Honokiol Research Articles

A novel honokiol liposome: formulation, pharmacokinetics, and antitumor studies

It is necessary to discover a novel antitumor liposome with prolonged circulation time, high efficacy, and low cost. Here, we reported a liposomal honokiol (HNK) prepared with a new type of excipient, Kolliphor HS15, which was termed as HS15-LP-HNK. In addition, we employed PEGylated liposomal honokiol (PEG-LP-HNK) as positive control. The HS15-LP-HNK was prepared by thin-film hydration method. It was near-spherical morphology with an average size of 80.62 ± 0.72 nm (PDI = 0.234 ± 0.007) and a mean zeta potential of −3.91 ± 0.06 mv. In vivo studies exhibited no significant difference between HS15-LP-HNK and PEG-LP-HNK. The pharmacokinetic and biodistribution results showed that HS15-LP-HNK could improve the bioavailability and increase tumor accumulation of honokiol. Furthermore, HS15-LP-HNK could enhance antitumor efficacy of honokiol with low toxicity. In summary, HS15-LP-HNK is promising in tumor targeted drug delivery system.

Genome-wide screening of budding yeast with honokiol to associate mitochondrial function with lipid metabolism.

Honokiol (HNK), an important medicinal component of Magnolia officinalis, is reported to possess pharmacological activities against a variety of diseases. However, the molecular mechanisms of HNK medicinal functions are not fully clear. To systematically study the mechanisms of HNK action, we screened a yeast mutant library based on the conserved nature of its genes among eukaryotes. We identified genes associated with increased resistance or sensitivity to HNK after mutation. After functional classification of these genes, we found that most HNK-resistant strains in the largest functional category were petites with mutations in mitochondrial genes, indicating that mitochondria were related to HNK resistance. Additional analysis showed that resistance of petite mutants to HNK was associated with upregulation of the ATP-binding cassette transporter Pdr5, which pumps out HNK. We also found that several HNK-sensitive mitochondria mutants were not petites, and had larger lipid droplets (LDs). Furthermore, HNK treatment on wild-type yeast cells seemed to disrupt mitochondrial morphology, induced triacylglycerol synthesis, and generated supersized LDs surrounded by mitochondria and endoplasmic reticulum (ER). These changes are also applied to atp7Δ mutant if no carbon resource was available. These results suggested that HNK treatment partly impaired normal mitochondrial function to form larger LDs by altering lipid metabolism.

Honokiol protects pulmonary microvascular endothelial barrier against lipopolysaccharide-induced ARDS partially via the Sirt3/AMPK signaling axis

AimsAcute respiratory distress syndrome (ARDS) is characterized by acute hypoxemia with diffuse alveolar damage and increased pulmonary microvascular permeability. Honokiol (HKL), the principal active ingredient of Chinese herb magnolia officinalis, protected the lung of experimental ARDS models via attenuation of inflammation and oxidative stress. However, whether HKL has protective effects against the dysfunction of pulmonary microvascular endothelial barrier and the potential mechanisms remain unclear. Main methodsIn the present study, we examined the levels of plasma Angiopoietin-2 (Ang-2) in ARDS patients, explored the effects of HKL on the vascular endothelial barrier at the ARDS animal and cell levels. Key findingsOur data showed that compared with the healthy controls, circulating Ang-2 level was higher in the patients with ARDS, and were usually supposed to be positively related to the severity of ARDS. Moreover, HKL effectively inhibited lung inflammatory injury and microvascular leakage, and improved ARDS mice survival. HKL also inhibited the expression of Ang-2, ICAM-1 and VCAM-1, and restored the expression of Sirt3, β-Catenin and VE-Cadherin. Furthermore, HKL improved ECs survival and inhibited the apoptosis of ECs. The inhibition of Ang-2 expression in vitro by HKL is accompanied by the upregulation of Sirt3 and AMPK phosphorylation. SignificanceOur data demonstrated that HKL protected pulmonary microvascular endothelial barrier against LPS-induced ARDS at least in part through activating the Sirt3/AMPK signaling and inhibiting the Ang-2 expression. Thus, our findings show that the activation of Sirt3 signaling is a potential mechanism for the protective effects of HKL on vascular barrier.

