{"id":51793,"date":"2023-09-30T11:46:33","date_gmt":"2023-09-30T11:46:33","guid":{"rendered":"https:\/\/biomedpharmajournal.org\/?p=51793"},"modified":"2023-10-07T08:34:49","modified_gmt":"2023-10-07T08:34:49","slug":"antitumor-activity-of-selenium-in-ehrlich-ascites-carcinoma-bearing-mice","status":"publish","type":"post","link":"https:\/\/biomedpharmajournal.org\/staging\/vol16no3\/antitumor-activity-of-selenium-in-ehrlich-ascites-carcinoma-bearing-mice\/","title":{"rendered":"Antitumor Activity of Selenium in Ehrlich Ascites Carcinoma Bearing Mice"},"content":{"rendered":"\n

Introduction<\/strong><\/p>\n\n\n\n

The liver is the largest and one of the most essential organs in our bodies, serving as the primary regulatory organ for several physiological and biochemical processes ; angiogenesis is one of the fundamental physiological and pathological processes involved in carcinogenesis, progression, and metastasis, according to a large body of evidence1<\/sup>. Several growth hormones and cytokines are important in modulating the physiological process of angiogenesis. 2<\/sup> In almost all malignancies, tumor angiogenesis, which is the growth of new blood vessels to feed nutrients to tumor cells that are dividing quickly, is a key step in the tumor metastasis route3<\/sup>. The damage and fibrosis of the liver caused by tumor angiogenesis is one of the characteristics of cancer that is connected to liver fibrosis and inflammation in chronic liver disease, and that it also promotes the growth of hepatocellular carcinoma (HCC)4<\/sup>. However, the relationship between tumor angiogenesis and hepatitis remains unknown. As a result, a thorough understanding of the relationship between tumor angiogenesis and inflammatory liver illnesses is required for the development of cutting-edge therapeutic options for tumor-associated hepatitis5<\/sup>. This lethal disease-related syndrome is caused by a combination of growth factors and cytokines5<\/sup>. Hypoxia activates numerous pro-angiogenic pathways, which promote capillary growth by secreting a variety of angiogenic growth factors, including the well-studied vascular endothelial growth factor (VEGF), placental growth factor (PIGF), fibrosis-associated transforming growth factor-beta (TGF-beta), and inflammation-associated tumor necrosis growth factor (TNF-beta), via the mitochondria6<\/sup>. In addition to these angiogenic growth factors, several inflammatory cytokines play an important role in liver inflammation and fibrosis7<\/sup>. Addressing tumor angiogenesis will be a promising therapeutic option for treating individuals with tumor-associated liver fibrosis and inflammation2<\/sup>. Several studies have revealed that angiogenesis is a key characteristic of several malignancies, including HCC8<\/sup>. Under normal physiological conditions, angiogenesis allows immune cells to migrate from one organ to another while delivering food and oxygen. It also stimulates the healing of tissue injury and the repair of damaged tissues during the course of tissue homeostasis9<\/sup>. However, the total tumor cell count and tumor volume increased as a result of the EAC cells’ rapid multiplication in the peritoneal cavity, generating a hypoxic environment in the adjacent microenvironment. As a result, many angiogenic growth factors and cytokines are produced, including VEGF, PLGF, TNF, and TGF, stimulating endothelial cells10<\/sup>. These components have been found to have an important role in the angiogenesis of HCC11,<\/sup> 12<\/sup>. It has been proposed that EAC tumor-induced peritoneal angiogenesis and newly created capillaries can transport angiogenic and inflammatory markers to the liver and may promote the activation of stellate cells, Kupffer cells, and mast cells that are linked to the liver, resulting in liver damage. associated inflammation signalling13<\/sup>. Previous investigations indicated that angiogenesis pathways are crucial to the development of hepatitis, fibrosis, and HCC14<\/sup>.<\/p>\n\n\n\n

It has been demonstrated that selenium, an essential trace mineral present in both organic and inorganic chemical forms, is vital for sustaining mammalian cells in their ideal physiological state. It has demonstrated chemopreventive ability against the emergence of tumors as well as different types of environmental stress15<\/sup>. Recent studies suggested that the use of Se in combination with conventional chemotherapy medications and therapeutic hormones can disclose cancer, even though Se is in clinical trials for the chemoprevention of prostate, colon, and lung cancer16<\/sup>. Se’ beneficial and harmful effects have a very narrow window; therefore its long- term supplementation for preventative and therapeutic reasons is constrained16<\/sup>.<\/p>\n\n\n\n

As a result, the current study assessed the impact of\nchanging angiogenic regulators on the development\nof tumors and the\nindicators of inflammation and liver damage. Ehrlich cells were used as\na tumor carcinoma model in mice\nto study the impact of selenium\nin vivo.<\/p>\n\n\n\n

