Immunogenic cell death: the role of SAGE
Immunogenic cell death: the role of SAGE
Introduction
Millions of cells are always being removed in the human body not only to regulate and coordinate the body’s biochemical pathways but also to keep the cells healthy. This particular systematic removal of cells occurs through a selective mechanism called cell death or apoptosis process. Apoptosis is not a new concept in the understanding of the biological workings of the body. This homeostatic death of body cells has been defined as a tolerogenic or a non-immunogenic event for many [1]. However, new discoveries have unearthed the existence of a different concept of cell death – the Immunogenic Cell Death (ICD). Unlike in Apoptosis, in ICD, the death of a cell stimulates a response from the host’s immune system against antigens of a dead cell, and this mechanism of cell death may play an especially important role in cancer cells [2]. This discovery has prompted medical trials of ICD in the fight against cancer cells in lab animals. The efficacy of this immunogenic cell death in cancer therapy has been tested in mouse and rat models. Elaborate experiments that have taken into account differences in species and strains have been performed in this context, proving evidence that immune responses to tumors can define the efficiency of anti-cancer treatment [3].
In humans, the discovery of ICD shows significant promise in addressing cancerous body cells and tissues. In particular, studies show that human tumor cells when exposed to anthracycline – a cancer drug – appear to be significantly immunogenic [4]. This feature of the cells may contribute to why it has been successfully used as an efficient drug for treating cancer patients [4]. In the treatment of cancer, anthracyclines have been known to be efficiently active against several tumors in the body. They are however much more efficient against cell solid tumors. Despite their effectiveness against body tumors, there has been a significant medical hindrance to the use of these drugs because of their unintended toxicity to the body and ability to induce resistance in cancer cells. Tumor resistance to anthracycline is due to several reasons, one of which includes the P-glycoprotein-mediated resistance. In addition to that, anthracyclines can produce hydroxyl free radicals which account for both its anti-tumor and toxicity effects, especially to myocardial tissues which are easily damaged by free radicals. These drugs release free radicals because they contain chemotherapeutic Quinone substances [27]. While anthracyclines have several side effects to the body, typical symptoms include acute myelosuppression, cumulative dose-dependent cardiotoxicity, nausea, vomiting, and inflammation of the mucous membrane of the mouth (stomatitis) [27]. Further studies show that anthracyclines can even have side effects such as cardiomyopathy, which is irreversible and often leads to congestive heart failure [27]. Another study conducted by Volkova et al. in 2011 on the same indicated that cardiotoxicity caused by chemotherapy are of two types; type I or type II [33]. Type I cardiotoxicity may be directly attributed by ICD and is irreversible while type II is caused by cardiomyocyte dysfunction which is reversible. Understanding the cardiotoxic activities of anthracyclines must, therefore, include the development of strategies to reduce or ameliorate the aforementioned cardiac damages in patients [33].
Using anthracycline for chemotherapy stimulates a complex immune response with the help of dendritic cells (DCs) to present a cytotoxic antigen from dying tumors, which leads to increased recruitment of cytotoxic T-lymphocytes. However, they have been shown to induce immunogenic death of cells by affecting the translocation of Heat Shock Proteins (HSP70) in the cells. They carry out this translocation function by stimulating the cellular release of Extracellular Vesicles (EV). Studies show that EV plays crucial roles in promoting as well as maintaining cancer cell dissemination and progression in cancer patients [28]. In cancer therapy, EVs act as the natural delivery systems of anti-cancer drugs in and out of tumor cells [28].
HSP70 is commonly integrated into artificial bilayer openings of the cell ion conductance pathways. It is also usually translocated into the cell plasma membrane after the cell is under stress. This cellular translocation recognizes the HSP70 released into the Extracellular Environment surrounding the cell. Outside the cell, they exist in a Membrane-Associated form that is then used to activate cell Macrophages [28]. Heat Shock Proteins (HSPs) are the intracellular chaperones responsible for instigating cell recovery from stress. However, unlike HSPs that are intracellular in nature (located inside the cell), the HSP70s which are the stress-induced versions of HSPs have been found outside the cell. They are capable of activating the body immune cells and are known to share the characteristics of protein found in the cell membrane [28].