Honokiolinhibits the proliferation and invasion abilityof pancreatic cancer cells and the mechanism

Objective To observe the effect and mechanism of honokiol on the proliferation and invasion of SW1990cell. Methods SW1990 were treated with honokiol, Cell Counting Kit-8 (CCK-8) assay was used to detect the cell proliferation. cell apoptotic rate was detected by flow cytometry, cell invasion ability was evaluated by tumour invasion assay; and the mRNA and protein expression of E-cadherin and Wnt/β-cateninsignal were detected by reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting, respectively. Results Honokiol inhibited the growth of SW1990 cell in a time-dependent and concentration-dependent manner. With the increase of honokiol concentration, the late stage apoptosis of SW1990 were (2.10±0.44)%, (5.25±1.39)%, (25.75±1.00)%, (60.19±3.14)%, respectively (F=572.336, P=0.000). The number of invaded cells were (170.40±12.20), (95.20±6.98), (54.80±5.63), (21.40±4.93) cells, respectively (F=321.703, P=0.000). The mRNA and protein expression of β-catenin, Vimentin, B-cell lymphoma/leukemia (bcl)-xl, Survivin, c-Myc were down-regulated and bax, Caspase-3, E-cadherin were up-regulated in a concentration-dependent manner, which was detected by RT-PCR and Western blotting. Conclusion Honokiol inhibitedproliferation and invasion of SW1990 cell obviously, which may be through down-regulating the Wnt/β-catenin signalpathway. Key words: Honokiol; Pancreatic cancer; Proliferation; Apoptosis; Invasion

Abstract 2337: Effect of Honokiol and Indol-3-carbinol in viability and apoptosis of colorectal cancer cell lines treated with Orexin-A

Abstract Introduction: Colorectal cancer (CRC) is the third leading cause of cancer deaths worldwide. Recently, much attention has been given to nutraceuticals. They are being investigated for prevention and treatment of cancer. It has been shown that they have antitumoral properties without secondary effects, and could be used as a complement to conventional therapy for CRC. Orexin-A (OXA) is a hypothalamic neuropeptide with various functions in central nervous system and peripheral tissues. Orexin receptor 1 (OX1R) is expressed in CRC tissues. Activation of OX1R by OXA in CRC cell lines triggers apoptosis and was proposed as a new alternative for therapy. The aim of this study was to evaluate different nutraceuticals that could decrease survival and increase apoptosis of CRC cells lines in synergism with OXA. Material and Methods: Two CRC cell lines were obtained from ATCC: Caco2 and SW480. Cell lines were treated with Honokiol (HK) or Indol-3-carbinol (I3C) for 24h at different doses with or without 100nM OXA. Cell viability was determined using CellTiter Kit (Promega). Caspase 3/7 mediated apoptosis activity was assessed using ApoOne Kit (Promega). Real-time qPCR was performed to determine the effect of nutraceuticals on OX1R expression. Results: In this study, HK decreases cellular viability on average of 47% in Caco2 and 80% SW480 cells lines, at 25uM, 50uM or 100uM. In Caco2 cell line an increase in caspase 3/7 mediated apoptosis of 60% and 76% was observed at 25uM and 50uM, respectively, unlike what was observed in SW480 in which there was an inhibition of 80% at the different doses used. The combined treatment of 50uM HK with 100nM OXA does not produce a synergistic effect in cell viability or apoptosis compared to use of HK alone in both cell lines. HK produces an inhibition in the OX1R expression that explains the lack of synergism with OXA. I3C produces a decrease of 25% to 95% of cell viability according to doses used with an increase in apoptosis of 6 % to 38% in Caco2 cell lines. The combined effect of 500uM I3C with 100nM OxA produces a synergistic effect of 95% decrease on cell viability, with an increase of OX1R expression, which is not explained by an increase in apoptosis mediated by caspase 3/7. Conclusions: We found that HK and I3C can inhibit viability and induce apoptosis in Caco2 and SW480 cells. In Caco2 cell line this can be explained by an increase in 3/7 caspase-mediated apoptosis, unlike that observed for SW480 treated with HK. Only 13C has a synergistic effect with OXA on cell viability in Caco2 cell lines but was independent of caspase 3/7 mediated apoptosis at doses and times used. Treatments using new doses and times are necessary to improve response. In our results, I3C might be a better therapeutic agent than HK for CRC. In vivo studies and clinical trials are further required to evaluate physiologic efficacies of the combination treatment. Fondecyt 1140012. Citation Format: Ana Maria Wielandt, Cyntia Villarroel, Elea Gros, Mauricio Moreno, Claudia Hurtado, Udo Kronberg, Francisco Lopez-Kostner. Effect of Honokiol and Indol-3-carbinol in viability and apoptosis of colorectal cancer cell lines treated with Orexin-A [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2337.

