Although cytotoxic chemotherapy is widely used against epithelial ovarian cancer (EOC), adverse side effects and emergence of resistance can limit its utility. Therefore, new drugs with systemic delivery platforms are urgently needed for this disease. In this study, we developed linalool-incorporated nanoparticles (LIN-NP) as a novel anticancer agent. We prepared LIN-NPs by the self-assembly water-in-oil-in-water (w/o/w) emulsion method. LIN-NP–mediated cytotoxicity and apoptosis was assessed in EOC cells, and the role of reactive oxygen species (ROS) generation as the mechanism of action was evaluated. In addition, therapeutic efficacy of LIN-NP was assessed in cell lines and patient-derived xenograft (PDX) models for EOC. LIN-NPs had significant cytotoxicity and apoptotic activity against EOC cells, including A2780, HeyA8, and SKOV3ip1. LIN-NP treatment increased apoptosis in EOC cells through ROS generation and a subsequent decrease in mitochondrial membrane potential and increase in caspase-3 levels. In addition, 100 mg/kg LIN-NPs significantly decreased tumor weight in the HeyA8 (P < 0.001) and SKOV3ip1 (P = 0.006) in vivo models. Although treatment with 50 mg/kg LIN-NP did not decrease tumor weight compared with the control group, combination treatment with paclitaxel significantly decreased tumor weight compared with paclitaxel alone in SKOV3ip1 xenografts (P = 0.004) and the patient-derived xenograft model (P = 0.020). We have developed LIN-NPs that induce ROS generation as a novel anticancer agent for EOC. These findings have broad applications for cancer therapy. Mol Cancer Ther; 15(4); 618–27. ©2016 AACR.

Although epithelial ovarian cancer (EOC) has poor prognosis, more than 70% of patients exhibit a favorable initial response to primary treatment, including surgery and chemotherapy (1). However, successful management of advanced-stage or recurrent EOC is challenging because of the emergence of cancer cells that are resistant to cytotoxic drugs (2, 3). Therefore, novel therapeutic approaches are urgently needed for the treatment of this disease.

The terpenoid alcohol linalool is a natural substance that is biosynthesized by a host of plants, specifically herbs, spices, and fruits (4, 5) and is potentially cytotoxic and able to enhance immune responses (4). Moreover, linalool promotes doxorubicin influx in cancer cells, thus increasing the concentration and enhancing the antitumor activities of doxorubicin (6). However, its mechanism of action and therapeutic efficacy are not well understood in solid tumors, including EOC. Moreover, possible limitations to the use of linalool in human cancer research are its water-insolubility and the requirement for a relatively high dose to achieve effective anticancer effects. To overcome these limitations, effective drug-delivery systems for linalool are needed. Nanoparticles (NP) are an attractive and promising way to target tumor tissue by enhanced permeability and retention (EPR) effects that potentially lead to enhanced drug concentrations in target tissue while limiting access to normal tissues as well as an increased half-life of the drug in circulation after administration (7, 8).

In this study, we developed a novel linalool-incorporated nanoparticle (LIN-NP) system and evaluated its anticancer efficacy and the possible mechanism of action using in vitro and in vivo models of EOC.

Cell lines and culture conditions

The human EOC cell lines HeyA8, A2780, and SKOV3ip1 (A.K. Sood, MD Anderson Cancer Center, 2014) were maintained and propagated in RPMI 1640 supplemented with 10% fetal bovine serum and 0.1% gentamicin sulfate (Gemini Bioproducts; ref.9). The cell lines were routinely tested for the absence of Mycoplasma and virus infections for hepatitis, sendai, and vesivirus from Samsung Medical Center (Ref. No. 2014-1089R, South Korea). The cells were free of virus infection and inspected at 2014. The identity of the cell lines was confirmed using STR fingerprinting. All in vitro and in vivo experiments were conducted when cells reached 70% to 80% confluency.