Materials and methods<\/strong><\/p>\n\n\n\n

Materials<\/strong><\/p>\n\n\n\n

Doxorubicin ( DOX) was purchased from the pharmacy.<\/p>\n\n\n\n

culture media was obtained from Invitrogen\u2010Life Technologies. <\/p>\n\n\n\n

selenite (Na2<\/sub>SeO3<\/sub>) was purchased from Merck Chemical Inc. (Darmstadt, Germany).<\/p>\n\n\n\n

All other using chemicals were HPLC grade and purchased from Sigma-Aldrich, Germany.<\/p>\n\n\n\n

In vitro study<\/strong><\/p>\n\n\n\n

Cytotoxic effect on human cell lines<\/strong><\/p>\n\n\n\n

The mitochondrial dependent\nreduction of yellow MTT (3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyl tetrazolium bromide) to purple formazan was used to determine cell viability.<\/p>\n\n\n\n

Procedure<\/strong><\/p>\n\n\n\n

All procedures were carried out in a sterile environment\nwith the use of a Laminar flow biosafety cabinet Class II A2.HepG2\ncells were suspended\nin DMEM (Dulbecco’s Modified Eagle Medium) with high glucose and stable glutamine,\n1% antibiotic, and 5% fetal bovine serum at 37 o<\/sup>C in a CO2 incubator\n(Sartorius stedium,biotech).<\/p>\n\n\n\n

Cells were seeded at a density of 10×103<\/sup> cells\/well\nin fresh complete growth medium in 96-well plastic\nplates at 37 o<\/sup>C\nfor 24 h under 5% CO2 either alone (negative control) or with different concentrations of drugs and Doxorubicin\n(DOX) as a positive control group, yielding a final concentration of (50, 25, 12.5, 6.25, 3.125, 1.5625 mg\/ml).\nAfter 48 hours, the medium was aspirated,\nand 20 \u03bcl of MTT salt\n(2.5g\/ml) was added to each well before incubating for another four hours at 37oC with 5% CO2. To\ndissolve the generated crystals and terminate the reaction, 200 \u03bcl of 10% Sodium dodecyl\nsulphate (SDS) in 0.01 mole HCL was added\nto each well, and the plate\n  was\nthen incubated overnight in a dark environment.17, 18<\/sup>.<\/p>\n\n\n\n

The absorbance was then measured\nusing a microplate reader at 595nm and a reference wavelength of 620nm. Viability\nwas calculated as follow:<\/p>\n\n\n\n

 Viability = absorbance of drug \/ absorbance of\ncontrol x 100<\/p>\n\n\n\n

Cytotoxicity = 100- viability<\/p>\n\n\n\n

IC 50 was calculated from the relation between the\ndifferent concentration of the drug and cell viability for each drug\nconcentration.<\/p>\n\n\n\n

The IC 50 was computed using the relationship between\ndrug concentration and cell viability for each drug concentration18<\/sup>.<\/p>\n\n\n\n

LD50 estimation based\non in vitro IC50 value<\/p>\n\n\n\n

Using the following formula, the in vivo LD50 of acute\noral toxicity was calculated from the in vitro IC50:\nlog LD50 = 0.372 log IC50 (g\/mL)\n+ 2.02419<\/sup>.<\/p>\n\n\n\n

In vivo study<\/strong><\/p>\n\n\n\n

Experimental Animals<\/strong><\/p>\n\n\n\n

Adult female Swiss albino mice (25-30g) were procured from the\nNational Research Centre’s Animal\nHouse in Giza, Egypt. They were maintained in stainless steel cages in a controlled environment (temperature 20 \u00b1 2\n\u00baC) with regular\nlaboratory diet and ad libitum\nwater at the National Research Centre’s Animal House in Giza, Egypt. . The National Institutes of\nHealth Guide for Care and Use of Laboratory Animals (Publication No. 85-23,\nupdated 1985) was followed for animal procedures, and the experiment followed\nthe recommendations and criteria of the National Research Centre’s (NRC)\nethical committee.<\/p>\n\n\n\n

Transplantation of a tumor<\/strong><\/p>\n\n\n\n

Dr. Gklien kindly\nprovided an EAC cell line, which was maintained in experimental female\nSwiss albino mice via intraperitoneal injection of 2.5 x 106<\/sup> cells\nper mouse. Fluid tumor was detected after\n5-7 days of EAC cell\ninoculation20,<\/sup> 21<\/sup>.<\/p>\n\n\n\n

Experimental design<\/strong><\/p>\n\n\n\n

Thirty-two Swiss albino mice were weighted and\ntheir weight volumes were recorded to compare with their weight after the\nexperimental period; they were then randomly assigned to four experimental\ngroups (8 mice per group) as appeared in chart 1 and categorised as follows: <\/p>\n\n\n\n

Group 1:\n healthy mice.<\/p>\n\n\n\n

 Group II : EAC cell line was administered\nintraperitoneally into healthy mice. <\/p>\n\n\n\n

