Bone morphogenetic proteins induce expression of metalloproteinases in melanoma cells and fibroblasts

Bone morphogenetic proteins are secreted growth factors which belong to the TGFbeta super family. In recent studies, we showed that the expression of BMP-4 and -7 is induced in melanoma cells in comparison to normal melanocytes. Functional analyses revealed that BMPs are inevitable factors for migration and invasion processes of melanoma cells; however, the role of BMPs in degradation and remodelling of the extracellular matrix remained unknown. We discovered that melanoma cell clones with reduced BMP activity, generated by stable transfection with an antisense BMP-4 construct or with the BMP inhibitor chordin, showed reduced expression of MMP-1, -2, -3 and -9. Moreover, BMPs displayed paracrine effects on stromal fibroblasts. Treatment of fibroblasts with BMP-2 or -4 led to increased MMP-1, -2, -3 and -13 expression. These data show that BMPs play an important role in dissemination of tumour cells from the primary tumour, either by enhancing the matrix degrading capacity of melanoma cells themselves or by stimulating tumour surrounding fibroblasts to induce expression of matrix metalloproteinases.     

Bone morphogenetic protein (BMP)-2 induces apoptosis in human myeloma cells

BMPs (bone morphogenetic proteins), members of the transforming growth factor (TGF)-beta superfamily, are a group of related proteins which are capable of inducing the formation of cartilage and bone, but are now regarded as multifunctional cytokines. However, little is known about their role in hematopoiesis. Recently, we found a novel function of BMPs to hematopoietic cells in that BMP-2 induces apoptosis not only in human myeloma cell lines, but also in primary samples from patients with multiple myeloma in vitro. BMP-2 caused cell cycle arrest in the G1 phase which was associated with accumulation of p21CIP1/WAF1 and p27KIP1, and the subsequent apoptosis of myeloma cells. Further analysis showed that BMP-2 induced down-regulation of Bcl-X(L) through the inactivation of STAT3, resulting in the induction of apoptosis in myeloma cells. We conclude that BMP-2 may have the potential to be one of the novel therapeutic agents for treatment in patients with multiple myeloma because of the beneficial effects on both myeloma cells and bone diseases. In this review, we summarize data concerning BMPs and BMP-2-induced apoptosis of myeloma cells including our own recent experimental data.     

Bone morphogenetic protein-6 promotes osteoblastic prostate cancer bone metastases through a dual mechanism

Prostate cancer frequently metastasizes to bone where it forms osteoblastic lesions through unknown mechanisms. Bone morphogenetic proteins (BMP) are mediators of skeletal formation. Prostate cancer produces a variety of BMPs, including BMP-6. We tested the hypothesis that BMP-6 contributes to prostate cancer-induced osteosclerosis at bone metastatic sites. Prostate cancer cells and clinical tissues produced BMP-6 that increased with aggressiveness of the tumor. Prostate cancer-conditioned medium induced SMAD phosphorylation in the preosteoblast MC3T3 cells, and phosphorylation was diminished by anti-BMP-6 antibody. Prostate cancer-conditioned medium induced mineralization of MC3T3 cells, which was blocked by both the BMP inhibitor noggin and anti-BMP-6. Human fetal bones were implanted in severe combined immunodeficient mice and after 4 weeks, LuCaP 23.1 prostate cancer cells were injected both s.c. and into the bone implants. Anti-BMP-6 or isotype antibody administration was then initiated. Anti-BMP-6 reduced LuCaP 23.1-induced osteoblastic activity, but had no effect on its osteolytic activity. This was associated with increased osteoblast numbers and osteoblast activity based on bone histomorphometric evaluation. As endothelin-1 has been implicated in bone metastases, we measured serum endothelin-1 levels but found they were not different among the treatment groups. In addition to decreased bone production, anti-BMP-6 reduced intraosseous, but not s.c., tumor size. We found that BMP-2, BMP-4, BMP-6, and BMP-7 had no direct effect on prostate cancer cell growth, but BMP-2 and BMP-6 increased the in vitro invasive ability of prostate cancer cell. These data show that prostate cancer promotes osteoblastic activity through BMP-6 and that, in addition to its bone effects, suggest that BMPs promote the ability of the prostate cancer cells to invade the bone microenvironment.     

