A P53-related microRNA model for predicting the prognosis of hepatocellular carcinoma patients.

Studies have shown that microRNAs (miRNAs) play a vital role in tumor progression and patients' prognosis. Therefore, we aimed to construct a miRNA model for forecasting the survival of hepatocellular carcinoma (HCC) patients. The gene expression data of 433 patients with HCC from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus public databases were remined by survival analysis and receptor manipulation characteristic curve (ROC). A prognostic model including six miRNAs (hsa-mir-26a-1-3p, hsa-mir-188-5p, hsa-mir-212-5p, hsa-mir-149-5p, hsa-mir-105-5p, and hsa-mir-132-5p) were constructed in the training dataset (TCGA, n = 333). HCC patients were stratified into a high-risk group and a low-risk group with significantly different survival (median: 2.75 vs. 8.93 years, log-rank test p < .001). Then we proved its performance of stratification in another independent dataset (GSE116182, median: 2.55 vs 6.96 years, log-rank test p = .008). Cox regression analysis showed that the prognostic model was an independent prognostic indicator for HCC patients. Then time-dependent ROC analyses were performed to test the prognostic ability of the model with that of TNM staging, we found the model had a better performance, especially at 5 years (AUC = 0.76). Functional prediction showed that the genes targeted by the six prognostic miRNAs in the prognostic model were highly expressed in the P53-related pathway. In conclusion, we constructed a prognostic miRNA model that could indicate the survival of HCC patients.

Effects of PEG-liposomal oxaliplatin on apoptosis, and expression of Cyclin A and Cyclin D1 in colorectal cancer cells.

Oxaliplatin is one of the agents used against colorectal cancer. Using PEG-liposome encapsulated oxaliplatin may enhance the accumulation of drugs in tumor cells, inducing apoptosis. However, the mechanism of action of PEG-liposome encapsulated oxaliplatin remains unclear. SW480 human colorectal cancer cells were treated with empty PEG-liposomes, free oxaliplatin or PEG-liposomal oxaliplatin. Cell cycle and apoptosis were assessed using fluorescence confocal microscopy and terminal deoxynucleotidyl transferase-mediated dUTP-fluorescein nick-end-labeling (TUNEL). Western blotting was used to analyze the expression of pro-apoptotic, anti-apoptotic and cyclin proteins. We found that PEG-liposomal oxaliplatin induced a stronger apoptotic response than empty PEG-liposomes or free oxaliplatin. Moreover, expression of Cyclin D1 increased, whereas expression of Cyclin A decreased after treatment with PEG-liposomal oxaliplatin. Furthermore, the cell cycle was arrested in the G1 phase. The results presented here indicate that PEG-liposome entrapment of oxaliplatin enhances the anticancer potency of the chemotherapeutic agent. The effect of PEG-liposomal oxaliplatin on apoptosis of SW480 human colorectal cancer cells may be through regulation of expression of Cyclin A or Cyclin D1, as well as pro-apoptotic and anti-apoptotic proteins.

Tubeimoside-1 induces G2/M phase arrest and apoptosis in SKOV-3 cells through increase of intracellular Ca²âº and caspase-dependent signaling pathways.

Tubeimoside-1 (TBMS1) extracted from Bolbostemma paniculatum (Maxim), is a traditional Chinese herb with anticancer potential. It induces apoptosis in a number of human carcinoma cell lines, but the mechanism has remained unclear. In the present study, we investigated the pro-apoptotic activity of TBMS1 against SKOV-3 cell lines and the underlying mechanisms. Treatment with TBMS1 resulted in dose- and time-dependent inhibition of proliferation, led to arrest in phase G2/M of the cell cycle and increased the levels of intracellular Ca²âº. Furthermore, TBMS1 up-regulated the levels of the glucose-regulated protein 78/immunoglobuin heavy chain binding protein (GRP78/Bip), C/EBP homologous protein (CHOP), Bax, and cleaved caspase-3 and down-regulated the levels of Bcl-2. It was shown to be linked to activation of the extracellular signal-regulated kinase (ERK) 1 and 2 signal transduction pathway. A decrease in Bcl-2/Bax ratio with increased expression of caspase-3, and intracellular Ca²âº provide compelling evidence that TBMS1-induced apoptosis is mediated by the mitochondrial pathway. The results of the present study suggest that TBMS1 has immense potential in cancer prevention and therapy based on its antiproliferative and apoptosis inducing effects.

