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Therapeutic targeting of polo-like kinase 1 using RNA-interfering nanoparticles (iNOPs) for the treatment of non-small cell lung cancer.

McCarroll JA, Dwarte T, Baigude H, Dang J, Yang L, Erlich RB, Kimpton K, Teo J, Sagnella SM, Akerfeldt MC, Liu J, Phillips PA, Rana TM, Kavallaris M - Oncotarget (2015)

Bottom Line: Polo-like kinase 1 (PLK1) is a serine-threonine protein kinase which is overexpressed in cancer cells, and plays a major role in regulating tumor growth.Notably, iNOP-7 delivered siRNA to orthotopic lung tumors in mice, and administration of iNOP-7-PLK1 siRNA reduced lung tumor burden.These novel data show that iNOP-7 can deliver siRNA against PLK1 to NSCLC cells, and decrease cell proliferation both in vitro and in vivo. iNOP-7-PLK1 siRNA may provide a novel therapeutic strategy for the treatment of NSCLC as well as other cancers which aberrantly express this gene.

View Article: PubMed Central - PubMed

Affiliation: Children's Cancer Institute, Lowy Cancer Research Centre, Randwick, UNSW Australia (UNSW), NSW, Australia.

ABSTRACT
Non-small cell lung cancer (NSCLC) remains the most common cause of cancer death worldwide due its resistance to chemotherapy and aggressive tumor growth. Polo-like kinase 1 (PLK1) is a serine-threonine protein kinase which is overexpressed in cancer cells, and plays a major role in regulating tumor growth. A number of PLK1 inhibitors are in clinical trial; however, poor tumor bioavailability and off-target effects limit their efficacy. Short-interfering-RNA (siRNA) holds promise as a class of therapeutics, which can selectively silence disease-causing genes. However, siRNA cannot enter cells without a delivery vehicle. Herein, we investigated whether RNAi-interfering nanoparticles could deliver siRNA to NSCLC cells and silence PLK1 expression in vitro and in vivo. iNOP-7 was non-toxic, and delivered siRNA with high efficiency to NSCLC cells. iNOP-7-PLK1 siRNA silenced PLK1 expression and reduced NSCLC growth in vitro. Notably, iNOP-7 delivered siRNA to orthotopic lung tumors in mice, and administration of iNOP-7-PLK1 siRNA reduced lung tumor burden. These novel data show that iNOP-7 can deliver siRNA against PLK1 to NSCLC cells, and decrease cell proliferation both in vitro and in vivo. iNOP-7-PLK1 siRNA may provide a novel therapeutic strategy for the treatment of NSCLC as well as other cancers which aberrantly express this gene.

No MeSH data available.


Related in: MedlinePlus

iNOP-7 siRNA delivery to NSCLC cells in vitro(A) Representative agarose gel showing free unmodified siRNA (siRNA, 60 ng) alone (lane 2), iNOP-7 alone (lane 3), or increasing amounts of iNOP-7 complexed to siRNA (1:1-8:1 w/w ratio with siRNA) (lanes 5-9). M = molecular weight marker. (B) A graph showing PLK1 knockdown in H1299 cells, 24h post-transfection with increasing amounts of iNOP-7 complexed to PLK1 siRNA (100 nM). Cells incubated in culture media or transfected with PLK1 siRNA complexed to lipofectamine 2000 (L2K) served as controls, n = 3; bars, mean ± SE. *p < 0.01, **p < 0.001. (C) A graph showing no significant toxicity in NSCLC cells (H460, Calu-6) or normal human fibroblasts (MRC-5) when treated with iNOP-7 alone or complexed to non-silencing siRNA (8:1 w/w) 24h post-transfection. Cells incubated in culture medium served as control, n = 3; bars, mean ± SE. (D) Flow cytometry histograms (I and II) and confocal images (III-VI) demonstrating cell uptake of fluorescently labeled-siRNA (green) in NSCLC (H460 and Calu-6) cells complexed to iNOP-7 (8:1 w/w) 24h post-transfection. Fluorescent siRNA (Green), cell membrane (Red), and nuclear DNA (Blue), n = 3 (white arrows show the location of siRNA).
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Figure 3: iNOP-7 siRNA delivery to NSCLC cells in vitro(A) Representative agarose gel showing free unmodified siRNA (siRNA, 60 ng) alone (lane 2), iNOP-7 alone (lane 3), or increasing amounts of iNOP-7 complexed to siRNA (1:1-8:1 w/w ratio with siRNA) (lanes 5-9). M = molecular weight marker. (B) A graph showing PLK1 knockdown in H1299 cells, 24h post-transfection with increasing amounts of iNOP-7 complexed to PLK1 siRNA (100 nM). Cells incubated in culture media or transfected with PLK1 siRNA complexed to lipofectamine 2000 (L2K) served as controls, n = 3; bars, mean ± SE. *p < 0.01, **p < 0.001. (C) A graph showing no significant toxicity in NSCLC cells (H460, Calu-6) or normal human fibroblasts (MRC-5) when treated with iNOP-7 alone or complexed to non-silencing siRNA (8:1 w/w) 24h post-transfection. Cells incubated in culture medium served as control, n = 3; bars, mean ± SE. (D) Flow cytometry histograms (I and II) and confocal images (III-VI) demonstrating cell uptake of fluorescently labeled-siRNA (green) in NSCLC (H460 and Calu-6) cells complexed to iNOP-7 (8:1 w/w) 24h post-transfection. Fluorescent siRNA (Green), cell membrane (Red), and nuclear DNA (Blue), n = 3 (white arrows show the location of siRNA).

