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ORIGINAL ARTICLE
Year : 2020  |  Volume : 16  |  Issue : 6  |  Page : 1506-1516

Pro-apoptotic and anti-neoplastic impact of luteolin on solid Ehrlich carcinoma.bearing mice exposed to gamma radiation


Department of Radiation Biology, National Centre for Radiation Research and Technology, Atomic Energy Authority, Cairo, Egypt

Date of Submission13-Dec-2019
Date of Decision08-Apr-2020
Date of Acceptance18-Jun-2020
Date of Web Publication18-Dec-2020

Correspondence Address:
Khaled Shaaban Azab
Department of Radiation Biology, Radiation Research Division National Centre for Radiation Research and Technology, Atomic Energy Authority, Cairo
Egypt
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/jcrt.JCRT_1116_19

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 > Abstract 


Background: Cancer remains a major health issue and the second foremost root of morbidity worldwide behind cardiovascular diseases. Apoptosis had linked to the eradication of possibly malignant cells, hyperplasia, and tumor progression.
Objective: The present study is an endeavor to evaluate the influence of luteolin, a modifier to apoptotic regulator on the tumor growth and the tumor cell sensitivity to ionizing radiation in Ehrlich solid tumor-bearing mice (E).
Materials and Methods: Mice were immunized with Ehrlich carcinoma cells (2.5 × 106 cells/mouse), received consecutive equal doses of luteolin, 1.25 mg/mouse/day and exposed to 6.5 Gy of whole-body gamma irradiation (0.46 Gy/min).
Results: Luteolin markedly suppresses the developing of tumor in E mice group or mice which bearing tumor with exposure to radiation (E + R group) which has collimated with significant inhibition in protein expression of inflammatory molecules cyclooxygenase 2 and the concentration of (prostaglandin E2). Also, matrix metalloproteinase-2, 9 proteins concentrations significantly decreased with amelioration in apoptotic regulators (Caspase-3 and Granzyme-B activities). The expression of signal transducer and activator of transcription (STAT) and tumor necrosis factor-alpha genes meliorated significantly. Besides, the level of oxidant/antioxidant (reduced glutathione/malondialdehyde) markedly improved. Obviously, the most reduction of changes in all measured parameters has appeared in tumor bearing mice, injected with luteolin and exposed to gamma radiation (E + Luteolin + R group).
Conclusion: It could be suggested that luteolin has a potential beneficial effect against cancer. This could be due to its ability on the induction of apoptosis, inhibition of inflammatory response, downregulation of angiogenic factors as well as increase sensitivity of tumor cells to gamma radiation.

Keywords: Caspase 3, granzyme B, reduced glutathione, inflammatory markers, ionizing radiation, luteolin, malondialdehyde, matrix metalloproteinases, signal transducer and activator of transcription-3, tumor


How to cite this article:
Azab KS, El Fatih NM, El Tawill G, El Bakary NM. Pro-apoptotic and anti-neoplastic impact of luteolin on solid Ehrlich carcinoma.bearing mice exposed to gamma radiation. J Can Res Ther 2020;16:1506-16

How to cite this URL:
Azab KS, El Fatih NM, El Tawill G, El Bakary NM. Pro-apoptotic and anti-neoplastic impact of luteolin on solid Ehrlich carcinoma.bearing mice exposed to gamma radiation. J Can Res Ther [serial online] 2020 [cited 2021 Nov 27];16:1506-16. Available from: https://www.cancerjournal.net/text.asp?2020/16/6/1506/303886




 > Introduction Top


Cancer is a typical growth of cells' fallouts from the accumulation of inherited and/or acquired genetic mutations causing oncogene activation or inactivation. It leads to deregulation of the normal cellular program for cell division, cell differentiation, deregulated balance of cell proliferation and cell death, and finally developing a cell population which can conquer tissues and metastasize to detached sites, causing substantial illness and demise of the host.[1]

Nearly 25% of the universally malignancies are associated by long-lasting inflammation, where changed levels of pro-inflammatory and pro-angiogenic factors were detected in diverse cancers. Activation of key inducers of pro-inflammatory factors, including the transcription factors STAT3 and nuclear factor kappa beta (NF-β), leads to enhancement of cell proliferation, apoptotic evasion, invasion, and metastasis, as well as angiogenesis.[2]

The first barrier for an invading epithelial tumor is the basement membrane. Degradation of the basement membrane and the extracellular matrix (ECM) in the tissue of surrounding by tumor is an essential process in invasion and metastasis.[3] Whereas, the matrix metalloproteinases (MMPs), a potent proteolytic enzyme, play key roles in membrane degradation process, leading to alterations in tissue architecture, a hallmark of the malignant disease.[4]

Apoptosis is a molecular event by which cell termination occurs after performing its physiological roles. It plays an essential role in growth, advance immune response, and dell jobless or abnormal cells in organism. Apoptosis conserves tissue homeostasis through intricate process prompted by a variety of provocations, including cytokines, hormones, toxic insults, and viruses.[5]

