chapter 5 results and discussion synthesis and...
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Chapter 5 RESULTS AND DISCUSSION Synthesis and Characterization of Gold Nanoparticle Embedded 3,6-dihydroxyflavone Three main factors in nanoparticle preparation should be considered: selection of the solvent, use of environmentally benign reducing agent and use of non toxic material for stabilizing agent. In green nanoparticle synthesis, water is commonly used as environmentally benign solvents replacing toxic organic solvents [Bansal et al. 2008]. Biological entities (Flavonoids) have been reported serving as both reducing and stabilizing agent for green synthesis of metallic nanoparticles [Thakkar et al. 2010]. Whole plant extracts rich in polyphenols are powerful reducing agents for the production of gold nanoparticles. In addition to the whole plant extract, pure compound isolated from the plant like flavonoids are being utilized for nanoparticle synthesis [Padmanaban et al. 2012]. An aqueous solution of colloidal gold was prepared by reducing tetrachloroauric acid with 3,6-dihroxyflavone. The reduction of chloroaurate occurred through the transfer of electron from hydroxyl group of 3,6-dihroxyflavone to the Au3+ ion leading to the formation of Au0. This metallic gold then nucleates and grows to form gold nanoparticles, and is subsequently kept and stabilized by 3,6-dihroxyflavone [Wangoo et al. 2008]. Gold nanoparticle embedded 3,6-dihydroxyflavone was synthesized using chemical reduction method and was subjected for following steps of characterization.
Fig. 5.1: Synthesis of gold nanoparticle embedded 3,6-dihydroxyflavone
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Ultraviolet-Visible analysis UV-Vis method was used to ascertain the formation and stability of gold nanoparticles [Khoee et al. 2012]. The change in colour of the solution (yellow to light red) by the addition of NaAuCl4 (100 μl; 0.1M), producing a broad peak in the range of 425-520 nm, indicates the particles are mono dispersed.
Fig. 5.2: UV-Vis spectra of 3,6-dihydroxyflavone & gold nanoparticle embedded 3,6-dihydroxyflavone The appearance of light red colour is attributed to surface plasmon resonance arising from free conduction electrons induced by an interacting electromagnetic field [Song et al. 2008] and excitation of surface plasmon vibrations in the structure of gold nanoparticle embedded 3,6-dihydroxy flavone. The comparative spectra of 3,6-dihydroxyflavone and gold nanoparticle embedded 3,6-dihydroxyflavone is shown in (Fig. 5.2). X-ray diffraction analysis The X-ray diffraction pattern for 3,6-dihydroxyflavone does not show any characteristic peaks due to its amorphous nature while in case of gold nanoparticle embedded 3,6-dihydroxyflavone three characteristic peaks correspond to 111, 200 and 220 of Au located at 2Ө = 38.29°, 44.43° and 64.68°, respectively were observed, confirming that the sample is composed of crystalline gold phase. Using Scherer’s equation, t = kλ/BCosӨ, where t is crystallite size, B, full width at half maxima, k, shape factor (0.9 for spherical particles) and λ incident wavelength of X-ray (1.548 A°), average particle size of synthesized material was 12 nm. The comparative XRD pattern of 3,6-dihydroxyflavone and gold nanoparticle embedded 3,6-dihydroxyflavone is shown in (Fig. 5.3).
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Fig. 5.3: XRD of a) 3,6-dihydroxyflavone & b)Gold nanoparticle embedded 3,6-dihydroxyflavone Scanning electron microscope analysis SEM micrographs of 3,6-dihydroxyflavone and gold nanoparticle embedded 3,6-dihydroxy flavone were recorded. 3,6-dihydroxy flavone showed larger size of aggrigated rhombic crystal while uniform needle type morphology was observed in case of gold nanoparticle embedded flavonoid (Fig. 5.4). This difference may be ascribed to low temperature, polarity of solvent during synthesis of the compound and characteristic (111) peak observed in XRD pattern.
Fig.5.4: SEM image of a) 3,6-dihydroxyflavone & b) Gold nanoparticle embedded 3,6-dihydroxyflavone
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Transmission electron microscope analysis TEM micrographs of 3,6-dihydroxy flavone were of larger size, irregular shape while gold nanoparticle embedded 3,6-dihydroxyflavone of nucleated cell type of morphology with 6-12 nm particle size (Fig. 5.5).
Fig.5.5: TEM image of a) 3,6-dihydroxyflavone & b) Gold nanoparticle embedded 3,6-dihydroxyflavone Energy dispersive X-ray spectroscopy EDAX Spectrometry was used to further confirm the presence of gold in the synthesized material with no other contaminants. The appearance of optical adsorption peak at approximately 2.30 keV is the characteristic for the adsorption of gold nanocrystallites. The current profile of EDAX of gold nanoparticle embedded 3,6-dihydroxyflavone showed strong gold atom signals around 2.30, 9, 10.30, 11.30, and 12.30 keV (Fig. 5.6). Our experimental findings are in the harmony of the earlier reports on the preparation of gold embedded extract particles from the plant products [Huang et al. 2007, Parshar et al. 2009 and Tamizhamudu et al. 2011].
Fig. 5.6a: EDAX spectrum
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Fig. 5.6b: EDAX spectrum analysis chart
AFM analysis AFM images of native and gold nanoparticle embedded 3,6-dihydroxyflavone were recorded in plain and 2D view. Native 3,6-dihydroxyflavone showed rough surface with larger particle size while, gold nanoparticle embedded 3,6-dihydroxyflavone showed continuous and uniformly distributed particle with small size (Fig. 5.7).
Fig.5.7: AFM image of a) 3,6-dihydroxyflavone & b)Gold nanoparticle embedded 3,6-dihydroxyflavone
Spectrum: Gold nanoparticle embedded 3,6-dihydroxyflavone Element Series unn. C norm. C Atom. C Error [wt.%] [wt.%] [wt.%] [wt.%] ---------------------------------------------------------------------------------------------------------- Copper K-series 45.16 45.16 13.48 01.50 Carbon K-series 54.63 54.63 86.25 02.20 Gold L-series 00.00 00.00 00.01 00.00 Oxygen K-series 00.22 00.22 00.26 00.10 --------------------------------------------------------------------------------------------------------- Total: 100.00 100.00 100.00
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In vitro Antioxidant Activity of Dietary Compounds: Combination and Nanotech Enhancement DPPH (2, 2-Diphenyl-1-Picrylhydrazyl) Assay The antioxidative effect of dietary phytochemical (3,6-dihydroxyflavone), antioxidant (lutein), sensitizer (selenium methyl selenocysteine) and gold nanoparticle embedded 3,6-dihydroxyflavone single and in combination were tested for their DPPH radical scavenging effect using reference antioxidant ascorbic acid (Vit. C) as a positive control in the concentrations range (10-100 µg/ml). These radicals are known to have damaging effect to almost every biological molecule found in living cells [Erlejman et al. 2004 and Moon et al. 2009]. DPPH is a stable nitrogen-centered free radical, the color of which changes from violet to yellow upon the reduction by either the process of hydrogen or electron donation. If free radicals have been scavenged, solution will become yellow because of formation of α-α diphenyl-β-picryl hydrazine. The extent of discolouration indicates the amount of DPPH radical scavenged and the test sample shows antioxidant activity. Substances which are able to perform this reaction can be considered as antioxidants and, therefore, radical scavengers [Dehpour et al. 2009].
