Potential Utilization of Bystander / Abscopal-Mediated ...

Current Signal Transduction Therapy, 2007, 2, 000-000

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Potential Utilization of Bystander / Abscopal-Mediated Signal Transduction Events in the Treatment of Solid Tumors Matthew E. Peters1,2, Mohammed M. Shareef1, Seema Gupta1, Marianna Zagurovskaya-Sultanov1, Munira Kadhim3, Mohammed Mohiuddin4 and Mansoor M. Ahmed1,* 1

Weis Center for Research, Geisinger Clinic, Danville, PA 17822, USA; 2Eberly College of Science, Pennsylvania State University, University Park, PA 16802, USA; 3Genomic Instability Group, Medical Research Council, Harwell, Oxford, UK; 4Geisinger-Fox Chase Cancer Center at The Henry Cancer Center, Geisinger Clinic, Wilkes-Barre, PA 18711, USA Abstract: A transformed cell among a group of normal cells exerts a dynamic influence for clonal growth and mass transformation. Likewise, a treatment-induced damaged cell might exert deleterious signal to either neighboring or distal cells. These signals that elicit either transformation or cell death are classified under two independent phenomena. These two phenomena are called (1) Abscopal effect and (2) Bystander effect. There are several agents that have been reported to induce abscopal and bystander effects. Ionizing radiation and ultraviolet radiation are prime inducers of abscopal and bystander effects. In addition, localized therapies for tumor control such as gene therapy approaches, prodrug conversion based chemotherapy approaches, and surgical procedures are significant inducers of either abscopal or bystander effects. The proposed mechanisms that have been reported in literature clearly indicate pivotal roles of cytokine and ceramide signaling leading to the activation of pro-survival proteins and/or pro-apoptotic proteins. Together these pathways provide distinct differences between abscopal and bystander effects that are of particular interest in modern cancer therapeutics. The most exciting future direction of bystander/ abscopal biology in terms of cancer therapeutics will potentially arise from the use of stem cells. In this review, a critical evaluation of potential benefits of abscopal / bystander effects mediated signaling pathways in relation to cancer therapeutics are discussed in detail.

Key Words: Abscopal, bystander, radiation, cancer therapeutics, carcinogenesis, signal transduction. I. INTRODUCTION Extensive research in the biology of cancer has led to the development of different treatment strategies in the management of solid tumors. Some of these treatment strategies have proven effective, while others have shown poor outcomes. Moreover, certain treatment strategies are proven better in the treatment of particular malignancies, while having ineffectiveness in other malignancies. Whether or not a tumor will respond to a particular treatment strategy relies on a number of factors. Such factors include intrinsic genetic alterations of the tumor cells, tumor hypoxia, and extent of vascularization. Other major factors contributing toward resistance are often induced by the therapy itself. In addition to the aforementioned challenges, other events, such as shedding of cells following surgery, or transformations initiated in normal tissue following localized radiotherapy, can potentially cause more challenges in the management of solid tumors. It is important to note that the initiation of carcinogenesis process and challenges posed in cancer therapeutics depend on two major phenomena. These two major phenomena have been demonstrated using in vitro, as well as in vivo experimental approaches. One of these phenomena, called the bystander effect, can cause deleterious effect to cells that neighbor around the treated / damaged cells. The other phenomena, called the abscopal *Address correspondence to this author at the Weis Center for Research, Geisinger Clinic, Lab 121A, 100 N. Academy Avenue, Danville, PA 178222616, USA; Tel: (570) 214-3972; Fax: (570) 214-9861; E-mail: [email protected] 1574-3624/07 $50.00+.00

effect, can cause deleterious effect to cells that are distally located from the treated / damaged cells. The deleterious effect on the normal cells often leads to transformation and genomic instability whereas the deleterious effect on tumor cells often leads to growth inhibition and apoptosis. It is important to understand the basis behind each of these phenomena, as well as the correct context in which each is applied to. a) Bystander Effect The medical definition of bystander, taken from Dorland’s Medical Dictionary, is “that which is only incidentally involved in a process.” The bystander effect arises in cells that are “in the right place at the right time.” To further append to the simplistic nature of the bystander effect, the factors released are thought to travel away from the damaged cells in a manner similar to a diffusion process [1]. For example, bystander effects arise in cells that are not directly exposed to radiation during radiotherapy [1]. These surrounding, or more appropriately termed bystander, cells experience biological effects as a consequence of signals being sent out from the irradiated cells nearby. It should be noted, however, that all deleterious effects caused by radiation should not be called bystander effects [2]. Bystander effects occur by two main mechanisms Fig. (1). The first mechanism is through direct intercellular communication using gap junctions. This type of communication is commonly called Gap Junctional Intercellular Communication, GJIC [3]. The second mechanism depends on secretory factors. Some examples of these factors are interleukin©2007 Bentham Science Publishers Ltd.

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Fig. (1). Bystander/Abscopal effect. (Left) Illustration of therapy-induced abscopal effect that is mediated by circulating humoral factors that may potentially have deleterious effect on metastatic sites. (Right) Illustration of therapy-induced bystander effect that is mediated through secreted factors and gap junctional intercellular communication (GJIC).

6, TNF-, TGF-, and TRAIL. There is evidence to support the role of both GJIC and secretory factors in eliciting a bystander signaling response, and it appears that the resulting bystander effects might differ depending on cell type [4]. b) Abscopal Effect The abscopal effect has many similarities to the bystander effect, but key differences also exist. In 1953, R.H. Mole coined the term abscopal effect after observing systemic changes in an animal at sites separate from the area of localized radiotherapy [5]. He derived the word abscopal from the Latin words ab meaning “position away from” and scopos meaning “mark or target for shooting at”. Abscopal effects arise in cells that are distant from the site of applied therapy Fig. (1). Mole claimed that no one cell in the body works alone and that all cells are interdependent on one another. Thus, if one cell in the body is damaged, for example due to radiation exposure, the functional network in the whole organism will be affected [6]. This offers a key difference between the bystander effect (in which targeting of distal cells is not important) and the abscopal effect. Mole’s abscopal effect has since been reported in a large array of malignancies. These include, but are not limited to: chronic lymphocytic leukemia, lymphoma, melanoma, and adenocarcinoma of the esophagus [7-10]. The concept of the abscopal effect was initially challenged because it was rarely seen after radiation therapy. This can be explained by the fact that the abscopal effect is not always recognized and rarely reported [6]. Since then, many studies both in vivo and in vitro have demonstrated the existence of abscopal effects. This review focuses on the potential utilization of bystander/abscopal effects in different therapeutic approaches to eliminate malignant cells while protecting the normal tissue. In order to understand the difference between bystander and abscopal effects, these phenomena need to be analyzed together, not in isolation. To say that only bystander, or

only abscopal, effects are initiated after a particular type of applied radiation, or after a specific form of chemotherapy, would not be meaningful. Although this review does not provide an exhaustive information of pathways involved in these two effects, a general frame of mechanisms involved in these two phenomena are discussed in detail. II. INDUCERS OF BYSTANDER/ABSCOPAL EFFECT There are many potential inducers of the bystander effect and the abscopal effect. While evidence for the bystander effect has been established through in vivo, as well as in vitro studies, the studies demonstrating abscopal effect are mostly from in vivo experiments. The following treatments are established modes of management in solid tumor therapy and are found to potentially elicit both the phenomena of bystander and/or abscopal effects: radiation, surgery, chemotherapy, gene therapy, chemo-gene therapy, radio-gene therapy, and photodynamic therapy Figs. (2) and (3). a) Radiation Ionizing radiation (IR) is a known inducer of both bystander and abscopal effects. Moreover, bystander and abscopal effects have critical implications concerning the central dogma of radiation biology. The accepted, classical dogma of radiation biology states that a local radiation treatment cannot, and does not, have a systemic effect [11]. It asserts that the genetic changes caused by an addition of energy to a DNA molecule come directly from, and are caused directly by, the radiation exposure (and from any shortterm oxy-radicals formed) [4]. Further any damage caused by the irradiation will be repaired by natural cellular mechanisms within one to two cell generations [12]. Interestingly, the biology of bystander and abscopal effects challenges the foundation of this concept. Radiation-induced bystander effects were first observed in Chinese hamster ovary cells exposed to -particle irradiation [13]. This observation, made by Nagasawa and Little,

Potential Utilization of Bystander / Abscopal-Mediated Signal

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Fig. (2). Bystander effects and Responses. Inducers of bystander effects leading to various responses that cause either pro-death or prosurvival outcomes.

found an enhanced frequency of sister chromatid exchanges (SCE) in 20-40% of the cells when only 0.1-1% of the nuclei were irradiated by an -particle [14]. Since then, bystander effects induced by radiation have been observed in two forms based on whether the cells are exposed to high LET or low LET radiation [15]. Following high LET radiation (such as -particle irradiation), GJIC seems to be induced [16, 17],

whereas following low LET radiation (such as irradiation) GJIC does not seem to be necessary and thus secretory factors may play an important role [18]. Apart from genomic instability and clastogenicity, cell proliferation and cell death have been reported in unirradiated cells following radiation exposure [19].

