The development of original approaches in cancer treatment to arrive new level of success in the continuous battle against cancer required searching deeply in the relative tiny details. One of the interesting tricky points is the relation between estrogens and breast cancer. Our study focused on the aromatase enzyme a rate limiting enzyme in the pathway of estrogen synthesis. Likewise, the present work comprises the examination of calcitriol (vit D3-OH) or/and low dose gamma irradiation as novel approaches targeting the adjustment of aromatase expression, reducing the tumor proliferation and overcoming the disadvantage of using ordinary aromatase inhibitors.

While, aromatase (CYP19) catalyzes the synthesis of estrogens from androgenic precursors and ovaries are the principal source of circulating estrogens in premenopausal women, many other tissues including the breast express aromatase and hence have the capacity to locally synthesize estrogens [34]. Tissue specific promoters differentially regulated by various transcription factors are controlled the aromatase expression in these different situates [35]. Lønning et al. stated that circulating pool of estrogens may be more important for breast carcinogenesis than local estrogen synthesis upregulated in postmenopausal benign breast tissue or malignant breast tumors [36].

In the present study, the data obtained pointed out to significantly higher (p<0.05) mRNA expression of aromatase in solid tumor of murine model (EST bearing mice) compared with its corresponding values in the other mice groups (Table 2). These results could highlight the relation between locally synthesized estrogen and the development of cancer. Zhao et al. disclosed the hyper proliferation and neoplastic damage caused by estrogen and its metabolites [37].

Worth mention, mouse model is not being an exact mirror of estrogen production in humans since mouse aromatase is expressed in fewer tissues than human aromatase [38]. However; this was going over in our work as a highly expressed aromatase gene was observed in solid tumor of mice inoculated with EAC cells (Table 2). The upregulation of aromatase gene expression could be attributed to the presence of multiple exons available for use in aromatase gene. The regulation of tissue specific aromatase gene by alternative uses of these exons has been identified [39]. During cancer development exon 1 termed exon I.4 is often toggled from exon I.4 (1b) to exon I.3 (1c) or exon PII (1d), (1b) which causes enhancement of aromatase expression in cancer-associated adipocytes and fibroblasts. Moreover, the aromatase gene is further regulated at both the transcriptional and post-transcriptional levels through protein kinase A (PKA)-, protein kinase C (PKC)-, and tyrosine kinase receptor-mediated signaling pathways that employ prostaglandin E2 and class 1 cytokines. Epigenetic modifications of the aromatase gene and microRNA mediated aromatase regulation may play a critical role in cancer progression. Several genetic polymorphisms in the aromatase gene may be prognostic factors of disease and may influence response to aromatase inhibitors.

Lately, inhibition of aromatase has become one of the key strategies in breast cancer treatment. In the present work, we recorded a significant decrease in the expression of aromatase in EST mice treated with calcitriol or/and exposed to low dose gamma irradiation (LDR) with remarkable advantage of mice received Cal and exposed to LDR (Table 2). The results obtained signalize obviously the capability of calcitriol to reduce aromatase gene expression even applied lonely or with LDR. As an inception, calcitriol could exert anti proliferative, pro-apoptotic and pro-differentiation actions in several malignant cells including   breast cancer cells. Thoroughly, it can inhibit the expression of CYP19A1 gene encoding aromatase enzyme that catalyzes estrogen synthesis from androgenic precursors. In addition, calcitriol repress nuclear receptor responsible for estrogen actions; Era [40]. These mutual actions (opposing the estrogen effect and inhibition of aromatase mRNA expression) trim down the levels of the estrogenic hormones as well as their biological activities within the breast [41]. It was previously mentioned the presence of different promoters distributed over a regulatory region of the CYP19A1 gene permit regulation of the aromatase gene in a tissue selective manner [42]. The main beneficial action of calcitriol is its ability to selectively diminished aromatase transcription. Promoters responsible for aromatase gene expression not only differ between normal tissues but also between normal and   cancer tissue. So, tissue-selective CYP19A1 promoters have been proposed as a possible strategy for development of selective aromatase modulators (SAM) [43]. Wheler et al. mentioned that AIs, a main therapeutic approach to treat breast cancer, prevent progression or recurrence of cancer breast in postmenopausal women after primary therapy have a serious disadvantageous of inhibiting estrogen synthesis at any sites as bone [44,45].

