Evaluation of Apoptotic Activity of Modified Carvacrol and Anticancer Peptide Against AGS Gastric Cancer Cell Line

IntroductionTreating stomach cancer remains a major challenge. There have been many reports of the positive effect of modified carvacrol and anti-cancer peptides on gastric cancer. The main purpose of this study was to determine and design a fusion modified carvacrol and anti-cancer peptide against AGS gastric cancer cell line using flowcytometry technique.MethodDetermination of cell lethality was conducted with different concentrations of the modified carvacrol and anti-cancer peptides against AGS cell line by MTT technique. Treatment of AGS cells with different concentrations of components to evaluate the apoptosis of AGS cells was performed by flow cytometry. In this study used. The MTT test performed according to protocol with different concentrations of components.ResultsThe results showed that the percentage of cells treated with modified carvacrol and anti-cancer peptides at a concentration of 80μg/ml with total apoptosis was 46.91% and increased by 36.25% compared to untreated cells.ConclusionDue to the anticancer properties of modified carvacrol with anticancer peptide, the above combination can be used to treat and induction apoptotic activity against gastric cancer cell line.

Lactate induces aberration in the miR-30a–DBF4 axis to promote the development of gastric cancer and weakens the sensitivity to 5-Fu

Background Gastric cancer (GC) is one of the most common malignancies, molecular mechanism of which is still not clear. Aberrant expression of tumor-associated genes is the major cause of tumorigenesis. DBF4 is an important factor in cancers, although there is yet no report on its function and molecular mechanism in GC. Methods The expression of DBF4 in tumor tissues or cells of GC was detected by qRT-PCR and western blotting. Gastric cancer cell line MGC-803 and AGS were transfected with DBF4 siRNA or overexpression vector to detect the function of DBF4 in proliferation, migration and the sensitivity to 5-Fu with CCK-8 assay, colony formation assay, transwell assay, and wound healing assay. miR-30a was found to be the regulator of DBF4 by online bioinformatics software and confirmed with qRT-PCR, western blot and dual-luciferase reporter assays. Results In our study, increased expression of DBF4 in GC tissues was first identified through The Cancer Genome Atlas (TCGA) and later confirmed using specimens from GC patients. Furthermore, functional experiments were applied to demonstrate that DBF4 promotes cell proliferation and migration in GC cell lines, moreover weakens the sensitivity of MGC803 and AGS cells to 5-Fu. We further demonstrated that miR-30a showed significantly lower expression in GC cells and inhibited the expression of DBF4 through 3ʹ-UTR suppression. Furthermore, rescue experiments revealed that the miR-30a-DBF4 axis regulated the GC cell proliferation, migration and the sensitivity to 5-Fu. The important composition in tumor microenvironment, lactate, may be the primary factor that suppressed miR-30a to strengthen the expression of DBF4. Conclusions Taken together, our study was the first to identify DBF4 as a regulator of cell proliferation and migration in GC. Furthermore, our study identified the lactate-miR-30a-DBF4 axis as a crucial regulator of tumor progression and the tumor sensitivity to 5-Fu, which maybe serve useful for the development of novel therapeutic targets.

Ethyl acetate extract of Elephantopus mollis Kunth induces apoptosis in human gastric cancer cells

Background Gastric cancer is one of the most leading causes of cancer death worldwide. Therefore, treatment studies have been being conducted, one of which is screening of novel agents from medicinal herbs. Elephantopus mollis Kunth (EM) belonging to Asteraceae family is a perennial herb with several therapeutic properties including anticancer activity. However, the effect of this species on gastric cancer has not been reported yet. In this study, cytotoxicity of different EM crude extracts was investigated on AGS gastric cancer cell line. Besides, the effects of extract on nuclear morphology, caspase-3 activation, and gene expression were also explored. Results The results showed that ethyl acetate extract exhibited a remarkably inhibitory ability (IC50 = 27.5 μg/ml) on the growth of AGS cells, while causing less toxicity to normal human fibroblasts. The extract also induced apoptotic deaths in AGS cells as evidenced by cell shrinkage, formation of apoptotic bodies, nuclear fragmentation, caspase-3 activation, and the upregulation of BAK and APAF-1 pro-apoptotic genes related to mitochondrial signaling pathway. Specifically, BAK and APAF-1 mRNA expression levels showed 2.57 and 2.71-fold increases respectively. Conclusions The current study not only proved the anti-gastric cancer activity of EM ethyl acetate extract but also proposed its molecular mechanism. The extract could be a potential candidate for further investigation.

