Glioma stem cells (GSCs) are believed to contribute to glioblastoma multiforme (GBM) propagation and treatment resistance. Tanshinone IIA possesses anticancer and anti-inflammatory activities. This study aimed to determine the inhibitory effect of tanshinone IIA on human GSCs
Glioblastoma multiforme (GBM, grade IV astrocytoma) is considered to be the most highly malignant brain tumor (
Cancer stem cells (CSCs) are a subpopulation of tumor cells with the ability to undergo self-renewal and propagate the tumor population (
Tanshinone IIA is a lipophilic diterpene isolated from
Previous studies have shown that CSCs are maintained by inflammatory signaling pathways and the microenvironment (
In the present study, we investigated the effect of serum-free medium (SFM) on GSC formation and examined the effects of tanshinone IIA on human GSCs
Human WJ1 (
Total RNA was harvested from cells using TRIzol reagent (CWBIO). PCR was performed on cDNA generated by HIFI-MMLV reverse transcriptase (CWBIO) in a total reaction volume of 20 μl according to the manufacturer’s instructions. The sequences of forward and reverse oligonucleotide primers, specific to the chosen candidates and housekeeping genes, were used as follows: CD133 forward, 5′-CGACTGAGACCCAACATC-3′ and reverse, 5′-CCCTTTT GATACCTGCTACGAC-3′; GFAP forward, 5′-CGATCAACT CACCGCCAACA-3′ and reverse, 5′-GTGGCTTCATCTGCT TCCTGTC-3′; GAPDH forward, 5′-ACCACAGTCCATGCCA TCAC-3′ and reverse, 5′-TCCACCACCCTGTTGCTGTA-3′. All data were normalized to GAPDH transcript levels.
Western blotting was performed as previously described (
To evaluate the tumorigenicity of the GBMS cells, 1×104, 1×105, 1×106 of GBM cells and 1×102, 1×103, 1×104 of GBMS cells were suspended in 50 μl Matrigel (BD Biosciences) and 50 μl PBS, and then cells were implanted bilaterally into the flank of BALB/c nu/nu mice. Animals were maintained under standard conditions according to the guidelines of the Institutional Animal Care and Use Committee of Sichuan University. Tumor incidence was recorded 3 times weekly after the injection. Mice were maintained on a standard rodent chow and had free access to water. Tumors of the euthanized mice were collected, and their size and weight were measured.
Single-cell suspensions of GBMS were seeded in a 96-well plate at a density of 100 cells per well. Cells were treated with tanshinone IIA at concentrations of 0.25, 0.5 and 1.0 μg/ml respectively. Each concentration was repeated in 6 wells. After a 7-day incubation at 37°C in a humidified 5% CO2 atmosphere, the number of neurospheres in each well was quantified under a microscope. Three independent experiments were performed.
The 3-[4,5-dimethylthiazol-2-yl]-2, 5 diphenyltetrazolium bromide (MTT) assay was performed as previously described (
In order to determine apoptosis in GBMS cells, the Annexin V-FITC apoptosis detection kit (KeyGen Biotech, Nanjing, China) was used according to the manufacturer’s instructions. The extent of apoptosis in the samples was measured by flow cytometry using BD FACSCalibur flow cytometer and CellQuest software. Three independent experiments were performed.
The flank xenografts were established by subcutaneous injections of 104 GSCs per flank (5 mice per group). Animals were maintained under standard conditions according to the guidelines of the Institutional Animal Care and Use Committee of Sichuan University. After one week of inoculation, the tumor-bearing mice were randomized into four groups, each having 5 mice. The mice of the three treatment groups were injected with 10, 20 and 40 mg/kg of tanshinone IIA i.p. 3 times a week for 8 weeks, respectively, and the control mice were injected with an equal volume of blank dissolvant. In the experimental process, mice were weighed, and tumor volumes were assessed each week. At the end of the experiment, the mice were sacrificed by carbon dioxide asphyxiation and tumors were harvested, weighed and examined. The tumor inhibitory rate was calculated using the following formula: Tumor inhibitory rate (%) = (mean tumor weight of the control mice − mean tumor weight of the treated mice)/average tumor weight of the control mice × 100%.
