Activated TRPA1 plays a therapeutic role in TMZ resistance in glioblastoma by altering mitochondrial dynamics

Cell culture and drug treatment

U251 cells and SHG-44 cells (Shanghai Institutes for Biological Sciences, China Academy of Science, Shanghai) were cultured in high-glucose Dulbecco’s modified Eagle’s medium (HyClone, Logan, UT, USA) with 10% fetal bovine serum (BioInd, Kibbutz Beit Haemek, Israel), 100 U/ml penicillin and 100 μg/ml streptomycin (Thermo Fisher Scientific, Waltham, MA, USA) at 37 °C in 5% CO2.

On the basis of observations in a preliminary study, U251 cells and SHG-44 cells were treated with 100 μΜ temozolomide (MedChemExpress, MCE, New Jersey, USA) for 24 h to cause cell injury. The cells were pretreated with 100 μM TRPA1 agonist (PF-4840154, MCE) or 100 μM TRPA1 antagonist (HC030031, MCE) for 1 h and then cultured with vehicle (culture medium) or TMZ for another 24 h.

Cell viability assay

Cell Counting Kit-8 (CCK-8; Dojindo, Kumamoto, Japan) assays were performed to measure the effects of the vehicle, TMZ, and a TRPA1 agonist and inhibitor on cell proliferation. Briefly, cells were cultured at a density of 5 × 104 cells/well in a 96-well plate, incubated overnight at 37 °C, and then treated with vehicle or other irritants. The treated cells were incubated for 24 h and washed with PBS. Then, following the CCK-8 kit manufacturer’s protocol, the cells were incubated with the working solution of CCK-8 for 2 h at 37 °C. The absorbance was measured at 450 nm with a iMark microplate reader (Molecular Devices, Sunnyvale, USA).

Measurement of intracellular ROS and mitochondrial ROS levels

DCFH-DA (D6883, Sigma–Aldrich, St. Louis, MO, USA) was used to detect intracellular ROS generation through fluorescence microscopy with a fluorescence plate reader. Briefly, in each group, cells were seeded at a density of 5 × 104 in 96-well black plates in 6 parallel wells. After 24 h, the cells were stained with 10 μM DCFH-DA at 37 °C for 15 min in the dark. Then, every well was washed three times with PBS, and the level of intracellular ROS was determined by microscopy (Leica, Germany) or by a Flexstation®2 fluorescence plate reader (Molecular Devices, San Jose, CA, USA) at excitation and emission wavelengths of 488 nm and 525 nm, respectively.

The activity levels of the mitochondrial ROS in cells were measured by MitoSOX Red (Invitrogen) assay, a redox-sensitive fluorescent probe that targets mitochondria in cells. In each group, cells were seeded at a density of 5 × 104 in 96-well black plates with 6 parallel wells. After 24 h, the cells were stained with 5 mmol/ml MitoSOX Red probe at 37 °C in the dark for 10 mins. Then, every well was washed twice with PBS, and red fluorescence was detected with a Flexstation®2 fluorescence plate reader at excitation and emission wavelengths of 510 nm and 580 nm, respectively.

Measurement of Ca2+ influx

Intracellular Ca2+ levels in the cells were determined according to the manufacturer’s instructions for a Fluo-4 AM calcium assay kit (Beyotime, Jiangsu, China). Briefly, in each group, the cells were seeded at a density of 5 × 104 in 96-well black plates with 6 parallel wells. The experimental groups were pretreated with agonist or antagonist for 1 h, and then, all groups were exposed to TMZ for 2 mins. The cells were washed three times and stained with 5 μM Fluo-4 AM at 37 °C in the dark for 30 mins. Then, the cells in every well were washed three times with PBS, and the level of fluorescence was determined at excitation and emission wavelengths of 488 nm and 516 nm, respectively, by microscopy (Leica) with a Flexstation®2 fluorescence plate reader.

Western blot (WB) assay

The expression levels of DRP1, MFF, OPA1, MFN2, BAX, BCL2, TRPA1 and GAPDH were detected by WB assay. Total cell proteins in every group were extracted with RIPA buffer (Beyotime). Protein concentrations were determined with a BCA protein assay kit (Beyotime). Total protein samples were separated by 10% SDS–PAGE, and then, the proteins were transferred to a PVDF membrane. Finally, 5% nonfat milk dissolved in Tris-buffered saline with Tween 20 (TBST, BioTNT, Shanghai, China) was used to block the membrane for 4 h. The membrane was tailored according to the molecular weight of the target protein, then the cropped membrane was incubated with primary antibodies against DRP1, MFF, OPA1, MFN2, BAX, BCL2, TRPA1, MGMT, Cleaved Caspase-3, Caspase-3 and GAPDH (all diluted 1:1000, Cell Signaling Technology, Boston, MA, USA) overnight at 4 °C. Horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (1:15000, Cell Signaling Technology) and secondary antibodies were incubated with the membrane at room temperature for 2 h. Then, the membrane was washed 3 times in TBST and visualized with an enhanced chemiluminescent (ECL) detection system.

Reverse transcription–polymerase chain reaction (RT–PCR)

RT–PCR was used to test the mRNA levels of antioxidants (MnSOD, HO-1 and NQO1). Total RNA was extracted from cells with TRIzol reagent (TaKaRa, Dalian, Liaoning, China). The concentration of the total RNA was determined with an ultraviolet spectrophotometer. The Prime Script™ RT Master Mix Kit (TaKaRa) was used to conduct reverse transcription according to the manufacturer’s instructions. RT–PCR was performed by Power Green qPCR Mix (TaKaRa) and the ABI ViiATM 7 System. The specific primers (Table 1) for MnSOD, HO-1, NQO1 and β-actin were generated by BioTNT (Shanghai, China). All samples were assayed in triplicate, and the values were normalized to the level of β-actin.

Table 1 The sequences of forward and reverse primers of antioxidants (HO-1, NQO1 and MnSOD) and β-actinMeasurement of GSH levels

Intracellular glutathione (GSH) levels in the cells were determined according to the manufacturer’s instructions for a GSH and GSSG Assay Kit (Beyotime, Jiangsu, China). Briefly, in each group, the cells were seeded at a density of 1 × 106 in 6-well plates with 6 parallel wells. The experimental groups were pretreated with agonist or antagonist for 1 h, and then, all groups were exposed to TMZ for 24 h. The cells were washed with PBS. Then, supernatant of lysed cells was collected and tested as required, and the level of fluorescence was determined at wavelengths of 412 nm, by microscopy (Leica) with a Flexstation®2 fluorescence plate reader.

Cell transduction with lentivirus

A recombinant lentiviral vector overexpressing short hairpin RNA (shRNA) was designed and constructed by Zorin (Shanghai, China). Human U251 cells were infected with 107 TU/mL (multiplicity of infection [MOI] = 10) lentivirus-mediated shRNA (TRPA1+/+) or negative control shRNA (Con) for 12 h. The cells were collected to determine their interference efficiency by WB assay.

Statistical analysis

Statistical analysis was performed with GraphPad Prism (version 7; GraphPad Software, Inc., San Diego, CA). One-way ANOVA with Bonferroni’s post hoc test (for equal variance) or Dunnett’s T3 post hoc test (for unequal variance) was performed for comparisons among multiple groups. P < 0.05 was considered to be statistically significant.

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