Inhibitory Effect of Multimodal Nanoassemblies against Glycative and Oxidative Stress in Cancer and Glycation Animal Models

In recent years, there has been a progress in the study of glycation reaction which is one the possible reason for multiple metabolic disorders. Glycation is a nonenzymatic reaction between nucleic acids, lipids, and proteins resulting into the formation of early glycation products that may further lead to the accumulation of advanced glycation end products (AGEs). The precipitation of AGEs in various cells, tissues, and organs is one of the factors for the initiation and progression of various metabolic derangements including the cancer. The AGE interaction with its receptor “RAGE” activates the inflammatory pathway; yet, the downregulation of RAGE and its role in these pathways are not clear. We explore the effect of anticancer novel nanoassemblies on AGEs to determine its role in the regulation of the expression of RAGE, NFƙB, TNF-α, and IFN-γ. This paper is based on the in vivo and in vitro study in glycation and lung cancer model systems. Upon the treatment of nanoassemblies in both the model systems, we observed a protective effect of nanoassemblies over the inhibition of glycative and oxidative stress via mRNA expression analysis. The mRNA expression results corroborated with the reactive oxygen species (ROS), carboxy-methyl-lysine (CML), and fluorescence studies. In this study, we found that the presence of common factors for glycation and lung cancer is oxidative and glycative stress. This oxidation and glycation might be responsible for the initiation of inflammation which may further lead to uncontrolled growth of cells leading to cancer. This can be a strong association between lung cancer and glycation reaction. The intervention of the anticancer and antiglycation effects of multimodal nanoassemblies throughout the study promises a new pathway for cancer research.


Introduction
Glycation is the nonenzymatic reaction between free amino groups of proteins and nucleic acids and free carbonyl groups of reducing sugars which damage or alter the protein struc-anticancer mononucleotide (trifluorothymidine, TFT). The size and shape of the nanoassemblies were examined by transmission electron microscopy. Human serum albumin (HSA) was used as a drug delivery carrier to construct a drug, and an approachable model is necessary for targeting drug delivery in cancer therapy. Also, HSA is an attractive model used to design multifunctional nanoassemblies for the theranostic approach. It acts as a drug carrier for a long time either by direct genetic fusion or conjugation or by a noncovalent association of a drug with the protein. The synthesized nanoassemblies, viz., PFT-HSA-TFT-Cy7 and PFT-Hcy-HSA-Cy7, were gifted by our collaborators, Professor Tatyana S. Godovikova and Professor Vladimir Silnikov, from ICBFM, RAS, and Novosibirsk, Russia.
There is a gradual increase in lung cancer subjects in the past decade which prompt us to perform this study [17]. We choose to perform the antiglycation study by comparing it with the anticancer activity. This has been monitored in lung cancer and glycation animal model. In our prior experiments, we developed two in vivo models based on the glycation and lung cancer model and one in vitro model based on lung cancer A-549 cell line [18]. Previously, the anticancer study was done on lung cancer cell line A-549 and breast cancer cell line MCF-7 in the presence of PFT-Hcy-HSA-Cy7 [19]. The antiglycation effects were examined by measuring fluorescent and nonfluorescent AGEs, while the anticancer effects were examined by observing inhibition in tumor growth, H&E staining, cell proliferation, and intracellular reactive oxygen species (ROS). Besides, the receptor of advanced glycation end products (RAGE) and AGEs activates the inflammatory reactions involved in the generation and progression of cancer. The AGE-RAGE axis leads to the activation of transcription factors, cytokines, and other interleukins that modify the signaling pathways by inducing the oxidation and inflammation [20]. The AGE-RAGE interaction mediates cell stress and triggers the inflammatory pathways by activating the signal transduction pathway (Scheme 1). Hence, we aim to target the signaling pathways which are involved in the initiation of cancer such as NFƙB, TNF-α, and IFN-γ. We analyzed the gene expression of NFƙB, TNF-α, and IFN-γ in the presence or absence of PFT-HSA-TFT-Cy7 and PFT-Hcy-HSA-Cy7.

Ethics Statement.
The animals were treated with the utmost care, and treatments were done with minimum pain. The rats and mice (6 per cage) were housed and acclimatized to the animal house environment for 10 days at room temperature (25°C) with 12 hour light and dark cycles. The in vivo work was approved by the Institutional Animal Ethical Committee (IAEC) of Integral University, Lucknow, India (1213/GO/ac/2008/CPCSEA).  [18]. All the animal groups were kept on 10% glucose for the next 24 hours to prevent hypoglycemia. The treatment group was given with 5 μM concentration of both the nanoassemblies that is PFT-HSA-TFT-Cy7 and PFT-Hcy-HSA-Cy7. Fasting blood glucose (FBG) was checked after 24 hours of each dosing of alloxan. The procedure for the development of the glycation animal model as well as its treatment procedure with or without the presence of nanoassemblies has been provided in Table 1. All the rats were anesthetized using diethyl ether and sacrificed. Blood was collected by heart puncture. A sample of the kidney tissue was preserved in RNA later.

