Repurposing Combination Therapy of Voacamine with Vincristine for Down Regulation of HIF-1α/FASN Co-Axis and PHD2 Activation in ER+ Mammary Neoplasia

Background: The current study was attempted to inquest the role of combination therapy of Voacamine and Vincristine for the prevention of mammary gland carcinoma through prolyl hydroxylase ‐ 2 activation. The prolyl hydroxylase ‐ 2 activation leads the downregulation of hypoxia ‐ inducible factor ‐ 1α and fatty acid synthase. Over expression of hypoxia inducible factor-1α and fatty acid synthase is previously reported in solid tumor of mammary gland. Methods: After screening a battery of natural compounds which were similar to vincristine, vocamine was selected as a possible prolyl hydroxylase ‐ 2 activator and justify its activity using 7, 12-Dimethylbenz[a]anthracene induced rat model. The combination therapy was evaluated for cardiac toxicity using hemodynamic prole. The angiogenic markers were evaluated using carmine staining. Monotherapy and combination therapy were also evaluated for liver and kidney toxicity through haematoxylin and eosin staining. The combination therapy also delineated the markers of oxidative stress favorably. Afterwards, the disruption of fatty acids was evaluated using gas chromatography. Results: The immunoblotting analysis validated that combination therapy has a potential to switch on the prolyl hydroxylase ‐ 2 activity and thus initiate proteolytic degradation of hypoxia ‐ inducible factor ‐ 1α and its consequence effects. The combination therapy also stimulated programmed cell death (apoptosis) in rapidly dividing cancer cells. Conclusion: The present study explores the role of voacamine in activation of prolyl hydroxylase ‐ 2 which can decrease over expression of hypoxia ‐ inducible factor ‐ 1α and fatty acid synthase in cells of mammary gland carcinoma. the high dose monotherapy and with high dose combination therapy when compared to the normal control. QT interval was observed to be decreased in DC, combination low and high dose in TC whereas it was observed to be increased with monotherapy (T1 and T2). A raised level of R amplitude was observed in TC group when compared to all the treatment groups. S amplitude was decreased in DC, T1 and T4 while it was found to be increased with VIN high dose treatment group. T amplitude decreased with DMSO treatment but increased with high dose of monotherapy. A small decrease in heart rate was observed with DMSO administered group but sharp decrease in heart rate was observed with DMBA administration in TC group. Heart rate was even more decrease in all the treatment groups (T1, T2, T3, and T4) with the institution of therapy when compared to the NC, DC and TC. JT interval increased with monotherpapy but opposite trend of RR interval observed in all the treatment groups. Q amplitude was increase with VIN high dose but no change observed with other treatments. T peak was greatly increased with high dose of combination therapy but small increase in T peak also observed with monotherapy. metabolites responsible for variation and class separation between normal control vs toxic control and Normal control vs treatment groups (T1, T2, T3 and T4). The metabolic biomarkers were screened based upon the VIP score values > 1.0 (derived from PLS-DA modeling, showing discrimination signicance) and then tested (using univariate and student t-test) for statistical signicance based on p-value < 0.05. The up ( ↑ ) and down ( ↓ ) arrows represent, respectively, increased and decreased metabolite levels within the groups compared to controls. dodecyl gel electrophoresis , PVDF: polyvinylidene diuoride, SREBP-1c: Sterol regulatory element binding protein-1c, TC: Toxic control, NC: Normal control, BC: Bowmans capsule, G: Glomerulus, PCT: Proximal convoluted tubule, DCT: Distal convoluted tubule, PUFA: Poly unsaturated fatty acids, LDL: Low density lipoprotein, VLDL: Very low density lipoproteins, GC-FID: Gas chromatography-Flame ionization detector, LD: Lactiferous duct, AD: Adipocytes, DE: Ductal epithelium, He: Hepatocytes, Sn: Distorted sinusoids, ROS: Reactive oxygen species, GLUT-1: Glucose transporter one, VEGF: Vascular endothelial growth factor BC,G,PCT and DCT; VIN treated rat kidney showing extensive damage to BC,G,PCT and DCT; Ee & F,f-kideny of rats treated with combination therapy of VOA and VIN showing even more damage to BC,G, and to microtubules(PCT&DCT); G,g-kideny of rat treated 3%DMSo showing normal architecture of kidney. dBC- damaged bowmans capsule, dG-damaged glomeruls, PCT-proximal convoluted tubule, DCT-distal convoluted tubule 2C: Effect of VOA and VIN therapy on rat Liver A1-G1&A2-G2-Histologgy of rat treated with VOA nad VIN at 4Xand 40X. A1-A2-liver section of NC rat showing normal architecture of liver lobules(Lo), hepatocutes(He), sinusoids(Sn) and central vein(CV); B1-B2-liver section of TC rats showing dilated central vein, damaged He, distorted Sn and Lo, C1-C2-liver section of VOA treated rat showing little dilation of CV, intact lobular structure, no distortion of Sn and with normal He, D1-D2-liver section of VIN treated rats showing dilated CV, distended portal triads(PT), enhancement in sinusoidal space; E1-E2 and F1-F2-liver section of rat treated with low and high dose combination therapy of VAO and VIN showing extensive distortion of Sn, damage to He, enlargement of central and portal triads and disruption of lobular structure; G1-G2-liver section of rat treated with 3%DMSo showing normal liver architecture like normal.


