Chemical synthesis of La1 isolated from the venom of the scorpion Liocheles australasiae and determination of its disulfide bonding pattern

La1 is a 73‐residue cysteine‐rich peptide isolated from the scorpion Liocheles australasiae venom. Although La1 is the most abundant peptide in the venom, its biological function remains unknown. Here, we describe a method for efficient chemical synthesis of La1 using the native chemical ligation (NCL) strategy, in which three peptide components of less than 40 residues were sequentially ligated. The peptide thioester necessary for NCL was synthesized using an aromatic N‐acylurea approach with Fmoc‐SPPS. After completion of sequential NCL, disulfide bond formation was carried out using a dialysis method, in which the linear peptide dissolved in an acidic solution was dialyzed against a slightly alkaline buffer to obtain correctly folded La1. Next, we determined the disulfide bonding pattern of La1. Enzymatic and chemical digests of La1 without reduction of disulfide bonds were analyzed by liquid chromatography/mass spectrometry (LC/MS), which revealed two of four disulfide bond linkages. The remaining two linkages were assigned based on MS/MS analysis of a peptide fragment containing two disulfide bonds. Consequently, the disulfide bonding pattern of La1 was found to be similar to that of a von Willebrand factor type C (VWC) domain. To our knowledge, this is the first report of the experimental determination of the disulfide bonding pattern of peptides having a single VWC domain as well as their chemical synthesis. La1 synthesized in this study will be useful for investigation of its biological role in the venom. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.     

Anticancer activity of paroxetine in human colon cancer cells: Involvement of MET and ERBB3

The concept of drug repositioning has recently received considerable attention in the field of oncology. In the present study, we propose that paroxetine can be used as a potent anticancer drug. Paroxetine, one of the selective serotonin reuptake inhibitors (SSRIs), has been widely prescribed for the treatment of depression and anxiety disorders. Recently, SSRIs have been reported to have anticancer activity in various types of cancer cells; however, the underlying mechanisms of their action are not yet known. In this study, we investigated the potential anticancer effect of paroxetine in human colorectal cancer cells, HCT116 and HT‐29. Treatment with paroxetine reduced cell viability, which was associated with marked increase in apoptosis, in both the cell lines. Also, paroxetine effectively inhibited colony formation and 3D spheroid formation. We speculated that the mode of action of paroxetine might be through the inhibition of two major receptor tyrosine kinases – MET and ERBB3 – leading to the suppression of AKT, ERK and p38 activation and induction of JNK and caspase‐3 pathways. Moreover, in vivo experiments revealed that treatment of athymic nude mice bearing HT‐29 cells with paroxetine remarkably suppressed tumour growth. In conclusion, paroxetine is a potential therapeutic option for patients with colorectal cancer.     

A proteomics approach to identifying novel protein targets involved in erinacine A–mediated inhibition of colorectal cancer cells’ aggressiveness

Erinacine A, a major active component of a diterpenoid derivative isolated from Hericium erinaceus mycelium, has been demonstrated to exert anticancer effects. Herein, we present an investigation of the molecular mechanism of erinacine A induction associated with cancer cells’ aggressive status and death. A proteomic approach was used to purify and identify the differentially expressed proteins following erinacine A treatment and the mechanism of its action in apoptotic and the targets of erinacine A. Our results demonstrate that erinacine A treatment of HCT‐116 and DLD‐1 cells increased cell cytotoxicity and reactive oxygen species (ROS) production as well as decreased cell proliferation and invasiveness. Ten differentially displayed proteins were determined and validated in vitro and in vivo between the erinacine A‐treated and untreated groups. In addition, erinacine A time‐dependent induction of cell death and inhibitory invasiveness was associated with sustained phosphorylation of the PI3K/mTOR/p70S6K and ROCK1/LIMK2/Cofilin pathways. Furthermore, we demonstrated that erinacine A–induced HCT‐116 and DLD‐1 cells viability and anti‐invasion properties by up‐regulating the activation of PI3K/mTOR/p70S6K and production of ROS. Experiments involving specific inhibitors demonstrated that the differential expression of cofilin‐1 (COFL1) and profilin‐1 (PROF1) during erinacine A treatment could be involved in the mechanisms of HCT‐116 and DLD‐1 cells death and decreased aggressiveness, which occurred via ROCK1/LIMK2/Cofilin expression, with activation of the PI3K/mTOR/p70S6K signalling pathway. These findings elucidate the mechanism of erinacine A inhibiting the aggressive status of cells by activating PI3K/mTOR/p70S6K downstream signalling and the novel protein targets COF1 and PROF1; this could be a good molecular strategy to limit the aggressiveness of CRC cells.     

