Combination Of Honokiol Research Articles

Bioinformatics and In Vitro Study Reveal ERα as The Potential Target Gene of Honokiol to Enhance Trastuzumab Sensitivity in HER2+ Trastuzumab-Resistant Breast Cancer Cells

Trastuzumab resistance presents a significant challenge in the treatment of HER2+ breast cancer, necessitating the investigation of combination therapies to overcome this resistance. Honokiol, a compound with broad anticancer activity, has shown promise in this regard. This study aims to discover the effect of honokiol in increasing trastuzumab sensitivity in HER2+ trastuzumab-resistant breast cancer cells HCC1954 and the underline mechanisms behind. A bioinformatics study performed to explore the most potential target hub gene for honokiol in HER2+ breast cancer. Honokiol, trastuzumab and combined treatment cytotoxicity activity was then evaluated in both parental HCC1954 and trastuzumab resistance (TR-HCC1954) cells using MTT assay. The expression levels of these hub genes were then analyzed using qRT-PCR and those that could not be analyzed were subjected to molecular docking to determine their potential. Honokiol showed a potent cytotoxicity activity with an IC50 of 41.05 μM and 69.61 μM in parental HCC1954 and TR-HCC1954 cell line respectively. Furthermore, the combination of honokiol and trastuzumab resulted in significant differences in cytotoxicity in TR-HCC1954 cells at specific concentrations. Molecular docking and the qRT-PCR showed that the potential ERα identified from the bioinformatics analysis was affected by the treatment. Our results show that honokiol has the potential to increase the sensitivity of trastuzumab in HER2+ trastuzumab resistant breast cancer cell line HCC1954 by affecting regulating estrogen receptor signaling. Further research is necessary to validate these findings.

Honokiol induces apoptosis and autophagy in dexamethasone-resistant T-acute lymphoblastic leukemia CEM-C1 cells

ABSTRACT Objective: To study whether and, if so, how honokiol overcome dexamethasone resistance in DEX-resistant CEM-C1 cells. Methods: We investigated the effect of honokiol (0–20 µM) on cell proliferation, cell cycle, cell apoptosis and autophagy in DEX-resistant CEM-C1 cells and DEX-sensitive CEM-C7 cells. We also determined the role of c-Myc protein and mRNA in the occurrence of T-ALL associated dexamethasone resistance western blot and reverse transcription-qPCR (RT-qPCR) analysis. Results: Cell Counting Kit (CCK)−8 assay shows that DEX-resistant CEM-C1 cell lines were highly resistant to dexamethasone with IC50 of 364.1 ± 29.5 µM for 48 h treatment. However, upon treatment with dexamethasone in combination with 1.5 µM of honokiol for 48 h, the IC50 of CEM-C1 cells significantly decreased to 126.2 ± 12.3 µM, and the reversal fold was 2.88. Conversely, the IC50 of CEM-C7 cells was not changed combination of dexamethasone and honokiol as compared to that of CEM-C7 cells treated with dexamethasone alone. It has been shown that honokiol induced T-ALL cell growth inhibition by apoptosis and autophagy via downregulating cell cycle-regulated proteins (Cyclin E, CDK4, and Cyclin D1) and anti-apoptotic proteins BCL-2 and upregulating pro-apoptotic proteins Bax and led to PARP cleavage. Honokiol may overcome dexamethasone resistance in DEX-resistant CEM-C1 cell lines via the suppression of c-Myc mRNA expression. Conclusion: The combination of honokiol and DEX were better than DEX alone in DEX-resistant CEM-C1 cell lines. Honokiol may regulate T-ALL-related dexamethasone resistance by affecting c-Myc.