PEG-liposomal oxaliplatin induces apoptosis in human colorectal cancer cells via Fas/FasL and caspase-8.

Since cellular uptake of PEG [poly(ethylene glycol)]-liposomal L-OHP (oxaliplatin) induces bioactive changes in CRC (colorectal cancer), we have investigated its apoptotic effect and anticancer mechanism. Human CRC SW480 cells were treated with PEG-liposomal L-OHP and a caspase-8 inhibitor [Z-IETD-FMK (benzyloxycarbonyl-Ile-Glu-Thr-dl-Asp-fluoromethylketone)]. Apoptosis was measured by FCM (flow cytometry) and TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling) assay. Expression of Fas/FasL and cytochrome c was detected using FCM and an immunofluorescence assay. Expression of caspase-8, Bid, caspase-9, caspase-7 and activated caspase-3 (P17) was examined by Western blot analyses. The results indicated that PEG-liposomal L-OHP (28 µg/ml L-OHP) induced marked apoptosis in SW480 cells compared with 28 µg/ml free L-OHP. The expression levels of Fas, FasL, cytochrome c, caspase-9, caspase-7 and activated caspase-3 proteins were up-regulated, with a corresponding increase in apoptosis; however, expression of caspase-8 and Bid were down-regulated as apoptosis increased. When cells were treated with Z-IETD-FMK, apoptosis was inhibited, but there was little impact on the expression of Fas, FasL, cytochrome c, Bid, caspase-9, caspase-7 and activated caspase-3. These findings indicate that PEG-liposomal L-OHP enhances the anticancer potency of the chemotherapeutic agent; moreover, Fas/FasL and caspase-8 signalling pathways play a key role in mediating PEG-liposomal L-OHP-induced apoptosis.

Tubeimoside I sensitizes cisplatin in cisplatin-resistant human ovarian cancer cells (A2780/DDP) through down-regulation of ERK and up-regulation of p38 signaling pathways.

Cisplatin (CDDP) is a major chemotherapeutic drug used in the treatment of human ovarian cancer. Tubeimoside I (TBMS1) has also shown potent antitumor and antitumor-promoting effects, and may offer a promising new approach in the effective treatment of CDDP-resistant human ovarian cancers. This study aimed to investigate the effect of TBMS1 in sensitizing CDDP in CDDP-resistant human ovarian cancer cells (A2780/DDP). A variety of methods were employed to measure cell apoptosis, p38, ERK1/2 and glutathione S-transferase (GST)-π expressions. It was found that TBMS1 combined with CDDP promoted cell apoptosis, decreased proliferation activity and increased cytosolic Ca2+ levels. Bcl-2 protein expression was down-regulated but Bax was up-regulated. Moreover, GST-π mRNA and protein expression were decreased. TBMS1 reduced the resistance of the cells to CDDP-induced cytotoxicity. Both the p38 inhibitor (SB203580) and the ERK1/2 inhibitor (PD98059) effectively blocked this effect. These results suggest that TBMS1 can effectively sensitize CDDP in CDDP-resistant human ovarian cancer cells through the down-regulation of the ERK1/2 and the up-regulation of the p38 signaling pathways.

Tetramethylpyrazine inhibits migration of SKOV3 human ovarian carcinoma cells and decreases the expression of interleukin-8 via the ERK1/2, p38 and AP-1 signaling pathways.

