Selective neutralization of IL-12 p40 monomer induces death in prostate cancer cells via IL-12–IFN-γ
Madhuchhanda Kundua, Avik Roya, and Kalipada Pahana,b,1
aDepartment of Neurological Sciences, Rush University Medical Center, Chicago, IL 60612; and bDivision of Research and Development, Jesse Brown Veterans Affairs Medical Center, Chicago, IL 60612
Edited by Xiaojing Ma, Weill Medical College, New York, NY, and accepted by Editorial Board Member Carl F. Nathan September 18, 2017 (received for review April 12, 2017)
Cancer cells are adept at evading cell death, but the underlying p40 and p402 and developed an ELISA to monitor these cytokines mechanisms are poorly understood. IL-12 plays a critical role in the separately (10). Mouse squamous (KLN), prostate (TRAMP), early inflammatory response to infection and in the generation of breast (4T1), and liver hepatoma (Hepa) cells were cultured under T-helper type 1 cells, favoring cell-mediated immunity. IL-12 is com- serum-free condition for 48 h, followed by measurement of the posed of two different subunits, p40 and p35. This study underlines levels of p40, p402, IL-12, and IL-23 by sandwich ELISA. In gen- the importance of IL-12 p40 monomer (p40) in helping cancer cells to eral, the levels of IL-12 and IL-23 were very low compared with escape cell death. We found that different mouse and human cancer p40 and p402 in each of these cell lines (Fig. 1 A–C). Interestingly, cells produced greater levels of p40 than p40 homodimer (p402), the level of p40 was much higher than the levels of p402, IL-12, or IL-12, or IL-23. Similarly, the serum level of p40 was much greater IL-23 in TRAMP, 4T1, and Hepa cells (Fig. 1 A–C). However, in patients with prostate cancer than in healthy control subjects. levels of p40 and p402 were almost the same in KLN cells (Fig. 1A), Selective neutralization of p40, but not p402,bymAbstimulated suggesting the specificity of our finding. To confirm the presence of death in different cancer cells in vitro and in vivo in a tumor model. p40 in cancer cells, we adopted different techniques. First, we Interestingly, p40 was involved in the arrest of IL-12 receptor (IL-12R) monitored the level of p40 in the supernatants of TRAMP cells by IL-12Rβ1, but not IL-12Rβ2, in the membrane, and that p40 neutral- native PAGE followed by Coomassie staining and found the pres- ization induced the internalization of IL-12Rβ1 via caveolin and ence of a 40-kDa protein as the major secretory molecule (Fig. 1D).
caused cancer cell death via the IL-12–IFN-γ pathway. These studies Second, immunoblot analysis of supernatants of TRAMP cells with INFLAMMATION identify a role of p40 monomer in helping cancer cells to escape cell our p40 monomer mAb a3-3a showed the presence of p40 in IMMUNOLOGY AND death via suppression of IL-12Rβ1 internalization. supernatants (Fig. 1E). Finally, intracellular FACS analyses with p40 mAb a3-3a and p402 mAb a3-1d after live/dead cell exclusion prostate cancer | IL-12 p40 monomer | IL-12Rb1 | IL-12 | IFN-γ (SI Appendix,Fig.S1) also showed that the level of p40 was sig- nificantly higher than p402 in TRAMP cells (Fig. 1 F and G). nderstanding the mechanisms by which cancer cells escape Next, we measured the level of p40 in different human cancer Udeath is an important area of research. Because IL-12 is an cell lines. Native PAGE analyses followed by Coomassie staining important molecule in terms of cell-mediated immunity (1), this of supernatants demonstrated that human hepatoma Hep3B H I J cytokine is always under scanner for the treatment of cancer (2, 3). (Fig. 1 ), prostate LNCaP (Fig. 1 ), and breast MCF-7 (Fig. 1 ) The IL-12 family of cytokines has four members, including p40 cancer cells produced significant levels of p40. ELISA of supernatants clearly indicated that LNCaP, MCF-7, and Hep3B monomer (p40), p40 homodimer (p402), IL-12 (p40:p35), and IL-23 cells expressed