Analysis of caspase-3 enzyme marker expression in oral squamous cell carcinoma: An Immunohistochemical study

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ANALYSIS OF CASPASE-3 ENZYME MARKER

EXPRESSION IN ORAL SQUAMOUS CELL CARCINOMA

- AN IMMUNOHISTOCHEMICAL STUDY

A Di ss ertat ion su bm itted

in part ial ful film ent of the r equir em ents for the degr ee of

MASTE R O F DENT AL SURGE RY

BRANCH – VI

ORAL PATHOLOGY AND MICROBIOLOGY

THE TAMIL NADU DR.M.G.R. ME DICAL UNI VERS ITY CHENNAI- 600032

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CERTIFICATE

This is to certi f y that the Diss ert ation entitl ed “ANALYSIS

O F CAS PASE -3 ENZYME MARKE R E XPRE SSION

IN ORAL SQ UAMO US CE LL CARCI NO MA - AN

IMMUNOHISTO CHEMI CAL ST UDY" b y Dr. K. SH ARI FA Post

Graduat e student MDS O ral Maxil lofaci al Pathology and

Mi crobiology , Madha Dent al Coll ege & Hospit al -Chennai - 69.

Submitt ed to Tamil nadu Dr. M.G.R . M edi cal Universit y the MDS

Degree Exami nat ion Apri l 2018 is bonafide res earch work carri ed

out b y her under m y super visi on and gui dance

Guid ed B y

Dr. G. SI VAKUMAR, M.D.S.,

Professor and Head

Departm ent of Oral Maxillofaci al P at hol ogy & Mi crobi ology

Madha Dent al Coll ege & Hospit al,

Chennai - 600 069

MADH A DE NT AL COLLE GE AND H OSPITAL

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ENDORSEMENT BY HEAD OF THE DEPARTMENT /

HEAD OF THE INSTITUTION

This is to certi f y t hat the Diss ertat ion entitl ed “ANALYS IS

O F CAS PASE -3 E NZYME EXPRSSI ON IN O RAL SQ UAMO US

CELL CARCI NO MA -AN-I MMUNOHIS TOCHE MICAL ST UDY”

is a bonafi de work done b y Dr. SH ARI FA. K, Pos t Graduat e

student (2015 -2018) in the Department of Oral and Maxillofaci al

Pathology, under the guidance of Dr. G. SI VAKUMAR, MDS .,

Professor, Departm ent of Oral and Maxi llofacial Pathol ogy, Madha

Dental Col lege and Hospital, C hennai – 600 069.

Dr. G. SI VAKUMAR, M.D.S. Dr. M.C. SAI NATH, M.D.S.

Professor and Head PGDCR, MB A

Departm ent of Oral Maxillofaci al Princi pal

Pathology & Microbi ology

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DECLARATION

I, Dr. K. SH ARI FA , do hereb y decl are that the Diss ert ation

titled "ANAL YSIS O F CAS PASE -3 ENZYME MARKER

EXPRESSION I N O RAL SQUAMOUS CELL CARCINO MA – AN

IMMUNOHISTO CHEMI CAL ST UDY" was done in the

Departm ent of Oral Maxillofacial Pat hology & Mi crobi ology,

Madha Dent al Coll ege & Hospital, C hennai - 600 069. I have

utilized the facil iti es provi ded in the M adha Dent al College for the

stud y in parti al ful fillment of the requi rem ents for the degree of

Mast er of Dent al Surger y in t he speci alt y of Oral M axillofa cial

Pathology & Mi crobiology (Branch V I) M adha Dent al C ollege,

Cheenai -69 duri ng the course peri od 2015-2018 under the

concept ual izati on and guidance of m y di ssert ation guide, Profess or

Dr. G. SI VAKUMAR, MDS.

I declare that no part of t he Diss ert ation will be ut ilized for

gai ning fi nanci al assist ance for res earch or ot her prom otions

without obt aini ng prior permis sion from Departm ent of Oral &

Maxillo Faci al P athology, M adha Dent al Co llege & Hos pit al.

I al so declare that no part of t his work will be publis hed

either in the print or el ectroni c m edi a except with thos e who have

been acti vel y involved in thi s diss ertat ion work and I fi rml y affi rm

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Chennai 600 069, but wit h the ves ted ri ght that I s hal l be cited as

the author(s ).

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ACKNOWLEDGEMENT

I woul d li ke to extend m y si ncere gratit ude and thanks t o our

bel oved Chai rman, Managing Di rector and Vi ce Ch ai rpers on of

Madha Dental College and Hospit al, Kundrathur, Chennai -69 for

thei r conti nuous support , help and encouragem ent duri ng m y post

graduati on t o accom plish m y academic carri er.

M y sincere and heart felt thanks to Dr. M.C. SAINATH ,

M.D.S., our Pri nci pal and P rofessor of Orthodontics, Madha Dental

College and Hos pi tal, Chennai – 69, for his continuous and

enormous support and for his const ant encouragem ent and advice .

I express m y deep s ens e of gratitude and great honour t o m y

respected guide, P rofess or Dr. G. SIVAKUMAR MDS., Head of

Departm ent of Oral Maxillofaci al Pathol ogy, M adha Dent al College

& Hospit al , Chennai - 69, for his as tut e, conceptual ization

guidance, support and encouragem ent throughout m y post graduate

cours e and t o bring t his diss ert ati on to a success ful compl eti on.

I owe m y thanks and great honour to Dr. R.

SARANGARAJAN, M.D.S., P rofess or, Departm ent of Oral

Maxillofaci al P athology, M adha Dent al C ollege & Hospit al,

Chennai - 69, for helpi ng me with his valuable and timel y

suggesti ons and encouragem ent.

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Pathology, M adha Dental Coll ege & Hospital, Chennai - 69, for

hel ping m e wit h his valuable and timel y s uggest i ons and

encouragem ent .

I owe m y t ha nks and great honour to Dr. M. SI VAKUMAR,

M.D.S., R eader , Department of Oral Maxillofacial P at hology,

Madha Dent al Coll ege & Hospital , Chennai - 69, for hel pi ng me

with his valuabl e and tim el y suggestions and encouragement.

I owe m y t hanks and great hono ur to Dr. G.KESEVAN,

M.D.S., Sr.Lecture , Departm ent of Oral Maxillofacial Pat hology,

with his valuabl e and tim el y suggestions and encouragement.

I owe m y t hanks and great honour to Dr. R. RAGHI NI ,

M.D.S., S r. Lect ure, Department of Oral M axillofaci al P athology,

with her valuabl e and tim el y suggestions and encouragement.

I take t his opport uni t y t o express m y gratitude to m y fri end’s

coll eagues and m y fam il y m embers for thei r val uabl e hel p and

suggesti ons throughout thi s st ud y.

I s eek the bl essi ng of the almi ght y God without whose

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TRIPARTITE AGREEMENT

This agreement herein aft er t he “Agreement ” is ent ered i nt o

on thi s... day of J anuar y 2018 bet ween the M adha Dental

College and Hos pit al repres ent ed b y i ts Prin cipal havi ng address at

Madha Dent al College and Hospit al, C hennai -69, (hereafter re ferred

to as , ’the coll ege’)

And Dr. G. SIVAKUMAR aged 46 years worki ng as

profess or and HOD at the Coll ege, having residence address at

25A, 3rd C ross St reet, Sheno y Nagar,Chennai -600030, Tamilnadu

(Herei n after referred to as the ‘P rincipal Aut hor’)

And Dr. K. SHARI FA aged 39 years currentl y stud yi ng as

post graduat e student in Departm ent of Oral M axillofaci al Pat hology

& Mi crobi ology i n Madha Dental Coll ege and Hospit al (Herein after

referred to as the ‘P G/Research s tudent and Co- Aut hor’).

