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Glucose starvation-mediated inhibition of salinomycin induced autophagy amplifies cancer cell specific cell death.

Jangamreddy JR, Jain MV, Hallbeck AL, Roberg K, Lotfi K, Łos MJ - Oncotarget (2015)

Bottom Line: Numerous studies show that Salinomycin not only reduces tumor volume, but also decreases tumor recurrence when used as an adjuvant to standard treatments.Our in vitro studies further demonstrate that the combined use of 2-Fluoro 2-deoxy D-glucose, or 2-deoxy D-glucose with Salinomycin is lethal in cancer cells while the use of Oxamate does not improve cell death-inducing properties of Salinomycin.Thus, this study underlines the potential use of Salinomycin as a cancer treatment, possibly in combination with short-term starvation or starvation-mimicking pharmacologic intervention.

View Article: PubMed Central - PubMed

Affiliation: Department of Clinical & Experimental Medicine (IKE), Division of Cell Biology, Integrative Regenerative Medical Center (IGEN), Linköping University, Linköping, Sweden.

ABSTRACT
Salinomycin has been used as treatment for malignant tumors in a small number of humans, causing far less side effects than standard chemotherapy. Several studies show that Salinomycin targets cancer-initiating cells (cancer stem cells, or CSC) resistant to conventional therapies. Numerous studies show that Salinomycin not only reduces tumor volume, but also decreases tumor recurrence when used as an adjuvant to standard treatments. In this study we show that starvation triggered different stress responses in cancer cells and primary normal cells, which further improved the preferential targeting of cancer cells by Salinomycin. Our in vitro studies further demonstrate that the combined use of 2-Fluoro 2-deoxy D-glucose, or 2-deoxy D-glucose with Salinomycin is lethal in cancer cells while the use of Oxamate does not improve cell death-inducing properties of Salinomycin. Furthermore, we show that treatment of cancer cells with Salinomycin under starvation conditions not only increases the apoptotic caspase activity, but also diminishes the protective autophagy normally triggered by the treatment with Salinomycin alone. Thus, this study underlines the potential use of Salinomycin as a cancer treatment, possibly in combination with short-term starvation or starvation-mimicking pharmacologic intervention.

No MeSH data available.


Related in: MedlinePlus

Assessment of cell death mechanism triggered by Salinomycin – effects of starvation(A) PC3 cells were grown to reach full confluency for 48h, using normal media (10% FBS and 4.5 g/L glucose). The cell culture medium for control cells was replaced with normal medium, where as the experimental samples were provided with media containing 1% FBS and 0.75 g/L glucose, after washing with PBS. Salinomycin was added to experimental samples 1h after respective media replacements, and 24h-incubation followed. The forms of cell death, and the effects of serum- or glucose starvation on Salinomycin's toxicity were assessed by flow cytometry. Apoptosis was detected by Po-Pro staining, and necrosis was assessed by 7AAD-staining. (B) To confirm that apoptosis may have been induced by Salinomycin under starvation conditions PARP1-cleavage (indicator of active caspase-3 or -7) was detected by Western blotting in cells prestarved as in “A”, and then treated with Salinomycin for 24h. (C and D) To check which caspases were involved in Salinomycin-induced cell death, under normal, or starvation conditions, the activity of caspases-3, -8 and -9 was assessed by flow cytometry, in a similar experimental settings as in “A”, (N=3, *p<0.05, **p<0.01). Control sample were non-treated cells but handled and stained similarly to experimental samples. The control ‘mean fluorescence intensity’ (MFI) was considered as 100% and a relative change in MFI among test samples was calculated accordingly.
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Figure 5: Assessment of cell death mechanism triggered by Salinomycin – effects of starvation(A) PC3 cells were grown to reach full confluency for 48h, using normal media (10% FBS and 4.5 g/L glucose). The cell culture medium for control cells was replaced with normal medium, where as the experimental samples were provided with media containing 1% FBS and 0.75 g/L glucose, after washing with PBS. Salinomycin was added to experimental samples 1h after respective media replacements, and 24h-incubation followed. The forms of cell death, and the effects of serum- or glucose starvation on Salinomycin's toxicity were assessed by flow cytometry. Apoptosis was detected by Po-Pro staining, and necrosis was assessed by 7AAD-staining. (B) To confirm that apoptosis may have been induced by Salinomycin under starvation conditions PARP1-cleavage (indicator of active caspase-3 or -7) was detected by Western blotting in cells prestarved as in “A”, and then treated with Salinomycin for 24h. (C and D) To check which caspases were involved in Salinomycin-induced cell death, under normal, or starvation conditions, the activity of caspases-3, -8 and -9 was assessed by flow cytometry, in a similar experimental settings as in “A”, (N=3, *p<0.05, **p<0.01). Control sample were non-treated cells but handled and stained similarly to experimental samples. The control ‘mean fluorescence intensity’ (MFI) was considered as 100% and a relative change in MFI among test samples was calculated accordingly.

