COX2: A Prognostic Inammogenic Marker Drives Cervical Carcinogenesis In Vivo Through NFκB/IAP/p53Axis

Cycloxygenase2, a prostaglandin synthesizing enzyme is a key player in inammation-induced vasculogenesis that enables tumor growth. This study explores the central role of COX2 and its relative prosurvival proteins in evoking inammatory events during development of an in vivo cervical cancer model upon chronic treatment with 3-methylcholanthrene (3MC; a chemical carcinogen) in virgin-female Swiss Albino mice. Chronic painting of mice cervix with 3MC solution triggered the persistent expression and activity of COX2; eventuating in overexpression of major prosurvival molecules (NFκB, XIAP, survivin, GM-CSF1) and proliferative antigens (Ki67, PCNA). COX2-arbitrated prosurvival signaling subsequently deranged the expression proles of tumor supressor proteins (p53/Acetyl-p53, p21, Rb) within the cervix. COX2 helmed molecular alterations successively surged leukocyte inux within cervix; catering in localized inammation which gradually distorted its tissue architecture. Cervical carcinogenesis was further braced by higher levels of systemic-ROS and RNS, escalated iNOS activity and compromised anti-oxidant enzyme capacities, which were accompanied by splenomegaly. Additionally, circulation of blood-leucocytes with damaged DNA throughout the mice body, envisaged the impact of cervix-limited inammation upon the mice physiology. Conclusively, the present study deciphered the role of COX2 effectuated NFκB/IAP/p53 functions in sequestering the contributors of localized and systemic inammogenesis for propelling 3MC-mediated cervical carcinogenesis in vivo. reports, the present study was designed to investigate the mediatory role of COX2 in domineering the inception and progression of cervical carcinogenesis within Swiss Albino mice upon chronic treatment with 3methylcholanthrene (3MC), a potent chemical carcinogen of PAH family. This study systematically dismantled the role of COX2 in sequestering inammogens to drive the activation of prosurvival signaling nexus for promoting cervical carcinogenesis in vivo. electrophoretically separated using electrophoresis Tris, 192mM glycine, 10% SDS) electro-transferred with of a transfer buffer Tris, 192mM glycine, 10% Methanol). These membranes were blocked with 5% (w/v) BSA solution, washed with Tris Buffered Saline (TBS; pH-7.5; 25mM Tris.HCl, 150mM NaCl) and incubated overnight with primary antibodies at 4 0 C under constant shaking. Blots were thereafter washed with TBST Buffer solution (TBS; tween 20) for 4 times and subsequently incubated with alkaline phosphatase conjugated secondary antibodies (GeneTex, 1:500dilutions in TBS) at 4 0 C for 2h, followed by TBST washing (4times) and incubation with the chromogenic substrate 5-bromo, 4-chloro, 3-indoylphosphate/ Nitro-Blue tetrazolium (BCIP/NBT; for visualizing protein expressions in the form of bands. β-actin was used as a loading control protein. These experiments were performed in triplicate.


