Level of serum enzymes and electrocardiogram in healthy rabbits after injection of ICD-85 as an anticancer agent

Our previous in vivo studies confirmed that ICD-85, as an anticancer agent, was able to prevent further growth of breast tumors and expand the life expectancy of mice with breast cancer. Blood collection was carried out before, 1, 3, and 6 hours after ICD-85 injection. Sera were used to determinate the cardio and hepatic enzymes levels, including ALT, AST, LDH, CPK, and Ck-MB. Coagulation factors such as PT and PTT were also assayed. ECGs of all rabbits were recorded during the experiment. ECG results showed that the injection of 50 and 100 micro g/kg ICD-85 into healthy rabbits has no significant effect on heart function while the injection of 150 to 200 micro g/kg ICD-85 caused ECG wave changes and mild bradycardia without toxic effects on heart. After ICD-85 injection [concentrations below 100 micro g/kg], no significant increase was observed in liver and cardiac enzymes [ALT, AST, LDH, CPK, and CK-MB]. However, the concentration of 150 micro g/kg and above caused a rise in the enzymes. Comparison of the PT and PTT before and after ICD-85 injection showed no significant clotting time at any concentrations below 200 micro g/kg. Based on the results obtained in the present study as well as our previous reports, ICD-85 at concentrations below 100 micro g/kg seems to have no significant effect on the serum enzymes as indicators of hepatotoxicity and cardiotoxicity in healthy rabbits. However, to confirm this conclusion, more detailed surveys on heart and liver is needed to be carried out

Cytotoxic effect of Iranian vipera lebetina snake venom on HUVEC cells

Envenomation by heamotoxic snakes constituted a critical health occurrence in the world. Bleeding is the most sever consequence following snake bite with viperid and crothalid snakes. It is believed that the degradation of vascular membrane caused hemorrhage; in contrast, some suggested that direct cytotoxicity has role in endothelial cell disturbances. This study was carried out to evaluate the direct toxicity effect of V. lebetina crude venom on Human Umbilical Vein Endothelial Cells [HUVECs]. The effect of V. lebetina snake venom on HUVECs growth inhibition was determined by MTT assay and neutral red uptake assay. The integrity of cell membrane through LDH release was measured with the Cytotoxicity Detection Kit. Morphological changes of endothelial cells were also evaluated using a phase contrast microscope. In MTT assay, crude venom showed a cytotoxic effect on endothelial cells which was confirmed by the effect observed with neutral red assay. Also, crude venom caused changes in the integrity of cell membrane by LDH release. The morphological alterations enhanced in high concentration results in total cells number reduced. V. lebetina venom showed potential direct cytotoxic effects on human endothelial cells in a manner of concentration-dependent inhibition

In vivo and in vitro anti-angiogenesis effect of venom-derived peptides [ICD-85]

Agiogenesis is the development of new blood vessels from pre-existing vasculatures. Although essential in the physiological process, it becomes pathological in various diseases including cancer. Preventing the formation of new blood vessels causes reductions in tumor size and metastasis. This study has been undertaken to elucidate the anti-angiogenesis effects of ICD-85 [derived peptides from venom]. We evaluated the ICD-85 anti-angiogenesis activity by the in vivo CAM assay and in vitro tube formation assay of human umbilical vein endothelial cells [HUVECs]. The anti-proliferative activity of ICD-85 was also determined through MTT assay on HUVECs. Results of this study revealed the anti-proliferative activity of ICD-85 on the HUVEC cell line with an IC50 of 12 micro g/mL. The in vivo CAM assay also clearly showed the prevention of new vascular formation when the chick embryos were exposed to 0.15 micro g/disc of ICD-85. In vitro tube formation assay of HUVECs also showed the complete prevention of capillary tube formation on 18 micro g/mL. Based on the results obtained in this study, ICD-85 has anti-angiogenesis activity as shown by the prevention of capillary tube formation and the CAM assay

Comparative cytotoxic evaluation of free and sodium alginate nanoparticle-encapsulated ICD-85 on primary lamb kidney cells