Group\nlll : mice with EAC were given selenium (as Na2<\/sub>SeO3<\/sub>\ndissolved in water) orally for ten days22, 23<\/sup> at a dose one-tenth of\nthe LD50.  <\/p>\n\n\n\n

Group IV : mice with EAC were given DOX (1\/20 of the LD50) .<\/p>\n\n\n\n\n\n
\"\"<\/td>\n\n

Chart 1: Experimental study appeared groups and duration of the experiment<\/strong><\/p>\n

<\/p>\n

Click here to view Chart<\/a><\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n

Following the 10-day study period, rats were\nfasted overnight and blood was collected from the orbital vein, centrifuged at\n4000 rpm for 20 minutes, and the separated plasma was stored at -80 o<\/sup>C for\nsubsequent examination. The comet assay was\nperformed on blood, and other biochemical parameters such as plasma caspase 3,\nAlpha fetoprotein (AFP), tumor necrosis factor-alpha (TNF-alpha), and Bcl2 were\ndetermined using an ELISA technique with commercial kits from R&D Systems\nGmbH (Wiesbaden, Germany), according to the manufacturer\u2019s instructions .  In addition blood transaminases (ALT and AST)\nwere estimated colorimetrically using spectroUV-VIS Double Beam UVD-3500  .EAC fluid volume was sucked  and measured \nits volume by ml using  a syringe.\n<\/p>\n\n\n\n

Comet test<\/strong><\/p>\n\n\n\n

The comet test was created to detect cellular DNA\ndamage24. Lymphocytes were separated and washed\nin pH 7.4 phosphate buffered saline (PBS). Ten microliters of cells were\nsuspended in 75 l of 0.5% low\nmelting agarose to pipette on microscope slides with a layer of 1% agarose,\nspread with a cover slip, and\nsolidified for 5 minutes on an ice-cold flat tray. After removing the cover slip, the slides were immersed in cold\nlysis solution for 1 hour, followed by 40 minutes of electrophoresis at 25 V, 300 mA, before being carefully removed\nfrom the tank and washed three times\nwith 0.4 M Trizma base at pH 7.5 for 10 minutes. Each slide received 20\nmicroliters of ethidium bromide (10\ng\/ml). The slides were examined at 40 magnification with a fluorescence microscope (Leica Microsystems, CMS GM b H, Wetzlar,\nGermany. Model DM 2500) and power Max. 160 W equipped with a 549 nm\nexcitation filter and a 590 nm barrier filter. The “comet appearance” of damaged cells was\nseen, with a brilliantly fluorescent head and a tail to one side generated by DNA strand breaks that will\ndraw away during electrophoresis. The percentage of damage was calculated by counting the damaged\ncell out of 100 cells each slide.<\/p>\n\n\n\n

Statistical analysis<\/strong><\/p>\n\n\n\n

The statistical package\nfor the social\nsciences (SPSS) application, version 16, and Microsoft Excel\n2007 were used to conduct data analysis. The data were displayed as\nmeans standard error (SE). The\nsignificance of the difference in results was calculated using one-way ANOVA and\nthe Student’s t-test. A statistically\nsignificant difference was defined as P 0.05.<\/p>\n\n\n\n

Results<\/strong><\/p>\n\n\n\n

The in vitro\nstudy indicated that , IC 50 for selenium was 11.585 \u03bcg\/ml as appeared in\ntable, and 0.816 \u03bcg\/ml for doxorubicin (\ntable1,2&fig1,2) .<\/p>\n\n\n\n\n\n
\"\"<\/td>\n\n

Figure 1: IC50 for different concentration of selenium against HepG2<\/strong><\/p>\n

<\/p>\n

Click here to view Figure<\/a><\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n

Table 1: Cell viability and cytotoxicity of selenium\u2019 different concentrations against HepG2<\/strong><\/p>\n\n\n\n\n\n\n\n\n\n\n\n
\n

