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EVALUATION OF THE VIABILITY OF PTEN
TRANSFECTED MDA-MB – 468 BREAST CANCER
CELLS AGAINST ETOPOSIDE USING MTT ASSAY
Amal Dhivahar S., Vismaya M. R., Neethu Krishna and Dr. Ananda
Mukherjee
EVOGEN 2K19
ABSTRACT
The PTEN (Phosphatase and Tensin homolog deleted on chromosome TEN) is a tumor suppressor gene that negatively controls the
phosphoinositide 3‐kinase signalling pathway for regulation of cell proliferation and cell survival. MDA-MB-468 human breast cancer
cells lack estrogen receptors, over-express epidermal growth factor (EGF) receptors, and are growth inhibited by EGF. Etoposide being
an extensive chemotherapeutic drug is employed to treat several human cancers and remains one of the most highly prescribed
anticancer drugs in the world. In our experiment we made an attempt to examine the viability of four PTEN recombinant plasmid
transfected MDA-MB-468 breast cancer cells, namely, the Enhancer green fluorescent protein – C terminal 1 (EGFPC1), wild type (WT),
nuclear export signal (NES) and the nuclear localization signal (NLS) plasmids, by testing them against the anticancer drug, Etoposide.
The transfection process was carried out using the Lipofectamine 3000 (L3000) and P3000 enhancer reagents in the ratio 1:1 and was
confirmed by fluorescent microscopy. To measure the viability of the transfected cells, we used the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyl tetrazolium bromide) assay and administered the drug at two different concentrations, 0.26µl and 0.65µl per 1.3ml of the
media, to each of the four categories of the transfected cells. After an incubation period of 6 hours, the absorbance readings were
measured using a UV spectrophotometer at 570nm and the % viability values calculated were found to be statistically significant (p ≤
0.05). From both the cases of treatment, it became evident that the breast cancer cells exhibit a greater viability percentage when the
PTEN gene was localized in their nucleus, via pEGFPC1-NLS-PTEN, before the Etoposide treatment.
Keywords: PTEN, MDA-MD-468 cells, Etoposide, recombinant plasmids, MTT assay.
The PTEN Gene
 tumor suppressor gene
 Somatic mutations in PTEN - inactivation - occur in
multiple sporadic tumor types (Baker 2007).
 Germline mutations of PTEN - the inherited hamartoma
and cancer predisposition syndrome - Cowden disease
(Chow and Baker 2006).
 the central negative regulator of the
Phosphatidylinositol-3-kinase (PI3K) signal transduction
cascade (Engelman, Luo, and Cantley 2006).
Proposed Nuclear and Cytoplasmic Function and Regulation of PTEN.
Retrieved from DOI: https://doi.org/10.1016/j.cell.2006.12.023
Cell
Culture
Transfection
Etoposide
Treatment
MTT
Assay
Results
Work Flow
Materials and Methods
Cell Culture
The basal A (Triple-negative Breast cancer) TNBC cell line MDA-MB-468 TNBC grows with a grape cluster like
pattern.
“Because these 3D cultures allow for interaction between the cell and the (Extra cellular matrix) ECM, they are a
more physiological system in which to study the cell lines and their response to therapies” (Chavez J. Kathryn,
Garimella V. Sireesha 2012).
Media Composition
 RPMI 1640 media
 10% FBS - high content of embryonic growth promoting factors
 0.2% HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) buffer - extra buffering capacity (pH 7.2
thro’ 7.6)
 Insulin - to remove or reduce serum in culture and as a growth factor.
Cell Enumeration
Total volume of the culture in the centrifuge tube = 2.75 ml
No. of cells counted = 55 cells (Hemacytometer)
Therefore, the No. of cells present in 2.75ml of the culture media
= 55 x 2.75 x 104 = 1.5125 x 106
The culture was then centrifuged at 1000 rpm for 5 minutes.
The pellet thus obtained was re-suspended in 1 ml of the media.
Hence we obtained a culture media of concentration 1.5125x106
cells/ml.
