[Zexie Decoction regulates Akt/TFEB signaling pathway to promote lipophagy in hepatocytes].

Taking lipophagy as the breakthrough point, we explored the mechanism of Zexie Decoction(ZXD) in improving lipid metabolism in the hepatocyte model induced by palmitic acid(PA) and in the animal model induced by high-fat diet(HFD) on the basis of protein kinase B(Akt)/transcription factor EB(TFEB) signaling pathway. Co-localization was carried out for the microtubule-associated protein light chain 3(LC3) plasmid labeled with green fluorescent protein(GFP) and lipid droplets(LDs), and immunofluorescence co-localization for liver LC3 of HFD mice and perilipin 2(PLIN2). The results showed that ZXD up-regulated the expression of LC3, reduced lipid accumulation in hepatocytes, and increased the co-localization of LC3 and LDs, thereby activating lipo-phagy. Western blot results confirmed that ZXD increased autophagy-related protein LC3Ⅱ/LC3Ⅰ transformation ratio and lysosome-associated membrane protein 2(LAMP2) in vivo and in vitro and promoted the degradation of sequestosome-1(SQSTM1/p62)(P<0.05). The results above jointly explained that ZXD regulated lipophagy. Furthermore, ZXD activated TFEB expression(P<0.05) and reversed the PA-and HFD-induced decrease of TFEB nuclear localization in hepatocytes(P<0.05). Meanwhile, ZXD activated liver TFEB to up-regulate the expression of the targets Lamp2, Lc3 B, Bcl2, and Atg5(P<0.05). Additionally, ZXD down-regulated the protein level of p-Akt upstream of TFEB in vivo and in vitro. In conclusion, ZXD may promote lipophagy by regulating the Akt/TFEB pathway.

LncRNA-cCSC1 promotes cell proliferation of colorectal cancer through sponging miR-124-3p and upregulating CD44.

LncRNA-cCSC1 is highly expressed in colorectal cancer (CRC). The study was designed to evaluate the function and mechanism of lncRNA-cCSC1 in cell proliferation of CRC. RT-PCR was used to measure the expression levels of lncRNA-cCSC1 in CRC cell lines. CCK-8, colony formation, EdU staining, flow cytometry and Western blot were performed to examine the effect of interference with lncRNA-cCSC1 expression on cell proliferation. miR-124-3p and the target genes of miR-124-3p were investigated using bioinformatics analysis and verified by dual-luciferase reporter, RT-PCR and Western blot. Rescue experiments were carried out to confirm the role of miR-124-3p in cell proliferation of CRC. Our results showed that cell proliferation of CRC was promoted by lncRNA-cCSC1 upregulation and inhibited by lncRNA-cCSC1 downregulation. In addition, miR-124-3p is predicted to be the target of lncRNA-cCSC1 and is negatively correlated with lncRNA-cCSC1. Moreover, the addition of miR-124-3p mimics or inhibitor reversed the effects induced by lncRNA-cCSC1 overexpression or silencing on cell proliferation of CRC. Additionally, lncRNA-cCSC1 regulated the expression level of CD44, a target gene of miR-124-3p. Finally, we studied the effects of the lncRNA-cCSC1/miR-124-3p axis on CD44. These results indicate that lncRNA-cCSC1 promotes cell proliferation of CRC through sponging miR-124-3p and upregulating CD44.

High expression of CASK correlates with progression and poor prognosis of colorectal cancer.

