Dominique Gales

The Chemokine CXCL8 in Carcinogenesis and Drug Response

Although the functions of chemokines in the regulation of immune processes have been studied in some detail, the role of these biomolecules in cancer is not fully understood. Chemokines mediate migration of immune cells and other functions related to immunity. They are also involved in oncogenesis and in tumor progression, invasion, and metastasis through mechanisms similar to their roles in immune functions. Various chemokines also promote cell proliferation and resistance to apoptosis of stressed cells. Consequently, chemokines and their receptors present potential therapeutic targets for anticancer drugs. The chemokine CXCL8, also known as interleukin-8 (IL8), is a proinflammatory molecule that has functions within the tumor microenvironment. Due to its potent angiogenic effects and the activity of the chemokine and its receptors in the promotion of invasion and metastasis, CXCL8 and its receptors are now considered as attractive targets for cancer therapy. This review relates the current understanding of the regulation, signaling, and functions of CXCL8 that contribute to tumor growth and metastasis, and of its role in drug response.

Variable NF-κB pathway responses in colon cancer cells treated with chemotherapeutic drugs.

BackgroundThe nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signaling pathway is activated in cells exposed to various stimuli, including those originating on the cell surface or in the nucleus. Activated NF-κB signaling is thought to enhance cell survival in response to these stimuli, which include chemotherapy and radiation. In the present effort, we determined which anticancer drugs preferentially activate NF-κB in colon cancer cells.MethodsNF-κB reporter cells were established and treated with 5-fluorouracil (5-FU, DNA/RNA damaging), oxaliplatin (DNA damaging), camptothecin (CTP, topoisomerase inhibitor), phleomycin (radiomimetic), or erlotinib (EGFR inhibitor). The activation of NF-κB was assessed by immunofluorescence for p65 translocation, luciferase assays, and downstream targets of NF-κB activation (cIAP2, and Bcl-XL) were evaluated by immunoblotting, by ELISA (CXCL8 and IL-6 in culture supernatants), or by gene expression analysis.ResultsColon cancer cells responded variably to different classes of therapeutic agents, and these agents initiated variable responses among different cell types. CPT activated NF-κB in SW480 colon cancer cells in a dose-dependent manner, but not in HCT116 cells that were either wild-type or deficient for p53. In SW480 colon cancer cells, NF-κB activation by CPT was accompanied by secretion of the cytokine CXCL8, but not by up-regulation of the anti-apoptotic genes, cIAP2 or Bcl-XL. On the contrary, treatment of HCT116 cells with CPT resulted in up-regulation of CXCR2, a receptor for CXCL8, without an increase in cytokine levels. In SW480 cells, NF-κB reporter activity, but not cytokine secretion, was inhibited by SM-7368, an NF-κB inhibitor.ConclusionThe results show that, in response to cancer therapeutic agents, NF-κB activation varies with the cellular make up and that drug-induced NF-κB activation may be functionally uncoupled from anti-apoptotic outcomes found for other stimuli. Some cancer cells in a heterogeneous tumor tissue may, under therapeutic pressure, release soluble factors that have paracrine activity on neighboring cells that express the cognate receptors.

Effects of consumption of whole grape powder on basal NF-κB signaling and inflammatory cytokine secretion in a mouse model of inflammation

Dietary consumption of polyphenol-rich fruits, such as grapes, may reduce inflammation and potentially prevent diseases linked to inflammation. Here, we used a genetically engineered murine model to measure Nuclear Factor kappa-light-chain-enhancer of activated B cells (NF-κB) activity and pro-inflammatory cytokine secretion to test the hypothesis that oral consumption of whole grape formulation reduces inflammatory signaling in the body. NF-κB luciferase reporter mice were divided into two groups, one which was fed an experimental diet formulated with 4% (w/w) whole grape powder (WGP) or another which was fed a control diet formulated with 3.6% glucose/fructose (w/w) combination. Simulated inflammation was induced in the mice by intraperitoneal injection of lipopolysaccharide (LPS). In vivo imaging was used to determine the effect of each diet on NF-κB activity. We found that there were no significant differences in weight gain between the WGP and control diet groups. However, there was a statistically significant (p<0.0001) difference in the progression of basal levels of NF-κB signaling between mice fed on control or WGP diet. There were no significant differences in NF-κB reporter indices between WGP- and control-diet groups after either acute or repeated inflammatory challenge. However, terminal blood collection revealed significantly (p<0.01) lower serum concentrations of the inflammatory cytokines Interleukin-6 (IL-6) and Tumor Necrosis Factor alpha (TNFα) only among WGP diet mice subjected to acute inflammatory challenge. Overall, these data suggest that while diets supplemented with WGP may suppress steady-state low levels of inflammatory signaling, such a supplementation may not alleviate exogenously induced massive NF-κB activation.

