Abstract
Triple-negative breast cancer (TNBC) is a high-risk subtype of breast cancer with high capacity for metastasis and lacking of therapeutic targets. Our previous studies indicated that cystathionine γ-lyase (CSE) may be a new target related to the recurrence or metastasis of TNBC. Downregulation of CSE could inhibit the growth and metastasis of TNBC. The purpose of this study was to investigate the activity of the novel CSE inhibitor I194496 against TNBC in vivo and in vitro. The anticancer activity of I194496 in vitro were detected by MTS, EdU, and transwell assays. Methylene blue assay was used to determine the H2S level. Western blot was performed to analyze the expression of related pathway proteins. Xenograft tumors in nude mice were used to analyze the anticancer activity of I194496 in vivo. I194496 exerted potent inhibitory effects than l-propargylglycine (PAG, an existing CSE inhibitor) on human TNBC cells and possessed lower toxicity in normal breast epithelial Hs578Bst cells. I194496 reduced the activity and expression of CSE protein and the release of H2S in human TNBC cells. Meanwhile, the protein levels of PI3K, Akt, phospho (p)-Akt, Ras, Raf, p-ERK, p-Anxa2, STAT3, p-STAT3, VEGF, FAK, and Paxillin were decreased in human TNBC cells administrated with I194496. Furthermore, I194496 showed more stronger inhibitory effects on human TNBC xenograft tumors in nude mice. I194496 could inhibit the growth of human TNBC cells via the dual targeting PI3K/Akt and Ras/Raf/ERK pathway and suppress the metastasis of human TNBC cells via down-regulating Anxa2/STAT3 and VEGF/FAK/Paxillin signaling pathways. CSE inhibitor I194496 might become a novel and potential agent in the treatment of TNBC.
Highlights
Triple-negative breast cancer (TNBC) is a high-risk subtype of breast cancer with high capacity for metastasis and lacking of therapeutic targets
To confirm the inhibitory effect of I194496 on cystathionine γ-lyase (CSE), we firstly examined the direct inhibition of CSE by I194496 using the recombinant CSE protein and observed that I194496 possessed significant inhibitory activity against CSE protein with IC50 of 0.79 mM (Supplementary Fig. 1C)
We further confirmed the inhibitory effect of I194496 on CSE protein activity by the investigation of the production of H2S, and we found that I194496 significantly inhibited production of H 2S in MDA-MB-231 and MDA-MB-468 cells (Fig. 2C,D)
Summary
Triple-negative breast cancer (TNBC) is a high-risk subtype of breast cancer with high capacity for metastasis and lacking of therapeutic targets. Our previous studies indicated that cystathionine γ-lyase (CSE) may be a new target related to the recurrence or metastasis of TNBC. The purpose of this study was to investigate the activity of the novel CSE inhibitor I194496 against TNBC in vivo and in vitro. I194496 reduced the activity and expression of CSE protein and the release of H2S in human TNBC cells. The protein levels of PI3K, Akt, phospho (p)-Akt, Ras, Raf, p-ERK, p-Anxa[2], STAT3, p-STAT3, VEGF, FAK, and Paxillin were decreased in human TNBC cells administrated with I194496. Our previous studies indicated that the high-expression of CSE protein promoted the proliferation and metastasis of TNBC MDA-MB-231 c ells[18,19]. We detected the effects of I194496 on tumor growth in nude mice bearing human TNBC xenografts.
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