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Para-toluenesulfonamide induces tongue squamous cell carcinoma cell death through disturbing lysosomal stability.

Liu Z, Liang C, Zhang Z, Pan J, Xia H, Zhong N, Li L - Anticancer Drugs (2015)

Bottom Line: Para-toluenesulfonamide (PTS) has been implicated with anticancer effects against a variety of tumors.Next, the effects of PTS on cell viability, invasion, and cell death were determined.Lysosomal integrity assay and western blot showed that PTS increased lysosomal membrane permeabilization associated with activation of lysosomal cathepsin B.

View Article: PubMed Central - PubMed

Affiliation: aDepartment of Head and Neck Oncology bState Key Laboratory of Oral Diseases cDepartment of Oral and Maxillofacial Surgery, West China Hospital of Stomatology, Sichuan University, Chengdu dState Key Laboratory of Respiratory Disease, Guangzhou Institute of Respiratory Diseases, First Affiliated Hospital, Guangzhou Medical University, Guangzhou, China.

ABSTRACT
Para-toluenesulfonamide (PTS) has been implicated with anticancer effects against a variety of tumors. In the present study, we investigated the inhibitory effects of PTS on tongue squamous cell carcinoma (Tca-8113) and explored the lysosomal and mitochondrial changes after PTS treatment in vitro. High-performance liquid chromatography showed that PTS selectively accumulated in Tca-8113 cells with a relatively low concentration in normal fibroblasts. Next, the effects of PTS on cell viability, invasion, and cell death were determined. PTS significantly inhibited Tca-8113 cells' viability and invasive ability with increased cancer cell death. Flow cytometric analysis and the lactate dehydrogenase release assay showed that PTS induced cancer cell death by activating apoptosis and necrosis simultaneously. Morphological changes, such as cellular shrinkage, nuclear condensation as well as formation of apoptotic body and secondary lysosomes, were observed, indicating that PTS might induce cell death through disturbing lysosomal stability. Lysosomal integrity assay and western blot showed that PTS increased lysosomal membrane permeabilization associated with activation of lysosomal cathepsin B. Finally, PTS was shown to inhibit ATP biosynthesis and induce the release of mitochondrial cytochrome c. Therefore, our findings provide a novel insight into the use of PTS in cancer therapy.

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Para-toluenesulfonamide (PTS) induces lysosomal membrane permeabilization (LMP) and cathepsin B activation. (a) Tca-8113 cells were treated with 40 μmol/l PTS or dimethyl sulfoxide for 1 h; the ultrastructural changes were observed using a transmission electron microscope. The secondary lysosomes are shown (arrows) (bar=1 μm). (b) Plots of LMP after 40 μmol/l PTS for 1 h in Tca-8113 cells. (c) Western blot analysis of Tca-8113 cells after 40 μmol/l PTS treatment for 1 h. Cleaved, active cathepsin B and total cathepsin B are shown. β-Actin was used as a loading control. Data represent the mean±SD of three independent experiments. *P<0.05.
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Figure 3: Para-toluenesulfonamide (PTS) induces lysosomal membrane permeabilization (LMP) and cathepsin B activation. (a) Tca-8113 cells were treated with 40 μmol/l PTS or dimethyl sulfoxide for 1 h; the ultrastructural changes were observed using a transmission electron microscope. The secondary lysosomes are shown (arrows) (bar=1 μm). (b) Plots of LMP after 40 μmol/l PTS for 1 h in Tca-8113 cells. (c) Western blot analysis of Tca-8113 cells after 40 μmol/l PTS treatment for 1 h. Cleaved, active cathepsin B and total cathepsin B are shown. β-Actin was used as a loading control. Data represent the mean±SD of three independent experiments. *P<0.05.

Mentions: To investigate the mechanisms of PTS-induced cell death, we used transmission electron microscopy analysis to observe the changes in ultrastructures of Tca-8113 cells. As shown in Fig. 3a, cells showed shrunk nuclei and an irregular nuclear membrane after 40 μmol/l PTS treatment. More secondary lysosomes with high electron density were observed after PTS treatment. Lysosomal instability was proposed to control the fate of cells either through activation of apoptosis or necrosis 18,19. We therefore performed a lysosomal integrity assay to assess the effects of PTS on lysosomal membrane integrity. Using the UMBG assay for β-galactosidase activity, PTS was shown to significantly increase free enzyme activity in a dose-dependent manner, which suggests that PTS induces lysosomal membrane permeabilization (LMP) and lysosomal damage (Fig. 3b). LMP leads to release intralysosomal proteases such as cathepsin B and D and chymotrypsin B, which are suggested to be essential downstream effectors of caspases 20. Western blot showed PTS treatment significantly increased cleaved cathepsin B expression (Fig. 3c). These results suggest that the anticancer ability of PTS might be attributed to inducing lysosomal instability and activating lysosome-mediated cell death.