A novel combination therapy with Cabozantinib and Honokiol effectively inhibits c-Met-Nrf2-induced renal tumor growth through increased oxidative stress

Receptor tyrosine kinase (RTK), c-Met, is overexpressed and hyper active in renal cell carcinoma (RCC). Most of the therapeutic agents mediate cancer cell death through increased oxidative stress. Induction of c-Met in renal cancer cells promotes the activation of redox-sensitive transcription factor Nrf2 and cytoprotective heme oxygenase-1 (HO-1), which can mediate therapeutic resistance against oxidative stress. c-Met/RTK inhibitor, Cabozantinib, has been approved for the treatment of advanced RCC. However, acquired drug resistance is a major hurdle in the clinical use of cabozantinib. Honokiol, a naturally occurring phenolic compound, has a great potential to downregulate c-Met-induced pathways. In this study, we found that a novel combination treatment with cabozantinib + Honokiol inhibits the growth of renal cancer cells in a synergistic manner through increased production of reactive oxygen species (ROS); and it significantly facilitates apoptosis-and autophagy-mediated cancer cell death. Activation of c-Met can induce Rubicon (a negative regulator of autophagy) and p62 (an autophagy adaptor protein), which can stabilize Nrf2. By utilizing OncoDB online database, we found a positive correlation among c-Met, Rubicon, p62 and Nrf2 in renal cancer. Interestingly, the combination treatment significantly downregulated Rubicon, p62 and Nrf2 in RCC cells. In a tumor xenograft model, this combination treatment markedly inhibited renal tumor growth in vivo; and it is associated with decreased expression of Rubicon, p62, HO-1 and vessel density in the tumor tissues. Together, cabozantinib + Honokiol combination can significantly inhibit c-Met-induced and Nrf2-mediated anti-oxidant pathway in renal cancer cells to promote increased oxidative stress and tumor cell death.

Honokiol inhibits the growth of hormone-resistant breast cancer cells: its promising effect in combination with metformin.

Primary and metastatic breast cancers still represent an unmet clinical need for improved chemotherapy and hormone therapy. Considerable attention has been paid to natural anticancer compounds, especially lignans. The study aimed to evaluate the activity of several lignans against breast cancer cells and assess the effect of leading lignans on signaling pathways in combination with metformin. Human breast cancer cell lines MCF7 (hormone-dependent), MDA-MB-231, and SKBR3 (hormone-independent) were used. A hormone-resistant MCF7/hydroxytamoxifen (HT) subline was obtained by long-term cultivation of the MCF7 line with hydroxytamoxifen. Antiproliferative activity was assessed by the MTT test; the expression of signaling pathway proteins was evaluated by immunoblotting analysis. We evaluated the antiproliferative activity of lignans in breast cancer cells with different levels of hormone dependence and determined the relevant IC50 values. Honokiol was chosen as the leading compound, and its IC50 ranged from 12 to 20 μM, whereas for other tested lignans, the IC50 exceeded 50 μM. The accumulation of cleaved PARP and a decrease in the expression of Bcl-2 and ERα in MCF7/HT were induced following the combination of honokiol with metformin. Honokiol demonstrated significant antiproliferative activity against both hormone-dependent breast cancer cells and lines with primary and acquired hormone resistance. The combination of honokiol with metformin is considered an effective approach to induce death in hormone-resistant cells. Honokiol is of interest as a natural compound with antiproliferative activity against breast cancers, including resistant tumors.

Identification of potential target genes of honokiol in overcoming breast cancer resistance to tamoxifen.

