Algae-meditated route to cuprous oxide (Cu₂O) nanoparticle: differential expression profile of MALAT1 and GAS5 LncRNAs and cytotoxic effect in human breast cancer

Abstract

Background

Breast cancer (BC), as the most widely recognized disease in women worldwide, represents about 30% of all cancers impacting women. This study was aimed to synthesize Cu₂O nanoparticles from the cystoseira myrica algae (CM-Cu₂O NPs) assess their antimicrobial activity against pathogenic bacteria and fungi. We evaluated the expression levels of lncRNAs (MALAT1 and GAS5) and apoptosis genes (p53, p27, bax, bcl2 and caspase3), their prognostic roles.

Methods

In this study, CM-Cu₂O NPs synthesized by cystoseira myrica algae extraction used to evaluate its cytotoxicity and apoptotic properties on MDA-MB-231, SKBR3 and T-47D BC cell lines compared to HDF control cell line. The CM-Cu₂O NPs was characterized by UV–Vis spectroscopy, Fourier transform infrared spectroscopy (FTIR), X-ray diffraction (XRD), Transmission electron microscopy (TEM) and Scanning electron microscopy (SEM). The antimicrobial activity of CM-Cu₂O NPs was assessed against pathogenic bacteria, staphylococcus aureus (S. aureus) PTCC 1112 bacteria as a standard gram-positive bacteria and pseudomonas aeruginosa (P. aeruginosa) PTCC 1310 as a standard gram-negative bacterium. Expression profile of MALAT1 and GAS5 lncRNAs and apoptosis genes, i.e., p27, bax, bcl2 and caspase3 genes, were calculated utilizing qRT-PCR. The changes in the expression levels were determined using the DDCT method.

Results

MALAT1 was upregulated in MDA-MB-231, SKBR3 and T-47D BC (p < 0.01), while GAS5 was downregulated in SKBR3 and T-47D cell lines tested compared with HDF control cell line (p < 0.05) was found. The results revealed that, p27, bax and caspase3 were significantly upregulated in BC cell lines as compared with normal cell line. Bcl2 expression was also significantly increased in MDA-MB-231 and T47D cell lines compared with normal cell line, but bcl2 levels were downregulated in SKBR3 cell line.

Conclusions

Our results confirm the beneficial cytotoxic effects of green-synthesized CM-Cu₂O NPs on BC cell lines. This nanoparticle decreased angiogenesis and induces apoptosis, so we conclude that CM-Cu₂O NPs can be used as a supplemental drug in cancer treatments. Significantly, elevated circulating lncRNAs were demonstrated to be BC specific and could differentiate BC cell lines from the normal cell lines. It was demonstrated that lncRNAs used in this study and their expression profiles can be created as biomarkers for early diagnosis and prognosis of BC. Further studies utilizing patients would give recognizable identification of lncRNAs as key players in intercellular interactions.

Introduction

It is believed that natural compounds obtained from the plant materials are benign and easily metabolized drug in comparison with other synthetic medicinal compounds (Gnanavel et al. 2017). The secondary metabolites obtained from the plant materials have been used to develop drugs (Siegel et al. 2013). Approximately one-fourth of the total medicinal compounds consumed by the developed countries are natural compounds (Rasmussen et al. 2010). At present, the main cancer treatment procedures such as alkylating agents, antimetabolites and other different cancer therapy methods have several side effects because of the incapability of differentiating the normal cell and cancer causing cells that results in toxicity (Ahmad et al. 2013). It is believed that using nanomaterials to treat cancer is a significant procedure to discover the modern cancer drugs (Vinardell and Mitjans 2015). It has been reported that nanoparticles are useful structured modern medicines for treating cancer due to their nanoscale sized feature that results in increased drug effectiveness and sustained release of drug material (Kumar et al. 2012). The brown alga Cystoseira myrica is one of the leading species of brown macroalgae in the Persian Gulf that could be collected economically (Naddafi and Saeedi 2009). Many studies have been done to characterize the metal binding features of many kinds of biomass, but no study has been conducted on brown macro alga Cystoseira myrica until now.

The Cu₂O NPs have distinctive features in the field of nanoscale technological aspect (Gnanavel et al. 2017). The Cu₂O NPs have narrow band gap of 1.7 eV that develops their application in the field of superconductors (Bednorz and Müller 1986; Malandrino et al. 1997), solar energy transformation, synthesis of organic and inorganic nanostructure composites (Vijay Kumar et al. 2001), gas sensors (Ishihara et al. 1998), magnetic resistant equipment’s (Zheng et al. 2000), antifungal, antimicrobial, anti-biotic agents (Borkow et al. 2009). The Cu₂O NPs are also applied in pesticide research due to their biocidal features (Borkow and Gabbay 2012) and antibacterial agent (Kumar et al. 2015). It is possible to achieve Cu₂O NPs by microwave irradiations (Wang et al. 2002), sol–gel method (Zhang et al. 2001), electrochemical technique (Yin et al. 2001), thermal decomposition (Gnanavel et al. 2017) and alkoxide supported method (Carnes et al. 2002). These methods have numerous disadvantages chiefly due to the usage of harmful chemicals, high energy utilization and problems to purify the nanoparticles (Ghidan et al. 2016). The process of toxic free green materials like plant extract and microorganisms (Parashar et al. 2009) to prepare nanoparticles would be advantageous for applications in pharmaceutical and drug discovery safety due to toxic free chemicals technique to prepare the nanoparticles (Kumar et al. 2013).

