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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 downregulates the expression of survivin and XIAP and inhibits the proliferation of cancer cells.Notes: (A) PANC-1 cells were transfected with either scramble siRNA or survivin siRNA for the indicated durations. Expression of different proteins was determined by Western blotting. The numbers under each blot are the intensities of the blot relative to that of the control (scramble). (B) Cancer cells were transfected with either empty plasmid or pSur/AS-Sur for the indicated durations. The expression of survivin, XIAP, and Bcl-2 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). (C) Cancer cells were transfected with pSur/AS-Sur for 4 days. Cell proliferation was evaluated using the BrdU proliferation assay. “***” denotes statistical significance with a P-value <0.001 between the testing groups. (D) PANC-1 and HUVECs were transfected with either an empty plasmid or pSur/AS-Sur for 4 days. Cytotoxicity was evaluated using the LDH assay. “**” denotes statistical significance with a P-value <0.01 between the testing groups. “NS” denotes no statistical significant difference between the testing groups. (E) Expression of survivin in KB-TAX50 and MCF7-TamC3 cells was determined by Western blotting. (F) Expression of MDR1 in KB and KB-TAX50 cells was determined by RT-PCR. (G) KB-TAX50 and MCF7-TamC3 cells were transfected with pSur/AS-Sur for 4 days. Cell proliferation was evaluated using the BrdU proliferation assay. “***” denotes statistical significance with a P-value <0.001 between the testing groups.Abbreviations: HUVEC, human umbilical vein endothelial cell; RT-PCR, reverse transcription-polymerase chain reaction; siRNA, small interfering RNA; h, hours; BrdU, Bromodeoxyuridine; XIAP, X-linked inhibitor of apoptosis protein; pSur/AS-Sur, survivin promoter-driven antisense survivin.
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f3-ott-9-2601: Delivery of pSur/AS-Sur downregulates the expression of survivin and XIAP and inhibits the proliferation of cancer cells.Notes: (A) PANC-1 cells were transfected with either scramble siRNA or survivin siRNA for the indicated durations. Expression of different proteins was determined by Western blotting. The numbers under each blot are the intensities of the blot relative to that of the control (scramble). (B) Cancer cells were transfected with either empty plasmid or pSur/AS-Sur for the indicated durations. The expression of survivin, XIAP, and Bcl-2 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). (C) Cancer cells were transfected with pSur/AS-Sur for 4 days. Cell proliferation was evaluated using the BrdU proliferation assay. “***” denotes statistical significance with a P-value <0.001 between the testing groups. (D) PANC-1 and HUVECs were transfected with either an empty plasmid or pSur/AS-Sur for 4 days. Cytotoxicity was evaluated using the LDH assay. “**” denotes statistical significance with a P-value <0.01 between the testing groups. “NS” denotes no statistical significant difference between the testing groups. (E) Expression of survivin in KB-TAX50 and MCF7-TamC3 cells was determined by Western blotting. (F) Expression of MDR1 in KB and KB-TAX50 cells was determined by RT-PCR. (G) KB-TAX50 and MCF7-TamC3 cells were transfected with pSur/AS-Sur for 4 days. Cell proliferation was evaluated using the BrdU proliferation assay. “***” denotes statistical significance with a P-value <0.001 between the testing groups.Abbreviations: HUVEC, human umbilical vein endothelial cell; RT-PCR, reverse transcription-polymerase chain reaction; siRNA, small interfering RNA; h, hours; BrdU, Bromodeoxyuridine; XIAP, X-linked inhibitor of apoptosis protein; pSur/AS-Sur, survivin promoter-driven antisense survivin.

Mentions: Next, we evaluated the specificity of pSur/AS-Sur in targeting survivin in A549, MDA-MB-231, and PANC-1 cells. At the molecular level, survivin stabilizes XIAP through physical interactions with Smad/DIABLO, and the targeting survivin has been shown to downregulate XIAP expression in cancer cells.32,33 Here, Western blot analysis revealed that the downregulation of survivin expression by siRNA decreased XIAP expression as expected (Figure 3A). Similar to the results of cancer cells treated with survivin siRNA, delivery of pSur/AS-Sur concurrently downregulated the expression of survivin and XIAP in A549, MDA-MB-231, and PANC-1 cells in a time-dependent manner (Figure 3B).


