Scutellarin regulates MAPK/ERK signalling in nasopharyngeal cancer via the apoptotic and ROS induced DNA damage

Highlights

• •NPC - significant risk to public health in areas where it is endemic.
• •Scutellarin fights cancer by acting on a variety of signaling pathways.
• •NPC cells were exposed to scutellarin (20 and 30 μM/ml).
• •Proliferation and apoptosis were evaluated.
• •Inducing apoptosis via the signaling pathways.
• •Scutellarin may be effective conventional therapeutic drug.

Abstract

Nasopharyngeal carcinoma (NPC) occurs frequently, and NPC poses a significant risk to public health in areas where it is endemic. Better care is needed because NPC is associated with considerable morbidity and mortality. A natural anticancer substance called scutellarin fights cancer by acting on a variety of signalling pathways. Nevertheless, little is known about the underlying apoptotic and anti-proliferative actions of scutellarin. The current study aimed to determine the molecular effects of in vitro scutellarin on CNE1 human NPC cells through mechanisms such as cell proliferation, anti-inflammatory, and anti-apoptotic effects. NPC cells were exposed to scutellarin (20 and 30 μM/ml), and their proliferation and apoptosis were evaluated using the MTT assay, AO/EB, Rh-123, DCFH-DA, DAPI, and PI staining, cell adhesion, cell migration, and western blot analysis. We evaluated putative molecular pathways, MAPKs/NF-κB signaling, MMP, and intracellular ROS, cell proliferation regulatory proteins. By generating intracellular ROS, causing MMP loss and inducing apoptosis via the signalling pathways of TNF-α, COX-2, iNOS, and IL-6, pRB, cyclin-D1, CDK4/CDK6, and MAPKs/NF-κB, it has been found that scutellarin may reduce the proliferative, inflammatory, migratory, and invasive capacity of NPC cells. Our research supports the MAPKs/NF-κB pathway as a therapeutic target and suggests that it may play a key role in mediating the scutellarin actions against nasopharyngeal cancer malignancy. In summary, scutellarin may be an effective conventional therapeutic drug in preventing the progression of NPC.

Graphical abstract

Introduction

Nasopharyngeal carcinoma (NPC), which arises from the epithelial cells of the nasopharynx, is one type of head and neck cancer [1]. The pathogenesis of NPC is significantly influenced by Epstein-Barr (EBV) infections and genetic anomalies [2]. Some parts of South Asia, the Middle East, and North Africa have an annual incidence of 15 to 50 cases per 100,000 people; in these regions, it is far more common [3]. In China, there are up to 21 instances per 100,000 people. It's also more common among Inuit people from Alaska and Canada [4]. Morbidity and death rates are high in NPC. The likelihood of an NPC spreading to neighbouring organs is increased by its physical closeness to the cervical lymph nodes. NPC is distinguished by its primary causes of death, which are distant metastases, recurrence, and undifferentiated carcinoma [5]. Currently, people with progressing NPC live longer thanks to radiation and treatment. Relative survival rate is used to compare individuals with the same type and stage of cancer to the general population. The 5-year death rate for patients with nasopharyngeal cancer is usually only 50–60 % due to the long-term side effects of chemotherapy and radiation, as well as the frequency of both local and distant metastases. Most patients have unacceptable toxicity from conventional medicines, which only offer temporary relief. Thus, there is an immediate need for a different, more effective, and safer corrective strategy. It's possible that medicinal plants could be developed into pharmaceuticals [6].

Natural medicines derived from plants are effective in treating cancer and chemo preventing it because of their low toxicity and anti-cancer properties [7]. Scutellarin is a type of flavonoid that is glucuronide and is isolated from many plants, including Erigeron breviscapus (vant.) Hand-Mazz, Scutellaria altissima L, and Scutellaria barbata D. Don [8]. There have been suggestions that SL has apoptotic, anti-inflammatory, and anti-tumor biological properties [9]. Scutellarin causes multiple myeloma cells to undergo apoptosis by inducing the generation of ROS [10]. It has been demonstrated that scutellarin suppresses carcinomas, including colorectal, hepatic, breast, and tongue cancers [[11], [12], [13], [14]]. Scutellarin stops the growth of cancer cells by inducing apoptosis and cell cycle arrest through a variety of biochemical mechanisms [15]. It has also been found that in prostate cell malignancy, scutellarin stops cell division by stopping the G2/M mobile cycle phase [16]. Scutellarin inhibits AKT signalling and increases ERK1/2 activation, which leads to the death of lung cancer cells [17]. Uncertainty surrounds the anti-cancer activity of scutellarin and its underlying regulation mechanism in NPC.

