Benzidine promotes the stemness of bladder cancer stem cells via activation of the Sonic hedgehog pathway

Abstract

Substantial evidence suggests that cancer stem cells (CSCs) are the main cause of the initiation, progression and recurrence of tumors. Benzidine has been identified as a risk factor for bladder cancer. The aim of the present study was to investigate the effects of benzidine on bladder CSCs (BCSCs) and the possible mechanism underlying its action. The bladder cancer cell lines UM-UC-3 and EJ were maintained in serum-free medium and cells forming three-dimensional spheres were characterized as BCSCs. The sphere-forming cells were exposed to different concentrations of benzidine and vismodegib, and western blotting was performed to evaluate the expression of markers associated with CSCs and the Sonic hedgehog (SHH) signaling pathway. Flow cytometry was used to detect the distribution of cells in different phases of the cell cycle, and immunofluorescence staining was used to detect the protein expression of CD44. The results revealed that the levels of BCSC markers, namely CD133, CD44, aldehyde dehydrogenase 1-A1, Nanog and octamer-binding transcription factor-4, in the cell spheres were markedly elevated compared with those in cells cultured in serum-supplemented medium. Furthermore, benzidine increased the expression of BCSC markers and promoted the sphere-forming ability of the cells. In addition, it was observed that benzidine activated the SHH pathway, while inhibition of the Shh pathway using vismodegib diminished the promoting effects of benzidine on BCSCs. The findings of the present study indicate that benzidine promoted the stemness of BCSCs via activation of the SHH pathway, which may support further exploration of the molecular basis of the association between benzidine exposure and bladder oncogenesis.

Benzidine Induces Proliferation of UMUC3 Bladder Cancer Cells Through Activation of ERK5 Pathway

“…Substantial epidemiological researches concerning the association between benzidine and BC have been completed since 1950s [15]. In vitro and in vivo researches have assessed the genetic expression variations related to acute benzidine exposure [12,[16][17][18][19][20][21][22]. Previously, we reported that benzidine treatment could result in increased cellular proliferation, EMT alteration and MAPK activation in SV-HUC-1 and T24 cells [12,[17][18][19][20][21].…”

“…Previously, we reported that benzidine treatment could result in increased cellular proliferation, EMT alteration and MAPK activation in SV-HUC-1 and T24 cells [12,[17][18][19][20][21]. In addition, the enhancement of the stemness of BC stem cells was discovered after benzidine treatment [22]. Nonetheless, the causal links by which benzidine induced the developmental process of BC are still elusive.…”

“…Certain proofs have revealed that ERK5-mediated signal transduction facilitates the development of many tumors like gastric [40], bladder [12, 23], colon [41], and lung cancers [42]. ERK5 can be stimulated by receptor tyrosine kinases and other stimulating substances like benzidine [12, 17–22]. Mechanistic studies indicated that phosphorylated ERK5 transfered from the cytoplasm to the nucleus, and then regulated the activity of transcriptional factors to modulate cell proliferation [13].…”

“…The abnormal expression of ERK5 was detected in oncocytes, like stomach carcinoma MGC-803 cells, and it induced an enhanced ability of cell proliferation and colony formation, therefore contributing to increased cell growth [40]. Moreover, certain researches revealed that ERK5 regulated benzidine-mediated urocystic EMT and that ERK5 inhibition reversed EMT alterations [12, 22]. In a mouse model, fibroblast-specific ERK5 deficiency enhanced tumor vessel formation and increased the number of activated fibroblast, and it exacerbated tumor progression [41].…”

“…ERK5 can be stimulated by receptor tyrosine kinases and other stimulating substances like benzidine [12,[17][18][19][20][21][22]. Mechanistic studies indicated that phosphorylated ERK5 transfered from the cytoplasm to the nucleus, and then regulated the activity of transcriptional factors to modulate cell proliferation [13].…”