BTG1 Overexpression Might Promote Invasion and Metastasis of Colorectal Cancer via Decreasing Adhesion and Inducing Epithelial-Mesenchymal Transition.

BTG (B-cell translocation gene) could inhibit cell proliferation, metastasis, and angiogenesis and regulate cell cycle progression and differentiation in a variety of cancer cell types. To clarify the role of BTG1 in invasion and metastasis, its expression was compared with the clinicopathological parameters of colorectal cancer by bioinformatics and immunohistochemical analyses. We also overexpressed BTG1 in HCT-15 cells and examined its effects on adhesion, migration, and metastasis with their related molecules screened. BTG1 mRNA expression was negatively correlated with its promoter methylation in colorectal cancer (P < 0.05). Among them, cg08832851 and cg05819371 hypermethylation and mRNA expression of BTG1 were positively related with poor prognosis of the colorectal cancer patients (P < 0.05). BTG1 expression was found to positively correlate with depth of invasion, venous invasion, lymph node metastasis, distant metastasis, and TNM staging of colorectal cancer (P < 0.05) but negatively with serum levels of CEA and CA19-9 (P < 0.05). According to the TCGA database, BTG1 mRNA expression was lower in well-, moderately, and poorly differentiated than mucinous adenocarcinomas and positively correlated with ras or BRAF mutation (P < 0.05). Kaplan-Meier analysis showed the negative correlation between BTG1 mRNA expression and overall survival rate of all cancer patients (P < 0.05). BTG1 overexpression weakened adhesion and strengthened migration and invasion of HCT-15 cells (P < 0.05). There was E-cadherin hypoexpression, N-cadherin and MMP-9 hyperexpression, Zeb1 and Vimentin mRNA overexpression, a high expression of CEA mRNA and protein, and a strong secretion of CEA in BTG1 transfectants, compared with the control or mock. It was suggested that BTG1 expression might promote invasion and metastasis by decreasing adhesion, and inducing epithelial-mesenchymal transition.

Valproic Acid Increased Autophagic Flux in human Multiple Myeloma Cells in Vitro.

BACKGROUND: To investigate the effects of valproic acid (VPA) on autophagic flux in multiple myeloma (MM) cells. METHODS AND RESULTS: Cell proliferation was assayed by the Cell Counting Kit-8 assay. The qRT-PCR was used to measure the expressions of LC3-II at mRNA level. Autophagic flux was measured by LC3-II turnover using western blot analysis and flow cytometry using the fluorescent dye Cyto-ID. An assay using the RFP-GFP-LC3 tandem construct was performed to monitor autophagic flux. Cell proliferation assay showed that VPA could inhibit the proliferation of MM cells and the inhibitory effects were enhanced with the extension of time. The qRT-PCR and western blot showed that the expression level of LC3-II in the VPA plus CQ group was significantly higher than that in CQ group. Cyto-ID autophagy test showed that the intracellular average fluorescence intensity in VPA plus CQ group was significantly higher than that in control and VPA group (all p < 0.001). The results of RFP-GFP-LC3 tandem construct showed that the numbers of yellow puncta and red puncta in VPA group was higher than that in control group. CONCLUSIONS: VPA could inhibit the proliferation of MM cells and the inhibitory effects were enhanced with the extension of time. VPA could enhance autophagic flux in MM cells, and the increase of autophagosomes was caused by autophagy enhancement rather than inhibition. These findings provided rationale for the treatment of MM with VPA.

The Suppressing Effects of Dkk3 Expression on Aggressiveness and Tumorigenesis of Colorectal Cancer.

