L. Di Giambattista Lattanzi

1st Italian1st Italian Wokshop on Wokshop on UltraViolet Techniques UltraViolet Techniques and Applicationsand Applications

““UVB-INDUCED EFFECTS ON UVB-INDUCED EFFECTS ON JURKAT CELLS: A FILTER JURKAT CELLS: A FILTER

PROVIDED BY A VEGETABLE PROVIDED BY A VEGETABLE MIXTURE”MIXTURE”

CollaboratorsCollaborators

P. Grimaldia, S.Gaudenzia, M. Grandid, S. Morronec, D. Pozzic, I. Silvestric and A. Congiu Castellanoa

aDepartment of Physics, Sapienza-RomacDepartment of Experimental Medicine, Sapienza-RomadPoliambulatory La Torre, Torino

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What is the cellular response to UV radiation?

“UV radiation has a variety of physiological and biological effects on the organism including the transformation to malignancy, immune suppression, cellular aging, and the induction of DNA repair and apoptosis……”

…………The cellular response is multifactorial!!

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1

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We studied the induction of apoptosis in Jurkat cells by UVB radiation (wavelength 290–320 nm) at a dose of 310 mJ/cm2. We combined Fourier transform infrared (FTIR) spectroscopy with flow cytometry to determine whether the combination of both techniques could provide new and improved information about cell modifications. To do this, we looked for correspondences and correlations between spectroscopy and flow cytometry data and found three highly probable spectroscopic markers of apoptosis.

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0’ 20’

60’ 120’

210’

420’

An example of result in the protein region (1800-1480 cm-1)…1 2

3

Pozzi et al, Rad. Res. 168, 698-705 (2007)

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Now……..we have studied the induction of apoptosis in Jurkat cells by three different treatments: UVB radiation, vegetable mixture and vegetable mixture plus UVB radiation. Following the same experimental methodology, we have analysed and investigated the possible biological effects induced in tumoral line cells.At irradiation dose and drug concentrations utilized,we have found that the apoptotic process of this cell line is modulated by the drug which seems to filter the UVB radiation.

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THE TREATMENT: vegetable mixture

The mixture contains substances extracted from a lichen (Evernia Prunastri) and other vegetable substances (Epilobium and Urtica dioica).

Recent studies show how the products of lichens are potential filters of UV radiation, thanks to their absorption in this region and to the antioxidant power.

Evernia Prunastri

Evernia Prunastri contains a lichen metabolite, Usnic Acid that has a good UV light filtering power.

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THE TREATMENTS: vegetable mixture plus UVB radiationThe use of this mixture inhibits synergically the apoptotic process induced by UVB radiation in the Jurkat cell line.We have analysed the following cellular samples:

Cells treated with the drug in different concentrations (5,20,30 μl)

Cells radiated with UVB-dose 310 mJ/cm2 at 30 cm from the UV-source

UVB radiated cells for 30 minutes after treatment with the drug in different concentrations (5, 20, 30 μl)

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WHAT IS THE APOPTOSIS ?

The apoptosis is an endogenous program of cellular suicide:

it is a physiological process

it is very different from necrosis

its misfunctioning is at the basis of many diseasesThe apoptotic process also plays a key role in various biological functions.

The difference between Apotosis and Necrosis

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THE APOPTOTIC PROCESS

The morphological changes that the cell undergoes during apoptosis are:

cytoplasmic and nuclear condensation reduction of the cell volume maintenance of the membrane integrity formation of blebs or apoptotic bodies

The process causes no inflammation of the surrounding tissue.

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THE OPTICAL MICROSCOPY: The morphology of Jurkat cells at 210 minutesCTR UV

B

DRUG +UVB

DRUG

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THE RESULTS: Flow Cytometry

1 2

3 40

5

10

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20

UVB 30 l + UVB

% N

ecr

otic

ce

lls

Time[min]060210360

010

20

30

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UVB 30 l + UVB

% A

po

pto

tic c

ells

Time[m

in] 0

60

210

3600102030

40

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UVB 30 l+UVB

% V

iab

le c

ells

Time[min] 0

60210

360

0

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40

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24

0

UVB 30 l + UVB 30 l

% V

iab

le c

ells

Time[hour]

ANALYSE: Flow cytometry

Sample injection

To collection

Catcher Tube Moves in and out stream

To waste

Apoptosis was assessed using annexin V-FITC apoptosis kit (Bender Med. Systems) or propidium iodide PI (DNA probe) and flow cytometry.

