Qiu PC et al. (2020), Reduction of SCUBE3 by a new marine-derived ast...

ECB-ART-49691

Oncogenesis 2020 Aug 06;98:71. doi: 10.1038/s41389-020-00252-4.

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Reduction of SCUBE3 by a new marine-derived asterosaponin leads to arrest of glioma cells in G1/S.

Qiu PC , Lu YY , Zhang S , Li H , Bao H , Ji YQ , Fang F , Tang HF , Cheng G .

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Many saponins are characterized as exhibiting a wide spectrum of antitumor activities at low concentrations. Most of the previous studies that aimed to understand the mechanisms underlying anticancer saponins have focused on numerous classical signaling pathways. However, at the oncogene level, little is known about the action of saponins, especially asterosaponin. In this study, CN-3, a new asterosaponin isolated from the starfish Culcita novaeguineae, decreased the proliferation of U87 and U251 cells at low doses in a dose- and time-dependent manner. Microarray analysis revealed CN-3 significantly induced the differential expression of 661 genes that are related to its antiglioma effect in U251. Nine downregulated genes (SCUBE3, PSD4, PGM2L1, ACSL3, PRICKLE1, ABI3BP, STON1, EDIL3, and KCTD12) were selected, for further verification of their low expression. Then, shRNA transfection and high-content screening were performed and significantly decreased U251 cell proliferation rate was only observed for the SCUBE3 knockdown. qPCR confirmed SCUBE3 was highly expressed in U251 and U87 cells, and had medium expression levels in U373 cells. Real-time cellular analysis using iCELLigence demonstrated that SCUBE3 is an oncogene in U251 and U87 cells, with knockdown of SCUBE3 inhibiting U251 and U87 cell proliferation while, conversely, SCUBE3 overexpression promoted their proliferation. Afterward, SCUBE3 protein was found to have high expression in primary glioma specimens from patients examined by immunohistochemistry but low expression in normal brain. PathScan ELISA analysis in conjunction with TEM observation demonstrated that the effect of SCUBE3 knockdown in U251 does not appear to be related to the induction of apoptosis. Employing CCK-8, iCELLigence, flow cytometry, western blotting, and shRNA transfection (knockdown and overexpression) experiments, we reveal that the reduction of SCUBE3 expression, induced by CN-3, mediated both inhibition and G1/S arrest of U251 via the Akt/p-Akt/p53/p21/p27/E2F1 pathway.

