- Email: [email protected]
CC motif chemokine ligand 19 suppressed colorectal cancer in vivo accompanied by an increase in IL-12 and IFN-γ
Biomedicine & Pharmacotherapy 69 (2015) 374–379
Available online at
ScienceDirect www.sciencedirect.com
Original article
CC motif chemokine ligand 19 suppressed colorectal cancer in vivo accompanied by an increase in IL-12 and IFN-g Jun Lu a,1, Junjun Ma a,1, Wei Cai a, Xiongzhi Wangpu a, Hao Feng b, Jingkun Zhao a, Shaopei Guan a, Yaping Zong a,*, Aiguo Lu a,* a b
Shanghai Minimally Invasive Surgery Center, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, No. 197 Second Ruijin Road, Shanghai, China University of Munich School of Medicine, Munich, Germany
A R T I C L E I N F O
A B S T R A C T
Article history: Received 26 November 2014 Accepted 11 December 2014
In this study we investigate the role of CC motif chemokine ligand 19 (CCL19) to colorectal cancer (CRC) in vivo. We injected different dose of recombinant mouse CCL19 (rmCCL19) in the tumor site of the model of transplanted tumor. Result shows that rmCCL19 can suppress CRC tumorigenesis and growth in vivo, and it can also prolong overall survival of mice. Quantitative reverse transcription-polymerase chain reaction and enzyme linked immunosorbent assay results showed that the interferon-g (IFN-g) and interleukin-12 (IL-12) levels in the tumors and plasma were significantly enhanced after processing with rmCCL19. ß 2014 Elsevier Masson SAS. All rights reserved.
Keywords: Colorectal cancer Chemokine CCL19 Immunotherapy
1. Introduction Colorectal cancer (CRC) is the second most common cancer and the fourth most common cause of cancer-related death worldwide [1]. There are many means of adjuvant therapy to cure CRC after surgical resection, including radiotherapy, chemotherapy, traditional Chinese medicine therapy, etc. However, these therapeutic methods still lack targeting and specificity, and they can induce patients’ serious systemic side effects [2,3]. Immunotherapy, which means recruiting and activating the body’s own immune cells to kill tumor cells, is given more and more attentions in recent years [4–6]. We have revealed the antitumor efficacy of CC motif chemokine ligand 19 (CCL19) in CRC in vitro [7]. And our later research certified that this efficacy may work via inhibiting wnt/b-catenin signaling pathway (data not shown). But whether CCL19 can suppress CRC in vivo is rarely reported. CCL19, however, was reported to have the ability to recruit DCs, which was highly expressed its specific receptor (CCR7) [8]. And CCL19 has been frequently reported to have the antitumor efficacy via recruiting dendritic cells (DCs) [9–11]. So we became interested in whether CCL19 can suppress CRC in vivo by mediating antitumor immunity of DCs. In order to eliminate CCL190 direct effects on the tumor cells, we chose LoVo to be applied to our
* Corresponding authors. E-mail addresses: [email protected] (Y. Zong), [email protected] (A. Lu). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.biopha.2014.12.032 0753-3322/ß 2014 Elsevier Masson SAS. All rights reserved.
research, which have been proved weak response after upregulating or downregulating CCL19 in vitro [7].
2. Materials and methods 2.1. Cell lines and culture conditions CRC cell line LoVo was purchased from the American Type Culture Collection (Manassas, VA, USA). And it was cultured by F12K containing 10% heat-inactivated fetal bovine serum (FBS) (Invitrogen, Carlsbad, CA, USA). 2.2. Nude mice Male BALB/C nude mice (4 weeks) were purchased from the Shanghai Laboratory Animal Center (Chinese Academy of Sciences, Shanghai, China) and kept in a specific pathogen-free laboratory (Shanghai Jiao Tong University School of Medicine). All studies were conducted with the approval and guidance of the Animal Ethics Committee (Shanghai Jiao Tong University School of Medicine). 2.3. Best dose of recombinant mouse CCL19 (rmCCL19) was screened LoVo (5 106/100 ml) cells were subcutaneously inoculated into the right flank of 25 nude mice to construct the tumor-bearing model. After neoplasia (1 week), these mice were randomized into 5 groups and numbered 1–5. Then different doses of rmCCL19