Whereas, calcitriol could inhibit the production of estrogen receptors as well as suppress other growth signaling pathway selectively in tumor cells [46]. Furthermore, the improvement recorded in aromatase expression in mice exposed to low dose gamma radiation (Table 2) could be due to enhancement of the immune responses because there are no consequential evidences of direct effect of low level of gamma radiation on the machinery of aromatase gene expression. However, LDR may   potentiate the action of Cal on the unique exon’s arrangement in the structure of aromatase gene.

Just about the same issue, our experimental results identify significant increases in the protein expression of COX2 and PGE2 synthesis synchronous with highly expressed aromatase mRNA (Tables 2-4). We hypothesized the correlation between inflammatory response and aromatase gene expression in the development of cancer. We expected the contribution of Inflammation/ Aromatase axis to the increased risk of hormonal developing cancer. One could attribute the changes induced in COX 2 and PGE2 protein to the inflammatory burden implicated after inoculation of EAC cells in the right thigh of normal mice which in turn work to enhance aromatase expression. In agreement with this view Grivennikov et al. reported that the inflammatory responses play influential roles at different stages of tumor development, including initiation, promotion, malignant conversion, invasion, and metastasis [47]. The most, if not all, solid malignancies trigger an intrinsic inflammatory response that builds up a pro tumorigenic micro environment [35]. Tak and Firestein, nominate nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) as a link between inflammation, immunity to cancer development and progression [48]. NF-κB, highly activated at sites of inflammation in diverse diseases, can induce transcription of pro inflammatory cytokines, chemokines, adhesion molecules, matrix metalloproteinases (MMPs), COX2 [49,50].

Cyclooxygenases COX1 and COX2 are rate limiting enzymes in the production of PGE2 from arachidonic acid. The major pathophysiological functions of   PGE2 are bringing out pyrexia, pain sensation, and inflammation. So, the analgesic and anesthetic effects of the most widely used non-steroidal anti-inflammatory drugs (NSAIDs) are thought to be driven by inhibition of the production of PGE2. Going to the point, COX-2-derived PGE2 working to stimulates the cyclic adenosine monophosphate (cAMP) to PKA signal transduction pathway that activate CYP19 transcription resulting in increased aromatase expression [51]. Looking upon EST mice received doses of Cal or/and exposed to LDR, significant decreases in the levels of COX2 and the concentration of PGE2 were observed (Table 3,4). Calcitriol might play a noticeable role in preventing the exaggeration of inflammation in the tumor tissue of murine mice. Han et al. stated that pro inflammatory cytokines and COX 2 proteins are significant remarks of in situ active inflammatory responses [52]. Meanwhile the decreases happened in the COX2 protein expression and PGE2 concentration in EST mice received calcitriol treatment denotes its anti-inflammatory capacity. It could be suggested that calcitriol barricade the COX2 inducible cytokines in tumor tissue and consequently checked the production of COX2 in tumor tissue. The COX2 enzymatic activity upregulation could be attributed to the activation of several tumor necrosis factor alfa (TNF-α) dependent intracellular signaling pathway which plays a key role in the control of COX-2 induction. Different protein kinases and transcriptional factors that are greatly regulated by TNF-α are contributed to COX2 upregulation. The expression of COX2 mRNA and protein is often enhanced in various human cell types by inflammatory cytokines such as TNF-α [28]. As COX2 is the key enzyme in pathway of production of pro inflammatory PGs, synthesis and biological actions of PGs significantly withdrawn [41]. Furthermore, calcitriol could decrease PGE2 via enhancing the expression of 15-Hydroxyprostaglandin dehydrogenase (15-PGDH), the enzyme initiating PG catabolism [53].

Besides, low-dose radiation can be beneficial to living organisms, since the mild oxidative stress induced by low-dose radiation induces adaptive responses, including enhanced cell damage repair/protection [3,54]. Thus, the decreases in COX2 expression and PGE2 concentration in EST mice exposed to low dose gamma radiation could be interpreted in the view of the hormetic effect of LDR. Further, in EST mice exposed to LDR and received calcitriol demonstrate prevalent reduction of COX2 and PGE2 expression holding up the view of LDR advancing action of calcitriol.