Preparation of LIN-NPs

LIN-NPs were prepared using the self-nanoemulsifying drug-delivery system (SNEDDS) with polyoxyethylene sorbitan monooleate 20 or 80 (Tween 20 or Tween 80; refs.10, 11). Briefly, a predetermined amount of linalool was added to Tween 20 (4% v/v) or Tween 80 (4% v/v) solution, and the LIN-NPs were spontaneously formed with constant stirring at room temperature. Following incubation at 4°C for 40 minutes, LIN-NPs were isolated using a dialysis membrane (Thermo Biofuge) to remove unreacted Tween 20 or Tween 80 at 4°C for 24 hours. The LIN-NPs were stored at 4°C until further use. The linalool concentration in the LIN-NPs was determined by measuring the absorbance intensity using a UV spectrophotometer at 295 nm and final concentrations were calculated based on a standard curve of linalool. Size and zeta potential of LIN-NPs were measured by light scattering with a particle size analyzer and Zeta Plus (Brookhaven Instrument Co.), respectively. The physical morphology of the LIN-NPs was observed using transmission electron microscopy (TEM). The release of linalool from LIN-NPs and their stability were measured according to previously reported methods (12).

Cytotoxicity assay

The cytotoxic effects of LIN-NPs were determined by the MTT assay as described previously (13). Cells were plated in 96-well plates (3,000 cells/well for HeyA8 and 4,000 cells/well for SKOV3ip1 and A2780) in triplicate and incubated overnight at 37°C in a 5% CO2 incubator. Following incubation, cells were washed, placed in serum-free medium, and then treated with PBS (control) or LIN-NPs for 24, 48, or 72 hours. Each sample was assayed in triplicate and the experiment was repeated three times.

In vitro and in vivo apoptosis assays

The relative percentage of apoptotic cells was assessed using phycoerythrin (PE), annexin V, and 7-amino-actinomycin (7-AAD) staining (BD Biosciences), as previously described (14). Briefly, EOC cells (1×105 cells/mL) were pelleted, washed twice in PBS, and resuspended in binding buffer containing PE Annexin V and 7-AAD (5 μL per 105 cells). Samples were incubated in the dark for 15 minutes at room temperature before analysis by flow cytometry. In addition, we measured active caspase-3 activity in tumor tissue after in vivo tumor therapy using an ELISA assay kit (#KHO1091; Invitrogen). The tissues were lysed with lysis buffer, and apoptotic activity was determined according to the manufacturer's protocol.

ROS generation

To determine ROS generation after LIN-NP treatment, A2780, SKOV3ip1, and HeyA8 cells were incubated for 16 hours after addition of LIN-NPs. The cells were washed and loaded with 2 μmol/L CM-H2DCFDA in HBSS for 30 minutes at 37°C. The buffer was replaced with culture medium with or without 4 mmol/L LIN-NPs, and the cells were incubated for 30 minutes at 37°C. After treatment, the chamber slides were mounted onto a cover glass with Gel/Mount and images were obtained by fluorescence microscopy (15). To determine changes in intracellular ROS generation, tumor cells were stained with 2′, 7′-dichlorofluorescein-diacetate (DCFH-DA). Briefly, EOC cells were treated with 2 mmol/L or 4 mmol/L LIN-NPs for 24 hours. In addition, the cells in the 4 mmol/L LIN-NPs treatment group were pretreated with the ROS inhibitor N-acetylcysteine (NAC, 20 mmol/L) for 2 hours before LIN-NP treatment. After treatment, cells were incubated with 10 μmol/L DCFH-DA in the dark for 30 minutes and the cells were analyzed for DCF fluorescence by flow cytometry. The average data were obtained in triplicate of the experiment (n = 3).

Flow cytometric determination of mitochondrial membrane potential (ΔΨm)

Dissipation of ΔΨm is one of the most important mechanisms of apoptosis. As the mitochondrial membrane potential is reduced, mitochondrial permeability transition pores open, leading to release of cytochrome c and other proapoptotic molecules from the intermembrane space to the cytosol. Finally, caspase-3 is activated, leading to apoptosis (16). Therefore, to assess whether ΔΨm was affected after LIN-NP treatment, we measured ΔΨm in tumor cells by flow cytometry using rhodamine 123, which is a mitochondria-specific fluorescent dye. Briefly, the cells were treated with 2 mmol/L or 4 mmol/L LIN-NPs for 24 hours. The cells in the ROS inhibitor group were pretreated with 20 mmol/L NAC for 2 hours before LIN-NP treatment. After treatment, the cells were incubated with rhodamine 123 (10 μmol/L) at 37°C in the dark for 20 minutes and analyzed by flow cytometry. The average data were obtained in triplicate of the experiment (n = 3).

