Christina Voelkel-Johnson

Resistance of prostate cancer cells to soluble TNF-related apoptosis-inducing ligand (TRAIL/Apo2L) can be overcome by doxorubicin or adenoviral delivery of full-length TRAIL.

Tumor necrosis factor–related apoptosis-inducing ligand (TRAIL/Apo2L) has been shown to induce apoptosis in malignant cells without harming normal cells. To determine the antitumor potential of TRAIL against prostate cells, we undertook a comprehensive study that included eight prostate cancer cells lines (CWR22Rv1, Du145, DuPro, JCA-1, LNCaP, PC-3, PPC-1, and TsuPr1) and primary cultures of normal prostate epithelial cells (PrEC). Cells were tested for susceptibility to soluble TRAIL in the presence or absence of the chemotherapeutic agent doxorubicin. TRAIL was also delivered by an adenoviral vector. Our results reveal that Du145, DuPro, LNCap, TsuPr1, and PrEC were resistant to 100 ng/mL TRAIL. JCA-1 and PPC-1 were slightly sensitive (20% killing) and PC-3 and CWR22Rv1 exhibited the highest sensitivity to TRAIL (30% and 50% killing, respectively). The combination of 10 ng/mL TRAIL with doxorubicin resulted in 60–80% cytotoxicity in seven of eight prostate cancer cells. TRAIL-mediated apoptosis involved cleavage of Bid, caspase-3, and PARP, and required caspase-8 and -9 activity. Full-length TRAIL delivered by an adenoviral vector (AdTRAIL-IRES-GFP) killed prostate cancer cell lines and PrEC without requisite doxorubicin cotreatment. Therefore, expression of the transgene from a tissue-specific promotor would make gene therapy with AdTRAIL-IRES-GFP a possibility.

Doxorubicin pretreatment sensitizes prostate cancer cell lines to TRAIL induced apoptosis which correlates with the loss of c-FLIP expression.

Tumor necrosis factor-related apoptosis inducing ligand (TRAIL/Apo2L) can induce receptor-mediated apoptosis in prostate cancer cell lines that have been co-treated with the chemotherapeutic agent doxorubicin (Voelkel-Johnson C, et al. Cancer Gene Therapy 2002; 9:164–172). In this study, we report that pretreatment with doxorubicin is sufficient to sensitize cells to TRAIL. To identify possible targets of doxorubicin, we analyzed levels of several Bcl-2 family members, TRAIL receptors and the anti-apoptotic protein c-FLIP. Doxorubicin did not affect steady state levels of Bax, Bcl-2 and Bcl-XL in the majority of the prostate cancer cell lines. TRAIL receptor mRNAs (DR4, DR5, and DcR2) were induced by doxorubicin but these changes were not reflected at the protein level. In contrast, in response to doxorubicin, levels of c-FLIP, particularly FLIPS, decreased in all cell lines tested. The decrease in c-FLIPS correlated with onset and magnitude of caspase-8 and PARP cleavage in PC3 cells. In two TRAIL resistant cell lines, DU145 and LNCaP, treatment with TRAIL alone resulted in processing of c-FLIPL and initiated abortive caspase-8 proteolysis. TRAIL treatment did not affect levels of c-FLIPS in Du145 and LNCaP cells and did not result in PARP cleavage. Therefore, our results suggest that doxorubicin- mediated down regulation of c-FLIPS predisposes cells to TRAIL-induced apoptosis. Key Words: TRAIL, Doxorubicin, Apoptosis, Prostate cancer, c-FLIP

Ceramide synthase 6 modulates TRAIL sensitivity and nuclear translocation of active caspase 3 in colon cancer cells

