Combination Treatment of Citral Potentiates the Efficacy of

Aug 25, 2017 - The results indicated that citral was an effective additive for HIPEC with pirarubicin for colorectal cancer, which warrant further eff...
0 downloads 0 Views 9MB Size
Download PDF
Article pubs.acs.org/molecularpharmaceutics

Combination Treatment of Citral Potentiates the Efficacy of Hyperthermic Intraperitoneal Chemoperfusion with Pirarubicin for Colorectal Cancer Zhiyuan Fang,†,‡,# Yu Wang,†,# Hao Li,†,# Shuaishuai Yu,§ Ziying Liu,† Zhichao Fan,† Xiaomin Chen,† Yuying Wu,† Xuebo Pan,*,† Xiaokun Li,*,† and Cong Wang*,† †

School of Pharmaceutical Sciences, Wenzhou Medical University, Wenzhou, Zhejiang 325030, China Key Laboratory of Regenerative Biology, South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510530, China § Department of Biology, School of Laboratory Medicine and Life Science, Wenzhou Medical University, Wenzhou, Zhejiang 325030, China ‡

ABSTRACT: Citral is a widely used penetration enhancer that has been used to assist the delivery of drugs through the skin. In this study we aimed to investigate the effectiveness of combination treatments of citral with hyperthermic intraperitoneal chemotherapy (HIPEC) for colorectal cancer and to unravel the underlying mechanism by which citral increased the efficacy of HIPEC. In vitro experiments indicated that citral increased cytoplasmic absorption of pirarubicin and potentiated the effects of pirarubicin on colorectal cancer cells to induce apoptosis. Intracellular reactive oxygen species (ROS) activity was elevated after single or combo treatments with pirarubicin, leading to compromised NF-κB signaling. Therefore, the results suggested that the effects of citral were mediated by increasing cell permeability and ROS productions. Furthermore, the colorectal xenograft model was used to evaluate the efficacy of the combo treatment at the histological and molecular levels, which showed that the cotreatment with citral for colorectal cancer increased the efficacy of HIPEC with pirarubicin with respect to both ascite control and tumor load. The results indicated that citral was an effective additive for HIPEC with pirarubicin for colorectal cancer, which warrant further effort to explore the translational application of this new treatment regimen. KEYWORDS: hyperthermic intraperitoneal chemoperfusion, citral, pirarubicin, apoptosis, colorectal carcinoma

1. INTRODUCTION Colorectal carcinoma (CRC) is a key public health issue accounting for the third most commonly diagnosed malignancy and the fourth leading cause of cancer-related death in the world.1 Conventional treatment for CRC is surgical resection combined with systemic chemotherapy, which is not effective for peritoneal metastasis of the cancer.2 Recently, cytoreductive surgery integrated with hyperthermic intraperitoneal chemotherapy (CRS-HIPEC) has been adopted to improve survival rates and quality of life in selected patients.3−5 Taking advantage of slow peritoneal clearance, and therefore, maintenance of a higher concentration of chemotherapeutic drug in the peritoneal cavity, HIPEC is able to eradicate microtumor nodules and micrometastases that cannot be resected by conventional surgery.6 However, patients with abdominal malignancies still had a high rate of recurrence even after the CRS-HIPEC regime because of the incomplete cytoreduction due to low penetration of the chemotherapeutic drugs. Therefore, an improved penetration of the drug is needed to increase the efficacy of HIPEC.7−10 © 2017 American Chemical Society

The delivery of chemotherapy drugs in HIPEC generally relies on the activity of penetration and diffusion of the drug. In intraperitoneal chemoperfusion, the depth of penetration normally is within 1.5 mm from the peritoneal surface, which is less than 3−5 mm predicted by a theoretical model.11,12 Penetration enhancers can be a promising additive to improve the efficacy of HIPEC through increasing the drug penetration by reducing the barrier resistance of cellular lipid and/or tight junctions without damaging cell viability.13,14 Currently, multiple penetration enhancers, including synthetic solvents, azones, pyrrolidones, and surfactants are widely used to improve the uptake of drugs into the circulation and subsequently increase the efficacy.15 Due to their lipophilic nature and low molecular weights, essential oils have been used as penetration enhancers that have Received: Revised: Accepted: Published: 3588

July 30, 2017 August 23, 2017 August 25, 2017 August 25, 2017 DOI: 10.1021/acs.molpharmaceut.7b00652 Mol. Pharmaceutics 2017, 14, 3588−3597

