Apoptotic activity of caged xanthones from Garcinia hanburyi in cholangiocarcinoma cell lines

AIM:To investigate the growth inhibitory mechanism of four caged xanthones from Garcinia hanburyi in cholangiocarcinoma(CCA) KKU-100 and KKU-M156 cells.METHODS:Four caged xanthones,selected on the basis of their anticancer potency and chemical structure diversities(i.e.isomorellin,isomorellinol,forbesione and gambogic acid) were used in this study.Growth inhibition of these caged xanthones was determined using the sulforhodamine B assay.Induction of apoptosis was assessed by observing cell morphology,ethidium bromide and acridine orange staining and DNA fragmentation assay.Levels of apoptotic-related gene and protein expressions were determined by a real-time reverse transcriptase polymerase chain reaction and Western blotting analysis,respectively.RESULTS:The compounds were found to inhibit growth of both cell lines in a dose-dependent manner and also showed selective cytotoxicity against the cancer cells when compared with normal peripheral blood mononuclear cells.Growth suppression by these compounds was due to apoptosis,as evidenced by the cell morphological changes,chromatin condensation,nuclear fragmentation,and DNA ladder formation.At the molecular level,these compounds induced down-regulation of Bcl-2 and survivin proteins with up-regulation of Bax and apoptosisinducing factor proteins,leading to the activation of caspase-9 and-3 and DNA fragmentation.The functional group variations did not appear to affect the anticancer activity with regard to the two CCA cell lines;however,at a mechanistic level,isomorellinol exhibited the highest potency in increasing the Bax/Bcl-2 protein expression ratio(120 and 41.4 for KKU-100 and KKU-M156,respectively) and in decreasing survivin protein expression(0.01 fold as compared to control cells in both cell lines).Other activities at the molecular level indicate that functional groups on the prenyl side chain may be important.CONCLUSION:Our findings for the first time demonstrate that four caged xanthones induce apoptosis in CCA cells which is mediated through a mitochondriadependent signaling pathway.

Establishment and characterization of a cholangiocarcinoma cell line (RMCCA-1) from a Thai patient

AIM: To establish and characterize a new cell line derived from peripheral cholangiocarcinoma of a Thai patient.METHODS: The peripheral cholangiocarcinoma specimen surgically obtained from the patient was aseptically processed by washing and mincing before culturing in Ham’s F12 medium containing 10% fetal bovine serum. After 3 mo, when the cell line has become homogeneous and stabilized, several features were investigated, including growth characteristics, immunofluorescence staining for cytokeratins, expression of tumor markers, chromosomal analysis by G-banding and multicolour fluorescence in situ hybridization (mFISH), in vitro migration and invasion characteristics. RESULTS: The RMCCA-1 cell line has been established. These cells proliferated as a monolayer with a population doubling time of 48 h. Immunofluorescence staining showed positive staining for human cytokeratin 7 and 19 verifying the biliary epithelial origin. RMCCA-1 secreted carbohydrate antigen 19-9 (CA19-9), but insignificant levels of carcinoembryonic antigen (CEA) and α-fetoprotein (AFP). Chromosome analysis identified aneuploidy karyotypes with a modal chromosome number of 59. RMCCA-1 exhibited a low level of in vitro invasiveness, but a high degree of motility. The cell line exhibited a significant number of chromosomal aberrations as shown by mFISH and G-banding methods.CONCLUSION: A new cell line derived from peripheral cholangiocarcinoma of a Thai patient has been established. This cell line shows a low level of in vitro invasiveness, but a high degree of motility. It will serve as a valuable tool for further studies on tumor biology, molecular pathogenesis, metastatic mechanism and response to therapeutic drugs of cholangiocarcinoma.

