Research Article Intrinsic Apoptosis Pathway in Fallopian ...downloads.hindawi.com/journals/tswj/2014/928036.pdf · Research Article Intrinsic Apoptosis Pathway in Fallopian Tube

Research ArticleIntrinsic Apoptosis Pathway in FallopianTube Epithelial Cells Induced by Cladribine

Ewelina Wawryk-Gawda Patrycja ChyliNska-Wrzos Marta Lis-SochockaKamila Bulak and Barbara JodBowska-Jwdrych

Chair and Department of Histology and Embryology with Experimental Cytology Unit Medical University of LublinRadziwiłłowska 11 Street 20-080 Lublin Poland

Correspondence should be addressed to Ewelina Wawryk-Gawda ewelinawawrykwppl

Received 3 July 2014 Revised 8 September 2014 Accepted 12 September 2014 Published 5 November 2014

Academic Editor Irene Lorand-Metze

Copyright copy 2014 Ewelina Wawryk-Gawda et alThis is an open access article distributed under theCreativeCommonsAttributionLicense which permits unrestricted use distribution and reproduction in anymedium provided the originalwork is properly cited

Cladribine is a purine nucleoside analogwhich initiates the apoptoticmechanismwithin cellsMoreover the available data confirmsthat cladribine with the participation of the p53 protein as well as the proapoptotic proteins from the Bcl-2 family also inducesthe activation of the intrinsic apoptosis pathway However while there has been a lot of research devoted to the effect of cladribineon lymphatic system cells little is known about the impact of cladribine on the reproductive system The aim of our study wasto evaluate apoptosis in oviduct epithelial cells sourced from 15 different female rats In so doing the sections were stained withcaspases 3 9 and 8 Results suggest that cladribine also induces apoptosis in the oviduct epithelial cells by way of the intrinsicpathway Indeed the discontinuing of the administration of cladribine leads to a reduction in the amount of apoptotic cells in theoviduct epithelium

1 Introduction

Cladribine (2-chloro-21015840-deoxyadenosine 2-CdA) belongs tofamily of purine nucleoside analogs (PNA) This group ofdrugs was synthesized in the 1970s for the treatment ofhematologic diseases The use of these for this purpose con-tributed to observations of disturbances within lymphaticcells in people with a deficiency of deaminase adenosine(ADA) With these people the excessive accumulation inlymphatic cells of deoxyadenosine-51015840-triphosphate (dATP)brought about the destruction of these cells and an immunedeficiency was observed A similar effect was seen on lym-phatic cells incubated along with a synthetic derivative oftriphosphate adenosine Because of the substitution of thehydrogen atom with a chlorine atom in position 2 in thedeoxyadenosine ring cladribine is resistant to ADA actionCladribinersquos action is connected with the toxic impact ofits main metabolite 2-chlordeoxyadenosine triphosphate (2-CdATP) on cells This effect induces their apoptosis

The cladribine induced apoptotic mechanism has beenthe subject of intense research and most of the data obtainedconfirms the activation of the intrinsic apoptosis pathway by

way of the participation of the p53 protein and the proapop-totic proteins from the Bcl-2 family There has been a lot ofresearch devoted to cladribine action on lymphatic systemcells The results reveal that cladribine permanently lowersCD4+ and CD8+ lymphocyte levels as well as B lymphocytevalues and to a smaller degree the level of NK cells while theeffect on monocytes and neutrophils appears to be of tran-sient character Apart from the direct impact on the numberof lymphocytes cladribine also acts on the proinflammatorycytokines inhibiting their secretion [1 2] What is moreits suppressive action on lymphatic system cells permits theuse of cladribine in treating hematologic malignancies andautoimmune diseases For the last 30 years cladribine hasbeen used in the treatment of hairy cell leukemia (HCL)mdashwith good results in most cases In addition the positiveeffects of cladribine in the treatment of chronic lympho-cytic leukemia (CLL) have been described [3] as has itsefficacy in treating non-Hodgkin lymphoma (NHL) [3]mantle cell lymphoma (MCL) [4] acute myeloid leukemia(AML) [5] myelodysplastic syndromes (MDS) [6] chronicmyeloid leukemia (CML) [7] Waldenstrom macroglobu-linemia (WM) [8] histiocytosis (in children as well [9])

Hindawi Publishing Corporatione Scientific World JournalVolume 2014 Article ID 928036 9 pageshttpdxdoiorg1011552014928036

2 The Scientific World Journal

astrocytomas [10 11] multiple sclerosis (MS) [2 12 13] mastcell disease [14] psoriasis (including psoriatic arthritis [15])systemic lupus erythematosus (SLE) [16] angioedema andanaphylaxis [17] The treatment of younger individuals evenchildren with cladribine appears to be a major factor instimulating a more exact recognition of its actions due to thepossibility of being able to observe long-term reactions [18ndash21] By way of these studies the many side effects inducedby cladribine have been noticedThese are connectedmainlywith the suppressive effects of 2-CdA on the bone marrowThe consequences of myelosuppression include leukopeniathrombocytopenia and anemia The adverse side effects ofcladribine administration also include bacterial viral andfungal infections neuropathy cardiac insufficiency nephrop-athy disorders of the gastrointestinal tract musculoskeletaldisorders local skin reactions teratogenicity and also sec-ondary malignancies such as AML NHL [22ndash26]

