Optical and biochemical methods for the identification of cellular modifications during malignant transformations icon

Optical and biochemical methods for the identification of cellular modifications during malignant transformations




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Optical and biochemical methods for the identification of cellular modifications during malignant transformations


Sergey B. Yermolenko*a, Olexander G. Prydij a, Stefan G. Guminetsky a, Artem V. Motrich a,

Ion Gruiab, Maria Iuliana Gruiac, Teodora Ştefănescud

a Chernivtsi National University, Ukraine; bUniversity of Bucharest, Faculty of Physics, Romania;

cInstitute of Oncology Bucharest, Romania; dHigh School ”C.A. Rosetti” Bucharest, Romania


Abstract

This paper follows to combine optical and biochemical techniques for identification the cell membrane transformation in the dynamic of growth and development of are experimental solid tumour. An increase of the biochemical parameters during to the 14th day of the tumoral growth followed by a decrease of the investigated reactions dove by the absence (in our case) of the polyunsaturated fatty acids from the membrane lipids was shown. It was established than a combination between the polarimetry method with biochemical essays can deliver new date to identify the cell transformation.

Keywords: polarization, biological tissue, malignant tumor, spectropolatimetry.



  1. Introduction


The malignant transformation of normal cell is not a rare event, considering that any cell in the normal cycle division has the potential to become malignant. However most people go through life without a cancer, the detection and control of the body are able to stop the cells to be transformed in tumors [1-4].

Although malignant tumor cells take birth in normal cells, their morphology is much changed from the cells of origin. Optical microscopy studies have allowed to shape characteristics of cellular abnormalities.

Biological membranes plays a crucial role in all cellular phenomena. The main membranes constituents are lipids and proteins.

One of the major progress made in biological and biochemical studies of biological membranes is that they are not static entities with rigid structures. And maintaining the structure of liquid crystal, both lipids and proteins have freedom of movement. Flow can be regarded as disorderly movement of individual carbohydrate chains of molecules double layer. Mobility and rigidity of protein and lipid molecules is favored by temperature and high degree of unsaturated carbohydrate links and the length of carbohydrate chains. Decreased fluidity can be observed experimentally by lowering the temperature. If a high percentage of fat molecules in the membrane are unsatisfied, the melting temperature membrane is lower than body temperature. In this way a part of the double layer is fluid, and the polar groups of lipid molecules can move freely.




Figure 1. Cell membrane.


*yeserg@rambler.ru; phone/fax +30372244730;



After the malignant transformation, the cytoplasm membrane is the first modified structure.

Early detection of malignant transformation is a goal of modern medicine and to this end there is an impressive number of approaches from the scientific field that tries to identify early changes preceding malignant transformation in order to establish a correct diagnosis.

This paper aims to combination of optical and biochemical techniques for identifying the changes in membrane dynamics of growth and development of experimental solid tumors.


  1. ^ BIOCHEMICAL EXPERIMENTAL PART


Tumors obtained in murine models are considered experimental and currently an important tool in the elucidation of malignant mechanisms, no one perfect model for clinical oncology. Transplanted tumors in animals experience are different from human tumors in several respects: the rate of growth much faster, less heterogeneous cell expressed, develop-invasive and infiltration capacity much lower metastasis (1). A major disadvantage of transplanted tumors is the appearance of subpopulations with differentiated cell biological behavior from parental cells.

Despite these many shortcomings, murine tumors transplanted in conjunction with in vitro models (cell culture, tissue and organs) to continue support of the biological basis for various cancer studies.

Walker 256 carcinosarcoma is a standard used tumor in the preclinical screening of anti-substances, as well as in various experimental models of chemo-radio-immunotherapy (4).

Percentage of holding a subcutaneous tumor grafts is 80-90%. In Wistar rats the metastasis are rare, and the invasion of loco-regional nodes occurs in the terminal phase of tumor growth when tumor necrosis phenomena are emphasized.

Researches were conducted on male Wistar rats with average weight of 110g, clinically healthy, from the animal house of the Institute of Oncology from Bucharest. Tumoral implant was performed with a suspension of cells, about 1x107 cells in 1.0 ml saline buffer subcutaneous injection in the right flank. At 7 days after the cells inoculation the first clinical manifestations appears, tumor was tangible after 14 days.