We have previously shown that the death receptor ligand TRAIL (tumor necrosis factor-related apoptosis-inducing ligand) induces an increase of intracellular C16-ceramide in sensitive SW480 but not in resistant SW620 cells. Resistance in SW620 cells was overcome by exogenous ceramide, leading us to propose that defective ceramide signaling contributes to TRAIL resistance. In this study we found that the increase in C16-ceramide in SW480 cells was inhibited by fumonisin B1, an inhibitor of ceramide synthases (CerS). Protein analysis revealed that TRAIL-resistant SW620 cells expressed lower levels of ceramide synthase 6 (CerS6, also known as longevity assurance homologue 6), which prompted us to investigate the effect of CerS6 modulation on TRAIL phenotype. RNAi against CerS6 resulted in a specific and significant decrease of the C16-ceramide species, which was sufficient to inhibit TRAIL-induced apoptosis. In cells with decreased levels of CerS6, caspase-3 was activated but failed to translocate into the nucleus. CerS6 localized primarily to the perinuclear region, suggesting this enzyme may be important in regulation of nuclear permeability. Moderate elevation in CerS6 expression was sufficient to reverse TRAIL resistance in SW620 cells. These results suggest that modulation of CerS6 expression may constitute a new therapeutic strategy to alter apoptotic susceptibility.

Vorinostat and sorafenib increase CD95 activation in gastrointestinal tumor cells through a Ca2+ - de novo ceramide - PP2A - ROS dependent signaling pathway

The targeted therapeutics sorafenib and vorinostat interact in a synergistic fashion to kill carcinoma cells by activating CD95, and this drug combination is entering phase I evaluation. In this study, we determined how CD95 is activated by treatment with this drug combination. Low doses of sorafenib and vorinostat, but not the individual drugs, rapidly increased reactive oxygen species (ROS), Ca(2+), and ceramide levels in gastrointestinal tumor cells. The production of ROS was reduced in Rho zero cells. Quenching ROS blocked drug-induced CD95 surface localization and apoptosis. ROS generation, CD95 activation, and cell killing was also blocked by quenching of induced Ca(2+) levels or by inhibition of PP2A. Inhibition of acidic sphingomyelinase or de novo ceramide generation blocked the induction of ROS; however, combined inhibition of both acidic sphingomyelinase and de novo ceramide generation was required to block the induction of Ca(2+). Quenching of ROS did not affect drug-induced ceramide/dihydro-ceramide levels, whereas quenching of Ca(2+) reduced the ceramide increase. Sorafenib and vorinostat treatment radiosensitized liver and pancreatic cancer cells, an effect that was suppressed by quenching ROS or knockdown of LASS6. Further, sorafenib and vorinostat treatment suppressed the growth of pancreatic tumors in vivo. Our findings show that induction of cytosolic Ca(2+) by sorafenib and vorinostat is a primary event that elevates dihydroceramide levels, each essential steps in ROS generation that promotes CD95 activation.

Doxorubicin increases the effectiveness of Apo2L/TRAIL for tumor growth inhibition of prostate cancer xenografts

BackgroundProstate cancer is a significant health problem among American men. Treatment strategies for androgen-independent cancer are currently not available. Tumor necrosis factor-related apoptosis-inducing ligand (Apo2L/TRAIL) is a death receptor ligand that can induce apoptosis in a variety of cancer cell lines, including androgen-independent PC3 prostate carcinoma cells. In vitro, TRAIL-mediated apoptosis of prostate cancer cell lines can be enhanced by doxorubicin and correlates with the downregulation of the anti-apoptotic protein c-FLIP. This study evaluated the effects of doxorubicin on c-FLIP expression and tumor growth in combination with Apo2L/TRAIL in a xenograft model.MethodsIn vitro cytotoxic effects of TRAIL were measured using a MTS-based viability assay. For in vivo studies, PC3 prostate carcinoma cells were grown subcutaneously in athymic nude mice and tumor growth was measured following treatment with doxorubicin and/or Apo2L/TRAIL. c-FLIP expression was determined by western blot analysis. Apoptosis in xenografts was detected using TUNEL. Statistical analysis was performed using the student t-test.ResultsIn vitro experiments show that PC3 cells are partially susceptible to Apo2L/TRAIL and that susceptibility is enhanced by doxorubicin. In mice, doxorubicin did not significantly affect the growth of PC3 xenografts but reduced c-FLIP expression in tumors. Expression of c-FLIP in mouse heart was decreased only at the high doxorubicin concentration (8 mg/kg). Combination of doxorubicin with Apo2L/TRAIL resulted in more apoptotic cell death and tumor growth inhibition than Apo2L/TRAIL alone.ConclusionsCombination of doxorubicin and Apo2L/TRAIL is more effective in growth inhibition of PC3 xenografts in vivo than either agent alone and could present a novel treatment strategy against hormone-refractory prostate cancer. The intracellular mechanism by which doxorubicin enhances the effect of Apo2L/TRAIL on PC3 xenografts may be by reducing expression of c-FLIP.