Till now little has been known about the impact ofcladribine on the reproductive system Therefore it seemsappropriate to study this influence [27] In our previousstudies we have shown the toxic effect of cladribine action onOSE (ovarian surface epithelium) cells [28] The oviduct isan important organ of the female reproductive system Therole it plays in reproduction is dependent on the uptake ofthe oocyte released from the ovaries and consecutively itstransportation to the uterus Thus its action is crucial inproviding the proper conditions for fertilization and the sur-vival of the fertilized egg [29] For performing all the above-mentioned functions keeping the correct structure andproper functioning of all the elements forming the oviductis essential as factors causing structural or functional lesionsof the oviduct may lead to infertility [30]

In the present study we evaluated the impact of cladribineon the oviduct epithelium of healthy rat females at the doseapplied in humans for hematologic malignancies Becausenumerous studies show that the mechanism of 2-CdA actionis based on the induction of apoptosis in our study weanalyzed the expression of effector caspase 3 in the cells of thesurface epithelium of the oviduct after cladribine applicationMoreover in order to assess the pathway of apoptosis wecarry out an analysis of caspase 9 expression (which is anintrinsic pathwaymarker) and caspase 8 (which is a mediatorof the extrinsic way of apoptosis) Furthermore to assess thereversibility of the changes caused by cladribine in the epithe-lial cells we also tested material 4 weeks after 2-CdA therapy

2 Materials and Methods

21 Experimental Model White femaleWistar rats were usedfor the study These were 3- to 4-month-old females withan average body weight of 275 g Cladribine was appliedsubcutaneously interchangeably in the right and left side skinfold at the site of the lumbar spine (the study was approvedby the Local Bioethics Commission of Medical Universityof Lublin-number 1262001) During the experiment theanimals were housed in cages of a surface area of 05m2with a preserved circadian cycle (12 hday 12 hnight) airtemperature around 21∘C and relative humidity around 60

The rats received standard LSM feed and water withoutlimitations Before starting the experiment a cytologicalsmear drawn from each female was analyzed to determinethe phase of their estrous cycle In all the studied femalesthe estrous cycle lasted 4 days and consisted of four phasesproestrus estrus metestrus and diestrus On the first day ofthe fourth cycle drug administration was started

22 Research Groups The animals were randomly placedinto 3 groups one control group (C) and 2 study groups(A and B) each of which consisted of 5 individuals Theanimals in the study groups were given the drug at a doseof 010mgkg BW24 h for 7 consecutive days at exactly thesame time The dose used corresponded to the therapeuticdose used in the treatment of proliferative disorders of thelymphatic system in humans The control group animalsreceived only food and water without limitations In order tointroduce a stress-inducing factor as that of the study groupsrats in the control group were administered physiologicalsaline subcutaneously in a volume corresponding to theamount of drug administered to rats in the study groupsDecapitation of the animals of both group A and controlgroup was conducted 24 hours after the last injection thatis on the fourth day of the cycle (the final stage of diestrus)Decapitation of the animals of B study groupwas conducted 4weeks after the last injection Their oviducts were then takenfor histomorphological and immunohistochemical tests

23 Histological Tests (HampE Staining ImmunohistochemicalTests) After fixing the material in 10 formalin the organswere embedded in paraffin blocks The material was thentailored into 5 micrometers thick sectionsThe histomorpho-logical evaluation of tissues stained with hematoxylin andeosin (H + E) was performed with the use of a light micro-scope at 200x 400x and 1000x magnification Immunohis-tochemical tests were then done using antibodies directedagainst caspases 3 8 and 9 (manufactured by Sigma) Theexposure of the antigenic sites was performed thermallyby incubation in citrate buffer solution with pH = 6 in amicrowave oven at 800W for 3 cycles lasting 5 minutes eachTo inhibit endogenous peroxidase activity 03 perhydrol(H2O2) in methanol was used Normal Serum was also used

to block the nonspecific bindings of antigenThematerial wasincubated in a primary antibody diluted as recommended bythe manufacturer (1 100) overnight at 4∘C To visualize thereaction diaminobenzidine (DAB) solution and hematoxylincoloration were used In the negative control experimentswere conducted in a similar manner but omitting the specificprimary antibody The material was evaluated with the useof light microscope at 200x and 400x magnification Thepercentage of cells with positive expression was calculated inthe 300 cells of 3 randomly selected microscopic fields (inmagnification of 400x) Moreover the intensity of expressionof individual proteins of fallopian tube epithelial cells wascomparedThis intensity was graded as low (+) intermediate(++) and high (+++) by using the BX4 image analysissystem manufactured by Olympus with a DP 25 digitalcamera and the CellD software In addition the percentage

The Scientific World Journal 3

(a) (b) (c)

Figure 1 Section of the oviduct epithelium C control group A study group B study group (mag x400) and HampE staining The oviductepithelium showed no discernible pathological changes

of cells of the given intensity expression in each analyzedfield was calculatedThe obtained tests results were subjectedto statistical analysis with the use of Statistica 100 softwareThe chi-square (1205942) test was applied to compare the meanof proteins indicating positive and negative cells in groupsA statistical analysis of the intensity of protein expression ingroups was performed with the use of Kruskal-Wallis TestIn this regard a probability (119875) value less than 005 wasconsidered statistically significant