Anthracyclines are also known to cause an HSPs translocation that in turn can activate primary antibodies that can lead to immunogenic cell death. They are known to induce specifically the translocation of HSP90 and calreticulin to the surface of tumor cells. Unlike the HSP70, cellular HSP90s are three Endoplasmic Reticulum (ER) chaperones that are translocated from the tumor cytosol to the cell surface during ER stress. They are also translocated in cells challenged by factors such as Ultra-violet (UV) irradiation, microbial stimulation, and extreme exposure to drugs. In normal cells, HSP70s are the primary intra-cell ATPase molecules that are responsible for mediating and controlling the proper folding of synthesized proteins, the translocation of substances in and out of the cell, and the disassembly of stress-induced protein groups [36]. Their translocation across membranes into the cellular plasma membrane, however, occur after cell stress. Following the stress, the HSPs are forced into the external cell environment and are integrated into membrane-associated cell particles responsible for activating Macrophages [36]. The translocation occurs through entropic pulling that occurs due to entropy loss by the excluded HSP70 molecules. The loss of entropy then allows the HSP70s to convert the ATP energy released during cell hydrolysis into pull-force that enables them to translocate. Therefore utilizing entropic pulling, the HSP70 molecules are capable of accelerating protein unfolding and pulling the translocating substances across the cell intra and extracellular environments [36].
Once the HSP90 is in the extracellular space, they assume immunogenic characteristics that are involved in determining whether a cancer cell is targeted for cell death. When applied to clinical treatment, this cellular mechanism is an important target avenue for the elimination of cancerous cells by the body’s immune system. However, studies show that underlying autoimmune conditions such as multiple sclerosis and rheumatoid arthritis can sometimes be exacerbated by the release of these chaperones [29]. Therefore, extreme caution and monitoring are required when using extracellular chaperones as potential cancer immunotherapies in the treatment of tumors especially if the patient has an autoimmune disease [29].
HSP70, HSP90, and calreticulin serve as signals for dendritic cells to engulf tumor cells. The cells that are undergoing immunogenic cell death induced by anthracyclines also release the late apoptotic marker, high mobility group box1 (HMGB1), which can bind to several pattern recognition receptors such as Toll-like receptor 2 (TLR2) and 4 (TLR4), and receptor for advanced glycosylation end products (RAGE). HMGB1 release facilitates antigen presentation and further uptake of tumor cells by dendritic cells [5]. The secretion of ATP is also a characteristic feature of immunogenic cell death. ATP interacts with the pyrogenic receptor, P2rx7, on the surface of dendritic cells. Such mechanisms have been demonstrated in the laboratory in mice and humans, such as in clinical responses to chemotherapy with the loss of Toll-like receptor 4 and the pyrogenic receptor, P2rx7 [4].
Immunogenic cell death (ICD) is a result of autophagy and premortem endoplasmic reticulum (ER) stress, which exposes calreticulin (CRT) on the plasma membrane’s outer leaflet at its pre-apoptotic stage with adenosine triphosphate (ATP) [6]. Moreover, during ICD, dying cells also release HMGB1 during the permeabilization of membranes during secondary necrosis. Accordingly, CRT binds to CD91 on the pyrogenic receptor, P2RX7, and HMGB1 binds to TLR4; these bindings stimulate dendritic cell (DC) recruitment into the tumor microenvironment facilitating tumor-antigen engulfment by DCs and presenting the antigen to T-lymphocytes. These processes result in an immune response mediated by interferon (IFN- γ) and cytotoxic CD8+ T lymphocytes (CTLs), which lead to the eradication of chemotherapy-resistant tumor cells [6]. Several studies have considered and analyzed the question of why certain cytotoxic chemotherapeutics result in CRT exposure with ATP’s secretion and HMGB1 release while others may not [7]. In this regard, ICD, therefore, requires the appearance of two types of stress: Autophagy and ER stress.
CRT, which is inside of the ER during chemotherapy-induced ER stress, transfers itself to the outer surface of the plasma membrane (known as ecto-CRT) before the onset of apoptotic cell death [7]. Ecto-CRT appears to be a potent signal of engulfment because it recognizes and destroys tumor cells by dendritic cells. However, the CRT exposure seems to affect the efficiency of chemotherapy in vitro studies negatively; thus, the ER stress-dependent signal of the immune system appears to be an essential aspect of anti-cancer responses [7]. It is also noteworthy that anthracyclines that induce the ecto-CRT causes ICD. Pro-apoptotic agents, such as mitomycin C do not induce ecto-CRT or ICD. In the absence of CRT, ICD is not activated by anthracyclines, while exposure to drugs or pharmacological agents favoring CRT’s translocation can produce ICD. The same tumor cells must be exposed to ecto-CRT to induce apoptosis; if non-tumor cells are exposed to ecto-CRT, then an anti-tumor immune response will not be elicited [35].