Para-toluenesulfonamide induces tongue squamous cell carcinoma cell death through disturbing lysosomal stability.

Liu Z, Liang C, Zhang Z, Pan J, Xia H, Zhong N, Li L - Anticancer Drugs (2015)

Para-toluenesulfonamide (PTS) induces lysosomal membrane permeabilization (LMP) and cathepsin B activation. (a) Tca-8113 cells were treated with 40 μmol/l PTS or dimethyl sulfoxide for 1 h; the ultrastructural changes were observed using a transmission electron microscope. The secondary lysosomes are shown (arrows) (bar=1 μm). (b) Plots of LMP after 40 μmol/l PTS for 1 h in Tca-8113 cells. (c) Western blot analysis of Tca-8113 cells after 40 μmol/l PTS treatment for 1 h. Cleaved, active cathepsin B and total cathepsin B are shown. β-Actin was used as a loading control. Data represent the mean±SD of three independent experiments. *P<0.05.
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Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4588602&req=5

Figure 3: Para-toluenesulfonamide (PTS) induces lysosomal membrane permeabilization (LMP) and cathepsin B activation. (a) Tca-8113 cells were treated with 40 μmol/l PTS or dimethyl sulfoxide for 1 h; the ultrastructural changes were observed using a transmission electron microscope. The secondary lysosomes are shown (arrows) (bar=1 μm). (b) Plots of LMP after 40 μmol/l PTS for 1 h in Tca-8113 cells. (c) Western blot analysis of Tca-8113 cells after 40 μmol/l PTS treatment for 1 h. Cleaved, active cathepsin B and total cathepsin B are shown. β-Actin was used as a loading control. Data represent the mean±SD of three independent experiments. *P<0.05.
Mentions: To investigate the mechanisms of PTS-induced cell death, we used transmission electron microscopy analysis to observe the changes in ultrastructures of Tca-8113 cells. As shown in Fig. 3a, cells showed shrunk nuclei and an irregular nuclear membrane after 40 μmol/l PTS treatment. More secondary lysosomes with high electron density were observed after PTS treatment. Lysosomal instability was proposed to control the fate of cells either through activation of apoptosis or necrosis 18,19. We therefore performed a lysosomal integrity assay to assess the effects of PTS on lysosomal membrane integrity. Using the UMBG assay for β-galactosidase activity, PTS was shown to significantly increase free enzyme activity in a dose-dependent manner, which suggests that PTS induces lysosomal membrane permeabilization (LMP) and lysosomal damage (Fig. 3b). LMP leads to release intralysosomal proteases such as cathepsin B and D and chymotrypsin B, which are suggested to be essential downstream effectors of caspases 20. Western blot showed PTS treatment significantly increased cleaved cathepsin B expression (Fig. 3c). These results suggest that the anticancer ability of PTS might be attributed to inducing lysosomal instability and activating lysosome-mediated cell death.

Bottom Line: Para-toluenesulfonamide (PTS) has been implicated with anticancer effects against a variety of tumors.Next, the effects of PTS on cell viability, invasion, and cell death were determined.Lysosomal integrity assay and western blot showed that PTS increased lysosomal membrane permeabilization associated with activation of lysosomal cathepsin B.

View Article: PubMed Central - PubMed

Affiliation: aDepartment of Head and Neck Oncology bState Key Laboratory of Oral Diseases cDepartment of Oral and Maxillofacial Surgery, West China Hospital of Stomatology, Sichuan University, Chengdu dState Key Laboratory of Respiratory Disease, Guangzhou Institute of Respiratory Diseases, First Affiliated Hospital, Guangzhou Medical University, Guangzhou, China.

ABSTRACT
Para-toluenesulfonamide (PTS) has been implicated with anticancer effects against a variety of tumors. In the present study, we investigated the inhibitory effects of PTS on tongue squamous cell carcinoma (Tca-8113) and explored the lysosomal and mitochondrial changes after PTS treatment in vitro. High-performance liquid chromatography showed that PTS selectively accumulated in Tca-8113 cells with a relatively low concentration in normal fibroblasts. Next, the effects of PTS on cell viability, invasion, and cell death were determined. PTS significantly inhibited Tca-8113 cells' viability and invasive ability with increased cancer cell death. Flow cytometric analysis and the lactate dehydrogenase release assay showed that PTS induced cancer cell death by activating apoptosis and necrosis simultaneously. Morphological changes, such as cellular shrinkage, nuclear condensation as well as formation of apoptotic body and secondary lysosomes, were observed, indicating that PTS might induce cell death through disturbing lysosomal stability. Lysosomal integrity assay and western blot showed that PTS increased lysosomal membrane permeabilization associated with activation of lysosomal cathepsin B. Finally, PTS was shown to inhibit ATP biosynthesis and induce the release of mitochondrial cytochrome c. Therefore, our findings provide a novel insight into the use of PTS in cancer therapy.

Show MeSH
Related in: MedlinePlus