P-MAPA and Interleukin-12 Reduce Cell Migration/Invasion and Attenuate the Toll-Like Receptor-Mediated Inflammatory Response in Ovarian Cancer SKOV-3 Cells: A Preliminary Study.

Immunotherapies have emerged as promising complementary treatments for ovarian cancer (OC), but its effective and direct role on OC cells is unclear. This study examined the combinatory effects of the protein aggregate magnesium-ammonium phospholinoleate-palmitoleate anhydride, known as P-MAPA, and the human recombinant interleukin-12 (hrIL-12) on cell migration/invasion, apoptosis, toll-like receptor (TLR)-mediated inflammation, and cytokine/chemokine profile in human OC cell line SKOV-3. P-MAPA and IL-12 showed cancer cell toxicity under low doses after 48 h. Although apoptosis/necrosis and the cell cycle were unchanged by the treatments, P-MAPA enhanced the sensitivity to paclitaxel (PTX) and P-MAPA associated with IL-12 significantly reduced the migratory potential and invasion capacity of SKOV-3 cells. P-MAPA therapy reduced TLR2 immunostaining and the myeloid differentiation factor 88 (MyD88), but not the TLR4 levels. Moreover, the combination of P-MAPA with IL-12 attenuated the levels of MyD88, interferon regulatory factor 3 (IRF3) and nuclear factor kappa B (NF-kB p65). The IL-12 levels were increased and P-MAPA stimulated the secretion of cytokines IL-3, IL-9, IL-10, and chemokines MDC/CCL22 and, regulated on activation, normal T cells expressed and secreted (RANTES)/CCL5. Conversely, combination therapy reduced the levels of IL-3, IL-9, IL-10, MDC/CCL22, and RANTES/CCL5. Collectively, P-MAPA and IL-12 reduce cell dynamics and effectively target the TLR-related downstream molecules, eliciting a protective effect against chemoresistance. P-MAPA also stimulates the secretion of anti-inflammatory molecules, possibly having an immune response in the OC microenvironment.

Carbon Nanotube as a Tool for Fighting Cancer.

In 2015, cancer was the cause of almost 22% of deaths worldwide. The high frequency of relapsing diseases and metastasis requires the development of new diagnostic and therapeutic approaches, and the use of nanomaterials is a promising tool for fighting cancer. Among the more extensively studied nanomaterials are carbon nanotubes (CNTs), synthesized as graphene sheets, whose spiral shape is varied in length and thickness. Their physicochemical features, such as the resistance to tension, and thermal and electrical conductivity, allow their application in several fields. In this review, we show evidence supporting the applicability of CNTs in biomedical practice as nanocarriers for drugs and immunomodulatory material, emphasizing their potential for use in cancer treatment.

TLR9 stimulation induces increase in fungicidal activity of human dendritic cells challenged with Paracoccidioides brasiliensis.

Microorganisms killing by dendritic cells (DCs) is an important effector mechanism during innate immune response, as it can avoid dissemination of infection during migration of these cells toward draining lymph nodes. However, this function depends on pattern recognition receptors (PRRs) to which the microorganism will bind in these cells. Regarding this, TLR9 activation, by stimulating the oxidative metabolism, induces increase in microbicidal activity of these cells. Accordingly, we showed that DCs treatment with a TLR9 agonist results in an increase in fungicidal activity of these cells against the fungus Paracoccidioides brasiliensis (Pb), which however, was not associated to higher H2O2 levels.

Role of Dectin-1 receptor on cytokine production by human monocytes challenged with Paracoccidioides brasiliensis.

