Antiapoptotic Bcl-2 protein as a potential target for cancer therapy: A mini review

Abstract

Bcl-2, an antiapoptotic protein, is considered as a potential target in cancer treatment since its oncogenic potential has been proven and is well documented. Antisense technology and RNA interference (RNAi) have been used to reduce the expression of the Bcl-2 gene in many types of cancer cells and are effective as adjuvant therapy along with the chemotherapeutic agents. The lack of appropriate delivery systems is considered to be the main hurdle associated with the RNAi. In this review, we discuss the antiapoptotic Bcl-2 protein, its oncogenic potential, and various approaches utilized to target Bcl-2 including suitable delivery systems employed for successful delivery of siRNA.

Keywords::

• Antisense oligonucleotide
• apoptosis
• Bcl-2
• cancer
• drug delivery
• RNA interference

Introduction

Though etiology and molecular pathways involved in certain diseases like cancer have been understood over the last three decades, treating cancer is still a challenging task for physicians. Identifying novel targets responsible for tumor induction and growth would greatly help scientists to discover new anticancer molecules. The components of molecular pathways involved in cancer are well identified and studied. These components can be the potential targets for developing effective therapies against cancer. Researchers are focusing on the development of molecules, either synthetic or derived from natural sources, which directly target such components in cancerous cells without affecting the normal cells.

Apoptosis, a naturally occurring phenomenon and often considered programmed cell death, plays a very crucial role in physiological processes such as development, maintaining tissue homeostasis, and defence mechanisms in diseases that occur in higher organisms, from fetal development to the adult lifetime, (Hetts 1998). The disruption or dysregulation of apoptotic pathways contribute to the development of certain diseases like neurodegenerative disorders (Mattson 2000, Thompson 1995), tumor development (Reed 1999), and also in some of the autoimmune disorders (Lorenz et al. 2000). Apoptosis can be elicited by either of two pathways, namely the intrinsic pathway and the extrinsic pathway (Figure 1). The intrinsic pathway, also called the mitochondrial or stress pathway, is the most utilized pathway for triggering apoptosis in cells. The intrinsic pathway is initiated upon cell damage, activation of oncogene, and as a response to some stress or deprivation of nutrients. This review discusses the role of the antiapoptotic Bcl-2 (B cell CLL/lymphoma 2) protein in cancer progression, the technical aspects of targeting the Bcl-2 protein, and delivery systems available for utilizing these approaches for targeting Bcl-2.

Figure 1. Pathways of apoptosis: Intrinsic and Extrinsic.

Antiapoptotic Bcl-2 protein and Bcl-2 protein family

Bcl-2, the antiapoptotic protein from the Bcl-2 family of proteins, can be considered as a regulator of the intrinsic pathway of apoptosis. Initially, the Bcl-2 gene was identified and described as a proto-oncogene in 1984 in human follicular B-cell lymphoma cells carrying translocated Bcl-2 gene to the immunoglobulin heavy chain locus t(14;18)(q32;q21) (Marzo and Naval 2008, Hockenbery et al. 1990). The Bcl-2 gene is located at the long (q) arm of chromosome 18 at position 21.3 (Medicine, UNLO). The family of Bcl-2 proteins contains two classes of proteins, proapoptotic and antiapoptotic. Death or survival of the cell depends on the balance between proapoptotic and antiapoptotic proteins (Marzo and Naval 2008). These groups of proteins share one of the four homologous regions known as Bcl homology (BH) domains (BH1–BH4). Antiapoptotic members of the Bcl-2 family include all the four BH domains except Mcl-1, which includes BH-1, 2, and 3. The proapoptotic proteins Bax and Bak contain three domains (BH-1, 2, and 3), and Bad, Bim, Bik, PUMA, NOXA, Bmf, and Hrk contain only the BH3 domain (the BH3-only subfamily) (Heiser et al. 2004). Some of the Bcl-2 family members contain a carboxy terminal transmembrane (TM) region (Figure 2) (Kroemer 1997). The Bcl-2 protein (molecular weight of 26 kDa) is found on mitochondrial outer membrane, endoplasmic reticulum and nuclear envelope. It binds with Bax and prevents the release of cytochrome c from mitochondria and the formation of Apaf-1 and apoptosome, thereby preventing the activation of proteolytic demolition in cells (Adams and Cory 1998, Elmore 2007). Knock down of Bcl-2 gene in mice displayed early death after birth because of extended apoptosis in cells of the thymus and spleen (Veis et al. 1993). Cell survival depends on the level of Bcl-2 protein in cells.

