Long noncoding RNA HOTAIR silencing inhibits invasion and proliferation of human colon cancer LoVo cells via regulating IGF2BP2.

Colon cancer is one of the most life-threatening malignancies worldwide. Long noncoding RNA (lncRNA) HOX transcript antisense RNA (HOTAIR) is a cancer-associated biomarker involved in the metastasis and prognosis of several cancers. However, whether and how HOTAIR affects colon cancer progression is still unclear. Consequently, we used RNA interference to knock down HOTAIR to explore its effects on human colon cancer cells. The dual luciferase reporter gene assay was initially used for testify the regulating relationship between lncRNA HOTAIR and insulin-like growth factor 2 mRNA-binding protein 2 (IGF2BP2). We determined the expressions of HOTAIR, IGF2BP2, E-cadherin, and vimentin. Meanwhile, cell growth, cycle and apoptosis, migration, and invasion were assayed. LoVo cells were transplanted into nude mice, and tumor formation and microvessel density were evaluated. LncRNA HOTAIR positively regulated IGF2BP2. Besides, the expressions of HOTAIR and E-cadherin and the apoptosis were increased, while the expressions of IGF2BP2 and vimentin, the growth, invasion and migration of LoVo cells, the average tumor weight, and microvessel density value were decreased. Of importance, overexpressed IGF2BP2 could reverse the above impacts. Taken together, the current study indicates that silencing of HOTAIR could inhibit the invasion, proliferation, and migration, and promote apoptosis of colon cancer LoVo cells through suppressing IGF2BP2 and the epithelial-mesenchymal transition.

Regulatory T-cell density and cytotoxic T lymphocyte density are associated with complete response to neoadjuvant paclitaxel and carboplatin chemoradiotherapy in gastric cancer.

Regulatory T-cell density and cytotoxic T lymphocyte density are crucial in regulating antitumor immune responses. Tumor infiltration has marked therapeutic effects in gastric carcinoma, and there is evidence that chemoradiotherapy (CRT) exhibits an immune-mediated component. In the present study, the density of CD4+ and CD8+ cells were evaluated in post-CRT surgical samples from 68 patients with gastric cancer using immunohistochemistry. The associations between T-cell density, cytotoxic T lymphocyte density and clinical survival rate were also analyzed. Cytotoxic T lymphocyte density was associated with gastric carcinoma regression grade and regulatory T-cell density was also associated with gastric carcinoma regression grade. Of the patients who had a pathologic complete response, 84 and 76% were found to have a high CD3+ and CD4+ cell density, which was significantly different to patients who had a low CD3+ and CD4+ cell density. High cytotoxic T lymphocyte density was also associated with improved survival rates of patients with gastric cancer. In conclusion, these outcomes indicated that regulatory T-cell density and cytotoxic T lymphocyte density in the tumor microenvironment were associated with the response to neoadjuvant CRT and may represent a therapeutic target for gastric cancer.

Effects of Glut1 gene silencing on proliferation, differentiation, and apoptosis of colorectal cancer cells by targeting the TGF-ß/PI3K-AKT-mTOR signaling pathway.

