Honokiol Treatment Research Articles

Honokiol abrogates leptin-induced tumor progression by inhibiting Wnt1-MTA1-β-catenin signaling axis in a microRNA-34a dependent manner.

Obesity greatly influences risk, progression and prognosis of breast cancer. As molecular effects of obesity are largely mediated by adipocytokine leptin, finding effective novel strategies to antagonize neoplastic effects of leptin is desirable to disrupt obesity-cancer axis. Present study is designed to test the efficacy of honokiol (HNK), a bioactive polyphenol from Magnolia grandiflora, against oncogenic actions of leptin and systematically elucidate the underlying mechanisms. Our results show that HNK significantly inhibits leptin-induced breast-cancer cell-growth, invasion, migration and leptin-induced breast-tumor-xenograft growth. Using a phospho-kinase screening array, we discover that HNK inhibits phosphorylation and activation of key molecules of leptin-signaling-network. Specifically, HNK inhibits leptin-induced Wnt1-MTA1-β-catenin signaling in vitro and in vivo. Finally, an integral role of miR-34a in HNK-mediated inhibition of Wnt1-MTA1-β-catenin axis was discovered. HNK inhibits Stat3 phosphorylation, abrogates its recruitment to miR-34a promoter and this release of repressor-Stat3 results in miR-34a activation leading to Wnt1-MTA1-β-catenin inhibition. Accordingly, HNK treatment inhibited breast tumor growth in diet-induced-obese mouse model (exhibiting high leptin levels) in a manner associated with activation of miR-34a and inhibition of MTA1-β-catenin. These data provide first in vitro and in vivo evidence for the leptin-antagonist potential of HNK revealing a crosstalk between HNK and miR34a and Wnt1-MTA1-β-catenin axis.

Multifunctional effects of honokiol as an anti-inflammatory and anti-cancer drug in human oral squamous cancer cells and xenograft

The aim of this study was to investigate anti-inflammatory and anti-cancer effects of honokiol (HK) in two oral squamous cancer cell carcinoma (OSCC) cell lines, HN22 and HSC4, through the regulation of inducible nitric oxide synthase (iNOS) and endoplasmic reticulum resident protein 44 (ERp44). Griess assay, zymography, and quantitative PCR were performed to study iNOS expression and subsequent nitric oxide (NO) production in OSCC cell lines. Liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based proteomic analysis was used to elucidate the proteins associated with ER stress and cellular cytotoxic response induced by HK. Pull-down assay and molecular modeling were performed to better understand how HK interacts with ERp44. In vitro and in vivo experiments in which ERp44 expression was knocked down were performed to better understand the effects of ERp44 on a cellular level and anti-cancer effects of HK. Expression levels of iNOS and subsequent NO secretion were reduced in OSCC cell lines treated with HK. ERp44 was significantly decreased in OSCC cell lines by HK treatment. HK directly bound to ERp44, and ERp44 knock-down significantly inhibited oral cancer cell proliferation and colony formation. Moreover, HK treatment effectively inhibited tumor growth and ERp44 levels in BALB/c nude mice bearing HN22 cell xenografts. Our findings suggest that HK inhibited inflammation and induced apoptosis by suppressing both iNOS/NO and ERp44 expression in HN22 and HSC4 cells and xenograft tumors, and thus could be a potent anti-inflammatory and anti-cancer drug candidate for human oral cancer treatment.

Honokiol inhibits melanoma stem cells by targeting notch signaling.

Melanoma is an aggressive disease with limited therapeutic options. Here, we determined the effects of honokiol (HNK), a biphenolic natural compound on melanoma cells and stemness. HNK significantly inhibited melanoma cell proliferation, viability, clonogenicity and induced autophagy. In addition, HNK significantly inhibited melanosphere formation in a dose dependent manner. Western blot analyses also demonstrated reduction in stem cell markers CD271, CD166, Jarid1b, and ABCB5. We next examined the effect of HNK on Notch signaling, a pathway involved in stem cell self-renewal. Four different Notch receptors exist in cells, which when cleaved by a series of enzymatic reactions catalyzed by Tumor Necrosis Factor-α-Converting Enzyme (TACE) and γ-secretase protein complex, results in the release of the Notch intracellular domain (NICD), which then translocates to the nucleus and induces target gene expression. Western blot analyses demonstrated that in HNK treated cells there is a significant reduction in the expression of cleaved Notch-2. In addition, there was a reduction in the expression of downstream target proteins, Hes-1 and cyclin D1. Moreover, HNK treatment suppressed the expression of TACE and γ-secretase complex proteins in melanoma cells. To confirm that suppression of Notch-2 activation is critical for HNK activity, we overexpressed NICD1, NICD2, and performed HNK treatment. NICD2, but not NICD1, partially restored the expression of Hes-1 and cyclin D1, and increased melanosphere formation. Taken together, these data suggest that HNK is a potent inhibitor of melanoma cells, in part, through the targeting of melanoma stem cells by suppressing Notch-2 signaling.

Effect of honokiol on the induction of drug-metabolizing enzymes in human hepatocytes.

