Trichinella spiralis ESP inhibits tumor cell growth by regulating the immune response and inducing apoptosis

Although Trichinella (T. causes it has a strong and has for and Our previous study results showed that T. spiralis infection can inhibit the growth of liver cancer cells, but the specic mechanism has not been BALB/c mice injected with H22 cells and then infected with T. spiralis were used to detect tumor inhibition rate. Cell proliferation and apoptosis of H22 cells treated with excretory-secretory product (ESP) were measured by Cell-Counting Kit 8 (CCK-8) and Flow Cytometry (FCM). The expression of apoptosis-related genes in H22 cells and tumor tissues was detected by western blotting and real-time quantitative PCR (qPCR). IL-2, IFN-γ and IL-4 production in the spleens were measured by qPCR and enzyme-linked immunosorbent assay(ELISA). study, we evaluated the expression of relevant cytokines with quantitative real-time PCR (qPCR) and enzyme-linked immunosorbent assay (ELISA). H22 cell inhibition and apoptosis induced by ESP were assessed by using the CCK-8 method and ow cytometry (FCM). The expression of relevant genes was detected by qPCR and Western blotting to determine whether apoptosis occurs via the mitochondrial pathway. This study interprets a possible antineoplastic mechanism on the basis that T. spiralis infection may regulate the expression of cytokines and promote apoptosis in tumor cells.

nonsurgical treatment methods [2][3][4][5][6], neither surgical treatment nor nonsurgical treatment can effectively improve the outcome of liver cancer patients, so further research is necessary to nd a better treatment for liver cancer.
Considerable studies indicate a negative correlation between the prevalence of some parasite infections and cancer, as these infections interfere with tumor growth. Therefore, parasite antigens seem to be promising candidates for cancer immunotherapy. Toxoplasma gondii excreted/secreted antigens play a role in the suppression of B16 tumor growth by downregulating the CD4 + CD25 + Treg population while upregulating the NK cell population in tumor-bearing mice. Plasmodium infection can change the tumor microenvironment and inhibit the growth of lung cancer cells [7]. In addition, Echinococcus granulosus, Taenia crassiceps and Trypanosoma cruzi were also reported to inhibit the growth of breast cancer, colon cancer, colitis-associated colorectal cancer, mammary cancer, melanoma and other cancers [8][9][10][11][12][13]. The antitumor effect of Trichinella spiralis (T. spiralis) cannot be ignored either. As a unique in ammation modulator, T. spiralis ensure its survival and immune dialogue with the host by releasing excretorysecretory product (ESP) [14]. T. spiralis infection was shown to inhibit B16-F10 melanoma cell growth and metastasis by reducing the production of CXCL9 and CXCL10 in a mouse model [15]. Similar inhibitory effects were also found with the myeloma cell line SP2/0 [16]. In addition, T. spiralis muscle larvae (ML) ESP can induce apoptosis in small cell lung cancer H446 cells through the mitochondrial pathway in vitro [17].
To study the inhibitory effect of T. spiralis infection on liver cancer, human hepatoma H7402 cells had been used in our experiments. Although results had shown that the T. spiralis protein A200711 has the ability to induce apoptosis in H7402 cells [18], no speci c mechanism for the antitumor effect on liver cancer has been found. In this study, we evaluated the expression of relevant cytokines with quantitative real-time PCR (qPCR) and enzyme-linked immunosorbent assay (ELISA). H22 cell inhibition and apoptosis induced by ESP were assessed by using the CCK-8 method and ow cytometry (FCM). The expression of relevant genes was detected by qPCR and Western blotting to determine whether apoptosis occurs via the mitochondrial pathway. This study interprets a possible antineoplastic mechanism on the basis that T. spiralis infection may regulate the expression of cytokines and promote apoptosis in tumor cells.

Methods
Preparation of T. spiralis ESP ICR mice infected with T. spiralis for thirty-ve days were sacri ced, ML were collected according to the method of Beiting et al. [19], and adult worms at 6 days post infection (Ad6) were collected according to the method of Sun et al. [20]. The collected ML and Ad6 were washed multiple times with physiological saline containing 500 U/ml mycillin, transferred to RPMI 1640 medium containing 500 U/ml mycillin at a density of 5000 ML/ml and then cultured in a 5% CO 2 incubator at 37°C for 24 h. Subsequently, the culture supernatant was collected by centrifugation, ltered using a 0.22-μm lter, concentrated and stored at -80°C.

