Stellettin B-Induced Oral Cancer Cell Death via Endoplasmic Reticulum Stress-Mitochondrial Apoptotic and Autophagic Signaling Pathway

Abstract

Oral squamous cell carcinoma (OSCC) affects tens of thousands of people worldwide. Despite advances in cancer treatment, the 5-year survival rate of patients with late-stage OSCC is low at 50-60%. Therefore, the development of anti-OSCC therapy is necessary. We evaluated the effects of marine-derived triterpene stellettin B in human OC2 and SCC4 cells. Stellettin B dose-dependently decreased the viability of both cell lines, with a significant reduction in OC2 cells at ≥0.1 µM at 24 and 48 h, and in SCC4 cells at ≥1 µM at 24 and 48 h. Terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL)-positive cells were significantly observed at 20 µM of stellettin B at 48 h, with the overexpression of cleaved caspase3 and cleaved poly(ADP-ribose) polymerase (PARP). Moreover, mitochondrial respiratory functions were ablated by stellettin B. Autophagy-related LC3-II/LC3-I ratio and Beclin-1 proteins were increased, whereas p62 was decreased. At 20 µM at 48 h, the expression levels of the endoplasmic reticulum (ER) stress biomarkers calnexin and BiP/GRP78 were significantly increased and mitogen-activated protein kinase (MAPK) signaling pathways were activated. Further investigation using the autophagy inhibitor 3-methyladenine (3-MA) demonstrated that it alleviated stellettin B-induced cell death and autophagy. Overall, our findings show that stellettin B induces the ER stress, mitochondrial stress, apoptosis, and autophagy, causing cell death of OSCC cells.

Keywords: BiP/GRP78; ER stress; autophagy; mitochondrial stress; stellettin B.

Figure 1

The effects of stellettin B on cytotoxicity in OC2 and SCC4 cells. (A) Morphological images of OC2 and SCC4 cells after exposure to 0, 5, 10, or 20 µM stellettin B for 24 and 48 h. Phase-contrast microscopy was performed to observe the cells. Scale bar = 100 μm. (B) Stellettin B treatment affected the viability of human oral squamous cell carcinoma (OSCC) cells at 0, 0.1, 1, 5, 10, 20, 40, 80, 100, and 200 µM at 24 and 48 h, using the crystal violet assay. (C) The same stellettin B concentrations and inhibition (%) converted in the sigmoidal curve of OC2 and SCC4 cells for 24 and 48 h. Error bars represent the mean ± standard error (SE). The data are shown for a minimum of three independent trials in triplicate analyzed using Student’s t-test to determine significance; * p < 0.05 and ** p < 0.01 relative to the control (untreated cells).

Figure 2

Stellettin B-induced cell cycle arrest and apoptosis in human OSCC cells. (A) Analysis of cell cycle distribution of OC2 cells treated with stellettin B was assisted with propidium iodide (PI) staining-based flow cytometry. The cells were exposed to 0, 2.5, 5, 10, 20, and 40 µM stellettin B for 24 h. The peaks in the flowcharts indicate the cell cycle phases of sub-G1, G1, S, and G2/M. (B) Histograms displaying the proportion of each phase of the cell cycle under the indicated stellettin B concentration in OC2 cells. (C) OC2 and SCC4 cells were exposed to 0, 5, 10, and 20 µM stellettin B for 48 h and then stained with TUNEL dye (emitting green fluorescence) and 4,6-diamidino-2-phenylindole (DAPI) (showing blue fluorescence). The cells were placed under a fluorescence microscope for observation. Scale bar = 100 μm. (D) Quantified levels of TUNEL positive cells. (E) Western blot analysis of the expression of the apoptosis-related protein, cleaved caspase 3, PARP, and cleaved PARP, in OC2 and SCC4 cells treated with 0, 5, 10, and 20 µM stellettin B for 48 h. (F) Quantified levels of cleaved caspase 3, PARP, and cleaved PARP by comparing with β-actin. Original, uncropped images of the Western blots are displayed in Supplementary Figure S1A. β-actin was blotted as an internal control, and error bars stand for the mean ± SE. The data are shown for a minimum of three independent trials in triplicate analyzed using Student’s t-test to determine significance; * p < 0.05 and ** p < 0.01 relative to the control (untreated cells).

