studies

HEPATOLOGY, VOLUME 62, NUMBER 1 (SUPPL) AASLD ABSTRACTS 377A
327
Histone 3 lysine 9 trimethylation (H3K9me3) and
inflammasome as novel senescent associated markers
of drug induced premature senescence in hepatocellular
carcinoma cell lines
Bijoya Sen 1 , Tarique Anwar 2 , Chhagan Bihari 3 , Archana Rastogi 3 ,
Nirupma Trehanpati 1 , Sukriti Sukriti 1 , Shvetank Sharma 1 , Shiv K.
Sarin 4 , Gayatri Ramakrishna 1 ; 1 Dept of Research, Institut of Liver
and Biliary Scienc, Delhi, India; 2 Center for DNA fingerprinting
and diagnostics, Hyderabad, India; 3 department of pathology,
institute of liver and biliary sciences, New Delhi, India; 4 institute of
liver and biliary sciences, New Delhi, India
Abstract Chemotherapeutic drugs can induce premature
senescence in cancerous cells. The p53-p21 and pRb-p16 axis
are the most well studied mechanisms involved in induction
of senescence. However, these pathways are also associated
with cell cycle arrest conditions of quiescence, differentiation
and also cell death. Purpose of this study was to evaluate
novel markers specific to premature senescence. Hepatocellular
carcinoma cell lines (Huh7, Hep3B and HepG2) when
treated with low dose of doxorubicin, a well known chemotherapeutic
agent, showed permanent cell cycle arrest in G2/M
phase. Compared to proliferating cells, the senescent cells
showed enlarged cytomorphology, increased expression of
cyclin dependent kinase (p21) and positivity for SA-β-galactosidase,
all indicative of premature senescence. To analyze
a novel structural marker associated with senescence, transmission
electron microscopy (TEM) was done which showed
accumulation of autophagic vacuoles, condensed chromatin
and prominent mitochondrial-endoplasmic reticulum (ER)
coupling which were conspicuosly absent in non-senescent
hepatocytes. The changes in chromatin was further evaluated
by immunofluorescence microscopy which showed presence of
heterochromatic foci only in the senescent cells. Hetrochromatin
formation in turn involves histone modification and senescent
cells showed a significant increase in inactive chromatin mark
viz., H3K9 trimethylation compared to non-senescent cells. Further,
microarray based gene expression analysis was done to
identify novel pathways uniquely associated with senescence
which showed a differential increase in cytokine-chemokine
response. This in turn was supported by the fact that the secretome
of senescent cell was highly effective in closure of wound
as assessed by the wound healing assay, unlike the control
cell secretome. The active senescent secretome was associated
with increased transcript level of PyCard/Asc, and IL-1β indicative
of inflammasome activation. In conclusion we have now
identified inflammasomes and H3K9 trimethylation as specific
senescence associated markers. This project was funded by
Dept of Biotechnology, India.
328
Netrin-1 Promotes Liver Cancer Cells Collective Invasion
in a 3D Cell Culture Model
Ping Han 1 , Yu Fu 2 , Jingmei Liu 1 , Dongxiao Li 1 , Yunwu Wang 1 ,
Dean Tian 1 , Wei Yan 1 ; 1 Gastroenterology, Tongji Hospital, Tongji
Medical College, Huazhong University of Science and Technology,Wuhan,China,
Wuhan, China; 2 Gastroenterology, Union Hospital,
Tongji Medical College, Huazhong University of Science and
Technology,Wuhan,China, Wuhan, China
Collective invasion is the new understanding of tumor metastasis,
while the mechanism needs to be further studied. Our
previous studies showed that Netrin-1 promoted the invasion
ability of liver cancer cells(LCCs). Immunohistochemical studies
showed that LCCs invaded surrounding tissue as collective
invasion. The expression of Netrin-1 in these LCCs was
increased. In this study, we performed a 3D cell culture model
to further confirm the role of Netrin-1 in LCCs collective invasion.
We designed a 3D cell culture model, after centrifuge,
5×10 6 cells were resuspended by 120μl 10% sucrose solutions,
and quickly mixed with equal volume of 0.5% hydrogel
solution( BeaverNano ), the cells were then seeded in a Class
Bottom Cell Culture Dish(NEST), and allowed to grow for six
days. Then, cells were observed under a confocal microscopy.
The number and distance of collective invasion in LCCs were
evaluated after enhanced or silenced expression of Netrin-1.
The N-cadherin shRNAs were used to verify the role of N-cadherin-based
junctions in Netrin-1 promoted collective invasion.
We found the number and distance of invaded SK-Hep1 cells,
a high metastasis potential cell line, were significantly more
than Huh7 cells with low metastasis potential, the normal liver
cell line LO2 cells nearly had no cell invaded. Meanwhile,
the invaded cells had significant more expression of Netrin-1.
Overexpression of Netrin-1 increased collective invasion in the
3D cell culture model and elevated N-cadherin expression in
Huh7 cells, while stable knockdown of Netrin-1 remarkably
inhibited collective invasion and decreased N-cadherin expression
in SK-Hep1 cells. Interestingly, knockdown N-cadherin in
Huh7 cells significantly diminished Netrin-1 promoted liver cancer
cell collective invasion. These results suggest that Netrin-1
enhances N-cadherin junctions to promote LCCs collective invasion,
subsequently may increase LCCs metastasis.(This study
is supported by the National Natural Science Foundation of
China (81472311), the Fundamental Research Funds for the
Central Universities (2014ZHYX020,2014TS077), the Project
sponsored by SRF for ROCS, SEM (2014-1685) and Hubei
Province health and family planning scientic research project
(WJ2015Q006))
Collective invasion of SK-Hep1 cells in 3D cell culture
Disclosures:
The following authors have nothing to disclose: Ping Han, Yu Fu, Jingmei Liu,
Dongxiao Li, Yunwu Wang, Dean Tian, Wei Yan
Disclosures:
The following authors have nothing to disclose: Bijoya Sen, Tarique Anwar,
Chhagan Bihari, Archana Rastogi, Nirupma Trehanpati, Sukriti Sukriti, Shvetank
Sharma, Shiv K. Sarin, Gayatri Ramakrishna