Mentions: In our initial experiments we have used MTT assay, which only assesses cell survival, cell proliferation, but cannot distinguish between apoptosis and necrosis. To assess the form of cell death initiated by Salinomycin under tested experimental conditions, we have employed the flow-cytometry-based Po-Pro/7-AAD assay. High Po-Pro staining is indicative for apoptosis, whereas high 7-AAD signal indicates compromised cell membranes and necrosis; also late apoptotic cells stain with for both dyes. Under glucose starvation, and in the presence of Salinomycin, dying cells stained with both Po-Pro and 7-AAD, thus indicating apoptosis induction under such experimental conditions (Fig. 5A). To further elucidate the molecular mechanism of cell death under combined starvation and Salinomycin treatment, we checked for signs of executioner-caspase-3 (or -7) activity by using the cleavage of PARP1 as indicator (Fig. 5B). Indeed, Salinomycin treatments amplified glucose starvation-triggered PARP1-cleavage. To gain more insight into the molecular mechanism of cell death, under tested experimental conditions, we directly measured the activity of caspases-3, -8, and -9 by flow cytometry. Combination of glucose starvation and Salinomycin potentiated the activation of all three caspases tested, thus confirming that the combination of starvation and Salinomycin induces caspase-dependent cell death (Fig. 5C and 5D).


Glucose starvation-mediated inhibition of salinomycin induced autophagy amplifies cancer cell specific cell death.

Jangamreddy JR, Jain MV, Hallbeck AL, Roberg K, Lotfi K, Łos MJ - Oncotarget (2015)

Assessment of cell death mechanism triggered by Salinomycin – effects of starvation(A) PC3 cells were grown to reach full confluency for 48h, using normal media (10% FBS and 4.5 g/L glucose). The cell culture medium for control cells was replaced with normal medium, where as the experimental samples were provided with media containing 1% FBS and 0.75 g/L glucose, after washing with PBS. Salinomycin was added to experimental samples 1h after respective media replacements, and 24h-incubation followed. The forms of cell death, and the effects of serum- or glucose starvation on Salinomycin's toxicity were assessed by flow cytometry. Apoptosis was detected by Po-Pro staining, and necrosis was assessed by 7AAD-staining. (B) To confirm that apoptosis may have been induced by Salinomycin under starvation conditions PARP1-cleavage (indicator of active caspase-3 or -7) was detected by Western blotting in cells prestarved as in “A”, and then treated with Salinomycin for 24h. (C and D) To check which caspases were involved in Salinomycin-induced cell death, under normal, or starvation conditions, the activity of caspases-3, -8 and -9 was assessed by flow cytometry, in a similar experimental settings as in “A”, (N=3, *p<0.05, **p<0.01). Control sample were non-treated cells but handled and stained similarly to experimental samples. The control ‘mean fluorescence intensity’ (MFI) was considered as 100% and a relative change in MFI among test samples was calculated accordingly.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4496345&req=5

