| Background:In recent years, incidence and mortality of pancreatic cancer from malignant tumor were showing obvious rising trend. Due to the lack of early onset in clinical characteristic, clinical applications, screening is still lack of effective means at the same time. The most is to belong to middle-late diagnosis of pancreatic cancer and has highly malignant degree, lesser resection rate, easy to secondary recurrence, poor prognosis, low survival rate, etc. The etiology, development and mechanism of invasion and metastasis of pancreatic cancer has made some progress on basic research. In the surgery, neoadjuvant therapy and target therapy, new technologies, methods and drugs have already been found. Looking for pancreatic cancer gene therpy targets has been the research hotspot in the field of pancreatic cancer. In the molecular targeted therapies, there are still many problems to be solved.In the present studies, toll-like receptors(TLRs), not only innatural immunity, but also the connection aspects between the innate and actquired immune, play an important role.Chronic inflammation by promoting the evolution of tumors especially found that TLRs have also expressed in both inside and outside some certain tumor cell membranes. Those researches have great significance on occurrence, development and treatment of tumor.TLR7 is one of the important members of TLRs family. Recently, more researches show that TLR7 is mainly expressed in certain types of tumor cells, including lung cancer, kidney cancer, bladder cancer, skin cancer and colorectal cancer cells. Generally, the pancreatic cancer development is involved in the genetics and its microenvironment. TLR7 is one of the pattern recognition receptors which has been identified as primary sensors of bacterial and viral components. TLR7 could trigger a series of signal pathways by binding the single-strand RNA or small molecular chemicals, such as imiquimod, resiqumod, gardiquimod and its derivatives. Effects of TLR7 agonists on the cancer cells prove to be conflicting, however, there are lots of details remained unknown. Thus, the effects of TLR agonists on tumor cells need to be carefully investigated before their application in immunotherapy. TLR7 agonist has applied experimental and clinical research, but there is no literature reports about relations between gardiquimod and pancreatic cancer. The effects of TLR7 agonist on pancreatic cancer cells needs to be carefully examined.In this study, we observed the effects and molecular mechanism of the TLR7 agonists(gardiquimod)on the proliferation, migration and apoptosis of pancreatic cancer cell line(Bx PC-3). Our result will also provide a new insight of the pathogenesis of the pancreatic cancer, and may lay foundation on the new treating way in clinical.Objective: To explore the effects of the activation of TLR7 on the proliferation, migration and apoptosis of pancreatic cancer cell line, Bx PC-3, and then uncover the mechanism.Methods: The human primary pancreatic carcinoma Bx PC-3 cells were cultured, Real-time PCR and Western-blot were used to detect expression of TLR7 in m RNA and protein level. Bx PC-3 cells were treated with various concentration(0 μg/m L, 1.5 μg/m L, 3.0μg/m L, 5.0 μg/m L) of the ligands of TLR7 namely gardiquimod for different time(1d, 2d, 3d, 4d, 5d), MTS and flowcytometry methods were used to analyze the activation of TLR7 on the proliferation of Bx PC-3 cells. Bx PC-3 cells were treated with 3μg/ml concentration of the ligands of TLR7 for 1d and following 6d, the wound-healing were used to detect the effect of gardiquimod on migration. Bx PC-3 cells were treated with gardiquimod( 3μg/m L) of the ligands of TLR7 for different time( 1-5d), the flowcytometry was used to detect the effect of gardiquimod on apoptosis. Bx PC-3 cells were cultured and treated with various concentration of the ligands of TLR7 gardiquimod for different time(0min, 12 min, 30 min, 1h, 3h, 6h, 12 h, 24 h, 48h), and following 12 h, the Western-blot were used to detect the expression of cyclin B1, cyclin E, Bcl-2 and Bax, and then analysis the effects and mechanism of gardiquimod on the proliferation, migration and apoptosis of pancreatic cancer cell line, Bx PC-3.Results: The results of Real-time PCR and Western blot highlighted that the expression of TLR7 on pancreatic cancer cell line(Bx PC-3) was lesser than peripheral blood monouclear cells(PBMC), the expression in the latter is as 20.12±7.254 times as the former, with statistical significance( P < 0.01). The MTS, flowcytometry and wound-healing methods investigated that the ligands of TLR7 gardiquimod suppressed the proliferation of Bx PC-3 cells at a dose-dependent and time-dependent ways, with statistical significance(P < 0.05); Gardiquimod induced the apoptosis of Bx PC-3 cells at a time-dependent ways, with statistical significance(P < 0.05);Gardiquimod inhibited the migration of Bx PC-3 cells at a time-dependent ways, and the percents of wound-healing was only 60% after 6d, with statistical significance(P <0.01). Western-blot results showed that gardiquimod could dwonregulate the expression of the cyclin B1, cyclin E and Bcl-2, meanwhile upregulate the expression of the Bax. Gardiquimod can obviously raise expression of the Bax after 6 hour.Conclusion: We found the expression of TLR7 on pancreatic cancer cell line(Bx PC-3). TLR7 agonist gardiquimod could inhibit the proliferation, migration and induced the apoptosis of the Bx PC-3 cells. This suggested that the effect of gardiquimod on the proliferation and apoptosis of Bx PC-3 may partially depend on the regulation of cyclin B1, cyclin E, Bcl-2 and Bax expression. |
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