The effect of Annexin A5 overexpression on invasiveness and expression of the genes involved in epithelial-mesenchymal transition of HCT 116 cell line.

Epithelial-to-mesenchymal transition (EMT) plays a critical role in colorectal cancer (CRC) metastasis. In the present study, we evaluated the effects of annexin A5 (ANXA5) overexpression on invasiveness as well as the expression of genes involved in EMT of HCT 116 cell line. PCMV6-AC-IRES-GFP plasmid harboring ANXA5 cDNA was constructed. HCT 116 cell line was transfected with recombinant plasmids using Lipofectamine 3000. Fluorescent microscopy was used to determine the efficiency of plasmid transfection. Cell viability was determined using the MTT assay. HCT 116 cell migration was evaluated using wound healing assay and transwell migration assay. Real-time quantitative polymerase chain reaction (RT-qPCR) was used to measure the expression of genes involved in EMT. The results of RT-qPCR showed overexpression of ANXA5 compared to the control group. ANXA5 overexpression had no significant effects on cell viability but significantly decreased the rate of wound closure in the wound healing assay as well as the number of migrated cells in transwell assay. Furthermore, ANXA5 overexpression decreased the expression of N-cadherin, Snail, Slug, MMP-2, and MMP-9 while the expression of E-cadherin increased following ANXA5 overexpression. However, VEGF expression did not significantly change after ANXA5 overexpression. Results of the present study suggest that ANXA5 overexpression might have inhibitory effects on the metastasis of CRC through modulating the expression of EMT- related genes.

Anti-lung Cancer Marine Compounds: A Review.

Lung cancer is one of the most common and lethal cancers in human beings. Lung cancer has been divided into two major types: small cell lung cancer (SCLC) and non-small cell lung carcinoma (NSCLC). Current drugs suffer from various side effects, and the insufficient efficacy of present treatments creates a desire for better more efficient new drugs. This review compares the diversity of marine-derived bioactive compounds from different marine species. Some of the natural products from marine resources are in different stages of clinical trials. By the way, most of them have been studied in vitro and in vivo. Additionally, in this review, the mechanisms of action of marine-derived anti-lung cancer components on lung cancer cell lines have been reviewed. In addition, considering growing rate and the high costs of cancer research, attention must be paid to some aspects of targeting and developing anti-lung cancer drug. In better words, like the other therapeutic strategies that have their particular challenges and weak points, several challenges about marine-derived anti-lung cancer components which exist for scientists for doing research are explained. Moreover, as the attentions in the field of cancer therapy are focused on designing and developing new anticancer strategies for the treatment of cancer in the future, the application of marine-derived anti-lung cancer components in the field of future cancer therapy and their role in future anticancer strategies are briefly discussed.

The Hydroalcoholic Extract of Saffron Protects PC12 Cells against Aluminum-Induced Cell Death and Oxidative Stress in Vitro.

BACKGROUND: Aluminum (Al) exposure is among the environmental risk factors that may involve in the pathogenesis of neurodegenerative diseases. Oxidative stress has a critical role in the Al-induced toxicity. Saffron is a plant with potent radical scavenging and anti-oxidative properties. This investigation was designed to evaluate the possible protective effects of saffron extract (SE) on aluminum maltolate (Almal)-induced oxidative stress and apoptosis in PC12 cell line. METHODS: In this in vitro study, PC12 cells were divided into four groups including control, Almal (500 µM), Almal+SE (50 µg/ml), and Almal+SE (100 µg/ml). After 48 hours of treatment with Almal in the absence and presence of SE, cell viability and apoptosis were determined using MTT (3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide) assay and Annexin V flow cytometry, respectively. Catalase activity was determined as an index of oxidative stress. Statistical analyses were performed using one-way ANOVA (SPSS version 16.0). P<0.05 was accepted as a statistically significant difference between groups. RESULTS: Almal decreased the PC12 cells viability dose-dependently (IC50=500µM). Co-treatment of 50 and 100 µg/ml of SE with 500 µM of Al increased cell viability to 79% (P=0.04) and 86% (P=0.02) of the control group, respectively. Al also increased PC12 cells apoptosis and catalase activity to 37 and 2.7 folds of those of the control group (P<0.001 and =0.001respectively). 100 µg/ml of SE blunted the effects of Al on the increased cell apoptosis (P=0.02) and changes in the catalase activity (P=0.003). CONCLUSION: SE has protective effects against Al-induced apoptosis and oxidative stress and may possess therapeutic values in the treatment of Al-neurotoxicity.

Segmental Duplications as a Complement Strategy to Short Tandem Repeats in the Prenatal Diagnosis of Down Syndrome.