This study aims to investigate the effects of glucose transport l (Glut1) gene on proliferation, differentiation, and apoptosis of colorectal cancer (CRC) cells by regulating the TGF-ß/PI3K-AKT-mTOR signaling pathway. Immunohistochemistry was conducted to detect the positive Glut1 expression. Normal human CRC epithelial cells (CCD-18Co) and CRC cell line HCT116 were grouped into the control, blank, negative control (NC), and shGlut1-1 groups. RT-qPCR and Western blotting were performed to detect the expressions of Glut1, TGF-ß1, PI3K, AKT, PTEN, mTOR, Bcl-2, and Bax. Protein expression of phosphorylated-PI3K (p-PI3K), p-S473-AKT, p-S389-S6K1, p-T70-4EBP1, Cleaved caspase-3 and Cleaved-PARP were detected. MTT assay, flow cytometry, and colony formation assay were performed in order to detect cell viability, cell cycle, and apoptosis, respectively. The positive expression rate of Glut1 in CRC tissues was 75% ± 8%, while in the adjacent normal tissues it was 0%. In comparison to adjacent normal tissues, CRC tissues had increased Glut1, TGF-ß1, PI3K, AKT, mTOR, and Bcl-2 expressions, and p-PI3K, p-S473-AKT, p-S389-S6K1, and p-T70-4EBP1 expressions; and decreased PTEN, Bax, Cleaved caspase-3, and Cleaved-PARP expressions. In comparison with the blank and NC groups, cells in the shGlut1-1 group showed decreased Glut1, TGF-ß1, PI3K, AKT, mTOR, and Bcl-2 expressions, and p-PI3K, p-S473-AKT, p-S389-S6K1, and p-T70-4EBP1 expressions; and increased PTEN, Bax, Cleaved caspase-3, and Cleaved-PARP expressions, along with more arrested cells in C0/C1 phase than in S phase and slower cell growth. These results suggested that silencing the Glut1 gene inhibited proliferation and promoted apoptosis of CRC cells by inactivating TGF-ß/PI3K-AKT-mTOR signaling pathway.

S100B suppresses the differentiation of C3H/10T1/2 murine embryonic mesenchymal cells into osteoblasts.

S100 calcium-binding protein B (S100B) is expressed and released by adipocytes, and is positively correlated with body mass index, however, the direct effects of S100B on adipocytes remain unclear. Bone marrow­derived mesenchymal stem cells have the capacity to differentiate into osteoblasts and adipocytes, which is important for bone metabolism. The current study aimed to determine the effect of S100B on adipogenesis and osteogenesis. The mouse embryo cell line C3H/10T1/2 was used to build cell models with varying levels of S100B protein expression. Western blot analysis was performed to assess the expression of various marker proteins. Oil red O staining and alizarin red S staining were used to detect adipogenesis and osteogenesis, respectively. S100B overexpression was associated with a significant increase in oil red O staining and a significant reduction in alizarin red S staining. Runt­related transcription factor­2 and bone morphogenetic protein 2 expression levels were significantly increased in the S100B underexpression group, however not in the S100B overexpression group. By contrast, the expression levels of the adipogenesis markers peroxisome proliferator­activated receptor Î³ and CCAAT­enhancer­binding protein α was significantly increased in the S100B overexpression group, however not in the S100B underexpression group. Osteogenesis stimulation increased extracellular signal­regulated kinase (ERK) phosphorylation, and adipogenesis stimulation increased c­Jun N­terminal kinase (JNK) phosphorylation. The results suggest that S100B inhibits osteogenesis, however stimulates adipogenesis. The ERK pathway is involved in the regulation of osteogenesis, whereas the JNK pathway is involved in the regulation of adipogenesis.

Raf kinase inhibitor protein mediated signaling inhibits invasion and metastasis of hepatocellular carcinoma.

BACKGROUND: Hepatocellular carcinoma (HCC) is the most common type of liver cancer with high mortality and poor prognosis. Mitogen-activated protein kinase (MAPK) and nuclear factor kappa B (NF-κB) signaling pathways have been implicated in promoting tumor cell proliferation and invasion of HCC cells. METHODS: As a potential inhibitor of tumor metastasis, the role of Raf kinase inhibitor protein (RKIP) in HCC development and the functional relevance with MAPK and NF-κB signaling pathways were investigated. The levels of RKIP expression were examined in human HCC tissues and correlated with tumor stages and metastatic status. Function of RKIP in cellular proliferation, migration, invasion and apoptosis was investigated in HCC cell lines by either overexpressing or knocking down RKIP expression. Mouse xenograft model was established to assess the effect of RKIP expression on tumor growth. RESULTS: Our results demonstrated decreased RKIP expression in HCC tissues and a strong correlation with tumor grade and distant metastasis. Manipulation of RKIP expression in HCCLM3 and HepG2 cells indicated that RKIP functioned to inhibit HCC cell motility and invasiveness, and contributed to tumor growth inhibition in vivo. Mechanistic studies showed that the function of RKIP was mediated through MAPK and NF-κB signaling pathways. However, cell type-dependent RKIP regulation on these two pathways was also suggested, indicating the complex nature of signaling network. CONCLUSION: Our study provides a better understanding on the molecular mechanisms of HCC metastasis and sets the foundation for the development of targeted therapeutics for HCC.

