Peritumoral Administration Research Articles

RETRACTED ARTICLE: Injectable hydrogel encapsulating Cu2MnS2 nanoplates for photothermal therapy against breast cancer

BackgroundIn order to explore the possibility of treating breast cancer by local photo-therapy, a photothermal agents loaded in situ hydrogel was established. In detail, The Cu2MnS2 nanoplates were prepared by one-pot synthesis and, the thermosensitive Pluronic F127 was used as the hydrogel matrix. The Cu2MnS2 nanoplates and the hydrogel were characterized by morphous, particle size, serum stability, photothermal performance upon repeated 808 nm laser irradiation as well as the rheology features. The therapeutic effects of the Cu2MnS2 nanoplates and the hydrogel were evaluated qualitatively and quantitatively in 4T1 mouse breast cancer cells. The retention, photothermal efficacy, therapeutic effects and systemic toxicity of the hydrogel were assessed in tumor bearing mouse model.ResultsThe Cu2MnS2 nanoplates with a diameter of about 35 nm exhibited satisfying serum stability, photo-heat conversion ability and repeated laser exposure stability. The hydrogel encapsulation did not negatively influence the above features of the photothermal agent. The nanoplates loaded in situ hydrogel shows a phase transition at body temperature and, as a result, a long retention in vivo.ConclusionsThe photothermal agent embedded hydrogel played a promising photothermal therapeutic effects in tumor bearing mouse model with low systemic toxicity after peritumoral administration.

CuS Nanodot-Loaded Thermosensitive Hydrogel for Anticancer Photothermal Therapy.

The purpose of this research is to establish an injectable hydrogel encapsulating copper sulfide (CuS) nanodots for photothermal therapy against cancer. The CuS nanodots were prepared by one-pot synthesis, and the thermosensitive Pluronic F127 was used as the hydrogel matrix. The CuS nanodots and the hydrogel were characterized by morphous, particle size, serum stability, photothermal performance upon repeated 808 nm laser irradiation, and rheology features. The effects of the CuS nanodots and the hydrogel were evaluated qualitatively and quantitatively in 4T1 mouse breast cancer cells. The retention, photothermal efficacy, therapeutic effects, and systemic toxicity of the hydrogel were assessed in tumor bearing mouse model. The CuS nanodots with a diameter of about 8 nm exhibited satisfying serum stability, photoheat conversion ability, and repeated laser exposure stability. The hydrogel encapsulation did not negatively influence the above features of the photothermal agent. The nanodot-loaded hydrogel shows a phase transition at body temperature and, as a result, a long retention in vivo. The photothermal-agent-embedded hydrogel played a promising photothermal therapeutic effect in the tumor bearing mouse model with low systemic toxicity after peritumoral administration.

Abstract 3824: Antitumor activity of REGN4659, a fully human anti-CTLA-4 monoclonal antibody, against MC38.Ova tumors grown in immunocompetent human CTLA-4 knock-in mice