Imexon-induced apoptosis in multiple myeloma tumor cells is caspase-8 dependent.

PURPOSE: Imexon is a 2-cyanoaziridine agent that has been shown to inhibit growth of chemotherapy-sensitive myeloma cells through apoptosis with decreased cellular stores of glutathione and increased reactive oxygen species (ROS). We examined the mechanism of imexon cytotoxicity in a diverse panel of dexamethasone and chemotherapy-sensitive and -resistant myeloma cell lines. EXPERIMENTAL DESIGN: We examined cellular cytotoxicity, apoptosis, and changes in redox state in dexamethasone-sensitive (C2E3), dexamethasone-resistant (1-310 and 1-414), chemotherapy-sensitive (RPMI-8226), and chemotherapy-resistant (DOX-1V and DOX-10V) myeloma cell lines. RESULTS: We found significant cytotoxicity after 48-h incubation with imexon (80-160 microM) in dexamethasone and chemotherapy-sensitive and -resistant myeloma cell lines in a time- and dose-dependent manner. The mechanism of imexon cytotoxicity in all cell lines was related to induction of apoptosis with the presence of cleaved caspase-3. Moreover, after imexon exposure in C2E3 and 1-414 cell lines, we demonstrated caspase-8-dependent apoptosis. Bcl-2:bax was proapoptotic with imexon in C2E3, whereas bcl-2:bax was independent of steroid resistance, chemotherapy sensitivity, and chemotherapy resistance. Depletion of intracellular glutathione was documented in RPMI-8226 at high imexon concentrations (>or=225 microM) but not in other cell lines. Furthermore, ROS were found in C2E3, RPMI-8226, and 1-310 only at high imexon concentrations, whereas a sensitive marker of oxidative DNA damage, 8-hydroxydeoxyguanosine, was not increased in any cell line. CONCLUSIONS: Our results demonstrate that imexon has significant broad antimyeloma activity that is mediated through apoptotic mechanisms that is not dependent on production of ROS. Moreover, we have identified a mechanism of cytotoxicity in dexamethasone-sensitive and -resistant myeloma cells induced by imexon that is caspase-8 dependent.     

Growth inhibition and apoptosis of myeloma cells by the CDK inhibitor flavopiridol.

Although myeloma shows responsiveness in intensive chemotherapy, overall survival remains less than 40% at 2 years. Since myeloma appears to be dependent on cytokines, such as IL-6, we hypothesized that targeting signal transduction molecules could effectively treat myeloma. Two myeloma cell lines U266 and RPMI-8226 and CD38+ myeloma cells were studied by immune complex kinase assay or anti-phosphotyrosine blot for evidence of constitutive activation of tyrosine kinases. Growth arrest and apoptosis were evaluated in these two cell lines following their treatment with specific kinase inhibitors. We found that a variety of Src and Janus kinases were present and constitutively active in U266 and RPMI-8226 cells. Inhibitors of both Src and Janus kinases were inferior to the cyclin-dependent kinase inhibitor, flavopiridol, in inducing both growth arrest with GI50 of 100 nM and apoptosis in both cell lines and CD38+ myeloma cells. Although, flavopiridol did not affect cyclin D1 and cyclin A levels, it inhibited Mcl-1 and Bcl-2 protein levels and cyclin-dependent kinase 2 activity. Flavopiridol is a well-tolerated drug, currently in phase I-II trials for a variety of tumors. A clinical trial using flavopiridol should be performed in patients with myeloma. Its mechanism of action may involve targets other than the cyclin-dependent kinases.     