Experimental
Procedure for the Treatment of Nanoassemblies in the Lung Cancer Animal Model. The anticancer activity of the nanoassemblies in the lung cancer ani-mal model has been done by the oral dosing of both nanoassemblies at 1 μM and 3 μM. We developed the lung cancer animal model in Swiss albino mice with NDEA and PB [18]. The experimental procedure of the development of lung cancer, as well as the inhibitory effect of nanoassemblies, has been briefly explained in Table 2. After 50 days, all the mice were anesthetized using diethyl ether and sacrificed. Blood was collected through heart puncture. The lung tissue was isolated and preserved in 10% formalin and RNA later separately. The fragments from the lung tumur tissues were embedded in paraffin blocks and sectioned at 3-5 μm for histological analyses, and then each histological slide from each block was stained with hematoxylin (H) and eosin (E). Each experimental group was examined carefully using a light microscope with magnification at 40x.

Experimental
Procedure for the Treatment of Nanoassemblies in the vitro Model System. The A-549 lung cancer cell line (5000 cells/well) in 100 μl of complete medium was seeded in 96-well plates. After 4 hours of Table 1: Stepwise method for the inhibitory effect of nanoassemblies in the glycation animal model.
Step Experiment (dosing) (50 days) Group 1 (n = 6) Group 2 (n = 6) Group 3 (n = 6) Group 4 (n = 6) Step  incubation allowing the cells to attach to the bottom of the well, the medium was renewed for a further 24 hours of incubation along with the addition of 25 mM of BSA-MG-AGEs (100 μg) or unmodified BSA (100 μg). The BSA-MG-AGEs were prepared by our previous method (18), and this is a glycation reaction of a protein bovine serum albumin (BSA) and methylglyoxal (MG). The wells were treated with different concentrations of PFT-HSA-TFT-Cy7 and PFT-Hcy-HSA-Cy7 at 1 μM, 3 μM, and 5 μM, respectively. All the experiments were performed in duplicate. The cells were then lysed with 100 μl/well of TRIzol Reagent. The cell lysates were collected in microcentrifuge tubes and stored at -20°C. For cell proliferation effects, 30,000 cells were seeded in the six-well plate containing 2 ml of media. After 4 hours of incubation allowing the cells to attach to the bottom of the well, the media was renewed for a further 24 hours of incubation along with the addition of 25 mM of BSA-MG-AGEs (100 μg) or unmodified BSA (100 μg) in the presence of PFT-HSA-TFT-Cy7 and PFT-Hcy-HSA-Cy7 at 1 μM, 3 μM, and 5 μM, respectively. Cell counts were taken by using hemocytometer as previously suggested [21].
2.6. Estimation of Oxidative Stress with or without the Presence of PFT-HSA-TFT-Cy7 and PFT-Hcy-HSA-Cy7. The oxidative stress in the glycation, as well as the lung cancer animal model in the presence of nanoassemblies, has been detected through estimation of the carbonyl content and malondialdehyde (MDA) content. The carbonyl content was determined in the serum samples of the glycation animal model as well as the lung cancer animal model along with their treatment group, respectively, by using 2,4-dinitrophenylhydrazine (2,4 DNPH). The absorbance was read at 360 nm, and the carbonyl content was determined by using the extinction coefficient of 22,000 M −1 cm −1 [22]. Similarly, the thiobarbituric acid reactive substance (TBARS) assay is to perform for the detection of the MDA content, which is formed during the intermediate stage of the glycation reaction also. 100 μl serum sample was diluted with 500 μl of phosphate buffer saline (PBS). One ml TBA-TCA-HCl reagent was added to each diluted sera sample at room temperature. The pink supernatant was observed after boiling the reaction for 15 minutes at 100°C. The pink supernatant was read at 535 nm. The concentration was plotted by the obtained absorbance against the standard graph. The optical density of the pink color formed