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Effects of Fenugreek on Lung Cancer: In Vitro Study

Authors:

Abstract

Lung cancer is the leading cause (account for 18%) of cancer death in both men and women world-wide.The overall 5-year survival rate for all stages combined is disappointing (15%). The aim of this study is to evaluate the possible cytotoxic effects of fenugreek on lung cancer cell line and to determine its IC50 alone and in combination with cisplatin. And to study the effects of fenugreek on the expression of each of p53 and EGFR QU-DB lung cancer cells were cultured in Eagle's MEM culture media supplemented with 5% FBS and antibiotics. The cells were seeded in 96 well plate and the cytotoxic effects of each of cisplatin [25-0.195 μl/ml (or μg/ml)] and fenugreek [300-1.1719 μl/ml (each one μl is extracted from 25 μg of dried seed)] was determined using neutral red uptake (NRU) assay for 24, 48, and 72 hours in comparison with their corresponding control groups. Combined effect of each of fenugreek and cisplatin was determined also using NRU assay. Cytotoxicity was further assessed by trypan blue exclusion assay at IC50 of each agent for 48 hours duration. Immunocytochemistry assay was performed also to detect EGFR and p53 expression. Cisplatin induced a directly proportional, dose-dependent and time-dependant cytotoxic effect with an IC50 of 8.5 μg/ml and 7.3 μg/ml after 48 hrs and 72 hrs of exposure respectively. Significant differences (p<0.05) were observed in optic density of cisplatin group from that of the control for all tested concentrations. Fenugreek extract also induced a directly proportional, dose-dependent and time-dependant cytotoxic effect in experiments with 48 hrs and 72 hrs of exposure with an IC50 of 88.25 μl/ml and 125 μl/ml respectively (each one μl is extracted from 25 μg of dried seed). While it produces a growth enhancing effect in 24 hrs exposure experiment. Significant differences (p<0.05) were observed in optic density of fenugreek from that of the control at concentrations of 37.5 μl/ml and above. Fenugreek produces an antagonistic action when combined with cisplatin, combination index (CI) >1.3. Cisplatin highly significantly (p<0.005) increased EGFR expression at different concentrations. While fenugreek extract highly significantly (p<0.005) reduced EGFR expression at 300 μl/ml (each one μl is extracted from 25 μg of dried seed). Cisplatinand fenugreek highly significantly (p<0.005) decreased the expression of P53. Monotherapy of fenugreek have anticancer effect on lung cancer cell line, but an antagonizing effect to cisplatin when combined with it. Fenugreek may have a beneficial therapeutic effect in decreasing EGFR expression and decreasing mutant p53 expression. Further study is recommended to explore the effect of fenugreek on other cell cycle proteins and to study their potential beneficial therapeutic effects in vivo.
Medical Journal of Babylon-Vol. 12- No. 1 -2015 ٢٠١٥ - لوﻷا ددﻌﻟا-رﺷﻋ ﻲﻧﺎﺛﻟا دﻠﺟﻣﻟا -ﺔﯾﺑطﻟا لﺑﺎﺑ ﺔﻠﺟﻣ
116
Effects of Fenugreek on Lung Cancer / In Vitro Study
Azher Abdul-Hafidh Jabir* Haider Sabah Kadhim** Adeeb A. Alzubaidy**
* College of Dentistry, Babylon University, Hilla
** College of Medicine, Al-Nahrain University, Baghdad
Received 4 November 2014 Accepted 1 December 2014
Abstract
Lung cancer is the leading cause (account for 18%) of cancer death in both men and women world-wide.The
overall 5-year survival rate for all stages combined is disappointing (15%).
The aim of this study is to evaluate the possible cytotoxic effects of fenugreek on lung cancer cell line and to
determine its IC50 alone and in combination with cisplatin. And to study the effects of fenugreek on the expression
of each of p53 and EGFR
QU-DB lung cancer cells were cultured in Eagle's MEM culture media supplemented with 5% FBS and antibiotics.
The cells were seeded in 96 well plate and the cytotoxic effects of each of cisplatin [25-0.195 µl/ml (or µg/ml)] and
fenugreek [300-1.1719 µl/ml (each one µl is extracted from 25 µg of dried seed)] was determined using neutral red
uptake (NRU) assay for 24, 48, and 72 hours in comparison with their corresponding control groups.
Combined effect of each of fenugreek and cisplatin was determined also using NRU assay. Cytotoxicity was
further assessed by trypan blue exclusion assay at IC50 of each agent for 48 hours duration. Immunocytochemistry
assay was performed also to detect EGFR and p53 expression.
Cisplatin induced a directly proportional, dose-dependent and time-dependant cytotoxic effect with an IC50 of 8.5
µg/ml and 7.3 µg/ml after 48 hrs and 72 hrs of exposure respectively. Significant differences (p<0.05) were
observed in optic density of cisplatin group from that of the control for all tested concentrations.
Fenugreek extract also induced a directly proportional, dose-dependent and time-dependant cytotoxic effect in
experiments with 48 hrs and 72 hrs of exposure with an IC50 of 88.25 µl/ml and 125 µl/ml respectively (each one µl
is extracted from 25 µg of dried seed). While it produces a growth enhancing effect in 24 hrs exposure experiment.
Significant differences (p<0.05) were observed in optic density of fenugreek from that of the control at
concentrations of 37.5 µl/ml and above.
Fenugreek produces an antagonistic action when combined with cisplatin, combination index (CI) >1.3.
Cisplatin highly significantly (p<0.005) increased EGFR expression at different concentrations. While fenugreek
extract highly significantly (p<0.005) reduced EGFR expression at 300 µl/ml (each one µl is extracted from 25 µg
of dried seed).
Cisplatinand fenugreek highly significantly (p<0.005) decreased the expression of P53.
Monotherapy of fenugreek have anticancer effect on lung cancer cell line, but an antagonizing effect to cisplatin
when combined with it.
Fenugreek may have a beneficial therapeutic effect in decreasing EGFR expression and decreasing mutant p53
expression.
Further study is recommended to explore the effect of fenugreek on other cell cycle proteins and to study their
potential beneficial therapeutic effects in vivo.