Introduction
Cycloxygenase2 (COX2) is preeminent in in ammogenesis of cancers. Formally termed Prostaglandin H 2 Synthase-2 enzyme, COX2 catalyses the rate-limiting step of mitochondrial fatty acid oxidation concerning the conversion of arachidonic acid to biologically active prostanoids which are propagators of in ammation [1,2].
These prostanoids maintain COX2 expression to activate ROS/AKT/activator protein1 [3] and switch on the kinase-cascades [4,5] for enabling malignant transformations. Besides, COX2 promotes 'angiogenesis', the hallmark episode of new blood vasculature growth in solid tumors which assists them to sustain independently. Vascular Endothelial Growth Factor (VEGF), the key regulator of 'angiogenic switch' is also instructed by COX2 for undertaking endothelial growth [6,7]. Therefore, researches delving into contributions of COX2 in various neoplastic progressions are on rise.
With a chromosomal location of 1q31, COX2 gene is highly inducible in nature [8] due to which it gets overtly expressed for organizing in ammatory events only upon viral oncogenic inductions, chemical carcinogenic triggers or in pathological conditions developed due to chronic viral or bacterial infections [9,10]. In the incidences of Human Papilloma Virus (HPV) mediated cancers like cervical and oral carcinomas, extended viral latency period was found to be closely associated with COX2 generated non-compensatory in ammation states [11]. Similarly, persistent xenobiotics exposure induced COX2 activities which reportedly enhanced skin in ammations; bringing forth carcinogenesis [12]. Recent experimental reports are evidential for the prerequisite need of COX2 in cancer stem cell differentiation and proliferation [13,14]. The Polycyclic Aromatic Hydrocarbons (PAHs) of lower molecular weight emitted along with cigarette smoke were found to effectuate p38 MAP-Kinase to cause COX2 mRNA overproduction. As a consequence, eicosanoid signalling got kickstarted within the lung epithelium for launching carcinogenic changes [15]. Often in lymphomas, COX2 observably upgraded proliferative, angiogenic and invasive potentials of leukocytes [16,17]. Multiple cancers are characterized by genetic aberrations along with polymorphisms in their COX2 gene [18][19][20][21][22].
Several interesting documentations are supportive of COX2 being the central under pinner of in ammation, angiogenesis and tumorigenic progression [23,24]. COX2 abetted in ammation becomes the 'formative-ground' of a strong prosurvival signalling network, characterized by deteriorated tumor suppressor activities. Generally, it is a common observation that gain-of-function mutations in the effector genes of the Ras-MAPK and the PI3K/AKT-signalling pathways principally dictate over the malignant transformation processes. Reportedly, COX2 could potentiate many cancers for phenotypically mimicking the effects of these two pathways; even when the concerned driver mutations are absent [25]. In the hypoxic tumor cores, rise in expression of Hypoxia Inducible Factor-1 (HIF-1) collaterally accelerates COX2 kinetics for permitting the expressions of Nuclear Factor κB (NFκB) [26] and its effector-Inhibitor of Apoptosis Proteins (IAPs) [27]. COX2-derived prostanoids further activate MAPK pathway to allow VEGF translation; aiding in vasculogenesis [28,29]. Additionally, COX2 adorns functionality by repressing the tumor suppressors such as p53, p21 or Rb over activation of Bcl 2 , the antiapoptotic protein [30,31]. The relentless cytokine storm following COX2 activation relays dysregulated in ammatory signals which alter the functional differentiation of immune cells for promoting tumorigenesis [32]. COX2 and the related cytokines are held responsible for switching the developmental fate of 'tumorinhibiting' M1 macrophages to 'tumor-promoting' M2 macrophages or Tumor Associated Macrophages (TAM) [33,34]. Therefore, COX2 and its effectors empower a growing tumor mass with the armour of immune evasion [35]. Inevitably, COX2 is the prima facie requirement for promoting autonomous tumor growth.
With regard to the prevalent reports, the present study was designed to investigate the mediatory role of COX2 in domineering the inception and progression of cervical carcinogenesis within Swiss Albino mice upon chronic treatment with 3methylcholanthrene (3MC), a potent chemical carcinogen of PAH family. This study systematically dismantled the role of COX2 in sequestering in ammogens to drive the activation of prosurvival signaling nexus for promoting cervical carcinogenesis in vivo.

Animal Maintenance
Virgin female Swiss Albino mice (Mus musculus; 5-6 weeks old; weight: 23-25gms) obtained from the Central Animal Facility of CNCI were housed in polyvinyl cages within well-ventilated rooms under ideal conditions (temperature: 22 0 C; relative humidity: 50-60%; 12 hour day/night cycle). Prior to treatment initiation, the animals were subjected to a two-week acclimation period during which all the female and male mice were kept in complete isolation from each other. This induced pheromone in uenced oestrous cycle synchrony thereby nullifying the hormonal interferences [36]. Standard guidelines laid down by the Institutional Animal Ethics Committee (IAEC) certi ed by the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), New Delhi, India, were strictly followed for undertaking all animal experimentation.

