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العنوان
Enhancement of broccoli’s chemo-preventive agent sulforaphane production using plant tissue culture techniques /
المؤلف
Amer, May Ahmed.
هيئة الاعداد
باحث / مي أحمد عامر
مشرف / ثريا رشاد محمد
مشرف / عبدالفتاح بدر محمد بدر
مشرف / رؤفة أحمد عبدالرحمن
مشرف / منال علي شلبي
تاريخ النشر
2021.
عدد الصفحات
279 p. :
اللغة
الإنجليزية
الدرجة
الدكتوراه
التخصص
علوم النبات
تاريخ الإجازة
1/1/2021
مكان الإجازة
جامعة عين شمس - كلية البنات - النبات
الفهرس
Only 14 pages are availabe for public view

from 276

from 276

Abstract

Broccoli (Brassica oleracea var. italica) is one of the most important cruciferous vegetables that belongs to the family Brassicaceae. Broccoli has high content of glucosinolates which have positive potential in cancer treatment. The present investigation was carried out to study the effects of different treatments of abiotic and biotic elicitors on the production and accumulation of sulforaphane and the expression of myrosinase gene from broccoli plant which triggers the synthesis of sulforaphane. In addition, the cytotoxic effect of various sulforaphane treatments on the breast cancer cell line MDA-MB-231 was investigated. Moreover, the transcription levels of the apoptosis marker genes: Bcl-2, Bax, Caspase-3, Caspase-8 and Caspase-9 genes were the main objective of this work. To study these effects, the following experiments were carried out:
• Germination of broccoli seeds under aseptic conditions to produce sterile seedlings and the establishment of hairy root cultures from broccoli seedlings using leaf explants.
• Treatment of 7-days old seedlings and hairy root culture with different concentrations of the elicitors: zinc sulfate, copper sulfate, salicylic acid and methyl jasmonate.
• Extraction of sulforaphane from control and treated cultures, determination of sulforaphane by LC-MS.
• Determination of the myrosinase genes expression in control seedlings and hairy root cultures and elicitors treated seedlings and hairy root cultures using real-time PCR.
• Selection of the highest sulforaphane producing cell lines.
• Determination of the cytotoxic effect of sulforaphane treatments on MDA-MB-231 breast cancer cell line viability using neutral red uptake assay.
• Analysis of apoptosis marker genes transcription using real-time PCR.
The Murashige and Skoog (MS) medium was used for germination of broccoli seeds under aseptic condition in the laboratory to study the effects of different elicitors treatments on the production of sulforaphane in 7-days old seedlings and also in hairy root cultures produced from infected leaf disks of 21 days old seedlings of broccoli infected with strain A4 of Agrobacterium rhizogenes that were also exposed to the treatments of elicitors. Four elicitors were used in this work: copper sulfate (CuSO4), zinc sulfate (ZnSO4), as abiotic elicitors at concentrations of 0, 4 µM, 8 µM and 16 µM for 8 hrs and 16 hrs and methyl jasmonate (MeJA) and salicylic acid (SA) as biotic elicitors at concentrations of 0, 5 µM, 10 µM, 20 µM, 40 µM and 80 µM for 24 hrs and 48 hrs.
Sulforaphane was extracted from control and treated seedlings and in control and hairy root cultures of broccoli and analyzed by the Liquid chromatograph Mass Spectrometer (LC-MS). The results showed that the higher amount of sulforaphane was produced in seedlings treated with 16 µM CuSO4 and 8 µM ZnSO4, after 8 hrs of elicitation and with 80 µM SA and 40 µM of MeJA after 24 hrs of elicitation compared to the control and other treatments of elicitation. While in treated hairy roots, sulforaphane scored the higher amounts at 16 µM ZnSO4 and 4 µM CuSO4 after 16 hrs of elicitation and at 80 µM SA and 40 µM of MeJA after 24 hrs of elicitation.
Real-time PCR (qRT-PCR) was used to evaluate the myrosinase gene expression associated with sulforaphane production. The major finding of this evaluation is that increased production of sulforaphane is clearly associated with over-expression of the myrosinase gene. According to these results, the highest percentage of myrosinase gene up-regulation was detected in seedlings exposed to 4 µM ZnSO4 after 8 hrs of treatment and in seedlings exposed to 4 µM CuSO4 after 16 hrs of treatment. The highest percentage of gene up-regulation was also recorded in seedlings treated with 5 µM SA after 48 hrs of treatment and in seedlings exposed to 80 µM MeJA after 24 hrs of treatment. While, in the treated hairy root cultures, the highest percentage of myrosinase gene up-regulation was detected at 4 µM ZnSO4 after 16 hrs of treatment and in hairy root cultures treated with 4 µM CuSO4 after 8 hrs of treatment. The highest percentage of gene up-regulation was also recorded in hairy root cultures exposed to 40 µM of SA after 24 hrs of treatment and at 5 µM MeJA after 48 hrs of treatment.
The cytotoxic effect of sulforaphane treatments on the viability of the breast cancer cell line MDA-MB-231 was investigated by using neutral red uptake assay. The obtained results showed that, sulforaphane extract obtained from broccoli seedlings treated with MeJA has higher inhibitory effect on the viability of the MDA-MB-231 breast cancer cell line than sulforaphane extract obtained from broccoli seedlings treated with other elicitors. The inhibitory effect was increased by using purified sulforaphane. On the other hand, sulforaphane extract obtained from broccoli hairy roots treated with CuSO4 showed higher inhibitory effect on the MDA-MB-231 breast cancer cell line than sulforaphane extract obtained from broccoli hairy roots treated with other elicitors.
The transcription level of the apoptosis marker genes: Bcl-2, Bax, Caspase-3, Caspase-8 and Caspase-9, were analyzed using real-time PCR. The results showed that all sulforaphane treatments down-regulated the antiapoptotic gene Bcl-2 transcription, while up-regulating the transcription of the proapoptotic genes: Bax, Caspase-3, Caspase-8 and Caspase-9.
In brief, sulforaphane from broccoli seedlings and hairy root cultures caused an enhanced expression of Bax as well as triggering the down regulation of Bcl-2 with the subsequent promotion of the apoptotic activity in MDA-MB-231 breast cancer cells. The present study has established an association between elevated expression of Bax, Caspase-3, Caspase-8, Caspase-9 and decreased expression of Bcl-2 indicating their involvement in the cell apoptosis cascade.
In conclusion, the abiotic (CuSO4, ZnSO4) and biotic (SA and MeJA) elicitors generally increased sulforaphane production in broccoli seedlings and hairy root cultures. The MeJA was more effective as an elicitor for the induction of sulforaphane production in broccoli seedlings than in broccoli hairy root cultures. The anti-cancer effect of different sulforaphane treatments against MDA-MB-231 cell line showed that the sulforaphane extract obtained from broccoli seedlings treated with MeJA has higher inhibitory effect particularly after purification on the silica column. On the other hand, CuSO4 broccoli hairy roots elicitation was more effective as an elicitor for the induction of sulforaphane production in hairy roots. Sulforaphane extracted from broccoli treated hairy roots has more inhibitory effect than sulforaphane extracted from broccoli treated seedlings. Increased sulforaphane production may enhance its anti-cancer activity and thus induce apoptosis in MDA-MB-231 breast cancer cells. The analysis of apoptosis gene transcription showed that all sulforaphane treatments down-regulated the antiapoptotic gene Bcl-2 transcription, while up-regulating the proapoptotic genes Bax, Caspase-3, Caspase-8 and Caspase-9. These results may be an insight into cancer treatment using sulforaphane from broccoli plant.