PLD4 Promoted M1 Macrophages To Perform An Anti-tumor Effect In Colon Cancer Cells

AimPhospholipase D4(PLD4) was a newly identified protein that belonged to PLD protein family. Due to lacking of PX and PH domain, PLD4 did not exhibit an enzyme activity of PLD. Animal experiments on PLD4 indicated that although PLD4 was widely expressed in body, the expression of PLD4 in immune related tissues as liver, spleen, brain and lymph node was higher than the other organization. Furthermore, PLD4 that expressed in the microglia in brain could affect its activation after treated with lipopolysaccharide(LPS). A large of evidences indicated that macrophages played a key role in the recognition and abolished cancer cells. Though microglia and tumor associated macrophages had many immunological similarities, the expression and function of PLD4 in tumor associated macrophages was unclear. In the present study, we analyzed the expression and function of PLD4 in colon cancer. Method(1) PLD4 was really presented in colon cancer tissuesImmunohistochemical was used to examine the distribution of PLD4 in 110 colon cancer specimens under different pathological and clinical stages. And CD68 was used as a cell surface marker of the macrophages in colon cancer tissues. In continuous paraffins sections, the relationship of CD68 positive macrophages and PLD4 positive cells were analysised by immunohistochemical. Western blot was used to confirm the expression of PLD4 in 12 human surgical specimens and their matched adjacent normal intestinal epithelia.(2) THP-1 cells as a cell model induced into tumor-associated macrophagesFor preparation of M1-polarized THP-1 macrophages, 50 ng phorbol myristate acetate(PMA) was added to THP-1 cells for 48 h, followed plusing 20 ng/ml interferon-γ(IFN-γ) and 100ng/ml lipopolysaccharide(LPS) for the following 40 h. and for M2-polarized THP-1 macrophages, 50 ng PMA was added to THP-1 cells for 48 h, followed by plusing 20 ng/ml interleukin-4(IL-4) for the following 40 h. To determine the macrophages activation levels among the different induced condition in vitro, the amount of IL-1, IL-8, IL-6 and tumor necrosis factor-α(TNF-α) in the culture medium was measured by human instant ELISA kit(3) PLD4-positive cells were the M1-polarized TAMs derived from THP-1 cellsWestern blot analysis was used to detect the expression of PLD4 in macrophages induced before. To detect the expression of PLD4 m RNA, Total RNA was extracted from M1 cells by Trizol Plus RNA Purification Kit. Isolated total RNA was then reverse into c DNA by RT-PCR according to the manufacturer’s instructions.(4) The function of PLD4 in M1 macrophagesTo analysis of the PLD4 impacted on the M1 phenotype cells, using PLD4 specific small interfering RNA(si RNA) to inhibite the expression of PLD4. After M1 cells treated with si RNA, the decreased expression of PLD4 was confirmed by western blot. The relative PLD4 m RNA level both in PLD4-si RNA-treated cells and NG-si RNA treatment for 24 h was detected by QRT-PCR. We detected the secretion of IL-1, IL-6, and TNF-α pro-inflammatory cytokines which could reflect the activation of macrophages in different subtype macrophages and si RNA-interference M1 macrophages.(5) PLD4 impacted on M1 Macrophages in Colon Cancer CellsFurthermore, after co-cultured with si RNA-interference and NG-si RNA treatment of M1 macrophages condition memdium for 24 h, the cell proliferation, apoptosis, cycle and of HT-29, HCT-116, SW-620 colon cancer cells were deceted by CCK8 kits, cell apoptosis kits and cell cycle kits, respectively. Results(1) PLD4 was really presented in colon cancer tissuesImmunohistochemical analysis revealed the main expression of PLD4 was located in colon cancer mesenchymal and lymph nodes compared with normal tissues. At the same time, we analysis the relation between CD68-positive macrophages and PLD4-positive cells, and concluded that PLD4 could exist in partly macrophages in colon cancers. And immunohistochemistry showed strong PLD4 signals in the cytoplasm. Interestingly, the positive rate of PLD4 expression was associated with clinical staging of colon cancer, not pathological stage, age, gender and metalasis. The result in 12 human surgical specimens by Western blot showed that both primary colon carcinoma tissues and their matched adjacent normal intestinal epithelia existed the expression of PLD4, however, the expression of PLD4 in primary colon carcinoma tissues had a higher level than that in control.(2) PLD4-positive cells were the M1-polarized TAMs derived from THP-1Western blot analysis of macrophages homogenates exhibited that mainly M1 cells existed PLD4-related bands of 48 kd, and the expression of PLD4 m RNA in M1 cells was confirmed by RT-PCR at bands of 300 bp. The expression of IL-1, IL-6, IL-8, TNF-α in macrophages, M1 and M2 cells were detected by ELISA. The result showed all the pro-inflammation factors in M1 cells were up-regulated compared to macrophages and M2 cells.(3) PLD4-si RNA interference could reduce the activation of M1 macrophagesA marked decrease of PLD4 protein and m RNA level were found in si RNA-treated cells compared with control by western blot, QRT-PCR, respectively. The si RNA interference showed temporarily reduced release of IL-1, IL-6, TNF-αin M1 cells which indicated that the activation of macrophages was inhibited.(4) PLD4 stimulated M1 macrophages to develop anti-tumor effects in cancer cellsThen three tumor cells cultured with the inhibition of PLD4 in M1 cells condition memdium showed more proliferation abilities, shorter cell cycle and less apoptosis than those of untreated macrophages. These results indicated that PLD4 could be involved in activation process of M1 phenotype TAMs, and played an important role in the progress of colon cancer. Conclution(1) PLD4 was really presented in macrophages in colon cancer tissues;(2) Our findings highlighted a new pattern of tumor-associated macrophages activation which PLD4 could play an important role in this activation signals deliver;(3) PLD4 promoted M1 macrophages to perform an anti-tumor effect in colon cancer cells.