Mammary adenocarcinoma (Ehrlich ascites carcinoma [EAC]), adapted to ascites form, is widely used in cancer studies as an experimental tumor to explore the in vivo anticancer activity of different natural and synthetic chemical compounds. It is characterized by great transplantable capacity, high rate of shorter life span, absolute malignancy, and absence of tumor transplantation antigen.[6]

The ionizing radiation (IR) like gamma rays and that employ largely in different cancer researchers have the ability to activate both pro- and anti-proliferation cell signals leading to a case of an inequality in cell fate decision which ended by cell death.[7]

Recently, the dietary procedures to fight lingering diseases including cancer, recommend dependence on consumption of plants that are rich in antioxidants, including carotenoids and flavonoids.[2] Flavonoids exhibit various beneficial properties in the several disease conditions, including cardiovascular disease, neurodegenerative disorders, and cancer.[8] This study is interested in luteolin (3', 4', 5, 7-tetrahydroxyfavone), a widely distributed naturally occurring flavonoid found in many medicinal plants as well as fruits and vegetables. Epidemiological signals advocate that luteolin may have a significant capacity to diminish hazard of acute myocardial infarction. Luteolin holds a diversity of biological and pharmacological activities, such as antioxidant, anti-inflammatory, antimicrobial, anti-allergic, antiplatelet, and a number of other activities.[9]

The fallouts of the combining of exposure to gamma radiation and chemical agent in order to affect the cell signals in the microenvironment of tumor cells might determine greatly the benefit of using this agent. Therefore, this study is oriented to assess the antitumor luteolin work modality as a pro-apoptotic molecule in mice bearing Ehrlich solid tumor with exposure to IR. Experimental results emerged from in vitro assessment of EAC response to luteolin or/and radiation is concerned with the cytotoxic capacity of the treatments, only.


 > Materials and Methods Top


Experimental animals

In the present study, we have used female Swiss albino mice (20–25 g) attained from the National Cancer Institute (NCI) (Cairo University, Giza, Egypt). Animals were housed in plastic cages and maintained under standard conditions of temperature, humidity, and 12 h light/dark cycles. They were provided with a commercial standard pellet diet and water ad libitum. Mice were left for an acclimatization period of 7 days before the start of the experiment. All animal procedures were performed agreeing to the rules for Care and Usage of Laboratory Animals and approved institutional guidelines were followed.

Chemicals

Luteolin (3', 4', 5, 7-tetrahydroxyfavone) was purchased from Sigma-Aldrich (Gillingham, UK). Luteolin was liquefied in dimethyl sulfoxide; DMSO (1:10 v/v sterilized saline) and each mouse conventional a daily i.p. dose of 50 mg/kg body weight for 30 successive days.[10]

Radiation facility

Whole-body γ-irradiation of mice was performed with a Canadian gamma cell-40, (137Cs) housed in National Center for Radiation Research and Technology (NCRRT), Cairo, Egypt at a dose rate of 0.46 Gy/min. 137Cs is accommodated inside a cylindrical stainless-steel double capsule. First, the animals were categorized. Second, the animals were placed in a canister with good ventilation. Lastly, animals were exposed to whole-body 6.5 Gy gamma radiation.

Tumor transplantation

In the current study, cell line of EAC was used as a prototypical of solid carcinoma by inoculation in the right thigh of albino mice. The parent line was supplied as a gift from the Egyptian NCI, Cairo University. Human breast cancer is the source of EAC cells upon adapted to raise in female Swiss albino mice. The cell line of EAC was preserved by intraperitoneal injection (i.p.) of 2.5 million cells per animal. Bright line hemocytometer was used to count the EAC before i.p. injection, and the dilution was completed using physiological sterile saline solution. In order to develop Ehrlich solid tumor (E) in thigh, 0.2 ml of EAC cells (2.5 × 106 cells/mouse) was inoculated subcutaneously (s.c.) in the right thigh of the lower limb of the female mouse.[11],[12]

Animals categorization

In this study, we used 100 mice weighing about 20–25 g. All the experiments were conducted under national research center guidelines for the usage and attention for laboratory animals and were approved by an independent ethics committee of the NCRRT.

The animals were categorized into five equal groups of twenty mice each as follows:

  • Group (1): Control (C): Mice neither treated nor irradiated
  • Group (2): (E): Mice bearing solid Ehrlich tumor
  • Group (3): (E + Luteolin): Mice bearing solid Ehrlich tumor were injected i.p. with luteolin for 30 uninterrupted days at 1 day after EAC inoculation
  • Group (4): (E + R): Mice bearing solid Ehrlich tumor were subjected to 6.5 Gy whole body γ-irradiation (acute dose) at 1 day after EAC inoculation
  • Group (5): (E+ Luteolin +R): Mice bearing solid Ehrlich tumor were injected with luteolin as in group 3 along with a single whole-body γ-irradiation at a dose level of 6.5 Gy 30 min after the first injection of luteolin.


