Multiwalled carbon nanotubes inhibit cell migration and invasion by destroying actin cytoskeleton via mitochondrial dysfunction in ovarian cancer cells

Characterization of MWCNT

Commercial MWCNT dispersion exhibited black appearance (Figure 1A). The optical absorption spectra of MWCNT revealed around 260 nm of absorption peaks (Figure 1B). TEM images showed the complete and visible tubular structure (Figure 1C).

Figure 1. Characterization of multiwalled carbon nanotubes (MWCNTs). (A) The appearance of commercial MWCNTs. (B) The ultraviolet visible absorption spectra of MWCNTs. (C) The representative images of MWCNTs using transmission electron microscopy.

Effect of MWCNT on cytotoxicity in SKOV3 cells

MTT assay showed that MWCNT significantly inhibited cell viability in dose-dependent manner both at 24 and 48 h, and MWCNT with a dose of less than 100 μg/mL showed more than 50% of cell viability (Figure 2A). Another ovarian cancer cell line A2780 and used MTT to detect the effect of different concentrations of MWCNTs on cell viability. Consistently, colony formation results also revealed that compared with control cells, the clone number was obviously decreased in SKOV3 cells with MWCNT in dose-dependent manner (Figure 2B). In addition, flow cytometry analysis found that MWCNT treatment prominently reduced the cell number of G0/G1 phage, while increased cell number of S phage in dose-dependent manner (p < 0.05, Figure 2C). Meanwhile, the rate of apoptotic cells was remarkably increased after MWCNT treatment in dose-dependent manner at 24 h in SKOV3 cells compared with control cells (p < 0.05, Figure 2D).

Figure 2. Multiwalled carbon nanotubes (MWCNTs) inhibits tumor growth in SKOV3 cells. (A) Cell viability of SKOV3 cells treated with different doses of MWCNTs at 24h and 48 h by MTT assay. (B) Clone number of SKOV3cells treated with different doses of MWCNTs at 24h by colony formation assay. (C) Cell cycle of SKOV3 cells treated with different doses of MWCNTs at 24h by flow cytometry analysis. (D) Cell apoptosis rate of SKOV3 cells treated with different doses of MWCNTs at 24h and 48 h by flow cytometry analysis. *P < 0.05, and **P < 0.01 vs control cells (0 μg/mL MWCNTs).

Effect of MWCNT on tumor metastasis in SKOV3 cells

Wound healing assay showed that MWCNT significantly decreased the wound closure and inhibited wound healing rate of SKOV3 cells in dose- and time-dependent manner (p < 0.05, Figure 3A), suggesting a reduced migration potential after MWCNT treatment in SKOV3 cells. Transwell assay also revealed that cell migration and invasion were dramatically inhibited in SKOV3 cells treated with MWCNT compared with control cells (p < 0.05, Figure 3B).

Figure 3. Multiwalled carbon nanotubes (MWCNTs) inhibits tumor metastasis in SKOV3 cells. (A) The wound closure and wound healing rate of SKOV3 cells treated with different doses of MWCNTs at 0, 6, 12 h and 24 h by wound healing assay. (B) Cell migration and migration rates in SKOV3 cells treated with different doses of MWCNTs by Transwell assay. *P < 0.05, **P < 0.01, and ***P < 0.001 vs control cells.

Effect of MWCNT on actin cytoskeleton of SKOV3 cells

Actin cytoskeleton is essential for cell migration and invasion; thus, actin cytoskeleton of SKOV3 cells was observed under confocal microscopy. As shown in Figure 4, the cellular cytoplasm of control cell exhibited the well-arranged actin filaments in thick bundles. In contrast, MWCNT treatment damaged the structure of actin cytoskeleton of SKOV3 cells in dose-dependent manner (Figure 4).

Figure 4. Multiwalled carbon nanotubes (MWCNTs) disrupts actin cytoskeleton of SKOV3 cells. Actin cytoskeleton of SKOV3 cells treated with different doses of MWCNTs under confocal microscopy.

