In this study, the effect of COX-2 inhibition on NO release of BCG-treated macrophages was investigated. COX-2 inhibition shows a dual response on the NO release of BCG treated raw cells and this result suggest that COX-2 inhibition might follow both NO dependent/independent pathway in BCG-treated raw cells. Despite the fact that manufacturers call XTT assay as cell proliferation kit, our result shows that XTT assay only measures cell viability depending on mitochondrial activity of cells. Notably, BCG triggers mitochondrial activity in macrophages which appears as an increase in cell viability in XTT assay. Interestingly eventhough BCG induces COX-2, when combined with celecoxib, a dose-dependent decrease in raw cell viability was observed which indicates high toxicity due to co-treatment. Celecoxib might also exhibit anti-tumour activity in both COX-2 dependent and independent pathways since its action does not correlate with COX-2 inhibition always.
4.1 BCG-induced COX-2 inhibition follows NO dependent pathways
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Our findings show inhibition of COX-2 activity (by celecoxib) inhibiting BCG induced NO activity in Raw cells [fig-9(c)]. Immunoblot analysis also shows inhibition of NOS-2 and COX-2 expression by celecoxib at 40ÂµM dose [fig-9(d)]. These datas demonstrates the previous results which suggests that BCG-induced COX-2 expression follows NO-dependent pathways in macrophages (30). Another study shows that NF-kÎ² binding sites are present in the promoter regions of both isoforms which supports promotes tumour growth (44). On the other hand, NOS2 was unexpressed in western blot at BCG dose (5*106 CFU/ml) [lane-1 in fig-8(d)] as well as in [lane-5 in fig-7(d)] in western blot eventhough increased NO levels from Raw cells at same BCG dose were observed [fig 7(c)]. Since NOS is the predominant precursor of NO production, NO levels could be increased [(fig-7&9(c)] but NOS2 mRNA expression would have lowered to catalyze NO formation and hence would not have been detected in western blot [fig-7&9(d)].
4.2 BCG-induced COX-2 inhibition might also follow NO in-dependent pathways
Conversely, we also have results showing no inhibition of BCG triggered NO levels in Raw cells by celecoxib [fig-10(a)] and even BCG induced NOS2 expression is seen at high celecoxib doses-30&40ÂµM [fig-10(d)] which is contradictory to fig-9(d).These results correlates with the idea that BCG induced COX-2 expression not only involves NOS2 dependent pathway, it can also act through some NOS2 independent pathway. As reported in a study, BCG induces COX-2 expression in macrophages through both NO-dependent and independent pathways by activation of NF-kÎ² by Notch1-PI-3K signaling cascades and ERK1/2, P38 MAPKs respectively (30). Apart from this, the interaction between COX-2 and NOS2 differs in each cell line, tissues and pathophysiological conditions (41). Co-expression between these two genes and its byproduct are also identified in several human cancers like Hepatocellular carcinoma, non-small cell lung cancer and colorectal cancer (45,46,47). Thus, the cross-talk between NOS2 and COX-2 demands further investigation of these genes to identify the additional factors associated for NOS2 upregulation in BCG-treated macrophages.
4.3 BCG triggers mitochondrial activity in macrophages
High BCG doses have a cytotoxic effect on cells (34), however increased doses of BCG failed to show a notable decrease in cell viability when performed by XTT test [fig-7(a)]. Accordingly, the live raw cells counted manually from same XTT plate, using trypan blue test shows a sharp decline in cell viability at highest BCG dose-10*10^6 CFU/ml. Theory behind XTT assay suggests that the increased number of live cells correlates with more mitochondria by formation of more orange-coloured formazan. Since BCG is anti-proliferative and strange results were observed in XTT viability test which relies on mitochondrial activity, staining was performed for further investigation. The increased viability suggested by XTT test in fact is increased mitochondrial activity represented by larger and brighter cells in immunofluorescent staining and few cells at highest BCG dose (10*10^6 CFU/ml) indicates BCG toxicity (fig-8). Hence this result indicates that BCG, at appropriate dosage triggers the mitochondrial activity which ultimately shows a proliferative effect in XTT results, eventhough truly there is not an increase in cell viability.
4.4 BCG induces NO in macrophages
Theoretically, it is a well known fact that NO is a critical signalling and effector molecule in macrophage cytotoxicity post BCG immunotherapy and several studies prove that BCG induces NO activity in macrophages. Chemiluminescence results [fig-7(c)] replicates the previous published data that increased BCG dose corresponds to elevated levels of NO release. Convincing evidence in fig-7 (b-c) shows that low NO levels promotes cell growth whereas high concentrations exhibit cytotoxic effects. This hypothesis supports an earlier study which states that low concentrations of NO might stimulate NF-kÎ² leading to NOS2 transcription and high NO levels results in activation of apoptosis inducing factors (48). These results thereby elucidate the involvement of NO in anti- tumor mechanism of BCG in macrophages.