Figure 2. Walker cells DNA-labeled with PI - fluorescent red; x20 objective.





Figure 3. Walker cells - with acridine orange color; x20 objective.

Tumoral volume for each tumor is calculated in part by the relationship:


, (1)


where a - large diameter of tumor (mm); b - diameter of small tumor (mm); tegument thickness - 0.52 (mm).





Figure 4. Walker carcinoma 256.


Tumor is well vascularize with dilated vessels near the numerous outbreaks of necrosis.


Lipid Peroxidation is a classic example of the reaction mechanism chained radicals. Although the autooxidation or lipids peroxidation under the action of reactive oxygen species is a widely accepted phenomenon, mechanisms and consequences of its interpretation is very controversial due to the great diversification. This can be explained by the composition of extremely varied quality and quantity of lipids in polyunsaturates fatty acids by different experimental conditions. Lipid peroxides were assessed by measuring the concentration of MDA, end product of lipid degradation.


Table 1.





14 days

17 days

24 days

Ser

6,07 µmol /100 ml

9,74 µmol /100 ml

7,83 µmol /100 ml

Peritumoral tissue

4,0 µmol /100 ml

5,26 µmol /100 ml

6,19 µmol /100 ml

Tumoral tissue

7,76 µmol /100 ml

9,91 µmol /100 ml

7,93 µmol /100 ml


Action of active oxygen metabolites in the structural proteins or enzymes cause distortion thereof. Interactions most frequently occur in thiol groups of proteins which in turn may undergo oxidation or

A factor determined the reaction of oxidation of protein SH-groups by the SRO is deleted. Exposureof thiol groups of proteins is essential. This explains the increased sensitivity to oxidation of denatured proteins (this property is used as an effective method of identifying content in these groups and to identify secondary and tertiary structure of proteins.

Total albuminic thiol groups were determined by reaction with acid-5.5 `ditio-bis (2-nitrobenzoic) Ellman reagent, DTNB noted.


Table 2.





14 days

17 days

24 days

Ser

566 µmol /l

215 µmol /l

104 µmol /l

Peritumoral tissue

640 µmol /l

540 µmol /l

218 µmol /l

Tumoral tissue

701 µmol /l

495 µmol /l

344 µmol /l


Oxidative stress may be defined as a dynamic imbalance between the production in excess of reactive oxygen species to the detriment of their capture and neutralization of the endogenous antioxidant systems. When installed, oxidative stress may be reversible and is a production of free radicals in excess, but for a shorter period, or when action is irreversible for a long time, the concentration of free radicals and the higher effects are disastrous for the integrity and functioning of cell.


  1. ^ EXPERIMENTAL MODEL IN VIVO.


Preparation of biological samples.

Blood was collected by cardiac puncture (without anticoagulant), centrifuge at 3000 rpm for 15 minutes. Supernatantul (serum) was used for analysis.

After taking each sample tumoral tissue and liver were obtained homogeneous tissues grown by grinding with saline (at 1 g of tissue were added 10 ml saline). After centrifugation at 3000rpm was used for analysis supernatantul.


^ Determination of lipid peroxides.

Reagents and materials:

UV-VIS spectrometer;

Acid tiobarbituric 0.7% acetic acid in 50% trichloracetic acid 20%;

Buffer solution acetic acid - sodium acetate, 50mmoli, pH = 7;

Samples of blood, tissue and tumoral liver tissue.


Method.

Determination of lipid peroxides was performed in serum, tissue and tumoral liver tissue. In two tubes add the following reagents for sample 1.25 mL trichloracetic acid, 0.25 mL serum and 0.5 mL acid tiobarbituric and add blank pentreu same reagents, except serum, which in Loulé bidistilată add water.

After homogenization of content, eprubetele the heat on water bath for 30 minutes. After cooling are centrifugal to 3000 rpm for 15 minutes. Read sinks samples (supernatantului) at 532 nm against water.

Calculul s-a realizat prin aplicarea relaţiei:

The calculation was performed by applying the relationship:


(Aproba – Ablanc) x 60= mol MDA/100mL, (2)


where: A sample = sample sinks,

A sinks blank = blank map.


