We utilized a transgenic mouse model exhibiting DOX-inducible regulation of the mutant human Ki-rasG12C
allele in lung tissue to determine the potential chemopreventive effects of curcumin on lung tumor progression in early stage lesions. Previous studies from our laboratory demonstrated that treatment of CCSP-rtTA/Ki-rasG12C
mice with DOX for 9–12 months resulted in the development of hyperplastic foci and small (≤1 mm), benign, well-differentiated ADs that did not progress to more severe tumor phenotypes (32
). Analysis of signaling events downstream of RAS revealed that mutant RASCYS12
signaled to a subset of its downstream effectors, as we saw increased levels of extracellular signal-regulated kinases (ERK) and p38 phosphorylation but no activation of the AKT and JUN kinase (JNK) pathways (32
). This appears to account for the benign phenotype of the lung lesions. We compared the effects of curcumin, which is currently in clinical trials as a chemopreventive agent for colon cancer, to the known lung tumor promoter BHT and the known lung cancer chemopreventive agent sulindac in order to validate the bitransgenic model for use in chemopreventive studies. BHT mediates its tumor-promoting effects by creating an inflammatory environment in the lung (35
), whereas sulindac inhibits several mediators of oxidative stress (40
Co-treatment of bitransgenic mice with DOX and BHT resulted in a statistically significant (P
0.01) increase in tumor multiplicity, as assessed by counting the number of visible surface tumors, in the DOX/BHT animals compared with the DOX only treated group (). Introduction of curcumin into the diets of DOX and DOX/BHT-treated mice caused similar increases in lung tumor multiplicity as observed in mice treated with DOX/BHT (). Indeed, much to our surprise, there were no statistically significant differences between the DOX/BHT treated and DOX/BHT/curcumin and DOX/curcumin-treated bitransgenic mice (analysis of variance; P
0.6), indicating that curcumin exhibited tumor promoter activity comparable with that seen with BHT. Only five lung lesions were detected among the control groups—one lesion was detected in two individual untreated bitransgenic mice, one lesion was detected in two different DOX-treated monotransgenic Ki-rasG12C
mice and one lesion was detected in a BHT-treated bitransgenic mouse that did not receive DOX. The only significant difference in body weights between the various treatment groups was a 1.3-fold increase in body weight of DOX/curcumin male-treated mice relative to the other treatment groups at euthanasia.
Fig. 1. Average tumor multiplicity of mice at 45 weeks after the initiation of DOX treatment. The average tumor multiplicity for each treatment group is expressed as number of tumors per mouse/total number of mice with tumors ±SE. DOX only treated group (more ...)
Microscopic examination of lung sections from 12 individual mice for each treatment group revealed that there was an increase in adenoma (62 ± 7%, 61 ± 8% and 47 ± 10%) and large atypical adenoma, defined as ADs with a small focus of more anaplastic looking cells, and adenocarcinoma development (4, 5 and 2%) in the DOX/BHT, DOX/curcumin and DOX/BHT/curcumin-treated groups, respectively, compared with the DOX only treated group, where only 19 ± 12.4% of lesions were classified as ADs (). In contrast, while co-treatment of DOX/BHT mice with 80 p.p.m. of the non-steroidal anti-inflammatory agent sulindac in the diet had little or no effect on the average lung tumor multiplicity of the DOX/BHT bitransgenic mice after 9 months, sulindac prevented tumor progression. As shown in , distribution of hyperplastic foci and ADs in DOX/BHT/sulindac and DOX/sulindac-treated mice resembled that seen in DOX only treated mice. Sulindac has been shown to inhibit lung tumor progression in a variety of murine models (40
). Thus, the CCSP-rtTA/Ki-rasG12C
mice respond appropriately to known lung tumor-promoting and chemopreventive agents.
Fig. 2. Percent microscopic lesions grouped by tumor stage and treatment group. Percent proliferative lesions are expressed as the total number of lesions for each histological type within each treatment group/total number of proliferative lesions for each treatment (more ...)
Photomicrographs of representative lung sections are shown in . shows lung tissue from normal lung and demonstrates the lack of proliferative lesions or atypia in control lung tissue. Upon treatment of the CCSP-rtTA/Ki-rasG12C
bitransgenic mice with DOX, we noted the development of hyperplastic lesions within the lungs of treated animals (), consistent with our previous observations (32
). demonstrate the severe proliferative lesions seen in DOX/BHT, DOX/curcumin and DOX/BHT/curcumin bitransgenic animals.