3 Results

31 Histomorphological Assessment of the Oviduct Epitheliumin HampE Staining The oviduct epithelium of the analyzedgroups of animals in the histomorphological study showedno discernible pathological changes under the light micro-scope at 400x magnification (Figure 1) Moreover the epithe-lium lining of the oviducts of the tested females exhibitednormal properties of single layer cylindrical epitheliumMoreover in the mucous membrane we observed multiplefolds directed along the line of the oviduct The epitheliumwas created by two types of cells ciliated cells and secretorycells as well as scarce pig cells We did not observe thesupporting cells The abundant ciliated cells had numerouslong cilia on their free surface The nuclei of the ciliated cellswere oval and contained a small amount of heterochromatinand 1 or 2 nucleoli of spherical contour The cytoplasm ofthese ciliated cells was poorly eosinophilic In several singlecells we were able to observe mitotic divisions Slightly lessnumerous were the secretory cells These were characterizedbymore strongly eosinophilic cytoplasm and by having a con-siderable quantity of extended intracellular structures Thenuclei of these cells had an oval shape and had 2-3 nucleoliAll cells of the epithelium rested onwell-defined basal lamina

32 Immunohistochemical Evaluation of Caspase Expressionsin Oviduct Epithelial Cells The positive expression of cas-pases 3 and 9 was seen to be significantly different among theanalyzed groups (119875 lt 005 in the 1205942 test) In this respect pos-itive immunoprecipitation of caspases 3 and 9 was observedmore frequently in the study groups than in the control group(Figure 2) However positive expression of caspase 8 was rarein all three groups (119875 gt 005 in the 1205942 test)

0 20 40 60 80 100

Caspase 3

Caspase 9

Caspase 8

77

91

11

67

71

12

23

18

12

Control groupB study groupA study group

()

Figure 2 Percentage of positive caspases expression in cells ofoviduct epithelium Positive expression of caspase 8 was rare in allthree groups Positive immunoprecipitation of caspases 3 and 9 wasobserved more frequently in the study groups than in the controlgroup

321 Expression of Caspase 3 (Figure 3) Regarding the ex-pression of caspase 3 (Figures 3 and 4) in the oviduct epithelialcells of the control group we observed just low expressionIn group B however positive caspase expression occurredmuch more frequently and an intermediate intensity of thisexpression was observed in 16 of all cells (Figure 4) We didnot observe a high intensity of caspase 3 expression in theB group rather the highest intensity expression of this wasobserved in group A with the animals decapitated 24 hoursafter last cladribine injection

322 Expression of Caspase 9 (Figure 5) A high intensity ofcaspase 9 expression (Figure 5) was observed mainly in thestudy groups This expression was most frequently observedin study groupAmore rarely in group B and the rarest in thecontrol group (Figure 5) However a low intensity of caspase9 expression was observed in the 60 of cells of the oviductof the animals of group A (Figure 4) Moreover in this groupintermediate intensity was observed in 12 of cells and highintensity in around 14 of cells High intensity was observed

4 The Scientific World Journal

(a) (b) (c)

Figure 3 Expression of caspase 3 in oviduct epithelial cells of the control group (C) as well as study groups A and B (mag x400)The highestintensity expression in group A the lowest in the control group

0 10 20 30 40 50 60 70

55

51

18

60

58

14

11

20

16

5

23

9

4

12

3

0

0

7

3

1

12

()

Control groupB study groupA study group

3lowast

3lowastlowast

3lowastlowastlowast

Caspase

Caspase

Caspase

Caspase

Caspase

Caspase

Caspase

8lowast

9lowast

9lowastlowast

9lowastlowastlowast

Figure 4 Intensity of caspase expression in particular researchgroup The expression of caspase 8 was low (lowast) in all groups Theexpression of caspases 3 and 9 was at the higher (intermediate (lowastlowast)and high (lowastlowastlowast)) level in the study groups than in the control group

more rarely in group B and only in 1 of cells in the controlgroup

323 Expression of Caspase 8 (Figure 6) In thematerial takenfrom the animals of all the examined groups a similar quan-tity of cells was observed that displayed positive expressionof caspase 8 (Figure 6) Both in the material obtained fromanimals subjected to the action of cladribine and in the tissuesof animals given only a saline solution a minute percentageof positive immunoprecipitates was evident within the cellsof the oviduct epithelium In addition the intensity of theircolour staining was low (Figure 4) A stronger expressionwas not observed with regard to caspase 8 in the epitheliumof the oviducts of any group of animals Both among theexperimental groups and the control group and between theindividual experimental groups a characteristic differencein the appearance of the positive immunohistochemicalreactionwas not observed Indeed in all three analyzed visual

Table 1The differences in caspase 9 and caspase 8 expression in theoviduct epitheliummdasha probability (119875) value in chi-square (1205942) test

Caspase 9expression

versus caspase 8expression in Astudy group

Caspase 9expression

versus caspase 8expression in Bstudy group

Caspase 9expression

versus caspase8 expression incontrol group

chi-square(1205942) test 119875 lt 00001 119875 lt 00001 119875 = 00530

fields the rate of the essential relationship in the 1205942 testamounted to 119875 gt 005

33 Caspase 9 Expression versus Caspase 8 Expression In thestudied material we observed positive immunoprecipitatesof mainly caspase 9 while positive expression of caspase 8appearedmore rarely (Figure 2) In our work we demonstrat-ed a statistically significant difference between the appear-ances of the positive expression of caspase 9 and caspase 8 inthe cells of the epithelium of the oviduct of animals subjectedto the action of cladribine In contrast the difference in thedegree of caspase precipitation in the epithelium of oviductsof control grouprsquos animals (Table 1) was not statisticallysignificant Moreover the intensity of the expression ofcaspase 9 was much higher in the examined tissues of studygroups A and B compared with the expression of caspase 8(119875 lt 005 in the Kruskal-Wallis Test Figure 4)