Selenium concentration (\u03bcg\/ml)<\/strong><\/p>\n<\/td>\n

\n

Cell viability %<\/strong><\/p>\n<\/td>\n

\n

Cytotoxicity %<\/strong><\/p>\n<\/td>\n

\n

IC50<\/strong><\/p>\n

( \u03bcg\/ml)<\/strong><\/p>\n<\/td>\n

\n

LD50<\/strong><\/p>\n

( mg\/kg b.w.)<\/strong><\/p>\n<\/td>\n<\/tr>\n

\n

50<\/p>\n<\/td>\n

\n

7<\/p>\n<\/td>\n

\n

93<\/p>\n<\/td>\n

\n

11.585<\/p>\n<\/td>\n

\n

262.887<\/p>\n<\/td>\n<\/tr>\n

\n

25<\/p>\n<\/td>\n

\n

37<\/p>\n<\/td>\n

\n

63<\/p>\n<\/td>\n<\/tr>\n

\n

12.5<\/p>\n<\/td>\n

\n

48<\/p>\n<\/td>\n

\n

52<\/p>\n<\/td>\n<\/tr>\n

\n

6.25<\/p>\n<\/td>\n

\n

54<\/p>\n<\/td>\n

\n

46<\/p>\n<\/td>\n<\/tr>\n

\n

3.125<\/p>\n<\/td>\n

\n

60<\/p>\n<\/td>\n

\n

40<\/p>\n<\/td>\n<\/tr>\n

\n

1.562<\/p>\n<\/td>\n

\n

62<\/p>\n<\/td>\n

\n

38<\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n\n
\"\"<\/td>\n\n

Figure 2: IC50 for different concentration of DOX against HepG2<\/strong><\/p>\n

<\/p>\n

Click here to view Figure<\/a><\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n

Table 2: Cell viability and cytotoxicity of doxorubicin\u2019 different concentrations against HepG2<\/strong><\/p>\n\n\n\n\n\n\n\n\n\n\n\n
\n

Doxorubicin concentration (\u03bcg\/ml)<\/strong><\/p>\n<\/td>\n

\n

Cell viability %<\/strong><\/p>\n<\/td>\n

\n

Cytotoxicity %<\/strong><\/p>\n<\/td>\n

\n

IC50<\/strong><\/p>\n

( \u03bcg\/ml)<\/strong><\/p>\n<\/td>\n

\n

LD50<\/strong><\/p>\n

( mg\/kg b.w.)<\/strong><\/p>\n<\/td>\n<\/tr>\n

\n

5<\/p>\n<\/td>\n

\n

2<\/p>\n<\/td>\n

\n

93<\/p>\n<\/td>\n

\n

0.816<\/p>\n<\/td>\n

\n

97.982<\/p>\n<\/td>\n<\/tr>\n

\n

2.5<\/p>\n<\/td>\n

\n

31<\/p>\n<\/td>\n

\n

63<\/p>\n<\/td>\n<\/tr>\n

\n

1.25<\/p>\n<\/td>\n

\n

46<\/p>\n<\/td>\n

\n

52<\/p>\n<\/td>\n<\/tr>\n

\n

0.625<\/p>\n<\/td>\n

\n

51<\/p>\n<\/td>\n

\n

46<\/p>\n<\/td>\n<\/tr>\n

\n

0.3125<\/p>\n<\/td>\n

\n

55<\/p>\n<\/td>\n

\n

40<\/p>\n<\/td>\n<\/tr>\n

\n

0.156<\/p>\n<\/td>\n

\n

58<\/p>\n<\/td>\n

\n

38<\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n

Table (3): showed significant increase in body weight\ngain with a percentage increase of 55.96 %\nin ehrlich bearing mice related to control .While medication of mice\nwith EAC treated with Se and\nDOX showed an\ninsignificant change in body weight with percentages reached to -6.12 and- 8.92% respectively from EAC group . The percent of change in the volume of EAC ascites fluid in Se and DOX treated mice were -13.39\nand -15.68 respectively (Table 4). Significant elevation in the mortality rate of EAC mice with percentage increase\namounted 40% relative to control level . While ,marked\nreduction in EAC treated mice with Se (30%) was recorded. However ,the mortality rate in DOX \u2013treated EAC mice\nshowed insignificant difference compared to untreated EAC bearing mice (Table 4) . Additionally, Table (6): declared noticeable elevation in ALT, AST in\nEAC bearing mice relative to control group . Treated EAC mice with Se and DOX\nrevealed an improvement of these\nvalues . TNF-\u03b1 ,caspase 3, AFP and Bcl2 in plasma of EAC bearing mice were significantly increased\nin EAC compared to control\n. However, the treatment with either Se or DOX showed marked\namelioration in their\nlevels (Table 7). Significant increase\nin percentage of DNA\ndamage as recorded by the test of comet in EAC bearing mice compared to control mice . Howevere ,treated mice with Se\nshowed marked significant reduction in DNA damage % which is significantly lower compared to the recorded for DOX treated group\n(Table 8, Fig.3).<\/p>\n\n\n\n

Table 3: The mean value of body weight (g) of mice in different groups<\/strong><\/p>\n\n\n\n\n\n\n\n\n
\n

