Tumor budding and the prognosis of patients with metastatic colorectal cancer: a meta-analysis.

PURPOSE: Tumor budding has been suggested to be associated with poor survival of patients with colorectal cancer (CRC). However, it is unclear whether the association remains in patients with metastatic CRC (mCRC). The aim of the systematic review and meta-analysis was to investigate the potential predictive role of tumor budding for the prognosis of patients with mCRC. METHODS: PubMed, Embase, Cochrane Library, and Web of Science were searched for relevant observational studies comparing the survival outcomes between mCRC patients with high versus low tumor budding. Data collection, literature searching, and statistical analysis were conducted independently by two authors. Using a heterogeneity-incorporating random-effects model, the results were pooled. RESULTS: In this meta-analysis, 1503 patients from nine retrospective cohort studies were included. Pooled results showed that compared to those with low tumor budding, mCRC patients with high tumor budding were associated with a poor progression-free survival (hazard ratio (HR), 1.65; 95% confidence interval (CI), 1.31 to 2.07, p < 0.001; I2 = 30%) and overall survival (HR, 1.60; 95% CI, 1.33 to 1.93; p < 0.001; I2 = 0%). Influencing analysis by excluding one study at a time showed consistent results (p all < 0.05). Subgroup analyses showed consistent results in tumor budding evaluated from the primary cancer and metastases, in studies with a high tumor budding defined as ≥ 10 or 15 and ≥ 5 buds/HPF and in studies analyzed with univariate and multivariate regression models (p for subgroup difference all > 0.05). CONCLUSION: A high-degree tumor budding may be associated with poor prognosis in patients with mCRC.

Karyopherin Subunit Alpha 1 Enhances the Malignant Behaviors of Colon Cancer Cells via Promoting Nuclear Factor-κB p65 Nuclear Translocation.

BACKGROUND/AIMS: Aberrant nuclear factor-κB p65 (NF-κB p65) nuclear import commonly occurs in multiple cancers, including colon cancer. According to BioGRID, we noted that Karyopherin subunit alpha 1 (KPNA1), an important molecular transporter between the nucleus and the cytoplasm, may interact with NF-κB p65. KPNA1 itself is highly expressed in colon adenocarcinoma samples (N = 286) based on The Cancer Genome Atlas (TCGA) database. We aimed to explore the role of KPNA1 in colonic carcinogenesis and to determine whether NF-κB p65 nuclear translocation was involved. METHODS: KPNA1 expressions at mRNA and protein levels were analyzed in colon cancer tissues. The regulatory effect of KPNA1 on malignant biological properties was detected in SW480 and HCT116 colon cancer cells. Coimmunoprecipitation and immunofluorescence were performed to verify the relationship between KPNA1 and NF-κB p65. KPNA1 ubiquitination was also preliminarily investigated. RESULTS: KPNA1 was firstly confirmed as a significantly upregulated gene in our collected clinical colon cancer samples (N = 35). KPNA1 depletion inhibited cell proliferation, induced cell cycle arrest, and diminished migratory and invasive capacity of SW480 and HCT116 cells. Colon cancer cells overexpressing KPNA1 acquired more aggressive behaviors. KPNA1 acted as a transporter to induce the nuclear accumulation of NF-κB p65, thereby activating NF-κB signaling pathway in colon cancer cells. Furthermore, HECT, C2, and WW Domain-Containing E3 Ubiquitin (HECW2) interacted with KPNA1 to induce its ubiquitination. KPNA1 labeled with polyubiquitins was degraded through ubiquitin-proteasome system. CONCLUSION: The present study uncovers a role of KPNA1-NF-κB p65 axis in promoting colonic carcinogenesis.

Centromere Protein A (CENPA) Regulates Metabolic Reprogramming in the Colon Cancer Cells by Transcriptionally Activating Karyopherin Subunit Alpha 2 (KPNA2).

