Evaluation of venom diversity and antivenom quality from the venom of long-term captive vs recently wild captured Pseudocerastes persicus snake: An In vitro and In vivo study.

Snakebite envenomation is a life-threatening condition and antivenoms are used as the most effective treatment. Venom obtained from snakes in long-term captivity showed some variations in comparison to the venom of the wild snakes. The objective of this study is to compare the venom of the Pseudocerastes persicus under long-term captivity and wild conditions as well as the antivenom obtained from these venoms. We have analyzed venom samples and produced trivalent antivenoms using the venom of long-term captive (LTC) or recently wild-captured (RWC) Pseudocerastes persicus, and RWC Macrovipera lebetina, and Echis carinatus. The HPLC analysis revealed that the RWC snakes' venom had three peaks that were not present in the LTC snake's venom. Further analysis using MALDI-TOF and MS/MS showed that the fraction with a retention time (RT) of 14 min contained a toxin from the Kunitz-type serine protease inhibitor (KUT) class, while the fraction with RT 21 a peptide identified within the snake venom metalloproteinase (SVMP) class. The third peak was identified as a sphingolipid. Interestingly, the in vivo preclinical tests showed no significant differences in the effectiveness of the antivenoms. which could be due to the cross-immunogenicity or cross-reactivity between different toxins in the venom. According to our results, small variations in the venom composition of a species do not lead to a decrease in the efficacy of the polyvalent antivenom.

Natural agents as wound-healing promoters.

The management of acute and chronic wounds resulting from diverse injuries poses a significant challenge to clinical practices and healthcare providers. Wound healing is a complex biological process driven by a natural physiological response. This process involves four distinct phases, namely hemostasis, inflammation, proliferation, and remodeling. Despite numerous investigations on wound healing and wound dressing materials, complications still persist, necessitating more efficacious therapies. Wound-healing materials can be categorized into natural and synthetic groups. The current study aims to provide a comprehensive review of highly active natural animal and herbal agents as wound-healing promoters. To this end, we present an overview of in vitro, in vivo, and clinical studies that led to the discovery of potential therapeutic agents for wound healing. We further elucidated the effects of natural materials on various pharmacological pathways of wound healing. The results of previous investigations suggest that natural agents hold great promise as viable and accessible products for the treatment of diverse wound types.

The p75 neurotrophin receptor inhibitor, LM11A-31, ameliorates acute stroke injury and modulates astrocytic proNGF.

The precursor form of nerve growth factor (proNGF) is essential to maintain NGF survival signaling. ProNGF is also among endogenous ligands for p75 neurotrophin receptor (p75ntr). Mounting evidence implies that p75ntr signaling contributes to neural damage in ischemic stroke. The present study examines the therapeutic effect of the p75ntr modulator LM11A-31. Adult mice underwent transient distal middle cerebral artery occlusion (t-dMCAO) followed by LM11A-31 treatment (25 mg/kg, i.p., twice daily) either for 72 h post-injury (acute phase) or afterward till two weeks post-stroke (subacute phase). LM11A-31 reduced blood-brain barrier permeability, cerebral tissue injury, and sensorimotor function in the acute phase of stroke. Ischemic brain samples showed repressed proNGF/P75ntr signaling and Caspase 3 activation in LM11A-31 treated mice, where we observed less reactive microglia and IL-1ß production. LM11A-31 (20-80 nM) also mitigated neural injury induced by oxygen-glucose deprivation (OGD) in sandwich co-cultures of primary cortical neurons (PCN) and astrocytes. This concurred with JNK/PARP downregulation and reduced caspase-3 cleavage in the PCNs and was associated with repressed proNGF generation in astrocytes. Further in vitro experiments indicated human proNGF suppresses the pro-inflammatory phenotype in microglial cultures, as determined by a sharp decline in HMGB-1 production and moderate arginase-1 upregulation. Despite significant protection in acute stroke, LM11A-31 treatment did not improve cortical atrophy and sensorimotor function in the subacute phase. Our findings provide preclinical evidence supporting LM11A-31 as a promising therapy for acute stroke injury. Further investigations may elucidate if reduced astrocytic proNGF, an endogenous reservoir of pro-neurotrophins, may restrict the therapeutic window.

