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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 selectively induces apoptosis in prostate cancer cells. (A) Shikonin treatment inhibits prostate cancer cell viability without affecting normal prostate epithelial cells. DU-145, PC-3 and PrEC cells were treated with various doses (0.5, 1, 2.5, 5 and 10 μM) of Shikonin for 24 h and CCK-8 assay was used for the measurement of cell viability. (B) Shikonin treatment induces DNA fragmentation in prostate cancer cells. Cells were treated with various doses (0.5,1,2.5, 5 and 10 μM) of Shikonin for 24 h and TUNEL assay was done using flow cytometry and the % TUNEL positive cells were measured. (C) Shikonin treatment induces PARP activation. Cells were treated with 2.5 μM of Shikonin for 24 h and the cell lysates were analyzed by western blotting for the detection of cleaved PARP, a marker for PARP activation. (D) Shikonin treatment induces caspase-3 activation. Cells were treated with various doses of Shikonin (0.5, 1, 2.5, 5 and 10 μM) for 48 h and measurement of caspase-3 activity was done using the CASPASE-3 Colorimetric Activity Assay kit. Data is expressed in means ± SEM and represents the results of three independent experiments (p < 0.05).
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Fig1: Shikonin treatment selectively induces apoptosis in prostate cancer cells. (A) Shikonin treatment inhibits prostate cancer cell viability without affecting normal prostate epithelial cells. DU-145, PC-3 and PrEC cells were treated with various doses (0.5, 1, 2.5, 5 and 10 μM) of Shikonin for 24 h and CCK-8 assay was used for the measurement of cell viability. (B) Shikonin treatment induces DNA fragmentation in prostate cancer cells. Cells were treated with various doses (0.5,1,2.5, 5 and 10 μM) of Shikonin for 24 h and TUNEL assay was done using flow cytometry and the % TUNEL positive cells were measured. (C) Shikonin treatment induces PARP activation. Cells were treated with 2.5 μM of Shikonin for 24 h and the cell lysates were analyzed by western blotting for the detection of cleaved PARP, a marker for PARP activation. (D) Shikonin treatment induces caspase-3 activation. Cells were treated with various doses of Shikonin (0.5, 1, 2.5, 5 and 10 μM) for 48 h and measurement of caspase-3 activity was done using the CASPASE-3 Colorimetric Activity Assay kit. Data is expressed in means ± SEM and represents the results of three independent experiments (p < 0.05).

Mentions: Hormone refractory prostate cancer cells (DU-145 and PC-3) were treated with various doses of Shikonin (0.5, 1, 2.5, 5 and 10 μM) for 24 h. Cell viability was determined using the CCK-8 assay kit. The results indicated that the IC50 dose of Shikonin was ~ 5.0 μM in DU-145 cells and ~4.5 μM in PC-3 respectively. Consistent with earlier studies shikonin inhibited viability of a variety of non-prostate cancer cell types while sparing the normal cell types (Additional file 2: Figure S1). Furthermore, Shikonin inhibited proliferation of prostate cancer cells in a dose dependent manner (Additional file 2: Figure S2). While no significant (p > 0.05) effect on cell viability was observed in normal prostate epithelia cells (PrEc). Since a significant (~42%, p < 0.05) decrease in the cell viability was observed in both these cell lines treated with 2.5 μM of dose of Shikonin (Figure 1A), we selected this dose for the mechanistic experiments of the study. DNA fragmentation a characteristic of the apoptotic cells was quantified by the flowcytometry based TUNEL assay in Shikonin treated cells. The results depicted a significant (p < 0.05) increase in the TUNEL positive cells with Shikonin treatment (Figure 1B). Cleavage of PARP, a highly conserved 116 kDa nuclear poly (ADP-ribose) polymerase, is implicated in the apoptotic response of cells. Immunoblotting experiments clearly showed that Shikonin treatment induced PARP activation characterized by the cleaved PARP fragment in DU-145 and PC-3 cells, interestingly we did not observed any PARP clevage in PrEcs (Figure 1C). Activation of caspase-3 like proteases play a crucial role in apoptotic cell death, therefore we next determined the caspase-3 activities in Shikonin treated and DU-145 and PC-3 cells. A ~5.8-fold increase in the caspase-3 activities over control cells was observed in these cell lines (Figure 1D; p < 0.05). Collectively, these results indicated that shikonin inhibited proliferation of the prostate cancer cells through induction of apoptotic cell death.Figure 1