Tumor volume monitoring

Tumor volume was measured at different time intervals during the experimental period by a Vernier caliper on the 7th, 15th, 22th, and 30th days from EAC inoculation during the experimental period. The size of the solid tumor was calculated using formula (A × B2 × 0:52), where A and B are the longest and the shortest diameter of the tumor, respectively.[13]

Mice were sacrificed at the end of the experiment. The skeletal muscle (normal control), tumor tissues, and blood serum were collected for biochemical investigations.

Biochemical assay

RNA extraction and real-time measurable polymerase chain reaction

Total cellular RNA was extracted from frozen right thigh muscles and solid EC tumors consuming the RNeasy® Mini kit according to the manufacturer's instructions (Qiagen, Hilden, Germany). The purity and concentration of total RNA were assessed by measuring absorbance at 260 and 280 nm, respectively, in a spectrophotometer (Nano Drop 2000; Thermo Fisher Scientific, USA). First-strand complementary DNA (cDNA) was synthesized using Thermo Scientific™ RevertAid™ First-Strand cDNA Synthesis Kit (Fermentas, Thermo Fisher Scientific Inc, Runcorn, UK). Real-time polymerase chain reaction (PCR) amplification and analysis were performed in an optical 96-well plate in ABI PRISM 7500 Fast Sequence Detection System Thermal Cycler (Applied Biosystems, Foster City, CA, USA) using Power SYBR® Green PCR Master Mix (Applied Biosystems). The amplification protocol consisted of 40 cycles (denaturation at 95°C for 15 s, annealing at 55°C for 20 s, and extension at 72°C for 20 s). The primer used for STAT3, tumor necrosis factor-alpha (TNF-α), and glyceraldehyde-3-phosphate dehydrogenase (endogenous reference gene) is represented in [Table 1]. The relative expression of selected genes was determined by the ΔΔCT method.[14]
Table 1: STAT3, tumor necrosis factor-α, and glyceraldehyde-3-phosphate dehydrogenase primers for real-time polymerase chain reaction

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Western blot analysis of signal transducer and activator of transcription-3, P-signal transducer and activator of transcription-3

A section of tissue was homogenized with RIPA buffer containing 5 mol·L–1NaCl, 1 mmol·L–1 phenylmethylsulfonyl fluoride, 10% deoxycholic acid, 10% sodium dodecyl sulfate (SDS), and 1 mol­L–1Tris (pH 8.6). The tissue lysate was centrifuged at Xg for 20 min at 4°C. The lysate was then collected and protein concentration was determined with a BCA protein assay kit (Thermo Fisher Scientific). An aliquot of 7.5 g protein of each sample was denatured, then each sample was loaded on 8% SDS – polyacrylamide gels for separation by electrophoresis and then relocatedon nitrocellulose membrane (Amersham Bioscience, Piscataway, NJ, USA) using a semidry transfer apparatus (Bio-Rad, Hercules, Calif.). The membranes were incubated at 4°C overnight with 5% milk blocking buffer containing 10 mmol·L–1Tris–HCl (pH 7.4), 150 mmol·L–1NaCl, and Tris-buffered saline with 0.05% Tween-20 (TBST). The membranes were then washed with TBST and incubated on a roller shaker at 4°C overnight with a 1:1000 dilution of antibodies anti-STAT-3 (total and phosphorylated; Thermo Fisher Scientific). The filters were washed and subsequently probed with horseradish-peroxidase-conjugated goat anti-mouse immunoglobulin (Amersham. Life Science). Chemiluminescence detection was performed with the Amersham detection kit, according to the manufacturer's protocols, and exposed to X-ray film. The quantity of studied protein was measured by densitometric analysis of the autoradiograms by means of a scanning laser densitometer (Biomed Instruments). Results were normalized against β-actin protein expression (as the housekeeping protein).[15]

Gelatin zymography for matrix metalloproteinase 2, 9 detections

The presence and activity of specific MMP species (MMP-2 and 9) were initially detected in the serum using substrate (gelatin) gel electrophoresis.[16] A buffer of 4% SDS, 0.15 mol/L Tris (pH 6.8), 20% glycerol and 0.5% (w/v) bromophenol blue was added to the serum sample. Serum samples mixed with buffer were directly added to 10% SDS–acrylamide gel comprising 0.1% (w/v) gelatin (Sigma) and separated by running on a mini-gel apparatus at 15 mA/gel, and then gels were gently astounded in a 2.5% Triton X-100 solution for 30 min at room temperature (RT). Gels were then incubated overnight at 37°C in substrate buffer containing 50 mmol/L Tris–HCl (pH 8), 5 mmol/L CaCl2 and 0.02% NaN3. Gels were subsequently stained for 30 min in 0.5% Coomassie Blue R-250 dissolved in a 1:3:6 solutions of acetic acid, methanol, and water. The gel was recorded for the presence/absence MMP activity by a blinded assessor and photographed. MMP-2 and MMP-9 could be detected on the SDS gel as transparent bands.

