Phenethyl Isothiocyanate Inhibits in Vivo Growth of subcutaneous
Xenograft Tumors of Human Malignant Melanoma A375.S2 Cells
Wei-Ya Ni1,#, Hsu-Feng Lu 2,#, Shu-Chun Hsu3, Yu-Ping Hsiao4,5, Kuo-Ching Liu6,Jia-You Liu7, Bin-Chuan Ji8, Shu-Ching Hsueh9, Fang-Ming Hung10,* and Jing-Gung
Chung3,11,*
1Department of Food and Beverage management, NanKai University of Technology,
Nan-Tou County, Taiwan
2Department of Clinical Pathology, Cheng Hsin General Hospital, Taipei, Taiwan 3Department of Biological Science and Technology, China Medical University,
Taichung 404, Taiwan
4Institute of Medicine, Chung Shan Medical University, Taichung 402, Taiwan
5Department of Dermatology, Chung Shan Medical University Hospital, Taichung
402, Taiwan
6Department of Medical Laboratory Science and Biotechnology, China Medical
University, Taichung 404, Taiwan
7Department of Clinical Pathology, Cheng Hsin General Hospital, Taipei, Taiwan 8Division of Chest Medicine, Department of Internal Medicine, Changhua Christian
Hospital, Changhua 500, Taiwan
9Department of Clinical Pathology, Cheng Hsin General Hospital, Taipei, Taiwan 10Department of Surgical Intensive Care Unit, Far Eastern Memorial hospital, Taipei,
Taiwan
11Department of Biotechnology, Asia University, Taichung 413, Taiwan
#Both authors contributed equally to this work.
Running title: PEITC induces inhibit tumor growth of human melanoma A375.S2 cells
*Correspondence to Jing-Gung Chung, Ph.D., Department of Biological Science and Technology, China Medical University. No 91, Hsueh-Shih Road, Taichung 404,
Taiwan. Tel.: +886 4 22053366 ext 2161. Fax: +886 4 22053764. E-mail:
jgchung@mail.cmu.edu.tw
*Correspondence to Dr. Fang-Ming Hung, Department of Surgical Intensive Care Unit, Far Eastern Memorial hospital, No. 21, Sec.2, Nan-Ya south Rd. Banciao 220, Taipei, Taiwan
Abstract
Numerous studies have been shown that phenethyl isothiocyanate (PEITC) induces apoptosis in various human cancer cell lines, however, there is no report to show PEITC inhibit the tumor growth of A375.s cell xenograft in nude mice. Herein, we investigated the effects of PEITC on the growth of xenograft A375.S2 cell tumor on nude BLAB/c mice in vivo. For subcutaneous tumor growth study, A375.S2 cancer cells were inoculated subcutaneously into the lower flanks of each mouse. After 7 day cancer cell inoculation, each mouse had produce one palpable tumor and then were randomly divided into three groups by using intraperitoneal injections of PEITC (0, 20 and 40 mg/kg). Results from in vivo experimentals indicated that PEITC did nor significantly affect the body weight of xenograft A375.S2 cell tumor in nude BLAB/c mic, however, it did decreased the tumor weight.
Introduction
Skin cancer melanoma is becoming more common and continues to increase by approximately 4% annually (1) and resulting in increased mortality especial in Western countries (2). In the worldwide, melanoma accounts for only 4% of all skin cancers but this disease causes the greatest number of skin cancer-related deaths (3). The current treatment for melanoma or skin cancer such as surgery, radiation, chemotherapy, or a combination of radiotherapy with chemotherapy, however, the patient with these diseases, the mortality remains high except earlier treatment (4). Thus, numerous studies have focused on to find new compounds from natural product for treating cancer.
It was demonstrated that dietary intake of cruciferous vegetables (broccoli, cabbage, watercress, and cauliflower) can reduced the risk of various types of malignancies (5) and it can play protective roles in different diseases (6, 7). Cruciferous vegetables contain organic isothiocyanates (ITCs) which have been shown it may reduce the development of various malignancies (8) and to inhibit cancer formation (9). Phenethyl ITC (PEITC) is one of the ITC family compounds and it has cancer chemopreventive activity (10).
PEITC induced apoptosis in lung cancer cells (11), leukemia cells (12), colon cancer cells (13), breast cancer cells (14) prostate cancer cells (15), osteogenic sarcoma cells (16), ovarian cancer cells (17) and oral squamous carcinoma cells (18). Novel combinations such as metformin and PEITC show promise in expanding ovarian cancer therapies and overcoming the high incidence of cisplatin resistant cancers (19). The combination of PEITC and taxol exhibits a synergistic effect on growth inhibition in breast cancer cells (20). It is reported that PEITC has anti-angiogenic effects in a chemically induced breast cancer animal model (21). Recently, it was reported that PEITC can be used to induce expression of damaged DNA binding protein 2 (DDB2), and that expression of DDB2 is critical for effective response of tumors to PEITC (22). Although numerous studies have been shown that PEITC induced cytotoxic effects on many cancer cell lines, however, there are no reports to show PEITC affect human malignant melanoma cells in vivo. Thus, in the present studies, we investigated the effects of PEITC on tumor growth of human melanoma A375.S2 cells and results indicated that PEITC inhibit A375.S cell growth in vivo.
Materials and Methods Chemicals and reagents
Phenethyl Isothiocyanate (PEITC) and dimethyl sulphoxide (DMSO) were purchased from Sigma-Aldrich Corp. (St. Louis, MO, USA). Minimum Essential Medium (MEM), L-glutamine, fetal bovine serum, penicillin-streptomycin, and trypsin-EDTA were purchased from Gibco BRL (Grand Island, NY, USA).
