A small molecule redistributes iron in ferroportin-deficient mice and patient-derived primary macrophages.

Deficiencies of the transmembrane iron-transporting protein ferroportin (FPN1) cause the iron misdistribution that underlies ferroportin disease, anemia of inflammation, and several other human diseases and conditions. A small molecule natural product, hinokitiol, was recently shown to serve as a surrogate transmembrane iron transporter that can restore hemoglobinization in zebrafish deficient in other iron transporting proteins and can increase gut iron absorption in FPN1-deficient flatiron mice. However, whether hinokitiol can restore normal iron physiology in FPN1-deficient animals or primary cells from patients and the mechanisms underlying such targeted activities remain unknown. Here, we show that hinokitiol redistributes iron from the liver to red blood cells in flatiron mice, thereby increasing hemoglobin and hematocrit. Mechanistic studies confirm that hinokitiol functions as a surrogate transmembrane iron transporter to release iron trapped within liver macrophages, that hinokitiol-Fe complexes transfer iron to transferrin, and that the resulting transferrin-Fe complexes drive red blood cell maturation in a transferrin-receptor-dependent manner. We also show in FPN1-deficient primary macrophages derived from patients with ferroportin disease that hinokitiol moves labile iron from inside to outside cells and decreases intracellular ferritin levels. The mobilization of nonlabile iron is accompanied by reductions in intracellular ferritin, consistent with the activation of regulated ferritin proteolysis. These findings collectively provide foundational support for the translation of small molecule iron transporters into therapies for human diseases caused by iron misdistribution.

Hinokitiol Inhibits Breast Cancer Cells In Vitro Stemness-Progression and Self-Renewal with Apoptosis and Autophagy Modulation via the CD44/Nanog/SOX2/Oct4 Pathway.

Breast cancer (BC) represents one of the most prevalent malignant threats to women globally. Tumor relapse or metastasis is facilitated by BC stemness progression, contributing to tumorigenicity. Therefore, comprehending the characteristics of stemness progression and the underlying molecular mechanisms is pivotal for BC advancement. Hinokitiol (ß-thujaplicin), a tropolone-related compound abundant in the heartwood of cupressaceous plants, exhibits antimicrobial activity. In our study, we employed three BC cell lines (MDA-MB-231, MCF-7, and T47D) to assess the expression of stemness-, apoptosis-, and autophagy-related proteins. Hinokitiol significantly reduced the viability of cancer cells in a dose-dependent manner. Furthermore, we observed that hinokitiol enhances apoptosis by increasing the levels of cleaved poly-ADP-ribose polymerase (PARP) and phospho-p53. It also induces dysfunction in autophagy through the upregulation of LC3B and p62 protein expression. Additionally, hinokitiol significantly suppressed the number and diameter of cancer cell line spheres by reducing the expression of cluster of differentiation44 (CD44) and key transcription factors. These findings underscore hinokitiol's potential as a therapeutic agent for breast cancer, particularly as a stemness-progression inhibitor. Further research and clinical studies are warranted to explore the full therapeutic potential of hinokitiol in the treatment of breast cancer.

Enhancing Bioactivity through the Transfer of the 2-(Hydroxymethoxy)Vinyl Moiety: Application in the Modification of Tyrosol and Hinokitiol.

Utilizing Density Functional Theory (DFT) calculations at the B3LYP/QZVP level and incorporating the Conductor-like Polarizable Continuum Model (C-PCM) for solvation, the thermodynamic and chemical activity properties of 21-(hydroxymethoxy)henicosadecaenal, identified in cultured freshwater pearls from the mollusk Hyriopsis cumingii, have been elucidated. The study demonstrates that this compound releases formaldehyde, a potent antimicrobial agent, through dehydrogenation and deprotonation processes in both hydrophilic and lipophilic environments. Moreover, this polyenal exhibits strong anti-reductant properties, effectively scavenging free radicals. These critical properties classify the pearl-derived ingredient as a natural multi-functional compound, serving as a coloring, antiradical, and antimicrobial agent. The 2-(hydroxymethoxy)vinyl (HMV) moiety responsible for the formaldehyde release can be transferred to other compounds, thereby enhancing their biological activity. For instance, tyrosol (4-(2-hydroxyethyl)phenol) can be modified by substituting the less active 2-hydroxyethyl group with the active HMV one, and hinokitiol (4-isopropylotropolone) can be functionalized by attaching this moiety to the tropolone ring. A new type of meso-carrier, structurally modeled on pearls, with active substances loaded both in the layers and the mineral part, has been proposed.