Abstract Cytotoxic T-lymphocyte associated antigen 4 (CTLA-4) is an inhibitory checkpoint receptor that is expressed by activated conventional T cells and regulatory T cells and negatively regulates T cell activation. CTLA-4 plays a key role in restraining the adaptive immune response of T cells against a variety of antigens, including self-antigens. However, in the tumor microenvironment, the co-inhibitory CTLA-4 receptor contributes to tumor immune escape. CTLA-4 shares its two ligands, CD80 and CD86, which are largely expressed on antigen-presenting cells, with CD28, a co-activator of T cells; however, CTLA-4 binds CD80 and CD86 with higher affinity than CD28. Blockade of CTLA-4 elicits an effective antitumor immunity in preclinical animal models and has demonstrated favorable clinical results in several solid malignancies. REGN4659 is a human IgG1 antibody that binds to the extracellular domain of human and monkey CTLA-4 with high affinity and specificity. REGN4659 blocks CTLA-4 interaction with CD80 and CD86 ligands and enhances the cytokine release of activated primary human T cells in vitro. The effects of treatment with REGN4659 on the subcutaneous growth of a mouse adenocarcinoma tumors expressing chicken ovalbumin (MC38.Ova) were evaluated in immunocompetent mice genetically engineered to express a human CTLA-4 chimeric protein from the endogenous mouse locus (CTLA-4 hum/hum knock-in). Prophylactic treatment with REGN4659 significantly suppressed MC38.Ova tumor growth in CTLA-4 knock-in mice in a dose dependent manner, and improved mouse survival. Because patients treated systemically with CTLA-4 blockade have suffered from immune-related adverse events, we investigated the efficacy of a peritumoral low-dose administration of a mouse-reactive surrogate anti-CTLA-4 antibody in an attempt to minimize systemic exposure. Peritumoral injection of anti-CTLA-4 antibody was as efficacious as intraperitoneal delivery at inducing anti-tumor immunity. Peritumoral anti-CTLA-4 antibody delivery was associated with lower circulating antibody levels but nonetheless caused systemic antitumor immunity as revealed by growth retardation of concomitantly implanted distant tumors. The mechanism of action and robust anti-tumor efficacy of REGN4659 supports its clinical development for the treatment of human cancers. Citation Format: Elena Burova, Omaira Allbritton, Susannah Brydges, William Poueymirou, Robert Durso, Douglas MacDonald, Ella Ioffe, Markus Mohr, William Olson, Gavin Thurston. Antitumor activity of REGN4659, a fully human anti-CTLA-4 monoclonal antibody, against MC38.Ova tumors grown in immunocompetent human CTLA-4 knock-in mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3824.

Localized co-delivery of collagenase and trastuzumab by thermosensitive hydrogels for enhanced antitumor efficacy in human breast xenograft

Modulation of the collagen-rich extracellular matrix (ECM) in solid tumors by the treatment with collagenase has been proved effective in enhancement of the interstitial transport and antitumor efficacy of antibodies. We, therefore, developed a PLGA-PEG-PLGA polymer-based thermosensitive hydrogel, which incorporated a HER2-targeted monoclonal antibody trastuzumab and collagenase (Col/Tra/Gel) for peritumoral administration. HER2-positvie BT474 tumor-bearing mice were selected as a model. The Col/Tra/Gel showed the continuous and biphasic release of protein drugs for 9 days in vitro. NIR imaging studies demonstrated a long-term retention of Col/Tra/Gel hydrogel in the peritumoral area for over 20 days. Treatment with Col/Tra/Gel reduced the collagen density and enhanced apoptotic cell death in tumor tissue, resulting in superior treatments with increased efficacy and reduced toxicity compared with other control groups. Moreover, a quarter-dose of Col/Tra/Gel exhibited a better antitumor efficacy than that of intravenous injection of clinical trastuzumab formulation. This localized co-delivery system offers a potential strategy for the modulation of dense ECM and enhancement of antibody efficacy.

IFN-β augments the anti-tumor effects of PD-1 Abs in melanoma: The possible immunotherapy for metastatic melanoma

Together with regulatory T cells (Tregs), tumor-associated macrophages (TAMs) play roles in maintaining the tumor microenvironment. Since IFNβ has been clinically used for the treatment of malignant melanoma in Japan, first, we investigated its immunomodulatory effects during B16F10 melanoma growth. B16F10 melanoma cells (2 × 105 cells/mouse) were subcutaneously injected into C57BL/6 mice, and when the tumor had reached 5 mm in diameter, we administered IFN-β. We found that peritumoral administration of IFN-β significantly decreased tumorinfiltrating Tregs, using qRT-PCR and flowcytometry analysis. Next, we examined the chemokines in the tumor, which revealed that IFN-β reduced the expression of Tregs-related chemokines (CCL17, CCL22), which led to the suppression of the recruitment of Tregs in B16F10 melanoma. Moreover, to validate the production of chemokines, we isolated CD11b+ cells by MACS, analyzed the chemokine production, and confirmed the decrease of CCL22 in the IFN-β treated group. Since the administration of IFN-β augments the expression of PD-1 on TILs, the co-administration of anti-PD-1 Ab augmented the therapeutic effect of IFN-β on the treatment of B16F10 melanoma. In parallel with the mouse model, in human, IFN-β decreased the production of Th2-related chemokines and augmented the production of Th1-related chemokines from monocyte-derived M 2 macrophages. Since these immunomodulatory effects of IFN-β on macrophages were also observed in the lesional skin of human in-transit melanoma, we started phase Ib study to determine the safety dose of IFN-β in combination with anti-PD1 Ab therapy. Our present data suggest one of the possible use of IFN-β in combination with anti-PD-1 Ab for the treatment of melanoma patients.

