Restoration of antitumor immunity through selective inhibition of myeloid derived suppressor cells by anticancer therapies

Accumulating evidence suggests that the success of some anticancer therapies not only relies on their direct cytotoxicity on tumor cells but also on their ability to promote anticancer immune responses. However, immunosuppressive cells such as Myeloid Derived Suppressor Cells (MDSC) that are generated during tumor progression blunt antitumor immune responses and thus represent a major obstacle to the clinical implementation of immunotherapy protocols. We have recently identified 5-Fluorouracil (5-FU) as an anticancer agent that selectively induced MDSC apoptotic cell death in vitro and in vivo. The elimination of MDSC by 5-FU increased IFNγ secretion by tumor specific CD8(+) T cells infiltrating the tumor and promoted T-cell dependent antitumor responses in vivo, suggesting that some anticancer therapies can reverse tumor-mediated immunosuppression. Here, we review the molecular pathways leading to the induction of MDSC in cancer and discuss how different anticancer agents successfully target these cells in vivo, thereby restoring potent anticancer immunity.     

Molecular targets and signaling pathways regulated by interleukin (IL)-24 in mediating its antitumor activities

Cancer remains a major health issue in the world and the effectiveness of current therapies is limited resulting in disease recurrence and resistance to therapy. Therefore to overcome disease recurrence and have improved treatment efficacy there is a continued effort to develop and test new anticancer drugs that are natural or synthetic - (conventional chemotherapeutics, small molecule inhibitors) and biologic (antibody, tumor suppressor genes, oligonucleotide) product. In parallel, efforts for identifying molecular targets and signaling pathways to which cancer cells are “addicted” are underway. By inhibiting critical signaling pathways that is crucial for cancer cell survival, it is expected that the cancer cells will undergo a withdrawal symptom akin to “de-addiction” resulting in cell death. Thus, the key for having an improved and greater control on tumor growth and metastasis is to develop a therapeutic that is able to kill tumor cells efficiently by modulating critical signaling pathways on which cancer cells rely for their survival.Currently several small molecule inhibitors targeted towards unique molecular signaling pathways have been developed and tested in the clinic. Few of these inhibitors have shown efficacy while others have failed. Thus, targeting a single molecule or pathway may be insufficient to completely block cancer cell proliferation and survival. It is therefore important to identify and test an anticancer drug that can inhibit multiple signaling pathways in a cancer cell, control growth of both primary and metastatic tumors and is safe.One biologic agent that has the characteristics of serving as a potent anticancer drug is interleukin (IL)-24. IL-24 suppresses multiple signaling pathways in a broad-spectrum of human cancer cells leading to tumor cell death, inhibition of tumor angiogenesis and metastasis. Additionally, combining IL-24 with other therapies demonstrated additive to synergistic antitumor activity. Clinical testing of IL-24 as a gene-based therapeutic for the treatment of solid tumors demonstrated that IL-24 is efficacious and is safe. The unique features of IL-24 support its further development as an anticancer drug for cancer treatment.In this review we summarize the current understanding on the molecular targets and signaling pathways regulated by IL-24 in mediating its anticancer activity.     

Chemotherapy induces ATP release from tumor cells

Chemotherapy can induce anticancer immune responses. In contrast to a widely extended prejudice, apoptotic cell death is often more efficient in eliciting a protective anticancer immune response than necrotic cell death. Recently, we have found that purinergic receptors of the P2X7 type are required for the anticancer immune response induced by chemotherapy. ATP is the endogenous ligand that has the highest affinity for P2X7. Therefore, we investigated the capacity of a panel of chemotherapeutic agents to induce ATP release from cancer cells. Here, we describe that multiple distinct anticancer drugs reduce the intracellular concentration of ATP before and during the manifestation of apoptotic characteristics such as the dissipation of the mitochondrial transmembrane potential and the exposure of phosphatidylserine residues on the plasma membrane. Indeed, as apoptosis progresses, intracellular ATP concentrations decrease, although even advanced-stage apoptotic cells still contain sizeable ATP levels. Only when cells enter secondary necrosis, the ATP concentration falls to undetectable levels. Concomitantly, a wide range of chemotherapeutic agents causes the release of ATP into the extracellular space as they induce tumor cell death. Hence, ATP release is a general correlate of apoptotic cell death induced by conventional anticancer therapies.     