Study on effect and mechanism of Honokiol enhancing tumor necrosis factor-related apoptosis inducing ligand against hepatocellular carcinoma HepG2 cells via activating JNK signaling pathway

Objective: To investigate the effect and intrinsic mechanism of Honokiol (HNK) enhanced tumor necrosis factor-related apoptosis inducing ligand (TRAIL) on hepatocellular carcinoma HepG2 cells. Methods: HepG2 cells were routinely cultured. Firstly, a concentration gradient of HNK was given to observe its effect on the vitality of tumor cells. Western blot were used to detect change in the expression levels of c-jun N-terminal kinase (JNK), death receptor 4 (DR4), and DR5.Secondly, we observed the effect of combined drugs (HNK and TRAIL) on the vitality of tumor cells. Apoptosis-related protein expression levels were detected to determine the apoptosis condition. Thirdly, JNK inhibitor SP600125 was used to block the JNK pathway, and it was evaluated whether JNK signaling pathway mediated the DR4 and DR5 levels and finally, the subcutaneous tumor model of nude mice was constructed, and enhancement effect of HNK on TRAIL was confirmed in vivo. Results: Cell vitality was decreased (P < 0.05) in a dose-dependent manner after treatment with gradient HNK. Combined effect of TRAIL and HNK was superior to that of single drug administration (P < 0.05). Western blot analysis showed that pJNK level increased after HNK treatment and TRAIL receptor DR4 and DR5 expression were up-regulated. Combined application of HNK and TRAIL, B lymphocyte tumor factor 2 (BCL2) decreased significantly while Bcl2 related X protein (Bax) increased significantly. Blocking JNK pathway by SP600125, the expression of DR4 and DR5 decreased (P < 0.05), Bax expression decreased and Bcl2 expression increased compared with HNK+TRAIL group. In vivo results showed that TRAIL inhibited the growth of subcutaneous tumors, and enhanced inhibition effect in combination with TRAIL and HNK. Conclusion: HNK may enhance the inhibitory effect of TRAIL on HepG2 cells by activating JNK pathway and then upregulating the expression of DR4 and DR5.

Protective effects of honokiol against oxidative stress-induced apoptotic signaling in mouse podocytes treated with H2O2.

Honokiol (HNK), an important bioactive compound purified from Magnolia officinalis Cortex, has been demonstrated to have manifold beneficial anti-oxidative, anti-inflammatory, anti-bacterial and antitumor pharmacological effects. In the present study, the association of HNK in the signaling mechanism associated with hydrogen peroxide (H2O2)-induced apoptosis of cultured mouse podocytes was investigated. HNK did not cause significant changes in podocyte viability when its concentration remained below 20 µM. MTS assay and flow cytometry confirmed that H2O2 significantly enhanced the rates of apoptosis while produce significant reduction in viability of podocytes. Following 24 h of pre-treatment with different concentrations of HNK, the viability of adherent podocytes increased and apoptosis significantly decreased in a dose-dependent manner below 20 µM. Reverse transcription-polymerase chain reaction and western blot results indicated that HNK significantly decreased the expression of mRNA and cleaved protein of caspase-3 and caspase-9 in podocytes pre-treated with H2O2. Furthermore, phosphorylation of the signaling molecules protein kinase B (Akt) and extracellular signal-regulated kinase (Erk) 1/2 appeared to increase following HNK treatment. In conclusion, HNK largely eliminated the role of promoting podocyte apoptosis in an oxidative stress environment, which was a protective factor on podocytes cultured with H2O2. The anti-oxidative stress mechanisms of HNK are partly due to suppressing the expression of caspase-3 and caspase-9 and upregulating phosphorylated-Akt and -Erk 1/2.

Honokiol Alleviates Cognitive Deficits of Alzheimer's Disease (PS1V97L) Transgenic Mice by Activating Mitochondrial SIRT3.

