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, 116 (1), 164-76

Flavonoids Activated Caspases for Apoptosis in Human Glioblastoma T98G and U87MG Cells but Not in Human Normal Astrocytes

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Flavonoids Activated Caspases for Apoptosis in Human Glioblastoma T98G and U87MG Cells but Not in Human Normal Astrocytes

Arabinda Das et al. Cancer.

Abstract

Background: Human glioblastoma is a deadly brain cancer that continues to defy all current therapeutic strategies. The authors induced apoptosis in human glioblastoma T98G and U87MG cells after treatment with apigenin, (-)-epigallocatechin, (-)-epigallocatechin-3-gallate (EGCG), and genistein, which did not induce apoptosis in human normal astrocytes.

Methods: Induction of apoptosis was examined using Wright staining and ApopTag assay. Production of reactive oxygen species (ROS) and increase in intracellular free Ca(2+) were measured by fluorescent probes. Analysis of mRNA and Western blotting indicated increases in expression and activities of the stress kinases and cysteine proteases for apoptosis. JC-1 showed changes in mitochondrial membrane potential (DeltaPsi(m)), and use of specific inhibitors confirmed activation of kinases and proteases in apoptosis.

Results: Treatment of glioblastoma cells with apigenin, (-)-epigallocatechin, EGCG, or genistein triggered ROS production that induced apoptosis with phosphorylation of p38 mitogen-activated protein kinase (MAPK) and activation of the redox-sensitive c-Jun N-terminal kinase 1 pathway. Pretreatment of cells with ascorbic acid attenuated ROS production and p38 MAPK phosphorylation. Increases in intracellular free Ca2+ and activation of caspase-4 indicated involvement of endoplasmic reticulum stress in apoptosis. Other events in apoptosis included overexpression of Bax, loss of DeltaPsi(m), mitochondrial release of cytochrome c and Smac into the cytosol, down-regulation of baculoviral inhibitor-of-apoptosis repeat-containing proteins, and activation of calpain, caspase-9, and caspase-3. (-)-Epigallocatechin and EGCG also induced caspase-8 activity. Apigenin, (-)-epigallocatechin, EGCG, and genistein did not induce apoptosis in human normal astrocytes.

Conclusions: Results strongly suggest that flavonoids are potential therapeutic agents for induction of apoptosis in human glioblastoma cells.

Figures

FIGURE 1
FIGURE 1
Morphological and biochemical features of apoptosis in T98G and U87MG cells. Treatments (24 h): control (CON), 50 µM APG, 50 µM EGC, 50 µM EGCG, and 50 µM GST. (A) Wright staining. (B) ApopTag assay. Arrows indicate apoptotic cells. (C) Bar diagram to show percent apoptosis based on ApopTag assay.
FIGURE 2
FIGURE 2
Determination of ROS production and p38 MAPK phosphorylation in T98G and U87MG cells. Treatments (0, 30, 60, 90, 120, 150, 180, and 1440 min) in the presence of 5 µM 2,7-dichlorofluorescin diacetate (DCF-DA): control (CON), 50 µM APG, 50 µM EGC, 50 µM EGCG, and 50 µM GST. (A) ROS production in T98G cells. (B) ROS production in U87MG cells. Western blotting to show levels of phosphorylation of p38 MAPK (p-p38 MAPK), p38 MAPK, and β-actin in (C) T98G cells and (D) U87MG cells after the treatments (24 h): control (CON), 10 µM ascorbic acid (Asc), 50 µM APG, 10 µM Asc (1-h pretreatment) + 50 µM APG, 50 µM EGC, 10 µM Asc (1-h pretreatment) + 50 µM EGC, 50 µM EGCG, 10 µM Asc (1-h pretreatment) + 50 µM EGCG, 50 µM GST and 10 µM Asc (1-h pretreatment) + 50 µM GST. Pretreatment with Asc prevented formation of p-p38 MAPK.
FIGURE 3
FIGURE 3
Western blotting for the levels of p-JNK1, survival factors (p-Akt and p-Bcl-2), and inflammatory factors (NF-κB and COX-2) in T98G and U87MG cells. Treatments (24 h): control (CON), 50 µM APG, 50 µM EGC, 50 µM EGCG, and 50 µM GST. Protein levels of p-JNK1, p-Akt, total Akt, Bcl-2, NF-κB, COX-2, and β-actin.
FIGURE 4
FIGURE 4
Determination of caspase-8 activation and activity in T98G and U87MG cells. Treatments (24 h): control (CON), 50 µM APG, 50 µM EGC, 50 µM EGCG, and 50 µM GST. (A) Western blotting to show levels of caspase-8, β-actin, tBid, and COX4. (B) Colorimetric determination of caspase-8 activity.
FIGURE 5
FIGURE 5
Examination of components involved in mitochondrial pathway of apoptosis in T98G and U87MG cells. Treatments (24 h): control (CON), 50 µM APG, 50 µM EGC, 50 µM EGCG, and 50 µM GST. (A) Determination of mRNA levels of bax and bcl-2 by qualitative RT-PCR as well as real-time quantitative RT-PCR. (B) Western blotting for examination of Bax and Bcl-2 expression at protein levels and determination of Bax:Bcl-2 ratio based on Western blotting. JC-1 ratio (590 nm/530 nm) in (C) T98G and (D) U87MG cells after the treatments for different times (30, 60, 120, 180, 240, 300, 360, 420, 480, 540, 600, 660, and 1440 min). (E) Western blotting to show levels of cytochrome c, Smac, COX4, caspase-9, and β-actin. (F) Determination of caspase-9 activity using a colorimetric assay. (G) Qualitative RT-PCR and (H) Western blotting to show mRNA and protein levels of BIRC-2 to BIRC-5 and β-actin.
FIGURE 6
FIGURE 6
Examination of increase in intracellular free [Ca2+] and activation of caspase-4, calpain, and caspase-3 in T98G and U87MG cells. Treatments (24 h): control (CON), 50 µM APG, 50 µM EGC, 50 µM EGCG, and 50 µM GST. (A) Determination of intracellular free [Ca2+]. (B) Western blotting to show levels of caspase-4, inactive and active calpain, calpastatin, spectrin breakdown product (SBDP), inactive and active caspase-3, ICAD, CAD, and β-actin. (C) Determination of caspase-3 activity by a colorimetric assay.
FIGURE 7
FIGURE 7
Pretreatment with selective inhibitors prevented flavonoid mediated cell death in T98G and U87MG cells. Treatments (24 h): control (CON), 50 µM APG, 50 µM EGC, 50 µM EGCG, and 50 µM GST without any inhibitor and with 1-h pretreatment with 5 µM Asc, 10 µM caspase-8 inhibitor II, 10 µM caspase-9 inhibitor I, 10 µM calpeptin (calpain-specific inhibitor), or 10 µM caspase-3 inhibitor IV. Determination of percent viability in (A) T98G cells and (B) U87MG cells.
FIGURE 8
FIGURE 8
Effects of flavonoids on human normal astrocytes (HNA). Treatments (24 h): control (CON), 50 µM APG, 50 µM EGC, 50 µM EGCG, and 50 µM GST. (A) Determination of percent cell viability by trypan blue dye exclusion test. (B) Determination of ROS production in HNA at different time intervals (0, 30, 60, 90, 120, 150, 180, and 1440 min). (C) Colorimetric assays for determination of the caspase-8, caspase-9, and caspase-3 activities.

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