Cancer Gene Therapy

FIGURES AND TABLES

FROM:

Adenovirus-mediated PTEN treatment combined with caffeine produces a synergistic therapeutic effect in colorectal cancer cells

Yuji Saito, Began Gopalan, Abner M Mhashilkar, Jack A Roth, Sunil Chada, Louis Zumstein and Rajagopal Ramesh

BACK TO ARTICLE
Figure 1 - Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, please contact help@nature.com or the author

Figure 1.

Isobologram analysis of the effects of treatment with two-agent combinations on colorectal cell lines. Cells seeded in 96-well plates were treated with various concentrations of Ad-PTEN, caffeine, Ad-PTEN, or Ad-Luc and caffeine. At 72 hours after treatment, isobolograms at IC50 levels were generated in colorectal cancer cell lines (HCT116dotp53 (+ / +), HCT116dotp53 (- / -), SW480, DLD-1) (a) and (c), and normal colorectal fibroblast cells (CCD-18Co), (b), treated with combinations of Ad-PTEN and caffeine (a) and (b), or Ad-Luc and caffeine (c).

Full figure and legend (129K)
Figure 2 - Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, please contact help@nature.com or the author

Figure 2.

Induction of apoptosis by treatment with combinations of Ad-PTEN and caffeine. (a) The numbers of cells at phase sub-G0/G1 (apoptotic cells) in colorectal cancer cells HCT116dotp53 (+ / +) and normal colorectal fibroblast cells CCD-18Co were analyzed by flow cytometry 72 hours after treatment with PBS, Ad-Luc, Ad-PTEN, caffeine, or combinations of caffeine with Ad-Luc or Ad-PTEN. Data represent the means of two experiments. Error bars denote standard error (SE). (b) Apoptotic analysis of HCT116dotp53 (+ / +) cancer cells and CCD-18Co normal cells by Hoechst 33258 staining was performed 72 hours after treatment with PBS, caffeine, or combinations of Ad-PTEN and caffeine. Treatment with combinations of Ad-PTEN and caffeine induced apoptosis in cancer cells but not in normal cells. Arrows indicate apoptotic cells (magnification times 400).

Full figure and legend (232K)
Figure 3 - Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, please contact help@nature.com or the author

Figure 3.

Induction and abrogation of G2/M cell-cycle arrest due to overexpression of PTEN and treatment with caffeine. (a) HCT116dotp53 (+ / +) colorectal cancer cells and (b) CCD-18Co normal colorectal fibroblast cells were treated with PBS, Ad-Luc, Ad-PTEN, caffeine, combinations of caffeine with Ad-Luc or Ad-PTEN, or 20 muM LY294002. Cells were harvested 72 hours after treatment and cell-cycle analysis was performed by using flow cytometry. In total 20,000 events were captured for each treatment, and the data are shown as histograms. The cell-cycle phase is represented on the X-axis. Data were generated in duplicate; the average values are shown. Bars denote standard error (SE).

Full figure and legend (247K)
Figure 4 - Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, please contact help@nature.com or the author

Figure 4.

Signaling pathways regulated by PTEN overexpression and caffeine. HCT116dotp53 (+ / +) colorectal cancer cells and CC18-Co normal cells were treated with PBS, Ad-Luc, Ad-PTEN, caffeine, combinations of caffeine with Ad-Luc or Ad-PTEN, or 10 muM U0126. At 48 hours after treatment, cells were harvested and examined by Western blot analysis. (a) G1 and G2/M phase-associated proteins. (b) Phosphorylation status of p44/42MAPK. The corresponding beta-actin levels are shown as a loading control. (c) The numbers of cells at sub-G0/G1 (apoptotic cells) in HCT116dotp53 (+ / +) colorectal cancer cells were analyzed by flow cytometry 72 hours after treatment with PBS, Ad-Luc, Ad-PTEN, caffeine, or combinations of caffeine with Ad-Luc, Ad-PTEN, or Ad-PTEN and10 muM U0126. Data represent the means of duplicate experiments. Error bars denote standard error (SE).

Full figure and legend (229K)
Figure 5 - Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, please contact help@nature.com or the author

Figure 5.

Abrogation of G2/M cell-cycle arrest due to overexpression of PTEN or ionized radiation combined with caffeine. HCT116dotp53 (+ / +) colorectal cancer cells were (a) treated with Ad-PTEN or combination of Ad-PTEN and caffeine, or (b) exposed to 2 Gy IR or a combination of IR and caffeine. Cells were harvested 24 hours after treatment and cell-cycle analysis was performed by using flow cytometry. In total, 20,000 events were captured for each treatment, and the data are shown as histograms. The cell-cycle phase is represented on the X-axis. Data were generated in duplicate; the average values are shown. Bars denote standard error (SE).

Full figure and legend (44K)
Figure 6 - Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, please contact help@nature.com or the author

Figure 6.

Effect of treatment with combinations of IR and caffeine. HCT116dotp53 (+ / +) colorectal cancer cells were treated with PBS, Ad-PTEN, a combination of Ad-PTEN and caffeine, 2 Gy IR, a combination of IR and caffeine, or 10 muM U0126. (a) At 72 hours after treatment, isobolograms at IC50 levels were generated for cells treated with the combination of IR and caffeine. (b) The numbers of cells at sub-G0/G1 (apoptotic cells) were analyzed by flow cytometry on days 3 and 5 after treatment. Data represent the means of duplicate experiments. Error bars denote standard error (SE). (c) At 48 hours after treatment, cells were harvested and phosphorylation status of p44/42MAPK examined by Western blot analysis. The corresponding beta-actin levels are shown as a loading control.

Full figure and legend (162K)
Table 1 - Therapeutic effect produced in various cell lines when treated with a combination of Ad-PTEN and caffeine is independent of PTEN and p53 status - Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, please contact help@nature.com or the author

Table 1 - Therapeutic effect produced in various cell lines when treated with a combination of Ad-PTEN and caffeine is independent of PTEN and p53 status.

Full table
BACK TO ARTICLE