Growth inhibition induced by short hairpin RNA to silence survivin gene in human pancreatic cancer cells.

BACKGROUND: Survivin is known to be overexpressed in various human malignancies, including pancreatic cancer, and mediates cancer cell proliferation and tumor growth, so the regulation of this molecule could be a new strategy for treating pancreatic cancer. In this study, short hairpin RNAs (shRNAs) specific to survivin were introduced into human pancreatic cancer Patu8988 cells to investigate the inhibitory effects on survivin expression and cell proliferation in vitro and in vivo. METHODS: Three kinds of shRNA specific to the survivin gene were designed and cloned into eukaryotic expression plasmid pGenesil-1 vector. Subsequently the recombinant plasmids were transfected into human pancreatic cancer Patu8988 cells with lipfectamineTM 2000 reagent. The mRNA and protein expressions of survivin in the transiently transfected Patu8988 cells were determined by RT-PCR, flow cytometry, and Western blotting analysis. The proliferation inhibition rates of stably transfected Patu8988 cells were determined by MTT assay. The antitumor activities of the three kinds of survivin-shRNA plasmids were evaluated in BALB/c nude mice inoculated with Patu8988 cells and bearing human pancreatic cancer. RESULTS: The three survivin-shRNA plasmids named pGenesil-1-survivin-1, pGenesil-1-survivin-2 and pGenesil-1-survivin-1+2 (with double interfering RNA sites) were successfully constructed, and were confirmed by restriction enzyme cutting and sequencing. At 48 hours after transfection, the expression of survivin mRNA and protein was inhibited in Patu8988 cells transfected with pGenesil-1-survivin-1, pGenesil-1-survivin-2, and pGenesil-1-survivin-1+2 when compared with that of either pGenesil-1-NC (with scrambled small interfering RNA) transfected cells or control cells (P<0.05). The MTT results showed that the proliferation rates of Patu8988 cells stably transfected with survivin-shRNA plasmids were reduced when compared with that of either pGenesil-1-NC transfected cells or control cells (P<0.01). Furthermore, when Patu8988 cells stably transfected with survivin-shRNA were injected into BALB/c nude mice, tumor growth was dramatically lower and the tumor was smaller than that of either pGenesil-1-NC transfected cells or control cells (P<0.01). The inhibitory effect of pGenesil-1-survivin-1 was the best among the three kinds of survivin-shRNA plasmids, but no combination of inhibitory effects was found in pGenesil-1-survivin-1+2. CONCLUSIONS: shRNAs specific to survivin have gene silencing effects and inhibit pancreatic cancer cell proliferation. shRNA activity against survivin could be of potential value in gene therapy for pancreatic cancer. However, shRNAs with double combining sites did not significantly enhance the interference compared with single site shRNAs, therefore further studies on this are needed.

Wound edge protector for prevention of surgical site infection in laparotomy: an updated systematic review and meta-analysis.

BACKGROUND: An updated meta-analysis based on randomized controlled trials (RCTs) was conducted to evaluate the efficacy of wound edge protector (WEP) in the prevention of surgical site infection (SSI) in patients undergoing laparotomies. METHODS: Meta-analysis was conducted using Review Manager 5.2. The pooled risk ratio was estimated with random-effect model. Medline, Embase, the Cochrane library, reference lists and conference proceedings were data sources. Two independent reviewers screened studies for inclusion and data extraction. Eligible trials were RCTs enrolling patients accepting laparotomies to assess the effectiveness of WEP. RESULTS: Eleven RCTs totalling 2344 patients met the inclusion criteria. Six trials (1589 patients) testing the single-ring design WEP did not show a statistically significant reduction in SSI of laparotomy (RR 0.76, 95% CI 0.51-1.12). Pooled analysis of the five trials (755 patients) that tested the effect of dual-ring design WEP on SSI showed a significant reduction (RR 0.29, 95% CI 0.15-0.55). The combined data of the 11 trials favoured the effect of WEP (RR 0.58, 95% CI 0.39-0.87). Analysis adjusted by the degrees of contamination revealed that WEP is effective in reducing the incidence of SSI after laparotomy of contamination incision (RR 0.43, 0.26-0.72) but failed to demonstrate such effect in clean/contaminated and dirty incisions (RR 0.72, 95% CI 0.43-1.21; RR 0.82, 95% CI 0.43-1.55, respectively). CONCLUSIONS: Our exploratory meta-analysis suggests that WEP reduces the incidence of SSI in patients receiving laparotomies, especially in the circumstance of dual-ring WEP and in contaminated incisions. In order to fully assess the effectiveness of WEP, large-scale and well-designed RCTs are still needed in the future.

Increased activity of CHK enhances the radioresistance of MCF-7 breast cancer stem cells.