Abstract 5910: Influence of 3D- cultured prostate cancer and stromal cells on drug response

Background: Response to chemotherapeutic drugs presents a significant challenge to effective treatment of advanced prostate cancer. Exploration of therapeutic response has focused largely on the tumor cell. However, evidence suggests mechanisms that involve the tumor microenvironment mediate response to therapeutics. Therefore, it is imperative to mimic the in vivo tumor microenvironment, to bridge the gap between in vitro studies and the development of therapeutics. The concept of three-dimensional (3D) cell culture and co-culture of multiple cell types is critical to predict therapeutic efficacy and outcome. Thus, these studies sought to determine the distinct responses to anti-cancer drugs in 3D cell spheroids. Methods: In this study, we utilized metastatic prostate cancer cells (PC3) and fluorescently labeled prostate bone stromal cell line (HS27a) to generate tumor spheroids. Tumor spheroid viability was measured at 72 and 120 h using the CellTiter-Glo 3D assay after treatment with three common anti-cancer drugs, Docetaxel (4ηM), Enzalutamide (30μM), and Bicalutamide (30μM).Treatments were compared with spheroids originated from HS27a and PC3 cells alone and in co-culture. The cell viability percentage was normalized using the untreated cells as 100% cell survival. Results: Spheroid formation increased resistance to Docetaxel, Enzalutamide, and Bicalutamide. However, PC3 spheroid cells was significantly higher compared to HS27a and co-culture spheroids. For all three spheroids, as the size of the spheroids increase, cellular resistance to anti-cancer and hormone drugs increased. Conclusion: The findings in this study demonstrated that the use for 3D-cell cultures influence many aspects of PCa behavior including, proliferation rates, and response to chemotherapeutics. Note: This abstract was not presented at the meeting. Citation Format: Dominique N. Gales, Sonni Miller, Temesgen Samuel, Clatyon Yates. Influence of 3D- cultured prostate cancer and stromal cells on drug response [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5910. doi:10.1158/1538-7445.AM2017-5910

Bromoethylindole (BEI-9) redirects NF-κB signaling induced by camptothecin and TNFα to promote cell death in colon cancer cells

Chemotherapeutic regimens containing camptothecin (CPT), 5-fluorouracil, and oxaliplatin are used to treat advanced colorectal cancer. We previously reported that an indole derivative, 3-(2-bromoethyl)indole (BEI-9), inhibited the proliferation of colon cancer cells and suppressed NF-κB activation. Here, we show that a combination of BEI-9 with either CPT or tumor necrosis factor alpha (TNFα) enhances cell death. Using colorectal cancer cells, we examined the activation of NF-κB by drugs, the potential of BEI-9 for inhibiting drug-induced NF-κB activation, and the enhancement of cell death by combination treatments. Cells were treated with the chemotherapeutic drugs alone or in combination with BEI-9. NF-κB activation, cell cycle profiles, DNA-damage response, markers of cell death signaling and targets of NF-κB were evaluated to determine the effects of single and co-treatments. The combination of BEI-9 with CPT or TNFα inhibited NF-κB activation and reduced the expression of NF-κB-responsive genes, Bcl-xL and COX2. Compared to CPT or BEI-9 alone, sequential treatment of the cells with CPT and BEI-9 significantly enhanced caspase activation and cell death. Co-treatment with TNFα and BEI-9 also caused more cytotoxicity than TNFα or BEI-9 alone. Combined BEI-9 and TNFα enhanced cell death through caspase activation and cleavage of the switch-protein, RIP1 kinase. BEI-9 reduced the expression of COX2 both alone and in combination with CPT or TNF. We postulate that BEI-9 enhances the effects of these drugs on cancer cells by turning off or redirecting NF-κB signaling. Therefore, the combination of BEI-9 with drugs that activate NF-κB needs to be evaluated for clinical applications.

Abstract 1286: Cytokine response of stromal cells to prostate cancer chemotherapeutics