Figure 2. Members of the Bcl-2 protein family.

Oncological Potential of Bcl-2

It was observed that expression of the Bcl-2 protein is enhanced significantly in many types of cancers. Bcl-2 stimulates cancer cell survival rather than inducing proliferation. This concept led the researchers to understand that a disrupted or impaired apoptosis pathway may play a crucial role in tumor development. Disruption of the apoptotic pathway due to mutation is often observed in many cancer types (Lowe and Lin 2000). Overexpression of the Bcl-2 gene protects cells from undergoing apoptosis and enhances the survival of the cells in prostate (Raffo et al. 1995), breast (Joensuu et al. 1994), and bladder (Miyake et al. 1999) cancer, acute and chronic leukemia (Campos et al. 1993), follicular lymphoma (Reed et al. 1988), B-cell lymphoma, renal (Gobé et al. 2002), and neuroblastoma (Dole et al. 1994). Induction of apoptosis in cancerous cells can be a link between cancer genetics and treatment (Pan et al. 1997, Johnstone et al. 2002). Many chemotherapeutic agents used in the treatment of cancer induce apoptosis in response to drug-induced cell damage (Brown and Attardi 2005, Fesik 2005). Defects in the apoptotic pathway also contribute to the drug resistance among cancer cells. The role of the Bcl-2 protein in developing resistance to drug has been proven in certain classes of cancers (Davis et al. 2003, Schmitt et al. 2000, Sartorius and Krammer 2002).

Many research groups have proved the oncogenic potential of Bcl-2 in tumor development and metastasis, described elsewhere (Hockenbery 1994, Marzo and Naval 2008). Reed et al. (1988) demonstrated the oncogenic potential of Bcl-2 by the gene transfer approach in follicular lymphoma, the most common human B cell malignancy (Reed et al. 1988). The role of Bcl-2 in B-cell survival and follicular lymphoma was explored in transgenic mice in which Bcl-2 overexpression was targeted to B and T lymphocytes, which caused follicular hyperplasia or T-cell lymphomas (McDonnell et al. 1989). Bcl-2 contributes to oncogenesis with the synergistic effect of other oncogenes like c-myc and ras (Strasser et al. 1990, Reed et al. 1990, Cory et al. 1999). To evaluate this synergistic effect of Bcl-2 with c-myc oncogenes, Bcl-2 was transfected through retro virus into the bone marrow of transgenic mice overexpressing the c-myc oncogene. Bcl-2 co-operated with c-Myc to promote the transformation of B cell precursors (Vaux et al. 1988). However, Bcl-2 alone did not promote proliferation but inhibited apoptosis induced by cytokine withdrawal (Leskov et al. 2012). Sierra A. et al., 1996, reported that Bcl-2 expression was strongly associated with both loss of apoptosis and lymph node metastases (Sierra et al. 1996). Donatella Del Bufalo et al. 1997, demonstrated that Bcl-2 overexpression augmented both the tumorigenicity and metastatic potential of the breast cancer cell line MCF7 ADR cells, by injecting MCF7 ADR into nude mice by intravenous or subcutaneous injection after inserting human Bcl-2 gene in the cells (Del Bufalo et al. 1997). Miyake et al., 1999, also proved that the metastatic potential of the human bladder cancer cell line KoTCC-1/BH, transfected with overexpressed Bcl-2 gene in nude mice, was significantly enhanced with improved invasive ability and cell motility (Miyake et al. 1999).

Targeting Bcl-2 for cancer therapy

The role of Bcl-2 in tumor development and metastasis makes it the most promising target for cancer treatment. Induction of apoptosis in cancerous cells by inhibiting the overexpressed Bcl-2 protein can be an attractive and alternate therapy for cancer treatment. Molecules that suppress the expression of the Bcl-2 gene may be considered as a new therapeutic class of drugs in cancer treatment (Wang et al. 2003). The Bcl-2 gene is not only involved in the development of cancer but also in the mechanism whereby cancer cells develop resistance to chemotherapeutic agents (Leber et al. 2010). Molecules which suppress the Bcl-2 gene expression and sensitize the cells to conventional chemotherapeutic agents are also gaining more attention as an adjuvant therapy. Several research groups utilizing this approach have synthesized a few molecules that inhibit Bcl-2 proteins. Some of them are already in clinical trials and have shown a promising response (Table I) (Kang and Reynolds 2009), while some researchers exploited the gene silencing approach, utilizing antisense technology and RNA interference, to inhibit Bcl-2 gene expression.

