Synergistic anti-proliferative and apoptotic effect of NVP-BEZ235 and curcumin on human SH-SY5Y neuroblastoma cells.

Neuroblastoma, a tumor of the sympathetic nervous system, is one of the most common tumors found in children. Most patients develop resistance to therapy and show poor prognosis, thus there is a need of novel therapeutic agents for the treatment of neuroblastoma. NVP-BEZ235 is a dual Phosphatidylinositol 3-kinase (PI3K)/mammalian target of rapamycin (mTOR) kinase inhibitor that induces apoptosis and suppresses the growth of cancer. Curcumin acts as an anticancer agent in certain cancers. This study investigated the synergetic effect of NVP-BEZ235 and curcumin against neuroblastoma SH-SY5Y cell line. In the current study, the synergic effect of NVP-BEZ235 and curcumin in SH-SY5Y was examined in terms of the cell growth by cell viability and colony forming assay, cell cycle and apoptotic cell death by flow cytometry and mRNA expression levels by quantitative Real Time Polymerase Chain Reaction (qRT-PCR). Curcumin, NVP-BEZ235 or a combination of both, showed cytotoxicity in a dose and time dependent manner in SH-SY5Y cells. 10 µM curcumin and 200 nM NVP-BEZ235 were chosen as combination therapy, as the combination index showed synergism. Colony forming assay showed decrease in cell growth in combination group. The cell cycle distribution for combination group demonstrated a decrease in G0/G1 phase at 48 h. Annexin V showed an anticancer effect in combination group when compared to control group. Moreover, qRT-PCR results showed a significant increase in caspase 3, caspase 7, Bax and p53 genes, while a decrease in Bcl-2 gene expression levels. These findings suggest that combination therapy of NVP-BEZ235 and curcumin may be a promising therapeutic candidate for treatment of neuroblastoma.

Synergistic anti-cancer effect of sodium pentaborate pentahydrate, curcumin and piperine on hepatocellular carcinoma cells.

Hepatocellular carcinoma (HCC) is a leading cause of cancer-related death in the world. Poor prognosis of HCC patients is a major issue, thus, better treatment options for patients are required. Curcumin (Cur), hydrophobic polyphenol of the plant turmeric, shows anti-proliferative, apoptotic, and anti-oxidative properties. Boron is a trace element which is essential part of human nutrition. Sodium pentaborate pentahydrate (NaB), a boron derivative, is an effective agent against cancer. In the current study, we performed in vitro experiments and transcriptome analysis to determine the response of NaB, Cur, piperine (Pip) and their combination in two different HCC cell lines, HepG2 and Hep3B. NaB and Cur induced cytotoxicity in a dose and time dependent manner in HepG2 and Hep3B, whereas Pip showed no significant toxic effect. Synergistic effect of combined treatment with NaB, Cur and Pip on HCC cells was observed on cytotoxicity, apoptosis and cell cycle assay. Following in vitro studies, we performed RNA-seq transcriptome analysis on NaB, Cur and Pip and their combination on HepG2 and Hep3B cells. Transcriptome analysis reveals combined treatment of NaB, Cur and Pip induces anti-cancer activity in both of HCC cells.

Targeting prostate cancer with docetaxel-loaded peptide 563-conjugated PEtOx-co-PEI30%-b-PCL polymeric micelle nanocarriers.

Prostate cancer is a global disease that negatively affects the quality of life. Although various strategies against prostate cancer have been developed, only a few achieved tumor-specific targeting. Therefore, a special emphasis has been placed on the treatment of cancer using nano-carrier-encapsulated chemotherapeutic agents conjugated with tumor-homing peptides. The targeting strategy coupling the drugs with nanotechnology helps to overcome the most common barriers, such as high toxicity and side effects. Prostate-specific membrane antigen has emerged as a promising target molecule for prostate cancer and shown to be targeted with high affinity by GRFLTGGTGRLLRIS peptide known as peptide 563 (P563). Here, we aimed to assess the in vitro and in vivo targeting efficiency, safety, and efficacy of P563-conjugated, docetaxel (DTX)-loaded polymeric micelle nanoparticles (P563-PEtOx-co-PEI30%-b-PCL-DTX) against prostate cancer. To this end, we analyzed the cytotoxic activity of P563-PEtOx-co-PEI30%-b-PCL and P563-PEtOx-co-PEI30%-b-PCL-DTX by a cell proliferation assay using PNT1A and 22Rv1 cells. We have also determined the targeting selectivity of P563-PEtOx-co-PEI30%-b-PCL-FITC by flow cytometry and assessed the induction of cell death by western blot and TUNEL assays for P563-PEtOx-co-PEI30%-b-PCL-DTX in 22Rv1 cells. To investigate the in vivo efficacy, we administered DTX in the free form or in polymeric micelle nanoparticles to athymic CD-1 nu/nu mice 22Rv1 xenograft models and performed histopathological analyses. Our study showed that targeting prostate cancer with P563-conjugated PEtOx-co-PEI30%-b-PCL polymeric micelles could exert a potent anti-cancer activity with low side effects.

