Metabolic Assay Based Validation Of Cell Viability

Louisiana Tech University College of Engineering and Science

Metabolic Assay Based Validation Of Cell Viability To Inflammatory Stimuli

And Anti Cancer Drugs In Normal Astrocytes And Brain Tumor Cells

Presented by

Bharat Kumar Reddy Karumuri, B.Tech.Major Advisor: Dr. Mark A DeCoster

Cellular Neuroscience Lab 1


☤ Cancer ☤  Inflammation ☤ Metabolism ☤ MTT Assay ☤ EC50

Keywords Introduction Results Conclusions Materials and Methods

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☤ Cancer ☤  Inflammation and Cancer ☤ Role of Astrocytes in Inflammation ☤ Astrocytoma ☤ Apoptosis and Necrosis ☤ Drug Development process ☤ Metabolic Assay Based Validation of Drugs

Introduction Results Conclusions Materials and Methods

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☤ Cell Culture ☤ CRL-2303 ☤ Astrocytes

☤ Treatment of Cells ☤  Inflammatory Stimuli ☤ Anticancer Drugs

☤ Testing Procedures ☤ MTT Assay ☤ Protein Assay

☤ Experimental Setup


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Astrocytoma Cells ☤ Obtained from ATCC ☤ Product Number CRL-2303

Growth Media ☤ DMEM + 10% FBS + 0.5% P/S + 1% Amino Acid

Solution

Cells are grown at 37°C with 5% CO2

Cell Culture AstrocytesAstrocytoma Cells

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Astrocytes ☤  Isolated from primary rat brain cortical cells ☤ Contain 5.5 ± 0.9% microglia and 90-95% glial cells. ☤ Culturing cells in Astrocyte media for 2-3 passages

give purified astrocytes.

Growth Media ☤ Ham’s F-12K media + 5% HS + 5% FBS + 0.5% P/S

Cells are grown at 37°C with 5% CO2

Cell Culture AstrocytesAstrocytoma Cells

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☤  Inflammatory Stimuli ☤ Secreted Phospholipase A2 (sPLA2): belongs to a

superfamily of PLA2 enzymes, responsible for the generation of lysophospholipids and release of fatty acids. ☤ Glutamate (Glu): major excitatory neurotransmitter in

the CNS ☤ Staurosporine (STS): widely used apoptotic inducer.

STS inhibits the calcium dependent protein kinase. ☤ Caffeic Acid Penylethyl Ester (CAPE): inhibitor of

nuclear transcription factor NF-κβ.

Treatment of Cells Anticancer DrugsInflammatory Stimuli

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☤  Glutamate: cell death when treated in excess.

☤  sPLA2: causes inflammation

☤  CAPE Anti Inflammatory and anti carcinogenic

☤  STS: Cause cell death through apoptosis


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Neuron Glioma

Glia

Glu (+/-), sPLA2 (+), STS (-), CAPE (+)

Glu (+/-)

Glu (+/-), sPLA2 (+), STS (-), CAPE (-)


☤ Anticancer Drugs ☤ Menadione: analogue of vitamin K3

☤ Paclitaxel: potent anti-tumor drug ☤ Drug A: unknown novel-natural product


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☤ MTT Assay ☤ [3-(4,5-dimethylthiazol-2-yl)-2,5-

diphenyltetrazolium bromide] ☤ Powdered form, yellow in color, light sensitive ☤ 1.25mg/ml in Locke's solution, add 400 µL/well ☤ 75 minutes incubation (use aluminum foil)

Equilibration Exposure Assay

Seed Cells Add Test Compound Add Assay Reagent Record Data

Testing Procedures Protein AssayMTT Assay

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☤ MTT Assay (continued) ☤ Post incubation reagent is removed carefully ☤ Crystals are solubilized using 91% Isopropanol,

250 µL/well ☤  Transfer 200µL from each well to 96 well plate ☤ Measure Absorbance at 570nM


Seed Cells Add Test Compound Add Assay Reagent Record Data


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Advantages and Disadvantages of MTT Assay

Advantages: ☤  High throughput ☤  Easy to perform ☤  Quantitative

Disadvantages: ☤  Invasive – cells are dead ☤  Does not distinguish

between number of cells and metabolism ☤  Does not differentiate

between Apoptosis and Necrosis.

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☤ Protein Assay ☤ Cell lysate has unknown concentration of protein ☤ Prepare BSA standard soln. for Standard Curve


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Standard Curve

Record Data


Seed Cells Add Test Compound Add Assay Reagents

Protein Extraction

BSA Standard Solution

Serial Dilution

Unknown Concentration of Sample

Protein Estimation


☤ Protein Assay (continued) ☤ Add buffer solution and WST-8 Dye ☤ 20 µL (sample) + 180 µL (buffer soln.) + 20 µL

(WST-8 dye soln.) ☤ Mix it well and incubate for 30 minutes (aluminum

foil) ☤ Measure Absorbance at 640 nm ☤ Quantify the amount of Protein using standard

curve


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Experimental Setup

☤  Brian tumor is surrounded by normal astrocytes so there is a need to study the drug effects on both the normal astrocytes and brain cancer cells.

