Gambogic acid inhibits the growth of osteosarcoma cells in vitro by inducing apoptosis and cell cycle arrest

The natural product gambogic acid (GA) has been demonstrated to be a promising chemotherapeutic drug for some cancers because of its ability to induce apoptosis and cell cycle arrest. Until now, no studies have looked at the role of GA in osteosarcoma. In this study, we observed the effects of GA on the growth and apoptosis of osteosarcoma cells in?vitro. We found that GA treatment inhibits the proliferation of osteosarcoma cells by inducing cell cycle arrest. Moreover, we found that GA induces apoptosis in MG63, HOS and U2OS cells. Furthermore, we showed that GA treatment elevates the Bax/Bcl-2 ratio. GA mediated the G0/G1 phase arrest in U2OS cells; this arrest was associated with a decrease in phospho-GSK3-β (Ser9) and the expression of cyclin D1. Similarly, in MG63 cells, GA mediated G2/M cell cycle arrest, which was associated with a decrease in phospho-cdc2 (Thr 161) and cdc25B. Overall, our findings suggest that GA may be an effective anti-osteosarcoma drug because of its capability to inhibit proliferation and induce apoptosis of osteosarcoma cells.     

Targeting cyclin D1, a downstream effector of INI1/hSNF5, in rhabdoid tumors

Rhabdoid tumors (RTs) are aggressive and currently incurable pediatric malignancies. INI1/hSNF5 is a tumor suppressor biallelically inactivated in RTs. Our previous studies have indicated that cyclin D1 is a key downstream target of INI1/hSNF5 and genesis and/or survival of RTs in vivo is critically dependent on the presence of cyclin D1. In this report, we have tested the hypothesis that therapeutic targeting of cyclin D1 is an effective means of treating RTs. We found that RNA interference of cyclin D1 in rhabdoid cells was sufficient to induce G1 arrest and apoptosis. Furthermore, we found that pharmacological intervention with low micromolar concentrations of N-(4-hydroxyphenyl)retinamide (4-HPR), which downmodulates cyclin D1, induced G1 arrest and apoptosis in rhabdoid cell lines. 4-HPR in combination with 4-hydroxy-tamoxifen (4OH-Tam), synergistically inhibited survival as well as anchorage-dependent and -independent growth of rhabdoid cells and caused synergistic induction of cell cycle arrest and apoptosis. 4-HPR and tamoxifen exhibited synergistic growth inhibition of RTs in xenograft models in vivo. The effects of combination of drugs were correlated to the depletion of cyclin D1 levels both in in vitro and in vivo tumor models. These results demonstrate that 4-HPR and tamoxifen are effective chemotherapeutic agents for RTs. We propose that downmodulation of cyclin D1 is a novel and effective therapeutic strategy for RTs.     

Overexpression of LIMK1 promotes migration ability of multidrug-resistant osteosarcoma cells

Multidrug resistance (MDR) to chemotherapy is a major obstacle in the treatment of cancer and the resistance process is multifactorial. Studies on multidrug resistance mechanisms relied on the availability of cancer multidrug resistance cell lines that have been established. In this study we successfully established a multidrug resistance cell line MG63/VCR derived from human osteosarcoma cell line MG63 based on the induction by vincristine. MG63/VCR cells exhibited high resistance to vincristine and other anticancer drugs, accompanied by upregulated expression of MDR-associated genes MDR1, MRP1, and Bcl-2. Notably, we found that MG63/VCR cells exhibited higher migration ability compared to parental MG63 cells. Moreover, we demonstrated that LIMK1, a key regulator of actin cytoskeleton, was overexpressed at both mRNA and protein levels in MG63/VCR cells and the higher LIMK1 protein level was correlated with higher level of phosphorylated cofilin. In addition, knockdown of LIMK1 abolished the higher migration ability of MG63/ VCR cells. These results suggest that LIMK1 overexpression contributes to the invasion and metastasis of drug-resistant osteosarcoma and reveal LIMK as a novel therapeutic target for drug resistant osteosarcoma.     