The Effects of the Fraction Isolated from Iranian Buthotus shach Scorpion Venom on Synaptic Plasticity, Learning, Memory, and Seizure Susceptibility.

Epilepsy, as a neurological disease, can be defined as frequent seizure attacks. Further, it affects many other aspects of patients' mental activities, such as learning and memory. Scorpion venoms have gained notice as compounds with potential antiepileptic properties. Among them, Buthotus schach (BS) is one of the Iranian scorpions studied by Aboutorabi et al., who fractionated, characterized, and tested this compound using electrophysiological techniques in brain slices (patch-clamp recording). In the present study, the fraction obtained from gel electrophoresis was investigated through behavioral and electrophysiological assays. At first, ventricular cannulation was performed in rats, and then the active fraction (i.e., F3), carbamazepine, and the vehicle were microinjected into the brain before seizure induction by the subcutaneous (SC) injection of pentylenetetrazol (PTZ). Seizure behaviors were scaled according to Racine stages. Memory and learning were evaluated using the Y-maze and passive avoidance tests. Other groups entered evoked field potential recording after microinjection and seizure induction. Population spike (PS) and field excitatory postsynaptic potential (fEPSP) were measured. The F3 fraction could prevent the fifth stage and postpone the third stage of seizure compared to the control (carbamazepine) group. There was no significant improvement in memory and learning in the group treated with the F3 fraction. Also, PS amplitude and fEPSP slope increased significantly, and long-term potentiation was successfully formed after the high-frequency stimulation of the performant pathway. Our results support the antiepileptic effects of the F3 fraction of BS venom, evidenced by behavioral and electrophysiological studies. However, the effects of this fraction on memory and learning were not in the same direction, suggesting the involvement of two different pathways.

Correlation between coenzyme Q10 content and the nutrient sensors in AKI induced by Hemiscorpius lepturus envenomation.

Introduction: Acute kidney injury (AKI) may have a negative effect on mitochondrial hemostasis and bioenergetics as well as coenzyme Q10 (CoQ10) content. PGC-1α, AMPK, sirtuin 1 (Sirt1), and Sirt3, as the key metabolic regulators under nutritional stress, stimulate energy production via mitochondrial biogenesis during AKI. However, no report is available on the relationship between CoQ10 level and nutrient sensors in the pathophysiology of AKI caused by Hemiscorpius lepturus scorpion envenomation. Methods: Three doses of venoms (1, 5, and 10 mg/kg) were administered by subcutaneous (SC) injection to male albino mice. The animals were sacrificed 1 day or 7 days after administration of venom and their kidneys were collected to analyze gene expression involved in AKI, nutrient sensors, and apoptosis signaling activation by real-time polymerase chain reaction (PCR) and the measurement of CoQ10 level using the High-performance liquid chromatography (HPLC) method. Results: The data indicated a significant decrease in CoQ10 level after the administration of venom in 5 and 10 mg/kg. In addition, 1 day after the treatment, a significant over-expression of Sirt1 (5 and 10 mg/kg) was observed compared with normal mice. Overexpression of Sirt3 occurred 1 day and 7 days after treatment only at the dose of 5.0 mg/kg of venom. Furthermore, over-expression of AMPK as an important mitochondrial energetic sensor happened 1 day and 7 days after the injection of venom (5 mg/kg) (P < 0.01). The significant increase in the gene expression of caspase-9 and 3 after the injection of venom (5 and 10 mg/kg) confirmed the role of cell death signaling. Conclusion: The venom-induced energy-sensing pathways have a key role in gene expression of PGC-1α, AMPK, Sirt3, and CoQ10 content after venom-induced AKI.

Iranian Mesobuthus Eupeus Crude Venom Induces Selective Toxicity in Chronic Lymphocytic Leukemia B-Lymphocytes Through Lysosomal/Mitochondrial Dysfunction and Reactive Oxygen Species Formation.