Experimental design
Random allocation of 100 mice into three broad groups was accomplished on the basis of body weight. Each group was further classi ed into subgroups separated in two batches of 10 animals [No of mice (n) = 5 /cage (x2) for each subgroup]. Group I was kept as an 'untreated' control group where mice did not receive any treatment. Group III (groups 6-10) comprised of mice which had their cervix chronically painted with 3MC (MP Biomedicals, USA) solution prepared in Petroleum Ether (PET, Merck, Emplura, Germany) for respective intervals of 6, 12, 16, 24 and 30 weeks [37]. It was speci cally designated as 'treatment' group for studying cervical carcinogenesis along these time spans. To rule out any carcinogenic effect of the solvent (if any), Group II (groups 1-5) was assigned as a 'vehicle control' batch, with mice being solely treated with PET. Food and water was given ad libitum. Mice were periodically monitored for any visible health abnormalities and deaths. This purposeful procedure of mice grouping is well-represented in Table 1. After every mentioned treatment period, mice from all the three groups were sacri ced and their entire female reproductive tissue was excised out. In order to track the presence of any cervical tumor, these reproductive tissues were longitudinally opened up for examination through its upper vaginal (ectocervix) region. Cervix tissues (with or without tumor) were decapitated out for comparative studies. Besides cervix, blood, and spleen were also collected for experimental purposes.
Weekly body weight alterations in mice were recorded to assess the impact of chronic 3MC and PET treatments on their physiology with time. An orderly record of tumor incidences along with gradually acquired cervical dysplastic stages in the 3MC treated mice batches was also maintained.

Preparation of tissue lysates
Cervix tissues along with the adjoining tumor regions were dissected out, washed, and pooled separately from Group I, II and III mice. Tissue and tumor parts were dried, weighed and homogenized in Radio-Immunoprecipitation Assay Lysis buffer (RIPA;pH-8 comprising of 5M NaCl, 0.5M EDTA,1M Tris, NP-40,10% Sodiumdeoxycholate,10% SDS). The extracts were kept in ice for 30 min followed by sonication and centrifugation at 10,000g for 20 min at 4 0 C. The resulting supernatants were stored in chilled vials at -20 0 C.

Estimation of total protein
Total protein content of the tissue extracts was spectrophotometrically (VARIAN) estimated using 1X Bradford's reagent (HIMEDIA, PA, USA) against a standard curve of BSA. Absorbance was recorded at 595 nm with the experiment being repeated for 5 times.

Western Blot Analysis
The Quantitative estimation of COX2 activity COX2 enzyme (E.C 1.14.99.1) was spectrophotometrically quantitated at 590 nm using COX activity assay kit (Cayman Chemical, Cat No: 760151 Ann Arbor, MI, USA) as per the provided protocols. Results were represented in nmol/min/ml (U/mg of protein).

Isolation of blood leukocytes
One volume of mice blood collected aseptically from heart was mixed with three volumes of Solution A (pH-7.2; 0.87%NH 4 Cl in 10mM Tris HCl), ice incubated for 20 min and centrifuged at 400g for 20 min at 0 0 C. The supernatant was discarded and the pellets were again resuspended in Solution A followed by centrifugation at 400g for another 20 min at 0 0 C. Resulting pellets were suspended in Solution B (pH-7.2;0.25M mesoinositol, Single cell gel electrophoresis (SCGE or Comet assay) The clastogenic effect of 3MC on DNA was assessed following the standard laboratory protocol [41]. Concisely, a suspension of 0.6% (w/v) low melting agarose (LMA; Sigma-Aldrich, USA) and isolated leukocytes (1x10 4 cells) was smeared over a frosted microscopic glass slide which was priorly coated with the xative 0.75% (w/v) normal melting agarose (NMA; Lonza, USA). Following solidi cation at 4 0 C, cell and nuclear membranes were lysed in lysis buffer (pH-10; 2.5 M NaCl, 0.1 M Na 2 EDTA, 10 mM Tris, 0.3 M NaOH, 1%Triton X-100, and 10%DMSO). Exposed DNA from the lysed out leukocytes were unwound in a highly alkaline electrophoresis buffer (pH > 13.0; 300 mM NaOH, I mM Na 2 EDTA) prior to electrophoresis for 20 min (300 mA, 20 V). Slides were washed in neutralizing buffer (Tris 0.4 M, pH 7.5) thrice, stained with ethidium bromide ( nal concentration 40 µg/ml) and examined under a uorescence microscope (Leica). Image analysis, head DNA quanti cation, comet tail DNA length estimation and comet tail moment calculation were performed using Komet Software.