J. Lu et al. / Biomedicine & Pharmacotherapy 69 (2015) 374–379 Table 1 The tumor size of Group 1–5.
375
2.5. Cytokines in tumors were detected by quantitative reverse transcription-polymerase chain reaction (qRT-PCR)
Group
1
2
3
4
5
Dose (mg) Size (mm3)
0 1886 278
0.05 1463 170
0.1 1004 219
0.2 658 57
0.3 584 109
(R&D Systems, Minneapolis, MN, United States) were injected into the tumor site of each group (Group 1: 0 mg; Group 2: 0.05 mg; Group 3: 0.1 mg; Group 4: 0.2 mg; Group 5: 0.3 mg). And these processes were administered with 2 times/week. Mice were sacrificed after 4 weeks; the blood samples were collected from the tail vein. Then the tumors of mice were resected and measured. The volume was calculated by the formula (0.4 x2y), with x as the smaller diameter and y as the larger diameter. 2.4. The function of rmCCL19 in vivo was verified using the tumorigenesis models 30 nude mice were equally divided into 3 groups and labeled I, II, and III. And they were injected with LoVo cells as the same way as mentioned above. Different methods were performed in Group I–III as follows: Group I were injected PBS in the tumor injection site at the time of tumor inoculation and then 2 times/week; Group II were injected PBS 2 times in the first week, and injected 0.2 mg of rmCCL19 in the tumor site 2 times/week a week later; while Group III were injected 0.2 mg of rmCCL19 in the tumor injection site at the time of tumor inoculation and then 2 times/week. Mice were sacrificed after 4 weeks; the blood samples were collected from the tail vein, and then the tumors and tumors were also resected, respectively. Other 30 nude mice were performed to detect the overall survival. And they were grouped Group I0 –III0 and processed as the same as the mice in Group I–III.
Fig. 1. (A) Tumor model mice processed with different dose of rmCCL19. Group 1: 0 mg; Group 2: 0.05 mg; Group 3: 0.1 mg; Group 4: 0.2 mg; Group 5: 0.3 mg. (B) The tumors resected from each group of mice.
PCR-primers were designed as follows: interferon-g (IFN-g) sense primer: 50 -AGCCCTATTACAGCACAG-30 ; antisense primer: 50 TTCTAACAACAAGTATCCC-30 . Interleukin-12A (IL-12A) sense primer: 50 -TACTTACCCTCCAACTCCC-30 ; antisense primer: 50 -GACCCATTTCACATCTACTC-30 . Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) sense primer: 50 -GTTGCCTACGCAGGTCTT-30 ; antisense primer: 50 -CATAACGGCGGTTCATTC-30 . TRIzol reagent (Invitrogen, Carlsbad, CA, USA) was applied to extract the total RNA of nude mice tumors. cDNA was produced by RNA reverse transcription procedure. And qRT-PCR was processed according to the protocol: 95 8C for 15 min; then 35 cycles were conducted as follows: denaturation at 94 8C for 15 s, 55 8C for 30 s to be annealed, extension at 70 8C for 30 s to extend the DNA strand. GAPDH was used as the internal control. 2.6. Cytokines in tumors were detected by Immunoblot analysis Protein of nude mice tumors was extracted by Ripa Lysis Buffer (Sigma Aldrich, St Louis, MO, USA). Total protein levels were measured by standardized Western blot processes with the following antibodies: IFN-g (1 mg/mL, R&D Systems, Minneapolis, MN, USA); IL-12A (1:2000, Abcam, Cambridge, UK); and GAPDH (1:1000, Santa Cruz Biotechnology, Dallas, TX, USA). IFN-g and IL-12A levels were quantified by GAPDH. 2.7. Enzyme linked immunosorbent assay (ELISA) for the mice plasma 96-Well immunoassay microplates (Corning, Corning, NY, USA) were applied in the ELISA. And anti-IL-12 (Abcam, Cambridge, UK) and anti-IFN-g antibodies were diluted by alkalescent carbonate buffer solution (6 mg/mL). Then 100 ml/well antibody diluent was added into each well to coat on the immunoassay microplates. The incubations were performed for overnight at 4 8C. The mice plasma samples diluted at 1:100 were incubated in the antibody-coated
Fig. 2. The tumor size of Group 1–5. The differences between either two groups were significant in Group 1–4 (P < 0.05). But the difference between Group 4 and Group 5 showed no significance.
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J. Lu et al. / Biomedicine & Pharmacotherapy 69 (2015) 374–379
wells at 37 8C for 1 h followed by washing with PBST (PBS containing 0.1% Tween 20) for 3 times, and then incubated with HRP-labeled secondary antibody (Abcam, Cambridge, UK; 1:5000) at room temperature for 1 h followed by washing with PBST for 3 times. The 3,30 ,5,50 -tetramethylbenzidine (TMB) (Sigma–Aldrich, St Louis, MO, USA) solution containing 1% hydrogen peroxide was applied as the detecting reagent. The reaction was terminated by 2 M H2SO4. Finally, the relative protein levels were described as 450 nm optical density (OD) value.