☤  For the metabolic assay we require 600,000 cells/plate at a concentration of 25K/well.

☤  For Neurons to do on such a large scale was not feasible.

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Glioma

Normal Astrocytes

Neurons


☤ Results ☤ Treatment with sPLA2 ☤ Treatment with Glutamate ☤ Treatment with CAPE ☤ Treatment with Staurospourine ☤ Treatment with Menadione ☤ Treatment with Paclitaxel ☤ Treatment with Drug A


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Results sPLA2 Staurosporine CAPE Menadione Paclitaxel Drug AGlutamate

0 0.5

1 1.5

2 2.5

3

Abs

orba

nce

at 5

70 n

m

Average Absorbance

0 0.05 0.1

0.15 0.2

0.25 0.3

0.35 0.4

0.45

Abs

orba

nce

at 5

70nm

Average Absorbance

CRL-2303 Astrocytes

sPLA2: •  Pro inflammatory agent •  Astrocytes pre-treated with glutamate show more metabolism than the control

(untreated).

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CRL-2303

sPLA2: •  Lower concentration shows positive result but higher concentration showed

neuroprotective nature •  Biphasic nature doesn’t allow to predict the EC50

0.85

0.9

0.95

1

1.05

1.1

1.15 A

bsor

banc

e at

570

nm

Average Absorbance

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Glutamate: •  Excess glutamate causes inflammation •  Higher protein content at higher glutamate concentration •  Further Investigation of results could reveal metabolic pathway à

Apoptosis/Necrosis

0

0.5

1

1.5

2

2.5

Control Glutamate - 1 mM

Glutamate - 2.5 mM

Glutamate - 5mM

Abs

orba

nce

at 5

70 n

m

Average Absorbance

0 0.05 0.1

0.15 0.2

0.25 0.3

0.35

Control Glutamate - 1 mM

Glutamate - 2.5 mM

Glutamate - 5mM

Abs

orba

nce

at 6

40nm

Average Absorbance

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Protein Assay MTT Assay CRL-2303



Glutamate: •  EC50 for glutamate in glioma cells is 1.75 mM

Mean Effective Concentration Plot for Glutamate Treated Glioma Cells

y = -0.971x + 2.6589 0

0.2 0.4 0.6 0.8

1 1.2 1.4 1.6 1.8

2

0 1 2 3 4 5 6

Abs

orba

nce

at 5

70nm

Concentration of Glutamate in mM

Average Absorbance Average Absorbance at 50% Control

Linear (Average Absorbance)

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Staurosporine: •  Known apoptotic agent •  Note the scale difference •  Cells reverse to active stage after the stimulus is removed

Astrocytes CRL-2303

0 0.2 0.4 0.6 0.8

1 1.2 1.4

0 3.5 7.5 11 19.5

Abs

orba

nce

at 5

70 n

m

Time in Hrs

Control Staurosporine 2 µM Staurosporine 500 nM

0 0.05 0.1

0.15 0.2

0.25 0.3

0.35 0.4

Abs

orba

nce

at 5

70nm

Average Absorbance

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Staurosporine: •  EC50 for glioma is

0.359 µM •  EC50 for astrocytes is

1.609 µM. •  EC50 for astrocytes is

much higher than that of the glioma cellsà positive sign in testing the hypothesis.

•  Could be used to induce apoptosis (known).

y = -0.2458x + 0.6447

y = -0.078x + 0.2602

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0 0.5 1 1.5 2 2.5 3

Abs

orba

nce

at 5

70nm

Conentration of STS in µM

Average Absorbance of CRL-2303

Absorbance at 50 % Control for CRL-2303

Average Absorbance of Astrocytes

Absorbance at 50 % Control for Astrocytes

Linear (Average Absorbance of CRL-2303)

Linear (Average Absorbance of Astrocytes)

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CAPE: •  Inhibits the nuclear transcription factor NF-κB and known for its

anti-inflammatory and anti-carcinogenic properties. •  CAPE is dissolved in DMSO hence the corresponding control is

used to see the effect.

