Triptolide decreases cell proliferation and induces cell death in triple negative MDA-MB-231 breast cancer cells

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Abstract

Triple negative breast cancers (TNBCs) do not respond to conventional estrogen receptor/progesterone receptor/human epidermal growth factor receptor-2 targeted interventions due to the absence of the respective receptor targets. They are aggressive, exhibit early recurrence, metastasize, are more invasive in nature, and develop drug resistance. Some plant-derived substances have been screened and have gained attention as efficient anticancer drugs for TNBCs with few adverse effects. Here, we evaluate triptolide (concentrations in the range of 100 pM to 10 µM), a di-terpene tri-epoxide isolated from thunder god vine for its efficacy as anticancer drug in MDA-MB-231 TNBC cells. Cell proliferation and viability were assessed using 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) (MTS) assay and trypan blue exclusion assay, respectively. A flow cytometry-based apoptosis assay was performed by using fluorescein isothiocyanate (FITC)-conjugated annexin V and propidium iodide (PI). Western blotting was performed to determine the levels of apoptotic and autophagy proteins such as caspase 3, LC3B and SQSTM1/p62. Results indicate that in 72 h of 1 nM triptolide treatment, the percentage of cell proliferation in MDA-MB-231 cells declined to 49 ± 18.9% (mean ± standard deviation (SD)), whereas the proliferation rate did not drop below 80% in MCF-7 cells (non-TNBC cells which express the estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2) even at the highest concentration tested (10 µM). The MDA-MB-468 cells showed a similar trend to MDA-MB-231 cells. Furthermore, triptolide treatment for 72 h significantly decreased cell viability at concentrations above 10 nM. Apoptotic cell death assay in 72 h triptolide-treated MDA-MB-231 cells revealed 29.3 ± 10.57% of early apoptotic cells in comparison to the control group (4.61 ± 2.24%). Cell cycle analysis indicated accumulation of cells in sub G0 /G1 phase, indicating apoptosis. Western blot analysis in the 25 nM triptolide treatment group revealed induction of autophagy as shown by a significant decrease in the levels of autophagy marker p62 (by 0.2-fold p < 0.0001) and with an increase in the levels of LC3B-II (by 8-fold p < 0.05). An increase in the levels of the apoptotic marker cleaved caspase 3 (by 4-fold p < 0.05) was also observed in triptolide treated MDA-MB-231 cells. Our data shows that triptolide could be an efficient anticancer agent in the treatment of TNBCs.

Original languageEnglish
Article number163
JournalBiomolecules
Volume8
Issue number4
DOIs
Publication statusPublished - 1 Dec 2018

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Cell proliferation
Cell death
Cell Death
Cells
Cell Proliferation
Breast Neoplasms
Triple Negative Breast Neoplasms
Assays
Autophagy
Progesterone Receptors
Caspase 3
Estrogen Receptors
Pharmaceutical Preparations
Apoptosis
Cell Survival
Trypan Blue
Western Blotting
Flow cytometry
Propidium
Annexin A5

Keywords

  • Anticancer treatment
  • Apoptosis
  • Natural compounds
  • Triple negative breast cancers
  • Triptolide

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology

Cite this

@article{f97ca517882a47f7a336ead366babb20,
title = "Triptolide decreases cell proliferation and induces cell death in triple negative MDA-MB-231 breast cancer cells",
abstract = "Triple negative breast cancers (TNBCs) do not respond to conventional estrogen receptor/progesterone receptor/human epidermal growth factor receptor-2 targeted interventions due to the absence of the respective receptor targets. They are aggressive, exhibit early recurrence, metastasize, are more invasive in nature, and develop drug resistance. Some plant-derived substances have been screened and have gained attention as efficient anticancer drugs for TNBCs with few adverse effects. Here, we evaluate triptolide (concentrations in the range of 100 pM to 10 µM), a di-terpene tri-epoxide isolated from thunder god vine for its efficacy as anticancer drug in MDA-MB-231 TNBC cells. Cell proliferation and viability were assessed using 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) (MTS) assay and trypan blue exclusion assay, respectively. A flow cytometry-based apoptosis assay was performed by using fluorescein isothiocyanate (FITC)-conjugated annexin V and propidium iodide (PI). Western blotting was performed to determine the levels of apoptotic and autophagy proteins such as caspase 3, LC3B and SQSTM1/p62. Results indicate that in 72 h of 1 nM triptolide treatment, the percentage of cell proliferation in MDA-MB-231 cells declined to 49 ± 18.9{\%} (mean ± standard deviation (SD)), whereas the proliferation rate did not drop below 80{\%} in MCF-7 cells (non-TNBC cells which express the estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2) even at the highest concentration tested (10 µM). The MDA-MB-468 cells showed a similar trend to MDA-MB-231 cells. Furthermore, triptolide treatment for 72 h significantly decreased cell viability at concentrations above 10 nM. Apoptotic cell death assay in 72 h triptolide-treated MDA-MB-231 cells revealed 29.3 ± 10.57{\%} of early apoptotic cells in comparison to the control group (4.61 ± 2.24{\%}). Cell cycle analysis indicated accumulation of cells in sub G0 /G1 phase, indicating apoptosis. Western blot analysis in the 25 nM triptolide treatment group revealed induction of autophagy as shown by a significant decrease in the levels of autophagy marker p62 (by 0.2-fold p < 0.0001) and with an increase in the levels of LC3B-II (by 8-fold p < 0.05). An increase in the levels of the apoptotic marker cleaved caspase 3 (by 4-fold p < 0.05) was also observed in triptolide treated MDA-MB-231 cells. Our data shows that triptolide could be an efficient anticancer agent in the treatment of TNBCs.",
keywords = "Anticancer treatment, Apoptosis, Natural compounds, Triple negative breast cancers, Triptolide",
author = "Elizabeth Varghese and {Mathews Samuel}, Samson and Sharon Varghese and Sohaila Cheema and Ravinder Mamtani and Dietrich Busselberg",
year = "2018",
month = "12",
day = "1",
doi = "10.3390/biom8040163",
language = "English",
volume = "8",
journal = "Biomolecules",
issn = "2218-273X",
publisher = "Multidisciplinary Digital Publishing Institute (MDPI)",
number = "4",

