Targeting of FSP1 regulates iron homeostasis in drug-tolerant persister head and neck cancer cells via lipid-metabolism-driven ferroptosis.

BACKGROUND: Research has demonstrated that some tumor cells can transform into drug-tolerant persisters (DTPs), which serve as a reservoir for the recurrence of the disease. The persister state in cancer cells arises due to temporary molecular reprogramming, and exploring the genetic composition and microenvironment during the development of head and neck squamous cell carcinoma (HNSCC) can enhance our comprehension of the types of cell death that HNSCC, thus identifying potential targets for innovative therapies. This project investigated lipid-metabolism-driven ferroptosis and its role in drug resistance and DTP generation in HNSCC. METHODS: High levels of FSP1 were discovered in the tissues of patients who experienced relapse after cisplatin treatment. RNA sequencing indicated that a series of genes related to lipid metabolism were also highly expressed in tissues from these patients. Consistent results were obtained in primary DTP cells isolated from patients who experienced relapse. The Cancer Genome Atlas database confirmed this finding. This revealed that the activation of drug resistance in cancer cells is influenced by FSP1, intracellular iron homeostasis, and lipid metabolism. The regulatory roles of ferroptosis suppressor protein 1 (FSP1) in HNSCC metabolic regulation were investigated. RESULTS: We generated human oral squamous cell carcinoma DTP cells (HNSCC cell line) to cisplatin and observed higher expression of FSP1 and lipid-metabolism-related targets in vitro. The shFSP1 blockade attenuated HNSCC-DTP cell stemness and downregulated tumor invasion and the metastatic rate. We found that cisplatin induced FSP1/ACSL4 axis expression in HNSC-DTPC cells. Finally, we evaluated the HNSCC CSC-inhibitory functions of iFSP1 (a metabolic drug and ferroptosis inducer) used for neo-adjuvant chemotherapy; this was achieved by inducing ferroptosis in a patient-derived xenograft mouse model. CONCLUSIONS: The present findings elucidate the link between iron homeostasis, ferroptosis, and cancer metabolism in HNSCC-DTP generation and acquisition of chemoresistance. The findings may serve as a suitable model for cancer treatment testing and prediction of precision treatment outcomes. In conclusion, this study provides clinically oriented platforms for evaluating metabolism-modulating drugs (FSP1 inhibitors) and new drug candidates of drug resistance and ferroptotic biomarkers.

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.

γ-Mangostin isolated from Garcinia mangostana L. suppresses inflammation and alleviates symptoms of osteoarthritis via modulating miR-124-3p/IL-6/NF-κB signaling.

Osteoarthritis (OA) a disease associated with joints and become severe with age, due to softening, inflammation and degradation of cartilage in joints. The agents that can target OA is needed, specifically without any side effects. Garcinia mangostana L. (Mangosteen) a tropical fruit used to treat many skin and stomach associated ailments. γ- Mangostin (γ-MS) a key bioactive substance present in mangosteen. Here, we aimed to explore γ-MS potential in targeting the pro-inflammatory cytokine, factors and miRs in OA progression. Significantly, γ-MS suppresses the inflammatory cytokines (IL-6, TNF-α, and INF- γ) and factors (NF-κB, STAT3, and COX-2) which regulates/participate in the catabolic process of cartilage destruction. Result of Hematoxylin-eosin (H&E) staining of tissue sections of OA joints of γ-MS treated and non-treated mice confirm γ-MS improves the signs of injuries, and maintains the structural integrity of the articular cartilage (epiphyseal disk joints and bone marrow) and reduces inflammation. Mechanistically, γ-MS targets miR-98-5p and miR-124-3p which are found to suppress the expression IL-6 and NF-κB, respectively. But in OA these miRs are inhibited, especially miR-124-3p which regulates not only NF-κB but also TNF-α, IL-6 and MMP7. With a further investigation underway, γ-MS represents an important source for treating and managing OA.

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.

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.

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.

