Elevated Caspase-3 Research Articles

MicroRNA-153-3p enhances cell radiosensitivity by targeting BCL2 in human glioma

BackgroundGlioma is the most prevalent malignant tumor in human central nervous systems. Recently, the development of resistance to radiotherapy in glioma patients markedly vitiates the therapy outcome. MiR-153-3p has been reported to be closely correlated with tumor progression, but its effect and molecular mechanism underlying radioresistance remains unclear in glioma.MethodsThe expression of miR-153-3p was determined in radioresistant glioma clinical specimens as well as glioma cell lines exposed to irradiation (IR) using quantitative real-time PCR. Cell viability, proliferation and apoptosis were then evaluated by MTT assay, colony formation assay, Flow cytometry analysis and caspase-3 activity assay in glioma cells (U87 and U251). Tumor forming was evaluated by nude mice model in vivo. TUNEL staining was used to detect cell apoptosis in nude mice model. The target genes of miR-153-3p were predicted and validated using integrated bioinformatics analysis and a luciferase reporter assay.ResultsHere, we found that miR-153-3p was down-regulated in radioresistant glioma clinical specimens as well as glioma cell lines (U87 and U251) exposed to IR. Enhanced expression of miR-153-3p promoted the radiosensitivity, promoted apoptosis and elevated caspase-3 activity in glioma cells in vitro, as well as the radiosensitivity in U251 cell mouse xenografs in vivo. Mechanically, B cell lymphoma-2 gene (BCL2) was identified as the direct and functional target of miR-153-3p. Moreover, restoration of BCL2 expression reversed miR-153-3p-induced increase of radiosensitivity, apoptosis and caspase-3 activity in U251 cells in vitro. In addition, clinical data indicated that the expression of miR-153-3p was significantly negatively associated with BCL2 in radioresistance of glioma samples.ConclusionsOur findings suggest that miR-153-3p is a potential target to enhance the effect of radiosensitivity on glioma cells, thus representing a new potential therapeutic target for glioma.

Acrofolione A and B, acetophenone dimers from Acronychia pendunculata, induce an apoptotic effect on human NALM-6 pre-B cell leukaemia cells.

We investigated the apoptotic activities of acrofolione A (1) and B (2) isolated from Acronychia pedunculata against a human pre-B cell leukaemia cell line (NALM-6) to explore the apoptosis-related signalling molecules targeted by 1 and 2. The apoptosis effects of 1 and 2 in NALM-6 cells were investigated by TUNEL staining, annexin V, mitochondria membrane potential and caspase 3/7 activity. We carried out a protein array to explore the signalling molecules involved in apoptosis comprehensively. Acrofolione A (1) suppressed the growth of NALM-6, K562 and HPB-ALL cells (IC50 16.7 ± 1.9, 17.9 ± 0.3 and 10.1 ± 0.2 μm, respectively) more effectively than acrofolione B (2). Both compounds time-dependently increased the number of NALM-6 cells with abnormal nuclei, and increased the number of annexin V-positive cells and decreased the mitochondrial membrane potential of NALM-6 cells. Acrofolione A (1) markedly elevated caspase 3/7 activity and increased the number of TUNEL-positive cells. Cells treated with either compound showed enhanced expression of cleaved PARP and cleaved caspase 3 and 7, and reduced survivin protein levels. Acrofolione A (1) and B (2) may be useful in the treatment of various types of leukaemia.

Effects of liquid ionic silver concentration on caspase-3 and p53mt expressions in the oral mucosal epithelium of Wistar rats

Background: Silver, especially oxidized silver, has been used as a medicine considered to have bactericidal properties. In the present day, ionic silver (Ag+) is also used in the manufacture of cosmetics, socks, food containers, detergents, sprays and other products to prevent the spread of germs. Unfortunately, ionic silver is assumed to be toxic not only to bacteria, but also to humans and the environment. Therefore, it is essential to identify the optimum dosage of ionic silver considered safe by investigating the effects of ionic silver concentration on cell death through activation of mutant p-53 expression by caspase 3 in the oral epithelium. Purpose: This research aimed to analyze the effects of concentrated liquid ionic silver (Ag+) on caspase-3 and mutant p53 expression in the oral mucosal epithelium. Methods: This research constituted a laboratory-based experimental study with posttest-only design. The research subjects consisted of 28 Wistar rats, divided into four treatment groups, namely; KK (with Aquadest), KP 1 (with 5% liquid ionic silver), KP 2 (with 10% liquid ionic silver) and KP 3 (with 15% liquid ionic silver). Each rat was then treated orally with 0.5ml of liquid ionic silver at fixed concentrations twice a day for seven days. The Wistar rats were then terminated and their tissue samples processed by means of histopathological and immunohistochemical staining examination. The monoclonal Caspase-3 and mutant p53 expressions in each group were evaluated with the data being tabulated and analyzed statistically. Results: Mutant p53 expression was also found in the control group. Moreover, the higher the concentration of liquid ionic silver, the greater the elevated Caspase-3 and mutant p53 expressions. Conclusion: The concentration of liquid ionic silver plays an important role in elevating Caspase-3 and mutant p53 expressions.