From ancient times to the present-day animal venoms had been used as medicinal and therapeutic agents. Recently it has been reported that the scorpion venom is a potential source of active and therapeutic compounds to design potent drugs against variety of cancerous cells and other diseases. The current study aimed to evaluate the selective toxicity of Iranian Mesobuthus eupeus (IMe) crude venom as a potential source of anticancer compounds on cancerous CLL B-lymphocytes and normal lymphocytes. For this purpose, we isolated cancerous CLL B-lymphocytes and normal lymphocytes from chronic lymphocytic leukemia patients and healthy volunteers. Cancerous CLL B-lymphocytes and normal lymphocytes were treated with different concentration (0, 5, 10, 20, 40 and 80 µg/ml) of IMe crude venom for 12 hours and cytotoxicity, reactive oxygen species (ROS) production, collapse of mitochondrial membrane potential (MMP) and lysosomal membrane integrity were determined. The data demonstrated the significant cytotoxic effect of IMe crude venom on cancerous CLL B-lymphocytes, with a concentration value (IC50) that inhibits 50% of the cell viability of 60 µg/ ml after 12 h of incubation. MTT assay proved that the IMe crude venom is selectively toxic to cancerous CLL B-lymphocytes, and IMe crude venom induced selective cell death via activation of ROS formation and mitochondrial/lysosomal dysfunction. These finding showed that IMe crude venom has a selective mitochondrial/lysosomal-mediated cell death effect on cancerous CLL B-lymphocytes. Therefore, the IMe crude venom and its fractions may be promising in the future anticancer drug development for treatment of CLL and variety of cancers.

Isolation of the Anticoagulant and Procoagulant Fractions of the Venom of Iranian Endemic Echis carinatus.

Background: The venom of Echis carinatus contains both procoagulant and anticoagulant components that can either promote or block the blood coagulation cascade, and some of these components affect platelet function in different ways. Objectives: The present study focuses on setting up a procedure for the purification of crude venom and designing appropriate clotting tests in order to characterize the procoagulant and anticoagulant fractions of E. carinatus venom. Methods: Chromatographic methods, including gel filtration, ion-exchange chromatography, and reverse-phase high-performance liquid chromatography (HPLC), were applied for purifying these fractions. Coagulant activity testing, prothrombin time (PT), and activated partial thromboplastin time (APTT) were used to determine procoagulant and anticoagulant properties. For measuring molecular weight, 15% SDS-PAGE electrophoresis with a molecular weight standard ranging from 6.5 to 200 kDa was used. Results: We obtained five fractions named F1, F2, F3, F4, and F5. The F1 and F2 fractions showed procoagulant activity, and the F5 fraction had anticoagulant activity. The molecular weight of F2.4.2 from fraction F2 and F5.1 from fraction F5 were analyzed by SDS-PAGE electrophoresis under the reducing condition. These factors were identified as a single protein band at the end of purification. The molecular weights of these purified fractions were estimated to be 7.5 kDa and 38 kDa for F5.1(b) and F2.4.2(b), respectively. Conclusions: Our findings suggest an efficient and suitable procedure for the identification and purification of the procoagulant and anticoagulant factors of the venom of Iranian E. carinatus using the PT and APTT assays.

The Effect of Organizational Resilience and Strategic Foresight on Firm Performance: Competitive Advantage as Mediating Variable.

The pharmaceutical industry's performance in the global economy has been affected by the growing competition associated with globalization, economic liberalization, and the trade-related aspect of the intellectual property rights (TRIPS) agreement. To maintain performance, organizations need to consider strategic foresight (SF) and organizational resilience (OR) to anticipate future trends and survive crises. By proposing a conceptual framework, this study examines the relationship between organizational resilience, strategic foresight, competitive advantage (CA), and firm performance (FP). A conceptual framework was developed to assess the hypotheses in the pharmaceutical industry. Then, partial least squares structural equation modeling (PLS-SEM) was applied to investigate the relationships quantitatively. The results of structural equation modeling (SEM) based on the data generated from 202 completed questionnaires by the pharmaceutical companies in Iran demonstrate that OR, SF, and CA have significant positive impacts on FP. Moreover, CA partially mediates the relationship between OR and FP and also between SF and FP. The findings of this study enrich the existing literature by demonstrating that early detection of environmental change and resilient manner assist Iranian pharmaceutical firms to survive if joining the WTO. This is the first study that examines the direct and indirect effect of OR and SF on the FP, considering the mediating impact of CA. This investigation attempts to address the mechanisms through which OR and SF affect organizational performance, especially in the pharmaceutical industry.

