Biphenolic Compound Research Articles

Lipidomics reveals the potential mechanism of honokiol against adenine-induced chronic kidney disease.

Honokiol (HKL), a biphenolic compound, is derived from the bark of Magnolia officinalis, which is used in traditional Chinese medicine for gastrointestinal complaints. HKL has diverse pharmacological activities and has protective effects in various disease models. However, the role and mechanism of HKL in treating chronic kidney disease (CKD) remain unclear. This study was designed to investigate whether HKL can alleviate CKD and the potential mechanism by which it acts. Male Sprague-Dawley rats were fed 0.75% w/w adenine feed for 3 weeks to induce CKD. HKL was administered by gavage at a dose of 5 mg/kg/day for 4 weeks. Using a special kit, serum creatinine (Scr) and blood urea nitrogen (BUN) were measured. To assess renal pathology, periodic acid-Schiff and Masson’s trichrome staining were conducted. Renal lipid profiles were analyzed by ultra-high-performance liquid chromatography/mass spectrometry (UHPLC/MS). The results showed that the administration of HKL reduced Scr and BUN and alleviated renal tubular atrophy and tubulointerstitial fibrosis in an adenine-induced CKD rat model. By using lipidomics, we identified 113 lipids (47 lipids in negative ion mode, 66 lipids in positive ion mode) that could be significantly reversed by HKL treatment in CKD rat kidneys. Most of these lipids belonged to the phosphatidylcholine (PC), ceramide (Cer), phosphatidylethanolamine (PE), and triacylglycerol (TAG) classes. Moreover, HKL improved fatty acid oxidation in the kidneys of CKD rats. In conclusion, this study found that HKL can protect against adenine-induced CKD, possibly through the regulation of lipid metabolism.

Mitigation of honokiol on fluoride-induced mitochondrial oxidative stress, mitochondrial dysfunction, and cognitive deficits through activating AMPK/PGC-1α/Sirt3

Oxidative stress and mitochondrial dysfunction contribute greatly to fluoride-induced cognitive impairment and behavioural disorders. Honokiol, a natural biphenolic compound, possesses antioxidant and mitochondrial protective properties. The present study investigated the protective actions of honokiol on NaF-elicited cognitive deficits and elucidated the possible mechanism of honokiol-mediated protection. The results demonstrated that honokiol administration markedly attenuated fluoride-induced cognitive impairments and neural/synaptic injury in mice. Moreover, honokiol elevated the activity and expression of SOD2 and promoted mtROS scavenging through Sirt3 activation in NaF-treated mice and SH-SY5Y cell lines. Meanwhile, honokiol substantially lowered mtROS production by enhancing Sirt3-mediated mitochondrial DNA (mtDNA) transcription, thereby leading to significant increases in ATP synthesis and complex I activity. Further studies revealed that honokiol activated AMPK and upregulated the PGC-1α and Sirt3 protein expression in vivo and in vitro. Intriguingly, the protective actions of honokiol on oxidative stress and mitochondrial dysfunction were abolished by AMPK shRNA or Sirt3 shRNA. Notably, AMPK knockdown prevented the increase in PGC-1α and Sirt3 expression induced by honokiol, while Sirt3 shRNA suppressed Sirt3 signaling without significant effects on p-AMPK and PGC-1α expression. In conclusion, our findings indicate that honokiol mitigates NaF-induced oxidative stress and mitochondrial dysfunction by regulating mtROS homeostasis, partly via the AMPK/PGC-1α/Sirt3 pathway, which ultimately contributes to neuronal/synaptic injury and cognitive deficits.