Fig. (3). Abscopal effects and Responses. Inducers of abscopal effects leading to various responses that cause either pro-death or prosurvival outcomes.


Abscopal effects have been observed following IR exposure. Inflammatory signals produced by the irradiated tumor can circulate to distant sites and cause cell death of metastatic tumor tissue [20]. T-cells activated against tumor cells have enhanced specificity [21] and thus can target distant tumor cell deposits without affecting the surrounding normal tissue. Interestingly, the abscopal effect following radiation exposure is shown to be induced only following high dose of IR exposure. For example, using a mouse model it was found that an IR dose as high as 60 Gy was needed before a systemic anti-tumor response was initiated [22]. This is also true that clinically and high-dose GRID radiation therapy is effective in eliminating metastatic tumors particularly in patients with advanced tumors [23]. b) Surgery Surgical removal of tumorous tissue is one of the main therapies in the treatment of solid tumors [24]. Factors induced during surgical resection of tumor tissues can lead to either local recurrence of the tumor or metastatic tumor growth. Surgical removal of tumor tissue often leads to disruptions in the balance of cell apoptosis versus cell proliferation [25], ultimately inducing increased growth factor levels that promote the recurrence of the tumor [26]. Thus, although the mass of tumor tissue is removed, circulating tumor cells, coupled with humoral inflammatory responses, allow the survival of tumor cells leading to formation of metastatic attachments [24]. Growth of new tumor tissue at metastatic sites is a good example of abscopal effect, whereas any transformation of normal tissue around the site of tumor removal might form an example of bystander effect. Currently, the mechanisms involved in the bystander effect and abscopal effect initiated after surgery are scanty because of the lack of an appropriate in-vivo model. c) Chemotherapy Chemotherapy is often a systematically applied therapy and thus does not elicit bystander and abscopal effects. Localized chemotherapy, however, does have the potential to elicit effects outside of the treated area. One such form of localized chemotherapy, which has been used after surgical removal of brain tumor tissue, involves the use of biodegradable carmustine implants, or Gliadel™ wafers [27-29]. The wafers are said to release carmustine over a period of ~5 days and to completely dissolve 8 weeks post-implantation [29]. Furthermore, the spread of the drug is normally very small because it is absorbed into the tissue very quickly upon release. However, studies using primates have shown that after initial implantation, due to increased postoperative flows, the spread of the drug may be larger and to more distant parts of the brain [29]. This would suggest that the carmustine has the ability to cross the blood-brain barrier. Thus, an abscopal effect could arise in distal areas maybe even outside of the brain. This will be an excellent model to study the bystander or abscopal events associated with implants, however, till date no study has been documented. d) Gene Therapy Gene therapy is the transfer of nucleic acids into tumor, or normal, cells to eliminate or reduce tumor burden by direct cell-killing, immunomodulation, and/or correcting gene-

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tic errors to reverse malignant state. The advances that have been made in the past decade in the fields of gene transfer and molecular and immunobiology of tumorigenesis have brought to use direct gene transfer as anti-cancer treatment modality. Gene therapy for the treatment of cancer has a wide variety of potential uses and strategies. Strategies of gene therapy for cancer are given in examples as following: enhancing the immunogenicity of the tumor, for example by introducing genes that encode foreign antigens; enhancing immune cells to increase anti-tumor activity, for example by introducing genes that encode cytokines; inserting a “sensitivity” or ‘suicide’ gene into the tumor, for example by introducing gene that encodes herpes simplex virus thymidine kinase (HSV-tk); blocking the expression of oncogenes, for example by introducing the gene that encodes anti-sense to BCL-2 message; inserting a wild-type tumor suppressor gene, for example p53 or the cell cycle regulatory gene, retinoblastoma (Rb); protecting stem cells from the toxic effects of chemotherapy, for example by introducing the gene that confers multi-drug resistance (MDR-1); blocking the mechanisms by which tumors evade immunological destruction, for example by introducing the gene that encodes anti-sense IGF-1 message; killing tumor cells by inserting toxin genes under the control of a tumor-specific promoter, for example through the use of probasin or prostate specific antigen gene promoters that are activated in prostate tissue only; and killing tumor cells by inserting cytokines under the control of radiation or hypoxia inducible promoter, for example by using the Egr-1 gene promoter that is activated by ionizing radiation. Clinical trials using this approach in cancer are limited, however, considerable amount of work needs to be done to make gene therapy as an effective treatment modality. Although many genes involved in tumor formation have been identified, the ability to target these genes with 100% effectiveness has been elusive [30]. This treatment approach potentially gives rise to more of a bystander phenomenon than an abscopal effect. The presence of a bystander effect allows for only a subpopulation of tumor cells to be affected directly by the gene therapy with the rest of the cells experiencing significant indirect bystander effect. In a clinical study involving the use of p53 gene therapy for the treatment of non-small-cell lung cancer, an adenovirus vector containing the wild-type p53 gene was shown to be taken up by only a limited number of tumor cells, with a pro-death bystander effect being initiated in non-infected cells [31]. Similar type of bystander effect has been observed in gene therapies that involve pro-apoptotic molecules such as Fas Ligand, TRAIL, and Bax [30]. Although an abscopal effect has been observed following suicide gene therapy (see Chemo-gene therapy below), reports of observed abscopal effect following gene therapy alone is lacking. e) Chemo-gene therapy The idea of combining chemotherapies with gene therapies has lead to the idea of suicide gene therapy. The use of suicide gene therapy, or gene-directed enzyme prodrug therapy (GDEPT) has proved useful in the treatment of solid tumors [32]. GDEPT is a two-step process in which a particular gene coding for a foreign enzyme is delivered into tu-


mor cells using a vector followed by subsequent addition of a prodrug [32]. These prodrugs, also called tumor-activated prodrugs (TAPs), are not nearly as toxic to normal tissues as earlier drugs were because they are activated, selectively, by tumorous tissue [33]. Prodrugs can be activated in the normal physiological micro-environment of tumor cells (based on enzyme expression, hypoxia, etc.) that is distinct from the micro-environment of normal cells. This therapy may also involve delivery of prodrugs and enzymes targeted specifically to tumor cells, such as the GDEPT mentioned above, or by using antibody-directed enzyme prodrug therapy (ADEPT) [33]. Bystander effects elicited under this treatment process have demonstrated tremendous success for prodrug therapy [33] and without this phenomena the entire approach would be futile. The bystander effects initiated under this treatment strategy are very similar to the ones discussed above for simple gene therapy, except in this scenario, the prodrug concentration within the tumor tissue are vital to initiate the bystander effect [33]. This kind of bystander effect has been reported following suicide gene treatment in human cervical carcinoma [34], pancreatic cancer [35], and ovarian carcinoma [36] . Although much of the reports discuss the presence of bystander effects initiated by this type of system, abscopal effects have also been observed. Using one particular type of suicide gene therapy, the HSV-tk/Gancyclovir (GCV) system (discussed below), Kianmanesh et al. generated multiple tumors in the lobe regions of the livers of rats so that the separate tumors had no cell-to-cell contact [37]. Only one of the formed tumors was treated with the HSV-tk gene, but treatment with the GCV prodrug led to regression of not only the HSV-tk positive tumor, but also the HSV-tk negative tumors indicating the presence of a dynamic abscopal effect [37]. g) Radio-Gene Therapy Therapies combining radiotherapy and gene therapy (or suicide gene therapy) have been shown to elicit both bystander and abscopal effects. For example, in one study, the radiation-inducible promoter region of EGR-1 was linked to the gene encoding tumor necrosis factor- (TNF-) [38]. Following adenovirus delivery of this EGR-TNF construct into human tumor cells growing in nude mice, radiation therapy was administered. Inhibition of tumor tissue growth, without elevated harm to normal tissue, was increased during this treatment compared with either treatment administered alone [38]. Although this study did not specifically examine the occurrence of increased cell death in bystander cells, other studies have shown that combining radiotherapy and gene therapy leads to an increase in the bystander effect initiated when compared to gene therapy alone [39]. One such study, involving adenoviral TNF- gene therapy followed by radiation exposure, found that combining the therapies lead to a more than additive anti-tumor effect, possibly through some sort of bystander effect [40]. Obvious increase in abscopal effect has also been observed following combination of suicide gene therapy and radiotherapy. In a study involving a lung metastases model, 50% fewer metastatic lesions were observed when gene the-