One type of genetically programmable cell death is apoptosis. Tumor cells arise when apoptosis is dysregulated [18]. Anticancer medications are used to treat these cells in the event that they become malignant. The homeostasis of cells is significantly influenced by reactive oxygen species (ROS). Several studies have shown that increased intracellular ROS is typically associated with cancer cell death [19]. One well-known intracellular apoptotic signaling mechanism is the kinase cascade. It has been established that MAPKs and their upstream kinases, as well as external triggers, activate the apoptotic transducing pathway known as kinase [20]. It has been shown by kinase that ROS causes a variety of actions. These include the signaling pathways via which mitogen-activated protein kinases (MAPKs) induce cell death [21]. Family members of MAPK, p38, are significantly involved in cellular stress and are regulated by N-terminal c-Jun kinases (JNK) and extracellular signal kinases (ERK). They cause ROS to occur and are essential for the induction of apoptotic reactions to chemotherapeutic remedies [22]. Our goal in doing this experiment was to find out how scutellarin affected the growth, production of reactive oxygen species, and apoptosis of NPC cells. Additionally, we clarified the molecular NF-κB/MAPK signaling pathways that contribute to scutellarin's anti-cancer properties.

Materials and methods

Chemicals

Scutellarin (99.35 % of Purity) (Fig. 1), Roswell Park Memorial Institute (RPMI), antibiotics, fetal bovine serum (FBS), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), dichloro-dihydro-fluorescein diacetate (DCFH-DA), rhodamine-123 (Rh-123) and 4′,6-diamidino-2-phenylindole (DAPI), propidium iodide (PI), sodium dodysylsulphate (SDS), dimethyl sulphoxide (DMSO), phosphate buffered saline (PBS) and other biochemical reagents were obtained from Germany's Merck. Labome in the United States provided the primary and secondary antibodies. Bicinchoninic acid (BCA) protein assay kit purchased from Thermoscientific suppliers Pvt Ltd., USA.

Fig. 1.

Chemical structure of the Scutellarin.

Cell culture

The ATCC in the United States provided the human NPC CNE1 cell lines. Antibiotics and 10 % FBS were added to the RPMI-1640 media while the cell lines were being grown. Cell lines require CO₂ (5 %) at 37 °C in a humid environment to be maintained.

Cell viability by MTT assay

The MTT test was used to determine the toxicity of human NPC cells [23]. CNE1 cells were cultivated at 37 °C in a wet incubator with 5 % CO₂ after being seeded into 96 wells (1 × 10⁵ cells/well). Following an overnight storage period, the cells underwent a PBS wash before being exposed to scutellarin (2.5, 5, 10, 20, 30, 40, and 50 μM/ml) for a whole day. The treated cells were then given the MTT solution to add to them during the incubation time. After that, the mixture was allowed to sit for 4 h so that mitochondrial dehydrogenase could convert MTT into insoluble formazan crystals. DMSO was added to it to liquefy it. Through multifunctional reading plates, absorbance was tested at a wavelength of 490 nm (BD Biosciences, USA).

ROS measurement by DCFH-DA method

After being planted, human NPC cells (CNE1) were given a 24-hour growth period [24]. Subsequently, scutellarin at concentrations of 20 and 30 μM/ml was added and left for 24 h. Consequently, DCFH-DA (10 μM) was used to stain the control and treated cells, and they were then incubated for 30 min at 37 °C. PBS (Very cold) was collected and cleaned these cells twice in order to get rid of the stains. At 530 ± 12.50 emission nm and 485 ± 10 excitation nm, the fluorescence was measured.