Pseudomonas bohemica strain ins3 eliminates antibacterial hinokitiol from its culture broth.

A bacterial strain, Pseudomonas bohemica strain ins3 was newly isolated as a resistant strain against high concentrations of hinokitiol. This strain was revealed not only to show resistance but also completely remove this compound from its culture broth. In addition, its mechanism was revealed to be independent of conventional aromatic dioxygenases, ie catechol-1,2- or 2,3-dioxygenases.

At the dawn of novel aromatics: "On the Synthesis of Hinokitiol" by Tetsuo Nozoe et al.

A paper titled "On the Synthesis of Hinokitiol" appeared in this journal in 1950 and marked the beginning of a new research field of novel aromatics.

Antimicrobial surface processing of polymethyl methacrylate denture base resin using a novel silica-based coating technology.

OBJECTIVES: This study investigated the surface characteristics of denture base resin coatings prepared using a novel silica-based film containing hinokitiol and assessed the effect of this coating on Candida albicans adhesion and growth. METHODS: Silica-based coating solutions (control solution; CS) and CS containing hinokitiol (CS-H) were prepared. C. albicans biofilm formed on denture base specimens coated with each solution and these uncoated specimens (control) were analyzed using colony-forming unit (CFU) assay, fluorescence microscopy, and scanning electron microscopy (SEM). Specimen surfaces were analyzed by measuring the surface roughness and wettability and with Fourier-transform infrared (FT-IR) and proton nuclear magnetic resonance (1H NMR). Stability of coated specimens was assessed via immersion in water for 1 week for each group (control-1w, CS-1w, and CS-H-1w) followed by CFU assay, measurement of surface roughness and wettability, and FT-IR. RESULTS: CS-H and CS-H-1w contained significantly lower CFUs than those present in the control and control-1w, which was also confirmed via SEM. Fluorescence microscopy from the CS-H group identified several dead cells. The values of surface roughness from coating groups were significantly less than those from the control and control-1w. The surface wettability from all coating groups exhibited high hydrophobicity. FT-IR analyses demonstrated that specimens were successfully coated, and 1H NMR analyses showed that hinokitiol was incorporated inside CS-H. CONCLUSIONS: A silica-based denture coating that incorporates hinokitiol inhibits C. albicans growth on denture. CLINICAL RELEVANCE: We provide a novel antifungal denture coating which can be helpful for the treatment of denture stomatitis.

Inhibitory effects of hinokitiol on the development and pathogenicity of Colletotrichum gloeosporioides.

Postharvest anthracnose of mango fruit caused by Colletotrichum gloeosporioides is a devastating fungal disease, which causes tremendous quality deterioration and economic losses. Hinokitiol, an environmentally friendly natural compound, is effective in controlling a variety of postharvest fungal diseases. However, there is still a lack of research on the inhibitory effect of hinokitiol on C. gloeosporioides and its possible modes of action. In the present study, the activity of hinokitiol against C. gloeosporioides and its potential mechanisms involved have been investigated. We found that hinokitiol treatment could effectively inhibit the virulence of C. gloeosporioides to harvested mango fruit. After treatment with 8 mg/L hinokitiol, the mycelial growth of C. gloeosporioides was completely inhibited. When the concentration of hinokitiol reached 9 mg/L, the spore germination rate of C. gloeosporioides decreased to 2.43% after 9 h of cultivation. The inhibitory effect is mainly due to the attenuation in cell viability, and impairment in plasma membrane followed by leakage of cytoplasmic contents such as nucleic acids, proteins, and soluble carbohydrates, which ultimately leads to the destruction of cell structure. Furthermore, hinokitiol suppressed the expression of pathogenicity-related genes, leading to reduced infection activity. Collectively, these results suggest that hinokitiol may be an excellent bio-fungicides for the management of mango anthracnose.