The resistance of breast cancer to radiotherapy remains a major obstacle to successful cancer management. Radiotherapy may result in DNA damage and activate breast cancer stem cells. DNA damage may lead to activation of the checkpoint kinase (CHK) signaling pathway, of which debromohymenialdisine (DBH) is a specific inhibitor. Radiotherapy also increases the expression of phosphorylated CHK1/2 (pCHK1/2) in the breast cancer cell line, MCF-7, in vitro in a dose-dependent manner. DBH is a relatively stable effective inhibitor that significantly reduces pCHK1/2 expression and MCF-7 proliferation. Low-dose radiotherapy combined with DBH resulted in a higher MCF-7 inhibition rate compared with high-dose radiation alone. This result indicates that the inhibition of the CHK1/2 signal pathway may significantly reduce DNA damage within radiated cells. Radiotherapy may also regulate the proportion of CD44+/CD24- MCF-7 cancer stem cells in a dose- and time-dependent manner. However, the stem cell proportion of MCF-7 cells was significantly reduced by treatment with DBH. The inhibition is relatively stable and time dependent. Significant reductions were observed after 3 days of culture (P<0.01). The results of the present study indicate that the DBH-induced downregulation of CHK may provide a novel method of enhancing the effect of radiotherapy and reducing stem cell survival in the MCF-7 cell line.

TIGAR regulates DNA damage and repair through pentosephosphate pathway and Cdk5-ATM pathway.

Previous study revealed that the protective effect of TIGAR in cell survival is mediated through the increase in PPP (pentose phosphate pathway) flux. However, it remains unexplored if TIGAR plays an important role in DNA damage and repair. This study investigated the role of TIGAR in DNA damage response (DDR) induced by genotoxic drugs and hypoxia in tumor cells. Results showed that TIGAR was increased and relocated to the nucleus after epirubicin or hypoxia treatment in cancer cells. Knockdown of TIGAR exacerbated DNA damage and the effects were partly reversed by the supplementation of PPP products NADPH, ribose, or the ROS scavenger NAC. Further studies with pharmacological and genetic approaches revealed that TIGAR regulated the phosphorylation of ATM, a key protein in DDR, through Cdk5. The Cdk5-AMT signal pathway involved in regulation of DDR by TIGAR defines a new role of TIGAR in cancer cell survival and it suggests that TIGAR may be a therapeutic target for cancers.

Endoscopic subcutaneous mastectomy: A novel and effective treatment for gynecomastia.

The aim of this study was to evaluate the procedure for and efficacy of endoscopic subcutaneous mastectomy for gynecomastia. Endoscopic subcutaneous mastectomy was performed on 100 benign, palpable breast enlargements in 58 male patients who were followed-up for 15-63 months. The surgery was conducted with the insufflation of CO2 subdermally. No cases were converted to open surgery. The unilateral surgery time was 70-90 min. The mean volume of the resected tissue was 200 ml. All procedures were completed successfully, with satisfactory clinical effects and ideal esthetic results postoperatively. There were three cases (3%) of papillary epidermal partial necrosis; following removal of the dressing during the hospital stay, normal nipple sensation returned. Endoscopic subcutaneous mastectomy had good clinical effects and ideal cosmetic results and is an appropriate approach for gynecomastia.

[Combinational effects of K-ras and IGF-IR antisense oligodeoxynucleotide on proliferation and apoptosis of human pancreatic cancer Patu8988 cells].

BACKGROUND & OBJECTIVE: Point mutation of K-ras gene and overexpression of insulin-like growth factor receptor type 1 (IGF-IR) may contribute to the progression and aggressiveness of pancreatic cancer. Antisense oligodeoxynucleotide (ASODN) against K-ras mRNA and IGF-IR mRNA may inhibit the proliferation of pancreatic cancer cells. This study was to investigate the combinational effects of K-ras ASODN and IGF-IR ASODN on proliferation and apoptosis of human pancreatic cancer Patu8988 cells in vitro and in vivo. METHODS: K-ras gene point mutation in Patu8988 cells was detected by polymerase chain reaction using special sequence primers (PCR-SSP) and sequence analysis. According to the mutation style, K-ras ASODN was designed and composed. K-ras ASODN and IGF-IR ASODN were transfected into Patu8988 cells alone or in combination. Cell proliferation was analyzed by MTT and colony forming assay. The morphologic changes of Patu8988 cells were assessed under transmission electron microscope. The expression of K-ras and IGF-IR mRNA and protein in Patu8988 cells was measured by reverse transcription-polymerase chain reaction (RT-PCR) and flow cytometry (FCM). Cell apoptosis was determined by FCM. The combinational antitumor activity of K-ras ASODN and IGF-IR ASODN was evaluated in BALB/c nude mice bearing human pancreatic cancer inoculated with Patu8988 cells. RESULTS: The point mutation of K-ras gene at codon 12 was detected in Patu8988 cells, and the mutation style was GGT-->GTT. Either 2-32 microg/mL K-ras ASODN or IGF-IR ASODN inhibited proliferation and induced apoptosis of Patu8988 cells. This effect was more obvious when K-ras ASODN and IGF-IR ASODN were used in combination than used alone (P<0.01). In tumor-bearing mice, the inhibitory effect on the growth of transplanted pancreatic cancer was more obvious when K-ras ASODN and IGF-IR ASODN were used in combination than used alone (P<0.01). CONCLUSION: K-ras ASODN combined with IGF-IR ASODN could cooperatively inhibit the proliferation of Patu8988 cells and induce their apoptosis via down-regulating K-ras and IGF-IR expression.

