PLK1 inhibitor facilitates the suppressing effect of temozolomide on human brain glioma stem cells

Abstract

Glioblastoma is the most frequent and most aggressive brain tumour in adults. Temozolomide is an oral chemotherapy drug and one of the major components of chemotherapy regimens used as a treatment of some brain cancers. We examined the tolerance of stem cells isolated from glioma cell line U87 and U251 to temozolomide (TMZ) and explored the effect of PLK1 (Polo like kinase 1) protein expression on TMZ sensibility. In our results, the inhibitory effects of TMZ on glioma cells U87, U251 and its stem cells were confirmed to be dose dependent and time dependent. Compared with glioma cells, the glioma stem cells showed a greater degree of tolerance. As the concentration of TMZ increased, the expression of PLK1 protein increased in U87 cells, CD133⁺ U87 stem cells and CD133- U87 cells. The increase range of PLK1 protein was large in CD133⁺ U87 stem cells and small in CD133- U87 cells. TMZ treatment in cells with low PLK1 protein expression efficiently suppressed the cell proliferation and sphere formation, while G2/M arrest was strongly induced. What's more, TMZ and PLK1 inhibitor synergize to inhibit glioma growth in vivo. In conclusion, our results suggest that down-regulation of PLK1 protein enhanced the inhibition of TMZ on glioma stem cells, suggesting its clinical value to adverse TMZ resistance in glioma treatment.

Keywords: PLK1; BI2536; glioma stem cells; temozolomide.

Figure 1

U87 CD133+ cells and U251 CD133+ cells were more resistant to TMZ. A. Sorted by immunomagnetic beads, protein expression of CD133+ and CD133‐ U87 cells were determined with flow cytometry. Proportions of CD133+, CD44+, Nestin+ or CD24‐ cells were lined out, supporting that CD133+ U87 cells were glioma stem cells. B. The isolation and identification of CD133+ U251 glioma stem cells. C. Treated with different concentration of TMZ for 24 hours, cell viabilities of U87, CD133+ U87 and CD133‐ U87 cells were determined. The inhibition was dose‐dependent and U87 cells are more sensitive to TMZ compared with CD133+ U87 cells. C. Treated by 100 μM TMZ, U87, CD133+ U87 and CD133‐ U87 cells were collected at 24 h, 48 h and 72 h respectively. The inhibition was confirmed to be time ‐dependent and viability of CD133+ U87 cells reduced less than U87 cells. D. U251, CD133+ U251 and CD133‐ U251 cells were treated with different concentration of TMZ for 24 hours and then cell viabilities were measured by CCK‐8 assay. CD133+ U251 cells were more resistant to TMZ compared with CD133‐ U87 cells or U87 cells. E. Cell viability of U251, CD133+ U251 and CD133‐ U251 cells with 100 μM TMZ treatment for 24 h, 48 h and 72 h respectively. F. Clonogenesis ability of the U87, CD133+ U87 and CD133‐ U87 cells were tested by soft agarose assay after 40 μM TMZ treatments for 2 weeks. G. Cloning efficiency was measured by counting clone number growing in soft agar in U87, CD133+ U87 and CD133‐ U87 cells after exposing to 40 ?M TMZ for 2 weeks. H. Cloning efficiency of U251, CD133+ U251 and CD133‐ U251 cells after exposing to 40 μM TMZ for 2 weeks. Data was presented as Mean ± SD (standard deviation) from triple experiments. **P < 0.01 compared with CD133+ U87 cells or CD133+ U251 cells

Figure 2

TMZ Induced Cell Cycle Arrest of U87 CD133⁺ Cells and U251 CD133⁺ Cells. A, After treated by 100 μmol/L TMZ to U87 CD133⁺ cells, 40 μmol/L TMZ to U87 CD133‐ and U87 cells for 24 h, cells were collected and cell cycle was analysed using PI staining and flow cytometry. B, After treated by 100 μmol/L TMZ to U251 CD133⁺ cells, 40 μmol/L TMZ to U251 CD133‐ and U251 cells for 48 h, cells were collected and cell cycle was analysed using PI staining and flow cytometry. Data were presented as Mean ± SD (standard deviation) from triple experiments. **P < 0.01 compared with 0 μmol/L TMZ groups

Figure 3

PLK1 Expression was Probably Involved in TMZ Sensibility. A, After treated by different concentration of TMZ for 24 h, U87 cells, CD133⁺ U87 cells and CD133‐ U87 cells were harvested. Protein level of PLK1 was determined by Western blot. B, Analysed by ImageJ, PLK1 expression was quantified and shown in the histogram. Data were presented as Mean ± SD (standard deviation) from triple experiments. **P < 0.01 compared with untreated U87 cells. ^## P < 0.01 compared with untreated CD133⁺ U87 cells. ^&& P < 0.01 compared with untreated CD133‐ U87 cells