Dkk3 has been discovered during comparison of immortalized and parental cells. Its expression has been shown to reduce colony formation and induce apoptosis of cancer cells, acting as a tumor suppressor. Herein, we demonstrate that Dkk3 overexpression or protein treatment may inhibit colorectal cancer cell proliferation, migration, and invasion and that they may promote apoptosis and G2 phase arrest with hypoexpression of Bcl-2, cdc25B, cdc25c, N-cadherin, slug, and twist and hyperexpression of Bax and E-cadherin. This effect is consistent with that of recombinant Dkk3 exposure and blocked with anti-Dkk3 antibody. Dkk3 deletion in intestinal cells was not associated with the emergence of epithelial lesions; however, adenoma emerged after sodium desoxycholate treatment. At both mRNA and protein levels, Dkk3 expression was higher in normal than in cancer tissues (p<0.05). Dkk3 mRNA expression was negatively associated with its promoter methylation, growth pattern, differentiation, and favorable prognosis in the patients with colorectal cancer (p<0.05). Dkk3-related signal pathways in colorectal cancer included those of cellular adhesion and migration, melanogenesis, chemokine, Hedgehog, JAK-STAT, TOLL-like receptor, TGF-ß, MAPK, and calcium signaling (p<0.05). These findings indicate that Dkk3 expression levels can help assess cancer aggressiveness and patient prognosis. It might also suppress aggressive phenotypes and tumorigenesis as a molecular target in gene therapy.

The Roles of Beclin 1 Expression in Gastric Cancer: A Marker for Carcinogenesis, Aggressive Behaviors and Favorable Prognosis, and a Target of Gene Therapy.

Beclin 1 is encoded by Becn1, and plays a role in tumorigenesis, neurodegeneration, apoptosis and autophagy. Here, the aggressive phenotypes and relevant proteins were examined after Beclin 1 expression was altered in gastric cancer cells. We also observed the effects of Beclin 1 on gastric carcinogenesis using Becn1 knockout mice. Finally, clinicopathological significances of Beclin 1 expression were analyzed using meta- and bioinformatics analyses. Becn1 overexpression was found to inhibit proliferation, glucose metabolism, migration and invasion of gastric cancer cells, whereas its knockdown caused the opposite effects. Beclin 1 suppressed the tumor growth by decreasing proliferation and increasing apoptosis. The heterozygous abrogation of Becn1 in gastric pit, parietal and chief cells could not cause any epithelial lesion. Beclin 1-mediated chemoresistance was closely linked to the autophagy, Bax underexpression, and the overexpression of Bcl-2, LRP1, MDR1, and ING5. Bioinformatics analysis showed higher Becn1 mRNA expression in intestinal- than diffuse-type carcinomas (P<0.05), and in male than female gastric cancer patients (P<0.05). Becn1 hyperexpression was positively associated with both overall and progression-free survival rates of the cancer patients (P<0.05). Meta-analysis showed that down-regulated Beclin 1 expression in gastric cancer was positively with lymph node metastasis, TNM staging, dedifferentiation and poor prognosis (P<0.05). Becn1-related signal pathways in gastric cancer included prostate, lung, renal, colorectal, endometrial and thyroid cancers, glioma, and leukemia, the metabolism of amino acid, lipid and sugar, and some signal pathways of insulin, MAPK, TRL, VEGF, JAK-STAT, chemokine, p53, lysosome, peroxidome and ubiquitin-mediated protein degradation (P<0.05). These suggested that Beclin 1 might be considered as a potential marker of gastric carcinogenesis, aggressiveness and prognostic prediction, and as a target of gene therapy in gastric cancer.

[Effects of VPA on the Expression of Notch Signaling Pathway in Multiple Myeloma RPMI 8226 Cell Line].

OBJECTIVE: To investigate the effects of valproic acid(VPA) on the expression of intracellular domain of Notch1 (ICN1) and Hes1 in multiple myeloma RPMI 8226 cell line. METHODS: Experiments were divided into 4 group: blank control group and groups of cells treated with VPA of different concentration (2, 4, 8 mmol/L), the cell proliferation was detected by MTT method, RT-PCR was applied to detect the mRNA expression level of ICN1 and Hes1. Western blot was used to detect the protein expression of ICN1 and Hes1. RESULTS: The proliferation of the RPMI 8226 cell was obviously inhibited by different concentration of VPA (2, 4, 8 mmol/L) at the same time. The same concentration of VPA was used to treat RPMI8226 cell for different time (24, 48, 72 h), the cell proliferation was obviously inhibited. Compared with control group, the mRNA and protein expression of ICN1 was significantly depressed at different concentration of VPA(2, 4, 8 mmol/L) for 48 h (P<0.05). Compared with control group, the mRNA and protein expression of Hes1 was depressed at different concentration 2, 4, 8 mmol/L)of VPA for 48 h (P<0.05). CONCLUSION: VPA inhibits the proliferation of the RPMI 8226 cell in a time- and dose- dependent manner; VPA down-regulates the mRNA and protein expression level of ICN1 and Hes1 in RPMI8226 cell; thus VPA might inhibit cell proliferation possibly through the inhibition of Notch signaling pathway in multiple myeloma cells.