Gambogic acid-loaded PEG–PCL nanoparticles act as an effective antitumor agent against gastric cancer

Poor water solubility and side effects hampered the clinical application of gambogic acid (GA) in cancer therapy. Accordingly, GA-loaded polyethylene glycol-poly(ɛ-caprolactone) (PEG–PCL) nanoparticles (GA-NPs) were developed and administered peritumorally to evaluate their antitumor activity. The particle size, polydispersity index, encapsulation efficiency and loading capacity of GA-NPs were 143.78 ± 0.054 nm, 0.179 ± 0.004, 81.3 ± 2.5% and 14.8 ± 0.6%, respectively. In addition, GA-NPs showed excellent stability, good biocompatibility and sustained release profile. Endocytosis studies in vitro demonstrated that the GA-NPs were effectively taken up by tumor cells in a time-dependent manner. In vivo real-time imaging showed that the nanoparticles effectively accumulated within the tumor tissue after peritumoral administration. The cytotoxicity study revealed that the GA-NPs effectively inhibited the proliferation of gastric cancer cells. In vivo antitumor therapy with peritumoral injection of GA-NPs exhibited superior antitumor activity compared with free GA. Moreover, no toxicity was detected in any treatment group. Histological studies confirmed a lower cell density and a higher number of apoptotic cells in the GA-NPs group compared with the free GA group. Furthermore, the expression level of the cysteine proteases 3 precursor (pro-caspase3), a crucial component of cellular apoptotic pathways, was efficiently reduced in mice treated with GA-NPs. In conclusion, the GA-NPs system provided an efficient drug delivery platform for chemotherapy.

Inhibition of NF-κB Pathway and Modulation of MAPK Signaling Pathways in Glioblastoma and Implications for Lovastatin and Tumor Necrosis Factor-Related Apoptosis Inducing Ligand (TRAIL) Combination Therapy.

Glioblastoma is a common malignant brain tumor and it is refractory to therapy because it usually contains a mixture of cell types. The tumor necrosis factor-related apoptosis inducing ligand (TRAIL) has been shown to induce apoptosis in a range of tumor cell types. Previously, we found that two human glioblastoma cell lines are resistant to TRAIL, while lovastatin sensitizes these glioblastoma cells to TRAIL-induced cell death. In this study, we investigated the mechanisms underlying the TRAIL-induced apoptosis in human glioblastoma cell lines by lovastatin. Furthermore, we have confirmed the anti-tumor effect of combination therapy with lovastatin and TRAIL in the subcutaneous brain tumor model. We showed that lovastatin significantly up-regulated the expression of death receptor 5 (DR5) in glioblastoma cell lines as well as in tumor-bearing mice with peri-tumoral administration of lovastatin. Further study in glioblastoma cell lines suggested that lovastatin treatment could inhibit NF-κB and Erk/MAPK pathways but activates JNK pathway. These results suggest that lovastatin sensitizes TRAIL-induced apoptosis by up-regulation of DR5 level via NF-κB inactivation, but also directly induces apoptosis by dysregulation of MAPK pathway. Our in vivo study showed that local peri-tumoral co-injection of lovastatin and TRAIL substantially reduced tumor growth compared with single injection of lovastatin or TRAIL in subcutaneous nude mice model. This study suggests that combined treatment of lovastatin and TRAIL is a promising therapeutic strategy to TRAIL-resistant glioblastoma.