Statistical Analysis
The mean values of the control, vehicle control and treatment mice groups were compared by factorial Analysis of Variance (ANOVA). The relationship between the studied parameters was analysed by calculating Pearson's correlation coe cient using CORREL function of Microsoft Excel. Data were expressed as mean ± standard deviation (S.D.). p value calculations were performed using Prism GraphPad Software. * p < 0.005 and ** p < 0.01 were considered statistically signi cant in comparison to control batches.

Results
The optimum 3MC dose of 0.6 mg/ml was selected as the safest and e cacious treatment dose (< LD50) only after checking its impact on in vivo toxicity parameters namely SGPT, SGOT, ALP and creatinine levels (data not shown). Carcinogen preparation process is elaborated in Fig. 1A. The treatment durations of 6, 12, 16, 24 and 30 weeks were also accordingly identi ed. A clear schematic outline of the experimental design with appropriate timeline of animal sacri ce has been illustrated in Fig. 1B.
Emergence of tumor at the cervical region upon chronic 3MC treatment Prior to sacri ce, the experimental mice were preliminarily monitored for any apparent changes in the appearance of their ori ce and internal female reproductive organ owing to chronic treatment with PET or 3MC.
Relevant observations noted among mice of Group-I, II and III are comparatively depicted in Fig. 2A and 2B. Obvious progressive changes in the vaginal ori ce of 3MC treated mice ( Fig. 2Axi-xv) were an outcome of internal changes in their reproductive system (Fig. 2B). As indicated in Fig. 2Biv and v, remarkable alterations of the mice reproductive organs in 12 and 16 weeks of 3MC treatment mostly included mild thickening along with growth of new blood vasculature. Group-III mice from 16th week batches had a mildly swelled ori ce ( No such abnormalities were noted in the vehicle control and control mice neither amidst the treatment intervals nor after 24 weeks. Cytopathological changes in terms of increased 'keratinisation' of the cervical squamous epithelial cells bearing 'nuclear holes' (Fig. 2Cxii; indicated by arrows) became perceptible amongst 3MC treated mice in 12th week.
In ammation mediating leukocytes such as eosinophils, neutrophils and monocytes were also persistently apparent in the cervical in ltrations (Fig. 2Cxiii) during the entire treatment span. In the 24th and 30th weeks, the mice cervical exfoliates exhibited 'hyperchromatic nuclei' (Fig. 2Cxiv; indicated by arrows), assumed up a 'bizarre-structure' and harboured 'perinuclear halo' (Fig. 2Cxv, indicated by arrows) as signs of 'vacuolation'.
Cellular distortions of these types were clearly absent in PET treated groups. Acquired observations were comparable with control animals.
Noticeable cytopathological alterations in the cervix necessitated in exploration of its histology which vividly exhibited trans gurations as revealed from the presence of mild to severe cervical dysplasia ( Fig. 2Diii & iv) in mice exposed to 3MC for 6 and 12 weeks. Interestingly, development of 'invasive carcinoma' eventuated with extension of treatment upto 16 weeks (Fig. 2Dv). Furthering 3MC treatment to 24 weeks and beyond resulted in disorientation of cervical epithelial layers (Fig. 2Dvi). As clearly comprehensible from the micrographs, no such remodelled cervical tissue features were discerned amongst the vehicle control and control mice even after 24 weeks ( Fig. 2Di & ii). These aforementioned changes were in coherence with attainment of a cervical tumor. The rate of incidences of the attained dysplastic changes and tumor among Group-III mice is clearly shown in Table   2B. Moreover, chronic 3MC treatment affected the body weight of mice. The ndings tabulated in Table 2A depicted a gradual loss in bodyweight of these mice in 6weeks of 3MC treatment which continued till 12 weeks (25 ± 0.1716 gms). A signi cant weight reduction by 24 ± 0.0100gms was observed in the 16th week which further increased by 29.1067 ± 0.1940gms in 24th week and beyond. Therefore, the decreasing trend in body weight got violated by a consistent rise in the 30th week of 3MC treatment.
