Human immortalized nasopharyngeal epithelial cell line NP69 (BFN60870097, BLUEFBIO, Shanghai, China) and human NPC cell lines including C666-1 (BFN608006727, BLUEFBIO, Shanghai, China) and HK-1 (JNO-H0105, Jennio, Guangzhou, China) were cultured (37 ℃, 5% CO₂) in Dulbecco’s modified eagle medium (DMEM) (A4192101, Gibco, Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS) (16140071, Gibco). The cells used in this study were identified via short tandem repeat (STR) and the test results for mycoplasma were negative.

For the treatment of Sesamin alone, NP69, C666-1, and HK-1 cells were directly cultured (24 h) in Sesamin (10, 20, 50, 100, 200 µmol/L) (C₂₀H₁₈O₆, Purity: 99.70%, HY-N0121, MedChemExpress, Shanghai, China) and then collected for other experiments.

Then, HK-1 cells were assigned into 6 groups: Control (24-hour culture in DMEM with 10% FBS); N-acetyl-L-cysteine (NAC) (2-hour culture in 5 mM of NAC (S0077, Beyotime, Shanghai, China) and then 24-hour culture in DMEM with 10% FBS); 3-Methyladenine (3-MA) (2-hour culture in 3 mM 3-MA (M9281, Sigma, St. Louis, MO, USA) and then 24-hour culture in DMEM with 10% FBS); Sesamin (24-hour culture in 50 µmol/L Sesamin); Sesamin+NAC (2-hour culture in 5 mM of NAC and 24-hour culture in 50 µmol/L Sesamin); and Sesamin+3-MA (2-hour culture in 3 mM of 3-MA and 24-hour culture in 50 µmol/L Sesamin).

Methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay kit (M1020, Solarbio, Beijing, China) was used in the cell viability detection. After 24-hour treatment with Sesamin (different concentrations), NP69, C666-1, and HK-1 cells were incubated in a 96-well with the 90 µL fresh FBS-free medium and 10 µL MTT solution (4 h), followed by a 10-minute reaction with 110 µL formazan dissolvent. Finally, the absorbance (490 nm) was obtained by employing an Imark microplate reader (Bio-Rad, Hercules, CA, USA). Relative cell viability (%) = (experimental group OD – blank group OD)/(control group OD – blank group OD) $\times$ 100%; values were then normalized against controls.

Transwell assay was conducted in this part. Sesamin-treated C666-1 and HK-1 cells (both 1.5 $\times$ 10⁵) were suspended with 200 µL FBS-free DMEM. Transwell chambers (354234, Corning Life Sciences, Corning, NY, USA) were inserted into a 24-well plate. After the cells were placed into the chamber, 5% FBS-containing DMEM (700 µL) was added to the plate. After 24 h, the cells invading the lower chamber underwent fixation (4% paraformaldehyde fixative; P0099, Beyotime, Shanghai, China), and dyeing (crystal violet, 15 min; C110704, Aladdin, Shanghai, China). Finally, observation and data analysis were performed using the optical microscope (DM4M, Leica, Solms, Germany) and Image J 1.8.0 software (NIH, Bethesda, MD, USA), respectively. The comparison was performed using data normalization with the control group.

Dichlorodihydrofluorescein diacetate (DCFH-DA) staining was carried out for ROS detection. Sesamin/NAC/3-MA-treated C666-1 and HK-1 cells were subjected to washing with phosphate-buffered saline (PBS) thrice, followed by incubation with DCFH-DA staining buffer (S0033M, Beyotime, Shanghai, China) (30 min, 37 ℃). 2-(4-Amidinophenyl)-6-indolecarbamidine dihydrochloride (DAPI, Blue, C1002, Beyotime, Shanghai, China) was used to stain nuclei for 5 min. The observation was completed under a STELLARIS 5 laser confocal microscope ($\times$200, Leica, Solms, Germany). The ROS level is represented by the fluorescence intensity of DCF. The comparison was performed using data normalization with the control group. The quantitation of mean fluorescence intensity through ImageJ (version 1.48v, NIH, Bethesda, MD, USA) was used to compare the ROS changes.