Transformation by oncogenic RAS sensitizes human colon cells to TRAIL-induced apoptosis by up-regulating death receptor 4 and death receptor 5 through a MEK-dependent pathway

RAS oncogenes play a major role in cancer development by activating an array of signaling pathways, most notably mitogen-activated protein kinases, resulting in aberrant proliferation and inhibition of apoptotic signaling cascades, rendering transformed cells resistant to extrinsic death stimuli. However, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is able to kill specific tumor cells through the engagement of its receptors, death receptor 4 (DR4) and death receptor 5 (DR5), and the activation of apoptotic pathways, providing promising targets for anticancer therapies. In this study, we show that TRAIL induces cell death in human colon adenocarcinoma cells in a MEK-dependent manner. We also report a prolonged MEK-dependent activation of ERK1/2 and increased c-FOS expression induced by TRAIL in this system. Our study reveals that transformation of the colon cell line Caco-2 by Ki- and mainly by Ha-ras oncogenes sensitizes these cells to TRAIL-induced apoptosis by causing specific MEK-dependent up-regulation of DR4 and DR5. These observations taken together reveal that RAS-MEK-ERK1/2 signaling pathway can sensitize cells to TRAIL-induced apoptosis by up-regulating DR4 and DR5 and overall imply that TRAIL-based therapeutic strategies using TRAIL agonists could be used in cases of human colon cancers bearing RAS mutations.     

Exploiting death receptor signaling pathways for tumor therapy

Apoptosis or programmed cell death is a key regulator of physiological growth control and regulation of tissue homeostasis. Tipping the balance between cell death and proliferation in favor of cell survival may result in tumor formation. Moreover, current cancer therapies, e.g. chemotherapy, gamma-irradiation, immunotherapy or suicide gene therapy, primarily exert their antitumor effect by triggering an evolutionary conserved apoptosis program in cancer cells. For example, death receptor signaling has been implied to contribute to the efficacy of cancer therapy. Thus, failure to undergo apoptosis in response to anticancer therapy because of defects in death receptor pathways may result in resistance. Further insights into the mechanisms regulating apoptosis in response to anticancer therapy and how cancer cells evade cell death may provide novel opportunities for targeted therapeutics. Thus, agents designed to selectively activate death receptor pathways may enhance the efficacy of conventional therapies and may even overcome some forms of cancer resistance.     

Cell death in hematological tumors

Evasion of apoptosis is a hallmark of human cancers, for example in hematological malignancies. Apoptosis is an intrinsic cell death program that is crucial to maintain tissue homeostasis, for example in the hematopoietic system where there is a high turnover rate of cells. As a result, a decrease in the rate of apoptosis besides an increase in proliferation favors tumorigenesis as well as tumor progression. Further, the anti-leukemic action of current treatment approaches, including chemo-, radio- or immunotherapy, critically relies on intact cell death programs in cancer cells. Therefore, defects in apoptosis pathways are frequently associated with the resistance to anticancer therapies. In recent years, the identification and characterization of the molecules and pathways that are involved in the regulation and execution of cell death in leukemia and lymphoma cells have set the ground for the development of novel diagnostic tools and molecular therapeutics targeting apoptosis pathways in hematological malignancies.     

Lysosomes and lysosomal proteins in cancer cell death (new players of an old struggle)

Death of cancer cells influences tumor development and progression, as well as the response to anticancer therapies. This can occur through different cell death programmes which have recently been shown to implicate components of the acidic organelles, lysosomes. The role of lysosomes and lysosomal enzymes, including cathepsins and some lipid hydrolases, in programmed cell death associated with apoptotic or autophagic phenotypes is presented, as evidenced from observations on cultured cells and living animals. The possible molecular mechanisms that underlie the action of lysosomes during cell death are also described. Finally, the contribution of lysosomal proteins and lysosomes to tumor initiation and progression is discussed. Elucidation of this role and the underlying mechanisms will shed a new light on these 'old' organelles and hopefully pave the way for the development of novel anticancer strategies.     