Construction of tumor-bearing mice and grouping of experimental animals
Cryogenic vials containing H22 cells were removed from liquid nitrogen and placed into warm (37°C) water. After thawing, the cells were transferred into 5 ml of complete RPMI 1640 medium (containing 10% fetal calf serum and a 1% penicillin-streptomycin solution) and centrifuged at 1000 rpm for 5 min. The cells were then resuspended in complete RPMI 1640 medium for culturing. Once the cells had grown to 80% con uence in the culture dish, the cells were removed using 0.25% trypsin. After three generations, cells in the logarithmic phase were collected and subcutaneously injected into the armpit of BALB/c mice (1×10 5 cells per mouse) to construct a tumor-bearing mouse model. In this study, mice were randomly divided into four groups: the control group, tumor-bearing group, T. spiralis infection group and T. spiralis + tumor group (H22 cells were injected into the mice at 7 days post infection).

Calculation of the tumor inhibition rate
Mice were sacri ced by cervical dislocation on the 27th day after injection of H22 cells, the tumor was harvested, and the size of the solid tumor tissue was compared between the tumor-bearing group and the T. spiralis + tumor group. Then, the tumor tissue was weighed, and the tumor inhibition rate was calculated. Tumor inhibition rate (%) = (average tumor weight of tumor-bearing group-average tumor weight of T. spiralis + tumor group)/average tumor weight of tumor-bearing group × 100%.

CCK-8 assay
H22 cells in the logarithmic phase were seeded at 1000 cells/well in 96-well plates, and then different nal concentrations of ML or Ad6 ESP (0.05, 0.1, 0.2, and 0.4 mg/ml) were added to the wells. After 48 hours of incubation, cell proliferation was evaluated by using CCK-8 (Med Chem Express, American).
Generally, 10 µl of CCK-8 solution was added to each well, and the samples were incubated for one hour before the absorbance was measured at 450 nm. Each experiment was conducted ve times.

Detection of apoptosis
In this study, ow cytometry with Annexin V and PI staining was used to detect apoptosis. Cellular samples were harvested after incubation with ML or Ad6 ESP, and the density was adjusted to 1 × 10 6 cells/ml. After double staining with Annexin V and PI for 15 min at room temperature with the Annexin V-Alexa Apoptosis Detection Kit (Fcmacs, China), the samples were analyzed on a FACScan ow cytometer equipped with Cell Quest software (BD Biosciences, USA), and the results were used in apoptotic rate analyses.
Western blotting H22 cells incubated with ML or Ad6 ESP for 48 h and mouse spleen tissue collected at different time points (14, 21, 28, and 35 d) were treated with RIPA lysis buffer. A rabbit anti-mouse Bax polyclonal antibody was used at a 1:2000 dilution, a rabbit anti-mouse Bcl-2 monoclonal antibody was used at a 1:1000 dilution, and a rabbit anti-mouse Caspase 3 polyclonal antibody was used at a 1:500 dilution. A goat secondary antibody conjugated with horseradish peroxidase was used at a 1:2000 dilution. Imaging was performed using an ECL-based system. The protein expression level was normalized to the corresponding β-tubulin level in this study. qPCR RNA was extracted from H22 cells after coculturing with ESP for 48 h or from mouse spleen tissue collected at different time points (7, 14, 21, 28, and 35 d) in each experiment group using TRIzol and reverse transcribed using the PrimeScript RT Reagent Kit (Trans, China). The transcriptional levels of IL-2, IFN-γ, IL-4, Bax, Bcl-2, and Caspase-3 were normalized to those of the housekeeping gene GAPDH, and fold changes were determined by relative quanti cation (2^− ΔΔct ). The primers used for qPCR are listed in Table 1. At different time points, the splenocyte culture supernatant of each group was quantitatively analyzed using ELISA kits (R&D Systems, Inc., USA) for pro-in ammatory (IL-2 and IFN-γ) and anti-in ammatory (IL-4) cytokines.

Statistical analysis
All the data were analyzed with SPSS 20.0 software (SPSS, Inc., USA). All the data are presented as the mean ± SD, and ANOVA or a two-tailed Student's t-test was used to examine the statistical signi cance of comparisons of the means of different groups. P < 0.05 was accepted as statistically signi cant.

