| Esophageal cancer is one of the most malignant tumors in the world, which ranks the eighth in the incidence and the sixth in the mortality for tumors. Esophageal cancer is common with highly incidence rate in Asia and Southern America, especially in Japan and China with esophageal squamous cell carcinoma(ESCC) in most patients, which affects more than 450 000 people worldwide. And the main cause of mortality is local invasion and distant metastasis. Most patients are diagnosed at the advanced tumor stage and usually lose the chance of curative treatment. As a result, its mortality rate is almost equal to its incidence and the five-year survival rate is low at approximately 15%. Despite of advances in the surgical, radiation and chemotherapeutical treatment, it has no benefit on overall survival. Therefore, alternative strategies are needed.Currently, cancer immunotherapy has become a new trend, which could be another treatment pattern following three major tumor therapies including surgery, chemotherapy and radiotherapy. Cancer immunotherapy is to attack the tumor cells by using tumor antigens as targets, makes the patients suffer less trauma and is especially a better choice for patients with no chance of surgery. To identify effective target antigens is the most critical step in cancer immunotherapy. In order to avoid autoimmune diseases during immunotherapy, the specific tumor antigens are supposed to be only expressed highly in tumor tissues and not in normal tissues. Recently, cancer/testis antigens(CTA) are potentical tumor targets for their restricted expression pattens, which are expressed in various tumor cells, but not in healthy tissues except for the testis and placenta. Because testis is immunological privileged organ, without expressing HLA, there is no chance of igniting autoimmunity. Melanoma-associated antigens(MAGE) are a group of well-characterized members of CTA family and our research group directed by our professor has devoted to carrying research on MAGE family, including expression patterns, tumor targets and biological function. According to their expression patterns, MAGE gene family are classified as MAGE-Ⅰand MAGE-Ⅱ. MAGE-Ⅰ are only expressed in tumor tissures while MAGE-Ⅱ can be detected in normal tisures. Therefore, MAGE- Ⅰ belong to the CTA falimy and include MAGE-A, MAGE-B and MAGE-C subfamilies, among which MAGE-A compose of 12 members(MAGE-A1~MAGE-A12).To date, there is limited research on the expression profile, modulation and biological function of MAGE-A in esophageal cancer. Therefore, firstly we took use of PCR and enzyme digestion method to study the expression profile of MAGE-A subfamily members in five ESCC cell lines, and discovered MAGE-A expression patterns in ESCC. Secondly, with decitabine, a demethylating agent, we detected cell proliferation by MTT, cell migration by wound scratch assay, cell invasion capacity by transwell assay, cell cycle by flow cytometry(FCM). In addition, we observed the invasion-associated protein and signal pathway proteins after treatment with decitabine. And we found decitabine modulated MAGE-A expression profile and inhibited cell proliferation and invasion in ESCC cells. Finally, by use of si RNA to silence MAGE-A in Eca109 and KYSE170 cells, we studied the biological function of MAGE-A in ESCC cells, and the mechanism underling the biological roles. Part I Gene expression profile of MAGE-A in ESCC cell linesObjective: to investigate the gene expression profiles of MAGE-A subfamily such as MAGE-A1,-A2,-A3,-A4,-A6,-A8,-A9,-A10,-A11 in five ESCC cell lines including Eca109, KYSE170, TE1, TE10 and TE13.Methods: MAGE-A8,-A9,-A10 and-A11 were determined by conventional RT-PCR. In view of sharing most gene identity with each other, MAGE-A1,-A2,-A3,-A4 and-A6 were performed by multiplex semi-nested PCR and were identified by specific enzyme cut of the PCR products.Results1At least two MAGE-A members were expressed in each of five ESCC cells.2 MAGE-A subfamily were multiply expressed in the five ESCC