Resveratrol-based nano-formulations as an emerging therapeutic strategy for ovarian carcinoma: autophagy stimulation and SIRT-1/Beclin/MMP-9/P53/AKT signaling

Abstract

Background

Resveratrol (RVS) is a stilbene derivative polyphenolic compound extensively recognized for its anti-inflammatory, antioxidant and anti-aging properties, along with its enormous promise in carcinoma treatment. Unfortunately, the oral supplementation of RVS possesses physicochemical and pharmacokinetic constraints that hinder its effects, necessitating the development of suitable administration strategies to improve its effectiveness. As a result, the current study evaluates the use of resveratrol nano-formulations in ovarian cancer therapy. Ovarian cancer was induced in rats using (35 mg/kg BW) 20-Methyl cholanthrene (20-MC) followed by resveratrol and resveratrol nano-formulations therapy for one month.

Results

20-MC highlighted a noticeable alleviation in autophagy (ATF) biomarkers SIRT-1 and Beclin, inflammatory and apoptotic biomarkers MMP-9, P53 and AKT in addition to oxidative and nitrosative stress biomarkers TAC and NOX and ovarian cancer tumor biomarker CA-125.

Conclusions

Resveratrol and resveratrol nano-formulations modulated autophagy, inflammatory and oxidative stress biomarkers with the upper effect for resveratrol nano-formulations in competing 20-MC-induced ovarian cancer.

Introduction

Molecular targeted medicine has emerged as a prospective technique for overcoming the defect in specificity of chemotherapy, and nanotechnology emerges as a powerful strategy in this context. For cancer treatment, numerous smart drug delivery technologies, including liposomes or metallic nanoparticles, are currently being employed. Furthermore, there has been a growing emphasis on naturally existing bioactive compounds for chemoprevention and chemotherapy in various types of carcinoma, in addition, there is an obvious concentration on nano-therapy, in which nano-formulations cancer prevention is in an advanced progress (Navya et al. 2019). Alternative nano-formulations were compared to existing anticancer medications; among other things, they revealed more stability, efficacy, solubility and bio-distribution (Navya et al. 2019). A milestone in carcinoma therapy is the overview of nano-medicine in controlling tumor action and hindering its malignant characteristics. The foremost applications for NPs in carcinoma treatment was diagnosis, cargo delivery (genes or drugs) and non-invasive strategy for malignancy eradication. NPs possess important benefits in carcinoma treatment field, which support its use in oncology, in both clinical and preclinical stages. The advantages of NPs are briefly promoting therapeutic efficacy and reducing cytotoxicity (Dai et Al. 2024). NPs was utilized in phototherapy to enhance singlet oxygen production and hyperthermia for tumor ablation (Wang et al. 2024). NPs, engineered to redesign the tumor microenvironment, revealed prospective outcomes in promoting immunotherapy via immune modulation and targeted delivery by enhancing M1 macrophage polarization, reducing fibroblast activation, encouraging T cell infiltration and promoting dendritic cell maturation (Lu et al. 2024; Li et al. 2024).

ATF cellular machinery involves cytoplasmic substrates degradation in lysosomes via autophagosomes. The frequent elimination of old, damaged and useless cellular molecules via the consecutive translation among amino acids and proteins by protein synthesis, and autophagic protein degradation, aids cellular homeostasis. Developing researches demonstrated that autophagy plays a vital regulatory part in ovarian cancer (OC). Remarkably, microRNAs control gene expression in the posttranscriptional level and consequently can adjust the progression and development of OC via regulating autophagy.

Maturation of autolysosomes occur via the interaction with Beclin1 proteins. After fusion, autolysosomes carry dysfunctional organelles and misfolded proteins that are degraded by lysosomal acid hydrolases for recycling and metabolism in the substrate. Bovine ovarian granulosa cells (BGCs) as a model to elucidate autophagy reflected the role of miRNA-21-3p in bovine ovaries by preventing autophagy in BGCs by hindering AKT/mTOR signaling (Ding et al., 2024).

