Green-synthesized copper oxide nanoparticles induce apoptosis and up-regulate HOTAIR and HOTTIP in pancreatic cancer cells

Abstract

Aim: Cu₂O nanoparticles were synthesized using an extract from S. latifolium algae (SLCu₂O NPs). Their effect on PANC-1 cells and the expression of two drug resistance-related lncRNAs were evaluated in comparison with Arsenic trioxide.

Materials & methods: SLCu₂O NPs were characterized using XRD, SEM, and TEM microscopies. The effects of SLCu₂O NPs on cell cytotoxicity, cell cycle, and apoptosis, and expression of two drug resistance-related lncRNAs were examined using MTT assay, flow cytometry, and real-time PCR, respectively.

Results: SLCu₂O NPs demonstrated anti-cancer properties against PANC-1 cells comparable to Arsenic trioxide, and the expression of lncRNAs increased upon treatment with them.

Conclusion: SLCu₂O NPs demonstrate anti-cancer properties against PANC-1 cells; however, using gene silencing strategies along with SLCu₂O NPs is suggested.

Plain language summary

Article highlights.

• Copper nanoparticles have been proposed as a novel anti-cancer agent in cancer research including Pancreatic cancer.

• Copper oxide nanoparticles synthesis via physicochemical routs restricted their use in further cancer research or future clinical applications due to using highly toxic chemicals, high energy consumption, and high cost of synthesis.

• In this study, Cu₂O nanoparticles were synthesized using an extract from S. latifolium algae to address environmental concerns.

• The XRD patterns of the S. latifolium-derived Cu₂O nanoparticles showed the prominent peaks attributed to planes (110), (111), (200), (220), (311), and (222) that confirm the formation of the single-phase cubic structure with Pn-3m space group.

• Scanning electron microscopy confirm the spherical-cubic shape of S. latifolium-derived Cu₂O nanoparticles with the average size of 40–50 nm.

• S. latifolium-derived Cu₂O nanoparticles induced cell cytotoxicity on PANC-1 cells with IC₅₀ of 72.75 and 65.4 μg/ml at 24 and 48 h, respectively.

• S. latifolium-derived Cu₂O nanoparticles induced S-phase cell cycle arrest and apoptosis in PANC-1 cells.

• S. latifolium-derived Cu₂O nanoparticles showed comparable effects to arsenic trioxide in terms of cell cytotoxicity, colony formation, cell cycle arrest, and apoptosis.

• S. latifolium-derived Cu₂O nanoparticles increased the expression of two drug-resistant related lncRNAs, HOTAIR and HOTTIP, which may suggest using gene silencing strategies along with these nanoparticles.

1. Background

Pancreatic cancer (PC) is one of the deadliest cancers with 5-year survival rates of 4% [1]. Chemoresistance poses a formidable hurdle in effective PC treatment, with chemotherapy and targeted therapies exhibiting limited efficacy. The mechanism of chemoresistance in PC is very complex, and there are many uncertain cases [2]. Recently, the role of long non-coding RNAs (lncRNAs), notably HOTAIR and HOTTIP, in the development of chemoresistance in PC has been considered [3–6]. HOTAIR, transcribed from the antisense strand of the homeobox C gene on chromosome 12, exerts transcriptional regulatory functions [7]. HOTTIP encoded from a genomic region at the end of the 5′ of the HOXA locus on chromosome 7 and acts as an oncogenic lncRNA in almost all cancers [8]. Studies have highlighted the upregulation of HOTAIR and HOTTIP in PC tissues and cell lines. Their knockdown enhances the chemosensitivity of human PC cells to agents like gemcitabine, a commonly used chemotherapeutic drug [9,10]. Furthermore, the expression of these lncRNAs can be induced by chemotherapeutic agents that may suggest their role in drug resistance mechanism in PC. These lncRNAs might foster chemoresistance by impeding cell apoptosis, promoting epithelial–mesenchymal transition, augmenting cell self-renewal, evading cell cycle checkpoints, modulating cell autophagy, influencing DNA repair, altering efflux pump function, and shaping the tumor microenvironment [11].

Several chemotherapeutic drugs, such as gemcitabine/nab-paclitaxel and 5-fluorouracil/leucovorin with irinotecan and oxaliplatin, can improve the prognosis of pancreatic cancer. However, the development of chemoresistance still leads to poor clinical outcomes [12]. To overcome chemoresistance, researchers are focusing on finding new drugs or repurposing existing ones in combination with chemotherapeutic agents for the treatment of PC [13]. For example, arsenic trioxide (ATO), an FDA-approved drug for acute promyelocytic leukemia, has been shown to be effective alone or in combination with other conventional chemotherapy drugs on PC cells [14]. Thus, the search for new therapeutic substances continues to combat mechanisms underlie drug resistance in PC treatment [15].

