Induction of apoptosis and modulation of homologous recombination DNA repair pathway in prostate cancer cells by the combination of AZD2461 and valproic acid

Cancer therapies using defects in homologous recombination (HR) DNA repair pathway of tumor cells are not yet approved to be applicable in patients with malignancies other than BRCA1/2-mutated tumors. This study was designed to determine the efficacy of combination therapy of a histone deacetyl ase inhibitor, valproic acid (VPA) and a novel PARP inhibitor AZD2461 in both PC-3 (PTEN-mutated) and DU145 (PTEN-unmutated) prostate cancer cell lines. The Trypan blue dye exclusion assay an d the tetrazolium-based colorimetric (MTT) assay were performed to measure the cytotoxicity while combination effects were assessed based on Chou-Talalay's princi- ples. Flow-cytometric assay determined the type of cell deat h. The real-time PCR analysis was used to evaluate the alterations in mRNA levels of HR-related genes while their protein levels were measured using the ELISA method. γ -H2AX levels were determined as a marker of DNA damage. We observed a synergistic relationship between VPA and AZD2461 in all affected fractions of PC-3 cells (CI1.1). Annexin-V staining analysis revealed a significant induction of apoptosis when PC-3 cells were treated with VPA+AZD2461 ( p<0.05 ). Both mRNA and protein levels of Rad51 and Mre11 were significantly decreased in PC-3 cells co-treated with VPA+AZD2461 while enhanced H2AX phosphorylation was found in PC-3 cells after 12 and 24 hours of co-treatment ( p<0.05 ). Our findings established a preclinical rationale for selective targeting of HR repair pathways by a combination of VPA and AZD2461 as a mechanism for reducing the HR pathway sufficiency in PTEN -mutated prostate cancer cells

Evaluating cytotoxic effects of recombinant fragaceatoxin C pore forming toxin against AML cell lines

Objective(s): Current therapeutic strategies for cancer are associated with side effects and lack of specificity in treatments. Biological therapies including monoclonal antibodies and immune effectors have been the subject of multiple research projects. Pore-forming proteins may become the other biological strategy to overcome the problems associated with current treatments. But detailed mechanisms of their action on target membranes remained to be elucidated. We aimed to study the cytotoxic effects of recombinant form of fragaceatoxin C on AML cell lines HL-60 and KG-1. Materials and Methods: We cloned the FraC gene in pET-28a (+) bacterial expression vector and the expressed recombinant FraC protein was purified by affinity chromatography. Then, cytotoxic effects of the recombinant protein were examined on two AML cell lines, HL-60 and KG-1. Effects of serum and calcium ion were explored by hemolysis assay in more details. Results: Our results showed that the recombinant C-terminal polyhistidine-tagged FraC protein has potent cytotoxic effects on both AML cell lines, with IC50=5.6, and 4.6 µg.ml-1 for HL-60 and KG-1 cells, respectively. Serum showed dose-dependent and also time-dependent inhibitory effects on the hemolytic and cytotoxic activities of the FraC protein. Pre-incubation of the toxin with different concentrations of calcium ion also inhibited hemolytic activity of FraC toxin.Conclusion: Results of the present study showed that FraC has potential anti-tumor effects. By detailed investigation of the inhibition mechanism of serum and calcium effects in the future, it can be possible to design target sites for clinical applications of the toxin

Chaperone-Assisted Soluble Expression of a Humanized Anti-EGFR ScFv Antibody in E. Coli

Purpose: Formation of inclusion bodies is a considerable obstacle threatening the advantages of E. coli expression system to serve as the most common and easiest system in recombinant protein production. To solve this problem, several strategies have been proposed among which application of molecular chaperones is of remarkable consideration. The aim of this study was to evaluate the effects of molecular chaperones on soluble expression of aggregation-prone humanized single chain antibody. Methods: To increase the solubility of a humanized single chain antibody (hscFv), different chaperone plasmids including PG-tf2 (GroES- GroEL- tig), ptf16 (tig) and pGro7 (GroES- GroEL) were co-expressed in BL21 cells containing pET-22b- hscFv construct. The solubility of recombinant hscFv was analyzed by SDS-PAGE. After purification of soluble hscFv by Ni-NTA column, the biological activity and cytotoxicity of the recombinant protein were tested by ELISA and MTT assay, respectively. Results: SDS-PAGE analysis of the hscFv revealed that chaperone utility remarkably increased (up to 50%) the solubility of the protein. ELISA test and MTT assay analyses also confirmed the biological activity of the gained hscFv in reaction with A431 cells (OD value: 2.6) and inhibition of their proliferation, respectively. Conclusion: The results of this study revealed that co-expression of chaperones with hscFv leads to remarkable increase in the solubility of the recombinant hscFv, which could be of great consideration for large scale production of recombinant single chain antibodies