Background
The most commonly observed malignancy in females is the mammary gland carcinoma. It is reported that mammary gland cancer makes the one fourth of all the cancers diagnosed every year worldwide [1].
When growth and proliferation of mammary gland tissue is not under control, it divides and forms a lump of cells which transforms into malignant cancer if remain undiagnosed [2]. Despite of much progress in the eld of cancer radiation and chemotherapy, treatment and management of mammary gland carcinoma is still very di cult [3]. Moreover, failure of radiation and chemotherapy due to hypoxia in case of solid tumor of mammary gland is another encountered problem [4], Plethora of studies have revealed that hypoxia inducible factor-1α (HIF-1α) is the main component which takes various decisions to attain more hostile environment for proper growth and development of tumor cells even in the scarcity of oxygen [5]. It enhances the expressions of various genes involved in the metabolism of glucose and fatty acid synthesis pathway [6]. In order to synthesize their plasma membrane, rapidly dividing cancer cells require fatty acids, especially polyunsaturated fatty acids (PUFAs) in much larger quantities compared to the normal dividing cells [7]. Therefore, cancer cells adopt an alternative pathway to meet their fatty acid needs. Hence, the mammary gland cancer tissues utilize more glucose and glutamate for their production. So, from above discussion, it was clear that fatty acid plays a very important role in growth and development of mammary gland cancer cells as there is over expression of fatty acid synthase (FASN).
Prolyl hydroxylase-2 (PHD-2) is an important governor of HIF-1α and execute hydroxylation and nally degradation of HIF-1α in presence of oxygen and keep it to minimum level in normal cells [8]. But in case of hypoxic solid tumor, reduced level of oxygen deactivates the PHD-2 and activated HIF-1α. Earlier, our research group conjectured that chemical activation of PHD-2 could curtail the upsurged level of HIF-1α and FASN in mammary gland carcinoma [9][10][11].
In the present study, we are trying to demonstrate the role of natural compound Voacamine (VOA) in mammary gland chemoprevention through activation of PHD-2 and subsequently down regulation of HIF-1α and FASN.

Materials And Methods
In silico study A library of natural compounds was created using Zinc database. The compounds were structural similar with Vincristine (VIN). All the compounds were docked (Autodock 4.2) [12] with PHD-2 protein (PDB ID: 2G19) and about thirteen compounds were found to have good binding energy with PHD-2 protein. After analysis of all the compounds, VOA (Zinc ID169368472) was selected for in vivo study (Fig. 1A).
The study was continued up to 3 months. At the end of study, blood was collected from retro orbital plexus in order to study the metabolomics pro le of control and treatment groups. After blood withdrawal, animals were sacri ced under mild ether anesthesia by cervical dislocation. Abdominal cavity was opened though the median incision. Mammary gland was carefully separated from the skin with the help of sharp scissor and forceps and mounted whole on the slide in order to carry out carmine staining [13]. Liver and kidney was also separated and preserved in the − 20ºC to be used to assess the toxicity of drug molecules.