BC in women ranks among the most common malignant tumors; it is also second chief reason of mortality in industrialized countries (Arshi et al. 2018). In 2008, the American Cancer Society estimated that in 2014, roughly 182,500 women would be diagnosed with BC and 40,500 women would die because of BC (Ye et al. 2010). In Iran, the average age of diagnosis of BC is roughly 15 years lower than the reported cases in western countries (Mousavi et al. 2009). Given the increased number of cancer incidence (Asadzadeh Vostakolaei et al. 2013) and many cancer cases among young individuals in Iran, it is crucial to shed light on disease features and clinical outcomes to help health policy makers in terms of resource allocation, diagnosis, and treatment facilities (Vostakolaei et al. 2012). The etiology of BC is complex and multifactorial. It is believed that genetic, environmental, and reproductive factors are all involved in development of BC (Sapkota et al. 2013). The complex link between circadian rhythm (CR) disruption and BC development could be used as an instance of this etiologic complexity. It has been reported that there is a correlation between disruption in CR and the risk of BC development (Lie et al. 2011). Moreover, it has been shown that there is a link between several epigenetic changes and genomic polymorphisms in genes controlling and BC development (Erdem et al. 2017a; Zienolddiny et al. 2013). Besides, some studies argued that there a correlation between CR disruption and reduced telomere length in which short telomere length itself is linked with BC development (Erdem et al. 2017b).

Apoptosis is a form of programmed cell death signifying a serious tumor-suppressive mechanism activated in response to stress signals like DNA damage, growth factor deprivation, or endoplasmic reticulum (ER) stress (Thandapani and Aifantis 2017). The p53, which is believed to be a tumor suppressor, is mutated in about 50% of tumors, often through missense mutations happening in the DNA-binding domain. Apart from influencing the transcriptional activity of p53, these missense mutations are frequently linked with an increase in the half-life of p53 mutant p53 to accumulate in the tumor cells and often acquire a gain-of-function phenotype. The mutant p53 is not naturally stable, but some stressors influence the oncogenic activation and features of this protein. For instance, DNA damage and oncogene activation such as RAS mutations, c-myc, or p16^INK4a are capable of changing the oncogenic features of mutant p53 (Dibra et al. 2016). B-cell lymphoma-2 (Bcl-2) family proteins are the main architects in regulating the apoptotic threshold and are often deregulated in cancers (Delbridge and Strasser 2015). The ‘BCL-2-regulated’ or ‘intrinsic’ apoptotic pathway integrates stress and survival signaling to determine if a cancer cell will live or die. Actually, several pro-apoptotic members of the BCL-2 family have demonstrated tumor-suppression activity in mouse models of cancer and are lost or repressed in certain human cancers (Delbridge and Strasser 2015). A candidate gene to boost the sensitivity of tumors to conventional treatment is the proapoptotic gene Bax (BCL2 Associated X, Apoptosis Regulator)(Arafat et al. 2000). Bax is a protein of the Bcl-2 family promoting apoptosis (Oltval et al. 1993). Studies show that p53-mediated cell death is influenced partly through downstream interactions with Bax (Xiang et al. 1998). It has also been shown that Bax acts as a tumor suppressor gene (Yin et al. 1997). Moreover, mutations of Bax have been detected in several human tumors such as those derived from the breast, colon, and ovary (Arafat et al. 2000). Moreover, it has been shown that restoring wild-type Bax sensitizes tumor cells to apoptosis (Brimmell et al. 1998; Sakakura et al. 1996). Caspase 3 is the most extensively investigated of the effector caspases. It is involved significantly in both the death receptor pathway, initiated by caspase 8, and the mitochondrial pathway, involving caspase 9. Moreover, several studies reported that caspase 3 activation is obligatory for apoptosis induction in response to chemotherapeutic drugs, e.g., taxanes, 5-fluorouracil, and doxorubicin (O’Donovan et al. 2003). Caspase 3 is produced as an inactive 32-kDa proenzyme, which is cleaved at an aspartate residue to yield a 12-kDa and a 17-kDa subunit. Two 12-kDa and two 17-kDa subunits combine for the formation of the active caspase 3 enzyme. Caspase 3 cleaves various cellular substrates such as structural proteins (e.g., lamins) and DNA repair enzymes [e.g., poly (ADP-ribose) polymerase]. It also activates an endonuclease caspase-activated DNAse that causes the DNA fragmentation that is a feature of apoptosis (Stennicke and Salvesen 1998). P27 was first introduced as a cell cycle inhibitor in the cells arrested by transforming growth factor β, and the expression of p27 is regulated by growth inhibitory cytokines and contact inhibition (Matsunobu et al. 2004). P27 is one of cyclin-dependent kinase inhibitors inhibiting cyclin-dependent kinase activities causing cell cycle arrest. It has been reported that there an inverse correlation between a low level of p27 expression with the tumor progression or the poor prognosis in several human malignancies including breast, prostate, colon, and lung cancers (Matsunobu et al. 2004; Porter et al. 1997). A recent study using p27 knockout mice reported that p27 is involved significantly in inhibiting tumor development and showed the features of a tumor suppressor gene (Nakayama et al. 1996). Overexpression of p27 by replication-deficient recombinant adenovirus led to apoptosis and reduced malignancy potential in human breast, lung, and prostate cancer cell lines (Katner et al. 2002). Since EWS-Fli1 caused the down-regulation of p27 in vitro (Matsumoto et al. 2001), p27 expression could be clinically correlated with disease phenotype, therapeutic responsiveness, and patient result.