In the first case , the percentage of apoptotic cells was determined by the green fluorescence emitted by Annexin V-FITC bound to Phosphatidylserine (PS) exposed to the outer leaflet of the apoptotic cells membrane (flip-flop).

To distinguish the percentage of living, apoptotic or necrotic cells, propidium iodide (PI) was added to the cell.

In the second case, DNA content was assessed with propidium iodide (PI); it enters into damaged cells, intercalates into DNA staining them and emitting a red fluorescence.

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THE RESULTS: FTIR spectroscopy

Fourier transform infrared (FTIR) spectroscopy, which is based on the characteristic molecular vibrational spectra of cells, was used to investigate spectral differences between untreated and treated Jurkat cells.

Absorption peak (cm-1) Assignments

2960 (L1) –CH3 asymmetric stretching, lipids, proteins

2924 (L2) –CH2 asymmetric stretching, lipids, proteins

2872 (L3) –CH3 symmetric stretching, lipids, proteins

2852 (L4) –CH2 symmetric stretching, lipids, proteins

1646 (A1) amide I (–C=O stretching), proteins

1541 (A2) amide II (–N–H bending, –C–N stretching), proteins

1454 (P1) –CH2 scissoring/–CH3 asymmetric bending, proteins, lipids

1399 (P2) –COO- symmetric stretching, proteins, lipids

1236 (P3) -PO2- asymmetric stretching, nucleic acids, phospholipids

1085 (P4) –PO2- symmetric stretching, nucleic acids, phospholipids

1050 (P5) –C–O– stretching, carbohydrates

967 (P6) –PO4- symmetric stretching, nucleic acids

The Lipid (3000-2800 cm-1) and Protein – Nucleic Acids (1800 – 900 cm-1) regions have been analysed.

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AN EXAMPLE OF THE RESULTS: FTIR spectroscopyWe have analysed the ratio between the region area (2800-2865) cm-1 of symmetric CH2 groups (L4) and the region area (2865-2890) cm-1 of symmetric CH3 groups (L3) : CH2 / CH3 (parameter R1) symmetric ratio as function of time (between 0-360 minutes).Correlation between the parameter R1 and the % Apoptotic cells

A

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THE CONCLUSIONS

We have made a comparative study between the effects induced by UVB radiation on Jurkat cells and the effects on the same cells treated with different concentrations of the mixture. The mixture protects the cellular sample from the damage induced by the UVB radiation at the level of some structure in the lipid (3000-2800 cm-1) and the protein-nucleic acids (1800 – 900 cm-1) regions.

Cytofluorometry measurements showed that the ability of UVB radiation to induce apoptosis is attenuated by the treatment with Usnic Acid (lichen metabolite). The mixture can be considered as a potential filter for the UVB radiation.

The FTIR spectroscopy measures have enabled to detect structural changes in the cells related to the response to the stimulation of cells in the early stage of the apoptotic process. We have found some highly probable spectroscopic markers of this process by correlating spectroscopy and flow cytometry data.

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THE FUTURE OUTLOOK

Finally, the Fluorescence Microscopy is in progress in laboratory in order:

to observe and distinguish the different stages of the apoptotic process

to examine the autofluorescene of cellular sample treated with the mixture plus UVB radiation

to establish a stastical model of the distribution of the light peaks using the fluorescence image (software IMAGEJ)

THANKS

TREATMENT: UVB radiation

310 nm

The cell sample was irradiated for 30 minutes at a distance of 30 cm with a UV lamp (Philips, TL20W/12 RS SLV) whose emission maximum is at 310 nm.

The measured radiation dose that hits the sample is 310 mJ/cm2.

Irradiance on a surfacewithIrradiance average

Final value with

Wavelength [nm]

lR

PI

)(2)(

)cos()(

r

R

0

0

)(2

1

0

dII

lr

PI

0

0

2

)sin(

r

d

2arctan0

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