Table I. List of the molecules targeting Bcl-2 proteins.

Name	Target	Cancer	References
Depsipeptide	Bcl-2, Bcl-XL, and Mcl-1	Multiple myeloma cells	Khan et al. (2004)
Fenretinide	Bcl-2, Bcl-XL, and Mcl-1	Leukemia	Delia et al. (1995)
AT-101, (levo gossypol, Ascenta)	Bcl-2 and Mcl-1	Chronic lymphocytic leukemia (CLL), prostate cancer	MacVicar et al. (2008)
Apogossypol (BI97C1, sabutoclax or ONT-701)	Bcl-2, Bcl-XL, and Mcl-1	Solid tumors	Kitada et al. (2008)
Apogossypolone (BI97C10 and BI112D1)	Mcl-1, Bcl-2, Bcl-XL	Solid tumors	Wei et al. (2009, 2011)
ABT-737 (A-779024, Abbott Laboratories)	Bcl-2, Bcl-XL, and Bcl-w	Acute myeloid leukemia, multiple myeloma, lymphoma, CLL, acute lymphoblastic leukemia, and small-cell lung cancer	Konopleva et al. (2006), Chauhan et al. (2006), Tahir et al. (2007), Kang and Reynolds (2009)
ABT-263 (Oral version of ABT-737)	Bcl-2, Bcl-XL, and Bcl-w	small cell lung cancer, B-cell lymphoma and multiple myeloma	Tse et al. (2008)
GX15-070 (obatoclax, Gemin X)	Bcl-2, Bcl-XL, Bcl-w, and Mcl-1	CLL	Kang and Reynolds (2009)
HA14-1	Bcl-2	Glioblastoma, colon cancer	Manero et al. (2006), Sinicrope et al. (2004)
S1	Bcl-2 and Mcl-1	Liver cancer	Zhang et al. (2011), Song et al. (2012)

Antisense technology was widely used for transcription gene silencing before the discovery of ribozymes and RNAi (Kurreck 2003). Antisense oligonucleotides, single-stranded 15–25 base-long DNA molecules complementary to the mRNA of the targeted gene, are used to prevent transcription. When introduced into the cells, antisense oligonucleotides target and bind with the mRNA having a complementary sequence and inhibit the protein synthesis (Re 2000). There are several issues to overcome to enable the success of antisense technology and RNAi as therapeutic tools, including stability, delivery, cost, and toxicity.

A 35S-labeled phosphorothioate oligonucleotide (G3139, Genasense; Genta International Inc., Berkeley Heights, NJ) having 18 bases with sequences complementary to the first six codons of the open reading frame of Bcl-2 (G3139) showed potent antitumor effect against the DoHH2 lymphoma implanted in severe combined immunodeficient female BALB/c mice after single intravenous (i.v.) bolus administration or subcutaneous infusion (s.c) for 1 week (Cotter et al. 1996, Raynaud et al. 1997). The results from the preclinical study in mice were encouraging, and researchers conducted this phase I clinical trial of G3139 antisense oligonucleotide in nine patients and later in 21 patients with Bcl-2 positive relapsed non-Hodgkin's lymphoma. Bcl-2 antisense therapy showed good efficacy in patients, with improvement in biochemical and hematological symptoms and downregulation of the Bcl-2 protein in some patients (Webb et al. 1997, Waters et al. 2000). The G3139 antisense oligonucleotide was also evaluated in a phase I study with fludarabine (FL), cytarabine (ARA-C), and granulocyte colony-stimulating factor (G-CSF) (FLAG) salvage chemotherapy in 20 patients with refractory or relapsed acute leukemia. G3139 was administered safely with FLAG chemotherapy and efficiently downregulated Bcl-2 expression in 75% of the patients evaluated (Marcucci et al. 2003). The effect of G3139 was evaluated in prostate cancer in combination with docetaxel (Tolcher 2001), in small-cell lung cancer, in combination with carboplatin and etoposide (Rudin et al. 2004) and in small-cell lung cancer in combination with paclitaxel in phase I clinical trials (Rudin et al. 2002). Oblimersen sodium (Genasense), in combination with dacarbazine, was further evaluated in phase II clinical trials in advanced melanoma (Bedikian et al. 2006), in combination with dexamethasone and thalidomide in relapsed multiple myeloma (Badros et al. 2005), in combination with rituximab in recurrent B-cell non-Hodgkin lymphoma (Pro et al. 2008), and with carboplatin and etoposide along with or without a combination of oblimersen in small-cell lung cancer (Rudin et al. 2008). Phase III clinical trials were completed in combination with fludarabine and cyclophosphamide in recurrent or refractory chronic lymphocytic leukemia, with dexamethasone in recurrent or refractory multiple myeloma, with dacarbazine in metastatic malignant melanoma, and with docetaxel in non-small-cell lung carcinoma. Malignant melanoma did not show a survival benefit from G3139, according to reports of the phase-III clinical trial. Final results from other trials are still awaited (Kim et al. 2004).