Determination tiolilor. Reagents and materials:

  • UV-VIS spectrometer;

  • Reagent is prepared by dissolving 40 mg of hot acid bis (2 - nitro-benzoic) (DTNB) dissolved in 100 ml phosphate buffer solution pH = 8.0.

  • Samples of blood, tissue and tumoral liver tissue.

Method.

Determination was performed tiolilor serum, tissue and tumoral liver tissue.

In eprubetele evidence to be added: 0.15 mL serum, 4mL buffer solution and reagent blank in the tubes are replaced with the reagent with 0.5 mL buffer solution. Shake contents eprubetelor and read first absorption tubes for the content of the second tubes (witness) at 410 nm. Absorption obtained is multiplied by 2.06 (mmol / L).



  1. ^ THE LINEAR DICROISM OF BIOTISSUES WITH DIFFERENT TYPES.


In order to investigate thin tissue cuts of the prostate gland near one hundred cuts were produced. Unfortunately, the investigation was possible only on some of those cuts as a great amount of samples turned to be so small that the cutover of the beam exceeded their magnitudes. Despite this, the results of the investigation on different types of tumors (4-5 samples for each type) can be regarded as those that have general character [5-9].

The results of the investigation of the benign tumor tissues showed that the magnitude of the linear dichroism Δ is insignificant in the whole researched spectral range λ=280-840 nm and specific regularities in its change aren’t observed.

The results of the investigation of the malignant tumor tissues in the form of spectral dependence of the magnitude Δ for two samples are showed on the Figure 5.

On Fig.5 spectral dependences of the dichroism magnitude for two tissue samples of the esophagus with a malignant tumor are showed, where we can see that in the area λ<380 nm and λ>700 nm the linear dichroism is also lacking. Two maxima are observed: at λ=440 nm and λ≈530-540 nm, and the last one is not clear marked.





Figure 5. Spectral dependence of magnitude of the linear dichroism of the esophagus tissues:

1 – samples № 5, 2 – sample № 4.


The obtained results can be used for differential diagnostics of malignant or benign tumors using not difficult biochemical analyses, but a simple optical method. For its simplification one shouldn’t conduct measuring on the whole spectrum, it’s enough to measure the magnitude of the dichroic ratio only for two waves with characteristic length: - in the maximum of the spectral dependence ∆ and at = 700-800 nm, for which ∆=0. Their magnitude for both types of tumors is showed in fig. 1 in case with the prostate gland and in fig.2 – with the esophagus. It is noticed that the value of diachronic relation for malignant tumors for both wave lengths differ in 1,5-2,0 times in case with the prostate gland and in 6 -10% in case with the esophagus whereas for benign tumors of healthy tissues it doesn’t differ.


Table 3. The magnitude of dichroic ratio in case with the esophagus


№ of the sample

The type of epithelium

D, at λ=440 nm

D, at λ=800 nm

№ 4

A malignant tumor

1,062

1,0

№ 5

A malignant tumor

1,085

1,0

№ 1

healthy

1,00

1,009

№ 2

healthy

1,006

1,0

№ 3

healthy

1,00

1,0


The modeling of a cancer disease on rats gave the possibility to observe how the linear dichroism of muscle tissues changes during the process of a malignant tumor development. The results of the investigation on the example of one rat are showed in fig.5. Here we can see that the magnitude of the linear dichroism grow with the development of the cancer tumor especially in the maxima after seventeen days since contamination which are observed at λ=370 nm and λ=460 nm. In the area λ>500 nm ∆=0. ∆=0 for healthy tissue in the whole investigated spectral range.



  1. ^ EXPERIMENTAL SETUP.


For the conduction of such investigations a new installation was created which optical scheme is shown in Fig. 6



Fig.6. Structure functional scheme of the installation


Here ^ S – a stabilized source of radiation; К1a lighted condenser; D1 і D2 – an entrance and exit slot of the monochromator MUM; Об.1 і Об.2 – objective glasses; D3 і D4 – diaphragms; П – a polarizer, Nicol prism; З – an investigated sample; D –radiation detector: в in the area λ=330 – 600 nm ФЕП-39, in the area λ=600- 750 nm the silicon photodiode FD-24К; DV – digital voltmeter.

