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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) suppresses cell viability and invasion, and induces apoptosis and necrosis simultaneously. (a) Tca-8113 and human gingival fibroblast (HGF) cells were treated with 2.5–80 μmol/l PTS for 1 h. The effects of PTS on cell viability were assessed using MTT 72 h after treatment. (b) Tca-8113 and HGF cells were treated with 40 μmol/l PTS or dimethyl sulfoxide (DMSO) (control vehicle) for 1 h; the morphologic changes were observed using a phase-contrast microscope. The apoptotic body was shown (arrow) (bar=30 μm). (c) Tca-8113 cells were treated with 40–80 μmol/l PTS or DMSO for 1 h, the cytotoxic effects of PTS on Tca-8113 cells were assessed using the colony formation assay. (d) Tca-8113 cells were treated with 40–80 μmol/l PTS or DMSO for 1 h. Cells were stained with Annexin V–FITC and propidium iodide (PI), followed by flow cytometric analysis. (e) Tca-8113 cells were treated with 80 μmol/l PTS or DMSO for 1 h; PTS-induced necrosis was determined using the lactate dehydrogenase (LDH) release assay. (f) Tca-8113 cells were treated with 40 μmol/l PTS or DMSO for 1 h; the effects of PTS on cell migration were determined using a transwell migration assay. Data represent the mean±SD of three independent experiments. *P<0.05. MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide.
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Figure 2: Para-toluenesulfonamide (PTS) suppresses cell viability and invasion, and induces apoptosis and necrosis simultaneously. (a) Tca-8113 and human gingival fibroblast (HGF) cells were treated with 2.5–80 μmol/l PTS for 1 h. The effects of PTS on cell viability were assessed using MTT 72 h after treatment. (b) Tca-8113 and HGF cells were treated with 40 μmol/l PTS or dimethyl sulfoxide (DMSO) (control vehicle) for 1 h; the morphologic changes were observed using a phase-contrast microscope. The apoptotic body was shown (arrow) (bar=30 μm). (c) Tca-8113 cells were treated with 40–80 μmol/l PTS or DMSO for 1 h, the cytotoxic effects of PTS on Tca-8113 cells were assessed using the colony formation assay. (d) Tca-8113 cells were treated with 40–80 μmol/l PTS or DMSO for 1 h. Cells were stained with Annexin V–FITC and propidium iodide (PI), followed by flow cytometric analysis. (e) Tca-8113 cells were treated with 80 μmol/l PTS or DMSO for 1 h; PTS-induced necrosis was determined using the lactate dehydrogenase (LDH) release assay. (f) Tca-8113 cells were treated with 40 μmol/l PTS or DMSO for 1 h; the effects of PTS on cell migration were determined using a transwell migration assay. Data represent the mean±SD of three independent experiments. *P<0.05. MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide.

Mentions: To examine the effects of PTS on cell viability, Tca-8113 and HGF cells were treated with different doses of PTS for 1 h. As shown in Fig. 2a, PTS significantly reduced the viability of Tca-8113 cells in a dose-dependent manner. 80 μmol/l PTS treatment almost completely suppressed the number of viable cells. The inhibitory effects of PTS in HGF cells were significantly decreased compared with that in Tca-8113 cells, which is consistent with our HPLC results that HGF cells accumulated less intracellular PST. Observation of morphologic changes using a phase-contrast microscope showed that 40 μmol/l PTS treatment induced classical apoptotic features including cell shrinkage, nuclear condensation, cell density reduction, and apoptotic body formation (Fig. 2b). Colony formation assay further confirmed the cytotoxic effects of PTS on Tca-8113 cells (Fig. 2c). We next measured cancer cell death using flow cytometric analysis. PTS treatment for 1 h induced a significant increase in early apoptotic cells (Annexin V+/PI−) with 5.82, 20.2, and 64.1% for DMSO, and 40 and 80 μmol/l of PTS treatment. Late apoptotic/necrotic cells (Annexin V+/PI+) after DMSO, and 40 and 80 μmol/l of PTS treatment were 7.70, 11.5, and 15.9%, respectively. Viable cells (Annexin V−/PI−) decreased from 83.0% in the DMSO-treated group to 67.5 and 19.8% after 40 and 80 μmol/l of PTS treatment (Fig. 2d). The results of flow cytometric analysis showed that PTS induced cancer cell death by activating apoptosis and necrosis simultaneously. We further determined PTS-induced necrosis using the LDH release assay. LDH is a cytosolic enzyme that can be released into culture medium upon damage of the plasma membrane. Necrosis, which results in an early loss of plasma membrane integrity, can be determined using the LDH release assay 16,17. As shown in Fig. 2e, PTS induced a significant increase in the activity of LDH released from necrotic cells. 80 μmol/l PTS treatment for 1 h induced a two-fold increase in LDH release compared with the control group. In addition, we investigated the effects of PTS on the invasive ability of cancer cells. Using a transwell migration assay, we observed that 40 μmol/l PTS treatment for 1 h significantly reduced the invasive ability of Tca-8113 cells (Fig. 2f).


