Synergistic effect of the herbal mixture C5E on gemcitabine treatment in PANC­1 cells.

The present study aimed to determine the anticancer effect of the herbal mixture extract C5E in the pancreatic cancer cell line, PANC­1, in the absence or presence of gemcitabine treatment, a chemotherapeutic drug used for the treatment of pancreatic cancer. The anticancer effects of C5E, gemcitabine and C5E plus gemcitabine in PANC­1 cells following 72 h of treatment were investigated. The effect of each treatment on cell cycle arrest, apoptosis and the proportion of side population (SP) cells was determined using flow cytometric analysis following propidium iodide (PI), Annexin V­FITC/PI double staining and Hoechst 33342 staining, respectively. SP cells share similar characteristics to cancer stem­like cells, and a reduction in the SP is considered to be indicative of an anticancer effect. The percentage of SP cells and the cell viability of general PANC­1 cells were significantly decreased in response to all treatments. The percentage of SP cells was reduced from 8.2% (control) to 3.9, 7.2 and 5.1% following the treatment with C5E, gemcitabine and the co­treatment, respectively. All three treatments were discovered to inhibit cell viability by arresting the cell cycle at the S phase and promoted cell death by inducing early apoptosis, with the levels of apoptosis being increased from 1.9% (control) to 7.3, 2.5 and 12.0% following the treatment with C5E, gemcitabine and the co­treatment, respectively. The mRNA expression levels of sonic hedgehog, which is implicated in the development of certain types of cancer, were downregulated to a greater extent following the co­treatment with C5E and gemcitabine compared with the treatment with either C5E or gemcitabine alone. As the co­treatment with gemcitabine and C5E was more effective than each individual treatment, the present study suggested that the combined treatment may exhibit synergistic effects in PANC­1 cells.

PM014 attenuates radiation-induced pulmonary fibrosis via regulating NF-kB and TGF-b1/NOX4 pathways.

Radiation therapy is the mainstay in the treatment of lung cancer, and lung fibrosis is a radiotherapy-related major side effect that can seriously reduce patient's quality of life. Nevertheless, effective strategies for protecting against radiation therapy-induced fibrosis have not been developed. Hence, we investigated the radioprotective effects and the underlying mechanism of the standardized herbal extract PM014 on radiation-induced lung fibrosis. Ablative radiation dose of 75 Gy was focally delivered to the left lung of mice. We evaluated the effects of PM014 on radiation-induced lung fibrosis in vivo and in an in vitro model. Lung volume and functional changes were evaluated using the micro-CT and flexiVent system. Fibrosis-related molecules were evaluated by immunohistochemistry, western blot, and real-time PCR. A orthotopic lung tumour mouse model was established using LLC1 cells. Irradiated mice treated with PM014 showed a significant improvement in collagen deposition, normal lung volume, and functional lung parameters, and these therapeutic effects were better than those of amifostine. PM104 attenuated radiation-induced increases in NF-κB activity and inhibited radiation-induced p65 translocation, ROS production, DNA damage, and epithelial-mesenchymal transition. PM104 effectively alleviated fibrosis in an irradiated orthotopic mouse lung tumour model while not attenuating the efficacy of the radiation therapy by reduction of the tumour. Standardized herbal extract PM014 may be a potential therapeutic agent that is able to increase the efficacy of radiotherapy by alleviating radiation-induced lung fibrosis.

LXA4-FPR2 signaling regulates radiation-induced pulmonary fibrosis via crosstalk with TGF-ß/Smad signaling.

Radiation therapy is an important modality in the treatment of lung cancer, but it can lead to radiation pneumonitis, and eventually radiation fibrosis. To date, only few available drugs can effectively manage radiation-induced pulmonary fibrosis. Lipoxins are endogenous molecules exhibit anti-inflammatory and pro-resolving effects. These molecules play a vital role in reducing excessive tissue injury and chronic inflammation; however, their effects on radiation-induced lung injury (RILI) are unknown. In this study, we investigated the effects of lipoxin A4 (LXA4) on RILI using our specialized small-animal model of RILI following focal-ablative lung irradiation (IR). LXA4 significantly inhibited immune-cell recruitment and reduced IR-induced expression of pro-inflammatory cytokines and fibrotic proteins in the lung lesion sites. In addition, micro-CT revealed that LXA4 reduced IR-induced increases in lung consolidation volume. The flexiVentTM assays showed that LXA4 significantly reversed IR-induced lung function damage. Moreover, LXA4 downregulated the activities of NF-κB and the Smad-binding element promoters. The expression of FPR2, an LXA4 receptor, increased during the development of IR-induced pulmonary fibrosis, whereas silencing of endogenous LXA4 using an antagonist (WRW4) or FPR2 siRNA resulted in impaired development of pulmonary fibrosis in response to IR. Collectively, these data suggest that LXA4 could serve as a potent therapeutic agent for alleviating RILI.

Prognostic significance of E-cadherin and ZEB1 expression in intraductal papillary mucinous neoplasm.