Fig.5.8: DPPH assay: Mechanistic pathway
Radical scavenging activities of all the dietary compounds under study increased with increasing concentration exhibiting its dose dependant nature. Percent of inhibition for DPPH radical scavenging activity is presented in (Fig. 5.9).
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Each value is mean ± SD (n = 3). p < 0.05 (single and combination) versus ascorbic acid. Where VIT C: ascorbic acid, DHF: 3,6-dihydroxyflavone, GNDHF: gold nanoparticle embedded 3,6-dihydroxyflavone, LUT: lutein and MSC: selenium methyl selenocysteine
Fig. 5.9: Percentage inhibition of DPPH radical concentration dependency of dietary compounds, single and in combination against standard ascorbic acid A perusal of the data shows that at concentration 100 µg/ml, all three dietary compounds individually exhibited optimum percent of inhibition: 3,6-dihydroxyflavone (64.21 %), lutein (65.79 %) and selenium methyl selenocysteine (43.85 %) against ascorbic acid (96.28 %) as standard. The triple combination (1:1:1) exhibited maximum percent of inhibition (72.89 %) at same concentration level, indicating 14.94 % enhancement in antioxidant activity. Gold nanoparticle embedded 3,6-dihydroxyflavone individually showed maximum percent of inhibition (72.04 %) at the same concentration compared to native 3,6-dihydroxyflavone (64.21 %). The inclusion of gold nanoparticle embedded 3,6-dihydroxyflavone with other dietary nutrients lutein and selenium methyl selenocysteine further increased maximum inhibition (87.13 %) at the same concentration (100 µg/ml). Thus, nanotech enhancement obtained in the antioxidant activity is (29.23 %) of triplet combination involving gold nanoparticle embedded 3,6-dihydroxyflavone.
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Hydroxyl Radical (Fenton) Assay The hydroxyl radical formed in the FENTON reaction in the presence of reduced transition metals such as Fe2
+ and H2O2 which are known to be the most reactive of all the reduced forms of dioxygen is capable of damaging of almost every molecule found in living cells [Rollet-Labelle et al. 1998]. Deoxyribose was oxidized when exposed to hydroxyl radicals generated by Fenton reagent and the oxidation degradation can be detected by heating the products with thiobarbuteric acid (TBA).
Fig.5.10: Fenton assay: Mechanistic pathway
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The hydroxyl radical scavenging activity of all the dietary compounds in single and combination was determined. Increased radical scavenging activities with increasing concentration of the dietary compounds under study were noticed. Percent of inhibition for OH radical scavenging activity are tabulated in (Fig. 5.11).
Each value is mean ± SD (n = 3). p < 0.05 (single and combination) versus ascorbic acid. Where VIT C: ascorbic acid, DHF: 3,6-dihydroxyflavone, GNDHF: gold nanoparticle embedded 3,6-dihydroxyflavone, LUT: lutein and MSC: selenium methyl selenocysteine. Fig. 5.11: Percentage inhibition of OH radical concentration dependency of dietary compounds, single and in combination against standard ascorbic acid
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Data indicate that all the three dietary compounds individually exhibited optimum percent of inhibition: 3,6-dihydroxyflavone (62.11 %), lutein (63.85 %) and selenium methyl selenocysteine (41.62 %) against ascorbic acid (96.18 %) as standard at concentration 100 µg /ml. The triplet combination (1:1:1), exhibited maximum percent of inhibition (70.63 %) at same concentration of 100 µg/ml indicating (14.77 %) enhancement in antioxidant activity. Gold nanoparticle embedded 3,6-dihydroxyflavone individually showed maximum percent of inhibition (70.01 %) at the same concentration compared to the native 3,6-dihydroxyflavone (62.11 %). The inclusion of gold nanoparticle embedded 3,6-dihydroxyflavone with other dietary nutrients lutein and selenium methyl selenocysteine further increased the maximum inhibition (85.11 %) at the same concentration, with overall enhancement (26.61 %) in antioxidant activity. H2O2 Scavenging Assay H2O2 scavenging by dietary compounds may be attributed to donate electrons to H2O2, thus neutralizing it to water. The ability of these compounds effectively scavenges hydrogen peroxide [Sroka et al. 2003]. Radical scavenging activities of all the dietary compounds studied increased with increasing concentration. Percent of inhibition for H2O2 radical scavenging activity are shown in (Fig. 5.13).
Fig.5.12: H2O2 assay: Mechanistic pathway
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Data show that the dietary compounds individually exhibited optimum percent of inhibition; 3,6-dihydroxyflavone (60.11 %), lutein (61.85 %) and selenium methyl selenocysteine (40.02 %) against ascorbic acid (96.12 %) as standard at concentration 100 µg /ml. Among the combinations studied, the triple combination (1:1:1), exhibited maximum percent of inhibition (71.35 %) at the same concentration level, indicating 17.35 % enhancement in antioxidant activity.
Each value is mean ± SD (n = 3). p < 0.05 (single and combination) versus ascorbic acid. Where VIT C: ascorbic acid, DHF: 3,6-dihydroxyflavone, GNDHF: gold nanoparticle embedded 3,6-dihydroxyflavone, LUT: lutein and MSC: selenium methyl selenocysteine.
Fig. 5.13: Percentage inhibition of H2O2 radical concentration dependency of dietary compounds, single and in combination against standard ascorbic acid
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Gold nanoparticle embedded 3,6-dihydroxyflavone individually showed optimum percent of inhibition (71.08 %) compared to normal 3,6-dihydroxyflavone (60.11 %) at the same concentration level. The combination study with the inclusion of gold nanoparticle embedded 3,6-dihydroxyflavone with other dietary nutrients lutein and selenium methyl selenocysteine further increased maximum inhibition (83.10 %) at the same concentration. Thus, the overall enhancement could be gained in the antioxidant activity (25.45 %) of triplet combination having of gold nanoparticle embedded 3,6-dihydroxyflavone. Nitric Oxide Scavenging Assay Nitric oxide (NO) radical has also been involved in a variety of biological functions, including neurotransmission, vascular homeostasis, antimicrobial, and antitumor activities. Despite the possible beneficial effects of NO radical, its contribution is to oxidative damage. This is due to the fact that NO radical can react with superoxide to form the peroxynitrite anion, which is a potential oxidant that can decompose to produce OH radical and NO radical. The procedure is based on the principle that, sodium nitro-prusside in aqueous solution at physiological pH spontaneously generates nitric oxide which interacts with oxygen to produce nitrite ions that can be estimated using Griess reagent. Scavengers of nitric oxide compete with oxygen, leading to reduced production of nitrite ions.