Transfection
Reagents and Requirements:
 Lipofectamine 3000 (L3000)
 P3000 additional enhancer reagent
 Optimum media
 Recombinant plasmids
“It is to be noted that the ideally 0.2ng of the sample is taken for a 96-well plate and it is increased based upon
the percentage of the super coiled plasmid”
The Four plasmid vectors were employed: pEGFPC1 – 1.187µg/µl
pEGFPC1-WT-PTEN- 6µg/µl
pEGFPC1-NLS-PTEN – 4.5µg/µl
pEGFPC1-NES-PTEN - 4µg/µl
Plasmids &
Components
pEGFPC1 pWT-PTEN pNES-PTEN pNLS-PTEN
Plasmid
volume (µl)
3.75 0.8 1 1.5
P3000 volume
(µl)
9 9 9 9
Optimum
media volume
(µl)
13.55 16.5 16.3 15.8
L3000 volume
(µl)
0.9 0.9 0.9 0.9
Optimum
media volume
(µl)
3.31 3.31 3.31 3.31
Example:
There is 1.18µg of the plasmid for 1µl.
Therefore for 1µg of the plasmid DNA, we have
1/1.18µl
Similarly, as per the protocol,
for 0.6µg of DNA to present in a single well,
= (1/1.18) x 0.6
= 0.5085µl ≈ 3.8µl
Assuming for 6+1 wells = 7 wells,
= 0.6 x 7 = 4.2 ≈ 4.5µg
Therefore, for 4.5µg,
= (4.5/1.18)
= 3.75 ≈ 3.8µl
Etoposide treatment
First case: the first row of the transfected wells + added
with about 10µM/µl of Etoposide per well & the second
row of wells were left undisturbed to serve as the control
for about 1 whole day.
Second case: increased concentration (25µM/µl) &
decreased duration ( about half a day).
Case I:
Concentration of the Etoposide stock = 50Mm/ml
Concentration of the Etoposide working standard =
10µM/µl
Total volume of media in the 12 wells = (12+1) x100 =
1300µl = 1.3ml
V1N1 = V2N2
50 x 103µM/µl x V1 = 10µM/µl x 1.3ml
Therefore, V1 = (10 x 1.3) / (50 x 103) = 0.26 x 10-3ml =
0.26µl of Etoposide added to 1.3ml of media.
The setup is then incubated overnight (≈16 hours) at a
5% CO2 incubator.
Case II:
Concentration of the Etoposide stock = 50mM/ml
Concentration of the Etoposide working standard =
25µM/µl
Total volume of media in the 12 wells = (12+1) x100 =
1300µl = 1.3ml
V1N1 = V2N2
50 x 103µM/µl x V1 = 25µM/µl x 1.3ml
Therefore, V1 = (25 x 1.3) / (50 x 103) = 0.65 x 10-3ml =
0.65µl of Etoposide added to 1.3ml of media.
The setup is then incubated overnight (≈16 hours) at a
5% CO2 incubator.
MTT Assay
 We added 100µl of RPMI media to each well after
removing the old media (if there is presence of any
dead cells in the media)
 10µl of MTT solution was added to each well.
Preparation of the MTT reagent:
Weight of the empty glass bottle = 1037.6g
Weight of the empty glass bottle with the MTT powder
= 1062.7g
Weight of the MTT powder alone = 1062.7 – 1037.6 =
0025.1g = 25.1mg ≈25mg
Solubilizing buffer (pH: 4.7)
= 40% Dimethyl formamide (DMF) – 20ml
+ 2% Glacial acetic acid – 1ml
+ 16% Sodium Dodecyl Sulfate (SDS) – 8g
+ Mill-q water.
25mg of the MTT powder + 5ml of the solubilizing
buffer - filtered - transferred into 5 1ml MCTs (micro-
centrifuge tubes)
= 5 vials containing MTT reagent with the
concentration of 5mg/ml for each vial.