Results
Mice infected with T. spiralis showed tumor growth inhibition The mice in the T. spiralis + tumor group were injected with H22 cells on the 7th day after infection with T. spiralis (Fig. 1A). The injection time was the same in the tumor-bearing group, and the mice in the two groups were sacri ced 21 days after injection. The tumors in the T. spiralis + tumor group were signi cantly smaller than those in the tumor-bearing group ( Fig. 1B-C), and the tumor inhibition rate calculated from tumor weight was 61.06 ± 6.67% (Table 2). 14.34% (ML ESP), respectively ( Fig. 3A-C), which were both higher than the rate of the control group (P < 0.05) (Fig. 3D), indicating that T. spiralis ESP induce apoptosis in H22 cells.
To determine whether ESP induce apoptosis in H22 cells through the mitochondrial pathway, we tested the changes in the expression levels of mitochondria-related genes with qPCR and Western blotting in vitro and in vivo. Compared with those in the control group, the mRNA and protein expression levels of the pro-apoptotic genes Bax and caspase-3 in the experimental groups showed increases, while the mRNA and protein expression levels of the anti-apoptotic genes Bcl-2 both decreased (P < 0.05) (Figs. 4). The western blot results of tumor tissue apoptosis-related genes at different days (14,21,28,35) after T. spiralis infection are also consistent with the results in vitro (Fig. 5). Thus, the mitochondrial pathway may be a means by which ESP induce apoptosis in H22 cells.

T. spiralis infection inhibited tumor growth by regulating cytokine expression in vivo
ELISA results showed that the IL-2 and IFN-γ levels in the splenocyte culture supernatants of the T. spiralis group and T. spiralis + tumor group were higher than those of the control group and tumor-bearing group at 7, 14, and 21 days. The IL-4 concentration in the splenocyte supernatants of the T. spiralis group and T. spiralis + tumor group was high on the 14th and 21st days, and that of the tumor-bearing group gradually increased with time (Fig. 6A). The qPCR results were basically consistent with the ELISA results (Fig. 6B).