Hemodynamic analysis
For the assessment of cardiac toxicity due to DMBA, hemodynamic pro le was performed using AD Instrument. The animals were mounted on the wax trays after anesthetizing by injecting ketamine hydrochloride (50 mg/kg, i.m.) and diazepam (2.5 mg/kg, i.m.). In order to record the ECG signals, dorsal and ventral thorax skin was cleaned and sterilized with spirit and then platinum hook electrodes were xed on it. These electrodes had connections with the Bio-ampli e (ML-136) and channel power lab (ML-136). Both these instruments work together to convert analogue signals into digital signals (AD Instruments, Australia) which were stored in the hard disk of the system. O ine analysis of saved ECG signals was performed with Lab Chart Pro-8 (AD-Instruments, Australia) [14].
Further, ECG signals were also employed for HRV analysis. Firstly, a manual inspection of recorded signals was carried out in order to accurate detection of R-waves. Afterward, R waves per unit time were plotted in order to calculate the HR.. Then, HR was calculated by plotting the number of R-waves per unit time. Finally, Lab Chart Pro-8 (AD Instruments) employed to calculate the time and frequency domain parameters of HRV [15,16].

Carmine staining
For mammary gland whole mount analyses, mammary glands tissues were removed from rats and stretched over the glass slides. After drying, slides were placed in the Carnoy's xative solution [ethanol (60%), chloroform (30%) and glacial acetic acid (10%)] for 2 h and then washed with decreasing concentrations of ethanol (90%, 70%, 35%, and 15%) for 1 h. The slides were placed in the alum carmine stain (1 gm carmine dye and 2.5 gm aluminum potassium sulfate in 500 ml distill water) for 2 days. After 2 days, the Carmine stained slide were taken out and dehydrated with increasing concentration of ethanol (70%, 95% and 100%) and the lipids were removed by immersion in xylene for overnight period of time.
After that, slides were examined under the biological miscroscope at 4X in order to check the presence/absence of alveolar buds (ABs), terminal end duct (TED), terminal end bud (TEB) and lobules (LOs) [17,18].

Histopathological analysis
To examine the morphology of mammary gland, liver and kidney; Haemoxyline (H) and Eosin (E) staining was performed. Firstly, tissues were placed in 10% formaldehyde xing solution and then buried inside the wax cubes. Using the microtome, 5 µm sections were prepared and stained with H&E. Finally prepared tissue sections were examined under the digital biological microscope (BR-Biochem Life Sciences, N120, New Delhi, India) in order to visualization and imaging of stained tissue. Photographs were taken at 4X and 40X [19].