Calculation:
%Inhibition = ((ODcontrol – ODblank) - (ODtreated –
ODblank) ) x 100
%Viability = 100 - %Inhibition
Results and Discussion
Transfection – Fluorescent Microscopy
Green fluorescent images of the plasmids that have been successfully transfected into the MDA-MB-468
breast cancer cells.
A – pEGFPC1-PTEN;
B – pWT-PTEN;
C – pNES-PTEN;
D – pNLS-PTEN.
MTT Assay – UV Spectrophotometry
Case I:
Etoposide
Treatment
pEGFPC1 pWT pNES pNLS Blank
Control
1.3364 1.8298 1.5524 1.8499
0.6368
1.5481 1.8035 1.5693 1.8180
1.3025 1.7413 1.7104 1.8919
0.5140
Average 1.3957 1.7915 1.6107 1.8533
Treated
1.4193 1.5787 1.3721 1.6129
0.6888
1.3873 1.5209 1.2526 1.6776
1.3190 1.4683 1.4989 1.8111
Average 1.3752 1.5226 1.3745 1.7005 0.6132
Etoposide
Treatment
pEGFPC1 pWT pNES pNLS
Control - Blank 0.7825 1.1783 0.9975 1.2401
Treated - Blank 0.7620 0.9094 0.7613 1.0873
%Inhibition 2.05 26.89 23.63 15.28
%Viability 97.95 73.11 76.37 84.72
97.95
73.11
76.37
84.72
0
20
40
60
80
100
120
pEGFPC1-PTEN pWT-PTEN pNES-PTEN pNLS-PTEN
%
Viability
Plasmid transfected MDA-MB-468 cell line
MTT Assay - Case I
the pNLS-PTEN transfected
cell line exhibited higher
%viability compared to the
other PTEN plasmid
transfected cells with 10µM/µl
Etoposide treatment.
Etoposide
Treatment
pEGFPC1 pWT pNES pNLS Blank
Control
1.5870 1.931 1.7626 1.4432
0.423
1.7646 1.9143 1.7111 1.8765
1.5527 1.9014 1.9101 1.5752
0.2982
Average 1.6348 1.9156 1.7946 1.6316
Treated
1.2103 1.6363 1.5591 1.6613
0.5249
1.3072 1.6922 1.6606 1.5465
1.385 1.8281 1.7384 1.3085
Average 1.3008 1.7189 1.6527 1.5054 0.4154
Etoposide
Treatment
pEGFPC1 pWT pNES pNLS
Control - Blank 1.2194 1.5002 1.3792 1.2162
Treated - Blank 0.8854 1.3035 1.2373 1.0900
%Inhibition 33.4 19.67 14.19 12.62
%Viability 66.6 80.33 85.81 87.38
Case II:
66.6
80.33
85.81 87.38
0
10
20
30
40
50
60
70
80
90
100
pEGFPC1-PTEN pWT-PTEN pNES-PTEN pNLS-PTEN
%Viability
Plasmid transfected MDA-MB-468 cell line
MTT Assay - Case II
the pNLS-PTEN transfected
cell line exhibited greater
%viability compared to the
other transfected cells with
25µM/µl Etoposide treatment.
Conclusion
From both the cases of treatment, it is evident that the breast cancer
cells exhibit a greater viability percentage when the PTEN gene is
localised in their nucleus, via pEGFPC1-NLS-PTEN, prior to the
treatment with Etoposide. This shows that the PTEN gene plays a
significant role in the nucleus compared to other parts of the cell.
Thank
you
References:
Baker, Suzanne J. 2007. “PTEN Enters the Nuclear Age.” Cell 128(1): 25–28.
Chow, Lionel M.L., and Suzanne J. Baker. 2006. “PTEN Function in Normal and Neoplastic
Growth.” Cancer Letters 241(2): 184–96.
Chavez J. Kathryn, Garimella V. Sireesha, and Lipkowitz Stanley. 2012. “For Better Treatment
of Triple Negative Breast Cancer.” 32: 35–48.
Engelman, Jeffrey A, Ji Luo, and Lewis C Cantley. 2006. “The Evolution of Phosphatidylinositol
3-Kinases as Regulators of Growth and Metabolism.” Nature Reviews Genetics 7(8): 606–19.
https://doi.org/10.1038/nrg1879.