Establishment and characterization of an opisthorchiasis-associated cholangiocarcinoma cell line (KKU-100)

AIM To establish and dharacterize a nev cholangiocarcinoma cell line from a patient living in the Opisthorchis viverrini (O. viverrini) endemic area of Northeast Thailand.METHODS: Fresh liver biopsy and bile specimens were obtained from a 65-year-old Thai woman with cholangiocarcinoma of the porta hepatis. After digestion, the cells were cultured in Ham's F12 media. The established cell line was then characterized for growth kinetics, cell morphology, imm unocytochemistry and cytogenetics. Tumorigenicity of the cell line was determined by heterotransplanting in nude mice. RESULTS: The primary tumor was a poorly differentiated tubular adenocarcinoma. Examination of the bile revealed malignant cells with O. viverrini eggs. The cholangiocarcinoma cell line KKU-100 was established 4 mo after the primary culture-population doubling time was 72 h. KKU-100 possesses compact and polygonal-shapedepithelial cells. Immunocytochemically, this cell line exhibited cytokeratin, EMA, CEA, and CA125, but not α-fetoprotein (AFP), CA19-9, desmin, c-met, or p53. Such protein expressions parallel those of the primary tumor. Cytogenetic analysis identified aneuploidy karyotypes with a modal chromosome number of 78 and marked chromosomal structural changes. Inoculation of KKU-100 cells into nude mice produced a transplantable, poorly differentiated aden-ocarcinoma, similar to the original tumor.CONCLUSION: KKJ-100 is the first egg-proven, Opisthorchis- associated cholangiocarcinoma cell line, which should prove useful for further investigations of the tumor biology of this cancer.

DIFFERENCE IN BIOLOGICAL CHARACTERISTICS AND SENSITIVITY TO CHEMOTHERAPY AND RADIOTHERAPY BETWEEN INTRAHEPATIC AND EXTRAHEPATIC CHOLANGIOCARCINOMA CELLS IN VITRO

Objective To investigate and compare the biological characteristics and sensitivity to chemotherapy and radiotherapy of intrahepatic and extrahepatic cholangiocarcinoma cells in vitro.Methods The intrahepatic and extrahepatic cholangiocarcinoma cell lines were established,and cells with steady passage were chosen to study the biological characteristics including morphology,growth dynamics,chromosome,and levels of cancer antigen(CA)125,CA19-9,alpha-fetoprotein(AFP),and carcino-embryonic antigen(CEA).Meanwhile,MTT assay was used to determine the sensitivity of both kinds of cells to 6 chemotherapeutic drugs,including cisplatin,paclitaxel,harringtonine,5-fluorouracil,vincristine,and aclacimomycin,and the inhibitory rate of cells under the irradiation of 10 Gy ray was also measured.Results The intrahepatic cholangiocarcinoma cells were mostly fusiform in shape,and extrahepatic cholangiocarcinoma cells were mostly round or polygon in shape.Their doubling time was 26.3 hours and 23.1 hours,respectively.Their average number of chromosomes was 59(range,38-84)and 67(range,49-103),respectively.The chromosome karyotypes of most intrahepatic cholangiocarcinoma cells were hyperdiploid and hypotriploid,while hypertriploid was predominant in extrahepatic cholangiocarcinoma cells.The level of CA 125 in supernatant of extrahepatic cholangiocarcinoma cells increased obviously,while levels of other determined tumor markers in both kinds of cells were all within normal range.The intrahepatic cholangiocarcinoma cells were low sensitive to cisplatin and paclitaxel,but not sensitive to the other 4 chemotherapeutic drugs.The extrahepatic cholangiocarcinoma cells were high sensitive to cisplatin,but not sensitive to the other 5 drugs.Both kinds of cells had poor sensitivity to radiotherapy.Conclusions Intrahepatic and extrahepatic cholangiocarcinoma cells show differences in shape,doubling time,chromosome karyotype,tumor marker level,and chemosensitivity,whereas they both have poor radiosensitivity.Though they are similar in histopathology,they have different growth characteristics and have discrepancy in treatment and prognosis.

Honokiol-enhanced cytotoxic T lymphocyte activity against cholangiocarcinoma cells mediated by dendritic cells pulsed with damage-associated molecular patterns