Antioxidant markers
Frozen mammary gland tissue samples were thawed, precisely weighed and homogenized in 0.15M KCl. The mixture was centrifuged at 10000 rpm for 15 min. Supernatants were collected and kept in ice bath until analysis. The enzymatic assays of catalase, thiobarbituric acid reactive substances (TBARs), superoxide dismutase (SOD), protein carbonyl (PC) and glutathione (GSH) were determined performed according to the method described us earlier [20,21]. The reactivity of the enzymes with tissue samples was determined specrophotometrically (UV-Visible spectrophotometer Agilent Technologies, Carry 60).
Fatty acid methyl ester (FAME) analysis Frozen mammary gland tissue was accurately weighed and dissolved in mixture of chloroform: methanol (2:1) in order to make 0.5% tissue homogenate. The homogenized tissue was further sonicated at 4˚C up to 5 min and then ltered with Whatman lter paper. Methanol added at last to make up the nal volume. In the ltrate, 0.2 ml double distilled water was added to remove the non fatty components. The resultant mixture was left undisturbed for half an hour. After half, an hour, solution was centrifuged at 5000 rpm for 5 min. After centrifugation, lower lipid containing layer was conserved and the upper non fatty layer was discarded. In the subsequent step, methyl esters of the lipid sample were prepared by stirring the 0.75 gm of the sample with 2 ml hexane and 0.2 ml methanolic KOH(2N). The resultant mixture was vortexed for 15 min in order to separate into two layers. The FAME was present in the upper layer. It was carefully separated and used to analyze the lipid pro le in control, toxic and treatment groups [22,23].
1 H NMR study 1 H NMR was performed in blood serum samples. To remove precipitates, serum samples were thawed and then centrifuged. For data acquisition, 220 µl of supernatant was serum taken in the NMR tubes (Willmad Glass, USA). To make up the nal volume 440 µl in the NMR tubes, 220 µl NMR buffer solution (sodium phosphate saline of strength 20 mm of pH7.4 prepared in D2O) was added. After this, a 2 mm sealed tube called co-axial containing a 0.5 mM solution of 3-trimethylsilysily-(2,2,3,3-d4)-propionic acid (TSP) was inverted in 5 mm NMR tubes and 150 µl of the solution poured into it. It worked as internal reference standard. The prepared samples were analyzed with NMR spectrometer (Bruker NMR spectrometer), 800 MHz) and raw spectra was obtained as NMR peaks. Also, CPMG (Carr-Purcell-Meiboom-Gill) NMR spectra were recorded for each serum sample by adopting the Brukers standard pulse programme library sequence (cpmgpr1d) with saturation of the water peaks. The spectrum was further processed in Bruker software Topsin-v2.1 (Bruker BioSpin Gmb H, Silberstreinfen 476287 Rheinstetten Germany) and AMIX software in order to carry out spectral binning of the CPMG data. The binned data was then further submitted to MetaboAnalyst, in order to carry out multivariate analysis of metabolomic spectral data. Firstly, Principle Component Analysis (PCA) was performed in order to take initial overview of the metabolites in control, toxic and treatment groupd. Next, data again analyzed with Partial Least Squares Discriminant Analysis (PLS-DA) method to bring out the metabolites responsible for class separation and the class separation among the grouped animals. The data was Pareto scaled for both PCA and PLS-DA and strictly validated for statistical signi cance. The cross validation of the models was described by the R 2 and Q 2 parameters and the p-values ≤ 0.05 calculated with Mann Whitney test for pair wise comparisions) were assumed to be statistical signi cance [24,25].

Immunoblotting
For protein sample preparations 500 mg mammary gland tissue was weighted and completely homogenized in Radioimmunoprecipitation assay buffer (RIPA lysis buffer) and phenylmethylsulfonyl uoride (PMSF). The amount of protein in each sample was quanti ed with Bradford assay. After quanti cation, proteins were separated on 12.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel using the principle of Laemmli. In the subsequent step proteins separated on gel transferred on to the polyvinylidene di uoride (PVDF) membrane (IPVH 00010 Millipore, Bedford, MA USA). Proteins transferred on PVD membrane was blocked for 3 h with mixture of 5% BSA and 5% not fat skimmed milk prepared in TBST and followed by incubation at 4ºC with primary antibodies,[ SREBP-1c (SC-13551), HIF-1α (SC-13515), FASN (SC-55580), [(PHD2 (SC-67030)] for overnight period. β-actin was used as a standard reference. After overnight incubation with primary antibody, PVDF membrane was wiped three times with TBST and then incubated with HRP conjugated secondary antibodies [antimouse (SC-31430, Pierce Thermo Scienti c, USA), anti-rabbit (SC-2030), anti-goat (SC-2020)] at room temperature for 3 h [9,26]. At the end, membrane was washed one time with TBST and proteins blots were developed and analyzed in ChemiDoc XRS+ (Bio Rad).

Statistical analysis
The results were analyzed with Graph Pad Prism software (5.02). The values were presented as mean ± SD and the statistical signi cance was calculated by one way ANOVA followed by Bonferroni test. The values less than *p < 0.05, **p < 0.01, ***p < 0.001 were considered as statistically signi cant.

