Histological confirmation of ductal complex and adenocarcinoma in the pancreas of rat sacrificed at 24th weeks of DMBA implantation
To validate pancreatic carcinogenesis in the DMBA rat model, pancreas tissue of rats were sacrificed at the 24th week of DMBA implantation were histologically examined. Macroscopic inspection on pancreas specimens, which were sliced into 1-mm width after formalin-fixation and HE staining, revealed whitish nodules in Sect. 0,1,2,3,4,5 of pancreas head near the DMBA implanted sites, but not in Sect. 6–33 of body and tail of the pancreas (Fig. 2A). Microscopically, in all specimens from Sect. 0–5, both ductal complex and adenocarcinoma were colocalized. Representing histological images of ductal complex and adenocarcinoma areas in these nodules are shown in Fig. 2B and D, respectively, with each corresponding higher magnification appearance in the rectangle of Fig. 2C, E.
In the ductal complex area, its histological characteristics, as reported earlier [8]; multiple conglomerates of noninvasive microtubular structures, consisted of cells showing scanty mitosis, enlarged and condensed nucleus nuclei, and cytoplasmic atypia with associated inflammation and fibrosis were verified. Adenocarcinoma lesions were featured by massive invasive cells with aggregated nucleus nuclei of condensed chromatin, pseudostratification, and frequent mitosis (Fig. 2D, E) as reported previously [8].
Incidence of ductal complex and adenocarcinoma lesions during carcinogenesis induced by DMBA
Taking the histopathological characteristics of ductal complex and adenocarcinoma described above into account, both lesions during carcinogenesis were surveyed in the pancreas excised every 4 weeks after DMBA-implanting in group I rats (Fig. 1C), and the incidence of lesions was counted by defining the lesion as positive when at least one lesion was identified throughout the fields of one specimen. The first appearance of ductal complex occurred at week 16 of DMBA-implantation, with an incidence of 1/3 (33%); the incidence increased as the stage advanced; 2/3 (67%) at week 20 and 4/5 (80%) at week 24. In contrast, adenocarcinoma was seen only at a late stage, week 24, with an incidence of 3/5 (60%)(Table 1).
Table 1
Incidence of ductal complex and adenocarcinoma (group I)
Week
|
Ductal complex
|
Adenocarcinoma
|
Incidence (%)
|
Incidence (%)
|
4
|
0/3 (0)
|
0/3 (0)
|
8
|
0/3 (0)
|
0/3 (0)
|
12
|
0/3 (0)
|
0/3 (0)
|
16
|
1/3 (33)
|
0/3 (0)
|
20
|
2/3 (67)
|
0/3 (0)
|
24
|
4/5 (80)
|
3/5 (60)
|
Close relationship between KRAS point mutation and GSTP1 expression in ductal complex and adenocarcinoma induced by DMBA
Since the close correlation of KRAS mutation with high expression of GSTP1 in various human cancer tissues and cancer cells, including those of pancreas origin has been well described [6][9], we intended to confirm that this correlation is also found in our pancreas cancer model. In group 1 rats (Fig. 1C), a total of 7 ductal complex specimens; 1, 2, and 4 specimens from 16th week, 20th week, 24th week rats, respectively (Table 1) and 3 adenocarcinoma specimens from 24th week rats (Table 1) were micro-dissected and underwent for analyses of KRAS mutation at codon 12 by 2-step PCR-restriction enzyme fragment length polymorphism (RFLP). As control samples, 5 specimens from the pancreas head of 16 week-old normal rats were also analyzed for their KRAS mutations by RFLP.
In addition to bands with 75bp mobility, which were found in all control samples, those with 46bp mobility representing mutated KRAS were clearly identified in samples of ductal complex and adenocarcinoma (Fig. 3A). Incidences of 46bp band in samples of control, ductal complex, and adenocarcinoma were 0/5, 5/7, and 3/3, respectively (Fig. 3B).