Metal nanoparticles (MNs), derived from metal-containing compounds, have garnered attention due to their potential in overcoming chemoresistance. MNs can reduce chemoresistance by disrupting the cellular redox balance, which is the equilibrium between the generation and elimination of reactive oxygen species (ROS) in cancer cells [16]. Additionally, MNs could enhance drug sensitivity by altering the tumor microenvironment through the reorganization of the extracellular matrix and by activating the immune response [17].

Copper oxide nanoparticles as one of the metal nanoparticles, show selective cytotoxicity to several cancer cells and suppress tumor cell proliferation [18]. These nanoparticles can regulate signaling pathways and affect cell movements through ROS production [19]. However, the synthesis of copper oxide nanoparticles via physicochemical routes restricts their use in further cancer researches or future clinical applications due to using highly toxic chemicals, high energy consumption, and high cost of synthesis [20]. Hence, the demand for synthesizing copper oxide nanoparticles using simple, cost-effective, and environmentally friendly approaches, such as green synthesis using natural extracts as reducing agents, has surged [21]. Various biological sources have been explored for their capabilities in the biosynthesis of copper oxide nanoparticles [22,23].

Sargassum latifolium is a brown alga belonging to the order Fucales and mainly exists in tropical and temperate waters. S. latifolium has a good habitat for other marine plants and animals and often comes ashore in masses with flowing water [24]. The S. latifolium extracts have been used to synthesize different kinds of metal nanoparticles [25,26]. Yet, the precise cytotoxic effects of S. latifolium-derived Cu₂O nanoparticles compared with ATO on PC cells, alongside their impact on drug-resistant lncRNAs, remain largely unexplored.

The present study aimed to biosynthesize copper oxide nanoparticles using S. latifolium extract and evaluate their anti-cancer activity against PC cells in comparison to ATO, a known candidate for reducing chemoresistance in PC treatment research. Additionally, this study assessed the effects of biogenic copper (I) oxide (Cu₂O) nanoparticles on the expression of HOTAIR and HOTTIP lncRNAs.

2. Materials & methods

2.1. Materials

All chemicals, including copper (II) sulfate (CuSO₄), sodium citrate (Na₃C₆H₅O₇), and sodium carbonate (Na₂CO₃), were purchased from Merck, Germany. Arsenic (III) oxide was purchased from Sigma, USA. All culture vessels were obtained from SPL Life Science, South Korea.

2.2. Collecting S. latifolium & preparing algae extract

The brown macro-alga S. latifolium was collected from the Persian Gulf on the coast of Bandar Bushehr, Iran (28° 58′ N 50° 50′ E). The S. latifolium algae were washed in running tap water to remove filth and dust and rinsed with distilled water. Algae were then left to dry at room temperature for about 72 h and then were incubated in an oven at 40°C for 48 h. Using a mechanical grinder, the dried algae were ground into powder. Subsequently, 5 g of algae powder was added in to a 250 ml of Erlenmeyer flask containing 200 ml double-distilled water. Then, the Erlenmeyer flask was placed on a heater stirrer for 20 minutes at 100°C with gentle stirring using a magnet. At the end of the boiling time, the algae extract was passed through filter paper and then centrifuged at 6000 rpm for 10 minutes to obtain a clear solution [27,28].

2.3. Biosynthesis of Cu₂O NPs using Sargassum algae extract

The procedure for synthesizing Cu₂O NPs using S. latifolium algae extract is presented in Figure 1. Nanoparticles were synthesized using Benedict's solution and S. latifolium algae extract, as reported previously [29]. Benedict's solution was prepared as described before with slight modifications [30]. Briefly, 4 g of copper (II) sulfate salt was completely dissolved in 20 ml double-distilled water. Subsequently, 25 g of sodium citrate was added to the solution and completely dissolved, which turned the solution dark blue. Then, 10 g of sodium carbonate was added to the solution, and the volume of the final solution was increased to 40 ml with double-distilled water.

Figure 1.

The step-by-step procedure of the Cu₂O NPs biosynthesis using the S. latifolium algae extract.

To synthesize nanoparticles using S. latifolium algae extract, a beaker containing 40 ml of the Benedict's solution was placed on a heater stirrer at 100° C and the solution was mixed until it reached boiling point. Subsequently, 3 ml of S. latifolium algae extract was added dropwise using a micropipette in several steps. By adding the extract, the color of the solution gradually changed from blue to light green, and the reddish-orange particles appeared. Stabilizing the color of the solution indicates the end of the reaction. Then, the solution was centrifuged for 10 minutes at 6000 g to precipitate Cu₂O nanoparticles. The supernatant was discarded, and the nanoparticles were washed with double-distilled water and centrifuged at 6000 g. This step was repeated three-times. Finally, the precipitated nanoparticles were completely removed and placed in an oven at 60°C for 6 h to dry. For simplicity, the S. latifolium-derived Cu₂O nanoparticles were named SLCu₂O NPs in the rest of the manuscript.

