Supplementary material for the article: Živković, M. B.; Matić, I. Z.; Rodić, M. V.; Novaković, I. T.; Krivokuća, A. M.; Sladić, D. M.; Krstić, N. M. Anticancer Potential of New Steroidal Thiazolidin-4-One Derivatives. Mechanisms of Cytotoxic Action and Effects on Angiogenesis in Vitro. Journal of Steroid Biochemistry and Molecular Biology 2017, 174, 72–85. https://doi.org/10.1016/j.jsbmb.2017.07.031

Supplementary material for: [https://doi.org/10.1016/j.jsbmb.2017.07.031]Related to published version: [http://cherry.chem.bg.ac.rs/handle/123456789/2550]Related to accepted version: [http://cherry.chem.bg.ac.rs/handle/123456789/3191

Mutations in breast cancer susceptibility genes in BRCA1 and BRCA2 negative hereditary breast and ovarian cancer families in Serbia

Uvod: Nasledni karcinom čini 5-10% od svih slučajeva karcinoma dojke i prvenstveno je povezan sa štetnim mutacijama u visoko penetrabilnim genima BRCA1 i BRCA2. I pored intenzivnog istraživanja ostatka nasledne komponente, treći visoko penetrabilni gen u karcinomu dojke (BRCA3) još uvek nije pronadjen. Pod okriljem poligenog modela otkrivene su tri grupe alela koji doprinose riziku za karcinom dojke: visoko penetrabilni, koji povećavaju rizik na 40-85% (BRCA1, BRCA2, TP53), srednje penetrabilni (povećavaju rizik na 20-40%) kao što su CHEK2, RAD51, RAD51C i XRCC1, i učestale genske varijante koje su nisko penetrabilne. Pored štetnih mutacija u ovim genima, odredjene polimorfne varijante mogu uticati na efikasnost popravke grešaka u DNK i na nastanak karcinoma. Cilj: Obzirom da prisustvo mutacija u BRCA1/2 genima ne može da opiše celu genetičku osnovu naslednog karcinoma dojke, cilj ovog rada je istraživanje prisustva populaciono specifičnih mutacija u drugim genima (TP53, CHEK2, NBS1, RAD51, RAD51C i XRCC1) kod BRCA1/2 negativnog, naslednog karcinoma dojke u Srbiji. Utvrdjivanje asocijacije polimorfnih varijanti u ovim genima sa rizikom za nastanak karcinoma dojke, kao i njihove korelacije sa parametrima tumora i parametrima domaćina tumora, predstavlja dodatni cilj ovog rada. Materijal i metode: 57 ispitanika poreklom iz visokorizičnih porodica kod kojih je sekvenciranje BRCA1/2 gena dalo negativan rezultat (nasledni uzorak) i 107 pacijentkinja sa karcinomom dojke koji nije odredjen u odnosu na porodičnu istoriju bolesti (sistematski uzorak), uključeno je u ovu studiju. Kontrolnu grupu je činilo 114 briseva grlića materice zdravih žena sa normalnim Papa nalazom i bez prethodne istorije pre-kancerskih i kancerskih lezija...Background: 5-10% of all breast cancer cases are hereditary, and are often caused by deleterious mutations in high penetrance BRCA1 and BRCA2 genes. Despite significant efforts in unraveling the rest of the familial component, BRCA3 could not be identified. Under the polygenic model of inheritance, three groups of genetic predisposition alleles have been identified so far: high-risk genes, conferring 40–85% lifetime risk (BRCA1, BRCA2, TP53), moderate-risk genes (20–40% lifetime risk) including CHEK2, RAD51, RAD51C and XRCC1, and low-risk common alleles. Besides the deleterious mutations in these genes, certain polymorphic variants have functional significance in DNA repair pathways, and may be responsible for cancer development. Aim: Considering the fact that mutations in BRCA1/2 can’t explain the whole range of hereditary predisposition to breast cancer, we aimed to investigate the presence and frequency of population-specific mutations in other susceptibility genes (TP53, CHEK2, NBS1, RAD51, RAD51C and XRCC1) in BRCA1/2 negative hereditary breast cancer cases in Serbia. Determining association between polymorphic variants in these genes with breast cancer risk, as well as their correlation with patient’s characteristics and histological and pathological characteristics of breast cancer, were also the aims of this study. Material and methods: 57 subjects from high-risk families negative for small alterations in BRCA1/2 genes (hereditary group) and 107 breast cancer patients non-selected according to family history (consecutive group) were included in the study. The control group was consisted of 114 cervical smears of gynecological healthy women with normal Papa test results and without previous history of pre-cancer and cancer lesions. Large genomic rearrangements in BRCA1/2 genes were investigated by MLPA technique, while genotyping for specific CHEK2 mutations was done by allele specific PCR..

