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Shikonin selectively induces apoptosis in human prostate cancer cells through the endoplasmic reticulum stress and mitochondrial apoptotic pathway.

Gara RK, Srivastava VK, Duggal S, Bagga JK, Bhatt M, Sanyal S, Mishra DP - J. Biomed. Sci. (2015)

Bottom Line: Moreover, addition of antioxidants attenuated these effects.Shikonin also induced the mitochondrial apoptotic pathway mediated through the enhanced expression of the pro-apoptotic Bax and inhibition of Bcl-2, disruption of the mitochondrial membrane potential (MMP) followed by the activation of caspase-9, caspase-3, and PARP cleavage.The results suggest that shikonin could be useful in the therapeutic management of hormone refractory prostate cancers due to its modulation of the pro-apoptotic ER stress and mitochondrial apoptotic pathways.

View Article: PubMed Central - PubMed

Affiliation: Cell Death Research Laboratory, Endocrinology Division CSIR-Central Drug Research Institute, Lucknow, 226031, India. rgara@uthsc.edu.

ABSTRACT

Background: Despite the recent progress in screening and therapy, a majority of prostate cancer cases eventually attain hormone refractory and chemo-resistant attributes. Conventional chemotherapeutic strategies are effective at very high doses for only palliative management of these prostate cancers. Therefore chemo-sensitization of prostate cancer cells could be a promising strategy for increasing efficacy of the conventional chemotherapeutic agents in prostate cancer patients. Recent studies have indicated that the chemo-preventive natural agents restore the pro-apoptotic protein expression and induce endoplasmic reticulum stress (ER stress) leading to the inhibition of cellular proliferation and activation of the mitochondrial apoptosis in prostate cancer cells. Therefore reprogramming ER stress-mitochondrial dependent apoptosis could be a potential approach for management of hormone refractory chemoresistant prostate cancers. We aimed to study the effects of the natural naphthoquinone Shikonin in human prostate cancer cells.

Results: The results indicated that Shikonin induces apoptosis in prostate cancer cells through the dual induction of the endoplasmic reticulum stress and mitochondrial dysfunction. Shikonin induced ROS generation and activated ER stress and calpain activity. Moreover, addition of antioxidants attenuated these effects. Shikonin also induced the mitochondrial apoptotic pathway mediated through the enhanced expression of the pro-apoptotic Bax and inhibition of Bcl-2, disruption of the mitochondrial membrane potential (MMP) followed by the activation of caspase-9, caspase-3, and PARP cleavage.

Conclusion: The results suggest that shikonin could be useful in the therapeutic management of hormone refractory prostate cancers due to its modulation of the pro-apoptotic ER stress and mitochondrial apoptotic pathways.

No MeSH data available.


