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Overexpression of 17β-hydroxysteroid dehydrogenase type 10 increases pheochromocytoma cell growth and resistance to cell death.

Carlson EA, Marquez RT, Du F, Wang Y, Xu L, Yan SS - BMC Cancer (2015)

Bottom Line: Across disease states, increased HSD10 levels can have a profound and varied impact, such as beneficial in Parkinson's disease and harmful in Alzheimer's disease.In this study, we examined the tumor-promoting effect of HSD10 in pheochromocytoma cells.Our findings demonstrate that overexpression of HSD10 accelerates pheochromocytoma cell growth, enhances cell respiration, and increases cellular resistance to cell death induction.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology & Toxicology, University of Kansas, Lawrence, KS, 66047, USA. e086c574@ku.edu.

ABSTRACT

Background: 17β-hydroxysteroid dehydrogenase type 10 (HSD10) has been shown to play a protective role in cells undergoing stress. Upregulation of HSD10 under nutrient-limiting conditions leads to recovery of a homeostatic state. Across disease states, increased HSD10 levels can have a profound and varied impact, such as beneficial in Parkinson's disease and harmful in Alzheimer's disease. Recently, HSD10 overexpression has been observed in some prostate and bone cancers, consistently correlating with poor patient prognosis. As the role of HSD10 in cancer remains underexplored, we propose that cancer cells utilize this enzyme to promote cancer cell survival under cell death conditions.

Methods: The proliferative effect of HSD10 was examined in transfected pheochromocytoma cells by growth curve analysis and a xenograft model. Fluctuations in mitochondrial bioenergetics were evaluated by electron transport chain complex enzyme activity assays and energy production. Additionally, the effect of HSD10 on pheochromocytoma resistance to cell death was investigated using TUNEL staining, MTT, and complex IV enzyme activity assays.

Results: In this study, we examined the tumor-promoting effect of HSD10 in pheochromocytoma cells. Overexpression of HSD10 increased pheochromocytoma cell growth in both in vitro cell culture and an in vivo xenograft mouse model. The increases in respiratory enzymes and energy generation observed in HSD10-overexpressing cells likely supported the accelerated growth rate observed. Furthermore, cells overexpressing HSD10 were more resistant to oxidative stress-induced perturbation.

Conclusions: Our findings demonstrate that overexpression of HSD10 accelerates pheochromocytoma cell growth, enhances cell respiration, and increases cellular resistance to cell death induction. This suggests that blockade of HSD10 may halt and/or prevent cancer growth, thus providing a promising novel target for cancer patients as a screening or therapeutic option.

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Effect of HSD10-modification on CypD and how it may influence cancer cell growth and death. A. EV and HSD10 ov whole cell lysates were analyzed for CypD protein expression using immunoblotting. β-actin was used as the loading control, and CypD was normalized to actin (n = 4). B. Control shRNA and HSD10 shRNA whole cell lysates were analyzed for CypD protein expression using immunoblotting. β-actin was used as the loading control, and CypD was normalized to actin (n = 4). C. EV and HSD10 ov whole cell lysates were analyzed for HSD10-CypD complexes using co-immunoprecipitation. β-actin was used as the loading control for the input. The immunoblots demonstrate an increased HSD10-CypD interaction in PC-12 cells overexpressing HSD10 compared to EV cells. D. Confocal microscopy demonstrating immunofluorescence staining of HSD10 alone (red), CypD alone (green), and these two antigens co-localized (yellow) in EV and HSD10 ov cells. E. Immunofluorescence staining of HSD10 alone (red), mitochondrial marker SODII alone (green), and these two antigens co-localized (yellow) in HSD10 ov cells. F. Immunofluorescence staining of CypD alone (green), mitochondrial marker Hsp60 alone (red), and these two antigens co-localized (yellow) in HSD10 ov cells. Scale bar in F: 20 μm. G-H. Quantification of HSD10 and CypD fluorescence densities (depicted in D) displayed as fold increase (n = 4). Data presented as mean ± SE. *P < 0.01 versus transfected control group. I. Hypothetical mechanism of action for HSD10-mediated cancer cell growth. Top panel, cells overexpressing HSD10 bind to CypD and sequester it in the mitochondrial matrix, thereby avoiding cell death induction; this resistance allows for continued cancer cell proliferation. Bottom panel, cells under-expressing HSD10 cannot bind all of the available CypD; thus unbound CypD translocates to the IM where it induces cell death via MPTP opening.
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Fig6: Effect of HSD10-modification on CypD and how it may influence cancer cell growth and death. A. EV and HSD10 ov whole cell lysates were analyzed for CypD protein expression using immunoblotting. β-actin was used as the loading control, and CypD was normalized to actin (n = 4). B. Control shRNA and HSD10 shRNA whole cell lysates were analyzed for CypD protein expression using immunoblotting. β-actin was used as the loading control, and CypD was normalized to actin (n = 4). C. EV and HSD10 ov whole cell lysates were analyzed for HSD10-CypD complexes using co-immunoprecipitation. β-actin was used as the loading control for the input. The immunoblots demonstrate an increased HSD10-CypD interaction in PC-12 cells overexpressing HSD10 compared to EV cells. D. Confocal microscopy demonstrating immunofluorescence staining of HSD10 alone (red), CypD alone (green), and these two antigens co-localized (yellow) in EV and HSD10 ov cells. E. Immunofluorescence staining of HSD10 alone (red), mitochondrial marker SODII alone (green), and these two antigens co-localized (yellow) in HSD10 ov cells. F. Immunofluorescence staining of CypD alone (green), mitochondrial marker Hsp60 alone (red), and these two antigens co-localized (yellow) in HSD10 ov cells. Scale bar in F: 20 μm. G-H. Quantification of HSD10 and CypD fluorescence densities (depicted in D) displayed as fold increase (n = 4). Data presented as mean ± SE. *P < 0.01 versus transfected control group. I. Hypothetical mechanism of action for HSD10-mediated cancer cell growth. Top panel, cells overexpressing HSD10 bind to CypD and sequester it in the mitochondrial matrix, thereby avoiding cell death induction; this resistance allows for continued cancer cell proliferation. Bottom panel, cells under-expressing HSD10 cannot bind all of the available CypD; thus unbound CypD translocates to the IM where it induces cell death via MPTP opening.