Fig. 3. Photomicrographs of mouse lung tissue. Hematoxylin and eosin stains of tissue from control and treated mice. (A) Normal lung with typically thin alveolar walls. (B) Diffuse pneumocyte hyperplasia in a mouse treated with DOX. Alveolar walls are thickened (more ...)
Four to six representative slides from individual mice in each group were examined for evidence of leukocyte infiltration by hematoxylin and eosin staining and expression of COX-2 and NF-κβ by IHC. As shown in , lung tissue isolated from DOX-treated mice exhibited mild hyperplasia with little evidence of inflammation (panels A and B). There was minimal staining for either COX-2 or NF-κβ. In contrast, tumors isolated from DOX/BHT, DOX/curcumin and DOX/BHT/curcumin-treated mice (, and , respectively) exhibited inflammation with a high density of macrophages (cells with black dots visible in , panel B for example) which also appeared to be positive for staining for NF-κβ (, and ), whereas elevated COX-2 expression was seen in the endothelial cells in the tumor compartment (, and ). The inflammation appeared to be somewhat more severe in DOX/curcumin versus DOX/BHT-treated mice ( and , respectively) and was the most severe in mice treated with DOX/BHT/curcumin. Mice treated with DOX/BHT/sulindac exhibited milder inflammation, being similar to that seen in DOX/BHT mice (data not shown).
Fig. 4. Hematoxylin and eosin and immunohistochemical staining of lung tissue from DOX-treated mice. Panels (A) and (B) are ×10 and ×40 magnifications, respectively, of hematoxylin and eosin-stained slides showing relatively normal-appearing tissue (more ...)
Fig. 5. Hematoxylin and eosin and immunohistochemical staining of lung tissue from DOX/BHT-treated mice. Panels (A) and (B) are ×10 and ×40 magnifications, respectively, of hematoxylin and eosin-stained slides showing mild edema around the tumor; (more ...)
Fig. 6. Hematoxylin and eosin and immunohistochemical staining of lung tissue from DOX/curcumin-treated mice. Panels (A) and (B) are ×10 and ×40 magnifications, respectively, of hematoxylin and eosin-stained slides showing marked inflammation (more ...)
Fig. 7. Hematoxylin and eosin and immunohistochemical staining of lung tissue from DOX/BHT/curcumin-treated mice. Panels (A) and (B) are ×10 and ×40 magnifications, respectively, of hematoxylin and eosin-stained slides showing severe inflammation (more ...)
Based on the marked inflammation we observed in the tumor tissues with curcumin treatment and results obtained in the β-carotene chemoprevention trials, where patients in the trial exhibited higher incidences of lung cancer (42
) that have been attributed to the pro-oxidant effects of β-carotene in the lungs of smokers (44
), we hypothesized that curcumin may similarly exhibit pro-oxidant effects in the highly oxygenated environment of the lungs. To this end, we assessed the pro-oxidant effects of dietary curcumin exposure using a protein carbonylation assay as a biomarker for oxidative damage to proteins in the lungs of Ki-ras
-expressing mice. We examined the effects of curcumin after 1 week of dietary exposure as any observed effects could not be attributed to transient effects of curcumin but at the same time the exposure would be very early in the tumorigenic process. In addition, the levels of mutant Ki-ras
expression would be near maximal levels by 1 week of DOX treatment (32
). After 9 days of DOX treatment and 1 week of dietary curcumin exposure, we found that DOX/curcumin-treated mice exhibited a 33% increase (P
0.010) in the levels of modified protein relative to DOX-treated mice on the control diet (). These results suggest that, in the lungs of smokers, curcumin may increase the formation of reactive oxygen species to enhance tumor progression as a result of the curcumin-mediated oxidative damage to additional cellular macromolecules that act in concert with the genetic damage already prevalent in the lungs of smokers and ex-smokers.
Fig. 8. Curcumin-mediated increase in oxidative damage in lung tissue. CCSP-rtTA/Ki-rasG12C bitransgenic mice were treated with 500 μg/ml of DOX in the drinking water. Two days after the initiation of DOX treatment, mice were kept on the normal diet or (more ...)