2.8. Statistical analysis SAS 9.3 software (SAS Institute Inc., Cary, NC, USA) was applied to the statistical analysis. All the data were demonstrated as mean SD. Differences between the groups were evaluated by oneway ANOVA. Survival rate was assayed by Kaplan–Meier plots and compared by the log-rank test. Differences were considered significant at P < 0.05.
3. Results 3.1. RmCCL19 can suppress the growth of tumors in nude mice and 0.2 mg was the best dose The tumor size of Group 1–5 is shown in Table 1. Results showed that when the dose of rmCCL19 ranged from 0 mg to 0.2 mg, higher dose resulted in smaller size of tumor, and differences between either two groups were significant (P < 0.05). But the difference between 0.2 mg-Group and 0.3 mgGroup showed no statistical significance (P > 0.05) (Table 1 and Figs. 1 and 2). Considering economy and the side effects of rmCCL19 to mice, we selected 0.2 mg as the best dose to apply to our further research. Western blot and qRT-PCR results demonstrated that when the dose of rmCCL19 ranged from 0 to 0.2 mg, higher dose led to higher mRNA and protein levels of IFN-g and IL-12A in the mice tumors (Fig. 3A and B). ELISA results also showed that the dose of rmCCL19 was positively related with the IFN-g and IL-12 levels in mice
Fig. 3. (A) The protein expression of IFN-g and IL-12A in the tumors of Group 1–5 mice. (B) The mRNA levels of IFN-g and IL-12A in the tumors of Group 1–5 mice. (C) The IFN-g levels in the plasma of Group 1–5 mice by ELISA. (D) The IL-12 levels in the plasma of Group 1–5 mice by ELISA.
J. Lu et al. / Biomedicine & Pharmacotherapy 69 (2015) 374–379 Table 2 The tumor size of Group I–III.
377
difference between Group 4 and Group 5 showed no significance (P > 0.05, Fig. 3B–D).
Group
I
II
III
Treatment
PBS only
rmCCL19 only
Dose (mg) Frequency Size (mm3)
0 Every 3 days 2035 228
First week: PBS 2–4 weeks: rmCCL19 0.2 Every 3 days 597 68
0.2 Every 3 days 55 12
3.2. Recombinant CCL19 can suppress tumor tumorigenesis in nude mice and also prolong mice’s lifetime Tumors in the PBS-treated group (Group I) grew the fastest among Group I–III, while mice in Group II got smaller tumors than Group I (P < 0.05). However, few nude mice in Group III were seen obvious tumors. The differences of tumor size between either two groups were significant (P < 0.05, Table 2, Figs. 4 and 5A). So we supposed that rmCCL19 can suppress tumor cells tumorigenesis and growth in nude mice. The overall survival rate of mice in Group III0 was higher than other two groups. And mice in the Group I0 showed the shortest lifetime. The differences between either two groups were statistically significant (P < 0.05, Fig. 5B). Western blot and qRT-PCR results demonstrated that protein and mRNA levels of IFN-g and IL-12 in the Group I mice tumors were significantly lower than Group II and Group III (P < 0.05, Fig. 6A and B). The IFN-g and IL-12 protein levels in the plasma of Group I mice were also lower than Group II and Group III (P < 0.05, Fig. 6C and D).
4. Discussion
Fig. 4. (A) 3 groups of mice processed with different treatments which mentioned in the text. (B) The tumors resected from each group of mice.
plasma (Fig. 3C and D). The qRT-PCR and ELISA results also demonstrated that the differences between either two groups among Group 1–4 were statistically significant (P < 0.05). But the
Colorectal cancer (CRC) is one of the most common cancers worldwide [1]. And up to now, chemotherapy and radiotherapy are still the most effective therapies to advanced CRC patients after surgical resection. However, due to lack of targeting and specificity, these methods may induce patients’ serious systemic side effects [2,3]. Recently, tumor immunotherapy became hot-spot in cancer research [4–6]. Recruitment of dendritic cells (DCs) was one of the most frequently used methods in antitumor immunotherapy. DCs are the most powerful antigen-presenting cells, and they may mediate antitumor immunity by T-cell responses [12–14]. However, recent clinical researches have revealed that the DCs’ antitumor efficacy is more related with Natural Killer cells (NK cells) response after being stimulated by DCs than T-cell response [14–16]. CCL19, however, was reported to have the ability to recruit DCs, which was highly expressed its specific receptor (CCR7) [8]. And CCL19 has been frequently reported to have the antitumor efficacy via DCs [9–11].