Synthesis of Hinokitiol Sulfonate Derivatives and Their Anti-Oomycete and Nematicidal Activities.

In order to explore novel natural product-based anti-oomycete and nematicidal agents, sixteen unreported 2-sulfonyloxyhinokitiol derivatives were prepared using the principle of active splicing, and structurally confirmed by proton nuclear magnetic resonance (1 H-NMR), carbon-13 nuclear magnetic resonance (13 C-NMR), high-resolution mass spectrometry (HRMS), and melting point. Moreover, we evaluated the title compounds as anti-oomycete and nematicidal agents against two serious agricultural pests of Phytophthora capsici and Meloidogyne incongnita. Among the sixteen hinokitiol esters tested: (1) Compounds 3a and 3m exhibited the most potent anti-oomycete activity compared to zoxamide against P. capsici, and the median effective concentration (EC50 ) values of 3a, 3m, and zoxamide were 18.64, 21.11, and 23.15 mg/L, respectively; Further studies showed that the existence of seven membered ring and carbonyl group was the necessary condition for the high anti-oomycete activity of hinokitiol. (2) Compounds 3n and 3p exhibited more promising nematicidal activity than hinokitiol, and the median lethal concentration (LC50 ) values of 3n, 3p and 1 against M. incongnita were 0.2111, 0.2079, and 0.3933 mg/L, respectively. This result will pave the way for further modification of hinokitiol to develop potential new fungicides and nematicides.

Different Cell Responses to Hinokitiol Treatment Result in Senescence or Apoptosis in Human Osteosarcoma Cell Lines.

Hinokitiol is a tropolone-related compound isolated from the heartwood of cupressaceous plants. It is known to exhibit various biological functions including antibacterial, antifungal, and antioxidant activities. In the study, we investigated the antitumor activities of hinokitiol against human osteosarcoma cells. The results revealed that hinokitiol treatment inhibited cell viability of human osteosarcoma U-2 OS and MG-63 cells in the MTT assay. Further study revealed that hinokitiol exposure caused cell cycle arrest at the S phase and a DNA damage response with the induction of γ-H2AX foci in both osteosarcoma cell lines. In U-2 OS cells with wild-type tumor suppressor p53, we found that hinokitiol exposure induced p53 expression and cellular senescence, and knockdown of p53 suppressed the senescence. However, in MG-63 cells with mutated p53, a high percentage of cells underwent apoptosis with cleaved-PARP expression and Annexin V staining after hinokitiol treatment. In addition, up-regulated autophagy was observed both in hinokitiol-exposed U-2 OS and MG-63 cells. As the autophagy was suppressed through the autophagy inhibitor chloroquine, hinokitiol-induced senescence in U-2 OS cells was significantly enhanced accompanying more abundant p53 expression. In MG-63 cells, co-treatment of chloroquine increased hinokitiol-induced apoptosis and decreased cell viability of the treated cells. Our data revealed that hinokitiol treatment could result in different cell responses, senescence or apoptosis in osteosarcoma cell lines, and suppression of autophagy could promote these effects. We hypothesize that the analysis of p53 status and co-administration of autophagy inhibitors might provide more precise and efficacious therapies in hinokitiol-related trials for treating osteosarcoma.

Direct deuteration of hinokitiol and its mechanistic study.