Long non-coding RNAs (lncRNAs) are a category of non-coding RNAs, generally longer than 200 bp transcribed from the genome with several regulatory or unknown functions. It has been shown that lncRNAs are obligatory in normal cell and tissue development and differentiation as well as in the initiation and progression of various pathogenic conditions, including cancer (Hrdlickova et al. 2014). In this regard, dysregulated expression of lncRNAs has been reported in BC as well as in several other malignancies (Van Grembergen et al. 2016). Shedding light on the molecular biology of cancer, including that related to the function and behavior of lncRNAs, can help in early detecting as well as in designing targeted therapy for this multifaceted disorder (Hrdlickova et al. 2014). Growth arrest-specific 5 (GAS5) is an lncRNA 650 bases in length expressed at high levels during growth arrest. This lncRNA was originally isolated from NIH 3T3 cell line using subtraction hybridization in 1998 (Schneider et al. 1988). The human GAS5 has been classified as a member of the 59-terminal oligopyrimidine tract (59 TOP) gene family, and this gene is also the host of multiple small nucleolar RNAs within its 11 introns (Smith and Steitz 2015). At least four splice variants have so far been distinguished for GAS5, (Raho et al. 2000); it has been reported that this gene is capable of modulating cellular response to numerous apoptotic stimuli such as those prompted by various chemotherapeutic drugs, a concept recommending a mechanism connecting dysregulated GAS5 expression in cancer with prognosis (Nishimoto et al. 2013). MALAT1 (Metastasis Associated Lung Adenocarcinoma Transcript 1) is a highly conserved lncRNA that was first recognized as an upregulated lncRNA in lung cancer with a high tendency to metastasize (Ji et al. 2003). MALAT1 transcript is found in large quantities in mammalian cells, and the primary transcript is processed into two smaller RNAs: a long 6.7-kb transcript that localizes to the nuclear speckles (Bernard et al. 2010) and a tRNA-like small RNA (61 nt) that localizes to the cytoplasm (Wilusz et al. 2008). MALAT1 plays a role in regulating pre-mRNA alternative splicing, and its knockdown leads to cell-cycle arrest (Tripathi et al. 2010). MALAT1 is also essential for E2F target gene activation by repositioning E2F from polycomb bodies to transcriptionally active nuclear sites in a serum-dependent manner (Yang et al. 2011). Recently, two genome-wide association studies have reported that MALAT1, along with NEAT1, binds to the transcription start sites (TSSs) and to the gene bodies of those genes that are actively transcribed (West et al. 2014). It is believed that the overexpression of MALAT1 is linked to poor prognosis and shorter survival time in early-stage lung cancer (Wang et al. 2017).

We investigated the effect of CM-Cu₂O NPs on MDA-MB-231, SKBR3 and T-47D BC cell lines. Furthermore, we hypothesized that expression signatures of the profile of two lncRNAs (MALAT1 and GAS5) and four apoptosis genes (p53, p27, bax, bcl2 and caspase3) may be useful biomarkers for diagnosis, prognosis, or prediction of BC. The expression levels were evaluated by qRT-PCR.

Materials and methods

Materials

The brown macroalga was gathered from the Persian Gulf on the coast of Bandar Bushehr, Iran (28° 58′ N 50° 50′ E). Sigma Aldrich Chemicals (Ltd., Mumbai, India) provided the study with copper chloride (CuCl₂) and ethanol.

Preparing Cystoseira myrica leaf extract

The Cystoseira myrica were washed in running tap water for removing the filth and dust. The biomass was put to dry in the shade in a room temperature for about 72 h. By applying a mechanical grinder, the dried leaves materials were made into fine particles. Ethanol was applied by Soxhlet apparatus to extract the plant material for 4 h, which were subjected to rotary evaporator for removing the extra solvent. The concentrated ethanolic leaves extract of Cystoseira myrica were filtered and gathered for further process.

Preparing Cu₂O nanoparticles

Approximately 50 ml of freshly prepared 0.003 M hydrated CuCl₂ solution was mixed with 50 ml of ethanolic leaves extract of Cystoseira myrica and boiled for 3 h at 60 °C accompanied with incessant stirring. UV–visible spectroscopy was applied to monitor Cu₂O NPs formations. Because of the surface plasmon resonance excitation, the color turned from yellow to brown showing the formation of Cu₂O NPs. After observing the color change, the prepared content was put for centrifuging at 3000 rpm for 20 min. Distilled water was used to wash the formed pellets, which were kept in using crucible at 400 °C for 2 h.

Characterizing Cu₂O nanoparticles

Several analytical techniques like UV–visible Spectroscopy (Shimadzu UV-1800 PC, Japan) between the wavelength of 200–800 nm 62 were applied to confirm the formation of CM-Cu₂O NPs (Roopan et al. 2014). Advance Power XRD, model D8 (Bruker, Germany) was used to perform the X-Ray Diffractometer (XRD) analysis for the synthesized Cu₂O NPs. The Scherrer formula was applied for calculating the particle size determination using the formula, D = Kλ/βCosθ, where D shows the particle size, λ shows the wavelength, K denotes a Scherrer constant having value of 0.94, β is a half width maximum and θ shows the diffraction angle. FT-IR Spectroscopy (Bruker, Germany) was applied between the wave number of 400 cm⁻¹to 4000 cm⁻¹ to detect the functional group present in the synthesized Cu₂O NPs. Transmission electron microscopy (TEM) and Scanning Electron Microscope (SEM) were applied to determine the shape of the synthesized Cu₂O NPs.

Preparing microbial isolates

Tested organisms were common human pathogens, some of which are antibiotic resistant, involving staphylococcus aureus (S. aureus) PTCC 1112 bacteria as a standard gram-positive bacteria and Pseudomonas aeruginosa (P. aeruginosa) PTCC 1310 as a standard gram-negative bacterium obtained from the Iranian Research Organization for Science and Technology, Tehran, Iran. Isolates were kept fresh on nutrient agar plates (Oxoid) and on nutrient broth (Oxoid) with 15% glycerol and kept at − 4 °C for future use. Small inoculum of each of the pure, fresh cultures was added to 5 ml sterile distilled water making a 0.5 Mac Farland bacterial suspension, which was put for inoculation on Muller Hinton agar plate surfaces.

