Apoptosis Pathway Targeted by Thymoquinone in MCF7 Breast Cancer Cell Line
Array-based gene expression analysis is a powerful
tool to profile expression of genes and to generate information on
therapeutic effects of new anti-cancer compounds. Anti-apoptotic
effect of thymoquinone was studied in MCF7 breast cancer cell line
using gene expression profiling with cDNA microarray. The purity
and yield of RNA samples were determined using RNeasyPlus Mini
kit. The Agilent RNA 6000 NanoLabChip kit evaluated the quantity
of the RNA samples. AffinityScript RT oligo-dT promoter primer
was used to generate cDNA strands. T7 RNA polymerase was used to
convert cDNA to cRNA. The cRNA samples and human universal
reference RNA were labelled with Cy-3-CTP and Cy-5-CTP,
respectively. Feature Extraction and GeneSpring softwares analysed
the data. The single experiment analysis revealed involvement of 64
pathways with up-regulated genes and 78 pathways with downregulated
genes. The MAPK and p38-MAPK pathways were
inhibited due to the up-regulation of PTPRR gene. The inhibition of
p38-MAPK suggested up-regulation of TGF-ß pathway. Inhibition of
p38-MAPK caused up-regulation of TP53 and down-regulation of
Bcl2 genes indicating involvement of intrinsic apoptotic pathway.
Down-regulation of CARD16 gene as an adaptor molecule regulated
CASP1 and suggested necrosis-like programmed cell death and
involvement of caspase in apoptosis. Furthermore, down-regulation
of GPCR, EGF-EGFR signalling pathways suggested reduction of
ER. Involvement of AhR pathway which control cytochrome P450
and glucuronidation pathways showed metabolism of Thymoquinone.
The findings showed differential expression of several genes in
apoptosis pathways with thymoquinone treatment in estrogen
receptor-positive breast cancer cells.
[1] Subhasis, D., Kumar, D. K., Goutam, D., Ipsita, P., Abhijit, M., Sujata,
M., C, K. S. &Mahitosh, A. M.. Antineoplastic and Apoptotic Potential
of Traditional Medicines Thymoquinone and Diosgenin in Squamous
Cell Carcinoma. PloS One, 7(10), pp.e46641, 2012.
[2] Roepke, M., Diestel, A., Bajbouj, K., Walluscheck, D., Schonfeld, P.,
Roessner, A., Schneider-Stock, R., &Gali-Muhtasib, H. Lack of p53
augments Thymoquinone-induced apoptosis and caspase activation in
human osteosarcoma cells. Cancer BiolTher, 6(2), pp.160-169, 2007.
[3] El-Mahdy, M. A., Zhu, Q., Wang, Q. E., Wani, G. &Wani, A. A.
Thymoquinone induces apoptosis through activation of caspase-8 and
mitochondrial events in p53-null myoloblastic leukemia HL-60 cells. Int
J Cancer, 117pp.409-417, 2005.
[4] Arafa, El- S. A., Zhu, Q., Shah, Z. I., Wani, G., Barakat, B. M., Racoma,
I., El-mahdy, M. A. &Wani, A. A. Thymoquinone up-regulates PTEN
expression and induces apoptosis in doxorubicin-resistant human breast
cancer cells. Mutation Research/Fundamental and Molecular
Mechanisms of Mutagenesis, 706(1-2), pp.28-35, 2010.
[5] Gali-Muhtasib, H., Diab-Assaf, M., Boltze, C., Al-Hmaira, J., Hartig, R.,
Roessner, A., & Schneider-Stock, R. Thymoquinone extracted from
black seed triggers apoptotic cell death in human colorectal cancer cells
via a p53-dependent mechanism. Int J Oncol, 25(4), pp.857-66, 2004.
[6] Kaseb, A., Chinnakannu, K., Chen, D., Sivanandam, A., Tejwani, S.,
Menon, M., Dou, Q., & Reddy, G. Androgen receptor and E2F-1
targeted Thymoquinone therapy for hormone-refractory prostate cancer.
Cancer Res, 67(16), pp.7782-8, 2007.
[7] Woo, C. C., Loo, S. Y., Gee, V., Yap, C. W., Sethi, G., Kumar, A. P.,
Benny, T., &Huat, K. Anticancer activity of Thymoquinone in breast
cancer cells: Possible involvement of PPAR-[gamma] pathway.