Accumulating evidence has demonstrated that mitochondrial dysfunction is a prominent early event in the progression of Alzheimer's disease (AD). Whether protecting mitochondrial function can reduce amyloid-β oligomer (AβO)-induced neurotoxicity in PS1V97L transgenic mice remains unknown. In this study, we examined the possible protective effects of honokiol (HKL) on mitochondrial dysfunction induced by AβOs in neurons, and cognitive function in AD PS1V97Ltransgenic mice. We determined that HKL increased mitochondrial sirtuin 3 (SIRT3) expression levels and activity, which in turn markedly improved ATP production and weakened mitochondrial reactive oxygen species production. We demonstrated that the enhanced energy metabolism and attenuated oxidative stress of HKL restores AβO-mediated mitochondrial dysfunction in vitro and in vivo. Consequently, memory deficits in the PS1V97L transgenic mice were rescued by HKL in the early stages. These results suggest that HKL has therapeutic potential for delaying the onset of AD symptoms by alleviating mitochondrial impairment and increasing hyperactivation of SIRT3 in the pathogenesis of preclinical AD.

Honokiol Exerts Antidepressant Effects in Rats Exposed to Chronic Unpredictable Mild Stress by Regulating Brain Derived Neurotrophic Factor Level and Hypothalamus-Pituitary-Adrenal Axis Activity.

Honokiol (HNK), the main active component of Magnolia officinalis, has shown a variety of pharmacological activities. In the present study, we measured the antidepressant-like effects of HNK in a rat model of chronic unpredictable mild stress (CUMS) and explored its possible mechanisms. The antidepressant-like effects of HNK were assessed in rats by an open field test (OFT), sucrose preference test (SPT) and forced swimming test (FST). Then, serum levels of corticotrophin-releasing hormone (CRH), adrenocorticotropic hormone (ACTH) and corticosterone (CORT) and hippocampal brain-derived neurotrophic factor (BDNF) and glucocorticoid receptor α (GRα) levels were assessed to explore the possible mechanisms. We identified that HNK treatment (2, 4, and 8mg/kg) alleviated the CUMS-induced behavioural deficits. Treatment with HNK also normalized the CUMS-induced hyperactivity of the limbic hypothalamic-pituitary-adrenal (HPA) axis, as indicated by reduced CRH, ACTH and CORT serum levels. In addition, HNK increased the expression of GRα (mRNA and protein) and BDNF (mRNA and protein) in the hippocampus. These data confirmed the antidepressant-like effects of HNK, which may be related to its normalizing the function of the HPA axis and increasing the BDNF level in the hippocampus.

Synergistically Enhanced Antimetastasis Effects by Honokiol-Loaded pH-Sensitive Polymer-Doxorubicin Conjugate Micelles.

In an effort to prevent metastasis of breast tumor cells- at the same time of inhibiting tumor growth with less toxic side effects, honokiol (HNK) was encapsulated into pH-sensitive polymeric micelles based on the conjugate of poly(2-ethyl-2-oxazoline)-poly(d,l-lactide) (PEOz-PLA) with doxorubicin (DOX), denoted as PEOz-PLA-imi-DOX. PEOz-PLA-imi-DOX was successfully synthesized by connecting DOX to the hydrophobic end of PEOz-PLA via acid-cleavable benzoic imine linker. HNK-loaded conjugate micelles (HNK/PP-DOX-PM) with a size of 21 nm and homogeneous spherical shape exhibited high drug-loading capacity. PEOz-PLA-imi-DOX and HNK/PP-DOX-PM displayed faster release of DOX at pH 5.0 than at pH 7.4. As anticipated, PEOz-PLA-imi-DOX maintained cytotoxicity of DOX against MDA-MB-231 cells. The synergistically enhanced in vitro antitumor effect of HNK/PP-DOX-PM was confirmed by their synergetic inhibition of MDA-MB-231 cell growth. Furthermore, the efficient prevention of tumor metastasis by HNK/PP-DOX-PM was testified by in vitro anti-invasion, wound healing and antimigration assessment in MDA-MB-231 cells, and in vivo bioluminescence imaging in nude mice. The suppression of growth and metastasis of tumor cells by HNK/PP-DOX-PM was attributed to the synergistic effect of pH-triggered drug release and HNK-aroused inhibition of matrix metalloproteinases and epithelial-mesenchymal transition, respectively. In addition, HNK/PP-DOX-PM exhibited superior biosafety than physically encapsulated dual-drug micelles. Consequently, the fabricated HNK/PP-DOX-PM may have great potential for safe and effective suppression of tumor growth and metastasis.