Calcium/calmodulin-dependent serine protein kinase (CASK), which localizes at cell-cell adhesion sites and binds to the heparan sulfate proteoglycan syndecan-2, is involved in cell proliferation, cytoskeletal remodeling, and cell migration. To demonstrate the role of CASK in colorectal cancer (CRC) carcinogenesis, we examined the expression of CASK and its binding protein syndecan-2 in human CRC tissues. The expression of CASK was measured in CRC specimens and the controls from adenomas and normal mucosae by immunohistochemical staining and Western blot analysis. Syndecan-2 protein level was tested in CRC samples and the controls by Western blot analysis. The correlations between CASK expression and clinicopathological variables, including disease-free survival (DFS) and overall survival (OS), were analyzed. Compared to the controls, both CASK and syndecan-2 expression were enhanced in CRC tissues. Furthermore, high expression of CASK and syndecan-2 was significantly correlated with advanced tumor stage, lymphatic invasion, lymph node metastasis, vascular invasion, liver metastasis, and unresectable metastatic CRC. Survival analysis showed that patients with low CASK staining had a significantly better survival compared to patients with high CASK staining. In multivariate analysis, CASK overexpression, advanced tumor stage, lymph node metastasis, vasvular invasion, and liver metastasis were independent prognostic factors of poor DFS and OS. Our present study indicates that CASK overexpression is associated with an unfavorable prognosis. CASK is an independent prognostic factor for CRC, which suggests that it is a novel and crucial predictor for CRC metastasis.

[Effects of chlorogenic acid on the viability and HIF-1alpha mRNA expression of PC12 cells exposed to hypoxia].

OBJECTIVE: To investigate the effects of chlorogenic acid on the viability and HIF-1alpha mRNA expression of PC12 cells exposed to hypoxia. METHODS: PC12 cells were cultured in trigas incubator in order to establish the hypoxic condition. The effects of chlorogenic acid on the cells were evaluated by morphological observation, cell viability and LDH release assays as well as the examination of mRNA expression level of HIF-1alpha. RESULTS: Chlorogenic acid significantly improved the viability of cells exposed to hypoxia, decreased LDH release, arrested the cell cycle on G1 phase, and increased the gene expression level of HIF-1alpha. CONCLUSION: Chlorogenic acid protects PC12 cells from hypoxic damage by improving the expression of HIF-1alpha.

Paired-related homeobox 1 induces epithelial-mesenchymal transition in oesophageal squamous cancer.

BACKGROUND: It is unclear that paired-related homeobox 1 (PRRX1) induces epithelial-mesenchymal transition (EMT) in oesophageal cancer and the specific function of PRRX1 in oesophageal cancer metastasis. AIM: To assess the significance of PRRX1 expression and investigate the mechanism of EMT in oesophageal cancer metastasis. METHODS: Detect the expression of PRRX1 by immunohistochemistry in oesophageal tumour tissues and adjacent normal oesophageal tissues; the PRRX1 short hairpin RNA (shRNA) or blank vector lentiviral gene delivery system was transfected into cells; cell proliferation assay, soft agar colony formation assays, cell invasion and migration assays and animal studies were used to observe cells biological characteristics In vitro and in vivo; XAV939 and LiCl were used to alter the activity of Wnt/ß-catenin pathway. Immunofluorescence staining and western blot analysis were used to detect protein expression of EMT markers and Wnt/ß-catenin pathway. RESULTS: PRRX1 is expressed at high levels in oesophageal cancer specimens and is closely related to tumour metastasis in patients with oesophageal cancer. Regulation of PRRX1 expression might exert obvious effects on cell proliferation, especially the migration and invasion of oesophageal cancer cells. Moreover, silencing PRRX1 expression using a shRNA produced the opposite effects. In addition, when PRRX1 was overexpressed, inhibition of the Wnt/ß-catenin pathway with XAV939 negated the effect of PRRX1 on EMT, whereas when PRRX1 was downregulated, activation of the Wnt/ß-catenin pathway with LiCl impaired the effect on EMT. CONCLUSION: PRRX1 is upregulated in oesophageal cancer is closely correlated with cancer metastasis. Additionally, PRRX1 induces EMT in oesophageal cancer metastasis through activation of Wnt/ß-catenin signalling.

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.

A meta-analysis of the relationship between NAT2 polymorphism and colorectal cancer susceptibility.