Polyphenols (PP) are gaining popularity among active natural chemicals because to their high bioavailability and several therapeutic benefits, including anti-inflammatory, antioxidant, anticancer and anti-aging characteristics (Karakaya et al. 2019). In fact, the significance of PP as modulators of oxidative stress in cell carcinoma has received considerable attention. However, with limited stability as a restrictive element, nano-PP is regarded as an effective strategy for overcoming some limitations in safety, targeted power and pharmacokinetics concerns. Among this diverse group of chemicals, resveratrol has sparked significant scientific attention due to its anticancer and antioxidant properties (Summerlin et al. 2015). RVS is a vital antioxidant found in blueberries, peanuts and red grapes (Jeandet et al. 2021).

RVS (3,5,4′-trihydroxy-trans-stilbene) and is a natural stilbenoid. RVS is a phytoalexin, secondary metabolite of plants generated as an adaptive reaction to environmental stressors such as ultra violet irradiation, damage and fungal infections. Resveratrol has shown beneficial chemotherapeutic and chemo-preventive advantages against particular kinds of cancer by virtue of its broad antioxidant action (Oztürk et al., 2017).

RVS interfere with cancer initiates, expansion and progression in tumor microenvironment (Velmurugan et al. 2018 ; Huang et al. 2017). Resveratrol decreases tumor-derived NOS production, promotes tumor cell apoptosis, suppresses tumor migration and growth and prevents COX action. These detailed procedures have been confirmed in various kinds of cancer (Singh et al., 2013). In fact, the postulated resveratrol's anticancer potential includes pro-apoptotic and anti-proliferative activities and the power to control the expression of tumor suppressor microRNAs, NFƙB, nuclear respiratory factor-1 and 2, P53, peroxisome proliferator-activated receptor (PPAR) and pro-oncogenic genes. Although it is exceptional abilities (Zhang et al. 2019), RVS possess restrictions of low water solubility, inadequate oxidative stability and photosensitivity, which severely limit its applicability. As a result, nanotechnology may be an intriguing solution to overcome these constraints (Ahmadi et al., 2019).

In this regard, the present article confers the anticancer machinery of resveratrol and places special highlights on the prospective use of its nano-formulations as a developing cancer remedy, using liposomal nano-delivery systems against 20-MC-induced ovarian cancer while monitoring autophagy and inflammatory pathways.

Material and methods

Chemicals

Liposomal-resveratrol was supplied from Biolife (USA), whereas 20-MA and resveratrol were procured from Sigma-Aldrich Co (St. Louis, MO, USA). SIRT-1 and Beclin RT-PCR kits (Qiagen, USA). MMP-9, AKT and P53 ELISA kits (R&D Systems, USA). All of the chemicals used have a high analytical rank.

Animals and treatments

Western Albino rats (No.40: male) weighing 100–150 g from the National Research Center's animal house have been utilized in the present investigation. The animals were raised under controlled circumstances (22 5 °C, 55% humidity, and a 12-h light/dark cycle). Animals were provided with access to water and a typical diet. All animal care and treatment techniques closely follow to the ethical protocols and policies established by the Animal Care and Use Committee of the National Research Center (19–302) and the US National Institute of Health.

Experimental design

Proceeding acclimatization, animals were put in five cages (8 rats):

• G1: Control group administered Tween-80.

• G2: Administered 20-methylcholanthrene (IV, 30 mg/kg/10 days for 5 months) (ovarian cancer model)(Lin et al. 2011a).

• G3: Administered resveratrol (10 mg/kg BW) IP for 1 month.

• G4: Administered liposomal administered (3 mg/kg BW) IP for 1 month (Luo et al. 2013). 

• G5: Administered Gemzar (10 mg/kg BW) IP for 1 month (standard ovarian cancer drug).

Blood sampling and tissue preparation

Rats were weighed and sedated (with carbon dioxide). After gathering sera from the retro-orbital vein, samples were centrifuged at 5000 rpm for 10 min before being preserved at −80 °C. Rats were euthanized, and ovarian tissue was isolated and homogenized in phosphate buffered saline for further analysis.