Biochemical Pharmacology, 82(5), pp.464-75, 2011.
[8] Broker, L. E., Kruyt, F. A. E. &Giaccone, G. Cell Death Independent of
Caspases: A Review. Clin Cancer Res, 11(9), pp.3155-3162, 2005.
[9] Chen, L., Aann Mayer, J., Krisko, T. I., Speers, C. W., Wang, T.,
Hilsenbeck, S. G. & Brown, P. H. Inhibition of the p38 Kinase
Suppresses the Proliferation of Human ER-Negative Breast Cancer
Cells. Cancer Res 69(23), pp.8853-8861, 2009.
[10] Torres, M. P., Ponnusamy, M. P., Chakraborty, S., Smith, L. M., Das, S.,
Arafat, H. A., &Batra, S. K. Effects of Thymoquinone in the Expression
of Mucin 4 in Pancreatic Cancer Cells: Implications for the
Development of Novel Cancer Therapies. Mol Cancer Ther, 9(5),
pp.1419-1431, 2010.
[11] Mendoza, R. A., Moody, E. E., Enriquez, M. I., Mejia, S. M.,
&Thordarson, G. Tumourigenicity of MCF7 human breast cancer cells
lacking the p38α mitogen-activated protein kinase. J Endocrinol, 208(1),
pp.11-19.British Journal of Cancer, 92(1), pp.113-119, 2011.
[12] NCBI Resources. (2014).Gene. http://www.ncbi.nlm.
[13] Tvrdik, D., Skalova, H., Dundr, P., Povysil, C., Velenska, Z., Berkova,
A., Stanek, L. &Petruzelka, L., Apoptosis - associated genes and their
role in predicting responses to neoadjuvant breast cancer treatment. Med
SciMonit, 18(1), BR60-67, 2012.
[14] Wang, X., Wang, H., Figueroa, B., Zhang, W., Huo, C., Guan, Y.,
Zhang, Y., Bruey, J., Reed, J., &Friendlander, R. Dysregulation of
receptor interacting protein-2 and caspase recruitment domain only
protein mediates aberrant caspase-1 activation in Huntington's disease. J
Neurosci, 25(50), pp.11645-54.
[15] Wang, X., Wang, H., Figueroa, B., Zhang, W., Huo, C., Guan, Y.,
Zhang, Y., Bruey, J., Reed, J., &Friendlander, R. Dysregulation of
receptor interacting protein-2 and caspase recruitment domain only
protein mediates aberrant caspase-1 activation in Huntington's disease. J
Neurosci, 25(50), pp.11645-54.
[16] Marjaneh, M., Al-hassan, FM., &Shahrul H. Thymoquinoneregulates
gene expression levels in the estrogen metabolic and interferon pathways
in MCF7 breast cancer cells. Int. J Mol.Med, 33 (1):8-16, 2014.
[17] Hosgood, H. R., Baris, D., Zhang, Y., Zhu, Y., Zheng, T., Yeager, M.,
Welch, R., Zahm, S., Chanock, S., Rothman, N., &Lan, Q. Caspase
polymorphisms and genetic susceptibility to multiple myeloma.
HematolOncol, 26(3), pp.148-151, 2009.
[18] Smith, AL., Robin, TP., & Ford, Hl. Molecular pathways: targeting the
TGF-β pathway for cancer therapy. Clin Cancer Res, 18(17), pp.4514-
21, 2012.
[19] Dreesen, O. &Brivanlou, A. H. Signalling Pathways in Cancer and
Embryonic Stem Cells. Stem Cell Rev, 3(1), pp.7-17, 2007.
[20] Razandi, M., Oh, P., Pedram, A., Schnitzer, J., & Levin, E. R. ERs
associate with and regulate the production of caveolin: implications for
signalling and cellular actions. MolEndocrinol, 16(1), pp.100-15, 2002
[21] Zhang, G., Ahmed, N., Riley, C., Oliva, K., Barker, G., Quinn, M., &
Rice, G. Enhanced expression of peroxisome proliferator-activated
receptor gamma in epithelial ovarian carcinoma. British Journal of
Cancer, 92(1), pp.113-119, 2005.
[22] Xu, Y., Yang, G., & Hu, G. Binding of IFITM1 enhances the inhibiting
effect of caveolin-1 on ERK activation. ActaBiochimBiophys Sin
(Shanghai), 41(6), pp.488-94, 2009.