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Evaluation of the Viability of PTEN Transfected MDA-MB-468 Breast Cancer Cells against Etoposide using MTT Assay

  • 1. EVALUATION OF THE VIABILITY OF PTEN TRANSFECTED MDA-MB – 468 BREAST CANCER CELLS AGAINST ETOPOSIDE USING MTT ASSAY Amal Dhivahar S., Vismaya M. R., Neethu Krishna and Dr. Ananda Mukherjee EVOGEN 2K19
  • 2. ABSTRACT The PTEN (Phosphatase and Tensin homolog deleted on chromosome TEN) is a tumor suppressor gene that negatively controls the phosphoinositide 3‐kinase signalling pathway for regulation of cell proliferation and cell survival. MDA-MB-468 human breast cancer cells lack estrogen receptors, over-express epidermal growth factor (EGF) receptors, and are growth inhibited by EGF. Etoposide being an extensive chemotherapeutic drug is employed to treat several human cancers and remains one of the most highly prescribed anticancer drugs in the world. In our experiment we made an attempt to examine the viability of four PTEN recombinant plasmid transfected MDA-MB-468 breast cancer cells, namely, the Enhancer green fluorescent protein – C terminal 1 (EGFPC1), wild type (WT), nuclear export signal (NES) and the nuclear localization signal (NLS) plasmids, by testing them against the anticancer drug, Etoposide. The transfection process was carried out using the Lipofectamine 3000 (L3000) and P3000 enhancer reagents in the ratio 1:1 and was confirmed by fluorescent microscopy. To measure the viability of the transfected cells, we used the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyl tetrazolium bromide) assay and administered the drug at two different concentrations, 0.26µl and 0.65µl per 1.3ml of the media, to each of the four categories of the transfected cells. After an incubation period of 6 hours, the absorbance readings were measured using a UV spectrophotometer at 570nm and the % viability values calculated were found to be statistically significant (p ≤ 0.05). From both the cases of treatment, it became evident that the breast cancer cells exhibit a greater viability percentage when the PTEN gene was localized in their nucleus, via pEGFPC1-NLS-PTEN, before the Etoposide treatment. Keywords: PTEN, MDA-MD-468 cells, Etoposide, recombinant plasmids, MTT assay.
  • 3. The PTEN Gene  tumor suppressor gene  Somatic mutations in PTEN - inactivation - occur in multiple sporadic tumor types (Baker 2007).  Germline mutations of PTEN - the inherited hamartoma and cancer predisposition syndrome - Cowden disease (Chow and Baker 2006).  the central negative regulator of the Phosphatidylinositol-3-kinase (PI3K) signal transduction cascade (Engelman, Luo, and Cantley 2006). Proposed Nuclear and Cytoplasmic Function and Regulation of PTEN. Retrieved from DOI: https://doi.org/10.1016/j.cell.2006.12.023
  • 5. Materials and Methods Cell Culture The basal A (Triple-negative Breast cancer) TNBC cell line MDA-MB-468 TNBC grows with a grape cluster like pattern. “Because these 3D cultures allow for interaction between the cell and the (Extra cellular matrix) ECM, they are a more physiological system in which to study the cell lines and their response to therapies” (Chavez J. Kathryn, Garimella V. Sireesha 2012). Media Composition  RPMI 1640 media  10% FBS - high content of embryonic growth promoting factors  0.2% HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) buffer - extra buffering capacity (pH 7.2 thro’ 7.6)  Insulin - to remove or reduce serum in culture and as a growth factor. Cell Enumeration Total volume of the culture in the centrifuge tube = 2.75 ml No. of cells counted = 55 cells (Hemacytometer) Therefore, the No. of cells present in 2.75ml of the culture media = 55 x 2.75 x 104 = 1.5125 x 106 The culture was then centrifuged at 1000 rpm for 5 minutes. The pellet thus obtained was re-suspended in 1 ml of the media. Hence we obtained a culture media of concentration 1.5125x106 cells/ml.
