Antitumor and antioxidant effects of Clinacanthus nutans Lindau in 4 T1 tumor-bearing mice

Murine mammary carcinoma cell line, 4 T1 cells and RPMI-1640 were purchased from American Type Culture Collection (ATCC). Griess Reagent Kit was purchased from Molecular Probes, Eugene, OR. DuoSet ELISA Development System was purchased from R&D Systems, USA.

Clinacanthus nutans Lindau (C. nutans) was collected from TKC Herbal Nursery Sdn Bhd, Negeri Sembilan, Malaysia, and was classified as the whole plant of C. nutans by a science officer named Mr. Lim Chung Lu from the Phytomedicinal Herbarium at Institute of Bioscience (IBS), UPM before the sample was deposited in our laboratory (Voucher No. SK2775/15).

Murine mammary carcinoma cell line, 4 T1 cells were purchased from the American Type Culture Collection (ATCC, USA) and were maintained in RPMI-1640 medium supplemented with 1% penicillin-streptomycin, 1 mM sodium pyruvate, 2 mM glutamine and 10% FBS. Cells were placed in a CO₂ incubator with 95% humidity at 37 °C.

Six to eight-week-old female BALB/c mice were purchased from the Animal House of Faculty of Veterinary Medicine, Universiti Putra Malaysia. The mice were kept under a condition of 12-h dark/light cycle at 25 °C. The mice were fed with standard pellet diet and distilled water ad libitum. All the procedures involving mice were carried out in compliance with the regulations of the Animal Care and Use Committee (ACUC; UPM/IACUC/AUP-R086/2017), Universiti Putra Malaysia.

Mice were divided into 4 groups (n = 7), which consisted 1) control (without breast cancer, untreated), 2) untreated (with breast cancer, untreated) and two other groups of mice harboring breast cancer treated with 200 mg/kg (low-dose) and 1000 mg/kg (high-dose) of C. nutans extract. Similar doses were reported with normal behavior in a study by Lau et al., [18]. In this animal model, 1 × 10⁶ 4 T1 cells were subcutaneously inoculated, and 10 days of incubation were given for tumor growth prior to initiation of treatment. All treatments were administrated daily by oral gavage for 28 days. The size of tumors was measured using a vernier caliper with following the formula, V = (W x W x L)/2 where V is volume, W is width and L is length. After 28 days, mice were euthanized by cervical dislocation. Blood, tumors and vital organs such as liver, lung, spleen, bone marrow were collected for the following analyses. The weight of the tumors was recorded as well.

After mice euthanasia, the spleen was aseptically collected and excised for isolation of splenocytes for immunophenotyping as defined in the previous study [19]. Briefly, the spleen was prepared into single-cell suspension with Hank’s Balance Salt Solution (HBSS) containing 5 mM HEPES and 10% FBS by meshing with a 70 μm strainer. The splenocytes were incubated in lysis buffer (0.1 mM EDTA, 10 mM KHCO3, 0.15 M NH4Cl at pH 7.5) for 10 min at 4 °C for the removal of red blood cells. The cells were then rinsed with PBS before staining with appropriate antibodies (Abcam, USA) at 37 °C for 3 h. Next, the cells were rinsed with PBS twice before fixed with 1% paraformaldehyde (PFA). The cells were kept in the dark at 4 °C until analyzing through FACSCalibur flow cytometer (BD, USA).

The procedure has already been identified and carried out with minor modifications [20]. Briefly, the lung and liver were harvested and then cut into small fragments in sterile condition. The fragments were incubated in 5 ml enzyme cocktail containing 1X PBS, 1 mg/ml of hyaluronidase and 1 mg/ml of type I collagenase for 30 min at 37 °C. After incubation, the cell-containing solution was passed through a 70 mm cell strainer. The cells were pelleted down and washed with PBS twice. The cells were resuspended in 10 ml selection medium and incubated for 7 to 10 days at 37 °C, 5% CO_2, and 95% humidity. After incubation, the plate was prepared with fixing of methanol and stained with crystal violet to count the number of colonies formed per organ.

Bone marrow smears were prepared from the contents of the right femur, as previously described [21]. Briefly, bone marrows were flushed with 1X PBS and smeared across a clean glass slide. The slide was dried in air at room temperature prior to fixation with 100% methanol for 30 min and air-dried again before staining. The slide was then stained with Giemsa stain for 10 min and then air-dry.

Formalin-fixed paraffin-embedded sections of the tumor tissues were carried out as described in our previous study [22]. Briefly, the tumors were harvested and fixed in 10% neutral buffered formalin before being sent to the Histopathology Laboratory, Faculty of Veterinary Medicine, Universiti Putra Malaysia for hematoxylin and eosin staining. The stained tissue sections of 0.45 μm were examined under a microscope (Nikon).

The level of nitric oxide production was detected using the modified Griess assay [23]. Briefly, a mixture of 20 μl Griess reagent, 150 μl nitrite-containing sample and 130 μl distilled water were prepared. On the other side, a photometric reference sample was prepared by mixing 20 μl of Griess reagent and 280 μl of distilled water. The nitrite solutions were prepared by diluting the standard solution with distilled water. The absorbance of generic nitrite solutions was then measured in order to plot a standard curve of nitrite concentration against absorbance. The concentrations of nitrite corresponding nitrite concentrations corresponding to the absorbance of the samples were read from the standard plot.