Honokiol nanoparticles based on epigallocatechin gallate functionalized chitin to enhance therapeutic effects against liver cancer

This study aims to design a novel nano-sized anticancer drug delivery system that can enhance the therapeutic effects of the loaded drug. With this idea in mind, this work reported the design and characterization of epigallocatechin-3-gallate (EGCG) functionalized chitin (CH) derivative, and its application in nano-drug delivery system. The EGCG-functionalized CH (CE) polymer was firstly prepared and characterized. The nanoparticles (NPs) of CE-loaded honokiol (HK), which was prepared by ionic crosslinking, exhibited a size of 80 nm, zeta potential of +33.8 mV, and spherical morphology. The antitumor activity of the CE-HK NPs in vitro and in vivo was investigated and compared to free HK. As a result, the CE-HK NPs can effectively inhibited cell proliferation of HepG2 cell by inhibiting more cells in the G2/M phase and decreasing mitochondrial membrane potential. The CE-HK NPs (40 mg/kg) inhibited tumor growth by 83.55% (p < 0.05), which was far higher than the 30.15% inhibition of free HK (40 mg/kg). The proposed delivery system exhibits better tumor selectivity and growth reduction both in vitro and in vivo, and does not induce any side effects. Therefore, the CE-HK NPs may act as an effective delivery system of liver cancer agent HK.

Effects of Honokiol on airway inflammation in asthmatic mice exposed to PM2.5 and its mechanism

Objective To investigate the protective effect of Honokiol on the airway inflammation induced by particulate matter 2.5 (PM2.5) in the asthmatic mice and its mechanism. Methods Fifty male specific pathogen free(SPF) Balb/c mice were randomly divided into 5 groups.Group A: normal control group; group B: asthmatic model group; group C: PM2.5 exposure asthmatic group; group D: TAK-242 group; group E: Honokiol group.Asthmatic mouse models were established by ovalbumin (OVA) sensitization and challenge.On days 0 and 7, the mice in B-E groups were injected intraperitoneally with injection 100 mg/L OVA and aluminum hydroxide for sensitization; on days 14 to 21, 10 g/L OVA solution was given 30 min per day to challenge.During challenge phrase, the mice in C-E groups received intratracheal injection of PM2.5, every other day, 4 times totally.On this basis, the mice in group D received TAK-242 intraperitoneal injection, and the mice in group E received honokiol intragastric administration.Group A was given saline instead of OVA.Animals were sacrificed 24 h after the final inhalation challenge, and the bronchoalveolar lavage fluid(BALF) of the left lung was used for differential inflammatory cell counts.The expressions of Toll-like receptors 4(TLR4) and nuclear factor (NF)-κB at mRNA level were detected by real-time quantitative PCR.Flow cytometry analysis was performed to measure the levels of Th17 and Treg cells. Results Compared with group A, mice in group B and group C expressed more serious disorders of bronchial epithelial cells, alveolar wall congestion and edema, increased mucus secretion in the airway and infiltration of inflammatory cells in the lung, and those in group C were more obvious than those of group B and group E significantly reduced respiratory inflammation; compared with group A[(4.15±1.35)×108/L, 0.012 0±0.002 3], the total number of inflammatory cell counts [(16.79±5.62)×108/L and (24.58±13.46)×108/L], eosinophils proportions (0.113 8±0.022 3 and 0.197 8±0.084 9) in group B and group C, were significantly higher, and the differences were statistically significant(all P<0.05); The total number of inflammatory cell counts and eosinophils proportion in group E (8.56±3.28)×108/L and 0.041 5±0.013 5) were significantly lower than those in group C, and the differences were statistically significant(all P<0.05); The expressions of TLR4 mRNA and NF-κB mRNA in group B and C (1.85±0.56, 1.82±0.28 and 2.97±0.41, 2.83±0.32) were significantly higher, and the differences were statistically significant(all P<0.05); The expressions of TLR4 mRNA and NF-κB mRNA in group E(1.60±0.28, 1.54±0.25)was significantly lower than those in group C, and the differences were statistically significant(all P<0.05); the expressions of Th17 in group B and C [(2.89±0.61)% and (4.96±0.27)%] were significantly higher than those of group A[(1.03±0.35)%](all P<0.05); The expression of Th17 in group E[(1.83±0.23)%]was significantly lower than that of group C, and the differences were statistically significant(P<0.05); the expressions of Treg in group B and C [ (4.96±0.35)% and (2.27±0.41)%] were significantly lower than those of group A[(7.37±0.56)%], and the differences were statistically significant(all P<0.05); The expression of Treg in group E was significantly increased [(6.45±0.38)%]compared with that in group C, and the difference were statistically significant(P<0.05); and those of group D and E were improved remarkably. Conclusions Honokiol can relieve PM2.5 exposure of asthmatic airway inflammation through down-regulating the expression of TLR4 and NF-κB and Th17 and regulating the balance of Th17 and Treg cells. Key words: Asthma; Particulate matter; Toll-like receptors 4; Nuclear factor-κB; Honokiol