BACKGROUND AND OBJECTIVE: Although the association between N-acetyltransferase 2 (NAT2) polymorphism and colorectal cancer (CRC) susceptibility in humans has been extensively investigated, the results are contradictory. The aim of this study was to conduct a meta-analysis of published studies to quantitatively summarize the association between NAT2 polymorphism and risk of CRC. MATERIAL AND METHODS: Relevant studies that had investigated NAT2 polymorphism and CRC susceptibility were identified through a comprehensive search of Pubmed, EMBASE, Medline, Biosis, Wiley-Blackwell, ISI Web of Knowledge, CNKI, and Chinese Biomedicine Database until October 2011. After selection based on the inclusion and exclusion criteria, the relevant data were extracted from each study, and finally a meta-analysis was performed. RESULTS: Eight phenotype studies (791 cases and 1158 controls) and 45 genotype studies (13 875 cases and 18 879 controls) were included in the present meta-analysis. The pooling of phenotype studies showed no significant association between the NAT2 acetylator status and CRC susceptibility (rapid acetylator, OR, 1.32; 95% CI, 0.92-1.89, P=0.14; slow acetylator, OR, 0.76; 95% CI, 0.53-1.09, P=0.14). The combined ORs for rapid and slow acetylator status and CRC risk in genotype studies were 1.01 (95% CI, 0.94-1.08; P=0.86) and 0.99 (95% CI, 0.93-1.06; P=0.86), respectively. In the subgroup analysis by regions, no increased risks were found in Asians, Europeans, Americans, or Australasians. Pooling studies were also conducted on the groups of gender, specific tumor sites, and smoking status, but no significant association in genotype distribution between CRC and control was found as well. CONCLUSIONS: These results of our meta-analysis suggest that there is no overall association between NAT2 polymorphism and CRC susceptibility.

Localization of IL-17+Foxp3+ T cells in esophageal cancer.

The etiology of cancer is unclear. Recent studies indicate that some cytokines, such as interleukin (IL)-17, and regulatory T cells are involved in the development of cancer. This study aims to detect a subset of T cell, IL17+Foxp3+ T cell, in the pathogenesis of esophageal cancer (Eca). Twelve patients with squamous Eca were recruited in this study. The surgically removed Eca tissue was collected. Cells isolated from Eca tissue were analyzed by flow cytometry. The results showed that 2-10% Eca tissue-derived CD4(+) T cells expressed Foxp3; only 0.2-0.8% non-ca tissue-derived CD4(+) T cells expressed Foxp3. Further analysis showed that 3-15% Eca-isolated CD4(+) T cells were also IL-17 positive whereas only 0.4-1.5% non-ca tissue-isolated CD4(+) T cells were IL-17 positive. We also found that about 4.8-11.2% Foxp3(+) IL-17(+) T cells in isolated CD4(+) T cells from Eca tissue that were significantly less than in non-ca tissue derived CD4(+) T cells. Less than 1% Foxp3(+) IL-17(+) T cells in isolated CD4(+) T cells in both Eca patients and healthy controls. Treatment with hypoxia markedly increased the expression of IL-6 in peripheral CD68+ cells. Coculturing CD68+ cells and Foxp3+ T cells under hypoxic environment resulted in abundant expression of IL-17 in Foxp3+ T cells that could be blocked by pretreatment with either anti-IL-17 or anti-transforming growth factor beta antibodies. We conclude that IL-17+Foxp3+ T cells may contribute to the development of Eca.

[The study in lasting effectivity on conditionally replicating adenovirus shRNA mediating survivin gene silencing in colon carcinoma cell line HT-29].

