Effect of TRKA inhibitor GW441756 on the ability of pancreatic cancer cells to migrate towards neurites extended from DRG.

<p>A) Representative images (top panel: bright field; bottom panel: green fluorescence) of the migration of Mia PaCa-2 cells towards nerves treated with different concentrations of GW441756. Arrows indicate the cancer cells that migrated towards the neurites. B) Quantification of the effect on the migration of cancer cells using relative invasion index. *≤ 0.01; ** ≤ 0.001; *** ≤ 0.0001 (compared to no drug control).</p

Pixantrone: novel mode of action and clinical readouts

<p><b>Introduction</b>: Pixantrone is a first-in-class aza-anthracenedione approved as monotherapy for treatment of relapsed or refractory aggressive diffuse B-cell non-Hodgkin’s lymphoma (NHL), a patient group which is notoriously difficult to treat. It has a unique chemical structure and pharmacologic properties distinguishing it from anthracyclines and anthracenediones.</p> <p><b>Areas covered</b>: The chemical structure and mode of action of pixantrone versus doxorubicin and mitoxantrone; preclinical evidence for pixantrone’s therapeutic effect and cardiac tolerability; efficacy and safety of pixantrone in clinical trials; ongoing and completed trials of pixantrone alone or as combination therapy; and the risk of cardiotoxicity of pixantrone versus doxorubicin and mitoxantrone.</p> <p><b>Expert commentary</b>: Currently, pixantrone is the only approved therapy for multiply relapsed or refractory NHL, an area with few available effective treatment options. Pixantrone is currently being investigated as combination therapy with other drugs including several targeted therapies, with the ultimate goal of improved survival in heavily pretreated patients. In order for pixantrone to be acknowledged in the treatment of aggressive NHL, the perception of pixantrone as an anthracycline-like agent that has anthracycline-like activity and cardiotoxicity needs to be changed. Further data from ongoing clinical trials will help in confirming pixantrone as an effective and safe option.</p

Knocking down the expression of the NGF-TRKA signaling pathway genes using siRNA oligonucleotides.

<p>The cells were treated with the siRNA oligonucleotides for 72 hours and then subjected to Western blotting (A and B) or RT-PCR analysis (C and D) for NGF, p75^NTR and TRKA expression in Mia PaCa-2 (A and C) or BxPC-3 (B and D) cells. SF: siLentFect (transfection reagent), NT: Non-targeting siRNA control. *: p ≤ 0.05; ** ≤ 0.01.</p

Effect of NGF neutralizing antibody on the ability of pancreatic cancer cells to migrate towards neurites extended from dorsal root ganglia (DRG).

<p>A) Representative images (top panel: bright field; bottom panel: green fluorescence) of the migration of Mia PaCa-2 cells towards nerves treated with different concentrations of NGF neutralizing antibody. Arrows indicate the cancer cells that migrated towards the neurites. B) Quantification of the effect on the migration of cancer cells using relative invasion index. *: p ≤ 0.01; ** ≤ 0.001; *** ≤ 0.0001 (compared to no antibody control).</p

Knocking down the expression of the NGF-TRKA signaling pathway proteins or inhibiting the activity of TRKA reduces the growth and migratory activity of pancreatic cancer cells.

<p>A) Cell growth inhibition by siRNA treatment in Mia PaCa-2 and BxPC-3 cells. Cell viability was measure 72 hours post siRNA treatment and compared to Non-targeting (NT) siRNA control. B) Cell growth inhibition by TRKA inhibitor GW441756. Cells were treated with the inhibitor for 72 hours. C) siRNA knockdown of the expression of NGF, p75^NTR or TRKA reduced migration of pancreatic cancer cells. D) GW441756 inhibited the migration of pancreatic cancer cells in a dose dependent manner. *: p ≤ 0.05; ** ≤ 0.01 (compared to Non-targeting control).</p

Effect of siRNA knockdown of the expression of NGF, TRKA, or p75^NTR on the ability of pancreatic cancer cells to migrate towards neurites extended from DRG.