Fig. 5. (A) The tumor size of Group I–III, the differences between either two groups were significant (P < 0.05). (B) The overall survival of Group I0 –III0 , and the differences between either two groups also showed statistical significance (P < 0.05).
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Fig. 6. (A) The protein expression of IFN-g and IL-12A in the tumors of Group I–III mice. (B) The mRNA levels of IFN-g and IL-12A in the tumors of Group I–III mice. (C) The IFNg levels in the plasma of Group I–III mice by ELISA. (D) The IL-12 levels in the plasma of Group I–III mice by ELISA.
We proved that orthotopic rmCCL19 injection can suppress CRC in the tumor-bearing nude mice, and 0.2 mg was the best dose. Our further research also revealed that rmCCL19 can suppress tumor tumorigenesis and growth in vivo, and lead to a higher survival rate. We supposed that this antitumor efficacy may be related with accumulation of DCs in the tumor site which were recruited by CCL19. As nude mice are athymic and cellular immunity deficiency, we can rule out DCs’ antigen-presenting function in this antitumor effect. So we suppose that DCs-NK cells axis may play a crucial role in this antitumor response. To verify our speculation, we detected the IL-12 and IFN-g levels in the tumors and plasma of mice. IL-12 can be secreted by DCs, and it has the ability to mature and active the NK cells, which can further secrete IFN-g [17]. According to report, both IL-12 and IFN-g have the antitumor efficacy [18–20]. Our research revealed that IL-12 and IFN-g levels were enhanced in the tumor-bearing nude mice after orthotopic rmCCL19 injection, and the production seems to be dosedependent. So our research revealed the obvious antitumor efficacy of CCL19 in vivo accompanied by an increase in IL-12 and IFN-g, and this efficacy may be mediated by antitumor immune cells (DCs and NK cells). We hope our finding can be applied in the immunotherapy of CRC in the future.
5. Conclusion CCL19 can suppress CRC in vivo accompanied by an increase in IL-12 and IFN-g. Conflict of interest The authors declare that they have no conflicts of interest concerning this article. Acknowledgments This work was supported by grants from Shanghai National Science Foundation (124119a0900) and National Natural Science Foundation of China (81201625). References [1] Jemal A, Bray F, Center MM, Ferlay J, Ward E, Forman D. Global cancer statistics. CA Cancer J Clin 2011;61(2):69–90. [2] Ishikawa H, Onishi T, Kobayashi R, Ohashi Y, Suzuki K, Yamamoto N, et al. Effectiveness of steroids for the rash side effect of pemetrexed. Gan To Kagaku Ryoho 2013;40(1):75–8.
J. Lu et al. / Biomedicine & Pharmacotherapy 69 (2015) 374–379 [3] Iwai M, Kimura M, Yoshimura T, Yasuda T. Side effect analyses in consideration of renal functions for capecitabine-administered patients. Gan To Kagaku Ryoho 2012;39(5):783–6. [4] Couzin-Frankel J. Breakthrough of the year 2013. Cancer immunotherapy. Science 2013;342(6165):1432–3. [5] Nelson PJ, Muenchmeier N. Membrane-anchored chemokine fusion proteins: a novel class of adjuvants for immunotherapy. Oncoimmunology 2013;2(11): e26619. [6] Topalian SL, Weiner GJ, Pardoll DM. Cancer immunotherapy comes of age. J Clin Oncol 2011;29(36):4828–36. [7] Lu J, Zhao J, Feng H, Wang P, Zhang Z, Zong Y, et al. Antitumor efficacy of CC motif chemokine ligand 19 in colorectal cancer. Dig Dis Sci 2014;59(9):2153–62. [8] Dieu MC, Vanbervliet B, Vicari A, Bridon JM, Oldham E, Aı¨t-Yahia S, et al. Selective recruitment of immature and mature dendritic cells by distinct chemokines expressed in different anatomic sites. J Exp Med 1998;188:373–86. [9] Hamanishi J, Mandai M, Matsumura N, Baba T, Yamaguchi K, Fujii S, et al. Activated local immunity by CC chemokine ligand 19-transduced embryonic endothelial progenitor cells suppresses metastasis of murine ovarian cancer. Stem Cells 2010;28:164–73. [10] Hou JM, Zhao X, Tian L, Li G, Zhang R, Yao B, et al. Immunotherapy of tumors with recombinant adenovirus encoding macrophage inflammatory protein 3beta induces tumor-specific immune response in immunocompetent tumorbearing mice. Acta Pharmacol Sin 2009;30(3):355–63. [11] Hillinger S, Yang SC, Batra RK, Strieter RM, Weder W, Dubinett SM, et al. CCL19 reduces tumour burden in a model of advanced lung cancer. Br J Cancer 2006;94(7):1029–34. [12] Ilett EJ, Prestwich RJ, Melcher AA. The evolving role of dendritic cells in cancer therapy. Expert Opin Biol Ther 2010;10(3):369–79.
