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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

Shikonin induces ROS dependent mitochondrial apoptosis in prostate cancer cells. (A) Shikonin modulated mitochondrial membrane potential in DU-145 and PC-3 cells. The integrity of mitochondrial membrane potential (Δψ) was measured by JC-1 mitochondrial dye. Briefly DU-145 and PC-3 cells were treated with 2.5 μM for 0–24 h. Ratio of 590/530 nm was measured by flow cytometry as described in material and methods. Data is expressed in means ± SEM and represent the results of three independent experiments (p < 0.05). (B) Shikonin treatment alters expression of mitochondrial proteins in DU-145 and PC-3 cells. Cells were treated with Shikonin for 0-24 h, expression of indicated mitochondrial proteins were performed as described in Methods. Experiments were repeated minimum two-four times. (C) Shikonin induced ROS dependent mitochondrial apoptosis in DU-145 and PC-3. Cells were pretreatment with the antioxidants NAC (20 μM) GSH (10 mM) and Catalase (20 nM) and western bolting experiments were performed as described in Methods section. Data is represents the results of three independent experiments.
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Fig3: Shikonin induces ROS dependent mitochondrial apoptosis in prostate cancer cells. (A) Shikonin modulated mitochondrial membrane potential in DU-145 and PC-3 cells. The integrity of mitochondrial membrane potential (Δψ) was measured by JC-1 mitochondrial dye. Briefly DU-145 and PC-3 cells were treated with 2.5 μM for 0–24 h. Ratio of 590/530 nm was measured by flow cytometry as described in material and methods. Data is expressed in means ± SEM and represent the results of three independent experiments (p < 0.05). (B) Shikonin treatment alters expression of mitochondrial proteins in DU-145 and PC-3 cells. Cells were treated with Shikonin for 0-24 h, expression of indicated mitochondrial proteins were performed as described in Methods. Experiments were repeated minimum two-four times. (C) Shikonin induced ROS dependent mitochondrial apoptosis in DU-145 and PC-3. Cells were pretreatment with the antioxidants NAC (20 μM) GSH (10 mM) and Catalase (20 nM) and western bolting experiments were performed as described in Methods section. Data is represents the results of three independent experiments.

Mentions: As the previous results suggested that Shikonin treatment induced intracellular Ca2,+ calpain activation and generation of ROS in prostate cancer cells (Figures 2 and 3), we therefore explored whether Shikonin can activate ROS dependent ER stress in prostate cancer cells. Recent studies have established that drug induced increase in ROS generation, intracellular Ca2+ and activation of ER stress leads to mitochondrial apoptosis in cancer cells [26-28]. Therefore, we further examined ER stress associated proteins in these cell lines. First, the phosphorylation patterns of PERK and eIF2α were assessed, since PERK, an ER-resident transmembrane kinase, is known to auto phosphorylate its cytoplasmic kinase domain in response to accumulated unfolded proteins in the ER lumen and activated PERK is subsequently capable of phosphorylating several cytosolic proteins including eIF2α [29-32]. Western blot analysis revealed that treatment of cells with Shikonin (2.5 μM) led to an increase in the phosphorylation of PERK up to 24 h and eIF2α for up to 9 h of treatment (Figure 4A). The expression of GRP78/Bip, which serves as a gatekeeper to the activation of ER stress transducers [33,34] was also examined. The results indicated that treatment with Shikonin significantly increased the expression of GRP78/Bip and CHOP/GADD135 and led to a decrease in the expression of procaspase-3 were ROS dependent manner (Figure 4B). These results collectively suggested that the intracellular ROS were directly involved in the regulation of ER stress induced by shikonin in prostate cancer cells.Figure 3


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)

Shikonin induces ROS dependent mitochondrial apoptosis in prostate cancer cells. (A) Shikonin modulated mitochondrial membrane potential in DU-145 and PC-3 cells. The integrity of mitochondrial membrane potential (Δψ) was measured by JC-1 mitochondrial dye. Briefly DU-145 and PC-3 cells were treated with 2.5 μM for 0–24 h. Ratio of 590/530 nm was measured by flow cytometry as described in material and methods. Data is expressed in means ± SEM and represent the results of three independent experiments (p < 0.05). (B) Shikonin treatment alters expression of mitochondrial proteins in DU-145 and PC-3 cells. Cells were treated with Shikonin for 0-24 h, expression of indicated mitochondrial proteins were performed as described in Methods. Experiments were repeated minimum two-four times. (C) Shikonin induced ROS dependent mitochondrial apoptosis in DU-145 and PC-3. Cells were pretreatment with the antioxidants NAC (20 μM) GSH (10 mM) and Catalase (20 nM) and western bolting experiments were performed as described in Methods section. Data is represents the results of three independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Fig3: Shikonin induces ROS dependent mitochondrial apoptosis in prostate cancer cells. (A) Shikonin modulated mitochondrial membrane potential in DU-145 and PC-3 cells. The integrity of mitochondrial membrane potential (Δψ) was measured by JC-1 mitochondrial dye. Briefly DU-145 and PC-3 cells were treated with 2.5 μM for 0–24 h. Ratio of 590/530 nm was measured by flow cytometry as described in material and methods. Data is expressed in means ± SEM and represent the results of three independent experiments (p < 0.05). (B) Shikonin treatment alters expression of mitochondrial proteins in DU-145 and PC-3 cells. Cells were treated with Shikonin for 0-24 h, expression of indicated mitochondrial proteins were performed as described in Methods. Experiments were repeated minimum two-four times. (C) Shikonin induced ROS dependent mitochondrial apoptosis in DU-145 and PC-3. Cells were pretreatment with the antioxidants NAC (20 μM) GSH (10 mM) and Catalase (20 nM) and western bolting experiments were performed as described in Methods section. Data is represents the results of three independent experiments.
Mentions: As the previous results suggested that Shikonin treatment induced intracellular Ca2,+ calpain activation and generation of ROS in prostate cancer cells (Figures 2 and 3), we therefore explored whether Shikonin can activate ROS dependent ER stress in prostate cancer cells. Recent studies have established that drug induced increase in ROS generation, intracellular Ca2+ and activation of ER stress leads to mitochondrial apoptosis in cancer cells [26-28]. Therefore, we further examined ER stress associated proteins in these cell lines. First, the phosphorylation patterns of PERK and eIF2α were assessed, since PERK, an ER-resident transmembrane kinase, is known to auto phosphorylate its cytoplasmic kinase domain in response to accumulated unfolded proteins in the ER lumen and activated PERK is subsequently capable of phosphorylating several cytosolic proteins including eIF2α [29-32]. Western blot analysis revealed that treatment of cells with Shikonin (2.5 μM) led to an increase in the phosphorylation of PERK up to 24 h and eIF2α for up to 9 h of treatment (Figure 4A). The expression of GRP78/Bip, which serves as a gatekeeper to the activation of ER stress transducers [33,34] was also examined. The results indicated that treatment with Shikonin significantly increased the expression of GRP78/Bip and CHOP/GADD135 and led to a decrease in the expression of procaspase-3 were ROS dependent manner (Figure 4B). These results collectively suggested that the intracellular ROS were directly involved in the regulation of ER stress induced by shikonin in prostate cancer cells.Figure 3

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