Induction of Immune Responses and Phosphatidylserine Exposure by TLR9 Activation Results in a Cooperative Antitumor Effect with a Phosphatidylserine-targeting Prodrug.

Head and neck cancer is a major cancer type, with high motility rates that reduce the quality of life of patients. Herein, we investigated the effectiveness and mechanism of a combination therapy involving TLR9 activator (CpG-2722) and phosphatidylserine (PS)-targeting prodrug of SN38 (BPRDP056) in a syngeneic orthotopic head and neck cancer animal model. The results showed a cooperative antitumor effect of CpG-2722 and BPRDP056 owing to their distinct and complementary antitumor functions. CpG-2722 induced antitumor immune responses, including dendritic cell maturation, cytokine production, and immune cell accumulation in tumors, whereas BPRDP056 directly exerted cytotoxicity toward cancer cells. We also discovered a novel function and mechanism of TLR9 activation, which increased PS exposure on cancer cells, thereby attracting more BPRDP056 to the tumor site for cancer cell killing. Killed cells expose more PS in tumor for BPRDP056 targeting. Tumor antigens released from the dead cells were taken up by antigen-presenting cells, which enhanced the CpG-272-promoted T cell-mediated tumor-killing effect. These form a positive feed-forward antitumor effect between the actions of CpG-2722 and BPRDP056. Thus, the study findings suggest a novel strategy of utilizing the PS-inducing function of TLR9 agonists to develop combinational cancer treatments using PS-targeting drugs.

Virofree, an Herbal Medicine-Based Formula, Interrupts the Viral Infection of Delta and Omicron Variants of SARS-CoV-2.

Coronavirus disease 2019 (COVID-19) remains a threat with the emergence of new variants, especially Delta and Omicron, without specific effective therapeutic drugs. The infection causes dysregulation of the immune system with a cytokine storm that eventually leads to fatal acute respiratory distress syndrome (ARDS) and further irreversible pulmonary fibrosis. Therefore, the promising way to inhibit infection is to disrupt the binding and fusion between the viral spike and the host ACE2 receptor. A transcriptome-based drug screening platform has been developed for COVID-19 to explore the possibility and potential of the long-established drugs or herbal medicines to reverse the unique genetic signature of COVID-19. In silico analysis showed that Virofree, an herbal medicine, reversed the genetic signature of COVID-19 and ARDS. Biochemical validations showed that Virofree could disrupt the binding of wild-type and Delta-variant spike proteins to ACE2 and its syncytial formation via cell-based pseudo-typed viral assays, as well as suppress binding between several variant recombinant spikes to ACE2, especially Delta and Omicron. Additionally, Virofree elevated miR-148b-5p levels, inhibited the main protease of SARS-CoV-2 (Mpro), and reduced LPS-induced TNF-α release. Virofree also prevented cellular iron accumulation leading to ferroptosis which occurs in SARS-CoV-2 patients. Furthermore, Virofree was able to reduce pulmonary fibrosis-related protein expression levels in vitro. In conclusion, Virofree was repurposed as a potential herbal medicine to combat COVID-19. This study highlights the inhibitory effect of Virofree on the entry of Delta and Omicron variants of SARS-CoV-2, which have not had any effective treatments during the emergence of the new variants spreading.

Integrated therapy platform of exosomal system: hybrid inorganic/organic nanoparticles with exosomes for cancer treatment.

Recent studies have found that exosomes or extracellular vehicles (EVs) are associated with cancer metastasis, disease progression, diagnosis, and treatment, leading to a rapidly emerging area of exocrine vesicle research. Relying on the superior targeting function and bio-compatibility of exosomes, researchers have been able to deliver drugs to cancer stem cells deep within tumors in mouse models. Despite significant efforts made in this relatively new field of exosome research, progress has been held back by challenges such as inefficient separation methods, difficulties in characterization/tracking, and a lack of specific biomarkers. Therefore, current researches are devoted to combining nanomaterials with exosomes to improve these shortcomings. Adding inorganic/organic nanoparticles such as artificial liposomes and iron oxide can bring more drug options and various fluorescent or magnetic diagnostic possibilities to the exosome system. Moreover, the applications of exosomes need to be further evaluated under actual physiological conditions. This review article highlights the potential of exosome-biomimetic nanoparticles for their use as drug carriers to improve the efficacy of anticancer therapy.