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 selectively induces apoptosis in prostate cancer cells. (A) Shikonin treatment inhibits prostate cancer cell viability without affecting normal prostate epithelial cells. DU-145, PC-3 and PrEC cells were treated with various doses (0.5, 1, 2.5, 5 and 10 μM) of Shikonin for 24 h and CCK-8 assay was used for the measurement of cell viability. (B) Shikonin treatment induces DNA fragmentation in prostate cancer cells. Cells were treated with various doses (0.5,1,2.5, 5 and 10 μM) of Shikonin for 24 h and TUNEL assay was done using flow cytometry and the % TUNEL positive cells were measured. (C) Shikonin treatment induces PARP activation. Cells were treated with 2.5 μM of Shikonin for 24 h and the cell lysates were analyzed by western blotting for the detection of cleaved PARP, a marker for PARP activation. (D) Shikonin treatment induces caspase-3 activation. Cells were treated with various doses of Shikonin (0.5, 1, 2.5, 5 and 10 μM) for 48 h and measurement of caspase-3 activity was done using the CASPASE-3 Colorimetric Activity Assay kit. Data is expressed in means ± SEM and represents the results of three independent experiments (p < 0.05).
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4389804&req=5

Fig1: Shikonin treatment selectively induces apoptosis in prostate cancer cells. (A) Shikonin treatment inhibits prostate cancer cell viability without affecting normal prostate epithelial cells. DU-145, PC-3 and PrEC cells were treated with various doses (0.5, 1, 2.5, 5 and 10 μM) of Shikonin for 24 h and CCK-8 assay was used for the measurement of cell viability. (B) Shikonin treatment induces DNA fragmentation in prostate cancer cells. Cells were treated with various doses (0.5,1,2.5, 5 and 10 μM) of Shikonin for 24 h and TUNEL assay was done using flow cytometry and the % TUNEL positive cells were measured. (C) Shikonin treatment induces PARP activation. Cells were treated with 2.5 μM of Shikonin for 24 h and the cell lysates were analyzed by western blotting for the detection of cleaved PARP, a marker for PARP activation. (D) Shikonin treatment induces caspase-3 activation. Cells were treated with various doses of Shikonin (0.5, 1, 2.5, 5 and 10 μM) for 48 h and measurement of caspase-3 activity was done using the CASPASE-3 Colorimetric Activity Assay kit. Data is expressed in means ± SEM and represents the results of three independent experiments (p < 0.05).
Mentions: Hormone refractory prostate cancer cells (DU-145 and PC-3) were treated with various doses of Shikonin (0.5, 1, 2.5, 5 and 10 μM) for 24 h. Cell viability was determined using the CCK-8 assay kit. The results indicated that the IC50 dose of Shikonin was ~ 5.0 μM in DU-145 cells and ~4.5 μM in PC-3 respectively. Consistent with earlier studies shikonin inhibited viability of a variety of non-prostate cancer cell types while sparing the normal cell types (Additional file 2: Figure S1). Furthermore, Shikonin inhibited proliferation of prostate cancer cells in a dose dependent manner (Additional file 2: Figure S2). While no significant (p > 0.05) effect on cell viability was observed in normal prostate epithelia cells (PrEc). Since a significant (~42%, p < 0.05) decrease in the cell viability was observed in both these cell lines treated with 2.5 μM of dose of Shikonin (Figure 1A), we selected this dose for the mechanistic experiments of the study. DNA fragmentation a characteristic of the apoptotic cells was quantified by the flowcytometry based TUNEL assay in Shikonin treated cells. The results depicted a significant (p < 0.05) increase in the TUNEL positive cells with Shikonin treatment (Figure 1B). Cleavage of PARP, a highly conserved 116 kDa nuclear poly (ADP-ribose) polymerase, is implicated in the apoptotic response of cells. Immunoblotting experiments clearly showed that Shikonin treatment induced PARP activation characterized by the cleaved PARP fragment in DU-145 and PC-3 cells, interestingly we did not observed any PARP clevage in PrEcs (Figure 1C). Activation of caspase-3 like proteases play a crucial role in apoptotic cell death, therefore we next determined the caspase-3 activities in Shikonin treated and DU-145 and PC-3 cells. A ~5.8-fold increase in the caspase-3 activities over control cells was observed in these cell lines (Figure 1D; p < 0.05). Collectively, these results indicated that shikonin inhibited proliferation of the prostate cancer cells through induction of apoptotic cell death.Figure 1

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