Evaluation and analyses were accomplished via JC-1 staining and flow cytometry, respectively. Sesamin/NAC/3-MA-treated C666-1 and HK-1 cells were subjected to PBS washing (three times), followed by 20-minute incubation with JC-1 staining buffer (C2006, Beyotime, Shanghai, China) (37 ℃). Finally, cell MMP was dissected using a FACSCalibur^TM flow cytometer (BD Biosciences, Franklin Lake, NJ, USA).

Colony formation assays were performed. Sesamin/NAC/3-MA-treated C666-1 and HK-1 cells were washed with PBS three times. Then, the cells (1.0 $\times$ 10⁴) were resuspended in 2 mL 10% FBS-containing DMEM. A total of 200 µL cell resuspension in each well of a 6-well plate underwent a 14-day culture, and cell colonies were first fixed (4% paraformaldehyde fixative) and then stained (crystal violet) for 15 min. Following the removal of extra crystal violet using PBS three times, cell colonies were photographed with a camera (D-LUX7, Leica, Weztlar, Germany), and quantified using Image J 1.8.0 software. The comparison was performed using data normalization with the control group.

In this part, propidium iodide (PI) staining and flow cytometry were carried out. Sesamin/NAC/3-MA-treated C666-1 and HK-1 cells were washed with PBS three times, followed by 1-hour fixation (75% pre-cooling ethanol, M058838, MREDA, Beijing, China), 25-minute color development (PI buffer, ST511, Beyotime, Shanghai, China), and cell cycle distribution dissection (a flow cytometer).

Using Annexin-V-FITC/propidium iodide (PI) staining and flow cytometry, cell apoptosis was examined. Following PBS washing thrice, Sesamin/NAC/3-MA-treated C666-1 and HK-1 cells were exposed to Annexin-V-FITC (C1062S, Beyotime, Shanghai, China) and PI (30 min, darkness). Finally, dissection on cell cycle distribution was completed by utilising a flow cytometer.

Post PBS washing thrice, Sesamin/NAC/3-MA-treated C666-1 and HK-1 cells were exposed to NP-40 (P0013F, Beyotime, Shanghai, China) (15 min) on ice and centrifuged (20 min, 14,000 $\times$g) for total protein collection. After protein concentration determination (bicinchoninic acid assay (BCA) kit, P0009, Beyotime, Shanghai, China), 25 µg of proteins that were separated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels (P0052A, Beyotime, Shanghai, China) were transferred transferred to polyvinylidene difluoride membranes (ISEQ00010, Millipore, Billerica, MA, USA). After a 2-hour blockage (5% defatted milk, room temperature), membranes were incubated with primary (overnight, 4 ℃) and secondary (1.5 h, room temperature) antibodies (Abcam, Cambridge, UK): cleaved caspase-3 (1: 500, 17 kDa, ab2302), caspase-3 (1:5000, 32 kDa, ab32351), cleaved poly (ADP-ribose) polymerase 1 (PARP1) (1:5000, 25 kDa, ab32064), PARP1 (1:1000, 113 kDa, ab191217), Cyclin B1 (1:500, 55 kDa, ab72), microtubule-associated protein light chain 3 (LC-3)-I/II (1:3000, 16/18 kDa, ab51520), Beclin-1 (1:2000, 52 kDa, ab207612), P62 (1:10,000, 47 kDa, ab91526), and $\beta$-actin (1:5000, 42 kDa, ab8226), as well as rabbit (1:5000, ab205718) or mouse secondary antibody (1:5000, ab205719). The membrane was covered with developer solution (P0019, Beyotime, Shanghai, China) and the protein gray value was evaluated and anatomized using the Image Lab 3.0 Software (Bio-Rad, Hercules, CA, USA). The relative protein expression level = gray value of the target protein/gray value of $\beta$-actin. The comparison was performed using data normalization with the control group.