Corrigendum to "Honokiol Eliminates Human Oral Cancer Stem-Like Cells Accompanied with Suppression of Wnt/ß-Catenin Signaling and Apoptosis Induction".

[This corrects the article DOI: 10.1155/2013/146136.].

Honokiol inhibits sphere formation and xenograft growth of oral cancer side population cells accompanied with JAK/STAT signaling pathway suppression and apoptosis induction.

BACKGROUND: Eliminating cancer stem cells (CSCs) has been suggested for prevention of tumor recurrence and metastasis. Honokiol, an active compound of Magnolia officinalis, had been proposed to be a potential candidate drug for cancer treatment. We explored its effects on the elimination of oral CSCs both in vitro and in vivo. METHODS: By using the Hoechst side population (SP) technique, CSCs-like SP cells were isolated from human oral squamous cell carcinoma (OSCC) cell lines, SAS and OECM-1. Effects of honokiol on the apoptosis and signaling pathways of SP-derived spheres were examined by Annexin V/Propidium iodide staining and Western blotting, respectively. The in vivo effectiveness was examined by xenograft mouse model and immunohistochemical tissue staining. RESULTS: The SP cells possessed higher stemness marker expression (ABCG2, Ep-CAM, Oct-4 and Nestin), clonogenicity, sphere formation capacity as well as tumorigenicity when compared to the parental cells. Treatment of these SP-derived spheres with honokiol resulted in apoptosis induction via Bax/Bcl-2 and caspase-3-dependent pathway. This apoptosis induction was associated with marked suppression of JAK2/STAT3, Akt and Erk signaling pathways in honokiol-treated SAS spheres. Consistent with its effect on JAK2/STAT3 suppression, honokiol also markedly inhibited IL-6-mediated migration of SAS cells. Accordingly, honokiol dose-dependently inhibited the growth of SAS SP xenograft and markedly reduced the immunohistochemical staining of PCNA and endothelial marker CD31 in the xenograft tumor. CONCLUSIONS: Honokiol suppressed the sphere formation and xenograft growth of oral CSC-like cells in association with apoptosis induction and inhibition of survival/proliferation signaling pathways as well as angiogenesis. These results suggest its potential as an integrative medicine for combating oral cancer through targeting on CSCs.

Pterostilbene inhibits triple-negative breast cancer metastasis via inducing microRNA-205 expression and negatively modulates epithelial-to-mesenchymal transition.

Breast cancer is the leading cause of cancer-related deaths among females in economically developing countries. Greater than 95% of breast malignancies are of epithelial origin; the induction of epithelial-to-mesenchymal transition (EMT) has been shown to initiate the metastatic process in breast carcinoma and remains the key target for drug development. Here, we examine the anti-metastatic potential of pterostilbene in modulating EMT process in breast cancer cells both in vitro and in vivo. The differential invasive ability among MCF7, Hs578t and MDA-MB-231 breast cancer cell lines were closely correlated with the expression of EMT markers, determined by Western blots and Matrigel-coated transwells assay. Pterostilbene inhibited the migratory and invasive potential of triple-negative MDA-MB-231 and Hs578t cells, accompanied by the up-regulation of E-cadherin and down-regulation of Snail, Slug, vimentin and ZEB1. Mechanistic investigations revealed a significant up-regulation of miR-205, which resulted in the reduction of Src expression in pterostilbene-treated breast cancer cells. Importantly, pterostilbene suppressed tumor growth and metastasis in MDA-MB-231-bearing NOD/SCID mice by reducing Src/Fak signaling; this observation was consistent with the negative correlations between miR-205 and Src expression in both normal and malignant breast tissues. Our findings provide supports for the usage of pterostilbene as an inhibitor of EMT process and potential candidate for adjuvant therapy.

BlueBerry Isolate, Pterostilbene, Functions as a Potential Anticancer Stem Cell Agent in Suppressing Irradiation-Mediated Enrichment of Hepatoma Stem Cells.