Article

Molecular Pharmaceutics

(BD Pharmingen, San Diego, CA). The labeled cells were detected with a BD FACSCalibur flow cytometer (BD Biosciences, San Jose, CA). Western blots were analyzed with anti-Bax, Bcl-2, and Cleaved caspase 3 and were also used to detect apoptotic cells. 2.5. Western Blotting Analysis. Cells were lysed with the RIPA buffer (50 mM Tris, 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 0.1 mM EDTA, 0.05 mM PMSF, 2 μg/mL leupeptin, pH 7.4) containing a protease inhibitor cocktail (Thermo Scientific, Rockford, IL). The concentration of total protein was determined by BCA assay. The lysates were separated by SDS-PAGE and electrically blotted onto PVDF membranes. Rabbit anti-ERK (1:1000) and anti-GAPDH (1:1000) were purchased from Santa Cruz (Santa Cruz, CA). Rabbit anti-pERK (1:2000), anti-Bcl-2 (1:1000), anti-AKT (1:1000), anti-Bax (1:1000), anti-Cleaved-caspase-3 (1:1000), anti-pAKT (1:1000), anti-p65 (1:1000), anti-p-p65 (1:1000), anti-Ikkα/β (1:1,000), anti-p-Ikkα/β (1:1000), and anti-pStat3 (1:500) were purchased from Cell Signaling Technology (Beverly, MA). Specific bands recognized by aforementioned antibodies were visualized with the ECL Substrate Kit (Pierce, Rockford, IL). The intensity was quantified using the NIH ImageJ software (http://rsb.info. inh.gov/ij). 2.6. Transmission Electron Microscopy Analysis. Cells were fixed in 2.5% glutaraldehyde buffer at 4 °C overnight. After fixation, the cells were collected by centrifugation (1400g for 5 min), washed to remove residual fixative, and then placed in osmium tetroxide for 1 h before a series of acetone dehydration. Capsule infiltration was carried out using 50% acetone/50% resin solution and 100% resin for embedding. After preparing semithin plastic sections, methylene blue-azure II/basic fuchsin was used for nonen-bloc stained specimens. Cell organelles were visualized with a Hitachi transmission electron microscope. 2.7. Quantification of Reactive Oxygen Species (ROS). Cells (1 × 106 cells/well) were seeded in six-well plates and treated with citral (300 μM), pirarubicin (25 μM), combination or solvent control at 43 °C for 1 h. After the indicated time, cells were harvested and resuspended in 20 μM DCFH-DA. After incubation at 37 °C for 30 min, the production of reactive oxygen species (ROS) in samples with 50,000 cells was detected with a BD FACSCalibur flow cytometer (BD Biosciences, San Jose, CA). Fluorescence intensity ratio (RIF) was presented as the mean ± SD. Dihydroethidium (DHE) was added to the medium at a final concentration of 5 μM. After incubation at 37 °C for 30 min, the cells were washed with PBS for three times before observation. Red fluorescence was recorded by a confocal microscope (Zeiss LSM 510). 2.8. Chemotherapeutic Drug Treatment. For the colorectal peritoneal carcinomatosis (CRPC) experiments, mouse-derived colorectal cancer cells CT26 (5 × 106 cells in 0.5 mL of serum-free medium) were intraperitoneal inoculated into the flanks of Balb/C mice. Five days after the inoculation, these mice were randomly divided into four groups (n = 10). Mice in each group received HIPEC treatments with control, citral (300 μM), pirarubicin (25 μM), and combination (300 μM citral + 25 μM pirarubicin), respectively, for 1 h. To administer chemotherapeutic drugs, the mice were anesthetized with 1% pentobarbital sodium (15 mg/kg). Inflow and outflow tubes were inserted into the peritoneal cavity using two 15 G needles. Inflow needle was placed at the upper abdomen, and outflow needle was placed at the lower abdomen. The tubules

the ability to alter the structure of phospholipid bilayer and increase the fluidity and leakage of plasma membrane.16 Citral (3,7-dimethyl-2,6-octadienal) is a natural occurring terpenoid compound in essential oils. It has been extensively used to assist the delivery of drugs through skin.16−18 More interestingly, it has been recently shown that citral can induce apoptosis and autophagy in a caspase 3, p53, and Bcl-2 dependent manner and therefore has antitumor activity.19−22 These two distinct properties make citral an ideal enhancer for the HIPEC therapy. In this report, we demonstrated that citral was an effective additive for HIPEC in the treatment of colorectal cancer, which promoted the penetration of pirarubicin and inhibited NF-κB signaling and other prosurvival pathways.

2. EXPERIMENTAL SECTION 2.1. Animals. Female BALB/c mice (5 weeks old, 15 g/ mouse) were obtained from Shanghai SLAC Laboratory Animal Limited Liability Company (Shanghai, China) and were maintained in randomly assigned cohorts in the pathogen-free vivarium with temperature-controlled, light-cycled rooms of Wenzhou Medical University. All animal procedures were approved by the Institutional Animal Care and Use Committee of the Wenzhou Medical University. 2.2. Cell Viability Assay. HCT-116 colorectal cancer cells were purchased from American Type Culture Collection (ATCC). The cells were cultured in 1640 medium (Gibco, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS, Gibco, Carlsbad, CA, USA), 100 U of penicillin, and 100 μg/mL streptomycin in 5% CO2 incubators. No bacteria were observed under a microscope. No mycoplasma was detected with the mycoplasma detection kit. For cell viability assay, the cells were seeded at a density of 2 × 104 cells/well in 96-well plates and cultured overnight. After adding citral (Aladin, Shanghai, China) alone (0, 100, 300, and 600 μM) or combined with pirarubicin (5, 15, and 25 μM) to the culture media, the cells were transferred to 37 or 43 °C incubators for 1 h. After replacing the medium with fresh culture medium, the cells were cultured at 37 °C for 5 or 11 h. The MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) solution (10 μL of 2.5 mg/mL in PBS) was then added to each well followed by 4 h incubation following manufacturer’s instruction. The formazan produced in viable cells was measured with a plate-reader (BenchMark, Bio-Rad, CA) at 570 nm. All experiments were repeated at least three times, and the inhibition rate were calculated using SPSS version 21.0 (SPSS Inc., Chicago, IL). 2.3. Muscular Permeation Assay. Quadriceps muscles dissected from 6-week-old female BALB/c mice were randomly divided into four groups (six pieces per one group) for penetrating with citral (300 μM), pirarubicin (25 μM), combination (300 μM citral + 25 μM pirarubicin), and solvent control. All samples were kept in a 43 °C water bath incubator for 1 h. After the incubation, the muscles were frozen with dry ice and cryosectioned at 5 μm thickness for observation under a fluorescence microscope. 2.4. Apoptosis Assays. Cells (1 × 106 cells/well) were seeded in six-well plates and cultured overnight at 37 °C. Citral (300 μM), pirarubicin (25 μM), combination (300 μM citral + 25 μM pirarubicin), and solvent control were added to each well followed by hyperthermic treatment at 43 °C for 1 h. The medium was then replaced with fresh culture medium. The cells were then cultured at 37 °C for another 5 h. Apoptosis was analyzed with the FITC-Annexin V Apoptosis Detection kit 3589

