Mechanistic evaluation of a novel small molecule targeting mitochondria in pancreatic cancer cells

Abstract

Background: 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.

Methodology/principal findings: TP421 exhibited sub-micromolar IC(50) values in all the pancreatic cancer cell lines tested using MTT and colony formation assays. TP421 localized predominantly to mitochondria and induced G(0)/G(1) 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.

Conclusions/significance: 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.

Figure 1. TP421 significantly inhibits colony formation of pancreatic cancer cells.

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.

Figure 2. Effect of shorter exposure time on the cytotoxicity of TP421 in pancreatic cancer cells.

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.

Figure 3. TP421 cytotoxicity is selective for cancer cells.

(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.

Figure 4. TP421 arrests pancreatic cancer cells in G₀/G₁ phase of the cell cycle.

The effect of TP421 treatment on the cell cycle distribution of MIA PACa-2 cells was examined in a dose and time-dependent manner. Cells were untreated or treated with 0.1 and 1 µM TP421 for 24, 48 and 72 h. Histograms depicted are representative of three independent experiments.

Figure 5. TP421 accumulates in mitochondria.

(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.

Figure 6. TP421 causes oxidative stress in pancreatic cancer cells.

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.

Figure 7. TP421 causes activation of stress induced signaling pathways and a sustained activation of MEK and ERK.

Western blot analysis of the effect of TP421 treatment on the phosphorylation status of stress pathways. (A) MIA PaCa-2 cells were treated with 5 µM TP421 at increasing time points were probed for the phosphorylation of JNK1/2 and c-Jun. (B) MIA PaCa-2 and BxPC-3 cells were treated with 5 µM TP421 and probed for p-38 phosphorylation. Even loading for MIA PaCa-2 was verified using amido black total protein staining of membrane following transfer (data not shown) (C) MIA PaCa-2 cells were treated with 5 µM TP421 and probed for phosphorylation of MEK1/2 and Erk1/2.

Figure 8. Effect of TP421 treatment on DNA damage and apoptosis induction.

(A) BxPC-3 and MIA PaCa-2 cells treated with increasing concentrations of TP421 for 12–48 h were probed for the DNA damage marker phospho-H2A.X (S139). (B) MIA PaCa-2 cells treated with TP421 were probed for caspase activation and PARP-1 cleavage. (C) The effect of TP421 treatment on Bcl-2 and survivin levels was analyzed in MIA PaCa-2 cells.

Figure 9. TP421 affects the autophagic response in pancreatic cancer cells.

(A) BxPC-3 and MIA PaCa-2 cells were treated with 0.5 and 2.5 µM TP421 or chloroquine for 24 and 48 h and probed for Beclin1, p62, and LC3B-I/II. (B) LC3B puncta formation in MIA PaCa-2 cells treated with TP421 for 18 h alone or in combination with rapamycin or chloroquine. (C) MEF cells competent in autophagy (atg3+/+) and deficient in autophagy (atg3−/−) were treated with increasing concentrations of TP421 and percent cytotoxicity was determined by MTT. The data are mean ± SD from three independent experiments. * and ** indicate p<0.05 and p<0.001 respectively. (D) MEF atg3+/+ and −/− cells treated with TP421 for 24 h and probed for autophagy proteins LC3B-I/II and p62. (E) MIA PaCa-2 cells treated with TP421 for increasing durations of time and co-stained with LysoTracker Red (LTR).

Figure 10. TP421 decreases signaling via Src-FAK and inhibits cell migration.

(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.