2.4. Characterizing SLCu₂O NPs

The crystalline structure of SLCu₂O NPs was analyzed by advanced power x-ray diffraction (XRD), model D8 (Ultima IV Rigaku, Germany), with a Cu·K_α radiation source (1.54 Å) operating at 40 kV applied voltage and 30 mA current. XRD was performed at a scanning rate of 5° min^-1. The diffractogram was recorded between 2θ = 10° and 80°. Transmission electron microscopy (TEM) and scanning electron microscope (SEM) were applied to determine the shape and surface morphology of the SLCu₂O NPs.

2.5. Cell culture

Pancreatic cancer cell line (PANC-1) was purchased from the National Center for Genetic and Biological Resources (Tehran, Iran) and cultured in high glucose DMEM medium (BioIdea, Iran) supplemented with 10% FBS (Biosera, France). The cells were incubated at 37°C in a humidified incubator with 5% CO₂ and 95% humidity. To detach cells, the culture media was removed and cells were washed with PBS. Then, 1 ml trypsin (0.025%)/EDTA (0.01%) solution was added, and cells were incubated for 5 minutes. The effect of trypsin was blocked by adding 2 ml DMEM containing 10% FBS.

2.6. MTT assay

To assess the cytotoxicity of the SLCu₂O NPs, the MTT assay was used following the manufacturer's protocol (BioIdea, Iran). The optimal cell number for this assay was determined according to the manufacturer's instructions. To evaluate the cytotoxic effect of SLCu₂O NPs on PANC-1 cells, 8 × 10³ cells were cultured in each well of 96-well plate. After 24 h, the cells were exposed to different concentrations of SLCu₂O NPs and arsenic trioxide at 24 and 48 h. Subsequently, the culture media was replaced with 100 μl serum- and phenol red-free DMEM media and 10 μl of MTT solution (5 mg/ml) was added. Then, the cells were incubated in the incubator for 4 h. Afterward, 50 μl of dimethyl sulfoxide was used to lyse the cells and dissolve the purple crystals of formazan. Using a 96-well spectrophotometer, the optical density (OD) of the plate wells was read at 570 nm [31]. The cytotoxicity of samples on cells was expressed relative to the control cells. The MTT assay was performed three-times with six replicates per treatment. To calculate the half maximal inhibitory concentration (IC₅₀) of SLCu₂O NPs, six concentrations of SLCu₂O NPs (5, 10, 15, 30, 45 and 90 μg/ml) were used. The IC₅₀ value was determined using the linear regression equation Y = Mx + C, where Y was set to 50 and x was the IC₅₀ value.

2.7. Colony formation assay

The colony formation assay was performed as a golden standard method to evaluate the cell cytotoxicity. Briefly, 2.7 × 10⁴ PANC-1 cells were seeded into a 6-well plate and incubated for 24 h at 37°C. Cells were then exposed to arsenic trioxide and/or nanoparticles for 24 h at concentrations of 20 μM and 65 μg/ml or left untreated as control cells. Then the culture medium was removed, and cells were washed with PBS and trypsinized. Subsequently, 350 cells from each well in the first plate were transferred to the wells of a new 6-well plate. After 13 days, the cells were stained with violet crystal dye and colonies with over 50 cells were counted under a magnifying glass. We calculated the colonization efficiency (PE) and survival fraction (SF) using these equations. PE = (Number of cultured cells/ number of colonies formed) × 100, and SF = (number of cultured cells/ number of colonies formed after treatment) × PE.

