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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

Establishment of a NSCLC bioluminescent mouse model and iNOP-7 siRNA delivery in vivo(A) Panel I, a representative image and graph showing increased luciferase activity with increasing H1299-Luc NSCLC cell numbers, n = 3 experiments. Panel II, pseudocolor images of mice injected orthotopically with H1299-Luc cells showing an increase in lung bioluminescence at days 7-20 post-tumor cell injection. (B) Representative ex-vivo fluorescent images of lungs and lung tumors, liver and spleen collected from mice with H1299-Luc orthotopic lung tumors, 4h post-injection (intravenous) with fluorescent siRNA (Red) complexed to iNOP-7. Mice injected with iNOP-7-fluorescent siRNA showed high fluorescence in the lung and lung tumors, liver and spleen. Mice injected with fluorescent siRNA alone or PBS served as controls. (C) Panel I, micro-CT image (axial) showing the presence of a growing tumor within the lungs of a mouse prior to injection with iNOP-7-fluorescent siRNA (red-dotted line marks the area of the tumor within the lung). Panels II-IV, are representative confocal microscopy images of frozen sections of lung tumor, liver and spleen showing the presence of fluorescent siRNA when delivered by iNOP-7, 4h post-injection (white arrows mark the location of fluorescent siRNA). (D) Panel I, Immunohistochemical image demonstrating PLK1 expression in orthotopic H1299-Luc tumors growing in the lung of mice. Panel II, immunohistochemical image of the corresponding antibody isotype control.
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Figure 5: Establishment of a NSCLC bioluminescent mouse model and iNOP-7 siRNA delivery in vivo(A) Panel I, a representative image and graph showing increased luciferase activity with increasing H1299-Luc NSCLC cell numbers, n = 3 experiments. Panel II, pseudocolor images of mice injected orthotopically with H1299-Luc cells showing an increase in lung bioluminescence at days 7-20 post-tumor cell injection. (B) Representative ex-vivo fluorescent images of lungs and lung tumors, liver and spleen collected from mice with H1299-Luc orthotopic lung tumors, 4h post-injection (intravenous) with fluorescent siRNA (Red) complexed to iNOP-7. Mice injected with iNOP-7-fluorescent siRNA showed high fluorescence in the lung and lung tumors, liver and spleen. Mice injected with fluorescent siRNA alone or PBS served as controls. (C) Panel I, micro-CT image (axial) showing the presence of a growing tumor within the lungs of a mouse prior to injection with iNOP-7-fluorescent siRNA (red-dotted line marks the area of the tumor within the lung). Panels II-IV, are representative confocal microscopy images of frozen sections of lung tumor, liver and spleen showing the presence of fluorescent siRNA when delivered by iNOP-7, 4h post-injection (white arrows mark the location of fluorescent siRNA). (D) Panel I, Immunohistochemical image demonstrating PLK1 expression in orthotopic H1299-Luc tumors growing in the lung of mice. Panel II, immunohistochemical image of the corresponding antibody isotype control.

Mentions: To examine the therapeutic potential of iNOP-7-PLK1 siRNA in vivo, we developed H1299 NSCLC cells to stably express high levels of firefly luciferase (H1299-Luc) to allow for the measurement of tumor growth in an orthotopic NSCLC mouse model (Figure 5A, panel I). Importantly, lung tumors in this model grow in a microenvironment which closely mimics the human setting. Seven days post-tumor cell inoculation, tumors could be detected in the lungs of mice as evidenced by a strong bioluminescent signal (Figure 5A, panel II). The bioluminescent signal increased over time, and by day 20 diffuse lung tumor growth was observed. To assess whether iNOP-7 could deliver clinically-relevant amounts of siRNA to growing lung tumors, mice (15 days post-tumor cell inoculation) were administered systemically with iNOP-7 complexed to siRNA which was labeled with a near-infrared dye (AlexaFluor 647). Mice injected with PBS or fluorescent siRNA alone served as controls. Four hours post-injection, lungs (including lung tumor), liver and spleen were harvested, and ex-vivo fluorescent intensity measured. As expected, no fluorescence was detected in mice injected with fluorescent siRNA alone, indicating that the siRNA was eliminated from the body (Figure 5B). In contrast, mice injected with iNOP-7-siRNA complexes showed strong fluorescence in the lung and the surrounding tumor tissue as well as the liver and spleen (Figure 5B). Micro-CT imaging confirmed the presence of tumors growing within the lung of mice treated with iNOP-7-fluorescent siRNA (Figure 5C, panel I). Moreover, confocal microscopy confirmed the presence of fluorescent siRNA within tumor, liver and spleen tissue following delivery with iNOP-7 (Figure 5C, panels II-IV). Importantly, we also showed that human lung tumors growing in the mouse lung expressed our target protein PLK1 (Figure 5D). Finally, to confirm that iNOP-7 was non-toxic, we treated mice with iNOP-7 (8:1 w/w) or PBS (control). Twenty-four hours post-injection, lung, liver and spleen were harvested and gross histology assessed by hematoxylin and eosin staining. No obvious change in histology in the lung, liver or spleen was observed in mice treated with iNOP-7 when compared to controls (Supplementary Figure 5). Collectively, these results show for the first time that iNOP-7 can deliver siRNA to growing lung tumors in a cellular microenvironment which recapitulates many of the features observed in the human clinical setting.


