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. 2019 Jul 9;20(13):3366.
doi: 10.3390/ijms20133366.

Blockade of STAT3 Signaling Contributes to Anticancer Effect of 5-Acetyloxy-6,7,8,4'-Tetra-Methoxyflavone, a Tangeretin Derivative, on Human Glioblastoma Multiforme Cells

Yen-Po Cheng  1   2 , Shiming Li  3   4 , Wan-Ling Chuang  5 , Chia-Hsuan Li  2 , Guan-Jun Chen  2 , Ching-Chin Chang  2 , Chi-Hung R Or  6 , Ping-Yi Lin  7   8 , Chia-Che Chang  9   10   11   12
Affiliations

Blockade of STAT3 Signaling Contributes to Anticancer Effect of 5-Acetyloxy-6,7,8,4'-Tetra-Methoxyflavone, a Tangeretin Derivative, on Human Glioblastoma Multiforme Cells

Yen-Po Cheng et al. Int J Mol Sci. .

Abstract

Glioblastoma multiforme (GBM) is the most aggressive primary brain tumor with poor prognosis, largely due to resistance to current radiotherapy and Temozolomide chemotherapy. The constitutive activation of Signal Transducer and Activator of Transcription 3 (STAT3) is evidenced as a pivotal driver of GBM pathogenesis and therapy resistance, and hence, is a promising GBM drug target. 5-acetyloxy-6,7,8,4'-tetramethoxyflavone (5-AcTMF) is an acetylated derivative of Tangeretin which is known to exert anticancer effects on breast, colon, lung, and multiple myeloma; however, its effect on GBM remains elusive. Herein, we reported that 5-AcTMF suppressed the viability and clonogenicity along with inducing apoptosis in multiple human GBM cell lines. Mechanistic analyses further revealed that 5-AcTMF lowered the levels of Tyrosine 705-phosphorylated STAT3 (p-STAT3), a canonical marker of STAT3 activation, but also dampened p-STAT3 upregulation elicited by Interleukin-6. Notably, ectopic expression of dominant-active STAT3 impeded 5-AcTMF-induced suppression of viability and clonogenicity plus apoptosis induction in GBM cells, confirming the prerequisite of STAT3 blockage for the inhibitory action of 5-AcTMF on GBM cell survival and growth. Additionally, 5-AcTMF impaired the activation of STAT3 upstream kinase JAK2 but also downregulated antiapoptotic BCL-2 and BCL-xL in a STAT3-dependent manner. Moreover, the overexpression of either BCL-2 or BCL-xL abrogated 5-AcTMF-mediated viability reduction and apoptosis induction in GBM cells. Collectively, we, for the first time, revealed the anticancer effect of 5-AcTMF on GBM cells, which was executed via thwarting the JAK2-STAT3-BCL-2/BCL-xL signaling axis. Our findings further implicate the therapeutic potential of 5-AcTMF for GBM treatment.

