摘要
MUC1是黏蛋白家族中的跨膜糖蛋白,不仅具有润滑、保护及调节细胞间的粘附等机械作用,而且作为癌基因可导致正常细胞恶性转化。可溶性MUC1(MUC1/SEC)通过抑制DC细胞成熟而抑制Th1细胞,还发现其可通过调节天然免疫和T细胞免疫抑制肿瘤生长。我们的研究首次发现外源性MUC1串联重复序列多肽(MUC1多肽)能直接抑制T淋巴瘤Jurkat、肝癌SMCC7721、乳腺癌MCF-7、B淋巴瘤Raji和单核淋巴瘤U937细胞增殖。而且MUC1多肽也能抑制BABL/c小鼠Jurkat细胞皮下移植瘤的生长(P<0.05)。为研究MUC1多肽抑制肿瘤生长的机制,我们首先检测MUC1多肽是否诱导细胞凋亡。经Giemsa染色和Annexin V/PI双标记法检测结果显示未见明显的凋亡细胞。细胞周期分析结果显示MUC1多肽诱导Jurkat细胞生长抑制,细胞周期阻滞在G0/G1期。有研究报道MUC1/SEC可以与细胞膜上MUC1/Y结合。因此我们进一步调查MUC1多肽是否与细胞表面MUC1蛋白相互作用抑制肿瘤生长。流式细胞仪检测肿瘤细胞表面MUC1蛋白的表达结果显示,Jurkat、SMCC7721、MCF-7细胞表面MUC1蛋白表达较高,而Raji和U937细胞表达略低。应用抗MUC1多克隆抗体封闭Jurkat细胞表面MUC1表位后再用MUC1多肽刺激,结果发现MUC1多肽诱导的生长抑制几乎全部消失。在MUC1过表达试验中,发现MUC1多肽明显抑制稳定转染MUC1基因的B16-MUC1细胞生长,但对B16细胞和转染空载体的B16-neo细胞生长无明显作用。应用MUC1 siRNA沉默Jurkat细胞MUC1表达后,再用MUC1多肽刺激,结果发现MUC1多肽诱导的Jurkat细胞生长抑制作用消失。GST pull down实验显示MUC1多肽与细胞表面MUC1蛋白存在直接相互作用。上述结果证实外源性MUC1多肽抑制肿瘤细胞生长是通过结合细胞表面MUC1蛋白发挥作用,进一步揭示MUC1作为癌基因在细胞恶性转化中的作用。
本研究首次发现外源性MUC1串联重复序列多肽具有直接抑制肿瘤生长作用并揭示其作用机制,为恶性肿瘤治疗提供新的研究思路,为开发新的抗肿瘤药物奠定坚实的研究基础,为研究MUC1蛋白的生物学功能建立新的配体模型。
MUC1, a transmembrane glycoprotein of the mucin family, is expressed primarily on epithelial cells and aberrantly overexpressed on epithelial-derived tumor cells including breast cancer, lung cancer, ovarian cancer, prostate cancer, pancreatic cancer and so on and some hematological malignancies. Alternative splicing after MUC1 gene transcription produces 9 different forms of mRNA that encode various isoforms of MUC1 protein including MUC1/REP, MUC1/SEC and so on. MUC1/REP (designed as MUC1 or MUC1/TM) is membrane-bound and MUC1/SEC is secreted and both of them contain tandem repeats, however, unlike MUC1/REP, MUC1/SEC does not contain a cytoplasmic domain. In a previous study, the biological function of MUC1/REP is mainly confined to the lubrication and protective effects provided by this protein. Recent studies have indicated that MUC1 can act as an oncogene and induce cell malignant transformation. MUC1 is involved in activation of signals promotes cell proliferation, through interactions with members in MAP kinase pathway. Furthermore, MUC1 prevents apoptosis by assoicats with P53 and contributes to the transcriptional activation of P21. It is reported that MUC1/SEC can inhibit Th1 cells by supressing DC maturation and tumor growth by regulating innate immune and T cell immune.