  • 6. Transfection Reagents and Requirements:  Lipofectamine 3000 (L3000)  P3000 additional enhancer reagent  Optimum media  Recombinant plasmids “It is to be noted that the ideally 0.2ng of the sample is taken for a 96-well plate and it is increased based upon the percentage of the super coiled plasmid” The Four plasmid vectors were employed: pEGFPC1 – 1.187µg/µl pEGFPC1-WT-PTEN- 6µg/µl pEGFPC1-NLS-PTEN – 4.5µg/µl pEGFPC1-NES-PTEN - 4µg/µl
  • 7. Plasmids & Components pEGFPC1 pWT-PTEN pNES-PTEN pNLS-PTEN Plasmid volume (µl) 3.75 0.8 1 1.5 P3000 volume (µl) 9 9 9 9 Optimum media volume (µl) 13.55 16.5 16.3 15.8 L3000 volume (µl) 0.9 0.9 0.9 0.9 Optimum media volume (µl) 3.31 3.31 3.31 3.31 Example: There is 1.18µg of the plasmid for 1µl. Therefore for 1µg of the plasmid DNA, we have 1/1.18µl Similarly, as per the protocol, for 0.6µg of DNA to present in a single well, = (1/1.18) x 0.6 = 0.5085µl ≈ 3.8µl Assuming for 6+1 wells = 7 wells, = 0.6 x 7 = 4.2 ≈ 4.5µg Therefore, for 4.5µg, = (4.5/1.18) = 3.75 ≈ 3.8µl
  • 8. Etoposide treatment First case: the first row of the transfected wells + added with about 10µM/µl of Etoposide per well & the second row of wells were left undisturbed to serve as the control for about 1 whole day. Second case: increased concentration (25µM/µl) & decreased duration ( about half a day). Case I: Concentration of the Etoposide stock = 50Mm/ml Concentration of the Etoposide working standard = 10µM/µl Total volume of media in the 12 wells = (12+1) x100 = 1300µl = 1.3ml V1N1 = V2N2 50 x 103µM/µl x V1 = 10µM/µl x 1.3ml Therefore, V1 = (10 x 1.3) / (50 x 103) = 0.26 x 10-3ml = 0.26µl of Etoposide added to 1.3ml of media. The setup is then incubated overnight (≈16 hours) at a 5% CO2 incubator. Case II: Concentration of the Etoposide stock = 50mM/ml Concentration of the Etoposide working standard = 25µM/µl Total volume of media in the 12 wells = (12+1) x100 = 1300µl = 1.3ml V1N1 = V2N2 50 x 103µM/µl x V1 = 25µM/µl x 1.3ml Therefore, V1 = (25 x 1.3) / (50 x 103) = 0.65 x 10-3ml = 0.65µl of Etoposide added to 1.3ml of media. The setup is then incubated overnight (≈16 hours) at a 5% CO2 incubator.
  • 9. MTT Assay  We added 100µl of RPMI media to each well after removing the old media (if there is presence of any dead cells in the media)  10µl of MTT solution was added to each well. Preparation of the MTT reagent: Weight of the empty glass bottle = 1037.6g Weight of the empty glass bottle with the MTT powder = 1062.7g Weight of the MTT powder alone = 1062.7 – 1037.6 = 0025.1g = 25.1mg ≈25mg Solubilizing buffer (pH: 4.7) = 40% Dimethyl formamide (DMF) – 20ml + 2% Glacial acetic acid – 1ml + 16% Sodium Dodecyl Sulfate (SDS) – 8g + Mill-q water. 25mg of the MTT powder + 5ml of the solubilizing buffer - filtered - transferred into 5 1ml MCTs (micro- centrifuge tubes) = 5 vials containing MTT reagent with the concentration of 5mg/ml for each vial. Calculation: %Inhibition = ((ODcontrol – ODblank) - (ODtreated – ODblank) ) x 100 %Viability = 100 - %Inhibition
  • 10. Results and Discussion Transfection – Fluorescent Microscopy Green fluorescent images of the plasmids that have been successfully transfected into the MDA-MB-468 breast cancer cells. A – pEGFPC1-PTEN; B – pWT-PTEN; C – pNES-PTEN; D – pNLS-PTEN.