Selective anticancer strategies via intervention of the death pathways relevant to cell transformation

Apoptosis is an important physiological process that promotes tissue homeostasis by eliminating unnecessary or malfunctioning cells. Abnormality in this process contributes to tumorigenesis, as well as the resistance to cancer treatment by radiation and chemotherapy. Restoration of normal apoptosis would not only promote cancer cell death and halt tumor progression, but also increase the response to many current cancer therapies. Although apoptosis induction is an important principle of currently used radiation and chemotherapy treatment, uncovering the mechanisms that govern this process, and which are lost during transformation, represents an important direction for realizing improved therapies for the future. This article first briefly reviews aspects of current discovery strategies for new anticancer therapeutics based on intervening in cell death pathways, and then discusses in more detail several cancer-relevant death pathways, which are disabled during transformation and which can be targeted therapeutically. These include anoikis/cell adhesion; energy metabolism and the unfolded protein response. Finally, we introduce a new concept, which utilizes cancer-specific apoptosis induced by oncolytic viruses. The discussion of these topics involves novel targets, compounds and virotherapy.     

Endoplasmic reticulum stress,autophagic and apoptotic cell death,and immune activation by a natural triterpenoid in human prostate cancer cells

Though the current therapies are effective at clearing an early stage prostate cancer, they often fail to treat late-stage metastatic disease. We aimed to investigate the molecular mechanisms underlying the anticancer effects of a natural triterpenoid, ganoderic acid DM (GA-DM), on two human prostate cancer cell lines: the androgen-independent prostate carcinoma (PC-3), and androgen-sensitive prostate adenocarcinoma (LNCaP). Cell viability assay showed that GA-DM was relatively more toxic to LNCaP cells than to PC-3 cells (IC₅₀s ranged 45-55 µM for PC-3, and 20-25 µM for LNCaP), which may have occurred due to differential expression of p53. Hoechst DNA staining confirmed detectable nuclear fragmentation in both cell lines irrespective of the p53 status. GA-DM treatment decreased Bcl-2 proteins while it upregulated apoptotic Bax and autophagic Beclin-1, Atg5, and LC-3 molecules, and caused an induction of both early and late events of apoptotic cell death. Biochemical analyses of GA-DM-treated prostate cancer cells demonstrated that caspase-3 cleavage was notable in GA-DM-treated PC-3 cells. Interestingly, GA-DM treatment altered cell cycle progression in the S phase with a significant growth arrest in the G2 checkpoint and enhanced CD4 ⁺ T cell recognition of prostate tumor cells. Mechanistic study of GA-DM-treated prostate cancer cells further demonstrated that calpain activation and endoplasmic reticulum stress contributed to cell death. These findings suggest that GA-DM is a candidate for future drug design for prostate cancer as it activates multiple pathways of cell death and immune recognition.     

Celastrol inhibits growth and induces apoptotic cell death in melanoma cells via the activation ROS-dependent mitochondrial pathway and the suppression of PI3K/AKT signaling

Celastrol has been reported to possess anticancer effects in various cancers; however, the precise mechanism underlying ROS-mediated mitochondria-dependent apoptotic cell death triggered by celastrol treatment in melanoma cells remains unknown. We showed that celastrol effectively induced apoptotic cell death and inhibited tumor growth using tissue culture and in vivo models of B16 melanoma. In addition to apoptotic cell death in B16 cells, several apoptotic events such as PARP cleavage and activation of caspase were confirmed. Pretreatment with caspase inhibitor modestly attenuated the celastrol-induced increase in PARP cleavage and sub-G1 cell population, implying that caspases play a partial role in celastrol-induced apoptosis. Moreover, ROS generation was detected following celastrol treatment. Blocking of ROS accumulation with ROS scavengers resulted in inhibition of celastrol-induced Bcl-2 family-mediated apoptosis, indicating that celastrol-induced apoptosis involves ROS generation as well as an increase in the Bax/Bcl-2 ratio leading to release of cytochrome c and AIF. Importantly, silencing of AIF by transfection of siAIF into cells remarkably attenuated celastrol-induced apoptotic cell death. Moreover, celastrol inhibited the activation of PI3K/AKT/mTOR signaling cascade in B16 cells. Our data reveal that celastrol inhibits growth and induces apoptosis in melanoma cells via the activation of ROS-mediated caspase-dependent and -independent pathways and the suppression of PI3K/AKT signaling.     