A ginger derivative,zingerone—a phenolic compound—induces ROS‐mediated apoptosis in colon cancer cells (HCT‐116)

Zingerone (ZO), an active phenolic agent derived from Zingiber officinale (Ginger), has many pharmacological properties such as antioxidant, antiangiogenic, and antitumor. However, its potential value in cancer and the mechanism by which ZO wields its therapeutic effects remain obscure. Therefore, in this current study, we explored the effects of ZO on suppressing cell proliferation and enhancing apoptosis in colon cancer cells (HCT116). Our results indicated that ZO significantly enhances the production of reactive oxygen species, lipid peroxidation (thiobarbituric acid reactive substance [TBARS]), and loss of cell viability; and reduces mitochondrial membrane potential and antioxidant levels (SOD, CAT, and GSH) in ZO‐treated HCT116 cells in a dose‐dependent (2.5, 5, and 10 µM) manner. Furthermore, ZO induces oxidative stress‐mediated apoptosis as evidenced by apoptotic morphological changes predicted by AO/EtBr, Hoechst staining and further confirmed by comet assay. Moreover, immunoblotting techniques showed that ZO treatment effectively enhances Bax, caspase‐9, and caspase‐3 expressions and decreases the expression of Bcl‐2 in colon cancer cells. Together, our results evidenced that the antitumor effects of ZO reduce cell proliferation and stimulate apoptosis through modulating pro‐ and antiapoptotic molecular events in HCT116 colon cancer cells. Therefore, based on our findings, ZO may be used as a therapeutic agent for the treatment of colon cancer.     

ZEB1‐mediated vasculogenic mimicry formation associates with epithelial–mesenchymal transition and cancer stem cell phenotypes in prostate cancer

The zinc finger E‐box‐binding homeobox 1 (ZEB1) induced the epithelial–mesenchymal transition (EMT) and altered ZEB1 expression could lead to aggressive and cancer stem cell (CSC) phenotypes in various cancers. Tissue specimens from 96 prostate cancer patients were collected for immunohistochemistry and CD34/periodic acid–Schiff double staining. Prostate cancer cells were subjected to ZEB1 knockdown or overexpression and assessment of the effects on vasculogenic mimicry formation in vitro and in vivo. The underlying molecular events of ZEB1‐induced vasculogenic mimicry formation in prostate cancer were then explored. The data showed that the presence of VM and high ZEB1 expression was associated with higher Gleason score, TNM stage, and lymph node and distant metastases as well as with the expression of vimentin and CD133 in prostate cancer tissues. Furthermore, ZEB1 was required for VM formation and altered expression of EMT‐related and CSC‐associated proteins in prostate cancer cells in vitro and in vivo. ZEB1 also facilitated tumour cell migration, invasion and clonogenicity. In addition, the effects of ZEB1 in prostate cancer cells were mediated by Src signalling; that is PP2, a specific inhibitor of the Src signalling, dose dependently reduced the p‐Src⁵²⁷ level but not p‐Src⁴¹⁶ level, while ZEB1 knockdown also down‐regulated the level of p‐Src⁵²⁷ in PC3 and DU‐145 cells. PP2 treatment also significantly reduced the expression of VE‐cadherin, vimentin and CD133 in these prostate cancer cells. Src signalling mediated the effects of ZEB1 on VM formation and gene expression.     