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 downregulates the expression of survivin and XIAP and inhibits the proliferation of cancer cells.Notes: (A) PANC-1 cells were transfected with either scramble siRNA or survivin siRNA for the indicated durations. Expression of different proteins was determined by Western blotting. The numbers under each blot are the intensities of the blot relative to that of the control (scramble). (B) Cancer cells were transfected with either empty plasmid or pSur/AS-Sur for the indicated durations. The expression of survivin, XIAP, and Bcl-2 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). (C) Cancer cells were transfected with pSur/AS-Sur for 4 days. Cell proliferation was evaluated using the BrdU proliferation assay. “***” denotes statistical significance with a P-value <0.001 between the testing groups. (D) PANC-1 and HUVECs were transfected with either an empty plasmid or pSur/AS-Sur for 4 days. Cytotoxicity was evaluated using the LDH assay. “**” denotes statistical significance with a P-value <0.01 between the testing groups. “NS” denotes no statistical significant difference between the testing groups. (E) Expression of survivin in KB-TAX50 and MCF7-TamC3 cells was determined by Western blotting. (F) Expression of MDR1 in KB and KB-TAX50 cells was determined by RT-PCR. (G) KB-TAX50 and MCF7-TamC3 cells were transfected with pSur/AS-Sur for 4 days. Cell proliferation was evaluated using the BrdU proliferation assay. “***” denotes statistical significance with a P-value <0.001 between the testing groups.Abbreviations: HUVEC, human umbilical vein endothelial cell; RT-PCR, reverse transcription-polymerase chain reaction; siRNA, small interfering RNA; h, hours; BrdU, Bromodeoxyuridine; XIAP, X-linked inhibitor of apoptosis protein; 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

f3-ott-9-2601: Delivery of pSur/AS-Sur downregulates the expression of survivin and XIAP and inhibits the proliferation of cancer cells.Notes: (A) PANC-1 cells were transfected with either scramble siRNA or survivin siRNA for the indicated durations. Expression of different proteins was determined by Western blotting. The numbers under each blot are the intensities of the blot relative to that of the control (scramble). (B) Cancer cells were transfected with either empty plasmid or pSur/AS-Sur for the indicated durations. The expression of survivin, XIAP, and Bcl-2 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). (C) Cancer cells were transfected with pSur/AS-Sur for 4 days. Cell proliferation was evaluated using the BrdU proliferation assay. “***” denotes statistical significance with a P-value <0.001 between the testing groups. (D) PANC-1 and HUVECs were transfected with either an empty plasmid or pSur/AS-Sur for 4 days. Cytotoxicity was evaluated using the LDH assay. “**” denotes statistical significance with a P-value <0.01 between the testing groups. “NS” denotes no statistical significant difference between the testing groups. (E) Expression of survivin in KB-TAX50 and MCF7-TamC3 cells was determined by Western blotting. (F) Expression of MDR1 in KB and KB-TAX50 cells was determined by RT-PCR. (G) KB-TAX50 and MCF7-TamC3 cells were transfected with pSur/AS-Sur for 4 days. Cell proliferation was evaluated using the BrdU proliferation assay. “***” denotes statistical significance with a P-value <0.001 between the testing groups.Abbreviations: HUVEC, human umbilical vein endothelial cell; RT-PCR, reverse transcription-polymerase chain reaction; siRNA, small interfering RNA; h, hours; BrdU, Bromodeoxyuridine; XIAP, X-linked inhibitor of apoptosis protein; pSur/AS-Sur, survivin promoter-driven antisense survivin.
Mentions: Next, we evaluated the specificity of pSur/AS-Sur in targeting survivin in A549, MDA-MB-231, and PANC-1 cells. At the molecular level, survivin stabilizes XIAP through physical interactions with Smad/DIABLO, and the targeting survivin has been shown to downregulate XIAP expression in cancer cells.32,33 Here, Western blot analysis revealed that the downregulation of survivin expression by siRNA decreased XIAP expression as expected (Figure 3A). Similar to the results of cancer cells treated with survivin siRNA, delivery of pSur/AS-Sur concurrently downregulated the expression of survivin and XIAP in A549, MDA-MB-231, and PANC-1 cells in a time-dependent manner (Figure 3B).

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