Clone Formation Ability Research Articles

Effects of RSF-1 on proliferation and apoptosis of breast cancer cells.

Effect of interference with chromatin remodeling and spacing factor-1 (RSF-1) on proliferation and apoptosis of breast cancer cells was investigated. MCF-7 and SKBR-3 cells were cultured in vitro and were divided into 3 groups: control group, negative siRNA control group (NC) and RSF-1 siRNA group. Western blot analysis was used to detect the expression of RSF protein after interference. Cell Counting Kit-8 (CCK-8) method was used to detect the effect of RSF-1 siRNA on cell proliferation. Plate cloning assay was used to detect the effect of RSF-1 siRNA on cell clone formation ability. Annexin V/PI double staining method was used to detect the effect of RSF-1 siRNA on cell apoptosis. Effect of RSF-1 siRNA on nuclear factor-κB (NF-κB) and its downstream signaling pathway were detected by western blot analysis. Western blot analysis showed that RSF-1 siRNA significantly downregulated the expression of RSF-1 protein in MCF-7 and SKBR-3 cells at 72 h after transfection (P<0.01). Cell proliferation assay showed that RSF-1 siRNA significantly reduced the proliferation ability and clone formation ability of MCF-7 and SKBR-3 cells compared with the control group (P<0.01). Annexin V/PI double staining assay results showed that compared with the control group, RSF-1 siRNA significantly increased the apoptosis rate of MCF-7 and SKBR-3 cells (P<0.01). Helenalin and Rsf-1 siRNA significantly reduced the expression levels of p-p65, Bcl-2, and XIAP proteins (P<0.01). Interfering with the expression of RSF-1, gene can effectively inhibit the proliferation of MCF-7 and SKBR-3 cells and promote their apoptosis. RSF-1 can be used as a potential new therapeutic target for the treatment of breast cancer.

Hypoxia-inducible factor-1 promotes cancer progression through activating AKT/Cyclin D1 signaling pathway in osteosarcoma.

Osteosarcoma is an aggressive malignant neoplasm, which commonly afflicts patients of 20-30 years of age, and its morbidity has increased markedly in recent years. Certain genes and signal pathways have been identified to exert key roles in osteosarcoma progression. Here, we set out to characterize in more detail of the role of HIF-1/AKT/Cyclin D1 pathway in the progression of osteosarcoma. Immunohistochemistry, western blot and qPCR were used to test the protein or mRNA levels of HIF-1 in osteosarcoma tissues or adjacent nontumor tissues. MTT, clone formation, wound healing, Transwell, in vivo tumorigenesis, flow cytometry and western blot analysis were used to determine cell proliferation, clone formation ability, migration, invasion, tumorigenesis, and cell apoptosis in MG63 and U2OS cells, respectively. Immunoprecipitation and immunofluorescence assays were performed to investigate the protein-protein interaction between HIF-1α and proteins related to signal pathways. HIF-1 was overexpressed in osteosarcoma tissues and cell lines, which promoted cell proliferation, clone formation, migration, invasion and inhibited cell apoptosis. Results also demonstrated that HIF-1 combined with AKT, and there might be a positive loop between the two proteins of HIF-1 and AKT, then the protein-protein interaction up-regulated the expression of Cyclin D1 in protein level, but not mRNA level, made Cyclin D1 protein more stable, triggered cell proliferation, clone formation, tumorigenesis, but inhibited cell apoptosis. The present study showed that HIF-1 modulated Cyclin D1 expression might through shaping a positive loop with AKT proteins. Additionally, HIF-1α promoted the tumor cells growth, migration and invasion in osteosarcoma through the activation of the AKT/Cyclin D1 signal cascade. We proposed that HIF-1 could be served as a marker for distinguishing osteosarcoma and an effective therapeutic target for osteosarcoma.

SIX1 reduces the expression of PTEN via activating PI3K/AKT signal to promote cell proliferation and tumorigenesis in osteosarcoma.

Osteosarcoma is the most common form of primary malignant bone cancer which is most prevalent in children and adolescents. Dysregulated expressions of SIX1 and PTEN/PI3K/AKT have been demonstrated in bone malignancies including osteosarcoma. However, the mechanism of SIX1/PTEN/PI3K/AKT on osteosarcoma progression remains unknown. Therefore, this study aims to investigate the molecular mechanism of SIX1 and PTEN/PI3K/AKT on osteosarcoma progression. In this study, we first examined the expression of SIX1 and PTEN in human osteosarcoma tissues or blood samples and cell lines by immunohistochemistry, western blot analysis and qPCR. MTT, clone formation assay, wound healing assay, Transwell assay, in vivo tumorigenesis, flow cytometry and western blot were used to determine the function of SIX1/PTEN on cell proliferation, clone formation ability, migration, invasion, tumorigenesis, and cell apoptosis in SAOS2 and U2OS cells, respectively. Results showed that SIX1 was overexpressed in osteosarcoma tissues, blood samples and cell lines, whereas PTEN expression was reduced. SIX1 promoted cell growth, migration, invasion, and suppressed cell apoptosis. Up-regulation of SIX1 associated with reduced expression of PTEN and activation of PI3K/AKT signaling pathway. Down-regulated the expression of PTEN using gene transfer in U2OS and SAOS2 cells increased cell proliferation and inhibited cell apoptosis through activating PI3K/AKT signaling cascade. In addition, the tumorigenesis of U2OS and SAOS2 cells was suppressed when the cells were stably overexpressed SIX1 and PTEN simultaneously, compared with that in cells stably overexpressed SIX1 only. SIX1 promoted the progression of osteosarcoma via regulating PTEN/PI3K/AKT signaling cascade, which might provide a new potent therapeutic target for osteosarcoma.