Fig.5.14: NO radical assay: Mechanistic pathway
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A perusal of the data shows that at concentration 100 µg/ml, all the three dietary compounds individually exhibited maximum percent of inhibition; 3,6-dihydroxyflavone (61.24 %), lutein (60.85 %) and selenium methyl selenocysteine (42.11 %) against ascorbic acid (96.02 %) as standard. The triple combination (1:1:1), exhibited maximum percent of inhibition (69.09 %) at same concentration indicating 14.35 % enhancement in antioxidant activity. Gold nanoparticle embedded 3,6-dihydroxyflavone individually showed maximum percent of inhibition (69.01 %) at the same concentration compared to percent inhibition of normal 3,6-dihydroxyflavone (61.24 %). The inclusion of gold nanoparticle embedded 3,6-dihydroxyflavone with other dietary nutrients further increased maximum inhibition (84.02 %) at the same concentration level, exhibiting over all enhancement in the antioxidant activity (26.07 %). The percent inhibitions for NO radical scavenging activity are reported in (Fig. 5.15).
Each value is mean ± SD (n = 3). p < 0.05 (single and combination) versus ascorbic acid. Where VIT C: ascorbic acid, DHF: 3,6-dihydroxyflavone, GNDHF: gold nanoparticle embedded 3,6-dihydroxyflavone, LUT: lutein and MSC: selenium methyl selenocysteine.
Fig.5.15: Percentage inhibition of NO radical concentration dependency of dietary compounds, single and in combination against standard ascorbic acid
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A comparison of overall experiments (DPPH, Fenton, H2O2 and NO bioassays) conducted for the assessment of antioxidant activity has clearly pointed out that the percent inhibition of native 3,6-dihydroxyflavone (64.21±1.28, 62.11±1.11, 60.11±0.81 and 61.24±0.77) % can be successfully enhanced by 3,6-dihydroxyflavone embedded to gold nanoparticles reaching to the value of percent inhibition (72.04±1.17, 70.01±1.30, 71.08±0.80 and 69.01±1.50) %. The combination studies including of gold nanoparticle embedded 3,6-dihydroxyflavone with other dietary compounds further enhanced maximum inhibition to the value of (87.13±1.43, 85.11±1.31, 83.10±1.51 and 84.02±1.13) % at the same concentration level (100 µg/ml), thus, exhibiting over all enhancement of (29.23, 26.61, 25.45 and 26.07) % in the antioxidant activity (Fig. 5.16). The order of free radical scavenging activity was as follows: Vitamin C > (GNDHF: LUT: MSC) > (DHF: LUT: MSC) >GNDHF > LUT > DHF > MSC
In each case, the role of gold salt solution toward antioxidant activity has been checked separately and has not been found to reflect any noticeable antioxidant activity. The presence of flavonoidal content could have been adsorbed on the surface of nanoparticle induced in the catalytic reduction of Au3+ ions to Au0 nanoparticles with interaction of carbonyl groups or π-electrons. While conversion of C═O group of flavonoid to – C(O)═O group leads to the reduction of gold ions. Thus the presence of free electron movement in electron deficient gold nanoparticle embedded 3,6-dihydroxyflavone is found the basis of increasing the rate of free radical scavenging activity. However, oxidation products and the reaction mechanism are not well identified [Aleksandra et al. 2012 and Reddy et al. 2012]. The reduction of Au (III) to Au (0) is expected to occur through the oxidation of flavonoid hydroxyl to carbonyl groups. The proposed oxidation of flavonoid in presence of Au (III) is as follows: AuCl4
− + 3R–OH Au0 + 3R=O + 3H+ + 4Cl−
Each value is mean ± SD (n = 3). p < 0.05 combination versus ascorbic acid. Where VIT C: ascorbic acid, DHF: 3,6-dihydroxyflavone, GNDHF: gold nanoparticle embedded 3,6-dihydroxyflavone, LUT: lutein and MSC: selenium methyl selenocysteine.
Fig.5.16: Percentage inhibition of DPPH, FENTON, H2O2 & NO radicals of test compounds, in combination against standard ascorbic acid
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In vivo Antioxidant Studies of Dietary Compounds: Nanotech Enhancement In vivo antioxidative effect of gold nanoparticle embedded 3,6-dihydroxyflavone with antioxidant (lutein), sensitizer (selenium methyl selenocysteine) single and combination were tested in terms of downregulation level of reduced glutathione and an increased concentration of MDA per mg wet weight in Sarcoma 180 cancer cells induced female Balb/c mice liver. Cancer mediated modulation of down regulation level of reduced glutathione in liver The down regulation level of reduced glutathione in liver of experimental mice was investigated to determine the antioxidative effect of test groups against the oxidative stress induced by Sarcoma 180 cancer cells.
Each value is mean±SD (n=6). p > 0.01 vs. tumor control, p < 0.05 vs. normal control. Where Group I: NC – Saline treated
normal control, Group II: TC - Tumor Control, Group III: 2% DMSO - Dimethyl sulphoxide, Group IV: DOXO - Doxorubicin (1
mg/Kg b.w.), Group V: GNDHF - Gold nanoparticle embedded 3,6-dihydroxyflavone (8 µM; 5 mg/Kg b.w ), Group VI: LUT
– lutein (1.5 µM; 5 mg/Kg b.w), Group VII: MSC - Selenium methyl selenocysteine (10 µM; 5 mg/Kg b.w) and Group VIII:
GNDHF+LUT+MSC (8/1.5/10 µM; 5 mg/Kg b.w).
Fig. 5.17: Combination effect of the gold nanoparticle embedded 3,6-dihydroxyflavone with lutein and selenium methyl selenocysteine against standard doxorubicin on level of GSH per mg protein (μM) in normal and cancer tumor bearing mice at 29th day
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After induction of Sarcoma 180 cancer cells, the weights and the level of GSH of liver tissues of experimental mice were recorded. The liver weight of combination of GNDHF: LUT: MSC treated mice was found (1.16 gm) similar to the normal control group mice (1.23 gm) as compared to the tumor control mice (1.54 gm). Decreased concentration of reduced GSH (0.07 μM) per mg protein in tumor control group has been observed compared to normal control (0.27 μM) per mg protein. The reduced GSH level of test samples at dose of 5 mg/Kg body weight of mice were as follows: GNDHF (8 μM) - 0.12 μM; LUT (1.5 μM) – 0.10 μM; MSC (8 μM) – 0.09 μM; GNDHF: LUT: MSC (8/1.5/10 μM) – 0.15 μM against the reference drug doxorubicin at dose of 1 mg/Kg body weight of mice 0.22 μM shown in (Fig. 5.17). Subcutaneous induction of Sarcoma 180 cancer cell showed a significant lowering of reduced glutathione in liver compared to normal and reduced the scavenging of reactive oxygen species. Among the groups studied, optimum value of reduced GSH per mg protein is found to be in the order: Doxo reference > (GNDHF: LUT: MSC combination) > GNDHF > LUT > MSC.