BACKGROUND: Quantitative fluorescence-polymerase chain reaction (QF-PCR) is an inexpensive and accurate method for the prenatal diagnosis of aneuploidies that applies short tandem repeats (STRs) as a chromosome-specific marker. Despite its apparent advantages, QF-PCR is not applicable in all cases due to the presence of uninformative STRs. This study was carried out to investigate the efficiency of a method based on applying segmental duplications (SDs) in conjunction with STRs as an alternative to stand-alone STR-based QF-PCR for the diagnosis of Down syndrome. METHODS: Fifty amniotic fluid samples from pregnant women carrying Down syndrome fetuses, 9 amniotic fluid samples with 1 or without any informative STR marker (inconclusive), and 100 normal samples were selected from Shiraz, Iran, between October 2015 and December 2016. Analysis was done using an in-house STR-SD-based multiplex QF-PCR and the results were compared. Statistical analysis was performed using MedCalc, version 14. RESULTS: All the normal, Down syndrome, and inconclusive samples were accurately identified by the STR-SD-based multiplex QF-PCR, yielding 100% sensitivity and 100% specificity. Karyotype analysis confirmed all the cases with normal or trisomic results. CONCLUSION: The STR-SD-based multiplex QF-PCR correctly identified all the normal and trisomy 21 samples regardless of the absence of informative STR markers. The STR-SD-based multiplex QF-PCR is a feasible and particularly useful assay in populations with a high prevalence of homozygote STR markers.

The First Case of a Small Supernumerary Marker Chromosome 18 in a Klinefelter Fetus: A Case Report.

Small supernumerary marker chromosomes (sSMCs), or markers, are abnormal chromosomal fragments that can be hereditary or de novo. Despite the importance of sSMCs diagnosis, de novo sSMCs are rarely detected during the prenatal diagnosis process. Usually, prenatally diagnosed de novo sSMCs cannot be correlated with a particular phenotype without knowing their chromosomal origin and content; therefore, molecular cytogenetic techniques are applied to achieve this goal. The present study aimed to characterize an sSMC in a case of Klinefelter syndrome using an in-house microsatellite analysis method and fluorescent in situ hybridization (FISH) technique. Amniotic fluid was collected from a pregnant woman who was considered to have risk factors for trisomy higher than the screening cut-off. Karyotype analysis was followed by the amplification of different microsatellite loci and FISH technique. Karyotype analysis identified a fetus with an extra X chromosome and also an sSMC with unknown identity. Further investigation of the parents showed that the sSMC is de novo. Microsatellite amplification by quantitative fluorescent PCR (QF-PCR) and FISH analysis showed that the sSMC is a derivative of chromosome 18. Eventually, the patient decided to terminate the pregnancy. Here, the first case of the coincidence of sSMC 18 in a Klinefelter fetus is reported.

Sodium valproate ameliorates aluminum-induced oxidative stress and apoptosis of PC12 cells.

OBJECTIVES: According to recent studies, valproate shows some protection against oxidative stress (OS) induced by neurotoxins. Current investigation tried to determine the possible ameliorating effects of sodium valproate (SV) against aluminum (Al)-induced cell death, apoptosis, mitochondrial membrane potential (MMP), and OS in PC12 cells. MATERIALS AND METHODS: In this in vitro study, PC12 cells were treated with different concentrations of aluminum maltolate (Almal) with and without SV (50-400 µM). Cell viability was assessed by MTT assay. To measure quantitatively the effects of SV on Al-induced apoptosis and reactive oxygen species (ROS), flowcytometry using 7AAD/annexin-V and 2', 7'-dichlorofluorescein diacetate staining were employed, respectively. MMP was monitored using the retention of rhodamine 123. Catalase (CAT) activity was assayed by the rate of decomposition of hydrogen peroxide. RESULTS: Exposure of PC12 cells for 48 hr to Almal (125-2000 µM) significantly reduced cell viability (IC50=1090 µM), increased ROS generation and apoptosis, and reduced MMP and CAT activity. SV reduced the Almal-induced cell death and apoptosis. Furthermore, the effects of Almal on ROS generation, catalase activity, and MMP reduction were significantly diminished by SV. CONCLUSION: Data from this study suggest that SV can inhibit Al-induced cell death and apoptosis of PC12 cells via ameliorating OS.

Glycyrrhizic Acid Ameliorates Mitochondrial Function and Biogenesis Against Aluminum Toxicity in PC12 Cells.

Glycyrrhizic acid (GA) is the most effective ingredient in the root of licorice, with important pharmacological effects. We investigate the effects of GA on mitochondrial function and biogenesis in the aluminum toxicity in PC12 cell line. After pretreatment of PC12 cells with different concentrations of GA (5-100 µM), and specific concentration of aluminum maltolate (Almal,1000 µM) for 48 h, cell viability, reactive oxygen species (ROS), mitochondrial membrane potential (MMP), mitochondria mass, cytochrome c oxidase enzyme activity, and the ATP level of the cells were measured. The expression mRNA level of PGC-1α, NRF1, NRF2, and TFAM was confirmed by the real-time PCR quantitative method. The results showed that low concentrations of GA protected Almal-induced cell death in 48 h. It was also observed that GA reduced the ROS production and increased the ATP level. The activity of cytochrome c oxidase enzyme and also decrease of MMP were improved. In addition, GA significantly increased the expression of mitochondrial genes and mass against aluminum toxicity. GA can exert its protective effect against the toxicity of Almal through maintaining mitochondrial function and subsequently increasing energy metabolism and mitochondrial biogenesis. GA as a natural product can be considered as a supplement in neurodegenerative disease.