significantly higher levels of p40 than p402 (Fig. (p40:p19). In this era of science dominated by heterodimers, only IL- K L 23andIL-12werethoughttohavebiological functions. As a result, 1 ). Levels of IL-12 and IL-23 (Fig. 1 ) were also much lower compared with p40 (Fig. 1K). Furthermore, immunoblot analyses of p40 and p402 were considered as nonfunctional members of the IL- 12 family (4). However, we have demonstrated a proinflammatory Significance property of p402 (5–7) and delineated that biological functions of p402 are different from those of IL-12 and IL-23 (8, 9). Furthermore, after raising separate functional blocking mAb and ELISA against Different mouse and human cancer cells produce excess p40 mouse p402 and p40 (10), we have delineated that mAb against p402 monomer compared with p40 homodimer, IL-12, and IL-23. The protects mice against experimental allergic encephalomyelitis (11). serum level of p40 monomer is also much higher in patients Here, we demonstrate that different forms of cancer cells, excluding with prostate cancer than in healthy control subjects. Inter- lung cancer, are associated with specific elevation of p40. A selective estingly, this p40 monomer helps cancer cells to escape IL-12– increase of p40 was also seen in serum from patients with prostate IFN-γ–mediated death via suppression of IL-12 receptor-β1 cancer compared with age-matched controls. Interestingly, selective internalization. This report demonstrates a biological role of ablation of p40 by mAb stimulated cell death in different cancer cells p40 monomer, the so-called nonfunctional member of the and in vivo in transgenic adenocarcinoma of the mouse prostate IL-12 family of cytokines, in cancer cell survival. These results (TRAMP) tumor tissue. Furthermore, p40 suppressed the caveolin- also delineate that neutralization of p40 monomer may be a mediated internalization of IL-12 receptor (IL-12R) IL-12Rβ1and therapeutic target in prostate and other cancers, which are associated IL-12 signaling in TRAMP tumor cells, and these were associated with excessive production of p40. neutralized by p40 mAb. These results delineate a pathogenic role Author contributions: K.P. designed research; M.K. and A.R. performed research; M.K. of p40 whereby it helps cancer cells to evade cell death. and A.R. contributed new reagents/analytic tools; M.K., A.R., and K.P. analyzed data; and M.K. and K.P. wrote the paper. Results The authors declare no conflict of interest. Levels of IL-12 Family of Cytokines (p40, p402, IL-12, and IL-23) in This article is a PNAS Direct Submission. X.M. is a guest editor invited by the Different Mouse and Human Cancer Cell Lines. First, we monitored Editorial Board. the levels of cytokines in different cancer cell lines. It was not Published under the PNAS license. possibletoexaminetheroleofp40andp402 in the pathogenesis of 1To whom correspondence should be addressed. Email: [email protected]. any disease as a result of the unavailability of specific functional This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. blocking mAbs. Therefore, we generated neutralizing mAbs against 1073/pnas.1705536114/-/DCSupplemental.
www.pnas.org/cgi/doi/10.1073/pnas.1705536114 PNAS Early Edition | 1of6 Downloaded by guest on September 29, 2021 3.2015 (
Stimulation of Death Response in Different Mouse and Human Cancer Cells by Selective Neutralization of p40 by Monoclonal Antibody. Because, among the IL-12 family members, the level of p40 was the highest in most cancer cells, we examined its role in survival of cancer cells. It is often quite straightforward to consider a KO mouse model to in- vestigate the role of a molecule in any disease process. However, we − − cannot use p40 ( / ) mice in this case because knocking out the p40 gene will knock down IL-12, IL-23, p402, and p40. Therefore, to investigate the role of p402 and p40 in the life and death of different