Anti-proliferative and pro-apoptotic effects of bacterial
secreted cardiac protein
Incubation of MCF-7 human mammary carcinoma cells with growth media of
bacteria bearing construct under hypoxic condition for a 48-h period,
promoted both cell death and apoptosis. The results of MTT assay
demonstrated that administration of the growth media of bacteria bearing
construct, resulted in inhibition of growth in about 40% of MCF-7 cells
which was significantly higher compared to growth media of bacteria
without construct and PBS (25% and 5% inhibition respectively). (fig.
3A). The Annexin-V/7-AAD flow cytometry analysis also demonstrated that
treating MCF-7 cells with the growth media of bacteria bearing construct
significantly increases rate of apoptosis compare to bacteria without
construct and PBS treated group (Fig. 3B).
Preferential colonization
of E. coli BW25113 within tumor
Systemic administration of E. coli BW25113 in syngeneic mice bearing
spontaneous mammary tumors resulted in elicit and preferential
colonization of bacteria at tumor site. As depicted in figure 4A,
bacteria accumulated in tumor site with an approximately 15-fold greater
density compare to the liver. Furthermore, microscopic examination of
tissue slides also confirmed that E. coli was present in all analyzed
tumors. Accumulation of E. coli BW25113 was more significant at necrotic
sites of tumor.
Confirming hypoxia-activated expression of cardiac peptides
following tumor specific localization of E. coli Bw25113 usingIn
vivo fluorescence
reflectance imaging
E. coli BW25113 bearing cardiac hormone expressing constructs and PBS
were injected to the mice and images were collected immediately and 24
h, 3 days and 6 days post injection. As depicted in figure 4B, no
significant differences in emitted fluorescent intensity was observed
between two group during the first 24 h post injection. On day 3
however, emitted fluorescent from bacteria receiving group slightly grew
and become a bit higher than those observed in PBS receiving group.
Continuing similar trend, on day 6
post injection, emitted fluorescent from bacteria receiving group
reached to its maximum and was completely restricted to the tumor site.
Regarding other organelles, no significant changes in fluorescent
intensity was observed during image acquisition period neither in
control nor in bacteria receiving groups. These observations further
confirm site specific colonization of bacteria and expression of GFP as
an indicator of successful expression of construct in hypoxic condition.
Tumor growth retardation potential ofbacteria expressing cardiac
protein
The delay in tumor growth was evaluated over a 24-day period after the
administration of bacteria bearing construct with concentrations equal
to 107 and 108 cfu/g, bacteria
without construct in concentration equal to 108 cfu/g
and PBS as the control group. Based on results, administration of
bacteria without construct didn’t result in any significant tumor growth
retardation compared to PBS receiving group. Therefore, bacteria per se
were not capable of suppressing tumor growth speed. Contrarily,
administration of bacteria bearing constructs, even at concentration
equal to 107 cfu/g resulted in a significantly greater
delay in tumor growth compare to PBS group. Furthermore, increasing the
concentration of bacteria from 107 cfu/g to
108 cfu/g resulted in more enhanced tumor suppression.
As depicted in figure 5A, differences in delaying tumor growth between
two concentrations of bacteria bearing construct become significant on
day 15 from beginning of treatment. Furthermore, the differences between
delaying in tumor growth in group receiving concentrations of bacteria
equal to 108 cfu/g and PBS group become significant on
day 12. However, similar significancy for the group receiving bacteria
bearing construct at 107 cfu/g took place on day 15.
Based on these observations we concluded that first, the delay in tumor
growth observed with administration of bacteria bearing construct is
mainly due to the secretion of cardiac proteins. Second, increasing
concentration of administered bacteria may result in enhancement of
production and secretion of cardiac peptides. Administration of bacteria
bearing constructs at both concentrations also significantly enhanced
survival rate as observed in Kaplan-Meyer analysis (figure 5B).