Bacterial Expression of TMTP1-Fused L-Asparaginase for Targeting Leukemia and Metastatic Tumor Cells

L-asparaginase is recognized as a first-line anticancer drug for acute lymphoblastic leukemia (ALL); however, low-substrate specificity and exhibiting glutaminase activity cause various off-target toxicities on normal cells. In the following study, we functionalized wild-type asparaginase with the TMTP1 targeting peptide which specifically targets a variety of hematological and metastatic cancer cells. The peptide sequence was genetically added to the N-terminal end of the asparaginase using the restriction endonuclease-free cloning method. Wild-type and engineered asparaginases were expressed in E. coli and purified by Nickel affinity chromatography column. The in vitro activity of both types of enzymes was evaluated by Nessler’s method. The sequencing results showed that the TMTP1 sequence was added in the correct frame to the asparaginase. Wild-type and TMTP1-fused asparaginases were produced in a soluble state with the specific activity of 172 U/mg and 153 U/mg, respectively. The evidence from this study suggests that TMTP1-fused asparaginase could preserve its solubility and activity compared to the wild-type species and can be proposed for future research in anticancer therapies

Recombinant production and affinity purification of the FraC pore forming toxin using hexa-His tag and pET expression cassette

Objective(s): A newly-introduced protein toxin from a sea anemone, namely fragaceatoxin C is a protein with molecular weight of 20 kDa and pore-forming capability against cell membranes has recently grasped great attentions for its function. In this study, its coding sequence cloned as a fusion protein with His-tag for simple production and rapid purification. Materials and Methods: After PCR amplification using NcoI and HindIII-harboring primers, the gene fragment was cloned into pET-28a(+). Escherichia coli BL21 was used for expression of constructed vector and toxin expression was verified by SDS-PAGE. For one-step purification Ni-NTA sepharose  affinity chromatography was employed. For examination of purified toxin function, RBC hemolytic test was conducted. Results: The results showed that the FraC-coding gene was successfully cloned between NcoI and HindIII restriction sites and purified with affinity chromatography. Densitometric analysis represented the purity of approximately 97%. Hemolytic test indicated the purified FraC had remarkable lytic activity on RBC and almost lysed 50% of cells at the concentration value of 6.25 nM. Conclusion: The results indicated that not only purified toxin preserved its activity during expression and purification processes but also exerted its function at lower concentrations so that even the 0.09 nM displayed hemolytic effect

The Effect of the Crocus Sativus L. Carotenoid, Crocin, on the Polymerization of Microtubules, in Vitro

Objective(s): Crocin, as the main carotenoid of saffron, has shown anti-tumor activity both in vitro and in vivo. Crocin might interact with cellular proteins and modulate their functions, but the exact target of this carotenoid and the other compounds of the saffron have not been discovered yet. Microtubular proteins, as one of the most important proteins inside the cells, have several functions in nearly all kinds of cellular processes. The aim of this study was to investigate whether crocin affects microtubule polymerization and tubulin structure. Materials and Methods: Microtubules were extracted from sheep brains after two cycles of temperature-dependant assembly-disassembly in the polymerization buffer (PMG). Then phosphocellulose P11 column was used to prepare MAP-free tubulin. Turbidimetric assay of microtubules was performed by incubation of tubulins at 37 ºC in PIPES buffer. To investigate the intrinsic fluorescence spectra of tubulins, the emission spectra of tryptophans was monitored. To test the interaction of crocin with tubulin in more details, ANS has been used. Results: Crocin extremely affected the tubulin polymerization and structure. Ultraviolet spectroscopy indicated that crocin increased polymerization of microtubules by nearly a factor of two. Fluorescence spectroscopic data also pointed to significant conformational changes of tubulin. Conclusion: We showed that crocin increased tubulin polymerization and microtubule nucleation rate and this effect was concentration dependant. After entering cell, crocin can modulate cellular proteins and their functions. Concerning the results of this study, crocin would be able to affect several cell processes through interaction with tubulin proteins or microtubules