Honokiol (HON) inhibits epidermal growth factor receptor (EGFR) signaling and increases the activity of erlotinib, an EGFR inhibitor, in human head and neck cancers. In this study, using a bioinformatics approach and in vitro experiments, we assessed the target genes of HON against breast cancer resistance to tamoxifen (TAM). Microarray data were obtained from GSE67916 and GSE85871 datasets to identify differentially expressed genes (DEGs). DEGs common between HON-treated and TAM-resistant cells were analyzed by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses and protein-protein interaction (PPI) networks were constructed. Selected genes were analyzed for genetic alterations, expression, prognostic value, and receiver operating characteristics (ROC). TAM-resistant MCF-7 (MCF-7 TAM-R) cells were generated and characterized for their resistance toward TAM. A combination of HON and TAM was used for cytotoxicity and gene expression analyses. Molecular docking was performed using the Molecular Operating Environment software. PPI network analysis revealed that FN1, FGFR2, and RET were the top three genes with the highest scores. A genetic alteration study of potential target genes revealed MMP16 and ERBB4 as the genes with the highest alterations among the breast cancer samples. Pathway enrichment analysis of FGFR2, RET, ERBB4, SOX2, FN1, and MMP16 showed that the genetic alterations herein were likely to impact the RTK-Ras pathway. The expression levels of RET, MMP16, and SOX2 were strongly correlated with prognostic power, with areas under the ROC curves (AUC) ​​of 1, 0.8, and 0.8, respectively. The HON and TAM combination increased TAM cytotoxicity in MCF-7 TAM-R cells by regulating the expression of potential target genes ret, ERBB4, SOX2, and FN1, as well as the TAM resistance regulatory genes including HES1, VIM, PCNA, TP53, and CASP7. Molecular docking results indicated that HON tended to bind RET, ErbB4, and the receptor protein Notch1 ankyrin domain more robustly than its native ligand. HON could overcome breast cancer resistance to TAM, potentially by targeting FGFR2, RET, ERBB4, MMP16, FN1, and SOX2. However, further studies are required to validate these results.

Honokiol attenuate the arsenic trioxide-induced cardiotoxicity by reducing the myocardial apoptosis.

Despite advantages of arsenic trioxide (ATO) in oncological practice, its clinical applications have been hampered by severe cardiotoxicity. The general mechanism of ATO‐induced cardiotoxicity has been attributed to its damage to mitochondria, resulting in cardiac remodeling. Honokiol (HKL) is a naturally occurring compound derived from Magnolia bark. Previous studies have demonstrated that HKL exerts cardio‐protective effects on ischemia/reperfusion (I/R) or chemical‐induced cardiotoxicity by counteracting the toxic effects on mitochondria. The present study was conducted to investigate whether HKL pretreatment protects against ATO‐induced cardiac oxidative damage and cell death. For the in vitro study, we evaluated the effects of ATO and/or Honokiol on reactive oxygen species (ROS) production and apoptosis induction in primary cultured cardiomyocytes; for the in vivo study, BALB/c mice were administrated with ATO and/or HKL for a period of 4 weeks, myocardial apoptosis, cardiac function, and cardiac remodeling (cardiac hypertrophy and cardiac fibrosis) were assessed at the end of administration. Our results demonstrated Honokiol pretreatment alleviated the ATO‐induced boost in ROS concentration and the following apoptosis induction in primary cultured cardiomyocytes. In the mouse model, Honokiol pretreatment ameliorated ATO‐induced myocardial apoptosis, cardiac dysfunction, and cardiac remodeling. Collectively, these results indicated that Honokiol provide a protection against ATO‐induced cardiotoxicity by reducing mitochondrial damage. In addition, given that Honokiol has shown considerable suppressive effects on leukemia cells, our data also imply that ATO and Honokiol combination may possibly be a superior avenue in leukemia therapy.

Synergistic killing effect of paclitaxel and honokiol in non-small cell lung cancer cells through paraptosis induction.

Paclitaxel is an anticancer drug for the treatment of non-small cell lung cancer (NSCLC). However, drug-resistance remains a major problem. Honokiol is a natural component which has been found to exhibit anti-tumor activity. Paclitaxel and honokiol have been reported to be able to induce paraptosis. The aim of this study was to investigate whether honokiol can reverse paclitaxel resistance by inducing paraptosis in NSCLC cells. NSCLC cell lines H1650 (paclitaxel-sensitive), H1299 and H1650/PTX (intrinsic and acquired paclitaxel-resistant, respectively) were used to assess the cytotoxic effects of paclitaxel and honokiol. Light and transmission electron microscopy were performed to detect cytoplasmic vacuolation. In vitro cell viability and clonogenic survival assays, as well as in vivo xenograft assays were conducted to test synergistic killing effects of paclitaxel and honokiol on NSCLC cells. Western blotting, flow cytometry and immunofluorescence were performed to evaluate paraptosis-regulating mechanisms. We found that combination treatment with paclitaxel and honokiol synergistically killed H1650,H1299 and H1650/PTX cells by inducing paraptosis, which is characterized by cytoplasmic vacuolation. Moreover, paclitaxel/honokiol treatment resulted in a significant growth delay in H1299 xenograft tumors that showed extensive cytoplasmic vacuolation. Mechanistically, proteasomal inhibition-mediated endoplasmic reticulum (ER) stress and unfolded protein responses leading to ER dilation, and the disruption of intracellular Ca2+ homeostasis and mitochondrial Ca2+ overload resulting in mitochondrial disfunction, were found to be involved in paclitaxel/honokiol-induced paraptosis.Cellular protein light chain 3 (LC3) may play an important role in paclitaxel/honokiol induced cytoplasmic vacuolation and NSCLC cell death. Combination of honokiol and paclitaxel may represent a novel strategy for the treatment of paclitaxel-resistant NSCLC.