[23] Valeria, B., Larbi, A., Cruz, Hinojos, Jeannie, Z., Maureen, G., Mancini,
M., Zelton, D. S., & Mancini, M. A. Activation of Estrogen Receptor-α by E2 or EGF Induces Temporally Distinct Patterns of Large-Scale
Chromatin Modification and mRNA Transcription. PlosOne, 3, e2286,
2008.
[24] Finlay, T. M., Abdulkhalek, S., Gilmour, A., Guzzo, C., Jayanth, P.,
Amith, S. R., Gee, K., Beyaert, R. &Szewczuk, M. R. Thymoquinoneinduced
Neu4 sialidase activates NFκB in macrophage cells and proinflammatory
cytokines in vivo. Glycoconj J, 27(6), pp.583-600, 2010.
[25] Filardo, E. J, Quinn, J. A., Frackelton, AR. JR & Bland, KI. Estrogen
action via the G protein-coupled receptor, GPR30: stimulation of
adenylyl cyclase and cAMP-mediated attenuation of the epidermal
growth factor receptor-to-MAPK signalling axis. MolEndocrinol, 16(1),
pp.70-84, 2012.
[26] Albanito, L., Madeo, A., Lappano, R., Vivacqua, A., Rago, V., Carpino,
A., Oprea, T., Prossnitz, ER.,Musti, AM., Andò, S., Maggiolini, M. G
protein-coupled receptor 30 (GPR30) mediates gene expression changes
and growth response to 17beta-estradiol and selective GPR30 ligand G-1
in ovarian cancer cells. Cancer Res. 67(4):1859-66, 2007.
[27] Koka, P. S., Mondal, D., Schultz, M., Abdel-Mageed., & Agrawal, K. C.
Studies on molecular mechanisms of growth inhibitory effects of
Thymoquinone against prostate cancer cells: role of reactive oxygen
species. Experimental Biology and Medicine, 235(6), pp.751-760, 2010.
[1] Subhasis, D., Kumar, D. K., Goutam, D., Ipsita, P., Abhijit, M., Sujata,
M., C, K. S. &Mahitosh, A. M.. Antineoplastic and Apoptotic Potential
of Traditional Medicines Thymoquinone and Diosgenin in Squamous
Cell Carcinoma. PloS One, 7(10), pp.e46641, 2012.
[2] Roepke, M., Diestel, A., Bajbouj, K., Walluscheck, D., Schonfeld, P.,
Roessner, A., Schneider-Stock, R., &Gali-Muhtasib, H. Lack of p53
augments Thymoquinone-induced apoptosis and caspase activation in
human osteosarcoma cells. Cancer BiolTher, 6(2), pp.160-169, 2007.
[3] El-Mahdy, M. A., Zhu, Q., Wang, Q. E., Wani, G. &Wani, A. A.
Thymoquinone induces apoptosis through activation of caspase-8 and
mitochondrial events in p53-null myoloblastic leukemia HL-60 cells. Int
J Cancer, 117pp.409-417, 2005.
[4] Arafa, El- S. A., Zhu, Q., Shah, Z. I., Wani, G., Barakat, B. M., Racoma,
I., El-mahdy, M. A. &Wani, A. A. Thymoquinone up-regulates PTEN
expression and induces apoptosis in doxorubicin-resistant human breast
cancer cells. Mutation Research/Fundamental and Molecular
Mechanisms of Mutagenesis, 706(1-2), pp.28-35, 2010.
[5] Gali-Muhtasib, H., Diab-Assaf, M., Boltze, C., Al-Hmaira, J., Hartig, R.,
Roessner, A., & Schneider-Stock, R. Thymoquinone extracted from
black seed triggers apoptotic cell death in human colorectal cancer cells
via a p53-dependent mechanism. Int J Oncol, 25(4), pp.857-66, 2004.
[6] Kaseb, A., Chinnakannu, K., Chen, D., Sivanandam, A., Tejwani, S.,
Menon, M., Dou, Q., & Reddy, G. Androgen receptor and E2F-1
targeted Thymoquinone therapy for hormone-refractory prostate cancer.
Cancer Res, 67(16), pp.7782-8, 2007.
