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Doxorubicin is commonly considered to exert its anti-tumor activity by triggering apoptosis of cancer cells through DNA damage. Recent reports have shown that Doxorubicin elicits a marked heat shock response, and that either inhibition or silencing of heat shock proteins enhance the Doxorubicin apoptotic effect in neuroblastoma cells. In order to investigate whether Doxorubicin may also act through protein modification, we performed a proteomic analysis of ubiquitinated proteins. Here we show that nanomolar Doxorubicin treatment of neuroblastoma cells caused: (a) dose-dependent over-ubiquitination of a specific set of proteins in the absence of measurable inhibition of proteasome, (b) protein ubiquination patterns similar to those with Bortezomib, a proteasome inhibitor, (c) depletion and loss of activity of ubiquitinated enzymes such as lactate dehydrogenase and α-enolase, and (d) a decrease in HSP27 solubility, probably a consequence of its binding to denatured proteins. These data strongly reinforce the hypothesis that Doxorubicin may also exert its effect by damaging proteins. [ABSTRACT FROM AUTHOR]

OBJECTIVE To assess the presence of circulating prostate-specific antigen (PSA)-expressing cells in patients with prostate cancer or benign prostatic hyperplasia (BPH), and to determine their diagnostic usefulness using a highly sensitive quantitative real-time reverse-transcription polymerase chain reaction (qRT-PCR) method. PATIENTS, SUBJECTS AND METHODS Venous blood samples were obtained from 175 patients with prostate cancer (12 metastatic and 163 not metastatic), 49 with BPH, and 50 healthy volunteers. To improve the specificity and sensitivity of the qRT-PCR three innovative features were combined; a primer overlapping two adjacent exons to inhibit nonspecific amplification; a no-end-point first round amplification to increase the sensitivity; and a target-specific primer for the RT phase to increase the specificity. RESULTS The sensitivity of the method was 1 cell/mL of blood and the interassay coefficient of variation was 10.5%. None of the healthy subjects tested positively, while 9% of those with prostatic cancer and 14% with BPH had PSA-positive cells in the blood. There was a positive association between a positive test and the National Comprehensive Cancer Network classification in the patients with newly diagnosed prostate cancer ( P = 0.022). There were no additional statistically significant associations. CONCLUSION Our results strongly indicate that although there were no false-positive results and the sensitivity of the method was increased to maximal levels, a low frequency of positive results in patients with prostatic cancer and a high frequency of positive results in those with BPH seems to discourage the use of PSA-positive circulating cells in the search for a clinical diagnosis of prostate cancer. [ABSTRACT FROM AUTHOR]

Neuroblastoma (NB) and Ewing’s sarcoma (ES) represent the most common extracranial solid tumors of childhood. Heat shock proteins (HSP) are elevated in cancer cells and their over-expression was correlated to drug-resistance. In this work we identified the HSP by a sensitive proteomic analysis of NB and ES cell lines, then, we studied the HSP response to doxorubicin. Some identified HSP were constitutively more expressed in NB than in ES cells. Doxorubicin-stimulated HSP response only in NB cells. Quercetin was found to inhibit HSP expression depleting heat shock factor 1 (HSF1) cellular stores. Quercetin caused a higher anti-proliferative effect in NB (IC50: 6.9 ± 5.8 μmol/L) than in ES cells (IC50: 85.5 ± 53.1 μmol/L). Moreover, quercetin caused a very pronounced doxorubicin sensitizing effect in NB cells (241 fold IC50 decrease) and a moderate effect in ES cells. HSP involvement in NB cells sensitization was confirmed by the silencing of HSF1. Quercetin treatment and HSF1 silencing increased the pro-apoptotic effect of doxorubicin. In conclusion, the higher HSP levels, observed in NB cells, did not confer increased resistance to doxorubicin; on the contrary, HSP inhibition by quercetin or gene silencing caused higher sensitization to doxorubicin. These results may have a potential application in the treatment of NB. [ABSTRACT FROM AUTHOR]