Hinokitiol has a broad antibacterial activity against bacteria and fungi. While its biosynthetic pathway has been intensively studied, its dynamics in natural environments, such as biodegradation pathway, remain unclear. In this study, the authors report a direct deuterium labeling of hinokitiol as a traceable molecular probe to serve those studies. Hinokitiol was subjected to the H2-Pd/C-D2O conditions and deuterated hinokitiol was obtained with excellent deuteration efficiencies and in moderate yield. The 1H and 2H NMR spectra indicated that all ring- and aliphatic hydrogens except that on C-6 were substituted by deuterium. According to the substrate scope and computational chemistry, deuteration on tropolone ring was suggested to proceed via D+-mediated process, and which was supported by the results of the experiment with trifluoroacetic acid and Pd(TPP)4. On the other hand, the deuteration on aliphatic group was predicted to be catalyzed by Pd(II) species.

Hinokitiol Exhibits Antitumor Properties through Induction of ROS-Mediated Apoptosis and p53-Driven Cell-Cycle Arrest in Endometrial Cancer Cell Lines (Ishikawa, HEC-1A, KLE).

Hinokitiol is a natural tropolone derivative that is present in the heartwood of cupressaceous plants, and has been extensively investigated for its anti-inflammatory, antioxidant, and antitumor properties in the context of various diseases. To date, the effects of hinokitiol on endometrial cancer (EC) has not been explored. The purpose of our study was to investigate the anti-proliferative effects of hinokitiol on EC cells. Cell viability was determined with an MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay, and the quantification of apoptosis and reactive oxygen species (ROSs) was performed by using flow cytometry, while protein expression was measured with the Western blotting technique. Hinokitiol significantly suppressed cell proliferation through the inhibition of the expression of cell-cycle mediators, such as cyclin D1 and cyclin-dependent kinase 4 (CDK4), as well as the induction of the tumor suppressor protein p53. In addition, hinokitiol increased the number of apoptotic cells and increased the protein expression of cleaved-poly-ADP-ribose polymerase (PARP) and active cleaved-caspase-3, as well as the ratio of Bcl-2-associated X protein (Bax) to B-cell lymphoma 2 (Bcl-2). Interestingly, except for KLE cells, hinokitiol induced autophagy by promoting the accumulation of the microtubule-associated protein light chain 3B (LC3B) and reducing the sequestosome-1 (p62/SQSTM1) protein level. Furthermore, hinokitiol triggered ROS production and upregulated the phosphorylation of extracellular-signal-regulated kinase (p-ERK1/2) in EC cells. These results demonstrate that hinokitiol has potential anti-proliferative and pro-apoptotic benefits in the treatment of endometrial cancer cell lines (Ishikawa, HEC-1A, and KLE).

Thallium(I) Tropolonates: Synthesis, Structure, Spectral Characteristics, and Antimicrobial Activity Compared to Lead(II) and Bismuth(III) Analogues.

Synthesis, single-crystal X-ray determination diffraction and FT-IR, NMR (1H, 13C, 19F and 205Tl), UV-vis, and luminescence spectra characteristics were described for series of thallium(I) compounds: thallium(I) triflate (Tl(OTf)), 1:1 co-crystals of thallium(I) triflate and tropolone (Htrop), Tl(OTf)·Htrop, as well as simple thallium(I) chelates: Tl(trop) (1), Tl(5-metrop) (2), Tl(hino) (3), with Htrop, 5-methyltropolone (5-meHtrop), 4-isopropyltropolone (hinokitiol, Hhino), respectively, and additionally more complex {Tl@[Tl(hino)]6}(OTf) (4) compound. Comparison of their antimicrobial activity with selected lead(II) and bismuth(III) analogs and free ligands showed that only bismuth(III) complexes demonstrated significant antimicrobial activity, from two- to fivefold larger than the free ligands.

Hinokitiol reduces tumor metastasis by inhibiting heparanase via extracellular signal-regulated kinase and protein kinase B pathway.

