Investigation Of The Apoptotic And Anti-Proliferative Effects Of Farnesyltransferase Inhibitor In Mutant And Wild Type Colon Cancer Cell Lines

Kolon kanserinin kesin etiyolojisi bilinmemekle birlikte bazı genetik ve çevresel faktörlerin kolon kanserinde önemli rol oynadıkları yapılan çalışmalarla gösterilmiştir. Kolorektal kanserlerde mutasyon ile aktive olmuş K-Ras major olarak Ras/Raf/MEK/ERK ve Ras/PI3K/Akt sinyalizasyon yolakları üzerinden sağkalım ve hücre çoğalmasında görev alan genlerin transkripsiyonunu regüle etmektedir. K-Ras ın hücre zarına post-translasyonel dönüşümle bağlanmasının ilk basamağı olan prenilasyon aşaması, Ras aracılı sinyalizasyonun hız belirleyici basamağını oluşturmaktadır. LB42708, Ras proteinlerinin post-translasyonel dönüşümünün ilk basamağında görev alan farnesil transferaz ve geranil geranil transferaz enzimlerini inhibe ederek K-Ras proteinlerinin normal ve alternatif prenilasyon mekanizmalarını ortadan kaldırarak MAPK/ERK/p38 ve PI3K/Akt üzerinden hücre çoğalması ve sağkalım sinyallerini bloke etmektedir. Çalışmamızda, LB42708 ajanının belirli doz ve zaman aralığında uygulanması sonucunda p53 +/+ ve p53 -/- HCT-116 insan kolon kanser hücre hatlarında MTT yöntemi, AO/EtBr boyaması ve BrdU ELISA yöntemleriyle hücre canlılığı belirlenmiştir. Apoptotik oran LDH, DNA Fragmentasyon analizi ile doğrulandı. Apoptotik ve proliferatif sinyal iletim yolaklarında görev alan genlerin mRNA ifade düzeylerinin belirlenmesi için kantitatif Real-Time PCR yöntemi kullanıldı. Buna göre LB42708 nin, artan doza bağlı olarak anti-apoptotik genlerin (Bcl-2, Bcl-XL) ekspresyonunu azaltması, pro-apoptotik (Bax, Bak, Bim) genlerin ekspresyonunu artırması ve hücre döngüsünde G1/S geçişinde görev alan Siklin D1 ekspresyonunu azaltması ile hücrelerin apoptoza yönlendiğini ve ayrıca hücre proliferasyonunda görev alan c-Myc mRNA düzeylerini azaltması ile proliferasyonu azalttığı gen düzeyinde belirlenmiştir. Sonuç elde ettiğimiz bulgular, LB42708 uygulanması in vivo modellerde yapılacak çalışmalar ile kolorektal kanserlere karşı potansiyel antineoplastik ilaç olabileceği düşüncesini güçlendirmektedir.The main ethiology of colon cancers is unknown, however most studies have shown that some genetic and enviromental factors play important role on formation of colon cancers. In colorectal cancers, mutationally activated K-Ras, regulates genes transcription of cell proliferation and survival signals via Ras/Raf/MEK/ERK and Ras/PI3K/Akt pathways. The first and rate limiting step of post-translational modification leading K-Ras to anchor the inner surface of the cell membrane is the prenylation step. LB42708 is an farnesyltransferase and geranylgeranyltransferase inhibitor that inhibits normal and alternative prenylation of K- Ras protein, leading to the inhibition of cell survival and proliferation by arresting the MAPK/ERK/p38 and PI3K/Akt pathways. In this study we aimed to investigate the apoptotic and anti-proliferative effects of LB42708, at different doses at a period of time on p53 +/+ and p53 -/- HCT-116 colon cancer cell lines by MTT assay, AO/EtBr staining and BrdU ELISA methods and confirmed our results with LDH and DNA fragmentation analysis. We use RT-PCR analysis to evaluate the mRNA levels of the genes involved in apoptotic and proliferative signaling pathways. According to this, treatment with the increasing dose of LB42708 has shown that the transcription levels of anti-apoptotic genes (Bcl-2, Bcl-XL) had been decreased, pro-apoptotic genes (Bax, Bak, Bim) had been increased and cell cycle G1/S transition facilitator cyclin D1 had been decreased. Moreover, c-myc mRNA levels, which involves in cell proliferation, had been decreased. Our results along with in vivo models strengthen the idea LB42708 will be a potential antineoplastic drugs against colorectal cancer