Western blot analysis

Preparation of cultured cell lysates has been previously described (9). Protein concentrations were determined using a BCA Protein Assay Reagent Kit (Pierce Biotech) and 20-μg aliquots of protein were subjected to gel electrophoresis on 7.5% or 10% SDS-PAGE gels. Transfer to membranes and immunoblotting was performed as described previously. Protein bands were probed with antibodies specific for total-p38, phospho-p38 (p-p38), total-Akt, phospho-Akt (p-Akt), total-JNK, phospho-JNK (p-JNK; 1:1,000 dilution; Cell Signaling Technology); caspases 3, 8, and 9 (1:1,000 dilution, Abcam); β-actin (1:3,000 dilution, Santa Cruz Biotechnology). Bands were visualized by enhanced chemiluminescence using an ECL kit (Amersham Biosciences) according to the manufacturer's protocol.

Orthotropic in vivo model of EOC and tissue processing

Female BALB/c nude mice were purchased from Orient Bio and maintained as previously described (17). This animal study was reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of Samsung Biomedical Research Institute (Ref. No. H-A9-003) and Konkuk University (Ref. No. KU13157), which is accredited by the Association for the Assessment and Accreditation of Laboratory Animal Care International (AAALAC International) and abides by the Institute of Laboratory Animal Resources (ILAR) guide. The mice used for in vivo experiments were 5 to 6 weeks old. To produce tumors, HeyA8 (5×105 cells per 0.2 ml HBSS) and SKOV3ip1 (1×106 cells per 0.2 mL HBSS) cells were injected into the peritoneal cavity of mice. Seven days after intraperitoneal (i.p.) injection of tumor cells, the mice were randomly allocated to the following groups (n = 10 mice/group): (i) control (PBS treatment), (ii) 50 mg/kg LIN-NP, and (iii) 100 mg/kg LIN-NP. LIN-NPs were administered i.p. twice weekly at a dose of 50 mg/kg or 100 mg/kg body weight. Mice were monitored daily for adverse effects of therapy and were euthanized when any of the mice appeared moribund (4 to 5 weeks after cell injection depending on the cell line). Mouse weight, tumor weight, number of nodules, and distribution of tumors in mice were recorded at the time of euthanasia. Tissue specimens were fixed with either formalin or optimum cutting temperature (Miles, Inc.) or were snap frozen.

Immunohistochemistry

Immunohistochemical (IHC) analysis was performed on tumor tissue from mice that were treated by i.p. injection of LIN-NPs. Procedures for IHC analysis of markers of cell proliferation (Ki67), microvessel density (MVD; CD31), and apoptosis (caspase-3) were performed as described previously (9). For the tumor tissue ROS generation assay, we used an OxiSelect In Vitro ROS/RNS assay kit (#STA-347; Cell Biolab). ROS generation was determined by a fluorescence-based assay according to the manufacturer's protocol, and the ROS level was determined by comparison with DCF standards (15). All data were recorded in five random fields for each slide at ×200 magnification and were quantified by two investigators in a blinded fashion.

Therapeutic efficacy of combination of LIN-NP and paclitaxel

The mice used for in vivo experiments were 5 to 6 weeks old. To produce tumors, SKOV3ip1 cells (1×106 cells per 0.2 mL HBSS) were injected into the peritoneal cavity of mice. Seven days after injection of tumor cells into the peritoneal cavity, the mice were randomly allocated to the following groups (n = 10 mice/group): (i) control (PBS treatment), (ii) 50 mg/kg LIN-NP, (iii) 6 mg/kg paclitaxel, and (iv) 50 mg/kg LIN-NP + 6 mg/kg paclitaxel. Treatment of both LIN-NPs and paclitaxel began 1 week after i.p. injection of tumor cells into mice. LIN-NPs were administered i.p. twice weekly at a dose of 50 mg/kg body weight. Paclitaxel was diluted in PBS and injected i.p. once a week at a dose of 6 mg/kg to confirm a combination synergistic effect. Treatment continued until any of the mice became moribund (typically for 4 to 5 weeks depending on the tumor cells).

Therapeutic efficacy of LIN-NPs in patient-derived xenograft (PDX) model

To establish a PDX model of EOC, surgical tumor specimens of the patient were sliced into small pieces (less than 2–3 mm), implanted into the subrenal capsule of the left kidneys of mice, and propagated by serial transplantation (17). Mice were randomly allocated to the following groups (n = 10 mice/group): (i) 6 mg/kg paclitaxel and (ii) 6 mg/kg paclitaxel + 50 mg/kg LIN-NP. LIN-NPs were administered i.p. twice weekly at a dose of 50 mg/kg body weight. Paclitaxel was diluted in PBS and injected i.p. once a week at a dose of 6 mg/kg. Treatment continued until the mice became moribund. The mean survival of our PDX models for EOC is 6.5 month.