The karyopherin α2 subunit gene (KPNA2), an oncogene, is involved in metabolic reprogramming in cancer. This study aimed to explore the function of KPNα2 in the growth and glycolysis in colon cancer (CC) cells. Genes from the Oncomine database that were differentially expressed in multiple CC types were screened. Bioinformatics analysis suggested that KPNA2 was highly expressed in CC, and consequently, high expression of KPNA2 was detected in the CC cell lines. Down-regulation of KPNA2 reduced viability and DNA-replication ability, and increased apoptosis of HCT116 and LoVo cells. It also reduced glucose consumption, extracellular acidification rate, and the ATP production in cells. Centromere protein A (CENPA) was confirmed as an upstream transcription activator of KPNA2. There was significant H3K27ac modification in the promoter region of KPNA2. CENPA primarily recruited histone acetyltransferase general control of amino acid synthesis (GCN)-5 to the promoter region of KPNA2 to induce transcription activation. Overexpression of either CENPA or GCN-5 blocked the role of short hairpin KPNα2 and restored growth and glycolysis in CC cells. To conclude, the findings from this study suggest that CENPA recruits GCN-5 to the promoter region of KPNA2 to induce KPNα2 activation, which strengthens growth and glycolysis in, and augments the development of, CC.

Styrene-maleic acid copolymer-encapsulated carbon monoxide releasing molecule-2 (SMA/CORM-2) suppresses proliferation, migration and invasion of colorectal cancer cells in vitro and in vivo.

Although increasing evidence have confirmed that carbon monoxide release molecule-2(CORM-2) plays an active role in the treatment of inflammation and tumors, poor aqueous solubility and short CO-release duration restrict its extensive application. Our previous work synthesized styrene-maleic acid copolymer-encapsulated CORM-2 (SMA/CORM-2) to overcome above-mentioned deficiencies and demonstrated satisfactory effects in colitis. This study is to investigate the function of SMA/CORM-2 on colorectal cancer proliferation and metastasis. CCK-8 experiment is used to clarify the half maximal inhibitory concentration (IC50) of SMA/CORM-2 and to detect cell proliferation. Transwell assay coated with or without matrigel was to detect cell invasion and migration. Western blot was used to detect ß-catenin, AKT, p-AKT, VEGF, MMP-2 and MMP-9 proteins. At last, nude mice xenograft was used to further investigate the anti-tumor effect of SMA/CORM-2 in vivo. After SW480 and C26 cells were treated with 0.5 mg/ml SMA/CORM-2, CRC cells proliferation, migration and invasion were inhibited. In vivo, SMA/CORM-2 treatment remarkably suppressed tumor growth and lung metastasis in nude mice. Furthermore, the expression of ß-catenin, p-AKT, VEGF, MMP-2 and MMP-9 proteins could be down-regulated after SMA/CORM-2 treatment. SMA/CORM-2 exerted both in vitro and in vivo anti-proliferation and anti-metastatic effects, which may yield a novel therapeutic strategy for CRC.

Upregulation of heme oxygenase-1 in colorectal cancer patients with increased circulation carbon monoxide levels, potentially affects chemotherapeutic sensitivity.

BACKGROUND: Heme oxygenase-1 (HO-1) and its major product carbon monoxide (CO) are known to be involved in the development and progression of many tumors. The present study was to elucidate the expression and function of HO-1 in colorectal cancer (CRC), specially focusing on the circulation CO levels in CRC patients and the possible roles of HO-1 in chemoresistance of colon cancer cells. METHODS: One hundred and eighteen patients received resection for colorectal cancer and polyps at China Medical University Sheng Jing Hospital, were collected in this study. HO-1 expression in CRC tissues was analyzed by immnuohistochemical staining; circulation CO levels as carboxyhemoglobin (COHb) in CRC patients were analyzed by an ABL800 FLEX blood gas analyzer. HO-1 expression in murine colon cells C26 and human colon cancer cells HT29 and DLD1 under HO-1 inducer hemin and anticancer drug pirarubicin (THP) treatment was examined by RT-PCR, and the cell viability after each treatment was investigated by MTT assay. Data were analyzed by student's t-test or one-way ANOVA followed by Bonferroni t-test or Fisher's exact test. RESULTS: HO-1 expression in tumor tissues of CRC (61.0%) was significantly higher than in normal colorectal tissues and polyps tissues (29.7%, P < 0.01); well-differentiated CRC seemed to express more HO-1 (81.5%) than moderately/poorly-differentiated cancers (59.5%, P < 0.05). However, the nuclear HO-1 expression is apparently higher in moderately/poorly differentiated CRC than well-differentiated CRC probably suggesting a new mechanism of function involved in HO-1 in cancer. In parallel with HO-1 expression, circulation CO levels in CRC patients also significantly accelerated. Moreover, HO-1 expression/induction also related to the chemosensitivity of colon cells; HO inhibitor zinc protoporphyrin significantly increased cytotoxicities of THP (i.e., 2.6 - 5.3 folds compared to cells without zinc protoporphyrin treatment). CONCLUSIONS: These findings strongly suggested HO-1/COHb is a useful diagnostic and prognostic indicator for CRC, and inhibition of HO-1 may be a option to enhance the chemotherapeutic effects of conventional anticancer drugs toward CRC.

