PEITC Suppresses the Growth of Lewis Lung Cancer in Immune-Complete Mouse

Research Article

Ann Hematol Oncol. 2019; 6(9): 1267.

PEITC Suppresses the Growth of Lewis Lung Cancer in Immune-Complete Mouse

Liuyang He, Mingming Su, Yu Bai, Xiao Sun, Lei Xia, Chunjian Qi

Medical Research Center, The Affiliated Changzhou No. 2 People’s Hospital of Nanjing Medical University, China

*Corresponding author: Chunjian Qi, Medical Research Center, The Affiliated Changzhou No. 2 People’s Hospital of Nanjing Medical University, Changzhou, China

Received: July 08, 2019; Accepted: July 30, 2019; Published: August 06, 2019


Phenethyl Isothiocyanate (PEITC) isolated from cruciferous vegetables has gained wide attention in recent years due to its strong anticancer effects. Previously, numerous studies have showed that PEITC can induce excessive Reactive Oxygen Species (ROS) accumulation, mitochondrial dysfunction and suppression of stem-like cancer cells, thus leading to the apoptosis of cancer cells and the inhibition of tumor development. However, few studies have analyzed the effects of PEITC on the immune cells of tumor-bearing hosts. In the current study, we found that PEITC treatment slowed down the growth of Lewis lung cancer in immune-complete C57BL/6 mice rather than BALB/cnude mice. Based on the result, we focused on immune cells of Lewis Lung Cancer (LLC)-bearing C57BL/6 mice treated by the oral administration with PEITC and then detecting changes in the number of MDSCs (myeloid-derived suppressor cells), macrophages, T lymphocytes from tumor issues, Draining Lymph Nodes (DLNs) and spleens. Results from flow cytometry analysis and immunocytochemistry showed the number of tumor-infiltrating CD8+T cells of C57BL/6 mice receiving PEITC per day declined significantly when compared with that of untreated control mice, while intra-tumoral MDSCs, macrophages and CD4+T cells were unaffected. These findings suggest that PEITC can exert anticancer function by inhibiting CD8+T cells with immunomodulatory capacity. Our study offers a new theoretical basis for the clinical application of PEITC in lung cancer treatment.

Keywords: PEITC; Lewis lung cancer; Regulatory CD8+T cells; Mice model


With an increasing number of smokers and deterioration of the environment, the incidence rate of lung cancer has been increasing worldwide. It is predicted that more than one million Chinese will develop lung cancer yearly by 2025 according to the World Health Organization (WHO)’s estimates. Thus, measures should be taken to prevent and control this health-threatening cancer [1]. In addition to radiotherapy and chemotherapy, how to reactivate the autoimmunity of tumor patients is an effective therapeutic modality against cancer, as the emergence and development of tumors are closely related to the loss of immune function. The ability of immune cells to distinguish between normal cells and cancerous cells is attributed to tumor antigens that are specifically expressed on cancer cells. T effector cells are prime contributors to eradicate tumor cells. Particularly, cytotoxic CD8+T cells can recognize tumor antigens complexed with Major Histocompatibility Class I (MHC-I) on the Antigen- Presenting Cell (APC) surface, thus exerting specific immune-killing effects by generating apoptosis signals (FasL) and releasing perforin and granzymes. However, a relatively small group of CD8-expressing regulatory cells harbors immunosuppressive functions. This cell population has similarities to conventional CD4+Foxp3+Tregs in suppressing immune function and has been studied deeply as noncanonical regulatory cells [2].

JC-5411, a new-targeted antitumor drug produced by Wuxi JC Company Limited, mainly consists of the active component Phenylethyl Isothiocyanate (PEITC) isolated from cruciferous vegetables, such as cauliflower, radish and cabbage. PEITC, which is endowed with the Isothiocyanate (-N=C=S) structure in its molecule, has been widely investigated for its health-protecting benefits, especially regarding its strong cancer-treatment effects on various cancers, including oral cancer [3], head and neck cancer [4], brain glioblastoma [5], and prostate cancer [6]. Therefore, PEITC has been registered in phase III clinical trials in the USA to further confirm the safety and efficacy of tumor therapy. It has been reported that several different mechanisms are involved in preventing and treating cancers with PEITC. A previous study showed that PEITC and its analogs can cause ROS accumulation and redox imbalance in cancer cells to inhibit tumor growth [7]. In another study, only human sensoryacceptable doses of PEITC (10 mg per kg body) were proven to induce cell cycle arrest in the G1/S phase of p53-mutated oral cancer cells [3]. However, limited studies have reported on whether PEITC affects immune cells in mice with tumors. In the only published paper, MDSCs from Peripheral Blood Mononuclear Cells (PBMCs) of breast cancer xenografts in SCID/NSG mice were suppressed with PEITC oral treatment [8].

