A novel bioactive peptide derived from enzymatic hydrolysis of Ruditapes philippinarum: Purification and investigation of its free-radical quenching potential

We purified a novel antioxidant peptide from Ruditapes philippinarum (R. philippinarum) and investigated its free radical scavenging activities. To prepare the peptide, eight proteases were tested for enzymatic hydrolysis. α-chymotrypsin hydrolysate, which showed clearly superior hydroxyl radical scavenging activity (p < 0.05), were further purified using a flow filtration system and consecutive chromatographic methods. Finally, a novel antioxidant peptide was obtained, and the sequence was identified as Ser-Val-Glu-Ile-Gln-Ala-Leu-Cys-Asp-Met. The peptide from R. philippinarum effectively scavenged hydroxyl, DPPH, alkyl and superoxide radicals, with observed IC₅₀ values of 0.042, 0.091, 0.107 and 0.372 mg/ml, respectively. This is the first report of an antioxidant peptide derived from the hydrolysates of R. philippinarum which, further, possesses competitive free radical quenching potential.     

Antitumor effects and cell selectivity of temporin-1CEa, an antimicrobial peptide from the skin secretions of the Chinese brown frog (Rana chensinensis)

Many antimicrobial peptides from amphibian exhibit additional anticancer properties due to a similar mechanism of action at both bacterial and cancer cells. We have previously reported the cDNA sequence of the antimicrobial peptide temporin-1CEa precursor cloned from the Chinese brown frog Rana chensinensis. In this study, we purified, synthesized and structurally characterized temporin-1CEa from the skin secretions of R. chensinensis. The cytotoxicity and cell selectivity of temporin-1CEa were further examined on twelve human carcinoma cell lines and on normal human umbilical vein smooth muscle cells (HUVSMCs). Our results indicated that temporin-1CEa has the amino acid sequence of FVDLKKIANIINSIF-NH₂, and exhibits 50–56% identity with temporin family peptides from other frog species. The CD spectra for temporin-1CEa adopted a well-defined α-helical structure in 50% TFE/water solution. The results of MTT assay showed that temporin-1CEa exhibits cytotoxicity to all tested cancer cell lines in a concentration-dependent manner, being MCF-7 cells the most sensitive. Moreover, temporin-1CEa had lower hemolytic effect to human erythrocytes and had no significant cytotoxicity to normal HUVSMCs at concentrations showed potent antitumor activity. In summary, temporin-1CEa, an amphiphilic α-helical cationic peptide, may represent a novel anticancer agent for breast cancer therapy, considering its cancer cell selectivity and relatively lower cytotoxicity to normal cells.     

Construction and expression of sTRAIL–melittin combining enhanced anticancer activity with antibacterial activity in Escherichia coli

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), as an anticancer protein with tumor-selective apoptotic activity, has been examined for use in clinical application. Melittin, an antibacterial peptide isolated from the bee Apis mellifera, has shown strong cytotoxicity to both tumor and normal cells. To ameliorate the cytotoxicity of melittin on cells and enhance the activity of TRAIL on cancer cells, we constructed a novel fusion protein, sTRAIL–melittin, containing a small ubiquitin-related modifier (SUMO) tag and expressed this fusion protein in Escherichia coli. Data showed that expression of the soluble fusion protein with the SUMO tag was approximately 85 % of total target protein which was much higher than that without the SUMO tag (approximately 10 %); sTRAIL–melittin was easily purified using Ni-NTA affinity chromatography and the tag was removed easily using SUMO-specific protease. To assay anticancer activity and side effects, methyl thiazolyl tetrazolium, hemolytic, and apoptosis assays were employed. Results demonstrated that sTRAIL–melittin had cytotoxic and apoptotic activity in K562 leukemia cells and HepG2 liver carcinoma cells, while it had only a minimal effect on erythrocytes and normal HEK293 cells. This indicates that the cytotoxicity of sTRAIL–melittin in normal cells was low and the anticancer activity of the fusion protein in tumor cells was significantly enhanced compared with sTRAIL (P?<?0.01). Furthermore, we found that sTRAIL–melittin also showed antibacterial activity to Staphylococcus aureus due to the presence of the melittin domain. Therefore, TRAIL fused with an antibacterial peptide may be a promising novel TRAIL-based anticancer treatment strategy.     

