Association of PTEN expression with liver function and inflammatory changes in patients with liver cancer after chemotherapy

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1 ONCOLOGY LETTERS Assocition of PTEN expression with liver function nd inflmmtory chnges in ptients with liver cncer fter chemotherpy JIXIANG ZHOU nd XIAOLI LI Deprtment of Heptobiliry Surgery, Xingy Hospitl, Centrl South University, Chngsh, Hunn , P.R. Chin Received Mrch 28, 2018; Accepted September 7, 2018 DOI: /ol Abstrct. This study investigted the effects of phosphtse nd tensin homolog deleted on chromosome ten (PTEN) expression on liver function clssifiction, serum tumor mrkers nd liver function of ptients with liver cncer. A totl of 63 ptients with primry liver cncer treted in Xingy Hospitl (Chngsh, Chin) were retrospectively nlyzed. The difference in the PTEN expression levels in norml liver cells nd liver cncer cells ws compred vi immunohistochemistry. According to the expression level of PTEN in the ptient's pthologicl report, ptients were divided into PTEN positive nd PTEN negtive groups. The expression level of ech tumor mrker in serum of ptients ws observed, nd the ssocition of PTEN expression level with the serum tumor mrkers ws nlyzed. Moreover, the chnges in liver function nd inflmmtory fctors before nd fter chemotherpy were compred. Finlly, the reltionship between the PTEN expression level nd Child Pugh grding of the liver function ws detected. Compred with tht in norml liver cells, the positive expression rte of PTEN protein in liver cncer cells ws significntly decresed (P<0.05). No significnt difference ws found in the expression levels of serum tumor mrkers, except α fetoprotein (AFP) in liver cncer ptients between PTEN positive nd PTEN negtive group, indicting tht PTEN expression hs no significnt effects on serum tumor mrkers. The levels of lbumin (ALB), lkline phosphtse (ALP) nd prothrombin ctivity (PTA) were decresed significntly fter chemotherpy compred with those before chemotherpy (P<0.05). Besides, the levels of inflmmtory fctors were remrkbly reduced Correspondence to: Dr Xioli Li, Deprtment of Heptobiliry Surgery, Xingy Hospitl, Centrl South University, 87 Xingy Rod, Chngsh, Hunn , P.R. Chin E mil: xingylxl1216@126.com Key words: liver cncer, PTEN, Child Pugh grding, serum tumor mrkers, inflmmtory fctors fter che motherpy. PTEN expression ws negtively ssocited with liver function grding, nd the higher the PTEN expression, the lower the liver function grding ws. The low expression of PTEN hs certin ssocition with the occurrence nd grding of liver cncer. PTEN gene hs guiding significnce in predicting the occurrence, development nd prognosis of liver cncer. Introduction Liver cncer, including heptocellulr crcinom (HCC) nd intrheptic cholngiocrcinom, is one of the common mlignnt tumors of the digestive trct in Chin, with mortlity rte s high s 42.5%, second only to esophgel nd gstric cncer. The most importnt risk fctors for liver cncer re heptitis B virus nd lcoholism. According to the results of n epidemiologicl survey, the incresed content of orgnic chlorides nd DDT in drinking wter nd fltoxin in food re lso mjor fctors inducing liver cncer (1). It hs lso been reported tht the occurrence of liver cncer my be relted to the difference in gene expression levels in ptients, in which the bnorml ctivtion of Wnt/β ctenin signling pthwy nd expression deletion of the phosphtse nd tensin homolog deleted on chromosome ten (PTEN) nd other tumor suppressor genes cn medite the occurrence of liver cncer (2,3). PTEN, discovered in 1997, is tumor suppressor gene with dul specificity phosphtse ctivity locted on chromosome 10, which cn dephosphorylte downstrem phosphtidylinositol 3,4,5 trisphosphte in norml liver tissues, thus blocking the downstrem phosphtidylinositol 3 kinse/protein kinse B (PI3K/Akt) signling pthwy, nd negtively regulting the growth of liver cells. In cse of deletion or muttion of PTEN, however, its negtive regultory effect on downstrem pthwy will dispper or decline, nd the norml regultory mechnism for cell growth or metstsis will be lost, thereby inducing the occurrence or mlignnt trnsformtion of tumors (4,5). At present, it hs been confirmed tht the bnorml expression of PTEN is ssocited with the occurrence nd development of vriety of mlignnt tumors, such s gliom, endometril, liver nd prostte cncers (6,7). In this study, the expression of PTEN in norml liver cells nd liver cncer cells ws retrospectively