Honokiol, 2-(4-hydroxy-3-prop-2-enyl-phenyl)-4-prop-2-enyl-phenol, an active component of Magnolia officinalis and Magnolia grandiflora, exerts various pharmacological activities such as antitumorigenic, antioxidative, anti-inflammatory, neurotrophic, and antithrombotic effects. To investigate whether honokiol acts as a perpetrator in drug interactions, messenger ribonucleic acid (mRNA) levels of phase I and II drug-metabolizing enzymes, including cytochrome P450 (CYP), UDP-glucuronosyltransferase (UGT), and sulfotransferase 2A1 (SULT2A1), were analyzed by real-time reverse transcription polymerase chain reaction following 48-hour honokiol exposure in three independent cryopreserved human hepatocyte cultures. Honokiol treatment at the highest concentration tested (50 μM) increased the CYP2B6 mRNA level and CYP2B6-catalyzed bupropion hydroxylase activity more than two-fold in three different hepatocyte cultures, indicating that honokiol induces CYP2B6 at higher concentrations. However, honokiol treatment (0.5–50 μM) did not significantly alter the mRNA levels of phase I enzymes (CYP1A2, CYP3A4, CYP2C8, CYP2C9, and CYP2C19) or phase II enzymes (UGT1A1, UGT1A4, UGT1A9, UGT2B7, and SULT2A1) in cryopreserved human hepatocyte cultures. CYP1A2-catalyzed phenacetin O-deethylase and CYP3A4-catalyzed midazolam 1′-hydroxylase activities were not affected by 48-hour honokiol treatment in cryopreserved human hepatocytes. These results indicate that honokiol is a weak CYP2B6 inducer and is unlikely to increase the metabolism of concomitant CYP2B6 substrates and cause pharmacokinetic-based drug interactions in humans.

Honokiol affects melanoma cell growth by targeting the AMP-activated protein kinase signaling pathway

BackgroundMalignant melanoma is an aggressive form of skin cancer with limited effective therapeutic options. Melanoma research concentrates on maximizing the effect on cancer cells with minimal toxicity to normal cells. AMP-activated protein kinase (AMPK) is an important regulator of cellular energy homeostasis and has been shown to control tumor progression regulating the cell cycle, protein synthesis, and cell growth and/or survival. Honokiol (HNK) is a biphenolic compound derived from Magnolia officinalis, a plant that has been used in traditional Chinese and Japanese medicine for the treatment of various pathological conditions. Recent studies have shown that HNK has antitumor activity with relatively low toxicity. In this study, we demonstrated that the growth inhibitory effects of HNK on melanoma and melanoma cancer stem cells were mediated through the activation of AMPK and hence AMPK signaling in melanoma cells. MethodsWe determined the effects of HNK treatment on various melanoma cell lines. HNK-induced cell growth inhibitory effects were determined using hexosaminidase assay. Protein expression studies were done by immunoblotting. Primary spheroid assay was used to assess stemness by growing single suspension cells in ultralow attachment plates. ResultsHNK is highly effective in inhibiting melanoma cells by attenuating protein kinase B/mammalian target of rapamycin and AMPK signaling. HNK showed significant inhibition of the spheroid-forming capacity of melanoma cells and, hence, stemness. HNK significantly decreased the number and size of melanospheres in a dose-dependent manner. Western blot analyses showed enhanced phosphorylation of AMPK in melanoma cells. Furthermore, HNK decreased the cellular adenosine triphosphate pool in a dose-dependent manner with maximum effects observed at 48 hours. ConclusionsThe results suggest that HNK can target melanoma cells and mark them for cell death through AMPK signaling. Further studies are warranted for developing HNK as an effective chemopreventive/therapeutic agent in melanoma.

Abstract LB-61: Inhibiting “Dedifferentiation” in breast cancer cells using Honokiol - a plant derived polyphenol

Abstract Introduction: Dedifferentiation involves terminally differentiated cells reverting back to less differentiated cells such as precursor cells or stem cells within their own lineage allowing the cells to proliferate again before re-differentiation. Tumorigenesis involves uncontrolled cell proliferation and many host-related factors aid in dedifferentiation of tumor cells into stem cell like phenotypes hence providing a mechanism leading to tumor heterogeneity and aggressiveness. Pluripotency factors Oct4, Nanog and Sox2 have been implicated in dedifferentiation and are highly expressed in tumors. The importance of active constitutive agents in natural products has become increasingly apparent owing to their potential cancer chemopreventive and therapeutic properties. Honokiol (HNK) is a natural phenolic compound isolated from an extract of seed cones from Magnolia grandiflora. Recent studies from our laboratory demonstrated that HNK has suppressing effects on different aspects of cancer progression. The present study was designed to specifically examine the potential of HNK to inhibit pluripotency related transcription factors and reverse the process of dedifferentiation associated with uncontrolled cell proliferation in tumorigenesis. Results: Here, we provide evidence that HNK inhibits the ability of breast cancer cells to form mammospheres. HNK treatment inhibits dedifferentiation/pluripotency markers Oct4, Nanog, Sox2 in MCF-7, MDA-MB-231, T47D, MDA-MB-468 breast cancer cells. An analysis of underlying signaling mechanism reveal that HNK inhibits phosphorylation of Stat3 and the downregulation of pluripotency factors is mediated via inhibition of Stat3. Stat3 inhibitor, Stattic potentiates the effect of HNK while overexpression of constitutively active Stat3 interferes with HNK-mediated inhibition of Oct4, Nanog and Sox2. It is interesting to note that HNK treatment also increases the expression of upstream kinase and tumor suppressor LKB1. Utilizing LKB1-null cells and gain-of-function strategies, we show that HNK-mediated inhibition of Oct4, Nanog and Sox2 is regulated by tumor suppressor LKB1. The ability of HNK to inhibit mammosphere formation is abrograted in breast cancer cells stably-silenced for LKB1. In vitro and in vivo analyses show novel functional interactions between HNK, Stat3, LKB1 and dedifferentiation/pluripotency markers. Conclusions: Taken together, these studies provide evidence for a previously unrecognized cross-talk between HNK and dedifferentiation/pluripotency markers Oct4, Nanog, Sox2 via tumor suppressor LKB1-Stat3 axis in inhibiting “dedifferentiation” in breast cancer. Citation Format: Sonali Sengupta, Michael Y. Bonner, Jack L. Arbiser, Neeraj K. Saxena, Dipali Sharma. Inhibiting “Dedifferentiation” in breast cancer cells using Honokiol - a plant derived polyphenol. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-61. doi:10.1158/1538-7445.AM2014-LB-61

