Immunomodulatory effects of low dose chemotherapy and perspectives of its combination with immunotherapy

Authors

  • Mariana S. Nars,

    1. Department of Microbiology and Immunology, Institute of Biosciences, UNESP–Univ Estadual Paulista, Botucatu, São Paulo, Brazil
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  • Ramon Kaneno

    Corresponding author
    1. Department of Microbiology and Immunology, Institute of Biosciences, UNESP–Univ Estadual Paulista, Botucatu, São Paulo, Brazil
    2. Department of Pathology, School of Medicine, UNESP–Univ Estadual Paulista, Botucatu, São Paulo, Brazil
    • Departamento de Microbiologia e Imunologia, Instituto de Biociências de Botucatu, UNESP. Cx Postal 510, Distrito de Rubião Jr. s/n, 18618-970, Botucatu, São Paulo, Brazil
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    • Tel.: [551438800432], Fax: +[55-14-38153744]


  • Conflict of interest: Authors declare that there is no conflict of interest.

Abstract

Given that cancer is one of the main causes of death worldwide, many efforts have been directed toward discovering new treatments and approaches to cure or control this group of diseases. Chemotherapy is the main treatment for cancer; however, a conventional schedule based on maximum tolerated dose (MTD) shows several side effects and frequently allows the development of drug resistance. On the other side, low dose chemotherapy involves antiangiogenic and immunomodulatory processes that help host to fight against tumor cells, with lower grade of side effects. In this review, we present evidence that metronomic chemotherapy, based on the frequent administration of low or intermediate doses of chemotherapeutics, can be better than or as efficient as MTD. Finally, we present some data indicating that noncytotoxic concentrations of antineoplastic agents are able to both up-regulate the immune system and increase the susceptibility of tumor cells to cytotoxic T lymphocytes. Taken together, data from the literature provides us with sufficient evidence that low concentrations of selected chemotherapeutic agents, rather than conventional high doses, should be evaluated in combination with immunotherapy.

Abbreviations: BSA: body surface area-dosing; CEP: circulating endothelial precursor cells; CTL: cytolytic T cells; CTX: cyclophosphamide; DCs: dendritic cells; DOX: doxorubicin; LDM: low dose chemotherapy; MDSC: myeloid-derived suppressor cells; MTD: maximum tolerable dose; MTX: methotrexate; PTX: paclitaxel; Treg: regulatory T lymphocytes

According to the most recent epidemiological surveys of the World Health Organization, cancer is the third main cause of death worldwide, just after cardiovascular diseases and infectious and parasitic diseases, being the second ranked disease in most western countries.1 Data from the WHO and the International Agency for Research on Cancer indicates that 12.7 million new cases were reported in 2008 and 7.6 million deaths were attributed to this disease.2

The main types of human tumors comprise breast, lung, prostate and colon cancer and the conventional treatment for cancer includes surgery, chemotherapy and radiotherapy, which can be used alone or in combination with each other. Chemotherapy is the main modality of treatment being administrated both in inoperable cases and as adjuvant treatment following surgery. In fact, more than half of all people diagnosed with cancer receive chemotherapy as a neoadjuvant or adjuvant modality for preoperative or postoperative treatment, respectively.3 In this review, we report the effects of different antineoplastic chemotherapeutic agents on the immune system, emphasizing the immunomodulatory effects of moderate and low concentrations of drugs.

Conventional Chemotherapy

Conventional chemotherapy is based on the maximum tolerated dose (MTD), which corresponds to the highest dose of a drug that presents tolerable side effects. This strategy has been shown to be effective in curing or controlling the disease in a great number of patients but it is associated with significant short- and long-term toxicity and side effects, including myelosuppression, neutropenia, thrombocytopenia, increased risk of infection and bleeding, gastrointestinal dysfunctions, arthralgia, liver toxicity and damage to the cardiac and nervous system.3–5

