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Page 6 of 8 Ding et al. Hepatoma Res 2018;4:12 I http://dx.doi.org/10.20517/2394-5079.2018.07

β2GPI is synthesized by liver cells and plays roles in anticoagulation, cell clearance, and lipid metabolism
[4]
under normal physiological conditions . β2GPI is also involved in the pathogenesis of chronic viral
[12]
hepatitis, alcoholic liver disease, autoimmune liver disease, liver cirrhosis and liver cancer . A previous
study showed that a fraction with maximal apoH (β2GPI)-binding predominantly contained full Dane
[13]
[14]
particles in HBV patients . Gao et al. found there was a specific binding event between HBV and β2GPI.
Gao et al. [15,16] provided the first evidence that a protein existed on SMMC-7721 cell membrane that could
specifically bind β2GPI. The binding protein was later identified as annexin II. A previous study from our
[6]
lab , demonstrated strong β2GPI expression in hepatitis B-related HCC tissue. In addition, the combination
of β2GPI and HBsAg was shown to significantly activate NF-κB and expression of AFP, suggesting that
β2GPI may be involved in the pathogenesis of hepatitis B-related HCC. However, it is unknown whether
β2GPI directly interacts with HBsAg or if other proteins are involved in NF-κB activation.

[17]
β2GPI is physically closed in a circular conformation, with low activity . β2GPI opens and adopts a J-like
[17]
conformation and becomes active when combined with antibodies or anionic phospholipids. In a study ,
it was found that LPS opened β2GPI, exposed its binding sites in domain V, and interacted with β2GPI
to participate in physiology and pathology. The β2GPI and LPS complex relied on the TLR4 signaling
[9]
pathway to activate NF-κB in macrophages. A previous study from our lab found that LPS enhanced
signal transduction in β2GPI in liver cancer cells leading to activation of NF-κB, triggering downstream
signal transduction and increasing the expression of downstream factors. This activation was related with
LPS concentration. This suggests that LPS enhancement of β2GPI signal transduction may participate in the
development of liver cancer.

LPS, a component of the cell wall of gram-negative bacteria, is an important mediator of the host
inflammatory response to infection. A study of 169 patients with chronic hepatic disease found elevated
levels of LPS in 27%, 85%, and 41% of patients with chronic hepatitis, chronic hepatitis with acute
[18]
exacerbation and cirrhosis, respectively . In patients with chronic liver diseases, elevated levels of LPS
in the portal and/or systemic circulation are common because of increases in intestinal permeability and
bacterial translocation. LPS from gut microbiota contributed to HCC promotion by activating TLR4
signaling. Classically, TLR4 recognizes microbial lipids in homodimer configuration, thus activating various
intracellular signaling pathways, such as the NF-kB and MAPK pathways. TLR4 has been identified in HCC
and may play a role in progression of HCC. LPS-induced activation of TLR4 signaling promoted HCC cell
survival and proliferation associated with regulation of the activation of the NF-kB and MAPK pathways [19-22] .

In the present study, we demonstrated substantial activity of NF-kB in cells transfected with both β2GPI
and HBsAg and treated with LPS. Our data suggested that the combined action of β2GPI and HBsAg were
enhanced by LPS in the progression of carcinogenesis. Constitutive expression of NF-kB is emerging as a
hallmark of cancer. In fact, constitutive NF-kB activation is generally associated with cancer proliferation,
[23]
survival, chemoresistance, and progression of HCC .
NF-kB is another pro-inflammatory transcription factor that triggers downstream signal transduction and
increases expression of downstream factors. In the present study, inflammatory cytokines (TNF-α, IL-1β, and
AFP) were substantially elevated in cells transfected with both β2GPI and HBsAg and treated with LPS, more
so than by single transfections with either factor. The action of various inflammatory mediators is known
to occur in carcinogenesis. TNF-α has been postulated to have a crucial role in the pathogenesis of various
cancers. It is one of the most important pro-inflammatory cytokines involved in the growth, differentiation,
cellular function and survival of many cells. It is produced by several types of cells, including macrophages,
[24]
neutrophils, fibroblasts, keratinocytes, NK cells, T and B cells, and tumor cells . IL-1β is also known to
mediate several immune responses in HCV/HBV infection. There is a network of TNF-α and IL-1β secretion
[24]
and interactive bio-functions in immune responses .