A Novel Combination Treatment with Honokiol and Rapamycin Effectively Restricts c-Met-Induced Growth of Renal Cancer Cells, and also Inhibits the Expression of Tumor Cell PD-L1 Involved in Immune Escape.

The mTOR inhibitor Rapamycin has tumor inhibitory properties; and it is also used as an immunosuppressive agent after organ transplantation. However, prolonged Rapamycin treatment re-activates Akt and can promote cancer growth. Honokiol is a natural compound with both anti-tumorigenic and anti-inflammatory properties. Here, we assessed the anti-tumor effects of Rapamycin and Honokiol combination in renal cell carcinoma (RCC). Receptor tyrosine kinase c-Met-mediated signaling plays a major role in RCC growth. We observed that compared with Rapamycin alone, Rapamycin + Honokiol combination can effectively down-regulate c-Met-induced Akt phosphorylation in renal cancer cells; and it markedly inhibited Ras activation and cell proliferation and promoted G1 phase cell cycle arrest. The combination treatment significantly induced ROS generation and cancer cell apoptosis even when c-Met is activated. Importantly, Honokiol, but not Rapamycin, decreased c-Met-induced expression of the co-inhibitory molecule PD-L1, implied in the immune escape of renal cancer cells. In mouse renal cancer cells and Balb/c splenocytes co-culture assay, Rapamycin + Honokiol markedly potentiated immune-cell-mediated killing of cancer cells, possibly through the down-regulation of PD-L1. Together, Honokiol can effectively overcome the limitation of Rapamycin treatment alone; and the combination treatment can markedly restrict the growth of RCC, with particular importance to post-transplantation renal cancer.

Honokiol Restores Polymyxin Susceptibility to MCR-1-Positive Pathogens both In Vitro and In Vivo.

The emergence of the plasmid-mediated colistin resistance gene mcr-1 has led to serious multidrug-resistant (MDR) Enterobacteriaceae infections, which are a great threat to the clinic. This study aims to find an inhibitor of MCR-1 to reestablish the use of polymyxins against MDR Enterobacteriaceae infections. Here, we determined that the natural compound honokiol could enhance the efficacy of polymyxins against MDR Enterobacteriaceae infections by a checkerboard MIC assay, a time-kill assay, a combined disk test, Western blotting, molecular simulation dynamics, and mouse infection models. The MIC results indicated that honokiol can recover the sensitivity of polymyxins against MCR-1-positive Klebsiella pneumoniae and Escherichia coli (with a fractional inhibitory concentration index ranging from 0.09 ± 0.00 to 0.27 ± 0.06). Based on time-kill curve analysis, all of the tested bacteria were killed within 1 h following the combined therapy with honokiol and polymyxins. Molecular simulation dynamics results suggested that honokiol directly binds to the MCR-1 active region, reducing the biological activity of MCR-1. The combination of honokiol and polymyxins could increase the 40% protection rate and reduce the bacterial load on the thigh muscles of mice. Our study indicates that honokiol is a predominant natural compound whose combination therapy with polymyxins is very promising in future treatment options for MCR-1-positive Enterobacteriaceae infections.IMPORTANCE In the present study, honokiol could effectively inhibit the activity of MCR-1 and showed almost no cytotoxicity to MH-S cells. According to our results, the combination of honokiol and polymyxin had a clear synergistic effect against MCR-1-positive Enterobacteriaceae in vitro Combination therapy also showed a powerful therapeutic effect in vivo, which can significantly improve mouse livability, reduced the load of bacteria, and reduced pathological change. This combined therapy of small molecule compounds and antibiotics may not continue to induce new bacterial resistance, due to the fact that MCR-1 targeted by honokiol is not indispensable for the bacterial viability; on the other hand, it can reduce the dosage of combined antibiotics, and it is also a promising alternative therapy for the treatment of drug-resistant infections in the future.