RNA干扰抑制FANCD2基因对多药耐药骨髓瘤细胞株药物敏感性的影响

目的：探讨FA/BRCA（fanconi anemia/BRCA）中siRNA干扰对多药耐药骨髓瘤细胞株的影响.方法：体外培养多发性骨髓瘤细胞RPMI-8226细胞系,通过相应渐增浓度的马法兰药物处理,选择性获得多药耐药的子代肿瘤细胞株RPMI-8226/R细胞.MTT法检测RPMI-8226和RPMI-8226/R细胞马法兰、ADM、VCR、CTX、Ara-C、VP-16及DDP的敏感性.siRNA干扰抑制FA/BRCA途径FANCD2蛋白表达,观察多发性骨髓瘤多药耐药细胞株对烷化剂药物敏感性的变化.结果：成功构建针对FANCD2基因的siRNA,将其转染多药耐药骨髓瘤细胞株,可以抑制细胞中FANCD2基因表达,转染细胞较转染前细胞FANCD2基因表达降低,对烷化剂的敏感性升高.结论：人多发性骨髓瘤多药耐药细胞株RPMI-8226/R其多药耐药性的产生可能与FA/BRCA途径中FANCD2表达增强有关,可通过抑制FANCD2表达而逆转细胞的耐药性达到增强烷化剂对耐药肿瘤细胞的增殖抑制及诱导凋亡作用.沉默FANCD2表达可以提高多药耐药MM细胞对烷化剂的敏感性,而RNA干扰是一种沉默FANCD2表达的理想方法,因此它可能是一种有潜力的耐药MM辅助治疗新方法.     

Diverse biological effect and Smad signaling of bone morphogenetic protein 7 in prostate tumor cells

We found that bone morphogenetic protein (BMP) 7, a member of the BMP family, was strikingly up-regulated during the development of primary prostatic adenocarcinoma in the conditional Pten deletion mouse model. To determine the relevance of this finding to human prostate cancer, we examined the expression of BMPs and BMP receptors (BMPR) as well as the responsiveness to recombinant human BMP7 in a series of human prostate tumor cell lines. All prostatic cell lines tested expressed variable levels of BMP2, BMP4, and BMP7 and at least two of each type I and II BMPRs. In all cases, BMP7 induced Smad phosphorylation in a dose-dependent manner, with Smad5 activation clearly demonstrable. However, the biological responses to BMP7 were cell type specific. BPH-1, a cell line representing benign prostatic epithelial hyperplasia, was growth arrested at G1. In the bone metastasis-derived PC-3 prostate cancer cells, BMP7 induced epithelial-mesenchymal transdifferentiation with classic changes in morphology, motility, invasiveness, and molecular markers. Finally, BMP7 inhibited serum starvation-induced apoptosis in the LNCaP prostate cancer cell line and more remarkably in its bone metastatic variant C4-2B line. Each of the cell lines influenced by BMP7 was also responsive to BMP2 in a corresponding manner. The antiapoptotic activity of BMP7 in the LNCaP and C4-2B cell lines was not associated with a significant alteration in the levels of the proapoptotic protein Bax or the antiapoptotic proteins Bcl-2, Bcl-xl, and X-linked inhibitor of apoptosis. However, in C4-2B cells but not in LNCaP cells, a starvation-induced decrease in the level of survivin was counteracted by BMP7. Taken together, these findings suggest that BMPs are able to modulate the biological behavior of prostate tumor cells in diverse and cell type-specific manner and point to certain mechanisms by which these secreted signaling molecules may contribute to prostate cancer growth and metastasis.     

β-catenin inhibitors ICG-001 and pyrvinium sensitize bortezomib-resistant multiple myeloma cells to bortezomib

Although bortezomib (BTZ) displays efficacy in treating multiple myeloma (MM), BTZ resistance in MM patients has been reported. Meanwhile, treating BTZ resistant MM cells with β-catenin inhibitors have demonstrated the ability to reserve BTZ resistance. Thus, the present study aimed to investigate the synergistic effect of the β-catenin inhibitors, ICG-001 and pyrvinium (PP), with BTZ in the treatment of BTZ-resistant MM cells. Different concentrations of ICG-001 (0–32 µM) or PP (0–32 nM) were used to treat the BTZ-resistant RPMI-8226 (RPMI-8226BR) and BTZ-resistant KMS-11 (KMS-11BR) cell lines, followed by a BTZ combination treatment. Subsequently, cell viability and apoptosis in these two cell lines were determined by CCK-8 assay and flow cytometry, respectively. The proteins involved in the Wnt/β-catenin signaling pathway were detected using western blotting. The Wnt/β-catenin signaling pathway was activated in the RPMI-8226BR and the KMS-11BR cells. In addition, the cell viability of RPMI-8226BR and KMS-11BR cells were decreased following β-catenin inhibitor (ICG-001 and PP) treatment alone. Furthermore, the β-catenin inhibitors, ICG-001 and PP, plus BTZ combination treatment revealed a notable decrease in cell viability and a marked increase in cell apoptosis rate, compared with that in cells treated with ICG-001, PP or BTZ alone in the RPMI-8226BR and KMS-11BR cell lines. In conclusion, the β-catenin inhibitors, ICG-001 and PP not only increased apoptosis, but also sensitized BTZ-resistant MM cells to BTZ, indicating their potential therapeutic application in MM.     