rapy was followed by radiation therapy as compared to gene therapy alone [39]. These results begin to show the therapeutic possibilities that may arise through the exploitation and manipulation of bystander and abscopal effects via combined radiotherapy and gene therapy approaches. h) Photo-Dynamic Therapy Another possible inducer of a pro-apoptotic bystander effect is photo-dynamic therapy (PDT) [41-43]. PDT involves the use of a photosensitizer that produces singlet oxygen (1O2) when excited with visible light [42]. This treatment ultimately leads to destruction of cells through necrosis and/or apoptosis [43]. When exposed to PDT, dead cells form in clusters rather than in random locations. It is thought that signals arising in damaged cells can be communicated to undamaged cells and induce apoptosis [41]. Further, the bystander effect is stronger when the photosensitizer induces damage of the plasma membrane. One possible explanation for this is that any damaged-induced factors could “leak” out of the plasma membrane into the intercellular environment. Dahle et al. have ruled out the possible role of GJIC in the bystander effect initiated following PDT [43]. The GJIC inhibitor dieldrin was used to show that inhibiting GJIC did not stop the initiated bystander effect. The signaling pathways that give rise to this particular bystander effect are not known. Mediators such as nitric oxide, cytotoxic aldehydes, and lipid peroxidation are potentially implicated in this bystander mechanism [42]. Abscopal effects were not reported. Other Inducers While radiation therapy and suicide gene therapy are the two major areas in which bystander and abscopal effects have been studied extensively, other novel therapeutic strategies have been shown to activate these two responses. One such therapy, that elicits a bystander effect, is methionine deprivation therapy [44]. Human tumor cells need methionine to stay alive, but normal tissue cells can use homocysteine instead and thus are not dependent on methionine [45]. This allows strategic therapeutic intervention that can be devised to target cells for complete elimination of methionine leading to methionine deprivation-induced stress pathways [44]. Although numerous pathways are activated by this methionine stress, one particular activated cytokine called melanoma differentiation-associated gene 7, MDA-7, (ST16 or IL24) is involved in eliciting bystander effect. MDA-7 is part of the IL-10 family and has anti-tumor properties, inducing apoptosis in tumor cells [46]. Since MDA-7 does not cause cell death of the normal cells, the bystander effect elicited through it could be potentially used to target tumor cells [47, 48]. In fact, the normal cells can be infected with MDA-7 gene therapy to cause a dynamic bystander effect in the neighboring tumor cells for complete eradication. III. SIGNALING TRANSDUCTION CASCADES ELICITING BYSTANDER/ABSCOPAL EFFECT The molecular signal transduction mechanisms leading to bystander/abscopal effect will be useful in understanding some of the key differences between these two phenomena. These pathways involve numerous gene signaling events,


growth factors, cytokines, and gap junctions (connexins), as well as oxidative metabolism events Fig. (4). a) Effect of Genetic Background on Abscopal/Bystander Effects Bystander and abscopal signaling responses have been shown to be dependent on the genetic background of both the releasing and target cells. Although evidence for these factors has been accumulating over past decades, their exact nature has proven elusive, as have the mechanisms by which they cause distant bystander effects. One such mechanism might be through radiation-induced early genes. The most commonly radiation-induced early gene is early growth response gene-1 (EGR-1). Induction of EGR-1 promotes the level of transcription factors, such as MYC and NUR77; and it can also induce growth factors or their receptors, such as

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transforming growth factor-1 (TGF-1), tumor necrosis factor-alpha (TNF-), or epidermal growth factor receptor (EGFR) [49]. EGR-1 controls the cell cycle regulators, such as the retinoblastoma susceptibility gene Rb, cyclin D1, and p53 [50]. TNF- and TNF-related apoptosis-inducing ligand (TRAIL) are directly involved in apoptosis and are induced by ionizing radiation. Under ionizing radiation exposure, pro-survival factors such as NF-B and Bcl-2 are both downregulated by EGR-1. In addition, BAX, as a target gene of EGR-1, is upregulated (unpublished observation). To understand the molecular mechanisms involved in the bystander effect in small cell lung cancer cells and to identify the putative soluble clastogenic factor, our lab used lung carcinoma A549 and H460 cells, which are functionally EGR-1 positive and negative respectively. These two types of cells responded differentially after exposure to high dose

Fig. (4). Signaling Pathways and bystander/abscopal effect. Illustration of the role of TNF- and TGF- signaling in regulating cell death of epithelial and endothelial compartments of the tumor via abscopal (right) and bystander (left) effects. In a bystander scenario, applied therapy can induce the transcription factor EGR-1 that in turn induces cytokines such as TNF- and TGF-. TNF- can cause induction of caspases and can lead to cell death of the targeted cells and the bystander cells. The secreted TGF- can bind to the receptor of the bystander cell and cause a series of SMAD signaling which leads to a growth inhibitor effect. In a similar applied therapy situation, the induced TNF- or TGF- can cause similar types of signaling effects in distal epithelial and endothelial cells leading to abscopal effect. In particular, endothelial cells of the tumor compartment might elicit a different signaling pathway via TNF-, such as induction of ASMase leading to enrichment of LDL-ceramide that in turn will cause induction of apoptosis in the endothelial compartment.


radiation by secreting different cytokines (unpublished observations). EGR-1 positive cells responded by secreting TNF- and EGR-1 negative cells by TRAIL. It is known that TNF- and TRAIL are positively and negatively regulated, respectively, by EGR-1 [51] and our results validate this fact. Further, the cells expressing EGR-1 showed enhanced apoptosis following radiation. Interestingly, the cells that received medium from the irradiated cells lacking EGR-1 function had a higher incidence of bystander-induced cell death. This means that the cytokine TRAIL was implicated in this particular bystander-mediated cell killing. As another example, p53 expression seems to play an important role in the induction of abscopal effect [5]. Camphausen et al. showed using a mouse model that p53 function is required for the induction of an anti-tumoral abscopal effect following irradiation of normal tissue. When p53 null mice, or mice treated with pifithrin-alpha (which blocks p53 function), were subjected to localized radiation on the normal skin region, no abscopal effect was observed. These are some potential examples of gene signal transduction pathways regulating the responses elicited either by abscopal or bystander effect. b) Interleukin-6 (IL-6) and Abscopal Effect An important abscopal effect-mediated pathway that is present naturally between primary tumors and metastatic sites is the secretion of interleukin (IL)-6, a member of the cytokines family [6, 52]. This abscopal effect is commonly associated with overexpression of the IL-6 receptor [6]. IL-6 leads to cell proliferation and can lead to growth of cancerous tissue [53]. Further, IL-6 allows the primary tumor to survive and grow and if secreted it can maintain the cells at metastatic sites [52]. Thus, IL-6 secreted from the primary tumor can bind to the overexpressed IL-6 receptors on the distant tumor and cause the initiation of common IL-6 downstream pathways. It has been shown that IL-6 activates the p38 MAPK pathway that leads to activation of certain cell survival pathways [54]. Activation of serum and glucocorticoid stimulated kinase (SGK) by phosphorylation may play an important role in the cell survival mechanism initiated by the IL-6 pathway. Inhibiting the expression of SGK in cholangiocarcinoma cells by using siRNA lead to increased cell death following application of chemotherapeutic drugs [54]. It has also been shown that IL-6 leads to the activation of Janus kinases (JAKs) through receptor complexes [55]. This can lead to the induction of signal transducer and activator of transcription 3 (STAT3) and extracellular signal-regulated kinase 1/2 (ERK1/2). Interestingly, ERK activation is different in cancerous versus normal cell tissues. In hepatocellular carcinoma cells and normal liver specimens, the ERK activity was shown to be stimulated in a biphasic manner in cancerous cells and in a monophasic way in normal liver cells [56]. c) Bystander/Abscopal Pathways Arising from Radiation Therapy There are many reports that demonstrate the bystander and abscopal effects arising in response to radiation therapy. It has been documented that bystander/abscopal effect-


