Fatty Acid Synthase Inhibitors Induce Apoptosis in Non-Tumorigenic Melan-A Cells Associated with Inhibition of Mitochondrial Respiration

<div><p>The metabolic enzyme fatty acid synthase (FASN) is responsible for the endogenous synthesis of palmitate, a saturated long-chain fatty acid. In contrast to most normal tissues, a variety of human cancers overexpress FASN. One such cancer is cutaneous melanoma, in which the level of FASN expression is associated with tumor invasion and poor prognosis. We previously reported that two FASN inhibitors, cerulenin and orlistat, induce apoptosis in B16-F10 mouse melanoma cells <i>via</i> the intrinsic apoptosis pathway. Here, we investigated the effects of these inhibitors on non-tumorigenic melan-a cells. Cerulenin and orlistat treatments were found to induce apoptosis and decrease cell proliferation, in addition to inducing the release of mitochondrial cytochrome c and activating caspases-9 and -3. Transfection with FASN siRNA did not result in apoptosis. Mass spectrometry analysis demonstrated that treatment with the FASN inhibitors did not alter either the mitochondrial free fatty acid content or composition. This result suggests that cerulenin- and orlistat-induced apoptosis events are independent of FASN inhibition. Analysis of the energy-linked functions of melan-a mitochondria demonstrated the inhibition of respiration, followed by a significant decrease in mitochondrial membrane potential (ΔΨm) and the stimulation of superoxide anion generation. The inhibition of NADH-linked substrate oxidation was approximately 40% and 61% for cerulenin and orlistat treatments, respectively, and the inhibition of succinate oxidation was approximately 46% and 52%, respectively. In contrast, no significant inhibition occurred when respiration was supported by the complex IV substrate <i>N</i>,<i>N</i>,<i>N′</i>,<i>N′</i>-tetramethyl-<i>p</i>-phenylenediamine (TMPD). The protection conferred by the free radical scavenger N-acetyl-cysteine indicates that the FASN inhibitors induced apoptosis through an oxidative stress-associated mechanism. In combination, the present results demonstrate that cerulenin and orlistat induce apoptosis in non-tumorigenic cells <i>via</i> mitochondrial dysfunction, independent of FASN inhibition.</p></div

NAC pre-incubation protects melan-a cells from cerulenin or orlistat-induced apoptosis.

<p>Melan-a cells were pre-incubated with 5 mM NAC for 1 h, followed by treatment with 22 µM cerulenin for an additional 24 h (<b>A</b>) or 30 µM orlistat for 48 h (<b>B</b>). NAC was also present during the incubations with cerulenin or orlistat. Apoptosis was then determined by flow cytometry after Annexin V staining. The values represent the mean ± s.e.m of six independent experiments. *Significantly different from the respective control at <i>p</i><0.05. #Significantly different from the respective condition in the absence of NAC at <i>p</i><0.05.</p

Treatment of melan-a cells with FASN inhibitors leads to the release of mitochondrial cytochrome c and the activation of caspases-3 and -9 but not -8.

<p>Melan-a cells were treated with 22 µM cerulenin or 30 µM orlistat for 12 or 24 h, respectively; then, the release of cytochrome c was determined by flow cytometry (<b>A</b>). The cells were also treated with cerulenin or orlistat under the same conditions, and the activation of caspase-3 was estimated using FITC-DEVD-FMK (<b>B</b>). The activities of caspase-9 and -8 (<b>C</b> and <b>D</b>) were determined as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0101060#s2" target="_blank">Material and Methods</a>. The values represent the mean ± s.e.m of at least three independent experiments. *Significantly different from the respective control at <i>p</i><0.05.</p

Treatment with FASN inhibitors promotes the inhibition of respiration in melan-a cells.

<p>Oxygen consumption by the melan-a cells was measured after treatment for 24 h with 22 µM cerulenin (<b>A</b>) or for 48 h with 30 µM orlistat (<b>B</b>) using high-resolution respirometry (Oroboros) in a closed chamber equipped with a magnetic stirrer and temperature control set to 37°C. Approximately 2×10⁶ viable cells/ml were permeabilized with 15 µM of digitonin and then were added to 2 ml of reaction medium (described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0101060#s2" target="_blank">Materials and Methods</a>). Analyses of oxidative phosphorylation and respiratory activity of the mitochondria were made by sequential additions of 300 µM ADP, 2 µg/ml oligomycin, 100 nM carbonylcyanide p-trifluoromethoxyphenylhydrazone (FCCP), 5 mM succinate, 0.5 µM antimycin and 200 µM N,N,N′,N′-tetramethyl-p-phenylenediamine (TMPD) with 2 mM ascorbate (<b>A–D</b>). The activity of citrate synthase was measured in melan-a cells after 24 or 48 h of treatment with 22 µM cerulenin or 30 µM orlistat (<b>E</b>). The values represent the mean ± s.e.m of at least four independent experiments. *Significantly different from the respective control at <i>p</i><0.05.</p

Cerulenin and orlistat reduce cell viability and induce apoptosis in the melan-a cell line.