Overcoming acquired resistance of EGFR-mutant NSCLC cells to the third generation EGFR inhibitor, osimertinib, with the natural product honokiol.

The development of acquired resistance to osimertinib (Osim) (AZD9291 or TAGRISSOTM), an FDA‐approved third‐generation epidermal growth factor receptor (EGFR) inhibitor for the treatment of EGFR‐mutant nonsmall cell lung cancer (NSCLC), limits the long‐term benefits for patients. Thus, effective treatment options are urgently needed. To this end, we explored whether honokiol (HNK), a natural product with potential antitumor activity, may be used to overcome Osim resistance. The combination of HNK and Osim synergistically decreased the survival of several Osim ‐resistant cell lines with enhanced effects on inhibiting cell colony formation and growth and on inducing apoptosis. This combination also showed greater growth suppression of Osim‐resistant xenograft tumors including those with 19del, T790M, and C797S triple mutations in nude mice. Mechanistically, the augmented induction of apoptosis by the combination is largely due to enhanced Mcl‐1 reduction through facilitating its degradation. A synthetic HNK derivative exerted similar effects with greater efficacy. Our findings warrant further study of HNK and its derivatives in overcoming Osim resistance in the clinic.

Abstract 1277: Honokiol, a natural product, overcomes acquired resistance of EGFR mutant NSCLC cells to the third generation EGFR inhibitor, osimertinib

Abstract Osimertinib (AZD9291) is a FDA-approved EGFR inhibitor for treating non-small cell lung cancer (NSCLC) patients with activating EGFR mutations (first-line) or those who have become resistant to 1st generation EGFR inhibitors through the T790M mutation (second-line). The development of acquired resistance to osimertinib limits the long-term benefits of patients, and effective treatment options are urgently needed. We have identified that honokiol, a natural product with potential antitumor activity, may be used to overcome osimertinib resistance. In several osimertinib-resistant cell lines including HCC827/AR, PC-9/AR, PC-9/GR/AR and PC-9/3M (with EGFR 19del, T790M and C797S mutations), the combination of honokiol and osimertinib had synergistic effects on decreasing the survival of these cell lines. The combination inhibited cell colony formation and growth, and augmented induction of apoptosis of resistant cells. This combination enhanced reduction of Mcl-1, a critical mechanism by which osimertinib induces apoptosis in sensitive EGFR mutant NSCLC cells; this effect is likely due to enhanced Mcl-1 degradation since Mcl-1 was degraded much faster in cells exposed to the combination of honokiol and osimertinib than in DMSO-treated control cells. Moreover enforced overexpression of ectopic Mcl-1 abolished the enhanced effect of honokiol and osimertinib combination on induction of apoptosis, supporting a critical role of Mcl-1 reduction in inducing apoptosis of the osimertinib-resistant cells by this combination. Our findings warrant further study of this combination for overcoming osimertinib-resistance in vivo and potentially in the clinic. (This work was supported by the NIH/NCI R01 CA223220 to SYS and Winship lung cancer pilot award to SYS). Citation Format: Hongjing ZANG, Songqing Fan, Jack Arbiser, Taofeek K. Owonikoko, Suresh S. Ramalingam, Shi-Yong Sun. Honokiol, a natural product, overcomes acquired resistance of EGFR mutant NSCLC cells to the third generation EGFR inhibitor, osimertinib [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1277.

Inhibition of Herpes Simplex Virus-1 Replication by Natural Compound Honokiol.