Abstract LB-187: Novel mechanistic insights into the bioactive compound honokiol-mediated inhibition of epithelial to mesenchymal transition in breast cancer: Therapeutic modulation of miR-34a via tumor suppressor LKB1

Abstract Introduction: An emerging hypothesis is that epithelial-mesenchymal-transition (EMT) bestows metastatic potential to epithelial cancer cells enabling them to invade, migrate and subsequently disseminate to form distant metastases resulting in non-curable disease. Pharmacological inhibition of EMT can potentially lead to reversion of aggressive breast cancer cells to a more-differentiated epithelial phenotype by inducing mesenchymal-epithelial transition (MET) to prevent metastasis and diminish distant recurrence. The importance of active constitutive agents in natural products has become increasingly apparent owing to their potential cancer chemopreventive and therapeutic properties. Honokiol (HNK) is a natural phenolic compound isolated from an extract of seed cones from Magnolia grandiflora. Recent studies from our lab demonstrated that HNK has suppressing effects on different aspects of cancer progression. The present study was designed to specifically examine the potential of HNK to inhibit EMT and elucidate the molecular mechanism by which HNK inhibits EMT in breast cancer cells. Aim: To elucidate the mechanism by which HNK inhibits EMT in breast cancer cells. Results: Here, we provide molecular evidence that HNK inhibits EMT in breast cancer cells resulting in significant downregulation of mesenchymal marker proteins and concurrent upregulation of epithelial markers. In vitro and in vivo analyses show functional interactions between HNK, STAT3, and EMT-signaling components. Mechanistically, HNK inhibits recruitment of STAT3 on mesenchymal transcription factor ZEB1 promoter resulting in decreased ZEB1 expression and nuclear translocation. We also discover that HNK increases E-cadherin expression via STAT3-mediated release of ZEB1 from E-cadherin promoter. Since ZEB1 is a transcriptional repressor of miR-34a, we next examined the effect of HNK on miR-34a expression. Intriguingly, we found that HNK upregulates the levels of miR-34a in a time- and dose-dependent manner. It is interesting to note that HNK treatment also increases the expression of upstream kinase and tumor suppressor LKB1. Utilizing LKB1-null cells and gain-of-function strategies, we found that HNK-mediated increase in miR-34a is regulated by tumor suppressor LKB1. Conclusions: Taken together, these studies provide evidence for a previously unrecognized cross-talk between HNK and miR-34a via tumor suppressor LKB1 in inhibiting EMT in breast cancer. Citation Format: Dimiter B. Avtanski, Michael Y. Bonner, Timothy P. Tiutan, Jack L. Arbiser, Neeraj K. Saxena, Dipali Sharma. Novel mechanistic insights into the bioactive compound honokiol-mediated inhibition of epithelial to mesenchymal transition in breast cancer: Therapeutic modulation of miR-34a via tumor suppressor LKB1. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-187. doi:10.1158/1538-7445.AM2014-LB-187

Abstract 5494: The effect of honokiol combined with PPARγ agonists in regulation of cell growth and migration in human hepatoma cells