4 Discussion

Cladribine induces the apoptosis of cells and it penetratesinto the interior of the cell through the cellular membraneby way of nucleoside transporters (NT)This is the first phaseof the intracorporeal effects of this medicine Within the cellcladribine is converted into the active 2-CdATP metabolite[31 32] Its presence in the cell leads to a series of enzymaticand structural changes which upset the balance betweendamaging factors and repair mechanisms This results inthe end of the cellrsquos life Cladribine works both on activelyproliferating and on nonactively proliferating cells but thecytotoxic effect is multidirectional

The Scientific World Journal 5

(a) (b) (c)

Figure 5 Expression of caspase 9 in the oviduct epithelial cells of the control group C and study groups A and B (mag x400)The expressionof caspase 9 was more intensive in the study groups than in the control group

(a) (b) (c)

Figure 6 Expression of caspase 8 was at the low level in oviduct epithelial cells of control group (C) as well as study groups A and B (magx400)

With dividing cells the 2-CdA action is connectedmainlywith the inhibition of the deoxynucleotide synthesis thatplays amajor part in the replication and repair of DNA Apartfrom this 2-CdA suppresses the initiation of ribonucleotidereductase (RR) which is the enzyme that conditions thespeed of the synthesis of diphosphodeoxyribonucleotides(dNDP) The limiting of dNDP quantity upsets the balancein the nucleoside triphosphate pool (dNTP) which in turnis involved in activating endonucleases This brings aboutthe fracturing of one or both threads of DNA [31 33] Thiseffect is additionally strengthened through the inhibitionof repair of damaged DNA as a result of the inactivationthrough cladribine of enzymes such asI polymerase or DNApolymerases [31 32 34 35]

Apart from this synthetic nucleosides generated by wayof cladribine can join in the polynucleotide chain during thereplication of DNA and this can litigate this process or formdouble strand breaks (DSB) The presence of damage withinthe DNA contributes to the activation of the p53 proteinwhich initiates the repair mechanisms of the cell Howeverwhen damage is too extensive and their repair is not possiblethe p53 protein directs the cell towards apoptosis [31 36]In cases of the lack of sufficient p53 protein cladribine candirectly activate the Apaf-1 factor or lead to an increasedpermeability of the mitochondrial membrane to cytochromec by way of the independent caspases mechanismThe intra-cellular calcium ion concentration also probably plays a rolein the induction of apoptosis through cladribine [37 38]

A few studies indicate the presence of an extrinsic path-way for apoptosis engendered by way of the administrationof cladribine A membrane activation of death receptorsFASCD95 through 2-CdA was shown by Nomura et al ina line of human leukaemicMOLT-4 cells [39] Other possiblemeans of terminating the cell through 2-CdA is throughthe activation of the MAPK p38 line (p38 mitogen-activatedprotein kinase) or by way of ERK12 (extracellular signal-regulated kinases 1 and 2) [37 40]

In our study we assessed the effect produced by cladribineon the cells of the epithelium of the oviducts We also ascer-tained the standard pathway of the activation of the apoptosisthrough the participation of caspases Moreover by way ofimmunohistochemical staining we were able to determinethe expression of the caspase 3 effector which is responsiblefor the destructive changes in the cell and therefore isregarded as being amolecularmarker for apoptosis Initiatingcaspase 9 plays a role in the intrinsic pathway of the apoptosisbut in the extrinsic pathway the key role is played by the deathreceptor and by caspase 8 [41ndash43] Therefore determiningthis expression of those initiating caspases allowed us todetermine the pathway for the induction of the apoptosisthrough cladribine in the cells of the examined tissues

Although the epithelium of the oviducts that we analysedis characterized by a typical morphological construction forthe epithelium in the diestrus phase still in the immuno-histochemical examination differences in the expression ofthe proteins of the apoptosis line were discernible among

6 The Scientific World Journal

the individual research groups In our examination the mostintense expression of caspase 3 was observed in the cellsof the epithelium of the oviducts of animals belonging toexperimental group A This reveals the inductive action ofcladribine within the process of the programmed death ofthe examined epithelium cells What is more a difference inintensity of the expression of caspase 3 was shown in ourexamination to be statistically significant among individualexperimental groups as well as among experimental groupsand the control group

A similar effect was repeatedly observed to have beenexerted on unaffected and leukemic cells in the examinationsof material obtained from the tissues of affected and controlpatients as well as through cell cultures lines [32 37 42 44]In addition an increase in the activity of caspase 3 was shownin the majority of the research accompanying the effectivetreatment by way of cladribine administration of hyperplas-tic illnessesMoreover Ceruti et al demonstrated the increasein the activity of caspase 3 in astrocytoma cells subjected tocladribine action [11] Only in the research undertaken byGalmarini et al was an increase of the expression of caspase3 in cells incubated in the environment of cladribine notobserved in spite of the appearance of the death of the cellsby way of their dependency on the active p53 protein [45]