Antimicrobial assay

The agar well diffusion technique was applied for the antimicrobial activity in vitro (Vanden Berghe et al. 1991). A sterile cotton swab was used to spread the small inoculum of each of the prepared bacterial evenly onto Muller Hinton agar plate surfaces (Oxoid). Before inoculation, plates were put to rest at room temperature for the absorption of the microbial inoculums. A 5–7 mm diameter sterile cork borer was applied to make 4 equidistant wells on each MH plates corresponding to the dry and fresh S. aureus and P. aeruginosa. 100ll of the appropriate seaweed extracts was used to load the wells. Before inoculation, plates were put to rest at room temperature for 30 min for enhanced absorption; then, plates were put for incubation at 37 °C for 18–24 h. All tests were performed in duplicate. The inhibition zone around each well was determined to show the antimicrobial activity. Mean diameter values were determined from duplicate runs of each assay. The effectiveness of the target algal extracts was compared with standard antibiotic disks, methanol and acetone used as positive and negative controls, in the respective order.

Cell culture

The Institute of the Chinese Academy of Sciences (Shanghai, China) provided the study with the MDA-MB-231 (ATCC HTB-26), SKBR3 (ATCC HTB-30), T-47D (ATCC HTB-133) and HDF (ATCC PCS-201-012) cell lines. The MDA-MB-231, SKBR3 and T-47D cell lines were cultured and kept in RPMI-1640 medium (Sigma-Aldrich, St. Louis, MO, USA) supplemented with l-Glutamine (Sigma-Aldrich, St. Louis, MO, USA), 10% fetal bovine serum (Gibco, Carlsbad, CA, USA), 100 U/ml penicillin (Sigma-Aldrich, St. Louis, MO, USA), and 100 μg/ml streptomycin (Sigma-Aldrich, St. Louis, MO, USA) in a cell culture incubator at 37 °C, 5% CO₂, and 95% humidity. HDF cells were used as non-cancer cell group during the experiment (control cell).

Cell take-up and Cu’s delivery into cell medium

The cell take-up of nanoparticles and Cu delivery to the medium were dictated by inductively coupled plasma-mass spectrometry (ICP-MS) (Thermo Elemental X series 2, USA). For that, the exposed cells were harvested and counted, then cells were processed by treatment with nitric corrosive for 8 h in 37 °C (Abudayyak et al. 2020).

MTT assay

The viable MDA-MB-231 (ATCC HTB-26), SKBR3 (ATCC HTB-30), T-47D (ATCC HTB-133) and HDF (ATCC PCS-201-012) cell lines with a concentration of 5 × 103 (100 μl/well) were cultured in flat bottom 96-well plates for 24 h for performing the MTT assay; then, the supernatant was replaced by 100 μl RPMI medium containing CM-Cu₂O NPs (0, 5, 10, 15, 30, 45, 60, 75, 90 and 120 µg/ml). Experiments are performed in triplicate for each incubation time (24, 48, and 72 h). After the end of the incubations, the MTT solution, at the final concentration of 4 mg/ml in PBS, was added to each well and incubation was done for 4 h at 37 °C in a 5% CO₂ atmosphere; Next, the supernatant was once more replaced by DMSO (100 μl). After the end of assay, shaking was applied for 10 min and the plate was read against a blank reagent at 570 nm. The cytotoxicity of samples on cells was expressed as IC50 that is a concentration of the tested samples and reduces 50% of cellular growth than untreated control sample and was determined using the following formula:

$${\text{Growth inhibition }} = {\text{ OD control }} - {\text{ OD treated sample }} \times { 1}00/{\text{OD control}}.$$

Flow cytometry-based cell cycle assay

PI staining was performed for the cell cycle analysis followed by flow cytometry. Briefly, HDF, MDA-MB-231, SKBR3 and T-47D cells were treated with slightly different concentrations of nanoparticles (0, 5, 10, 15, 30, 45, 60, 75, 90 and 120 µg/ml). Treated and untreated control cells were harvested, washed double with phosphate-buffered saline (PBS), and glued in cold grain alcohol (70%) for four h at 4 °C. The mounted cells were washed, pelleted, resuspended in PBS, stained with an answer containing PI and RNase (40 μg/ml and 100 μg/ml, respectively) and incubated at 37 °C for thirty min until analysis. Flow cytometry was performed victimization associate Epics Altra flow cytometer (Beckman Coulter) at associate excitation wavelength of 488 nm and an emission wavelength of 610 nm. The information collected for 2 × 104 cells per condition were analyzed using WinMDI 2.8 software.

RNA Extraction and cDNA Synthesis

The RNX-Plus solution (SinaClon, Iran) was applied according to the manufacturer’s instructions to extract total RNA from cells. Thermo Scientific NanoDrop 2000 Spectrophotometer (Thermo Scientific, Germany) was applied to determine the purity and concentration of the extracted RNA; gel electrophoresis was done to verify the integrity of the RNA. After extracting RNA, a DNaseI treatment (EN0521, Fermentas, Germany) was carried out; 1 μg RNA was then applied for cDNA synthesis through the application of random hexamers as primers and Prime Script-RT kit (Takara, Japan).

Real-time qPCR

Specific primers (Table 1) and SYBR Premix Ex Taq II kit (Takara, Japan) were applied according to the manufacturer’s instruction for real-time qPCR. Rotor gene 6000 Corbett detection system was used for amplification. The thermal cycling condition was set as follows: an initial activation step for 5 min at 95 °C, followed by 40 cycles of 95 °C for 15 s and 65 °C for 1 min. No template controls (NTCs) were also applied in each run. A series of experiments was done with varying primer combinations (Table 1) to detect the best primer concentrations. Standard curves were prepared for each primer set using data from serially diluted samples for confirming the reaction efficacies. Melting curve analyses were also done for each primer set. Moreover, PCR products were electrophoresed on 2% agarose gel for confirming the product sizes. The GAPDH housekeeping gene was applied as a normalizer, and the HDF cell line as the control group for the cancer cell lines. Relative expressions were determined by applying the 2 − ΔΔCT method. The qPCR assays were performed in triplicate, and the data are presented as the mean ± SEM where applicable.