There is an urgent need to investigate the genetic changes that occur in intraductal papillary mucinous neoplasm (IPMN), which is a well-known precursor of pancreatic cancer. In this study, gene expression profiling was performed by removing unwanted variation to determine the differentially expressed genes (DEGs) associated with malignant progression of IPMN. Among the identified DEGs, zinc finger E-box binding homeobox 1 (ZEB1) and E-cadherin, a crucial regulator of epithelial-to-mesenchymal transition (EMT), was validated among identified DEGs. A total of 76 fresh-frozen tissues were used for gene expression profiling and formalin-fixed, paraffin-embedded blocks from 87 patients were obtained for immunohistochemical analysis. Loss of E-cadherin expression (p = 0.023, odd ratio [OR] = 4.923) and expression of ZEB1 in stromal cells (stromal ZEB1, p < 0.001, OR = 26.800) were significantly correlated with degree of dysplasia. The hazard of death was significantly increased in patients with loss of E-cadherin expression (hazard ratio [HR] = 13.718, p = 0.004), expression of epithelial ZEB1 (HR = 19.117, p = 0.001), and stromal ZEB1 (HR = 6.373, p = 0.043). Based on the results of this study, loss of E-cadherin and expression of stromal ZEB1 are associated with increased risk of malignant progression. Epithelial and stromal ZEB1, as well as E-cadherin may be strong predictors of survival in patients with IPMN. Our finding suggests that these EMT markers may be utilized as potential prognosticators and may be used to improve and personalize treatment of IPMN.

Antitumor effects of herbal mixture extract in the pancreatic adenocarcinoma cell line PANC1.

A recent study showned that complementary medicine is gradually gaining wide acceptance. In the present study, the herbal mixture extract (H3) composed of 3 oriental herbal plants was investigated for anticancer activity in vitro and in vivo. H3 inhibited PANC1 cell growth by promoting G0/G1 arrest (11% increase) and apoptotic cell death (9% increase). H3 also suppressed stem cell-like side population cells (4% decrease) and migration activity (24% decrease). In contrast, gemcitabine decreased side population cells and migration activity by 3 and 11%, respectively. These effects of H3 and gemcitabine were further studied by examining the expression of apoptosis-associated genes (CXCR4, JAK2 and XIAP) and stem cell-associated genes (ABCG2, POU5F1 and SOX2). We also found that H3 suppressed tumor growth by 46% in a PANC1­xenograft model, while gemcitabine caused a 36% decrease. The antitumor effects of H3 were confirmed by western blot analysis for COX-2 and cytochrome c expression. Furthermore, necrotic cell death and erythrocyte-containing cavities were detected in tumor tissue by hematoxylin and eosin (H&E) staining. Notably, the combinatorial therapy (H3 and gemcitabine) increased tumor growth compared to that in the control. In conclusion, the present study shows that H3 has promise as a therapeutic agent against pancreatic cancer and its cancer stem cells.

Silver wire amplifies the signaling mechanism for IL-1beta production more than silver submicroparticles in human monocytic THP-1 cells.

Silver materials have been widely used in diverse fields. However, their toxicity and their mechanism, especially in different forms, have not been studied sufficiently. Thus, cytotoxicity, apoptosis, and interleukin-1beta (IL-1ß) production were investigated using macrophage-like THP-1 cells in the presence of Ag microparticles (AgMPs, 2.7 µm), Ag submicroparticles (AgSMPs, 150 nm), and Ag wires (AgWs, 274 nm×5.3 µm). The levels of cytotoxicity, apoptosis, and IL-1ß production by AgWs were higher than those by the other two AgSMPs and AgMPs. This trend was also observed with each step of the signaling mechanism for IL-1ß production, which is a single pathway affiliated with ROS generation or lysosomal rupture or both, cathepsin B, caspase-1 (NALP3 inflammasome), and finally IL-1ß production in THP-1 cells. All these results suggest that, for development of safe and effective silver materials, the shape or form of silver materials should be considered, especially for macrophage cell lines because epithelial cell lines are not overly sensitive to silver materials.

Poly-γ-glutamic acid enhances the quality of recombant erythropoietin produced by CHO cells.

The effect of poly-γ-glutamic acid (γPGA), which is produced by Bacillus sp., on the production of recombinant erythropoietin (rEPO) by Chinese hamster ovary (CHO) cells in suspension culture was evaluated. The growth, viability, and productivity of recombinant CHO cells were investigated in a chemically defined medium with 50 and 500 kD γPGAs at 0.075% or with Pluronic F68. Cell growth with the two γPGAs was lower than that with Pluronic F68 but significantly higher than that without any additive (control). The effect of additives on rEPO productivity was 50 kDa γPGA > 500 kDa γPGA > Pluronic F68 > control. Using EPO-dependent F-36E cells, we found that the effect of additives on rEPO quality was 500 kDa γPGA > 50 kDa γPGA > control > Pluronic F68. γPGA has an enhancement effect on the quality of rEPO produced by CHO cells.

Involvement of oxidative stress and mitochondrial apoptosis in the pathogenesis of pelvic organ prolapse.