Apoptotic morphology analysis by AO/EB staining

In a dual staining experiment, the DNA-binding dyes acridine orange/ethidium bromide (AO/EB) were used for the morphologically apoptotic and necrotic cells [25]. Following a 48-hour exposure to scutellarin (20 and 30 μM/ml), the CNE1 cells were isolated, followed by a cold PBS wash, and five minutes of room temperature staining with a mixture of AO (100 mg/ml) and EB (100 mg/ml). The labelled CNE1 cells were examined under a fluorescence microscope with a 40x magnification. Four groups of CNE1 cells were identified: living cells with a normal green nucleus; early apoptotic cells with a bright green nucleus with chromatin that was condensed or fragmented; late apoptotic cells with orange-stained nuclei from chromatin condensation or fragmentation; and necrotic cells with uniformly orange-stained nuclei. There were 300 cells in each concentration for each experiment.

Effect of scutellarin apoptosis by staining with PI

Propidium iodide (PI) staining was used to assess the effectiveness of scutellarin in fragmenting DNA in human NPC cells (CNE1) [26]. For one day, CNE1 cells were treated with scutellarin at 20 and 30 μM/ml. The cells were NPC washed twice with PBS, static with 4 % paraformaldehyde for 20 min, cleaned, stained with PI, and incubated for another 20 min. The cells were then washed with PBS and examined under a fluorescence microscope to analyse their membrane potential.

Effect of scutellarin on apoptosis by staining with Rh-123 and DAPI

Rhodamine-123 (Rh123) and 4′,6-diamidino-2-phenylindole (DAPI) labelling were used to assess the apoptotic efficiency of scutellarin on human NPC cells (CNE1) [26]. For one day, CNE1 cells were treated with scutellarin at 20 and 30 μM/ml. The cells were NPC washed twice with PBS, static with 4 % paraformaldehyde for 20 min, cleaned, and stained with DAPI before being incubated for 20 min. After treatment, CNE1 cells were stained for 30 min at 37 °C using Rh-123. Rinse the cells twice with methanol to get rid of any remaining stains. The cells were then washed with PBS and examined under a fluorescence microscope to analyse the membrane potential.

Assay for cell adhesion

CNE-1 cells were primed with scutellarin (20 and 30 μM/ml) and suspended in culture media before being plated against a 24-well plate coated with fibronectin (500 μl/well). To remove any non-adherent cells, the well plate was gently rinsed twice with PBS after being incubated at 37 °C for 0–24 h. After fixing each well with 4 % paraformaldehyde, they were incubated for five minutes at 4 °C. Following well plate incubation, adherent cells were treated with scutellarin to halt the adhesion assay of CNE-1 cells, control, and TB (0.4 %) staining and counting under fluorescence microscopy [26].

Cell migration assay

The cell migration test is used to measure the healing of wounds and scratches. Vasconcelos and Cavaco-Paulo [27] procedure was followed when conducting the cell migration test. CNE-1 cells, which permit cell adhesion up to a 90 % confluent monolayer, were seeded into a 24-well plate. A confluent monolayer was then punctured through the middle of each well using a sterile micropipette tip (10 μl). Apply the media treated with scutellarin (20 and 30 μM/ml) for a full day after washing the well plate with PBS and eliminating any loose cells. This incubation length was selected based on the photocopying times of 60 and 100 min for the treated CNE-1 cells. Images were taken at 60-second intervals beginning with each treated dose and control at times 60 and 100 min. The monolayer of scratches. Using ImageJ, the scratch monolayer area was examined.

Western blot analysis

Human NPCs (CNE1) were cultured for 24 h after scutellarin (20 and 30 µM/ml) was introduced [25]. The cell lysates were produced using frozen lysis buffer, which ensures the presence of protease inhibitors, and western blotting analysis was performed. The BCA Protein Assay Kit was used to calculate the protein content. In a nutshell, a protein was transferred into a PVDF membrane. Following a 60-minute probe-prevention period, 1:1000 dilutions of primary antibodies against TNF-α, COX-2, iNOS, IL-6, CDK4, CDK6, Cyclin-D1, p-RB, RB, MMP-2, MMP-9, TIMP-1, P-JNK, JNK, p-p38, p38 MAPK, p-ERK, ERK, p-NF-κB, NF-κB, and GAPDH were added to the film. The film was then left overnight at 4 °C. Subsequent antibodies were then added. Protein detection was visualized and protein strands were stained. The measurement of protein bands was assessed.