Abstract Hepatocellular carcinoma (HCC) is one of the malignant cancers worldwide. At present, surgical resection is the best treatment for HCC. Despite some other options such as chemotherapy and radiotherapy, recurrence and metastasis are the major causes of increased mortality. It has been reported honokiol, a natural biphenolic compound, exerts inhibitory effects on the growth and migration of hepatoma cells. Most recently, it was found that honokiol functions as not only an RXR agonist but also a PPARγ agonist, and is capable of potentiating the activation of PPARγ in the presence of PPARγ agonist rosiglitazone in 3T3-L1 adipocytes and HLE human hepatoma cells. Moreover, PPARγ agonists exhibited inhibitory role in cell growth and migration in various cancers through PPARγ-dependent and -independent pathway. However, the combination effect and the molecular mechanism of cotreatment with honokiol and PPARγ agonists are still unclear in hepatoma cells. In this study, Hep3B, Mahlavu, and SK-Hep1 human hepatoma cells were treated with honokiol and/or PPARγ agonists. Honokiol and PPARγ agonists rosiglitazone and troglitazone inhibited cell growth in hepatoma cells by surforhodamine B assay, respectively. Moreover, cotreatment with honokiol and PPARγ agonist showed additive effect in cell growth inhibition. Furthermore, retrovirus-mediated PPARγ overexpression in SK-Hep1 cells resulted in higher sensitivity to honokiol treatment in cell growth inhibition compared to vector control. Additionally, PPARγ agonists rosiglitazone and troglitazone inhibited cell migration in Hep3B cells by wound healing and transwell assays. These results together suggest that honokiol combined with PPARγ agonist had additive effect in cell growth inhibition and migration of hepatoma cells. The molecular mechanism and potential application of this combination for treatment of hepatoma will be presented. Citation Format: Chin-Wen Chi, Hui-Tzu Hsu. The effect of honokiol combined with PPARγ agonists in regulation of cell growth and migration in human hepatoma cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5494. doi:10.1158/1538-7445.AM2014-5494

Intravenous administration of Honokiol provides neuroprotection and improves functional recovery after traumatic brain injury through cell cycle inhibition

Recently, increasing evidence has shown that cell cycle activation is a key factor of neuronal death and neurological dysfunction after traumatic brain injury (TBI). This study aims to investigate the effects of Honokiol, a cell cycle inhibitor, on attenuating the neuronal damage and facilitating functional recovery after TBI in rats, in an attempt to unveil its underlying molecular mechanisms in TBI. This study suggested that delayed intravenous administration of Honokiol could effectively ameliorate TBI-induced sensorimotor and cognitive dysfunctions. Meanwhile, Honokiol treatment could also reduce the lesion volume and increase the neuronal survival in the cortex and hippocampus. The neuronal degeneration and apoptosis in the cortex and hippocampus were further significantly attenuated by Honokiol treatment. In addition, the expression of cell cycle-related proteins, including cyclin D1, CDK4, pRb and E2F1, was significantly increased and endogenous cell cycle inhibitor p27 was markedly decreased at different time points after TBI. And these changes were significantly reversed by post-injury Honokiol treatment. Furthermore, the expression of some of the key cell cycle proteins such as cyclin D1 and E2F1 and the associated apoptosis in neurons were both remarkably attenuated by Honokiol treatment. These results show that delayed intravenous administration of Honokiol could effectively improve the functional recovery and attenuate the neuronal cell death, which is probably, at least in part, attributed to its role as a cell cycle inhibitior. This might give clues to developing attractive therapies for future clinical trials.

Effects of Honokiol on Sepsis-Induced Acute Kidney Injury in an Experimental Model of Sepsis in Rats

Acute kidney injury (AKI) is a severe complication of sepsis, which largely contributes to the high mortality rate of sepsis. Honokiol, a natural product isolated from Magnolia officinalis (Houpo), has been shown to exhibit anti-inflammatory and antioxidant properties. Here, we investigated the effects of honokiol on sepsis-associated AKI in rats subjected to cecal ligation and puncture (CLP). We found that the administration of honokiol improved the survival of septic rats. Periodic acid-Schiff stain revealed that the morphological changes of kidney tissues in CLP rats were restored after honokiol treatment. Furthermore, honokiol reduced CLP-induced oxidative stress and inflammatory cytokine production. The levels of nitric oxide (NO) and inducible NO synthetase (iNOS) were attenuated by honokiol in septic rats. Finally, honokiol inhibited CLP-induced activation of NF-κB signaling in CLP rats. Our findings suggest that honokiol might be used as a potential therapeutic agent for complications of sepsis, especially for sepsis-induced AKI.

Abstract P1-07-04: A novel bioactive approach to inhibit leptin-induced epithelial-mesenchymal transition in breast cancer

