MOLECULAR MEDICINE REPORTS 12: 6293-6299, 2015
Cytotoxic and apoptogenic effects of Strobilanthes crispa Blume extracts on nasopharyngeal cancer cells RHUN YIAN KOH1, YI CHI SIM2, HWEE JIN TOH2, LIANG KUAN LIAM2, RACHAEL SZE LYNN ONG2, MEI YENG YEW3, YEE LIAN TIONG4, ANNA PICK KIONG LING1, SOI MOI CHYE1 and KHUEN YEN NG3 1
Department of Human Biology, School of Medicine; 2School of Pharmacy and Health Sciences, International Medical University, Kuala Lumpur 57000; 3Jeffrey Cheah School of Medicine and Health Sciences, Monash University Malaysia, Bandar Sunway, Selangor 47500; 4School of Postgraduate Studies and Research, International Medical University, Kuala Lumpur 57000, Malaysia Received May 14, 2014; Accepted June 3, 2015 DOI: 10.3892/mmr.2015.4152 Abstract. The chemotherapeutic agents used to treat nasopharyngeal cancer (NPC) exhibit low efficacy. Strobilanthes crispa Blume is widely used for its anticancer, diuretic and anti‑diabetic properties. The present study aimed to determine the cytotoxic and apoptogenic effects of S. crispa on CNE‑1 NPC cells. A 3‑(4,5‑dimethylthiazol‑2‑yl)‑2,5 diphenyl tetrazolium bromide assay was used to evaluate the cytotoxic effects of S. crispa against CNE‑1 cells. The rate of apoptosis was determined using propidium iodide staining and caspase assays. Ethyl acetate, hexane and chloroform extracts of S. crispa leaves all exhibited cytotoxic effects on CNE‑1 cells, at a half maximal inhibitory concentration (IC50) of 119, 123.5 and 161.7 µg/ml, respectively. In addition, hexane, chloroform and ethyl acetate extracts of S. crispa stems inhibited CNE‑1 cell proliferation, at a IC50 of 49.4, 148.3 and 163.5 µg/ml, respectively. Flow cytometric analysis revealed an increased proportion of cells in the sub G1 phase and a decreased proportion of cells in the G2/M phase, following treatment with the extracts. However, the extracts did not alter the activities of caspase ‑3/7, ‑8 and ‑9. No cytotoxic effect was observed when the cells were treated with the methanol and water extracts of S. crispa stems and leaves. In conclusion, the S. crispa extracts were cytotoxic against CNE‑1 cells and these extracts were able to induce apoptosis, independent of caspase activation. Introduction Nasopharyngeal carcinoma (NPC) is a tumor, which arises from the epithelial cells of the nasopharynx. NPC can be
Correspondence to: Dr Rhun Yian Koh, Department of Human Biology, School of Medicine, International Medical University, 126 Jalan Jalil Perkasa 19, Bukit Jalil, Kuala Lumpur 57000, Malaysia E‑mail:
[email protected]
Key words: apoptosis, cytotoxicity, Strobilanthes crispa Blume, nasopharyngeal cancer
classified into three subtypes: Squamous cell carcinoma, non‑keratinizing carcinoma and undifferentiated carcinoma. The exact etiology of NPC remains to be elucidated, however, it has been suggested that Epstein‑Barr virus may be one of the causes of NPC, since it has been reported to be associated with epithelial cell transformation into NPC type 2 and 3 (1,2). In addition, type 2 (non‑keratinizing carcinoma) and 3 (undifferentiated carcinoma) NPCs are associated with increased titers of the virus (3). Diet, genetic factors, gender and ethnicity have also been regarded as possible risk factors of NPC. NPC is a rare malignancy in the majority of countries, with an incidence rate of 3 were considered to have high selectivity (21). Morphological assessment of cells. The CNE‑1 cells (1x10 6 cells per well of 6‑well plate) were treated with various extracts at their IC50 concentration for 72 h at 37˚C. Morphological changes of the cells were observed under an inverted microscope (Nikon Eclipse TS100; Nikon Corporation, Tokyo, Japan) and micrographs were captured. Determination of population doubling time (PDT). The PDT was calculated to estimate the duration of the cell cycle and compare the effects of the extracts. The cells were counted using a manual hemocytometer (Marienfeld, Lunda‑Königshofen, Germany). Briefly, 1x10 4 CNE‑1 cells were seeded into a 24‑well plate and treated with the various extracts at their IC50 concentration for 72 h at 37˚C. Subsequently, the number of cells were counted following staining with trypan blue (Gibco Life Technologies). The PDT was calculated by dividing the total duration (h) by the total number of generations. The number of generations was calculated by 3.32 (logNN‑logN1), where NN is the number of cells counted and N1 is the number of cells inoculated (22). Flow cytometric analysis. The cells (1x10 6 cells per well of 6‑well plate) were treated with the plant extracts at their respective IC50 concentrations for 72 h at 37˚C were harvested and fixed in 70% ethanol. Following two washes with cold phosphate‑buffered saline, 500 µl propidium iodide (20 µg/ml; Sigma‑Aldrich, St. Louis, MO, USA) and 500 µl RNase (Sigma‑Aldrich) was added to the cells. Flow cytometric analysis (FACScan; BD Biosciences, Franklin Lakes, NJ, USA) was performed following 30 min incubation, in order to investigate the rate of cell apoptosis. Caspase activity assay. The Apo‑ONE ® Homogeneous Caspase‑3/7 Assay kit was purchased from Promega Corporation (Madison, WI, USA) and the Caspase‑8 and‑9 Assay kits were obtained from Calbiochem (Merck KGaA, Darmstadt, Germany). The CNE‑1 cells were treated with the
MOLECULAR MEDICINE REPORTS 12: 6293-6299, 2015
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Table I. IC50 (µg/ml) and selectivity index values of extracts of Strobilanthes crispa on CNE‑1 and NRK‑52E cells. IC50 (µg/ml) ‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑ Plant material Extract/treatment CNE‑1 NRK‑52E Leaves Stems ‑
Hexane Chloroform Ethyl acetate Methanol Water Hexane Chloroform Ethyl acetate Methanol Water 5‑fluorouracil
Selectivity index
123.50±37.50 84.00±1.41 0.68 161.70±20.20 184.50±12.02 1.14 119.00±48.10 166.50±2.12 1.40 N/A N/A ‑ N/A N/A ‑ 49.40±8.00 11.00±2.83 0.22 148.30±23.20 N/A >1.35 163.50±16.30 174.00±5.66 1.06 N/A N/A ‑ N/A N/A ‑ 3.05±1.15 9.60±4.81 3.15
IC50 values are expressed as the mean ± standard deviation of three independent experiments. N/A, Not available; IC50; half maximal inhibitory concentration; SD, standard deviation.
different plant extracts at their IC50 concentrations for 72 h, following which the activities of caspase 3/7, ‑8 and ‑9 were measured using the above-mentioned kits. The detection of caspase activity was performed, according to the manufacturer's instructions. Statistical analysis. The results are presented as the mean ± standard deviation. The data were subjected to one‑way analysis of variance using GraphPad Instat version 3.0 (GraphPad Software, Inc., La Jolla, CA, USA). PLH> SC>LC>SEA extracts of S. crispa. Methanol and water extracts of the S. crispa leaves and stems caused no significant inhibition of cell growth of CNE‑1 cells. The cytotoxicity of the extracts on NRK‑52E cells was also assessed. Similar observations were noted in the NRK‑52E cells as the CNE‑1 cells. SH exhibited the most potent activity against the NRK‑52E cells, with the lowest IC50 value. However, it was not possible to determine the IC50 value of SC. Overall, all of the extracts in the present study exhibited a low SI, with values ranging between 0.22 and 1.40. The anticancer drug 5‑fluorouracil exhibited potent activity against CNE‑1 cell growth, with a low IC50 value (3.05±1.15 µg/ml) and high SI (3.15). Antiproliferative effects of S. crispa extracts. As shown in Fig. 1, all the plant extracts prolonged the PDT of the cells, with
Figure 1. Effects of Strobilanthes crispa extracts on the population doubling time of CNE‑1 nasopharyngeal carcinoma cells. Cells were treated with various plant extracts for 72 h, and the number of cells was counted to determine the population doubling time. Data are presented as the mean ± standard deviation of three independent experiments. *P