Measured biochemical parameters

Ovarian oxidative stress biomarkers

Total antioxidant capacity and nitric oxide were estimated via kits provided from Randox Company. Further, the absorbance was determined spectrophotometry at 505 nm (V-730 UV–visible spectrophotometer-Jasco Inc.) (Abdel-Megeed et al., 2020a).

mRNA gene expression of ovarian SIRT-1 and Beclin

Initially, total RNA was isolated from ovarian tissue using the RNA-easy mini-kit (Qiagen, Germany) and amplified using the RT-PCR kit (Qiagen, USA). The reaction was carried out in a total volume of 20 L of master mix. The thermal profile was as follows: 50 ^oC for 2 min, 95 ^oC for 10 min, and 95 ^oC for 45 to 60 ^oC for 30 s, 72 ^oC for 30 s, and 72 ^oC for 10 min (Agilent, MxPro qPCR, Mx3000P). The ∆∆CT fold change method was used to calculate gene analysis. SIRT-1 and Beclin primers (forward and reverse) illustrated in Table 1 (Kadry et al. 2023b; Abdel-Megeed, et al 2020a,  2020b).

Table 1 Primers sequence designed for RT-PCR gene expression

ELISA determination of ovarian CA-125, MMP-9, P53 and P-AKT

The activities of CA-125, MMP-9, P53, and P-AKT were measured using an enzyme-linked immunosorbent assay kit (R&D systems, MN, USA) according to the manufacturer's instructions. The experiment was then evaluated using a quantitative sandwich enzyme immunoassay. The microplate was then pre-coated with the appropriate antibodies. The immobilized antibody that attached to CA-125, MMP-9, P53, and P-AKT was then added, followed by the enzyme-linked secondary antibody specific for CA-125, MMP-9, P53, and P-AKT. The absorbance was then measured at 450 nm. The color intensity (at 450 nm) was measured (Agient BioTek Microplate reader, Neo2) (Kadry and Abdel-Megeed, 2022; Abdel-Megeed et al., 2020b).

Statistical analysis

The data were provided in terms of mean standard error of the mean (SEM). For the statistical analysis, GraphPad Instat 3 (GraphPad Software Inc., San Diego, CA, USA) was utilized. SPSS 16 was used to analyze the data using one-way ANOVA, followed by a post hoc Tukey's test. P-values of less than 0.05 were considered statistically significant.

Results

Modulating protein expression of CA-125

The protein expression of the ovarian cancer biomarker CA-125 was dramatically increased by 20-methyl cholanthrene. Meanwhile, resveratrol, liposomal-resveratrol, and Gemzar considerably improved CA-125 protein expression when compared to the control value, with liposomal-resveratrol outperforming the others (Fig. 1).

Fig. 1

Impact of resveratrol and liposomal-resveratrol on serum CA-125 post 20-MC-induced ovarian cancer. Data are expressed as means ± SEM (n = 8), P < 0.05. Groups having different letters are considered significantly different, while, groups having similar letters are not significantly different from each other

Modulating SIRT-1 and Beclin gene expression

The autophagy biomarkers SIRT-1 and Beclin were dramatically upregulated by 20-methylcholanthrene, indicating endoplasmic reticulum stress. Meanwhile, resveratrol, Liposomal-resveratrol, and Gemzar considerably modified these changed genes when compared to the control, with liposomal-resveratrol having the greatest impact (Figs. 2, 3).

