Background: A major problem with cisplatin treatment is the development of

Background: A major problem with cisplatin treatment is the development of acquired-drug resistance of the tumour cells. cisplatin-resistant sub-lines (P31res/H1299res) were incubated with VT-1 and/or cisplatin followed by determination of Gb3 expression cell viability apoptosis and signalling pathways. Results: Cells from your resistant sub-lines experienced elevated Gb3 expression compared with the parental cell lines and cisplatin Ginsenoside Rg3 further increased Gb3 expression whereas VT-1 reduced the percentage of Gb3-expressing cells. Combination of cisplatin and sub-toxic concentrations of VT-1 led to a super-additive increase of cytotoxicity and TUNEL staining especially in the cisplatin-resistant sub-lines. Blockade of Gb3 synthesis by a Gb3 synthesis inhibitor not only led to eradicated TUNEL staining of P31 cells but also sensitised P31res cells to the induction of apoptosis by cisplatin alone. Cisplatin- and VT-1-induced apoptosis involved the MAPK pathways with increased C-Jun N-terminal kinase and MAPK kinase-3 and -6 phosphorylation. Conclusions: We show the presence of Gb3 in acquired-cisplatin resistance in P31res and H1299res cells. Cisplatin up-regulated Gb3 expression in all cells and thus sensitised the cells to VT-1-induced cytotoxicity. A strong super-additive effect of combined cisplatin and a sub-toxic concentration of VT-1 in cisplatin-resistant malignant pleural mesothelioma cells were observed indicating a new potential clinical-treatment approach. (Lingwood cell death detection kit TMR reddish (Roche Mannheim Germany) was used. P31 and H1299 cells were cultured to about 80% confluence and the medium was thereafter changed Ginsenoside Rg3 to fresh medium made up of 0 or 5?mg?l-1 cisplatin and/or 0.1?… MDR1/PgP and Gb3 expression of cells and their resistant cell sub-lines Circulation cytometry showed a correlation between MDR1/PgP and Gb3 Ginsenoside Rg3 co-expression in P31res as well as H1299res cell sub-lines (Physique 4). P31res cells showed co-expression in two sub-fractions with one expressing ~10-fold expression of MDR1/PgP compared with Gb3. Incubation of the cells with 10?μmol?l-1 verapamil for 72?h before expression analysis did however not reduce the expression of MDR1/PgP or Gb3 (results not show). Physique 4 Circulation cytometry analysis of Gb3- and MDR1/Pgp expression of cisplatin-resistant cell sub-lines. (A) Cell surface expression of MDR1/PgP (ordinate) and Gb3 (abscissa) of P31res cells. (B) Cell surface expression of MDR1/PgP (ordinate) and Gb3 (abscissa) … We therefore also tested whether the more effective MDR1/PgP inhibitor cyclosporin A (10?μmol?l-1 incubated with the cells for 72?h) as well as PPMP (2?μmol?l-1) affected the co-expression of MDR1/PgP and Gb3. Un-expectantly cyclosporin A did not noticeably inhibit MDR1/PgP expression in any of Rabbit Polyclonal to ASC. the cell types but possibly Ginsenoside Rg3 the expression of Gb3 in the resistant sub-lines whereas PPMP as expected markedly reduced not only Gb3 expression in the resistant cell sub-lines but also of MDR1/PgP expression especially of the cells of the resistant cell lines with also high expression of Gb3 (Physique 5). Physique 5 Circulation cytometry analysis of Gb3- and MDR1/PgP expression of P31 and H1299 cells and their cisplatin-resistant sub-lines incubated with 10?μmol?l-1 cyclosporin A or 2?μmol?l-1 PPMP for 72?h. … VT-1 and cisplatin induction of MPM cell DNA fragmentation The TUNEL-staining assay showed no increase of DNA fragmentation in P31 Ginsenoside Rg3 cells after exposure to 0.1?μg?l-1 VT-1 for 72?h. A slight increase (to 17%) in DNA fragmentation was however noted in the P31res cells (Physique 6A). Cisplatin (5?mg?l-1) was sufficient to induce massive (to 78%) DNA fragmentation in P31 cells whereas there was no or limited effect (19%) in the resistant sub-line (P31res). The proportion of P31res cells with DNA fragmentation was dramatically increased (to 78% of the cells) by combined exposure to 5?mg?l-1 cisplatin and 0.1?μg?l-1 VT-1 but no further effect than that of cisplatin alone was noted in the P31 cells (Physique 6A). Physique 6 (A) Circulation cytometry analysis of TUNEL staining.