4.5 BCG induces COX-2 in macrophages
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Several researchers have demonstrated the association of COX-2 and its byproduct PGE2 with procarcinogenic effects in the progression of bladder carcinomas. COX-2, one of the major inflammatory mediators in TCC is believed to promote tumour progression by supporting angiogenesis (36). Therefore, elevated COX-2 levels can be used as an effective biomarker in bladder cancer to improve targeted therapy. Immunostaining (fig-12) and Western blot results [fig-7(d)] clearly indicate that BCG induces COX-2 expression in macrophages (fig-12) and our result goes in line with the previous published data (30). Inhibition of COX-2 after BCG stimulation would reduce the cytotoxicity induced by macrophage and its secreted factors.
4.6 Celecoxib decreases Raw cell viability in a dose-dependent manner Mechanistically, celecoxib is known to downregulate PI-3K, a positive regulator of COX-2, resulting in activation of caspase-9 finally leading to apoptosis (49).Â Celecoxib, also initiates apoptosis by cell cycle arrest on a dose-dependent manner (37) which matches our toxicity measures of the drug on cell viability (fig-5). The drug was also found to be very sensitive. If frozen, celecoxib appears to lose its toxicity and it was also noted that it is not stable in DMSO for more than a couple of weeks. Eventhough, effect of celecoxib on raw cells in fig-9(a) shows almost the same results as fig-5, small discrepancies are seen between the absorbance values in these XTT tests. Reasons for the difference in these experiments could be use of different small aliquots of celecoxib in batches (since the drug is not stable) or accuracy of experiments since fig-9(a) has large SEM.
4.7 Mode of action of Celecoxib
Mechanism of action of celecoxib is complex involving different inhibitory pathways to act as an anti-proliferative, anti-apoptotic and anti-angiogenic agent in varied cell types (43). COX-2 expression always doesn't correlate with the response of celecoxib [fig-9(d), 10(d)] and recent result reveals that celecoxib induces action by both COX-2 dependent and independent pathways in urinary bladder cancer cells (35). Other study, suggest that celecoxib induces apoptosis through activation of a novel mitochondrial pathway, where Bcl-2 family is inhibited (38). As shown in fig-9(a-b), the "only CXC" does not show the same results with XTT and trypan blue tests, which might be because of the alteration in mitochondrial activity of Raw cells (in XTT test) after addition of celecoxib (fig-11).
4.8 Co-treatment of BCG with celecoxib shows high toxicity
Our results suggest that celecoxib in addition with BCG, accelerates toxicity by decreasing cell viability as seen in both XTT and trypan blue exclusion viability tests [fig-7(a-b)]. This supports the results from a recent study which suggests that, combination of BCG and celecoxib increases the release of tumour infiltrating lymphocytes (TILs) in macrophages when compared to BCG alone in UCC mouse model (39). Hence, combination therapy of celecoxib and BCG which shows an increase in tumor efficacy turns out to be a promising therapeutic strategy for invasive bladder cancer patients.
4.9 Implications for future research
In future, since, celecoxib is known to have a half life of about 11 hours (43), a time course response of celecoxib in cell viability should be performed. The effect of NO inhibition on COX-2 using L-NAME should be performed since a positive feedback loop exists between these two isoforms (30). We also need to investigate whether supernatant from BCG treated raw cells, where COX-2 is inhibited can induce NOS-2 upregulation in urinary bladder cancer cells (MBT2 cells).
In conclusions, instillation of BCG in bladder induces a pro-inflammatory response which activates immune cells releasing various types of cytokines. Both COX-2 and NOS-2 stimulated by these cytokines seems to be co-regulated with each other. NO mediated regulation of COX-2 takes place through several pathways in macrophages (30) which ultimately results in different macrophageal responses to varied NO levels caused in the inflammatory response (40). In order to enhance the quality of life of bladder cancer patients, determining whether high NO levels are either advantageous to BCG treatment or not is essential. Also, identifying the molecular markers or regulators that modulate tumour microenvironment improves the overall efficacy of BCG therapeutic response. Thus, a better understanding of the BCG-induced tumour remission mechanism in macrophages is crucial for enhancing the treatment of bladder cancer by specific targeting of tumor microenvironment and adaptive responses.
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Majority of the Bladder Cancer cases include Carcinoma in situ (CIS) form. Eventhough, Bacillus Calmette Guerin (BCG) is considered as the golden standard treatment in CIS, only 70% of bladder cancer patients respond to treatment. The exact mechanism of how BCG eradicates tumour is still unknown. Earlier reports put forward the idea that BCG induces an inflammatory response which activates macrophages resulting in stimulation of cytotoxic factors. NO is believed to be responsible for the BCG-mediated cytotoxic effect in macrophages. Recent evidences suggest that a mutual cross-talk exists between NOS2 and COX-2 in cancer. The present thesis was aimed to determine the additional factors essential for NOS2 upregulation in BCG-treated macrophages by inhibiting COX-2 using celecoxib. As analyzed by NO measures and western blot, COX-2 inhibition had a dual impact on NO released by BCG treated Raw cells. In addition, a decrease in cell viability was seen in BCG-treated macrophages co-treated with celecoxib. Immunofluorescence staining results reveals an increase in the mitochondrial activity of BCG-treated macrophages, illustrating the varying cell viability results measured by XTT and Trypan blue exclusion tests. Thus, the molecular pathway between NOS2 and COX-2 should be explored further to investigate the role of NO in BCG immunotherapy.