Morphine improved the antitumor effects on MCF-7 cells in combination with 5-Fluorouracil.

BACKGROUND: The most frequently used opioid in cancer pain management is morphine which remains a cornerstone for the management of cancer pain, due to the largest experience existing among physicians and widely availability in a variety of formulation. Considering that analgesics on cancer pain is often under the condition of chemotherapy and 5-Fluorouracil (5-FU) is widely used today as a potent drug for the treatment of advanced cancers, whether analgesics such as morphine, interferes the chemotherapy such as 5-FU, arose as a considerable problem. METHODS: In this study, the MCF-7 breast cancer cells were used to determine the antitumor effects of the 5-FU in combination with morphine. The cell proliferation was determined by the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay and the apoptosis was determined by the Annexin V/PI staining and flow cytometry. The immunocytochemistry and western blot was used to determine the Bcl-2 and Bax expression. RESULTS: It was shown that in MCF-7 cells, the proliferation was inhibited, the apoptosis was promoted, the Bcl-2 expression was suppressed and the Bax expression was promoted by both 5-FU alone and morphine alone, while the superior effects were achieved in combination with the two drugs. CONCLUSION: These results suggest that the morphine may have the beneficial effects on the antitumor chemotherapy with 5-FU, in stead of interferential effects.

Valproic acid inhibits tumor angiogenesis in mice transplanted with Kasumi­1 leukemia cells.

Histone deacetylase (HDAC) inhibitors have been reported to inhibit tumor angiogenesis via the downregulation of angiogenic factors. Our previous in vitro studies demonstrated that valproic acid (VPA) exerted antitumor effects on Kasumi­1 cells, which are human acute myeloid leukemia cells with an 8;21 chromosome translocation. In the present study, the effects of VPA on tumor angiogenesis were investigated in mice transplanted with Kasumi­1 cells. Semi­quantitative reverse transcription­polymerase chain reaction, western blotting and immunohistochemistry were used to detect the expression of vascular endothelial growth factor (VEGF), VEGF receptor (VEGFR2) and basic fibroblast growth factor (bFGF). The tumor microvessel density was measured following staining with an anti­CD34 antibody. Chromatin immunoprecipitation was used to study the effect of VPA­induced histone hyperacetylation on VEGF transcription. An intraperitoneal injection of VPA inhibited tumor growth and angiogenesis in mice transplanted with Kasumi­1 cells. The mRNA and protein expression of VEGF, VEGFR2 and bFGF were inhibited by VPA treatment. In addition, VPA downregulated HDAC, increased histone H3 acetylation and enhanced the accumulation of hyperacetylated histone H3 on the VEGF promoters. The findings of the present study indicate that VPA, an HDAC inhibitor, exerts an antileukemic effect through an anti­angiogenesis mechanism. In conclusion, the mechanism underlying VPA­induced anti­angiogenesis is associated with the suppression of angiogenic factors and their receptors. VPA may increase the accumulation of acetylated histones on the VEGF promoters, which possibly contributes to the regulation of angiogenic factors.

[Effect of valproic acid against angiogenesis of Kasumi-1 xenograft tumor in nude mice].

