Abstract Background: Microglia polarization plays an important role in poststroke recovery. Inhibition of proinflammatory (M1) polarization and promotion of anti-inflammatory (M2) polarization of microglia are potential therapeutic strategies for inflammation reduction and neuronal recovery after stroke. Analgecine (AGC), the extracts of Vaccin a variola-inoculated rabbit skin, is used to treat patients with chronic low back pain due to degenerative vertebral disorders. Here, we evaluated the neuroprotective effect of AGC in stroke and investigate anti-inflammatory mechanism of AGC on microglia-mediated neural damage.Methods: Sprague-Dawley (SD) rats underwent 120 min of middle cerebral artery occlusion (MCAO) followed by reperfusion. We injected AGC intravenously into rats starting 3 h after the onset of MCAO. Then we investigated the effect of AGC on neurological impairment, neuronal loss and inflammatory cytokines. For in vitro study, we examined the effect of AGC on microglial polarization in oxygen-glucose deprivation/reperfusion (OGD/R) or LPS/IFN-γ induced microglia cells and further investigated neuroprotective effect of ACG in microglia-mediated neural damage based on the direct or indirect co-culture systems. Finally, TLR4/Myd88/ NF-κB pathway was detected in OGD/R-induced microglia cells with or without Myd88 siRNA transfection.Results: AGC treatment reduced the neurological deficits and suppressed neuronal loss. In terms of inflammatory cytokines, AGC inhibited the release of pro-inflammatory cytokines and elevated the content of anti-inflammatory cytokines in vivo (SD rats) and in vitro (microglia). We further showed that AGC promoted M1 to M2 phenotypic transition of microglia in OGD/R or LPS/IFN-γ induced microglia cells. Based on the direct or indirect co-culture systems, we found AGC indirectly inhibits LPS/IFN-γ-induced neuronal damage by modulating microglial polarization. Moreover, AGC suppressed the nuclear translocation of the phosphorylation of NF-κB p65 by inhibiting the TLR4/Myd88/TRAF6 but not TLR9 signaling. We also confirmed that AGC-regulated TLR4 inhibition partly dependent on Myd88 in a Myd88 depletion cell line.Conclusion: Our findings provide a new understanding of AGC in neuroprotection by inhibiting M1 microglial polarization and promoting anti-inflammation by suppressing TLR4 MyD88-dependent and MyD88-independent pathways. Thus, AGC treatment may represent a novel approach in inflammation reduction or poststroke recovery.
Abstract Background: The analytical process, especially reagent-related factors, demonstrates a significant effect on clinical chemistry analyzer. Therefore, it should have our attention both in total laboratory system (TLA) and modular automation (MA). The reagent-loading mode of modular analyzer influences testing turnaround time (tTAT) and the cost.Methods: Parameters were simulated by cobas 8000 workflow simulator for reagent-loading manner with at least three positions (Pattern 1), the module-parallel reagent-loading manner (Pattern 2) and the single-position loading mode (Pattern 3). Then, we chose Pattern 2 for Tuesday effect-based optimization verification.Results: tTAT, reagent on-line time, quality control cost and performance verification times all declined by 43%. Tuesday effect solved the repetitive problem for verification. With comprehensive comparison between Pattern 1, Pattern 2 and Pattern 3, Pattern 2 shows optimal performance in subsequent verification.Conclusions: The optimization of reagent-loading manner saved much workforce, and reduced the quality control cost and internal quality assessment consumption.Trial registration: This article did not report the results of a health care intervention on human participants. Trial registration was not needed here.
The aim of this study was to examine the changes in diurnal cortisol patterns and its associated factors among breast cancer patients over a 14-month follow up period.A total of 85 breast cancer patients were recruited to participate in this study. Assessments were performed at baseline (T0), T1 (the 2nd month), T2 (the 5th month), T3 (the 8th month), and T4 (the 14th month). Salivary cortisol was measured and the following questionnaires were administered: BDI-II depression scale, European Organization for Research and Treatment of Cancer Core Cancer Quality of Life Questionnaire (EORTC QLQ-C30) and its breast cancer-specific complementary measure (EORTC QLQ-BR23). Patients were grouped into flat and steep groups, according to the median of the diurnal cortisol slopes at the time of the transition period.Breast cancer patients in the flatter slope group at transition period demonstrated steeper slopes over the course of recovery from treatment and those in the steeper slope group at transition period continued with steeper slopes over the course of recovery. The greater breast cancer-related symptoms (side-effects, symptoms relating to breast and arm, and hair loss) were associated with the changes in flatter diurnal cortisol slopes during14-month follow up period.Diurnal cortisol patterns in flatter slope group at the transition period appear to have a trend of recovery with the passage of time over the course of recovery from treatment. Management of breast cancer symptoms could improve dysregulation of diurnal cortisol patterns among survivors.