Table 1 Sequences and optimized concentration of primers used in this study

Statistical analysis

GraphPad Prism 7.00 (GraphPad, La Jolla, CA, USA) was applied to analyze Data and statistical tests. Unpaired t-Test was used to compare the survival rates between treat group and the untreat group (control). Means were compared using a one-way analysis of variance (ANOVA), followed by a Tukey–Kramer post hoc test using a 95% confidence interval. Differences were considered significant at p < 0.05.

Results

Antimicrobial assay of CM-Cu₂O NPs

Figure 1 shows the in vitro antimicrobial activity of the dried seaweeds; CM-Cu₂O NPs, ciprofloxacin and cefalexin. Among the dried extracts, S. aureus and P. aeruginosa was the most effective displaying the largest inhibition zone especially with CM-Cu₂O NPs. Moreover, CM-Cu₂O NPs exhibited variable antibacterial influence against some isolates, but higher in comparison with the standard antibiotic disks being used (Table 2).

Fig. 1

Data for the Growth diameter (mm) after 72 h of incubation at temperature range of 15–30 °C

Table 2 IC50 values of HDF, MDA, SKBR and T47D cells lines incubated with CM-Cu₂O NPs for 24, 48 and 72 h

UV–visible spectral analysis of CM-Cu₂O NPs

The UV spectral analysis for the synthesized CM-Cu₂O NPs were done at regular interval of time. The color change from yellow to brown color primarily indicates the conversion of Cu into Cu₂O NPs. The UV spectrum of the synthesized CM-Cu₂O NPs evidently indicates the progression and stability, as shown in Fig. 2. The surface plasmon resonances of the CM-Cu₂O NPs were confirmed by the appearance of maximum absorbance at the 200 nm.

Fig. 2

UV–Visible spectrum of the synthesized CM-Cu₂O NPs

FT-IR analysis of CM-Cu₂O NPs

The FT-IR transmittance analysis was done for determining the functional groups present in the synthesized CM-Cu₂O NPs. The FT-IR analysis shows different characteristics peaks at 612.06 cm⁻¹, 1217.15 cm⁻¹, 1738.57 cm⁻¹ and 3016.89 cm⁻¹ between the range 600–4000 cm⁻¹ (Fig. 3). The slight broad band at 3016.89 cm⁻¹ corresponds to the N–H stretch due to amine group and the peak at 1738.57 cm⁻¹ shows the presence C=O bending due to the presence of aromatic secondary metabolites. The band at 1738.57 cm⁻¹ shows the presence of C–O bending due to the alkene group. The prominent peak at 612.06 cm⁻¹ confirms the presence the Cu–O vibration in the synthesized CuO NPs (Davoodi et al. 2013).

Fig. 3

FTIR spectrum of the synthesized CM-Cu₂O NPs

XRD, TEM and SEM analysis of CM-Cu₂O NPs

The X-ray diffractometer (XRD) analyses were carried out for the synthesized CM-Cu₂O NPs for verifying the crystalline nature of synthesized nanoparticles (Fig. 4). The XRD analysis of the Cu₂O NPs was interconnected with the Joint Committee on Powder Diffraction Standards (JCPDS) verifying the crystalline nature of Cu₂O NPs (JCPDS 96-901-5925). TEM (Fig. 5-A) and SEM (Fig. 5-B) were done to ascertain the morphological studies for the synthesized CM mediated Cu₂O NPs. The SEM analysis for the synthesized nanoparticles showed the formation of CM-Cu₂O NPs around 2 µm, 1 µm, 500 nm, 200 nm and 100 nm in an agglomerated cluster forms as shown in the (Fig. 5-B).

Fig. 4

XRD pattern of synthesized CM-Cu₂O NPs

Fig. 5

TEM (a) and SEM (b) images of synthesized CM-Cu₂O NPs

Cell take-up and Cu’s delivery into cell medium

The ICP-MS test results demonstrate that Cu particles were not distinguished in cell-free medium, while approximately 4% of the exposure dose was detected in the cells, which indicates the uptake of Cu₂O-NPs by HDF, MDA-MB-231, SKBR3 and T-47D cell lines following exposure for 24 h (Table 3). Cu₂ substance of the unexposed cell (NC) was additionally estimated for each cell line.

Table 3 Assessment of the cellular uptakes of CuO-NPs

CM-Cu₂O NPs-induced inhibition of proliferation

MTT: Cytotoxicity effect of CM-Cu₂O NPs was evaluated on four human cancer cells, i.e., HDF, MDA, SKBR and T47D. All cells were treated with different concentration of CM-Cu₂O NPs for incubation time of 24, 48 and 72 h. MTT was used to measure cell viability. The values of IC50 (µg/ml) were obtained for the cells. As presented in Table 2, the values of IC50 (µg/ml) were decreased in time of incubation dependent manner (Fig. 6a).

Fig. 6

Cytotoxicity of CM-Cu₂O NPs in HDF, MDA-MB-231, SKBR3 and T-47D cells. a CM-Cu₂O NPs decreased growth and proliferation of HDF, MDA, SKBR and T47D cells in concentration-dependent manner in all the time of incubation by MTT method and b Flow cytometry assays assess the apoptotic activity of paclitaxel-treated cells in HDF, MDA-MB-231, SKBR3 and T-47D at 24, 48 and 72 h post-treatment

PI: Following treatment of cells with different concentrations of CM-Cu₂O NPs, the findings revealed that the proliferation of MDA, SKBR and T47D cells was substantially inhibited at 15 μg/ml; these variations were statistically important compared to the corresponding values in the Cu-NPs (Fig. 6b).