Life or death? Autophagy in anticancer therapies with statins and histone deacetylase inhibitors

Autophagy, which could be either cytoprotective or cytotoxic, is often observed in tumor cells in response to chemotherapy. Understanding the signaling pathways involved in the regulation of autophagy represents a new direction in the development of anticancer therapies. Our recent studies investigate the role of autophagy in the anticancer effects of statins and histone deacetylase: inhibitors (HDACi). Inhibition of autophagy enhances the efficacy of statins, whereas autophagic cell death in cancer cells is induced by HDACi. Here, we will discuss the differential signaling pathways elicited by statins and HDACi. We find that AMPK, cytoplasmic p21 and Akt signaling are crucial determinants of cell fates (i.e., life or death) while autophagy is induced.     

HTLV-1 and apoptosis: role in cellular transformation and recent advances in therapeutic approaches

A universal cellular defense mechanism against viral invasion is the elimination of infected cells through apoptotic cell death. To counteract host defenses many viruses have evolved complex apoptosis evasion strategies. The oncogenic human retrovirus HTLV-1 is the etiological agent of adult-T-cell leukemia/lymphoma (ATLL) and the neurodegenerative disease known as HTLV-associated myelopathy/tropical spastic paraparesis (HAM/TSP). The poor prognosis in HTLV-1-induced ATLL is linked to the resistance of neoplastic T cells against conventional therapies and the immuno-compromised state of patients. Nevertheless, several studies have shown that the apoptotic pathway is largely intact and can be reactivated in ATLL tumor cells to induce specific killing. A better understanding of the molecular mechanisms employed by HTLV-1 to counteract cellular death pathways remains an important challenge for future therapies and the treatment of HTLV-1-associated diseases.     

Interconnections between apoptotic,autophagic and necrotic pathways: implications for cancer therapy development

The rapid accumulation of knowledge on apoptosis regulation in the 1990s was followed by the development of several experimental anticancer‐ and anti‐ischaemia (stroke or myocardial infarction) drugs. Activation of apoptotic pathways or the removal of cellular apoptotic inhibitors has been suggested to aid cancer therapy and the inhibition of apoptosis was thought to limit ischaemia‐induced damage. However, initial clinical studies on apoptosis‐modulating drugs led to unexpected results in different clinical conditions and this may have been due to co‐effects on non‐apoptotic interconnected cell death mechanisms and the ‘yin‐yang’ role of autophagy in survival versus cell death. In this review, we extend the analysis of cell death beyond apoptosis. Upon introduction of molecular pathways governing autophagy and necrosis (also called necroptosis or programmed necrosis), we focus on the interconnected character of cell death signals and on the shared cell death processes involving mitochondria (e.g. mitophagy and mitoptosis) and molecular signals playing prominent roles in multiple pathways (e.g. Bcl2‐family members and p53). We also briefly highlight stress‐induced cell senescence that plays a role not only in organismal ageing but also offers the development of novel anticancer strategies. Finally, we briefly illustrate the interconnected character of cell death forms in clinical settings while discussing irradiation‐induced mitotic catastrophe. The signalling pathways are discussed in their relation to cancer biology and treatment approaches.     