 <\/p>\n<\/td>\n

\n

Control<\/strong><\/p>\n<\/td>\n

\n

Ehrlich<\/strong><\/p>\n<\/td>\n

\n

Treated- selenium<\/strong><\/p>\n<\/td>\n

\n

Treated -DOX<\/strong><\/p>\n<\/td>\n<\/tr>\n

\n

Mean \u00b1SE<\/p>\n<\/td>\n

\n

26.16\u00b11.10<\/p>\n<\/td>\n

\n

40.80\u00b13.11a<\/sup><\/p>\n<\/td>\n

\n

38.30\u00b12.87a<\/sup><\/p>\n<\/td>\n

\n

37.16\u00b13.18a<\/sup><\/p>\n<\/td>\n<\/tr>\n

\n

% change from control group<\/p>\n<\/td>\n

\n

–<\/p>\n<\/td>\n

\n

+55.96<\/p>\n<\/td>\n

\n

+46.41<\/p>\n<\/td>\n

\n

+42.10<\/p>\n<\/td>\n<\/tr>\n

\n

%of change from ehrlich
group<\/p>\n<\/td>\n

\n

–<\/p>\n<\/td>\n

\n

–<\/p>\n<\/td>\n

\n

-6.12<\/p>\n<\/td>\n

\n

-8.92<\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

Data are expressed as mean \u00b1SE, where a: indicated significant at P \u22640.05 from the control, and b: indicated significant at P \u22640.05 from ehrlich group<\/p>\n\n\n

Table 4: The mean Volume (ml) of EAC ascites fluid of mice in different groups<\/strong><\/p>\n\n\n\n\n\n\n\n
\n

 <\/p>\n<\/td>\n

\n

Control<\/strong><\/p>\n

group<\/strong><\/p>\n<\/td>\n

\n

Ehrlich<\/strong><\/p>\n<\/td>\n

\n

Se-treated mice<\/strong><\/p>\n<\/td>\n

\n

DOX -treated mice<\/strong><\/p>\n<\/td>\n<\/tr>\n

\n

Mean \u00b1SD<\/p>\n<\/td>\n

\n

0.00<\/p>\n<\/td>\n

\n

15.30 \u00b10.56 a<\/sup><\/p>\n<\/td>\n

\n

13.25\u00b10.89a,b<\/sup><\/p>\n<\/td>\n

\n

12.90\u00b10.65a,b<\/sup><\/p>\n<\/td>\n<\/tr>\n

\n

%of change from<\/p>\n

EAC group<\/p>\n<\/td>\n

\n

—<\/p>\n<\/td>\n

\n

—<\/p>\n<\/td>\n

\n

-13.39<\/p>\n<\/td>\n

\n

-15.68<\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

Data are expressed as mean \u00b1SE, where a: indicated significant at P \u22640.05 from the control, and b: indicated significant at P \u22640.05 from ehrlich group<\/p>\n\n\n

Table 5: Mortality rate(%)post 10 days of treatment<\/strong><\/p>\n\n\n\n\n\n\n\n\n\n
\n<\/td>\n\n

Control<\/strong><\/p>\n

group<\/strong><\/p>\n<\/td>\n

\n

Ehrlich<\/strong><\/p>\n

group<\/strong><\/p>\n<\/td>\n

\n

Selenium<\/strong><\/p>\n

treated group<\/strong><\/p>\n<\/td>\n

\n

DOX treated group<\/strong><\/p>\n<\/td>\n<\/tr>\n

\n

Day 1<\/p>\n<\/td>\n

\n

10<\/p>\n<\/td>\n

\n

10<\/p>\n<\/td>\n

\n

10<\/p>\n<\/td>\n

\n

10<\/p>\n<\/td>\n<\/tr>\n

\n

Day 10<\/p>\n<\/td>\n

\n

9<\/p>\n<\/td>\n

\n

6<\/p>\n<\/td>\n

\n

7<\/p>\n<\/td>\n

\n

6<\/p>\n<\/td>\n<\/tr>\n

\n

Dead animals<\/p>\n<\/td>\n

\n

1<\/p>\n<\/td>\n

\n

4<\/p>\n<\/td>\n

\n

3<\/p>\n<\/td>\n

\n

4<\/p>\n<\/td>\n<\/tr>\n

\n

% of death<\/p>\n<\/td>\n

\n

10%<\/p>\n<\/td>\n

\n

40%<\/p>\n<\/td>\n

\n

30%<\/p>\n<\/td>\n

\n

40%<\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n

Table 6: Liver functions in different groups<\/strong><\/p>\n\n\n\n\n\n\n\n
\n

Groups<\/strong><\/p>\n<\/td>\n

\n

Control<\/strong><\/p>\n<\/td>\n

\n

Ehrlich group<\/strong><\/p>\n<\/td>\n

\n

Se treated group<\/strong><\/p>\n<\/td>\n

\n

DOX<\/strong><\/p>\n

treated group<\/strong><\/p>\n<\/td>\n<\/tr>\n

\n

ALT (U\/L)<\/p>\n<\/td>\n

\n

11.17\u00b11.00<\/p>\n<\/td>\n

\n

33.50\u00b12.00a<\/sup><\/p>\n<\/td>\n

\n

17.83\u00b11.21ab,c<\/sup><\/p>\n<\/td>\n

\n

21.33\u00b1 1.10ab<\/sup><\/p>\n<\/td>\n<\/tr>\n

\n

AST (U\/L)<\/p>\n

% change<\/p>\n<\/td>\n

\n

38.17\u00b12.50<\/p>\n<\/td>\n

\n

73.00\u00b14.22a<\/sup><\/p>\n<\/td>\n

\n

40.5\u00b13.11ab,c<\/sup><\/p>\n<\/td>\n

\n

48.50\u00b13.07ab<\/sup><\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

Data are expressed as mean \u00b1SE, where a: indicated significant at P \u22640.05 from the control group;b: indicated significant at P \u22640.05 from the ehrlich group;c: indicated significant at P \u22640.05 from the DOX treated group.<\/p>\n\n\n