DOI: 10.1021/acs.molpharmaceut.7b00652 Mol. Pharmaceutics 2017, 14, 3588−3597

Article

Molecular Pharmaceutics were connected to a water bag incubated in a 43 °C water bath. The “tubule-bag system” is a closed circuit driven by a peristaltic pump at 3 mL/min. A total of 5 mL of chemotherapy solution was used in the circulation, and the HIPEC was carried out for 60 min. All mice were recovered on a warming blanket. The mice were subcutaneously injected with butorphanol (0.1 mg/kg) for postoperation pain control. For the survival analysis, mice were housed as previously described after the HIPEC treatment. The general status of animals was recorded daily, and the body weight of each animal was documented every 4 days. Postmortem pathological examinations were carried for each mouse, including tumor size, tumor distributions, and ascites. 2.9. Statistical Analyses. Kaplan−Meier survival was analyzed using the log-rank test by SPSS software version 21.0 (SPSS Inc., IL, USA). Categorized variables were compared by chi square test (χ2) or Fisher’s exact test. Twosided p < 0.05 was considered statistically significant.

3. RESULTS 3.1. Citral Increases Cytotoxicity of Pirarubicin in Coleractal Cancer Cells under the Hyperthermal Condition. Since the activity of pirarubicin was more potent at 43 °C than at 37 °C,23 the effect of citral on promoting the antitumor activity of pirarubicin was tested at 37 and 43 °C for 6 and 12 h, separately (Figure 1A,B). Citral and pirarubicin alone or combination showed substantial cytotoxicity in colorectal cancer cells. At low concentrations, although no synergy effect was observed within 6 h after the treatment (Figure 1Aa), significant synergy was observed at 12 hours after the treatment (Figure 1Ba). The inhibition rate was 50% when cells were treated with 25 μM pirarubicin and 300 μM citral at 43 °C (Figure 1Ab,Bb). The inhibition rate of 300 μM citral was about 60% higher than that of 100 μM citral. However, at 43 °C, there was no statistical difference between the three groups with 600 μM citral in combination with 5, 15, or 25 μM pira (Figure 1Ab,Bb). This was likely due to the dominant effect of citral at such high concentration. In addition, the inhibition rates were independent of pirarubicin concentrations (Figure 1Ac,Bc). Together, the results showed that hyperthermia enhanced the cytotoxicity of pirarubicin and that the cytotoxicity was positively correlated with the concentration of citral and pirarubicin. 3.2. Citral Promotes the Penetration of Pirarubicin into Colorectal Cancer Cells. The penetration of pirarubicin was tested at 43 °C. As shown in Figure 2A, HCT-116 cells maintained normal morphology at 43 °C, indicating that the hyperthermia treatment did not affect the viability of the cells. However, treating with either pirarubicin (25 μM) and citral (300 μM) alone or the combination of the two drugs at the same concentrations induced shrinkage and detachment of cells. The majority of cells floated in the medium after the combination treatment, which was also quantitated (Figure 2A). The intensity of fluorescence in cells indicated the intracellular concentration of pirarubicin. It was clear that the influx of pirarubicin was enhanced by citral in a dose-dependent manner (Figure 2B). However, there was no significant increase of pirarubicin penetration when the concentration of citral was elevated from 300 to 600 μM, which was due to a high level of citral destroying the cell membrane, resulting in leaks of pirarubicin from the cells. In addition, citral also significantly improved the penetration of pirarubicin from 45.7 ± 4 to 83 ± 14.2 μm in muscle (P = 0.012). This suggested that muscle is

Figure 1. Citral potentiates the growth inhibition activity of pirarubicin in colorectal cancer cells under hyperthermal condition. HCT-116 cells were seeded in 96-well plates and cultured overnight. After adding citral alone or combined with pirarubicin to the culture media, the cells were transferred to 37 or 43 °C incubators for 1 h. After replacing the medium with fresh culture medium, the cells were cultured at 37 °C for another 5 or 11 h. (A) Cytotoxicity in HCT-116 cells after 6 h treatment. Citral increased the inhibition rate of pirarubicin at 43 °C than 37 °C (a). The inhibition rate was upregulated in dose dependently on citral and pirarubicin at 43 °C (b,c). (B) Cytotoxicity in HCT116 cells after 12 h treatment. The inhibition rate was strongly raised at 43 °C (a). The inhibition rate was upregulated in dose dependently on citral and pirarubicin at 43 °C (b,c).

an ideal model for assessing drug penetration (Figure 2C). Therefore, the dosages of 300 μM citral and 25 μM pirarubicin were selected for subsequent study. 3.3. Citral Enhances Pirarubicin To Induce Apoptosis in Colorectal Cancer Cells. To determine whether the cytotoxic effect of pirarubicin enhanced by citral was associated with apoptosis, the FITC-Annexin V based apoptosis analysis was used to label early apoptosis cells. As shown in Figure 3A, 300 μM citral and 25 μM pirarubicin increased apoptotic cells from 7.45% to 11.7% and 15.1%, respectively. The apoptotic cells reached to 18.7% after the combo treatment of pirarubicin and citral. The ultrastructure of the cells revealed by transmission electron microscopy further confirmed that the combo treatment induced apoptosis (Figure 3B). Pro-apoptosis events, such as chromatin condensation and sporadic cavitations were obvious after single treatment with citral or pirarubicin. The combo treatment induced cells undergo apoptosis as revealed by the disaggregated nucleolus and large amount of apoptotic bodies. Western blot analyses 3590