Honokiol is a pleiotropic natural compound isolated from Magnolia and has multiple biological and clinically relevant effects, including anticancer and antimicrobial function. However, the antiviral activity of honokiol has not yet been well studied. Here we showed that honokiol had no effect on herpes simplex virus-1 (HSV-1) entry, but inhibited HSV-1 viral DNA replication, gene expression and the production of new progeny viruses. The combination of honokiol and clinical drug acyclovir augmented inhibition of HSV-1 infection. Our results illustrate that honokiol could be a potential new candidate for clinical consideration in the treatment of HSV-1 infection alone or combination with other therapeutics.

Honokiol Inhibits DNA Polymerases β and λ and Increases Bleomycin Sensitivity of Human Cancer Cells.

A major concept to sensitize cancer cells to DNA damaging agents is by inhibiting proteins in the DNA repair pathways. X-family DNA polymerases play critical roles in both base excision repair (BER) and nonhomologous end joining (NHEJ). In this study, we examined the effectiveness of honokiol to inhibit human DNA polymerase β (pol β), which is involved in BER, and DNA polymerase λ (pol λ), which is involved in NHEJ. Kinetic analysis with purified polymerases showed that honokiol inhibited DNA polymerase activity. The inhibition mode for the polymerases was a mixed-function noncompetitive inhibition with respect to the substrate, dCTP. The X-family polymerases, pol β and pol λ, were slightly more sensitive to inhibition by honokiol based on the Ki value of 4.0 μM for pol β, and 8.3 μM for pol λ, while the Ki values for pol η and Kf were 20 and 26 μM, respectively. Next we extended our studies to determine the effect of honokiol on the cytotoxicity of bleomycin and temozolomide in human cancer cell lines A549, MCF7, PANC-1, UACC903, and normal blood lymphocytes (GM12878). Bleomycin causes both single strand DNA damage that is repaired by BER and double strand breaks that are repaired by NHEJ, while temozolomide causes methylation damage repaired by BER and O6-alkylguanine-DNA alkyltransferase. The greatest effects were found with the honokiol and bleomycin combination in MCF7, PANC-1, and UACC903 cells, in which the EC50 values were decreased 10-fold. The temozolomide and honokiol combination was less effective; the EC50 values decreased three-fold due to the combination. It is hypothesized that the greater effect of honokiol on bleomycin is due to inhibition of the repair of the single strand and double strand damage. The synergistic activity shown by the combination of bleomycin and honokiol suggests that they can be used as combination therapy for treatment of cancer, which will decrease the therapeutic dosage and side effects of bleomycin.

Synergistic Antioxidant and Anti-Inflammatory Effects between Modified Citrus Pectin and Honokiol.

Inflammation is a normal physiological process; however, dysregulation of this process may contribute to inflammatory-based chronic disorders and diseases in animals and humans. Therefore, the antioxidant and anti-inflammatory properties of natural products, often recognized in traditional medicine systems, represent therapeutic modalities to reduce or prevent uncontrolled inflammatory processes which in turn potentially ameliorate or prevent sequelae of inflammatory-based symptoms of chronic diseases. We have investigated the antioxidant and anti-inflammatory effects of honokiol (HNK) and modified citrus pectin (MCP) in vitro and examined whether the MCP : HNK combination has synergistic effects on antioxidant and anti-inflammatory properties. Although both HNK and MCP induced a dose-dependent increase in antioxidant activity, the latter has a consistently higher antioxidant effect. The MCP : HNK (9 : 1) combination induced a synergistic effect on antioxidant activity suggesting that the combination is significantly more efficacious than individual compounds. In mouse monocytes, the lipopolysaccharide- (LPS-) induced tumor necrosis-α (TNF-α) synthesis was significantly inhibited by HNK and the MCP : HNK combination in a dose-dependent manner and synergistic effects were clearly demonstrated with the combination on TNF-α inhibition. This combination effect was also evident on inhibition of nuclear factor-kappa B activity, cyclooxygenase-II activity, and lipid peroxidation in mouse monocytes. Further research into the combination is warranted.

Honokiol targets mitochondria to halt cancer progression and metastasis.