It is depicted, Combination of gold nanoparticle embedded 3,6-dihydroxyflavone, lutein and selenium methyl selenocysteine (8/1.5/10 μM) at dose of 5 mg/Kg body weight of mice has shown significant antioxidant activity. Free radicals and their biochemical reactions in each stage of the metabolic process are involved in cancer development [Kun-Young et al. 2003]. Antioxidants act as the primary line of defense against reactive oxygen species and suggest their usefulness in estimating the risk of oxidative damage induced during carcinogenesis. Glutathione, a potent inhibitor of neoplastic process plays an important role as an endogenous antioxidant system that is found particularly in high concentration in liver and is known to have key function in the protective process [Sinclair et al. 1990]. It is a non-protein cellular thiol which in conjunction with glutathione peroxydase has a regulatory role in cell proliferation. GSH and its dependent enzymes scavenge the electrophilic moieties involved in the cancer initiation and serves as marker for the evaluation of oxidative stress [Comporti et al. 1989 and Nam et al. 2008]. The level of reduced glutathione was depleted in cancer bearing mice which may be due to its utilization by the excessive amount of free radicals. Subcutaneous induction of Sarcoma 180 cancer cell showed a significant lowering of reduced glutathione in liver compared to normal control mice and reduced the scavenging of reactive oxygen species. Treatment with GNDHF: LUT: MSC combination was found to increase the GSH content in the liver as compared to cancer control animals. Our observation supports the fact that Sarcoma 180 cancer cells induced oxidative stress is due to the depletion of antioxidant system. Lipid per-oxidation in terms of increased concentration of MDA per mg wet weight Decreased concentration of MDA (0.47 nM) per mg wet weight in tumor control has been observed compared to normal control (0.52 nM). Concentration of MDA per mg wet weight at dose of 5 mg/Kg body weight of mice were as follows: GNDHF (8 μM) - 0.22 nM; LUT (1.5 μM) – 0.26 nM; MSC (8 μM) – 0.27 nM; GNDHF: LUT: MSC (8/1.5/10 μM) – 0.18 nM against the reference drug doxorubicin at dose of 1 mg/Kg body weight of mice 0.15 nM. Among the groups studied, optimum value of MDA per mg wet weight was found to be in the order: Doxo reference < GNDHF: LUT: MSC combination < GNDHF < LUT < MSC. Based on our observation, GNDHF: LUT: MSC combination (8/1.5/10 μM) at dose of 5 mg/Kg body weight of mice exhibited optimum antioxidant activity and rendered significant protection against oxidative stress induced by Sarcoma 180 cancer cells in liver tissues shown in (Fig. 5.18).
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The improper balance between ROMs (Reactive Oxygen Metabolites) and antioxidant defenses results in ‘oxidative stress’, which deregulates the cellular functions leading to various pathological conditions including cancer [Bandyopadhyay et al. 1999 and Adesegun et al. 2009]. The oxidative stress may lead to damage of the macromolecules such as lipids and can induce lipid peroxidation in vivo [Yagi et al. 1991].
Each value is mean±SD (n=6). p > 0.01 vs. tumor control, p < 0.05 vs. normal control. Where Group I: NC - Saline treated
normal control, Group II: TC - Tumor Control, Group III: 2% DMSO - Dimethyl sulphoxide, Group IV: DOXO – Doxorubicin (1 mg/Kg b.w), Group V: GNDHF - Gold nanoparticle embedded 3,6-dihydroxyflavone (8 µM; 5 mg/Kg b.w), Group VI: LUT -
lutein (1.5 µM; 5 mg/Kg b.w), MSC: Group VII: MSC - Selenium methyl selenocysteine (10 µM; 5 mg/Kg b.w) and Group
VIII: GNDHF+LUT+MSC (8/1.5/10 µM; 5 mg/Kg b.w).
Fig. 5.18: In vivo LPO study-effect of the gold nanoparticle embedded 3,6-dihydroxyflavone with lutein and selenium methyl selenocysteine (single and combination), against doxorubicin on conc of MDA nM per mg wet weight in normal and cancer tumor bearing mice at 29th day.
In cancer control group of mice the level of lipid peroxide in liver was significantly elevated, which was however reduced to near normal level in the GNDHF: LUT: MSC combination treated group animals. This reflects the decrease in free radical production and the subsequent reduction in oxidative stress, one of the main risk factors for the disease [Saha et al. 2011].
On the other hand the free radical scavenging enzyme catalase is present in all oxygen-metabolizing cells and its function is to provide a direct defense against the potentially damaging reactivities of superoxide and hydrogen peroxide. The inhibition of catalase activities was resulted of tumor growth [Marklund et al. 1982]. The administration of GNDHF: LUT: MSC combination (8/1.5/10 µM) at dose of 5 mg/Kg body weight of mice concentration increased the catalase levels, which along with the restoration of lipid peroxide and GSH content to near normal indicates the antioxidant and free radical scavenging property of GNDHF: LUT: MSC combination. Our experimental findings find support from other observations. For the reduction and stabilization of metallic nanoparticles, the use of various biological entities has received considerable attention in
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the field of Nanobiotechnology, Pharmaceutical and Biomedical applications [Park et al. 2011]. The unique kinetic propensity of phytochemicals to reduce the gold metal at macro and micro concentrations to the corresponding gold nanoparticles has been highly appreciated in the scientific world [Shankar et al. 2004, Chandran et al. 2006, Han et al.2007, Philip et al. 2009, Wang et al. 2009, Krpetic et al. 2009 and Katti et al. 2009]. The versatile phytochemical (Flavonoid) has been largely used for the preparation of gold nanoparticles. Flavonoidal compounds have been found powerful reducing and stabilizing agents for the production of gold nanoparticles [Shukla et al. 2008, Song et al. 2009, Jha et al. 2009, Kasthuri et al. 2009, Smitha et al. 2009, Thakkar et al. 2010, Dumur et al. 2011, Tripathi et al. 2012, Siddiqi et al. 2012, Jagganathan et al. 2007, Hseih et al. 2012 and Pongsuchat et al. 2012]. Flavonoids and polyphenolic compounds play a key role in bio fabrication or capping of gold nanoparticles. Nanoparticles embedded with biologically active phytochemicals might exert synergistic effects by combining their biological activities with those of nanoparticles [Park et al. 2011]. Dietary phytochemicals that contain functional groups like hydroxyl, phenols and aromatic ring provide synergistic power for the reduction of gold salts into their corresponding gold nanoparticle. Further, the coating of phytochemical on the gold nanoparticles, thus, paves an unprecedented process for the production and stabilization of gold nanoparticles simultaneously enhancing bioactivity in a singular green process.
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In vitro Antibreast Cancer Efficacy of Dietary Compounds: Combination and Nanotech Enhancement The phytochemicals selected for the present study were known for their anti-cancer activity at non toxic concentrations. Therefore, Inhibitory concentration (IC50) was experimentally evaluated (Fig. 5.19) for all the three native dietary products with respect to vehicle control and standard drug (doxorubicin). Based on (IC50) values 3,6-dihydroxyflavone (16 μM), selenium methyl selenocysteine (10 μM) and lutein (1.5 μM), different double and triple combinations of the three dietary products were designed and monitored for their cytotoxic effect against two human breast cancer cell lines (MCF-7 and MDA MB-468).
