Antrodia salmonea suppresses epithelial-mesenchymal transition/metastasis and Warburg effects by inhibiting Twist and HIF-1α expression in Twist-overexpressing head and neck squamous cell carcinoma cells.

Hseu JH ，Lin YA ，Pandey S ，Vadivalagan C ，Ali A ，Chen SJ ，Way TD ，Yang HL ，Hseu YC ... -  《-》

Coenzyme Q(0) inhibited the NLRP3 inflammasome, metastasis/EMT, and Warburg effect by suppressing hypoxia-induced HIF-1α expression in HNSCC cells.

Coenzyme Q0 (CoQ0), a quinone derivative from Antrodia camphorata, has antitumor capabilities. This study investigated the antitumor effect of noncytotoxic CoQ0, which included NLRP3 inflammasome inhibition, anti-EMT/metastasis, and metabolic reprogramming via HIF-1α inhibition, in HNSCC cells under normoxia and hypoxia. CoQ0 suppressed hypoxia-induced ROS-mediated HIF-1α expression in OECM-1 and SAS cells. Under normoxia and hypoxia, the inflammatory NLRP3, ASC/caspase-1, NFκB, and IL-1β expression was reduced by CoQ0. CoQ0 reduced migration/invasion by enhancing epithelial marker E-cadherin and suppressing mesenchymal markers Twist, N-cadherin, Snail, and MMP-9, and MMP-2 expression. CoQ0 inhibited glucose uptake, lactate accumulation, GLUT1 levels, and HIF-1α-target gene (HK-2, PFK-1, and LDH-A) expressions that are involved in aerobic glycolysis. Notably, CoQ0 reduced ECAR as well as glycolysis, glycolytic capability, and glycolytic reserve and enhanced OCR, basal respiration, ATP generation, maximal respiration, and spare capacity in OECM-1 cells. Metabolomic analysis using LC-ESI-MS showed that CoQ0 treatment decreased the levels of glycolytic intermediates, including lactate, 2/3-phosphoglycerate, fructose 1,6-bisphosphate, and phosphoenolpyruvate, and increased the levels of TCA cycle metabolites, including citrate, isocitrate, and succinate. HIF-1α silencing reversed CoQ0-mediated anti-metastasis (N-Cadherin, Snail, and MMP-9) and metabolic reprogramming (GLUT1, HK-2, and PKM-2) under hypoxia. CoQ0 prevents cancer stem-like characteristics (upregulated CD24 expression and downregulated CD44, ALDH1, and OCT4) under normoxia and/or hypoxia. Further, in IL-6-treated SG cells, CoQ0 attenuated fibrosis by inhibiting TGF-β and Collagen I expression and suppressed EMT by downregulating Slug and upregulating E-cadherin expression. Interesting, CoQ0 inhibited the growth of OECM-1 tumors in xenografted mice. Our results advocate CoQ0 for the therapeutic application against HNSCC.

Yang HL ，Chang CW ，Vadivalagan C ，Pandey S ，Chen SJ ，Lee CC ，Hseu JH ，Hseu YC ... -  《International Journal of Biological Sciences》

Coenzyme Q(0) defeats NLRP3-mediated inflammation, EMT/metastasis, and Warburg effects by inhibiting HIF-1α expression in human triple-negative breast cancer cells.

Coenzyme Q0 (CoQ0) is a derivative quinone from Antrodia camphorata (AC) that exerts anticancer activities. This study examined the anticancer attributes of CoQ0 (0-4 µM) on inhibited anti-EMT/metastasis and NLRP3 inflammasome, and altered Warburg effects via HIF-1α inhibition in triple-negative breast cancer (MDA-MB-231 and 468) cells. MTT assay, cell migration/invasion assays, Western blotting, immunofluorescence, metabolic reprogramming, and LC-ESI-MS were carried out to assess the therapy potential of CoQ0. CoQ0 inhibited HIF-1α expression and suppressed the NLRP3 inflammasome and ASC/caspase-1 expression, followed by downregulation of IL-1β and IL-18 expression in MDA-MB-231 and 468 cells. CoQ0 ameliorated cancer stem-like markers by decreasing CD44 and increasing CD24 expression. Notably, CoQ0 modulated EMT by upregulating the epithelial marker E-cadherin and downregulating the mesenchymal marker N-cadherin. CoQ0 inhibited glucose uptake and lactate accumulation. CoQ0 also inhibited HIF-1α downstream genes involved in glycolysis, such as HK-2, LDH-A, PDK-1, and PKM-2 enzymes. CoQ0 decreased extracellular acidification rate (ECAR), glycolysis, glycolytic capacity, and glycolytic reserve in MDA-MB-231 and 468 cells under normoxic and hypoxic (CoCl2) conditions. CoQ0 inhibited the glycolytic intermediates lactate, FBP, and 2/3-PG, and PEP levels. CoQ0 increased oxygen consumption rate (OCR), basal respiration, ATP production, maximal respiration, and spare capacity under normoxic and hypoxic (CoCl2) conditions. CoQ0 increased TCA cycle metabolites, such as citrate, isocitrate, and succinate. CoQ0 inhibited aerobic glycolysis and enhanced mitochondrial oxidative phosphorylation in TNBC cells. Under hypoxic conditions, CoQ0 also mitigated HIF-1α, GLUT1, glycolytic-related (HK-2, LDH-A, and PFK-1), and metastasis-related (E-cadherin, N-cadherin, and MMP-9) protein or mRNA expression in MDA-MB-231 and/or 468 cells. Under LPS/ATP stimulation, CoQ0 inhibited NLRP3 inflammasome/procaspase-1/IL-18 activation and NFκB/iNOS expression. CoQ0 also hindered LPS/ATP-stimulated tumor migration and downregulated LPS/ATP-stimulated N-cadherin and MMP-2/-9 expression. The present study revealed that suppression of HIF-1α expression caused by CoQ0 may contribute to inhibition of NLRP3-mediated inflammation, EMT/metastasis, and Warburg effects of triple-negative breast cancers.

