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Thioredoxin 1 modulates apoptosis induced by bioactive compounds in prostate cancer cells

View Article: PubMed Central - PubMed

ABSTRACT

Accumulating evidence suggests that natural bioactive compounds, alone or in combination with traditional chemotherapeutic agents, could be used as potential therapies to fight cancer. In this study, we employed four natural bioactive compounds (curcumin, resveratrol, melatonin, and silibinin) and studied their role in redox control and ability to promote apoptosis in androgen sensitive and insensitive prostate cancer cells. Here is shown that curcumin and resveratrol promote ROS production and induce apoptosis in LNCaP and PC-3. An increase in reactive species is a trigger event in curcumin-induced apoptosis and a consequence of resveratrol effects on other pathways within these cells. Moreover, here we demonstrated that these four compounds affect differently one of the main intracellular redox regulator, the thioredoxin system. Exposure to curcumin and resveratrol promoted TRX1 oxidation and altered its subcellular location. Furthermore, resveratrol diminished TRX1 levels in PC-3 cells and increased the expression of its inhibitor TXNIP. Conversly, melatonin and silibinin only worked as cytostatic agents, reducing ROS levels and showing preventive effects against TRX oxidation. All together, this work explores the effect of compounds currently tested as chemo-preventive agents in prostate cancer therapy, on the TRX1 redox state and function. Our work shows the importance that the TRX system might have within the differences found in their mechanisms of action. These bioactive compounds trigger different responses and affect ROS production and redox systems in prostate cancer cells, suggesting the key role that redox-related pathways might play in processes like differentiation or survival in prostate cancer.

No MeSH data available.


Related in: MedlinePlus

Effect of bioactive compounds on viability in prostate cancer cells. (A) Androgen-sensitive LNCaP and androgen-insensitive PC-3 were exposed to melatonin, silibinin, curcumin or resveratrol and MTT assay was performed after 48 incubation. Data are shown as mean±SEM. of six independent samples. Experiments were repeated at least three times. * p<0.05; ** p<0.01; *** p<0.001 versus CON. (B) Apoptosis was evaluated in LNCaP and PC-3 after 48 h incubation with IC50 of the compounds (1 mM melatonin, 50 µM silibinin, 25 µM curcumin and 50 µM resveratrol), using Annexin V-FITC assay by flow cytometry. (C) LNCaP cells were cultured with or without 1 mM melatonin, 50 µM silibinin, 25 µM curcumin or 50 µM resveratrol for 48 h. BCL-2 and BAX protein levels were assessed by western blot. GAPDH and β-Actin were employed as loading control. (D) Changes in cell morphology were examined under a phase contrast microscope with a 200× magnification and photographed (scale bar, 25 µm).
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f0005: Effect of bioactive compounds on viability in prostate cancer cells. (A) Androgen-sensitive LNCaP and androgen-insensitive PC-3 were exposed to melatonin, silibinin, curcumin or resveratrol and MTT assay was performed after 48 incubation. Data are shown as mean±SEM. of six independent samples. Experiments were repeated at least three times. * p<0.05; ** p<0.01; *** p<0.001 versus CON. (B) Apoptosis was evaluated in LNCaP and PC-3 after 48 h incubation with IC50 of the compounds (1 mM melatonin, 50 µM silibinin, 25 µM curcumin and 50 µM resveratrol), using Annexin V-FITC assay by flow cytometry. (C) LNCaP cells were cultured with or without 1 mM melatonin, 50 µM silibinin, 25 µM curcumin or 50 µM resveratrol for 48 h. BCL-2 and BAX protein levels were assessed by western blot. GAPDH and β-Actin were employed as loading control. (D) Changes in cell morphology were examined under a phase contrast microscope with a 200× magnification and photographed (scale bar, 25 µm).

Mentions: The effects of four bioactive compounds (melatonin, silibinin, curcumin and resveratrol) on the viability and proliferation of prostate cancer cells were evaluated using MTT assay (Fig. 1A). After 48 h treatment, the growth of androgen-sensitive LNCaP cells was significantly inhibited by melatonin 500 µM and 1 mM and silibinin 50 µM, while no significant effect was found in androgen-insensitive PC-3 cells (Fig. 1A). Curcumin and resveratrol inhibited the viability of both cell lines at concentrations over 10 and 20 µM, respectively. MTT assay cannot discriminate cell growth inhibition from cell death. Thus, annexinV staining in combination with flow cytometry quantification of stained cells was performed after culturing LNCaP and PC-3 cells with melatonin (1 mM), silibinin (50 µM), curcumin (25 µM) or resveratrol (50 µM) (Fig. 1B). Only incubation with curcumin and resveratrol induced a significant increase in apoptotic cells in both cell types. To confirm this, the levels of anti-apoptotic protein BCL2 and pro-apoptotic protein BAX were assessed by western blot. In LNCaP cells, a clear decrease in BCL2 and an increase in BAX protein levels were found after incubation with curcumin or resveratrol. On the contrary, an increased in BCL2 was observed after melatonin incubation while no significant differences were observed in cells treated with silibinin in BCL2/BAX ratio (Fig. 1C). Fig. 1D shows the morphology of LNCaP and PC-3 cells after incubation for 48 h with all compounds. The characteristic morphological features of apoptotic cells such as cell shrinkage, rounded shape and partial detachment were all observed after curcumin incubation in both cell types. Similarly, PC-3 cells undergoing resveratrol incubation showed morphological signs of apoptosis but those were preceded by cytoplasmic vacuolization. Significant changes in the morphology, including long processes (neurites) and rounded nuclei, typical features of neuroendocrine differentiation, were observed in cells cultured with melatonin and silibinin as previously reported [32].


