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ORIGINAL ARTICLE
Year : 2016  |  Volume : 12  |  Issue : 1  |  Page : 244-247

Dihydroartemisinin inhibits cell proliferation by induced G1 arrest and apoptosis in human nasopharyngealcarcinoma cells


1 Department of Otolaryngology, Ganzhou People's Hospital, Ganzhou, Jiangxi, 341000, China
2 Department of Otolaryngology, The Second Clinical College of Guangdong Medical College, Dongguan, Guangdong, China
3 The First Affiliated Hospital of Guangdong Medical College, Guangdong Medical College, Zhanjiang, Guangdong, 524023, China
4 The First Affiliated Hospital of Guangdong Medical College; Department of Otolaryngology, Guangdong Medical College, Zhanjiang, Guangdong, 524023, China

Date of Web Publication13-Apr-2016

Correspondence Address:
Guoqing Luo
Department of Otolaryngology, First Affiliated Hospital of Guangdong Medical College, Zhanjiang - 524023, Guangdong
China
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0973-1482.151855

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 > Abstract 


Context: Dihydroartemisinin (DHA) exhibits anticancer activity in a number of human cancer cells. However, it is still unknown whether DHA has anticancer effect on nasopharyngeal cancer (NPC) cells.
Aims: To investigate the anticancer activity of DHA on CNE-2 cells.
Materials and Methods: Cell cycleand invasion assay were used to detect the effect of DHA on CNE-2 cells. Protein molecular differences were examined by Western blot.
Results: DHA strongly inhibited cell proliferation by induced cell cycle G1 arrest and apoptosis in CNE-2 cells. In addition, cell motility, invasion, and colony formation were suppressed by DHA.
Conclusions: DHA shows significant anticancer effects on human NPC cells, and may be a preventive and therapeutic agent against NPC.

Keywords: Cancer, dihydroartemisinin, nasopharyngeal carcinoma, natural compound


How to cite this article:
Huang Z, Huang X, Jiang D, Zhang Y, Huang B, Luo G. Dihydroartemisinin inhibits cell proliferation by induced G1 arrest and apoptosis in human nasopharyngealcarcinoma cells. J Can Res Ther 2016;12:244-7

How to cite this URL:
Huang Z, Huang X, Jiang D, Zhang Y, Huang B, Luo G. Dihydroartemisinin inhibits cell proliferation by induced G1 arrest and apoptosis in human nasopharyngealcarcinoma cells. J Can Res Ther [serial online] 2016 [cited 2019 Nov 14];12:244-7. Available from: http://www.cancerjournal.net/text.asp?2016/12/1/244/151855




 > Introduction Top


Nasopharyngeal carcinoma is endemic in Southeast Asia and Southern China, where it is one of the most common cancers amongst men, with incidence rates of more than 20 cases per 100,000 individuals.[1],[2],[3] The etiology of nasopharyngeal cancer (NPC) is complex, including genetic, environmental, and viral factors.[4],[5],[6] Existing therapies for NPC usually involve the combined use of radiotherapy and systemic chemotherapy.[7] However; it induces a considerable incidence of side effect. Additionally, there is no effective treatment for those who have cancer recurrence and resistant to radiotherapy. Therefore, a possible treatment strategy is to screen traditional Chinese medicinal plants as antitumor agents with few side effects.[8],[9],[10],[11]

Artemisinin is an active principle isolated from traditional Chinese medicinal Artemisia annua, which contains an endoperoxide bridge structure.[12],[13] Artemisinin and its derivatives are very important antimalarial drugs in clinic with fewer adverse side effects. Recent studies have demonstrated that artemisinin and its derivatives have anticancer properties, and low toxicity.[14] The anticancer effects of artemisinin have been proposed to be apoptosisinduction,[15],[16] mediate cell cycle arrest,[15],[17] modulation of the expression of tumor-related genes,[18] and anti-angiogenesis.[19] Dihydroartemisinin (DHA) is one of the most active artemisinin derivative. It has much better water solubility and stronger antimalarial activity than other artemisinin derivatives. Moreover, DHA has showed important cytotoxicity to osteosarcoma, pancreatic cancer, and ovarian cancer.[20],[21],[22]

In the present study, we examined the anticancer activity of DHA on poorly-differentiated human NPC cells. Here, we report that DHAstrongly inhibited cell proliferation by induced cell cycle G1 arrest and apoptosis in CNE-2 cells.


 > Materials and Methods Top


Chemicals

DHA was obtained from Sigma-Aldrich (St Louis, MO). Roswell Park Memorial Institute (RPMI)-1640 medium and fetal bovine serum (FBS) were purchased from Gibco (Grand Island, NY). The antibodies against CDK2, CDK4, cyclin D1, cyclin-E, and p16 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). The antibody against beta-actin (b-actin), Hoechst staining kit, cell cycle and apoptosis analysis kit, and protein assay kit were obtained from Beyotime Biotechnology Corporation (Beyotime, Jiangsu, China).