Autophagy appears to be obligatory for stressed cells to be considered immunogenic. Autophagy-deficient tumor cells release less ATP as compared to their autophagy-competent counterparts, but inhibiting autophagy influences neither HMGB1 release nor CRT exposure. Autophagy is required for ATP secretion following chemotherapy treatment and immunogenic cell death. The decreased release of ATP causes autophagy-deficient tumor cells to stop the recruitment of dendritic cells and thus reduce immunogenic cell death. The opposite is the case for autophagy-competent tumor cells. This phenomenon can be corrected by the inhibition of extracellular ATP degrading enzymes. The inhibition of such enzymes causes an increase in ATP in tumor cells, which allow for the recruitment of dendritic cells [8]. Therefore, autophagy is essential for ICD so that ATP is released. Furthermore, increased concentrations of extracellular ATP provide potential to improve the efficiency of anti-neoplastic chemotherapies in conditions when autophagy is unavailable or disabled [8].
Considering the factors mentioned above, the importance of immunogenic cell death lies within its potential to improve and enhance current cancer treatments [9]. Chemotherapeutics can induce powerful combinations of the death and stress of tumor cells that would be immunogenic; moreover, the majority of such procedures have significantly high success rates to improve the status of cancer patients. In other words, the death of tumor cells in patients are nowadays becoming a considerably effective form of treatment, being able to stimulate an immune response aimed specifically to remove tumor cells, which results in further control and eradication of cancer cells [9]. In vitro, ICD has been seen in tumor cells by launching a response from the immune system and thereby protecting the body from tumor cells. For ICD occurring in vivo, it also elicits a local immune response with the recruitment of cognate and innate immune system effector cells into the tumor bed, which results in tumor growth inhibition. Scientists have considered various preclinical models and have provided evidence for these phenomena occurring in different immune system alterations [9].
During cell death, biomarkers are involved in the stimulation of the immune system, such as the HMGB1 protein along with its receptor, RAGE [10]. HMGB1 is a small protein that possesses three structural domains: two tandem HMG boxes and an acidic C-terminal tail separated by a short linker. This C-terminal tail of HMGB1 mediates its interaction with RAGE [11]. HMGB1 was discovered as a ligand for RAGE during a search for the potential binding partners of RAGE in the detergent extract of bovine lung acetone powder applied to Sepharose and heparin columns by elution in which eluent fractions were checked for RAGE binding activity. The eluent fractions were purified and sequenced and was found to be HMGB1 or amphoterin. To further confirm this interaction, recombinant amphoterin from rat was purified and was shown to bind to RAGE in the same manner. This binding was proved to be highly specific [12].
In particular, HMGB1 represents a nuclear protein that has a high association with chromatin, playing a vital role in transcription processes and cell regulation. Previous studies related to HMGB1 have reported that it can only be released during necrotic cell death; however, the latest research has proved that the release is also possible during late stages of apoptosis. Afterward, HMGB1 becomes firmly attached to the chromatin, also being released in the form of HMGB1-nuclesome complexes. HMGB1 functions as a danger associated molecular pattern (DAMP), which binds with the TLR4 to elicit signaling events that contribute to the effects of HMGB1.The efficacy of HMGB1 has been reported in conditions when nucleosomes, DNA or lipopolysaccharides (LPS) are bound to it. Its principal mechanism of functioning lies within binding to particular receptors on antigens or dendritic cells such as TLR4 or RAGE in association with LPS [10].
HMGB1 binding to RAGE leads to the activation of the IKK [inhibitor of NF-κB (IκB)] kinase complex that phosphorylates IκBα, which takes place when inactive NF-KB residues in the cell’s cytoplasm are bound to its inhibitor proteins. Inflammatory cytokines activate the IKB kinase complex, which is composed of IKK1, IKK2, and NF-Kb essential modulator (NEMO); HMBG1 binding to RAGE phosphorylation is therefore through the activated IKK pathway [30]. This phosphorylation induces the release of NF-κB and its translocation to the nucleus where it increases. Thus, the transcription of pro-inflammatory genes (such as interleukin-1 (IL-1), IL-6 and tumor necrosis factor) is induced, as shown in figure 1. As a result, phagocytized particles are intracellularly organized for presentation at the cell surface, which stimulates the cytotoxic response of T-lymphocytes, as a response to tumors [9].