Furthermore in our work undifferentiated stem cellsalso a known target of cladribine action were found in theepithelium of the oviductsThis was earlier described in a fewstudies [46ndash48] Chow in his study that was devoted to theimpact of cytostatics on cells in the different stage ofmaturityshowed that cladribine induces the apoptosis of progenitorCD34+CD38+ cells [46] However Lech-Maranda et alobserved a growth inhibition of granulocyte-macrophageprogenitor cells (CFU-GM) and Petzer et al revealed thesame in T-lymphocyte colony forming cells (CFU-TL) sub-jected to action of cladribine [47 48] What is more previousresearch showed that 2-CdA was suppressing the maturationof the precursors of erythrocytes and granulocytes and itsaction is less intensive towards undifferentiated stem cells ascompared with more directed progenitors of blood cells [46ndash48]

For the purpose of the verification of the pathway thatinduces apoptosis through cladribine we conducted an anal-ysis of the expression of caspase 9 and caspase 8 The resultsof these analyses confirmed previous data resulting from themajority of conducted examinations relating the activationof apoptosis by cladribine in the intrinsic pathway In ourwork the expression of caspase 9 in the cells of oviductepithelium of animals belonging to the experimental groupand similarly the expression of caspase 3 was much higherwhen compared with the remaining groups In additionthe positive expression of caspase 9 was also found in theexamined epithelium of the organs of decapitated animalsfour weeks following the administration of the last dose ofcladribine (group B) however its intensity was much lowerIn our work the expression of caspase 8 being a markerfor the receptor pathway of apoptosis in the studied cells ofthe epithelium appeared in a minor percentage of cells andin fact was very low Furthermore we did not observe astatistical difference between the appearance and intensity of

the one caspase in the individual research groups It is thuspossible to conclude that the supply of cladribine did notaffect the expression of caspase 8 in the studied material

In our study we also prove that after 4 weeks of break (7estrous cycles) in the 2-CdA therapy the intracellular changesare still present in the epithelium of the oviduct which is notsufficient time to plan the pregnancy

Our research also confirmed the earlier studies in whichthe increase in the activity of caspase 9 was effected by way ofthe influence of cladribine [41 42] However an increase inthe expression was not observed for caspase 8 This was sim-ilar to that mentioned by Nomura et al [39]The observationof a lower intensity of the expression of caspases 3 and 9 incells of the taken tissues 4 weeks following the last injectionconfirms prior reports pointing at the passing cytotoxic effectof cladribine on the organismrsquos healthy cells [40 49 50]

Current examinations concerning the action of cladribinewere conducted on lymphatic cells and little information isgiven as to 2-CdA action on the reproductive system Oneincidence involving one patient was described This was anindividual who 10 months following treatment with cladrib-ine for hairy cell leukaemia ended her self-administrationof oral contraceptives Next she became pregnant and gavebirth to a healthy child This indicated that her reproductivefunction was retained in spite of the administration of cyto-statics in the past [51]

The data regarding the influence of analogues of purinenucleosides on reproduction functions results undoubtedlyfrom the epidemiology of hairy cell leukaemia (HCL) Long-term observation of cladribine action is mainly conductedamongst men and women in the postmenopausal age sincethis is the group of individuals mostly affected by HCL Atpresent however cladribine is also applied in other diseas-esmdashsuch as in multiple sclerosis the myelodysplastic syn-drome or in histiocytosis These are recognized as affectingyoung people not to mention children therefore knowledgeof cladribinersquos influence on fertility seems to be essential[2 5 9 12 27]

The changes that we observed in the cells of the epithe-lium of the oviducts can have a significant effect on thecorrect functioning of the reproductive systemHowever thisexamination requires confirmation within human studiesPresumably it is believed that cladribine can weaken thereproduction abilities of women and men and act teratogeni-cally therefore it should not be administered to pregnantwoman or to women who are planning pregnancy [27 52]Wubah et al showed in a few examinations conducted onanimal models that the teratogenic action of cladribine isconnected with the increase in the activity of the p53 proteinwithin the cells of the foetus [53 54] However Lau et al statethat the damaging action on the foetus depended on the kindof species accepting the cladribine [52]

Demonstrating the toxic effect of the given compoundon the fertility is not straight forward on account of theabundance of factors influencing its pathogenesis Indeedobserving the menstruation regularity within patients or thepresence of Graafian follicles does not testify to their abilityto have an offspring Moreover examinations of the semensometimes do not also deliver enough data [55 56] However

The Scientific World Journal 7

ultrasound examinations allow researchers and doctors toevaluate the structure of generative organs and detect possiblepathological changes in reproduction abilities which could berestored by way of surgery [57 58] Moreover simultaneousapplication of various diagnostic methods along with thecontrol of the hormone economy can help in the assessmentof the impact of drugs for restoring the fertility of treatedpatients

5 Conclusions

(1) Exposing healthy organisms to the action of cladrib-ine does not bring about discernible structuralchanges in the epitheliumof the oviducts visible in thelight microscope

(2) Cladribine leads to the induction of apoptosis of theoviducts epithelial cells in the intrinsic pathway

(3) Changes produced by cladribine in fallopian tubeepithelial cells are transient but after 4weeks of break(7 estrous cycles) in the 2-CdA therapy the intracel-lular changes are still present in the epithelium of theoviduct

(4) Obtained findings require confirmation by way ofexaminations on human individuals in order toemploy the generated data as potential clinical targets

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

References

[1] B Laugel F Borlat L Galibert et al ldquoCladribine inhibitscytokine secretion by T cells independently of deoxycytidinekinase activityrdquo Journal of Neuroimmunology vol 240-241 pp52ndash57 2011

