Effects Of ABCG2 Gene Silencing On The Proliferative Ability In Vivo And Morphological Changes In Human Lung Adenocarcinoma Cell GLC-82

Background and objectivesLung cancer is one of the most common malignant tumors which is a big threat to human's health,and its morbidity and mortality rates continue to rise.Molecular, biological and immunological studies show that development of lung cancer is a complicated pathological process with interaction of multiple factors and multiple gene mutations.With evolution of scientific research,the treatment and management of lung cancer has improved a lot,but the 5-year survival rate is still below 15%. Since the detection,treatment and prognosis will not be optimistic without a clear understanding of the underlying mechanisms and effective therapeutic options,it is imperative to explore further the etio-pathogenesis and develop more potent means of prevention and cure.ABCG2 gene is a member of ATP-binding cassette transporter superfamily, participating in multidrug resistance(MDR).Because it was first discovered in MCF-7 breast cancer cell sub-line,the protein it encodes is also referred to as breast cancer resistance protein(BCRP).With more in-depth research,evidence has emerged linking ABCG2 and stem cell,suggesting a role as a potential marker of stem cell.However,research on distribution of ABCG2 in lung cancer tissue and cell lines indicate that it cannot be considered as a single marker of lung cancer and so most research on ABCG2 focuses on it's correlation with multidrug resistance and stem cells.Our previous study on "effect of ABCG2 on the biological behavior of lung cancer cell line"showes that ABCG2 gene silencing can induce slow proliferation in vitro,whether it's influence on the proliferative ability in vivo is unkown.To reveal the effect of ABCG2 gene silencing on the proliferative ability in vivo,this study was carried out based on subcutaneous tumor of ABCG2 gene silencing in human lung carcinoma cell.Furthermore,we use quantitative computerized image analysis and stereological method to show the morphological and ultrastructural changes in human lung carcinoma cell after ABCG2 gene silencing.That we can gain more insight into the relationship between this transporter and lung cancer,which could provide a more effectual strategy for prevention and cure of this dreaded disease.Materials and Methodsâ… .Effects of ABCG2 gene silencing on the proliferative ability in vivo of human lung carcinoma cell.(1) Human lung carcinoma cell lines GLC-82,GLC-82/EGFP⁺ and GLC-82/EGFP⁺/ ABCG2^- were implanted into subcutaneous tissue of nude mice to observe the process of tumorgenesis and compare proliferative ability in vivo.(2) Flow cytometry was used to detect the cell cycle of the heteroploid cells.(3) By immuno-histochemical methods(SP) the expressions of Ki-67,ABCG2, Bcl-2 and p53 was assessed.(4) Quantitative studies and morphometric analysis of AgNORs were conducted on tissues with AgNOR staining technique(Ploton). â…¡.Effects of ABCG2 gene silencing on morphological changes in human lung carcinoma cell.(1) Morphological cellular features of GLC-82/EGFP⁺/ABCG2^-,GLC-82/EGFP⁺, and GLC-82 were observed both in vitro and vivo,with cytoskeleton staining used in vitro cells.(2) Morphological parameters of cells(HE) both in vitro and vivo were examined by quantitative computerized image analysis.The parameters included Cells and nuclear included area(A_c,A_n),Circumference(C_c,C_n),Long axis(d_maj,c,d_maj,n), Short axis(d_min,c,d_min,n),Axis Ratio(Ra_c,Ra_n),Karyoplasmic ratio(Rnp),Form factor PE(PE_c,PE_n),Form factor AR(AR_c,AR_n),Regular form factor(RFF_c,RFF_n),and Form irregular index(FII_c,FII_n).(3) The images of subcutaneous tumor were photographed with electron microscopy and stereological analysis carried out using the Image-Pro-Plus analytic software. The parameters included volume density(V_v),surface density(S_v),ratio of surface to volume(Rsv),numerical density(N_v),mean volume(v),mean surface(s),ratio of nucleus to cytoplasm(Rnp),mean free distance(λ).Resultsâ… .Effects of ABCG2 gene silencing on the proliferative ability in vivo of human lung carcinoma cell.(1) Compared with tumors from GLC-82 and GLC-82/EGFP⁺,the tumorgenesis in GLC-82/EGFP⁺/ABCG2^- proceeded slowly with smaller volume tumors.