cells.3 MAGE-A subfamily were heterogeneously expressed in five ESCC cells.4 As for the individual members, MAGE-A2, A3, A8, A9, A10, A11 were expressed with a frequency at 80%, 40%, 20%, 100%, 80%, 40%, respectively.Conclusions:1 MAGE-A subafamily are expressed with high frequency in ESCC cell lines.2 MAGE-A subfamily are expressed in ESCC cell lines with multiple and heterogeneous pattern.3 MAGE-A2, A9, A10 are expressed with higher frequency suggesting as potential targets for esophageal cancer immunotherapy. Part II The modulation of decitabine on MAGE-A expressions and its effects on biological function on tumor cellsPart II Gene expression profile of MAGE-A in ESCC cell linesObjective: to investigate the modulation of decitabine on MAGE-A expression profile in Eca109 cells, study its effects on tumor cell proliferation and invasion and explore its mechanism.Methods: MAGE-A8,-A9,-A10 and-A11 were determined by conventional RT-PCR. In view of sharing most gene identity with each other, MAGE-A1,-A2,-A3,-A4,-A6 were performed by multiplex semi-nested PCR and were identified by specific enzyme cut of the PCR products. After decitabine treatment, MTT were used to evaluate the cell proliferation; Wound scratch assay was performed to dectect the tumor cell migration capability; Transwell matrix penetration assay were for cell invasive ability. And by use of FCM and western blotting, cell cycle analysis, cell invasion associated protein MMP2 and signal pathway protein NF-kB were detected.Results:1 Decitabine modulated the gene expression of MAGE-A members. The Results: baseline expression of MAGE-A in Eca109 cells were MAGE-A2,-A3,-A9, and-A10. After decitabine(0.5 μM) treatment, the positive gene expressions of MAGE-A8 and MAGE-A4 were observed and the expression of MAGE-A9 and-A10 were decreased.2 Decitabine inhibited cell proliferation in Eca109 cells. Decitabine(0.5 μM, 2.5 μM, 5 μM doses) caused time- and dose- dependentant inhibition of Eca109 cell proliferation. Different doses of decitabine(0.5 μM, 2.5 μM, 5 μM) showed with 15%, 17.6% and 18.2% proliferation inhibition at 24 h, respectively. And 72 hours later, it showed 18.18%( P<0.01)、 24.64%(P<0.05)、40.13%(P<0.05)proliferation inhibition.3 Decitabine inhibited cell migration in Eca109 cells. Eca109 cells treated with decitabine(0.5 mM) exhibited a decreased migration activity by 39.1%(P<0.05), 31.7%(P<0.01), 34%(P<0.01) at 6 h, 12 h and 24 h, respectively, as compared with control groups.4 Decitabine inhibited cell invasion in Eca109 cells. The invasive behavior of Eca109 cells at 48 h after decitabine(0.5 μM) treatment was decreased significantly by 47.2%(P<0.05)than untreated cells.5 Decitabine induced G2/M arrest in the cell cycle of Eca109. The number of cells going into the G2/M cell-cycle phase in decitabine and control were 31.59±0.89 and 27.84±0.22(P<0.05), respectvively. And cells in S cell-cycle phase were 25.68±0.83 and 32.03±1.73(P<0.05), respectively. There is no significant difference in G0/G1 phase.6 Decitabine decreases expression of NF-kB2 and MMP2. After treatment with 0.5 mM and 1 mM decitabine, NF-κB p- p100 and NF-κB p52 were decreased by 26.77%, 36.78%(P<0.05)and 34.98%、50.63%(P<0.05). MMP2 expression were also inhibited in 0.5 mM and 1 mM decitabine groups, and decreased by 20% and 24.26%(P<0.05), respectively.Conclusions:1 Low dose decitabine-induced G2/M arrest probably partly contributes to the inhibition of cell proliferation in Eca109 cells.2 Low dose decitabine significantly hampers the invasion in ESCC cell line probably by decreasing expression of MMP2, in which NF-kB2 signal pathway is involved.3 Low dose decitabine enhances the gene expression of MAGE-A8 and MAGE-A4, enlarging the candidates for tumor targets.4 It may pave the way for combining low dose decitabine with immunotherapy targeting MAGE-A for treating advanced esophageal squamous cell carcinoma. Part III The biological role of MAGE-A subfamily and its mechanismObjective: To study the biological role of MAGE-A subfamily on