Induction of multiple myeloma cancer stem cell apoptosis using conjugated anti-ABCG2 antibody with epirubicin-loaded microbubbles

BackgroundMultiple myeloma (MM) currently remains largely incurable. Cancer stem cells (CSCs) are believed to be responsible for drug resistance and eventual relapse. In this study, we exploited a novel agent to evaluate its inhibitory effect on MM CSCs.MethodsEpirubicin (EPI)-loaded lipid microbubbles (MBs) conjugated with anti-ABCG2 monoclonal antibody (EPI-MBs + mAb) were developed and their effect on MM 138−CD34− CSCs isolated from human MM RPMI 8226 cell line plus ultrasound exposure in vitro and in vivo in a nonobese diabetic/severe combined immunodeficient mouse model were assessed.ResultsEPI-MBs + mAb combined with ultrasound led to a significant decrease in the clone formation ability and the mitochondrial membrane potential along with an increase in MM CSC apoptosis. Moreover, treatment with EPI-MBs + mAb with ultrasound exposure remarkably inhibited the growth of MM CSC-derived tumors in xenograft nonobese diabetic/severe combined immunodeficient mice compared with a single agent or EPI-MBs + mAb without ultrasound exposure. The inhibitive efficacy was also correlated with an increased expression of caspase-3, Bax, and TUNEL and decreased expressions of PCNA, Bcl-2, and CD31.ConclusionsOur findings reveal that the EPI-MBs + mAb combined with therapeutic ultrasound may confer an effective approach for treatment of MM by induction of an apoptotic pathway in MM CSCs.

Functional transformation of N-myc downstream-regulated gene 4 in gastric cancer cells under oxygen stress

Objective To investigate the influence of hypoxia on the function of N-myc downstream-regulated gene 4 (NDRG4) in gastric cancer cells. Methods The hypoxia model in gastric cancer cells was constructed to investigate the effect of hypoxia on metastasis of gastric cancer cells using scratch healing assay and Transwell assay. The mRNA and protein expression levels of NDRG4 in gastric cancer cells subjected to hypoxia were also detected using Western blotting and reverse transcriptase-polymerase chain reaction (RT-PCR) respectively. NDRG4 overexpression was conducted in gastric cancer cells and the effect of NDRG4 on malignant phenotype of gastric cancer cells was explored through scratch healing assay, Transwell assay, cell growth curve and clone formation test under normoxic conditions. Results Hypoxia could obviously enhance the metastasis capability of gastric cancer cells. As compared with the normoxic state, the number of invasive cells in both BGC-823 and MGC-803 subjected to hypoxia for 24 h was notably increased (174.20±9.04 vs. 48.20±11.76, and 229.00±34.22 vs. 28.75±2.50, respectively, both P=0.000). Besides, hypoxia could also significantly increase the scratch healing rate in both BGC-823 and MGC-803 cells [(25.00±0.02)% vs. (15.69±0.04)%, and (32.03±0.04)% vs. (11.90±0.04)% respectively, P=0.025, 0.006 respectively]. Accompanied with enhanced metastasis potential of gastric cancer cells induced by hypoxia for 24 h, the protein level of NDRG4 was dramatically increased. However, the ectopic overexpression of NDRG4 could dramatically inhibit the metastasis capacity, sustain cell proliferation and decrease the clone-forming ability under the normoxic condition. Conclusion Upregulation of NDRG4 induced by hypoxia might play an important role during the progression of gastric cancer trigged by hypoxia. We suspected that abnormal accumulation and functional protein modification of NDRG4 induced by hypoxia maybe contribute to the function changes of NDRG4 under different oxygen concentrations. Key words: Gastric cancer; N-myc downstream-regulated gene 4; Hypoxia; Metastasis

TGF-β3 reduces apoptosis in ischemia-induced adipose-derived stem cells by enhancing DNA repair.