mediated pathways following radiotherapy will lead to either cell proliferation or cell death. Genes involved in such processes are discussed below in detail Figs. (2) (3) and (4). i) The Role of TGF-1 in Abscopal/Bystander-Mediated Signaling Pathways Many reports have shown induction of proliferation of cells surrounding irradiated cells following radiation exposure [14, 57-60]. Transforming growth factor-1 (TGF-1) seems to play an important role in eliciting such promitogenic effects in cells within and outside of the field of irradiation [61]. This is an important observation because TGF- is a negative growth-regulator, but in certain instances it elicits a pro-survival effect. In its pro-survival role, the induction of TGF-1 in unirradiated cells can be inhibited by superoxide dismutase, a superoxide radical scavenger, suggesting that radical oxygen species (ROS) may play a role in the initiation of this particular bystander effect [59]. It has been documented that the NADPH-oxidase/NF-B pathway may be involved in the production of these ROS [61]. In recent studies involving the treatment of non-irradiated cells with supernatants from -particle irradiated cells, TGF-1 was upregulated and cell proliferation ensued [61]. On the other hand, in another report when unirradiated cells were treated with heavy-ion irradiated cell supernatants, increased production of nitric oxide coincided with increases in proliferation [61, 62]. Further, an experiment using UV irradiation of HaCaT cells showed that either pro-mitogenic or proapoptotic events could be initiated in unirradiated cells with the response being mediated by nerve growth factor (NGF) [57]. An interesting possible explanation for these promitogenic events involves the correlation between a G1 arrest and increases in proliferation of irradiated tissue [63]. This arrest is well documented, but reports pertaining to increased cell growth following a transient G1 arrest is lacking. Perhaps, a similar cell cycle arrest mechanism might occur when non-irradiated cells are exposed to irradiated media. Further, TGF- has the ability to activate the phosphatidylinsitol 3-kinase (PI 3-K)/Akt pathway that can lead to a proliferative response [64, 65]. Activation of PI 3-K may lead to the secretion of TNF-, which induces cell proliferation [66]. Furthermore, it was found that Smad signaling is not disrupted in cells that do not induce apoptosis when exposed to TGF- [64]. This is important because it suggests that pro-survival signals are being induced by the secreted TGF-, rather than the pro-survival response being merely a result of missing pro-apoptotic Smad pathways. Another example is B cell chronic lymphocytic leukemia (B-CLL), in which inhibition of apoptosis is associated with failure to downregulate Akt and NF-B activity [67]. Furthermore, when the PI 3-K pathway was active in B-CLL cells, the anti-apoptotic protein Bcl-xL could not be downregulated, and apoptosis did not result. Thus, it could very well be the ability of TGF- to initiate the PI 3-K/Akt pathway that leads to these pro-survival effects. ii) The Role of the Clusterin Gene in Prosurvival Bystander Signaling Klokov et al. have published a review article on clusterin gene expression and bystander effects [58]. The secreted


form of clusterin (sCLU) leads to cytoprotective functions in bystander cells following low dose IR (> 0.02 Gy) [68]. The nuclear form of this same gene (nCLU) is a pro-apoptotic protein that is activated after higher doses of IR (> 1 Gy) [69]. CLU is classified as a sulfated glycoprotein, and it is found in mammalian tissues [58]. Interestingly, TGF-1 has been shown to activate CLU expression through transcription, possibly through the use of c-FOS [70]. ROS, as well as interleukin-8, which are induced in cells exposed to radiation, have also been shown to activate the CLU gene expression. Thus, it appears that the CLU gene can be activated after oxidative stress [71]. This provides an intriguing link between ROS, TGF-1, and pro-mitogenic bystander effects. In one experiment using human lung fibroblasts, it was found that not only did IR cause cell proliferation in unirradiated cells, but it also led to decreases in p21 and p53 expression within these cells [61]. p53 is known to repress the activity of CLU, and thus its downregulation would lead to increases in CLU expression. Further, p53 acts as a regulator for the production of nCLU versus sCLU [58]. Klokov et al. suggested that p53 performs these regulatory functions by controlling the amount of activated sCLU present. The ratio of functional sCLU to nCLU may determine the activation of pro-survival, or pro-death, pathways. To understand the role of sCLU in bystander kinetics, it has been shown that not only can sCLU bind a large number of biomolecules, but it also binds to a number of cellular membrane receptors [58]. This means that damage signals, such as proteins secreted from an irradiated cell, could be picked up by a receptor, such as the gp330/megalin receptor (which is known to interact with sCLU) [72], and internalized through non-professional phagocytosis into a bystander cell [73]. iii) Nitric Oxide (NO) Pathways and Radioresistance in Unirradiated Cells Another factor that goes hand-in-hand in survival events of cells experiencing abscopal and bystander effects is radioresistance. Decreased radiosensitivity has been seen in unirradiated cells following relatively high dose, low-LET IR exposure [59, 74]. It has also been shown, in numerous studies, that radioresistance, as well as the ability to overcome the effects of a challenge dose of irradiation, can develop in unirradiated cells following release of factors from irradiated cells [60, 75]. Two explanations for these results are that the bystander cells experience a jump in their ability to repair sublethal DNA damage or that these cells fail to maintain normal cell cycle checkpoints [76]. Without these checkpoints, the cells, which may be damaged, fail to enter the cell death pathway as normal and thus are allowed to survive. It has been reported, most importantly in vivo [77], that agents induced through nitric oxide (NO) pathways can act as “whole-body radiation protector” [74]. NO can lead to the induction of heat-shock proteins (HSPs) and p53 proteins [78, 79]. Both of these proteins lead to organismal adaptive responses to stressors, including certain forms of irradiation [80]. More specifically, an HSP known as HSP72 results in immunity of cells against the cytotoxic effects of irradiation [81]. p53 on the other hand helps to keep the genomic integrity of cells in response to injury [82]. In a study by Ma-

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tsumoto et al. using human glioblastoma cell line A-172, it was found that NO secreted from cells irradiated with X-rays caused induction of HSP72 and p53 in unirradiated cells [74]. One theory, that coincides with what has already been mentioned in this review, is that NO may lead to the secretion of cytokines or growth factors [83] (such as TGF-1) from the irradiated cells, which can lead to the induction of HSP72 and p53 gene expression in non-irradiated cells. Another possible pathway that may be activated by NO, or its byproducts, causes induction of guanylic acid cyclase, which activates cGMP, and subsequently increases the levels of HSP72 and p53 protein [84]. A possible explanation for how this induction of HSP72 and p53 leads to radio-resistance in bystander cells may be attributed to the G1-phase cell cycle arrest that was mentioned earlier [74]. It has been shown that G1 arrest occurs in human carcinoma cell lines following production of NO [85]. During this arrest, accumulation of HSP72 and p53 can occur, and this accumulation is a result of the activation of DNA repair activities [74]. Perhaps, these DNA repair processes are not only capable of repairing current DNA damage, but can also prevent, or quickly repair, damage caused by subsequent radiation. This leads to what appears to be a sort of “inoculation” against radiation damage and thus possible cancer initiation. iv) Activation of the Immune System and Pro-Apoptotic Abscopal Effect Apart from the pro-survival events that can arise in tumor cells following radiation exposure, more therapeutically positive, pro-apoptotic, tumor killing pathways can be elicited by radiotherapy. The documentation demonstrating proapoptotic responses in unirradiated cells is enormous [4, 6, 14-16, 19, 20, 57, 59, 60, 86-90]. Most evidence for proapoptotic abscopal effects following radiation exposure involve activation of the immune system [6, 20]. Radiation has been shown to induce T-cell activation in response to tumor antigens [6]. These T-cells are then free to circulate in the immune system and attack tumor cells at distal sites. Dendrite cells may play a pivotal role in this abscopal effect [20]. Cancer cells have poor immunogenicity and thus do not trigger activation of surrounding T-cells on their own. Dendrite cells can receive tumor antigens from cancer cells during what is known as “cross-priming” [91, 92]. The dendrite cells are then able to present the tumor antigens and, along with major histocompatibility complex (MHC) class I molecules, lead to the activation of CD8+ T cells. Demaria et al. used Fms-like tyrosine kinase receptor 3 ligand (Flt3-L) in combination with local radiotherapy to show an abscopal effect in cell line 67NR (a mouse-derived mammary carcinoma cell line) [20]. Flt3-L is a growth factor that results in the production of dendrite cells [93]. A local radiotherapy treatment of 2 Gy was combined with Flt3-L treatment and resulted in anti-tumor effects locally and further controlled tumor growth at distal sites [20]. It is known that IR-induced apoptosis occurs through the interaction of Fas and its ligand [94]. There is evidence that the abscopal effects may originate with plasma membrane-


derived vesicles containing these Fas molecules which are exfoliated from cells that are in the direct field of the IR [89]. v) Unknown Factors Lead to a Pro-Apoptotic Bystander Effect


level of circulating vascular endothelial growth factor (VEGF) were observed following surgical removal of tumors and most of it was produced by platelets and leukocytes [100]. Although not specifically stated, any growth at metastatic sites could accurately be considered an abscopal effect. VEGF has been shown to cause tumor growth by disrupting pathways involved in prevention of tumor formation at metastatic sites [101].