2 2 ZHOU nd LI: CORRELATION OF PTEN WITH CHANGES IN LIVER CANCER nlyzed to investigte the possible ssocitions of PTEN with the incidence of liver cncer, liver function grding nd liver function. Ptients nd methods Generl ptient dt. A totl of 63 ptients dignosed with primry liver cncer vi preopertive puncture or postopert ive pthologicl dignosis in Xingy Hospitl were enrolled, including 37 mles nd 26 femles ged yers with n verge of 48.5±8.6 yers. The hospitliztion durtion ws yers with n verge of 4.6±2.7 yers. All ptients underwent surgicl resection fter preopertive chemotherpy. No bdominl viscerl metstsis nd lymph node metstsis were found, nd there were no other tumors, except liver cncer, during opertion. The study ws pproved by the Ethics Committee of Xingy Hospitl, Centrl South University (Chngsh, Chin). Signed informed consents were obtined from the ptients or the gurdins Reserch methods. All ptients enrolled in this study were treted with preopertive chemotherpy using cispltin (CDDP) once per month (3 4 times s one course of tretment). After 2 3 courses of chemotherpy, the ptients tht stisfied the surgicl indictions received surgicl resection. After opertion, the specimens were fixed in 40% formlin t 25 C for 24 h, collected, dehydrted, nd prepred into 3 µm sections, followed by routine hemtoxylin nd eosin (H&E) stining nd immunohistochemicl stining. The positive control ws set. The tissue ws blocked with 10% FBS t 20 C for 10 min, then incubted with rbbit nti-humn PTEN monoclonl ntibody (ct. no. 9559, 1:50; Cell Signling Technology, Inc., Dnvers, MA, USA) t 4 C for 12 h, nd wshed with PBS for 10 min, 3 times. The tissue ws then incubted with secondry ntibody SignlStin Boost IHC Detection Regent (HRP, rbbit) (ct. no. 8114, 1:500; Cell Signling Technology, Inc.) t 20 C for 1 h. Serum tumor mrkers, blood biochemicl fctors nd inflmmtory fctors in ptients were detected vi chemiluminescence. Fsting venous blood ws drwn from ptients t 1 week before nd fter chemotherpy, nd the superntnt ws tken fter centrifugtion t 6,000 x g t 4 C for 15 min for on-mchine detection. Before chemotherpy, the liver function of ptients ws scored ccording to the Child Pugh grding: grde A, 5 6 points; grde B, 7 9 points; nd grde C, points. Observtion indexes. Interprettion of results: PTEN displyed brown yellow prticles in immunohistochemistry, nd the expression of PTEN in liver cncer cells ws observed under high power field by light microscope (x100): PTEN ++, percentge of positive cells >50%; PTEN +, percentge of positive cells >10 50%; nd PTEN, percentge of positive cells <10%. In this study, the percentge of positive cells >10% indicted positive PTEN expression. Sttisticl methods. GrphPd 5.0 softwre (GrphPd Softwre, Inc., L Joll, CA, USA) ws used for the sttisticl nlysis of dt. t test ws used for mesurement dt nd Chi-squre test for enumertion dt. One wy nlysis of vrince ws used for multiple comprisons nd Tukey's test Tble I. Comprison of PTEN protein expression in norml liver tissues nd liver cncer tissues. PTEN [n (%)] Type of tissue + Norml liver tissues (n=63) 3 (4.76) 60 (95.23) Liver cncer tissues (n=63) 34 (53.97) 29 (46.03) χ P vlue PTEN, phosphtse nd tensin homolog deleted on chromosome ten. Tble II. Comprison of serum tumor mrker levels with PTEN expression before opertion (men ± SD). Serum tumor mrker PTEN ( ) PTEN (+) AFP (ng/ml) 167.4± ±32.1 CA125 (U/ml) 143.5± ±13.6 CEA (ng/ml) 31.4± ±3.2 CA199 (U/ml) 51.3± ±4.8 CA153 (U/ml) 45.6± ±5.7 P<0.05 vs. PTEN negtive group. PTEN, phosphtse nd tensin homolog deleted on chromosome ten; AFP, α fetoprotein. ws the post hoc test used. P<0.05 suggested tht the difference ws sttisticlly significnt. Results Expression of PTEN in norml liver tissues nd liver cncer tissues. In the 63 specimens of this study, the positive expression rte of PTEN ws 95.23% in norml liver tissues, but only 46.03% in liver cncer tissues, suggesting tht the positive expression rte of PTEN in liver cncer tissues ws significntly decresed, nd the difference ws sttisticlly significnt (P<0.05) (Tble I). Comprison of serum tumor mrker levels between the two groups of ptients. After chemotherpy, liver cncer ptients were divided into PTEN positive nd PTEN negtive groups ccording to the