Utjecaj dsRNA i cisplatine na povećanje osjetljivosti stanica tumora grkljana čovjeka na zračenje

master's thesis

• Title (english): Effects of dsRNA and cisplatin on sensitization of human larynx carcinoma cells to radiation
• Author: Antonio Kobeščak
• Mentor: Karmela Barišić (mentor)
• Committee member: Karmela Barišić
• Committee member: Sandra Šupraha Goreta
• Granter: University of Zagreb; Faculty of Pharmacy and Biochemistry; (Department of medical biochemistry and haematology); Zagreb
• Defense date and country: 2015-09-28, Croatia
• Scientific / art field, discipline and subdiscipline: BIOMEDICINE AND HEALTHCARE; Pharmacy; Pharmacy

Abstract

Karcinomi pločastog epitela glave i vrata predstavljaju šesti najčešći tumor u svijetu, a preživljenje bolesnika i ishodi
terapije nisu zadovoljavajući. U nedavnim su istraživanjima na stanicama ovih te mnogih drugih tumora nađeni Tolllike
receptori (TLR). TLR omogućuju domaćinu prepoznavanje niza molekularnih sljedova podrijetlom od patogena (PAMP) pri čemu aktiviraju prirođeni i stečeni imunosni odgovor. TLR3 je unutarstanični receptorski protein koji veže dvolančanu RNA (dsRNA). Nađeno je da je funkcionalan TLR3 izražen u stanicama tumora metastaza karcinoma ždrijela te da njegova aktivacija uzrokuje apoptozu stanica tumora. Stoga je u ovom istraživanju ispitan učinak pojedinačne i kombinirane primjene liganda TLR3, poli(I:C), s citostatikom cisplatinom i γ-zračenjem na preživljenje i regulaciju staničnog ciklusa stanica tumora grkljana. U istraživanju je korištena stanična linija SQ20B razdvojena u dvije podpopulacije; klonove s normalno eksprimiranim TLR3 i klonove u kojima je korištenjem shRNA utišan gen za TLR3. Testom formiranja kolonija pokazano je da poli(I:C) uzrokuje smanjenje preživljenja stanica karcinoma grkljana za oko 50 %, dok stanice s utišanim genom za TLR3 pokazuju veću otpornost i preživljenje nakon tretmana citostaticima. Kombinirana terapija imala je presnažan učinak, što se može riješiti smanjenjem doza. Analizom pomoću protočnog citometra, uz bojenje stanične DNA propidijevim jodidom, utvrđeno je da poli(I:C) povećava udio stanica SQ20B u fazi G2/M staničnog ciklusa u stanicama s funkcionalnim TLR3, dok je u onima s
utišanim TLR3 taj broj podjednak u odnosu na kontrolu. Cisplatina je u većini slučajeva maskirala učinak poli(I:C) kada je primijenjena u kombinaciji. Rezultati ovoga istraživanja pokazuju kako bi se kombinacija poli(I:C), cisplatine i γ-zračenja mogla koristiti u liječenju bolesnika s tumorima koji izražavaju funkcionalni TLR3, uz korištenje relativno niskih doza lijekova i zračenja za postizanje zadovoljavajućeg protutumorskog učinka. Međutim, prije konačnih zaključaka valja provesti dodatna istraživanja o molekularnim mehanizmima opaženih rezultata.

Abstract (english)

Head and neck squamous cell carcinoma represents the sixth most common cancer in the world, while patients'
survival and therapy outcome remain unsatisfying. In recent studies Toll-like receptors (TLRs) have been found in
these and various other human tumor cells. TLRs enable their host to recognize a series of molecular motifs derived
from pathogens (PAMPs) by activating innate and adaptive immune responses. TLR3 is an intracellular protein
receptor that binds double-stranded RNA (dsRNA). It has been reported that the functional TLR3 is expressed in
pharyngeal metastatic tumor cells and its activation drives them to apoptosis. Therefore, in this study we tested the
effect of individual and combined application of TLR3 ligand, poly(I:C), with antitumor drug cisplatin and γ-
irradiation on survival and cell cycle regulation of larynx carcinoma cells. In this study we used SQ20B cell line stably
transfected with control shRNA and shRNA for TLR3. Thus we obtained cells with functional TLR3 and cells with
silenced gene for TLR3. Results from colony forming assay show that poly(I:C) reduces survival of larynx carcinoma
cells by approximately 50 %, while cells with TLR3 knock-down show greater resistance and survival after cytotoxic
treatment. Combined therapy proved to be too strong, and requires dose reduction. Cell cycle distribution was
analyzed after cell staining with propidium iodide using a flow cytometer. Poly(I:C) increased the arrest of SQ20B
cells with functional TLR3 in phases G2/M of the cell cycle, while cell cycle distribution of cells with TLR3 knockdown
was similar to control. Cisplatin masks the effect of poly(I:C) in most cases when applied in combination. The
results of this study demonstrate how the combination of poly(I:C), cisplatin and γ-radiation could be used in the
treatment of patients with tumors that express functional TLR3. This allows the usage of lower doses of drugs and
radiation for maximizing the antitumor effect while limiting their toxicities. However, further investigation on
molecular mechanisms of the observed effects is required before final conclusions.

• Keywords: TLR3; tumori glave i vrata; poli(I:C); cisplatina; stanični ciklus
• Keywords (english): TLR3; head and neck cancer; poly(I:C); cisplatin; cell cycle
• Language: croatian
• URN:NBN: urn:nbn:hr:163:030091
• Study programme: Title: Pharmacy; Study programme type: university; Study level: integrated undergraduate and graduate; Academic / professional title: magistar/magistra farmacije (magistar/magistra farmacije)
• Type of resource: Text
• File origin: Born digital
• Access conditions: Open access
• Created on: 2016-02-18 16:43:36