Honokiol Research Articles

Deep sequencing of Magnoliae officinalis reveals upstream genes related to the lignan biosynthetic pathway

Magnoliae officinalis is the plant source of houpo, a widely used traditional Chinese medicine to treat symptoms of gastrointestinal diseases. Its main active components, magnolol (MG) and honokiol (HK), have excellent pharmacological actions, but little research has focused on the functional genes involved in the MG and HK metabolic pathways. In this study, using RNA-seq and gene expression profile, we present the first transcriptome characterization of M. officinalis leaves, twigs and stems. Based on similarity search against nonredundant protein databases, 30,660 contigs had at least a significant alignment to existing public database. Pathway analysis showed that 8707 contigs were assigned to 317 KEGG pathways. A second skeleton pathway with 14 putative homologous genes was also identified as involved in lignan biosynthesis. Expression profiles of these 14 genes showed that leaves and twigs seem to have higher transcript levels for lignan components than in stem tissue; this result was then verified by qRT-PCR. Our work will immensely facilitate metabolic research on lignan biosynthesis in M. officinalis.

Mitochondrial dysfunction in cancer chemoprevention by phytochemicals from dietary and medicinal plants.

Cancer chemoprevention, a scientific term coined by Dr. Sporn in the late seventies, implies use of natural or synthetic chemicals to block, delay or reverse carcinogenesis. Phytochemicals derived from edible and medicinal plants have been studied rather extensively for cancer chemoprevention using preclinical models in the past few decades. Nevertheless, some of these agents (e.g., isothiocyanates from cruciferous vegetables like broccoli and watercress) have already entered into clinical investigations. Examples of widely studied and highly promising phytochemicals from edible and medicinal plants include cruciferous vegetable constituents (phenethyl isothiocyanate, benzyl isothiocyanate, and sulforaphane), withaferin A (WA) derived from a medicinal plant (Withania somnifera) used heavily in Asia, and an oriental medicine plant component honokiol (HNK). An interesting feature of these structurally-diverse phytochemicals is that they target mitochondria to provoke cancer cell-selective death program. Mechanisms underlying cell death induction by commonly studied phytochemicals have been discussed rather extensively and thus are not covered in this review article. Instead, the primary focus of this perspective is to discuss experimental evidence pointing to mitochondrial dysfunction in cancer chemoprevention by promising phytochemicals.

Honokiol suppresses pancreatic tumor growth, metastasis and desmoplasia by interfering with tumor–stromal cross-talk

The poor clinical outcome of pancreatic cancer (PC) is largely attributed to its aggressive nature and refractoriness to currently available therapeutic modalities. We previously reported antitumor efficacy of honokiol (HNK), a phytochemical isolated from various parts of Magnolia plant, against PC cells in short-term in vitro growth assays. Here, we report that HNK reduces plating efficiency and anchorage-independent growth of PC cells and suppresses their migration and invasiveness. Furthermore, significant inhibition of pancreatic tumor growth by HNK is observed in orthotopic mouse model along with complete-blockage of distant metastases. Histological examination suggests reduced desmoplasia in tumors from HNK-treated mice, later confirmed by immunohistochemical analyses of myofibroblast and extracellular matrix marker proteins (α-SMA and collagen I, respectively). At the molecular level, HNK treatment leads to decreased expression of sonic hedgehog (SHH) and CXCR4, two established mediators of bidirectional tumor-stromal cross-talk, both in vitro and in vivo . We also show that the conditioned media (CM) from HNK-treated PC cells have little growth-inducing effect on pancreatic stellate cells (PSCs) that could be regained by the addition of exogenous recombinant SHH. Moreover, pretreatment of CM of vehicle-treated PC cells with SHH-neutralizing antibody abolishes their growth-inducing potential on PSCs. Likewise, HNK-treated PC cells respond poorly to CM from PSCs due to decreased CXCR4 expression. Lastly, we show that the transfection of PC cells with constitutively active IKKβ mutant reverses the suppressive effect of HNK on nuclear factor-kappaB activation and partially restores CXCR4 and SHH expression. Taken together, these findings suggest that HNK interferes with tumor-stromal cross-talk via downregulation of CXCR4 and SHH and decreases pancreatic tumor growth and metastasis.