Ubiquitin-specific peptidase 15 regulates the TFAP4/PCGF1 axis facilitating liver metastasis of colorectal cancer and cell stemness.

The tumor recurrence and metastasis of colorectal cancer (CRC) are responsible for most of CRC-linked mortalities. It is an urgent need to deeply investigate the pathogenesis of CRC metastasis and look for novel targets for its treatment. The current study aimed to investigate the effects of ubiquitin-specific peptidase 15 (USP-15) on the CRC progression. In vivo, a mouse model of liver metastasis of CRC tumor was established to investigate the role of USP-15. In vitro, the migrated and invasive abilities of CRC cells were assessed by transwell assay. Cell stemness was evaluated by using sphere formation assay. The underlying mechanism was further explored by employing the co-immunoprecipitation, dual luciferase reporter assay, oligonucleotide pull-down assay, and chromatin immunoprecipitation assay. The results showed that USP-15 was upregulated in CRC patients with liver metastasis and high metastatic potential cell lines of CRC. Loss of USP-15 repressed the epithelial-to-mesenchymal transition (EMT), migration, invasion, and stemness properties of CRC cells in vitro. Downregulation of USP-15 reduced the liver metastasis of mice in vivo. USP-15 upregulation obtained the contrary effects. Subsequently, USP-15 deubiquitinated transcription factor AP-4 (TFAP4) and enhanced its protein stability. TFAP4 could transcriptionally activated polycomb group ring finger 1 (PCGF1). The pro-cancer effects of USP-15 were rescue by the knockdown of TFAP4 or PCGF1. In conclusions: USP-15 facilitated the liver metastasis by the enhancement of cell stemness and EMT in CRC, which was at least partly mediated by the deubiquitination of TFAP4 upon the upregulation of PCGF1.

Protection from inflammatory bowel disease and colitis-associated carcinogenesis with 4-vinyl-2,6-dimethoxyphenol (canolol) involves suppression of oxidative stress and inflammatory cytokines.

Oxidative stress is associated with various pathological processes including inflammatory bowel disease, which is a major cause of colon cancer. Here, we examined the antioxidative and anti-inflammatory effects of 4-vinyl-2,6-dimethoxyphenol (canolol), a potent antioxidant compound obtained from crude canola oil. Oral administration of 2% dextran sulfate sodium (DSS) resulted in the progression of colitis with shortening of the large bowel length. Administering a diet containing canolol significantly suppressed pathogenesis; diarrhea markedly improved and the length of large bowel returned to almost normal. Pathological examination clearly revealed improvement of colonic ulcers. Production of inflammatory cytokines, i.e. interleukin-12 and tumor necrosis factor-α, was significantly increased during this pathological process; their production was markedly inhibited by canolol. In the azoxymethane/DSS-induced colon cancer model, mice receiving canolol had a reduced occurrence of cancer, to 60%, compared with control mice, 100% of which had colon cancer. The numbers of tumors in each mouse were also significantly reduced in mice receiving the canolol-containing diet (5.6±2.0) compared with azoxymethane/DSS control mice (10.8±4.2). No apparent toxicity of canolol was observed. Moreover, inflammatory cytokines (i.e. cyclooxygenase-2, inducible nitric oxide synthase and tumor necrosis factor-α) and oxidative responding molecules, i.e. heme oxygenase-1, in colon were suppressed during this treatment. In a mouse colon 26 solid tumor model, canolol significantly suppressed cyclooxygenase-2 expression; however, no significant tumor growth inhibition was observed, suggesting that canolol preferably shows chemopreventive effects during the stages of initiation/promotion. Canolol may, thus, be considered a potential cancer preventive agent or supplement.

