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. 2015 Jun 25;10(6):e0131441.
doi: 10.1371/journal.pone.0131441. eCollection 2015.

The Synergistic Effect of Combination Progesterone and Temozolomide on Human Glioblastoma Cells

Fahim Atif  1 , Neil R Patel  1 , Seema Yousuf  1 , Donald G Stein  1
Affiliations

The Synergistic Effect of Combination Progesterone and Temozolomide on Human Glioblastoma Cells

Fahim Atif et al. PLoS One. .

Abstract

Glioblastoma multiforme (GBM) is the most common and most aggressive malignant brain tumor. Despite optimal treatment and evolving standard of care, the median survival of patients diagnosed with GBM is only 12-15 months. In this study, we combined progesterone (PROG) and temozolomide (TMZ), a standard chemotherapeutic agent for human GBM, to test whether PROG enhances the antitumor effects of TMZ and reduces its side effects. Two WHO grade IV human GBM cells lines (U87MG and U118MG) and primary human dermal fibroblasts (HDFs) were repeatedly exposed to PROG and TMZ either alone or in combination for 3 and 6 days. Cell death was measured by MTT reduction assay. PROG and TMZ individually induced tumor cell death in a dose-dependent manner. PROG at high doses produced more cell death than TMZ alone. When combined, PROG enhanced the cell death-inducing effect of TMZ. In HDFs, PROG did not reduce viability even at the same high cytotoxic doses, but TMZ did so in a dose-dependent manner. In combination, PROG reduced TMZ toxicity in HDFs. PROG alone and in combination with TMZ suppressed the EGFR/PI3K/Akt/mTOR signaling pathway and MGMT expression in U87MG cells, thus suppressing cell proliferation. PROG and TMZ individually reduced cell migration in U87MG cells but did so more effectively in combination. PROG enhances the cytotoxic effects of TMZ in GBM cells and reduces its toxic side effects in healthy primary cells.

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Conflict of interest statement

Competing Interests: A US patent (# US 8,435,972 B2) was issued to FA and DGS on May 7, 2013, for the use of PROG and compositions related thereto for the treatment of neurogenic tumors specially neuroblastoma and glioblastoma. At present, there are no commercial or financial claims related to the patent. The Allen and Company is an investment firm with no commercial interests in pharmacology or any other matter relating to our research. The Marcus Foundation is a philanthropic foundation supporting research in developmental psychobiology and other related areas. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Therefore, they don’t alter the authors’ adherence to PLoS One policies on sharing data and materials.