This study was aimed to investigate the effect of valproic acid (VPA), a histone deacetylase inhibitor, on angiogenesis of acute myeloid leukemia in vivo and vitro, and to explore its molecular mechanism. Human t (8;21) AML cell line Kasumi-1 cells were treated with VPA at different concentration for 3 d, the mRNA and protein expression levels of Ang1 and Ang2 were determined by semi-quantitative RT-PCR and Western blot respectively. Nude mice model with xenograft Kasumi-1 tumor was established by subcutaneous inoculation of Kasumi-1 cells. The CD34, Ang1 and Ang2 protein levels were analyzed by immunohistochemistry method. The mRNA and protein expression levels of Ang1, Ang2 and VEGF were determined by semi-quantitative RT-PCR and Western blot. The results showed that in vitro, VPA at 3 mmol/L downregulated the Ang mRNA relative expression level for Ang1 from 0.360 ± 0.116 to 0.040 ± 0.008, Ang2 from 0.540 ± 0.049 to 0.146 ± 0.038. The animal experiment further verified that VPA 500 mg/kg, ip, for 14 d, reduced the relative expression of Ang1, Ang2 and VEGF mRNA and proteins in Kasumi-1 tumor of nude mice, and reduced microvascular density in xenograft tumor of nude mice (8.470 ± 0.300 vs 2.600 ± 0.200). It is concluded that VPA significantly inhibits tumor angiogenesis through the regulation of angiopoietins, thereby inhibits the proliferation and metastasis of leukemia cells.

[Inhibitory effect of valproic acid on xenografted Kasumi-1 tumor growth in nude mouse and its mechanism].

OBJECTIVE: To investigate in vivo inhibitory effect of histone deacetylase (HDAC) inhibitor valproic acid (VPA) on xenografted Kasumi-1 tumor in nude mice and its mechanism. METHODS: Xenografted Kasumi-1 tumor mouse model was established by subcutaneous inoculation of Kasumi-1 cells. Xenotransplanted nude mice were assigned into control or VPA treatment groups. Volume of the xenografted tumors was measured and compared between the two groups. Terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling (TUNEL) was applied to detection of tumor cell apoptosis. The gene expression of GM-CSF, HDAC1, Ac-H3 and survivin was studied with semi-quantitative RT-PCR and Western blotting. ChIP method was used to assay the effects of VPA on acetylation of histone H3 within GM-CSF promoter region. RESULTS: (1) VAP significantly inhibited xenografted Kasumi-1 tumor growth. The calculated inhibition rate was 57.25%. (2) Morphologic study showed that VPA induced differentiation and apoptosis of Kasumi-1 tumor cells. The apoptosis index of VAP treatment group [(3.661 +/- 0.768)%] was significantly higher than that of control group [(0.267 +/- 0.110)%]. (3) Comparing to those in control group, the level of nuclear HDAC1 protein was significantly decreased, the Ac-H3 protein expression level was increased, the mRNA and protein expression levels of GM-CSF and acetylation of histone H3 were remarkably increased, and the gene expression level of survivin significantly decreased in VPA treatment group. CONCLUSION: VAP significantly inhibits xenografted Kasumi-1 tumor growth and induces tumor cell differentiation and apoptosis. The mechanism may be decrease of survivin gene expression, inhibition of nuclear expression of HDAC, promotion of histone protein acetylation level and acetylation of histone H3 within GM-CSF promoter region, and increase of GM-CSF transcription.

[Effects of histone deacetylase inhibitor on the expression of angiogenesis related factors in Kasumi-1 leukemic cell line].

OBJECTIVE: To investigate the effects of two histone deacetylase (HDAC) inhibitors, valproic acid (VPA) and TSA, on the expression of vascular endothelial growth factor (VEGF) and its receptor KDR of the leukemia cell line Kasumi-1 cells, and to explore their potential mechanism in leukemia angiogenesis. METHOD: Kasumi-1 cells were treated with VPA and TSA at different concentrations for 3 days. The mRNA and protein expression levels of VEGF and KDR were determined by semi-quantitative RT-PCR and Western blot, and the bFGF mRNA by semi-quantitative RT-PCR. RESULTS: As compared with that of control groups, VPA at 3 mmol/L downregulated the VEGF mRNA expression level for VEGF(121) from 0.632 ± 0.014 to 0.034 ± 0.004 and for VEGF(165) from 0.526 ± 0.021 to 0.015 ± 0.001, for KDR mRNA from 0.258 ± 0.034 to 0.038 ± 0.000, and for bFGF mRNA from 0.228 ± 0.017 to 0.086 ± 0.015. TSA downregulated the VEGF mRNA and KDR mRNA at concentration of 100 nmol/L, but its effect on bFGF mRNA only at higher concentration. CONCLUSION: HDAC inhibitors might inhibit the leukemia angiogenesis by regulating the expression of VEGF and its recptor.

