Targeting the Neuropilin-1 receptor with Ovatodiolide and progress in using periodontal ligament organoids for COVID-19 research and therapy.

The discovery of SARS-CoV-2 RNA in the periodontal tissues of patients who tested positive for COVID-19, 24 days post the initial symptom onset, indicates the oral cavity could serve as a viral reservoir. This research aims to investigate the antiviral capabilities of Ovatodiolide, introducing a novel periodontal ligament organoid model for the study of SARS-CoV-2. We have successfully established a reliable and expandable organoid culture from the human periodontal ligament, showcasing characteristics typical of epithelial stem cells. This organoid model enables us to delve into the lesser-known aspects of dental epithelial stem cell biology and their interactions with viruses and oral tissues. We conducted a series of in vitro and ex vivo studies to examine the inhibitory impacts of Ova on SARS-CoV-2. Our findings indicate that Ovatodiolide molecules can bind effectively to the NRP1 active domain. Our study identifies potential interaction sites for Ovatodiolide (OVA) within the b1 domain of the NRP1 receptor. We generated point mutations at this site, resulting in three variants: Y25A, T44A, and a double mutation Y25A/T44A. While these mutations did not alter the binding activity of the spike protein, they did impact the concentration of OVA required for inhibition. The inhibitory concentrations for these variants are 15 µM for Y25A, 15.2 µM for T44A, and 25 µM for the double mutant Y25A/T44A. In addition, in vitro inhibition experiments demonstrate that the EC50 of Ova against the main protease (Mpro) of the SARS-CoV-2 virus is 7.316 µM. Our in vitro studies and the use of the periodontal ligament organoid model highlight Ovatodiolide's potential as a small molecule therapeutic agent that impedes the virus's ability to bind to the Neuropilin-1 receptor on host cells. The research uncovers various pathways and biochemical strategies through which Ovatodiolide may function as an effective antiviral small molecule drug.

Anti-Oxidant, Anti-Mutagenic Activity and Safety Evaluation of Antrocin.

Antrocin is a novel compound isolated from Antrodia cinnamomea, and is classified as a sesquiterpene lactone. The therapeutic efficacy of antrocin has been studied, and it has shown an antiproliferative effect on various cancers. The aim of this study was to evaluate the anti-oxidant activity, potential genotoxicity, and oral toxicity of antrocin. Ames tests with five different strains of Salmonella typhimurium, chromosomal aberration tests in CHO-K1 cells, and micronucleus tests in ICR mice were conducted. The results of anti-oxidant capacity assays showed that antrocin has great anti-oxidant activity and is a moderately strong antimutagenic agent. In the results of the genotoxicity assays, antrocin did not show any mutagenic potential. In the 28-day oral toxicity test, Sprague Dawley rats were gavaged with 7.5 or 37.5 mg/kg of antrocin for 28 consecutive days. In addition, 7.5 mg/kg sorafenib, an anti-cancer drug, was used as a positive control for toxicity comparison. At the end of the study, antrocin did not produce any toxic effects according to hematology, serum chemistry, urine analysis, or histopathological examinations. According to the results of the genotoxicity and 28-day oral toxicity study, antrocin, at a dose of 37.5 mg/kg, did not cause adverse effects and can be a reference dose for therapeutic agents in humans.

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.

Application of Multiple Strategies to Improve the Production of the Potential Cancer Drug 4-Acetylantroquinonol B (4-AAQB) by the Rare Fungus Antrodia cinnamomea.

