Pifithrin-μ, an inhibitor of heat-shock protein 70, can increase the antitumor effects of hyperthermia against human prostate cancer cells.

Hyperthermia (HT) improves the efficacy of anti-cancer radiotherapy and chemotherapy. However, HT also inevitably evokes stress responses and increases the expression of heat-shock proteins (HSPs) in cancer cells. Among the HSPs, HSP70 is known as a pro-survival protein. In this study, we investigated the sensitizing effect of pifithrin (PFT)-μ, a small molecule inhibitor of HSP70, when three human prostate cancer cell lines (LNCaP, PC-3, and DU-145) were treated with HT (43°C for 2 h). All cell lines constitutively expressed HSP70, and HT further increased its expression in LNCaP and DU-145. Knockdown of HSP70 with RNA interference decreased the viability and colony-forming ability of cancer cells. PFT-μ decreased the viabilities of all cell lines at one-tenth the dose of Quercetin, a well-known HSP inhibitor. The combination therapy with suboptimal doses of PFT-μ and HT decreased the viability of cancer cells most effectively when PFT-μ was added immediately before HT, and this combination effect was abolished by pre-knockdown of HSP70, suggesting that the effect was mediated via HSP70 inhibition. The combination therapy induced cell death, partially caspase-dependent, and decreased proliferating cancer cells, with decreased expression of c-Myc and cyclin D1 and increased expression of p21(WAF1/Cip), indicating arrest of cell growth. Additionally, the combination therapy significantly decreased the colony-forming ability of cancer cells compared to therapy with either alone. Furthermore, in a xenograft mouse model, the combination therapy significantly inhibited PC-3 tumor growth. These findings suggest that PFT-μ can effectively enhance HT-induced antitumor effects via HSP70 inhibition by inducing cell death and arrest of cell growth, and that PFT-μ is a promising agent for use in combination with HT to treat prostate cancer

The Roles of ROS and Caspases in TRAIL-Induced Apoptosis and Necroptosis in Human Pancreatic Cancer Cells

<div><p>Death signaling provided by tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) can induce death in cancer cells with little cytotoxicity to normal cells; this cell death has been thought to involve caspase-dependent apoptosis. Reactive oxygen species (ROS) are also mediators that induce cell death, but their roles in TRAIL-induced apoptosis have not been elucidated fully. In the current study, we investigated ROS and caspases in human pancreatic cancer cells undergoing two different types of TRAIL-induced cell death, apoptosis and necroptosis. TRAIL treatment increased ROS in two TRAIL-sensitive pancreatic cancer cell lines, MiaPaCa-2 and BxPC-3, but ROS were involved in TRAIL-induced apoptosis only in MiaPaCa-2 cells. Unexpectedly, inhibition of ROS by either <i>N</i>-acetyl-L-cysteine (NAC), a peroxide inhibitor, or Tempol, a superoxide inhibitor, increased the annexin V-/propidium iodide (PI)⁺ early necrotic population in TRAIL-treated cells. Additionally, both necrostatin-1, an inhibitor of receptor-interacting protein kinase 1 (RIP1), and siRNA-mediated knockdown of RIP3 decreased the annexin V^-/PI⁺ early necrotic population after TRAIL treatment. Furthermore, an increase in early apoptosis was induced in TRAIL-treated cancer cells under inhibition of either caspase-2 or -9. Caspase-2 worked upstream of caspase-9, and no crosstalk was observed between ROS and caspase-2/-9 in TRAIL-treated cells. Together, these results indicate that ROS contribute to TRAIL-induced apoptosis in MiaPaCa-2 cells, and that ROS play an inhibitory role in TRAIL-induced necroptosis of MiaPaCa-2 and BxPC-3 cells, with caspase-2 and -9 playing regulatory roles in this process.</p></div

No crosstalk among caspase-2/-9 and ROS in TRAIL-treated cancer cells.

<p>(A) MiaPaCa-2 and BxPC-3 cells were cultured with TRAIL (50 ng/mL) with or without caspase-2 inhibitor (20 μM) for 12 h, and the protein expression levels of caspase-3, -8, and -9 were evaluated by immunoblot. α-Tubulin was used as the control. (B) MiaPaCa-2 and BxPC-3 cells were cultured with TRAIL (50 ng/mL) with or without caspase-9 inhibitor (20 μM) for 12 h, and the protein expression of caspase-2 was evaluated by immunoblot. β-Actin was used as the control. (C) MiaPaCa-2 and BxPC-3 cells were cultured with TRAIL (50 ng/mL) with or without NAC (10 mM) for 12 h, and the protein expression of caspase-8, -2, and -9 was evaluated by immunoblot. β-Actin was used as the control. (D) MiaPaCa-2 and BxPC-3 cells were cultured with TRAIL (50 ng/mL) with the indicated inhibitors (20 μM). As a vehicle control, an equal volume of DMSO was added. After 6 h for MiaPaCa-2 and 12 h for BxPC-3, these cells were cultured with carboxy-H₂DCFDA for 30 min and examined for ROS levels by flow cytometry. The number represents the mean fluorescence intensity. (E) Data represent the mean of three culture wells. MFI: mean fluorescence intensity. *<i>P</i><0.05, N.S., not significant.</p

ROS-dependent apoptosis in TRAIL-treated MiaPaCa-2 cells.