Gastric cancer stem cells survive in stress environments via their autophagy system

Cancer stem cells (CSCs) play an important role in the progression of carcinoma and have a high potential for survival in stress environments. However, the mechanisms of survival potential of CSCs have been unclear. The aim of this study was to clarify the significance of autophagy systems of CSCs under stress environments. Four gastric cancer cell line were used. Side population (SP) cells were sorted from the parent cells, as CSC rich cells. The expression of stem cell markers was examined by RT-PCR. The viability of cancer cells under starvation and hypoxia was evaluated. The expression level of the autophagy molecule LC3B-II was examined by western blot. The numbers of autophagosomes and autolysosomes were counted by electron microscope. SP cells of OCUM-12 showed a higher expression of stem cell markers and higher viability in starvation and hypoxia. Western blot and electron microscope examinations indicated that the autophagy was more induced in SP cells than in parent cells. The autophagy inhibitor significantly decreased the viability under the stress environments. These findings suggested that Cancer stem cells of gastric cancer might maintain their viability via the autophagy system. Autophagy inhibitors might be a promising therapeutic agent for gastric cancer.

Pulsed Magnetic Fields Cause hes1 and hsa-Circ-0068530 Expression Changes in Gastric Cancer Cell Line and Human Normal Fibroblast Cell Line

Background: In recent years, the relationship between cancer cells and electromagnetic radiation has received much attention. Objectives: The present study aimed to evaluate the effects of different intensities of electromagnetic fields on gastric cancer cell lines (AGS). Methods: After preparing AGS and Hu02 (normal) cell lines, they were exposed to magnetic flux densities of 0.25, 0.5, 1, and 2 millitesla (mT) for 18 h. The cell viability was studied by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The expression levels of hes1 and hsa-circ-0068530 RNAs were studied by the quantitative Real-time-PCR technique. Results: The inhibition of gastric cancer cell line growth was observed under the influence of electromagnetic fields at different intensities. However, they did not affect the viability of normal cells. A sharp increase in the expression of hes1 and hsa-circ-0068530 genes was observed in normal cells exposed to 2 mT electromagnetic fields. Conclusions: In general, it can be concluded that the effect of electromagnetic fields on gastric cancer cells depends on their intensity. Magnetic flux densities of 0.25 and 0.5 mT had anti-cancer effects and magnetic flux density of 2 mT showed carcinogenic effects.

Evaluation of Apoptotic Activity of Protein Fusion Enterocin A- Pyocin R-Lactocin, Bacteriocins and Specific Ligand of Gastric Cancer Cell Line (AGS)

Conventional antimicrobial and anti-cancer treatments, including chemotherapy, surgery, radiation therapy, etc., have all been associated with antimicrobial resistance (AMR) and side effects on healthy cell damage. Recently, bacteriocins have emerged as a promising option in antimicrobial and anticancer therapies.In this study, EntA-PynR-Lac gene sequence was obtained using bioinformatics software. The designed fusion gene was synthesized and cloned into the pET22b expression vector and transferred to the engineered E. coli BL21 bacterium for expression of the recombinant protein. The three-dimensional structures and stability of the designed recombinant protein were evaluated. Confirmation and purification of the recombinant protein was performed by Western blotting and nickel column chromatography and determination of cell lethality of different concentrations of the recombinant protein against AGS cell line by MTT technique was conducted. Treatment of cells with different concentrations of protein to evaluate the apoptosis of AGS cells was performed by flow cytometry. The results showed that the percentage of cells treated with recombinant protein at a concentration of 80µg/ml with total apoptosis was 46.91% and increased by 36.25% compared to untreated cells. Due to the anti-apoptotic properties of fusion protein, it can be used to inhibit cancer cells as a therapeutic supplement and prevention.