Dithiocarbamate prodrugs activated by prostate specific antigen to target prostate cancer

Disulfiram in conjunction with copper has been shown to be a potent anticancer agent. However, disulfiram’s therapeutic potential in prostate cancer is hindered by off-target effects due to its reactive and nucleophilic thiol-containing component, diethyldithiocarbamate (DTC). To minimize undesirable reactivity, we have strategically blocked the thiol moiety in DTC with a cleavable p-aminobenzyl (pAB) group linked to peptide substrates recognized by prostate specific antigen (PSA). Here we report the synthesis and evaluation in cancer cell models of two PSA-activatable prodrugs: HPD (Ac-HSSKLQL-pAB-DTC and RPD (RSSYYSL-pAB-DTC). In vitro exposure to PSA was found to trigger activation of HPD and RPD to release diethyldithiocarbamate, and both prodrugs were found to induce toxicity in prostate cancer cells, with HPD showing the most promising selectivity. With copper supplementation, the IC₅₀ of HPD was 1.4 µM in PSA-expressing LNCaP cells, and 11 µM in PC3 cells that do not express PSA. These studies demonstrate the utility of using peptide recognition handles to direct the activity of dithiocarbamate prodrugs for selective cytotoxicity of cancer cells.     

Identification of Novel Cytotoxic Peptide KENPVLSLVNGMF from Marine Sponge <Emphasis Type="Italic">Xestospongia testudinaria</Emphasis>, with Characterization of Stability in Human Serum

Resistance and side effects are common problems for anticancer drugs used in chemotherapy. Thus, continued research to discover novel and specific anticancer drugs is obligatory. Marine sponges hold great promise as a source of potent cytotoxic peptides with future applications in cancer treatments. This study aimed to purify and identify cytotoxic peptides from the protein hydrolysates of the giant barrel sponge Xestospongia testudinaria, guided by a cytotoxicity assay based on the human cervical cancer cell line (HeLa). Comparison among trypsin, chymotrypsin, papain and alcalase hydrolysates of X. testudinaria revealed papain hydrolysate (PH) to be the most active. PH was purified consecutively by membrane ultrafiltration, gel filtration chromatography, and reversed-phase high performance liquid chromatography (RP-HPLC). Following liquid chromatography-tandem mass spectrometric analysis, two peptides were identified from the most cytotoxic RP-HPLC fraction: KENPVLSLVNGMF and LLATIPKVGVFSILV. Between the two, only the synthetic peptide KENPVLSLVNGMF showed cytotoxicity toward HeLa cells in a dose-dependent manner. KENPVLSLVNGMF (EC₅₀ 0.67 mM) was 3.8-fold more cytotoxic compared with anticancer drug 5-fluorouracil (EC₅₀ 2.56 mM). Furthermore, KENPVLSLVNGMF show only marginal 5% cytotoxicity to Hek293, a non-cancerous, human embryonic kidney cell line, when tested at 0.67 mM. The half-life of the peptide was 3.2?±?0.5 h in human serum in vitro, as revealed by RP-HPLC analyses. These results suggest that KENPVLSLVNGMF identified from X. testudinaria papain hydrolysate has potential applications as peptide lead in future development of potent and specific anticancer drugs.     

