Antcin B and Its Ester Derivative from Antrodia camphorata Induce Apoptosis in Hepatocellular Carcinoma Cells Involves Enhancing Oxidative Stress Coincident with Activation of Intrinsic and Extrinsic Apoptotic Pathway

The triterpenoids methylantcinate B (MAB) and antcin B (AB), isolated from the medicinal mushroom Antrodia camphorata, have been identified as strong cytotoxic agents against various type of cancer cells; however, the mechanisms of MAB- and AB-induced cytotoxicity have not been adequately explored. This study investigated the roles of caspase cascades, reactive oxygen species (ROS), DNA damage, mitochondrial disruption, and Bax and Bcl-2 proteins in MAB- and AB-induced apoptosis of hepatocellular carcinoma (HCC) HepG2 cells. Here, we showed that MAB and AB induced apoptosis in HepG2 cells, as characterized by increased DNA fragmentation, cleavage of PARP, sub-G1 population, chromatin condensation, loss of mitochondrial membrane potential, and release of cytochrome <i>c</i>. Increasing the levels of caspase-2, -3, -8, and -9 activities was involved in MAB- and AB-induced apoptosis, and they could be attenuated by inhibitors of specific caspases, indicating that MAB and AB triggered the caspase-dependent apoptotic pathway. Additionally, the enhanced apoptotic effect correlates with high expression of Fas, Fas ligand, as well as Bax and decreased protein levels of Bcl-_XL and Bcl-2, suggesting that both the extrinsic and intrinsic apoptosis pathways were involved in the apoptotic processes. Incubation of HepG2 cells with antioxidant enzymes superoxide dismutase and catalase and antioxidants <i>N</i>-acetylcysteine and ascorbic acid attenuated the ROS generation and apoptosis induced by MAB and AB, which indicate that ROS plays a pivotal role in cell death. NADPH oxidase activation was observed in MAB- and AB-stimulated HepG2 cells; however, inhibition of such activation by diphenylamine significantly blocked MAB- and AB-induced ROS production and increased cell viability. Taken together, our results provide the first evidence that triterpenoids MAB and AB induced a NADPH oxidase-provoked oxidative stress and extrinsic and intrinsic apoptosis as a critical mechanism of cause cell death in HCC cells

Additional file 2: Figure S1. of Ovatodiolide suppresses colon tumorigenesis and prevents polarization of M2 tumor-associated macrophages through YAP oncogenic pathways

YAP1 overexpression (OE) leads to increased stemness in colon cancer cells. (A) A significantly increased CD133+ cell percentage was found in the YAP1 OE cells as compared to their wild-type counterparts as demonstrated by our flow cytometric analysis. (B) YAP1-overexpressing HCT116 and DLD-1 cells were found to form a significantly higher number of tumor spheres (*P ≤ 0.05, **P ≤ 0.01). (C) Comparative western blots of wild-type and YAP1 overexpressing HCT116 and DLD-1 cells. An increased expression of β-catenin, NF-kB, vimentin, and Kras is associated with YAP1 overexpression. (PPTX 396 kb

Additional file 1: Figure S1. of Honokiol inhibits sphere formation and xenograft growth of oral cancer side population cells accompanied with JAK/STAT signaling pathway suppression and apoptosis induction

Honokiol (5 μM) and siSTAT3 did not affect the cell viability of SAS cells in the 24-h wound healing assay. (A) SAS cells were treated with honokiol (5 μM) for 24 h in the same culture condition shown in Fig. 6b for wound healing assay. The cell viability was then determined by the quantitative staining of cellular proteins by sulforhodamine B. (B) After transfection with siSTAT3 and analysis for the expression of STAT3, the SAS cells were seeded into 6-well plate in the same condition shown in Fig. 6b for wound healing assay. After 24 h of incubation, the cell viability was then determined by the quantitative staining of cellular proteins by sulforhodamine B. (TIFF 1240 kb

Additional file 1: Figure S2. of Ovatodiolide suppresses colon tumorigenesis and prevents polarization of M2 tumor-associated macrophages through YAP oncogenic pathways

YAP1 expression is associated with M1-M2 polarization. (A) YAP1 silencing in macrophages (M0) leads to decreased M2 polarization. Real-time PCR analysis demonstrates that M2 markers, TGF-b1 and Ym2, were significantly reduced while one of the M1 marker iNOS was significantly increased, in the macrophages generated in the presence of DLD-1 cells. (B) YAP1-overexpressing colon cancer cells also promote the polarization of M2 genotype in THP-1 cells. Experiments were performed at least three times. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. The primer sequences used in the q-PCR experiments are shown. (PPTX 109 kb

A Vaccine Targeted at CETP Alleviates High Fat and High Cholesterol Diet-Induced Atherosclerosis and Non-Alcoholic Steatohepatitis in Rabbit