Mentions: Next, we sought to determine the mechanism behind the ability of HSD10 to regulate cancer cell growth and cell death resistance associated with mitochondrial function. We examined the relationship between HSD10 and CypD, which is a modulator of MPTP-opening and cell death induction under stress conditions. We postulate that HSD10 aids in cancer cell resistance by preventing MPTP-induced cell death via enhanced binding to CypD. Using immunoblot analysis, we determined that CypD protein expression remains similar between HSD10 ov cells and EV cells (Figure 6A). This result is consistent with other studies of CypD in cancer [32]. Interestingly, CypD protein expression was significantly reduced in HSD10 shRNA cells compared with control shRNA cells (Figure 6B). This indicates that while CypD expression remains level during HSD10 overexpression, it is negatively impacted by HSD10 reduction. We suggest that, due to the reductions in both HSD10 and CypD, cancer cells become more susceptible to cell death induction.Figure 6


Overexpression of 17β-hydroxysteroid dehydrogenase type 10 increases pheochromocytoma cell growth and resistance to cell death.

Carlson EA, Marquez RT, Du F, Wang Y, Xu L, Yan SS - BMC Cancer (2015)

Effect of HSD10-modification on CypD and how it may influence cancer cell growth and death. A. EV and HSD10 ov whole cell lysates were analyzed for CypD protein expression using immunoblotting. β-actin was used as the loading control, and CypD was normalized to actin (n = 4). B. Control shRNA and HSD10 shRNA whole cell lysates were analyzed for CypD protein expression using immunoblotting. β-actin was used as the loading control, and CypD was normalized to actin (n = 4). C. EV and HSD10 ov whole cell lysates were analyzed for HSD10-CypD complexes using co-immunoprecipitation. β-actin was used as the loading control for the input. The immunoblots demonstrate an increased HSD10-CypD interaction in PC-12 cells overexpressing HSD10 compared to EV cells. D. Confocal microscopy demonstrating immunofluorescence staining of HSD10 alone (red), CypD alone (green), and these two antigens co-localized (yellow) in EV and HSD10 ov cells. E. Immunofluorescence staining of HSD10 alone (red), mitochondrial marker SODII alone (green), and these two antigens co-localized (yellow) in HSD10 ov cells. F. Immunofluorescence staining of CypD alone (green), mitochondrial marker Hsp60 alone (red), and these two antigens co-localized (yellow) in HSD10 ov cells. Scale bar in F: 20 μm. G-H. Quantification of HSD10 and CypD fluorescence densities (depicted in D) displayed as fold increase (n = 4). Data presented as mean ± SE. *P < 0.01 versus transfected control group. I. Hypothetical mechanism of action for HSD10-mediated cancer cell growth. Top panel, cells overexpressing HSD10 bind to CypD and sequester it in the mitochondrial matrix, thereby avoiding cell death induction; this resistance allows for continued cancer cell proliferation. Bottom panel, cells under-expressing HSD10 cannot bind all of the available CypD; thus unbound CypD translocates to the IM where it induces cell death via MPTP opening.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4384325&req=5