Wnt pathway activation and ABCB1 expression account for attenuation of Proteasome inhibitor-mediated apoptosis in multidrug-resistant cancer cells

Multiple drug resistance (MDR) is a major obstacle to attenuating the effectiveness of chemotherapy to many human malignancies. Proteasome inhibition induces apoptosis in a variety of cancer cells and is recognized as a novel anticancer therapy approach. Despite its success, some multiple myeloma patients are resistant or become refractory to ongoing treatment by bortezomib suggesting that chemoresistant cancer cells may have developed a novel mechanism directed against the proteasome inhibitor. The present study aimed to investigate potential mechanism(s) of attenuation in a MDR cell line, MES-SA/Dx5. We found that compared to the parental human uterus sarcoma cell line MES-SA cells, MES-SA/Dx5 cells highly expressed the ABCB1 was more resistant to MG132 and bortezomib, escaping the proteasome inhibitor-induced apoptosis pathway. The resistance was reversed by co-treatment of MG132 and the ABCB1 inhibitor verapamil. The data indicated that ABCB1 might play a role in the efflux of MG132 from the MES-SA/Dx5 cells to reduce MG132-induced apoptosis. Furthermore, the canonical Wnt pathway was found activated only in the MES-SA/Dx5 cells through active β-catenin and related transactivation activities. Western blot analysis demonstrated that Wnt-targeting genes, including c-Myc and cyclin D1, were upregulated and were relevant in inhibiting the expression of p21 in MES-SA/Dx5 cells. On the other hand, MES-SA cells expressed high levels of p21 and downregulated cyclin D1 and caused cell cycle arrest. Together, our study demonstrated the existence and participation of ABCB1 and the Wnt pathway in an MDR cell line that attenuated proteasome inhibitor-induced apoptosis.     

Effect of As2O3 on cell cycle progression and cyclins D1 and B1 expression in two glioblastoma cell lines differing in p53 status

Recent clinical studies have demonstrated that As2O3 is an effective drug in the treatment of acute promyelocytic leukemia (APL) by inducing apoptosis and inhibiting the proliferation of leukemia cells both in vitro and in vivo. As a novel anticancer agent for the treatment of solid cancer, As2O3 is promising, but no experimental investigations of its efficacy on glioblastoma have been conducted at concentrations that may be achieved clinically. In addition, the cell proliferation and cell cycle regulating mechanism of As2O3 has not yet to be clarified, especially in solid cancers. We investigated the effect of As2O3 on proliferation and cell cycle regulation with change in cyclins in two human glioblastoma cell lines differing in p53 status (U87MG-wt; T98G-mutated). Sensitivity to As2O3 varied depending on the dose with the IC50 of the U87MG and T98G cells being 1.78 and 3.55 microM, respectively. Analysis by laser scanning cytometry (LSC) indicated that As2O3 inhibited the proliferation of the two cell lines via cell cycle arrest both at the G1 and G2 phases. To address the mechanism of the antiproliferative effect of As2O3, we examined its effect on cell cycle-related proteins by means of LSC, confocal microscopy and Western blot analysis. As2O3 induced an increase in p53 level and a decrease in level of cyclin B1 combined with cell arrest at G2/M in both cell lines. Cell arrest in G1, however, was associated with a decline in cyclin D1 expression only in the wt U87MG cells. As2O3 also induced apoptosis of U87MG cells as evidenced by the presence of cells with fractional DNA content ( cell populations). The present evidence that As2O3 at relatively low concentration effectively inhibited proliferation of U87MG and T98G cells in vitro, suggests that the drug may be considered for in vivo testing on animal models and possibly clinical trials on glioma patients.     

Conditional transformation of rat embryo fibroblast cells by a cyclin D1-cdk4 fusion gene

Cyclin D1 gene overexpression is a frequent event in a number of human cancers. These observations have led to the suggestion that cyclin D1 alterations might play a role in the etiology of cancer. This possibility is supported by the finding that transfection of mammalian cells with cyclin D1 can accelerate progression through the G1 phase of the cell cycle. Moreover, cyclin D1 can function as an oncogene by cooperating with activated Ha-ras to transform primary rat embryo fibroblasts (REFs). In addition, cyclin D1 transgenics develop hyperplasia and neoplasia of the thymus and mammary gland. We have constructed a novel fusion gene consisting of full-length human cyclin D1 and cdk4 genes. This fusion gene was expressed in insect cells and the fusion protein was shown to be enzymatically active. The fusion gene was expressed in mammalian cells under the control of tet-repressor. This fusion gene immortalized primary REFs, and cooperated with activated Ha-ras to transform primary REFs, in terms of anchorage-independent growth in vitro and formation of tumors in vivo. Utilizing a tet-regulated gene expression system, we have shown that proliferation of stably transfected primary REFs in vitro and in vivo is dependent on the continued expression of the cyclin D1-cdk4 fusion gene. These cell lines could be useful in the discovery of novel cancer therapeutics to modulate cyclin D1.cdk4 activity.     