[7] Woo, C. C., Loo, S. Y., Gee, V., Yap, C. W., Sethi, G., Kumar, A. P.,
Benny, T., &Huat, K. Anticancer activity of Thymoquinone in breast
cancer cells: Possible involvement of PPAR-[gamma] pathway.
Biochemical Pharmacology, 82(5), pp.464-75, 2011.
[8] Broker, L. E., Kruyt, F. A. E. &Giaccone, G. Cell Death Independent of
Caspases: A Review. Clin Cancer Res, 11(9), pp.3155-3162, 2005.
[9] Chen, L., Aann Mayer, J., Krisko, T. I., Speers, C. W., Wang, T.,
Hilsenbeck, S. G. & Brown, P. H. Inhibition of the p38 Kinase
Suppresses the Proliferation of Human ER-Negative Breast Cancer
Cells. Cancer Res 69(23), pp.8853-8861, 2009.
[10] Torres, M. P., Ponnusamy, M. P., Chakraborty, S., Smith, L. M., Das, S.,
Arafat, H. A., &Batra, S. K. Effects of Thymoquinone in the Expression
of Mucin 4 in Pancreatic Cancer Cells: Implications for the
Development of Novel Cancer Therapies. Mol Cancer Ther, 9(5),
pp.1419-1431, 2010.
[11] Mendoza, R. A., Moody, E. E., Enriquez, M. I., Mejia, S. M.,
&Thordarson, G. Tumourigenicity of MCF7 human breast cancer cells
lacking the p38α mitogen-activated protein kinase. J Endocrinol, 208(1),
pp.11-19.British Journal of Cancer, 92(1), pp.113-119, 2011.
[12] NCBI Resources. (2014).Gene. http://www.ncbi.nlm.
[13] Tvrdik, D., Skalova, H., Dundr, P., Povysil, C., Velenska, Z., Berkova,
A., Stanek, L. &Petruzelka, L., Apoptosis - associated genes and their
role in predicting responses to neoadjuvant breast cancer treatment. Med
SciMonit, 18(1), BR60-67, 2012.
[14] Wang, X., Wang, H., Figueroa, B., Zhang, W., Huo, C., Guan, Y.,
Zhang, Y., Bruey, J., Reed, J., &Friendlander, R. Dysregulation of
receptor interacting protein-2 and caspase recruitment domain only
protein mediates aberrant caspase-1 activation in Huntington's disease. J
Neurosci, 25(50), pp.11645-54.
[15] Wang, X., Wang, H., Figueroa, B., Zhang, W., Huo, C., Guan, Y.,
Zhang, Y., Bruey, J., Reed, J., &Friendlander, R. Dysregulation of
receptor interacting protein-2 and caspase recruitment domain only
protein mediates aberrant caspase-1 activation in Huntington's disease. J
Neurosci, 25(50), pp.11645-54.
[16] Marjaneh, M., Al-hassan, FM., &Shahrul H. Thymoquinoneregulates
gene expression levels in the estrogen metabolic and interferon pathways
in MCF7 breast cancer cells. Int. J Mol.Med, 33 (1):8-16, 2014.
[17] Hosgood, H. R., Baris, D., Zhang, Y., Zhu, Y., Zheng, T., Yeager, M.,
Welch, R., Zahm, S., Chanock, S., Rothman, N., &Lan, Q. Caspase
polymorphisms and genetic susceptibility to multiple myeloma.
HematolOncol, 26(3), pp.148-151, 2009.
[18] Smith, AL., Robin, TP., & Ford, Hl. Molecular pathways: targeting the
TGF-β pathway for cancer therapy. Clin Cancer Res, 18(17), pp.4514-
21, 2012.
[19] Dreesen, O. &Brivanlou, A. H. Signalling Pathways in Cancer and
Embryonic Stem Cells. Stem Cell Rev, 3(1), pp.7-17, 2007.
[20] Razandi, M., Oh, P., Pedram, A., Schnitzer, J., & Levin, E. R. ERs
associate with and regulate the production of caveolin: implications for
signalling and cellular actions. MolEndocrinol, 16(1), pp.100-15, 2002
[21] Zhang, G., Ahmed, N., Riley, C., Oliva, K., Barker, G., Quinn, M., &
Rice, G. Enhanced expression of peroxisome proliferator-activated
receptor gamma in epithelial ovarian carcinoma. British Journal of
Cancer, 92(1), pp.113-119, 2005.