Honokiol inhibits EMT-mediated motility and migration of human non-small cell lung cancer cells in vitro by targeting c-FLIP.

Honokiol (HNK) is a natural compound isolated from the magnolia plant with numerous pharmacological activities, including inhibiting epithelial-mesenchymal transition (EMT), which has been proposed as an attractive target for anti-tumor drugs to prevent tumor migration. In this study we investigated the effects of HNK on EMT in human NSCLC cells in vitro and the related signaling mechanisms. TNF-α (25 ng/mL) in combination with TGF-β1 (5 ng/mL) was used to stimulate EMT of human NSCLC A549 and H460 cells. Cell proliferation was analyzed using a sulforhodamine B assay. A wound-healing assay and a transwell assay were performed to examine cell motility. Western blotting was used to detect the expression levels of relevant proteins. siRNAs were used to knock down the gene expression of c-FLIP and N-cadherin. Stable overexpression of c-FLIP L (H157-FLIP L) or Lac Z (H157-Lac Z) was also performed. Treatment with TNF-α+TGF-β1 significantly enhanced the migration of A549 and H460 cells, increased c-FLIP, N-cadherin (a mesenchymal marker), snail (a transcriptional modulator) and p-Smad2/3 expression, and decreased IκB levels in the cells; these changes were abrogated by co-treatment with HNK (30 μmol/L). Further studies demonstrated that expression level of c-FLIP was highly correlated with the movement and migration of NSCLC cells, and the downstream effectors of c-FLIP signaling were NF-κB signaling and N-cadherin/snail signaling, while Smad signaling might lie upstream of c-FLIP. HNK inhibits EMT-mediated motility and migration of human NSCLC cells in vitro by targeting c-FLIP, which can be utilized as a promising target for cancer therapy, while HNK may become a potential anti-metastasis drug or lead compound.

Synergistic effect of honokiol and 5‐fluorouracil on apoptosis of oral squamous cell carcinoma cells

5-Fluorouracil (5-FU) is an essential chemotherapeutic agent for oral squamous cell carcinoma (OSCC). However, toxic side effects have limited its role in OSCC therapy. The aim of this study was to explore whether combination therapy with 5-FU and honokiol (HNK), a small natural organic molecule shown to induce apoptosis in OSCC cells, could enhance the anticancer activity of 5-FU without notably increasing its toxicity. 5-FU and/or HNK were used to treat OSCC cells both in vitro and in vivo. The therapeutic effect and underlying mechanisms were evaluated by cell viability assay, flow cytometry, OSCC xenograft mouse model, and Western blot. Tumor tissue apoptosis was detected by terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay. Toxicity was assessed following hematoxylin and eosin staining. Exposure to HNK + 5-FU produced a synergistic cytotoxic effect on OSCC cells. Both HNK and 5-FU could induce apoptosis through the mitochondria-mediated intrinsic pathway, and their specific signaling pathways were different. In the mouse OSCC xenograft model, treatment with 5-FU + HNK substantively retarded tumor growth, as compared to treatment with either drug individually. TUNEL analysis further confirmed that the superior in vivo antitumor efficacy of 5-FU + HNK was associated with enhanced stimulation of cell apoptosis. Notably, HNK did not increase the toxicity of 5-FU. These findings suggest that HNK and 5-FU exert a synergistic therapeutic effect on OSCC by inducing apoptosis. HNK might thus enhance the clinical therapeutic efficacy of 5-FU without increasing its toxicity.