Statistical analysis

The Mann–Whitney U test was used to evaluate significance and to compare differences among the groups for both in vitro and in vivo assays. All statistical tests were two-sided, and P values less than 0.05 were considered to be statistically significant. SPSS software (version 17.0; SPSS) was used for all statistical analyses.

Anticancer effects of natural linalool

We first tested the in vitro cytotoxicity of natural linalool against EOC cell lines using the MTT assay (Fig. 1A–C). Linalool treatment significantly decreased cell viability in all EOC cell lines. In addition, we performed in vivo experiments for natural linalool using a HeyA8 orthotopic mouse model. To produce tumors, HeyA8 cells (5×105 cells per 0.2 mL HBSS) were injected into the peritoneal cavity of mice. Seven days after injection of tumor cells into the peritoneal cavity, the mice were randomly allocated to the following groups (n = 10 mice/group): (i) control (PBS treatment), (ii) 300 mg/kg natural linalool, and (iii) 600 mg/kg natural linalool. Treatment began 1 week after i.p. injection of tumor cells into mice. LIN-NPs were administered i.p. 600 mg/kg natural linalool treatment significantly decreased tumor weight compared with control (P = 0.047; Fig. 1D). However, mouse weight at the end of treatment was significantly lower in the linalool treatment group compared with the control in a dose-dependent manner (Fig. 1E). These results suggested that natural linalool at a relatively high dose might have serious toxicity in mice; therefore, we aimed to develop a delivery system to increase therapeutic doses in the tumor and decrease unwanted side effects.

Figure 1.

Cytotoxicity of linalool in HeyA8 (A), SKOV3ip1 (B), and A2780 cells (C). D, therapeutic efficacy of linalool in the HeyA8 tumor model; *, P < 0.047. E, distribution of mouse weight after linalool treatment (control vs. 300 mg/kg linalool treatment; *, P = 0.004, control vs. 600 mg/kg linalool treatment; **, P < 0.001). Results, mean ± SD; n = 10 mice per group. Statistical tests were two-sided, and P values were evaluated using ANOVA.

Figure 1.

Cytotoxicity of linalool in HeyA8 (A), SKOV3ip1 (B), and A2780 cells (C). D, therapeutic efficacy of linalool in the HeyA8 tumor model; *, P < 0.047. E, distribution of mouse weight after linalool treatment (control vs. 300 mg/kg linalool treatment; *, P = 0.004, control vs. 600 mg/kg linalool treatment; **, P < 0.001). Results, mean ± SD; n = 10 mice per group. Statistical tests were two-sided, and P values were evaluated using ANOVA.

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Characteristics of LIN-NPs

We developed LIN-NPs by the self-nanoemulsifying drug delivery system (SNEDDS) using different emulsifying agents (Tween 20 or Tween 80). The size of the LIN-NPs was 246 nm for Tween 20 and 93 nm for Tween 80 (Fig. 2A); the histograms are shown in Fig. 2B. Additionally, the loading efficiency of linalool into the LIN-NPs was as high as 98%, as confirmed by UV-vis spectrometry (Fig. 2C). The morphology of the LIN-NPs was determined by TEM, which revealed a spherical shape (Fig. 2D). The release pattern of linalool from the LIN-NPs is shown in Fig. 2E. While initial size of Tween 20 LIN-NPs was increased, Tween 80 LIN-NPs were stable at 4°C (Fig. 2F). Therefore, we selected Tween 80 LIN-NPs for subsequent experiments.

Figure 2.

Physicochemical properties of LIN-NPs. Size (A) and size distribution (B) of LIN-NPs. C, loading efficiency of linalool into LIN-NPs. D, morphology of Tween 80 LIN-NPs as determined by TEM. Scale bars, 1 μm (top) and 200 nm (bottom). E, release of linalool from LIN-NPs at 37°C. F, stability of LIN-NPs at 4°C. Results, mean ± SD.

Figure 2.

