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REVIEW ARTICLE
Year : 2015  |  Volume : 52  |  Issue : 1  |  Page : 11-21
 

Applicability of RNA interference in cancer therapy: Current status


Department of Pharmaceutical Biotechnology, Amity Institute of Pharmacy, Amity University, Noida, Uttar Pradesh, India

Date of Web Publication3-Feb-2016

Correspondence Address:
S Maduri
Department of Pharmaceutical Biotechnology, Amity Institute of Pharmacy, Amity University, Noida, Uttar Pradesh
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0019-509X.175598

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 » Abstract 

Cancer is a manifestation of dysregulated gene function arising from a complex interplay of oncogenes and tumor suppressor genes present in our body. Cancer has been constantly chased using various therapies but all in vain as most of them are highly effective only in the early stages of cancer. Recently, RNA interference (RNAi) therapy, a comparatively new entrant is evolving as a promising player in the battle against cancer due to its post-transcriptional gene silencing ability. The most alluring feature of this non-invasive technology lies in its utility in the cancer detection and the cancer treatment at any stage. Once this technology is fully exploited it can bring a whole new era of therapeutics capable of curing cancer at any stage mainly due to its ability to target the vital processes required for cell proliferation such as response to growth factors, nutrient uptake/synthesis, and energy generation. This therapy can also be used to treat stage IV cancer, the most difficult to treat till date, by virtue of its metastasis inhibiting capability. Recent research has also proved that cancer can even be prevented by proper modulation of physiological RNAi pathways and researchers have found that many nutrients, which are a part of routine diet, can effectively modulate these pathways and prevent cancer. Even after having all these advantages the potential of RNAi therapy could not be fully tapped earlier, due to many limitations associated with the administration of RNAi based therapeutics. However, recent advancements in this direction, such as the development of small interfering RNA (siRNA) tolerant to nucleases and the development of non-viral vectors such as cationic liposomes and nanoparticles, can overcome this obstacle and facilitate the clinical use of RNAi based therapeutics in the treatment of cancer. The present review focuses on the current status of RNAi therapeutics and explores their potential as future diagnostics and therapeutics against cancer.


Keywords: Cancer, micro-RNA, oncogenes, RNA interference, small interfering RNA, short hairpin RNA, tumor suppressor genes


How to cite this article:
Maduri S. Applicability of RNA interference in cancer therapy: Current status. Indian J Cancer 2015;52:11-21

How to cite this URL:
Maduri S. Applicability of RNA interference in cancer therapy: Current status. Indian J Cancer [serial online] 2015 [cited 2018 Dec 14];52:11-21. Available from: http://www.indianjcancer.com/text.asp?2015/52/1/11/175598



 » Introduction Top


Due to the advancements in the area of reverse genetics the cause of uncontrolled proliferative ability of cancer cells has been traced back to either increased expression of oncogenes or the decreased expression of tumor suppressor genes.[1] This discovery has given rise to a whole new area of research, which aims at correcting this dysregulated gene expression. By virtue of the advancements in this area of research, it has been found that disturbances in the physiological RNAi pathways are the main cause for this dysregulated gene expression.[2],[3],[4] The physiological RNAi pathways regulate the gene expression with the aid of micro-RNAs (miRNAs), which destroy the expressed messenger RNA (mRNA) [Figure 1] and ultimately lead to post-transcriptional gene silencing.[5],[6] Thus, the level of expression of any gene is regulated by the levels of miRNA produced against the mRNA of that gene. Due to this, the root cause of the dysregulation of gene expression in cancer cells was found to be either the increased production of miRNAs against tumor suppressor genes or decreased production of miRNAs against oncogenes.[7] This discovery led to the production of synthetic siRNAs, which resemble physiological miRNAs and can be administered into the cancer cells to compensate for the decreased production of miRNAs against oncogenes.[8],[9],[10],[11],[12],[13] However, since siRNAs exert their effect only for short durations [14] the researchers started administering them as short hairpin RNAs (shRNAs) expressed by viral expression vectors [15] [Figure 2]. These shRNAs, which are formed as the expression products of viral vectors are then processed by Dicer enzyme complex in cells into active siRNAs [Figure 2]. As these viral vectors can express the shRNAs for a long period of time, the gene silencing effect of the siRNAs formed from these shRNAs can be sustained for a long duration of time.[16],[17],[18],[19] The recent developments in the non-viral gene delivery systems further increased the applications of this technology by facilitating the targeted delivery of these interfering RNAs to cancer cells.[20],[21] All these developments have revolutionized RNAi based therapy as an effective and reliable alternative to chemotherapy for curing cancer. This review focuses on the current status of RNAi therapy in treatment of cancer and applicability of this technology in various areas of cancer therapy such as cancer diagnosis, treatment, and prevention along with a glance on the limitations of this technology and the recent developments facilitating its clinical application for the treatment of cancer.
Figure 1: Production of pre-miRNA in the nucleus by transcription and its subsequent processing into miRNA in the cytoplasm followed by the destruction of target mRNA by miRNA leading to post-transcriptional gene silencing