Discovery of honokiol thioethers containing 1,3,4-oxadiazole moieties as potential α-glucosidase and SARS-CoV-2 entry inhibitors

Honokiol, isolated from a traditional Chinese medicine (TCM) Magnolia officinalis, is a biphenolic compound with several biological activities. To improve and broaden its biological activity, herein, two series of honokiol thioethers bearing 1,3,4-oxadiazole moieties were prepared and assessed for their α-glucosidase and SARS-CoV-2 entry inhibitory activities. Among all the honokiol thioethers, compound 7l exhibited the strongest α-glucosidase inhibitory effect with an IC50 value of 18.9 ± 2.3 µM, which was superior to the reference drug acarbose (IC50 = 24.4 ± 0.3 µM). Some interesting results of structure–activity relationships (SARs) have also been discussed. Enzyme kinetic study demonstrated that 7l was a noncompetitive α-glucosidase inhibitor, which was further supported by the results of molecular docking. Moreover, honokiol thioethers 7e, 9a, 9e, and 9r exhibited potent antiviral activity against SARS-CoV-2 pseudovirus entering into HEK-293 T-ACE2h. Especially 9a displayed the strongest inhibitory activity against SARS-CoV-2 pseudovirus entry with an IC50 value of 16.96 ± 2.45 μM, which was lower than the positive control Evans blue (21.98 ± 1.98 μM). Biolayer interferometry (BLI) binding and docking studies suggested that 9a and 9r may effectively block the binding of SARS-CoV-2 to the host ACE2 receptor through dual recognition of SARS-CoV-2 spike RBD and human ACE2. Additionally, the potent honokiol thioethers 7l, 9a, and 9r displayed relatively no cytotoxicity to normal cells (LO2). These findings will provide a theoretical basis for the discovery of honokiol derivatives as potential both α-glucosidase and SARS-CoV-2 entry inhibitors.

Honokiol: A naturally occurring lignan with pleiotropic bioactivities.

Honokiol is the dominant biphenolic compound isolated from the Magnolia tree, and has long been considered as the active constituent of the traditional Chinese herb, 'Houpo', which is widely used to treat symptoms due to 'stagnation of qi'. Pharmacological studies have shown that honokiol possesses a wide range of bioactivities without obvious toxicity. Honokiol protects the liver, kidneys, nervous system, and cardiovascular system through reducing oxidative stress and relieving inflammation. Moreover, honokiol shows anti-diabetic property through enhancing insulin sensitivity, and anti-obese property through promoting browning of adipocytes. In vivo and in vitro studies indicated that honokiol functions as an anti-cancer agent through multiple mechanisms: inhibiting angiogenesis, promoting cell apoptosis, and regulating cell cycle. A variety of therapeutic effects of honokiol may be associated with its physiochemical properties, which make honokiol readily cross the blood brain barrier and the blood-cerebrospinal fluid barrier, with high bioavailability. In the future, more clinical researches on honokiol are needed to fully authenticate its therapeutic values.

Honokiol Affects Stem Cell Viability by Suppressing Oncogenic YAP1 Function to Inhibit Colon Tumorigenesis.

Honokiol (HNK) is a biphenolic compound that has been used in traditional medicine for treating various ailments, including cancers. In this study, we determined the effect of HNK on colon cancer cells in culture and in a colitis-associated cancer model. HNK treatment inhibited proliferation and colony formation while inducing apoptosis. In addition, HNK suppressed colonosphere formation. Molecular docking suggests that HNK interacts with reserve stem cell marker protein DCLK1, with a binding energy of −7.0 Kcal/mol. In vitro kinase assays demonstrated that HNK suppressed the DCLK1 kinase activity. HNK also suppressed the expression of additional cancer stem cell marker proteins LGR5 and CD44. The Hippo signaling pathway is active in intestinal stem cells. In the canonical pathway, YAP1 is phosphorylated at Ser127 by upstream Mst1/2 and Lats1/2. This results in the sequestration of YAP1 in the cytoplasm, thereby not allowing YAP1 to translocate to the nucleus and interact with TEAD1-4 transcription factors to induce gene expression. However, HNK suppressed Ser127 phosphorylation in YAP1, but the protein remains sequestered in the cytoplasm. We further determined that this occurs by YAP1 interacting with PUMA. To determine if this also occurs in vivo, we performed studies in an AOM/DSS induced colitis-associated cancer model. HNK administered by oral gavage at a dose of 5mg/kg bw for 24 weeks demonstrated a significant reduction in the expression of YAP1 and TEAD1 and in the stem marker proteins. Together, these data suggest that HNK prevents colon tumorigenesis in part by inducing PUMA-YAP1 interaction and cytoplasmic sequestration, thereby suppressing the oncogenic YAP1 activity.