2.8. Evaluation of apoptotic & necrotic cells by Annexin V-FITC/ PI staining

Labeling of cells with fluorescein isothiocyanate (FITC)-conjugated Annexin V (Annexin V-FITC) together with propidium iodide (PI) is a common method to measure apoptosis and necrosis using flow cytometry. Annexin V-FITC can recognize phosphatidylserine on apoptotic cells, whereas PI as a DNA binding dye can only enter cells when their membranes are ruptured. The apoptosis detection kit (Biolegend, USA) was used to measure apoptotic and necrotic cells following the manufacturer's protocol. Briefly, 2 × 10⁵ cells were cultured in a 6-well plate until reaching 70% confluence. Cells were then treated with 20 μM Arsenic trioxide or 65 μg/ml Cu₂O nanoparticles or left untreated as control cells for 48 h. Then, the cells were washed twice with phosphate buffer saline, and 4 × 10⁵ cells resuspended in 200 μl Annexin V binding buffer. Subsequently, 100 μl cell suspension was transferred to new microtube and 10 μl of PI and 5 μl of Annexin V-FITC solution was added and incubated for 15 min at room temperature. Finally, 400 μl binding buffer was added. The stained cells were analyzed using flow cytometry (BD Biosciences, USA) and FlowJo software to measure healthy cells (PI^-,Annexin-V^-), early apoptotic cells (PI^-,Annexin-V⁺), necrotic cells (PI⁺,Annexin-V^-), and necrotic or late apoptotic cells (PI⁺,Annexin-V⁺). The samples were analyzed by flow cytometry. All tests were performed in triplicates.

2.9. Flow cytometry-based cell cycle assay

Using flow cytometry, the cell cycle can be examined based on the DNA content of the cell using PI staining. To perform this test, the dye solution was prepared by mixing 20 μl of PI (1 mg/ml), 1 μl of Triton-X100 and 20 μl of RNaseA (5 mg/ml) and 950 μl PBS. 2 × 10⁵ cells were seeded in a 96 well-plate. The cells were harvested 48 h using trypsin after treatment with 20 μM arsenic trioxide or 65 μg/ml nanoparticles. After centrifugation at 200 g, the cells were washed with cold PBS and fixed with 70% ethanol. After removal of ethanol, 300 μl of PI dye solution was added to the cell precipitate and cells were analyzed using flow cytometry. All tests were performed in triplicates. Using FlowJo software, the percentage of the cell population at different stages of the cell cycle was determined.

2.10. Real-time PCR

To analyze the expression of HOTAIR and HOTTIP lncRNAs real time PCR was performed. To do this, 2 × 10⁵ cells were seeded in a 6-well plate. The cells were treated for 24 h with 20 μM arsenic trioxide or 65 μg/ml nanoparticles, then the supernatant was removed and washed with PBS. RNA extraction was performed using RNX plus solution (Sinaclone, Iran) according to the manufacturer's instructions. The extracted RNAs were treated with DNase I before cDNA synthesis. In order to synthesize cDNA, the Sinaclon First Strand cDNA Synthesis Kit-50T was used.

Real-time PCR was performed using the Rotor Gene Q system (Qiagen, USA), 2X RealQ Plus 2x Master Mix Green Without ROX (Ampliqon, Denmark), and 10 ng of cDNA template. The following primers were used in the PCR reactions: HOTTIP: Forward primer: 5′-CTGGTGAGGGGAGCTGAGTACG-3′, Reverse primer: 5′-GTCAGCGGCCAACCTTGATGC-3′; HOTAIR: Forward Primer: 5′-AGCACCCACCCAGGAATCCAC-3′, Reverse primer: 5′-AGGGTCCCACTGCATAATCACTCC-3′; ACTB: Forward primer: 5′-AGCCTTCCTTCCTGGGCATGG-3′; Reverse primer: 5′-AGCACTGTGTTGGCGTACAGGTC-3′. The thermal reaction conditions were as follows: initial denaturation at 95°C for 15 min, followed by 35 cycles of denaturation at 95°C for 15 s, annealing at 60°C for 30 s, and extension at 72°C for 20 s. Real-time PCR was carried out in the final reaction volume of 20 μl. To control DNA contamination, a no reverse transcribed RNA sample was used, and a negative control sample containing no cDNA template was used to check PCR false amplification for each gene amplification in each run of real-time PCR.

2.11. Statistical analysis

Statistical analyses were conducted using GraphPad Prism software (version 9). All data were presented as means ± standard deviation (SD) of three independent biological experiments. Comparisons between groups were performed using analysis of variance (ANOVA) followed by post-hoc tests or independent t-tests, as appropriate. Gene expression was analyzed by the 2^-ΔΔCt method. Differences were considered significant at p <0.05.

3. Results

3.1. XRD analysis confirmed the formation of SLCu₂O NPs

The XRD patterns of the SLCu₂O nanoparticles are shown in Figure 2. The prominent peaks attributed to planes (110), (111), (200), (220), (311), and (222) show the formation of the single-phase cubic structure with the Pn-3m space group according to the standard Cu₂O powder diffraction data (JCPDS card no. 01-074-1230). The XRD analysis with the Rietveld refinement through the Material Analysis Using Diffraction (MAUD) program confirmed a single-phase formation without evidence of a sondary phase of cubic structure with the experimental lattice parameter (a = 4.25Å), calculated density (6.18 g/cm³), measured density (5.92 g/cm³) and cell volume (76.87 10⁶ pm³).