Graptopetalum paraguayense Extract Ameliorates Proteotoxicity in Aging and Age-Related Diseases in Model Systems.

Declines in physiological functions are the predominant risk factors for age-related diseases, such as cancers and neurodegenerative diseases. Therefore, delaying the aging process is believed to be beneficial in preventing the onset of age-related diseases. Previous studies have demonstrated that Graptopetalum paraguayense (GP) extract inhibits liver cancer cell growth and reduces the pathological phenotypes of Alzheimer's disease (AD) in patient IPS-derived neurons. Here, we show that GP extract suppresses ß-amyloid pathology in SH-SYS5Y-APP695 cells and APP/PS1 mice. Moreover, AMP-activated protein kinase (AMPK) activity is enhanced by GP extract in U87 cells and APP/PS1 mice. Intriguingly, GP extract enhances autophagy in SH-SYS5Y-APP695 cells, U87 cells, and the nematode Caenorhabditis elegans, suggesting a conserved molecular mechanism by which GP extract might regulate autophagy. In agreement with its role as an autophagy activator, GP extract markedly diminishes mobility decline in polyglutamine Q35 mutants and aged wild-type N2 animals in C. elegans. Furthermore, GP extract significantly extends lifespan in C. elegans.

Identification of NSP3 (SH2D3C) as a Prognostic Biomarker of Tumor Progression and Immune Evasion for Lung Cancer and Evaluation of Organosulfur Compounds from Allium sativum L. as Therapeutic Candidates.

The multi-domain non-structural protein 3 (NSP3) is an oncogenic molecule that has been concomitantly implicated in the progression of coronavirus infection. However, its oncological role in lung cancer and whether it plays a role in modulating the tumor immune microenvironment is not properly understood. In the present in silico study, we demonstrated that NSP3 (SH2D3C) is associated with advanced stage and poor prognoses of lung cancer cohorts. Genetic alterations of NSP3 (SH2D3C) co-occurred inversely with Epidermal Growth Factor Receptor (EGFR) alterations and elicited its pathological role via modulation of various components of the immune and inflammatory pathways in lung cancer. Our correlation analysis suggested that NSP3 (SH2D3C) promotes tumor immune evasion via dysfunctional T-cell phenotypes and T-cell exclusion mechanisms in lung cancer patients. NSP3 (SH2D3C) demonstrated a high predictive value and association with therapy resistance in lung cancer, hence serving as an attractive target for therapy exploration. We evaluated the in silico drug-likeness and NSP3 (SH2D3C) target efficacy of six organosulfur small molecules from Allium sativum using a molecular docking study. We found that the six organosulfur compounds demonstrated selective cytotoxic potential against cancer cell lines and good predictions for ADMET properties, drug-likeness, and safety profile. E-ajoene, alliin, diallyl sulfide, 2-vinyl-4H-1,3-dithiin, allicin, and S-allyl-cysteine docked well into the NSP3 (SH2D3C)-binding cavity with binding affinities ranging from -4.3~-6.70 A and random forest (RF) scores ranging from 4.31~5.26 pKd. However, S-allyl-cysteine interaction with NSP3 (SH2D3C) is unfavorable and hence less susceptible to NSP3 ligandability. In conclusion, our study revealed that NSP3 is an important onco-immunological biomarker encompassing the tumor microenvironment, disease staging and prognosis in lung cancer and could serve as an attractive target for cancer therapy. The organosulfur compounds from A. sativum have molecular properties to efficiently interact with the binding site of NSP3 and are currently under vigorous preclinical study in our laboratory.

Honeysuckle Aqueous Extracts Induced let-7a Suppress EV71 Replication and Pathogenesis In Vitro and In Vivo and Is Predicted to Inhibit SARS-CoV-2.