Adipose-derived stem cells (ADSCs) possess good proliferative and differentiative abilities, making then a promising candidate for the treatment of cartilage defects. However, local ischemia often causes apoptosis in ADSCs. Transforming growth factor-β3 (TGF-β3) is often used as a chondrogenic differentiation cytokine whose function in apoptosis is unclear. The aim of the present study was to investigate the role of TGF-β3 in ischemia-induced ADSC apoptosis. In the present study, the phenotypes and multipotent differentiation properties of human ADSCs at passage 3 were analyzed using flow cytometry and cytochemical staining. ADSCs were cultured in a serum- and glucose-free medium under hypoxic conditions with or without exogenous TGF-β3 treatment. The apoptosis rate was measured using a TUNEL array and Annexin V/propidium iodide staining. The expression of apoptosis-associated proteins was measured using western blotting. The results revealed ADSCs cultured in normal condition have multi-lineage differentiation potential and high levels of cluster of differentiation (CD)29, CD44 and CD105 expression. Furthermore, ADSCs weakly express CD14, CD34 and CD45, with strong clone formation and migration abilities. Serum deprivation under hypoxic conditions resulted in mitochondria-mediated apoptosis in ADSCs, which was attenuated by exogenous TGF-β3 treatment via upregulation of poly ADP-ribose polymerase (PARP). The results of the present study indicate that TGF-β3 is able to protect ADSCs from ischemia-induced apoptosis via PARP-associated DNA damage repair.

FGFR inhibitor AZD4547 can enhance sensitivity of esophageal squamous cell carcinoma cells with epithelial‑mesenchymal transition to gefitinib.

Activation of fibroblast growth factor receptor (FGFR) signaling occurs in various cancers, including esophageal squamous cell carcinoma (ESCC), however, the effect of targeting FGFR in ESCC is not clear. Herein, we examined the phosphorylation level of FGFR1Y654 (p‑FGFR1) in ESCC cell lines and tumor tissues, as well as the cancer cell killing effects of gefitinib and FGFR inhibitor AZD4547 in combination form or alone in ESCC cells. Immunohistochemistry staining was used to detect the expression level of p‑FGFR1 in 87 ESCC specimens. The effects of gefitinib and FGFR inhibitor AZD4547 on ESCC cells were analyzed by CCK‑8 assay, flow cytometry and western blotting assays. Twenty‑six patients diagnosed with esophageal squamous cell carcinoma (ESCC) (29.9%) were observed with a high level of p‑FGFR1. The proportion of lesions located in the lower segment of the esophagus was significantly higher in the high p‑FGFR1 level group (26.9 vs. 8.2%, P=0.003). The IC50 values of gefitinib alone and in combination with 500nM AZD4547 were 22.9±2.1 and 4.13±0.12µM in TE10 cells, and 9.85±5.5 and 3.21±0.76µM in EC9706 cells, respectively. The combination of AZD4547 and gefitinib induced robust apoptosis and decreased clone formation ability compared to gefitinib monotherapy in the TE10 cells. TE10 cells exhibited a mesenchymal phenotype, with a higher level of p‑FGFR1 and p‑AKT than that in EC9706 cells. AZD4547 and gefitinib co‑treatment resulted in a significant decrease in the level of p‑AKT in TE10 cells and a complete inhibition of phosphorylation of ERK1/2 in EC9706 cells. Collectively, AZD4547 can improve sensitivity of ESCC cells to gefitinib.

Artesunate inhibits proliferation of glioblastoma cells by arresting cell cycle

Glioblastoma is a common brain tumor and the overall survival rate of the patients is very low, so it is an effective way to develop the potential chemotherapy or adjuvant chemotherapy drugs in glioblastoma treatment. As a well-known antimalarial drug, artesunate(ARTs) has clear side effects, and recently it has been reported to have antitumor effects, but rarely reported in glioblastoma. Different concentrations of ARTs were used to treat the glioblastoma cells, and then the inhibitory effect of ARTs on glioblastoma proliferation was detected by MTT assay; Ki67 immunofluorescence assay was used to detect the proliferation of cells; Soft agar experiment was used to explain the clonal formation abilities in vitro; Flow Cytometry was used to detect the cell cycle; and Western blot assay was used to determine the expression of key cell cycle protein. MTT assay results indicated that ARTs-treated glioblastoma cell A172, U251, U87 were significantly inhibited in a time-and-dose dependent manner as compared to the control group(DMSO treatment group). Soft agar experiment showed that ARTs could significantly reduce the clonal formation ability of glioblastoma. Furthermore, Flow cytometry analysis showed that ARTs could obviously increase the cell proportion in G₀/G₁ phase and reduce the cell proportion in S phase. Western blot results showed that the expressions of cell cycle-related proteins CDK2, CDK4, cyclin D1 and cyclin B1 were all obviously down-regulated. Above all, ARTs may inhibit the proliferation of glioblastoma cells by arresting cell cycle in G₀/G₁ phase through down-regulating the expression of CDK2, CDK4, cyclin D1, cyclin B1. These results may not only provide a novel method for rediscovering and reusing ARTs but also provide a new potential drug for treating glioblastoma.

Fasudil inhibits proliferation and migration of Hep-2 laryngeal carcinoma cells.