cell proliferation and invasion in Eca109 and KYSE170 cells and explore the underling mechanisms.Methods: According to si RNA silencing technique, the si RNA pools were used to silence all MAGE-A members, which included six si RNA duplexes targeting the identical sequences of MAGE-A subfamily. By use of RT-PCR and western blotting, the silencing effects of MAGE-A si RNA were evaluated. After MAGE-A silencing, MTT was used to evaluate the cell proliferation; Wound scratch assay was performed to dectect the tumor cell migration capability; Transwell chamber with/without gel was set for evaluating cell invasive ability. And by western blotting, cell invasion associated protein MMP2 and signal pathway protein p38 were detected. Further, we used SB203580 to block the p38 signal pathway and detected the cell invasive ability and the expression of MMP2 and c-Myc in ESCC cells.Results:1 MAGE-A si RNA silencing inhibited the MAGE-A expression at m RNA and protein levels. Both cells transfected with MAGE-A si RNA at 50 n M and 100 n M significantly reduced the expression of MAGE-A both at m RNA and protein levels. And cells transfected with 100 n M showed better inhibition of MAGE-A, suggesting 100 n M as proper concentration for lateral experiments.2 Silencing MAGE-A inhibited cell proliferation. The cell viabilities were moderately reduced by 11.26%(P<0.05)and 12.61%(P<0.05)at 48 h after transfection with 100 n M MAGE-A si RNA in Eca109 and KYSE170 cells, respectively.3 Silencing MAGE-A inhibited cell migration. Cells transfected with 100 n M MAGE-A si RNA showed reduced cell migration ability by 32.97% and 41.15%(P<0.05)at 24 h after line scratching in Eca109 and KYSE170 cells respectively.4 Silencing MAGE-A inhibited cell invasion. Cells transfected with 100 n M MAGE-A si RNA showed reduced cell invasion by 32% and 44.55%(P<0.05)in no gel transwell chamber assay while by 51.5% and 39.48%(P<0.05)in gel transwell chamber assay.5 Silencing MAGE-A down-regulated expression of MMP2, c-Myc and phosphorylated-p38. The expression of MMP2 and c-Myc were decreased by 19.12% and 61.11%(P<0.05)in Eca109 cells. The expression of c-Myc were decreased by 47.67%(P<0.05)in KYSE170 cells. And the level of phosphorylated p38(p-p38) in both cells were observed down-regulated by 27.6% and 39.45%(P<0.05)respectively at 48 h after transfection with MAGE-A si RNA, suggesting that p38 MAPK pathway was probably involved in the decreased invasion ability of ESCC cells following MAGE-A silencing in which MMP2 and c-Myc were likely the downstream effectors.6 Block of p38 MAPK signaling pathway by SB203580 inhibited cell invasion and the expression of MMP2 and c-Myc. Both cells treated with SB203580 at 2mm showed decreased cell invasion by 31.67% and 28.2%(P<0.05)as compared with control. Additionally, a statistically significant lower expression of MMP2 by 19.3%(P<0.05)in Eca109 cells was observed, as well as down-expressed c-Myc by 27.65% and 28.2%(P<0.05)respectively in Eca109 and KYSE170 cells after treatment with SB203580 at 2mm. In addition, after SB203580, there is no significant differences in the expression of p-p38/p38 and p100/p52 in both cells. This suggest that MAGE-A silencing and block of p38 signal pathway could lead to the similar effects in tumor cells. And possibly MAGE-A silencing may inhibited tumor cells through p38 MAPK signal pathway, which induced the down-regulated expression of MMP2 and c-Myc.Conclusions:1 MAGE-A silencing inhibits ESCC cell proliferation, migration and invasion.2 MAGE-A silencing inhibits the expression of c-Myc and MMP2 in ESCC cells.3 MAGE-A silencing inhibits the expression of phosphorylated p38, suggesting a possibility for involving p38 signal pathway.4 P38 MAPK pathway is involed in tumor cell inhibition which is led by MAGE-A silencing.5 MAGE-A silencing inhibits tumor proliferation and especially invasion in ESCC cells by modulating the expression of c-Myc and MMP2 through the p38 MAPK signaling pathway. |
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