Marsdenia tenacissima extract induces apoptosis and suppresses autophagy through ERK activation in lung cancer cells

BackgroundMarsdenia tenacissima is an herb medicine which has been utilized to treat malignant diseases for decades. The M. tenacissima extract (MTE) shows significant anti-proliferation activity against non-small cell lung cancer (NSCLC) cells, but the underlying mechanisms remain unclear. In this study, we explored the potential anti-proliferation mechanisms of MTE in NSCLC cells in relation to apoptosis as well as autophagy, which are two critical forms to control cancer cell survival and death.MethodsThe proliferation of H1975 and A549 cells was evaluated by MTT assay. Cell apoptosis was assessed by Annexin V and PI staining, Caspase 3 expression and activity. Autophagy flux proteins were detected by Western blot with or without autophagy inducer and inhibitor. Endogenous LC3-II puncta and LysoTracker staining were monitored by confocal microscopy. The formation of autophagic vacuoles was measured by acridine orange staining. ERK is a crucial molecule to interplay with cell autophagy and apoptosis. The role of ERK on cell apoptosis and autophagy influenced by MTE was determined in the presence of MEK/ERK inhibitor U0126.ResultsThe significant growth inhibition and apoptosis induction were observed in MTE treated NSCLC cells. MTE induced cell apoptosis coexisted with elevated Caspase 3 activity. MTE also impaired autophagic flux by upregulated LC3-II and p62 expression. Autophagy inducer EBSS could not abolish the impaired autophagic flux by MTE, while it was augmented in the presence of autophagy inhibitor Baf A1. The autophagosome–lysosome fusion was blocked by MTE via affecting lysosome function as evidenced by decreased expression of LAMP1 and Cathepsin B. The molecule ERK became hyperactivated after MTE treatment, but the MEK/ERK inhibitor U0126 abrogated autophagy inhibition and apoptosis induction caused by MTE, suggested that ERK signaling pathways partially contributed to cell death caused by MTE.ConclusionOur results demonstrate that MTE caused apoptosis induction as well as autophagy inhibition in NSCLC cells. The activated ERK is partially associated with NSCLC apoptotic and autophagic cell death in response to MTE treatment. The present findings reveal new mechanisms for the anti-tumor activity of MTE against NSCLC.

Costimulation of Murine Osteoblasts with Interferon-γ and Tumor Necrosis Factor-α Induces Apoptosis through Downregulation of Bcl-2 and Release of Cytochrome c from Mitochondria.

During chronic inflammation from diseases, such as periodontal disease, the proinflammatory cytokines interferon-gamma (IFNγ) and tumor necrosis factor-α (TNFα) alter bone remodeling. To elucidate the underlying molecular mechanisms, we investigated the effect of IFNγ and TNFα on the proliferation and survival of clonal MC3T3-E1 mouse osteoblasts. We found that although IFNγ or TNFα alone affected cell growth and survival only marginally, costimulation with both synergistically inhibited cell growth and reduced cell viability. The diminished cell viability was due to apoptosis, as indicated by increased TUNEL staining and elevated caspase 3, 8, and 9 activities. Western blot also showed that costimulation with IFNγ and TNFα elicited cytochrome c release and downregulated B cell lymphoma 2 (Bcl-2) expression without affecting Bcl-2-associated X (Bax) protein expression. Furthermore, stable Bcl-2 overexpression significantly alleviated cell death following costimulation. Collectively, these results suggested that IFNγ and TNFα elicited osteoblast apoptosis via cytochrome c release from damaged mitochondria, caspase activation, and Bcl-2 downregulation.