Honeysuckle (Lonicera japonica Thunb) is a traditional Chinese medicine (TCM) with an antipathogenic activity. MicroRNAs (miRNAs) are small non-coding RNA molecules that are ubiquitously expressed in cells. Endogenous miRNA may function as an innate response to block pathogen invasion. The miRNA expression profiles of both mice and humans after the ingestion of honeysuckle were obtained. Fifteen overexpressed miRNAs overlapped and were predicted to be capable of targeting three viruses: dengue virus (DENV), enterovirus 71 (EV71) and SARS-CoV-2. Among them, let-7a was examined to be capable of targeting the EV71 RNA genome by reporter assay and Western blotting. Moreover, honeysuckle-induced let-7a suppression of EV71 RNA and protein expression as well as viral replication were investigated both in vitro and in vivo. We demonstrated that let-7a targeted EV71 at the predicted sequences using luciferase reporter plasmids as well as two infectious replicons (pMP4-y-5 and pTOPO-4643). The suppression of EV71 replication and viral load was demonstrated in two cell lines by luciferase activity, RT-PCR, real-time PCR, Western blotting and plaque assay. Furthermore, EV71-infected suckling mice fed honeysuckle extract or inoculated with let-7a showed decreased clinical scores and a prolonged survival time accompanied with decreased viral RNA, protein expression and virus titer. The ingestion of honeysuckle attenuates EV71 replication and related pathogenesis partially through the upregulation of let-7a expression both in vitro and in vivo. Our previous report and the current findings imply that both honeysuckle and upregulated let-7a can execute a suppressive function against the replication of DENV and EV71. Taken together, this evidence indicates that honeysuckle can induce the expression of let-7a and that this miRNA as well as 11 other miRNAs have great potential to prevent and suppress EV71 replication.

ALDH2 deficiency induces atrial fibrillation through dysregulated cardiac sodium channel and mitochondrial bioenergetics: A multi-omics analysis.

Point mutation in alcohol dehydrogenase 2 (ALDH2), ALDH2*2 results in decreased catalytic enzyme activity and has been found to be associated with different human pathologies. Whether ALDH2*2 would induce cardiac remodeling and increase the attack of atrial fibrillation (AF) remains poorly understood. The present study evaluated the effect of ALDH2*2 mutation on AF susceptibility and unravelled the underlying mechanisms using a multi-omics approach including whole-genome gene expression and proteomics analysis. The in-vivo electrophysiological study showed an increase in the incidence and reduction in the threshold of AF for the mutant mice heterozygous for ALDH2*2 as compared to the wild type littermates. The microarray analysis revealed a reduction in the retinoic acid signals which was accompanied by a downstream reduction in the expression of voltage-gated Na+ channels (SCN5A). The treatment of an antagonist for retinoic acid receptor resulted in a decrease in SCN5A transcript levels. The integrated analysis of the transcriptome and proteome data showed a dysregulation of fatty acid ß-oxidation, adenosine triphosphate synthesis via electron transport chain, and activated oxidative responses in the mitochondria. Oral administration of Coenzyme Q10, an essential co-factor known to meliorate mitochondrial oxidative stress and preserve bioenergetics, conferred a protection against AF attack in the mutant ALDH2*2 mice. The multi-omics approach showed the unique pathophysiology mechanisms of concurrent dysregulated SCN5A channel and mitochondrial bioenergetics in AF. This inspired the development of a personalized therapeutic agent, Coenzyme Q10, to protect against AF attack in humans characterized by ALDH2*2 genotype.

Honeysuckle (Lonicera japonica) and Huangqi (Astragalus membranaceus) Suppress SARS-CoV-2 Entry and COVID-19 Related Cytokine Storm in Vitro.

