Combined treatment of IL-1α and TNF-α potentiates the antitumour effect of hyperthermia

Vasoactive cytokines, such as IL-1α and TNF-α, modulate the homeostatic state at the endothelial surface and cause various types of pathological damage in vascular systems. We investigated the potential therapeutic effects of IL-1α and TNF-α in combination with hyperthermia on SCK tumours grown in the legs of A/J mice. We first determined the effect of cytokines on tumour blood perfusion with the ⁸⁶Rb uptake method. When the host mice were given an i.p. injection of 25 μg/kg IL-1α or 50 μg/kg TNF-α, the tumour blood perfusion markedly declined to 46 and 82% of control, respectively. The combination of IL-1α and TNF-α reduced the ⁸⁶Rb uptake to 41% of control. Hyperthermia at 42·5°C for 1 h reduced the tumour blood flow to 71% of control. The tumour blood perfusion decreased further to 20% of control when the tumours were heated for 1 h at 42.5°C starting 4h after the injection of both IL-1α and TNF-α. The changes in clonogenic cell numbers in SCK tumours, as determined by the in vivo-in vitro assay, following various treatments was also investigated. At 4h after an i.p. injection of 25 μg/kg IL-1α or 50 μg/kg TNF-α, the clonogenicity of SCK tumours significantly decreased to 29 or 37% of control, respectively. Heating at 42.5°C for 1h caused a decline in the clonogenic cell number to 30% of control. When both IL-1α and TNF-α were given and tumours were heated 4h later at 42·5°C for 1h, the clonogenic cell number markedly declined to 0.4% of control. The time needed for control tumours to reach 4x their initial volume was about 3 days, and treatment with IL-1α or hyperthermia alone induced a tumour delay growth by about 1 day. The combined injection of IL-1α and TNF-α followed by a heating at 42·5°C for 1h delayed the tumour growth by 6 days. The results in this study suggest that prior impairment of blood circulation by the combined treatment of IL-1α and TNF-α potentiates hyperthermic damage in tumours.     

CRP, TNFα, IL-1ra, IL-6, IL-8 and IL-10 in blood serum of colorectal cancer patients

Blood serum cytokines: TNFalpha, IL-1ra, IL-6, IL-8, IL-10 as well as CRP were investigated in patients with colorectal cancer, prior treatment and 1, 10 and 42 days after surgery. There was an increase of the levels of CRP, IL-6 and IL-10 in most patients 24 hours after surgery. The levels of IL-1ra were elevated in patients in stage C and in several patients in stage B of the disease and there was a decrease of circulating TNFalpha in stage B patients. On day 10 and 42 after surgery, the levels of cytokines followed various patterns.     

Hepatitis B Virus X Protein Up-regulates TNF-α and IL-1β Secretion of Macrophages

Objective: To provide the experimental basis for further studying the molecular transformation mechanism of Hepatitis B virus (HBV) X protein (HBx) on hepatocellular carcinoma. Methods: Reconstructed plasmid pcDNA3.1( )-HBx was transfected into THP-1 macrophages. Expression of HBx was assayed in macrophages lysate by Western-blotting, and TNF-α and IL-1β contents were detected respectively by ELISA. All the data were analyzed by SPSS13.0. Results: In THP-1macrophages, the pcDNA3.1( )-HBx plasmid expressed HBx with a molecular weight of about 17 KDa demonstrated by Western-blotting. The secreted TNF-α and IL-1β from macrophages were determined by ELISA, the results from analysis of all groups showed as following: control group was different from LPS group and pcDNA3.1( ) group (P<0.01), and so was pcDNA3.1( )-HBx group; but there was no obvious difference between pcDNA3.1( ) group and LPS group (P>0.05), all of which indicated that transient overexpression of HBx enhanced LPS-induced production of TNF-α and IL-1β by macrophages. Conclusion: Transient overexpression of HBx up-regulates LPS-induced TNF-α and IL-1β secretion of macrophages.     

Effect of topical chamomile on immunohistochemical levels of IL-1β and TNF-α in 5-fluorouracil-induced oral mucositis in hamsters

Purpose

The aim of the present study was to evaluate the effect of topical chamomile and corticosteroid treatment on the profile of tissue cytokines (IL-1β and TNF-α) in 5-fluorouracil-induced oral mucositis in hamsters.

