Therapeutic targeting of hepatocellular carcinoma cells with antrocinol, a novel, dual-specificity, small-molecule inhibitor of the KRAS and ERK oncogenic signaling pathways.

Until recently, sorafenib has been the only treatment approved by the U.S. Food and Drug Administration for patients with advanced hepatocellular carcinoma (HCC). Some patients, however, exhibit resistance to this treatment and subsequently experience cancer progression, recurrence, or death. Therefore, identifying a new alternative treatment for patients with little or no response to sorafenib treatment is vital. In this study, we explored the therapeutic potential and underlying molecular mechanism of antrocinol ((3aS,4R,6aS,10aR)-4-(hydroxymethyl)-7,7-dimethyldecahydro-1H-naphtho[1,8a-c]furan-1-one) in patients with HCC. The results indicated that antrocinol was more therapeutically effective than antrocin, Stivarga, and sorafenib against HCC cell lines. Antrocinol also substantially suppressed the expression of KRAS-GTP, p-MEK1/2, p-ERK1/2, and p-AKT in the Huh7 cell line. Additionally, antrocinol-induced apoptosis in the Huh7 cell line, inhibited the formation of tumorspheres, and suppressed the expression of cancer stem cell markers CD133, KLF4, CD44, OCT4, SOX2, and c-Myc. Animal studies revealed that antrocinol alone considerably suppressed tumor growth in nonobese diabetic/severe combined immunodeficient mice inoculated with Huh7 tumorspheres. It also synergistically enhanced the anticancer effect of sorafenib, resulting in enhanced suppression of tumor growth (p < 0.001) and tumorsphere formation (p < 0.001). In tumor samples resected from mice treated with antrocinol alone or in combination with sorafenib, immunohistochemical analysis revealed an increase in BAX expression and a decrease in ERK and AKT protein expression. To the best of our knowledge, this is the first report of the anti-HCC activity of antrocinol. With its higher therapeutic efficacy than that of sorafenib, antrocinol is a candidate drug for patients with HCC who demonstrate little or no response to sorafenib treatment.

Ovatodiolide and antrocin synergistically inhibit the stemness and metastatic potential of hepatocellular carcinoma via impairing ribosome biogenesis and modulating ERK/Akt-mTOR signaling axis.

Activation of mitogen-activated protein kinase (MAPK) and PI3K signaling confers resistance against sorafenib, a mainstay treatment for advanced hepatocellular carcinoma (HCC). Antrocin and ovatodiolide constitute as the most potent secondary metabolites isolated from Antrodia camphorata and Anisomeles indica, respectively. Both natural compounds have recently gained a lot of attention due to their putative inhibition of MAPK and PI3K signaling in various solid cancers. However, whether their combination is effective in HCC remains unknown. Here, we investigated their effect, alone or in various combinations, on MAPK and PI3K signaling pathways in HCC cells. An array of in vitro study were used to investigate anticancer and stemness effects to treat HCC, such as cytotoxicity, drug combination index, migration, invasion, colony formation, and tumor sphere formation. Drug effect in vivo was evaluated using mouse xenograft models. In this study, antrocin and ovatodiolide synergistically inhibited the SNU387, Hep3B, Mahlavu, and Huh7 cell lines. Sequential combination treatment of Huh7 and Mahlavu with ovatodiolide followed by antrocin resulted stronger cytotoxic effect than did treatment with antrocin followed by ovatodiolide, their simultaneous administration, antrocin alone, or ovatodiolide alone. In the Huh7 and Mahlavu cell lines, ovatodiolide→antrocin significantly suppressed colony formation and proliferation as well as markedly downregulated ERK1/2, Akt, and mTOR expression. Inhibition of ERK1/2 and Akt/mTOR signaling by ovatodiolide→antrocin suppressed ribosomal biogenesis, autophagy, and cancer stem cell-like phenotypes and promoted apoptosis in Huh7 and Mahlavu cells. The sorafenib-resistant clone of Huh7 was effectively inhibited by synergistic combination of both compound in vitro. Eventually, the ovatodiolide→antrocin combination synergistically suppressed the growth of HCC xenografts. Taken together, our findings suggested that ovatodiolide→antrocin combination may represent potential therapeutic approach for patients with advanced HCC.

Urokinase plasminogen activator induces epithelial-mesenchymal and metastasis of pancreatic cancer through plasmin/MMP14/TGF-ß axis, which is inhibited by 4-acetyl-antroquinonol B treatment.

