Level of Serum Enzymes and Electrocardiogram in Healthy Rabbits after Injection of ICD-85 as an Anticancer Agent.

BACKGROUND: 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. METHODS: 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. RESULTS: ECG results showed that the injection of 50 and 100 µg/kg ICD-85 into healthy rabbits has no significant effect on heart function while the injection of 150 to 200 µg/kg ICD-85 caused ECG wave changes and mild bradycardia without toxic effects on heart. After ICD-85 injection (concentrations below 100 µg/kg), no significant increase was observed in liver and cardiac enzymes (ALT, AST, LDH, CPK, and CK-MB). However, the concentration of 150 µg/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 µg/kg. CONCLUSION: Based on the results obtained in the present study as well as our previous reports, ICD-85 at concentrations below 100 µg/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.

Cytotoxicity of ICD-85 NPs on Human Cervical Carcinoma HeLa Cells through Caspase-8 Mediated Pathway.

The biological application of nanoparticles (NPs) is a rapidly developing area of nanotechnology that raises new possibilities in the treatment of human cancers. The cytotoxicity was evaluated by MTT and LDH assays. The apoptotic effect of free ICD-85 and ICD-85 NPs on HeLa cells was assessed using caspase-8 colorimetric assay. The MTT assay showed that ICD-85 NPs could enhance the in-vitro cytotoxicity against HeLa cells compared to the free ICD-85. The IC50 value at 72 h was reduced from 25 ± 2.9 µg/mL for free ICD-85 to 15.5 ± 2.4 µg/mL for ICD-85 NPs. However, LDH assay demonstrated that ICD-85 has dose-dependent cytotoxicity on HeLa cells while ICD-85 NPs exhibited weaker cytotoxicity on same cells. The results also indicate that ICD-85-induced apoptosis on HeLa cells is associated with the activation of caspase-8. Moreover, caspase-8 assay analysis demonstrated that the ICD- 85 NPs induced a higher apoptotic rate in HeLa cells compared to free ICD-85. Our results demonstrated that the encapsulation of ICD-85 enhances its anti-proliferative effects. Taken together, these results suggest that the delivery of ICD-85 in nanoparticles may be a promising approach for the treatment of the cancer.

The association of -475 and -631 interleukin-2 gene polymorphism with multiple sclerosis in Iranian patients.

OBJECTIVE: Multiple sclerosis (MS) is a chronic autoimmune disease due to demyelination of the central nervous system. It is believed that cytokines are involved in the pathogenesis of MS. The interleukin-2 (IL2) gene is powerful functional candidate that is involved in immune regulation and operation. In this study, for the first time, we investigated the effect of -475 A/T and -631 G/A IL2 polymorphisms on MS disease in Iranian patients. MATERIALS AND METHODS: In this case-control study, 100 MS patients (mean age: 32.95 ± 6.51 years, age range: 20-42 years) selected according to McDonald criteria, and 100 ethnically, sex and age matched healthy controls (mean age: 29 ± 7.8 years, age range: 20-52 years) with no personal or family history of autoimmune diseases were studied. The restriction fragment length polymorphism-polymerase chain reaction (RFLP-PCR) method was applied to define different alleles and genotypes of IL2 promoter single nucleotide polymorphism -475 A/T as well as -631 G/A among individuals. χ(2) was calculated and Fisher's exact test was applied to analyze the obtained data. The value of p < 0.05 was considered significantly . RESULTS: Evaluation of the -475 IL2 revealed that T allele and A/T genotype are present in 2% and 4% of MS patients, respectively, whereas T allele was absent in control samples. The comparison between alleles and genotypes in MS patients and healthy controls was not significant (p=0.1). For the -631 position, 1% and 2% of MS patients carried A allele and A/G heterozygote genotypes, respectively. All control samples had G allele and G/G genotype. The differences between patients and controls were not significant (p=0.4). Moreover, our results showed a very low frequency of T at -475 and A at -631 IL2 position in each of the two groups. CONCLUSION: Both -475 and -631 IL2 polymorphisms were higher in MS patients as compared to controls, but the frequency differences were not significant. Based on these data, it is suggested that the -475 and -631 IL2 polymorphisms as functional promoter position may be involved in IL2 expression and regulation. To find out the exact effect of the mentioned SNPs on susceptibility to MS, study on a larger sample size is suggested.

Cytotoxic effect of ICD-85 (venom-derived peptides) on HeLa cancer cell line and normal LK cells using MTT Assay.

BACKGROUND: 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. METHODS: Cells were exposed to various concentrations (8 × 10-4 to 5.6 × 10 µg/ml) 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. RESULTS: Data analysis showed that ICD-85 has a dose-dependent cytotoxic effect on HeLa cells with an inhibitory concentration 50% (IC50) of 26.62 ± 2.13 µg/ml at 24 hours, 27.33 ± 2.35 µg/ml at 48 hours, and 28.13 ± 2.52 µg/ml 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. DISCUSSION: ICD-85 has a dose-dependent cytotoxic effect on HeLa cancer cells in contrast with its negligible effect on normal LK cells.

Necrotic Effect versus Apoptotic Nature of Camptothecin in Human Cervical Cancer Cells.

BACKGROUND: 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. METHODS: The anti-proliferative 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. RESULTS: 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 µg/ml. However the significant (p<0.05) increase happens in LDH activity at concentrations 1×10(-1)µg/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. CONCLUSION: Camptothecin as an anticancer drug may have anti-proliferative 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 1×10(-1) µg/ml.

Extracellular Caspase-8 Dependent Apoptosis on HeLa Cancer Cells and MRC-5 Normal Cells by ICD-85 (Venom Derived Peptides).

BACKGROUND: 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. METHODS: 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. RESULTS: The results show that Inhibitory Concentration 50% (IC50) value of ICD-85 for HeLa cells at 24 h was estimated and found to be 25.32±2.15 µg/mL. Furthermore, treatment of HeLa cells with ICD-85 at concentrations of 1.6×10 and 2.6×10 µg/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 2.6×10µg/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. CONCLUSION: 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.