Apoptotic parameters (enzyme-linked immune sorbent assay detection)

Enzyme-linked immune sorbent assay (ELISA) for levels of Granzyme-B and Caspase-3 were determined by using ELISA Kits (Rand D systems) according to the manufacturer's instructions on the supernatants of sample tissue homogenates. In brief, microplates were coated with 100 μl/well of capture antibody, and then they were incubated overnight at 4°C. After washes, the plates were blocked with assay diluent at RT for 1 h. One hundred microliters of a serum sample were added to each well of the plate, followed by incubation for 2hat RT. The working detector was added into each well, and the plate was incubated for an additional 1 h at RT before the addition of the substrate solution. The reaction was stopped by adding stop solution. The absorbance was read using an ELISA reader. The concentrations were considered from the standard curve according to the instructions in the protocol.

Oxidant-antioxidant status

Lipid peroxidation in thigh muscle and tumor tissue was measured by thiobarbituric acid assay, which is based on malondialdehyde (MDA) reaction with thiobarbituric acid forming thiobarbituric acid reactive substances (TBARS), a pink-colored complex exhibiting a maximum absorption at 532 nm.[17] The reduced glutathione (GSH) content in tissue was determined photometrically at 412 nm using 5, 5-dithiobis-2-nitrobenzoic acid.[18]

Inflammatory marker

Cyclooxygenase 2 (COX-2) enzyme activities were detected spectrophotometrically in tissue extract. Cyclooxygenases catalyze the producing of prostaglandins E (PGE) from arachidonic acid. The COX element changes arachidonic acid to prostaglandinG2 (PGG2) whereas the peroxidase component reduces the PGG2 to the corresponding alcohol, PGH2. In this kit, the TMPD is the reducing agent, TMPD gets oxidized, so electrons flow from the TMPD to PGG2 and the appearance of the oxidized TMPD is observed at 590 nm. The rate of increase in absorbance was taken for calculating the activity of the enzyme.[19]

Prostaglandin E2 (PGE2) concentration was measured by using several steps standard assay PGE2 in vitro competitive ELISA kit for the precise quantifiable measurement of PGE2 in tissue homogenate. A mouse IgG antibody has been pre-coated onto 96-well plates.

Standards or test samples are added to the wells, along with alkaline phosphatase (AP) Conjugated-PGE2 antibody.

Afterward incubation para-Triphenylphosphate (pNpp) substrate is added after washing away the excess reagents and is catalyzed by AP to produce a yellow color. The intensity of the yellow coloration is inversely proportional to the amount of PGE2 captured in the plate.

Statistics

Statistical analysis was performed by one-way analysis of variance followed by Duncan's multiple range test by using the Statistical Package of social science version 20.0 for windows (IBM SPSS statistics). P = 0.05 was considered as a level of significance.


 > Results Top


Impact of luteolin and/or γ-irradiation exposure on tumor volume

Using of luteolin according to the present study protocol resulted in a noticeable retarding in tumor growth in E +luteolin group mice and E+ luteolin +R group mice when compared with E mice at the selected time intervals. Besides, the exposure to whole-body γ-irradiation produce a maximum growth repression, especially on the 30th day [Figure 1].
Figure 1: Impact of luteolin on tumor proliferation in different animal groups. E: Mice inoculated with Ehrlich Ascites Carcinoma R: Mice irradiated with gamma radiation. E + luteolin: Mice inoculated with Ehrlich Ascites Carcinoma and treated with luteoliin. Each value represents the mean of 6 records ± S.E

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Impact of luteolin and/or γ-irradiation exposure on relative expression of signal transducer and activator of transcription-3 and tumor necrosis factor-alpha

Significant upregulation was detected in the expression of STAT3 and TNF-α gene in solid E tumors, compared to non-E-bearing mice (Control). In contrast, treatment of E-bearing mice with luteolin resulted in a significant reduction in the expression of STAT3 and TNF-α gene, compared to untreated E-bearing mice. Further, exposure of E-bearing mice to γ-irradiation either alone or in combination with Luteolin produced a more pronounced inhibition of STAT3 and TNF-α gene expression in tumor cells [Figure 2]a and [Figure 2]b.
Figure 2: (a and b) Relative mRNA expression of signal transducer and activator of transcription-3 and tumor necrosis factor-alpha in the right thigh muscle (control) or solid E tumor tissues in different groups. E: Mice inoculated with Ehrlich Ascites Carcinoma R: Mice irradiated with gamma radiation. E + luteolin: mice inoculated with Ehrlich Ascites Carcinoma and treated with luteoliin. Each value represents the mean of 6 records ± S. E. Disparate letters (a and b) over histogram column represents the significant differences between animal groups at P < 0.05

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Impact of luteolin and/or γ-irradiation exposure on signal transducer and activator of transcription-3 and signal transducer and activator of transcription-3 proteins