RNA interference (RNAi) is a natural biological mechanism in which double-stranded RNA (dsRNA) intrudes with the gene expression having a homologous sequence with the dsRNA, when introduced. RNAi is widely used to regulate gene expression in many studies, like basic research, functional genomics, drug discovery, drug validation, and transgenic designing. RNAi-induced gene silencing can be achieved by utilizing small interfering RNA (siRNA) of 21–23 nucleotide length that is inherently produced from long dsRNA by the enzyme Dicer, which has RNase-III-like activity. These siRNA are integrated into a multi-protein RNA-inducing silencing complex (RISC). The duplex siRNA is unwound by helicase of RISC, causing the formation of sense and antisense strands. The antisense strand and RISC directed to its complementary mRNA and by the action of the exonuclease and endonuclease domain of RISC, targeted mRNA is cleaved by the action of the exonuclease and endonuclease domain of RISC (Figure 3) (Elbashir et al. 2001).

Figure 3. Mechanism of RNA interference.

Many research groups have selected the Bcl-2 gene as the target, utilizing RNAi technology in developing treatment for many cancer types. siRNA specific for the Bcl-2 gene can be designed utilizing many available bioinformatics tools. In 2002, Takashi Futami et al. used the siRNA expression system-based vector-targeting Bcl-2 gene in HeLa cells to induce apoptosis. The siRNA vectors made HeLa cells more sensitive to doxorubicin treatment (Futami et al. 2002). Silencing of Bcl-2 in melanoma cells by specific siRNA led to an increase in apoptotic cell death and inhibition of cell growth. siRNA-targeting Bcl-2 in combination with cisplatin showed a massive increase in apoptotic cell death (Wacheck et al. 2003). Thereafter, the siRNA-targeting Bcl-2 gene was intensively used in pancreatic (Ocker et al. 2005), liver (Feng et al. 2006, Warmann et al. 2008), breast (Akar et al. 2008), ovarian (Saad et al. 2008), gastric (Hao et al. 2007), and bladder (Kunze et al. 2008) cancer, and in human glioblastoma (George et al. 2009a, 2009b) and neuroblastoma (Shen et al. 2012), in vitro and in vivo.

Various studies show that siRNA and antisense nucleotide-based nucleic acid drugs are very effective in controlling the expression of genes such as Bcl-2. These nucleic acid-based drugs can be effective only if they can reach their target present within the cell. However, a number of factors hinder the in vivo applications of these drugs (Yin et al. 2013, Draz et al. 2014).

• Charge: Nucleic acid based drugs are negatively charged. Therefore, these drugs cannot cross the cell membrane easily.

• Stability: Upon entry into the systemic circulation, siRNA is recognized by the immune system and is rapidly cleared. siRNA can pass through the glomerular filtration barrier and therefore undergoes rapid renal clearance. These drugs are degraded by the highly acidic endosomes and serum nucleases.

• Off-target effect: Binding siRNA with other mRNA rather than the targeted mRNA could result in prevention of translation of genes coding for essential proteins. These negatively charged nucleotides can also bind with serum proteins, resulting in decreased bioavailability.