Effect of MWCNT on mitochondrial function of SKOV3 cells

The mitochondrial membrane potential results showed that control cells mainly presented JC-1 aggregates (red fluorescence), while increased JC-1 monomers (green fluorescence) and reduced JC-1 aggregates were observed in cells treated with MWCNT in dose-dependent manner (Figure 5A). This indicated that the treatment of MWCNTs caused a decrease in mitochondrial membrane potential. Consistently, when SKOV3 cells exposed to MWCNT for 24 h, the activities of mitochondrial electron transfer chain complexes I-V were significantly decreased in dose-dependent manner compared with un-treated cells (Figure 5B). Here, we detected the level of p-NF-κB and p-p38-MAPK, which related to mitochondrial function. MWCNTs might alter the function of mitochondria by activating MAPK signaling and NF-κB signaling. The OCR detection showed that cells treated with MWCNTs have lower mitochondrial respiration.

Figure 5. Multiwalled carbon nanotubes (MWCNTs) disrupted mitochondrial function of SKOV3 cells. (A) The mitochondrial membrane potential in SKOV3 cells treated with different doses of MWCNTs. (B) The activities of mitochondrial respiratory chain complexes I-V detected by commercially kits.

Characterization of MWCNTs

MWCNTs with > 95% purity were purchased from XFNANO Materials Tech Co. Ltd (Nanjing, Jiangsu, China). MWCNTs were dissolved in 1% DMSO and MWCNTs suspension was prepared by sonication machine. The ultraviolet (UV) visible spectra of MWCNTs was measured by UV visible light absorption spectroscopy (Biochrom, Cambridge, UK). The morphology of MWCNTs was observed by transmission electron microscopy (TEM; HT7700; Hitachi, Japan).

Cell culture and treatment

Ovarian cancer cell line SKOV3 and A2780 were obtained from Shanghai Obio Technology Co., Ltd, and then maintained in complete DMEM medium (Gibco, Carlsbad, CA, USA) under 37° C and 5% CO₂. SKOV3 and A2780 cells were exposed to different doses of MWCNT.

MTT assay

SKOV3 and A2780 cells were grown in 96-well plates, and then underwent different doses of MWCNT (1, 5, 20, 50, 100, and 200 μg/mL). After conventional incubation for 24 and 48 h, each well was added with 10 μl of MTT (5 mg/mL, Sigma) for another 4 h. Afterwards, 100 μL of dimethyl sulfoxide was added. Microplate spectrophotometer was used to evaluate cell viability based on the absorbances at 470 nm.

Colony-forming assay

SKOV3 cells were grown in 6-well plates at a density of 400 cells/well, and then underwent different dose of MWCNT (1, 5, 20, 50, 100, and 200 μg/mL) for 14 days under standard culture conditions. Then cells were fixed with absolute methanol, and incubated with crystal violet. Ultimately, the number of colonies was counted.

Flow cytometry assay

Cell cycle and apoptosis were evaluated by flow cytometry assay. SKOV3 cells were treated with different doses of MWCNT (20, 50, and 100 μg/mL) for 24 h. Trypsin was used to digest cells with various treatments, and then cells were harvested. For cell cycle assay, cells were incubated in 70% ethanol on ice for 2 h, and then stained with propidium iodide (PI) for 30 min at 37° C. For cell apoptosis, FITC-Annexin V Apoptosis kit was used. Briefly, cells were rinsed with PBS, and resuspended with Binding Buffer. Next, the cells underwent the incubation of FITC-Annexin V and PI with cells for 15 min, in turn. Flow cytometer (BD, CA, USA) was used to measure cell cycle and calculate the number of apoptotic cells.

Wound healing assay

SKOV3 cells were inoculated in 6-well plates. After growing to 60% of confluence, cells were wounded by scratching the vertical lineation using pipette tips, followed by culturing in DMEM without serum. Subsequently, cells were treated with 0, 20 and 100 μg/mL of MWCNT, respectively. The distance of the scratch was observed at 0, 6, 12 and 24 h using a light microscope (Olympus, Japan).