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) suppresses cell viability and invasion, and induces apoptosis and necrosis simultaneously. (a) Tca-8113 and human gingival fibroblast (HGF) cells were treated with 2.5–80 μmol/l PTS for 1 h. The effects of PTS on cell viability were assessed using MTT 72 h after treatment. (b) Tca-8113 and HGF cells were treated with 40 μmol/l PTS or dimethyl sulfoxide (DMSO) (control vehicle) for 1 h; the morphologic changes were observed using a phase-contrast microscope. The apoptotic body was shown (arrow) (bar=30 μm). (c) Tca-8113 cells were treated with 40–80 μmol/l PTS or DMSO for 1 h, the cytotoxic effects of PTS on Tca-8113 cells were assessed using the colony formation assay. (d) Tca-8113 cells were treated with 40–80 μmol/l PTS or DMSO for 1 h. Cells were stained with Annexin V–FITC and propidium iodide (PI), followed by flow cytometric analysis. (e) Tca-8113 cells were treated with 80 μmol/l PTS or DMSO for 1 h; PTS-induced necrosis was determined using the lactate dehydrogenase (LDH) release assay. (f) Tca-8113 cells were treated with 40 μmol/l PTS or DMSO for 1 h; the effects of PTS on cell migration were determined using a transwell migration assay. Data represent the mean±SD of three independent experiments. *P<0.05. MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide.
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Figure 2: Para-toluenesulfonamide (PTS) suppresses cell viability and invasion, and induces apoptosis and necrosis simultaneously. (a) Tca-8113 and human gingival fibroblast (HGF) cells were treated with 2.5–80 μmol/l PTS for 1 h. The effects of PTS on cell viability were assessed using MTT 72 h after treatment. (b) Tca-8113 and HGF cells were treated with 40 μmol/l PTS or dimethyl sulfoxide (DMSO) (control vehicle) for 1 h; the morphologic changes were observed using a phase-contrast microscope. The apoptotic body was shown (arrow) (bar=30 μm). (c) Tca-8113 cells were treated with 40–80 μmol/l PTS or DMSO for 1 h, the cytotoxic effects of PTS on Tca-8113 cells were assessed using the colony formation assay. (d) Tca-8113 cells were treated with 40–80 μmol/l PTS or DMSO for 1 h. Cells were stained with Annexin V–FITC and propidium iodide (PI), followed by flow cytometric analysis. (e) Tca-8113 cells were treated with 80 μmol/l PTS or DMSO for 1 h; PTS-induced necrosis was determined using the lactate dehydrogenase (LDH) release assay. (f) Tca-8113 cells were treated with 40 μmol/l PTS or DMSO for 1 h; the effects of PTS on cell migration were determined using a transwell migration assay. Data represent the mean±SD of three independent experiments. *P<0.05. MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide.
Mentions: To examine the effects of PTS on cell viability, Tca-8113 and HGF cells were treated with different doses of PTS for 1 h. As shown in Fig. 2a, PTS significantly reduced the viability of Tca-8113 cells in a dose-dependent manner. 80 μmol/l PTS treatment almost completely suppressed the number of viable cells. The inhibitory effects of PTS in HGF cells were significantly decreased compared with that in Tca-8113 cells, which is consistent with our HPLC results that HGF cells accumulated less intracellular PST. Observation of morphologic changes using a phase-contrast microscope showed that 40 μmol/l PTS treatment induced classical apoptotic features including cell shrinkage, nuclear condensation, cell density reduction, and apoptotic body formation (Fig. 2b). Colony formation assay further confirmed the cytotoxic effects of PTS on Tca-8113 cells (Fig. 2c). We next measured cancer cell death using flow cytometric analysis. PTS treatment for 1 h induced a significant increase in early apoptotic cells (Annexin V+/PI−) with 5.82, 20.2, and 64.1% for DMSO, and 40 and 80 μmol/l of PTS treatment. Late apoptotic/necrotic cells (Annexin V+/PI+) after DMSO, and 40 and 80 μmol/l of PTS treatment were 7.70, 11.5, and 15.9%, respectively. Viable cells (Annexin V−/PI−) decreased from 83.0% in the DMSO-treated group to 67.5 and 19.8% after 40 and 80 μmol/l of PTS treatment (Fig. 2d). The results of flow cytometric analysis showed that PTS induced cancer cell death by activating apoptosis and necrosis simultaneously. We further determined PTS-induced necrosis using the LDH release assay. LDH is a cytosolic enzyme that can be released into culture medium upon damage of the plasma membrane. Necrosis, which results in an early loss of plasma membrane integrity, can be determined using the LDH release assay 16,17. As shown in Fig. 2e, PTS induced a significant increase in the activity of LDH released from necrotic cells. 80 μmol/l PTS treatment for 1 h induced a two-fold increase in LDH release compared with the control group. In addition, we investigated the effects of PTS on the invasive ability of cancer cells. Using a transwell migration assay, we observed that 40 μmol/l PTS treatment for 1 h significantly reduced the invasive ability of Tca-8113 cells (Fig. 2f).

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