ﺔﺋرﻟا نﺎطرﺳ ﻰﻠﻋ ﺔﺑﻠﺣﻟا تارﯾﺛﺄﺗ/جﺎﺟزﻟا ﻲﻓ ﺔﺳارد
ﺔﺻﻼﺧﻟا
وﻫ ِ
ﺔﺋرﻟا نﺎطرﺳ نا ُ
بﺑﺳﻟا ُ
يدﺎﯾﻘﻟا) ﻲﻟاوﺣ١٨ (% ُ
بّ
ﺑﺳُ
ﯾ ﻪﻧا ثﯾﺣ ،مﻟﺎﻌﻟا لوﺣ ءﺎﺳﻧﻟاو لﺎﺟرﻟا نﻣ ًﻼﻛ ﻲﻓ نﺎطرﺳﻟا بﺑﺳﺑ ةﺎﻓوﻠﻟ١,٤ ﺔﻟﺎﺣ نوﯾﻠﻣ
ُ
لﺎﻣﻶﻟ بّ
ﯾﺧﻣ ًﺔﻛرﺗﺷﻣ ضرﻣﻟا لﺣارﻣ ّ
لُ
ﻛﻟ ِ
تاوَ
َ
ﺳ ﺔﺳﻣﺧﻟ ﻲﻟﺎﻣﺟﻻا ِ
ءﺎﻘﺑﻟا لدﻌﻣ ناو ،ﺔﻧّ
ﺳﻟﺎﺑ ةﺎﻓو)١٥ .(%
ﻣﺗﺣﻣﻟا ﺔﯾوﻠﺧﻟا ﺔﯾﻣﺳﻟا ِ
تارﯾﺛﺄﺗﻟا مﯾﯾَﺗﻟ ةدﺣ ﻰﻠﻋ ﺎﻬﻧﻣ لﻛﻟ جﺎﺟزﻟا ﻲﻓ فﺻﻧﻠﻟ ﺔﯾوﻠﺧﻟا ﺔﯾﻣﺳﻟا ﺔﻋرﺟﻟا رﯾرَْﺗﻟو ِ
ﺔﺋرﻟا ِ
نﺎطرﺳﻟ يوﻠﺧ
طَ
ﺧ ﻰﻠﻋ ﺔﺑﻠﺣﻠﻟ ﺔﻠ
نﯾﺗﻼﺑﺳﯾﺳﻟا رﺎﻘﻋ ﻊﻣ جزﺎﻣﺗﻟﺎﺑو . ِ
رﯾﺑﻌﺗ ﻰﻠﻋ ﺎﻬﻧﻣ ّ
لُ
ِ
تارﯾﺛﺄﺗ ﺔَ
ﺳارِ
دﻟ كﻟذﻛوp53 وEGFR .
عوـﻧ ِ
ﺔـﺋر ِ
نﺎطرـﺳ ﺎـﯾﻼﺧ تـﻋرزQU-DB عوـﻧ نـﻣ تﺑﻧﺗـﺳﻣ ﻲـﻓEagle's MEM ﺔﺑـﺳﻧﺑ ﻊﯾـﺿرﻟا لـﺟﻌﻟا لـﺻﻣﺑ لـ
َ
ُ
ﻣﻟا٥ %ﺔـﯾوﯾﺣﻟا تادﺎـﺿﻣﻟﺎﺑو .
Medical Journal of Babylon-Vol. 12- No. 1 -2015 ٢٠١٥ - لوﻷا ددﻌﻟا-رﺷﻋ ﻲﻧﺎﺛﻟا دﻠﺟﻣﻟا -ﺔﯾﺑطﻟا لﺑﺎﺑ ﺔﻠﺟﻣ
117
تاذ عرز ﺔﺣﯾﻔﺻ ﻲﻓ ﺎﯾﻼﺧﻟا تﻋرز٩٦ زـﯾﻛرﺗﺑ نﯾﺗﻼﺑـﺳﯾﺳﻟا نـﻣ لـﻛﻟ ﺔـﯾوﻠﺧﻟا ﺔﯾﻣـﺳﻟا ِ
تارﯾﺛﺄـﺗﻟا تددـﺣو ةرـﻔﺣ]٢٥-٠,١٩٥ رـﺗﻠﯾﻠﻣ لـﻛﻟ رﺗﻟورﻛﯾﺎـﻣ) وا
رـــﺗﻠﯾﻠﻣ لـــﻛﻟ مارﻏورﻛﯾﺎــﻣ [( ﺔـــﺑﻠﺣﻟاو]٣٠٠-١,١٧١٩ ـــﺗﻠﯾﻠﻣ لـــﻛﻟ رﺗﻟورﻛﯾﺎـــﻣ ر) نـــﻣ رﺗﻟورﻛﯾﺎـــﻣ لـــﻛ صﻼﺧﺗــﺳا مـــﺗ٢٥ ﺔـــﻓﺎﺟﻟا روذـــﺑﻟا نـــﻣ مارﻏورﻛﯾﺎـــﻣ [(
ﺔﺳﯾﺎﻘﻣ مادﺧﺗﺳﺎﺑﻟا ﺔﻐﺑﺻ لﯾﺛﻣﺗ ِ
رﻣﺣﻷا طﺑﻘﺔﻟدﺎﻌﺗﻣﻟا(NRU) ل٢٤ ,٤٨و ،٧٢
ِ
ﺔـﻘﺑﺎطﻣﻟا ِ
ﺔﯾـﺳﺎﯾﻘﻟا مﻬﻌﯾﻣﺎـﺟﻣﺑ ﺔﻧرﺎﻘﻣﻟﺎﺑ ﺔﻋﺎﺳ . تارﯾﺛﺄـﺗﻟا تددـﺣ كﻟذـﻛ
ِ
رﻣﺣﻷا طﺑﻗ ﺔﺳﯾﺎﻘﻣ مادﺧﺗﺳﺎﺑ نﯾﺗﻼﺑﺳﯾﺳﻟاو ﺔﺑﻠﺣﻠﻟ ﺔﻛرﺗﺷﻣﻟا ِ
دﯾﺎﺣﻣﻟا(NRU) .
قرزﻻا نﺎــﺑﯾرﺗﻟا دﺎﻌﺑﺗــﺳا ِ
ﺔـﺑرﺟﺗ مادﺧﺗــﺳﺎﺑ دــﻌﺑأ لﻛــﺷﺑ تـَ
ّ
ُ
ﻗ ﺔــﯾوﻠﺧﻟا ﺔﯾﻣــﺳﻟا ِ
تارﯾﺛﺄـﺗﻟا نا(trypan blue) ﻲــﻓ فــﺻﻧﻠﻟ ﺔــﯾوﻠﺧﻟا ﺔﯾﻣـﺳﻟا ﺔــرﺟﻟا دــﻧﻋ
ِ
ةّ
دﻣﻟ لﻣﺎﻋ ّ
لُ
ﻛﻟ جﺎﺟزﻟا٤٨
ِ
ﺔﻋﺎﺳ .ﯾﺑﻌﺗ فﺎﺷِﻛﻹ ﺔ
ِ
ﻋﺎﻧَ
ﻣﻟا ﺔﯾﺟوﻟوﺗﯾﺳﻟا ُ
ءﺎﯾْ
ﻣﯾِ
ﻛﻟا ﺔﺑرﺟﺗ ﺎﺿﯾا تﯾرﺟا ِ
رEGFR وp 53.