[Analysis of gastric cancer tissues genome methylation by DNA methylation chip].

OBJECTIVE: To detect the methylation status of gastric cancer tissue genome by DNA methylation chip. METHODS: Methylation status of 6 samples of gastric cancer tissues and their matched adjacent tissues was analyzed using methylated DNA immunoprecipitation(MeDIP) combined with NibleGen chip. Significantly different methylated genes in promoter region and CpG island between two tissues were searched. Functions of these significantly different methylated genes were analyzed by Gene Ontology and Pathway assays. RESULTS: In gene promoter regions, 113 significantly different methylated genes were identified in gastric cancer tissues, such as SHP1, FGF8 and CSF2RA, while 161 significantly different methylated genes were identified in their matched adjacent tissues, such as TNF, IGF2 and BMP7. In the CpG islands, 123 significantly different methylated genes were identified in gastric cancer tissues, such as WNT2B, JAK2 and TPT1, while 139 significantly different methylated genes were identified in their matched adjacent tissues, such as TNFRSF4, HOXC8 and NFYA. These genes located on different chromosomes. In gastric cancer tissues, the 1st and the 4th chromosomes had the most (both 11), the 18th and the 20th chromosomes had the least(both 1). In matched adjacent normal tissues, the 11th chromosome had the most (17), and no significantly different methylated gene was found on Y chromosome. These genes involved in many functions, such as protein phosphorylation, regulating cellular catabolism, ion transport, enzyme activity, transcriptional regulation, cell division, cell cycle regulation, and signal transduction. CONCLUSIONS: There are significant differences between gastric cancer tissues and their matched adjacent tissues in DNA methylation. DNA methylation genes locate on different chromosomes, and their number and distribution vary widely. These genes may be associated with many pathways in carcinogenesis.

Phenological adaptations in Ficus tikoua exhibit convergence with unrelated extra-tropical fig trees.

Flowering phenology is central to the ecology and evolution of most flowering plants. In highly-specific nursery pollination systems, such as that involving fig trees (Ficus species) and fig wasps (Agaonidae), any mismatch in timing has serious consequences because the plants must balance seed production with maintenance of their pollinator populations. Most fig trees are found in tropical or subtropical habitats, but the dioecious Chinese Ficus tikoua has a more northerly distribution. We monitored how its fruiting phenology has adapted in response to a highly seasonal environment. Male trees (where fig wasps reproduce) had one to three crops annually, whereas many seed-producing female trees produced only one fig crop. The timing of release of Ceratosolen fig wasps from male figs in late May and June was synchronized with the presence of receptive figs on female trees, at a time when there were few receptive figs on male trees, thereby ensuring seed set while allowing remnant pollinator populations to persist. F. tikoua phenology has converged with those of other (unrelated) northern Ficus species, but there are differences. Unlike F. carica in Europe, all F. tikoua male figs contain male flowers, and unlike F. pumila in China, but like F. carica, it is the second annual generation of adult wasps that pollinate female figs. The phenologies of all three temperate fig trees generate annual bottlenecks in the size of pollinator populations and for female F. tikoua also a shortage of fig wasps that results in many figs failing to be pollinated.

Proliferative and apoptotic effects of andrographolide on the BGC-823 human gastric cancer cell line.