Whereas t he ‘P G/Research student as part of hi s curri cul um

undert akes to res earch "CAS PASE 3 EXPRESSION IN ORAL

SQUAMO US CEL L CARCI NO MA" for whi ch purpose the

PG/Pri ncipal aut hor shall act as princi pal author and the coll ege

shal l provide the requisit e infras truct ure based on avail abil it y and

also provide facil it y to t he PG/Research student as to the extent

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Whereas the parti es, b y t his agreem ent have mut ual l y agreed

to the various issues incl uding in particul ar the cop yri ght and

confidenti alit y iss ues that aris e in thi s regard.

Now thi s agreem ent witness es as foll ows:

1. The parti es agree that all the R es earch mat eri al and owners hip

therein shall becom e the vested ri ght of t he col l ege, including in

parti cul ar all t he cop yri ght i n the lit erat ure including t he stud y,

research and all other rel at ed papers.

2. To the extent that the college has l egal ri ght to do go, shall grant

to licens e or assi gn the cop yri ght do ves ted with it for m edi cal

and/or comm erci al usage of i nt erest ed persons/ entiti es subject to

a reasonable t erms/ conditi ons i ncl udi ng ro yalt y as deem ed b y the

coll ege.

3. The ro yal t y s o recei ved b y the coll ege s hall be s hared equall y b y

all t he parties

4. The PG/Research s tudent and PG /P rinci pal Aut hor s hal l under no

circumst ances deal with the cop yri ght , Confi denti al information

and know – how generat ed during t he cours e of res earch/study i n

an y manner what soever, whil e s hall sol e vest wit h the m anner

whatsoever and for an y purpose wit hout the express writ ten

consent of the coll ege.

5. All expens es pert aining to t he res earch s hall be decided upon b y

the principal i nves ti gat or/Co -Author or borne sol e b y t he

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6. The coll ege shall provide all infrastruct ure and ac ces s facil i ties

withi n and in other instit utes to the extent pos sibl e. This

incl udes pati ent inte ractions, introductor y lett ers,

recommendation lett ers and such other acts required in t his

regard.

7. The pri n cipal author shall s uit abl y gui de the st udent R es earch

ri ght from selecti on of the R es earch Topi c and Area till its

com plet ion. However the s el ection and conduct of res earch, t opi c

and area res earch b y the student researcher under guidance from

the pri ncipal author shall be subject to the prior approv al ,

recommendations and comm ents of t he Ethical Committ ee of the

coll ege constit ut ed for t his purpose.

8. It i s agreed t hat as regards other aspects not covered under t his

agreem ent , but whi ch pertain to the res earch undertaken b y the

student R es earcher, unde r gui dance from the P rinci pal aut hor,

the deci sion of t he college shall be bi ndi ng and final

9. If an y disput e aris es as to t he m att ers rel ated or connect ed t o this

agreem ent herei n, it shal l be referred to arbit ration in accordance

with the provisi ons of the Arbit ration and Concil iati on Act ,

1996.

In wit ness whereof the parties hereinabove m enti oned have on

this the da y month and year herein above ment ioned s et thei r hands

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College repres ent ed b y it s

Prin cipal PG Student

Witn ess es Stud en t Guid e

1.

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ABSTRACT

BACKGRO UND

Oral cancer is the m ost common m ali gnanc y of head and neck

region its ranking 6th position of all m ali gnanc y . The most

comm onl y occuri ng sites are tongue, buccal m ucos a, floor of the

mouth, lip and gingi va. Sm oki ng tobacco and pan chewing are t he

etiol ogi c al fact or for t he occurence of oral squamous cell

carcinoma. Inhi bition or imbal ance of apoptos is or programm ed cel l

death leads to cancer. Two pathwa ys are involved in apopt osis 1.

Int rins ic or mitochondri al pat hwa y 2. Extrinsi c pathwa y. Caspase -3

enz ym es or involved in t he apopt osis .

AI M

To Stud y t he Imm unohist ochem ical expression of cas pase -3 in

oral s quamous cell carci nom a.

MATE RI ALS AND METHO DS

In our pres ent study, total l y 25 s am pl es were i ncluded to

det ect immunohis tochemi cal expres sion of ca s pase-3. Twent y,

formalin fixed, paraffi n em bedded wax tissues hi stopat hologi call y

diagons ed oral squamous cell carcinoma were incl uded as cas e

(group I). Five normal buccal and gingi val tis sue form ali n fix ed,

paraffin embedded wax tissues included as cont rol (group II).

Tissues were secti oned 0.5m icron thi cknes s and stai ned with

caspas e-3 antibod y. The res ults were obt ained and st ati sticall y

anal yz ed b y using M ann -Whit ne y t est .

RESULTS

Increas ed expressi on of caspas e -3 was s een in epithelium and

connecti ve tis sue of oral s quamous cell carci nom a ti ssue. In most of

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CONCLUS ION

The results of our pres ent stud y demonstrat ed t hat increased

caspas e-3expression was s een in oral squamous cel l carcinoma

tissues than t hat of the norm al mucosa. These fi ndi ngs i ndi cat e the

caspas e-3 express ion respons e to prognos is of t he cancer and thi s

has to be explored in a larger s ampl e for further s tudi es .

KE Y WORDS

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INTRODUCTION

Oral squamous cell carcinoma (OSCC ) i s the most frequent oral

mali gnanc y of head & neck accounting for 90% of all m ali gnanci es, [ 1 ,

2 ] _{and oft en pres ent a clini cal di agnost ic chall enge, part icularl y i n its}

earl y st ages of devel opm ent. The incidence of OSCC differs widel y i n

various parts of the world, and ranges from 2 -10 per 1, 00,000 i n a

year.

The South Asian countries such as S ril a nka, Indi a, Pakist an and

Bangladesh have shown a hi gh incidence rat e ( 3 , 4 ). Despit e advances in

preventi on, diagnosi s, and treatm ent , the 5 - year survi val rate has not

improved over the past 30 years. [ 5 ] Surve ys in Indi a have s hown that

about 80% of oral carcinom as are preceded b y oral potentiall y

mali gnant oral di sorders, most oft en, persist ent leukopl akia (the most

comm on pot enti all y mali gnant l esion of the oral mucosa or oral s ub

mucous fi brosis (OSMF), which predominantl y affect s the peopl e of

South -East ori gin. ( 6 )

The most comm on caus es for the devel opment of oral Squam ous

cell carcinom a were us e of tobacco or bet el qui d and the regul ar

consumption of al coholi c beverages . Infection of hum an papi l lomavirus

(HP V) geno -t ypes, and person eati ng l es s am ount of fresh fruits and

veget abl es were als o caus ati ve of occurence of oral Squamous cel l

carcinoma [ 7,8 ]. The mut ageni c effects of tobacco, alcohol, bet el quid

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Introduction

and are accel erat ed by the concurrent use of two or more of thes e

agents [ 8 ]_.

Compare to women men were most com monl y affect ed b y oral

squamous cell carci nom a because of hi gh ri sk habi ts . The probabilit y

of devel oping oral S quamous cell carcinom a i ncreases wit h t he period

of exposure to risk factors, and increasi ng age whi ch adds the furt her

dimension of age -rel at ed m ut ageni c and epi geneti c changes .

The vent ral s urface of t he tongue and t he floor of the m out h are

the m ost comm on s i tes affected b y squamous cell carcinoma becaus e

the y are lined b y thi n non -keratinized epithel ium. C arci nogens readil y

penet rate t his thin epithel ium to reach the progeni tor cell com partm ent,

as well as carci nogens s uch as t obacco products and al cohol i n

soluti on, co nst ant l y accumul at e in the fl oor of the mouth and bat he the

tissues of the floor of the m outh and the vent ral aspect of t ongue [ 9 ].

The s tage of advancem ent of oral Squam ous cell carcinoma at the tim e

of diagnosis is t he m ost im portant prognostic factor [ 1 0 ].

Oral Squam ous cel l carcinoma i s most frequentl y di agnos ed l at e

in the cours e of the diseas e becaus e affect ed persons fai l to s eek tim el y

professi onal advice, either becaus e t he y do not understand t he

si gni fi cance of earl y si gns and s ym pt om s , or because the y are i gnorant

of the health im pli cations . The cours e of oral Squam ous cell carci nom a

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Introduction

met ast asis , M -di stant met ast asi s) of the prim ar y t umor correlat es wel l

with the s urvival rat e [ 8 ]_.

