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Chalcones found in fruits and vegetables have promising cancer chemopreventive properties. This study attempts to identify the anticancer efficacies of chalcone flavokawain B (FKB) in the rhizomes of Alpinia pricei Hayata by examining key molecular events in non‐small‐cell lung cancer (A549) cells. Our results indicated that in human A549 cells, FKB (0–15 μg/ml) decreases cell viability and colony formation, dysregulates the Bax:B‐cell lymphoma 2 ratio and increases apoptotic DNA fragmentation. Mitochondrial (caspase‐9/‐3 and poly ADP ribose polymerase [PARP]) signaling was found to be involved in FKB‐induced apoptosis. In addition, FKB‐induced reactive oxygen species (ROS) generation, and N‐acetylcysteine attenuated FKB‐induced apoptotic cell death. Moreover, FKB triggered autophagy, as evidenced by the improved acidic vesicular organelle formation, lipidated light chain 3 (microtubule‐related light chain 3) accumulation, and ATG7 expression and the decreased mammalian target of rapamycin phosphorylation. Furthermore, FKB suppressed ROS‐mediated ATG4B expression. Inhibiting autophagy using 3‐methyladenine/chloroquine diminished FKB‐induced cell death, indicating that autophagy is triggered as a death mechanism by FKB. In summary, FKB has a crucial role in the execution and propagation of ROS‐mediated apoptotic and autophagic cell death of lung adenocarcinoma cells. [ABSTRACT FROM AUTHOR]

Abstract: Ethnopharmacological relevance: Antrodia camphorata (AC) is well known in Taiwan as a traditional Chinese medicinal fungus. However, the anticancer activity of AC against human HER-2/neu-overexpressing ovarian cancers is poorly understood. Materials and methods: The aim of this study is to investigate whether a submerged fermentation culture of AC can inhibit human ovarian carcinoma cell (SKOV-3) proliferation by suppressing the HER-2/neu signaling pathway. Cell viability, colony formation, DCFH-DA fluorescence microscopy, western blotting, HER-2/neu immunofluorescence imaging, flow cytometry, and TUNEL assays were carried out to determine the anti-cancer effects of AC. Results: MTT and colony formation assays showed that AC induced a dose-dependent reduction in SKOV-3 cell growth. Immunoblot analysis demonstrated that HER-2/neu activity and tyrosine phosphorylation were significantly inhibited by AC. Furthermore, AC treatment significantly inhibited the activation of PI3K/Akt and their downstream effector β-catenin. We also observed that AC caused G2/M arrest mediated by down-regulation of cyclin D1, cyclin A, cyclin B1, and Cdk1 and increased p27 expression. Notably, AC induced apoptosis, which was associated with DNA fragmentation, cytochrome c release, caspase-9/-3 activation, PARP degradation, and Bcl-2/Bax dysregulation. An increase in intracellular reactive oxygen species (ROS) was observed in AC-treated cells, whereas the antioxidant N-acetylcysteine (NAC) prevented AC-induced cell death, HER-2/neu depletion, PI3K/Akt inactivation, and Bcl-2/Bax dysregulation, indicating that AC-induced cell death was mediated by ROS generation. Conclusions: These results suggest that AC may exert anti-tumor activity against human ovarian carcinoma by suppressing HER-2/neu signaling pathways. [Copyright &y& Elsevier]