Expression of apoptosis genes in BC cell lines

According to qPCR analysis on RNA samples from cancer cell lines, all of apoptosis genes, i.e., p53, p27, bax, bcl2 and caspase3 genes, were overexpressed in MDA-MB-231, SKBR3 and T-47D BC cell lines in comparison with HDF control cell line (Fig. 7).

Fig. 7

Expression levels of apoptosis genes, i.e., p53, p27, bax, bcl2 and caspase3 genes in BC cell lines for incubation time of 24, 48 and 72 h compared to normal cell line. qRT-PCR was used to quantify the expression levels of p53, p27, bax, bcl2 and caspase3 genes in BC cell lines. * Significant at the 0.05 level; **significant at the 0.001 level; ***significant at the 0.0001 level. Error bars show the minimum and maximum variables

LncRNA Expression Profiles in BC cell lines

The qRT-PCR was applied to assess the transcriptional status of MALAT1 and GAS5 lncRNAs in BC cell lines for incubation time of 24, 48 and 72 h. In comparison with the average expression in normal cell line, MALAT1 was upregulated in MDA-MB-231, SKBR3 and T-47D BC cell lines and GAS5 was upregulated in SKBR3 cell line tested (Fig. 8). While GAS5 exhibited lower expression levels in MDA-MB-231 and T-47D cell lines. While MALAT1 showed a fairly similar overexpression pattern in 24 h SKBR3 cell line and GAS5 showed lower expression levels in 72 h cell lines. We found a significant difference for MALAT1 and GAS5 lncRNAs (p < 0.001).

Fig. 8

Expression levels of lncRNAs in BC cell lines for incubation time of 24, 48 and 72 h compared to normal cell lines. qRT-PCR was used to quantify the expression levels of GAS5 and MALAT1 lncRNAs in BC cell lines. Error bars show the minimum and maximum variables

Correlations between the Expression of the lncRNAs

Table 4 shows the correlations between lncRNAs expression. There were no significant correlations between the lncRNAs expression. Error bars show the minimum and maximum variables.

Table 4 Correlation between LncRNA Expression

Correlations between the expression of the apoptosis genes

Table 5 shows the correlations among apoptosis genes (p53, p27, bax, bcl2 and caspase3). There were significant correlations between the lncRNAs expression (p > 0.05).

Table 5 Correlation between apoptosis genes

Correlations between lncRNAs expression and apoptosis genes

Table 6 shows the correlations between lncRNAs expression (MALAT1 and GAS5) and apoptosis genes (p53, p27, bax, bcl2 and caspase3). There was a significant correlation between lncRNAs expression and apoptosis genes (p > 0.05).

Table 6 Correlation between lncRNA expression and apoptosis parameters

Discussion

Cancer is believed to be one of the chief causes of death all around the globe. Undoubtedly, cancer patients’ care has been improved by the approach of modern drug-targeted therapeutics. However, advanced metastasized cancer is still untreatable. Therefore, it is necessary for look for a reliable and more efficient chemoprevention and treatment to develop the facilities and reduce treatment costs for cancer care. Cancer chemoprevention with natural phytochemical compounds is a novel technique for preventing and curing cancer (Wang et al. 2012). In the present study, our research groups have biosynthesized the stable and cost-effective CM-Cu₂O NPs. The synthesis of crystalline CM-Cu₂O NPs were confirmed by various analytical techniques like UV–Vis, FTIR, XRD and SEM. Further the synthesized CM-Cu₂O NPs were screened for anticancer activity on BC cell lines by MTT assay. The obtained result inferred that the synthesized CM-Cu₂O NPs demonstrated high anticancer cytotoxicity on BC cell lines (MDA, SKBR and T47D).

Copper oxide nanoparticles are applied increasingly for various purposes like antimicrobial preparations, heat transfer fluids, semiconductors or intrauterine contraceptive devices (Chen et al. 2011). Copper oxide nanoparticles have two key forms including CuO and Cu₂O. Some studies have shown the toxic influence of CuO associated with the production of reactive oxygen species (ROS), lipid peroxidation, and DNA damage (Fahmy and Cormier 2009) except for Cu₂O. Thus, it is essential to investigate the toxicity of Cu₂O NP that is believed to be a source of water-borne copper-containing species (Singh and Turner 2009). The solubility of nanoparticles is dangerous for the toxicity of these particles and their influences on ecosystems (Midander et al. 2009). Yet, some studies reported that higher stability could help the nanoparticles for penetrating, accumulating and persisting to a large extent in an organism (Midander et al. 2009). However, it is still not clear as to what extent the toxicity of particles can be ascribed to the soluble or released metal fraction or by the specific particle. It is believed that in the mechanism of the toxicity of CuNPs, the cellular membrane is ruptured (Karlsson et al. 2013). Because there is a higher O2 concentration in the cell membrane compared to the cell media, CuNPs are oxidized as they go through the cell membrane, producing copper ions. Thus, the cellular rupture could be the result of the metal release process into the cell, which results in the production of H2O2 locally at the cell membrane (Vanwinkle et al. 2009). Note that rupturing of the cellular membrane is not only caused by the released copper ion into the cell, but also is brought about by the direct exposure of human alveolar epithelial cell line A549 to the copper ions resulting in less toxic damage to the membrane (Karlsson et al. 2008; Midander et al. 2009). Note that copper ion release process from CuNPs into the vicinity of membrane surface is a serious stage in CuNPs toxicity. Other studies have used carbon-coated CuNPs and CuCl₂, both of which did not bring about membrane rupture, while CuNPs induced rupturing of the cell membrane (Minocha and Mumper 2012). It could be said that the ultimate fate of Cu-metal nanoparticles after going into the body is degradation and production of Cu2 + ions. Thus, after exerting their cytotoxic effect on cancer cell and being released as ions, they will not be cytotoxic, since Cu2 + ion is not cytotoxic in concentrations lower than 500 μM.

