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Isorhapontigenin (ISO) inhibits stem cell-like properties and invasion of bladder cancer cell by attenuating CD44 expression


Cancer stem cells (CSC) are highly associated with poor prognosis in cancer patients. Our previous studies report that isorhapontigenin (ISO) down-regulates SOX2-mediated cyclin D1 induction and stem-like cell properties in glioma stem-like cells. The present study revealed that ISO could inhibit stem cell-like phenotypes and invasivity of human bladder cancer (BC) by specific attenuation of expression of CD44 but not SOX-2, at both the protein transcription and degradation levels. On one hand, ISO inhibited cd44 mRNA expression through decreases in Sp1 direct binding to its promoter region-binding site, resulting in attenuation of its transcription. On the other hand, ISO also down-regulated USP28 expression, which in turn reduced CD44 protein stability. Further studies showed that ISO treatment induced miR-4295, which specific bound to 3′-UTR activity of usp28 mRNA and inhibited its translation and expression, while miR-4295 induction was mediated by increased Dicer protein to enhance miR-4295 maturation upon ISO treatment. Our results provide the first evidence that ISO has a profound inhibitory effect on human BC stem cell-like phenotypes and invasivity through the mechanisms distinct from those previously noted in glioma stem-like cells.

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Actinomycin D


Alkaline phosphatase


Bafilomycin A1


Bladder cancer


Chromatin immunoprecipitation




Cancer stem cells


Dimethyl sulfoxide


Deubiquitinating enzymes


Fetal bovine serum


Immunoglobulin G




Muscle-invasive bladder cancer




Non-coding RNA


Non-muscle-invasive bladder cancer


Reverse transcription-polymerase chain reaction


Wide type


3′-Untranslated regions


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This work was partially supported by Grants from NIH/NCI CA177665, CA217923, CA229234, CA165980, and NIH/NIEHS ES000260.

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Fig S1.

T24T(vector) and T24T(CD44) cells were treated with ISO or DMSO at indicated concentrations for 7 days to determine impact on sphere formation ability (JPEG 650 kb)

Fig S2.

(A) T24T cells were treated with Bafomycin A1 (5 nM) or ISO (20 μM) alone or Bafomycin A1+ ISO, which tested whether CD44 protein accumulated by Bafomycin A1. (B) Cell sphere formation by cells transfected with Flag-USP28 or vector. Transfectants were exposed to 20 μM ISO for 7 days before analyses using microscopy (JPEG 778 kb)

Fig S3.

(A) Schematic representation of transcription factor binding sites in human CD44 promoter region from -1133 to -1. (B & C) FOXO1 or c-MYC did not affect CD44 protein expression. (D) T24T(vector) and T24T(GFP-Sp1) cells were treated with/without ISO (20 μM) to determine sphere formation ability. (E) T24T(nonsense) and T24T(shSp1) cells were used in sphere formation assay as described in Methods (JPEG 900 kb)

Fig S4.

(A) usp28 mRNA expression levels in T24T cells were determined by RT-PCR after cells were treated with 20 μM ISO for indicated time periods. (B) T24T cells were treated with/without 20 μM ISO in the presence of CHX for indicated time periods, then underwent extraction; extracts were then analyzed by Western blot to assess USP28 protein expression. (C) Schematic representation of potential miRNA-binding sites in USP28 3’UTR were analyzed with TargetScan software. (D) Schematic sequence of intact miR-4295-binding site in wide-type USP28 3’UTR and its mutation of USP28 3’UTR luciferase reporter (JPEG 912 kb)

Fig S5.

Invasivity of T24T(vector) and T24T(miR-4295 inhibitor) cells treated with or without 20 µM ISO for 24 hours was evaluated using a BD BioCoat TM Matrigel TM Invasion Chamber. After incubation for 24 hours, cells were fixed and stained and took pictures as described in Methods (JPEG 838 kb)

Fig S6.

(A) T24T(miR-4295 promoter) stable transfectants were treated with 20 μM ISO for indicated time periods; thereafter, miR-4295 promoter luciferase activity was measured (via Dual-Luciferase Reporter Assay System). (B) T24T cells treated with 20 μM ISO or vehicle control - both along with ActD - for the time periods indicated. Total RNA was isolated and subjected to qRT-PCR evaluation of miR-4295 expression levels. (C) Cell sphere formation abilities of T24T(vector) and T24T(shDicer) cells treated with or without 20 µM ISO were determined as described in Methods (JPEG 811 kb)

Fig S7.

(A) Kaplan-Meier estimation about has-mir-4295 of overall survival (OS) in bladder cancer (BC) patients from the database. Overall survival (OS) curves showing that patients with high miR-4295 expression (n=564) was related with better OS, compared with lower expression of miR-4295 (n=186). (B) Kaplan-Meier estimation about USP28 of overall survival (OS) in bladder cancer (BC) patients from the database. Overall survival (OS) curves showing that patients with high USP28 expression (n=294) were positively associated with decreased survival of BC patients, lower expression of miR-4295 (n=191) was associated with longer survival (JPEG 833 kb)

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Supplementary material 13 (JPEG 986 kb)

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Luo, Y., Tian, Z., Hua, X. et al. Isorhapontigenin (ISO) inhibits stem cell-like properties and invasion of bladder cancer cell by attenuating CD44 expression. Cell. Mol. Life Sci. 77, 351–363 (2020).

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  • Isorhapontigenin
  • Bladder cancer
  • Stem cell-like properties
  • CD44
  • USP28