The selective inhibition of nuclear PKCζ restores the effectiveness of chemotherapeutic agents in chemoresistant cells

The atypical protein kinase C (PKC) isoform zeta (PKCζ) has been implicated in the intracellular transduction of mitogenic and apoptotic signals by acting on different signaling pathways. The key role of these processes in tumorigenesis suggests a possible involvement of PKCζ in this event. PKCζ is activated by cytotoxic treatments, inhibits apoptotic cell death and reduces the sensitivity of cancer cells to chemotherapeutic agents. Here, using pharmacological and DNA recombinant approaches, we show that oxidative stress triggers nuclear translocation of PKCζ and induces resistance to apoptotic agents. Accordingly, chemoresistant cells show accumulation of PKCζ within the nucleus, and a nuclear-targeted PKCζ transfected in tumor cells decreases sensitivity to apoptosis. We thus developed a novel recombinant protein capable of selectively inhibiting the nuclear fraction of PKCζ that restored the susceptibility to apoptosis in cells in which PKCζ was enriched in the nuclear fraction, including chemoresistant cells. These findings establish the importance of PKCζ as a possible target to increase the effectiveness of anticancer therapies and highlight potential sites of intervention.     

Caspase-driven cancer therapies: Navigating the bridge between lab discoveries and clinical applications

Apoptosis is the cell's natural intrinsic regulatory mechanism of normal cells for programmed cell death, which plays an important role in cancer as a classical mechanism of tumor cell death causing minimal inflammation without causing damage to other cells in the vicinity. Induction of apoptosis by activation of caspases is one of the primary targets for cancer treatment. Over the years, a diverse range of natural, synthetic, and semisynthetic compounds and their derivatives have been investigated for their caspase-mediated apoptosis-induced anticancer activities. The review aims to compile the preclinical evidence and highlight the critical mechanistic pathways related to caspase-induced cell apoptosis in cancer treatment. The focus is placed on the key components of the mechanisms, including their chemical nature, and specific attention is given to phytochemicals derived from natural sources and synthetic and semisynthetic compounds. 180+ compounds from the past two decades with potential as anticancer agents are discussed in this review article. By summarizing the current knowledge and advancements in this field, this review provides a comprehensive overview of potential therapeutic strategies targeting apoptosis in cancer cells. The findings presented herein contribute to the ongoing efforts to combat cancer and stimulate further research into the development of effective and targeted anticancer therapies.     

Targeting FcαRI on polymorphonuclear cells induces tumor cell killing through autophagy

Neutrophils are the most abundant circulating FcR-expressing WBCs with potent cytotoxic ability. Currently, they are recognized as promising effector cells for Ab-mediated immunotherapy of cancer, because their capacity to kill tumor cells is greatly enhanced by tumor Ag-specific mAbs. The FcαRI represents the most potent FcR on neutrophils for induction of Ab-mediated tumor cell killing. However, the mechanisms of cell death that are induced are poorly understood. Because these mechanisms can be used for modulation of anticancer treatment, we investigated the tumor cell death induced by neutrophil-mediated Ab-dependent killing via FcαRI. Human mammary carcinoma cells were efficiently killed when incubated with human neutrophils and tumor-specific FcαRI bispecific or IgA Abs. Interestingly, we observed characteristics of autophagy such as autophagic structures by electron microscopy and LC3B(+) autophagosomes in different human epithelial carcinoma cells, which resulted in tumor cell death. To a lesser extent, necrotic features, such as cellular membrane breakdown and spillage of intracellular content, were found. By contrast, apoptotic features including fragmented nuclei, Annexin V-positivity, and presence of cleaved caspase-3 were not observed. These findings indicate that neutrophils mainly facilitate autophagy to induce tumor cell death rather than the more commonly recognized apoptotic cell death mechanisms induced by NK cells or cytotoxic T cells. This knowledge not only reveals the type of tumor cell death induced in neutrophil-mediated, Ab-dependent cellular cytotoxicity, but importantly opens up additional perspectives for modulation of anticancer therapy in, for example, apoptosis-resistant tumor cells.     