Sorafenib Activates CD95 and Promotes Autophagy and Cell Death via Src Family Kinases in Gastrointestinal Tumor Cells

Sorafenib and vorinostat interact in a synergistic fashion to kill carcinoma cells by activating CD95; the present studies have determined how sorafenib and vorinostat individually contribute to CD95 activation. Sorafenib (3–6 μmol/L) promoted a dose-dependent increase in Src Y416, ERBB1 Y845 and CD95 Y232/Y291 phosphorylation, and Src Y527 dephosphorylation. Low levels of sorafenib-induced (3 μmol/L) CD95 tyrosine phosphorylation did not promote surface localization whereas sorafenib (6 μmol/L), or sorafenib (3 μmol/L) and vorinostat (500 nmol/L) treatment promoted higher levels of CD95 phosphorylation which correlated with DISC formation, receptor surface localization, and autophagy. CD95 (Y232F, Y291F) was not tyrosine phosphorylated and was unable to localize plasma membrane or induce autophagy. Knockdown/knockout of Src family kinases abolished sorafenib-induced CD95 tyrosine phosphorylation, DISC formation, and the induction of cell death and autophagy. Knockdown of platelet-ived growth factor receptor-β enhanced Src Y416 and CD95 tyrosine phosphorylation, which correlated with elevated CD95 plasma membrane levels and autophagy, and with a reduced ability of sorafenib to promote CD95 membrane localization. Vorinostat increased reactive oxygen species levels, and in a delayed NFκB-dependent fashion, those of FAS ligand and CD95. Neutralization of FAS-L did not alter the initial rapid drug-induced activation of CD95; however, neutralization of FAS-L reduced sorafenib + vorinostat toxicity by ∼50%. Thus, sorafenib contributes to CD95 activation by promoting receptor tyrosine phosphorylation, whereas vorinostat contributes to CD95 activation via the initial facilitation of reactive oxygen species generation and subsequently of FAS-L expression. Mol Cancer Ther; 9(8); 2220–31. ©2010 AACR.

An antibody against DR4 (TRAIL-R1) in combination with doxorubicin selectively kills malignant but not normal prostate cells.

Prostate cancer is a major health problem among American men and new treatment approaches are needed. Tumor necrosis factor related apoptosis-inducing ligand (TRAIL/Apo2L) is a death ligand that can induce apoptosis in some but not all cancer cells. Resistance to TRAIL-mediated apoptosis can be overcome by radiation or chemotherapy. The effect of doxorubicin/TRAIL combination therapy was compared among PC3 cells, normal prostate epithelial (PrEC) and stromal (PrSC) cells and cell viability measured by MTS assay. Combination of doxorubicin and TRAIL caused cytotoxicity in all cells tested, although PrSC were more resistant. There was no correlation between TRAIL phenotype and expression of c-FLIP, caspases or TRAIL decoy receptors, although PrSC failed to express DR4. A DR4-specific antibody, which behaved as an agonist in combination with doxorubicin, selectively induced cell death in malignant but not normal prostate cells. Although normal PrEC expressed DR4 as determined by western blot, flow cytometry revealed that only maligant prostate cancer cells (PC3, JCA-1) and not PrEC’s exhibited DR4 surface expression. Therefore, combination of doxorubicin and an antibody to DR4 might have therapeutic potential for the treatment of prostate cancer by selectively targeting malignant prostate cells.