Related in: MedlinePlus

ER stress inhibition attenuates Shikonin induced mitochondrial apoptosis. (A) DU-145 and PC-3 cells were treated with either Shikonin (2.5 μM) alone for 24 h or pretreated with ER stress inhibitor Salubrinal (10 μM) for 24 h and subsequently treated with Shikonin. Protein expression was measured by western blotting as described in the Methods section. Shikonin treatment resulted in a marked reduction in the expression of Bcl-2 and an increase in the levels of Bax and cleaved PARP when compared with the controls. Pretreatment with ER stress inhibitor Salubrinal led to the increased expression of Bcl-2 and the decreased expression of Bax and cleaved PARP. (B) Pretreatment with caspase inhibitors reversed Shikonin induced caspase activation. Treatment of cells with Shikonin (2.5 μM) for 24 h resulted in a marked reduction of shikonin induced caspase-9 and caspase-3 activities. (C) Inhibition of ER suppressed shikonin induced casapase activity. Prostate cancer cells were pretreated with Salubrinal (10 μM), caspase 3 and 9 activity were performed by colorimetric assay as described in material and method section. (D) ER stress regulates mitochondrial membrane potential in Shikonin treated DU-145 and PC-3 cells. DU-145 and PC-3 cells were treated with either Shikonin (2.5 μΜ) alone or along with pretreatment with salubrinal (10 μM) for 24 h, and then incubated with mitochondria specific dye JC-1 (10 m g/ml aft 37°C for 15 min), and the florescence was monitored using fluorimeter (Excitation 530 nm/Fluorescence 590 nm) for measurement of the mitochondrial membrane potential. Shikonin treatment significantly reduced mitochondrial membrane potential whereas pretreatment of ER stress inhibitor reversed this effect. Data is expressed in means ± SEM and represents the results of three independent experiments.
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Fig6: ER stress inhibition attenuates Shikonin induced mitochondrial apoptosis. (A) DU-145 and PC-3 cells were treated with either Shikonin (2.5 μM) alone for 24 h or pretreated with ER stress inhibitor Salubrinal (10 μM) for 24 h and subsequently treated with Shikonin. Protein expression was measured by western blotting as described in the Methods section. Shikonin treatment resulted in a marked reduction in the expression of Bcl-2 and an increase in the levels of Bax and cleaved PARP when compared with the controls. Pretreatment with ER stress inhibitor Salubrinal led to the increased expression of Bcl-2 and the decreased expression of Bax and cleaved PARP. (B) Pretreatment with caspase inhibitors reversed Shikonin induced caspase activation. Treatment of cells with Shikonin (2.5 μM) for 24 h resulted in a marked reduction of shikonin induced caspase-9 and caspase-3 activities. (C) Inhibition of ER suppressed shikonin induced casapase activity. Prostate cancer cells were pretreated with Salubrinal (10 μM), caspase 3 and 9 activity were performed by colorimetric assay as described in material and method section. (D) ER stress regulates mitochondrial membrane potential in Shikonin treated DU-145 and PC-3 cells. DU-145 and PC-3 cells were treated with either Shikonin (2.5 μΜ) alone or along with pretreatment with salubrinal (10 μM) for 24 h, and then incubated with mitochondria specific dye JC-1 (10 m g/ml aft 37°C for 15 min), and the florescence was monitored using fluorimeter (Excitation 530 nm/Fluorescence 590 nm) for measurement of the mitochondrial membrane potential. Shikonin treatment significantly reduced mitochondrial membrane potential whereas pretreatment of ER stress inhibitor reversed this effect. Data is expressed in means ± SEM and represents the results of three independent experiments.

Mentions: To investigate the mitochondrial apoptotic events involved in shikonin-induced apoptosis, the levels of the antiapoptotic protein Bcl-2 and the pro-apoptotic protein Bax were analyzed. Western blot analysis showed that the treatment of DU-145 and PC-3 cells with Shikonin resulted in a marked reduction in the expression of Bcl-2 and increased the level of Bax and cleaved PARP when compared with untreated cells (Figure 6A). These results suggested that shikonin alters the levels of pro- and anti-apoptotic proteins of the Bcl-2 family in a manner that contributes to the shikonin-induced apoptosis. Salubrinal is an inhibitor of the serine/threonine phosphatase PP1 and inhibits eIF2α dephosphorylation that in turn blocks ER stress-induced cell death [35,36]. The inhibition of ER stress in DU-145 and PC-3 cells by salubrinal led to the increased expression of Bcl-2 and decreased the levels of Bax and cleaved PARP, in contrast to cells treated with Shikonin alone, indicating that Shikonin treatment -induced the mitochondrial apoptotic pathway (Figure 6A). Similarly, pretreatment with caspase-9 and caspase-3 inhibitors (Figure 6B) or the ER stress inhibitor Salubrinal (Figure 6C) significantly inhibited the Shikonin induced caspase activation in DU-145 and PC-3 prostate cancer cells. Furthermore, cell viability assays with pretreatment of either the pan caspase inhibitor (Z-VAD-FMK) or the caspase-9 (Z-LEHD-FMK)or caspase-3 inhibitor (Z-DEVD-FMK) confirmed a sequential activation of caspases upon Shikonin treatment (Additional file 2: Figure S3). Further to define the role of ROS-Calcium Stress mediated modulation of mitochondrial membrane potential, we had pretreated prostate cancer with ROS scavengers (NAC,GSH or Catalase) and the calpain inhibitor calpeptin as described in material methods. Our results suggested that both ROS and intracellular calcium signaling are crucial for mitochondrial apoptosis in prostate cancer cells. Additionally we also observed that either inhibition of caspase 9, 3 or Salubrinal prevented Shikonin induced reduction of cell viability (Figure 7A), and formation of apoptotic bodies in these cells (Figure 7B) as a marker of apoptotic cell death. Collectively these results indicated that ER stress played an critical role in the shikonin induced apoptosis of prostate cancer cells.Figure 6


Shikonin selectively induces apoptosis in human prostate cancer cells through the endoplasmic reticulum stress and mitochondrial apoptotic pathway.