In this study, we firstly found that the exogenous MUC1 tandem repeat peptide (MUC1 peptide) could directly inhibit proliferation of various tumor cells such as T cell lymphoma Jurkat, hepatocellular carcinoma SMMC7721, breast cancer MCF-7, B cell lymphoma Raji and monocytic lymphoma U937 cells in vitro and investigated the inhibitory mechanism of MUC1 peptide on tumor cells growth. Inhibitory effect of MUC1 peptide on tumor cells growth
To investigate inhibitory effect of MUC1 peptide on Jurkat, SMCC7721, MCF-7, Raji and U937 cell growth, tumor cells were co-cultured with recombinant MUC1 peptide at 0, 2.5, 5, 10 and 20μg/ml and the viable cell numbers were counted by Trypan Blue. The result showed that recombinant MUC1 peptide inhibited growth of Jurkat, SMMC7721, MCF-7, Raji and U937 cells in dose-dependent manner. GST protein control in the concentration of 2.5, 5, 10, 20μg/ml was designed to exclude the effect of some of components in E. coli. And the result showed that no measurable effect on growth of these cells which mentioned above at all concentration of GST protein, compared to the negative control group P>0.05. The growth of Jurkat cells were inhibited most significantly with the inhibition ratio being 41.1%±6.0%. Hence, Jurkat cells will be defined as the focus in the following studies. Further study showed that synthetic MUC1 peptide also inhibited the growth of Jurkat cell in dose-dependent manner and its inhibitory ratio was slightly higher than recombinant MUC1 peptide. Jurkat cells were stimulated with recombinant and synthetic MUC1 peptides in different times. The result showed that recombinant and synthetic MUC1 peptide inhibited growth of Jurkat cells in time-dependent manner. These results confirmed that MUC1 peptide inhibited the growth of tumor cells.
Antitumor effect of synthetic and recombinant MUC1 peptide in vivo
The growth of Jurkat cells were inhibited most significantly, so we established a tumor animal model by injecting Jurkat cells s.c. into BABL/c mice, to investigate the antitumor effect of recombinant and synthetic MUC1 peptide in vivo. To suppress mouse immunity, firstly BABL/c mice were irradiatied with 6 Gy X-ray, then inoculated with 4×106 Jurkat cells per animal s.c. in the back. The next day of tumor injection, the mice were administered with an immunosuppressive reagent, FTY-720 3 mg/kg every day by oral for 4 days to maintain immunosupressed state. All of mice developed tumors that were identified by HE staining. This established animal model was used to observate the antitumor effect of recombinant and synthetic MUC1 peptide. As soon as Jurkat cells were inoculated, PBS or 60μg of recombinant or synthetic MUC1 peptide per animals was injected through tail vein, once every other day for 3 times. These mice were sacrificed on the 10th day after tumor injection and the volume of tumors was measured by calipers. The result showed that mice had a smaller tumor size when treated with recombinant or synthetic MUC1 peptide, compared with the control group significantly (P < 0.05). This suggested that recombinant and synthetic MUC1 peptide could inhibit growth of Jurkat cells in BABL/c mice.
MUC1 peptide induced G0/G1 phase arrest of Jurkat cell cycle
We found that MUC1 peptide significantly inhibited growth of Jurkat cell in vitro and in vivo. In order to investigate inhibitory mechanism of MUC1 peptide, firstly, we detected whether MUC1 peptide can induce apoptosis. Jurkat cells that stimulated for 48 h with MUC1 peptide were stained by Giemsa staining and Annexin V/PI. The result showed that apoptotic cells were not detected and suggested that MUC1 peptide could not inhibited the growth of Jurkat by apoptosis. In order to study the effect of MUC1 peptide on cell cycle phase, we analyzed Jurkat cell cycle by flow cytometry and found that MUC1 peptide inhibited growth of Jurkat cells and induced G0/G1 phase arrest.