Long Noncoding RNA SNHG1 Promotes Neuroinflammation in Parkinson’s Disease via Regulating miR-7/NLRP3 Pathway

Parkinson’s disease (PD) is the second most common neurodegenerative disorders. Neuroinflammation plays an important role in the pathogenesis of PD. Long noncoding RNA small nucleolar RNA host gene 1 (SNHG1) was elevated in the brain specimens of PD patients and MPP+-treated SH-SY5Y cells. The expression of mouse Snhg1 and miR-7 was firstly determined in lipopolysaccharide (LPS)-induced BV2 cells. The role and mechanism of SNHG1 in the neuroinflammation of PD were investigated using gain- and loss-of function approaches both in vitro and in vivo. Snhg1 expression was elevated, whereas miR-7 reduced in LPS-induced BV2 cells. Upregulation of Snhg1 elevated, and Snhg1 knockdown suppressed LPS-induced BV2 microglial activation and inflammation. miR-7 reversed, while anti-miR-7 further enhanced the effects of Snhg1 on BV2 cells. Furthermore, we found that Snhg1 functioned as a competing endogenous RNA for miR-7 to regulate nod-like receptor protein 3 (NLRP3) expression, leading to the activation of NLRP3 inflammasome. In the microglial culture supernatant transfer model, knockdown of Snhg1 or NLRP3 in LPS-stimulated BV2 cells inhibited primary neurons from apoptosis and elevated caspase-3 activity. Additionally, Snhg1 was increased in MPTP-induced PD mouse models. Downregulation of Snhg1 elevated miR-7 expression, suppressed the activation of microglia and NLRP3 inflammasome as well as dopaminergic neuron loss in the midbrain substantia nigra pars compacta in MPTP-treated mice. In conclusion, our study suggests that SNHG1 promotes neuroinflammation in the pathogenesis of PD via modulating miR-7/NLRP3 pathway.

Abstract 25: Neuroligin 4X: A neural cell adhesion molecule, in breast cancer

Abstract Purpose: Neuroligins are neural cell adhesion molecules that are implicated in heterotopic cell adhesion. Our previous studies using a selection from a combinatorial random peptide library against breast and pancreatic cancer cell lines identified several peptides mimicking neuroligin (NLGN-1, 3 and NLGN4X respectively). In this study, we investigated the expression, relevance and functional significance of Neuroligin 4X in human breast cancer. Methods: NLGN4X expression data for all breast cancer cell lines in the Cancer Cell Line Encyclopedia were analyzed. The correlation between NLGN4X levels and clinicopathologic parameters were established within Oncomine datasets. As a proof-of- principle, we evaluated survival by generating Kaplan–Meier plots using publicly available microarray datasets. To examine the expression of NLGN4X, immunohistochemistry of breast cancer tissue arrays was conducted. Additionally, flow cytometry and immunofluorescence staining were performed to investigate NLGN4X expression in MDA-MB-231, MCF-7, and MCF-7 treated with TGFβ. To observe the effect of NLGN4X gene knockdown, MDA-MB-231 cells were transfected with NLGN4X-specific siRNA. NLGN4X gene expression was analyzed by RT-PCR, western blot and flow cytometry. Post transfection, wound healing and cell viability assays were performed to determine the effect of NLGN4X knockdown on migration and proliferation. Apoptotic outcomes were examined through detection of caspase activation and Annexin V-FITC methods by using flow cytometry. Results: NLGN4X showed abundant expression in breast cancer tissues. The evaluation of bioinfomatic datasets revealed that NLGN4X expression was higher in triple negative breast cancer (TNBC) and in metastatic tissues. Interestingly, high NLGN4X expression correlated with a decrease in relapse free-survival in TNBC. RT-PCR, flow cytometry and immunofluorescence validated that NLGN4X expression was high in MDA-MB-231 as well as MCF-7 TGFβ, which suggests that NLGN4X is associated with the mesenchymal phenotype. Knockdown of NLGN4X expression by siRNA significantly decreased cell proliferation(P<0.001) and migration(P<0.05) in MDA-MB-231 in conjunction with the induction of apoptosis as determined by annexin staining, elevated caspase 3/7 and cleaved PARP by flow cytometry. Conclusion: Our findings suggest that NLGN4X could represent a novel biomarker and therapeutic target. This is the first study to link the expression of neuronal cell adhesion molecules, neuroligins, to breast cancer. We intend to continue the investigation of the role of NLGN4X to elucidate the mechanistic role of this adhesion protein in breast cancer progression and metastasis. Citation Format: Henry J. Henderson, Karanam Balasubramanyam, Rajeev Samant, Komal Vig, Shree R. Singh, Clayton Yates, Deepa Bedi. Neuroligin 4X: A neural cell adhesion molecule, in breast cancer [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 25.

