The Effect Of VEGF-C On Pancreatic Cancer Lymph Node Metastasis And The Correlation With Prognosis

PartⅠThe different expression of VEGF-C on primary tumor and lymph node metastasis of nude mice with orthotopic implantation for the human pancreatic cancer cellObjective:To investigate the different expression of vascular endothelial growth factor C (VEGF-C) on pancreatic cancer cell PANC-1 derived from primary tumor and spontaneous lymph node metastasis.Methods:Establishment of the spontaneous lymph node metastasis model in nude mice with orthotopic implantation for the human pancreatic cancer cell line PANC-1. Then, isolate and culture the pancreatic cancer cell from primary tumor and spontaneous lymph node metastasis. The different expression of VEGF-C on pancreatic cancer cell was detected by RT-PCR, Western Blot, ELISA.Results: The rate of tumor take was 100.0%. The rate of the spontaneous lymph node metastasis was 62.5%. The rate of the spontaneous hepatic metastasis was 25.0%. Not to see lung metastasis.The pancreatic cancer cell PANC-1 isolated from primary tumor and spontaneous lymph node metastasis were designated PANC-1-PRO and PANC-1-LN, separately. The mRNA levels of VEGF-C were 0.61±0.15 and 0.87±0.11 respectively; The protein levels of VEGF-C were 0.65±0.17 and 0.88±0.09 respectively inside 2 cells; Then the VEGF-C levels in culture supernatants were (1403.67±128.15) pg/ml and (1682.37±156.73) pg/ml. The levels of VEGF-C in PANC-1-LN cell were higher than PANC-1-PRO cell, the differentiation were statistical significance (t=3.1255,2.9062,3.0782,P=0.0141,0.0197,0.0152).Conclusions:The expressive level of VEGF-C on pancreatic cancer cell PANC-1 derived from spontaneous lymph node metastasis was higher than cell derived from primary tumor, the expression of VEGF-C on different organ was discrepancy.PartⅡEffect on apoptosis of pancreatic cancer derived from lymph node metastasis with antisense oligonucleotide of VEGF-CObjective:To investigate the effect of VEGF-C antisense oligonucleotide on pancreatic cancer cell PANC-1 derived from primary tumor and spontaneous lymph node metastasis.Methods: Establishment of the spontaneous lymph node metastasis model in nude mice with orthotopic implantation for the human pancreatic cancer cell line PANC-1. Then, isolate and culture the pancreatic cancer cell from primary tumor and spontaneous lymph node metastasis (PANC-1-PRO and PANC-1-LN). The cells were randomized into 3 groups: Control group, scramble-sense oligonucleotide (SODN) group and antisense oligonucleotide (ASODN) group. The effect of transfection on the expressive VEGF-C and apoptosis of pancreatic cancer cells were detected by RT-PCR, Western Blot, ELISA, flow cytometer and TUNEL in vitro and vivo.Results:After treatment in vitro, the VEGF-C level of mRNA, protein and culture supernatants on ASODN group of PANC-1-PRO and PANC-1-LN were decreased than control group and SODN group (P<0.01). In 3 groups of PANC-1-PRO and PANC-1-LN, the mRNA levels of apoptosis related gene bcl-2 were 0.67±0.12, 0.69±0.14, 0.61±0.11 and 0.56±0.16, 0.59±0.18, 0.27±0.17 separately. Only ASODN group of PANC-1-LN was decreased significantly (t=3.0428,3.1659,P=0.0124,0.0101). The rates of early apoptosis were (3.51±1.38)%, (4.79±2.16)%, (5.33±2.18)% in PANC-1-PRO, the differentiation were not statistical significance (t=1.7279,0.4310,P=0.1147,0.6756); But the rates were (2.83±1.01)%, (4.98±2.05)%, (13.22±2.17)% in PANC-1-LN, ASODN group was elevated, the differentiation were statistical significance (t=10.6329,6.7613,P=0.0000,0.0000);After treatment in vivo, the serum VEGF-C level of ASODN group were decreased than control group and SODN group in model(P<0.05). The volume of tumor, rates and parameter of lymph node metastasis all were not statistical differentiation (P>0.05). The rates of apoptosis were (1.29±0.53)%, (1.98±0.77)%, (2.01±0.80)% in 3 groups of primary tumor, the differentiation were not statistical significance (t=2.1221,0.0764,P=0.0522,0.9402); But in groups of lymph node metastasis, the rates of apoptosis were (1.78±0.49)%, (1.96±0.68)%, (4.38±1.02)% respectively, the rates of ASODN group was elevated, the differentiation were statistical significance (t=6.4987,5.2301,P=0.0000,0.0001).Conclusions:To inhibit the expression of VEGF-C on pancreatic cancer derived from lymph node metastasis can decreased the level of bcl-2, and promoted it to apoptosis, but no effect on pancreatic cancer derived from primary tumor. It is suggested that the effect of VEGF-C also have organic differentiation. It is mechanism of lymph node metastasis in pancreatic cancer.PartⅢThe correlation between the serum concentration of VEGF-C and prognosis in unrecetable pancreatic cancerObjective:To investigate the correlation between the serum concentration of VEGF-C and prognosis in unrecetable pancreatic cancer.Methods:In this study, select 35 unrecetable local advanced pancreatic cancer cases in Pancreatic Surgical Center of Union Hospital, Wuhan, China, from Dec. 2004 to Aug. 2005. We review karnofsky performance scale index (KPS), diameter of tumor and serum concentration of CA19-9. And the serum concentration of VEGF-C detected by ELISA, 10 health cases as to control. Survival rate and curves were calculated and drown by Kaplan-Meier. Survival rates in different groups were processed with Log-rank. Univariance and multiplevariance analysis were used to screen by Cox congression.Results:The mean serum concentration of VEGF-C was (1309.23±542.05) pg/ml, control group was (256.13±96.59) pg/ml, the differentiation were statistical significance (P<0.05).The patient year, diameter of tumor, position of tumor, KPS, the serum concentration of VEGF-C and CA19-9were univariance analysed by Cox congression. KPS, the serum concentration of CA19-9 and VEGF-C were all correlated with prognosis in unrecetable pancreatic cancer (χ2=7.208,6.908,3.867,P=0.007,0.029,0.049).In multiplevariance analysis by Cox congression (backward), SLE was 0.10, SLS was 0.11, the sequence of markers stepwise backward to model: Step 1 was VEGF-C (χ2=3.738,P=0.053), step 2 was VEGF-C (χ2=4.873,P=0.027), step 3 was KPS (χ2=5.274,P=0.022).In groups of KPS <70 and≥70分, The median survival were 6.60 and 10.11 months, the survival rates of 1 year were 21.43% and 33.33% respectively. As the median of VEGF-C was 1280pg/ml, in groups of≤1280pg/ml and >1280pg/ml, the median survival were 11.26 and 6.29 months, the survival rates of 1 year were 50.00% and 5.88% respectively, the differentiation were statistical significance (χ2=4.0400,9.4000,P=0.0443,0.0022). But, in groups of CA19-9≤200U /ml and >200U/ml, the median survival were 9.99 and 7.76 months, the survival rates of 1 year were 37.50% and 21.05% respectively, the differentiation were not statistical significance (χ2=1.9100,P=0.1667). Conclusions:KPS of the patients were independent factor of prognosis in unrecetable pancreatic cancer. The serum concentration of VEGF-C and CA19-9 were yet correlated with prognosis, and can used as aid marker for judgement of prognosis.