Molecular evidence of IGFBP-3 dependent and independent VD3 action and its nonlinear response on IGFBP-3 induction in prostate cancer cells.

BACKGROUND: Clinical trials have been conducted to clarify the beneficial effects of VD3 (1α,25-dihydroxy vitamin D3, also known as calcitriol) treatment in prostate cancer. However, the molecular mechanisms underlying these effects are not fully understood. Recent studies on IGFBP-3 have indicated its intracellular functions in cell growth and apoptosis. The aim of this study was to confirm the benefits of low-dose VD3 treatment and clarify the molecular mechanisms underlying these beneficial effects in prostate cancer cells. METHODS: The molecular effects of simultaneous treatment of LNCaP cells and their genetically modified cell lines with low concentration of docetaxel and VD3 were biologically and biochemically analyzed. To further determine the effects of VD3 treatment on IGFBP-3 induction system, cells were temporarily treated with VD3 in combination with a transcriptional inhibitor or protein synthesis inhibitor. Bcl-2 protein and its mRNA behavior were also observed in Igfbp-3 expression-modified LNCaP cells to determine the involvement of IGFBP-3 in the suppression of Bcl-2 by VD3 treatment. RESULTS: Changes in IGFBP-3 expression levels in LNCaP cells indicated that it mediated the inhibition of cell growth induced by VD3 treatment. IGFBP-3 was also found to be a mediator of the enhanced cytotoxicity of prostate cancer cells to VD3 in combination with the anti-cancer drug. We further identified the distinct property of the IGFBP-3 induction system, wherein temporal VD3 stimulation-induced prolonged IGFBP-3 expression and VD3 treatment-induced increase in IGFBP-3 expression were optimized based on the protein concentration rather than the mRNA concentration. Meanwhile, Bcl-2 expression was down-regulated by VD3 treatment in an IGFBP-3-independent manner. CONCLUSION: These findings indicate the molecular mechanisms of IGFBP-3 induction stimulated by VD3 and IGFBP-3 independent Bcl-2 suppression by VD3 treatment in prostate cancer cells. The results could prompt a re-evaluation of VD3 usage in therapy for patients with prostate cancer.

Development of a dendritic cell-targeting lipopeptide as an immunoadjuvant that inhibits tumor growth without inducing local inflammation.

Materials used for the past 30 years as immunoadjuvants induce suboptimal antitumor immune responses and often cause undesirable local inflammation. Some bacterial lipopeptides that act as Toll-like receptor (TLR) 2 ligands activate immune cells as immunoadjuvants and induce antitumor effects. Here, we developed a new dendritic cell (DC)-targeting lipopeptide, h11c (P2C-ATPEDNGRSFS), which uses the CD11c-binding sequence of intracellular adhesion molecule-1 to selectively and efficiently activate DCs but not other immune cells. Although the h11c lipopeptide activated DCs similarly to an artificial lipopeptide, P2C-SKKKK (P2CSK4), via TLR2 in vitro, h11c induced more effective tumor inhibition than P2CSK4 at low doses in vivo with tumor antigens. Even without tumor antigens, h11c lipopeptide significantly inhibited tumor growth and induced tumor-specific cytotoxic T cells. P2CSK4 was retained subcutaneously at the vaccination site and induced severe local inflammation in in vivo experiments. In contrast, h11c was not retained at the vaccination site and was transported into the tumor within 24 hr. The recruitment of DCs into the tumor was induced by h11c more effectively, while P2CSK4 induced the accumulation of neutrophils leading to severe inflammation at the vaccination site. Because CD11b+ cells, but not CD11c+ cells, produced neutrophil chemotactic factors such as macrophage inflammatory protein (MIP)-2 in response to stimulation with TLR2 ligands, the DC-targeting lipopeptide h11c induced less MIP-2 production by splenocytes than P2CSK4. In this study, we succeeded in developing a novel immunoadjuvant, h11c, which effectively induces antitumor activity without adverse effects such as local inflammation via the selective activation of DCs.

Deflection of vascular endothelial growth factor action by SS18-SSX and composite vascular endothelial growth factor- and chemokine (C-X-C motif) receptor 4-targeted therapy in synovial sarcoma.

