Plant-Based Biosynthesis of Copper/Copper Oxide Nanoparticles: An Update on Their Applications in Biomedicine, Mechanisms, and Toxicity

Plants are rich in phytoconstituent biomolecules that served as a good source of medicine. More recently, they have been employed in synthesizing metal/metal oxide nanoparticles (NPs) due to their capping and reducing properties. This green synthesis approach is environmentally friendly and allows the production of the desired NPs in different sizes and shapes by manipulating parameters during the synthesis process. The most commonly used metals and oxides are gold (Au), silver (Ag), and copper (Cu). Among these, Cu is a relatively low-cost metal that is more cost-effective than Au and Ag. In this review, we present an overview and current update of plant-mediated Cu/copper oxide (CuO) NPs, including their synthesis, medicinal applications, and mechanisms. Furthermore, the toxic effects of these NPs and their efficacy compared to commercial NPs are reviewed. This review provides an insight into the potential of developing plant-based Cu/CuO NPs as a therapeutic agent for various diseases in the future

Development and Evaluation of 1′-Acetoxychavicol Acetate (ACA)-Loaded Nanostructured Lipid Carriers for Prostate Cancer Therapy

1′-acetoxychavicol acetate (ACA) extracted from the rhizomes of Alpinia conchigera Griff (Zingiberaceae) has been shown to deregulate the NF-ĸB signaling pathway and induce apoptosis-mediated cell death in many cancer types. However, ACA is a hydrophobic ester, with poor solubility in an aqueous medium, limited bioavailability, and nonspecific targeting in vivo. To address these problems, ACA was encapsulated in a nanostructured lipid carrier (NLC) anchored with plerixafor octahydrochloride (AMD3100) to promote targeted delivery towards C-X-C chemokine receptor type 4 (CXCR4)-expressing prostate cancer cells. The NLC was prepared using the melt and high sheer homogenization method, and it exhibited ideal physico-chemical properties, successful encapsulation and modification, and sustained rate of drug release. Furthermore, it demonstrated time-based and improved cellular uptake, and improved cytotoxic and anti-metastatic properties on PC-3 cells in vitro. Additionally, the in vivo animal tumor model revealed significant anti-tumor efficacy and reduction in pro-tumorigenic markers in comparison to the placebo, without affecting the weight and physiological states of the nude mice. Overall, ACA-loaded NLC with AMD3100 surface modification was successfully prepared with evidence of substantial anti-cancer efficacy. These results suggest the potential use of AMD3100-modified NLCs as a targeting carrier for cytotoxic drugs towards CXCR4-expressing cancer cells

Geranylated 4-Phenylcoumarins Exhibit Anticancer Effects against Human Prostate Cancer Cells through Caspase-Independent Mechanism.

Geranylated 4-phenylcoumarins, DMDP-1 & -2 isolated from Mesua elegans were investigated for anticancer potential against human prostate cancer cells. Treatment with DMDP-1 & -2 resulted in cell death in a time and dose dependent manner in an MTT assay on all cancer cell lines tested with the exception of lung adenocarcinoma cells. DMDP-1 showed highest cytotoxic efficacy in PC-3 cells while DMDP-2 was most potent in DU 145 cells. Flow cytometry indicated that both coumarins were successful to induce programmed cell death after 24 h treatment. Elucidation on the mode-of-action via protein arrays and western blotting demonstrated death induced without any significant expressions of caspases, Bcl-2 family proteins and cleaved PARP, thus suggesting the involvement of caspase-independent pathways. In identifying autophagy, analysis of GFP-LC3 showed increased punctate in PC-3 cells pre-treated with CQ and treated with DMDP-1. In these cells decreased expression of autophagosome protein, p62 and cathepsin B further confirmed autophagy. In contrary, the DU 145 cells pre-treated with CQ and treated with DMDP-2 has reduced GFP-LC3 punctate although the number of cells with obvious GFP-LC3 puncta was significantly increased in the inhibitor-treated cells. The increase level of p62 suggested leakage of cathepsin B into the cytosol to trigger potential downstream death mediators. This correlated with increased expression of cathepsin B and reduced expression after treatment with its inhibitor, CA074. Also auto-degradation of calpain-2 upon treatment with DMDP-1 &-2 and its inhibitor alone, calpeptin compared with the combination treatment, further confirmed involvement of calpain-2 in PC-3 and DU 145 cells. Treatment with DMDP-1 & -2 also showed up-regulation of total and phosphorylated p53 levels in a time dependent manner. Hence, DMDP-1 & -2 showed ability to activate multiple death pathways involving autophagy, lysosomal and endoplasmic reticulum death proteins which could potentially be manipulated to develop anti-cancer therapy in apoptosis resistant cells

Autophagy inhibitor chloroquine enhanced ACA-induced cytotoxicity through apoptosis.