Simple Summary: Melanoma is the most dangerous type of skin cancer that develops from the pigment-producing cells known as melanocytes. One of the primary causes of melanoma is ultraviolet light (UV) exposure in those with low levels of the skin pigment melanin. Flavokawain B (FKB) is a naturally occurring chalcone, which is known to possess anti-proliferative pharmacological activity on various cancer cells. However, the effect of FKB on the anti-melanoma pharmacological role has not been investigated. Therefore, in this study, we explored the anti-melanoma properties of FKB on human melanoma cells and the cell death mechanisms that were mediated through the induction of reactive oxygen species (ROS) were investigated via in vitro and in vivo approaches. Our study results support promising application prospects of FKB in the treatment of human melanoma cancer. Melanoma is the most prevalent type of skin cancer with high mortality rates. This study demonstrates the in vitro and in vivo anticancer properties of chalcone flavokawain B (FKB) induced ROS-mediated apoptosis and autophagy in human melanoma (human epithelial melanoma cell line A375 and/or human skin lymph node derived melanoma cell line A2058) cells. Cell viability was calculated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and the expression patterns of various apoptosis, autophagy-associated proteins were determined by Western blot methods. Annexin V was detected by flow cytometry, whereas acidic vesicular organelles (AVOs) and intracellular ROS levels were measured by fluorescence microscopy. The in vivo anticancer properties of FKB were evaluated by xenografting the A375 cells into nude mice. The results convey that FKB inhibited cell viability, B-Raf proto-oncogene, serine/threonine kinase (BRAF)/extracellular signal-regulated kinase (ERK) expression in human melanoma cells. Caspase-3 activation, poly (ADP-ribose) polymerase (PARP) cleavage pathway, and Bcl2 associated X (Bax)/B-cell lymphoma 2 (Bcl-2) dysregulation were involved in the execution of apoptosis. Moreover, FKB-induced autophagy was observed through increased microtubule-associated protein 1A/1B-light chain 3B (LC3-II) accumulation and AVOs formation, which was also associated with an increase in sequestosome 1 (SQSTM1/p62), decreased protein kinase B (AKT)/mammalian target of rapamycin (mTOR) expressions, and dysregulated Beclin-1/Bcl-2 levels. Autophagy inhibitors [3-methyladenine (3-MA)/chloroquine (CQ)] and LC3 silencing suppressed FKB-induced apoptosis by decreasing caspase-3 in melanoma cells. The antioxidant N-acetylcysteine (NAC) diminished FKB-induced apoptotic and autophagic cell death. However, the inhibition of apoptosis decreased FKB-induced autophagy (LC3-I/II). The in vivo study confirmed that FKB inhibited melanoma growth in A375-xenografted nude mice. This study concluded that FKB is critically associated with the execution and generation of ROS-modulated apoptotic and autophagic cell death of melanoma cells. FKB also repressed tumor growth in xenografted nude mice. Therefore, flavokawain B might be a potential anti-tumor agent in human melanoma treatment. [ABSTRACT FROM AUTHOR]

Simple Summary: Among various kinds of treatment strategies for cancers, combination therapy has attracted significant attention due to its beneficial effects than the individual effects of the same compounds. Based on this idea, this study has investigated the synergistic effects of combination treatment of a natural anti-cancer agent flavokawain B (FKB) and a chemotherapeutic agent Doxorubicin on human gastric cancer cells and the underlying molecular mechanisms were deciphered through in vitro and in vivo approaches. Experimental data obtained in this study provided promising application prospects of FKB + Doxrubicin combination treatment in human gastric cancer cells. 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. [ABSTRACT FROM AUTHOR]