Synovial sarcoma (SS) is a malignant soft-tissue tumor characterized by the recurrent chromosomal translocation SS18-SSX. Vascular endothelial growth factor (VEGF)-targeting anti-angiogenic therapy has been approved for soft-tissue sarcoma, including SS; however, the mechanism underlying the VEGF signal for sarcomagenesis in SS is unclear. Here, we show that SS18-SSX directs the VEGF signal outcome to cellular growth from differentiation. Synovial sarcoma cells secrete large amounts of VEGF under spheroid culture conditions in autocrine fashion. SS18-SSX knockdown altered the VEGF signaling outcome, from proliferation to tubular differentiation, without affecting VEGF secretion, suggesting that VEGF signaling promoted cell growth in the presence of SS18-SSX. Thus, VEGF inhibitors blocked both host angiogenesis and spheroid growth. Simultaneous treatment with VEGF and chemokine (C-X-C motif) (CXC) ligand 12 and CXC receptor 4 inhibitors and/or ifosfamide effectively suppressed tumor growth both in vitro and in vivo. SS18-SSX directs the VEGF signal outcome from endothelial differentiation to spheroid growth, and VEGF and CXC receptor 4 are critical therapeutic targets for SS.

Favine/CCDC3 deficiency accelerated atherosclerosis and thrombus formation is associated with decreased MEF2C-KLF2 pathway.

Currently, no mouse models manifest calcification and thrombus formation, which is frequently associated with human atherosclerosis. We demonstrated that lack of Favine/CCDC3 in apoE knockout mice accelerated atherosclerosis accompanied by large cholesterol crystals and calcification, and also promoted thrombus formation in the left ventricle and arteries. Circulating Favine was detectable in WT mouse plasma. RNA-sequencing analysis of aortae in DKO mice showed similar gene expression patterns of human atherosclerosis with unstable and vulnerable plaques. Importantly, human FAVINE mRNA expressions were lower in atheroma plaque than in adjacent intact aortic tissue and decreased with the progression of atherosclerosis. Pathway analysis of aortae in DKO mice suggested the decrease of the MEF2C-KLF2-mediated transcriptional pathway. Favine insufficiency and its attenuated downstream pathways may increase atherosclerosis progression with calcification and thrombus, which have not previously been fully modeled in experimental animals. Favine and its downstream pathways may have therapeutic potential for atherosclerosis.

Role of Meis1 in mitochondrial gene transcription of pancreatic cancer cells.

Pre-B-cell leukemia transcription factor (PBX)/three-amino-acid loop extension (TALE) class transcription factors [PBX1-4, Meis homeobox (Meis) 1-3, pbx/knotted 1 homeobox (Prep) 1, 2] are involved in tumorigenesis and metastasis. To investigate further the function of PBX/TALE class transcription factors, mRNA expression profile after downregulation of each mRNA expression by siRNA transfection in pancreatic cancer cell line, Panc-1, was examined. Downregulation of Meis1 resulted in downregulation of mitochondrial genes, but those of PBX1 and PBX2 did not. Quantitative reverse transcription polymerase chain reaction confirmed downregulation of mitochondrial genes by Meis1 siRNA transfection. Chromatin immunoprecipitation assay revealed the binding of Meis1 to the mitochondrial promoter region that contained the putative Meis1 binding site. Luciferase reporter assay showed the increase of luciferase activity of a construct containing the Meis1 binding site compared with that with shorter fragment without Meis1 binding region. These findings indicate that Meis1 works as a transcription factor for mitochondrial genes in pancreatic cancer cells.

Clinical utility of apparent diffusion coefficient (ADC) values in patients with prostate cancer: can ADC values contribute to assess the aggressiveness of prostate cancer?

PURPOSE: To retrospectively evaluate the relationship between apparent diffusion coefficient (ADC) values and Gleason score (GS) in prostate cancer. METHODS: A total of 60 patients who underwent radical prostatectomy for clinically localized prostate cancer were selected for this study. Diffusion-weighted magnetic resonance (MR) images were obtained using a 1.5 T system. ADC values were analyzed between three groups: GS of 6 or less (n = 7); GS of 7 (n = 37); and GS of 8 or higher (n = 16). ADC values of the three GS groups were statistically analyzed in order to determine the relationship with GS. In the 37 patients with GS = 7 the difference in ADC values between GS 3+4 and GS 4+3 was analyzed. RESULTS: Median ADC values (10⁻³ mm² /s) of the three GS groups were 1.04 (GS = 6 or less), 0.867 (GS = 7), and 0.729 (GS = 8 or higher). Although there was considerable overlap among the groups, the differences in ADC were statistically significant (P < 0.0001). There was a significant inverse correlation between GS and ADC values (z = -0.437, P < 0.0005). Median ADC values (10⁻³ mm² /s) of GS 3+4 and GS 4+3 patients were 0.88 and 0.814, respectively (P < 0.05). CONCLUSION: ADC values showed a negative correlation with GS. Pathologically, however, there was considerable intrasubject heterogeneity.