cancer cells, the only feasible approach was to use neutralizing mAbs against these molecules. The p40 mAb a3-3a, but not p402 mAb a3-1d, increased LDH (lactate dehydrogenase) release (Fig. 2 A– D) and decreased MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] (Fig. 2 E–H) in TRAMP (Fig. 2 B and F), 4T1 (Fig. 2 C and G), and Hepa (Fig. 2 D and H)cells.Onthe contrary, p40 mAb had no effect on LDH or MTT in KLN lung cancer cells, indicating the specificity of the effect. To monitor death of tumor cells from another perspective, we measured calcium influx through the t-type calcium channel. Interestingly, treatment of dif- ferent cancer cells with p40, but not p402, mAb displayed a reduced t-type calcium influx in TRAMP (Fig. 2J), 4T1 (Fig. 2K), and Hepa (Fig. 2L) cells. Again, p40 mAb remained unable to modulate t-type calcium influx in KLN cancer cells (Fig. 2I). Accordingly, TUNEL (Fig. 2M) and Annexin V labeling (Fig. 2N) followed by quantitative analyses (Fig. 2 O and P) reinforced that neutralization of p40, but not p402, stimulated death in TRAMP, 4T1, and Hepa cancer cells. However, p40 mAb had no effect on the apoptosis of KLN cancer cells. Similar to mouse cancer cells, neutralization of p40, but not p402, induced cell death in human LNCaP (SI Appendix,Fig.S3A Fig. 1. Levels of IL-12 family of cytokines in different cancer cells. Levels of and D), MCF-7 (SI Appendix,Fig.S3B and E), and Hep3B (SI p40 and p402 (A), IL-12 (B), and IL-23 (C) were measured in supernatants of Appendix,Fig.S3C and F)cancercells. mouse tumor cells (KLN, TRAMP-C2, 4T1, and Hepa) by ELISA. Results are mean ± SD of three different experiments (aP < 0.001 vs. p40 measured in respective Regression of Tumor Growth and Stimulation of Death Response in tumor cells). (D) TRAMP-C2 supernatants were passed through a 10-kDa cut column followed by native PAGE and Coomassie blue staining. The p40 band was Vivo in Tumor Tissues by Specific Neutralization of p40. Next, we detected by comparing with pure p40 protein (far left column). (E) Native PAGE examined the effect of p40 mAb on tumor size and the death of immunoblot analyses of p40 in the supernatants of TRAMP-C2 from three sep- tumor tissue in vivo when TRAMP cells were grown as a tumor in
arate experiments. (F) Intracellular FACS assay of p40 and p402 in cultured the flank of male C57BL/6 mice. When the tumor had reached 0.8– TRAMP-C2 cells after live/dead cell exclusion. (G) Mean fluorescence intensity 1 mm in size, mice were treated with p40 mAb a3-3a at an i.p. dose < (MFI) of intracellular p40 and p402 was calculated (*P 0.01 vs. p40; NS, not of 2 mg/kg body weight twice per week for 2 wk. The tumor size was significant). Native PAGE followed by Coomassie staining was performed to recorded every alternate day. After 2 wk, tumors were labeled with detect p40 in supernatants of human cancer cells [Hep3B (H); LnCAP (I); MCF-7 IR dye 800-conjugated 2-deoxy-D-glucose via tail vein injection and (J)]. Levels of p40 and p402 (K) and IL-12 and IL-23 (L) were measured in super- natants of Hep3B, LnCAP, and MCF-7 cells by sandwich ELISA. Results are mean ± then imaged in a LI-COR Odyssey IR scanner. Interestingly, we SD of three different experiments (aP < 0.001 vs. p40 measured in respective observed that administration of p40 mAb significantly reduced the tumor cells). Native PAGE immunoblot analyses of p40 and other IL-12 members size of tumors as evident from whole-animal IR images (Fig. 3A)and of cytokines (M) in supernatants of Hep3B, LnCAP, and MCF-7 cells. images of excised tumors (Fig. 3B). From the tumor regression curve, it was clear that the size of tumors in the p40 mAb-treated group decreased steadily and significantly [F1,7 = 16.78 (>Fc = 5.59); P < different supernatants with anti-human pan-IL-12p40/p70 antibody 0.01 (P = 0.004); Bonferroni post hoc test] compared with the con- also showed that all three