KIF18b-dependent hypomethylation of PARPBP gene promoter enhances oxaliplatin resistance in colorectal cancer.

As the new platinum drug oxaliplatin has been widely used in clinical treatment of colorectal cancer (CRC), oxaliplatin resistance has become a burning problem. In this study, higher expression of PARP-1 binding protein (PARPBP) was detected in oxaliplatin-resistant CRC (OR-CRC) cells than in non-resistant cells. Further research showed that kinesin family member 18 b (KIF18b) induced the overexpression of PARPBP, sustaining oxaliplatin resistance in OR-CRC cells. Through exploring the PARPBP gene promoter, we found that SP1-recruited DNMT3b methylated PARPBP promoter to suppress transcription in CRC cells, and increased KIF18b attenuated the recruitment of DNMT3b to PARPBP promoter by directly interacting with SP1 in OR-CRC cells. Clinical analysis suggested a positive relationship between KIF18b and PARPBP in CRC tissues and indicated poor prognosis in CRC patients with high level of KIF18b or PARPBP. In summary, KIF18b-induced PARPBP contributes to the resistant phenotype of OR-CRC.

E2F1/SP3/STAT6 axis is required for IL-4-induced epithelial-mesenchymal transition of colorectal cancer cells.

Colorectal cancer (CRC) is a type of cancer with a mortality rate among the highest worldwide owing to its high rate of metastasis. Therefore, inflammation-associated metastasis in the development of CRC is currently a topic of considerable interest. In the present study, the pro-inflammatory cytokine interleukin-4 (IL-4) was identified to promote the epithelial-mesenchymal transition (EMT) of CRC cells. However, the enhancing effect of IL-4 was more evident in HCT116 cells compared with in RKO cells. Accordingly, an increased expression level of STAT6 was observed in HCT116 cells compared with RKO cells. Further investigations identified that E2F1 was required for maintaining the level of signal transducer and activator of transcription 6 (STAT6) in HCT116 cells. Mechanistically, E2F1 induced specificity protein 3 (SP3) directly by binding to the promoter of the STAT6 gene and activating its transcription in CRC cells. As a result, phosphorylation-activated STAT6 increased the expression of several EMT drivers, including zinc finger E-box-binding homeobox (Zeb)1 and Zeb2, which serve a critical function in IL-4-induced EMT. Rescue experiments further confirmed that IL-4-induced EMT relied on an intact E2F1/SP3/STAT6 axis in CRC cells. Finally, analysis of clinical CRC specimens revealed a positive correlation between E2F1, SP3 and STAT6. The ectopically expressed E2F1/SP3/STAT6 axis indicated a poor prognosis in patients with CRC. In conclusion, the E2F1/SP3/STAT6 pathway was identified to be essential for IL-4 signaling-induced EMT and aggressiveness of CRC cells.

Application of next-generation sequencing to characterize novel mutations in clarithromycin-susceptible Helicobacter pylori strains with A2143G of 23S rRNA gene.

BACKGROUND: Clarithromycin (CLR) resistance has become a predominant factor for treatment failure of Helicobacter pylori eradication. Although the molecular mechanism of CLR resistance has been clearly understood in H. pylori, it is lack of evidence of other genes involved in drug resistance. Furthermore, the molecular mechanism of phenotype susceptible to CLR while genotype of 23S rRNA is mutant with A2143G is unclear. Here, we characterized the mutations of CLR-resistant and -susceptible H. pylori strains to explore bacterial resistance. METHODS: In the present study, the whole genomes of twelve clinical isolated H. pylori strains were sequenced, including two CLR-susceptible strains with mutation of A2143G. Single nucleotide variants (SNVs) were extracted and analyzed from multidrug efflux transporter genes. RESULTS: We did not find mutations associated with known CLR-resistant sites except for controversial T2182C outside of A2143G in the 23S rRNA gene. Although total SNVs of multidrug efflux transporter gene and the SNVs of HP0605 were significant differences (P ≤ 0.05) between phenotype resistant and susceptible strains. There is no significant difference in SNVs of RND or MFS (HP1181) family. However, the number of mutations in the RND family was significantly higher in the mutant strain (A2143G) than in the wild type. In addition, three special variations from two membrane proteins of mtrC and hefD were identified in both CLR-susceptible strains with A2143G. CONCLUSIONS: Next-generation sequencing is a practical strategy for analyzing genomic variation associated with antibiotic resistance in H. pylori. The variations of membrane proteins of the RND family may be able to participate in the regulation of clinical isolated H. pylori susceptibility profiles.

CREB1 directly activates the transcription of ribonucleotide reductase small subunit M2 and promotes the aggressiveness of human colorectal cancer.

As the small subunit of Ribonucleotide reductase (RR), RRM2 displays a very important role in various critical cellular processes such as cell proliferation, DNA repair, and senescence, etc. Importantly, RRM2 functions like a tumor driver in most types of cancer but little is known about the regulatory mechanism of RRM2 in cancer development. In this study, we found that the cAMP responsive element binding protein 1 (CREB1) acted as a transcription factor of RRM2 gene in human colorectal cancer (CRC). CREB1 directly bound to the promoter of RRM2 gene and induced its transcriptional activation. Knockdown of CREB1 decreased the expression of RRM2 at both mRNA and protein levels. Moreover, knockdown of RRM2 attenuated CREB1-induced aggressive phenotypes of CRC cells in vitro and in vivo. Analysis of the data from TCGA database and clinical CRC specimens with immunohistochemical staining also demonstrated a strong correlation between the co-expression of CREB1 and RRM2. Decreased disease survivals were observed in CRC patients with high expression levels of CREB1 or RRM2. Our results indicate CREB1 as a critical transcription factor of RRM2 which promotes tumor aggressiveness, and imply a significant correlation between CREB1 and RRM2 in CRC specimens. These may provide the possibility that CREB1 and RRM2 could be used as biomarkers or targets for CRC diagnosis and treatment.

