Volume 30, Issue 2 (4-2023)
RJMS 2023, 30(2): 0-0 |
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Research code: 00000
Ethics code: IR.IAU.TMU.REC.1397.167
Clinical trials code: 00000
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Hashemi dehaghi M, Noorbazargan H, Akbari N, Eslami M. Cytotoxic effects of Grammosciadium platycarpum on HT-29 colon cancer cells and analysis of caspase3 and MMP2 genes expression using Real-time PCR. RJMS 2023; 30 (2)
URL: http://rjms.iums.ac.ir/article-1-6378-en.html
URL: http://rjms.iums.ac.ir/article-1-6378-en.html
, Maryam.eslami2010@gmail.com
Abstract: (84 Views)
Background: Colon cancer or growth of cancer cells in the rectum is the second most common cancer in the world and the third leading cause of cancer death. Grammosciadium platycarpum has antioxidant and anti-cancer properties due to its biologically active compounds. Therefore, the aim of this study was to investigate the toxicity of Grammosciadium platycarpum extract against HT-29 colon cancer cell line and to evaluate the expression change of MMP2 and caspase3 genes in treated cells
.Methods: In this study, extraction was performed from the leaves of Grammosciadium platycarpum. Grammosciadium platycarpum was purchased from the plant bank of Iran Biological Resources Center and then approved by the botanical department with the herbarium number of 1342. In this study, gastric cancer cell line (HT-29) was purchased from the National Center for Genetic and Biological Resources of Iran and was cultured. Then, HT-29 cancer cell lines were treated by different concentrations of the extract for 24, 48 and 72 hours and the toxicity effects of the extract were evaluated by MTT colorimetric method. Expression of caspase3 and MMP2 genes was examined by real-time PCR. For this purpose, all RNA cells were extracted using RNX-PLUS kit (Sinagen, Iran) and cDNA synthesis using Revert kit AidTM First Strand cDNA Synthesis Kit (Fermentas, USA) was performed. In this study, Real-Time PCR model ABI step one (Applied Biosystems, Foster City, CA, USA) was used. Cell migration was assessed by scratch test.
Results: According to the results obtained in 24 hours, with increasing concentration, cell viability decreased so that at a concentration of 450 μg / ml, only 6.66% of cells were alive. The highest rate of cell death compared to the control group at 72 and was related to the concentration of 450 µg / ml that nearly 98% of cells were destroyed. The highest cell viability rate was related to the concentration of 3.5 µ g / ml and in 24 hours, about 94% of the cells survived. After 72 hours, the lowest concentration of the extract (3.5 µg / ml) had a lethal effect on cancer cells, indicating the dependence of cell survival on both time factor and extract concentration. The IC50 value of Grammosciadium platycarpum extract was calculated on HT-29 cell line for 24, 48 and 72 hours. The results of the experiment showed that the IC50 concentration of Grammosciadium platycarpum extract after 24 hours of treatment was 86.86 µg / ml, after 48 hours of treatment 54.88 µg / ml and after 72 hours of treatment 12.12 µg / ml. It shows that the concentration of IC50 value decreases with increasing time, that means the lethality of the extract of Grammosciadium platycarpum boosts with increasing time. The results show that the expression level of caspase3 gene compared to the reference gene in 24, 48 and 72 hours was 1.54, 1.94 and .3.1, respectively, which indicates a significant increase in the expression of this gene at 48 and 72 hours compared to the control group. Also, the increase in caspase 3 gene expression in 24 hours was not significant compared to the control group. In addition, the expression levels of MMP2 gene at 24, 48 and 72 hours were 0.89, 0.8 and 0.54, respectively. Significant decrease in gene expression was observed at 48 and 72 hours compared to the control group but the decrease in expression at 24 hours was not significant compared to the control group. Scratch test results show a marked reduction in cell migration after treatment with plant extract after 72 hours.
Conclusion: The results of the present study showed that Grammosciadium platycarpum extract has the ability to inhibit the growth and proliferation of HT-29 cancer cells, which is done by inducing apoptosis through a non-caspase-3 pathway in cancer cells. This extract is also able to inhibit the invasion of colon cancer cells by inhibiting cell growth and reducing cell migration. The maximum activity of this extract was at 72 hours and its effect was increased with increasing both time and concentration factors. Therefore, the present study shows that Grammosciadium platycarpumextract has cytotoxic properties and can be used as an effective alternative in the treatment of colon cancer.
.Methods: In this study, extraction was performed from the leaves of Grammosciadium platycarpum. Grammosciadium platycarpum was purchased from the plant bank of Iran Biological Resources Center and then approved by the botanical department with the herbarium number of 1342. In this study, gastric cancer cell line (HT-29) was purchased from the National Center for Genetic and Biological Resources of Iran and was cultured. Then, HT-29 cancer cell lines were treated by different concentrations of the extract for 24, 48 and 72 hours and the toxicity effects of the extract were evaluated by MTT colorimetric method. Expression of caspase3 and MMP2 genes was examined by real-time PCR. For this purpose, all RNA cells were extracted using RNX-PLUS kit (Sinagen, Iran) and cDNA synthesis using Revert kit AidTM First Strand cDNA Synthesis Kit (Fermentas, USA) was performed. In this study, Real-Time PCR model ABI step one (Applied Biosystems, Foster City, CA, USA) was used. Cell migration was assessed by scratch test.
Results: According to the results obtained in 24 hours, with increasing concentration, cell viability decreased so that at a concentration of 450 μg / ml, only 6.66% of cells were alive. The highest rate of cell death compared to the control group at 72 and was related to the concentration of 450 µg / ml that nearly 98% of cells were destroyed. The highest cell viability rate was related to the concentration of 3.5 µ g / ml and in 24 hours, about 94% of the cells survived. After 72 hours, the lowest concentration of the extract (3.5 µg / ml) had a lethal effect on cancer cells, indicating the dependence of cell survival on both time factor and extract concentration. The IC50 value of Grammosciadium platycarpum extract was calculated on HT-29 cell line for 24, 48 and 72 hours. The results of the experiment showed that the IC50 concentration of Grammosciadium platycarpum extract after 24 hours of treatment was 86.86 µg / ml, after 48 hours of treatment 54.88 µg / ml and after 72 hours of treatment 12.12 µg / ml. It shows that the concentration of IC50 value decreases with increasing time, that means the lethality of the extract of Grammosciadium platycarpum boosts with increasing time. The results show that the expression level of caspase3 gene compared to the reference gene in 24, 48 and 72 hours was 1.54, 1.94 and .3.1, respectively, which indicates a significant increase in the expression of this gene at 48 and 72 hours compared to the control group. Also, the increase in caspase 3 gene expression in 24 hours was not significant compared to the control group. In addition, the expression levels of MMP2 gene at 24, 48 and 72 hours were 0.89, 0.8 and 0.54, respectively. Significant decrease in gene expression was observed at 48 and 72 hours compared to the control group but the decrease in expression at 24 hours was not significant compared to the control group. Scratch test results show a marked reduction in cell migration after treatment with plant extract after 72 hours.
Conclusion: The results of the present study showed that Grammosciadium platycarpum extract has the ability to inhibit the growth and proliferation of HT-29 cancer cells, which is done by inducing apoptosis through a non-caspase-3 pathway in cancer cells. This extract is also able to inhibit the invasion of colon cancer cells by inhibiting cell growth and reducing cell migration. The maximum activity of this extract was at 72 hours and its effect was increased with increasing both time and concentration factors. Therefore, the present study shows that Grammosciadium platycarpumextract has cytotoxic properties and can be used as an effective alternative in the treatment of colon cancer.
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