Methods

Thirty-six hamsters were randomly separated into three groups (12 animals each): Group I—without treatment (control); Group II—treatment with chamomile (Ad-Muc^®); and Group III—treatment with corticosteroid (betamethasone elixir- Celestone^®). The animals received an intraperitoneal injection of 5-fluorouracil on Days 0 and 2. On Days 3 and 4, the buccal mucosa was scratched and therapy was initiated on Day 5. Three animals from each group were killed on Days 0, 5, 10, and 14 and the buccal mucosa was removed. The streptavidin–biotin complex method was used to delineate the in situ distribution, localization, and semiquantitative analysis of IL-1β and TNF-α. Data from the semiquantitative analysis of immunohistochemical staining were comparatively analyzed using the Kruskal–Wallis test, followed by Dunn’s multiple comparisons test.

Results

The distribution and localization of IL-1β and TNF-α immunolabeling were similar. These proteins exhibited a diffuse pattern distributed throughout the connective tissue. The epithelium and adipose tissue were negative for both proteins. The semiquantitative analysis revealed that immunolabeling of IL-1β and TNF-α increased in all groups with the development of mucositis. On Day 10 (period of peak mucositis), the group treated with chamomile had lower scores for both pro-inflammatory cytokines.

Conclusions

Treatment with topical chamomile reduced the tissue levels of IL-1β and TNF-α, thereby demonstrating anti-inflammatory action in oral mucositis in hamsters.     

Role of ethnic variations in TNF-α and TNF-β polymorphisms and risk of breast cancer in India

TNF-α and -β, the multi-functional pro-inflammatory cytokines, are known to play important roles in both tumor progression and destruction based on their concentrations. Growth factors and various stimuli such as cytokines regulate proliferation of the breast epithelial cells. Therefore, the polymorphisms in the genes encoding these signaling molecules could affect the risk of breast cancer. We have investigated selected genetic polymorphisms in TNF-α promoter (rs1800629, −308 G>A and rs361525, −238 G>A) and TNF-β intron 1 (rs909253, +252 A>G) in ethnically two different case–control groups from India. The study included 200 cases and 200 controls from an Indo-European (North Indian) group, and 265 cases and 237 controls from a Dravidian (South Indian) group. Genotyping of a total of 902 individuals was done by direct DNA sequencing. None of the polymorphisms showed significant association with breast cancer in the Indo-European group; however, all the three polymorphisms showed strong association with breast cancer in the Dravidian group. Further, sub-group analysis in the Indo-European group showed no significant difference between pre-menopausal cases and controls or between post-menopausal cases and controls at any of the loci analyzed. However, all the polymorphisms in the Dravidian group were significantly associated with pre-menopausal but not with post-menopausal breast cancer. In conclusion, TNF-α and -β polymorphisms are strongly associated with breast cancer in the Dravidian but not in the Indo-European group.     

Role of cytokines (TNF-α, IL-1β and KC) in the pathogenesis of CPT-11-induced intestinal mucositis in mice: effect of pentoxifylline and thalidomide

Introduction Irinotecan (CPT-11) is an inhibitor of DNA topoisomerase I and is clinically effective against several cancers. A major toxic effect of CPT-11 is delayed diarrhea; however, the exact mechanism by which the drug induces diarrhea has not been established. Purpose Elucidate the mechanisms of induction of delayed diarrhea and determine the effects of the cytokine production inhibitor pentoxifylline (PTX) and thalidomide (TLD) in the experimental model of intestinal mucositis, induced by CPT-11. Materials and methods Intestinal mucositis was induced in male Swiss mice by intraperitoneal administration of CPT-11 (75 mg/kg) daily for 4 days. Animals received subcutaneous PTX (1.7, 5 and 15 mg/kg) or TLD (15, 30, 60 mg/kg) or 0.5 ml of saline daily for 5 and 7 days, starting 1 day before the first CPT-11 injection. The incidence of delayed diarrhea was monitored by scores and the animals were sacrificed on the 5th and 7th experimental day for histological analysis, immunohistochemistry for TNF-α and assay of myeloperoxidase (MPO) activity, tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and KC ELISA. Results CPT-11 caused significant diarrhea, histopathological alterations (inflammatory cell infiltration, loss of crypt architecture and villus shortening) and increased intestinal tissue MPO activity, TNF-α, IL-1β and KC level and TNF-α immuno-staining. PTX inhibited delayed diarrhea of mice submitted to intestinal mucositis and reduced histopathological damage, intestinal MPO activity, tissue level of TNF-α, IL-1β and KC and TNF-α immuno-staining. TLD significantly reduced the lesions induced by CPT-11 in intestinal mucosa, decreased MPO activity, TNF-α tissue level and TNF-α immuno-staining, but did not reduce the severity of diarrhea. Conclusion These results suggest an important role of TNF-α, IL-1β and KC in the pathogenesis of intestinal mucositis induced by CPT-11.     

