Semaphorin 3A lytic hybrid peptide binding to neuropilin-1 as a novel anti-cancer agent in pancreatic cancer

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 cells. In this study, we have generated a novel type hybrid peptide, semaphorin 3A lytic (Sema3A-lytic), which is composed of two functional amino acid domains: a sequence derived from Sema3A that binds to neuropilin-1 (NRP1) and a cytotoxic lytic peptide. We found that this hybrid peptide had cytotoxic activity against NRP1-positive pancreatic cancer cell lines such as BxPC-3 and Panc-1, whereas the peptide did not affect the viability of normal cells in vitro. It was also found by affinity analysis that Sema3A peptide binds to NRP1, and two arginines (372R and 377R) in Sema3A peptide are involved in the interaction with NRP1 protein. In addition, confocal microscopy analysis revealed that Sema3A-lytic peptide could not penetrate normal cells regardless of the presence of NRP1 mRNA, suggesting that the ability of Sema3A-lytic peptide to concentrate adjacent to the cell membrane by binding to NRP1 with the target-binding moiety contributes to its selective cytotoxic activity. These results indicate that Sema3A-lytic hybrid peptide would be a possible anti-cancer agent for treatment of human pancreatic cancer.     

Characterization of antilytic peptide antibody: application for the detection of lytic‐based hybrid peptide in serum samples

We previously reported that a novel targeted drug termed hybrid epidermal growth factor receptor (EGFR)‐lytic peptide, made by chemical conjugation of targeted binding peptide and cell‐killing, lytic‐peptide components, has selective cytotoxic activity that allows it to discriminate between normal and cancer cells. In addition, in vivo analysis revealed that this hybrid peptide displays significant antitumor activity in a xenograft model of human breast and pancreatic cancer in mice. Here, we characterized antilytic peptide antibody, which was raised from rabbit serum using the antigen of lytic peptide conjugated with keyhole limpet hemocyanin. It was found that antilytic peptide antibody is specific to the lytic peptide as assessed by both ELISA and surface plasmon resonance analysis and can also bind to EGFR‐lytic peptide. Epitope mapping analysis using Biacore showed that two successive lysine regions in the lytic‐peptide sequence are significant for recognition by this antibody. In addition, it was shown that this antibody can detect lytic‐based hybrid peptide in serum samples from mouse blood and also in cultured breast cancer MDA‐MB‐231 cell samples by immunocytochemical staining experiments. It was found that the maximum concentrations of this peptide in serum were reached within 15–30 min of i.v. administration of EGFR‐lytic peptide to mice. These results indicate that this antibody will be a useful tool for the detection of lytic‐based peptides to investigate their in vivo stability and pharmacokinetics. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.     

Synthesis and biological evaluation of irreversible EGFR tyrosine kinase inhibitors containing pyrido[3,4-d]pyrimidine scaffold

In the present study, a new class of compounds containing pyrido[3,4-d]pyrimidine scaffold with an acrylamide moiety was designed as irreversible EGFR-TKIs to overcome acquired EGFR-T790M resistance. The most promising compound 25h inhibited HCC827 and H1975 cells growth with the IC₅₀ values of 0.025?μM and 0.49?μM, respectively. Meanwhile, 25h displayed potent inhibitory activity against the EGFR^L858R (IC₅₀?=?1.7?nM) and EGFR^L858R/T790M (IC₅₀?=?23.3?nM). 25h could suppress EGFR phosphorylation in HCC827 and H1975 cell lines and significantly induce the apoptosis of HCC827 cells. Additionally, compound 25h could remarkably inhibit cancer growth in established HCC827 xenograft mouse model at 50?mg/kg in vivo. These results indicated that the 2,4-disubstituted 6-(5-substituted pyridin-2-amino)pyrido[3,4-d]pyrimidine derivatives can serve as effective EGFR inhibitors and potent anticancer agents.     