Abstract 4118: Honokiol inhibits lung cancer brain metastasis through inhibition of STAT3 signaling pathway

Abstract Lung cancer is the leading cause of cancer death in the United States, and metastasis to lymph-node (LN) and distal organs especially brain leads to sever complications and are the major cause of death. Preventing lung cancer development and metastases is important strategy to reduce lung cancer mortality. Honokiol (HNK), a natural compound present in magnolia bark extracts, has a good bioavailability profile and recently has also been shown to readily cross both the blood-brain barrier (BBB). We previously demonstrated that HNK can prevent and inhibit squamous lung cancer cell carcinoma both in-vitro and in-vivo. In the current study, we evaluated the anti-metastatic effects of HNK in both LN and brain murine models of lung tumor metastasis. The H2030-BrM3 (BR-brain seeking) lung tumor cells stably transfected with luciferase and GFP were orthotopically injected into lung for LN metastases, or directly to the left ventricle of mouse heart for brain metastases, LN or brain metastasis was monitored using a non-invasive IVIS bio-luminescent imaging system. To test the efficacy of HNK in preventing tumor cells migrating to LN or brain, mice were given 10 mg/kg per body weight HNK by aerosol (for lung to LN metastasis model) or oral gavage starting the day after tumor cell injection, our results reveal that HNK significantly prevented the metastasis of lung cancer cells to LN and brain. Our results demonstrate that HNK decreased LN metastases incidence to 30% compare to 100% in control mice, and inhibited brain metastasis near 70% compare to control. Furthermore, analysis of HNK's mechanism of action by utilizing both RNA sequencing and receptor tyrosine kinase assay suggests that its effect is primarily mediated by inhibiting STAT3 pathway. HNK specifically inhibits STAT3 phosphorylation, and knock-down STAT3 totally abrogated the anti-metastasis effects of HNK in brain metastatic lung cancer cell lines, PC9-BrM3 and H2030-BrM3. These finding suggest that HNK could provide novel chemopreventive or therapeutic options for preventing both lung tumor progression and lung cancer brain metastasis. Citation Format: Yongik Lee, Jing Pan, Qi Zhang, Yian Wang, Ming You. Honokiol inhibits lung cancer brain metastasis through inhibition of STAT3 signaling pathway. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4118.

Honokiol targets mitochondria to halt cancer progression and metastasis.

Cancer continues to be the leading cause of death worldwide. Plants have a long history of use in the treatment of cancer. Honokiol (HNK) is an important bioactive compound found in the bark of Magnolia tree, and has been shown to inhibit cancer growth and metastasis in many cell types in vitro and in animal models. Resistance to chemotherapy and radiotherapy is the major obstacle for cure of cancer. Combination of HNK with many traditional chemotherapeutic drugs as well as radiation sensitizes cancer cells to apoptotic death, suggesting that HNK not only directly inhibits primary cancers and metastasis, but also has potential to overcome drug resistance. Ultimately, this may mean that HNK could be combined with traditional chemotherapies administered at lower doses to significantly reduce toxicity, meanwhile enhance efficacy. As a natural compound, HNK is composed of polyphenols and has been described in many studies targeting multiple key cell signaling molecules. Mitochondria are the main hub for cellular energy production and play an important role in cell survival, and are the key target identified for HNK to mediate cancer cell death, survival, and metastasis. In this review, we have summarized different aspects of HNK's anti-cancer effects from recent accumulated literature, as well as the underlying molecular mechanisms. This review is primarily focused on the effects of HNK on epidermal growth factor receptor (EGFR) and signal transduction and activator of transcription 3 (STAT3) signaling, as well as the broader regulation of mitochondrial function and cancer cell metabolism.

Effect of Chemical Profiling Change of Processed Magnolia officinalis on the Pharmacokinetic Profiling of Honokiol and Magnolol in Rats.