For many malignancies, radiation therapy remains the second option only to surgery in terms of its curative potential. However, radiation-induced tumor cell death is limited by a number of factors, including the adverse response of the tumor microenvironment to the treatment and either intrinsic or acquired mechanisms of evasive resistance, and the existence of cancer stem cells (CSCs). In this study, we demonstrated that using different doses of irradiation led to the enrichment of CD133(+) Mahlavu cells using flow cytometric method. Subsequently, CD133(+) Mahlavu cells enriched by irradiation were characterized for their stemness gene expression, self-renewal, migration/invasion abilities, and radiation resistance. Having established irradiation-enriched CD133(+) Mahlavu cells with CSC properties, we evaluated a phytochemical, pterostilbene (PT), found abundantly in blueberries, against irradiation-enriched CSCs. It was shown that PT treatment dose-dependently reduced the enrichment of CD133(+) Mahlavu cells upon irradiation; PT treatment also prevented tumor sphere formation, reduced stemness gene expression, and suppressed invasion and migration abilities as well as increasing apoptosis of CD133(+) Mahlavu CSCs. Based on our experimental data, pterostilbene could be used to prevent the enrichment of CD133(+) hepatoma CSCs and should be considered for future clinical testing as a combined agent for HCC patients.

A preclinical evaluation of antimycin a as a potential antilung cancer stem cell agent.

Drug resistance and tumor recurrence are major obstacles in treating lung cancer patients. Accumulating evidence considers lung cancer stem cells (CSCs) as the major contributor to these clinical challenges. Agents that can target lung CSCs could potentially provide a more effective treatment than traditional chemotherapy. Here, we utilized the side-population (SP) method to isolate lung CSCs from A549 and PC-9 cell lines. Subsequently, a high throughput platform, connectivity maps (CMAPs), was used to identify potential anti-CSC agents. An antibiotic, antimycin A (AMA), was identified as a top candidate. SP A549 cells exhibited an elevated stemness profile, including Nanog, ß -catenin, Sox2, and CD133, and increased self-renewal ability. AMA treatment was found to suppress ß -catenin signaling components and tumor sphere formation. Furthermore, AMA treatment decreased the proliferation of gefitinib-resistant PC-9/GR cells and percentage of SP population. AMA demonstrated synergistic suppression of PC-9/GR cell viability when combined with gefitinib. Finally, AMA treatment suppressed tumorigenesis in mice inoculated with A549 SP cells. Collectively, we have identified AMA using CMAP as a novel antilung CSC agent, which acts to downregulate ß -catenin signaling. The combination of AMA and targeted therapeutic agents could be considered for overcoming drug resistance and relapse in lung cancer patients.

Honokiol Eliminates Human Oral Cancer Stem-Like Cells Accompanied with Suppression of Wnt/ ß -Catenin Signaling and Apoptosis Induction.

Honokiol, an active compound of Magnolia officinalis, exerted many anticancer effects on various types of cancer cells. We explored its effects on the elimination of cancer stem-like side population (SP) cells in human oral squamous cell carcinoma SAS cells. The sorted SP cells possessed much higher expression of stemness genes, such as ABCG2, ABCC5, EpCAM, OCT-4, CD133, CD44, and ß -catenin, and more clonogenicity as compared with the Non-SP cells. After 48 h of treatment, honokiol dose dependently reduced the proportion of SP from 2.53% to 0.09%. Apoptosis of honokiol-treated SP cells was evidenced by increased annexin V staining and cleaved caspase-3 as well as decreased Survivin and Bcl-2. Mechanistically, honokiol inhibited the CD44 and Wnt/ ß -catenin signaling of SP cells. The Wnt signaling transducers such as ß -catenin and TCF-4 were decreased in honokiol-treated SP cells, while the ß -catenin degradation promoting kinase GSK-3 α / ß was increased. Consistently, the protein levels of ß -catenin downstream targets such as c-Myc and Cyclin D1 were also downregulated. Furthermore, the ß -catenin-related EMT markers such as Slug and Snail were markedly suppressed by honokiol. Our findings indicate honokiol may be able to eliminate oral cancer stem cells through apoptosis induction, suppression of Wnt/ ß -catenin signaling, and inhibition of EMT.

