Molecular effects of taxol and caffeine on pancreatic cancer cells.

Pancreatic cancer is the fifth leading cause of cancer related deaths in the United States. Despite many recent advances in the treatment modalities, the mortality rate still remains very high. Paclitaxel (Taxol) and Caffeine have been used for the treatment of this disease, however the molecular mechanisms of these agents are not fully understood, which may be partly responsible for the failure of these agents in the treatment of pancreatic cancer. Human pancreatic adenocarcinoma cell lines, HPAC and PANC-1 containing wild-type and mutant p53 respectively, were used to investigate the effects of Taxol and Caffeine on cell growth, and their effects on the modulation of cell cycle and apoptosis related genes. Protein extracts from these cells treated with 100 nM of Taxol or 4 mM of Caffeine were subjected to Western blot analysis for this study. Drug treated cells were also analyzed to calculate the number of cells undergoing apoptosis. Dose and time dependent growth inhibition was observed in both PANC-1 and HPAC cells when treated with either Taxol or Caffeine. Western blot analysis showed an up-regulation of p21WAF1 in both cell lines treated with either Taxol or Caffeine. Furthermore, down-regulation of cyclin B and cdk1 was observed in Taxol and Caffeine treated HPAC cells. However, the results were drastically different in PANC-1 cells where cyclin B was down regulated only by Caffeine treatment and the level of cdk1 protein was undetectable in this cell line. Moreover, up-regulation of p53 and down-regulation of Bcl-2 was observed only in HPAC cells treated with Taxol. Apoptotic cell death analysis showed increasing number of cells undergoing apoptosis between 24 and 48 h of Caffeine treatment, however only Taxol showed greater than 50% cells under-going apoptosis only in HPAC cells. The up-regulation of p21WAF1 and down-regulation of cyclin B and cdk1 suggest their possible roles in G2/M cell cycle arrest caused by both Taxol and Caffeine as reported earlier. From these results we conclude that the differential molecular changes observed in this study may determine the cellular effects of these agents on pancreatic adenocarcinoma cells and that the effects of chemotherapeutic agents may be determined by the endogenous status of p53 mutation and, in turn, may determine the therapeutic effects of these agents in the treatment of pancreatic cancer.

p53 gene alterations in prostate cancer after radiation failure and their association with clinical outcome: a molecular and immunohistochemical analysis.

This study evaluates the prevalence of p53 gene mutations in prostate cancer in salvage prostatectomies after radiation failure using single strand conformational polymorphism (SSCP) and direct sequencing of the polymerase chain reaction (PCR) product. Findings were correlated with immunohistochemically (IHC) detectable p53 expression in residual prostate cancer. The usefulness of p53 as a marker of clinical outcome was evaluated. Thirty-three cases were available for molecular and immunohistochemical analysis. Immunohistochemical stains for p53 were performed with clone DO7. PCR-SSCP for mutations in the coding region of p53 DNA (exons 4-9) was performed on all immunopositive cases and 12 of 23 immunonegative cases. All samples with an SSCP shift were sequenced for the respective exon. Patients were evaluated for biochemical failure for 1-82 months (median 38 months) following surgery. Immunohistochemical p53 reactivity was noted in 10 of 33 (30%) patients. Among p53 immunopositive cases SSCP shifts were seen in 7 of 10 (70%) samples with 5 of the 7 (71%) showing p53 mutations. Univariate analysis revealed abnormal expression of p53 protein by immunohistochemistry to be a significant predictor of poorer outcome (p = 0.025, log rank), however this was not independent of pathologic stage, surgical margin status and Gleason score. The presence of p53 gene mutations by PCR-SSCP and direct sequencing did not predict for outcome. In our study 30% of prostate cancers at the time of salvage prostatectomy after radiation failure expressed immunohistochemically detectable p53. PCR-SSCP and sequencing shows that not all of these cases have detectable mutations in the most frequent mutation sites (exons 4-9). Clinical failure is more common in the group of prostate cancer patients with abnormal p53 immunoreactivity.