Kalantuboside B (KB), a natural bufadienolide derivative extracted from the succulent plant Kalanchoe tubiflora , is well-known for its cardiotonic, immunomodulatory, and anti-inflammatory properties. In this study, we tested in vitro and in vivo anti-cancer efficacy with low concentrations of KB (5–30 ng/mL; 8.7–52.2 nM) on A2058 melanoma cells; and for the molecular mechanisms that underlie them. KB significantly inhibited the cell viability and colony formation via arresting the cell cycle at G2/M phase. There was an association with a decrease in Cyclin A/B1, Cdc25C, and Cdc2 expressions. Further, this treatment indicated the induction of apoptosis, DNA fragmentation, cytochrome c release, and caspase-3, -8, -9, and -12 activation, and PARP cleavage, which shows that mitochondrial, death-receptor, and ER-stress signaling pathways are involved. KB-induced autophagy was apparent from enhanced LC3-II accumulation, GFP-LC3 puncta, and AVO formation. Surprisingly, KB-mediated cell death was potentiated by 3-MA and CQ to suggest the role of autophagy as a cytoprotective mechanism. Moreover, KB-treated A2058 cells enhanced intracellular ROS generation and antioxidant NAC prevented apoptosis and reversed cytoprotective autophagy. Interestingly, KB-induced apoptosis (PARP cleavage) and cytoprotective autophagy (LC3-II accumulation) were mediated by the up-regulation of the ERK signaling pathway. It was also shown that KB promoted cytoprotective autophagy by a calcium dependent-p53 downregulation pathway. In vivo data showed that KB suppressed tumor growth significantly in A2058-xenografted nude mice. A Western blot indicated cell-cycle inhibition (cyclin A reduction), apoptosis induction (PARP cleavage and Bcl-2 inhibition), and cytoprotective autophagy (LC3-II upregulation and p53 downregulation) in KB-treated A2058-xenografted mice. Our findings suggested that KB-induced ROS pathway plays a role in mediating the apoptosis and cytoprotective autophagy in human melanoma cells. Thus, KB is considered to be a putative anti-tumor agent. Image 1 • Kalantuboside B (KB, 8.7–52.2 nM) shows anti-tumor activity in human melanoma cells. • KB inhibited melanoma cell proliferation via G 2 /M cell-cycle arrest and apoptosis. • KB induced autophagy in A2058 cells as a cell-survival mechanism. • ROS pathway plays a role in KB-mediated apoptosis and cytoprotective autophagy. • KB mediated the suppression of A2058 xenograft tumor growth in athymic nude mice. [ABSTRACT FROM AUTHOR]

Antrodia salmonea (AS) exhibits anticancer activities against various cancers. This study investigated the anticancer activities of AS on human glioblastoma (GBM8401 and U87MG) cells both in vitro and in vivo and explained the underlying molecular mechanism. MTT, colony formation, migration/invasion assay, immunoblotting, immunofluorescence, TUNEL, Annexin V/PI staining, AO staining, GFP-LC3 transfection, TEM, qPCR, siLC3, DCFH2-DA assay, and xenografted-nude mice were used to assess the potential of AS therapy. AS treatment retarded growth and suppressed colony formation in glioblastoma cells. AS attenuates EMT by suppressing invasion and migration, increasing E-cadherin expression, decreasing Twist, Snail, and N-cadherin expression, and inhibiting Wnt/β-catenin pathways in GBM8401 and U87MG cells. Furthermore, AS induced apoptosis by activating caspase-3, cleaving PARP, and dysregulating Bax and Bcl-2 in both cell lines. TUNEL assay and Annexin V/PI staining indicated AS-mediated late apoptosis. Interestingly, AS induced autophagic cell death by LC3-II accumulation, AVO formation, autophagosome GFP-LC3 puncta, p62/SQSTM1 expression, and ATG4B inhibition in GBM8401 and U87MG cells. TEM data revealed that AS favored autophagosome and autolysosome formation. The autophagy inhibitors 3-MA/CQ and LC3 knockdown suppressed AS-induced apoptosis in glioblastoma cells, indicating that the inhibition of autophagy decreased AS-induced apoptosis. Notably, the antioxidant N -acetylcysteine (NAC) inhibited AS-mediated ROS production and AS-induced apoptotic and autophagic cell death. Furthermore, AS induced ROS-mediated inhibition of the PI3K/AKT/mTOR signaling pathway. AS reduced the tumor burden in GBM8401-xenografted nude mice and significantly modulated tumor xenografts by inducing anti-EMT, apoptosis, and autophagy. AS could be a potential antitumor agent in human glioblastoma treatment. [ABSTRACT FROM AUTHOR]