Hepatocellular carcinoma (HCC) is the 5th most common cancer worldwide with approximately 564,000 new cases diagnosed every year . Patients with HCC have poor prognosis, with few treatment options available . Therefore, the development of novel strategies by identifying key targets at a molecular level is critical to cure HCC. Recent studies suggest that the aggressiveness, responsiveness to therapy and prognosis of HCC are closely linked to the Bcl-2 family members [1, 3].
The Bcl2 family members have homology clustered within four conserved Bcl2 homology (BH) domains: BH1, BH2, BH3 and BH4, with only the antiapoptotic proteins, Bcl2, Bcl-XL, Bcl-w and A1, bearing the NH2-terminal BH4 domain . In contrast, Mcl-1 has a helical BH4-like domain which is located between the PEST region and the BH3 domain . The proapoptotic family members can be divided into two subgroups based on the presence of BH domains: the BH123 multidomain proteins (i.e. Bax and Bak) and the BH3-only molecules [6–8]. Recent studies suggest that there are two different subgroups in the BH3-only members. One group, including Bid and Bim, can function both directly to bind and activate Bax as well as indirectly to counteract the inhibition of Bax or Bak by antiapoptotic members including Bcl2 and Bcl-XL. Other BH3-only proteins (i.e. Bad, Bik, Noxa and PUMA) lack the ability to directly activate Bax but can oppose the action of antiapoptotic family members. Thus, both direct and indirect functions of BH3-only proteins may initiate apoptosis via selective interaction of its BH3 domain with an extended hydrophobic groove on the antiapoptotic Bcl2-like proteins and/or facilitate a conformational change in the multidomain proapoptotic proteins (i.e. Bax and Bak), which induce a death effect by promoting their insertion into mitochondrial membranes and oligomerization [6–8]. Bcl2 and related antiapoptotic proteins block the progression of a death signal by preventing Bax/Bak oligomerization .
Mcl-1 is a major antiapoptotic member of the Bcl2 family, which is crucial for liver development and hepatocellular homeostasis [10, 11]. Mcl-1 is also an oncoprotein that promotes the development of cancer [12, 13]. Importantly, Mcl-1 is overexpressed in about 50% of HCC patients , suggesting that Mcl-1 is a potential therapeutic target for some patients with HCC. In contrast to Bcl2 and Bcl-XL, Mcl-1 is rapidly inducible with a shorter half-life and seems to be more widely expressed in HCC [1, 9, 14]. Mcl-1 is mainly localized to the outer mitochondrial membrane via its C-terminal TM domain [15, 16]. Several residues, including serine (S) 64, threonine (T) 92, S155, S159 and T163, have been identified as the potential phosphorylation sites following various stimuli [15, 17–19]. However, phosphorylation of Mcl-1 at different site(s) distinctly regulates Mcl-1 protein turnover and its anti-apoptotic function [17–19]. For example, ERK1/2-mediated T163 site phosphorylation of Mcl-1 prolongs the half-life of Mcl-1, which leads to its increased antiapoptotic function [12, 18]. We have recently discovered that nicotine induces Mcl-1 phosphorylation at T163 in association with increased chemoresistance of human lung cancer cells . In contrast, GSK-3β-mediated Mcl-1 phosphorylation at the S159 site facilitates Mcl-1 ubiquitination and degradation leading to decreased survival activity . It has been suggested that the BH3-only protein Bim can directly activate Bak, leading to mitochondrial dysfunction and apoptosis . Thus, Mcl-1 may exert its antiapoptotic function via direct interaction with Bim leading to suppression of Bim-triggered Bak activation.
Bile is produced by the liver and consists of phospholipids and bile salts forming mixed micelles. Bile salts constitute a major portion of bile and are secreted by hepatocytes into the canaliculi. Hepatocellular bile salt secretion is mediated by an ATP-binding cassette (ABC) transporter named bile salt export pump (BSEP), which drives and maintains enterohepatic circulation of bile salts . Bile salts have carcinogenic roles because bile salts not only induce DNA damage but also stimulate cell survival and proliferation [22–25]. Thus, accumulation of bile salts or bile acids in hepatocytes by some mechanisms may lead to chronic liver damage, proliferation or development of HCC [24–26]. Bile acid (i.e. deoxycholate) has been reported to enhance Mcl-1 expression through activation of the EGFR/Raf-1 signaling pathway in KMBC cells (a human cholangiocarcinoma cell line), which may participate in cholangiocyte carcinogenesis by inhibiting apoptosis . It is currently unclear whether bile salts also regulate Mcl-1 in HCC cells through a similar or distinct mechanism(s).The conjugated bile salt, glycochenodeoxyocholic acid (GCDA), has been reported to activate PI3K/AKT and MAPK ERK1/2 signaling pathways in association with increased survival and proliferation of SEG-1 cells (a Barrett's adenoma cell line) [24, 25]. However, the downstream survival substrate(s) is not yet identified. Here we demonstrate that GCDA-activated ERK1/2 induces Mcl-1 phosphorylation at T163, which stabilizes Mcl-1 protein to enhance its antiapoptotic function. Additionally, GCDA can also induce apurinic/apyrimidinic (AP) sites of DNA lesions. These properties of GCDA may contribute to the development of HCC and/or chemoresistance.