Association of epigenetic inactivation of the WRN gene with anticancer drug sensitivity in cervical cancer cells

The Werner (WRN) gene codes for a DNA helicase that contributes to genomic stability and has been identified as the gene responsible for progeria. Recent studies have shown reduced WRN expression due to aberrant DNA hypermethylation in cancer cells. Furthermore, WRN expression is thought to affect sensitivity to DNA topoisomerase I inhibitors in cancer therapy. In this study, we examined the relationship between aberrant DNA hypermethylation of WRN and the sensitivity of cervical cancer cells to anticancer drugs. DNA was extracted from samples from 22 patients with primary cervical cancer and 6 human cervical cancer-derived cell lines. Aberrant DNA hypermethylation was analyzed by methylation-specific PCR. WRN expression in cultured cells before and after addition of 5-aza-2-deoxycytidine, a demethylating agent, was examined using RT-PCR. The sensitivity of cells to anticancer drugs was determined using a collagen gel droplet embedded culture drug sensitivity test (CD-DST). siRNA against WRN was transfected into a cervical cancer-derived cell line with high WRN expression. Changes in drug sensitivity after silencing WRN were determined by CD-DST. Aberrant DNA hypermethylation and decreased expression of WRN were detected in 7/21 cases of primary cervical cancer and in two cervical cancer-derived cell lines. These two cell lines showed high sensitivity to CPT-11, a topoisomerase I inhibitor, but became resistant to CPT-11 after treatment with 5-aza-2-deoxycytidine. Transfection of siRNA against WRN increased the sensitivity of the cells to CPT-11. Aberrant DNA hypermethylation of WRN also increased the sensitivity of cervical cancer cells to CPT-11. Therefore, epigenetic inactivation of this gene may be a biomarker for selection of drugs for the treatment of cervical cancer. This is the first report to show a relationship between the methylation of the WRN gene and sensitivity to CPT-11 in gynecological cancers.     

Peripheral nerve conduction abnormalities precede morphological alterations in an experimental rat model of sepsis

Purpose

The pathological mechanisms of critical illness polyneuropathy (CIP), an acute neuromuscular disorder, remain unknown. In this study, we evaluated nerve and vascular properties that might account for electrophysiological abnormalities, including reduced nerve conduction amplitude, in the early phase of CIP.

Methods

Rats were administered intravenous saline (C-group; n = 31) or lipopolysaccharide (3 mg/kg/day; L-group; n = 30) for 48 h. Subsequently, tracheotomy was performed and sciatic nerves exposed bilaterally. A catheter was inserted into the left internal carotid artery to measure the mean arterial pressure (MAP). Nerve conduction velocity (NCV), nerve blood flow (NBF), evoked amplitudes, chronaxie, rheobase, and the absolute refractory period (ARP) were measured from the sciatic nerves. Degeneration, myelination, and neutrophil infiltration were examined in the sciatic nerves using histology and electron microscopy.

Results

The NBF (C-group 25 ± 3 ml/100 g/min, L-group 13 ± 3 ml/100 g/min, p < 0.001) was lower in the L-group, but the MAP was similar between groups (C-group 119 ± 17 mmHg, L-group 115 ± 18 mmHg, p = 0.773). LPS also caused a severe reduction in amplitude (C-group 0.9 ± 0.2 mV, L-group 0.2 ± 0.1 mV, p < 0.001), while latency and NCV were not affected. Of note, response amplitudes partially recovered with an increase in stimulus intensity. LPS treatment increased the rheobase and decreased the chronaxie (rheobase: C vs L-group; 0.35 ± 0.07 vs 1.29 ± 0.66 mA, p < 0.001; chronaxie 171 ± 24 vs 42 ± 20 µs, p < 0.001), while ARP was unchanged. No primary axonal degeneration or inflammatory infiltration was observed.

Conclusions

Our findings suggest that primary electrophysiological deterioration is due to threshold alterations rather than morphological alterations after 48 h of LPS treatment.

     

Variability of Myocardial Repolarization in Pediatric Patients with a Ventricular Septal Defect

In patients with a ventricular septal defect, left-to-right shunting increases the left ventricular preload. This pathological change affects myocardial depolarization and repolarization and has the potential to evoke arrhythmogenic substrates. We examined the effect of ventricular septal defects on myocardial repolarization by investigating the variability in the repolarization interval. This retrospective study included 19 patients (mean age, 1.8 ± 2.1 years) who underwent surgical closure (mean left-to-right shunt ratio, 2.60 ± 0.55) and 26 age-matched healthy controls from 2008 to 2015. Using preoperative electrocardiograms, we studied two electrocardiographic parameters (heart rate-corrected repolarization and variability of repolarization) and four repolarization intervals (QT, JT, J point to T peak [JTp], and T peak to T end [Tp-e] intervals). The variability index (VI) was calculated from the logarithm of the ratio of the repolarization parameter variance to heart rate variance. The various measures were compared between the patients and controls, and significant differences were found in the corrected QT, JTp, and Tp-e intervals (p < 0.05). The VI of the four intervals also showed significant differences (patients vs. controls: QTVI, ?0.55 ± 0.61 vs. ?1.10 ± 0.53; JTVI, ?0.33 ± 0.60 vs. ?0.86 ± 0.57; JTpVI, ?0.15 ± 0.78 vs. ?0.73 ± 0.56; Tp-eVI, 0.75 ± 0.70 vs. 0.11 ± 0.73, respectively; p < 0.05). No correlation was found between the QTVI and corrected QT interval using linear regression analysis. These repolarization characteristics provide not only electrophysiological indices but also a new index with which to assess the pathophysiology of congenital heart disease.     