OBJECTIVE: To investigate the lasting effect of conditionally replicating adenovirus shRNA on survivin gene silencing in colon carcinoma cell lineHT-29. METHODS: We transfected the colon cancer HT-29 cell with Ad-delE1b55KD-shRNA/Survivin-EGFP (control shRNA vector was the replication defective adenovirus called as Ad-shRNA/Survivin-EGFP). The expressions of EGFP, survivin mRNA and protein in HT-29 were detected at 1st day, 7th day, 14th day and 28th day after cell transfection. RESULTS: The expression of EGFP in HT-29 was high at 7th day after cell transfection but survivin mRNA and protein expressions inhibited in each experiment group, among which Ad-delE1b55KD-shRNA/Survivin-EGFP group showed the most high EGFP expression; at 14th day, the expression of EGFP went down but the survivin mRNA and protein expressions upregulated obviously in replication defective adenovirus group and liposome vector group, however EGPF expression in HT-29 cell was still high and the survivin mRNA and protein expressions were still inhibited in Ad-delE1b55KD-shRNA/Survivin-EGFP group; at 28th day after cell HT-29 transfected, the expression of EGPF and the expression inhibition of survivin mRNA and protein disappeared in Ad-shRNA/Survivin-EGFP and liposome groups, but it was not in Ad-delE1b55KD-shRNA/Survivin-EGFP group (P < 0.05). CONCLUSION: Conditionally replicating adenovirus shRNA can mediate the survivin gene silencing with long-lasting effects on colon carcinoma cell lines HT-29.

Sestrin 2 suppresses cells proliferation through AMPK/mTORC1 pathway activation in colorectal cancer.

Sestrin 2 is a conserved antioxidant protein that reduces reactive oxygen species (ROS) and inhibits mammalian target of rapamycin complex 1 (mTORC1). We previously showed that sestrin 2 is abnormally decreased in colorectal cancer (CRC). To elucidate the molecular mechanism behind the potential contribution of sestrin 2 to CRC, we used a lentiviral expression vector system to determine the effects of sestrin 2 overexpression on human CRC cells. We found that sestrin 2 overexpression decreased ROS production, inhibited cell growth, and stimulated apoptosis in two CRC cell lines. In parallel, expression of the proliferation marker PCNA was decreased, proapoptotic caspase 3, 7, and 9 levels were increased, and expression of the anti-apoptotic protein survivin was reduced. Sestrin 2 overexpression also activated the adenosine monophosphate-activated protein kinase (AMPK) pathway, and suppressed mTORC1 signaling. Treating CRC cells with compound C, an AMPK inhibitor, reversed or attenuated changes in proliferation, apoptosis, and signaling proteins of the AMPK/mTORC1 axis. In a xenograft mouse model, CRC growth was attenuated by sestrin 2 overexpression. These results suggest that sestrin 2 suppresses CRC cell growth through activation of the AMPK/mTORC1 pathway and induction of apoptosis, and could be a novel pharmacological target for the treatment of CRC.

Decreased expression of sestrin 2 predicts unfavorable outcome in colorectal cancer.

Sestrin 2 is a conserved antioxidant protein that is involved in p53­dependent antioxidant defenses and protects cells against oxidative stresses. The present study was conducted to examine the expression of sestrin 2 in colorectal cancer (CRC) and investigate a possible relationship between sestrin 2 expression and prognosis in CRC. The expression of sestrin 2 in human CRC tissues and cell lines was evaluated by immunohistochemical or immunofluorescent staining and western blot analysis. The correlations between sestrin 2 expression in human CRC tissues and clinicopathological variables, including overall survival (OS) and disease­free survival (DFS), were analyzed. Both human CRC tissues and cell lines showed a decreased expression of sestrin 2. Furthermore, a low expression of sestrin 2 was significantly correlated with advanced tumor stage, lymphatic invasion, lymph node metastasis, vascular invasion and liver metastasis. Survival analysis showed that patients with low sestrin 2 staining had a significantly worse DFS and OS. Additionally, early or advanced stage CRC patients with a low expression of sestrin 2 had a shorter survival. In univariate analysis, the patients with low sestrin 2 expression, advanced tumor stage, lymphatic invasion, lymphatic node metastasis, vascular invasion, liver metastasis and peritoneal metastasis had shorter OS and DFS. In multivariate analysis, only low sestrin 2 expression, advanced tumor stage, lymphatic node metastasis, vascular invasion and liver metastasis remained as independent prognostic factors of poor OS and DFS. The findings suggested that a decreased expression of sestrin 2 is associated with an unfavorable prognosis, which suggests that it is a novel and crucial predictor for CRC metastasis.

PEG-liposomal oxaliplatin combined with nuclear factor-κB inhibitor (PDTC) induces apoptosis in human colorectal cancer cells.