Non-responsiveness to chemotherapeutic drugs presents a significant challenge to the effective treatment of advanced prostate cancer. Exploration of the therapeutic response in prostate cancer has largely focused on the epithelial tumor cells. However, evidence suggests that the tumor microenvironment may modulate response to chemotherapeutics. The tumor microenvironment consists of immune, fibroblastic, and vascular cells, as well as extracellular matrix (ECM) and stromal-derived soluble factors, all of which contribute to interactions within the tumor microenvironment. It has been demonstrated that tumor cells cross-talk with stromal cells, to establish a protective niche at primary and distant sites and to facilitate survival of cancer cells. Such tumor-stroma cell cross-talk often involves secreted factors, such as cytokines. Previous studies have illustrated the importance of cytokines and cytokine receptors as modulators of drug response. In this study, we sought to identify cytokine factors expressed in response to drugs clinically used to control prostate cancer. To obtain the expression profiles, we conducted a human cytokine and chemokine array, which consisted of 84 specific genes. Analysis of the prostate cancer and stromal cells treated with Docetaxel (4nM), Enzalutamide (30μM) and Bicalutamide (30μM) revealed differential expression patterns. The effect of chemotherapeutics on the expression of the cytokine IL-16 and its cognate receptors CD9 and CCR5 in prostate cancer and stromal cell lines was further examined. We found IL-16 expression increased several fold following chemotherapeutic treatment in stromal cell lines compared to prostate cancer cell lines. To validate gene expression, we examined the expression of IL-16 and its receptors by RT-PCR. All experiments were performed in triplicates. Student t- tests were used to determine the statistical significance between groups. IL-16 expression increased following exposure to chemotherapeutics in stromal cell lines. CCR5 expression showed a decrease in PC3 prostate cancer cells but no significant changes were observed in LNCaP cells. CD9 expression showed an increase in both LNCaP and PC3 cell lines. Collectively, these results indicate that chemotherapy of prostate cancer may alter the expression of IL-16 and its receptors. We hypothesize that IL-16 is a soluble stromal factor that is expressed in response to anticancer drugs, and may have paracrine activity on neighboring cells that express the cognate receptors. Citation Format: Dominique Gales, Clayton C. Yates, Temesgen Samuel. Cytokine response of stromal cells to prostate cancer chemotherapeutics. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1286.

Abstract 3783: Co-treatment of metastasis-derived colon cancer cells with bromoethyl indole (BEI) enhances camptothecin or TNF alpha-induced cell death

Colorectal cancer is still the third most deadly cancer in the United States. Currently, there are limited choices of targeted anticancer agents that are durably effective against colorectal cancer. Therefore, chemotherapeutic regimens containing the drugs Camptothecin (CPT), 5-FU and Oxaliplatin remain the mainstays for the treatment of advanced colorectal cancer. We recently found that the indole-derivative small compound Bromoethyl Indole (BEI) potently inhibits the proliferation of colon cancer cells and also suppresses NF-kB activation. In this study, we investigated if the combination of BEI with either CPT or TNFα would enhance cell death in vitro. Metastasis-derived parental or engineered NF-kB-reporter colon cancer cells were used to examine 1) the activation of NF-kB signaling by clinically used drugs, 2) the potential for BEI to inhibit drug-induced NF-kB activation, and 3) the potential for combination treatment to enhance cell death in vitro. Cells were treated with either the drugs alone or in combination with BEI at varying concentrations. NF-kB-inducing concentration of CPT (0.6 to 2.5 μM), TNFα (25 to 50 ng/ml), and BEI concentrations of 1 to 50 μM range were tested. Cell cycle profiles and cell death markers were assessed to determine the effects of single or co-treatments. The expressions of Bcl-xL and cIAP2 (BIRC3) proteins were examined to monitor the targets of NF-kB activation. Our results show that 1) both TNFα and CPT induce NF-kB signaling in metastasis-derived colon cancer cells, 2) combination of BEI and TNFα or CPT inhibits such a drug-induced NF-kB activation and reduces the expression of NF-kB responsive genes, 3) sequential treatment of the cells with CPT and BEI delivers the best outcome, increasing cell death by up to 3-fold compared to either CPT or BEI alone, and 4) co-treatment of the cells with TNFα and BEI increases cell death by up to 2-fold compared to either TNFα or BEI alone, 5) BEI is most effective in inducing cell death at concentrations between 2 and 10 μM. We anticipate that BEI will improve the therapeutic index of some chemotherapeutic drugs. Therefore, the potential benefit for combination of BEI with NF-kB activating drugs needs to be evaluated in vivo. Citation Format: Rupak Chowdhury, Sonni A. Miller, Dominique Gales, Jason White, Upender Manne, Temesgen Samuel. Co-treatment of metastasis-derived colon cancer cells with bromoethyl indole (BEI) enhances camptothecin or TNF alpha-induced cell death. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3783.