<p>(A) Representative fluorescence photomicrograph (400 × magnification) illustrating the acidic vesicular organelles in A549 and SK-LU-1 cell lines after treatment with ACA in presence or absence of CQ. Data were presented as relative fluorescence intensity in comparison to untreated cells ± SD of three independent experiments. ** <i>p</i> < 0.01 and *** <i>p</i> < 0.001 statistically different in comparison to untreated. # <i>p</i> < 0.05 statistically different in comparison to CQ. (B) Representative fluorescence photomicrograph (400 × magnification) illustrating the GFP-LC3-II punctate formation in A549 and SK-LU-1 cell lines upon exposure to co-treatment of CQ and ACA. Data were represented as mean percentage of cells with GFP-LC3-II punctate ± SD of three independent experiments. *** <i>p</i> < 0.001 statistically different in comparison to untreated. # <i>p</i> < 0.05 and ### <i>p</i> < 0.001 statistically different in comparison to CQ. (C) Effect of CQ on LC3-I/LC3-II protein expression after pre-treated with CQ prior ACA treatment in A549 and SK-LU-1 cell lines. Data were represented as normalized intensity ± SD of three independent experiments. * <i>p</i> < 0.05, ** <i>p</i> < 0.01 and *** <i>p</i> < 0.001 statistically different in comparison to untreated. # <i>p</i> < 0.05 and ## <i>p</i> < 0.01 statistically different in comparison to CQ. (D) Effect of CQ on the cell viability of ACA-treated A549 and SK-LU-1 cell lines. Data represented as mean percentage of cell viability ± SD for three independent experiments. *** <i>p</i> < 0.001 statistically different in comparison to untreated. ### <i>p</i> < 0.001 statistically different in comparison to ACA. (E) Effect of CQ on ACA-induced apoptotic cells in A549 and SK-LU-1 cell lines. Representative annexin V-FITC/PI scatter plots of 1 × 10⁴ cells after 24 h of treatment. Data represented as mean percentage of apoptotic cells ± SD for three independent experiments. *** <i>p</i> < 0.001 statistically different in comparison to untreated. # <i>p</i> < 0.05 and ### <i>p</i> < 0.001 statistically different in comparison to ACA.</p

Autophagy effect of ACA on A549 and SK-LU-1 cell lines.

<p>(A) Representative fluorescence photomicrograph (400 × magnification) illustrating the acidic vesicular organelles in A549 and SK-LU-1 cell lines after treated with ACA for 0, 3, 6, 12, and 24 h. Data were presented as relative fluorescence intensity in comparison to untreated cells ± SD. * <i>p</i> < 0.05, ** <i>p</i> < 0.01, and *** <i>p</i> < 0.001 statistically different in comparison to untreated. (B) Representative fluorescence photomicrograph (400 × magnification) illustrating the GFP-LC3-II punctate formation in A549 and SK-LU-1 cell lines upon exposure to ACA. Data were represented as mean percentage of cells with GFP-LC3-II punctate ± SD of three independent experiments. * <i>p</i> < 0.05, ** <i>p</i> < 0.01, and *** <i>p</i> < 0.001 statistically different in comparison to untreated. (C) Protein expression of LC3-I/II and p62 after ACA treatment in A549 and SK-LU-1 cell lines. Data were represented as mean normalized intensity ± SD of three independent experiments. * <i>p</i> < 0.05, ** <i>p</i> < 0.01, and *** <i>p</i> < 0.001 statistically different in comparison to untreated.</p

ACA inhibited cell viability of A549 and SK-LU-1 cell lines.

<p>(A) Chemical structure of 1’S-1’-acetoxychavicol acetate (ACA). (B) The cell viability of MCF 10A, A549 and SK-LU-1 cells lines after exposure to ACA (0–30 μM) for 24 h was assessed using MTT assay. Data represented as mean percentage of cell viability ± SD for three independent experiments. (C) The cell viability of A549 and SK-LU-1 cells lines after exposure to IC₅₀ of ACA (25 μM for SK-LU-1 and 30 μM for A549 cells) on respective cell lines for 0–24 h was assessed using MTT assay. Data represented as mean percentage of cell viability ± SD for three independent experiments. * <i>p</i> < 0.05 and ** <i>p</i> < 0.01 statistically different in comparison to 0 h (D) Representative photomicrograph (200 × magnification) of A549 and SK-LU-1 cell lines upon ACA treatment. Arrow indicates the cytoplasmic vacuole.</p

Sequence of three unique 27 mer siRNA duplexes targeting <i>LC3</i> (OriGene, USA).