For quantification of MDA level, this procedure was adapted from the procedure by Samiaa et al., [24]. A mixture of 200 μl sample, 800 μl of PBS, 25 μl of butylated hydroxytoluene (BTH) and 500 μl of trichloroacetic acid (TCA) was prepared in a 50 ml tube and incubated on ice for 2 h. After incubation, the tube was centrifuged for 15 min at room temperature prior to mixing 1 ml of the supernatant with 75 μl of 0.1 M EDTA and 250 μl of thiobarbituric acid (TBA) in 1 M NaOH. The mixture was boiled for 15 min and cooled down to room temperature. The absorbance was measured at 532 nm and 600 nm using a spectrophotometer (Beckman Coulter, USA).

The level of IL-2 and Interferon-γ (IFN-γ) secretions were assessed from the serum mice. The samples were collected and analysed using the DuoSet ELISA Development System (R&D Systems, USA). Designated capture antibodies (Mouse IL-2 Capture Antibody and IFN-γ Capture Antibody) were diluted to working concentration in PBS without carrier protein. The procedures were performed in accordance with the manufactural protocol. Briefly, 96-well plates were coated with 100 μl per well of the diluted Capture Antibodies and incubated overnight at room temperature. The following day, the plates were washed three times with Wash Buffer. The plates were then blocked with Block buffer at room temperature for 1 h. Next, 100 μl of serum in Reagent Diluent was added in each well and incubated for 2 h. The solutions were aspirated, washed three times and added 100 μl of detection antibodies were applied. The plates were then incubated for another 2 h. Next, each well was incubated with Streptavidin-HRP at room temperature for 20 min. The plates were washed three times before incubation with Substrate Solution for 20 min. Lastly, the reaction was stopped by adding Stop Solution and the plates were read at 450 nm and 570 nm using a microplate reader (Azure Biosystems).

After 28 days of treatment, the high-dose of methanol extract of C. nutans significantly reduced the volume and weight of the tumor in mice. The weight of tumors was reduced in the low-dose group (1.396 ± 0.251 g) and high-dose group (1.338 ± 0.327 g) compared to the untreated group (1.565 ± 0.357 g) as rendered in Fig. 1a. Similarly, in Fig. 1b, the volume of tumors in the untreated group was 391.0 ± 26.7 mm³, whereas in the low-dose of treatment group, the weight decreased to 370.1 ± 24.9 mm³. Tumor volume also significantly decreased from 391.0 ± 26.7 mm³ to 302.2 ± 40.3 mm³ in the high-dose of C. nutans–treated group.

Several antibodies for immune system marker antibodies (CD3, CD4, CD8, and NK1.1) were used to identify the C. nutans activity on splenocyte cell populations. As rendered in Fig. 2, the population of CD4/CD3 and CD8/CD3 cells was found to increase significantly, independent of low-dose and high-dose of C. nutans treatments compared to the untreated group. There was also a similar pattern in the NK1.1/CD3 population in low-dose and high-dose treatments relative to the untreated group even not significantly different. To further refine the anti-metastatic effect of C. nutans, the secretion of cytokines (IL-2 and IFN-γ) were measured, as conferred with Fig. 3. The expression levels of IL-2 and IFN-γ have increased in both low-dose and high-dose of C. nutans treatments compared to the untreated group. As rendered in Fig. 3, the expression level of IL-2 was fewer in the untreated group (471.1 ± 46.3 pg/ml) compared to low-dose and high-dose of C. nutans groups were 508.9 ± 34.2 pg/ml and 525.7 ± 28.5 pg/ml, respectively. In addition, the level of IFN-γ in the untreated group (187.2 ± 3.24 pg/ml) was decreased to 209.7 ± 12.3 pg/ml and 352.4 ± 24.9 pg/ml in the low-dose- and high-dose-treated mice.

To elucidate the inflammation effect of C. nutans in the tumors, the level of nitrite oxide (NO) was measured. The level of NO was decreased in both low-dose and high-dose of C. nutans treatment groups compared to the untreated group. As presented in Fig. 4, the expression level of NO was decreased from 0.080 ± 0.025 μM/mg in the untreated group to 0.054 ± 0.013 μM/mg in low-dose of treatment and 0.044 ± 0.010 μM/mg in high-dose of C. nutans treatment. To determine the antioxidant properties of C. nutans methanol extract, while, the level of MDA was also elucidated. As shown in Fig. 5, the MDA level in the untreated group was 0.017 ± 0.001 nM/mg, whereas in the low-dose and high-dose of C. nutans treatments were 0.013 ± 0.001 nM/mg and 0.0083 ± 0.001 nM/mg, respectively.

To determine the anti-metastatic activity of the C. nutans in vivo, the tumor sections were stained with hematoxylin and eosin (H&E). As rendered in Fig. 6, abnormal mitotic figures and coarse chromatin data were seen more visible and frequently in the untreated group compared to the low-dose and high-dose of treatment. In addition, the number of mitotic cells decreased in both the low-dose and high-dose of treatment as shown in Fig. 6b. On the other hand, the clonogenic assay was established to elucidate the anti-metastatic properties of C. nutans further. As shown in Fig. 7, the number of colonies produced in the liver and lung with a low-dose of treatment was significantly decreased while no colonies were formed at a high-dose of treatment. The presence of large and irregular cells was reported as metastatic cells in the bone marrow assay as shown in Fig. 8 which has been found only in the untreated group.