Fig. 2

Impact of resveratrol and liposomal-resveratrol on ovarian SIRT-1 gene expression post 20-MC-induced ovarian cancer. Data are expressed as means ± SEM (n = 8), P < 0.05. Groups having different letters are considered significantly different, while, groups having similar letters are not significantly different from each other. Β-actin was used as reference gene

Fig. 3

Impact of resveratrol and liposomal-resveratrol on ovarian Beclin-1 gene expression post 20-MC-induced ovarian cancer. Data are expressed as means ± SEM (n = 8), P < 0.05. Groups having different letters are considered significantly different, while, groups having similar letters are not significantly different from each other. Β-actin was used as reference gene

Modulating protein expression of MMP-9, P-AKT and P53

20-Methyl cholanthrene significantly increased MMP-9 protein expression while decreasing P53. Meanwhile, resveratrol, liposomal-resveratrol, and Gemzar greatly improved these changed proteins when compared to the control value, with liposomal-resveratrol outperforming the others (Figs. 4, 5 and 6).

Fig. 4

Impact of resveratrol and liposomal-resveratrol on ovarian MMP-9 protein expression post 20-MC-induced ovarian cancer. Data are expressed as means ± SEM (n = 8), P < 0.05. Groups having different letters are considered significantly different, while, groups having similar letters are not significantly different from each other

Fig. 5

Impact of resveratrol and liposomal-resveratrol on ovarian P53 protein expression post 20-MC-induced ovarian cancer. Data are expressed as means ± SEM (n = 8), P < 0.05. Groups having different letters are considered significantly different, while, groups having similar letters are not significantly different from each other

Fig. 6

Impact of resveratrol and liposomal-resveratrol on ovarian AKT-1 protein expression post 20-MC-induced ovarian cancer. Data are expressed as means ± SEM (n = 8), P < 0.05. Groups having different letters are considered significantly different, while, groups having similar letters are not significantly different from each other

Modulating oxidative stress biomarkers

20-Methyl cholanthrene increased the oxidative stress biomarker NOX while decreasing the anti-oxidative stress biomarker Total antioxidant capacity, indicating an oxidative stress and redox imbalance. Meanwhile, resveratrol, liposomal-resveratrol, and Gemzar considerably modified these changed biomarkers as compared to the control, with liposomal-resveratrol having the greatest impact (Figs. 7, 8).

Fig. 7

Impact of resveratrol and liposomal-resveratrol on ovarian NOX level post 20-MC-induced ovarian cancer. Data are expressed as means ± SEM (n = 8), P < 0.05. Groups having different letters are considered significantly different, while, groups having similar letters are not significantly different from each other

Fig. 8

Impact of resveratrol and liposomal-resveratrol on ovarian TAC level post 20-MC-induced ovarian cancer. Data are expressed as means ± SEM (n = 8), P < 0.05. Groups having different letters are considered significantly different, while, groups having similar letters are not significantly different from each other

Discussion

Autophagy is an auto-destructive mechanism that guarantees the survival or promotes the apoptosis of abnormal cells, going ahead the environments of damage, stress and pathogenic poison. Furthermore, autophagy results in the removal of misfolded protein, malfunctional organelles, and the elimination of pathogenic molecules (Elshaer et al. 2018).

Herein, 20-MC elevated autophagy biomarkers including SIRT-1, Beclin and AKT gene expression on the other hand reduced tumor suppressor P53 meanwhile, resveratrol and liposomal-resveratrol modulated these altered genes with the superiority of liposomal-resveratrol. This is consistent with a research conducted on 50 animals treated with 20-MA, in which cystic tumors were developed in 17 cases and solid tumors in 20 cases. The time lapse required for tumor developing was at least 20 weeks, and the longer the observation period, the more tumors generated. After 30 weeks, the development rate of solid tumors was 22.5% (Toshi et al., 1973).

