Involvement of Met receptor pathway in aggressive behavior of colorectal cancer cells induced by parathyroid hormone-related peptide

BACKGROUND Parathyroid hormone-related peptide(PTHrP)plays a key role in the development and progression of many tumors.We found that in colorectal cancer(CRC)HCT116 cells,the binding of PTHrP to its receptor PTHR type 1(PTHR1)activates events associated with an aggressive phenotype.In HCT116 cell xenografts,PTHrP modulates the expression of molecular markers linked to tumor progression.Empirical evidence suggests that the Met receptor is involved in the development and evolution of CRC.Based on these data,we hypothesized that the signaling pathway trigged by PTHrP could be involved in the transactivation of Met and consequently in the aggressive behavior of CRC cells.AIM To elucidate the relationship among PTHR1,PTHrP,and Met in CRC models.METHODS For in vitro assays,HCT116 and Caco-2 cells derived from human CRC were incubated in the absence or presence of PTHrP(1-34)(10-8 M).Where indicated,cells were pre-incubated with specific kinase inhibitors or dimethylsulfoxide,the vehicle of the inhibitors.The protein levels were evaluated by Western blot technique.Real-time polymerase chain reaction(RT-qPCR)was carried out to determine the changes in gene expression.Wound healing assay and morpho logical monitoring were performed to evaluate cell migration and changes related to the epithelialmesenchymal transition(EMT),respectively.The number of viable HCT116 cells was counted by trypan blue dye exclusion test to evaluate the effects of irinotecan(CPT-11),oxaliplatin(OXA),or doxorubicin(DOXO)with or without PTHrP.For in vivo tests,HCT116 cell xenografts on 6-wk-old male N:NIH(S)_nu mice received daily intratumoral injections of PTHrP(40μg/kg)in 100μL phosphate-buffered saline(PBS)or the vehicle(PBS)as a control during 20 d.Humanitarian slaughter was carried out and the tumors were removed,weighed,and fixed in a 4%formaldehyde solution for subsequent treatment by immunoassays.To evaluate the expression of molecular markers in human tumor samples,we studied 23 specimens obtained from CRC patients which were treated at the Hospital Interzonal de Graves y Agudos Dr.JoséPenna(Bahía Blanca,Buenos Aires,Argentina)and the Hospital Provincial de Neuquén(Neuquén,Neuquén,Argentina)from January 1990 to December 2007.Seven cases with normal colorectal tissues were assigned to the control group.Tumor tissue samples and clinical histories of patients were analyzed.Paraffin-embedded blocks from primary tumors were reviewed by hematoxylin-eosin staining technique;subsequently,representative histological samples were selected from each patient.From each paraffin block,tumor sections were stained for immunohistochemical detection.The statistical significance of differences was analyzed using proper statistical analysis.The results were considered statistically significant at P<0.05.RESULTS By Western blot analysis and using total Met antibody,we found that PTHrP regulated Met expression in HCT116 cells but not in Caco-2 cells.In HCT116 cells,Met protein levels increased at 30 min(P<0.01)and at 20 h(P<0.01)whereas the levels diminished at 3 min(P<0.05),10 min(P<0.01),and 1 h to 5 h(P<0.01)of PTHrP treatment.Using an active Met antibody,we found that where the protein levels of total Met decreased(3 min,10 min,and 60 min of PTHrP exposure),the status of phosphorylated/activated Met increased(P<0.01)at the same time,suggesting that Met undergoes proteasomal degradation after its phosphorylation/activation by PTHrP.The increment of its protein level after these decreases(at 30 min and 20 h)suggests a modulation of Met expression by PTHrP in order to improve Met levels and this idea is supported by our observation that the cytokine increased Met mRNA levels at least at 15 min in HCT116 cells as revealed by RT-qPCR analysis(P<0.05).We then proceeded to evaluate the signaling pathways that mediate the phosphorylation/activation of Met induced by PTHrP in HCT116 cells.By Western blot technique,we observed that PP1,a specific inhibitor of the activation of the protooncogene protein tyrosine kinase Src,blocked the effect of PTHrP on Met phosphorylation(P<0.05).Furthermore,the selective inhibition of the ERK 1/2 mitogen-activated protein kinase(ERK 1/2 MAPK)using PD98059 and the p38 MAPK using SB203580 diminished the effect of PTHrP on Met phosphorylation/activation(P<0.05).Using SU11274,the specific inhibitor of Met activation,and trypan blue dye exclusion test,Western blot,wound healing assay,and morphological analysis with a microscope,we observed the reversal of cell events induced by PTHrP such as cell proliferation(P<0.05),migration(P<0.05),and the EMT program(P<0.01)in HCT116 cells.Also,PTHrP favored the chemoresistance to CPT-11(P<0.001),OXA(P<0.01),and DOXO(P<0.01)through the Met pathway.Taken together,these findings suggest that Met activated by PTHrP participates in events associated with the aggressive phenotype of CRC cells.By immunohistochemical analysis,we found that PTHrP in HCT116 cell xenografts enhanced the protein expression of Met(0.190±0.014)compared to tumors from control mice(0.110±0.012;P<0.05)and of its own receptor(2.27±0.20)compared to tumors from control mice(1.98±0.14;P<0.01).Finally,assuming that the changes in the expression of PTHrP and its receptor are directly correlated,we investigated the expression of both Met and PTHR1 in biopsies of CRC patients by immunohistochemical analysis.Comparing histologically differentiated tumors with respect to those less differentiated,we found that the labeling intensity for Met and PTHR1 increased and diminished in a gradual manner,respectively(P<0.05).CONCLUSION PTHrP acts through the Met pathway in CRC cells and regulates Met expression in a CRC animal model.More basic and clinical studies are needed to further evaluate the PTHrP/Met relationship.