COVID-19 is threatening human health worldwide but no effective treatment currently exists for this disease. Current therapeutic strategies focus on the inhibition of viral replication or using anti-inflammatory/immunomodulatory compounds to improve host immunity, but not both. Traditional Chinese medicine (TCM) compounds could be promising candidates due to their safety and minimal toxicity. In this study, we have developed a novel in silico bioinformatics workflow that integrates multiple databases to predict the use of honeysuckle (Lonicera japonica) and Huangqi (Astragalus membranaceus) as potential anti-SARS-CoV-2 agents. Using extracts from honeysuckle and Huangqi, these two herbs upregulated a group of microRNAs including let-7a, miR-148b, and miR-146a, which are critical to reduce the pathogenesis of SARS-CoV-2. Moreover, these herbs suppressed pro-inflammatory cytokines including IL-6 or TNF-α, which were both identified in the cytokine storm of acute respiratory distress syndrome, a major cause of COVID-19 death. Furthermore, both herbs partially inhibited the fusion of SARS-CoV-2 spike protein-transfected BHK-21 cells with the human lung cancer cell line Calu-3 that was expressing ACE2 receptors. These herbs inhibited SARS-CoV-2 Mpro activity, thereby alleviating viral entry as well as replication. In conclusion, our findings demonstrate that honeysuckle and Huangqi have the potential to be used as an inhibitor of SARS-CoV-2 virus entry that warrants further in vivo analysis and functional assessment of miRNAs to confirm their clinical importance. This fast-screening platform can also be applied to other drug discovery studies for other infectious diseases.

Bioactivity Evaluation of a Novel Formulated Curcumin.

Curcumin has been used as a traditional medicine and/or functional food in several cultures because of its health benefits including anticancer properties. However, poor oral bioavailability of curcumin has limited its oral usage as a food supplement and medical food. Here we formulated curcumin pellets using a solid dispersion technique. The pellets had the advantages of reduced particle size, improved water solubility, and particle porosity. This pellet form led to an improvement in curcumin's oral bioavailability. Additionally, we used the C-Map and Library of Integrated Network-Based Cellular Signatures (LINCS) Unified Environment (CLUE) gene expression database to determine the potential biological functions of formulated curcumin. The results indicated that, similar to conventional curcumin, the formulated curcumin acted as an NF-κB pathway inhibitor. Moreover, ConsensusPathDB database analysis was used to predict possible targets and it revealed that both forms of curcumin exhibit similar biological functions, including apoptosis. Biochemical characterization revealed that both the forms indeed induced apoptosis of hepatocellular carcinoma (HCC) cell lines. We concluded that the formulated curcumin increases the oral bioavailability in animals, and, as expected, retains characteristics similar to conventional curcumin at the cellular level. Our screening platform using big data not only confirms that both the forms of curcumin have similar mechanisms but also predicts the novel mechanism of the formulated curcumin.

Assessing the therapeutic potential of Graptopetalum paraguayense on Alzheimer's disease using patient iPSC-derived neurons.

Alzheimer's disease (AD) is the most common type of dementia and also one of the leading causes of death worldwide. However, the underlying mechanisms remain unclear, and currently there is no drug treatment that can prevent or cure AD. Here, we have applied the advantages of using induced pluripotent stem cell (iPSC)-derived neurons (iNs) from AD patients, which are able to offer human-specific drug responsiveness, in order to evaluate therapeutic candidates for AD. Using approach involving an inducible neurogenin-2 transgene, we have established a robust and reproducible protocol for differentiating human iPSCs into glutamatergic neurons. The AD-iN cultures that result have mature phenotypic and physiological properties, together with AD-like biochemical features that include extracellular ß-amyloid (Aß) accumulation and Tau protein phosphorylation. By screening using a gene set enrichment analysis (GSEA) approach, Graptopetalum paraguayense (GP) has been identified as a potential therapeutic agent for AD from among a range of Chinese herbal medicines. We found that administration of a GP extract caused a significantly reduction in the AD-associated phenotypes of the iNs, including decreased levels of extracellular Aß40 and Aß42, as well as reduced Tau protein phosphorylation at positions Ser214 and Ser396. Additionally, the effect of GP was more prominent in AD-iNs compared to non-diseased controls. These findings provide valuable information that suggests moving extracts of GP toward drug development, either for treating AD or as a health supplement to prevent AD. Furthermore, our human iN-based platform promises to be a useful strategy when it is used for AD drug discovery.

