Augmentation of Docetaxel-Induced Cytotoxicity in Human PC-3 Androgen-Independent Prostate Cancer Cells by Combination With Four Natural Apoptosis-Inducing Anticancer Compounds.

Docetaxel (DTX) is the treatment of choice for metastatic castration-resistant prostate cancer. However, developing drug resistance is a significant challenge for achieving effective therapy. This study evaluated the anticancer and synergistic effects on DTX of four natural compounds (calebin A, 3'-hydroxypterostilbene, hispolon, and tetrahydrocurcumin) using PC-3 androgen-resistant human prostate cancer cells. We utilized the CellTiter-Glo® luminescent cell viability assay and human PC-3 androgen-independent prostate cancer cells to determine the antiproliferative effects of the four compounds alone and combined with DTX. Cytotoxicity to normal human prostate epithelial cells was tested in parallel using normal immortalized human prostate epithelial cells (RWPE-1). We used cell imaging and quantitative caspase-3 activity to determine whether these compounds induce apoptosis. We also measured the capacity of each drug to inhibit TNF-α-induced NF-kB using a colorimetric assay. Our results showed that all four natural compounds significantly augmented the toxicity of DTX to androgen-resistant PC-3 prostate cancer cells at IC50. Interestingly, when used alone, each of the four compounds had a higher cytotoxic activity to PC-3 than DTX. Mechanistically, these compounds induced apoptosis, which we confirmed by cell imaging and caspase-3 colorimetric assays. Further, when used either alone or combined with DTX, the four test compounds inhibited TNF-α-induced NF-kB production. More significantly, the cytotoxic effects on normal immortalized human prostate epithelial cells were minimal and non-significant, suggesting prostate cancer-specific effects. In conclusion, the combination of DTX with the four test compounds could effectively enhance the anti-prostate cancer activity of DTX. This combination has the added value of reducing the DTX effective concentration. We surmise that calebin A, 3'-hydroxypterostilbene, hispolon, and tetrahydrocurcumin were all excellent drug candidates that produced significant antiproliferative activity when used alone and synergistically enhanced the anticancer effect of DTX. Further in vivo studies using animal models of prostate cancer are needed to confirm our in vitro findings.

Phenolic and Steroidal Metabolites from the Cultivated Edible Inonotus hispidus Mushroom and Their Bioactivities.

A chemical study on the fruiting bodies of cultivated edible mushroom Inonotus hispidus resulted in 14 metabolites including three new hispolon congeners, named inonophenols A-B and one new lanostane triterpenoid, named inonoterpene A. These structures were identified by NMR, high-resolution electrospray ionization mass spectrometry (HRESIMS), and electronic circular dichroism (ECD) data analysis. All metabolites were assessed for neurotrophic, anti-inflammatory, and antioxidative activities. Among them, inonophenols B and C were the most active in promoting PC-12 cell neurite outgrowth at a concentration of 10 µM. The phenolic derivatives reduced NO generation by lipopolysaccharide (LPS)-induced BV-2 microglial cells by suppressing the expression of toll-like receptor-4 (TLR-4) and the nuclear factor-kappa-B (NF-κB) signaling pathway as well as the inflammatory mediators including inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2). Moreover, the phenolics showed antioxidant effects in DPPH scavenging assay with the IC50 values of 9.82-21.43 µM. These findings showed that I. hispidus may be a new source of neurotrophic and protective agents against neurodegenerative disorders.

Natural substances to potentiate canonical glioblastoma chemotherapy.

Glioblastoma multiforme (GBM) is the most frequent primary malignant brain tumour prevalent in humans, that exhibits aggressive cell proliferation and rapid invasion of normal brain tissue. Despite aggressive therapeutic approaches consisting of maximum safe surgical resection followed by radio-chemotherapy with temozolomide (TMZ), more than 95% of GBM patients die within 5 years after diagnosis. In most cases, the therapy is not able to counteract the growth and invasiveness of the tumour, which relapses after an interval of time that varies from patient to patient. An increasing number of evidence indicates that natural substances exhibited effective anti-tumour functions and might be successfully used in the treatment of GBM. This review summarizes some natural substances: lactoferrin, hispolon, aloe-emodin and tea tree oil; all these show a growth inhibition and synergistic effect when together with TMZ, (the most commonly used alkylating drug for the treatment of glioblastoma) were administered to U87MG glioblastoma cell line in vitro and in murine animal model. U87MG cell growth was monitored by daily cell count after treatments with the substances mentioned above and growth analysis showed that all drugs significantly decrease proliferation of U87MG in a time- and dose-dependent manner. FACS analysis demonstrates a block of cell cycle in S, G2/M or G0/G1 phases. These substances mediate multiple processes including apoptosis by releasing the inducing factor: PARP. Natural compounds, in combination with conventional chemotherapy TMZ, are a powerful approach to improve the effectiveness of brain cancer treatment.