Elimination of cancer stem-like "side population" cells in hepatoma cell lines by chinese herbal mixture "tien-hsien liquid".

There are increasing pieces of evidence suggesting that the recurrence of cancer may result from a small subpopulation of cancer stem cells, which are resistant to the conventional chemotherapy and radiotherapy. We investigated the effects of Chinese herbal mixture Tien-Hsien Liquid (THL) on the cancer stem-like side population (SP) cells isolated from human hepatoma cells. After sorting and subsequent culture, the SP cells from Huh7 hepatoma cells appear to have higher clonogenicity and mRNA expressions of stemness genes such as SMO, ABCG2, CD133, ß-catenin, and Oct-4 than those of non-SP cells. At dose of 2 mg/mL, THL reduced the proportion of SP cells in HepG2, Hep3B, and Huh7 cells from 1.33% to 0.49%, 1.55% to 0.43%, and 1.69% to 0.27%, respectively. The viability and colony formation of Huh7 SP cells were effectively suppressed by THL dose-dependently, accompanied with the inhibition of stemness genes, e.g., ABCG2, CD133, and SMO. The tumorigenicity of THL-treated Huh7 SP cells in NOD/SCID mice was also diminished. Moreover, combination with THL could synergize the effect of doxorubicin against Huh7 SP cells. Our data indicate that THL may act as a cancer stem cell targeting therapeutics and be regarded as complementary and integrative medicine in the treatment of hepatoma.

Pro-cellular survival and neuroprotection of citrus flavonoid: the actions of hesperetin in PC12 cells.

Hesperetin protects cells against oxidative stress by diverse mechanisms including receptor-mediated actions. PGC-1α and seladin-1 provide potential targets for intervention in oxidative stress-associated neurodegeneration. PC12 cells express TrkA and estrogen receptor (ER). It is known that TrkA triggers the MAPK/ERK1, PI-3 K/Akt, PLCγ/PKC and cAMP/PKA pathways, and membrane ER triggers the MAPK/ERK1, PKA, Akt/PKB or PKC pathway. Using PC12 cells and immunoblotting, we show that hesperetin induces the rapid (15 min) and sustained (~24 h) expression of PGC-1α (regulated by CREB) and seladin-1 (regulated by ER); hesperetin activates PI-3 K, PKA, PKC, ERK1 and CREB, and it induces PI-3 K, PKA, PGC-1α and seladin-1 via both ER and TrkA; any inhibitor of PI-3 K, PKA or PKC effectively suppresses the activation of ERK1 and CREB as well as the induction of PGC-1α and seladin-1; ERK1 inhibitors effectively suppress hesperetin-induced CREB activation and PGC-1α expression, but have no effect on the induction of seladin-1. This study reveals that hesperetin triggers ER- and TrkA-mediated parallel pathways, collaborating to induce proteins regulated by different transcriptional factors. This novel mechanism explains why hesperetin, although it is known to have relatively low antioxidant and estrogen activities, can exhibit multiple neuroprotective effects.

Apoptotic Cell Death and Inhibition of Wnt/ß-Catenin Signaling Pathway in Human Colon Cancer Cells by an Active Fraction (HS7) from Taiwanofungus camphoratus.