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Figure 2: Expression of shRNA by viral expression vector and its subsequent processing into siRNA in the cytoplasm followed by the destruction of target mRNA by siRNA leading to post-transcriptional gene silencing

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RNAi in cancer diagnosis

As the initiation of cancer is directly related to dysregulation of physiological RNAi pathways. The circulating miRNAs whose physiological levels fluctuate during cancer can be used as a biomarker for the diagnosis of cancer.[22],[23] Many such miRNAs whose physiological levels fluctuate during cancer have been identified in recent times and are shown in [Table 1] and [Table 2]. The physiological levels of some of these miRNAs show great selectivity and specificity towards cancer and can be considered as the most promising biomarkers for cancer diagnosis.
Table 1: miRNA biomarkers identified on the basis of their elevated levels in blood of cancer patients

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Table 2: miRNA biomarkers identified on the basis of their reduced levels in blood of cancer patients

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The miR-195 is one such miRNA whose elevated blood levels were found to be breast cancer specific. When used for detecting non-invasive and early stage breast cancer; researchers were able to differentiate breast cancer from other cancers and from controls with a sensitivity of 88% and specificity of 91%.[26]

Similarly, significantly elevated levels of two circulating miRNAs; miR-375 and miR-141 were found to be prostate cancer specific. The efficacy of these miRNAs was proved through an independent validation study wherein, prostate cancer was diagnosed in 72 patients using these miRNAs as the biomarkers.[27]

In another such study involving 24 ovarian cancer patients, a group of three miRNAs, that is, miR-21, miR-92, and miR-93 were found to be significantly over expressed in the serum from ovarian cancer patients compared to controls.[29]

Along with the above stated RNAi based biomarkers, miR-21 can also be used as a non-specific biomarker in cancer detection as its serum levels are elevated in many different cancers.[22],[27],[28],[29]

RNAi in treatment of cancer

Due to the recent advances in RNAi technology, this technology can now be used to treat cancers in any stage of its progression, that is, from stages 0 to IV.[36]

RNAi for the treatment of carcinoma in situ(stage-0)

Carcinoma in situ (CIS), often referred by doctors as pre-cancer; is a stage where the cancer is just originating. It is a localized phenomenon with no potential for tumor formation and metastasis; however, can transform into a true cancer if left untreated. It is the stage where the normal cells have just begun undergoing transformation into neoplastic cells and hence CIS is called as the stage-0 of cancer. As the recent advances in cancer research have proved that cancer initiates from a group of cells known as cancer stem cells, which are self-renewable.[37],[38] CIS can be considered as the stage where the cancer stem cells are formed from normal stem cells, due to the dysregulation caused by acquired epigenetic variations. This dysregulation in turn leads to unlimited self-renewal ability of these cells.[39] The main reason for the altered behavior of normal stem cells and their subsequent transformation into cancer stem cells was found to be the disturbances in RNAi pathways.[40] This statement is further supported by the recent identification of miRNAs in cancer stem cells, which have the ability to function as oncogenic and tumor suppressor miRNAs.[7] Contrary to the oncogenes which code for proteins that promote uncontrolled cell proliferation, the oncogenic miRNAs down-regulate tumor suppressor or other genes involved in the cell differentiation and promote tumor formation.[29] Similarly the tumor suppressor miRNAs down-regulate different genes that code for proteins with oncogenic activity and prevent tumor formation.[29] Hence, by developing an RNAi based therapy targeted at the suppression of oncogenic miRNAs or elevation of tumor suppressor miRNAs in cancer stem cells, cancer can be cured at CIS stage itself.

Since the cancer stem cells have been identified in many different types of cancers such as leukemia and solid tumors of breast, brain, pancreas, colon and head and neck cancers,[40] the above discussed approach for the cancer treatment can be applied for the treatment of many different types of cancers.

