Augmenter Of Liver Regeneration Potentiates Doxorubicin Anticancer Efficacy By Reducing The Expression Of ABCB1 And ABCG2 In Hepatocellular Carcinoma

Background & Aims : Hepatocellular carcinoma?HCC?is the fifth most common cancer and the third leading cause of cancer-related death worldwide.Even more seriously,the prevalence of HCC is increasing.Despite the recent improvement in the earlier diagnosis of HCC with imaging techniques,its treatment is still a challenge to both physicians and patients.Depending on the stage,there are several treatment modalities for HCC,including surgery,radiotherapy,and chemotherapy.A challenging issue for HCC chemotherapy is that HCC is highly chemoresistant,displaying multi-drug resistance?MDR?to functionally and structurally different anticancer drugs.Augmenter of liver regeneration?ALR?,previously also known as ?hepatic stimulator substance?,was initially reported as a factor that promotes liver regeneration in rats after partial hepatectomy.Experimental and clinical studies have shown that alternations in ALR expression are closely associated with several liver diseases.Enhanced ALR mRNA and protein expression are found in the livers of patients with cirrhosis,cholangiocellular carcinoma,and HCC.It has also been reported that ALR expression correlates inversely with the HCC tumor grade and its metastasis status.Although HCC metastasis has been longly believing with increased drug resistance,little information is yet available to explain the relationship between ALR and HCC in terms of chemoresistance.In this study,we are aiming to elucidiate the link between ALR and chemoresistance in HCC and identify the poetinal mechanism that it might be dealt with.Methods:1.To clarify the link between ALR and chemoresistance in HCC,HepG2 cells were stably transfected with ALR or ALR-shRNA;the mock-transfected and stably vector-transfected cells were used as the controls.Cells were treated with increasing doses?0,0.5,1,2,4,8,or 16 ?g/ml?of doxorubicin for 48 h;the survival rates were assessed with MTS method,and accordingly the cell inhibition rates and IC₅₀ values were calculated.The long-term effects of chemoresistance were also tested with a colony-forming assay.And to test whether the ALR-associated antitumor effect was specific to HCC cells,other sorts of HCC cells,including Bel-7402,Hep3 B,and Huh7 cells,were employed;transfected with ALR-containing plasmid and reated with doxorubicin for 48 h,and cytotoxicity was detected with an MTS colorimetric assay.2.To confirm that ALR increases the antitumor effect of doxorubicin in vivo,equal amounts of ALR-tx or ALR-shRNA cells were injected under the left armpits of NOD/SCID mice,and untransfected HepG2 cells were injected under the right armpit of each mouse as the control.The administration of 1.8 mg/kg doxorubicin?ip 3q × 7?retarded the growth of the ALR-tx xenografts.The tumors were excised after treatment,and the relative tumor volumes,increment rates and tumor inhibition as well were calculated.The microstructures of the tumor tissues from each group were observed after HE staining and the ultrastructure was observed with transmission electronic microscope?TEM?.3.After confirmation of the ALR relation with antitumor effect,the caspase-activation-induced apoptosis in ALR-transfected HCC cells was explored.The cells were treated with or without 1.88 ?g/ml doxorubicin(IC₅₀ of Vector-tx)for 24 h or 48 h.Caspase activity was detected with the Caspase-Glo 3/7 Assay Kit,and cleaved PARP was analyzed with western blotting and apoptosis was analyzed with flow cytometry.4.Since chemoresistance is closely related with the sufficient retention of an anti-cancer drug within cells,the cells were treated with doxorubicin for 2 h,and then the intracellular retention of doxorubicin was analyzed with a high-content analyzer and a laser scanning confocal microscope.5.To elucidiate a mechanism responsible for the ALR-related antitumor effect,expressions and activities MDR-ABC transporter were further investigated.Total mRNA was extracted from human HCC HepG2 cells,Mock,Vector-tx,ALR-tx,and ALR-shRNA cells,and the expressions of several members of MDR family such as ABCB1,ABCC1,ABCC10,and ABCG2 genes were quantified with qRT–PCR and western blotting.The alternations of ALR,ABCB1,and ABCG2 expressions in the xenografts were also determined with western blotting.6.Whether alternations of ABCB1 and ABCG2 expressions were resulted from ALR transfection becomes a subsequent interesting topic to be explored.To do so,ALR-shRNA cells were transfected with the ALR plasmid.After transfection for 24 h,the expressions of ALR,ABCB1,and ABCG2 were analyzed with western blotting.Cells were pretreated or not with 3 ?M verapamil,and then treated with 2.5 ?M doxorubicin for 2 h.The intracellular retention of doxorubicin