When these results were analyzed along the carcinogenesis stages, mutation of KRAS become positive after the 20th week of DMBA implantation; for ductal complex, 1/2 at week 20 and 4/4 at week 24 and for adenocarcinoma, 3/3 at 24 weeks (Table 2). GSTP1 expression in these lesions of group I rats were then examined by immunostaining. Representative staining patterns are shown in Fig. 4. In clear contrast to staining of HE specimens; Fig. 4A, D, G and negative controls which stained with non-specific antibody, those of both duct epithelium of ductal complex (Fig. 4E) and cancerous cell of adenocarcinoma (Fig. 4H) from rats implanted with DMBA for 24 weeks, showed distinct positive staining for GSTP1.
Table 2
Incidence of mutated KRAS, GSTP1 expression in ductal complex and adenocarcinoma (Group1)
|
No (week)
|
Mutated
KRAS
|
GSTP1 expression
|
Normal pancreas
tissue
|
1
|
|
-
|
-
|
2
|
|
-
|
-
|
3
|
|
-
|
-
|
4
|
|
-
|
-
|
5
|
|
-
|
-
|
|
|
|
0/5 (0%)
|
0/5 (0%)
|
Ductal complex
|
1
|
(16)
|
-
|
-
|
2
|
(20)
|
-
|
+
|
3
|
(20)
|
+
|
+
|
4
|
(24)
|
+
|
+
|
5
|
(24)
|
+
|
+
|
6
|
(24)
|
+
|
+
|
7
|
(24)
|
+
|
+
|
|
|
|
5/7 (71.4%)
|
6/7 (85.7%)
|
Adenocarcinoma
|
1
|
(24)
|
+
|
+
|
2
|
(24)
|
+
|
+
|
3
|
(24)
|
+
|
+
|
|
|
|
3/3 (100%)
|
3/3 (100%)
|
Then, immunostaining analyses of GSTP1 were conducted along carcinogenesis stages, defining it as positive when staining patterns shown in Fig. 4E or H were evident in any area of specimens. While all specimens from normal pancreas were negative for staining, those of ductal complex from rats after 20 weeks of DMBA treatment were positive; 2/2 at 20th week and 4/4 at 24th week and those of adenocarcinoma at 24th week were all positive; 3/3 (Table 2).
Effect of HGBPE on the generation of ductal complex and adenocarcinoma at 16 weeks (II, III group) and 36 weeks (IV, V group) after DMBA-embedding
Incidence of ductal complex and adenocarcinoma was examined after administrating HGBPE to DMBA embedded rats. At week 16 of DMBA-embedding, incidences of the ductal complex in group III (control) that was administrated only vehicle and in group II that was administrated HGBPE were 50% (5/10) and 10% (1/10) respectively, suggesting significant suppression (p = 0.05) of ductal complex formation by HGBPE (Table 3). Adenocarcinoma was not developed in both groups. At 36 weeks of DMBA-embedding, incidences of the ductal complex in V group and in IV group were 72.2% (13/18) and in 25.0% (5/20), respectively, with a statistical significance of p = 0.0036 (Table 3). Further, incidences of adenocarcinoma in V group and in IV group were 50.0% (9/18) and15.0% (3/20) respectively with significant difference (p = 0.0247). Collectively, these results suggest that GSTP1 inhibitor, HGBPE is a potent chemopreventive agent against DMBA induced pancreas cancer.
Table 3
Preventive effect of HGBPE on generation of ductal complex and adenocarcinoma (Group II-V)
|
Ductal complex
|
|
Adenocarcinoma
|
|
Week
|
HGBPE
|
Vehicle
|
p
|
HGBPE
|
Vehicle
|
p
|
16
|
1/10
(10.0%)
|
5/10
(50.0%)
|
0.05
|
0/10
(0.0%)
|
0/10
(0.0%)
|
|
36
|
5/20
(25.0%)
|
13/18
(72.2%)
|
0.0036
|
3/20
(15.0%)
|
9/18 (50.0%)
|
0.02
|
HGBPE, O1-Hexadecyl-γ-glutamyl-S-benzylcysteinyl-D-phenylglycine-Ethylester |