Delivery of curcumin within emulsome nanoparticles enhances the anti-cancer activity in androgen-dependent prostate cancer cell.

BACKGROUND: Curcumin, a dietary polyphenol isolated from turmeric, is a potent phytochemical possessing intrinsic anticancer activities against various cancer types including prostate cancer. However, low water solubility and bioavailability of the compound are major challenges against its medical use. The objective of this study is to evaluate the therapeutic potential of curcumin-loaded emulsome nanoparticular system, i.e. CurcuEmulsomes, for the treatment of androgen dependent LNCaP prostate cancer cell line. METHODS AND RESULTS: The antiproliferative effect of both free curcumin and CurcuEmulsome were investigated comparatively on LNCaP and PNT1A cells. Cell viability data indicates that the inhibition in proliferation of LNCaP cells becomes more effective when curcumin is provided with its emulsome formulation rather than its free form. Corresponding to a therapeutic index of 2.25, Half maximal inhibitory (IC50) and cytotoxic (CC50) concentrations of CurcuEmulsomes for LNCaP and PNT1A cells were estimated as 17.1 µM and 38.6 µM, respectively. The fluorescence signal of autofluorescence curcumin was preserved within the CurcuEmulsomes at 72 h after the treatment. Thus, CurcuEmulsomes prolonged biological activity of curcumin. Induced apoptotic cell death and stimulated cell cycle arrest at G2/M phase were attributed to antiproliferative activity of CurcuEmulsomes. Treatment of LNCaP cells with CurcuEmulsomes increased expression of caspase-3 significantly by 11.76-fold, whereas decreased cyclin D1, Bcl-2 and AR expression levels significantly by of 0.18, 0.06 and 0.46-fold, respectively. CONCLUSIONS: Presented safety and anticancer activity of CurcuEmulsomes on LNCaP cell line highlights the potential of CurcuEmulsomes to benefit intrinsic anticancer activities of curcumin in androgen dependent prostate cancer therapy.

Integrated transcriptome and in vitro analysis revealed anti-proliferative effect of sodium perborate on hepatocellular carcinoma cells.

BACKGROUND: Hepatocelular carcinoma is one of the leading cancer types with no effective cure as poor prognosis is still a challenging aspect. Thus, alternative therapeutics are necessary to control hepatocelular carcinoma. Boron derivatives such as boric acid (BA), sodium perborate tetrahydrate (SPT) and sodium pentaborate pentahydrate (NaB) have been discovered to have anti-cancer effect. This study investigated the anti-proliferative effects of SPT against hepatocelular carcinoma (HCC) using in vitro and transcriptome approaches. METHODS: Cytotoxic level of SPT on cell survival were detected using MTS assay. The apoptotic cell death and cell cycle arrest was determined using Annexin V/PI and cell cycle assay, respectively. Transcriptome analysis was performed using RNA-seq, followed by functional and KEGG pathway enrichment analysis. qPCR was used to validate the different genes. RESULTS: SPT treated HepG2 and Hep3B cells induced cytotoxicity having IC50 values of 1.13 mM and 0.91 mM, respectively. SPT caused mitotic arrest in G0/G1 phase at 48 h and subsequent apoptotic cell death. RNA-seq revealed a total number of 822 and 1075 differentially expressed genes (DEGs) which after SPT treatment in HepG2 and Hep3B cells, respectively. Functional and KEGG pathway enrichment results suggested that there are several genes involved to induce apoptosis related pathways. The DEGs in p53 signaling pathway may have closely relationships to the cells apoptosis caused by SPT treatment. qPCR results validated dynamic changes in p53 signaling pathway, DNA replication and cell cycle related genes, such as CDKN1A, SERPINE1, PMAIP1, MCM3, MCM5 and MCM6. CONCLUSION: In vitro experiments and RNA-seq analysis show anti-proliferative and apoptotic effect of SPT in HCC cells. Further studies might help in understanding the molecular mechanisms of SPT.

Delivery of doxorubicin loaded P18 conjugated-poly(2-ethyl-oxazoline)-DOPE nanoliposomes for targeted therapy of breast cancer.