Table 7: Serum levels of TNF-\u03b1, caspase 3,AFP, and Bcl\u20102 in different groups:<\/strong><\/p>\n\n\n\n\n\n\n\n\n\n
\n

Groups \/ Markers<\/strong><\/p>\n<\/td>\n

\n

Control group<\/strong><\/p>\n<\/td>\n

\n

Ehrlich group<\/strong><\/p>\n<\/td>\n

\n

Se- treated group<\/strong><\/p>\n<\/td>\n

\n

DOX t-<\/strong><\/p>\n

reated group<\/strong><\/p>\n<\/td>\n<\/tr>\n

\n

TNF-\u03b1 ( ng\/L)<\/p>\n<\/td>\n

\n

5.30\u00b1 0.70<\/p>\n<\/td>\n

\n

14.72\u00b1 1.00 a<\/sup><\/p>\n<\/td>\n

\n

11.22\u00b1 0.50 a.b<\/sup><\/p>\n<\/td>\n

\n

12.90\u00b1 0.77 a,b<\/sup><\/p>\n<\/td>\n<\/tr>\n

\n

Caspase 3(ng\/ml)<\/p>\n<\/td>\n

\n

0.49 \u00b1 0.02<\/p>\n<\/td>\n

\n

1.30 \u00b1 0.04 a<\/sup><\/p>\n<\/td>\n

\n

0.72\u00b1 0.05 a.b<\/sup><\/p>\n<\/td>\n

\n

0.79 \u00b1 0.05 a,b<\/sup><\/p>\n<\/td>\n<\/tr>\n

\n

AFP(IU\/ml)<\/p>\n<\/td>\n

\n

0.41\u00b1 0.01<\/p>\n<\/td>\n

\n

1.50\u00b1 0.07 a<\/sup><\/p>\n<\/td>\n

\n

0.72 \u00b1 0.02 a,b<\/sup><\/p>\n<\/td>\n

\n

0.81 \u00b1 0.03 a,b<\/sup><\/p>\n<\/td>\n<\/tr>\n

\n

Bcl\u20102 (ng\/ml)<\/p>\n<\/td>\n

\n

4.47 \u00b1 0.04<\/p>\n<\/td>\n

\n

9.32 \u00b1 0.05 a<\/sup><\/p>\n<\/td>\n

\n

5.70 \u00b1 0.03 a,b<\/sup><\/p>\n<\/td>\n

\n

6.30\u00b1 0.05 a,b<\/sup><\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

Data are expressed as mean \u00b1SE, where a: indicated significant at P \u22640.05 from the control group;b: indicated significant at P \u22640.05 from the ehrlich group;c: indicated significant at P \u22640.05 from the DOX treated group.<\/p>\n\n\n

Table 8: Percentage of DNA damage in different studied groups<\/strong>.<\/p>\n\n\n\n\n\n\n
\n

groups<\/strong><\/p>\n<\/td>\n

\n

Control group<\/strong><\/p>\n<\/td>\n

\n

Ehrlich group<\/strong><\/p>\n<\/td>\n

\n

Se- treated group<\/strong><\/p>\n<\/td>\n

\n

DOX- treated group<\/strong><\/p>\n<\/td>\n<\/tr>\n

\n

DNA %<\/p>\n<\/td>\n

\n

2.20\u00b10.33<\/p>\n<\/td>\n

\n

32.00\u00b13.60a<\/sup><\/p>\n<\/td>\n

\n

16.<\/sup>.00\u00b11.03a,b,c<\/sup><\/p>\n<\/td>\n

\n

22.00\u00b12.30a,b<\/sup><\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

Data are expressed as mean \u00b1SE, where a: indicated significant at P \u22640.05 from the control group;b: indicated significant at P \u22640.05 from the ehrlich group;c: indicated significant at P \u22640.05 from the DOX treated group.<\/p>\n\n\n\n
\"\"<\/td>\n\n

Figure 3: DNA damage in different studied groups appeared the percent of damage in A) control group, B) EAC group, C) se treated group and D) DOX treated group.<\/strong><\/p>\n

<\/p>\n

Click here to view Figure<\/a><\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n

