Combination Treatment of Citral Potentiates the Efficacy of

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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 Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.7b00652 • Publication Date (Web): 25 Aug 2017 Downloaded from http://pubs.acs.org on August 28, 2017

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Molecular Pharmaceutics

Combination treatment of citral potentiates the efficacy of hyperthermic intraperitoneal chemoperfusion with pirarubicin for colorectal cancer

Zhiyuan Fang1, 2#, Yu Wang1#, Hao Li1#, Shuaishuai Yu3, Ziying Liu1, Zhichao Fan1, Xiaomin Chen1, Yuying Wu1, Xuebo Pan1*, Xiaokun Li1*, Cong Wang1*

1 School of Pharmaceutical Sciences, Wenzhou Medical University, Wenzhou, Zhejiang, China 325030 2 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, China 510530 3 Department of Biology, School of Laboratory Medicine and Life Science, Wenzhou Medical University, Wenzhou, Zhejiang, China 325030 *To whom correspondence may be addressed: School of Pharmaceutical Sciences, Wenzhou

Medical

University,

Wenzhou,

Zhejiang,

China

325030.

Tel:

0577-86597330, Fax: 0577-86699350. Cong Wang, E-mail: [email protected], Xuebo

Pan,

E-mail:

[email protected]

and

Xiaokun

Li,

Email:

[email protected]. #

Equally contributed to the manuscript.

Running title: Citral for effective HIPEC Keywords: Hyperthermic intraperitoneal chemoperfusion, Citral, Pirarubicin, Apoptosis, Colorectal carcinoma

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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 co-treatment 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.

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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 micro tumor nodules and micro metastases 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 . 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 cells 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. 3



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Due to their lipophilic nature and low molecular weights, essential oils have been used as penetration enhancers that have 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 occurred 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, 20 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 4


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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 penicillin and 100 µg/ml streptomycin in 5 % CO2 incubators. No bacterial 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 µM, 100 µM, 300 µM, 600 µM) or combined with pirarubicin (5 µM, 15 µM, 25 µM) to the culture media, the cells were transferred to 37°C 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 2.5 mg/ml in PBS) was then added to each well following by 4 hours incubation following manufactured 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 weeks old female BALB/c mice were randomly divided into 4 groups (6 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 hour. After the incubation, the muscles were frozen with dry ice and cryosectioned at 5µm thickness for observation under a fluorescence microscope.

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2.4 Apoptosis assays Cells (1×106 cells/well) were seeded in 6-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 following 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 (BD Pharmingen, San Diego, CA). The labeled cells were detected with a BD FACSCalibur flow cytometer (BD Biosciences, San Jose, CA). Western blot were analyzed with anti-BAX, BCL-2, and cleaved Caspase 3 were also used to detect apoptotic cells.

2.5 Western Blotting Analysis Cells were lysed with the RIPA buffer (50mM Tris, 150mM NaCl，1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 0.1mM 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 (1:1,000), and anti-GAPDH (1:1,000) were purchased from Santa Cruz (Santa Cruz, CA). Rabbit anti-pERK (1:2,000), anti-Bcl-2 (1:1,000), anti-AKT (1:1,000), anti-Bax (1:1,000), anti-cleaved-caspase-3 (1:1,000), anti-pAKT (1:1,000), anti-p65 (1:1,000), anti-p-p65 (1:1,000), anti-Ikkα/β (1:1,000), anti- p-Ikkα/β (1:1,000) 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 Image J software (http://rsb.info.inh.gov/ij). 6


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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 (1400 g for 5 minutes), 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 semi-thin plastic sections, methylene blue-azure II/basic fuchsin was used for non-en-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 6-well plates and treated with citral (300 µM), pirarubicin (25 µM) or combination at 43°C for 1 h. After the indicated time, cells were harvested and re-suspended in 20 µM DCFH-DA. After incubation at 37°C for 30 minutes, the production of reactive oxygen species (ROS) in samples with 50,000 cells were 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 minutes, 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 7



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For the colorectal peritoneal carcinomatosis (CRPC) experiments, mouse-derived colorectal cancer cells CT26 (5×106 cells in 0.5 ml serum-free medium) were intraperitoneal inoculated into the flanks of Balb/C mice. Five days later the inoculation, these mice were randomly divided into 4 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 hour. 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 were connected to a water bag incubated in a 43 °C water bath. The “tubule-bag system” is a closed circuit that driven by a peristaltic pump at 3 ml/min. A total of 5 ml 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 post-operation 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. Post mortem pathological examinations were carried for each mouse, including tumor size, tumor distributions, and ascites. Depth of tissue penetration was analyzed in frozen tissue section under a fluorescence microscope one hour after the treatments.

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 8


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(χ2) or Fisher’s exact test. Two-sided p

Combination Treatment of Citral Potentiates the Efficacy of

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