expression of PTEN. There ws no sttisticlly significnt difference in the levels of serum tumor mrkers between the two groups of ptients fter chemotherpy (P>0.05), but the level of α fetoprotein (AFP) in PTEN positive group ws obviously lower thn tht in PTEN negtive group (P<0.05) (Tble II). Comprison of chnges in liver function of ptients before nd fter chemotherpy. After chemotherpy, the expression levels of serum lnine trnsminse (ALT) nd sprtte trnsminse (AST) in ptients were slightly incresed, which might be relted to side effects of chemotherpy drugs on the liver. The results were not sttisticlly different from those before chemotherpy. After chemotherpy, the levels of

3 ONCOLOGY LETTERS 3 Tble III. Comprison of chnges in the liver function of ptients before nd fter chemotherpy (men ± SD). Serum biochemicl Before After index of liver chemotherpy chemotherpy ALT (U/l) 387.5± ±110.7 AST (U/l) 396.4± ±104.3 ALB (U/l) 38.5± ±6.2 ALP (g/l) 237.8± ±57.1 PTA (%) 134.9± ±18.6 P<0.05 vs. before chemotherpy. ALT, lnine trnsminse; AST, sprtte trnsminse; ALB, lbumin; ALP, lkline phosphtse; PTA, prothrombin ctivity. Figure 1. PTEN expression in norml liver tissues (x100). PTEN, phosphtse nd tensin homolog deleted on chromosome ten. Tble IV. Assocition between PTEN expression nd liver function clssifiction. Positive PTEN Child Pugh grding expression [n (%)] n A 11 (78.57) 14 B 13 (46.43) 28 C 5 (23.8) 21 P<0.05 vs. grde A. PTEN, phosphtse nd tensin homolog deleted on chromosome ten. Tble V. Comprison of serum inflmmtory fctors in ptients with liver cncer before nd fter chemotherpy (men ± SD). Inflmmtory Before After fctor chemotherpy chemotherpy Figure 2. PTEN expression in liver cncer tissues (x100). PTEN, phosphtse nd tensin homolog deleted on chromosome ten. IL 1 (ng/l) 5.46± ±0.19 b IL 6 (ng/l) ± ±9.26 b IL 2 (µg/ml) ± ±91.3 IL 12 (pg/ml) ± ±86.47 IL 10 (ng/ml) ± ±69.7 b MIF (ng/ml) 25.42± ±4.31 b P<0.05, b P<0.001 vs. before chemotherpy. IL, interleukin. lbumin (ALB), lkline phosphtse (ALP) nd prothrombin ctivity (PTA) in ptients were obviously decresed, nd there were sttisticlly significnt differences compred with those before chemotherpy (P<0.05) (Tble III). Assocition between PTEN expression nd liver function clssifiction. There were vrying degrees of chnges in the liver function of ptients with liver cncer, mong which the PTEN positive rte ws 78.57% in ptients in grde A, 46.43% in ptients in grde B nd 23.8% in ptients in grde C. With the increse of Child Pugh grding of ptients with liver cncer, the positive expression rte of PTEN in ptients Figure 3. PTEN expression in liver cncer tissues fter chemotherpy (x100). PTEN, phosphtse nd tensin homolog deleted on chromosome ten. ws decresed, displying negtive ssocition between them (Tble IV). Comprison of serum inflmmtory fctors in ptients with liver cncer before nd fter chemotherpy. Detection results of serum biochemicl indexes in ptients t 1 week before nd fter chemotherpy mnifested tht the levels of inflmmtory fctors in peripherl blood of ptients fter chemotherpy

4 4 ZHOU nd LI: CORRELATION OF PTEN WITH CHANGES IN LIVER CANCER were remrkbly lower thn those before chemotherpy, nd differences were sttisticlly significnt (P<0.05 or P<0.001) (Tble V). Assocition between PTEN expression nd liver cncer tissues. Immunohistochemicl results reveled tht PTEN ws minly loclized in the cytoplsm, nd lso slightly in the nucleus. Compred with those in norml liver tissues, the expression of PTEN in liver cncer tissues before nd fter chemotherpy ws obviously reduced (Figs. 1 3). Discussion PTEN is tumor suppressor gene with dul specificity phosphtse ctivity, which is involved in the growth, prolifertion, dhesion, migrtion, differentition nd poptosis of norml cells nd plys importnt roles in these processes (8,9). Therefore, the role of PTEN gene deletion nd muttion in tumorigenesis hs ttrcted the ttention of mny reserchers. At present, certin progress hs been mde in the reserch on the phosphorylse ctivity nd substrte of PTEN in tumor cells nd the role of PTEN in signl trnsduction pthwy in vrious tumor cells (10). Through the in vitro nerve cell experiment, Weng et l hve confirmed tht PTEN leds to the decline in the ctivtion nd phosphoryltion of glycogen synthse kinse 3 (GSK3) through the PI3K/AKT signling pthwy. Due to the decresed phosphoryltion of GSK3 for downstrem