BackgroundRho-kinase signal pathway is a new target for cancer therapy. Fasudil, a selective Rho-kinase inhibitor, is found to exert antitumor effects on several types of cancer, but whether fasudil has antitumor effects on laryngeal carcinoma is still unknown. The aim of this study was to determine the effects of fasudil on laryngeal carcinoma and explore the underlying molecular mechanisms in this process.MethodsAfter treatment with fasudil, changes in biological behaviors, including the growth, proliferation, clone formation, apoptosis, and migration of human laryngeal carcinoma cells (Hep-2 cells) were observed. The influences on apoptotic protease activity factor-1 (APAF-1)-mediated apoptosis pathway and the activities of matrix metalloproteinases (MMP-2 and MMP-9) were measured by Western blotting and gelatin zymography assay.ResultsHalf-maximal inhibitory concentration of fasudil to Hep-2 cells was ~3.40×103 µM (95% CI: 2.53–4.66×103 µM). Moreover, fasudil treatment significantly decreased the ability of growth, proliferation, clone formation, and migration of Hep-2 cells, while remarkably increased the apoptosis rate. Furthermore, the expressions of APAF-1, caspase-9, and caspase-3 significantly increased in fasudil treatment group. Meanwhile, fasudil led to a remarkable decrease in the expressions and activities of MMP-2 and MMP-9.ConclusionOur findings first demonstrate that fasudil not only inhibits the proliferation of laryngeal carcinoma cells through activating APAF-1-mediated apoptosis pathway, but also prevents migration by inhibiting the activities of MMP-2 and MMP-9. Therefore, fasudil is an attractive antitumor drug candidate for the treatment of laryngeal carcinoma.

The role of aldehyde dehydrogenase 1A1 in B-cell non-Hodgkin's lymphoma.

Previously we showed that aldehyde dehydrogenase1A1 (ALDH1A1) is a new mediator for resistance of DLBCL to CHOP and a facility predictor of clinical prognosis. In the present study, knockdown and inhibitor of ALDH1A1 were applied to identify the role of ALDH1A1 in Raji cells. CCK-8 and clone formation assay were applied to determine the CHOP sensitivity and clone formation ability. Caspase colorimetric assay and AnnexinV/FITC staining was performed to determine the degree of apoptosis. Western blot analysis was used to detect the NF-κB/STAT3 signaling proteins and apoptotic-associated proteins. Real-time quantitative PCR (RT-PCR) was used to identify the differential expression of ALDH1A1 between NHL patients and healthy donors. We demonstrated that inhibition of ALDH1A1 increased the sensitivity of Raji cells to CHOP, as indicated by increased cytotoxicity, reduced clonogenicity, activated caspase-3/-9, decreased NF-κB/STAT3 signaling and increased pro-apoptosis signaling, ad increased apoptosis rate. Moreover, we found high ALDH1A1 expression was associated with poor prognosis in NHL patients. Our data revealed the critical role of ALDH1A1 in NHL and provides a theoretical basis for the use of ALDH1A1 inhibitors in NHL patients.

Role of long non-coding RNA SNHG1 in occurrence and progression of ovarian carcinoma.

To investigate the expression of human long non-coding ribonucleic acid (lncRNA) small nucleolar RNA host gene 1 (SNHG1) in epithelial ovarian carcinoma tissues and its effects on the in vitro proliferation, apoptosis, invasion and metastasis of ovarian carcinoma cells, and to investigate its possible mechanism. The expressions of SNHG1 in 20 pairs of epithelial ovarian carcinoma tissues and para-carcinoma normal tissues were detected by Real-time fluorescence quantitative polymerase chain reaction (qRT-PCR). The expressions of SNHG1 in normal ovarian epithelial cells (IOSE25) and ovarian carcinoma cells (CAOV-3, SKOV-3, ES2 and A2780) were further detected. The knockdown efficiency of SNHG1 small interfering RNA (siRNA) in SKOV-3 cells was detected via qRT-PCR. Moreover, the effects of SNHG1 knockdown on proliferation, migration and apoptosis of SKOV-3 cells were detected by cell counting kit 8 (CCK8) proliferation assay, clone formation assay, transwell migration assay and flow cytometry. Finally, the expressions of apoptosis-related proteins, epithelial-mesenchymal transition (EMT)-related proteins and matrix metalloproteinases (MMPs) in control group and interference group were detected by Western blotting. The expression level of lncRNA SNHG1 in ovarian carcinoma tissues was significantly higher than that in para-carcinoma normal tissues. After lncRNA SNHG1 knockdown in SKOV-3 cells, the cell proliferation and clone formation abilities were significantly inhibited. The apoptosis assay proved that inhibiting lncRNA SNHG1 could promote the apoptosis of SKOV-3 cells. Besides, Western blotting revealed that the expressions of pro-apoptotic proteins in interference group were significantly upregulated compared with those in control group. Wound-healing assay and transwell migration assay showed that the down-regulation of lncRNA SNHG1 could inhibit the invasion and metastasis of SKOV-3 cells, whose mechanism was related to the inhibition of EMT process and down-regulation of expressions of MMPs. LncRNA SNHG1 is highly expressed in ovarian carcinoma, which can promote the growth, invasion and metastasis of ovarian carcinoma cells. The down-regulation of SNHG1 can inhibit the proliferation, invasion and metastasis of SKOV-3 cells. Inhibiting the expression of SNHG1 may be a potentially effective means of treating ovarian carcinoma.