Gara RK, Srivastava VK, Duggal S, Bagga JK, Bhatt M, Sanyal S, Mishra DP - J. Biomed. Sci. (2015)

ER stress inhibition attenuates Shikonin induced mitochondrial apoptosis. (A) DU-145 and PC-3 cells were treated with either Shikonin (2.5 μM) alone for 24 h or pretreated with ER stress inhibitor Salubrinal (10 μM) for 24 h and subsequently treated with Shikonin. Protein expression was measured by western blotting as described in the Methods section. Shikonin treatment resulted in a marked reduction in the expression of Bcl-2 and an increase in the levels of Bax and cleaved PARP when compared with the controls. Pretreatment with ER stress inhibitor Salubrinal led to the increased expression of Bcl-2 and the decreased expression of Bax and cleaved PARP. (B) Pretreatment with caspase inhibitors reversed Shikonin induced caspase activation. Treatment of cells with Shikonin (2.5 μM) for 24 h resulted in a marked reduction of shikonin induced caspase-9 and caspase-3 activities. (C) Inhibition of ER suppressed shikonin induced casapase activity. Prostate cancer cells were pretreated with Salubrinal (10 μM), caspase 3 and 9 activity were performed by colorimetric assay as described in material and method section. (D) ER stress regulates mitochondrial membrane potential in Shikonin treated DU-145 and PC-3 cells. DU-145 and PC-3 cells were treated with either Shikonin (2.5 μΜ) alone or along with pretreatment with salubrinal (10 μM) for 24 h, and then incubated with mitochondria specific dye JC-1 (10 m g/ml aft 37°C for 15 min), and the florescence was monitored using fluorimeter (Excitation 530 nm/Fluorescence 590 nm) for measurement of the mitochondrial membrane potential. Shikonin treatment significantly reduced mitochondrial membrane potential whereas pretreatment of ER stress inhibitor reversed this effect. Data is expressed in means ± SEM and represents the results of three independent experiments.
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Fig6: ER stress inhibition attenuates Shikonin induced mitochondrial apoptosis. (A) DU-145 and PC-3 cells were treated with either Shikonin (2.5 μM) alone for 24 h or pretreated with ER stress inhibitor Salubrinal (10 μM) for 24 h and subsequently treated with Shikonin. Protein expression was measured by western blotting as described in the Methods section. Shikonin treatment resulted in a marked reduction in the expression of Bcl-2 and an increase in the levels of Bax and cleaved PARP when compared with the controls. Pretreatment with ER stress inhibitor Salubrinal led to the increased expression of Bcl-2 and the decreased expression of Bax and cleaved PARP. (B) Pretreatment with caspase inhibitors reversed Shikonin induced caspase activation. Treatment of cells with Shikonin (2.5 μM) for 24 h resulted in a marked reduction of shikonin induced caspase-9 and caspase-3 activities. (C) Inhibition of ER suppressed shikonin induced casapase activity. Prostate cancer cells were pretreated with Salubrinal (10 μM), caspase 3 and 9 activity were performed by colorimetric assay as described in material and method section. (D) ER stress regulates mitochondrial membrane potential in Shikonin treated DU-145 and PC-3 cells. DU-145 and PC-3 cells were treated with either Shikonin (2.5 μΜ) alone or along with pretreatment with salubrinal (10 μM) for 24 h, and then incubated with mitochondria specific dye JC-1 (10 m g/ml aft 37°C for 15 min), and the florescence was monitored using fluorimeter (Excitation 530 nm/Fluorescence 590 nm) for measurement of the mitochondrial membrane potential. Shikonin treatment significantly reduced mitochondrial membrane potential whereas pretreatment of ER stress inhibitor reversed this effect. Data is expressed in means ± SEM and represents the results of three independent experiments.
Mentions: To investigate the mitochondrial apoptotic events involved in shikonin-induced apoptosis, the levels of the antiapoptotic protein Bcl-2 and the pro-apoptotic protein Bax were analyzed. Western blot analysis showed that the treatment of DU-145 and PC-3 cells with Shikonin resulted in a marked reduction in the expression of Bcl-2 and increased the level of Bax and cleaved PARP when compared with untreated cells (Figure 6A). These results suggested that shikonin alters the levels of pro- and anti-apoptotic proteins of the Bcl-2 family in a manner that contributes to the shikonin-induced apoptosis. Salubrinal is an inhibitor of the serine/threonine phosphatase PP1 and inhibits eIF2α dephosphorylation that in turn blocks ER stress-induced cell death [35,36]. The inhibition of ER stress in DU-145 and PC-3 cells by salubrinal led to the increased expression of Bcl-2 and decreased the levels of Bax and cleaved PARP, in contrast to cells treated with Shikonin alone, indicating that Shikonin treatment -induced the mitochondrial apoptotic pathway (Figure 6A). Similarly, pretreatment with caspase-9 and caspase-3 inhibitors (Figure 6B) or the ER stress inhibitor Salubrinal (Figure 6C) significantly inhibited the Shikonin induced caspase activation in DU-145 and PC-3 prostate cancer cells. Furthermore, cell viability assays with pretreatment of either the pan caspase inhibitor (Z-VAD-FMK) or the caspase-9 (Z-LEHD-FMK)or caspase-3 inhibitor (Z-DEVD-FMK) confirmed a sequential activation of caspases upon Shikonin treatment (Additional file 2: Figure S3). Further to define the role of ROS-Calcium Stress mediated modulation of mitochondrial membrane potential, we had pretreated prostate cancer with ROS scavengers (NAC,GSH or Catalase) and the calpain inhibitor calpeptin as described in material methods. Our results suggested that both ROS and intracellular calcium signaling are crucial for mitochondrial apoptosis in prostate cancer cells. Additionally we also observed that either inhibition of caspase 9, 3 or Salubrinal prevented Shikonin induced reduction of cell viability (Figure 7A), and formation of apoptotic bodies in these cells (Figure 7B) as a marker of apoptotic cell death. Collectively these results indicated that ER stress played an critical role in the shikonin induced apoptosis of prostate cancer cells.Figure 6

