CXCR1/2 Chemokine Network Regulates Melanoma Resistance to Chemotherapies Mediated by NF-κB.

BACKGROUND: Cancer-related inflammation is recognized as a driver for tumor progression and chemokines are important players in both inflammation and the progression of many cancer types. CXC chemokines, especially CXCL8, have been implicated in melanoma growth and metastasis, while less is known for their roles in drug resistance. METHODS: We generated drug-resistant cells by continuous exposure to chemotherapeutic drugs and analyzed the mechanism(s) of therapy resistance in malignant melanoma. RESULTS: We report chemotherapies induced upregulation of a variety of chemokines in the CXCR1/CXCR2 network by an NF-κB-dependent mechanism. Notably, analysis of the drug-resistant melanoma cell line selected after prolonged exposure to chemotherapeutic drug dacarbazine revealed higher levels of CXCL8 and CXCR2 compared with parent cells as a signature of drug resistance. CXCR2 neutralization markedly improved sensitivity to dacarbazine in melanoma cells. CONCLUSION: These data provide insights into what drives melanoma cells to survive after chemotherapy treatment, thus pointing to strategies for developing combined drug therapies for combating the problem of chemotherapy resistance in melanoma.

Secreted semaphorin 5A suppressed pancreatic tumour burden but increased metastasis and endothelial cell proliferation.

BACKGROUND: Our earlier reports demonstrated that membrane-bound semaphorin 5A (SEMA5A) is expressed in aggressive pancreatic cancer cells and tumours, and promotes tumour growth and metastasis. In this study, we examine whether (1) pancreatic cancer cells secrete SEMA5A and (2) that secreted SEMA5A modulates certain phenotypes associated with tumour progression, angiogenesis and metastasis through various other molecular factors and signalling proteins. METHODS AND RESULTS: In this study, we show that human pancreatic cancer cell lines secrete the extracellular domain (ECD) of SEMA5A (SEMA5A-ECD) and overexpression of mouse Sema5A-ECD in Panc1 cells (not expressing SEMA5A; Panc1-Sema5A-ECD; control cells - Panc1-control) significantly increases their invasion in vitro via enhanced ERK phosphorylation. Interestingly, orthotopic injection of Panc1-Sema5A-ECD cells into athymic nude mice results in a lower primary tumour burden, but enhances the micrometastases to the liver as compared with Panc1-control cells. Furthermore, there is a significant increase in proliferation of endothelial cells treated with conditioned media (CM) from Panc1-Sema5A-ECD cells and a significant increase in microvessel density in Panc1-Sema5A-ECD orthotopic tumours compared with those from Panc1-control cells, suggesting that the increase in liver micrometastases is probably due to increased tumour angiogenesis. In addition, our data demonstrate that this increase in endothelial cell proliferation by Sema5A-ECD is mediated through the angiogenic molecules - interleukin-8 and vascular endothelial growth factor. CONCLUSION: Taken together, these results suggest that a bioactive, secreted form of Sema5A-ECD has an intriguing and potentially important role in its ability to enhance pancreatic tumour invasiveness, angiogenesis and micrometastases.

CXCR1 and CXCR2 enhances human melanoma tumourigenesis, growth and invasion.

The aggressiveness of malignant melanoma is associated with differential expression of CXCL-8 and its receptors, CXCR1 and CXCR2. However, the precise functional role of these receptors in melanoma progression remains unclear. In this study, we investigate the precise functional role of CXCR1 and CXCR2 in melanoma progression. CXCR1 or CXCR2 were stably overexpressed in human melanoma cell lines, SBC-2 (non-tumourigenic) and A375P (low-tumourigenic) exhibiting low endogenous expression of receptors. Functional assays were performed to study the resulting changes in cell proliferation, motility and invasion, and in vivo tumour growth using a mouse xenograft model. Our data demonstrated that CXCR1- or CXCR2-overexpressing SBC-2 and A375P melanoma cells had enhanced proliferation, chemotaxis and invasiveness in vitro. Interestingly, CXCR1 or CXCR2 overexpression in SBC-2 cells induced tumourigenicity, and A375P cells significantly enhanced tumour growth as examined in vivo. Immunohistochemical analyses showed significantly increased tumour cell proliferation and microvessel density and reduced apoptosis in tumours generated from CXCR1- or CXCR2-overexpressing melanoma cells. CXCR1- or CXCR2-induced modulation of melanoma cell proliferation and migration was observed to be mediated through the activation of ERK1/2 phosphorylation. Together, these studies demonstrate that CXCR1 and CXCR2 play essential role in growth, survival, motility and invasion of human melanoma.

