Apoptotic induction and anti-metastatic activity of eugenol encapsulated chitosan nanopolymer on rat glioma C6 cells via alleviating the MMP signaling pathway

https://doi.org/10.1016/j.jphotobiol.2019.111773Get rights and content

Highlights

  • Gliomas are one of the most recurrent types of primary intracerebral cancer which are tending to be exceedingly fatal.

  • Eusingle bondCs nanopolymer was assessed by evaluating the protein expression epithelial mesenchymal transition inducer protein.

  • Eusingle bondCs nanopolymers inhibits the expression of NF-κB & EMT protein thereby induces apoptosis.

Abstract

Glioma is the prime cause of cancer allied mortality in adolescent people and it accounts about 80% of all malignant tumours. Eugenol is a major bioactive constituent present in the essential oils with numerous pharmacological benefits including nueroprotective activity. The major drawback of eugenol is its extreme volatile property and oxygen sensitivity therefore we increased the efficacy of drug; eugenol by encapsulating with chitosan polymer. Eugenol loaded chitosan polymer (Eusingle bondCs) was characterized using FTIR, XRD, SEM, HR-TEM analysis and the encapsulation, drug release efficacy was assessed at in vitro condition. The induction of autophagy and anticancer efficacy of Eusingle bondCs on glioma cells was evaluated with rat C6 glioma cells using MTT assay, acridine orange staining, immunocytochemical analysis of NFκβ protein expression and FLOW cytometric analysis. The anti-metastatic property of Eu-CS was assessed by immunoblotting and RT-PCR analysis of epithelial mesenchymal transition protein expression in Eusingle bondCs treated rat C6 glioma cells. Our characterization analysis proves that Eusingle bondCs possess essential physical and functional properties of copolymer to be utilized as a drug. Further the MTT analysis and AO staining confirms even in the presence of oncogenic inducer and autophagic inhibitors, Eusingle bondCs exhibits apoptotic potency on rat C6 glioma cells. The result of immunocytochemical studies depicts the inhibition of NFκβ protein expression and flow cytometry studies confirm apoptosis induction by Eusingle bondCs. The inhibition of metastasis by Eusingle bondCs was proven by the decrease in epithelial mesenchymal transition protein expression in

Eu-Cs treated rat C6 glioma cells. Over all our results authentically confirms eugenol loaded chitosan nanopolymer persuasively induces apoptosis and inhibits metastasis in rat C6 glioma cells.

Introduction

Glioma is a deadliest disease affecting central nervous system causing recurrent primary tumours of glial cells. They accounts about 80% of all malignant tumours and are classified into various types such as astrocytoma, oligodendeogliomas, ependymomas [[1], [2], [3], [4]]. The most malignant and recurrent type of gliomas are glioblastoma multiforme which is classified as Grade IV astrocytoma by World Health Organization (WHO) and reports about 60% of adult brain tumours [2,5]. Gliomas are the prime basis of cancer allied deaths in young people, it occurrence is evidenced more pronounced in men compared to women [6,7]. The survival rate of glioblastoma multiforme patients is pitiable that only 3–5% patients possess beyond 5 years of survival [8]. Surgery pursues by radiotherapy and intake of temozolomide is the current effective therapy prescribed to glioma patients [9]. But the high invasive property of gliobastoma cells restricts the surgical therapy since it invades the normal cells upon surgical resection causing recurrence of cancer. Several researches were intended to discover a drug to treat glioma but most of them are not potent and also renders drastic side effects like nausea, body weakness, headache, and vomiting, high fever and hair loss.

