Abstract Background Cancer immunotherapies aimed at activating immune system, especially by blocking immune checkpoints, have become a successful modality for treating patients with advanced cancers. However, its clinical practice is frequently conceded by high outcomes, low initial response rates and severe side effects. New strategies are necessary to complement and advance this biological therapy. Erzhi Pills (EZP) have diverse pharmaceutical effects including immune regulation, anti-tumor and anti-senescence. We hypothesized that EZP could exert its antitumor effect through immunomodulation. Purpose The aim of this study was to investigate the effects of EZP on anti-tumor activities, and define its molecular mechanisms. Methods By applying melanoma model with high immune infiltrates, we determined the anti-melanoma effect of EZP. To identify whether this effect was mediated by direct targeting tumor cells, cell viability and apoptosis were examined in vitro. Network pharmacology analysis was used to predict the potential mechanisms of EZP for melanoma via immune response. Flow cytometry, immunohistochemistry (IHC), enzyme-linked immunosorbent assay (ELISA) and crystal violet (CV) experiments were performed to detect T cell infiltrations and functions mediated by EZP. The mechanism of EZP was further investigated by western blotting both in vivo and in vitro. Results The administration of EZP significantly inhibited tumor weight and volume. EZP extract could only slightly reduce cell viability and induce melanoma apoptosis. Network pharmacology analysis predicted that JAK-STAT signaling pathway and T cell receptor signaling pathway might be involved during EZP treatment. Flow cytometry and IHC analyses showed that EZP increased the number of CD4^+ T cells and enhanced the function of CD8^+ T cells. In co-culture experiments, EZP elevated killing ability of T cells. Western blotting showed that EZP treatment reduced PD-L1 signaling pathway. Conclusion These findings indicated that EZP exerted anti-melanoma effects by inducing apoptosis and blocking PD-L1 to activate T cells. EZP might represent a promising candidate drug for cancer immunotherapies. Keywords: EZP, Melanoma, PD-L1, T cells, Cancer immunotherapies 1. Introduction Cancer cells can evade from immune surveillance by binding immune checkpoint-specific ligands to prevent immunogenic cell death [[39]1,[40]2]. During the last decade, cancer immunotherapies aimed at activating immune system to recognize and eliminate tumor cells, especially by blocking immune checkpoints, have become a successful modality for treating patients with advanced cancers [[41]3,[42]4]. Programmed cell death receptor 1 (PD-1) and programmed cell death ligand 1 (PD-L1) checkpoint inhibitors are widely applied in clinic to attack malignant cells. Meanwhile, the clinical practice of immunotherapy is frequently conceded by high outcomes, low initial response rates and severe side effects [[43]5,[44]6]. New strategies are necessary to complement and advance this biological therapy. Traditional medicines are gaining increasing popularity in cancer treatment due to low toxic effects, lack of drug resistance, and convenient administration [[45][7], [46][8], [47][9],[48][7], [49][8], [50][9],[51][7], [52][8], [53][9]]. Erzhi pills (EZP) are composed of Ligustri lucidi fructus (LLF, Ligustrum lucidum Ait.) and Ecliptae herba (EH, Eclipta prostrata L.) on the ratio of 1: 1 and have diverse pharmaceutical effects including immune regulation and anti-senescence [[54][10], [55][11]]. In the clinical practices, EZP was used to prevent and treat breast cancer [[56]12]. Network pharmacology predicted that EZP could inhibit skin cutaneous melanoma. Given the immunoregulatory, anti-aging and antineoplastic effects reported for EZP, we hypothesized that EZP could exert its antitumor effect through immunomodulation. The aim of this study is to define molecular mechanisms of EZP using melanoma model to supply pharmacological proof supporting its clinical employment. 