Eighteen female BALB/c nude mice (5–6 weeks old and 20 $\pm$ 2 g) were fed in the same specific pathogen-free environment with a 12-hour light/dark cycle, and diet obtained ad libitum. After a 5-day acclimation, three groups (n = 6/group) were set up based on the animals: the Model, the Sesamin-L, and the Sesamin-H.

Then, all mice received injection with 100 µL PBS containing 1 $\times$ 10⁶ HK-1 cells via the right forelimb (above axillary fossa). After the cells were grown in the mice, gavage (once a day for 14 days) was separately performed on the Model, Sesamin-L, and Sesamin-H groups with 100 µL sterile normal saline (M051054, MREDA, Beijing, China), 50 mg/kg Sesamin, and 100 mg/kg Sesamin. Body weight and tumor volume measurements were performed every other day, and the tumor volume was calculated as follows: major diameter (L) $\times$ (minor diameter)² (W²)/2 mm³. On the last day, after anesthesia (5%, 50 mg/kg sodium pentobarbital; B005, Jiancheng Bioengineering, Nanjing, China) and euthanization (cervical dislocation), the tumors were collected, photographed, weighed, and further used for immunohistochemical analysis.

After being photographed and weighed, the tumor tissue underwent fixation (24 h, paraformaldehyde fixative, P885233, MREDA, Beijing, China), transparentization (xylene, M056094, MREDA, Beijing, China), and paraffin embedding (M060593, MREDA, Beijing, China). A total of 4 µm tissue slices were prepared and dewaxed. Then, the slices were placed in an antigen repair buffer (M052013, MREDA, Beijing, China) and incubated with Ki-67 (1:500, ab15580, Abcam, Cambridge, UK) or cleaved caspase-3 (1:500, #9661, CST, Boston, MA, USA) antibodies (overnight, 4 ℃), and reacted (1 h) with rabbit antibody (ab205718, Abcam, Cambridge, UK). After DBA (SFQ004, 4A Biotech, Beijing, China) and hematoxylin (M1428, MREDA, Beijing, China) stainings, the slices were transparentized using xylene and sealed with neutral gum (M051989, MREDA, Beijing, China). Lastly, the tissue image ($\times$200) was recorded with an automatic DMLA microscope (Leica, Wetzlar, Germany). The comparison was performed using data normalization with the Model group.

Multi-group comparisons were completed by employing one-way ANOVA. All statistical analyses were implemented using GraphPad Prism 8.0 software (GraphPad Software, Inc., San Diego, CA, USA), with the final data described using Mean $\pm$ Standard Deviation. Tukey’s multiple comparison test and Dunnett’s multiple comparison test were used for statistical analyses. p 0.05 was determined to be of statistical significance.

Fig. 1A exhibited the chemical structure of Sesamin. The means of how Sesamin influences the viability of normal NP69 cells (Fig. 1B), as well as NPC C666-1 cells (Fig. 1C) and HK-1 cells (Fig. 1D), were determined. The results showed that 10/20/50 µmol/L Sesamin did not affect NP69 cell viability (Fig. 1B), while 100/200 µmol/L Sesamin reduced its viability (p 0.05; Fig. 1B). In the NPC C666-1 and HK-1 cells, 20/50/100/200 µmol/L Sesamin significantly weakened the viability (p 0.05; Fig. 1C,D). Therefore, 20 and 50 µmol/L Sesamin were applied for later assays. Besides, according to evaluation results, the inhibited proliferation and promoted apoptosis of Sesamin-induced NPC cells were found (Fig. 1E–H, p 0.001). Collectively, Sesamin may reduce viability and proliferation while inducing the apoptosis of NPC cells.

Fig. 1.

Sesamin reduced viability and proliferation while enhancing apoptosis of NPC cells. (A) The chemical structure of Sesamin. (B–D) Viability of NP69 cells (B), C666-1 cells (C), and HK-1 cells (D) after Sesamin treatment (MTT assay). (E,F) Proliferation of C666-1 cells (E) and HK-1 cells (F) after Sesamin treatment (colony formation assay). (G,H) Apoptosis of C666-1 cells (G) and HK-1 cells (H) after Sesamin treatment (flow cytometry). (^*p 0.05, ^**p 0.01,^*⁣**p 0.001, vs. Control, n = 3). NPC, nasopharyngeal carcinoma; MTT, methylthiazolyldiphenyl-tetrazolium bromide.