Discussion<\/strong><\/p>\n\n\n\n

We seek to describe the novel molecular mechanism of HCC related with liver disease using the EAC mouse model and the therapeutic role of selenium based on this experimental observation. As a result, this model can be utilized to investigate new signaling systems, as well as offer additional insight on how cancer causes hepatitis and related liver illness. Few additional research have revealed that EAC tumor metastasizes to multiple organs, which may be related to the activation of angiogenic pathways, which can cause liver inflammation and fibrosis by various angiogenic growth factors as well as various cytokines2<\/sup>. Tumor angiogenesis and its detrimental consequences in comorbidities are known to be the primary causes of cancer-related liver dysfunction25<\/sup>. Our findings were comparable, and we believe that inflammation-induced hepatitis may be a cause of HCC-related mortality, even after therapeutic recovery26<\/sup>. Cancer cells, as previously stated, activate angiogenesis by raising the expression of AFP, Caspase 3, Bcl2, and<\/p>\n\n\n\n

the release of\nangiogenic growth factors such as VEGF, TNF\u03b1, and TGF \u03b1, as well as different cytokines27<\/sup>, as evidenced\nby these findings.\nAngiogenic factors and previously generated\ncytokines enter the portal circulatory system and portal vein before\nreaching the liver, where they bind\nto particular receptors and activate numerous signal transduction pathways.\nThis pathogenic event then activates hepatic\nstellate cells, Kupffer\ncells, and mast cells, which are the mediators of hepatic\ninflammation, fibrosis, and, in other words, liver damage14<\/sup>. Activation\nof these hepatic stellate cells, on\nthe other hand, produces chemokines that promote angiogenesis, inflammation, and fibrosis.\nEGF and EGFR implicated in\nvarious biological activities; including mitogenic and angiogenic purposes;\nEGF-mediated signaling pathway was shown to play a key role in motivating\nproliferation of microvascular endothelial cells and also lymphatic endothelial\ncells . EGF can act through both paracrine and autocrine mechanisms to\nfacilitate the expression of key proteases on endothelial cells to remodel\nsurrounding extracellular matrix permitting endothelial cell migration,\nregulate the expression of VEGF and other growth factors, and induce\nangiogenesis via PI3k, MAPK, and eNOS pathways in a VEGF-independent 28<\/sup>.\nThe increase in TNF\u03b1 levels in our results may be related to its participation\nin the neovascularization process. TNF\u03b1 is a significant\ninflammatory mediator that causes a variety of\nalterations in endothelial cells (EC) , including the activation of adhesion\nmolecules, integrines, and matrix\nmetalloproteinases29<\/sup>. Different types of cancer\nhave altered levels\nof pro-inflammatory and pro-angiogenic proteins, and\nTNF-expression has been associated to tumor differentiation, invasiveness, and angiogenesis30<\/sup>.\nAccording to the results presented, an increase in TNF\u03b1 is associated by an increase\nin tumor volume.\nThe tumor’s ability\nto develop, as well as its invasiveness and metastatic ability, is\nenhanced by neovasculaturization15<\/sup>.Furthermore, a rise in tumor volume in mice with EAC is related\nwith an increase in BcL2,\nAFP,  Caspase 3, and DNA damage (Table 7,8).<\/p>\n\n\n\n

As we discovered in our in vivo investigation, body weight increases with cancer growth (as seen by the current findings, which reveal a considerable increase in EAC ascitic fluid volumes due to inflammation (infiltration of immune cells) and concomitant fibrosis (collagen fiber accumulation). These pathological alterations damage hepatocytes’ and the liver’s overall physiological function. In support of these findings in liver damage, our biochemical assays demonstrated elevated blood levels of AST and ALT in EAC tumor-bearing mice compared to control mice. Another published study31 backed up our hypothesis. The increased level of liver enzyme activity in serum in EAC tumor-bearing mice definitely suggested liver injury. These new findings further suggest that the EAC tumor promotes peritoneal angiogenesis and is involved in the circulation of proangiogenic substances, which may contribute to the advancement of liver inflammation and fibrosis32<\/sup>. The considerable rise in AFP in the current study suggests that AFP reduces tumor immunity and promotes tumor development, decreasing cancer immunity and increasing tumor 33<\/sup>.Based on our findings, there is a considerable rise in TNF\u03b1, BcL2, and Caspse<\/p>\n\n\n\n

3 in the serum of EAC-bearing mice compared to the control. These biomarkers are considered key regulators of inflammation and fibrosis. These indicators are thought to be important regulators of inflammation and fibrosis. Overall, elevated expression of these markers in EAC mouse serum can cause various clinical outcomes, such as hepatitis-like symptoms in breast cancer patients. This is the most likely source of liver inflammation and fibrosis in mice with EAC tumors 2<\/sup>. According to the findings, it was discovered that higher tumor volume in EAC- bearing mice is connected with a significant decrease in antioxidant biomarkers SOD, CAT and GSH15<\/sup> .    <\/p>\n\n\n\n