Abstract Introduction: Molecular effects of obesity, a well-established risk factor for breast cancer progression, are mediated by adipocytokine leptin. Recent studies from our lab reveal that leptin induces epithelial-mesenchymal transition (EMT) and tumorsphere formation via concomitant activation of Akt/GSK3b and MTA1/Wnt1 signaling axes leading to β catenin activation. Given the important role of leptin in breast cancer growth and metastasis, novel strategies to antagonize biological effects of leptin are much desired. We showed previously that honokiol, a bioactive polyphenol from Magnolia grandiflora, inhibits breast carcinogenesis. The present study provides first evidence for the efficacy of honokiol against oncogenic effects of leptin including EMT. Methods: Efficacy of honokiol to inhibit oncogenic effects of leptin was evaluated by using clonogenicity, anchorage–independent growth, matrigel-invasion and spheroid-migration assays. RT-PCR, western blot and immunofluorescence analyses were used to examine the molecular changes associated with EMT as well as underlying molecular pathways. Functional importance of MTA1/Wnt1/ β catenin axis was examined by using overexpression, phospho-deficient constructs and specific inhibitors. Finally, mouse xenografts, immuniohistochemical, RT-PCR and western blot analysis of tumors was used. Results: Honokiol treatment circumvents leptin-induced EMT-associated phenotypic changes. A biochemical hallmark of EMT is loss of expression of epithelial markers with a concurrent increase in mesenchymal marker expression. Honokiol elicits increased expression of E-cadherin, occludin and keratin-18 (epithelial markers) and decreased expression of vimentin, fibronectin and N-cadherin (mesenchymal markers) in leptin-treated cells providing molecular evidence for reversal of EMT. Honokiol also inhibits expression and nuclear translocation of transcriptional effectors of EMT; Snail, Slug, Zeb1 and Zeb2. Analysis of underlying molecular mechanisms reveals that Honokiol effectively inhibits leptin-induced MTA1/Wnt1/ β catenin axis. Furthermore, using nontoxic doses of honokiol, we show that honokiol treatment effectively inhibits leptin-induced breast tumorigenesis in vivo. Analysis of breast tumors show that honokiol treatment reverses leptin-associated signaling (MTA1/Wnt1/ β catenin axis) along with decreased expression of mesenchymal markers and increased expression of epithelial markers. Conclusions: In this study, we demonstrate for the first time that HNK is able to abolish leptin-induced EMT and provide in vitro and in vivo evidence for the integral role of a previously unrecognized crosstalk between honokiol and MTA1/Wnt1/ β catenin axis. Thus, HNK may be used as a non-toxic and non-endocrine rational therapeutic strategy for breast carcinoma in obese patients with high leptin levels. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P1-07-04.

English

The drug oxaliplatin is important in the chemotherapy of colorectal carcinoma, but its toxicity, especially dose-related neurosensory toxicity, is not well tolerated. We investigated if honokiol could augment the anti-tumor effect of oxaliplatin in colon cancer HT-29 cells in vitro and determined if honokiol could be used with oxaliplatin to decrease its dose. Cell proliferation, apoptosis, and prostaglandin E2 (PGE2) and vascular endothelial growth factor (VEGF) levels were investigated. Expression of cyclo-oxygenase 2 (COX-2), VEGF, AKT/p-AKT, extracellular signal-related kinase (ERK)1/2/p-ERK1/2, nuclear factor kappa B (NF-κB), P65/p-P65, and caspase-3 was examined. Honokiol or oxaliplatin alone suppressed the proliferation of HT-29 cells in a concentration-dependent manner. HT-29 cells were more sensitive to oxaliplatin treatment in the presence of honokiol. Oxaliplatin combined with honokiol improved the rate of HT-29 cell apoptosis and reduced PGE2 and VEGF secretion levels. Expression of COX-2 and VEGF protein and phosphorylation of AKT, ERK1/2, NF-κB and P65 were also inhibited, caspase-3 levels were upregulated after honokiol treatment. Therefore, honokiol can be combined with oxaliplatin in the chemotherapy of colorectal carcinoma, this combination allows a reduction in oxaliplatin dose, and thereby reduces its adverse effects, and may also enhance the chemotherapeutic effect of oxaliplatin for this disease.   Key words: Honokiol, colorectal cancer, apoptosis, oxaliplatin, nuclear factor-kappa B (NF-κB).

Honokiol augments the anti-cancer effects of oxaliplatin in colon cancer cells

Oxaliplatin is an important drug in the chemotherapy of colorectal carcinoma, but its toxicity, especially dose-related neurosensory toxicity, is not well tolerated. In this study, we investigated whether honokiol could augment the anti-tumor effect of oxaliplatin in colon cancer HT-29 cells in vitro and whether honokiol could be used with oxaliplatin to decrease oxaliplatin dose. We used the normal colon cells, human colonic epithelial cells (HCoEpiCs) as control cells. Cell proliferation, apoptosis, prostaglandin E2 (PGE2) and vascular endothelial growth factor (VEGF) levels were also investigated. Expression levels of cyclo-oxygenase 2 (COX-2), VEGF, AKT/p-AKT, extracellular signal-related kinase (ERK)1/2/p-ERK1/2, nuclear factor kappa B (NF-κB) P65/p-P65, and caspase-3 were measured. Honokiol or oxaliplatin suppressed the proliferation of HT-29 cells in a concentration-dependent manner, but only high concentrations of honokiol would suppress the proliferation of HCoEpiCs. HT-29 cells were more sensitive to oxaliplatin treatment in the presence of honokiol. Oxaliplatin combined with honokiol improved the apoptosis rate of HT-29 cell and reduced PGE2 and VEGF secretion levels. Expression levels of COX-2 and VEGF protein and phosphorylation of AKT, ERK1/2, and NF-κB P65 were also inhibited. Caspase-3 levels were upregulated after honokiol treatment. Therefore, honokiol can be used in combination with oxaliplatin in the chemotherapy of colon cancer. This combination allows a reduction in oxaliplatin dose, and thereby reduces its adverse effects. It may also enhance the chemotherapeutic effect of oxaliplatin for this disease.