Expression of interleukin 8 and its receptors in human colon carcinoma cells with different metastatic potentials.

PURPOSE: In the present study, we examined the expression of a multifunctional cytokine, interleukin 8 (IL-8), and its receptors, CXCR1 and CXCR2, in human colon carcinoma cells with different metastatic potentials and determined their role in modulating phenotypes associated with metastasis. EXPERIMENTAL DESIGN: IL-8, CXCR1, and CXCR2 protein and mRNA expression were examined using ELISA, immunocytochemistry, and reverse transcription-PCR in human colon carcinoma cells with different metastatic potentials. IL-8-mediated proliferation, migration, and tumor-endothelial cell interaction were analyzed. RESULTS: IL-8 mRNA and protein expression was very low in Caco2 cells but elevated in KM12C cells and very high in KM12L4 cells, suggesting an association between the IL-8 production and metastatic potential. Similarly, CXCR1 and CXCR2 expression was lower in Caco2 cells than in low and high metastatic KM12C and KM12L4 cells. The recombinant human IL-8 enhanced the proliferation of colon carcinoma cells. Furthermore, proliferation of low and high metastatic cells expressing different levels of IL-8 was inhibited by neutralizing antibodies to IL-8, CXCR1, and CXCR2. We observed significant differences in the invasive potential of colon carcinoma cells expressing different levels of IL-8. In addition, we observed that IL-8 modulates adhesion of tumor cells to endothelial cells in an autocrine and paracrine manner. CONCLUSION: Our present data suggest an association between constitutive expression of IL-8 and aggressiveness in human colon carcinoma cells and the possible role of IL-8 in modulating different metastatic phenotypes associated with progression and metastasis.

Expression of interleukin-8 in human metastatic endometrial carcinoma cells and its regulation by inflammatory cytokines.

In the present study, we analyzed the expression of a multifunctional cytokine, interleukin-8 (IL-8), in metastatic endometrial carcinoma cells. Our data demonstrate that human serous papillary endometrial adenocarcinoma (SPEC) and human endometrial adenocarcinoma (HEC) cells expressed steady-state IL-8-specific mRNA transcript and secreted IL-8 protein. The levels of IL-8 mRNA in SPEC-2 cells established from stage IV serous papillary adenocarcinoma were three-fold higher as compared to endometrial adenocarcinoma cells, HEC-1 A, established from stage IA endometrial cancer. Further, we observed higher levels of IL-8 mRNA and protein expression in the metastatic variants of SPEC-2 and HEC-1A cells as compared to the parent cell lines, demonstrating that IL-8 expression was associated with metastatic potential. Further, the treatment of endometrial carcinoma cells with inflammatory cytokines, IL-1beta and tumor necrosis factor-alpha (TNF-alpha), demonstrated that IL-1beta and TNF-alpha induced IL-8 expression in endometrial cancer cells. IL-1beta was a more potent inducer of IL-8 expression than TNF-alpha in our studies. These data demonstrate that constitutive and induced IL-8 expression in endometrial carcinoma cells might be an important regulatory mechanism of tumor growth and metastasis.

IL-8 expression in malignant melanoma: implications in growth and metastasis.

This review article has described briefly studies supporting the concept that IL-8 expression and its regulation by inflammatory cytokines like IL-1 may play an important role in controlling the phenotypes associated with melanoma progression and metastasis. It is clear from the experiments presented here that IL-8 is an important autocrine multifunctional cytokine that modulates melanoma/cell proliferation, migration by induction of extracellular matrix degradation enzymes and induces neovascularization, all of which are critical for melanoma growth and metastasis. In addition, their expression in melanoma tumor specimens suggests an association between IL-8 expression and tumor aggressiveness. Further, inflammatory cytokines produced by either tumor cells or stromal cells may regulate IL-8 expression, which can control melanoma growth and enhance our current knowledge regarding melanoma progression and metastasis. Understanding these events and their significance will allow us to design novel therapeutic approaches for treatment of melanoma.

Immune dysfunction despite high levels of immunoregulatory cytokine gene expression in autologous peripheral blood stem cell transplanted non-Hodgkin's lymphoma patients.