A significant increase in protein expression of STAT-3 and P-STAT-3 has been observed in the E group compared to the control group. On the other hand, treating of bearing tumor mice by luteolin resulted in a significant reduction for STAT-3 and P-STAT-3 protein expression, compared to E mice group. Further, exposure of tumor bearing mice to radiation combining with luteolin produced more pronounced reticence in the protein expression for STAT-3 and P-STAT-3 [Figure 3]a and [Figure 3]b.
Figure 3: (a and b) Protein expression of (a) signal transducer and activator of transcription-3 and (b) P-STAT-3 (Western blot analysis in the right thigh muscle (control) or solid EC tumor tissues in different groups. E: Mice inoculated with Ehrlich Ascites Carcinoma R: Mice irradiated with gamma radiation. E + luteolin: Mice inoculated with Ehrlich ascites carcinoma and treated with luteoliin. Each value represents the mean of 6 records ± S. E. Disparate letters (a and b,.) over the histogram columns represents the significant differences between animal groups at P < 0.05

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Impact of luteolin and/or γ-irradiation exposure on reduced glutathione and thiobarbituric acid reactive substances concentration (cellular redox tone)

The results obtained illustrated in [Figure 4]a and [Figure 4]b displayed a significant recovery in normal tissue redox tone upon administration of luteolin or/and radiation. The concentration of glutathione was significantly decreased (P < 0.05) in the tumor cells of mice bearing solid tumor (E+ luteolin, E + R, and E+ luteolin +R) when compared with untreated tumor-bearing mice (E). In addition, the level of liver MDA measured as TBARS concentration was markedly increased whereas the utmost changes were recorded in E+ luteolin +R mice group.
Figure 4: (a and b) Impact of luteolin and/or γ-irradiation exposure on tumor cells GSH (μmol/g tissue) (a) and MDA (nmol/g tissue) (b) concentrations in different mice groups in different animal groups. E: Mice inoculated with Ehrlich Ascites Carcinoma R: Mice irradiated with gamma radiation. E + luteolin: Mice inoculated with Ehrlich Ascites Carcinoma and treated with luteoliin. Each value represents the mean of 6 records ± S.E. Disparate letters (a and b,.) over the histogram columns represents the significant differences between animal groups at P < 0.05

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Impact of luteolin and/or γ-irradiation exposure on inflammatory markers

[Figure 5]a and [Figure 5]b elucidate the impact of luteolin or/and γ-radiation on inflammatory markers in mice tumor (COX2 activity and PGE2 concentration, respectively). The results obtained reveals a significant drop in COX2 activity and PGE2 concentration in tumor tissue of mice treated with luteolin alone or luteolin and γ-irradiation when compared with their equivalents in E mice. However, significant elevation was observed in mice of E+ R group. Drops in sharpness of changes was observed in E+ luteolin +R mice when compared the values of COX2 activity and PGE2 concentration with their values in E+ luteolin mice group.
Figure 5: (a and b) Impact of luteolin and/or γ-irradiation exposure on tumor cells cyclooxygenase 2 activity (U/mg tissue) and prostaglandin E2 concentration (Pg/mg tissue) in different mice groups in different animal groups. E: Mice inoculated with Ehrlich Ascites Carcinoma R: Mice irradiated with gamma radiation. E + luteolin: Mice inoculated with Ehrlich Ascites Carcinoma and treated with luteoliin. Each value represents the mean of 6 records ± S.E. Disparate letters (a and b,.) over the histogram columns represents the significant differences between animal groups at P < 0.05

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Impact of luteolin and/or γ-irradiation exposure on apoptotic regulators molecules Caspase3 and Granzyme B

The data demonstrate a significant upregulation (P < 0.05) of Caspase3 and Granzyme B enzymes activities in the tumor cells of E+ luteolin and E+ luteolin +R mice groups when compared to their corresponding values in E mice. The changes are more obvious in E+ luteolin +R mice group comparing to other tumor-bearing groups and was treated by luteolin or was exposed to gamma radiation [Figure 6]a and [Figure 6]b.
Figure 6: (a and b) Impact of luteolin and/or γ-irradiation exposure on tumor cells Caspase3 (Pg/mg tissue) and Granzyme B concentrations in different mice groups in different animal groups. E: Mice inoculated with Ehrlich Ascites Carcinoma R: Mice irradiated with gamma radiation. E + luteolin: Mice inoculated with Ehrlich Ascites Carcinoma and treated with luteoliin. Each value represents the mean of 6 records ± S. E. Disparate letters (a and b,.) over the histogram columns represents the significant differences between animal groups at P < 0.05

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Impact of luteolin and/or γ-irradiation exposure on angiogenic factors (Serum MMP-2 and MMP-9)

A significant increase (P < 0.05) in MMP2 and MMP9 concentration were observed in samples isolated from Ehrlich bearing mice. The pretreatment of mice bearing tumor by luteolin according to the present protocol induced remarkable reduction in MMP2 and MMP9 concentration as compared to their concentration in Ehrlich bearing mice. A more pronounced decrease in MMP2 and MMP9 concentration were recorded in E + R mice group comparing to E mice group [Figure 7] and [Table 2].
Figure 7: Gelatin zymography, the activity of serum matrix metalloproteinase-2, and matrix metalloproteinase-9 in different groups (n = 5). Lane M: Protein marker; and lanes 1–5, control, E, E + luteolin, E +R, E + luteolin +R, respectively