BACKGROUND Cholangiocarcinoma or biliary tract cancer has a high mortality rate resulting from late presentation and ineffective treatment strategy. Since immunotherapy by dendritic cells (DC) may be beneficial for cholangiocarcinoma treatment but their efficacy against cholangiocarcinoma was low. We suggest how such antitumor activity can be increased using cell lysates derived from an honokioltreated cholangiocarcinoma cell line (KKU-213L5). AIM To increase antitumour activity of DCs pulsed with cell lysates derived from honokiol-treated cholangiocarcinoma cell line (KKU-213L5). METHODS The effect of honokiol, a phenolic compound isolated from Magnolia officinalis, on choangiocarcinoma cells was investigated in terms of the cytotoxicity and the expression of damage-associated molecular patterns (DAMPs). DCs were loaded with tumour cell lysates derived from honokiol-treated cholangiocarcinoma cells their efficacy including induction of T lymphocyte proliferation, proinflammatory cytokine production and cytotoxicity effect on target cholangiocarcinoma cells were evaluated. RESULTS Honokiol can effectively activate cholangiocarcinoma apoptosis and increase the release of damage-associated molecular patterns. DCs loaded with cell lysates derived from honokiol-treated tumour cells enhanced priming and stimulated T lymphocyte proliferation and type I cytokine production. T lymphocytes stimulated with DCs pulsed with cell lysates of honokiol-treated tumour cells significantly increased specific killing of human cholangiocarcinoma cells compared to those associated with DCs pulsed with cell lysates of untreated cholangiocarcinoma cells. CONCLUSION The present findings suggested that honokiol was able to enhance the immunogenicity of cholangiocarcinoma cells associated with increased effectiveness of DC-based vaccine formulation. Treatment of tumour cells with honokiol offers a promising approach as an ex vivo DC-based anticancer vaccine.

High expression of ErbB2 contributes to cholangiocarcinoma cell invasion and proliferation through AKT/p70S6K

AIM:To compare the impact of ErbB2 on cell invasion and proliferation in cholangiocarcinoma(CCA) cell lines.METHODS:Level of endogenous ErbB2 expression in three CCA cell lines,namely HuCCA-1,KKU-100 and KKU-M213,was determined by real-time reversetranscriptase polymerase chain reaction.Two ErbB2 inhibitory methods,a small molecule ErbB2 kinase inhibitor(AG825) and siRNA,were used to disrupt ErbB2 function in the cell lines.CCA cell invasion,motility and proliferation under ErbB2-disrupted conditions were detected using Transwell and 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide assays.In addition,ErbB2 downstream effectors were investigated by Western blotting analysis.RESULTS:Suppression of ErbB2 activity,using a specific kinase inhibitor(AG825),reduced invasion,motility and proliferation of all three CCA cell lines.The ability of this drug to inhibit neoplastic properties(invasion,motility and proliferation) increased concomitantly with the level of ErbB2 expression.Similarly,knockdown of ErbB2 level by siRNA inhibited cell invasion and proliferation of KKU-M213,a high-ErbB2-expressing cell,better than those of the lower-ErbB2-expressing cells,HuCCA-1 and KKU-100.Thus,both inhibitory methods indicated that there is more ErbB2-dependency for malignancy of the high-ErbB2-expressing cell,KKU-M213,than for that of low-ErbB2-expressing ones.In addition,interrupting ErbB2 activity decreased phosphorylation of AKT and p70S6K,but not extracellular signal-regulated kinase 1/2,in the high-ErbB2-expressing CCA cell line.CONCLUSION:Our data indicated that high ErbB2 expression enhances CCA invasion,motility and proliferation via the AKT/p70S6K pathway,which suggests the possibility of targeting these molecules for CCA therapy.

Effect of mutant p27^(kip1) gene on human cholangiocarcinoma cell line, QBC_(939)

AIM:To investigate the effects of exogenously mutated p27kip1 (p27) on proliferation and apoptosis of human cholangiocarcinoma cell line, QBC939 in vivo.METHODS: Adenviral vectors were used to transfect mutated p27 cDNA into human QBC939 cell line. Expression of p27 was detected by RT-PCR. Western blot. Cell growth, morphological change, cell cycle, apoptosis and cloning formation were determined by MTT assay and ? ow cytometry.RESULTS: The expression of p27 protein and mRNA was increased signifi cantly in QBC939 cell line transfected with Ad-p27mt. The transfer of Ad-p27mt could signifi cantly inhibit the growth of QBC939 cells, decrease the cloning formation rate and induce apoptosis. p27 over expression caused cell cycle arrest at G0/G1 phase 72 h after infection with Ad-p27mt.CONCLUSION: p27 may cause cell cycle arrest at G0/G1 phase and subsequently lead to apoptosis. Recombinant adenovirus expressing mutant p27 may be potentially useful in gene therapy for cholangiocarcinoma.