Current anti-cancer drug therapy results in systemic side effects due to non-specific uptake by normal healthy noncancerous tissues. To alleviate this difficulty, many attempts have been devoted to the development of new delivery systems such as polymeric Nanoparticles [NPs]. In this study, we prepared ICD-85 NPs based on sodium alginate and analyzed the cytotoxic activity of ICD-85 NPs relative to free ICD-85 on primary lamb kidney cells. ICD-85 loaded sodium alginate nanoparticles were prepared by ionic gelation method and were characterized by the particle size, size distribution and Fourier Transform Infrared [FT-IR] spectroscopy. The in vitro cytotoxicity was evaluated by MTT assay and membrane integrity was evaluated by measuring Lactate Dehydrogenase [LDH] activity. The morphological alterations of untreated and treated cells were assessed by light inverted microscope. MTT assay showed that ICD-85 NPs could significantly decrease the in vitro cytotoxicity on primary lamb kidney cells compared to the free ICD-85. The IC[10] value at 72 hours was increased from 9 +/- 2.7 microg/ml for free ICD-85 to 52 +/- 4.3 microg/ml for ICD-85 NPs. LDH assay demonstrated that free ICD-85 had dose-dependent cytotoxicity on primary lamb kidney cells while ICD-85 NPs exhibited significantly decreased cytotoxicity at equivalent concentrations. Moreover, morphological analysis showed no significant difference between control and treated cells with ICD-85 NPs. Based on the results obtained in the present study it can be concluded that encapsulation of ICD-85 with sodium alginate nanoparticles can reduce its necrotic effect on primary lamb kidney cells

Extracellular caspase-8 dependent apoptosis on hela cancer cells and MRC-5 normal cells by ICD-85 [venom derived peptides]

Our previous studies revealed an inhibitory effect of ICD-85 [venom derived peptides] on MDA-MB231 and HL-60 cell lines, through induction of apoptosis. The purpose of this study was to investigate apoptosis-induced mechanism on HeLa and MRC-5 cells by ICD-85 through activation of caspase-8. Cell viability, cytosolic enzyme Lactate Dehydrogenase [LDH] and cell morphology were assessed under unexposed and ICD-85 exposed conditions.Caspase-8 activity was assayed by caspase-8 colorimetric assay Kit. The results show that Inhibitory Concentration 50% [IC[50]] value of ICD-85 for HeLa cells at 24 h was estimated and found to be 25.32 +/- 2.15microg/ml. Furthermore, treatment of HeLa cells with ICD-85 at concentrations of 1.6x10 and 2.6x10microg/ml did not significantly increase LDH release. Morphological changes in HeLa cells on treatment with ICD-85 compared with untreated HeLa cells consistent with an apoptotic mechanism of cell death, such as cell shrinkage which finally results in the generation of apoptotic bodies. However, when MRC-5 cells were exposed to ICD-85, no significant changes in cell morphology and LDH were observed at concentrations below 2.6x10microg/ml. Also, the apoptosis-induction mechanism by ICD-85 on HeLa cells was found through activation of caspase-8 and the activity of caspase-8 in HeLa cells was 1.5 folds more than its activity on MRC-5 cells. Therefore, the apoptosis-induced mechanisms by ICD-85 are through activation of caspase-8 and concerning the least cytotoxic effect on MRC-5 cells, ICD-85 may be used as anticancer compound to inhibit growth of cancer cells.

Cytotoxic effects of two Iranian scorpions odontobuthusdoriae and bothutus saulcyi on five human cultured cell lines and fractions of toxic venom

Scorpion venom toxicity is of major concern due to its influence on human activities and public health. We investigated the in-vitro process of cell death caused by two Iranian scorpions Odontobuthus doriae and Bothutus salceyi venom on human cell lines. The aim of this study was to provide further information about triggering cell death and suggestion of methods for the elimination of unwanted cells such as tumor cells. The cytotoxicity and apoptosis induced by effect of scorpion venoms on five established eukaryotic cell lines are analyzed on different human cell lines. All cultured cell lines were incubated with varying doses of scorpion venom for 24 h at 37°C. Control culture was treated with an equal amount of SFM. The percentage of cell survival was measured using the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium [MTT] colorimetric assay. Our data demonstrated that Bothutus saulcyi, does not show cytotoxic effect on any of the used cell lines. Odontobuthus doriae, however, has resulted a dose dependent cytotoxic effect with maximum at 1 ug/mL on 1321N1 glioma like cell line. Then the cytotoxic venom of O. doriae was fractionated using Sephadex G50 gel chromatography. The toxic fractions on mouse used to Cytotoxicity assay on 1321 N1 cell line and data demonstrated that, the fraction F3 showed a dose dependent Cytotoxicity assay. Further studies to explode the mode of action of these venoms are recommended and purification of the toxic fraction should be done

Cytotoxicity effect of ICD-85 [Venom - derived peptides] on promyelocytic cancer cell lines [HL-60]