Localized multidrug co-delivery by injectable self-crosslinking hydrogel for synergistic combinational chemotherapy.

Significant progress has been made in the use of injectable hydrogels as drug carrier systems to treat cancers by the peritumoral-localized co-delivery of multiple drugs with different therapeutic mechanisms. In this study, a novel, injectable self-crosslinking HA-SH hydrogel was able to concurrently encapsulate multiple drugs (sorafenib, doxorubicin, and metformin) to enhance chemotherapy efficacy. The hydrogel was relatively stable under physiological conditions and could quickly and directly release the loaded drugs to the tumor site in a reductive tumor microenvironment. The in vitro antitumor activity and cell-apoptosis assay demonstrated that the hydrogel loaded with multiple drugs (Gel + DS or Gel + DSM) showed obvious synergistic effects against breast cancer cells. The combinational chemotherapy enhanced the sensitivities of tumor cells and promoted tumor cell apoptosis after peritumoral administration. Compared with the single drug-loaded hydrogel, the hydrogel co-loaded with multiple drugs (Gel + DSM) showed the best tumor growth inhibition. Moreover, the monitoring of mice weight and ex vivo histological analysis of the main organs indicated that localized treatment with the hydrogel co-loaded with multiple drugs (Gel + DSM) obviously relieved the systemic toxicity and showed promise for inhibiting tumor metastasis, suggesting the superiority and potential application prospects of the injectable, self-crosslinking hydrogel co-loaded with multiple drugs.

RECOMBINANT TUMOR NECROSIS FACTOR-THYMOSIN-A1: THE IMPACT ON THE EFFECTIVENESS OF NEOADJUVANT CHEMOTHERAPY AND ANGIOGENESIS IN BREAST CANCER

The article covers the results of clinical application of a domestic drug based on recombinant tumor necrosis factor-alpha-thymosin-alpha1 (TNF-T) combined with chemotherapy FAC and РА during neoadjuvant treatment of patients with breast cancer IIB-IIIB stage. In contrast to the standard subcutaneous application method, the authors proposed peritumoral administration of TNF-T to maximize the drug's action directly on the tumor, peritumoral tissue. TNF-T 200000 IU peritumorally on days 1-5 of each treatment course showed significant increase in objective effect rate, including increase in frequency of complete regressions, and decrease in frequency and intensity of chemotherapy complications. Morphological examination revealed an increase rate of medical pathomorphosis grade III-IV, decreased number of microvessels in tumor in comparison with primary tumors

Codelivery of doxorubicin-containing thermosensitive hydrogels incorporated with docetaxel-loaded mixed micelles enhances local cancer therapy

Doxorubicin (DOX) thermosensitive hydrogels (TSHs) incorporated with docetaxel (DOC)-loaded mixed micelles were developed to co-deliver these two drugs through a TSH system, DH700kMF-13.5/M-DocLF, to improve local cancer therapy and reduce side effects. First, Pluronics-based DOC-loaded mixed micelles were developed and optimized. The optimal formulation designated as M-DocLF was composed of 1mg/g docetaxel, 15mg/g Pluronic F127 (PF127), and 45mg/g Pluronic L121 (PL121). Rheological tests showed that DH700kMF-13.5/M-DocLF was an injectable flowing solution, which formed a nonflowing gel at body temperature. After intratumoral (IT) or peritumoral (PT) administration, DH700kMF-13.5/M-DocLF demonstrated efficient growth inhibition of CT-26 tumors in a Balb/c mice model. The tumor inhibitory rate after IT administration of DH700kMF-13.5/M-DocLF was 92.4%, followed by 85.8%, 75.6%, 62.9%, 50.6%, and 49.5% for DH700kMF-15, free DOX, F-13.5/M-DocLF, Tynen (DOC solution), and M-DocLF, respectively. Furthermore, PT administration of DH700kMF-13.5/M-DocLF resulted in similar efficacies. Pharmacokinetic and biodistribution studies showed that after subcutaneous (SC) and IT administration of the designated formulations, smaller amounts of DOX and DOC were absorbed from the local SC or tumor sites into systemic circulation, probably reducing their systemic toxicity. Tumor retention of DOX and DOC in biodistribution studies further revealed that co-delivery of these two drugs in DH700KMF-13.5/M-DocLF potentially enhanced the efficacy of tumor inhibition. In conclusion, our in situ injectable DOX and DOC TSH is a potential dual drug delivery system, which can enhance the efficacy of cancer chemotherapy with minimal side effects and reduced chemoresistance.