Characterization of a novel rat cholangiocarcinoma cell culture model-CGCCA

AIM:To characterize a culture model of rat CCA cells,which were derived from a transplantable TTA-induced CCA and designated as Chang Gung CCA(CGCCA).METHODS:The CGCCA cells were cultured at in vitro passage 12 times on a culture dish in DMEM medium.To measure the doubling time,103 cells were plated in a 96-well plate containing the growth medium.The cells were harvested 4 to 10 d after seeding,and astandard MTT assay was used to measure the growth.The phenotype of CACCA cell and xenograft was determined by immunohistochemical study.We also determine the chromosomal alterations of CGCCA,G-banding and spectral karyotyping studies were performed.The CGCCA cell line was transplanted into the nude mice for examining its tumorigenicity.2-Deoxy-2-(18F)fluoro-Dglucose(FDG) autoradiography was also performed to evaluate the FDG uptake of the tumor xenograft.RESULTS:The doubling time for the CGCCA cell line was 32 h.After transplantation into nude mice,FDG autoradiography showed that the tumors formed at the cell transplantation site had a latency period of 4-6 wk with high FDG uptake excluding necrosis tissue.Moreover,immunohistochemical staining revealed prominent cytoplasmic expression of c-erb-B2,CK19,c-Met,COX-Ⅱ,EGFR,MUC4,and a negative expression of K-ras.All data confirmed the phenotypic features of the CGCCA cell line coincide with the xenograft mice tumors,indicating cells containing the tumorigenicity of CCA originated from CCA.In addition,karyotypic banding analysis showed that the diploid(2n) cell status combines with ring and giant rod marker chromosomes in these clones;either both types simultaneously appeared or only one type of marker chromosome in a pair appeared in a cell.The major materials contained in the marker chromosome were primarily identified from chromosome 4.CONCLUSION:The current CGCCA cell line may be used as a non-K-ras effect CCA model and to obtain information and reveal novel pathways for CCA.Further applications regarding tumor markers or therapeutic targeting of CCA should be addressed accordingly.

Induction of biliary cholangiocarcinoma cell apoptosis by ^103pd cholangial radioactive stent γ-rays

Background In recent years, interventional tumor therapy, involving implantation of intra-cholangial metal stents through percutaneous trans-hepatic punctures, has provided a new method for treating cholangiocarcinoma, ^103Pd cholangial radioactive stents can concentrate high radioactive dosages into the malignant tumors and kill tumor cells effectively, in order to prevent re-stenosis of the lumen caused by a relapsed tumor. The aim of the present study was to investigate the efficacy of y-rays released by the ^103pd biliary duct radioactive stent in treating cholangiocarcinoma via induction of biliary cholangiocarcinoma cell apoptosis. Methods A group of biliary duct cancer cells was collectively treated with a dose of y-rays. Cells were then examined by the 3-（4,5-dimethyl thiazol-2-yl）-2,5-diphenyl terazolium-bromide （MTF） technique for determining the inhibition rate of the biliary duct cancer cells, as well as with other methods including electron microscopy, DNA agarose gel electrophoresis, and flow cytometry were applied for the evaluation of their morphological and biochemical characteristics. The growth curve and the growth inhibition rate of the cells were determined, and the changes in the ultrastructure of the cholangiocarcinoma cells and the DNA electrophoresis bands were examined under a UV-lamp. Results The y-ray released by ^103Pd inhibited cholangiocarcinoma cell growth, as demonstrated when the growth rate of the cells was stunned by a y-ray with a dosage larger than 197.321 MBq. Typical features of cholangiocarcinoma cell apoptosis were observed in the 197.321 MBq dosage group, while cell necrosis was observed when irradiated by a dosage above 245.865 MBq. DNA agarose gel electrophoresis results were different between the 197.321 MBq irradiation dosage group, the 245.865 MBq irradiation dosage group, and the control group. Conclusions ^103pd radioactive stents which provide a radioactive dosage of 197.321 MBq are effective in the treatment of cholangiocaminoma; ^103pd radioactive stents should be useful for the clinical treatment of cholangiocarcinoma.