2. Methods 2.1. Chemicals and reagents EZP (Z36021716) was obtained from Jiangxi Yaodu Zhangshu Pharmaceutical Co., Ltd (Jiangxi, China). Dacarbazine (DTIC, HY-B0078) and polyethylene glycol 300 (PEG300, HY-Y0873) were purchased from MedChemExpress (NJ, USA). Dimethylsulfoxide (DMSO, 20–139), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, M2128), tribromoethanol ([57]T48402) and tert-amylalcohol (152463) were obtained from Sigma-Aldrich (MO, USA). Recombinant murine interferon-γ (IFN-γ, 315-05) was purchased from PeproTech (NJ, USA). Phosphate-buffered saline (PBS, P1020), 4 % paraformaldehyde (PFA, P1110), ethylene diamine tetraacetic acid (EDTA, E8040) and red blood cell (RBC, R1010) lysis buffer were provided from Solarbio (Beijing, China). 0.9 % sterile saline (1501251) was purchased from Guangdong Otsuka Pharmaceutical Co., Ltd (Guangdong, China). Bovine serum albumin (BSA, 4240GR100) was purchased from Biofroxx (Einhausen, Germany). Tween‐20 (T6335) was provided from Macklin (Shanghai, China). Immunohistochemistry (IHC) kit (SA1022) was purchased from Boster (Wuhan, China). 2.2. Extract preparation EZP extract was prepared by the Tianjin State Key Laboratory of Modern Chinese Medicine of Tianjin university of Traditional Chinese Medicine. 2.3. Animals A total of 40 clean specific pathogen-free grade female C57BL/6 mice (weight, 20–25 g; age, 6–8 weeks) were purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd. (Beijing, China). The protocols for in vivo study were approved by the Ethics Committee of Tianjin University of Traditional Chinese Medicine (approval number TCM-LAEC2021214, registered on February 10, 2021). Animal rooms were maintained a standard rearing environment at a temperature of 23 ± 2 °C, 50 ± 10 % humidity and a 12-h light/dark cycle. Free food and water intake and adaptive feeding was allowed for a week prior to initiation. 2.4. Cell culture and drug treatment Jurkat cells (BNCC338675) were purchased from Beina Bio (Beijing, China). A375 human melanoma cells and B16F10 mouse melanoma cells were derived from our laboratory depository. A375 and B16F10 cells were cultured in DMEM with high glucose containing 1 % penicillin/streptomycin (P/S) and 10 % heat-inactivated fetal bovine serum (HI-FBS) at 37 °C in 5 % CO[2] incubator. Jurkat cells were maintained in RPMI 1640 containing 10 % HI-FBS and 1 % P/S. Cell culture reagents were purchased from Biological Industries (BI, Kibbutz, Israel) unless otherwise mentioned. Some western blotting experiments, A375 and B16F10 cells adjusted the cell density to 1.5 × 10^5 cells/mL were seeded into 6-well plates and treated with 0.1 % DMSO and 1 μg/mL EZP extract for 48 h. To induce the maximum expression of PD-L1, A375 cells (1 × 10^5 cells/mL) were cultures into 96-well plates and treated with 10 ng/mL IFN-γ for 24 h in culture media. Afterwards, A375 cells were administrated with 0.1 % DMSO, different EZP extract concentrations (0.01, 0.1, 1 μg/mL) for 48 h. 2.5. Melanoma model and treatment Animals were randomly divided into five groups of eight animals each: one control group and four treatment groups. B16F10 mouse melanoma cells (6 × 10^4 cells) in 200 μL PBS were injected subcutaneously into the right flank of C57BL/6 female mice. Following tumor inoculation, the control group was treated with 0.9 % sterile saline whereas the other groups received DTIC and varying doses of EZP. EZP was dissolved in 0.9 % sterile saline and administered by gavage at 0.59, 1.17 and 2.34 g/kg/day for 14 consecutive days. As a positive control, DTIC (70 mg/kg) was suspended in a 50:50 mix of 0.9 % sterile saline and PEG300 and provided to mice by intraperitoneal injection every 2 days on fourth day after inoculation. Treatments were started immediately after the injection of the B16F10 cells and continued until day 14, when mice were sacrificed. Tumor length (L) and width (W) were measured using a calliper, and the tumor volume was calculated as L × W^2 × 0.5. At the end of the experiment, tumor tissues were collected, weighed, frozen in liquid nitrogen and stored at −80 °C until use or 4 % PFA fixed and paraffin embedded for subsequent sectioning. Meanwhile, single cell suspensions of spleen were prepared. 