Mutation spectrum in human colorectal cancers and potential functional relevance.

BACKGROUND: Somatic variants, which occur in the genome of all cells, are well accepted to play a critical role in cancer development, as their accumulation in genes could affect cell proliferations and cell cycle. METHODS: In order to understand the role of somatic mutations in human colorectal cancers, we characterized the mutation spectrum in two colorectal tumor tissues and their matched normal tissues, by analyzing deep-sequenced transcriptome data. RESULTS: We found a higher mutation rate of somatic variants in tumor tissues in comparison with normal tissues, but no trend was observed for mutation properties. By applying a series of stringent filters, we identified 418 genes with tumor specific disruptive somatic variants. Of these genes, three genes in mucin protein family (MUC2, MUC4, and MU12) are of particular interests. It has been reported that the expression of mucin proteins was correlated with the progression of colorectal cancer therefore somatic variants within those genes can interrupt their normal expression and thus contribute to the tumorigenesis. CONCLUSIONS: Our findings provide evidence of the utility of RNA-Seq in mutation screening in cancer studies, and suggest a list of candidate genes for future colorectal cancer diagnosis and treatment.

MORC4 plays a tumor-promoting role in colorectal cancer via regulating PCGF1/CDKN1A axis in vitro and in vivo.

MORC family CW-type zinc finger 4 (MORC4) possessing nuclear matrix binding domains has been observed to be involved in multiple cancer development. By digging three gene expression omnibus (GEO) gene microarrays (GSE110223, GSE110224 and GSE24514), we found that MORC4 was overexpressed in colorectal cancer (CRC) samples (log2 Fold change >1, p < 0.05). We aimed to investigate the role of MORC4 in CRC malignant behaviors, with an emphasis on polycomb group ring finger 1 (PCGF1)/cyclin-dependent kinase inhibitor 1A (CDKN1A) axis. Firstly, we confirmed MORC4 as an upregulated gene in 60 pairs of frozen CRC and adjacent normal samples. MORC4 overexpression increased proliferation and metastasis, and decreased apoptosis in SW480 and HT29 cells, which was diminished by the knockdown of PCGF1, a transcriptional repressor of CDKN1A (a potent cyclin-dependent kinase inhibitor). MORC4 was further identified as a novel molecule that interacted with PCGF1 via coimmunoprecipitation. MORC4 itself did not substantially suppress CDKN1A transcriptional activity, but it augmented PCGF1's effect on CDKN1A. Additionally, MORC4 acted as the substrate of HECT, C2, and WW domain-containing E3 ubiquitin protein ligase 2 (HECW2) and was degraded through ubiquitin-proteasome system. Collectively, our work suggested that MORC4 accelerated CRC progression via governing PCGF1/CDKN1A signaling.

Artificial Downregulation of Ribosomal Protein L34 Restricts the Proliferation and Metastasis of Colorectal Cancer by Suppressing the JAK2/STAT3 Signaling Pathway.

The highly conserved ribosomal protein L34 (RPL34) has been reported to play an essential role in the progression of diverse malignancies. RPL34 is aberrantly expressed in multiple cancers, although its significant in colorectal cancer (CRC) is currently unclear. Here, we demonstrated that RPL34 expression was higher in CRC tissues than in normal tissues. Upon RPL34 overexpression, the ability of proliferation, migration, invasion, and metastasis of CRC cells were significantly enhanced in vitro and in vivo. Furthermore, high expression of RPL34 accelerated cell cycle progression, activated the JAK2/STAT3 signaling pathway, and induced the epithelial-to-mesenchymal transition (EMT) program. Conversely, RPL34 silencing inhibited the CRC malignant progression. Utilizing immunoprecipitation assays, we identified the RPL34 interactor, the cullin-associated NEDD8-dissociated protein 1 (CAND1), which is a negative regulator of cullin-RING ligases. CAND1 overexpression reduced the ubiquitin level of RPL34 and stabilized RPL34 protein. CAND1 silencing in CRC cells resulted in a decrease in the ability of proliferation, migration, and invasion. CAND1 overexpression promoted CRC malignant phenotypes and induced EMT, and RPL34 knockdown rescued CAND1-induced CRC progression. In summary, our study indicates that RPL34 acts as a mediator, is stabilized by CAND1, and promotes proliferation and metastasis, in part, through the activation of the JAK2/STAT3 signaling pathway and induction of EMT in CRC.