Fungal recognition by Dectin-1 receptor triggers a series of cellular mechanisms involved in a protective activation of the immune system. In this study, we aimed to evaluate the participation of Dectin-1 receptor in the induction of IL-8, TNF-α, IL-12, IL-10 and IL-17A secretion by human monocytes activated with different cytokines, and challenged in vitro with Paracoccidioides brasiliensis (P. brasiliensis). Our results show that monocytes challenged with P. brasiliensis (Pb265) are able to produce IL-12, IL-8, IL-17, IL-10 and TNF-α. Dectin-1 receptor blockage decreased the IL-12, IL-17, IL-10 and TNF-α levels indicating the participation of such receptor in the induction of these cytokines. Only IL-8 production was not affected by the blockage. Cells activation with different cytokines showed that GM-CSF was able to induce secretion of all cytokines and the receptor blockage prior to the challenge also decreased the cytokine secretion, except IL-8. Monocytes activated with TNF-α promoted IL-8, IL-10 and TNF-α production, whereas stimulation with IFN-γ promoted mainly IL-12 and TNF-α. Thus, these findings bring new and important knowledge about Dectin-1 participation in cytokines production by monocytes challenged with Pb265.

Stryphnodendron adstringens and purified tannin on Pythium insidiosum: in vitro and in vivo studies.

BACKGROUND: Pythium insidiosum is the etiological agent of pythiosis, an emerging life-threatening infectious disease in tropical and subtropical regions. The pathogen is a fungus-like organism resistant to antifungal therapy, for this reason, most cases need extensive surgical debridments as treatment, but depending on the size and anatomical region of the lesion, such approach is unfeasible. We investigate the fungicidal effect and toxicity of crude bark extract of Stryphnodendron adstringens and commercially available tannin on Pythium insidiosum both in vitro and in vivo. METHODS: Standardized fragments of mycelia of fifteen isolates of P. insidiosum were tested with different concentrations of bark extract (10 to 30% v/v) and tannin (0.5, 1.0 and 1.5 mg/mL). For in vivo study, fifteen rabbits were experimentally infected with zoospores of P. insidiosum and treated by oral and intralesional applications of bark extract and tannin. Acute toxicity tests with both substances were also performed in rats. RESULTS: In vitro studies showed fungicidal effect for both substances at different concentrations and the SEM showed alteration on the cell wall surface of the pathogen. All infected rabbits developed a firm nodular mass that reached around 90 mm2 ninety days after inoculation, but neither the intralesional inoculation of tannin, nor the oral administration of crude extract and tannin were able to promote remission of the lesions. CONCLUSIONS: Lesions developed by rabbits presented an encapsulated abscess being quite different of naturally acquired pythiosis, which is characterized by ulcerated lesions. Since no toxicity was observed in rats or rabbits inoculated with these products, while in vitro experiments showed direct antifungal effect, therapeutic activity of S. adstringens and tannin should be clinically tested as an alternative for healing wounds in naturally acquired pythiosis.

Ability of Salmonella spp. to produce biofilm is dependent on temperature and surface material.

Salmonella, one of the most important pathogens transmitted by food, especially poultry, has the ability to form biofilms on surfaces. Its adhesion can be influenced by different physicochemical properties of these surfaces, while Salmonella uses fimbriae and produces cellulose as the main matrix components of biofilms. Their synthesis is co-regulated by a LuxR-type regulator, the agfD (aggregative fimbriae, curli), and adrA genes, respectively. Thus, this study investigated the production of biofilm by Salmonella spp. isolated from raw poultry (breast fillet), purchased in Botucatu, Sao Paulo, Brazil, on glass, polyvinyl chloride, and stainless steel at different temperatures (16°, 20°, 28°, and 35°C). We analyzed the frequency of the agfD and adrA genes and the rdar morphotype at 28°C and 35°C in isolated strains. We found Salmonella in 112 of 240 poultry samples (46.7%), and 62 strains previously isolated from the same kind of food were included in the study on biofilm development, gene expression, and rdar morphotype. All of them were positive for both genes, and 98.3% were able to produce biofilm in at least one temperature. The rates of rdar morphotype at 28°C and at 35°C were 55.2% (96 strains) and 2.3% (4 strains), respectively. Glass was the best material to avoid biofilm production, while Salmonella grew even at 16°C on stainless steel. These results point out the need for more effective sanitizing processes in the slaughter plants in order to avoid the permanence of these bacteria in food and eventual human foodborne diseases.

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

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.

Melatonin enhances cell death and suppresses the metastatic capacity of ovarian cancer cells by attenuating the signaling of multiple kinases.