ﻟاو ﺔﻋرﺟﻟا ﻰﻠﻋ ةدﻣﺗﻌﻣو ةرﺷﺎﺑﻣ ﺔﯾﺑﺳﻧ ﺔﯾوﻠﺧ ﺔﯾﻣِ
تارﯾﺛﺄﺗ نﯾﺗﻼﺑﺳﯾﺳﻟا ثدﺣا و جﺎـﺟزﻟا ﻲـﻓ فﺻﻧﻠﻟ ﺔﯾوﻠﺧﻟا ﺔﯾﻣﺳﻟا ﺔﻋرﺟﻟا تﻧﺎﻛو تﻗ)IC50
(يوﺎـﺳﺗ
٨,٥ و رـﺗﻠﯾﻠﻣ لـﻛﻟ مارﻏورﻛﯾﺎـﻣ٧,٣ ةدـﻣﻟ ضﯾرــﻌﺗﻟا دـﻌﺑ رـﺗﻠﯾﻠﻣ لـﻛﻟ مارﻏورﻛﯾﺎــ٤٨ و٧٢ ﻲﻟاوـﺗﻟا ﻰــﻠﻋ ﺔﻋﺎـﺳ . تـظﺣوﻟ ﺎـﻬﺑ دــﺗﻌﻣ تﺎـﻓﻼﺗﺧإ)p <
0.05 (ﺔﺑّ
رﺟُ
ﻣﻟا زﯾﻛارﺗﻟا ّ
لُ
ﻛﻟ ِ
ةرطﯾﺳﻟا ﺔﻋوﻣﺟﻣﻟ ﺔﻠﺛﺎﻣﻣﻟا كﻠﺗ ْ
نِ
ﻣ نﯾﺗﻼﺑﺳﯾﺳﻟا ﺔﻋوﻣﺟﻣﻟ ِ
ﺔﯾرﺻﺑﻟا ِ
ﺔﻓﺎﺛﻛﻟا ﻲﻓ.
ﺔﻋرﺟﻟا ﻰﻠﻋ ةدﻣﺗﻌﻣو ةرﺷﺎﺑﻣ ﺔﯾﺑﺳﻧ ﺔﯾوﻠﺧ ﺔﯾﻣﺳ ِ
تارﯾﺛﺄﺗ ﺔﺑﻠﺣﻟا صﻠﺧﺗﺳﻣ ثدﺣا تﻗوﻟاو ةدـﻣﻟ ضﯾرـﻌﺗﻟا دﻌﺑ٤٨ و٧٢ ـﺳﻟا ﺔـﻋرﺟﻟا تـﻧﺎﻛو ﺔﻋﺎـﺳ ﺔﯾﻣ
جﺎﺟزﻟا ﻲﻓ فﺻﻧﻠﻟ ﺔﯾوﻠﺧﻟا)IC50
( يوﺎﺳﺗ٨٨,٢٥ و رﺗﻠﯾﻠﻣ لﻛﻟ رﺗﻟورﻛﯾﺎﻣ١٢٥ ﻲﻟاوﺗﻟا ﻰﻠﻋ رﺗﻠﯾﻠﻣ لﻛﻟ رﺗﻟورﻛﯾﺎﻣ) نـﻣ رﺗﻟورﻛﯾﺎﻣ لﻛ صﻼﺧﺗﺳا مﺗ
٢٥ ﺔﻓﺎﺟﻟا روذﺑﻟا نﻣ مارﻏورﻛﯾﺎﻣ ( ارﯾﺛﺄﺗ صﻠﺧﺗﺳﻣﻟا ثدﺣا ﺎﻣﻧﯾﺑوﻣﻧﻠﻟ ﺎﻧﺳﺣﻣ ةدﻣﻟ ضﯾرﻌﺗﻟا دﻌﺑ٢٤ ﺔﻋﺎﺳ . ﺎﻬﺑ دﺗﻌﻣ تﺎﻓﻼﺗﺧإ تظﺣوﻟ)p<0.05 (
زﯾﻛارﺗﻠﻟ ِ
ةرطﯾﺳﻟا ﺔﻋوﻣﺟﻣﻟ ﺔﻠﺛﺎﻣﻣﻟا كﻠﺗ ْ
نِ
ﻣ ﺔﺑﻠﺣﻟا ﺔﻋوﻣﺟﻣِ
ﺔﯾرﺻﺑﻟا ِ
ﺔﻓﺎﺛﻛﻟا ﻲﻓ٣٧,٥ قوـﻓ ﺎـﻣﻓ رـﺗﻠﯾﻠﻣ لـﻛﻟ رﺗﻟورﻛﯾﺎﻣ .لـﻠﻗ دـﻘﻟ ﺔـﺑﻠﺣﻟا صﻠﺧﺗـﺳﻣ نـﻣ
رﯾﺛﺄﺗا كرﺗﺷﻣﻟا مادﺧﺗﺳﻻا دﻧﻋ نﯾﺗﻼﺑﺳﯾﺳﻟﻪﻌﻣ رﺷؤﻣ نﺎﻛ ثﯾﺣ ﺔﻛارﺷﻟا(CI) نﻣ رﺑﻛا)١,٣.( ﺎﻬﺑ ادﺗﻌﻣ ةدﺎﯾز نﯾﺗﻼﺑﺳﯾﺳﻟا ثدﺣا(p<0.005) رﯾﺑﻌﺗﺑ
EGFR ﺎـﻬﺑ ادـﺗﻌﻣ ﺎﻧﺎـﺻﻘﻧ ﺔـﺑﻠﺣﻟا صﻠﺧﺗـﺳﻣ ثدـﺣا ﺎـﻣﻧﯾﺑ ،ﺔـﻔﻠﺗﺧﻣ زﯾﻛارﺗ ةدﻌﻟ(p<0.005) رـﯾﺑﻌﺗﺑ EGFR ﺎـﯾﻠﻌﻟا زـﯾﻛارﺗﻟا دـﻧﻋ ﺎـﺻوﺻﺧ]٣٠٠
رﺗﻠﯾﻠﻣ لﻛﻟ رﺗﻟورﻛﯾﺎﻣ) نﻣ رﺗﻟورﻛﯾﺎﻣ لﻛ صﻼﺧﺗﺳا مﺗ٢٥ ﺔﻓﺎﺟﻟا روذﺑﻟا نﻣ مارﻏورﻛﯾﺎ.[( ﺎﻧﺎﺻﻘﻧ ﺔﺑﻠﺣﻟا صﻠﺧﺗﺳﻣ و نﯾﺗﻼﺑﺳﯾﺳﻟا نﻣ لﻛ ثدﺣا
ﻪـﺑ ادـﺗﻌﻣ(p<0.005) رــﯾﺑﻌﺗﺑp 53 رﻓﺎـطﻟا. رــﻬظا ت ﺔـﺑﻠﺣﻟا)ةدـﺣ ﻰــﻠﻋ ( ترــﻬظا ﺎـﻬﻧﻛﻟو ،ِ
ﺔــﺋرﻟا ِ
نﺎطرـﺳﻟ يوـﻠﺧ طــﺧ ﻰـﻠﻋ نﺎطرـّ
ﺳﻠﻟ ةدﺎــﺿﻣ ُ
تارﯾﺛﺄـﺗ
ﻛارﺷ ﻪﻌﻣ تﻣدﺧﺗﺳا ﺎﻣدﻧﻋ نﯾﺗﻼﺑﺳﯾﺳﻠﻟ ﺔﺿﻫﺎﻧﻣ تارﯾﺛﺄﺗ. رﯾﺑﻌﺗ ضﯾﻔﺧﺗ ناp 53 و رﻓﺎطﻟاEGFR دﯾﻔﻣ ﻲّ
ﺟﻼﻋ رﯾﺛﺄﺗ ﻪَﻟ ﺎﻣ
ُ
رَ
ﻟ ﺔﺑﻠﺣﻟا ﺔطﺳاوﺑ .
