t-CA treatment diminished UVA-induced photoaging/collagen degradation, and protected structural integrity of the skin.

Chalcone flavokawain B (FKB) possesses a chemopreventive and anti-cancer activity. Doxorubicin is a chemotherapeutic DNA intercalating agent widely used in malignancy treatment. The present study investigated whether synergistic effects exist between the combination of FKB (1.25–5 µg/mL) and doxorubicin (0.5 µg/mL) on the apoptosis and autophagy in human gastric cancer (AGS) cells, and the possible in vitro and in vivo mechanisms. The MTT assay measured cell viability. Various apoptotic-, autophagy-associated protein expression was determined by the Western blot technique. FKB+doxorubicin synergy was estimated by the Chou-Talalay combination index (CI) method. In vivo studies were performed on BALB/c mice. Results showed that compared to FKB/doxorubicin treatments, low doses of FKB+doxorubicin suppressed AGS cell growth. FKB potentiated doxorubicin-induced DNA fragmentation, apoptotic cell death, and enhanced doxorubicin-mediated mitochondrial, death receptor pathways. FKB+doxorubicin activated increased LC3-II accumulation, p62/SQSTM1 expression, and AVO formation as compared to the FKB/doxorubicin alone treatments indicating autophagy in these cells. The death mechanism in FKB+doxorubicin-treated AGS cells is due to the activation of autophagy. FKB+doxorubicin-mediated dysregulated Bax/Bcl-2, Beclin-1/Bcl-2 ratios suggested apoptosis, autophagy induction in AGS cells. FKB+doxorubicin-induced LC3-II/AVOs downregulation was suppressed due to an apoptotic inhibitor Z-VAD-FMK. Whereas, 3-methyladenine/chloroquine weakened FKB+doxorubicin-induced apoptosis (decreased DNA fragmentation/caspase-3). Activation of ERK/JNK may be involved in FKB+doxorubicin-induced apoptosis and autophagy. FKB+doxorubicin-triggered ROS generation, but NAC attenuated FKB+doxorubicin-induced autophagic (LC3 accumulation) and apoptotic (caspase-3 activation and PARP cleavage) cell death. FKB+doxorubicin blocked gastric cancer cell xenografts in nude mice in vivo as compared to FKB/doxorubicin alone treatments. FKB and doxorubicin wielded synergistic anti-tumor effects in gastric cancer cells and is a promising therapeutic approach.

TGF‐β1 plays a crucial role in the pathogenesis of vascular fibrotic diseases. Chalcones are reportedly cancer chemo‐preventive food components that are rich in fruits and vegetables. In this study, flavokawain A (FKA, 2‐30 μM), a naturally occurring chalcone in kava extracts, was evaluated for its anti‐fibrotic and antioxidant properties in TGF‐β1‐stimulated vascular smooth muscle (A7r5) cells, as well as its underlying molecular mechanism of action. Immunofluorescence data showed down‐regulated F‐actin expression with FKA treatment in TGF‐β1‐stimulated A7r5 cells. Western blotting demonstrated that FKA treatment suppressed the expression of α‐SMA and fibronectin proteins under TGF‐β1 stimulation. Findings from wound‐healing and invasion experiments showed that FKA inhibits TGF‐β1‐mediated migration and invasion. Western blotting demonstrated that treatment with FKA down‐regulated MMP‐9 and MMP‐2 and up‐regulated TIMP‐1 expression. Further evidence showed that FKA decreased TGF‐β1‐mediated phosphorylation and the transcriptional activity of Smad3. TGF‐β1‐induced excessive ROS production was remarkably reversed by FKA treatment in A7r5 cells, and inhibition by FKA or N‐acetylcysteine (NAC) substantially diminished TGF‐β1‐induced p‐Smad3 activation and wound‐healing migration. Interestingly, FKA‐mediated antioxidant properties were associated with increased nuclear translocation of Nrf2 and elevated antioxidant response element (ARE) luciferase activity. Activation of Nrf2/ARE signaling was accompanied by the induction of HO‐1, NQO‐1 and γ‐GCLC genes in FKA‐treated A7r5 cells. Notably, silencing of Nrf2 (siRNA transfection) significantly diminished the FKA‐mediated antioxidant effects, indicating that FKA may inhibit TGF‐β1‐induced fibrosis through suppressing ROS generation in A7r5 cells. Our results suggested that anti‐fibrotic and antioxidant activities of the chalcone flavokawain A may contribute to the development of food‐based chemo‐preventive drugs for fibrotic diseases.