PURPOSE: Biomechanical weakness of the pelvic supportive structures has been proposed to be a cause of pelvic organ prolapse. However, the molecular mechanism involved in these changes is not completely understood. In this investigation we evaluated oxidative stress biomarkers in the uterosacral ligaments of women with pelvic organ prolapse and compared them with those of women with normal support. In addition, mitochondrial apoptosis was examined. MATERIALS AND METHODS: Samples were collected from 26 women with advanced stage pelvic organ prolapse and 29 age matched controls. The expression levels of 8-OHdG and 4-hydroxy-2-nonenal in the uterosacral ligaments were measured using immunohistochemistry. To assess mitochondrial apoptosis we performed TUNEL assay, immunohistochemistry for cleaved caspase-3 and cytochrome c, and Western blot analyses for cleaved caspase-3 and caspase-9. RESULTS: The mean percentage of cells immunopositive for 8-OHdG, 4-hydroxy-2-nonenal, TUNEL, cleaved caspase-3 and cytochrome c in the uterosacral ligaments was significantly higher in patients with pelvic organ prolapse than in controls. Similarly, Western blot analysis revealed increased expression of cleaved caspase-3 and caspase-9 in patients with pelvic organ prolapse. Correlation analyses revealed significant positive correlations between the percentage of cells immunopositive for 8-OHdG or 4-hydroxy-2-nonenal and markers of mitochondrial apoptosis. Analyzing by pelvic organ prolapse quantification system stage according to C point, the mean percentage of cells immunopositive for 8-OHdG, 4-hydroxy-2-nonenal and cytochrome c was significantly higher in patients with pelvic organ prolapse compared to controls, regardless of stage. However, the mean percentage of TUNEL and cleaved caspase-3 positive cells was significantly higher only in patients with stage III or IV pelvic organ prolapse. CONCLUSIONS: Oxidative stress and increased mitochondrial apoptosis may contribute to the pathological process of pelvic organ prolapse.

Increased BCR promoter DNA methylation status strongly correlates with favorable response to imatinib in chronic myeloid leukemia patients.

To define the correlation between BCR promoter DNA methylation and response to imatinib in chronic myeloid leukemia (CML), we investigated BCR promoter DNA methylation in three groups of subjects. The first group included chronic phase patients enrolled in an imatinib dose escalation trial. In the trial, patients who failed to achieve optimal response with 400 mg/day (suboptimal responders) received an escalated imatinib dose. The level of BCR promoter DNA methylation was quantitated at baseline six months after dose escalation. The second group included patients who achieved complete cytogenetic remission after receiving 400 mg/day of imatinib (optimal responders), and the third group were the healthy controls. In the suboptimal responders, an increased BCR promoter DNA methylation at six months compared with the baseline was related to a rapid reduction in the BCR-ABL/ABL transcript level following dose escalation (p=0.001) and a longer time to treatment failure (TTFx) of the dose-escalated imatinib (p=0.008). When multivariate analysis was performed with regard to the baseline BCR-ABL transcript level, baseline BCR promoter DNA methylation, and a change in the BCR promoter DNA methylation following dose escalation, the increase in the BCR promoter DNA methylation following dose escalation was an independent predictive factor for TTFx of dose-escalated imatinib (hazard ratio, 0.294; p=0.015). The baseline BCR promoter DNA methylation level in the suboptimal responders was lower than that in BCR promoter DNA methylation in the optimal responders (p=0.001) and healthy controls (p<0.001). In both the optimal and suboptimal responders, BCR promoter DNA methylation had an inverse correlation with the duration of the 400 mg/day imatinib use. In conclusion, increased BCR promoter DNA methylation strongly correlates with a more favorable imatinib response in CML patients.

GSTT1 copy number gain is a poor predictive marker for escalated-dose imatinib treatment in chronic myeloid leukemia: genetic predictive marker found using array comparative genomic hybridization.

In a study population of 45 patients who were previously enrolled in an imatinib dose escalation trial, genome-wide screening for regions of genetic gains and losses was performed using array comparative genomic hybridization (aCGH). Early molecular response (EMR), defined as >50% reduction in the ratio of BCR-ABL1 to ABL1 within 6 months after dose escalation, was a major endpoint for analysis. After aCGH analysis, copy number change of four genes was investigated in 52 patients as a validation. Copy number gain in 16p11.2 was more frequently observed in patients with EMR than in patients who failed to achieve EMR (P = 0.034). A tendency for increased copy number in 22q11.23 in patients without EMR and for decreased copy number in 17q12 in patients with EMR was observed (P = 0.072 and P = 0.070, respectively). For GSTT1, in 22q11.23, copy number gain was observed in patients without EMR (P = 0.035). GSTT1 copy number gain was related to short time to treatment failure (TTFx) in patients without BCR-ABL1 mutations (P = 0.007). In multivariate analysis, GSTT1 copy number gain was an independent predictive factor for short TTFx (P = 0.020). We conclude that chromosome regions 16p11.2, 22q11.23, and 17q12 are potential locations related to response in imatinib dose escalation therapy for CML. GSTT1 copy number gain is a genetic change affecting outcome in this setting.