Whereas most of the evidence for pro-apoptotic abscopal effects involves clear-cut activation of the immune system, a definite mechanism for pro-apoptotic bystander effects has yet to be discovered. Gap junctions have been shown to be important in the mechanism underlying death-inducing bystander effect following radiotherapy. More specifically, connexin43 seems to play a role in the GJIC initiated in unirradiated cells following radiation exposure [95]. Several factors also are known to play an important role in bystander effects arising from low LET radiation exposure [18] but are yet to be identified and thus the pathways involved have yet to be resolved. Although this is a very disappointing fact, current research may identify the factors that underlie the radiation-induced pro-apoptotic bystander effect which may include TGF-, TNF-, and TRAIL.

Increased levels of von Willebrand factor (vWf) are commonly seen following surgery. vWf along with its ligand, glycoprotein Ib, has been shown to increase tumorplatelet aggregation and thus formation of metastasis [102]. Together, the production of VEGF and vWf following surgical removal of tumor tissue can lead to an abscopal effect which elicits metastatic formation. These two factors, when combined, produce a situation in which formation of metastasis is favored and tumor destruction is inactivated.

For example, it has been shown that medium from irradiated cells can induce the following effects in unirradiated cells: 1) an efflux of intracellular calcium, 2) the disruption of the membrane potential of mitochondria, and 3) increases in reactive oxygen species [96]. All of these events are considered to be early events in apoptotic cascades, but more specific pathways arising from bystander effects following radiation exposure have yet to be discovered. Of importance is that some of the biological outcomes arising from these pro-apoptotic bystander effects are known. Of particular interest is the concept of genomic instability and its connection to bystander effect [97, 98] Fig. (2). There is also evidence of mitochodnrial changes which are indicative of apoptosisinitiation in cells treated with medium from irradiated cells [99, 100] Fig. (2). These concepts will be discussed later in relevance to other biological outcomes from bystander/ abscopal effect.

Although abscopal and bystander effects have been observed in gene therapy, radio-gene therapy, and photodynamic therapy, the specific pathways underlying the production of these effects has not been well documented. As far as gene therapy is concerned, the pathways should be similar to those that are involved with prodrugs in suicide gene therapy, which will be discussed shortly. Radio-gene therapy has had limited clinical applicability and thus findings from studies specifically focusing on the abscopal/bystander pathways are scanty. Although PDT may be a successful treatment modality, the pathways arising from its initiated bystander effect are unclear. As mentioned earlier, chemotherapy causes weak bystander/abscopal effect and only localized chemotherapy may possibly lead to initiation of intense effects and subsequent pathways in bystander / abscopal effects.

d) Bystander/Abscopal Pathways Arising from Surgery

f) Bystander/Abscopal Pathways Arising from ChemoGene Therapy

The growth of recurrent tumors following surgery has been shown in many different models including: human prostatic adenocarcinoma, human colorectal adenocarcinoma, and numerous mouse models [24]. Coffey et al. showed in multiple models that the PI 3-K/AKT pathway is involved in the progressive growth of recurrent tumors. This is the pathway discussed above leading to similar pro-survival events in bystander cells following radiotherapy. In this case, the subunits of the class Ia PI 3-K molecular complex are changed. The expression of the gene encoding the regulatory subunit, p85, is downregulated, whereas the expression of the gene encoding the catalytic subunits, p110, is upregulated [24]. These changes lead to increases in Akt phosphorylation within the cells that eventually lead to the growth of the recurrent tumors. Thus, it may be the changes in the expression of genes encoding the class Ia PI 3-K molecular complex that leads to increased intrinsic resistance and re-growth of tumor tissue following surgery. Pathways underlying the abscopal effect initiated after tumor removal have also been suggested. Increases in the

e) Unknown Pathways Lead to Bystander/Abscopal Effects Following Chemotherapy, Gene Therapy, RadioGene Therapy, and Photodynamic Therapy

The bystander response initiated by chemo-gene therapy, or more specifically suicide gene therapy, seems to be intense as shown by Hong et al [103]. Using Escherichia coli purine nucleoside phosphorylase (PNP) expression in human cancer cells and the clinically approved prodrug fludarabine phosphate (F-araAMP), potent regression was observed in xenografts when 95 to 97.5% of the cells within the tumor micro-environment were bystander cells. This demonstrates that only a small number of tumor cells harboring adenoviral infection is necessary to cause such a dynamic response. Another example is Ara-C that can insert into replicating DNA and induce DNA strand break, thus leading to the apoptosis [104]. Ara-C alone has limited effectiveness in the treatment of solid tumors, but it leads to enhanced apoptosis when combined with deoxycytidine kinase gene expression (dCK). When animals with tumors expressing the dCK gene were treated with Ara-C, 88% of them were cured [104]. It is thought that the dCK gene enhances the ability of the transfected cells to metabolize Ara-C into its cytotoxic form. The curative possibilities involving suicide gene therapy are si-


gnificant and depend on successful initiation of the bystander effect. The suicide gene therapy that is best studied, and that has shown to have both bystander and abscopal effects, involves HSV-tk gene therapy combined with the intra-peritoneal GCV prodrug, commonly called the HSV-tk/GCV therapy system [6, 35, 36, 95, 105-108]. Cells transfected with the HSV-tk gene are able to produce thymidine kinase that can phosphorylate the GCV prodrug and convert it into the mono-phosphate form (GCV-MP) [109]. Further, cellular kinases convert GCV-MP into di-phosphate (GCV-DP) and triphosphate forms (GCV-TP) [107]. This GCV-TP is toxic and blocks DNA replication by inhibiting DNA polymerases by inserting GCV-MP into the nascent strand leading to apoptosis [110]. Since rapid DNA synthesis is not present in normal cells, uptake of phosphorylated GCV is not observed within these cells [108]. In tumor cells, direct incorporation of GCV-TP can cause cell death via increased chromosomal aberrations and sister chromatid exchanges [111]. The presence of a bystander effect using this HSVtk/GCV system was first observed in vivo by Culver et al. in 1992 [112]. Tumors were grown in mice using a 50/50 mixture of HSV-tk transfected cells and wild-type cells. Complete tumor regression in 93% of the mice was observed after application of GCV despite the fact that only half of the cells contained the HSV-tk gene. Gap junctional intercellular communication, and the expression of certain connexins, is instrumental in the bystander effect initiated from the HSV-tk/GCV system [95]. Using ovarian cancer tumor cells, Zhang et al. showed that there is a relationship between the expression of connexin43 and the bystander effect induced by the HSV-tk/GCV system. All-trans retinoic acid (ALTRA) has been shown to improve the bystander effect of this system [113]. ALTRA enhances GJIC, but it does so in a manner that does not lead to the upregulation of connexin43 [114]. This would indicate that ALTRA enhances GJIC in the post-transcriptional phase. Other studies have documented that this bystander effect may be mediated by the entering of apoptotic vesicles released from dying HSV-tk positive cells into neighboring cells via phagocytosis, ultimately leading to their death [115]. Additionally, secreted cytotoxic substances, such as GCVTP, have been observed in the medium and these substances could be taken up by surrounding cells leading to apoptosis [116]. Whatever the pathways involved, these bystander effects are a pivotal backbone for the success of the suicide gene therapy. It was once thought, by many researchers, that cell-cell contact, and thus GJIC, was necessary for all unique “bystander” effects observed in the HSV-tk/GCV system [107]. The presence of abscopal effect following GCV treatment proves that in some cases, gap junctional intercellular communication is not necessary [37]. A recent in vivo study showed regression of heptacellular carcinoma transduced tumors following GCV treatment as well as regression of non-transduced tumors at distant sites [105]. Furthermore, the occurrence of an abscopal effect was 92% in an experiment by Agard et al. using multiple tumors implanted into