Discussion
In this study, mice were injected with H22 cells on the 7th day after infection with T. spiralis and sacri ced 21 days after injected. It was observed that the T. spiralis + tumor group showed signi cant inhibition of tumor growth compared with the tumor-bearing group, and the inhibition rate was 61.06 ± 6.67%. To further explore the mechanism by which T. spiralis infection inhibits tumor growth, we examined changes in cytokine expression and those in H22 cell proliferation inhibition and apoptosis induced by ESP.
Different parasites can exert antitumor effects through different mechanisms. Protozoans such as Toxoplasma gondii and Trypanosoma cruzi have an antitumor effect on some types of cancer cells through an antiangiogenic capacity, immune response reactivation and apoptosis induction. On the other hand, Taenia crassiceps is able to regulate the cancer-promoting in ammatory response. Echinococcus granulosus has different antitumor mechanisms, such as immune response reactivation and antiproliferative effects on transformed cells [21]. However, the antitumor mechanism of T. spiralis has not yet been clearly clari ed. According to several studies, the antitumor mechanism of T. spiralis can be divided into two types: one is regulation of the host's immune response, and the other is acting directly on tumor cells through arrest of the cell cycle and apoptosis induction in tumor cells. The antitumor effect of T. spiralis on a melanoma model established by subcutaneous injection of B16-F10 cells was achieved by complex changes in the regulation of cytokine pro les, including those of CXCL9, CXCL10, and CXCL13 [15]. A crude extract of T. spiralis inhibited cell proliferation through arrest of the cell cycle in the G1 or S phase in the human chronic myeloid leukemia cell line K562 and the hepatoma cell line H7402 [18,22]. T. spiralis ML ESP can induce apoptosis in H446 cells through the mitochondrial pathway [17]. This study explained the mechanism of the antitumor effect of T. spiralis on H22 liver cancer cells from the two perspectives of regulating the expression of host cytokines and inducing apoptosis.
At present, most studies have concluded that T. spiralis induces a mixed Th1/Th2 response during the intestinal phase and predominance of the Th2 response during the muscle phase. In the early stage of infection, T. spiralis induces an increase in the levels of the Th1 cytokines IL-2 and IFN-γ, which subsequently transforms into increases in the levels of the Th2 cytokines IL-4 and IL-13 with the migration of newborn larvae and formation of cysts [23,24]. IL-2 and IFN-γ are two important antitumor cytokines. IL-2 can stimulate natural killer (NK) cells, natural killer T (NKT) cells and B cells, playing an important role in antitumor immunity [25]. IFN-γ has strong antitumor and immunomodulatory effects and can inhibit tumor angiogenesis and tumor development [26]. The results of this experiment showed that the expression of antitumor-related cytokines such as IL-2 and IFN-γ increased during the acute phase of T. spiralis infection, which might inhibit the proliferation of tumor cells in the early stage of tumor growth.
One of two classical cell apoptosis pathways is the mitochondrial apoptotic pathway, also known as the intrinsic apoptotic pathway. The apoptosis-related genes bcl-2 and bax can control the release of cyt-c and activation of caspase-3, thereby mediating cell survival or death [27]. Bax can translocate to the mitochondrial membrane after stimulation by a death signal, and then permeable pores are formed in the mitochondrial membrane, disrupting the concentrations of ions and proteins inside and outside the membrane and releasing cyt-c and other proapoptotic factors [28]. In the presence of dATP, cyt-c combines with Apaf-1 to form an apoptotic body and recruits procaspase-9 to oligomerize [29], thereby activating caspase-3 and initiating the caspase cascade reaction [30]. Overexpression of bcl-2 can block the process of apoptosis in three ways. One is the formation of a heterodimer with bax, which inhibits the translocation and dimerization of bax, closes the permeable pores, blocks the release of cyt-c and inhibits the activation of caspase-3 to effectively inhibit cell apoptosis [31]. Second, bcl-2 binds to Apaf-1 and inhibits its function, preventing the activation of procaspase-9 to achieve an antiapoptotic effect [32].
Third, overexpression of bcl-2 can cause the accumulation of glutathione in the nucleus, leading to changes in the redox balance in the nucleus, preventing intracellular Ca 2+ out ow, inhibiting the release of cyt-c, and thereby inhibiting the activation of caspase-3 and interrupting the process of apoptosis. To date, there are few studies on the mechanism of apoptosis induction by T. spiralis. Some researchers have found that the expression of apoptosis-related factors in muscles increases during cyst formation [33]. Other researchers have suggested that T. spiralis ML ESP induce apoptosis through the outer caspase-dependent apoptotic pathway or mitochondrial pathway [34,35]. Here, we evaluated the changes in the expression of mitochondrial apoptosis-related genes. We found that stimulating tumor cells with ML or Ad6 ESP in vitro increased the expression of the proapoptotic gene Bax and decreased the expression of the antiapoptotic gene Bcl-2, thereby regulating the increase in the caspase-3 level and ultimately leading to apoptosis. Experiment results in vivo consisted with that in vitro. Thus, T. spiralis ESP activate intrinsic mitochondrial pathways in the process of inducing apoptosis in H22 cells. However, the involved components that participate in the antitumor effect of ESP are not yet clear, although during the formation of T. spiralis cysts, the p53 protein, which regulates the cell cycle and induces apoptosis, is expressed, which may lead to the inhibition of tumor growth [36].
T. spiralis ESP, which are di cult to collect, comprise a complex protein mixture, and their toxic effects on the body need to be demonstrated. If ESP are to be developed into a new drug for cancer treatment, it is necessary to further explore the components that exert the antitumor effect. Fortunately, through proteomic evaluation of ESP, it has been discovered that there are antitumor-related components, but this has not been con rmed experimentally. In the future, we may select these proteins in ESP for veri cation and hope to develop a new candidate for cancer treatment.

Conclusions
This study provides important data showing negative relationship between T. spiralis infection and H22 cell growth. T. spiralis infection could inhibit H22 cell growth by inducing apoptosis in vivo and in vitro. In addition, the Th1 cytokines induced in the early stage of T. spiralis infection also has anti-tumor effects.
Understanding the underlying anti-tumor mechanism of T. spiralis infection will provide new ideas for treatment of liver cancer. Availability of data and materials The datasets used or analysed during the current study are available from the corresponding author on reasonable request.

Competing Interests
The authors disclosed no conflicts of interest regarding this publication.    protein was obtained from the H22 cells and expression of Bax, Bcl-2, Caspase-3 was detected by Western blotting using the β-tubulin gene as a reference for normalization of protein expression. H22 cells not cocultured were ESP were used as control.

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