Signal transducer and activator of transcription factor 3 (STAT3) plays an important role in the proliferation and angiogenesis in human glioma. Previous research indicated that saw palmetto extract markedly inhibited the proliferation of human glioma cells through STAT3 signal pathway. But its effect on tumor metastasis and antiangiogenesis is not clear. This study is to further clear the impact of saw palmetto extract on glioma cell metastasis, antiangiogenesis, and its mechanism. TUNEL assay indicated that the apoptotic cells in the saw palmetto treated group are higher than that in the control group (
Human gliomas which originate from neural stromal cells are the most common and malignant brain tumor in human [
The growth and proliferation of glioma cells are highly dependent on angiogenesis [
Signal transducer and activator of transcription factor 3 (STAT3) with tyrosine phosphorylation in signal pathway exists in the cell holder, regulating the expression of a variety of functional protein, cell proliferation, and apoptosis. The present study has proven that there is a close relationship between STAT3 and cell adhesion molecules, extracellular matrix degrading enzymes, tumor angiogenesis, metastasis through MMP, VEGF, and other related gene interactions [
Saw palmetto (SR) is a kind of palm plant in Southeast American. In in vitro experiments people found that saw palmetto extract can effectively inhibit the proliferation with dose dependent relationship in human breast cancer cells [
As mentioned, saw palmetto extract regulates the proliferation of tumor cells by inhibiting the STAT3 signaling pathway, but its effect on tumor metastasis and antiangiogenesis is not clear. The purpose of this study is through researching the effect of saw palmetto extract on glioma cell migration related protein and marker of angiogenesis to further clear the impact of saw palmetto extract on glioma cell metastasis, antiangiogenesis, and its mechanism.
30 healthy SD male rats were purchased from China Medical University Department of Experimental Animal, which weighed 250–300 g. The rats were randomly divided into three groups: control group, low dose SR group, and high dose SR group. Human glioma cell lines U87 and U251 were purchased from Beijing Dingguochangsheng Biotech Co., Ltd. (Beijing, China).
Saw palmetto extract was purchased from Yongyuan Bio-technology, Co., Ltd. (Xi’an, China). TUNEL kit, rabbit anti-B-cell lymphoma-2 (Bcl-2), anti-CD34, anti-MMP-2, anti-PARP, and anti-pSTAT3 antibodies were purchased from the Beyotime Institute of Biotechnology (Shanghai, China).
Human glioma cell lines, U87 and U251, were grown in a 25 cm2 cell culture bottle containing Dulbecco’s modified Eagle’s medium (DMEM), supplemented with 10% fetal bovine serum, 100 IU/mL penicillin, and 100
Blood cell counting plate and cover glass were cleaned with 95% alcohol. Take 1
The vigorous growing cell was collected; the cell concentration was adjusted to
TUNEL assay was utilized to detect the apoptosis in glioma tissues. The tissues were fixed with 10% formalin, were dehydrated, were transparent, and were embedded in paraffin; then the specimens were cut into 5–8
All specimens were promptly fixed in 10% buffered formalin and embedded in paraffin. 4
pSTAT3, Bcl-2, CD34, MMP-2, and PARP positive nuclei were brown or dark brown. The immunoreactive positive cells were determined by the average optical density value of ImageJ image analysis software.
All the experimental data were processed by SPSS 19 statistical analysis software; the values are expressed in mean ± standard deviation. Comparison between groups was analyzed by one-way ANOVA, with statistically significant difference in
The apoptosis of glioma cell induced by saw palmetto was detected by TUNEL assay. As shown in Figure
The apoptosis of glioma cell induced by saw palmetto through TUNEL assay. (a) is the control group, (b) is the low dose group in which the rats received administration of 50 mg/kg saw palmetto extract every day for 4 weeks, and (c) is the high dose group in which the rats received administration of 300 mg/kg saw palmetto extract every day for 4 weeks. (d) is the histogram to evaluate the apoptosis cell induced by saw palmetto of above three groups.
Previous study suggested that saw palmetto induced growth arrest and apoptosis of prostate cancer cells by the inhibition of STAT3 signal pathway. We adopted immunohistochemistry assay to measure the expression of pSTAT3 in glioma tissue. The results revealed that the optical density value is
The expression of pSTAT3 in the glioma tissue. (a) is the control group, (b) is the low dose group in which the rats received administration of 50 mg/kg saw palmetto extract every day for 4 weeks, and (c) is the high dose group in which the rats received administration of 300 mg/kg saw palmetto extract every day for 4 weeks. (d) is the histogram to evaluate the expression of pSTAT3 in the three groups.
The previous study certified that MMP-2 is associated with the metastasis of tumor. We detected the expression of MMP-2 to evaluate the effect of saw palmetto on the metastasis of glioma. We adopted immunohistochemistry assay to measure the expression of MMP-2 in glioma tissue. The results revealed that the optical density value is
The expression of MMP-2 in the glioma tissue. (a) is the control group, (b) is the low dose group in which the rats received administration of 50 mg/kg saw palmetto extract every day for 4 weeks, and (c) is the high dose group in which the rats received administration of 300 mg/kg saw palmetto extract every day for 4 weeks. (d) is the histogram to evaluate the expression of MMP-2 in the three groups.