Long Non-Coding RNA CCAT2 Activates RAB14 and Acts as an Oncogene in Colorectal Cancer.

Here, we investigated the clinicopathological and prognostic potential of the long noncoding RNA Colon Cancer-Associated Transcript 2 (CCAT2) in human colorectal cancer (CRC). We used qPCR to quantify CCAT2 levels in 44 pairs of CRC tissues and adjacent nontumor and healthy colon mucosa tissues, and in several CRC cell lines (SW620, SW480, HT-29, LOVO, HCT116 and DLD-1) and normal human colorectal epithelial cells (HFC). We assessed the effects of CCAT2 overexpression or knockdown on the proliferation, migration and invasion by SW620 and LOVO cells using CCK-8, transwell, and wound-healing assays, respectively. We also investigated the potential interaction between CCAT2 and TAF15 through RNA pull down and rescue experiments. Lastly, we evaluated the expression of the cell cycle progression markers and GSK3ß signaling pathway proteins using Western blotting. Our results showed that CCAT2 was upregulated in CRC tissues and cell lines as com-pared to controls. Ectopic expression of CCAT2 promoted CRC cell proliferation, migration and invasion, likely through direct interaction with TAF15, transcriptional activation of RAB14, and activation of the AKT/GSK3ß signaling pathway. In vivo, CCAT2 promoted CRC cell growth and metastasis in nude mice. Taken together, these results highlight the actions of CCAT2 as a CRC oncogene.

Analysis of the Clinicopathological Characteristics of Stage I-III Colorectal Cancer Patients Deficient in Mismatch Repair Proteins.

PURPOSE: To investigate the clinicopathological characteristics of stage I-III colorectal cancer (CRC) patients with deficient mismatch repair (dMMR) protein. PATIENTS AND METHODS: A retrospective analysis of 61 patients with stage I-III CRC confirmed by immunohistochemistry as dMMR after radical resection at Shenjing Hospital of China Medical University from May 2017 to June 2019 was performed. A total of 183 stage I-III CRC patients with proficient mismatch repair (pMMR) protein from the same period were randomly selected as a control group. The clinicopathological data of the two groups were investigated. RESULTS: There were significant differences between the two groups in age, sex, site of onset, maximum diameter of tumor, T stage, tumor differentiation, and histological type (P < 0.05). No significant difference was detected in nerve vessel invasion, cancer nodules, the N stage or the TNM stage. In the dMMR group, 41 patients (66.13%) showed PMS2/MLH1 deletion, and the number of MSH2/MSH6 deletion is 21 patients (33.87%). Among them, 34 patients (54.84%) had PMS2 and MLH1 deficiency. In total, 16 patients (25.81%) had MSH2 and MSH6 deficiency. A total of 5 patients (8.06%) showed simply PMS2 deletion and 5 patients (8.06%) showed simply MSH6 deletion. In total, 2 patients (3.23%) showed concurrent loss of PMS2, MLH1 and MSH2. No significant difference were found (P > 0.05) in the above factors among dMMR CRC patients with different MMR proteins deletions. CONCLUSION: Our results show that dMMR status may be more likely exist in female and younger (≤55 years) patients with a greater tumor burden (>5cm), right colon, T4 stage disease, poor differentiation and mucinous adenocarcinoma. Loss of PMS2 and MLH1 is the most common pattern of MMR protein expression deficiency, followed by concurrent deletion of MSH2 and MSH6.

Knockdown of SFRS9 Inhibits Progression of Colorectal Cancer Through Triggering Ferroptosis Mediated by GPX4 Reduction.

