Retinoids induce differentiation and downregulate telomerase activity and N-Myc to increase sensitivity to flavonoids for apoptosis in human malignant neuroblastoma SH-SY5Y cells

Abstract

Human malignant neuroblastoma is characterized by poor differentiation and uncontrolled proliferation of immature neuroblasts. Retinoids such as all-trans-retinoic acid (ATRA), 13-cis-retinoic acid (13-CRA), and N-(4-hydroxyphenyl) retinamide (4-HPR) at low doses are capable of inducing differentiation, while flavonoids such as (-)-epigallocatechin-3-gallate (EGCG) and genistein (GST) at relatively high dose can induce apoptosis. We used combination of retinoid and flavonoid for controlling growth of malignant neuroblastoma SH-SY5Y cells. Cells were treated with a retinoid (1 microM ATRA, 1 microM 13-CRA, or 0.5 microM 4-HPR) for 7 days and then with a flavonoid (25 microM EGCG or 25 microM GST) for 24 h. Treatment of cells with a low dose of a retinoid for 7 days induced neuronal differentiation with downregulation of telomerase activity and N-Myc but overexpression of neurofilament protein (NFP) and subsequent treatment with a relatively high dose of a flavonoid for 24 h increased apoptosis in the differentiated cells. Besides, retinoids reduced the levels of inflammatory and angiogenic factors. Apoptosis was associated with increases in intracellular free [Ca2+], Bax expression, cytochrome c release from mitochondria and activities of calpain and caspases. Decreases in expression of calpastatin (endogenous calpain inhibitor) and baculovirus inhibitor-of-apoptosis repeat containing (BIRC) proteins (endogenous caspase inhibitors) favored apoptosis. Treatment of SH-SY5Y cells with EGCG activated caspase-8, indicating induction of the receptor-mediated pathway of apoptosis. Based on our observation, we conclude that combination of a retinoid and a flavonoid worked synergistically for controlling the malignant growth of human neuroblastoma cells.

Figure 1

Morphological and biochemical features of neuronal differentiation in SH-SY5Y cells following treatment with retinoids. Treatment with retinoid (7 days): control (CTL), 1 μM ATRA, 1 μM 13-CRA and 0.5 μM 4-HPR. (a) Methylene blue staining to identify morphological features of neuronal differentiation. (b) Application of TRAP assay to determine any decrease in the telomerase activity. (c) Western blotting to examine levels of hTERT, N-Myc, NFP and ß-actin.

Figure 2

Morphological and biochemical features of apoptosis in SH-SY5Y cells. Treatment with retinoid (7 days) and flavonoid (24 h): CTL, 25 μM EGCG, 25 μM GST, 1 μM ATRA, 1 μM ATRA + 25 μM EGCG, 1 μM ATRA + 25 μM GST, 1 μM 13-CRA, 1 μM 13-CRA + 25 μM EGCG, 1 μM 13-CRA + 25 μM GST, 0.5 μM 4-HPR, 0.5 μM 4-HPR + 25 μM EGCG and 0.5 μM 4-HPR + 25 μM GST. (a) Wright staining for morphological features of apoptosis. (b) ApopTag assay for labeling of DNA fragmentation in apoptotic cells. (c) Bar diagram to show the percentage of apoptosis. Significant difference between control (CTL) and EGCG is indicated by #. Significant difference between CTL and GST is indicated by *.

Figure 3

Western blotting to examine the levels of inflammatory and angiogenic factors in SH-SY5Y cells. Treatment with retinoid (7 days) and flavonoid (24 h): CTL, 25 μM EGCG, 25 μM GST, 1 μM ATRA, 1 μM ATRA + 25 μM EGCG, 1 μM ATRA + 25 μM GST, 1 μM 13-CRA, 1 μM 13-CRA + 25 μM EGCG, 1 μM 13-CRA + 25 μM GST, 0.5 μM 4-HPR, 0.5 μM 4-HPR + 25 μM EGCG and 0.5 μM 4-HPR + 25 μM GST. Representative Western blots to show levels of NF-κB, VEGF and ß-actin.

Figure 4

Determination of caspase-8 activation and activity in SH-SY5Y cells. Treatment with retinoid (7 days) and flavonoid (24 h): CTL, 25 μM EGCG, 25 μM GST, 1 μM ATRA, 1 μM ATRA + 25 μM EGCG, 1 μM ATRA + 25 μM GST, 1 μM 13-CRA, 1 μM 13-CRA + 25 μM EGCG, 1 μM 13-CRA + 25 μM GST, 0.5 μM 4-HPR, 0.5 μM 4-HPR + 25 μM EGCG and 0.5 μM 4-HPR + 25 μM GST. (a) Representative Western blots to show levels of caspase-8 and ß-actin. (b) Determination of caspase-8 activity by colorimetric assay.

Figure 5

Examination of mitochondrial pathway of apoptosis in SH-SY5Y cells. Treatment with retinoid (7 days) and flavonoid (24 h): CTL, 25 μM EGCG, 25 μM GST, 1 μM ATRA, 1 μM ATRA + 25 μM EGCG, 1 μM ATRA + 25 μM GST, 1 μM 13-CRA, 1 μM 13-CRA + 25 μM EGCG, 1 μM 13-CRA + 25 μM GST, 0.5 μM 4-HPR, 0.5 μM 4-HPR + 25 μM EGCG and 0.5 μM 4-HPR + 25 μM GST. (a) The representative gel pictures with RT-PCR products to show the mRNA levels of baxα, bcl-2α and ß-actin. (b) Representative Western blots to show levels of Bax, Bcl-2, cytochrome c, COX4, caspase-9 and ß-actin. (c) Determination of caspase-9 activity using a colorimetric assay. (d) Representative gel pictures with RT-PCR products to show the mRNA levels of BIRC-2 to BIRC-8 and ß-actin.

Figure 6

Determination of intracellular free [Ca²⁺] and activities of calpain and caspase-3 in SH-SY5Y cells. Treatment with retinoid (7 days) and flavonoid (24 h): CTL, 25 μM EGCG, 25 μM GST, 1 μM ATRA, 1 μM ATRA + 25 μM EGCG, 1 μM ATRA + 25 μM GST, 1 μM 13-CRA, 1 μM 13-CRA + 25 μM EGCG, 1 μM 13-CRA + 25 μM GST, 0.5 μM 4-HPR, 0.5 μM 4-HPR + 25 μM EGCG and 0.5 μM 4-HPR + 25 μM GST. (a) Determination of percentage of increase in intracellular-free [Ca²⁺]. (b) Representative gel pictures with RT-PCR products to show the mRNA levels of m-calpain, calpastain and ß-actin. (c) Representative Western blots to show the levels of m-calpain, calpastatin, SBDP and ß-actin. (d) Determination of caspase-3 activity using a colorimetric assay.