Our previous studies revealed an inhibitory effect of ICD-85; a venom derived peptide has been extracted by researchers of Razi vaccine and serum research institute, on breast cancer cell line [MDA-MB231]. The present study was undertaken to evaluate the anti proliferative effect of HL60 cells. The effect of ICD-85 on proliferation of HL-60 cancer cells was determined using the MTT assay. A quantitative cytotoxicity assay based on the release of lactate dehydrogenase [LDH; EC 1.1.1.27] was performed 24 h after exposure to various concentrations of ICD-85. The morphological changes of ICD-85 treated HL-60 cancer cells were observed under electron microscope. ICD-85 induced marked concentration inhibition of cancer cell proliferation with an IC50 value of 0.04 micro g/ml following 24 h incubation. Electron microscopic findings of ICD-85 treated cells showed nuclear material condensation, endoplasmic reticulum dilation, mitochondria swelling or degradation, increased cytoplasmic vacuoles, reduction or disappearance in cytoplasmic process and decreased nuclear/cytoplasmic ratio. LDH determination of cultured media of HL60 cells exposed to various concentration of ICD-85 revealed no significant changes in LDH levels compared with untreated cells. The results of present study indicated that ICD-85 inhibited the cancer cell proliferation by inducing apoptosis rather than necrosis

Necrotic effect versus apoptotic nature of camptothecin in human cervical cancer cells

Functional defects in mitochondria are involved in the induction of cell death in cancer cells. The process of programmed cell death may occur through the mechanisms of apoptosis. Several potential lead molecules such as Camptothecin [CPT] and its analogues have been isolated from plants with anticancer effect. The aim of the present study was to understand the necrotic effect versus apoptotic nature of CPT in HeLa cancer cells. The antiproliferative activity of CPT was estimated through 3-[4, 5- Dimethyl thiazol-2-yl]-2, 5-diphenyl tetrazolium bromide [MTT] assay and DNA fragmentation analysis using gel electrophoresis. Lactate Dehydrogenase [LDH] activity and cell morphology were assessed under control and CPT exposed conditions to evaluate the necrotic effect of CPT. The results showed that CPT inhibited the proliferation of HeLa cells in a dose-dependent manner with an Inhibitory Concentration 50% [IC50] of 0.08 +/- 0.012 microg/ml. However the significant [P<0.05] increase happens in LDH activity at concentrations 1x10-1microg/ml and above. Morphological changes showed that CPT in low concentrations induced an apoptotic mechanism of cell death, such as cell shrinkage and characteristic rounding of dying cells, while at high concentrations showed necrosis changes. The characteristic DNA ladder formation of CPT-treated cells in agarose gel electrophoresis confirmed the results obtained by light microscopy and LDH assay. Camptothecin as an anticancer drug may have antiproliferative effect on HeLa cancer cells in low concentrations, through its nature of induction of apoptosis. The border line between necrotic effect and apoptotic nature of CPT in HeLa cancer cells has been found to be at concentration of 1x10-1 microg/ml

Cytotoxic effect of ICD-85 [venom-derived peptides] on HeLa cancer cell line and normal LK cells using MIT assay

Cancer is the fifth leading cause of death worldwide. There are considerable efforts to identify naturally occurring substances for use as new drugs in cancer therapy. Some components of animal venoms have been identified that possess substantial anticancer properties. In our previous studies, the cytotoxic effects of ICD-85 [venom-derived peptides] have been reported on HL-60 and MDA-MB231 cell lines. This has prompted us to investigate the comparative cytotoxic effects of ICD-85 on the HeLa cell line and normal lamb kidney [LK] cells. Cells were exposed to various concentrations [8 x 10[4] to 5.6 x 10 microg/mI] of ICD-85 at various incubation times [24, 48 and 72 hours]. Cell viability was measured by the MTT assay. A morphological study was also carried out using an inverted microscope. Caspase-8 activity was assayed by the Caspase-8 Colorimetric Assay Kit in HeLa cells that were exposed to ICD-85 for 48 hours. Data analysis showed that ICD-85 has a dose-dependent cytotoxic effect on HeLa cells with an inhibitory concentration 50% [IC[50]] of 26.62 +/- 2.13 microg/mI at 24 hours, 27.33 +/- 2.35 microg/mI at 48 hours, and 28.13 +/- 2.52 microg/mI at 72 hours. Results also indicated that the cytotoxic effect of ICD-85, at 48 and 72 hours incubation times did not show significant alteration compared to 24 hours of exposure. Interestingly, the minimum concentration of ICD-85 which showed a cytotoxic effect on LK cells was found to be 3500-fold less than the minimum concentration that showed a cytotoxic effect on the HeLa cancer cells. While morphological analysis revealed a significant difference that included the characteristic rounding of dying cells by treatment with ICD-85 compared with untreated HeLa cells, this difference was not observed in normal cells. ICD-85 increased caspase-8 activity in HeLa cells after 48 hours of exposure. ICD-85 has a dose-dependent cytotoxic effect on HeLa cancer cells in contrast with its negligible effect on normal LK cells