Molecular and morphological evidence of hybridization between native Ruditapes philippinarum and the introduced Ruditapes form in Japan

Marine aquaculture and stock enhancement are major causes of the introduction of alien species. A good example of such an introduction is the Japanese shortneck clam Ruditapes philippinarum, one of the most important fishery resources in the world. To meet the domestic shortage of R. philippinarum caused by depleted catches, clams were imported to Japan from China and the Korean peninsula. The imported clam is an alien species that has a very similar morphology, and was misidentified as R. philippinarum (hereafter, Ruditapes form). We genotyped 1,186 clams of R. philippinarum and R. form at four microsatellite loci, sequenced mitochondrial DNA (COI gene fragment) of 485 clams, 34 of which were R. variegatus, and measured morphometric and meristic characters of 754 clams from 12 populations in Japan and China, including the Ariake Sea and Tokyo Bay, where large numbers of R. form were released. Our analyses confirmed that R. form was from the genus Ruditapes, and the genetic differentiation between R. philippinarum and R. form was distinct, but small, compared with five bivalve outgroups. However, R. form had distinct shell morphology, especially larger numbers of radial ribs on the shell surface, suggesting that R. form might be a new Ruditapes species or a variation of R. philippinarum that originated from southern China. A genetic affinity of the sample from the Ariake Sea to R. form was found with the intermediate shell morphology and number of radial ribs, and the hybrid proportion was estimated at 51.3 ± 4.6 % in the Ariake Sea.     

Identification of Prostate-Specific G-Protein Coupled Receptor as a Tumor Antigen Recognized by CD8+ T Cells for Cancer Immunotherapy

Background

Prostate cancer is the most common cancer among elderly men in the US, and immunotherapy has been shown to be a promising strategy to treat patients with metastatic castration-resistant prostate cancer. Efforts to identify novel prostate specific tumor antigens will facilitate the development of effective cancer vaccines against prostate cancer. Prostate-specific G-protein coupled receptor (PSGR) is a novel antigen that has been shown to be specifically over-expressed in human prostate cancer tissues. In this study, we describe the identification of PSGR-derived peptide epitopes recognized by CD8⁺ T cells in an HLA-A2 dependent manner.

Methodology/Principal Findings

Twenty-one PSGR-derived peptides were predicted by an immuno-informatics approach based on the HLA-A2 binding motif. These peptides were examined for their ability to induce peptide-specific T cell responses in peripheral blood mononuclear cells (PBMCs) obtained from either HLA-A2⁺ healthy donors or HLA-A2⁺ prostate cancer patients. The recognition of HLA-A2 positive and PSGR expressing LNCaP cells was also tested. Among the 21 PSGR-derived peptides, three peptides, PSGR3, PSGR4 and PSGR14 frequently induced peptide-specific T cell responses in PBMCs from both healthy donors and prostate cancer patients. Importantly, these peptide-specific T cells recognized and killed LNCaP prostate cancer cells in an HLA class I-restricted manner.

Conclusions/Significance

We have identified three novel HLA-A2-restricted PSGR-derived peptides recognized by CD8⁺ T cells, which, in turn, recognize HLA-A2⁺ and PSGR⁺ tumor cells. The PSGR-derived peptides identified may be used as diagnostic markers as well as immune targets for development of anticancer vaccines.     

Purification and characterization of anti-tubercular and anticancer protein from Staphylococcus hominis strain MANF2: In silico structural and functional insight of peptide

The present context was investigated to purify and characterize anti-tubercular as well as anticancer protein from fermented food associated Staphylococcus hominis strain MANF2. Initially, the anti-tubercular potency of strain MANF2 was assessed against Mycobacterium tuberculosis H37Rv using luciferase reporter phase assay which revealed pronounced relative light unit (RLU) reduction of 92.5 ± 1.2%. The anticancer property of strain MANF2 was demonstrated against lung cancer (A549) and colon cancer (HT-29) cell lines using MTT assay which showed reduced viabilities. Anti-tubercular activities of the purified protein were observed to be increased significantly (P < 0.05) ranging from 34.6 ± 0.3 to 71.4 ± 0.4% of RLU reduction. Likewise, the purified protein showed significantly (P < 0.05) reduced viabilities of A549 and HT-29 cancer cells with IC₅₀ values of 46.6 and 48.9 µg/mL, respectively. The nominal mass of the purified protein was found to be 7712.3 Da as obtained from MALDI-TOF MS/MS spectrum. The protein showed the sequence homology with 1–336 amino acids of Glyceraldehyde-3-phosphate dehydrogenase from Staphylococcus sp., thus, categorizing as a new class of Glyceraldehyde-3-phosphate dehydrogenase-like protein. The amino acid sequence of the most abundant peptide (m/z = 1922.12) in the purified protein was obtained as ‘KAIGLVIPEIDGKLDGGAQRV’ and it was identified as peptide NMANF2. In silico tools predicted significant stereo-chemical, physiochemical, and functional characteristics of peptide NMANF2. In a nutshell, protein purified from strain MANF2 can certainly be used as an ideal therapeutic agent against tuberculosis and cancer (lung and colon).     