In general, S quamous cell carci nom as of the post erior part of the

oral cavit y are more likel y t o m et ast asiz e to regional l ym ph nodes t han

squamous cell carci nom as of the ant eri or part of the oral cavit y [ 7 ]_.

Small well -di fferent iat ed, low -grade oral Squamous cell carci nom as

usuall y m et ast asiz e to regi onal l ym ph nodes onl y aft er invading

connecti ve tiss ue, muscle or bone. On the other hand, poorl y

-differenti at ed, hi gh -grade oral S quamous cell carcinomas are

biologicall y more aggressi ve and tend to met ast asiz e to regi onal l ym ph

nodes earl y i n the cours e of the dis ease. [ 7 ]

In t he l ast 30 years , the 5 - year survival rat e of patients wit h oral

Squam ous cell carcinoma has not i mproved despi te advances i n

diagnos tic t echniques and i mprovem ents in t reat ment modaliti es.

Indeed, t he i nci dence and preval ence of oral SCC are i ncreas ing,

parti cul arl y i n younger persons [ 9 ].

Apoptosi s or Programmed C ell Deat h (P CD) was fi rst des cri bed

b y Kerr et al. ( 1 1 ) whi ch are cha racteri zed b y speci fi c morphologi cal

and biochemi cal changes of d ying cel ls, including cell s hrinkage,

nuclear condensation and fragm entat ion, d ynami c m em brane bl ebbing

and loss of adhesi on to nei ghbors or to extracellul ar matrix ( 1 2 ).

Biochem ical changes include chrom os omal DNA cl eavage in to

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Introduction

number of int racellular s ubst rat e cl eavages b y speci fic prot eol ysis ( 1 3 ,

1 4 )_.

Apoptosi s (t ype I PCD) is the m ajor t ype of cell death that

occurs when DNA dam age is irreparabl e two pathwa ys initiate

apoptos is, both of which activat e the executi oner caspas es 3, 6, and 7.

The first pat hwa y is the i ntri nsi c or mit ochondri al, pathwa y where the

mitochondria cont rol the ini tiation of apoptosis .

Apoptot ic sti muli i nduce the rel eas e of c ytochrom e c and other

apoptoti c regul ators from the int erm embrane space of mitochondria. In

the c yt os ol, c yt ochrome c, AP AF -1, ATP , and t he init iat or procaspas e 9

forms the apoptos ome, whi ch affect s the cl eavage of effectors

caspas es. The Bcl -2 fam il y of prot ei ns regul at es the mi tochondri al

perm eabilit y.

The s econd pathwa y is known as the extrinsi c pathwa y, whi ch is

medi at ed b y various deat h receptors on the cell surface. Thes e

recept ors , once acti vat ed b y speci f i c l i gands, initi at e t he recruitment of

FAS-ass ociat ed deat h dom ai n prot ein and procaspas es 8 and 10 t o the

death dom ain, whi ch forms t he deat h inducing si gnal complex and

prom ot es t he acti vati on of caspas e 8. [ 1 5 ]

Apoptosis occurs normall y duri n g development and aging and as

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Introduction

Apoptosi s al so occurs as a defense m echanism such as i n immune

reactions or when cells are dam aged b y diseas e or noxious agents . ( 1 6 )

Although there are a wide vari et y of stim uli and conditi ons , both

ph ysi ological and pathological , that can tri gger apopt osis , not all cells

will necess aril y di e in respons e to the same stim ulus . Irradiati on or

drugs used for cancer chem otherap y res ults in DNA dam age of some

cells, whi ch can lead to apoptot ic death through a p53-dependent

pat hwa y. S om e hormones , such as corti costeroi ds, m a y lead to

apoptoti c death of some cells (e.g. th ym oc yt es ) alt hough other cells are

unaffect ed or even st imulated.

Some cell s expres s Fas or TNF receptors that can lead to

apoptos is vi a li gands binding and prot ein cros s -linki ng. Ot her cell s

have a default death pat hwa y t hat must be bl ocked b y a s urvi val fact or

such as a horm one or growth factor. There is also t he issue of

distinguis hing apo pt osis from necrosis , two process es that can occur

independent l y, s equential l y, as well as si multaneous l y.( 1 7 )

In s ome cas es it ’s the t ype of stimul i and/or t he degree of sti muli

that det ermines t he cells death b y apopt osis or necrosis . At low dos es,

sev eral of inj uri ous stimuli such as heat, radiat ion, h ypoxia and

c yt otoxic anti cancer drugs can induce apoptos is but thes e s ame stimuli

can caus e necrosis at hi gher doses . Final l y, apoptosi s is a coordi nated

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Introduction

group of c ys tei ne proteas es call ed “cas pases ” and a compl ex cas cade of

events that link the i nitiating stim uli t o t he final demis e of the cell.

Caspas es ( c ys teinal aspart at e-s peci fi c prot eas es ) are enz ymes

whi ch utiliz e a cat al yti c c ys t eine to cleave thei r pept ide subst rat es

aft er s peci fi c aspart at e residues . The first cas pase was dis covered in

1992 and becaus e of its function was named i nt erl eukin -1-“convert ing

enz ym e ( ICE) but was l ater renam ed to caspas e -1. In 1993, C ed -3 from

C. el eg ans was found to be hom ologous to IC E and the corresponding

hum an protein CPP 32 (lat er nam ed caspase -3) was found in 1994. The

official caspas e nom encl ature was deci ded on in 1996 t o all eviat e the

confusion t hat went along wit h the dis cover y of ten differe nt cas pases

(som e wit h mul tipl e nam es ). ( 1 8 )

Caspas es are divided int o t wo mai n categori es bas ed on t hei r

functi on: apoptotic caspas es and infl amm ator y caspas es . The apoptoti c

caspas es are further divided into t wo categori es based on the tim e of

ent r y int o the apoptoti c cas cade: init iator caspas es and effector

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[image:21.595.116.485.68.381.2]

Introduction

Fig1; Domain arran gemen t of mammali an caspas es

The effectors caspas es -3, -6, and -7, are found as inactive di mers

in the cell. The y are activat ed once an initi ator caspas e cl eaves t heir

inters ubunit linkers. Because t he y do not requi re death s caffolds fo r

dimer form ation, t hei r prodom ains are s hort and lack t he C AR D

(cas pase acti vat ion and recruitm ent domain) or DED (deat h effectors

dom ain), dom ains t ypi cal of initi ator c aspases. Thei r prodomains are,

however, l ikel y to be involved i n t argeting wit hin the cell.( 1 9 )

The activati on of caspas es comm its the cel l to apoptosi s. The

main hall marks of apoptos is include rounding of cel ls , ret raction from

nei ghbors , m embrane blebbing t o form vesi cl es call ed apopt otic bodi es ,

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Introduction

DNA to approximat el y 200 bp fragment s, and translocation of

phos phatid yl s eri ne (PS) to the ext ernal s urface of cell s as an “eat m e”

si gn al t o phagoc yt es .

The apoptoti c caspases are necess ar y for conferring all of t hes e

phenot ypes. In addi tion to t he s ys t em atic dism ant ling of the cell ,

caspas es are al so i nvolved in produci ng “find-me” s i gnals to caus e

chemo taxis of phagoc yt es to apoptotic cells ( 2 0 ). The recrui tment of

phagoc yt es keeps the cells from rel easing t heir cont ents i nto

extracell ular space and activati ng an im mune respons e which could be

harmful t o t he tiss ue.

When the num ber of apoptoti c cells is t oo great for con sumpt ion

b y phagoc yt es, Secondar y necrosis can occur. When this happens, the

cell rel eases its cont ents i nto the ext racel lular space. However, immune

cells are som ehow able to recogniz e the cell s undergoing apoptos is

(and s econdar y necrosis ) di fferentl y from the necroti c cel ls . This i s

likel y due t o the act ion of caspas es. C aspases keep danger -ass oci at ed

molecul ar patt erns (DAMP s) and al armi ng from being activat ed. ( 2 1 )

This can be thought of as a “tol erat e m e” si gnal .