As indicated below (Fig 1), the cell cycle (life cycle) is regulated and controlled by a series of a complex and interconnecting network of signaling pathways comprising of numerous of protein-based regulatory agents. If some of these essential proteins are altered or there are defects in their signaling mechanisms, normal cell growth regulatory mechanisms can break down and possibly lead to the development of cancer. Tumor growth can also occur if extracellular agents bind to the receptor sites of these cell growth regulatory proteins and thereby activating growth pathways that lead to the proliferation of tumor cells. Figure 1 shows that the High-mobility group box1 protein (HMGB1) does not only function as a nuclear factor crucial for cellular transcription but is also an important cytokine that helps mediate in the cell response to factors such as injury, inflammation, and, in this case, tumor infection. It also links with RAGE in forming an autophagy cell response at multiple levels.
Figure 1: The HMGB1-RAGE pathway.
Image as retrieved from: Lotze & Tracey, 2005
Recent studies have shown that the DAMPs released through stressed cells with apoptosis and necrosis appears to be essential for a patient’s response to successful chemotherapy. In this regard, research also reports that knockdown or neutralization of TLR4 or HMGB1 is associated with significant reduction of an immune reaction both in vivo and in-vitro, along with a weak outcome for therapy [11]. Still, it remains uncertain whether HMGB1’s serum levels have a significant predictive or prognostic association with cancer [11].
Besides, it has also been shown that HMGB1 not only launches the protective reactions of T-lymphocytes but can also stimulate tumor invasiveness and neo-angiogenesis [14]. In vitro experiments have demonstrated that suppression of HMGB1 and RAGE by antisense S-oligodeoxynucleotide imparts tumor proliferation and migration. The same has been observed in animals where the suppression of RAGE-HMGB1 interaction induces the activation of mitogen-activated protein kinase pathway and NF-kB pathway and thus inhibits tumor growth. The downstream signaling components of HMGB1 need to be elucidated to counteract this effect. HMGB1 and RAGE’s overexpression is vital for tumor metastasis, according to evidence obtained in the colon and gastric cancer. However, RAGE’s serum levels have been reported to decline significantly in some forms of cancer. Therefore, there is a double role of RAGE, as it is involved in the DAMP cellular signal transmission as a surface receptor, and may also correlate with high cellular expression of RAGE [15].
Further sections of this research paper consider important biomarkers as an inevitable part of the cell death process, with the growth of tumors and immunogenicity represented in various forms of cancer to identify their role and importance in the prediction and prognosis of responses to different types of chemotherapy during different phases of cancer treatment.
Chemotherapy is considered to be an immunosuppressive treatment due to the depletion of NK-cells and T-lymphocytes as they become functionally inactive [16]. As a result, the immune system of patients after chemotherapy is severely impaired, which often leads to the growth of residual tumors and infectious complications. In most cases, taking chemotherapeutic drugs or receiving radiotherapy treatment include apoptosis induction, which mediates the cytotoxic effects of the treatment. Such methods have been considered to be non-immunogenic and non-inflammatory. Still, current research has discovered that the “danger signals” from cell death resulted from chemotherapy could potentially be antigen-specific T-lymphocyte immunity, which is TLR4/HMGB1-dependent [16].
Studies have also shown that chemo-radiation can lead to local HMGB1 upregulation in patients with esophageal squamous cell carcinoma (ESCC), asHMGB1’s high expression results in better overall survival of patients [13]. Additionally, in vitro studies have reported that there are significant variations in the production of HMGB1 after chemo-radiation procedures with ESCC cell lines; moreover, almost the same numbers of stressed cells have had no influence on this finding. Still, these studies have reported that the HMGB1-induced immune responses after chemo-radiation procedures may significantly affect the status of ESCC patients [17].
Cancer patients with tumors of gastric, lung, rectal and esophageal cancers are treated with chemo-radiotherapy, and the outcomes of treatment are patient-specific. For example, the same treatment may yield a positive result for some patients, but only elicit weak responses from others. In cases where immunogenic cell death mechanisms have been detected after chemotherapy, treatments might have prognostic value, having the ability to predict the chemotherapy’s success with the help of respective measurements of HMGB1 and CRT. Another application would be that ICD may enhance therapeutic benefits by combining ICD with therapies that activate the immune system of patients [18].