Pituitary Adenylate Cyclase Activating Polypeptide (PACAP1–38) Exerts Both Pro and Anti-Apoptotic Effects on Postnatal Retinal Development in Rat

PACAP1–38, a ubiquitous and multifunctional regulator has been in the focus of neurotoxicity research due to its impressive neuroprotective potential. Although the literature extensively demonstrated its repressive effect on the apoptotic machinery in neurodegenerative models, there is a striking absence of analysis on its role in normal development. We performed quantitative analyses on caspase activity in developing retina upon 100, 50, 25 or 1 pmol intravitreal PACAP1–38 injection from postnatal day 1 (P1) through P7 in Wistar rats. Retinas were harvested at 6, 12, 18, 24 or 48 h post-injection. Apoptotic activity was revealed using fluorescent caspase 3/7 enzyme assay, western blots and TUNEL assay. Unexpectedly, we found that 100 pmol PACAP1–38 increased the activity of caspase 3/7 at P1 and P5 whereas it had no effect at P7. At P3, as a biphasic effect, PACAP1–38 repressed active caspase 3/7 at 18 h post-injection while increased their activity in 24 h post-injection. Amounts, smaller than 100 pmol, could not inhibit apoptosis whereas 50, 25 or 1 pmol PACAP1–38 could evoke significant elevation in caspase 3/7 activity. TUNEL-positive cells appeared in the proximal part of inner nuclear as well as ganglion cell layers in response to PACAP1–38 treatment. The fundamental novelty of these results is that PACAP1–38 induces apoptosis during early postnatal retinogenesis. The dose as well as stage-dependent response suggests that PACAP1–38 has a Janus face in apoptosis regulation. It not only inhibits development-related apoptosis, but as a long-term effect, facilitates it.

Nickel-induced neurodegeneration in the hippocampus, striatum and cortex; an ultrastructural insight, and the role of caspase-3 and α-synuclein

Human overexposure to nickel (Ni) emanating from the increasing application of Ni compounds in modern technology is a major public health concern. Nickel has been shown to be teratogenic, immunotoxic, genotoxic and carcinogenic. The current knowledge on Ni neurotoxicity is still relatively limited. We have previously demonstrated that Ni treatment alters cognitive and locomotor behaviors, induces oxidative stress and neurodegeneration in brains of rats. In this study, we examine the ultrastructural changes to neurons in the hippocampus, striatum and cortex of the brain following Ni treatment, as well as attempt to delineate the roles for caspase-3 and α-synuclein in Ni-induced neurodegeneration. Rats were treated with either saline, 10 or 20 mg/kg of nickel chloride for 4 weeks via oral gavage. Electron microscopy analysis revealed ultrastructural alterations in neurons of the hippocampus, striatum and cortex following Ni treatment. Mitochondria structural integrity within neurons were markedly compromised. We also detected elevated caspase-3 activity in hippocampus and striatum, as well as overexpression of α-synuclein in the cortex following Ni treatment. Our study demonstrates that mitochondria are a key target in Ni-induced neurodegeneration. Additionally, we implicate apoptotic pathway via caspase-3 action as the executioner and perturbation of α-synuclein expression in Ni-induced neurodegeneration.

Migration and invasion in B16-F10 mouse melanoma cells are regulated by Nrf2 inhibition during treatment with ionizing radiation.

Nuclear factor erythroid 2-related factor 2 (Nrf2) serves a critical role in carcinogenesis. The present study examined the effect of Nrf2 on the proliferation and invasion of melanoma cells that were treated with ionizing radiation. B16-F10 mouse melanoma cells were exposed to various doses of ionizing radiation for different time periods. Small interfering (si)RNAs targeting Nrf2 were transfected into B16-F10 cells, and cell proliferation, invasion and apoptosis were detected by Transwell, MTT or western blot assays. The expression of Nrf2 and its downstream heme oxygenase 1 (HO-1) was analyzed by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and western blotting. HO-1 activity was also examined. Ionizing radiation stimulated Nrf2 expression, increased caspase-3 expression, and reduced the viability, migration and invasion of B16-F10 mouse melanoma cells. Transfection with Nrf2 siRNA was able to inhibit Nrf2 and HO-1 expression in B16-F10 mouse melanoma cells that were treated by ionizing radiation. Inhibition of Nrf2 further reduced cell viability, invasion and migration, and elevated caspase-3 expression in B16-F10 mice melanoma cells that were treated by ionizing radiation. In summary, treatment with ionizing radiation was able to stimulate Nrf2 expression and regulate cell viability, invasion and migration of B16-F10 cells. A combination of Nrf2 knockdown and ionizing radiation treatment exerted a synergistic effect on migration, invasion and apoptosis in B16-F10 murine melanoma cells.