[2] T P Leist and R Weissert ldquoCladribine mode of action andimplications for treatment of multiple sclerosisrdquo Clinical Neu-ropharmacology vol 34 no 1 pp 28ndash38 2011

[3] K U Chow M J Rummel E Weidmann et al ldquoInduction ofapoptosis by 2-chloro-2rsquodeoxyadenosine (2-CdA) alone and incombination with other cytotoxic drugs synergistic effects onnormal and neoplastic lymphocytes by addition of doxorubicinand mitoxantronerdquo Leukemia and Lymphoma vol 36 no 5-6pp 559ndash567 2000

[4] D J Inwards P A S Fishkin D W Hillman et al ldquoLong-term results of the treatment of patients with mantle celllymphomawith cladribine (2-CDA) alone (95-80-53) or 2-CDAand rituximab (N0189) in the North Central Cancer TreatmentGrouprdquo Cancer vol 113 no 1 pp 108ndash116 2008

[5] AWierzbowska T RobakA Pluta et al ldquoCladribine combinedwith high doses of arabinoside cytosine mitoxantrone and G-CSF (CLAG-M) is a highly effective salvage regimen in patientswith refractory and relapsed acute myeloid leukemia of thepoor risk a final report of the Polish Adult Leukemia GrouprdquoEuropean Journal of Haematology vol 80 no 2 pp 115ndash1262008

[6] F M Laurencet G B Zulian C Cabrol and D C Bet-ticher ldquoMyelodysplastic syndrome with biclonal monosomy7 and trisomy 8 after treatment with cladribine (2-chloro-2-deoxyadenosine) and involved field radiation therapyrdquo CancerGenetics and Cytogenetics vol 99 no 1 pp 85ndash89 1997

[7] G Martinelli P L Zinzani and P Farabegoli ldquoPurine analogs(fludarabine and 2-chlorodeoxyadenosine) as apoptosis-induc-ing drugs in CML therapyrdquo Haematologica vol 81 no 3 pp286ndash287 1996

[8] M Rourke K C Anderson and I M Ghobrial ldquoReview ofclinical trials conducted in Waldenstrom macroglobulinemiaand recommendations for reporting clinical trial responses inthese patientsrdquo Leukemia and Lymphoma vol 51 no 10 pp1779ndash1792 2010

[9] HMottl J Stary M Chanova M Nekolna E Drahokoupilovaand V Smelhaus ldquoTreatment of recurrent Langerhans cell his-tiocytosis in children with 2-chlorodeoxyadenosinerdquo Leukemiaand Lymphoma vol 47 no 9 pp 1881ndash1884 2006

[10] S Ceruti E Beltrami P Matarrese et al ldquoA key role forcaspase-2 and caspase-3 in the apoptosis induced by 2-chloro-21015840-deoxy-adenosine (Cladribine) and 2-chloro-adenosine inhuman astrocytoma cellsrdquoMolecular Pharmacology vol 63 no6 pp 1437ndash1447 2003

[11] S Ceruti C Franceschi D Barbieri et al ldquoApoptosis inducedby 2-chloro-adenosine and 2-chloro-2rsquo-deoxy-adenosine in ahuman astrocytoma cell line differential mechanisms andpossible clinical relevancerdquo Journal of Neuroscience Researchvol 60 no 3 pp 388ndash400 2000

[12] H-P Hartung ldquoNew oral therapies may offer improved treat-ment options for patients with multiple sclerosisrdquo CurrentOpinion in Neurology vol 22 no 1 pp S10ndashS14 2009

[13] R Vosoughi and M S Freedman ldquoTherapy of MSrdquo ClinicalNeurology and Neurosurgery vol 112 no 5 pp 365ndash385 2010

[14] K J Aichberger W R Sperr K V Gleixner A Kretschmer andP Valent ldquoTreatment responses to cladribine and dasatinib inrapidly progressing aggressive mastocytosisrdquo European Journalof Clinical Investigation vol 38 no 11 pp 869ndash873 2008

[15] B Eibschutz S M Baird M H Weisman et al ldquoOral 2-chlo-rodeoxyadenosine in psoriatic arthritis a preliminary reportrdquoArthritis and Rheumatism vol 38 no 11 pp 1604ndash1609 1995

[16] A Bernknopf K Rowley and T Bailey ldquoA review of systemiclupus erythematosus and current treatment optionsrdquo Formu-lary vol 46 no 5 pp 178ndash194 2011

[17] E J Dutta T M Habermann G L Hoos and C R WeilerldquoTreatment of idiopathic urticaria angioedema and anaphy-laxis with cladribinerdquo Journal of Allergy and Clinical Immunol-ogy vol 109 no 1 supplement 1 p S335 2002

[18] G Biswas A Khadwal B Arora et al ldquoActivity and toxicityof 2-CDA in Langerhans cell histiocytosis a single institutionalexperiencerdquo Indian Journal of Cancer vol 44 no 4 pp 137ndash1412007

[19] C E Dearden M Else and D Catovsky ldquoLong-term resultsfor pentostatin and cladribine treatment of hairy cell leukemiardquoLeukemia and Lymphoma vol 52 no 2 pp 21ndash24 2011

[20] K L McClain ldquoDrug therapy for the treatment of Langerhanscell histiocytosisrdquo Expert Opinion on Pharmacotherapy vol 6no 14 pp 2435ndash2441 2005