Honokiol Inhibits Tumor Necrosis Factor-α-Stimulated Rat Aortic Smooth Muscle Cell Proliferation via Caspase- and Mitochondrial-Dependent Apoptosis

This study aims to investigate the effects of honokiol on proliferation, cell cycle, and apoptosis in tumor necrosis factor (TNF)-α-induced rat aortic smooth muscle cells (RASMCs). We found that honokiol treatment showed potent inhibitory effects on TNF-α-induced RASMC proliferation, which were associated with G0/G1 cell cycle arrest and downregulation of cell cycle-related proteins, including cyclin D1, cyclin E, cyclin-dependent kinase (CDK)2 and CDK4. Furthermore, honokiol treatment led to the release of cytochrome c into cytosol and a loss of mitochondrial membrane potential (ΔΨm), as well as a decrease in the expression of Bcl-2 and an increase in the expression of Bax. Treatment with honokiol also reduced TNF-α-induced phosphorylation of p38, extracellular signal-regulated kinase 1/2, and c-Jun N-terminal kinase. Taken together, our results suggest that honokiol suppresses TNF-α-stimulated RASMC proliferation via caspase- and mitochondria-dependent apoptosis and highlight the therapeutic potential of honokiol in the prevention of cardiovascular diseases.

Honokiol thwarts gastric tumor growth and peritoneal dissemination by inhibiting Tpl2 in an orthotopic model

Honokiol is known to suppress the growth of cancer cells; however, to date, its antiperitoneal dissemination effects have not been studied in an orthotopic mouse model. In the present study, we evaluated the antiperitoneal dissemination potential of Honokiol in an orthotopic mouse model and assessed associations with tumor growth factor-β1 (TGFβ1) and cells stimulated by a carcinogen, N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). Our results demonstrate that tumor growth, peritoneal dissemination and peritoneum or organ metastasis of orthotopically implanted MKN45 cells were significantly decreased in Honokiol-treated mice and that endoplasmic reticulum (ER) stress was induced. Honokiol-treated tumors showed increased epithelial signatures such as E-cadherin, cytokeratin-18 and ER stress marker. In contrast, decreased expression of vimentin, Snail and tumor progression locus 2 (Tpl2) was also noted. TGFβ1 and MNNG-induced downregulation of E-cadherin and upregulation of Tpl2 were abrogated by Honokiol treatment. The effect of Tpl2 inhibition in cancer cells or endothelial cells was associated with inactivation of CCAAT/enhancer binding protein B, nuclear factor kappa-light-chain-enhancer of activated B cell and activator protein-1 and suppression of vascular endothelial growth factor. Inhibition of Tpl2 in gastric cancer cells by small interfering RNA or pharmacological inhibitor was found to effectively reduce growth ability and vessel density in vivo. Honokiol-induced reversal of epithelial-to-mesenchymal transition (EMT) and ER stress-induced apoptosis via Tp12 may involve the paralleling processes. Taken together, our results suggest that the therapeutic inhibition of Tpl2 by Honokiol thwarts both gastric tumor growth and peritoneal dissemination by inducing ER stress and inhibiting EMT.

The natural product honokiol inhibits calcineurin inhibitor-induced and Ras-mediated tumor promoting pathways

Although calcineurin inhibitors (CNIs) are very useful in preventing allograft rejection, they can mediate a rapid progression of post-transplantation malignancies. The CNI cyclosporine A (CsA) can promote renal tumor growth through activation of the proto-oncogene ras and over-expression of the angiogenic cytokine VEGF; the ras activation also induces over-expression of the cytoprotective enzyme HO-1, which promotes survival of renal cancer cells. Here, we show that the natural product honokiol significantly inhibited CsA-induced and Ras-mediated survival of renal cancer cells through the down-regulations of VEGF and HO-1. Thus, honokiol treatment may help to prevent tumor-promoting effects of CsA in transplant patients.

Abstract 3662: Honokiol affects stem cell viability in part through suppression of Hippo signaling pathway.

Abstract Background: Despite therapeutic advances, colon cancer remains the second leading cause of death in the United States. Cancer stem cells are implicated in resistance to radiation and chemotherapy. Honokiol is a biphenolic compound that has been used in the traditional Chinese Medicine for treating various ailments. The current study is designed to determine whether honokiol affected colon cancer stem cells and to identify a mechanism. Method: Colon cancer 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, HCT116 xenografts were developed in the flanks of nude mice. Immunohistochemistry was performed for CD31, stem cell markers and Hippo signaling proteins. Results: Honokiol induced a significant dose-dependent inhibition of proliferation and colony formation of the two colon cancer cell lines, but not that of the normal cells. Honokiol treatment also induced the colon cancer cells to undergo apoptosis. To demonstrate honokiol effects on stem cells, we performed colosphere assays. Honokiol significantly reduced the number and size of colospheres, suggesting effects on stem cells. In addition, colon cancer stem cell marker proteins DCLK1, LGR5, CD44 and SOX-9 were also decreased. Further proof was obtained by flow cytometry analyses, where honokiol reduced the number of DCLK1+ cells. We next determined whether signaling pathways are affected, for this, we tested the effect of honokiol on Hippo signaling pathway, which is an active pathway in intestinal stem cells. Honokiol significantly decreased the phosphorylation of Hippo pathway proteins Mst1/2, Lats1/2 and YAP1. Furthermore, honokiol 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 honokiol-mediated growth suppression. To determine the effect of honokiol on tumor growth in vivo, nude mice harboring HCT116 tumor xenografts in their flanks were administered the compound intraperitoneally every day for 21 days. Honokiol treatment significantly inhibited tumor xenograft growth, with notably lower tumor volume and weight. Microvessel density, based on CD31 staining was also significantly lower in the tumors following honokiol treatment when compared to controls. Western blot and immunohistochemistry analyses demonstrated significant inhibition in the expression of stem marker and Hippo signaling proteins in the honokiol-treated xenograft tissues. Conclusion: Together, these data suggest that honokiol is a potent inhibitor of colon cancer that targets stem cells by inhibiting Hippo signaling pathway. Citation Format: Dharmalingam Subramaniam, Sivapriya Ponnurangam, Shahid Umar, Satish Ramalingam, Roy A. Jensen, Shrikant Anant. Honokiol affects stem cell viability in part through suppression of Hippo signaling pathway. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3662. doi:10.1158/1538-7445.AM2013-3662