BACKGROUND: The current standard therapy for metastatic pancreatic cancer is ineffective, necessitating a new treatment approach for prognosis improvement. The urokinase-plasmin activator (uPA) is a critical factor in epithelial-mesenchymal transition (EMT) and cancer metastasis, but its underlying mechanisms in pancreatic cancer remains elusive. METHODS: We investigated uPA expression in our pancreatic cancer cohort. A bioinformatics approach was used to further determine the role of uPA in pancreatic cancer. We employed MiaPaCa-2 and PANC-1 cell lines to investigate how uPA regulates EMT and metastasis in pancreatic cancer and present a novel approach aimed at inhibiting uPA in pancreatic cancer. RESULTS: We observed that higher uPA mRNA expression was significantly associated with overall-poor survival and progression-free survival in pancreatic cancer. uPA was highly expressed in tumor tissue. Gene set enrichment analysis revealed a positive association between uPA mRNA expression and EMT and transforming growth factor ß (TGF-ß) signaling pathways. Moreover, shRNA-mediated uPA gene knockdown reduced plasmin, MMP14, and TGF-ß activation, leading to the inhibition of PANC-1 cells' EMT marker expression, migration, invasion, and cell viability. Notably, 4-acetyl-antroquinonol B (4-AAQB) treatment suppressed MiaPaCa-2 and PANC-1 cell migratory and invasive abilities by inhibiting the uPA/MMP14/TGF-ß axis through upregulation of miR-181d-5p. In the xenograft mouse model of orthotropic pancreatic cancer, 4-AAQB treatment has reduced tumor growth and metastasis rate by deactivating uPA and improving the survival of the mice model. CONCLUSION: Accordingly, to extent of our knowledge and previous studies, we demonstrated that 4-AAQB is an anti Pan-Cancer drug, and may inhibit pancreatic cancer EMT and metastasis and serve as a new therapeutic approach for patients with late-stage pancreatic cancer.

Ovatodiolide inhibits the oncogenicity and cancer stem cell-like phenotype of glioblastoma cells, as well as potentiate the anticancer effect of temozolomide.

BACKGROUND: Ovatodiolide (Ova), a major bioactive diterpenoid isolate of Anisomeles indica has drawn considerable attention lately as an effective anticancer agent with several published works demonstrating its tumor-inhibitory activity in various cancer types. PURPOSE: In this study, we examined the modulatory effect of Ova on the oncogenicity, proliferation, and cancer stem cell-like traits of glioblastoma (GBM) cells, as well as investigated the underlying molecular mechanism for the anticancer activity of Ova in GBM cell lines, U-87MG and GBM8401. METHODS: The antiproliferative, apoptotic, and stemness-attenuating effects of Ova were evaluated using the sulforhodamine B (SRB) colorimetric assay, western blot and fluorescent immunocytochemistry. Cell apoptosis was analyzed based on variation in the expression levels of Bcl-2 family of regulator proteins Bax, Bak, Bcl-2 and Bcl-xL. RESULTS: Ova induced the apoptosis of the U-87MG and GBM8401 cells, as well as effectively inhibited the proliferation and motility of the GBM cell lines in a dose- and time-dependent manner. Ova-induced apoptosis correlated with increased Bax/Bcl-2 ratio, while inhibition of tumor cell migration and colony formation was associated with reduced Slug, Vimentin, NCadherin and ß-catenin protein expression and increased E-Cadherin. In addition, exposure to Ova inhibited tumorsphere formation, elicited downregulation of CD44, CD133, Sox2, and Oct4, as well as correlated with dysregulation of the JAK2-STAT3 signaling pathway. Furthermore, we showed for the first time to the best of our knowledge that Ova potentiate the chemotherapeutic effect of Temozolomide. CONCLUSION: Taken together, our findings demonstrate the anticancer potential of Ova in GBM and its efficacy in the treatment of GBM as monotherapy and in combination with Temozolomide.

Ovatodiolide suppresses nasopharyngeal cancer by targeting stem cell-like population, inducing apoptosis, inhibiting EMT and dysregulating JAK/STAT signaling pathway.

BACKGROUND: Treatment for metastatic nasopharyngeal carcinoma (NPC) is challenging. Till now, a truly effective chemotherapy regimen for NPC has not yet been identified. These clinical observations prompted us to investigate a potential drug as alternative option for treating. PURPOSE: This study evaluated the inhibitory effects of Ovatodiolide (Ova), on tumorigenic and cancer stem cell characteristics of NPC cells. METHODS: Two NPC cell lines (NPC-BM1 and NPC-BM2) were used to examine the anticancer effects of Ova and the molecular mechanism underlying these activities by using sulforhodamine B cytotoxicity assay, western blot, immunofluorescence, migration, colony and tumorsphere formation assays. RESULTS: Ova significantly inhibited the viability of BM1 and BM2 cells, downregulated Bcl-xL and Puma, and upregulated Bax/Bad expression levels. Ova dose-dependent suppressed migratory/invasive potential of NPC cells, and reduced ability to form colonies. Ova-induced apoptosis correlated with increased Bax/Bcl-xL ratio while NPC motility and colony formation inhibition were associated with reduced expression of p-FAK, p-PXN, F-actin, and Slug proteins and increased E-cadherin. Furthermore, ova inhibited NPC tumorsphere formation, associated with decreased SOX2, OCT4 and JAK-STAT signaling pathway. Ova also attenuated NPC stem cell tumorigenicity, inhibited tumor growth, and enhanced the sensitivity of NPC cells to cisplatin treatment, in vivo. CONCLUSIONS: Our results demonstrated the anticancer efficacy of Ova in NPC and its potential as a putative inhibitor of JAK2 and STAT3, which are essential in tumorigenesis of NPC. Further development of Ova is encouraged.