To achieve sufficient antitumor activity for colorectal carcinoma, optimization of the therapeutic regimen is of great importance. The aim of the present study was to investigate the effects of polyethylene glycol (PEG)-liposomal oxaliplatin (L-OHP) on the induction of apoptosis in human colorectal cancer SW480 cells and how the nuclear factor-κB (NF-κB) pathway may contribute to mediating PEG-liposomal L-OHP-induced apoptosis. PEG-liposomal L-OHP was prepared and used to treat colorectal cancer SW480 cells. SW480 cell uptake of liposomes was observed by laser focus or SEM. Apoptosis was measured by flow cytometry (FCM) and terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling (TUNEL) assay. Expression of NF-κB, Bcl-2, Bax and activated caspase-3 (P17) was examined by western blot analyses. The results indicated that PEG-liposomal L-OHP induced apoptosis. When pretreated with pyrrolidine dithiocarbamate (PDTC), PEG-liposomal L-OHP induced a significant apoptotic response. Moreover, apoptosis was associated with concentration of PDTC. Expression of protein p-P65, Bcl-2 was downregulated, but Bax and P17 were upregulated. These findings indicate that PEG-liposomal L-OHP enhances the anticancer potency of the chemotherapeutic agent. Moreover, NF-κB signaling pathways may contribute to mediating PEG-liposomal L-OHP-induced apoptosis.

Liposomal delivery and polyethylene glycol-liposomal oxaliplatin for the treatment of colorectal cancer (Review).

Oxaliplatin is effective for the treatment of advanced colorectal cancer; however, its application is restricted due to its dose-limiting toxicity. Liposomes are sphere-shaped vesicles consisting of one or more phospholipid bilayers. Liposomes as drug carriers are characterized by delayed release, lesion targeting and may be used as a drug-delivery system to decrease the side effects of cytotoxic drugs. Active targeting modification of liposomes may change the biological distribution of the anticancer agents, reduce or reverse multidrug resistance of tumor cells and enhance the effects of anticancer therapy. Based on the characteristics mentioned above, the aim of the present review was to demonstrate that polyethylene glycol-liposomes containing oxaliplatin may offer advantages for the treatment of colorectal cancer in clinical practice.

Effect of curcumin on human colon cancer multidrug resistance in vitro and in vivo.

OBJECTIVE: To determine whether curcumin reverses the multidrug resistance of human colon cancer cells in vitro and in vivo. METHODS: In a vincristine-resistant cell line of human colon cancer, the cell viability of curcumin-treated cells was determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Rhodamine123 efflux was evaluated to detect P-glycoprotein transporter activity, and expression of the multidrug resistance protein 1 and survivin genes was analyzed by reverse transcription polymerase chain reaction and western blotting. In addition, xenograft mouse tumors were grown and treated with curcumin. The morphology of the xenografts was investigated by hematoxylin-eosin staining. The in vivo expression of the multidrug resistance gene and P-glycoprotein and survivin genes and proteins was observed using reverse transcription-polymerase chain reaction and western blotting, respectively. RESULTS: Curcumin was not obviously toxic to the vincristine-resistant human colon cancer cells at concentrations less than 25 µM, but the growth of cells was significantly inhibited. At concentrations greater than 25 µM, curcumin was toxic in a concentration-dependent manner. The sensitivity of cells to vincristine, cisplatin, fluorouracil, and hydroxycamptothecin was enhanced, intracellular Rhodamine123 accumulation was increased (p<0.05), and the expression of the multidrug resistance gene and P-glycoprotein were significantly suppressed (p<0.05). The combination of curcumin and vincristine significantly inhibited xenograft growth. The expression of the multidrug resistance protein 1 and survivin genes was significantly reduced in xenografts of curcumin-treated mice and mice treated with both curcumin and vincristine relative to control mice. CONCLUSION: Curcumin has strong reversal effects on the multidrug resistance of human colon carcinoma in vitro and in vivo.

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.

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.