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	Fig. 1. A low concentration of CN-3 inhibited glioma cell proliferation.a The 1H–1H COSY and key HMBC correlations of the asterosaponin CN-3; b IC50 value of 1.59 μM CN-3 for U251 cells (n = 3, 48 h); c IC50 value of 1.418 μM CN-3 for U87 cells (n = 3, 48 h); d IC40 (1.42 μM) was used as a low dose and IC70 (2.47 μM) was used as a high dose of CN-3 for treating U251 cells (n = 3, p < 0.05 compared with normal U251 cells); e IC40 (1.34 μM) was used as a low dose and IC70 (2.05 μM) was used as a high dose of CN-3 for treating U87 cells (n = 3, p < 0.05 compared with normal U87 cells).
	Fig. 2. Microarray and qPCR indicate CN-3 suppressed SCUBE3 expression.a Between normal U251 cells (n = 2) and CN-3-treated U251 cells (n = 4), 452 genes were upregulated, and 209 genes were downregulated (GSE108343); b qPCR confirmed significant downregulation of nine genes (SCUBE3, PSD4, PGM2L1, ACSL3, PRICKLE1, ABI3BP, STON1, EDIL3, and KCTD12) in U251 cells treated with 1.42 μM CN-3 for 48 h (n = 3, p < 0.05, p < 0.01, and **p < 0.001 compared with normal U251 cells).
	Fig. 3. SCUBE3 silence inhibited U251 cell proliferation.a Effects of lentivirus-mediated silencing of nine genes in U251 cells as examined using fluorescence microscopy. The representative pictures shown are from one of three independent experiments; b 5 days of continuous counting of the cell numbers by HCS (n = 3); c 5 days of continuous counting of the cell numbers fold change by HCS (n = 3, p < 0.05 compared to blank-shctrl U251); d 5 days of continuous CCK-8 assays (n = 3, p < 0.05 compared with blank-shctrl U251); e 5 days of continuous CCK-8 assays, fold change (n = 3, p < 0.05 compared with blank-shctrl U251); f the efficiency of knockdown SCUBE3 by shRNA was verified in U251 cells by western blot at 96 h (n = 3; “+” represents positive, and “−” represents negative compared with blank-shctrl U251); g the efficiency of knockdown SCUBE3 by shRNA was verified in U251 cells by qPCR at 96 h (n = 3, **p < 0.001 compared with blank-shctrl U251).
	Fig. 4. SCUBE3 is an oncogene in U251 and U87 cells.a Data in the Human Protein Atlas (https://www.proteinatlas.org/) showed SCUBE3 was enhanced in some cell lines; b SCUBE3 was highly expressed in U251 and U87 cells and showed moderate expression in U373 cells (∆Ct = SCUBE3 − GAPDH, if ∆Ct ≤ 12, then SCUBE3 was considered highly expressed; if 12 < ∆Ct < 16, then SCUBE3 was considered to show moderate expression; if ΔCt ≥ 16, then SCUBE3 was considered to show low expression, n = 3, p < 0.05 and p < 0.01 compared with ΔCt in U251); c iCELLigence showed knockdown of SCUBE3 inhibited U87 cell proliferation, and overexpression of SCUBE3 promoted U87 cell proliferation (n = 3, p < 0.05 compared with blank-shctrl-U87); d iCELLigence showed knockdown of SCUBE3 inhibited U251 cell proliferation (n = 3, p < 0.05 compared with blank-shctrl-U251); e iCELLigence showed overexpression of SCUBE3 promoted U251 cell proliferation and rescued the inhibition of U251 induced by CN-3 (n = 3, p < 0.05 compared with blank-shctrl-U251).
	Fig. 5. SCUBE3 knockdown may not induce caspase-dependent apoptosis in U251 cells.a Golgi swelling and endoplasmic reticulum swelling were observed in SCUBE3-knockdown U251 (pointed by arrows) under TEM; b a PathScan Stress and Apoptosis Signaling Antibody Array Kit was used for overall detection of 18 signaling molecules in down-shSCUBE3-transfected U251 cells. The expression level was calculated relative to the positive control signal as a gray value (n = 3, p < 0.05, *p < 0.01 compared with blank-shctrl group).
	Fig. 6. SCUBE3 knockdown arrested G1/S transition for CN-3 in U251 cells.a 1.42 μM CN-3 arrested G1/S transition in U251 cells examined by flow cytometry (n = 3, *p < 0.001 compared with normal U251); b knockdown and overexpression of SCUBE3 revealed SCUBE3-mediated G1/S transition in U251 cells, and SCUBE3 overexpression rescued the arrest induced by CN-3 or SCUBE3 silencing (n = 3, p < 0.01 and ***p < 0.001 compared with blank-shctrl group); c in U251 cells, western blot results showed knockdown of SCUBE3 increased p53, p27, and p21 and decreased Akt, p-Akt, and E2F1. Remarkably, SCUBE3 knockdown and 1.42 μM CN-3 decreased p-Akt in U251 cells, while overexpression of SCUBE3 abolished this decrease (n = 3; “+” represents positive, and “−” represents negative).
	Fig. 7. SCUBE3 protein was low expression in normal brain but enhanced in tumor specimens from glioma patients.a Kaplan–Meier survival curves (http://kmplot.com/analysis/) revealed when SCUBE3 was prognostic and favorable or unfavorable for the high and low expression group in pan-cancer analysis; b the Human Protein Atlas (https://www.proteinatlas.org/) was used to generate an anatomogram of SCUBE3 expression in human tissue, which pointed out that SCUBE3 was not highly expressed in brain; c immunohistochemical staining of SCUBE3 protein expression (brown area) in the normal brain and tumor specimens from glioma patients (n = 3, magnification 10 × 40).
	Fig. 8. A schematic diagram of the molecular mechanism by which CN-3 arrests U251 cells in G1/S.