The carcinogen 20-methylcholanthrene (20-MCA) induces cancers in almost all animal species. This chemical is a polycyclic aromatic hydrocarbon (PAH), which has been linked to both animal and human cancers. These compounds are commonly present in the environment, in water, cigarette smoke, food and motor combustion. (20-MCA) is converted enzymatically into a number of metabolites, some of which are reactive. 20-MCA has been employed as a successful animal model in the study of chemoprevention and carcinogenesis in a vast set of enzymes implicated in carcinogenic metabolism. Cell carcinoma is the worldwide cause of mortality globally, and the failure of traditional chemotherapy to significantly reduce mortality suggests that alternative techniques are urgently needed. Resveratrol's ability to cause autophagy apoptosis by triggering autophagy in apoptotic-deficient cells is a plausible technique for its potential application in cancer treatment. The most prevalent resveratrol signaling pathways involve autophagy induction via enhanced stimulation of MAPK and SIRT1 and hindering Akt/mTOR as noticed in NSCLC. RVS can promote autophagy and accelerate the degradation of p62, as well as inhibit the Nrf2/ARE pathway (Wang et al. 2018). Resveratrol increased PTEN expression while decreased the phosphorylation of AKT in breast cancer. Resveratrol inhibits stromal interaction molecule 1 (STIM1) and inactivates the mTOR pathway in prostate and lung cancers (LCs), whereas it promotes autophagy in colorectal cancer by triggering ROS release and promoting caspases-8 and 3 (Miki et al. 2012; Chang et al. 2017). However, it activates Beclin-1 in ovarian cancer. In conclusion, exaggerated autophagy causes cancer cell apoptosis (Alayev et al. 2015).

RVS has been shown in cellular regulatory mechanisms to induce apoptosis by conjugation with v3 integrin and activating (ERK)1/2 via MAPK-kinase (Elshaer et al. 2018). RVS also interferes in c-Jun and p38 kinases, which has been proven to be the clue in ovarian, breast and prostate carcinoma (Yang et al. 2011). Stimulated ERK1/2 causes P53 phosphorylation in various cell lines for cancer (Lin et al. 2011b), and that RVS can trigger P53-correlated apoptosis. Actually, p53 can be linked to DNA in its activated state and is involved in apoptosis. Furthermore, RVS can increase COX-2 nuclear accumulation via p53-correlated apoptosis in ovarian, and breast cancer (Jang et al. 2022).

In this study, 20-MC increased NOX, MMP, and AKT levels, whereas decreasing TAClevels; resveratrol and liposomal-resveratrol regulated their levels with the superiority of liposomal-resveratrol. Angiogenesis is an essential for cancer progression and growth as cancer cells demand blood vessels for nutrition and oxygen to proliferate and metastasize. Elevation in matrix metalloproteinase (MMP) expression is important for angiogenic process. RVS can decrease endothelial cell migration and adhesion by decreasing MMP-2 and NO action along neo-angiogenesis. This technique is especially useful in chemoprevention. Resveratrol also inhibits the accumulation of HIF-1α and increases thrombospondin-1 (TSP1), a natural inhibitor of angiogenesis (Trapp et al. 2010). NO is involved in cell angiogenesis and proliferation, both can promote tumor metastasis and growth (Trapp et al. 2010).

Metastasis is the mechanism by which cancer cells move to healthy tissues, typically via the lymph or blood. By the way, RVS reduce MMP-9&2, AKT, α-SMA, P-PI3K and Smad-2&3 expressions (Sun et al., 2019). These factors are associated with the TGF-1-induced EMT, and RVS can suppress MDA231 cell migration via EMT. Importantly, tumor invasion and metastasis are linked to the EMT pathway in ovarian cancer (Guarino, 2007).

Resveratrol increased P51 and 53 and α-galactosidase in human NSCLC at a low dose. Thus, the processes by which resveratrol might cause accelerated aging have been linked to an increase in DNA double-strand breaks, ROS generation and reducing GSH (Kumar et al., 2015).

As previously stated, the majority of resveratrol's therapeutic advantages are associated with its anti-inflammatory, antioxidant and neuro-protective properties (Carrizzo et al. 2013).