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Table 2: Serum matrix metalloproteinase-2, 9 activities in different animal groups

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 > Discussion Top


Although radiotherapy advocated as an effective strategy in the treatment of cancers, radiation resistance is considered as one of the major causes of radiotherapy failure and subsequent tumor reversion. Enhancing the responsiveness of tumors to radiation using radiosensitizing agents, is a hopeful tactic to increase the effectiveness of radiation therapy.[2] As an advance cancer therapeutic conventions, this study was commenced to gauge the antitumor activity of luteolin together with γ-radiation against solid EC tumors in female mice. The diversity of molecular marks was analyzed in order to explore the primary mechanisms by which luteolin repressed the growth of the solid tumor or/and enhanced the radiosensitivity of cancer cells to γ-irradiation.

Our data obtained reveals a substantial reduction in the tumor growth observed in murine (Solid tumor bearing mice) groups treated with luteolin alone or with radiation with advantage in case of combining the two treatments. This is accompanied by several alterations for tumorigenic mediators, pro-inflammatory cytokines, inflammatory markers, angiogenic markers as well as redox tone in tumor cells. Apparently, in solid tumor bearing mice (untreated) the STAT-3 as one of the major mediator of tumorigenesis suffer from significant increase either on the level of gene expression or on the level of STAT-3 and p-STAT3 protein expression. Also, there is a significant increase for expression of pro-inflammatory cytokine TNF-α gene. Further, the inflammatory markers COX2 and PGE2 have been upregulated in the time of decreases in the apoptotic mediators (caspase3 and Granzyme B) and increases in angiogenic markers (MMP 2 and 9). In parallel, several disturbances in redox tone for tumor cells have been recorded.

Tumor cells acquire specific functions necessary for tumor growth and dissemination. Our results pointed to the instigation of STAT-3 gene expression followed by an overproduction of STAT-3 and STAT3 proteins (PSTAT-3) which could be prompted to tumorigenesis and pave to tumor proliferation [Figures 1, 2a, b and 3a,b]. Huang et al., reported that STAT3 is mainly activated in malignant tumors and has important roles in multiple aspects of cancer aggressiveness.[20] The STAT3 signaling distributed in diverse cancers is an argument of convergence for activation of many protein kinases. In addition, the constitutive activation of STAT3 plays a critical role in tumor proliferation.[21] The increases in TNF-α mRNA [Figure 2b] could be contributed to the activation of the STAT-3. TNF-α is the significant cytokine for tumor advancement in mouse skin and very possibly for carcinogenesis in humans as well.[22] Evidence in our work supports the hypothesis that attribute TNF-α rise to induction of STAT-3. Suleman et al. exhibit the correlation between STAT-3 and TNF-α in many cases. They stated that during preconditioning by TNFα, activation of the STAT-3 is essential in ischemia whereas ischemic cardiac preconditioning involves both STAT-3 and Akt during reperfusion.[23] Also, STAT3 and NF-γB signaling molecules, which are known to be involved in the TNF-α pathway appear to be upregulated upon treating by TNF-α. TNF-α treatment increased STAT3 phosphorylation and induced the degradation of IγBα, which activates NF-γB.[24]

Spending of luteolinin the present study successfully ameliorates the changes in STAT3 gene expression and also the active form of PSTAT3 reported in Ehrlich solid tumor bearing mice. In addition, the expression of TNF-α was significantly downregulated compared to untreated EST mice. Song et al.[25] indicated that luteolin selectively executes STAT3 over activated in cancer cells that are frequently drug resistant. Luteolin expressively repressed STAT3 phosphorylation and diminished the expression of STAT3 targeting gene Mcl-1, Survivin, and Bcl-xl. Quieting of SHP-1, a protein tyrosine phosphatase, eradicated the inhibitory effect of luteolin on STAT3 and cell apoptosis, suggesting that SHP-1 is vital in luteolin-mediated cellular function. Moreover, this luteolin effect of STAT3 dephosphorylation by SHP-1 involved in HSP-90, which endangered STAT3 phosphorylation by forming HSP-90/STAT3 complex. Thus, luteolin subdued STAT3 activation through disorderly the binding of HSP-90 to STAT3, which encouraged its interaction to SHP-1, ending by the dephosphorylation of STAT3.

