Preclinical Evaluation of Novel Triphenylphosphonium Salts with Broad-Spectrum Activity

BACKGROUND: Recently, there has been a surge of interest in developing compounds selectively targeting mitochondria for the treatment of neoplasms. The critical role of mitochondria in cellular metabolism and respiration supports this therapeutic rationale. Dysfunction in the processes of energy production and metabolism contributes to attenuation of response to pro-apoptotic stimuli and increased ROS production both of which are implicated in the initiation and progression of most human cancers. METHODOLOGY/PRINCIPAL FINDINGS: A high-throughput MTT-based screen of over 10,000 drug-like small molecules for anti-proliferative activity identified the phosphonium salts TP187, 197 and 421 as having IC₅₀ concentrations in the submicromolar range. TP treatment induced cell cycle arrest independent of p53 status, as determined by analysis of DNA content in propidium iodide stained cells. In a mouse model of human breast cancer, TP-treated mice showed significantly decreased tumor growth compared to vehicle or paclitaxel treated mice. No toxicities or organ damage were observed following TP treatment. Immunohistochemical staining of tissue sections from TP187-treated tumors demonstrated a decrease in cellular proliferation and increased caspase-3 cleavage. The fluorescent properties of analog TP421 were exploited to assess subcellular uptake of TP compounds, demonstrating mitochondrial localization. Following mitochondrial uptake cells exhibited decreased oxygen consumption and concomittant increase in mitochondrial superoxide production. Proteomics analysis of results from a 600 target antibody microarray demonstrated that TP compounds significantly affected signaling pathways relevant to growth and proliferation. CONCLUSIONS/SIGNIFICANCE: Through our continued interest in designing compounds targeting cancer-cell metabolism, the Warburg effect, and mitochondria we recently discovered a series of novel, small-molecule compounds containing a triphenylphosphine moiety that show remarkable activity in a panel of cancer cell lines as well as in a mouse model of human breast cancer. The mechanism of action includes mitochondrial localization causing decreased oxygen consumption, increased superoxide production and attenuated growth factor signaling

The role of covalent dimerization on the physical and chemical stability of the EC1 domain of human E-cadherin

The objective of this work was to evaluate the solution stability of the EC1 domain of E-cadherin under various conditions. The EC1 domain was incubated at various temperatures (4, 37, and 70 °C) and pH values (3.0, 7.0, and 9.0). At pH 9.0 and 37 or 70 °C, a significant loss of EC1 was observed due to precipitation and a hydrolysis reaction. The degradation was suppressed upon addition of DTT, suggesting that the formation of EC1 dimer facilitated the EC1 degradation. At 4 °C and various pH values, the EC1 secondary and tertiary showed changes upon incubation up to 28 days, and DTT prevented any structural changes upon 28 days of incubation. Molecular dynamics simulations indicated that the dimer of EC1 has higher mobility than does the monomer; this higher mobility of the EC1 dimer may contribute to instability of the EC1 domain

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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

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

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

<p>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.</p

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

<p>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.</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