Each value is mean ± SD (n=3). MSC: Selenium methyl selenocysteine, DHF:3,6-dihydroxyflavone,LUT:Lutein,GNDHF: Gold nanoparticle embedded3,6-dihydroxyflavonel.
Fig. 5.19: IC50 values of DHF, GNDHF, LUT & MSC In vitro MTT, TBE and SRB assays Cytotoxicity in terms of percent inhibition was monitored after 48 h incubations by MTT, TBE and SRB bioassays. Current bioassays for measuring cytotoxicity are based on alterations of plasma membrane permeability and the consequent release of components into the supernatant. In case of trypan blue exclusion assay, the dead cells uptake the dye while the viable cells are excluded [Baliga et al. 1996]. In MTT assay, dead cells are unable to metabolize yellow tetrazolium salt while viable cells metabolize yellow tetrazolium salt into purple formazan crystal [Kumar et al. 2009] and sulphorhodamine B is an anionic aminoxanthene dye that forms an electrostatic complex with the basic amino acid residues of proteins under moderately acidic conditions, which provides a sensitive linear response [Perez et al. 1993]. All the three bioassays (Fig. 5.20) are likely to provide real picture of percent inhibition of dietary products and hence selected for the present study.
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Fig. 5.20: Schematic presentation of the working of MTT, TBE and SRB bioassays
The combination study of dietary products; flavonoid: 3,6-dihydroxy flavone, sensitizer: selenium methyl selenocysteine and antioxidant: lutein was tested for their cytotoxic efficacy against MCF-7 and MDA-MB-468 breast cancer cell lines using reference anticancer drug (doxorubicin) as a positive control. MCF-7 cells are well characterized estrogen receptor positive, non invasive differentiated mammary epithelium cells while MDA-MB-468 are highly invasive, estrogen independent and dedifferentiated
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breast carcinoma cell line, therefore selected for the present study. The pattern of combinations considered for the study has been presented schematically in (Table 5.1) Table.5.1: Schematic pattern of the combination study of three native dietary compounds against doxorubicin as standard drug
Table 5.2a: In vitro cytotoxic effects of single and combination treatments of 3,6-dihydroxyflavone, lutein and selenium methyl selenocysteine (Single, double and triple combination) on MCF-7 breast cancer cell line using MTT, TBE and SRB bioassays
Each value is mean ± SD (n = 3). p < 0.05 (single and combination) versus doxorubicin. Where Doxo: doxorubicin, DHF: 3,6-dihydroxyflavone, LUT: lutein and MSC: selenium methyl selenocysteine.
Table 5.2b: In vitro cytotoxic effects of single and combination treatments of 3,6-dihydroxyflavone, lutein and selenium methyl selenocysteine (Single, double and triple combination) on MDA-MB-468 breast cancer cell line using MTT, TBE and SRB bioassays.
Treatment MTT assay TBE assay SRB assay Control (0) 0 0 0
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Doxo (2) 50±1.39 50.05±1.39 51.15±1.39
DHF(16) 50.26±1.4 51.04±1.4 51.86±1.4
MSC (10) 50.01±1.42 50.84±1.42 51.34±1.42
LUT(1.5) 50.05±1.42 50.52±1.42 50.82±1.42
MSC+LUT(10/1.5) 52.62±1.43 52.23±1.42 52.44±1.41
DHF+MSC (16/10) 53.61±1.44 53.45±1.42 53.27±1.42
DHF+LUT (16/1.5) 54.87±1.39 54.51±1.43 54.31±1.43
DHF+MSC+LUT (16/10/1.5) 68.25±1.42 68.94±1.4 68.12±1.44
Each value is mean ± SD (n = 3). p < 0.05 (single and combination) versus doxorubicin . Where Doxo: doxorubicin, DHF: 3,6-dihydroxyflavone, LUT: lutein and MSC: selenium methyl selenocysteine.
Test compounds Combination pattern Doxorubicin (standard) X
3,6-dihydroxyflavone X X X X
Lutein X X X
Selenium methyl selenocysteine X X X
Treatment MTT assay TBE assay SRB assay Control (0) 0 0 0
V. control (0) 0 0 0
Doxo (2) 50.01±0.82 51.81±0.83 52.01±0.83
DHF(16) 50.32±0.78 51.86±0.8 52.16±0.82
MSC (10) 50.02±0.72 51.25±0.76 52.89±0.79
LUT(1.5) 50.14±0.87 51.44±0.8 52.96±0.82
MSC+LUT(10/1.5) 54.16±0.75 54.64±0.8 54.92±0.84
DHF+MSC (16/10) 57.51±0.78 57.41±0.81 57.82±0.83
DHF+LUT (16/1.5) 57.11±0.83 57.21±0.82 57.23±0.88
DHF+MSC+LUT (16/10/1.5) 71.53±0.88 72.23±0.83 72.07±0.87
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A perusal of the data (Table 5.2a & b) shows that doublet combination (MSC and LUT) did not show effective percent of inhibition (54.16 %, 54.64 % and 54.92 %) in MCF-7 and (52.62 %, 52.23 % and 52.44 %) in MDA-MB 468 cell lines for MTT, TBE and SRB respectively. However, the inclusion of target flavonoid in doublet combinations, (DHF + MSC) and (DHF + LUT) exhibited no noticeable enhancement in percentage of inhibition (57.51 %, 57.11 %, 57.41 %, 57.21 %, 57.82 %, 57.23 %) in MCF-7 and (53.61 %, 54.87 %, 53.45 %, 54.51 %, 53.27 %, 54.31 %) in MDA-MB-468 cell line by MTT, TBE and SRB bioassays respectively. The triple combination (DHF+MSC+LUT) exhibited appreciable inhibition (71.53 %, 72.23 % and 72.07 %) in MCF-7 and (68.25 %, 68.94 % and 68.12 %) in MDA-MB-468 cell lines by all the bioassays. However, the percent inhibition in case of MCF-7 is more pronounced compared to MDA-MB-468 breast cancer cell line. In order to observe, any nanotech reinforcement in percent inhibition, native 3,6-dihydroxyflavone was replaced by gold nanoparticle embedded 3,6-dihydroxyflavone (GNDHF) and monitored for in vitro cytotoxic activity under similar experimental conditions. The pattern of combinations considered for the study has been presented schematically in (Table 5.3). Data (Table 5.4a & b) show that double combination (MSC and LUT), in this case also, did not exhibit any marked change in percent of inhibition in both the cell lines for each assay conducted. The inclusion of gold nanoparticle embedded 3,6-dihydroxyflavone in double combinations, (GNDHF + MSC) and (GNDHF + LUT) exhibited tendency of enhancing the percentage of inhibition (62.15 %, 62.11 % , 62.14 %, 62.21 %, 62.28 %, 62.82 %) in MCF-7 and (59.81 %, 60.78 %, 59.54 %, 60.15 %, 59.72 %, 60.13 %) in MDA-MB-468 cell lines by MTT, TBE and SRB bioassays respectively. Table.5.3: Schematic pattern of the nanotech and combination enforcement study of test compounds against doxorubicin as standard drug on the basis of their IC50 values
Table 5.4a: In vitro cytotoxic effects of single and combination treatments of gold nanoparticle embedded 3,6-dihydroxyflavone, lutein and selenium methyl selenocysteine (Single, double and triple combination) on MCF-7 breast cancer cell line using MTT, TBE and SRB bioassays
Each value is mean ± SD (n = 3). p < 0.05 (single and combination) versus doxorubicin. Where Doxo: doxorubicin, GNDHF: gold nanoparticle embedded 3,6-dihydroxyflavone, LUT: lutein and MSC: selenium methyl selenocysteine.
