Discussion
Based on observed results, it is elicit that E. coli BW25113 is capable
of expressing and secreting cardiac peptides including LANP, VD and KP
as well as GFP as an indicator protein under the control of hypoxic or
anaerobic condition. Secreted cardiac peptides by interacting with their
receptors on carcinoma cells result in induction of apoptosis and cell
death. The main advantage of presented approach is selectivity in both
targeting tumor site and inducing cytotoxicity only in hypoxic areas of
tumor. As demonstrated in sections collected from tumor tissue and
liver, bacteria preferentially localized in tumor site with a
significantly greater density. Furthermore, activation of protein
expression only at hypoxic condition, prohibits expression of cardiac
proteins and observation of undesired side effects at normally perfused
body organs including liver, lung, heart, etc. Other studies, including
the one performed by Stritzker et al., have also demonstrated that
different strains of e. coli are capable of preferentially colonizing in
tumor site (13).
Based on the study performed by Kim et al, contrary to the salmonella
typhimurium which is frequently applied in bacterial tumor targeting, E.
coli strain K-12 (MG1655) is not capable of suppressing tumor growth per
se (14). Our results also clearly demonstrated that administration of e.
coli BW25113 even at high doses didn’t significantly suppressed tumor
growth. As the only difference between bacteria bearing construct and
untransformed ones is the capability of expressing cardiac proteins,
differences in tumor growth delay may be attributed to the expressed
cardiac peptides under hypoxic condition of tumor.
Several in vitro studies have clearly indicated the cytotoxic effects of
cardiac peptides against malignant cells. Based on studies performed by
Vesely et al, four cardiac peptides including LANP, KP, VD and ANP
demonstrate significant anti-proliferative effects against breast,
prostate, lung, brain and renal carcinomas (15). Reported by Sun et al.,
VD and KP demonstrate their anti-cancer activities in prostate cancer
through inhibiting RAS activity (16). Furthermore, LANP is capable of
inhibiting 80-90% of MEK1/2 and ERK1/2 activity in prostate cancer
cells (17, 18). As ERK plays an important role in controlling apoptosis,
inhibition of its phosphorylation and activation by LANP may partly
explain its pro-apoptotic mechanism of action (19).
Currently, multiple studies have addressed the role of cytokines in
cancer proliferation, invasion and metastasis. For instance, it has been
shown that high circulating levels of IL-1β and IL-6 are unfavorable
prognostic indicators and directly interrelate with higher rate of
recurrence and clinical stage in patients with breast cancer (20-22).
Contrarily, overexpression of INF-γ has shown to promote cell death
through up-regulating caspases and demonstrate significant
antiangiogenic activity (23-25). Administration of bacteria bearing
construct resulted in a complex effect on expression of pro-inflammatory
cytokines. Many studies have shown that cardiac peptides demonstrate an
anti-inflammatory behavior. For instance, it has been shown that ANP
could down regulate expression of TNF-α which has an important role in
alloreactivity of T-cells (26, 27). Furthermore, ANP can also enhance
expression of IL-10 which in turn, may result in downregulation of IL-2
and proliferation of T-cells (28, 29). IL-6 concentration has also been
shown to decline in response to administration of ANP (29). Finally,
administration of ANP together with LPS decreases production of IL-12 by
dendritic cells and generates fewer INF-γ positive T cells and much more
IL-4 positive ones (30). Consistent with these reports, here we also
observed a significant decline in peripheral blood concentrations of
pro-inflammatory cytokines including IL-1a, IL-1b, IL-6, IL-12 and
TNF-a. contrarily the concentrations of IL-10 and INF-γ were
significantly increased in response to therapy.
Angiogenesis is a firmly controlled process, crucial for tumor expansion
beyond 2 to 3 mm in diameter and a critical step of tumor invasion and
metastasis (31). Different studies have now suggested a critical role
for MMP9 in formation of a proper microenvironment for promotion of
tumor growth and angiogenesis through enhancing the association between
VEGF and VEGFR2 (32, 33). This mainly takes place through the
proteolytic cleavage and subsequent release of extracellular matrix
(ECM) bound VEGF by MMP9 activity which is an important step of
angiogenic switch. Based on Bergers et al. inhibitors of MMP9
effectively suppressed tumor growth and reduced angiogenic switching
(34). It has also been shown that MMP-cleaved VEGF plays an important
role in tumors vascular patterning (7, 35). Based on previous reports,
cardiac peptides effectively reduce expression of VEGF in vitro (7). In
present study, we demonstrated that administration of bacteria
expressing cardiac peptides under hypoxic condition could reduce MMP-9
expression in vivo. In parallel, VEGFR2-triggered angiogenesis is
considered as a hallmark of cancer progression and metastasis (36, 37).
Consistent with previous studies, we also found that expression of
VEGFR2 was significantly reduced in tumors from mice receiving bacteria
bearing cardiac peptides expressing construct (7).
Results from different reports show that high tumor micro-vessel density
measured by immunohistochemical analysis of CD31 expression, is
correlated with higher rates of lymph node metastasis and poor prognosis
(38-40). In addition, MMP-9 has widely been mentioned as an important
driver of cancer’s malignant progression, invasion and metastasis (41).
Also, determination of proportion of cancer cells undergoing
proliferation by immunohistochemical analysis of Ki-67, has shown that
high Ki-67 expression is associated with increased risk of metastasis
development as well as poor overall survival (42). As administration of
bacteria expressing cardiac peptides could effectively reduce expression
of Ki-67, CD31 and MMP-9, it may be an effective therapeutic for
preventing from invasion and future development of metastasis. Finally,
presence of TILs (especially CD8+ TILs), which demonstrate effective
anti-tumoral activity in tumor sites, has shown to be together with a
favorable prognosis value in different neoplasms (43-46). Therefore,
significant increase in survival rate as observed with Kaplan-Meyer
analysis may be partly attributed to the higher number of CD8+ TILs in
tumor site, as well as lower expression values of above-mentioned
markers.