Zerumbone-loaded nanostructured lipid carrier induces apoptosis in human colorectal adenocarcinoma (Caco2) cell line

The incorporation of zerumbone (Zer) into nanostructured lipid carrier (NLC) is hypothesized to increase the efficacy of the drug. Nanostructured lipid carrier has sustained-drug release characteristics and is able to improve the solubility and bioavailability of the lipophilic drug. In this study, the anti-cancer effect of Zer was tested on human colorectal adenocarcinoma (Caco-2) cell line. The effect of Zer, zerumbone-loaded nanostructured lipid carrier (Zer-NLC) and NLC on the Caco-2 cell viability were determined using the MTT assay. The treatment concentration ranges from 0 to 120 μM at four different time intervals (i.e., 0 h, 24 hrs, 48 hrs and 72 hrs) were evaluated. At 24 hrs, the half-growth inhibitory concentration (GI50) of Zer-NLC (i.e., 4.25 μM) is lower than that of Zer (i.e., 23.75 μM). However, Zer outperformed the Zer-NLC at the subsequent time points. Similar trend was observed in other parameters including the cytostatic concentration (CC) and half-lethal concentration 50 (LC50). Phase contrast imaging and AO/PI fluorescence staining were performed at the CC and LC50 values. The morphological changes and the apoptosis features could be seen in cells treated with Zer and Zer-NLC while cells treated with NLC showed minor morphological changes. The cells treated with Zer-NLC demonstrated a slightly slower progression of apoptosis, which could be due to the controlled release of Zer from the NLC matrix. It was concluded that the incorporation of Zer into NLC did not compromise the potency and efficacy of the drug

Zerumbone-loaded nanostructured lipid carrier induces apoptosis of canine mammary Adenocarcinoma cells

Canine mammary gland tumor (CMT) is the most common tumor in intact female dog. Zerumbone (ZER) has promising anticancer properties, but plagued with poor water solubility, poor absorption, bioavailability, and delivery to target tissues. To solubilize, ZER was loaded into nanostructured lipid carrier (NLC) to produce ZER-loaded NLC (ZER-NLC). Te objectives of this study were to determine the antiproliferative efect and the mode of cell death induced by ZER-NLC and ZER on a canine mammary gland tumor (CMT) adenocarcinoma primary cell line. Tere was no signifcant diference (p>0.05) between ZER-NLC and ZER treatments in the inhibition of CMT cell proliferation; thus, the loading of ZER into NLC did not compromise the cytotoxic efect of ZER. Microscopically, ZER-NLC- and ZER-treated CMT cells showed apoptotic cell morphology. ZER-NLC and ZER treatments signifcantly downregulated the antiapoptotic Bcl-2 and upregulated the proapoptotic Bax gene expressions in CMT cells. Both ZER-NLC and ZER-treated CMT cells showed signifcant (p<0.0001) increases in caspase-8, -9, and -3/7 protein activities. In conclusion, ZER-NLC induced CMT cell death via regulation of Bcl-2 and Bax gene expressions and caspase activations, indicating the involvement of both the intrinsic and extrinsic pathways of apoptosis. Tis study provided evidences for the potential of ZERNLC as an anticanine mammary gland adenocarcinoma chemotherapy

Induction of cell cycle arrest and apoptosis by copper complex Cu(SBCM)₂ towards oestrogen-receptor positive MCF-7 breast cancer cells

Copper complexes have the potential to be developed as targeted therapy for cancer because cancer cells take up larger amounts of copper than normal cells. Copper complex Cu(SBCM)2 has been reported to induce cell cycle arrest and apoptosis towards triple-negative breast cancer cells. Nevertheless, its effect towards other breast cancer subtypes has not been explored. Therefore, the present study was conducted to investigate the effect of Cu(SBCM)₂ towards oestrogen-receptor positive MCF-7 breast cancer cells. Growth inhibition of Cu(SBCM)₂ towards MCF-7 and human non-cancerous MCF-10A breast cells was determined by MTT assay. Morphological changes of Cu(SBCM)2-treated-MCF-7 cells were observed under an inverted microscope. Annexin V/PI apoptosis assay and cell cycle analysis were evaluated by flow cytometry. The expression of wild-type p53 protein was evaluated by Western blot analysis. The intracellular ROS levels of MCF-7 treated with Cu(SBCM)₂ were detected using DCFH-DA under a fluorescence microscope. The cells were then co-treated with Cu(SBCM)₂ and antioxidants to evaluate the involvement of ROS in the cytotoxicity of Cu(SBCM)2. Docking studies of Cu(SBCM)2 with DNA, DNA topoisomerase I, and human ribonucleotide reductase were also performed. The growth of MCF-7 cells was inhibited by Cu(SBCM)2 in a dose-dependent manner with less toxicity towards MCF-10A cells. It was found that Cu(SBCM)₂ induced G2/M cell cycle arrest and apoptosis in MCF-7 cells, possibly via a p53 pathway. Induction of intracellular ROS was not detected in MCF-7 cells. Interestingly, antioxidants enhance the cytotoxicity of Cu(SBCM)2 towards MCF-7 cells. DNA topoisomerase I may be the most likely target that accounts for the cytotoxicity of Cu(SBCM)₂

