br To show that p has an essential role
To show that p53 has an essential role in response to 8-AHN treatment, RNAi studies were carried out to knock down endogenous p53 expression using TP53-specific siRNA. FCM showed that silencing of p53 by siRNA can partially rescue cells from 8-AHN-induced cell apoptosis (Fig. 5C). Summarized these data, we can draw a conclusion that up-regulation and activation of p53 was essential for 8-AHN-in-duced MPP+ Iodide arrest and apoptosis.
3.6. 8-AHN inhibited colorectal cancer tumor xenograft growth in vivo
To evaluate the role of 8-AHN in tumor proliferation in vivo, we examined the ability of 8-AHN to suppress the growth of HCT116 tumor xenografts in nude mice. Mice were treated with 8-AHN 10 mg/kg every other day for 2 weeks. 8-AHN significantly inhibited the growth of tumor xenografts (Fig. 6A). The excised tumors from the control group weighed between 550 and 750 mg, whereas these from the 10 mg/kg body 8-AHN-treated animals averaged 300 mg (Fig. 6B). In addition, treatment with 8-AHN at 10 mg/kg body resulted in a significant de-crease in tumor volume compared to the control group (Fig. 6D). Meanwhile, there was no significant loss in body weight in the animals (Fig. 6C). Immunohistochemistry staining of excised tumor sections also revealed a higher expression of BAX and p53, but decreased expression of cyclin B and PCNA in 8-AHN-treated tumors (Fig. 6E). H&E staining of the major organs collected at the end of the study also suggested no major organ-related toxicities (Fig. 6F). Together, these results in-dicated that treatment with 8-AHN potently suppresses tumor growth without aﬀecting normal tissues in mice via proliferation suppression and apoptosis induction.
Cell cycle control is the major regulatory mechanism in the cell growth process. Many cytotoxic agents and/or DNA-damaging agents arrest the cell cycle at the G0/G1, S, or G2/M phase and then induce apoptosis. Several studies have shown that various cytotoxic drugs in-duce G2/M phase accumulation (Dofe et al., 2017; Zhang et al., 2017). G2 to M phase progression is regulated by a number of the cyclin/cy-clin-dependent kinase (CDK) family. In particularly, activation of the cyclin B/CDK1 complex is required for transition from G2 to the M phase (Abraham, 2001; Pabla et al., 2012; Yan et al., 2016).
Additionally, up to date, p53 is a well-known tumor suppressor gene of human cancer, which is essential for cell apoptosis and the cell cycle process (Moonen et al., 2018). Activated p53 can bind to specific DNA sequences in the promoter region of its target genes, including p21, B-cell lymphoma 2 (BCL2)-associated X protein (Bax), p53 up-regulated modulator of apoptosis (PUMA) and growth arrest and DNA damage (GADD45) (Amin et al., 2015; Yaswen et al., 2015). On the cell cycle, European Journal of Pharmacology 854 (2019) 256–264
p21 was a universal CDK inhibitor, which will inhibit CDK1/cyclin B complexation and thereby prevent G2/M transition (Chung et al., 2002; Khalid et al., 2014). Gadd45 separates the CDK1/cyclin B complex and accelerates the degradation of cyclin B (Jin et al., 2000; Zhan et al., 1999). In addition, p53 has inhibitory eﬀect on cyclin B as well (Taylor and Stark, 2001). As a result, activated p53 can induce G2/M cell cycle arrest. On the apoptosis, activated p53 elevated the expression of p53 target genes FAS, BAX. Afterwards, both mitochondrial apoptosis pathway and death receptor apoptosis pathway are activated. The re-sult is that caspase 3 was activated and leaded to apoptosis. As a con-sequence, activated p53 can induce apoptosis. In our research, 8-AHN displays an antitumor eﬀect through cell cycle arrest and apoptosis in colorectal cells via activating p53.
In addition, p53 mutated in ∼50% of all malignant neoplasm and it is reported that the mutation of p53 can cause alteration of growth arrest and deficient apoptosis. In contrast to these suppressive roles, mutant p53 loses these functions and serves as an oncogene by physi-cally interacting with other proteins and thus modulating their cellular function. For example, the interaction between mutant p53 and p63 results in the decreased tumor suppressive activity of p63 (Freed-Pastor and Prives, 2012). This is why p53 wild type colorectal cancer cells (HCT116 and LOVO) are more sensitive to 8-AHN than p53 mutated colorectal cancer cells (HCT15 and SW480). At present, many studies have shown that p73 took the place of p53 in p53 mutated cells (Hong et al., 2014; Tiwary et al., 2011). p73 plays an equivalent role of p53 in the cell cycle and apoptosis. Maybe this is the reason why 8-AHN took eﬀect in p53 mutated colorectal cancer cells (HCT15 and SW480).