BACKGROUND: Andrographolide has been shown to have anticancer activity on diverse cancer cell lines representing different types of human cancers. The aim of this research was to investigate the anticancer and apoptotic effects of andrographolide on the BGC-823 human gastric cancer cell line. METHODS: Cell proliferation and IC50 were evaluated using MTT assay, cell-cycle analysis with flow cytometry apoptotic effects with Annexin-V/propidium iodide double-staining assay, and morphologic structure with transmission electron microscopy. Immunohistochemistry and reverse-transcription PCR was used to analyze Bcl-2, Bax, and caspase-3 expressions. RESULTS: Andrographolide showed a time- and concentration-dependent inhibitory effects on BGC-823 cell growth. Compared to controls, the number of cells in the G0-G1-phase increased significantly, S and G2-M-phase cells decreased after 48 hours of treatment with andrographolide, and both early and late apoptotic rates increased significantly compared to the controls, all in a concentration-dependent manner. Bax and caspase-3 expressions were markedly increased, and Bcl-2 expression was decreased. CONCLUSIONS: Andrographolide inhibits BGC-823 cell growth and induces BGC-823 cell apoptosis by up-regulating Bax and caspase-3 expressions and down-regulating Bcl-2 expression. Andrographolide may be useful as a potent and selective agent in the treatment of human gastric cancers.

[Effect of andrographolide on proliferation and apoptosis of gastric cancer BGC-823 cells].

OBJECTIVE: To investigate the effect of andrographolide (AD) on proliferation, cell cycle and apoptosis of human gastric cells line BGC-823. METHODS: MTT assay, flow cytometry and Annexin-V/PI double-staining flow cytometry assay were used to evaluate the effect of AD on proliferation, cell cycle and apoptosis of BGC-823 cells respectively. Optical microscope and transmission electron microscopy were used to observe the cell morphological changes. RESULTS: A time- and concentration-dependent proliferative inhibition effect of AD was demonstrated in BGC-823 cells. AD concentration lower than 7.5 mg/L possessed weak inhibitory effect,while concentration between 15.0-60.0 mg/L possessed higher inhibitory effect. The concentration higher than 60.0 mg/L had no significant increase of inhibitory effect. IC50 of AD at 24, 48 and 72 h was (35.3±4.3), (25.5±3.5) and (18.2±2.7) mg/L respectively. Compared with the negative control group, the number of G0/G1 phase cells increased significantly (P<0.05), while the number of S and G2/M phase cells decreased after incubation with AD for 48 h, and the alteration was in a concentration-dependent manner. AD arrested BGC-823 cells at the G0/G1 phase of the cell cycle. After incubation with 7.5, 10.0 and 15.0 mg/L AD for 24 h, the early apoptotic rates of BGC-823 cells were (19.3±4.7)%, (29.4±4.1)% and (52.7±6.7)% respectively, and the late apoptotic rates were (10.8±1.8)%, (10.9±4.7)% and (14.7±4.8)% respectively. Both the early apoptotic rates and the late apoptotic rates increased significantly compared to the control group (all P<0.05),and the alteration was in a concentration-dependent manner. CONCLUSION: Andrographolide can inhibit BGC-823 cells proliferation, arrest BGC-823 cells in G0/G1 phase and induce apoptosis, and may be a potential traditional Chinese medicine with anti-cancer effect.

Perifosine induces cell apoptosis in human osteosarcoma cells: new implication for osteosarcoma therapy?

Despite the advances of adjuvant chemotherapy and significant improvement of survival, the prognosis for patients with osteosarcoma is generally poor. The search for more effective anti-osteosarcoma agents is necessary and urgent. Here we report that perifosine induces cell apoptosis and growth inhibition in cultured human osteosarcoma cells. Perifosine blocks Akt/mTOR complex 1 (mTORC1) signaling, while promoting caspase-3, c-Jun N-terminal kinases (JNK), and p53 activation. Further, perifosine inhibits survivin expression probably by disrupting its association with heat shock protein-90 (HSP-90). These signaling changes together were responsible for a marked increase of osteosarcoma cell apoptosis and growth inhibition. Finally, we found that a low dose of perifosine enhanced etoposide- or doxorubicin-induced anti-OS cells activity. The results together suggest that perifosine might be used as a novel and effective anti-osteosarcoma agent.

Co-administration phenoxodiol with doxorubicin synergistically inhibit the activity of sphingosine kinase-1 (SphK1), a potential oncogene of osteosarcoma, to suppress osteosarcoma cell growth both in vivo and in vitro.