A single replacement of histidine to arginine in EGFR-lytic hybrid peptide demonstrates the improved anticancer activity

We previously reported that novel targeted “hybrid peptide” in which epidermal growth factor receptor (EGFR) binding peptide was conjugated with lytic-type peptide had selective cytotoxic activity to EGFR expressing cancer cell lines, and in vivo analysis revealed that this EGFR-lytic peptide displayed significant antitumor activity in a xenograft model of human breast cancer which was resistant to tyrosine kinase inhibitor drugs. As an attempt to improve the selective anticancer activity of EGFR-lytic peptide, we modified the EGFR-binding peptide through introducing the mutation of amino acid according to biophysical analysis by biomolecular interaction and circular dichroism (CD) spectra. When cytotoxic activity of EGFR-lytic or EGFR(2R)-lytic hybrid peptides was investigated in various human cancer and normal cell lines, it was demonstrated that EGFR(2R)-lytic, in which second histidine (H) of EGFR-binding peptide was replaced to arginine (R) had 1.2–1.9-fold higher cytotoxic activity than that of original EGFR-lytic peptide. In vivo analysis also revealed that this modified peptide displayed significant antitumor activity at as low as 1 mg/kg dosage. These results suggest that mutated arginine on EGFR-lytic peptide produces higher binding ability to EGFR on cancer cells, and thereby the improved anticancer activity.     

Identification and structural characterization of a new pro-apoptotic cyclic octapeptide cyclosaplin from somatic seedlings of Santalum album L.

Small cyclic peptides exhibiting potent biological activity have great potential for anticancer therapy. An antiproliferative cyclic octapeptide, cyclosaplin was purified from somatic seedlings of Santalum album L. (sandalwood) using gel filtration and RP-HPLC separation process. The molecular mass of purified peptide was found to be 858 Da and the sequence was determined by MALDI-ToF-PSD-MS as ‘RLGDGCTR’ (cyclic). The cytotoxic activity of the peptide was tested against human breast cancer (MDA-MB-231) cell line in a dose and time-dependent manner. The purified peptide exhibited significant antiproliferative activity with an IC₅₀ 2.06 μg/mL. In a mechanistic approach, apoptosis was observed in differential microscopic studies for peptide treated MDA-MB-231 cells, which was further confirmed by mitochondrial membrane potential, DNA fragmentation assay, cell cycle analysis and caspase 3 activities. The modeling and docking experiments revealed strong affinity (kcal/mol) of peptide toward EGFR and procaspase 3. The co-localization studies revealed that the peptide sensitizes MDA-MB-231 cells by possibly binding to EGFR and induces apoptosis. This unique cyclic octapeptide revealed to be a favorable candidate for development of anticancer agents.     

