bafilomycin-a and Carcinoma

Increasing resistance to cisplatin as well as the severity of the adverse effects limit the use of this drug, particularly at high doses. Evidence has implicated the importance of autophagy in cancer resistance as well as the fact that various chemotherapy agents induce autophagy in cancer cells. We therefore aimed to first assess the role of autophagy in cisplatin treatment and second to assess whether a nontoxic concentration of cisplatin, together with autophagy inhibition, is able to maintain its cancer-specific cytotoxic action.. Three human cervical cell lines were used: a noncancerous ectocervical epithelial cell line (Ect1/E6E7) and 2 cancerous cervical cell lines (HeLa and CaSki). Autophagy was monitored through the presence of the classical protein markers LC-3 II and p62 under basal and treatment conditions, and inhibited using bafilomycin and autophagy protein 5 (ATG5) siRNA under treatment conditions. Cell death was analyzed through examination of the apoptotic markers PARP and caspase-3 through Western blotting, as well as the Caspase-Glo assay to confirm caspase-3/7 activity. Cervical biopsies were analyzed for the presence of LC-3 using Western blotting and immunofluorescence to determine if a correlation between autophagic levels and the progression of the disease exists.. Cervical cancer cells exhibit increased basal autophagic levels in comparison to the noncancerous counterparts. Cisplatin treatment enhanced autophagic activity in all 3 cell lines. Inhibition of this autophagic response together with cisplatin treatment leads to significant increases in cancer cell death. Expression profiles of LC-3 in normal, premalignant (low-grade and high-grade squamous intraepithelial lesion), and cancerous cervical tissue revealed that autophagy is significantly up-regulated in HSILs and carcinoma cervical tissue, which emphasized the role of autophagy in the progression of the disease.. The inhibition of autophagy improves the cytotoxicity of a nontoxic concentration of cisplatin and provides a promising new avenue for the future treatment of cervical cancer.

Topics: Antineoplastic Agents; Autophagy; Autophagy-Related Protein 5; Carcinoma; Cisplatin; Drug Resistance, Neoplasm; Enzyme Inhibitors; Female; HeLa Cells; Humans; Macrolides; Microtubule-Associated Proteins; RNA-Binding Proteins; RNA, Small Interfering; Squamous Intraepithelial Lesions of the Cervix; Uterine Cervical Neoplasms

In this study we show that diindolylmethane (DIM) induces autophagy in ovarian cancer cells by regulating endoplasmic reticulum (ER) stress and AMPK. Treatment of SKOV-3, OVCAR-3 and TOV-21G ovarian cancer cells with varying concentrations of DIM for 24 hours resulted in a concentration dependent induction of autophagy as measured by flowcytometry. Electron microscopy confirmed the presence of autophagosomes in DIM treated cells. Western blot analysis showed that DIM treatment increased the expression of LC3B, a hall mark of autophagy as well as p62 and Atg 12 proteins that are accumulated during autophagy. Autophagy inhibitors bafilomycin or chloroquine inhibited DIM induced autophagy. Furthermore, DIM treatment significantly increased the expression of ER stress regulators such as Grp78, IRE1 and GADD153. Cycloheximide or ER stress inhibitor mithramycin not only blocked ER stress proteins that were activated by DIM but also autophagy. Silencing Grp78 or GADD 153 significantly blocked the expression of LC3B and p62 indicating that autophagy in our model is mediated by ER stress. Knocking out LC3B inhibited DIM induced autophagy. DIM treatment increased the cytosolic calcium levels which lead to the activation of AMPK in our model. Chelating cytosolic calcium with BAPT-AM abrogated not only the phosphorylation of AMPK but also prevented DIM induced autophagy. Inhibiting AMPK by a chemical inhibitor or siRNA blocked the induction of LC3B or p62, indicating that DIM mediated autophagy requires activation of AMPK. Oral administration of DIM significantly suppressed SKOV-3 tumor xenografts in nude mice. Activation of ER stress and autophagy were observed in the tumors of DIM treated mice. Taken together, these results suggest that induction of autophagy by DIM in ovarian cancer cells was associated with ER stress and AMPK activation.

Topics: Adaptor Proteins, Signal Transducing; AMP-Activated Protein Kinase Kinases; Autophagy; Autophagy-Related Protein 12; Calcium Signaling; Carcinoma; Cell Line, Tumor; Chloroquine; Endoplasmic Reticulum; Endoplasmic Reticulum Chaperone BiP; Endoplasmic Reticulum Stress; Endoribonucleases; Female; Heat-Shock Proteins; Humans; Indoles; Macrolides; Membrane Proteins; Microtubule-Associated Proteins; Ovarian Neoplasms; Protein Kinases; Protein Serine-Threonine Kinases; RNA, Small Interfering; Sequestosome-1 Protein; Small Ubiquitin-Related Modifier Proteins; Transcription Factor CHOP

The presence of an H+/K(+)-ATPase and its contribution to the regulation of intracellular pH (pHi) was investigated in Caco-2 cells. The H+/K(+)-ATPase was detected immunologically using the monoclonal antibody 5-B6, which was raised against hog gastric H+/K(+)-ATPase. Cell pH was determined using the pH-sensitive dye 2',7'-bis(carboxyethyl)-carboxyfluorescein. Control pHi, measured in HCO(3-)-free medium, was 7.62 +/- 0.03 (n = 27) when cells were cultured for 14 days and decreased to 7.40 +/- 0.03 (n = 18) after 35 days in culture. Recovery of pHi following a NH+4/NH3 pulse could be reduced by either 100 microM SCH 28080 or 1 mM amiloride, or by removing extracellular Na+. The inhibitory effects of SCH 28080 and amiloride were additive, demonstrating the involvement of a gastric-like H+/K(+)-ATPase and a Na+/H+ exchanger in regulating pHi. Recovery rates at pHi 6.8 were not significantly different in cells cultured for up to 21 days, but were significantly lower in cells cultured for 28 and 35 days. This decrease in recovery rate was due to a decrease in the SCH-28080-insensitive recovery, indicating a reduction of the relative importance of Na+/H+ exchange to the recovery. Recovery of pHi was also inhibited by 1 mM N-ethylmaleimide. However, it is unlikely that N-ethyl-maleimide inhibited a vacuolar type of H+-ATPase, since bafilomycin A1 had no effect on pHi recovery. In conclusion, Caco-2 cells contain a SCH-28080-sensitive mechanism for regulating pHi, which is most conveniently studied after 28 days in culture, when the relative contribution of a Na+/H+ exchanger to pHi regulation is decreased.

Topics: Amiloride; Ammonium Chloride; Anti-Bacterial Agents; Anti-Ulcer Agents; Carcinoma; Colonic Neoplasms; Ethylmaleimide; H(+)-K(+)-Exchanging ATPase; Humans; Hydrogen-Ion Concentration; Imidazoles; Macrolides; Sodium Chloride; Tumor Cells, Cultured