<p>A) Representative images (top panel: bright field; bottom panel: green fluorescence) of the migration of Mia PaCa-2 cells towards nerves treated with siRNA oligonucleotides targeting the indicated genes. Arrows indicate the cancer cells that migrated towards the neurites. B) Quantification of the effect on the migration of cancer cells using relative invasion index. SF: siLentFect (transfection reagent), NT: Non-targeting siRNA control. ** ≤ 0.001; *** ≤ 0.0001 (compared to Non-targeting siRNA control).</p

Reciprocal signaling between pancreatic cancer cells and neurites extended from the PC-12 cells is reduced when the NGF-TRKA signaling pathway is disrupted.

<p>A) Treatment with the NGF neutralizing antibody or the TRKA inhibitor (GW441756) reduces the neurite outgrowth induced by conditional medium from pancreatic cancer cells. B) Quantification of neurite outgrowth assay shown in A. Conditioned Media (CM) collected from MiaPaCa2 or BxPC3 cells were treated with a neutralizing NGF antibody and a TRKA inhibitor and neurite (yellow arrows) extension from the PC-12 cells was visualized under a bright field microscope. Recombinant NGF protein was used as a positive control. *≤ 0.01; ** ≤ 0.001; *** ≤ 0.0001 (compared to Conditioned Media).</p

Inhibition of ROCK1 kinase modulates both tumor cells and stromal fibroblasts in pancreatic cancer

<div><p>ROCK, or Rho-associated coiled coil-containing protein kinase, is a member of the AGC kinase family and has been shown to play a role in cell migration, ECM synthesis, stress-fiber assembly, and cell contraction. Increased ROCK expression has been reported in multiple pathological conditions, including cancer. Here, we report increased expression of ROCK 1 in pancreatic tumor epithelial cells as well as in cancer associated fibroblasts (CAF). In our analysis, 62% of tumor samples exhibited ≥2+ in staining intensity by IHC analysis, versus 40% of adjacent normal tissue samples (P<0.0001). Thus, we hypothesized that ROCKs may play a significant role in pancreatic cancer progression, and may serve as a suitable target for treatment. We report a low frequency (4/34) amplification of the ROCK1 gene locus at chromosome 18q11.1 in pancreatic ductal adenocarcinoma (PDAC) patient tissue samples by aCGH analysis. Inhibition of ROCK kinase activity by a small molecule inhibitor (fasudil) resulted in moderate (IC₅₀s of 6–71 μM) inhibition of PDAC cell proliferation, migration, and activation of co-cultured stellate cells. In the KPC mouse model for pancreatic cancer, fasudil decreased tumor collagen deposition. This translated to an enhanced overall survival of the mice and an increase in gemcitabine uptake. Though fasudil may target both the tumor epithelial cells and the CAFs, our findings are consistent with the hypothesis that inhibition of tumor stroma enhances drug penetration and efficacy in PDAC. Overall, our data suggests that ROCK1 may serve as a potential therapeutic target to enhance current treatment regimens for pancreatic cancer.</p></div

Knockdown of ROCK1 by siRNA in pancreatic cancer cell inhibits cell proliferation.

<p>A) ROCK1 was detected in pancreatic cancer cell lines (PANC-1, Mia PaCa-2, SU.86.86, BxPC3, AsPC-1, and HS766T), the immortalized normal pancreatic ductal epithelial cell line (HPDE6), and the cancer associated fibroblasts (CW-1) by Western blotting. B) Western blotting analysis of ROCK1 knockdown by siRNA over the course of 72 hours. (C) Western blotting analysis of ROCK1 knockdown by siRNA (72 hour treatment) in two cell lines, SU.86.86 and PANC-1. (D) Growth curves of pancreatic cancer cells (PANC-1 and SU.86.86) treated with siRNA to ROCK1. * P < 0.001 (compared to the untreated control). UT, untreated; tR, transfection reagent only; NT, non-targeting; ASD, cell death (positive) control; R1, ROCK1 siRNA1; R2, ROCK1 siRNA 2.</p

Effects of fasudil treatment on tumor stroma in KPC mice.

<p>A) Pancreatic tumor tissues from vehicle, gemcitabine, or combination of gemcitabine plus fasudil treated KPC mice were harvested and stained for various stromal markers. Representative images are shown of H&E staining, α-SMA, Desmin, CD31, Collagen I, and Movat's pentachrome staining.</p