The stem of Magnoliae officinalis (MO) cortex is always preliminarily processed before being applied in traditional Chinese medicine. The definite bioavailability of honokiol (HO) and magnolol (MA) in processed MO (PMO) and the effect of chemical profiling change on the pharmacokinetics of HO and MA are always a greater challenge compared with those of MO. Compared with that of MO, the pharmacokinetic profiling of HO and MA in the PMO was significantly changed and the mean Tmax of HO and MA was increased by 31 and 50% (P < 0.05), respectively; the mean AUC0-t and Cmax of HO were increased by 36 and 24% (P < 0.05), respectively. Subsequently, the chemical profiling of MO and PMO was investigated by a simple and rapid LC-Q/TOF-MS coupled with multivariate analysis method. Principal component analysis and hierarchical cluster analysis of the chromatographic data demonstrated that the chemical profiling of PMO was significantly different from that of MO. Eight marker components including six alkaloids (magnocurarine, magnoflorine, roemerine and three unidentified peaks) and two lignans (obovatol and MA) were screened out by partial least-squares discriminant analysis. The results indicated that the changes of eight marker components of PMO may have an effect on the pharmacokinetic profiles of HO and MA.

MTOR signaling pathway is inhibited downstream of the cyclophilin D-mediated mitochondrial permeability transition in honokiol-triggered regulated necrosis.

Honokiol (HNK) is a pharmacologically active small molecule that is isolated from the traditional Chinese medicinal herb, houpu. It may induce diversified types of regulated cell death, which are dependent on different cell types and varying concentrations of therapeutic agent. We previously reported that HNK triggers a cyclophilin D (CypD)-mediated regulated necrosis in various cell lines at certain concentrations (two‑fold higher than its half maximal inhibitory concentration). Subsequent study revealed that HNK induced cell death transition from early apoptosis to regulated necrosis in parallel with the increase of HNK dose. In the current study, a lower concentration of HNK (30 µg/ml) than previously reported also induced simplex CypD‑mediated mitochondrial permeability transition (MPT)‑associated regulated necrosis in the HEK‑293 human embryonic kidney cell line. HNK, at concentration of 30 µg/ml, induced necrotic cell death in HEK‑293 cells, which was demonstrated by positive staining for propidium iodide. No DNA ladder patterns or apoptotic bodies were detected in cells that underwent this type of necrotic cell death. Caspase‑8 and ‑3 were not activated during the process of HNK‑induced necrosis. In addition, pan‑caspase inhibitor, z‑VAD‑fmk and receptor‑interacting protein 1 inhibitor, necrostatin‑1 did not inhibit HNK‑induced necrosis. However, CypD inhibitor, cyclosporin A (CsA), blocked HNK‑induced necrosis. These findings indicate that 30 µg/ml HNK induced simplex CypD-mediated MPT‑associated regulated necrosis in HEK‑293 cells. Furthermore, the findings demonstrated that during HNK-triggered regulated necrosis the mammalian target of rapamycin (mTOR) signaling pathway is also inhibited. Pretreatment with CsA, therefore, inhibits HNK‑triggered regulated necrosis and reverses dephosphorylation of Akt, eIF4E‑binding protein 1 and S6 kinase. This indicated that the mTOR signaling pathway is effective downstream of the CypD‑mediated MPT and before the onset of plasma membrane breakdown during the regulated necrosis process. Therefore, it has been demonstrated for the first time, to the best of our knowledge, that the mTOR signaling pathway was inhibited downstream of the CypD-mediated MPT in the process of HNK-induced regulated necrosis.

Abstract B02: Honokiol attenuates stemness, growth and metastasis of pancreatic tumor cells: A novel natural agent for therapy and prevention