Statistical analysis

Version 8.0.1 of GraphPad Prism was utilized to statistically evaluate the information. All groups were compared using the unpaired Student's t-test or the one-way analysis of variance (ANOVA) test. A p-value of less than 0.5 was considered significant.

Results

Effect of scutellarin on CNE1 cell cytotoxicity

Scutellarin on cell cytotoxicity and cell inhibition was evaluated using MTT analysis on two CNE1 (NPC) cell lines at various doses (2.5. 5, 10, 20, 30, 40, and 50 μM/ml) (Fig. 2A and B). The viability of NPC cells was not significantly affected by scutellarin at concentrations lower than 10 μM at SL doses of 30, 40, and 50 μM/ml, CNE1 cell growth was effectively reduced at p < 0.05. According to the findings, scutellarin has concentration-dependent cytotoxic and anti-proliferative actions on CNE1. After calculating the IC₅₀ value of scutellarin, concentrations of 20, and 30 μM/ml were chosen for more research.

Fig. 2.

(A and B) Effect of scutellarin on cell viable and inhibition number were analyzed by MTT assay in CNE1 cells. SCC131 cells were collected for total cell viability and inhibition were measurements, which were treated with scutellarin at various concentrations (2.5–50 µm/ml) for 24 h using MTT dye. Data were presented as mean±SD asterisks indicating statically different experiments (*P < 0.05) compared to control.

Effect of scutellarin on the intracellular ROS accumulation in NPC cells

Intracellular ROS are created when a variety of stimuli induce involuntary cell death and stop the cell cycle. NPC cells' intracellular ROS level was dramatically elevated (P < 0.05) by scutellarin (20 and 30 µM/ml) for a full day. Cells treated with DCFH-DA were examined under a fluorescent microscope. In comparison to untreated control CNE1 cells, the fluorescence intensity of ROS was shown to be elevated with 20, and 30 µM of scutellarin (Fig. 3A).

Fig. 3.

(A) In NPC cells, scutellarin promotes the build-up of ROS. Scutellarin (20 and 30 µM/ml) was added to human CNE1 cells and left for 24 hours. Scale bar = 50 μm. Under an inverted fluorescent microscope, the DCFH-DA fluorescence accumulation was located. (B) When using AO/EB staining to analyze nuclear morphology, scutellarin caused apoptosis. Scale bar = 50 μm. After being exposed to scutellarin at concentrations of 20 and 30 µM/mL for a whole day, the CNE1 cells were stained with AO/EB and their fractured nuclei and apoptotic cells were observed under a fluorescence microscope. C) Using PI staining to analyze nuclear morphology, scutellarin caused apoptosis. Scale bar = 50 μm. After being exposed to scutellarin at concentrations of 20 and 30 µM/mL for a whole day, the CNE1 cells were stained with PI and analyzed under a fluorescence microscope to detect apoptotic cells. For the experiments, the results were presented as mean±SD. *P < 0.05 denotes significance in relation to the untreated control group.

Effect of scutellarin on apoptotic activation of NPC cells

AO/EB staining was applied to the NPC cells utilized in the research of cell apoptosis. After adding 20 and 30 μM/ml doses of scutellarin to cells, the cells underwent an overnight incubation period during which time the same morphological changes, such as nuclear fragmentation and chromatin fragment condensation that are indicative of the cell mechanistic stages of death, were seen, along with cytoplasm shrinkage. The outcomes are displayed in Fig. 3B Minor nuclear changes were observed in non-treated NPC cells. NPC cells treated with 20 μM/ml of scutellarin showed green at the early apoptotic stage, while cells treated with 30 μM/ml of scutellarin showed orange at the late apoptotic stage, with a significant p < 0.05.

Effect of scutellarin on apoptosis by staining with propidium iodide staining

Only the vehicle control NPC cells (CNE1) exhibited propidium iodide (PI) staining (Fig. 3C). Fig. 3C illustrates the substantial (p < 0.05) fluorescence densities that were observed in response to scutellarin treatment at different concentrations (20 and 30 μM/ml). CNE1 cells treated with scutellarin showed signs of apoptosis, including chromatin condensation. When combined, the brightly fluorescent apoptotic nuclei in many segregated masses of chromatin were plainly apparent, suggesting that the concentration was between IC₅₀ and 20 μM/ml. Scutellarin may significantly change the nucleus shape of CNE1 cells by causing apoptosis.