Elimination of chemoresistant multiple myeloma clonogenic colony-forming cells by combined treatment with a plasma cell-reactive monoclonal antibody and a P-glycoprotein-reactive monoclonal antibody

Patients with multiple myeloma (MM) commonly become refractory to chemotherapy despite a favorable response to induction treatment. We examined the effectiveness of a previously characterized plasma cell-reactive monoclonal antibody, MM4, in eliminating MM clonogenic colony-forming cells (CCC) with a multidrug-resistant (MDR) phenotype. Experiments were performed using MM cell lines that exhibit 6 (RPMI 8226/DOX6)- and 40 (RPMI 8226/DOX40)-fold resistance to doxorubicin (DOX). Both lines were selected from the chemosensitive MM line RPMI 8226/S and were cross-resistant to mitoxantrone, acronycine, etoposide, and vincristine. Surface marker analysis conducted in this study showed that DOX6 and DOX40 overexpressed the MDR1 gene product p170. Both MDR lines remained reactive to the plasma cell-reactive monoclonal antibodies MM4 and PCA-1 and expressed the relevant cytoplasmic immunoglobulin light chain. Treatment with MM4 and rabbit complement (C') was equally cytotoxic to RPMI 8226/S [80 +/- 5.6% (SD)], DOX6 [74 +/- 8.5], and DOX40 cells [75 +/- 11.3%], based on short-term chromium release studies. Furthermore, MM4 + C' deleted up to 3 logs of CCC colonies from chemosensitive and MDR lines (RPMI 8226/S, 99.87 +/- 0.11%; DOX6, 99.91 +/- 0.08%; DOX40, 99.55 +/- 0.44%). By comparison, the P-glycoprotein-reactive monoclonal antibody MRK-16 and C' inhibited tumor colony formation of MDR cells (8226/DOX6, 95.71 +/- 2.51%; 8226/DOX40, 99.61 +/- 0.43%) but affected that of chemosensitive cells only slightly (8.9 +/- 17.8%). In an attempt to optimize the depletion of myeloma CCC, MM4 was used together with MRK-16. This approach resulted in uniform depletion of myeloma clonogenic colony-forming cells from the chemosensitive (98.32 +/- 1.53%, n = 4) and MDR lines (8226/DOX6, 98.83 +/- 0.08%, n = 4; 8226/DOX40 99.29 +/- 0.62, n = 7) but did not result in enhanced CCC depletion. When DOX40 cells were mixed with normal bone marrow (BM) in the ratio of 90:10 (BM:MM), either MM4 or MRK-16 and C' depleted MM colonies (98.8 +/- 0.71% and 98.10 +/- 1.0%, respectively) without affecting the majority of BM progenitor cells. These observations suggest that either MM4 or MRK-16 is useful for depleting MDR myeloma clonogenic colony-forming cells.     

Combination therapy with interleukin-6 receptor superantagonist Sant7 and dexamethasone induces antitumor effects in a novel SCID-hu In vivo model of human multiple myeloma.