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

Co-delivery of honokiol, a constituent of Magnolia species, in a self-microemulsifying drug delivery system for improved oral transport of lipophilic sirolimus

Sirolimus is recognized as a P-glycoprotein (P-gp) substrate with poor water-solubility. To improve its solubility and bioabsorption, self-microemulsifying drug delivery systems (SMEDDS) containing a novel P-gp inhibitor, honokiol, were prepared. The aim of this work was to evaluate the enhanced transport of sirolimus SMEDDS as well as the roles of honokiol. In situ single-pass intestinal perfusion and in vitro human colon adenocarcinoma (Caco-2) cell models were applied to study the effects of honokiol within SMEDDS on the transport of sirolimus. The results indicated that a combination of honokiol with sirolimus in SMEDDS did not significantly alter the particle size, polydispersity index and release of drugs. In addition, the absorption rate constant (Ka) as well as the effective permeability coefficients (Peff) of sirolimus in situ intestinal absorption, and the apparent permeability coefficients (Papp) of sirolimus in caco-2 cells were significantly enhanced by cremophor EL-based SMEDDS with honokiol as compared with those of SMEDDS without honokiol. Rhodamine123 uptake rate in caco-2 cells and in vitro cytotoxicity of sirolimus were enhanced by honokiol in SMEDDS indicating a substantial P-gp inhibition of honokiol. In conclusion, coadministration of honokiol with poor soluble P-gp substrate in SMEDDS, could serve as a favorable approach for oral delivery.

Chemopreventive effects of combination of honokiol and magnolol with α-santalol on skin cancer developments

α-Santalol is active component of sandalwood oil and has been shown to have chemopreventive effects against chemically and UVB-induced skin cancer development in mice. α-Santalol is also shown to have skin permeation enhancing effects. Honokiol and magnolol isolated from Magnolia officinalis bark extract have also been shown to have chemopreventive effects against chemically and UVB-induced skin cancer in mice. This study was conducted to investigate the combination effects of α-santalol, honokiol and magnolol to study any additive/synergistic effects to lower the doses required for chemoprevention. Pretreatment of combinations of α-santalol with honokiol and magnolol significantly decreased tumor multiplicity upto 75% than control, α-santalol, honokiol and magnolol alone in SKH-1 mice. Combination of α-santalol with honokiol and magnolol also decreased cell viability, proliferation, and enhanced apotosis in comparison to α-santalol, honokiol and magnolol alone in Human epidrmoid carcinoma A431 cells. Overall, the results of present study indicated combinations of α-santalol with honokiol and magnolol could provide chemoprevention of skin cancer at lower doses than given alone.

Honokiol radiosensitizes colorectal cancer cells: enhanced activity in cells with mismatch repair defects.

DNA mismatch repair is required for correcting any mismatches that are created during replication and recombination, and a defective mismatch repair system contributes to DNA damage-induced growth arrest. The colorectal cancer cell line HCT116 is known to have a mutation in the hMLH1 mismatch repair gene resulting in microsatellite instability and defective mismatch repair. Honokiol is a biphenolic compound that has been used in traditional Chinese medicine for treating various ailments including cancer. This study was designed to test the hypothesis that honokiol enhances the radiosensitivity of cancer cells with mismatch repair defect (HCT116) compared with those that are mismatch repair proficient (HCT116-CH3). We first determined that the combination of honokiol and γ-irradiation treatment resulted in dose-dependent inhibition of proliferation and colony formation in both cell lines. However, the effects were more pronounced in HCT116 cells. Similarly, the combination induced higher levels of apoptosis (caspase 3 activation, Bax to Bcl2 ratio) in the HCT116 cells compared with HCT116-CH3 cells. Cell cycle analyses revealed higher levels of dead cells in HCT116 cells. The combination treatment reduced expression of cyclin A1 and D1 and increased phosphorylated p53 in both cell lines, although there were significantly lower amounts of phosphorylated p53 in the HCT116-CH3 cells, suggesting that high levels of hMLH1 reduce radiosensitivity. These data demonstrate that honokiol is highly effective in radiosensitizing colorectal cancer cells, especially those with a mismatch repair defect.