Also, luteolin inhibited TNFα-induced activation of nuclear transcription factor-kappa B (NF-γB), the main survival factor in TNFα signaling. As a result, luteolin suppressed the expression of NF-γB-targeted antiapoptotic genes, including A20 and cellular inhibitor of apoptosis protein-1.[26]

Moreover, our results display a significant elevation in inflammatory markers (COX2 activity and PGE2 concentration) in the group of E mice [Figure 5a and b]. A noteworthyexpanse of research specifies that the cyclooxygenase/PGE2 pathway of inflammation subsidizes to the development and progression of a diversity of cancers. The inflammatory response is a defense mechanism initiated due to tissue injury, of any nature, through the action of a variety of inflammatory mediators. Mediators of inflammation, such as cytokines, PGE, COX enzymes and MMPs could persuaded genetic and epigenetic changes, causing clampdown of tumor suppressor genes through DNA methylation and posttranslational modifications.[27]

However, the treatment of EST mice with luteolin alone or with radiation exposure displayed significant decreases in the inflammatory molecules (COX2 and PGE2) when compared with the E mice group [Figure 5a and b]. This could be attributed to the diversity of bioactive properties of this edible plant flavonoid. Harris et al., reported that luteolin inhibits multiple cancer-related signal transduction pathways, including MAPK, NF-γB, and Akt. It inhibited COX-2 protein expression and PGE2expression. The effects of luteolin are not due to toxicity and may be related to an inhibition of reactive oxygen species or effects on genes controlling cell morphology.[28]

COX2/PGE2 inflammatory mechanisms can direct to the development and progression of cancer. The up-regulated inflammatory pathway could be endorsed by the elevation of TNF-αexpression as described in the present work. The pro-inflammatory effects of TNF-α are principally due to its aptitude to activate NF-γB. Nearly all cell types, when exposed to TNF-α, activate NF-γB, leading to the expression of inflammatory genes. Over 400 genes have been identified that are regulated by NF-γB activation. These include COX-2, lipoxygenase-2, cell-adhesion molecules, anti-apoptotic proteins, inflammatory cytokines, chemokines, and inducible nitric oxide synthase. Also, the induction of genes encoding NF-γB-dependent anti-apoptotic molecules in the tumor microenvironment by TNF-α produced from tumor cells or inflammatory cells can promote tumor cell survival.[29]

COX-2 can be tempted by interferon gamma, and TNF-α, pro-inflammatory cytokines, leading to increased PGE2 production.[30] PGE2 contributes to tumor advancement through the reactive oxygen species (ROS) cohort, provoking oncogenic transcriptional factors, repressing anti-tumor immune responses,[31] and stimulating angiogenesis.[32]

Whereas, high GSH concentration and high MDA recorded in E mice group [Figure 4a and b] could participate largely in tumor growth. Increase antioxidant capacity (GSH) and the rise of peroxidation outcome (MDA) of tumor reflect the proximity of the tumor to survive. Cancer cells are extremely revised to elevated levels of ROS by triggering antioxidant pathways. Kim et al. conveyed that change in redox balance and deregulations of redox signaling are mutual marks of cancer development and resistance to treatment.[33] The rising of antioxidant capacity is associated with cancer cells in order to maintain ROS homeostasis and evade cancer cell death.[34] This could bring in an explanation of increased GSH level emanated in the present study in sync with increased of MDA; the action products of the abundant release of ROS in the tumor cell.

Targeting the ROS signaling pathways and redox mechanisms elaborate in cancer development are new probable approaches to stop cancer. Many anticancer drugs targeted the increase of ROS level in tumor cells which in turn triggers the cascades leads to death. Thus, the release of ROS in tumor cells in our results is considered aneradicate achievement induced by luteolin or/and radiation.

The data expanded in the present study drive us to adopt the consideration that luteolin's redox regulation activity is involved in its cellular effects and it is the key to evaluating its potential as an anticancer agent. Current dietary plans to fight chronic diseases, including cancers, recommend increasing intake of plant foods that are rich in antioxidants.[2] Because oxidative stresses are closely linked to mutagenesis and carcinogenesis, luteolin, as an antioxidant, might turn as a chemo preventive agent to guard cells from various forms of oxidant stresses and thus prevent tumor transformations. On the other hand, the pro-oxidant possessions of luteolin may be intricate in its aptitude to persuade tumor cell apoptosis, which is attained partly through direct oxidative impairment of DNA, RNA, and/or protein in the cells.[35]

Increased expression of MMPs noticed in our results [Figure 6] is predictive of tumor aggressiveness and metastasis. Recently, MMPs have been considered to be avital factor in prompting epithelial–mesenchymal transition (EMT). MMPs (a family of enzymes involved in matrix remodeling) represented as a key player in these processes, permitting tumor cells to change the ECM and to release cytokines, growth factors, and other cell-surface molecules, ultimately facilitating protease-dependent tumor progression.[36] It was described that MMP-9 expression is a communal phenomenon in pancreatic cancer, which can be controlled by a number of factors, including some inflammatory stimuli. TNF-α can upregulate it and increase the invasiveness of pancreatic cancer cells. Our results demonstrate the sync between increases in TNF-α and upregulation of MMPs. Furthermore, Xianmin et al. reported that COX-2/PGE2 may surge cell invasiveness and metastasis through MMP-9.[37]