Heparanase cleaves the extracellular matrix by degrading heparan sulfate that ultimately leads to cell invasion and metastasis; a condition that causes high mortality among cancer patients. Many of the anticancer drugs available today are natural products of plant origin, such as hinokitiol. In the previous report, it was revealed that hinokitiol plays an essential role in anti-inflammatory and anti-oxidation processes and promote apoptosis or autophagy resulting to the inhibition of tumor growth and differentiation. Therefore, this study explored the effects of hinokitiol on the cancer-promoting pathway in mouse melanoma (B16F10) and breast (4T1) cancer cells, with emphasis on heparanase expression. We detected whether hinokitiol can elicit anti-metastatic effects on cancer cells via wound healing and Transwell assays. Besides, mice experiment was conducted to observe the impact of hinokitiol in vivo. Our results show that hinokitiol can inhibit the expression of heparanase by reducing the phosphorylation of protein kinase B (Akt) and extracellular regulated protein kinase (ERK). Furthermore, in vitro cell migration assay showed that heparanase downregulation by hinokitiol led to a decrease in metastatic activity which is consistent with the findings in the in vivo experiment.

New therapeutic strategy of hinokitiol in haemorrhagic shock-induced liver injury.

Haemorrhagic shock and resuscitation (HS/R) may cause global ischaemia-reperfusion injury, which can result in systemic inflammation, multiorgan failure (particularly liver failure) and high mortality. Hinokitiol, a bioactive tropolone-related compound, exhibits antiplatelet and anti-inflammatory activities. Targeting inflammatory responses is a potential strategy for ameliorating hepatic injury during HS/R. Whether hinokitiol prevents hepatic injury during HS/R remains unclear. In the present study, we determined the role of hinokitiol following HS/R. The in vivo assays revealed that hinokitiol markedly attenuated HS/R-induced hepatic injury. Hinokitiol could inhibited NF-κB activation and IL-6 and TNF-α upregulation in liver tissues. Moreover, hinokitiol reduced caspase-3 activation, upregulated Bax and downregulated Bcl-2. These findings suggest that hinokitiol can ameliorate liver injury following HS/R, partly through suppression of inflammation and apoptosis. Furthermore, the in vitro data revealed that hinokitiol significantly reversed hypoxia/reoxygenation (H/R)-induced cell death and apoptosis in the primary hepatocytes. Hinokitiol prevented H/R-induced caspase-3 activation, PPAR cleavage, Bax overexpression and Bcl-2 downregulation. Moreover, hinokitiol attenuated H/R-stimulated NF-κB activation and reduced the levels of IL-6 and TNF-α mRNAs, suggesting that hinokitiol can protect hepatocytes from H/R injury. Collectively, our data suggest that hinokitiol attenuates liver injury following HS/R, partly through the inhibition of NF-κB activation.

Hinokitiol suppresses growth of B16 melanoma by activating ERK/MKP3/proteosome pathway to downregulate survivin expression.

Metastasis is the major cause of treatment failure in patients with cancer. Hinokitiol, a metal chelator derived from natural plants, has anti-inflammatory and antioxidant activities as well as anticancer effects. We investigated the potential anticancer effects of hinokitiol in metastatic melanoma cell line B16-F10. Exposure of the melanoma B16-F10 cells to hinokitiol significantly inhibited colony formation and cell viability in a time and concentration-dependent manner. The hinokitiol-treated cells exhibited apoptotic features in morphological assay. Results from Western blot and immunoprecipitation showed that hinokitiol treatment decreased survivin protein levels and increased suvivin ubiquitination. Pretreatment with proteosome inhibitors effectively prevented hinokitiol-induced decrease in survivin expression, implying that ubiquitin/proteosome pathway involved in hinokitiol-reduced survivin expression. Hinokitiol rapidly induced ERK phosphorylation followed by a sustained dephosphorylation, which accompanied with an increase in expression of tumor suppressor MKP-3 (mitogen-activated protein kinase phosphatase-3). Inhibition of hinokitiol-induced ERK activation by MEK inhibitor U0126 completely blocked expression of MKP-3. More importantly, inhibition of MKP-3 activity by NSC 95397 significantly inhibited hinokitiol-induced ERK dephosphorylation, ubiquitination and downregulation of survivin. These results suggested that hinokitiol inhibited growth of B16-F10 melanoma through downregulation of survivin by activating ERK/MKP-3/proteosome pathway. Hinokitiol-inhibition of survivin may be a novel and potential approach for melanoma therapy. Hinokitiol can be useful for developing therapeutic agent for melanoma.