Is there any potential management against COVID-19? A systematic review and meta-analysis.

PURPOSE: A recent survey has shown that the COVID-19 pandemic has culminated in dramatical and critical treatment particularly in acute infected patients. In fact, this systematic review-meta-analysis was directly pertained to estimation at the efficient value of some clinical managements to confront the COVID-19 infection. METHODS: Pubmed, Embase, Scopus, Cochrane, and Scholar databases were searched from inception to July 1, 2020, to identify studies reporting the current treatment process and medications (e.g. hydroxychloroquine, antiviral therapy, convalescent plasma, and immunomodulatory agents) for COVID-19. A random-effects model meta-analysis was performed to calculate the relative risk (RR) with 95% confidence intervals (CI). The outcomes of this study were the frequency of negative conversion cases, clinical improvements, mechanical ventilation demand, intensive care unit (ICU) entry, and mortality. The standard treatment refers to the published guidelines and specialist experience which varies in different articles, and the proposed treatment refers to the kind of interest suggested in the included studies. RESULTS: A number of 45 articles met the eligibility criteria (out of 6793 articles). Among them, 26 articles involving 3263 patients were included in quantitative analysis. Anti-COVID-19 interventions could significantly increase clinical improvement (RR 1.17, 95% CI 1.08-1.27; I2 = 49.8%) and reduce the mortality rate (RR 0.58, 95% CI 0.35-0.95; I2 = 74.8%). Although in terms of negative conversion, ICU entry, and mechanical ventilation demand, clinical intervention had no beneficial effect. The clinical effect of immunomodulatory agents (especially tocilizumab and anakinra) was noticeable compared to other medications with RR of 0.22 (95% CI 0.09-0.53; I2 = 40.9%) for mortality and 1.25 (95% CI 1.07-1.46; I2 = 45.4%) for clinical improvement. Moreover, Antivirals (RR 1.13, 95% CI 1.01-1.26; I2 = 47.0%) and convalescent plasma therapy (RR 1.41, 95% CI 1.01-1.98; I2 = 66.6%) had significant beneficial effects on clinical improvement. CONCLUSION: Based on our findings, all the included interventions significantly declined the mortality and enhanced clinical improvements with no effect on negative conversion and mechanical ventilation demand. Especially, immunomodulators and plasma therapy showed favorable outcomes. An evaluation on the efficacy of proposed treatment against COVID-19.

Studies on the cholinesterases inhibiting compounds from the Cassiopea andromeda venom.

Cholinesterase inhibitors find application in the combat and care of several diseases, especially AD. Jellyfish venoms are the most promising sources of potent cholinesterase inhibitors. Therefore, it is of interest to study cholinesterases inhibiting compounds from the Cassiopea andromeda venom. We report bioactive compounds using the GC-MC method followed by molecular modeling and docking data analysis. The GC-MS analysis of the crude venom led to the identification of seven bioactive compounds (C1-C7), comprising the steroidal alkaloids, phenolic and carotenoid derivatives. The venom exhibited inhibitory activities against the cholinesterase enzymes. The compound C2, a Dioxolane steroid, displayed the strongest inhibition on both AChE and BChE activities for further consideration.

The effects of Hemiscorpius lepturus induced-acute kidney injury on PGC-1α gene expression: From induction to suppression in mice.

Hemiscorpius lepturus envenomation induces acute kidney injury (AKI) through hemoglubinoria and mitochondrial dysfunction. Mitochondria supports ATP production to promote the regulation of fluid and electrolyte balance. Mitochondrial homeostasis in different metabolic environments can be adjusted by overexpression of PGC-1α. High reactive oxygen species (ROS) production after H. lepturus envenomation and heme oxygenase-1 (HO-1) overexpression causes ATP depletion as well as mitochondrial homeostasis disruption, which lead to progression in renal diseases. The present study aims to evaluate the role of venom induced-AKI in modulating mitochondrial function in cell death and metabolic signaling associated with PPAR-α, PGC-1α, and Nrf-2 as the main transcription factors involved in metabolism. Based on the data, two significant events occurred after envenomation: reduction of gl glutathione level and overexpression of the cytoprotective enzyme HO-1. Apaoptosis induction is associated with a significant decrease in the transcription of PPAR-α, PGC-1α and Nrf-2 after administrating lethal dose of venom (10 mg/kg). Furthermore, at the lower doses of venom (1 and 5 mg/kg), with a significant recovery accompanied with PGC-1α upregulation occurs after AKI. As the findings indicate, PGC-1α has a key role in restoring the mitochondrial function at the recovery phase of mouse model of AKI, which highlights the PGC-1α as a therapeutic target for venom induced-AKI prevention and treatment.