Fig6: Effect of HSD10-modification on CypD and how it may influence cancer cell growth and death. A. EV and HSD10 ov whole cell lysates were analyzed for CypD protein expression using immunoblotting. β-actin was used as the loading control, and CypD was normalized to actin (n = 4). B. Control shRNA and HSD10 shRNA whole cell lysates were analyzed for CypD protein expression using immunoblotting. β-actin was used as the loading control, and CypD was normalized to actin (n = 4). C. EV and HSD10 ov whole cell lysates were analyzed for HSD10-CypD complexes using co-immunoprecipitation. β-actin was used as the loading control for the input. The immunoblots demonstrate an increased HSD10-CypD interaction in PC-12 cells overexpressing HSD10 compared to EV cells. D. Confocal microscopy demonstrating immunofluorescence staining of HSD10 alone (red), CypD alone (green), and these two antigens co-localized (yellow) in EV and HSD10 ov cells. E. Immunofluorescence staining of HSD10 alone (red), mitochondrial marker SODII alone (green), and these two antigens co-localized (yellow) in HSD10 ov cells. F. Immunofluorescence staining of CypD alone (green), mitochondrial marker Hsp60 alone (red), and these two antigens co-localized (yellow) in HSD10 ov cells. Scale bar in F: 20 μm. G-H. Quantification of HSD10 and CypD fluorescence densities (depicted in D) displayed as fold increase (n = 4). Data presented as mean ± SE. *P < 0.01 versus transfected control group. I. Hypothetical mechanism of action for HSD10-mediated cancer cell growth. Top panel, cells overexpressing HSD10 bind to CypD and sequester it in the mitochondrial matrix, thereby avoiding cell death induction; this resistance allows for continued cancer cell proliferation. Bottom panel, cells under-expressing HSD10 cannot bind all of the available CypD; thus unbound CypD translocates to the IM where it induces cell death via MPTP opening.
Mentions: Next, we sought to determine the mechanism behind the ability of HSD10 to regulate cancer cell growth and cell death resistance associated with mitochondrial function. We examined the relationship between HSD10 and CypD, which is a modulator of MPTP-opening and cell death induction under stress conditions. We postulate that HSD10 aids in cancer cell resistance by preventing MPTP-induced cell death via enhanced binding to CypD. Using immunoblot analysis, we determined that CypD protein expression remains similar between HSD10 ov cells and EV cells (Figure 6A). This result is consistent with other studies of CypD in cancer [32]. Interestingly, CypD protein expression was significantly reduced in HSD10 shRNA cells compared with control shRNA cells (Figure 6B). This indicates that while CypD expression remains level during HSD10 overexpression, it is negatively impacted by HSD10 reduction. We suggest that, due to the reductions in both HSD10 and CypD, cancer cells become more susceptible to cell death induction.Figure 6

Bottom Line: Across disease states, increased HSD10 levels can have a profound and varied impact, such as beneficial in Parkinson's disease and harmful in Alzheimer's disease.In this study, we examined the tumor-promoting effect of HSD10 in pheochromocytoma cells.Our findings demonstrate that overexpression of HSD10 accelerates pheochromocytoma cell growth, enhances cell respiration, and increases cellular resistance to cell death induction.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology & Toxicology, University of Kansas, Lawrence, KS, 66047, USA. e086c574@ku.edu.

ABSTRACT

Background: 17β-hydroxysteroid dehydrogenase type 10 (HSD10) has been shown to play a protective role in cells undergoing stress. Upregulation of HSD10 under nutrient-limiting conditions leads to recovery of a homeostatic state. Across disease states, increased HSD10 levels can have a profound and varied impact, such as beneficial in Parkinson's disease and harmful in Alzheimer's disease. Recently, HSD10 overexpression has been observed in some prostate and bone cancers, consistently correlating with poor patient prognosis. As the role of HSD10 in cancer remains underexplored, we propose that cancer cells utilize this enzyme to promote cancer cell survival under cell death conditions.

Methods: The proliferative effect of HSD10 was examined in transfected pheochromocytoma cells by growth curve analysis and a xenograft model. Fluctuations in mitochondrial bioenergetics were evaluated by electron transport chain complex enzyme activity assays and energy production. Additionally, the effect of HSD10 on pheochromocytoma resistance to cell death was investigated using TUNEL staining, MTT, and complex IV enzyme activity assays.

Results: In this study, we examined the tumor-promoting effect of HSD10 in pheochromocytoma cells. Overexpression of HSD10 increased pheochromocytoma cell growth in both in vitro cell culture and an in vivo xenograft mouse model. The increases in respiratory enzymes and energy generation observed in HSD10-overexpressing cells likely supported the accelerated growth rate observed. Furthermore, cells overexpressing HSD10 were more resistant to oxidative stress-induced perturbation.

Conclusions: Our findings demonstrate that overexpression of HSD10 accelerates pheochromocytoma cell growth, enhances cell respiration, and increases cellular resistance to cell death induction. This suggests that blockade of HSD10 may halt and/or prevent cancer growth, thus providing a promising novel target for cancer patients as a screening or therapeutic option.

Show MeSH
Related in: MedlinePlus