Effects of tamoxifen-loaded erythropoietin-coated nanostructured lipid carrier on breast cancer cells and rat mammary gland tumour

Nanomedicine is an emerging and fast developing area in the medical field, especially in the treatment of cancers. Most chemotherapy drugs have the limitation of poor drug water solubility which hinders their drug efficacy. The incorporation of drugs into nanoparticulated carriers had improved the low water solubility and efficacy of antibreast cancer drugs. In this study, the nanostructured lipid carrier (NLC) was loaded with tamoxifen (TAM) and coated with erythropoietin (EPO) to produce EPOTAMNLC, and the anticancer effects of this drug delivery system was determined. For comparison the TAM-loaded NLC (TAMNLC) was also developed. These nanoparticulated carriers were produced by using the high pressure homogeniser method and physiochemically and morphologically characterised using the dynamic light scattering technique, zetasizer, and transmission electron microscopy. The thermodynamic interaction between EPO and TAMNLC was investigated through the fluorescent spectroscopy and isothermal titration calorimetry while their binding efficiency was obtained through sodium dodecyl sulfate polyacrylamide gel electrophoresis. The elucidation of oestrogen and erythropoietin receptors status on MCF-7 and LA7 cells was determined through immunocytochemistry staining. The cytotoxic effect, mode of cell death and cell cycle arrest caused by treatment with EPOTAMNLC and TAMLC toward MCF-7 and LA7 cells was determined by the 3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and flow cytometry. The in vivo effects of EPO-TAMNLC and TAMNLC were determined in the normal and mammary gland tumour rats by using doses 1.25 mg/kg BW, 2.5 mg/kg BW and 5 mg/kg BW and treated through intravenously. The rat mammary gland tumour was induced by injecting 6 × 106 LA7 cells into the mammary fat pad. Toxicity parameters included serum kidney and liver function parameters and the histology of the kidneys, liver, heart, lungs, spleen, and bone marrow. The EPO-TAMNLC formulation was stable with particle size of 55.39±0.98 nm, zeta potential of -1.58±0.47 mV, and polydispersity index of 0.19±0.01. Based on ultrastructural analysis, the nanoparticles were spherical. The binding interaction of EPO and TAMNLC shown was spontaneous with positive enthalpy. It was shown the binding efficiency of EPO to TAMNLC was highest at pH 7.2 at 55.43%. The immunocytochemistry staining revealed that these cells are positive for oestrogen (ER) and erythropoietin receptors (EpoRs). The in vitro toxic effect of EPO-TAMNLC and TAMNLC on MCF-7 and LA7 cells was timedependent with the GI50 of 4.8 μM, 5.1 μM, 2.5 μM and 2.5 μM respectively which postulated to occur through the targeting of the ER and EpoRs in the cancer cells. However, both drug carrier systems did not significant (P>0.05) affect the viability of the normal MCF-10A and HDFa cells. Flow cytometry study showed that EPOTAMNLC induced apoptosis and G0/G1 cell cycle arrest in the cancerous MCF-7 and LA7 cell lines. For the in vivo part, normal rats treated with intravenous EPO– TAMNLC and TAMNLC did not show evidence of toxicity from the treatments, suggesting that EPO-TAMNLC and TAMNLC are safe for parenteral use and the LD50 exceed 5 mg/kg BW. Both EPO-TAMNLC and TAMNLC, while significantly (p<0.05) reducing the mammary gland tumour size in rats, achieved sustain antitumour effect and are more effective anti-tumour agents than oral TAM. In conclusion, EPO-TAMNLC is a promising targeted anticancer drug formulation for treatment of ER-positive breast cancers

Development of erythropoietin receptor-targeted drug delivery system against breast cancer using tamoxifen-loade nanostructured lipid carriers