Antibacterial activity of hinokitiol against both antibiotic-resistant and -susceptible pathogenic bacteria that predominate in the oral cavity and upper airways.

Hinokitiol, a component of the essential oil isolated from Cupressaceae, possesses antibacterial and antifungal activities and has been used in oral care products. In this study, the antibacterial activities of hinokitiol toward various oral, nasal and nasopharyngeal pathogenic bacteria, including Streptococcus mutans, Streptococcus sobrinus, Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans, Prevotella intermedia, Fusobacterium nucleatum, methicillin-resistant and -susceptible Staphylococcus aureus, antibiotic-resistant and -susceptible Streptococcus pneumoniae, and Streptococcus pyogenes were examined. Growth of all these bacterial strains was significantly inhibited by hinokitiol, minimal inhibitory concentrations of hinokitiol against S. mutans, S. sobrinus, P. gingivalis, P. intermedia, A. actinomycetemcomitans, F. nucleatum, methicillin-resistant S. aureus, methicillin-susceptible S. aureus, antibiotic-resistant S. pneumoniae isolates, antibiotic-susceptible S. pneumoniae, and S. pyogenes being 0.3, 1.0, 1.0, 30, 0.5, 50, 50, 30, 0.3-1.0, 0.5, and 0.3 µg/mL, respectively. Additionally, with the exception of P. gingivalis, hinokitiol exerted bactericidal effects against all bacterial strains 1 hr after exposure. Hinokitiol did not display any significant cytotoxicity toward the human gingival epithelial cell line Ca9-22, pharyngeal epithelial cell line Detroit 562, human umbilical vein endothelial cells, or human gingival fibroblasts, with the exception of treatment with 500 µg/mL hinokitiol, which decreased numbers of viable Ca9-22 cells and gingival fibroblasts by 13% and 12%, respectively. These results suggest that hinokitiol exhibits antibacterial activity against a broad spectrum of pathogenic bacteria and has low cytotoxicity towards human epithelial cells.

Hinokitiol ablates myofibroblast activation in precancerous oral submucous fibrosis by targeting Snail.

Oral submucous fibrosis (OSF) is a precancerous condition with symptoms of limited mouth opening and areca nut chewing habit has been implicated in its pathogenesis. Hinokitiol, a natural tropolone derived from Chamacyparis taiwanensis, has been reported to improve oral lichen planus and inhibit various cancer cells. Here, we showed that hinokitiol reduced the myofibroblast activities in fBMFs and prevented the arecoline-induced transdifferentiation. Treatment of hinokitiol dose-dependently downregulated the myofibroblast markers as well as various EMT transcriptional factors. In particular, we identified that Snail was able to bind to the E-box in the α-SMA promoter. Our data suggested that exposure of fBMFs to hinokitiol mitigated the hallmarks of myofibroblasts, while overexpression of Snail eliminated the effect of hinokitiol. These findings revealed that the inhibitory effect of hinokitiol on myofibroblasts was mediated by repression of α-SMA via regulation of Snail and showed the anti-fibrotic potential of hinokitiol in the treatment of OSF.

Hinokitiol Inhibits Migration of A549 Lung Cancer Cells via Suppression of MMPs and Induction of Antioxidant Enzymes and Apoptosis.