[Reversed effect of valproic acid on transcription inhibition of AML1-ETO fusion protein of kasumi-1 leukemic cell line].

This study was aimed to investigate the mechanism of histone deacetylase (HDAC) inhibitor, valproic acid (VPA), reversing transcription inhibition of AML1-ETO fusion protein in Kasumi-1 cell line. The mRNA expressions of AML1-ETO, AML1 and cyclin D2 were detected by semi-quantitation RT-PCR after treating kasumi-1 cells with VPA at different doses/and different time points. The results indicated that the mRNA expression of AML1-ETO showed no obvious change, when kasumi-1 cells were treated with VPA. Compared with control group, the expression level of AML1 mRNA significantly increased in a dose-dependent manner. Compared with control group, the expression level of cyclin D2 mRNA significantly decreased when kasumi-1 cells had been treated with 3 mmol/L VPA as well as kasumi-1 cells were treated with different concentrations of VPA for 3 days. In conclusion, VPA could remove transcription inhibition of AML1-ETO fusion protein, increase transcription of AML1 and down-regulate mRNA expression of AML1 target gene cyclin D2 through HDAC inhibiting activity.

[5-Aza-2'-deoxycytidine enhances differentiation and apoptosis induced by phenylbutyrate in Kasumi-1 cells].

OBJECTIVE: To investigate whether phenylbutyrate (PB) combined with 5-aza-2'-deoxycytidine (5-Aza-CdR)could inhibit transcription repression and induce t(8;21) acute myelogenous leukemia (AML) Kasumi-1 cells to differentiate and undergo apoptosis. METHODS: Kasumi-1 cells were treated with PB and 5-Aza-CdR at different concentrations in suspension culture. Cellular proliferation was determined by the MTT assay, expression of myeloid-specific differentiation antigen and cell cycles were analyzed by flow cytometry. Cell apoptosis were assessed using AnnexinV/PI staining and flow cytometry. RESULTS: Treatment of Kasumi-1 cells with PB caused a dose-dependent inhibition of proliferation, with an IC(50) of 2.3 mmol/L. When combined with 5-Aza-CdR, PB resulted in a greater growth inhibition with an IC(50) of 1.95 mmol/L. Treatment of Kasumi-1 cells with PB resulted in cell cycle arrest at G(0)/G(1), while combined treatment with PB and 5-Aza-CdR led to cell cycle arrest at G(2)/M. Expression of myeloid cell differentiation antigens CD11b and CD13 induced by PB was enhanced when Kasumi-1 cells were pretreated with low dose of 5-Aza-CdR. High, but not low, concentrations of 5-Aza-CdR could enhance early apoptosis of Kasumi-1 cells induced by PB. CONCLUSION: Phenylbuty rate, when combined with 5-Aza-CdR, inhibits AML cell in vitro proliferation and increases apoptosis in a synergistic fashion.

[Combination of phenylbutyrate and 5-Aza-2'deoxycytidine inhibits human Kasumi-1 xenograft tumor growth in nude mice].