Design,synthesis, molecular modeling and biological evaluation of novel Benzoxazole-Benzamide conjugates via a 2-Thioacetamido linker as potential anti-proliferative agents,VEGFR-2 inhibitors and apoptotic inducers

A novel series of 2-thioacetamide linked benzoxazole-benzamide conjugates 1–15 was designed as potential inhibitors of the vascular endothelial growth factor receptor-2 (VEGFR-2). The prepared compounds were evaluated for their potential antitumor activity and their corresponding selective cytotoxicity was estimated using normal human fibroblast (WI-38) cells. Compounds 1, 9–12 and 15 showed good selectivity and displayed excellent cytotoxic activity against both HCT-116 and MCF-7 cancer cell lines compared to sorafenib, used as a reference compound. Furthermore, compounds 1 and 11 showed potent VEGFR-2 inhibitory activity. The cell cycle progression assay showed that 1 and 11 induced cell cycle arrest at G2/M phase, with a concomitant increase in the pre-G1 cell population. Further pharmacological studies showed that 1 and 11 induced apoptosis and inhibited the expression of the anti-apoptotic Bcl-2 and Bcl-xL proteins in both cell lines. Therefore, compounds 1 and 11 might serve as promising candidates for future anticancer therapy development.     

Attachment of a Frog Skin-Derived Peptide to Functionalized Cerium Oxide Nanoparticles

Peptide Brevinin-2R (B2R) has derived from frog skin secretions and possesses cytotoxic effects on cancer cells. Beside, cerium oxide nanoparticle (CNP) has antioxidant properties and could be used in anticancer studies. The purpose of this study is to investigate antioxidant and cytotoxicity of cerium oxide nanoparticle conjugated with B2R. First, cerium oxide nanoparticles were amine functionalized and peptide attached to it through establishment of peptide bond. Then, (1,1-diphenyl-2-picrylhydrazyl) DPPH and 2, 2′-azinobis (3-ethylbenzothiazoline-6-sulphonic acid) diammonium salt (ABTS) radical scavenging activities of CNP-B2R were determined. MTT assay were used in order to compare cytotoxicity of CNP- B2R on two different cell lines. HFLF-pI5 cell and A549 cell lines were selected as representative of normal and cancer cells, respectively. Also, the cytotoxic effects of CNP, B2R and CNP-B2R were investigated on A549 cell line. Results of antioxidant evaluations showed that the antioxidant activity of CNP-Peptide increased at higher concentration of CNP-B2R with IC₅₀ of 0.2 and 0.54 (mg/mL) for DPPH and ABTS assays, respectively. The results of cytotoxicity effects showed that CNP-B2R was more potent in killing tumor cell line in comparison with normal cell line. Cytotoxicity of CNP, B2R and CNP-B2R demonstrated that CNP-B2R and B2R had the lower cell viability effects compared to CNP. Our findings showed cytotoxicity of CNP-B2R against cancer cell lines in comparison with normal cells indicating the potential anticancer properties of CNP-B2R.     

Antitumor effects of snake venom chemically modified Lys49 phospholipase A2-like BthTX-I and a synthetic peptide derived from its C-terminal region

The present work evaluates both in vitro and in vivo antitumor activity of BPB-modified BthTX-I and its cationic synthetic peptide derived from the 115–129 C-terminal region. BPB-BthTX-I presented cytotoxicity of 10–40% on different tumor cell lines, which were also susceptible to the lytic action of the synthetic peptide. Injection of the modified protein or the peptide in mice, 5 days after transplantation of S180 tumor cells, reduced 30 and 36% of the tumor size on day 14th and 76 and 79% on day 60th, respectively, when compared to the untreated control group. Thus, these antitumor properties might be of interest in the development of therapeutic strategies against cancer.     