Physicochemical properties of LIN-NPs. Size (A) and size distribution (B) of LIN-NPs. C, loading efficiency of linalool into LIN-NPs. D, morphology of Tween 80 LIN-NPs as determined by TEM. Scale bars, 1 μm (top) and 200 nm (bottom). E, release of linalool from LIN-NPs at 37°C. F, stability of LIN-NPs at 4°C. Results, mean ± SD.

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Effects on LIN-NPs on cell viability and apoptosis in EOC cells

We next assessed in vitro cell viability after LIN-NP treatment in EOC cells. LIN-NPs showed significant cytotoxicity in HeyA8, SKOV3ip1, and A2780 cells in a dose-dependent manner (Fig. 3A–C). Treatment with LIN-NPs (2 mmol/L) also induced a significant increase in apoptosis in these cells in a time-dependent manner (Fig. 3D–F). LIN-NP resulted in potent cell apoptosis and showed a uniform effect in EOC cell lines.

Figure 3.

Cytotoxicity of LIN-NPs in HeyA8 (A), SKOV3ip1 (B), and A2780 (C) tumor cells as determined by the MTT assay. Apoptosis assay of LIN-NPs (2 mmol/L) in HeyA8 (control vs. 72-hour treatment; *, P < 0.001; D), SKOV3ip1 (control vs. 72-hour treatment; *, P < 0.001; E), and A2780 (control vs. 72-hour treatment; *, P < 0.001; F) tumor cells. Results, mean ± SD.

Figure 3.

Cytotoxicity of LIN-NPs in HeyA8 (A), SKOV3ip1 (B), and A2780 (C) tumor cells as determined by the MTT assay. Apoptosis assay of LIN-NPs (2 mmol/L) in HeyA8 (control vs. 72-hour treatment; *, P < 0.001; D), SKOV3ip1 (control vs. 72-hour treatment; *, P < 0.001; E), and A2780 (control vs. 72-hour treatment; *, P < 0.001; F) tumor cells. Results, mean ± SD.

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Effect of LIN-NPs on ROS generation and mitochondrial membrane potential (ΔΨm) in EOC cells

Most essential oil components, and in particular linalool, can enhance apoptosis by increasing ROS generation (18). We found that LIN-NP treatment increased ROS generation in EOC cells compared with control (Fig. 4A). We also assessed the level of ROS generation after LIN-NP treatment by flow cytometry. LIN-NP–treated cells showed a significant increase in ROS generation compared with controls (Fig. 4B). Notably, the LIN-NP + NAC (ROS inhibitor) group showed weak generation of ROS after LIN-NP treatment (Fig. 4B). This result clearly demonstrated that LIN-NP treatment significantly increased ROS generation in the tumor cells. We next determined the effect of LIN-NPs on mitochondrial membrane potential (ΔΨm) in EOC cells. The levels of ΔΨm in cells treated with LIN-NPs were significantly decreased compared with the group with inhibition of ROS generation (LIN-NP + NAC), which indicated that ROS generation after LIN-NP treatment promoted disruption of ΔΨm (Fig. 4C).

Figure 4.

LIN-NP treatment induces ROS generation in EOC cell lines. A, ROS generation after LIN-NP treatment in EOC cell lines. Magnification ×100. B, ROS generation after LIN-NP treatment in EOC cell lines. Cells were treated with 2 mmol/L or 4 mmol/L LIN-NP in the presence or absence of 20 mmol/L NAC for 24 hours and then analyzed by flow cytometry (control vs. 4 mmol/L LIN-NP; *, P < 0.001). Data, mean ± SD (n = 3). C, mitochondrial membrane potential (ΔΨm) in EOC cell lines treated with LIN-NP. Cells were treated with LIN-NP at different concentrations in the presence or absence of 20 mmol/L NAC for 24 hours and analyzed by flow cytometry (control vs. 4 mmol/L LIN-NP; *, P < 0.001, n = 3). D, expression of proteins involved in the apoptotic pathway was analyzed by Western blot analysis. E, schematic diagram of the mechanism of action of LIN-NP via ROS-induced apoptosis.

Figure 4.