Cell cycle analysis assay

CNE-2, a poorly-differentiated human NPC cell line, was supplied by the Institute of Biochemistry and Molecular Biology of Guangdong Medical College (Dongguan, China). The cells were seeded (1 × 106 cells/well) in six-well plates with 10% FBS/RPMI-1640 and incubated overnight at 37°C in a 5% CO2 incubator. Cells were then starved in serum-free medium for 24 h followed by treatment for 24 h with DHA (0–40 µM) in 10% FBS/RPMI-1640. The cells were trypsinized and then washed twice with cold PBS, and fixed with ice-cold 70% ethanol at −20°C overnight. Cells were then washed twice with PBS, incubated with 20 mg/ml RNase A and 200 mg/ml propidium iodide in PBS at room temperature for 30 min in the dark, and subjected to flow cytometry using the FACSCalibur flow cytometer. Data were analyzed using ModFit LT (Verity Software House, Inc., Topsham, ME).

Western blot analysis

After the cells (5 × 106) were cultured in a 10-cm dish overnight, they were treated with DHA (0–40 µM) for 24 h and then harvested. The harvested cells were disrupted, and the supernatant fractions were boiled for 5 min. The protein concentration was determined using a dye-binding protein assay kit as described in the manufacturer's manual. Lysate protein (30–50 µg) was subjected to 10% Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and electrophoretically transferred to a polyvinylidene difluoride membrane (GE Healthcare). After blotting, the membrane was incubated with the specific primary antibody at 4°C overnight. Protein bands were visualized by the enhanced chemiluminescence (ECL) solution after hybridization with the horseradish peroxidase (HRP)-linked secondary antibody.

Assessment of nuclear apoptotic morphology

Staining with Hoechst 33,258 (1,1'-dioctadecyl-3, 3, 3',3'-tetramethylindocarbocyanine) was used to observe apoptotic nuclei in a similar procedure as described above. Cells were stained with Hoechst 33,258 staining solution according to the manufacturer's instructions provided with Apoptosis, Hoechst Staining Kit. Stained nuclei were assessed under a laser scanning confocal microscope (TCS-SP2, Leica Microsystems, Heidelberg, Germany).

Wound healing assay

Cells were seeded (2.5 × 104 cells/well) in 24-well plates. After the cells were allowed to attach and reach 80% subconfluency, a scratch was performed through the cell monolayer using a yellow pipet tip and then they were treated with DHA in serum-free mediumfor 24 or 48 h. Cells were washed with PBS before photographs of the scratch area were taken in treated and untreated cells using a charge-coupled device camera (C2400; NEC, Hawthorne, CA) connected to an inverted microscope (TS100-F, Nikon, Japan).

Invasion assay

Invasion assay was performed using matrigel invasion chambers (Becton Dickinson, Sunnyvale, CA, USA). 4 × 104 cells untreated or treated with DHA were seeded into the upper well of the chamber containing serum-free culture medium. The lower well was filled with culture medium containing 10% FCS. After 24 hours, cells on the upper surface of the well were removed and cells on the lower surface were fixed in 95% ethanol and stained with 0.1% crystal violet. Then, the transmigrated cells were counted using a microscope. For each experiment, 10 random high power fields were counted.

Colony formation assay

Cells (1 × 103) were seeded in 10 cm culture plates and cultured in serum-free medium in the absence (control) or presence of DHA. After 10 days of incubation, the cells were stained with 0.1% crystal violet. The number of colonies was counted under a microscope.

Statistical analyses

All data are expressed as means ± standard deviation (SD) of at least three independent experiments. Statistical differences were evaluated by one-way analysis of variance (ANOVA) using commercially available software (SPSS 17.0, SPSS Inc, Chicago, IL). P< 0.05 were considered statistically significant.


 > Results Top


DHA inhibits proliferation of CNE-2 cells by inducing cell cycle G1 phase accumulation and apoptosis

To examine whether treatment with DHA influences cancer cell proliferation, we employed Cell Counting Kit-8 (CCK-8) assays.[23] Low dose (1 μM) treatment with DHA reduced cell growth after 72 h by 26%. An even stronger effect was observed after incubation with 10 or 40 μM DHA, reducing cell growth by at least 36–54%. To determine whether the inhibitory effect on cell proliferation was caused by modulation of cell cycle phase, we investigated the effect of DHA on cellcycle progression. Results showed that the high dose of DHA treatment for 24 h results in an increased accumulation of cells in G1 phase [Figure 1]a. The regulation of cell cycle is primarily controlled by a family of cyclin/cyclin-dependent kinase (CDK) complexes. To confirm DHA induced G1 arrest, we tested the effect of DHA on G1 phase-related protein expression. Western blot data showed decrease in CDK2, CDK4, cyclin D1, and cyclin E and increase in p16 protein level after DHA treatment for 24 h [Figure 1]b.
Figure 1: Cell proliferation and cell cycle. (a) DHA induces significant G1 arrest. Cells were starved in serum-free medium for 24 h and then treated with DHA for 24 h. Cell cycle analysis was performed using flow cytometry. Data are shown as mean ±SD. The asterisks (*) indicate a significant difference (P < 0.05) between groups treated with DHA and the group treated with DMSO. (b) DHA decreases CDK2, CDK4, cyclin D1, and cyclin E and increases p16 protein level. Cells were treated with DHA for 24 h and harvested. The levels of cell cycle related proteins were determined by Western blot analysis. DMSO = dimethyl sulfoxide, DHA = dihydroartemisinin

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Cellular homeostasis, the balance between cell proliferation and cell death is controlled by cell cycle and apoptosis. These results indicated DHA strongly inhibited proliferation of CNE-2 cells by inducing cell cycle G1 phase accumulation and apoptosis.