The sample З is fastened in the limb and can turn round in the beam from 0% to 360% relative to the area of radiation polarization after the polarizer. It gives the possibility to determine the magnitude of the minimum transmission (at the predominant orientation of the sample structure elements parallel to the area of polarization) - τ0 and maximum transmission (at the predominant orientation of the sample structure elements perpendicularly to the area of polarization) - τ90 for each given wave length λ by the monochromator. In order to obtain the absolute factor values of the transmission in the direct beam the investigated sample is taken out of the beam zone – the digital voltmeter registers the magnitude of the signal norm І0.



  1. ^ EXPERIMENTAL REZULTS AND DISCUSSION.


In the capacity of researched objects the samples of thin muscle tissue cuts of rats on the different stages of the cancer disease development were used: 7, 17, 40, 47, 52 days since the cancer cells were implanted. As this procedure was held on five rats, there were only 25 samples for this investigation. For the demonstration of the methodology of the investigation and the processing of the research results we will observe the concrete sample of one rat after 7 days since the contamination. The process of measuring is the following. The investigated sample is fastened in the limb with the help of diaphragm D4 we regulate the diameter of the beam so that its magnitude won’t go beyond the borders of the sample during its reversion on the full circle from 0% to 360%. For all the samples the beam’s diameter was equal and was 6 mm. Turning the sample relative to the polarizer we find on the digital voltmeter the minimal Іmin and maximum Іmax value of signal, then the sample is taken sideways out of beam zone and is registered the magnitude of the signal norm І0. Similar measurements are held for each fixed wave length that is set on the barrel of the monochromator.

The corresponding transmission factors are figured out:

; , (3)

the magnitude of the linear dichroism

, (4)

the mean factor of the sample transmission

. (5)

The results of the investigation and calculations for the given example are showed in tabl.4.

Table 4. The values of the calculated magnitudes for one of the rats after 7 days since the contamination.


λ, nm

Іmax

Іmin

I0

Δ

τс

330

161

151

346

0,0289

0,451

350

463

426

978

0,0376

0,454

370

1087

1018

2279

0,0303

0,462

400

3170

2967

6650

0,0305

0,461

430

1240

1157

2581

0,032

0,464

450

2036

1887

4041

0,037

0,485

500

567

512

1113

0,0494

0,485

550

704

646

1342

0,0467

0,503

600

363

339

690

0,0348

0,509

650

498

469

958

0,0302

0,505

700

598

569

1180

0,0246

0,495

750

630

603

1219

0,0221

0,506


The investigation of the given sample on the spheric photometer showed that the magnitude Δ is practically for all λ close to zero, except the value on λ=350 nm, for which Δ=0,010 at τс=0,888. According to the facts in the table 4 these λ meet the maxima in the spectral dependence Δ. It means that for the diffuse divergent samples on the spheric photometer at small values Δ it is impossible to reveal it as there is no way to take into account the diffuse returned radiation which except skipped current is also registered by the spheric photometer. Considerably bigger values τсер indicate that which were obtained on the spheric photometer which conditioned not only by the directed skipped current but also by scattered radiation [10-12].

The results of the investigation for each rat on different stages of cancer disease development were recorded in the table which is similar to table 4. As not all the samples were good for the investigation (heterogeneity of the beam thickness, insufficient size of the sample and as the result the radiation beam with the diameter of 6 mm went out of these sizes), that’s why we managed to obtain only the full set of tables for different stages of the cancer disease development only for three rats.

The similar regularity is noticed for all the rats: the linear dichroism grows practically in the investigated spectral range with the growth of the cancer disease time about to 47-50 days, to 52 days its decrease is noticed.

It can be explained by the fact that with the growth of the cancer tumor its structure arrangement grows to some range (47-50 days), and then destructive processes begin since 50 days. Besides we can observe that the magnitude Δ considerably depends on the wavelength λ and the more the longer is the cancer disease time.

Moreover two maxima are observed in the beginning of the process (7-17 days) in most cases in the dependence Δ=f(λ): the first is at λ≈430 nm and the second is at λ≈500 nm; in the end of the process (47 days) – there is only one maximum in the area λ≈450 - 500 nm. It is better seen when we conduct the average of the values Δ on five rats on five investigated stages of the cancer disease development. The results of these calculations are showed in graphs on fig.7.