A case of intrahepatic clear cell cholangiocarcinoma

Intrahepatic clear cell cholangiocarcinoma is very rareonly 8 cases have been reported.A 56-year-old Japanese man with chronic hepatitis B infection was diagnosed with a 2.2 cm hepatocellular carcinoma on imaging,and hepatic segmentectomy was performed.Histopathologically,the tumor cells had copious clear cytoplasm and formed glandular structures or solid nests.These pathological findings suggested the tumor was a clear cell variant of intrahepatic cholangiocarcinoma.Particular stains and radiological images suggested that the cause of the clear cell change had been glycogen,not mucin nor lipid.On immunohistochemical staining,cytokeratin(CK)7 and CK19 were positive,whereas CK20 was negative.Vimentin was detected on the cell membranes,and CD56 was focally positive.The patient was given adjuvant chemotherapy and is currently free from the tumor 7 mo postoperatively.Careful follow-up with adequate postoperative supplementary chemotherapy is necessary because the characteristics of this type of tumor are unknown.

Anti-apoptosis Effect of Decoy Receptor 3 in Cholangiocarcinoma Cell Line TFK-1

Background： Decoy receptor 3 （DcR3） is a protein with anti-apoptotic effect that belongs to the tumor necrosis factor receptor superfamily. DcR3 is highly expressed in a variety of malignant tumors including cholangiocarcinoma and its expression was found to be related to the clinical stage, the invasion, and tile metastasis of the tumor. This in vitro study aimed to investigate the effect ofdownregulated expression of DcR3 on cell viability, cell apoptosis, and cell cycle in cholangiocarcinoma cell line TFK-1. Methods： Three different cell lines were cultured： human cholangiocarcinoma TFK-I, human biliary epithelial carcinoma HuCCT-1, and human cholangiocarcinoma RBE. The cholangiocarcinoma cell line with the highest expression of DcR3 was selected for further investigation. The expression of DcR3 was silenced/knocked down by transfection with DcR3-siRNA in the selected cell line. Various biological phenotype parameters such as cell viability, apoptosis, and cell cycle were observed. Results： The mRNA and protein levels of DcR3 were measured in the three cell lines, and TFK-1 was selected. After the treatment with DcR3-siRNA for 48 h, DcR3 mRNA and protein expression in the treatment group were 38.45% （P 〈 0.01 ） and 48.03% （P 〈 0.05） of that of＇the control, respectively. It was found that the cell viability decreased to 61.87% of the control group （P 〈 0.01 ） after the downregulation of DcR3 in cholangiocarcinoma cell line TFK- 1 by transfection with DcR3-siRNA, while tile percentage ofapoptotic cells was 2.98 times as compared with the control group （P 〈 0.05）. Compared with the control group the ratio of G0/G1 increased, and the ratio of G2/M dccreased in the treatment group. However, the differences were not statistically significant. Conclusions： The effect of DcR3 on the growth and apoptosis ofcholangiocarcinoma has been demonstrated. DcR3 is not only a predictive marker for malignant tumor but it is also likely to be a potential target for cancer gene therapy. Further studies should focus on exploring the binding ligand of DcR3, the signaling pathway involved, and the molecular mechanism for the regulation of DcR3 expression in cholangiocarcinoma.

Clinicopathological analysis of 14 patients with combined hepatocellular carcinoma and cholangiocarcinoma

BACKGROUND:Combined hepatocellular carcinoma and cholangiocarcinoma (cHCC-CC) is an uncommon subtype of primary hepatic carcinoma,and its prognosis is poor.This study was undertaken to investigate the prognosis and the clinicopathological characteristics of cHCC-CC,including their possible cellular origin.METHODS:Among 852 patients with a primary hepatic carcinoma who underwent hepatectomy from January 1998 to April 2008 at our hospital,cHCC-CC was identified in 14 patients The clinicopathological characteristics of the 14 patients were analyzed retrospectively.The expression of the liver stem cell markers (c-kit,CD90,CD133 and CK19) in the tumor tissue was detected by immunohistochemistry,and the Kaplan-Meier method was used to evaluate survival.RESULTS:Among the 14 patients,9 presented with abdominal pain,3 with anorexia and debilitation,and the remaining two patients were asymptomatic.The mean age was 53.6±3.0 (range 38-74) years.Among the included patients,11 had an elevated serum alpha-fetoprotein level,13 were infected with hepatitis B virus,9 had vascular invasion and 1 had lymph node metastasis The average diameter of the tumors was 9.9±1.1 (range 5.0-16.0) cm.The median overall survival time was 7.9±1.0 months In addition,the presence of the liver stem cell markers,c-kit CD90,CD133 and CK19 was 71.4%,85.7%,92.9% and 78.6% respectively.All four markers were simultaneously expressed in eight cases.CONCLUSIONS:cHCC-CC has aggressive characteristics and the prognosis is extremely dismal.The high expression of liver stem cell markers in the tumor tissue suggests that these tumors may derive from liver stem cells.