BACKGROUND: Ovarian cancer is a highly aggressive disease that is frequently diagnosed in advanced stages. Melatonin, with its numerous antitumor properties, holds great promise in cancer treatment. Herein, we investigated the effects of melatonin on apoptosis, cell migration, and kinase levels in human ovarian carcinoma SKOV-3 cells and determined whether these effects are mediated by the activation of the MT1 receptor. METHODS: SKOV-3 cells were exposed to different concentrations of melatonin based on the presence of MT1 receptor, and we also performed specific silencing of the melatonin receptor gene MTNR1A. RESULTS: Our findings revealed that melatonin reduced cell viability as shown by the MTT assay, and flow cytometry analysis showed increased rates of apoptosis and necrosis in all melatonin-treated cells. Melatonin significantly decreased the migratory and invasive capacities of the cells. Propidium iodide labeling indicated that melatonin induced cell cycle arrest by reducing DNA content in the S and G2/M phases in SKOV-3 cells. Additionally, the levels of AKT, ERK1/2, JNK, CREB, p70S6K, STAT3/5, and p38 MAP kinase involved in cell survival, proliferation, motility, and stress responses were depressed by melatonin and further reduced after MT1 knockdown. These molecules were found to be associated with lower overall survival in ovarian cancer patients. CONCLUSIONS: Melatonin had obvious oncostatic actions on ovarian cancer cells, and MT1 receptor knockdown intensified its antitumor effect. The inhibition of the MT1 receptor resulted in a substantial reduction in the migratory and invasive capacities of the cells, suggesting its potential as a therapeutic target for the treatment of ovarian cancer.

Immunomodulatory properties of nanostructured systems for cancer therapy.

Based on statistical data reported in 2020, cancer was responsible for approximately 10 million deaths. Furthermore, 17 million new cases were diagnosed worldwide. Nanomedicine and immunotherapy have shown satisfactory clinical results among all scientific and technological alternatives for the treatment of cancer patients. Immunotherapy-based treatments comprise the consideration of new alternatives to hinder neoplastic proliferation and to reduce adverse events in the body, thereby promoting immune destruction of diseased cells. Additionally, nanostructured systems have been proven to elicit specific immune responses that may enhance anti-tumor activity. A new generation of nanomedicines, based on biomimetic and bioinspired systems, has been proposed to target tumors by providing immunomodulatory features and by enabling recovery of human immune destruction capacity against cancer cells. This review provides an overview of the aspects and the mechanisms by which nanomedicines can be used to enhance clinical procedures using the immune modulatory responses of nanoparticles (NPs) in the host defense system. We initially outline the cancer statistics for conventional and new treatment approaches providing a brief description of the human host defense system and basic principles of NP interactions with monocytes, leukocytes, and dendritic cells for the modulation of antitumor immune responses. A report on different biomimetic and bioinspired systems is also presented here and their particularities in cancer treatments are addressed, highlighting their immunomodulatory properties. Finally, we propose future perspectives regarding this new therapeutic strategy, highlighting the main challenges for future use in clinical practice.

Expression of inflammatory cytokines in mesenchymal stem cells derived from proximal humerus fractures.

Background: Mesenchymal stem cells (MSCs) are an excellent treatment option for a wide variety of orthopaedic conditions. This study aimed to establish if bone marrow MSCs obtained from proximal humerus fractures can be an alternative source for obtaining primary cultures of human MSCs. Methods: Human bone marrow was obtained during osteosynthesis surgeries on closed proximal humerus fractures within 48 hours of injury. MSCs were harvested using the Ficoll gradient separation protocol and in vitro cultured until the third passage. Then, the cells were immunophenotyped by flow cytometry using stem cell specific surface markers. The cells were also induced to differentiate into osteoblasts and adipocytes for the characterization and confirmation of MSCs. The production of cytokines interleukin (IL)-1ß, IL-6, IL-8, IL-10, tumor necrosis factor α (TNF-α), and interferon γ (IFN-γ) was assessed using enzyme-linked immunosorbent assay (ELISA) in the supernatant of the cultures after 3, 5 or 7 days. Results: Immunophenotyping showed high expression of the stem cell surface markers CD73, CD90, and CD105 and negative or very low expression of CD34, CD45, CD11b, CD19, and human leukocyte antigen (HLA)-DR. The bone marrow derived MSCs were able to differentiate into osteoblasts and adipocytes. The quantification of secreted cytokines revealed that IL-8 was the most produced cytokine, followed by IL-6 and IL-10 at similar quantities and lower levels of IL-1ß. TNF-α and IFN-γ were not detected. Conclusions: Proximal humerus fractures can be an alternative source for the collection of bone marrow MSCs. The cytokine production of these cells is very similar to the production profile of fracture haematomas previously reported and may be used for improving bone repair.