Figure 2.

Characterization of SLCu₂O NPs. The XRD analysis with the Rietveld refinement through MAUD program confirmed a single-phase crystal structure.

Also, the average crystallite size (D) of sample was calculated using the Williamson–Hall equation [32,33]:

$$\beta \cos \left(\theta \right)=\frac{k\lambda }{D}+4\epsilon \sin \left(\theta \right)$$

where β is the full-width at half maximum (FWHM) of the XRD peaks, θ is the Bragg angle, λ is the x-ray radiation wavelength, and k is the shape factor changing in the range from 0.9 to 1.15 (≈0.9 for spherical-shaped crystallites). The average crystallite size of SLCu₂O nanoparticles was found to be 22.2 nm.

3.2. SEM confirmed the spherical shape of SLCu₂O NPs

The morphological analyses of the specific sample SLCu2O nanoparticles were carried out using scanning electron microscopy (SEM). The SEM micrograph is provided in Figure 3A. The average size of the nearly spherical-cubic nanoparticles was found to be in the range of 40–50 nm, although the nanoparticles are agglomerated.

Figure 3.

Electronic microscopic images of SLCu₂O NPs. (A) SEM images of Cu₂O NPs synthesized in the presence of sargassum algae extract at the 100 Kx magnification. (B) TEM image of SLCu₂O NPs at the 50 nm scale.

3.3. TEM confirmed that SLCu2O NPs mean particle size is less than 50 nm

Figure 3B shows the transmission electron microscopy (TEM) image of the SLCu₂O nanoparticles. It is evident from this figure that the majority of the particles are rounded with the diameters less than 100 nanometers (mean particle size is less than 50 nm). Considering the matter that each particle contains several crystallites, the particle size obtained by SEM and TEM images confirms the validity of the crystallite size calculated by XRD.

3.4. SLCu₂O NPs induced cell cytotoxicity on PANC-1 cells

MTT assay was performed to study the cytotoxic effect of the SLCu₂O NPs on PANC-1 cells. The results showed that Cu₂O NPs reduced the viability of PANC-1 cells in a concentration-dependent manner at 24 and 48 h (Figure 4A). A significant increase in cell cytotoxicity was observed in concentrations over 45 μg/ml. The IC₅₀ of SLCu₂O NPs on PANC-1 cells was 72.75 and 65.4 μg/ml at 24 and 48 h, respectively. The cytotoxic effect of SLCu₂O NPs was compared with ATO on PANC-1 cells. The ATO IC₅₀ at 48 h was found to be 20 μM. As shown in Figure 4B, similar cytotoxic effect was observed at the IC₅₀ for SLCu₂O NPs and ATO at 48 h. Also, it was observed that the combination of SLCu2O NPs with ATO (65 μg/ml NP and 20 μM ATO) exhibited higher toxicity compared with the individual treatments of ATO and SLCu2O NPs at 48 h (Figure 4B).

Figure 4.

Cytotoxic effect of SLCu₂O NPs and ATO. (A) The OD (mean ± SD) absorbances of dissolved formazan in cells treated with different concentration of Cu₂O NPs (n = 5). (B) The OD absorbances (mean ± SD) of dissolved formazan in cells treated with Cu₂O NPs or/and ATO (n = 5). SLCu₂O NPs showed synergistic cytotoxic effect in combination with ATO. (C) SLCu₂O NPs decrease the number and size of colonies relative to untreated cells. (D) The mean ± SD of survival fraction among Cu₂O NPs or ATO treated-cells versus untreated cells. SLCu₂O NPs and ATO shows comparable effect on the survival fraction of PANC-1 cells.

**p <0.01; ns: Not significant; SD: Standard deviation.

3.5. SLCu₂O NPs showed comparable effect to ATO on PANC-1 cell colony formation ability

In order to investigate the cytotoxic effect of SLCu₂O NPs on PANC-1 cells, colony formation was performed as a gold standard assay (Figure 4C). As shown in Figure 4D, treatment of cells with 65 μg/ml SLCu₂O NPs decreased the survival fraction by 45%. This effect was similar to the effect of 20 μM ATO which decreased the survival fraction by 40%.