Diverse amide analogs of sulindac for cancer treatment and prevention

Sulindac is a non-steroidal anti-inflammatory drug (NSAID) that has shown significant anticancer activity. Sulindac sulfide amide (1) possessing greatly reduced COX-related inhibition relative to sulindac displayed in vivo antitumor activity that was comparable to sulindac in a human colon tumor xenograft model. Inspired by these observations, a panel of diverse sulindac amide derivatives have been synthesized and their activity probed against three cancer cell lines (prostate, colon and breast). A neutral analog, compound 79 was identified with comparable potency relative to lead 1 and activity against a panel of lymphoblastic leukemia cell lines. Several new series also show good activity relative to the parent (1), including five analogs that also possess nanomolar inhibitory potencies against acute lymphoblastic leukemia cells. Several new analogs identified may serve as anticancer lead candidates for further development.     

Synthesis and cytotoxicity evaluation of novel indolylpyrimidines and indolylpyrazines as potential antitumor agents

Novel indolylpyrimidines and indolylpyrazines have been synthesized as potential antitumor agents. They were screened in a panel of 60 human tumor cell lines in vitro. Compounds 7, 9, 10, 15, 21 exhibited efficiently cytotoxic activities with GI(50) values in the low micromolar range against a variety of human cancer cell lines. 2,4-Bis(3'-indolyl)pyrimidine 8 displayed selective cytotoxic activity against IGROV1 tumor cell line with the GI(50) value below 0.01 microM.     

Evaluation of the Antitumor Activity of Dacomitinib in Models of Human Bladder Cancer

Members of the human epidermal growth factor receptor (HER) family play a significant role in bladder cancer progression and may underlie the development of chemotherapy resistance. Dacomitinib is an irreversible tyrosine kinase inhibitor with structural specificity for the catalytic domains of epidermal growth factor receptor (EGFR), HER2 and HER4 that has exhibited vigorous efficacy against other solid tumors. We evaluated the antitumor activity of dacomitinib in human bladder cancer cell lines expressing varying levels of HER family receptors. These cell lines also were established as bladder cancer xenografts in nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice to assess dacomitinib activity in vivo. Significant cytotoxic and cytostatic effects were noted in cells expressing elevated levels of the dacomitinib target receptors with apoptosis and cell cycle arrest being the predominant mechanisms of antitumor activity. Cells expressing lower levels of HER receptors were much less sensitive to dacomitinib. Interestingly, dacomitinib was more active than either trastuzumab or cetuximab in vitro, and exhibited increased growth inhibition of bladder tumor xenografts compared with lapatinib. Pharmacodynamic effects of dacomitinib included decreased E-cadherin (E-cad) expression, reduction of EGFR and extracellular signal-regulated kinase (ERK) phosphorylation and reduced mitotic count. Dacomitinib also inhibited tumor growth in a chemotherapy-resistant xenograft and, when combined with chemotherapy in a sensitive xenograft, exhibited superior antitumor effects compared with individual treatments. Evaluation in xenograft-bearing mice revealed that this combination was broadly feasible and well tolerated. In conclusion, dacomitinib exhibited pronounced activity both as a single agent and when combined with chemotherapy in human bladder cancer models. Further investigation of dacomitinib in the preclinical and clinical trial settings is being pursued.     

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.     

Targeted delivery of insulin-modified immunoliposomes in vivo

The purpose of this study was to investigate the effect of liposomes conjugated with insulin to the surface on circulation time, biodistribution, and antitumor activity after intravenous injection in tumor-bearing mice. Immunoliposomes were constructed with insulin, which was covalently linked to liposomes containing anticancer drugs. In order to investigate the targeting performance of insulin-modified immunoliposomes (SILs) in vivo, plasma pharmacokinetics, biodistribution, and antitumor activity were tested. In comparison with nontargeted liposomes (SLs), SILs were cleared faster from circulation as a result of greater liver and tumor uptake. In addition, SILs retarded the growth of the tumor effectively, compared with the ZTO injection or SL. This is the first time for selective in vivo targeting of tumor vessels using insulin-modified immunoliposomes. SILs are candidate drug-delivery systems for therapeutic anticancer approaches.     