  • 11. MTT Assay – UV Spectrophotometry Case I: Etoposide Treatment pEGFPC1 pWT pNES pNLS Blank Control 1.3364 1.8298 1.5524 1.8499 0.6368 1.5481 1.8035 1.5693 1.8180 1.3025 1.7413 1.7104 1.8919 0.5140 Average 1.3957 1.7915 1.6107 1.8533 Treated 1.4193 1.5787 1.3721 1.6129 0.6888 1.3873 1.5209 1.2526 1.6776 1.3190 1.4683 1.4989 1.8111 Average 1.3752 1.5226 1.3745 1.7005 0.6132 Etoposide Treatment pEGFPC1 pWT pNES pNLS Control - Blank 0.7825 1.1783 0.9975 1.2401 Treated - Blank 0.7620 0.9094 0.7613 1.0873 %Inhibition 2.05 26.89 23.63 15.28 %Viability 97.95 73.11 76.37 84.72
  • 12. 97.95 73.11 76.37 84.72 0 20 40 60 80 100 120 pEGFPC1-PTEN pWT-PTEN pNES-PTEN pNLS-PTEN % Viability Plasmid transfected MDA-MB-468 cell line MTT Assay - Case I the pNLS-PTEN transfected cell line exhibited higher %viability compared to the other PTEN plasmid transfected cells with 10µM/µl Etoposide treatment.
  • 13. Etoposide Treatment pEGFPC1 pWT pNES pNLS Blank Control 1.5870 1.931 1.7626 1.4432 0.423 1.7646 1.9143 1.7111 1.8765 1.5527 1.9014 1.9101 1.5752 0.2982 Average 1.6348 1.9156 1.7946 1.6316 Treated 1.2103 1.6363 1.5591 1.6613 0.5249 1.3072 1.6922 1.6606 1.5465 1.385 1.8281 1.7384 1.3085 Average 1.3008 1.7189 1.6527 1.5054 0.4154 Etoposide Treatment pEGFPC1 pWT pNES pNLS Control - Blank 1.2194 1.5002 1.3792 1.2162 Treated - Blank 0.8854 1.3035 1.2373 1.0900 %Inhibition 33.4 19.67 14.19 12.62 %Viability 66.6 80.33 85.81 87.38 Case II:
  • 14. 66.6 80.33 85.81 87.38 0 10 20 30 40 50 60 70 80 90 100 pEGFPC1-PTEN pWT-PTEN pNES-PTEN pNLS-PTEN %Viability Plasmid transfected MDA-MB-468 cell line MTT Assay - Case II the pNLS-PTEN transfected cell line exhibited greater %viability compared to the other transfected cells with 25µM/µl Etoposide treatment.
  • 15. Conclusion From both the cases of treatment, it is evident that the breast cancer cells exhibit a greater viability percentage when the PTEN gene is localised in their nucleus, via pEGFPC1-NLS-PTEN, prior to the treatment with Etoposide. This shows that the PTEN gene plays a significant role in the nucleus compared to other parts of the cell. Thank you References: Baker, Suzanne J. 2007. “PTEN Enters the Nuclear Age.” Cell 128(1): 25–28. Chow, Lionel M.L., and Suzanne J. Baker. 2006. “PTEN Function in Normal and Neoplastic Growth.” Cancer Letters 241(2): 184–96. Chavez J. Kathryn, Garimella V. Sireesha, and Lipkowitz Stanley. 2012. “For Better Treatment of Triple Negative Breast Cancer.” 32: 35–48. Engelman, Jeffrey A, Ji Luo, and Lewis C Cantley. 2006. “The Evolution of Phosphatidylinositol 3-Kinases as Regulators of Growth and Metabolism.” Nature Reviews Genetics 7(8): 606–19. https://doi.org/10.1038/nrg1879.