Interleukin-8 (IL-8) expression by melanoma cells may influence their metastatic capabilities. Tetramethylpyrazine (TMP) from Ligusticum wallichil Franch. possesses anti-inflammatory and antitumor activities. It has recently been suggested that autocrine IL-8 may play a role in tumor cell survival, invasion and migration. The role of TMP in association with IL-8 in the tumor cell migratory process remains unclear. The purpose of the present study was to determine whether TMP influences the migratory ability of a human ovarian carcinoma cell line (SKOV3) via regulation of IL-8 expression in vitro. Cell counts showed that treatment of SKOV3 with TMP (25-100 µg/ml) for 24 h did not decrease cell numbers, while an effect of TMP on the down-regulation of the expression of IL-8 was observed. In addition, migration of SKOV3 cells was suppressed after treatment with TMP (25-100 µg/ml) for 24 h. Therefore, expression of IL-8 by SKOV3 cells correlates with their metastatic potential. Western blot analysis revealed that ERK1/2 and p38 phosphorylation was blocked by TMP. Furthermore, IL-8 mRNA expression was inhibited significantly after co-incubation with PD98059 (ERK inhibitor) and SB203580 (p38 inhibitor), respectively. Notably, these changes were the results of activator protein-1 (AP-1) activity suppression rather than that of NF-κB. Our data suggest that TMP may inhibit tumor cell invasion and migration, at least in part, through its down-regulation of IL-8 expression. Our results provide evidence that anti-inflammation plays an important role in integrative cancer therapies.

PEG-liposomal oxaliplatin potentialization of antitumor efficiency in a nude mouse tumor-xenograft model of colorectal carcinoma.

The non-selectivity of chemotherapeutics between normal tissue and pathological sites poses a challenge for the treatment strategy for advanced colorectal carcinoma. To obtain sufficient antitumor activity, optimization of the therapeutic regimen is of great importance. We investigated PEG-liposomal oxaliplatin potentialization of antitumor efficiency in a nude mouse tumor-xenograft model of colorectal carcinoma. A tumor-bearing nude mouse model, intravenous injections of (Dio)-labeled PEG-liposomes via tail vein and fluorescence imaging with in vivo imaging system were employed. Mice were treated with free L-oHP, PEG-liposomal L-oHP via the tail vein, followed by analysis of the accumulation of L-oHP in tumor tissues by high-performance liquid chromatography (HPLC), observation of the tumor volume and the survival rate. Apoptosis and proliferation of tumors were detected by TUNEL assay and immunohistochemistry. The mRNA and protein levels of Bcl-2, Bax, caspase-3 (P17) and Ki-67 were determined by RT-PCR and Western blotting. Fluorescence imaging with in vivo imaging showed PEG-liposome targeting in tumor tissues. After intravenous injections of PEG-liposomal oxaliplatin, tumor tissue maximum accumulation of L-oHP was 9.37 ± 0.79 µg/g at 24 h; The tumor volume was significantly suppressed, and mice showed longer survival, compared with the free oxaliplatin group. Apoptosis increased, but proliferation decreased in tumor tissues. The mRNA expression of Bcl-2 and Ki-67 was down-regulated, while Bax and caspase-3 expression was up-regulated. Protein expression of Bcl-2 was down-regulated, while Bax and P17 expression was up-regulated. The results indicate that PEG-liposomal oxaliplatin can improve antitumor efficiency in a nude mouse tumor-xenograft model of colorectal carcinoma.

[The mechanism and treatment phases chosen of glycine for inhibition lipopolysaccharide induced Kupffer cells activation].

OBJECTIVE: To explore the possible mechanism and optimal treatment phase of glycine for inhibition lipopolysaccharide (LPS) induced Kupffer cells (KCs) activation. METHODS: The KCs were isolated from 40 BALB/c mice and divided into four groups: the endotoxin group, the prevention group, the early treatment group and the later treatment group (n = 10). The endotoxin group was treated with 10 mg/L LPS, and in the other three groups, glycine (1 mmol/L) was given 24 h before, or at 0 h or 4 h respectively after LPS stimulation. At 0 h, 1 h, 2 h, 6 h and 12 h after LPS stimulation, the mRNA levels and protein expression of interleukin-1 receptor associated kinase-4 (IRAK-4) were determined by reverse transcription polymerase chain reaction (RT-PCR) and Western blot respectively, and nuclear factor-kappaB (NF-kappaB) activities as well as tumor necrosis factor alpha (TNF-alpha) levels were also detected by enzyme-linked immunosorbent assay (ELISA). RESULTS: The climax values of IRAK-4, NF-kappaB and TNF-alpha were significantly higher in the endotoxin group and the later treatment group than that in the other two groups (t = 3.17, 4.33, 2.47, 126.73, P < 0.01). CONCLUSION: The results indicated that prophylactic or simultaneous treatment with glycine could effectively inhibit LPS-induced KCs activation by inhibiting IRAK-4 expression.