Figure 3

Stellettin B-induced down-regulation of mitochondrial respiration in OC2 cells at 0, 5, 10, 20 μM. (A) Measurement of OCRs and time (min) curve. (B) Analysis of basal respiration. (C) Analysis of ATP production. (D) Analysis of proton leak. (E) Analysis of maximal respiration capacity. (F) Analysis of spare respiration capacity. (G) Analysis of non-mitochondrial respiration. (H) Analysis of extracellular acidification rate. The data are shown for a minimum of three independent trials in triplicate analyzed using Student’s t-test to determine significance; ** p < 0.01 relative to the stellettin B untreated groups.

Figure 4

The effects of stellettin B on the levels of ER stress-related protein biomarkers calnexin and BiP/GRP78. OC2 and SCC4 cells were exposed to 0–20 µM stellettin B for 24 and 48 h. (A) Western blot analysis of the expression of ER stress proteins BiP/GRP78 and calnexin. (B) Quantified levels of BiP/GRP78 and calnexin. Original, uncropped Western blots images are displayed in Supplementary Figure S2. β-actin was used as an internal control. The data are shown for a minimum of three independent trials in triplicate analyzed using Student’s t-test to determine significance; * p < 0.05 relative to the stellettin B untreated groups.

Figure 5

Stellettin B regulated MAPK signaling. (A) Western blotting for p38, p-p38, ERK, pERK, JNK, and pJNK levels in OC2 and SCC4 cells treated with 0–20 µM stellettin B for 24 and 48 h. (B) Quantification of the MAPKs. Original, uncropped Western blot images are displayed in Supplementary Figure S3. β-actin was blotted as an internal control, and error bars stand for the mean ± SE. The data are shown for a minimum of three independent trials in triplicate analyzed using Student’s t-test to determine significance; * p < 0.05 and ** p < 0.01 relative to the control (untreated cells).

Figure 6

The effects of stellettin B on autophagy-related AVOs and autophagy proteins p62, LC3-I/II, and Beclin-1. (A) The fluorescence intensity of AO measured for detecting autophagy at 0–20 µM stellettin B for 24 and 48 h using a Beckman CytoFLEX flow cytometer. (B) Quantified levels of AVO-positive cells after exposure to 0–20 µM stellettin B for 24 and 48 h were analyzed using Beckman CytoExpert flow analysis software (v.2.5., Indianapolis, IN, USA). (C) OC2 and SCC4 cells were exposed to 0-20 µM stellettin B for 24 and 48 h. Western blot analysis was performed with the use of antibodies against autophagy-related proteins LC3, p62, Beclin-1, and β-actin. (D) Quantified levels of the proteins in Figure 3C. Original, uncropped images of the Western blots are displayed in Supplementary Figure S1B. β-actin was used as an internal control, and error bars stand for the mean ± SE. The data are shown for a minimum of three independent trials in triplicate analyzed using Student’s t-test to determine significance; * p < 0.05 and ** p < 0.01 relative to the control (untreated cells).

Figure 7

The rescue effects of the autophagy inhibitor, 3-MA, on stellettin B-induced cell viability and autophagic proteins. (A) OC2 and SCC4 cells were pre-exposed to 1 mM 3-MA prior to exposure to 10 µM stellettin B for 48 h. Cell viability was determined using MTT-based assay. (B) Autophagy was evaluated by conducting immunofluorescence staining of LC3 proteins (showing green signal), while the nuclei were labeled with DAPI (showing blue signal). Scale bar = 50 μm. On the merged images, the white squared areas are enlarged and placed on the right shown as zoomed-in. (C) Western blotting for autophagy-related proteins p62 and LC3-I/II in OC2 and SCC4 cells after 48 h treatment. (D) Quantified levels of p62 and LC3-I/II. Original, uncropped Western blot images are displayed in Supplementary Figure S4, and error bars stand for the mean ± SE. The data are shown for a minimum of three independent trials in triplicate analyzed using Student’s t-test to determine significance; * p < 0.05 and # p < 0.05 relative to the 3-MA untreated and stellettin B untreated groups, respectively.

Figure 8

Proposed mechanisms of stellettin B-induced anti-oral cancer effect.