ErbB-2 induces the cyclin D1 gene in prostate epithelial cells in vitro and in vivo

The receptor tyrosine kinase ErbB-2 plays an important role in the regulation of growth factor-induced signal transduction cascades in the epithelium, and ErbB-2 is frequently overexpressed in epithelial tumors. Our previous studies on clinical prostate cancer specimens indicated that ErbB-2 expression was increased in patients undergoing hormone ablation therapy. We had also shown that the critical cell cycle regulatory gene cyclin D1 and its promoter were targets of proliferative signaling in prostate cancer cell lines, and that cyclin D1 was required for ErbB-2-induced mammary tumorigenesis. In the current studies, we found that increased ErbB-2 membrane expression correlated with increased nuclear cyclin D1 staining in clinical prostate cancer specimens, and that expression of ErbB-2 was capable of inducing cell cycle progression in human prostate cancer cell lines. We further showed that ErbB-2 induced the cyclin D1 promoter in DU145 cells, and that small interfering RNA knockdown of cyclin D1 protein levels blocked a significant proportion of the heregulin-induced cell cycle progression in LNCaP cells. Probasin promoter-targeted expression of an activated ErbB-2 isoform induced cyclin D1 expression in the mouse prostate, commensurate with prostate intraepithelial neoplasia. Together, these in vitro and in vivo studies identify cyclin D1 as a critical downstream target of ErbB-2 in the prostate epithelium, both of which are possible therapeutic targets for cancer intervention. Furthermore, our novel mouse model provides a useful platform for ongoing in vivo investigations of ErbB-2 signaling in the prostate epithelium.     

Critical role of cyclin D3 in TSH-dependent growth of thyrocytes and in hyperproliferative diseases of the thyroid gland

We report that cyclin D3 is rate limiting for G1 progression in thyroid follicular cells and that its constitutive upregulation by chronic stimulation of the TSH/cAMP pathway plays a role in human and experimental hyperproliferative diseases of the thyroid gland. These conclusions are supported by in vitro and in vivo studies. In rat thyrocytes (PC Cl 3 cells), cyclin D3 expression is enhanced in response to activation of the TSH/cAMP pathway. Interference with the expression of G1 cyclins (in particular cyclin D3) by the antisense methodology strongly reduced TSH-dependent proliferation of PC Cl 3 cells, indicating that proper progression through G1 requires cyclin D3. Accordingly, PC Cl 3 cells engineered to overexpress cyclin D3 (PC-D3 cells) show enhanced growth rate and elude hormone-dependence and contact inhibition. Using an animal experimental model of thyroid stimulation, we demonstrate that cyclin D3 is a key mediator of TSH-dependent proliferation of thyroid follicular cells also in vivo. Cyclin D3 protein levels were higher in the thyrocytes from glands of propylthiouracil-treated rats compared with control animals. The increase in cyclin D3 expression occurred after the propylthiouracil-induced increase in TSH levels and preceded the burst of cell proliferation. Finally, we found that cyclin D3 protein is expressed in a fraction of human goiters but it is strongly overexpressed in most follicular adenomas.     

SB-431542 and Gleevec inhibit transforming growth factor-beta-induced proliferation of human osteosarcoma cells

Transforming growth factor-beta (TGF-beta) has growth-stimulating effects on mesenchymal cells and several tumor cell lines. The signaling pathway for this effect is, however, not well understood. We examined how TGF-beta stimulates proliferation of MG63 human osteosarcoma cells. Two distinct type I receptors for TGF-beta, ALK-1 and ALK-5, were expressed and functional in MG63 cells. Of these two receptors, ALK-5 appears to be responsible for the growth stimulation because expression of constitutively active ALK-5, but not ALK-1, stimulated proliferation of MG63 cells. SB-431542 (0.3 microM), a novel inhibitor of ALK4/5/7 kinase, suppressed TGF-beta-induced growth stimulation. DNA microarray analysis as well as quantitative real-time PCR analysis of RNAs from TGF-beta-treated cells demonstrated that several growth factors, including platelet-derived growth factor AA, were induced in response to TGF-beta in MG63 cells. Gleevec (1 microM) as well as AG1296 (5 microM) inhibited TGF-beta-induced growth stimulation of MG63 cells, suggesting that platelet-derived growth factor AA was mainly responsible for the growth-stimulatory effect of TGF-beta. We also examined the mechanisms of perturbation of growth-suppressing signaling in MG63 cells. We found that expression of c-Myc, which is down-regulated by TGF-beta in many other cells, was up-regulated in MG63 cells, suggesting that up-regulation of c-Myc expression may be the mechanism canceling growth-suppressing signaling of TGF-beta in MG63 cells.     

