Manoalide Induces Intrinsic Apoptosis by Oxidative Stress and Mitochondrial Dysfunction in Human Osteosarcoma Cells

Abstract

Osteosarcoma (OS) is the most common primary malignant bone tumor that produces immature osteoid. Metastatic OS has a poor prognosis with a death rate of >70%. Manoalide is a natural sesterterpenoid isolated from marine sponges. It is a phospholipase A2 inhibitor with anti-inflammatory, analgesic, and anti-cancer properties. This study aimed to investigate the mechanism and effect of manoalide on OS cells. Our experiments showed that manoalide induced cytotoxicity in 143B and MG63 cells (human osteosarcoma). Treatment with manoalide at concentrations of 10, 20, and 40 µM for 24 and 48 h reduced MG63 cell viability to 45.13-4.40% (p < 0.01). Meanwhile, manoalide caused reactive oxygen species (ROS) overproduction and disrupted antioxidant proteins, activating the apoptotic proteins caspase-9/-3 and PARP (Poly (ADP-ribose) polymerase). Excessive levels of ROS in the mitochondria affected oxidative phosphorylation, ATP generation, and membrane potential (ΔΨ_m). Additionally, manoalide down-regulated mitochondrial fusion protein and up-regulated mitochondrial fission protein, resulting in mitochondrial fragmentation and impaired function. On the contrary, a pre-treatment with n-acetyl-l-cysteine ameliorated manoalide-induced apoptosis, ROS, and antioxidant proteins in OS cells. Overall, our findings show that manoalide induces oxidative stress, mitochondrial dysfunction, and apoptosis, causing the cell death of OS cells, showing potential as an innovative alternative treatment in human OS.

Keywords: antioxidants; apoptosis; inflammation; mitochondria; mitochondrial respiratory chain; osteosarcoma; oxidative stress; reactive oxygen species.

Figure 1

Manoalide influences cell viability, apoptosis, DNA fragmentation, and intrinsic apoptosis pathways in OS cells. (A) The MTT assay was used to determine the effect of manoalide. MG63 cells were treated with manoalide concentrations of 0, 0.1, 1, 5, 10, 20, and 40 µM for 24 and 48 h. Cell viability was expressed as the percentage of viable cells after drug treatment compared to untreated cells. (B) For 24 and 48 h, 143B cells were treated with various concentrations of manoalide. Cell viability was expressed as the percentage of viable cells after drug treatment compared to untreated cells. (C) Human OS cells (MG63) were treated with different concentrations of manoalide (0, 0.1, 1, 5, 10, 20, and 40 µM) for 24 and 48 h, and the drug-inhibitory ability was represented by s−curves. (D) Human OS cells (143B) were treated with different concentrations of manoalide (0, 0.1, 1, 5, 10, 20, and 40 µM) for 24 and 48 h and the drug-inhibitory ability was represented by s−curves. (E) After the treatment of MG63 with 0, 0.1, 1, 5, and 10 µM of manoalide for 24 h, the degree of apoptosis in the MG63 cells was determined using Annexin V/PI staining on a flow cytometry device. A four−quadrant dot plot created with CytExpert analysis software is shown in the figure. (F) With the use of CytExpert analysis software, the lower right quadrant shows that the apoptosis is in the early stage, and the upper right quadrant shows that the apoptosis is in the late stage, and the sum is produced as a bar graph. (G) Whole−cell lysate proteins were loaded for Western blot utilizing caspase−9, apoptosis Western blot cocktail, and GAPDH antibody after treatment with 0, 0.1, 1, 5, and 10 µM of manoalide in MG63 cells for 24 h. Blot figures were cropped from different gels, and PVDF membranes were subjected to the same conditions. Full Western blot figures are shown in Supplementary Figure S1B. The protein levels of cleaved PARP (H), procaspase−9/cleaved caspase−9 (I), and procaspase−3/cleaved caspase−3 (J) were quantified using ImageJ software and normalized to that of actin or GAPDH and were expressed as fold changes. Each bar represents the mean ± SE (n = 3) of three independent experiments, and the results were analyzed using Student’s t-test. * p < 0.05 and ** p < 0.01 relative to the control (0 µM manoalide).