Silica nanoparticle-induced oxidative stress and mitochondrial damage is followed by activation of intrinsic apoptosis pathway in glioblastoma cells.

IntroductionRecently, the focus of oncological research has been on the optimization of therapeutic strategies targeted at malignant diseases. Nanomedicine utilizing silicon dioxide nanoparticles (SiNPs) is one such strategy and is rapidly developing as a promising tool for cancer diagnosis, imaging, and treatment. Nevertheless, little is known about the mechanisms of action of SiNPs in brain tumors.Materials and methodsHere, we explored the effects of 5–15 nm SiNPs in the human glioblastoma cell line LN229. In this respect, MTT assays, microscopic observations, flow cytometry analyses, and luminescent assays were performed. Moreover, RT-qPCR and Western blot analyses were done to determine gene and protein expressions.ResultsWe demonstrated that SiNPs triggered evident cytotoxicity, with microscopic observations of the nuclei, annexin V–fluorescein isothiocyanate/propidium iodide staining, and elevated caspase 3/7 activity, suggesting that SiNPs predominantly induced apoptotic death in LN229 cells. We further showed the occurrence of oxidative stress induced by enhanced reactive oxygen-species generation. This effect was followed by deregulated expression of genes encoding the antioxidant enzymes SOD1, SOD2, and CAT, and impaired mitochondria function. SiNP- induced mitochondrial dysfunction was characterized by membrane-potential collapse, ATP depletion, elevated expression of BAX, PUMA, and NOXA with simultaneous downregulation of BCL2/BCL2L1, and activation of caspase 9. Moreover, RT-qPCR and Western blot analyses demonstrated increased levels of the endoplasmic reticulum stress markers GRP78, GRP94, and DDIT3, as well as strongly increased expressions of the IL1B and COX2 genes, suggesting activation of endoplasmic reticulum stress and a proinflammatory response.ConclusionsAltogether, our data indicate that in LN229 cells, SiNPs evoke cell death via activation of the intrinsic apoptosis pathway and suggest that other aspects of cellular function may also be affected. As such, SiNPs represent a potentially promising agent for facilitating further progress in brain cancer therapy. However, further exploration of SiNP long-term toxicity and molecular effects is necessary prior to their widespread application.

Inhibitory Eefects of the novel tyrosine kinase inhibitor BGJ398 against human leukemic cell line KG-1 cells

目的探讨针对FGFR1的酪氨酸激酶抑制剂BGJ398对人急性髓系白血病细胞株KG-1细胞的影响及可能的作用机制。方法CCK-8法检测BGJ398对KG-1细胞增殖的影响；流式细胞术Annexin Ⅴ/PI双重染色法检测BGJ398对细胞凋亡的影响；RQ-PCR检测细胞凋亡相关基因的表达；Western blot法检测凋亡相关蛋白、FGFR1OP2-FGFR1融合蛋白及信号通路分子磷酸化水平的表达变化。结果BGJ398能有效抑制KG-1细胞增殖，抑制率呈剂量依赖性升高，并能诱导细胞凋亡。BGJ398作用KG-1细胞48 h后，与对照组比较凋亡相关基因Bcl-2表达下降（0.342±0.054对1.026±0.165，t=3.94，P=0.017），caspase-3表达上调（0.456±0.189对1.008±0.091，t=16.44，P<0.001），差异均有统计学意义。与对照组相比，BGJ398作用组caspase-3活化蛋白表达增加，同时Bcl-2蛋白表达下调；FGFR1OP2-FGFR1融合蛋白水平及AKT、S6K1磷酸化水平下调，差异均有统计学意义（P值均<0.01），但ERK磷酸化表达水平无明显改变。结论BGJ398能有效抑制KG-1细胞增殖并诱导细胞凋亡，其机制可能与抑制FGFR1表达、下调Bcl-2水平、促进Caspase-3活化及抑制AKT和S6K磷酸化有关。

Bio-Effects of TiO2 Nanoparticles on Human Colorectal Cancer and Umbilical Vein Endothelial Cell Lines