Survival effect of probiotics in a rat model of colorectal cancer treated with capecitabine

BACKGROUND Probiotics are used to manage a number of gastrointestinal disorders due to their beneficial properties.Clinical reports showed that probiotics also improve the life quality of patients with colorectal cancer(CRC)subjected to oncologic treatment.In a CRC animal model,probiotics supplementation has the potential to decrease the formation of aberrant crypts and ameliorate tumor malignancy,enhancing the antitumor effect of 5-fluorouracil(5-FU)chemotherapy.Based on these data,we hypothesize that the administration of probiotics impact positively in the overall survival and life quality of rats with CRC under the treatment of capecitabine,which is the pro drug of 5-FU.AIM To evaluate the probiotics effects in a rat CRC model treated with capecitabine and followed until the end of life.METHODS 1,2-Dimethylhidrazine dihydrochloride(1,2-DMH)was employed as carcinogen inductor of CRC.Fifty male Wistar-Lewis rats were randomly assigned to one of five following groups:Control(n=5),Control+probiotics(Control-P group,n=5),1,2-DMH alone(DMH group,n=10),1,2-DMH+capecitabine(DMH-C group,n=10),1,2-DMH+probiotics(DMH-P group,n=10)and 1,2-DMH+capecitabine+probiotics(DMH-C-P group,n=10).All parametric data were expressed as the mean±SD.The statistical significance of differences was analyzed using one-way ANOVA.Data were analyzed with InfoStat software.The results were considered statistically significant at P<0.05.Overall survival was evaluated with the Kaplan-Meier estimator with the log-rank test.RESULTS The data of mean overall survival for DMH,DMH-P,DMH-C,DMH-C-P,Control and Control-P groups were 250 d[95%confidence interval(CI):242.5-253.1],268 d(95%CI:246.3-271.4),380 d(95%CI:337.8-421.9),480 d(95%CI:436.9-530.7),588 d(95%CI:565.8-609.3)and 590 d(95%CI:564.3-612.9),respectively,with a significant difference between DMH-C and DMH-C-P groups(P=0.001).Comparing all groups by Kaplan-Meier estimator,we found a significantly different in the overall survival of DMH and DMH-P groups respect to DMH-C(P=0.001)and DMH-C-P(P=0.001)groups;interestingly,there were no meaningful differences between Control,Control-P and DMH-C-P groups(P=0.012).The tendency of change in body weight gain of the rats at 90 d of finishing DMH administration was similar in Control group compared with DMH-C and DMHC-P groups;however,and of relevance,DMH-C-P group has experienced a higher body weight gain at the end of animal’s life than DMH-C group(P=0.001).In DMH-C-P group we found a positive effect of probiotics in clinical manifestations since diarrhea,constipation and blood stool were absenting.Also,the tumor burden was lower in DMH-C-P than DMH-C,DMH-P or DMH groups(1.25 vs 1.81 vs 3.9 vs 4.8 cm2,respectively).DMH-C and DMH-C-P groups showed only mucinous carcinoma type while in other DMH groups the tumor types were variable.However,mucinous carcinoma from DMH-C-P group showed invasion until muscularis propria layer.Interestingly,metastatic lymph node was observed in DMH,DMH-C and DMH-P groups but not in DMH-C-P.All animals in Control group died from natural causes without objective injuries.All animals of DMH and DMH-P groups died from tumor complications(i.e.,obstruction or intestinal perforation);however,this cause was seen only in 44.5%of DMH-C and DMH-C-P groups CONCLUSION Probiotics administration improves life quality of rats with CRC under capecitabine treatment and also has a positive effect in the overall survival of these animals treated with this drug.