Elucidation of the mechanisms of chemo-resistance and implementation of strategies to overcome it will be pivotal to improve the survival for osteosarcoma (OS) patients. We here suggest that sphingosine kinase-1 (SphK1) might be the key factor contributing to chemo-resistance in OS. Our Western-blots and immunohistochemistry results showed that SphK1 is over-expressed in multiple clinical OS tissues. Over-expression of SphK1 in OS cell line U2OS promoted its growth and endorsed its resistance against doxorubicin, while knocking-down of SphK1 by shRNA inhibited U2OS cell growth and increased its sensitivity to doxorubicin. Co-administration phenoxodiol with doxorubicin synergistically inhibited SphK1 activity to trigger cellular ceramide accumulation, and achieved synergistic anti-OS growth effect, accompanied with a significant increased of apoptosis and cytotoxicity. Increased cellular level of ceramide by the co-administration induced the association between Akt and Protein Phosphatase 1 (PP1) to dephosphorylate Akt, and to introduce a constitutively active Akt (CA-Akt) restored Akt activation and diminished cell growth inhibition. Further, phenoxodiol and doxorubicin synergistically activated apoptosis signal-regulating kinase 1(ASK1)/c-jun-NH2-kinase (JNK) signaling, which also contributed to cell growth inhibition. Significantly, the role of SphK1 in OS cell growth and the synergistic anti-OS effect of phenoxodiol and doxorubicin were also seen in a mice OS xenograft model. In conclusion, our data suggest that SphK1 might be a critical oncogene of OS and co-administration phenoxodiol with doxorubicin synergistically inhibited the activity of SphK1 to suppress osteosarcoma cell growth both in vivo and in vitro.

[Expression of Raf kinase inhibitor protein and its significance in invasion and metastasis of hepatocellular carcinoma].

OBJECTIVE: To investigate the expression of RKIP, p65 and pERK in hepatocellular carcinoma (HCC) and theIr correlation with invasion and metastasis of HCC. METHODS: Reverse transcription polymerase chain reaction (RT-PCR) was used to detect the expression of RKIP mRNA. The expression levels of RKIP, p65 and pERK proteins in HCC tumor and peritumoral tissues were determined by immunohistochemistry and Western blot analysis. Statistical analysis was performed to determine the relationship between their expression and clinicopathological parameters. RESULTS: RKIP protein expression level (RKIP/actin) was 0.579 ± 0.380 in HCCs, 1.178 ± 0.659 in peritumoral tissues and 1.115 ± 0.442 in normal liver tissues. The pERK protein level was 1.023 ± 0.478, 0.605 ± 0.367 and 0.461 ± 0.293, p65 protein level was 0.83 ± 0.376, 0.63 ± 0.337 and 0.466 ± 0.345, respectively. Immunohistochemistry analysis showed that the RKIP positive rates in HCCs, peritumoral tissues and normal liver tissues, were 22.2%, 86.0%, and 93.8%, positive rates of p65 were 73.6%, 56.0% and 37.5%, positive rates of pERK were 65.3%, 38.0% and 31.3%, respectively. Statistical analysis revealed that there was a significant difference in RKIP protein expression levels (P < 0.05), but no significant difference in RKIP mRNA expression levels (P > 0.05) among HCC tumors, peritumoral tissues and normal liver tissues. The p65-positive and pERK-positive rates were higher in tumor tissues than that in peritumoral tissues and in normal liver tissues (P < 0.05), but RKIP-positive rates were lower in tumor tissues than that in paritumoral tissues and normal liver tissues (P < 0.05). RKIP protein expression levels were significantly lower in HCCs with intrahepatic or lymphatic metastasis than that in without. The RKIP positive rates in moderately and well differentiated HCCs were significantly higher than that in poorly differentiated HCCs. There was a relationship between RKIP and pERK expressions (P = 0.04), but RKIP expression was not correlated with p65 expression in HCCs (P = 0.143). CONCLUSIONS: Our findings indicate that the down-regulation of RKIP expression may serve as a predictive marker for HCC development, progression and metastasis, which may contribute to the elevated ERK activity. The inhibiting effect of RKIP on invasion and metastasis of liver cancer cells may be due to the down-regulation of pERK expression rather than p65 expression.