was examined with laser scanning confocal microscopy and with a high-content analysis.7.Given the critical role of the AKT/Snail signaling pathway indrug resistance,we explored the expression of Snail,phosphorylation of Akt at Ser473 and Thr308.Results:1.As a control,the vector-transfected cells exhibited an IC₅₀ at 1.88 ?g/ml,while,the ALR-tx cells presented lower dose of IC₅₀ at 1.23 ?g/ml and the IC₅₀ of the ALR-shRNA cells was increased to 2.87 ?g/ml.Additionally,colony-forming assay revealed that the chemoresistance of HepG2 cells was significantly reduced in the ALR-tx cells,while the ALR-shRNA cells had an increased capacity to form colonies.The ALR-tx cells appeared more sensitive to doxorubicin than the control cells.This case remains similar in other HCC cell lines.2.With the treatment of doxorubicin,the tumor inhibition rates were 54.12%,82.37%,and 9.94% for the Mock,ALR-tx,and ALR-shRNA mice,respectively.And tumor growth rate was significantly lower in the ALR-tx mice than in the ALR-shRNA mice during the 21 days doxorubicin treatment.HE staining showed that,comparing with the control group,focal necrosis was readily observed in the Mock group,whereas marked necrosis was seen in the ALR-tx group.On the contrary,the knockdown of ALR expression?ALR-shRNA?reduced doxorubicin-induced necrosis in the tumor tissues.Ultrastructural observations showed that,after treatment with doxorubicin for 21 days,typical apoptotic nuclei were observed,along with the chromatin condensed into several large masses close to the nuclear envelope,whereas in the ALR-tx samples,the nuclear membrane was markedly disrupted and karyolysis was clearly apparent.3.After 24 h of doxorubicin treatment,caspase-3 activity was markedly increased in the ALR-tx cells,while the changes of other cell lines were scarcely;48 h posterior to the treatment,all the cells showed higher caspase-3 activity,and this increase exhibited remarkably hampered in the ALR-shRNAcells.Consistent with this,treatment with doxorubicin also resulted in cleavage of PARP protein,a marker of cellular apoptosis,in the ALR-tx cells at 24 h;and this increase was all eviated in the ALR-shRNA cells at 48 h compared with other cell lines.And after treatment with doxorubicin for 24 h,the apoptotic rate in the Mock cells and vector-transfected cells clearly increased by 20.3% and 11.5%,respectively.However,the ALR-transfected cells displayed a significantly higher apoptotic rate of 38.3%.Likewise,when the cells were treated with doxorubicin for 48 h,the apoptotic rates in the Mock cells and vector-transfected cells were 32.7% and 28.9%,respectively,whereas that in the ALR-transfected cells was 39.4%.4.After treated with different doses of doxorubicin?2.5,5 or 10 ?M?for 2 h,HepG2 cells displayed different drug intracellular retention rates,among them,the retention seemed to be the highest in the ALR-tx cells,and the retention appeared largely declined in the ALR-shRNA cells,i.e.doxorubicin was less accumulatedin the ALR-knockdown cells compared with the ALR-tx cells.We also observed the intracellular retention of doxorubicin?2.5 ?M?by fluoresce microscope.The amount of doxorubicin,represented by the red fluorescent signal,clearly increased in the intracellular compartment of ALR-tx cells and nuclei as well,and this increased appeared declined when ALR was knocked down.Verapamil,a classic MDR efflux pump inhibitor,was used as a positive control.The doxorubicin accumulation in the Bel-7402 cells subjected to ALR plasmid or ALR shRNA transfection exhibited an identical pattern to that was in HepG2 cells.5.Among the four genestested,only the expressions of ABCB1 and ABCG2 changed significantly in the ALR-tx and shRNA knockdown hepatocytes.The transfection of cells with ALR reduced the expression of ABCB1 and ABCG2,whereas ALR knockdown increased the ABCG2 levels.The expressions of ALR,ABCB1 and ABCG2 in the xenografts were identical to that seen in HepG2 cells.6.After ALR rescue,the expressions of ABCB1 and ABCG2 were significantly suppressed?by 39.7% and 33.6%,respectively?compared with those in the ALR-shRNA cells.Simultaneously,the intracellular level of doxorubicin increased more than 2-fold after ALR rescue.7.Western blot analysis showed that the transfection of ALR in HepG2 cell would inhibit the phosphorylation of Akt at Ser473 and Thr308,and the level of Snail was decreased.And the phosphorylation of AKT and the expression of Snail were increased in cells treated with ALR shRNA.Conclusions: Overexpression of ALR in cancerous hepatocytes improves their sensitivities to antitumor drugs by increasing the retention of intracellular drugs,at least partly through the inhibition of ABCB1 and ABCG2 expression.In the future,ALR may be used to improve the overall prognosis of HCC when it is combined with common chemotherapeutic drugs.