Table2A: Impact of 3MC treatment upon body weight of mice.  The reproductive tissues isolated from these mice batches appeared to be strangled with blood vessels (Fig. 5B; indicated by arrows). This kind of vasculogenic changes are product of carcinogenesis-enabling vehement in ammatory reactions, which thereafter prompted for the subsequent exploration of the responsible molecular interactome involving COX2. According to western blot results (Fig. 5D), the cervical COX2 expression in mice heightened along with 3MC treatment extension. An elevated COX2 level in the tumor core was characteristically noted from the study of differential COX2 expression patterns in various tumor regions (Cortex-Tumor COR ; Cortico-medullary-Tumor COR/MED; Medulla-Tumor MED ). Further assessment of the functional status of COX2 was performed by IHC wherein COX2 was found to be spatially distributed in the brown stained nucleus and cytoplasm (Fig. 5C) of the cervical tissue sections obtained from 3MC treated mice in the 16th and 24th weeks.
These ndings endorsed an increasing trend in cervical COX2 activity (Fig. 5E). A signi cantly escalated COX2 activity (12th week: 3.8 ± 0.0091 U/mg; 16th week: 5.72 ± 0.016 U/mg) in mice cervix continued to rise consistently even in 24th and 30th weeks of 3MC treatment. This cumulative increase in expression and activity of COX2 protein was found to be very strongly correlated (r = 0.9750; p < 0.005) among the 3MC treated groups (Table 3A). COX2 activities among vehicle control mice were at par with the control data. In ammation mediated COX2 activation propagated into subsequent overexpression of NFκB, GM-CSF1 and Ki67 A gain in expression of NFκB, GM-CSF1 and Ki67 (Fig. 6A) as obtained from western blot results delineated the pathway taken by activated COX2 for promoting carcinogenesis. To establish the possibility of COX2 being the culprit in upregulating NFκB (p50/p65), GM-CSF1 and Ki67, correlation coe cient were calculated and represented in a tabulated format (Table 3A). Evidentially, a strong positive correlation between each of these parameters was mathematically obtained which further strengthened the notion.
An earlier experimental observation attained by the team with the same model revealed the presence of an upregulated cervical IL6 and IL8 activities alongside high expressions [37]. These are also the upstream activators of COX2 that fuel a plethora of prosurvival signalling molecules. Consequentially, with further study elevated Granulocyte Macrophage Colony Stimulating Factor1 (GM-CSF1) expressions as evident from the incremental band intensity patterns (Fig. 6A, lower panel), corroborated with the frequent ux of neutrophils in the cervix (Fig. 3C). This justi ed the conviction that IL6, IL8 and GM-CSF1 cytokines along with an upregulated COX2 resulted in escalation of NFκB expressions (Fig. 6A, upper panel). In alliance with COX2 expressions, NFκB (p50/p60) subunits were observed to be very frequently localized within the nucleus at the tissue level (Fig. 6B, upper panel). In the micrographic excerpts of IHC tissue sections sampled from mice subjected to 16 and 24 weeks of 3MC treatment, positive nuclear staining for both p65 and p50 subunits of the protein was attained. High intensity positive nuclear staining for p50 and p65 enumerating to 78.77% (8/10 mice) and 82.03% (8/10 mice) were scored amongst the 16th week mice groups (Fig. 6B, lower panel). Respective protein expression scores stoking to about 80.23% (8/10 mice) and 86.23% (8/10 mice) were further quantitated amongst mice in 24 weeks of 3MC treatment (Fig. 6B, lower panel). Moreover, Ki67 expression patterns in the whole tissue proteins were fairly high (Fig. 6A, upper panel). Such a distinctively active functional status of the protein was absent amongst vehicle control and control mice during this interval.