Manipulating Sirtuin 3 pathway ameliorates renal damage in experimental diabetes

More effective treatments for diabetic nephropathy remain a major unmet clinical need. Increased oxidative stress is one of the most important pathological mechanisms that lead to kidney damage and functional impairment induced by diabetes. Sirtuin 3 (SIRT3) is the main mitochondrial deacetylase and critically regulates cellular reactive oxygen species (ROS) production and detoxification. Honokiol is a natural biphenolic compound that, by activating mitochondrial SIRT3, can carry out anti-oxidant, anti-inflammatory and anti-fibrotic activities. Here, we sought to investigate the renoprotective effects of honokiol in BTBR ob/ob mice with type 2 diabetes. Diabetic mice were treated with vehicle or honokiol between the ages of 8 and 14 weeks. Wild-type mice served as controls. Renal Sirt3 expression was significantly reduced in BTBR ob/ob mice, and this was associated with a reduction in its activity and increased ROS levels. Selective activation of SIRT3 through honokiol administration translated into the attenuation of albuminuria, amelioration of glomerular damage, and a reduction in podocyte injury. SIRT3 activation preserved mitochondrial wellness through the activation of SOD2 and the restoration of PGC-1α expression in glomerular cells. Additionally, the protective role of SIRT3 in glomerular changes was associated with enhanced tubular Sirt3 expression and upregulated renal Nampt levels, indicating a possible tubule-glomerulus retrograde interplay, which resulted in improved glomerular SIRT3 activity. Our results demonstrate the hitherto unknown renoprotective effect of SIRT3 against diabetic glomerular disease and suggest that the pharmacological modulation of SIRT3 activity is a possible novel approach to treating diabetic nephropathy.

Antidepressant-like mechanism of honokiol in a rodent model of corticosterone-induced depression.

Depression is closely linked to hypothalamus-pituitary-adrenal axis hyperactivity. Honokiol, a biphenolic lignan compound obtained from the traditional Chinese medicine Magnolia officinalis, can reduce the activity of the hypothalamus-pituitary-adrenal axis and improve depression-like behavior caused by hypothalamus-pituitary-adrenal axis hyperactivity. The current study investigated the specific mechanism of action of this effect. A depression model was established by repeated injections of corticosterone to study the antidepressant-like effect of honokiol and its potential mechanism. Honokiol prevented the elevated activity of the hypothalamus-pituitary-adrenal axis and the depression-like behavior induced by corticosterone. Treatment with honokiol resulted in greater glucocorticoid receptor mRNA expression, greater glucocorticoid receptor-positive expression, and a greater ratio of glucocorticoid receptor to the mineralocorticoid receptor in the hippocampus. Moreover, honokiol treatment led to lower levels of interleukin-1β in serum and the positive expression of the interleukin-1β receptor in the hippocampus. These results demonstrate that the antidepressant-like mechanism of honokiol, which has effects on inflammatory factors, may act through restoring the typical activity of the hypothalamus-pituitary-adrenal axis by regulating the glucocorticoid receptor-mediated negative feedback mechanism and the balance between glucocorticoid and mineralocorticoid receptors.

Honokiol Enhances TRAIL-Mediated Apoptosis through STAMBPL1-Induced Survivin and c-FLIP Degradation.