LIN-NP treatment induces ROS generation in EOC cell lines. A, ROS generation after LIN-NP treatment in EOC cell lines. Magnification ×100. B, ROS generation after LIN-NP treatment in EOC cell lines. Cells were treated with 2 mmol/L or 4 mmol/L LIN-NP in the presence or absence of 20 mmol/L NAC for 24 hours and then analyzed by flow cytometry (control vs. 4 mmol/L LIN-NP; *, P < 0.001). Data, mean ± SD (n = 3). C, mitochondrial membrane potential (ΔΨm) in EOC cell lines treated with LIN-NP. Cells were treated with LIN-NP at different concentrations in the presence or absence of 20 mmol/L NAC for 24 hours and analyzed by flow cytometry (control vs. 4 mmol/L LIN-NP; *, P < 0.001, n = 3). D, expression of proteins involved in the apoptotic pathway was analyzed by Western blot analysis. E, schematic diagram of the mechanism of action of LIN-NP via ROS-induced apoptosis.

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To confirm the effect of LIN-NP on apoptosis, we examined the expression of several known proapoptotic regulators by Western blot analysis. Expression levels of p-p38 and p-JNK were significantly increased in 1 hour and then decreased following LIN-NP treatment compared with control, whereas levels of p-Erk, caspase-9, and caspase-3 were significantly increased, suggesting that LIN-NP treatment induced an increase in tumor cell apoptosis through activation of caspase-3 (Fig. 4D). Consequently, LIN-NP treatment might increase cell apoptosis by decreasing the mitochondrial membrane potential and increasing caspase-3 levels after ROS generation in EOC cells (Fig. 4E).

Therapeutic efficacy of LIN-NPs in EOC cell line xenograft

To evaluate the effect on in vivo tumor growth, we performed therapy experiments with LIN-NPs using orthotopic HeyA8 or SKOV3ip1 EOC models. In the HeyA8 model, 100 mg/kg LIN-NP significantly decreased tumor weight compared with 50 mg/kg LIN-NP (62%, P < 0.001) and control (58%, P < 0.001; Fig. 5A). In the SKOV3ip1 model, 100 mg/kg LIN-NP significantly decreased tumor weight compared with 50 mg/kg LIN-NP (70%, P = 0.041) and control (74%, P = 0.017; Fig. 5B). There were no differences in the mean body weight of mice (Fig. 5A and B), feeding habits, or behavior between the groups, suggesting that there were no overt therapy-related toxicities (Supplementary Fig. S1). In addition, we measured ROS generation in the tumor tissue of LIN-NP–treated groups and found that 100 mg/kg LIN-NP significantly increased ROS generation in tumor tissues in both models compared with control or 50 mg/kg LIN-NP (Fig. 5A and B, right).

Figure 5.

Therapeutic efficacy of LIN-NPs against ovarian cancer growth. Treatment with LIN-NPs was started 1 week after intraperitoneal injection of HeyA8 (A) and SKOV3ip1 (B) tumor cells in mice. LIN-NPs were injected i.p. twice weekly at doses of 50 mg/kg and 100 mg/kg body weight. Treatment was continued until mice in any group became moribund. Tumor nodules and mouse weight were measured. ROS generation in tumor tissue after LIN-NP treatment was measured by a fluorescence-based assay (right). Immunohistochemistry for markers of cell proliferation (Ki67, magnification ×200), MVD (CD31), and apoptosis (caspase-3) were performed on HeyA8 (C) and SKOV3ip1 (D) tumor tissues following treatment with LIN-NPs (bar, 50 μm). All analyses were performed in five random fields for each slide. Results, mean ± SD; n = 10 mice per group. Statistical tests were two-sided, and P values were evaluated using ANOVA analysis.

Figure 5.

Therapeutic efficacy of LIN-NPs against ovarian cancer growth. Treatment with LIN-NPs was started 1 week after intraperitoneal injection of HeyA8 (A) and SKOV3ip1 (B) tumor cells in mice. LIN-NPs were injected i.p. twice weekly at doses of 50 mg/kg and 100 mg/kg body weight. Treatment was continued until mice in any group became moribund. Tumor nodules and mouse weight were measured. ROS generation in tumor tissue after LIN-NP treatment was measured by a fluorescence-based assay (right). Immunohistochemistry for markers of cell proliferation (Ki67, magnification ×200), MVD (CD31), and apoptosis (caspase-3) were performed on HeyA8 (C) and SKOV3ip1 (D) tumor tissues following treatment with LIN-NPs (bar, 50 μm). All analyses were performed in five random fields for each slide. Results, mean ± SD; n = 10 mice per group. Statistical tests were two-sided, and P values were evaluated using ANOVA analysis.