Interleukin-6 (IL-6) protects multiple myeloma cells against apoptosis induced by glucocorticoids. Here, we investigated whether inhibition of the IL-6 signaling pathway by the IL-6 receptor superantagonist Sant7 enhances the in vivo antitumor effects of dexamethasone on the IL-6-dependent multiple myeloma cell line INA-6. For this purpose, we used a novel murine model of human multiple myeloma in which IL-6-dependent INA-6 multiple myeloma cells were directly injected into human bone marrow implants in severe combined immunodeficient (SCID) mice (SCID-hu). The effect of in vivo drug treatments on multiple myeloma cell growth was monitored by serial determinations of serum levels of soluble IL-6 receptor (shuIL-6R), which is released by INA-6 cells and served as a marker of tumor growth. In SCID-hu mice engrafted with INA-6 cells, treatment with either Sant7 or dexamethasone alone did not induce significant reduction in serum shuIL-6R levels. In contrast, the combination of Sant7 with dexamethasone resulted in a synergistic reduction in serum shuIL-6R levels after 6 consecutive days of treatment. Gene expression profiling of INA-6 cells showed down-regulation of proliferation/maintenance and cell cycle control genes, as well as up-regulation of apoptotic genes in multiple myeloma cells triggered by Sant7 and dexamethasone combination. In vitro colony assays showed inhibition of myeloid and erythroid colonies from normal human CD34(+) progenitors in response to dexamethasone, whereas Sant7 neither inhibited colony growth nor potentiated the inhibitory effect of dexamethasone. Taken together, these results indicate that inhibition of IL-6 signaling by Sant7 significantly potentiates the therapeutic action of dexamethasone against multiple myeloma cells, providing the preclinical rationale for clinical trials of Sant7 in combination with dexamethasone to improve patient outcome in multiple myeloma.     

BMP signals inhibit proliferation and in vivo tumor growth of androgen-insensitive prostate carcinoma cells

Prostate cancer is one of the most common cancers in men. Several lines of evidence have suggested that bone morphogenetic protein (BMP) signals play important roles in the generation and progression of prostate cancers. In the present study, we show that BMP-7 inhibits the proliferation of androgen-insensitive PC-3 and DU-145 prostate cancer cells in a medium containing 1% fetal bovine serum, observed as decreased incorporation of [(3)H]thymidine and decreased cell number. Cell cycle analysis by flow cytometry showed an increased fraction of cells in the G1 phase and subsequent decrease in both S and G2/M phase after BMP-7 stimulation. BMP-7 caused an upregulation of the cyclin-dependent kinase inhibitor (CDKI) p21(CIP1/WAF1), and decreased the activity of Cdk2, leading to hypophosphorylation of Rb proteins. Furthermore, in order to evaluate the impact of BMP signals on prostate tumor growth, we generated the PC-3 cell lines expressing a constitutively active BMP type I receptor (constitutively active (c.a.) activin receptor-like kinase (ALK)-6) in a tetracycline (Tet)-regulated manner. Tet/doxycycline-regulated expression of c.a.ALK-6 resulted in the inhibition of in vitro cell proliferation and reduction of the size of tumors derived from the PC-3 cells subcutaneously injected into immune-deficient mice. Collectively, these findings suggest that BMP signals inhibit growth and proliferation of prostate tumor cells through induction of CDKI. Furthermore, this is the first report of a role for BMP signaling in reducing growth kinetics of androgen-insensitive prostate tumors.     

The aberrant promoter methylation of BMP3b and BMP6 in malignant pleural mesotheliomas

Bone morphogenetic proteins (BMPs) belong to the transforming growth factor-beta superfamily. Recent studies have showed that aberrant methylation of BMP genes is present in several types of human cancer. We examined the expression and methylation status of BMP3b and BMP6 in malignant pleural mesotheliomas (MPMs). The expression status of BMP3b, and BMP6 mRNAs were examined in seven MPM cell lines by RT-PCR assay. The expression of BMP3b was completely suppressed in 2 and partially suppressed in 2 of 7 cell lines and expression of BMP6 was partially suppressed in 2 cell lines. Methylation status of BMP3b in cell lines was determined by methylation-specific assay to find aberrant methylation in 6 cell lines which include 4 cell lines with suppressed BMP3b expression. Partial methylation of BMP6 was found in 2 cell lines whose expression was partially suppressed. Treatment with 5-Aza-dC restored BMP3b expression in methylated cell lines. Next, we examined the methylation status in 57 surgically resected MPM cases and found aberrant methylation of BMP3b in 9 (53%) out of 17 cases from Japan and 3 (8%) of 40 cases from USA and that of BMP6 in 4 (24%) cases from Japan and 12 (30%) cases from USA, showing significant difference in frequency of BMP3b methylation between MPMs of the two countries (P=0.0004). Our study indicated that BMP3b and BMP6 genes were suppressed by DNA methylation and methylation of BMP3b is significantly frequent in Japanese MPMs, suggesting its pathogenic role and the ethnic difference in MPMs.     