Proliferation of estrogen-responsive mouse tumor cell line B-1F stimulated by Saiboku-to, but inhibited by Scutellaria baicalensis, a component of Saiboku-to.

We have demonstrated that the proliferation of estrogen-responsive mouse Leydig tumor cell line B-1F is induced via suppression of 5-lipoxygenase activity followed by decrease of leukotrienes (LTs). Additionally, it has been reported that LTD4 induces apoptosis in B-1F cells. In this study, we examined effects of Saiboku-to, a traditional Chinese medicine having suppressive activities for LT production and release, on the proliferation. Saiboku-to promoted, but Scutellaria baicalensis, one of components (herbs) of Saiboku-to, significantly inhibited the proliferation of B-1F cells in vitro and in vivo. The action of Scutellaria baicalensis in B-1F cells was studied in more detail. Although Scutellaria baicalensis consists of flavonoids, iridoids, volatile oils and others, it and its major constituents had no direct effect on estrogen binding sites in B-1F cells. B-1F cells treated with Scutellaria baicalensis showed morphological changes such as nuclear aggregation and fragmentation. DNA fragmentation was also observed, indicating that Scutellaria baicalensis induces apoptosis in B-1F cells and that it or its constituents might be a good resource for searching new drugs, especially anti-cancer drugs. Moreover, Saiboku-to promoted B-1F cell proliferation, but Scutellaria baicalensis inhibited it, showing complexity of action of traditional Chinese medicines.

Stat4 suppresses the proliferation of connective tissue-type mast cells.

Mast cells are the progeny of hematopoietic stem cells, and murine mast cells are usually divided into two distinct populations, mucosal mast cells (MMCs) and connective tissue-type mast cells (CTMCs). We previously reported that CTMCs expressed signal transducer and activator of transcription (Stat) 4, but MMCs did not. Stat4 is also expressed in T cells and plays important roles in their homeostasis. In the present study, we show that Stat4 is involved in the homeostasis of CTMCs. The number of skin CTMCs increased in Stat4-deficient Balb/c mice, but that of gastric MMCs did not, when compared to those in control Balb/c(+/+) mice. The comparison between cultured Stat4-deficient CTMCs and cultured Balb/c(+/+) CTMCs revealed that cell cycle progression and cyclin D3 expression in the cultured Stat4-deficient CTMCs were enhanced in a Stat3 activation-dependent manner. This phenotype was explained by upregulation of KitL-induced interleukin (IL)-6 acting in an autocrine manner in cultured Stat4-deficient CTMCs. These results show that Stat4 suppresses the proliferation of CTMCs by controlling IL-6 via an autocrine mechanism.

Blocking CD147 induces cell death in cancer cells through impairment of glycolytic energy metabolism.

CD147 is a multifunctional transmembrane protein and promotes cancer progression. We found that the anti-human CD147 mouse monoclonal antibody MEM-M6/1 strongly induces necrosis-like cell death in LoVo, HT-29, WiDr, and SW620 colon cancer cells and A2058 melanoma cells, but not in WI-38 and TIG-113 normal fibroblasts. Silencing or overexpression of CD147 in LoVo cells enhanced or decreased the MEM-M6/1 induced cell death, respectively. CD147 is known to form complex with proton-linked monocarboxylate transporters (MCTs), which is critical for lactate transport and intracellular pH (pHi) homeostasis. In LoVo cells, CD147 and MCT-1 co-localized on the cell surface, and MEM-M6/1 inhibited the association of these molecules. MEM-M6/1 inhibited lactate uptake, lactate release, and reduced pHi. Further, the induction of acidification was parallel to the decrease of the glycolytic flux and intracellular ATP levels. These effects were not found in the normal fibroblasts. As cancer cells depend on glycolysis for their energy production, CD147 inhibition might induce cell death specific to cancer cells.