Angiogenesis is very important in the tumor cell proliferation and metastasis. As known, CD34 is a good marker to reflect the density of blood vessels in the tumor. In order to study the effect of saw palmetto on angiogenesis, we measure the CD34 by immunohistochemistry method. The results revealed that the optical density value is
The expression of CD34 in the glioma tissue. (a) is the control group, (b) is the low dose group in which the rats received administration of 50 mg/kg saw palmetto extract every day for 4 weeks, and (c) is the high dose group in which the rats received administration of 300 mg/kg saw palmetto extract every day for 4 weeks. (d) is the histogram to evaluate the expression of CD34 in the three groups.
Furthermore, the proapoptotic effect of saw palmetto was explored by immunohistochemistry assay. The Bcl-2 and PARP (poly ADP-ribose polymerase) were detected in this study. In the study of cell apoptosis, PARP as the DNA repair enzyme which can repair the DNA is a core member of apoptosis. It plays an important role in DNA damage repair and apoptosis. PARP can be used as the hallmark of apoptosis. The average optical density is
The expression of PARP in the glioma tissue. (a) is the control group, (b) is the low dose group in which the rats received administration of 50 mg/kg saw palmetto extract every day for 4 weeks, and (c) is the high dose group in which the rats received administration of 300 mg/kg saw palmetto extract every day for 4 weeks. (d) is the histogram to evaluate the expression of PARP in the three groups.
The expression of Bcl-2 in the glioma tissue. (a) is the control group, (b) is the low dose group in which the rats received administration of 50 mg/kg saw palmetto extract every day for 4 weeks, and (c) is the high dose group in which the rats received administration of 300 mg/kg saw palmetto extract every day for 4 weeks. (d) is the histogram to evaluate the expression of Bcl-2 in the three groups.
Human gliomas are tumors of glial origin of central nervous system (CNS) and they are the most common primary tumors, accounting for ~46% of intracranial tumors and ~2% of adult tumors. The glioma is characterized by its invasive growth and is difficult to be treated via surgery. In addition, the human glioma cell is not sensitive to chemotherapy and radiotherapy.
In previous years, studies have focused on chemical compounds which derived from plants that possess pharmacological activity in antitumor therapy [
The effective ingredients of saw palmetto extract, as matured dry fruit of saw palm, are fatty acids. A foreign study reported that saw palmetto extract could inhibit the proliferation and induction of apoptosis in prostate cancer cells [
Glioma is one of the most common malignant tumors with the highest degree of vessels in intracranial tumor. It is typical angiogenesis dependent tumor which makes its invasion and recurrence rate significantly higher than those of other intracranial tumors [
Study on the mechanism of glioma angiogenesis and antiangiogenesis has become a new method for the treatment of glioma in recent years. In this study we adopted immunohistochemistry assay to detect CD34, MMP-2, and other indicators in glioma tissue. The results suggested that CD34 optical density value of the experimental group was higher than that of control group. As is known, CD34 is a good marker to reflect the density of blood vessels in the tumor. CD34 is closely related to tumor occurrence, development, and prognosis [
Signal transducer and activator of transcription factor 3 (STAT3) is a bifunctional protein coupled with tyrosine phosphorylation signal pathway, which exists in the cell holder and regulates the expression of a variety of related functional protein, cell proliferation, and apoptosis [
The tyrosine of STAT3 is activated under the combination cytokines, growth factors, and hormones with its receptor. STAT3 combined with Janus kinase (JAK) in the cytoplasm caused its tyrosine and JAK tyrosine phosphorylation. The STAT3 is activated after the tyrosine phosphorylation. After STAT3 activated, STAT3 forms homo- or heterodimer in cytoplasm and quickly enters the nucleus to combine with the specific gene promoter on the transcription of target genes, such as the antiapoptotic genes Bcl-2 and Mcl21, cell cycle control genes c-myc and cyclinDl, and angiogenesis related genes VEGF. Studies show that STAT3 phosphorylation at tyrosine 705 is abnormal aggregation in malignant glioma cells, particularly in glioblastoma. The expression of pY705-STAT3 is positively correlated with tumor grade and is one of the poor prognosis factors in survival analysis [
As mentioned, matrix metalloproteinases (MMPs) are a key enzyme in the degradation of extracellular matrix. The study found that STAT3 can bind to MMP-2 promoter to enhance the expression of MMP-2 [
In summary, we found that saw palmetto extract was an important phytotherapeutic drug against the human glioma through STAT3 signal pathway. Saw palmetto extract has been widely used to treat benign prostatic hyperplasia and androgenic alopecia in clinic; the related adverse reactions included ejaculatory disorders, postural hypotension, dizziness, headache, gastrointestinal disorders, rhinitis, fatigue, and asthenia, but the side-effects were limited [
The authors declare that there are no competing interests regarding the publication of this paper.
Yuqin Che conceived and designed the study. Hong Ding and Jinglian Shen performed immunohistochemistry. Yang Yang dealt with data analysis. Hong Ding and Yuqin Che wrote and revised the paper. All authors read and approved the final paper.
This study is supported by a grant from the Liaoning Provincial Natural Science Foundation of China (no. 2013010078-401).