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<p>Results of TP421 proteomics screen for proteins whose total abundances were decreased in response to TP421 treatment.</p

Mechanistic Evaluation of a Novel Small Molecule Targeting Mitochondria in Pancreatic Cancer Cells

<div><p>Background</p><p>Pancreatic cancer is one of the deadliest cancers with a 5-year survival rate of 6%. Therapeutic options are very limited and there is an unmet medical need for safe and efficacious treatments. Cancer cell metabolism and mitochondria provide unexplored targets for this disease. We recently identified a novel class of triphenylphosphonium salts, TP compounds, with broad- spectrum anticancer properties. We examined the ability of our prototypical compound TP421– chosen for its fluorescent properties – to inhibit the growth of pancreatic cancer cells and further investigated the molecular mechanisms by which it exerts its anticancer effects.</p><p>Methodology/Principal Findings</p><p>TP421 exhibited sub-micromolar IC₅₀ values in all the pancreatic cancer cell lines tested using MTT and colony formation assays. TP421 localized predominantly to mitochondria and induced G₀/G₁ arrest, ROS accumulation, and activation of several stress-regulated kinases. Caspase and PARP-1 cleavage were observed indicating an apoptotic response while LC3B-II and p62 were accumulated indicating inhibition of autophagy. Furthermore, TP421 induced de-phosphorylation of key signaling molecules involved in FAK mediated adhesion that correlated with inhibition of cell migration.</p><p>Conclusions/Significance</p><p>TP421 is a representative compound of a new promising class of mitochondrial-targeted agents useful for pancreatic cancer treatment. Because of their unique mechanism of action and efficacy further development is warranted.</p></div

Percent distribution of DNA content per cell cycle phase of several pancreatic cancer cell lines in response to treatment with TP421 and close analogs.

<p>Percent distribution of DNA content per cell cycle phase of several pancreatic cancer cell lines in response to treatment with TP421 and close analogs.</p

TP421 causes oxidative stress in pancreatic cancer cells.

<p>The effect of TP421 treatment on the levels of (A) H₂O₂ and (B) mitochondrial O₂⁻ in BxPC-3 and MIA PaCa-2 cells were measured using the fluorescent probes Amplex Red and MitoSOX Red, respectively. (C) Effect of antioxidant pretreatment on the cytotoxicity of TP421 in PANC-1 cells. *, ** and *** indicate p<0.05, p<0.005 and p<0.001 respectively.</p

Effect of shorter exposure time on the cytotoxicity of TP421 in pancreatic cancer cells.

<p>Dose response survival curves for cell lines treated with TP421 comparing continuous exposure (closed squares) to 0.25 h drug exposure followed by a media wash out (open squares) and further incubation. Total incubation time is indicated in parentheses. The data are mean ± SD from three independent experiments. TP421 concentrations are plotted on a Log base 10 scale.</p

TP421 accumulates in mitochondria.

<p>(A) PANC-1 cells treated with TP421 and stained with MitoTracker Red (MTR) reveal extensive co-localization of TP421 and mitochondrial marker dye. (B) PANC-1 cells pretreated with FCCP show non-mitochondrial TP421 localization. (C) Reduced cytotoxicity of TP421 in MIA PaCa-2 cells pretreated with FCCP is seen at 24, 48 and 72 h. The data are mean ± SD from three independent experiments. * indicates p<0.01.</p

IC₅₀ values (µM) for TP421 and close structural analogs in pancreatic cancer cell lines.

<p>IC₅₀ values (µM) for TP421 and close structural analogs in pancreatic cancer cell lines.</p

TP421 decreases signaling via Src-FAK and inhibits cell migration.

<p>(A) MIA PaCa-2 cells treated with 5 µM TP421 for indicated time and probed for de-activating phosphorylation (Y527) and the activating phosphorylation (Y416) of Src. (B) MIA PaCa-2 and BxPC-3 cells were treated with 5 µM TP421 and probed for phosphorylation of FAK. (C) Effect of 5 µM TP421 treatment on phosphorylation status of p130Cas and Paxillin proteins downstream of Src activation. (D) 5 µM TP421 treatment decreases the phosphorylation of Smad1/5/8 in MIA PaCa-2 cells. (E) Effect of 24 h TP421 treatment of serum starved MIA PaCa-2 cells on their ability to migrate through a Boyden Chamber setup. (F) TP421 treated PANC-1 cells are inhibited from migrating into denuded area of the wound.</p

TP421 cytotoxicity is selective for cancer cells.

<p>(A) Growth of normal HFF-1 cells is unaffected while the pancreatic cancer MIA PaCa-2 cells show extensive death following 72 h exposure to escalating doses of TP421. (B) TP421 induces greater cell death in three pancreatic cancer cell lines as compared to HFF-1 cells as measured by trypan blue exclusion. (C) Proliferation of MIA PaCa-2 but not HFF-1 is greatly inhibited by TP421 in the alamar blue assay. Three independent experiments were conducted, representative images are shown in (A), mean ± SD are plotted in (B) and (C). *, **, *** and **** indicate p-value <0.05, p<0.01, p<0.001 and p<0.00005 respectively.</p

TP421 significantly inhibits colony formation of pancreatic cancer cells.

<p>Effect of 24 h drug exposure on colony forming ability of (A) MIA PaCa-2 and (B) BxPC-3. Images are representative of three independent experiments.</p