Figure 2

Effect of manoalide on the ROS and oxidative stress defense enzymes in MG63 cells. (A) MitoSOX^TM Red fluorescent dye was used to determine the fluorescence intensity of mitochondrial O₂•−, which was observed by flow cytometry in the MG63 cells treated with 0, 0.1, 1, 5, and 10 µM of manoalide for 4 h. (B) Manoalide-induced mitochondrial O₂•− accumulation in mitochondria was quantified by CytExpert software analysis of the gated range of 8 × 10³–10⁶ single-parameter histograms. (C) The fluorescence intensity of iROS was determined using CM-H₂DCFDA green fluorescent dye and detected by flow cytometry in the MG63 cells treated with 0, 0.1, 1, 5, and 10 µM of manoalide for 4 h. (D) Manoalide-induced iROS accumulation in the cell was quantified by CytExpert software analysis of the gated range of 5 × 10³–10⁷ single-parameter histograms. (E) The fluorescence intensity of O₂•− and •OH levels in the mitochondria and nucleus was determined using CellROX^® Green fluorescent dye and detected by flow cytometry in the MG63 cells treated with 0, 0.1, 1, 5, and 10 µM of manoalide for 4 h. (F) Manoalide-induced O₂•− and •OH levels in the mitochondria and nucleus accumulation in the cell were quantified by CytExpert software analysis of the gated range of 6 × 10⁵–10⁷ single-parameter histograms. (G) In the treatment with 0, 0.1, 1, 5, and 10 µM of manoalide in MG63 cells for 24 h, whole-cell lysate proteins were loaded for Western blot by using SOD2, oxidative stress defense (catalase, SOD1, TRX, smooth muscle actin) Western blot cocktail, and GAPDH antibody. Blot figures were cropped from different gels, and PVDF membranes were subjected to the same conditions. Full Western blot figures are shown in Supplementary Figure S1C. The protein levels of catalase (H), thioredoxin (H), SOD1 (I), and SOD2 (I) were quantified using ImageJ software and normalized to that of alpha smooth muscle actin or GAPDH and were expressed as fold changes. Each bar represents the mean ± SE (n = 3) of three independent experiments, and the results were analyzed using Student’s t-test. * p < 0.05 and ** p < 0.01 relative to the control (0 µM manoalide). ROS: reactive oxygen species; iROS: intracellular ROS; H₂DCFDA: 2′,7′-dichlorodihydrofluorescein diacetate; SOD1: superoxide dismutase 1; SOD2: superoxide dismutase 2; TRX: thioredoxin; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; PVDF: polyvinylidene difluoride.

Figure 3

Effect of manoalide on the oxygen consumption rate and OXPHOS enzymatic complex protein in MG63 cells. (A) OCR values (pmoles/min/mg protein) and time (minutes) graphs were measured after MG63 cells were treated with various concentrations of manoalide for 4 h, followed by continuous injection of Seahorse XF Cell Mito Stress kit reagents, including oligomycin, FCCP, and antimycin/rotenone. In MG63 cells treated with 0, 0.1, 1, 5, and 10 μM manoalide, the parameters quantified and analyzed were (B) basal respiratory capacity, (C) ATP production (coupled respiration), (D) maximal respiratory capacity, (E) spare respiratory capacity, and (F) non-mitochondrial respiratory capacity. These data were quantified by normalizing cellular protein concentrations. (G) In the treatments with 0, 0.1, 1, 5, and 10 µM of manoalide in MG63 cells for 24 h, whole-cell lysate proteins were loaded for Western blot by using total OXPHOS human WB antibody cocktail and GAPDH antibody. Blot figures were cropped from different gels, and PVDF membranes were subjected to the same conditions. Full Western blot figures are shown in Supplementary Figure S2A. The protein levels of NDUFB8 (H), SDHB (I), UQCRC2 (I), COX II (J), and ATP5A (J) were quantified using ImageJ software and normalized to that of GAPDH and were expressed as fold changes. Each bar represents the mean ± SE (n = 3) of three independent experiments, and the results were analyzed using Student’s t-test. * p < 0.05 and ** p < 0.01 relative to the control (0 µM manoalide). OXPHOS: oxidative phosphorylation; OCR: oxygen consumption rate; FCCP: Carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone; WB: Western blotting; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; PVDF: polyvinylidene difluoride; NDUFB8: NADH dehydrogenase [ubiquinone] 1 beta subcomplex subunit 8; SDHB: succinate dehydrogenase complex iron sulfur subunit B; UQCRC2: ubiquinol–cytochrome C reductase core protein 2; COX II: cytochrome c oxidase subunit II; ATP5A: ATP synthase subunit alpha.