Effect of scutellarin on mitochondrial mediated apoptosis during NPC cells

The impact of scutellarin (20 and 30 µM/ml) on the morphology of NPC cells was examined using DAPI labeling (Fig. 4). Adducts across dual-DNA strands are known to be produced by DAPI staining. SL-induced apoptosis was demonstrated by multiple indicators, including condensed nuclei shape, nuclear body disintegration, and loss of membrane integrity, as compared to untreated NPC cells. These findings suggest that SL inhibits CNE1 cell proliferation and induces apoptosis. A possible electric anomaly in MMP is caused by the molecules and ion permeability (Fig. 4). Rh-123 staining causes the mitochondrial membrane to become depolarized. As a result, apoptosis occurs promptly. Neighbouring active mitochondria are stained with the lipophilic cationic fluorescent dye Rh-123, emphasizing the vital function that mitochondria play in the apoptotic model.

Fig. 4.

Scutellarin causes human NPC cells to undergo apoptosis. Scale bar = 50 μm. Scutellarin (20 and 30 µM/ml) was applied to human NPC cells for a full day. Apoptosis was observed by DAPI staining, uptake, and Rh-123. photomicrograph of the CNE1 cells' combined Rh-123 and DAPI staining, taken under a fluorescent microscope and preserved for 24 h.

Effect of scutellarin on cell adhesion in CNE-1 cells

The establishment of tumor cell metastasis is significantly influenced by the adhesion of CNE-1 cells and extracellular matrix (ECM). In CNE-1 cells, integrin controls cell attachment and signalling. The effect of scutellarin silencing on CNE-1 cells' adherence to extracellular matrix proteins has been investigated in this work. After 60 or 100 min, the quantity of adhering CNE-1 cells significantly reduced (P < 0.05) with the scutellarin suppression treatment in comparison to the control (Fig. 5A). Tumor cell proliferation and/or metastasis may be inhibited by the modulatory effects of cell adhesion caused by scutellarin silencing. The adherence of CNE-1 cells treated with scutellarin (20 and 30 µm/ml) was dramatically reduced by 50 % and 70 %, respectively, in comparison to the control (Fig. 5A).

Fig. 5.

A) Scutellarin inhibited the adherence of CNE-1 cells. CNE-1 cells adherence force was evaluated by adherence assay in 60 and 100 min. The attached cells were fixed and stained, and ten random fields were counted. Shown is the number of adherent cells from three independent experiments. This assay was representative of three experiments performed in triplicate, and the values are designed as a graph. Mean denuded zone was measured. For the experiments, the results were presented as mean±SD. *P < 0.05 denotes significance about the untreated control group. B) Confluent monolayers of each CNE-1 cell line were wounded by scratching the surface as uniformly as possible with a micropipette tip. CNE-1 cells were treated to scutellarin (20 and 30 µm/ml). After 12 and 24 h of incubation, the wound surface was evaluated by photography and stated as the width of the remaining wounded surface area relative to the initial wound surface area. This assay was representative of three experiments performed in triplicate, and the values are designed as a graph. Mean denuded zone was measured. For the experiments, the results were presented as mean±SD. *P < 0.05 denotes significance about the untreated control group.

Effect of scutellarin inhibits invasion and migration of CNE-1 cells

The effect of scutellarin on the wound invasion and migration potential of CNE-1 cells was investigated by executing wound healing procedures after 0 and 24 h of scutellarin treatment for different concentrations to assess the pharmaceutical ability of scutellarin against CNE-1 cells. Scutellarin treatment was found to have a dose-dependent effect on the amount of CNE-1 cell migration into the free gap (wound) (Fig. 5B). According to the findings, scutellarin 30 µm/ml significantly reduced (P < 0.05) the CNE-1 cells' movement intensity by 80.05 % over 24 h. Furthermore, the effects of scutellarin administration at 0 h (migration and invasion) on CNE-1 cells were assessed using fibronectin-coated chamber tests. Cell invasion and migration were reduced by 63 % at 20 µm of scutellarin (Fig. 5B). This finding suggests that scutellarin, at various dosages, significantly reduced the ability of CNE-1 cells to invade new cells and migrate between them. CNE-1 cell invasion and migration were assessed using a trans well test.