Thymic stromal lymphopoietin (TSLP) inhibits human colon tumor growth by promoting apoptosis of tumor cells.

Thymic stromal lymphopoietin (TSLP) has recently been suggested in several epithelial cancers, either pro-tumor or anti-tumor. However, the role of TSLP in colon cancer remains unknown. We here found significantly decreased TSLP levels in tumor tissues compared with tumor-surrounding tissues of patients with colon cancer and TSLP levels negatively correlated with the clinical staging score of colon cancer. TSLPR, the receptor of TSLP, was expressed in all three colon cancer cell lines investigated and colon tumor tissues. The addition of TSLP significantly enhanced apoptosis of colon cancer cells in a TSLPR-dependent manner. Interestingly, TSLP selectively induced the apoptosis of colon cancer cells, but not normal colonic epithelial cells. Furthermore, we demonstrated that TSLP induced JNK and p38 activation and initiated apoptosis mainly through the extrinsic pathway, as caspase-8 inhibitor significantly reversed the apoptosis-promoting effect of TSLP. Finally, using a xenograft mouse model, we demonstrated that peritumoral administration of TSLP greatly reduced tumor growth accompanied with extensive tumor apoptotic response, which was abolished by tumor cell-specific knockdown of TSLPR. Collectively, our study reveals a novel anti-tumor effect of TSLP via direct promotion of the apoptosis of colon cancer cells, and suggests that TSLP could be of value in treating colon cancer.

THE POSSIBILITY OF USING TUMOR NECROSIS FACTOR-THYMOSIN-α1 IN NEOADJUVANT CHEMOTHERAPY OF BREAST CANCER

The article covers the results of clinical application of a domestic drug based on recombinant tumor necrosis factor-alphathymosin-alpha1 (TNF-T) combined with chemotherapy FAC and РА during neoadjuvant treatment of patients with breast cancer IIB-IIIB stage. In contrast to the standard subcutaneous application method, the authors proposed peritumoral administration of TNF-T to maximize the drug’s action directly on the tumor, peritumoral tissue. TNF-T 200000 IU peritumorally on days 1–5 of each treatment course during chemotherapy showed significant increase in objective effect rate, including increase in frequency of complete regressions, and decrease in frequency and intensity of chemotherapy complications. Morphological examination revealed an increase rate of medical pathomorphosis grade III-IV, decreased number of microvessels in tumor in comparison with primary tumors. Study of immune status of patients in dynamics shows that the proposed method of peritumoral application of recombinant hybrid protein of tumor necrosis factor-alpha- thymosin-alpha1 has an immunocorrective effect because of its effect on T- and B-cell component of the immune system.

A modified sentinel node and occult lesion localization (SNOLL) technique in non-palpable breast cancer: a pilot study

