ISSN 2093-6966 [Print], ISSN 2234-6856 [Online] Journal of Pharmacopuncture 2019;22[2]:102-108 DOI: https://doi.org/10.3831/KPI.2019.22.013 Original article Antiproliferative Effects of Free and Encapsulated Hypericum Perforatum L. Extract and Its Potential Interaction with Doxorubicin for Esophageal Squamous Cell Carcinoma Issa Amjadi1, Mohammad Mohajeri2, Andrei Borisov3 , Motahare-Sadat Hosseini4* 1 Department of Biomedical Engineering, Wayne State University, Detroit, United States 2 Department of Medical Biotechnology, Mashhad University of Medical Sciences, Mashhad, Iran 3 Department of Biomedical Engineering, Wayne State University, Detroit, United States 4 Biomaterials Group, Department of Biomedical Engineering, Amirkabir University of Technology, Tehran, Iran Key Words Results: Free drugs and nanoparticles significantly inhibited KYSE30 cells by 55-73% and slightly affected doxorubicin, hypericum perforatum extract, nano- normal cells up to 29%. The IC50 of Dox NPs and HP particles, esophageal squamous cell carcinoma, drug NPs was ~ 0.04-0.06 mg/mL and ~ 0.6-0.7 mg/mL, re- resistance spectively. Significant decrease occurred in cyclin D1 expression by Dox NPs and HP NPs (P < 0.05). Expo- Abstract sure of KYSE-30 cells to combined treatments includ- ing both Dox and HP extract significantly increased the Objectives: Esophageal squamous cell carcinoma level of cyclin D1 expression as compared to those with (ESCC) is considered as a deadly medical condition that individual treatments (P < 0.05). affects a growing number of people worldwide. Target- ed therapy of ESCC has been suggested recently and Conclusion: Dox NPs and HP NPs can successfully and required extensive research. With cyclin D1 as a thera- specifically target ESCC cells through downregulation peutic target, the present study aimed at evaluating the of cyclin D1. The simultaneous use of Dox and HP ex- anticancer effects of doxorubicin (Dox) orHypericum tract should be avoided for the treatment of ESCC. perforatum L. (HP) extract encapsulated in poly(lac- tic-co-glycolic acid) (PLGA) nanoparticles on the ESCC 1. Introduction cell line KYSE30. Cancer is described as a disease where a group of Methods: Nanoparticles were prepared using double tumor types are developed through an uncontrolled emulsion method. Cytotoxicity assay was carried out growth of abnormal cells. Upon the rapid growth, to measure the anti-proliferation activity of Dox-load- pathological outcomes such as tissue fractures are es- ed (Dox NPs) and HP-loaded nanoparticles (HP NPs) tablished which cannot be overcome by normal cells. against both cancer and normal cell lines. The mRNA Cancer constitutes the second cause of death across gene expression of cyclin D1 was evaluated to validate the globe and its incidence is steadily on the rise [1, the cytotoxicity studies at molecular level. 2]. Esophageal cancer is a male-dominant aggressive malignancy, while ranking eighth among cancers and sixth in mortality [3, 4]. It has two most-common Received: Aug 06, 2018 Reviewed: Feb 13, 2019 Accepted: May 20, 2019 types, including squamous cell carcinoma and ade- This is an Open-Access article distributed under the terms of the Creative Commons *Corresponding Author Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/) Motahare-Sadat Hosseini. Biomaterials Group, Department of Biomedical Engineering, which permits unrestricted noncommercial use, distribution, and reproduction in any Amirkabir University of Technology, Tehran, Iran. medium, provided the original work is properly cited. Tel: +98-915-245-0301 E-mail: [email protected] This paper meets the requirements of KS X ISO 9706, ISO 9706-1994 and ANSI/NISO Z39.48-1992 (Permanence of Paper). ⓒ 2019 Korean Pharmacopuncture Institute http://www.journal.ac Journal of Pharmacopuncture 2019;22[2]102-108 http://www.journal.ac 103 nocarcinoma [5]. The former (i.e., esophageal squamous have a lower prevalence of gastric ulcers [22]. Quercetin cell carcinoma; ESCC) is primarily reported from Asia and I3,II8-biapigenin, known as the two main oil extracts [6]. In spite of progress in surgical resection and adjuvant of HP, demonstrated special anti-inflammatory and gas- chemoradiotherapy, affected patients still tend to have troprotective actions [23]. More recently, a case report by poor prognoses. Although, there are individual differenc- Karaarslan et al. provided a perspective on the anti-tumor es in ESCC formation, determination of gene expression properties of HP with possible mechanisms and patho- in response to certain anticancer drugs could promote the logical evidence in three patients with gastrointestinal design of targeted ESCC therapy [7]. Cyclin D1 appears cancer. They observed long-term HP use, extensive host to be mostly enhanced and over-expressed in ESCC, sug- reaction, and tendency to develop barrier against tumor gesting that it acts as a central player in the disease. In- that can denote a morphological sign of its anti-tumor re- deed, cyclin D1 amplification induces tumorigenesis as sponse [24]. well as tumor growth [8]. In our previous studies