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[13] Sun JC, Pan K, Chen MS, Wang QJ, Wang H, Ma HQ, et al. Dendritic cellsmediated CTLs targeting hepatocellular carcinoma stem cells. Cancer Biol Ther 2010;10(4):368–75. [14] Karimi K, Boudreau JE, Fraser K. Enhanced antitumor immunity elicited by dendritic cell vaccines is a result of their ability to engage both CTL and IFN gamma-producing NK cells. Mol Ther 2008;16(2):411–8. [15] Di Nicola M, Zappasodi R, Carlo-Stella C, Mortarini R, Pupa SM, Magni M, et al. Vaccination with autologous tumor-loaded dendritic cells induces clinical and immunologic responses in indolent B-cell lymphoma patients with relapsed and measurable disease: a pilot study. Blood 2009;113(1):18–27. [16] Viaud S, Terme M, Flament C, Taieb J, Andre´ F, Novault S, et al. Dendritic cell-derived exosomes promote natural killer cell activation and proliferation: a role for NKG2D ligands and IL-15Ralpha. PLoS ONE 2009;4(3): e4942. [17] Bontkes HJ, Ruizendaal JJ, Kramer D, Meijer CJ, Schreurs MW, Hooijberg E, et al. Interleukin-12 increases proliferation and interferon-gamma production but not cytolytic activity of human antigen-specific effector memory cytotoxic T lymphocytes: power of the effect depends on the functional avidity of the T cell and the antigen concentration. Hum Immunol 2005;66(11):1137–45. [18] Zapala L, Wolny R, Wachowska M, Jakobisiak M, Lasek W. Synergistic antitumor effect of JAWSII dendritic cells and interleukin 12 in a melanoma mouse model. Oncol Rep 2013;29:1208–14. [19] Wallin RP, Sundquist VS, Bra˚kenhielm E, Cao Y, Ljunggren HG, Grandien A. Angiostatic effects of NK cell-derived IFN-g counteracted by tumour cell Bcl-xL expression. Scand J Immunol 2014;79(2):90–7. [20] Miller CH, Maher SG, Young HA. Clinical use of interferon-gamma. Ann N Y Acad Sci 2009;1182:69–79.
Available online at
ScienceDirect www.sciencedirect.com
Original article
CC motif chemokine ligand 19 suppressed colorectal cancer in vivo accompanied by an increase in IL-12 and IFN-g Jun Lu a,1, Junjun Ma a,1, Wei Cai a, Xiongzhi Wangpu a, Hao Feng b, Jingkun Zhao a, Shaopei Guan a, Yaping Zong a,*, Aiguo Lu a,* a b
Shanghai Minimally Invasive Surgery Center, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, No. 197 Second Ruijin Road, Shanghai, China University of Munich School of Medicine, Munich, Germany
A R T I C L E I N F O
A B S T R A C T
Article history: Received 26 November 2014 Accepted 11 December 2014
In this study we investigate the role of CC motif chemokine ligand 19 (CCL19) to colorectal cancer (CRC) in vivo. We injected different dose of recombinant mouse CCL19 (rmCCL19) in the tumor site of the model of transplanted tumor. Result shows that rmCCL19 can suppress CRC tumorigenesis and growth in vivo, and it can also prolong overall survival of mice. Quantitative reverse transcription-polymerase chain reaction and enzyme linked immunosorbent assay results showed that the interferon-g (IFN-g) and interleukin-12 (IL-12) levels in the tumors and plasma were significantly enhanced after processing with rmCCL19. ß 2014 Elsevier Masson SAS. All rights reserved.
Keywords: Colorectal cancer Chemokine CCL19 Immunotherapy
1. Introduction Colorectal cancer (CRC) is the second most common cancer and the fourth most common cause of cancer-related death worldwide [1]. There are many means of adjuvant therapy to cure CRC after surgical resection, including radiotherapy, chemotherapy, traditional Chinese medicine therapy, etc. However, these therapeutic methods still lack targeting and specificity, and they can induce patients’ serious systemic side effects [2,3]. Immunotherapy, which means recruiting and activating the body’s own immune cells to kill tumor cells, is given more and more attentions in recent years [4–6]. We have revealed the antitumor efficacy of CC motif chemokine ligand 19 (CCL19) in CRC in vitro [7]. And our later research certified that this efficacy may work via inhibiting wnt/b-catenin signaling pathway (data not shown). But whether CCL19 can suppress CRC in vivo is rarely reported. CCL19, however, was reported to have the ability to recruit DCs, which was highly expressed its specific receptor (CCR7) [8]. And CCL19 has been frequently reported to have the antitumor efficacy via recruiting dendritic cells (DCs) [9–11]. So we became interested in whether CCL19 can suppress CRC in vivo by mediating antitumor immunity of DCs. In order to eliminate CCL190 direct effects on the tumor cells, we chose LoVo to be applied to our
* Corresponding authors. E-mail addresses: [email protected] (Y. Zong), [email protected] (A. Lu). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.biopha.2014.12.032 0753-3322/ß 2014 Elsevier Masson SAS. All rights reserved.