Result
Effect of drugs and toxicants upon hemodynamic pro le Hemodynamic pro le was measured by ECG ( Fig. 1B) and HRV ( Table 1). The results were analyzed online by MetaboAnalyst selecting PCA from chemometric analyses. 2D score plot of PCA of NC and treatment groups depicted a very good separation among the groups (Fig. 1C). DMSO treated group was moving away from NC groups which indicates DMSO administration somehow affecting the cardiac activity. DMBA administration also displaced the TC control group. Treatment with monotherpay with VOA (T1) and VIN (T2) further displaced away from the NC. But treatment with combination therapy displaced T3 and T4 even far away from the NC that clearly indicates cardiac toxicity of the drug. To further con rm the cardio toxicity of the drugs, data was again analyzed by Box-Cum Whisker plot (Fig. 1C). A signi cant difference in R and P amplitude was observed in TC group when compared to the NC and treatment groups. P amplitude increased in all the groups when compared to the NC group with more pronounced effect in VIN treated group. Duration of P wave signi cantly increased with VOA (T1) but remain same in all the other groups. PR interval increased with high dose monotherapy of VOA, VIN and with high dose combination therapy when compared to the normal control. QT interval was observed to be decreased in DC, combination low and high dose in TC whereas it was observed to be increased with monotherapy (T1 and T2). A raised level of R amplitude was observed in TC group when compared to all the treatment groups. S amplitude was decreased in DC, T1 and T4 while it was found to be increased with VIN high dose treatment group. T amplitude decreased with DMSO treatment but increased with high dose of monotherapy. A small decrease in heart rate was observed with DMSO administered group but sharp decrease in heart rate was observed with DMBA administration in TC group. Heart rate was even more decrease in all the treatment groups (T1, T2, T3, and T4) with the institution of therapy when compared to the NC, DC and TC. JT interval increased with monotherpapy but opposite trend of RR interval observed in all the treatment groups. Q amplitude was increase with VIN high dose but no change observed with other treatments. T peak was greatly increased with high dose of combination therapy but small increase in T peak also observed with monotherapy. Time domain  Histopathology H&E staining was performed to view the microscopic architecture of mammary gland tissue of control and toxic treated rats. On DMBA administration, signi cance damage to the lactiferous duct (LD), adipocytes (AD) along with distorted myoepithelium (ME) and ductal epithelium (DE) was observed in TC group when compared to the NC ( Fig. 2A-A2-G2). On initiation of therapy with VOA and VIN (as monotherapy and combination therapy) granted a signi cant protection to the mammary gland tissue as evidenced by the regeneration of lactiferous duct (LD), brous stromal tissue (FST) and adipocytes. A better improvement was observed with the combination therapy comparable to normal. Not any kind of cellular damage was observed in the DMSO (3%) treated animals. This further gives a clue that DMSO at this concentration has no side effects if used as a solvent.

H&E staining of liver and kidney tissue
Results of H&E staining of rats treated with DMBA caused signi cant damage to the Bowmans capsule (BC) and glomerules (G) (Fig. 2B). The space between BC and G was observed to be increased and structure of proximal convoluted tubules (PCT) and distal convoluted tubule (DCT) observed to be largely distorted in TC when compared to the NC (Fig. 2B-B1

NMR
The collected serum samples were analyzed in 1H NMR to predict change in serum metabolomics and the results are presented in the Fig. 3 (A, B and C . Monotherapy also worked well in lactate reduction but no signi cant change in lactate was observed. The level of Glutamate was reduced in T1, T2 and T3 but increased in T4. Glutamine was remain unchanged in T1 but reduced in T2, T3and T4. As far as PUFAs are considered, a linearity pattern of reduction was observed with monotherapy and combination therapy. Gas chromatography FAME analysis of mammary gland tissue of control and treatment groups were analyzed by GC-FID as mentioned in material and method section. A signi cant change in fatty acid composition of TC animals were observed when compared with NC. Higher levels of PUFAs were observed in TC after DMBA administration. On treatment with VOA and VIN monotherapy as well as with combination therapy, a signi cant reduction in synthesis of PUFAs was noted (Fig. 4).

Western Blotting
Both After DMBA administration, a signi cant change in expression of anti apoptotic (Bcl-XL) and proapoptotic (BAD, BAX) proteins were noted in toxic control animals. Also, the increased expression of VDAC, Apaf-1, and Caspase 9 were observed in toxic control. But VOA and VIN therapy imparted signi cant protection, worked well to re-store the changes (Fig. 5B).