Ferroptosis, a newly discovered form of programmed cell death characterized by lipid peroxidation, crafts a new perspective on cancer treatment. Serine and arginine rich splicing factor 9 (SFRS9) is frequently described as a proto-oncogene in cervical and bladder cancer. However, the role of SFRS9 in colorectal cancer (CRC) and whether SFRS9 exerts its function associated with ferroptosis is largely unknown. Herein, we found that the expression of SFRS9 mRNA and protein in the CRC tissues was obviously higher than that in the paracancerous tissues. Function assays revealed that SFRS9 overexpression (SFRS9-OE) significantly promoted cell viability, cell cycle progression and colony formation of CRC cells. While SFRS9 knockdown by shRNAs transfection inhibited these progressions. Furthermore, cell death and lipid peroxidation induced by ferroptosis inducers erastin and sorafenib were suppressed by SFRS9-OE. Bioinformatics analysis indicated that SFRS9 can bind to peroxidase 4 (GPX4) mRNA which is a central regulator of ferroptosis. Western blot showed that GPX4 protein expression was clearly elevated upon SFRS9-OE, while it was decreased in SFRS9-inhibited CRC cells. RNA immunoprecipitation experiment was carried out in HCT116 cells to confirm the binding of SFRS9 and GPX4 mRNA specifically. SiGPX4 transfection reversed the inhibitory effects of SFRS9-OE on the erastin and sorafenib-induced ferroptosis. Consistent with our in vitro observations, SFRS9 promoted the growth of tumors while SFRS9 knockdown significantly inhibited tumor growth in nude mice. In conclusion, SFRS9 represents an obstructive factor to ferroptosis by upregulating GPX4 protein expression, and knocking down SFRS9 might be an effective treatment for CRC.

Inhibition of SRSF9 enhances the sensitivity of colorectal cancer to erastin-induced ferroptosis by reducing glutathione peroxidase 4 expression.

Ferroptosis, a newly iron-dependent form of cell death, is often accompanied by the damage of membrane lipid peroxide. Recently, the ferroptosis inducer erastin has been reported to exhibit potential anti-cancer activities. The aim of this study was to investigate the effects of SRSF9 on the sensitivity of colorectal cancer (CRC) to erastin and explore the underlying molecular mechanism. Short hairpin RNAs (shRNAs) or SRSF9 overexpression vector (SRSF9-OE) was transfected into erastin-induced human CRC cells to inhibit or overexpress SRSF9. Results showed that SRSF9 inhibition promoted the cell death induced by erastin, conversely, SRSF9 overexpression augmented the resistance to erastin-induced death in human CRC cells. SRSF9 decreased lipid peroxide damage which was a key event during erastin-induced ferroptosis in human CRC cells. Furthermore, we found that SRSF9 inhibition increased erastin-induced ferroptosis by downregulating GPX4 level. In an In vivo study, SRSF9 shRNA or SRSF9-OE stably transfected human CRC cells were subcutaneously injected into the right flank of nude mice. SRSF9 overexpression partly abolished the tumor growth inhibition and ferroptosis induced by erastin. Our data indicated SRSF9's regulation of GPX4 as an essential mechanism driving CRC tumorigenesis and resistance of erastin-induced ferroptosis. This molecular mechanism may provide a novel method for improving the sensitivity of CRC to erastin.

A seven immune-related lncRNA signature predicts the survival of patients with colon adenocarcinoma.

This study aimed to explore immune-related lncRNAs for predicting the overall survival of patients with colon adenocarcinoma. RNA-sequencing data were downloaded from the TCGA data portal. The immune-related lncRNAs with differential expression were identified with Cox and LASSO regression analysis. With the stepwise regression analysis, a seven lncRNA signature was established for calculating the Risk Score with following formula: Risk Score = [Expression level of AC027307.2 * (0.156)] + [Expression level of AC074117.1 * (0.294)] + [Expression level of AC103702.2 * (-0.025)] + [Expression level of CYTOR * (0.205)] + [Expression level of LINC02381 * (0.251)] + [Expression level of MIR200CHG * (0.052)] + [Expression level of SNHG16 * (-0.101)]. The Risk Score was validated with survival analysis, achieving moderate area under the curve (AUC) of receiver operating characteristic (ROC) curve over 0.7. GSEA and immune-cell abundance analysis further supported the involved lncRNAs were immune-relevant. Finally, the prognosis prediction efficacy was verified with clinical samples with an AUC of 0.674 in ROC curve. Both the Risk Score and involved immune-related lncRNAs presented promising clinical significance.

Augmentation of EPR Effect and Efficacy of Anticancer Nanomedicine by Carbon Monoxide Generating Agents.