Tamoxifen (TAM) has been used in the treatment of breast cancers and is supplemented with erythropoietin (EPO) to alleviate the cancer-related anemia. The purported deleterious effects caused by the use of EPO with chemotherapeutic agents in the treatment of cancer-related anemia vary across studies and remain controversial. The use of nanoparticles as a drug delivery system has the potential to improve the specificity of anticancer drugs. In this study, we simultaneously incorporated two pharmacological active ingredients in one nanocarrier to develop EPO-conjugated TAM-loaded lipid nanoparticles (EPO-TAMNLC), a targeted delivery system, to enhance the cytotoxic activity while reducing the side effects of the ingredients. The effect of temperature in modulating the thermodynamic parameters associated with the binding of EPO and TAMNLC was assessed using isothermal titration calorimetry, while the unfolding of EPO structure was determined using fluorescence-quenching approach. The association efficiency of EPO and TAMNLC was 55.43%. Unlike binding of albumin to TAMNLC, the binding of EPO to TAMNLC occurred through endothermic and entropy-driven reaction. The EPO-TAMNLC formulation was stable because of the hydrophobic interaction and the high free energy, suggesting the spontaneity of the interactions between EPO and TAMNLC. The EPO-TAMNLC enhanced the in vitro cytotoxicity of TAM to MCF-7 cells. The EPO surface-functionalized TAMNLC could sequentially deliver EPO and TAM as well as improving site-specific delivery of these therapeutic compounds

Enhanced anti-mammary gland cancer activities of tamoxifen-loaded erythropoietincoated drug delivery system

Nanomedicine is an emerging area in the medical field, particularly in the treatment of cancers. Nanostructured lipid carrier (NLC) was shown to be a good nanoparticulated carrier for the delivery of tamoxifen (TAM). In this study, the tamoxifen-loaded erythropoietin-coated nanostructured lipid carriers (EPO-TAMNLC) were developed to enhance the anti-cancer properties and targetability of TAM, using EPO as the homing ligand for EPO receptors (EpoRs) on breast cancer tissue cells. Tamoxifen-loaded NLC (TAMNLC) was used for comparison. The LA7 cells and LA7 cell-induced rat mammary gland tumor were used as models in the study. Immunocytochemistry staining showed that LA7 cells express estrogen receptors (ERs) and EpoRs. EPO-TAMNLC and TAMNLC significantly (p<0.05) inhibited proliferation of LA7 in dose- and time-dependent manner. EPO-TAMNLC induced apoptosis and G0/G1 cell cycle arrest of LA7 cells. Both drug delivery systems showed anti-mammary gland tumor properties. At an intravenous dose of 5 mg kg-1 body weight, EPO-TAMNLC and TAMNLC were not toxic to rats, suggesting that both are safe therapeutic compounds. In conclusion, EPO-TAMNLC is not only a unique drug delivery system because of the dual drug-loading feature, but also potentially highly specific in the targeting of breast cancer tissues positive for ERs and EpoRs. The incorporation of TAM into NLC with and without EPO coat had significantly (p<0.05) improved specificity and safety of the drug carriers in the treatment of mammary gland tumors

Zerumbone-Loaded Nanostructured Lipid Carrier Induces Apoptosis of Canine Mammary Adenocarcinoma Cells

Canine mammary gland tumor (CMT) is the most common tumor in intact female dog. Zerumbone (ZER) has promising anticancer properties, but plagued with poor water solubility, poor absorption, bioavailability, and delivery to target tissues. To solubilize, ZER was loaded into nanostructured lipid carrier (NLC) to produce ZER-loaded NLC (ZER-NLC). The objectives of this study were to determine the antiproliferative effect and the mode of cell death induced by ZER-NLC and ZER on a canine mammary gland tumor (CMT) adenocarcinoma primary cell line. There was no significant difference (p>0.05) between ZER-NLC and ZER treatments in the inhibition of CMT cell proliferation; thus, the loading of ZER into NLC did not compromise the cytotoxic effect of ZER. Microscopically, ZER-NLC- and ZER-treated CMT cells showed apoptotic cell morphology. ZER-NLC and ZER treatments significantly downregulated the antiapoptotic Bcl-2 and upregulated the proapoptotic Bax gene expressions in CMT cells. Both ZER-NLC and ZER-treated CMT cells showed significant (p<0.0001) increases in caspase-8, -9, and -3/7 protein activities. In conclusion, ZER-NLC induced CMT cell death via regulation of Bcl-2 and Bax gene expressions and caspase activations, indicating the involvement of both the intrinsic and extrinsic pathways of apoptosis. This study provided evidences for the potential of ZER-NLC as an anticanine mammary gland adenocarcinoma chemotherapy