The Anti-tumor Effect Of Lipoxin A₄ On Hepatocarcionoma And Possible Mechanisms

PartⅠLipoxin A4 inhibited the EMT, proliferation, migration, angiogenesis and metastasis and promoted the apoptosis of hepatocarcinoma cellsBackgroundIt was reported that chronic inflammation has close relationship with the genesis and progress of malignant tumors. Hepatocarcinoma is one of the most popular malignant tumors worldwide with rapid growth, early metastasis and high mortality. What's more, it is widely acceptable that hepatocarcinoma has close relationship with chronic hepatitis. As we all known, tumor microenvironment plays an important role in the progression of solid malignant tumors, moreover, tumor associated macrophages (TAMs) which was a critical part of microenvironment was proposed as a major contributor to tumor progression.Lipoxin (LX), the 'stop signal' for inflammation, which is generated via biosynthetic pathways involving the dual lipoxygenation of arachidonic acid is one of the most important anti-inflammatory or inflammatory pro-resolving mediators in our bodies. Our precious study confirmed that Lipoxin A4 (LXA4) could inhibit the activities of NF-κB in HepG2 cells and RAW264.7 cells. These data provided evidence for our study on the anti-hepatocarcinoma effect of LXA4.ObjectiveTo investigate the effect of LXA4 on the EMT, apoptosis, proliferation, migration and angiogenesis of human hepatocarcinoma cell line (HepG2) and underlying mechanisms in vitro and investigate the effect of BML-111, the analogue of LXA4 on the proliferation, angiogenesis, inflammatory cell infiltration, invasion and metastasis of tumor cells in the primary tumor tissue of H22 bearing mice and underlying mechanisms in vivoMethodsWe simulated inflammatory environment by lipopolysaccharide (LPS) or activated macrophage-conditioned media (ACM), and firstly investigated the effect LXA4 of on the EMT of HepG2 cells by HE staining, immunofluorescence, western blotting and RT-PCR; and then investigated the effect of LXA4 on the apoptosis of HepG2 cells by Hoechst staining, immunofluorescence and Flow cytometry; and following investigated the effect of LXA4 on the proliferation of HepG2 cells by MTT, RT-PCR and western blotting; wound healing test and ELISA method were undertaken to investigate the effect of on the migration and VEGF production of HepG2 cells respectively; finally, we investigated the effect of BML-111 on the invasion, angiogenesis, apoptosis, proliferation, inflammatory cell infiltration and metastasis of tumor cells in the primary tumor tissue of H22 bearing mice.Results1. LXA4 effectively inhibited the EMT of HepG2 cells. By HE staining test, we found that HepG2 cells became dissociated from the epithelial clusters. However, cells recovered to normal after LXA4 was administrated, and Boc-2, the specific inhibitor of LXA4 receptor, could attenuate the effect. Immunofluorescence test found that E-cadherin expression decreased after LPS treatment, and Vimentin and Fibronectin expression increased inversely. However, when LXA4 was administrated, E-cadherin expression increased and Vimentin and Fibronectin expression decreased inversely. Additionally, P53 down regulated E-cadherin expression. Western blotting results were the same as immunofluorescence. RT-PCR test showed that Slug and Twist regulated the process of EMT. 2. LXA4 promoted the apoptosis of HepG2 cells. The apoptosis decreased and Bcl-2 expression increased after LPS treatment. After LXA4 was administrated, apoptosis dramatically increased and Bcl-2 expression decreased. Flow cytometry assay showed that 50nM LXA4 could effectively promote the apoptosis of HepG2 cells.3. LXA4 could effectively inhibit the proliferation of LPS-stimulated HepG2 cells. MTT assay showed that 100,200,400nM LXA4 could effectively inhibit HepG2 cell proliferation. We detected nucleolin (C23) and nucleostemin (NS) expression meanwhile. Immunofluorescence and RT-PCR showed that LXA4 could inhibit C23 and NS expression, and western blotting showed that the inhibitory effect of LXA4 was in dose-dependent manner. Moreover, we found that LXA4 inhibited the proliferation of LPS-stimulated HepG2 cells via inhibiting NF-κBp65. Additionally, we found that the inhibitory effect of LXA4 on proliferation diminished after knocking down FPRL1.4. Clinic specimen of HCC patients were studied, and we found that there was much more infiltration of leucocytes and macrophages in HCC tissue than in surrounding tissue. It was also found out that HCC tissue expressed higher VEGF.5. LXA4 effectively inhibited the proliferation of ACM-stimulated HepG2 cells. MTT assay showed that ACM could promote the proliferation while LXA4 could inhibit this