Honokiol is a natural biphenolic compound extracted from traditional Chinese medicine Magnolia species, which have been known to display various biological effects including anti-cancer, anti-proliferative, anti-angiogenic, and anti-metastatic activities in cancer cells. Here, we found that honokiol sensitizes cancer cells to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis through downregulation of anti-apoptotic proteins survivin and c-FLIP. Ectopic expression of survivin and c-FLIP markedly abolished honokiol and TRAIL-induced apoptosis. Mechanistically, honokiol induced protein degradation of c-FLIP and survivin through STAMBPL1, a deubiquitinase. STAMBPL1 interacted with survivin and c-FLIP, resulted in reduction of ubiquitination. Knockdown of STAMBPL1 reduced survivin and c-FLIP protein levels, while overexpression of STAMBPL1 inhibited honokinol-induced survivin and c-FLIP degradation. Our findings provided that honokiol could overcome TRAIL resistance through survivin and c-FLIP degradation induced by inhibition of STAMBPL1 expression.

Honokiol post-treatment ameliorates myocardial ischemia/reperfusion injury by enhancing autophagic flux and reducing intracellular ROS production

Honokiol (HKL) is a natural low-molecular-weight biphenolic compound derived from the bark of magnolia trees. Previous studies indicate that HKL exerts potent cardioprotective effects on ischemia/reperfusion (I/R) injury; however, evidence of the further relationship between HKL posttreatment and myocardial I/R injury has not been clearly found. In our study, we explored the protective effect of HKL post treatment on myocardial I/R injury in C57BL/6 mice. We also demonstrated that HKL significantly reduced cellular reactive oxygen species production and attenuated mitochondrial damage in neonatal rat cardiomyocytes exposed to hypoxia/reoxygenation (H/R). In addition, HKL was found to enhance autophagy during I/R or H/R; these effects could be partially blocked by the autophagic flux inhibitor chloroquine. Moreover, our results suggested that enhanced autophagic flux is associated with the Akt signaling pathway. Collectively, our results indicate that HKL posttreatment alleviates myocardial I/R injury and suggest a critical cardioprotective role of HKL in promoting autophagic flux.

Honokiol induces apoptosis and suppresses migration and invasion of ovarian carcinoma cells via AMPK/mTOR signaling pathway.

Honokiol, a natural biphenolic compound, exerts anticancer effects through a variety of mechanisms on multiple types of cancer with relatively low toxicity. Adenosine 5′-phosphate-activated protein kinase (AMPK), an essential regulator of cellular homeostasis, may control cancer progression. The present study aimed to investigate whether the anticancer activities of honokiol in ovarian cancer cells were mediated through the activation of AMPK. Honokiol decreased cell viability of 2 ovarian cancer cell lines, with an half-maximal inhibitory concentration value of 48.71±11.31 µM for SKOV3 cells and 46.42±5.37 µM for Caov-3 cells. Honokiol induced apoptosis via activation of caspase-3, caspase-7 and caspase-9, and cleavage of poly-(adenosine 5′-diphosphate-ribose) polymerase. Apoptosis induced by honokiol was weakened by compound C, an AMPK inhibitor, suggesting that honokiol-induced apoptosis was dependent on the AMPK/mechanistic target of rapamycin signaling pathway. Additionally, honokiol inhibited the migration and invasion of ovarian cancer cells. The combined treatment of honokiol with compound C reversed the activities of honokiol in wound healing and Matrigel invasion assays. These results indicated that honokiol may have therapeutic potential in ovarian cancer by targeting AMPK activation.

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.

Inhibitory Effects of Honokiol on the Voltage-Gated Potassium Channels in Freshly Isolated Mouse Dorsal Root Ganglion Neurons.