Selenium treatment showed a significant improvement in different biomarkers examined in EAC-bearing mice related to control group. These findings can be explained by the fact that Se can inhibit hepatocarcinoma angiogenesis in rats by down-regulating the expression of TNF- and VEGF34, and it also inhibits TNF- in human umbilical vein endothelial cells [HUVEC], which leads to suppression of MMP-2 and MMP-9 activities15. Sodium selenite and other different forms of selenium might be able to inhibit cancer metastasis and primary tumor growth in several cancers in animals 15<\/sup>. Many researchers have observed that inhibiting antioxidant mechanisms in rat blood and tissues after irradiation35 and in irradiated carrier mice by EAC15<\/sup> is associated with an increase in lipid peroxide products. Numerous investigations, on the other hand, have revealed that tumor growth can generate antioxidant disruptions in some tumor host tissues15<\/sup>. In fact, tumor growth may be to blame for the depletion of antioxidants in the liver as well as an increase in the quantity of lipid peroxidation products36<\/sup>. Past lipid peroxidation appears to be begun by the extraction of hydrogen from lipid molecules by lipid radiolysis products, <\/strong>resulting in permeability disruptions due to variations in membrane proteins and polysaccharides. It has also been shown that the rate of LPx increase following irradiation is proportional to the radiation dose and time37<\/sup>.When oxidative damage caused by tumor development is severe, ROS scavenging enzymes (SOD & CAT) and GSH are degraded15<\/sup>. In turn, free radicals and oxidative stress enhance the expression of TNF- and AFP, which are involved in the angiogenesis process and contribute to tumor formation. Furthermore, the anticancer efficacy of several Se compounds in the EAC mouse model has previously been described16<\/sup>. Se as adjuvant therapy with cyclophosphamide shown significant anticancer and antioxidant benefits in EAC-bearing mice16<\/sup>.Thus, Se played an important role in the reduction of Bcl2 in EAC cells, which may lead to the induction of mitochondria-mediated apoptosis by altering mitochondrial permeability and releasing specific apoptotic proteins such as cytochrome c 38<\/sup>; this, in turn, increases the cascade of caspases in EAC cells, particularly caspase-3, known as the death protein. 39<\/sup>, 40<\/sup>. These pathways have been approved in tumor cell execution in vitro in human colon cancer cells and human oral squamous cell carcinoma, respectively. Furthermore, our findings support the findings of Jin et al41<\/sup>, who discovered a strong apoptotic effect on colorectal cancer cells in vitro and in vivo related with Bcl-2\/Bax\/Caspase-3 signaling. These findings were supported by a recent experiment42<\/sup> that validated these apoptotic processes in human prostate cancer cells in vitro. The most recent data also revealed higher DNA damage in EAC mice, as well as the modulatory action of Se. This could be because Se can impact the level of p53 in EAC cells; also, the P53 gene is recognized as the tumor suppressor gene and is in charge of DNA repair or damage in cells38<\/sup>. In cancer cells, the p53 gene is mutated, and the p53 proteins are changed into mutant p53 proteins that supply energy and dietary antioxidants to cancer cells, making them more resistant to chemotherapy medicines. and growing their numbers38<\/sup>. Finally, the data reported here show the establishment of a novel evidence-based mechanism for analyzing Ehlish-induced tumor angiogenesis in mice. In this mouse EAC model, we also demonstrated how tumor angiogenesis contributes to the activation of liver enzymes, the elevation of TNF-\u03b1, AFP, Bcl2, caspase 3, as well as an increase in DNA damage, and ultimately leads to the development of hepatitis due to inflammatory cell infiltration and collagen deposition. As a result, our findings may give more evidence that inhibiting tumor angiogenesis may be a promising therapeutic strategy in the treatment of liver impairment associated with advanced hepatitis. Furthermore, Se was able to lower inflammatory markers as well as Bcl2 activity, which supported an increase in caspases, particularly caspase-3, which caused cancer cells to be executed.<\/p>\n\n\n\n

Conclusion<\/strong><\/p>\n\n\n\n

From the in vitro <\/em>study (using HepG2 cell line) we can calculate LD50 that was used in determination of using selenium dose. This work appeared that Ehrlich Ascites Carcinoma (EAC) elevated liver functions and the inflammatory markers as appeared by measuring TNF-\u03b1, AFP in addition to increasing DNA damage, BcL2 and AFP; whereas treatment with selenium could attenuate these disturbances . We can consider selenium as a promising agent that can regulat angiogenesis and the development of tumors in liver inflammation;  and we recommended more studies on different types of cancer.<\/p>\n\n\n\n

Conflict of Interest<\/strong><\/p>\n\n\n\n

There is no conflict of Interest<\/p>\n\n\n\n

References<\/strong><\/p>\n\n\n\n

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