Aberrant activation of Wnt/ß-catenin signaling plays an important role in the development of colon cancer. HS7 is an active fraction extracted from Taiwanofungus camphoratus, which had been widely used as complementary medicine for Taiwan cancer patients in the past decades. In this study, we demonstrated the effects of HS7 on the growth inhibition, apoptosis induction, and Wnt/ß-catenin signaling suppression in human colon cancer cells. HS7 significantly inhibited proliferation of HT29, HCT116, and SW480 colon cancer cells in a dose- and time-dependent manner. The apoptosis induction was evidenced by DNA fragmentation and subG1 accumulation, which was associated with increased Bax/Bcl-2 ratio, activation of caspase-3 and cleavage of PARP. By using Tcf-dependent luciferase activity assay, HS7 was found to inhibit the ß-catenin/Tcf transcriptional activities. In addition, HS7 strongly suppressed the binding of Tcf complexes to its DNA-binding site shown in electrophoretic mobility shift assay. This inhibition was further confirmed by the decreased protein levels of Tcf-4 and ß-catenin. The ß-catenin/Tcf downstream target genes, such as survivin, c-myc, cyclin D1, MMP7, and MT1-MMP involved in apoptosis, invasion, and angiogenesis were also diminished as well. These results indicate that Taiwanofungus camphoratus may provide a benefit as integrative medicine for the treatment of colon cancer.

Dietary phenolic acids attenuate multiple stages of protein glycation and high-glucose-stimulated proinflammatory IL-1beta activation by interfering with chromatin remodeling and transcription in monocytes.

This study examined the effects of dietary phenolic acids on individual stages of protein glycation and utilized monocyte cultures to assess whether these phytochemicals modulate the activation of proinflammatory cytokine under high glucose (HG, 15 mmol/L) conditions mimicking diabetes. In vitro glycation assays showed that a number of phenolic acids exerted inhibitory effects on the glycation reaction and its subsequent crosslinking. Phenolic acids, especially methoxyphenolic acids, prevented increase in both levels of the interleukin-1beta (IL-1beta) and oxidative stress caused by HG. The effect appeared to be mediated by modulation of the protein kinase C/nuclear factor-kappaB axis. Chromatin immunoprecipitation demonstrated for the first time that HG increased the recruitment of nuclear factor-kappaB p65 and CREB-binding protein to the IL-1beta promoter. Interestingly, HG also increased histone acetylation and methylation within the IL-1beta promoter and decreased histone deacetylase activities in monocytes, thus facilitating chromatin remodeling and transcription. Such inappropriate inflammatory responses were found to be controlled effectively by treatment with methoxyphenolic compounds. In conclusion, this study suggests that phenolic acids could exert their anti-inflammatory activities as antiglycation agents and as modifiers of signaling pathways. It provides evidence for a novel mechanism by which phenolics supplementation might have additional protective effects against diabetic complications.

Antihypertensive effects of Hsian-tsao and its active compound in spontaneously hypertensive rats.

The present study aimed to investigate the effects of Hsian-tsao (Mesona procumbens Hemsl.) and its active compound on blood pressure, lipid peroxidation and total antioxidant status of spontaneously hypertensive rats (SHRs). Male SHRs were orally administrated either a water extract of Hsian-tsao (WEHT) (1.0 g/kg) or caffeic acid (CA) [0.1 g/kg of body weight (BW)] on a daily basis for 6 weeks. The results indicated that both hepatic and plasmatic malondialdehyde concentration were increased and total liver glutathione (GSH) levels and antioxidant enzyme activities were decreased in SHRs when compared to the control Wistar Kyoto rats at the end of the trail. In SHRs, oral administration of WEHT or CA for 6 weeks reduced blood pressure as well as plasma and hepatic malondialdehyde levels and increased hepatic antioxidant enzyme activities when compared to SHRs control rats. Reverse transcriptase-polymerase chain reaction results indicated that the changes in hepatic antioxidant enzyme mRNA levels by WEHT or CA were similar to those noted in the enzyme activity levels. The hepatic levels obtained from WEHT or CA-administrated rats had significantly greater oxygen radical absorbance capacity values and total GSH levels than those of control rats. Following oral administration of CA, phenolic acid was detected in the plasma, and C(max) value after 1.0 h administration was 0.92 micromol/L. These findings indicate that a supplement of Hsian-tsao may prevent development of increased blood pressure and enhance the total antioxidant status in vivo.