promotion. RT-PCR and immunofluorescence showed that LXA4 could inhibit ACM-stimulated up-regulation of C23 and NS, and western blotting showed that these effects were in dose-dependent manner. More importantly, LXA4 could inhibit the translocation of NF-κBp65 as well as the degradation of IκBα, and SP600125, the specific inhibitor of JNK could attenuate the effect of LXA4.6. LXA4 could inhibit LPS or ACM-stimulated migration and VEGF production. LPS or ACM could speed up HepG2 cell migration while LXA4 could inhibit these effects. LPS promoted the VEGF production from 1023.4 pg/ml to 1285.0 pg/ml (P<0.05) however,200nM and 400nM LXA4 could effectively decrease it to 748.5 and 547.5 pg/ml (P<0.05) respectively. U937/ACM enhanced VEGF production from 578.7 pg/ml to 745.6 pg/ml (P<0.05), when LXA4 administrated, VEGF was as low as 547.5 pg/ml which was significantly lower than ACM treated cells.7. LXA4 could inhibit the EMT of ACM-stimulated HepG2 cells. HE staining showed that ACM derived from U937, THP-1, RAW264.7 and PBMCs could all promote EMT while LXA4 could inhibit these effects.8. The inhibitory effect of BML-111 on the primary tumor and metastatic tumor of H22 bearing mice. For PBS group, the borderline between the tumor tissue and its surrounding tissue was not clear, the capsule was not complete, tumor tissue invaded into the fat tissue, but for BML-111 group, the borderline of the tumor tissue and its surrounding tissue was very clear, the capsule was very complete. For PBS group, the tumor was alive with vascular, had no necrosis, and the atypia of tumor cells was very obvious, but for BML-111 group, the tumor was short of vascular, had necrosis, and the atypia of tumor cells was not obvious. Then TUNEL assay was applied on tumor section, and we found that apoptosis was more obvious after BML-111 treatment. For PBS group, the expression of NS located in the internal of tumor tissues, but for BML-111 group, the tumor tissue had no expression, and the expression of NS located in the borderline between the tumor tissue and normal tissue which was alive with inflammation cells. In addition, the tumor tissue in BML-111 group expressed less VEGF. We next investigated the influence of BML-111 on the inflammatory cell infiltration in tumor tissue. CD68 and CD45 were chosen as the markers for macrophages or leukocyte, respectively. BML-111 could obviously inhibit macrophage and leukocyte infiltration. Finally, we found that there were several metastatic tumors in lung tissue of PBS group mice, but for BML-111 group, the lung tissue was very clear, no metastatic tumors could be found.9. The anti-metastasis mechanism of LXA4. In vitro experiment, immunofluorescence showed that LPS-stimulated osteopontin (OPN) up-regulation could be inhibited by LXA4, and RT-PCR results showed that the inhibitory effect of LXA4 could be attenuated by PD98059, the inhibitor of ERK. LXA4 could inhibit MMP9 expression meanwhile. Western blotting results showed that 50,100,200 and 400nM LXA4 could all up-regulate E-cadherin expression and down-regulate Vimentin and P53 expression. However,50nM LXA4 had the most significant inhibitory effect on P53 expression while 200nM LXA4 had the most significant regulation on E-cadherin and Vimentin expression. In vivo experiment, we found that BML-111 inhibited osteopontin (OPN) and P53 expression and increased E-cadherin expression in the primary tumor tissue of H22 bearing mice.Conclusions1. LXA4 could effectively inhibit the EMT of LPS-stimulated HepG2 cells, Boc-2 could attenuate this effect;2. LXA4 could promote the apoptosis of LPS-stimulated HepG2 cells;3. LXA4 could effectively inhibit the proliferation of LPS-stimulated HepG2 cells via NF-κB pathway;4. LXA4 could effectively inhibit the proliferation of ACM-stimulated HepG2 cells via NF-κB and JNK pathways;5. LXA4 could effectively inhibit the EMT of ACM-stimulated HepG2 cells;6. LXA4 could effectively inhibit the migration and VEGF production of LPS or ACM-stimulated HepG2 cells7. BML-111 could inhibit the proliferation, angiogenesis, invasion and inflammatory cell infiltration and promote the apoptosis of hepatocarcinoma cells in the primary tumor tissue of H22 bearing mice;8. LXA4 and BML-111 inhibited metastasis via down-regulating OPN, P53, MMP9 and up-regulating E-cadherin expression. Moreover, ERK pathway was involved in the mechanism of anti-metastasis. PartⅡThe anti-tumor effect of Lipoxin A4 on hepatocarcinoma by down-regulating Nrf2 via ERK pathwayBackgroundThe genesis and progress of tumor is a multiple and complex process. Chemical agents including those that induce reactive oxygen species (ROS) play an important role in the process of killing tumor cells. Nuclear