2.6. MTT assays Cell viability was detected using MTT assays. In brief, A375 and B16F10 cells in logarithmic growth phase were seeded in 96-well plates and treated with DMSO or different EZP extract concentrations (0.001, 0.01, 0.1, 1 μg/mL) for 24, 48 and 72 h. The supernatant was discarded and then 20 μL MTT reagent (5 mg/ml) was added to each well and incubated for 4 h and then terminated by adding 150 μL DMSO and incubating at 37 °C in the dark for 15 min. OD value was assessed by measuring the absorbance at 570 nm (Molecular Device Flex Station® 3, Silicon Valley, USA). 2.7. Western blotting Shortly, 20 μg proteins from tumors or cultured cells were separated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then electrotransferred onto polyvinylidene fluoride membranes. Next, membranes were blocked in Tris-buffered saline-Tween-20 (TBS-T) (5 % non-fat powdered milk, 0.2 % Tween 20) and washed thrice with TBS-T (2 % Tween 20). The membranes were incubated with the primary antibodies against PD-L1 (A18103, Abclonal, Wuhan, China), caspase-3 (#14220), cleaved-caspase-3 (#9664), B-cell lymphoma-2 (Bcl-2, #3498), Bcl-2 like 1 (Bcl-xl), Bcl-2 associated X protein (Bax, #2772), signal transducer and activator of transcription (STAT) 1 (#14994), phospho-STAT1 (p-STAT1, #9167), nuclear factor kappa-B (NF-κB) p65 (RELA, #8242) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (#5174, Cell Signaling Technology, CST, MA, USA) in 5 % BSA in TBS-T overnight at 4 °C and followed by exposure to the secondary horseradish peroxidase-labeled goat anti-rabbit IgG (H + L) (ZB-2301, Beijing ZSGB-BIO Technology Co., Ltd., Beijing, China) for 1 h at room temperature. Then the blots were developed by Super ECL Detection Reagent (36208ES60, YEASEN, Shanghai, China). The protein bands were photographed using a gel imaging system (Amersham Imager 680, GE, Boston, USA). Finally, results were analyzed by image J software (Image J, NIH). 2.8. Network pharmacology The drug-likeness and the physicochemical properties of the selected hits of EZP were evaluated using PubChem database ([58]https://pubchem.ncbi.nlm.nih.gov/), SwissADME database ([59]www.swissadme.ch) and TCMSP database ([60]http://tcmspw.com/tcmsp.php). Related targets of EZP were selected using TCMSP database and SwissTargetPrediction database ([61]http://www.swisstargetprediction.ch/). Targets of melanoma and immune response were collected using GeneCards database ([62]https://www.genecards.org/) and comparative toxicogenomics database (CTD, [63]http://ctdbase.org/). We intersected the obtained drug targets with the targets associated with melanoma and immune response, and obtained a Venn diagram of the common targets. Based on the results of protein-protein interaction (PPI) in STRING database ([64]https://string-db.org), the Cytoscape 3.7.2 software was used to construct the PPI network. Based on the PPI network, the plug-in “CytoHubba” was used to calculate the degree values of nodes in the network. Gene ontology (GO) functional enrichment analysis and kyoto encyclopedia of genes and genomes (KEGG) pathway enrichment analysis were performed using the DAVID database ([65]https://david.ncifcrf.gov/). 2.9. Flow cytometry Splenocytes were isolated by mechanical disruption from the freshly obtained spleen of each group mice and passed through a 70 μm cell strainer to prepare cell suspensions. After removal of RBC using RBC lysis buffer, cell suspensions were surface labeled with mouse antibodies for 30 min at 4 °C. After staining, the cells were washed once with PBS and resuspended in staining buffer (PBS, 2 mM EDTA, 2 % HI-FBS). Finally, samples were assessed using BD Accuri™ C6 plus (BD Biosciences, CA, USA) and analyses were performed using FlowJo. PerCP/Cyanine5.5 anti-mouse CD3 antibody (E-AB-F1013J), FITC anti-mouse CD8a antibody (E-AB-F1104C) and PE anti-mouse CD4 antibody (E-AB-F1097D) were obtained from Elabscience (Wuhan, China). 