BackgroundThe spread of mammographic screening programs has allowed an increasing amount of early breast cancer diagnosis. A modern approach to non-palpable breast lesions requires an accurate intraoperative localization, in order to achieve a complete surgical resection. In addiction, the assessment of lymph node status is mandatory as it represents a major prognostic factor in these patients. The aim of this study is to evaluate the reliability of a modified technical approach using a single nanocolloidal radiotracer to localize both sentinel node and breast occult lesion.MethodsTwenty-five patients with a single non-palpable breast lesions and clinically negative axilla were enrolled. In the same day of surgery, patients underwent intratumoral and peritumoral administration of 99mTc-labeled nanocolloid tracer under sonographic guidance. A lymphoscintigraphy was performed to localize the sentinel lymph node and its cutaneous projection was marked on the skin in order to guide the surgeon to an optimal incision. During surgery an hand-held gamma-detection probe was used to select the best surgical access route and to guide localization of both occult breast lesion and sentinel lymph node. After specimen excision, the surgical field was checked with the gamma-probe to verify the absence of residual sources of significant radioactivity, thereby ensuring a radical treatment in a single surgical session and minimizing normal tissue excision.ResultsBoth targeted breast lesion and sentinel lymph node were localized and removed at the first attempt in every patients and histopathological diagnosis of malignancy was confirmed in 25/26 samples. Non-palpable lesions were included within the surgical margins in all patients and in all samples surgical margins were free from neoplastic infiltration thus avoiding any further reintervention. Only two patients showed metastatic involvement of sentinel lymph node.ConclusionsThe modified sentinel node and occult lesion localization (SNOLL) technique performed with a single injection of nanocolloidal radiotracer has shown an excellent intraoperative identification rate of both non-palpable lesion and sentinel lymph node. This procedure offers, as opposed to standard techniques, an accurate, simple and reliable approach to the management of non-palpable breast cancer.

IL-1βR-dependent priming of antitumor CD4+ T cells and sustained antitumor immunity after peri-tumoral treatment with MSU and mycobacteria

Local immune-activating therapies seek to improve the presentation of tumor antigen, thereby promoting the activation of antitumor CD8+ T cells and delaying tumor growth. Surprisingly, little is known about the ability of these therapies to stimulate antitumor CD4+ T cells. We examined tumor-specific CD4+ T cell responses after peri-tumoral administration of the TLR3 agonist polyinosinic-polycytidylic acid (poly I:C), or the danger signal monosodium urate crystals in combination with Mycobacterium smegmatis (MSU + Msmeg) in mice. Both treatments delayed tumor growth, however, only MSU + Msmeg induced proliferation of tumor-specific CD4+ T cells in the draining lymph node (dLN). In line with the proliferation data, administration of MSU + Msmeg, but not poly I:C, enhanced the infiltration of CD4+FoxP3− T cells into the tumor, increased their capacity to produce IFNγ and TNF-α, and decreased PD-1 expression on tumor-infiltrating CD8+ T cells. Induction of CD4+ T cell proliferation by treatment with MSU + Msmeg required IL-1βR signaling, as it was blocked by administration of the IL-1βR antagonist Anakinra. In addition, treatment with Anakinra or with anti-CD4 also reversed the increased survival after tumor challenge in MSU + Msmeg treated mice. Thus, peri-tumoral treatment with MSU + Msmeg results in IL-1βR-dependent priming of antitumor CD4+ T cells in the LN, with consequent superior activation of CD4+ and CD8+ T cells within the tumor, and sustained antitumor activity.

METABOLIC MODULATION OF ANTITUMOR ACTIVITY OF CYTOSTATICS IN EXPERIMENT AND IN CLINICAL PRACTICE

In the experimental intraperitoneal chemotherapy with cyclophosphamide for sarcoma 45 and peritumoral administration of the factors inhibiting the Krebs cycle (diphenhydramine and ATP) histological, ultrastructural and immunocytometric indicators characterizing the development of the involutive processes of the tumor and its regression were studied. Modeling of metabolic microenvironment on the background of systemic neoadjuvant chemotherapy in patients with the stages T3-4N0-3M0 breast cancer led to an increase in antitumor effect. Assessment of tumor response was correlated with cytochemical indicators of the status of the key enzymes of the Krebs cycle (SDH, -GPDH) in lymphocytes of peritumoral area.