One of the main features of antineoplastic chemotherapeutic agents is their cytotoxic activity on highly proliferative cells, implying that its effect on proliferating hematopoietic and immune cells leads to the depression of both natural and adaptive defense mechanisms. In fact, the myelosuppressive effect of several drugs is well described.6–10 As recently reviewed by Shurin et al.10 the main immunosuppressive effect of high dose chemotherapy is the depletion of T lymphocytes. Both CD4+ and CD8+ are affected by cytotoxic drugs but the normal levels of peripheral CD4+ cells are more slowly restored than CD8+.11,12 In addition, many commonly used agents cause dysfunction of natural killer (NK)13 and γδT cells,14 impairing the antitumor immune surveillance. Although these cells are involved in the innate immunity, the modulatory effect of paclitaxel (PTX), for instance, seems to be dependent on the fail in their activation by IL-2.15

Dendritic cells are also sensitive to high dose chemotherapy and the effects of several cytotoxic drugs have been reported. In fact, such drugs can interfere on antigen presentation processes,16 reduce cell mobilization17 and downregulate the expression of cell surface markers such as CD80 and CD86.18 Such effects on the immunocompetent cells may enable tumor escape, thus allowing the proliferation of chemo-resistant variants.19,20

To avoid extreme side effects and immunosuppression, administration of cytotoxic drugs are followed by 3–4-week intervals to allow hematopoiesis and restoration of peripheral leukocyte count. However, the low blood levels or absence of medication during these resting intervals can also allow the growth of drug-resistant tumor cell clones. Then, despite an initially impressive tumor regression or even remission following chemotherapy, regrowth or recurrence is quite common in metastatic cancer.21 As a consequence, it is estimated that 45% of cancer patients are unable to control the disease.22

MTD is calculated by the body surface area-dosing (BSA) method, a calculation that can lead to an unpredictable variation in the therapeutic effect, due to either overdosing or underdosing. Overdosing is easily detected due to the increased toxicity, whereas underdosing is more difficult to recognize and may occur in 30% or more of patients receiving standard regimen. Therefore, those underdosed patients are at risk of a significantly reduced anticancer effect of the medication.23 For instance, a survival reduction of almost 20% was observed in breast cancer patients treated with cyclophosphamide (CTX), doxorubicin and 5-fluorouracil at adjuvant BSA-based concentrations.24,25 The authors considered the possibility that a large proportion of these patients had been underdosed, which may have facilitated the development of drug-resistance.

Several mechanisms can be associated with the development of drug resistance including the expression of ATP-binding cassette family transporters, that can exclude cytotoxic drugs from target cells26,27; development of resistance to apoptosis related to Bcl-228,29 and Notch receptor expression30,31 as well as immunosuppression32–34 that can allow the development of “dormant tumor stem cells.” Another important phenomenon for developing drug resistance is the epigenetic changes in tumor cells. For instance, resistance of colorectal tumor cells to 5-fluorouracil (5-FU) is hypothesized to be acquired by DNA hypermethylation. In fact, treatment of 5-FU-resistant colon cancer cells (HCT-8) with low dose of 5-aza-deoxycytidine, prevents DNA hypermethylation and restores their sensitivity to 5-FU.35 This effect was also demonstrated in vivo by treating nude mice bearing 5-FU-resistant tumor cells. Treatment with low doses of AZA overcame the resistance to 5-FU.36 Therefore, there is a great interest in minimizing toxicity while increasing drug efficacy,37 suggesting the necessity for different approaches.

