Doxycycline (DC) belongs to the tetracycline family of antibiotics and has been used clinically for over 5 decades. Despite advances in understanding the molecular pathogenesis of pancreatic cancer, no chemotherapy course has shown significant effectiveness. Hence new treatments are needed. In this study we report the proapoptotic effects of DC in 2 pancreatic adenocarcinoma cell lines, T3M4 and GER. Cell proliferation was measured using the SRB protein dye. Induction of apoptosis was detected using ELISA. Caspase activation was detected using either immunoblotting or a colorimetric assay based on cleavage of caspase-associated substrates. Expression of proteins and post-translational modifications were determined using immunoblotting. Treatment of pancreatic cancer cells with DC reduces their proliferation. This reduction is, at least partly, due to increased caspase-dependent apoptosis involving activation of caspase3, caspase7, caspase8, caspase9, caspase10 and increased levels of FADD. Inhibition of caspase8 or caspase10 but not caspase9 significantly decreases DCinduced apoptosis in both cell lines. Furthermore treatment of pancreatic cancer cells with DC increases protein levels of Bax and phosphorylation of members of the p38MAPK pathway such as p38MAPK, MKK3/6 and MAPKAPK2. These results provide an insight into mechanisms behind the pro-apoptotic effects of DC in pancreatic cancer cells. ' 2006 Wiley-Liss, Inc.
Aim: Thermal isoeffective dose (TID) has not been convincingly validated for application to predict biological effects from rapid thermal ablation (e.g., using >55 C). This study compares the classical method of quantifying TID (derived from hyperthermia data) with a temperature-adjusted method based on the Arrhenius model for predicting cell survival in vitro, after either 'rapid' ablative or 'slow' hyperthermic exposures. Methods: MTT assay viability data was obtained from two human colon cancer cell lines, (HCT116, HT29), subjected to a range of TIDs (120-720 CEM 43) using a thermal cycler for hyperthermic (>2 minutes, <50 C) treatments, or a novel pre-heated water bath based technique for ablative exposures (<10 seconds, >55 C). TID was initially estimated using a constant R CEM>43 C ¼0.5, and subsequently using R CEM (T), derived from temperature dependent cell survival (injury rate) Arrhenius analysis. Results: 'Slow' and 'rapid' exposures resulted in cell survival and significant regrowth (both cell lines) 10 days post-treatment for 240 CEM 43 (R CEM>43 C ¼0.5), while 340-550 CEM 43 (R CEM>43 C ¼0.5) delivered using 'rapid' exposures showed 12 ± 6% viability and 'slow' exposures resulted in undetectable viability. Arrhenius analysis of experimental data (activation energy DE ¼ 5.78 ± 0.04 Â 10 5 J mole À1 , frequency factor A ¼ 3.27 ± 11 Â 10 91 sec À1) yielded R CEM ¼0.42 Ã e 0.0041 Ã T which better-predicted cell survival than using R CEM> 43 C ¼0.5. Conclusions: TID calculated using an R CEM (T) informed by Arrhenius kinetic parameters provided a more consistent, heating strategy independent, predictor of cell viability, improving dosimetry of ablative thermal exposures. Cell viability was only undetectable above 305 ± 10 CEM 43 using this revised measure.
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