[21] E Wawryk-Gawda P Chylinska-Wrzos B Jodłowska-Jędrychand M Lis-Sochocka ldquoUse of selected purine analogs in neo-plastic and autoimmune diseasesrdquo Annales Universitatis MariaeCurie-Sklodowska Sectio DDD Pharmacia vol 24 no 4 pp29ndash47 2011

8 The Scientific World Journal

[22] Y Chubar and M Bennett ldquoCutaneous reactions in hairy cellleukaemia treated with 2-chlorodeoxyadenosine and allopuri-nolrdquoTheBritish Journal of Haematology vol 122 no 5 pp 768ndash770 2003

[23] M S Rossini E M de Souza M L Cintra K B Pagnano AC Chiari and I Lorand-Metze ldquoCutaneous adverse reactionto 2-chlorodeoxyadenosine with histological flame figures inpatients with chronic lymphocytic leukaemiardquo Journal of theEuropean Academy of Dermatology and Venereology vol 18 no5 pp 538ndash542 2004

[24] R Hassan M Gupta W Kern and H Ozer ldquoAcute myeloidleukemia following treatment with cladribine for hairy cellleukemia a case report and review of the literaturerdquo Leukemiaand Lymphoma vol 45 no 10 pp 2149ndash2152 2004

[25] A Marczak D Łubgan T Robak and Z Jozwiak ldquoInfluence of2-chlorodeoxyadenosine (cladribine) on human erythrocytesrdquoThe International Journal of Biochemistry amp Cell Biology vol 36no 8 pp 1645ndash1654 2004

[26] A Saven C Burian J A Koziol and L D Piro ldquoLong-termfollow-up of patients with hairy cell leukemia after cladribinetreatmentrdquo Blood vol 92 no 6 pp 1918ndash1926 1998

[27] T Tadmor ldquoPurine analog toxicity in patients with hairy cellleukemiardquo Leukemia and Lymphoma vol 52 no 2 pp 38ndash422011

[28] M Jędrych E Wawryk-Gawda B Jodłowska-Jedrych PChylinska-Wrzos and L Jasinski ldquoImmunohistochemical eval-uation of cell proliferation and apoptosis markers in ovariansurface epithelial cells of cladribine-treated ratsrdquo Protoplasmavol 250 no 5 pp 1025ndash1034 2013

[29] H Abe ldquoRegional variations in the ultrastructural featuresof secretory cells in the rat oviductal epitheliumrdquo AnatomicalRecord vol 240 no 1 pp 77ndash85 1994

[30] A Aflatoonian B Baghianimoghadam P Partovi et al ldquoA newclassification for female infertilityrdquo Clinical and ExperimentalObstetrics and Gynecology vol 38 no 4 pp 379ndash381 2011

[31] J B Johnston ldquoMechanism of action of pentostatin and cladrib-ine in hairy cell leukemiardquoLeukemia and Lymphoma vol 52 no2 pp 43ndash45 2011

[32] E Van Den Neste S Cardoen B Husson et al ldquo2-Chloro-21015840-deoxyadenosine inhibits DNA repair synthesis and potentiatesUVC cytotoxicity in chronic lymphocytic leukea B lympho-cytesrdquo Leukemia vol 16 no 1 pp 36ndash43 2002

[33] T Robak A Korycka L M Ewa and P Robak ldquoCurrent statusof older and new purine nucleoside analogues in the treatmentof lymphoproliferative diseasesrdquo Molecules vol 14 no 3 pp1183ndash1226 2009

[34] G Nocentini ldquoRibonucleotide reductase inhibitors new strate-gies for cancer chemotherapyrdquo Critical Reviews in Oncol-ogyHematology vol 22 no 2 pp 89ndash126 1996

[35] M de Campos-Nebel I Larripa and M Gonzalez-Cid ldquoNon-homologous end joining is the responsible pathway for therepair of fludarabine-induced DNA double strand breaks inmammalian cellsrdquo Mutation Research vol 646 no 1-2 pp 8ndash16 2008

[36] A Korycka ldquoPerspectives of therapeutical application of newpurine nucleoside analoguesrdquoActaHaematologica Polonica vol39 no 4 pp 727ndash741 2008 (Polish)

[37] D M Conrad M R J Robichaud J S Mader et al ldquo2-Chloro-21015840-deoxyadenosine-induced apoptosis in T leukemia cells ismediated via a caspase-3-dependent mitochondrial feedbackamplification looprdquo International Journal of Oncology vol 32no 6 pp 1325ndash1333 2008

[38] P Perez-Galan I Marzo P Giraldo et al ldquoRole of caspasesand apoptosis-inducing factor (AIF) in cladribine-inducedapoptosis of B cell chronic lymphocytic leukemiardquo Leukemiavol 16 no 10 pp 2106ndash2114 2002

[39] Y Nomura O Inanami K Takahashi A Matsuda andM Kuwabara ldquo2-Chloro-21015840-deoxyadenosine induces apoptosisthrough the FasFas ligand pathway in human leukemia cell lineMOLT-4rdquo Leukemia vol 14 no 2 pp 299ndash306 2000

[40] C Smal S LisartMMaerevoet A Ferrant F Bontemps and EvanDenNeste ldquoPharmacological inhibition of theMAPKERKpathway increases sensitivity to 2-chloro-21015840-deoxyadenosine(CdA) in the B-cell leukemia cell line EHEBrdquo BiochemicalPharmacology vol 73 no 3 pp 351ndash358 2007