human cancer cells produced a higher trol group (Fig. 3C). On the contrary, control hamster IgG had no level of p40 compared with other IL-12 cytokines (Fig. 1M). such effect (Fig. 3C). Next, we monitored apoptosis in these tumor tissues. Our TUNEL results clearly showed that the population of Levels of p40, p402, and IL-12 in Serum of Patients with Prostate TUNEL-positive cells in the p40 mAb-treated tumors was higher Cancer. To understand the significance of our finding, we mea- than in control or IgG-treated tumors (Fig. 3D), suggesting that sured levels of p40, p402, and IL-12 in the serum of patients with neutralization of p40 by p40 mAb is capable of inducing apoptosis in prostate cancer (n = 11) and healthy control subjects (n = 10). tumor tissues. To further confirm this finding, we monitored the There was no significant difference between patients with pros- mRNA expression of different apoptosis-related genes in treated and tate cancer and healthy controls in terms of age and race (Table untreated tumor tissues by using a custom gene array. Gene array F = 1), but the level of p40 was significantly higher [ 1,18 74.8235 (Fig. 3E) followed by real-time PCR analysis of individual genes (Fig. >F = P < P = ( c 4.42); 0.0001 ( 0.0000000792)] in the serum of 3F) clearly indicated that p40 mAb treatment significantly elevated patients with prostate cancer compared with healthy controls the expression of apoptosis-related genes such as caspase 3, caspase SI Appendix A (Table 1 and , Fig. S2 ). In contrast, the level of p402 7, caspase 8, caspase 9, BAD, BID, cytochrome C, BAK, and p53. was significantly higher [F1,18 = 43.4154 (>Fc = 4.42); P < 0.0005 (P = 0.0000345)] in healthy controls relative to prostate cancer Enlargement of Tumor by Supplementation of p40. Because neu- cases (Table 1 and SI Appendix, Fig. S2B). On the contrary, we tralization of p40 induced the death of cancer cells and reduced did not observe any significant difference in IL-12 levels [F1,18 = tumor size, we investigated whether supplementation of p40 had
2of6 | www.pnas.org/cgi/doi/10.1073/pnas.1705536114 Kundu et al. Downloaded by guest on September 29, 2021 Table 1. Levels of p40, p402, and IL-12 in serum of patients with prostate cancer and control subjects
Samples Age, y Race/ethnicity Gleason score PSA, ng/mL Cancer stage p40, pg/mL p402, pg/mL IL-12, pg/mL
PC-1 55 White 6–7 13.15 III 3,471 ± 318 ND 4 ± 0.4 PC-2 58 Asian 8–9 42.4 III 2,962 ± 732 330 ± 290 4 ± 0.084 PC-3 56 White 8–9 28.1 III 4,246 ± 1,272 618 ± 478 6 ± 1.76 PC-4 67 White 7 22.48 III 3,337 ± 2,206 514 ± 68 3 ± 0.753 PC-5 61 White 7–8 29.0 III 2,799 ± 291 ND 3 ± 2.93 PC-6 56 White 8–9 56.4 III 2,494 ± 1,251 454 ± 88 6 ± 2.49 PC-7 60 White 8–9 24.6 III 3,590 ± 1,822 ND 3 ± 2.83 PC-8 59 Caucasian 7 4 IV 3,446 ± 1,082 ND 4 ± 1.37 PC-9 52 White 6 28.4 II 1,490 ± 543 ND 4.9 ± 0.21 PC-10 76 Caucasian 9 6.52 IV 3,704 ± 1,460 ND 5 ± 3.81 PC-11 71 Caucasian NA 102.79 IV 2,184 ± 751 ND 7 ± 1.61 Control-1 65 White Healthy Healthy Healthy 1,060 ± 855 1,164 ± 328 3 ± 0.448 Control-2 63 White Healthy Healthy Healthy 1,018 ± 333 729 ± 530 6 ± 0.097 Control-3 83 White Healthy Healthy Healthy 866 ± 540 2,361 ± 235 3 ± 0.097 Control-4 63 White Healthy Healthy Healthy 863 ± 359 1,991 ± 785 5 ± 0.98 Control-5 61 White Healthy Healthy Healthy 1,055 ± 768 1,884 ± 433 6 ± 0.098 Control-6 57 Hispanic Healthy Healthy Healthy 1,198 ± 395 2,148 ± 539 10 ± 1.69 Control-7 70 White Healthy Healthy Healthy 1,343 ± 728 1,765 ± 507 3 ± 0.53 Control-8 67 White Healthy Healthy Healthy 606 ± 401 1,534 ± 634 6 ± 2.05 Control-9 66 White Healthy Healthy Healthy 1,003 ± 293 1,192 ± 325 6 ± 1.22 Control-10 52 Asian Healthy Healthy Healthy 865 ± 107 626 ± 200 10 ± 4.5