ِ
رﯾﺛﺄﺗ فﺎﺷْ
ِْ
ﺳﻹ دﻌﺑا تﺎﺳاردﺑ ﻲﺻوﻧ
ِ
مﺳﺟﻟا لﺧاد ِ
ةدﯾﻔﻣﻟا ِ
ﺔﯾّ
ﺟﻼﻌﻟا ِ
ﺔﻠﻣﺗﺣﻣﻟا مﻬِﺗارﯾﺛﺄﺗَ
ﺳارِ
دﻟو ِ
ﺔﯾﻠﺧﻟا ِ
ةرود نﻣ ىرﺧا تﺎﻧﯾﺗورﺑ ﻰﻠﻋ ﺔﺑﻠﺣﻟا.
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Introduction
ung cancer is the leading cause
(18%) of cancer death in both
men and women world-wide,
causing 1.4 million deaths per year (1, 2).
The overall 5-year survival rate for all
stages combined is a disappointing (15%)
(3,2,4).
Chemotherapy with cisplatin is associated
with many adverse side effects, such as
nephrotoxicity, ototoxicity, bone marrow
suppression and neurotoxicity (5).
The dominant oncogens that are
frequently involved in lung cancer include
c-MYC, K-RAS, EGFR (epidermal
growth factor receptor), and HER-2/neu.
The commonly deleted or inactivated
tumor suppressor genes include p53
(protein 53 or tumor protein 53), RB,
p16INK4a, and multiple loci on
chromosome 3p. (6, 3,4).
Published reports reveal that DG
inhibits proliferation and induces
apoptosis in a wide variety of human
tumor cells: colon, breast, prostate and
liver as well as osteosarcoma and
leukemia (7, 8).
Trigonellinehas been found to be an
efficient inhibitor of a transcription factor
called nuclear factor E2-related factor 2
(Nrf2), capable of blocking Nrf2-
dependent proteasome activity and
thereby apoptosis protection in pancreatic
cancer cells (9).
It was said that the holy prophet
Muhammad (piece on him and on his aal)
said: “treat yourselves with fenugreek; if
my nation knows what is in fenugreek
they would treat themselves with it even if
it cost an equal weight of gold to its
weight" (10). This famous saying
indicates that fenugreek contain valuable
remedies against a wide range of diseases.
The magnitude of EGFR expression
correlated with increased tumor
chemoresistance and radioresistance in a
variety of in vivo tumors, including
murine carcinoma, squamous cell
carcinoma, ovarian adenocarcinoma,
hepatocarcinoma, and adenosquamous
carcinoma (11, 12, 13).
A number of human cancers, including
colon and lung carcinomas, as well as
L
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osteosarcomas, are associated with either
a missing or mutated p53 gene.
Aims of the study:
To evaluate the possible cytotoxic
effects of fenugreek on QU-DB lung
cancer cell line when used alone and
when used in combination to cisplatin,
and to study the effects of fenugreek on
the expression of p53 and EGFR in these
cells.
Materials and Methods
Lung cancer cell line:
Human lung cancer cell line "QU-DB"
was purchased from national cell bank of
Iran (NCBI),Pasteur Institute of Iran in
Tehran/ Iran, (NCBI Code: C565) (13).
It is a large cell carcinoma cell line. It was
cultured in DMEM + 10% fetal bovine
serum (FBS) at the NCBI and was
adopted in this study in Eagle's MEM
+5% FBS (15).
Preparation of plants' and drugs' stock
solution for cytotoxic investigation
Seeds of fenugreek were obtained from
traditional market and were identified by
the resources division of botany
directorate / Abugraib /Baghdad/ Iraq.
Plant extract was done according to the
procedure mentioned by (16). Briefly,
crude plant seeds (fenugreek) were
pulverized, weighed (2.5 g),
macerated/homogenized and extracted in
10 ml of absolute ethanol for 7 days at 4
°C. The whole solution was then
centrifuged for 2 minutes at 5000 rpm.
Each 1 ml of the supernatant was
subsequently diluted to 10 ml with Hank's
balanced salt solution (HBSS) + 5 mM
HEPES, pre-adjusted to a pH of 7.4 with
0.1 N NaOH. The resultant solution was
filtered through 0.45 micron and then
through 0.2 micron millipore filters.
Cisplatin was used as a solution taken
from the provided vial for intravenous
injection in a concentration of 1mg/ml
(which contains sodium=30 mmol/L).
Sodium chloride (SC) solution (0.18%
which contains sodium=30 mmol/L) was
prepared from sodium chloride solution
(0.9% which contains 150 mmol/L
sodium).
Ethanol (Eth) control stock solution was
prepared by diluting 1 ml of absolute
ethanol (99.9 %) to 10 mL with HBSS + 5
mM {(N-[2-hydroxyethylpiperazine-N′-
[2-ethanesulfonic acid]) (HEPES)}, pre-
adjusted to a pH of 7.4 with 0.1 N NaOH
(16).
The final stock solutions of all the above
mentioned agents were then kept in sterile
dark glass containers and kept in
refrigerator at 4 °C until use.
Seven serial dilutions of each
experimental agent were prepared from
the stock solution in order to span about
250-fold concentration gradient with the
highest final plating concentration set at
300 µl/ml (each one µl is extracted from
25 µg of dried seed) for fenugreek and 25
µl/ml (µg/ml) for cisplatin.
Assessment of cytotoxicity by neutral
red uptake (NRU) assay
The NRU assay was carried out as
previously described (17, 18&19).
Briefly, after incubation of cells with
serial concentrations of tested agents for
desired time interval, the medium was
removed and the cells were incubated
with fresh medium containing 40 µg/ml
neutral red dye for 3 h. The medium was
removed and the plate was rapidly rinsed
with a mixture of 1% CaCl2 / 0.5%
formaldehyde. The dye was extracted into
supernatant with 0.2 ml of solution of 1%
acetic acid/50% ethanol. After agitation
on a microtiter plate shaker (for few
minutes), the optic density (OD) of the
extracted dye was measured at 540 nm
with a microplate spectrophotometers.