Apoptosis or cell suicide is a highly regulated process happening in almost all living cells. In apoptosis, a series of molecular events is activated that results in cell death characterized by cellular, morphological and biochemical variations including cell shrinkage, chromatin condensation and nuclear fragmentation, membrane blebbing, caspase activation, and the formation of membrane bound vesicles termed as apoptotic bodies (Balachandran et al. 2014). Two key pathways are involve in apoptosis. The first, called the extrinsic or cytoplasmic pathway, is activated through the Fas death receptor, a member of the tumor necrosis factor (TNF) receptor superfamily (Zapata et al. 2001). The second pathway is the intrinsic or mitochondrial pathway that when stimulated results in the release of cytochrome c from the mitochondria and activation of the death signal (Hockenbery et al. 1990). Both pathways converge to a final common pathway and trigger a cascade of proteases called caspases that cleave regulatory and structural molecules, terminating in the death of the cell. The main pathway resulting in the execution of the death signal is the activation of a series of proteases termed caspases. Not all caspases play a role in apoptosis. Caspases are cysteine-aspartic proteases families involved significantly in apoptosis, necrosis and inflammation. Caspases are regulated at a post-translational level making certain that they can be quickly triggered. The caspases implicated in apoptosis can be further categorized into two functional subgroups according to their known or hypothetical roles in the process: initiator caspase (caspases-2, -8, -9, and -10) and effector caspases (caspases-3, -6, and -7). The caspases that have been well described are caspases-3, -6, -7, -8 and -9 (Mancini et al. 1998). Caspase-3 is considered as a key protease triggered during the early stages of apoptosis (Walsh et al. 2008). Caspases are triggered in a sequential cascade of cleavages from their inactive forms and the active caspase-3 proteolytically cleaves and triggers other caspases and other relevant target molecules in the cytoplasm or nucleus (Brentnall et al. 2013; Walsh et al. 2008). The process of cell death could involve discharging cytochrome c from the mitochondria leading consequently to apoptosis through triggering the caspases. Together, these data recommend a linear and specific activation cascade between caspase-9 and caspase-3 in response to cytochrome c discharged from the mitochondria (Shalini et al. 2015). Activation of caspase-9, in turn, cleaves effector caspases such as caspase-3, -6, and -7 (Porter and Jänicke 1999; Shalini et al. 2015). Then the effector caspases cleave their target proteins and culminate in the systematic death of the cell. In this pathway of apoptosis, caspase-3 and caspase-9 could be the key factors, since their activities affects the process of apoptosis as well as the cell death type (Balachandran et al. 2014). Caspase-3 then could be capable of cleaving a series of proteins such as PARP, a DNA repair enzyme, nuclear lamins, gelsolin, and fodrin (Chang and Yang 2003). In the mitochondria, the tBid efficiently triggers Bax, leading to the release of cytochrome c and mitochondrial dysfunction. This alternative is a crosstalk between the receptor and mitochondria mediated pathways that is capable of amplifying the caspase activation required for apoptosis. Actually, more recent studies have turned their attention to multiple signal transduction cascades activating cells to experience apoptosis (Lockshin and Zakeri 2004). Caspase 3 is a potential marker to predict response or resistance to chemotherapeutic agents in BC (O’Donovan et al. 2003). In fact, recent data from BC cell lines confirm this hypothesis. For instance, Blanc et al. (2000) argued that caspase 3 was necessary for procaspase 9 processing and cisplatin-induced apoptosis in MCF7 BC cells. Applying the same cell lines, Yang et al.(Yang et al. 2001) showed that transfection with cDNA for caspase 3 resulted in doxorubicin- and etoposide-induced apoptosis. In comparison with other genes involved in apoptosis (e.g., p53 and the Bcl-2 family), studies on caspase expression in BC are scarce.

It is believed that p27 is an important factor of the G₁- to S-phase transition in the cell cycle that is involved in tumor progression (Matsunobu et al. 2004). P27 is a member of the Cip/Kip family of cyclin-dependent kinase inhibitors present at high levels in quiescent cells (Sherr and Roberts 1995). The gene encoding p27 is seldom mutated; however, p27 is functionally inactivated in most human cancers through improved p27 proteolysis, through sequestration by cyclin D/cyclin-dependent kinase complexes, and by cytoplasmic mislocalization (Filipits et al. 2009). Guan et al. (2010) reported that further studies were required to shed light on the role of cytoplasmic p27 in BC. Porter et al. (1997) assayed both p27 and cyclin E levels in 246 primary BC of women under 45 years of age. Patients whose BCs showed both low p27 and elevated cyclin E proteins had the highest mortality, and multivariate analysis demonstrated that both levels of p27 and cyclin E were independent predictors of overall survival. Tan et al. (1997) reported that the prognostic value of p27 in 202 patients with BCs was less than 1 cm in size (T1a,b) in comparison with clinicopathological features and other parameters such as p53, c-erb-B2, Ki-67, cdc25B, and the density of microvessels. This study revealed that low p27 level, established as less than 50% of the tumor cells being positive for p27, was not a dependent risk factor on multivariate analysis and was linked to a 3.4-fold increased risk of death, especially in node-negative tumors. The p53 tumor suppressor gene inhibits tumourigenesis in response to physiological and environmental stress and is involved in cell cycle progression, apoptosis and repair of DNA damage. P53 may play a role in transcriptional regulation of pro-apoptotic genes linked to intrinsic and extrinsic pathways (Liu et al. 2014). Triggered p53 increases the expression of p21 in DNA damaged cells and influences expression of p27 (Balachandran et al. 2014). The loss of the p53 function shows the most common genetic change known in human cancers. Haldar et al. argued the down-regulation of Bcl-2 at both the protein and mRNA levels was induced due to the overexpression of mutant p53 in BC (MCF-7) cell line (Haldar et al. 1994). Some studies have confirmed the involvement of Bcl-2 in the control of apoptosis; recent experimental studies have confirmed that Bcl-2 is implicated in regulating the cell cycle and proliferation (Bonnefoy-Berard et al. 2004). The Bcl-2 deficient T cells showed an accelerated cell cycle progression (Linette et al. 2002). However, the cells overexpressing the Bcl-2 gene product not only revealed a delayed start of apoptosis but also a quick arrest in the G1 phase of the cell cycle (Marvel et al. 1994) indicating that Bcl-2 is involved in the transition from G0/G1 to S phase (Bonnefoy-Berard et al. 2004). Vairo et al. also confirmed the Bcl-2 hindered the cell cycle entry by increasing the p27 and p130 levels (Vairo et al. 2000). Greider et al. reported that Bcl-2 could not hinder the S phase entry in p27 null cells indicating that p27 is necessary for the cell cycle regulation of Bcl-2 (Greider et al. 2002). According to these experimental findings, Bcl-2 directly increased the level of p27 (Greider et al. 2002; Linette et al. 2002; Marvel et al. 1994), thus regulating the cell cycle and progression (Bonnefoy-Berard et al. 2004). Triggering p53 induces up-regulation of pro-apoptotic Bax but not anti-apoptotic Bcl-2. P53 mediated apoptosis play a role in triggering Fas and the intrinsic mitochondrial pathway leading to the activation of caspases-8 and -9 (Liebermann et al. 2007). Now, the cancer biology requires identifying new active chemotherapy drugs capable of acting as lead compounds for effective drug development. Thus, the BCL2/BAX ratio within a cell is a critical determinant of apoptosis and a reduced BCL2/BAX ratio is a factor causing endometrial cells susceptible to apoptosis. Some studies believe that dysfunction of the p53/BCL2/BAX apoptosis signaling pathway is involved in tumorigenesis and tumor progression (Argiris et al. 2006; Vaskivuo et al. 2000).