(2) Flow cytometry results showed no difference in the percentage of heteroploid cell numbers in G₂ among the 3 groups.The cell numbers in G₁ increased in GLC-82/EGFP⁺/ABCG2^- group while the cell numbers in S decreased.(3) Immunohistochemistry results indicated that ABCG2 protein expressed on the tumors of GLC-82/EGFP⁺/ABCG2^- group attenuated.Image analysis demonstrated that the Positive Unity(PU) of ABCG2 protein in GLC-82/EGFP⁺/ABCG2^- group was lower than GLC-82/EGFP⁺,GLC-82 groups with significant difference.Ki-67 proliferation index of GLC-82/EGFP⁺/ABCG2^- cells in vivo was significantly lower than GLC-82/EGFP⁺ cells and GLC-82 cells(P＜0.05).There were no significant differences in Bcl-2 and p53 expression among the 3 groups.(4) Image analysis revealed significant differences in AgNORs parameters with lesser number of AgNORs and total AgNORs areas but higher mean AgNORs areas in the cells of GLC-82/EGFP⁺/ABCG2^- group.AgNORs in GLC-82/EGFP⁺/ ABCG2^- group were most mixed type,while dispersed type were the main pattern in GLC-82/EGFP⁺ and GLC-82 group.â…¡.Effects of ABCG2 gene silencing on morphological changes in human lung carcinoma cell.(1) GLC-82/EGFP⁺/ABCG2^- exhibited visible differences in size and shape appearing mainly spindle shaped,whereas GLC-82/EGFP⁺,GLC-82 cells were spherical,oval or polygonal.(2) Cytoskeleton staining results showed the cytoskeleton of GLC-82/EGFP⁺/ ABCG2^- to be fine and spread throughout the cytoplasm,while that of GLC-82/ EGFP⁺,GLC-82 was coarse and clumped around the nucleus.(3) Image analysis of H&E stained cell slide revealed that when compared with GLC-82/EGFP⁺,GLC-82 group,the values of d_maj,c,C_c,Ra_c,d_maj,n,Ra_n,Ac,, Acp,FII_c,FII_n in GLC-82/EGFP⁺/ABCG2^- cell were the highest,but its PE_c,AR_c, RFF_c,PE_n,AR_n,RFF_n,R_np were the lowest.(4) Histologically,the volume of the cells in GLC-82/EGFP⁺/ABCG2^- group was increased with clear cytoplasm or presence of vacuoles inside.Negative AB-PAS staining ruled out the presence of mucus inside the cytoplasm.The nuclei of GLC-82/EGFP⁺/ABCG2^- cells exhibited marked pleomorphism with numerous bizarre mitoses.Image analysis revealed that the values of PE_n,AR_n,RFFn and R_np decreased while FII_n,A_c,Acp increased.(5) Image analysis of HE stained tumor tissue revealed that when compared with GLC-82/EGFP⁺,GLC-82 group,the values of FII_n,A_n,Acp,in GLC-82/EGFP⁺/ ABCG2^- cell increased,but its PE_n,AR_n,RFF_n,R_np decreased(6) Ultrastructural examination showed obvious pleomorphism of nucleus in both GLC-82/EGFP⁺/ABCG2^-,GLC-82/EGFP⁺ groups with numerous lysosomes.The characteristic changes in GLC-82/EGFP⁺/ABCG2^- group were mitochondrial swelling and dilatation of endoplasmic reticulum,with presence of specific "albuminoid material" inside the cytoplasm crushing the nucleus.(7) Three-dimensional quantificational results of the ultrastructure revealed that the V_v,S_v,R_np of nucleus decreased withλincreased in GLC-82/EGFP⁺/ABCG2^-group;the value of V_v,S_v for mitochondrion was higher andλlower,V_v for endoplasmic reticulum was higher in GLC-82/EGFP⁺/ABCG2^- group.Conclusions:1.ABCG2 gene silencing can effectively suppress the proliferative ability of lung carcinoma cells in vivo:the tumorgenesis of GLC-82/EGFP⁺/ABCG2^- cells proceeded slowly with smaller volume tumors,indicating that ABCG2 gene is significant to the proliferate of GLC-82 cell in vivo and it could be a curative target to be deeply investigated.2.Ki-67 proliferation index decreased in GLC-82/EGFP⁺/ABCG2^- group;Flow cytometry results showed that most percent of cells in GLC-82/EGFP⁺/ABCG2^-group were inhibit in G1;number of AgNORs and total AgNORs areas but higher mean AgNORs areas in the cells of GLC-82/EGFP⁺/ABCG2^- group.AgNORs in GLC-82/EGFP⁺/ABCG2^- group were most mixed type,while dispersed type were the main pattern in GLC-82/EGFP⁺ and GLC-82 group. 3.After ABCG2 gene silencing the morphology of GLC-82 cell changed:the lung cancer cells in vitro transform into spindle shaped cells.In vivo,the cell body dilated with nucleus heteromorphism(V_v,S_v,R_np decreased andλ,increased) mitochondrial swelling(V_v,S_v increased andλdecreased) and dilatation of endoplasmic reticulum(V_v increased and Rsv decreased) ultrastructurally.Yhe volume of the cells in GLC-82/EGFP⁺/ABCG2^- group was increased with clear cytoplasm and nucleus heteromorphism histologically.