Hispolon Methyl Ether, a Hispolon Analog, Suppresses the SRC/STAT3/Survivin Signaling Axis to Induce Cytotoxicity in Human Urinary Bladder Transitional Carcinoma Cell Lines

Abstract

Bladder cancer is a leading human malignancy worldwide. Signal transducer and activator of transcription (STAT) 3 is an oncogenic transcription factor commonly hyperactivated in most human cancers, including bladder cancer. Notably, preclinical evidence has validated STAT3 blockade as a promising therapeutic strategy for bladder cancer. Hispolon Methyl Ether (HME) is a structural analog of hispolon, an anticancer component of the medicinal mushroom Phellinus linteus. Thus far, HME’s anticancer activity and mechanisms remain largely unknown. We herein report HME was cytotoxic, more potent than cisplatin, and proapoptotic to various human bladder transitional carcinoma cell lines. Of note, HME blocked STAT3 activation, evidenced by HME-elicited reduction in tyrosine 705-phosphorylated STAT3 levels constitutively expressed or induced by interleukin-6. Significantly, HME-induced cytotoxicity was abrogated in cells expressing a dominant-active STAT3 mutant (STAT3-C), confirming STAT3 blockage as a pivotal mechanism of HME’s cytotoxic action. We further revealed that survivin was downregulated by HME, while its levels were rescued in STAT3-C-expressing cells. Moreover, survivin overexpression abolished HME-induced cytotoxicity, illustrating survivin as a central downstream mediator of STAT3 targeted by HME. Lastly, HME was shown to lower tyrosine 416-phosphorylated SRC levels, suggesting that HME inhibits STAT3 by repressing the activation of SRC, a STAT3 upstream kinase. In conclusion, we present the first evidence of HME’s anti-bladder cancer effect, likely proceeding by evoking apoptosis through suppression of the antiapoptotic SRC/STAT3/survivin signaling axis.

Blockade of the SRC/STAT3/BCL-2 Signaling Axis Sustains the Cytotoxicity in Human Colorectal Cancer Cell Lines Induced by Dehydroxyhispolon Methyl Ether

“…The pBabe-based vectors for ectopic expression of N-terminal hemagglutinin epitope (HA)-tagged dominant-active STAT3 mutant (STAT3-C), BCL-2 , and N-terminal HA-tagged v-src were respectively coined as pBabe-HA-STAT3-C, pBabe-BCL-2, and pBabe-HA-v-src and have been described previously [ 32 , 33 , 34 ]. To generate pBabe-derived retroviral particles, 2.5 μg of the pBabe-based plasmids were transfected into 293-T cells (70–80% confluency) along with the plasmids expressing gag-pol (2.5 μg) and VSVG proteins (0.5 μg) to assemble retroviral particles.…”

“…Immunoblotting was executed based on our reported protocol [ 33 , 34 ]. Primary antibodies against cleaved PARP (#9541), HA-tag (#3724), phospho-STAT3 (Y705) (#9145), phospho-JAK2 (Y1007/1008) (#3776), JAK2 (#3230), phospho-Src (Y416) (#6743), and Src (#2108) were purchased from Cell Signaling Technology (Boston, MA, USA).…”

Methoxyhispolon Methyl Ether, a Hispolon Analog, Thwarts the SRC/STAT3/BCL-2 Axis to Provoke Human Triple-Negative Breast Cancer Cell Apoptosis In Vitro

“…TNBC cells (7 × 10 3 cells/well) seeded in 96-well culture plates were subject to treatment with MHME (0, 6.25, 12.5, 25, 50 µM) for 24 h and 48 h, followed by assessment of cell viability using CellTiter 96 ® AQueous One Solution Cell Proliferation Assay (MTS) assay (Promega; Madison, WI, USA) as previously described [25][26][27][28] (Figure S1).…”

“…TNBC cells (4 × 10 5 cells) were treated with MHME (0, 25, 50 µM) for 24 h, followed by seeding 2 × 10 2 of MHME-treated cells onto 6-well plates to grow into colonies in drug-free media for 10~14 days. The TNBC colonies were revealed by 1% crystal violet staining, and then the numbers of colonies were scored as stated previously [25][26][27][28].…”

“…Owing to Hispolon's promising anti-cancer activity on varied human cancers, numerous Hispolon analogs generated by chemical modifications of Hispolon's aromatic ring have been investigated for their potential anti-cancer effects and possible molecular targets, such as histone deacetylase and NF-κB, using in silico approaches [21][22][23][24]. Along this line, our laboratory has reported the first evidence about the in vitro anti-bladder cancer effect of Hispolon methyl ether (HME) [25] and the in vitro anti-colorectal cancer activity of Dehydroxyhispolon methyl ether (DHME), along with the underlying mechanisms of action [26,27]. Still, the anti-cancer properties with the underlying mechanisms of action for most Hispolon analogs remain to be explored.…”