Effect of polymorphisms of IL-1β and TNF-α genes on CpG island hyper methylation (CIHM) in the nonneoplastic gastric mucosa

CpG island hyper methylation (CIHM) is one of the major events in gastric carcinogenesis. To evaluate the influence of host genetic factors in CIHM related carcinogenesis, we investigated the association between common polymorphisms in IL-1β and TNF-α genes, with CIHM status in the nonneoplastic gastric mucosa. Polymorphisms in the IL-1β gene (-31T>C and -511C>T) and the TNF-α gene (-857C>T) were genotyped in 385 cancer-free subjects. CIHM of four candidate genes: p16 (INK4a), p14 (ARF), E-cadherin (CDH1), and death-associated protein kinase (DAP-kinase), were determined by methylation-specific-polymerase chain reaction (MSP). CIHM high was defined as two or more CpG islands methylated. CIHM of all four genes and CIHM high were significantly associated with Helicobacter pylori infection status. In over all, significant marginal association was found between IL-1β-511 TT genotype and reduced susceptibility to CIHM of DAP-kinase (adjusted OR = 0.48, 95% CI = 0.29-0.78) and CIHM high (adjusted OR = 0.53, 95% CI = 0.32-0.86). This association was more enhanced in subjects 65 yr or younger age. We also found positive association between TNF-α-857T carrier and increased susceptibility to CIHM of CDH (adjusted OR = 1.78, 95% CI = 1.01-3.16), and CIHM high (adjusted OR = 1.86, 95% CI = 1.04-3.33) in the same generation. The mean number of CIHM was lower in subjects with IL-1β-511TT genotype, while the mean number was higher in subjects with TNF-α-857 T carrier especially in subjects 65 yr and younger patients. IL-1β-511 TT genotype is associated with reduced susceptibility to CIHM especially in younger generation. Furthermore, the TNF-α-857T carrier is associated with increased susceptibility of CIHM in the same generation.     

ERβ and PEA3 co-activate IL-8 expression and promote the invasion of breast cancer cells

Metastasis represents the major remaining cause of mortality in human breast cancer. Interleukin-8 (IL-8), a proinflammatory chemokine, plays an important role during tumor angiogenesis and metastasis. In this study, we found that IL-8 and ERβ showed positive association. Overexpression of ERβ or PEA3 could up-regulate IL-8 promoter activity, mRNA and secretion; silencing of ERβ or PEA3 decreased IL-8 mRNA and secretion. ERβ and PEA3 increased IL-8 expression through binding to the IL-8 promoter and increased cell invasion. HER2 could increase ERβ and PEA3 expression and their binding to the IL-8 promoter. We conclude that ERβ and PEA3 play important roles in tumor invasion by regulating IL-8 expression, and HER2 maybe the upstream of ERβ and PEA3 - IL-8 pathway.     

Overall Survival and Clinicopathological Characteristics of Patients with Breast Cancer in Relation to the Expression Pattern of HER-2, IL-6, TNF-α and TGF-β1

The present study was conducted to investigate the prognostic significance of co-expression patterna of HER-2,IL-6, TNF-a and TGF-β1 in breast cancer, by correlating the number of markers with positive expression withclinicopathological characteristics indicative of tumor progression and overall survival. One hundred thirtyconsecutive patients with primary breast cancer were prospectively included and evaluated. Serum concentrationsof the above markers were measured by ELISA. Median split was used to subdivide patients with marker positiveor negative expression. The presence of ≥3 positive markers was independently associated with extended lymphnode (>3) involvement (aOR, 11.94, p=0.001) and lymphovascular invasion (aOR, 12.04, p=0.018), increasingthe prognostic significance of each marker considered separately. Additional prognostic information regardingsurvival was also provided; as the number of positive markers increased, a gradually reduction of survival timewas observed. In addition, patients with 4 positive markers had significantly shorter survival (25 vs 39 months,p=0.006) and a more than 4 fold increased risk of death (aHR, 4.35, p=0.003) compared to patients with 3 positivemarkers. Our findings suggest that the coexpression pattern of these four markers could be used clinically as auseful marker for tumor extension and outcome of breast cancer.     