Antrocin, a bioactive component from Antrodia cinnamomea, suppresses breast carcinogenesis and stemness via downregulation of ß-catenin/Notch1/Akt signaling.

BACKGROUND: We identified increased ß-catenin and Atk expression was associated with drug resistance and poor prognosis in breast cancer patients using public databases. Antrocin treatment suppressed breast tumorigenesis and stemness properties. HYPOTHESIS/PURPOSE: We aimed to provide preclinical evidence for antrocin, an active component of Antrodia cinnamomea, as a potential small-molecule drug for treating drug-resistant breast cancer. METHODS: Various in vitro assays including SRB, Boyden chamber, colony formation, drug combination index and tumor sphere generation were used to determine the anti-cancer and stemness effects of antrocin. Mouse xenograft models were used to evaluate antrocin's effect in vivo. RESULTS: Antrocin treatment suppressed the viability, migration colony formation and mammosphere generation. Antrocin-mediated anti-cancer effects were associated with the decreased expression of oncogenic and stemness markers such as ß-catenin, Akt and Notch1. A sequential regimen of antrocin and paclitaxel synergistically inhibit breast cancer viability in vitro and in vivo. CONCLUSION: Our preclinical evidence supports antrocin's ability of inhibiting tumorigenic and stemness properties in breast cancer cells. Further develop of antrocin should be encouraged; the combined use of antrocin and paclitaxel may also be considered for future clinical trials.

Investigation of ovatodiolide, a macrocyclic diterpenoid, as a potential inhibitor of oral cancer stem-like cells properties via the inhibition of the JAK2/STAT3/JARID1B signal circuit.

BACKGROUND: The cancer stem cells (CSCs) have been shown to play key roles in the oral cancer initiation, distant metastasis, the development of chemoresistance and recurrence after treatment. Therefore, the inhibition of oral CSCs has been the target for therapeutic development. PURPOSE: In this study, we investigated the anti-CSCs potential of Ovatodiolide (Ova), a diterpenoid isolate of Anisomeles indica, in vitro and in vivo. METHODS: Oral CSCs were treated with Ova, and the expression of pluripotency factors Oct4, Sox-2, and Nanog were evaluated by western blot. Effect of Ova on self-renewal capacity and clonogenicity were assessed with the sphere formation and clonogenic assay in CSCs model derived from oral cancer cell. The effect of Ova was also investigated in a mouse xenograft model obtained by injecting nude mice with oral CSCs cells. RESULTS: We demonstrated that Ova significantly and dose-dependently suppressed oral cancer cell viability and colony formation; Ova markedly inhibited the ALDH1 activities and reduced the CD44high/ALDHrich cell sub-population. Additionally, Ova suppressed orosphere formation by down-regulating CD133, Klf4, Oct4A, Nanog and JARID1B expression. Furthermore, Ova-mediated anti-cancer effects were associated with the dose-dependent reduction in the expression levels of STAT3, p-STAT3, pJAK2, pAKT and pERK1/2 protein. Moreover, Ova synergistically enhanced the anticancer effect of cisplatin against the SAS, FaDu, HSC-3 and TW2.6 orospheres. Ova significantly attenuated the tumor-initiating potential of orosphere in mouse xegnograft model. CONCLUSION: These results demonstrate that Ova effectively suppressed oral tumorigenesis and stemness properties via JAK2/STAT3 signaling. Ova may be considered for future clinical usage.

Downregulation of Cancer Stemness by Novel Diterpenoid Ovatodiolide Inhibits Hepatic Cancer Stem Cell-Like Traits by Repressing Wnt/[Formula: see text]-Catenin Signaling.

