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. 2021 Aug;46(2):166.
doi: 10.3892/or.2021.8117. Epub 2021 Jun 24.

Dehydrocrenatidine extracted from Picrasma quassioides induces the apoptosis of nasopharyngeal carcinoma cells through the JNK and ERK signaling pathways

Ming-Chang Hsieh  1 , Yu-Sheng Lo  2 , Yi-Ching Chuang  2 , Chia-Chieh Lin  2 , Hsin-Yu Ho  2 , Ming-Ju Hsieh  2 , Jen-Tsun Lin  3
Affiliations

Dehydrocrenatidine extracted from Picrasma quassioides induces the apoptosis of nasopharyngeal carcinoma cells through the JNK and ERK signaling pathways

Ming-Chang Hsieh et al. Oncol Rep. 2021 Aug.

Abstract

Nasopharyngeal carcinoma (NPC) is an indicator disease in Asia due to its unique geographical and ethnic distribution. Dehydrocrenatidine (DC) is a β‑carboline alkaloid abundantly present in Picrasma quassioides (D. Don) Benn, a deciduous shrub or small tree native to temperate regions of southern Asia, and β‑carboline alkaloids play anti‑inflammatory and antiproliferative roles in various cancers. However, the mechanism and function of DC in human NPC cells remain only partially explored. The present study aimed to examine the cytotoxicity and biochemical role of DC in human NPC cells. The MTT method, cell cycle analysis, DAPI determination, Annexin V/PI double staining, and mitochondrial membrane potential examination were performed to evaluate the effects of DC treatment on human NPC cell lines. In addition, western blotting analysis was used to explore the effect of DC on apoptosis and signaling pathways in related proteins. The analysis results confirmed that DC significantly reduced the viability of NPC cell lines in a dose‑ and time‑dependent manner and induced apoptosis through internal and external apoptotic pathways (including cell cycle arrest, altered mitochondrial membrane potential, and activated death receptors). Western blot analysis illustrated that DC's effect on related proteins in the mitogen‑activated protein kinase pathway can induce apoptosis by enhancing ERK phosphorylation and inhibiting Janus kinase (JNK) phosphorylation. Notably, DC induced apoptosis by affecting the phosphorylation of JNK and ERK, and DC and inhibitors (SP600125 and U0126) in combination restored the overexpression of p‑JNK and p‑ERK. To date, this is the first study to confirm the apoptosis pathway induced by DC phosphorylation of p‑JNK and p‑REK in human NPC. On the basis of evidence obtained from this study, DC _targeting the inhibition of NPC cell lines may be a promising future strategy for NPC treatment.

Keywords: MAPK pathway; apoptosis; dehydrocrenatidine; nasopharyngeal cancer.