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	Fig. 1. A low concentration of CN-3 inhibited glioma cell proliferation.a The 1H–1H COSY and key HMBC correlations of the asterosaponin CN-3; b IC50 value of 1.59 μM CN-3 for U251 cells (n = 3, 48 h); c IC50 value of 1.418 μM CN-3 for U87 cells (n = 3, 48 h); d IC40 (1.42 μM) was used as a low dose and IC70 (2.47 μM) was used as a high dose of CN-3 for treating U251 cells (n = 3, p < 0.05 compared with normal U251 cells); e IC40 (1.34 μM) was used as a low dose and IC70 (2.05 μM) was used as a high dose of CN-3 for treating U87 cells (n = 3, p < 0.05 compared with normal U87 cells).
	Fig. 2. Microarray and qPCR indicate CN-3 suppressed SCUBE3 expression.a Between normal U251 cells (n = 2) and CN-3-treated U251 cells (n = 4), 452 genes were upregulated, and 209 genes were downregulated (GSE108343); b qPCR confirmed significant downregulation of nine genes (SCUBE3, PSD4, PGM2L1, ACSL3, PRICKLE1, ABI3BP, STON1, EDIL3, and KCTD12) in U251 cells treated with 1.42 μM CN-3 for 48 h (n = 3, p < 0.05, p < 0.01, and **p < 0.001 compared with normal U251 cells).
	Fig. 3. SCUBE3 silence inhibited U251 cell proliferation.a Effects of lentivirus-mediated silencing of nine genes in U251 cells as examined using fluorescence microscopy. The representative pictures shown are from one of three independent experiments; b 5 days of continuous counting of the cell numbers by HCS (n = 3); c 5 days of continuous counting of the cell numbers fold change by HCS (n = 3, p < 0.05 compared to blank-shctrl U251); d 5 days of continuous CCK-8 assays (n = 3, p < 0.05 compared with blank-shctrl U251); e 5 days of continuous CCK-8 assays, fold change (n = 3, p < 0.05 compared with blank-shctrl U251); f the efficiency of knockdown SCUBE3 by shRNA was verified in U251 cells by western blot at 96 h (n = 3; “+” represents positive, and “−” represents negative compared with blank-shctrl U251); g the efficiency of knockdown SCUBE3 by shRNA was verified in U251 cells by qPCR at 96 h (n = 3, **p < 0.001 compared with blank-shctrl U251).
	Fig. 4. SCUBE3 is an oncogene in U251 and U87 cells.a Data in the Human Protein Atlas (https://www.proteinatlas.org/) showed SCUBE3 was enhanced in some cell lines; b SCUBE3 was highly expressed in U251 and U87 cells and showed moderate expression in U373 cells (∆Ct = SCUBE3 − GAPDH, if ∆Ct ≤ 12, then SCUBE3 was considered highly expressed; if 12 < ∆Ct < 16, then SCUBE3 was considered to show moderate expression; if ΔCt ≥ 16, then SCUBE3 was considered to show low expression, n = 3, p < 0.05 and p < 0.01 compared with ΔCt in U251); c iCELLigence showed knockdown of SCUBE3 inhibited U87 cell proliferation, and overexpression of SCUBE3 promoted U87 cell proliferation (n = 3, p < 0.05 compared with blank-shctrl-U87); d iCELLigence showed knockdown of SCUBE3 inhibited U251 cell proliferation (n = 3, p < 0.05 compared with blank-shctrl-U251); e iCELLigence showed overexpression of SCUBE3 promoted U251 cell proliferation and rescued the inhibition of U251 induced by CN-3 (n = 3, p < 0.05 compared with blank-shctrl-U251).
	Fig. 5. SCUBE3 knockdown may not induce caspase-dependent apoptosis in U251 cells.a Golgi swelling and endoplasmic reticulum swelling were observed in SCUBE3-knockdown U251 (pointed by arrows) under TEM; b a PathScan Stress and Apoptosis Signaling Antibody Array Kit was used for overall detection of 18 signaling molecules in down-shSCUBE3-transfected U251 cells. The expression level was calculated relative to the positive control signal as a gray value (n = 3, p < 0.05, *p < 0.01 compared with blank-shctrl group).
	Fig. 6. SCUBE3 knockdown arrested G1/S transition for CN-3 in U251 cells.a 1.42 μM CN-3 arrested G1/S transition in U251 cells examined by flow cytometry (n = 3, *p < 0.001 compared with normal U251); b knockdown and overexpression of SCUBE3 revealed SCUBE3-mediated G1/S transition in U251 cells, and SCUBE3 overexpression rescued the arrest induced by CN-3 or SCUBE3 silencing (n = 3, p < 0.01 and ***p < 0.001 compared with blank-shctrl group); c in U251 cells, western blot results showed knockdown of SCUBE3 increased p53, p27, and p21 and decreased Akt, p-Akt, and E2F1. Remarkably, SCUBE3 knockdown and 1.42 μM CN-3 decreased p-Akt in U251 cells, while overexpression of SCUBE3 abolished this decrease (n = 3; “+” represents positive, and “−” represents negative).
	Fig. 7. SCUBE3 protein was low expression in normal brain but enhanced in tumor specimens from glioma patients.a Kaplan–Meier survival curves (http://kmplot.com/analysis/) revealed when SCUBE3 was prognostic and favorable or unfavorable for the high and low expression group in pan-cancer analysis; b the Human Protein Atlas (https://www.proteinatlas.org/) was used to generate an anatomogram of SCUBE3 expression in human tissue, which pointed out that SCUBE3 was not highly expressed in brain; c immunohistochemical staining of SCUBE3 protein expression (brown area) in the normal brain and tumor specimens from glioma patients (n = 3, magnification 10 × 40).
	Fig. 8. A schematic diagram of the molecular mechanism by which CN-3 arrests U251 cells in G1/S.