Yang HL ，Lin PY ，Vadivalagan C ，Lin YA ，Lin KY ，Hseu YC ... -  《-》

In vitro and in vivo anti-tumor activity of Antrodia salmonea against twist-overexpressing HNSCC cells: Induction of ROS-mediated autophagic and apoptotic cell death.

Head and neck squamous cell carcinoma (HNSCC) is a relatively common malignancy, characterized by lethal morbidity. Herein, we attempted to investigate the autophagy/apoptosis activities of the submerged fermented broths of Antrodia salmonea (AS) in HNSCC Twist-overexpressing (OECM-1 and FaDu-Twist) cells. AS (0-150 μg/mL) effectively reduced cell viability, colony formation, and downregulated Twist expression in OECM-1 and FaDu-Twist cells compared to FaDu cells. AS- induced apoptosis was mainly associated with activation of caspase-3, PARP cleavage, increased expression of VDAC-1 and disproportionation of Bax/Bcl-2. Annexin V/PI staining suggested late apoptosis induction by AS treatment. AS exhibits enhanced autophagy process mediated via LC3-I/II accumulation, increased acidic vesicular organelles (AVOs) formation and p62/SQSTM1 expression feeding into the apoptotic program. However, pre-treatment with autophagy blockers 3-MA and CQ significantly diminished AS-induced cell death. Additionally, suppression of AS-induced ROS release by treatment with antioxidant N-acetylcysteine (NAC) resulted in reduction of apoptotic and autophagic cell death. In vivo studies strengthened the above observations and showed that AS effectively reduced the tumor volume and tumor weight in OECM-1-xenografted nude mice. This study discovered that Antrodia salmonea exhibits a novel anti-cancer mechanism which could be harnessed as a new potent drug for HNSCC treatment.

Yang HL ，Lin YA ，Pandey S ，Liao JW ，Way TD ，Yeh YL ，Chen SJ ，Hseu YC ... -  《-》

CSC-3436 inhibits TWIST-induced epithelial-mesenchymal transition via the suppression of Twist/Bmi1/Akt pathway in head and neck squamous cell carcinoma.

Head and neck squamous cell carcinoma (HNSCC) is one of the leading causes of cancer deaths worldwide, especially in male. With poor prognosis, significant portions of patients with HNSCC die due to cancer recurrence and tumor metastasis after chemotherapy and targeted therapies. The HNSCC FaDu cell ectopic expression of Twist, a key transcriptional factor of epithelial-mesenchymal transition (EMT), which triggers EMT and results in the acquisition of a mesenchymal phenotype, was used as the cell model. Our results demonstrated that treatment with newly synthesized 2-(3-hydroxyphenyl)-5-methylnaphthyridin-4-one (CSC-3436), a flavonoid derivative, elicited changes in its cell morphology, upregulated E-cadherin messenger RNA and protein expression, downregulated N-cadherin, vimentin, and CD133 (a marker associated with tumor-initiating cells) in FaDu-pCDH-Twist cells. Moreover, CSC-3436 exposure reduced B cell-specific Moloney murine leukemia virus integration site 1 (Bmi1) expression regulated by Twist and further suppressed the direct co-regulation of E-cadherin by Twist and Bmi1. Interestingly, CSC-3436 reduced EMT, cancer stemness, and migration/invasion abilities through the inhibition of the Twist/Bmi1-Akt/β-catenin pathway. Most importantly, our findings provided new evidence that CSC-3436 played a crucial role in therapeutic targeting to Bmi1 and its molecular pathway in HNSCC, and it will be valuable in prognostic prediction and treatment.

Lai YJ ，Yu WN ，Kuo SC ，Ho CT ，Hung CM ，Way TD ，Chen CT ... -  《-》