[41] M Chmielewski K Linke andM Zabel ldquoMetody wykrywaniazjawiska apoptozy w komorkach wątrobowych in situ (Meth-ods of detecting the phenomenon of apoptosis in liver cellsin situ)rdquo Nowiny Lekarskie vol 77 no 3 pp 223ndash226 2008(Polish)

[42] A Klopfer A Hasenjager C Belka K Schulze-Osthoff BDorken and P T Daniel ldquoAdenine deoxynucleotides fludara-bine and cladribine induce apoptosis in a CD95Fas receptorFADD and caspase-8-independent manner by activation of themitochondrial cell death pathwayrdquoOncogene vol 23 no 58 pp9408ndash9418 2004

[43] A Sznarkowska R Olszewski and J Zawacka-Pankau ldquoPhar-macological activation of tumor suppressor wild-type p53 as apromising strategy to fight cancerrdquo Postępy Higieny i MedycynyDoswiadczalnej vol 64 pp 396ndash407 2010 (Polish)

[44] L Bastin-Coyette C Smal S Cardoen P Saussoy E van DenNeste and F Bontemps ldquoMechanisms of cell death induced by2-chloroadenosine in leukemic B-cellsrdquo Biochemical Pharma-cology vol 75 no 7 pp 1451ndash1460 2008

[45] C M Galmarini N Voorzanger N Falette et al ldquoInfluence ofp53 and p21WAF1 expression on sensitivity of cancer cells tocladribinerdquo Biochemical Pharmacology vol 65 no 1 pp 121ndash129 2003

[46] K U Chow S Boehrer J Bojunga et al ldquoInduction of apoptosisby cladribine (2-CdA) gemcitabine and other chemotherapeu-tic drugs on CD34+CD38+ and CD34+CD38- hematopoieticprogenitor cells selective effects of doxorubicin and 2-CdAwithprotection of immature cellsrdquo Leukemia and Lymphoma vol 43no 2 pp 377ndash384 2002

[47] E Lech-Maranda A Korycka and T Robak ldquoInfluence ofgemcitabine (2rsquo2rsquo-difluorodeoxycytidine) and 2-chlorodeox-yadenosine on growth of normal and leukemic cells in vitrordquoEuropean Journal of Haematology vol 65 no 5 pp 317ndash3212000

[48] A L Petzer R Bilgeri U Zilian et al ldquoInhibitory effect of 2-chlorodeoxyadenosine on granulocytic erythroid and T-lym-phocytic colony growthrdquo Blood vol 78 no 10 pp 2583ndash25871991

[49] C A M Idink-Mecking D J Richel I Vermes M R Schaaf-sma C Reutelingsperger and C Haanen ldquoEx vivo evidenceof lymphocyte apoptosis in hairy cell leukemia induced by 2-chlorodeoxyadenosine treatmentrdquo Annals of Hematology vol76 no 1 pp 25ndash29 1998

[50] A Szmigielska-Kaplon P Smolewski M Najder and T RobakldquoEvaluation of apoptosis induced in vitro by cladribine (2-CdA) combined with anthracyclines in lymphocytes frompatients with B-cell chronic lymphocytic leukemiardquo Annals ofHematology vol 81 no 9 pp 508ndash513 2002

The Scientific World Journal 9

[51] R Z Orlowski ldquoSuccessful pregnancy after cladribine therapyfor hairy cell leukemiardquo Leukemia and Lymphoma vol 45 no1 pp 187ndash188 2004

[52] C Lau M G Narotsky D Lui et al ldquoExposure-disease con-tinuum for 2-chloro-21015840-deoxyadenosine (2-CdA) a prototypeteratogen induction of lumbar hernia in the rat and speciescomparison for the teratogenic responsesrdquo Teratology vol 66no 1 pp 6ndash18 2002

[53] J A Wubah M M Ibrahim D Nguyen M M Pisano and TB Knudsen ldquoTeratogen-induced eye defects mediated by p53-dependent apoptosisrdquo Current Biology vol 6 no 1 pp 60ndash691996

[54] J A Wubah R Woodrow Setzer C Lau J H Charlap andT B Knudsen ldquoExposure-disease continuum for 2-chloro-21015840-deoxyadenosine a prototype ocular teratogen 1 Dose-responseanalysisrdquo Teratology vol 64 no 3 pp 154ndash169 2001

[55] E Petru ldquoFertility preservation and infertilitytreatment inbreast cancerpatientsrdquoWiener Medizinische Wochenschrift vol16 pp 160ndash487 2010

[56] B Valanis W Vollmer K Labuhn and A Glass ldquoOccupationalexposure to antineoplastic agents and self-reported infertilityamong nurses and pharmacistsrdquo Journal of Occupational andEnvironmental Medicine vol 39 no 6 pp 574ndash580 1997

[57] M Hajishafiha T Zobairi V R Zanjani M Ghasemi-Rad ZYekta and N Mladkova ldquoDiagnostic value of sonohysterogra-phy in the determination of fallopian tube patency as an initialstep of routine infertility assessmentrdquo Journal of Ultrasound inMedicine vol 28 no 12 pp 1671ndash1677 2009

[58] B J van Voorhis ldquoUltrasound assessment of the uterus andfallopian tube in infertile womenrdquo Seminars in ReproductiveMedicine vol 26 no 3 pp 232ndash240 2008

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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