Effect of scutellarin protein expression on inflammatory signaling in NPC cells

It was observed that scutellarin (20 and 30 µM/ml) treated CNE1 cells significantly (P < 0.05) down-regulated the levels of inflammatory proteins; TNF-α, COX-2, iNOS and IL-6 compared to the control cells. These findings indicate that scutellarin has a strategic governing action on NPC cell cycle (Fig. 6A).

Fig. 6.

A) Effect of scutellarin on western blotting protein expression status of TNF-α, COX-2, IL-6 and iNOS protein in CNE1 cells. Scutellarin (20 and 30 µM/ml) was added to human CNE1 cells and left for 24 h. The western blot technique was used to assess the expression of the proteins TNF-α, COX-2, IL-6, and iNOS. B) Effect of scutellarin on western blotting protein expression status of CDK4, CDK6, cyclin-D1, p-RB, and RB protein in CNE1 cells. C) Scutellarin (20 and 30 µM/ml) was added to human CNE1 cells and left for 24 h. The western blot technique was used to assess the expression of the proteins MMP-2, MMP-9, and TIMP-1, and GAPDH. For triplicate rats in each group *p < 0.05 and the control cells, bars are expressed as mean±SD.

Effect of scutellarin on protein expression in NPC cells

In comparison to control cells, it was shown that CNE1 cells treated with scutellarin (20 and 30 µM/ml) had significantly lower (P < 0.05) levels of critical cell-cycle regulatory proteins, including cyclin-D1, CDK4, CDK6, and pRB. These results suggest that SL controls the NPC cell cycle strategically (Fig. 6B).

Effect of scutellarin suppressive action on MMP-2, −9, and TIMP-1 signalling pathways

When scutellarin was applied to NPC cells (CNE1), the levels of MMP-2, MMP-9 were down-regulated and TIMP-1 were up-regulated in contrast to the untreated control cells (Fig. 6C). The CNE1 cells protein expression was considerably (p < 0.05) MMP-2, MMP-9 were reduced and TIMP-1 increased upon exposure to 20 and 30 µM/ml of scutellarin.

Effect of scutellarin suppressive action on MAPKs and NF-κB signaling pathways

When scutellarin was applied to NPC cells (CNE1), the expressions of JNK, p38, ERK, and NF-κB were down-regulated in contrast to the untreated control cells (Fig. 7). The CNE1 cells protein expression was considerably (p < 0.05) reduced upon exposure to 20 and 30 µM/ml of scutellarin. This suggests that NF-κB signaling and MAPK inhibition reduced cell invasion, metastasis, and inflammation. It further suggests that MAPKs pathways are involved in the apoptosis of NPC cells driven by scutellarin.

Fig. 7.

On NPC human cells, scutellarin inhibits the MAPKs/NF-κB signaling pathway. For twenty-four hours, scutellarin (20 and 30 µM/ml) was added to human CNE1 cells. Western blotting was used to evaluate the p-JNK, JNK, p-p38, p38, p-ERK, ERK, p-NF-κB, NF-κB and GAPDH protein expression. For triplicate rats in each group *p < 0.05 and the control cells, bars are expressed as mean±SD.

Discussion

High morbidity endemic NPCs are primarily seen in certain regions and among certain ethnic groups [1,2]. Therapeutic effectiveness is limited by the early metastasis of NPC and the toxic side effects of current therapeutic approaches [4]. A diverse reservoir of bioactive chemicals found in plants may be essential for safer chemotherapy for non-polychromatic cancer [28,29]. In vitro NPC cells (CNE1) were used in this work to demonstrate scutellarin's anti-cancer properties. The results of the cytotoxicity research showed that the viability of CNE1 cells decreased dose-dependently. These results corroborate previous findings that scutellarin inhibits the growth of human hepatocellular HepG2 cancer cells [11] and breast melanoma cells. MCF-7 [12].

