Assessment of the Inhibitory Effects of Ficin-hydrolyzed Gelatin Derived from Squid (Uroteuthis duvauceli) on Breast Cancer Cell Lines and Animal Model.

Marine novel natural products have been applied for cancer therapy. Enzyme-digested gelatin hydrolysates have proven to serve as promising sources of potent biologically active peptides. Potential anti-breast cancer properties of the extracted Ficin-digesterd gelatin hydrolysate from Indian squid (Uroteuthis duvauceli) was extensively characterized by cellular and animal models. Gelatin was extracted from squid skin, hydrolyzed by Ficin, and characterized by standard physico-chemical methods. Ficin-digested gelatin hydrolysate was used at various doses of 0-0.1 mg/mL for assessment of MCF-7 and MDA-MB-231 breast cancer cells versus HUVEC normal cells. Cytotoxicity, phase-contrast morphological examination, apoptosis/necrosis, clonal-growth, cell-migration, Matrix-metalloproteinases (MMPs) zymography, and Western blotting were used for cellular assessments. For animal studies, breast tumor-induced BALB/c mice received hydrolyzed gelatin regimen, followed by tumor size/growth and immune-histochemical analyses. Significant inhibition of MCF-7 and MDA-MB-231 with no cytotoxicity on HUVEC cells were detected. Apoptosis was increased in cancer cells, as revealed by elevated ratio of cleaved caspase-3 and PARP. MMP-2 and MMP-9 activities in both cancer cells were diminished. In mice, gelatin hydrolysate prevented weight loss, decreased tumor size, induced p53, and down-regulated Ki67 levels. These findings suggest that Ficin-digested gelatin hydrolysate could be a beneficial candidate for novel breast cancer therapies.

Growth inhibition of MDA-MB-231 cell line by peptides designed based on uPA.

Interaction between urokinase-type plasminogen activator (uPA) and its receptor (uPAR) plays an important role in the progression of numerous cancer types including breast cancer by promoting tumor initiating, proliferation, invasion and metastasis. Hence, disruption of this interaction inhibits their downstream cascades and subsequently tumor growth. For this, we created two series of 8 and 10 amino acids linear peptides, derived from uPA binding region to target uPAR and studied the inhibition of proliferation in MDA-MB-231 cell line. Results revealed that all of the 10-mer peptides inhibited breast cancer cell proliferation significantly with maximum 40% inhibition of 103 peptides. Meanwhile, none of the 8-mer peptides showed significant toxicity. Current results indicate that the linear 10-mer peptides which mimic a small part of a sequence of a binding domain of uPA to uPAR could be exploited to design a novel class of anti-cancer agents.

Bypassing the need for pre-sensitization of cancer cells for anticancer TRAIL therapy with secretion of novel cell penetrable form of Smac from hA-MSCs as cellular delivery vehicle.

TNF-related apoptosis inducing ligand (TRAIL) is a novel anticancer agent with selective apoptosis-inducing activity on cancer cells. However, many malignant tumors still remain unresponsive. Although cells can bypass apoptosis by different functions, the defect in the blocking role of second mitochondria-derived activator of caspase (Smac) on X-linked inhibitor of apoptosis protein (XIAP) is known to be an important hub for immortal characteristic of malignant cells. Actually, XIAP is known as an apoptosis inhibitor. To date, the sensitization of cancerous cells to TRAIL was successfully performed with different protocols, mainly through blocking XIAP with Smac administration. However, all these sensitization methodologies need to be performed prior to TRAIL administration on cancerous cells which in turn limit their practical application in clinics. Therefore, we hypothesized that concurrent expression of Smac and TRAIL on human adipose-derived mesenchymal stem cells (hA-MSC-ST) could both sensitize and destroy cancerous cells. To this aim, we generated hA-MSC-ST, secreting a novel cell penetrable form of Smac and a trimeric form of TRAIL. Indeed, the cell penetrable form of Smac obviates the need for any pretreatment of cancerous cells. Our data depicted that individual overexpression of TRAIL or Smac in different breast cancer cell types induced limited or no apoptosis, respectively. Conversely, their concomitant overexpression markedly increased cell death even for a resistant type of breast cancer cells, MCF-7. Notably, we observed no cytotoxicity of our methodology on normal cells. In summary, this is the first demonstration that a dual approach using simultaneous overexpression of a cell penetrable form of Smac and TRAIL sensitize and promote apoptotic process even in resistant breast cancer cells.