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)

Establishment of a NSCLC bioluminescent mouse model and iNOP-7 siRNA delivery in vivo(A) Panel I, a representative image and graph showing increased luciferase activity with increasing H1299-Luc NSCLC cell numbers, n = 3 experiments. Panel II, pseudocolor images of mice injected orthotopically with H1299-Luc cells showing an increase in lung bioluminescence at days 7-20 post-tumor cell injection. (B) Representative ex-vivo fluorescent images of lungs and lung tumors, liver and spleen collected from mice with H1299-Luc orthotopic lung tumors, 4h post-injection (intravenous) with fluorescent siRNA (Red) complexed to iNOP-7. Mice injected with iNOP-7-fluorescent siRNA showed high fluorescence in the lung and lung tumors, liver and spleen. Mice injected with fluorescent siRNA alone or PBS served as controls. (C) Panel I, micro-CT image (axial) showing the presence of a growing tumor within the lungs of a mouse prior to injection with iNOP-7-fluorescent siRNA (red-dotted line marks the area of the tumor within the lung). Panels II-IV, are representative confocal microscopy images of frozen sections of lung tumor, liver and spleen showing the presence of fluorescent siRNA when delivered by iNOP-7, 4h post-injection (white arrows mark the location of fluorescent siRNA). (D) Panel I, Immunohistochemical image demonstrating PLK1 expression in orthotopic H1299-Luc tumors growing in the lung of mice. Panel II, immunohistochemical image of the corresponding antibody isotype control.
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Figure 5: Establishment of a NSCLC bioluminescent mouse model and iNOP-7 siRNA delivery in vivo(A) Panel I, a representative image and graph showing increased luciferase activity with increasing H1299-Luc NSCLC cell numbers, n = 3 experiments. Panel II, pseudocolor images of mice injected orthotopically with H1299-Luc cells showing an increase in lung bioluminescence at days 7-20 post-tumor cell injection. (B) Representative ex-vivo fluorescent images of lungs and lung tumors, liver and spleen collected from mice with H1299-Luc orthotopic lung tumors, 4h post-injection (intravenous) with fluorescent siRNA (Red) complexed to iNOP-7. Mice injected with iNOP-7-fluorescent siRNA showed high fluorescence in the lung and lung tumors, liver and spleen. Mice injected with fluorescent siRNA alone or PBS served as controls. (C) Panel I, micro-CT image (axial) showing the presence of a growing tumor within the lungs of a mouse prior to injection with iNOP-7-fluorescent siRNA (red-dotted line marks the area of the tumor within the lung). Panels II-IV, are representative confocal microscopy images of frozen sections of lung tumor, liver and spleen showing the presence of fluorescent siRNA when delivered by iNOP-7, 4h post-injection (white arrows mark the location of fluorescent siRNA). (D) Panel I, Immunohistochemical image demonstrating PLK1 expression in orthotopic H1299-Luc tumors growing in the lung of mice. Panel II, immunohistochemical image of the corresponding antibody isotype control.
Mentions: To examine the therapeutic potential of iNOP-7-PLK1 siRNA in vivo, we developed H1299 NSCLC cells to stably express high levels of firefly luciferase (H1299-Luc) to allow for the measurement of tumor growth in an orthotopic NSCLC mouse model (Figure 5A, panel I). Importantly, lung tumors in this model grow in a microenvironment which closely mimics the human setting. Seven days post-tumor cell inoculation, tumors could be detected in the lungs of mice as evidenced by a strong bioluminescent signal (Figure 5A, panel II). The bioluminescent signal increased over time, and by day 20 diffuse lung tumor growth was observed. To assess whether iNOP-7 could deliver clinically-relevant amounts of siRNA to growing lung tumors, mice (15 days post-tumor cell inoculation) were administered systemically with iNOP-7 complexed to siRNA which was labeled with a near-infrared dye (AlexaFluor 647). Mice injected with PBS or fluorescent siRNA alone served as controls. Four hours post-injection, lungs (including lung tumor), liver and spleen were harvested, and ex-vivo fluorescent intensity measured. As expected, no fluorescence was detected in mice injected with fluorescent siRNA alone, indicating that the siRNA was eliminated from the body (Figure 5B). In contrast, mice injected with iNOP-7-siRNA complexes showed strong fluorescence in the lung and the surrounding tumor tissue as well as the liver and spleen (Figure 5B). Micro-CT imaging confirmed the presence of tumors growing within the lung of mice treated with iNOP-7-fluorescent siRNA (Figure 5C, panel I). Moreover, confocal microscopy confirmed the presence of fluorescent siRNA within tumor, liver and spleen tissue following delivery with iNOP-7 (Figure 5C, panels II-IV). Importantly, we also showed that human lung tumors growing in the mouse lung expressed our target protein PLK1 (Figure 5D). Finally, to confirm that iNOP-7 was non-toxic, we treated mice with iNOP-7 (8:1 w/w) or PBS (control). Twenty-four hours post-injection, lung, liver and spleen were harvested and gross histology assessed by hematoxylin and eosin staining. No obvious change in histology in the lung, liver or spleen was observed in mice treated with iNOP-7 when compared to controls (Supplementary Figure 5). Collectively, these results show for the first time that iNOP-7 can deliver siRNA to growing lung tumors in a cellular microenvironment which recapitulates many of the features observed in the human clinical setting.

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