• Toxicity: siRNA is capable of activating Toll-like receptors, leading to increase in the cytokine levels. Nuclease-cleaved RNA can activate innate immunity.

Therefore, it is necessary to develop and adopt proper techniques so that these nucleic acid-based drugs are able to reach their targets. The following section discusses some of the currently tested techniques, with a focus on nanoparticulate-based delivery systems useful in assisting these drugs reach their targets (Figure 4).

Figure 4. Various delivery systems for siRNA and delivery antisense oligonucleotides.

Virus-based delivery system

Viruses are “naturally evolved vehicles” that have the highest ability to easily transfer their genetic material into the hosts. Due to this, a number of viral vector systems have been screened for possible use as drug carriers to deliver genes of interest. The viruses are selected based on the safety, ability to carry foreign genes, transfection efficiency, and gene expression. Among the various virus groups, retroviruses have been the virus of choice for delivery systems. Nearly 40% of the clinical trials involved in studies using viral vector systems utilize recombinant retroviruses as the carrier. Other viral vector systems include the adenovirus, adeno-associated virus, herpes simplex virus, and lentivirus. These viral vectors are well characterized and easy to manipulate (Walther and Stein 2000).

T7 vector-based delivery systems

T7 vector-based delivery systems are useful to obtain a sufficient amount of mRNA within the target cells. These vectors can be made to specifically target Bcl-2 by preparing the “T7 promoter-driven siRNA expression vector system (Bcl-2/T7)”. These kinds of vectors are utilized in reducing the level of Bcl-2 mRNA (Holle et al. 2004).

Virosomes

Virosomes are prepared by incorporating proteins derived from the envelope of the virus. This delivery system is useful for the cytosolic release of drugs (Felnerova et al. 2004). Detailed descriptions of the virus as a drug carrier for delivery of nucleic acid-based drugs have been well documented (Walther and Stein 2000, El-Aneed 2004).

Chemical modifications

One way of successfully using siRNA in vivo is through suitable chemical modification. siRNA can be modified in a number of ways, to increase their stability and efficacy. Replacing oxygen atoms (which are not involved in bridging two ribonucleotides) present in the phosphate backbone, with atoms of sulfur, helps in preparing siRNA with ‘phosphorothioate’ linkages. This technique increases the resistance against serum nucleases. Similarly, if the replacing atom is ‘boron’, then siRNA will contain ‘boranophosphate’ linkages. This siRNA is believed to produce a better gene-silencing effect even at a lower concentration, in addition to the increased stability in serum. Bridging 2’ ribose carbon with 4’ carbon results in methylene linkages which produce nucleotides with higher affinity binding and better efficacy. This kind of siRNA is often referred to as “Locked nucleic acid (LNA)” nucleotides (Corey 2007). Readers can refer the article “Chemical modification: the key to clinical application of RNA interference?” by David R. Corey, for complete explanation on various approaches available (Corey 2007).

Conjugates

The stability of genetic material such as siRNA can also be improved by preparing them in the form of conjugates which can improve pharmacokinetic properties, particularly the half-life and volume of distribution. Cholesterol-siRNA conjugates are found to be absorbed better by the tissues (Wolfrum et al. 2007, Shim and Kwon 2010). Through suitable modification and by using Bcl-2-specific siRNA, this approach could be adopted to target various cancer types.

Cationic polymers

Poly(L-lysine) (PLL)

PLL is a biodegradable lysine-containing linear polypeptide. The main drawback of a PLL-based delivery system is the rapid clearance from the plasma (El-Aneed 2004). However, PLL can be easily grafted to make it suitable for specific applications (Lollo et al. 2002). PLL can be even grafted using materials such as silica, to make it suitable for oral delivery of genes (Badros et al. 2005). Modified PLL is useful in delivering antisense oligonucleotides specific for Bcl-2 (Read et al. 2000). Modified PLL is also useful as a delivery system for co-administration of siRNA and anticancer agents. This combination drug strategy is effective in downregulating the expression of the Bcl-2 gene and enhancing the effect of the chemotherapeutic agent (Zheng et al. 2013).

