Breast cancer is the most common malignancy in women and the second leading cause of cancer death in women. Triple negative breast cancer (TNBC) subtype is a breast cancer subset without ER (estrogen receptor), PR (progesterone receptor) and HER2 (human epidermal growth factor receptor 2) expression, limiting treatment options and presenting a poorer survival rate. Thus, we investigated whether histone deacetylation inhibitor (HDACi) could be used as potential anti-cancer therapy on breast cancer cells. In this study, we found TNBC and HER2-enriched breast cancers are extremely sensitive to Panobinostat, Belinostat of HDACi via experiments of cell viability assay, apoptotic marker identification and flow cytometry measurement. On the other hand, we developed a bioluminescence-based live cell non-invasive apoptosis detection sensor (NIADS) detection system to evaluate the quantitative and kinetic analyses of apoptotic cell death by HDAC treatment on breast cancer cells. In addition, the use of HDACi may also contribute a synergic anti-cancer effect with co-treatment of chemotherapeutic agent such as doxorubicin on TNBC cells (MDA-MB-231), but not in breast normal epithelia cells (MCF-10A), providing therapeutic benefits against breast tumor in the clinic.
Anaplastic thyroid carcinoma (ATC) and squamous thyroid carcinoma (STC) are both rare and advanced thyroid malignancies with a very poor prognosis and an average median survival time of 5 months and less than 20% of affected patients are alive 1 year after diagnosis. The clinical management of both ATC and STC is very similar because they are not particularly responsive to radiotherapy and chemotherapy. This inspired us to explore a novel and effective clinically approved therapy for ATC treatment. Histone deacetylase inhibitor (HDACi) drugs are recently FDA-approved drug for malignancies, especially for blood cell cancers. Therefore, we investigated whether an HDACi drug acts as an effective anticancer drug for advanced thyroid cancers. Cell viability analysis of panobinostat treatment demonstrated a significant IC50 of 0.075 µM on SW579 STC cells. In addition, panobinostat exposure activated histone acetylation and triggered cell death mainly through cell cycle arrest and apoptosis-related protein activation. Using CRISPR/Cas9 to knock out HDAC1 and HDAC2 genes in SW579 cells, we observed that the histone acetylation level and cell cycle arrest were enhanced without any impact on cell growth. Furthermore, HDAC1 and HDAC2 double knockout (KO) cells showed dramatic cell apoptosis activation compared to HDAC1 and HDAC2 individual KO cells. This suggests expressional and biofunctional compensation between HDAC1 and HDAC2 on SW579 cells. This study provides strong evidence that panobinostat can potentially be used in the clinic of advanced thyroid cancer patients.
Since imatinib (Glivec or Gleevec) has been used to target the BCR-ABL fusion protein, chronic myeloid leukemia (CML) has become a manageable chronic disease with long-term survival. However, 15%–20% of CML patients ultimately develop resistance to imatinib and then progress to an accelerated phase and eventually to a blast crisis, limiting treatment options and resulting in a poor survival rate. Thus, we investigated whether histone deacetylase inhibitors (HDACis) could be used as a potential anticancer therapy for imatinib-resistant CML (IR-CML) patients. By applying a noninvasive apoptosis detection sensor (NIADS), we found that panobinostat significantly enhanced cell apoptosis in K562 cells. A further investigation showed that panobinostat induced apoptosis in both K562 and imatinib-resistant K562 (IR-K562) cells mainly via H3 and H4 histone acetylation, whereas panobinostat targeted cancer stem cells (CSCs) in IR-K562 cells. Using CRISPR/Cas9 genomic editing, we found that HDAC1 and HDAC2 knockout cells significantly induced cell apoptosis, indicating that the regulation of HDAC1 and HDAC2 is extremely important in maintaining K562 cell survival. All information in this study indicates that regulating HDAC activity provides therapeutic benefits against CML and IR-CML in the clinic.
