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Delivery of a survivin promoter-driven antisense survivin-expressing plasmid DNA as a cancer therapeutic: a proof-of-concept study.

Lin KY, Cheng SM, Tsai SL, Tsai JY, Lin CH, Cheung CH - Onco Targets Ther (2016)

Bottom Line: Western blot analysis showed that liposomal delivery of pSur/AS-Sur successfully downregulated the expression of survivin in A549, MBA-MB-231, and PANC-1 cells in vitro.In addition, delivery of pSur/AS-Sur induced autophagy, caspase-dependent apoptosis, and caspase-independent apoptosis as indicated by the increased LC3B-II conversion, autophagosome formation, caspase-9/-3 and poly(ADP-ribose) polymerase-1 cleavage, and apoptosis-inducing factor nuclear translocation in A549, MBA-MB-231, and PANC-1 cells.In conclusion, the results of this study suggest that delivery of a survivin promoter-driven antisense survivin-expressing plasmid DNA is a promising way to target survivin and to treat survivin-expressing cancers in the future.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, College of Medicine, National Cheng Kung University, Tainan, Taiwan, ROC.

ABSTRACT
Survivin is a member of the inhibitor-of-apoptosis proteins family. It is overexpressed in many different cancer types but not in the differentiated normal tissue. In addition, overexpression of survivin promotes cancer cell survival and induces chemotherapeutic drug resistance, making it an attractive target for new anticancer interventions. Despite survivin being a promising molecular target for anticancer treatment, it is widely accepted that survivin is only a "semi-druggable" target. Therefore, it is important to develop a new strategy to target survivin for anticancer treatment. In this study, we constructed a novel survivin promoter-driven full-length antisense survivin (pSur/AS-Sur) expression plasmid DNA. Promoter activity assay revealed that the activity of the survivin promoter of pSur/AS-Sur correlated with the endogenous expression of survivin at the transcriptional level in the transfected A549, MDA-MB-231, and PANC-1 cancer cells. Western blot analysis showed that liposomal delivery of pSur/AS-Sur successfully downregulated the expression of survivin in A549, MBA-MB-231, and PANC-1 cells in vitro. In addition, delivery of pSur/AS-Sur induced autophagy, caspase-dependent apoptosis, and caspase-independent apoptosis as indicated by the increased LC3B-II conversion, autophagosome formation, caspase-9/-3 and poly(ADP-ribose) polymerase-1 cleavage, and apoptosis-inducing factor nuclear translocation in A549, MBA-MB-231, and PANC-1 cells. Importantly, liposomal delivery of pSur/AS-Sur was also capable of decreasing the proliferation of the survivin/MDR1 coexpressing multidrug-resistant KB-TAX50 cancer cells and the estrogen receptor-positive tamoxifen-resistant MCF7-TamC3 cancer cells in vitro. In conclusion, the results of this study suggest that delivery of a survivin promoter-driven antisense survivin-expressing plasmid DNA is a promising way to target survivin and to treat survivin-expressing cancers in the future.

No MeSH data available.


Related in: MedlinePlus

Delivery of pSur/AS-Sur modulates autophagy in cancer cells.Notes: (A) Cancer cells were transfected with either empty plasmid or pSur/AS-Sur for the indicated durations. The conversion of LC3B-II and the expression of γH2AX were examined by Western blotting. Equal protein loading was verified by actin. The numbers under each blot are the intensities of the blot relative to that of the control (empty plasmid). (B) Cancer cells were transfected with EGFP-tagged LC3B with or without pSur/AS-Sur cotransfection for 48 hours. LC3 puncta formation in cells (~200 cells) was observed under a fluorescence microscope. The number of puncta present in cells was analyzed using the ImageJ software. (C) Cancer cells were transfected with pSur/AS-Sur for 48 hours and then stained with MDC. The number of puncta present in cells (~200 cells) was analyzed using the ImageJ software. “*,” “**,” and “***” denote statistical significance with P-values <0.05, <0.01, and <0.001, respectively, between the testing groups.Abbreviations: h, hours; MDC, monodansylcadaverine; pSur/AS-Sur, survivin promoter-driven antisense survivin.
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f4-ott-9-2601: Delivery of pSur/AS-Sur modulates autophagy in cancer cells.Notes: (A) Cancer cells were transfected with either empty plasmid or pSur/AS-Sur for the indicated durations. The conversion of LC3B-II and the expression of γH2AX were examined by Western blotting. Equal protein loading was verified by actin. The numbers under each blot are the intensities of the blot relative to that of the control (empty plasmid). (B) Cancer cells were transfected with EGFP-tagged LC3B with or without pSur/AS-Sur cotransfection for 48 hours. LC3 puncta formation in cells (~200 cells) was observed under a fluorescence microscope. The number of puncta present in cells was analyzed using the ImageJ software. (C) Cancer cells were transfected with pSur/AS-Sur for 48 hours and then stained with MDC. The number of puncta present in cells (~200 cells) was analyzed using the ImageJ software. “*,” “**,” and “***” denote statistical significance with P-values <0.05, <0.01, and <0.001, respectively, between the testing groups.Abbreviations: h, hours; MDC, monodansylcadaverine; pSur/AS-Sur, survivin promoter-driven antisense survivin.