factor E2-related factor 2 (Nrf2) is involved in this process, and induces the production of phaseⅡdetoxification and antioxidant enzymes, such as NAD(P)H: quinone oxidoreductase-1 (NQO1), heme oxygenase-1 (HO-1), glutathione peroxidase (GPx), manganese superoxide dismutase (MnSOD) and glutamate-cystein ligase catalytic subunit (GCLC), which protect tumor cells from injury. Our precious study showed that Lipoxin A4 (LXA4) could regulate the production of ROS in RAW264.7 cells. Based on that, we suppose that LXA4 could suppress hepatocarcinoma via regulating Nrf2.ObjectiveTo investigate the effect of LXA4 on the regulation of Nrf2 and haseⅡdetoxification and antioxidant enzymes and investigate whether LXA4 could increase the sensitivity of Vincristine (VCR) on HepG2 cells as wellMethodsThe expression of a series of phaseⅡdetoxification and antioxidant enzymes was detected in liver cancer tissue and in surrounding tissue. The effects of LXA4 on the translocation of Nrf2, the production of ROS, the activity of GPx and MnSOD were also detected in HepG2 cells. RNA knockdown experiment was applied to investigate whether the inhibitory of LXA4 on Nrf2 was FPRL1-dependent. The combination of VCR and LXA4 was to investigate whether LXA4 could increase the sensitivity of chemotherapy of VCR. Results1. The expression of Nrf2 and itsⅡdetoxification and antioxidant enzymes was higher in liver cancer tissue than in its surrounding tissue. We found that the expression of Nrf2 and itsⅡdetoxification and antioxidant enzymes including NQO1, HO-1, GPx, MnSOD and GCLC was obviously higher in liver cancer tissue than in its surrounding tissue detected by RT-PCR and immunohistochemistry. The positive rate of Nrf2 was 93.43%±0.05 which was significantly higher than that (3.93%±0.02) in its surrounding tissue. What's more, liver cancer tissue expressed more ERK2, C23 and less Caspase-3.2. BML-111 inhibited the expression of Nrf2 in the tumor tissue of H22-bearing mice. The positive rate of Nrf2 in PBS group was 69.07%±0.07 which was significantly higher than that (8.44%±0.03) in BML-111 group.3. LXA4 inhibited the expression of Nrf2 phaseⅡdetoxification and antioxidant enzymes in HepG2 cells induced by LPS via ERK and FPRL1 pathway. LXA4 inhibited the expression of Nrf2, NQO1, HO-1, GPX, MnSOD and GCLC at mRNA level, the specific inhibitor of ERK, PD98059 attenuated these effects. Moreover, we found that LXA4 could down-regulate the expression of C23 and up-regulate Caspase-3 at mRNA level. That LPS induced up-regulation of Nrf2 in HepG2 cells were abolished by silencing FPRL1 expression with specific siRNA.4. LXA4 inhibited the translocation of Nrf2 in HepG2 cells induced by LPS. Immunofluorescence analysis showed that Nrf2 was mainly localized in the cytoplasm of control cells. Nuclear translocation of Nrf2 was increased by LPS treatment after 24h. Western blotting analysis showed that Nrf2 was mainly detected in the nuclear fractions in both LPS-treated and LPS+LXA4 treated cells, Nrf2 was dramatically increased in the nucleus of cells treated with LPS, and these effects could be inhibited by LXA4. 5. LXA4 decreased the activity of SOD and GPx. We found that LXA4 could decrease the activity of SOD and GPx in LPS+ LXA4 group compared with LPS group, but PD98059 could attenuate the effect of LXA4.6. LXA4 increased the production of ROS. We found that LXA4 could increase the production of ROS while LPS could decrease it, and the effect of LXA4 could be attenuated by PD98059.7. LXA4 increased the sensitivity of chemotherapeutics via ERK pathway. Morphological observation showed that VCR could effectively promote the death of HepG2 cells; however, this effect was more obvious when cells were co-treated with LXA4 while PD98059 could attenuate this effect. MTT assay showed that the combination of VCR and LXA4 had the best inhibitory effect; what's more, the inhibitory effect existed until the 6th day. C23, Nrf2, ERK, pERK at protein level were also detected. As shown in Fig.6C, we found that the phosphorylation of ERK was positively related with Nrf2 expression, and LXA4 could down-regulate Nrf2 which was regulated by the phosphorylation of ERK. Moreover, the combination of VCR and LXA4 could decrease C23 expression more effectively than VCR alone.Conclusions1. Nrf2 was abnormally activated in HCC tissue.2. BML-111 could effectively inhibit Nrf2 expression in the primary tumor tissue of H22 bearing mice.3. LXA4 could down regulate Nrf2 expression in HepG2 cells via ERK pathway, and the underlying mechanism was inhibiting the translocation of Nrf2.4. LXA4 could increase the sensitivity of chemotherapy of VCR, and the underlying mechanism was inhibiting the phosphorylation of ERK.