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1

Proces składania RNA jako cel oddziaływania terapeutycznego

100%

Paluszczak J.

Farmacja Polska

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2019

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vol. 75

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issue 11

605-616

EN

Gene transcription leads to the generation of pre-mRNA molecules which contain both coding sequences (exons) and intervening non-coding sequences (introns). The primary transcript needs further processing which involves the excision of introns and ligation of exons. This process is called RNA splicing. Nearly all primary transcripts undergo alternative forms of splicing, which may lead to exon skipping or intron inclusion in the final mRNA. Thus, the translation of alternatively spliced RNA molecules results in the formation of slightly different proteins, which may, in some cases, exert antagonistic activity. Splicing is a multistage process which is conducted by a complex machinery comprising small nuclear RNA molecules and many proteins called splicing factors. The process undergoes precise regulation by means of cis acting internal RNA sequences and trans acting protein factors which may either enhance or silence the splicing of an exon. Many diseases are associated with aberrations of alternative splicing and its modulation may be used therapeutically, e.g. for the treatment of spinal muscular atrophy (SMA) or Duchenne muscular dystrophy (DMD). This article presents current knowledge of the ways of pharmacological modulation of alternative splicing. The focus is on the use of those therapeutics which have been already approved for clinical application or have entered clinical trials. The chemistry and mechanism of action of specific splice switching oligonucleotides is presented. Nusinersen promotes exon inclusion during splicing of SMN2 and is used for SMA treatment. On the other hand, eteplirsen is an oligonucleotide promoting exon skipping during splicing of mutated DMD and has been conditionally approved for DMD treatment. Moreover, small molecule modulators of alternative splicing (e.g. branaplam) are also described. The dynamic developments in this field should result in the approval of new drugs acting by the modulation of alternative splicing in the nearest future.

PL

Powstająca w wyniku transkrypcji genu cząsteczka pre-mRNA zawiera odcinki kodujące (eksony) poprzedzielane odcinkami niekodującymi (intronami). Pierwotny transkrypt wymaga obróbki, która polega między innymi na wycinaniu intronów i łączeniu eksonów. Proces ten nazywany jest składaniem RNA. W przypadku niemal wszystkich transkryptów, składanie pre-mRNA przebiega w komórkach alternatywnymi drogami, na przykład prowadząc do wykluczenia jednego z eksonów z ostatecznego transkryptu, lub włączenia jednego z intronów. Proces składania RNA przeprowadzany jest przez skomplikowaną maszynerię złożoną z małych cząsteczek RNA i białek, i podlega precyzyjnej regulacji. Wiele chorób związanych jest z nieprawidłowościami alternatywnego składania RNA, a modulacja tego procesu może być wykorzystana terapeutycznie, między innymi w leczeniu rdzeniowego zaniku mięśni (SMA, ang. spinal muscular atrophy) czy dystrofii mięśniowej Duchenne’a (DMD). Celem artykułu jest przedstawienie wiedzy na temat farmakologicznych możliwości wpływania na proces składania RNA. Nacisk położono na scharakteryzowanie tych terapeutyków, które zostały już zarejestrowane do użytku klinicznego, lub które są w trakcie zaawansowanych badań klinicznych. Zaprezentowano budowę chemiczną i mechanizm działania oligonukleotydowych przełączników składania RNA. Nusinersen stymuluje włączanie eksonu 7 w trakcie składania SMN2 i jest wykorzystywany w leczeniu SMA. Eteplirsen, który stymuluje pomijanie eksonu 51 w trakcie składania zmutowanych wariantów DMD, został warunkowo dopuszczony do leczenia DMD w USA. Opisano także drobnocząsteczkowe modulatory alternatywnego składnia RNA, np. branaplam. Dynamiczny rozwój tego obszaru badań stwarza szansę na wprowadzone w najbliższej przyszłości do lecznictwa kolejnych leków, których mechanizm działania oparty będzie na modulacji alternatywnego składania RNA.

2

THE EFFECT OF NICLOSAMIDE ON THE HEAD AND NECK CARCINOMA CELLS SURVIVAL AND THE EXPRESSION OF WNT/β-CATENIN SIGNALING AND GLYCOLYSIS PATHWAY COMPONENTS

51%

Kleszcz R., Paluszczak J., Baer-Dubowska W.