Caspas es are als o invol ved in t urni ng off trans cript ion and

transl ation. This keeps an y infecti ng vi ral parti cl es from repli cating

using the host ’s m achiner y and t he y also fragment t he Gol gi, ER, and

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Introduction

differenti ati on of eryt hrobl ast s, skel et al muscl e, bone marrow stromal

stem cells , and neural st em cells.

Caspas e -3 has several ot her non -apopt otic functions in nerve

cells. In additi on to neural cell differenti ation, cas pase -3 has also been

impli cat ed in neuronal mi grati on and plas ticit y, axon pruning, and

s ynaps e eli mination .Cas pases have been shown to pl a y a role in cell

mi grati on and invas ion under certain circumst ances. The y can als o

induce the nei ghbori ng cells to proliferat e and the repl ace d ying cells

in a process call ed apoptosis -induced proliferati on. ( 2 3 ) Thes e rol es of

caspas es have impl i cations for cancer s uch as moderat e act ivati on of

caspas es could, cause cancer to progress rather than regress .

Caspas e -3 is a m ember of the c ys t eine proteas e famil y and pl a ys

a criti cal rol e in the regulation of programm ed cell death (apoptosis ).

Caspas e -3 expression has been ext ensi vel y st udi ed in m an y cancers and

the positive correl ation bet ween its expressi on and a favorabl e

prognosis has been report ed in s everal cancers, suggesti ng its use as a

prognosti c m arker for cancers. In oral squamous cell carci nom as

(OSCCs ), increas ed caspas e -3 express ion has been s hown to invers el y

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Aim and Objectives

AIM AND OBJECTIVES

 To evaluat e t he expressi on of cas pase 3 enz ym e marker i n oral

squamous cell carcinoma.

 To evaluat e the expression of cas pase3 enz yme m arker in norm al

oral m ucos a.

 To com pare the expressi on of caspas e3 enz ym e m arker i n oral

squamous cell carcinoma and normal oral mucosa.

 To aid in as ses sing the prognosis of the oral squamous cell

(25)

Materials and Methods

MATERIALS AND METHODS

STUDY DESI GN:

A cross s ectional st ud y was perform ed to det ermine the

expressi on of caspas e3 enz ym e m arker i n Oral squam ous cel l

carcinoma tiss ue. These tiss ues were obt ained from t went y (20)

pati ent s with l esi ons cli ni cal l y and hist opathol ogi call y confirm ed o f

Oral s quamous cel l carcinoma and Five (5) heal th y i ndivi duals

com pri sed the cont rol group.

STUDY S UBJECTS:

Twent y (20) cas es of oral s quamous cell carcinoma were select ed

and constit uted as Group I. A predet ermined cli ni cal cas e sheet was

used to record all the cas es and a det ail ed cas e hist or y i ncl uding age,

sex, occupation, past medi cal and dental histor y along with t he hist or y

of habits and drugs were recorded.

This was followed by general exami nati on and int ra oral

examinati on. Incis ional biops y of s uffi ci ent widt h and depth to ensure

incl usion of connect ive ti ssue from t he affected mucos a was done and

trans ferred to 10% buffered form ali n. Onl y l esions, which were

diagnos ed hist o pathologicall y as squamous cell carcinoma, were

incl uded i n t he stud y.

Five (5) patients who report ed t o the outpati ent departm ent of

(26)

constitut ed the cont rol group Group II. Norm al buccal and gingival

mucosa adjacent t o t he si te of s urger y wa s bi opsi ed. The ti ss ues t aken

were immedi atel y t rans ferred to 10% buffered form alin for furt her

processi ng and after adequate fixation; paraffin blocks of t he tiss ues

were m ade.

Informed consent was obt ained from all t he subj ects enrolled in

the s tud y a nd Inst itutions of Et hi cal Committ ee permi s sion was

(27)

IMMUNOHISTO CHEMIST RY PROCEDURE:

Armamentariu m:

 Microt ome

 Autocl ave

 Hot ai r oven

 Coupli ng j ars

 Measuring j ar

 Refri gerator

 Wei ghi ng m achi ne

 C ycl omixer

 APES coat ed slides [3 –Amino Prop yl t ri Ethox y Sil ane]

 Slide carri er

 Slide warmer

 Alumi num foi l

 Micropipett es

 Toot h forceps

 Elect ronic tim er

 Beakers

 Rectangul ar s teel trays with gl ass rods

 Sterile gauz e

 Cover slips

(28)

Reagents for IH C

1. Conc.HC L

2. Lax bro s oluti on

3. Acetone

4. Cit rate buffer [Ph -6]

5. TR IS buffer s ali ne [Ph 7.2 -7.4]

6. 3 %(H2O2)

7. Dei oniz ed distil led wat er

8. Haem atox yli n

9. Eosin

10. Al cohol (70%)

11. Abs olut e al cohol

(29)

Antib odies us ed:

1. P rim ar y anti bod y

Monoclonal mous e anti caspas e 3(C ell Si gnalling)

2. S econdar y ant ibod y

Citrate buffer

3. Chromogen DAB (3 - Diam inobenzidine Tet ra H yrochooride)

PRO CEDURE :

Pre m ade APES coated s lides were us ed. Tiss ue secti on of 0.5

micron t hickness were m ade in a rotar y semi aut omat ic mi crotom e and

the ri bbon of tis sue secti ons were transferred on to the APES [3

Amino P rop yl t ri –Ethox y Sil ane] coat ed sli de from the tis su e

float ation bath such that t wo tiss ue bits com es on t o the sli de with a

space in between. One of the tiss ue s ect ions was l abel ed positive [P]

control and the other as negative [ N] cont rol.

IMMUNOHISTO CHEMIST RY PROCEDURE:

The sli des with the ti ssue s ection were t reat ed with three changes

of x yl ene t o remove paraffin wax and then the y were put in de s cendi ng

grades of al cohol and lat er reh ydrat ed wi th wat er. Then the sl ides were

trans ferred to cit rate buffer and autoclaved for anti gen retri eval at 15

lbs pressure for 15 minut es. The sli des were washed in two changes of

distill ed wat er and were dipped in 3 changes of Tri -Phosphat e buffer

sali ne (TBS) for 5 minut es each. The sl ides were then wiped careful l y

without touching the tissue s ection wit h gauz e in order t o rem ove

(30)

anti bodi es added l at er do not spread and are rest ri ct ed to t he ci rcl e.

Blocking reagent was added t o both t he specim ens and incubat ed for 5

minut es. The s ections taken out were washed in t hree changes of col d

TBS for 5 mi nut es in each. Then the slides were wiped carefull y

without t ouching the tissue s ection to remove exces s TBS . The prim ar y

anti bod y, CASP ASE 3 Mouse Monoclonal Anti bod y was added to [P]

positi ve tis sue on the slide and then TBS was added to the [N] negative

tissue. The slides were incubat ed for 1 hour.

The s ect ions taken out were washed i n three changes of cold

TBS for 5 minut es in each to remove t he excess antibod y. Then the

slides were wi ped carefull y without t ouching the t issue s ection to

rem ove excess TBS. Then a drop of bi otn yl ated li nk from s econdar y

anti bod y kit was added on both the sections and the sl ides were

incubated for 20 minutes . Lat er slides were washed i n three changes of

col d TBS for 5 m inut es each and were wiped carefull y without

touching the ti ssue section to remove excess TBS. Then a drop of

Strept avidin - Horse Radish Peroxidas e (HRP) from the secondar y

anti bod y kit was added on both the sections and the sl ides were

incubated for 10 mi nutes . The s ections were washed in three changes of

col d TBS for 5 mi nut es in each and we re wi ped carefull y wit hout

touching the tiss ue s ection to remove excess TBS.