In this study, we examined the expression of apoptosis genes (p53, p27, bax, bcl2 and caspase3) in MDA-MB-231, SKBR3 and T-47D BC cell lines compared to HDF control cell line. The results revealed that p53, p27, bax and caspase3 were significantly upregulated in BC cell lines as compared with normal cell line (Fig. 7). On the other hand, bcl2 expression was also significantly increased in MDA-MB-231 and T47D cell lines compared with normal cell line, but bcl2 levels were downregulated in SKBR3 cell line. As a limitation of our study, differential expression in protein for p53, p27, bax, bcl2 and caspase3, which may reduce the accuracy of the results. A main reason for the application of a cytotoxic chemotherapeutic agent is its potential for inducing apoptosis and cell death in cancer cells (Hannun et al. 1997), because during apoptosis, the cells die with no inflammatory responses (Satchell et al. 2003). Statistics show that BC has developed uncontrollably necessitating the discovery and development of a novel anticancer agent having less side effects for normal cells (Parkin et al. 2009).

lncRNAs have been brought to light lately because of their confirmed involvement in the molecular pathobiology of several cancers (Zhao et al. 2014). BC has been observed in women of different ages, but that affecting younger women is of poorer prognosis (Santos et al. 2014). The present study assessed the expression levels of two lncRNAs, i.e., MALAT1 and GAS5 in the MDA-MB-231, SKBR3 and T-47D BC cell lines. According to our expression analyses, MALAT1 was upregulated, while GAS5 was downregulated in cell lines (Fig. 8). As discussed above, some studies have confirmed poorer prognosis for BC (Arshi et al. 2018). Some studies have shown that GAS5 has a tumor-suppressive role by controlling mammalian cell apoptosis, and its downregulation is reportedly is involved in tumor formation (Mourtada-Maarabouni et al. 2009). Moreover, there is a link between GAS5 low expression levels and a poor prognosis in head and neck squamous cell carcinoma (Gee et al. 2011). Thus, our finding of significant GAS5 downregulation in SKBR3 and T-47D cell lines tested compared to HDF cell line may explain the molecular mechanism causing a poorer prognosis of BC in this age group.

Conclusions

It was shown in this study that the leaves of Cystoseira myrica can be an unconventional resource for the green synthesis of CM-Cu₂O NPs. The analytical techniques like UV–Vis, XRD, TEM and SEM were applied to characterize the nature of the synthesized CM-Cu₂O NPs. It was also shown that the CM-Cu₂O NPs synthesized from the leaves of Cystoseira myrica have significant anticancer activity in BC cell lines. Moreover, identifying and isolating the biologically active compound from the leaves extract of Cystoseira myrica could facilitate the discovery of novel anticancer drug. The superior effectiveness and lower toxicity of new anticancer drug are mostly because of applying nanosized Cu₂O based drug designing molecules. According to new studies, various natural products are capable of inducing the apoptosis of cancer cells, and inhibiting metastasis and tumor cell growth highlighting the application and advantage of these natural compounds as of new medical treatment of human cancer. According to in vitro studies, CM-Cu₂O NPs, which is commonly used among current cell lines utilize in this study, is an anticancer substance with proapoptotic characteristics in terms of human cancer cells. Our study obviously showed that CM-Cu₂O NPs alters the expression level of some critical genes in various human cancer cells. These findings show that CM-Cu₂O NPs could probably be used as a therapeutic anticancer drug. Previous research works and the data in our analysis show that dysregulation of lncRNA expression is a main component in the tumorigenesis process. Yet, the expression pattern of these RNAs is different in various BC cell lines, an idea shedding light on the significance of considering the variable pathobiology of tumors upon the application of therapeutic approaches. Accordingly, the present study particularly revealed that lncRNAs evaluated in this study show different levels of expression in BCs developed in BC cell lines compared to normal cell line. Specifically, previous studies supported the fact that different expression of one of these differentially expressed lncRNAs, i.e., GAS5, could shed light on the worse result of BC in patients at molecular levels. These findings can also be used to define future therapeutic regimens.