The average of the results of the replicates
for each concentration was then obtained.
The cytotoxic effect of each tested
agent was evaluated based on
percentage inhibition values calculated
according to the following formula:
Percentage of inhibition (%) = OD
control OD tested agent
x 100%
OD control
Cytotoxicity of each tested agent is
expressed as 50% inhibitory
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concentrations (IC50) value. The IC50
value is the concentration of tested agents
that causes 50% inhibition or cell death,
averaged from the above mentioned
experiments, and was obtained by plotting
the percentage inhibition versus
concentration of tested agents (20).
Combination test using (NRU) assay
Interaction between cisplatin and
fenugreekwas evaluated by the
isobolographic analysis (a dose-oriented
geometric method of assessing drug
interactions) (21, 22).
Immunocytochemistry (ICC)
Detection of EGFR and p53 expression
was done using immunocytochemical
procedure (23). Briefly, tissue-culture
flasks with cells in exponential phase of
growth (~85% confluent) were selected
each time for ICC as follows:
After exposure to serial concentrations
of tested agents, the plate was incubated
for 48 hours. After incubation, medium
was removed and cells were stained
according to manufacturer's protocol
{Expose mouse specific AP (red)
detection IHC kit, 2012} with the use of
Carazzi's haematoxylin preparation (24)
as a counter stain.
Few drops of glycerol were added to
cover cells in each well and prevent
drying until the time of photographing.
Five sites for each concentration (each
well) were photographed in 2 powers
(10X and 40 X). The color intensity of 5
cells per each site (for EGFR) or 10 cells
(for p53) was measured using digimizer
software (25) and the average of all cells
was taken as the final result for that
concentration of the tested agent for
comparison with those obtained from
control group.
Statistical Analysis
The data were expressed in tables [as
mean ± standard error of the mean
(SEM)] and in figures. Statistical analysis
was done for data of 48 hrs exposure
(n=6) using unpaired student's t-test.
Values with p 0.05 were considered
significant (26).
Results
Cytotoxicity assay and combination
test:
Cytotoxicity assay:
Cisplatinand fenugreek showed directly
proportional cytotoxic effects on lung
cancer (QU-DB) cells with increased
concentration of each agent:
1. Effects of cisplatin {0.1953-25 µl/ml
(µg/ml)} on QU-DB lung cancer cells:
Microscopic assessment:
Microscopic examination of all
experiments of cisplatin (i.e., after 24 hrs,
48 hrs, and 72 hrs of exposure), showed
an obvious decrease in number of viable
cells and an obvious increase in number
of unviable cells in comparison with the
control (SC) see figure (1).
Neutral red uptake (NRU) assay:
At the end of each experiment, the
measured optic density of the extracted
dye was decreased with increasing
concentration of cisplatin in comparison
with the control (SC) group. For the 24
hrs experiment and 72 hrs experiment,
there was an obvious decrease in mean
optic density (n=3) for all concentrations
when being compared to the
corresponding concentrations of the
control (SC) group.
After 48 hrs of exposure, a significant
decrease (p<0.05) was detected in mean
optic density (n=6) for all concentrations
when being compared to the
corresponding concentrations of the
control (SC).
Plotting the values of percentage of
growth inhibition against cisplatin
concentration for 24, 48, and 72 hrs
experiments (figure -2) reveals that about
90% inhibition was achieved in all
experiments at cisplatin concentration of
25 µl/ml (µg/ml), with IC50 of 8.5 and 7.3
µl/ml g/ml) after 48 hrs and 72 hrs of
exposure respectively.
2. Effects of fenugreek (1.17188-300
µl/ml) on QU-DB lung cancer cells:
Microscopic assessment
Microscopic examination showed that
24 hrs exposure to fenugreek did not
obviously affect cell viability, while
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examination after 48 hrs and 72 hrs
revealed a marked decrease in number of
viable cells and an obvious increase in
number of unviable cells in comparison
with the control (Eth) group as shown in
figure (3).
Neutral red uptake (NRU) assay
At the end of 24 hrs experiment, the
mean optic density (n=3 for each
concentration) of the extracted dye of
fenugreek (FG) group was increased in
comparison with that of the control (Eth)
group giving negative values for
percentage of growth inhibition (except
for the first concentration 1.172 µl /ml)
which indicate that more viable cells are
present after exposure to fenugreek.
On the other hand, the optic density was
decreased with increasing concentration
of fenugreek in comparison with the
control (Eth) group in 48 hrs and 72 hrs
experiments. In 48 hrs experiment, (n=6)
a significant decrease (p 0.05) was
detected at concentration of 37.5 µl/ml
and above when being compared to the
corresponding dilutions of the control (p ≤
0.05).
Plotting the values of percentage of
growth inhibition against fenugreek
concentration for 24, 48, and 72 hrs
experiments (figure-4) reveals that
percentage of growth inhibition was
directly proportional to fenugreek
concentration for 48 hrs and 72 hrs
experiments, with IC50 of (88.25 µl/ml)
and (125 µl/ml) respectively. On the other
hand, fenugreek did not affect the growth
of cells after 24 hrs exposure.
Combination tests:
When combining fenugreek with
cisplatinat IC50 (8.5 µl/ml) (Table-1),
there was an obvious decrease in mean
optic density (n=4 for each concentration)
from that of the control (Eth + SC at 8.5
µl/ml) group.
Isobolographic analysis of Interactions
between cisplatin and fenugreek for 70%
toxicity showed antagonistic effects of
fenugreek when combined with cisplatin
as shown in table (2).
Immunocytochemistry (ICC)
Microscopic examination of the
stained QU-DB lung cancer cells revealed
that EGFR expression was totally
cytoplasmic, while that of p53 was
primarily nuclear, for that reason the color
intensity was measured from these areas
accordingly.
1.Effects of cisplatinand fenugreek on
the expression of EGFR:
Cisplatin obviously increased EGFR
expression, and this increment was highly
significant (p<0.005) at concentrations of
6.25, 25 and 50 µl/ml (µg/ml) (figure -5).
On the other hand, fenugreek generally
decrease EGFR expression and this
decrement was highly significant
(p<0.005) at a concentration of 300 µl/ml
(figure-6).
Figure (7) shows the microscopically
noticeable changes mentioned above at
the highest concentrations of each tested
agent.
2.Effects of cisplatinand fenugreek on
the expression of p53:
Cisplatin generally decreased p53
expression, and this decrement was highly
significant (p<0.005) at concentrations of
25 and 50 µl/ml (µg/ml). On the other
hand, cisplatincauses an increased p53
expression at lower concentrations and
this increment was significant at
concentration of 12.5 µl/ml (µg/ml)
(Figure -8).
Fenugreek generally decrease p53
expression and this decrement was highly
significant (p<0.005) at concentrations of
75 µl/ml (figure -9).
