رسائل دكتوراة
English
2020
The Effect of Combined Action of Oncolytic Measles Virus Vaccine with Cyperus rotundus on Breast Cancer Cell Line
أحمد مجيد حمزة, سليمان عجاج عبد الله, صفاء عبد لطيف
كلية العلوم-جامعة الأنبار · العراق
This project considers as an explorer study for the activity of oncolytic measles virus vaccine and Cyperus rotund us phenolic extract alone and in combination against local (AMJ13) and global (MCF-7 and CAL-51) breast cancer cell lines (In vitro). First, the MeV vaccine was multiplied in veroh-slam cells, and thenTCID50of the virus was calculated. The TCID50 was (5.3x107). The cytotoxicity of MeV on cancer cells and normal cells (HBL-100) was evaluated at certain virus MOIs (20, 10, 5, 3, 1, 0.5 and 0.1) after 72h of infection to calculate the IC50using GraphPad prism software version 7.0. Cell viability was estimated using MTT assay. The IC50s for cancer cellsAMJ13,MCF-7, CAL-51 and normal cellsHBL-100 were (3.527, 5.079, 9.171and 22.53) respectively. These results indicated that the local breast cancer cells (AMJ13) are more sensitive to the MeV vaccine which required the least number of viruses for 50% growth inhibition in comparison to the international cell lines. Furthermore, the result showed that MeV vaccine has a negligible cytotoxic effect on the non-cancerous cell HBL-100. By other hand, the phenolic compounds of C. rotund us rhizomes were extracted, and GC-MS analysis revealed that it contains six phenolic compounds (4-Mercaptophenol, Benz[e]indene-3,7-diol1, 4-Methanoazulen-7-ol, Orcinol, phenanthren-3-ol, and 2H-3,3'-dimethoxy-[1,1'-biphenyl]-4,4'-diol). The cytotoxicity ofC. Rotund us phenolic extract was evaluated at certain concentrations (400,200, 100, 75, 50 and 25) μg /ml to calculate the IC50after 48h of treatment. The IC50s for cancer cellsMCF-7,AMJ13, CAL-51 and normal cellsHBL-100 were (135.3, 148.3, 218.6and 329.6) respectively. A combination of measles virus vaccine and C. rotund us phenolic extract were used to make two dilutions higher and lower than the IC50value of each treatment. Nine groups (A, B, C, D, E, F, G, Hand I) of combinations used in this study to assessment the synergistic effect. Combination index (CI) for each mixture was determined by
using compusyn software over a 48-hrs treatment. Compared with the results of growth inhibition (GI) caused by MeV or phenolic extract alone, the combination of the two treatments revealed a significant (P<0.05) increase in growth inhibition among all groups of combination. For MCF-7 cells the GIof MeV alone was calculated at MOI=3, 5 and 7 which were (32±1.852, 43.1±2.425 and 52.4±3.538);respectively, and phenolic extract alone at concentrations75, 123.7 and 200μg/ml which were (40.866±2.442, 54.366±2.984 and63.53±2.055);respectively. The combinations of the three doses of MeV and C. rotund us phenolic extract were also tested at nine groups (7+200,7+123.7, 7+75, 5+200, 5+123.7, 5+75, 3+200, 3+123.7, and 3+75) which were (75.3±3.966, 77.233±4.038, 71.066±5.341, 72.1±1.744,68.766±1.429, 62.23±2.66, 66.3±2.179, 58.3±1.513 and 49.433±1.250);respectively. The results showed a synergistic GI among the groups B, C, E, and F (CI=0.623, 0.703, 0.837 and 0.872);respectively. For AMJ13cells the GI of Me Valone was calculated at MOI=2, 3.527 and 5 which were (34.6±2.193, 43.833±2.237 and 54.633±2.203);respectively, and phenolic extract alone at concentrations 75, 148.4 and 200μg/ml which were (41.7±1.375, 54.6±2.551 and 60.9±4.015);respectively. The GIof combination groups (5+200, 5+148.4, 5+75, 3.527+200, 3.527+148.4, 3.527+75, 2+200, 2+148.4, and 2+75) were (78.967±0.603, 69.033±3.853, 64.433±0.945, 69.76±1.975, 71.1±1.212, 60.7±1.803, 65.467±1.804, 61.33±1.935 and 48.6±2.030);respectively. The results showed a synergistic GI among the groups A, C, E, and F (CI=0.582, 0.899, 0.706 and 0.877);respectively. For CAL-51cells the GI of MeV alone was calculated at MOI=7, 9.171and 11 which were (36.067±2.501, 41.2±1.277 and 45.966±2.206);respectively, and phenolic extract alone at concentrations150, 235 and 300μg/ml which were (40.5±1.114, 51.533±1.779 and 57.433±2.65);respectively. The GI of combination groups (11+300, 11+235, 11+150, 9.171+300, 9.171+235, 9.171+150, 7+300, 7+235, and 7+150) were (71.52±2.65, 65.66±1.956, 61±1.87, 68.13±0.6, 66.3±2.326, 62.03±2.859, 64.7±2.66, 61.23±2.608 and 52.76±5.49);respectively. The results showed a synergistic GI among the groups A, E, and F (CI=0.831, 0.86 and 0.811) respectively. The apoptotic effect of MeV alone, C. rotund us phenolic extract alone and in combination on breast cancer cell lines determined using PI/AO stain. The result of apoptosis showed an increase the mean percentage of apoptotic cells in MCF-7, AMJ13, and CAL-51 when treated with MeV vaccine
)45.95%, 48.39% and 42.49%);respectively and C. rotund us phenolic extract (53.22%, 54.3% and 56.85%);respectively when compared to control groups (6.07%, 6.31% and 6.04 %);respectively. On the other hand, the combination of MeV and phenolic extract reflect an elevation in apoptotic effect (74.78%,78.3% and 68.66%) respectively when compared with the effect of each of them alone and with the control groups. The qRT-PCR used to quantitatively evaluate the gene expression of three different proteins (CASP8, CASP9, and P53).Gene expression fold change (2^-ΔΔCt) was calculated in relation to the housekeeping gene (GAPDH) of each exposure groups in compare to control groups. Analysis of gene expression results in comparison with the control groups revealed that MeV increasesCASP-8, but decreasesCASP-9 gene expression in all tested breast cancer cell lines, while P53 upregulated in MCF-7 and AMJ13 cells only. Phenolic extract induced CASP-8 upregulation in AMJ13 and CAL-51 only, whereas CASP-9 and P53 upregulated in MCF-7 and CAL-51 only. Combination result of tow treatments showed an increasing in CASP-8 gene expression in all cell lines, increasing in CASP-9 gene expression in MCF-7 and CAL-51 only, whereas our results showed an increasing in P53 gene expression in MCF-7 and AMJ13 only