Analysis of colorectal cancer glyco‐secretome identifies laminin β‐1 (LAMB1) as a potential serological biomarker for colorectal cancer

The high mortality rate in colorectal cancer is mostly ascribed to metastasis, but the only clinical biomarker available for disease monitoring and prognosis is the carcinoembryonic antigen (CEA). However, the prognostic utility of CEA remains controversial. In an effort to identify novel biomarkers that could be potentially translated for clinical use, we collected the secretomes from the colon adenocarcinoma cell line HCT‐116 and its metastatic derivative, E1, using the hollow fiber culture system, and utilized the multilectin affinity chromatography approach to enrich for the secreted glycoproteins (glyco‐secretome). The HCT‐116 and E1 glyco‐secretomes were compared using the label‐free quantitative SWATH‐MS technology, and a total of 149 glycoproteins were differentially secreted in E1 cells. Among these glycoproteins, laminin β‐1 (LAMB1), a glycoprotein not previously known to be secreted in colorectal cancer cells, was observed to be oversecreted in E1 cells. In addition, we showed that LAMB1 levels were significantly higher in colorectal cancer patient serum samples as compared to healthy controls when measured using ELISA. ROC analyses indicated that LAMB1 performed better than CEA at discriminating between colorectal cancer patients from controls. Moreover, the diagnostic performance was further improved when LAMB1 was used in combination with CEA.     

A novel peptide from alpha5 helix of Asterina pectinifera cyclin B conjugated to HIV-Tat(49-57) with cytotoxic and apoptotic effects against human cancer cells

A series of novel peptides from various motifs of Asterina pectinifera cyclin B and their derivatives conjugated to HIV-Tat(49-57) were designed and synthesized. Their bioactivities on two human cancer cell lines were determined. Among them, Tat-a5 (KAQIRAMECNILGRKKRRQRRR) exhibited significant cytotoxic effects on cancer cell lines EC-9706 and HCT-116. Tat-a5 could arrest cancer cells at G(2)/M phase and make them apoptotic. Our results suggested that Tat-a5 could be a novel leading peptide with anticancer activity.     

Inhibition of proteasomal degradation of Mcl-1 by cobalt chloride suppresses cobalt chloride-induced apoptosis in HCT116 colorectal cancer cells

Cobalt promotes apoptosis in multiple cell systems, however, the molecular mechanisms that influence cobalt-induced apoptosis are not fully understood. We investigated mechanisms of cobalt chloride induced apoptosis in HCT116 colorectal cancer cells. Cobalt chloride induced dose dependent apoptosis in HCT116 cells (250–750 μM) which, at higher concentrations (500–750 μM), was associated with an increase in the expression of the Bcl-2-related Mcl-1 survival protein. Cobalt chloride caused the accumulation of higher molecular weight ubiquitin-conjugates of Mcl-1 in intact HCT116 cells and inhibited the activity of the trypsin-like site of the 20S proteasome in an in vitro assay. Although siRNA-mediated knockdown of Mcl-1 increased apoptosis in HCT116 cells, the combination of Mcl-1 siRNA and cobalt chloride induced very high levels of cell killing. Therefore, inhibition of the proteasome by cobalt chloride leads to the accumulation of Mcl-1 which acts to limit cobalt chloride induced apoptosis.     

STIP overexpression confers oncogenic potential to human non‐small cell lung cancer cells by regulating cell cycle and apoptosis

Sip1/tuftelin‐interacting protein (STIP), a multidomain nuclear protein, is a novel factor associated with the spliceosome, yet its role and molecular function in cancer remain unknown. In this study, we show, for the first time, that STIP is overexpressed in non‐small cell lung cancer (NSCLC) tissues compared to adjacent normal lung tissues. The depletion of endogenous STIP inhibited NSCLC cell proliferation in vitro and in vivo, caused cell cycle arrest and induced apoptosis. Cell cycle arrest at the G2/M phase was associated with the expression and activity of the cyclin B1‐CDK1 (cyclin‐dependent kinase 1) complex. We also provide evidence that STIP knockdown induced apoptosis by activating both caspase‐9 and caspase‐3 and by altering the Bcl‐2/Bax expression ratio. RNA sequencing data indicated that the MAPK mitogen‐activated protein kinases, Wnt, PI3K/AKT, and NF‐κB (nuclear factor kappa‐light‐chain‐enhancer of activated B cells) signalling pathways might be involved in STIP‐mediated tumour regulation. Collectively, these results suggest that STIP may be a novel potential diagnostic and therapeutic target for NSCLC.     

