Influence of three BALB/c substrain backgrounds on the skin tumor induction efficacy to DMBA and TPA cotreatment

Abstract

Differences in responsiveness of BALB/c substrains have been investigated in various fields, including diabetes induction, corpus callosum deficiency, virus-induced demyelinating disease, aggressive behavior and osteonecrosis. However, induction efficacy of skin tumor remains untried. We therefore investigated the influence of BALB/c substrain backgrounds on the skin tumor induction efficacy in response to DMBA (7,12-Dimethylbenz[a]anthracene) and TPA (12-O-tetradecanoylphorbol-13-acetate) cotreatment. Alterations in the levels of tumor growth related factors, histopathological structure, and the expression to tumor related proteins were measured in three BALB/c substrains (BALB/cKorl, BALB/cA and BALB/cB) after exposure to DMBA (25 μg/kg) and three different doses of TPA (2, 4 and 8 μg/kg). The average number and induction efficacy of tumors in response to DMBA+TPA treatment were significantly greater in the BALB/cKorl substrain than in BALB/cA and BALB/cB. However, cotreatment with DMBA+TPA induced similar responses for body and organ weights of all three substrains. Few differences were detected in the serum analyzing factors, while similar responsiveness was observed for blood analyzing factors after DMBA+TPA treatment. Furthermore, the three BALB/c substrains exhibited similar patterns in their histopathological structure in DMBA+TPA-induced tumors. The expression levels of apoptotic proteins and tumor related proteins were constantly maintained in all three BALB/c substrains treated with DMBA+TPA. In addition, the responsiveness to cisplatin treatment was overall very similar in the three BALB/c substrains with DMBA+TPA-induced tumors. Taken together, these results indicate that genetic background of the three BALB/c substrains does not have a major effect on the DMBA+TPA-induced skin carcinogenesis and therapeutic responsiveness of cisplatin, except induction efficacy.

Keywords: BALB/c; BALB/cKorl; Cisplatin; DMBA+TPA; Skin tumor; Substrains.

Fig. 1

Experimental scheme and average tumor number in the three BALB/c substrains. a After applying single dose DMBA, three different doses of TPA were applied to the shaved back skin, twice weekly for 20 weeks. Also, one group of BALB/c mice was administered DMBA and medium dose TPA, with subsequent intraperitoneal injection of cisplatin as anti-tumor drug. b Average number of tumors. Only papilloma with tumor size 2 cm or more were counted, and are represented in the graph. Data represent the mean ± SD. *, p < 0.05 compared to the No treated group. #, p < 0.05 compared to the Vehicle treated group

Fig. 2

Tumor growth and incidence in three BALB/c substrains after DMBA and TPA cotreatment. a Morphological images of the tumor developed on the back skin of three BALB/c substrains treated with DMBA and TPA at 22 weeks. b Tumor incidence calculated as described in Materials and Method

Fig. 3

Body and organ weight of three BALB/c substrains after DMBA and TPA cotreatment. After final treatment in a BALB/cKorl, b BALB/cA and c BALB/cB, five internal organs, including liver, lung, kidney, thymus and spleen, were collected from mice of subset groups, and their weights were measured in duplicate. Data represent the mean ± SD. *, p < 0.05 compared to the No treated group. #, p < 0.05 compared to the Vehicle treated group

Fig. 4

Serum parameters of three BALB/c substrains after DMBA and TPA cotreatment. After final treatment in a BALB/cKorl, b BALB/cA and c BALB/cB, serum levels of four parameters (AST, ALT, ALP, LDH) and Ca were measured, as described in Materials and Methods. Data represent the mean ± SD. *, p < 0.05 compared to the No treated group. #, p < 0.05 compared to the Vehicle treated group

Fig. 5

Histopathological analysis of skin and tumor tissue. After harvesting the skin and tumors from DMBA+TPA treated BALB/c substrains, the histopathological changes in slide sections of skin (left column) and tumor tissue (right column) were identified by staining with hematoxylin and eosin and observation at 200× magnification

Fig. 6

Protein expressions of p27 and p53 in tumor samples from the DMBA+TPA treated a BALB/cKorl, b BALB/cA, and c BALB/cB substrains. Expression levels of p53 and p27 proteins were determined by Western blot analysis using HRP-conjugated anti-rabbit IgG antibodies. Band intensity of each protein was measured using an imaging densitometer, and expressions of the two proteins were calculated relative to the intensity of β-actin protein. Data represent the mean ± SD. *, p < 0.05 compared to the No treated group. #, p < 0.05 compared to the Vehicle treated group

Fig. 7

Protein expressions of Bax, Bcl-2 and Caspase-3 in tumor samples from the DMBA+TPA treated a BALB/cKorl, b BALB/cA and c BALB/cB substrains. Expression levels of Bax, Bcl-2 and Caspase-3 proteins were determined by Western blot analysis using HRP-conjugated anti-rabbit IgG antibodies. Band intensity of each protein was measured using an imaging densitometer, and expressions of the three proteins were calculated relative to the intensity of β-actin protein. Data represent the mean ± SD. *, p < 0.05 compared to the No treated group. #, p < 0.05 compared to the Vehicle treated group