Low Dose Metronomic Chemotherapy

One of the alternative approaches is low-dose metronomic (LDM) chemotherapy, which uses much lower doses of chemotherapeutics and may decrease the disadvantages of conventional cytotoxic chemotherapy.19,20 The difference between this approach and simple underdosing is that the former involves more frequent administration38–40 to maintain the serum levels of antineoplastic activity and to avoid myelosuppression and other side effects. It has attracted attention given that it may reduce the risk of developing drug resistance while reducing the toxic effects.41

One of the main antitumor mechanisms of LDM is the antiangiogenic activity, since endothelial precursor cells are more sensitive to cytotoxic drugs than tumor cells themselves.42,43 Such effect was shown to be enhanced in vivo by combination with antiangiogenic agents. In fact, it was observed in animal models that the association of antineoplastic drugs with antiangiogenic agents such as anti-VEGF, anti-VEGFR, β-cyclodextrin tetradecasulfate, tetrahydrocortisol and endoglin significantly increased the survival of tumor-bearing animals.44–47 The absence of side effects and a visible decrease in tumor size were associated with the treatments.44,48,49 MTD and LDM chemotherapy have opposite effects on the mobilization and viability of circulating endothelial precursor (CEP) cells, with LDM being more effective than MTD at destroying these cells involved in tumor angiogenesis.50

In vitro antiangiogenic activity of LDM docetaxel has also been shown, while its in vivo effectiveness was demonstrated in a murine gastric tumor model. It was observed that it affected microvessel growth without exerting a toxic effect on nude mice.51

Interestingly, looking back at the history of pediatric cancer therapy, several successful approaches could be classified as metronomic chemotherapy but instead are known as “maintenance therapy.”37 For instance, pediatric oncologists have shown that daily mercaptopurine and weekly methotrexate administrations constitute a successful combination against leukemia.20 The main knowledge on LDM is based on the studies with CTX and PTX, but the feasibility of using other medications alone or in combination has also been investigated.

Cyclophosphamide

CTX is an alkylating agent that is converted into its active metabolite, derivative phosphoramide mustard, in the liver. This metabolite inhibits DNA replication by forming interstand and intrastrand DNA crosslinking. Currently, it is the best-known clinical metronomic drug52 and it was observed that LDM CTX increases the frequency of apoptotic CEPs, while high-dose CTX mobilizes viable ones giving a significant contribution to the growth of experimental lymphoma.53 Examples of the clinical use of LDM CTX include the combination of weekly vincristine and low-dose daily CTX that was shown to be effective against neuroblastoma.54 Combination of LDM CTX with celecoxib in heavily pretreated patients contributed to an impressive duration of stable disease with no toxicity,45 whereas this combination is also effective in treating refractory high-grade lymphoma.55 The combination of CTX plus weekly vinblastine and rofecoxib is effective for a subset of Hodgkin's disease patients.56 CTX has also been used with low-dose methotrexate in the treatment of early breast cancer and the inhibition of angiogenesis is caused by a decrease in vascular endothelial growth factor.57

Effects of CTX on the immune response and combination with immunotherapy

Regulatory T cells and myeloid-derived suppressor cells (MDSC) are in higher number in the tumor microenvironment and can facilitate tumor immune escape, since they affect the functioning of effective CTLs and NK cells.58,59 Tumor-bearing mice show an increased prevalence of Tregs not only at the tumor site but also in the peripheral blood and lymph nodes when compared with normal controls.60–62 Otherwise, selective depletion of these cells may be accomplished following low-dose CTX administration. The administration of this drug at 45-day intervals block the renewal of Tregs in mice with multiple myeloma and enable the restoration of an efficient immune response against the tumor cells, thereby leading to prolonged survival and prevention of disease recurrence. Then, low-dose CTX induces beneficial immunomodulatory effects in the immunotherapy context,61–63 but more frequent administrations at 7- or 21-day intervals did not improve the therapeutic effect, since the mice developed multiple myeloma at a higher incidence.51

Intraperitoneal injection of CTX significantly suppresses bilateral growth of murine colon cancer cells (CT-26). Association of this drug with doxorubicin decreased the tumor growth inducing the remotely implanted cells, indicating the development of systemic tumor-specific T cell response.64 In fact, such combination increased the frequency of IFN-γ-producing cells and decreased that the expression of FoxP3 and TGF-β within the remote tumor site.