Adiponectin deficiency enhances colorectal carcinogenesis and liver tumor formation induced by azoxymethane in mice.

AIM: To investigate the causal relationship between hypoadiponectinemia and colorectal carcinogenesis in in vivo experimental model, and to determine the contribution of adiponectin deficiency to colorectal cancer development and proliferation. METHODS: We examined the influence of adiponectin deficiency on colorectal carcinogenesis induced by the administration of azoxymethane (AOM) (7.5 mg/kg, intraperitoneal injection once a week for 8 wk), by using adiponectin-knockout (KO) mice. RESULTS: At 53 wk after the first AOM treatment, KO mice developed larger and histologically more progressive colorectal tumors with greater frequency compared with wild-type (WT) mice, although the tumor incidence was not different between WT and KO mice. KO mice showed increased cell proliferation of colorectal tumor cells, which correlated with the expression levels of cyclooxygenase-2 (COX-2) in the colorectal tumors. In addition, KO mice showed higher incidence and frequency of liver tumors after AOM treatment. Thirteen percent of WT mice developed liver tumors, and these WT mice had only a single tumor. In contrast, 50% of KO mice developed liver tumors, and 58% of these KO mice had multiple tumors. CONCLUSION: Adiponectin deficiency enhances colorectal carcinogenesis and liver tumor formation induced by AOM in mice. This study strongly suggests that hypoadiponectinemia could be involved in the pathogenesis for colorectal cancer and liver tumor in human subjects.

Expression of p21Cip1/Waf1 and p27Kip1 in small cell neuroendocrine carcinoma of the uterine cervix.

Small-cell neuroendocrine carcinoma of the uterine cervix (SCCC), a rare but malignant cervical neoplasm, has a highly aggressive phenotype that requires more intensive treatment than other cervical tumors. Immunohistochemical methods were used to compare the expression of p21Cip1/Waf1 and p27Kip1 in SCCC and squamous cell carcinoma, the most common type of cervical cancer. In SCCC, p21 expression was significantly reduced compared with squamous cell carcinoma, whereas expression of p27 was similar in both carcinomas. Reduced expression of p21 could be a helpful diagnostic marker and may contribute to the invasive phenotype of SCCC.

Methylcobalamin inhibits fibroblast growth factor-8 stimulated proliferation and induces apoptosis in Shionogi carcinoma cells.

SC-3 cells, an androgen-dependent mouse mammary carcinoma cell line, in response to androgen stimuli, induces the secretion of fibroblast growth factor (FGF-8), which in turn increases the proliferation of these cells. We have shown previously that methylcobalamin (MeCbl) decreases the levels of FGF-8 mRNA in SC-3 cells stimulated by testosterone, inhibiting the proliferation of SC-3 cells and inducing apoptosis. In the present study, we analyzed the effects of MeCbl on SC-3 cell proliferation in response to exogenous addition of FGF-8. Thymidine incorporation showed a significant decrease in SC-3 cells cultured with MeCbl. Immunocytochemistry for single-stranded DNA (ssDNA) and DNA fragmentation analysis demonstrated that MeCbl induced apoptosis in SC-3 cells, even in the presence of FGF-8. These results show that the addition of FGF-8 stimulates the proliferation of SC-3 cells under the androgen-depleted condition and that MeCbl might be able to interfere with FGF-8 action.

Promotion of malignant phenotype after disruption of the three-dimensional structure of cultured spheroids from colorectal cancer.

Individual and small clusters of cancer cells may detach from the edges of a main tumor and invade vessels, which can act as the origin of metastasis; however, the mechanism for this phenomenon is not well understood. Using cancer tissue-originated spheroids, we studied whether disturbing the 3D architecture of cancer spheroids can provoke the reformation process and progression of malignancy. We developed a mechanical disruption method to achieve homogenous disruption of the spheroids while maintaining cell-cell contact. After the disruption, 9 spheroid lines from 9 patient samples reformed within a few hours, and 3 of the 9 lines exhibited accelerated spheroid growth. Marker expression, spheroid forming capacity, and tumorigenesis indicated that stemness increased after spheroid disruption. In addition, the spheroid forming capacity increased in 6 of 11 spheroid lines. The disruption signature determined by gene expression profiling supported the incidence of remodeling and predicted the prognosis of patients with colorectal cancer. Furthermore, WNT and HER3 signaling were increased in the reformed spheroids, and suppression of these signaling pathways attenuated the increased proliferation and stemness after the disruption. Overall, the disruption and subsequent reformation of cancer spheroids promoted malignancy-related phenotypes through the activation of the WNT and ERBB pathways.