Keywords: 5-acetyloxy-6,7,8,4′-tetramethoxyflavone; BCL-2; BCL-xL; STAT3; apoptosis; glioblastoma multiforme; polymethoxyflavone; tangeretin.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Inhibitory effect of 5-AcTMF on cell growth and survival of multiple human GBM cell lines. (A) Cytotoxic effect of 5-AcTMF on GBM cells. Human GBM cell lines GBM8401, U-87 MG, and T98G were treated with various concentrations (0, 25, 50, 100, and 200 μM) of 5-AcTMF for 48 h, followed by MTS assay for evaluation of cell viability. Data were represented as mean ± SD from three independent experiments. The statistic difference between 5-AcTMF-treated and drug-untreated cells were indicated (***: p < 0.001). (B) Colony formation capacity of 5-AcTMF-treated GBM cells. Colonies of GBM cell lines were revealed by crystal violet staining and then scored 10 days after 5-AcTMF treatment. Data were represented as mean ± SD from three independent experiments (**: p < 0.01; ***: p < 0.001). (C) Proapoptotic effect of 5-AcTMF on GBM cells. All GBM cell lines were subject to immunoblot analysis for the levels of cleaved PARP (c-PARP) following 24 h-treatment with 5-AcTMF. The levels of GAPDH were used as a loading control. The numbers beneath c-PARP blots indicate the relative density of each c-PARP immunoblot signal compared to that of 5-AcTMF-untreated control.
Figure 2
Figure 2
5-AcTMF suppresses constitutive and inducible activation of STAT3 in GBM cells. (A) 5-AcTMF lowers the levels of Tyrosine 705-phosphorylated STAT3 (p-STAT3) levels in GBM cell lines. GBM8401, U-87 MG, and T98G cells were treated with indicated concentrations of 5-AcTMF for 24 h, followed by immunoblotting for the levels of p-STAT3 and total STAT3. (B) Blockage of IL-6-induced STAT3 activation in GBM cells by 5-AcTMF. T98G cells were treated with 5-AcTMF for 24 h without or with stimulation by IL-6 (200 ng/mL) for 30 min. The levels of p-STAT3, total STAT3, BCL-2, and BCL-xL were examined by immunoblotting thereafter. (C) Inhibition of JAK2 activation by 5-AcTMF. T98G cells after 24 h-treatment with 5-AcTMF were subjected to immunoblotting for the levels of Tyrosine 1007/1008-phophorylated JAK2 (p-JAK2) and total JAK2. Either GAPDH or β-actin were used as the control for equal loading.
Figure 3
Figure 3
Essential role of STAT3 suppression in the anti-GBM effect of 5-AcTMF. (A) Constitutive STAT3 activation impaired 5-AcTMF-elicited apoptosis. T98G clones stably carrying an empty vector or the vector expressing an N-terminal HA-tagged dominant-active STAT3 mutant (HA-STAT3-CA) were treated with 5-AcTMF for 24 h, followed by immunoblotting for the levels of HA (to confirm the expression of HA-STAT3-CA) and cleaved PARP (c-PARP). α-tubulin was used as a loading control. (B) Blockage of STAT3 activation is required for the proapoptotic action of 5-AcTMF on GBM cells. T98G vector or STAT3-CA stable clones were treated with 5-AcTMF for 24 h and then subjected to Annexin V/PI dual staining analysis using flow cytometry. Annexin V-positive cell populations were scored as apoptotic cells and shown as the percentage of total cell population. Data were represented as mean ± SD. (C) Constitutive STAT3 activation protects GBM cells from 5-AcTMF-induced suppression of clonogenicity. T98G vector or STAT3-CA stable clones were treated with 5-AcTMF and then subjected to analysis for colony formation capacity. Colony numbers were scored and shown as the percentage of colonies formed in 5-AcTMF-untreated controls. Data represented as mean ± SD. ***: p < 0.001.
Figure 4
Figure 4
5-AcTMF downregulates antiapoptotic BCL-2 and BCL-xL through suppression of STAT3. (A) Constitutive STAT3 activation prevents 5-AcTMF-elicited downregulation of BCL-2 and BCL-xL. T98G vector or STAT3-CA stable clones were treated with indicated doses of 5-AcTMF for 24 h, and the levels of HA, BCL-2 and BCL-xL were determined by immunoblotting thereafter. α-tubulin was used as a loading control. (B) Blockage of proteasome-dependent degradation is not likely involved in 5-AcTMF-induced downregulation of BCL-2 and BCL-xL. T98G cells were treated with 100 μM of 5-AcTMF for 24 h without or with co-treatment of MG132 (20 μM) added 2 h before harvesting lysates. The levels of BCL-2 and BCL-xL in each treatment were determined using immunoblotting thereafter. β-actin served as the loading control.
Figure 5
Figure 5
Essential role of BCL-2 downregulation in the proapoptotic effect of 5-AcTMF on GBM cells. (A) BCL-2 ectopic expression prevents 5-AcTMF-elicited PARP cleavage in GBM cells. T98G vector or BCL-2 stable clones were treated with indicated doses of 5-AcTMF for 24 h, and then the levels of PARP cleavage and BCL-2 were revealed by immunoblotting. β-actin was used as a loading control. (B) Ectopic BCL-2 expression antagonizes the cytotoxic effect of 5-AcTMF on GBM cells. Cell viability of T98G vector or BCL-2 stable clones after 24 h-treatment with 5-AcTMF (0, 50, 100 μM) was measured by MTS assay. (C) BCL-2 overexpression represses the proapoptotic effect of 5-AcTMF on GBM cells. T98G vector or BCL-2 stable clones treated with indicated doses of 5-AcTMF for 24 h were subjected to flow cytometry analysis for the levels of Annexin V-positive (apoptotic) cell population. ***: p < 0.001.
Figure 6
Figure 6
Requirement of BCL-xL downregulation for the proapoptotic effect of 5-AcTMF on GBM cells. (A) BCL-xL ectopic expression abolished 5-AcTMF-induced PARP cleavage in GBM cells. T98G vector or HA-BCL-xL stable clones were treated with indicated doses of 5-AcTMF for 24 h, and then subjected to immunoblotting for the levels of HA, c-PARP and BCL-xL. β-actin was used as the control for equal loading. (B) Ectopic BCL-xL expression alleviates 5-AcTMF-mediated cytotoxicity to GBM cells. T98G vector or HA-BCL-xL stable clones were exposed to indicated doses of 5-AcTMF for 24 h, followed by cell viability evaluation using MTS assay. (C) BCL-xL overexpression mitigates 5-AcTMF’s proapoptotic action on GBM cells. T98G vector or BCL-2 stable clones treated with 5-AcTMF (0, 50, 100 μM) for 24 h were then subjected to flow cytometry analysis for the levels of Annexin V-positive (apoptotic) cell population. ***: p < 0.001.
Figure 7
Figure 7
Schematic model of the mechanism of action underlying 5-AcTMF-elicited anticancer effect on GBM cells established in this study. Briefly, 5-AcTMF thwarts GBM cell survival and clonogenicity via repressing STAT3 activation, likely through inhibition of STAT3 upstream kinase JAK2, to downregulate STAT3 transcriptional targets BCL-2 and BCL-xL, thereby leading to the induction of GBM cell apoptosis.
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