Original ArticlesNanoliposomal formulation encapsulating celecoxib and genistein inhibiting COX-2 pathway and Glut-1 receptors to prevent prostate cancer cell proliferation
Introduction
Although there has been tremendous progress in the development of novel strategies for prostate cancer treatment, however, it still remains as one of the most challenging health burdens for men globally. As per the statistics of the year 2012, there were over 1 million new prostate cancer patients, accounting for 8% of all the new cancer cases worldwide [1]. In recent past, there has been a significant development in the realm of molecular biology, immunology, and target identification, which have produced a rich library of new targets, signaling pathways, and antibodies for immunotherapies. Additionally, investigations in the domain of new drug development have led to the identification of novel anticancer drugs. However, despite utilizing these tools, the successful handling of prostate cancer has not been realized so far. Although it is well known that the possible major obstacle is the early detection of prostate cancer biomarkers, however, target-dependent delivery of anticancer agents remains at the top of the list.
Among several anticancer drugs developed for inhibition of prostate cancer progression, celecoxib is one of the key drug types, which has shown excellent results in several studies [[2], [3], [4], [5], [6], [7]]. Celecoxib is a cyclooxygenase (COX-2) dependent nonsteroidal anti-inflammatory drug (NSAID) generally prescribed to reduce pain and inflammation. As an inducible enzyme, COX-2 modulates the production of prostaglandin E2 and thus reported to be overexpressed in different types of cancers including breast, colon, lung, melanoma, and prostate [2,3]. In prostate cancer cells, COX-2 has been considered as a key target, which plays an important role in the development of 50–70% of prostate tumors [8,9]. Celecoxib is known to inhibit COX-2 expression, which leads to the reduction in the cellular prostaglandin E2 leading to the compensatory increase in COX-2 protein concentration in cells [2,10]. The role of prostaglandin E2 has been linked with proliferation, invasiveness, angiogenesis, avoiding apoptosis, and production of tumor-inducing eicosanoids [11,12]. Further, it has been reported that in human studies the effect of celecoxib is limited, which could be due to its efficacy variation from androgen dependence to independence. A study reported by Zheng et al. revealed that the xenografted tumors generated from androgen-dependent LNCaP cells, upon treatment with celecoxib, regressed initially, however, tumors eventually progressed androgen dependence and thus started to develop further [13]. Despite the broad spectrum use of celecoxib in many cancer types, its administration is limited due to the safety concerns, potentially to serious toxicity to healthy individuals including increased risk of cardiovascular events including myocardial infarction [14,15]. When compared with other NSAID, celecoxib, at dosages greater than suggested clinically, showed a lower incidence of symptomatic ulcers and related complications along with other clinically important toxic effects [16]. Therefore, it may be suggested to use the lowest possible concentrations of celecoxib for prostate cancer treatment.
It is well documented that cancer cells have rapid metabolism with high glucose consumption to fuel the metabolism. Considering this characteristic, cancer tissues are imaged by PET (Positron Emission Tomography), where a radiolabelled (18F)-2-Fluoro-2-deoxy-d-glucose (FDG) is used. This glucose analog is rapidly taken up by cancer cells, which are identified using radioactive signals. The rapid glucose transport in cancer cells is facilitated by glucose transporter (Glut) proteins, spanning across the cell membrane. Although there are several types of Glut proteins, Glut-1 proteins have been considered to be involved in glucose transport among cancer cell membrane. Further, to investigate the role of Glut-1 receptors over glucose uptake, Singh et al. [17] have developed two types of gold nanoclusters coated with BSA and glucose and followed the pattern of internalization of these probes in cancerous and noncancerous skin cells. They reported that glucose coated nanoclusters were not taken up by noncancerous (HaCaT) cells, however, specifically taken up by cancerous (A431) cells through Glut-1 receptors. It was also found that the uptake of glucose coated nanoclusters was cell membrane potential independent while BSA coated nanoclusters were found internalize by membrane potential dependent manner. Studies involving humans suggest that high levels of Glut-1 expression in tumors are associated with poor survival [18]. Hypoxia is also reported to increase Glut-1 levels and thus glucose uptake. Further, Chandler et al. have reported that Glut-1 receptors are highly expressed in human prostate cancer cells and are associated with the Golgi body, possibly offering glucose supply to Golgi for by-product incorporation into the prostate secretion [19]. Thus targeting Glut-1 could be a novel strategy for detection and treatment of prostate cancer. Genistein is a well-known inhibitor of the Glut-1 protein, and thus induce the formation of ROS and association with AMPK signaling pathway activation. Considering the above discussion, genistein could be considered as a novel chemotherapeutic agent for prostate cancers. Although genistein alone may not be able to induce significant suppression to prostate cancer progression, however, combined with some known anti-prostate cancer agent could offer excellent results. Hwang et al. have reported that genistein combined with 5-Fluorouracil can effectively inhibit the progression of colon cancer cells, where genistein acts as cell sensitizer supporting 5-Fluorouracil to abolish the up-regulated COX-2 and prostaglandin secretion [20].