The expression of oncogenic miRNAs can be suppressed by administering an anti-miRNA oligonucleotide into the cancer cells. Recently, miR-21 in breast cancer cells was suppressed by administering anti-miR-21 oligonucleotide into breast cancer cells, which reduced cell growth in vitro and tumor growth in vivo.[41]

The expression of tumor suppressor miRNAs can be increased by artificially administering the vectors possessing the ability to express miRNA in cancer stem cells. By administering vectors with the ability to express miRNA instead of miRNA itself, the action of tumor suppressor miRNA can be sustained for a longer period of time. Recently, miR-34a expressing lentivirus was administered into pancreatic cancer stem cells which inhibited cancer cell growth and tumorsphere formation.[42]

The success of this therapeutic strategy led to the discovery of the many miRNAs with oncogenic [Table 3] and tumor suppressor [Table 4] effects in cancer stem cells.
Table 3: Oncogenic miRNAs discovered in cancer stem cells

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Table 4: Tumor suppressor miRNAs discovered in cancer stem cells

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RNAi for the treatment of cancers in stages I-III

The stages I-III of cancer have been classified on the basis of the status of tumor, that is, its size, severity and more the size and severity of tumor, the higher will be the stage number assigned to it and vice versa.[36] There has been a lot of progress in recent times in the development of RNAi therapies, which inhibit the tumor growth and in some cases probably revert the tumor to an earlier stage. The RNAi therapeutic strategy for treatment of cancers in these stages is based on silencing those genes which code for factors, which promote tumor growth. These factors can be growth factors, transport proteins, signaling molecules or any other factor, which in some way or the other promotes cancer cell proliferation and subsequent tumor growth.

Angiopoietin-1 can be considered as one such factor as it promotes the formation of tumor vasculature. The growth of tumor almost entirely depends on the blood supply to the tumor by tumor vasculature. Hence, tumor vasculature can be considered to directly support the tumor growth. Thus by blocking tumor angiogenesis, the tumor growth can be inhibited. This therapeutic strategy proved to be very effective in a recent study where the adenovirus based Angiopoietin-1 targeted siRNA expression system was tested on esophageal cancer cell line Eca109 in vitro and on athymic nude mice model for esophageal cancer in vivo leading to reduced tumor growth and reduced blood vessel formation.[52]

Tumor growth can also be stopped by inhibiting nutrient uptake/synthesis by cancer cells. In one such study, zinc uptake by cancer cells was inhibited by blocking the expression of a zinc transporter called ZIP4, coded by SLC39A4 gene. This gene coding for zinc transporter was silenced using a shRNA with the aid of a retroviral vector in pancreatic cell lines APSC-1 and BxPC-3 in vitro and in pancreatic cancer mouse models in vivo. This led to a reduction in cellular proliferation in vitro and reduced tumor growth and metastasis in vivo.[53] Similarly, the use of anti-arginosuccinate synthase shRNA led to cell death in MCF-7 and HELa cell lines due to the blockade of arginine synthesis by permanent down regulation of the gene coding for the enzyme arginosuccinate synthetase. Maximum effect was produced when, the gene down-regulation was coupled with depletion of intracellular stores of arginine by simultaneous administration of recombinant arginine deaminase.[54]

Similarly, the tumor growth can also be inhibited by silencing genes, which code for the proteins that support cancer cell proliferation. In one such study, the gene coding for nucleophosmin (an abundant and ubiquitously expressed phosphoprotein with an important role in cellular replication) was silenced by administration of an anti-nucleophosmin shRNA into K562 leukemic cell line using a plasmid vector p-Genesil-1. This led to cell cycle arrest in G1 phase and also increased the apoptosis in K562 cells.[55] In yet another study, gastrin; which so far is known only as a gastric acid secretion promoting peptide hormone, was found to promote the growth of pancreatic cancer. Thus, the silencing of the gene coding for gastrin using three different shRNAs and a siRNA against gastrin mRNA led to reduced cellular proliferation in BxPC-3 pancreatic cells in vitro and reduced tumor growth in subcutaneous and orthotropic athymic nude mice models in vivo.[56]