Acta Poloniae Pharmaceutica - Drug Research

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2019

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vol. 76

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issue 4

661-669

EN

The aim of this study was to evaluate the effect of niclosamide, an antihelminthic drug recently identified as potential anti-cancer agent, on head and neck squamous carcinoma cells (HNSCC) viability, cell cycle distribution and apoptosis. The expression of key components of Wnt (CTNNB1, GSK-3β, CCND1, c-MYC, MMP7, BIRC5, Axin2) and glycolysis (GLUT1, MCT1, HK2, PFKM, PKM2, PDHA1, PDK1, LDHA) pathways was also examined to assess possible involvement in niclosamide anti-carcinogenic activity. HNSCC cells (FaDu, BICR6, H314 lines) were used in the research. Niclosamide treatment affected hypopharyngeal FaDu cells to the most extent (IC50 = 0.40 µM), while H314 cells derived from the floor of mouth were the least sensitive (IC50 = 0.94 µM). In FaDu cells the increased percentage of the cells in the S phase was observed along with the induction of apoptosis. Treatment with niclosamide in FaDu cells reduced the expression of MMP7 and the majority of glycolytic genes except increased LDHA. These results indicate that niclosamide is efficient inhibitor of HNSCC cells viability, however this effect depends on the cell type. In FaDu cells, the most sensitive to its anti-proliferative effect and prone to cell cycle arrest and apoptosis, this effect might be related to slightly modulation of canonical Wnt signaling and increased expression of LDHA.

3

THE COMPARISON OF THE EFFECTS OF PANOBINOSTAT AND PKF118-310 ON β-CATENIN-DEPENDENT TRANSCRIPTION IN HEAD AND NECK SQUAMOUS CELL CARCINOMA CELL LINES

51%

Paluszczak J., Kleszcz R., Witczak O., Krajka-Kuźniak V.

Acta Poloniae Pharmaceutica - Drug Research

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2020

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vol. 77

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issue 1

77-88

EN

Advanced head and neck squamous cell cancers (HNSCC) have unfavorable prognosis and new therapeutic options are necessary to improve treatment outcomes. The Wnt pathway plays an important role in the pathogenesis and progression of HNSCC. The aim of this study was to assess the effects of a histone deacetylase inhibitor – panobinostat on Wnt-dependent gene expression and on cell migration. Cell viability in HNSCC cell lines (BICR6, CAL27, FaDu, H314, SCC-25) was evaluated by MTT assay. The expression of β-catenin-target genes was assessed by qPCR and TCF/LEF-dependent reporter assay. Protein content was evaluated by Western blot. Cell migration was analyzed by the wound healing assay. Panobinostat showed differential modulation of gene expression. It reduced the level of Axin2 in CAL27 and SCC-25 cells but upregulated its expression in BICR6 and H314 cell lines. Moreover, it diminished the expression of MMP7 in BICR6, H314 and CAL27 cell lines. In contrast, the inhibitor of β-catenin transcriptional activity – PKF118-310 down-regulated the expression of β-catenin-target genes in HNSCC cell lines. Interestingly, panobinostat had opposite effects on cell migration in CAL27 and FaDu where it inhibited or stimulated migration, respectively. On the other hand, PKF118-310 reduced cell migration. The anti-cancer effects of panobinostat in HNSCC cells are rather not related to the inhibition of Wnt signaling. PKF118-310 attenuates Wnt signaling, but only in a limited number of HNSCC cell lines. Importantly, the inhibition of Wnt pathway reduces the capacity of cells for migration suggesting that it may potentially therapeutically reduce cell invasion.

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2 Acta Poloniae Pharmaceutica - Drug Research

1 Farmacja Polska

1 2020

2 2019

3 Paluszczak J.

2 Kleszcz R.

1 Baer-Dubowska W.

1 Krajka-Kuźniak V.

1 Witczak O.

Proces składania RNA jako cel oddziaływania terapeutycznego

THE EFFECT OF NICLOSAMIDE ON THE HEAD AND NECK CARCINOMA CELLS SURVIVAL AND THE EXPRESSION OF WNT/β-CATENIN SIGNALING AND GLYCOLYSIS PATHWAY COMPONENTS

THE COMPARISON OF THE EFFECTS OF PANOBINOSTAT AND PKF118-310 ON β-CATENIN-DEPENDENT TRANSCRIPTION IN HEAD AND NECK SQUAMOUS CELL CARCINOMA CELL LINES