As pointed out previously, the exposure of calreticulin dictates the immunogenicity of the death of cancer cells. ICD, on the other hand, leads to changes in the composition of cell surfaces occurring in temporal sequences, leading to the stimulation and presentation of tumor antigens [31]. Currently, ICD is being studied as a potential benefit for the treatment of colon cancer [22]. Research findings related to treating cancer with the help of ICD, such as the studies of the American Cancer Society between 2011 and 2013, shows that the actual types of treatment of colon cancer have not changed significantly [19]. Even taking into consideration that the number of previously mentioned analytical and practical studies reported the positive use of ICD in treating multiple types of cancer, during that period, the most general method of treatment for colon cancer was surgery and chemotherapy, depending on the status of cancer and the patient [19].
In particular, in colon cancer, during the carcinoma in situ stage of cancer, the growth of abnormal cells is eliminated by a colonoscopy or polypectomy. Segments of the colon resection are performed in cases of significant sizes of tumors. During the local stage of cancer, surgical resection is used for tumor removal. In cases where cancer has not reached the lymph nodes, corresponding surgical resection of the colon’s segment that contains the tumor may be the only treatment needed. If the cancer is suspected to emerge in the colon again, chemotherapy or radiotherapy could also be applied. Otherwise, once cancer reaches the lymph nodes, all the measures as mentioned above are performed to eliminate cancer; the difference is that the infected lymph nodes are also removed with the tumor. As for chemotherapy, it is based on fluorouracil (5-FU), which is an active drug to improve patients’ survival and reduce the recurrence of colon cancer [20]. In cases of considerably high tumor growth rates, radiotherapy could also be applied.
Often, surgery is aimed to remove colon blockage to prevent further complications during cancer metastasis. Still, during this stage, surgery is not recommended for any patient. Instead, radiotherapy, chemotherapy or biologically targeted procedures should be given to prolong the patient’s survival along with providing symptom relief. Recently, three monoclonal antibody therapies have been approved by the US Food and Drug Administration (FDA) to provide treatment for metastatic colon cancer. For example, Avastin (bevacizumab) have been used to block the blood flow to the specified tumor. Erbitux (cetuximab) and Vectibix (panitumumab) have been used to eliminate the effects of hormone-like factors that influence colon cancer. Nevertheless, some cancer patients with aggressive tumors have had no benefited from these drugs [21].
Recent studies further show that apoptosis can only be immunogenic under certain conditions. However, immune effector cells during the pre-apoptotic stage can attack pre-apoptotic tumor cells. Segments derived from the damaged DNA damage can also produce signals that can lead to the immunogenicity of the tumor cell, such as p53 activation. ER elements such as eukaryotic initiation factor 2A (EIF2a) phosphorylation produce similar signals as well. Tumor cells that are responding to chemotherapy, therefore, release immune-stimulatory factors that stimulate innate immune effector cells. It is therefore anticipated that the mechanisms governing the immunogenicity of cell death will have a significant impact on the design of anticancer therapies [34]. It is clear that the previously widespread methods of treating colon cancer have been treated with the help of surgeries and chemotherapies or radiation treatments that also could not guarantee a complete recovery from cancer. Thus, the following sections of this paper will consider how ICD could improve the situation with cancer treatments as well as the survivability of cancer patients.
The Benefits of ICD in the treatment of colon cancer
While there are numerous advantages of using ICD in the treatment of tumors in cancer patients, one of the most significant benefits of using ICD is that they play a substantial role in restoring cellular calreticulin (CRT). This function is important as it precedes cell apoptosis and is a vital sign of knowing when the cell death is about to commence. This feature is particularly important in the treatment of cancer as studies show that when tumor cells are dying, they tend to instigate cellular anticancer immune response by directly exposing the calreticulin (CRT) (the cell ERp57 complex on the surface of the tumor cell. This innate cellular response against tumor cells is the basis for chemotherapeutic interventions of selective killing of malignant tissues and cells and containing the spread of the diseases in the host’s body.
