Accumulation of prohibitin is a common cellular response to different stressing stimuli and protects melanoma cells from ER stress and chemotherapy-induced cell death

Melanoma is responsible for most deaths among skin cancers and conventional and palliative care chemotherapy are limited due to the development of chemoresistance. We used proteomic analysis to identify cellular responses that lead to chemoresistance of human melanoma cell lines to cisplatin. A systems approach to the proteomic data indicated the participation of specific cellular processes such as oxidative phosphorylation, mitochondrial organization and homeostasis, as well as the unfolded protein response (UPR) to be required for the survival of cells treated with cisplatin. Prohibitin (PHB) was among the proteins consistently accumulated, interacting with the functional clusters associated with resistance to cisplatin. We showed PHB accumulated at different levels in melanoma cell lines under stressing stimuli, such as (i) treatment with temozolomide (TMZ), dacarbazine (DTIC) and cisplatin; (ii) serum deprivation; (iii) tunicamycin, an UPR inducer. Prohibitin accumulated in the mitochondria of melanoma cells after cisplatin and tunicamycin treatment and its de novo accumulation led to chemoresistance melanoma cell lines. In contrast, PHB knock-down sensitized melanoma cells to cisplatin and tunicamycin treatment. We conclude that PHB participates in the survival of cells exposed to different stress stimuli, and can therefore serve as a target for the sensitization of melanoma cells to chemotherapy.


Isoelectric focusing (IEF) and SDS-PAGE
An IPG-Phor electrophoretic system was used with 18 cm pH 4-7 gradient strips (GE Healthcare). The strip was reduced with 65 mM DTT and alkylated with 100 mM iodoacetamide. One mg protein was applied during rehydration (Rabilloud, 1994). Electrophoresis was carried out at 50 mA/strip and 150 V for 1 h, 500 V for 1 h, 1000 V for 1 h, and 8000 V until 70,000 Vh were accumulated. SDS-PAGE was carried out in an EttanDALTsix electrophoretic system (GE Healthcare Life Sciences) using homogeneous 12.5% polyacrylamide gels of 1.5 mm thickness. The IEF strip was placed on top of the gel with 0.5% agarose. Electrophoresis was carried out at 20 mA/ gel, 300 V, 6 W, at 10 o C until the bromophenol left the gel after about 15 h [11]. Gels were fixed in 40% v/v ethanol containing 10% v/v acetic acid for 1 h, washed in water and stained with 0.1% w/v colloidal CBB-250 (Serva, Germany) containing 2% w/v phosphoric acid, 10% w/v ammonium sulfate, and 20% v/v methanol for 24 h. After staining, gels were briefly washed in 25% v/v methanol and stored in 20% w/v ammonium sulfate at 4ºC until use.
Gels were scanned with the ImageScanner acquisition system (GE Health Care) using ImageMaster-2D software. Comparisons between 2 D maps of cells treated or not with cisplatin were made between spots of three replicate gels of cells treated or not with cisplatin. Reproducibility of the location of the same spots on pairs of gels was 86 and 91% and the number of spots not detected in other gels was 14, 61, and 31. These spots were of low intensity. Variations in pI and molecular mass were 0.3 and 2.0%, respectively. Ninety percent or more of all three spots detected at the same location had high staining intensity of >400. The precision of the measured intensity was <±40% for 20 spots in triplicate gels. Therefore plus or minus 2-fold was used to define protein accumulation or reduction and used to compare the same spot before and after cisplatin treatment.
Spots were cut out of the gel and eluted with 0.1 M NH 4 CO 3 , pH 8.0, containing 50% acetonitrile (CAN) until the stain was extracted. The extract was rehydrated with 5 µl of 1 M NH 4 CO 3 containing 0.4µg trypsin (Promega) and incubated for 24 h at 37 o C. Peptides were eluted 3 times with 100 µl of 50% ACN containing 2.5% trifluoroacetic acid (TFA). The peptides were desalted on C 18 resin in a pipette tip and analyzed with a MALDI-TOF spectrometer (model LR, Micromass). Peptides were incorporated into a matrix with 1% CHCA, 50% ACN and 0.2% TFA, evaporated and ionized. The mass spectrometer was calibrated with the following peptides: angiotensin I (1297.

Databank searches
Ions identified by MS were analyzed with the MS-Fit tool (Protein Prospector -http://prospector.ucsf. edu) using the Swiss-Prot databank for human-mouse proteins. The parameters used for the search were 0.2 Da for permitted mass error and one missed cleavage site for trypsin hydrolysis specificity. Proteins were identified on the basis of minimum sequence coverage of more than 15%. Functional protein classification was based on level 5 of the Gene Ontology classification, available at http// source.stanford.edu.

JC-1 and Annexin-V
For annexin-V analyses, cells were incubated with Annexin-V/FITC (Invitrogen) in presence of binding buffer (10 mmol/L HEPES, 140 mmol/L NaCl, 2.5 mmol/L CaCl2 x 2H2O, and 0.1% bovine serum albumin). Cell suspension was then transferred to microtubes and incubated on ice, in the dark, for 15 min. 4 µg/mL of propidium iodide were added and samples were immediately analyzed by flow cytometry using FACSCalibur (Beckton and Dickson®, BD Biosciences, San Jose, CA, USA). For JC-1 staining, cells were incubated with 200nM of JC1 in 10% FBS in medium for 15 minutes at 37°C. Cell suspension was then transferred to microtubes and immediately analyzed by flow cytometry. Loss of ™ψm was measured in the right lower quadrant.