OBJECTIVE: In the present studies, we examined the role of immunoregulatory cytokine gene expression in immune reconstitution following high-dose chemotherapy and peripheral stem cell transplantation. MATERIALS AND METHODS: We analyzed the steady-state mRNA cytokine levels and the immune phenotype and function in the peripheral blood mononuclear cells from intermediate-grade non-Hodgkin's lymphoma patients prior to and following high-dose chemotherapy and peripheral stem cell transplantation. RESULTS: Significantly higher mRNA levels of both type 1 and type 2 cytokines and monokines were observed in patients undergoing high-dose chemotherapy and peripheral stem cell transplantation as compared with normal healthy individuals. Pretransplant mRNA levels of interleukin-2, -4, -8, -10, interferon-gamma and tumor necrosis factor-alpha were significantly higher than in normal individuals. In addition, on days 30 and 100 following transplantation interleukin-10 levels were significantly increased compared with pretreatment levels. In contrast, the levels of interleukin-2 mRNA and interferon-gamma were decreased significantly on day 365 compared with pretransplant levels. CONCLUSIONS: The high levels of cytokine mRNA transcripts, both prior to and following peripheral stem cell transplantation, were not due to an increased cellular frequency; rather, they appear to be due to abnormal cellular activation. However, T-cell function is significantly depressed compared with normal donors, which is associated with significantly higher levels of cellular-dependent T cell inhibitory activity and, we suggest herein, high levels of interleukin-10, a type 2 cytokine.

Expression of interleukin-8 in primary and metastatic malignant melanoma of the skin.

It was recently demonstrated that interleukin-8 (IL-8) is produced by cultured melanoma cells and acts as an essential autocrine growth factor. Earlier studies from our laboratory demonstrated a direct correlation between IL-8 expression in human variant cell lines and their metastatic potential in nude mice. In the present study we examined the expression of IL-8 in human malignant melanomas using immunohistochemistry to correlate IL-8 levels with disease stage. None of the radial growth phase (RGP) tumours (melanoma in situ) expressed IL-8. In contrast, 50% of the vertical growth phase (VGP) tumours (invasive melanoma), which have a high risk of metastasis, showed IL-8 immunoreactivity. Further, all the metastatic lesions analysed showed intense staining for IL-8; the levels were higher than those observed in the primary skin tumours. In summary, the data suggest an association between the expression of IL-8 and the metastatic phenotype.

Fas-FasL-mediated CD4+ T-cell apoptosis following stem cell transplantation.

We report the preferential expression of Fas on CD4+ T cells and Fas ligand (FasL) on monocytes and their potential role in the selective loss of CD4+ T cells in breast cancer patients undergoing high-dose chemotherapy and peripheral blood stem cell transplantation (PSCT). A high frequency of apoptotic CD4+ T cells (28-51%) is observed during the first 100 days after PSCT concomitant with a significant increase in monocyte frequency and FasL expression (11.6-23%) on monocytes. The preferential expression of Fas on CD4+ T cells (73-92%) in the peripheral blood (PB) of these patients is associated with a significantly higher frequency of CD4+ T-cell apoptosis compared with CD8+ T cells (28-47%) and CD4+ T cells (46 +/- 5.7%) in normal PB. These data suggest that "primed" Fas+ CD4+ lymphocytes interact with activated monocytes that express FasL, resulting in apoptosis, leading to deletion of CD4+ T cells, an inversion in the CD4:CD8 T-cell ratio, and immune dysfunction. The prevention of CD4+ T-cell apoptosis and improved immune reconstitution by the manipulation of PB stem cell products, blockade of Fas-FasL interactions, or cytokine support after transplantation may be important adjuvant immunotherapeutic strategies in patients undergoing high-dose chemotherapy and PSCT.

Expression of interleukin-10 in isolated CD8+ T cells and monocytes from growth factor-mobilized peripheral blood stem cell products: a mechanism of immune dysfunction.

Previous reports showed the abnormal activation of immune cells in growth factor-mobilized peripheral blood stem cell (PBSC) products, which might be responsible for depressed T cell responsiveness to mitogens compared with normal peripheral blood mononuclear cells (PBMC). In the present study, the mRNA expression levels of interleukin (IL)-2, IL-4, IL-10, and interferon-gamma (IFN-gamma) were significantly higher in CD4+ and CD8+ T cells from mobilized PBSC products compared with CD4+ and CD8+ cells from normal peripheral blood (PB). The mRNA expression levels of IL-4 and IL-10 were significantly higher in CD8+ compared with CD4+ cells from PBSC products. However, the expression of IL-2 and IFN-gamma mRNA transcripts was similar in the CD4+ and CD8+ T cells from PBSC products. The levels of IL-10, IL-8, and tumor necrosis factor (TNF)-alpha mRNA were also significantly higher in monocytes isolated from PBSC products compared with monocytes isolated from normal PB. Expression of IL-10-specific mRNA in monocytes also was significantly higher than the levels observed in CD8+ cells from PBSC products. We suggest that both CD4+ and CD8+ cells in the PBSC products are highly activated. However, their response to phytohemagglutinin (PHA) mitogenesis is depressed in part because of IL-10 expression by CD8+ cells and monocytes in addition to the higher levels of monocyte-dependent T cell inhibitory activity. These data demonstrate that aberrant IL-10 expression in the CD8+ T cells and monocytes present in PBSC products may represent a possible mechanism of immune dysfunction in patients after high-dose chemotherapy (HDT) and peripheral blood stem cell transplantation (PBSCT).

