Induction of neural differentiation in rat C6 glioma cells with taxol

Abstract

Background: Glioblastoma is a common and aggressive type of primary brain tumor. Several anticancer drugs affect GBM (glioblastoma multiforme) cells on cell growth and morphology. Taxol is one of the widely used antineoplastic drugs against many types of solid tumors, such as breast, ovarian, and prostate cancers. However, the effect of taxol on GBM cells remains unclear and requires further investigation.

Methods: Survival rate of C6 glioma cells under different taxol concentrations was quantified. To clarify the differentiation patterns of rat C6 glioma cells under taxol challenge, survived glioma cells were characterized by immunocytochemical, molecular biological, and cell biological approaches.

Results: After taxol treatment, not only cell death but also morphological changes, including cell elongation, cellular processes thinning, irregular shapes, and fragmented nucleation or micronuclei, occurred in the survived C6 cells. Neural differentiation markers NFL (for neurons), β III-tubulin (for neurons), GFAP (for astrocytes), and CNPase (for oligodendrocytes) were detected in the taxol-treated C6 cells. Quantitative analysis suggested a significant increase in the percentage of neural differentiated cells. The results exhibited that taxol may trigger neural differentiation in C6 glioma cells. Increased expression of neural differentiation markers in C6 cells after taxol treatment suggest that some anticancer drugs could be applied to elimination of the malignant cancer cells as well as changing proliferation and differentiation status of tumor cells.

Keywords: Glioma; neural differentiation; taxol.

Figure 1

The morphological effect of C6 cells after 48 h taxol treatment. C6 cells were cultured in medium composed of Dubecco's modified Eagle's medium and (A) 10% FBS and with different concentrations of taxol, including (B)10, (C) 20, (D) 50, (E) 100, (F) 200, (G) 500, and (H) 1000 nmol/L for 48 h. The morphological images of the cells were recorded by phase contrast microscopy. Bar scale = 100 μm. (I) Cell viability was measured by MTT assay after 48‐h incubation. (J) Real‐time PCR data showed that apparent increases in mRNA levels of tubb3 (β III‐tubulin), gfap, and cnp when taxol concentration reached 100 nmol/L. mRNA of nestin was downregulated in taxol‐treated C6 cells. Data are presented as the mean ± SEM for three experiments in each group.

Figure 2

The expression of neural differentiation markers induced by taxol in C6 cells as demonstrated by immunofluorescent staining. Control (untreated) C6 cells and 100 nmol/L taxol‐treated C6 cells were analyzed by immunofluorescent staining at 48 h. In control cells, (A) the neural stem cell marker nestin was detected, (B) very few cells carrying GFAP protein could be found, and (C) immunoreactivites for β III‐tubulin and CNPase were difficult to detect. (D–F) The immunoreactivities for neuronal marker β III‐tubulin, astrocyte marker GFAP, and oligodendrocyte marker CNP, were clearly identified in the C6 cells after treated with taxol. These results indicate that expression of the neural differentiation markers were induced when the cells were challenged by taxol. Bar scale = 25 μm.

Figure 3

Characterizations of the mRNA and protein levels of β III‐tubulin, GFAP, CNPase, and nestin in taxol‐ and dbcAMP‐treated C6 cells by real‐time PCR and Western blot analysis. (A) mRNA levels of tubb3, gfap, and cnp were upregulated but nestin mRNA was downregulated in taxol‐treated C6 cells compared to the control group. The mRNA levels of target genes were calibrated based on the levels of gapdh mRNA. In dbcAMP‐treated C6 cells, mRNA level of gfap, cnp, and nestin were upregulated compared to control group. Data are presented as the mean ± SEM for three experiments in each group. *P < 0.01, unpaired Student's t‐test. (B) The immunoreactivities of β III‐tubulin, GFAP, CNPase proteins were upregulated while nestin was downregulated in taxol‐treated C6 cells. Nestin was downregulated in taxol‐treated C6 cells, while the immunoreactivities of β III‐tubulin were dramatically upregulated and the protein levels of GFAP and CNPase were also increased. In dbcAMP‐treated group, the immunoreactivities of GFAP and CNPase proteins were obviously increased but β III‐tubulin was not detectable compared to control group. The experiments were repeated three times and the results of a typical experiment are shown.

Figure 4

Percentages of nestin‐, β III‐tubulin‐, GFAP‐, and CNPase‐positive cells relative to the total number of taxol‐ and dbcAMP‐treated C6 cells. Neural cell‐specific molecular markers in control, taxol‐, and dbcAMP‐treated C6 cells were examined by immunocytochemical staining and then the immunostained cells were quantified. The percentages of nestin‐positive cells in taxol‐ and dbcAMP‐treated samples were significantly less than that in the control group. On the contrary, the percentages of neural differentiated cells in drug‐treated cells were significantly greater than that in the control groups. Data are shown as mean ± SEM for three experiments in each group. *P < 0.01, unpaired Student's t‐test.

Figure 5

Coexpression of β III‐tubulin and NFL detected in taxol‐treated C6 cells by immunofluorescent staining. (Α) β III‐tubulin and NFL proteins were not detectable in control cells. (B) Coexpression of β III‐tubulin (green) and NFL (red) proteins was found in 100 nmol/L taxol‐treated C6 cells. (C) NFL was not detectable in dbcAMP‐treated cells. Cell nuclei are labeled in blue. Bar scale = 25 μm.