Dehydrocrenatidine Extracted from Picrasma Quassioides Induces the Apoptosis of Nasopharyngeal Carcinoma Cells Through the JNK and ERK Signaling Pathways

ONCOLOGY REPORTS 46: 166, 2021 Dehydrocrenatidine extracted from Picrasma quassioides induces the apoptosis of nasopharyngeal carcinoma cells through the JNK and ERK signaling pathways MING‑CHANG HSIEH1,2, YU‑SHENG LO3, YI‑CHING CHUANG3, CHIA‑CHIEH LIN3, HSIN‑YU HO3, MING‑JU HSIEH3‑6 and JEN‑TSUN LIN6‑8 1School of Medical Laboratory and Biotechnology, Chung Shan Medical University; 2Department of Clinical Laboratory, Chung Shan Medical University Hospital, Taichung 40201; 3Oral Cancer Research Center, Changhua Christian Hospital, Changhua 500; 4Institute of Medicine, Chung Shan Medical University, Taichung 40201; 5Graduate Institute of Biomedical Sciences, China Medical University, Taichung 404; 6Post Baccalaureate Medicine, National Chung Hsing University, Taichung 402; 7Division of Hematology and Oncology, Department of Medicine, Changhua Christian Hospital, Changhua 500; 8School of Medicine, Chung Shan Medical University, Taichung 40201, Taiwan, R.O.C. Received March 16, 2021; Accepted June 2, 2021 DOI: 10.3892/or.2021.8117 Abstract. Nasopharyngeal carcinoma (NPC) is an indicator membrane potential, and activated death receptors). Western disease in Asia due to its unique geographical and ethnic distri‑ blot analysis illustrated that DC's effect on related proteins bution. Dehydrocrenatidine (DC) is a β‑carboline alkaloid in the mitogen‑activated protein kinase pathway can induce abundantly present in Picrasma quassioides (D. Don) Benn, apoptosis by enhancing ERK phosphorylation and inhibiting a deciduous shrub or small tree native to temperate regions of Janus kinase (JNK) phosphorylation. Notably, DC induced southern Asia, and β‑carboline alkaloids play anti‑inflamma‑ apoptosis by affecting the phosphorylation of JNK and ERK, tory and antiproliferative roles in various cancers. However, and DC and inhibitors (SP600125 and U0126) in combination the mechanism and function of DC in human NPC cells remain restored the overexpression of p‑JNK and p‑ERK. To date, this only partially explored. The present study aimed to examine the is the first study to confirm the apoptosis pathway induced by cytotoxicity and biochemical role of DC in human NPC cells. DC phosphorylation of p‑JNK and p‑REK in human NPC. On The MTT method, cell cycle analysis, DAPI determination, the basis of evidence obtained from this study, DC targeting Annexin V/PI double staining, and mitochondrial membrane the inhibition of NPC cell lines may be a promising future potential examination were performed to evaluate the effects strategy for NPC treatment. of DC treatment on human NPC cell lines. In addition, western blotting analysis was used to explore the effect of DC on apop‑ Introduction tosis and signaling pathways in related proteins. The analysis results confirmed that DC significantly reduced the viability The most common malignant tumor of nasopharyngeal of NPC cell lines in a dose‑ and time‑dependent manner and epithelial cells is nasopharyngeal carcinoma (NPC) (1). It induced apoptosis through internal and external apoptotic is relatively rare in the world and has a unique geographical pathways (including cell cycle arrest, altered mitochondrial distribution in Asia, particularly in East and Southeast Asia. According to the records of the International Agency for Research on Cancer, approximately 129,000 new cases of nasopharyngeal cancer occurred in 2018, accounting for only Correspondence to: Dr Ming‑Ju Hsieh, Oral Cancer Research 0.7% of all cancers diagnosed in 2018 (2). NPC pathogenesis Center, Changhua Christian Hospital, 135 Nanxiao Street, Changhua is closely related to Epstein‑Barr virus (EBV) infection, County, Changhua 500, Taiwan, R.O.C. human papillomavirus infection, genetic susceptibility, and E‑mail: [email protected] consumption of salted fish. Furthermore, cancer‑derived EBV DNA circulating in plasma has been identified as a tumor Dr Jen‑Tsun Lin, Division of Hematology and Oncology, Department of Medicine, Changhua Christian Hospital, 135 Nanxiao Street, marker for NPC with 96% sensitivity (3,4). The anatomical Changhua County, Changhua 500, Taiwan, R.O.C. location of NPC makes it difficult to access for surgery and E‑mail: [email protected] is highly sensitive to radiation. Therefore, radiotherapy (RT) is the treatment of choice for non‑metastatic NPC (5). Key words: dehydrocrenatidine, nasopharyngeal cancer, apoptosis, Intensity‑modulated radiation therapy is the most commonly MAPK pathway recommended radiation method because of its excellent local control. For locally advanced NPC, concurrent RT and chemo‑ 2 HSIEH et al: DEHYDROCRENATIDINE INDUCES NPC APOPTOSIS therapy is recommended as first‑line treatment (6). Studies tions (0, 25, 50 and 100 µM) of DC were prepared