As depicted in Fig. 2A,B, the migration rates of both NPC cells C666-1 (Fig. 2A) and HK-1 (Fig. 2B) were diminished by Sesamin (p 0.001). Also, the cell cycle distribution after Sesamin induction (Fig. 2C,D) was confirmed to be decreased in the S phase (p 0.05) and increased in the G0–G1 phase (p 0.05) of the C666-1 cells (Fig. 2C) and HK-1 cells (Fig. 2D), which indicated that Sesamin inhibited the cell cycle progression of NPC cells and hindered the cell cycle in G0–G1 phase.

Fig. 2.

Sesamin inhibited the migration and cell cycle progression of NPC cells. (A,B) Migration of C666-1 cells (A) and HK-1 cells (B) after Sesamin treatment (transwell assay). Scale bars, 50 µm. (C,D) Cell cycle distribution of C666-1 cells (C) and HK-1 cells (D) after Sesamin treatment (flow cytometry). (^*p 0.05, ^**p 0.01, ^*⁣**p 0.001, vs. Control, n = 3). NPC, nasopharyngeal carcinoma.

The quantification of apoptosis (cleaved caspase-3 and cleaved PARP1)- and cell cycle (Cyclin B1)-related factors was determined (Fig. 3A–H). The results show that due to Sesamin treatment, cleaved caspase-3/caspase-3 and cleaved PARP1/PARP1 levels in C666-1 cells (Fig. 3A–C) and HK-1 cells (Fig. 3E–G) were up-regulated (p 0.01), while Cyclin B1 level in C666-1 cells (Fig. 3A,D) and HK-1 cells (Fig. 3E,H) were down-regulated (p 0.001). Furthermore, autophagy-related factors (LC3-I, LC3-II, Beclin-1, and P62) were also quantified (Fig. 3I–P). In response to Sesamin treatment, LC3-II/LC3-I ratio and Beclin-1 level in C666-1 cells (Fig. 3I–K) and HK-1 cells (Fig. 3M–O) were elevated (p 0.01), while P62 level in C666-1 cells (Fig. 3I,L) and HK-1 cells (Fig. 3M,P) were deceased (p 0.05). This evidence further confirmed that Sesamin promoted NPC cell apoptosis and autophagy.

Fig. 3.

Sesamin influenced expressions of factors related to apoptosis/cell cycle/autophagy in NPC cells. (A–H) Expressions of apoptosis-related (cleaved caspase-3, caspase-3, cleaved PARP1, and PARP1) and cell cycle-related (Cyclin B1) factors in C666-1 cells (A–D) and HK-1 cells (E–H) after Sesamin treatment (Western blot assay). (I–P) Expressions of autophagy-related factors (LC3-I, LC3-II, Beclin-1, and P62) in C666-1 cells (I–L) and HK-1 cells (M–P) after Sesamin treatment (Western blot assay). $\beta$-actin acted as an internal control. (^*p 0.05, ^**p 0.01, ^*⁣**p 0.001, vs. Control, n = 3). NPC, nasopharyngeal carcinoma; PARP1, cleaved poly (ADP-ribose) polymerase 1.

Next, the ROS level in NPC cells after Sesamin induction was evaluated (Fig. 4A,B) and the results exhibited that the ROS level in the C666-1 cells (Fig. 4A) and the HK-1 cells (Fig. 4B) was remarkably up-regulated by Sesamin (p 0.05). Besides, as illustrated in Fig. 5A,B, the MMP in the two cells were both decreased after Sesamin treatment (p 0.001). These data proved Sesamin may influence NPC cells by mediating excessive production of ROS.

Fig. 4.