Astrocytes CRL-2303

0 0.2 0.4 0.6 0.8

1

Abs

orba

nce

at 5

40nm

Average Absorbace

0 0.05 0.1

0.15 0.2

0.25 0.3

0.35

Abs

orba

nce

at 5

70nm

Average Absorbance

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CAPE: •  EC50 for glioma is

14.21 µM •  EC50 for astrocytes

id 65.20 µM. •  EC50 for astrocytes

is approximately four fold to that of the glioma cells.

y = -0.0023x + 0.2793

y = -0.0084x + 0.5055

0

0.1

0.2

0.3

0.4

0.5

0.6

0 20 40 60 80 100

Aver

age

Abs

orba

nce

at 5

70nm

Concentration of CAPE in µM

Average Absornbance of Astrocytes Absorbance at 50 % Control fro Astrocytes Average Absorbance of CRL-2303 Absorbance at 50 % control for CRL-2303 Linear (Average Absornbance of Astrocytes) Linear (Average Absorbance of CRL-2303)

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Menadione: •  Chemotherapeutic drug •  Significant variation in the absorbance value in between

25 µM and 10 µM

Astrocytes CRL-2303

0 0.1 0.2 0.3 0.4 0.5

Abs

orba

nce

at 5

70nm

Average Absorbance

0 0.1 0.2 0.3 0.4 0.5

Abs

orba

nce

at 5

70nm

Average Absorbance

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Menadione: •  EC50 for glioma is

17.66 µM •  EC50 for astrocytes is

18.75 µM. •  EC50 for both cell are

similar. •  Difficult to treat

glioma without affecting normal cells.

•  May not be a good prospect for further drug screening.

y = -0.022x + 0.6061

y = -0.0128x + 0.423

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0.4

0.45

0 5 10 15 20 25 30

Abs

orba

nce

at 5

70nm

Concentration of Minadione in µM

Average Absorbance of CRL-2303 Absorbance at 50 % Controlfor CRL-2303 Average Absorbance of Astrocytes Absorbance at 50 % Control for Astrocytes Linear (Average Absorbance of CRL-2303) Linear (Average Absorbance of Astrocytes)

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Paclitaxel: •  Known anti cancer drug

CRL-2303

0 0.2 0.4 0.6 0.8

1 1.2

Abs

orba

nce

at 5

70nm

Average Absorbance

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Paclitaxel: •  Tested in presence

and absence of Glutamate.

•  Pre-treatment with Glu has very little effect

•  EC50 for CRL-2303 is 7.19 µM.

•  Px has low EC50 value for glioma which makes it a good prospect.

y = -0.0468x + 0.8101

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

0 2 4 6 8 10

Abs

orba

nce

at 5

70nm

Concentration of Paclitaxel iin µM

Average Absorbance

Average Absorbance at 50 % Control

Linear (Average Absorbance)

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Drug A: •  Natural herbal product •  Unknown dried plants

Astrocytes CRL-2303

0

0.2

0.4

0.6

0.8

1

1.2

Control A - 50x A- 100x A - 250x A - 500x A - 1000x

Abs

orba

nce

at 5

70nm

Average Absorbance

0 0.05 0.1

0.15 0.2

0.25 0.3

0.35 0.4

Control A - 50x A - 100x A - 250x A - 500x

Abs

orba

nce

at 5

70nm

Average Absorbance

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Drug A: •  Tested for its anti-cancer

properties. •  EC50 for CRL-2303 is

74.77 x. •  EC50 for astrocytes is

58.95 x. •  Note: “x” is the times

dilution higher x means lower dilution.

•  Low concentration of rug A can be used in cancer cells for 50 % cell death than the normal astrocytes.

y = 0.0072x - 0.0671

y = 0.0019x + 0.0411

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0 50 100 150 200

Abs

rorb

ance

at 5

70nm

Concentration of Drug A in x fold (x = dilution factor)

Average Absorbance of CRL-2303 Absorbance at 50 % Control for CRL-2303 Average Absorbance of Astrocytes Absorbance at 50 % Control for Astrocytes Linear (Average Absorbance of CRL-2303) Linear (Average Absorbance of Astrocytes)

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Conclusion: ☤ CAPE has shown significant result so it can be

elevated further for advanced analysis. ☤  Staurospourine and Drug A have shown promise

to kill the cancer cells; further experiments can show which of these compounds can be elevated to next level. ☤  Successfully studied the effect of anticancer

drugs and inflammatory stimuli on astrocytoma and normal astrocytes by metabolic assay.


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☤ Mean Effective Concentrations of different compounds for Glioma and Astrocytes


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Future Scope: ☤ Comparative study between the morphological

and metabolic analysis can extend the scope of research. ☤  Co-culturing the normal and cancerous cells

would create much more dynamic environment for invitro testing. ☤  3D cell culture experiments could lead to a better

understanding of the cancer metabolism.


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Acknowledgements: Dr. Mark A. DeCoster Dr. Steven A. Jones Dr. Teresa Murray Dr. David K. Mills Cellular Neuroscience LAB members

Acknowledgements

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Thank you Questions ??

Questions

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