Figure 4

Effects of manoalide on ΔΨ_m and the expression levels of mitochondrial dynamic proteins in MG63 cells. (A) The fluorescence intensity of ΔΨ_m in MG63 cells treated with 0, 0.1, 1, 5, and 10 µM of manoalide after 4 h was evaluated using DiOC6 green fluorescent dye and identified by flow cytometry. (B) The manoalide-induced reduction in ΔΨ_m was quantified by CytExpert software analysis of the gated range of 8 × 10⁵–10⁷ single-parameter histograms. (C) After treatment with 0, 0.1, 1, 5, and 10 µM of manoalide for 4 h in MG63 cells, JC-1 labeling by flow cytometry revealed a decrease in red fluorescence, indicating mitochondrial depolarization. A four-quadrant dot plot created with CytExpert analysis software is shown in the figure. (D) Through analysis of the selected range of the quadrant plot, CytExpert quantitated JC-1 signals. The aggregated and monomer JC-1 amount was determined using the high and low ΔΨ_m (upper right and lower right) quadrants. (E) The treatments with 0, 0.1, 1, 5, and 10 µM of manoalide in MG63 cells for 24 h: whole-cell lysate proteins were loaded for Western blot by using OPA1, DRP1, and GAPDH antibodies. Blot figures were cropped from different gels, and PVDF membranes were subjected to the same conditions. Full Western blot figures are shown in Supplementary Figure S2B. The protein levels of OPA1 (F) and DRP1 (G) were quantified using ImageJ software and normalized to that of GAPDH and were expressed as fold changes. Each bar represents the mean ± SE (n = 3) of three independent experiments, and the results were analyzed using Student’s t-test. * p < 0.05 and ** p < 0.01 relative to the control (0 µM manoalide). DiOC6: 3,3′-dihexyloxacarbocyanine iodide; ΔΨ_m: mitochondrial membrane potential; JC-1: 5′,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolyl-carbocyanine iodide; OPA1: optic atrophy 1; DRP1: dynamin-related protein 1; GAPDH; glyceraldehyde-3-phosphate dehydrogenase; PVDF: polyvinylidene difluoride.

Figure 5

Pretreatment with antioxidant NAC partially rescued apoptosis, iROS accumulation, and antioxidant enzymatic protein induced by manoalide in MG63 cells. (A) Using the “Apoptosis Western Blot Cocktail” antibody, Western blot analyses of cells treated with or without NAC and manoalide were performed. Blot figures were cropped from different gels, and PVD membranes were subjected to the same conditions. Full Western blot figures are shown in Supplementary Figure S2C. The protein levels of cleaved PARP and procaspase-3 (B) were quantified using ImageJ software and expressed as fold changes when adjusted to actin levels. (C) The fluorescence intensity of iROS in MG63 cells treated with or without NAC and manoalide for 24 h was evaluated using CM-H₂DCFDA green fluorescent dye and identified by flow cytometry. (D) Quantitation of the DCF detection signals by CytExpert software analysis of the gated range of 5 × 10³–10⁷ single-parameter histograms. (E) Western blot analyses of cells treated with or without NAC and manoalide using Oxidative Stress Defense (catalase, SOD1, TRX, smooth muscle actin) Western Blot Cocktail, SOD2, and GAPDH antibody. Blot figures were cropped from different gels, and PVD membranes were subjected to the same conditions. Full Western blot figures are shown in Supplementary Figure S2D. The protein levels of catalase (F), TRX (F), SOD1, and SOD2 (G) were quantified using ImageJ software and normalized to that of actin, and GAPDH was expressed as the fold change. Each bar represents the mean ± SE (n = 3) of three independent experiments, and the results were analyzed using ANOVA. * p < 0.05, **; p < 0.01 relative to the control group (without NAC and manoalide), and ^# p < 0.05 relative to the experimental group with 10 μM of manoalide alone. iROS: intracellular ROS; NAC: N-acetylcysteine; PARP: poly(ADP-ribose) polymerase; SOD1: superoxide dismutase 1, TRX: thioredoxin.

Figure 6

Hypothetical scheme for manoalide-mediated oxidative stress and mitochondrial dysfunction in osteosarcoma cells. mtROS: mitochondrial ROS; iROS: intracellular ROS; nROS: nuclear ROS; SOD2: superoxide dismutase 2, Mn-SOD; mitochondrial manganese superoxide dismutase; SOD1: superoxide dismutase 1, Cu-ZnSOD; TRX: thioredoxin; OXPHOS: oxidative phosphorylation; ATP: adenosine triphosphate; ΔΨm: mitochondrial membrane potential; PARP: poly(ADP-ribose) polymerase; NAC: N-acetylcysteine).