Bottom Line: Moreover, addition of antioxidants attenuated these effects.Shikonin also induced the mitochondrial apoptotic pathway mediated through the enhanced expression of the pro-apoptotic Bax and inhibition of Bcl-2, disruption of the mitochondrial membrane potential (MMP) followed by the activation of caspase-9, caspase-3, and PARP cleavage.The results suggest that shikonin could be useful in the therapeutic management of hormone refractory prostate cancers due to its modulation of the pro-apoptotic ER stress and mitochondrial apoptotic pathways.

View Article: PubMed Central - PubMed

Affiliation: Cell Death Research Laboratory, Endocrinology Division CSIR-Central Drug Research Institute, Lucknow, 226031, India. rgara@uthsc.edu.

ABSTRACT

Background: Despite the recent progress in screening and therapy, a majority of prostate cancer cases eventually attain hormone refractory and chemo-resistant attributes. Conventional chemotherapeutic strategies are effective at very high doses for only palliative management of these prostate cancers. Therefore chemo-sensitization of prostate cancer cells could be a promising strategy for increasing efficacy of the conventional chemotherapeutic agents in prostate cancer patients. Recent studies have indicated that the chemo-preventive natural agents restore the pro-apoptotic protein expression and induce endoplasmic reticulum stress (ER stress) leading to the inhibition of cellular proliferation and activation of the mitochondrial apoptosis in prostate cancer cells. Therefore reprogramming ER stress-mitochondrial dependent apoptosis could be a potential approach for management of hormone refractory chemoresistant prostate cancers. We aimed to study the effects of the natural naphthoquinone Shikonin in human prostate cancer cells.

Results: The results indicated that Shikonin induces apoptosis in prostate cancer cells through the dual induction of the endoplasmic reticulum stress and mitochondrial dysfunction. Shikonin induced ROS generation and activated ER stress and calpain activity. Moreover, addition of antioxidants attenuated these effects. Shikonin also induced the mitochondrial apoptotic pathway mediated through the enhanced expression of the pro-apoptotic Bax and inhibition of Bcl-2, disruption of the mitochondrial membrane potential (MMP) followed by the activation of caspase-9, caspase-3, and PARP cleavage.

Conclusion: The results suggest that shikonin could be useful in the therapeutic management of hormone refractory prostate cancers due to its modulation of the pro-apoptotic ER stress and mitochondrial apoptotic pathways.

No MeSH data available.


Related in: MedlinePlus