Silencing of ENO1 by shRNA Inhibits the Proliferation of Gastric Cancer Cells

α-Enolase is a significant subunit of enolase and acts as a glycolytic enzyme responsible for catalyzing the conversion of 2-phosphoglycerate to phosphoenolpyruvate in the anaerobic glycolysis pathway. The research about their role is known little in tumor invasion and metastasis. This research analyzed the effect of α-enolase in proliferation and progression of human gastric cancer. The constructed PLKO.1-ENO1 shRNA vector was transfected into 293 T cells and used to infect gastric cancer cells, MKN45, by using lentivirus method. Negative controls were generated by infection with viruses containing empty vector PLKO.1-scramble-shRNA by the same protocol and using wild-type MKN45 cells as blank control. The silencing effect was confirmed by reverse transcription polymerase chain reaction and Western blotting at messenger RNA and protein levels, respectively. Cell proliferation and chemosensitivity were tested by methyl-thiazolyl-tetrazolium assay. Cell apoptosis was tested by flow cytometry. The cell line α-enolase short hairpin RNA stabling silence α-enolase was successfully constructed. In the α-enolase short hairpin RNA cell lines, messenger RNA and protein expression of α-enolase were significantly lower than those in negative control and blank control groups. The proliferation and clone formation ability were significantly inhibited, cell apoptosis was increased significantly, and the inhibition rate of chemotherapy drugs was increased (P < .05). Our data provide strong evidence that α-enolase short hairpin RNA interference vector can effectively suppress the proliferation and increase chemosensitivity of MKN45 cells, which may provide a novel gene therapy for gastric cancer.

The CircRNA-ACAP2/Hsa-miR-21-5p/ Tiam1 Regulatory Feedback Circuit Affects the Proliferation, Migration, and Invasion of Colon Cancer SW480 Cells

Background/Aims: Circular RNAs (circRNAs), a type of RNA that is widely expressed in human cells, have essential roles in the development and progression of cancer. CircRNAs contain microRNA (miRNA) binding sites and can function as miRNA sponges to regulate gene expression by removing the inhibitory effect of an miRNA on its target gene. Methods: We used the bioinformatics software TargetScan and miRanda to predict circRNA-miRNA and miRNAi-Mrna interactions. Rate of inhibiting of proliferation was measured using a WST-8 cell proliferation assay. Clone formation ability was assessed with a clone formation inhibition test. Cell invasion and migration capacity was evaluated by performing a Transwell assay. Relative gene expression was assessed using quantitative real-time polymerase chain reaction and relative protein expression levels were determined with western blotting. circRNA and miRNA interaction was confirmed by dual-luciferase reporter and RNA-pull down assays. Results: In the present study, the miRNA hsa-miR-21-5p was a target of circRNA-ACAP2, and T lymphoma invasion and metastasis protein 1 (Tiam1) was identified as a target gene of hsa-miR-21-5p. CircRNA-ACAP2 and Tiam1 were shown to be highly expressed in colon cancer tissue and colon cancer SW480 cells, but miR-21-5p was expressed at a low level. SW480 cell proliferation was suppressed when the expression of circRNA-ACAP2 and Tiam1 was decreased and the expression of miR-21-5p was increased in vivo and in vitro. SW480 cell migration and invasion were also inhibited under the same circumstance. The circRNA-ACAP2 interaction regulated the expression of miR-21-5p, and miR-21-5p regulated the expression of Tiam1. Down-regulation of circRNA-ACAP2 promoted miR-21-5p expression, which further suppressed the transcription and translation of Tiam1. Conclusion: The present study shows that the circRNA-ACAP2/hsa-miR-21-5p/Tiam1 regulatory feedback circuit could affect the proliferation, migration, and invasion of colon cancer SW480 cells. This was probably due to the fact that circRNA-ACAP2 could act as a miRNA sponge to regulate Tiam1 expression by removing the inhibitory effect of miR-21-5p on Tiam1 expression. The results from this study have revealed new insights into the pathogenicity of colon cancer and may provide novel therapeutic targets for the treatment of colon cancer.