Cell-mediated immunity and expression of MHC class I and class II molecules in dogs naturally infected by canine transmissible venereal tumor: Is there complete spontaneous regression outside the experimental CTVT?

The canine transmissible venereal tumor (CTVT) is a transplantable cancer with the ability evade the immune system, despite strict immune surveillance of the host; in this context, the relationship between inflammatory infiltrate and CTVT prognosis is not entirely understood. Natural canine transmissible venereal tumors of 22 dogs were evaluated for tumor/host interaction through clinical and epidemiological data, cyto-histopathological and cytogenetic findings and, mainly, cell-mediated immune response. We performed analysis on dogs with naturally acquired disease to provide information from the study of CTVT biology in its natural course, as the clinical evolution of the natural tumor in the host is not yet as well known as in the laboratory. Populations for T cell labeling (CD3+ CD4+ CD8+), B cells, NK cells, and macrophages were analyzed by flow cytometry in blood and tumor samples and expressions of MHC class I and class II molecules were quantified by immunohistochemistry and compared mainly between the phases of progression and regression in the natural CTVT. Dogs were also treated with vincristine sulfate and evaluated for chemotherapeutic response. Chemotherapy was effective in 88% of cases and there was no recurrence of the disease 12 months after the cure. Tumor cells displayed a numerical chromosomal variation between 54 and 72, not correlating with the host genotype. Although a greater expression of MHC molecules [18.6 ± 5.8% class I (P < 0.004) and 38.5 ± 6.5% class II (P < 0.003)] was observed in the regression phase, no significant effect was observed between the clinical phase of the tumor and cellular immune response in the analysis by flow cytometry (P > 0.05). We also found no correlation between cytological subtype of the tumor (plasmacytoid, lymphocytoid and mixed) and cellular immune response, suggesting that there is no difference in tumor immunogenicity. Here, we found no immunological evidence to support the theory of the immune-induced complete spontaneous regression in CTVT.

Cytotoxic Activity and Lymphocyte Subtypes in Mice Selected for Maximal and Minimal Inflammatory Response after Transplantation of B16F10 and S91 Melanoma Cells.

AIRmax and AIRmin mice strains were selected according to the intensity of their acute inflammatory responsiveness. Previous studies have shown that AIR mice differ in their resistance to chemically induced skin tumors and in the development of melanoma metastases, in addition to differences in neutrophil and NK cells activity. In the present work, we aimed to evaluate whether the difference of susceptibility to murine melanoma is associated with NK cytotoxic activity against Yac.1 cells and lymphocyte subsets. Mice were subcutaneously inoculated with B16F10 or S91 melanoma cells. After 7, 14, or 30 days, the animals were euthanized to analyze the number of lymphocyte subsets, cytotoxic activity, and number of cytokine-producing spleen cells. AIRmax mice presented a higher number of CD4+/CD25+ cells than that of AIRmin mice following inoculation of B16F10 cells, whereas inoculation of S91 cells reduced CD4+/CD25+ and increased TCD8+ cell subsets in the AIRmax mice. AIRmax mice had a higher number of interleukin (IL)-10- and IL-12-producing cells and a lower number of interferon-γ-producing cells than those of AIRmin mice at 30 days. The cytotoxic activity of nonadherent spleen cells was similar in both the AIR strains. These results suggest that melanoma cells can induce different responses in AIR mice, possibly owing to alterations in regulatory mechanisms, such as the action of CD4+/CD25+ regulatory T cells and IL-10, in AIRmax mice.

Chemotherapeutic agents in noncytotoxic concentrations increase antigen presentation by dendritic cells via an IL-12-dependent mechanism.