Abstract 299: Targeting epithelial-mesenchymal transition in breast cancer cells using Honokiol, a natural phenolic compound.

Abstract Introduction: The epithelial-mesenchymal transition (EMT) presents a critical step in the acquisition of metastatic state. Tumor cells undergoing EMT not only exhibit increased migration and invasion potential but also acquire increased resistance to chemotherapy and radiation therapy. Hence, EMT is an attractive target for therapeutic interventions directed against tumor metastasis. Honokiol (HNK) is a natural phenolic compound isolated from an extract of seed cones from Magnolia grandiflora. Recent studies from our lab show that honokiol inhibits breast carcinogenesis. The present study is designed to systematically elucidate if nontoxic lower doses of honokiol can be used to specifically inhibit EMT in breast cancer cells. Results: Our studies show that HNK inhibits EMT in human breast cancer cells. Exposure of estrogen-independent and estrogen-responsive breast cancer cells to nontoxic lower doses of HNK results in significant downregulation of mesenchymal marker proteins (Fibronectin, Vimentin) along with simultaneous upregulation of epithelial markers (E-cadherin, Cytokeratin-18). HNK also inhibits expression and nuclear translocation of transcriptional modulators of EMT-related genes (Snail, ZEB1/2 and Twist). Experimental EMT induced by exposure to TGFβ and TNFα in a spontaneously immortalized nontumorigenic human mammary epithelial cell line (MCF-10A) was also completely reversed by HNK treatment as evidenced by morphological changes (inhibition of fibroblast-like appearance, decreased pseudopodia and actin reorganization to membrane-bound location in TGFβ and TNFα treated MCF10A cells) as well as molecular changes (downregulation of Fibronectin, Vimentin, Snail, Zeb1/2 and upregulation of E-cadherin and Cytokeratin-18). Mechanistically, HNK inhibits MTA1-Wnt1 axis leading to stabilization and nuclear translocation of β-catenin to inhibit EMT. Analysis of breast tumors treated with honokiol show modulation of EMT markers and inhibition of key molecules of MTA1-Wnt1-β-catenin axis corroborating our in vitro findings. Conclusions: Taken together, these data provide the first in vitro and in vivo evidence of the potential of HNK as a novel, non-toxic and non-endocrine therapeutic strategy for breast carcinoma. MTA1, Wnt1 and β-catenin appear to be novel biomarkers associated with HNK treatment. Citation Format: Dimiter B. Avtanski, Arumugam Nagalingam, Panjamurthi Kupusamy, Neeraj K. Saxena, Dipali Sharma. Targeting epithelial-mesenchymal transition in breast cancer cells using Honokiol, a natural phenolic compound. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 299. doi:10.1158/1538-7445.AM2013-299

Abstract 5497: A novel bioactive approach to inhibit leptin-induced epithelial-mesenchymal transition in breast cancer.