General anesthesia by propofol infusion for delivery by cesarean section compared with sevoflurane anesthesia using bispectral index (BIS) monitoring

BACKGROUND: We compared the postoperative effects of propofol and sevoflurane used for anesthesia during elective cesarean section, using bispectral index (BIS) monitoring. METHODS: Fourteen parturients were randomized into two groups (Propofol group; P group, Sevoflurane group; S group). All patients received thiopental and suxamethonium for induction and had orotracheal intubation in rapid sequence. All patients received 1% sevoflurane until delivery. In the P group, sevoflurane was stopped after delivery and propofol infusion was started, to achieve target BIS values below 60; the infusion rate was 6. 17 +/- 0.98 mg x kg(-1) x h(-1). Patients in the S group received 1% sevoflurane. All patients were given fentanyl and vecuronium as required. RESULTS:There were no significant differences between the two groups in their times for beginning drinking, walking, eating or removal of urinary catheters after the operations. There were no significant differences in total blood loss or intraoperative dose of oxytocin, but postoperative and total doses of oxytocin were significantly higher in those in the P group. CONCLUSIONS: Comparing the effects of propofol and sevoflurane on the postoperative condition of parturients, no differences could be detected. We can choose to use propofol instead of sevoflurane for cesarean section.     

Indomethacin overcomes doxorubicin resistance with inhibiting multi-drug resistance protein 1 (MRP1)

Drug resistance continues to be a serious problem in cancer therapy. We investigated whether indomethacin, which inhibited cyclooxygenases, would overcome doxorubicin resistance in K562/ADR leukemia cells. Indomethacin at 10 μM increased the cytotoxicity of doxorubicin, as well as vincristine in K562/ADR. Both multi-drug resistant protein1 (MRP1) and P-glycoprotein were overexpressed in K562/ADR cells when compared with K562 parent cells (K562/P). Expression of MRP1 mRNA and protein, but not P-glycoprotein, was significantly decreased in K562/ADR cells after indomethacin treatment. Indomethacin treatment increased 5(6)-carboxyfluorescein diacetate (CFDA) efflux, as well as decreased accumulation in K562/ADR cells. The activity of the MRP1 promoter decreased after indomethacin treatment in Hela cells. These data strongly suggest that the cyclooxygenase inhibitor, indomethacin, increased the cytotoxicity of doxorubicin with decreasing expression of MRP1 through inhibition of MRP1 promoter activity.     

Effects of N-nitrosofenfluramine, a component of Chinese dietary supplement for weight loss, on CD-1 mice

Many cases of hepatopathy including deaths have frequently occurred after ingestion of Chinese dietary supplements for weight loss containing N-nitrosofenfluramine (N-fen), a nitroso derivative of fenfluramine (Fen), which was used for the treatment of obesity in the United States. Since Fen decreases appetite by decreasing the serotonin level and exhibits an antibiotic effect, N-fen may have been added, expecting a similar effect. Thus, we synthesized N-fen and orally administered it to mice, and investigated its effect on the liver as well as on the cerebral serotonin nervous system to investigate whether N-fen exhibits an anorectic effect. Three doses of N-fen were orally administered once daily to mice for 1 week. No significant changes in body weight, food intake, and general condition were noted. The liver and kidney weights were significantly increased. On blood chemistry, alkaline phosphatase, aspartate aminotransferase, and alanine aminotransferase activities were increased, and total bilirubin and albumin were slightly decreased. On histopathological examination, acidophilic changes and mild cellular swelling were noted in the liver. The liver drug-metabolizing enzyme (P-450) level was significantly higher. The effect of N-fen on the serotonin (5HT) nervous system was examined by quantitative autoradiography of the mouse brain, and it was found that N-fen did not decrease the 5HT nerve activity. Effects of reuptake and release of monoamine neurotransmitters [dopamine (DA), 5HT, and norepinephrine (NE)] were investigated. N-fen inhibited a little 5HT reuptake, and did not inhibit reuptakes of DA and NE. Moreover, N-fen did not affect release of the three monoamines. The above findings suggested that N-fen did not exhibit a serotonin nerve fiber-mediated anorectic effect in mice, but induced hepatopathy.