research, which have been proved weak response after upregulating or downregulating CCL19 in vitro [7].
2. Materials and methods 2.1. Cell lines and culture conditions CRC cell line LoVo was purchased from the American Type Culture Collection (Manassas, VA, USA). And it was cultured by F12K containing 10% heat-inactivated fetal bovine serum (FBS) (Invitrogen, Carlsbad, CA, USA). 2.2. Nude mice Male BALB/C nude mice (4 weeks) were purchased from the Shanghai Laboratory Animal Center (Chinese Academy of Sciences, Shanghai, China) and kept in a specific pathogen-free laboratory (Shanghai Jiao Tong University School of Medicine). All studies were conducted with the approval and guidance of the Animal Ethics Committee (Shanghai Jiao Tong University School of Medicine). 2.3. Best dose of recombinant mouse CCL19 (rmCCL19) was screened LoVo (5 106/100 ml) cells were subcutaneously inoculated into the right flank of 25 nude mice to construct the tumor-bearing model. After neoplasia (1 week), these mice were randomized into 5 groups and numbered 1–5. Then different doses of rmCCL19
J. Lu et al. / Biomedicine & Pharmacotherapy 69 (2015) 374–379 Table 1 The tumor size of Group 1–5.
375
2.5. Cytokines in tumors were detected by quantitative reverse transcription-polymerase chain reaction (qRT-PCR)
Group
1
2
3
4
5
Dose (mg) Size (mm3)
0 1886 278
0.05 1463 170
0.1 1004 219
0.2 658 57
0.3 584 109
(R&D Systems, Minneapolis, MN, United States) were injected into the tumor site of each group (Group 1: 0 mg; Group 2: 0.05 mg; Group 3: 0.1 mg; Group 4: 0.2 mg; Group 5: 0.3 mg). And these processes were administered with 2 times/week. Mice were sacrificed after 4 weeks; the blood samples were collected from the tail vein. Then the tumors of mice were resected and measured. The volume was calculated by the formula (0.4 x2y), with x as the smaller diameter and y as the larger diameter. 2.4. The function of rmCCL19 in vivo was verified using the tumorigenesis models 30 nude mice were equally divided into 3 groups and labeled I, II, and III. And they were injected with LoVo cells as the same way as mentioned above. Different methods were performed in Group I–III as follows: Group I were injected PBS in the tumor injection site at the time of tumor inoculation and then 2 times/week; Group II were injected PBS 2 times in the first week, and injected 0.2 mg of rmCCL19 in the tumor site 2 times/week a week later; while Group III were injected 0.2 mg of rmCCL19 in the tumor injection site at the time of tumor inoculation and then 2 times/week. Mice were sacrificed after 4 weeks; the blood samples were collected from the tail vein, and then the tumors and tumors were also resected, respectively. Other 30 nude mice were performed to detect the overall survival. And they were grouped Group I0 –III0 and processed as the same as the mice in Group I–III.
Fig. 1. (A) Tumor model mice processed with different dose of rmCCL19. Group 1: 0 mg; Group 2: 0.05 mg; Group 3: 0.1 mg; Group 4: 0.2 mg; Group 5: 0.3 mg. (B) The tumors resected from each group of mice.
