İnsan kolorektal kanser hücrelerinde Pistacia eurycarpa ekstraktlarıyla kombine 5-florourasilin artırılmış antiproliferatif ve apoptotik etkileri

Abstract

Kolorektal adenokarsinoma, kolon ve rektumu etkileyen yaygın bir kanser türüdür. Genellikle kolonda oluşan poliplerin yayılması ile gelişir. Kolorektal kanser vakalarında, 5-Florourasil (5-FU) klinik tedavinin temel basamağını oluşturur. Ancak 5-FU’nun ciddi yan etkilere sahip olması, 5-FU temelli yeni tedavilerin geliştirilmesini gerekli kılmaktadır. Pistacia eurycarpa farklı tıbbi etkilere sahip bir bitkidir. Burada sunulan çalışmada, P.eurycarpa ekstraktlarının tek ve 5-FU ile kombine olarak uygulandığında, HT-29 insan kolorektal kanser hücreleri üzerindeki antiproliferatif ve apoptotik etkileri incelendi. Hücre canlılığı MTT analizi ile belirlendi. Bitki kstraktları ile 5-FU arasındaki sinerjizmi belirlemek için izobologram analizi yapıldı. Apoptozun belirlenmesinde CDDE yöntemi kullanıldı. İnsan VEGF Elisa yöntemi ile VEGF miktarı kantitatif olarak ölçüldü. Ayrıca, hücreler 24 saat boyunca, belirlenen IC50değerlerinde ekstraktlar ve kombinasyonlar ile tedavi edildiğinde, bazı apoptotik ve anti-apoptotik gen ve proteinlerin (p53, Bax, Bcl-2, p38 MAPK, mTOR, PTEN ve Akt) ekspresyon düzeylerindeki değişiklikler, gerçek zamanlı polimeraz zincir reaksiyonu (qRT-PCR) ve western blot analizleri ile değerlendirildi. P. eurycarpa hekzan, kloroform ve metanol ekstreleri ile 5-FU'nun IC50 dozlarının kombine tedavisinde hücre canlılıkları sırasıyla % 23.5, % 21.2 ve % 11.9 olarak belirlendi (p<0.0001). P. eurycarpa hekzan, kloroform ve metanol ekstreleri, 5-FU ile ombinasyon halinde apoptozu 8.6, 9.2 ve 11.8 kat arttırdı ve kontrol grubuna kıyasla VEGF miktarını 76.2, 75.1 ve 64.7 pg/ml'ye düşürdü. Apoptotik ve anti-apoptotik genlerdeki ve proteinlerin ekspresyon düzeylerindeki değişiklikler hemen hemen tüm tedavi gruplarında istatistiksel olarak anlamlı bulundu. Tüm bu sonuçlar, P. eurycarpa ekstraktlarınının ve bu ekstraktların 5-FU ile kombinasyonunun, kolorektal kanser hücrelerinin canlılığını önemli ölçüde azalttığını ve hücreleri apoptoza yönlendirdiğini göstermektedir.

Keywords

• Pistacia eurycarpa
• kanser
• 5-Florourasil
• apoptoz
• p53

Enhanced antiproliferative and apoptotic effects of 5-fluorouracil by combined with Pistacia eurycarpa Extracts on human colorectal cancer cells

Abstract

Colorectal adenocarcinoma is a common type of cancer that affects the colon and rectum. It usually develops by spreading polyps in the colon. In cases of colorectal cancer, 5-Fluorouracil (5-FU) constitutes the basic step of clinical treatment. However, the fact that 5-FU has serious side effects necessitates the development of new therapies based on 5-FU. Pistacia eurycarpa is a plant with different medical effects. In this study, the antiproliferative and apoptotic effects of P.eurycarpa extracts alone or combined with 5-FU on HT-29 human colorectal cancer cells were investigated. Cell viability was determined by MTT analysis. Isobologram analysis was performed to determine the synergism between plant extracts and 5-FU. CDDE method was used to determine apoptosis. VEGF was quantitatively measured by human VEGF Elisa method. Moreover, when cells were treated with extracts and combinations at determined IC50 values for 24 hours, changes in expression levels of some apoptotic and anti-apoptotic genes and proteins (p53, Bax, Bcl-2, p38 MAPK, mTOR, PTEN and Akt) were evaluated by real time polymerase chain reaction (qRT-PCR) and western blot analysis. In combined treatment at IC50 doses of P. eurycarpa hexane, chloroform and methanol extracts with 5-FU, cell viability was determined as 23.5%, 21.2% and 11.9%, respectively (p <0.0001). Compared to the control, P. eurycarpa hexane, chloroform and methanol extracts in combination with 5-FU, increased the apoptosis 8.6, 9.2 and 11.8 fold and reduced the amount of VEGF to 76.2, 75.1 and 64.7 pg/ml, respectively. Changes in expression levels of apoptotic and anti-apoptotic genes and proteins were found to be statistically significant in almost all therapies. All these results had showed that, the P.eurycarpa extracts and combination of these extracts with 5-FU dramatically reduced the viability of colorectal cancer cells and directed the cells to apoptosis.

Keywords

• Pistacia eurycarpa
• cancer
• 5-Fluorouracil
• apoptosis
• p53

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