Abstract Pancreatic cancer (PC) is the fourth leading cause of cancer-related death in the United States with a five year survival rate at just 6.0%. Its poor clinical outcome is largely attributed to its aggressive and metastatic nature as well as refractoriness to currently available therapeutic modalities. Hence, there is a critical need to develop novel and more effective treatments for this malignancy. We previously reported that honokiol (HNK), a phytochemical isolated from the leaves and bark of Magnolia grandiflora, suppresses the growth of PC cells. Moreover, HNK sensitized PC cells to gemcitabine by preventing its activation of NF-kB pathway. Here, we have further analyzed the effects of HNK on clonogenicity, stemness, tumorigenicity and metastasis by performing in vitro and in vivo functional assays. Furthermore, we have conducted biochemical, histological and immunohistochemical analyses on vehicle and HNK-treated tumor cells and xenografts to examine the mechanism of action and molecular targets of HNK. We report that HNK treatment reduces the clonogenicity and stemness of PC cells as well as limits their capability to migrate and invade through the extracellular matrix. Our animal studies demonstrate that HNK significantly impedes the growth of pancreatic tumors and reduces the incidence as well as extent of metastases. Histological and immunohistochemical analyses suggest a decrease in desmoplasia, which is a key feature of pancreatic adenocarcinomas, acting as a physical barrier for drug delivery. At the molecular level, we have observed that HNK decreases the expression of hedgehog pathway through suppression of SHH production in PC cells. Further, we demonstrate a suppression of CXCR4 (a receptor for stromal cell derived chemokine, CXCL12) after treatment with HNK in a dose- and time-dependent manner. This is highly significant considering the known functions of SHH/CXCR4 signaling axes in growth, invasion, metastasis and therapeutic resistance of pancreatic tumor cells. Using transient transfection of constitutively active IKKB; mutant (pCMV-IKKB; S177E S181E) to neutralize the suppressive effect of HNK on NF-kB activation, we reveal its partial role in downregulation of CXCR4 and SHH expression. Together, our findings suggest that HNK likely impacts multiple molecular pathways to confer its anti-tumor activity and thus support its potential utility as a novel agent for PC therapy and/or prevention. Citation Format: Courey A. Averett, Arun Bhardwaj, Sumit Arora, Sanjeev Srivastava, Seema Singh, JE Carter, Ajay P. Singh. Honokiol attenuates stemness, growth and metastasis of pancreatic tumor cells: A novel natural agent for therapy and prevention. [abstract]. In: Proceedings of the Fourth AACR International Conference on Frontiers in Basic Cancer Research; 2015 Oct 23-26; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2016;76(3 Suppl):Abstract nr B02.

Protective effect of honokiol against LPS-induced lung injury via attenuation of matrix metalloproteinase-9 and oxidative stress

Despite high morbidity and mortality, no effective options are available for the treatment of acute lung injury (ALI). Therefore, the present study investigated the protective effect of honokiol (HK) on ALI via determination of its effect on several key biomarkers. The results of the study showed that HK significantly inhibited the infiltration of neutrophils and protein leakage induced by lipopolysaccharide (LPS) (p&lt;0.05). The pretreatment with HK considerably boosted the endogenous antioxidant defense system to counteract the oxidative stress in LPS-induced ALI by elevating the levels of superoxide dismutase (SOD), catalase (CAT) and glutathione (GSH). Moreover, the activity of toxic mediators, such as myeloperoxidase (MPO), and lipid peroxidation were significantly inhibited upon treatment with HK. In order to examine the mechanism of action of HK, its effect was quantified using matrix metalloproteinase-9 (MMP-9) activity in bronchoalveolar lavage fluid (BALF) by gelatin zymography. Pretreatment with HK considerably suppressed the activation of MMP-9 in a concentration-dependent manner. These findings suggest that HK protects from lung injury via inhibition of MMP-9, and by enhancing the activity of the endogenous antioxidant defense system.

Encapsulation of honokiol-loaded nanoparticles in lecithin microbubbles for targeted tumor therapy

This study aimed to develop a new drug delivery system that combines honokiol-loaded albumin nanoparticles (HKNs) with perfluorocarbon-filled microbubbles (MBs) to improve the target delivery of honokiol (HK).

In vitro synergism of magnolol and honokiol in combination with antibacterial agents against clinical isolates of methicillin-resistant Staphylococcus aureus (MRSA).