DOI: 10.1021/acs.molpharmaceut.7b00652 Mol. Pharmaceutics 2017, 14, 3588−3597

Article

Molecular Pharmaceutics

Figure 2. Intracellular concentration of pirarubicin is increased by citral treatment. HCT-116 cells treated with citral alone or combined with pirarubicin were incubated at 43 °C for 1 h. After replacing the medium with fresh culture medium, the cells were cultured at 37 °C for another 5 h (A,B). (A) Majority of cells floated in the medium after the combination treatment, which was quantitated in figure Ae: (a) control, (b) citral, (c) pirarubicin, and (d) combination. (B) Cells photographed under the fluorescence microscopy indicated that the intensity of fluorescence was elevated in dose dependently on pirarubicin combined with citral treatment. (C) Quadriceps muscles penetrated with citral, pirarubicin, combination, and solvent control were kept in a 43 °C water bath incubator for 1 h. After the incubation, the muscles were frozen and cryosectioned for observation under a fluorescence microscope: (a) control, (b) citral, (c) pirarubicin, and (d) combination.

DHE, although no significant differences were observed between the pirarubicin and the combo groups (Figure 5B). The results further demonstrate the role of citral in boosting the activity of chemotherapeutic drugs with respect to inducing oxidative stress and cell death. 3.6. Improved HIPEC with Pirarubicin by Citral Coadministration on Xenografts Derived from Colorectal Cancer Cells. HCT116 are human originated cells. It is difficult to establish xenografts from this cell line. Therefore, we chose mouse-derived colorectal cancer cells CT26 for the CRPC experiments. Since in vitro experiments indicated that the efficacy of the drug at 43 °C was better that at 37 °C, we decided to test the treatment under the hyperthermia condition. To evaluate the efficacy of the combo treatment with pirarubicin and citral, mice bearing CRPC were divided into four groups with 10 mice per group for the following treatments: control, citral, pirarubicin, and combo treatments. The treatments were started at day five after the implantations. Five days after the treatment, tumor nodules were detectable in control mice by palpation. The averaged body weight of the mice before HIPEC treatment was 16.3 ± 1.5 g. At day 30 after the tumor implantations, only two mice survived in the control group (median survival time: 18 ± 2.1 day), two mice survived in the citral group (median survival time: 22.7 ± 2.1 day), six mice survived in the pirarubicin group (median survival time: 26.8 ± 1.7 day), and eight mice survived in the combo group (median survival 29.4 ± 1.3 day). The autopsy examination revealed that the citral group had smaller tumor mass than the control group (Figure 6A). Averaged body weights of the mice in the four groups were 19.1 ± 1.6 g (control), 19.3 ± 2.5 g (citral), 19.4 ± 1.4 g (pira), and 15.5 ± 1 g (citral + pira), respectively (Figure 6B). The pirarubicin group only had small tumor nodules. The combo group had no obvious tumor nodule in most mice. Consistent with the tumor load, the volume of ascites was significantly reduced in the

confirmed that both single and combo treatments decreased expression of antiapoptotic molecules (Figure 3C). 3.4. Citral Enhances Pirarubicin To Suppress Inflammatory Signaling in Colorectal Cancer Cells. To further explore mechanisms underlying the citral enhanced cytotoxic effect of pirarubicin, the expression and activation of multiple cell signaling pathways related to inflammation were analyzed. The results revealed that the NF-κB pathway was inactivated after the treatment. Either single or combination treatment downregulated the expression of Ikkα and Ikkβ (Figure 4A). In addition, the phosphorylation of Akt, Stat-3, and ERK was also suppressed by the combination treatment (Figure 4A). However, only the combination of citral and pirarubicin inhibited the phosphorylation of Ikkα/β. Although the expression of p65 was not affected by all the treatment, the nuclear localization of phosphorylated p65 was compromised in the double treated groups (Figure 4B). The result suggested that the combination at the hyperthermia condition was able to inhibit the NF-κB pathway. 3.5. Citral Treatment Elevates ROS Production in Colorectal Cancer Cells. To access the ROS production after drug administration, dichloro-dihydro-fluorescein diacetate (DCFH-DA) and dihydroethidium (DHE) staining were used to detect the superoxide in colorectal cancer cells. Although DCFH-DA is not a specific indicator for ROS levels, it has been routinely used for measuring intracellular H2O2 and, therefore, redox contents.24 DHE stains superoxida, peroxide, and peroxynitrite.25 Therefore, the two methods were used to measure ROS in this study. As shown in Figure 5A with DCFH-DA staining by flow cytometry, single treatment with either citral or pirarubicin only increased the average fluorescence intensity in the cells to 9.7% and 13.1%, respectively. However, the combo treatment with citral and pirarubicin increased the ROS level to 43.6% (Figure 5A). Similar results were also observed by fluorescent staining of 3591

DOI: 10.1021/acs.molpharmaceut.7b00652 Mol. Pharmaceutics 2017, 14, 3588−3597

Article

Molecular Pharmaceutics

Figure 3. Citral promotes the pirarubicin-induced apoptosis in a dose-dependent manner in colorectal cancer cells. HCT-116 cells treated with different administration were incubated at 43 °C for 1 h. After replacing the medium with fresh culture medium, the cells were cultured at 37 °C for 11 h. (A) Apoptosis was analyzed with the FITC-Annexin V apoptosis detection kit by FACS: (a) control, (b) citral, (c) pirarubicin, and (d) combination. (B) Ultrastructure was analyzed by a transparent electrical microscope. Citral obviously increased the nuclear rupture stimulated by pirarubicin (d), which was more severe than citral (b) and pirarubicin (c) single groups. (C) Cells were lysed and expression of apoptotic related protein Bcl-2, Bax, and Cleaved-caspase 3 was detected by Western blotting: (a) control, (b) citral, (c) pirarubicin, and (d) combination.