Serum samples were obtained from Discovery Life Sciences. Each sample was analyzed for p40, p402, and IL-12 three times by ELISA. NA, not available; ND, not detectable; p40, p40 monomer; p402, p40 homodimer; PC, prostate cancer. INFLAMMATION the opposite effects. In fact, at a dose of 20 ng/mL, recombinant up-regulated expression of IFN-γ.MTT(SI Appendix,Fig.S7A and IMMUNOLOGY AND p40 increased the survival of TRAMP cells as shown by MTT (SI C)andLDH(SI Appendix,Fig.S7B and D) assays revealed that Appendix,Fig.S4A)andLDHrelease(SI Appendix,Fig.S4B). p40 mAb treatment induced death in Hepa (SI Appendix,Fig.S7A Accordingly, recombinant p40 significantly increased the size of and B)and4T1(SI Appendix,Fig.S7C and D) cells via IFN-γ. tumors as evident from whole-animal IR scanning (SI Appendix, We also observed that p40 mAb-treated tumor tissue ex- Fig. S4C) and images of excised tumors (SI Appendix,Fig.S4D). It pressed more IFN-γ mRNA (SI Appendix,Fig.S8A–C)and is clear from the tumor growth curve that the size of tumors in the protein (SI Appendix,Fig.S8D and E) compared with control p40-treated group increased significantly [F1,4 = 9.99 (>Fc = 7.7); and IgG-treated tumors. In contrast, recombinant p40-treated γ P < 0.05 (P = 0.03) at 100 ng per mouse and F1,4 = 10.78 (>Fc = tumor tissue expressed less IFN- protein compared with con- 7.7); P < 0.05 (P = 0.03) at 200 ng per mouse on week 8] compared trol (SI Appendix,Fig.S8D). IFN-γ-neutralizing antibody also with the control group (SI Appendix,Fig.S4E). On the contrary, abrogated tumor-suppressing activity of p40 mAb [F1,7 = 13.7 >F = P < P = heat-inactivated p40 had no such stimulatory effect [F1,4 = 0.055 ( c 5.59); 0.01 ( 0.004); Bonferroni post hoc test] SI Appendix (
Kundu et al. PNAS Early Edition | 3of6 Downloaded by guest on September 29, 2021 endocytic vesicles in the membrane (Fig. 5C, Bottom). In contrast, we did not observe increased membrane levels of β-actin in p40- treated TRAMP cells (Fig. 5C). These results suggest that p40, but neither p402 nor p70, may be involved in arresting IL-12Rβ1inthe membrane. However, there was no difference in IL-12Rβ1in whole-cell extract when TRAMP cells were treated with these cytokines (Fig. 5D), negating the possibility of induction of IL- 12Rβ1 level by p40 monomer. Consistently, the p40 mAb abro- gated p40-mediated increase in IL-12Rβ1 in membrane of TRAMP cells (Fig. 5E), suggesting that p40 is indeed involved in the mem- brane arrest of IL-12Rβ1. Pan-cadherin was analyzed to monitor the purity of the membrane fraction (Fig. 5F, Top). The level of β-actin was higher in p40 mAb-treated cells, suggesting that the absence of p40 may induce the formation of endocytic vesicles in TRAMP cells (Fig. 5F, Bottom). However, again, there was no difference in the level of total IL-12Rβ1 between cells treated with p40 with or without p40 mAb and cells treated with p40, suggesting that p40 mAb treatment does not down-regulate the expression of IL-12Rβ1 in TRAMP cells (Fig. 5G). Together, these results suggest that excess p40 released by TRAMP cells inhibit IL-12 signaling by suppressing the internalization or endocytosis of IL-12Rβ1.
Fig. 2. Monoclonal antibody-mediated neutralization of p40, but not p402, induces death of different mouse cancer cells. KLN (A and E), TRAMP-C2 (B and F), 4T1 (C and G), and Hepa (D and H) cells were treated with mAbs against
p40 and p402 for 48 h under serum-free conditions followed by monitoring of LDH release (A–D)andMTT[(E–H) *P < 0.01 vs. control]. t-type calcium influx was performed in KLN (I), TRAMP-C2 (J), 4T1 (K), and Hepa (L) cells. t-type cal- cium influx was measured in the presence of 1 M KCl. TUNEL assay (M)and phycoerythrin-tagged Annexin V staining (N) were performed in KLN, TRAMP- C2, 4T1, and Hepa cells. TUNEL-positive (O) and phycoerythrin-tagged Annexin V-positive (P) cells were counted in 10 different images per group and then plotted as percentages of control. All results are mean ± SD of three different experiments (*P < 0.001 vs. respective controls for TUNEL and Annexin V assays).