to treat NPC have indicated that simultaneous chemotherapy and RT may cells in subsequent experiments and were incubated for 24 h. increase treatment‑related toxicity and reduce willingness to undergo treatment, which may cause some patients to stop Cell viability. The effect of DC on cell growth was determined using RT (7). Therefore, providing appropriate treatment using the MTT method (20). First, NPC‑BM, NPC‑039 and guidelines and reducing drug toxicity can further increase the RPMI‑2650 cell lines were seeded on a plate (1x104 cells/well), cure rate of cancer. treated with various concentrations of DC, and cultured at 37˚C Natural compounds extracted from plants are used as for 24 h. Then, the culture medium was removed, MTT reagent traditional medicines for treating various diseases, including (final concentration of 0.5 mg/ml) was added to each well, and various cancer types. In addition, natural medicine use has cells were incubated in 5% (v/v) CO2 at 37˚C for >4 h. After relatively low toxicity. Traditional Chinese pharmacopoeia centrifugation, the supernatant was removed. Then, DMSO has used Picrasma quassioides (D. Don) Benn (PQ), a was carefully added to each well to dissolve formazan crystals deciduous shrub or small tree native to temperate regions for measurement. The absorbance was measured at 595 nm by of southern Asia, for the treatment of inflammation, micro‑ using an ELISA microplate reader. Each experimental condi‑ bial infections, and fever. PQ produces various compound tion was repeated three times, and the data were analyzed for types, such as alkaloids (mainly β‑carboline and cathinone at least three independent experimental results. alkaloids), bitter components, and triterpenoids (8). The β‑carboline alkaloids extracted from PQ, which feature Colony formation assays. In a suitable medium, the cell line anti‑inflammatory and antitumor activity, are widely used in was seeded on a 6‑well cell culture plate at a concentration of medical treatment (9‑13). 1x104 cells as described in a previous study (21). Then, the cells Dehydrocrenatidine (DC) is a β‑carboline alkaloid were evenly distributed and incubated followed by culturing abundantly present in PQ (14). Zhao et al demonstrated that with various DC concentrations. The incubation medium was β‑carboline alkaloids (the main active ingredient of medicinal changed twice a week, and the medium was removed after plants) exert anti‑inflammatory effects through the inhibition of two weeks. The colonies were further fixed with formalin and the iNOS pathway (15). The β‑carboline enantiomer extracted stained with 0.5% crystal violet. Finally, a stereo microscope from PQ was found to decrease cell viability and inhibit the was used to count the total number of colonies and colonies proliferation of various cancer cells, such as liver, cervical, and consisting of >50 cells. breast cancer cells (10,14,16,17). Zhao et al demonstrated that the analgesic effect of DC may be achieved through the inhibi‑ Cell cycle analysis. The cells seeded on the plate tion of neuronal excitability (14). Zhang et al showed that the (1x104 cells/well) were treated with various DC concentrations Janus kinase (JAK) inhibitor DC inhibits JAK2 in the tumori‑ and cultured at 37˚C for 24 h as previously described (22). genesis of solid tumors constitutively activated through signal Following the same drug treatment method as specified in transduction and by transcription activator 3 (18). However, previous studies, after the cells were collected through centrif‑ the molecular targeting effect of DC on human NPC cells is ugation and fixed in ethanol, the ethanol was eliminated and unclear. The present study aimed to examine the cytotoxicity the cells were suspended in Muse cell cycle kit reagents and and biochemical role of DC in human NPC cells. placed in the dark at room temperature. Finally, flow cytom‑ etry was used to analyze the cell cycle distribution results. Materials and methods DAPI staining. The NPC cells (1x104 cells/well) were grown Cell culture. Human NPC cell lines [including NPC‑039 and on glass coverslips and then treated with various DC concen‑ NPC‑BM (19)] were provided by Dr Jen‑Tsun Lin, Department trations for 24 h as described in previous research (20). The of Hematology and Oncology, Changhua Christian Hospital. method used for cell processing was the same as that in a RPMI‑2650 head and neck squamous cell line was obtained previous study; according to fixation and permeabilization, from Japanese Collection of Research Bioresources Cell the DAPI dye was applied to stain cells in the dark. Nuclear Bank (JCRB Cell Bank, Osaka, Japan) cultured in Eagle's morphological changes associated with apoptosis were Minimum Essential Medium (Gibco BRL; Thermo Fisher evaluated in at least 500 cells. The resulting

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