Antrodia cinnamomea Enhances Chemo-Sensitivity of 5-FU and Suppresses Colon Tumorigenesis and Cancer Stemness via Up-Regulation of Tumor Suppressor miR-142-3p.

5-Fluorouracil (5-FU) regimen remains the backbone of the first-line agent to treat colon cancer, but often these patients develop resistance. Cancer stem cells (CSC's) are considered as one of the key contributors in the development of drug resistance and tumor recurrence. We aimed to provide preclinical evidence for Antrodiacinnamomea (AC), as a potential in suppressing colon cancer CSC's to overcome 5-FU drug-resistant. In-vitro assays including cell viability, colony formation, AC + 5-FU drug combination index and tumor sphere generation were applied to determine the inhibitory effect of AC. Mouse xenograft models also incorporated to evaluate in vivo effect of AC. AC treatment significantly inhibited the proliferation, colony formation and tumor sphere generation. AC also inhibited the expression of oncogenic markers (NF-κB, and C-myc), EMT/metastasis markers (vimentin and MMP3) and stemness associated markers (ß-catenin, SOX-2 and Nanog). Sequential treatment of AC and 5-FU synergized and reduces colon cancer viability both in vivo and in vitro. Mechanistically, AC mediated anti-tumor effect was associated with an increased level of tumor suppressor microRNAs especially, miR142-3p. AC can be a potent synergistic adjuvant, down-regulates cancer stemness genes and enhances the antitumor ability of 5-FU by stimulating apoptosis-associated genes, suppressing inflammation and metastasis genes through miR142-3p in colon cancer.

Graptopetalum paraguayense Inhibits Liver Fibrosis by Blocking TGF-ß Signaling In Vivo and In Vitro.

BACKGROUND AND AIMS: Liver fibrosis is the excessive accumulation of extracellular matrix proteins, including collagen, which occurs in most types of chronic liver diseases. Advanced liver fibrosis results in cirrhosis, liver failure, and portal hypertension. Activated hepatic perivascular stellate cells, portal fibroblasts, and myofibroblasts of bone marrow origin have been identified as major collagen-producing cells in the injured liver. These cells are activated by fibrogenic cytokines, such as TGF-ß1. The inhibition of TGF-ß1 function or synthesis is a major target for the development of antifibrotic therapies. Our previous study showed that the water and ethanol extracts of Graptopetalum paraguayense (GP), a Chinese herbal medicine, can prevent dimethylnitrosamine (DMN)-induced hepatic inflammation and fibrosis in rats. METHODS: We used rat hepatic stellate HSC-T6 cells and a diethylnitrosamine (DEN)-induced rat liver injury model to test the potential mechanism of GP extracts and its fraction, HH-F3. RESULTS: We demonstrated that GP extracts and HH-F3 downregulated the expression levels of extracellular matrix (ECM) proteins and inhibited the proliferation and migration via suppression of the TGF-ß1 pathway in rat hepatic stellate HSC-T6 cells. Moreover, the HH-F3 fraction decreased hepatic collagen content and reduced plasma AST, ALT, and γ-GT activities in a DEN-induced rat liver injury model, suggesting that GP/HH-F3 has hepatoprotective effects against DEN-induced liver fibrosis. CONCLUSION: These findings indicate that GP/HH-F3 may be a potential therapeutic agent for the treatment of liver fibrosis. The inhibition of TGF-ß-mediated fibrogenesis may be a central mechanism by which GP/HH-F3 protects the liver from injury.

Modified Sijunzi decoction can alleviate cisplatin-induced toxicity and prolong the survival time of cachectic mice by recovering muscle atrophy.

