Cordycepin inhibits the proliferation of malignant peripheral nerve sheath tumor cells through the p53/Sp1/tubulin pathway

Abstract

Neurofibromatosis type 1 (NF1) is one of the most common hereditary neurocutaneous disorders. In addition to skin pigmentation and cutaneous neurofibroma, some patients developed the plexiform neurofibroma since birth. Plexiform neurofibroma has abundant Schwann cells, fibroblasts, mast cells, blood vessels, and connective tissues, which increases the risk of developing a malignant peripheral nerve sheath tumor (MPNST). MPNST is a highly invasive cancer with no effective therapeutic agent. Cordycepin or 3'-deoxyadenosine is an extract from cordyceps militaris, which has been reported as an anti-inflammation and anti-tumor agent. Herein, we evaluated cordycepin's anti-proliferative effect on MPNST cell lines both in vitro and in vivo. Cordycepin inhibited the MPNST cell growth with an arrest of cell cycle at G2/M and S phases. The administration of naringin and pentostatin, inhibitors for adenosine deaminase (ADA), enzyme responsible for cordycepin degradation, did not show a synergistic effect in MPNST cells treated with cordycepin. However, the combined treatment enhanced the decrease of tumors in xenograft mouse model. Immunoblotting showed a decreased level of p53 protein in all MPNST cell lines, but S462TY cells. After cordycepin treatment, the levels of ERK, survivin, pAKT, and Sp1 proteins also decreased. The level of tubulin, but not actin or GAPDH, decreased in a dose-dependent manner. The microtubule network which is composed of tubulins was markedly decomposed in those treated MPNST cells. To elucidate the epigenetic control of transcription, ChIP-qPCR assay of the Sp1 and tubulin promoter regions revealed decreased Sp1 binding. The incorporation of 3'-doexyadenosine is detrimental for the process of poly(A) tail elongation. The poly(A) tail length assay showed the tail length in Sp1 and tubulin transcripts decreased in the treated cells. Nevertheless, the administration of SP1 protein to the treated cells could not rescue them completely. Furthermore, the p53-knocked-down cells (S462TY) where the expression of both p53 and Sp1 was suppressed, were vulnerable to cordycepin. The p53 protein could ameliorate the effect. In summary, cordycepin is effective to inhibit the growth of MPNST, probably through the pathway of p53/Sp1/tubulin.

Keywords: ChIP; Cordycepin; MPNST; p53/Sp1/tubulin; poly(A) tail length.

Figure 1

Cell survival in MPNST cell lines after treatment of cordycepin. A. The cell viability (presented in percentage of control) of normal human Schwann cells (HSC) and different MPNST cell lines (S462TY, ST8814, STS26T, and T265) after treated with different concentrations (presented in μM) of cordycepin. B, C. The survival of MPNST cells after the treatment of naringin, cordycepin or the combination of both. Two concentrations of naringin, 100- (1B) and 500-µM (1C) were used to assess any synergistic effect when combined with cordycepin.

Figure 2

Flow cytometry analysis of MPNST cells after treatment of Cordycepin. A. Flow cytometry analysis of ST8814 cells with/without the treatment of 600 µM of cordycepin. The cell populations at different stages were counted according to the intensity of the propidium iodide (PI), which stained the DNA. The classification of cell stage was dependent on the amount of cellular DNA reflected by the PI intensity. The P5 stage is at G0/G1, P6 is S, and P7 is G2/M phase. Cell populations of ST8814 at P5, P5 and P7 stages did not have a significant change in the control group. However, the populations in P6 and P7 stages increased significantly along with the treatment time. B. The changes of cell population percentages to represent the partition of different phases in cell cycle in HSC and four different MPNST cells after treatment of 600 µM of cordycepin for 24 hrs. The cell population in G2/M and S phases were significantly increased in HSC and MPNST cell lines.

Figure 3

Tumor size of the xenograft mouse model after treatment of cordycepin, naringin or the combination of both. A. The nude mice bearing xenograft tumor were subjected to be assessed of the tumor size to evaluate the effect of cordycepin. There are four groups of xenograft mice. The mice received the total volume of 100 μl of PEG (Control group, N = 7), 67 mg/kg of cordycepin (Cordycepin group, N = 7), 50 mg/kg of naringin (Naringin group, N = 9), and the combination of cordycepin and naringin (Combined group, N = 11). The medications were injected intraperitoneally in every other day. B. The tumor size was represented by the percentage of the original tumor volume (mm³), which was calculated as, maximum length (mm) × (perpendicular width (mm)²)/2. The size of tumor was recorded during the treatment course, up to 30 days. In addition to evaluating the tumor size, the body weight and activity of animals were also assessed.

Figure 4

Western blotting for the specific protein levels in MPNST cells after treatment with different concentrations of cordycepin. (A) Immunoblotting of phosphorylated ERBB2 (p-ERBB2), ERBB2, surviving, hTERT, p-p65, p65, p21^WARF1, p-ERK, ERK, p53, p-AKT, AKT, Sp1 and GAPDH proteins in the normal human Schwann (HSC) and MPNST cells (STS26T, ST8814, T265, and S462TY). The cells were treated with 300 μM of cordycepin. (B) Immunoblotting of the candidate proteins as shown in (A) in HSC and MPNST cells before and after treatment of 600 μM of cordycepin.