Poly(ethylenimine) (PEI)

Among the cationic polymers, PEI is the most widely used. This is mainly due to its ability to buffer a wide pH range. PEI is highly positively charged due to the presence of nitrogen atoms. Molecules such as DNA and RNA are negatively charged. Therefore, these molecules are easily complexed by the highly positively charged PEI. Although there is no clear proof on the mechanism by which PEI is able to resist highly acidic conditions, it is believed that these PEI complexes are able to escape lysosomal degradation due to the “proton sponge” effect (Benjaminsen et al. 2013). Cytotoxicity, activation of immune cells, slow release of siRNA, and the tendency to form aggregates are some of the limitations associated with PEI-mediated genetic delivery systems (Wen and Meng 2014). Among the various factors, viz., particle size, molecular weight, degree of branching, etc., it is believed that the high positive charge on the PEI contributes to its toxicity (El-Aneed 2004, Shen et al. 2013). Therefore, approaches such as PEGylation help in reducing the toxicity. PEGylation further improves the stability and biocompatibility of the complexes (Shen et al. 2013).

Polyamidoamine

The ease with which the charge density and molecular weight can be altered has made polyamidoamine one of the preferred carrier systems for the delivery of siRNA. The amines present at the surface of the polyamidoamine aid in binding with the siRNA. The tertiary amine present in the interior of the polyamidoamine helps in the escape from endosomal degradation (Wen and Meng 2014).

Cationic (amphipathic) peptides (CP)

CP are amphiphilic in nature and therefore have the ability to undergo conformational change when exposed to acidic conditions. This helps them to escape lysosomal degradation and deliver their cargoes. The positively charged amino acids present in the cationic peptides help them to condense the negatively charged materials such as DNA and siRNA and deliver inside the cell (El-Aneed 2004). CP are particularly useful when used along with other carriers such as cationic polymers (Kichler et al. 2006). The properties that CP should possess for the delivery of nucleic acid-based drugs include solubility in aqueous solutions, presence of positive charge (preferably amphipathic in nature), cell membrane destabilizing ability, and short size (preferably less than 30 amino acids) (Kichler et al. 2006, Verdurmen and Brock 2011). If it is decided that CP will be used as a drug carrier, the CP should be carefully selected as CP possess intrinsic biologic properties (Verdurmen and Brock 2011).

Gold nanoparticles (GNP)

Interest in the use of GNP as a delivery system for nucleic acid-based drugs, particularly siRNA, has steadily increased, with the first report published in 2006 (Lytton-Jean et al. 2011). siRNA can be conjugated with GNP either through chelation of gold-thiol or through electrostatic interaction. Newly synthesized GNP are negatively charged. These negatively charged GNP are surface-coated with suitable materials/polymers such as PEI, so that they acquire positive charge. They can then bind with siRNA through electrostatic interaction (Lytton-Jean et al. 2011, Wen and Meng 2014). Scavenger receptors present on the cell surface help in internalization of GNP. The circulation time of GNP improves upon PEGylation or attachment with a suitable ligand (Wen and Meng 2014). GNP are able to protect siRNA from nucleases and shield them from the immune system (Lytton-Jean et al. 2011).

Iron oxide-based nanoparticles (INP)

INP are extensively used as diagnostic agents and also in hypothermic-based cancer treatment. Among various types of iron oxides available, magnetite (Fe₃O₄) and maghemite (γ-Fe₂O₃) are widely used. This is due to their biocompatibility, stability within biological conditions, and low cost of preparation (Figuerola et al. 2010). Recent studies show that INP could be used as carriers in delivering cargo specifically into the target site. These nanocarriers are even useful to target specific proteins such as Bcl-2. Coupling the oncopeptide NuBCP-9 with INP allows specific targeting of Bcl-2. These INP are particularly useful in delivering levoform proteins and peptides to the targets lying within the cell (Kumar et al. 2014). Although the aqueous solubility of INP is the main limitation, this is easily overcome through surface coating of these nanoparticles (Figuerola et al. 2010). Reports even suggest that ultra-small superparamagnetic iron oxide (USPIO) can be easily synthesized through suitable modification in the method of synthesis and subsequent coating with amphiphilic polymers (Yang et al. 2013). These USPIO are made Bcl-2-specific by binding them with Bcl-2 monoclonal antibody using a N-hydroxysuccinimide/ethyl(diethylaminopropyl) carbodiimide-based method (Yang et al. 2013).