Figure-10 shows the microscopically
noticeable changes mentioned above at
the highest concentrations of each tested
agent.
Discussion
Attention is now being directed to find
novel anticancer agents (with their
possible mechanisms of action) alone and
in combination with conventional
anticancer agents (27and 28).
Cell culture provide a good tool for
testing novel agents, as the results
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obtained from different experiments are
both accurately and reproducible.
In the US NCI plant screening program
for testing crude extract, the
recommended incubation time is between
48 and 72 hours (29, 30). For that reason,
the schedule of this study was focused
mainly on 48 hours; finding a novel
anticancer agent effective on cancer cells
in 48 hrs duration is more significant than
finding another one effective in 72 hrs.
Fotakis and Timbrell (32) reported that
NRU and MTT {3-(4,5-dimethylthiazol-
2-yl)-2,5-diphenyl tetrazolium bromide}
are the most sensitive cytotoxicity assay
that show statistically significant
difference between the treated cells and
the controls, especially in detecting
early toxicity.
The two important chemical constituents
of fenugreek with medicinal value; i.e.
diosgenin and trigonelline together with
many other active phytochemicals were
demonstrated to have anticancer activity
when applied separately on different cell
lines including lung cancer cell lines (7
and 9).
Diosgenin, furanones, dioscin,
protodioscin and trigonelline have been
shown to have anticancer activity in mice,
against breast, and colon cancer (33).
Cytotoxic effects of cisplatin on QU-DB
lung cancer cells
The significant decrease (p < 0.05) in
mean optic density readings of all applied
concentrations of cisplatin from those of
the control in 48 hrs experiment and the
obvious changes in viable cell estimate
(figure -1) and growth inhibition (figure-
2) signify directly proportional dose-
dependent cytotoxic effect of cisplatin.
In addition to similarity in pattern
(directly proportional, dose-dependent),
the lower readings of cisplatin in 24 hrs
experiment and the higher readings in 72
hrs experiment (figure-2) from those of
the 48 hrs experiment indicate the time-
dependant effects of the drug. These
results coincide with the known cytotoxic
effects of cisplatin on lung cancer cells (5,
34).
Cytotoxic effects of Fenugreek on QU-
DB lung cancer cells
In the present study, the significant
decrease (p < 0.05) in mean optic density
readings of concentrations 37.5 µl/ml and
above of fenugreekfrom those of the
control in 48 hrs experiment and the
apparent changes in viable cell estimate
and growth inhibition signify directly
proportional dose-dependent cytotoxic
effect of fenugreek. Furthermore, the
noticeable decrease in mean optic density
readings of fenugreek for concentrations
less than 37.5 µl/ml from those of the
control in 48 hrs experiment supports the
above finding
Chen, et al., (7) and Li, et al., (8) showed
that diosgenin, a steroidal saponin present
in fenugreek can inhibit proliferation, and
induce apoptosis in various tumor cells.
These effects are mediated through cell-
cycle arrest, disruption of Ca2+
homeostasis, activation of p53, release of
apoptosis-inducing factor, and modulation
of caspase-3 activity (35 and 36).
Furthermore, Shishodia and Aggarwal,
(37) stated that diosgenin inhibits
osteoclastogenesis, invasion, and
proliferation through the downregulation
of Akt, IκB kinase activation and NF-κB-
regulated gene expression.
Shabbeer, et al., (38) showed that
protodioscin (Another active agent
identified in fenugreek extract) induces
cell death and morphological change
indicative of apoptosis in the leukemic
cell line H-60, but not in gastric cancer
cell line.
On other hand, the negative values of
growth inhibition in 24 hrs experiment
point to the growth enhancing effect of
fenugreek (because optic density is
proportional to the number of viable
cells).This result may point to the
different nutrients that are available in
fenugreek seeds including proteins, amino
acids and vitamins (39). In contrast,
another study (40) showed that fenugreek
alone did not enhance cell viability; yet,
the combined therapy decreased the toxic
effects of cisplatin on Vero cells.
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Furthermore, another study (41)
demonstrate the potential protective effect
of fenugreek seeds against 7,12-
dimethylbenz (α) anthracene (DMBA)-
induced breast cancer in rats. Also, a
chloroform extract of fenugreek seeds was
found to stimulate the proliferation of
MCF-7 (estrogen receptor positive breast
carcinoma) cells, and the latter action is
possibly due to the in vitro estrogenic
activities of fenugreek (42). Another study
detected that trigonelline has similar
results on breast cancer cells (43).
Growth inhibition results for 72 hrs
experiment were consistently lower than
the corresponding results in 48 hrs
experiment (Figure-4). This time-
dependent decrement in cytotoxicity is
similar to the dose-dependent decrement
in cytotoxicity found by Shabbeer, et al.,
(38); they found that the growth inhibitory
effects of fenugreek extract on primary
prostate or htert-immortalized prostate
cells was lost when the dose was
increased. These two findings may again
support the growth enhancing effect of
different nutrients that are available in
fenugreek seeds including proteins, amino
acids and vitamins.
Combination test using (NRU) assay
It is important to investigate the
combined effects of novel agents with
standard therapy (cisplatin in this study)
to know the expected behavior of new
agents when they are rationally introduced
in combination with conventional therapy.
As the value of the combination index for
the above combination was more than 1.3,
this value indicates antagonistic action of
fenugreek to cisplatin (Table 2).
Sakret al., (44) showed that aqueous
fenugreek seed extract ameliorates
adriamycin-induced cytotoxicity and
testicular alterations in albino rats.
Immunocytochemistry (ICC) for p53
and EGFR
Epidermal growth factor receptor
(EGFR) is one of the dominant oncogens
that are frequently involved in lung cancer
(3and 4). EGFR is highly expressed in
lung cancer, and plays an important role
in tumor growth, infiltration and
metastasis (45). EGFR tyrosine kinase
inhibitors are known to contribute
considerably to the extension of
progression-free survival in EGFR-mutant
non-small cell lung cancer. Nevertheless,
a significant percentage of lung cancer
patients do not respond to anti-EGFR
agents and secondary resistance after
initial benefit is a challenging reality
faced by clinicians (46).
The highly significant (p< 0.005) increase
in EGFR expression noticed mainly at
higher concentration (figure -5) may give
a clue to two possibilities; firstly: this
increase in expression may indicate the
resistance of QU-DB lung cancer cells to
chemotherapy by cisplatin. Golding et al.,
(47) and Baiet al., (48) showed that the
up-regulation of the wild-type EGFR or
the expression of its mutants is associated
with resistance of tumor cells to both
chemo- and radiotherapy and poor clinical
outcomes. Secondly: it may indicate the
potent cytotoxic action of cisplatin which
induces a compensatory increase in EGFR
expression to overcome the acute insult on
the cells by cisplatin (treatment-induced
repair mechanisms) (49). EGFR
modulates DNA repair after radiation-
induced damage through association with
the catalytic subunit of DNA protein
kinase (DNA-PKcs) (50).
