Sponsored by M.D.,
Ph.D. Kutushov. Israel
Conducted by:
HPBM
Harlan Pharmacological & Biological
Monitoring Kiryat Weizmann, Rehovot, Israel
April 2003
During April 2003, HPBM performed In Vitro various compounds, supplied by Idealiza.
HPBM experimental program examined the effect of the compounds on proliferation rate of HFF and A357 cell-lines using Thymidine incorporation study.
HPBM experimental program was conducted according to the experimental design that was agreed by the Sponsor.
As cells grow in culture, they divide and intrinsic characterizations and response to various environmental signals. In order to evaluate proliferation rate 3H-thymidine uptake into cells measured. The radioactive thyimidine is incorporated to the DNA that newly formed. The higher the proliferative response, the higher number of counts in the respective culture will be.
If 3H-thymidine uptake is not detected, we can only assume that cells do not proliferate. Cells may be viable but arrest in a certain phase of the cell cycle. To differentiate between the two possibilities, a metabolic activity measurement should be performed (i.e Alamar blue™ assay).
In culture, the cell cycle distribution is normal. Thus, in a certain moment various cells may be found in different cell-cycle phases. This may cause variability in results due to dissimilar thyimidine uptake by the cells in each culture. Therefore, standard deviation (SD) in this assay is high. In order to decrease variations, 3H-Thymidine was present for a period of 48 hours enabling cells to complete at least one cell-cycle.
The normal cells chosen for this test were HFF (human forskin fibroblast). Being similar to embryonic cells, HFF proliferation rate is high as the transformed cells A375 (human malignant melanoma). However, HFF proliferation rate does not reflect normal cell proliferation rates which are relatively low particularly after differentiation processes. HFF are utilized only since they enable us to culture normal cells in-vitro. Despite their similarity to malignant cells in their proliferation rate, HFF represent normal cells at the molecular level within the cells.
4.1 Test compounds
Tested substances (1-4) were supplied by the sponsor and dissolved to
104M in RPMI1640 medium. Compounds were added in a 1:10 dilution to the wells to obtain 10-5M, 10-7M and 10-9M, the tested final concentrations.
4.2 Controls
Positive control – Doxorubicin, in the aboved indicated concentrations. Negative control – Medium
4.3 Buffers
Washing buffer: PBS + 0.01% Triton X100.
Lysis buffer: 50mM Tris-HCl ph=8, 150mM NaCl, 1% Np-40, 2mM EDTA.
4.4 Cell lines
HFF (human forskin fibroblast) cells were grown in DMEM high glucose medium, supplemented with 10% FBS, 2mM L-glutamine,1% Penicilline/streptomycin, 1% non essential amino acid and 0.26% Sodium bicarbonate.
A375 (human malignant melanoma) cells were grown in RPMI1640 medium, supplemented with 10% FBS, 2mM L-glutamine, 1%Penicilline/streptomycin and 1% non essential amino acid.
5.1 Cells proliferation assay:
HFF and A375 were grown in in 75 cm2 culture flasks. The culture medium was changed every other day and the day before the experiment. For Subculture, the medium was removed and the cells were detached from the culture flasks with 0.25% trypsin–EDTA. Culture medium with fetal bovine serum was added to stop trypsinization. The cultures were kept at 37°C in an atmosphere of 5% CO2 and 100% humidity.
Cells were diluted at a density of 5,000 cells/well in 96 well microscintilation plate (Packard). Culture was in the logarithmic phase at the whole time of the experiment.
A day after seeding cells, 1-4 substances were added to the plates to 10-5M, 10-7M and 10-9M concentrations. Each plate column (8 wells) was used to test one substance in one concentration.
Two days later, for each well in the culture plate 3[H]-Thymidine (NET-027
Thymidine[methyl-3H] from NEN, 6.7Ci/mmol) was added to final concentration of 0.4 uCi/well. Plates were returned to the incubator for 48 hours and than harvested.
5.2 Cells harvesting
Medium was collected and cells were washed twice with PBS containing 0.01% TritonX100. Cells were lysed with 20µl Lysis Buffer per well for 30 minutes with shaking. Radioactivity was determined after an addition of 180µl Scintillation liquid (Microscint 20TM, Packard).
5.3 Data calculation:
Data represent the mean amount of Thymidine counted per well (CPM). Average and standard deviation (SD) were calculated for each plate column (8 wells). In order to compare between HFF and A375 cells, the average of negative control
(medium) column of either HFF or A375 was presented as 100% and all other tested concentrations were respectively translated.