<p>Melan-a cells were treated with increasing concentrations of cerulenin or orlistat for 24 or 48 h, respectively; cell viability was determined using trypan blue (<b>A</b> and <b>B</b>) or MTT assays (<b>C</b> and <b>D</b>), and apoptosis was determined by flow cytometry after Annexin V staining (<b>E</b> and <b>F</b>). The values represent the mean ± s.e.m of at least three independent experiments. *Significantly different from the respective control at <i>p</i><0.05.</p

FASN inhibitors result in decreased ΔΨm and increased superoxide production in melan-a cells.

<p>Melan-a cells were treated with 22 µM cerulenin or 30 µM orlistat for 6 or 24 h, respectively; then, approximately 2×10⁶ viable cells/ml were permeabilized with 15 µM digitonin. ΔΨm was estimated by Safranin fluorescence. The arrows indicate the addition of 15 µM digitonin, 100 µM ADP, 5 µM carboxyatractyloside (CAT) and 1 µM CCCP (<b>A</b> and <b>B</b>, representative of at least three independent experiments). Melan-a cells were also treated with 22 µM cerulenin or 30 µM orlistat for 24 or 48 h, respectively and also incubated in the presence or absent of NAC; the cells were then washed and probed with 5 µM MitoSOX (<b>C</b>). The values represent the mean ± s.e.m of four independent experiments. *Significantly different from the respective control at p<0.05. #Significantly different from the respective condition in the absence of NAC at <i>p</i><0.05.</p

FASN inhibitors blocked cell cycle progression in non-tumorigenic cells.

<p>Melan-a cells were treated with 22 µM cerulenin or 30 µM orlistat for 24 or 36 h, respectively. Then, the percentage of cells in each phase of the cell cycle was determined by flow cytometry after PI staining (<b>A</b>). Western blot analysis of protein extracts prepared from cerulenin- and orlistat-treated melan-a cells revealed the accumulation of the p21WAF1/Cip1 tumor suppressor protein (<b>B</b>). The data were normalized using beta-actin as a loading control. The values represent the mean ± s.e.m of at least five independent experiments. *Significantly different from the respective control at <i>p</i><0.05.</p

FASN silencing does not induce apoptosis in melan-a cells.

<p>Melan-a cells were either transfected with specific siRNAs (siRNA), treated with the transfection reagent alone (mock) or maintained in culture with equimolar concentrations of a nonspecific control oligo (control). Cells were incubated for 48 h. FASN protein content was determined by Western blot analysis (<b>A</b>), and apoptosis was estimated by flow cytometry after Annexin V staining (<b>B</b>). The values represent the mean ± s.e.m of three independent experiments.</p

Agrin and Perlecan Mediate Tumorigenic Processes in Oral Squamous Cell Carcinoma

<div><p>Oral squamous cell carcinoma is the most common type of cancer in the oral cavity, representing more than 90% of all oral cancers. The characterization of altered molecules in oral cancer is essential to understand molecular mechanisms underlying tumor progression as well as to contribute to cancer biomarker and therapeutic target discovery. Proteoglycans are key molecular effectors of cell surface and pericellular microenvironments, performing multiple functions in cancer. Two of the major basement membrane proteoglycans, agrin and perlecan, were investigated in this study regarding their role in oral cancer. Using real time quantitative PCR (qRT-PCR), we showed that agrin and perlecan are highly expressed in oral squamous cell carcinoma. Interestingly, cell lines originated from distinct sites showed different expression of agrin and perlecan. Enzymatically targeting chondroitin sulfate modification by chondroitinase, oral squamous carcinoma cell line had a reduced ability to adhere to extracellular matrix proteins and increased sensibility to cisplatin. Additionally, knockdown of agrin and perlecan promoted a decrease on cell migration and adhesion, and on resistance of cells to cisplatin. Our study showed, for the first time, a negative regulation on oral cancer-associated events by either targeting chondroitin sulfate content or agrin and perlecan levels.</p></div

Treatment with chondroitinase ABC decreased SCC-9 LN-1 cell adhesion to extracellular matrix, but not SCC-9 LN-1 cell migration and increased sensibility of SCC-9 LN-1, SCC-9 and A431 cells to cisplatin.

<p>(A) SCC-9 LN-1 had a lower ability to adhere to extracellular matrix proteins (Matrigel) after treatment with 0.1 U/ml of chondroitinase for 4 h/37°C in serum free media (n = 3, triplicate, Student’s <i>t</i>-test, * indicates p<0.05). (B) SCC-9 LN-1 was treated with 0.1 U/ml for 4 h/37°C in serum free media were seeded in the upper chamber of 96-well transwell plates (n = 3, triplicate). RPMI media, which was supplemented with 1% FBS, was added in the lower chamber. (C) SCC-9 LN-1, SCC-9 and A431 cells treated with increasing concentrations of cisplatin (0–100 µM) for 48 h in the presence of 0.1 U/ml of chondroitinase showed increased sensibility to cisplatin, calculated by a non-linear regression of a dose-response curves (log[µM cisplatin] vs normalized response). Data are expressed as means ± SD from one independent experiment.</p