Figure 5

Altered levels of tubulin and the defects of microtubule network after cordycepin treatment. (A) Change of α-, β-tubulin, and GAPDH concentrations in MPNST cells after cells been treated with different concentrations (100, 300, and 600 μM) of cordycepin. The level of tubulin decreased in a dose dependent manner. However, there is no change of concentration in GAPDH. (B) Immunocytochemistry to observe the changes of tubulin level after cordycepin treatment. After 24 hours of treatment of DMSO (control group, left panel) or 600 μM of cordycepin (cordycepin group, right panel), cells were fixed and stained with α-tubulin (green) and DAPI (blue) to examine the changes of tubulin expression. Scale bar, 50 μm. Cell types: Human Schwann Cells (HSC), STS26T, ST8814, and S462TY. (C) Changes of area of microtubule network after treatment of cordycepin. The green area from (B) represented the microtubule network in cytosol. Using Image J software, the stained area of each cell before and after treatment can be calculated. The student t-test was employed to compare between the two groups and the p value was also presented.

Figure 6

Sp1 transcription factor involves in regulation of tubulin expression after treatment of cordycepin. A. The binding sites of transcriptional factors (Sp1, red; p53, black) in promoter region (0~-500 nucleotide) of the genes, Sp1, TUBA1B, TUBA1A, ACTB and GAPDH genes. B. The value of ΔΔC_T of Sp1 and TUBA1B genes after the treatment of 600 μM of cordycepin in HSC and among different MPNST cell lines, S462TY, ST8814, STS26T and T265. The ΔΔC_T value was obtained from the subtraction of ΔC_T value between before and after treatment. The quantitative PCR was performed using the template DNA which was precipitated (ChIP) by anti-Sp1 antibody. C. The poly(A) tail length of the mRNA of Sp1 and TUBA1B genes in HSC, S462TY and STS26T cells was measured and compared before and after treatment of 600 μM of cordycepin. The cluster of peaks, like shark teeth represents the poly(A) tail which was from the PCR product to amply the 3’end and poly(A) of the candidate gene. The size of poly(A) tail length and the semi-quantitation of the PCR product were assessed using peak scanner software.

Figure 7

The Sp1 level in tissues from xenograft mouse model after cordycepin treatment (A, B), and from the human patient tissues (C). (A) The level of Sp1 protein in the xenograft tumor tissues from the poly-ethylene glycol (PEG) and cordycepin treated mice. The immunoblotting with anti-Sp1 antibody (green) and DAPI (blue) was observed by the fluorescence microscopy. (B) Mean gray value of the stained tissue slices from the xenograft tumor shown in (A). To analyze the immunohistochemistry staining density of the tissue slice, a semi-quantitation method to measure the mean gray value, defined as integrated density divided by area were used. The values calculated from the tissue slices of PEG treated (N = 18) and cordycepin treated mice (N = 36) were presented. The student t-test was employed to compare between the two groups. The mean gray value was significantly decreased in the xenograft tumor from cordycepin treated mice (P = 0.001187). (C) The level of Sp1 protein in the MPNST, benign neurofibroma and normal human tissues. Immunostaining of Sp1 protein (green) and DAPI (blue) was observed by the confocal microscopy.

Figure 8

Sp1 associated with p53 proteins increase the survival of MPNST cells after treatment of cordycepin. A. Cell survival after the treatment of different concentrations of mithramycin A. The S462TY and STS26T cells were subjected to MTT assay after the treatment of 0, 25, 50, 100, 200, 400, and 800 μM of mithramycin A. B. Cell viability of S462TY and STS26T cells after the treatment of cordycepin and the combined treatment of cordycepin, Sp1 and p53 proteins. The cells were incubated with 600 μM cordycepin for 24 hours before MTT assay. Three more groups with combined treatment were also subjected to cell survival assay. In addition to cordycepin, one group combined with 500 ng/mL of Sp1 recombinant protein, one with 1000 ng/mL of p53 protein and one with both proteins. The student t-test was employed to compare between the two groups (*, P < 0.05, **, P < 0.01). C. Knockdown the expression of Sp1 and p53 mRNA transcripts of S462TY cells and the cell survival assay in these cells after treatment of 600 μM of cordycepin. The student t-test was employed to compare between the two groups (**, P < 0.01). D. Cell survival of human Schwann cell (HSC) and S462TY cells after the treatment of pifithrin-α. Left panel, cell viability of HSC and S462TY cells after the combined treatment of cordycepin and different concentrations (20, 50, 100, 200 and 500 µM) of pifithrin-α. Right panel, cell viability of HSC and S462TY cells after the treatment of 200 µM of pifithrin-α, 600 µM of cordycepin or both combination. The student t-test was employed to compare between the two groups (*, P < 0.05, **, P < 0.01). E. Cell survival of STS26T and SY8814 cells after the treatment of 600 μM of cordycepin along with different concentrations (30, 300 and 1000 ng/mL) of recombinant human p53 protein. The MTT assay has been performed after incubation of 24 hours. The student t-test was employed to compare between the two groups (*, P < 0.05, **, P < 0.01, ***, P < 0.001).