Studies of MUC1 peptide interaction with MUC1 protein on the cell surface
MUC1, as an oncogene, plays an important role in inducing cell malignant transformation. Some studies show that P-selectin glycoprotein ligand 1 is a mucin-like glycoprotein and can bind to P-selectin through the appropriate post-translational modification. And some studies have shown that MUC1/SEC can bind to MUC1/Y on the cell surface. So we presume whether MUC1 peptide can inhibit growth of Jurkat cell by binding to MUC1 protein on its surface. To confirm this assumption, firstly, we analyzed expression of MUC1 protein on the surface of tumor cells, then, we used anti-MUC1 antibody to block the MUC1 protein on cell surface, furthermore MUC1 gene overexpression, gene silencing and GST pull down were used to analyze MUC1 protein interacted with MUC1 peptide.
1. Expression of MUC1 protein on the cell surface
We detected expression of MUC1 protein on the surface of tumor cells with an anti-MUC1 mAb against the extracellular TR domain (HMPV) by flow cytometry. The result showed that MUC1 protein was highly expressed on Jurkat, SMMC7721 and MCF-7 cells in 56.3%, 72.1% and 62.8% respectively; however, it was less expressed on Raji and U937 cells in 16.0% and 35.3%.
2. Effect of recombinant MUC1 peptide on Jurkat cell growth after the epitope of MUC1 is blocked with anti-MUC1 antibody
In order to study whether MUC1 peptide can interact with MUC1 protein on the surface of Jurkat cells, the binding sites of MUC1 protein on the surface of Jurkat cells were firstly blocked with anti-MUC1 antibody for 1h and then these cells were cultured with recombinant MUC1 peptide. The results showed that inhibitory effects of recombinant MUC1 peptide were almost completely abolished after antibody treatment. This suggested that recombinant MUC1 peptide might bind to MUC1 protein on its surface to inhibiti Jurkat cell growth.
3. Recombinant MUC1 peptide inhibited growth of overexpression MUC1 B16-MUC1 cells
To further confirm the binding site of the MUC1 peptide, MUC1 gene overexpression technology had been designed. The full-length MUC1 gene containing 22 tandem repeat sequence was inserted into pcDNA3 vector and transfected into mouse melanoma B16 cells. The B16 cells (B16-MUC1) that stably expressed MUC1 protein were obtained by the selection of G418. The expression of MUC1 protein in B16-MUC1 cells was identified by flow cytometry and the result showed that the positive cells reached to 96.7%. B16-MUC1, B16-neo and B16 cells were co-cultured with recombinant MUC1 peptide and found that recombinant MUC1 peptide inhibited growth of B16-MUC1 cells, compared with that of control group (P <0.01); but no significant effect was found in the B16-neo and B16 cells. These results further suggested that MUC1 peptide inhibited the growth of Jurkat cells by binding to MUC1 protein on its surface.
4. Effect of recombinant and synthetic MUC1 peptide on Jurkat cells in MUC1 knock-down
To further confirm that MUC1 peptide interacts with MUC1 protein on its surface to inhibit growth of Jurkat cells, MUC1 of Jurkat cells were silenced using MUC1 siRNA, and effect of recombinant and synthetic MUC1 peptides on Jurkat cells in MUC1 knock-down were observated. Firstly, according to two target sites in MUC1 mRNA sequence (GATCCCAGCACCGACTACT and ACCTCCAGTTTAATTCCT C), we designed two pairs of DNA template, inserted into pSilencerTM 3.1-H1 neo vector and obtained pSH1Si-MUC1/A and pSH1Si-MUC1/B vectors for MUC1 siRNA expression. We determined the sequences of MUC1 siRNA by sequencing and confirmed that the sequences are same as expected. Then, the recombinant vectors were electroporated into Jurkat cells by 300 V, 950μF. Efficient knock-down of MUC1 was detected by RT-PCR and flow cytometry. Results demonstrate that by 24 h, there is a 50% reduction in MUC1 surface expression; the most effective knock-down was observed at 72 h, where MUC1 expression is nearly undetectable; by 96 h, MUC1 expression reappears.