ETHNOPHARMACOLOGICAL RELEVANCE: Sijunzi decoction is a well-known traditional Chinese medicine (TCM) commonly used for invigorating vital energy and for the enhancement of immunity. Modified Sijunzi decoctions have been extensively used to treat cachexia and improve the quality of life of cancer patients undergoing chemotherapy. AIM OF THE STUDY: This study was aimed to provide comprehensive evidence for the anti-cachectic effect of a modified Sijunzi decoction (Zhen-Qi; ZQ-SJZ) and characterize its anti-cachectic mechanism, especially in cisplatin-induced muscle atrophy. MATERIALS AND METHODS: We employed a Lewis lung carcinoma (LLC)-induced cancer cachectic mouse model to demonstrate the anti-cachectic effect of ZQ-SJZ. Moreover, we provided an in vitro C2C12 myotube formation model to investigate the effect of ZQ-SJZ in hampering cisplatin-induced muscle atrophy. RESULTS: The administration of ZQ-SJZ can recover tumor- and/or cisplatin-induced body weight loss, intestinal mucosal damage, as well as forelimb grip strength and myofiber size. The administration of ZQ-SJZ also significantly prolonged the survival of LLC-induced cachectic mice under cisplatin treatment. Mechanistically, ZQ-SJZ increased the levels of myogenic proteins, such as myosin heavy chain (MyHC) and myogenin, and decreased the atrophy-related protein, atrogin-1, in cisplatin-treated C2C12 myotubes in vitro. In addition, cisplatin-induced mitochondria dysfunction could be hampered by the co-administration of ZQ-SJZ, by which it recovered the cisplatin-mediated decrease in PGC-1α and PKM1 levels. CONCLUSIONS: The administration of ZQ-SJZ can recover tumor- and/or cisplatin-induced cachectic conditions and significantly prolong the survival of LLC-induced cachectic mice under cisplatin treatment. The profound effect of ZQ-SJZ in hampering tumor- and/or cisplatin-induced cachexia may be due to its modulation of the mitochondrial function and subsequent myogenesis. Taken together, these results demonstrated the anti-cachectic mechanism of ZQ-SJZ and its potential use as a palliative strategy to improve the efficacy of chemotherapy.

MART-10, the new brand of 1α,25(OH)2D3 analog, is a potent anti-angiogenic agent in vivo and in vitro.

BACKGROUND: Angiogenesis is the hall marker for cancer growth and metastasis. Thus, anti-angiogenesis emerges as a new way to treat cancer. 1α,25(OH)2D3 is recently getting popular due to the non-mineral functions, which have been applied fore cancer treatment. The newly-synthesized 1α,25(OH)2D3 analog, MART-10, has been proved to be much more potent than 1α,25(OH)2D3 regarding inhibiting cancer cells growth and metastasis without inducing hypercalcemia in vivo. In this study, we aimed to investigate the effect of MART-10 and 1α,25(OH)2D3 on angiogenesis in vitro and in vivo. METHODS AND RESULTS: MART-10 and 1α,25(OH)2D3 were able to repress VEGFA-induced human umbilical vein endothelial cells (HUVECs) migration, invasion and tube formation, but not proliferation, with MART-10 much more potent than 1α,25(OH)2D3. The Chick Chorioallantoic Membrane (CAM) assay and matrigeal angiogenesis assay further confirmed the in vivo more potent anti-angiogenesis effect of MART-10. MART-10 inhibited the VEGFA-induced HUVECs angiogenesis process through downregulation of Akt and Erk 1/2 phosphorylation. The VEGFA-VEGFR2 (VEGF receptor 2) axis is the main signal transducing pathway to stimulate angiogenesis. A positive autocrine manner was found for the first time in HUVECs as treated by VEGFA, which induced VEGFA expression and secretion, and VEGFR2 expression. MART-10 and 1α,25(OH)2D3 were demonstrated to be able to repress this positive autocrine manner, thus inhibiting angiogenesis. CONCLUSIONS: MART-10 and 1α,25(OH)2D3 both are effective anti-angiogenesis agents. Given MART-10 is much more potent than 1α,25(OH)2D3 and active in vivo without obvious side effect, MART-10 should be deemed as a promising anti-cancer agent.

Gene Expression Profiling and Pathway Network Analysis Predicts a Novel Antitumor Function for a Botanical-Derived Drug, PG2.