PCR-primers were designed as follows: interferon-g (IFN-g) sense primer: 50 -AGCCCTATTACAGCACAG-30 ; antisense primer: 50 TTCTAACAACAAGTATCCC-30 . Interleukin-12A (IL-12A) sense primer: 50 -TACTTACCCTCCAACTCCC-30 ; antisense primer: 50 -GACCCATTTCACATCTACTC-30 . Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) sense primer: 50 -GTTGCCTACGCAGGTCTT-30 ; antisense primer: 50 -CATAACGGCGGTTCATTC-30 . TRIzol reagent (Invitrogen, Carlsbad, CA, USA) was applied to extract the total RNA of nude mice tumors. cDNA was produced by RNA reverse transcription procedure. And qRT-PCR was processed according to the protocol: 95 8C for 15 min; then 35 cycles were conducted as follows: denaturation at 94 8C for 15 s, 55 8C for 30 s to be annealed, extension at 70 8C for 30 s to extend the DNA strand. GAPDH was used as the internal control. 2.6. Cytokines in tumors were detected by Immunoblot analysis Protein of nude mice tumors was extracted by Ripa Lysis Buffer (Sigma Aldrich, St Louis, MO, USA). Total protein levels were measured by standardized Western blot processes with the following antibodies: IFN-g (1 mg/mL, R&D Systems, Minneapolis, MN, USA); IL-12A (1:2000, Abcam, Cambridge, UK); and GAPDH (1:1000, Santa Cruz Biotechnology, Dallas, TX, USA). IFN-g and IL-12A levels were quantified by GAPDH. 2.7. Enzyme linked immunosorbent assay (ELISA) for the mice plasma 96-Well immunoassay microplates (Corning, Corning, NY, USA) were applied in the ELISA. And anti-IL-12 (Abcam, Cambridge, UK) and anti-IFN-g antibodies were diluted by alkalescent carbonate buffer solution (6 mg/mL). Then 100 ml/well antibody diluent was added into each well to coat on the immunoassay microplates. The incubations were performed for overnight at 4 8C. The mice plasma samples diluted at 1:100 were incubated in the antibody-coated
Fig. 2. The tumor size of Group 1–5. The differences between either two groups were significant in Group 1–4 (P < 0.05). But the difference between Group 4 and Group 5 showed no significance.
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J. Lu et al. / Biomedicine & Pharmacotherapy 69 (2015) 374–379
wells at 37 8C for 1 h followed by washing with PBST (PBS containing 0.1% Tween 20) for 3 times, and then incubated with HRP-labeled secondary antibody (Abcam, Cambridge, UK; 1:5000) at room temperature for 1 h followed by washing with PBST for 3 times. The 3,30 ,5,50 -tetramethylbenzidine (TMB) (Sigma–Aldrich, St Louis, MO, USA) solution containing 1% hydrogen peroxide was applied as the detecting reagent. The reaction was terminated by 2 M H2SO4. Finally, the relative protein levels were described as 450 nm optical density (OD) value.
2.8. Statistical analysis SAS 9.3 software (SAS Institute Inc., Cary, NC, USA) was applied to the statistical analysis. All the data were demonstrated as mean SD. Differences between the groups were evaluated by oneway ANOVA. Survival rate was assayed by Kaplan–Meier plots and compared by the log-rank test. Differences were considered significant at P < 0.05.
3. Results 3.1. RmCCL19 can suppress the growth of tumors in nude mice and 0.2 mg was the best dose The tumor size of Group 1–5 is shown in Table 1. Results showed that when the dose of rmCCL19 ranged from 0 mg to 0.2 mg, higher dose resulted in smaller size of tumor, and differences between either two groups were significant (P < 0.05). But the difference between 0.2 mg-Group and 0.3 mgGroup showed no statistical significance (P > 0.05) (Table 1 and Figs. 1 and 2). Considering economy and the side effects of rmCCL19 to mice, we selected 0.2 mg as the best dose to apply to our further research. Western blot and qRT-PCR results demonstrated that when the dose of rmCCL19 ranged from 0 to 0.2 mg, higher dose led to higher mRNA and protein levels of IFN-g and IL-12A in the mice tumors (Fig. 3A and B). ELISA results also showed that the dose of rmCCL19 was positively related with the IFN-g and IL-12 levels in mice
Fig. 3. (A) The protein expression of IFN-g and IL-12A in the tumors of Group 1–5 mice. (B) The mRNA levels of IFN-g and IL-12A in the tumors of Group 1–5 mice. (C) The IFN-g levels in the plasma of Group 1–5 mice by ELISA. (D) The IL-12 levels in the plasma of Group 1–5 mice by ELISA.
J. Lu et al. / Biomedicine & Pharmacotherapy 69 (2015) 374–379 Table 2 The tumor size of Group I–III.
377
difference between Group 4 and Group 5 showed no significance (P > 0.05, Fig. 3B–D).
