Biochemical and Biophysical Research Communications
ASPP2 enhances chemotherapeutic sensitivity through the down-regulation of XIAP expression in a p53 independent manner in hepatocellular carcinoma
Introduction
Hepatocellular carcinoma (HCC), the most common primary liver cancer with a poor prognosis, has a limited treatment option [1]. Sorafenib, a multi-targeted tyrosine kinase inhibitor, remains the major drug approved for systemic treatment in advanced HCC for pro-apoptotic activity [2]. Unfortunately, only limited patients show a regression of the tumor after the initiation of sorafenib administration [3]. A huge number of HCC patients are resistant to sorafenib which display a poor prognosis with reduced survival as their tumor progresses. At present, a number of studies and trials performed to fund the underlying mechanisms involved in the drug resistance to sorafenib in HCC. Any molecular and regents modulate the sensitivity of sorafenib bring hopes to HCC patients.
X-linked inhibitor of apoptosis protein (XIAP), baculoviral inhibitor of apoptosis repeats (BIR) in the N-terminal and RING Zn finger in the C-terminal, is the most pivotal member of the IAP family. XIAP inhibited both initiator caspases (e.g., caspase-8 and caspase-9) and effector caspases (e.g., caspases-3 and caspases-7) by an unequal mechanism. BIR2 and the linker region of BIR2 in the XIAP inhibit the activity of caspase-3 and caspase-7, and BIR3 along is enough to suppress caspase-9 [4,5]. RING Zn finger domain has an E3 ligase activity and potentiate the ubiquitination of targets [6]. Together, BIR and RING Zn finger domain in the XIAP endue the capacity for inhibiting all the caspases that induce apoptosis [7,8].
However, mechanisms that regulate XIAP expression remains a litter understood, and XIAP is high express in HCC. Though, p53 accumulation and XIAP depletion are known as a dual mechanism induce apoptosis of cancer cells [9], apoptosis induced by the down-regulation of XIAP dependent on cellular p53 status [10]. Regulators of p53 may thus also have a role in affecting cellular responses to XIAP. Among those, apoptosis-stimulating protein of p53 2(ASPP) is a potential candidate. ASPP2 promotes the pro-apoptotic transcriptional activity of p53 by promoting the binding of p53 to the downstream pro-apoptotic cofactor (e.g., PUMA, Fas/CD95 and Noxa) promoter region thought to be an effective regulator of p53 [11,12]. Though the mechanism is a litter known, low ASPP2 expressed malignant tumor does have a poor sensitivity to chemotherapy [13,14].
In this study, we confirmed that ASPP2 facilitated chemotherapy-induced cell death in vitro and vivo independent of p53 via suppressing XIAP expression. XIAP, suppress by ASPP2, facilitated sorafenib induced apoptosis in ASPP2 over-expressed mice. In HCC patients, XIAP suppressed in ASPP2 highly expressed tumor adjacent normal tissues, suggesting that the increased XIAP in ASPP2 low-expressed liver tumors might have contributed to chemoresistance following chemotherapy.
Section snippets
Cell culture
Human HCC cell lines wild-type p53 HepG2 and p53 null Hep3B were cultured in Minimum Eagle’s medium (Gibco™). ASPP2 expression was knockdown by shASPP2-lentivirus and vector-lentivirus infection. Cells infected with ASPP2-Adenovirus, and vector-Adenovirus for the over-expression of ASPP2.
Immunoblot assay
Cells harvested and lysed in a cold RIPA buffer for 30 min. The supernatant separated in SDS-PAGE gels, and separated proteins then transferred to a PVDF membrane. Membranes incubated with appropriate primary
ASPP2 suppress the expression of XIAP in HCC
Previous reports suggest that ASPP2 plays an important role in inducing tumor cells apoptosis including p53 dependent and independent pathway, but the mechanism was a litter understood. We run multiple genes qRT-PCR array with ASPP2 overexpression cells and ASPP2 shut down cells in both wtP53 cells-HepG2 and p53 null cells-Hep3B. The multiple genes qRT-PCR results showed that XIAP expression was strong influenced by ASPP2 expression following sorafenib stress for 24 h (Fig. 1 A, B, E and F).
Discussion
Various molecular mechanisms, transporter pumps, oncogenes, tumor suppressor genes, mitochondrial alteration, DNA repair, autophagy, epithelial-mesenchymal transition (EMT), cancer stemess and exosome, were associated with chemoresistance. Cross talks are essential [16,17]. p53, tumor suppressor gene, an indispensable gene in chemoresistance of advanced tumors, facilitates chemotherapeutic drug-induced apoptosis by regulating the expression of anti-apoptotic genes (MDM2, p21Waf1/Cip1 and Fas)
Funding sources
This work was supported by the National Institutes of Health grants [81272266, 81361120401]; and the Capital Health Research and Development Special grants [2018-1-1151].
Declaration of interest
The authors report no conflicts of interest.
Acknowledgments
We are very grateful to Charles D. Lopez for ASPP2+/- mice.
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