Breast cancer is a major health threat world wide. It is the top cancer in women both in the developed and the developing world, comprising 16% of all female cancers. Optimal systemic treatment (adjuvant therapy) after breast cancer surgery is a crucial factor in reducing mortality in women with breast cancer, however a significant number of them still develop metastatic diseases and respond only transiently to conventional treatments leading to eventual mortality. So understanding the mechanism of drug resistance would help us to manage these patients and improved
their prognosis. Connective tissue growth factor (CTGF) expression is elevated in advanced breast cancer and promotes breast cancer metastasis. In the present study, we examined whether CTGF expression could confer drug resistance in human breast cancer. In breast cancer patients who received neoadjuvant chemotherapy, CTGF expression inversely correlated with chemotherapy response. Overexpression of CTGF in MCF7 cells (MCF7/CTGF) enhanced clonogenic ability, cell viability and resistance to apoptosis upon exposure to doxorubicin and paclitaxel. Reducing the CTGF level in MDA-MB-231 (MDA231) cells by antisense CTGF cDNA (MDA231/AS cells) decreased these effects. CTGF overexpression resulted in resistance to doxorubicin- and paclitaxel-induced apoptosis by up-regulation of Bcl-xL and cIAP1. Furthermore, CTGF overexpression resulted in activation of the ERK1/2 pathway. Inhibition of ERK1/2 effectively reversed the resistance to apoptosis as well as the up-regulation of Bcl-xL and cIAP1 in MCF7/CTGF cells. We also found that a C-terminal domain peptide from CTGF could exert similar activities to full-length CTGF in activation of ERK1/2, up-regulation of Bcl-xL/cIAP1 and resistance to apoptosis. We conclude that CTGF expression could confer resistance to chemotherapeutic agents through ERK1/2-mediated Bcl-xL/cIAP1 up-regulation of a survival pathway.
Convallatoxin is widely used as a cardiac glycoside in acute and chronic congestive heart-failure and paroxysmal tachycardia, with many effects and underlying protective mechanisms on inflammation and cellular proliferation. However, convallatoxin has not been investigated in its antioxidant effects and lifespan extension in Caenorhabditis elegans. In this study, we found that convallatoxin (20 μM) could significantly prolong the lifespan of wild-type C. elegans up to 16.3% through daf-16, but not sir-2.1 signalling and increased thermotolerance and resistance to paraquat-induced oxidative stress. Convallatoxin also improved pharyngeal pumping, locomotion, reduced lipofuscin accumulation and reactive oxygen species levels in C. elegans, which were attributed to hormesis, free radical-scavenging effects in vivo, and up-regulation of stress resistance-related proteins, such as SOD-3 and HSP-16.1. Furthermore, aging-associated genes daf-16, sod-3, and ctl-2 also appeared to contribute to the stress-resistance effect of convallatoxin. In summary, this study demonstrates that convallatoxin can protect against heat and oxidative stress and extend the lifespan of C. elegans, pointing it as a potential novel drug for retarding the aging process in humans.
Abstract Background: With more hormonal therapies (HT) based treatment (tx) available, predictive markers that could lead to a selection of the optimal tx is necessary. The predictive role of ctDNA mutations in ER+/HER2- MBC after prior HT is less well characterized in Asian patients (pts). Methods: ER+/HER2- MBC pts starting HT based salvage tx after refractory to at least one-line of HT were eligible. ctDNA was extracted from pre- and post-tx plasma and prepared for next-generation sequencing (NGS) analysis. The targeted NGS mutations included regions of ESR1 ligand-binding domain, PIK3CA mutation hotspots, and TP53 mutation hotspots. 96% of the samples were sequenced at an average depths >10000x using the Ion Torrent platform. Progression-free survival (PFS) was defined from the start of the salvage tx to the date of progression. Results: From 2015/08 to 2019/04, a total of 129 and 70 pts treated with HT based tx had pre- and post-tx ctDNA tested, respectively. The median age is 60 (32-92). 