Attenuation of Lipopolysaccharide-Induced Acute Lung Injury by Hispolon in Mice, Through Regulating the TLR4/PI3K/Akt/mTOR and Keap1/Nrf2/HO-1 Pathways, and Suppressing Oxidative Stress-Mediated ER Stress-Induced Apoptosis and Autophagy.

The anti-inflammatory effect of hispolon has identified it as one of the most important compounds from Sanghuangporus sanghuang. The research objectives were to study this compound using an animal model by lipopolysaccharide (LPS)-induced acute lung injury. Hispolon treatment reduced the production of the pro-inflammatory mediator NO, TNF-α, IL-1ß, and IL-6 induced by LPS challenge in the lung tissues, as well as decreasing their histological alterations and protein content. Total cell number was also reduced in the bronchoalveolar lavage fluid (BALF). Moreover, hispolon inhibited iNOS, COX-2 and IκB-α and phosphorylated IKK and MAPK, while increasing catalase, SOD, GPx, TLR4, AKT, HO-1, Nrf-2, Keap1 and PPARγ expression, after LPS challenge. It also regulated apoptosis, ER stress and the autophagy signal transduction pathway. The results of this study show that hispolon regulates LPS-induced ER stress (increasing CHOP, PERK, IRE1, ATF6 and GRP78 protein expression), apoptosis (decreasing caspase-3 and Bax and increasing Bcl-2 expression) and autophagy (reducing LC3 I/II and Beclin-1 expression). This in vivo experimental study suggests that hispolon suppresses the LPS-induced activation of inflammatory pathways, oxidative injury, ER stress, apoptosis and autophagy and has the potential to be used therapeutically in major anterior segment lung diseases.

Developing Hispolon-based novel anticancer therapeutics against human (NF-κß) using in silico approach of modelling, docking and protein dynamics.

Hispolon is a polyphenolic compound derived from black hoof mushroom (Phellinus linteus) or shaggy bracket mushroom (Inonotus hispidus) which induces the inhibition of cancer-promoting nuclear factor-kappa beta (NF-κß) complex. To develop more potent lead molecules with enhanced anticancer efficiency, the mechanism of hispolon-mediated nuclear factor-κß inhibition has been investigated by molecular modelling and docking. Ten derivatives of hispolon (DRG1-10) have been developed by pharmacophore-based design with a view to enhance the anticancer efficacy. Hispolon and its derivatives were further screened for different pharmacological parameters like binding free energy, drug likeliness, absorption-digestion-metabolism-excretion (ADME), permeability, mutagenicity, toxicity and inhibitory concentration 50 (IC50) to find a potent lead molecule. Based on pharmacological validation, comparative molecular dynamics (MD) simulations have been performed for three lead molecules: Hispolon, DRG2 and DRG7 complexed with human NF-κß up to 50 ns. By analysing different factors like root mean square deviation (RMSD), root mean square fluctuation (RMSF), radius of gyration (Rg), solvent-accessible surface area (SASA) and principal component analysis (PCA), Gibb's free energy plots DRG2 have more binding efficiency compared to hispolon and DRG7. In RMSD plot, hispolon-bound NF-κß has the most deviation within a range between 0.125 and 0.45 nm, and DRG2-bound complex showed the range between 0.125 and 0.25 nm. The residues of NF-κß responsible for hydrophobic interactions with ligand, e.g. Met469, Leu522 and Cys533, have the lowest fluctuation values in DRG2-bound complex. The average Rg fluctuation for DRG2-bound NF-κß has been recorded under 2.025 nm for most of the simulation time which is much less compared to hispolon and DRG7. Gibb's free energy plots also define the highest stability of DRG2-bound NF-κß. Communicated by Ramaswamy H. Sarma.

