The effect of the combination of OncoVexGALV/CD and chemotherapy (mitomycin, cisplatin, gemcitabine) on bladder tumour cell (EJ, TCCSUP-G, T24, KU19-19) proliferation was assessed by calculating combination-index (CI) values using CalcuSyn software (Biosoft, Ferguson, MO). Derived from the median-effect principle of Chou and Talalay (Chou and Talalay 1984), the CI provides a quantitative measure of the degree of interaction between two or more agents. A CI of 1 denotes an additive interaction, >1 antagonism and <1 synergy. Analysis using the CalcuSyn programme performs optimally when data is collected from a constant ratio combination design experiment. Typically the combination agents are chosen at their equipotent ratio (eg at the ratio of their IC50s). The human bladder cancer cell lines were infected with OncoVEXGALV/CD or treated with one of the chemotherapeutical agents (mitomycin, cisplatin, gemcitabine) or both in combination where the equipotent ED ratio of each agent was mixed for combination. The cellswere measured by the MTS assay after 2 days. Our results showed synergistic effect when OncoVEXGALV/CD was combined with mitomycin in EJ, T24 and KU19-19 cells (Figure 4.3.1-3). Whereas the combination effect of OncoVEXGALV/CD with cisplatin or gemcitabine was antagonistic in EJ, T24 and TCCSUP-G cells (Figure 4.3.4-9) (Table 4.3.1). In order to evaluate whether this antagonistic effect is related to the Herpes Simplex Virus instead of to the implanted GALV and CD genes we have tested the backbone OncoVEXGFP in combination with cisplatin or gemcitabine in EJ cells. Results showed antagonistic effect both with cisplatin or gemcitabine (Table 4.3.1).
Figure 4.3.1 EJ cells were plated at1 x 104 per well of a 96-well tray in appropriate FGM. Doubling dilution series were prepared from OncovexGALVCD and mitomycin in 2% FCS medium. Equipotent ED ratio of each agent was mixed for combination. 100μL of appropriate dilution was added to each well and incubate 48hr. After 2 days the cells were measured by the MTS assay (A). The isobologram curves (B) and the combination indexes were prepared by using CalcuSyn programme (Biosoft, Ferguson, MO).
Experiments were repeated at least three times. The figure shows a representative experiment, where the error bars are standard deviations.
0.8 ED90
0.73 ED75
0.67 ED50
Combination index
0 20 40 60 80 100 120
0.12/0.8
0.06/0.4
0.03/0.2
0.015/0.1
0.0075/0.05
% of survival
GALVCD MOI Mitomycin C GALV+Mitomycin C
A
B
MOI/uM
Figure 4.3.2 T24 cells were plated at 1 x 104 per well of a 96-well tray in appropriate FGM. Doubling dilution series were prepared from OncovexGALVCD and mitomycin in 2% FCS medium. Equipotent ED ratio of each agent was mixed for combination. 100μL of appropriate dilution was added to each well and incubate 48hr. After 2 days the cells were measured by the MTS assay (A). The isobologram curves (B) and the combination indexes were prepared by using CalcuSyn programme (Biosoft, Ferguson, MO).
Experiments were repeated at least three times. The figure shows a representative experiment, where the error bars are standard deviations.
0.72 ED90
0.63 ED75
0.78 ED50
Combination index
0 10 20 30 40 50 60 70 80 90 100
0.944/0.712
0.472/0.356
0.236/0.178
0.118/0.089
0.059/0.044
%of survival.
GALVCD MOI Mitomycin C GALV+Mitomycin C
A
B
MOI/uM
Figure 4.3.3 KU19-19 cells were plated at 1 x 104 per well of a 96-well tray in appropriate FGM. Doubling dilution series were prepared from OncovexGALVCD and mitomycin in 2% FCS medium. Equipotent ED ratio of each agent was mixed for combination. 100μL of appropriate dilution was added to each well and incubate 48hr.
After 2 days the cellswere measured by the MTS assay (A). The isobologram curves (B) and the combination indexes were prepared by using CalcuSyn programme (Biosoft, Ferguson, MO). Experiments were repeated at least three times. The figure shows a representative experiment, where the error bars are standard deviations.