4. DISCUSSION Cytoreductive surgery performed along with perioperative HIPEC has been considered as a new therapeutic modality to treat CRPC, which benefits in preventing residual tumor cells to be trapped in the postoperative fibrin adhesions.26 Importantly, a great benefit has been obtained by combining one adjuvant therapeutic chemo-drug with a golden standard regimen, such as doxorubicin, cis-platinum, or paclitaxel. In the recent decades, more and more natural-derived compounds were found to posses effective anticancer activity,27,28 which can be a good alterative adjuvant in combination with standardized cancer drugs. In this study, we showed that citral greatly benefits pirarubicin based HIPEC both in vitro and in vivo. The potentiated cytotoxic effect was due to its activity to increase cell permeability and therefore facilitates penetration of anticancer drugs. High concentration of citral can disorganize the highly ordered cell membrane and lead to leaking of the plasma

combo group (Figure 6C). Furthermore, the weight of tumor in the combo group was lower than those in the individual treatments (Figure 6D). Statistical analyses revealed that the combo HIPEC showed great survival benefit compared with single citral alone (p < 0.01), although the median survival of the combo group was not statistically different from that of pirarubicin alone (p = 0.331). Furthermore, seven mice in the control group developed metastasis, and four mice in the citral group, four mice in the pirarubicin group, and two mice in the combo group developed ascites (Figure 6E). As shown in Figure 7, facilitated by citral, pirarubicin penetrated through basal lamina and cell membranes, where it induces apoptosis of tumor cells. This finding indicates the addition of citral to HIPEC with pirarubicin not only increasing potency to suppress tumor growth but also inhibiting tumor dissemination. Together, it brings notable survival benefit for the tumorbearing mice. 3592

DOI: 10.1021/acs.molpharmaceut.7b00652 Mol. Pharmaceutics 2017, 14, 3588−3597

Article

Molecular Pharmaceutics

Figure 4. Downregulation of NF-κB signal pathway after the combination treatment. Citral alone or combined with pirarubicin was added to each well followed by hyperthermic treatment at 43 °C for 1 h. After replacing the medium with fresh culture medium, the cells were cultured at 37 °C for 11 h. (A) Cells were lysed with RIPA for Western blot detecting. Pirarubicin combined with citral inactivated the NF-κB pathway. Either single or combination treatment downregulated the expression of Ikkα and Ikkβ. The combination of citral and pirarubicin inhibited the phosphorylation of Ikkα/β and p-65. (B) Nuclear and cytoplasma protein were separated for Western blotting. Citral-supplied hyperthermal chemotherapy was able to inhibit the phosphorylation of Akt, Stat-3, and ERK.

membrane of the cell. Pirarubicin is a pro-apoptotic agent, and the combination of citral significantly promoted tumor cell apoptosis. The ROS has been reported to modulate p-AKT and NF-κB signaling pathways, which changes expression of Bcl-2/ Bax and leads to the cell nuclear break down ultimately. Many essential oils have been reported to induce tumor cell death by similar mechanisms.29 The ROS generation disrupts mitochondrial transmembrane potential (ΔΨm), increases the release of cytochrome c to the cytosol, and activates tumor suppressor genes, including p53, Akt, NF-κB, AP-1, and MAPK.29−31 ROS induced by hydrogen peroxide under hyperthermic condition has been reported to decrease tumor cells growth in vivo.32 Therefore, the beneficial activities of citral likely come from two aspects. On the one hand, citral helps pirarubicin to penetrate tumor cells, which not only increases the intracellular drug concentration but also facilitates pirarubicin to reach tumors barricaded by tissues. On the other hand, an increase of ROS production caused by citral also enhances the pirarubicin mediated apoptosis through blocking Akt, NF-κB, and ERK signal pathways. Besides, citral and ROS are also able to kill noncycling cells, which is a good complementation to traditional chemo-drugs that target cycling cells. After intraarterial hepatic (i.a.h) administration, pirarubicin reaches a higher tumor concentration in the rabbit VX2 tumor model even at a lower systemic exposure, suggesting that it has a pharmacokinetic advantage.33 Pirarubicin followed three similar plasma concentration curves, which could be fitted by a two-compartment model with successive half-lives of 22.0 min and 12.7 h. Total plasma clearance of the drug was 90 L/h/m2 and total volume of distribution was 1380 L/m2.34 Multiple pharmacological activities of citral have been documented, including anti-inflammatory35 and anticancer activities.21,22 The anticancer activity has been demonstrated

Figure 5. Citral elevates ROS production in colorectal cancer cells. Cells treated with citral (300 μM), pirarubicin (25 μM), or combination at 43 °C for 1 h were stained with DCFH-DA (A) and DHE (B). (A) Cells were harvested and resuspended in 20 μM DCFH-DA incubating at 37 °C for 30 min. The production of reactive oxygen species (ROS) was detected with a flow cytometer. (a) Control, (b) citral, (c) pirarubicin, and (d) combination. (B) Dihydroethidium (DHE) was added to the medium at a final concentration of 5 μM for 30 min incubation at 37 °C before observation under confocal microscopy: (a) control, (b) citral, (c) pirarubicin, and (d) combination.

in multiple cancer models, including leukemic, breast cancer, and lymphoma cells.19,36−38 Some of these effects are attributed to their lipophilic nature and low molecular weights of essential oils that allow them to cross cell membranes, alter the phospholipid layers, increase the membrane fluidity, and lead to leakage of the cell membrane.16 The increased cell membrane fluidity can cause a disruption of the mitochondrial transmembrane potential (ΔΨm), which increases the release of cytochrome c to the cytosol and induces apoptosis by a Bcl-2 family-dependent manner.30 In addition, essential oils also act as pro- or antioxidants, affecting the cellular redox state,39,40 which ultimately changes intracellular ROS level. In this study, we also demonstrated that the peritoneal carcinomatosis was increased by citral significantly. In the control group, all mice 3593