The highly significant (p<0.005)
decrement in EGFR expression for
fenugreek (figures-6) may point to its
beneficial effect in reducing the resistance
to chemotherapy and radiotherapy. This is
the first report about the effect of
fenugreek on the expression of EGFR.
The opposite effect of fenugreek to that of
cisplatin in regard to EGFR expression
may explain its antagonistic effect to
cisplatin obtained from combination test
by NRU assay (Table -2).
The oncogen p53 is one of the commonly
deleted or inactivated tumor suppressor
genes involved in lung cancer. p53
mutations are common to both small cell
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and non-small cell lung carcinomas (3 and
4).
Normally functioning (wild-type) p53
gene protects the body from cells that
contain DNA damage and mutations. For
that reason, p53 gene and its product p53
protein have been described as “the
guardian of the genome” (6). After
chronic exposure to tobacco-related
carcinogens, p53 gene mutation within the
bronchial epithelium is relatively
common, leading to impaired function of
the p53 protein (51).
Wild-type p53 protein has a very short
half-life and thus is present in only minute
amounts, generally below the detection
level of immunocytochemical methods. In
contrast, mutant p53 proteins are much
more stable than wild-type p53 proteins,
and have a much longer half-life and tend
to accumulate to a high level in tumor
cells; therefore, if a p53 protein is
detectable by immunocytochemistry, it is
generally considered to be a mutant form
(52 and 53).
Loss of the p53 function may cause
resistance to apoptosis that leads to
treatment failure to DNA-damaging
agents (54).
Thus in the present study, the highly
significant (p<0.005) decrease in p53
expression for cisplatin, and fenugreek
(figures 3-20, 3-22 and 3-23) may point to
the beneficial effects of these agents in
reducing the mutant p53 and (possibly)
the recovery of the normal wild type p53.
Both p53 dependent expression of
caspases 6 and 7 and p53-independent
activation of caspases through Bax/Bak
mediated release of cytochrome C
contribute to cisplatin induced tubular
epithelial cell death (55).
In addition to cisplatin's known
mechanism of action as an alkylating
agent, it also causes elevated levels of
wild-type p53 and P21 in a dose-
dependent manner (56).
Shabbeer, et al., (38) showed that a
fenugreek extract down regulates mutant
p53 in DU-145 cells. Raju, et al., (57)
stated that possible mechanisms that could
be involved in the inhibition of HT-29
(human colon cancer) cells by diosgenin
(a fenugreek extract) could be those
relating to modulation of cyclooxygenase-
2 and the activation of nuclear factor-κB,
p53, or p21 expression.
Conclusion
1. Fenugreek has an in vitro cytotoxic
effect against lung cancer with an IC50 of
88.25 µl/ml and 125 µl/ml for 48 hrs and
72 hrs of exposure respectively. While it
produces a protective effect in 24 hrs
exposure experiment.
2. Fenugreek produces an antagonistic
action when combined with cisplatin,
combination index (CI) >1.3.
3. The reduced EGFR expression after
exposure to fenugreek may point to its
possible beneficial effect in reducing the
resistance to chemotherapy and
radiotherapy.
4. The decreased expression of mutant
p53 by fenugreek signifies its beneficial
effect in restoring normal p53 functions.
Medical Journal of Babylon-Vol. 12- No. 1 -2015 ٢٠١٥ - لوﻷا ددﻌﻟا-رﺷﻋ ﻲﻧﺎﺛﻟا دﻠﺟﻣﻟا -ﺔﯾﺑطﻟا لﺑﺎﺑ ﺔﻠﺟﻣ
124
Conc.
µl/ml 0.1953 12.5 25
Control
Cisplatin
DB* cells in comparison to control (SC) group -Effect of cisplatin on QU :Figure (1)
48 hrs after exposure to serial concentrations of cisplatin. White arrow (viable cell),
black arrow (unviable cells)
* = large cell lung cancer cell line.
Figure (2): Effects of cisplatin on the growth of QU-DB* cells after 24, 48, and 72 hrs of
exposure as evidenced by NRU¶ assay.
* = large cell lung cancer cell line.
¶ = Neutral red uptake
0
10
20
30
40
50
60
70
80
90
100
0 5 10 15 20 25 30
Growth inhibition (%)
Concentration (µl/ml )
24
hr
48
hr
72
hr
Medical Journal of Babylon-Vol. 12- No. 1 -2015 ٢٠١٥ - لوﻷا ددﻌﻟا-رﺷﻋ ﻲﻧﺎﺛﻟا دﻠﺟﻣﻟا -ﺔﯾﺑطﻟا لﺑﺎﺑ ﺔﻠﺟﻣ
125
300
150 1.172
Conc.
µl/ml
Control
Fenugreek
Figure (3): Effect of fenugreek on QU-DB* cells in comparison to the control (Eth) group 48
hrs after exposure to serial concentrations of fenugreek. White arrow (viable cell), black arrow
(unviable cell)
* = large cell lung cancer cell line.
Figure (4): Effects of fenugreek on the growth of QU-DB* cells after 24, 48, and 72 hrs of
exposure as evidenced by NRU¶ assay.( Negative value indicates growth enhancing effect)
* = large cell lung cancer cell line.
¶ = Neutral red uptake
-20
-10
0
10
20
30
40
50
60
70
80
90
0 100 200 300 400
Growth inhibition (%)
Concentration (µl/ml of media)
24
hr
48
hr
72
hr
Medical Journal of Babylon-Vol. 12- No. 1 -2015 ٢٠١٥ - لوﻷا ددﻌﻟا-رﺷﻋ ﻲﻧﺎﺛﻟا دﻠﺟﻣﻟا -ﺔﯾﺑطﻟا لﺑﺎﺑ ﺔﻠﺟﻣ
126
Table 1: Combined effects of fenugreek (FG) and cisplatin (CIS) at IC50 of CIS (8.5 µl/ml)
on the growth of QU-DB* cells in comparison to their controls (Eth and SC respectively)
after 48 hrs as evidenced by NRU¶ assay.
Concentration
of Eth and of FG
(µl /ml)
Optic density (mean ± SEM) (n=4) Growth
inhibition(%)
Eth + SC at 8.5 µl/ml FG+CIS at 8.5
µl/ml
18.75 0.259 ± 0.009 0.091 ± 0.007 64.89
37.5 0.260 ± 0.015 0.088 ± 0.010 66.35
75 0.266 ± 0.021 0.077 ± 0.005 71.03
150 0.250 ± 0.021 0.066 ± 0.010 73.45
* = large cell lung cancer cell line.
¶ = Neutral red uptake
Table 2: Combination index (CI) values of the interaction between cisplatin (CIS) and
fenugreek (FG) against QU-DB* cells after 48 hrs as evidenced by NRU¶ assay.
