Exploring long-term protection of normal human fibroblasts and epithelial cells from chemotherapy in cell culture

Abstract

Killing of proliferating normal cells limits chemotherapy of cancer. Several strategies to selectively protect normal cells were previously suggested. Here we further explored the protection of normal cells from cell cycle-specific chemotherapeutic agents such as mitotic inhibitors (MI). We focused on a long-term cell recovery (rather than on a short-term cell survival) after a 3-day exposure to MI (paclitaxel and nocodazole). In three normal human cell types (RPE, NKE, WI-38t cells) but not in cancer cells with mutant p53, pre-treatment with nutlin-3a, a non-genotoxic inducer of wt p53, caused G1 and/or G2 arrest, thus preventing lethal mitotic arrest caused by MI and allowing normal cells to recover after removal of MI. Rapamycin, an inhibitor of the nutrient-sensing mTOR pathway, potentiated the protective effect of nutlin-3a in normal cells. Also, a combination of rapamycin and metformin, an anti-diabetic drug, induced G1 and G2 arrest selectively in normal cells and thereby protected them from MI. A combination of metformin and rapamycin also protected normal cells in low glucose conditions, whereas in contrast it was cytotoxic for cancer cells. Based on these data and the analysis of the literature, we suggest that a rational combination of metformin and rapamycin can potentiate chemotherapy with mitotic inhibitors against cancer, while protecting normal cells, thus further increasing the therapeutic window.

Figure 1. Nutlin-3a protects cells by preventing mitotic arrest caused by nocodazole

(A) Drug treatment schedule: MI was added 1 day after nutlin-3a. 3days later all drugs were washed off and cells were allowed to recover for 6 d and then counted. (B) Selective protection of WI-38t, but not MDA-MB-231 cells, from the cytotoxic effect of nocodazole. 10,000 cells were plated per well (in 6 well plates), treated as shown in panel A and counted after 6 days. The results are shown as % of control, in log scale. (C) Prevention of toxic mitotic arrest by non-toxic G1 and G2 arrest. WI-38-t cells were pre-treated with 2.5 μM nutlin-3a, and then were treated with 200 nM nocodazole. After 24 hours, cells were microphotographed (right panel), collected and analyzed by flow cytometry.

Figure 2. Protective induction of p53/p21 by nutlin-3a in WI-38t cells

A. Immunoblot. WI-38t cells were pre-treated with indicated concentrations of nutlin-3a (2.5 μM and 10 μM), and then treated with 200 nM nocodazole. The next day, cells were lysed and immunoblot was performed as described in the Methods section. B. 50, 000 cells were plated per well (in 6 well plates) and treated as shown in Fig. 1A. The results present cell numbers expressed as % of control in log scale. Note: Cell numbers in Nutlin+Noco exceeded plated cell numbers almost 10-fold, indicating active cell proliferation after drug removal (not shown).

Figure 3. Nutlin-3a protects normal retinal pigment epithelial (RPE-19) cells but not MDA-MB-231 cells

10,000 cells were plated and treated the next day with 2.5 μM nutlin-3a or left untreated. The next day, cells were treated with either 200 nM nocodazole, 50 nM Taxol (paclitaxel) or left untreated. Cells were counted 6 days after wash because control cells reached confluence at that time. The results are shown as % of control, in log scale. Note: Nutlin+Noco, final cell numbers exceeded plated cell numbers, indicating cell proliferation after drug removal.

Figure 4. Nutlin-3a prevents toxic mitotic arrest by causing non-toxic G2 arrest in RPE cells

RPE (A) and MDA-MB-231 (B) cells were plated in 6-well plates at 50,000/well. Cells were either pretreated with 2.5 μM nutlin-3a or left untreated before addition of 200 nM nocodazole. After 24 hours treatment with nocodazole, cells were microphotographed (left panels), collected and analyzed by flow cytometry (right panels).

Figure 5. Cytoprotection of RPE cells with nutlin-3a, rapamycin and metformin 10,000 RPE cells were plated in normal (1g/L) glucose medium (panel A) and low (0.5 g/L) glucose medium (panel B)

The next day, cells were treated with either 2.5 μM nutlin-3a (Nutlin), 1 and 3 mM metformin (M 1 and M 3), 1 nM rapamycin (R) or left untreated. The next day, cells were treated with either 200 nM nocodazole (Noco) or 50 nM paclitaxel (PTX). 3 days later, the cells were washed and cultured in normal glucose for an additional 10 days and then were counted. The results are shown as % of control (no treatment). Note: in control cells reached confluence during the experiment.

Figure 6. Cytoprotection of WI-38t cells by combining rapamycin and metformin 10,000 WI-38t cells were plated in normal glucose medium

The next day, cells were treated with either 1 mM or 3 mM metformin (M 1 and M 3) and 1 nM or 100 nM rapamycin (R 1 and R 100) or left untreated (control). The next day, cells were treated with 200 nM nocodazole (Noco) as indicated. After the 3 days, the cells were washed and cultured for an additional 10 days and then were counted. The results are shown as % of control (no treatment). Note: in control cells reached confluence during the experiment.

Figure 7. A combination of rapamycin and metformin prevents mitotic arrest in WI-38t cells

50,000 WI-38t and MDA-MB-231 cells were plated in 6-well plates and pre-treated with a combination of 100 nM rapamycin and 3 mM metformin (R+M). The next day, cells were treated with 200 nM nocodazole. After 24 hours, cells were microphotographed (left panels), collected and flow cytometry was performed (right panels).

Figure 8. Protection of NKE cells from nocodazole and paclitaxel in normal glucose and low glucose

10,000 NKE cells were plated in either 1g/L glucose or 0.5g/L glucose (low). Cells were pre-treated for 24 hrs with either 2.5 μM nutlin-3a, 1 and 10 nM rapamycin (R1 and R10), 3 mM metformin (M) alone or in combination and then were treated with either 200 nM nocodazole (Noco) or 50 nM Taxol (PTX). After 3 days, cells were washed and cultured for 6 days in fresh medium before counting.

Figure 9. Cyclotherapy: protection of normal cells and unshielding of cancer cells

Figure 10. Proposal for potential clinical trials