Korean J Physiol Pharmacol.  2024 May;28(3):219-227. 10.4196/kjpp.2024.28.3.219.

Novel artesunate-metformin conjugate inhibits bladder cancer cell growth associated with Clusterin/SREBP1/FASN signaling pathway

• Lin P
• Yang X
• Wang L
• Zou X
• Mu L
• Xu C
• Yang X

• KMID: 2555664
• DOI: http://doi.org/10.4196/kjpp.2024.28.3.219

Abstract

Bladder cancer remains the 10th most common cancer worldwide. In recent years, metformin has been found to have potential anti-bladder cancer activ- ity while high concentration of IC₅₀ at millimolar level is needed, which could not be reached by regular oral administration route. Thus, higher efficient agent is urgently demanded for clinically treating bladder cancer. Here, by conjugating artesunate to metformin, a novel artesunate-metformin dimer triazine derivative AM2 was designed and synthesized. The inhibitory effect of AM2 on bladder cancer cell line T24 and the mechanism underlying was determined. Anti-tumor activity of AM2 was assessed by MTT, cloning formation and wound healing assays. Decreasing effect of AM2 on lipogenesis was determined by oil red O staining. The protein expressions of Clusterin, SREBP1 and FASN in T24 cells were evaluated by Western blotting. The results show that AM2 significantly inhibited cell proliferation and migration at micromolar level, much higher than parental metformin. AM2 reduced lipogenesis and down-regulated the expressions of Clusterin, SREBP1 and FASN. These results suggest that AM2 inhibits the growth of bladder cancer cells T24 by inhibiting cellular lipogenesis associated with the Clusterin/SREBP1/FASN signaling pathway.

Keyword

Artesunate; Lipogenesis; Metformin; Urinary bladder neoplasms

Figure

• 

  Fig. 1 The synthetic route and mass spectrometry results of AM2. (A) Free metformin obtained from metformin hydrochloride is esterified with artesunate. Reagents and conditions: (a) DCM, NaOH, rt, 2 h; (b) DCM, EDCI, DMAP, rt, 12 h; (B) Mass detection result of AM2.

• 

  Fig. 2 The anti-proliferation activities of AM2 on a variety of bladder cancer cells. (A) RT4, UMUC3, T24, and HUVEC cells were treated with 0, 0.25, 0.5, 1, and 2 μM of AM2 for 72 h, then observed the cell viability. (B) Comparison of the inhibitory effects of AM2 on cell proliferation in various of bladder cancer cells and HUVEC cells. (C–E) T24 cells were treated with different concentration of AM2, metformin and cisplatin for 72 h, then observed the cell viability. Data are presented as the mean ± SD of three independent experiments. ns, no significance. ****p < 0.0001.

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  Fig. 3 The inhibition of colony formation and migration of AM2 on T24 cells. (A) Evaluation of colony suppression by AM2. (B) Quantification of the colony formation. OD values were scanned at a wavelength of 550 nm. (C, D) Inhibitory migration effect of AM2 on T24 cells for 12, 24, 48 h. Scale bar was 200 μm. Data are presented as the mean ± SD of three independent experiments. ns, no significance. **p < 0.01, ***p < 0.001, ****p < 0.0001.

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  Fig. 4 AM2 regulated lipogenesis in T24 cells. (A) Oil red O staining images of T24 treated with different concentrations of AM2. (B) Effect of AM2 on the expression of FASN, SREBP1 and Clusterin in T24 cells. (C) The expression level of FASN protein. (D) The expression level of Clusterin protein. (E) The expression level of p-SREBP1. (F) The expression level of mature forms of n-SREBP1. Data are presented as the mean ± SD of three independent experiments. ns, no significance. *p < 0.5, **p < 0.01, ***p < 0.001.

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