master's thesis
Effects of dsRNA and cisplatin on sensitization of human larynx carcinoma cells to radiation

Antonio Kobeščak (2015)
Sveučilište u Zagrebu
Farmaceutsko-biokemijski fakultet
Zavod za medicinsku biokemiju i hematologiju
Metadata
TitleUtjecaj dsRNA i cisplatine na povećanje osjetljivosti stanica tumora grkljana čovjeka na zračenje
AuthorAntonio Kobeščak
Mentor(s)Karmela Barišić (thesis advisor)
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.
KeywordsTLR3 head and neck cancer poly(I:C) cisplatin cell cycle
Parallel title (English)Effects of dsRNA and cisplatin on sensitization of human larynx carcinoma cells to radiation
Committee MembersKarmela Barišić
Sandra Šupraha Goreta
GranterSveučilište u Zagrebu
Farmaceutsko-biokemijski fakultet
Lower level organizational unitsZavod za medicinsku biokemiju i hematologiju
PlaceZagreb
StateCroatia
Scientific field, discipline, subdisciplineBIOMEDICINE AND HEALTHCARE
Pharmacy
Pharmacy
Study programme typeuniversity
Study levelintegrated undergraduate and graduate
Study programmePharmacy
Academic title abbreviationmag. pharm.
Genremaster's thesis
Language Croatian
Defense date2015-09-28
Parallel 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.
Parallel keywords (Croatian)TLR3 tumori glave i vrata poli(I:C) cisplatina stanični ciklus
Resource typetext
Access conditionOpen access
Terms of usehttp://rightsstatements.org/vocab/InC/1.0/
URN:NBNhttps://urn.nsk.hr/urn:nbn:hr:163:030091
CommitterPetra Gašparac