Effects of imatinib mesylate in mouse models of multiple sclerosis and in vitro determinants.

Experimental autoimmune encephalomyelitis (EAE) is a mouse model for multiple sclerosis (MS), This autoimmune disease is mainly mediated by adaptive and innate immune responses that lead to an inflammatory demyelination and axonal damage. Imatinib mesylate is a selective protein tyrosine kinase inhibitor with immunomodulatory properties that abrogates multiple signal transduction pathways in immune cells. In the present research, our aim was to test the therapeutic efficacy of imatinib in experimental model of MS. We performed EAE induction in 23 female C57 mice by myelin oligodendrocyte glycoprotein-35-55 (MOG35-55) in Complete Freund's Adjuvant (CFA) emulsion and used imatinib for treatment of EAE. The clinical evaluation and histopathology were assessed. Also for in vitro analysis, we used U-87 MG, C6 and WEHI-164 cell lines to evaluate the inhibitory effects of imatinib in cell proliferation, as well as pro-inflammatory cytokines (TNF-α, IL-1ß, IL-6) and matrix metalloproteinase (MMP) secretion. Our findings demonstrated that this drug had beneficial effects on EAE by attenuation in the severity and a delay in the onset of disease. In vitro, imatinib inhibited cell proliferation, MMP-2 expression and activity and also attenuated the production of proinflammatory cytokines. Imatinib with its potential therapeutic effects and immunomodulatory properties may be considered, after additional necessary tests and trials, for treatment of MS.

Lithium chloride protects PC12 pheochromocytoma cell line from morphine-induced apoptosis.

BACKGROUND: Opioid drugs are considered as important members of drugs of abuse. Opioid abusers are more likely to be infected which may be due to apoptotic effects of the drugs on immune cells. Furthermore, there are some reports on the apoptotic effect of morphine on neural cells. In the present study, the effect of morphine and lithium on apoptosis in PC12 cell line (as a model of neural cells) was examined. METHODS: We used 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, annexin V-fluorescein isothiocyanate test and quantitative real time RT-polymerase chain reaction for detection of necrosis and apoptosis (programmed cell death). RESULTS: PC12 cells were exposed to different concentrations of morphine for six, 12, 24, 48, and 96 hours. Quantitative real-time RT-polymerase chain reaction revealed that mRNA expression of BAX (proapoptotic element) increased while a decrement in the mRNA expression of BCL-2 (protective element) was observed after six hours (but not after 12 or 24 hours) exposure to morphine. Furthermore, the results of MTT assay and annexin V-fluorescein isothiocyanate test indicated that morphine exposure causes an increase in the percentage of apoptotic and necrotic cells, respectively. Interestingly, the results of MTT assay and annexin V-fluorescein isothiocyanate test were observed 12 and 24 hours after morphine exposure. Thus, it can be concluded that alteration in mRNA expression is an early event rather than as a consequence of apoptosis or necrosis. On the other hand, lower concentrations of lithium elicit protective effect against apoptosis in some of mammalian cells while the higher concentrations are toxic. Despite large body of evidences on the protective effect of lithium, elucidation of downstream events are still unknown. In the present study, 72-hour preincubation of PC12 cells with 1.2 mM lithium chloride reversed the effects of morphine on the mRNA expression of BAX and BCL-2. Furthermore, the results of real time RT-polymerase chain reaction were supported by annexin V-fluorescein isothiocyanate test and MTT assay. CONCLUSION: The protective effect of lithium on the morphine-induced cytotoxicity is mediated via down-regulation of BAX and up-regulation of BCL-2 mRNA expression.