Mentions: In the preceding section our data strongly supports PLK1 siRNA inhibition as a potential therapeutic strategy against NSCLC cells. However, an effective siRNA delivery vehicle is required. We previously reported on the synthesis of a novel modified dendrimer nanoparticle (iNOP-7) which could deliver RNAi molecules to the liver of mice and silence the expression of key genes or miRNAs involved in regulating cholesterol metabolism [19–22]. However, whether iNOP-7 could deliver siRNA to tumor cells in vitro and in vivo was unknown. First, we examined how much iNOP-7 was required to fully self-assemble with siRNA via an electrostatic interaction using agarose gel electrophoresis. Non-complexed siRNA migrated to the bottom of the gel (Figure 3A). In contrast, increasing amounts of iNOP-7 (2:1-8:1 w/w) complexed to siRNA prevented its migration, confirming our previous studies showing that upon simple mixing iNOP-7 was able to rapidly complex with siRNA [19–21] (Figure 3A). It was evident from these results that at 2:1 (w/w) iNOP-7 could complex approximately 50% siRNA, while at the higher amounts of 4:1-8:1 (w/w) iNOP-7 fully complexed siRNA (Figure 3A). To examine whether the differing iNOP-7-siRNA ratios possessed gene silencing activity, NSCLC cells were transfected with increasing amounts of iNOP-7 [2:1-8:1 (w/w)] complexed to a fixed amount of PLK1 siRNA (100 nM). Cells treated with PLK1 siRNA complexed to lipofectamine 2000 (L2K) or culture medium alone served as controls. iNOP-7 complexed to PLK1 siRNA at all of the chosen ratios [2:1-8:1 (w/w)] displayed gene silencing activity in NSCLC cells (Figure 3B). Notably, iNOP-7-PLK1 siRNA at the ratios of 4:1-8:1 (w/w) silenced PLK1 gene expression by >70% (p < 0.001), which was comparable to L2K (Figure 3B). Based on these results iNOP-7 at an 8:1 (w/w ratio with siRNA) was further characterized as a siRNA delivery vehicle for NSCLC cells.


Therapeutic targeting of polo-like kinase 1 using RNA-interfering nanoparticles (iNOPs) for the treatment of non-small cell lung cancer.

McCarroll JA, Dwarte T, Baigude H, Dang J, Yang L, Erlich RB, Kimpton K, Teo J, Sagnella SM, Akerfeldt MC, Liu J, Phillips PA, Rana TM, Kavallaris M - Oncotarget (2015)