4-Acetylantroquinonol B (4-AAQB) was identified in the rare fungus Antrodia cinnamomea and has been proven to be a potential therapeutic agent for cancer treatment. But the extraction of 4-AAQB from the fruit body led to a low yield and limited its further application in the pharmaceutical field. In this work, 4-AAQB production was enhanced in the submerged fermentation by the combination of exogenous additives, surfactants with the in situ extractive fermentation. 4-Methylbenzoic acid was proven to be an efficient additive for the accumulation of 4-AAQB by Antrodia cinnamomea, while 2% (w/v) Tween-80 added on the first day as surfactant and 30% (w/v) oleic acid added on the sixteenth day as extractant were the most available couples for 4-AAQB production in the in situ extractive fermentation. The combination of these multiple strategies resulted in the yield of 4-AAQB to 17.27 mg/g dry cell weight with a titer of 140 mg/L, which was the highest titer of 4-AAQB reported so far. It showed that the combination of these strategies had a significant promotion on 4-AAQB production by A. cinnamomea, which laid a good foundation for its large-scale production and also provided a viable method for the cultivation of other rare fungi.

Identification of a Novel Theranostic Signature of Metabolic and Immune-Inflammatory Dysregulation in Myocardial Infarction, and the Potential Therapeutic Properties of Ovatodiolide, a Diterpenoid Derivative.

Myocardial infarction (MI) is a multifactorial global disease, recognized as one of the leading causes of cardiovascular morbidity and mortality. Timely and correct diagnoses and effective treatments could significantly reduce incidence of complications and improve patient prognoses. In this study, seven unconventional differentially expressed genes (DEGs) (MAN2A2, TNFRSF12A, SPP1, CSNK1D, PLAUR, PFKFB3, and CXCL16, collectively termed the MTSCPPC signature) were identified through integrating DEGs from six MI microarray datasets. The pathological and theranostic roles of the MTSCPPC signature in MI were subsequently analyzed. We evaluated interactions of the MTSCPPC signature with ovatodiolide, a bioactive compound isolated from Anisomeles indica (L.) Kuntze, using in silico molecular docking tools and compared it to specific inhibitors of the members of the MTSCPPC signature. Single-cell transcriptomic analysis of the public databases revealed high expression levels of the MTSCPPC signature in immune cells of adult human hearts during an MI event. The MTSCPPC signature was significantly associated with the cytokine-cytokine receptor interactions, chemokine signaling, immune and inflammatory responses, and metabolic dysregulation in MI. Analysis of a micro (mi)RNA regulatory network of the MTSCPPC signature suggested post-transcriptional activation and the roles of miRNAs in the pathology of MI. Our molecular docking analysis suggested a higher potential for ovatodiolide to target MAN2A2, CSNK1D, and TNFRSF12A. Collectively, the results derived from the present study further advance our understanding of the complex regulatory mechanisms of MI and provide a potential MI theranostic signature with ovatodiolide as a therapeutic candidate.

Identification and semisynthesis of (-)-anisomelic acid as oral agent against SARS-CoV-2 in mice.

(-)-Anisomelic acid, isolated from Anisomeles indica (L.) Kuntze (Labiatae) leaves, is a macrocyclic cembranolide with a trans-fused α-methylene-γ-lactone motif. Anisomelic acid effectively inhibits SARS-CoV-2 replication and viral-induced cytopathic effects with an EC50 of 1.1 and 4.3 µM, respectively. Challenge studies of SARS-CoV-2-infected K18-hACE2 mice showed that oral administration of anisomelic acid and subcutaneous dosing of remdesivir can both reduce the viral titers in the lung tissue at the same level. To facilitate drug discovery, we used a semisynthetic approach to shorten the project timelines. The enantioselective semisynthesis of anisomelic acid from the naturally enriched and commercially available starting material (+)-costunolide was achieved in five steps with a 27% overall yield. The developed chemistry provides opportunities for developing anisomelic-acid-based novel ligands for selectively targeting proteins involved in viral infections.

Genotoxicity and 28-day repeated dose oral toxicity study of ovatodiolide in rats.