The hierarchical tumor propagation or cancer stem cells (CSCs) model of carcinogenesis postulates that like physiologic adult stem cell (ASC), the CSCs positioned at the apex of any tumor population form the crux of tumor evolution with a constitutive regenerative capacity and differentiation potential. The propagation and recurrence of the characteristically heterogeneous and therapy-resistant hepatocellular carcinoma (HCC), adds to accumulating evidence to support this CSCs model. Based on the multi-etiologic basis of HCC formation which among others, focuses on the disruption of the canonical Wnt signaling pathway, this study evaluated the role of cembrane-type phytochemical, Ovatodiolide, in the modulation of the Wnt/[Formula: see text]-catenin pathway, and its subsequent effect on liver CSCs' activities. Our fluorescence-activated cell sorting (FACS) and quantitative RT-PCR analyses of side population (SP) indicated that CD133+ cells were [Formula: see text]-catenin-overexpressing, more aggressive, and resistant to the conventional anticancer agents, Cisplatin and Doxorubicin, when compared to [Formula: see text]-catenin-downregulated group. We demonstrated that marked upregulation of [Formula: see text]-catenin and its downstream targets effectively enhanced hepatosphere formation, with an associated induction of CD133, OCT4 and Sox2 expression and also caused an significant enhancement of HCC proliferation. However, treatment with Ovatodiolide induced downregulation of [Formula: see text]-catenin and its downstream effector genes, abolished hepatosphere formation and reversed the [Formula: see text]-catenin-associated enhancement of HCC growth. In summary, we demonstrated for the first time that Ovatodiolide suppressed the canonical Wnt signaling pathway, and inhibited the generation of liver CSCs; Thus, projecting Ovatodiolide as a putatively effective therapeutic agent for anti-HCC target therapy.

Ovatodiolide isolated from Anisomeles indica induces cell cycle G2/M arrest and apoptosis via a ROS-dependent ATM/ATR signaling pathways.

Ovatodiolide was isolated from the traditional Chinese medicinal herb Anisomeles indica, possesses anti-bacterial and anti-inflammatory properties; however, the anti-cancer activity and its mechanisms have been limitedly reported. This study aimed to examine the effect and molecular action of ovatodiolide in lung cancer cells. Cell cycle distribution and reactive oxygen species (ROS) generation were measured by flow cytometry. Apoptosis was detected by propidium iodide/annexin V staining and TUNEL assay. DNA damage was investigated by comet assay and γ-H2AX staining. Caspase activity was determined using caspase fluorometric kits. Moreover, protein levels were examined by western blot. Ovatodiolide provoked reactive oxygen species generation and DNA damage, as well as inhibited cell growth and induced apoptosis in human lung cancer A549 and H1299 cell lines. DNA damage-related molecules, ATM/ATR and CHK1/CHK2 were activated by ovatodiolide. Moreover, ovatodiolide-mediated G2/M arrest was associated with the decrease of Cyclin B1 and CDC25C levels, and increase of p21WAF1/CIP1 expression. Additionally, ovatodiolide-triggered apoptosis was through both intrinsic and extrinsic pathways characterized by the elevating PUMA, Bax, and DR5 proteins, decreasing Bcl-2 and Mcl-1, and activating caspase-8, caspase-9 and caspase-3. Caffeine, an ATM/ATR inhibitor, rescued ovatodiolide-mediated cell cycle arrest and apoptosis, but not reactive oxygen species generation. Nevertheless, antioxidant N-acetyl-cysteine completely blocked ovatodiolide-mediated molecular events, G2/M arrest, and apoptosis. These observations suggest that ovatodiolide stimulates reactive oxygen species generation, causes oxidative stress and DNA damage; subsequently, provokes DNA damage signaling pathways, eventually leads to block cell cycle at G2/M phase and trigger apoptosis in lung cancer A549 and H1299 cells.

Antcin-H Isolated from Antrodia cinnamomea Inhibits Renal Cancer Cell Invasion Partly through Inactivation of FAK-ERK-C/EBP-ß/c-Fos-MMP-7 Pathways.

Antcin-H, a natural triterpene, is purified from a famous anticancer medicinal mushroom, Antrodia cinnamomea, in Taiwan. This study showed that antcin-H inhibited the growth of human renal carcinoma 786-0 cells; the IC50 value (for 48 h) was 170 µM. Besides, the migration and invasion of 786-0 cells were suppressed by antcin-H under noncytotoxic concentrations (<100 µM); these events were accompanied by inhibition of FAK and Src kinase activities, decrease of paxillin phosphorylation, impairment of lamellipodium formation, and upregulation of TIMPs and downregulation of MMPs, especially MMP-7 expression. Luciferase reporter assay showed that antcin-H repressed the MMP-7 promoter activity, in parallel to inhibiting c-Fos/AP-1 and C/EBP-ß transactivation abilities. Moreover, antcin-H suppressed the activity of ERK1/2 and decreased the binding ability of C/EBP-ß and c-Fos on the upstream/enhancer region of MMP-7 promoter. Overall, this study demonstrated that the anti-invasive effect of antcin-H in human renal carcinoma 786-0 cells might be at least in part by abrogating focal adhesion complex and lamellipodium formation through inhibiting the Src/FAK-paxillin signaling pathways and decreasing MMP-7 expression through suppressing the ERK1/2-AP-1/c-Fos and C/EBP-ß signaling axis. Our findings provide the evidence that antcin-H may be an active component existing in A. cinnamomea with anticancer effect.