Internalization of IL-12Rβ1 in Tumor Cells by Selective Neutralization of p40. The IL-12 signaling pathway is initiated by the interaction between IL-12 and IL-12R, which is a heterodimer of IL-12Rβ1 and IL-12Rβ2. A functional IL-12R has been reported to be in- ternalized after successful binding with its ligand IL-12 (18). Therefore, we examined if p40 monomer was involved in the ar- resting of IL-12R on TRAMP cells to negate the IL-12 signaling pathway. Interestingly, our FACS analyses revealed that the treatment with p40, but not with p402 or p70, increased the surface expression of IL-12Rβ1 in TRAMP cells (Fig. 5A, i–iv). In contrast, Fig. 3. Regression of TRAMP tumor in vivo in mice by p40 mAb. (A) Eight- to 10-wk-old male C57BL/6 mice (n = 5 per group) were injected s.c. with a p40 did not have any effect on the surface expression of IL-12Rβ2 SI Appendix suspension of 1 million TRAMP-C2 cells. After approximately 6 wk, when ( ,Fig.S12). Furthermore, treatment with p40 mAb, tumors were 0.8–1 mm in size, mice were treated with p40 mAb (Middle) but not IgG, down-regulated the membrane level of IL-12Rβ1 and hamster IgG (Right) at a dose of 2 mg/kg body weight twice per week. (Fig. 5A, v and vi), suggesting the involvement of p40 in arresting After 2 wk, tumors were labeled with Alexa 800-conjugated 2-deoxy-D- IL-12Rβ1 on the membrane. To further confirm, we performed glucose dye via tail vein injection and then imaged in a LI-COR Odyssey IR immunoblot analyses of IL-12Rβ1 in the membrane fraction of imaging system. Results were compared with the control group (Left). TRAMP cells. Interestingly, we found that treatment with p40, but (B) Tumors were excised from the flank of all groups of mice. Five mice were β included in each group. (C) Tumor size was monitored every alternate day. not with p402 or IL-12, increased the presence of IL-12R 1inthe ± membrane (Fig. 5B). Pan-cadherin was analyzed to check the Results are mean SEM of five different mice. (D) TUNEL assay in control, IgG, and p40 mAb-treated tumors (green, β-actin; red, TUNEL). (E) Custom purity of the membrane fraction (Fig. 5C, Top). Surprisingly, we β mRNA array for 12 different apoptotic genes in control and p40 mAb- found increased levels of -actin in membrane fractions of p402- treated group, which was then plotted with Heat Map Explorer software. and p70-treated TRAMP cells, suggesting that the treatment with (F) Real-time mRNA analyses of 12 apoptotic genes. Results are mean ± SEM p402 or p70 is possibly associated with increased formation of of five mice (*P < 0.05 vs. control and **P < 0.01 vs. control).
4of6 | www.pnas.org/cgi/doi/10.1073/pnas.1705536114 Kundu et al. Downloaded by guest on September 29, 2021 is known that dendritic cells and macrophages, cells that are able to secrete heterodimeric IL-12 or IL-23, always produce an excess of p40 as monomer or homodimer (i.e., p402)(4).However,bi- ological functions of p402 and p40 remained unknown as a result of the unavailability of specific neutralizing antibodies. Here, we demonstrate that different cancer cells produce ex- cess p40 compared with p402, IL-12, and IL-23, and that p40 is involved in cancer cell survival. Our conclusion is dependent on the following observations. First, the level of p40 was much higher in mouse (TRAMP, 4T1, and Hepa) as well as human (LNCaP, Hep3b, and MCF-7) cancer cells compared with p402, IL-12, and IL-23. On the contrary, we did not notice this selective increase in p40 in KLN mouse lung cancer cells, indicating the specificity of this finding. Second, serum of patients with prostate cancer also contained greater levels of p40 compared with healthy control subjects. Third, neutralization of p40, but not p402,induced INFLAMMATION IMMUNOLOGY AND
Fig. 4. Role of IFN-γ in p40 mAb-mediated death of TRAMP-C2 cells. (A)Cells were treated with p40 mAb and control IgG for 6 h, followed by real-time PCR analysis for IFN-γ mRNA expression (aP < 0.01 vs. control). (B) Levels of IFN-γ were measured in supernatants by ELISA after 24 h of treatment (aP < 0.001 and bP < 0.01 vs. control). (C) Intracellular IFN-γ FACS was performed after live/dead cell exclusion. Phycoerythrin (PE) control was included to show the specificity. (D) MFI of IFN-γ was calculated. (E) Immunocytochemical analyses of IFN-γ (green) in TRAMP cells after 24 h of treatment. Results represent three independent experiments. (F) Levels of IL-10 were measured by ELISA after 24 h of treatment (aP < 0.05 vs. control). (G) TUNEL assay in control cells and cells treated with p40 mAb, IgG, p40 mAb plus different doses of IFN-γ-neutralizing Ab, p40 mAb plus IgG, and IFN-γ Ab TRAMP cells after 48 h of treatment. Under similar treatment conditions, LDH (H) and MTT (I) assays were performed. Results are mean ± SD of three different experi- ments (aP < 0.01 vs. control; bP < 0.01 vs. p40 mAb-treated cells).