Test compounds Combination pattern Doxorubicin (standard) X
Gold nanoparticle embedded 3,6-dihydroxyflavone X X X X
Lutein X X X
Selenium methyl selenocysteine X X X
Treatment MTT assay TBE assay SRB assay Control (0) 0 0 0
V. control (0) 0 0 0
Doxo (2) 50.01±0.82 51.81±0.83 52.01±0.83
GNDHF(8) 50.05±0.9 51.32±0.92 52.87±0.92
MSC (10) 50.02±0.72 51.25±0.76 52.89±0.79
LUT(1.5) 50.14±0.87 51.44±0.8 52.96±0.82
MSC+LUT(10/1.5) 54.16±0.75 54.64±0.8 54.92±0.84
GNDHF +MSC (8/10) 62.15±0.79 62.14±0.81 62.28±0.85
GNDHF +LUT (8/1.5) 62.11±0.85 62.21±0.83 62.32±0.86
GNDHF+MSC+LUT (8/10/1.5) 90.27±0.89 90.51±0.85 91.57±0.81
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The combination (GNDHF + MSC + LUT) exhibited significant (p < 0.05) enhancement in the percent of inhibition (90.27 %, 90.51 % and 91.57 %) in MCF-7 and (80.17 %, 80.27 % and 80.67 %) in MDA-MB-468 cell lines in all the bioassays respectively (Table 5.4a & b). However, in this case also, percent inhibition was found more pronounced (p < 0.05) in MCF-7 compared to MDA-MB-468 cell lines in all the bioassays studied. Table 5.4b: In vitro cytotoxic effects of single and combination treatments of gold nanoparticle embedded 3,6- dihydroxyflavone, lutein and selenium methyl selenocysteine (Single, double and triple combination) on MDA-MB-468 breast cancer cell line using MTT, TBE and SRB bioassays
Treatment MTT assay TBE assay SRB assay
Control (0) 0 0 0
V. control (0) 0 0 0
Doxo (2) 50±1.39 50.05±1.39 51.15±1.39
GNDHF(8) 50±1.41 50.52±1.41 51.02±1.41
MSC (10) 50.01±1.42 50.84±1.42 51.34±1.42
LUT(1.5) 50.05±1.42 50.52±1.42 50.82±1.42
MSC+LUT(10/1.5) 52.62±1.43 52.23±1.42 52.44±1.41
GNDHF+MSC (8/10) 59.81±1.43 59.54±1.43 59.72±1.42
GNDHF+LUT (8/1.5) 60.78±1.43 60.15±1.42 60.13±1.43
GNDHF+MSC+LUT (8/10/1.5) 80.17±1.42 80.27±1.43 80.67±1.43
Each value is mean ± SD (n = 3). p < 0.05 (single and combination) versus doxorubicin. Where Doxo: doxorubicin, GNDHF: gold nanoparticle embedded 3,6-dihydroxyflavone, LUT: lutein and MSC: selenium methyl selenocysteine.
Percent inhibition resulting from all the in vitro bioassays demonstrates that the (GNDHF + MSC + LUT) combination is the efficient candidate as cytotoxic bioagent (antibreast cancer activity) against the cell lines MCF-7 and MDA-MB-468.
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In vivo Antitumor Studies of Dietary Compounds: Nanotech Combination Injection of Sarcoma 180 cancer cells subcutaneously into Balb/c mice was followed by 29 days observation, monitoring the mean body weight, tumor volume (Fig. 5.21) and tumor growth delay of all the experimental groups shown in (Table 5.5). The body weights of the control and treated mice were determined periodically to assess non-specific toxicity of test samples. The average body weights of the control and GNDHF, LUT and MSC (single and combination) treated mice did not differ significantly by one-way ANOVA suggesting that GNDHF, LUT and MSC administration did not cause weight loss. The mice in GNDHF, LUT and MSC treated group appeared healthy and did not show any other sign of non-specific toxicity, such as food and water withdrawal and impaired movement. Average tumor volume in the tumor control group and test samples treated group are depicted in (Table 5.5), and can be ordered as: Doxo reference < (GNDHF: LUT: MSC combination) < GNDHF < LUT < MSC < tumor control. Similar trend was also observed when tumor delay time in all the experimental groups has been studied. Overall, significant reduction in tumor volume and tumor delay time was found in (GNDHF: LUT: MSC) combination treated mice. Table 5.5: Combination effect of gold nanoparticle embedded 3,6-dihydroxyflavone with lutein and selenium methyl selenocysteine against standard doxorubicin on body weight, tumor volume and tumor delay time of Balb/c mice after induction of Sarcoma 180 cancer cells
Groups Body weight ±SD(gm)
Tumor Volume ± SD (mm3)
Tumor delay time (days)
Group I: NC 30.4±1.07 - -
Group II: TC 28.0±1.20 126.9±1.14 0
Group III: 2% DMSO 31.2±1.12 121.2±1.05 0
Group IV: DOXO (1 mg/Kg b.w) 25.6±1.17 004.0±1.15 7
Group V: GNDHF (8 μM; 5 mg/Kg b.w) 27.05±1.18 104.7±1.16 3
Group VI: LUT (1.5 μM; 5 mg/Kg b.w) 28.02±1.19 109.2±1.16 3
Group VII: MSC (10 μM; 5 mg/Kg b.w) 28.27±1.09 112.1±1.15 3
Group VIII: GNDHF+LUT+MSC (8/1.5/10 μM; 5 mg/Kg b.w) 29.17±1.16 091.0±1.19 5
Each value is mean±SD (n=6). p > 0.01 vs. tumor control, p < 0.05 vs. normal control. NC: Normal Control, TC: Tumor Control, GNDHF: gold nanoparticle embedded 3,6-dihydroxyflavone, LUT: lutein, MSC: Selenium methyl selenocysteine, DMSO: Dimethyl sulphoxide, DOXO: Doxorubicin
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Fig. 5.21: Tumors in female Balb/c mice a) Tumor control b) DOXO (1mg/Kg b.w.) c)GNDHF (8 µM; 5 mg/Kg b.w.) d)LUT (1.5 µM; 5 mg/Kg b.w.) e)MSC (10 µM; 5 mg/Kg b.w.) & f) GNDHF: LUT : MSC (8/1.5/10 μM; 5 mg/Kg b.w.) treated at 29th day
