Additional file 2: Table S2. of Minnelide effectively eliminates CD133+ side population in pancreatic cancer

Tumorigenicity with MIA PaCa-2, CSM and 12 T cells. (DOC 28 kb

Additional file 6: of Minnelide effectively eliminates CD133+ side population in pancreatic cancer

IC50 values for triptolide of MIA PaCa-2 and CD133 + and CD133 -  of CSM and 12T groups. (TIF 705 kb

Additional file 1: Table S1. of Minnelide effectively eliminates CD133+ side population in pancreatic cancer

Expression of stemness genes in CSM and 12 T cells (Fold change in expression over CSM cells). SEM is represented within parenthesis. (DOC 30 kb

Boletín de Segovia: Número 90 - 1862 julio 25

Copia digital. Madrid : Ministerio de Cultura. Subdirección General de Coordinación Bibliotecaria, 200

MUC1c Regulates Cell Survival in Pancreatic Cancer by Preventing Lysosomal Permeabilization

<div><h3>Background</h3><p>MUC1 is a type I transmembrane glycoprotein aberrantly overexpressed in various cancer cells including pancreatic cancer. The cytosolic end of MUC1 (MUC1-c) is extensively involved in a number of signaling pathways. MUC1-c is reported to inhibit apoptosis in a number of cancer cells, but the mechanism of inhibition is unclear.</p> <h3>Method</h3><p>Expression of MUC1-c was studied in the pancreatic cancer cell line MIAPaCa-2 at the RNA level by using qRTPCR and at the protein level by Western blotting. MUC1-c expression was inhibited either by siRNA or by a specific peptide inhibitor, GO-201. Effect of MUC1-c inhibition on viability and proliferation and lysosomal permeabilization were studied. Association of MUC1-c with HSP70 was detected by co-immunoprecipitation of MUC1-c and HSP70. Localization of MUC1-c in cellular organelles was monitored by immunofluorescence and with immuno- blotting by MUC1-c antibody after subcellular fractionation.</p> <h3>Results</h3><p>Inhibition of MUC1-c by an inhibitor (GO-201) or siRNA resulted in reduced viability and reduced proliferation of pancreatic cancer cells. Furthermore, GO-201, the peptide inhibitor of MUC1-c, was effective in reducing tumor burden in pancreatic cancer mouse model. MUC1-c was also found to be associated with HSP70 in the cytosol, although a significant amount of MUC1 was also seen to be present in the lysosomes. Inhibition of MUC1 expression or activity showed an enhanced Cathepsin B activity in the cytosol, indicating lysosomal permeabilization. Therefore this study indicates that MUC1-c interacted with HSP70 in the cytosol of pancreatic cancer cells and localized to the lysosomes in these cells. Further, our results showed that MUC1-c protects pancreatic cancer cells from cell death by stabilizing lysosomes and preventing release of Cathepsin B in the cytosol.</p> </div

MUC1-c associates with HSP70 and localizes in the lysosomes.

<p>Immunoprecipitation with MUC1 and HSP70 showed the two proteins to be associated (A). Lanes 1, 2 and 3 show total protein levels, HSP70 and MUC1 in the flowthrough and the two proteins immunoprecipitated by HSP70 antibody respectively. Lanes 4 and 5 show flowthrough and immunoprecipitates by MUC1 antibody. MUC1-c was found to be localized to the lysosomes in MIAPaCa-2 cells, though almost equal amounts of MUC1-c were also cytosolic (B). Lamp2, a resident lysosomal protein, and actin, a predominantly cytosolic protein, were used as fractionation markers. Lane 1 is the cytosolic fraction. Lane 2 is a crude lysosomal fraction, which is further enriched in Lane 3. HSP70, though associated with MUC1, was seen to be present predominantly in the cytosol. Immunofluorescence confirmed co-localization of MUC1-c and Lamp2 in lysosomes (C).</p

Inhibition of MUC1 expression or activity results in reduced proliferation and cell viability.

<p>MUC1 expression was inhibited by siRNA in MIAPaCa-2 (A) Lanes 1–3 show control MIAPaCa-2, MIAPaCa-2 transfected with non-specific siRNA and MUC1 siRNA respectively. Both proliferation (B) and viability (C) were reduced with decreased expression of MUC1 in MIAPaCa-2 cells. On treatment with MUC1-C activity inhibitor GO-201, which inhibits the signaling activity of this protein, no change was seen in expression levels of MUC1 in MIAPaCa-2 cells (D). Lane 1 was untreated MIAPaCa-2 cells and lane 2 was MIAPaCa-2 treated with GO-201. Proliferation (E) and viability (F) were seen to be decreased. Similarly, in another cell line, AsPC-1, transfection with siRNA showed decreased protein levels (G) where Lane 1 is control AsPC-1, Lane 2: non-specific siRNA transfected AsPC-1 and Lane 3 is MUC1 siRNA-transfected AsPC1. Both proliferation (H) and viability (I) were reduced. On treatment with the inhibitor GO-201, there was no change in protein levels (J): Lane 1 was untreated AsPC-1 cells and lane 2 was AsPC-1 treated with GO-201. Proliferation (K) and viability (L) were seen to be reduced. Data are expressed as mean+/−SEM of 3 independent experiments. *<i>P</i><.05 (<i>t</i> test) as compared with controls.</p

MUC1 is expressed in most pancreatic cancer cell lines and mouse models.

<p>mRNA expression levels of MUC1 in several pancreatic cancer cell lines relative to non-tumorigenic HPDEC (A). Data are expressed as mean+/−SEM of 3 independent experiments. *<i>P</i><.05 (<i>t</i> test) as compared with controls. Protein expression levels of MUC1-c in different pancreatic cancer cell line and non-tumorigenic HPDEC (B).</p

Evaluation of metastasis in the mouse model for pancreatic cancer.

<p>Evaluation of metastasis in the mouse model for pancreatic cancer.</p

Minnelide: A Novel Therapeutic That Promotes Apoptosis in Non-Small Cell Lung Carcinoma In Vivo

<div><p>Background</p><p>Minnelide, a pro-drug of triptolide, has recently emerged as a potent anticancer agent. The precise mechanisms of its cytotoxic effects remain unclear.</p> <p>Methods</p><p>Cell viability was studied using CCK8 assay. Cell proliferation was measured real-time on cultured cells using Electric Cell Substrate Impedence Sensing (ECIS). Apoptosis was assayed by Caspase activity on cultured lung cancer cells and TUNEL staining on tissue sections. Expression of pro-survival and anti-apoptotic genes (<i>HSP70</i>, <i>BIRC5, BIRC4, BIRC2, UACA, APAF-1</i>) was estimated by qRTPCR. Effect of Minnelide on proliferative cells in the tissue was estimated by Ki-67 staining of animal tissue sections.</p> <p>Results</p><p>In this study, we investigated <i>in</i><i>vitro</i> and <i>in</i><i>vivo</i> antitumor effects of triptolide/Minnelide in non-small cell lung carcinoma (NSCLC). Triptolide/Minnelide exhibited anti-proliferative effects and induced apoptosis in NSCLC cell lines and NSCLC mouse models. Triptolide/Minnelide significantly down-regulated the expression of pro-survival and anti-apoptotic genes (<i>HSP70</i>, <i>BIRC5, BIRC4, BIRC2, UACA</i>) and up-regulated pro-apoptotic <i>APAF-1</i> gene, in part, via attenuating the NF-κB signaling activity.</p> <p>Conclusion</p><p>In conclusion, our results provide supporting mechanistic evidence for Minnelide as a potential in NSCLC.</p> </div