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rat liver lobes [117]. Only one of the tumors implanted into the liver was subjected to adenoviral HSV-tk gene transfection and subsequent GCV treatment regressed both the tumors. This study also found that CD8 + lymphocytes play an important role in this particular abscopal effect [117]. While the bystander effect makes suicide gene therapy successful as a concept, the abscopal effect helps to make it successful as a treatment. g) Bystander Pathways Arising from MDA-7 Expression As was mentioned earlier, the MDA-7 gene, and the protein it encodes, can elicit a strong bystander effect. There is evidence that MDA-7 protein expression during the development of melanomas is downregulated, thus allowing for the progress of the cancer [118]. Reintroducing MDA-7 using a replication-deficient adenoviral vector (Ad-MDA-7) has been shown to cause apoptosis of the tumor tissue. Instead of using an adenoviral vector, methionine-deprivation can be used to naturally induce upregulation of MDA-7 in tumor cells. Of interest, it has been reported that MDA-7 will cause apoptosis independently of p53 functional status [119]. MDA-7 is a pro-Th1 cytokine and leads to the release of TNF-, IL-6, IFN-, IL-12, and GM-CSF [120]. MDA-7 also has anti-angiogenic activity and it can reverse the effects of bFGF, IL-8, and VEGF [121]. It is thought that this antiangiogenic activity occurs through the receptors for interleukin-22 and interleukin 20, known as IL-22R1 and IL-20R1, respectively [122]. This pathway, particularly because it arises after methionine-stress, can easily be used in conjunction with other therapies to treat solid tumors. IV. BIOLOGICAL OUTCOMES OF BYSTANDER/ABSCOPAL EFFECT: THERAPEUTICAL POTENTIAL The resultant biological outcomes from the bystander/abscopal effect can potentially be exploited in solid tumor therapeutics. Certain treatments can lead to the reversal, and/or inhibition, of some of the pro-survival pathways listed above, while others can compliment the pro-apoptotic responses. The ultimate goal of these treatments is to establish a stronger therapeutic outcome. As mentioned earlier, bystander/abscopal-mediated signaling events can lead to varied biological outcomes. These events can be arranged broadly into two categories. The first category involves effects that lead to the elimination of malignant cells through processes such as: reproductive death, apoptosis, regression, and further elimination of metastasis. The second category involves effects that might potentially cause genomic instability within cells and induce carcinogenesis Figs. (2) and (3); Table 1. Particularly, the first category, can be exploited to combine with other therapeutic modalities in order to eliminate both primary tumors and metastatic deposits concomitantly (Table 1). a) Pro-Therapeutic Biological Outcomes As mentioned above, some therapies such as radiotherapy, chemotherapy, gene therapy, suicide gene therapy, radio-gene therapy, photodynamic therapy, and methioninedeprivation therapy can lead to pro-apoptotic events in which cancerous tissue is eliminated without killing normal tissue.


Table 1.


A List of Inducers that will Elicit Specific Responses Through Bystander/Abscopal Effects

Inducers

Outcome

Response

Ref.

Pro-mitogenic

increased sister chromatid exchanges, TGF- induction, nitric oxide induction, nerve growth factor induction, and/or increased sCLU expression gap junctional intercellular communication, and/or secretion of factors (TNF-, TGF-, TRAIL)

[13, 14, 57-62, 68]

Radiation Bystander

Pro-apoptotic

[16-18, 95, 99, 100]

Bystander/Abscopal

Radio-resistance

induction of nitric oxide pathways

[39, 60, 74, 75]

Abscopal

Pro-apoptotic

activation of immune system (T-Cell act.)

[6, 20, 22]

Bystander

Recurrent tumor growth

PI-3K molecular complex changes

[24]

Abscopal

Growth at metastatic sites

vWf and/or VEGF secretion

[100-102]

Bystander

Pro-apoptotic

Gap junctional intercellular communication and/or secretion of apoptotic vesicles

[33, 35, 36, 95, 103, 112-116]

Abscopal

Pro-apoptotic

Death of tumor cells at distal sites (lymphocyte activation)

[37, 105, 117]

Bystander

Pro-apoptotic

TNF- secretion

[38, 39]

Abscopal

Pro-apoptotic

increased cell death at distal sites

[38]

Pro-apoptotic

possible release of nitric oxide, cyotoxic aldehydes, lipid peroxidation

[41-43]

Pro-apoptotic

induciton of MDA-7

[47, 48, 118]

Surgery

Chemo-gene therapy

Radio-gene therapy

Photo-dynamic Therapy Bystander Methionine-stress Bystander

One important biological outcome that has been observed in cells experiencing a bystander effect is mitochondrial changes which are indicative of initiation of cell death [99]. Apoptosis-inducing chemicals can lead to increased mitochondrial proliferation and thus an increase in the mitochondrial mass present within cells. ROS which, as listed above, are involved in the bystander effect and also lead to increases in mitochondrial mass [123]. This is interesting because it provides a way to measure the induction of apoptosis in unirradiated cells.

lacking. One possible mechanism for increasing at least the bystander effect elicited by suicide gene therapy is to enhance the GJIC [107]. Another possibility, that has been successful for the treatment of human pleural mesothelioma, involves infecting eukaryote cells with HSV-tk in vitro and then injecting them near the tumor of the patient [124]. No side effects were observed when this approach was adopted and the injected cells seemed to play an important role in conferring bystander/abscopal effects.

i) Increasing the Therapeutic Effectiveness of Suicide Gene Therapy

Another strategy involves combining these gene therapy methods with radiation therapy (similar to radio-gene therapy that was discussed earlier). Chhikara et al. showed that treating a prostate tumor in mice with the HSV-tk/GCV system followed by radiation exposure of 5 Gy resulted not only in decreased tumor growth, but also prolonged overall survival of the animal [125]. This indicates that perhaps the best way to increase the effectiveness of suicide gene therapy and prodrug use is to combine with radiotherapy and thus exploit the radiation-induced pathways as well.

Human clinical trials involving suicide gene therapy have led to disappointing results [107]. While the GDEPT systems do appear to be safe, their anti-tumor abilities seem to be

Furthermore, this prodrug application by itself can lead to radio-sensitization in tumor cells making them prime targets for the use of radiation therapy [126]. In fact, some prodrugs

Apoptosis, reproductive death, regression, and elimination of metastasis have all been observed following therapies in which abscopal/bystander effects are observed. Salient strategies to enhance the effects of bystander/abscopal events in response to various treatment approaches are discussed below.


are already being used without gene therapy but in conjunction with radiotherapy. A few examples are cyclophosphamide (CPA), irinotecan (CPT-11), gemcitabine (dFdC) and mitomycin C (MMC) [126]. All of the chemotherapeutic drugs listed above have prodrug counterparts, but not all prodrugs have such counterparts. These therapies gave successful results, but the abscopal/bystander effects initiated by combining pro-drugs with radiotherapy are not known. ii) MDA-7 Gene Therapy/ Methionine-Stress Therapy in Combination with Radiotherapy The MDA-7 gene has been shown to elicit some interesting pro-therapeutic bystander effects. The bystander signaling responses initiated by the secretory form of MDA-7 can be combined as an adjuvant for radiation therapy [127]. Because the bystander responses of MDA-7 can be elicited either by (1) infection of non-transformed cells by Ad-MDA-7 virus, or by (2) methionine stress, the possibility of developing a therapeutic strategy in combination with radiation is plausible. MDA-7 has been shown to be a radio-sensitizer [128] and thus when combined with radiotherapy, increased cell death, as well as increased bystander responses, should ensue. This again indicates that using therapies known to elicit bystander effect in combination with radiotherapy can lead to increased cell death without harming surrounding normal tissue. b) Pro-Carcinogenic Biological Outcomes While the strategies outlined above involve the therapeutic biological outcomes arising from bystander/abscopal effect, there are also strategies available that may lead to a decrease in the carcinogenesis initiated by bystander/abscopal effects. One of the main concepts dealing with how abscopal/bystander pro-carcinogenic effects can lead to increased or sustained tumor growth is genomic instability. i) Genomic Instability Genomic instability has been observed in cells after exposure to chemicals and radiation [129]. The phenomenon of genomic instability involves the appearance of: chromosomal alterations, sister chromatid exchanges, micronuclei, gene mutations, and other potentially harmful effects in cells after the initial exposure to a potential mutagen. It is thought that inability to activate cellular responses to deal with the induced DNA damage may lead to the cells being able to replicate without first repairing their DNA [130]. It has been shown that p53 status can be an important factor in the onset of genomic instability as the rate of genomic instability is higher in p53 null mice than in p53 wild-type mice [130]. p53 is known to control radiation-induced apoptosis and thus its inactivation should lead to a less efficient “cleaning-up” of cells that have experienced some sort of genomic alteration. Of vast importance is that genomic instability has been observed in bystander cells in vivo [131, 132]. This means that bystander effects and genomic instability can occur at the same time within a cellular system. If the bystander/abscopal effect that has been initiated following a parti-

