5 -FU/UPRT G ENE T HERAPY
5.5 Synergistic Effect in Combination Therapy
Caspase gene expression was increased in Ag NPs treated cells (lane 2 and 4) than untreated control in lane 1 and 3 of Figure 5.17 C, whereas the corresponding signals of bcl-2, an anti-apoptotic gene expression was considerably reduced in both the cell types (Figure 5.17 C). These data confirmed the caspase mediated apoptosis in Ag NPs treated cells. Here, β-actin was used as an internal control.
A
Figure 5.17: Detection of Ag NPs induced apoptosis by cellular DNA fragmentation ELISA, DNA laddering and apoptotic gene expression. For DNA fragmentation ELISA (A), the BrdU-labeled 2 ×104 cells/well were incubated with Ag NPs (11.0 µg/mL) for 2, 4 and 6 h, respectively. The amount of fragmented DNA as measured by absorbance at 450 nm increased with time of incubation. For laddering (B), DNA extracted from cells at 12 h of Ag NPs treatment was resolved on 1.2%
agarose gel electrophoresis. Lane 1: λ /EcoR I + Hind III marker; lane 2: untreated
control BHK21 cells; lane 3: 11.0 µg/mL of Ag NPs treated BHK21 cells;
lane 4: untreated control HT29 cells; lane 5: 11.0 µg/mL of Ag NPs treated HT29 cells. (C), Ag NPs treated cells were harvested, RNA was isolated and RT-PCR was performed for caspase-3, bcl-2 and β-actin genes. Lane: 1 and 3 were for untreated control BHK21 & HT29 cells; 2 and 4 for Ag NPs (11.0 µg/mL) treated BHK21 &
HT29 cells, respectively.
combination therapy as compared to 5-FU or Ag NPs alone, which indicated that cell death, was due to regulated event, like apoptosis and not due to necrosis. The apoptosis was synergized in combination therapy, which was supported by cellular DNA fragmentation ELISA (Figure 5.22 B) and DNA laddering experiments (Figure 5.22 C). When Ag NPs were used in combination with 5-FU more number of cells undergo apoptosis as compared to Ag NPs or 5-FU treatment alone (Figure 5.22 B).
The results shown in Figure 5.22 B, confirmed that the BrdU DNA release due to apoptosis greatly increased in combination treatment on UPRT transduced cells. The results of 11 µg/mL Ag NPs on UPRT transduced cells without 5-FU treatment was similar that of 11 µg/mL Ag NPs on non-transduced cells.
DNA ladders of the corresponding treated samples, as represented in Figure 5.22 C, confirmed apoptosis. Ag NPs or 5-FU alone induced DNA ladders were also shown in lane 7 and 14 of Figure 5.22 C. DNA fragmentation during apoptosis was quantified by ELISA. It was observed that the Ag NP mediated- apoptosis involved caspase and bcl-2 signaling molecules (Figure 5.17 C). The combination of Ag NPs on UPRT expressing cells upon 5-FU treatment enhanced apoptosis by synergy of the two systems involving caspase signaling.
In summary, the antibacterial property of Ag NPs was investigated using GFP expressing E. coli bacteria as a prototype. GFP expressing E. coli has the advantage of easy morphological identification by fluorescence microscopy and the possibility of a noninvasive detection method, whereas the other wild-type bacterial strains relied essentially on viability tests, which are time-consuming and error prone. Furthermore, the direct effect of Ag NPs on DNA and protein migration profiles was also studied.
GFP expressing E. coli was established as a reliable model system to study the antibacterial efficacy of NPs.
Figure 5.18: Construction of UPRT vector and confirmation of UPRT gene transfer and expression by PCR and RT-PCR analysis. A, schematic of UPRT vector*
construction. B, PCR & C, RT-PCR analysis. Lane 1: λ DNA/EcoR I + Hind III marker; lane 2: control UPRT PCR product (651bp); lane 3: untransfected control BHK21 cells; lane 4: UPRT transfected BHK21cells; lane 5: untransfected control HT29 cells; lane 6: UPRT transfected HT29 cells.