2.10. Immunohistochemistry IHC stainings of CD4, CD8 and granzyme B (GZMB) were performed on paraffin-embedded tumor sections. After deparaffinization, rehydration, antigen retrieval, endogenous peroxidase inactivation and blocking non-specific binding, the 5 μm-thick sections were incubated with primary anti-CD4, anti-CD8 and GZMB antibodies overnight at 4 °C. Then, the slides were incubated with a corresponding secondary antibody and photomicrographs were taken utilizing a Nikon Eclipse E400 microscope (Nikon, Tokyo, Japan). Quantitative IHC data for CD4, CD8 and GZMB marker was calculated by counting the number of CD4^+, CD8^+ and GZMB^+ cells in three fields at 400 × magnification. CD4 (D7D2Z, #25229), CD8 (D4W2Z, #98941) and GZMB (E5V2L, #44153) antibodies were purchased in CST. 2.11. Co-culture of Jurkat and A375 cells We constructed an in vitro co-culture model to model the tumor microenvironment. Briefly, cultures containing either Jurkat, A375 cells or both were established in 12-well plates containing DMEM with 10 % FBS and 1 % P/S. As target cells, A375 cells were seeded in 96-well plates at a density of 5 × 10^5/well and treated with 0.1 % DMSO and different EZP extract doses (0.01, 0.1, 1 μg/mL) for 4 h. Next, A375 cells were stimulated with IFN-γ (10 ng/mL) for 24 h. Jurkat cells, acting as effector cells, were stimulated by simultaneously activating the T-cell receptor/CD3 complex and CD28 coreceptor with purified anti-human CD3 antibody (317301) and purified anti-human CD28 antibody (302901, BioLegend, CA, USA). For co-cultures, Jurkat cells were added to the target cells at an effector-to-target (E:T) ratio of 10:1 and were allowed to attach for 72 h at 37 °C in 5 % CO[2] incubator. 2.12. Crystal violet staining After 72 h of culture, the co-cultured cells were harvested for further analysis. For the crystal violet (CV) staining, cells were fixed with 4 % PFA and stained with 0.1 % CV (C0121, Beyotime, Shanghai, China) for 10 min and washed with distilled H[2]O to removed excessive staining solution. Finally, Images were captured by a Nikon Eclipse Ts2 microscope (Nikon, Tokyo, Japan) and counted by ImageJ. 2.13. Lactate dehydrogenase assay Culture supernatants were collected after 72 h of co-culture. Lactate dehydrogenase (LDH) was measured by the LDH assay kit (A020-1, Nanjing Jiancheng Bioengineering Institute, Nanjing, China) according to the manufacturer's instructions. The absorbance value of each well was measured with Molecular Device Flex Station® 3 at 450 nm. The harvested media were used for immunoblotting and enzyme immunoassay. 2.14. Enzyme-linked immunosorbent assay The co-culture supernatants were collected for enzyme-linked immunosorbent assay (ELISA). Interleukin-2 (IL-2) was performed using the human IL-2 ELISA kit (JYM0146Hu, Wuhan jiyinmei Biotechnology Co., Ltd., Wuhan, China) according to manufacturer's guidelines. 2.15. Statistical analysis All data are presented as means ± standard deviation (SD). The differences between the two groups were analyzed by performing unpaired Student's t-tests. GraphPad prism 8.0 was used for the statistical analysis. P value of less than 0.05 was considered statistically significant. 3. Results 3.1. EZP administration inhibited melanoma growth Considering EZP possessed pharmacological effects of tonics, immune regulation and anti-tumor activity , we testified the preventive efficacy of EZP on tumor growth in vivo using melanoma-bearing C57BL/6 mice ([66]Fig. 1A). The administration of EZP (0.59, 1.17, 2.34 g/kg) significantly inhibited tumor weight and volume ([67]Fig. 1B–E), indicating EZP provided potential benefit in retarding melanoma development. Fig. 1. [68]Fig. 1 [69]Open in a new tab EZP provided therapeutic benefit in B16F10 tumor-bearing mice. (A) A schematic view of the treatment plan. C57BL/6 mice were implanted with B16F10 mouse melanoma cells (6 × 10^4 cells) in 200 μL PBS and received EZP (0.59, 1.17, 2.34 g/kg) treatment or DTIC (70 mg/kg). (B) Photograph of tumor tissues. (C) Summary of weight data of B16F10 tumors harvested after euthanizing the mice. (D) Plots of tumor volumes and mice body weight measured every 2 days. (E) Summary of volume data of B16F10 tumors harvested after euthanizing the mice. Data are expressed as mean ± SD. n = 8. **P < 0.01 and ***P < 0.001 vs saline. EZP, erzhi pill; DTIC, dacarbazine. To identify whether this anti-melanoma effect was mediated by direct targeting tumor cells, two melanoma cell lines (B16F10 and A375) were applied to examine the viability of melanoma