Serum levels of IL-6 and IL-1β can predict the efficacy of gemcitabine in patients with advanced pancreatic cancer

Background:

With this study, we sought to characterise the impact of pro-inflammatory cytokines on the outcomes of gemcitabine monotherapy (GEM) in patients with pancreatic cancer (PC).

Methods:

Treatment-naive patients with advanced PC and no obvious infections were eligible for enrolment. All of the patients were scheduled to undergo systemic chemotherapy. Serum pro-inflammatory cytokines were measured using an electro-chemiluminescence assay method before chemotherapy. High cytokine levels were defined as values greater than the median. Clinical data were collected prospectively.

Results:

Sixty patients who received GEM were included in the analysis. High IL-6 and IL-1β levels were poor prognostic factors for overall survival in a multivariate analysis (P=0.011 and P=0.048, respectively). Patients with both a high IL-6 level and a high IL-1β level exhibited shortened overall and progression-free survival, a reduction in the tumour control rate, and a high dose intensity of GEM compared with patients with low levels of both IL-6 and IL-1β.

Conclusion:

The serum levels of IL-6 and IL-1β predict the efficacy of GEM in patients with advanced PC.     

Intratumoral delivery of encapsulated IL-12, IL-18 and TNF-α in a model of metastatic breast cancer

Intratumoral (i.t.) cytokine release through the use of poly-lactic acid microspheres (PLAM) holds tremendous potential for the immunotherapy of breast cancer as it harnesses the immunologic potential of autologous tumor in a clinically feasible and minimally toxic manner. We examined the potential of combinations of i.t. IL-12, IL-18 and TNF-α PLAM to generate a tumor-specific immune response and improve outcome in a model of metastatic breast cancer. Balb/c mice with established 4T1 mammary carcinomas were treated with a single injection of BSA, IL-12, IL-18 or TNF-α-loaded PLAM alone or in combination after spontaneous metastases occurred. Combined treatment with IL-12 and TNF-α PLAM was superior to all other treatments, including the triple combination of IL-12, IL-18 and TNF-α in ablation of the primary tumor, eradicating distant disease and enhancing survival. Simultaneous delivery of IL-12 and TNF-α was superior to sequential delivery of IL-12 followed by TNF-α, but not TNF-α followed by IL-12. In vivo lymphocyte depletion studies established that the effects of IL-12 alone are mediated primarily by NK cells, while the combination of IL-12 and TNF-α is dependent upon CD8+ T-cells. Only the combination of IL-12 and TNF-α results in an increase in both CD4+ and CD8+ T-cells and a reduction in CD4+CD25+ cells. While there was no change in the dendritic cell population, IL-12 and TNF-α resulted in a dramatic increase in DC maturation and antigen presentation. Neoadjuvant immunotherapy with simultaneous intratumoral delivery of IL-12 and TNF-α PLAM augments DC antigen presentation and increases cytotoxic T-cells without increasing regulatory T-cells, resulting in a T-cell based anti-tumor immune response capable of eradicating disseminated disease. The addition of IL-18 did not improve the efficacy.     

Down-regulation of transforming growth factor-β and interleukin-10 secretion from malignant glioma cells by cytokines and anticancer drugs

The effect of treatment with interleukin-1 (IL-1), interferon- (IFN-), vincristine, and etoposide was evaluated on the secretion of transforming growth factor- (TGF-) and IL-10 and the expression of major histocompatibility complex (MHC) class I, intercellular adhesion molecule-1 (ICAM-1), and CD80 molecules by malignant glioma cells. Five malignant glioma cell lines were treated with IL-1, IFN-, and/or anticancer agents (vincristine and etoposide). Combined treatment with IL-1 and IFN- caused greater inhibition of TGF- secretion compared to treatment with IFN-, and almost the same levels of inhibition as treatment with vincristine and etoposide. The greatest inhibition of TGF- secretion was achieved by treatment with all agents. Low levels of IL-10 secretion were determined in two out of five malignant glioma cell lines. This IL-10 secretion was inhibited by treatment with IL-1, IFN-, vincristine, and/or etoposide. Treatment with both cytokines and anticancer agents increased the expression of MHC class I and ICAM-1 in all tumor cell lines. The mean increase of expression of MHC class I was 50% and that of ICAM-1 was 12-fold. No tumor cell lines expressed CD80 molecules on the cell surface, and no treatment caused CD80 expression. These results suggest that TGF- and IL-10 secretion by malignant glioma cells can be suppressed by treatment with a combination of IL-1, IFN-, vincristine, and etoposide, and the treatment up-regulates MHC class I and ICAM-1 expression on tumor cells. These results have implications for immunotherapy and chemotherapy in patients with malignant tumors.     