Two biological active fractions isolated from buthotus schach [bs] scorpion venom examined on striated muscle preparation, in-vitro

Buthotus schach is one of the most dangerous scorpions in tropical part of Iran. The effects of its crude venom at 1, 3, 10 microg/mL and its obtained fractions by gel filtrations were investigated on neuromuscular transmission. CBC and MHD indirectly and directly stimulated preparations techniques were used to study their possible pre or post junctional activities. At 3 and 10 microg/ mL [not at 1 microg/mL], BS venom caused initiall increase in twitch height followed by blockage due to large contraction that responded gradually at the same time. Contracture responses to exogenous Ach [1-2 mM, 30 sec] and Carb [30-40 microM, 60 sec] in the presence of the venom were not increased which does show no anticholinstrease effects. Furthermore Contracture response to KC1 [20-40 mM, 30 sec] does changed exposure to venom in CBC preparations. On the other hand the effects of the venom in response to directly stimulated preparations was shallower than in indirect stimulated preparations. So in agreement with KCL response BS venom affects mostly prejunctionally to facilitate the neurotransmitter release rather than postjunctionally effects. To access bioactive components, seven fractions were collected by gel filtrations techniques. Among the fractions F[6], LD[50]=21 microg < F[4], LD[50]= 35.5 microg < Venom LD[50]= 84 microg per mice were more toxic respectively. Both fractions show the same effects but stronger than venom on twitch height responses in indirectly stimulated CBC preparations. Finally, according to our results venom as well as fractions F4 and F6 act mostly prejunctionally on Ach release. More attempt is carrying out to study their effects on ion channel activities

Induction of apoptosis in human leukemia cell line [HL60] by animal's venom derived peptides [ICD-85]

Our previous studies revealed an inhibitory effect of ICD-85 [Venom derived peptides] on breast cancer cell line MDA-MB231. ICD-85 was also confirmed by in-vivo studies to suppress the breast tumor in mice. However, the exact mechanism of ICD-85 was unknown. Hence, the present study was undertaken to assess the mechanism of ICD-85 effect as an anti-proliferative agent of cancer cells. The effect of ICD-85 on proliferation of HL-60 cancer cells was determined by using the MTT assay. The morphological changes of ICD-85 treated HL-60 cells were observed under transmission electron microscope [TEM] DNA fragmentation analysis was also carried out using gel electrophoresis. ICD-85 induced the marked inhibition of HL60 cell proliferation with an IC[50]-value of 0.04 |ug/mL following 24 h of incubation. ICD-85 treated cells when compared with untreated cells, showed nuclear material condensation, endoplasmic reticulum dilation, mitochondria swelling or degradation, increased cytoplasmic vacuoles, reduction or disappearance in cytoplasmic process and decreased nuclear/cytoplasmic ratio was observed. The characteristic DNA ladder formation of ICD-85-treated cells in agarose gel electrophoresis confirmed the results obtained through the electron microscopy. The results of the present study indicated that ICD-85 inhibited the cancer cell proliferation by inducing cell apoptosis

Preparation and biodistribution study of a [99m]Tc-labeled toxic fraction of Iranian mesobuthus eupeus scorption venom

Iranian scorpion species are classified in Buthidae and Scorpionidae with 16 genera and 25 species. In Iran, similar to other parts of the world, there are a few known species of scorpions responsible for severe envenoming. Mesobuthus eupeus is the most common species in Iran. Its venom contains several toxin fractions which can affect the ion channel. In this study purification, labeling and biological evaluation of Mesobuthus eupeus scorpion venom are described. To separate different venom fractions, soluble venom was loaded on a chromatography column packed with sephadex G50 gel then the fractions were collected according to UV absorption at 280 nm wavelength. Toxic fraction [F3] was loaded on anionic ion exchanger resin [DEAE] and then on a cationic resins [CM]. Finally toxic fraction F319 was labeled with [99m]Tc and radiochemical analysis was determined by paper chromatography. The biodistribution was studied after injection into normal mice. Toxic fraction of venom was successfully obtained in purified form. Radiolabeling of venom was performed at high specific activity with radiochemical purity more than 95% which was stable for more than 4 h. Biodistribution studies in normal mice showed rapid clearance of compound from blood [2.64% ID at 4 h] and tissues except the kidneys [27% ID at 4 h]. As tissue distribution studies are very important for clinical use, results of this study suggest that [99m]Tc labeling of venom can be a useful tool for in vivo studies and is an excellent approach to follow the process of biodistribution and kinetics of toxins