Effect of hypoxia-inducible factor 1-α on Survivin in colorectal cancer.

Colorectal cancer is a one of the most common malignancies. Hypoxia-inducible factor 1-α (HIF1-α) and Survivin play important roles in tumor development; however, the literature currently contains few reports on the relationship between them in colorectal cancer. In this study, we investigated the effect of HIF1-α on Survivin in colorectal cancer. Immunohistochemical staining was used to detect the expression of HIF1-α and Survivin in colorectal cancer tissue from 32 patients. Colon adenocarcinoma SW480 cells were cultured under normoxia and hypoxic conditions, and the expression of HIF1-α and Survivin was detected by RT-PCR and Western blotting. We also silenced HIF1-α in order to detect the expression of Survivin and cell apoptosis. In an in vivo xenograft tumor model, the effect of HIF1-α on cancer development and Survivin was evaluated by the measurment of tumor volume and immunohistochemical analysis. Analysis revealed that HIF1-α (75%) and Survivin (68.75%) were both overexpressed in colorectal cancer, and that their expression was correlated. They were also expressed in SW480 cells under conditions of normoxia, and exhibited a significant increase in expression under hypoxic conditions. The inhibition of HIF1-α by RNA interference decreased the expression of Survivin and led to the apoptosis of the SW480 cell line. In the in vivo xenograft tumor model, the expression of HIF1-α and Survivin was decreased in the siHIF1-α group, and the tumor volume (586.67±41.63 mm3) was much smaller than that in the negative interference (1374.67±85.87 mm3) and saline-treated (1382.80±28.42 mm3) groups. Our results indicate that HIF1-α is an important regulator of Survivin expression and has great potential capacity for cancer therapeutics.

Oncolytic adenovirus mediated Survivin RNA interference and 5-fluorouracil synergistically suppress the lymphatic metastasis of colorectal cancer.

Colorectal cancer is one of the leading malignancies in the world. The mortality is mainly caused by tumor metastasis. It is urgent to find new therapeutics against metastatic colorectal cancer. We constructed an adenovirus carrying a Survivin targeted shRNA, tested its effects alone or with 5-fluorouracil (5-FU) both in vitro and in a nude mouse xenograft model. Results showed that the recombinant adenovirus reduced the expression of Survivin effectively, and administration of virus together with 5-FU inhibited cancer cell metastasis both in vitro and in vivo at concentrations at which each agent alone was ineffective. We conclude Survivin targeted virotherapy together with 5-FU chemotherapy may be a promising treatment for metastatic colorectal cancer.

Oncolytic adenovirus mediated Survivin knockdown by RNA interference suppresses human colorectal carcinoma growth in vitro and in vivo.

BACKGROUND: Colorectal cancer is a one of the most common alimentary malignancies. Survivin has been proved by many studies to be an ideal target for cancer gene therapy because of its strong anti-apoptotic effect. The reduction of Survivin expression by means of chemically synthesized small interfering RNA or small hairpin RNA expressed from plasmid and resulted growth inhibition of cancer cells had been proved by many studies including ours, but the transfection efficiency was not encouraging. So for the first time we constructed the Survivin shRNA into an oncolytic adenovirus, tested its effects on colorectal cancer cell lines and nude mice xenograft model. METHODS: In this study, we constructed an oncolytic adenovirus with a Survivin targeted small hairpin RNA and a reporter gene (ZD55-Sur-EGFP). The expression of Survivin mRNA and protein were analyzed by RT-PCR and western blot. The cell growth and apoptosis were tested by in vitro cytopathic assay, MTT assay and flow cytometry respectively. The effect of the constructed virus on xenograft model was evaluated by tumor volume and western blot analysis. RESULTS: ZD55-Sur-EGFP replicated in cancer cells specifically, reduced the expression of Survivin mRNA and protein expression effectively (P < 0.0001), induced cancer cell apoptosis and inhibited SW480 cell growth both in vitro and in vivo significantly. CONCLUSION: We conclude Survivin RNA interference combining with oncolytic adenovirus virotherapy to be a promising treatment for colorectal cancer.