Discussion
PUFA's are crucial for plasma membrane synthesis in rapidly dividing cancer cells of mammary gland tissue and dietary sources alone are insu cient to accomplish this [27]. Therefore, cancer cells must take over an alternative fatty acid synthesis pathway and HIF-1α helps them to adapt this [28]. Previous studies have reported that HIF-1α acts in a very smart way and modify the tumor microenvironment in such a way that indirectly enhances the fatty acid synthesis, required for the synthesis of plasma membrane and to furnish other purposes in cancer cells [29]. Considering, the role of HIF-1α and FASN, the present study was undertaken to downregulate HIF-1α by activating PHD-2 with natural drugs VOA alone and in combination with VIN.
Cardiac toxicity is very common risk factor in mammary gland carcinoma patient. For the analysis of cardiac toxicity, hemodynamic pro le of animals was performed (Fig. 1B, Table 1). Normal ECG and HRV was recorded in case of NC groups when administered vehicle for 3 months. DMSO is a universal solvent for hydrophobic drugs and its cardio toxic affect is well established [30]. DMSO administration decreased the heart rate, S, ST and T interval and increased the RR and P amplitude in DMSO control group.
Decrease in HRV after VIN treatment is already reported in previous studies [31] and the same was also re ected in the present study. A large perturbation in ECG and HRV parameters were observed with both low and high dose of combination therapy. Hence, the concomitant administration of VOA with VIN/DMSO would have further exacerbated the cardiotoxic effect (Fig. 1C).
Hypoxia in solid tumors plays important role in angiogenesis which is necessary to accomplish increasing demand of oxygen and other nutrients. Various studies have already reported that increased level of HIF-1α stimulates angiogenesis in tumor cells [32,33]. In the present study, results of the carmine staining manifest an increase in the AB count and lobules in the toxic control group which clearly depicts the formation of neovascularization ( Fig. 2A-A1-G1). But treatment with monotherapy and combination therapy of VOA and VIN reduced the AB count and no of lobules in the experimental animals which indicates suppression of angiogenesis after initiation of therapy. Therapy with VOA and VIN might have regulated HIF-1α pathway to abolish angiogenesis.
Histopatology of mammary gland tissue of carcinogen treated rats have shown extensive damage to the lactiferous duct (LD), adipocytes (AD), ductal epithelium (DE) and myoepithelium as reported in the previous studies [34]. Extensive damage to the micro architecture of mammary tissue like LD, AD, DE and myoepithelium was observed after DMBA administration in TC control ( Fig. 2A-A2-G2). A revert effect upon the same was exhibited with VOA and VIN treatment. This indicates VOA and VIN would be working at cellular level to stop tumor progression. Histopathological examination of mammary gland tissue further witnesses the protective action of therapy.
Liver and kidney is the vital and sensitive organ in the body which are responsible for the detoxi cation and excretion of drugs. Continuous detoxi cation and excretion of harmful drug molecules specially those which belongs to the anticancer class, can cause harm to these organs and ultimately becomes the reason of nephrotoxicity and hepatotoxicity if dose is not monitored adequately. Most of the anticancer drugs are reported to have varying degree of nephrotoxicity and hepatotoxicity in patients taking them [35]. Nephrotoxicities due to chemotherapy is marked by the necrosis of the proximal convoluted tubule (PCT), distal convoluted tubule (DCT) epithelial cells and injury to the Bowman's capsule, ultimately kidney failure [36]. Histopathological examination of mammary glands of treated rats revealed the same type of damage to the microarchitecture of kidney after DMBA administration (Fig. 2B). Subsequent treatment with VIN monotherapy further exacerbated the renal toxicity as evidenced by the greater damage to the PCT, DCT and Bowmans capsule as its nephorotoxicity is already reported [37]. It would be pertinent to mention that no renal toxicity was noted in rats treated with monotherapy of VOA which con rms its safety in renal failure. Even more damage to kidney was exhibited by both low and high dose of combination therapy marked by the necrosis of the renal tubular epithelial cell, loop of Henle, and glomerulus attributed to the high drug accumulation, either of the two. Since, VOA is already known to have Pgp (e ux pump) inhibitory effect [38], it might have helped intracellular pooling of VIN in the nephron of experimental animals, can be the one possible cause of nephrotoxicity (Fig. 2B).
Liver is the site where most of the drugs undergoes their rst pass metabolism (except from those administered through parenteral route [39]. Normal functioning of the liver affected due to continuous exposure to high concentration of cytotoxic substances which then leads to liver failure in some patients. Liver toxicity is marked by dilation of central vein, damages to hepatocytes (He), distorted sinusoids (Sn), and lobules (Lo) which is very well evident in DMBA treated animals (Fig. 2C). Monotherapy of VOA and VIN worked well to stop further damage to liver of experimental animals which documents the liver safety of both drugs at the given dose. But histology of combination therapy treated rat's depicted large damage to the liver organ which again proves the VIN /VOA accumulation, or either of two into the hepatocytes and consequently resultant hepatotoxicity.
Various studies have reported the role of ROS in development of cancer manifested by the increase in TBARs, PC and reduction in the activity of SOD, GSH and Catalase [40,41]. Same type of manifestations in the antioxidant markers were also noted in DMBA treated rats. Interestingly, both, monotherapy as well as combination therapy effectively restored the TBARs, SOD and other associated antioxidant markers. From this we can expect, restoration of antioxidant activity could be the one possible mechanism behind the anticancer potential of the drug ( Table 2).