Inhibition of human breast cancer Matrigel invasion by Streptolysin O activation of the EGF receptor ErbB1

Streptolysin O (SLO) is a protein cytotoxin derived from Group A beta-hemolytic streptococci that associates with membranes and permeabilizes cells. Oxidation inactivates SLO, eliminating the characteristic hemolytic and cytotoxic activities. However, oxidized SLO produces beneficial therapeutic effects in vivo on scleroderma, scar formation and wound healing. Here we report that oxidized SLO also significantly inhibited invasion by human metastatic breast cancer MDA-MB-231 cells through Matrigel in an in vitro model of metastatic disease. This dose-dependent response corresponded to selective SLO activation of epidermal growth factor receptor (EGFR) ErbB1. SLO and EGF were equally selective in activation of EGFR, but EGF elicited larger relative increases in phosphorylation at various sites, especially pronounced for Tyr845. Addition of SLO did not affect either ERK1/2 or Akt kinases and altered the expression of only 10 of 84 metastasis-related genes in MDA-MB-231 cells. Neither SLO nor EGF promoted growth of several human breast cancer cell lines. Knockdown of EGFR by siRNA ablated the inhibitory effect of SLO on cancer cell invasion, showing SLO selectively activated ErbB1 kinase to reduce invasion without increasing cell growth. The results suggest SLO might have promise as a new therapy to inhibit metastasis.     

以胰岛素样生长因子受体1基因为靶的反义寡核苷酸体内外抗肿瘤研究

以胰岛素样生长因子受体－1基因为靶筛选抗肿瘤药物.根据IGF1RmRNA的二级结构设计了9条反义寡核苷酸药物,以脂质体介导进行转染,MTT染色计算细胞生长抑制率,从中筛选出一条序列并对之进行优化,最后以最佳序列进行体外作用持续时间及体内细胞生长抑制率分析.结果表明该序列在体内外具有良好的抗肿瘤活性,具有剂量依赖性关系,且对荷瘤裸鼠无明显的毒性.IGF1R可作为肿瘤治疗的新靶点.     

6-Oxooxazolidine–quinazolines as noncovalent inhibitors with the potential to target mutant forms of EGFR

Despite the remarkable benefits of gefitinib, the clinical efficacy is eventually diminished due to the acquired point mutations in the EGFR (T790M). To address this unmet medical need, we demonstrated a strategy to prepare a hybrid analogue consisting of the oxooxazolidine ring and the quinazoline scaffold and provided alternative noncovalent inhibitors targeting mutant forms of EGFR. Most of the derivatives displayed moderate to good anti-proliferative activity against gefitinib-resistant NCI-H1975. Some of them exhibited potent EGFR kinase inhibitory activities, especially on EGFR^T790M and EGFR^L858R kinases. SAR studies led to the identification of a hit 9a that can target both of the most common EGFR mutants: L858R and T790M. Also, 9a displayed weaker inhibitory against cancer cell lines with low level of EGFR expression and good chemical stability under different pH conditions. The work presented herein showed the potential for developing noncovalent inhibitors targeting EGFR mutants.     

Studies on anticancer activities of antimicrobial peptides

In spite of great advances in cancer therapy, there is considerable current interest in developing anticancer agents with a new mode of action because of the development of resistance by cancer cells towards current anticancer drugs. A growing number of studies have shown that some of the cationic antimicrobial peptides (AMPs), which are toxic to bacteria but not to normal mammalian cells, exhibit a broad spectrum of cytotoxic activity against cancer cells. Such studies have considerably enhanced the significance of AMPs, both synthetic and from natural sources, which have been of importance both for an increased understanding of the immune system and for their potential as clinical antibiotics. The electrostatic attraction between the negatively charged components of bacterial and cancer cells and the positively charged AMPs is believed to play a major role in the strong binding and selective disruption of bacterial and cancer cell membranes, respectively. However, it is unclear why some host defense peptides are able to kill cancer cells when others do not. In addition, it is not clear whether the molecular mechanism(s) underlying the antibacterial and anticancer activities of AMPs are the same or different. In this article, we review various studies on different AMPs that exhibit cytotoxic activity against cancer cells. The suitability of cancer cell-targeting AMPs as cancer therapeutics is also discussed.     