Effect of API-1 and FR180204 on cell proliferation and apoptosis in human DLD-1 and LoVo colorectal cancer cells

Elmazoglu, Zubeyir/0000-0003-4527-8834WOS: 000385579200033PubMed: 27698814The activation of the phosphatidylinositol-3 kinase/v-akt murine thymoma viral oncogene homolog (Akt) and mitogen activated protein kinase kinase/extracellular signal-regulated kinase (ERK) pathways are implicated in the majority of cancers. Selective inhibition of Akt and ERK represents a potential approach for cancer therapy. Therefore, the present study aimed to investigate the apoptotic and anti-proliferative effects of the novel and selective Akt inhibitor 4-amino-5,8-dihydro-5-oxo-8--D-ribofuranosyl-pyrido[2,3-d]pyrimidine-6-carboxamide (API-1) and selective ERK1/2 inhibitor FR180204 (FR) alone and in combination on colorectal cancer (CRC) cells (DLD-1 and LoVo). In addition, the effects of API-1 and FR on Akt and ERK signaling pathways were also investigated. The effects of the agents on DLD-1 and LoVo cells were evaluated in terms of cell viability, cytotoxicity, DNA synthesis rate, DNA fragmentation and caspase-3 activity levels. In addition, quantitative reverse transcription-polymerase chain reaction and western blot analysis were performed to examine relevant mRNA and protein levels. The present study observed that the combination of FR with API-1 resulted in significant apoptosis and cytotoxicity compared with any single agent alone in a time-dependent manner in these cells. Also, treatment with FR and API-1 in combination decreased the expression levels of B-cell lymphoma-2 (BCL2), Bcl-2-like1, cyclin D1 and cMYC, and increased the expression levels of BCL2-associated X protein and BCL2 antagonist/killer via phosphorylated Akt and phosphorylated ERK1/2 downregulation. The combination of Akt and ERK1/2 inhibitors resulted in enhanced apoptotic and anti-proliferative effects against CRC cells. The present study hypothesizes that the combination of FR and API-1 in CRC cells may contribute toward potential anti-carcinogenic effects. Additional analyses using other cancer cell lines and animal models are required to confirm these findings in vitro and in vivo.Scientific and Technological Research Council of TurkeyTurkiye Bilimsel ve Teknolojik Arastirma Kurumu (TUBITAK) [111S054]The present study was supported by The Scientific and Technological Research Council of Turkey (grant number 111S054)

General toxicity assessment of the novel aldose reductase inhibitor cemtirestat

Cemtirestat, 3-mercapto-5H-[1,2,4]-triazino[5,6-b]indole-5-acetic acid was recently designed and patented as a highly selective and efficient aldose reductase inhibitor endowed with antioxidant activity. The aim of the present study was to assess the general toxicity of cemtirestat using in silico predictions, in vitro and in vivo assays. ProTox-II toxicity prediction software gave 17 “Inactive” outputs, a mild hepatotoxicity score (0.52 probability) along with a predicted LD50 of 1000 mg/kg. Five different cell lines were used including the immortalized mouse microglia BV-2, the primary human fibroblasts VH10, the insulinoma pancreatic β-cells INS-1E, the human colon cancer cells HCT116 and the human immortalized epithelial endometrial cell lines HIEEC. In contrast to the clinically used epalrestat, cemtirestat showed remarkably low cytotoxicity in several different cell culture viability tests such as MTT proliferation assay, neutral red uptake, BrdU incorporation, WST-1 proliferation assay and propidium iodide staining followed by flow cytometry. In a yeast spotting assay, the presence of cemtirestat in incubation of Saccaromyces cerevisiae at concentrations as high as 1000 µM did not affect cell growth rate significantly. In the 120-day repeated oral toxicity study in male Wistar rats with daily cemtirestat dose of 6.4 mg/kg, no significant behavioral alterations or toxicological manifestations were observed in clinical and pathological examinations or in hematological parameters. In summary, these results suggest that cemtirestat is a safe drug that can proceed beyond preclinical studies