Another benefit of using ICD in the treatment of cancers is that it boosts the manufacture and secretion of ATP molecules in the cells. They also excite the release of HMGB1 in the cell [22]. The death of cancer cells depends on the initial stimulus. In the cells, the initiating stimulus is of two types: immunogenic stimulus and non-immunogenic stimulus. Immunogenic stimulus results in immunogenic cell death and involves altering biochemical compositions on cell surfaces to release chemical mediators. This kind of cell death occurs in a defined temporal sequence within the cell. It is typically induced by the presence of pathogen-associated molecular pattern (PAMPs). The biomedical implications of the above benefits suggest that cancer treatment is only optimal when the drugs or chemotherapy administered stimulates ICD on the targeted cell. ICD, in turn, initiates a bodily response that functions to control and eliminate residual tumor parts. They also show that the failure of the targeted tumors to emit any immunogenic response from the cell such as CRT exposure can render the anti-cancer treatment ineffective. This immunogenic response against cancers lead primarily to the translocation of endo-CRT (endogenous CRT) to the surface of the cell (ecto-CRT) and is the mechanisms through which anticancer cytostatic agents such as anthracyclines.
Unlike non-immunogenic cellular death, immunogenic apoptosis of tumors happens by inducing sufficient cell response by activating both the dendritic cells (DCs) and T Cells [22]. This process occurs when the anticancer agents such as anthracycline cause stress to the Endoplasmic Reticulum (ER) leading the stressed ER to react by producing rationed portions of Cellular Reactive Oxygen Species (ROSs) that is essential for initiating ICD. In immunogenic cellular response, the process is usually characterized by the presence of DAMPs – Damage-Associated Molecular Patterns key of which are the Calreticulin (CRT), Heat-Shock Proteins (HSPs), and amphoterin (HMGB1) and ATP.
Different from immunogenic cell death, non-immunogenic cell death is caused by a non-immunogenic stimulus and is physiological in nature. This type of cell death is usually characterized by modifications of the cytoplasmic organelles and the reduction of cellular volume. It is caused by the cell’s ability to recognize the presence of Pathogen Associated Molecular Patterns (PAMPs) and DAMPs in the host’s system and the ensuing cellular reaction to them [22[. This responsive attitude is one of the ways other the immunogenicity that cells deploy against tumors and to protect the unaffected cells from infection. These foreign particles once in the body induce an immune response from the cellular antigens and determine the cellular engulfment of the identified apoptotic bodies. Medical observation shows that non-immunogenic cell death occurs through rounding-up of the affected cell, retraction of cell pseudopods, karyorrhexis (the fragmentation of the nucleus of the cell), and alterations in the cell plasma membrane. These cellular non-immunogenic responses are triggered by intrinsic or extrinsic immune response and through cellular stimulation with or without caspase activation.
Like in tolerogenic cell death, ICD also leads to changes and the release of cellular biochemical mediators. However, this series of events occurs in particular sequences. Firstly, the cells release tumor antigens. Secondly, the antigens are presented to the T Cells. Thirdly, the T Cells become stimulated by dendritic cell receptors. Lastly, signals produced by the surface of the cell regulates the after-stimulation activities of the dendritic cells. Through such a process, studies show that ICD constitutes one of the crucial cellular pathways in the body responsible for the activation of the immune system in cancerous cells [32]. Because of its complex path, the ICD model is used in most cancer therapies. Because of the adverse impacts of chemotherapy in the treatment of cancer, scientists have devised some ways to reduce these effects. One such strategy is the restoration of the depleted CRT exposed during chemo sessions through the co-administration of EIF2A phosphatase and its associated regulatory subunit 15A using inhibitors protein phosphatase 1 (PP1) [32]. These proteins have the ability to translocate CRTand and thus, allow the phosphorylation of EIF2A to the surface of the cell from the ER lumen martens regardless of overt ER stress [22,32]. CT26 cells, a colon carcinoma cell line, have been treated with etoposide (a chemotherapy drug), as it is used for a variety of malignant tumors. This drug is a topoisomerase II inhibitor that alone shows CRT exposure, but combined with other inhibitors, such as salubrinal and calyculin A, results in CRT exposure [22].
Such co-administration promotes the phosphorylation of EIF2A, which has high potential in recovering regulated death cell immunogenicity resulted from anti-cancer agents which do not traditionally stimulate CRT exposure but lead to an increased secretion of ATP and HMGB1 release. Moreover, it has been shown that the inability to stimulate CRT’s translocation to the plasma membrane’s outer leaflet can be corrected with the help of administering recombinant and exogenous CRT. Moreover, in vitro, its absorption on malignant cells attached to cells that are not immunogenic has been reported as completely restoring the ability of these cells to provide an effective treatment [22].