PG2 is a botanical drug that is mostly composed of Astragalus polysaccharides (APS). Its role in hematopoiesis and relieving cancer-related fatigue has recently been clinically investigated in cancer patients. However, systematic analyses of its functions are still limited. The aim of this study was to use microarray-based expression profiling to evaluate the quality and consistency of PG2 from three different product batches and to study biological mechanisms of PG2. An integrative molecular analysis approach has been designed to examine significant PG2-induced signatures in HL-60 leukemia cells. A quantitative analysis of gene expression signatures was conducted for PG2 by hierarchical clustering of correlation coefficients. The results showed that PG2 product batches were consistent and of high quality. These batches were also functionally equivalent to each other with regard to how they modulated the immune and hematopoietic systems. Within the PG2 signature, there were five genes associated with doxorubicin: IL-8, MDM4, BCL2, PRODH2, and BIRC5. Moreover, the combination of PG2 and doxorubicin had a synergistic effect on induced cell death in HL-60 cells. Together with the bioinformatics-based approach, gene expression profiling provided a quantitative measurement for the quality and consistency of herbal medicines and revealed new roles (e.g., immune modulation) for PG2 in cancer treatment.

Using the gene expression signature of scutebarbalactone VN isolated from Scutellaria barbata to elucidate its anticancer activities.

Bioassay-guided fractionation led to the discovery of a novel neo-clerodane diterpenoid, scutebarbalactone VN (BalA: 8,13-epoxy-3-en-7-hydroxy-6,11-O- dibenzoyl-15,16-clerodanolide), from the methanol extract of the whole-plant of Vietnamese Scutellaria barbata D. Don. A microarray technique combined with bioinformatic analyses showed that BalA could inhibit cell cycle pathways by downregulating genes such as CDC25A and AURKA. BalA also showed the potential to reactivate downregulated genes in hepatocellular carcinoma cells and genes in antioxidant pathways such as HMOX1 and HSPA1A. Querying Connectivity map 2.0 resulted in a match of the BalA-modulated gene signature with that of 10 known compounds, most of which are currently marketed chemotherapy drugs. The highest matching scores belonged to lomustine, semustine, and withaferin A. Lomustine and semustine were found to alkylate DNA and RNA, while withaferin A inhibits nuclear factor kappa B (NF-κB) activity. A luciferase reporter assay was also conducted on 293/NF-κB human embryonic kidney cells that had been transfected with the NF-κB-luciferase plasmid to verify the anticancer activity of BalA. The assay showed that BalA effectively blocked NF-κB with an IC50 of 38.6 ± 0.05 µM.

Evaluation of the medicinal herb Graptopetalum paraguayense as a treatment for liver cancer.

BACKGROUND: Hepatocellular carcinoma (HCC) is the fifth most common malignancy and the third most common cause of cancer-related death worldwide. Sorafenib is the only drug for patients with advanced-stage hepatocellular carcinoma (HCC) that has been shown to confer a survival benefit to patients with HCC; however, it has many side effects. Thus, alternate therapeutic strategies with improved safety and therapeutic efficacy for the management of HCC should be developed. METHODS AND FINDINGS: We demonstrate that an extract of Graptopetalum paraguayense (GP) down-regulated the expression levels of several onco-proteins, including AURKA, AURKB, and FLJ10540, in HCC cells. To isolate the active components in the GP extracts, we prepared extracts fractions and assessed their effects on the expression of onco-proteins in HCC cells. The fraction designated HH-F3 was enriched in active ingredients, exhibited cytotoxic effects, and suppressed the expression of the onco-proteins in HCC cells. The structure of the main active compound in HH-F3 was found to be similar to that of the proanthocyanidin compounds derived from Rhodiola rosea. In addition, a distinct new compound rich in 3, 4, 5-trihydroxy benzylic moieties was identified in the HH-F3 preparations. Mechanistic studies indicated that HH-F3 induced apoptosis in HCC cells by promoting the loss of mitochondrial membrane potential and the production of reactive oxygen species. HH-F3 also enhanced PTEN expression and decreased AKT phosphorylation at Ser473 in a concentration-dependent manner in HCC cells. Moreover combination of GP or HH-F3 and sorafenib synergistically inhibits the proliferation of Huh7 cells. The treatment of a rat model with diethylnitrosamine (DEN)-induced liver cancer with extracts of GP and HH-F3 decreased hepatic collagen contents and inhibited tumor growth. CONCLUSIONS: These results indicate that GP extracts and HH-F3 can protect the liver by suppressing tumor growth; consequently, these compounds could be considered for the treatment of HCC.