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cular type of therapy leads to a pro-carcinogenic effect and at the same time provides protection to tumor cells both at primary treated site as well as distal sites, with increased genomic instability, then the possibility of developing a recurrent tumor formation is high. However, there have been strategies developed that can help to eliminate the pro-survival pathways induced by abscopal/ bystander effects. These include: eliminating the IL-6 pathway that protects cells at metastatic sites, and the use of antisense oligonucleotides (ASOs) to downregulate clusterin gene expression. ii) Targeting IL-6 May Lead to Regression at Metastatic Sites Therapeutically, surgical removal of the primary tumor can cause death of cancerous cells at distant sites [6]. Further, removing the primary tumor can inhibit the induction of the MAPK and ERK pathways mediated through IL-6 within the cells in both the primary tumor and at metastatic sites. With the elimination of tumor, the initial IL-6 production, as well as the initiation of downstream pathways, no longer occurs. By inhibiting the abscopal effect mediated through IL-6, tumor growth at distal sites decreases and may regress [133]. The supply of interleukin-6 can be abrogated using antagonist therapy [134]. In multiple melanoma, super-antagonist Sant7 has been shown to induce apoptosis and inhibit the cell proliferation caused by IL-6 [135]. Sant7 prevents IL-6 from binding to its receptor [134]. Combining Sant7 treatment with glucocorticoids, such as dexamethasone, overcomes the resistance of IL-6 and successfully kills tumor cells. Although no studies have demonstrated the effects of IL-6mediated inhibition of abscopal effects, in theory, downregulation of the secreted IL-6, and its ability to bind to its receptor, should eliminate the protection elicited to metastatic sites by the primary tumor. Thus, after application of Sant7, distal tumor cells begin to undergo apoptosis. iii) Inhibiting the Bystander/Abscopal Effect-Mediated by PI 3-K/Akt Pathway The PI 3-K pathway is important in both the pro-survival bystander effect initiated following radiotherapy and the growth of recurrent tumor following surgical removal. Although specific inhibition of PI 3-K and Akt has yet to be achieved, inhibiting downstream targets of the PI 3-K/Akt pathway, as well as using the protein kinase mammalian target of rapamycin, mTOR, have proven to eliminate some of the cell survival effects [136]. Since bystander cells can experience the effects of the PI 3-K/Akt pathway following activation of TGF-, this type of therapy offers opportunities to inhibit pro-survival events not only in treated tumor cells, but also in untreated tumor cells. Rapamycin inhibits the function of mTOR and other pathways that eventually contribute to cell survival [137]. Importantly, rapamycin has the ability to induce apoptosis in cancer cells with minimal toxicity to surrounding normal tissue [138]. Furthermore, rapamycin has been shown to increase the effectiveness of cytotoxic agents such as cisplatin [139] and doxorubicin [140]. Pre-clinical study results have shown that tumors in which the PI-3K/Akt pathway is important for cell survival may be sensitive to the rapamycin therapy [136, 141, 142].


iv) The Use of ASOs to Downregulate Clusterin Gene Expression It was discussed earlier that CLU gene expression following low dose IR causes activation of cell survival pathways. These pathways can lead to the protection of tumor cells with increased genomic instability [58]. Once a normal tissue has developed a certain amount of genomic instability it can transform into a malignant tissue. The down-regulation of p53 function and up-regulation of sCLU can cause increased cell proliferation and cell cycle pertubations. Indeed, the accumulation of sCLU has been correlated to incidence of cancer [58]. Therefore, a therapeutic intervention could be developed that prevents the activation of sCLU following radiation exposure and inhibit cancer formation. Therapeutic interventions involving clusterin gene targeting have been studied over the past couple of years [143147]. ASOs have been shown to downregulate clusterin gene expression [144]. It has been shown to increase apoptosis following treatment with radiation, as well as chemotherapy [145]. Pro-apoptotic events following down-regulation of the clusterin gene using ASOs have been demonstrated for a number of malignancies including: breast cancer [146], renal cell cancer [144], human bladder cancer [147], and prostate cancer [143, 145]. For hormone-refractory prostate cancer in particular, the use of gene therapy by adenoviral-mediated p53 gene transfer, in combination with ASO treatment has been shown to inhibit tumor progression in mice [143]. Furthermore, if this treatment was followed by mitoxanthrone treatment, complete eradication was observed in 60% of the tumors and 100% of the formed lymph node metastases. These results show that targeting of the clusterin gene is very plausible and could easily lead to apoptosis in bystander cells. V. EXPLOITING BYSTANDER/ABSCOPAL EFFECT IN THE TREATMENT OF SOLID TUMORS a)

GRID Therapy

Therapeutic interventions for large and bulky tumors have proved problematic due to injury of surrounding normal tissue [23]. Normal tissue tolerance is not large enough to


deal with irradiation of a tumor with too large an area (> 8 cm). The idea of using altered fractionation of radiation to allow treatment of these bulky tumors has not been very successful [148]. A new innovative technique that our group has performed extensive research on, referred to as GRID therapy, or spatially fractionated radiation therapy (SFGRT), exploits both the abscopal and bystander effects in treated tumor tissue. This technique involves applying radiation such that not all of the tissue is exposed to 100% of the dose Fig. (5). High doses (15 – 20Gy) of radiation, strong enough to overcome radio-resistance of tumor cells, can be applied using GRID therapy without damaging, or more importantly killing, surrounding tissue [149]. The abscopal effects induced by GRID therapy are similar to those produced by high-dose open-field radiation (unpublished data). One of our studies evaluated and compared the tumor regressing effects of low energy high-dose spatially fractionated GRID radiation (SFGRT) with conventional ionizing radiation therapy (IRT) in a human lung adenocarcinoma, A549, model tumor system. Tumors were developed in both right (RT) and left (LT) flanks of the nude mice. It was found that GRID therapy led to the induction of more pro-apoptotic factors than open field radiation. Also, the mice treated with high dose SFGRT showed increased cell death in the unirradiated tumor when compared to the mice that received open field radiation. When SFGRT (7.5 Gy) in one flank was combined with conventional IR (5 doses at 2 Gy) in the other flank, the tumor growth delay was prolonged. Levels of two pro-apoptotic proteins, EGR-1 nad Bax were elevated in the unirradiated cells in mice receiving GRID therapy and no change in Bcl-2 expression was seen. Increased regression of the untreated tumor in the animals in which the tumor in the other flank was exposed to SFGRT suggests that the high-dose SFGRT may release tumoricidal factors such as cytokines (TNF or TRAIL) systemically in the mice which may cause an abscopal tumor regression response with increased apoptosis. In relation to GRID therapy, the abscopal effect deals with regression of tumors at sites that do not recieve SFGRT. Bystander effects, on the other hand, are observed in the cells that do not receive the full dose of SFGRT in primary

Fig. (5). SFGRT in abscopal/bystander effect. An illustration demonstrating the utilization of spatially-fractionated GRID radiation therapy (SFGRT) in eliciting bystander/abscopal effect within the directly-treated tumor, as well as in a distant tumor.