It is crucial to know that for ICD to be effective, it requires ATP stimulation to restore its levels of concentrations. While the reaction of anthracycline some amounts of ATP are secreted by the CT26, these molecules lack vital components such as Beclin1, Atg5 or Atg7 that are necessary for the cell autophagy [23]. Nevertheless, this shortcoming can be fixed with the help of ARL67156 co-administration, in other words, the administration of extracellular nucleotidase inhibitors. These inhibitors work by constituting favorable conditions required for the further stimulation of the regulated cell death (RCD) in ATP absent environments. Clinical studies show that chemotherapeutic agents are only able to fight cancer cells by inducing the cell’s Immunogenic Cell Death (ICD) activities by converting the targeted tumor into the vaccine that stimulates intracellular (Anti-cancer) body response against the residual cancer cells. This activity is the mechanism through which anthracyclines work against cancer in the body – their ability to induce ICD [23]. Therefore, biological defects that hamper cellular recognition of the ICD-related inducers such as the Danger Associated Molecular Pattern (DAMPs) by the host cell can actively reduce the efficacy of ICD-dependent chemotherapy. Similarly, particular defects in the targeted cancer cells that in any way can lead to their incapacity to produce ICD-related DAMPs can also cause cell-restorative failure. Thus according to Michaud et al. (2014), tumor cells unable to mount proper premoterm autophagic response cannot secret the required ATP molecules. This innate inability also means that such cells cannot also draw the myeloid cells into the tumor surface after chemotherapy procedures. As a result of this, body tumor cells lacking the essential autophagy genes such as Atg5 or Atg7 or from which these necessary genes have been deleted show clinical symptoms of a reduced cell anticancer immune response capacity and growth levels to ICD-induced chemotherapy treatment [23].
Several studies have been performed to ascertain the role of the above Autophagy-dependent anticancer cell response in cancer therapies. One such study is the 2014 Michaud et al.’s study in which tumor cells obtained from a mouse was subjected to a series of chemotherapeutic treatments. Firstly, the cells were treated with the ICD induced in vitro until approximately 70% of the study cells showed signs of apoptosis [23]. The cells were then cleaned and subcutaneously injected into the selected histocompatible and immunocompetent study mice and left for one week after which they then injected with a second dose of tumor cells into their contralateral flank. After close monitoring and observation, the mice showed no sign of tumor growth. This study results showed that the dying cancer cells provided a protective anticancer immune response against the second dose thus containing and subsequently eliminating the tumor [23]. Secondly, an immunodeficient cancer suffering mice were treated with the ICD inducer in vivo with the study results showing active immune response against the tumor after the ICD induction. This results showed that the therapeutic effects of cancer drugs and treatment are in large part due to the active contribution of the host immune system [23]. Such clinical observation shows that anticancer chemotherapies do induce not only cytostasis (growth arrest) and cytotoxicity (death) of targeted tumor cells but also stimulate an anticancer immune response from the host in the treatment of cancer. Consequently, successful ICD-induced chemotherapies initiate cellular immunosurveillance mechanisms that allow the host’s immune system to fight the tumors.
In the tradition administration of anthracycline, the associated drug’s autophagy pharmacological activation does not fulfill the requirements for cancer cell immunogenicity. In conditions where anti-cancer agents are combined with molecules that perform the up-regulation of autophagic flux, RCD may convert to ICD with high levels of proximity [23]. This effect triggers a cytotoxic response that further acts as a trigger for the host immune system. Research findings on the role of RCD further show that it provokes the release of Type I IFN in the cell, which plays a crucial role in ICD [23,24]. Studies also show that the mechanism of cancer response to the different types of anthracyclines is through three paths, the TLR3-elicited signal transduction, the paracrine type I IFN signaling, and the secretion of chemokine ligand 10 [24]. Therefore, when IFN or TLR3 is not present (IFNAR1−/− and TLR3−/−) in the target cell, exposure to anthracyclines are not able to act as treatment since there is no effect. Nevertheless, the inability of these cells to perform ICD is also possible to correct with the co-administration of recombinant CXCL10 (motif chemokine 10) or recombinant type I IFNs. Therefore, with the help of these reactions, even non-immunogenic cells could turn immunogenic [24]. However, more clinical and experimental evidence for these reactions is also required to understand better this.