Traditional methods of cancer treatment involve single anticancer agent prescribed to patients and that have realized to have limited success due to the toxicity to healthy cells and resistance to cancer cells. Such obstacles are more pronounced due to the administration of a high dose of anticancer agents in order to achieve enhanced efficacy. A well-known example is a use of Vemurafenib (PLX4032) for the treatment of melanoma. Although the drug showed excellent melanoma regression activity, however, the long-term use eventually led to the resistance in melanoma cells [21]. Further investigation revealed that repeated exposure of a drug led to the development of an alternative survival pathway and thus express suitable cell surface receptors. Such events are common in the strategies involving single drug treatment because targeting individual signaling pathway of cancer cell survival may not be enough to achieve high therapeutic efficacy. Owing to the multi-gene abnormality, the survival of cancer cells is regulated by multiple signaling pathways, therefore, formulations containing multiple drugs (targeting multiple pathways) could be the strategy for cancer treatment with better results.
Nanomaterials, such as nanoliposomes, micelles, polymeric, metallic, and other nanostructures, have shown excellent results in encapsulating multiple anticancer agents and delivering them at the desired tumor site [[22], [23], [24], [25], [26]]. Nanomaterials-based drug delivery agents also offer the controlled release of drugs in the desired ratio and long-term circulation in the blood, which further enhances the treatment efficacy. Drug delivering nanocarriers offer better therapeutic efficacy due to the “Enhanced Permeable and Retention” (EPR) effect exhibited by nanomaterials of ∼100 nm diameter. This passive targeting is mainly facilitated by leaky vasculature in cancer tissues, which unlike normal healthy vessels, have ∼600–800 nm wide gaps among adjacent endothelial cells. Such faulty vascular system with poor lymphatic drainage enables drug delivering nanomaterials to extravasate into the extravascular space and accumulate in tumor tissues [27,28]. It has been reported that due to EPR effect ∼10 fold increase in drug retention occurs in tumor region compared to free drugs [29]. PEGylation (stealthing) of the nanoparticles are suggested to make them “invisible” to the macrophages and phagocytes leading to the long circulation time in blood [30,31].
Approaches using co-delivery strategy for prostate cancer treatment are limited and have used non-specific drugs. Patil et al. have reported the co-encapsulation of mitomycin C and doxorubicin in liposome conjugated with folate receptors. This formulation showed enhanced toxicity to LNCaP prostate cancer cell line and KB cells [32,33]. Another study reported the use of a combination of doxorubicin and chloroquine (as chemosensitizer). Their result showed that the prostate tumor growth was arrested during the three-week administration period without any pervasive side effects [34]. Recently, there have been increasing attention towards the development of multifunctional nanocarriers which can combinatorially deliver the chemotherapeutic agents and produce maximum therapeutic efficiency. Such strategies using selected drugs can overcome multidrug resistance as well as inhibit the anti-apoptotic property of cancer cells and achieve synergistic anticancer effects.
In this work, we report the synthesis of a multifunctional liposome-based nanocarrier system encapsulating celecoxib and genistein. The prostate cancer cell-based assays suggest the inhibition of proliferation of cancer cells and no effect to normal fibroblasts cells. The mechanistic investigation revealed that both of these drugs deregulate the key signaling pathways of prostate cancer cells needed for their growth and metastasis. Inhibition of the expression of COX-2 and Glut-1 receptors thereby decreased glucose intake were some of the vital processes, which induce the prostate cancer cell apoptosis.
Section snippets
Preparation of liposomes containing celecoxib and genistein
The synthesis of nanoliposomes was performed by following a method described by Gowda et al. [25] with slight modification. Briefly, the liposomes were prepared by using l-α-phosphatidylcholine (eggPC) and 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N- [methoxy(polyethylene glycol)-2000] ammonium salt (DPPE-PEG-2000) in chloroform at 95:5 mol % with 25 mg/mL total lipid concentration (Avanti Polar Lipids). Chloroform from the lipid mixture was removed by blowing N2 gas and the so obtained
Synthesis and characterization of nanoliposomes encapsulating celecoxib and genistein
Recently, liposomes-based nanoparticles (nanoliposomes) have shown tremendous success towards improved therapies including delivery of therapeutic drugs/genes at the targeted site, with minimum obstacles to tissue uptake and improved pharmacokinetics and pharmacodynamics. Considering these advantages, several liposome-based strategies are already approved for clinical trials [35]. However, most of these strategies are based on single-agent delivery, which still faces some of the limitations
Conclusion
In this study, we have developed celecoxib and genistein encapsulating nanoliposomes, which represent a unique class of anticancer agent with the potential of being used to treat several types of cancer including prostate. The prepared nanoliposomes are ∼100 nm in diameter, which is considered as preferred size to be readily internalized by cancer cells. The encapsulated drugs, celecoxib, and genistein retain their anticancer properties and in fact, work synergistically to decrease the
Conflicts of interest
Authors declare no conflict of interest.
Acknowledgement
Authors acknowledge Jilin University. China for the necessary support and facility to carry out this project.
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