Simultaneous determination of Magnolol and Honokiol by amino acid ionic liquid synchronous fluorescence spectrometry

A novel method based on amino acid ionic liquids (AAILs) as an additive synchronous fluorescence spectrometry is proposed for simultaneous determination of magnolol (MN) and honokiol (HN) in traditional Chinese medicine Houpu. The overlapping fluorescence spectrum of MN and HN could be completely separated in the AAILs medium. Experiment parameters (the type and concentration of AAILs, pH values and temperature) were discussed. The detection limits of MN and HN reached 1.46ng/mL, 0.92ng/mL and the recovery rates ranged from 98.6%–100.7%, 99.7%–100.6%, respectively. This methods was successfully employed for simultaneously determination of MN and HN in real samples. No significant differences could be found in the results of this method and the pharmacopoeia of People's Republic of China 2015 (Ch.P.2015). The experiment mechanisms were discussed by the Gaussian simulation and fluorescence quantum yield.

Hyaluronic Acid-Modified Micelles Encapsulating Gem-C12 and HNK for Glioblastoma Multiforme Chemotherapy.

Glioblastoma multiforme (GBM), a prevalent brain cancer with high mortality, is resistant to the conventional single-agent chemotherapy. In this study, we employed a combination chemotherapy strategy to inhibit GBM growth and addressed its possible beneficial effects. The synergistic effect of lauroyl-gemcitabine (Gem-C12) and honokiol (HNK) was first tested and optimized using U87 cells in vitro. Then, the hyaluronic acid-grafted micelles (HA-M), encapsulating the optimal mole ratio (1:1) of Gem-C12 and HNK, were prepared and characterized. Cell-based studies demonstrated that HA-M could be transported into cells by a CD44 receptor-mediated endocytosis, which could penetrate deeper into tumor spheroids and enhance the cytotoxicity of payloads to glioma cells. In vivo, drug-loaded HA-M significantly increased the survival rate of mice bearing orthotopic xenograft GBM compared with the negative control (1.85-fold). Immunohistochemical analysis indicated that the enhanced efficacy of HA-M was attributed to the stronger inhibition of glioma proliferation and induction of apoptosis. Altogether, our findings showed advantages of combination chemotherapy of GBM using HA-grafted micelles.

Honokiol induces apoptosis and autophagy via the ROS/ERK1/2 signaling pathway in human osteosarcoma cells in vitro and in vivo

Osteosarcoma is the most common primary malignant tumor of bone, the long-term survival of which has stagnated in the past several decades. In the present study, we investigated the anticancer effect of honokiol (HNK), an active component isolated and purified from the magnolia officinalis on human osteosarcoma cells. Our results showed that honokiol caused dose-dependent and time-dependent cell death in human osteosarcoma cells. The types of cell death induced by honokiol were primarily autophagy and apoptosis. Furthermore, honokiol induced G0/G1 phase arrest, elevated the levels of glucose-regulated protein (GRP)−78, an endoplasmic reticular stress (ERS)-associated protein, and increased the production of intracellular reactive oxygen species (ROS). In contrast, reducing production of intracellular ROS using N-acetylcysteine, a scavenger of ROS, concurrently suppressed honokiol-induced cellular apoptosis, autophagy, and cell cycle arrest. Consequently, honokiol stimulated phosphorylation of extracellular signal-regulated kinase (ERK)1/2. Furthermore, pretreatment of osteosarcoma cells with PD98059, an inhibitor of ERK1/2, inhibited honokiol-induced apoptosis and autophagy. Finally, honokiol suppressed tumor growth in the mouse xenograft model. Taken together, our results revealed that honokiol caused G0/G1 phase arrest, induced apoptosis, and autophagy via the ROS/ERK1/2 signaling pathway in human osteosarcoma cells. Honokiol is therefore a promising candidate for development of antitumor drugs targeting osteosarcoma.