Then a drop of freshl y prepared DAB (3 -Di ami nobenzi dine Tet ra

(31)

and counter st ained with hematox ylin. The slides were pl aced in a t ra y

with t ap wat er for bluing. Then the sli des were t rans ferred to 70%

al cohol 100% alcohol and two changes of x yl ene. The tiss ue secti ons

were then mounted with DP X and were then obs erved under the

micros cope. Throughout t he procedure, care was taken not t o dr y the

tissues.

POSITI VE CONT ROL:

Tonsil carci noma speci men ti ss ues were fixed, process ed,

embedded, secti oned and s tai ned i n t he s am e m anner and was us ed as a

positi ve cont rol . One posit ive control tissue slide was incl uded for

(32)

IHC PRO CE DURE FL OW CHART :

APES coat ed slides with 2 paraffin em bedded ti ssues

↓

Placed in x yl ene thri ce (5 mi nut es each)

↓

Placed in 100% isopropanol (5 minut es)

↓

Placed in 70% i sopropanol (5 minutes)

↓

Washed in distill ed wat er t hri ce (5 minut es )

↓

Placed in 3% h ydrogen peroxide ( 20 mi nutes )

↓

Washed in distill ed wat er t hri ce (5 minut es each)

↓

Kept i n a cit rat e buffer P h 6 and then autocl aved at 21ºc with 121psi

for Anti gen ret ri eval fol lowed b y bench cooling for 40 minut es

↓

Washed in TBS thri ce (5 m inut es each)

↓

Blocking reagent was added to both the s pecim ens and then i ncubat ed

at room t emperat ure in an enclosed h ydrated cont ainer (5 mi nutes )

↓

(33)

Prim ar y anti bod y was added to t he stud y specim en and incubated in an

enclos ed h ydrat ed contai ner (1 hour)

↓

Washed in TBS thri ce (5 m inut es )

↓

Secondar y antibod y was added and incubat ed i n an encl os ed hydrat ed

contai ner (20 mi nut es)

↓

Washed in TBS thri ce

↓

Strept avidin HRP was added and i ncubat ed in an enclos ed h yd rat ed

contai ner

(10 minute s)

↓

Washed in TBS thri ce (5 m inut es each)

↓

DAB was added and incubated i n an encl osed h ydrat ed cont ai ner

(5mi nut es )

↓

Washed in TBS thri ce (5 m inut es )

↓

Stained wit h hem atox ylin (1 minut es )

↓

Washed in running wat er

(34)

Placed in ammoni a (1 minute)

↓

Washed in running wat er

↓

Placed in 70% al cohol (5 mi nut es )

↓

Placed in 100% alcohol (5 minut e)

↓

Placed in x yl ene twi ce (5 mi nut e each)

↓

Slides were mount ed using DPX

↓

(35)

Statistical Analysis

STATISTICAL ANALYSIS

The Norm alit y test s Kolm ogorov -Sm irnov and S hapi ro -Wilks

tests results reveal t hat vari abl es do NOT follow Norm al di stribution.

Therefore to anal ys e the data Non param etric m ethods are applied. To

com pare values bet ween cas es and Controls M ann Whitney t es t is

applied. To anal ys e the data SPSS (IBM SPSS Stat isti cs for Windows,

Versi on 22.0, Arm onk, NY: IB M Corp. Releas ed 2013) is used.

Signi ficance l evel is fixed as 5% (α = 0.05).

 Independent s ample T -Test was done t o compare m ean values

bet ween Groups .

 Chi-S quare t est t o compare proportions between C as es and

Controls .

 Mann Whitne y test was done to compare the st ai ning intensit y

(36)

Review of Literature

REVIEW OF LITERATURE

The nam e caspas e is short for c yst ein yl as part at e -specifi c

prot eas e. C as pases ( c yst ei nal asp art at e -s peci fic prot eases) are enz ym es

that utilize a cat al yt ic c ys t eine to cl eave thei r pepti de subs trat es aft er

speci fi c aspart at e residues . Caspas es are the t erminal prot eas es

invol ved in apoptosi s, as well as being involved in i nflam mati on.( 2 5 )

The first caspas e was dis covered in 1992 and becaus e of its

functi on it was nam ed as i nt erl eukin -1-“converting enz ym e (IC E) ( 2 6 ,

2 7 ) _{but was l at er renamed to caspas e -1. In 1993, C ed -3 from C . el egans}

was found t o be hom ologous to ICE ( 2 8 ) and t he correspondi ng hum an

prot ei n CPP32 (later named cas pase -3) was found i n 1994 ( 2 9 ).The

official caspas e nom encl ature was deci ded on in 1996 t o all eviat e the

confusion t hat went along wi th the di scover y of t en different caspas es

and s om e with m ulti ple names.

Caspas es are express ed as proenz ymes (z ymogens ) call ed

procaspas es , whi ch t hen becom e act ivated to the mat ure cas pas e form .

Procaspas e st ructure can be divided i nto three dom ains such as an N

-terminal prodom ain, a l arge subunit , and a sm all subunit and t hei r first

step in ma turati on is dimeriz ation. Then, prot eol yt ic processi ng

rem oves the prodomain and cl eaves a loop call ed the intersubunit

(37)

.

Fig 2.Procasp as e -3 mod el and crystal s tru cture of caspase -3. Active

site l oop col oring: yellow DL1, r ed DL2, cyan DL20, blu eDL 3, tan DL4

The s econdar y st ruct ure of mature cas pas es consis t of six core

-strands in a sl i ghtl y twist ed sheet in each m onom er, wi th t wo m ain

heli ces on one face (the “front ”) of t he protei n and three heli ces on the

other face (the “back”) of the prot ei n (Fi g 2). The fi rst four core

strands and heli ces 1–3 form the large subunit, whereas the last two

core st rands and heli ces 4–5 form the s mall er subunit. The di mer

interface consi sts of the final st rand from each monom er, si de b y side

in an anti parallel m anner. The two monomers are rel ated through a C 2

[image:37.595.93.543.81.427.2]

(38)

the other m onom er. Five l oops are important for the form ati on of the

active sit e and once the int ersubunit li nker is cl eaved, the two halves

of the cl eaved linker are called L2 and L20. Acti ve sit e loops L1, L2,

L3, and L4 com e from one monom er, and loop L20 comes from t he

other. The catal yt i c c yst ei ne is a part of loop L2, whil e the catal yt i c

histidi ne is part of a loop extending from the C t erm inal end of 3. ( 1 8 )

Caspas es are not onl y requi red for apopt osis, but it al so be required for

regul ating non apopt otic functions i n cert ain cell t ypes .( 3 0 )

.

Pei -Feng Liu et al conduct ed a st ud y for the expression levels of

cl eaved caspas e-3, caspas e-8, and caspas e-9 on ti ssue mi croarra ys

consisti ng of s ampl es from 246 oral squamous cell carci nom a (OSCC )

pati ent s (tongue) b y immunohist ochemistr y. The prot ei n levels o f

cl eaved cas pase -3, caspas e-8, and caspas e-9 in tum or ti ss ues were

si gni fi cantl y hi gher when compared to those in adjacent norm al ti ssues

(al l p<0.001).

The expression l evels of cas pase -8 in t umors were el evat ed in

pati ent s with l ymph node invasi on and moreover, pos itive expres sion

of cl eaved caspas e -3 was associ at ed with short er dis eas e -free survival

(DFS ) in OTSCC pati ents with moderate di fferenti ation a nd l ym ph

node invasi on. Com bination of either positivel y cl eaved caspase -3 or

hi gher caspas e-3 expressi on or both was associ at ed with poor DFS.

The y concluded that co -expres sion l evel of cleaved caspas e -3 and

(39)

parti cul ar i n thos e pati ents with certain tum or st ages and cell

differenti ati on status .( 3 1 )

Huang et al. Evaluat ed caspas e 3 expressions b y

immunohist ochem ical anal ys is in two cohorts of hum an cancer patients,

in which the first cohort, i n cl uded 39 head and neck cancer patients

treat ed wit h radiot herap y or chemo -radiotherap y whil e the s econd

cohort i ncl uded 48 advanced st age breast cancer pati ent s . Results

showed higher cl eaved (activat ed) caspas e 3 l evels in the tum or

samples and a hi gh er rat e of t umor recurrence; hence t he y concluded

that el evat ed cas pas e 3 l evels were s een i n cancer pati ent s.( 3 2 ) .