Cancer cells stemness: A doorstep to targeted therapy

Recent advances in research on cancer have led to understand the pathogenesis of cancer and development of new anticancer drugs. Despite of these advancements, many tumors have been found to recur, undergo metastasis and develop resistance to therapy. Accumulated evidences suggest that small population of cancer cells known as cancer stem cells (CSC) are responsible for reconstitution and propagation of the disease. CSCs possess the ability to self-renew, differentiate and proliferate like normal stem cells. CSCs also appear to have resistance to anti-cancer therapies and subsequent relapse. The underlying stemness properties of the CSCs are reliant on multiple molecular targets such as signaling pathways, cell surface molecules, tumor microenvironment, apoptotic pathways, microRNA, stem cell differentiation, and drug resistance markers. Thus an effective therapeutic strategy relies on targeting CSCs to overcome the possible tumor relapse and chemoresistance. The targeted inhibition of these stem cell biomarkers is one of the promising approaches to eliminate cancer stemness. This review article summarizes possible targets of cancer cell stemness for the complete treatment of cancer.     

Preferential targeting of apoptosis in tumor versus normal cells

Elimination of cancer cells by early apoptosis is preferred over other forms of cell growth inhibition. Apoptosis directly leads to tumor regression and reduces risks of selecting more aggressive and/or drug-resistant phenotypes that are often responsible for tumor regrowth and treatment failure. Although DNA damage by anticancer drugs is commonly recognized as an apoptotic stimulus, there is enormous variability in the magnitude and timing of such effects. Especially potent and rapid apoptosis seems to be a hallmark of various alkylating anticancer drugs that are regarded as DNA-reactive agents but are observed to react mainly with cellular proteins. Our studies with such dual-action drugs (irofulven, oxaliplatin) suggest that not only DNA damage, but also protein damage, contributes to apoptosis induction. DNA damage is well known to initiate death-signaling pathways leading to mitochondrial dysfunction. Protein damage, in turn, can distort cell redox homeostasis, which facilitates apoptosis execution. Such dual effects can be particularly lethal to tumor cells, which tend to function under pro-oxidative conditions. In contrast to tumor cells that are highly susceptible, normal cells show marginal apoptotic responses to the dual action drugs. This protection of normal cells might reflect their greater ability to buffer pro-oxidative changes and quickly restore redox homeostasis, despite substantial drug uptake and macromolecular binding. Importantly, by targeting the death process at multiple points, DNA- and protein-damaging drugs can be less vulnerable to various bypass mechanisms possible with single targets. The reviewed studies provide a proof of concept that differential apoptosis targeting in cancer versus normal cells can be a basis for tumor selectivity of anticancer drugs.     

Combination treatment with arsenic trioxide and irradiation enhances cell-killing effects in human fibrosarcoma cells in vitro and in vivo through induction of both autophagy and apoptosis

The traditional treatments for fibrosarcoma have limited efficacy. Therefore, new therapeutic strategies and/or new adjuvant drugs still need to be explored. Accumulating evidence indicates that programmed cell death (PCD) is closely related to anticancer therapy. Many studies have shown that tumor cells treated with anticancer drugs experience the induction of type I PCD, apoptosis, and type II PCD, autophagy. In the present study, we investigated the anticancer effects of ionizing radiation (IR) combined with arsenic trioxide (ATO) in human fibrosarcoma cells in vitro and in xenograft tumors in SCID mice in vivo. We found that IR increased the population of HT1080 cells in the G₂/M phase in a time-dependent manner within 9 h. IR treatment combined with ATO at this time point induced a significantly prolonged G₂/M arrest and consequently enhanced cell death. Furthermore, damage of mitochondria membrane potential could be involved in the underlying mechanisms. The enhanced cytotoxic effect of combined treatment occurred due to the increased induction of more autophagy and apoptosis through the inhibition of Akt and the activation of ERK1/2 signaling pathways in HT1080 cells. The combined treatment of HT1080 cells pretreated with Z-VAD or 3-MA resulted in a significant reduction in AO-positive cells, apoptotic cells and cytotoxicity. In in vivo studies, the combination of IR and ATO significantly reduced the tumor volume in SCID mice that had received a subcutaneous injection of HT1080 cells. The data suggest that a combination of IR and ATO could be a new potential therapeutic strategy for the treatment of fibrosarcoma.