As it is also a proven fact that cancer cells, due to their rapid proliferating nature; are prone to severe damage upon problems in DNA repair mechanisms. Apoptosis can be induced in cancer cells by subjecting them to genotoxic stress by disrupting their DNA repair mechanisms. In a recent study based on this principle, the DNA repair mechanism of various prostate cancer cell lines was disrupted by silencing the gene coding for the enzyme Uracil DNA glycosylase. The gene was silenced using a combination of four different siRNAs. This gene silencing in turn led to an increased expression of various tumor suppressors which, reduced cell proliferation and increased apoptosis.[57]

Tumor growth can also be prevented by apoptosis induction in cancer cells by disrupting/preventing the energy generation in cancer cells. This ultimately leads to apoptosis by creating a state of cellular starvation. Adenine nucleotide translocase-2 (ANT-2) is one such enzyme which is involved in Adenosine triphosphate (ATP) generation in cells and unlike all other isoforms of ANT found in humans, ANT-2 works in an opposite fashion and is up-regulated in cancer. In a study based on this observation, the ANT-2 gene expression was silenced in MCF-7 and MDA-MB-231 breast cancer cell lines and SK-OV-3 and SNU8 ovarian cancer cells by administration of a combination of three different siRNAs against ANT-2 using a DNA based expression vector. The silencing of ANT-2 by RNAi led to reduced cell proliferation and induced apoptosis in vitro and inhibited tumor growth in subcutaneous breast cancer models of Balb/c nude mice in vivo.[58]

Due to the recent developments in RNAi technology like the development of multiple cassette expression vectors, which facilitate the co-expression of different shRNAs.[59] It is now possible to use RNAi to silence two or more genes simultaneously. This paved a way for the development of a new class of RNAi based therapy called as the combinatorial RNAi (coRNAi). As the name indicates coRNAi includes the use of a combination of shRNAs to post-transcriptionally silence a set of genes, which code for various factors promoting tumor growth.[60] coRNAi is much more efficient when compared to conventional RNAi monotherapies as, it can more efficiently prevent tumor growth due to its ability to simultaneously silence a set of genes instead of silencing a single gene.[60] For example, in a recent study on laryngeal squamous carcinoma, it was shown that coRNAi approach employing three shRNAs against vascular endothelial growth factor, TERT, and Bcl-x1 had more prominent inhibitory effect on the laryngeal squamous carcinoma cells in vitro and in vivo than an RNAi mono-therapy.[61],[62]

In another such study, the potential of coRNAi to treat chronic myeloid leukemia (CML) was analyzed by targeting the constitutively active Bcr-Abl tyrosine kinase and the downstream signaling molecules Shp2, Stat5, and Gab2.[63],[64] Bcr-Abl modulates intracellular signaling cascades, thereby enhancing survival and proliferation of leukemic cells.[65] As the treatment of CML with a drug that inhibits the Bcr-Abl oncoprotein leads to the selection of drug-resistant cancer cells, especially in advanced disease.[66] In the said study, the downstream signaling molecules were targeted as an alternative therapeutic option. Increased inhibitory efficacy was obtained with lentiviral vectors expressing 2 versus a single shRNA, without loss of specificity. Interestingly, simultaneous knockdown of Shp2 and Stat5 triggered the specific depletion of Bcr-Abl-expressing cells, indicating that it is not necessary to knock down the Bcr-Abl oncoprotein itself.[67]

In a more recent study, the survival period of intracerebral glioblastoma mouse models was increased by simultaneous administration of siRNAs against epithelial growth factor receptor and Akt-2. The increase in survival period was more with the coRNAi therapy than the RNAi monotherapy.[68]

Along with the above discussed approaches RNAi can also be used to enhance antitumor immunity and make tumor cells prone to immune destruction. The antitumor immunity can be enhanced, either by making tumor cells more susceptible to the effector cells in the immune system or it can also be enhanced by potentiating the cells of the immune system against the tumor cells, by which they can efficiently recognize and destroy them. The former approach has been proved to be very efficient in a recent study where in, the tumor cells were made susceptible to immune destruction by inducing the expression of potent antigens on the surface of tumor cells. This was achieved by RNAi mediated inhibition of nonsense-mediated mRNA decay, which led to the expression of new antigenic determinants on tumor cells which in turn led to their immune mediated rejection.[69] Even though the concept of potentiating the cells of the immune system against the tumor cells by RNAi mediated suppression of negative feedback control has been extensively reviewed.[70],[71],[72] Most of these reviews discuss mainly the possibility of using RNAi to potentiate cells of the immune system and any study with a substantial result in this direction is yet to be reported. Some of the reported studies that have been carried out in this direction mainly deal with, the use of RNAi in potentiating the immune response that is generated upon the administration of a secondary immunogenic agent such as a cancer vaccine.[73],[74] However, these studies do not deal with the direct use of potentiated immune cells for enhancing the antitumor efficiency.