Ovatodiolide is a bioactive cembrane-type diterpenoid isolated from Anisomeles indica (L.) Kuntze. It has been proven that ovatodiolide is anti-inflammatory, anti-tumorigenic, anti-melanogenic and attenuates asthma by regulating signaling pathways. The aim of this study was to evaluate the safety of ovatodiolide by conducting genotoxicity tests and 28-day oral toxicity tests in rats. Genotoxicity assays were conducted by using a bacterial reverse mutation test and mammalian chromosomal aberration test to assess whether ovatodiolide causes reverse mutations and mutagenicity with or without metabolism activation. For the in vivo mammalian erythrocyte micronucleus test, mice were administered a single dose of 0, 250, 500 or 1000 mg/kg b.w. ovatodiolide by single gavage. In the acute oral toxicity test, rats were given a single dose of ovatodiolide 1000 mg/kg b.w. by single gavage. In the 28-day oral toxicity test, groups were divided into a control, ovatodiolide 10, 25 and 50 mg/kg b.w. The results showed that there was no mutagenicity in the bacterial reverse mutation test or the mammalian chromosomal aberration test with or without S9 fraction. Ovatodiolide did not produce an increase in micronucleated reticulocytes in the micronucleus test. The results revealed that the acute oral toxicity of ovatodiolide is over 1000 mg/kg b.w. in rats. Moreover, 10, 25 and 50 mg/kg b.w. of ovatodiolide did not cause a significant effect in rats. According to the results of the genotoxicity and oral toxicity studies in rats, ovatodiolide did not produce any adverse effects, and the tested doses can serve as clinical references.

4-Acetyl-Antroquinonol B Improves the Sensitization of Cetuximab on Both Kras Mutant and Wild Type Colorectal Cancer by Modulating the Expression of Ras/Raf/miR-193a-3p Signaling Axis.

The KRAS mutation is one of the leading driver mutations in colorectal cancer (CRC), and it is usually associated with poor prognosis and drug resistance. Therapies targeting the epidermal growth factor receptor (EFGR) are widely used for end-stage CRC. However, patients with KRAS mutant genes cannot benefit from this therapy because of Ras signaling activation by KRAS mutant genes. Our previous study revealed the anti-proliferative effect of 4-acetyl-antroquinonol B (4-AAQB) on CRC cells, but whether the drug is effective in KRAS-mutant CRC remains unknown. We screened CRC cell lines harboring the KRAS mutation, namely G12A, G12C, G12V and G13D, with one wild type cell line as the control; SW1463 and Caco-2 cell lines were used for further experiments. Sulforhodamine B assays, together with the clonogenicity and invasion assay, revealed that KRAS-mutant SW1463 cells were resistant to cetuximab; however, 4-AAQB treatment effectively resensitized CRC cells to cetuximab through the reduction of colony formation, invasion, and tumorsphere generation and of oncogenic KRAS signaling cascade of CRC cells. Thus, inducing cells with 4-AAQB before cetuximab therapy could resensitize KRAS-mutant, but not wild-type, cells to cetuximab. Therefore, we hypothesized that 4-AAQB can inhibit KRAS. In silico analysis of the publicly available GEO (GSE66548) dataset of KRAS-mutated versus KRAS wild-type CRC patients confirmed that miR-193a-3p was significantly downregulated in the former compared with the latter patient population. Overexpression of miR-193a-3p considerably reduced the oncogenicity of both CRC cells. Furthermore, KRAS is a key target of miR-193a-3p. In vivo treatment with the combination of 4-AAQB and cetuximab significantly reduced the tumor burden of a xenograft mice model through the reduction of the expression of oncogenic markers (EGFR) and p-MEK, p-ERK, and c-RAF/p-c-RAF signaling, with the simultaneous induction of miR-193a-3p expression in the plasma. In summary, our findings provide strong evidence regarding the therapeutic effect of 4-AAQB on KRAS-mutant CRC cells. Furthermore, 4-AAQB effectively inhibits Ras singling in CRC cells, through which KRAS-mutant CRC can be resensitized to cetuximab.

4-Acetylantroquinonol B induced DNA damage response signaling and apoptosis via suppressing CDK2/CDK4 expression in triple negative breast cancer cells.