Distinctive time-schedule administration of low-dose CTX is beneficial for breaking immune tolerance while its modulatory effect on DC-based immunotherapy leads to increased survival of tumor-bearing mice, which can be further improved by elimination of Tregs.65 These results suggest that the DC-based antitumor treatment of patients might be improved by simultaneous depletion of Tregs using a low dose of CTX. Moreover, this combination significantly improves the survival of tumor-bearing mice, when compared with DC vaccination or CTX alone. Despite the well-documented effects of CTX on Treg elimination, these cells are rapidly reconstituted in both secondary lymphoid tissues and at the tumor site, which could discourage new approaches in this field. Further attempts to prolong Treg depletion have yielded no additional benefit.66

It is generally believed that the antitumor immunity induced by the combination of chemotherapy and immunotherapy depends on the homeostatic role of immunotherapy acting during the recovery phase that follows the chemotherapy mediated myelolymphodepletion.67 The ability of this drug to reverse the systemic and in situ DC paralysis in tumor-bearing hosts and its ability to mobilize early proliferating DC progenitors are other crucial mechanisms for enhancing antitumor immunity.66 In this aspect, it was observed that CTX administration restores myelopoiesis, leading to reversion of systemic and in situ tumor-induced DC paralysis. Moreover, CTX mobilized proliferating early DC progenitors to the tumor site where they yielded new DCs.

These several findings are relevant for understanding the multifaceted mechanisms behind the beneficial effects of CTX on augmenting antitumor immunity. In other words, although this drug shows myelosuppressive activity and eliminates the tumor-infiltrating DCs, it is able to mobilize early DC precursors. These precursor cells proliferate rapidly on the periphery and increase the presence of new APCs that can prime de novo T cell responses.66

Thus, CTX has a direct impact on DC-T cell interactions in a tumor-bearing host that ultimately lead to a better priming of tumor-specific T cells. These observations support the notion that mobilization of early DC precursors to sites of ongoing immune response and their differentiation into mature DCs can lead to optimal T cell priming.68,69 Therefore, CTX can influence the DC homeostasis both in situ and systemically through its unique ability to mobilize DC precursors.66

Another important finding on the immune effects of CTX was that a single administration of low-dose CTX (50 mg/kg) in tumor-bearing mice prior to immunization with DC increases the frequency of IFN-γ secreting antitumor CTLs.61 This is in agreement with the fact that different chemotherapeutic agents at very low concentrations increase the ability of DCs to induce T cell proliferation.70

Considering that the use of monoclonal antibodies is the more prominent modality of immunotherapy, Garcia et al.71 have reported remarkable findings in a phase II study of low-dose CTX and bevacizumab against recurrent, refractory ovarian cancer. They have shown that the administration of CTX increased the probability of progression free survival of bevacizumab-treated patients. Combination was not free of toxicity and indeed, lymphopenia was one of most common side effects showed by patients.

Several groups have made efforts to develop safe and effective antitumor vaccines using cytokine genes inserted into viral vectors. In this direction, Malvicini et al.72 observed in a murine model of colorectal carcinoma that the combination of CTX with an IL-12-encoding adenovirus vector induces complete regression in more than 50% of tumor-bearing mice. Such treatment was able to decrease the number of peripheral and spleen Treg, at the same time that it increased the numbers of activated DC and IFN-γ-secreting CD4+ lymphocytes.

The beneficial effects of CTX in augmenting antitumor immunity can be due to the combination of its ability to eliminate Tregs, reset the DC homeostasis in tumor-bearing host and restore the homeostasis-driven expansion of pre-existing tumor-specific effector T cells.60,73,74