Background:Due to the possible biomedical potential of nanoparticles, titanium dioxide nanoparticles (TiO2 NPs) have received great attention in cancer research. Although selectivity of cytotoxicity with TiO2 NPs in various cells is clinically significant comparisons of cancer and non-cancer cells have been limited. Therefore, we here studied exposure to TiO2 NPs in colorectal cancer cells (CRCs) and human umbilical vein endothelial cells (HUVECs).Methods:After characterization of TiO2 NPs, culture and treatment of cells (HCT116, HT29 and HUVEC), viability was assessed by MTT assay and in terms of morphological features. Acridine orange (AO) and propidium iodide (PI) assays were carried out to estimate the incidence of apoptosis. The RT-PCR method was also employed to evaluate the expression of P53, Bax, Bcl-2 and Caspase 3.Results:Exposure to increasing concentrations of TiO2 NPs enhanced overall cell survival of HCT116 cells and reduced the Bcl-2 and Caspase 3 expression while the ratio of Bax/Bcl-2 was down-regulated. TiO2 NPs at 400 and 50 μg/ml concentrations suppressed cell proliferation and induced apoptosis of HT29 cells and also up-regulated P53 and Bax at the mRNA level, enhanced the Bax/Bcl-2 ratio and eventually up-regulated Caspase 3 mRNA. Although, inhibition of cell proliferation in HUVECs was seen at 200 and 400 μg/ml TiO2 NPs, it was not marked.Conclusion:TiO2 NPs have selective bio-effects on exposed cells with dose- and cell-dependent influence on viability. Cell proliferation in HCT116 as a metastatic colorectal cancer cell line appeared to be stimulated via multiple signaling pathways, with promotion of apoptosis in less metastatic cells at 50 and 400 μg/ml concentrations. This was associated with elevated P53, Bax and Caspase 3 mRNA and reduced Bcl-2 expression. However, TiO2 NPs did not exert any apparent significant effects on HUVECs as hyperproliferative angiogenic cells.

The regulatory role of SLP-2 and mechanism on CCBE1 gene expression in rectal carcinoma and adjacent lymphatic tube tissues.

The incidence of rectal carcinoma (RC) has been increasing recently, and becomes the second most common digestive tumors besides gastric cancer, with a rise in the incidence of RC in younger populations. The early diagnosis and treatment are thus critical for the improvement of survival rate and life quality of patients. Stomatin-like protein 2 (SLP-2) is a type of membrane factor, which is generally found highly expressed in various tumors. Collagen and calcium-binding EGF domain (CCBE1) belongs to lymphatic tube genesis factor. The regulatory role of SLP-2 gene on CCBE1 expression in RC tumor and adjacent lymphatic tube tissues, however, has not been studied. 52 RC patients were recruited, and tumor and adjacent lymphatic tube tissues were collected. Real-time PCR, western blotting and immunohistochemistry (IHC) staining were used to analyze SLP-2 and CCBE1 expressions. Human lymphatic endothelial cells (LECs) were cultured in vitro and were assigned to control, scramble, and SLP-2 siRNA group. MTT assay was used to detect cell proliferation, while caspase 3 activity was detected. SLP-2 and CCBE1 levels were significantly elevated in tumor lymphatic tissues, compared to that in adjacent tissues. Statistically positive correlation between SLP-1 and CCBE2 was found (p<0.05). The downregulation of SLP-2 by siRNA inhibited cell proliferation, elevated caspase3 activity, and decreased CCBE1 expression (p<0.05 compared to control group). SLP-2 is up-regulated in RC lymphatic tissues, and is positively correlated with the level of CCBE1, which provides the academic the basis for the development of medicine targeting SLP-2 in the anti-rectal carcinoma therapy.

Neuroligin 4X overexpression in human breast cancer is associated with poor relapse-free survival.

The molecular mechanisms involved in breast cancer progression and metastasis still remain unclear to date. It is a heterogeneous disease featuring several different phenotypes with consistently different biological characteristics. Neuroligins are neural cell adhesion molecules that have been implicated in heterotopic cell adhesion. In humans, alterations in neuroligin genes are implicated in autism and other cognitive diseases. Until recently, neuroligins have been shown to be abundantly expressed in blood vessels and also play a role implicated in the growth of glioma cells. Here we report increased expression of neuroligin 4X (NLGN4X) in breast cancer. We found NLGN4X was abundantly expressed in breast cancer tissues. NLGN4X expression data for all breast cancer cell lines in the Cancer Cell Line Encyclopedia (CCLE) was analyzed. Correlation between NLGN4X levels and clinicopathologic parameters were analyzed within Oncomine datasets. Evaluation of these bioinfomatic datasets results revealed that NLGN4X expression was higher in triple negative breast cancer cells, particularly the basal subtype and tissues versus non-triple-negative sets. Its level was also observed to be higher in metastatic tissues. RT-PCR, flow cytometry and immunofluorescence study of MDA-MB-231 and MCF-7 breast cancer cells validated that NLGN4X was increased in MDA-MB-231. Knockdown of NLGN4X expression by siRNA decreased cell proliferation and migration significantly in MDA-MB-231 breast cancer cells. NLGN4X knockdown in MDA-MB-231 cells resulted in induction of apoptosis as determined by annexin staining, elevated caspase 3/7 and cleaved PARP by flow cytometry. High NLGN4X expression highly correlated with decrease in relapse free-survival in TNBC. NLGN4X might represent novel biomarkers and therapeutic targets for breast cancer. Inhibition of NLGN4X may be a new target for the prevention and treatment of breast cancer.