Comparison of monocyte-dependent T cell inhibitory activity in GM-CSF vs G-CSF mobilized PSC products.

This study compares the immune properties of peripheral blood stem cell (PSC) products mobilized with different hematopoietic growth factors (HGFs) as well as apheresis products and peripheral blood leukocytes (PBL) from normal individuals. We found that monocytes in mobilized PSC products appear to inhibit T cell function independent of whether granulocyte colony-stimulating factor (G-CSF) or granulocyte-macrophage colony-stimulating factor (GM-CSF) was used for mobilization. In addition, the GF used to mobilize the stem cell product may be less important to the CD4:CD8 ratio than the extent of prior chemotherapy, as we found an inverse correlation between chemotherapy and the CD4:CD8 ratio. In other observations, all apheresis products, whether mobilized or unmobilized, contained significantly more monocytes compared to normal PBL. The mononuclear cells (MNC) from G-CSF or GM-CSF mobilized PSC products had a similar T cell phytohemagglutinin (PHA) mitogenic response that was significantly lower (P = 0.001 and P = 0.005, respectively) than non-mobilized apheresis products. We also examined the T cell inhibitor (TI) activity of the MNC from the PSC products for allogeneic lymphocyte proliferation and found that PSC products significantly reduced the proliferation of allogeneic PBL to PHA. A significant correlation (P = 0.001, r = 0.517) between the frequency of monocytes and TI activity also was observed.

Immunoregulatory cytokines in bone marrow and peripheral blood stem cell products.

In these studies, we compared the phenotype, function, and expression of type 1, type 2, and monocyte-associated cytokine mRNA transcripts in autologous bone marrow (BM) and growth factor-mobilized peripheral blood stem cell (PSC) products. These studies demonstrate that lymphocytes and monocytes in stem cell products are abnormally activated, expressing significantly higher levels of interleukin (IL)-2, 4 and 10, interferon gamma (IFN-gamma), and tumor necrosis factor alpha (TNF-alpha), but not IL-8, as compared to normal peripheral blood mononuclear cells (PBMC). In addition, the levels of IL-2, IL-10 and TNF-alpha are significantly higher in mobilized PSC as compared to BM products. The high cytokine levels are unexpected as T cell function in stem cell products is depressed. PSC products have high levels of T cell inhibitory activity, which directly correlates with IL-10 expression, both of which are mechanisms that might be involved in the immune dysfunction within stem cell products used for autologous stem cell transplantation. These data demonstrate that: (1) immune cells in autologous BM and PSC products are activated with the expression of high levels of type 1 and type 2 cytokines as well as monokines; (2) PSC products contain a high frequency of monocytes which mediate T cell inhibitory activity; and (3) despite the high levels of cytokine expression, T cell function in stem cell products is depressed. The significance of these immune abnormalities within stem cell products for myeloid and lymphoid recovery following autologous stem cell transplantation remains to be determined.

Regulation of interleukin 8 expression in human malignant melanoma cells.