Group
I
II
III
Treatment
PBS only
rmCCL19 only
Dose (mg) Frequency Size (mm3)
0 Every 3 days 2035 228
First week: PBS 2–4 weeks: rmCCL19 0.2 Every 3 days 597 68
0.2 Every 3 days 55 12
3.2. Recombinant CCL19 can suppress tumor tumorigenesis in nude mice and also prolong mice’s lifetime Tumors in the PBS-treated group (Group I) grew the fastest among Group I–III, while mice in Group II got smaller tumors than Group I (P < 0.05). However, few nude mice in Group III were seen obvious tumors. The differences of tumor size between either two groups were significant (P < 0.05, Table 2, Figs. 4 and 5A). So we supposed that rmCCL19 can suppress tumor cells tumorigenesis and growth in nude mice. The overall survival rate of mice in Group III0 was higher than other two groups. And mice in the Group I0 showed the shortest lifetime. The differences between either two groups were statistically significant (P < 0.05, Fig. 5B). Western blot and qRT-PCR results demonstrated that protein and mRNA levels of IFN-g and IL-12 in the Group I mice tumors were significantly lower than Group II and Group III (P < 0.05, Fig. 6A and B). The IFN-g and IL-12 protein levels in the plasma of Group I mice were also lower than Group II and Group III (P < 0.05, Fig. 6C and D).
4. Discussion
Fig. 4. (A) 3 groups of mice processed with different treatments which mentioned in the text. (B) The tumors resected from each group of mice.
plasma (Fig. 3C and D). The qRT-PCR and ELISA results also demonstrated that the differences between either two groups among Group 1–4 were statistically significant (P < 0.05). But the
Colorectal cancer (CRC) is one of the most common cancers worldwide [1]. And up to now, chemotherapy and radiotherapy are still the most effective therapies to advanced CRC patients after surgical resection. However, due to lack of targeting and specificity, these methods may induce patients’ serious systemic side effects [2,3]. Recently, tumor immunotherapy became hot-spot in cancer research [4–6]. Recruitment of dendritic cells (DCs) was one of the most frequently used methods in antitumor immunotherapy. DCs are the most powerful antigen-presenting cells, and they may mediate antitumor immunity by T-cell responses [12–14]. However, recent clinical researches have revealed that the DCs’ antitumor efficacy is more related with Natural Killer cells (NK cells) response after being stimulated by DCs than T-cell response [14–16]. CCL19, however, was reported to have the ability to recruit DCs, which was highly expressed its specific receptor (CCR7) [8]. And CCL19 has been frequently reported to have the antitumor efficacy via DCs [9–11].
Fig. 5. (A) The tumor size of Group I–III, the differences between either two groups were significant (P < 0.05). (B) The overall survival of Group I0 –III0 , and the differences between either two groups also showed statistical significance (P < 0.05).
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Fig. 6. (A) The protein expression of IFN-g and IL-12A in the tumors of Group I–III mice. (B) The mRNA levels of IFN-g and IL-12A in the tumors of Group I–III mice. (C) The IFNg levels in the plasma of Group I–III mice by ELISA. (D) The IL-12 levels in the plasma of Group I–III mice by ELISA.
We proved that orthotopic rmCCL19 injection can suppress CRC in the tumor-bearing nude mice, and 0.2 mg was the best dose. Our further research also revealed that rmCCL19 can suppress tumor tumorigenesis and growth in vivo, and lead to a higher survival rate. We supposed that this antitumor efficacy may be related with accumulation of DCs in the tumor site which were recruited by CCL19. As nude mice are athymic and cellular immunity deficiency, we can rule out DCs’ antigen-presenting function in this antitumor effect. So we suppose that DCs-NK cells axis may play a crucial role in this antitumor response. To verify our speculation, we detected the IL-12 and IFN-g levels in the tumors and plasma of mice. IL-12 can be secreted by DCs, and it has the ability to mature and active the NK cells, which can further secrete IFN-g [17]. According to report, both IL-12 and IFN-g have the antitumor efficacy [18–20]. Our research revealed that IL-12 and IFN-g levels were enhanced in the tumor-bearing nude mice after orthotopic rmCCL19 injection, and the production seems to be dosedependent. So our research revealed the obvious antitumor efficacy of CCL19 in vivo accompanied by an increase in IL-12 and IFN-g, and this efficacy may be mediated by antitumor immune cells (DCs and NK cells). We hope our finding can be applied in the immunotherapy of CRC in the future.
5. Conclusion CCL19 can suppress CRC in vivo accompanied by an increase in IL-12 and IFN-g. Conflict of interest The authors declare that they have no conflicts of interest concerning this article. Acknowledgments This work was supported by grants from Shanghai National Science Foundation (124119a0900) and National Natural Science Foundation of China (81201625). References [1] Jemal A, Bray F, Center MM, Ferlay J, Ward E, Forman D. Global cancer statistics. CA Cancer J Clin 2011;61(2):69–90. [2] Ishikawa H, Onishi T, Kobayashi R, Ohashi Y, Suzuki K, Yamamoto N, et al. Effectiveness of steroids for the rash side effect of pemetrexed. Gan To Kagaku Ryoho 2013;40(1):75–8.
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