One obstacle to the successful delivery of nanodrugs into solid tumors is the heterogeneity of an enhanced permeability and retention (EPR) effect as a result of occluded or embolized tumor blood vessels. Therefore, the augmentation of the EPR effect is critical for satisfactory anticancer nanomedicine. In this study, we focused on one vascular mediator involved in the EPR effect, carbon monoxide (CO), and utilized two CO generating agents, one is an extrinsic CO donor (SMA/CORM2 micelle) and another is an inducer of endogenous CO generation via heme oxygenase-1 (HO-1) induction that is carried out using pegylated hemin. Both agents generated CO selectively in solid tumors, which resulted in an enhanced EPR effect and a two- to three-folds increased tumor accumulation of nanodrugs. An increase in drug accumulation in the normal tissue did not occur with the treatment of CO generators. In vivo imaging also clearly indicated a more intensified fluorescence of macromolecular nanoprobe in solid tumors when combined with these CO generators. Consequently, the combination of CO generators with anticancer nanodrugs resulted in an increased anticancer effect in the different transplanted solid tumor models. These findings strongly warrant the potential application of these CO generators as EPR enhancers in order to enhance tumor detection and therapy using nanodrugs.

Melatonin protects liver from intestine ischemia reperfusion injury in rats.

AIM: To investigate the protective effect of melatonin on liver after intestinal ischemia-reperfusion injury in rats. METHODS: One hundred and fifty male Wistar rats, weighing 190-210 g, aged 7 wk, were randomly divided into melatonin exposure group, alcohol solvent control group and normal saline control group. Rats in the melatonin exposure group received intraperitoneal (IP) melatonin (20 mg/kg) 30 min before intestinal ischemia-reperfusion (IR), rats in the alcohol solvent control group received the same concentration and volume of alcohol, and rats in the normal saline control group received the same volume of normal saline. Serum samples were collected from each group 0.5, 1, 6, 12, and 24 h after intestinal IR. Levels of serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were measured with an auto-biochemical analyzer. Serum TNF-alpha was tested by enzyme-linked immunosorbent assay (ELISA). Malondialdehyde (MDA) in liver was detected by colorimetric assay. Pathological changes in liver and immunohistochemical straining of ICAM-1 were observed under an optical microscope. RESULTS: The levels of ALT measured at various time points after intestinal IR in the melatonin exposure group were significantly lower than those in the other two control groups (P < 0.05). The serum AST levels 12 and 24 h after intestinal IR and the ICAM-1 levels (%) 6, 12 and 24 h after intestinal IR in the melatonin exposure group were also significantly lower than those in the other two control groups (P < 0.05). CONCLUSION: Exotic melatonin can inhibit the activity of ALT, AST and TNF-alpha, decrease the accumulation of MDA, and depress the expression of ICAM-1 in liver after intestinal IR injury, thus improving the liver function.

Protective effect of ischemic preconditioning on hepatic ischemia-reperfusion injury by advancing the expressive phase of survivin in rats.

BACKGROUND: Survivin is a new and important gene in the regulation of apoptosis. It is very important to explore the effect of the expression of survivin protein caused by ischemia-reperfusion (IR) injury. The effect of IR injury caused by ischemic preconditioning (IP) on the liver in rats and the relation between the protective effect of IP and the expression of survivin are unclear. METHODS: One hundred and fifty male Wistar rats (weighing 190-210 g, aged 6-7 weeks) were divided into three groups at random: ischemic preconditioning (IP), ischemia-reperfusion (IR) and sham-operation (SO). Sample specimens were collected from each group at 6, 12, 24, 48, and 72 hours after reperfusion. Serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were measured by an automatic biochemical analyzer. Malondialdehyde (MDA) in liver tissue was measured. Pathological changes in the liver and immunohistochemical staining for survivin were determined with an optical microscope. RESULTS: The ALT levels in the IP and IR groups after reperfusion at each time were higher than those in the SO group (P<0.05), whereas after reperfusion for 6 and 12 hours, the ALT levels in the IP group were lower than those in the IR group (P<0.05). The AST levels in all IP and IR groups were higher than those in the SO group (P<0.05), whereas after reperfusion for 12, 24, 48 and 72 hours, the AST levels in the IP group were lower than those in the IR group (P<0.05). The MDA concentrations after reperfusion in the IP group were lower than those in the IR group (P<0.05), though the MDA concentrations in the IP and IR groups increased in contrast to those in the SO group after reperfusion at each time (P<0.05). After reperfusion for 12, 24, 48 and 72 hours, the number of survivin-positive cells was larger in the IP and IR groups than in the SO group (P<0.05). After reperfusion for 12, 24, and 48 hours the number of survivin-positive cells in the IP group increased compared with that in the IR group (P<0.05). CONCLUSIONS: IR increases the protein expression of survivin in liver tissue. IP inhibits the accumulation of MDA, advances the expressive phase of survivin protein in hepatic tissue, and improves liver function.