Sesamin increased ROS production. (A,B) The ROS level in C666-1 cells (A) and HK-1 cells (B) after Sesamin treatment (DCFH-DA staining). Scale bars, 100 µm. (^*p 0.05, ^**p 0.01, ^*⁣**p 0.001, vs. Control, n = 3). ROS, reactive oxygen species; DCFH-DA, dichlorodihydrofluorescein diacetate.

Fig. 5.

Sesamin decreased MMP of NPC cells. (A,B) The mitochondrial membrane potential of C666-1 cells (A) and HK-1 cells (B) after Sesamin treatment (flow cytometry). (^*⁣**p 0.001, vs. Control, n = 3). MMP, mitochondrial membrane potential; NPC, nasopharyngeal carcinoma.

To ascertain whether Sesamin influences NPC cells by regulating ROS production and autophagy, the scavenger of ROS (NAC) and the inhibitor of autophagy (3-MA) were used to pre-treat the cells before Sesamin treatment. As depicted in Fig. 6A, the ROS production in HK-1 cells was decreased by 3-MA (p 0.01) and increased by Sesamin (p 0.001), while post-co-treatment with Sesamin and NAC or 3-MA, the effect of Sesamin on ROS production was weakened by NAC or 3-MA (p 0.05). Meanwhile, the influence of Sesamin on decreasing MMP of HK-1 cells was also attenuated by NAC and 3-MA, respectively (Fig. 6B, p 0.001). Furthermore, the proliferation (Fig. 7A) and cell cycle distribution (Fig. 7B) were determined. The inhibiting role of Sesamin in HK-1 cell proliferation was weakened by both NAC and 3-MA (Fig. 7A, p 0.01). As for the cell cycle distribution (Fig. 7B), NAC and 3-MA increased the S phase level (p 0.05), while Sesamin decreased the S phase and G2 phase level (p 0.01) and increased the G0–G1 phase level (p 0.001) of the cells. Notably, after treatment with Sesamin and NAC or Sesamin and 3-MA, the influence of Sesamin on the cell cycle distribution was counteracted by NAC or 3-MA (p 0.05). All the discoveries indicated that Sesamin mediated proliferation and cell cycle distribution of NPC cells by regulating ROS production and autophagy.

Fig. 6.

Inhibitors of ROS and autophagy neutralized the effect of Sesamin on ROS production, and MMP of the NPC cells. (A) The ROS level in HK-1 cells after being co-treated with Sesamin and NAC or 3-MA (DCFH-DA staining). Scale bars, 100 µm. (B) Mitochondrial membrane potential of HK-1 cells after being co-treated with Sesamin and NAC or Sesamin and 3-MA (flow cytometry). (^*p 0.05, ^**p 0.01, ^*⁣**p 0.001, vs. Control; ^#⁢#⁢#p 0.001, vs. NAC; ^{^⁢^⁢^}p 0.001, vs. 3-MA; ^&p 0.05, ^&⁣&&p 0.001, vs. Sesamin, n = 3). NPC, nasopharyngeal carcinoma; ROS, reactive oxygen species; NAC, n-acetyl-L-cysteine.

Fig. 7.

Inhibitors of ROS and autophagy neutralized the effect of Sesamin on proliferation, and cell cycle progression of the NPC cells. (A) Proliferation of HK-1 cells after being co-treated with Sesamin and NAC or 3-MA (colony formation assay). (B) Cell cycle distribution of HK-1 cells after being co-treated with Sesamin and NAC or 3-MA (flow cytometry). (^*p 0.05, ^**p 0.01, ^*⁣**p 0.001, vs. Control; ^#p 0.05, ^#⁢#p 0.01, ^#⁢#⁢#p 0.001, vs. NAC; ^{^}p 0.05, ^{^⁢^}p 0.01, ^{^⁢^⁢^}p 0.001, vs. 3-MA; ^&p 0.05, ^&&p 0.01, ^&⁣&&p 0.001, vs. Sesamin, n = 3). NPC, nasopharyngeal carcinoma; ROS, reactive oxygen species.