Analysis of Quinolinequinone Analogs with Promising Cytotoxic Activity against Breast Cancer

It is quite challenging to find out bioactive molecules in the vast chemical universe. Quinone moiety is a unique structure with a variety of biological properties, particularly in the treatment of cancer. In an effort to develop potent and secure antiproliferative lead compounds, five quinolinequinones ( AQQ1-5 ) described previously have been selected and submitted to the National Cancer Institute (NCI) of Bethesda to envisage their antiproliferative profile based on the NCI Developmental Therapeutics Program. According to the preliminary in vitro single-dose anticancer screening, four of five quinolinequinones ( AQQ2-5 ) were selected for five-dose screening and they displayed promising antiproliferative effects against several cancer types. All AQQs showed a excellent anticancer profile with low micromolar GI₅₀ and TGI values against all leukemia cell lines, some non-small cell lung and ovarian cancer, most colon, melanoma, and renal cancer, and in addition to some breast cancer cell lines. AQQ2-5 reduced the proliferation of all leukemia cell lines at a single dose and five additional doses, as well as some non-small cell lung and ovarian cancer, the majority of colon cancer, melanoma and renal cancer, and some breast cancer cell lines. This motivated us to use in vitro, in silico, and in vivo technologies to further investigate their mode of action. We investigated the in vitro cytotoxic activities of the most promising compounds, AQQ2 and AQQ3 , in HCT-116 colon cancer, MCF7 and T-47D breast cancer, and DU-145 prostate cancer cell lines, and HaCaT human keratinocytes. Concomitantly, IC₅₀ values of AQQ2 and AAQ3 against MCF7 and T-47D cell lines of breast cancer, DU-145 cell lines of prostate cancer, HCT-116 cell lines of colon cancer, and HaCaT human keratinocytes were determined. AQQ2 exhibited anticancer activity through the induction of apoptosis and caused alterations in the cell cycle. In silico pharmacokinetic studies of all analogs have been carried out against ATR, CHK1, WEE1, CDK1, and CDK2. In addition to this, in vitro ADME and in vivo pharmacokinetic profiling for the most effective AAQ ( AAQ2 ) have been studied.     

Biological effects of a de novo designed myxoma virus peptide analogue: evaluation of cytotoxicity on tumor cells

Background

The Resonant Recognition Model (RRM) is a physico-mathematical model that interprets protein sequence linear information using digital signal processing methods. In this study the RRM concept was employed for structure-function analysis of myxoma virus (MV) proteins and the design of a short bioactive therapeutic peptide with MV-like antitumor/cytotoxic activity.

Methodology/Principal Findings

The analogue RRM-MV was designed by RRM as a linear 18 aa 2.3 kDa peptide. The biological activity of this computationally designed peptide analogue against cancer and normal cell lines was investigated. The cellular cytotoxicity effects were confirmed by confocal immunofluorescence microscopy, by measuring the levels of cytoplasmic lactate dehydrogenase (LDH) and by Prestoblue cell viability assay for up to 72 hours in peptide treated and non-treated cell cultures. Our results revealed that RRM-MV induced a significant dose and time-dependent cytotoxic effect on murine and human cancer cell lines. Yet, when normal murine cell lines were similarly treated with RRM-MV, no cytotoxic effects were observed. Furthermore, the non-bioactive RRM designed peptide RRM-C produced negligible cytotoxic effects on these cancer and normal cell lines when used at similar concentrations. The presence/absence of phosphorylated Akt activity in B16F0 mouse melanoma cells was assessed to indicate the possible apoptosis signalling pathway that could be affected by the peptide treatment. So far, Akt activity did not seem to be significantly affected by RRM-MV as is the case for the original viral protein.