Breast cancer, a heterogeneous disease, has the highest incidence rate and is a major cause of death in females worldwide. Drug delivery by using nanotechnology has shown great promise for improving cancer treatment. Nanoliposomes are known to have enhanced accumulation ability in tumors due to prolonged systemic circulation. Peptide 18 (P18), a tumor homing peptide targeting keratin-1 (KRT-1), was previously shown to have high binding affinity towards breast cancer cells. In this study, we investigate the ability of P18 conjugated PEtOx-DOPE nanoliposomes (P18-PEtOx-DOPE) for the targeted delivery of doxorubicin to AU565 breast cancer model. Toxicology studies of PEtOx-DOPE nanoliposomes performed on normal breast epithelial cells (MCF10A), showed minimal toxicity. Doxorubicin delivery by P18-PEtOx-DOPE to AU565 cells induces cytotoxicity in a dose and time dependent manner causing mitotic arrest in G2/M phase at 24 h. Anti-cancer activity of P18-PEtOx-DOPE-DOX nanoliposomes on AU565 cells was detected by Annexin V/PI apoptosis assay. In terms of in vivo antitumor efficacy, P18-PEtOx-DOPE-DOX nanoliposomes administration to AU565 CD-1 nu/nu mice model showed significant decrease in tumor volume suggesting that DOX delivered by these nanoliposomes elicited a strong antitumor response comparable to the free delivery of doxorubicin. Overall, our results offered preclinical proof for the use of P18-PEtOx-DOPE-DOX nanoliposomes in KRT-1+ breast cancer therapy.

Design and evaluation of peptide-18-targeted nanoliposomes constructed by poly(2-oxazoline)-DOPE for doxorubicin delivery.

AIMS: The aim of this study is to develop targeted nanoliposome formulations to provide efficient treatment for breast cancer. In this study, peptide 18-modified poly(2-ethyl-2-oxazoline)-dioleoylphosphatidylethanolamine (P18-PEtOx-DOPE), was synthesised to construct nanoliposomes. METHODS: Doxorubicin (DOX) was encapsulated into the nanoliposomes by ethanol injection method. Particle size and polydispersity index were measured by dynamic light scattering. Zeta potential was determined by electrophoretic laser Doppler anemometry. The shape of the nanoliposomes was examined by transmission electron microscope. Specific bindings of P18-PEtOx-DOPE nanoliposomes were demonstrated on AU565 cells by confocal microscopy and flow cytometry studies. RESULTS: DOX-loaded nanoliposomes with particle diameter of 150.00 ± 2.84 nm and PDI of 0.212 ± 0.013 were obtained. PEtOx-DOPE and PEtOx-DOPE nanoliposomes are non-toxic on HUVEC, HEK293 and hMSC cells for 48 h. Furthermore, P18-PEtOx-DOPE nanoliposomes demonstrated specificity towards AU565 cells with high binding affinity. CONCLUSIONS: As a result, DOX-loaded P18-PEtOx-DOPE nanoliposomes can serve as favourable candidates in breast cancer targeted therapy.

A robust optimization approach for the breast cancer targeted design of PEtOx-b-PLA polymersomes.

The equipping of nanoparticles with the peptide moiety recognizing a particular receptor, enables cell or tissue-specific targeting, therefore the optimization of the targeted nanoparticles is a key factor in the formulation design process. In this paper, we report the optimization concept of Doxorubicin encapsulating PEtOx-b-PLA polymersome formulation equipped with Peptide18, which is a breast cancer recognizing tumor homing peptide, and the unveiling of the cell-specific delivery potential. The most dominant formulation parameters, which are the polymer to Doxorubicin mass ratio (w/w) and the aqueous to organic phase ratio (v/v), were optimized using Central Composite Design (CCD) based Response Surface Methodology. The characteristics of optimum polymersome formulation were determined as the hydrodynamic diameter of 146.35 nm, the PDI value of 0.136, and the encapsulation efficiency of 57.11% and TEM imaging, which are in agreement with the DLS data, showed the spherical morphology of the polymersomes. In order to demonstrate the breast cancer-specific delivery of targeted polymersomes, the flow cytometry and confocal microscopy analyses were carried out. The targeted polymersomes were accumulated 8 times higher in AU565 cells compared to MCF10A cells and the intracellular Doxorubicin was almost 10 times higher in AU565 cells. The CCD-mediated optimized targeted polymersomes proposed in this report holds the promise of targeted therapy for breast cancer and can be potentially used for the development of novel treatments.

The Utilization of Poly(2-ethyl-2-oxazoline)-b-Poly(ε-caprolactone) Ellipsoidal Particles for Intracellular BIKDDA Delivery to Prostate Cancer.