The cationic cell-penetrating KT2 peptide promotes cell membrane defects and apoptosis with autophagy inhibition in human HCT 116 colon cancer cells

The anticancer activity of cationic antimicrobial peptides (AMPs) has become more interesting because some AMPs have selective recognition against cancer cells. However, their antitumor properties and underlying mechanisms in cancer cells have not been clearly understood. In this study, we evaluated the effects of KT2 (lysine/tryptophan-rich AMP) on the cellular uptake and internalization mechanism, cell viability, surface charge of the cell membrane, membrane integrity, apoptotic cell death, and autophagy in human HCT 116 colon cancer cells. We found that KT2 interacted with the cell membrane of HCT 116 cells and was internalized into HCT 116 cells via clathrin-mediated and caveolae-mediated endocytosis mechanisms. The interaction of KT2 with cells caused cell membrane structure change, elevated membrane permeability, and KT2 also affected the lipid component. The results of atomic force microscopy showed cellular membrane defects of KT2-treated cells. The internalized KT2 induced nuclear condensation and apoptotic cell death. It elevated the apoptotic factor levels including those of cytochrome c and apoptosis-inducing factor. Furthermore, KT2 inhibited autophagy by the suppression of autophagy-related 5, autophagy-related 7, autophagy-related 16 like 1, and Beclin-1 proteins. In conclusion, these results revealed the cytotoxicity of cationic KT2 against HCT 116 cells and may help to clarify the interactions between cationic AMPs and cancer cells.     

A novel microtubule depolymerizing colchicine analogue triggers apoptosis and autophagy in HCT‐116 colon cancer cells

Colchicine is a tubulin‐binding natural product isolated from Colchicum autumnale. Here we report the in vitro anticancer activity of C‐ring modified semi‐synthetic derivative of colchicine; N‐[(7S)‐1,2,3‐trimethoxy‐9‐oxo‐10‐(4‐phenyl‐piperidin‐1‐yl)‐5,6,7,9 tetrahydrobenzo[a]heptalen‐7‐yl]acetamide ( 4h ) on colon cancer HCT‐116 cell line. The compound 4h was screened for anti‐proliferative activity against different human cancer cell lines and was found to exhibit higher cytotoxicity against colon cancer cell lines HCT‐116 and Colo‐205 with IC₅₀ of 1 and 0.8 μM respectively. Cytotoxicity of the compound to the normal fR2 breast epithelial cells and normal HEK293 human embryonic kidney cells was evaluated in concentration and time‐dependent manner to estimate its selectivity for cancer cells which showed much better selectivity than that of colchicine. Compound 4h induced cell death in HCT‐116 cells by activating apoptosis and autophagy pathways. Autophagy inhibitor 3‐MA blocked the production of LC3‐II and reduced the cytotoxicity in response to 4h , but did not affect apoptosis, suggesting thereby that these two were independent events. Reactive oxygen species scavenger ascorbic acid pretreatment not only decreased the reactive oxygen species level but also reversed 4h induced cytotoxicity. Treatment with compound 4h depolymerized microtubules and the majority of cells arrested at the G2/M transition. Together, these data suggest that 4h has better selectivity and is a microtubule depolymerizer, which activates dual cell‐death machineries, and thus, it could be a potential novel therapeutic agent in cancer therapy. Copyright © 2016 John Wiley & Sons, Ltd.     