Voltage-gated potassium (KV) currents, subdivided into rapidly inactivating A-type currents (I A) and slowly inactivating delayed rectifier currents (I K), play a fundamental role in modulating pain by controlling neuronal excitability. The effects of Honokiol (Hon), a natural biphenolic compound derived from Magnolia officinalis, on KV currents were investigated in freshly isolated mouse dorsal root ganglion neurons using the whole-cell patch clamp technique. Results showed that Hon inhibited I A and I K in concentration-dependent manner. The IC50 values for block of I A and I K were 30.5 and 25.7µM, respectively. Hon (30µM) shifted the steady-state activation curves of I A and I K to positive potentials by 17.6 and 16.7mV, whereas inactivation and recovery from the inactivated state of I A were unaffected. These results suggest that Hon preferentially interacts with the active states of the I A and I K channels, and has no effect on the resting state and inactivated state of the I A channel. Blockade on K+ channels by Hon may contribute to its antinociceptive effect, especially anti-inflammatory pain.

Magnolol Attenuates Concanavalin A-induced Hepatic Fibrosis, Inhibits CD4+ T Helper 17 (Th17) Cell Differentiation and Suppresses Hepatic Stellate Cell Activation: Blockade of Smad3/Smad4 Signalling.

Magnolol is a pharmacological biphenolic compound extracted from Chinese herb Magnolia officinalis, which displays anti-inflammatory and antioxidant effects. This study was aimed at exploring the potential effect of magnolol on immune-related liver fibrosis. Herein, BALB/c mice were injected with concanavalin A (ConA, 8mg/kg/week) up to 6weeks to establish hepatic fibrosis, and magnolol (10, 20, 30mg/kg/day) was given to these mice orally throughout the whole experiment. We found that magnolol preserved liver function and attenuated liver fibrotic injury invivo. In response to ConA stimulation, the CD4+ T cells preferred to polarizing towards CD4+ T helper 17 (Th17) cells in liver. Magnolol was observed to inhibit Th17 cell differentiation in ConA-treated liver in addition to suppressing interleukin (IL)-17A generation. Hepatic stellate cells were activated in fibrotic liver as demonstrated by increased alpha smooth muscle actin (α-SMA) and desmin. More transforming growth factor (TGF)-β1 and activin A were secreted into the serum. Magnolol suppressed this abnormal HSC activation. Furthermore, the phosphorylation of Smad3 in its linker area (Thr179, Ser 204/208/213) was inhibited by magnolol. In vitro, the recombinant IL-17A plus TGF-β1 or activin A induced activation of human LX2 HSCs and promoted their collagen production. Smad3/Smad4 signalling pathway was activated in LX2 cells exposed to the fibrotic stimuli, as illustrated by the up-regulated phospho-Smad3 and the enhanced interaction between Smad3 and Smad4. These alterations were suppressed by magnolol. Collectively, our study reveals a novel antifibrotic effect of magnolol on Th17 cell-mediated fibrosis.

Magnolol protects against trimethyltin-induced neuronal damage and glial activation in vitro and in vivo

Trimethyltin (TMT), an organotin with potent neurotoxic effects by selectively damaging to hippocampus, is used as a tool for creating an experimental model of neurodegeneration. In the present study, we investigated the protective effects of magnolol, a natural biphenolic compound, on TMT-induced neurodegeneration and glial activation in vitro and in vivo. In HT22 murine neuroblastoma cells, TMT induced necrotic/apoptotic cell death and oxidative stress, including intracellular reactive oxygen species (ROS), protein carbonylation, induction of heme oxygenase-1 (HO-1), and activation of all mitogen-activated protein kinases (MAPKs) family proteins. However, magnolol treatment significantly suppressed neuronal cell death by inhibiting TMT-mediated ROS generation and activation of JNK and p38 MAPKs. In BV-2 microglial cells, magnolol efficiently attenuated TMT-induced microglial activation via suppression of ROS generation and activation of JNK, p38 MAPKs, and nuclear factor-κB (NF-κB) signaling. In an in vivo mouse study, TMT induced massive neuronal damage and enhanced oxidative stress at day 2. We also observed a concomitant increase in glial cells and inducible nitric oxide synthase (iNOS) expression on the same day. These features of TMT toxicity were reversed by treatment of magnolol. We observed that p-JNK and p-p38 MAPK levels were increased in the mouse hippocampus at day 1 after TMT treatment and that magnolol blocked TMT-induced JNK and p38 MAPK activation. Magnolol administration prevented TMT-induced hippocampal neurodegeneration and glial activation, possibly through the regulation of TMT-mediated ROS generation and MAPK activation.