Paclitaxel

PTX is a mitotic inhibitor that causes cell cycle arrest by stabilizing tubulin in microtubules. It is another useful agent for LDM therapy given its broad spectrum antitumor activity and ability to in vitro inhibit endothelial cell functions that are fundamental for angiogenesis. In fact, comparing MTD and LDM therapies with PTX, it can be observed that the latter presents stronger antiangiogenic and antilymphangiogenic activities than the former in breast cancer metastases. Since breast cancers spread predominantly through the lymphatic system,75 the antilymphangiogenic activity of LDM PTX can be considered a fundamental feature of this schedule. This drug overcomes the angiogenic role of VEGF-A and reduces the rate of prostate cancer growth in rats by 24% and the vascularized area by 35%.41 However, the benefits of LDM and antiangiogenic therapies are generally limited by acquired resistance76; since, these regimens might lead to selection of hypoxia-resistant tumor cell populations as a mechanism of tumor escape.77 In addition, patients under LDM PTX show less pronounced side effects such as body weight loss and leukopenia than those treated with MTD. This reduced toxicity makes it possible to use LDM as an adjuvant therapy for prolonged periods against advanced metastatic breast cancer.78

Immunomodulatory effects of PTX and combination with immunotherapy

In a murine lung carcinoma model, the combination of low-dose PTX followed by intratumoral injection of DC vaccine is much more efficient for the treatment than either DC vaccine or PTX alone. The combination was shown to inhibit tumor growth, induce tumor infiltration by CD4+ and CD8+ T cell and induce a tumor-specific immune response in regional lymph nodes. The chosen concentration of PTX showed no toxicity to bone marrow cells and, in addition, was able to stimulate DC maturation and function and prevent the inhibitory activity of tumor cells on DC maturation and motility.67 These observations supported the design of intratumoral administration of DCs after pretreatment of tumor-bearing mice with LDM PTX.79 Such combination inhibits the s.c. development of murine lung cancer, increasing the infiltration of CD4+ and CD8+ cells. Increased survival was also correlated with release of IFN-γ by draining lymph nodes lymphocytes and local production of MCP-1 and IP-10. Similar results were observed in the tumor progression of a breast cancer model, which growth was delayed in contrast with the poor activity of drug alone.80

Immunotherapy based on the administration of cytokines is one of the goals of several groups. It was observed, for instance, that the incubation of tumor cells with granulocyte-macrophage colony-stimulating factor (GM-CSF) induces cell surface modification.81

Low-dose PTX combined with the GM-CSF-surface-modified tumor-cell vaccine effectively enhances tumor regression when the vaccine is administered after PTX. This drug mainly enhances the antitumor immune response at the priming time, being the administration of the lower doses (4 and 20 mg/kg) prior to the vaccine superior to post-vaccine treatment. However, the frequency of administrations depends on the dose of the drug, since at higher doses (40 mg/kg) no significant differences were identified between the administration sequences.81 The administration of the low-dose PTX followed by the vaccine induced a high degree of CD8+ T cell infiltration in tumor tissue, suggesting the induction of tumor-specific immune response. The combination treatment also induced a higher number of tumor-specific IFN-γ CD8+ T cells than vaccine alone. Low-dose PTX is sufficiently potent to induce phagocytosis of tumor antigens by DCs and to enhance their maturation and priming of antitumor immune response.81

It is important to note that high PTX concentrations induce DC apoptosis, whereas low concentrations not only diminish apoptosis of these cells but also block the inhibitory effect of the tumor on DC maturation. The effect of this type of treatment may be enhanced by selecting the proper sequence for various drug-vaccine combinations based on the immunomodulatory mechanisms of each treatment.81 Moreover, MTD is more effective at inducing tumor apoptosis and there is a significant difference between these two schedules concerning the apoptosis index of tumor cells.82 However, even though the destruction of tumor cells is the main goal of the treatments, lack of apoptosis can play a favorable role as will be discussed below.