BackgroundMethicillin-resistant Staphylococcus aureus (MRSA) is a problematic pathogen posing a serious therapeutic challenge in the clinic. It is often multidrug-resistant (MDR) to conventional classes of antibacterial agents and there is an urgent need to develop new agents or strategies for treatment. Magnolol (ML) and honokiol (HL) are two naturally occurring diallylbiphenols which have been reported to show inhibition of MRSA. In this study their synergistic effects with antibacterial agents were further evaluated via checkerboard and time-kill assays.MethodsThe susceptibility spectrum of clinical MRSA strains was tested by the disk diffusion method. The minimal inhibitory concentrations (MICs) and minimal bactericidal concentrations (MBCs) of ML and HL were assayed by broth microdilution. The synergy was evaluated through checkerboard microdilution and time-killing experiments.ResultsML and HL showed similar activity against both MSSA and MRSA with MIC/MBC at 16 ~ 64 mg/L, with potency similar to amikacin (AMK) and gentamicin (GEN). When they were used in combination with conventional antibacterial agents, they showed bacteriostatic synergy with FICIs between 0.25 ~ 0.5, leading to the combined MICs decreasing to as low as 1 ~ 2 and 1 ~ 16 mg/L for ML (HL) and the agents, respectively. MIC50 of the combinations decreased from 16 mg/L to 1 ~ 4 mg/L for ML (HL) and 8 ~ 128 mg/L to 2 ~ 64 mg/L for the antibacterial agents, which exhibited a broad spectrum of synergistic action with aminoglycosides (AMK, etilmicin (ETM) and GEN), floroquinolones (levofloxacin (LEV), ciprofloxacin and norfloxacin), fosfomycin (FOS) and piperacillin. The times of dilution (TOD, the extent of decreasing in MIC value) were determined up to 16 for the combined MIC. A more significant synergy after combining was determined as ML (HL) with AMK, ETM, GEN and FOS. ML (HL) combined with antibacterial agents did not show antagonistic effects on any of the ten MRSA strains. Reversal effects of MRSA resistance to AMK and GEN by ML and HL were also observed, respectively. All the combinations also showed better dynamic bactericidal activity against MRSA than any of single ML (HL) or the agents at 24 h incubation. The more significant synergy of combinations were determined as HL (ML) + ETM, HL + LEV and HL + AMK (GEN or FOS), with △LC24 of 2.02 ~ 2.25.ConclusionML and HL showed synergistic potentiation of antibacterial agents against clinical isolates of MRSA and warrant further pharmacological investigation.

Genotoxic assessment of Crataegus oxyacantha fruits extract in cells of mice

Self-assembled pectin nanoparticles was prepared and evaluated for delivering the hydrophobic drug, honokiol (HK), to HepG2 cells. These hydrophobic drug-loaded nanoparticles were developed without using any surfactant and organic solvent. Hydroxypropyl-β-cyclodextrin (HCD) was used to fabricate an inclusion complex with HK (HKHCD) to increase the solubility of the drug and thus facilitate its encapsulation and dispersion in the pectin nanoparticles. Investigation of the in vitro release indicated that the drug-loaded nanoparticles exhibited a higher drug release rate than free honokiol and an effective sustained-release. Cytotoxicity, cell apoptosis and cellular uptake studies further confirmed that the pectin nanoparticles with galactose residues generated higher cytotoxicity than free honokiol on HepG2 cells which highly expressed asialoglycoprotein receptors (ASGR). Nevertheless, these findings were not observed in ASGR-negative A549 cells under similar condition. Therefore, pectin nanoparticles demonstrated a specific active targeting ability to ASGR-positive HepG2 cells and could be used as a potential drug carrier for treatment of liver-related tumors.

Comparative pharmacokinetics and brain distribution of magnolol and honokiol after oral administration of Magnolia officinalis cortex extract and its compatibility with other herbal medicines in Zhi-Zi-Hou-Po Decoction to rats.

