The effect of C3G, DDP, or their combination on the body weight of nude mice
The change in body weight is the fundamental method to detect acute toxicity of any test substance. Generally, the acute toxicity of a drug is evaluated by examining whether the body weight of the mice decreases sharply after 72 h of treatment22. We proceeded to investigate the potential therapeutic benefits of C3G with DDP in vivo using mice xenograft models.
Tumors were allowed to grow for one week, after which mice were treated with C3G, DDP, or C3G+DDP for another 2 weeks (Fig. 1A). At the end of the study, we observed that DDP caused a reduction in the animal’s body weight when compared with the model group (P < 0.05; Fig. 1B). There was no significant change in body weight in mice when we compared the C3G or C3G+DDP group, with the model group (P > 0.05). In addition, the mice in the C3G and the C3G+DDP groups were more lively and active than the mice in the DDP group. These results indicate that anti-cancer drug (DDP) affected the body weight in mice with tumours and that C3G alleviated, to some extent, the weight loss in tumour bearing mice.
The effect of C3G, DDP, or their combination on tumour weight and tumour growth
At the end of the experimental period, tumour tissues were excised and their volumes and weighst were measured. As shown in Figures 2A and 2B, the tumour growth was suppressed in all the treatment groups, relative to that in the model group. The tumour volumes of the combination group were significantly smaller than in the model group (P < 0.01), which indicates that the inhibitory effects on tumour volumes were much stronger by the combination treatment of C3G and DDP.
Tumour weight in all treatment groups decreased significantly relative to those in the model group on day 22 (P < 0.01; Fig. 2C). A combination of C3G (40 mg/kg/d) and DDP 3 mg/mg once in 3 days) decreased the tumour weights form 610 ± 56.25 mg in the model group to 253.2 ± 49.51 mg (P < 0.01) in the treatment group. As shown in Figure 2D, compared with the model group, the tumour growth was inhibited by 40.49%, 50.15%, and 58.49%, in the C3G, DDP and C3G+DDP groups, respectively. Thus, the combination of C3G and DDP inhibited tumour growth 8.34% more than DDP alone. In summary, the combination treatment could significantly reduce tumour weight and growth, suggesting that the anti-cancer effect of the combined treatment was significantly stronger than that of DDP alone.
The effect of C3G, DDP, or their combination on cell apoptosis of the xenograft tumour
The persistence of tumour cells in the xenograft indicates that they have evaded the host immune system; therefore, an extraneous treatment is required to control uncontrolled proliferation23. We analysed the induction of apoptosis using the TUNEL assay, wherein fragmented DNA, a characteristic of apoptotic cells, is used to identify apoptotic cells. Cells fluorescing red owing to fragmented DNA indicates apoptotic cells. Apoptosis was observed in all treated groups, compared with that of the model group. As shown in Figure 3A, the area of fluorescence in the xenograft tumours significantly increased in mice treated with the combination of drugs (C3G + DDP) compared with that in mice treated with either C3G or DDP alone. Figure 3B shows that the percentage of cells undergoing apoptosis increased upon treatment with either C3G or DDP alone compared with the model. The percentage of apoptotic cells in the tumours of mice treated with the combination of drugs (C3G + DDP) was 15.85% more than in the tumours of mice treated with either drug alone.
The effect of C3G, DDP, or their combination on apoptotic proteins in xenograft tumour
To demonstrate the pro-apoptotic effects of C3G alone or in combination with DDP on xenograft tumour, we analysed the expression levels of Bax, Bcl-2, and cleaved caspase-3, known as apoptosis regulators. As shown in Figure 4, the xenograft tumour cells were stained with DAB that could specifically bind to Bax, Bcl-2, and cleaved caspase-3. The related proteins were imaged as blue fluorescent areas. The results indicate that the percentage of Bcl-2 protein-positive cells significantly decreased in all treatment groups compared to that of the model (P < 0.05; Fig. 4C and 4D). In contrast, the percentage of Bax were significantly increased by 3.77%, 3.47%, and 7.23% and those of cleaved caspase-3 by 3.03%, 2.83%, and 4.92% in the C3G, DDP and the C3G+DDP groups, respectively, compared with model group (all P < 0.05; Fig. 4A, 4B, 4E, and 4F).
The effect of C3G, DDP, or their combination on the phosphatidyl inositol 3-kinase PI3K/AKT/mTOR pathway in the xenograft tumour
The PI3K/AKT/mTOR pathway is involved in the regulation of cell proliferation and apoptosis24-26. Activation of the PI3K/AKT/mTOR signalling pathway is critical for tumour cell growth and survival in several solid cancers27. Therefore, to understand the molecular processes underlying the effects of C3G and DDP on xenograft tumour, we examined the expression of the signalling proteins involved in the PI3K/AKT/mTOR pathway.
The results indicate that the percentage of p-AKT, p-PI3K, and p-mTOR protein-positive cells were less in all treatment groups compared with the C3G+DDP group; less by 4.57%, 6.04%, and 6.16%, in the C3G, DDP, and C3G+DDP groups, respectively (Fig. 5). Furthermore, the percentage of these signalling-proteins-positive cells may explain why the combinational treatment exhibited stronger apoptotic effects in tumour tissues than those exhibited by the treatment with C3G or DDP alone. This study suggests that the observed apoptosis in the xenograft tumour apoptosis caused by DDP or C3G is regulated by t p-AKT, p-PI3K, and p-mTOR.