DOI: 10.1021/acs.molpharmaceut.7b00652 Mol. Pharmaceutics 2017, 14, 3588−3597

Article

Molecular Pharmaceutics

Figure 6. Improved HIPEC with pirarubicin by citral coadministration for colorectal carcinoma in mice. CT26 cells were intraperitoneally inoculated into the flanks of Balb/C mice for CRPC model. Five days after the inoculation, mice received HIPEC treatments with control, citral (300 μM), pirarubicin (25 μM), and combination (300 μM citral + 25 μM pirarubicin), respectively, for 1 h. At day 30 after the tumor implantations, mice were sacrificed for further analysis. (A) Peritoneal carcinomatosis of mice after different treatments. (B) Body weight of mice before sacrifice. (C) Volume of ascites in abdominal cavity in different groups. (D) Weight of tumors after different treatments. (E) Kaplan−Meier survival curve after HIPEC procedure (p = 0.003, comb vs control, p = 0.011, comb vs citral, p = 0.331, comb vs pira): (a) control, (b) citral, (c) pirarubicin, and (d) combination.

the mice developed tumors in the group treated with combo. Besides the significant survival benefit, the citral based regime has also been proved to be effective in ascite control in the

developed tumors after the implantation of the CRC cells. In the group treated with pirarubicin or citral alone, about 70 and 80% of mice developed tumors. However, only about 30% of 3594

DOI: 10.1021/acs.molpharmaceut.7b00652 Mol. Pharmaceutics 2017, 14, 3588−3597

Article

Molecular Pharmaceutics

Figure 7. Schematic of citral induced influx of pirarubicin. The orderly lipid bilayer is disturbed by the insertion of citral, creating a crevice for the quick pass of pirarubicin. Pirarubicin permeates from peritoneal cavity through basal lamina to other tissues and cells following the pirarubicin concentration gradient. The increased intracellular pirarubicin potentiates apoptosis of tumor cells.

Notes

present study. Considering the good biocompatibility of citral, its usage will not only reduce the dosage of chemo drug but also bring out a better outcome of the treatment. In conclusion, the present study showed feasibility and effectiveness of citral additive in HIPEC for the treatment of colorectal peritoneal carcinomatosis. Since incomplete eradication of remnant tumor cells by HIPEC is the most serious limitation in the current management of peritoneal metastasis, this new modality offers several advantages over traditional HIPEC in that it (i) enhances penetration of chemo-drug through tissue and eradicates remnant tumor nodules, (ii) increases intracellular concentration of anticancer drugs, (iii) induces the generation of endogenous ROS and is effective for noncycling tumor cells, and (iv) is safe to combine with other chemo-drugs with less side effects. Nevertheless, more data are necessary to determine the morbidity, mortality, and long-term oncological outcome of this approach.

■

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS This work was supported by the Natural Science Foundation of Zhejiang Province of China (LY16H140004), the National Natural Science Foundation of China (31371470, 81270761, 81602246) to C.W., the Natural Science Foundation of Ningbo City (2015A610198), the National Natural Science Foundation of China (81602608) to Z.Y.F., and the Talent project foundation of Wenzhou Medical University (QTJ14030).

■

ABBREVIATIONS MTT, thiazolyl blue; BCA, bicinchonininc acid; PVDF, polyvinylidene fluoride; ECL, enhanced chemiluminescence; HIPEC, hyperthermic intraperitoneal chemotherapy; ROS, reactive oxygen species; DCFH-DA, 2′,7′-dichlorofluorescin diacetate; DHE, dihydroethidium; PMSF, phenylmethanesulfonyl fluoride

AUTHOR INFORMATION

■

Corresponding Authors

*Tel: 0577-86597330. Fax: 0577-86699350. E-mail: cwang@ wmu.edu.cn. *E-mail: [email protected]. *E-mail: [email protected].

REFERENCES

(1) Torre, L. A.; Bray, F.; Siegel, R. L.; Ferlay, J.; Lortet-Tieulent, J.; Jemal, A. Global cancer statistics, 2012. Ca-Cancer J. Clin. 2015, 65 (2), 87−108. (2) Jayne, D. G.; Fook, S.; Loi, C.; Seow-Choen, F. Peritoneal carcinomatosis from colorectal cancer. Br. J. Surg. 2002, 89 (12), 1545−50. (3) Kuijpers, A. M.; Mirck, B.; Aalbers, A. G.; Nienhuijs, S. W.; de Hingh, I. H.; Wiezer, M. J.; van Ramshorst, B.; van Ginkel, R. J.; Havenga, K.; Bremers, A. J.; de Wilt, J. H.; Te Velde, E. A.; Verwaal, V. J. Cytoreduction and HIPEC in the Netherlands: nationwide longterm outcome following the Dutch protocol. Ann. Surg Oncol 2013, 20 (13), 4224−30.

ORCID

Cong Wang: 0000-0002-2184-6879 Author Contributions

# These authors contributed equally to the manuscript. Z.F., C.W., and X.L. designed the experiments; Y.W., H.L., X.P., S.Y., Z.L., Z.F., and X.C. performed the cell biology experiments; Y.W., H.L., and Y.S. performed the in vivo experiments; and X.P. and C.W. wrote the paper.