Chronic Microcystin-LR Exposure Induces Hepatocarcinogenesis via Increased Gankyrin in Vitro and in Vivo

Background/Aims: Our recent study indicated that the serum microcystin-LR (MC-LR) level is positively linked to the risk of human hepatocellular carcinoma (HCC). Gankyrin is over-expressed in cancers and mediates oncogenesis; however, whether MC-LR induces tumor formation and the role of gankyrin in this process is unclear. Methods: We induced malignant transformation of L02 liver cells via 35 passages with exposure to 1, 10, or 100 nM MC-LR. Wound healing, plate and soft agar colony counts, and nude mice tumor formation were used to evaluate the tumorigenic phenotype of MC-LR-treated cells. Silencing gankyrin was used to confirm its function. We established a 35-week MC-LR exposure rat model by twice weekly intraperitoneal injection with 10 μg/kg body weight. In addition, 96 HCC patients were tested for tumor tissue gankyrin expression and serum MC-LR levels. Results: Chronic low-dose MC-LR exposure increased proliferation, mobility, clone and tumor formation abilities of L02 cells as a result of gankyrin activation, while silencing gankyrin inhibited the carcinogenic phenotype of MC-LR-treated cells. MC-LR also induced neoplastic liver lesions in Sprague-Dawley rats due to up-regulated gankyrin. Furthermore, a trend of increased gankyrin was observed in humans exposed to MC-LR. Conclusion: These results suggest that MC-LR induces hepatocarcinogenesis in vitro and in vivo by increasing gankyrin levels, providing new insight into MC-LR carcinogenicity studies.

The study of suppression and action mechanism of malignant phenotype by downregulating expression of histone deacetylase in prostate cancer cell

Objective Histone deacetylase (HDAC) promoted growth and metastasis of prostate cancer cell and it’s action mechanism, however, remain unclear. The aim of present study is investigate the suppression and action mechanism of malignant phenotype by downregulating expression of histone deacetylase (HDAC1 and HDAC2) in prostate cancer cell. Methods Prostate cancer cell lines, LNCaP and DU145 cells were transfected with siHDAC using liposome 2000 or treated with MHY219, named as LNCaP-siHDAC, DU145-siHDAC cells negative control LNCaP-NC small interfering RNA (siRNA), DU145-NCsiRNA cells, and the expressions of HDAC1, HDAC2, HDAC3, matrix metalloproteinase (MMP)-1, MMP-2, MMP-9 and TIMP-1 protein were determined by Western blotting and the levels of MMP-1, MMP-2, MMP-9 and TIMP-1 mRNA were detected using Real time RT-PCR. Cell viability, clone formation ability and metastasis were monitored using MTT, clone formation assay and Transwell respectively in LNCaP and DU145 cells. Results The relative expression levels of HDAC1 and HDAC2 protein was respectively 0.22±0.01, 0.25±0.02 in LNCaP-siHDAC cell and 0.15±0.01, 0.30±0.03 in DU145-siHDAC cell, and significantly lower than LNCaP-NCsiRNA (1.00±0.05, FLNCaP=289.440, P=0.008) and DU145--NCsiRNA (1.00±0.03, FDU145=336.125, P=0.002). The relative expression levels of HDAC1 and HDAC2 protein was respectively 0.53±0.04, 0.60±0.02 in LNCaP cell and 0.58±0.04, 0.66±0.05 in DU145 cell treated with MHY219, and significantly lower than Untreated group (FLNCaP=147.158, P=0.022; FDU145=134.573, P=0.035). The results from the MTT indicated that cell viability was respectively (40.38±4.16)% in LNCaP-siHDAC cell and (21.42±5.26)% in DU145-siHDAC cell with statistical differences compared with LNCaP-NCsiRNA cell [(98.47±6.70)%, FLNCaP=125.627, P=0.004] and DU145-NCsiRNA cell [(99.15±7.00)%, FDU145=137.560, P=0.000]. The cell viability was respectively (61.75±7.12)% and (45.00±5.53)% in LNCaP cell and DU145 cell treated with MHY219, and with statistical differences compared with Untreated group LNCaP [(99.34±7.12)%, FLNCaP=88.392, P=0.032] and DU145 [(98.58±8.05)%, FDU145=103.10, P=0.017]. The results from clone formation assay shown that clone formation rate was respectively (26.37±3.50)% in LNCaP-siHDAC cell and (19.48±2.33)% in DU145-siHDAC cell, and with statistical differences compared with LNCaP-NCsiRNA cell [(97.20±7.88)%, FLNCaP=287.653, P=0.000] and DU145-NCsiRNA cell [(99.23±9.52)%, FDU145=315.042, P=0.000]. The clone formation rate was respectively (43.88±4.12)% and (438.05±6.11)% in LNCaP cell and DU145 cell treated with MHY219, and with statistical differences compared with Untreated group LNCaP [(100.00±5.24)%, FLNCaP=203.540, P=0.018] and DU145 [(97.70±8.74)%, FDU145=264.110, P=0.010]. Transwell results indicated that the cell number of metastasis was respectively (102.5±7.5) in LNCaP-siHDAC cell and (95.2±4.7) in DU145-siHDAC cell and significantly lower LNCaP-NCsiRNA cell (236.8±12.7, FLNCaP=316.047, P=0.00) and DU145-NCsiRNA cell (278.5±18.6, FDU145=357.052, P=0.000). Western blotting and Real time RT-PCR results shown that downegulated HDAC1 or (and) HDAC2 expression notable suppressed the expression of MMP-1and MMP-2 in protein and mRNA levels, but TIMP-1 expression was significantly enhanced in LNCaP and DU145 cells. Conclusion The suppression of malignant phenotype by downregulating expression of HDAC1 and HDAC2 in prostate cancer cell. Downregulated HDAC expression can inhibited malignant phenotype and it’s action mechanism was achieved at least partly by regulating expression of MMP-1, MMP-2 and TIMP-1 expression in LNCaP and DU145 cells. Key words: Prostate cancer; Histone deacetylase; Matrix metalloproteinase; Malignant phenotype; Action mechanism