A recombinant trans-membrane protein hMnSOD–R9 inhibits the proliferation of cervical cancer cells in vitro

Human manganese superoxide dismutase (hMnSOD) is a new type of cancer suppressor. Nonamer of arginine (R9) is an efficient protein transduction domain (PTD). The aim of the study was to improve the transduction efficiency of hMnSOD and investigate its activity in vitro. In this study, we designed, constructed, expressed, and purified a novel fusion protein containing the hMnSOD domain and R9 PTD (hMnSOD–R9). The DNA damaged by Fenton’s reagent was found to be significantly reduced when treated with hMnSOD–R9. hMnSOD–R9 fusion protein was successfully delivered into HeLa cells. The MTT assay showed that proliferation of various cancer cell lines were inhibited by hMnSOD–R9 in a dose-dependent manner. In addition, the cell cycle of HeLa cells was arrested at the sub-G0 phase by hMnSOD–R9. hMnSOD–R9 induced apoptosis of HeLa cells in a dose-dependent manner. With hMnSOD–R9 treatment, Bax, JNK, TBK1 gene expression was increased and STAT3 gene expression was gradually down-regulated in HeLa cells. We also found that apoptosis was induced by hMnSOD–R9 in HeLa cells via up-regulation of cleaved caspase-3 and down-regulation phospho-STAT3 pathway. These results indicated that hMnSOD–R9 may provide benefits to cervical cancer treatment.     

Cationic Antimicrobial Peptides Derived from <Emphasis Type="Italic">Crocodylus siamensis</Emphasis> Leukocyte Extract,Revealing Anticancer Activity and Apoptotic Induction on Human Cervical Cancer Cells

Known antimicrobial peptides KT2 and RT2 as well as the novel RP9 derived from the leukocyte extract of the freshwater crocodile (Crocodylus siamensis) were used to evaluate the ability in killing human cervical cancer cells. RP9 in the extract was purified by a combination of anion exchange column and reversed-phase HPLC, and its sequence was analyzed by mass spectrometry. The novel peptide could inhibit Gram-negative Vibrio cholerae (clinical isolation) and Gram-positive Bacillus pumilus TISTR 905, and its MIC values were 61.2 µM. From scanning electron microscopy, the peptide was seen to affect bacterial surfaces directly. KT2 and RT2, which are designed antimicrobial peptides using the C. siamensis Leucrocin I template, as well as RP9 were chemically synthesized for investigation of anticancer activity. By Sulforhodamine B colorimetric assay, these antimicrobial peptides could inhibit both HeLa and CaSki cancer cell lines. The IC₅₀ values of KT2 and RT2 for HeLa and CaSki cells showed 28.7–53.4 and 17.3–30.8 µM, while those of RP9 were 126.2 and 168.3 µM, respectively. Additionally, the best candidate peptides KT2 and RT2 were used to determine the apoptotic induction on cancer cells by human apoptosis array assay. As a result, KT2 and RT2 were observed to induce apoptotic cell death in HeLa cells. Therefore, these results indicate that KT2 and RT2 with antimicrobial activity have a highly potent ability to kill human cervical cancer cells.     

Some population parameters of Ruditapes philippinarum (Bivalvia,Veneridae) on the southern coast of the Marmara Sea,Turkey

Ruditapes philippinarum, a venerid clam, is a dominant species in the sandy and muddy areas in the coastal waters of the Marmara Sea. Intensive commercial harvesting of this species is conducted in these regions. We studied the population dynamics of R. philippinarum on the southern coast of the Marmara Sea (Band?rma). Samples were collected on a monthly basis between September 2012 and August 2013. Seasonal von Bertalanffy growth parameters using the length–frequency distribution of R. philippinarum were estimated at L _∞ = 67.50 mm and K = 0.33 year^?1, and the seasonal oscillation in growth rate was 0.53. The slowest growth period was in January. The growth performance index and potential lifespan were 3.182 and 8.06 years, respectively. The growth relationship was confirmed to have a positive allometric pattern. The average total mortality rate was estimated to be 0.777 year^?1, whereas the natural and fishing mortality rates were 0.539 and 0.238 year^?1, respectively. The current exploitation rate of R. philippinarum was 0.306. The recruitment pattern peaked during June–August, and spawning occurred between May and August. The results of this study provide valuable information on the status of R. philippinarum stocks.     

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.     