Antineoplastic chemotherapeutic agents may indirectly activate dendritic cells (DCs) by inducing the release of "danger" signals from dying tumor cells. Whereas the direct cytotoxic or inhibitory effect of conventional chemotherapy on DCs has been reported, modulation of DC function by chemotherapeutic agents in low noncytotoxic concentrations has not yet been investigated. We have tested the effects of different classes of antineoplastic chemotherapeutic agents used in low noncytotoxic concentrations on the Ag-presenting function of DCs. We revealed that paclitaxel, doxorubicin, mitomycin C, and methotrexate up-regulated the ability of DCs to present Ags to Ag-specific T cells. Stimulation of DC function was associated with the up-regulation of expression of Ag-processing machinery components and costimulatory molecules on DCs, as well as increased IL-12p70 expression. However, the ability of DCs treated with paclitaxel, methotrexate, doxorubicin, and vinblastine to increase Ag presentation to Ag-specific T cells was abolished in DCs generated from IL-12 knockout mice, indicating that up-regulation of Ag presentation by DCs is IL-12-dependent and mediated by the autocrine or paracrine mechanisms. At the same time, IL-12 knockout and wild-type DCs demonstrated similar capacity to up-regulate OVA presentation after their pretreatment with low concentrations of mitomycin C and vincristine, suggesting that these agents do not utilize IL-12-mediated pathways in DCs for stimulating Ag presentation. These findings reveal a new mechanism of immunopotentiating activity of chemotherapeutic agents-a direct immunostimulatory effect on DCs (chemomodulation)-and thus provide a strong rationale for further assessment of low-dose chemotherapy given with DC vaccines for cancer treatment.

Inhibiting autophagy to prevent drug resistance and improve anti-tumor therapy.

Cytotoxic drugs remain the first-line option for cancer therapy but the development of drug-resistance by tumor cells represents a primary obstacle for successful chemotherapy. Autophagy is a physiological mechanism of cell survival efficiently used by tumor cells to avoid cell death and to induce drug-resistance. It is a macromolecular process, in which cells degrade and recycle intracellular substrates and damaged organelles to alleviate cell stress caused by nutritional deprivation, hypoxia, irradiation, and cytotoxic agents, as well. There is evidence that autophagy prevents cancer during the early steps of carcinogenesis, but once transformed, these cells show enhanced autophagy capacity and use it to survive, grow, and facilitate metastasis. Current basic studies and clinical trials show the feasibility of using pharmacological or molecular blockage of autophagy to improve the anticancer therapy efficiency. In this review, we overviewed the pathways and molecular aspects of autophagy, its role in carcinogenesis, and the evidence for its role in cancer adaptation and drug-resistance. Finally, we reviewed the clinical findings on how the autophagy interference helps to improve conventional anticancer therapy.

Gutting it Out: Developing Effective Immunotherapies for Patients With Colorectal Cancer.

Risk factors for colorectal cancer (CRC) include proinflammatory diets, sedentary habits, and obesity, in addition to genetic syndromes that predispose individuals to this disease. Current treatment relies on surgical excision and cytotoxic chemotherapies. There has been a renewed interest in immunotherapy as a treatment option for CRC given the success in melanoma and microsatellite instable (MSI) CRC. Immunotherapy with checkpoint inhibitors only plays a role in the 4%-6% of patients with MSIhigh tumors and even within this subpopulation, response rates can vary from 30% to 50%. Most patients with CRC do not respond to this modality of treatment, even though colorectal tumors are frequently infiltrated with T cells. Tumor cells limit apoptosis and survive following intensive chemotherapy leading to drug resistance and induction of autophagy. Pharmacological or molecular inhibition of autophagy improves the efficacy of cytotoxic chemotherapy in murine models. The microbiome clearly plays an etiologic role, in some or most colon tumors, realized by elegant findings in murine models and now investigated in human clinical trials. Recent results have suggested that cancer vaccines may be beneficial, perhaps best as preventive strategies. The search for therapies that can be combined with current approaches to increase their efficacy, and new knowledge of the biology of CRC are pivotal to improve the care of patients suffering from this disease. Here, we review the basic immunobiology of CRC, current "state-of-the-art" immunotherapies and define those areas with greatest therapeutic promise for the future.