SU6668 inhibits the proliferation and motility of colorectal cancer cells by inducing cycle arrest, and promotes their apoptosis

Background: The multi-targeted angiogenesis inhibitor SU6668 has demonstrated promising therapeutic effects on the progression of hematologic malignancies and certain solid tumors. Previous studies reported that SU6668 inhibited the liver metastasis of both human and mouse colon cancer xenografts via its antiangiogenic effect. This study was conducted to determine the effect of SU6668 on the proliferation, migration, and invasion of colorectal cancer cells and their rates of apoptosis. Methods: SW480 and SW620 cells were treated with various concentrations of SU6668 (15, 30, and 45 μM, respectively), and their proliferation was examined by CCK-8 and EdU assays. Changes in the clone formation ability of colorectal cancer cells treated with SU6668 were assessed with colony formation assays. The effects of SU6668 on cell cycle progression and apoptosis were detected by flow cytometry. Hoechst staining was used to determine the rates of apoptotic cell death. Western blot was used to analyze the relative levels of Bax, caspase-3 and Bcl-2 protein expression. Matrigel invasion assays were used to assess the effects of SU6668 on colorectal cancer cell migration and invasion capabilities. Results: SU6668 inhibited the proliferation and clone formation ability of colorectal cancer cells in a dose- and time-dependent manner. SU6668 also induced G0/G1 cell cycle arrest and cell apoptosis. The expression levels of apoptosis-related proteins (Bax and caspase-3) were enhanced, while the level of Bcl-2 expression was significantly decreased in SU6668-treated colorectal cancer cells. SU6668 significantly inhibited the migration and invasion of colorectal cancer cells. Conclusions: SU6668 can inhibit the proliferation, migration, and invasion of colorectal cancer cells, as well as their ability for colony formation. SU6668 inhibited cell proliferation by inducing G1 cell cycle arrest and induced apoptosis by activating Bax and caspase-3.

Expression of annexin A1 protein in esophageal carcinoma and its clinical significance

Objective To investigate the biological role of Annexin A1 protein in carcinogenesis and progression of esophageal carcinoma. Methods Annexin A1 protein level was analyzed by immunohistochemical assay in 94 cases of esophageal squamous carcinoma tissues and surrounding normal tissues. Annexin A1 overexpression and knockdown stable cell lines in human immortalized esophageal epithelial cell line SHEE were constructed by lentivirus. Cell proliferation of Annexin A1 overexpression and knockdown stable cell lines were analyzed by cell counting kit-8 (CCK-8). Cell invasion ability of Annexin A1 overexpression and knockdown stable cell lines were analyzed by transwell method. Cell colony forming ability in stable cell lines was analyzed by soft agar assay. Tumor formation in mice was analyzed by cell transplantation. Results Compared with the normal esophageal tissues, the expression levels of Annexin A1 protein in esophageal cancer tissues increased significantly (P=0.004). Compared with the control cell lines, proliferation (P=0.026) and invasion (P=0.017) ability in Annexin A1 protein overexpression cell lines significantly increased, while in the Annexin A1 protein knockdown cell lines, cell proliferation (P=0.019) and invasion (P=0.023) ability significantly decreased. Compared with the control cell lines, the clonal formation ability of Annexin A1 overexpression cell lines significantly enhanced(P=0.022), while the ability of clones formation in Annexin A1 protein knockdown cell line significantly decreased (P=0.035). In vivo tumor formation test showed that the tumor formation rate in Annexin A1 protein overexpressing cell lines significantly increased compared with control cell lines (P=0.001). Conclusion Annexin A1 protein overexpressed in esophageal carcinoma tissues, which was involved in the growth, invasion and tumorigenesis of esophageal cancer cells. Key words: Annexin A1 protein; Esophageal carcinoma; Proliferation; Invasion; Tumorigenesis

Effects of pituitary adenylate cyclase activating polypeptide on small intestinal INT 407 cells

