Examination of cytotoxicity of Crotalus adamanteus and Crotalus scutulatus scutulatus venom on human skin melanoma and ovarian carcinoma cell lines

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Awarding institution
  • University of Strathclyde
Date of award
  • 2016
Thesis identifier
  • T14326
Person Identifier (Local)
  • 201264698
Qualification Level
Qualification Name
Department, School or Faculty
  • Cancer is global health problem that is responsible for the highest number of human mortalities worldwide. Cancers originates from aberrations in genes that are responsible for maintaining tissue homeostasis. These genetic aberrations render some unique qualities to cancer cells, such as uncontrolled proliferation, apoptosis evasion, angiogenesis, insensitivity to growth signals and metastasis to other parts of the body, which make the condition challenging for the current medical practices. Efforts for eradication of cancer have been made since the dawn of civilisation and have resulted in some promising outcomes such as complete cure of a few types of cancer, improvement in patient’s quality of life and increasing survival rates but the adverse effects associated with cancer treatment are mostly life threatening in the form of initiation of other tumours, effects on the vital body organs and anxiety. In order to introduce better treatment options in terms of less adverse effects, cost effectiveness and cancer specificity, the pursuit of a satisfactory cancer cure is still ongoing. Nature has always been a source of some important remedies such as antimalarial, antibiotics and neuromuscular agents which were used to successfully cope with contemporary health challenges and that is why scientists have always turned to nature to find answers to the existing medical problems. Snakes have held a fascination for mankind since early civilisation, probably because they are responsible for substantial mortality and morbidity around the world. Snake venom mainly comprises of a cocktail of a large number of enzymatic and non-enzymatic proteins, which affect the vital physiological systems. Due to their biological consequences, efforts are being made to translate snake venom toxins into potential therapeutics for various medical conditions. Since the emergence of captopril, the classic example of a venom based drug, various components of the venom have been isolated, identified, characterised and their associated pharmacology is established and have been used a platform for development of pro/anti-coagulants, analgesics and anti-cancer entities. In addition to other enzymes, L-amino acid oxidases (LAAO) are present in significant amounts in snake venom. They are a group of flavoenzymes, known to catalyse oxidative deamination of L-amino acid substrates, in order to form α-keto acids, hydrogen peroxide and ammonia.The aim of this study was to evaluate the cytotoxic effects of Crotalas adamanteus (Cad) and Crotalus scutulatus scutulatus (Css) snake venom on a human skin melanoma cell line (A375) and human ovarian cancer cell line and to establish the mode of cell death along with isolation and identification the active components. In order to determine the enzymatic activity of the whole venom, substrate solution containing 250 μM L-leucine (L-Leu) was incubated with 20 μg/mL of Cad and Css whole venom for 4 hours (h) at 37 °C. Both the venom samples were then fractionated using size exclusion chromatography and the fractions were lyophilised. Fractionation resulted in 5 peaks from each venom. As previously reported, LAAO are inactivated under high pH or low temperature as consequence of conformational changes in prosthetic group of the enzyme. Therefore, the fractions obtained were heated at pH 5 for 30 minutes (min) for reactivation and the enzyme activity assay was repeated to identify the fraction containing LAAO. Fraction 1 from Cad, called as Cad F1, whereas, fraction 2 of Css whole venom (Css F2) showed LAAO activity which indicated by the significant (P=0.005) metabolism of L-Leu. Cad F1 and Css F2 were further fractionated using cation exchange chromatography (CEC), producing 4 and 6 peaks, respectively. The reactivation step and LAAO activity assay were repeated to identify the LAAO in both the separated fractions. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) was used to establish the purity and estimate the molecular weights (MW) of the purified enzymes. The presence of single band on the gel, after SDS-PAGE, indicated the purity of the extracted components. LAAO purified from Cad was designates as Lcad and had MW of 75 kDa, and the LAAO purified from Css had MW of 50 kDa and was designated as CsL. MW determined for Lcad and CsL are similar to MW reported for LAAO of Cad and Css by other researchers. Michaelis-Menten equation used to determine the maximum velocity of the reaction (vmax) and Michaelis-Menten constant (KM) which shows the substrate turn over for the enzymes. vmax for Cad (0.8911±.023 μM.minˉ¹.mgˉ¹), Cad F1 (0.8163±0.026 μM.minˉ¹.mgˉ¹) and Lcad (0.7897±0.023 μM.minˉ¹.mgˉ¹) showed a gradual decrease which was also observed in KM values for Cad (137.7±19.9 μM), Cad F1 (124.2±19.19.1μM) and Lcad (131.4±20.7 μM). Similarly, decrease in vmax values which were 0.9630±0.034, 0.7841±0.018, 0.07051±0.011 μM.minˉ¹.mgˉ¹ for Css, Css F2 and CsL, respectively, and KM values (221±31.1, 204.6±18.8 and 210.8±13.2 μM for Css, Css F2 and CsL, respectively) was also observed. These observations referred to the fact that LAAO partially and gradually lose their enzymatic activity when reactivated after every lyophilisation step. LAAO are of great interest in pharmacological research because of their reputed therapeutic properties, including apoptotic-inducing