Silencing of Long Non-coding RNA HOTAIR Suppresses Reverses Epithelial-Mesenchymal Transition in AGS Gastric Cancer Cell Line by Downregulation of Fibronectin 1 and Claudin-4

Background: Gastric cancer is the second reason for cancer mortality worldwide, with a high capacity for metastasis. Long non-coding RNAs (lncRNAs) are recently described as lengthy transcripts with no open reading frame. The lncRNAs play an important role in critical cellular and molecular pathways, including cell cycle, growth, differentiation, and apoptosis. Therefore, it is not surprising that abnormal expression of lncRNAs may be involved in human cancers. The HOX antisense intergenic RNA (HOTAIR) is a highly cited lncRNAs whose altered expression has been reported in a variety of human cancers such as gastric cancer. Epithelial to mesenchymal transition (EMT) is a cellular route in which an epithelial phenotype of the cells can be changed into the mesenchymal state. The signaling pathways involved in EMT are related to cancer metastasis and recurrence of gastric cancer. Methods: The present study aimed to investigate the effect of HOTAIR gene silencing on expression levels of fibronectin 1 (FN1) and claudin-4 (CLDN4) genes, two important markers of EMT, in AGS cellular model of gastric cancer. The AGS cells were exposed to the HOTAIR-specific siRNA for 48 hours. The extracted RNAs were subjected to complementary DNA synthesis and real-time PCR. Data were analyzed using 2−ΔΔCt method. Cells with no siRNA treatment were considered control set. The P-value < 0.05 was considered statistically significant. Results: The observed data showed that the expression levels of two EMT markers FN1 and CLDN4, were significantly decreased after HOTAIR silencing. Conclusions: This study demonstrates that HOTAIR can regulate the EMT signaling pathway through critical EMT factors like FN1 and CLDN4 transcripts. However, a long way remains to apply this finding in therapeutic approach, and further experiments are needed.

Autophagy induced by H. pylori VacA regulated the survival mechanism of the SGC7901 human gastric cancer cell line

Background Vacuolating cytotoxin (VacA) is an important virulence factor of Helicobacter pylori (H. pylori). It was previously believed that VacA can trigger the cascade of apoptosis on mitochondria to lead to cell apoptosis. Recently, it was found that VacA can induce autophagy. However, the molecular mechanism by which VacA induces autophagy is largely unknown. Objective We aimed to explore the molecular mechanism of autophagy induced by H. pylori in gastric cancer cells and the effect of autophagy on the survival of gastric cancer cells. Methods The autophagy of human gastric cancer cell line SGC7901 was detected by Western blot and RT-PCR in the treatment of VacA protein of H. pylori. The relationship between autophagy and reactive oxygen species (ROS) in the proliferation of gastric cancer cells were studied by gene expression silences (siRNA) and CM-H2DCFDA (DCF) staining. Results The results showed that VacA protein secreted by H. pylori in the supernatant stimulated autophagy in SGC7901 cells. After VacA protein treatment, the mRNA expressions of BECN1, ATG7 and PIK3C3, were up-regulated. ATG7 silencing by siRNA inhibited VacA-induced autophagy. Furthermore, our data demonstrated that VacA protein increased ROS levels. Addition of the antioxidant N-acetyl-l-cysteine (NAC) suppressed the levels of ROS, leading to inhibition of autophagy. Conclusions H. pylori VacA is a key toxin that induces autophagy by increased ROS levels. And our findings demonstrated that VacA significantly inhibited proliferation in SGC7901 cells.