Thioredoxin 1 modulates apoptosis induced by bioactive compounds in prostate cancer cells
Effect of bioactive compounds on viability in prostate cancer cells. (A) Androgen-sensitive LNCaP and androgen-insensitive PC-3 were exposed to melatonin, silibinin, curcumin or resveratrol and MTT assay was performed after 48 incubation. Data are shown as mean±SEM. of six independent samples. Experiments were repeated at least three times. * p<0.05; ** p<0.01; *** p<0.001 versus CON. (B) Apoptosis was evaluated in LNCaP and PC-3 after 48 h incubation with IC50 of the compounds (1 mM melatonin, 50 µM silibinin, 25 µM curcumin and 50 µM resveratrol), using Annexin V-FITC assay by flow cytometry. (C) LNCaP cells were cultured with or without 1 mM melatonin, 50 µM silibinin, 25 µM curcumin or 50 µM resveratrol for 48 h. BCL-2 and BAX protein levels were assessed by western blot. GAPDH and β-Actin were employed as loading control. (D) Changes in cell morphology were examined under a phase contrast microscope with a 200× magnification and photographed (scale bar, 25 µm).
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f0005: Effect of bioactive compounds on viability in prostate cancer cells. (A) Androgen-sensitive LNCaP and androgen-insensitive PC-3 were exposed to melatonin, silibinin, curcumin or resveratrol and MTT assay was performed after 48 incubation. Data are shown as mean±SEM. of six independent samples. Experiments were repeated at least three times. * p<0.05; ** p<0.01; *** p<0.001 versus CON. (B) Apoptosis was evaluated in LNCaP and PC-3 after 48 h incubation with IC50 of the compounds (1 mM melatonin, 50 µM silibinin, 25 µM curcumin and 50 µM resveratrol), using Annexin V-FITC assay by flow cytometry. (C) LNCaP cells were cultured with or without 1 mM melatonin, 50 µM silibinin, 25 µM curcumin or 50 µM resveratrol for 48 h. BCL-2 and BAX protein levels were assessed by western blot. GAPDH and β-Actin were employed as loading control. (D) Changes in cell morphology were examined under a phase contrast microscope with a 200× magnification and photographed (scale bar, 25 µm).
Mentions: The effects of four bioactive compounds (melatonin, silibinin, curcumin and resveratrol) on the viability and proliferation of prostate cancer cells were evaluated using MTT assay (Fig. 1A). After 48 h treatment, the growth of androgen-sensitive LNCaP cells was significantly inhibited by melatonin 500 µM and 1 mM and silibinin 50 µM, while no significant effect was found in androgen-insensitive PC-3 cells (Fig. 1A). Curcumin and resveratrol inhibited the viability of both cell lines at concentrations over 10 and 20 µM, respectively. MTT assay cannot discriminate cell growth inhibition from cell death. Thus, annexinV staining in combination with flow cytometry quantification of stained cells was performed after culturing LNCaP and PC-3 cells with melatonin (1 mM), silibinin (50 µM), curcumin (25 µM) or resveratrol (50 µM) (Fig. 1B). Only incubation with curcumin and resveratrol induced a significant increase in apoptotic cells in both cell types. To confirm this, the levels of anti-apoptotic protein BCL2 and pro-apoptotic protein BAX were assessed by western blot. In LNCaP cells, a clear decrease in BCL2 and an increase in BAX protein levels were found after incubation with curcumin or resveratrol. On the contrary, an increased in BCL2 was observed after melatonin incubation while no significant differences were observed in cells treated with silibinin in BCL2/BAX ratio (Fig. 1C). Fig. 1D shows the morphology of LNCaP and PC-3 cells after incubation for 48 h with all compounds. The characteristic morphological features of apoptotic cells such as cell shrinkage, rounded shape and partial detachment were all observed after curcumin incubation in both cell types. Similarly, PC-3 cells undergoing resveratrol incubation showed morphological signs of apoptosis but those were preceded by cytoplasmic vacuolization. Significant changes in the morphology, including long processes (neurites) and rounded nuclei, typical features of neuroendocrine differentiation, were observed in cells cultured with melatonin and silibinin as previously reported [32].

View Article: PubMed Central - PubMed

ABSTRACT

Accumulating evidence suggests that natural bioactive compounds, alone or in combination with traditional chemotherapeutic agents, could be used as potential therapies to fight cancer. In this study, we employed four natural bioactive compounds (curcumin, resveratrol, melatonin, and silibinin) and studied their role in redox control and ability to promote apoptosis in androgen sensitive and insensitive prostate cancer cells. Here is shown that curcumin and resveratrol promote ROS production and induce apoptosis in LNCaP and PC-3. An increase in reactive species is a trigger event in curcumin-induced apoptosis and a consequence of resveratrol effects on other pathways within these cells. Moreover, here we demonstrated that these four compounds affect differently one of the main intracellular redox regulator, the thioredoxin system. Exposure to curcumin and resveratrol promoted TRX1 oxidation and altered its subcellular location. Furthermore, resveratrol diminished TRX1 levels in PC-3 cells and increased the expression of its inhibitor TXNIP. Conversly, melatonin and silibinin only worked as cytostatic agents, reducing ROS levels and showing preventive effects against TRX oxidation. All together, this work explores the effect of compounds currently tested as chemo-preventive agents in prostate cancer therapy, on the TRX1 redox state and function. Our work shows the importance that the TRX system might have within the differences found in their mechanisms of action. These bioactive compounds trigger different responses and affect ROS production and redox systems in prostate cancer cells, suggesting the key role that redox-related pathways might play in processes like differentiation or survival in prostate cancer.

No MeSH data available.


Related in: MedlinePlus