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Antigenotoxic studies The effect of the test samples on percent of chromosomal aberration was measured in terms of chromatid breaks, centric rings, acrocentric association, acentric fragments, intracalary deletion, minutes and total abnormal metaphases (Fig. 5.23). Percent of aberrant metaphase in various groups were in the range as follows: Cancerous control (77%) > Doxo treated (69 %) > MSC (10 μM): 57 % > LUT (1.5 μM): 45 % > GNDHF (8 μM): 31 %> GNDHF: LUT: MSC combination (8/1.5/10 μM): 17 % (Table 5.6). Table 5.6: Combination effect of gold nanoparticle embedded 3,6-dihydroxyflavone with lutein and selenium methyl selenocysteine against standard doxorubicin on percent of aberrant metaphases in the bone marrow of Balb/c mice after induction of Sarcoma 180 cancer cells
S No
Group Chromosome aberration (%)
CB CR FR ACA ICD AC MIN
1 NC 12.50 ±1.14 0 ± 0.0 25.00 ± 1.13 12.50 ± 1.02 0.0 ± 0.0 12.50 ± 1.05 0.0 ± 0.0
2 TA 12.59 ±1.85 8.14 ± 1.85 17.03 ± 2.09 38.51 ± 2.14 11.85 ± 1.98 9.62 ± 1.65 2.96 ± 1.74
3 2%DMSO 21.93 ±1.78 7.74 ± 1.98 9.03 ± 2.41 29.03 ± 2.10 9.67 ± 2.14 9.67 ± 1.65 5.64 ± 1.57
4 DOXO(1mg/Kg b.w) 25.11 ±1.85 7.44 ± 1.65 8.37 ± 1.84 31.62 ± 1.78 9.30 ± .98 10.23 ± 1.56 5.11 ± 1.64
5 GNDHF(5mg/Kg b.w) 12.63 ±0.94 5.26 ± 0.68 7.36 ± 0.84 16.84 ± 0.98 6.31 ±0.75 10.52 ± 0.72 5.26 ± 0.86
6 LUT (5mg/Kg b.w)) 14.14 ±0.68 5.05 ± 0.92 7.07 ± 0.84 18.18 ± 0.65 6.06 ±0.94 11.11 ± 0.81 5.05 ± 0.75
7 MSC (5mg/Kg b.w) 14.70 ±1.78 3.92 ± 1.58 7.84 ± 1.46 19.60 ± 1.34 7.84 ±1.52 12.74 ± 1.64 4.9 ± 1.74
8 GNDHF+LUT+MSC (5mg/Kg b.w)
7.69 ±1.78 4.32 ± 1.95 6.69 ± 2.1 15.38 ± 2.15 5.98 ±2.08 5.76 ± 1.80 3.84 ± 1.71
Each value is mean±SD (n=6). p > 0.01 vs. tumor control, p < 0.05 vs. normal control. NC: Normal Control, TC: Tumor Control, GNDHF: gold nanoparticle embedded 3,6-dihydroxyflavone, LUT: lutein, MSC: Selenium methyl selenocysteine, DMSO: Dimethyl sulphoxide, DOXO: Doxorubicin, CB: Chromatid Breaks, CR: Chromatid Rings, FR: Fragments, ACA: Acrocentric Association, ICD: Intercalary Deletion, AC: Acentric Association, MIN: Minutes The combination of GNDHF: LUT: MSC exhibited maximum reduction in all the chromosomal aberrations studied in bone marrow cells compared to tumor control and reference doxorubicin. Most chromosomal aberrations are deleterious and result in cell death. However, some types (reciprocal translocations, small deletions, and inversions) can lead to alter gene function(s) without accompanying loss in cell viability [Dkhil et al. 2011]. Alternation in gene function occurring as a
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result of several different kinds of cancer [Mitelman et al. 1983 and Yoshida et al. 2008], indicating the probable involvement of chromosomal aberrations in carcinogenesis. The significant chromosomal aberrations observed were mainly in the form of chromatid breakages (deletion, gap, break and fragments). Damage to chromosomes after the G1 stage of the cell cycle causes chromatid lesions only, and results in chromatid breakage [Moore et al. 1981 and Febrer et al. 2008]. Therefore, it may be possible to conclude that combination of GNDHF: LUT: MSC exerts its clastogenic effect after the G1 stage of the cell cycle. Antimutagenic studies The effect of various treatments on Sarcoma 180 cancer induced mice was determined in terms of micronucleated polychromatic erythrocytes (PCEs) and normochromatic erythrocytes (NCEs) per 1000 cells. Percent of PCE and NCE in various groups were in the range as follows: cancerous control (87.7 - 94.5 %), Doxo treated (37.1 -40.6 %), GNDHF (8 μM): 16.8-20.2 %, LUT (1.5 μM): 16.9-19.9 %, MSC (10 μM): 15.4-19.1 %, and GNDHF: LUT: MSC (8/1.5/10 μM): 5.1-5.9% (Fig. 5.22). The GNDHF: LUT: MSC combination treated mice was significantly (p > 0.01) reduced the micronuclei in PCEs and NCEs comparable with tumor control and doxorubicin treated group. This reduction could be due to either direct cytotoxicity or micronuclei formation and heavy DNA damages leading to cell death or apoptosis [Krishna et al. 2000, Valette et al. 2002, Ouanes et al. 2003 and Shahrim et al. 2006]. Nevertheless, the mean number of PCEs frequencies in the positive control receiving doxorubicin showed significant increase. These findings demonstrate the validity of the experiment and the sensitivity of the animal strains to a clastogenic agent. The combination of GNDHF: LUT: MSC reduces the frequency of micronuclei per polychromatic (PCEs) and normochromatic erythrocytes (NCEs) compared to standard drug and tumor control group.
Each value is mean±SD (n=6). p > 0.01 vs. tumor control, p < 0.05 vs. normal control. NC: Normal Control, TC: Tumor Control, DOXO treated (1mg/Kg b.w.), GNDHF (8µM; 5mg/Kg b.w.), LUT (1.5µM; 5mg/Kg b.w.), MSC (10µM; 5 mg/Kg b.w.), COMB (GLM) - GNDHF:LUT:MSC (8/1.5/10 µM; 5 mg/Kg b.w.).
Fig. 5.22: Combination effect of gold nanoparticle embedded 3,6-dihydroxyflavone with lutein and selenium methyl selenocysteine against doxorubicin on percentage Micronucleated polychromatic and Normochromatic erythrocytes in normal and cancer tumor bearing mice at 29th day
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Fig. 5.23: Chromosomal aberration in a) Normal control b) Tumor control c) DOXO treated (1mg/Kg b.w.) d) GNDHF (8 µM; 5 mg/Kg b.w.) e) LUT (1.5 µM; 5 mg/Kg b.w.) f) MSC (10 µM; 5 mg/Kg b.w.) & g) GNDHF:LUT:MSC (8/1.5/10 µM; 5 mg/Kg b.w.)