Characterization of Functional Effects of Two New Active Fractions Isolated From Scorpion Venom on Neuronal Ca2+ Spikes: A Possible Action on Ca2+-Dependent Dependent K+ Channels.

INTRODUCTION: It is a long time that natural toxin research is conducted to unlock the medical potential of toxins. Although venoms-toxins cause pathophysiological conditions, they may be effective to treat several diseases. Since toxins including scorpion toxins target voltage-gated ion channels, they may have profound effects on excitable cells. Therefore, elucidating the cellular and electrophysiological impacts of toxins, particularly scorpion toxins would be helpful in future drug development opportunities. METHODS: Intracellular recording was made from F1 cells of Helix aspersa in the presence of calcium Ringer solution in which Na+ and K+ channels were blocked. Then, the modulation of channel function in the presence of extracellular application of F4 and F6 toxins and kaliotoxin (KTX; 50 nM and 1 µM) was examined by assessing the electrophysiological characteristics of calcium spikes. RESULTS: The two active toxin fractions, similar to KTX, a known Ca2+-activated K+ channel blocker, reduced the amplitude of AHP, enhanced the firing frequency of calcium spikes and broadened the duration of Ca2+ spikes. Therefore, it might be inferred that these two new fractions induce neuronal hyperexcitability possibly, in part, by blocking calcium-activated potassium channel current. However, this supposition requires further investigation using voltage clamping technique. CONCLUSION: These toxin fractions may act as blocker of calcium-activated potassium channels.

Induction of two independent immunological cell death signaling following hemoglobinuria -induced acute kidney injury: In vivo study.

The main important clinical signs in acute kidney injury (AKI) after sever Hemiscorpius lepturus envenomation in patients is associated with proteinuria, hemolysis and hemoglobinuria. Unfortunately, our limited knowledge of molecular cell death mechanism in H. lepturus induced AKI restricts the development of desirable therapeutics. So, in the present study, the potential role of necroptosis and ferroptosis in H. lepturus induced AKI were investigated in male albino mice. The animals were administrated by SC injection of venom (1, 2.5, 5 and 10 mg/kg) based on LD50 determination. After 1 and 7 days, urinalysis, stereological assessments and gene expression of Ngal, Tnf-α, Tlr-4, Ripk3, Mlkl and Acsl4 were evaluated by real time PCR. Our data revealed that upregulation of renal Ngal expression is associated with the gene over expression of Tnf-α, Tlr-4, Ripk3 and Mlkl in venom treated kidneys. We observed that the Malondialdehyde (MDA) level was increased in dose-dependent manner similar to Acsl4 gene over expression suggesting a main role of ferroptosis in hemoglobinuria mediated AKI following envenomation. Moreover, transcriptional enhancement of Tlr-4and Tnf-α receptor can cause phosphorylation of Ripk3-Mlkl complex, collapse of membrane potential and DAMPs release which intensified the inflammation cytokines in kidney. Taken together, it supposes co-existence of two separate pathways of regulated necrosis and inflammatory environment provides a promising outlook in prevention and management of hemoglobinuria induced AKI following envenomation in clinical practice.