BAI,A novel cyclin‐dependent kinase inhibitor induces apoptosis in A549 cells through activation of caspases and inactivation of Akt

Previously, we have synthesized a novel cyclin‐dependent kinase (CDK) inhibitor, 2‐[1,1′biphenyl]‐4‐yl‐N‐[5‐(1,1‐dioxo‐1λ⁶‐isothiazolidin‐2‐yl)‐1H‐indazol‐3‐yl]acetamide (BAI) and reported its anti‐cancer activity in head and neck cancer cells. In this study, we further evaluated the effect of BAI on growth of various human cancer cell lines, including A549 (nonsmall cell lung cancer), HCT116 (colon), and Caki (kidney). Profoundly, results of XTT and clonogenic assays demonstrated that BAI at nanomolar concentrations (20–60 nM) inhibited growth of A549, HCT116, and Caki cells, suggesting the anti‐cancer potency. We show that BAI induced a dose‐dependent apoptotic cell death in these human cancer cells, as measured by fluorescence‐activated cell sorting (FACS). Interestingly, further biochemical analysis showed that treatment with BAI at 20 nM induced apoptosis in A549 cells in association with activation of caspases, cleavage of phospholipase C‐γ1 (PLC‐γ1), and inhibition of Akt in A549 cells. Importantly, pharmacological inhibition study revealed that pretreatment with z‐VAD‐fmk, a pan caspase inhibitor strongly blocked the BAI‐induced apoptosis in A549 cells. Transfection analysis with Akt cDNA encoding constitutively active Akt further addressed the significance of Akt inhibition in the BAI‐induced apoptosis in A549 cells. Notably, disruption of the PI3K/Akt pathway by LY294002, a PI3K/Akt inhibitor potentiated apoptosis in A549 cells by BAI at a subcytotoxic concentration. These findings collectively suggest that BAI potently inhibits growth of A549, HCT116, and Caki cells, and that the BAI‐induced apoptosis in A549 cells is associated with activation of caspases, and inhibition of Akt. J. Cell. Biochem. 114: 282–293, 2013. © 2012 Wiley Periodicals, Inc.     

Purinergic signaling as potential target of thiamethoxam-induced neurotoxicity using silver catfish (<Emphasis Type="Italic">Rhamdia quelen</Emphasis>) as experimental model

Resveratrol is a polyphenolic compound in many edible foods including grapes, peanuts, and berries. Several studies have revealed the beneficial effects of resveratrol against various diseases such as heart disease, diabetes, obesity, neurological disorders, and cancer. A recent study showed that resveratrol inhibits the proliferation of HCT116 human colorectal cancer cells in three-dimensional culture (3DC) via induction of luminal apoptosis in HCT116 cell spheroids. In this study, we showed that a novel compound, caffeic acid-adducted resveratrol, has a stronger inhibitory effect on the growth of HCT116 cell spheroids in 3DC than resveratrol. It showed almost the same inhibitory efficacy as 5-fluorouracil, a conventional anticancer drug. We further showed that the resveratrol derivative did not affect the growth of HKe3 cell spheroids derived from HCT116 cells by disruption of the activating mutant KRAS gene. These results suggest that the resveratrol derivative inhibits the growth of HCT116 cell spheroids via inhibition of an oncogenic KRAS-mediated signaling pathway.     

Genomic and tumor biological aspects of the anticancer nicotinamide phosphoribosyltransferase inhibitor FK866 in resistant human colorectal cancer cells

Cancer cells' resistance to drugs remains an important problem affecting cancer treatment strategies. We previously studied the nicotinamide phosphoribosyltransferase (NAMPT) inhibitor FK866's resistance mechanisms in the human colorectal cancer HCT116 cells. We established an acquired FK866-resistant cell line, HCT116R^FK866. In this study, we investigated gene mutations in parental HCT116 and HCT116R^FK866 cells using exome sequencing technology. The results indicated cluster genes related to NAD⁺ biosynthesis (including NAMPT), DNA repair, and ATP-binding cassette transporters were differentially altered in these cells. Interestingly, HCT116R^FK866 cells, which are resistant to other class NAMPT inhibitors, were more sensitive to the anticancer 5-fluorouracil and cisplatin and γ-ray irradiation compared to parental HCT116 cells. This higher sensitivity appears to cause a genetic change in the identified gene clusters by resistance to the NAMPT inhibitor FK866. Collectively, these novel findings provide a better understanding of anticancer candidate NAMPT inhibitors with regard to resistance mechanisms and cancer chemotherapy strategies.     

