Increased Apoptosis Cell Research Articles

Vitamin D ameliorates asthma-induced lung injury by regulating HIF-1α/Notch1 signaling during autophagy.

Herein, we aimed to determine the effect of vitamin D (Vit D) and underlying mechanisms on asthma‐induced lung injury via regulation of HIF‐1α/Notch1 (hypoxia‐inducible factor 1 alpha/neurogenic locus notch homolog protein 1) signaling during autophagy. We established an asthma mouse model using respiratory syncytial virus (RSV) nasal drop combined with ovalbumin (OVA) atomization. Mice were treated with different Vit D concentrations. Pathological changes and cell apoptosis were examined using hematoxylin–eosin (HE) staining and TUNEL (terminal deoxynucleotidyl transferase‐mediated deoxyuridine triphosphate (dUTP) nick end‐labeling) assay, respectively. Additionally, periodic acid–Schiff (PAS) and Masson's trichrome staining solutions were used to examine changes in lung tissue. Immunofluorescence determined LC 3B (microtubule‐associated protein 1 light chain 3B) expression in lung tissues, whereas western blotting and immunohistochemistry were used to evaluate other proteins, including HIF‐1α and Notch1. Compared with the normal group, the asthma model group exhibited pathological lung tissue deterioration, elevated fibrosis, increased apoptosis cell numbers, and upregulated autophagy. Vitamin D supplementation ameliorated pathological changes and fibrosis in the lung tissue. Furthermore, Vit D treatment significantly suppressed apoptotic cell numbers and autophagy while enhancing the HIF‐1α/Notch1 pathway. Given the HIF‐1α/Notch1 agonistic activity, Vit D treatment inhibited apoptosis cell numbers, which were increased following asthma‐induced upregulation of autophagy. Vitamin D improved asthma‐induced lung tissue injury by suppressing autophagy via regulation of HIF‐1α/Notch1 signaling in vivo.

Assessment of oral ciprofloxacin impaired gut barrier integrity on gut bacteria in mice

Gut bacteria and gut barrier plays important roles in body homeostasis. Ciprofloxacin (CPFX) is widely used to treat bacterial infections. However, whether high dosage of CPFX has side effects on gut barrier integrity is still unclear. Our results indicated that the High CPFX treatment (1 mg/ml) caused weight loss, nervousness, anorexia, and increased apoptosis cells in gut, but less influence was observed in the Low CPFX group (0.2 mg/ml). Meanwhile, the High CPFX treatment impaired tight junction molecules Ocln/ZO-1 level and down-regulated antibacterial genes expression (reg3γ, pla2g2α and defb1). Further, the High CPFX treatment increased pro-inflammatory cytokine IL-1β in intestinal tract, decreased IL-17A of duodenum but increased IL-17A of colon at day 37. In addition, the gut bacterial diversity and richness behaved significantly loss regarding CPFX treatment, especially in the High CPFX group during the experiment. Indole exhibited sharply decline in both Low and High CPFX groups at day 7, and the High CPFX mice needed longer time on restoring indole level. Meanwhile, CPFX treatment strongly decreased the concentrations of butyric acid and valeric acid at day 1. Correlation analysis indicated that the linked patterns between the key bacteria (families Bacteroidales_S247, Ruminococcaceae and Desulfovibrionaceae) and metabolites (indole and butyric acid) were disturbed via the CPFX treatment. In conclusion, the High CPFX treatment impaired the gut barrier with the evidence of reduced expression of tight junction proteins, increased apoptosis cells and inflammatory cells, decreased the bacterial diversity and composition, which suggesting a proper antibiotic-dosage use should be carefully considered in disease treatment.