is directly proportional to the concentration of serum MDA in the given sample. The AGE-induced formation of intracellular ROS of the in vitro model was measured by using dichlorodihydrofluorescein diacetate (H2DCFDA) as described previously with slight modification [23]. A549 cells were seeded in 96-well plates (3000 cell/well) and incubated at 37°C with 5% CO2 for about 24 h. The cells were treated with 100 μg/ml of 25 mM bovine serum albumin AGE (BSA-AGEs) samples for 24 h in the presence of both the nanoassemblies at 1 μM, 3 μM, and 5 μM, respectively. After incubation, DCFDA was added (10 μM), and the plate was incubated for 30 min in dark. DCFDA was removed, and wells were washed once with PBS and finally, cells were suspended in 100 μl of PBS. The plate was read at 485/528 (excitation/emission) at 100% sen-sitivity, and readings were presented as relative fluorescence unit for ROS generation. The cells without treatment were considered as control. For CML detection, we used the commercially available ELISA kit for all the three model systems with or without the presence of nanoassemblies. The treated and nontreated serum samples and cell lysates were added in the wells after dilution with PBS in the ratio of 1 : 100. After incubation, the kit protocol has been followed until the end of the reaction. The reaction was stopped by the addition of an acidic stop solution, and absorbance is measured at 450 nm.
2.8. RNA Isolation and Quantification. Total number of RNA was extracted from the tissue of the lung cancer and glycation model system. The isolation was done by using the TRIzol Reagent method. The minced tissue sample and cell lysates were centrifuged at 14,000 rpm for 10 minutes at 4°C. Transfer the supernatant in a new tube containing chloroform in the ratio of 1 : 3. Keep the sample at room temperature after shaking vigorously for 30 seconds. Repeat the centrifugation process once again. Now, carefully transfer the supernatant in a fresh tube containing ice chilled isopropanol. Incubate the mixture for 15 minutes at room temperature. Again, repeat the centrifugation process. Wash the RNA pellet with 80% ethanol. Use DEPC treated water to dissolve the RNA pellet finally. The concentration of isolated RNA from tissues samples and cell lysates was quantified by Nanodrop. Blank was set through a solvent (DEPC treated water) used to dissolve RNA. RNA sample was mixed by gentle vortexing and inversion and placed 1 μl RNA solution 2.9. cDNA Synthesis. cDNA synthesis was done as per the manufacturer's (Thermo scientific) protocol. Thermo scientific c-DNA synthesis kit is a complete system for efficient synthesis of first-strand cDNA. The final volume was 20 μl.
The procedure for cDNA synthesis from a kit is as given below in Table 3. Add the following reagent on ice in a sterile condition after thawing properly. Mix gently and spin it for proper mixing. The reaction is extended at its first stage for 62 minutes at 42°C. At the second stage, the reaction is heated at 70°C for 5 minutes to inactivate the enzyme.  Table 4. The desired primer sequences were designed through Primer 3.0 software.
2.11. Real-Time PCR. The real-time PCR analysis of the tissue samples and cell lysates was assisted after the isolation and quantification of RNA followed by the synthesis of cDNA. The reaction volume is 13 μl per reaction following the RT-PCR standard condition, i.e., 95°C for 5 min at hold stage, 95°C for 30 sec, 59°C for 30 sec, and 72°C for 35 sec×40 cycles at PCR stage, and 95°C for 20°sec, 60°C for 60°sec, and 95°C for 30 sec at melting stage. The cycle of the threshold (Ct) value of each sample was calculated by using the Livak method for the fold change expression, i.e., 2 -ΔΔCt .
2.12. Statistical Analysis. Data are presented as mean ± SD, and statistical significance of the data was determined by Microsoft Excel (version 2010). One-way ANOVA was