Figures

Fig 1
Fig 1. Effect of individual repeated treatment with TMZ and PROG on the viability of U87MG and U118MG cell lines.
Cells were grown in 24-well plate and repeatedly treated with PROG and TMZ at different concentration for 3 and 6 days. For repeated exposure, culture medium was replaced daily and the drugs were added to the medium every day. On day 4 and 7, cell viability test was performed using MTT reduction assay. PROG and TMZ stocks were prepared in absolute DMSO and further diluted in culture medium. The final concentration of DMSO was kept at <5μl/ml. Data are expressed as means ± SD of three separate replication experiments (n = 3 each). Statistically significant difference: *P<0.05 compared with control group.
Fig 2
Fig 2. Effect of combined repeated treatment with PROG and TMZ on the viability of U87MG and U118MG cell lines.
Cells were grown in 24-well plate and repeatedly treated with PROG and TMZ at different concentration for 3 and 6 days. For repeated exposure, culture medium was replaced daily and the drugs were added to the medium every day. On day 4 and 7, cell viability test was performed using MTT reduction assay. PROG and TMZ stocks were prepared in absolute DMSO and further diluted in culture medium. The final concentration of DMSO was kept at <5μl/ml. Data are expressed as means ± SD of three separate replication experiments (n = 3 each). Statistically significant difference: *P<0.05 compared with control group; # P<0.05 compared to T100 alone group. P5 = PROG (5 μM); P80 = PROG (80 μM); T100 = TMZ (100 μM).
Fig 3
Fig 3. Individual and combined treatment effect of PROG and TMZ on the viability of primary human dermal fibroblasts (HDF).
Cells were grown in 24-well plate and repeatedly treated with PROG and TMZ at different concentration for 3 and 6 days. For repeated exposure, culture medium was replaced daily and the drugs were added to the medium every day. On day 4 and 7, cell viability test was performed using MTT reduction assay. PROG and TMZ stocks were prepared in absolute DMSO and further diluted in culture medium. The final concentration of DMSO was kept at <5μl/ml. Data are expressed as means ± SD of three separate replication experiments (n = 3 each). Statistically significant difference: # P<0.05 compared to control group; *P<0.05 compared to T100 alone. P5 = PROG (5 μM); P10 = PROG (10 μM); P40 = PROG (40 μM); P80 = PROG (80 μM); T100 = TMZ (100 μM).
Fig 4
Fig 4. Individual treatment effect of PROG on U87MG cell migration in a wound healing assay.
Cells were grown in multi-well plates and pre-treated with PROG alone at different concentrations for 2 h. A scratch/wound was formed with a 200-μl tip and the cells were incubated with PROG for the next 24 h. Photographs (4x) were taken at 0 h and 24 h post-wound formation. In the vehicle group, a large number of cells migrated from both sides to heal the wound at 24 h compared to 0 hr. PROG decreased U87MG cell migration 24 h after wound formation compared to vehicle. Representative photomicrographs from three separate replication experiments (n = 3 each).
Fig 5
Fig 5. Combined treatment effect of PROG and TMZ on U87MG cell migration in a wound healing assay.
Cells were grown in multi-well plates and pre-treated with (A) PROG alone and (B) in combination with TMZ at different concentrations for 2 h. A scratch/wound was formed with a 200-μl tip and the cells were incubated with PROG, TMZ or their combinations for the next 24 h. Photographs (4x) were taken at 0 h and 24 h post-wound formation. In the vehicle group, a large number of cells migrated from both sides to heal the wound at 24 h compared to 0 hr. PROG and TMZ individually decreased U87MG cell migration 24 h after wound formation compared to vehicle. The combination of the highest concentrations of both the drugs inhibited cell migration better than either drug alone. Representative photomicrographs from three separate replication experiments (n = 3 each).
Fig 6
Fig 6. Effect of PROG and TMZ on the PI3k/Akt/mTOR signaling pathway in U87MG and U118MG cells.
Tumor cells (U87MG and U118MG) were seeded (0.5 x 106) in 60-mm petri dishes and kept under starvation overnight prior to drug exposure. Cells were repeatedly exposed to different concentrations of PROG and/or TMZ for 3 days. Protein samples (50 μg) were separated under reducing and denaturing conditions by 4–20% acrylamide Criterion gel and analyzed for EGFR, pAkt, total Akt and mTOR expression. The density of each P-Akt band was normalized with the density of corresponding total Akt band. β-actin was used as a loading control for densitometry. Representative Western blot and densitometric analysis of the expression of EGFR, phospho-Akt (Ser473) and mTOR in (A) U87MG and (B) U118MG cell lines. Data are expressed as means ± SD from two separate replication experiments (n = 3 samples each). Statistically significant difference: *P<0.05 compared to control; # P<0.05 compared to T100 alone.
Fig 7
Fig 7. Effect of PROG and TMZ on the proliferation and the expression of MGMT in U87MG and U118MG cells.
Tumor cells (U87MG and U118MG) were seeded (0.5 x 106) in 60-mm petri dishes and kept under starvation overnight prior to drug exposure. Cells were repeatedly exposed to different concentrations of PROG and/or TMZ for 3 days. Protein samples (50 μg) were separated under reducing and denaturing conditions by 4–20% acrylamide Criterion gel and analyzed for EGFR, pAkt, total Akt and mTOR expression. The density of each P-Akt band was corrected for variance in loading, using the density of the corresponding total Akt. β-actin was used as a loading control for densitometry. (A) Representative Western blot and densitometric analysis of the expression of proliferation marker PCNA. (B) Expression of MGMT in U87MG and U118MG cells, and (C) Inhibitory effect of PROG on MGMT in U118MG cells. Data are expressed as means ± SD from two separate replication experiments (n = 3 samples each). Statistically significant difference: *P<0.05 compared to control; # P<0.05 compared to T100 alone.
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This research was supported in part by gifts from Allen and Company, The Marcus Foundation, The Laney Graduate School of Emory University and the Stein Family Research Fund.