In our study, we first compared the curative effect of JC-5411 between Lewis Lung Cancer (LLC)-bearing C57BL/6 mice and BALB/c-nude mice. After treating with JC-5411 by oral gavage in established tumor-bearing mice, we found that JC-5411 treatment retarded the growth of tumors in C57BL/6 mice compared with that in BALB/c-nude mice. Next, we focused on the effects of JC-5411 oral administration on multiple leukocytes from the tumor issues, Draining Lymph Nodes (DLNs) and spleens in C57BL/6 mice with Lewis lung cancer. Our results showed that PEITC had an inhibitory effect on tumor-infiltrating suppressive CD8+T cells to delay tumor development.

Materials and Methods

Chemicals, mice and cell lines

JC Pharmaceutical Company Limited (Wuxi, Jiangsu, China) kindly provided JC-5411. Female SPF experimental C57BL/6 mice and BALB/c-nude mice, aged 6-8 weeks, were purchased from Changzhou Cavens Company. All mice were fed with free access to food and drinking water and were maintained under specific pathogen-free conditions. The Lewis Lung Cancer line (LLC) was obtained from the American Type Culture Collection (ATCC) and cultured in Roswell Park Memorial Institute-1640 Medium (RPMI- 1640; Thermo-Fisher Scientific), supplemented with 10% heatinactivated fetal bovine serum (FBS; Thermo-Fisher Scientific). The cells were incubated at 37oC under a humidified atmosphere of 5% CO2. Fluorescein-conjugated mouse-specific antibodies against CD11b, CD4, CD8, Gr-1 and F4/80 for flow cytometry analysis were purchased from BD Pharmingen. The rabbit monoclonal antibody against CD8 for immunocytochemistry and immunofluorescence was purchased from Abcam (ab217344), and goat anti-rabbit IgG cross-adsorbed secondary antibody conjugated with Alexa Fluor 568 was obtained from Thermo-Fisher Scientific.

Animal experiments

Viable Lewis lung cancer cells were counted and suspended in phosphate-buffered saline (PBS; Thermo-Fisher Scientific) at a concentration of 5×106 cells/ml. Next, 100 μl of cells suspended in PBS (including 5×105 cells) were subcutaneously injected into the flanks of each mouse. After the tumor became measurable, the mice were randomly assigned to two groups (control group vs. treatment group). The mice in the treatment group were orally administered with 200 μl of 5 mg/ml of JC-5411 once per day, while the mice in the control group were fed normally without treatment. The tumor masses were measured with a caliper three times per week, and the tumor dimensions were calculated according to the formal V=1/2*A*B2, where A is the vertical length of the tumor and B is the horizontal length of the tumor. At the end of the experiment, all the mice were sacrificed by CO2 overdose in compliance with relevant laws and institutional guidelines. The tumors from two groups of mice were dissected out and weighed. Spleens and Draining Lymph Nodes (DLNs) of mice were dissected out simultaneously for subsequent experiments.

Cell preparation and flow cytometry analysis of leukocytes

Tumor issues from control mice and treated mice were finely minced and digested with collagenase. Briefly, small minced tumor pieces were rinsed twice with PBS solution and were digested on a rotating platform at 37oC for 30 min in 50 ml of 5% FBS/RPMI medium containing 0.25 g of collagenase, 0.1 g of hyaluronidase and 0.0075 g of DNase. After digestion, the tissues were filtered to remove insoluble fiber. The tumor single-cell suspension was washed with 1× Red Blood Cell (RBC) lysis buffer to remove red blood cells. The tumor cells were collected and washed with PBS for surface marker staining. Fluorescein-conjugated Mouse-Specific Antibodies (mAbs) against CD11b, CD4, CD8, Gr-1 and F4/80 were used to label leukocytes. Labeled cells were incubated on ice for 30 min in the dark, followed by washing and analysis by flow cytometry (BD Biosciences) to determine the cell phenotype.