Chronic myelogenous leukemia (CML) is the most common type of leukemia in adults, and more than 90% of CML patients harbor the abnormal Philadelphia chromosome (Ph) that encodes the BCR-ABL oncoprotein. Although the ABL kinase inhibitor (imatinib) has proven to be very effective in achieving high remission rates and improving prognosis, up to 33% of CML patients still cannot achieve an optimal response. Here, we used CRISPR/Cas9 to specifically target the BCR-ABL junction region in K562 cells, resulting in the inhibition of cancer cell growth and oncogenesis. Due to the variety of BCR-ABL junctions in CML patients, we utilized gene editing of the human ABL gene for clinical applications. Using the ABL gene-edited virus in K562 cells, we detected 41.2% indels in ABL sgRNA_2-infected cells. The ABL-edited cells reveled significant suppression of BCR-ABL protein expression and downstream signals, inhibiting cell growth and increasing cell apoptosis. Next, we introduced the ABL gene-edited virus into a systemic K562 leukemia xenograft mouse model, and bioluminescence imaging of the mice showed a significant reduction in the leukemia cell population in ABL-targeted mice, compared to the scramble sgRNA virus-injected mice. In CML cells from clinical samples, infection with the ABL gene-edited virus resulted in more than 30.9% indels and significant cancer cell death. Notably, no off-target effects or bone marrow cell suppression was found using the ABL gene-edited virus, ensuring both user safety and treatment efficacy. This study demonstrated the critical role of the ABL gene in maintaining CML cell survival and tumorigenicity in vitro and in vivo. ABL gene editing-based therapy might provide a potential strategy for imatinib-insensitive or resistant CML patients.
OBJECTIVE Effective perioperative hand antisepsis is crucial for the safety of patients and medical staff in surgical rooms. The antimicrobial effectiveness of different antiseptic methods, including conventional hand scrubs and waterless hand rubs, has not been well evaluated. DESIGN, SETTING, AND PARTICIPANTS A randomized controlled trial was conducted to investigate the effectiveness of the 3 antiseptic methods among surgical staff of Taipei Medical University-Shuang Ho Hospital. For each method used, a group of 80 participants was enrolled. INTERVENTION Surgical hand cleansing with conventional 10% povidone-iodine scrub, conventional 4% chlorhexidine scrub, or waterless hand rub (1% chlorhexidine gluconate and 61% ethyl alcohol). RESULTS Colony-forming unit (CFU) counts were collected using the hand imprinting method before and after disinfection and after surgery. After surgical hand disinfection, the mean CFU counts of the conventional chlorhexidine (0.5±0.2, P<0.01) and waterless hand rub groups (1.4±0.7, P<0.05) were significantly lower than that of the conventional povidone group (4.3±1.3). No significant difference was observed in the mean CFU count among the groups after surgery. Similar results were obtained when preexisting differences before disinfection were considered in the analysis of covariance. Furthermore, multivariate regression indicated that the antiseptic method (P=.0036), but not other variables, predicted the mean CFU count. CONCLUSIONS Conventional chlorhexidine scrub and waterless hand rub were superior to a conventional povidone-iodine product in bacterial inhibition. We recommend using conventional chlorhexidine scrub as a standard method for perioperative hand antisepsis. Waterless hand rub may be used if the higher cost is affordable. Infect Control Hosp Epidemiol 2017;38:417-422.
