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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 treatment induces ROS generation, increase in Intracellular Calcium in prostate cancer cells. (A-B) Shikonin induces ROS generation in prostate cancer cells. DU-145 (A) and PC-3 (B) cells were seeded in 6-well plates overnight in the presence or absence of Shikonin, and incubated with DCF-DA 50 μM for 30 min, washed three time with ice cold PBS. Generation of ROS was measured using flourimetry as per standard protocol. (C) Shikonin induces increase in intracellular Calcium in Shikonin treated prostate cancer cells. The level of intracellular calcium in DU-145 and PC-3 cells were determined using flourimetry technique in cells using the Fluo-4AM (50 μM/ml) dye in Shikonin treated prostate cancer cells as described in material and methods. (D) Shikonin treatment induces calpain activity in prostate cancer cells. Shikonin treated prostate cancer cells were analyzed for calpain activity as described in as described in material and methods. (E-F) Antioxidant pretreatment reverses shikonin induced increase in the intracellular calcium. DU-145 and PC-3 cells were pretreated with NAC (20 μM), GSH (10 mM) or Catalase (20 nM) for 2 hours and the ROS inhibitor rescued intracellular calcium release was measured as described in the Methods section. Values are represented as Mean ± SEM, from three independent experiments.
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Fig2: Shikonin treatment induces ROS generation, increase in Intracellular Calcium in prostate cancer cells. (A-B) Shikonin induces ROS generation in prostate cancer cells. DU-145 (A) and PC-3 (B) cells were seeded in 6-well plates overnight in the presence or absence of Shikonin, and incubated with DCF-DA 50 μM for 30 min, washed three time with ice cold PBS. Generation of ROS was measured using flourimetry as per standard protocol. (C) Shikonin induces increase in intracellular Calcium in Shikonin treated prostate cancer cells. The level of intracellular calcium in DU-145 and PC-3 cells were determined using flourimetry technique in cells using the Fluo-4AM (50 μM/ml) dye in Shikonin treated prostate cancer cells as described in material and methods. (D) Shikonin treatment induces calpain activity in prostate cancer cells. Shikonin treated prostate cancer cells were analyzed for calpain activity as described in as described in material and methods. (E-F) Antioxidant pretreatment reverses shikonin induced increase in the intracellular calcium. DU-145 and PC-3 cells were pretreated with NAC (20 μM), GSH (10 mM) or Catalase (20 nM) for 2 hours and the ROS inhibitor rescued intracellular calcium release was measured as described in the Methods section. Values are represented as Mean ± SEM, from three independent experiments.

Mentions: There is a close relationship between ROS and Ca2+ in apoptotic signal transduction pathways [20]. Shikonin is known to influence intracellular Ca2+ [20] and ROS generation in different cancer cells types [21-23] but similar modulation of intracellular Ca2+ in hormonal refractory prostate cancer cells has not been elucidated. Our results indicated that Shikonin treatment induces time dependent ROS generation, which could be rescued with pretreatment of antioxidants (NAC, GSH and Catalase) in both DU-145 (Figure 2A) and PC-3 (Figure 2B) cells. ROS mediated free intracellular Ca2+ is known to be involved in ER stress induced apoptotic signaling [24]. Therefore we further investigated for these changes, in DU-145 and PC-3 cell, treated with Shikonin. We observed a marked increase in the intracellular Ca2+ levels (Figure 2C) and Calcium-Activated Neutral Protease (Calpain) activity as early as 60 min (Figure 2D) in shikonin treated DU-145 and PC-3 cells. Pretreatment with ROS inhibitors (NAC, GSH and Catalase) attenuated ROS-induced intracellular Ca2+ levels in DU-145 (Figure 2E) and PC-3 cells (Figure 2F). Excessive generation of ROS renders the cells oxidatively stressed and impairs membrane proteins, leading to mitochondrial dysfunction and apoptotic cell death [25]. Shikonin treatment induced significant loss of mitochondrial membrane potential (Figure 1A) and modulated pro-anti-apoptotic mitochondrial proteins expression (Figure 1B) in these cells. Our western blot analysis showed that shikonin activated expression of the upstream proteins Bax, cytochrome c as well as downstream caspases 7 and AIF in both prostate cancer cell types (Figure 1B). Moreover, pre-incubation with ROS inhibitors (NAC, GSH and Catalase) attenuated these effect in both cell types (Figure 1C). Collectively these results indicated that Shikonin treatment induces ROS generation, increases in intracellular calcium levels and ROS-dependent mitochondrial apoptosis in prostate cancer cells.Figure 2