BACKGROUND: Triple-negative breast cancer (TNBC) has a more aggressive phenotype and poorer prognosis than hormone receptor (HR+) and human epidermal growth factor receptor (HER2 -) subtypes. Inhibition of cyclin-dependent kinase (CDK)4 and CDK6 was successful in patients with advanced metastatic HR+/HER2- breast cancer, but those with TNBC exhibited low or no response to this therapeutic approach. This study investigated the dual therapeutic targeting of CDK2 and CDK4 by using 4-acetyl-antroquinonol B (4-AAQB) against TNBC cells. METHODS: We examined the effects of CDK2, CDK4, and CDK6 inhibition through 4-AAQB treatment on TNBC cell lines and established an orthotropic xenograft mouse model to confirm the in vitro results of inhibiting CDK2, CDK4, and CDK6 by 4-AAQB treatment. RESULTS: High expression and alteration of CDK2 and CDK4 but not CDK6 significantly correlated with poor overall survival of patients with breast cancer. CDK2 and CDK4 were positively correlated with damage in DNA replication and repair pathways. Docking results indicated that 4-AAQB was bound to CDK2 and CDK4 with high affinity. Treatment of TNBC cells with 4-AAQB suppressed the expression of CDK2 and CDK4 in vitro. Additionally, 4-AAQB induced cell cycle arrest, DNA damage, and apoptosis in TNBC cells. In vivo study results confirmed that the anticancer activity of 4-AAQB suppressed tumor growth through the inhibition of CDK2 and CDK4. CONCLUSION: The expression level of CDK2 and CDK4 and DNA damage response (DDR) signaling are prominent in TNBC cell cycle regulation. Thus, 4-AAQB is a potential agent for targeting CDK2/4 and DDR in TNBC cells.

Ovatodiolide Suppresses Oral Cancer Malignancy by Down-Regulating Exosomal Mir-21/STAT3/ß-Catenin Cargo and Preventing Oncogenic Transformation of Normal Gingival Fibroblasts.

Oral squamous cell carcinoma (OSCC) is among the most commonly diagnosed malignancies in the world. Patients with OSCC often develop treatment resistance, resulting in a poor prognosis. Mounting evidence indicates that interactions between cancerous cells and other components of the tumor microenvironment (TME) determine their response to treatment. Herein, we examined the role of cancer stem cell-derived extracellular vesicles (CSC_EVs) generated from CAL27 and SCC-15 OSCC cells in the development of cisplatin (CDDP) resistance. We demonstrated that CSC_EVs enhance CDDP resistance, clonogenicity, and the tumorsphere formation potential of OSCC cells. Our bioinformatics analyses revealed that OSCC_EVs are enriched with microRNA (miR)-21-5p and are associated with increased metastasis, stemness, chemoresistance, and poor survival in patients with OSCC. Mechanistically, enhanced activity of CSC_EVs was positively correlated with upregulated ß-catenin, phosphatidylinositol-3 kinase (PI3K), signal transducer and activator of transcription 3 (STAT3), mammalian target of rapamycin (mTOR), and transforming growth factor (TGF)-ß1 messenger (m)RNA and protein expression levels. CSC_EVs also conferred a cancer-associated fibroblast (CAF) phenotype on normal gingival fibroblasts (NGFs), with the resultant CAFs enhancing the oncogenicity of OSCC cells. Interestingly, treatment with ovatodiolide (OV), the bioactive component of Anisomeles indica, suppressed OSCC tumorigenesis by reducing the cargo content of EVs derived from CSCs, suppressing self-renewal, and inhibiting the NGF-CAF transformation by disrupting EV-TME interactions. Moreover, by suppressing miR-21-5p, STAT3, and mTOR expressions in CSC_EVs, OV re-sensitized CSCs to CDDP and suppressed OSCC tumorigenesis. In vivo, treatment with OV alone or in combination with CDDP significantly reduced the tumor sphere-forming ability and decreased EV cargos containing mTOR, PI3K, STAT3, ß-catenin, and miR-21-5p. In summary, our findings provide further strong evidence of OV's therapeutic effect in OSCC.

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.