The spleens from mice were ground with a syringe and were washed with PBS. Red blood cells were removed with RBC lysis buffer as described above. The collected single-cell suspensions were stained with mAbs to investigate the effects of JC-5411 on leukocytes.

The Draining Lymph Nodes (DLNs) were ground as spleens without lysis with RBC lysis buffer. The single-cell suspensions were washed with PBS and stained with mAbs for 30 min on ice, followed by analysis using a FACS Canto TM flow cytometer (BD Biosciences). All the samples were analyzed using FlowJo software.

Immunocytochemistry and immunofluorescence

The tumor issues covered with cryo-embedding media were stored at -80oC for sectioning. After the tissue blocks were completely frozen, they were transferred to a cryostat (-20oC) for sectioning and the temperature of the frozen tissue blocks was allowed to equilibrate to that in the cryostat for 15 min. Using a cryotome, the tissue blocks were sectioned into a thickness of 6-8 μm, and sections were placed onto glass slides with positive charges. The glass slides loaded with tissue sections were immersed in precooled methanol at -20oC for 10 min to fix the tissue sections, followed by washing with PBS twice for 5 min. The glass slides were incubated in 0.3% H2O2 dissolved in methyl alcohol at room temperature for 10 min to inactivate endogenous peroxidase and then washed in PBS twice for 5 min each. Subsequently, 100 μl of blocking buffer (5% NGS in TBST) was added onto the sections of the slides, which were then were incubated at room temperature for 1 hour. Excess blocking buffer was drained off from the slides, which were then rinsed with PBS 3 times (5 min each). Next, 100 μl of primary antibodies diluted in blocking buffer (1:400) was added dropwise onto the sections on the slides, which were then incubated in a humidified chamber at room temperature overnight. On the following day, the slides were washed with PBS 3 times to remove excess primary antibodies. Thereafter, 100 μl of diluted secondary antibodies was applied to the sections after equilibration to room temperature. The slides were incubated in a humidified incubator at 37oC for 40 min, followed by washing with PBS for 5 min. Next, 100 μl of DAB substrate solution was applied to reveal the color of the antibody staining. The color developing time was controlled by observation with a microscope. When the desired color intensity of background was reached, the DAB solution was removed quickly, and then the slides were completely washed with running tap water for 10 min.

For immunofluorescence, the tissue blocks were sectioned and fixed for the preparation of immunofluorescence as described above. Prediluted anti-CD8 alpha antibody (1:500 of rabbit monoclonal antibody against CD8) was applied onto the sections of slides. The glass slides loaded with stained tissue sections were incubated at 4oC in the dark overnight. On the following day, the tissue sections were stained with goat anti-rabbit IgG secondary antibody (1:1000) for 40 min, and living cells were stained using diluted DAPI solution (1:500) for 5 min to identify cells. The glass slide was immersed in PBS and washed twice for 5 min each. The CD8+T cells of sections were studied by fluorescence microscopy (U-RFL-T; Olympus).

Statistical analysis

GraphPad Prism 6.0 was used to conduct statistical analysis. The results of animal experiments were represented as the mean values ± SD with a minimum value of n=3. The data were analyzed using Student’s t-test. The differences between groups were considered statistically significant at p‹0.05.


JC-5411 treatment suppresses the growth of tumors in C57BL/6 mice with Lewis Lung Cancer (LLC), but not in BALB/c-nude mice.

To determine whether JC-5411 could exert anti-cancer effects through the immune system in vivo, 5×105 LLC cells cultured in vitro were subcutaneously injected into the inguinal regions of each C57BL/6 female mouse and BALB/c-nude female mouse lacking the thymus, respectively. Once the tumor was palpable, the mice were randomly and equally divided into two groups (control group and JC-5411-treated group). Control mice were fed normally, while mice of the treated group were administered intragastrically one time every day with JC-5411 at a dose of 200 μl (5 mg/ml) additionally. Compared with the control groups, C57BL/6 mice treated with JC- 5411 had smaller tumor volumes and lighter tumor weights at the end of experiment (Figure 1a), while there were no significant differences in the tumor growth and tumor weight between JC-5411-treated BALB/c-nude mice and control BALB-nude mice (Figure 1b). These results indicated that JC-5411 had an inhibitory effect on tumor growth in C57BL/6 mice but no substantial effect on tumor-bearing nude mice, suggesting that JC-5411 could exert immune systemmediated anti-cancer efficiency in vivo.