Effects of tetrahydrobiopterin on coronary vascular reactivity in atherosclerotic human coronary arteries.

OBJECTIVES: Reduced nitric oxide (NO) bioavailability is a key mechanism in the development of endothelial dysfunction. The NO synthase cofactor, tetrahydrobiopterin (BH4), increases NO availability, yet its effect in the human coronary circulation, particularly following PCI, remains uncertain. This study was designed to evaluate the effects of intracoronary BH4 in human coronary arteries with non-critical coronary artery disease or following percutaneous coronary intervention (PCI). METHODS: The study group consisted of 57 stable patients, 10 of which were controls. Active drug was administered in 47 patients, with either de novo non-critical coronary disease (non-stent group; n=25) or following PCI (stent group; n=22). Coronary blood flow (CBF) was measured (0.014-inch Doppler flow wire) in each of these groups in response to sequential intracoronary infusions of acetylcholine (Ach, 10(-7) & 10(-6) M), BH4 (250 microg/min & 500 microg/min) and a co-infusion of BH4 (500 microg/min) and Ach (10(-7) & 10(-6) M). The primary endpoint evaluated the % change in CBF to Ach compared to co-infusion of Ach and BH4. RESULTS: Mean age was 60+/-10 years (M 45:F 12). Regarding the primary hypothesis, no difference was observed between Ach response compared to co-infusion of BH4 and Ach in the % change in CBF in either the non-stent group (Ach 97+/-122%, Ach/BH4 87+/-95%) or the stent group (Ach 77+/-105%, Ach/BH4 55+/-97%). CONCLUSIONS: In native non-critical coronary artery disease or following PCI, coronary microvascular endothelial function is not improved by co-administration of Ach and BH4.

Assessment of left ventricular diastolic suction in dogs using wave-intensity analysis.

Two apparently different types of mechanisms have emerged to explain diastolic suction (DS), that property of the left ventricle (LV) that tends to cause it to refill itself during early diastole independent of any force from the left atrium (LA). By means of the first mechanism, DS depends on decreased elastance [e.g., the relaxation time constant (tau)] and, by the second, end-systolic volume (V(LVES)). We used wave-intensity analysis (WIA) to measure the total energy transported by the backward expansion wave (I(W-)) during LV relaxation in an attempt to reconcile these mechanisms. In six anesthetized, open-chest dogs, we measured aortic, LV (P(LV)), LA (P(LA)), and pericardial pressures and LV volume by orthogonal ultrasonic crystals. Mitral velocity was measured by Doppler echocardiography, and aortic velocity was measured by an ultrasonic flow probe. Heart rate was controlled by pacing, V(LVES) by volume loading, and tau by isoproterenol or esmolol administration. I(W-) was found to be inversely related to tau and V(LVES). Our measure of DS, the energy remaining after mitral valve opening, I(W-DS), was also found to be inversely related to tau and V(LVES) and was approximately 10% of the total "aspirating" energy generated by LV relaxation (i.e., I(W-)). The size of the Doppler (early filling) E wave depended on I(W-DS) in addition to I(W+), the energy associated with LA decompression. We conclude that the energy of the backward-going wave generated by the LV during relaxation depends on both the rate at which elastance decreases (i.e., tau) and V(LVES). WIA provides a new approach for assessing DS and reconciles those two previously proposed mechanisms. The E wave depends on DS in addition to LA decompression.

Effects of left ventricular contractility and coronary vascular resistance on coronary dynamics.

Wave-intensity analysis, which separates upstream from downstream events and defines their interaction, has been used to study the effects of changes in left ventricular (LV) contractility (E(max)) and left circumflex coronary artery resistance (R(LCx)) on the coronary systolic flow impediment (CSFI). In 10 anesthetized, open-chest dogs, we measured coronary, aortic, and LV pressures, coronary velocity (Flowire), and flow. E(max) was increased by paired pacing and R(LCx) was modulated by intracoronary infusions of vasodilators (adenosine and nitroglycerin) and a vasoconstrictor (phenylephrine). When both E(max) and R(LCx) were varied, CSFI and the energy of the backward-going compression wave (I(W-)) were greatest at the highest levels of E(max) and the lowest levels of R(LCx). I(W-) was proportional to the CSFI. We conclude that contractility and coronary resistance change CSFI by modulating the backward-going compression wave.