The function of BTG3 in colorectal cancer cells and its possible signaling pathway.

PURPOSE: B-cell translocation gene 3 (BTG3) has been identified as a candidate driver gene for various cancers, but its specific role in colorectal cancer (CRC) is poorly understood. We aimed to investigate the relationship between expression of BTG3 and clinicopathological features and prognosis, as well as to explore the effects and the role of a possible BTG3 molecular mechanism on aggressive colorectal cancer behavior. METHODS: BTG3 expression was assessed by immunohistochemistry (IHC) on specimens from 140 patients with CRC. The association of BTG3 expression with clinicopathological features was examined. To confirm the biological role of BTG3 in CRC, two CRC cell lines expressing BTG3 were used and BTG3 expression was knocked down by shRNA. CCK-8, cell cycle, apoptosis, migration, and invasion assays were performed. The influence of BTG3 knockdown was further investigated by genomic microarray to uncover the potential molecular mechanisms underlying BTG3-mediated CRC development and progression. RESULTS: BTG3 was downregulated in colorectal cancer tissues and positively correlated with pathological classification (p = 0.037), depth of invasion (p = 0.016), distant metastasis (p = 0.024), TNM stage (p = 0.007), and overall survival (OS) and disease-free survival (DFS). BTG3 knockdown promoted cell proliferation, migration, invasion, relieved G2 arrest, and inhibited apoptosis in HCT116 and LoVo cells. A genomic microarray analysis showed that numerous tumor-associated signaling pathways and oncogenes were altered by BTG3 knockdown. At the mRNA level, nine genes referred to the extracellular-regulated kinase/mitogen-activated protein kinase pathway were differentially expressed. Western blotting revealed that BTG3 knockdown upregulated PAK2, RPS6KA5, YWHAB, and signal transducer and activator of transcription (STAT)3 protein levels, but downregulated RAP1A, DUSP6, and STAT1 protein expression, which was consistent with the genomic microarray data. CONCLUSIONS: BTG3 expression might contribute to CRC carcinogenesis. BTG3 knockdown might strengthen the aggressive colorectal cancer behavior.

Naringenin induces laxative effects by upregulating the expression levels of c-Kit and SCF, as well as those of aquaporin 3 in mice with loperamide-induced constipation.

Constipation is a common affliction which causes discomfort and affects the quality of life of affected individuals. Naringenin (NAR), a natural flavonoid widely found in citrus fruits and tomatoes, has been reported to exhibit various pharmacological effects, such as anti-inflammatory, anti-atherogenic, anti-mutagenic, hepatoprotective and anticancer effects. Increasing evidence has indicated that NAR has potential for use in the treatment of constipation. Thus, the aim of this study was to evaluate the laxative effects of NAR in mice with loperamide-induced (Lop-induced) constipation. The data indicated that NAR relieved Lop-induced constipation in mice based on the changes of fecal parameters (numbers, weight and water content), the intestinal charcoal transit ratio and the histological alteration. ELISA revealed that NAR regulated the production levels of gastrointestinal metabolic components, such as motilin (MTL), gastrin (Gas), endothelin (ET), substance P (SP), acetylcholinesterase (AChE) and vasoactive intestinal peptide (VIP) in serum. The expression levels of enteric nerve-related factors, glial cell line-derived neurotrophic factor (GDNF), transient receptor potential vanilloid 1 (TRPV1), nitric oxide synthase (NOS), c-Kit, stem cell factor (SCF) and aquaporin 3 (AQP3) were examined by western blot analysis and RT-PCR analysis. The results of this study suggest that NAR relieves Lop-induced constipation by increasing the levels of interstitial cells of Cajal markers (c-Kit and SCF), as well as AQP3. Thus, NAR may be effective as a candidate in patients suffering from lifestyle-induced constipation.