RNAi for treatment of cancers in stage IV

Stage IV is the final stage of cancer and mainly represents metastatic tumors. These tumors are the deadliest and are very difficult to treat and only a few of the currently available cancer therapeutics are efficient enough to cure cancer in stage IV. Hence, it is very important to first block the metastasis of cancer cells while treating the metastatic tumors. Due to the fact that the metastasis is a multistep cascade, RNAi can be used to block metastasis at many different steps such as the epithelial mesenchymal transition (EMT), cellular migration, and cellular invasion. These steps can be blocked using the RNAi to target different factors that are essential for these steps such as the transcription factors, signaling molecules, and chemoattractants.

Since, EMT is the first step in the multistep metastatic cascade,[75] cancer cell metastasis can be efficiently prevented by blocking EMT. As loss of cellular adhesion is very important for EMT, it can be blocked by promoting the transcription of proteins, which promote cellular adhesion.[75] In one such study, EMT was inhibited in HeLa cells by silencing of genes coding for E-cadherin transcriptional repressors ZEB1 and ZEB2 by RNAi, using the miRNAs belonging to miR-200 family.[76] Similarly, EMT can also be blocked by blocking the signaling pathways that promote it. In one such study, EMT was blocked in A549 cells by silencing a gene coding for Forkhead Box M1 (FOXM1) by RNAi, using miR-134.[77]

Even if, the cancer cells undergo EMT, RNAi can be used to block metastasis by preventing cancer cell migration by silencing the genes which code for proteases which lyse the extra cellular matrix and facilitate cellular migration. In a recent study, a gelatinase called matrix metalloproteinase-2 (MMP-2) was found to promote metastasis in malignant melanoma by facilitating cellular migration. When MMP-2 was silenced using adenovirus mediated transfer of anti MMP-2 siRNA, it successfully prevented metastasis in nude mice spinal metastatic malenoma model.[78] Encouraged by the initial results, the researchers tried out coRNAi approach to silence a combination of genes coding for proteases. In one such study, a cysteine protease cathepsin B and MMP-2 were silenced simultaneously by administration of siRNAs against these proteases using a bicistronic pcDNA3 vector construct. This inhibited tumor growth and reduced metastasis in human meningiomal IOMM-Lee cells induced intracranial tumor model in athymic mice.[79] Same bicistronic vector construct was also used to express shRNA for urokinase plasminogen activator and MMP-2, and when it was tested in the same animal model, it inhibited tumor growth and reduced metastasis.[80] In a similar study, the proteoglycan production by myoepithelial cells of salivary adenoid cystic carcinoma (SACC) was blocked by silencing the gene coding for the rate limiting enzyme in the production of proteoglycans, that is, xylosyltransferase-1 gene. It led to reduced metastasis in BALB/C nude mice models of SACC induced with SACC-M-WJ4, SACC-M-HK and SACC-M cell lines.[81]

Similarly, cancer cell migration can also be stopped by targeting actin regulatory proteins. Since the contractility of actin fiber is the main mechanism behind cellular mobility, the cancer cell migration can be stopped by targeting actin regulatory proteins. In one such study, the migration of two human breast cancer cell lines, that is, MCF-7 and MDA MB-231 was repressed in vitro by using miR-200c to target actin regulatory proteins FHOD1 and PPM1F.[82]

RNAi can also be used to efficiently prevent the invasion of a new tissue by cancer cells even if, the cancer cells have successfully migrated to that tissue. This can be achieved by RNAi based transcription suppression of those proteins, which are required for the binding of cancer cells to new tissues. In one such study, the cellular invasion of head and neck squamous cell carcinoma cells was inhibited in vitro by targeting laminin-332 by RNAi, using miR-218.[83]

Invasion of healthy tissues and organs by cancer cells can also be prevented by targeting the chemoattraction between cancer cells and target organs. In one such study, the invasion of healthy tissues by A-498; renal cell carcinoma cells (RCC) was prevented by silencing of CXCR4 gene by RNAi.[84] This gene codes for the CXCR4 G-protein coupled receptor on the surface of RCC cells. By silencing this gene, the chemoattraction between this receptor and stromal derived factor 1 was blocked which in turn prevented the invasion of healthy tissues by RCC cells.[84]