Hispolon Suppresses LPS- or LTA-Induced iNOS/NO Production and Apoptosis in BV-2 Microglial Cells.

Hispolon (HIS) is an active polyphenol compound derived from Phellinus linteus (Berkeley & Curtis), and our previous study showed that HIS effectively inhibited inflammatory responses in macrophages [Yang, L.Y., S.C. Shen, K.T. Cheng, G.V. Subbaraju, C.C. Chien and Y.C. Chen. Hispolon inhibition of inflammatory apoptosis through reduction of iNOS/NO production via HO-1 induction in macrophages. J. Ethnopharmacol. 156: 61-72, 2014]; however, its effect on neuronal inflammation is still undefined. In this study, HIS concentration- and time-dependently inhibited lipopolysaccharide (LPS)- and lipoteichoic acid (LTA)-induced inducible nitric oxide (NO) synthase (iNOS)/NO production with increased heme oxygenase (HO)-1 proteins in BV-2 microglial cells. Accordingly, HIS protected BV-2 cells from LPS- or LTA-induced apoptosis, characterized by decreased DNA ladder formation, and caspase-3 and poly(ADP ribose) polymerase (PARP) protein cleavage in BV-2 cells. Similarly, the NOS inhibitor, N-nitro-L-arginine methyl ester (NAME), inhibited LPS- or LTA-induced apoptosis of BV-2 cells, but neither NAME nor HIS showed any inhibition of NO production or cell death induced by the NO donor, sodium nitroprusside (SNP), indicating the involvement of NO in the inflammatory apoptosis of microglial cells. Activation of c-Jun N-terminal kinase (JNK) and nuclear factor (NF)-[Formula: see text]B contributed to LPS- or LTA-induced iNOS/NO production and apoptosis of BV-2 cells, and that was suppressed by HIS. Additionally, HIS possesses activity to induce HO-1 protein expression via activation of extracellular signal-regulated kinase (ERK) in BV-2 cells, and application of the HO inhibitor, tin protoporphyrin (SnPP), or knockdown of HO-1 protein by HO-1 small interfering (si)RNA significantly reversed HIS inhibition of NO production and cell death in BV-2 cells stimulated by LPS. Results of an analysis of the effects of HIS and two structurally related chemicals, i.e. dehydroxy-HIS (D-HIS) and HIS-methyl ester (HIS-ME), showed that HIS expressed the most potent inhibitory effects on iNOS/NO production, JNK activation, and apoptosis in BV-2 microglial cells activated by LPS with increased HO-1 protein expression. Overall these results suggested that HIS possesses inhibitory activity against LPS- or LTA-induced inflammatory responses including iNOS/NO production and apoptosis in BV-2 microglial cells and that the mechanisms involve upregulation of the HO-1 protein and downregulation of JNK/NF-[Formula: see text]B activation. A critical role of hydroxyl at position C3 in the anti-inflammatory actions of HIS against activated BV-2 microglial cells was suggested.

Hispolon from Phellinus linteus possesses mediate caspases activation and induces human nasopharyngeal carcinomas cells apoptosis through ERK1/2, JNK1/2 and p38 MAPK pathway.

Hispolon, a phenol compound isolated from Phellinus linteus (PL), possesses anti-inflammatory, antiproliferative, and antioxidant effects. However, the effects of hispolon on human nasopharyngeal carcinomas have yet to be evaluated. Here, the molecular mechanism by which hispolon anticancer effects in human nasopharyngeal carcinomas cells was investigated. The results showed that hispolon significantly inhibited cell proliferation of HONE-1 and NP-039 cell lines. Furthermore, hispolon induced apoptosis through caspases-3, -8, and -9 activations and PARP cleavage in dose- and time-dependent manner in HONE-1 and NP-039 cells. Moreover, hispolon also showed that increase phosphorylation of ERK1/2, p38 MAPK and JNK1/2 in dose- and time-dependent manner by western blot analysis. However, hispolon-induced activation of the caspase-3, -8 and -9 significantly abolished by inhibition of p38 MAPK and JNK1/2 specific inhibitors. In this study, we determine that the effects of hispolon on the apoptosis and related regulation mechanism in HONE-1 and NPC-039 cells takes place. Our findings revealed that hispolon may be a useful candidate as a chemotherapeutic agent for NPC therapy.