Minimum acceptable sensitivity of intraoperative gamma probes used for sentinel lymph node detection in melanoma patients

The aim of this study was to determine the suspension level for the sensitivity of an intraoperative scintillation gamma probe in the detection of the sentinel lymph node (SLN) in melanoma patients.Thirty-eight consecutive patients with melanoma were enrolled in the study during a 12-month period and underwent lymphatic scintigraphy after the peritumoral intradermal administration of about 14 MBq of 99mTc-nanocolloids. The SLNs were successfully removed during the surgical intervention about 4 h later.To identify and localize the SLN, a scintillation NaI(Tl) collimated probe was used. Predictably, the probe sensitivity decreased as the photopeak energy window was progressively narrowed, from 6.9 ± 0.7 counts per second (cps)/kBq (designated as the ‘optimum,’ or ‘OPT,’ sensitivity) to 2.5 ± 0.3 cps/kBq (LOW sensitivity) and to 1.4 ± 0.2 cps/kBq (VLOW sensitivity).Maximum lymph node count rates (cps) were determined for the foregoing energy windows prior to skin incision (PREOPT, PRELOW, PREVLOW, respectively) and in vivo after incision (INVOPT, INVLOW, INVVLOW).Forty-three SLNs were removed with a mean source-to-detector distance of 46 ± 24 mm (min 12 mm, max 92 mm). Four SLNs could not have been detected using PRELOW. This figure would have decreased to 34, with nine undetectable lymph nodes, with PREVLOW. One SLN could not have been identified using INVLOW and four could not have be identified using INVVLOW.In the clinical scenario of SLN detection in melanoma patients, a system sensitivity of 2.5 cps/kBq represents a suspension level, that is, a level under which the equipment must be suspended from clinical use and the poor performance must be investigated.

The chemokine CCL5 induces CCR1-mediated hyperalgesia in mice inoculated with NCTC 2472 tumoral cells

Although the expression of the chemokine receptor CCR1 has been demonstrated in several structures related to nociception, supporting the nociceptive role of chemokines able to activate it, the involvement of CCR1 in neoplastic pain has not been previously assessed. We have assayed the effects of a CCR1 antagonist, J113863, in two murine models of neoplastic hyperalgesia based on the intratibial injection of either NCTC 2472 fibrosarcoma cells, able to induce osteolytic bone injury, or B16-F10 melanoma cells, associated to mixed osteolytic/osteoblastic bone pathological features. The systemic administration of J113863 inhibited thermal and mechanical hyperalgesia but not mechanical allodynia in mice inoculated with NCTC 2472 cells. Moreover, in these mice, thermal hyperalgesia was counteracted following the peritumoral (10–30μg) but not spinal (3–5μg) administration of J113863. In contrast, hyperalgesia and allodynia measured in mice inoculated with B16-F10 cells remained unaffected after the administration of J113863.The inoculation of tumoral cells did not modify the levels of CCL3 at tumor or spinal cord. In contrast, although the concentration of CCL5 remained unmodified in mice inoculated with B16-F10 cells, increased levels of this chemokine were measured in tumor-bearing limbs, but not the spinal cord, of mice inoculated with NCTC 2472 cells. Increased levels of CCL5 were also found following the incubation of NCTC 2472, but not B16-F10, cells in the corresponding culture medium.The intraplantar injection of CCL5 (0.5ng) to naïve mice evoked thermal hyperalgesia prevented by the coadministration of J113863 or the CCR5 antagonist, d-Ala-peptide T-amide (DAPTA), demonstrating that CCL5 can induce thermal hyperalgesia in mice through the activation of CCR1 or CCR5. However, contrasting with the inhibitory effect evoked by J113863, the systemic administration of DAPTA did not prevent tumoral hyperalgesia. Finally, the peritumoral administration of an anti-CCL5 antibody completely inhibited thermal hyperalgesia evoked by the inoculation of NCTC 2472 cells.