Glucocorticoid effects on myeloma cells in culture: correlation of growth inhibition with induction of glucocorticoid receptor messenger RNA

Glucocorticoids are widely used for the treatment of multiple myeloma. To investigate the direct actions of glucocorticoids on myeloma cells, we have used three cell lines of human multiple myeloma, OPM-1, OPM-2, and RPMI 8226. When growth curves of these cells were examined, OPM-1 cells were resistant, while OPM-2 were sensitive to dexamethasone (DEX). In cultures of OPM-2 cells, addition of DEX led to virtual cessation of growth, with only 16% of the residual cells viable after 4 days. RPMI 8226 appeared to be slightly sensitive, showing some slowing of growth for several days in DEX, with later recovery. Viabilities of OPM-1 and RPMI 8226 cells were not affected. Secretion of immunoglobulin (Ig-lambda) was also partially suppressed, by 30% in OPM-2 and 14% in OPM-1. No significant suppression was observed in RPMI 8226. To explore the mechanism of these differential responses to the steroid, glucocorticoid receptor (GR) was examined. Binding assays showed high affinity binding sites in all three cell lines: 64 +/- 11 fmol/10(6) cells in OPM-1, 78 +/- 14 in OPM-2, and 62 +/- 16 in RPMI 8226. Nuclear transfer of GR and DNA-cellulose binding after heat activation appeared similar in all three cell lines. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of cytosol proteins labeled with [3H]dexamethasone mesylate showed a GR of Mr 95,000 in all three. When GR mRNA was studied in these cells, all of them had GR mRNA of approximately 7 kilobases, but OPM-2 and RPMI 8226 had 3 times more GR mRNA than OPM-1. OPM-2 GR mRNA was induced 2-fold by DEX treatment at 5 x 10(-9) M or greater. OPM-1 GR mRNA was much less sensitive, with no response at less than 10(-6) M DEX and only 1.5-fold induction at that concentration. These results demonstrate that some myeloma cells can be killed by a direct action of glucocorticoids. The quantity and affinity of GR in the cells were not predictive of this response. Therefore, we propose that the resistance of OPM-1 and the relative resistance of RPMI 8226 to glucocorticoid inhibition of cell growth is by post-receptor mechanisms. The high sensitivity of induction of GR mRNA in OPM-2 may correlate with glucocorticoid-evoked cell kill.     

Overexpression of BMP-2/4, -5 and BMPR-IA associated with malignancy of oral epithelium

The aim of the present study was to determine the relationships between bone morphogenetic proteins (BMPs), BMP receptor type IA and carcinogenesis of oral epithelium. A retrospective study was performed on material obtained from oral mucosa, including nine cases of normal mucosa (NB), eight cases of nonspecific chronic inflammation (NCI), seven cases of hyperkeratosis (HK), five cases of squamous cell papilloma (SCP), 29 cases of squamous cell carcinoma (SCC) with various grades of differentiation and 10 cases of epithelium adjacent to carcinoma (EAC). Six cases of NB from hard palate (NHP) were chosen as a control group. The benign groups consisted of NCI, HK and SCP. The antibodies against BMP-2/4, -5, receptor BMPR-IA and purified bovine BMP (bBMP-McAb) were utilised using an immunocytochemical method. The results demonstrated that the immunostaining of BMP-2/4, BMP-5, BMPR-IA and bBMP-McAb was weak and not consistent in normal and benign groups. The immunoreactivity level was independent of the clinical and pathological grading of SCC. All cases of SCC showed positive staining for BMP-2/4, BMP-5, BMPR-IA and bBMP-McAb except for three cases and one case of SCC which negatively stained for BMP-2/4 and BMP-5, respectively. The staining intensity and proportion of the positively stained cells were markedly increased in SCC when compared with that of the normal and benign groups except for EAC. The metastatic carcinoma cells in lymph nodes were strongly and positively stained for BMP-2/4 and BMP-5 when compared with the primary lesions. Our results indicate that there was an overexpression of BMP-2/4, BMP-5, bBMP-McAb and BMPR-IA in the high-risk premalignant and malignant lesions of oral epithelium. Our findings suggest that BMP-2/4 and BMP-5 but not BMPR-IA might be involved in the metastasis of oral carcinoma cells.