Numerous studies have reported that myriad changes occur in a biological system under diseases condition which can be detected in the biological uids like blood serum. With this goal serum metabolic study was carried out to extract biomarkers and to understand the interplay between molecular and cellular components (Fig. 3A,B,C) [42,43]. Previous studies have demonstrated that a hypoxic tumor cells utilize more and more glucose to meet its energy and for biomass accumulation [44,45]. Several studies have reported that tumor cells produce high amount of lactate and glutamate that impart bene t to the tumor cells in various ways like fatty acid biosynthesis, immune protection, angiogenesis and invasiveness [46]. The metabolic pro le of DMBA treated rats have shown high level of lactate and glutamate and that of PUFAs which is in accordance to the previous studies i.e excess lactate/glutamate is converted into fatty acids. Decreased level of glucose further a rms the above ndings. Interestingly, reveres chronological order of above metabolites were observed with VOA and VIN (monotherapy as well as with combination therapy) i.e. decreased level of lacate, glutamate and PUFAs were noted all in the treatment groups. It would be pertinent to mention that monotherapy with VOA and high dose combination therapy of VOA(1 mg/kg) and VIN(1 mg/kg) provided much better fatty acid synthesis inhibitory action compared to the VIN monotherapy and combination low dose. Serum metabolomics analysis of present clearly established a relationship between glycolysis, lactate, glutamate and fatty acid synthesis production (Table 3).
The list of metabolites responsible for variation and class separation between normal control vs toxic control and Normal control vs treatment groups (T1, T2, T3 and T4). The metabolic biomarkers were screened based upon the VIP score values > 1.0 (derived from PLS-DA modeling, showing discrimination signi cance) and then tested (using univariate and student t-test) for statistical signi cance based on p-value < 0.05. The up (↑) and down (↓) arrows represent, respectively, increased and decreased metabolite levels within the groups compared to controls.
The list of metabolites responsible for variation and class separation between normal control vs toxic control and Normal control vs treatment groups (T1, T2, T3 and T4). The metabolic biomarkers were screened based upon the VIP score values > 1.0 (derived from PLS-DA modeling, showing discrimination signi cance) and then tested (using univariate and student t-test) for statistical signi cance based on p-value < 0.05. The up (↑) and down (↓) arrows represent, respectively, increased and decreased metabolite levels within the groups compared to controls.
The list of metabolites responsible for variation and class separation between normal control vs toxic control and Normal control vs treatment groups (T1, T2, T3 and T4). The metabolic biomarkers were screened based upon the VIP score values > 1.0 (derived from PLS-DA modeling, showing discrimination signi cance) and then tested (using univariate and student t-test) for statistical signi cance based on p-value < 0.05. The up (↑) and down (↓) arrows represent, respectively, increased and decreased metabolite levels within the groups compared to controls.
We further performed the FAME analysis of mammary gland tissue to a rm the raised level of fatty acids after DMBA, VOA and VIN treatment. Similar story was also observed in the FAME analysis of mammary gland tissue i.e high level PUFAs were detected toxic control and very low level of PUFAS were noted in the treatment groups (Fig. 4). Results of FAME analysis further evidences that excess lactate was utilized in fatty acid synthesis. No PUFAs like toxic control were observed in FAME analysis of treated animals which clearly indicates VOA and VIN have an inhibitory action on fatty acid synthesis.
Immunoblotting were further performed to investigate the effect of therapy on the proteins of hypoxic and fatty acid synthetic pathway. Several studies have con rmed that the expression of HIF-1α increase in oxygen scarcity which enhances the expression of other genes that indirectly bene ts the tumor cells in various ways [47,48]. A study conducted by P.Maxwell et al on the wild type (wt) Hepa-1 cells and derivatives c4,c31 and Rc4 proved that HIF-1α plays crucial role in GLUT one transporter and VEGF [48]. Another study conducted by Wendi Sun et al on HeLa, HCT116 and on cultured human primary epithelial cells showed that HIF-1α increased the level of SREBP-1c and FASN [49]. Intriguingly, similar type of trend was also observed in the present study i.e. the level of HIF-1α, SREBP and FASN expression was up regulated while PHD2 expression was down regulated after DMBA treatment (Fig. 5A). An, opposite effect on the same was observed after the initiation of VOA/VIN therapy. It is already reported that PHD2 is negative regulator of HIF-1α and activation of PHD2 alone can downregulate all its downstream effects [50]. This a rms that VOA and VIN might have activated PHD2 and subsequently the level of HIF-1α along with FASN proteins would have dwindled as it was hypothesized.
Several studies have reported that failure of apoptosis in normal cells is an indication of cancer transformation [51][52][53]. Decrease in anti-apoptotic proteins (Bcl-XL and Bcl-2) and increase in proapoptotic proteins (BAD,BAX) indicates normal functioning mitochondrial apoptotic pathway [7]. Result of immunoblotting shows increase in expression of Bcl-XL and decrease in BAX and BAD proteins in toxic control after DMBA administration. Furthermore, expression of VDAC, Apaf-1 and caspase9 were also found elevated which proves the failure of mitochondrial apoptotic pathway. Treatment with monotherapy as well as with combination therapy of VOA and VIN restored the apoptotic pathway in treatment groups which is evidenced by the increase level of cytochrome-c as well (Fig. 5B).

