| Part I THE EFFECT AND UNDERLYING MECHANISM OF LEPTIN ON INVASION IN GASTRIC CANCER CELLSBackgroundGastric cancer (GC) ranks as the second leading cause of cancer-related death worldwide. It is highly aggressive and rapidly metastasizes, which contributes to its high mortality rate. However, the exact effect and mechanism remains unclear. Multiple factors are involved in this process, including obesity, an important risk factor in GC. Leptin is the product of obese gene and implicated in tumorigenesis. Leptin overexpression is closely correlated with GC invasion, but its exact effect and the underlying mechanism in tumorigenesis remains poorly understood.The tumor cells lyses the extracellular matrix via synthesizing a number of matrix metalloproteinascs, which is one of the key steps in the tumor invasion and metastasis. Membrane type1-matrix metalloproteinase (MT1-MMP), which is a surface-anchored "master switch" proteinase, can directly decompose ECM, activate a variety of enzyme substrate, indirectly breaks down a variety of ingredients in the ECM and plays crucial roles in tumor invasion. MT1-MMP is closely related to invasion and metastasis in gastric cancer. The premise of this molecular’s effect is intracellular generating and transferred to the cell membrane. So there may be a variety of potential targets for early intervention for its activity. Recent years, many studies have demonstrated that leptin can regulate the expression of MMP-2, MMP-7and MMP-13in breast cancer and glioma, promotes the tumor invasion and metastasis. But the mechanism of leptin on the generation and surface localization of MT1-MMP in GC has not been elucidated.From what has been discussed above, based on the research progress of domestic and overseas and the research in the field of prophase research foundation, we observed the effect of leptin on MT1MMP expression and invasion of gastric cancer cell in vitro, explored the role of leptin receptor downstream signaling molecules Akt, ERK1/2and STAT3in mediating kinesin KIFs of MT1MMP-membrane expression of function, collected the specimens of gastric cancer tissue and cancer adjacent tissues, detect the expression of those above molecules, analyzed the relationship with clinical parameter. This study will further clarify the mechanism of gastric cancer invasion and metastasis, as well as provide potential therapeutic targets for early gastric cancer control.Objective1. Observation of leptin on gastric cancer cell invasion and the regulation of MT1-MMP expression.2. Analysis of KIF1B in leptin regulates the role of MT1-MMP-expression in gastric cancer cells and the effect on gastric cancer cell invasion.3. Illustrating the role of the leptin receptor downstream signaling pathway in gastric cancer cells in regulating MT1-MMP-generating and the role of KIF1B in regulating the expression of MMPs.4. Detection the expression of leptin, KIF1B and MT1-MMP in gastric cancer and cancer adjacent tissues, and analyzing the relationship of the three molecules and the relationship with clinical parameters.Methods1. The effect of leptin on gastric cancer cell invasion.GC cells (AGS, MKN-45, MKN-28) were treated with leptin (0,50,100,250,500 ng/ml) for24hours, transwell was used to detect GC cell invasion, determining the optimal concentration of leptin role. GC cells were divided into control, leptin, anti-leptin, leptin+anti-leptin. Collagenase invasive experiments detected the effects of leptin on gastric cancer cell invasion.2. The effect of leptin in generation of MT1-MMP in gastric cancer cells.GC cells were divided into control、leptin、anti-leptin、leptin+anti-leptin, cultured for24hours at37℃, RT-PCR was employed to detect MT1-MMP at mRNA level, western blot was employed to detect MT1-MMP at protein level. Flow cytometry and cell surface biolyation were used to measure cell surface MT1-MMP. Gelatin enzyme spectrum was used to detect the expression of MMP-2in gastric cancer cell culture supernatant.3. Detection the expression of KIFs molecule in gastric cancer cells after the effect of leptin, observing its role in leptin regulates MT1-MMP expression and its role in gastric cancer cell invasion.ACS cells were divided into control, leptin, anti-leptin and leptin+anti-leptin, RT-PCR was used to detect MT1-MMP at mRNA level, screening those cells that KIFs molecular expression changes. Western Blot was used to detect MTl-MMP at protein level.SiRNA koocked down KIFs molecular, GC cells were divided into control> con-siRNA、KIFs-siRNA、Leptin、Leptin+Con-siRNA and Leptin+KIFs-siRNA, cultured for24hours at37℃. RT-PCR was used to detect MTl-MMP at mRNA level. Western Blot was used to detect MT1-MMP at protein level, flow cytometry