Lowering of Blood Glucose by PFT-HSA-TFT-Cy7 and
PFT-Hcy-HSA-Cy7. The level of FBG was measured as a strong factor in the glycation animal model. The % decrease in the FBG has been observed as 4.6%, 15.73%, 31.60%, and 61.74% (group 3) respective to the control group with the treatment of PFT-HSA-TFT-Cy7 at week 1 st , week 2 nd , week 3 rd , and week 4 th . The % decrease in the level of FBG in the presence of PFT-Hcy-HSA-Cy7 has been calculated as 4.06%, 10.11%, 31.32%, and 57.48% (group 4) respective to the control group at 1 st week, 2 nd week, 3 rd week, and 4 th week (Figure 1(a)).  (Figure 1(c)). Tumor growth was characterized by a tumor node which that was almost negligible in the treatment groups. A marked reduction in tumor node was seen in Figure 1 (Figure 2(b)).  (Figure 4(c)). PFT-Hcy-HSA-Cy7 shows 2.2%, 26.66%, and 31.54% inhibition as compared to their respective controls at 1 μM, 3 μM, and 5 μM, respectively (Figure 4(d)). The nonfluorescent AGEs, i.e., CML, have been largely known AGEs found in food. Previously, we observed that MG which is a potent precursor of AGEs can be able to further generate CML in glycation and lung cancer animal model [16]. This observation encourages us to go through the inhibitory activity of the provided nanoassemblies against CML. We found % decrease in the CML content as 33.33% and 35.00% with the treatment of PFT-HSA-TFT-Cy7 and PFT-Hcy-HSA-Cy7 at 5 μM in the glycation animal model ( Figure 5(b)). The lung cancer animal model showed Similarly, in the same model, we observed 23.01% and 27.33% inhibitory activity in the presence of PFT-Hcy-HSA-Cy7 at 1 μM and 3 μM, respectively ( Figure 5(a)). After the successful in vivo inhibition of CML, we further escalated our study to in vitro CML content inhibition. The maximum inhibition in the CML content was recorded as 38.7% and 33.77% with the treatment of PFT-HSA-TFT-Cy7 and PFT-Hcy-HSA-Cy7 as compared to their respective control ( Figure 5(c)).

Inhibition of Tumor
3.6. RAGE Expression in All the Three Samples with or without the Presence of PFT-HSA-TFT-Cy7 or PFT-Hcy-HSA-Cy7. The RAGE mRNA levels were increased in the tis-sue sample of the glycation animal model as compared to its control group, with an average increase of 3.0-fold (P < 0:008 ) (Figure 6(b)), while the RAGE mRNA levels of the tissue sample of the cancer animal model were increased as compared to its control group with an average increase of 2.9-fold (P < 0:001) (Figure 6(a)). The RAGE expression was significantly upregulated in the BSA-MG-AGEs/A549 lung cancer cell line as compared to the control group, with an average increase of 9.7-fold (P < 0:001) (Figure 6(c)). Similarly, the treatment group of the glycation model system in the presence of PFT-HSA-TFT-Cy7 and PFT-Hcy-HSA-Cy7 shows significantly downregulated level of RAGE mRNA, with an average decrease in fold change as 2.3-fold and 2.0-fold at 5 μM of concentration, respectively (Figure 6(b)). While on the other hand, the treatment group of the cancer tissue with

NFκB Expression in All the Three Samples with or without the Presence of PFT-HSA-TFT-Cy7 or PFT-Hcy-HSA-Cy7.
The NFκB mRNA levels were increased in the tissue sample of the glycation animal model as compared to its control group, with an average increase of 3.9-fold ( Figure 6(b)), while the NFκB mRNA levels of the tissue sample of the cancer animal model were increased as compared to its control group with an average increase of 4.1-fold (P < 0:001) (Figure 6(a) 3.9. IFN-γ Expression in All the Three Samples with or without the Presence of PFT-HSA-TFT-Cy7 or PFT-Hcy-HSA-Cy7. The IFN-γ mRNA levels were increased in the tissue sample of the glycation animal model as compared to its control group, with an average increase of 1.9-fold (P < 0:005) (Figure 6(b)), while the IFN-γ mRNA levels of the tissue sample of the cancer animal model were increased as compared to its control group with an average increase of 5.7-fold (P < 0:005) (Figure 6(a)). Similarly, the IFN-γ expression was significantly upregulated in the BSA-MG-AGEs/A549 lung cancer cell line as compared to the control group, with an average increase of 9.7-fold ( Figure 6(f)). The treatment group of the glycation animal model in mediation of PFT-HSA-TFT-Cy7 showed downregulation. PFT-HSA-TFT-Cy7 and PFT-Hcy-HSA-Cy7 show significantly downregulated level of IFN-γ mRNA, with an average decrease in fold change as 1.59-fold and 1.3-fold at 5 μM of concentration ( Figure 6(b)). While on the other hand, the treatment group of the cancer tissue with PFT-HSA-TFT-Cy7 and PFT-Hcy-HSA-Cy7 shows a significant decrease in level of IFN-γ mRNA, with an average decrease in fold change as 3.3-fold and 3.2-fold at 1 μM and 3.0-fold and 1.7-fold at 3 μM concentration of nanoassemblies (Figure 6(a)). The treatment group of the in vitro model system shows 6.6-fold, 5.5-fold, and 2.8-fold change in the presence of PFT-HSA-TFT-Cy7 at 1 μM, 3 μM, and 5 μM, respectively, with respect to their control ( Figure 6(f)). Similarly, the treatment group of the in vitro model system in the presence of PFT-Hcy-HSA-Cy7 showed 5.9-fold, 3.6-fold, and 2.9 fold at 1 μM,

