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.