Abstract Introduction: Molecular effects of obesity, a well-established risk factor for breast cancer progression, are mediated by adipocytokine leptin. Recent studies from our lab reveal that leptin induces epithelial-mesenchymal transition (EMT) and tumorsphere formation via concomitant activation of Akt/GSK3β and MTA1/Wnt1 signaling axes leading to β-catenin activation. Given the important role of leptin in breast cancer growth and metastasis, novel strategies to antagonize biological effects of leptin are much desired. We showed previously that honokiol (HNK), a bioactive polyphenol from Magnolia grandiflora, inhibits breast carcinogenesis. The present study provides first evidence for the efficacy of HNK against oncogenic effects of leptin including EMT. Methods: Efficacy of HNK to inhibit oncogenic effects of leptin was evaluated by using clonogenicity, anchorage-independent growth, matrigel invasion and spheroid-migration assays. RT-PCR, Western blot and immunofluorescence analyses were used to examine the molecular changes associated with EMT as well as underlying molecular pathways. Functional importance of MTA1/Wnt1/β-catenin axis was examined by using overexpression, phospho-deficient constructs and specific inhibitors. Finally, mouse xenografts, immuniohistochemical, RT-PCR and Western blot analysis of tumors was used. Results: HNK treatment circumvents leptin-induced EMT-associated phenotypic changes. A biochemical hallmark of EMT is loss of expression of epithelial markers with a concurrent increase in mesenchymal marker expression. HNK elicits increased expression of E-cadherin, occludin and cytokeratin-18 (epithelial markers) and decreased expression of vimentin, fibronectin and N-cadherin (mesenchymal markers) in leptin-treated cells providing molecular evidence for reversal of EMT. HNK also inhibits expression and nuclear translocation of transcriptional effectors of EMT: Snail, Slug, Zeb1 and Zeb2. Analysis of underlying molecular mechanisms reveals that HNK effectively inhibits leptin-induced MTA1/Wnt1/β-catenin axis. Furthermore, using nontoxic doses of HNK, we show that HNK treatment effectively inhibits leptin-induced breast tumorigenesis in vivo. Analysis of breast tumors show that HNK treatment reverses leptin-associated signaling (MTA1/Wnt1/β-catenin axis) along with decreased expression of mesenchymal markers and increased expression of epithelial markers. Conclusions: In this study, we demonstrate for the first time that HNK is able to abolish leptin-induced EMT and provide in vitro and in vivo evidence for the integral role of a previously unrecognized crosstalk between honokiol and MTA1/Wnt1/ β catenin axis. Thus, HNK may be used as a non-toxic and non-endocrine rational therapeutic strategy for breast carcinoma in obese patients with high leptin levels. Citation Format: Dimiter B. Avtanski, Arumugam Nagalingam, Panjamurthy Kuppusamy, Neeraj K. Saxena, Dipali Sharma. A novel bioactive approach to inhibit leptin-induced epithelial-mesenchymal transition in breast cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5497. doi:10.1158/1538-7445.AM2013-5497

Abstract 5555: Honokiol inhibits tumor growth in a xenograft melanoma mice model and activates the apoptotic signaling pathway in vivo.

Abstract Melanoma is the deadliest type of skin cancer. For adults 25-29 years old, melanoma is the most common cancer, while it is the second most common cancer for 15-29 years old. Limited therapies are available for advanced stages; therefore, there is a need for development of new treatments for melanoma. Honokiol is a plant lignan extracted from the magnolia bark tree. Studies from our laboratory showed its chemopreventive effects in a UVB-induced skin cancer model, as well as in vitro pro-apoptotic effects in A431 squamous cancer cells, UACC-62 and SKMEL-2 human melanoma cell lines. In this study, the antineoplastic effects of honokiol were evaluated in vivo against the malignant melanoma cell lines UACC-62 that harbors mutated BRAF, CDK4i, PTEN and SKMEL-2 cells that presents NRAF, and TP53 mutated genes. The present study investigated the mechanism of honokiol-induced antitumoral effects in human melanoma UACC-62 and SKMEL-2 cells in two xenograft melanoma models in mice. UACC-62 and SKMEL-2 melanoma cells were subcutaneously grafted in nude mice, treatment groups received honokiol 50 mg/kg thrice a week and tumor growth was followed. After five to seven weeks, animals were euthanized and tumor tissue collected for gene profiling and western blots. The genetic expression of apoptotic biomarkers was assessed by qRT-PCR by using PCR arrays for the apoptotic pathway (SABiosciences, CA). After subcutaneous grafting of UACC-62 or SKMEL-2 human melanoma cells in the nude mice, the intraperitoneal administration of honokiol at 50 mg/kg body weight for five to seven weeks caused a significant reduction of tumor growth. Honokiol activated the apoptotic pathway in UACC-62 cells xenograft tumors in mice, as attested by the significant up-regulation of BAK1, BAX, CARD8, CASP10, CASP2, CASP6, CASP7, FADD, TNFRSF10B, TNFRSF25, TP53 and TP53BP2 transcripts. In the western blots from the UACC-62 cells tumors, there was an increase in the expression of CASP3, CASP7, CASP8, CASP6, cleaved CASP6 and CASP10 proteins in the honokiol treated group. In the tumors originated from the SKMEL-2 melanoma cells, a significant up-regulation of BAD, BAK1, BAX, NOD1, CARD6, CASP7, FADD, TNFRSF21 and TRAF3 transcripts was observed. The activation of the apoptotic pathway in the SKMEL-2 cells tumors by honokiol was further confirmed by western blots in the tumor lysates, where it was observed an increase in the activation of caspases and cleavage of PARP in the honokiol treated animals. Honokiol treatment exerted significant antitumor effects, including the activation of the apoptotic signaling pathway in UACC-62 and SKMEL-2 human melanoma cells in xenograft tumors in mice. These findings suggest that honokiol is a good candidate for further studies as a treatment for malignant melanoma. This work is supported by a Translational Cancer Center Research Grant, funded as 2010 Research Initiative Center by the State of South Dakota. Citation Format: Ruth F. Guillermo, Sreevidya Santha, Jonathan Stevens, Emily Coughlin, Pious Patel, Radhey S. Kaushik, Chandradhar Dwivedi. Honokiol inhibits tumor growth in a xenograft melanoma mice model and activates the apoptotic signaling pathway in vivo. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5555. doi:10.1158/1538-7445.AM2013-5555