A comparative proteomic study of secretomes in kaempferitrin-treated CTX TNA2 astrocytic cells.

ETHNOPHARMACOLOGICAL RELEVANCE: Kaempferitrin is extracted in significantly high quantities from the leaves of Cinnamomum osmophloeum (C.O) and Bauhinia forficata, and are used as an antidiabetic herbal remedy in China and Brazil. Commercial product using dry Cinnamomum osmophloeum leaves has been sold locally in Taiwan. Oral administration of kaempferitrin reduced blood sugar in diabetic rats. AIM OF THE STUDY: Though previously demonstrated to activate the classical insulin signaling pathways, a mechanism for kaempferitrin is still not fully understood. Also, studies on kaempferitrin on immune related cells have been inconclusive, and people consuming extract containing kaempferitrin often happen to be at high risk of diabetes and neurodegenerative diseases. Therefore, for kaempferitrin to be used every day, a comprehensive study is needed. MATERIALS AND METHODS: Astrocytic cell line was used as a model to test the differentially regulated secretomes, to test kaempferitrin effect on CNS glia, on pro-inflammatory cytokines, and to test how different the mechanism of kaempferitrin is from that of insulin. CTX TNA2 astrocytic cells were differentially treated with and without 10 µM kaempferitrin for 24 h, and the conditioned medium was collected. For the proteomic study, protein in conditioned medium was trypsin digested, and resulting peptides in kaempferitrin/non-treated sample pair were differentially dimethyl labeled. The labeled peptides were further fractionated by StageTip-based strong-exchange method before LC-MS/MS analyses. Levels of interesting proteins were verified using Western or Eliza. C.O. leaf crude extract treated samples were included for a comparison of effects of purified kaempferitrin vs. kaempferitrin containing crude extract. RESULTS AND CONCLUSIONS: Data were obtained via ProteomeXchange with identifier PXD002814. It was found that no pro-inflammatory cytokines or inhibitory ECM were elevated upon treatment of kaempferitrin or a crude extract of C.O. leaves. This suggests that prolonged use of kaempferitrin containing herbs may not increase pro-inflammatory reaction. LDL-R trafficking between the cell membrane and the extracellular niche was regulated by kaempferitrin toward reduced secretion. Our proteomic study also demonstrated that molecules related to plasma membrane recycling were regulated by kaempferitrin. Our discoveries provide evidence that link kaempferitrin regulation for LDL-R and membrane recycling to the blood lipid regulation by the C.O. leaves extract. However, these proteins were differently regulated when cells were treated with crude extract. This demonstrates that the molecular interactions within crude extract of herbs are complex and may not act similar to the compound purified from the crude extract.

Antrodia cinnamomea sensitizes radio-/chemo-therapy of cancer stem-like cells by modulating microRNA expression.

ETHNOPHARMACOLOGICAL RELEVANCE: The discovery of many tissue-specific cancer stem cells (CSCs) continues to attract scientific attention. These CSCs are considered to be associated with chemo- and radio-resistance, and consequently, failure of conventional anticancer therapies. The recent demonstration of several microRNAs as enhancers of tumorigenicity via modulation of epithelial-mesenchymal transition and cancer stemness, makes them putative novel therapeutic target in oncology. Antrodia cinnamomea is a Chinese traditional medicine with several biological functions including anti-inflammation, antioxidant, and cancer prevention. However, the anti-CSC capability of A. Cinnamomea is not clear yet. AIM OF THE STUDY: To investigate the inhibitory effect of A. cinnamomea mycelium and extract on CSCs derived from various human cancer cell lines using our in-house therapeutics and human genome-wide miRNA screening panels. MATERIALS AND METHODS: A broad range of human cancer cell lines, including the acute monocytic leukemia (THP-1), glioblastoma multiforme (GBM 8401), lung carcinoma (A549), breast adenocarcinoma (MDA-MB-231), hepatoblastoma (HepG2), colorectal adenocarcinoma (SW620), and foreskin fibroblast (HS68), were exposed to A. cinnamomea in this study. CD133+ CSCs generated from the cell lines were characterized and isolated by flow cytometry, effect of chemo- and radiotherapy was assessed using the MTT assay, while the RT-PCR and human genome wide qRT-PCR determined the differential gene expression patterns. A comparative analysis of the anticancer effect of A. cinnamomea and Cisplatin, Taxol, or irradiation was also performed. RESULTS: Our results indicated that A. cinnamomea mycelium and its ethyl acetate extracts showed anti-proliferation effects against all types of CSCs, especially the lung, breast, and head and neck squamous cell carcinoma CSCs. Furthermore, CSCs treatment with A. cinnamomea combined with irradiation or chemotherapeutics demonstrated significant anti-cancer effect. We also established an association between the CSC-inhibitory effect of A. cinnamomea and significant downregulation of several microRNAs and cancer stemness expression levels in brain and breast CSCs. More importantly, higher CD133 expression is associated with poor prognosis in glioblastoma and breast cancer patients. CONCLUSION: Herein, we demonstrate the putative role of A. cinnamomea as an effective ethnopharmacologic therapeutic agent for cancer treatment.