cyclin D1, more cyclin D1 is ccumulted in cells, thus rresting cells in G1 phse (11). According to results of reserch on glioblstom, PTEN cn inhibit cell growth nd migrtion vi regulting focl dhesion kinse (FAK) nd p130 cs tyrosine phosphoryltion (12,13). In brest cncer cells, PTEN, through ffecting the insulin receptor IRS 1 phosphoryltion nd the formtion of IRS 1/Grb2/SOS complex, blocking Gb1 migrtion, nd other pthwys, cn inhibit mitogen ctivted protein kinse (MAPK) ctivity, nd reduce the positive regultory effects of MAPK signling pthwy on cell growth, prolifertion nd differentition (14). It hs lso been reported tht PTEN plys role in prostte cncer, in which PTEN cn increse the sensitivity of prostte cncer cells to poptosis receptor nd drug medited Fs ssocited protein with deth domin (FADD) dependent poptosis signling pthwy, mking brekthrough in the tretment of prostte cncer (15). Therefore, PTEN gene hs close ssocition with the occurrence nd development of tumors. It hs been reported tht the positive expression rte of PTEN protein in HCC tissues is mrkedly lower thn tht in pr crcinom norml tissues, nd even not expressed in cncer tissues (16). The expression of PTEN is negtively ssocited to HCC pthologicl grding nd the presence of cncer thrombus. The positive expression rtes of PTEN in ptients in Child Pugh grde C nd B re significntly lower thn tht in ptients in grde A, nd HCC ptients with low or negtive PTEN protein expression re often ccompnied by the elevted level of AFP nd the metstsis of cncer cells. However, there re no definite reports on the reltionships of the positive expression rte of PTEN with HCC size, serum AFP level nd pthologicl typing (17). The results of this study mnifested tht the expression of PTEN in liver cncer tissues ws minly locted in the cytoplsm, nd slightly in the nucleus, which re consistent with the results previously reported in the literture (18 20). Compred with tht in norml liver tissues, the expression of PTEN in HCC ws remrkbly decresed, nd the difference ws sttisticlly significnt (P<0.05). No significnt differences were found in the expression levels of serum tumor mrkers, except AFP, in HCC ptients with negtive PTEN expression compred with those in HCC ptients with positive PTEN expression, indicting tht PTEN expression bsiclly hs no effect on the levels of serum tumor mrkers in HCC, but the expression of AFP is significntly incresed in HCC ptients with negtive PTEN expression. Whether PTEN is relted to the expression level of AFP nd the wy in which it ffects the expression of AFP remin to be further investigted. Moreover, there ws no significnt chnge in blood biochemicl indexes in ptients fter chemotherpy. The levels of ALT nd AST in HCC ptients were slightly incresed fter chemotherpy, which ws possibly due to the side effects of chemotherpy drugs on the liver. The expression levels of ALB, ALP nd PTA nd inflmmtory fctors (IL 1 nd IL 2) were obviously decresed fter chemotherpy, nd there were sttisticlly significnt differences compred with those before chemotherpy (P<0.05). The expression of PTEN ws negtively ssocited with the Child Pugh grding of liver function, which ws obviously lower in HCC ptients in grde B nd C thn tht in ptients in grde A, indicting tht the expression of PTEN hs certin effect on the liver function of HCC ptients. In conclusion, the ssocition of the expression of PTEN with the liver function clssifiction, serum tumor mrkers nd liver function of HCC ptients is described bove, providing experimentl dt for predicting the clinicl progression nd prognosis of HCC. PTEN cn serve s potentil gene in the biologiclly trgeted therpy of liver cncer. Acknowledgements Not pplicble. Funding This study ws supported by the Hunn Nturl Sciences Foundtion Key Progrm (no. 2017JJ3504). Avilbility of dt nd mterils The dtsets used nd/or nlyzed during the present study re vilble from the corresponding uthor on resonble request. Authors' contributions JZ nd XL drfted the mnuscript. They lso recorded nd nlyzed the expression of PTEN nd the liver function. Both uthors red nd pproved the finl mnuscript. Ethics pprovl nd consent to prticipte The study ws pproved by the Ethics Committee of Xingy Hospitl, Centrl South University (Chngsh, Chin). Signed informed consents were obtined from the ptients or the gurdins.

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