Stevioside mediated chemosensitization studies and cytotoxicity assay on breast cancer cell lines MDA-MB-231 and SKBR3

Cancer is one of the most impacting life-threatening disease for the human populace. Hence, over the years we have seen a consistent interest to study and investigate new treatments to cure and prevent this disease. Medicinal plants have played a progressive part in treatment since many years. In this research study, we have explored the cytotoxicity effect of purified bioactive compound isolated from Stevia rebaudiana leaves and the key mechanism responsible for apoptosis in human breast cancer cells. The anticancer properties of Stevia rebaudiana leaves has been suggested in earlier literature. Hence, the aim of this study was to investigate the cytotoxicity of purified stevioside in human breast cancer cell lines MDA-MB-231 and SKBR3. Results showed that purified stevioside inhibited the growth of cancerous cell lines. The IC50 obtained after treatment with stevioside on cancer cells MDA-MB-231 and SKBR3 are 55 µM and 66 µM respectively. This shows purified stevioside is capable of inducing apoptosis indicating its promising anticancer activity. However, so far chemosensitization effects of stevioside on breast cancer have not been fully explained by other studies. Hence, additionally, this study also evaluates the chemosensitization potential of stevioside in combination with 5-FU. This research study shows the importance of Stevia rebaudiana as a good source of bioactive compounds with high anti-cancer property.     

Scalable Production of Recombinant Membrane Active Peptides and Its Potential as a Complementary Adjunct to Conventional Chemotherapeutics

The production of short anticancer peptides in recombinant form is an alternative method for costly chemical manufacturing. However, the limitations of host toxicity, bioactivity and column purification have impaired production in mass quantities. In this study, short cationic peptides were produced in aggregated inclusion bodies by double fusion with a central protein that has anti-cancer activity. The anticancer peptides Tachiplicin I (TACH) and Latarcin 1 (LATA) were fused with the N- and C-terminus of the MAP30 protein, respectively. We successfully produced the recombinant TACH-MAP30-LATA protein and MAP30 alone in E. coli that represented 59% and 68% of the inclusion bodies. The purified form of the inclusion bodies was prepared by eliminating host cell proteins through multiple washing steps and semi-solubilization in alkaline buffer. The purified active protein was recovered by inclusive solubilization at pH 12.5 in the presence of 2 M urea and refolded in alkaline buffer containing oxides and reduced glutathione. The peptide-fusion protein showed lower CC₅₀ values against cancer cells (HepG2, 0.35±0.1 μM and MCF-7, 0.58±0.1 μM) compared with normal cells (WRL68, 1.83±0.2 μM and ARPE19, 2.5±0.1 μM) with outstanding activity compared with its individual components. The presence of the short peptides facilitated the entry of the peptide fusion protein into cancer cells (1.8 to 2.2-fold) compared with MAP30 alone through direct interaction with the cell membrane. The cancer chemotherapy agent doxorubicin showed higher efficiency and selectivity against cancer cells in combination with the peptide- fusion protein. This study provides new data on the mass production of short anticancer peptides as inclusion bodies in E. coli by fusion with a central protein that has similar activity. The product was biologically active against cancer cells compared with normal cells and enhanced the activity and selective delivery of an anticancer chemotherapy agent.     

Heavy Metal Concentrations in Surface Sediments and Manila Clams (Ruditapes philippinarum) from the Dalian Coast, China after the Dalian Port Oil Spill

We conducted an investigation of heavy metal concentrations in Manila clams (Ruditapes philippinarum) and surface sediments after the Dalian Port oil spill. Samples were collected from three mariculture zones (Jinshitan, Dalijia, and Pikou) along the Dalian coast. Heavy metal concentrations in R. philippinarum were consistent and ranked in decreasing order of Zn > Cu > As > Cr > Pb > Cd > Hg, while concentrations in surface sediments were ranked as Zn > Cr > Cu > Pb > As > Cd > Hg, respectively. Bioaccumulation of Zn, Cd, and Hg had obviously occurred in R. philippinarum. Statistically significant correlations (p?<?0.05) between concentrations of Pb, Cd, and Hg in R. philippinarum and in surface sediments were observed. Except for Cr and As, heavy metal concentrations in R. philippinarum were well within the legal limits for human consumption.     