and cell surface biolyation were used to measure cell surface MT1-MMP. GC cells were divided into control, Leptin, KIF1B-siRNA, MT1-MMP-siRNA, Leptin+KIF1B-siRNA, Leptin+MT1-MMP-siRNA, Leptin+KIF1B-siRNA+MTl-MMP-siRNA, transwell was used to detect the in vation changes of gastric cancer cells.4. The influence of leptin on the interaction of KIFs and MTl-MMP.Immune coprecipitation was used to analyze the KIFs. Building MT1-MMP-eukaryotic expression vector (pcDNA3.1-MT1-MMP), then transecting gastric cancer cells. Immune coprecipitation was used to detect the influence of leptin on the interaction of KIFs and MT1-MMP.5. The role of Leptin receptor downstream signaling molecules AKT, ERK1/2, STAT3in leptin mediates KIFs adjust MT1-MMP expression.Leptin AGS gastric cancer cells at different times (0,15,30,60minutes), Western blot was used to detect the level of different signal path (STAT3, P-STAT3, AKT, P-AKT, ERK1/2and P-ERK1/2).GC cells were divide into control, leptin, LY294002, U0126, cucurbitacin I, Leptin+LY294002, Leptin+U0126, Leptin+Cucurbitacin I, Western Blot was used to detect the level of KIFs and MT1-MMP, cell surface biolyation was used to measure cell surface MT1-MMP.6. Detection of the expression of leptin, MT1-MMP and KIFs in gastric cancer tissue, and analyzing the correlation of the three moleculars and relationship with clinical parameters.86gastric cancer tissue and cancer adjacent tissues obtained from Qilu hospital of Shandong university, immunohistochemical and western blot were used to detect the level of leptin, MT1-MMP and KIFs in gastric cancer tissue, analyzing the correlation of the three moleculars and relationship with clinical parameters..Results1. Leptin promoted the invasion of gastric cells. Matrigel assay indicated that leptin (0,50,100,250,500ng/mL) promoted the invasion of gastric cancer cells in a dose-dependent manner, and anti-leptin antibody (10ug/mL) can abrogate this effect. Leptin (100ng/mL) can significantly induce the invasion of gastric cancer cells (AGS (3.81±0.39-fold,P=0.006).006)>MKN-45(2.76±0.23-fold, P=0.005)>MKN-28(1.83±0.18-fold, P=0.016). Meanwhile, leptin promoted the invasion of gastric cancer cells in collagen.2. Leptin enhanced the membrane localization of MT1-MMP, which subsequently promoted invasion of gastric cancer cells.Both the mRNA (3.01±0.39-fold, P=0.0\0) and protein levels of MT1-MMP were upregulated by leptin (100ng/ml; mRNA (3.01±0.39-fold,P=0.010), protein (3.77±0.99-fold, P=0.009)). Immunoprecipitation and flow cytometry results showed that the surface expression of MT1-MMP was enhanced by leptin, which can be released by the anti-leptin antibody. Meanwhile, the expression of MMP-2was also upregulated by leptin (AGS:1.73±0.1-fold, P=0.006; MKN-45:1.55±0.09-fold, P=0.008; MKN-28:1.33±0.07-fold,P=0.013).3. Leptin enhanced the interaction between KIF1B and MT1-MMP, which consequently contributed to the membrane localization of MT1-MMP.The RT-PCR results indicated that the expression of KIF3A, K1F3B and KIF5B were not influenced (P>0.05) by leptin (100ng/ml) treatment. However, both the mRNA (3.34±0.12-fold,P=0.007) and protein (3.52±0.18-fold,P=0.002) level of KIF1B were significantly upregulated, which can be blocked by anti-leptin antibody, suggesting that leptin may modulate the MT1-MMP expression via KIF1B. Indeed, although the mRNA and protein levels were not influenced (P>0.05), the leptin-induced surface expression of MT1-MMT was reduced by the KIF1B-siRNA (48.1%±4.0%, P=0.003). Consistently, the leptin-induced invasion of gastric cancer cells could be blocked by MT1-MMP-siRNA (62.8%±4.5%, P=0.005) or KIF1B-siRNA (45.2%±6.5%,P=0.016), and their combination decreased the gastric cancer cells invasion by up to (71.1%±3.7%,P<0.001).4. The effect of leptin on the interaction between KI FIB and MT1-MMP.The immunoprecipitation results showed K.IF1B and MT1-MMP interacted with each other in vivo, which can be significantly augmented by leptin. We set up different time points (6h,12h,24h and48h) to detect the effect of leptin, and found there is no significant change on the interaction between the two molecules at6h. The promoted interaction appeared at12h, and after48h, and the expression of MT1-MMP was higher than KIF1B, suggesting that the transport of MT1-MMP by KIF1B could be promoted by leptin.5. The AKT pathway was involved in the modulation of leptin on KIF1B and MT1-MMP.The leptin signal can activate three downstream signaling pathways:STAT3, HRK1/2and AKT, whose activation reached a peak at15min,30min and30min, respectively. However, the STAT3pathway inhibitor cucurbitacin I did not affect the expression of KIF1B, MT1-MMP and surface MT1-MMP. Whereas the AKT pathway