Zhi-Zi-Hou-Po decoction (ZZHPD) is one of the famous antidepressant Chinese formulas and is composed of Magnolia officinalis cortex (HP), Gardenia jasminoides Ellis (ZZ) and Citrus aurantium L. (ZS). Magnolol (MN) and honokiol (HN) from HP are the major active ingredients responsible for the therapeutic effects of ZZHPD. The aim of this study is to compare the pharmacokinetics and rat brain distribution of MN and HN after oral administration of HP extract and its compatibility with other herbal medicines in ZZHPD by HPLC-FLD. Compared with the HP group, Tmax (time to reach peak drug concentration in plasma) and AUC(0-τ) significantly increased in the ZZHPD and HP-ZZ groups. There was little change in the HP-ZS group in comparison with the HP group, which indicated that ZZ promotes absorption extent and defers the absorption rate of MN. The different compatibility of ZZHPD had a different degree of impact on the concentration of MN and HN in brain. The concentration of MN significantly increased in the HP-ZZ group while it decreased in the HP-ZS group compared with the HP group, which explained the concentration of compounds being slightly greater in the ZZHPD group than in the HP group. HP mixed with other medicines resulted in a decrease in HN concentration in the brain, particularly HP compatible with ZS. The results could be helpful for revealing the compatibility mechanism and providing clinical medication guidance for ZZHPD.

Honokiol activates LKB1-miR-34a axis and antagonizes the oncogenic actions of leptin in breast cancer

Leptin, a major adipocytokine produced by adipocytes, is emerging as a key molecule linking obesity with breast cancer therefore, it is important to find effective strategies to antagonize oncogenic effects of leptin to disrupt obesity-cancer axis. Here, we examine the potential of honokiol (HNK), a bioactive polyphenol from Magnolia grandiflora, as a leptin-antagonist and systematically elucidate the underlying mechanisms. HNK inhibits leptin-induced epithelial-mesenchymal-transition (EMT), and mammosphere-formation along with a reduction in the expression of stemness factors, Oct4 and Nanog. Investigating the downstream mediator(s), that direct leptin-antagonist actions of HNK; we discovered functional interactions between HNK, LKB1 and miR-34a. HNK increases the expression and cytoplasmic-localization of LKB1 while HNK-induced SIRT1/3 accentuates the cytoplasmic-localization of LKB1. We found that HNK increases miR-34a in LKB1-dependent manner as LKB1-silencing impedes HNK-induced miR-34a which can be rescued by LKB1-overexpression. Finally, an integral role of miR-34a is discovered as miR-34a mimic potentiates HNK-mediated inhibition of EMT, Zeb1 expression and nuclear-localization, mammosphere-formation, and expression of stemness factors. Leptin-antagonist actions of HNK are further enhanced by miR-34a mimic whereas miR-34a inhibitor results in inhibiting HNK's effect on leptin. These data provide evidence for the leptin-antagonist potential of HNK and reveal the involvement of LKB1 and miR-34a.

Encapsulation of honokiol into self-assembled pectin nanoparticles for drug delivery to HepG2 cells

Self-assembled pectin nanoparticles was prepared and evaluated for delivering the hydrophobic drug, honokiol (HK), to HepG2 cells. These hydrophobic drug-loaded nanoparticles were developed without using any surfactant and organic solvent. Hydroxypropyl-β-cyclodextrin (HCD) was used to fabricate an inclusion complex with HK (HKHCD) to increase the solubility of the drug and thus facilitate its encapsulation and dispersion in the pectin nanoparticles. Investigation of the in vitro release indicated that the drug-loaded nanoparticles exhibited a higher drug release rate than free honokiol and an effective sustained-release. Cytotoxicity, cell apoptosis and cellular uptake studies further confirmed that the pectin nanoparticles with galactose residues generated higher cytotoxicity than free honokiol on HepG2 cells which highly expressed asialoglycoprotein receptors (ASGR). Nevertheless, these findings were not observed in ASGR-negative A549 cells under similar condition. Therefore, pectin nanoparticles demonstrated a specific active targeting ability to ASGR-positive HepG2 cells and could be used as a potential drug carrier for treatment of liver-related tumors.