Conclusion
Results of western blotting shows that HIF-1α, SREBP-1c and FASN were signi cantly raised in TC group that was observed to be decreased in subsequent treatments groups (treated with VOA and VIN). Western blotting has evidenced the increased expression of SREBP-1c which then enhanced the FASN expression and thus of more and more synthesis of PUFAs resulted to construct the plasma membrane and to meet energy requirements. Treatment with VOA and VIN decreased in level of lactate, glutamate.
From this it can be concluded that HIF-1α increased the intracellular lactate acidosis to activate SREBP-1c in order to enhance fatty acid synthesis that was reverted back when HIF-1α went degradation upon PHD2 to activation with VOA and VIN therapy. All in all, VOA and VIN activated PHD2 which down regulated HIF-1α, reduced lactate acidosis, inactivated SREBP-1c and FASN to check fatty acid synthesis needed for plasma membrane synthesis by the rapidly dividing breast cancer cells (Fig. 6).    SD. Comparisons were made on the basis of one way ANOVA followed by Bonferroni multiple test. All groups were compared to the DMBA treated group (*p<0.05, **p<0.01, ***p<0.001). 5B: Effect of VOA and VIN on mitochondrial apoptotic markers. Values are presented as mean + SD. Comparisons were made on the basis of one way ANOVA followed by Bonferroni multiple test. All groups were compared to the DMBA treated group (*p<0.05, **p<0.01, ***p<0.001).

Figure 6
Mechanism of VOA and VIN to inhibit fatty acid synthesis in DMBA induced mammary gland carcinoma of albino wistar rats. Hypoxia activated HIF-1α enhance lactate acidosis in tumor microenvironment.