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 treatment induces ROS generation, increase in Intracellular Calcium in prostate cancer cells. (A-B) Shikonin induces ROS generation in prostate cancer cells. DU-145 (A) and PC-3 (B) cells were seeded in 6-well plates overnight in the presence or absence of Shikonin, and incubated with DCF-DA 50 μM for 30 min, washed three time with ice cold PBS. Generation of ROS was measured using flourimetry as per standard protocol. (C) Shikonin induces increase in intracellular Calcium in Shikonin treated prostate cancer cells. The level of intracellular calcium in DU-145 and PC-3 cells were determined using flourimetry technique in cells using the Fluo-4AM (50 μM/ml) dye in Shikonin treated prostate cancer cells as described in material and methods. (D) Shikonin treatment induces calpain activity in prostate cancer cells. Shikonin treated prostate cancer cells were analyzed for calpain activity as described in as described in material and methods. (E-F) Antioxidant pretreatment reverses shikonin induced increase in the intracellular calcium. DU-145 and PC-3 cells were pretreated with NAC (20 μM), GSH (10 mM) or Catalase (20 nM) for 2 hours and the ROS inhibitor rescued intracellular calcium release was measured as described in the Methods section. Values are represented as Mean ± SEM, from three independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Fig2: Shikonin treatment induces ROS generation, increase in Intracellular Calcium in prostate cancer cells. (A-B) Shikonin induces ROS generation in prostate cancer cells. DU-145 (A) and PC-3 (B) cells were seeded in 6-well plates overnight in the presence or absence of Shikonin, and incubated with DCF-DA 50 μM for 30 min, washed three time with ice cold PBS. Generation of ROS was measured using flourimetry as per standard protocol. (C) Shikonin induces increase in intracellular Calcium in Shikonin treated prostate cancer cells. The level of intracellular calcium in DU-145 and PC-3 cells were determined using flourimetry technique in cells using the Fluo-4AM (50 μM/ml) dye in Shikonin treated prostate cancer cells as described in material and methods. (D) Shikonin treatment induces calpain activity in prostate cancer cells. Shikonin treated prostate cancer cells were analyzed for calpain activity as described in as described in material and methods. (E-F) Antioxidant pretreatment reverses shikonin induced increase in the intracellular calcium. DU-145 and PC-3 cells were pretreated with NAC (20 μM), GSH (10 mM) or Catalase (20 nM) for 2 hours and the ROS inhibitor rescued intracellular calcium release was measured as described in the Methods section. Values are represented as Mean ± SEM, from three independent experiments.
Mentions: There is a close relationship between ROS and Ca2+ in apoptotic signal transduction pathways [20]. Shikonin is known to influence intracellular Ca2+ [20] and ROS generation in different cancer cells types [21-23] but similar modulation of intracellular Ca2+ in hormonal refractory prostate cancer cells has not been elucidated. Our results indicated that Shikonin treatment induces time dependent ROS generation, which could be rescued with pretreatment of antioxidants (NAC, GSH and Catalase) in both DU-145 (Figure 2A) and PC-3 (Figure 2B) cells. ROS mediated free intracellular Ca2+ is known to be involved in ER stress induced apoptotic signaling [24]. Therefore we further investigated for these changes, in DU-145 and PC-3 cell, treated with Shikonin. We observed a marked increase in the intracellular Ca2+ levels (Figure 2C) and Calcium-Activated Neutral Protease (Calpain) activity as early as 60 min (Figure 2D) in shikonin treated DU-145 and PC-3 cells. Pretreatment with ROS inhibitors (NAC, GSH and Catalase) attenuated ROS-induced intracellular Ca2+ levels in DU-145 (Figure 2E) and PC-3 cells (Figure 2F). Excessive generation of ROS renders the cells oxidatively stressed and impairs membrane proteins, leading to mitochondrial dysfunction and apoptotic cell death [25]. Shikonin treatment induced significant loss of mitochondrial membrane potential (Figure 1A) and modulated pro-anti-apoptotic mitochondrial proteins expression (Figure 1B) in these cells. Our western blot analysis showed that shikonin activated expression of the upstream proteins Bax, cytochrome c as well as downstream caspases 7 and AIF in both prostate cancer cell types (Figure 1B). Moreover, pre-incubation with ROS inhibitors (NAC, GSH and Catalase) attenuated these effect in both cell types (Figure 1C). Collectively these results indicated that Shikonin treatment induces ROS generation, increases in intracellular calcium levels and ROS-dependent mitochondrial apoptosis in prostate cancer cells.Figure 2

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