Next, we investigated mechanisms by which neutralization of p40 induced the internalization of IL-12Rβ1. Receptor internalization primarily involves clathrin-dependent and caveolin-dependent path- ways. To examine the involvement of clathrin or caveolin, we used two pharmacological inhibitors, filipin and chlorpromazine (cpm), Fig. 5. Neutralization of p40 by mAb stimulates the internalization of IL-12Rβ1
respectively. Interestingly, pretreatment with filipin, but not cpm, in TRAMP-C2 cells. (A) Cells were treated with p40 (20 ng/mL), p402 (20 ng/mL), significantly inhibited the membrane internalization of IL-12Rβ1in p70 (20 ng/mL), p40 mAb (0.5 μg/mL), and mouse IgG (0.5 μg/mL) for 2 h in serum- p40 mAb-treated TRAMP cells (Fig. 5 H and I), suggesting that p40- free conditions followed by FACS analyses of IL-12Rβ1 in control cells (i) and cells β treated with p70 (ii), p40 (iii), p402 (iv), p40 mAb (v), and IgG (vi). Immunoblot mediated internalization of IL-12R 1 occurs via a caveolin-sensitive β and clathrin-independent pathway. Immunofluorescence analysis analyses of membrane-bound IL-12R 1(B), pan-cadherin (pCAD) (C), and total IL-12Rβ1(D)inp40-,p402-,p70-,p40mAb-,andIgG-treatedTRAMPcells.Im- further confirmed that the p40 mAb-mediated internalization of munoblot analyses of membrane-bound IL-12Rβ1(E), pCAD (F), and total IL-12Rβ1 in TRAMP cells is caveolin-dependent (Fig. 5 J–L). IL-12Rβ1(G) in p40 cytokine- and p40 mAb-treated TRAMP cells. Results represent three independent experiments. Immunoblot analyses of IL-12Rβ1(H)inthe Discussion membrane fraction of p40 mAb-treated TRAMP cells pretreated with 5 μM filipin IL-12 (p35:p40) is an important cytokine for triggering cell- or 2 μM cpm for 2 h. Immunoblot results were normalized with pCAD immuno- mediated immune response (4). It is produced mainly by antigen- blot (Bottom). (I) Relative density of immunoblot analyses normalized with pCAD. ± < presenting cells upon activation through Toll-like receptors and by Results are mean SD of three different experiments (*P 0.001 vs. p40 mAb). + Immunocytochemical analyses of IL-12 Rβ1 (red) and caveoiln-1 (Cav-1; green) in interactions with CD4 T cells (4, 19). Eventually, p40 was shown (J) control, (K)p40mAb-,and(L) p40 mAb plus filipin-treated TRAMP cells. Nuclei to pair with p19 to form a newly discovered cytokine, IL-23 (20). were stained with DAPI. (M) Schematic presentation by which neutralization of Either p19 or p35 is constitutively expressed in many cell types. It p40 induces cell-mediated immunity in TRAMP cells.