3.6. SLCu₂O NPs induced the S phase arrest in PANC-1 cells

Evaluating the effects of SLCu₂O nanoparticles on cell cycle progression is crucial due to the propensity of cancer cells to proliferate uncontrollably. To study the impact of SLCu₂O NPs on the cell cycle of PANC-1 cells, the cells were treated with SLCu₂O NPs at a concentration of 65 μg/ml. After 48 h, the cells were stained with PI and analyzed using flow cytometry. Figure 5A & B shows SLCu₂O NPs induced the S phase arrest in PANC-1 cells. Figure 5A shows the histograms of cell distribution in different phases of cell cycle and Figure 5B represents the percentage of cell distribution in different phases of cell cycle. As shown in Figure 5B, the treatment with SLCu₂O NPs significantly reduced the population of PANC-1 cells in G₁ phase by 17.88%. Indeed, the mean percentage of G1 phase-cell population was 49.65% among untreated cells, whereas the mean percentage of G1 phase-cell population was 31.77 among treated cells. A similar effect was observed when the ATO-treated cells were analyzed by flowcytometry. Also, SLCu₂O NPs increased the population of S-phase cell population by 24.96%. This effect was similar to that of ATO which also increased the population of S-phase PANC-1 cells by 21.41% (23.78% among control cells vs 52.46% among treated cells).

Figure 5.

The effect of SLCu₂O NPs on PANC-1 cell cycle. (A) the histogram diagrams of cells. (B) The mean ± SD percentage of cell populations among SLCu₂O NPs or ATO treated-cells versus untreated cells (n = 3). SLCu₂O NPs induces S-phase cell cycle arrest in PANC-1 cells similar to ATO effect.

**p <0.01; n: Number of replicate; SD: Standard deviation.

3.7. SLCu₂O NPs induced apoptosis in PANC-1 cells

To study the impact of SLCu₂O NPs on PANC-1 cell death, flow cytometry analysis was conducted 48 h after cell treatment using Annexin V-PI staining. Figure 6A & B shows that SLCu₂O NPs induced apoptosis in PANC-1 cells. Figure 6A represents the dot plot which shows that SLCu₂O NPs induced early and late apoptosis in comparison with the untreated cells. As shown in Figure 6B, the mean percentage of apoptotic cells (early and late apoptosis) in control cells was 4.55%, while in cells treated with SLCu₂O NPs was 39.6%. Also, the mean percentage of apoptotic cells was 27.2% among ATO-treated cells. Furthermore, as depicted in Figure 6A, the most dead cells among ATO-treated cells at 48 h were early apoptotic cells, whereas most dead cells among SLCu₂O NPs-treated cells were necrotic/late apoptotic cells. This data shows that the cytotoxic effect of SLCu₂O NPs in PANC-1 cells results in cell apoptosis.

Figure 6.

The effect of SLCu₂O NPs on PANC-1 cell apoptosis and drug resistant-related lncRNAs expression. (A) Flowcytometry analysis of Cu₂O NPs and ATO-treated cells compared with untreated cells at 48 h. (B) The mean ± SD percentage of apoptotic cells among Cu₂O NPs or ATO-treated cells and untreated cells (n = 3). (C) Alterations in the mean fold changes ± SD of HOTAIR and HOTTIP lncRNAs in Cu₂O NPs and ATO-treated cells (n = 3). (SD = standard deviation, n = number of replicates, *p <0.05; **p <0.01; ***p <0.001).

3.8. SLCu2O NPs increased the expression of HOTTIP & HOTAIR LncRNAs

In order to investigate the effect of SLCu₂O NPs on the expression of two drug-resistant lncRNAs, real-time PCR were performed. As shown in the Figure 6C, the expression of HOTAIR and HOTTIP was increased by about 2.4 and 2.8-fold, respectively in SLCu₂O NPs-treated cells. This effect was comparable to the expression alterations of these two lncRNAs in cells treated with ATO.

4. Discussion

New anti-cancer drugs for PC treatment are being actively pursued through various initiatives. Copper oxide NPs have been found to exhibit anti-cancer effects against various types of cancer, including PC. Advancing research in this field requires affordable and environmentally friendly methods to synthesize copper oxide NPs. The abundance of algae and their metal ion reduction ability make them a promising candidate for green metal NP synthesis [34]. This study is the first to evaluate the anti-cancer activity of S. latifolium-derived Cu₂O NPs and their mechanisms of action against PANC-1 PC cells.

In this study, our data demonstrated that S. latifolium algal extract can be utilized as a reducing and capping agent to synthesize Cu₂O NPs. This is consistent with previous studies that have shown the potential of algal extracts in the biosynthesis of metal NPs [29,34,35]. Although the components of S. latifolium algal extract were not characterized in the present study, the successful synthesis of SLCu₂O NPs suggests that S. latifolium algal extract could contain active compounds such as carbohydrates, vitamins, fats, polyunsaturated fatty acids, antioxidants, pigments, and phycobilins, which have been theoretically described as agents for reducing metal ions and stabilizing nanoparticle synthesis [36].