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Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Figure 1.
Figure 1.
DC causes cytotoxicity in human NPC cells. (A-C) Human NPC cell lines (including NPC-039 and NPC-BM) and RPMI-2650 were individually treated with various concentrations (0, 25, 50, and 100 µM) of DC for 24, 48, and 72 h. Cell viability was analyzed using MTT analysis. (D and E) In the colony formation assay, NPC-039 and NPC-BM cell lines were evenly distributed and incubated and then cultured with various DC concentrations (0, 25, 50, and 100 µM). The incubation medium was changed twice a week, and the medium was removed after 2 weeks. (F) The IC50 values of NPC cells after treatment with DC. *P<0.05 vs. the control group. NPC, nasopharyngeal carcinoma; DC, dehydrocrenatidine; IC50, half maximal inhibitory concentration.
Figure 2.
Figure 2.
DC-mediated cell cycle arrest in human NPC cell lines (including NPC-039 and NPC-BM). (A) NPC-039 and NPC-BM cell lines were treated with DC (0, 25, 50, and 100 µM) to measure and predict the phase distribution of the cell cycle (G0/G1, S, and G2/M) by using flow cytometry. (B) Distribution of cell cycle phases in the NPC-039 and NPC-BM cell lines was detected and compared with the control group. (C and D) Western blotting for the determination of cell cycle marker-related proteins showed the levels of cyclin A, cyclin B, cyclin D3, CDK4, CDK6, p-cdc2, Myt1, p-WEE1, and p-Rb. β-actin was used as an internal standard for protein expression, and the results of protein levels were normalized to β-actin for quantification. *P<0.05 vs. the control group. NPC, nasopharyngeal carcinoma; DC, dehydrocrenatidine; CDK, cyclin-dependent kinase; Myt1, myelin transcription factor 1; WEE1, WEE1 G2 checkpoint kinase; Rb, retinoblastoma protein; p-, phosphorylated.
Figure 3.
Figure 3.
DC induces cell apoptosis in human NPC cell lines (including NPC-039 and NPC-BM). (A) DAPI staining was used to analyze the level of nuclear counterstaining. The characteristic morphology of blebbing nuclei was detected by using fluorescence microscopy. Nuclei condensation and fragmentation are indicated by red arrows. (B) The results of A were quantified by assessing the total cell nuclei with DNA condensation. (C) Annexin V/PI staining was used to reveal DC-induced apoptosis. (D) The percentage distribution of apoptotic cells including early and late apoptotic states. *P<0.05 vs. the control group. NPC, nasopharyngeal carcinoma; DC, dehydrocrenatidine.
Figure 4.
Figure 4.
DC treatment leads to the apoptosis of human NPC cell lines (including NPC-039 and NPC-BM) through the mitochondrial and death receptor pathways. (A) Mitochondrial membrane potential was analyzed after treatment with DC (0, 25, 50, and 100 µM) by using the Muse Cell Analyzer. Quantitative results were analyzed using Muse Cell software and compared with the control group. (B and C) Protein levels of the Fas pathway and the TNF pathway including FADD, TNF-R1, DcR2, cleaved-RIP, and DR5 were detected by western blotting. β-actin was used as an internal standard for protein expression, and the results of protein levels were normalized to β-actin for quantification. *P<0.05 vs. the control group. NPC, nasopharyngeal carcinoma; DC, dehydrocrenatidine; FADD, FAS-associated death domain protein; TNF-R1, tumor necrosis factor receptor 1; DcR2, decoy receptor 2; RIP, ribosome-inactivating protein; DR5, death receptor 5.
Figure 5.
Figure 5.
DC promotes apoptosis through the regulation of apoptosis-related proteins in human NPC cell lines (including NPC-039 and NPC-BM) through extrinsic and intrinsic caspase cell signaling pathways. (A and B) After treatment of NPC-039 and NPC-BM cell lines for 24 h with DC, caspase-3/7 was detected using the Muse caspase-3/7 kit. The level of caspase-3/7 activation was quantitatively analyzed in the treatment group and compared with the control group. (C and D) The activated form of apoptosis proteins was detected through western blotting, including cleaved caspase-3, −8, and −9 and cleaved PARP proteins. (E and F) Expression levels of related proteins, including Bax, Bak, t-Bid, Bcl-xL, and Bcl-2 proteins, were determined and quantified through Western blotting. *P<0.05 vs. the control group. (G and H) Cell lines were pre-treated with Z-VAD-FMK (20 µM) for 1 h, then with treated DC (50 µM) for 24 h. The activated form of apoptosis proteins was detected through western blotting, including cleaved caspase-3, −8, and −9 and cleaved PARP proteins. Protein levels were determined through densitometry, with β-actin as an internal standard for protein expression. Results of all protein levels were normalized to β-actin for quantification compared with the control, *P<0.05 vs. the control group; #P<0.05 vs. DC treatment alone group. NPC, nasopharyngeal carcinoma; DC, dehydrocrenatidine; Bax, BCL2 associated X, apoptosis regulator; Bak, BCL2 antagonist/killer 1; Bid, BH3-interacting domain death agonist; Bcl-xL, B-cell lymphoma-extra large; Bcl-2, B-cell lymphoma 2; PARP, poly(ADP-ribose) polymerase.
Figure 6.
Figure 6.
DC regulates the protein expression of the MAPK pathway in human NPC cell lines (including NPC-039 and NPC-BM). (A) Analysis and quantification of expression levels of AKT, p38, ERK1/2, and JNK1/2 proteins through western blotting. (B) Protein levels were analyzed through densitometry; β-actin was used as an internal standard for protein expression, and all proteins were normalized to β-actin. *P<0.05 vs. the control group. (C-H) NPC-039 and NPC-BM cell lines were pretreated with each MAPK inhibitor, including AKT inhibitor (LY294002), ERK1/2 inhibitor (U0126), and JNK1/2 inhibitor (SP600125), for 1 h and then cotreated with or without DC for 23 h. Analysis and quantitative regulation of protein expression, including cleaved caspase-3, −8, and −9 and cleaved PARP protein, through western blotting. Protein levels were determined through densitometry, with β-actin as an internal standard for protein expression. Results of all protein levels were normalized to β-actin for quantification compared with control, *P<0.05 vs. the control group; #P<0.05 vs. DC treatment alone group. NPC, nasopharyngeal carcinoma; DC, dehydrocrenatidine; PARP, poly(ADP-ribose) polymerase; p-, phosphorylated; AKT, protein kinase B (PKB); p38, p38 mitogen-activated protein kinases; ERK, extracellular signal-regulated kinases; JNK, c-Jun N-terminal kinase.
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Grants and funding

This study was supported by grants from National Science Council, Taiwan (grant no. MOST 109-2314-B-371-005) and Changhua Christian Hospital (grant no. 109-CCH-MST-161).
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