In Vitro Cytotoxic and Proapoptotic Activities of Anatolian Macrovipera Lebetina Obtusa (Dwigubski, 1832) Crude Venom on Cultured K562 Human Chronic Myelogenous Leukemia Cells.

Abstract

In the context of searching for anticancer compounds in natural products, snake venom is one of the important sources for peptide/ protein based bioactive molecules. Proteins and peptides with anticancer activity were purified and identified from snake venoms. The aim of the present study was to determine the in vitro cytotoxicity of Macrovipera lebetina obtusa (Blunt-Nosed Viper) crude venom from southeastern Anatolia against K562 human chronic myelogenous leukemia (CML) cells by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and Adenosine tripohsphate (ATP) assays. Additionally, the apoptosis induction was assessed by morphological evaluation and immunohistochemical analysis for activated caspase-3. For histopahtological evaluation, haematoxylineosin, giemsa and papanicolau stains were used in combination. M. l. obtusa venom showed dose-dependent toxicity against K562 cells after 72 h treatment with different concentrations of crude venom. IC50 values were 0.45 and 0.37 µg/mL for MTT and ATP assays, respectively. Nuclear fragmentation and condensation, apoptotic bodies and activation of caspase-3, as an induction of apoptosis were also observed in K562 cells. Since apoptosis-inducing compounds are important for the treatment of cancer, further studies on Anatolian M. l. obtusa venom could result in the purification and identification of new proteins and peptides, which might have therapeutic value for the treatment of CML. Keywords: Anticancer, Apoptosis, Blunt-nosed viper, Macrovipera lebetina obtusa, Snake venom ÖZET Anadolu'da Yayılış Gösteren Macrovipera lebetina obtusa (Dwigubski, 1832) Zehrinin K562 İnsan Kronik Myeloid Lösemi Hücreleri Üzerinde in vitro Sitotoksik ve Apoptotik Aktiviteleri Doğal ürünlerde antikanser bileşiklerin araştırılması kapsamındaki çalışmalarda kullanılan önemli biyoaktif peptit/protein kaynaklarından biri de yılan zehridir. Yılan zehirlerinden antikanser etkili protein ve peptitler saflaştırılmış ve tanımlanmıştır. Bu çalışmanın amacı, güneydoğu Anadolu bölgesinde bulunan Macrovipera lebetina obtusa (Koca Engerek) ham zehrinin K562 insan kronik myeloid lösemi (KML) hücreleri üzerinde in vitro sitotokik etkisinin 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) ve Adenosine tripohsphate (ATP) testleri ile belirlenmesidir. Ek olarak, morfolojik değerlendirme ve aktif kaspaz-3 immünohistokimyasal analizi ile zehrin apoptotik etkisi de değerlendirilmiştir. Histopatolojik değerlendirme için hematoksilen-eozin, gimza ve papanikolau boyaları kullanılmıştır. M. l. obtusa zehrinin farklı konsantrasyonlarıyla 72 saatlik inkübasyon sonucunda K562 hücrelerine karşı doza bağlı toksisite görülmüştür. IC50 değerleri MTT ve ATP testleriyle sırasıyla 0.45 and 0.37 µg/mL olarak hesaplanmıştır. Ayrıca apoptoz uyarımının göstergeleri olarak nükleer fragmentasyon ve yoğunlaşma, apoptotik veziküller ve kaspaz-3 aktivasyonu gözlenmiştir. Apoptoza neden olan bileşiklerin kanser tedavisinde önemli bir yeri olması göz önünde bulundurulduğunda, Anadolu'da bulu...

Cytotoxic Activities of the Crude Venoms of Macrovipera lebetina lebetina from Cyprus and M. l. obtusa from Turkey (Serpentes: Viperidae) on Human Umbilical Vein Endothelial Cells

“…Crude venom of M. l. obtusa showed dose-dependent cytotoxicity against some cancer cell lines, kidney epithelial cells from African green monkey (Vero), and human embriyonic kidney 293 cells (HEK-293) in previous studies (Samel, Trummal, Siigur, & Siigur, 2012;Ozen, İğci, Yalçin, Goçmen, & Nalbantsoy, 2015;Jahromi, Mirakabadi, & Kamalzadeh, 2016;Süzergöz et al 2016;Oghalaie, Kazemi-Lomedasht, Zareinejad, & Shahbazzadeh, 2017). Moreover, dose-dependent cytotoxic activity of M. l. lebetina venom against L929 mouse fibroblast cell line was also reported previously (Nalbantsoy et al, 2012).…”

Effect of Temperature on the Protein Profile of Macrovipera Lebetinus (Blunt-Nosed Viper) Venom: A Preliminary Study

“…The protein profile of M. lebetinus venom as obtained by 2D-PAGE in the present study is consistent with a previous study by Igci and Demiralp [8]. Earlier studies have shown that the most abundant proteins classes found in M. lebetinus venom are SVMP, SVSP, PLA 2 , CLP, LAAO, and disintegrin [8,[22][23][24], which are responsible for Its pathogenesis and biological activities [9][10][11][12].…”

“…The following protein families have been identified in the venom of M. lebetinus up to date: snake venom metalloproteinase (SVMP), snake venom serine proteinase (SVSP), phospholipase A 2 (PLA 2 ), ʟ-amino acid oxidase (LAAO), hyaluronidase, 5´-nucleotidase, phosphodiesterase (PDE), C-type lectin-like protein (CLP), cysteine-rich secretory protein (CRISP), vascular endothelial growth factor (VEGF), nerve growth factor (NGF), disintegrin, Kunitz-type serine protease inhibitor, bradykinin-potentiating peptide (BPP), and natriuretic peptide [7,8]. These proteins are responsible for the biological activities of M. lebetinus venom such as cytotoxic, antimicrobial, antiaggregant, anticancer, anti/procoagulant, necrotic and hemorrhagic activities [9][10][11][12].…”

Investigating the cytotoxic effects of the venom proteome of two species of the Viperidae family (Cerastes cerastes and Cryptelytrops purpureomaculatus) from various habitats

“…via blockage of integrins, induction of apoptosis or necrosis pathways and disruption of the cell cycle (Calderon et al, 2014;Göçmen et al, 2015a;Sciani and Pimenta, 2017;Yalcın et al, 2014;Zainal Abidin et al, 2018). This correlates to some extent with chemotherapeutic agents that are currently being used in clinics, making venoms attractive sources of future anti-cancer agents (Calderon et al, 2014;Meyer et al, 2004;Nalbantsoy et al, 2017;Suzergoz et al, 2016).…”