3595

DOI: 10.1021/acs.molpharmaceut.7b00652 Mol. Pharmaceutics 2017, 14, 3588−3597

Article

Molecular Pharmaceutics (4) Glehen, O.; Gilly, F. N.; Boutitie, F.; Bereder, J. M.; Quenet, F.; Sideris, L.; Mansvelt, B.; Lorimier, G.; Msika, S.; Elias, D. Toward curative treatment of peritoneal carcinomatosis from nonovarian origin by cytoreductive surgery combined with perioperative intraperitoneal chemotherapy: a multi-institutional study of 1,290 patients. Cancer 2010, 116 (24), 5608−18. (5) Riss, S.; Mohamed, F.; Dayal, S.; Cecil, T.; Stift, A.; BachleitnerHofmann, T.; Moran, B. Peritoneal metastases from colorectal cancer: patient selection for cytoreductive surgery and hyperthermic intraperitoneal chemotherapy. Eur. J. Surg Oncol 2013, 39 (9), 931−7. (6) Flessner, M. F. The transport barrier in intraperitoneal therapy. Am. J. Physiol Renal Physiol 2005, 288 (3), F433−42. (7) Simkens, G. A.; van Oudheusden, T. R.; Luyer, M. D.; Nienhuijs, S. W.; Nieuwenhuijzen, G. A.; Rutten, H. J.; de Hingh, I. H. Serious Postoperative Complications Affect Early Recurrence After Cytoreductive Surgery and HIPEC for Colorectal Peritoneal Carcinomatosis. Annals of surgical oncology 2015, 22 (8), 2656−62. (8) Konigsrainer, I.; Horvath, P.; Struller, F.; Forkl, V.; Konigsrainer, A.; Beckert, S. Risk factors for recurrence following complete cytoreductive surgery and HIPEC in colorectal cancer-derived peritoneal surface malignancies. Langenbecks Arch Surg 2013, 398 (5), 745−9. (9) Coccolini, F.; Catena, F.; Glehen, O.; Yonemura, Y.; Sugarbaker, P. H.; Piso, P.; Montori, G.; Ansaloni, L. Complete versus incomplete cytoreduction in peritoneal carcinosis from gastric cancer, with consideration to PCI cut-off. Systematic review and meta-analysis. Eur. J. Surg Oncol 2015, 41 (7), 911−9. (10) Ahmed, S.; Levine, E. A.; Randle, R. W.; Swett, K. R.; Shen, P.; Stewart, J. H.; Votanopoulos, K. I. Significance of diaphragmatic resections and thoracic chemoperfusion on outcomes of peritoneal surface disease treated with cytoreductive surgery (CRS) and hyperthermic intraperitoneal chemotherapy (HIPEC). Ann. Surg Oncol 2014, 21 (13), 4226−31. (11) Los, G.; Mutsaers, P. H.; van der Vijgh, W. J.; Baldew, G. S.; de Graaf, P. W.; McVie, J. G. Direct diffusion of cisdiamminedichloroplatinum(II) in intraperitoneal rat tumors after intraperitoneal chemotherapy: a comparison with systemic chemotherapy. Cancer Res. 1989, 49 (12), 3380−4. (12) El-Kareh, A. W.; Secomb, T. W. A theoretical model for intraperitoneal delivery of cisplatin and the effect of hyperthermia on drug penetration distance. Neoplasia 2004, 6 (2), 117−27. (13) Williams, A. C.; Barry, B. W. Penetration enhancers. Adv. Drug Delivery Rev. 2004, 56 (5), 603−18. (14) Som, I.; Bhatia, K.; Mohd, Y. Status of surfactants as penetration enhancers in transdermal drug delivery. J. Pharm. BioAllied Sci. 2012, 4 (1), 2−9. (15) Lashmar, U. T.; Hadgraft, J.; Thomas, N. Topical application of penetration enhancers to the skin of nude mice: a histopathological study. J. Pharm. Pharmacol. 1989, 41 (2), 118−22. (16) Herman, A.; Herman, A. P. Essential oils and their constituents as skin penetration enhancer for transdermal drug delivery: a review. J. Pharm. Pharmacol. 2015, 67 (4), 473−85. (17) Mutalik, S.; Parekh, H. S.; Davies, N. M.; Nayanabhirama, U. A combined approach of chemical enhancers and sonophoresis for the transdermal delivery of tizanidine hydrochloride. Drug Delivery 2009, 16 (2), 82−91. (18) Ali, A.; Trehan, A.; Ullah, Z.; Aqil, M.; Sultana, Y. Matrix type transdermal therapeutic systems of glibenclamide: Formulation, ex vivo and in vivo characterization. Drug Discoveries Ther. 2011, 5 (1), 53−9. (19) Zeng, S.; Kapur, A.; Patankar, M. S.; Xiong, M. P. Formulation, Characterization, and Antitumor Properties of Trans- and Cis-Citral in the 4T1 Breast Cancer Xenograft Mouse Model. Pharm. Res. 2015, 32 (8), 2548−58. (20) Liu, Y.; Whelan, R. J.; Pattnaik, B. R.; Ludwig, K.; Subudhi, E.; Rowland, H.; Claussen, N.; Zucker, N.; Uppal, S.; Kushner, D. M.; Felder, M.; Patankar, M. S.; Kapur, A. Terpenoids from Zingiber officinale (Ginger) induce apoptosis in endometrial cancer cells through the activation of p53. PLoS One 2012, 7 (12), e53178.