Abstract 3468: Antitumor effect by inhibition of PI3K/AKT/mTOR and RAS/MAPK pathways in human oral squamous cell carcinoma cells

Abstract Molecular target drugs are used in the treatment of a variety of malignant tumors, but this approach to developing novel therapies for oral squamous cell carcinoma (OSCC) has lagged behind the progress seen for other cancers. The PI3K/AKT/mTOR and RAS/MAPK pathways are important pathways for the growth and survival of cancer cells. These pathways also have been activated such as AKT and ERK overexpression in OSCC. In this study, we investigated the antitumor effect of PI3K/mTOR inhibitor, NVP-BEZ235 and MEK1/2 inhibitor, Trametinib, in human OSCC cells. First, we confirmed the inhibitory effect of NPV-BEZ235 and Trametinib in PI3K/AKT/mTOR and RAS/MAPK pathways by Western blotting. NPV-BEZ235 and Trametinib were reduced phosphorylation of AKT, p70S6K, and MEK1/2 in OSCC cells. Next, we examined the anti-proliferative effect of NVP-BEZ235 and Trametinib using 4 human OSCC cell lines by WST-8 assay. NVP-BEZ235 and Trametinib significantly inhibited the growth of all OSCC cells in a dose-dependent manner in vitro. Further, we investigated potential synergistic effects of inhibiting both the PI3K/AKT/mTOR and RAS/MAPK pathways, by treating with NVP-BEZ235 and Trametinib. The anti-proliferative effect of this combination was measured in OSCC cells by calculating the Combination Index. Combination treatment with NVP-BEZ235 and Trametinib showed a synergistic effect on the cell growth of all OSCC cells in vitro. Subsequently, we analyzed the effect of NBP-BEZ235 and Trametinib on the cell cycle of green fluorescent protein (GFP)-SAS cells by flow cytometry. Treated with NVP-BEZ235 showed no enhanced effect compared to control. Treatment of Trametinib resulted in the decrease in percentage of cells in G2 phase and increase in G1 phase. This indicated that the anti-proliferative effect of Trametinib was due to G1 arrest. Furthermore, treatment of Trametinib increased apoptosis cells in GFP-SAS cells. Next, we assessed the effect of NVP-BEZ235 and Trametinib on the in vivo growth of GFP-SAS cells. GFP-SAS tumor-bearing mice were treated with single, combination, or vehicle by oral daily. Treatment with NVP-BEZ235 and Trametinib significantly reduced the size of tumors compared with vehicle control. However, combination treatment showed no synergistic effect compared to single treatment. Finally, we investigated the gene expression changes with combination treatment using microarray analysis. The suppression of PI3K/AKT/mTOR and RAS/MAPK pathways caused overexpression of genes related to cell cycle and DNA-repair. These results suggest that activation of PI3K/AKT/mTOR and RAS/MAPK pathways support the growth of OSCC cells and NVP-BEZ235 and Trametinib may be a useful therapeutic strategy for OSCC. Citation Format: Norihiko Tokuzen, Koh-ichi Nakashiro. Antitumor effect by inhibition of PI3K/AKT/mTOR and RAS/MAPK pathways in human oral squamous cell carcinoma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3468.

P2.03a-064 Inhibition and Exploitation of Aldehyde Dehydrogenase 1 as a Cancer Stem Cell Marker to Overcome Cisplatin Resistant NSCLC: Topic: Clinical Trials

The root of therapeutic resistance is hypothesized to be the presence a rare CSC population within the tumor population which survives chemotherapeutic treatment and has the potential to recapitulate a heterogenic tumor. Aldehyde dehydrogenase 1 (ALDH1) is involved the catalytic conversion of vitamin A (retinol) to retinoic acid and has been identified as a CSC marker in a number of solid malignancies. FACS analysis of an isogenic panel of matched parent (PT) and cisplatin resistant (CisR) NSCLC cell lines identified ALDH1 as a promising CSC-marker associated with cisplatin resistance across NSCLC histologies. The H460 CisR subline was separated by FACS into ALDH1-positive and negative subpopulations and subcutaneously injected into NOD/SCID mice to assess tumor initiation and growth. ALDH1 was inhibited in vitro within the cell lines using two pharmacological ALDH1 inhibitors, DEAB and disulfiram, alone and in combination with cisplatin. Cell lines were treated in vitro with retinol and all-trans retinoic acid (ATRA) to exploit the vitamin A/retinoic acid axis in which ALDH1 is involved. The CisR sublines showed significantly greater ALDH1 activity relative to their PT counterparts. In vivo subcutaneous injection of ALDH1-positive and negative subpopulations revealed no significant difference in tumor initiation or growth rate. ALDH1 inhibition in combination with cisplatin significantly decreased clonogenic and proliferative competencies and increased apoptosis cell death compared to cisplatin alone. Vitamin A supplementation and ATRA treatment in combination with cisplatin showed similar re-sensitizing effects. This pharmacological CSC depletion in conjunction with cisplatin treatment resulted in re-sensitization of cisplatin resistant cells to the cytotoxic effects of cisplatin. These data suggest vitamin A supplementation or the addition of ATRA or an ALDH1 inhibitor to the cisplatin-based chemotherapeutic regimen may be of clinical benefit in overcoming tumor recurrence and cisplatin resistance.