Gluconeogenesis, lipogenesis, and HBV replication are commonly regulated by PGC-1α-dependent pathway.

PGC-1α, a major metabolic regulator of gluconeogenesis and lipogenesis, is strongly induced to coactivate Hepatitis B virus (HBV) gene expression in the liver of fasting mice. We found that 8-Br-cAMP and glucocorticoids synergistically induce PGC-1α and its downstream targets, including PEPCK and G6Pase. Also, HBV core promoter activity was synergistically enhanced by 8-Br-cAMP and glucocorticoids. Graptopetalum paraguayense (GP), a herbal medicine, is commonly used in Taiwan to treat liver disorders. Partially purified fraction of GP (named HH-F3) suppressed 8-Br-cAMP/glucocorticoid-induced G6Pase, PEPCK and PGC-1α expression and suppressed HBV core promoter activity. HH-F3 blocked HBV core promoter activity via inhibition of PGC-1α expression. Ectopically expressed PGC-1α rescued HH-F3-inhibited HBV surface antigen expression, HBV mRNA production, core protein levels, and HBV replication. HH-F3 also inhibited fatty acid synthase (FASN) expression and decreased lipid accumulation by down-regulating PGC-1α. Thus, HH-F3 can inhibit HBV replication, gluconeogenesis and lipogenesis by down-regulating PGC-1α. Our study indicates that targeting PGC-1α may be a therapeutic strategy for treatment of HBV infections. HH-F3 may have potential use for the treatment of chronic hepatitis B patients with associated metabolic syndrome.

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.

Graptopetalum paraguayense ameliorates chemical-induced rat hepatic fibrosis in vivo and inactivates stellate cells and Kupffer cells in vitro.

BACKGROUND: Graptopetalum paraguayense (GP) is a folk herbal medicine with hepatoprotective effects that is used in Taiwan. The aim of this study was to evaluate the hepatoprotective and antifibrotic effects of GP on experimental hepatic fibrosis in both dimethylnitrosamine (DMN)- and carbon tetrachloride (CCl(4))-induced liver injury rats. METHODS: Hepatic fibrosis-induced rats were fed with the methanolic extract of GP (MGP) by oral administration every day. Immunohistochemistry, biochemical assays, and Western blot analysis were performed. The effects of MGP on the expression of fibrotic markers and cytokines in the primary cultured hepatic stellate cells (HSCs) and Kupffer cells, respectively, were evaluated. RESULTS: Oral administration of MGP significantly alleviated DMN- or CCl(4)-induced liver inflammation and fibrosis. High levels of alanine transaminase, aspartate transaminase, bilirubin, prothrombin activity and mortality rates also decreased in rats treated with MGP. There were significantly decreased hydroxyproline levels in therapeutic rats compared with those of the liver-damaged rats. Collagen I and alpha smooth muscle actin (α-SMA) expression were all reduced by incubation with MGP in primary cultured rat HSCs. Furthermore, MGP induced apoptotic cell death in activated HSCs. MGP also suppressed lipopolysaccharide-stimulated rat Kupffer cell activation by decreasing nitric oxide, tumor necrosis factor-α and interleukin-6 production, and increasing interleukin-10 expression. CONCLUSIONS: The results show that the administration of MGP attenuated toxin-induced hepatic damage and fibrosis in vivo and inhibited HSC and Kupffer cell activation in vitro, suggesting that MGP might be a promising complementary or alternative therapeutic agent for liver inflammation and fibrosis.