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Histopathological studies: Tumor micro vessel density evaluation Accumulating evidences [Folkman et al. 1999, Saaristo et al. 2000, Folkman et al. 2002 and Chekenya et al. 2002] demonstrate that tumor growth and lethality are dependent on angiogenesis. An observation of histological slides shown in (Fig. 5.24) exhibits the decrease in tumor growth in mice by the GNDHF, LUT and MSC treatment which may be attributed to decreased host angiogenesis. A marked and dense microvasculature was observed in the control tumors. Tumors treated with GNDHF: LUT: MSC combination (29.19 %) and doxorubicin (23.2 %) had significantly fewer micro-vessels compared with the GNDHF (41.23 %), LUT (43.4 %), MSC (38.31 %) and tumor control (58.2 %). Angiogenesis inhibition observed with GNDHF: LUT: MSC combination treatment is indicative of drug accumulation in the tumor and decreased tumor micro-vessel density which is further associated to the suppression of angiogenic vascularization, inhibited tumor cell proliferation and increased tumor cell apoptosis.
Fig. 5.24: Tumor histology of a) Tumor control b) DOXO treated (1 mg/Kg b.w.) c)GNDHF (5 mg/Kg b.w.) d)LUT (5 mg/Kg b.w.) e)MSC (5 mg/Kg b.w.) & f) GNDHF: LUT: MSC (8/1.5/10 μM; 5 mg/Kg b.w.)
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Synergism and Nanotech Enhancement in Anticancer Bioefficacy of Dietary Compounds Due to the complex pharmacological actions, the mechanism of the enhanced bioefficacy of combination and nanotech enforcement of dietary products is not easy to predict. Based on our experimental findings and pertinent information available on the topic, attempt has been made to synthesize the hypothesis to explain the observed findings. The fate of a drug after administration in vivo is determined by a combination of several processes, such as distribution, metabolism and elimination when given intravenously or absorption, distribution, metabolism and elimination when an extravascular route is used. The result depends mainly on the physicochemical properties of the drug and therefore on its chemical structure. The in vivo transport of an anticancer drug will be governed by the physiological (pressure), physicochemical (composition, structure) properties of the interstitium and the physicochemical properties of the molecule itself (size, configuration, charge, hydrophobicity). Physiological barriers at the tumor level (poorly vascularized tumor regions, acidic environment, high interstitial pressure and low micro vascular pressure) as well as at the cellular level (altered activity of specific enzyme systems, altered apoptosis regulation and transport based mechanisms) and in the body (distribution, biotransformation and clearance of anticancer agent) must be overcome to deliver anticancer agents to tumor cells [Brigger et al. 2002]. 3,6-dihydroxyflavone has strong cytotoxicity and induce apoptosis in breast cancer cells [Chang et al. 2008] through miR-21 and miR-34a associated way [Chang et al. 2012]. MiR-34a expression is sufficient to induce apoptosis through accepted p53-dependent mechanism [Pang et al. 2010]. The main cellular event, sensitized by selenium methyl selenocysteine, is the tumor suppression via p53 mechanism [Lanfear et al. 1994]. Lutein, being antioxidant has potential to quench free radicals [steele et al. 2000] and thus promotes significant inhibition of malignant cell growth. Our in vitro experimental findings that flavonoid together with selenium methyl selenocysteine and lutein synergistically enhance cytotoxicity in human breast cancer cell lines, are in support of the above facts. The combination has an ability to penetrate the cell membrane and internalize within the cellular matrix. The novel combination is with broad-spectrum anticancer activity and triggered dose-dependent apoptosis in human breast cancer MCF-7 and MDAMB-468 cell lines. Therefore, we hypothesize [Medhe et. al. 2013] mitochondria and death receptor-mediated apoptosis p53 pathway to explain observed combination effects of (dietary flavonoid: 3,6-dihydroxy flavone, sensitizer: selenium methyl selenocysteine antioxidant: lutein) as follows in (Fig. 5.25). Nanoparticles can target tumor cells by permeation and retention effect, imposed by angiogenic vessels and improper lymphatic flow [Ruoslahti et al. 2010]. Therefore, the nanoparticles can accumulate selectively inside the cancerous cells at higher concentrations than the normal cells. The accumulation mechanism of intravenously injected nanoparticles in cancer tissues relies on a passive diffusion or convection across the hyper permeable tumor vasculature. Additional retention of the colloidal particles in the tumor inter-stitium is due to the compromised clearance via lymphatic’s. The release of the drug content inside the tumoral interstitium may be achieved by controlling the nanoparticle structure, activity of compound used for nanoparticle synthesis and the way by which the drug is associated with the carrier (adsorption or encapsulation) [Serpe et al. 2004]. This so-called enhanced permeability and retention effect results in an important intra tumoral drug accumulation that is even higher than that observed in plasma and other tissues [Maeda et al. 2001].
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Fig. 5.25: Proposed mechanism of three dietary compounds for anticancer activity
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Colloidal nanoparticles incorporating anticancer agents can overcome resistances to drug action, increasing the selectivity of drugs towards cancer cells and reducing their toxicity towards normal cells. Nanoparticles loaded with anticancer agents can successfully increase drug concentration in cancer tissues and also act at cellular levels, enhancing antitumor efficacy. They can be endocytosed or phagocytosed by cells, with resulting cell internalization of the drug. Nanoparticles, likely, have a better safety profile than free anticancer agents when acting on normal tissue. Nanoparticles can be targeted to the particulate region of capillary endothelium, to concentrate the drug within a particular organ and allow it to diffuse from the carrier to the target tissue. However, the contribution of conventional nanoparticles to enhance anticancer drugs efficacy is limited to targeting tumors at the organs of the mononuclear phagocytes system organ. Owing to their very short circulation time (the mean half-life of conventional nanoparticles is 3–5 min after intravenous administration) addressing anticancer drug-loaded nanoparticles to other tumoral tissues is not enough. The contribution of conventional nanoparticles to enhance anticancer drugs efficacy is limited to targeting tumors at the organs of the mononuclear phagocytes system organ. Moreover, penetration of such a carrier system across the tumoral endothelium would be minimum, leading to subtherapeutic concentrations of the drug near the cancer cells. This biodistribution can be of benefit for the chemotherapeutic treatment of mononuclear phagocytes system localized tumors. Nanoparticles conjugated with an antibody against a specific tumor antigen have been developed to obtain selective drug delivery systems for the treatment of tumors expressing a specific tumor antigen. Folinic acid has some advantages over transferring or antibodies as a ligand for long-circulating carriers because it is a much smaller molecule that is unlikely to interact with opsonins and can be coupled easily to a poly (ethylene glycol) (PEG) chain without loss of receptor-binding activity. This targeting strategy has also been applied to long-circulating nanoparticles prepared from a cyanoacrylate-based polymer. Folate grafted to PEG cyanoacrylate nanoparticles has a ten-fold higher apparent Conventional Chemotherapeutic Drug Nanoparticles for Cancer Treatment affinity for the folate-binding protein than the free folate. Indeed, the particles represent a multivalent form of the ligand folic acid and folate receptors are often disposed in clusters. Thus conjugated nanoparticles could display a stronger interaction with the surface of malignant cells [Stella et al. 2000].