Preliminary investigation of methylation status of microRNA-124a in spinal cords of rat fetuses with congenital spina bifida

Objective: We investigated the expression of microRNA-124a and its methylation status in the spinal cords of rats with congenital spina bifida versus rats with normal fetuses.Methods: Real-time quantitative reverse transcription-polymerase chain reaction was used to compare the expression of microRNA-124a in the spinal cords of 42 rats with all-trans retinoic acid induced congenital spina bifida and 42 rats with normal fetuses. The DNA methylation status in the promoter region of miRNA-124a was detected using methylation specific-PCR.Results: Compared with rats with normal fetuses, expression of microRNA-124a was significantly decreased in rats with congenital spina bifida fetuses. The percentages of spinal cords with DNA hypermethylation in the microRNA-124a promoter were 81% and 14% in the congenital spina bifida and normal control groups, respectively. The difference was statistically significant. Further apoptosis testing revealed increased apoptosis cell numbers in the congenital spina bifida samples. Meanwhile, the phosphorylated mitogen-activated protein kinase protein expression level dramatically decreased in the congenital spina bifida samples.Conclusion: Aberrant DNA methylation was responsible for down-regulation of microRNA-124a by regulating the mitogen-activated protein kinase pathway, suggesting that microRNA-124a is a potential diagnostic biomarker in congenital spina bifida.

Novel Bcl‐2 Inhibitors: Discovery and Mechanism Study of Small Organic Apoptosis‐Inducing Agents

Apoptosis as a novel target for cancer chemotherapy has generated an intense demand for new apoptosis-inducing agents. The newly revealed role of protein families involved in the apoptosis pathway, and resistance to cytotoxic therapies have opened new avenues for the development of novel anticancer strategies. We have established a novel strategy to rapidly obtain protein-targeted, instead of conventional DNA-targeted, apoptosis inducers as antitumor leads. First, a novel organic non-DNA intercalative compound S1 (8-oxo-3-thiomorpholin-4-yl-8H-acenaphtho[1,2-b]pyrrole-9-carbonitrile, M(W) = 331) was found with an IC50 of 10(-7)-10(-8) microM against diverse cancer cell lines. Further biological evaluation demonstrated that it was an apoptosis-inducer both in vivo and in vitro. The treatment of hydroperitoneum hepatoma cells (H22 cell line) with S1 at various concentrations (from 0.01 to 10 microM) for 24 h triggered these cells to enter the apoptosis process. The antitumor efficiency was also tested in the H22 xenotransplant models in mice. At a dosage of 0.3 mg kg(-1), S1 exhibited significant antitumor activity with a much longer survival time, a decrease in tumor size, and increased apoptosis cells in tumor tissue. More importantly, studies of the molecular mechanism of apoptosis induction by S1 revealed that S1 inactivated the Bcl-2 protein by binding to it, depolarizing the mitochondrial membrane, and then activating caspase 9, followed by caspase 3. Finally, structure-based virtual modification was performed by computer modeling. As a result, a derivative, S2 (8-oxo-3-[(thienylmethyl)amino]-8H-acenaphtho[1,2-b]pyrrole-9-carbonitrile, M(W) = 341) was identified that possessed a lower binding energy to Bcl-2, and demonstrated better antitumor potency, even on the Bcl-2-overexpressing human acute myeloid leukemia (HL-60) cells (IC50 = 1.3 microM) in vitro. S1 and S2 are the well-defined Bcl-2 inhibitors that give us a promising platform for the development of new therapeutic agents.