treated tumor [23]. In another study performed by our group, a grid block was constructed that contained 256 holes (50% open and 50% blocked) which was used as collimator in Xray linear accelerator [23]. Compared to the dose received through the grid holes, the blocked regions only received about 25-30% of the SFGRT. The GRID set-up was used to treat 71 patients with different types of advanced bulky tumors. Of the 71 patients who underwent treatment, 62.5% had a clinical complete response, and 50% had a pathological complete response. Also of interest is that 92% of the patients treated with GRID therapy and an external beam radiation dose of 4000 cGy or greater responded to the treatment [23]. Our clinical outcomes coincide with our data dealing with abscopal effects and showed that combining GRID therapy with conventional IR synergizes for potentiation of bystander factors (unpublished data). More importantly, the radiation dose applied during the GRID therapy is high enough to initiate the production of cytokines involved in the bystander response [23]. In this case, we hypothesized that this bystander effect was mediated by TNF- and the ceramide pathway. To further test this hypothesis, our group tested the possible involvement of not only TNF- in this bystander response, but also TGF- [149]. In this study, 34 patients with at least one large and bulky tumor were treated with SFGRT. The results showed induction of TNF- and downregulation of TGF-1. TNF- leads to tumor killing and radiation sensitization of cells, whereas TGF-1 offers protection to normal tissues surrounding the irradiated tumor [149]. There was also a strong correlation between the induction of TNF- and complete clinical response of the tumor, but no strong correlation was observed between complete clinic response and TGF-1 induction. This is a very conducive situation since SFGRT deactivates possible cell protective pathways in unirradiated cells (through TGF-1) while upregulating the proapoptotic functions (through TNF-). One of the goals of another study published by our group [150] was to determine the effect of this treatment on serum ceramide content and to investigate possible involvement of ceramide in tumor regression after SFGRT. Serum ceramide and secretory SMase (S-SMase) were quantified in 11 patients before and at 24, 48 and 72 h after the dose of 15 Gy. Furthermore, LDL particles were isolated from the serum and their apoptotic ability was tested in human endothelial cells by terminal deoxyuridine transferase mediated nucleotide end labeling (TUNEL) assay. 67% (6/8) of the patients with partial (PR) or complete (CR) response showed statistically significant increase in serum ceramide levels. Of the non-responders in the study, none showed an elevation in ceramide. S-SMase activity underwent similar changes. Low-density lipoproteins (LDL) particles from serum of patients collected 72 hours after SFGRT sensitized the endothelial cells to undergo apoptosis in response to 5 Gy radiation that by itself had only modest effect on cell death. Independent elevation of ceramide content of endothelial cells that were otherwise resistant to radiation-induced cell death also was sufficient to sensitize these cells to apoptosis. Serum S-SMase activity and ceramide content increased following SFGRT and correlated with the clinical response. Apparently, these changes are in the LDL-associated ceramide

Peters et al.

and may contribute to better tumor reduction after SFGRT due to the ability of LDL-derived ceramide to sensitize endothelial cells for apoptosis. Overall, GRID therapy is an optimal strategy that initiates precisely the type of simultaneous abscopal effect and bystander effect needed for tumor death. It appears that combining GRID therapy with conventional IR therapy may be the best way to deal with multiple tumor sites as well as treat bulky tumors. This would resolve many of the current issues concerning the treatment of bulky tumors through the use of a relatively simple grid design. b) Stem Cell Gene/Suicide Gene Therapy The use of stem cells has lead to some novel development of cancer therapies [151, 152]. In children, bone marrow transplantation is already part of cancer therapy when it comes to high-risk malignancies [152]. Important in the context of this paper is stem cells that have been used in certain suicide gene therapy approaches [151] which of course can give rise to bystander/abscopal effects. Davidoff et al. created hematopoietic cells modified to express a truncated, soluble form of Flk-1 (tsFlk-1). This tsFlk-1 has the ability to block the function of VEGF [153, 154] and thus can potentially inhibit tumor metastasis. Of vast importance is that these cells were shown to remain stable in mice, without selection for destruction, for at least 6 months [152]. This means that application of stem cells encoded with a particular gene could potentially remain active, and perhaps circulating, within the body for extended periods of time. Also, the stem cells that have endothelial cell precursor function could be genetically altered, so that they may no longer be recruited for tumor-induced neovascularization [152]. A main feature of most cancer types is neo-vasculogenesis [151]. Thus, the majority of endothelial progenitor cells present within the body are attracted towards the tumor tissue for neo-vascularization. Embryonic endothelial progenitor cells (eEPCs) can be genetically altered, can lead to an unlimited supply of cells lacking MHC I antigens, and are not targeted by non-activated natural killer cells [151]. All of these features make eEPCs a perfect carrier for a suicide gene that could successfully target tumors and metastasis without being destroyed by the immune system. Wei et al. showed that 77% to 90% of implanted eEPCs could successfully target lung tumor nodules [151]. They also showed the induction of a cytotoxic bystander effect in vitro following application of eEPCs containing the suicide gene suicide gene Escherichia coli cytosine deaminase (CD)/ uracil phosphoribosyltransferase (UPRT). An additional study in mice found that application of CD/UPRT eEPCS followed by subsequent addition of prodrug 5-flourouracil (5-FU) lead to a significant increase in the survival of the mice containing multiple lung metastases [151]. This shows that while the potential certainly exists for the use of stem cells in cancer therapeutics, extensive research is warranted to successfully exploit the uniqueness of stem cells. VI. THE IMPACT OF ABSCOPAL/BYSTANDER EFFECTS ON RISK ESTIMATION Another important fact that needs to be considered when evaluating the significance of abscopal and bystander signa-


ling in therapeutics is risk estimation. In radiation therapy, for instance, only the tissues that are in the path of the beam have traditionally been considered during risk estimation [2]. Thus, only the tissues which can tolerate the smallest dose of radiation and which will be directly in the path of the beam, are taken into consideration when planning the therapy. As has been mentioned throughout this review, bystander effects, and more importantly abscopal effects, can take the resulting damage elicited by these therapies and spread its effects to tissues far away from where the therapy was applied. A good example of how bystander and abscopal effects change risk estimation involves the use of fractionated radiotherapy. Although fractionated therapy leads to less total cell death in normal tissue (in relation to the total dose), the factors secreted systematically each time are equally as toxic [155]. In other words, while the normal tissue receiving the direct exposure will be spared, the cells receiving these signals that have not been exposed directly, including normal tissue cells, will respond to each fraction [2]. This means that apoptosis could be induced in cells not even considered during the risk estimation following each fraction of radiation exposure.


ABBREVIATIONS 5-FU

=

5-fluorouracil

ADEPT

=

Antibody-directed enzyme prodrug therapy

ALTRA =

All-trans retinoic acid

ASOs

=

Antisense oligonucleotides

CD

=

Escherichia coli cytosine deaminase

eEPCs

=

Embryonic endothelial progenitor cells

EGR-1

=

Early growth response gene-1

GCV

=

Gancyclovir

GDEPT

=

Gene-directed enzyme prodrug therapy

GJIC

=

Gap junctional intercellular communication

HSPs

=

Heat shock proteins

IL-6

=

Interleukin-6

LDL

=

Low-density lipoproteins

LNT

=

Linear no threshold

MDR-1

=

Multi-drug resistance

The linear no threshold model (LNT model) is currently used when estimating risks related to ionizing radiation at low doses [3]. This model holds that the responses arising from irradiation are additive and thus cells should not respond to individual doses, but the dose as a whole. This model was developed based on the effects observed in survivors of atomic bomb exposure and the low-dose region was developed by estimation. The LNT model is used to determine the risk of developing cancer after radiation exposure, even after exposure to low doses [3]. As was mentioned earlier, cells experiencing abscopal and bystander effects do not respond in accordance to this LNT model. Thus, a new model, which takes into account of these bystander and abscopal effects, especially at low doses (such as proposed by Nikjoo and Khvostunov [3]) will be more appropriate.

MHC

=

Major histocompatibility complex

nCLU

=

Nuclear form of clusterin

NO

=

Nitric oxide

PDT

=

Photodynamic therapy

ROS

=

Reactive oxygen species

SCE

=

Sister chromatid exchanges

sCLU

=

Secreted form of clusterin

SFGRT

=

Spatially-fractionated GRID radiation therapy

HSV-tk

=

Herpes simplex virus thymidine kinase

SGK

=

Serum and glucocorticoid stimulated kinase

CONCLUSION

TAP

=

Tumor-activated prodrugs

In day-to-day interaction between body cells and their environments, there is a significant amount of interaction that occurs with neighboring cells through secretion of factors and GJIC leading to bystander effect and to distant cells via humoral factors causing abscopal effect. Although bystander/abscopal effects can be potentially deleterious in inducing transformation, they also have therapeutic potential that has not been exploited. Hence, established treatment strategies and novel treatment strategies should incorporate into the design for potential use of bystander/abscopal effect. Radiation therapy seems to be one of the important modalities through which bystander/abscopal effects can be utilized. The most promising effects might come from embryonic stem cells, which act as potent releasers of bystander/abscopal factors. Thus, all therapeutic modalities should consider the use of potent inducers of bystander/abscopal effect and release of factors that cause bystander/abscopal effect for enhanced therapeutic efficacy.

TUNEL

=

Terminal deoxyuridine transferase mediated nucleotide end labeling

UPRT

=

Uracil phosphoribosyltransferase

VEGF

=

Vascular endothelial growth factor

vWf

=

Von Willebrand factor

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