Use of Single-Chain Antibody Derivatives for Targeted Drug Delivery

Abstract Single-chain antibodies (scFvs), which contain only the variable domains of full-length antibodies, are relatively small molecules that can be used for selective drug delivery. In this review, we discuss how scFvs help improve the specificity and efficiency of drugs. Small interfering RNA (siRNA) delivery using scFv-drug fusion peptides, siRNA delivery using scFv-conjugated nanoparticles, targeted delivery using scFv-viral peptide-fusion proteins, use of scFv in fusion with cell-penetrating peptides for effective targeted drug delivery, scFv-mediated targeted delivery of inorganic nanoparticles, scFv-mediated increase of tumor killing activity of granulocytes, use of scFv for tumor imaging, site-directed conjugation of scFv molecules to drug carrier systems, use of scFv to relieve pain and use of scFv for increasing drug loading efficiency are among the topics that are discussed here

Paclitaxel and curcumin coadministration in novel cationic PEGylated niosomal formulations exhibit enhanced synergistic antitumor efficacy

Abstract Background The systemic administration of cytotoxic chemotherapeutic agents for cancer treatment often has toxic side effects, limiting the usage dose. To increase chemotherapeutic efficacy while reducing toxic effects, a rational design for synergy-based drug regimens is essential. This study investigated the augmentation of therapeutic effectiveness with the co-administration of paclitaxel (PTX; an effective chemotherapeutic drug for breast cancer) and curcumin (CUR; a chemosensitizer) in an MCF-7 cell line. Results We optimized niosome formulations in terms of surfactant and cholesterol content. Afterward, the novel cationic PEGylated niosomal formulations containing Tween-60: cholesterol:DOTAP:DSPE-mPEG (at 59.5:25.5:10:5) were designed and developed to serve as a model for better transfection efficiency and improved stability. The optimum formulations represented potential advantages, including extremely high entrapment efficiency (~ 100% for both therapeutic drug), spherical shape, smooth-surface morphology, suitable positive charge (zeta potential ~ + 15 mV for both CUR and PTX), sustained release, small diameter (~ 90 nm for both agents), desired stability, and augmented cellular uptake. Furthermore, the CUR and PTX kinetic release could be adequately fitted to the Higuchi model. A threefold and 3.6-fold reduction in CUR and PTX concentration was measured, respectively, when the CUR and PTX was administered in nano-niosome compared to free CUR and free PTX solutions in MCF-7 cells. When administered in nano-niosome formulations, the combination treatment of CUR and PTX was particularly effective in enhancing the cytotoxicity activity against MCF-7 cells. Conclusions Most importantly, CUR and PTX, in both free form and niosomal forms, were determined to be less toxic on MCF-10A human normal cells in comparison to MCF-7 cells. The findings indicate that the combination therapy of PTX with CUR using the novel cationic PEGylated niosome delivery is a promising strategy for more effective breast cancer treatment

HB5 aptamer-tagged graphene oxide for co-delivery of doxorubicin and silibinin, and highly effective combination therapy in breast cancer

Abstract Using a chemotherapeutic agent, such as doxorubicin (DOX), with a natural agent, such as silibinin (Sili), is highly valuable to minimize systemic toxicity. However, Sili and DOX face disadvantages, such as low aqueous solubility and poor bioavailability. Here, we have engineered a drug delivery cargo by decorating carboxylated graphene oxide (cGO) with an aptamer, HB5, for simultaneous delivery of DOX and Sili as a combination therapy against MCF-7 and SK-BR-3 breast cancer cells. The resulting Apt-cGO displayed a typical sheet-like nanostructure with a broad surface. The maximum entrapment efficiency was 70.42% and 84.22% for Sili and DOX, respectively. When the Apt-cGO-DOX-Sili nanocomposites were selectively taken up by breast cancer cells, the interaction between cGO and drugs was cleaved, causing releasing both Sili and DOX into the tumor cells, respectively. Compared to free drugs, Apt-cGO-DOX-Sili nanocomposites displayed higher cytotoxicity in vitro. Apt-cGO-DOX-Sili nanocomposites potentially suppressed some cancer cell survival signals. They accelerated cell apoptosis and increased Rb levels as well as reduced Akt, mTOR, NF-κB, and CDK2 levels. In conclusion, the developed Apt-cGO-DOX-Sili can be suggested as a simple and efficient drug delivery approach for breast chemotherapy