Chronic 3MC treatment induced COX2 enabled IAP activations
In order to assess the strength of this prosurvival signalling in overriding apoptotic cues the disposition of XIAP and survivin was analysed in the cervical tissues of 3MC treated mice. Western blot results as showcased in  (Table 3A).Absence of these ndings in the vehicle control and control animals were imperative of the involvement of NFκB /COX2/IAP axis in development of cervical neoplasia in 3MC treated mice.

COX2 activation immortalized cervical cells by facilitating Ki67 and PCNA expressions
The spatial distribution of proliferative antigens Ki67 and PCNA was studied by IHC in order to assess the proliferative potentials of the cervical epithelium as a culmination of NFκB /COX2/IAP functions. Positive staining of cellular nucleus in all the differential cervical epithelial layers was noted amongst the tissue sections of mice subjected to 3MC treatment for 16 and 24 weeks or more (Fig. 8A). These patterns were delimited only to the undifferentiated proliferative basal epithelial cell layers of the control and vehicle control groups. Clear manifestations of uniform staining for Ki67 and PCNA of all cells in the cervical epithelium were scored and illustrated comparatively in Fig. 8B.

In ammation induced systemic stress generates free radicals by disrupting antioxidant defence mechanism
To further nd out the systemic impact of chronic cervical 3MC treatment in mice, ROS and RNS levels were quanti ed in the blood leukocytes and peritoneal macrophages. Relative interpretations of these ndings were undertaken with respect to iNOS and the total antioxidant enzyme activities. A signi cant appraisal in ROS levels were noted in 3MC treated mice from 16th week onwards (Fig. 9A). These observations were coherent with that of RNS levels among these same mice groups (Fig. 9B). Nitrite generation was documented to rise from 16th week (3.813 ± 0.00686 µM/10 6 cells/min) which was found to attain peak by 30 weeks of 3MC treatment. Both of these ndings coincided with the activity trends of iNOS as shown in Fig. 9C where the enzyme activity was found to produce citrulline of about 11.89 ± 0.15311% in the 16th week speci cally. These parameters were found to be subletting each other for promoting systemic stress as revealed from their signi cantly strong positive correlations (Table 3B). All these ndings necessitated to look into the activities of the total antioxidant enzyme capacities. Graphical overview clearly depicted gradual rise in free radical scavenging activity up to 12th and 16th week of treatment ( Fig. 9D), which subsequently declined by 24 and 30 weeks. Relatively, in the vehicle control groups the free radical scavengers were found to function properly with exposure to PET like that of control one. The subtly increasing trend of anti-oxidant enzyme activities holistically helped to quench the PET generated free radicals which went unquenched in the carcinogen treated groups.

Cervical in ammation imposed systemic genotoxic stress eventually deregulating key tumor suppressor proteins
To ascertain the genotoxic pro le upon 3MC exposure mediated in ammatory stress, comet assay was performed with the leukocytes isolated from mice blood of all the three groups. Fluorescent microscopic images exhibited the distribution patterns of damaged DNA in terms of DNA migration (comet formation) among different groups (Fig. 10Ai-xv). The results upheld the appearance of distinct DNA-tails among the 3MC treated groups with incremental time period. Among the PET treated mice, presence of such damage was indistinct just like that of the untreated animals. Concomitant rise in the comet tail moment as calculated by KOMET software is represented graphically in Fig. 10B. Results were indicative of the impact of free radical mediated stress upon the DNA (Table 3B).
Blood bearing leukocytes harbouring damaged DNA also drain into cervical region which was under eventual transformation by the hiked COX2 activities. Continual in ammation driving changes mediated by COX2 accompanied subsequent reduction in expression pro les of tumor supressors at the protein levels in the mice cervical tissue extracts. Western blot data as depicted in Fig. 10C