Establishment of a progesterone-sensitive cell line from human lung cancer.

For deveplopment and function of the lung, progesterone (Prog) fulfils important roles. In a recent report, immunolocalization of Prog and estrogen receptors in non-small cell lung carcinomas were examined and it was shown that the Prog receptor might be a potent prognostic factor. In the present study, a cell line with the sensitivity to Prog was established from a human lung cancer and the growth mechanism was analyzed. The proliferation of established SN96-42 cells was sensitive to Prog and antiprogesterone RU38486 inhibited their proliferation stimulated by Prog. Exposure of these cells to Prog resulted in a decreased formation of leukotriene (LT). The 5-lipoxygenase inhibitor (5-LOX), AA861, effectively stimulated SN96-42 cell proliferation and 5-LOX-catalyzed product(s), especially LTC4, inhibited SN96-42 cell proliferation caused by Prog. Prog-sensitive enhancement of SN96-42 cell proliferation is at least partly mediated through an inhibition of LT formation and these data suggest that 5-LOX and LTs play important roles in SN96-42 cell proliferation stimulated by Prog.

Nuclear expression of STAT5 in intraductal papillary mucinous neoplasms of the pancreas.

Intraductal papillary mucinous neoplasms (IPMNs) are noninvasive lesions of the pancreas and classified as intraductal papillary mucinous adenomas (IPMAs), borderline IPMNs, and intraductal papillary mucinous carcinomas (IPMCs). Expression patterns of the specific genes alter during IPMN progression. Based on the evidence that signal transducers and activators of transcription (STAT) 5 play important roles in tumor development, we tested STAT5 expression in IPMAs, borderline IPMNs, and IPMCs by immunohistochemical method. STAT5 frequently expressed in the nuclei of tumor cells of borderline IPMNs or IPMCs but was not observed in those of IPMAs. Nuclear expression of STAT5 protein correlated to the Ki-67 labeling index of the examined IPMNs. STAT5 protein could contribute to the progression and proliferation of IPMNs.

Development of effective tumor immunotherapy using a novel dendritic cell-targeting Toll-like receptor ligand.

Although dendritic cell (DC)-based immunotherapy shows little toxicity, improvements should be necessary to obtain satisfactory clinical outcome. Using interferon-gamma injection along with DCs, we previously obtained significant clinical responses against small or early stage malignant tumors in dogs. However, improvement was necessary to be effective to largely developed or metastatic tumors. To obtain effective methods applicable to those tumors, we herein used a DC-targeting Toll-like receptor ligand, h11c, and examined the therapeutic effects in murine subcutaneous and visceral tumor models and also in the clinical treatment of canine cancers. In murine experiments, most and significant inhibition of tumor growth and extended survival was observed in the group treated with the combination of h11c-activated DCs in combination with interferon-gamma and a cyclooxygenase2 inhibitor. Both monocytic and granulocytic myeloid-derived suppressor cells were significantly reduced by the combined treatment. Following the successful results in mice, the combined treatment was examined against canine cancers, which spontaneously generated like as those in human. The combined treatment elicited significant clinical responses against a nonepithelial malignant tumor and a malignant fibrous histiocytoma. The treatment was also successful against a bone-metastasis of squamous cell carcinoma. In the successful cases, the marked increase of tumor-responding T cells and decrease of myeloid-derived suppressor cells and regulatory T cells was observed in their peripheral blood. Although the combined treatment permitted the growth of lung cancer of renal carcinoma-metastasis, the marked elevated and long-term maintaining of the tumor-responding T cells was observed in the patient dog. Overall, the combined treatment gave rise to emphatic amelioration in DC-based cancer therapy.

Inhibition by interleukin-18 of the growth of Dunn osteosarcoma cells.