Hinokitiol, a natural monoterpenoid from the heartwood of Calocedrus formosana, has been reported to have anticancer effects against various cancer cell lines. However, the detailed molecular mechanisms and the inhibiting roles of hinokitiol on adenocarcinoma A549 cells remain to be fully elucidated. Thus, the current study was designed to evaluate the effect of hinokitiol on the migration of human lung adenocarcinoma A549 cells in vitro. The data demonstrates that hinokitiol does not effectively inhibit the viability of A549 cells at up to a 10 µM concentration. When treated with non-toxic doses (1-5 µM) of hinokitiol, the cell migration is markedly suppressed at 5 µM. Hinokitiol significantly reduced p53 expression, followed by attenuation of Bax in A549 cells. A dose-dependent inhibition of activated caspase-9 and -3 was observed in the presence of hinokitiol. An observed increase in protein expression of matrix metalloproteinases (MMPs) -2/-9 in A549 cells was significantly inhibited by hinokitiol. Remarkably, when A549 cells were subjected to hinokitiol (1-5 µM), there was an increase in the activities of antioxidant enzymes catalase (CAT) and superoxide dismutase (SOD) from the reduction in cells. In addition, the incubation of A549 cells with hinokitiol significantly activated the cytochrome c expression, which may be triggered by activation of caspase-9 followed by caspase-3. These observations indicate that hinokitiol inhibited the migration of lung cancer A549 cells through several mechanisms, including the activation of caspases-9 and -3, induction of p53/Bax and antioxidant CAT and SOD, and reduction of MMP-2 and -9 activities. It also induces cytochrome c expression. These findings demonstrate a new therapeutic potential for hinokitiol in lung cancer chemoprevention.

Hinokitiol induces DNA demethylation via DNMT1 and UHRF1 inhibition in colon cancer cells.

BACKGROUND: DNA hypermethylation is a key epigenetic mechanism for the silencing of many genes in cancer. Hinokitiol, a tropolone-related natural compound, is known to induce apoptosis and cell cycle arrest and has anti-inflammatory and anti-tumor activities. However, the relationship between hinokitiol and DNA methylation is not clear. The aim of our study was to explore whether hinokitiol has an inhibitory ability on the DNA methylation in colon cancer cells. RESULTS: MTT data showed that hinokitiol had higher sensitivity in colon cancer cells, HCT-116 and SW480, than in normal colon cells, CCD18Co. Hinokitiol reduced DNA methyltransferase 1 (DNMT1) and ubiquitin-like plant homeodomain and RING finger domain 1 (UHRF1) expression in HCT-116 cells. In addition, the expression of ten-eleven translocation protein 1 (TET1), a known DNA demethylation initiator, was increased by hinokitiol treatment. ELISA and FACS data showed that hinokitiol increased the 5-hydroxymethylcytosine (5hmC) level in the both colon cancer cells, but 5-methylcytosine (5mC) level was not changed. Furthermore, hinokitiol significantly restored mRNA expression of O6-methylguanine DNA methyltransferase (MGMT), carbohydrate sulfotransferase 10 (CHST10), and B-cell translocation gene 4 (BTG4) concomitant with reduction of methylation status in HCT-116 cells. CONCLUSIONS: These results indicate that hinokitiol may exert DNA demethylation by inhibiting the expression of DNMT1 and UHRF1 in colon cancer cells.

Molecular interactions of the inclusion complexes of hinokitiol and various cyclodextrins.

The aim of this study was to prepare inclusion complexes of hinokitiol (HT)/α-cyclodextrin (α-CD) and HT/ß-cyclodextrin (ß-CD) by cogrinding and to evaluate the differences in their formation. The physical properties of the preparation were evaluated by Job's plot, phase solubility studies, differential scanning calorimetry, powder X-ray diffraction, solid fluorescence spectra, and infrared absorption spectra. Intermolecular interaction in the solid state was confirmed to be in the ratios HT/α-CD = 1/2 and HT/ß-CD = 1/1. Results indicated that the dissolution property of HT was improved by inclusion in the complexes HT/α-CD and HT/ß-CD compared with HT crystals. The 1H-1H ROESY NMR spectrum of HT/α-CD showed that part of the seven-membered ring of HT and the isopropyl group of HT was linked to the wider edges of the two α-CDs. In HT/ß-CD, the seven-membered ring of HT interacted with the narrower edge of ß-CD and the isopropyl group of HT interacted with the wider edges. This structure of inclusion complexes was attributed to the difference in the cavity diameter of the CD and was thought to influence the dissolution properties.