M2 Macrophages Mediate the Resistance of Gastric Adenocarcinoma Cells to 5-Fluorouracil through the Expression of Integrin ß3, Focal Adhesion Kinase, and Cofilin.

Tumor microenvironment components dictate the growth and progression of various cancers. Tumor-associated macrophages are the most predominant cells in TME and play a major role in cancer invasiveness. Gastric cancer is one of the most common cancers in Asia, and recently, various cases of resistance to fluorouracil treatment have been reported. In this study, we investigated the role of alternatively activated macrophages in the resistance of AGS gastric cancer cells to fluorouracil. THP-1 cells were polarized using recombinant human IL-4, then were cocultured with AGS cells treated with fluorouracil. Cell viability, Western blot, immunofluorescence, and cell invasion were performed for this investigation. Our results demonstrated that polarized macrophages initiated the survival of treated AGS cells and induced the resistance in AGS by upregulating the expression of integrin ß3, focal adhesion protein (FAK), and cofilin proteins. These results reveal that integrin ß3, focal adhesion protein (FAK), and cofilin proteins are potential targets for the improvement of fluorouracil efficacy in gastric cancer treatment.

Induction of apoptosis and G1 phase cell cycle arrest by Aster incisus in AGS gastric adenocarcinoma cells.

In recent decades, various bioactive compounds from plants have been investigated for their potential use in the treatment of diseases in humans. Aster incisus extract (AIE) is the extract of a common plant that is mostly found in Asia. It has traditionally been used for medicinal purposes in South Korea. In this study, we evaluated the potential anticancer effects of a methanolic extract of Aster incisus in a normal human cell line (HaCaT keratinocytes) and in 4 different types of human cancer cell lines (A549, lung cancer; Hep3B, liver cancer; MDA­MB­231, breast cancer; and AGS, gastric cancer). The HaCaT, A549, Hep3B, MDA­MB­231 and AGS cells were treated with various concentrations of AIE and following treatment, cell survival was evaluated. Additional analyses, such as WST-1 assay, western blot analysis, DAPI staining, flow cytometry, immunofluorescence staining and wound healing assay were performed to elucidate the mechanisms and pathways involved in the cell death induced by AIE. Treatment with AIE induced morphological changes and considerably reduced the viability of the both normal and cancer cell lines. Further analysis of the AGS gastric cancer cells revealed that AIE led to the induction of apoptosis and a high accumulation of cells in the G1 cell phase following treatment with AIE in a dose-dependent manner. The results also revealed that AIE successfully suppressed the migration of the AIE-treated AGS cells. The results of western blot analysis indicated that AIE increased the expression of pro-apoptotic proteins, particularly Bid, Bad, Bak, cytochrome c, apoptosis inducing factor (AIF), cleaved caspase­3, -8 and -9 and cleaved poly(ADP-ribose) polymerase (PARP). Additionally, AIE decreased the expression of the anti-apoptotic proteins, Bcl-2 and Bcl-xL. On the whole, the findings of this study demonstrate that AIE induces apoptosis through the activation of the caspase­dependent pathway mediated by the mitochondrial pathway and by arresting the cell cycle in AGS cells.

Anti-Inflammatory Effects of Aster incisus through the Inhibition of NF-κB, MAPK, and Akt Pathways in LPS-Stimulated RAW 264.7 Macrophages.

Aster incisus is a common flower found in almost all regions of South Korea. In the current study, we investigated the potential antioxidant and anti-inflammatory properties of the Aster incisus methanol extract in LPS-stimulated RAW 264.7 cells. We analyzed the phytochemicals contained in the extract by GC-MS. GC-MS results showed that the Aster incisus extract contains 9 known compounds. Later on, DPPH assay, WST-1 assay, nitric oxide (NO) assay, Western blot, and RT-PCR were conducted to investigate the anti-inflammatory effects of the extract. Our WST-1 assay results revealed that Aster incisus did not affect the viability of all tested cell lines up to a concentration of 200 µg/ml; therefore, lower concentrations (50 µg/ml and 150 µg/ml) were used for further assays. Aster incisus scavenged DPPH and inhibited the production of NO. Aster incisus also reduced significantly the production of inflammation-related enzymes (iNOS, Cox-2) and cytokines (TNFα, IL-1ß, and IL-6) and the gene expression of the proinflammatory cytokines. Additionally, further Western blot results indicated that Aster incisus inhibited the expression of p-PI3K, p-IκBα, p-p65 NF-κB, p-ERK1/2, p-SAPK/JNK, and p-Akt. Our results demonstrated that Aster incisus suppressed the expression of the inflammation mediators through the regulation of NF-κB, MAPK, and Akt pathways.

Cyperus amuricus induces G1 arrest and apoptosis through endoplasmic reticulum stress and mitochondrial signaling in human hepatocellular carcinoma Hep3B cells.