Agent IC50 IC70 IC70 at IC50 of
CIS
CI Interpretation
FG 88.25 173.6 62.5 1.72 antagonism
* = large cell lung cancer cell line.
¶ = Neutral red uptake
Figure (5): Effects of cisplatin (48 hrs exposure)
on mean EGFR¶ expression (1/intensity) in QU-
DB* cells stained by immunocytochemistry.
¶ = epidermal growth factor receptor,
* = large cell lung cancer cell line.
= highly significant (p<0.005)
Figure (6): Effects of fenugreek (48 hrs exposure)
on mean EGFR¶ expression (1/intensity) in QU-DB*
cells stained by immunocytochemistry.
¶ = epidermal growth factor receptor,
* = large cell lung cancer cell line.
= highly significant (p<0.005)
1.5
1.7
1.9
2.1
2.3
2.5
2.7
2.9
50
25
12.5
6.25
3.125
Mean EGFR expression (1/Intensity)
Concentration µl/ml
Cisplatin
Control (SC
(
1.5
1.7
1.9
2.1
2.3
2.5
2.7
2.9
3001507537.518.75
Mean EGFR expression (1/Intensity)
Concentration µl/ml
Fenugreek
Control (Eth
(
Medical Journal of Babylon-Vol. 12- No. 1 -2015 ٢٠١٥ - لوﻷا ددﻌﻟا-رﺷﻋ ﻲﻧﺎﺛﻟا دﻠﺟﻣﻟا -ﺔﯾﺑطﻟا لﺑﺎﺑ ﺔﻠﺟﻣ
127
Cisplatin Control (SC)
fenugreek
Control (Eth)
-
A
-
-
B
-
Figure (7): Effects of cisplatin 50 µl/ml (A) and fenugreek300 µl/ml (B) on EGFR expression.
Figure (8): Effects of cisplatin (48 hrs exposure) on
mean p53¶ expression (1/intensity) in QU-DB* cells
stained by immunocytochemistry.
¶ = protein 53,
* = large cell lung cancer cell line.
= highly significant (p<0.005)
= significant (p<0.05)
Figure (9): Effects of fenugreek (48 hrs exposure)
on mean p53¶ expression (1/intensity) in QU-DB*
cells stained by immunocytochemistry.
¶ = protein 53,
* = large cell lung cancer cell line.
= highly significant (p<0.005)
Cisplatin
Control
fenugreek
Control
-
A
-
-
B
-
Figure (10): Effects of cisplatin 50 µl/ml (A) and fenugreek 300 µl/ml (B) on p53 expression
1.7
1.9
2.1
2.3
2.5
2.7
502512.56.253.125
Mean p53 expression (1/Intensity)
Concentration µl/ml
Cisplatin
Control (SC
(
1.7
1.9
2.1
2.3
2.5
2.7
300
150
75
37.5
18.75
Mean p53 expression (1/Intensity)
Concentration µl/ml
Fenugreek
Control (Eth
(
Medical Journal of Babylon-Vol. 12- No. 1 -2015 ٢٠١٥ - لوﻷا ددﻌﻟا-رﺷﻋ ﻲﻧﺎﺛﻟا دﻠﺟﻣﻟا -ﺔﯾﺑطﻟا لﺑﺎﺑ ﺔﻠﺟﻣ
128
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... TQ was more effective than alpha-hederin against HEp-2 cells (Rooney & Ryan, 2005). Alfa-hederin a constituent of N. sativa showed anticancer effect on LL/2 (Lewis Lung carcinoma) in BDF1 mice (Jabir et al., 2015). Table 5. ...
... Alpha-hederin Antitumor against LL/2 (Lewis lung carcinoma) (Jabir et al., 2015) HEp-2 cellular model TQ and alpha-hederin 6-40 μM 25-150 μM Inhibited cell proliferation, induced apoptosis and necrosis (Rooney & Ryan, 2005) Lung carcinoma cells Carvacrol Inhibited IL-25, IL-33 and TSLP release, TLRs, SOCS1 and SHIP1 expression and miRNAs, reduced protein levels of TLR4 (Khosravi & Erle, 2016) Abbreviations: HNE, Human neutrophil elastase; ppm, part/milion. ...
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This book provides you with the essential knowledge and skills to undertake and understand evidence-based clinical research. This book is invaluable for researchers and clinicians engaged in a wide range of research studies. A practical, comprehensive, step-by-step guide is provided - from study design, required sample size, selecting the correct statistical test, checking test assumptions, conducting and interpreting statistics, interpretation of effect sizes and P values, to how best report results for presentation and publication. The SPSS commands for methods of statistical analyses frequently conducted in the health care literature are included such, as t-tests, ANOVA, regression, survival analysis, diagnostic and risk statistics etc. In addition, the most relevant corresponding output and interpretation is presented, with clear and concise explanations. Each chapter includes worked research examples with real data sets that can be downloaded. Critical appraisal checklists are also included to help researchers systemically evaluate the results of studies. This new edition includes a new chapter on longitudinal data that includes both a repeated measures and mixed models approach. Furthermore, all commands and output have been updated to IBM Statistics SPSS version 21 and SigmaPlot version 13.
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Background: Cisplatin is a widely used anticancer drug that may induce serious toxicity in normal tissues including the kidneys. In recent times, there has been a surge in the popularity of herbal/traditional medicine. Vero cells, derived from kidney cells of green monkeys, have been used to study cell growth, differentiation, and cytotoxicity induced by different agents or conditions. Aim of work: This study aimed at elucidating the protective effect of the aqueous extract of fenugreek on cisplatin-induced toxicity in the Vero cell line. Materials and methods: Cultured Vero cells were divided into four groups. In group I untreated Vero cells were taken as controls; in group II Vero cells were incubated with 25 μg/ml cisplatin; in group III the cells were incubated with an aqueous extract of fenugreek (20 μg/ml) and in group IV both cisplatin and fenugreek were added simultaneously to Vero cells. The cultured cells of all groups were incubated for 24 and 48 h. Morphological, morphometric, and cytotoxic studies were conducted. Results: On Coomassie staining, cells of group II were seen to be enlarged with the appearance of cytoplasmic vacuoles. A highly significant increase in their nuclear and cytoplasmic areas was observed after 48 h. Compared with group I, the cells from group III revealed a highly significant decrease in nuclear area after 24 h with no significant difference after 48 h. Compared with group II, the cells from group IV showed a decrease in cytoplasmic vacuolization and a highly significant decrease in the nuclear and cytoplasmic areas after 48 h. The mean absorbance using the sulforhodamine B assay was decreased in group II but increased in groups III and IV in a time-dependent manner. Conclusion: Fenugreek alone did not enhance cell viability; yet, the combined therapy decreased the toxic effects of cisplatin on Vero cells. Hence, fenugreek might represent an advisable adjuvant therapy for the protection of tissues sensitive to cisplatin toxicity. Further studies on the effects of fenugreek on the cellular structure are also recommended.