Honokiol suppresses proliferation and induces apoptosis via regulation of the miR‑21/PTEN/PI3K/AKT signaling pathway in human osteosarcoma cells.

Honokiol (HNK) is a small biphenolic compound, which exerts antineoplastic effects in various types of cancer. However, the mechanism underlying the antitumor effects of HNK in osteosarcoma (OS) cells is not yet fully understood. Emerging evidence has indicated that microRNAs (miRNAs/miRs) serve key roles in numerous pathological processes, including cancer. It has previously been reported that Chinese medicinal herbs harbor anticancer properties via modulating miRNA expression. Therefore, the present study aimed to determine whether HNK could suppress OS cell growth by regulating miRNA expression. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and flow cytometric analysis were used to evaluate the cell proliferation and apoptosis in human OS cells after treatment with HNK, respectively. The results demonstrated that HNK inhibits proliferation and induces apoptosis of human OS cells in a dose-dependent manner. Furthermore, HNK-induced apoptosis was characterized by upregulation of proapoptotic proteins, including cleaved-caspase-3, cleaved-poly (ADP-ribose) polymerase and B-cell lymphoma 2 (Bcl-2)-associated X protein, and downregulation of the anti-apoptotic protein Bcl-2. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) verified that HNK was able to induce aberrant expression of miRNAs in human OS cells, and miR-21 was one of the miRNAs that was most significantly downregulated. To further investigate miR-21 function, the present study validated that HNK reduces miR-21 levels in a dose-dependent manner. In addition, restoration of miR-21 expression abrogated the suppressive effects of HNK on OS cells. Luciferase assay and western blot analysis identified that miR-21 inhibits the expression of phosphatase and tensin homolog (PTEN) by directly targeting its 3′-UTR. Notably, HNK was able to suppress the phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT) signaling pathway; however, it was reactivated by miR-21 overexpression. Taken together, these data indicated that HNK may inhibit proliferation and induce apoptosis of human OS cells by modulating the miR-21/PTEN/PI3K/AKT signaling pathway. Therefore, miR-21 may be considered a potential therapeutic target for the treatment of osteosarcoma with HNK.

Hyaluronic acid modified daunorubicin plus honokiol cationic liposomes for the treatment of breast cancer along with the elimination vasculogenic mimicry channels

Background: Breast cancer is an alarming global public health problem and a main cause of cancer-related death in women. Systemic chemotherapy is the most widely used treatment for breast cancer. However, current chemotherapy treatments are far from desirable due to poor targeting specificity, severe side effects and vasculogenic mimicry (VM).Purpose: Hyaluronic acid (HA)-modified daunorubicin plus honokiol (HNK) cationic liposomes were prepared and characterised for treatment of breast cancer by eliminating VM.Methods: HA-modified daunorubicin plus HNK cationic liposomes were prepared by a thin-film hydration method. Evaluations were performed on MCF-7 cells and MDA-MB-435S cells, which are human breast cancer cells, and xenografts of MDA-MB-435S cells.Results: In vitro results revealed that the HA-modified daunorubicin plus HNK cationic liposomes enhanced the cellular uptake and destroyed VM channels. In vivo results demonstrated that the liposomes prolonged the circulation time in the blood, obviously accumulated in the tumour region, and enhanced the overall anticancer effects. Action mechanisms were related to down-regulation of VM protein indicators including FAK, EphA2, MMP-2 and MMP-9.Conclusions: The prepared HA-modified daunorubicin plus HNK cationic liposomes may serve as a promising therapeutic strategy for the treatment of breast cancer.

