IL-17E, a proinflammatory cytokine, has antitumor efficacy against several tumor types in vivo - PubMed (original) (raw)
IL-17E, a proinflammatory cytokine, has antitumor efficacy against several tumor types in vivo
Tania Benatar et al. Cancer Immunol Immunother. 2010 Jun.
Abstract
Interleukin-17E (IL-17E) belongs to a novel family of cytokines that possess significant homology to IL-17. IL-17E has potent inflammatory effects in vitro and in vivo. Overexpression of IL-17E in mice results in a T helper-2 (Th2)-type immune response, which includes the expansion of eosinophils through the production of IL-5, and elevated gene expression of IL-4 and IL-13 in multiple tissues. In this study, we show that IL-17E has antitumor activity in vivo, a previously unrecognized function of IL-17E. Antitumor efficacy of IL-17E was examined in a variety of human tumor xenograft models, including melanoma, breast, lung, colon, and pancreatic cancers. Injection of recombinant IL-17E every other day resulted in significant antitumor activity in these tumor models. In addition, the combination of IL-17E with chemotherapy or immunotherapy agents showed an enhanced antitumor efficacy in human tumor xenograft models in mice as compared to either agent alone. Antitumor activity was demonstrated using different routes of administration, including intraperitoneal, intravenous, and subcutaneous injection. Anticancer activity was shown for both mouse and human forms of IL-17E, which have a high degree of sequence identity. Tumor-bearing mice treated with IL-17E showed a significant increase in serum levels of IL-5 and increased numbers of eosinophils in peripheral blood compared to the control group. Spleens isolated from IL-17E-treated mice showed a significant increase in eosinophils that correlated with antitumor activity of IL-17E in a dose-response manner. Finally, we demonstrate that B cells are necessary for IL-17E-mediated antitumor activity and that IL-17E was found to activate signaling pathways in B cells in vitro. Taken together, these data demonstrate that IL-17E has antitumor activity in vivo, and support further investigation of the potential clinical use of IL-17E as an anticancer agent.
Figures
Fig. 1
Antitumor activity of mouse IL-17E given by different routes of administration. Human melanoma C8161 cells were subcutaneously injected into the right flank of CD-1 nude mice. Five days later, the mice were administered with 0.1 ml of PBS or mouse IL-17E (0.04 mg/kg) every 2 days either a intraperitoneally (ip) or b intravenously (iv) until the end of experiment. Tumors were removed and weights were measured (n = 10; *P < 0.05)
Fig. 2
Comparison of antitumor activities of mouse and human IL-17E in vivo. Human melanoma C8161 cells were subcutaneously injected into the right flank of CD-1 nude mice. Five days later, the mice were administered with 0.1 ml of PBS, mouse IL-17E (0.04 mg/kg), or human IL-17E (0.04 mg/kg) every 2 days until end of experiment. Tumors were removed and weights were recorded (n = 10; *P < 0.05)
Fig. 3
Antitumor activity of human IL-17E with subcutaneous administration. a Human melanoma C8161 cells were subcutaneously injected into the right flank of CD-1 nude mice. Five days later, hIL-17E was administered subcutaneously on the opposite flank into groups of mice (n = 10) at 0.04 mg/kg every 2 days for 4 weeks. At the end of the study, tumors were excised and weighed. b Dose–response antitumor activity of IL-17E. C8161 tumor-bearing CD-1 nude mice were injected subcutaneously into the opposite flank with IL-17E (0.144, 0.016, and 0.0018 mg/kg) or PBS every 2 days until the endpoint of experiment. At each timepoint, tumors were measured using calipers, and sizes were calculated. Each dose of hIL-17E was diluted in a final volume of 100 ul PBS for injection. A control group of mice were treated with 100 ul PBS. Standard errors of mean tumor volumes are shown
Fig. 4
Antitumor activity of human IL-17E against various human tumors, alone and in combination with cancer therapeutics. a–e Plots of sizes of tumor xenografts in CD-1 mouse models over the course of the study. Tumor sizes (mm3) were measured at each point shown in the growth curves. For each study, hIL-17E was administered by intravenous (i.v.) injection at 0.04 mg/kg every 2 days. Doses and routes of administration for cancer therapeutics are shown below. Control groups were given 100 ul PBS by i.v. injection every 2 days. All treatments were administered for the duration of the study. For combination studies, each agent was administered at the same dose level used for single-agent experiments. a Human colon adenocarcinoma (HT-29). Mice (n = 10 per group) were treated with hIL-17E, CPT-11 (20 mg/kg every 5 days, i.v.), Avastin (0.4 mg/kg every 2 days, i.v.), or combinations of CPT-11 and IL-17E or Avastin and IL-17E. b Human non-small cell lung carcinoma (NSCLC) (H460). Mice were treated with hIL-17E, Tarceva (100 mg/kg in 200 μl of vehicle solution, one time per day, oral), Taxotere (10 mg/kg in 100 μl of vehicle solution, one time per week, i.v.), or combinations of IL-17E and Tarceva or IL-17E and Taxotere. c Human melanoma (C8161). Mice were treated with hIL-17E, DTIC (80 mg/kg per mouse, one time per week, i.p) or a combination of IL-17E + DTIC. d Human breast adenocarcinoma (MDA-MB-435). Mice were treated with hIL-17E, Taxol (10 mg/kg in 100 μl of vehicle solution, one time per week, i.v.), cisplatin (4 mg/kg in 100 μl of vehicle solution, one time per week, i.v.), or combinations of IL-17E and Taxol or IL-17E and cisplatin. e Human pancreatic carcinoma (MIA PaCa-2). Mice were treated with hIL-17E, gemcitabine (100 mg/kg, one time per week, i.v.) or a combination of both agents. Standard error measurements for each data point did not exceed 25% of the mean tumor volume