It is well recognized that pathological and physiological central intracellular signals are associated with intracellular ROS. Increased ROS and genomic ambiguity are related. It is a factor in the development of tumors [30,31]. According to the current study, scutellarin caused mitochondrial-mediated apoptosis in CNE1 cells, reduced MMP, and raised intracellular ROS levels. It has previously been shown that treatment with scutellarin causes human colon cancer cells to produce more ROS, which interferes with MMP, causes apoptosis, and reduces the growth of HCT116 cells. Scutellarin may impair mitochondrial processes by producing excessive amounts of ROS, which are the primary cause of death for HCT116 cells [32]. In apoptosis, ROS and mitochondria both demonstrated a crucial role [33]. However, new research indicates that natural anti-cancer substances may cause cancerous cells to undergo apoptosis through the generation of accumulated ROS. In malignancies, ROS further induces MMP distraction, which results in cell death [34]. Therefore, intracellular ROS production and tumor cell death can be enhanced by natural anti-cancer medicines like scutellarin.

Tumor necrosis factor alpha (TNF-α) has been involved in inflammatory processes and to possess pleiotropic features. The TNF-α receptor-associated death domain also makes use of TNF receptor-related factor which it uses to activate IκBα kinase (IKK-α) through receptor-interacting protein, which causes IκBα to be phosphorylated, ubiquitinated, and destroyed. IκBα degradation leads to the activation of nuclear factor-κB (NF-κB). Following NF-κB activation, TNF increases the expression of genes linked to apoptosis [35]. Qureshi et al. [36]. have shown that curcumin inhibits phospholipases, NF-κB, lipoxygenase, and cyclooxygenase-2 (COX-2). The reduction of significant inflammatory cytokines, including as TNF-α, IL-16, and inducible nitric oxide synthase (iNOS), is associated with the extract's anti-inflammatory properties. Similarly, in our current study scutellarin downregulated the TNF-α, COX-2, iNOS and IL-6 in the HCT-1 cells in concluded that scutellarin inhibited the inflammatory responses to control the nasopharyngeal cancer.

The disruption of apoptosis in cancer cells has been thoroughly studied, and one strategy for creating anticancer drugs is to trigger apoptosis in malignant cells. We demonstrate that CNE1 cells treated with succinylcholine underwent dose and time-dependent sequences of apoptotic events, including loss of cell viability, inter-nucleosomal DNA breaks, and accumulation of G1/G0 phase. The processes linked to scutellarin-induced apoptotic cell death in CNE1 cells are also described in this work; the great majority of these cells are employed as apoptosis biomarkers. Two main mechanisms govern apoptosis: membrane death receptor and mitochondria. We proved that scutellarin management of CNE1 cells led to accumulation of the G1/G0 phase, loss of cell viability, and internucleosomal DNA breakage, as well as dose and time dependent sequences of apoptotic events as revealed by DAPI, PI, and AO/EB staining. The processes linked to scutellarin-induced apoptotic cell death in CNE1 cells are also described in this work; the great majority of these cells are employed as apoptosis biomarkers. Membrane death receptor (DR) and mitochondrial processes are the two main regulators of apoptosis.

In vitro NPC cells were used in our investigation to evaluate the molecular anti-cancer properties of scutellarin. According to our findings, scutellarin suppressed the two CNEs cells by enhancing apoptosis in cells and preventing them from progressing past the G0/G1 stage. It is commonly recognized that a key therapeutic strategy for the treatment of cancer is the induction of apoptosis in tumor cells [37]. Changes in the cell cycle can impact viability and raise metabolism [38]. At the moment, we observed that scutellarin decreased the expression levels of pRb, CDK 4, CDK 6, and cyclin-D1. A switch involving cyclins, CDKs, and CDK4/6 is part of the cell cycle regulation process. Cyclin-D phosphorylates protein RB.

They initiate evolution in the G1 phase restraint socket. CDK4/6/Cyclin-D acts in the late-middle G1 phase requisite. In the phosphorylation Rb gene product (pRb), which restricts a constraint on progression at the late G1 restriction point chief controller of the G1/S switch [39]. Modifications of any constituent of this route, such as hyperexpression that cyclin-D associated CDKs or CDK mutations that alter binding at p16, lead to phosphorylation of Rb and ensure phase G1 through S progression [40]. These variations have been established in many human cancers, suggesting that cell cycle pathway inactivation plays a significant role in cancer pathogenesis.