A cell-permeable dominant-negative survivin protein induces apoptosis and sensitizes prostate cancer cells to TNF-α therapy

Background

Survivin is a member of the inhibitor-of-apoptosis (IAP) family which is widely expressed by many different cancers. Overexpression of survivin is associated with drug resistance in cancer cells, and reduced patient survival after chemotherapy and radiotherapy. Agents that antagonize the function of survivin hold promise for treating many forms of cancer. The purpose of this study was to investigate whether a cell-permeable dominant-negative survivin protein would demonstrate bioactivity against prostate and cervical cancer cells grown in three dimensional culture.

Results

A dominant-negative survivin (C84A) protein fused to the cell penetrating peptide poly-arginine (R9) was expressed in E. coli and purified by affinity chromatography. Western blot analysis revealed that dNSurR9-C84A penetrated into 3D-cultured HeLa and DU145 cancer cells, and a cell viability assay revealed it induced cancer cell death. It increased the activities of caspase-9 and caspase-3, and rendered DU145 cells sensitive to TNF-α via by a mechanism involving activation of caspase-8.

Conclusions

The results demonstrate that antagonism of survivin function triggers the apoptosis of prostate and cervical cancer cells grown in 3D culture. It renders cancer cells sensitive to the proapoptotic affects of TNF-α, suggesting that survivin blocks the extrinsic pathway of apoptosis. Combination of the biologically active dNSurR9-C84A protein or other survivin antagonists with TNF-α therapy warrants consideration as an approach to cancer therapy.     

Investigating the antiangiogenic potential of Rumex vesicarius (humeidh), anticancer activity in cancer cell lines and assessment of developmental toxicity in zebrafish embryos

Recent trends in anticancer therapy is to use therapeutic agents which not only kill the cancer cell, but are less toxic to surrounding normal cells/tissue. One approach is to cut the nutrient supply to growing tumor cells, by blocking the formation of new blood vessels around the tumor. As the phytochemicals and botanical crude extracts have proven their efficacy as natural antiangiogenic agents with minimum toxicities, there is need to explore varieties of medicinal plants for novel antiangiogenic compounds.Rumex vesicarius L. (Humeidh), is an annual herbal plant with proven medicinal values. The antiangiogenic potential, and developmental toxicity of humeidh in experimental animal models has never been studied before. The crude extracts were prepared from the roots, stems, leaves and flowers of Rumex vesicarius L. in methanol, chloroform, ethyl acetate and n-hexane. The developmental toxicity screening in zebrafish embryos, has revealed that Rumex vesicarius was not toxic to zebrafish embryos. The chloroform stem extract showed significant level of antiangiogenic activity in zebrafish angiogenic assay on a dose dependent manner. Thirty five (35) bioactive compounds were identified by gas chromatography mass spectrophotometry (GC–MS) analysis in the stem extract of Rumex vesicarius. Propanoic acid, 2-[(trimethylsilyl)oxy]-, trimethylsilyl ester, Butane, 1,2,3-tris(trimethylsiloxy), and Butanedioic acid, bis(trimethylsilyl) ester were identified as major compound present in the stem of R. vasicarius.The anticancer activity of roots, stem, leaves and flowers crude extract was evaluated in human breast cancer (MCF7), human colon carcinoma (Lovo, and Caco-2), human hepatocellular carcinoma (HepG2) cell lines. Most of the crude extracts did not show significant level of cytotoxicity in tested cancer cells line, except, chloroform extract of stem which exhibited strong anticancer activity in all tested cancer cells with IC₅₀ values in micro molar range.Based on these results, it is recommended that formulation prepared from R. vesicarius can further be tested in clinical trials in order to explore its therapeutic potential as an effective and safe natural anticancer product.