Hepatoprotective activities of Antrodia camphorata and its triterpenoid compounds against CCl4-induced liver injury in mice.

ETHNOPHARMACOLOGY RELEVANCE: Antrodia camphorata (AC) is a rare and precious fungus indigenous to Taiwan used as a traditional medicine for the treatment of liver injury. Triterpenoids are the major bioactive constituents of A. camphorata and have been reported to possess hepatoprotective activities. To meet the increasing demand, artificial cultivation techniques have been developed. AIM OF THE STUDY: This study aims to evaluate the hepatoprotective activities of AC samples derived from different cultivation techniques and to dissect the main active triterpenoid compounds. MATERIALS AND METHODS: The ethanol extracts of five batches of AC samples, including wild growing fruiting bodies, cutting wood culture fruiting bodies, dish cultures, cutting wood culture mycelia, and submerged fermentation mycelia were orally administered (50mg/kg or 200mg/kg) to ICR mice for 7 days. On the last day, CCl4 (0.2%, 7mL/kg, i.p.) was used to induce liver injury, and the activities of serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were determined 24h after the injection. Moreover, a HepG2 cell model treated with CCl4 (0.35%) was used to screen the protective activities of 29 AC triterpenoids. After incubation for 6h, viabilities of the cells were tested using MTS assay. The in vivo hepatoprotective activities of antcin B and antcin K were further studied on the mice model by ALT and AST tests and histopathologic examinations. To elucidate the mechanisms, the mRNA levels of iNOS, COX2, TNF-α and IL-1ß, and the protein levels of NF-κB (p65/p-p65), iNOS and COX2 in liver tissues were determined. RESULTS: The wild growing or cutting wood culture fruiting bodies, and the dish cultures of AC showed more potent activities than the mycelia (P<0.001). At 20µM, 16 of 29 triterpenoids showed significant protective activities, increasing HepG2 cell viability from 46% of the CCl4 group to >90%. Antcin B and antcin K could dose-dependently (10 or 50mg/kg, 7 days, i.g.) decrease the serum levels of ALT and AST, and decrease the incidence of liver necrosis. The effects of 50mg/kg of antcin K or antcin B were almost identical to those of 100mg/kg silymarin. Furthermore, qRT-PCR and Western blotting analyses revealed they could down-regulate IL-1ß, TNF-α, iNOS, COX-2 and NF-κB in liver tissues at both transcriptional and translational levels. CONCLUSION: The results indicate that cultivation techniques remarkably affect the hepatoprotective activities of AC. Antcin K and antcin B are the major hepatoprotective compounds of A. camphorata, and the mechanism is related with anti-inflammation. Given its high natural abundance and good oral absorption, antcin K could be a promising drug candidate for liver injury.

Aloe-Emodin Enhances Tamoxifen Cytotoxicity by Suppressing Ras/ERK and PI3K/mTOR in Breast Cancer Cells.

Aloe-emodin (AE) is derived from Aloe vera and rhubarb (Rheum palmatum) and exhibits anticancer activities via multiple regulatory mechanisms in various cancers. AE can also enhance the anticancer efficacy of cisplatin, doxorubicin, docetaxel, and 5-fluorouracil; however, its effects remain poorly characterized. MCF-7, MDA-MB-231, MDA-MB-468, BT-474, and HCC-1954 breast cancer cell lines were treated with the indicated conditions of AE, and cell viability assays were performed. The expression levels of signaling proteins were determined by western blot analysis, intracellular reactive oxygen species (ROS), cell cycle distributions, and rates of apoptosis as estimated by flow cytometry. In comparison with other cells, MCF-7 cells were more sensitive to AE treatment; AE enhanced the cytotoxicity of 9[Formula: see text][Formula: see text]g/ml tamoxifen by reducing EGFR, ER[Formula: see text], Ras, ERK, c-Myc, and mTOR protein expression and blocking PI3K and mTOR activation. Finally, although co-treatment of AE with tamoxifen increased intracellular ROS, there were no effects on cell cycle progression. Besides facilitating tamoxifen-induced cell death, AE also enhanced the antiproliferative activity of tamoxifen by blocking Ras/ERK and PI3K/mTOR pathways in breast cancer cells, thus demonstrating the chemosensitizing potential of AE.