Other chemotherapeutic agents

Effects of metronomic therapy with other common chemotherapeutics such as 5-FU, gencitabine and oxaliplatin have also been evaluated. In contrast with CTX, gencitabine showed no effect on Treg, but selectively reduces MDSC (CD11b+/Gr1+ cells)83,84 and enhances the antitumor activity of CD8+ T and NK cells.84

Correale's group has analyzed the efficacy of these drugs associated with monoclonal antibodies based immunotherapy and reported that treatment of high-risk non-small cell lung cancer patients with cisplatin plus etoposide promoted total or partial remission of disease in 45.2% of the patients enrolled in a phase II trial. Similarly, to previously described drugs, the activity of this metronomic schedule was attributed to changes on tumor neoangiogenesis.85 Such effect was further reported to be enhanced by combination with the anti-VEGF humanized antibody bevacizumab.86 However, this chemoimmunotherapy combination was not completely safe and some patients presented side effects such as hematological changes, mucosal toxicity, alopecia, infections, thromboembolia and mood depression.86,87

Another chimeric antibody Cetuximab, directed to the receptor for EGF, was associated with irinotecan, leucovorin and 5-FU for treating colon cancer patients. This combination was able to increase the expression of antigens by tumor cells. Immunological effects included increased CTL activity, probably due to the enhanced susceptibility of tumor cells to endocytosis by DC.88

Immunomodulatory Effects of Ultra-Low Concentrations of Chemotherapeutics

It was observed that ultra-low noncytotoxic concentrations of selected antineoplastic agents are able to modulate the immune system. Recent findings suggest that some chemotherapeutic drugs can improve the efficacy of immunotherapy by different means. Therefore, many efforts have been directed toward the development of immunotherapeutic approaches to enable the immune system to respond specifically against cancer.20,89

Dendritic cells have different sensitivities to conventional cytotoxic agents. In this aspect, it was reported that PTX and other chemotherapeutic agents could up-regulate DC functions in vitro.79 Studies from our group showed that low noncytotoxic concentrations of doxorubicin, methotrexate, mitomycin C and PTX directly up-regulate the ability of DCs to present antigens for Ag-specific T cells in vitro. These drugs significantly up-regulate the expression of CD80, CD86, CD40 and MHC class II molecules on DCs.90 In addition, we observed that chemotherapeutic agents up-regulate APC function of DCs. In vitro treatment of DCs with doxorubicin, PTX or methotrexate increases their ability to present ovalbumin (OVA) to OVA-specific CD8+ T cells.90 This treatment also up-regulates the expression of the antigen processing machinery (APM) components, including immunoproteasomes (LMP7 and LMP10) and chaperones such as tapasin and calmodulin (delta).90

Furthermore, IL-12 might also play an important role in this phenomenon as a signal 3 molecules91,92 and, indeed, doxorubicin (DOX), methotrexate (MTX), PTX and vinblastine increase spontaneous IL-12 expression in treated DCs.90 In fact, in a murine model, the increased ability of DCs to present antigens to Ag-specific T cells after treatment with such drugs is abolished in DCs generated from IL-12 knockout mice. It indicates that up-regulation of this phenomenon is IL-12-dependent and mediated by autocrine or paracrine mechanisms. As like as human DC, murine cells are also up regulated by doxorubicin methotrexate and PAC to express the mature DC phenotype and increase their ability to stimulate the allogeneic response in the mixed lymphocyte reaction.70 Interestingly, higher concentrations of mitomycin C (up to 6.0 μM) induce the generation of tolerogenic DCs, which express low levels of CD80 and CD86 and display weak activity in the MLR assay.18

Increased expression of CD40 molecules on DCs treated with methotrexate and mitomycin C is in agreement with their increased ability to stimulate T cell proliferation in the MLR assay. However, this correlation was not seen for other tested drugs, suggesting the importance of other mechanisms involved in up-regulation of antigen-presenting function of DCs by chemomodulation.70 Expression of CD40 on DCs is essential for their interaction with T lymphocytes and development of efficient Th1 responses.93 Because expression of CD40 on DCs and the CD40-mediated DC function are suppressed during tumor progression,94 its up-regulation by nontoxic chemotherapy should support the development of antitumor immunity in tumor-bearing hosts. In addition, CD40 ligation protects human and murine DCs from tumor-induced apoptosis by inducing expression of antiapoptotic proteins from the Bcl-2 family.95–97 Therefore, a combination of chemotherapy plus immunotherapy has been discussed and requires more studies to support its widespread clinical use.