In our study, we found a strong correlation between ROS and scutellarin-induced cell cycle arrest. Comparable outcomes have been reported in renal cancer cells (RCC) treated with a high dose of scutellarin and stopped in the G0/G1 phase. Additionally, Cyclin-D1 and CDK2 expression have been discovered to be suppressed by scutellarin. These two led to G1 S shift and increased p21, which resulted in phase G0/G1 arrest RCC cells [41]. On the other hand, Deng et al. [42] have shown that scutellarin suppressed cyclin-B1 and CDK1 expression in Hela cell culture, hence causing cell cycle G2/M arrest. According to Cao et al. [43]. cyclin-B1 and CDK2 decline in prostate cancer cells at the G2/M phase due to scutellarin-enhanced cell cycle inhibition. Therefore, we postulated that in order for scutellarin to exhibit its anti-cancer effects, it may activate distinct molecular processes in various cancer types [44]. p38, JNK, and ERK1/2 facilitate MAPK signaling, which is important for the mechanism of cell proliferation, diversity, transformation, and assembly of pro-inflammatory mediators and NF-κB inflection [45]. The MAPK signaling cascade starts the transcription of cellular inflammatory retort accompanying genes arbitrated by NF-κB and pro-inflammatory molecules synthesis. The NF-κB pathway has been found to stop apoptosis through death receptors and stimulate the proliferation of malignant cells [46]. Constitutive instigation of NF-κB has been noted in diverse cancer cells.

Growth arrest was presumably induced by scutellarin's underlying mode of action, which involved the inhibition of MAP kinases and NF-κB. These in turn cause cyclin-D1, p-Rb, and G1-correlated CDKs (CDK4/CDK6) to drop [47]. According to earlier research, scutellarin may limit the phosphorylation of MAPKs by obstructing the upstream kinase. Increased endogenous ROS levels have been linked to down-regulated MAPK signaling and have been shown to induce ROS-mediated mitochondrial dysfunction-associated apoptosis in human cancer cells [48]. MMPs and TIMPs were identified in many neoplasms, including cancers of head and neck, lung, breast, and colon cancer. In most research, a strong relation between MMPs and TIMP expression created a prognostic impact in cancer cells [49]. Similarly in the current study showed that the scutellarin was applied to NPC cells (CNE1), the expressions of MMP-2, MMP-9 were down-regulated and TIMP-1 were up-regulated control cancer cells. The CNE1 cell's protein expression was MMP-2, MMP-9 were reduced and TIMP-1 increased upon exposure to 20 and 30 µM/ml of scutellarin. −2 and MMP-9 expression were observed in the treatment of scutellarin in nasopharyngeal cancer therapy. Our findings validate that scutellarin inhibited the expression of multiple components of MAPKs and NF-κB signaling. It also impeded the growth and transition of NPC cells from the G1 to the S phase of the cell cycle. Additionally, we advise conducting in vivo research using a mouse model to examine the bioavailability of scutellarin and the rate oftumor inhibition in mice tumor xenograft model.

Conclusions

In conclusion, the current in vitro investigation demonstrated that scutellarin effectively reduced viability, blocked MMP, and initiated apoptosis in CNE1 human NPC cells. Through the buildup of ROS and the downregulation of TNF-α, COX-2, iNOS, and IL-6, scutellarin revealed its cytotoxicity. Scutellarin stopped MMP, which damaged DNA and decreased the expression of MAPKs, NF-κB, MMP-2 and MMP-9 and increased TIMP-1. The goal of this study was to investigate the mechanism underlying scutellarin's anti-cancer properties. The findings indicate that scutellarin suppresses the proliferation, inflammation of NPC cells as well as angiogenic signalling via MAPKs/NF-κB signalling. Thus, we draw the conclusion that scutellarin could be a potential medication for treating nasopharyngeal cancer. In future study will be carried out in the in vivo model experiments both biochemical, molecular and computational study, it’s used in future to study or discover the innovative drug for the therapeutic applications

Funding

None.

Ethical statement

None.

CRediT authorship contribution statement

Jingda Xu: Writing – original draft, Methodology, Investigation, Formal analysis, Conceptualization. Guanzheng Wu: Methodology, Investigation. Lu Wang: Validation, Project administration. Qingfeng Zhang: Resources, Formal analysis. Periyannan Velu: Validation, Data curation. Annamalai Vijayalakshmi: Validation, Formal analysis. Gang Chen: Writing – review & editing, Supervision.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Data availability

The data sets used and/or analyzed during the current study are available from the corresponding author on reasonable request.