Discussion
Cancer is a leading health problem globally with around 14 million new cases recorded each year [24]. Hyperglycemia, energy imbalance, chronic inflammation, and insulin resistance are underlying mechanisms of cancer development. Moreover, AGEs are also involved in cancer progression. AGEs regulate cancer progression by activating its receptor RAGE. In our previous study, we established a link between glycation and lung cancer animal model [24]. While in this study, we used new multimodal nanoassemblies which are anticancerous in nature and designed as fluorinated nucleotide conjugated with dually labeled albumin. The report on its anticancer activity has been previously published [25], while in the current research, we are trying to find out its antiglycation property since we believe that AGE reduction might combat the cancer risk as well. Cancer is uncontrollable while AGEs are not; therefore, it is better to control cell growth by choosing an alternate pathway which might be an intermediate link for cancer growth. AGEs are supposed to be one of them [18,26]. We evaluated the inhibitory effect of PFT-HSA-TFT-Cy7 and PFT-Hcy-HSA-Cy7 on glucose in our glycation animal model. The maximum inhibition in the FBG level was observed on the fourth week which was recorded to be 61.7% by PFT-HSA-TFT-Cy7 (5 μM) and 57.65% by PFT-Hcy-HSA-Cy7 (5 μM) (Figure 1(a)). Furthermore, we identified its effect against glycative stress by measuring nonfluorescent AGEs "CML" and fluorescent AGEs. The inhibition of CML in the glycation animal model was 33.33% in the presence of PFT-HSA-TFT-Cy7 at 5 μM concentration. Meanwhile, the inhibition via PFT-Hcy-HSA-Cy7 was recorded to be 35% against CML at the same concentration ( Figure 5

BioMed Research International
MDA content with the treatment of both nanoassemblies that is PFT-HSA-TFT-Cy7 and PFT-Hcy-HSA-Cy7 at 5 μM, respectively (Figure 2(b)). Also, our in vitro experiments using A549 lung cancer cell line showed successful inhibition of glycative and oxidative stress under the influence of PFT-HSA-TFT-Cy7 and PFT-Hcy-HSA-Cy7. We observed a successful inhibition of ROS production compared to control. The sample treated with PFT-HSA-TFT-Cy at 5 μM showed 39.56% maximum inhibition in the ROS level compared to BSA-MG-AGEs. On the other hand, the sample treated with PFT-Hcy-HSA-Cy7 at 5 μM showed 50.09% maximum inhibition in ROS level compared to BSA-MG-AGEs (Figure 3(b)). In addition, we observed 26.65% and 31.54% maximum inhibition of fluorescent AGEs in the cell lysates with PFT-HSA-TFT-Cy and PFT-Hcy-HSA-Cy7, respectively, at 5 μM concentration (Figures 4(c) and 4(d)).
RAGE alters cell signaling pathways and contributes to endothelial dysfunction via tumor necrosis factor-α (TNFα). The interaction of AGEs with RAGE sets off a cascade of events leading to the modulation of the gene expression and loss of vascular and tissue homeostasis, the processes that contribute to cardiovascular diseases [26] lung cancer [27], pancreatic cancer [28], hepatocellular carcinoma [29], and various other forms of cancer as well [30,31]. Hence, Since AGEs are implicated in type 2 diabetes mellitus as reported by us and others [32,33], there is a strong link between diabetes and cancer [34]. Diabetes is associated with increased risk for some cancers like the liver, pancreas, endometrium, colon, breast, and bladder. The association between diabetes and some cancers may partly be due to shared risk factors between the two diseases, such as AGEs, aging, obesity, diet, and physical inactivity. Possible mechanisms for a direct link between diabetes and cancer include hyperinsulinemia, hyperglycemia, and inflammation [34]. This is the preliminary study in which we have seen the expression study at mRNA level only, and some glycation, oxidation, and inflammatory markers were only studied. This study further warrants to study the expression of the pathways at protein levels too by western blotting experiments.

Conclusion
Our findings from this study indicate that the nanoassemblies may play a protective role in cancer by inhibiting glycative and oxidative stress. This study is the first attempt to evaluate the antiglycation and anticancer activity simultaneously in the presence of newly synthesized nanoassemblies, i.e., PFT-HSA-TFT-Cy7 and PFT-Hcy-HSA-Cy7. In conclusion, our study suggests that preventing the formation of AGEs inside the body might lower the risk of cancer growth.

Data Availability
The data used to support the findings of this study are included within the article.

Conflicts of Interest
The authors declare that they have no conflicts of interest.