Eugenol, 4-allyl-2-methoxyphenol is an imperative bioactive constituent present in the essential oils especially oils extracted from Eugenia caryophyllata. Eugenia sps were extensively grown in countries such as Indonesia and Madagascar. Eugenol is also reported to be present in most of the aromatic plants such as Cinnamomum sps, Ocimum sps [10,11]. Eugenol is extensively utilized in a food and cosmetic manufacturing and the daily intake of 2.5 mg/kg eugenol for humans was approved by the Food and Agriculture Organization (FAO) and WHO [12,13]. Eugenol possess various pharmaceutical properties such anti-inflammatory, antimicrobial, antiviral, antiseptic, anticonvulsive [14,15]. It also acts as nephroprotectant, antihelemthic, antioxidant at lower concentration and at higher concentration induces reactive oxygen species which targets the cancer cell rendering anticancer activity [[16], [17], [18], [19]]. Studies also confirmed that eugenol protects the mouse neuronal cells from glutamate induced excitotoxicy, 6-hydroxydopamine neurotoxicity and ischemic induced toxicity. It also effectively inhibited the chemical induced neurotoxicity in rats [20,21] which clearly depicts eugenol crosses the blood-brain barrier and it is acts as potent neuroprotectant. The major disadvantage of eugenol is its volatile nature and oxygen sensitive which hinders its usage in pharmaceutics [22,23].

Nanoencapsulation technique gained the attention of pharmaceutical industries which extensively increases the stability of drug, water solubility and enhances the targeted drug delivery in a feasible manner [24]. Reports suggest that encapsulation protects highly volatile phenolic such as carvacrol, caffeine, cinnamaldehyde from humidity, heat and UV light exposure [25,26]. Various types of materials are used to encapsulate the drug but chitosan, polysaccharide are widely used material to capsulate vitamins, drugs, proteins etc. Due it is cationic nature it easily capsulate the drug and also its non compatible, non toxic and biodegradable nature claims it to be the ideal candidate use for encapsulating drug [23,27,28]. The present research investigation was planned to examine the ameliorating potency of synthesized eugenol loaded nanocomposite nanopolymer using chitosan against the rat C6 glioma cells via suppressing the signaling pathway.

Section snippets

Chemicals

Chitosan, and glacial acetic acid was obtained from central drug house Pvt. Ltd., India. Ammonium per sulphate (APS) and eugenol drug was procured from Sigma Aldrich, India. Dulbecco's Modified Eagle's Medium (DMEM), fetal bovine serum (FBS), Trysin-EDTA, Antibiotic-Antimycotic mixtures and other chemicals were bought in SigmaAldrich, USA. Thiazolyl blue tetrazolium bromide is procured from Abcam, USA. Whole other chemicals utilized in current investigation were of high quality diagnostic range.

Functional Group Characterization of Eusingle bondCs Nanopolymer

Fig. 1 depicts the results of FTIR analysis of chitosan, eugenol and eugenol loaded CS nanopolymeric composites. Chitosan exhibits an absorption peak at 3613, 2986 and 1692 cm−1 which were assigned to OH, Csingle bondH and Cdouble bondO groups respectively. The 1310, 1055 and 900 cm−1 peaks displays the Csingle bondH, Csingle bondO stretching vibration of chitosan functional groups (Fig. 1a). Fig. 1b represents the FTIR absorption pattern of eugenol and eugenol loaded chitosan nanopolymer (Eusingle bondCs) shown absorption peaks at 3510, 2935 cm−1

Discussion

Glioma is the most common type of cancers occurs in central nervous system out of which glioblastoma multiforme, cancer of astrocytes accounts about 60% of all brain tumours [31]. The foremost therapies used to treat most of the cancers are alkylating chemo drug combined with radiotherapy but the potency of such drug to treat brain tumours are questionable because of the exclusive nature of brain tumour which renders resistance to most of the drugs [32]. The penetration of drug along the blood

Conclusion

In this study, we synthesized and characterized chitosan nanopolymer encapsulated with phytochemical eugenol. Our FTIR, XRD and electroscopic analysis proves Eusingle bondCs nanopolymer is an ideal nanoparticle possessing the properties for a nanomedicine. Chitosan nanopolymer effectively controlled the release of eugenol and Eusingle bondCs nanopolymer significantly inhibited the cell viability of C6 rat glioma cells. Eusingle bondCs nanopolymers inhibits the expression of NF-κB protein thereby induces apoptosis and it also

Declaration of Competing Interest

The authors declare no conflict of interest.

Acknowledgments

The authors would also like to extend their sincere appreciation to the Deanship of Scientific Research at King Saud University for its funding of this research through the Research Group Number (RG-1435-081).

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