Honokiol enhanced the antitumor activity of Sorafenib through inhibiting glycolysis

Objective To investigate the synergetic inhibitory effects of Honokiol and Sorafenib on hepatocellular carcinoma cell line HepG2 in vitro. Methods The expression of lactate dehydrogenase A (LDHA) in liver cancer tissue was detected by immunohistochemistry (IHC), the glycolysis level was evaluated by detecting the expression of LDHA in tumor cells and the level of lactate in the culture supernatants by immunofluorescence (IF) and colorimetry, respectively. The inhibitory effect of honokiol combined with sorafenib on growth of tumor cells was tested by methyl thiazol tetrazolium (MTT) and clonogenic assays, and apoptosis was examined by annexin V staining and caspase-3 activity detecting. Results LDHA expression increased in hepatocellular carcinoma as compared with normal liver tissues (3.74±0.98 vs. 1.92±0.63) (P=0.024). Treatment with 2.5 μmol/L Sorafenib for 2 days induced enhancement of aerobic glycolysis, reflecting by an increased levels of LDHA in cells and lactate in cell supernatants [from (17.23±3.42) mmol/L to (28.34±8.19) mmol/L](P=0.001). As compared with Sorafenib monotherapy, the significant reduction of cell growth rate [(74.35±13.64)% vs. (38.16±5.75)%](P=0.008) and increase of apoptosis rate (47% vs. 18%) (P=0.029) were observed in the tumor cells treated with Sorafenib plus Honokiol. Conclusion Honokiol enhances the killing effect of Sorafenib on hepatoma cell line HepG2, which may be related to the inhibition of tumor cell glycolysis and induction of apoptosis. Key words: Carcinoma, hepatocellular; Sorafenib; Lactate dehydrogenase A; Glycolysis; Honokiol

Honokiol Ameliorates Myocardial Ischemia/Reperfusion Injury in Type 1 Diabetic Rats by Reducing Oxidative Stress and Apoptosis through Activating the SIRT1-Nrf2 Signaling Pathway.

Reducing oxidative stress is a crucial therapeutic strategy for ameliorating diabetic myocardial ischemia/reperfusion (MI/R) injury. Honokiol (HKL) acts as an effective cardioprotective agent for its strong antioxidative activity. However, its roles and underlying mechanisms against MI/R injury in type 1 diabetes (T1D) remain unknown. Since SIRT1 and Nrf2 are pivotal regulators in diabetes mellitus patients suffering from MI/R injury, we hypothesized that HKL ameliorates diabetic MI/R injury via the SIRT1-Nrf2 signaling pathway. Streptozotocin-induced T1D rats and high-glucose-treated H9c2 cells were exposed to HKL, with or without administration of the SIRT1 inhibitor EX527, SIRT1 siRNA, or Nrf2 siRNA, and then subjected to I/R operation. We found that HKL markedly improved the postischemic cardiac function, decreased the infarct size, reduced the myocardial apoptosis, and diminished the reactive oxygen species generation. Intriguingly, HKL remarkably activated SIRT1 signaling, enhanced Nrf2 nuclear translocation, increased antioxidative signaling, and decreased apoptotic signaling. However, these effects were largely abolished by EX527 or SIRT1 siRNA. Additionally, our cellular experiments showed that Nrf2 siRNA blunted the cytoprotective effects of HKL, without affecting SIRT1 expression and activity. Collectively, these novel findings indicate that HKL abates MI/R injury in T1D by ameliorating myocardial oxidative damage and apoptosis via the SIRT1-Nrf2 signaling pathway.

Honokiol Protects against Anti-β1-Adrenergic Receptor Autoantibody-Induced Myocardial Dysfunction via Activation of Autophagy.

Myocardial diseases are prevalent syndromes with high mortality rate. The exploration of effective interference is important. Anti-β1-adrenergic receptor autoantibody (β1-AAB) is highly correlated with myocardial dysfunction. The actions and underlying mechanisms of honokiol (HNK) in β1-AAB-positive patients await to be unraveled. In this study, we established a rat model of β1-AAB positive with myocardial dysfunction. Cardiac function following β1-AR-ECII administration was analyzed using the VisualSonics Vevo 770 High-Resolution In Vivo Imaging System. The levels of autophagy-related proteins were detected by Western blotting. Our data revealed that HNK reversed β1-AAB-induced effects and protected myocardial tissues from dysfunction. After HNK treatment, the cardiac contractile ability increased and the LDH activity decreased. HNK attenuated myocardial degeneration. In addition, HNK promoted the activation of the AMP-dependent protein kinase/Unc-51-like autophagy activating kinase (AMPK/ULK) pathway and activated autophagy. These results suggest that HNK protects against β1-AAB-induced myocardial dysfunction via activation of autophagy and it may be a potentially therapeutic compound for β1-AAB-positive myocardial diseases.