Ana Fl avia et al conduct ed a retrospective s tud y i n 120 pati ent s

whi ch included 20 oral l eukopl aki a cases (16 wit h and 4 without

d yspl asi a) 20 actinic cheliti s cases( 15 with and 5 without dys pl asi a)

40 oral s quamous cell carcinoma cases ( 20 intraoral squamous cell

carcinomas and 20 lower li p Squamous cell carcinomas) and 40

infl amm ator y fi brous h yperpl as ia cases (20 int ra o ral infl amm ator y

fibrous h yper pl asi a and 20 lower lip infl amm atory fibrous

h yperpl asi a) b y immunohi stochemi cal anal ys is with anti cl eaved

caspas e-3 anti bod y. Their resul ts s howed that cleaved cas pase 3 l evels

were hi gher i n int ra oral Squamous cell ca rcinom as .Hence the y

concl uded t hat cas pas e 3 l evel s were el evat ed i n oral squamous cell

(40)

Shuxia LI ,Yanqi et al conducted a st ud y to investi gat e t he impact

of survivin and cas pas e -3 on apoptosis and angiogenesis i n oral cancer

pati ent s, whi ch included a tot al of 16 oral l eukopl aki a cas es of

moderat e epitheli al d yspl asi a, 12 oral leukopl aki a cas es of s evere

epit hel ial d ys plasi a, 17 cases of m oderat el y di fferenti at ed oral

squamous cel l carcinom a and 10 cas es of normal oral mucos a.

Im mu nohi stochemi cal anal ys is was done t o det ect the expressi on levels

of survi vin, caspas e -3, and caspas e inhibi tor factor V III in l es ions from

each group and t he y reveal ed an i ncreased expression l evel of survivin

in oral cancer tiss ues when compared wi th the norm al mucos a and al so

concl uded t hat the expres sion of C as pas e -3 was decreas ed during

mali gnant t rans form ation.( 3 4 )

Janaki raman etal conduct ed a stud y on R NA -bi nding prot ein Hu

Anti gen-R (HuR ) that cont rol s post -t rans cri ptional gene regul ation and

und ergoes st ress -act ivat ed caspas e -3 dependent cl eavage i n cancer

cells. The y said t hat the C aspas es are defined as either tumor

suppressors or oncogenes b y executing cell deat h in norm al cells or b y

undergoi ng s evere s uppression in som e cancers cel ls and t he Absence

of caspas e-3 was report ed i n mult ipl e cancers including breast cancer,

hepat ocell ular and prostate carci nom as. In addition to t hat , inact ive

caspas e-3 was reported in both oral carcinomas and normal oral

epit hel ial tis sues and thei r results clea rl y showed that over express ed

COX-2 inhi bits the cl eavage of caspas e -3 and HuR i n oral cancer

(41)

Hague A et al Conduct ed a stud y to det erm ine alt eration of caspas e -3

expressi on duri ng oral carcinogenesis.C aspase -3 expressi on i n normal

oral epit hel ium and oral s quamous cell carcinoma were com pared b y

incl uding 39 s ampl es of norm al oral epi theli um and 54 oral squamous

cell carcinomas i n their st ud y and the results showed t hat S quamous

cell carcinomas had more int ens e cas pas e -3 s tai ning than nor m al

epit hel ium. In t he oral squamous cell carcinoma seri es, there was

si gni fi cantl y more int ens e caspas e 3 expression i n nucl ear and

c yt opl asmi c stai ning with increasi ng S TNMP st age.( 3 6 )

Coutinho-Camill o et al conducted a res earch stud y to anal yz e t he

expressi on profi les of 23 prot eins that have been li nked to the

inhibit ion (Bcl -2, Bcl-x, Bcl-x L, Bcl -2-relat ed protei n A1, BAG -1, and

survivin) and prom otion (Bak, Bax, Bi m/Bod, Bim -Long, Bad, Bi d,

PUMA, Apaf -1, cas pas e-2, caspas e -3, cas pas e -6, cas pase -7, caspas e -8,

caspas e-9, cas pase -10, Smac/D IAB LO, and c yt ochrom e c) of apopt osis

in oral squamous cell carci nom a and t he y incl ude Two -hundred and

twent y nine cas es of oral squamous cell carci nom a, b y using

immunohist ochem ical method anal ys is, results were quanti fi ed on an

aut omat ed imaging s ys tem and thei r res ults showed that t he caspas e -3

was express ed hi gher in s quamous cel l carci nom a and it was correl at ed

with l ymphnode m et ast asis .. ( 3 7 )

Liang Pang et al eval uat ed the decreas ed mRNA expressi on of

caspas e-3, caspas e -8 and caspas e-9 in Hum an Oral Squamous C ell

(42)

West ern blot ass a ys . Aft er ursodeox ycholi c acid ( UDCA) t reat ment , the

mRNA and prot ein ex pressi on of cas pase -3, caspas e -8 and caspas e-9

were increas ed com pare to t he cont rol cells, and t hese changes were

increased in a concentration -dependent manner.( 3 8 )

Poomsawat et al invest i gated the expression of survi vin and caspas e

3 in 114 s ampl es including oral squam ous cell carcinoma, oral

leukopl aki a( with and wi thout d ys pl as ia) and norm al oral mucosa and

were immunohi sto chemi call y anal yz ed and t hei r results showed that

the numbers of c yt oplas mic and nucl ear expres sion of cas pas e 3 i n

OSCC were s i gni fi cantl y hi gher t han t hat of norm al mucos a and oral

leukopl aki a (wi th and without d ysplasia) so t he y concl uded that

el evated expres sion of caspas e 3 in oral s quamous cell carcinoma

indi cat es the advanced stage of oral cance r.( 3 9 )

Nagaraj et al conducted a research stud y on h ypoxia medi at ed

apoptos is in oral squamous cel l carcinom a and showed that oral cance r

cells from pri mar y tumors and l ym ph node m etast asis undergo

apoptos is aft er 24 to 48 hours of h ypoxia. He also reveal ed that during

h ypoxi c growth, an increas e i n t he caspas e -3 prot eol yt ic activit y

accompani ed b y t he cl eavage of PARP (pol y ADP -ribose. pol ym eras e)

was obs erved in oral squamous cell carci nom a.( 4 0 )

Veeran varmal et al conduct ed a st ud y i n 112 s am ples whi ch included

(43)

b y imm unohist ochemical anal ys is using cas pase 3 antibod y. The

percentage of positi ve cells were calcul at ed using Image J software and

the res ult showed t hat cas pas e‑3 expression epit hel ial cell s ranged

from 11% to 38% i n the norm al oral m ucosa and 0 -8% i n the oral

squamous cell carcinom a, and hence the y concluded t hat the

decreas ed expressi on of cas pase -3 in diseas e progression refl ects its

rol e i n the mal i gnant trans form ation.( 4 1 )

D Andressk is et al inves ti gat ed cas pas e -3 and caspas e-8 prot ein

expressi on in 87 cas es of pri mar y tongue squamous cell carcinoma b y

immunohist ochem ical anal ys is and di gi tal im aging anal ys i s as sa y.

