. In this study, we focused on the effects of flavopiridol on chemically-induced rat lung adenocarcinoma, osteosarcoma and malignant fibrous histiocytoma (MFH) cell lines showing different pattern of p16INK4a status. The data demonstrated that flavopiridol inhibited cellular growth in a dose- and time-dependent manner, inducing apoptosis within 24 h in all cell lines at a concentration of 300 nM. The growth inhibition rate was the greatest for lung adenocarcinoma cells, lacking p16INK4a expression associated with methylation-mediated gene silencing; 83% at a concentration of 300 nM for 72-h treatment; while the growth of osteosarcoma and MFH cells, both expressing p16INK4a, were inhibited at similar levels; 54-61% for osteosarcoma and 61-64% for MFH cell lines. Then, we further investigated the influence of p16INK4a induction upon the effect of flavopiridol in p16INK4a-deficient lung adenocarcinoma cells. 5-aza 2'-deoxycytidine (5-Aza-CdR) induced p16INK4a expression and inhibited cellular growth in lung adenocarcinoma at a similar level to that with flavopiridol treatment. After the induction of p16INK4a expression by 5-Aza-CdR, the growth inhibition rates of flavopiridol in the p16INK4a-induced lung adenocarcinoma cells could not achieve comparable inhibition to that in the p16INK4a-deficient cells; the efficacy was reduced compared to original p16INK4a-deficient cells at each concentration of 50, 100 and 500 nM for 72-h treatment. These data indicate that flavopiridol shows cell type specific inhibition and possibly acts in a more compensatory manner for endogenous p16INK4a function in tumor cells having the aberrations of p16INK4a gene.

Expression of the p16INK4a gene and methylation pattern of CpG sites in the promoter region in rat tumor cell lines.

Loss of p16(INK4a) protein expression has frequently been related to DNA methylation in association with gene silencing. Although the methylation status of exon1alpha for p16(INK4a) involvement in various cancers has been extensively analyzed, it has been pointed out that some inconsistencies existed in its relationship to gene silencing of p16(INK4a). In this study, we focused on the expression and methylation status in the regions of nt -478 to -201, containing a putative TATA box (nt -401 to -396), and nt -233 to 26, both in a recently cloned 5' upstream region of rat p16(INK4a). We showed that rat lung adenocarcinoma RLCNR did not express the p16(INK4a) gene, whereas rat osteosarcoma COS1NR and malignant fibrous histiocytoma MFH1NR both expressed it at levels similar to normal fibroblasts, even though the region of nt -233 to 26 was hypermethylated in COS1NR rather than RLCNR. In contrast, the CpG islands near the putative TATA box region were consistently methylated in RLCNR, but not in COS1NR and MFH1NR, as well as in normal fibroblasts. Treatment with 5-aza 2'-deoxycytidine induced expression of p16(INK4a) gene in RLCNR after 48 h, but no changes were observed in COS1NR and MFH1NR. The results indicated that methylation of CpG islands near a TATA box region played a critical role for gene silencing of the rat p16(INK4a) gene, rather than that of other regions.

Growth inhibition of rat osteosarcoma and malignant fibrous histiocytoma cells by tyrosine kinase inhibitor STI571.

STI571 is a 2-phenylaminopyrimide derivative that was designed as an Abl tyrosine kinase inhibitor, but it is also effective against platelet-derived growth factor receptor (PDGFR) and c-Kit tyrosine kinase. Recent studies have demonstrated that STI571 inhibits the growth of several tumors in which PDGF or c-kit play an important role in tumor pathogenesis. We have recently established rat osteosarcoma and malignant fibrous histiocytoma (MFH) cell lines. RT-PCR analysis revealed that MFH and osteosarcoma cell lines expressed high and very low levels of PDGFR alpha respectively, and that all cell lines expressed similar levels of PDGFR beta. The level of c-kit mRNA expression were almost negligible hardly in all cell lines. The effect of STI571 on cellular growth measured by MTS colorimetric dye reduction showed that the growth of each cell line was inhibited in a dose- and time-dependent manner. STI571 (10 microM) inhibited the rates of cell growth of MFH cells by up to 40% and of osteosaroma cells by only to 20% after 72 hours. These data suggested that STI571 tyrosine kinase inhibitor plays a role in blocking or slowing the rate of growth of MFH and osteosarcoma cells expressing tyrosine kinase type receptor.