Clinicopathological significance of chemotactic factor IL-8, MCP-1 and MIP-1α expressions in gallbladder carcinoma

Objective:Gal bladder carcinoma was one of the malignant tumors in the digestive system, characterized by high recurrence and invasion. Recent research indicates that chemotactic factors such as IL-8, ...     

Metastatic cells can escape the proapoptotic effects of TNF-α through increased autocrine IL-6/STAT3 signaling

The liver is a common site for cancer metastases in which the entrance of tumor cells has been shown to trigger a rapid inflammatory response. In considering how an inflammatory response may affect metastatic colonization in this setting, we hypothesized that tumor cells may acquire resistance to the proapoptotic and tumoricidal effects of TNF-α, a cytokine that is elevated in a proinflammatory tissue microenvironment. In this study, we investigated molecular mechanisms by which such resistance may emerge using tumor cells in which the overexpression of the type I insulin-like growth factor receptor (IGF-IR) enhanced the inflammatory and metastatic capacities of poorly metastatic cells in the liver. Mechanistic investigations in vitro revealed that IGF-IR overexpression increased cell survival in the presence of high levels of TNF-α, in a manner associated with increased autocrine production of interleukin-6 (IL)-6. In turn, tumor cell-derived IL-6 induced gp130 and IL-6R-dependent activation of STAT3, leading to reduced caspase-3 activation and apoptosis. We found that IL-6 production and cell death resistance were dose dependent with increasing TNF-α levels. In addition, RNA interference-mediated knockdown of either IL-6 or gp130 that established a blockade to autocrine STAT3 induction was sufficient to abolish the prosurvival effect of TNF-α and to inhibit liver metastasis. Taken together, our findings define an IGF-IR-mediated mechanism of cancer cell survival that is critical for metastatic colonization of the liver.     

Expression of TNF-α leader sequence renders MCF-7 tumor cells resistant to the cytotoxicity of soluble TNF-α

Transmembrane TNF-α (tmTNF-α) contains a leader sequence (LS) that can be phosphorylated and cleaved at its cytoplasmic portion, inducing IL-12 production. We observed that the breast cancer cell line MDA-MB-231 expressing transmembrane TNF-α (tmTNF-α) at high level was resistant to soluble TNF-α (sTNF-α)-induced cytotoxicity, accompanied by constitutive NF-κB activation. In contrast, MCF-7 cells expressing tmTNF-α at very low level were sensitive to sTNF-α-induced cell death and had no detectable NF-κB activation. Consistently, siRNA-mediated tmTNF-α knockdown blocked NF-κB activation and rendered MDA-MB-231 sensitive. To test our hypothesis that TNF-LS may play an important role in determining the sensitivity of tumor cells to sTNF-α, we stably transfected MCF-7 cells with TNF-LS. We found that transfection of TNF-LS or wild-type TNF-α containing LS constitutively activated NF-κB and conferred the cytotoxic resistance of MCF-7 cells, while transfection of a mutant tmTNF-α lacking the cytoplasmic segment of LS neither activated NF-κB nor affected the sensitivity. However, NF-κB inhibitor PDTC suppressed NF-κB activation and reconstituted sensitivity of TNF-LS/MCF-7 cells. To check whether TNF-LS is required to be cleaved or internalized for NF-κB activation to occur, we used signal peptide peptidase inhibitor (Z-LL)₂-ketone and receptor internalization inhibitor MDC to treat cells. Interestingly, both inhibitors increased TNF-LS expression on the cell surface and enhanced NF-κB activation. These results indicate that membrane-anchored TNF-LS contributes to constitutive activation of NF-κB and resistance to sTNF-α-induced cell death. Therefore, TNF-LS appears to be responsible for tmTNF-α-induced resistance in the breast cancer cells. D. Yan and N. Qin have contributed equally to this work.