Phenylpropanoids isolated from Piper sarmentosum Roxb. induce apoptosis in breast cancer cells through reactive oxygen species and mitochondrial-dependent pathways

The aim of the present study is to isolate bioactive compounds from the roots of Piper sarmentosum and examine the mechanism of action using human breast cancer cell line (MDA-MB-231). Bioassay guided-fractionation of methanolic extract led to the isolation of asaricin (1) and isoasarone (2). Asaricin (1) and isoasarone (2) had significant cytotoxicity towards MDA-MB-231. MCF-10A (human normal breast epithelial cells) cells are less sensitive than MDA-MB-231, but they respond to the treatment with the same unit of measurement. Both compounds increase reactive oxygen species (ROS), decrease mitochondrial membrane potential (MMP) and enhance cytochrome c release in treated MDA-MB-231 cells. Isoasarone (2) markedly elevated caspase -8 and -3/7 activities and caused a decline in nuclear NF-κB translocation, suggesting extrinsic, death receptor-linked apoptosis pathway. Quantitative PCR results of MDA-MB-231 treated with asaricin (1) and isoasarone (2) showed altered expression of Bcl-2: Bax level. The inhibitory potency of these isolates may support the therapeutic uses of these compounds in breast cancer.

Effects of cyclin D1 gene silencing on cell proliferation, cell cycle, and apoptosis of hepatocellular carcinoma cells.

This study aims to investigate the effects of Cyclin D1 silencing on cell cycle, cell proliferation, and apoptosis of hepatocellular carcinoma cells (HCC). Cells were divided into the blank group, negative control group (HCC cells transfected with control shRNA), Cyclin D1 shRNA group (HCC cells transfected with Cyclin D1 shRNA), and the normal group (human normal liver L-02 cells). Expressions of Cyclin D1, Caspase-3, Bcl-2, and C-myc were detected by RT-qPCR and Western blotting. Cell proliferation was detected by Cell Counting Kit-8. Cell cycle and apoptosis were detected by flow cytometry. Tumor xenograft in nude mice was performed to detect in vivo tumorigenesis. HCC tissues and HCC cells exhibited elevated expression levels of Cyclin D1. Cyclin D1 expression levels was found to be correlated with tumor size and tumor staging. Compared with the normal group, the blank group showed enhanced cell proliferation, a reduction in the amount of cells in G0/G1 phase, increased number cells in S and G2/M phase, reduced apoptosis, elevated expressions of Cyclin D1, Bcl-2, and C-myc, decreased Caspase-3 activity and significant tumorigenicity. In comparison with the blank group, the Cyclin D1 shRNA group revealed weakened cell proliferation, reduced cells in S and G2/M phase, increased cells in G0/G1 phase, increased Annexin V positive cell ratio, decreased expression of Cyclin D1, Bcl-2, and C-myc, elevated Caspase-3 activity and inhibited tumorigenicity. In conclusion, Cyclin D1 gene silencing suppresses cell proliferation and inhibits cell apoptosis, which may be a new target approach in the treatment and management for HCC.

Triptolide induces Sertoli cell apoptosis in mice via ROS/JNK-dependent activation of the mitochondrial pathway and inhibition of Nrf2-mediated antioxidant response.