Here, we report the molecular regulation of interleukin (IL)-8 expression in human melanoma cells. The inflammatory cytokines IL-1beta and tumor necrosis factor-alpha (TNF-alpha) up-regulated IL-8 expression, in a time- and concentration-dependent manner, in three metastatic melanoma variants, SBC-2 (nonmetastatic), A375P (low metastatic), and A375SM (high metastatic), by increased transcription of the IL-8 gene, leading to increased levels of IL-8 mRNA and protein production. Furthermore, we report that IFN-alpha and IFN-beta did not inhibit steady-state IL-8 production. However, IFN-alpha and IFN-beta inhibited IL-1beta or TNF-alpha-mediated up-regulation of IL-8 mRNA. In addition, IFN-beta demonstrated a more potent inhibitory effect at a lower concentration than did IFN-alpha. Both pretreatment and simultaneous treatment of melanoma cells with IFN-alpha or IFN-beta inhibited the IL-1beta and TNF-alpha up-regulation of IL-8 mRNA levels. This inhibition was at the transcriptional levels and was unaffected by a protein synthesis inhibitor, suggesting that this did not require de novo protein synthesis. Further, modulation of IL-8 levels by IL-1beta, alone or in combination with IFN-beta, affected the proliferation of melanoma cells. In summary, our data suggest that the up-regulation of IL-8 expression in melanoma cells is regulated at the transcriptional level and is rapidly and specifically inhibited by IFN-alpha or IFN-beta, independent of de novo protein synthesis, perhaps due to a transient modification of a preexisting factor(s).

Monocyte activation by an oral immunomodulator (bestatin) in lymphoma patients following autologous bone marrow transplantation.

Bestatin (ubenimex), an inhibitor of aminopeptidase, is an oral immunomodulator that binds to CD13 (aminopeptidase N) on macrophages/monocytes. To examine its immunomodulatory effect after high-dose therapy and autologous bone marrow transplantation (BMT), a dose-finding phase Ib trial was conducted with 30 Hodgkin's disease and non-Hodgkin's lymphoma patients who received no drug (control), 10 and 30 mg (low dose), or 90 and 180 mg (high dose) of bestatin daily for 60 days following autologous BMT. Bestatin administration was initiated when the absolute neutrophil count was greater than 250/mm3 on 2 consecutive days. The serum neopterin levels, an indicator of monocyte/macrophage activation, increased in the high-dose group compared to the control group (not significantly) and the low-dose group (significantly). Similarly, the colony-stimulating activity in the sera was significantly increased in the high-dose group compared to the control and low-dose groups. We also examined the expression of cell-surface markers on monocytes in these patients by fluorescent cytometry analysis. There was no significant difference either in the frequency or absolute number of monocytes (CD14+) among the three groups at any time. However, a significant increase in the frequency of CD16(FcgRIII)-positive monocytes (a marker of activation) was observed in the high-dose group compared to controls from day 14 to day 60 after the start of bestatin administration. Further, the frequency of HLA-DR+ monocytes (another marker of activation) was significantly increased in the high-dose group. These results indicate that bestatin at higher doses (90 and 180 mg daily), but not lower doses, activates macrophages/monocytes, as demonstrated by phenotypic marker (HLA-DR and CD16) up-regulation, and this provides augmentation of neopterin and colony-stimulating activity in the serum of patients following autologous BMT.

Immunologic phenotype and function in human bone marrow, blood stem cells and umbilical cord blood.

The T cell-mediated antineoplastic activity observed following allogeneic transplantation and the suggestion of improved therapeutic efficacy by autologous peripheral stem cell transplantation (PSCT) as compared to autologous bone marrow transplantation (ABMT) for non-Hodgkin's lymphoma (NHL) stimulated our interest in the immunologic competence of stem cell products. We report the immune phenotype and function of normal peripheral blood (PB) cells, bone marrow (BM) cells from normal donors and cancer bearing patients, GM-CSF-mobilized and apheresed blood mononuclear cells from NHL patients, unmobilized apheresed mononuclear cells from normal volunteers and umbilical cord blood (CB). The analyses include three-color fluorescent cytometry of the major hematologic and immunologic phenotypes as well as natural killer (NK) activity, natural suppressor (NS) activity, and phytohemagglutinin (PHA) and pokeweed (PWM) mitogenesis. These studies demonstrated an increased frequency of T cells in apheresis products as compared to BM and CB products. Specifically, the mobilized PSC had significant increases in CD3+, CD4+, CD45RO+ and CD56+ cells relative to BM cells. In addition, the frequency of TCR gamma/delta + cells in all the stem cell products, with the exception of CB, were also increased compared to normal peripheral blood leukocytes (PBL). However, all the stem cell products had a significant depression in T (PHA mitogenesis) and B (PWM mitogenesis) cell function. The depression in immune cell functionality, in the PSC products was perhaps due to the high frequency of monocytes which appeared to be increased due to both mobilization and apheresis. The frequency of the NK cell phenotype (CD56) but not function was increased in the mobilized PSC products, while the NK cell function in the BM products from cancer patients but not normal donors was depressed as compared to normal PBL. In summary, there are significant differences in the cellular phenotypes and immunologic competence among the various stem cell products with potential therapeutic implications.