Figure 7. The spectral magnitude dependence of the linear dichroism which is mean for all the rats:

  1. 7 days after the contamination; 2) 17 days after the contamination; 3) 40 days after the contamination; 4) 47 days after the contamination; 5) 52 days after the contamination.




  1. CONCLUSIONS.



The following conclusions are made on the results of the investigation:

  1. It is researched that in all the cases the linear dichroism appears in biotissues (the human esophagus, the muscle tissue of rats) with the cancer disease the magnitude of which depends on the type of the tissue and on the time of the cancer process development.

  2. The phenomenon of the linear dichroism formation has a selective character: maximum values Δ are observed in the area λ=410 – 430 nm and in the area λ=500 – 530 nm; for the wavelenth λ< 300 nm and λ> 750 nm Δ is almost zero or zero.

  3. The linear dichroism magnitude depends on the thickness of samples that’s why at thicknesses d=10-12 mkm when the transmission is 80 % and more, it doesn’t become apparent at the measurements on the spheric photometer, in this case it is better to conduct the transmission investigation in the direct beam.

  4. As the linear dichroism is lacking for healthy tissues, then the obtained results can have diagnostic values with the purpose of detection and estimation of the stage of the cancer disease development.



References


  1. Heppner, G. H. Tumor heterogeneity// Cancer Res., 44, 2259 – 2265 (1984).

  2. Daniel S. Martin, M. Earl Balis, Bernard Fisher, Emil Frei, Emil J Freireich, Gloria H. Heppner, James F. Holland, Janet A. Houghton, Peter J. Houghton, Randall K. Johnson, Arnold Mittelman, Youcef Rustum, Robert C. Sawyer, Franz A. Schmid, Robert L. Stolfi, and Charles W. Young Role of Murine Tumor Models in Cancer Treatment Research// Cancer Res, 46, 2189-2192, April (1986).

  3. Chan, S. H., Perussia, B., Gupta, J. W., Kobayashi, M., Pospisil, M., Young, H. A., Wolf, S. G., Young, D., Clark, S. C. & Trinchieri, G. J. Exp. Med. 173, 869- 879 (1991).

  4. Mu J., Zou J.-P., Yamamoto N., Tsutsui T., Tai X.-G., Kobayashi M., Herrmann S., Fujiwara H. & Hamaoka T. Cancer Res. 55, 4404-4408 (1995).

  5. S.C. Cowin. How is a tissue built? // J. Biomed. Eng., 122, 553-568 (2000).

  6. Handbook of Coherent-Domain Optical Methods. Biomedical Diagnostics, Environmental and Material Science/ ed. V.Tuchin. - Kluwer Academic Publishers, (2004).

  7. W.B.Wang, J.H.Ali, J.H.Vitenson, J.M.Lombardo, and R.R.Alfano, “Spectral polarization imaging of human prostate tissues”, Proc. SPIE, 3917,75 (2000) .

  8. X. Wang, G. Yao, and L.-H. Wang. Monte Carlo model and single-scattering approximation of polarized light propagation in turbid media containing glucose // Appl. Opt., 41, 792–801 (2002).

  9. X. Wang and L.-H. Wang. Propagation of polarized light in birefringent turbid media: a Monte Carlo study // J. Biomed. Opt., 7, 79–290 (2002).

  10. A.G.Ushenko, and V.P.Pishak. Laser Polarimetry of Biological Tissue. Principles and Applications // in Coherent-Domain Optical Methods. Biomedical Diagnostics, Environmental and Material Science / ed. V.Tuchin. - Kluwer Academic Publishers, 67-72 (2004).

  11. A.G. Ushenko “Stokes-correlometry of biotissues”, Laser Physics,10, 6, 1-7 (2000).

  12. O. V. Angelsky, Yu. Y. Tomka, A. G. Ushenko, Ye. G. Ushenko, S. B. Yermolenko, Yu. A. Ushenko. “
    2-D tomography of biotissue images in pre-clinic diagnostics of their pre-cancer states”, Proc. SPIE, 5972, 158-162 (2005).

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