Abstract 3689: Targeting cell survival pathways with the combination of honokiol and resveratrol for prevention of head and neck cancer

Abstract Natural dietary agents have drawn great attention for cancer prevention because of their wide safety margin and ready availability. However, single agent intervention has failed to bring the expected outcomes in clinical trials. In the current study, we investigated a combinatorial approach using two natural compounds, honokiol (isolated from the bark of the magnolia tree) and resveratrol (a major constituent of red wines and grapes), and found that their combination at low doses (at which single agents induce minimal cell growth inhibition) strongly inhibited cell growth as measured by SRB assay. Moreover, the combination of low dose honokiol (which alone induces 10-15% apoptosis) with low dose resveratrol (also induces ∼10% apoptosis) strongly increased apoptosis (to 60-80%) in multiple head and neck cancer cell lines as evidenced by annexin V-PE staining and PARP cleavage. Interestingly, normal human fibroblasts (BJ) or immortalized normal bronchial epithelial cells (BEAS-2B) were resistant to apoptosis induced by the combination of the two compounds (less than 10% apoptosis). To understand the mechanism of apoptosis, we fractionated cytosolic and mitochondrial fractions to study the release of cytochrome c from the mitochondria to the cytosol, and found that the combination of honokiol and resveratrol more efficiently caused release of cytochrome c compared with either single agent alone. Further mechanistic studies suggested that the combination of the two agents inhibited phospho-STAT3 and phospho-AKT and reduced NF-κB protein level more efficiently than either single agent. Moreover, although single agent treatment has only minimal effect on expression of NF-κB and the STAT3 targets Bcl-2, Mcl-1 and survivin, the combination of the two agents strongly inhibited expression of these proteins, further supporting the involvement of mitochondria-mediated apoptosis. However, expression of Bcl-xL, another proapoptotic Bcl-2 protein remained unchanged after treatment with the compounds. We also found transient inhibition of phospho S6 kinase, a downstream target of p-AKT. NF-κB, phospho-STAT3 and phospho-AKT pathways are frequently over-active in head and neck cancer, suggesting that the combination of honokiol and resveratrol might be an effective strategy for the prevention of head and neck cancer and providing an important rationale for future preclinical and clinical development. (Supported by U01CA101244, R01CA112643, and P50CA128613 to DMS) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3689. doi:10.1158/1538-7445.AM2011-3689

Honokiol Inhibits Epidermal Growth Factor Receptor Signaling and Enhances the Antitumor Effects of Epidermal Growth Factor Receptor Inhibitors

This study aimed to investigate the utility of honokiol, a naturally occurring compound, in the treatment of head and neck squamous cell carcinoma (HNSCC) as well as its ability to target the epidermal growth factor receptor (EGFR), a critical therapeutic target in HNSCC, and to enhance the effects of other EGFR-targeting therapies. Human HNSCC cell lines and the xenograft animal model of HNSCC were used to test the effects of honokiol treatment. Honokiol was found to inhibit growth in human HNSCC cell lines, with 50% effective concentration (EC(50)) values ranging from 3.3 to 7.4 micromol/L, and to induce apoptosis, as shown through Annexin V staining. These effects were associated with inhibition of EGFR signaling, including downstream inhibition of mitogen-activated protein kinase, Akt, and signal transducer and activator of transcription 3 (STAT3), and expression of STAT3 target genes, Bcl-X(L) and cyclin D1. Furthermore, honokiol enhanced the growth inhibitory and anti-invasion activity of the EGFR-targeting agent erlotinib. Although HNSCC xenograft models did not show significant inhibition of in vivo tumor growth with honokiol treatment alone, the combination of honokiol plus cetuximab, a Food and Drug Administration-approved EGFR inhibitor for this malignancy, significantly enhanced growth inhibition. Finally, HNSCC cells rendered resistant to erlotinib retained sensitivity to the growth inhibitory effects of honokiol. These results suggest that honokiol may be an effective therapeutic agent in HNSCC, in which it can augment the effects of EGFR inhibitors and overcome drug resistance.