Discussion
In spite of therapeutic advancements, higher expression status of COX2 in cervical cancer surmounts in therapy resistance followed by disease relapse. Traditionally being a HPV mediated cancer the viral oncoproteins E5, E6 and E7 were found to turn on 'amphiregulin', the COX2 activator either in a ligand dependent or independent manner via MAPK or PI3K/Akt pathways [42,43]. In coherence with HPV mediated cervical cancers, the present model also exhibited an escalated COX2 kinetics. Hence, the role of COX2 as an orchestrator of dysregulated in ammation in inducing cervical neoplastic changes upon chronic treatment with 3MC is well highlighted in this study. Concomitant rise in expression and activity of COX2 accelerated expressions of prosurvival molecules like NFκB (p50/p65), GM-CSF1, XIAP and survivin within the mice cervix tissue with prolongation of treatment duration. This clearly annotated for the presence of a 'positive feedback loop' of prosurvival signalling molecules which paved a way for cervical neoplasia (Table 3A). Further experimentation revealed convenient regulation by COX2 in generation of a molecular interactome involving erratically expressed tumor suppressor proteins (p53, p21 and Rb) and upregulated prosurvival proteins which majorly included NFκB, XIAP and survivin in 3MC driven cervical carcinogenesis. This eventually rendered an impact in transformation of the reproductive organ anatomy, cytopathology, histopathology and biochemistry of the carcinogen treated mice. COX2 and p53 are antagonistic in function because the latter is known to positively regulate 'thrombospondin-1' which directly inhibits angiogenesis by shutting down the angiogenic effector molecule, VEGF [28,29]. Hypoxia induced functional facilitation of p53 is a common occurrence within a tumor lump, alongside activation of NFκB and COX2. However, during carcinogenic discourse, growth aiding cues received in terms of hiked iNOS activity was reported to encourage NFκB and COX2 for overriding the p53 action so as to progress with neoplasia [44,45]. In agreement with these previously published reports, the tumor obtained after 24 weeks of carcinogen treatment in this present study also exhibited a high COX2 and NFκB expressions in the tumor core with concomitantly high COX2 activity. IHC results fostering nuclear accumulation of p65 and p50 subunits of NFκB was indicative of its active functional stature. Nonetheless, an eventual decline in p53 correspondingly accompanied by rise in IAPs with time was an alibi to COX2 being the silent umpire of the interplay between these promoters of carcinogenesis.
Another interesting observation of the present study was formation of enlarged spleen (splenomegaly) which was speci cally observed in animals at 16th week of 3MC treatment and onwards. Documentation of the cumulative experimental ndings had unveiled the treatment period of 16th week as the 'crucial-interim' for the development of invasive cancer among these mice. Enlarged spleen showcased an altered histopathology which was characterized by the presence of eosinophil in itrations and enhanced number of 'germinal centres'; the hubs which nourish and train the naïve blood leukocytes to become immunologically competent [46].
Continual carcinogenic treatment in mice for 30weeks caused outbursts of free radicals to mediate systemic stress as evident from escalated iNOS activity, indiscriminately increased ROS and RNS levels which conjugatively compromised scavenging capacity of the antioxidant enzymes. Impact of these multiple oxidative hits was apparent in the form of concurrent damage incurred upon the leukocyte DNA as observed from Comet Assay (Table 3B) Overall ndings have been schematically represented in Fig. 11.

Conclusion
The in vivo model established here aunts a microenvironment which has upregulated prosurvival molecules under the command of COX2. Thus, it can be treated as a model to address issues of therapy resistance that stymies cervical cancer treatment. This referral model throws paramount light upon ways to target COX2 and its several unknown effectors as prognostic and diagnostic biomarkers for planning a better treatment rationale.    Expression patterns (lower panel) of tumor suppressor proteins (p53, p21, Rb and Acetylated p53) as studied by western blot analysis speci cally in the cervix tissue lysates of Group III mice. Experiments are repeated twice