<div><p>Low HDL-C levels are associated with atherosclerosis and non-alcoholic steatohepatitis, and increased levels may reduce the risk of these diseases. Inhibition of cholesteryl ester transfer protein (CETP) activity is considered a promising strategy for increasing HDL-C levels. Since CETP is a self-antigen with low immunogenicity, we developed a novel CETP vaccine (Fc-CETP₆) to overcome the low immunogenicity of CETP and for long-term inhibition of CETP activity. The vaccine consists of a rabbit IgG Fc domain for antigen delivery to antigen-presenting cells fused to a linear array of 6 repeats of a CETP epitope to efficiently activate B cells. Rabbits were fed a high fat/cholesterol (HFC) diet to induce atherosclerosis and NASH, and immunized with Fc-CETP₆ vaccine. The Fc-CETP₆ vaccine successfully elicited anti-CETP antibodies and lowered plasma CETP activity. The levels of plasma HDL-C and ApoA-I were higher, and plasma ox-LDL lower, in the Fc-CETP₆-immunized rabbits as compared to the unimmunized HFC diet-fed rabbits. Pathological analyses revealed less lipid accumulation and inflammation in the aorta and liver of the Fc-CETP₆-immunized rabbits. These results show that the Fc-CETP₆ vaccine efficiently elicited antibodies against CETP and reduced susceptibility to both atherosclerosis and steatohepatitis induced by the HFC diet. Our findings suggest that the Fc-CETP₆ vaccine may improve atherosclerosis and NASH and has high potential for clinical use.</p></div

Additional file 2: of A phase I clinical study of immunotherapy for advanced colorectal cancers using carcinoembryonic antigen-pulsed dendritic cells mixed with tetanus toxoid and subsequent IL-2 treatment

The results of T cell proliferation assay. (XLS 93 kb

Vaccination with Fc-CETP₆ attenuates high HFC diet-induced steatosis and NASH.

<p>(A) Representative liver sections stained with H&E (magnification, x200). Liver section stained with H&E from control rabbits showing NASH characteristics, such as ballooning degeneration, infiltrating inflammatory cells and Mallory hyaline were observed in the control (magnification, x200). (B) Expression of RAM-11 (magnification, upper panel x100 and lower panel x400) of the liver from control and Fc-CETP₆ rabbits. (C) Expression of NF-kB (magnification, upper panel x100 and lower panel x400) of the liver from control and Fc-CETP₆ rabbits. (D) Expression of inflammation related genes in control rabbits (black bars) and Fc-CETP₆ rabbits (white bars). The bar graphs represent the average (C: control group n = 7; Fc-CETP₆ group n = 8) for each group with standard errors. Values are the mean ± SEM. *p<0.05; ***p<0.001 compared to the control.</p

MOESM3 of Active fraction (HS7) from Taiwanofungus camphoratus inhibits AKT-mTOR, ERK and STAT3 pathways and induces CDK inhibitors in CL1-0 human lung cancer cells

Additional file 3. Apoptosis induction in CL1-0 lung cancer cells by HS7 at dose of 50 Οg/mL

Vaccination with Fc-CETP₆ reduces Aortic lesions and inflammation (experiment 1).

<p>(A) Representative Sudan IV-stained aortic specimens at the end of week 24. (B) Quantification of the aortic lesion area. The bar graphs represent the average (N: normal group n = 4; C: control group n = 7; Fc-CETP₆ group n = 8) for each group with standard errors. *<i>p</i><0.05 compared to the control. (C) Expression of NF-κB and RAM-11 (magnification, x200) of the aorta from normal rabbits, control rabbits, and Fc-CETP₆ rabbits. (D) Quantification of the RAM-11 and NF-κB positive area of aorta. The bar graphs represent the average (N: normal group n = 4; C: control group n = 7; Fc-CETP₆ group n = 8) for each group with standard errors. The positive-stained area was calculated as the (stained area/total area) x 100. Values are the mean ± SEM. *<i>p</i><0.05 compared to the control.</p

Measurement of plasma anti-CETP titers and CETP activity in rabbits immunized with Fc-CETP₆ (experiment 1).

<p>(A) Plasma antibody titers against CETP. Plasma samples were collected as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111529#s2" target="_blank">material and methods</a> (Control group n = 7; Fc-CETP₆ group n = 8) and diluted 2000-fold and the anti-CETP titer measured by ELISA. (B) Plasma CETP activity. Plasma CETP activity was measured and expressed as a percentage of that in the pre-vaccination sample (Control group n = 7; Fc-CETP₆ group n = 8). Values are the mean ± SEM for the control rabbits fed the HFC diet and the rabbits fed the HFC diet and immunized with Fc-CETP₆. *<i>p</i><0.05 compared to the control at the same time.</p