cells under the treatment of EZP. As shown in [70]Fig. 2A and B, EZP extract could only slightly reduce cell viability. We further detected the expressions of apoptosis-related proteins after treatment with 1 μg/mL EZP extract for 48 h based on the results of cell viability. In B16F10 cells, expression levels of cleaved-caspase-3 and Bax were significantly upregulated, while the levels of Bcl-2 and Bcl-xl were downregulated ([71]Fig. 2C). In A375 cells, expression levels of cleaved-caspase-3 and NF-κB p65 were significantly upregulated, while the levels of Bcl-xl were downregulated ([72]Fig. 2D). These results showed that EZP induced melanoma apoptosis by inhibiting anti-apoptotic proteins and activating pro-apoptotic proteins. The above data suggested that EZP restrained melanoma growth associated with induced cell death. Fig. 2. [73]Fig. 2 [74]Open in a new tab In vitro antitumor activity of EZP extract against melanoma cells. (A and B) Cell proliferation ability of B16F10 (A) and A375 (B) was detected using MTT assay at 24, 48, and 72 h after treatment with different concentrations of EZP (0.001, 0.01, 0.1, 1 μg/mL) for 24, 48, and 72 h n = 7–12. (C and D) Caspase-3, cleaved-caspase-3, Bcl-2, Bax, Bcl-xl and NK-κB p65 protein expression levels with GAPDH serving as a loading control (cleaved-caspase-3 expression levels versus caspase-3) were analyzed by western blotting and quantified after B16F10 (C) and A375 (D) cells treating with 1 μg/mL EZP extract for 48 h n = 3. Data are expressed as mean ± SD. *P < 0.05, **P < 0.01 and ***P < 0.001 vs DMSO. DMSO, dimethylsulfoxide; Bcl-2, B-cell lymphoma-2; Bax, Bcl-2 associated X protein; Bcl-xl, Bcl-2 like 1; NF-κB, nuclear factor kappa-B; GAPDH, glyceraldehyde 3-phosphate dehydrogenase. 3.2. A network pharmacology approach to predict potential mechanisms of EZP To predict potential mechanisms of EZP for melanoma via immune response, network pharmacology was applied as an invaluable tool to describe and analyze its medicinal properties of complex natural products [[75]13,[76]14]. Through searching the PubChem, SwissADME, TCMSP and SwissTargetPrediction databases to obtain active compounds and prediction targets of EZP, combining the compounds information of each database and eliminating the repeated targets, a total of 435 active targets of EZP were obtained. Next, we searched CTD and GeneCards database and screened the known target genes related to melanoma and immune response ([77]Fig. 3A). The intersection of 435 predicted EZP targets, 20765 melanoma targets and 15607 targets of immune response was obtained in Venn diagram to obtain 406 common targets ([78]Fig. 3B). These 406 common targets were considered as potential targets of EZP for the treatment of melanoma via immune response. Fig. 3. [79]Fig. 3 [80]Open in a new tab Network pharmacology of EZP for melanoma via immune response. (A) The overall workflow of network pharmacological study of EZP. (B) VENN diagram of the number of target genes of EZP, melanoma and immune response and their common targets. (C and D) PPI analysis. (C) PPI network of common target. (D) Barplot graph of the key common targets of degree in the PPI network. (E) GO functional enrichment analysis. (F) KEGG pathway enrichment analysis. PPI, protein-protein interaction; GO, gene ontology; KEGG, kyoto encyclopedia of genes and genomes. Following this lead, a PPI network was constructed using the STRING database and then processed with Cytoscape 3.7.2 software ([81]Fig. 3C). According to the results of PPI network analyses using CytoHubba to calculate degree value, Jun Proto-Oncogene, AP-1 transcription factor subunit (JUN), STAT1, STAT3, NF-κB, RELA, hypoxia inducible factor 1 subunit alpha (HIF-1α), AKT serine/threonine kinase 1 (AKT1) and phosphoinositide-3-kinase regulatory subunit 1 (PI3KR1) were the key nodes ([82]Fig. 3D). The GO analysis revealed that EZP affects a list of genes associated with cell proliferation, cell cycle, cell apoptosis and T cell activation, and the KEGG pathways analysis stated that JAK-STAT signaling pathway and T cell receptor signaling pathway might be involved during EZP treatment ([83]Fig. 3E and F). STAT1, and STAT3 