Abstract B11: Stromal response to prostate cancer therapeutics

Docetaxel (D) is a clinically used front-line chemotherapeutic agent for castration resistant advanced prostate cancer (Pca). However, its long-term benefits are limited, with most patients eventually progressing because of inherent or acquired drug resistance. The treatment of advanced Pca is a significant challenge and there are no effective treatments that stably suppress the disease. The mechanisms behind the resistance to Docetaxel are not fully understood. It is well accepted that tumor microenvironment is essential for tumor cells survival, cancer progression and metastasis. However, the mechanisms by which tumor cells interact with their surrounding at different stages of cancer development or during chemotherapy are largely unidentified. The central goal is to identify the cellular and biochemical responses of stromal cells in tumor microenvironment with which prostate cancer cells interact. Specifically, the interaction of prostate cancer cells with stromal cells of the prostate or the bone microenvironment in the presence of clinically used cancer therapeutics will be examined. Key regulators of these interactions will also be identified. Such regulators include cytokines, growth factors, proteins, and their receptors. By identifying these regulators and their contribution to tumor drug response, novel therapeutics targeted to the microenvironment can be developed. Therefore, we hypothesize that the response of prostate stromal cells exposed to chemotherapeutic agents could modify drug response or therapeutic outcome of prostate cancer. Cellular proliferation was analyzed by MTT assay. The IC50 of Docetaxel for WPMY-1 (normal stromal), LNCaP (androgen dependent), DU-145 (androgen independent), and HS27A (bone stromal) showed inhibitory effects between 2-10nM. Cell cycle profiles were assessed by flow cytometry. The analysis showed that Docetaxel (2-50nM) induced prominent G2-M arrest in prostate and stromal cells within 24 hours. Gene expression profiling of prostate cancer and stromal cells lines were evaluated. To obtain the expression profiles, we conducted a human cytokines and chemokines array, which consisted of 84 specific genes. Analysis of the prostate cancer and stromal cells treated with Docetaxel revealed differential expression patterns. These results suggest Docetaxel exhibits strong anti-proliferative effects and analysis of the gene array suggest regulation of genes associated with, cell growth, survival, proliferation, metastasis, angiogenesis, and apoptosis. Note: This abstract was not presented at the conference. Citation Format: Dominique Nicole Gales, Khalda Fadlalla, Upender Manne, Clayton Yates, Temesgen Samuel. Stromal response to prostate cancer therapeutics. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Drug Sensitivity and Resistance: Improving Cancer Therapy; Jun 18-21, 2014; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(4 Suppl): Abstract nr B11.

Abstract C39: Docetaxel-induced growth and cell cycle phenotypes in prostate stromal cells

Background: Docetaxel (D) is a clinically used front-line chemotherapeutic agent for castration resistant advanced prostate cancer. However, its long-term benefits are limited with most patients eventually progressing because of inherent or acquired drug resistance. The treatment of advanced prostate cancer is a significant challenge and there are no effective treatments that stably suppress the disease. The mechanisms behind resistance to Docetaxel are not fully understood. Since in vivo Docetaxel cytotoxicity is directed not only to cancer cells, we hypothesize that the response of prostate stromal cells exposed to chemotherapeutic agents could modify drug response or therapeutic outcome in prostate cancer. One of cellular responses to chemotherapy is NF-kB activation and inflammatory signaling. NF-κB activation has been proposed as an important event in PCa development, castration-resistant progression and resistance to chemotherapy. Objective: In this study, we examined the response of prostate stromal (WPMY- 1) cells to Docetaxel. Current Results: Cellular proliferation was analyzed by MTT assay. The IC50 of Docetaxel for WPMY-1 cells was 10nM. Cell cycle profiles were assessed by flow cytometry. The analysis showed that Docetaxel (10-100nM) induced prominent G2-M arrest in these stromal cells within 24 hours, especially in the higher dose ranges. Interestingly lower doses of Docetaxel (10nM), in contrast to higher doses, consistently resulted in accumulation of sub-G1 population (28% 10nM vs 6% in 100nM) of cells suggestive of fragmented DNA associated with apoptotic process. We show that Docetaxel exhibits strong anti-proliferative effects on prostate stromal WPMY-1 cells. Conclusions: These preliminary results show that stromal cells are sensitive to Docetaxel. Why lower doses of the drug induce sub-G1 (apoptotic) accumulation while higher doses preferentially induce G2-M arrest is currently unknown. Future Steps: We will compare responses to D between prostate cancer cells and stromal cells in individual and co-culture models. NF-kB activity following D treatment or radiation, and the expression of genes in NF-kB pathway in D treated cells will be examined. Citation Format: Dominique Gales, Brionna McMcmeans, Temesgen Samuel. Docetaxel-induced growth and cell cycle phenotypes in prostate stromal cells. [abstract]. In: Proceedings of the Sixth AACR Conference: The Science of Cancer Health Disparities; Dec 6–9, 2013; Atlanta, GA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2014;23(11 Suppl):Abstract nr C39. doi:10.1158/1538-7755.DISP13-C39