Conclusions/Significance

Our findings indicate the successful application of the RRM concept to design a bioactive peptide analogue (RRM-MV) with cytotoxic effects on tumor cells only. This 2.345 kDa peptide analogue to a 49 kDa viral protein may be suitable to be developed as a potential cancer therapeutic. These results also open a new direction to the rational design of therapeutic agents for future cancer treatment.     

Design of a Tumor Homing Cell-Penetrating Peptide for Drug Delivery

The major drawbacks with conventional cancer chemotherapy are the lack of satisfactory specificity towards tumor cells and poor antitumor activity. In order to improve these characteristics, chemotherapeutic drugs can be conjugated to targeting moieties e.g. to peptides with the ability to recognize cancer cells. We have previously reported that combining a tumor homing peptide with a cell-penetrating peptide yields a chimeric peptide with tumor cell specificity that can carry cargo molecules inside the cells. In the present study, we have used a linear breast tumor homing peptide, CREKA, in conjunction with a cell-penetrating peptide, pVEC. This new chimeric peptide, CREKA–pVEC, is more convenient to synthesize and moreover it is better in translocating cargo molecules inside cancer cells as compared to previously published PEGA–pVEC peptide. This study demonstrates that CREKA–pVEC is a suitable vehicle for targeted intracellular delivery of a DNA alkylating agent, chlorambucil, as the chlorambucil–peptide conjugate was substantially better at killing cancer cells in vitro than the anticancer drug alone.     

Synthesis of some quinazolinones inspired from the natural alkaloid L-norephedrine as EGFR inhibitors and radiosensitizers

A set of quinazolinones synthesized by the aid of L-norephedrine was assembled to generate novel analogues as potential anticancer and radiosensitizing agents. The new compounds were evaluated for their cytotoxic activity against MDA-MB-231, MCF-7, HepG-2, HCT-116 cancer cell lines and EGFR inhibitory activity. The most active compounds 5 and 6 were screened against MCF-10A normal cell line and displayed lower toxic effects. They proved their relative safety with high selectivity towards MDA-MB-231 breast cancer cell line. Measurement of the radiosensitizing activity for 5 and 6 revealed that they could sensitize the tumour cells after being exposed to a single dose of 8 Gy gamma radiation. Compound 5 was able to induce apoptosis and arrest the cell cycle at the G2-M phase. Molecular docking of 5 and 6 in the active site of EGFR was performed to gain insight into the binding interactions with the key amino acids.     

Synthesis and cytostatic properties of structure-simplified analogs of dolastatin 15.

The linear peptide dolastatin 15 (1), a potent antineoplastic constituent from the shell-less mollusk Dolabella auricularia, has been selected as the lead compound for developing novel antitumor drugs. Recently LU103793 (2), a synthetic and structure-simplified analog of dolastatin 15, has been demonstrated to be highly cytotoxic [IC50 = 0.1 nM; M. De Arruda, C.A. Cocchiaro, C.M. Nelson, C. M. Grinnel, B. Janssen, A. Haupt & T. Barlozzari (1995) Cancer Res. 55, 3085-3092]. Both compounds have been undergoing human cancer clinical trials in Europe and North America. Based on the novel structure of LU103793, a series of analogs modified at the N-terminal dolavalyl moiety and -Pro-Pro-benzylamide unit was developed. These synthesized analogs were tested using a sulforhodamine B (SRB) assay for the drug-screening program at NCI on a variety of human cancer cell lines. As expected, most analogs exhibited potent and selective growth inhibition against leukemia. Analog 18 was specifically active against HL-60 and K-562 cell lines (GI50s: 0.05 microM and 0.07 microM, respectively) while analogs 14 and 17 were selectively potent against prostate and breast cancer cell lines (GI50s at micromolar levels). However, all analogs were less potent than 2 as growth inhibitors of some breast and colon cancer cell lines (e.g. MDA-MB-435 and HT-29). We believe that modification of novel marine natural products as synthetic analogs might show particular promise for developing novel anticancer candidates with moderate specificity.