J Vet Sci.  2011 Dec;12(4):309-317. 10.4142/jvs.2011.12.4.309.

Aerosol delivery of kinase-deficient Akt1 attenuates Clara cell injury induced by naphthalene in the lungs of dual luciferase mice

• Minai-Tehrani A
• Park YC
• Hwang SK
• Kwon JT
• Chang SH
• Park SJ
• Yu KN
• Kim JE
• Shin JY
• Kim JH
• Kang B
• Hong SH
• Cho MH

• KMID: 1365013
• DOI: http://doi.org/10.4142/jvs.2011.12.4.309

Abstract

Conventional lung cancer therapies are associated with poor survival rates; therefore, new approaches such as gene therapy are required for treating cancer. Gene therapies for treating lung cancer patients can involve several approaches. Among these, aerosol gene delivery is a potentially more effective approach. In this study, Akt1 kinase-deficient (KD) and wild-type (WT) Akt1 were delivered to the lungs of CMV-LucR-cMyc-IRES-LucF dual reporter mice through a nose only inhalation system using glucosylated polyethylenimine and naphthalene was administrated to the mice via intraperitoneal injection. Aerosol delivery of Akt1 WT and naphthalene treatment increased protein levels of downstream substrates of Akt signaling pathway while aerosol delivery of Akt1 KD did not. Our results showed that naphthalene affected extracellular signal-regulated kinase (ERK) protein levels, ERK-related signaling, and induced Clara cell injury. However, Clara cell injury induced by naphthalene was considerably attenuated in mice exposed to Akt1 KD. Furthermore, a dual luciferase activity assay showed that aerosol delivery of Akt1 WT and naphthalene treatment enhanced cap-dependent protein translation, while reduced cap-dependent protein translation was observed after delivering Akt1 KD. These studies demonstrated that our aerosol delivery is compatible for in vivo gene delivery.

Keyword

aerosol delivery; Akt1; Clara cell; ERK; luciferase activity; naphthalene

MeSH Terms

Administration, Inhalation
Aerosols
Animals
Gene Expression Regulation
Gene Knockdown Techniques
Gene Therapy/*methods
Gene Transfer Techniques
Genes, Reporter
Injections, Intraperitoneal
Luciferases/genetics/*metabolism
Lung Diseases/*chemically induced
Male
Mice
Mice, Transgenic
Naphthalenes/administration & dosage/*toxicity
Proto-Oncogene Proteins c-akt/*administration & dosage/genetics/*metabolism

Figure

• 

  Fig. 1 Effects of Akt1 wild type (WT) and naphthalene on p70S6K, p-p70S6K, and extracellular signal-regulated kinase (ERK)1/2 protein expression. (A) Western blot and (B) densitometric analysis of p-p70S6K, p70S6K, and ERK1/2 expression. Con: control, Vec: vector, Akt1: Akt1 WT was delivered to the mice, Na: mice that received naphthalene alone, Akt + Na: mice exposed to both Akt1 WT and naphthalene. Data are presented as the mean ± SE (n = 3). *p < 0.05 indicating a significant difference compared to the corresponding control.

• 

  Fig. 2 Effects of Akt1 WT and naphthalene on eukaryotic initiation factor (eIF)4E and p-eIF4E protein expression. (A) Western blot and (B) densitometric analysis of eIF4E and p-eIF4E expression. (C) Effects of Akt1 on pulmonary luciferase activity in the dual reporter mice. Con: control, Vec: vector, Akt: Akt1 WT was delivered to the mice, Na: mice that received naphthalene only, Akt + Na: mice that were exposed to both Akt1 WT and naphthalene. Data are presented as the mean ± SE (n = 3). *p < 0.05 indicating a significant difference compared to the corresponding control.

• 

  Fig. 3 Immunohistochemical staining of the lungs with anti-ERK antibody. (A) Control, (B) mice exposed to both Akt1 WT and naphthalene, and (C) mice exposed to naphthalene only. ×200.

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  Fig. 4 Immunohistochemical staining of lungs with anti-p-eIF4E antibody. (A) Control, (B) mice exposed to both Akt1 WT and naphthalene, (C) mice exposed to naphthalene only, and (D) mice exposed to Akt1 WT. ×200.

• 

  Fig. 5 Immunohistochemical staining of lungs with anti-p-p70S6K antibody. (A) Control, (B) mice exposed to both Akt1 WT and naphthalene, (C) mice exposed to naphthalene only, and (D) mice exposed to Akt1 WT. ×200.

• 

  Fig. 6 Effects of the Akt1 KD mutant and naphthalene on p70S6K, p-p70S6K, and ERK1/2 protein expression. (A) Western blot and (B) densitometric analysis of p-pS6K, p70S6K, and ERK1/2 expression. Con: control, Vec: vector, KD: Akt1 KD was delivered to the mice, Na: mice that received naphthalene only, KD + Na: mice that were exposed to both Akt1 KD and naphthalene. Data are expressed as the mean ± SE (n = 3). *p < 0.05 indicating a significant difference compared to the corresponding control.

• 

  Fig. 7 Effects of Akt1 KD and naphthalene on eIF4E and p-eIF4E protein expression. (A) Western blot and (B) densitometric analysis of eIF4E and p-eIF4E expression in the lung of the luciferase mice. (C) Effects of Akt1 on pulmonary luciferase activity in the dual reporter mice. Con: control, Vec: vector, KD: Akt1 KD was delivered to the mice, Na: mice that received naphthalene only, KD + Na: mice that were exposed to both Akt1 KD and naphthalene. Data are expressed as the mean ± SE (n = 3).

• 

  Fig. 8 Histopathology of the lungs. (A) Vector control, (B) mice exposed to Akt1 WT, (C) mice exposed to Akt1 KD, (D) mice exposed to both Akt1 WT and naphthalene, (E) mice exposed to Akt1 KD and naphthalene, and (F) mice exposed to naphthalene only. H&E stain, ×400.

• 

  Fig. 9 Immunohistochemical staining of lungs with anti-CC10 antibody. (A) Mice exposed to naphthalene only and (B) mice exposed to both Akt1 KD and naphthalene. ×200.

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