Expression of MMP-2, 3, 9, and 28 in EMT by the loss of integral E-cadherin, augmented motility and invasiveness, downregulation of epithelial signs, and upregulation of mesenchymal indicators. Studies established that there is aparticipation of Rac signaling for cytoskeletal reordering and in arbitrating integrin signaling. Several reports have verified that ROS can be generated by integrin-Rac pathway, bringingout tumor cell migration and invasion. MMP-3–persuaded EMT seems to be intermediated via initiation of ROS and augmented appearance of the Rac1b. The ROS-quenching agent N-acetyl cysteine effectively subdued the MMP-3–induced EMT.[38]

Many studies had shown that cancer cells that were attributable to metastasis to distant organs prompt high levels of MMP-9.Therefore, MMP-9 is not only important in identifying invasion symptoms and diagnosis, but also a promising therapeutic target to prevent cancer invasion and metastasis.[39] Luteolin administration significantly down-regulate the increases in the protein expression of MMP2 and MMP9 [Figure 6] could be due to its inhibitory effect on the key points in the pathways leading to tumor metastases. Kumari et al. stated that inhibitors of ROS orMMP-3 could be useful in the setback of EMT or the killing of EMT-type cells or cancer stem cells resulting in decreased tumor aggressiveness.[34]

The disturbance in the angiogenic and apoptotic regulators leads to tumor proliferation and growth, which was clearly demonstrated by the increase in EC tumor volume along the experimental period in the undertaken study [Figures 1, 6 and 7]. Neovascularization enhances the ability of the tumor to grow and increases its invasiveness and metastatic ability.[40] In addition, the significant decline in the level of apoptotic molecules (caspase-3 and granzyme-B) in the solid EC tumors, contrasted to not EC-bearing mice [Figure 7 and Table 2] potentiates tumor proliferation. Caspase-mediated apoptosis is a major focus in the ground of cancer growth inhibition because activation of proteolytic caspase cascade is a critical component in the execution in apoptotic cell death.[41] The decreases in the Granzyme B could participate directly in the inhibition of Caspase3 and apoptotic process. Elmore stated thatGranzyme B can directly activate caspase-3. Thus, the contradictory signaling pathways are circumvented and there is direct initiation of the execution phase of apoptosis.[42] Granzyme B is the most powerful pro-apoptotic granzyme, as its ability to cleave target cell proteins mimics the caspases.[43] In addition, granzyme B can utilize the mitochondrial pathway for augmentation of the death signal by specific cleavage of pro-apoptotic protein and induction of cytochrome C release. Our results pointed to Luteolin induction of caspase-dependent and granzyme-b pathway. Luteolin as a natural agent, established antitumor aptitudes in the present solid tumor-bearing murine model through the suppression of essential molecular marks elaborated in proliferation, inflammation, and tumor invasiveness, with decisive induction of apoptosis. In addition, this compound demonstrated a synergistic potential to enhance the sensitivity of tumor cells to IR, furthermore, the combined use of luteolin with γ-irradiation may represent an effective way to decrease the dose of radiation taken.

Moreover, intrusion of cellular signaling by ROS may also subsidize to luteolin-induced apoptosis in tumor cells. Luteolin-induced oxidative stress causes suppression of the NF-γB pathway while it prompts JNK activation, which reinforce TNF-induced cytotoxicity in lung cancer cells.[35]

One of the important observations is the significance of the combination of gamma radiation and luteolin administration over the action of luteolin alone on the majority of the parameters subjected to an investigation [Figures 1-7].

It was argued that IR enhances both pro- and anti-proliferative signal pathways making a discrepancy in cell providence decision. IR is fit to control some genes and factors elaborated in cell-cycle progression, survival and/or cell death, DNA repair and inflammation modifying an intracellular radiation-dependent response. Radiation therapy could be contributed to modulation of anti-tumor immune responses and the modification of tumor and its microenvironment.[44] The complex relationship between IR, inflammation and immune responses in cancer could participate largely in the figure progressed in the present work.

IR altered the homeostatic balance between survival and cell death by activation of both pro-and antiproliferative signal pathways, which are regulated by several genes and factors involved in cell cycle progression, DNA repair, inflammation, and cell death induction.[45] Also, reduction of the incidence of distant metastases and improvement of survival by governing locoregional recurrence could ben emerged in response to IR.[46]


 > Conclusion Top


The treatment of EC-bearing mice with luteolin significantly reduced the growth of solid tumors, and a synergistic effect was demonstrated following exposure to γ-irradiation. Luteolin emanated as important dietary flavonoid with strong antioxidant, chemo preventive, and anti-inflammatory activities. It enhances the endogenous defense against oxidative stress, which is attributed to its aptitude not only to donate hydrogen to the harmful free radicals to prevent the oxidative damage but also to directly react with ROS and the cascades adjustment of inflammatory responses, enhancement of pro-apoptotic regulators and inhibition of angiogenic signals.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7]
 
 
    Tables

  [Table 1], [Table 2]



 

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