OBJECTIVE: To investigate the tumor suppression efficacy of histone deacetylase inhibitor, phenylbutyrate (PB), in combination with DNA methylation inhibitor 5-Aza-2-deoxycytidine (5-Aza-CdR) in the treatment of Kasumi-1 xenograft tumor in nude mice and its mechanism. METHODS: The nude mice model of Kasumi-1 xenograft tumor was established by subcutaneous inoculation. Latency of tumor formation, the ability of Kasumi-1 cells pre treated with PB to form the xenograft tumor, and the tumor suppression activity of PB and 5-Aza-CdR by intraperitoneal injection in xenografted mice model were detected. Cell differentiation and cell cycle parameters of the tumor cells were analyzed by flow cytometry analysis, apoptosis by TUNEL in situ hybridization, and tumor microvessel density (MVD) by immunohistochemistry study. RESULTS: The latency of tumor formation in mice with or without previous lienectomy was 17 approximately 23 and 40 approximately 50 days, respectively. Tumor cells xenografted could not be found in other tissues than in inoculation area, and still harbored the specific t(8;21) and AML1-ETO fusion gene. When the xenografted mice models treated with PB, 5-Aza-CdR, or both, the tumor growth inhibition rates were 49.07%, 25.69% and 87.46% (P < 0.05), the apoptosis indexes (AI) of tumor cells were (2.25 +/- 0.85)%, (1.32 +/- 0.68)%, and (5.41 +/- 1.56)% (P < 0.05), and the microvessel densities (MVD) were 21.69 +/- 6.25, 28.34 +/- 4.24 and 9.48 +/- 3.21 (P < 0.01), respectively. All the data above were significantly different from that in control (P < 0.05). The expression of CD11b and CD13 antigen of the tumor cells was increased in xenografted mice model treated with PB when compared with the control \[(12.08 +/- 1.02)% and (54.91 +/- 2.72)%\], respectively (P < 0.01), and tumor cells showed a cell cycle arrest with increased G(0)/G(1)-phase cells and decreased S-phase cells. CONCLUSION: PB inhibited the growth of Kasumi-1 xenograft tumor by inducing tumor cell apoptosis and differentiation, and suppressing its angiogenesis in vivo. 5-Aza-CdR could significantly enhance the antitumor activity of PB.

[Effect of phenylbutyrate, a histone deacetylase inhibitor, on differentiation and apoptosis of Kasumi-1 cells].

OBJECTIVE: To explore the blockade effect of phenylbutyrate (PB), a histone deacetylase inhibitor, on the in vitro biological function of AML1/ETO to reverse its transcription repression and induce Kasumi-1 cells to differentiate and apoptosis. METHODS: Kasumi-1 cells were treated with PB at different concentrations in suspension culture. Cell proliferation was analysed by MTT assay, morphological changes by light and electron microscopy, expression of myeloid-specific differentiation antigen and cell cycle by flow cytometry, cell apoptosis by annexin V staining, agarose gel electrophoresis and flow cytometry. RESULTS: PB treatment caused a dose-dependent inhibition of the cell proliferation. The IC(50) was about 2.3 mmol/L. PB treatment led to a progressive decline in the fraction of S-phase cells and increase in G(0)/G(1) cells. PB induced a time- and dose-dependent increase in expression of myeloid cell surface protein CD(11b) and CD(13). A dose-dependent increase in early apoptosis for 2 days treatment, late apoptosis for 3 days treatment. The DNA ladder of apoptosis was observed on agarose gel electrophoresis for 5 days treatment. Morphological features of monocytoid differentiation and apoptosis were seen on Wright-Giemsa staining smears. CONCLUSION: PB treatment could inhibit proliferation of Kasumi-1 cells, induce partial differentiation, apoptosis and accumulation of cells in G(0)/G(1) phase.

[Analysis and identification of transcriptional repression domain of ETO].

OBJECTIVE: To further verify the transcriptional repression domains in ETO and their relationship with histone deacetylase (HDAC). METHODS: Either of the ETO two zinc fingers was mutated respectively by site-directing mutagenesis. The truncation fragments of ETO were amplified by polymerase chain reaction (PCR) and cloned into eukaryotic expression plasmid pFA-CMV. By the means of DNA transfection and analysis of the transcription derived from the promoter of reporter gene, the transcriptional regulation domains of ETO was determined. RESULTS: The expression plasmids carrying truncated ETO and ETO with point mutation at either zinc finger were successfully constructed. Two repression domains were found within ETO, which were located at two zinc finger motifs and 275 - 487 amino acid residues, respectively. CONCLUSION: The transcription repression by ETO was mediated by two separated domains and closely associated with HDAC, which may be used as therapeutic target for acute myeloid leukemia M(2b).