Honokiol inhibits bladder tumor growth by suppressing EZH2/miR-143 axis.

The oncoprotein EZH2, as a histone H3K27 methyltransferase, is frequently overexpressed in various cancer types. However, the mechanisms underlying its role in urinary bladder cancer (UBC) cells have not yet fully understood. Herein, we reported that honokiol, a biologically active biphenolic compound isolated from the Magnolia officinalis inhibited human UBC cell proliferation, survival, cancer stemness, migration, and invasion, through downregulation of EZH2 expression level, along with the reductions of MMP9, CD44, Sox2 and the induction of tumor suppressor miR-143. Either EZH2 overexpression or miR-143 inhibition could partially reverse honokiol-induced cell growth arrest and impaired clonogenicity. Importantly, it was first revealed that EZH2 could directly bind to the transcriptional regulatory region of miR-143 and repress its expression. Furthermore, honokiol treatment on T24 tumor xenografts confirmed its anticancer effects in vivo, including suppression tumor growth and tumor stemness, accompanied by the dysregulation of EZH2 and miR-143 expressions. Our data suggest a promising therapeutic option to develop drugs targeting EZH2/miR-143 axis, such as honokiol, for bladder cancer treatment.

Effect of curcumin (Curcuma longa extract) on LPS-induced acute lung injury is mediated by the activation of AMPK.

Curcumin, a biphenolic compound extracted from turmeric (Curcuma longa), possesses potent anti-inflammatory activity. The present study investigated whether curcumin could increase 5' adenosine monophosphate-activated protein kinase (AMPK) activity in macrophages and modulate the severity of lipopolysaccharide (LPS)-induced acute lung injury. Macrophages were treated with curcumin and then exposed (or not) to LPS. Acute lung injury was induced by intratracheal administration of LPS in BALB/c mice. Curcumin increased phosphorylation of AMPK and acetyl-CoA carboxylase (ACC), a downstream target of AMPK, in a time- and concentration-dependent manner. Curcumin did not increase phosphorylation of liver kinase B1, a primary kinase upstream of AMPK. STO-609, an inhibitor of calcium(2+)/calmodulin-dependent protein kinase kinase, diminished curcumin-induced AMPK phosphorylation, but transforming growth factor-beta-activated kinase 1 inhibitor did not. Curcumin also diminished the LPS-induced increase in phosphorylation of inhibitory κB-alpha and the production of tumor necrosis factor alpha (TNF-α), macrophage inflammatory protein (MIP)-2, and interleukin (IL)-6 by macrophages. Systemic administration of curcumin significantly decreased the production of TNF-α, MIP-2, and IL-6 as well as neutrophil accumulation in bronchoalveolar lavage fluid, and also decreased pulmonary myeloperoxidase levels and the wet/dry weight ratio in mice subjected to LPS treatment. These results suggest that the protective effect of curcumin on LPS-induced acute lung injury is associated with AMPK activation.

Abstract 1893: Honokiol prevents colonic tumorigenesis and affects stem cell viability by affecting oncogenic YAP1 function