Radiation therapy increases the secretion of HMGB1 and thus induces ICD. In colorectal cancer patients, the levels of HMGB1 in serum following radiation therapy are increased. Chemo-radiation is known to increase antigen-specific responses [25], thus, the interaction of HMGB1 and its receptors has a role in promoting immunogenic cell death following radiation therapy in colorectal cancer patients. This interaction can be coupled with radiation therapy for the treatment of cancer [18]. HMGB1’s expression levels vary significantly depending on the type of tumor, which may also control tumor progression. For example, some malignant cells are known to not only release an insufficient amount of HMGB1 but also release HMGB1 that is incompatible with RAGE and TLR4 activation in immune cells [26]. In humans, colon cancer cells have been shown to secrete ATP, which provides CRT exposure with type I IFN production but show minimal levels of HMGB1 release. This mechanism of action has also been shown not to be able to induce an adequate immune response. Therefore, HMGB1 drives ICD. However, further research is still needed to enhance the current ICD mechanisms not only for colon cancer but also for other types of cancers as well.
RAGE: HMGB1 interaction as evidence for treating colon cancer
In a study by Fahmueller et al. [25], ICD and RAGE implications were shown specifically for the treatment of colon cancer [25]. These scientists chose 49 patients with colorectal cancer who also had liver metastases. The patients received treatment in the form of radioembolization (RE) for two years. Additionally, all patients had undergone surgical resection along with adjuvant chemotherapy, and all had liver metastases [25]. During the treatment procedure of RE therapy and after it, patients did not receive any additional cytotoxic or cytostatic therapy [25]. Fahmueller et al. added CYFRA 21-1, CA 19-9 and CEA tests to the patients to take into consideration nucleosome activity in the liver. These analyzes included C-reactive protein (CRP), lactate dehydrogenase (LDH), alanine aminotransferase (ALT), aspartate aminotransferase (AST), choline, lipase and amylase esterase and alkaline phosphatase (AP) due to their prognostic abilities in univariate analyzes [25].
Fahmueller et al. collected blood samples 24 hours before RE and 48 hours after receiving RE. They found that the concentration of HMGB1 was evaluated for each patient according to a sandwich ELISA method. Sandwich ELISA also evaluated the RAGE levels of the patients. HMGB1 serum concentrations were increased significantly in 24 hours after RE returning to pre-therapeutic levels 48 hours later. On the contrary, the levels of RAGE were decreased in both 24 and 48 hours after RE, with the activity of DNase remaining nearly at stable levels [25]. Before receiving RE, there was a correlation between HMGB1 and CEA, but not with nucleosomes, the activity of DNAse, or RAGE. In 24 hours after receiving RE, there was a high correlation of HMGB1 and the nucleosomes of cell death biomarkers. Additionally, post-RE correlated with CEA and CYFRA 21-1, but did not correlate with DNAse or RAGE. The same correlations have been registered for HMGB1 with CEA and with nucleosomes 48 hours after RE [25].
RE responses after three months led to only 14 patients showing either some levels of remission or no changes in their disease. The rest of them were reported as deceased or developed progressive disease either in the liver or other organs. [25]. Analyzing these ICD markers, the HMGB1 levels before therapy have been reported as considerably higher for non-responsive patients in comparison to responsive patients. The levels of DNAse and RAGE, however, have not been reported to significant change in this patient pool. However, the radiologic response of the patients to therapy significantly correlated with their time of survival. Moreover, it has been reported that high levels of serum HMGB1 were closely related to low survivability of the patients with the rest of the parameters showing no significant association [25]. Thus, it can be concluded that HMGB1 has been proved an important marker for the prognosis of colorectal cancer. Moreover, Fahmueller et al. have stated that HMGB1 levels along with CRP in the liver at 24 hours post-RE were the most effective predicting model.
Conclusion
HMGB1 is a promising marker that has been reported to stimulate significantly immune system responses against malignant tissues. In colon cancer particularly, HMGB1 has been shown to invade cancer tissue and create vital cancer-fighting metastases. Additionally, RAGE appears to be a relevant receptor for stimulating the immune response. It plays significant roles in the control of tumor cell invasiveness. However, research is still wanting in the understanding of RAGE in cancer cell marking. In Fahmueller et al.’s study, all the participants used were in the late stages of their disease and hence had lower rates of survivability. However, high disease progress was seen. For that reason, another study should be conducted on that field using patients at earlier stages of cancer this time. This undertaking could help confirm the effectiveness of the biomarkers for prognosis. Even though ICD has been presented in the discussion above as potentially beneficial aspect of treating cancer, especially colon cancer, it is imperative to note that additional research is still required on the matter because it has only been used in a clinical trial only. Nevertheless, the research findings on the prognostic and preventive value of ICD such as RAGE, HMGB1, and DNase suggests that the potential of these ICD markers in treating cancer should not be under-estimated.
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