Mentions: Recent studies showed that downregulation of survivin by either pharmacological inhibitor or siRNA induces autophagy in cancer cells.33,35,36 In particular, our previous study revealed that downregulation of survivin by YM155 induced autophagy and autophagy-dependent DNA damage in breast cancer cells.33 To further confirm the target specificity of pSur/AS-Sur, we determined whether delivery of pSur/AS-Sur can also induce autophagy and DNA damage in cancer cells. The Western blot analysis revealed that downregulating survivin expression by siRNA increased LC3B-II conversion and γ-H2AX expression, which is a molecular marker for autophagy and DNA damage, respectively, as expected (Figure 3A). Similar to these results, delivery of pSur/AS-Sur also increased LC3B-II conversion and γH2AX expression in A549, MDA-MB-231, and PANC-1 cells in a time-dependent manner (Figure 4A). To further confirm the effect of pSur/AS-Sur on autophagy modulation, cancer cells were cotransfected with pSur/AS-Sur and a plasmid that overexpresses the EGFP-tagged LC3B and formation of autophagosome/autophagolysosome was determined by fluorescence microscopy. Fluorescence microscopy revealed that EGFP-LC3B was overexpressed and equally distributed inside the cells under pSur/AS-Sur-free conditions (Figure 4B). Delivery of pSur/AS-Sur significantly increased the formation of EGFP-LC3B punctate (green fluorescent punctate) in A549, MDA-MB-231, and PANC-1 cells, indicating the formation of autophagosome and/or autophagolysosome (Figure 4B). Cancer cells were also stained with MDC to further confirm the formation of AVOs, which is also a marker for autophagy, in cells with pSur/AS-Sur delivery. MDC is a fluorescent compound widely used for the detection of AVOs, including lysosome and autophagolysosome. Increased number and/or size of punctate formed in the MDC-stained cells are considered as the formation of AVOs. Fluorescence microscopy again revealed that delivery of pSur/AS-Sur significantly increased the number of AVOs present in cells, indicating the induction of autophagy, further supporting that delivery of pSur/AS-Sur can modulate autophagy in survivin-expressing cancer cells (Figure 4C).


Delivery of a survivin promoter-driven antisense survivin-expressing plasmid DNA as a cancer therapeutic: a proof-of-concept study.

Lin KY, Cheng SM, Tsai SL, Tsai JY, Lin CH, Cheung CH - Onco Targets Ther (2016)

Delivery of pSur/AS-Sur modulates autophagy in cancer cells.Notes: (A) Cancer cells were transfected with either empty plasmid or pSur/AS-Sur for the indicated durations. The conversion of LC3B-II and the expression of γH2AX were examined by Western blotting. Equal protein loading was verified by actin. The numbers under each blot are the intensities of the blot relative to that of the control (empty plasmid). (B) Cancer cells were transfected with EGFP-tagged LC3B with or without pSur/AS-Sur cotransfection for 48 hours. LC3 puncta formation in cells (~200 cells) was observed under a fluorescence microscope. The number of puncta present in cells was analyzed using the ImageJ software. (C) Cancer cells were transfected with pSur/AS-Sur for 48 hours and then stained with MDC. The number of puncta present in cells (~200 cells) was analyzed using the ImageJ software. “*,” “**,” and “***” denote statistical significance with P-values <0.05, <0.01, and <0.001, respectively, between the testing groups.Abbreviations: h, hours; MDC, monodansylcadaverine; pSur/AS-Sur, survivin promoter-driven antisense survivin.
© Copyright Policy
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4862386&req=5