In our study, the XRD pattern of the synthesized NPs displayed peaks consistent with pure Cu₂O NPs without any peaks corresponding to Cu or CuO NPs [37]. This data contrasts with some previous studies that used a green route to synthesize copper NPs, resulting in a mixture of Cu/CuO/Cu₂O NPs [38,39]. The variance in our results compared with prior research may stem from differences in the biological sources of reducing and stabilizing agents employed in our study versus those in previous studies [40].

The SEM and TEM microscopic images of SLCu₂O NPs revealed their spherical morphology, with most particles measuring less than 50 nm in diameter. These findings are in line with Soureshjani et al.'s study [29], which also reported spherical Cu₂O NPs derived from S. latifolium. However, it's worth noting that the average size of Cu₂O NPs in their study was smaller than those derived in our study. This discrepancy could potentially be attributed to slight differences in pH, precursor concentration, reaction time, exposure time, and temperature between our study and Soureshjani et al.'s investigation [29].

Here, in this study, our data indicated that SLCu₂O NPs have cytotoxic effects on PANC-1 cells with an IC₅₀ of 65.4 μg/ml at 48 h. Additionally, the colony formation assay further confirmed the cytotoxicity of SLCu₂O NPs on PANC-1 cells. While there are no previous reports specifically addressing the cytotoxic effect of Cu₂O NPs on PANC-1 cells, Kodasi et al. demonstrated cytotoxic effects of Cu₂O nanoparticles synthesized using kiwi fruit juice on breast cancer MCF-7 cells, with an IC₅₀ of 6.25 μg/ml at 48 h. Furthermore, the S. latifolium- derived Cu₂O NPs showed cytotoxic effects on K562 chronic myeloid leukemia cells, with an IC₅₀ of 30 μg/ml at 48 h [29]. The difference in IC₅₀ might be attributed to variation in drug sensitivity of different cell lines that were used in mentioned studies. Nonetheless, collectively, these reports support our findings regarding the cytotoxicity of SLCu₂O NPs.

In another study, Benguigui et al. [15] reported that chemically synthesized CuO NPs showed a cytotoxic effect on PANC-1 cells with an IC₅₀ of 20 μg/ml at 24 h. Consequently, it appears that chemically synthesized CuO NPs are more cytotoxic to PANC-1 cells compared with SLCu₂O NPs. The higher cytotoxicity of CuO NPs compared with SLCu₂O NPs on PANC-1 cells could be attributed to differences in their stability and mechanism of action [41,42], which could result in distinct patterns of cellular damage.

Our study revealed that SLCu₂O NPs possess anti-cancer activity by inducing cell cycle arrest and apoptosis. This finding is supported by recent researches demonstrating the anti-cancer effects of copper oxide nanoparticles against various types of cancer cells. For instance, Karakus et al. reported that oolong tea extract-mediated CuO/Cu₂O nanoparticles induced cell death and apoptosis in colon cancer (HCT116) and breast cancer cells (MCF-7) [39]. Bai et al. demonstrated that copper nanoparticles fabricated using Acroptilon repens leaf extract exhibit anti-lung cancer activity through cell cycle arrest, apoptosis, and modulation of mTOR signaling [43]. Additionally, Kodasi et al. showed the anti-cancer activity of Cu₂O nanoparticles against MCF-7 breast cancer cells, primarily through apoptosis and inhibition of cell cycle progression [44]. Shinde et al. fabricated heterogeneous Cu/CuO/Cu₂O NPs using groundnut shell extract and reported their anti-cancer activity on MCF-7 breast cancer cells by inducing oxidative DNA damage [38]. Mahmood et al. demonstrated that copper oxide nanoparticles synthesized by Annona muricata extract exert anti-cancer activity by inducing apoptosis through triggering caspase 3 and 9 expression in breast cancer cells [45].

Our results indicated that the SLCu₂O NPs caused S-phase cell cycle arrest and apoptosis. Agents inducing cell cycle arrest can trigger apoptosis and offer potential therapeutic avenues for cancer treatment, either alone or in combination with other therapies [46]. Apoptosis is important in cancer therapies as helps prevent uncontrolled tumor growth while minimizing damage to healthy tissues [47]. Benguigui et al. [15] reported that CuO NPs induced G2/M cell cycle arrest and apoptosis in PANC-1-derived tumor-initiating cells. Wang et al. [48] reported that CuO NPs induced G2/M arrest and apoptosis in PC-3 cells by modulating the Wnt signaling pathway. Thit et al. [49] demonstrated that CuO NPs induced G2/M arrest in epithelial cells from Xenopus laevis. In contrast, Chen et al. [50] reported that carbon quantum dots/Cu₂O composite induced S-phase arrest and apoptosis in SKOV3 ovarian cancer cells. The different effects of copper NPs on cell cycle arrest may be due to the varying cytotoxicity mechanisms of Cu (I) and Cu (II) [51].