inhibitor LY294002can reduce the leptin induced total MT1-MMP, KIF1B and surface MT1-MMP expression by (84.7%±6.8%, P=0.005),(81.5%±8.5%,P=0.012) and (76.0%±8.9%, P=0.01), respectively. The ERK1/2inhibitor partly suppressed the MT1-MMP (53.4%±5.6%, P=0.003), KIF1B (47.6%±4.9%, P=0.011) induced by leptin, but the surface MT1-MMP was not influenced (P>0.05), suggesting that the AKT pathway was involved in the KIF1B-dependent MT1-MMP surface transport.6. Leptin, MT1-MMP and KIF1B were all overexpressed in gastric cancer tissue and positively correlated with lymph nodes metastasis and clinical stage and most importantly, with one another.The immunohistochemistry results indicated that in gastric carcinoma, the expression of leptin (59.3%,51/86), MT1-MMP (63.9%,55/86) and KIF1B (68.6%,59/86) were much stronger than that of normal tissues, and their expression were significantly correlated with one another (Leptin vs MT1-MMP:P<0.001; Leptin vs KIF1B: P=0.039; MT1-MMP vs KIF1B:P<0.001). Most importantly, the expression of leptin, MT1-MMP and KIF IB were positively correlated with lymph nodes metastasis and clinical stage, suggesting a regulatory relationship among the three proteins in vivo.Conclusion1. Leptin is the effective intracellular activator of MT1-MMP.2. Leptin’s promotion of the gastric cancer cellMT1-MMP membrane expression is a KIF1B dependent process, involved in gastric cancer cell invasion process.3. To clarify the role of downstream of leptin receptors in leptin AKT signaling pathways in boosting MTI-MMP expression, providing a support to control for early gastric cancer invading potential diagnostic and treatment strategies and data.SignificanceThis study illuminates the effect and underlying mechanism of leptin on MTI-MMP, and futher explore the role of this process in the invasion of gastric cancer cells. This will help to identify the mechanism of gastric cancer invasion and control the activity of MTI-MMP in gastric cancer. Part II THE EFFECT AND UNDERLYING MECHANISM OF LEPTIN ON MIGRATION IN GASTRIC CANCER CELLSBackgroundGastric cancer (GC) ranks as the secondary leading cause of cancer-related death in the world. Adipocytes provide fatty acids for rapid tumor growth, and the dysfunction of lipid metabolism can lead to the pathogenesis of human GC. Leptin is an adipokine of the obesity (ob) gene, and its overexpression is closely correlated with GC metastasis, but its exact effect and the underlying mechanism in tumor progression remain unclear. Our previous findings reveal that leptin may promote GC cells invasion by upregulation of MT1-MMP. MT1-MMP may cleave intercellular adhesion molecule-1(ICAM-1), and then enhance tumor cells migration. ICAM-1is overexpressed and plays crucial roles in tumor metastasis. This study aimed to characterize the influence of leptin on ICAM-1expression in GC and elucidate its underlying molecular mechanism.We previously determined that leptin could promote the migration of GC through upregraulation the expression of MT1-MMP,and other dates suggested that MT1-MMP could cut ICAM-1and promote the migration of tumor cells,but the exact mechanism of how leptin act on ICAM-1remains unclear.Overall,based on the worldwide study progress and the results we discovered previously,we collected GC tissue samples and detected the expression of leptin and ICAM-1,then analyzed the correlation between leptin and clinical parameters.And we also observed the influence of leptin on the effects of ICAM-1expression,discussed the function of ICAM-1in the process of GC migration.We finally observed the function of Rho/ROCK in ICAM-1expression regulated by leptin simultaneously provided potential therapy target for the control of GC in early period.Objective1. To detect the expression of leptin and ICAM-1in GC tissues and analyze the corelation between them and the clinical parameters.2. To observe the impacts of leptin on the migration of GC. 3. To observe the effects of leptin on ICAM-1expression, then to discuss the function of leptin in the process of GC migration.4. To elucidate the function of Rho/RPCK pathway in ICAM-1expression changes regulated by leptin.Methods1. ICAM-1and leptin were both high expressed in GC tissue, had significant correlation, and they were closely related to clinical stages and migration.Archived paraffin-embedded GC tissues and matched adjacent normal gastric tissues were collected from84patients who underwent surgery for primary gastric carcinoma. The expression of leptin and ICAM-1were detected by immunohistochemistry, and the correlation of two proteins was further analyzed. Correlations of leptin and ICAM-1expression with clinicopathologic factors were analyzed.2. The impact of leptin