Cancer cell metastasis can also be blocked by blocking the signaling pathways that stimulate metastasis. In one such recent study, Src and its downstream signaling molecules STAT3 and cMyc were simultaneously silenced using siRNA against them. This led to reduced tumor formation and metastasis in human breast cancer model of NOD (Non-obese diabetic)/SCID (Severe combined immunodeficiency) mice induced with MDA-MB-435S breast cancer cells.[85] In another study, down regulation of heparinase synthesis through administration of siRNA led to inhibition of metastasis in SGC-7901 gastric cancer cell lines due to RNAi.[86]

RNAi in Improving the efficiency of anticancer drugs

Even though RNAi therapy alone is very potent in curing cancer, it can also be used in combination with a drug therapy wherein it can potentiate the drug action in various ways like sensitizing cancer cells toward the drug or by suppressing the ability of cancer cells to develop resistance towards the drug.

Some chemotherapeutic agents show dose dependent toxicities which limit their use wherein, the therapeutically effective dose is almost always associated with side effects. In case of such drugs, the side effects can be reduced by reducing the dose, but then it would lead to loss of effectiveness. Another way around, the dose of these drugs can be reduced by sensitizing the cancer cells toward them, thereby, reducing the load on the drug. This can be achieved by silencing the production of any of the cancer promoting factor which in turn, leads to reduction in the required dose of the drug. One such successful attempt was with doxorubicin where in, the siRNA silencing of telomerase reverse transcriptase in breast cancer cells combined with doxorubicin administration almost doubled the cancer cell mortality when compared to both therapies individually in MCF-7 and MDA-MB-453 breast cancer cell lines in vitro and in BALB/C nude mice models in vivo.[87] In a similar study, the inhibition of an anti-apoptotic factor, myeloid cell leukemia-1 by siRNA increased the apoptotic efficiency of epirubicin and 5-flourouracil in human hepatoma cell lines Hep3B, HepG2, and Huh7.[88]

RNAi can also be used to identify the genes coding for the factors, responsible for the loss of sensitivity towards chemotherapeutic agents. These identified genes can then be silenced and the cancer cells can be sensitized toward drugs. In a recent study, gene coding for check point kinase was found to be one such gene and its silencing using a siRNA, sensitized human pancreatic cell lines MIA paca-2 and BxPC-3 toward gemcitabine therapy.[89]

Even though the effectiveness of chemotherapeutic agents can be increased by silencing of genes coding for proteins inducing drug resistance, the effectiveness of a chemotherapeutic agent can also be enhanced by delivering the genes coding for the factors which promotes the uptake of said agent. Thus, the combination of both these strategies can yield promising results. In one such attempt, adenoviral systems expressing shRNA against MDR-1 and sodium iodide symporter were administered into human colorectal cancer cell line HCT-15. It led to an increased uptake of radioactive iodine-125 along with reduced resistance towards doxorubicin.[90]

RNAi can also be used to potentiate drugs for the treatment of those cancers in which the cancer cell proliferation is promoted by a peptide of external origin such as viral peptides. Some of these viral peptides not only promote cancer cell proliferation but also induce drug resistance. Epstein-Barr virus (EBV) is one such virus and it promotes cell proliferation in EBV positive gastric cancer cells. In a recent study, the proliferation of EBV positive gastric cancer cell line SNU-719 was effectively suppressed by siRNA silencing of viral latent membrane protein-2A. This also led to simultaneous enhancement of the anti-proliferative effect of 5-flourouracil on these cells.[91]

RNAi based preventive therapy for cancer

Preventive therapy is a treatment that is intended to prevent a medical condition from occurring and as far as cancer is concerned, preventive therapy is one of the areas of active research.[92] With respect to cancer, the term preventive therapy can be used to state its treatment even before it occurs. As this is not possible, the only effective way of preventing cancer is by avoiding the dysregulation of physiological RNAi pathways. Even though some drugs are capable of achieving this,[93] it is completely impractical and in some cases impossible to administer drugs on a regular basis throughout the life time. Thus, the researchers worldwide arrived at a conclusion that for any substance to be proved as a good oncoprotectant, it must be easily available, edible and must not require any special handling or storage and must be easily procurable. Of all the substances/molecules known to prevent dysregulation of physiological RNAi pathways certain biomolecules of plant and animal origin were found to be one of the best oncoprotectants [94] and their administration on a regular basis is also easy as most of these are found in regular fruits and vegetables. Some of the nutrients which have been proved to possess oncoprotectant activity include:

  • Retinoic acid an active metabolite of vitamin A has been proved to alter the miRNA levels in various cells lines, including acute promyelocytic leukemia [95] and neuroblastoma [96] cell lines. In experiments conducted using retinoic acid on cells procured from acute promyelocytic leukemia patients, eight miRNA were reported to be upregulated (miR-15a, miR-15b, miR-16-1, let-7a-3, let-7c, let-7d, miR-223, miR-342, and miR-107) and one downregulated (miR-181b) in human promyelocytic cell line.[95] Thus, administration of vitamin A in adequate quantities can protect against cancer besides other actions
  • Vitamin E is another essential nutrient whose modulatory effects on miRNA expression have been studied. Reduction in levels of miR-122a and miR-125b (miRNAs proposed to be involved in hepatocellular carcinoma [HCC])[97] was observed in rats fed on vitamin-E deficient diet. Thus, inclusion of adequate amount of vitamin-E in diet might exert regulatory action through modulation of miRNA expression and ultimately influence the processes required for cancer prevention Curcuma longa (or turmeric), a spice of utmost importance in Indian kitchen alters cancer signaling pathways through its regulation of miRNA expression. Curcumin (diferuloylmethane) was found to up-regulate 11 and down-regulate 18 miRNAs as reported from studies conducted on human pancreatic cancer cells. Curcumin also inhibits the transcriptional regulation of miR-21 thus evading tumor growth, in vivo metastasis and invasion.[98] These actions in turn contribute towards the anti-cancer effects of curcumin
  • Genistein, an isoflavone from soybeans has been proved to be a potent cancer prevention agent. It inhibits miR-27a expression and promotes the expression of the miR-27a in human uveal melanoma cells besides its other documented actions. Thus, consumption of this nutrient might serve as means to prevent cancer [99]
  • Resveratrol found in grapes, red wine, peanuts, and some berries also shows anticancer activity by modulating the miRNAs regulating the gene coding for transforming growth factor-β, which plays a very important role in tumor growth [100],[101],[102]
  • Oleic acid has been reported to up-regulate miRNA-21 which alters phosphatase and tensin homolog (a phosphoinositide phosphatase) levels in human HCC cells, suggesting a procarcinogenic activity of oleic acid [103]
  • Short chain fatty acids formed by microbial anaerobic fermentation of dietary fiber in the large intestine,[104] is a class of dietary factors which might prevent colorectal cancer as it has been reported to down-regulate miR-17, miR-20a, miR-20b, miR-93, miR-106a, and miR-106b and modulate p21 gene expression in human colon cancer cell line HCT-116.[105]


Thus by the regular and adequate consumption of nutrients with the ability to modulate physiological RNAi pathways, the cancer can be effectively prevented.

Limitations preventing the clinical application of RNAi for treatment of cancer

Even though, the RNAi based therapy has got enormous potential to cure cancer; the clinical RNAi therapy is still far away from being a reality. The main reason for this are the many limiting factors associated with the administration of si/shRNAs with the primary one being their susceptibility to tissue and plasma nucleases, which cleave them and make them inactive.[106] Along with this the pharmacokinetic and bio-distribution profiling of systemically administered siRNAs shows their half-life to be only around few minutes during the distribution phase,[106],[107] and their distribution pattern was also found to be very random with accumulation in most tissues, particularly the kidney and liver.[108],[109] This acts both as a barrier and an opportunity for RNAi based therapeutics. On the opportunity side the ability of kidney and liver to accumulate siRNAs can be used for RNAi based therapy of cancers arising in these tissues. However, in most cases this random bio-distribution only leads to unwanted effects, due to silencing of off-targets,[110] which has been a major limitation to the clinical use of RNAi therapeutics. It is very important to find a solution to this off-targeting because, recent research has shown that as little as 11 nucleotide match between the target and the siRNA can result in off-target knockdown.[111] More recent research has shown that the gene silencing by siRNA entirely depends on the binding of siRNA to a 6-7 base long region on mRNA called as the seed region.[112],[113] This further reduces the need of complementarity which a siRNA needs to silence a gene, which in turn increases the risk of off-targeting.