Ovatodiolide of Anisomeles indica Exerts the Anticancer Potential on Pancreatic Cancer Cell Lines through STAT3 and NF-κB Regulation.

Pancreatic cancer is the eighth leading cause of cancer death worldwide. Patients with pancreatic cancer are normally diagnosed at an advanced stage and present poor survival rate. Ovatodiolide (OV), a bioactive macrocyclic diterpenoid isolated from Anisomeles indica, showed cytotoxicity effects in pancreatic cancer cells by inhibiting cell proliferation and inducing apoptosis. Moreover, not only were cell adhesion and invasion markedly suppressed in a dose-dependent manner, but the mRNA expression of matrix metalloproteinase-9 (MMP-9) and focal adhesion kinase (FAK) was also significantly decreased. Western blot analysis indicated that OV potently suppressed the phosphorylation of STAT-3 and its upstream kinase including ERK1/2, P38, and AKT Ser473. Meanwhile, OV inactivated the nuclear factor kappa B (NF-κB) by inhibiting IκB kinase (IKK α/ß) activation and the subsequent suppression of inhibitor of kappa B (IκB) phosphorylation. These results demonstrated that OV could potentially inhibit Mia-PaCa2 cancer cells proliferation and induce apoptosis through modulation of NF-κB and STAT3 pathway. Moreover, OV suppressed cell invasiveness and interfered with cell-matrix adhesion in Mia-PaCa2 cancer cells by reducing MMP-9 and FAK transcription through suppressing NF-κB and STAT3 pathway. Taken together, our findings reveal a new therapeutic and antimetastatic potential of ovatodiolide for pancreatic cancer remedy.

Metabolites identification and multi-component pharmacokinetics of ergostane and lanostane triterpenoids in the anticancer mushroom Antrodia cinnamomea.

Antrodia cinnamomea is a precious medicinal mushroom popularly used for adjuvant cancer therapy in Taiwan. Its major bioactive constituents are ergostane and lanostane triterpenoids. Although clinical trials for A. cinnamomea have been recently initiated, its metabolism remains unclear. The present study aims to elucidate the metabolism and pharmacokinetics of A. cinnamomea in rats. After oral administration of an ethanol extract, 18 triterpenoids and 8 biotransformed metabolites were detected in rats plasma by UHPLC/qTOF-MS. Four of the metabolites were prepared by semi-synthesis and fully identified by NMR, while the others were tentatively characterized by comparing with the metabolites of single compounds (antcins B, C, H and K). Furthermore, a multi-component pharmacokinetic study of A. cinnamomea was carried out to monitor the plasma concentrations of 14 triterpenoids (ergostanes 1-3, 5-8, 14-16; lanostanes 9, 10, 17, 19) and 2 metabolites (M5, M6) by LC/MS/MS in rats after oral administration of the ethanol extract (1.0 g/kg). The results showed that ergostanes and Δ(7,9(11)) lanostanes, but not Δ(8) lanostanes, could get into circulation. The low-polarity ergostanes (antcins B and C) undertook hydrogenation (C-3 or C-7 carbonyl groups) or hydroxylation to produce polar metabolites. High-polarity ergostanes (antcins H and K) and Δ(7,9(11)) lanostanes were metabolically stable. We also discovered that ergostanes and lanostanes showed remarkably different pharmacokinetic patterns. The ergostanes were generally absorbed and eliminated rapidly, whereas the lanostanes remained in the plasma at a low concentration for a relatively long time. The results indicate that high-polarity ergostanes are the major plasma-exposed components of A. cinnamomea, and may play an important role in its therapeutic effects.

Selective apoptotic cell death effects of oral cancer cells treated with destruxin B.