Abstract Background: Despite advances in early detection, colon cancer remains the second leading cause of death in the United States. We are focused on developing dietary prevention strategies. HNK (HNK) is a biphenolic compound that is used in the traditional Chinese Medicine for treating various ailments. The current study is designed to determine whether HNK affected colon cancer stem cells and to identify a mechanism. Method: Colon cancer (CRC) cell lines HCT116 and SW480 and normal colon epithelial cells were used in the study. Cell growth was measured by hexoseaminidase and clonogenicity assays. Apoptosis was determined by measuring caspase 3/7 activities. Colosphere formation assay and FACS sorting were used for stem cells. For in vivo effects, we used the AOM/DSS-induced colonic tumorigenesis model. Immunohistochemistry was determined for stem cell markers and Hippo signaling proteins. Results: HNK induced a significant dose-dependent inhibition of proliferation and colony formation of the two CRC lines, but induced apoptosis. HNK did not affect the normal cells. To demonstrate HNK effects on stem cells, we performed colosphere assays. HNK significantly reduced the number and size of colospheres, suggesting effects on stem cells. In addition, colon stem cell marker proteins DCLK1, LGR5, and CD44 were also decreased. Further proof was obtained by flow cytometry analyses, where HNK reduced the number of DCLK1+ cells. We next determined whether stem cell signaling is affected. For this, we looked at the Hippo signaling pathway, which is active in intestinal stem cells. The key effector protein of this pathway, YAP1 is also oncogenic in many cancer types. In the canonical Hippo signaling pathway, YAP1 function is inhibited. When YAP1 is phosphorylated at Ser127 by the action of upstream Mst1/2 and Lats1/2 kinases, it is sequestered in the cytoplasm where it is degraded, thereby inhibiting downstream gene expression. HNK significantly reduced YAP1 levels. Furthermore, HNK inhibited the expression of YAP interacting proteins TEAD1, TEAD2, and TEAD4. On the other hand, ectopic expression of the TEAD1 partially rescued the cells from HNK-mediated growth suppression. To determine the in vivo effect of HNK on AOM/DSS induced colonic tumorigenesis, HNK were oral gavaged at a dose of 5mg/kg bw for 24 weeks. HNK treatment significantly reduced the colonic tumor numbers and size. Western blot and immunohistochemistry analyses demonstrated significant inhibition in the expression of stem marker proteins, oncogenic YAP1 phosphorylation and TEAD1 in the HNK-treated AOM/DSS colonic tumor tissues. Conclusion: Together, these data suggest that HNK prevents colonic tumorigenesis that targets stem cells by inhibiting oncogenic YAP1 in Hippo signaling pathway. Citation Format: Dharmalingam Subramaniam, Sivapriya Ponnurangam, Deep Kwatra, Gaurav Kaushik, Prabhu Ramamoorthy, Satish Ramalingam, Ossama Tawfik, Scott J. Weir, Subhash Padhye, Dan A. Dixon, Shahid Umar, Roy A. Jensen, Shrikant Anant. Honokiol prevents colonic tumorigenesis and affects stem cell viability by affecting oncogenic YAP1 function. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1893. doi:10.1158/1538-7445.AM2015-1893

Abstract 3517: Honokiol inhibits bladder tumor growth by suppressing EZH2/miR-143 axis