Transient expression of cyclin D1 is sufficient to promote hepatocyte replication and liver growth in vivo.

Cyclin D1 regulates mitogen-dependent progression through G(1) phase in cultured cells, and its overexpression in malignant cells is thought to contribute to autonomous proliferation in vivo. However, previous studies in cell lines have not demonstrated that cyclin D1 is sufficient to trigger cell replication. In this study, we found that transient transfection of adult hepatocytes with cyclin D1 stimulated assembly of active cyclin D1/cdk4 complexes, robust hepatocyte proliferation, and liver growth in the intact animal. After several days, hepatocyte proliferation was inhibited despite the persistence of high levels of cyclin D1 and cyclin E, suggesting that endogenous antiproliferative mechanisms were induced. Our data suggest that this antiproliferative response includes the marked up-regulation of p21, which in turn inhibits cyclin D1/cdk4 and cyclin E/cdk2 complexes. This study offers further evidence that cyclin D1 plays a pivotal role in the regulation of hepatocyte proliferation in the liver. Furthermore, this model may offer a unique system to study the normal cellular response to cyclin D1 expression in vivo.     

Specific positive and negative effects of FLIP on cell survival in human prostate cancer

We demonstrate here for the first time novel positive and negative effects of the FLICE-like inhibitory protein (FLIP) on human prostate cancer cell survival. A proteaosome inhibitor, MG132, mediated cell cycle arrest at G2/M and apoptosis through p38 activation. Interestingly, FLIP was stabilized by MG132 and interacted with Raf-1, resulting in enhancement of p38 signals and cytotoxicity. In contrast, overexpression of FLIP inhibited ubiquitylation and proteasomal degradation of beta-catenin, resulting in increase of the target gene cyclin D1, colony formation and invasive activity. Immunohistochemical analysis and in vitro experiments in primary culture showed FLIP to be overexpressed, statistically associated with expression of beta-catenin/cyclin D1 in metastatic cells, the FLIP/beta-catenin/cyclin D1 signals contributing to colony formation and invasion, which were canceled by FLIP knock down. In contrast, MG132-induced cytotoxicity including apoptosis was strongly inhibited by reduction of FLIP. Taken together, the results indicate that FLIP plays an important role in development of metastatic prostate cancer by inhibiting proteasomal degradation of beta-catenin, whereas it is mainly involved in proteasome inhibitior-mediated cell cycle arrest and apoptosis through activating the Raf-1/p38 pathway. Furthermore, proteasome inhibitors may be effective drugs for advanced prostate cancers overexpressing FLIP.     

Sequential and progressive cyclin expression in human osteosarcoma cells - diagnostic and therapeutic implications

Cyclin proteins in association with cyclin-dependent protein kinase (Cdk) subunits function to execute critical cell cycle transitions; dysregulation of these enzymes may play a pivotal role in human tumorigenesis. In this study we characterize the successive expression of cyclins D1, E, A, and B, as well as a novel cyclin-like protein, in synchronized human MG-63 osteosarcoma cells. The physiological activation, subunit configuration, and subcellular localization of cyclin D1 during the G1 phase of the cell cycle is characterized in additional detail. An essential role for cyclin D1 in osteosarcoma cell proliferation is inferred by the efficacy of an antisense oligo-deoxynucleotide strategy directed against this locus. In addition, we report on the amplification and comparative over-expression of cyclin D1 in a Ewing's sarcoma cell line. These findings support a sequential model of cyclin expression and suggest that examination of the levels of specific cyclins may provide valuable diagnostic and/or prognostic information in the evaluation of proliferative disorders.     

A splice variant of Skp2 is retained in the cytoplasm and fails to direct cyclin D1 ubiquitination in the uterine cancer cell line SK-UT.