Anti-PSMA monoclonal antibody increases the toxicity of paclitaxel carried by carbon nanotubes.

Multiple-wall carbon nanotubes (CNTs) were functionalized with polyethyleneimine in order to incorporate paclitaxel (PTX), the first line chemotherapeutic agent for prostate cancer. These particles were then covered with antibodies for the prostate-specific membrane antigen (PSMA), to address them to prostate cancer cells. LNCaP prostate cancer cells (PSMA+), HCT-116 and CaCo-2 colon cancer cells (PSMA-), as well as human peripheral monocytes and lymphocytes (PSMA-), were in vitro exposed to fluorescent CNT composites. The interaction/adherence of those composites to target cells was analyzed by fluorescence microscopy and flow cytometry, showing a diffuse interaction of CNTs and CNT-PTX with all cell types. Analysis of cytotoxicity revealed that both prostate (PSMA+) and colorectal cancer cells (PSMA-) were more susceptible to PTX complexed with CNTs than to pure PTX or CNTs alone, while the incorporation of anti-PSMA (CNT-PTX-PSMA) improved the toxicity on LNCaP cells but not on PSMA- targets. No toxicity was observed in human monocytes and lymphocytes but composites induced phenotypical changes in monocytes. Our results demonstrate the feasibility of using anti-PSMA antibody to address drug-loaded CNT to cancer cells as a strategy for improving the effectiveness of antineoplastic agents.

Anti-EGFR-Coated Gold Nanoparticles In Vitro Carry 5-Fluorouracil to Colorectal Cancer Cells.

: The aim of the current study is to present a strategy to improve the efficiency of 5-fluorouracil (5-FU), which is widely used as antineoplastic agent against solid tumors-based on the use of gold nanocarriers to overcome the resistance of colorectal cancer cells. 5-FU was loaded on gold nanoparticles (AuNP) coated with anti-EGFR antibodies in order to target them towards colorectal cancer cells that overexpress epidermal growth factor receptors (EGFR). Physicochemical characterization has shown that AuNP size was approximately 20 nm and that AuNP functionalization led to spherical nanoparticles. Flow cytometry allowed observing that some compounds synthesized by our research group have induced apoptosis/necrosis and impaired the proliferation of colon cancer cell lines 'HCT-116' and 'HT-29'. The antibody/drug combination in AuNP (AuNP 5FU EGFR) has improved the apoptosis rate and impaired cell proliferation in both cell lines, regardless of the exposure time. Overall, these results have shown that AuNP functionalization with monoclonal antibodies focused on delivering 5-FU to tumor cells is an exciting strategy against colorectal cancer.

P-MAPA activates TLR2 and TLR4 signaling while its combination with IL-12 stimulates CD4+ and CD8+ effector T cells in ovarian cancer.

AIMS: Ovarian cancer (OC) is the most lethal gynecological malignancies and many women develop chemoresistance associated with the inflammatory process. We investigated the effects of P-MAPA and IL-12 on the inflammatory and immune responses in a chemically-induced OC model. MAIN METHODS: OCs were induced with 7,12-dimethylbenz(a)anthracene into the ovarian bursa, and the animals were given P-MAPA (5 mg/kg bw., i.p., twice a week), or IL-12 (300 ng/kg bw., i.p., one a week) for 60 days, or both P-MAPA and IL-12. Immunohistochemistry, western blot, flow cytometry, and multiplex assay were used to examine the effectiveness of immunotherapies in OC. KEY FINDINGS: The combinatory therapy improved the general OC features, reducing inflammatory cells and adipocyte accumulation, in addition to revealing a soft and mobile tissue with no adherences and peritoneal implants. P-MAPA treatment increased the levels of TLR2, TLR4 and TRIF in OCs while decreasing the number of regulatory T (Treg) cells. Additionally, the association of P-MAPA with IL-12 significantly increased the number of CD4+ and CD8+ T effector cells in draining lymph nodes. Regarding the inflammatory mediators, P-MAPA enhanced the levels of the pro-inflammatory cytokine IL-17 while P-MAPA+IL-12 increased the levels of IL-1ß. Treatment with IL-12 enhanced the cytokine levels of IL-17, TNF-α, IL-1ß, and IL-2 in addition to the chemokine MIP-1α. SIGNIFICANCE: We conclude that P-MAPA upregulated TLR2 and TLR4 signaling, possibly activating the non-canonical pathway, while attenuating the tumor immunosuppression. Also, the combination of P-MAPA with IL-12 improves the antitumor immunoresponse, opening a new therapeutic approach for fighting OC.