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To determine the potential mechanisms underlying the efficacy of LIN-NP therapy in tumor tissues, we examined tumor tissues for markers of tumor MVD (CD31), and cell apoptosis (caspase-3). In both models, treatment with 100 mg/kg LIN-NP resulted in significant inhibition of MVD compared with 50 mg/kg LIN-NP and control (Fig. 5C and D). The percentage of apoptotic cells in the 100 mg/kg LIN-NP group was significantly increased compared with the 50 mg/kg LIN-NP group and control (Fig. 5C and D).

Combination effects of LIN-NPs and paclitaxel in EOC cell line xenograft and PDX models

Because paclitaxel is one of the most effective chemotherapeutic agents for EOC, we assessed the combination effect with LIN-NPs in SKOV3ip1 xenografts. To check whether combination treatment has a synergistic or additive effect we selected 50 mg/kg LIN-NP treatment, which did not show any therapeutic effects compared with control (Fig. 5A and B). The combination of 50 mg/kg LIN-NP and 6 mg/kg paclitaxel significantly decreased tumor weight compared with control (80%, P < 0.001), 50 mg/kg LIN-NP (79%, P < 0.001), and paclitaxel alone (41%, P = 0.040; Fig. 6A). In addition, the combination significantly decreased MVD compared with the control (P < 0.001), LIN-NPs (P = 0.002), and paclitaxel (P = 0.047); and significantly increased apoptosis compared with the control (P < 0.001), LIN-NPs (P < 0.001), and paclitaxel (P = 0.002; Fig. 6B).

Figure 6.

Therapeutic efficacy of combination therapy with LIN-NPs and paclitaxel in SKOV3ip1 and PDX tumor models. Treatment was initiated 1 week after i.p. injection of tumor cells into SKOV3ip1-bearing mice. LIN-NPs were injected i.p. twice weekly at a dose of 50 mg/kg body weight. Paclitaxel was diluted in PBS and injected i.p. once a week at a dose of 6 mg/kg. Treatment was continued until mice in any group became moribund. A, therapeutic efficacy for combination of LIN-NPs and paclitaxel. B, immunohistochemistry analyses to evaluate cell proliferation (Ki67, magnification ×200; scale bar, 50 μm), MVD (CD31), and apoptosis (caspase-3) were performed on SKOV3ip1 tumor tissues following treatment with L-NPs and paclitaxel. All analyses were performed in five random fields for each slide. Results, mean ± SD; n = 10 mice per group. C, therapeutic efficacy of 50 mg/kg LIN-NP and 6 mg/kg paclitaxel in a PDX model that was developed by subrenal implantation of human ovarian cancer tissues (*, P < 0.02). D, representative images of PDX tumor after treatment.

Figure 6.

Therapeutic efficacy of combination therapy with LIN-NPs and paclitaxel in SKOV3ip1 and PDX tumor models. Treatment was initiated 1 week after i.p. injection of tumor cells into SKOV3ip1-bearing mice. LIN-NPs were injected i.p. twice weekly at a dose of 50 mg/kg body weight. Paclitaxel was diluted in PBS and injected i.p. once a week at a dose of 6 mg/kg. Treatment was continued until mice in any group became moribund. A, therapeutic efficacy for combination of LIN-NPs and paclitaxel. B, immunohistochemistry analyses to evaluate cell proliferation (Ki67, magnification ×200; scale bar, 50 μm), MVD (CD31), and apoptosis (caspase-3) were performed on SKOV3ip1 tumor tissues following treatment with L-NPs and paclitaxel. All analyses were performed in five random fields for each slide. Results, mean ± SD; n = 10 mice per group. C, therapeutic efficacy of 50 mg/kg LIN-NP and 6 mg/kg paclitaxel in a PDX model that was developed by subrenal implantation of human ovarian cancer tissues (*, P < 0.02). D, representative images of PDX tumor after treatment.

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PDX models are attractive preclinical animal models for drug development. We therefore developed a PDX model through subrenal implantation of human ovarian cancer tissues (17). In this study, we used a PDX model of a FIGO stage IIIC serous papillary adenocarcinoma, grade II. The patient was a 61-year-old woman who received primary debulking surgery followed by adjuvant chemotherapy with paclitaxel–carboplatin for 6 cycles, and recurrence was detected at the 8-month post-therapy follow-up. She is now undergoing second-line chemotherapy. We treated the EOC PDX model for 4 weeks starting 2 months after the subrenal implantation (passage 5). The combination of 50 mg/kg LIN-NP and 6 mg/kg paclitaxel showed significant inhibition of tumor growth compared with paclitaxel alone (80%, P < 0.001; Fig. 6C and D).