In addition, HK-1 cell apoptosis was determined (Fig. 8A) and the results showed that Sesamin-induced changes in apoptosis were counteracted by NAC and 3-MA (p 0.001). Meanwhile, NAC and 3-MA abrogated Sesamin-induced up-regulated cleaved caspase-3/caspase-3 (Fig. 8B,C, p 0.01) and cleaved PARP1/PARP1 (Fig. 8B,D, p 0.05) and down-regulated Cyclin B1 (Fig. 8B,E, p 0.001). As for the expressions of autophagy-related factors (Fig. 8F–I), LC3-II/LC3-I ratio (Fig. 8F,G) was decreased by NAC (p 0.001) or 3-MA (p 0.001), which also further weakened the promoting effect of Sesamin on LC3-II/LC3-I ratio (p 0.01). Sesamin-induced up-regulation in Beclin-1 (Fig. 8F,H, p 0.001) and down-regulation in P62 (Fig. 8F,I, p 0.001) were all weakened by NAC or 3-MA (p 0.01). All the findings proved that Sesamin regulated NPC cell apoptosis by impacting apoptosis/autophagy-related factors.

Fig. 8.

Inhibitors of ROS and autophagy abrogated Sesamin-triggered changes in the apoptosis and expressions of apoptosis/autophagy-related factors in NPC cells. (A) HK-1 cell apoptosis after being co-treated with Sesamin and NAC or 3-MA (flow cytometry). (B–E) The expressions of apoptosis- (cleaved caspase-3/caspase-3 and cleaved PARP1/PARP1) and cell cycle (Cycle B1)-related factors in HK-1 cells after being co-treated with Sesamin and NAC or 3-MA (Western blot assays). (F–I) The expressions of autophagy-related factors (LC3-I, LC3-II, Beclin-1, and P62) in HK-1 cells after being co-treated with Sesamin and NAC or 3-MA (Western blot assay). $\beta$-actin functioned as an internal control in Western blot assays. (^**p 0.01, ^*⁣**p 0.001, vs. Control; ^#p 0.05, ^#⁢#p 0.01, ^#⁢#⁢#p 0.001, vs. NAC; ^{^}p 0.05, ^{^⁢^}p 0.01, ^{^⁢^⁢^}p 0.001, vs. 3-MA; ^&p 0.05, ^&&p 0.01, ^&⁣&&p 0.001, vs. Sesamin, n = 3). NPC, nasopharyngeal carcinoma.

Finally, to further verify the influence of Sesamin in vivo, the xenografted tumor mice model was established and treated with Sesamin. As shown in Fig. 9A, Sesamin treatment significantly inhibited the increases in tumor volume (p 0.001, Fig. 9A) and weight (p 0.001, Fig. 9D) without side effects on mouse body weight (Fig. 9B). Fig. 9C displayed solid tumor. In addition, Sesamin treatment visibly decreased the Ki-67 level (p 0.05, Fig. 9E,G) and augmented cleaved caspase-3 level (p 0.05, Fig. 9F,H) in the tumor tissues. All these discoveries further proved that Sesamin promoted the apoptosis of NPC cells and inhibited NPC growth.

Fig. 9.

Sesamin down-regulated Ki-67 and up-regulated cleaved caspase-3 in NPC tumors, as well as inhibited the growth of NPC in vivo. (A) Tumor volume subcutaneously growing in nude mice on days 0, 2, 4, 6, 8, 10, 12, or 14, n = 6. (B) The body weight of the mice on days 0, 2, 4, 6, 8, 10, 12, or 14 when tumor subcutaneously grown in nude mice, n = 6. (C) Pictures of xenografted tumor subcutaneously grown in nude mice for 14 days, n = 3. (D) Tumor weight after the tumor subcutaneously grown in nude mice for 14 days, n = 3. (E,G) Ki-67 expression in the tumor tissues (immunohistochemical analysis). Scale bars, 100 µm. (F,H) Cleaved caspase-3 expression in the tumor tissues (immunohistochemical analysis). (^*p 0.05, ^**p 0.01, ^*⁣**p 0.001, vs. Model, n = 3). Scale bars, 100 µm. NPC, nasopharyngeal carcinoma.