<p>(A) MiaPaCa-2 and BxPC-3 cells were cultured with TRAIL (50 ng/mL) with or without NAC (10 mM) for 24 h. After staining with annexin V-FITC/PI, flow cytometric analysis was performed. The numbers represent the proportions of each subset. (B) The percentages of annexin V⁺ cells are shown. (C) Both cell lines were cultured with TRAIL (50 ng/mL) with or without Tempol (1 mM) for 24 h and analyzed by flow cytometry. (D) The percentages of annexin V⁺ cells are shown. All data points shown represent the mean of three culture wells. *<i>P</i><0.05, **<i>P</i><0.01, N.S., not significant.</p

RIP3-dependent necroptosis in TRAIL-treated BxPC-3 cells under ROS inhibition.

<p>(A) The expression of RIP3 and RIP1 protein was examined in four cancer cell lines. β-Actin was used as a control. (B) MiaPaCa-2 and BxPC-3 cells were transfected with control siRNA or RIP3 siRNA. Three days after transfection, the cells were harvested and examined for RIP3 and RIP1 protein expression. (C) BxPC-3 cells transfected with either control siRNA or RIP3 siRNA 3 days prior were cultured with TRAIL. After 48 h, cell viability was determined by the WST-8 assay. (D) BxPC-3 cells transfected with either control siRNA or RIP3 siRNA 3 days prior were cultured with TRAIL and with either NAC (10 mM) or Tempol (1 mM) for 24 h. After staining with annexin V-FITC/PI, flow cytometric analysis was performed. The numbers represent the proportions of each subset. (E) The percentages of annexin V^-/PI⁺ cells and annexin V⁺ cells were calculated. All data points shown represent the mean of three culture wells. *<i>P</i><0.05, **<i>P</i><0.01.</p

The roles of caspases in apoptosis and necroptosis of TRAIL-treated cells.

<p>(A) MiaPaCa-2 and BxPC-3 cells were cultured with TRAIL (50 ng/mL) for 12 h, and protein expression levels of caspase-3, caspase-8, caspase-9, and caspase-2 were evaluated by immunoblot. α-Tubulin was used as the control. (B) MiaPaCa-2 and BxPC-3 cells were cultured with TRAIL (50 ng/mL) in the presence of a panel of caspase inhibitors (20 μM) for 24 h. After staining with annexin V-FITC/PI, flow cytometric analysis was performed. The numbers represent the proportions of each subset. The percentages of annexin V⁺ cells (C) and annexin V^-/PI⁺ cells (D) were determined by flow cytometry. All data points shown represent the mean of three culture wells. *<i>P</i><0.05, **<i>P</i><0.01. panCi, pan-caspase inhibitor; C9i, caspase-9 inhibitor; C8i, caspase-8 inhibitor; C2i, caspase-2 inhibitor. Vehicle controls received an equal volume of DMSO.</p

TRAIL-induced necroptosis in human pancreatic cancer cells under ROS inhibition.

<p>(A) The percentages of annexin V^-/PI⁺ cells were calculated based on the results of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0127386#pone.0127386.g002" target="_blank">Fig 2</a>. (B) Both cell lines were cultured with TRAIL (50 ng/mL) and NAC (10 mM), with or without necrostatin-1 (20 μM) for 24 h. After staining with annexin V-FITC/PI, flow cytometric analysis was performed. The numbers represent the proportions of each subset. (C) The percentages of annexin V^-/PI⁺ cells were calculated. All data points shown represent the mean of three culture wells. *<i>P</i><0.05, **<i>P</i><0.01.</p

ROS production in TRAIL-sensitive human pancreatic cancer cell lines.

<p>(A) Four human pancreatic cancer lines and PrEC cells were cultured in the presence of TRAIL. After 48 h, cell viability was determined by the WST-8 assay. The data shown represent the mean of three wells. (B) The expression of DR4, DR5, DcR1, and DcR2 in five cell lines was examined by flow cytometry. The line represents staining with mAb specific to either DR4 or DR5, followed by a FITC-conjugated secondary antibody. Solid gray represents staining with FITC-conjugated anti-mouse IgG alone. Regarding the expression of DcR1 and DcR2, the line represents staining with mAb specific to DcR1 and DcR2; solid gray represents staining with isotype-matched FITC-conjugated anti-mouse IgG. (C) Five cell lines were cultured with TRAIL (50 ng/mL). After 6 h for MiaPaCa-2 and 12 h for the other four lines, these cells were cultured with carboxy-H₂DCFDA (50 μM) for 30 min and examined for their ROS levels by flow cytometry. The number represents the mean fluorescence intensity. (D) The data shown represent the mean of three wells. MFI: mean fluorescence intensity. *<i>P</i><0.05, **<i>P</i><0.01, N.S., not significant.</p