ETHNOPHARMACOLOGY RELEVANCE: Cyperus amuricus (C. amuricus), belongs to the family Cyperaceae, was used to exert wound healing, diuretic, astringent and other intestinal problems in oriental medicine. However, only a few studies have reported its anticancer activities. AIM OF THE STUDY: In this study, we determined the activity of C. amuricus on ER stress-induced cell death and G1 cell cycle arrest in human hepatocellular carcinoma (HCC) Hep3B cells. MATERIALS AND METHODS: The in vitro cell proliferation assay of C. amuricus was tested on Hep3B and human embryonic kidney HEK293 cells. Subsequently, the cell cycle distribution in the indicated stages using flow cytometric analysis, the expression of cell cycle-related proteins by western blot analysis and immunofluorescence detection of p21CIP1/WAF1 were determined for the comprehensive identification of cell cycle arrest in Hep3B cells. The effect of C. amuricus on the expression of apoptosis-related proteins in Hep3B cells was also performed by western blot analysis. Furthermore, induction of the ER stress mediators in C. amuricus-treated Hep3B cells were observed by western blot analysis, intracellular Ca2+ mobilization assay and immunofluorescence detection of caspase-12. RESULTS: C. amuricus strongly exhibited cytotoxic activity on Hep3B cells, but not on HEK293 cells. C. amuricus affected the phosphorylation levels of endoplasmic reticulum sensors and increased the expression of GRP78/BiP and CHOP, resulting in the accumulation of unfolded proteins in the ER and triggering the unfolded protein response. These changes occurred by the elevation of intracellular Ca2+ concentrations, which contributed to ER stress-induced apoptosis in C. amuricus-treated Hep3B cells. C. amuricus also coordinated the stimulation of ER chaperones, which initiated G1 cell cycle arrest through the induction of CDKIs and the inhibition of cyclins and CDKs. Furthermore, C. amuricus triggered apoptosis through the activation of mitochondrial-dependent pathway in Hep3B cells. CONCLUSIONS: Our results suggest that C. amuricus is an effective apoptosis inducing agent for Hep3B cells via the G1 arrest, ER stress and mitochondrial mediated apoptotic pathways.

The herbal medicine Cyperus amuricus inhibits proliferation of human hepatocellular carcinoma Hep3B cells by inducing apoptosis and arrest at the G0/G1 cell cycle phase.

Cyperus amuricus (C. amuricus) is one of the most common herbs in Oriental folk medicine for exerting astringent, diuretic, wound healing and other intestinal problems. However, little is known about the molecular mechanism of C. amuricus on anticancer activity. In the present study, the underlying mechanism of the anticancer effect of C. amuricus were elucidated. The methyl alcohol extract from the whole plant of C. amuricus exhibited cytotoxicity against Hep3B cells, but not against A549 and HaCaT cells. Consistent with an acceleration of the sub-G1 phase, downregulation of cdc25A, cyclin D1 and cyclin E, CDK4 and 2 as well as E2F-1, phospho-Rb, with concomitant of upregulation of p21CIP1/WAF1, p27KIPI and p16INK4a proteins, as evidenced by the appearance of cell cycle arrest, were detected in C. amuricus-treated Hep3B cells. Additionally, the sequential activation of various caspases (cleaved caspase-8, -9, -3, -7 and -6, and cleaved PARP) and the changed expression of other proteins related to the apoptosis pathway were observed after C. amuricus exposure. An increment in the pro-apoptotic proteins (Bim, tBid, Bax and Bak) and a reduction of anti-apoptotic protein (Bcl-2) regulate Hep3B cell death by controlling the permeability of mitochondrial membranes and the release of cytochrome c from mitochondria into the cytosol with Apaf-1 after C. amuricus treatment. This is the first study indicating the potential of C. amuricus as a complementary agent for prevention and treatment of human liver cancer.

The Anti-inflammatory Effect of Gnaphalium affine Through Inhibition of NF-κB and MAPK in Lipopolysaccharide-Stimulated RAW264.7 Cells and Analysis of Its Phytochemical Components.

Gnaphalium affine is an annual herbaceous plant that is used as a traditional medicine in some Latin American and Asian countries. However, systematic studies on its anti-inflammatory activity and signaling pathways have not yet been reported. In this study, we investigated the anti-inflammatory effect of G. affine methanol extract in lipopolysaccharide (LPS)-stimulated RAW 264.7 murine macrophage cells and fractioned the methanol extract into hexane, chloroform, ethyl acetate (EtOAc), butyl alcohol (BuOH), and distilled water (DW) by measuring the generation of nitric oxide (NO). G. affine inhibited the generation of NO and prostaglandin E2. The chloroform-soluble fraction most effectively inhibited LPS-stimulated NO production. We also examined the cytotoxicity of G. affine in three normal cell lines: RAW264.7, HEK293, and HaCaT. Cell viability assays showed that the methanol extract and chloroform-soluble fraction of G. affine had no cytotoxic effect on normal cell lines. The expression of pro-inflammatory mediators was also investigated. Western blotting and immunofluorescence showed that G. affine reduces the expression of iNOS, COX-2, and MAPKs, as well as activation of NF-κB in LPS-stimulated RAW264.7 cells. RT-PCR showed that G. affine also negatively regulates inflammatory cytokines at the gene expression level. Taken together, G. affine exerts its anti-inflammatory activity through inhibition of NO generation as a result of inhibiting NF-κB and MAPKs-related inflammatory signaling pathways. In addition, the result of GC-MS analysis revealed the presence of nineteen different types of constituents including guaiacol in the chloroform-soluble fraction of G. affine.