Hispolon inhibition of inflammatory apoptosis through reduction of iNOS/NO production via HO-1 induction in macrophages.

ETHNOPHARMACOLOGICAL RELEVANCE: Phellinus linteus (Berkeley & Curtis), a well-known medical fungus, has long been used as a traditional medicine in Oriental countries to treat various diseases, and hispolon (HIS) is one of its bioactive components. HIS is known to possess potent antineoplastic and antiviral properties; however, its effect on inflammatory apoptosis is still undefined. MATERIALS AND METHODS: RAW264.7 macrophages were incubated with HIS for 30 min followed by LPS, LTA, or PGN stimulation for 12h. The expression of indicated proteins AP-1 and NF-κB transcriptional activities was examined by Western blotting using specific antibodies. Levels of NO and ROS were examined by Griess reaction, and DCHF-DA staining via flow cytometric analysis, respectively. AP-1 and NF-κB transcriptional activities were detected by luciferase reporter assay. Knockdown of HO-1 protein expression was performed by transfection of macrophages with HO-1 siRNA. Pharmacological inhibitors including ROS scavenger NAC, JNK inhibitor SP600125, NF-κB inhibitor BAY117082 were applied for mechanism study. RESULTS: HIS showed concentration-dependent inhibition of LPS, LTA, and PGN-induced iNOS protein expressions and NO production by RAW264.7 macrophages. Accordingly, HIS protected RAW264.7 cells from LPS-, LTA-, and PGN-induced apoptosis. Increased HO-1 by HIS was detected at both protein and mRNA levels along with an increase in intracellular peroxide, and this was inhibited by the translational inhibitor, cycloheximide (CHX), the transcriptional inhibitor, actinomycin D (Act D), and the reactive oxygen species scavenger, N-acetylcysteine (NAC). A mechanistic study indicated that inhibition of c-Jun N-terminal kinase (JNK) protein phosphorylation, and activator protein (AP)-1 and nuclear factor (NF)-κB activation were involved in the anti-inflammatory actions of HIS in macrophages. A structure-activity relationship analysis showed that HIS expressed the most potent effect of inhibiting iNOS and apoptosis elicited by LPS, LTA, and PGN with a significant increase in HO-1 protein in macrophages. CONCLUSIONS: Evidence supporting HIS prevention of inflammatory apoptosis via blocking NO production and inducing HO-1 protein expression in macrophages is provided, and the hydroxyl at position C3 is a critical substitution for the anti-inflammatory actions of HIS.

Estrogenic and anti-estrogenic activities of hispolon from Phellinus lonicerinus (Bond.) Bond. et sing.

Hispolon was the main antitumor active ingredient in Phellinus sensu lato species. In order to confirm the dual regulating estrogenic ingredient and obtain more effective natural estrogen replacement drugs, hispolon was separated from Phellinus lonicerinus (Bond.) Bond. et sing. Hispolon exhibited significant anti-proliferative effect against estrogen-sensitive ER (+) MCF-7 cells in the absence of estrogen, and exhibits antagonistic effects on 17ß-estradiol (E2)-induced MCF-7 cell proliferation when E2 and the different concentrations of hispolon were treated simultaneously. Hispolon also inhibited the proliferation of estrogen-negative ER (-) MDA-MB-231 cells at the concentration of 5.00×10(-5) M. The yeast two-hybrid experiments showed that hispolon had strong and non-selective effects on the estrogen receptor (ER) α and ERß at a concentration of 1.00×10(-6) M. The ERß-binding ability of hispolon was larger than ERα in the concentration range of 1.00×10(-9) M and 1.00×10(-7) M. Hispolon could increase the body weight coefficient, serum E2 and progesterone contents in immature female mice at dose of 9.10×10(-6) mol/kg, and increase coefficient of thymus and spleen in mice. The Gscores of hispolon-ERα and hispolon-ERß docked complexes were -7.93 kcal/mol and -7.79 kcal/mol in docking simulations. Hispolon presented dual regulating estrogenic activities, which showed estrogenic agonist activity at low concentration or lack of endogenous estrogen, and the estrogenic antagonistic effect was stimulated at high concentrations or too much endogenous estrogen. Hispolon could be used for treating the estrogen deficiency-related disease with the benefit of non-toxic to normal cells, good antitumor effects and estrogenic activity.