Mutations in breast cancer susceptibility genes in BRCA1 and BRCA2 negative hereditary breast and ovarian cancer families in Serbia

Uvod: Nasledni karcinom čini 5-10% od svih slučajeva karcinoma dojke i prvenstveno je povezan sa štetnim mutacijama u visoko penetrabilnim genima BRCA1 i BRCA2. I pored intenzivnog istraživanja ostatka nasledne komponente, treći visoko penetrabilni gen u karcinomu dojke (BRCA3) još uvek nije pronadjen. Pod okriljem poligenog modela otkrivene su tri grupe alela koji doprinose riziku za karcinom dojke: visoko penetrabilni, koji povećavaju rizik na 40-85% (BRCA1, BRCA2, TP53), srednje penetrabilni (povećavaju rizik na 20-40%) kao što su CHEK2, RAD51, RAD51C i XRCC1, i učestale genske varijante koje su nisko penetrabilne. Pored štetnih mutacija u ovim genima, odredjene polimorfne varijante mogu uticati na efikasnost popravke grešaka u DNK i na nastanak karcinoma. Cilj: Obzirom da prisustvo mutacija u BRCA1/2 genima ne može da opiše celu genetičku osnovu naslednog karcinoma dojke, cilj ovog rada je istraživanje prisustva populaciono specifičnih mutacija u drugim genima (TP53, CHEK2, NBS1, RAD51, RAD51C i XRCC1) kod BRCA1/2 negativnog, naslednog karcinoma dojke u Srbiji. Utvrdjivanje asocijacije polimorfnih varijanti u ovim genima sa rizikom za nastanak karcinoma dojke, kao i njihove korelacije sa parametrima tumora i parametrima domaćina tumora, predstavlja dodatni cilj ovog rada. Materijal i metode: 57 ispitanika poreklom iz visokorizičnih porodica kod kojih je sekvenciranje BRCA1/2 gena dalo negativan rezultat (nasledni uzorak) i 107 pacijentkinja sa karcinomom dojke koji nije odredjen u odnosu na porodičnu istoriju bolesti (sistematski uzorak), uključeno je u ovu studiju. Kontrolnu grupu je činilo 114 briseva grlića materice zdravih žena sa normalnim Papa nalazom i bez prethodne istorije pre-kancerskih i kancerskih lezija...Background: 5-10% of all breast cancer cases are hereditary, and are often caused by deleterious mutations in high penetrance BRCA1 and BRCA2 genes. Despite significant efforts in unraveling the rest of the familial component, BRCA3 could not be identified. Under the polygenic model of inheritance, three groups of genetic predisposition alleles have been identified so far: high-risk genes, conferring 40–85% lifetime risk (BRCA1, BRCA2, TP53), moderate-risk genes (20–40% lifetime risk) including CHEK2, RAD51, RAD51C and XRCC1, and low-risk common alleles. Besides the deleterious mutations in these genes, certain polymorphic variants have functional significance in DNA repair pathways, and may be responsible for cancer development. Aim: Considering the fact that mutations in BRCA1/2 can’t explain the whole range of hereditary predisposition to breast cancer, we aimed to investigate the presence and frequency of population-specific mutations in other susceptibility genes (TP53, CHEK2, NBS1, RAD51, RAD51C and XRCC1) in BRCA1/2 negative hereditary breast cancer cases in Serbia. Determining association between polymorphic variants in these genes with breast cancer risk, as well as their correlation with patient’s characteristics and histological and pathological characteristics of breast cancer, were also the aims of this study. Material and methods: 57 subjects from high-risk families negative for small alterations in BRCA1/2 genes (hereditary group) and 107 breast cancer patients non-selected according to family history (consecutive group) were included in the study. The control group was consisted of 114 cervical smears of gynecological healthy women with normal Papa test results and without previous history of pre-cancer and cancer lesions. Large genomic rearrangements in BRCA1/2 genes were investigated by MLPA technique, while genotyping for specific CHEK2 mutations was done by allele specific PCR..

Anticancer potential of new steroidal thiazolidin-4-one derivatives. Mechanisms of cytotoxic action and effects on angiogenesis in vitro

The synthesis and cytotoxic activities determination of new steroidal mono- and bis(thiazolidin-4-ones) 4a-f and 5a-f have been performed. Their anticancer action was also evaluated in comparison to previously synthesized and reported corresponding steroidal thiosemicarbazones. All compounds were obtained as stereoisomeric mixtures with different configuration (E or Z) in the hydrazone moiety at the C-3 position. After several consecutive crystallizations diastereomerically pure major (5)-isomers of mono-thiazolidin-4-ones were isolated. The structure and stereochemistry of 2,4-thiazolidinedione,2-[(17-oxoandrost-4-en-3-ylidene)hydrazone] were confirmed by X-ray analysis. A pathway for the formation of thiazolidin-4-one ring was proposed. The steroid thiazolidinone derivatives examined in this study exerted selective concentration-dependent cytotoxic activities on six tested malignant cell lines. Ten out of twelve examined compounds exhibited strong cytotoxic effects on K562 cells (IC50 values from 8.5 mu M to 14.9 mu M), eight on HeLa cells (IC50 values ranging from 8.9 mu M to 15.1 mu M) while against MDA-MB-361 cells six compouds exerted similar or even higher cytotoxic action (IC50 values from 12.7 mu M to 25.6 mu M) than cisplatin (21.5 mu M) which served as a positive control. Eight of these ten compounds showed high selectivity in the cytotoxic action against HeLa and K562 cancer cell lines when compared with normal human fibroblasts MRC-5 and normal human PBMC. The study of mechanisms of the anticancer activity of the two selected compounds, mono- and bis(thiazolidin-4-one) derivatives of 19-norandrost-4-ene-3,17-dione 4a and 5a, revealed that both of these compounds induced apoptosis in HeLa cells through extrinsic and intrinsic signalling pathways. Treatment of EA.hy926 cells with sub-toxic concentrations of these compounds led to the inhibition of cell connecting and sprouting, and tube formation. The synthesized compounds exhibited poor antioxidant activity.Peer-reviewed manuscript:: [http://cherry.chem.bg.ac.rs/handle/123456789/3191]Supplementary material: [http://cherry.chem.bg.ac.rs/handle/123456789/3192