[Expression of CD14 and Toll-like receptor 4 on Kupffer cells and its role in ischemia-reperfusion injury on rat liver graft].

OBJECTIVE: To study the expression of lipopolysaccharide (LPS) receptor CD14 and Toll-like receptor 4 (TLR4) on Kupffer cells and its role in ischemia-reperfusion injury (IRI) on rat liver graft. METHODS: The Kupffer cells following IRI were isolated and divided into control, ischemia-reperfusion (IR), and anti-CD14 antibody group. The mRNA and protein expression of CD14 and TLR4, nuclear factor kappa B (NF-kappaB) activity and TNF-alpha level in supernatant were measured. RESULTS: The mRNA and protein expression of CD14 and TLR4 in IR group were significantly higher than those in control group (P < 0.01). The NF-kappaB activity and TNF-alpha level in IR group were significantly higher than those in control group (P < 0.01), and they greatly decreased after anti CD14 antibody treatment (compared with IR group, P < 0.05), but were still significantly higher than those in control group (P < 0.01). CONCLUSIONS: LPS following IRI could up-regulate CD14 and TLR4 gene and protein expression on Kupffer cells, and subsequently activate NF-kappaB to produce cytokines, but other signal transduction pathways might also participate in the IRI.

[Expression of CD14 protein in U937 cells induced by vitamin D3].

AIM: To observe the 1, 25-dihydroxy vitamin D3 (Vit D3) induced CD14 expression in human U937 cell line and the reaction of the cells to LPS stimulation following the induction. METHODS: U937 cells were cocultured with 0.1 mumol/L Vit D3 for 24 hours to induce the expression of CD14 gene and the sensitiveness of U937 cells to stimulation of LPS at various concentrations and for different times were observed. RESULTS: Vit D3 stably induced U937 cells to express CD14 mRNA and CD14 protein. The sensitivity of U937 cells to LPS stimulation increased notably after Vit D3 induction. It was demonstrated that low concentration of LPS stimulated the activation of NF-kappaB in U937/CD14 cells, and enhanced the transcription and expression of TNF-alpha gene, and even release of TNF-alpha into the culture supernatant. CONCLUSION: Vit D3 can induce U937 cells to express CD14 gene and CD14 protein and enhance the reactivity of U937/CD14 cells to LPS stimulation.

The U937 cell line induced to express CD14 protein by 1,25-dihydroxyvitamin D3 and be sensitive to endotoxin stimulation.

BACKGROUND: CD14 was first described as a differentiation antigen on the surface of myeloid lineage cells. It acts as a glycosylphosphatidylinositol (GPI)-anchored receptor for the complex of lipopolysaccharide (LPS) and plays a key role in the activation of LPS-induced monocytes. The purpose of this study was to observe the expression of CD14 protein and its gene in the human U937 promonocytic cell line when these cells were exposed to 1,25-dihydroxyvitamin D3(VitD3) and investigate their sensitivity to endotoxin stimulation. METHODS: U937 cells were exposed to (0.1 micromol) VitD3 for 24 hours and were induced to express the CD14 mRNA gene and CD14 protein, then their responses were observed when they were stimulated with different concentrations of LPS for different time. RESULTS: The U937 cells induced by VitD3 were found to stably express CD14 mRNA and CD14 protein. And CD14 protein enhanced the sensitivity of U937/CD14 cells to lipopolysaccharide (LPS) stimulation. NF-kappaB in U937/CD14 cells can be activated with low concentration of LPS (1 ng/ml-10 ng/ml), the TNF-alpha mRNA gene was induced, and then TNF-alpha was produced and released into the supernatant of culture. CONCLUSION: VitD3 can induce U937 cell to express the CD14 gene and CD14 protein and enhance the response of this type of cells to LPS stimulation.