(21) Dudai, N.; Weinstein, Y.; Krup, M.; Rabinski, T.; Ofir, R. Citral is a new inducer of caspase-3 in tumor cell lines. Planta Med. 2005, 71 (5), 484−8. (22) Dangkong, D.; Limpanasithikul, W. Effect of citral on the cytotoxicity of doxorubicin in human B-lymphoma cells. Pharm. Biol. 2015, 53 (2), 262−8. (23) Hahn, G. M.; Braun, J.; Har-Kedar, I. Thermochemotherapy: synergism between hyperthermia (42−43 degrees) and adriamycin (of bleomycin) in mammalian cell inactivation. Proc. Natl. Acad. Sci. U. S. A. 1975, 72 (3), 937−40. (24) Afri, M.; Frimer, A. A.; Cohen, Y. Active oxygen chemistry within the liposomal bilayer. Part IV: Locating 2′,7′-dichlorofluorescein (DCF), 2′,7′-dichlorodihydrofluorescein (DCFH) and 2′,7′dichlorodihydrofluorescein diacetate (DCFH-DA) in the lipid bilayer. Chem. Phys. Lipids 2004, 131 (1), 123−33. (25) Chen, J.; Rogers, S. C.; Kavdia, M. Analysis of kinetics of dihydroethidium fluorescence with superoxide using xanthine oxidase and hypoxanthine assay. Ann. Biomed. Eng. 2013, 41 (2), 327−37. (26) Elias, D.; Goere, D.; Blot, F.; Billard, V.; Pocard, M.; KohnehShahri, N.; Raynard, B. Optimization of hyperthermic intraperitoneal chemotherapy with oxaliplatin plus irinotecan at 43 degrees C after compete cytoreductive surgery: mortality and morbidity in 106 consecutive patients. Annals of surgical oncology 2007, 14 (6), 1818− 24. (27) Lesgards, J. F.; Baldovini, N.; Vidal, N.; Pietri, S. Anticancer activities of essential oils constituents and synergy with conventional therapies: a review. Phytother. Res. 2014, 28 (10), 1423−46. (28) Bhalla, Y.; Gupta, V. K.; Jaitak, V. Anticancer activity of essential oils: a review. J. Sci. Food Agric. 2013, 93 (15), 3643−53. (29) Gautam, N.; Mantha, A. K.; Mittal, S. Essential oils and their constituents as anticancer agents: a mechanistic view. BioMed Res. Int. 2014, 2014, 154106. (30) Saleh, A. M.; Aljada, A.; Rizvi, S. A.; Nasr, A.; Alaskar, A. S.; Williams, J. D. In vitro cytotoxicity of Artemisia vulgaris L. essential oil is mediated by a mitochondria-dependent apoptosis in HL-60 leukemic cell line. BMC Complementary Altern. Med. 2014, 14, 226. (31) Navarra, M.; Ferlazzo, N.; Cirmi, S.; Trapasso, E.; Bramanti, P.; Lombardo, G. E.; Minciullo, P. L.; Calapai, G.; Gangemi, S. Effects of bergamot essential oil and its extractive fractions on SH-SY5Y human neuroblastoma cell growth. J. Pharm. Pharmacol. 2015, 67 (8), 1042− 53. (32) Razavi, R.; Harrison, L. E. Thermal sensitization using induced oxidative stress decreases tumor growth in an in vivo model of hyperthermic intraperitoneal perfusion. Annals of surgical oncology 2010, 17 (1), 304−11. (33) Munck, J. N.; Riggi, M.; Rougier, P.; Chabot, G. G.; Ramirez, L. H.; Zhao, Z.; Bognel, C.; Ardouin, P.; Herait, P.; Gouyette, A. Pharmacokinetic and pharmacodynamic advantages of pirarubicin over adriamycin after intraarterial hepatic administration in the rabbit VX2 tumor model. Cancer Res. 1993, 53 (7), 1550−4. (34) Robert, J.; David, M.; Huet, S.; Chauvergne, J. Pharmacokinetics and metabolism of pirarubicin in advanced cancer patients. Eur. J. Cancer Clin. Oncol. 1988, 24 (8), 1289−94. (35) Lee, H. J.; Jeong, H. S.; Kim, D. J.; Noh, Y. H.; Yuk, D. Y.; Hong, J. T. Inhibitory effect of citral on NO production by suppression of iNOS expression and NF-kappa B activation in RAW264.7 cells. Arch. Pharmacal Res. 2008, 31 (3), 342−9. (36) Thomas, M. L.; de Antueno, R.; Coyle, K. M.; Sultan, M.; Cruickshank, B. M.; Giacomantonio, M. A.; Giacomantonio, C. A.; Duncan, R.; Marcato, P. Citral reduces breast tumor growth by inhibiting the cancer stem cell marker ALDH1A3. Mol. Oncol. 2016, 10 (9), 1485−1496. (37) Sanches, L. J.; Marinello, P. C.; Panis, C.; Fagundes, T. R.; Morgado-Diaz, J. A.; de-Freitas-Junior, J. C.; Cecchini, R.; Cecchini, A. L.; Luiz, R. C. Cytotoxicity of citral against melanoma cells: The involvement of oxidative stress generation and cell growth protein reduction. Tumor Biol. 2017, 39 (3), 1010428317695914. 3596

DOI: 10.1021/acs.molpharmaceut.7b00652 Mol. Pharmaceutics 2017, 14, 3588−3597

Article

Molecular Pharmaceutics (38) Hata, T.; Sakaguchi, I.; Mori, M.; Ikeda, N.; Kato, Y.; Minamino, M.; Watabe, K. Induction of apoptosis by Citrus paradisi essential oil in human leukemic (HL-60) cells. In Vivo 2003, 17 (6), 553−9. (39) Wei, A.; Shibamoto, T. Antioxidant/lipoxygenase inhibitory activities and chemical compositions of selected essential oils. J. Agric. Food Chem. 2010, 58 (12), 7218−25. (40) Tuttolomondo, T.; La Bella, S.; Licata, M.; Virga, G.; Leto, C.; Saija, A.; Trombetta, D.; Tomaino, A.; Speciale, A.; Napoli, E. M.; Siracusa, L.; Pasquale, A.; Curcuruto, G.; Ruberto, G. Biomolecular characterization of wild sicilian oregano: phytochemical screening of essential oils and extracts, and evaluation of their antioxidant activities. Chem. Biodiversity 2013, 10 (3), 411−33.

3597

DOI: 10.1021/acs.molpharmaceut.7b00652 Mol. Pharmaceutics 2017, 14, 3588−3597