Requirement of Krüppel-like factor 4 in preventing entry into mitosis following DNA damage

Previous studies indicate that Krüppel-like factor 4 (KLF4 or GKLF) controls the G1/S cell cycle checkpoint upon DNA damage. We present evidence for an equally important role of KLF4 in maintaining the integrity of the G2/M checkpoint following DNA damage. HCT116, a colon cancer cell line with wild type p53 alleles, underwent sustained G2 arrest up to 4 days after gamma-irradiation. In contrast, HCT116 cells null for p53 were able to enter mitosis following irradiation. Western blot analyses of irradiated HCT116 cells showed increased levels of p53, KLF4, and p21WAF1/CIP1 and decreased levels of cyclin B1 when compared with unirradiated controls. In contrast, the levels of cyclin B1 increased in irradiated HCT116 p53-/- cells, in which KLF4 failed to increase due to the absence of p53. When KLF4 was inhibited by small interfering RNA, irradiated HCT116 cells exhibited increased mitotic indices and a rise in cyclin B1 levels. Conversely, irradiated HCT116 p53-/- cells that were infected with KLF4-expressing adenoviruses demonstrated a concurrent reduction in mitotic indices and cyclin B1 levels. In each case, Cdc2 kinase measurements showed an inverse correlation between Cdc2 kinase activities and KLF4 levels. Co-transfection experiments showed that KLF4 repressed the cyclin B1 promoter through a specific GC-rich element. Moreover, chromatin immunoprecipitation experiments demonstrated that both KLF4 and HDAC were associated with the cyclin B1 promoter in irradiated HCT116 cells. We conclude that KLF4 is essential in preventing mitotic entry following gamma-irradiation and does so by inhibiting cyclin B1 expression.     

MicroRNA‐425‐5p regulates chemoresistance in colorectal cancer cells via regulation of Programmed Cell Death 10

Acquired chemoresistance represents a major obstacle in cancer treatment, the underlying mechanism of which is complex and not well understood. MiR‐425‐5p has been reported to be implicated tumorigenesis in a few cancer types. However, its role in regulating chemoresistance has not been investigated in colorectal cancer (CRC) cells. Microarray analysis was performed in isogenic chemosensitive and chemoresistant HCT116 cell lines to identify differentially expressed miRNAs. miRNA quantitative real‐time PCR was used to detect miR‐425‐5p expression levels between drug resistant and parental cancer cells. MiR‐425‐5p mimic and inhibitor were transfected, followed by CellTiter‐Glo^® assay to examine drug sensitivity in these two cell lines. Western Blot and luciferase assay were performed to investigate the direct target of miR‐425‐5p. Xenograft mouse models were used to examine in vivo function of miR‐425‐5p. Our data showed that expression of miR‐425‐5p was significantly up‐regulated in HCT116‐R compared with parental HCT116 cells. Inhibition of miR‐425‐5p reversed chemoresistance in HCT116‐R cells. Programmed cell death 10 (PDCD10) is the direct target of miR‐425‐5p which is required for the regulatory role of miR‐425‐5p in chemoresistance. MiR‐425‐5p inhibitor sensitized HCT116‐R xenografts to chemo drugs in vivo. Our study demonstrated that miR‐425‐5p regulates chemoresistance of CRC cells by modulating PDCD10 expression level both in vitro and in vivo. MiR‐425‐5p may represent a new therapeutic target for the intervention of CRC.     