[22] Xu, Y., Yang, G., & Hu, G. Binding of IFITM1 enhances the inhibiting
effect of caveolin-1 on ERK activation. ActaBiochimBiophys Sin
(Shanghai), 41(6), pp.488-94, 2009.
[23] Valeria, B., Larbi, A., Cruz, Hinojos, Jeannie, Z., Maureen, G., Mancini,
M., Zelton, D. S., & Mancini, M. A. Activation of Estrogen Receptor-α by E2 or EGF Induces Temporally Distinct Patterns of Large-Scale
Chromatin Modification and mRNA Transcription. PlosOne, 3, e2286,
2008.
[24] Finlay, T. M., Abdulkhalek, S., Gilmour, A., Guzzo, C., Jayanth, P.,
Amith, S. R., Gee, K., Beyaert, R. &Szewczuk, M. R. Thymoquinoneinduced
Neu4 sialidase activates NFκB in macrophage cells and proinflammatory
cytokines in vivo. Glycoconj J, 27(6), pp.583-600, 2010.
[25] Filardo, E. J, Quinn, J. A., Frackelton, AR. JR & Bland, KI. Estrogen
action via the G protein-coupled receptor, GPR30: stimulation of
adenylyl cyclase and cAMP-mediated attenuation of the epidermal
growth factor receptor-to-MAPK signalling axis. MolEndocrinol, 16(1),
pp.70-84, 2012.
[26] Albanito, L., Madeo, A., Lappano, R., Vivacqua, A., Rago, V., Carpino,
A., Oprea, T., Prossnitz, ER.,Musti, AM., Andò, S., Maggiolini, M. G
protein-coupled receptor 30 (GPR30) mediates gene expression changes
and growth response to 17beta-estradiol and selective GPR30 ligand G-1
in ovarian cancer cells. Cancer Res. 67(4):1859-66, 2007.
[27] Koka, P. S., Mondal, D., Schultz, M., Abdel-Mageed., & Agrawal, K. C.
Studies on molecular mechanisms of growth inhibitory effects of
Thymoquinone against prostate cancer cells: role of reactive oxygen
species. Experimental Biology and Medicine, 235(6), pp.751-760, 2010.
@article{"International Journal of Medical, Medicine and Health Sciences:69545", author = "M. Marjaneh and M. Y. Narazah and H. Shahrul", title = "Apoptosis Pathway Targeted by Thymoquinone in MCF7 Breast Cancer Cell Line", abstract = "Array-based gene expression analysis is a powerful
tool to profile expression of genes and to generate information on
therapeutic effects of new anti-cancer compounds. Anti-apoptotic
effect of thymoquinone was studied in MCF7 breast cancer cell line
using gene expression profiling with cDNA microarray. The purity
and yield of RNA samples were determined using RNeasyPlus Mini
kit. The Agilent RNA 6000 NanoLabChip kit evaluated the quantity
of the RNA samples. AffinityScript RT oligo-dT promoter primer
was used to generate cDNA strands. T7 RNA polymerase was used to
convert cDNA to cRNA. The cRNA samples and human universal
reference RNA were labelled with Cy-3-CTP and Cy-5-CTP,
respectively. Feature Extraction and GeneSpring softwares analysed
the data. The single experiment analysis revealed involvement of 64
pathways with up-regulated genes and 78 pathways with downregulated
genes. The MAPK and p38-MAPK pathways were
inhibited due to the up-regulation of PTPRR gene. The inhibition of
p38-MAPK suggested up-regulation of TGF-ß pathway. Inhibition of
p38-MAPK caused up-regulation of TP53 and down-regulation of
Bcl2 genes indicating involvement of intrinsic apoptotic pathway.
Down-regulation of CARD16 gene as an adaptor molecule regulated
CASP1 and suggested necrosis-like programmed cell death and
involvement of caspase in apoptosis. Furthermore, down-regulation
of GPCR, EGF-EGFR signalling pathways suggested reduction of
ER. Involvement of AhR pathway which control cytochrome P450
and glucuronidation pathways showed metabolism of Thymoquinone.
The findings showed differential expression of several genes in
apoptosis pathways with thymoquinone treatment in estrogen
receptor-positive breast cancer cells.
", keywords = "CARD16, CASP10, cDNA microarray, PTPRR,
Thymoquinone.", volume = "9", number = "1", pages = "87-5", }