Their resul ts showed that expressi ons of caspas e -3 prot eins were

obs erved 79 s amples (90.5 %).( 4 2 )

Anton io Bas con es –Martinez et al conduct ed a res earch stud y for t he

expressi on of p53, caspas e -3 Bcl -2, and ki-67 in prem ali gnant oral

epit hel ium and to st ud y i ts cl onal behavior 41tumors sam pl es with thei r

adj acent non -tum or epit hel ia were anal yzed b y imm unohistochemi cal

using m onoclona l antibodi es t hat recogni ze p53, caspas e -3, Bcl-2, and

Ki-67 and the y showed statisti call y si gni ficant expressi on of caspas e 3

in tum ors and its adj acent area( 4 3 )

SX Li et al was conduct ed a res earch st ud y to i nves ti gat e t he

expressi on of survi vin an d caspas e 3 i n oral squamous cel l carcinoma

and peritum oral tiss ues , by us ing E LISA and Western bl ot method

(44)

squamous cell carcinoma and 10 norm al tissue s am ples. Thei r result s

showed that n o di fference in expression of cas pase 3 i n peri tumoral

tissues and norm al ti ssues, but it was evi dentl y decreas ed in carcinoma

tissues.( 4 4 )

M.V.Fi andal o et al found that during t umor genesi s, si gni fi cant l oss or

inactivati on of caspas e leads to im pai red apoptosi s inducti on, thereb y

causing a dram ati c imbal ance in the growth d ynami cs and hence

resulti ng i n aberrant growth of human cancers .( 4 5 )

Andrej Cör et al conduct ed a stud y, i n 28 Head and Neck Squam ous

Cell C arcinom a (HNSCC) b y imm unohis tochemi cal anal ys is and t heir

results showed P ro -caspas e 3 expressi on reduced i n Head and Neck

Squam ous C ell C arci nom a. ( 4 6 )

Atsu shi Ku rabayas hi et al conduct ed a st ud y i n 76 esophageal

squamous cell carci noma b y imm unohist ochemi cal anal ys is and their

results showed that expressi on of caspas e 3 was si gnifi cantl y

ass oci at ed wit h poor prognosis .( 4 7 )

Qian Hu et al, conduct ed a stud y b y using i mmunohist ochemi cal

anal ys is, to obs erve the expressi on of cl eaved cas pase -3 i n 367 human

tumor s ampl es (gas tri c cancer: 97 cas es, ovari an cancer: 65 cas es ,

cervical cancer: 104 cases; col orect al cancer: 101 cas es ) and t he y

(45)

had a s i gni fi cant s hort er overall survi val tim e when com pared wit h

decreas ed levels of expres sion. ( 4 8 )

Heshiki et al conduct ed a res earch study for caspas e 3 expressi ons i n

30 pati ents who had recei ved surgi cal t reatm ent bet ween April 2007

and M arch 2014. The y i ncluded the pri mar y carcinom a sit es such as

the tongue ( n=10), maxillar y gi ngi va ( n=6), m andi bul ar gingi va ( n=5),

floor of the mouth ( n=3), and buc cal mucosa (n=6) and their resul ts

showed that all t he cases of oral squamous cell carci nom as

dem onst rated a posit ive expression of cl eaved caspas e -3.( 4 9 )

Manish a Sh rivastava et al condu cted a prospect ive pi lot stud y bas ed

upon the express ion of XIAP and caspas e 3 prot ei ns i n 11s s amples of

oral s quamous cell carcinoma. The res ults showed ampli fi cation of

caspas e-3 in 7 pure DNA sam pl es and hence the y concl uded that t he

oral cancer cells could be recognized by expression of cas pas e 3 as

wel l as it m a y have a furt her role in fut ure target therapy in oral

cancer pati ents.( 5 0 )

M Ols son et al revi ew of pri mar y breast tumor s ampl es obt ai ned from

pati ent s undergoing breast s urger y found that in approximat el75% of

the t umors as well as m orphologi call y norm al peritum oral tis sue

samples, cas pase -3 t rans cri pts and caspase -3 prot ein express ion were

(46)

Rein er et al st udi ed the expression of cas pas e -3 in knockout mice, and

concl uded t hat a cas pas e i s functi onall y delet ed in a cancer cell l ine

whil e it is al so required for cert ai n distinctive biochemical and

morphol ogi cal changes during apoptosi s .( 5 2 )

Ding YP et al conduct ed a stud y for the expres sion of surviving and

caspas e-3 in 17 oral squam ous cell carcinoma (OSCC ), 28 oral

leukopl aki a wit h d ysplasi a and 10 normal oral m ucos a cas es .The

results showed t hat Caspas e -3 was expressed in all the norm al mucosa

samples but it was obvious l y down -regul ated i n d ys pl asi a and oral

squamous cell carcinoma.( 5 3 )

Abdel et al conduct ed a st ud y in 25 l esional bi ops y s pecimens,

whi ch included 15 Cutaneous Li chen Planus (C LP ) , 10 Oral Li chen

Planus (OLP )] and 10 control spe cimens [5 norm al skin and 5 norm al

oral mucos a] b y i mmunohistochemi cal anal ysi s whi ch reveal ed an

increase cas pase -3 prot ein expression in lichen pl anus l esi ons .( 5 4 )

Jer –shyung hu ang et al conduct ed a stud y to eval uat ed caspas e -3

expressi on in tumor genesi s and its prognosi s i n 185 buccal m ucos a

squamous cell carci noma pat ients b y im munohistochemi cal stai ning

and thei r res ults showed that the expres sion l evels of cleaved cas pase

-3 in 185 buccal mucos a s quamous cell carci nom a tis sues were

si gni fi cantl y hi gher when com pared t o thos e in the tum or adjacent

(47)

Yu-Xia zh ang et al conduct ed a st ud y for cell viabilit y of tongue

squamous cel l carci nom a aft er the t reatm ent of st auros porine, and

thei r res ults showed that aft er treatment a tim e -dependent reduction

of survivi n and an activa t ion of caspas e-3 were obs erved .( 5 6 )

Zarin Zainul etal anal yz ed the express ion of cas pase 3,7,8,9 and 10

in oral squamous cel l carci nom a b y ti ssue mi cro arra y and t heir res ult

showed that caspas e 8 and 9 were prom inentl y express ed whil e

caspas e 3,7and 10ex press ed causal l y.( 5 7 )

Tetsu shi man e et al conduct ed a st udy in 119 oral squamous cell

carcinoma cas es b y usi ng imm unohistochemi cal anal ysis , thei r

results showed t hat apoptos is associ at ed speck like protei n appear in

wel l di fferenti at ed cancer cells. ( 5 8 )

Mutsuko Muk ai et al conduct ed a st udy, in hi s stud y resul ts showed

that caspas e -3 activation cont ribut es to the stres s-i nduced invasiv e

capacit y of thes e cancer cells .( 5 9 )

EA Sl ee et al showed in thei r review that the cells defi ci ent i n cas pase

-3 (either from CASP --3 null mi ce or due to a fram e s hi ft mut ation, i.e.

MCF-7 cells ) cl earl y di ed i n respons e to man y p ro -apoptoti c s timuli.( 6 0 )

Claudia Malh erios Coutinho -Camil o et al conduct ed a stud y in 229

tissues of oral squamous cell carcinoma b y imm unohist ochemi cal

anal ys is and t heir results showed t hat caspas e 3 was express ed

(48)

Eswaran Devarajan et al conduct ed a stud y for the caspas e 3

expressi ons in pri mar y breast tum or sampl es and normal breast

parench ym a sam pl es b y reverse transcriptas e -pol ym eras e, western blot

and nort hern bl ot anal ys is and t he y found that 75% of the t umor and

norm al peritumoral t issues l acked cas pas e3 prot ei n expression. Hence

thei r results suggest ed t hat the loss of caspas e3 expres sion represents

an import ant role in the survi val m echani sm of breast cancer.( 6 2 )

Yuan –Fen g Lin etal found in his res earch stud y t hat the Caspas e -3

down regul ation (C ASP3/DR) i n tum ors conferred resist ance to cancer

therap y and was si gnificant l y correl at ed wit h a poor prognosi s in

cancer pati ents.( 6 3 )

Rich ard Jager et al in his revi ewed that the caspas e -3 was det ect ed

less in colon and stomach cancer, Non -Hodgki n l ymphom a (NH L), and

hepat ocell ular carcinom a and whi le it was elevated in acute

m yel ogenous leukemia (AM L) breast and prost ate carcinoma. Hence

the y concl uded t h at the expres si on increased wit h tumor

progres sion.( 6 4 )

Pieter d e Heer Elza et al conduct ed a randomiz ed clini cal trial on

pre operat ed rect al biops ies and found that caspas e 3 act i vit y was

si gni fi cantl y l ower t han the norm al tis sue and hence the y concluded

that caspas e 3 acti vit y was an im portant denominator for local

(49)

J.Kani a etal conducted a st ud y i n gast ri c cancer, and t he y found t hat

caspas e 3 prot ein ex pressi on was undet ect abl e i n gast ri c cancer tiss ues

when com pare t o norm al m ucos a.( 6 6 )

Ulla Toramen etal conduct ed a stud y to evaluat e the cas pase 3 level in

non sm all cell lung carcinoma and the y found that caspas e expres sion

was seen in tum ors t issues and it s appearance was not associ ated with

(50)

Results

RESULTS

A tot al of 25 pat ient s were i ncluded in t his st ud y, comprisi ng of

2 groups. Group I was the st ud y group of 20 histologi call y confi rm ed

oral squam ous cell carci nom a patients . Group 2 was made of 5 norm al

pati ent s whose gingi val tiss ues were st udied.