Mauriporin, a Novel Cationic α-Helical Peptide with Selective Cytotoxic Activity Against Prostate Cancer Cell Lines from the Venom of the Scorpion Androctonus mauritanicus

Prostate cancer is the second most common cancer in men and the second leading cause of cancer-related deaths among men in the western world. Finding a cure for prostate cancer is urgently needed. Scorpion venoms are rich sources of biologically active peptides, among which the non-disulfide bridged peptides constitute an important group displaying multifunctional activities. The non-disulfide bridged scorpion venom peptides are rarely identified and poorly characterized so far. In this work, we report the molecular cloning and functional characterization of a novel non-disulfide bridged peptide from the venomous gland cDNA library of the Moroccan scorpion Androctonus mauritanicus. Named Mauriporin, the peptide was found to be composed of 48 residues and circular dichroism analysis revealed the peptide to display a well defined α-helical structure in membrane mimicking environments. A synthetic replicate of Mauriporin was found to exert potent selective cytotoxic and antiproliferative activity against prostate cancer cell lines (IC₅₀ 4.4–7.8 μM) when compared with non-tumorigenic cells. In this concentration range, Mauriporin produced also negligible degrees of hemolytic activities against mammalian erythrocytes. Apoptotic studies displayed that Mauriporin is not causing cell death through an apoptotic-mediated pathway but possibly through a necrotic mode of cell death. In conclusion Mauriporin may offer a novel therapeutic strategy in the treatment of prostate cancer considering its significant cytotoxic potency against prostate cancer cells and low toxicity to non-tumorigenic cells.     

Oxineur,a novel peptide from Caspian cobra Naja naja oxiana against HT-29 colon cancer

Colon cancer ranks fourth in mortality. This cancer is still an important clinical challenge worldwide due to its high prevalence and poor prognosis. Proteomic studies revealed that snake venom is a diverse and variable mixture of enzymatic and non-enzymatic proteins and peptides. Despite the toxic effects of these molecules, several proteins and peptides have been isolated that have practical applications and appear to induce apoptosis and prevent cell metastasis. In this study, we worked on cytotoxic effects and anticancer activity of Naja naja oxiana (Iranian Caspian cobra) snake venom components on HT-29 cell line colon cancer. Separated Fraction-5 by FPLC indicated the high cytotoxicity on HT-29 cell line colon cancer by MTT test. Further isolation of F5 by HPLC showed that the purified peak 2, nominated as Oxineur that contains a cytotoxic effect on HT-29 cells and reduces cell viability at 8 μg/ml to 4% in 24 h. Oxineur has the least cytotoxic effect on HEK-293 normal cells. Further studies on Oxineur peptide confirmed the apoptotic effects with high expression of CASP9 gene and DNA fragmentation in cancerous cells. The partial sequence of Oxineur revealed 71% homology with the neurotoxin II from Naja naja oxiana. Since our target molecule is a peptide in the molecular weight range of 7 kDa, it has potentially a therapeutic value.     

A Cancer Specific Cell-Penetrating Peptide,BR2, for the Efficient Delivery of an scFv into Cancer Cells

Cell-penetrating peptides (CPPs) have proven very effective as intracellular delivery vehicles for various therapeutics. However, there are some concerns about non-specific penetration and cytotoxicity of CPPs for effective cancer treatments. Herein, based on the cell-penetrating motif of an anticancer peptide, buforin IIb, we designed several CPP derivatives with cancer cell specificity. Among the derivatives, a 17-amino acid peptide (BR2) was found to have cancer-specificity without toxicity to normal cells. After specifically targeting cancer cells through interaction with gangliosides, BR2 entered cells via lipid-mediated macropinocytosis. Moreover, BR2 showed higher membrane translocation efficiency than the well-known CPP Tat (49–57). The capability of BR2 as a cancer-specific drug carrier was demonstrated by fusion of BR2 to a single-chain variable fragment (scFv) directed toward a mutated K-ras (G12V). BR2-fused scFv induced a higher degree of apoptosis than Tat-fused scFv in K-ras mutated HCT116 cells. These results suggest that the novel cell-penetrating peptide BR2 has great potential as a useful drug delivery carrier with cancer cell specificity.