Due to the fact that the human body is designed to destroy any externally administered RNA molecules; a strategy evolved to combat viral infections.[114],[115] The siRNAs are highly prone to immune destruction even when they are administered for therapeutic purposes. In fact, it has been identified by recent research that the immune system specifically recognizes siRNAs by identifying specific sequence motifs [116] in them and triggers an immune response against them. This ability of the immune system to specifically recognize and destroy the siRNAs is also limiting their clinical application.

Even if, the siRNAs cross all these barriers and reach the target cell, the magnitude and the duration of therapeutic effect is substantially effected by cellular uptake and subcellular distribution. It has been proven by recent research that the siRNAs enter the cell by endocytosis.[117] Which means that upon cellular uptake, the siRNA gets encapsulated inside the endosome and the siRNA needs to escape from the endosome before its fusion with lysosomes, in order to bind to the target mRNA. Hence, the endosomal trapping represents an important barrier for siRNA delivery.[106] Hence, to facilitate the clinical application of RNAi for the treatment of cancer, it is first necessary to find ways to overcome these limitations of RNAi based therapeutics.

Recent developments facilitating the clinical application of RNAi for cancer treatment

Considering the need to overcome various above discussed limitations of RNAi therapy, the recent research in this field has been focused on finding ways to overcome these limitations. As a result of these efforts, solutions have been found to many of these limitations, which are now facilitating the clinical application of RNAi therapy for the treatment of cancer. For example, the problem of degradation of siRNAs by the nucleases is rapidly being overcome by the chemical modification of siRNAs either by the use of chemically modified oligonucleotide building blocks for siRNA synthesis [118],[119] or by modification of synthesized siRNA by the addition of various functional residues.[120],[121],[122],[123],[124]

Similarly, the distribution half-life of siRNAs has also been increased by the conjugation of siRNAs to tocopherol,[125] cholesterol,[126] and other lipids. This conjugation of siRNAs to lipids has been shown to increase their distribution half-life by enhancing the binding of siRNAs to serum lipoproteins and/or albumin which resulted in their increased circulating lifetimes.[106],[125],[126] Along with this, great success has also been achieved in safe, efficient, and targeted delivery of interfering RNAs to cancer cells using non-viral vectors such as the liposomes and nanoparticles [20],[21],[127],[128] which improved their bio-distribution pattern by facilitating the localization of siRNAs in the target tissue. Similarly, the recent development of adenoviral vector that replicates only in p53 expression deficient cancer cells [129] has made cancer cell specific expression of shRNA a reality. This combined with the discovery about various modifications in the siRNA bases which could highly reduce the binding of siRNAs to off targets,[130] without compromising the degree of target silencing has led to a great reduction in the off-target knockdown by the administered siRNAs.

Recently, the problem of endosomal trapping of siRNAs has also been overcome by conjugation of siRNAs to endosomal release signal peptide [131] or nuclear localization signal peptide linked to a nanocarrier.[132]

All these developments have definitely paved a way for the clinical RNAi therapy for cancer treatment in the very near future. This statement is further supported by the ever increasing number of approved clinical trials [Table 5][133] that are being conducted to evaluate the efficiency of RNAi based therapeutics.
Table 5: RNAi therapeutics currently in clinical trials

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 » Conclusion Top


Even though, experimental evidences have shown that RNAi based cancer therapy can prove to be highly effective in treatment of cancer, there is still a long way to go before these therapeutics can successfully make a transition from laboratory promises to clinical realities. This is mainly due to the fact that this therapy is still in its exploration phase and will require further improvements before it can be used clinically. For example, to effectively use RNAi for cancer diagnosis there is a need to develop a microarray chip which can easily identify and quantify the cancer related miRNAs present in a sample of blood. Similarly, even after the development of many viral and non-viral vectors for the targeted delivery of RNAi therapeutics, the cell specific delivery of these therapeutics is still an issue that needs to be resolved, so as to facilitate its clinical application in the treatment of cancer. Compared to the other alternative forms of cancer treatment, the RNAi based therapeutics have developed at a much faster rate which is well illustrated by the fact that there are many RNAi based therapeutics in clinical trials just within 1½ decade of its discovery. Such an increasing growth pace of this therapy indicates that the day, when the full potential of RNAi based therapy as a non-invasive technology for complete cure of cancer would be realized, is not far away.

 
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    Figures

  [Figure 1], [Figure 2]
 
 
    Tables

  [Table 1], [Table 2], [Table 3], [Table 4], [Table 5]



 

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