An important application of superparamagnetic iron oxide (SPIO) is the possible use as a non-viral vector. Conjugation of SPIO further allows monitoring of the effectiveness of the therapy. Bcl-2-specific siRNA was found to be effective in controlling the expression of Bcl-2 in neuroblastoma cells when delivered along with a delivery system comprising a single-chain antibody conjugated with the PEG-PEI-SPIO system (Shen et al. 2013). Other miscellaneous, inorganic or metal-based particulate delivery systems used in the delivery of nucleic acid-based drugs include cyclodextrin, calcium phosphate, carbon nanotubes, silica, and silicon-based nanoparticles (Yin et al. 2013, Wen and Meng 2014, Draz et al. 2014).

Hydrogels

Hydrogels have a hydrophilic three-dimensional network capable of absorbing large quantities of water and biological fluids. They could be virtually made up of any hydrophilic polymers and can be modified easily for different shapes and properties (Draz et al. 2014, Hoare and Kohane 2008). Among the macro, micro, and nano hydrogels, nano hydrogels are extensively studied for possible application as a drug carrier for delivery of drugs into the cell. Due to their porous nature, hydrogels have high loading efficiency and can be easily employed for targeted delivery. This allows sustained release of drugs such as siRNA (Draz et al. 2014).

Cationic Liposomes

Cationic liposomes are one of the most studied nonviral-based delivery systems for nucleic acids and siRNA. Cationic lipids like DOTAP (1,2-dioleoyl-3-trimethylammonium-propane) and DOTMA (1,2-di-O-octadecenyl-3-trimethylammonium propane) have been extensively studied to deliver siRNA to target sites (Ma et al. 2005, Zhang et al. 2007). The cationic charge on the surface of particles helps to form stable complexes with negatively charged siRNA due to electrostatic interaction, which improves the stability of the complexes. Cationic liposomes will interact with the negatively charged cell membrane and easily get transfected, leading to enhanced cellular uptake. Activation of the immune response, the interaction with blood components and the removal by the reticuloendothelial system, leading to poor blood circulation, are considered as drawbacks for cationic liposomes. Cationic liposomes can be modified with polymers like polyethylene glycol (PEG) (Kim et al. 2010), peptides (Yang et al. 2014), galactose (Sonoke et al. 2011), transferrin (Nakase et al. 2005), and many other small molecules.

For effectively targeting Bcl-2, the potential drug candidates have to be delivered to their target sites. This is particularly challenging due to various factors including the presence of charged cell membranes and various efflux mechanisms present within the cell. Therefore, the small size of nanodelivery systems makes them useful in delivering the drugs into the cell. A viral-based delivery system is useful in achieving higher bioavailability. However, the chances of immune response have led to the search for newer non-viral based carriers. Although chemical modification and the use of conjugates helps in the delivery of drugs across the cell membrane, not all the drugs can be modified without affecting their activities. In such cases, nanoparticulate-based delivery systems are useful. In addition, nanoparticulate delivery systems provide more choice with respect to drug targeting. However, there is no complete information on nanoparticulate delivery systems, particularly their toxicity and degradation. Nevertheless, nanoparticulate delivery systems hold a great promise for targeting sites such as Bcl-2 present within the cell.

Conclusion

Cancer therapies have made a paradigm shift in approaches for proper understanding of cancer biology. The identification of genes having potential roles in the development of cancer has led to the selection of new targets for cancer therapy. One of the potential targets is the antiapoptotic Bcl-2 protein, proved to be a potential target due to its role in the development of cancer. Silencing the overexpressed Bcl-2 gene, preventing its translation, and using well-conceptualized RNAi and antisense technology, are proving to be viable and effective alternatives in cancer therapy. Developing formulations to deliver siRNA and antisense oligonucleotides to targeted sites is an issue. Many nanotechnology-based delivery systems are being developed and evaluated to achieve this goal. Hence, this approach holds a great promise for developing an alternative therapy for cancer or as an adjuvant therapy, thereby enhancing the effectiveness of existing anticancer drugs.

Acknowledgements

The authors would like to acknowledge the authorities of the Council of Scientific and Industrial Research (CSIR), New Delhi, for providing a fellowship to Mr. Hitesh Vitthal Jagani, and Manipal University for providing the necessary research facilities.

Declaration of interest

The authors report no declarations of interest. The authors alone are responsible for the content and writing of the paper.