Discussion
Apoptosis is a response of cells to internal and external signals under certain physiological and pathological circumstances, to maintain homeostasis [21]. Many anti-cancer drugs attack tumors by triggering apoptosis [22]. Mechanisms of drug-induced tumor apoptosis include altering cell signaling pathways, expression levels of tumor-suppressor oncogene products, and influencing other apoptosis-promoting and -inhibiting proteins. Anti-cancer drugs can also block the cell cycle and inhibit cell growth, while activating caspase cascades and modulating telomerase expression and activity [23–25].
As a newly found auxiliary transcription inhibition factor, HIPK2 has been suggested to affect many aspects of cancer. Studies showed that HIPK2 participates in a variety of signal transduction pathways, including p53 [26], Wnt/β-catenin [27], JNK [28], and hypoxiainducible factor [11, 29, 30]. Recent studies suggest that HIPK2 influences apoptosis through a variety of mechanisms, especially the p53-mediated apoptosis signaling cascade [19, 20]. p53 is the most important tumor-suppressor gene, and is implicated in regulation of apoptosis; its protein is activation is controlled by post-translational modifications, such as phosphorylation, acetylation, and interactions with other proteins. p53 phosphorylation not only stabilizes and enhances the transcription activity of p53, but also regulates its subcellular localization. p53 serine (Ser46) phosphorylation is critical to transcription of apoptosis-related genes. HIPK2 overexpression stabilizes and activatesp53 and promotes its binding to form the HIPK2–p53 complex, leading to Ser46 phosphorylation and increased apoptosis [31].
We conducted a retrospective analysis on 100 primary CRC tumor samples, and found that the average age of CRC diagnosis was 67.25 ± 11.91 years, which did not significantly vary by sex. Common symptoms of CRC include changes in bowel habits, hemafecia/melena, and abdominal pain or discomfort. Among them, hemafecia is the most common symptom, seen in 93.75% of patients with CRC. As for the clinicopathological features, the average tumor diameter was 5.31 ± 2.21 cm, with glandular cancer as the most common histology (91%), and ulcerative type as the major morphological type (37%). IHC analyses showed HIPK2 expression in normal colorectal mucosal tissues to be higher than in CRC samples. These data are consistent with previous reports showing a similar pattern for HIPK2 expressions in breast cancer and thyroid cancer [32–34]. Correlation analysis showed that HIPK2 expression was closely associated with Dukes staging and infiltration degrees, but not to sex, age, degree of differentiation, or lymph node metastasis.
We next tested VB’s anti-tumor activity in an in vivo mouse model of human CRC, and found VB to significantly inhibit xenograft tumor growth. IHC analyses showed heightened levels of pro-apoptotic proteins HIPK2, p53, Bax, and decreasedBcl-2 in VB-treated tumors. These results imply that VB promotes cancer cellapoptosis throughHIPK-2- and p53-related signaling. To study the mechanisms of this anti-cancer effect, we used VB to treat human CRC cell lines. As with the in vivo studies, VB had a remarkable anti-proliferative and apoptosis-promoting effect in HCT-116, HT-29, LoVo, and SW620 cells, in a time- and dose-dependent manner. In addition, this nicely correlates with the previous finding that VB induces genotoxic stress [35].
Reportedly, theHIPK2–p53 apoptotic pathway is downregulated in different human cancer cells [36–42]. In investigating the mechanisms that underpin VB-promoted apoptosis, we first learned that both in CRC tumors and cells, VB elevated HIPK2 protein levels. Additionally, levels of p53, p-p53 at Ser46, and downstream pro-apoptosis Bax protein were greatly boosted, whereas anti-apoptosis Bcl-2 protein expression was reduced, by VB treatment. Furthermore, the pro-apoptotic action of VB was obscured by a p53-specific inhibitor, which restored protein levels of p-p53 (Ser46), p53, Bax, and Bcl-2 to the untreated status. Interestingly, HIPK2 protein expression was not influenced. To summarize, our data suggest that VB promotes p53 phosphorylation and Bax expression and inhibits Bcl-2 expression by increasing HIPK2 levels in CRC, which leads to activation of theHIPK2–p53 signaling pathway and increased apoptosis.