on a nanoparticle drug delivery To treat both localized and metastasized cancers, the system, two different formulations were developed using main therapeutic approach is chemotherapy [2, 9, 10], Dox and HP as anticancer agents. The results indicated whereby specialized medicines with anticancer proper- that both drugs can be successfully loaded in the polymer- ties are infused through the blood vessels [11]. Recently, ic carriers made up of poly(lactic-co-glycolic acid) (PLGA) several clinically active anticancer drugs have been cre- [13, 25]. The present study was intended to investigate the ated and used for this purpose. The majority of cancer anticancer effects of these two complexes in an in vitro drugs fail to distinguish between cancer cells and normal model of ESCC (KYSE-30 cells). cells that culminates in adverse effects and deleterious reactions in tissues [10]. Targeted drug delivery is a pro- cess that a specific biomolecular agent is engineered to 2. Materials and Methods release with the purpose of improving product efficacy as well as patient safety [12, 13]. To this aim, many polymeric 2.1. Chemicals materials have been studied to encapsulate the drug and release it in a controlled fashion [11, 14, 15]. Additionally, Dox hydrochloride and PLGA 50:50 were respectively a drug delivery system based on polymers is more likely purchased from Sigma Chemical Company (USA) and to minimize these side effects [15], and it may enhance Boehringer Ingelheim (Germany). Dichloromethane drug distribution in the body, augment the drug speci- (DCM) and fully hydrolyzed (99.8%) poly vinyl alco- ficity, extend the duration of drug activity and ameliorate hol (PVA; Mn = 72000 g/mol and 99.8% hydrolysis) were in vivo degradation resistance [16]. In this regard, the en- purchased from Merck (Germany) without further puri- capsulation of anticancer agents into nanoparticles (NPs) fication. The HP extract was graciously provided by Prof. has garnered much global attention. Doxorubicin (Dox) Sefidkon (Research Institute of Forests and Rangelands, is a widely used drug with anticancer activities, chiefly Tehran, Iran). administered for the treatment or management of solid tumors [17]. Dox-induced toxicity and resistance are the major drawbacks in chemotherapy and clinical practice 2.2. Nanoparticle preparation [18]. Current evidence shows that it produces chemical- ly reactive molecules containing oxygen, able to trigger a Initially, HP extract (10 mg) was mixed with 5 mL of 70% series of unprecedented reactions that involve the heart, methanol under constant stirring in a water bath at 60°C kidney, liver, brain, and reproductive organs [18]. More to until it was completely dissolved. The other inner aque- the point, Dox has the ability to enter target cells through ous phase was Dox used at the same concentration of HP cellular membranes without taking advantage of distinct extract. Nanoparticles were prepared using the (should be transporters owning to its hydrophobic nature. However, omitted) double emulsion method, which was previously active efflux of the drug reduces its content inside cells by published. The drugs were emulsified in 50 mg of PLGA means of ATP-dependent efflux transporters. This event in 50 mL of DCM by sonication for 2 min. Afterwards, the is described as multidrug resistance that cancer cells re- solution was added to 3% PVA to form secondary emul- sist against the cytotoxicity caused by chemotherapeutic sion. After solvent evaporation, the particles were subject- drugs [18]. ed to centrifugation [13]. The size of resultant nanoparti- Hypericum perforatum L. (St. John's Wort; HP) is a com- cles with spherical morphology ranged between 100 and plex herbaceous drug from the Hypericaceae species 400 nm (data not shown), which was consistent with our [19] that contains a variety of anticancer components, previous reports [13, 25]. including hypericin, α-terpineol, betacarotene, caffeic acid, isoquercitin, kaempferol, gallic acid, limonene, ru- 2.3. Cell lines tin, and vanillic acid [13]. It was traditionally applied for the treatment of anxiety, depression, insomnia, water re- Two cancerous and normal cell lines were used in this tention, and gastritis. Also, HP extracts seem to decline study: the human esophageal squamous cell carcinoma oxidative stress, thereby inhibiting neurotoxicity, inflam- cells (KYSE-30; Cat No: 94072011) and normal cells (Cat mation, and gastrointestinal issues [20]. Rats treated with No: 85120602). They were purchased from the Cell Bank, doses of HP were found to decrease levels of blood and Pasteur Institute of Iran (Tehran, Iran). The second one bowel enzymes related to colonic inflammation [21], and served as the control group. The DMEM (Gibco, Germa- 104 http://www.journal.ac Journal of Pharmacopuncture 2019;22[2]102-108 ny) supplemented with 10% FBS (GIBCO, Germany), pen- mine a significant difference at P-values < 0.05. icillin (1% v/v), and streptomycin (1% v/v) was utilized to maintain the cells in an incubator set at 37°C and 5% CO2. 3. Results 2.4. Determination of cell viability 3.1.