Development of spontaneous melanoma in ret transgenic mice can be inhibited by their treatment with low dose PTX.98 This effect was observed to be associated with lower number of infiltrating MDSC in the tumor microenvironment. Michels et al.99 reported similar findings in C57BL/6 mice with PTX slightly decreasing the generation of MDSC from bone marrow precursor cells. Such decrease was not due to induction of apoptosis of these cells. It reinforces the evidence that PTX is able to modulate the immune system.

We also investigated the effect of ultra-low PTX concentrations on colon cancer cells. First, the DNA microarray showed that treatment of tumor cells induced transcriptional changes in several genes associated with both tumor immunogenicity and cell growth. Analysis of APM components of these cells showed that the treatment up regulated the expression of proteasomes, TAP, tapasin and calnexin. These results suggested to us that the tumor immunogenicity could be affected by such changes, which were confirmed by the increased ability of treated tumor cells to induce the generation of tumor-specific cytolytic T lymphocytes. In addition, PTX-treated tumor cells showed higher susceptibility to CTL activity than the wild type cells.100 This finding was reinforced by other authors who have observed that the in vitro treatment of murine colon cancer cells with low concentrations of PTX or doxorubicin increased their sensitivity to specific anti-p53 CTL.80 Authors obtained similar results treating human lung cancer cells (H332). Although no direct effect of PTX on murine lymphocytes was observed, doxorubicin negatively affected cell toxicity80 and human peripheral blood cells were sensitive to drug treatment. Such enhanced sensitivity of tumor cells to CTL cytotoxicity was attributed to the ability of PTX, doxorubicin and cisplatin to increase the cell permeability to Granzyme B.80

The dying tumor cells can release danger signals (damage-associated molecular pattern, DAMP) that lead to the activation of DCs and may facilitate the engulfing and processing of tumor antigens101,102; however, it is usually supposed that massive tumor cell death or apoptosis is nonimmunogenic. The molecular pathways of certain “immunogenic” forms of cancer cell death that cause DC activation have recently been defined. In this aspect, it was reported that DNA-damaging anthracyclines (idarubicin, doxorubicin and mitoxantrone) can induce the translocation of calreticulin to the surface of dying tumor cells, thus promoting phagocytosis by DCs. The surface exposure of calreticulin is an event that occurs only in immunogenic death, making it an important mediator in the uptake of tumor cells by DCs.103 Some danger signals, such as the high-mobility group box 1 (HMGB1) alarmin protein, are secreted by dying tumor cells, which interact with Toll-like receptor 4 (TLR4) expressed by DCs.

In addition, many chemotherapeutic agents, including CTX, induce production of uric acid caused by tumor cell death, which may activate DCs, thereby promoting tumor rejection.104 Moreover, the ability of vinblastine-treated mature DCs to induce CD8+ T cell responses is higher, when compared with untreated DCs.105

Direct effect of noncytotoxic concentrations of drugs on lymphocytes has not been reported except that doxorubicin can affect murine CTL, whereas PTX, doxorubicin and cisplatin can reduce the activity of human cytotoxic cells.80 Our findings on this subject indicate that lymphocytes are resistant to noncytotoxic concentrations of 5-fluorouracil, 5-aza-deoxycytidine and PTX. Differently of DC, lymphocyte functions are not modulated by such low concentrations of drugs (unpublished data), making DC the main target for chemoimmunemodulation.

Conclusion and Perspectives

Low-dose chemotherapy, including the use of noncytotoxic concentrations, metronomic schedule and noncontinuous low-dose administration of antineoplastic agents is an alternative for cancer chemotherapy. These different schedules minimize the side effects of conventional MTD and reduce the rise of resistant variants of tumor cells, which are the main disadvantages of classical treatment. The up-regulating effects on DCs allow us to suggest that the use of low concentrations of selected chemotherapeutics, rather than high doses, should be used in combination with immunotherapy to overcome the immunosuppression induced by cancer.

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