Establishment and characterization of Newcastle disease virus (NDV) persistently-infected colorectal cancer cell lines
Newcastle disease virus (NDV) is an avian paramyxovirus that causes Newcastle disease (ND) outbreak worldwide. For the last few decades, NDV has been studied extensively for its oncolytic effect in various cancer cell lines both in vitro and in vivo. For some strains, they have entered several ph...
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| Format: | Thesis |
| Language: | en |
| Published: |
2022
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| Subjects: | |
| Online Access: | http://psasir.upm.edu.my/id/eprint/122737/1/122737.pdf http://psasir.upm.edu.my/id/eprint/122737/ http://ethesis.upm.edu.my/id/eprint/18644 |
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| Summary: | Newcastle disease virus (NDV) is an avian paramyxovirus that causes
Newcastle disease (ND) outbreak worldwide. For the last few decades, NDV has
been studied extensively for its oncolytic effect in various cancer cell lines both
in vitro and in vivo. For some strains, they have entered several phases of clinical
trials. Recently, studies showed that NDV persistent infection in cancer cell lines
emerged which resulted in a development of populations of cancer cells that
resisted the oncolytic effect of the virus. Nevertheless, the impact of the
persistent infection on the cancer cells, phenotypically and genotypically, is yet
to be elucidate. Therefore, the aim of this study was to investigate the phenotypic
and genotypic of the NDV persistently-infected colorectal cancer (CRC) cell. To
achieve this objective, three CRC cell lines, namely, LoVo, RKO, and
HCT116p53+/+ were challenged with recombinant NDV expressing green
fluorescent protein (rAF-GFP). Following prolonged infection, NDV-resistant
subpopulation in each cell line emerged. These cell lines were designated as
LoVoPI, RKOPI and HCT116p53+/+PI, respectively. Flow cytometry analysis
showed that all these PI cells expressed GFP signal, despite diverse in
expression level, confirming the presence of actively replicating NDV within the
cells. During NDV reinfection, these PI cells resisted NDV-mediated oncolysis,
continued to grow, and produce infectious plaques-forming virus progenies.
The phenotypic characteristics of these PI cells such as invasiveness and
tumourigenicity were investigated via cell migration assay, colony formation
assay, and spheroid formation assay. Results revealed that all PI cells have
significantly reduced migration ability and formed lesser colonies compared to
that of their parental counterpart. In spheroid formation assay, LoVoPI and
HCT116p53+/+PI formed smaller size of spheroid compared to their parental cells
while RKOPI did not integrate into an intact spheroid, suggesting that persistent
infection of NDV negatively affect the tumourigenicity of the PI cells. Despite their resistant towards NDV infection, a subpopulation in all the mock-infected PI cells
were stained positive for Annexin V or 7-AAD, suggesting these PI cells exhibited
an elevated basal level of apoptotic cell populations. We further challenged the
PI cells with apoptosis-inducer 5-fluororacil (5-FU) and the results showed that
all the PI cells were significantly more sensitive to 5-FU treatment compared to
their parental cells.
One of the mechanism of NDV mediated oncolysis is via caspase-dependent
apoptotic pathway. None of the caspases were upregulated in PI cells
suggesting that other apoptotic mechanisms might be involved. Therefore, we
examined the expression of Bcl-2 family proteins including pro-survival Bcl-2
proteins (Bcl-2, Mcl-1 & Bcl-xL), pro-apoptotic Bcl-2 proteins (Bax, Bak & Bim)
and inhibitor of apoptosis (IAP) protein (Survivin) in these PI cells. Western blot
results revealed the elevated expression of the pro-survival Bcl-2 proteins and
Survivin proteins in LoVoPI and RKOPI cells, suggesting that these proteins
were critical in promoting the survival of PI cells. Knockdown of Survivin by small
interfering RNA (siRNA) enhanced the NDV-mediated apoptosis in RKOPI and
HCT116p53+/+PI but not in LoVoPI; knockdown of Bcl-2 however did not enhance
the NDV-mediated apoptosis in all PI cells. Overall, our study revealed that NDV
persistent infection may confer clinical benefits as the PI cells were attenuated
in their tumourigenicity and became more sensitive to apoptotic inducer. |
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