In this study, we developed a new linalool delivery method using a nanoparticle system prepared by a self-nanoemulsifying approach. The LIN-NPs had potent cytotoxic and apoptotic activities against EOC cells and robust antitumor effects in vivo without significant side effects. Moreover, a combination of LIN-NPs and paclitaxel significantly inhibited tumor growth compared with paclitaxel alone in cell lines and a PDX in vivo model of EOC. In addition, we found that LIN-NPs induced apoptosis in cancer cells by decreasing mitochondrial membrane potential and increasing caspase-3 levels after ROS generation.

The toxicities, side effects, and drug resistance associated with current cytotoxic chemotherapeutic agents have created a need for continued development of safer and more effective alternatives (6, 19). Some natural plant products can suppress cell proliferation, progression, migration, and invasion (20). The use of natural plant products with multiple medicinal effects may provide new and previously unrecognized opportunities. Linalool is a naturally occurring monoterpene alcohol with a pleasant odor that is present in more than 200 species of plants, including mint, laurel, cinnamon, and citrus fruits. Linalool is either a major or common compound of most herbal essential oils and is present in both green and black teas (4, 21). Although linalool is known to have cytotoxicity against cancer cells, its use in therapeutic applications has been limited by its water insolubility and possible toxic effects at higher doses. Moreover, the therapeutic mechanism of linalool as a chemotherapeutic agent has not been established.

In this study, we developed a novel systemic delivery platform for linalool treatment that could be beneficial in providing an enhanced therapeutic effect at the target site, increasing delivery efficiency, and ultimately increasing the therapeutic index. Nanoparticle systems are particularly attractive for clinical and biologic applications because they can carry an effective payload of the drug to the target disease site (20). Furthermore, because nanoparticle payloads are frequently located within the particles, their type and number may not affect their pharmacokinetics and biodistribution following administration. In addition, these nanoparticles have previously demonstrated significant efficacy against tumor models (22). Therefore, we developed LIN-NPs for systemic delivery in novel cancer therapeutic applications.

ROS generation has important physiological effects in normal cells, although enhanced production may lead to toxic effects. Several studies have shown that phytochemicals targeting ROS metabolism could selectively kill cancer cells because of the higher levels of endogenous active oxygen in tumor cells compared with normal cells. Excessive ROS generation can damage DNA in cancer cells, leading to cancer cell death and cell-cycle arrest and resulting in increased apoptosis (23, 24). In this study, we revealed the ability of LIN-NPs to increase ROS generation after treatment, which leads to increased cell apoptosis and ultimately triggers cell death in EOC cells.

No potential conflicts of interest were disclosed.

Conception and design: H.D. Han, S.K. Cho, D.-S. Bae, A.K. Sood, B.C. Shin, Y.-M. Park, J.-W. Lee

Development of methodology: S.K. Cho

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): Y.-J. Cho, S.K. Cho, B.-G. Kim

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): Y.-J. Cho, S.K. Cho, Y. Byeon, H.N. Jeon, B.-G. Kim, G. Lopez-Berestein, A.K. Sood

Writing, review, and/or revision of the manuscript: H.D. Han, Y.-J. Cho, G. Lopez-Berestein, A.K. Sood, B.C. Shin, J.-W. Lee

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): Y.-J. Cho

Study supervision: B.-G. Kim, D.-S. Bae, J.-W. Lee

Other (interpretation of effect of linalool on normal tissue of mice and taking photos): H.-S. Kim

Y.M. Park is supported by a National Research Foundation of Korea (NRF) grant funded by the Korean government (NRF-2012R1A2A1A03008433). H.D. Han is supported by a Basic Research Laboratory Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT & Future Planning (No. 2013R1A4A1069575). J.W. Lee is supported by a grant of the Korean Health Technology R&D project (HI14C1940) through the Korean Health Industry Development Institute (KHIDI; HI14C3418) funded by Ministry for Health and Welfare, Republic of Korea. J.W. Lee and H.D. Han are supported by a grant from the National R&D program for Cancer Control, Ministry for Health, Welfare and Family affairs, Republic of Korea (1520100).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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