Acetylcholinesterase inhibitory activity of a neurosteroidal alkaloid from the upside-down jellyfish Cassiopea andromeda venom

Abstract Natural compounds from marine organisms have been rarely studied for their acetylcholinesterase inhibitory activities. The aim of this study was to isolate novel compounds with antiAChE activity from the venom of upside-down jellyfish Cassiopea andromeda Forskål, 1775. The compounds of the fractionated venom on gel filtration chromatography were identified by analyzing gas chromatography-mass spectroscopy data. The structure of the isolated compound that showed the most potent antiAChE activity in a docking study was elucidated by different spectral data, including 1H NMR and 13C NMR. Three compounds, including a neurosteroidal alkaloid androtoxin B, were identified from two venom fractions. This neurosteroidal alkaloid showed strong acetylcholinesterase inhibitory activity (IC50 2.24 ± 0.1 µM) compared with the reference standard, galantamine. The results obtained by a docking study demonstrated that Androtoxin B had close contact with two of the three amino acid residues of the catalytic triad of acetylcholinesterase gorge and was accommodated within a peripheral hydrophobic pocket composed of numerous aromatic site chains. In conclusion, the isolated neurosteroidal alkaloid from Cassiopea andromeda was a potent antiAChE agent with strong binding to both the catalytic and peripheral sites of acetylcholinesterase that correlated well with the experimental data. Further studies are required to determine whether androtoxin B could be a potential treatment for Alzheimer's disease.

The Effects of Buthotus schach Scorpion Venom on Electrophysiological Properties of Magnocellular Neurons of Rat Supraoptic Nucleus.

Bothutous Schach (BS) scorpion venom consists of several polypeptides that could modulate ion channels. In this study, the effects of BS crude venom on passive and active electrophysiological properties of rat neurons in supraoptic nucleus (SON) of hypothalamus was investigated using whole-cell patch clamp technique. The results showed that bath application of BS venom produced significant change in passive properties of SON neurons, namely a decrease in resting membrane potential and an increase in input resistance of the cells. Also, significant change in active properties of SON neurons was shown after bath application of BS venom; including a decrease in the number of evoked action potential along with an increase in half width and decay time of action potential and a significant decrease in after-hyperpolarization amplitude. Finally, a decreased latency to the first spike accompanied by a lower current threshold to elicit the first spike was shown compared with the values before venom application. These effects are possibly through blocking different ion channels including potassium channels. Further experiments using different fractions of the venom is required to specify venom effects on various ion channels.

Naja Naja Oxiana Venom Fraction Selectively Induces ROS-Mediated Apoptosis in Human Colorectal Tumor Cells by Directly Targeting Mitochondria

Objective: To investigate the selective effect of Naja naja oxiana crude venom and its fractions on human colorectal cancer mitochondria to activate apoptosis signaling. Methods: Cells and mitochondria isolated from human cancerous and normal colorectal tissues exposed to N. oxiana crude venom and its fractions obtained from size-exclusion chromatography and then mitochondrial parameters related to up-stream cell death signalling such as reactive oxygen species formation, MMP, mitochondrial swelling, cytochrome c release and ATP content as mitochondrial parameters and activation of caspase3 and finally apoptosis/necrosis % were then assayed as cellular parameters. Result: Our findings indicated that crude venom (15, 30 and 60 µg/ml) and fraction 3; F3; (10, 20 and 40 µg/ml) of N. Oxiana venom induced a significant (p<0.05) increase of reactive oxygen species level, swelling of mitochondria, collapse of mitochondrial membrane potential (MMP), release of cytochrome c, activated caspase3 and decrease ATP content only in colon cancer tissue group but not from the healthy colon tissue group. Our results also showed that fraction 3 of venom decreased the percentage of viable cells and induced apoptosis in cancerous colorectal cells. Conclusion: F3 fraction of N. Oxiana venom is a suitable candidate for further studies as a new drug treatment of colorectal cancer due to its high capacity for induction of apoptosis signaling via mitochondrial pathway.

Hematological parameters on the effect of the jellyfish venom Cassiopea andromeda in animal models.

For the first time, we previously recorded an enormous population of the Cassiopea andromeda jellyfish that had increased dramatically from Bushehr coasts of Iran. The sub-acute toxicity of the jellyfish venom in rat organs was correspondingly carried out. The data presented in this paper relate to the in vivo and in vitro hematological effects of this venomous species of jellyfish venom.

Impact of Corporate Reputation on Brand Differentiation: An Empirical Study from Iranian Pharmaceutical Companies.