f4-ott-9-2601: Delivery of pSur/AS-Sur modulates autophagy in cancer cells.Notes: (A) Cancer cells were transfected with either empty plasmid or pSur/AS-Sur for the indicated durations. The conversion of LC3B-II and the expression of γH2AX were examined by Western blotting. Equal protein loading was verified by actin. The numbers under each blot are the intensities of the blot relative to that of the control (empty plasmid). (B) Cancer cells were transfected with EGFP-tagged LC3B with or without pSur/AS-Sur cotransfection for 48 hours. LC3 puncta formation in cells (~200 cells) was observed under a fluorescence microscope. The number of puncta present in cells was analyzed using the ImageJ software. (C) Cancer cells were transfected with pSur/AS-Sur for 48 hours and then stained with MDC. The number of puncta present in cells (~200 cells) was analyzed using the ImageJ software. “*,” “**,” and “***” denote statistical significance with P-values <0.05, <0.01, and <0.001, respectively, between the testing groups.Abbreviations: h, hours; MDC, monodansylcadaverine; pSur/AS-Sur, survivin promoter-driven antisense survivin.
Mentions: Recent studies showed that downregulation of survivin by either pharmacological inhibitor or siRNA induces autophagy in cancer cells.33,35,36 In particular, our previous study revealed that downregulation of survivin by YM155 induced autophagy and autophagy-dependent DNA damage in breast cancer cells.33 To further confirm the target specificity of pSur/AS-Sur, we determined whether delivery of pSur/AS-Sur can also induce autophagy and DNA damage in cancer cells. The Western blot analysis revealed that downregulating survivin expression by siRNA increased LC3B-II conversion and γ-H2AX expression, which is a molecular marker for autophagy and DNA damage, respectively, as expected (Figure 3A). Similar to these results, delivery of pSur/AS-Sur also increased LC3B-II conversion and γH2AX expression in A549, MDA-MB-231, and PANC-1 cells in a time-dependent manner (Figure 4A). To further confirm the effect of pSur/AS-Sur on autophagy modulation, cancer cells were cotransfected with pSur/AS-Sur and a plasmid that overexpresses the EGFP-tagged LC3B and formation of autophagosome/autophagolysosome was determined by fluorescence microscopy. Fluorescence microscopy revealed that EGFP-LC3B was overexpressed and equally distributed inside the cells under pSur/AS-Sur-free conditions (Figure 4B). Delivery of pSur/AS-Sur significantly increased the formation of EGFP-LC3B punctate (green fluorescent punctate) in A549, MDA-MB-231, and PANC-1 cells, indicating the formation of autophagosome and/or autophagolysosome (Figure 4B). Cancer cells were also stained with MDC to further confirm the formation of AVOs, which is also a marker for autophagy, in cells with pSur/AS-Sur delivery. MDC is a fluorescent compound widely used for the detection of AVOs, including lysosome and autophagolysosome. Increased number and/or size of punctate formed in the MDC-stained cells are considered as the formation of AVOs. Fluorescence microscopy again revealed that delivery of pSur/AS-Sur significantly increased the number of AVOs present in cells, indicating the induction of autophagy, further supporting that delivery of pSur/AS-Sur can modulate autophagy in survivin-expressing cancer cells (Figure 4C).

Bottom Line: Western blot analysis showed that liposomal delivery of pSur/AS-Sur successfully downregulated the expression of survivin in A549, MBA-MB-231, and PANC-1 cells in vitro.In addition, delivery of pSur/AS-Sur induced autophagy, caspase-dependent apoptosis, and caspase-independent apoptosis as indicated by the increased LC3B-II conversion, autophagosome formation, caspase-9/-3 and poly(ADP-ribose) polymerase-1 cleavage, and apoptosis-inducing factor nuclear translocation in A549, MBA-MB-231, and PANC-1 cells.In conclusion, the results of this study suggest that delivery of a survivin promoter-driven antisense survivin-expressing plasmid DNA is a promising way to target survivin and to treat survivin-expressing cancers in the future.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, College of Medicine, National Cheng Kung University, Tainan, Taiwan, ROC.

ABSTRACT
Survivin is a member of the inhibitor-of-apoptosis proteins family. It is overexpressed in many different cancer types but not in the differentiated normal tissue. In addition, overexpression of survivin promotes cancer cell survival and induces chemotherapeutic drug resistance, making it an attractive target for new anticancer interventions. Despite survivin being a promising molecular target for anticancer treatment, it is widely accepted that survivin is only a "semi-druggable" target. Therefore, it is important to develop a new strategy to target survivin for anticancer treatment. In this study, we constructed a novel survivin promoter-driven full-length antisense survivin (pSur/AS-Sur) expression plasmid DNA. Promoter activity assay revealed that the activity of the survivin promoter of pSur/AS-Sur correlated with the endogenous expression of survivin at the transcriptional level in the transfected A549, MDA-MB-231, and PANC-1 cancer cells. Western blot analysis showed that liposomal delivery of pSur/AS-Sur successfully downregulated the expression of survivin in A549, MBA-MB-231, and PANC-1 cells in vitro. In addition, delivery of pSur/AS-Sur induced autophagy, caspase-dependent apoptosis, and caspase-independent apoptosis as indicated by the increased LC3B-II conversion, autophagosome formation, caspase-9/-3 and poly(ADP-ribose) polymerase-1 cleavage, and apoptosis-inducing factor nuclear translocation in A549, MBA-MB-231, and PANC-1 cells. Importantly, liposomal delivery of pSur/AS-Sur was also capable of decreasing the proliferation of the survivin/MDR1 coexpressing multidrug-resistant KB-TAX50 cancer cells and the estrogen receptor-positive tamoxifen-resistant MCF7-TamC3 cancer cells in vitro. In conclusion, the results of this study suggest that delivery of a survivin promoter-driven antisense survivin-expressing plasmid DNA is a promising way to target survivin and to treat survivin-expressing cancers in the future.

No MeSH data available.


Related in: MedlinePlus