Age dis tributi on in study groups

In Group I .age di st ribution among the pati ent s ranged bet ween

25 to 75 years with mean age of 55.60 yrs .In Group II, age ranged

from 27 t o 30 years with mean age of 25.60 yrs .(Tabl e 1,G raph 1)

Gender distribu tion in s tudy groups

Out of 20 pat ients i n Group I consis ts of 15 were m al es and 5

were fem al es with a mal e: fem al e ratio of 3:1. In Group II, 3 were

mal es and 2 were femal es, wi th a mal e: fem ale rati o of 3:2 . (Tabl e 2,

Graph 2)

Site of biop sy in s tu dy groups

In Group I, bi opsi es were t aken from buccal mucos a for 12

pati ent s, alveol us or gingival for 4pat i ents , from the tongue for 4

pati ent s. In Group II all samp l es were t aken from the gi ngi val. (Table

(51)

Results

Comp arison of intensi ty of exp ression of Caspase3 in betw een

epith eliu m and con nective ti ssue of Group 1 and Group 2

Epitheliu m:

Group 1 – Out of the 20 tiss ue s ampl es , 7 (35%) showed mil d st aini ng

intensit y, 4 (20%) showed moderat e intens it y and 6 (30%) s howed

more int ensit y. Epi t heli al expressi on was not s een in 3 (15%) ti ssue

samples.

Group 2 – Out of 5 tiss ue s ampl es , 1(20%) s am ple showed mild

epit hel ial st aini ng and 4(80%) tis sue s ampl es was no t s howed an y

stai ning.

The di fference in epithel ial staining was stati sticall y si gni ficant

bet ween Group 1 and Group 2

(P-0.046)(Tabl e 4,Graph 4)

Conn ective tis sue:

Group 1 – out of the 20 tiss ue sam ples,9(45%) tiss ue s ampl es showed

mild expressi on,3(15%) ti ssue s am pl es showed m oderat e expressi on

and 1(5%) tissue showed more int ensit y i n stai ning. No expression was

seen i n 7 (35%) tis sue s amples.

Group 2 – No expression was seen in 5 t issue s amples.

The di fference in connecti ve tis sue st a i ning was s i gni fi cantl y bet ween

Group 1 and Group 2

(52)

Results

Mann -Whi tney T es t to comp are Epith eliu m grading b etween Cas es

and Con trols :

To compare t he s tai ning intensit y of epi theli um in bet ween oral

squamous cell carci nom a and norm al m ucosa Mann –Whi tne y t est was

perform ed. The p value for epithelium gradi ng was < 0.008 which was

hi ghl y st ati sticall y si gnificant . (Tabl e6, Graph6)

Mann -Whi tney Test to compare Conn ecti ve ti ssu e grading betw een

Cas es and Con tro ls :

To compare the st aini ng int ensit y of Connecti ve tissue in

bet ween oral s quam ous cell carci nom a and norm al mucos a Mann –

Whitne y t est was performed. The p value for Connect ive tiss ue grading

(53)

Tables and Graphs

TABLES AND GRAPHS

TABLE 1: AGE DIST RIB UTIO N BETWEE N GROUP1 AND

GROUP2

Group N Mean Age Std. Dev p-Value

Cas e 20 55.60 12.098

<0.001 Control 5 25.60 2.793

TABLE 1: AGE DIST RIB UTIO N BETWEE N GROUP1 AND

[image:53.595.86.538.193.679.2]

(54)

[image:54.595.85.515.123.299.2]

Tables and Graphs

TABLE 2: GENDER DIST RIB UTIO N BETWEE N TH E TWO

STUDY GROUPS

Gender

Group

p-Value

Cas e Control Total

N % N % N %

Fem ale 5 25.0% 2 40.0% 7 28.0%

0.597 Mal e 15 75.0% 3 60.0% 18 72.0%

Tot al 20 100.0% 5 100.0% 25 100.0%

GRAPH 2: GE NDER DISTRIB UTIO N BETWEE N TH E TWO

[image:54.595.92.545.326.709.2]

(55)

[image:55.595.83.517.94.326.2]

Tables and Graphs

TABLE 3: S ITE O F BIO PS Y I N THE S TUDY GROUPS

Site of biop sy

Group

p-Value

N % N % N %

Buccal M ucos a 10 50.0% 0 0.0% 10 40.0%

0.012 Floor of M outh 2 10.0% 0 0.0% 2 8.0%

Gingi va 4 20.0% 5 100.0% 9 36.0%

Tongue 4 20.0% 0 0.0% 4 16.0%

Tot al 20 100.0% 5 100.0% 25 100.0%

GRAPH 3: S ITE O F BIO PS Y I N THE S TUDY GROUPS

0 10 20 30 40 50 60 70 80 90 100

BM FM G T

50 10 20 20 0 0 100 0 P e rc e nt a ge Site

Site of biopsy

_Case

[image:55.595.92.547.340.708.2]

(56)

[image:56.595.85.515.127.355.2]

Tables and Graphs

TABLE4: DISTRI BUTIO N O F E PI THELIUM I NTENSITY O F

STAIN I N GROUP1 AND GRO UP2

Epitheliu m

inten sity

Group

p-Value

N % N % N %

INTENSE 6 30.0% 0 0.0% 6 24.0%

0.046

M ILD 7 35.0% 1 20.0% 8 32.0%

MODERATE 4 20.0% 0 0.0% 4 16.0%

NOSTA IN 3 15.0% 4 80.0% 7 28.0%

Tot al 20 100.0% 5 100.0% 25 100.0%

GRAPH 4: DIST RIBUTIO N O F E PI THELIUM INTENSITY O F

STAIN I N GROUP1 AND GRO UP2

0 10 20 30 40 50 60 70 80 90 100

NO STAIN MILD MODERATE INTENSE

15 35 20 30 80 20 0 0 P e rc e nt a ge

Epithelium intensity

Case

[image:56.595.95.544.433.761.2]

(57)

Tables and Graphs

TABLE 5: DIS TRIBUT ION O F CONNECTI VE TISSUE

INTENS ITY O F ST AIN I N GROUP1 AND GRO UP2

Conn ective

tissu e in tensi ty

Group

p-Value

N % N % N %

INTENSE 1 5.0% 0 0.0% 1 4.0%

0.087

M ILD 9 45.0% 0 0.0% 9 36.0%

MODERATE 3 15.0% 0 0.0% 3 12.0%

NOSTA IN 7 35.0% 5 100.0% 12 48.0%

Tot al 20 100.0% 5 100.0% 25 100.0%

GRAPH 5: DIS TRIBUT ION O F CO NNE CTI VE TISSUE

INTENS ITY O F STAI N IN GROUP 1 AND GROUP 2

0 10 20 30 40 50 60 70 80 90 100

NO STAIN MILD MODERATE INTENSE

35

45

15

5 100

0 0 0

P e rc e nt a ge Intensity

Connective tissue intensity

_Case

[image:57.595.83.544.114.763.2]

Analysis of caspase-3 enzyme marker expression in oral squamous cell carcinoma: An Immunohistochemical study