The Synergistic Effects of Celecoxib and Sodium Valproate on Apoptosis and Invasiveness Behavior of Papillary Thyroid Cancer Cell Line In-vitro.

Metastasis to lymph nodes and distant organs is the main challenge in the treatment of papillary thyroid cancer. In the current investigation, we aimed to evaluate the synergistic effects of celecoxib (CX) and sodium valproate (VPA) against cell survival, invasiveness properties, and expression of metalloproteinase-2 and -9 (MMP-2 and MMP-9) in papillary thyroid cancer cell line, B-CPAP cells. The effect of CX and VPA on B-CPAP cells viability and apoptosis were investigated using MTT assay and annexin V/7-AAD flowcytometry, respectively. The effects of the drugs on invasiveness properties of B-CPAP cells and expression of MMP-2 and MMP-9 were evaluated using transwell assay and real time PCR, respectively. MTT assay showed that CX and VPA decreased viability of B-CPAP cells dose dependently (IC50 32.4µM and 6.8 mM, respectively). Combination of CX (5 µM) and VPA (2.5 and 5 mM) increased apoptosis, and reduced cell migration and invasion of B-CPAP cell, synergistically. Real time PCR results showed that both CX (5 µM) and VPA (2.5 and 5 mM) reduced MMP-2 expression (P < 0.05) but had no significant effects on the expression of MMP-9. Our findings suggest that CX and VPA synergistically increase apoptosis and suppress migration and invasion of B-CPAP cells through inhibition of MMP-2 expression.

The protective effect of sodium benzoate on aluminum toxicity in PC12 cell line.

Sodium benzoate (SB) is one of the food additives and preservatives that prevent the growth of fungi and bacteria. SB has been shown to improve the symptoms of neurodegenerative disease such as Alzheimer's disease. The aim of this study was to evaluate the effect of SB on the cell survival and cellular antioxidant indices after exposure to aluminum maltolate (Almal) in PC12 cell line as a model of neurotoxicity. The cells exposed to different concentrations of SB (0.125 to 3 mg/mL) in the presence of Almal (500 µM) and cell viability, the level of reactive oxygen species (ROS), glutathione content and catalase activity were measured. The results showed that low concentrations of SB caused an increase in the cell survival, but cell viability was reduced in high concentrations. SB could neither prevent the level of ROS production nor change glutathione content. SB (0.5 mg/mL) significantly increased the catalase enzyme activity as compared to the Almal. This study suggested that SB did not completely protect the cell to aluminum-induced free radicals toxicity. Possibly SB improves the symptoms of neurodegenerative disease by other mechanisms.

Protective effects of nimodipine and lithium against aluminum-induced cell death and oxidative stress in PC12 cells.

OBJECTIVES: The role of aluminum (Al) in the pathogenesis of neurodegenerative diseases has been implicated in several studies. However, the exact mechanisms of cytotoxic effects of Al have not been elucidated yet. The aim of this study was to investigate the effect of L-type calcium channel antagonist, nimodipine (NM), and lithium chloride (LiCl) on Al-induced toxicity in PC12 cells. MATERIALS AND METHODS: PC12 cells were treated with Al-maltolate (Almal) in the presence and absence of different concentrations of NM (50-150 µm) and/or LiCl (0.5-1.0 mM) for 48 hr. Cell viability, apoptosis, and catalase (CAT) activity, a marker of oxidative stress, were then measured using MTT, flow cytometry and enzyme assay, respectively. RESULTS: The results showed that Almal, dose dependently induced cell death, apoptosis and CAT activity in the PC12 cells. NM significantly increased cell viability and decreased apoptosis and CAT activity of Almal-treated cells in a dose dependent mode. LiCl reduced CAT activity and increased cell viability in Almal-treated cells, without significant effect on apoptosis (P=0.74). CONCLUSION: These findings suggest that NM and Li may have benefits in the prevention of Al-induced cytotoxicity through decreasing oxidative stress.

Alpha synuclein protein is involved in Aluminum-induced cell death and oxidative stress in PC12 cells.

Increased expression and aggregation of α-synuclein (α-syn) protein plays a critical role in mediating the toxic effects of a number of neurodegenerative substances including metals. Thus, knockdown expression of α-syn is proposed as a possible modality for treatment of Parkinson disease (PD). Aluminum (Al) is a neurotoxic metal that contributes to pathogenesis of PD. The aim of this study was to investigate the role of α-syn protein in mediating Al-induced toxicity in PC12 cells. Specific α-syn small interference RNA (siRNA) was applied to knockdown the expression of α-syn protein in PC12 cells. The effects of different concentrations of Al-maltolate (Almal) were then evaluated on cell viability and oxidative stress in the α-syn downregulated cells. The results showed that Almal dose dependently induced apoptosis and increased malondialdehyde (MDA) and catalase activity in PC12 cells. Downregulation of α-syn protein significantly increased cell viability and decreased oxidative markers in Almal-treated cells. These findings suggest that α-syn protein may mediate Al-induced apoptosis and oxidative stress in PC12 cells.