To examine the usefulness of interleukin-18 (IL-18) in the treatment of osteosarcomas, the effect of IL-18 on the growth of Dunn osteosarcoma cells was investigated. Daily intraperitoneal (i.p.) injection of mouse recombinant IL-18 (2 microg/mouse) suppressed the growth of Dunn osteosarcoma cells transplanted subcutaneously (s.c.) into syngeneic C3H mice. This IL-18-induced suppression was not affected by simultaneous treatment with anti-asialo GM1 serum, which inactivates natural killer (NK) cells. However, IL-18 failed to suppress the growth of Dunn osteosarcoma cells transplanted into BALB/c-nude mice devoid of T lymphocytes or C3H-gld/gld mice deficient in functional Fas ligand (FasL). IL-18 also failed to suppress the growth of Dunn osteosarcoma cells in vitro, although expression of IL-18 receptor mRNA and MyD88 mRNA as well as Fas mRNA was detected by reverse transcriptase-polymerase chain reaction (RT-PCR). On the other hand, antimouse Fas antibody showed cytotoxicity against Dunn osteosarcoma cells in a dose-dependent manner in vitro. In addition, treatment of C3H mice with IL-18 enhanced the cytotoxic activity of CD8(+) T lymphocytes against Dunn osteosarcoma cells. These results indicate that IL-18 inhibits the growth of Dunn osteosarcoma cells in vivo by enhancing the cytotoxic activity of CD8(+) T lymphocytes through the FasL-Fas system.

Time trends for small gastric cancer in Japan.

BACKGROUND: Gastric cancer rates in Japan have been declining since the 1970s. The rate of differentiated carcinomas has decreased and that of undifferentiated carcinomas has increased. However, little is known about the time trends of small gastric cancer. The aim of this study was to investigate the trends of small gastric cancer over time in Japan. METHODS: We reviewed cases of small gastric cancer (less than 20 mm in diameter) in two groups of patients who entered the age range of 55-to-67 years 14 years apart: patients in cohort 1 (n = 66) were born between 1899 and 1912, and those in cohort 2 (n = 66) were born between 1926 and 1936. Between-group comparisons were made for macroscopic, microscopic, and histochemical findings. Mucin histochemical analysis was used to investigate gastric and nongastric phenotypes. Helicobacter pylori was also investigated by immunohistochemistry. RESULTS: There were significant decreases in the incidence of elevated carcinoma (20% in cohort 1 vs 6% in cohort 2; P < 0.05) and papillary adenocarcinoma (11% vs 2%; P < 0.05). The incidence of flat carcinomas was significantly increased (3% vs 15%; P < 0.05). The incidence of tumors surrounded by fundic gland mucosa increased (20% vs 29%), whereas that of tumors surrounded by intestinal metaplastic mucosa decreased (52% vs 41%). The rate of H. pylori infection in mucosa surrounding tumors was the same in both groups (35%). The incidence of tubular adenocarcinoma with gastric-type mucin was higher in cohort 2 (64%) than in cohort 1 (51%). CONCLUSION: The rate of tubular adenocarcinomas containing gastric type mucin has increased over time. These tumors had a tendency to develop in the fundic gland mucosa and to show less intestinal metaplasia. The H. pylori infection rate was unrelated to this time trend. In advanced gastric cancer, the differentiated carcinoma rate has decreased; however, in small gastric cancer, the rate of tubular adenocarcinoma containing gastric type mucin has increased. This suggests that tubular adenocarcinoma with gastric type mucin changes into poorly differentiated adenocarcinoma as tumors grow to advanced stages.

Inhibition of murine osteosarcoma cell proliferation by glucocorticoid.

The effects of glucocorticoid (GC) on the proliferation of Dunn Osteosarcoma (OS) cells were examined under in vitro culture conditions. Dexamethasone (Dex) inhibited the proliferation of Dunn OS cells in a dose-dependent manner, while the addition of anti-GC, RU486, to the culture medium in part recovered Dex-induced growth inhibition. The number of maximum binding sites (Bmax) and the dissociation constant (Kd) value of glucocorticoid receptor (GR) in Dunn OS cells were 19,560 sites/cell and 5.2 +/- 0.8 nM, respectively. RU486 competed with labeled Dex against GR at a concentration of 10(-6) M. Western blot analysis of [3H]Dex-mesylate-labeled cell homogenate and immunohistochemical staining against GR further confirmed the presence of GR. Dex treatment of Dunn OS cells resulted in apoptosis with the characteristic internucleosomal DNA cleavage shown by the DNA ladder pattern in agarose gel electrophoresis. These data demonstrate that GC inhibits the proliferation of Dunn OS cells via GR, for which one possible mechanism in vitro is induction of apoptosis.