0.69 ED90
0.68 ED75
0.78 ED50
Combination index
0 10 20 30 40 50 60 70 80 90 100
0.058/0.48
0.029/0.24
0.0145/0.12
0.00725/0.06
0.00365/0.03
% of survival.
GALVCD MOI Mitomycin C GALV+Mitomycin C
A
B
MOI/uM
Figure 4.3.4 EJ cells were plated at1 x 104 per well of a 96-well tray in appropriate FGM. Doubling dilution series were prepared from OncovexGALVCD and cisplatin in 2%
FCS medium. Equipotent ED ratio of each agent was mixed for combination. 100μL of appropriate dilution was added to each well and incubate 48hr. After 2 days the cells were measured by the MTS assay (A). The isobologram curves (B) and the combination indexes were prepared by using CalcuSyn programme (Biosoft, Ferguson, MO).
Experiments were repeated at least three times. The figure shows a representative experiment, where the error bars are standard deviations.
0
Figure 4.3.5 T24 cells were plated at 1 x 104 per well of a 96-well tray in appropriate FGM. Doubling dilution series were prepared from OncovexGALVCD and cisplatin in 2%
FCS medium. Equipotent ED ratio of each agent was mixed for combination. 100μL of appropriate dilution was added to each well and incubate 48hr. After 2 days the cells were measured by the MTS assay (A). The isobologram curves (B) and the combination indexes were prepared by using CalcuSyn programme (Biosoft, Ferguson, MO).
Experiments were repeated at least three times. The figure shows a representative experiment, where the error bars are standard deviations.
0
Figure 4.3.6 TCCSUP-G cells were plated at 1 x 104 per well of a 96-well tray in appropriate FGM. Doubling dilution series were prepared from OncovexGALVCD and cisplatin in 2% FCS medium. Equipotent ED ratio of each agent was mixed for combination. 100μL of appropriate dilution was added to each well and incubate 48hr.
After 2 days the cellswere measured by the MTS assay (A). The isobologram curves (B) and the combination indexes were prepared by using CalcuSyn programme (Biosoft, Ferguson, MO). Experiments were repeated at least three times. The figure shows a representative experiment, where the error bars are standard deviations.
0
Figure 4.3.7 EJ cells were plated at1 x 104 per well of a 96-well tray in appropriate FGM. Doubling dilution series were prepared from OncovexGALVCD and gemcitabine in 2% FCS medium. Equipotent ED ratio of each agent was mixed for combination. 100μL of appropriate dilution was added to each well and incubate 48hr. After 2 days the cells were measured by the MTS assay (A). The isobologram curves (B) and the combination indexes were prepared by using CalcuSyn programme (Biosoft, Ferguson, MO).
Experiments were repeated at least three times. The figure shows a representative experiment, where the error bars are standard deviations.
0
Figure 4.3.8 T24 cells were plated at1 x 104 per well of a 96-well tray in appropriate FGM. Doubling dilution series were prepared from OncovexGALVCD and gemcitabine in 2% FCS medium. Equipotent ED ratio of each agent was mixed for combination. 100μL of appropriate dilution was added to each well and incubate 48hr. After 2 days the cells were measured by the MTS assay (A). The isobolgram curves (B) and the combination indexes were prepared by using CalcuSyn programme (Biosoft, Ferguson, MO).
Experiments were repeated at least three times. The figure shows a representative experiment, where the error bars are standard deviations.
0
Figure 4.3.9 TCCSUP-G cells were plated at 1 x 104 per well of a 96-well tray in appropriate FGM. Doubling dilution series were prepared from OncovexGALVCD and gemcitabine in 2% FCS medium. Equipotent ED ratio of each agent was mixed for combination. 100μL of appropriate dilution was added to each well and incubate 48hr.
After 2 days the cellswere measured by the MTS assay (A). The isobologram curves (B) and the combination indexes were prepared by using CalcuSyn programme (Biosoft, Ferguson, MO). Experiments were repeated at least three times. The figure shows a representative experiment, where the error bars are standard deviations.
0
Table 4.3.1 The coadministration of OncovexGALVCD and mitomycin was synergistic whereas the coadministration of both OncovexGALVCD or OncovexGFP and cisplatin or gemcitabine was antagonistic. (NAD: no available data)