activities. The mechanism underlying the cytotoxicity has been mainly attributed to reactive oxygen species (ROS) production, which culminates in apoptosis via either caspase-dependent or apoptosis inducing factor (AIF) pathway. In order to confirm the cytotoxicity of Lcad/CsL and further elaborate on pharmacology underlying their cytotoxicity induced in human melanoma (A375) and human ovarian carcinoma (A2780) cell lines, several cell bases assays were carried out. For this purpose, cells of both cell lines were incubated with 20 μg/mL of Lcad and CsL at 37 °C for 4 h. Cell viability was measured by using CellTiter Blue® assay by incubating the cells treated with 0.15-20 μg/mL Lcad/CsL for 4 hours (h) with the dye at 37 °C and measuring the fluorescence at excitation/emisstion (ex/em) 560/590 nm. The potency of the separated proteins was determined by treating the cells with 0.15-20 μg/mL of cisplatin for 4 h at 37 °C. The cancer specificity of Lcad and CsL was established by incubating human non-cancer epithelial cell line (PNT2a) at 37 °C with 0.15-20 μg/mL of Lcad and CsL for 4 h. Morphological changes in the cells after Lcad/CsL treatment were observed under light upright microscope using 40X objective lens. Autophagy was evaluated by measuring the increase in acidic vesicular organelles (AVOs) by staining the Lcad/CsL treated cells with 100 μg/mL acridine orange (AO) and measuring the fluorescence at (ex/em) 490/650 nm with a microplate reader. Necrosis was evaluated by observing the morphological changes in the cells after Lcad/CsL treatment and, also, effect of the specific necrosis inhibitor, IM-54, on the cell death of by incubating cells with 10 μM of IM-54 prior to treating the cells with Lcad and CsL. Role of reactive oxygen species (ROS) was also investigated. In order to confirm the extracellular generation of ROS, the catalytic effect of Lcad and CsL on the growth medium L-amino acids was determine with/without additional L-Leu. Intracellular ROS production was measured with the help of cell permeant ROS stain 2,7-dichlorodihydrofluorescein diacetate (DCDHF) (10 μM). To establish whether cell death pathway initiated by Lcad/CsL proceeded through mitochondria, the effect of Lcad and CsL on the mitochondrial metabolic function and outer mitochondria membrane (OMM) integrity were determined. Mitochondrial function was determined by incubated the cells with 20 % (v/v) 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and measuring the absorbance at 560 nm. Tetramethylerhodamine ethyl ester (TMRE), to a final concentration 500 nm was incubated with the cells after Lcad/CsL treatment and the fluorescence was measured (ex/em: 545/590). Fluorescent substrate assay for caspase-3/7 was exploited to establish the activation of the effector caspases-3/7 in response to mitochondrial signalling. In order to have insight into the molecular mechanisms involved in the progression of cell death in response to Lcad and CsL treatment, western blotting was used to confirm the activation of caspase-8, Bid, apoptosis inducing factor (AIF) and poly-(ADP ribose)-polymerase (PARP).The cell viability assays showed that Lcad is cytotoxic as indicated by IC50 values 1.1±0.1 μg/mL and 1.95±0.2 μg/mL of Lcad on A375 and A2780 cell lines, respectively. Similarly, CsL cytotoxicity is denoted by the IC50 values of 2.46±0.2 μg/mL and 3.6±0.6 μg/mL on A375 and A2780, respectively. Cisplatin did not show any cytotoxic effect on both cancer cell lines after 4 h of incubation whereas Lcad and CsL did not affect the viability of PNT2a cells. Compared with the negative control, no significant increase in the increase of AO was observed which implied that intracellular acidity did not increase and there was basal level of AVOs. IM-54 did not delay cell death in the cells incubated with Lcad and CsL which proved that necrotic pathways were not activated by Lcad/CsL which is also supported by the loss in cellular volume rather than fragmentation of the treated cells, a hallmark of apoptosis. In addition, no extracellular or intracellular ROS generation was observed. Although there was significant (P<0.05) reduction in the mitochondrial metabolic activity was shown compared with the negative control, mitochondria were not uncoupled. The fluorescent substrate assay established that effector caspase-3/7 were not activated, suggesting that cell induced by Lcad/CsL is independent of effector caspases. Western blot confirmed the activation of caspase-8, translocation of Bid to mitochondria, translocation of AIF to nucleus and activation of PARP.This study concluded that Lcad/CsL are highly cytotoxic to human melanoma and human ovarian cancer cells lines, and are more potent than cisplatin. Lcad and CsL seem to be cancer-selective as inferred by the lack of cytotoxicity on PNT2a cell lines. It was established Lcad/CsL activate apoptotic pathway which is executed in caspase-independent way. It would appear that Lcad/CsL activates caspase-8, which was supposedly via activation of cell death receptors as there was no ROS production. Activated caspase-8 in turn signalled the translocation of Bid to mitochondria which resulted in the translocation of AIF to nucleus. However, mitochondria were not uncoupled in order for AIF to be released. The translocated AIF activates PARP, an enzyme responsible for DNA damage during cell death.The ROS-independent cell death and release of AIF without affecting the mitochondrial membrane integrity are rarely reported facts for snake venom LAAO which needs further investigation to understand variability of mechanism of action of LAAO from different resources which could be exploited in future for medical uses.
Advisor / supervisor
  • Ferro, Valerie
  • Rowan, Edward
Resource Type