Among the many processes regulating cell death, ceramide signaling is a vital component. We previously determined that acid ceramidase (AC) is upregulated in 60% of primary prostate cancer (PCa) tissues, suggesting that AC may play a role in tumor development. In order to determine the significance of AC elevation, stable clones of DU145 cells with AC overexpression (AC-EGFP) were generated. Compared to controls (EGFP), AC-EGFP cells exhibited enhanced cell proliferation and migration. Subcutaneous injection of AC-EGFP cells into Nu/Nu mice resulted in larger tumor volumes compared to EGFP controls. Moreover, using the MTS viability assay, AC-EGFP cells were more resistant to cell death induced by doxorubicin, cisplatin, etoposide, gemcitabine or C6-ceramide. Conversely, knock down of AC using siRNA, sensitized AC-EGFP cells to these drugs. In addition, mass spectroscopic analysis of sphingolipids indicated that long chain ceramide levels were decreased in AC-EGFP cells treated with either doxorubicin or etoposide. In conclusion, this study implicates AC as a critical regulator of PCa progression by affecting not only tumor cell proliferation and migration but also responses to drug therapy, suggesting AC as a potential therapeutic target in advanced PCa.

Radiation resistance in a subset of prostate tumors remains a challenge to prostate cancer radiotherapy. The current study on the effects of radiation on prostate cancer cells reveals that radiation programs an unpredicted resistance mechanism by upregulating acid ceramidase (AC). Irradiated cells demonstrated limited changes of ceramide levels while elevating levels of sphingosine and sphingosine-1-phosphate. By genetically downregulating AC with small interfering RNA (siRNA), we observed radiosensitization of cells using clonogenic and cytotoxicity assays. Conversely, AC overexpression further decreased sensitivity to radiation. We also observed that radiation-induced AC upregulation was sufficient to create cross-resistance to chemotherapy as demonstrated by decreased sensitivity to Taxol and C(6) ceramide compared to controls. Lower levels of caspase 3/7 activity were detected in cells pretreated with radiation, also indicating increased resistance. Finally, utilization of the small molecule AC inhibitor, LCL385, sensitized PPC-1 cells to radiation and significantly decreased tumor xenograft growth. These data suggest a new mechanism of cancer cell resistance to radiation, through upregulation of AC that is, in part, mediated by application of the therapy itself. An improved understanding of radiotherapy and the application of combination therapy achieved in this study offer new opportunities for the modulation of radiation effects in the treatment of cancer.

Treatment of different cancer cell lines with desipramine induced a time-and dose-dependent downregulation of acid ceramidase. Desipramine's effect on acid ceramidase appeared specific for amphiphilic agents (desipramine, chlorpromazine, and chloroquine) but not other lysomotropic agents such as ammonium chloride and bafilomycin A1, and was not transcriptionally regulated. The cathepsin B/L inhibitor, CA074ME, but not the cathepsin D inhibitor, pepstatin A, blocked desipramine's effect on acid ceramidase. Desipramine led to a more pronounced downregulation of sphingosine compared to ceramide suggesting acid ceramidase inhibition is important to desipramine's mechanism of action. This study reveals a new mechanism of action for desipramine.

Head and neck squamous cell cancers (HNSCC) are particularly aggressive and are resistant to many forms of treatment. Ceramide metabolism has been shown to play an important role in cancer progression and cancer resistance to therapy in many tumor models, including HNSCC. Here, we study the role of the ceramide-metabolizing enzyme acid ceramidase (AC) in therapeutic responses in HNSCC. First, we show that AC is over-expressed in 70% of head and neck squamous cell tumors compared with normal tissues, suggesting that this enzyme may play an important role in facilitating HNSCC growth. Next, comparison of three HNSCC cell lines with low, medium, and high levels of AC reveals an inverse correlation between the levels of AC and their response to exogenous C-6-ceramide. Furthermore, over-expression of AC in SCC-1 cells increased resistance to Fas-induced cell killing. Conversely, down-regulation of AC using specific AC small interfering RNA (siRNA) sensitized the SCC-1 cancer cell line to Fas-induced apoptosis. Finally, we show that the AC inhibitor LCL 204 can sensitize HNSCC cell lines to Fas-induced apoptosis both in vitro and in a xenograft model in vivo, suggesting that the combination of FasL gene therapy and LCL 204 may become a new treatment option for advanced-stage head and neck cancer.

Prostate cancer is the second most commonly diagnosed cancer in men. Recent evidence suggests that reduced expression of target protein antigens and human leukocyte antigen (HLA) molecules is the predominant immune escape mechanism of malignant prostate tumor cells. The purpose of this study was to investigate the prospect of antigen specific immunotherapy against prostate cancer via the HLA class II pathway of immune recognition. Here, we show for the first time that prostate cancer cells express HLA class II proteins that are recognized by CD4 þ T cells. Prostate tumor cells transduced with class II molecules efficiently presented tumor-associated antigens/peptides to CD4 þ T cells. This data suggests that malignant prostate tumors can be targeted via the HLA class II pathway, and that class II-positive tumors could be employed for direct antigen presentation, and CD4 þ T-cell mediated tumor immunotherapy.

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value could be identified to define what should constitute the extent of an 'extended PLND'.METHODS: We used a cohort of 1863 intermediate to high risk patients, selected for a PLND based on validated LNI risk stratification (Conrad S JU 2002). Between 1 and 29 nodes (mean 6.9, median 6) were removed. PSA values were 0.12 to 48.5 (mean 11.3, median 8.5). Biopsy Gleason sums were 6 or less in 519 (51.3%), 7 in 430 (42.5%) and 8-10 in 63 (6.2%) men. PSA, stage and Gleason were used as LNI predictors. To reduce overfit bias, 200 bootstrap resamples were used. Moreover, the multivariate nomogram method was used to graphically explore the increase in risk according to the extent of PLND after controlling for other covariates.RESULTS: LNI was diagnosed in 149 (14.7%). In univariate analyses, all variables statistically significantly predicted LNI (all p<0.001 ). The number of nodes was most informative (0.791 ), followed by Gleason (0.717), PSA (0673) and stage (0.607). The accuracy of a multivariate PSA, stage and Gleason sum-based model was 0.784 Vs 0.860, when total nodes were added (7.6% gain). The nomogram figure demonstrates that increasing number of total nodes (Totai#Nodes) directly increases the probability of LNI (in figure: PN+). For example, removal of 26 nodes instead of 5 results in 70 additional 'risks points', which increase from 10 to 90% the probability of LNI.CONCLUSIONS: The number of nodes represents the foremost determinant of LNI. A cut-off is difficult to determine, as LNI risk increases in a continuous fashion according to the number of nodes removed. However, our model accounts for this limitation and is capable of highly accurately predicting the probability of LNI, according to the number of removed nodes.