on GC migration.We employed AGS and MKN-45cell line in vitro and cocultured them with different dosages (0,50,100,250,500ng/ml) of leptin in a transwell system at37℃for a variety of time (0,6,12,24,36h), then we calculated the index of aggressiveness through observing the migration changes,and decided the optimal concentration of leptin in the process.3. The effect of leptin on ICAM-1expression in GC and the function of leptin in the process of GC migration.We separated GC (AGS and MKN-45) as control and leptin (100ng/ml) groups, then cultured at37℃for24h, then we employed RT-PCR and western blot to detect the expression the ICAM-1at both gene and protein levels, we also applied FCM to determine the expression of ICAM-1on GC membrane and ELISA technique to detect the sICAM-1expression in the GC culture supernatant.ICAM-1-siRNA was designed and transiently transfected in GC cells. We divided GC into Control, Leptin+con-siRNA, Leptin+ICAM-1-siRNA groups, and cultured them at37℃for24h, then determined the migration change with the help of transwell system.4. The role of Rho/ROCK pathway in ITAM-1expression regulated by leptin.After impact on ICAM-1by leptin for different time (0,15,30,60mins), G-LISA RhoA active detecting box was used to determine the activity of RhoA GTPase, western blot was applied to detect the expression of P-ROCK and ROCK in GC cells. GC cells were seperated as Control, Leptin, C3transferase (RhoA inhibitor), Y-27632(ROCKpathway inhibitor), Leptin+C3transferase, Leptin+Y-27632groups, cultured at37℃for24h. RT-PCR and western blot were used to detect the expression of ICAM-1in gene and protein levels. FCM was employed to determine the expression of ICAM-Ion GC cell membrane. ELISA kit was applied to detect the sICAM-1in the GC culture supernatant.Results1. Leptin and ICAM-1were high expressed in GC tissues, they had significant correlation and had a link to clinical stages and tumor migration.Immunohistochemical analysis revealed that leptin (48/84,57.1%) and ICAM-1(54/84,64.2%) were overexpressed in GC tissues, and they were positively correlated with each other (P<0.001), as well as with the clinical stage and lymphatic metastasis, which indicted that the two factors had potential coadjustment relationship.2. Leptin could promote the migration of GC.It is proved that leptin was able to promote the migration of GC (AGS and MKN-45) in a time and dosage dependent way.3. Lptin could increase the expression of ICAM-1, which led to the GC migration.Furthermore, leptin induced GC cell migration by upregulating ICAM-1expression (mRNA:4.06±0.54-fold for AGS, P<0.001;2.56±0.33-fold for MKN-45,P=0.005. Protein:3.07±0.25-fold for AGS, P<0.001;2.9±0.26-fold for MKN-45, P=0.003). Moreover, leptin could upregulate the expression of ICAM-1(AGS:from43.65%±2.42%to78.96%±2.09%,P<0.01; MKN-45:from35.35%±1.61%to59.64%±3.51%,<0.001) and sICAM-1expression (AGS, P<0.05; MKN-45, P<0.01), knockdown of ICAM-1by small interference RNA (siRNA) blocked this process (AGS:53.9%±3.6%,P-0.020; MKN-45:42.79%±3.78%, P=0.005).4. Rho/ROCK signal pathway took part in the control process of ICAM-1expression regulated by leptin. Leptin could promote the activity of Rho A on15min (AGS:1.2±0.2fold; MKN-45:1.1±0.1fold),30min (AGS:1.6±0.1fold; MKN-45:1.36±0.06fold) and60min (AGS:1.67±0.12fold; MKN-45:1.5±0.1fold. The phosphorylation level of ROCK also increased on15,30,60min.Notably, the surface expression of ICAM-1(AGS, P<0.001; MKN-45, P<0.01), as well as the soluble ICAM-1(sICAM-1)(AGS, P<0.05; MKN-45, P<0.01), was also enhanced by leptin. Moreover, leptin increased ICAM-1expression through Rho/ROCK pathway, which was attenuated by pharmacological inhibition of Rho (C3transferase) at0.25μg/ml (AGS, P<0.01; MKN-45, P<0.01) or inhibition of its downstream effector kinase Rho-associated protein kinase (ROCK)(Y-27632) at3.3μM (AGS, P<0.01; MKN-45, P<0.01), suggesting an essential role of Rho/ROCK pathway in this process.Conclusion1. Our findings indicate that leptin act as an effective activator and can enhance GC cell migration by increasing ICAM-1expression.2. To clarify the role of Rho/ROCK pathway in the process of ICAM expression change promoted by leptin may provide preliminary experimental clues for the development of new therapies against the metastasis of GC.SignificanceThis study illuminates the effect and underlying mechanism of leptin on the expression of ICAM-1, and further explores the role of this process in gastric cancer cell migration. Our findings will identify the relative mechanism of gastric cancer development, which help to find the new potential therapy target and optimize the therapy protocol. |
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