Nano-medicine are drug delivery systems that is highly being used to counteract the restrictions of drugs instability, poor bioavailability, large dosing requirements, rapid first pass metabolism and poor pharmacokinetics (Mudshinge et al. 2011; Jadhav et al. 2016). NPs encapsulated RVS protects resveratrol from UV light by enhancing bioavailability (Summerlin et al. 2015). On the other hand, liposomal-RVS in nude mice is efficient anti-proliferative agents in brain cancer cells via triggering apoptosis and limiting cell growth. RVS nano-formulation mixed with dequalinium-induced cell death via a mitochondrial pathway in two lung cancer cell lines. Javadi et al. (2018) discovered that liposomal-RVS possess stronger anti-proliferative action and also reduced naked mice tumor formation. Furthermore, the combination of folic acid + RVS-NPs reduced anti-apoptotic (BCL-2 and BCL-XL) genes and NF-kB, COX-2, p65, and pro-apoptotic (BAX and BAK), however a reduction in cancer cells survival rate and promotion in the cleaved caspase-3 expressions were also observed (Singh et al. 2018).

Polymeric NPs-RVS dramatically reduced cell viability in breast and lymph node cancer of the prostate (LNCaP) cells (Wang et al. 2017). Various processes were involved in this example, including apoptosis induction, cell cycle arrest at G1/S, phosphatidyl serine externalization, DNA damage, ROS generation, and mitochondrial membrane potential loss (Nassir et al., 2018; Serini et al. 2018). Casein micelle-RVS significantly reduced cell proliferation, tumor volume and growth biomarkers in breast cancer cells (El-Far et al. 2018). RVS-loaded lipid-core-nano-capsules also inhibited cancer growth in HT29 cells by triggering 36% cell death (Feng et al. 2017a, b). In murine melanoma cells, resveratrol-loaded nano-capsules decreased cell viability, tumor development and inhibited metastasis and lung bleeding (Carletto et al. 2016; Song et al., 2018; Medina-Aguilar et al. 2016). Another study found that encapsulating RVS in an SLN dose-dependent way inhibited proliferation, promoted apoptosis, arrest cell cycle at G0/G1 (Wang et al. 2016) and decreased cyclin D1 level in MDA-MB-231 cancer cells (Jiang et al. 2017; Kadry, 2019). Regardless of encouraging preclinical results, the replication of resveratrol's promise in clinical investigations has been impeded by its limited half-life, low solubility, instability and poor bioavailability. RVS is quickly metabolized to create sulfates and glucuronides thus eliminated in clinical trials (Sanna et al. 2013a, 2013b; Siddiqui et al. 2015; Shindikar et al. 2016). In this regard, various trials were invented to create nano-medicine techniques to accomplish acceptable bioavailability and boost effectiveness in an acceptable dose in order to counteract pharmacokinetic and physicochemical restrictions in clinical studies.

(Guo et al. 2013; Guo et al. 2010 discovered that nano-RVS elevated RVS concentrations in the ovary and delayed ovarian cancer growth in regard to free RVS. Tumor growth was inhibited by free and nano-resveratrol at 46% and 62%, respectively (Guo et al. 2010). Furthermore, the inclusion of resveratrol in SLN resulted in a decrease in cell proliferation, which is advantageous in the prevention of skin cancer. (Teskač and Kristl. 2010) found that resveratrol-SLN had higher solubility, stability, and intracellular delivery.

Sanna et al. (2012) created resveratrol-loaded poly(d,l-lactide-co-glycolide) nanoparticles coated with cationic chitosan (CS-) and anionic alginate (Alg-). Nanoparticles were effective in delivering RVS in an organized manner. RVS encapsulation provides considerable protection against light-sensitivity, indicating its impact in cancer treatment. When compared to free resveratrol, the impact of nano-RVS on human PC cells resulted in increased cytotoxicity (Sanna et al. 2013b; Nassir et al. 2018). Singh et al. (2017) found that RVS-NPs increased the power of NCI-H160 cells by decreasing the potential of mitochondrial membrane and cell variability while increasing reactive oxygen species, cytotoxicity and DNA mutilation. According to HPLC studies, RVS-NPs had longer half-life and higher bioavailability than RVS (Shangmei et al. 2024).

Conclusion

Nano-resveratrol could be a promising candidate for 20-MCA induced ovarian cancer therapy via regulating autophagy biomarkers (SIRT-1 and Beclin), inflammatory and apoptotic biomarkers (MMP-9, P53 and AKT).