Figure 4

The Effect of PLK1 on Tolerance of CD133⁺ U87 Stem Cells. A‐B, CD133⁺ U87 and CD133⁺ U251 stem cells were divided into blank group, control group, PLK1 inhibitor BI2536 group (cells treated with 0.5 nmol/L PLK1 inhibitor BI2536), PLK1 inhibitor Volasertib group (cells treated with 0.5 nmol/L PLK1 inhibitor Volasertib), pcDNA3.1 group, pcDNA3.1‐PLK1 group (cells transfected with pcDNA3.1‐PLK1), si‐NC group, PLK1‐siRNA1 group (cells transfected with PLK1‐specific siRNA1) and PLK1‐siRNA2 group (cells transfected with PLK1‐specific siRNA2). The total RNA was isolated and the expression of PLK1 mRNA was analysed by RT‐qPCR. **P < 0.01. C‐D, The total protein was analysed, and PLK1 protein expression was determined by Western blot in CD133⁺ U87 and CD133⁺ U251 stem cells. *P < 0.05, **P < 0.01. E‐F, Treated with PBS or 100 μmol/L TMZ for 24 h, cell viability of CD133⁺ U87 and CD133⁺ U251 stem cells was detected using CCK‐8 assay. TMZ efficiently suppressed the cell viability of CD133⁺ U87 stem cells with low PLK1 protein level. G‐H, PI staining and flow cytometry was performed to analyse the cell cycle of CD133⁺ U87 and CD133⁺ U251 stem cells in nine groups. TMZ induced stronger G2/M arrest in CD133⁺ U87 and CD133⁺ U251 stem cells with low PLK1 protein level. ^# P < 0.05 compared with control group which cells treated with PBS. **P < 0.01 compared with control group which cells treated with 100 μmol/L TMZ. ^& P < 0.05 compared with pcDNA3.1 group which cells treated with PBS. ^$ P < 0.05 compared with pcDNA3.1 group which cells treated with 100 μmol/L TMZ. ^## P < 0.01 compared with si‐NC group which cells treated with PBS. ^aa P < 0.01 compared with si‐NC group which cells treated with 100 μmol/L TMZ. Data was presented as Mean ± SD (standard deviation) from triple experiments

Figure 5

The Influence of TMZ on Sphere Formation of CD133⁺ U87 and CD133⁺ U251 Stem Cells was Investigated. A, During the sphere formation process, PBS or 100 μmol/L TMZ was added into the culture medium. After 8 d, photographs were taken to compare the sphere formation ability of CD133⁺ U87 stem cells. B, After centrifugation, sphere number was calculated. TMZ significantly reduced the sphere number in U87 stem cells with low PLK1 protein expression. C, After centrifugation, sphere number was calculated of CD133⁺ U251 stem cells. D, Sphere formation ability of CD133⁺ U251 stem cells after treated with PBS or 100 μmol/L TMZ for 8 d. Data were presented as Mean ± SD (standard deviation) from triple experiments. ^# P < 0.05 compared with control group which cells treated with PBS. **P < 0.01 compared with control group which cells treated with 100 μmol/L TMZ. ^& P < 0.05 compared with pcDNA3.1 group which cells treated with PBS. ^$ P < 0.05 compared with pcDNA3.1 group which cells treated with 100 μmol/L TMZ. ^## P < 0.01 compared with si‐NC group which cells treated with PBS. ^aa P < 0.01 compared with si‐NC group which cells treated with 100 μmol/L TMZ

Figure 6

Inhibition of PLK1 Enhanced Sensitivity to TMZ of CD133⁺ U87 Stem Cells In Vivo. Subcutaneous tumours generated from CD133⁺ U87 stem cells were allowed to reach a volume of 150‐200 mm³ and were treated with TMZ (50 mg/kg, intraperitoneal injection), BI2536 (40 mg/kg, intravenous injection), Volasertib (15 mg/kg, intravenous injection) or a combination of TMZ and BI2536, TMZ and Volasertib A, Tumour volumes in treatment groups. B, Tumour weights at 27 d after treatment. C, Representative HE staining of different groups was shown. D, The total PLK1 protein expression in tumour tissues was determined by Western blot. **P < 0.01 compared with Blank or control groups. ^## P < 0.01 compared with BI2536 or TMZ groups. ^aa P < 0.01 compared with Volasertib or TMZ groups