In the current study, we did not examine how SLCu₂O NPs cause cell cycle arrest and apoptosis. However, previous studies have proposed several mechanisms. Cu₂O NPs can increase intracellular ROS generation and oxidative stress, which lead to DNA damages and activates signaling pathways that cause cell cycle arrest, ultimately leading to cell death through apoptosis [52]. S-phase arrest could be caused by the activation of DNA damage response and replication stress through ATR kinase activity, leading to induction of p21 [53,54]. ROS induced by copper NPs are known to stimulate DNA damage response and replication stress by causing oxidative damage [55]. Additionally, it has been reported that copper oxide NPs can target lipoacylated proteins involved in the tricarboxylic acid cycle and induce another type of cell death named cuproptosis, although this was not assessed in our study [56].

This study found that the cytotoxic effect of SLCu₂O NPs is is comparable to that of ATO on PANC-1 cells, particularly in terms of cell apoptosis and cell cycle arrest. Additionally, SLCu₂O NPs were observed to enhance the toxicity of ATO. Given that both ATO and Cu₂O NPs have been reported to induce ROS [57], the increased cytotoxic effect of SLCu₂O NPs in combination with ATO may be attributed to the excessive generation of ROS.

In our study, we observed that SLCu₂O NPs increased the expression of two drug-resistant lncRNAs, HOTAIR and HOTTIP, similar to the effects of ATO. This finding aligns with the study by Tan et al.'s research [58], which demonstrated the upregulation of HOTAIR under ATO treatment in vivo and vitro settings. The up-regulation of drug-resistant-related lncRNAs in response to chemotherapeutic agents may be a part of the drug resistance mechanism. For example, Hu et al. reported that cisplatin treatment induces HOTAIR expression in bladder cancer cells through EGFR and NF-κB activation, leading to increased cisplatin resistance [59]. Upregulation of HOTAIR contributes to cisplatin resistance by inhibiting cell apoptosis, dysregulating cell cycle, enhancing epithelial-mesenchymal transition, autophagy, and self-renewal of cancer stem cells, interfering with DNA repair, and altering drug efflux pump mechanisms [11]. Similarly, Wang et al. found that Gemcitabine treatment causes resistance of pancreatic cancer stem-like cells via induction of HOTAIR, leading to promotion of proliferation and migration, reduced apoptosis rate, and increased chemoresistance [10]. Consistent with these prior studies, the increase in HOTAIR and HOTTIP expression in response to SLCu₂O NPs may be a mechanism employed by PANC-1 cells to enhance their drug resistance. To overcome drug resistance, it is suggested to explore the use of Cu₂O NPs in combination with gene silencing technologies for further research [9,60–62], although SLCu₂O NPs may still induce chemosensitivity by disturbing redox balance [16].

Our study also has some limitations that need to be addressed in future research. First, we did not evaluate the effect of SLCu₂O NPs on ROS production in the PANC-1 cell, even though past studies have indicated that copper NPs can cause cell toxicity and death by generating ROS and leading to DNA damage [19,63]. It is possible that SLCu₂O NPs may have a similar mechanism of action to ATO, generating ROS and affecting PANC-1 cells, but further studies are needed to confirm this. Additionally, our study was conducted solely in vitro, and we did not explore the therapeutic window for SLCu₂O NPs. However, a previous study reported that the minimum effective dose of CuO NPs against PANC-1 cells in mice was 1 mg/kg Cu, while a dose of 12.5 mg/kg Cu resulted in death of all mice [15]. Therefore, it is imperative to delve into the therapeutic window of Cu₂O NPs in future studies.

5. Conclusion

In conclusion, our study demonstrated the anti-cancer effects of SLCu₂O NPs against PANC-1 cells, which were comparable to the effects observed with ATO. The resemblance in anti-cancer efficacy highlights the potential of SLCu₂O NPs as a promising candidate for pancreatic cancer treatment. To enhance the effectiveness of pancreatic cancer therapy, future endeavors should focus on combining SLCu₂O NPs with strategies aimed at mitigating drug-resistant lncRNAs. This integrated approach holds promise for advancing pancreatic cancer treatment modalities.

Author contributions

Z Hosseini participated in data curation, formal analysis, and writing- original draft preparation. A Shadi, SJ Hosseini and H Nikmanesh contributed to conceptualization, review and editing. A Ahmadi contributed to the supervision of the study, data validation, writing-review and editing of the manuscript.

Financial disclosure

The authors have no financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.

Competing interests disclosure

The authors have no competing interests or relevant affiliations with any organization or entity with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.

Writing disclosure

No writing assistance was utilized in the production of this manuscript.