Effect of hypoxia inducible factor-1 antisense oligonucleotide on liver cancer.

Hepatocellular carcinoma (HCC) is one of the most frequent primary malignancies of the liver and is resistant to anticancer drugs. Hypoxia is a master cause of tumor resistance to chemotherapy. Hypoxia-inducible factor-one alpha (HIF-1α) plays a key role in the adaptive responses to hypoxic environments. HIF-1α is constitutively up-regulated in several tumor types might thus be implicated in tumor therapy resistance. We hypothesized that disruption of HIF-1α pathway could reverse the hypoxia-induced resistance to chemotherapy. In this report, we prepared DOTAP (a liposome formulation of a mono-cationic lipid N-[1-(2,3-Dioleoyloxy)]- N,N,N-trimethylammonium propane methylsulfate in sterile water) cationic liposomes containing an antisense oligonucleotide (AsODN) against HIF-1α. Gene transfer of antisense HIF-1α was effective in suppressing tumor growth, angiogenesis, and cell proliferation, and inducing cell apoptosis. Our results suggested that antisense HIF-1α therapy could be a therapeutic strategy for treating HCC.

[Epidemiological study on antibiotic resistance among Helicobacter pylori in Taizhou district, Zhejiang, 2010-2013].

OBJECTIVE: To study the infection status of Helicobacter pylori (H. pylori) and sensitivity to commonly used antibiotics in Taizhou district,Zhejiang province. METHODS: 39 099 cases aged between 5 and 95 years old (mean as 48.42 years) were involved during January 2010 to December, 2013 for this study. Sex ratio was 1 : 0.95. Yearly distribution of the number of cases were 5 031, 6 709, 11 902 and 15 457 in 2010, 2011, 2012 and 2013, respectively. Gastric mucosal specimens were collected and H. pylori strains were isolated and cultured in the same platform in Zhiyuan Medical Inspection Institute of Hangzhou. Resistance tests of all the H. pylori isolates were performed to 6 commonly used antibiotics:metronidazole, clarithromycin, amoxicillin, gentamicin, levofloxacin and furazolidone with the agar dilution method. The antibiotic resistance rates of H. pylori strains isolated during year 2010-2013 and the changing trends were analyzed. RESULTS: Resistance rates to levofloxacin and clarithromycin kept at higher level and the highest was in 2011 and then decreased in both 2012 and 2013 (P < 0.01). The resistance rates to both levofloxacin and clarithromycin reached the highest in 2011 (P < 0.01), and decreased thereafter, with no significant change in 2013 to 2012 (P > 0.05). CONCLUSION: Antibiotic resistance rate against metronidazole for HP isolate was highest. Resistance rate against amoxicillin and furazolidone, gentamicin was low. Clinical treatment should choose amoxicillin and furazolidone, gentamicin. The resistance rates to levofloxacin and clarithromycin had been seen at a significantly downward trend since 2011. However, the combined resistance rates to levofloxacin and clarithromycin did not seem to reduce since 2012.

Oxidative damage induced by chlorpyrifos in the hepatic and renal tissue of Kunming mice and the antioxidant role of vitamin E.

Chlorpyrifos is a broad-spectrum, chlorinated organophosphate pesticide employed for pest control in various agricultural and animal husbandries. Acute and chronic exposure to CPF can elicit several adverse effects, including oxidative stress. We investigated neurotoxicity of CPF-treated mice, and evaluated the antioxidant effect of vitamin E against oxidative stress and histological changes in the livers and kidneys of CPF-treated mice. Kunming mice were divided randomly into five exposure groups of six: (A) peanut oil; (B) 3mg/kg CPF; (C) 6 mg/kg CPF; (D) 12 mg/kg CPF; (E) vitamin E (100 mg/kg), 3h after administration of CPF (12 mg/kg) and used as a post-treatment group. Oral administration of high-dose groups (12 mg/kg) CPF led to a significant increase in levels of reactive oxygen species, DNA-protein crosslinks, 8-hydroxy-2-deoxyguanosine and malondialdehyde, decreases in acetylcholinesterase activity and glutathione level, as well as causing hepatic and renal histopathological change. Except for AChE activity levels, administration of vitamin E to CPF-treated mice restored these biochemical parameters to within normal levels, and resulted in overall improvement in damage to livers and kidneys. These data suggest that oxidative stress is involved in CPF-induced toxicity and that vitamin E can protect against the tissue damage induced by CPF.

Intraperitoneal injection of magnetic Fe3O4-nanoparticle induces hepatic and renal tissue injury via oxidative stress in mice.

Because of its unique magnetic properties, the iron oxide (Fe3O4) nanoparticle has been widely exploited and its application in various fields has promised immense benefits. However, doubts exist over the use of Fe3O4-nanoparticles in human beings. Thus, the aim of the current study was to find out the potential safety range of medical use. Twenty-five Kunming mice were exposed to Fe3O4-nanoparticles via intraperitoneal injection daily for 1 week at doses of 0, 5, 10, 20, and 40 mg/kg. Hepatic and renal tissues were sliced for physiological observation. Injuries were observed in the high-dose groups (20 and 40 mg/kg) compared with the control group (0 mg/kg). Biomarkers of reactive oxygen species, glutathione, malondialdehyde, DNA-protein crosslinks, and 8-hydroxy-2'-deoxyguanosine in the hepatic and renal tissues were detected. Injury to tissues and oxidative damage to cells at the molecular level was found. The safest dose recommended from the results of this study is 5 mg/kg, as we believe this to be an upper limit balancing the benefits and risks for sub-long-term exposure.