DHA suppresses migration, invasion, and colony formation of CNE-2 cells

We further investigated the effect of DHA on the migratory behavior of CNE-2 cells by performing wound healing assay. Here, we found that DHA treatment inhibited cell migration in CNE-2 cells [Figure 2]a. In addition, we performed trans-well assay to examine the effect of DHA on the invasive properties of CNE-2 cells. Our findings were comparable to the wound healing assay with a dramatically reduced invasiveness of cells after treatment with DHA [Figure 2]b.
Figure 2: Cell migration, invasion, and colony formation. (a) DHA suppresses migration. After cells were allowed to attach and reach 80% subconfluency, a scratch was performed through the cell monolayer using a yellow pipet tip and then they were treated with DHA in serum-free mediumfor 24 or 48 h. (b) DHA reduces cell invasion. Cell invasion was examined by modified Boyden chamber assay as described in Materials and Methods. Data are shown as means ± SD. The asterisk (*) indicates a significant difference (P < 0.05) between groups treated with DHA and the group treated with DMSO. (c) DHA suppresses colony formation. Colony formation assay was described as in Materials and Methods. Data are shown as means ± SD. The asterisk (*) indicates a significant difference (P < 0.05) between groups treated with DHA and the group treated with DMSO. SD = Standard deviation

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To investigate whether DHA may not only inhibit migration and invasion but also suppress colony formation in CNE-2 cells, soft agar assay was employed. Based on the numbers of cell colonies, treatment with DHA significantly inhibited colony formation [Figure 2]c.


 > Discussion Top


Nasopharyngeal carcinoma is a highly invasive and metastatic cancer in Southeast Asia and Southern China.[17] Until today, NPC is incurable malignant tumors. Therefore, new therapeutic strategies are constantly under investigation. Ideally, chemotherapeutic agents, especially naturally occurring pant products, are gaining more and more attention. Artemisinin is an effective antimalarial drug with low toxicity. Previous studies demonstrated that artemisinin and its derivatives inhibit the growth of various tumors,[15],[19],[20],[21],[22],[24] whereas, whether DHA exhibits therapeutic effects in NPC is still unknown. In the present study, we have shown a chemotherapeutic effect of DHA against NPC and identified a possible mechanism(s).

Cellular homeostasis is controlled by cell-cycle progression and apoptosis induction.[23] Unchecked proliferative potential involving deregulation of cell-cycle progression and apoptosis is generally described as a central progress in the development of tumor.[25],[26] Suppression of cell proliferation and induction of apoptosis are important therapeutic approaches. We examined the effects of DHA on CNE-2 cell proliferation, cell cycle, and apoptosis.[23] The data showed that inhibition of proliferation induced by DHA was associated with G1 cell cycle arrest and apoptosis induction. In addition, Ji et al., showed that DHA induced G2/M phase arrest by up regulation of cyclin D1 expression and concomitant down regulation of Cdc25B and cyclinB1 expressions.[21] Chen et al., suggested that treatment with DHA resulted in a dose-dependent G0/G1cell cycle arrest and regulated the expression of cyclins and CDKs, such as cyclin E, CDK2, CDK4, and p27Kip1 in pancreatic cancer.[20] In agreement with previous studies, we found that DHA up regulated p16 expression and down regulated the expression of CDK2, CDK4, cyclin D1, and cyclinE.

Many anticancer drugs cause cell death through the induction of apoptosis, which induce the intrinsic pathway and activate caspase in apoptosis progression. We determined the effects of DHA on caspase activity. Data showed DHA increased caspase-3 activity and suggested the activation of caspase-3 was involved in the DHA-induced cell apoptosis.[17] In addition to cell growth and death, treatment with DHA affected another hallmark of NPC, that is, migration, invasion, and colony formation. We have shown here DHA potently hampers migration, invasion, and colony formation of CNE-2 cells. Based on our study, DHA may be a good preventive or therapeutic agent against NPC.


 > Conclusion Top


In summary, DHA inhibits NPC cell proliferation, migration, invasion, and colony formation. The inhibition of proliferation occurs mainly through the induction of G1 accumulation and apoptosis. DHA may be aschemotherapeutic agent for NPC and other cancers, which needs further validation of results using animal/preclinical models.


 > Acknowledgments Top


This work was financially supported by the grant of Science and Technology Program of Guangdong Province, 93036 (To G. Luo).

 
 > References Top

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