A novel benzothiazole derivative YLT322 induces apoptosis via the mitochondrial apoptosis pathway in vitro with anti-tumor activity in solid malignancies.

Benzothiazole derivatives are known for various biological activities, and their potency in cancer therapy has received considerable attention in recent years. YLT322, a novel synthesized benzothiazole derivative, exhibits potent anti-tumor activity via inducing apoptosis both in vitro and in vivo. In this study, we found that YLT322 showed growth inhibition against a broad spectrum of human cancer cells and induced apoptosis of HepG2 cells in a dose- and time-dependent manner. The occurrence of its apoptosis was associated with activation of caspases-3 and -9, but not caspase-8. YLT322 increased the expression of Bax, decreased the expression of Bcl-2, and induced the release of cytochrome c which activates the mitochondrial apoptotic pathway. The down-regulation of phosphorylated p42/44 MAPK and phosphorylated Akt was also observed. Moreover, YLT322 suppressed the growth of established tumors in xenograft models in mice without obvious side effects. Histological and immunohistochemical analyses revealed an increase in TUNEL and caspase-3-positive cells and a decrease in Ki67-positive cells upon YLT322. These results suggest that YLT322 may be a potential candidate for cancer therapy

A novel VEGFR inhibitor ZLF-095 with potent antitumor activity and low toxicity

Angiogenesis plays a critical role in the survival, progression and metastasis of malignant tumors. Multiple factors are known to induce tumor angiogenesis, vascular endothelial growth factor (VEGF) is the most important one. Lenvatinib is an oral multi-kinase inhibitor of VEGFRs which has been approved for the treatment of various malignancies as the first-line agent by the Food and Drug Administration (FDA). It shows excellent antitumor efficacy in clinical practice. However, the adverse effects of Lenvatinib may seriously impair the therapeutic effect. Here we report the discovery and characterization of a novel VEGFR inhibitor (ZLF-095), which exhibited high activity and selectivity for VEGFR1/2/3. ZLF-095 displayed apparently antitumor effect in vitro and in vivo. We discovered that Lenvatinib could provoke fulminant ROS-caspase3-GSDME-dependent pyroptosis in GSDME-expressing cells by loss of mitochondrial membrane potential, which may be one of the reasons for Lenvatinib's toxicity. Meanwhile, ZLF-095 showed less toxicity than Lenvatinib by switching pyroptosis to apoptosis. These results suggest that ZLF-095 could become a potential angiogenesis inhibitor for cancer therapy

Effect of YLT322 on the expression levels of apoptosis-related proteins and the mitochondrial membrane potential (ΔΨ) of HepG2 cells.

<p><b>A.</b> The expression levels of pro-caspase-8, -3, -9, and their activated forms in HepG2 and Bel-7402 cells treated with YLT322 for 48 hours as assayed by western blot. <b>B.</b> HepG2 cells were treated with 2 µM YLT322 alone or in combination with Z-VAD-FMK (a general caspase inhibitor), Ac-LEHD-FMK (caspase-9 inhibitor) or Ac-IETD-FMK (caspase-8 inhibitor). Data are expressed as mean ± SD. for at least 3 independent experiments. (*p<0.05; **p<0.01; ***p<0.001). <b>C.</b> The mitochondrial membrane potential (ΔΨ) was detected by RH123 staining after treatment with YLT322 at varying concentrations of 0 µM (Dark blue), 0.5 µM (blue), 1 µM (orange), 2 µM (green) for 12 to 48 hours (Bars, SD; Column, mean; n, 3;*p<0.05; **p<0.01; ***p<0.001). <b>D.</b> The level of cytochrome <i>c</i> in the cytosol and mitochondrial fraction was analyzed by western blot analysis after incubation of cells with YLT322 for 48 hours. Expression of COX-4 served as the loading control for the mitochondria fraction. <b>E.</b> The protein levels of Bcl-2 and Bax were detected by western blot in HepG2 cells treated with YLT322 for 48 h. <b>F.</b> The mRNA level of Bax was examined by real-time RT-PCR in HepG2 cells treated with YLT322 for 48 h. (Bars, SD; Column, mean; n, 3;*p<0.05; **p<0.01; ***p<0.001). <b>G.</b> The expression levels of Akt, p44/42 MAPK and their phosphorylated forms were assayed by western blot in HepG2 cells treated with YLT322. <b>H.</b> HepG2 cells were treated with 2 µM YLT322 alone or in combination with LY294002 (PI3K/AKT inhibitor) or PD98059 (MEK/ERK inhibitor). Data shown are representative of three independent experiments.</p

The effect of YLT322 on cell morphology and viability of cancer cells.

<p><b>A.</b> Flow cytometric analysis of PI-stained HCC cell lines, including HepG2, SMMC-7721, Bel-7402 and Bel-7404, after treatment with 2 µM YLT322 for 48 hours. <b>B.</b> Statistical results of apoptosis assays presented as surviving cells (percentage of untreated control). Data are expressed as mean ± SD. for at least 3 independent experiments. (*p<0.05; **p<0.01; ***p<0.001). <b>C.</b> Fluorescence microscopy analysis of Hoechst 33342-stained HepG2 cells after incubation with varying concentrations of YLT322 (0 µM (a), 0.5 µM (b), 1 µM (c), 2 µM (d)) for 24 hours (×40). <b>D.</b> Flow cytometric analysis of cells stained with Annexin V-FITC/PI after treatment with various concentrations (0 µM, 0.5 µM, 1 µM, 2 µM) of YLT322 for 12 to 48 hours.</p

Inhibition of cell growth and colony formation in human cancer cell lines by YLT322 <i>in vitro</i>.

<p><b>A.</b> Cells (a:HepG2; b:Bel-7402; c: Bel-7404;d:SMMC-7721) were treated with concentrations of YLT322 ranging from 0.15 to 5.0 µM, for 12 to 48 hours and cell viability was determined by MTT assay . Data are expressed as mean ± SD. for at least 3 independent experiments. (*p<0.05; **p<0.01; ***p<0.001) <b>B.</b> Effects of varying concentrations of YLT322 on colony formation of HepG2 and Bel-7402 cells after two weeks treatment. <b>C.</b> Statistical results of colony-forming assays presented as surviving colonies (percentage of untreated control). Data are expressed as mean ± SD. for at least 3 independent experiments. (*p<0.05; **p<0.01; ***p<0.001).</p

The chemical structure of YLT322.

<p>The chemical structure of YLT322.</p

The proliferation inhibition of YLT322 in human tumor cell lines.

<p><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0063900#pone-0063900-t001" target="_blank">Table 1</a>. IC₅₀ values (µM) for inhibition of cell proliferation by 48- hour treatment with YLT322 (0–20 µM) or doxorubicine (0–40 µ M). Data are expressed as the mean from three experiments.</p