In vitro and in vivo evaluation of anisomycin against Ehrlich ascites carcinoma

Anisomycin eminently inhibits cell proliferation invitro. The aim of this study was to explore the potential of anisomycin to treat tumors invivo and its mechanism(s) of action. The results showed that peritumoral administration of anisomycin significantly suppressed Ehrlich ascites carcinoma (EAC) growth resulting in the survival of approximately 60% of the mice 90days after EAC inoculation. Enhancement of infiltrating lymphocytes was noted in the tumor tissue, which was dramatically superior to adriamycin. The growth inhibitory rate of EAC cells was enhanced with increasing concentrations of anisomycin, following an enhanced apoptotic rate. The total apoptotic rate induced by 160ng/ml of anisomycin was higher when compared to that induced by 500ng/ml of adriamycin. DNA breakage and nanostructure changes were also noted in the EAC cells. The levels of caspase-3 mRNA, caspase-3 and cleaved-caspase-3 proteins in the anisomycin‑treated EAC cells were augmented in a dose- and time-dependent manner, following the activation of caspase-8 and caspase-9, which finally triggered PARP cleavage. The cleaved-caspase-3, cleaved-caspase-8 and cleaved-caspase-9 proteins were mainly localized in the nuclei of the cells. These results indicate that anisomycin efficaciously represses invitro and invivo growth of EAC cells through caspase signaling, significantly superior to the effects of adriamycin. This suggests the potential of anisomycin for the treatment of breast cancer.

CCL2 released at tumoral level contributes to the hyperalgesia evoked by intratibial inoculation of NCTC 2472 but not B16-F10 cells in mice

The participation of the chemokine CCL2 (monocyte chemoattractant protein-1) in inflammatory and neuropathic pain is well established. Furthermore, the release of CCL2 from a NCTC 2472 cells-evoked tumor and its involvement in the upregulation of calcium channel α2δ1 subunit of nociceptors was demonstrated. In the present experiments, we have tried to determine whether the increase in CCL2 levels is a common property of painful tumors and, in consequence, the administration of a chemokine receptor type 2 (CCR2) antagonist can inhibit tumoral hypernociception. CCL2 levels were measured by ELISA in the tumoral region of mice intratibially inoculated with NCTC 2472 or B16-F10 cells, and the antihyperalgesic and antiallodynic effects evoked by the administration of the selective CCR2 antagonist RS 504393 were assessed. Cultured NCTC 2472 cells release CCL2 and their intratibial inoculation evokes the development of a tumor in which CCL2 levels are increased. Moreover, the systemic or peritumoral administration of RS 504393 inhibited thermal and mechanical hyperalgesia, but not mechanical allodynia evoked after the inoculation of these cells. Thermal hyperalgesia was also inhibited by the peritumoral administration of a neutralizing CCL2 antibody. In contrast, no change in CCL2 levels was observed in mice inoculated with B16-F10 cells, and RS 504393 did not inhibit the hypernociceptive reactions evoked by their intratibial inoculation. The peripheral release of CCL2 is involved in the development of thermal and mechanical hyperalgesia, but not mechanical allodynia evoked by the inoculation of NCTC 2472 cells, whereas this chemokine seems unrelated to the hypernociception induced by B16-F10 cells.

Revisiting CB1 Receptor as Drug Target in Human Melanoma

Previous studies have indicated the antitumoral effect of human melanocytes, human melanoma cell lines expressing CB1 receptor (CB1), and of the peritumoral administration of endocannabinoids. In the present study, we systematically screened several human melanoma cell lines for the expression of CNR1 and demonstrated transcription of the authentic gene. The product of CNR1, the CB1 protein, was found localized to the cell membrane as well as to the cytoskeleton. Further, the studied human melanoma cell lines expressed functional CB1 since physiological and synthetic ligands, anandamide (AEA), Met-F-AEA, ACEA and AM251 showed a wide range of biological effects in vitro, for example anti-proliferative, proapoptotic and anti-migratory. More importantly, our studies revealed that systemic administration of a stable CB1 agonist, ACEA, into SCID mice specifically inhibited liver colonization of human melanoma cells. Since therapeutic options for melanoma patients are still very limited, the endocannabinoid-CB1 receptor system may offer a novel target.