Fig. 5
Tumor weight measurements from efficacy studies with IL-17E alone and in combination with various anticancer drugs. At the end of the in vivo efficacy studies (described in Fig. 4), tumors were excised and weighed. Tumor weights for colon cancer, NSCLC, melanoma, breast cancer, and pancreatic cancer models showed a statistically significant reduction in tumor weights (*P < 0.05) compared to those from PBS-treated mice (a–e)
Fig. 6
B cells are required for IL-17E antitumor activity. Human melanoma C8161 cells were implanted subcutaneously in the right flank of SCID and CD1 nude mice. Five days later, mice were treated intravenously with either 0.1 ml of PBS or human IL-17E (0.04 mg/kg) every 2 days for the duration of the experiment. At each timepoint, tumors were measured using calipers, and sizes were calculated and plotted (a). At the end of the study tumors were excised and weighed (b). Tumors from SCID mice treated with IL-17E were not significantly reduced compared to tumors from PBS-treated SCID mice (P = 0.4), whereas tumors from IL-17E-treated CD-1 mice were significantly smaller relative to control (*P = 0.04 relative to PBS control)
Fig. 7
IL-17E stimulates eosinophil numbers in blood and spleen. CD-1 nude mice bearing C8161 human xenografts were given intraperitoneal injections of either 0.1 ml PBS or IL-17E (0.04 mg/kg). Blood (a) and spleens (b) were collected, red blood cells were removed, and cells were analyzed for eosinophils by flow cytometry using anti-CCR3-PE antibodies. c Splenic eosinophils in spleens from tumor-bearing mice treated with increasing doses of IL-17E. CD-1 nude mice with C8161 xenografts were treated with subcutaneous injections of PBS (0.1 ml) or human IL-17E (1.2, 0.4, and 0.04 μg per mouse; equivalent to 0.048, 0.016, and 0.0018 mg/kg, respectively) every 2 days for 4 weeks. CCR3+ cells were quantitated by flow cytometry as described above. Percentages of CCR3+ cells in the granulocyte population are shown in the graphs (**P < 0.01 compared to PBS control)
Fig. 8
Increased infiltration of eosinophils into tumors with IL-17E treatment. C8161 tumors from IL-17E-treated mice were collected and paraffin sections of tumors were prepared and stained for eosinophils using Sirius Red method. The number of eosinophils per square millimeter of tumor tissue (a) was determined by computer-assisted image analysis. Each bar in the graph represents the mean ± SE of determinations in six samples of the same treatment (**P < 0.05 compared to PBS control). b Representative micrographs of sections show stained eosinophils in tumors from mice treated with PBS or IL-17E
Fig. 9
Treatment of tumor-bearing mice with IL-17E results in induction of serum IL-5. Serum was collected from CD-1 nude mice engrafted with human tumors. a HT-29 human colon adenocarcinoma. Mice were treated with intravenous injections of either PBS (0.1 ml) or human IL-17E (0.04 mg/kg) every 2 days for 4 weeks. b C8161 human melanoma. Mice were treated with subcutaneous injections of PBS (0.1 ml) or human IL-17E (1.2, 0.4, and 0.04 μg per mouse) every 2 days for 4 weeks. IL-5 was quantitated in serum samples using an ELISA method. Results are expressed in pg IL-5/ml of serum
Fig. 10
IL-17E activates signaling pathways in B cells. WEHI-231 cells were stimulated in vitro (107/ml) for t = 0–30 min with 1.4 μg/ml of either human IL-17E or mouse IL-17E at 37°C. After cell lysis, 25 μg of protein lysate was loaded onto 4–12% SDS-PAGE gels followed by Western blotting using anti-phosphotyrosine antibody and HRP-conjugated anti-mouse-antibody. Blots were stripped and probed for GAPDH as a loading control. Molecular weight markers in kilodaltons (kDa) are indicated. A similar phosphorylation pattern was noted for both human IL-17E (a) and mouse IL-17E (b)
Fig. 11
Human and mouse IL-17E can activate NF-kappaB in B cells. a WEHI-231 cells (107/ml) were stimulated in vitro with 0.7 μg/ml of either hIL-17E or mIL-17E for t = 0, 2 or 5 min at 37°C. Cells were lysed in lysis buffer for 30 min on ice, followed by centrifugation for 10 min. After cell lysis, 30 μg of protein was separated onto 4–12% SDS-PAGE gels, followed by Western blotting with anti-phospho-IkB-alpha antibodies, followed by HRP-conjugated anti-rabbit antibodies, and developed using chemiluminescence. Blots were then stripped and reprobed with GAPDH together with HRP-conjugated anti-mouse antibodies
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