<p>Sequence of three unique 27 mer siRNA duplexes targeting <i>LC3</i> (OriGene, USA).</p

ACA-induced autophagy is independent of Beclin-1/PI3K complex in A549 and SK-LU-1 cell lines.

<p>(A) Protein expression of Beclin-1 after ACA treatment in A549 and SK-LU-1 cell lines. Data were represented as mean normalized intensity against GAPDH ± SD. *** <i>p</i> < 0.001 statistically different in comparison to untreated. (B) Representative fluorescence photomicrograph (400 × magnification) illustrating the acidic vesicular organelles in A549 and SK-LU-1 cell lines after treatment with ACA in presence or absence of 3-MA. Data were presented as relative fluorescence intensity in comparison to untreated cells ± SD of three independent experiments * <i>p</i> < 0.05 and ** <i>p</i> < 0.01 statistically different in comparison to untreated. # <i>p</i> < 0.05 and ## <i>p</i> < 0.01 statistically different in comparison to 3-MA. (C) Representative fluorescence photomicrograph (400 × magnification) illustrating the GFP-LC3-II punctate formation in A549 and SK-LU-1 cell lines upon exposure to co-treatment of 3-MA and ACA. Data were represented as mean percentage of cells with GFP-LC3-II punctate ± SD of three independent experiments. *** <i>p</i> < 0.001 statistically different in comparison to untreated. ### <i>p</i> < 0.001 statistically different in comparison to 3-MA. (D) Effect of 3-MA on LC3-I/LC3-II protein expression after pre-treatment of 3-MA prior to ACA treatment in A549 and SK-LU-1 cell lines. Data were represented as normalized intensity ± SD of three independent experiments. * <i>p</i> < 0.05, ** <i>p</i> < 0.01 and *** <i>p</i> < 0.001 statistically different in comparison to untreated. ## <i>p</i> < 0.01 and ### <i>p</i> < 0.001 statistically different in comparison to 3-MA. (E) Effect of 3-MA on the cell viability of ACA-treated A549 and SK-LU-1 cell lines. Data represented as mean percentage of cell viability ± SD for three independent experiments. * <i>p</i> < 0.05 and *** <i>p</i> < 0.001 statistically different in comparison to untreated.</p

Knockdown of <i>LC3</i> enhanced ACA-induced cytotoxicity through apoptosis.

<p>(A) Knockdown efficiency of the three unique 27 mer siRNA duplexes targeting <i>LC3</i> in A549 and SK-LU-1. Data were represented as mean percentage of relative intensity ± SD of three independent experiments. * <i>p</i> < 0.05 and *** <i>p</i> < 0.001 statistically different in comparison to negative control (NC). (B) Effect of si<i>LC3</i> on ACA-induced LC3-I/LC3-II protein expression in A549 and SK-LU-1 cell lines. Data were represented as normalized intensity ± SD of three independent experiments. *** <i>p</i> < 0.001 statistically different in comparison to negative control (NC). (C) Effect of si<i>LC3</i> on the cell viability of ACA-treated A549 and SK-LU-1 cell lines. Data represented as mean percentage of cell viability ± SD for three independent experiments. ** <i>p</i> < 0.01 and *** <i>p</i> < 0.001 statistically different in comparison to negative control (NC). # <i>p</i> < 0.05 and ### <i>p</i> < 0.001 statistically different in comparison to NC + ACA. (D) Effect of si<i>LC3</i> on ACA-induced apoptotic cells in A549 and SK-LU-1 cell lines. Representative annexin V-FITC/PI scatter plots of 1 × 10⁴ cells after 24 h of treatment. Data represented as mean percentage of apoptotic cells ± SD for three independent experiments. * <i>p</i> < 0.05, ** <i>p</i> < 0.01 and *** <i>p</i> < 0.001 statistically different in comparison to negative control (NC). ### <i>p</i> < 0.001 statistically different in comparison to NC + ACA.</p

Activation of proteins involved in caspase-independent pathways.

<p>(A) Cells were treated with DMDP-1 & -2 over 24 h, followed by examination of key proteins involved in multiple mode of cell death by western blotting. (B) Quantification of band intensities were determined by densitometry analysis and normalized to GADPH using the ImageJ v1.43 software. All results were presented as mean normalized intensity ±S.D. of three independent experiments.</p