*(Pro-promoter, Amp-Ampicillin resistance gene, ori- origin of replication, pAn-Poly adenylation)
Figure 5.19: MTS assay of 5-FU treated cells. BHK21 and HT29 cells were transduced with UPRT gene and treated with different concentrations of 5-FU.
BHK21 HT29
Figure 5.20: Confocal micrographs of AO/EB stained 5-FU treated cells. (A-C) &
(D-F) are the representative images of BHK21 cells and HT29 cells, respectively at 0, 8 and 24 h of 20 mM 5-FU treatment.
1 2 3 4
Figure 5.21: DNA laddering of 5-FU treated cells.BHK21 and HT29 cells were transfected with UPRT plasmid and subsequently treated with 20 mM of 5-FU for 12 h. Cellular DNA was extracted and subjected to agarose gel electrophoresis.
Lane 1: λ DNA/EcoR I + Hind III marker; lane 2: untreated control BHK21 cells;
lane 3: UPRT transfected BHK21 cells treated with 20 mM 5-FU; lane 4: UPRT transfected HT29 cells treated with 20 mM 5-FU.
Figure 5.22: LDH release and synergistic apoptosis due to combine therapy. A, Cells were treated with different combinations of Ag NPs and drug 5-FU for 6 h and LDH assay was performed to assess the LDH leakage in the medium. B, for synergistic apoptosis by cellular DNA fragmentation ELISA, the BHK21 and HT29 cells were labeled with BrdU and a portion of the labeled cells was transfected with UPRT vector. The UPRT transfected and non transfected BrdU labeled cells were treated with combinations of Ag NPs and 5-FU for 6 h. The amount of detected DNA fragments measured by absorbance at 450 nm was higher for combination treatment on UPRT expressed cells compared to the 5-FU, Ag NPs or 5-FU treatment on UPRT expression alone. Nos. 1: untreated controls, 2: 11.0 µg/mL of Ag NPs; 3: 20 mM 5-FU, 4: 11.0 µg/mL of Ag NPs with 20 mM 5-FU; 5: 20 mM 5-FU on UPRT transduced cells; 6: combine treatment of 11.0 µg/mL Ag NPs with 20 mM 5-FU on UPRT transduced cells. For synergistic effect on DNA laddering cellular DNA fragments of C, BHK21 cells and D, HT29 cells were analyzed by 1.2% agarose gel electrophoresis. Lane 1 & 8: λ DNA/EcoR I + Hind III marker; lane 2 & 9: control untreated; lane 3 & 10: treatment with 11.0 µg/mL of Ag NPs; lane 4 &11: treatment with 20 mM 5-FU; lane 5 & 12: combine treatment of 11.0 µg/mL of Ag NPs and 20 mM 5-FU; lane 6 & 13: treatment with 20 mM 5-FU on UPRT transduced cells;
lane 7 & 14: combine treatment of 11.0 µg/mL of Ag NPs with 20 mM 5-FU on UPRT transduced cells.
Furthermore, the concentration and time dependent induction of apoptosis in Ag NPs treated cancer HT29 as well as non-cancer BHK21 cells was established.
Experimental observations suggested Ag NPs further enhanced apoptosis of 5-FU/UPRT gene therapy system. The important feature of the Ag NPs has been to
sensitize cancer cells towards drug treatment even in absence of gene therapy. The switching behavior of Ag NPs could be useful in cancer treatment in conjunction with gene therapy. The major advantage in combination treatment of 5-FU/UPRT with Ag NPs is the individual component either 5-FU or Ag NPs can be used at much lower concentration than their IC50 values. Such treatment reduces development of resistance to any particular drug. For application in animal model, the Ag NPs and 5-FU/UPRT could be incorporated in delivery systems such as liposomes and virosomes for targeted delivery.