BackgroundAs cancer metastasis is the deadliest aspect of cancer, causing 90% of human deaths, evaluating the molecular mechanisms underlying this process is the major interest to those in the drug development field. Both therapeutic target identification and proof-of-concept experimentation in anti-cancer drug development require appropriate animal models, such as xenograft tumor transplantation in transgenic and knockout mice. In the progression of cancer metastasis, circulating tumor cells (CTCs) are the most critical factor in determining the prognosis of cancer patients. Several studies have demonstrated that measuring CTC-specific markers in a clinical setting (e.g., flow cytometry) can provide a current status of cancer development in patients. However, this useful technique has rarely been applied in the real-time monitoring of CTCs in preclinical animal models.MethodsIn this study, we designed a rapid and reliable detection method by combining a bioluminescent in vivo imaging system (IVIS) and quantitative polymerase chain reaction (QPCR)-based analysis to measure CTCs in animal blood. Using the IVIS Spectrum CT System with 3D–imaging on orthotropic-developed breast-tumor-bearing mice.ResultsIn this manuscript, we established a quick and reliable method for measuring CTCs in a preclinical animal mode. The key to this technique is the use of specific human and mouse GUS primers on DNA/RNA of mouse peripheral blood under an absolute qPCR system. First, the high sensitivity of cancer cell detection on IVIS was presented by measuring the luciferase carried MDA-MB-231 cells from 5 to 5x1011 cell numbers with great correlation (R2 = 0.999). Next, the MDA-MB-231 cell numbers injected by tail vein and their IVIS radiance signals were strongly corrected with qPCR-calculated copy numbers (R2 > 0.99). Furthermore, by applying an orthotropic implantation animal model, we successfully distinguished xenograft tumor-bearing mice and control mice with a significant difference (p < 0.001), whereas IVIS Spectrum-CT 3D–visualization showed that blood of mice with lung metastasis contained more than twice the CTC numbers than ordinary tumor-bearing mice. We demonstrated a positive correlation between lung metastasis status and CTC numbers in peripheral mouse blood.ConclusionCollectively, the techniques developed for this study resulted in the integration of CTC assessments into preclinical models both in vivo and ex vivo, which will facilitate translational targeted therapy in clinical practice.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-017-3419-x) contains supplementary material, which is available to authorized users.
Escherichia coli is the major Gram-negative bacterial pathogen in neonatal meningitis. Outer membrane protein A (OmpA) is a conserved major protein in the E. coli outer membrane and is involved in several host-cell interactions. To characterize the role of OmpA in the invasion of astrocytes by E. coli, we investigated OmpA-positive and OmpA-negative E. coli strains. Outer membrane protein A E44, E105, and E109 strains adhered to and invaded C6 glioma cells 10- to 15-fold more efficiently than OmpA-negative strains. Actin rearrangement, protein tyrosine kinase, and phosphoinositide 3-kinase activation were required for OmpA-mediated invasion by E. coli. In vitro infection of C6 cells and intracerebral injection into mice of the E44 strain induced expression of the astrocyte differentiation marker glial fibrillary acidic protein and the inflammatory mediators cyclooxygenase 2 and nitric oxide synthase 2. After intracerebral infection with E44, all C57BL/6 mice died within 36hours, whereas 80% of mice injected with E44 premixed with recombinant OmpA protein survived. Astrocyte activation and neutrophil infiltration were reduced in brain tissue sections in the mice given OmpA. Taken together, these data suggest that OmpA-mediated invasion plays an important role in the early stage of E.coli-induced brain damage, and that it may have therapeutic use in E. coli meningitis.
Severe acute respiratory syndrome (SARS) is a newly emergent human disease, which requires rapid diagnosis and effective therapy. Among antibody sources, immunoglobulin Y (IgY) is the major antibody found in chicken eggs and can be used as an alternative to mammalian antibodies normally used in research and immunotherapy. In this study, phage-expressing chicken monoclonal scFv antibody was chosen and characterized with phage display antibody technology. Truncated fragments of SARS-CoV spike protein were cloned in pET-21 vector and expressed in BL-21 Escherichia coli (E. coli) cells. After purification, the purity of these recombinant spike proteins was examined on SDS-PAGE and their identity verified with Western blot analysis using anti-his antibodies and sera from convalescent stage SARS-CoV-infected patients. Using these bacteria-derived proteins to immunize chickens, it was found that polyclonal IgY antibodies in the egg yolk and sera were highly reactive to the immunogens, as shown by Western blot and immunocytochemical staining analysis. A phage displaying scFv library was also established from spleen B cells of immunized chicken with 5 x 10(7) clones. After four panning cycles, the eluted phage titer showed a 10-fold increase. In sequence analysis with chicken germline gene, five phage clones reacted, with large dissimilarities of between 31 and 62%, in the complementarity-determining regions, one dominant phage 4S1 had strong binding to fragment Se-e, located between amino acid residues 456-650 of the spike protein and this particular phage had significantly strong binding to SARS-CoV-infected Vero E6 cells. Based on the results, we conclude that generating specific scFv-expressing phage binders with the phage display system can be successfully achieved and that this knowledge can be applied in clinical or academic research.
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