BACKGROUND: Recent studies have revealed that destruxins (Dtx) have potent cytotoxic activities on individual cancer cells, however, data on oral cancer cells especial human are absent. METHODS: Destruxin B (DB) was isolated and used to evaluate the selective cytotoxicity with human oral cancer cell lines, GNM (Neck metastasis of gingival carcinoma) and TSCCa (Tongue squamous cell carcinoma) cells, and normal gingival fibroblasts (GF) were also included as controls. Cells were tested with different concentrations of DB for 24, 48, and 72 h by MTT assay. Moreover, the mechanism of cytotoxicity was investigated using caspase-3 Immunofluorescence, annexin V/PI staining, and the expression of caspase-3, Bax, and Bcl-2 by western blotting after treated with different concentrations of DB for 72 h as parameters for apoptosis analyses. RESULTS: The results show that DB exhibited significant (p < 0.01) and selective time- and dose-dependent inhibitory effects on GNM and TSCCa cells viability but not on GF cells. The data suggested that DB is capable to induce tumor specific growth inhibition in oral GNM and TSCCa cancer cells via Bax/Bcl-2-mediated intrinsic mitochondrial apoptotic pathway in time- and dose-dependent manners. CONCLUSIONS: This is the first report on the anti-proliferation effect of DB in oral cancer cells. The results reported here may offer further evidences to the development of DB as a potential complementary chemotherapeutic target for oral cancer complications.

Purification of kavalactones from Alpinia zerumbet and their protective actions against hydrogen peroxide-induced cytotoxicity in PC12 cells.

This study found that fruit shells of shell ginger (Alpinia zerumbet) are a rich source of the kavalactones dihydro-5,6-dehydrokavain (DDK) and 5,6-dehydrokavain (DK). The fruit shell extraction with hexane resulted in good purity and higher yields of DDK and DK than did chloroform, ethanol, 10% ethanol, methanol or water. Additionally, this study examined the neuroprotective effects of DDK and DK against H2O2-induced cytotoxicity in PC12 cells and the possible molecular mechanisms involved. 16 h after stimulation with 400 µM H2O2, the viability (MTT reduction) of PC12 cells decreased while membrane damage (LDH release) was noticeably increased. However, pretreatment for 6 h with DDK and DK (1 µM, 5 µM, 10 µM and 50 µM) rescued PC12 cells from H2O2-induced cytotoxicity, as evidenced by decreased LDH release and increased cell viability. DDK and DK inhibit the MAPK family member p38, activate AKT, and reduce caspase-3 activity. DDK also reduced the oxidative status in H2O2-treated PC12 cells. Together, our data indicate that the A. zerumbet constituents, DDK and DK, exert a protective effect against oxidative stress-induced PC12 cell death and that the regulation of p-Akt and the p38 MAPK, and of oxidative states may be involved.

Destruxin B Isolated from Entomopathogenic Fungus Metarhizium anisopliae Induces Apoptosis via a Bcl-2 Family-Dependent Mitochondrial Pathway in Human Nonsmall Cell Lung Cancer Cells.

Destruxin B, isolated from entomopathogenic fungus Metarhizium anisopliae, is one of the cyclodepsipeptides with insecticidal and anticancer activities. In this study, destruxin B was extracted and purified by ion-exchange chromatography, silica gel chromatography, and semipreparative high-performance liquid chromatography. The potential anticancer effects and molecular mechanisms of destruxin B in human nonsmall cell lung cancer cell lines were characterized. Our results showed that destruxin B induced apoptotic cell death in A549 cells. This event was accompanied by the activation of caspase-2, -3, and -9. Moreover, destruxin B increased the expression level of proapoptotic molecule, PUMA, while decreased antiapoptotic molecule Mcl-1. Additionally, the translocation of Bax from cytosol to mitochondrial membrane was observed upon destruxin B treatment. Knockdown of Bax by shRNA effectively attenuated destruxin-B-triggered apoptosis in A549 cells. Interestingly, similar toxic effects and underlying mechanisms including caspase activation, upregulation of PUMA, and downregulation of Mcl-1 were also observed in a p53-null lung cancer H1299 cell line upon destruxin B treatment. Taken together, our findings suggest that destruxin-B-induced apoptosis in human nonsmall cell lung cancer cells is via a Bcl-2 family-dependent mitochondrial pathway.

Ovatodiolide inhibits the maturation of allergen-induced bone marrow-derived dendritic cells and induction of Th2 cell differentiation.

Ovatodiolide was a unique macrocyclic diterpenoid isolated from the traditional Chinese medicinal herb Anisomeles indica. The present study attempted to examine the ovatodiolide effects on dendritic cell (DC) maturation and immuno-stimulatory activities. The effects of ovatodiolide on DC surface molecule expression, cytokine production, and capacity to induce T-cell differentiation were examined in ovalbumin (OVA)/thymic stromal lymphopoietin (TSLP)-stimulated DCs. Ovatodiolide attenuated the expression of DC surface molecules CD80, CD86, histocompatibility complex (MHC) class II, and Th2 subset of CD4(+) T cells co-stimulatory molecule-OX40 ligand production. Additionally, ovatodiolide suppressed the CD4(+) T cells proliferation, and production of inflammatory cytokines interleukin (IL)-4, IL-5, and tumor necrosis factor (TNF)-α. This study may be useful to develop ovatodiolide as a therapeutic adjuvant.