Induction of the endoplasmic reticulum stress and autophagy in human lung carcinoma A549 cells by anacardic acid.

Anacardic acid (AA, 2-hydroxy-6-pentadecylbenzoic acid), a constituent of the cashew-nut shell, has a variety of beneficial effects on the treatment of cancer and tumors. However, the fact that AA induces ER stress and autophagy in cancer cell is not known. We investigated the effect of AA on ER-stress and autophagy-induced cell death in cancer cells. Because of our interest in lung cancer, we used the non-small cell lung adenocarcinoma A549 cells treated with 3.0 µg/ml of AA for this research. In this research we found that AA induces intracellular Ca(2+) mobilization and ER stress. AA induced the ER stress-inducing factors, especially IRE1α, and the hallmarks of UPR, Grp78/Bip and GADD153/CHOP. AA inhibited the expression of p-PERK and its downstream substrate, p-elF2α. We also demonstrated that AA induces autophagy. Up-regulation of autophagy-related genes and the appearance of autophagosome in transfected cells with green fluorescent protein (GFP)-LC3 and GFP-Beclin1 plasmids showed the induction of autophagy in AA-treated A549 cells. The morphological analysis of intracellular organelles by TEM also showed the evidence that AA induces ER stress and autophagy. For the first time, our research showed that AA induces ER stress and autophagy in cancer cells.

Anacardic acid induces mitochondrial-mediated apoptosis in the A549 human lung adenocarcinoma cells.

Anacardic acid (AA) is a constituent of the cashew nut shell and is known as an inhibitor of nuclear factor-κB (NF-κB). We investigated the cytotoxicity of AA on cancer cells and more experiments to reveal the cell death mechanism focused on A549 lung adenocarcinoma cells for our interest in lung cancer. To examine the molecular mechanism of cell death in AA treated A549 cells, we performed experiments such as transmission electron microscopy (TEM), western blot analysis, fluorescence-activated cell sorting (FACS), genomic DNA extraction and staining with 4',6-diamidino-2-phenylindole (DAPI). For the first time we revealed that AA induces caspase-independent apoptosis with no inhibition of cytotoxicity by pan-caspase inhibitor, Z-VAD-fmk, in A549 cells. Our results showed the possibility of mitochondrial-mediated apoptosis through the activation of apoptosis-inducing factor (AIF) and an intrinsic pathway executioner such as cytochrome c. This study will be helpful in revealing the cell death mechanisms and in developing potential drugs for lung cancer using AA.

Prospective impact of 5-FU in the induction of endoplasmic reticulum stress, modulation of GRP78 expression and autophagy in Sk-Hep1 cells.

Hepatocellular carcinoma (HCC) is one of the most aggressive malignant diseases and is highly resistant to conventional chemotherapy. Therefore, HCC requires more effective prevention and treatment strategies. 5-fluorouracil (5-FU) remains the most widely used chemotherapeutic drug for the treatment of gastrointestinal, breast, head and neck, and ovarian cancers. In pursuit of a novel effective strategy, we have evaluated the potential of 5-FU to promote endoplasmic reticulum (ER) stress and autophagy in Sk-Hep1 HCC cells. We found that 5-FU profoundly induces ER stress in Sk-Hep1 cells and upregulates p53 and activates CHOP/GADD153 and caspase-12. Activation of CHOP/GADD153 and caspase-12 promotes mitochondrial cell death in Sk-Hep1 cells followed by ER stress. Changes in calcium homeostasis and the protein folding machinery cause stress in the ER, leading to apoptotic cell death. Stress in the ER activates autophagy to remove the misfolded protein aggregates and recover from the stress environment. Our study demonstrates that 5-FU-induced ER stress suppresses autophagy and also downregulates GRP78 expression. Activation of autophagy followed by ER stress facilitates the cell survival response. Therefore, the inhibition of protective autophagy may provide a useful pharmacological target. Taken together, these results indicate that 5-FU-induced ER stress activates the mitochondrial apoptotic cell death pathway by downregulating GRP78 and protective autophagy proteins in Sk-Hep1 cells, raising the possibility of using 5-FU as a therapeutic agent to target human HCC.