Cyclin D1 is an important regulator of the transition from G1 into S phase of the cell cycle. The level to which cyclin D1 accumulates is tightly regulated. One mechanism contributing to the control of cyclin D1 levels is the regulation of its ubiquitination. SK-UT-1B cells are deficient in the degradation of D-type cyclins. We show here that p27, a substrate of the SCF(Skp2) ubiquitin ligase complex, is coordinately stabilized in SK-UT-1B cells. Further, we show that expression of Skp2 in SK-UT-1B cells rescues the cyclin D1 and p27 degradation defect observed in this cell line. These results therefore indicate that the SCF(Skp2) ubiquitin ligase complex affects the ubiquitination of cyclin D1. In addition, we show that SK-UT-1B cells express a novel splice variant of Skp2 that localizes to the cytoplasm and that cyclin D1 ubiquitination takes place in the nucleus. We propose that the translocation of Skp2 into the nucleus is required for the ubiquitination of cyclin D1 and that the absence of the SCF(Skp2) complex in the nucleus of SK-UT-1B cells is the mechanism underlying the ubiquitination defect observed in this cell line. Finally, our data indicates that differential splicing of F-box proteins may represent an additional level of regulation of the F-box mediated ubiquitination pathway.     

Insulin-like growth factor binding protein 5 suppresses tumor growth and metastasis of human osteosarcoma

Osteosarcoma (OS) is the most common primary malignancy of bone. There is a critical need to identify the events that lead to the poorly understood mechanism of OS development and metastasis. The goal of this investigation is to identify and characterize a novel marker of OS progression. We have established and characterized a highly metastatic OS subline that is derived from the less metastatic human MG63 line through serial passages in nude mice via intratibial injections. Microarray analysis of the parental MG63, the highly metastatic MG63.2 subline, as well as the corresponding primary tumors and pulmonary metastases revealed insulin-like growth factor binding protein 5 (IGFBP5) to be one of the significantly downregulated genes in the metastatic subline. Confirmatory quantitative RT-PCR on 20 genes of interest demonstrated IGFBP5 to be the most differentially expressed and was therefore chosen to be one of the genes for further investigation. Adenoviral mediated overexpression and knockdown of IGFBP5 in the MG63 and MG63.2 cell lines, as well as other OS lines (143B and MNNG/HOS) that are independent of our MG63 lines, were employed to examine the role of IGFBP5. We found that overexpression of IGFBP5 inhibited in vitro cell proliferation, migration and invasion of OS cells. Additionally, IGFBP5 overexpression promoted apoptosis and cell cycle arrest in the G1 phase. In an orthotopic xenograft animal model, overexpression of IGFBP5 inhibited OS tumor growth and pulmonary metastases. Conversely, siRNA-mediated knockdown of IGFBP5 promoted OS tumor growth and pulmonary metastases in vivo. Immunohistochemical staining of patient-matched primary and metastatic OS samples demonstrated decreased IGFBP5 expression in the metastases. These results suggest 1) a role for IGFBP5 as a novel marker that has an important role in the pathogenesis of OS, and 2) that the loss of IGFBP5 function may contribute to more metastatic phenotypes in OS.     

Validation of cyclin D1/CDK4 as an anticancer drug target in MCF-7 breast cancer cells: Effect of regulated overexpression of cyclin D1 and siRNA-mediated inhibition of endogenous cyclin D1 and CDK4 expression

Summary We have examined the role of cyclin D1 and cyclin-dependent kinase-4 (CDK4) in the cell cycle progression and proliferation of MCF-7 breast cancer cells. Forced expression of cyclin D1 using a tetracycline-regulated expression system, and suppression of endogenous cyclin D1 and CDK4 using small interfering RNA (siRNA) were used to validate this protein complex as a drug target in cancer drug discovery. Overexpression of cyclin D1 increased both phosphorylation of the retinoblastoma gene product (RB) and passage through the G1–S phase transition, resulting in increased proliferation of cells. When cyclin D1 expression was shut off, growth rates fell below those seen in control cell lines transfected with the vector, indicating an increased dependence on this protein for proliferation. Inhibition of endogenous cyclin D1 or CDK4 expression by RNA interference resulted in hypophosphorylation of RB and accumulation of cells in G1. These results support the prevailing view that pharmacological inhibition of cyclin D1/CDK4 complexes is a useful strategy to inhibit the growth of tumors. Furthermore, since MCF-7 cells appear to be dependent on this pathway for their continued proliferation, it is a suitable cell line to test novel cyclin D1/CDK4 inhibitors.