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TY - JOUR

T1 - Triptolide decreases cell proliferation and induces cell death in triple negative MDA-MB-231 breast cancer cells

AU - Varghese, Elizabeth

AU - Mathews Samuel, Samson

AU - Varghese, Sharon

AU - Cheema, Sohaila

AU - Mamtani, Ravinder

AU - Busselberg, Dietrich

PY - 2018/12/1

Y1 - 2018/12/1

N2 - Triple negative breast cancers (TNBCs) do not respond to conventional estrogen receptor/progesterone receptor/human epidermal growth factor receptor-2 targeted interventions due to the absence of the respective receptor targets. They are aggressive, exhibit early recurrence, metastasize, are more invasive in nature, and develop drug resistance. Some plant-derived substances have been screened and have gained attention as efficient anticancer drugs for TNBCs with few adverse effects. Here, we evaluate triptolide (concentrations in the range of 100 pM to 10 µM), a di-terpene tri-epoxide isolated from thunder god vine for its efficacy as anticancer drug in MDA-MB-231 TNBC cells. Cell proliferation and viability were assessed using 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) (MTS) assay and trypan blue exclusion assay, respectively. A flow cytometry-based apoptosis assay was performed by using fluorescein isothiocyanate (FITC)-conjugated annexin V and propidium iodide (PI). Western blotting was performed to determine the levels of apoptotic and autophagy proteins such as caspase 3, LC3B and SQSTM1/p62. Results indicate that in 72 h of 1 nM triptolide treatment, the percentage of cell proliferation in MDA-MB-231 cells declined to 49 ± 18.9% (mean ± standard deviation (SD)), whereas the proliferation rate did not drop below 80% in MCF-7 cells (non-TNBC cells which express the estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2) even at the highest concentration tested (10 µM). The MDA-MB-468 cells showed a similar trend to MDA-MB-231 cells. Furthermore, triptolide treatment for 72 h significantly decreased cell viability at concentrations above 10 nM. Apoptotic cell death assay in 72 h triptolide-treated MDA-MB-231 cells revealed 29.3 ± 10.57% of early apoptotic cells in comparison to the control group (4.61 ± 2.24%). Cell cycle analysis indicated accumulation of cells in sub G0 /G1 phase, indicating apoptosis. Western blot analysis in the 25 nM triptolide treatment group revealed induction of autophagy as shown by a significant decrease in the levels of autophagy marker p62 (by 0.2-fold p < 0.0001) and with an increase in the levels of LC3B-II (by 8-fold p < 0.05). An increase in the levels of the apoptotic marker cleaved caspase 3 (by 4-fold p < 0.05) was also observed in triptolide treated MDA-MB-231 cells. Our data shows that triptolide could be an efficient anticancer agent in the treatment of TNBCs.

AB - Triple negative breast cancers (TNBCs) do not respond to conventional estrogen receptor/progesterone receptor/human epidermal growth factor receptor-2 targeted interventions due to the absence of the respective receptor targets. They are aggressive, exhibit early recurrence, metastasize, are more invasive in nature, and develop drug resistance. Some plant-derived substances have been screened and have gained attention as efficient anticancer drugs for TNBCs with few adverse effects. Here, we evaluate triptolide (concentrations in the range of 100 pM to 10 µM), a di-terpene tri-epoxide isolated from thunder god vine for its efficacy as anticancer drug in MDA-MB-231 TNBC cells. Cell proliferation and viability were assessed using 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) (MTS) assay and trypan blue exclusion assay, respectively. A flow cytometry-based apoptosis assay was performed by using fluorescein isothiocyanate (FITC)-conjugated annexin V and propidium iodide (PI). Western blotting was performed to determine the levels of apoptotic and autophagy proteins such as caspase 3, LC3B and SQSTM1/p62. Results indicate that in 72 h of 1 nM triptolide treatment, the percentage of cell proliferation in MDA-MB-231 cells declined to 49 ± 18.9% (mean ± standard deviation (SD)), whereas the proliferation rate did not drop below 80% in MCF-7 cells (non-TNBC cells which express the estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2) even at the highest concentration tested (10 µM). The MDA-MB-468 cells showed a similar trend to MDA-MB-231 cells. Furthermore, triptolide treatment for 72 h significantly decreased cell viability at concentrations above 10 nM. Apoptotic cell death assay in 72 h triptolide-treated MDA-MB-231 cells revealed 29.3 ± 10.57% of early apoptotic cells in comparison to the control group (4.61 ± 2.24%). Cell cycle analysis indicated accumulation of cells in sub G0 /G1 phase, indicating apoptosis. Western blot analysis in the 25 nM triptolide treatment group revealed induction of autophagy as shown by a significant decrease in the levels of autophagy marker p62 (by 0.2-fold p < 0.0001) and with an increase in the levels of LC3B-II (by 8-fold p < 0.05). An increase in the levels of the apoptotic marker cleaved caspase 3 (by 4-fold p < 0.05) was also observed in triptolide treated MDA-MB-231 cells. Our data shows that triptolide could be an efficient anticancer agent in the treatment of TNBCs.

KW - Anticancer treatment

KW - Apoptosis

KW - Natural compounds

KW - Triple negative breast cancers

KW - Triptolide

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U2 - 10.3390/biom8040163

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