Transwell assay

Tumor cell invasion and migration were elevated by Transwell chambers (Corning). Concretely, the bottom compartment was added with DMEM supplemented with 10% FBS and MWCNT (20 and 100 μg/mL). SKOV3 cells were grown in the upper compartment coated with Matrigel Matrix and cultured in medium free of serum for 24 h. Subsequently, the cells in the bottom compartment were fixed and stained 4,6-diamidino-2-phenylindole. An inverted microscope (Olympus, Japan) was utilized to evaluate cell migration and invasion.

Cytoskeleton staining

SKOV3 cells were inoculated on cover slips, and then exposed to 0, 20, 50 and 100 μg/mL of MWCNT, respectively, for 24 h. The cells were then subjected to fixation using 4% paraformaldehyde and permeabilized treatment by 0.1% Triton X-100. Followed by blockage by 2% bovine serum albumin, the cells were stained with FITC-Phalloidin (actin, green) and DAPI (nucleus, blue), in turn. Finally, cell images were obtained using laser scanning confocal microscope (Olympus, Japan).

Mitochondrial membrane potential detection

Mitochondrial membrane potential depolarization was detected using commercial JC-1 kit (Beyotime). At high membrane potential, JC-1 shows red fluorescent aggregates; while JC-1 presents green fluorescent monomers at low membrane potential. Briefly, SKOV3 cells were treated with different doses of MWCNT (20, 50, and 100 μg/mL) for 24 h, and then stained with 1 ml of JC-1 dye at 37° C for 20 min. Next, cells were rinsed with JC-1 buffer, and observed under an inverted microscope (Olympus, Japan).

Activity measurements of mitochondrial respiratory chain complexes

SKOV3 cells were inoculated in 6-well plates, and treated with 0, 20, 50, and 100 μg/mL of MWCNT, respectively, for 24 h. Next, trypsin was used to digest cells with various treatments, and then cells were lysed. Subsequently, the activities of mitochondrial respiratory chain complexes I-V were detected by commercial kits (Jianchen, Nanjing, China).

Western blot analysis

The SKOV3 cells were lysed using radioimmunoprecipitation assay (RIPA) buffer (Beyotime, Shanghai, China). Total protein levels were quantified using a BCA Protein Assay kit (Pierce; Thermo Fisher Scientifc, Inc.) prior to performing immunoblot analysis. 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was used to separate protein samples (15 mg protein loaded per well) and then proteins were transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, Billerica, MA, USA). The membranes were blocked with 5% non-fat milk in Tris-buffered saline with 0.1% Tween 20 (TBST) at room temperature for 3 h, and then PVDF membranes were incubated with the corresponding primary antibodies (including anti- p-NF-κB, anti-p-p38-MAPK, anti-T-p38-MAPK, and anti-β-actin antibodies) at 4° C overnight. The membranes were washed three times with TBST, and then incubated with corresponding secondary antibodies at room temperature for 1 h. Finally, the protein bands were visualized using the enhanced chemiluminescence (ECL) protein detection kit (Millipore, MA, USA). The relative protein expression was analyzed using Image-Pro Plus software version 6.0 (Media Cybernetics, Inc., Rockville, MD, USA) and represented as the density ratio vs. β-actin.

Oxygen consumption rate (OCR) measurement

The Extracellular Flux Analyzer 8-well format (Seahorse Bioscience, Agilent Technologies, USA) was used to synchronously detect the oxygen consumption rate (OCR) in SKOV3 cells.

Briefly, cells were seeded onto XFp cell culture mini plates and cultured overnight. For OCR analysis, 1 μM oligomycin, 1 μM carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP) and 0.5 μM antimycin A (Sigma, USA) were added into special 8-well probe plates. All measurements were performed according to the manufacturer's instructions, and the number of cells in each well was normalized.

Statistical analysis

Data were presented as the mean ± SD. One-way ANOVA followed by multiple comparison was used for data comparisons based on SPSS software. P < 0.05 was considered significant.