Antitumor activity of cordycepin in murine malignant tumor cell line: An in vitro and in silico study

Highlights

• Cordycepin is found to be effective against DL cell line.
• Cordycepin induces significant apoptosis in DL cells, causing cytotoxicity.
• Cordycepin significantly interacts with anti-apoptotic receptors.
• Molecular dynamics study confirms cordycepin affinity for anti-apoptotic receptor.

Abstract

Cordycepin, also known as 3′-deoxyadenosine, is a substance similar to adenosine found in the entomopathogenic fungus Cordyceps militaris. This fungus is recognized for its remarkable therapeutic properties. This study seeks to investigate the antitumor activity of cordycepin in Dalton's lymphoma (DL) malignant cancer cell line using cytotoxicity and apoptosis as key parameters. Molecular docking (MD) and molecular dynamic simulation (MDS) were used to further validate the wet lab findings. Cytotoxicity study revealed that cordycepin induced significant cytotoxicity (P ≤ 0.05) in the DL cell line after 24 h of treatment. The efficacy of cordycepin as an apoptotic agent was substantiated by the highly promising results obtained through the apoptotic assay, utilizing the acridine orange/ethidium bromide (AO/EB) dual staining method. Cordycepin interacted with the active sites of antiapoptotic _target proteins (BCL-XL, SURVIVIN, BCL-2, BCL-B, HSP90, and IAP1), as demonstrated by MD study that corroborated the findings of cytotoxicity and apoptotic assays. The comparative examination of docking outcomes indicated that cordycepin exhibits higher affinity for HSP90 in comparison to other _targets. Molecular dynamics simulation for 120 ns further validated the findings of MD as cordycepin remained bound with HSP90 throughout the entire simulation time (120 ns) with favourable binding energy.

Introduction

Cordyceps militaris is an entomopathogenic fungus that grows on the larvae or pupae of insects and it belongs to the phylum Ascomycota, class Sordariomycetes and family Cordycipitaceae [1]. It is locally known as keeda ghas and in China, it is popularly called Dong-Chong-Xia-Cao which translates as winter worm summer grass [2]. In Ayurveda, cordyceps is considered a rasayana herb, which means it is believed to possess rejuvenating properties for prana, the life force. It promotes the well-being of the endocrine system and provides nourishment to the adrenal and reproductive glands. The use of Cordyceps sp. has garnered immense public attention in recent years because of its high price tag and rare availability as it is found at an altitude of approximately 14,000 ft in the Himalayan lands including Nepal, China, Tibet, and India [3,4]. This parasitic mushroom has been well-known in several other traditional medicines for several thousand years for exhibiting a multitude of pharmacological functions such as antioxidant, antitumor, antimetastatic, anti-inflammatory activities, immunopotentiation, immunosuppression, renoprotection, lung and liver protection, etc. [5,6,7,8].

C. militaris has a lot of bioactive compounds that give it medicinal and healing qualities. These include polysaccharides, essential amino acids, fats, flavonoids, phenolic substances, carotenoids, sterols, nucleosides, sterols, proteins, minerals, vitamins, etc. [9,10,4]. One of its potent active compounds, cordycepin or 3′-deoxyadenosine (9-(3-deoxy-β-d-ribofuranosyl) adenine), a nucleoside analogue (Fig. 1) was first extracted from Cordyceps militaris [11]. A purine (adenine) nucleoside and a ribose sugar (ribofuranose) moiety are joined together in the chemical structure of cordycepin via a β-N9-glycosidic bond. The chemical conflation of cordycepin is achieved through the replacement of the OH group at the 3′ position in the ribofuranosyl moiety with H, generating a deoxy analogue of adenosine [5,12]. Within the cellular realm, cordycepin undergoes a remarkable transformation, metamorphosing into 5′ mono-, di-, and tri-phosphates that inhibit the activity of specific enzymes, such as ribose phosphate pyrophosphokinase and 5-phosphoribosyl-1-pyrophosphate amidotransferase, which are used in the de novo biosynthesis of purines [13]. The presence of cordycepin in the cell causes premature termination of transcription due to the inclusion of cordycepin/ 3′-deoxyadenosine instead of adenosine during transcription due to their structural similarities [14]. Cordycepin is reported to possess many biological and pharmacological activities including anticancer, antiviral, immunostimulating activities, antithrombotic, anti-adipogenic, anti-ageing effects, etc. [6,15,16,17]. It is known to interfere with several biochemical and molecular processes, such as purine biosynthesis [13], DNA/RNA synthesis [14], and mTOR (mammalian _target of rapamycin) signaling transduction [18]. Therefore, in the past few decades, extensive research works using cordycepin have been carried out in different countries which has necessitated its large-scale production and this ever-increasing demand is being successfully met by lab based artificial cultivation of C. militaris [19,20]. The artificially grown cordyceps are also reported to show the same promising results and contain almost the same biochemical constituents like the natural ones [21].

Despite the significant role played by cordycepin in the anti-tumor activities the mechanism(s) by which cordycepin inhibits tumor growth is not yet clearly understood [22,23]. Hence, in the present study, the anticancer potential of cordycepin has been reported using cytotoxicity and apoptosis parameters in Dalton's lymphoma (DL) malignant cancer cell line. The findings were supported further by in silico investigations that included molecular docking simulation followed by molecular dynamics. Based on our findings, cordycepin may be a promising pharmacological candidate for the treatment of the most dreadful cancer.