Pituitary adenylate cyclase activating polypeptide (PACAP) is an endogenous neuropeptide having a widespread distribution both in the nervous system and peripheral organs including the gastrointestinal tract. It has been shown to exert actions on intestinal functions, mainly affecting glandular secretion and motility. PACAP has several different effects on cell survival depending on the cell type and the applied stimulus. Its influences on small intestinal epithelial cells are not yet elucidated, therefore the aim of the present study was to investigate the effects of PACAP on intestinal epithelial cells having high turnover (INT 407) against different harmful stimuli, such as oxidative stress, in vitro hypoxia and gamma radiation. We tested the effect of PACAP on proliferation and cell survival using MTT assay. Moreover, various cancer-related factors were evaluated by oncology array. PACAP did not influence the proliferation rate of INT 407 cells. Its cell survival-enhancing effect could be detected against oxidative stress, but not against in vitro hypoxia or gamma irradiation. Clonogenic survival assay was performed to analyze the effect of PACAP on clonogenic potential of cells exposed to gamma radiation. Surprisingly, PACAP enhanced the clone-forming ability decrease induced by irradiation. Western blot analysis of ERK1/2 phosphorylation was performed in order to obtain further information on the molecular background. Our data showed phospho-ERK1/2 suppression of PACAP in irradiated cells. Furthermore, the role of endogenous PACAP against oxidative stress was also investigated performing ADCYAP1 small interfering RNA transfection. We found significant difference in the cell vulnerability between cells undergoing silencing and cells without transfection suggesting the protective role of the endogenously present PACAP against oxidative stress in INT 407 cells. In summary, PACAP seems to be able to exert contradictory effects in INT 407 cells depending on the applied stressor, suggesting its regulatory role in the cellular household.

SHP-2 Activating Mutation Promotes Malignant Biological Behaviors of Glioma Cells.

BackgroundThis study investigated the mechanism underlying the activating mutation of SHP-2 in promoting malignant biological behaviors of glioma cells.Material/MethodsThe SHP-2 empty plasmid pcDNA3.1 and SHP-2 activating mutation plasmid pcDNA3.1 SHP-2 D61G mutant eukaryotic expression vectors were designed; stable SHP-2-expressing cells transfected with pcDNA3.1 SHP-2 D61G mutant were set as the mutation group; cells transfected with pcDNA3.1 were set as the empty vector group; and cells without transfection were set as the control group. The cell reproductive capacity in each group was measured by MTT method. The invasion ability of cells in vitro was detected by Transwell chamber assay, the cell apoptosis in each group was detected by Annexin-V/PE dual-staining method, and the clone formation ability of cells in vitro was detected by Tablet clone-forming assay. The activation of ERK1/2, ARK, and p38MAPK signal pathways in each group was determined by Western blot.ResultsAfter transfection, the expression of SHP-2 protein in the mutant group was significantly higher than that in the control group and empty vector group. The proliferation ability of transfected cells, the apoptosis rate, the invasion ability, and the expression levels of phosphorylated ERK1/2, AKT, and p38 in the mutation group was significantly higher than in the empty vector group and the control group (P<0.05). Moreover, the cell clone formation ability of the mutation group was obviously enhanced (P<0.05).ConclusionsThe activating mutation of SHP-2 can lead to malignant changes in biological behaviors of glioma cells, and the specific mechanism may be related to the activation of ERK1/2, AKT, and p38 signal pathway. SHP-2 protein may become a new target for anti-malignant transformation of glioma.

Effects of LncRNA-HOST2 on cell proliferation, migration, invasion and apoptosis of human hepatocellular carcinoma cell line SMMC-7721

The present study explored the effect of long non-coding RNA-human ovarian cancer-specific transcript 2 (LncRNA-HOST2) on cell proliferation, migration, invasion and apoptosis of human hepatocellular carcinoma (HCC) cell line SMMC-7721. HCC tissues and adjacent normal tissues from 162 HCC patients were collected. The HCC cell lines were assigned into the control group (regular culture), negative control (NC) group (transfected with siRNA) and experimental group (transfected with Lnc-HOST2 siRNA). Quantitative real-time PCR (qRT-PCR) was used to detect the expression of LncRNA-HOST2. Cell proliferation was detected by CCK-8 and colony-forming assays, cell apoptosis by flow cytometry and cell migration by Scratch test. Transwell assay was used to evaluate cell migration and invasion abilities. LncRNA-HOST2 expression in the HCC tissues increased 2–10 times than that in the adjacent normal tissues. Compared with the HL-7702 cell line, LncRNA-HOST2 expression in HepG2, SMMC-7721 and Huh7 cell lines was all up-regulated, but the SMMC-7721 cell had the highest Lnc-HOST2 expression. The LncRNA-HOST2 expression in the experimental group was down-regulated as compared with the control and NC groups. In comparison with the control and NC groups, cloned cells reduced, cell apoptosis increased, clone-forming ability weakened and inhibitory rate of colony formation increased in the experimental group. The cells migrating and penetrating into the transwell chamber were fewer in the experimental group than those in the control and NC groups. The experimental group exhibited slow wound healing and decreased cell migration area after 48 h. These findings indicate that LncRNA-HOST2 can promote cell proliferation, migration and invasion and inhibit cell apoptosis in human HCC cell line SMMC-7721.