Triptolide (TP), an oxygenated diterpene, has a variety of beneficial pharmacodynamic activities but its clinical applications are restricted due to severe testicular injury. This study aimed to delineate the molecular mechanisms of TP-induced testicular injury in vitro and in vivo. TP (5-50000 nmol/L) dose-dependently decreased the viability of TM4 Sertoli cells with an IC50 value of 669.5-269.45 nmol/L at 24 h. TP (125, 250, and 500 nmol/L) dose-dependently increased the accumulation of ROS, the phosphorylation of JNK, mitochondrial dysfunction and activation of the intrinsic apoptosis pathway in TM4 cells. These processes were attenuated by co-treatment with the antioxidant N-acetyl cysteine (NAC, 1 mmol/L). Furthermore, TP treatment inhibited the translocation of Nrf2 from cytoplasm into the nucleus as well as the expression of downstream genes NAD(P)H quinone oxidoreductase1 (NQO1), catalase (CAT) and hemeoxygenase 1 (HO-1), thus abrogating Nrf2-mediated defense mechanisms against oxidative stress. Moreover, siRNA knockdown of Nrf2 significantly potentiated TP-induced apoptosis of TM4 cells. The above results from in vitro experiments were further validated in male mice after oral administration of TP (30, 60, and 120 mg·kg-1·d-1, for 14 d), as evidenced by the detected indexes, including dose-dependently decreased SDH activity, increased MDA concentration, altered testicle histomorphology, elevated caspase-3 activation, apoptosis induction, increased phosphorylation of JNK, and decreased gene expression of NQO1, CAT and HO-1 as well as nuclear protein expression of Nrf2 in testicular tissue. Our results demonstrate that TP activates apoptosis of Sertoli cells and injury of the testis via the ROS/JNK-mediated mitochondrial-dependent apoptosis pathway and down-regulates Nrf2 activation.

IL-17A Enhances Microglial Response to OGD by Regulating p53 and PI3K/Akt Pathways with Involvement of ROS/HMGB1.

Cerebral ischemia-reperfusion injury (IRI) has a complex pathogenesis, and interleukin-17 (IL-17) is a newly identified class of the cytokine family that plays an important role in ischemic inflammation. An oxygen-glucose deprivation (OGD) model showed that IL-17A expression was significantly up-regulated in microglial cells. After IL-17A siRNA transfection, the inhibition of proliferation, and the increased apoptosis in microglial cells, induced by OGD/reperfusion, was improved, and the elevation of Caspase-3, Caspase-8, Caspase-9, and poly ADP ribose polymerase (PARP) activities was inhibited. Mass spectrometry demonstrated that IL-17A functioned through a series of factors associated with oxidative stress and apoptosis and regulated Caspase-3 activity and apoptosis in microglial cells via the p53 and PI3K/Akt signaling pathways. IL-17A, HMGB1, and ROS were regulated mutually to exhibit a synergistic effect in the OGD model of microglial cells, but the down-regulation of IL-17A or HMGB1 expression did not completely inhibit the production of ROS. These findings demonstrated that ROS might be located upstream of IL-17A and HMGB1 so that ROS can regulate HMGB1/IL-17A expression to affect the p53 and PI3K/Akt signaling pathways and therefore promote the occurrence of apoptosis in microglial cells. These findings provide a novel evidence for the role of IL-17A in ischemic cerebral diseases.

The ceramide pathway is involved in the survival, apoptosis and exosome functions of human multiple myeloma cells in vitro.

Multiple myeloma (MM) is characterized by the clonal proliferation of malignant plasma cells and refractoriness to traditional therapies. It has been shown that exosomes are involved in modulating the progression and the metastasis of cancers through microRNAs (miRs). Ceramide is a type of sphingolipid; the ceramide pathway of exosomal secretion has been shown to affect the apoptosis of cancer cells. But the role of this pathway in MM cell function, exosome function and miR regulation remains unknown. In this study, we showed that C6 ceramide (an exogenous ceramide supplement, 1.25-40 μmol/L) dose-dependently inhibited the proliferation and promoted the apoptosis in human MM OPM2 cell line, which were associated with elevated caspase 3/9 and PARP cleavage. We also found that C6 ceramide (5-20 μmol/L) dose-dependently stimulated exosome secretion and increased exosomal levels of tumor-suppressive miRs (miR 202, miR 16, miR 29b and miR 15a). Of note, exosomes from C6 ceramide-treated OPM2 cells could influence the proliferation and apoptosis of the recipient OPM2 cells, which correlated with increased tumor-suppressive exosomal miRs. In contrast, GW4869 (a ceramide inhibitor, 5-20 μmol/L) exerted the opposite effects on the regulation of MM function, exosome secretion and miR levels in MM exosomes. However, exosomes from GW4869-treated OPM2 cells had no effect on these miRs and the survival of targeted OPM2 cells. Taken together, our findings reveal that the ceramide pathway modulates MM survival, probably directly via the caspase pathway and indirectly via exosomal miR mechanisms.