Destruxin B Suppresses Drug-Resistant Colon Tumorigenesis and Stemness Is Associated with the Upregulation of miR-214 and Downregulation of mTOR/ß-Catenin Pathway.

BACKGROUND: Drug resistance represents a major challenge for treating patients with colon cancer. Accumulating evidence suggests that Insulin-like growth factor (IGF)-associated signaling promotes colon tumorigenesis and cancer stemness. Therefore, the identification of agents, which can disrupt cancer stemness signaling, may provide improved therapeutic efficacy. METHODS: Mimicking the tumor microenvironment, we treated colon cancer cells with exogenous IGF1. The increased stemness of IGF1-cultured cells was determined by ALDH1 activity, side-population, tumor sphere formation assays. Destruxin B (DB) was evaluated for its anti-tumorigenic and stemness properties using cellular viability, colony-formation tests. The mimic and inhibitor of miR-214 were used to treat colon cancer cells to show its functional association to DB treatment. In vivo mouse models were used to evaluate DB's ability to suppress colon tumor-initiating ability and growth inhibitory function. RESULTS: IGF1-cultured colon cancer cells showed a significant increase in 5-FU resistance and enhanced stemness properties, including an increased percentage of ALDH1+, side-population cells, tumor sphere generation in vitro, and increased tumor initiation in vivo. In support, using public databases showed that increased IGF1 expression was significantly associated with a poorer prognosis in patients with colon cancer. DB, a hexadepsipeptide mycotoxin, was able to suppress colon tumorigenic phenotypes, including colony and sphere formation. The sequential treatment of DB, followed by 5-FU, synergistically inhibited the viability of colon cancer cells. In vivo studies showed that DB suppressed the tumorigenesis by 5-FU resistant colon cells, and in a greater degree when combined with 5-FU. Mechanistically, DB treatment was associated with decreased the mammalian target of rapamycin (mTOR) and ß-catenin expression and an increased miR-214 level. CONCLUSION: We provided evidence of DB as a potential therapeutic agent for overcoming 5-FU resistance induced by IGF1, and suppressing cancer stem-like properties in association with miR-214 regulation. Further investigation is warranted for its translation to clinical application.

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 suppresses yes-associated protein 1-modulated cancer stem cell phenotypes in highly malignant hepatocellular carcinoma and sensitizes cancer cells to chemotherapy in vitro.

The cancer stem cells (CSCs) theory recently became a focus of heightened attention in cancer biology, with the proposition that CSCs may constitute an important therapeutic target for effective anticancer therapy, because of their demonstrated role in tumor initiation, chemo-, and radio-resistance. Liver CSCs are a small subpopulation of poorly- or undifferentiated liver tumor cells, implicated in tumorigenesis, metastasis, resistance to therapy and disease relapse, enriched with and associated with the functional markers corresponding to the CSCs-enriched side population (SP), high aldehyde dehydrogenase (ALDH) activity, and enhanced formation of in vitro liver CSCs models, referred to herein as hepatospheres. In this study, we found YAP1 was significantly expressed in the SP cells, as well as in generated hepatospheres compared to non-SP or parental HCC cells, at transcript and/or protein levels. In addition, downregulation of YAP1 expression levels by small molecule inhibitor and siRNA transfection, in the HCC cell lines, PLC/PRF/5 and Mahlavu, were associated with marked loss of ability to form hepatospheres and increased sensitivity to sorafenib. Consistent with the above, we demonstrated that YAP1 expression positively correlated with that of Sox2, Oct4, c-Myc and GRP78, markers of stemness and drug resistance. This is suggestive of YAP1's role as a modulator of cancer stemness, ER stress and chemoresistance. For the first time, we demonstrate that Ovatodiolide significantly attenuates YAP1 expression and subsequently suppressed YAP1-modulated CSCs phenotypes and associated disease progression, consistent with our previous finding in breast cancer. Taken together, our findings suggest that YAP1, highly expressed in malignant liver tumours, contributes to hepatocellular CSCs phenotype and is a molecular target of interest for CSCs targeted therapy in liver cancer patients.

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.