The chemopreventive effects of Protandim: modulation of p53 mitochondrial translocation and apoptosis during skin carcinogenesis

Abstract

Protandim, a well defined dietary combination of 5 well-established medicinal plants, is known to induce endogenous antioxidant enzymes, such as manganese superoxide dismutase (MnSOD). Our previous studies have shown through the induction of various antioxidant enzymes, products of oxidative damage can be decreased. In addition, we have shown that tumor multiplicity and incidence can be decreased through the dietary administration of Protandim in the two-stage skin carcinogenesis mouse model. It has been demonstrated that cell proliferation is accommodated by cell death during DMBA/TPA treatment in the two-stage skin carcinogenesis model. Therefore, we investigated the effects of the Protandim diet on apoptosis; and proposed a novel mechanism of chemoprevention utilized by the Protandim dietary combination. Interestingly, Protandim suppressed DMBA/TPA induced cutaneous apoptosis. Recently, more attention has been focused on transcription-independent mechanisms of the tumor suppressor, p53, that mediate apoptosis. It is known that cytoplasmic p53 rapidly translocates to the mitochondria in response to pro-apoptotic stress. Our results showed that Protandim suppressed the mitochondrial translocation of p53 and mitochondrial outer membrane proteins such as Bax. We examined the levels of p53 and MnSOD expression/activity in murine skin JB6 promotion sensitive (P+) and promotion-resistant (P-) epidermal cells. Interestingly, p53 was induced only in P+ cells, not P- cells; whereas MnSOD is highly expressed in P- cells when compared to P+ cells. In addition, wild-type p53 was transfected into JB6 P- cells. We found that the introduction of wild-type p53 promoted transformation in JB6 P- cells. Our results suggest that suppression of p53 and induction of MnSOD may play an important role in the tumor suppressive activity of Protandim.

Figure 1. Protandim suppressed DMBA/TPA induced apoptosis.

Skin tissues from each treatment group were collected at the end of the skin carcinogenesis study. Skin tissues were fixed and apoptotic cells were counted using light microscopy. The histological examination was confirmed with a pathologist (X.G.) and two-way analysis of variance (ANOVA) was used to assess the effects of TPA and Protandim on the number of apoptotic cells present per 100 cells. Tukey-Kramer method was used in the adjustment for multiple comparisons. Statistical software SAS system 9.3 (SAS Inc. Gary, NC) was used for two-way ANOVA data analysis. *, significantly different from Ctrl Veh: DMSO; #, significantly different from Ctrl/TPA. Ctrl: control basal diet (AIN-76A); Veh: Vehicle control (DMSO); Pro: Protandim-containing diet; TPA: 12-O-tetradecanoylphorbol-13-acetate.

Figure 2. Western blot analysis of p53 and Bax in mitochondrial fraction (left) and examination of apoptosis (right) in mouse skin epidermal tissues.

Succinate dehydrogenase subunit B (SDHB) served as the loading control. The levels of p53/Bax were normalized to that of SDHB. Statistical analysis was performed using one-way ANOVA (for multiple group comparison) followed by Newman-Keuls post-test. Ctrl, basal diet; Veh, vehicle control (DMSO); Pro, Protandim. *, p<0.05 when compared to DMBA/TPA group; #, significantly different from Ctrl/TPA.

Figure 3. The expression levels of p53 and Bax after TPA (100 nM) treatment in JB6 promotable (P+) and non-promotable (P-) cells.

TPA induced p53 activation and apoptosis only in P+ cells, not P- cells. Total cell lysate was used for the assay. The cells were grown in EMEM medium supplemented with 4% fetal bovine serum, 2 mM of L-glutamine, 50 µg/ml penicillin and 50 µg/ml streptomycin. 12-O-tetradecanoylphorbol-13-actetate (TPA) was prepared as a 20 nM stock solution in dimethylsulfoxide (DMSO). The TPA stock solution was diluted directly in the cell culture medium, with the resulting concentration being 100 nM. Ctrl: vehicle (0.1% DMSO) treatment for 24 h. *, p<0.05 when compared with the Ctrl group.

Figure 4

(A). The expression and activity levels of MnSOD between JB6 non-promotable (P-) and promotable (P+) cells. Mitochondrial fractions were used for the experiments. The NBT-BCS SOD inhibition assay was used to measure the MnSOD activity. The presence of MnSOD inhibited the NBT reduction. The data was plotted as percentage inhibition vs. protein concentration. One unit of activity was defined as the amount of protein needed to inhibit 50% of the NBT reduction rate. NaCN (5 mM) was used to measure MnSOD activity. Higher expression/activity levels of MnSOD were observed in JB6 P- cells compared to JB6 P+. SDHB served as the mitochondrial marker and loading control. (B). Detection of ROS levels in JB6 cells using H₂DCFDA staining. Cells grown in 96-well plates were incubated with TPA or Vehicle (0.1% DMSO) for 1 h following by incubation with 10 µM H₂DCFDA for 15 min. DCF fluorescence was detected using a fluorescence plate reader (Ex: 485 nm; Em: 528 nm). The fluorescent density was divided by the protein concentration for fair comparison. *, p<0.05 when compared to its control; #, p<0.05 when compared with the TPA group.