Results are expressed as means ± standard deviation. To evaluate the significant differences between two groups, the means were compared using the Student’s t-test. Multiple group comparisons were performed using one-way analysis of variance. The level of statistical significance was defined as P<0.05 for all tests. All statistical analyses were performed using SPSS 16.0 (SPSS, Inc., Chicago, IL, USA)
GBM cells exhibited adherent growth in SCM (
The level of the CD133 transcript was significantly higher in the GBMS when compared with the GBM cells. In contrast, the GFAP transcript was obviously lower in the spheres than that in the GBM cells (
To confirm the different tumor-initiating capabilities between GBMS and GBM cells
To determine whether tanshinone IIA suppresses GSC proliferation, we compared the inhibitory effect of tanshinone IIA on human GSCs, GBM and normal control (LO2) cells and under proper drug concentrations after treatment for 1–5 days, respectively. The inhibition of tanshinone IIA at various concentrations in these cells was determined as the ratio of the number of viable treated cells to the number of viable cells of the untreated controls. Tanshinone IIA exhibited a dose- and time-dependent inhibitory effect on GSCs, GBM and normal cells (
To test the inhibitory effect of tanshinone IIA on neurosphere formation in GSCs, we incubated single-cell suspensions of GBMS with escalating concentrations of tanshinone IIA and evaluated neurosphere formation on day 7. We found that there was a dose-dependent reduction in neurosphere formation after treatment with tanshinone IIA (
To investigate the inhibitory activity of tanshinone IIA on human GSCs
After treatment with tanshinone IIA, protein expression levels of GSC markers including CD133 and nestin and differentiation and neural lineage markers including GFAP for astrocytes and βIII-tubulin for neurons
Inflammatory cytokines play important roles in the maintenance and progression of CSCs. To understand the molecular mechanisms underlying the inhibitory effect of tanshinone IIA on GSCs, we examined protein expression profiles mediating inflammatory signaling pathways. Inflammatory cytokine IL6, STAT3 and the active form of STAT3 [(phospho-STAT3(tyrosine705) and phospho-STAT3(serine727)] in GSCs
GBM is an extremely lethal tumor that is highly resistent to therapy, and achieving a cure through surgical intervention is difficult. Previous research has shown that therapeutic resistance of GBM is caused by the selective survival of highly tumorigenic GSCs (
In the present study, we successfully isolated GBMS from the human GBM cell line WJ1. GBMS exhibited properties of GSCs, including the ability for self-renewal, exhibited pale with toluidine blue, expression of known GSC markers and high tumorigenicity
A previous study showed that GSCs play core roles in the therapeutic resistance of GBM (
Since CSCs play a core role in cancer recurrence, metastasis and high patient mortality, targeting and attenuating CSC stemness, induction of CSC differentiation and apoptosis are promising treatment avenues other than targeting tumor masses in the treatment of a malignant tumors. Friedman
It was well recognized that inflammatory cytokines and signaling pathways play pivotal roles in maintaining stem-like properties in human glioma cells (
Based on the findings in this experimental study, tanshinone IIA inhibits proliferation, attenuates stemness and induces apoptosis in GSCs
This research was supported by the National Natural Science Foundation of China (grant no. 31071197).
Morphology of GBM and GBMS cells. (A) GBM cells cultured in SCM grew as an adherent monolayer. (B) GBM cells cultured in SFM formed primary neurosphere-like colonies after 10–14 days.
GBMS exhibit characteristics of GSCs. (A) A higher CD133 and a lower GFAP transcript were noted in GBMS when coupled with those in GBM cells with GAPDH included as a reference control. (B) The histogram shows significantly higher CD133 and lower GFAP transcript in GBMS than those in the adherent GBM cells; **P<0.01. (C) Western blot analysis revealed higher CD133 and nestin, lower GFAP and β-tubilin III in GBMS than these levels in the adherent GBM cells with β-actin included as the reference control. (D) Tumor masses from the GBM group (implantation with 1×106, 1×105 and 1×104 GBM cells, respectively) and from the GBMS group (implanted with 1×104, 1×103 and 1×102 GBMS cells, respectively). The histogram shows that there was a significant difference in (E) mean tumor volume and (F) mean tumor weight between the GBMS and GBM group; *P<0.05.
Tanshinone IIA suppresses neurosphere formation and proliferation of GSCs. GBMS, GBM and LO2 (normal control) cells were treated with 0.125, 0.25, 0.5, 1.0 and 2.0 μg/ml of tanshinone IIA and incubated for 1, 2, 3, 4 and 5 days, respectively. Cell viability was detected by MTT assay, using the method indicated in Materials and methods. Tanshinone IIA exhibited a dose- and time-dependent inhibitory effect on (A) GBMS, (B) GBM and (C) normal control cells. Tanshinone IIA exhibited a more significant inhibitory effect in the GBMS cells than in the GBM and LO2 cells. (D) The half inhibitory concentration (IC50) values for the GSCs, GBM and normal cells after treatment with tanshinone IIA for 3 days are shown (72 h; *P<0.05, **P<0.01). (E) GSCs were plated in 96-well plates at a density of 100 cells/well and treated with the indicated concentrations of tanshinone IIA. Representative examples of neurospheres from GSCs on day 7 are shown. Tanshinone IIA decreased neurosphere number in a dose-dependent manner (magnification ×200). (F) The histogram shows that there was a significant decrease in the number of neurospheres after tanshinone IIA treatment in a dose-dependent manner; **P<0.01. Results are mean values ± SD of independent experiments performed in triplicate.
Tanshinone IIA inhibits human brain tumor growth initiated by GSCs
Tanshinone IIA regulates protein expression in connection with GSC stemness and differentiation. Human GSCs were treated with tanshinone IIA (0.25, 0.5 and 1.0 μg/ml) for 48 h, respectively. (A) Protein expression levels of CD133 and nestin were downregulated while (B) GFAP and βIII-tubulin were upregulated
Tanshinone IIA induces GSC apoptosis. (A) Following treatment with tanshinone IIA (1.0, 2.0 and 4.0 μg/ml) for 24 h, respectively, apoptosis in GSCs was detected by flow cytometry. (B) Apoptotic rate was calculated. (C) GSCs were treated with tanshinone IIA (1.0, 2.0 and 4.0 μg/ml) for 24 h, respectively. The levels of apoptotic proteins Bax and cleaved caspase-3 were upregulated, while the expression of anti-apoptosis protein Bcl2 was downregulated in a dose-dependent manner. (D) The histogram shows that there was a significant increase in Bax and cleaved caspase-3, and a significant decrease in Bcl2
Tanshinone IIA regulates protein expression mediating inflammatory signaling pathways in GSCs. (A) Western blot analysis of IL6, STAT3, phospho-STAT3(tyrosine705) and phospho-STAT3(serine727) proteins before and after tanshinone IIA (0.25, 0.5 and 1.0 μg/ml) treatment for 48 h. (B) The histogram shows that there was no change in STAT3 but a significant decrease in IL6, phospho-STAT3(tyrosine705) and phospho-STAT3(serine727)