Inner ear supporting cells protect hair cells by secreting HSP70

Mechanosensory hair cells are the receptor cells of hearing and balance. Hair cells are sensitive to death from exposure to therapeutic drugs with ototoxic side effects, including aminoglycoside antibiotics and cisplatin. We recently showed that the induction of heat shock protein 70 (HSP70) inhibits ototoxic drug-induced hair cell death. Here, we examined the mechanisms underlying the protective effect of HSP70. In response to heat shock, HSP70 was induced in glia-like supporting cells but not in hair cells. Adenovirus-mediated infection of supporting cells with Hsp70 inhibited hair cell death. Coculture with heat-shocked utricles protected nonheat-shocked utricles against hair cell death. When heat-shocked utricles from Hsp70-/- mice were used in cocultures, protection was abolished in both the heat-shocked utricles and the nonheat-shocked utricles. HSP70 was detected by ELISA in the media surrounding heat-shocked utricles, and depletion of HSP70 from the media abolished the protective effect of heat shock, suggesting that HSP70 is secreted by supporting cells. Together our data indicate that supporting cells mediate the protective effect of HSP70 against hair cell death, and they suggest a major role for supporting cells in determining the fate of hair cells exposed to stress.

The role of protein synthesis in cell cycling and cancer

Cell cycling and protein synthesis are both key physiological tasks for cancer cells. Here we present a model for how the elongation phase of protein synthesis, governed by elongation factor 2 and elongation factor 2 kinase, both modulates and responds to cell cycling. Within this framework we also discuss survivin, a protein with both pro‐mitotic and anti‐apoptotic roles whose persistence in the cell is tied to protein synthesis due to its short half‐life. Finally, we provide a brief overview of efforts of cancer researchers to target EF2 and EF2 kinase.

Resistance to TRAIL is associated with defects in ceramide signaling that can be overcome by exogenous C6-ceramide without requiring down-regulation of cellular FLICE inhibitory protein

Tumor necrosis factor–related apoptosis-inducing ligand (TRAIL) is a member of the tumor necrosis factor superfamily that selectively induces apoptosis in malignant cells. However, not all cancer cells are susceptible to TRAIL and mechanisms of resistance and new strategies to enhance sensitivity are an area of intense investigation. Glucose withdrawal or paclitaxel increase intracellular ceramide, down-regulate cellular FLICE inhibitory protein (cFLIP), and sensitize cells to TRAIL. Therefore, we investigated whether TRAIL resistance is due to ceramide levels and/or defects in ceramide generation following ligand binding. Colon cancer cells isolated from the primary tumor (SW480) and a subsequent metastasis (SW620) of the same patient have different sensitivities to TRAIL. Mass spectrometry was used to compare ceramide content in untreated and TRAIL-treated cells. Overall levels of ceramide were comparable in the cell lines but TRAIL-sensitive SW480 cells contained a higher percentage of C16-, and C18-ceramide and lower C24-ceramides than TRAIL-resistant SW620 cells. Upon TRAIL treatment, ceramide (primarily C16-ceramide) increased in SW480 but not SW620 cells. The increase in ceramide occurred with slow kinetics, paralleling caspase-3/7 activation. Combination of C6-ceramide with TRAIL resulted in apoptosis of SW620 cells. However, exogenous C6-ceramide did not affect levels of cFLIP nor did pretreatment sensitize cells to TRAIL. Exposure to TRAIL prior to ceramide was required to induce apoptosis, suggesting that ceramide plays a role in enhancing or amplifying TRAIL-mediated signaling. Our results suggest that ceramide plays a role in promoting TRAIL-mediated apoptosis and that TRAIL-resistant cancers may benefit from combination therapy with ceramide or agents that enhance ceramide accumulation.