The influence of company reputation or what is often referred to as corporate reputation on branding strategy and producing intangible asset for different industries has been researched in western countries, but there is a gap for the generalizability of findings to countries out of the United State and Europe. To establish the western researcher's external validity of theories in other countries and to obtain a better understanding of the influences of branding and company reputation on pharmaceutical business markets, the researchers applied this study for Iran, a country in the Middle East. The obtained results using SEM (by P.L.S. 2.0 software) showed a good relationship between value creation and brand differentiation (ß =0.360 and t-value = 3.167), between corporate communication and brand differentiation (ß = 0.022 and t-value = 3.668), and between strategic resources and brand differentiation (ß = 0.289 and t-value = 2.247). This study is a pioneering attempt in Iran to measure the impact of corporate reputation on brand differentiation strategy.

Anticancer Activity a of Caspian Cobra (Naja naja oxiana) snake Venom in Human Cancer Cell Lines Via Induction of Apoptosis.

Cancer is the leading cause of death worldwide. Current anticancer drugs involve various toxic side effects; efforts are ongoing to develop new anticancer agents especially from the screening of natural compounds. Present study investigated cytotoxic effects and mode of cell death induced by the Caspian cobra venom in some human cancer cell lines. Cytotoxic effects of snake venom toxins (SVT) were investigated via monitoring of morphological changes, MTT, trypan blue exclusion and LDH release assays. Mechanism of cell death was determined by AO/EtBr double staining, caspase-3 activity assay, flow cytometric analysis of apoptosis and mitochondrial membrane potential measurement. In morphological analysis, apoptotic alterations related to apoptosis such as cytoplasmic blebbing, chromatin condensation and irregularity in shape were seen. IC50 of SVT in HepG2, MCF7and DU145 cell lines were 26.59, 28.85 and 21.17µg/mL, respectively and significantly different from the MDCK normal cell line (IC50=47.1 µg/mL). AO/EtBr double staining showed the best apoptotic/necrotic ratio at 15 µg/mL after 48 h. LDH release showed no significant differences between 10 µg/mL SVT and cisplatin. Flowcytometric analysis confirms mitochondrial membrane potential loss and more than 95% apoptotic cell death at 15 µg/mL. Caspase-3 was significantly activated at doses higher than 2.5 µg/mL with a maximal activity at 10 µg/mL. Results from this study demonstrate that SVT induces mitochondrial and caspase-3 dependent apoptosis in cancer cell lines with minimum effects on studied normal cell. This potential might candidate this venom as a suitable choice for cancer treatment.

Cobra venom cytotoxins; apoptotic or necrotic agents?

Organs homeostasis is controlled by a dynamic balance between cell proliferation and apoptosis. Failure to induction of apoptosis has been implicated in tumor development. Cytotoxin-I (CTX-I) and cytotoxin-II (CTX-II) are two physiologically active polypeptides found in Caspian cobra venom. Anticancer activity and mechanism of cell death induced by these toxins have been studied. The toxins were purified by different chromatographic steps and their cytotoxicity and pattern of cell death were determined by MTT, LDH release, acridine orange/ethidium bromide (AO/EtBr) double staining, flow cytometric analysis, caspase-3 activity and neutral red assays. The IC50 of CTX-II in MCF-7, HepG2, DU-145 and HL-60 was 4.1 ± 1.3, 21.2 ± 4.4, 9.4 ± 1.8 µg/mL and 16.3 ± 1.9 respectively while the IC50 of this toxin in normal MDCK cell line was 54.5 ± 3.9 µg/mL. LDH release suddenly increase after a specific toxins concentrations in all cell lines. AO/EtBr double staining, flow cytometric analysis and caspase-3 activity assay confirm dose and time-dependent induction of apoptosis by both toxins. CTX-I and CTX-II treated cells lost their lysosomal membrane integrity and couldn't uptake neutral red day. CTX-I and CTX-II showed significant anticancer activity with minimum effects on normal cells and better IC50 compared to current anticancer drug; cisplatin. They induce their apoptotic effect via lysosomal pathways and release of cathepsins to cytosol. These effects were seen in limited rage of toxins concentrations and pattern of cell death rapidly changes to necrosis by increase in toxin's concentration. In conclusion, significant apoptogenic effects of these toxins candidate them as a possible anticancer agent.