Our data shows tumor micro environments rich with COX-2 and PGE2 (Table 3,4), elevated tumor apoptosis resistance manifested by decrease in cleaved caspase3 levels (Table 5) as well as tumor enlargement (Table 6).  The low concentration of Caspase3 observed in tumor tissue (addressing inhibition of apoptosis) might be considered a consequence event follow COX2 and PGE2 over expression. Favaloro et al. revealed that regulation of apoptosis is linked to several pathophysiological disorders, including autoimmune disorders, Alzheimer’s disease, and cancer [55]. During tumor transformation COX-2 over expression and activation caused increased production of PGE2, which can stimulate tumor growth by enhancing angiogenesis and invasiveness as well as inhibiting immune surveillance and apoptosis [56]. Further the over expressed COX2 up regulate Bcl2 localized to outer membrane of mitochondria leading to inhibition of pro apoptotic proteins Bax and Bak. These proapoptotic factors, work on mitochondrial membrane to promote permobilization and release of cytochrome c which is an important signal in apoptosis cascade.The mitochondrial release of cytochrome c in cytosol has been shown to be rate limiting for the activation of caspases and endonucleases [57]. Li et al. reported that cytosolic cytochrome c activates procaspase-9 by binding to Apaf1 in the presence of dATP, leading to caspase-9 activation and subsequent activation of downstream effector caspases, including caspase-3, with triggering of apoptosis [58].

He et al. stated that Caspases, a family of cysteine proteases that cleave their substrates on the carboxy-terminal side of specific aspartic acid residues [59]. These proteases are generally present in healthy cells as inactive zymogens, but when stimulated they undergo autolytic cleavage to become fully active. Cleaved caspase3 (an executioner caspase) is one of proteases or enzymes can kill the cell by degrading proteins indiscriminately. Thus, it could be mention that COX2 and PGE2 rich microenvironment hinders discharge of cytochrome c to cytosol and afterward suppress apoptotic cascade.

Standing on the previous we have two cohesive points to be followed in concern of calcitriol or and low dose gamma radiation impact against cancer proliferation in EST bearing mice. First which we already discuss is the Over expression of aromatase gene and second is the inhibition of apoptosis in response to highly expressed cycloxygenase2 and PGE2 which we are going to interpret.

The experimental data indicate that calcitriol administration or/and LDR exposure to EST mice induced significant increases in Caspase3 level compared with E mice (Table 5). The reported increases in caspase 3 tailored the promotion of apoptotic process in tumor cells after calcitriol administration. Krishnan et al. affirmed the anticancer effects of Calcitriol due to its ability to suppress proliferation and promotes apoptosis and differentiation of malignant cells and inhibits tumor angiogenesis and invasion [60].

Essentially most pathways which are leading to apoptosis are ultimately regulated by Bcl-2 family of proteins. The Bcl-2 family of proteins is divided into two subclasses that either promote (BclXS, Bax) or suppress (Bcl-2, Bcl-XL, Mcl-1) apoptosis. These proteins can form hetero- and homodimers and the ratio of apoptosis promoters to apoptosis suppressors is one determinant of cellular response. The increases in Bcl-2 expression were observed in a number of cancers including prostate, lung, and breast and is often associated with advanced stages of the disease [61]. Expected upregulation of Bcl2 could be confirmed by the over expression of COX2 and PGE2 (greatly linked toBcL2 prompting) recorded in the present study. On the other hand, the inhibition of this inflammatory signs due to Cal (Table 2,3) could prevent the up regulation of this pre survival proteins. As Bcl2 protein is critical in preventing the cell from initiating apoptosis, calcitriol have been sought to provide potential anti-cancer therapies by making cancer cells more susceptible to apoptosis-inducing agents.

In addition, the improvement in Caspase 3 expression in EST mice exposed to LDR could interpreted in the view of LDR hormetic effect. The LDR activation of apoptosis could be executed in cancer cells by different mechanisms like inhibition of inflammatory signs, inhibition of aromatase as well as promotion of proapoptotic proteins^3,4. Xin et al. reported that LDR induces apoptosis of tumor cells and has numerous beneficial effects on normal tissues, including radiation homeostasis and adaptive response [62].

In conclusion Calcitriol and low dose gamma irradiation could introduce transferable potentiating effect towards the cancer proliferation via selective suppression of tumor tissue m-RNA aromatase and amendment of inflammatory response which trigger apoptosis in tumor cells in murine model of EST. This may represent a prospective therapeutic policy for the management of solid tumor and decreasing the possibilities of tumor therapeutic resistance.

Conflicting Interests

The Authors declare that there is no conflict of interest.