iNOP-7 siRNA delivery to NSCLC cells in vitro(A) Representative agarose gel showing free unmodified siRNA (siRNA, 60 ng) alone (lane 2), iNOP-7 alone (lane 3), or increasing amounts of iNOP-7 complexed to siRNA (1:1-8:1 w/w ratio with siRNA) (lanes 5-9). M = molecular weight marker. (B) A graph showing PLK1 knockdown in H1299 cells, 24h post-transfection with increasing amounts of iNOP-7 complexed to PLK1 siRNA (100 nM). Cells incubated in culture media or transfected with PLK1 siRNA complexed to lipofectamine 2000 (L2K) served as controls, n = 3; bars, mean ± SE. *p < 0.01, **p < 0.001. (C) A graph showing no significant toxicity in NSCLC cells (H460, Calu-6) or normal human fibroblasts (MRC-5) when treated with iNOP-7 alone or complexed to non-silencing siRNA (8:1 w/w) 24h post-transfection. Cells incubated in culture medium served as control, n = 3; bars, mean ± SE. (D) Flow cytometry histograms (I and II) and confocal images (III-VI) demonstrating cell uptake of fluorescently labeled-siRNA (green) in NSCLC (H460 and Calu-6) cells complexed to iNOP-7 (8:1 w/w) 24h post-transfection. Fluorescent siRNA (Green), cell membrane (Red), and nuclear DNA (Blue), n = 3 (white arrows show the location of siRNA).
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Figure 3: iNOP-7 siRNA delivery to NSCLC cells in vitro(A) Representative agarose gel showing free unmodified siRNA (siRNA, 60 ng) alone (lane 2), iNOP-7 alone (lane 3), or increasing amounts of iNOP-7 complexed to siRNA (1:1-8:1 w/w ratio with siRNA) (lanes 5-9). M = molecular weight marker. (B) A graph showing PLK1 knockdown in H1299 cells, 24h post-transfection with increasing amounts of iNOP-7 complexed to PLK1 siRNA (100 nM). Cells incubated in culture media or transfected with PLK1 siRNA complexed to lipofectamine 2000 (L2K) served as controls, n = 3; bars, mean ± SE. *p < 0.01, **p < 0.001. (C) A graph showing no significant toxicity in NSCLC cells (H460, Calu-6) or normal human fibroblasts (MRC-5) when treated with iNOP-7 alone or complexed to non-silencing siRNA (8:1 w/w) 24h post-transfection. Cells incubated in culture medium served as control, n = 3; bars, mean ± SE. (D) Flow cytometry histograms (I and II) and confocal images (III-VI) demonstrating cell uptake of fluorescently labeled-siRNA (green) in NSCLC (H460 and Calu-6) cells complexed to iNOP-7 (8:1 w/w) 24h post-transfection. Fluorescent siRNA (Green), cell membrane (Red), and nuclear DNA (Blue), n = 3 (white arrows show the location of siRNA).
Mentions: In the preceding section our data strongly supports PLK1 siRNA inhibition as a potential therapeutic strategy against NSCLC cells. However, an effective siRNA delivery vehicle is required. We previously reported on the synthesis of a novel modified dendrimer nanoparticle (iNOP-7) which could deliver RNAi molecules to the liver of mice and silence the expression of key genes or miRNAs involved in regulating cholesterol metabolism [19–22]. However, whether iNOP-7 could deliver siRNA to tumor cells in vitro and in vivo was unknown. First, we examined how much iNOP-7 was required to fully self-assemble with siRNA via an electrostatic interaction using agarose gel electrophoresis. Non-complexed siRNA migrated to the bottom of the gel (Figure 3A). In contrast, increasing amounts of iNOP-7 (2:1-8:1 w/w) complexed to siRNA prevented its migration, confirming our previous studies showing that upon simple mixing iNOP-7 was able to rapidly complex with siRNA [19–21] (Figure 3A). It was evident from these results that at 2:1 (w/w) iNOP-7 could complex approximately 50% siRNA, while at the higher amounts of 4:1-8:1 (w/w) iNOP-7 fully complexed siRNA (Figure 3A). To examine whether the differing iNOP-7-siRNA ratios possessed gene silencing activity, NSCLC cells were transfected with increasing amounts of iNOP-7 [2:1-8:1 (w/w)] complexed to a fixed amount of PLK1 siRNA (100 nM). Cells treated with PLK1 siRNA complexed to lipofectamine 2000 (L2K) or culture medium alone served as controls. iNOP-7 complexed to PLK1 siRNA at all of the chosen ratios [2:1-8:1 (w/w)] displayed gene silencing activity in NSCLC cells (Figure 3B). Notably, iNOP-7-PLK1 siRNA at the ratios of 4:1-8:1 (w/w) silenced PLK1 gene expression by >70% (p < 0.001), which was comparable to L2K (Figure 3B). Based on these results iNOP-7 at an 8:1 (w/w ratio with siRNA) was further characterized as a siRNA delivery vehicle for NSCLC cells.

Bottom Line: Polo-like kinase 1 (PLK1) is a serine-threonine protein kinase which is overexpressed in cancer cells, and plays a major role in regulating tumor growth.Notably, iNOP-7 delivered siRNA to orthotopic lung tumors in mice, and administration of iNOP-7-PLK1 siRNA reduced lung tumor burden.These novel data show that iNOP-7 can deliver siRNA against PLK1 to NSCLC cells, and decrease cell proliferation both in vitro and in vivo. iNOP-7-PLK1 siRNA may provide a novel therapeutic strategy for the treatment of NSCLC as well as other cancers which aberrantly express this gene.

View Article: PubMed Central - PubMed

Affiliation: Children's Cancer Institute, Lowy Cancer Research Centre, Randwick, UNSW Australia (UNSW), NSW, Australia.

ABSTRACT
Non-small cell lung cancer (NSCLC) remains the most common cause of cancer death worldwide due its resistance to chemotherapy and aggressive tumor growth. Polo-like kinase 1 (PLK1) is a serine-threonine protein kinase which is overexpressed in cancer cells, and plays a major role in regulating tumor growth. A number of PLK1 inhibitors are in clinical trial; however, poor tumor bioavailability and off-target effects limit their efficacy. Short-interfering-RNA (siRNA) holds promise as a class of therapeutics, which can selectively silence disease-causing genes. However, siRNA cannot enter cells without a delivery vehicle. Herein, we investigated whether RNAi-interfering nanoparticles could deliver siRNA to NSCLC cells and silence PLK1 expression in vitro and in vivo. iNOP-7 was non-toxic, and delivered siRNA with high efficiency to NSCLC cells. iNOP-7-PLK1 siRNA silenced PLK1 expression and reduced NSCLC growth in vitro. Notably, iNOP-7 delivered siRNA to orthotopic lung tumors in mice, and administration of iNOP-7-PLK1 siRNA reduced lung tumor burden. These novel data show that iNOP-7 can deliver siRNA against PLK1 to NSCLC cells, and decrease cell proliferation both in vitro and in vivo. iNOP-7-PLK1 siRNA may provide a novel therapeutic strategy for the treatment of NSCLC as well as other cancers which aberrantly express this gene.

No MeSH data available.


Related in: MedlinePlus