Cell culture
The human malignant melanoma cancer cell line (A375.S2) was purchased from the Food Industry Research and Development Institute (Hsinchu, Taiwan). Cells were cultured in MEM supplemented with 10% FBS, 1% antibiotics (100 Units/ml penicillin and 100 μg/ml streptomycin) and 2 mM L-glutamine at 37°C in a humidified 5% CO2 and 95% air at one atmosphere incubator. The medium was
changed every 2 days (23).
Subcutaneous implantation of A375.S2 cells and treatment of PEITC
Thirty male BALB/c nude mice (6 weeks old) at the 25 g weight were purchased from the National Laboratory Animal Center (Taipei, Taiwan) and the animals were maintained in the Laboratory Animal Center of China Medical University and taken care as animal guideline (Affidavit of Approval of Animal Use Protocol) of China Medical University.
For subcutaneous tumor growth study, A375.S2 cancer cells (1×106 in 0.1 ml
Dulbecco’s PBS) were inoculated subcutaneously into the lower flanks of each mouse. After 7 day cancer cell inoculation, each mouse had produce one palpable tumor and then were randomly divided into three groups (n = 10/group). Group I of mice were given intraperitoneal injections of oliver oil only as a control group. Group II of mice were then given intraperitoneal injections of vehicle solution, 20 mg/kg PEITC. Group III of mice were then given intraperitoneal injections of vehicle solution, 40 mg/kg PEITC. All the mice were monitored weekly for tumor growth and were treated the above dose daily for up to 12 day before being weighed (24).
Statistical analysis
to compare the difference between control and PEITC treatments in each group. A p-value of less than 0.05 was considered to be statistically significant.
Results
In order to examine the effect of PEITC on tumor growth in vivo, we selected A375.S2 cell tumors grown subcutaneously in BLAB/c nude mice. In these experiments xenograft A375.S2 tumors were allowed to establish for 1 week before challenge 2 time per 2 day for total 12days with PEITC (20 or 40 mg/kg/mouse). The representative animal and associated tumor on the mice are showing in Figure 1A and B, the body weight are showing in Figure 1C, the representative isolated tumors and weight are showing in Figure 1D and E. Figure 1A, B and C indicated that PEITC did not affect the total body weight of mice after treated with PEITC, however, figure 1D and E indicated that PEITC significantly decreased the tumor weight. Figure 1D and E shows that 20 and 40 mg/kg PEITC was efficient at retarding growth of A375.S2 cell subcutaneous mouse tumors. Of note, PEITC treatment at both 20 mg/kg (Figure 1B) and 40 mg/kg (Figure 1C) did not provoke body weight loss in contrast untreated tumor bearing mice.
Discussion
Numerous studies have been shown that PEITC induced cytotoxicity effects on human cancer cell lines, in our previous studies, we also show that PEITC induced apoptosis in human oral squamous carcinoma HSC-3 cells and osteogenic sarcoma U-2 OS cells. (16, 18, 25). However, no available report to show PEITC affect xenograft A375.S2 cells tumor in nude mice, thus, herein, we are the first to show PEITC decreased the tumor weight and size of A375.S2 cell tumor in nude mice. Our previous studies also showed that PEITC induced cell cycle arrest in A375.S2 cells. It is well documented that cell cycle control represents a major regulatory mechanism of cell growth (25) and block the cell cycle have been recogned to be an effective strategy for the development of novel cancer therapies (26).
various human cancer cell lines in vitro and even in xenograft systems of human cancer cell tumors in vivo. In fact, many anticancer drugs have been used inclinical patients that were pbtained from natural plants. One of the often used and mentation in clinical patients is taxol (27) which induces apoptosis in various human cancer cell lines (28). Other agent is genistein which has been recognized to be a potential compounds for breast cancer chemoprevention (29) and it induce apoptosis (30). Our previous have shown that PEITC induced apoptosis in human prostate cancer DU 145 cells (31), osteogenic sarcoma U-2 OS cells (16) and colon cancer HT29 cells (32) and oral cancer HSC-3 cells (18). Furthermore, we also found that PEITC can promote immune responses in normal and WEHI-3 leukemia mice in vivo (33). Thus, based on our observations, we suggest PEITC may be promising in cancer therapy, however, we did not examined the effects of PEOITC on in vivo studies, therefore, herein, we showed that PEITC decreased the tumor size and weight in xenograft A375.S2 tumors in nude mice.
Acknowledgments
This work was supported by the grants CMU101-ASIA-23 from China Medical University, Taichung, Taiwan.
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Figure Legends
Figure 1. PEITC affect the subcutaneous implantation of A375.S2 cells on BLAB/c mice in vivo.
For subcutaneous tumor growth study, A375.S2 cancer cells (1×106 in 0.1 ml
Dulbecco’s PBS) were inoculated subcutaneously into the lower flanks of each mouse. After 7 day cancer cell inoculation, each mouse had produce one palpable tumor and then were randomly divided into three groups (n = 10/group). Group I of mice were given intraperitoneal injections of oliver oil only. Group II of mice were given intraperitoneal injections of vehicle solution, 20 mg/kg PEITC. Group III of mice were given intraperitoneal injections of vehicle solution, 40 mg/kg PEITC. All the mice were monitored weekly for tumor growth and were treated the above dose daily for up to 12 day before being weighed as described in Materials and Methods. The representative mice (A), representative tumors (B), total body weight (C), and tumor weight (D).*p<0.05 was recognized to be the sighnificant difference between control and PEITC treatment.