6.1 Anti-proliferation effect of compound no 1:
Figure 1: Effect of compound number 1 on HFF and A357 cells
As can be seen in figure 1 compound number 1 has anti-proliferative effect on both HFF and A375 cells in 10-5M, 10-7M and 10-9M tested concentrations. No significant difference can be seen between HFF and A375 cell-lines.
Anti-proliferation effect of compound no 2:
Figure 2: Effect of compound number 2 on HFF and A357 cells
In figure 2 compound number 2 shows evidence of dose-response effect. In concentrations of 10-5M and 10-7M low rates of proliferation were measured for HFF and A375 cells while, concentrations of 10-9M were not significantly different from the control group. Compound number 2, like compound number 1, did not influence selectively the proliferation level of either one of the cell-lines.
6.2 Anti-proliferation effect of compound no 3:
Figure 3: Effect of compound number 3 on HFF and A357 cells
Compound number 3 shows an indication of anti-proliferative effect in 10-5M concentration for both HFF and A375 cells. This effect does not seem to be statisticaly significantly from the controls, considering the SD values. No significant difference could be detected in 10-7M and 10-9M concentration, comparing to the controls in HFF and A375 cells.
6.3 Anti-proliferation effect of compound no 4:
Figure 4: Effect of compound number 4 on HFF and A357 cells
Compound number 4 has no effect on the proliferation-rate of neither on HFF nor A375 in the indicated tested concentrations.
6.4 Anti-proliferation effect of Doxorubicin:
Figure 5: Effect of Doxorubicin on HFF and A357 cells
Doxorubicin was used in this test as a positive control for anti-proliferation activity. From the literature it is known that the LD50 of Doxorubicin for HFF and A375 cells is in 10-8M concentration.
As figure 5 demonstrates an anti-proliferation activity was measured in 10-5M and 10-7M concentration of HFF and A375 cells. In 10-9M the proliferation rate was similar to the controls of HFF and A375. The pattern of the dose-response that was displayed by the Doxorubicin was similar to the one revealed by compound number 2. Doxorubicin affected HFF and A375 at the same extent in all tested concentrations.
The results above summarizes one test that screened 4 unknown com pounds for anti proliferation activity. Therefore, no calculations may be drown.
The anti-proliferation effect of the compounds was tested on normal cells ( HFF cells) compared to carcinoma cells (A375 cells).
We suggest two main indications from this test:
a. None of the compounds found to inhibit selectively A375 proliferation rate.
b. Compound number 1 and 2 may contain anti-proliferation activity in the tested concentrations, while compounds number 3 and 4 did show no significant effect on the proliferation rate.
Sponsored by M.D.,
Ph.D. Kutushov. Israel
Conducted by:
HPBM
Harlan Pharmacological & Biological
Monitoring Kiryat Weizmann, Rehovot, Israel
August 2005
1.1 Test items
The Sponsor supplied six compounds labeled A to F as dry powders. Prior to the assay all compounds were dissolved in DMSO to 1mg/ml concentration (accept D that was dissolved in PBS). Compounds were diluted with medium to final concentration of 10-9M in eight repeats for each assay.Figure 1 The effect of compounds A-F on the metabolic rate of MCF-7 cells.
MCF-7 cells were treated with 1nM compounds, 10μM Doxorubicin and 30μM positive controls and vehicle control (Medium+DMSO) for one day. Results were expressed as % of vehicle control Mean value ±SD. Alamar blue fluorescent signal was measured at excitation/emission 544nm/590nm. Raw data is presented in Appendix 1.
Figure 2 The effect of compounds A-F on the metabolic rate of A375 cells.
A375 cells were treated with 1nM compounds, 10μM Doxorubicin and 30μM positive controls and vehicle control (Medium+DMSO) for one day. Results were expressed as % of vehicle control Mean value ±SD. Alamar blue fluorescent signal was measured at excitation/emission 544nm/590nm. Raw data is presented in Appendix 1.
Figure 3 Anti proliferative effect of A-F compounds in MCF-7 and A375 cells
MCF-7 (A) and A375 (B) cells were treated with 1nM compounds A-F, positive controls (10μM Doxorubicin and 30μM Digoxin), and vehicle control (Medium+DMSO) for 3 days. Incorporation of 3H-Thymidine to proliferating cells is presented as % of vehicle control Mean value ±SD. CPM Raw data is presented in Appendix 2.
Table 1 Summary of Compounds A-F Activities on MCF-7 and A375 cells.
Summary of the results described above. + indicates activity, – indicates non-active.
Plan plate cultured with either MCF-7 cells or A375 cells.