Recombinant and synthetic MUC1 peptide was cultured with Jurkat cells of MUC1 knock-down for 48 h. The result showed that inhibitory effects of MUC1 peptide on Jurkat cells after MUC1 gene silencing were almost completely abolished. This demonstrated that the MUC1 peptide inhibited growth of Jurkat cells by binding to MUC1 protein on its surface.
5. GST pull down assay of the interaction of MUC1 protein on cell surface with MUC1 peptide
To further confirm MUC1 peptide interacted with MUC1 protein on the surface of Jurkat cell, GST pull down assay was carried out. MUC1-GST was binded to the Glutathione Sepharose 4B beads and reacted with lysates of Jurkat cells. The proteins in sample production were analyzed with anti-MUC1 GP1.4 or HMPV or MUC1/CT. The result showed that a protein, which interacted with the MUC1 peptide in the cell lysate, reacted with anti-MUC1 GP1.4 and HMPV and did not reacted with anti-MUC1/CT, suggested that it might be MUC1 protein. At the same time, MUC1 protein interacted with the MUC1 peptide also was detected in other cells. The results showed that the protein reacted with anti-MUC1 GP1.4 could be found in lysate of SMMC7721, MCF-7, Raji, U937 and actived T cells and could not be found in RAW264.7 and normal T cells.
These results described above indicated that MUC1 peptide inhibited tumor cell growth by binding to MUC1 protein on cell surface.
Recombinant MUC1 peptide has no significant effect on the growth of mouse monocyte-macrophage cell RAW264.7
To further illustrate MUC1 peptide induced growth inhibition of tumor cells by binding to MUC1 protein on its surface, we analyzed effect of MUC1 peptide on the MUC1-unexpressed cell growth. Fistly, we confirmed that mouse monocyte- macrophage cell RAW264.7 did not express MUC1 by RT-PCR; and then RAW264.7 cells were co-cultured with recombinant MUC1 peptide. The result showed that MUC1 peptide had no significant effect on the growth of RAW264.7 cells, compared with that of the control group (P> 0.05). This suggests that MUC1 protein on the cell surface is nessesary for recombinant MUC1 peptide-induced tumor cell growth inhibition.
Recombinant MUC1 peptide has no significant effect on the growth of actived T cells
It was reported that MUC1 was expressed on actived T cells, so we anazlyzed effect of recombinant MUC1 peptide on growth of normal T cells and actived T cells. Peripheral blood mononuclear cells (PBMCs) were separated from whole blood by Ficoll-Paque density gradient centrifugation, cultured with 5μg/ml PHA, then stimulated with 100 U/ml IL-2. Activated T cells harvested and used in the experiments. Normal T cells and actived T cells were co-cultured with recombinant MUC1 peptide respectively. The result showed that MUC1 peptide has no significant inhibitory effect on the growth of normal T cells and actived T cells, compared with the control group (P> 0.05). This suggested that normal cells might be different from tumor cells in proliferative and apoptotic signal transduction and gene regulation. MUC1 peptide inhibited growth of tumor cell might be assisted / participated with other elements or factors, expect interact with MUC1 protein on cell surface.
Conclusion: An exogenous MUC1 tandem repeat peptide (MUC1 peptide) can inhibit the growth of Jurkat, SMMC7721, MCF-7, Raji and U937 cells, and the most significant inhibition is found in Jurkat cell; MUC1 peptide has no significant effect on the growth of T cells and actived T cells. MUC1 peptide can induce G0/G1 phase arrest of Jurkat cell cycle. Exogenous MUC1 peptide can interact with MUC1 protein on cell surface and induce tumor cell growth inhibition, which further suggests that MUC1, as an oncogene, plays an important role in cell malignant transformation. In this study we demonstrate that an exogenous MUC1 peptide can directly inhibit growth of tumor cells and indicat its inhibitory mechanism, which will provide a novel strategy for the development of anti-cancer drugs and a novel ligand model for the study of biological functions of MUC1 protein.
引文
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