14%, 7%, 55%, and 19% of pts received HT only, HT + CDK4/6 inhibitor, HT + everolimus, and HT + metronomic chemotherapy, respectively. With mutation ctDNA > 0.5% as a threshold for positive calling, 79 (61.2%), 33 (25.6%), and 23 (17.8%) pts have at least one ESR1, PIK3CA, and TP53 mutation, respectively and 48 (37.3%) pts have >1 ESR1 mutation genotypes. When compared to other clinical trial data, Asian ER+ MBC pts had significantly higher ESR1 mutation rate as compared to the Western population (p < 0.001) (Table 1). Detectable PIK3CA and TP53 mutation pre-tx was significantly (median PFS 9.8 vs 4.8 months (mos), p= 0.002) and marginally (median PFS 7.9 vs 5.2 mos, p = 0.08) associated with shorter PFS, respectively; but neither the presence of at least one single or multiple clones of ESR1 mutation(s) was associated with PFS (p = 0.52). Conversely, pts without any detectable ctDNA mutation had marginally better PFS (median 12.0 vs 6.5 mos, p = 0.051). With respect to the impact of each ESR1 mutation genotype, the presence of E380Q is associated with significantly shorter PFS (median PFS 7.9 vs 3.4 mos, p = 0.033) while Y537S, D538G, and Y537C were not. When the threshold for the positive calling of ctDNA was raised to 2.5%, ESR1 Y537S mutation became a significant factor for shorter PFS (median PFS 9.1 vs 4.4 mos, p = 0.041); PIK3CA remained as a significant factor for shorter PFS (median 9.3 vs 4.4 mos, p <0.001). In the HT + everolimus cohort (n = 71), PIK3CA mutation ctDNA remained as a poor PFS factor (median PFS 5.9 vs 2.6 mos, p = 0.01) but neither TP53 or ESR1 mutations were significantly associated with PFS. When pre- and post-treatment ctDNA were included in the analysis, the emergence of ESR1 mutations was associated with a better PFS (p = 0.05) while the loss of ESR1 mutations or gain/loss of PIK3CA mutations are not associated with PFS in the HT-treated cohort. Conclusion: PIK3CA and ESR1 Y537S and E380Q mutations detected by ctDNA had predictive impact for late-line HT based tx but PIK3CA mutation is a better predictor marker than ESR1 mutation in pts treated with HT + everolimus. Table 1 A comparison of the proportion and distribution of ESR1 mutation genotypes among the Taiwan cohort and other clinical trial studiesStudiesESR1 mutation (%)% of ESR1-positive patients withY537SD538GY537CPALOMA3 (n = 360) Fribbens et al. 201691(25.3)25565SoFEA (n = 161) Fribbens et al. 201663(39.1)25465FERGI (n = 156) Spoerke et al. 201657(37.3)33545Taiwan cohort (n = 129)79(61.2)538153 Citation Format: Tom Wei-Wu Chen, Ming-Shen Dai, Dwang-Ying Chang, Ching-Hung Lin, I-Chun Chen, Ming-Yang Wang, Ling-Yi Huang, An Hsu, De-Wei Zhuo, Kien Thiam Tan, Yen-Jung Lu, Shu-Han Chang, Ann-Lii Cheng, Yen-Shen Lu. PIK3CA and ESR1 mutations detected in circulating tumor DNA (ctDNA) are predictive factors for late-line hormone-based therapies in ER+/HER2- metastatic breast cancer (MBC) [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P5-01-21.
A193 Effects of CTGF in drug resistance in breast cancer cells and the signal transduction pathways Breast cancer is the second most common women cancer and the forth most frequent cause of women cancer death in Taiwan. The use of adjuvant systemic chemotherapy has been clearly shown to provide survival benefit to all groups of patients regardless of menopausal, nodal or hormonal receptor status. Connective tissue growth factor (CTGF), which is also known as CTGF/CCN2, belongs to the CCN family. The biological properties of CCN proteins involve the stimulation of cellular proliferation, migration, adhesion, extracellular matrix formation, and also the regulation of angiogenesis and tumorigenesis. Over-expression of CTGF, WISP-1, and CYR61 in breast tumor cells have been linked to tumor size and lymph node metastasis, suggesting that these CCN proteins are involved in the progression of breast cancer. In the present study, we found that CTGF conferred drug resistance through the activation of the integrin avb3/FAK/ERK1/2 pathway. We examined the expression level of CTGF in several breast cancer cell lines and the combination effects of CTGF neutralizing antibody and chemotherapeutic agents. Co-treatment with CTGF-neutralizing antibody and chemo-therapeutic agents significantly increased the drug sensitivities of MDA231 and MDA435 cells which had high expression level of CTGF. This phenomenon was not found in T47D, BT483 and MCF-7 cells which had low CTGF expression level. In addition, we generated the CTGF-overexpression (MCF-7/CTGF) and antisense-CTGF expression (MDA231/AS) cells. The effects of CTGF in drug induced apoptosis were examined by TUNEL, PARP cleavage and flow cytometry assays. Since our unpublished data shows that Bcl-xL and cIAP1 were up-regulated by CTGF, we next inhibited the activation of integrin avb3 and ERK1/2 pathway by avb3-antibody and PD98059, respectively. Treatment with avb3-antibody or PD98059 decreased the CTGF-induced expression of both Bcl-xL and cIAP1, indicating that avb3/ERK pathway is required for CTGF to promote Bcl-xL and cIAP1 expression and, in addition to that, FAK phosphorylation is also increased by CTGF in which it might involved in avb3/ERK signaling pwthway. Restoring the expression of Bcl-xL or cIAP1 in MDA-231/AS CTGF cells conferred drug resistance. CTGF expression is also positively correlated with worse chemo-response in patients with breast cancer. Together, we showed that CTGF up-regulated Bcl-xL and cIAP1 via integrin avb3/FAK/ERK1/2 pathway which contributed the drug resistance in breast cancer.
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