Honokiol exhibits enhanced antitumor effects with chloroquine by inducing cell death and inhibiting autophagy in human non-small cell lung cancer cells.

Honokiol (HNK), a potential antitumor compound, has been widely studied in recent years. It induces apoptosis and affects autophagy in cancer cells, yet the mechanism of its antitumor efficacy remains obscure. Chloroquine (CQ), an autophagy inhibitor, is often applied to sensitize antitumor drugs in clinical trials. Here, we investigated the antitumor effect of HNK or CQ alone or in combination in non-small cell lung cancer (NSCLC) cells. Using an experimental approach, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) or sulforhodamine B (SRB) was used to determine the cytotoxicity of the agents. The expression levels of proteins were detected by western blotting. Apoptosis was examined via Annexin V-FITC and PI staining. H460 cell xenografts in nude mice were used to study the effects of HNK and/or CQ invivo. Transfection with siRNA was applied to knock down cathepsinD. The results demonstrated the enhanced effects of HNK combined with CQ on the inhibition of proliferation, induction of apoptosis invitro and the reduction in growth invivo. It was confirmed that HNK and/or CQ triggered apoptosis via a caspase-dependent manner. Furthermore, HNK significantly increased the expression of p62 and LC3-Ⅱ in the A549 and H460 cells and inhibited autophagy and induced apoptosis in a cathepsin D-involved manner. In conclusion, an enhanced antitumor effect was demonstrated following treatment with HNK combined with CQ by inhibiting autophagy and inducing apoptosis via a caspase-dependent and cathepsin D-involved manner. This combination may be a novel and useful antitumor approach for chemotherapy in NSCLC.

Anti-psoriatic effects of Honokiol through the inhibition of NF-κB and VEGFR-2 in animal model of K14-VEGF transgenic mouse

Honokiol (HK), a biphenolic neolignan isolated from Magnolia officinalis, has been reported to possess anti-inflammatory and anti-angiogenic activaties. In this study, our aim was to investigate anti-psoriatic activities of HK and the involved mechanisms. In vitro, the effects of HK on the regulation of Th1/Th2 and TNF-α-induced NF-κB (p65) activation were analyzed by respective FCS and immunofluorescence. Additionally, the K14-VEGF transgenic model was used for the in vivo study. ELISA and Q-PCR were performed to evaluate serum levels of Th1/Th2 cytokines and their corresponding mRNA expressions. Effects on VEGFR-2 and p65 activation, as well as other angiogenic and inflammatory parameters were studied by immunostainings. Importantly, we found that HK significantly decreased the ratio of Th1/Th2-expression CD4+ T cells and inhibited TNF-α-induced activation of NF-κB. The morphology and histological features of psoriasis were effectively improved by HK treatment. The expression of TNF-α and IFN-γ, and their corresponding mRNA levels were down-regulated and the expression of nuclear p65, VEGFR-2, as well as related phosphorylated proteins (p-VEGFR-2, p-ERK1/2, p-AKT and p-p38) were also suppressed. Overall, these results in our study suggested that HK exhibits anti-psoriatic effects through the inhibition of NF-κB and VEGFR-2.

The Pharmacokinetics and Tissue Distribution of Honokiol and its Metabolites in Rats.

Honokiol (HK) is the main bioactive compound isolated from the bark of Magnolia officinalis. The present work is the first to report the pharmacokinetics and distribution of HK and its two metabolites of hydroxylated HK conjugated with glucuronic and sulfuric acid (M1) and HK monoglucuronide (M2) in plasma, liver, kidney and brain following oral administration of HK (40mg/kg) to healthy Wistar rats. The results showed that only HK but not M1 or M2 was found in brain. Additionally, our work indicated that M2 not HK was the major compound in liver and plasma. The elimination of HK in liver, kidney and brain, and M2 in liver and kidney was more rapid than in plasma. The finding suggested that some of the pharmacological activity of HK might be generated by M2 but not HK.