Kundu et al. PNAS Early Edition | 5of6 Downloaded by guest on September 29, 2021 death in different mouse and humor cancer cells. Again, p40 mAb mediated immunity, IL-12 produced from antigen-presenting cells had no effect on the survival of KLN cells. Fourth, t-type calcium stimulates IFN-γ from T cells. In contrast, IL-12 generated from influx, a growth-supportive event in cancer cells, was significantly cancer cells in response to p40 neutralization is inducing IFN-γ reduced in TRAMP, 4T1, and Hepa cells, but not KLN cells, when production from the same cells in an autocrine fashion. A successful treated with p40 neutralizing antibody. Fifth, our TUNEL and interaction between IL-12 and IL-12R transmits the downstream Annexin-V staining experiments displayed more death in TRAMP, signal and then internalizes the receptor inside the cell. On the 4T1, and Hepa cells, but not KLN cells, after treatment with contrary, an unsuccessful interaction between IL-12 and IL-12R p40 mAb. Finally, i.p. injection of p40 mAb, but not IgG, signifi- leaves the receptor arrested in the membrane and unable to trans- cantly reduced the size of the tumors grown in the flank of male mit any downstream signal. Interestingly, we have found that p40 is C57BL/6 mice. This report demonstrates a biological role of involved in the membrane arrest of IL-12Rβ1, but not IL-12Rβ2. p40 monomer in cancer cell survival. As the serum of patients with prostate cancer also contained higher levels of p40 compared with Materials and Methods that of control subjects, these results indicate the possible thera- Animals and Regents. SI Appendix, SI Materials and Methods, provides further peutic prospect of p40 neutralization in cancer. details in regard to animals and regents, sandwich ELISA procedures, flow While investigating mechanisms behind p40-mediated killing of cytometry analysis with live/dead cell exclusion, tumor development and tumor cells, we observed up-regulation of IFN-γ, a major cytotoxic measurement, tissue preparation and immunohistochemistry, semiquantitative inflammatory cytokine (21), by p40 mAb in pure TRAMP cells. This RT-PCR and real-time PCR, FACS, and membrane isolation. Animal maintaining observation is striking, as T lymphocytes (22) and natural killer cells and experiments were in accordance with National Institutes of Health (23) are considered primary sources of IFN-γ. However, previous guidelines and were approved by the Institutional Animal Care and Use com- mittee (IACUC 14-019) of the Rush University of Medical Center, Chicago, IL. studies demonstrated that it can be produced by murine macro- phages (13) as well as epithelial cells (14, 15), prompting us to ex- γ MTT and LDH Assays. MTT and LDH assays were performed as described pre- amine IFN- production in TRAMP epithelial cells. We found that viously (26). TRAMP cells expressed very little IFN-γ in unstimulated conditions and that p40 mAb treatment stimulated the production of IFN-γ by TUNEL Assay. Following treatments with mAb against p40 or p402, TUNEL several fold. Moreover, cancer cells with epithelial origin such as assays were performed as described previously (26). 4T1 and hepatocellular origin such as Hepa also expressed significant amounts of IFN-γ when treated with p40 mAb, further suggesting Immunoblot Analyses. Immunoblot analyses were performed as described pre- that functional blocking of p40 could stimulate IFN-γ production in a viously (26) with the use of different primary antibodies (SI Appendix,TableS1). wide range of cancer cells. Consistent with the cytotoxic nature of IFN-γ, neutralization of this molecule abrogated p40 mAb-mediated Statistical Analysis. For tumor regression, quantitative data were presented as death of tumor cells and suppression of tumor growth in vivo in mice, the mean ± SEM. Statistical significance was accessed via one-way ANOVA demonstrating that p40 mAb induces death of cancer cells via IFN-γ. with Student–Newman–Keuls post hoc analysis. Other data were expressed γ as means ± SD of three independent experiments. Statistical differences Activation of the IL-12 signaling pathway induces IFN- pro- ’ duction in various cells via JAK-STAT pathway (4, 24, 25). Ac- between means were calculated by the Student s t test. A P value of less than 0.05 was considered statistically significant. cordingly, p40 mAb stimulated the production of IL-12 and IFN- γ in TRAMP cells, suggesting that the absence of p40 may favor ACKNOWLEDGMENTS. This study was supported by National Institutes of the interaction of IL-12 with IL-12R to turn on the transcription of Health Grants NS83054 and NS97426 and Veteran Affairs Merit Awards IFN-γ (24) in these cells. This is interesting because, in cell- I01BX002174 and 1I01BX003033.
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