Abstract EZH2, a histone H3K27 methyltransferase, plays an essential role in regulation of cell proliferation, survival, invasiveness and cancer stem cell (CSC) maintenance. The upregulation of the histone methyltransferase EZH2 is frequently detected in various cancer types, including urinary bladder cancer (UBC). However, the mechanisms underlying its role in cancer cell are not yet fully understood. Honokiol, a biologically active biphenolic compound isolated from the Magnolia spp, has been linked with anticancer activities; however, its mechanisms is poorly understood. In this study, we investigated the in vitro and in vivo effects of honokiol on UBC cells and whether honokiol can modulate EZH2 mediated network. By employing MTT, colony formation, wound healing, transwell invasion, Western blot, and Aldofluor stem cell detection assays, honokiol was shown to inhibit human UBC cell proliferation, survival, CSC maintenance, migration and invasion. Immunoblotting assay showed that the reduction of EZH2 expression level by honokiol was correlated with the decreases of MMP9, CD44, Sox2, Notch1. Forced expression of EZH2 reversed honokiol induced cell growth arrest and low clonogenicity, indicating that EZH2 is a major downstream target for honokiol in UBC cells. Since EZH2 is essential for gene expression repression, we screened 24 cancer related miRNAs and found that tumor suppressor miR-143 was the most induced miRNA by honokiol treatment. Further quantitative RT-PCR, chromatin immunoprecipitation (ChIP) and functional analysis revealed that EZH2 directly inhibited the expression of miR-143, whereas the inhibition of miR-143 partially rescued the EZH2 knockdown-induced cell growth arrest and reduced clonogenicity. Moreover, in vivo investigation using xenograft analysis in athymic nude mice indicated that honokiol treatment inhibited tumor progression, which was associated with reduced EZH2, CD44, Sox2, Notch1 and MMP9, and increased expression of miR-143. Altogether, our data indicated that honokiol inhibited human UBC tumor growth and aggressiveness through suppression of EZH2 function and induction of its target, miR-143, and that honokiol is promising candidate for chemoprevention and/or therapeutic treatment for bladder cancer. Citation Format: Jun Yan, Qing Zhang, Wei Zhao, Changxiao Ye, Cunjie Chang, Xiaojing Huang, Junlong Zhuang, Jiannan Song, Yangyan Cui, Isaac Eliaz, Bing Shen, Ruimin Huang, Hao Ying, Hongqian Guo. Honokiol inhibits bladder tumor growth by suppressing EZH2/miR-143 axis. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3517. doi:10.1158/1538-7445.AM2015-3517

Honokiol blocks and reverses cardiac hypertrophy in mice by activating mitochondrial Sirt3.

Honokiol (HKL) is a natural biphenolic compound derived from the bark of magnolia trees with anti-inflammatory, anti-oxidative, anti-tumor and neuroprotective properties. Here we show that HKL blocks agonist-induced and pressure overload-mediated, cardiac hypertrophic responses, and ameliorates pre-existing cardiac hypertrophy, in mice. Our data suggest that the anti-hypertrophic effects of HKL depend on activation of the deacetylase SIRT3. We demonstrate that HKL is present in mitochondria, enhances SIRT3 expression nearly two-fold and suggest that HKL may bind to SIRT3 to further increase its activity. Increased SIRT3 activity is associated with reduced acetylation of mitochondrial SIRT3 substrates, MnSOD and OSCP. HKL-treatment increases mitochondrial rate of oxygen consumption and reduces ROS synthesis in wild-type, but not in SIRT3-KO cells. Moreover, HKL-treatment blocks cardiac fibroblast proliferation and differentiation to myofibroblasts in SIRT3-dependent manner. These results suggest that HKL is a pharmacological activator of SIRT3 capable of blocking, and even reversing, the cardiac hypertrophic response.

Role of Curcumin in Modulating Plasma PON1 Arylesterase Activity and Susceptibility to LDL Oxidation in Oxidatively Challenged Wistar Rats

Curcumin (β-diketones diferuloylmethane), a biphenolic compound of Curcuma longa is reported to elicit a plethora of health protective effects. The present study was carried out to investigate the in vivo protective effect of curcumin (340 mg/kg b.w., oral) against HgCl2 induced oxidative stress in wistar rats. PON1 arylesterase activity, -SH value, and LDL oxidation susceptibility were determined in plasma of control, control+ curcumin, control+ HgCl2, HgCl2+ curcumin treated rats. Oxidative stress down-regulated PON1 activity, decreased -SH value and increased LDL oxidation susceptibility in plasma. Curcumin treatment significantly (p<0.05) increased PON1 activity, -SH value and reduced LDL oxidation susceptibility in healthy and HgCl2 treated rats. We show that curcumin protects PON1 activity, -SH value and LDL oxidation, and hypothesize that its supplementation may lower the incidence of those diseases where reduced PON1 activity and higher LDL oxidation is reported. Keywords: Curcumin, LDL oxidation, oxidative stress, plasma, PON1, thiol (-SH).