Figure 5: Assessment of cell death mechanism triggered by Salinomycin – effects of starvation(A) PC3 cells were grown to reach full confluency for 48h, using normal media (10% FBS and 4.5 g/L glucose). The cell culture medium for control cells was replaced with normal medium, where as the experimental samples were provided with media containing 1% FBS and 0.75 g/L glucose, after washing with PBS. Salinomycin was added to experimental samples 1h after respective media replacements, and 24h-incubation followed. The forms of cell death, and the effects of serum- or glucose starvation on Salinomycin's toxicity were assessed by flow cytometry. Apoptosis was detected by Po-Pro staining, and necrosis was assessed by 7AAD-staining. (B) To confirm that apoptosis may have been induced by Salinomycin under starvation conditions PARP1-cleavage (indicator of active caspase-3 or -7) was detected by Western blotting in cells prestarved as in “A”, and then treated with Salinomycin for 24h. (C and D) To check which caspases were involved in Salinomycin-induced cell death, under normal, or starvation conditions, the activity of caspases-3, -8 and -9 was assessed by flow cytometry, in a similar experimental settings as in “A”, (N=3, *p<0.05, **p<0.01). Control sample were non-treated cells but handled and stained similarly to experimental samples. The control ‘mean fluorescence intensity’ (MFI) was considered as 100% and a relative change in MFI among test samples was calculated accordingly.
Mentions: In our initial experiments we have used MTT assay, which only assesses cell survival, cell proliferation, but cannot distinguish between apoptosis and necrosis. To assess the form of cell death initiated by Salinomycin under tested experimental conditions, we have employed the flow-cytometry-based Po-Pro/7-AAD assay. High Po-Pro staining is indicative for apoptosis, whereas high 7-AAD signal indicates compromised cell membranes and necrosis; also late apoptotic cells stain with for both dyes. Under glucose starvation, and in the presence of Salinomycin, dying cells stained with both Po-Pro and 7-AAD, thus indicating apoptosis induction under such experimental conditions (Fig. 5A). To further elucidate the molecular mechanism of cell death under combined starvation and Salinomycin treatment, we checked for signs of executioner-caspase-3 (or -7) activity by using the cleavage of PARP1 as indicator (Fig. 5B). Indeed, Salinomycin treatments amplified glucose starvation-triggered PARP1-cleavage. To gain more insight into the molecular mechanism of cell death, under tested experimental conditions, we directly measured the activity of caspases-3, -8, and -9 by flow cytometry. Combination of glucose starvation and Salinomycin potentiated the activation of all three caspases tested, thus confirming that the combination of starvation and Salinomycin induces caspase-dependent cell death (Fig. 5C and 5D).

Bottom Line: Numerous studies show that Salinomycin not only reduces tumor volume, but also decreases tumor recurrence when used as an adjuvant to standard treatments.Our in vitro studies further demonstrate that the combined use of 2-Fluoro 2-deoxy D-glucose, or 2-deoxy D-glucose with Salinomycin is lethal in cancer cells while the use of Oxamate does not improve cell death-inducing properties of Salinomycin.Thus, this study underlines the potential use of Salinomycin as a cancer treatment, possibly in combination with short-term starvation or starvation-mimicking pharmacologic intervention.

View Article: PubMed Central - PubMed

Affiliation: Department of Clinical & Experimental Medicine (IKE), Division of Cell Biology, Integrative Regenerative Medical Center (IGEN), Linköping University, Linköping, Sweden.

ABSTRACT
Salinomycin has been used as treatment for malignant tumors in a small number of humans, causing far less side effects than standard chemotherapy. Several studies show that Salinomycin targets cancer-initiating cells (cancer stem cells, or CSC) resistant to conventional therapies. Numerous studies show that Salinomycin not only reduces tumor volume, but also decreases tumor recurrence when used as an adjuvant to standard treatments. In this study we show that starvation triggered different stress responses in cancer cells and primary normal cells, which further improved the preferential targeting of cancer cells by Salinomycin. Our in vitro studies further demonstrate that the combined use of 2-Fluoro 2-deoxy D-glucose, or 2-deoxy D-glucose with Salinomycin is lethal in cancer cells while the use of Oxamate does not improve cell death-inducing properties of Salinomycin. Furthermore, we show that treatment of cancer cells with Salinomycin under starvation conditions not only increases the apoptotic caspase activity, but also diminishes the protective autophagy normally triggered by the treatment with Salinomycin alone. Thus, this study underlines the potential use of Salinomycin as a cancer treatment, possibly in combination with short-term starvation or starvation-mimicking pharmacologic intervention.

No MeSH data available.


Related in: MedlinePlus