have been recognized as the key regulatory proteins within the JAK-STAT signaling axis. Additionally, JUN, NF-κB, RELA, AKT1, and PI3KR1 have been identified as the principal regulatory proteins involved in the T cell receptor signaling axis. Interestingly, it has become abundantly clear that these proteins play critical roles in activating T cell pathway [[84]15]. More experimental evidence is required to evaluate the potential immunopharmacological effects of EZP. 3.3. EZP increased the number of CD4^+ T cells and enhanced the function of CD8^+ T cells in vivo T cell responses could dictate tumor regression or progress. CD8^+ T cells have the ability to selectively detect and eradicate cancer cells by producing IFN-γ, GZMB and perforin. CD4^+ T cells help increase the cell-intrinsic anti-tumor activity of CD8^+ T cell by multiple complementary mechanisms [[85][16], [86][17], [87][18],[88][16], [89][17], [90][18],[91][16], [92][17], [93][18]]. Most cancer immunotherapies require T cells to perform anti-neoplastic effects. [94]Fig. 4A presents a flow cytometry gating strategy for CD4^+ and CD8^+ T cells. We observed the role of T cells in the inhibitory effects of EZP in melanoma growth. The administration of EZP significantly increased percentages of CD3^+CD4^+ T cells in spleen and the infiltration of CD4^+ T cells in tumor tissue ([95]Fig. 4B, C, 4E and 4F). However, EZP did not significantly affect the proportions of CD8^+ T cells in spleen and tumor ([96]Fig. 4E and G). We examined CD8^+ T cell function by evaluating intracellular expression of granzyme B. As illustrated in [97]Fig. 4E and H, EZP at clinically equivalent dose (2.34 g/kg) could elevate the expression of granzyme B, indicating EZP may exert its anti-tumor effect by enhancing function of CD8^+ T cells. Fig. 4. [98]Fig. 4 [99]Open in a new tab EZP increased the percentage of CD4^+ T cells and enhanced the function of CD8^+ T in vivo. (A–D) T cells were analyzed by flow cytometry. (A) The gate strategy for CD4^+ and CD8^+ T cells. Flow cytometry plots (B) and bar graphs present percentages of CD3^+CD4^+ T cells (C) and CD3^+CD8^+ T cells (D) in the spleen from each group mice. n = 5. (E–H) IHC staining for the tumor tissues. IHC images (E) and quantifications of CD4^+ T cells (F), CD8^+ T cells (G) and GZMB^+ cells (H) in tumor tissues. Scale bar, 100 μm. n = 3. Data are expressed as mean ± SD. *P < 0.05, **P < 0.01 and ***P < 0.001 vs DMSO. IHC, immunohistochemistry; GZMB, granzyme B. 3.4. EZP enhanced the cytotoxic activity of T cells To determine the extent of fratricide among T cells against melanoma cells, the killing assay was further performed in order to study T cell cytotoxicity using a co-culture model. Jurkat cell line is a human T-leukemic cell line suitable to mimic cultured T cells. The flow chart of the experimental design is shown in [100]Fig. 5A. After co-culture for 72 h, CV staining showed a greater number of surviving cells in the IFN-γ treatment alone group than EZP treatment group ([101]Fig. 5B). Moreover, compared with the IFN-γ treatment group, EZP increased LDH release in the culture supernatant ([102]Fig. 5C). IL-2 mainly produced by CD4^+ T cells and promoted CD8^+ T cells cytotoxicity activity is used to treat metastatic melanoma [[103]19]. In co-culture experiment, IFN-γ decreased the production of IL-2, while increased IL-2 secretion was observed after EZP treatment ([104]Fig. 5D). These results indicated that EZP could enhance the cytotoxic activity of T cells in vitro. Fig. 5. [105]Fig. 5 [106]Open in a new tab EZP enhanced the killing ability of T cells on melanoma cells. (A) Schematic of experimental design for co-culture experiments of Jurkat and A375 cells. Co-cultures at an effector-to-target (E:T) ratio of 10:1 were incubated for 72 h. (B) Crystal violet staining assay. (C) LDH release was measured by an LDH assay. (D) The secretion of IL-2 was measured in co-culture supernatants. n = 3–4. Data are expressed as mean ± SD. ^##P < 0.01 vs DMSO. *P < 0.05, **P < 0.01 and ***P < 0.001 vs IFN-γ group. LDH, lactate dehydrogenase assay; IFN-γ, interferon-γ. (For interpretation of the references to colour in this figure legend,