Antrodia salmonea is well known in Taiwan as a traditional Chinese medicinal fungus and has demonstrated antioxidant, anti-inflammatory, and anticancer effects. However, the anticancer activity of A. salmonea against human ovarian cancer is still elusive. Therefore, we investigated the antiovarian tumor activity of a fermented culture broth of A. salmonea and exhibits its underlying molecular mechanism. A. salmonea shows a significant effect on cell viability in human ovarian carcinoma (SKOV-3 or A2780) cell lines with an 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Increased terminal deoxynucleotidyl transferase dUTP nick-end labeling-positive cells and annexin V-propidium iodide stained cells indicate that A. salmonea induces late apoptosis in SKOV-3 cells. Notably, treatment with A. salmonea induced the following events: Apoptosis; caspase-3, -8, -9 and poly(ADP-ribose) polymerase activation; first apoptosis signal (Fas) and Fas ligand activation; Bid cleavage; and Bax2-B-cell lymphoma 2 dysregulation. The results show that A. salmonea-induced apoptosis was mediated by both mitochondrial and death receptor pathways. An increase in intracellular reactive oxygen species (ROS) was also observed in A. salmonea-treated cells, whereas the antioxidant N-acetylcysteine (NAC) prevented A. salmonea-induced cell death and DNA fragmentation, indicating that A. salmonea-induced apoptosis was mediated by ROS generation. Interestingly, A. salmonea-induced apoptosis is associated with the suppression of human epidermal growth factor receptor-2 (HER-2/neu) and phosphoinositide 3-kinase (PI3K)-protein kinase B (AKT) expression in HER-2/neu overexpressing SKOV-3 cells. NAC significantly prevented A. salmonea-induced HER-2/neu depletion and PI3K/AKT inactivation, indicating that A. salmonea-triggered apoptosis is mediated by ROS-inhibited HER-2/neu signaling cascades. To our knowledge, this is the first report describing the anticancer activity of this potentially beneficial mushroom against human ovarian carcinoma.

We reported in our previously executed studies that the fermented culture broth of Antrodia salmonea (AS), a mushroom used in Taiwanese folk medicine induced reactive oxygen species (ROS)‐mediated apoptosis in human ovarian carcinoma cells. In this study, we studied the anticancer efficacies of AS (0–240 μg/ml) by examining the key molecular events implicated in cell death associated with autophagy in SKOV‐3 and A2780 human ovarian carcinoma cells and clarified the fundamental molecular mechanisms. Treatment of ovarian carcinoma cells with AS‐induced autophagic cell death mediated by increased microtubule‐associated protein LC3‐II, GFP‐LC3 puncta, and acidic vesicular organelle (AVO) formation. These events are linked with the activation of p62/SQSTM1, the inhibition of ATG4B, the expression of ATG7, and the dysregulation of Beclin‐1/Bcl‐2 (i.e., B‐cell lymphoma 2). N‐acetylcysteine inhibited AS‐induced ROS generation, which in turn constricted AS‐induced LC3 conversion, AVO formation, and ATG4B inhibition, indicating ROS‐mediated autophagy cell death. In addition, the 3‐methyladenine (3‐MA) or chloroquine (CQ)‐induced autophagy inhibition decreased AS‐induced apoptosis. Additionally, apoptosis inhibition by Z‐VAD‐FMK, a pan‐caspase inhibitor, substantially suppressed AS‐induced autophagy. Furthermore, AS‐inhibited HER‐2/ neu and PI3K/AKT signaling pathways which were reversed by autophagy inhibitors 3‐MA and CQ. Thus, A. salmonea is a potential chemopreventive agent that is capable of activating ROS‐mediated autophagic cell death in ovarian carcinoma cells.