Prostate cancer is the most common cancer, which is about 15-20% among male cancers worldwide. As most common strategies such as radiotherapy, chemotherapy, or surgery alone can be unsuccessful in the treatment of prostate cancer, this study aims to develop a new approach to deliver newly generated proapoptotic gene, BIKDDA, to androgen independent prostate cancer cells, 22RV1, using new generation nanocarriers called ellipsoids. As far as it is known, this is the first study that assesses the ability of proapoptotic gene BIKDDA to induce apoptosis in prostate cancer cell. BIKDDA encapsulating PEtOx-b-PCL-based ellipsoids are fabricated by solvent-switch method, and their morphology, size, and BIKDDA content are characterized. Gene delivery efficiency of BIKDDA loaded PEtOx-b-PCL ellipsoids is demonstrated by analysis of BIK mRNA expression with real-time PCR. The apoptotic effect of PEtOx-b-PCL ellipsoids loaded with BIKDDA (EPs-BIKDDA) on 22RV1 is shown by Annexin V staining. The obtained results demonstrate that the treatment of 22RV1 cells with EPs-BIKDDA can significantly increase BIK mRNA levels by 4.5-fold leading to cell death. This study not only represents BIKDDA as a potential therapeutic strategy in prostate cancer but also the capacity of ellipsoids as promising in vivo gene delivery vehicles.

Curcumin- and Piperine-Loaded Emulsomes as Combinational Treatment Approach Enhance the Anticancer Activity of Curcumin on HCT116 Colorectal Cancer Model.

Combination chemotherapy, administrating two chemotherapeutic agents concurrently, comes into prominence, as the heterogeneity or the level of the disease necessitates a collaborative action. Curcumin, isolated from turmeric, and piperine, isolated from black long pepper, are two dietary polyphenols studied for their intrinsic anti-cancer properties against various cancer types including colorectal cancer (CRC). Furthermore, piperine improves the therapeutic effect of curcumin. Addressing this mutual behavior, this study combines curcumin and piperine within emulsome nanoformulations. Curcumin- (CurcuEmulsomes) and piperine-loaded emulsomes (PiperineEmulsomes) have established a uniform, stable, spherical dispersion with average diameters of 184.21 and 248.76 nm, respectively. The solid tripalmitin inner core achieved encapsulation capacities of up to 0.10 mg/ml curcumin and 0.09 mg/ml piperine content. While piperine treatment alone - in its both free and emulsome forms - showed no inhibition in the proliferation of HCT116 cells in vitro, its presence as the second drug agent enhanced curcumin's effect. Combination of 7 µM PiperineEmulsome and 25 µM CurcuEmulsome concentrations was found to be most effective with an inhibition of cell proliferation of about 50% viability. Cell cycle arrest at G2/M phase and induced apoptosis verified the improved anti-cancer characteristics of the therapy. While CurcuEmulsomes achieved a fourfold increase in Caspase 3 level, combination of treatment with PiperineEulsomes achieved a sixfold increase in the level of this apoptotic marker. Combinational treatment of HCT116 cells with CurcuEmulsomes and PiperineEmulsomes improved the anticancer activity of the compounds and highlighted the potential of the approach for further in vivo studies.

Cloning of intron-removed enolase gene and expression, purification, kinetic characterization of the enzyme from Theileria annulata.

Tropical theileriosis is a disease caused by infection with an apicomplexan parasite, Theileria annulata, and giving rise to huge economic losses. In recent years, parasite resistance has been reported against the most effective antitheilerial drug used for the treatment of this disease. This emphasizes the need for alternative methods of treatment. Enolase is a key glycolytic enzyme and can be selected as a macromolecular target of therapy of tropical theileriosis. In this study, an intron sequence present in T. annulata enolase gene was removed by PCR-directed mutagenesis, and the gene was first cloned into pGEM-T Easy vector and then subcloned into pLATE31 vector, and expressed in Escherichia coli cells. The enzyme was purified by affinity chromatography using Ni-NTA agarose column. Steady-state kinetic parameters of the enzyme were determined using GraFit 3.0. High quantities (~65 mg/l of culture) of pure recombinant T. annulata enolase have been obtained in a higly purified form (>95 %). Homodimer form of purified protein was determined from the molecular weights obtained from a single band on SDS-PAGE (48 kDa) and from size exclusion chromatography (93 kDa). Enzyme kinetic measurements using 2-PGA as substrate gave a specific activity of ~40 U/mg, K m: 106 µM, kcat: 37 s(-1), and k cat/K m: 3.5 × 10(5) M(-1) s(-1). These values have been determined for the first time from this parasite enzyme, and availability of large quantities of enolase enzyme will facilitate further kinetic and structural characterization toward design of new antitheilerial drugs.