Blocking drug-induced autophagy with chloroquine in HCT-116 colon cancer cells enhances DC maturation and T cell responses induced by tumor cell lysate.

Autophagy is an important mechanism for tumor escape, allowing tumor cells to recover from the damage induced by chemotherapy, radiation therapy, and immunotherapy and contributing to the development of resistance. The pharmacological inhibition of autophagy contributes to increase the efficacy of antineoplastic agents. Exposing tumor cells to low concentrations of select autophagy-inducing antineoplastic agents increases their immunogenicity and enhances their ability to stimulate dendritic cell (DC) maturation. We tested whether the application of an autophagy-inhibiting agent, chloroquine (CQ), in combination with low concentrations of 5-fluorouracil (5-FU) increases the ability of tumor cells to induce DC maturation. DCs sensitized with the lysate of HCT-116 cells previously exposed to such a combination enhanced the DC maturation/activation ability. These matured DCs also increased the allogeneic responsiveness of both CD4+ and CD8+ T cells, which showed a greater proliferative response than those from DCs sensitized with control lysates. The T cells expanded in such cocultures were CD69+ and PD-1- and produced higher levels of IFN-γ and lower levels of IL-10, consistent with the preferential activation of Th1 cells. Cocultures of autologous DCs and lymphocytes improved the generation of cytotoxic T lymphocytes, as assessed by the expression of CD107a, perforin, and granzyme B. The drug combination increased the expression of genes related to the CEACAM family (BECN1, ATGs, MAPLC3B, ULK1, SQSTM1) and tumor suppressors (PCBP1). Furthermore, the decreased expression of genes related to metastasis and tumor progression (BNIP3, BNIP3L, FOSL2, HES1, LAMB3, LOXL2, NDRG1, P4HA1, PIK3R2) was noted. The combination of 5-FU and CQ increases the ability of tumor cells to drive DC maturation and enhances the ability of DCs to stimulate T cell responses.

Chemomodulation of human dendritic cell function by antineoplastic agents in low noncytotoxic concentrations.

The dose-delivery schedule of conventional chemotherapy, which determines its efficacy and toxicity, is based on the maximum tolerated dose. This strategy has lead to cure and disease control in a significant number of patients but is associated with significant short-term and long-term toxicity. Recent data demonstrate that moderately low-dose chemotherapy may be efficiently combined with immunotherapy, particularly with dendritic cell (DC) vaccines, to improve the overall therapeutic efficacy. However, the direct effects of low and ultra-low concentrations on DCs are still unknown. Here we characterized the effects of low noncytotoxic concentrations of different classes of chemotherapeutic agents on human DCs in vitro. DCs treated with antimicrotubule agents vincristine, vinblastine, and paclitaxel or with antimetabolites 5-aza-2-deoxycytidine and methotrexate, showed increased expression of CD83 and CD40 molecules. Expression of CD80 on DCs was also stimulated by vinblastine, paclitaxel, azacytidine, methotrexate, and mitomycin C used in low nontoxic concentrations. Furthermore, 5-aza-2-deoxycytidine, methotrexate, and mitomycin C increased the ability of human DCs to stimulate proliferation of allogeneic T lymphocytes. Thus, our data demonstrate for the first time that in low noncytotoxic concentrations chemotherapeutic agents do not induce apoptosis of DCs, but directly enhance DC maturation and function. This suggests that modulation of human DCs by noncytotoxic concentrations of antineoplastic drugs, i.e. chemomodulation, might represent a novel approach for up-regulation of functional activity of resident DCs in the tumor microenvironment or improving the efficacy of DCs prepared ex vivo for subsequent vaccinations.