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

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

Boletín de Segovia: Número 90 - 1862 julio 25

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

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

Triptolide decreases proliferation and viability of NSCLC cells.

<p>A549 cells (right panel) and NCI-H460 cells (left panel) were treated with 25-200 nM of triptolide for times indicated. Proliferation (A, E) and viability (B, F), as well as BrdU incorporation (C, G) were significantly reduced of both cell lines were reduced; however, the cytotoxic effect of triptolide was more pronounced in NCI-H460 cells. The indicator of DNA repair, phosphorylated H2AX, was significantly increased in both cell lines with triptolide treatment as shown (D, H). <i>Columns</i>, mean, <i>bars</i>, SE. Statistical significance of results was calculated with the Student`s <i>t</i> test (N=3) *<i>P</i> = 0.05; **<i>P</i> = 0.005.</p

Minnelide leads to tumor regression in xenograft mouse models.

<p>In two xenograft mouse models, A549 (right panel) and NCI-H460 (left panel), tumor volumes were compared between minnelide treated (n = 10) and untreated groups (n = 10). Five days after tumor injection, mice began receiving daily intraperitoneal injections of minnelide at 0.42 mg/kg mouse weight. Control animals were injected with equivalent volumes of phosphate-buffered saline. Suppression of tumor growth occurred in the minnelide treated groups in comparison with the control groups in both xenograft models (A, E). Graphs (B and F) showed significantly decreased tumor volume from animals (A and E). Results were normalized to the untreated group for each cell line and expressed as the mean, <i>Columns</i>, <i>bars</i>, SE. Ki-67 protein expression was significantly decreased in the tumor tissue of minnelide treated group in both mouse models compare to saline treated groups (C and G). In concordance with decreased Ki-67 staining, TUNEL staining shown increased number of apoptotic cells in both mouse models (D and H). <i>Columns</i>, mean, <i>bars</i>, SE. Statistical significance of results was calculated with the Student`s <i>t</i> test (N=3) *<i>P</i> = 0.05; **<i>P</i> = 0.005.</p

Triptolide promotes pro-apoptotic condition in A549 and NCI-H460 cells.

<p>Triptolide treatment increased Apaf-1 mRNA expression level in both NSCLC cell lines (A and D). In parallel, caspase-9 activity was increased in both NSCLC cell lines (B and E). Level of UACA mRNA expression was decreased after triptolide treatment (C and F). <i>Columns</i>, mean, <i>bars</i>, SE. Statistical significance of results was calculated with the Student`s <i>t</i> test (N=3) *<i>P</i> = 0.05; **<i>P</i> = 0.005.</p

Triptolide down-regulates anti-apoptotic IAP genes in A549 and NCI-H460 cells.

<p>Triptolide significantly reduces the expression of IAP genes, (A and D) survivin, (B and E) XIAP and (C and F) c-IAP1. <i>Columns</i>, mean, <i>bars</i>, SE. Statistical significance of results was calculated with the Student`s <i>t</i> test (N=3) *<i>P</i> = 0.05; **<i>P</i> = 0.005.</p

Triptolide induces apoptosis in NSCLC cells.

<p>NSCLC cells were exposed to 100 nM and 200 nM of triptolide for 24 and 48 hours and caspase-3/-7 activities (A, C) and PARP cleavage (B, D) were assessed. Following triptolide treatment caspase-3/-7 activity and cleavage of PARP were significantly increased compare to untreated cells demonstrating apoptosis. <i>Columns</i>, mean, <i>bars</i>, SE. Statistical significance of results was calculated with the Student`s <i>t</i> test (N=3) *<i>P</i> = 0.05; **<i>P</i> = 0.005. Results were expressed after normalizing to cell viability.</p