Combined hepatocellular cholangiocarcinoma: Controversies to be addressed

Combined hepatocellular cholangiocarcinoma(CHC) accounts for 0.4%-14.2% of primary liver cancer cases and possesses pathological features of both hepatocellular carcinoma and cholangiocarcinoma. Since this disease was first described and classified in 1949, the classification of CHC has continuously evolved. The latest definition and classification of CHC by the World Health Organization is based on the speculation that CHC arises from hepatic progenitor cells. However, there is no evidence demonstrating the common origin of different components of CHC. Furthermore, the definition of CHC subtypes is still ambiguous and the identification of CHC subtype when a single tumor contains many components has remained unresolved. In addition, there is no summary on the newly recognized histopathology features or the contribution of CHC components to prognosis and outcome of this disease. Here we provide a review of the current literature to address these questions.

Multiple cells of origin in cholangiocarcinoma underlie biological,epidemiological and clinical heterogeneity

Recent histological and molecular characterization of cholangiocarcinoma(CCA) highlights the heterogeneity of this cancer that may emerge at different sites of the biliary tree and with different macroscopic or morphological features.Furthermore,different stem cell niches have been recently described in the liver and biliarytree,suggesting this as the basis of the heterogeneity of intrahepatic(IH)-and extrahepatic(EH)-CCAs,which are two largely different tumors from both biological and epidemiological points of view.The complexity of the organization of the liver stem cell compartments could underlie the CCA clinical-pathological heterogeneity and the criticisms in classifying primitive liver tumors.These recent advances highlight a possible new classification of CCAs based on cells of origin and this responds to the need of generating homogenous diagnostic,prognostic and,hopefully,therapeutic categories of IH-and EH-CCAs.

MUC13/HER2分子轴对胆管癌细胞恶性生物学行为和化疗耐药性的影响

目的:探讨黏蛋白13(MUC13)/人类表皮生长因子受体2(HER2)分子轴对胆管癌细胞(CCA)恶性生物学行为和化疗耐药性的影响。方法:选取2020年08月至2022年08月于本院就诊并接受手术治疗的60例CCA患者,免疫组化检测MUC13、HER2阳性表达;qRT-PCR检测CCA细胞系(QBC939、TFK-1、HuCCT-1)及胆管上皮细胞系HIBEpiC中MUC13、HER2 mRNA表达;然后以QBC939细胞为研究对象,设置对照组、sh-MUC13(MUC13的shRNA特异性抑制)组、sh-NC(阴性对照)组、sh-MUC13+pcDNA 3.1(空载体)组、sh-MUC13+pcDNA 3.1-HER2(HER2过表达载体)组;流式细胞仪检测上述各组QBC939细胞凋亡率变化;CCK-8检测各组QBC939细胞活力及耐药性;qRT-PCR检测各组QBC939细胞MUC13、HER2 mRNA表达;Transwell检测各组QBC939细胞侵袭及迁移;Western blot检测各组QBC939细胞MUC13、HER2、Bcl-2相关X蛋白(Bax)、细胞增殖相关核抗原(Ki-67)蛋白表达。结果:MUC13、HER2在癌组织中的阳性表达均显著增加(P<0.05);QBC939细胞中MUC13、HER2 mRNA表达增加最为显著(P<0.05);与对照组、sh-NC组相比,sh-MUC13组细胞增殖活力及耐药性、侵袭与迁移数、MUC13、HER2、Ki-67表达显著下降,凋亡率及Bax表达显著增加(P<0.05);与sh-MUC13+pcDNA 3.1组相比,sh-MUC13+pcDNA 3.1-HER2组细胞增殖活力、耐药性、侵袭与迁移数、MUC13、HER2、Ki-67表达显著增加,凋亡率及Bax表达显著下降(P<0.05)。结论:沉默MUC13表达有助于抑制QBC939细胞恶性生物学行为,降低其耐药性,可能与抑制HER2表达有关。

IDH1基因在肝内胆管癌细胞HuCCT1增殖与迁移中的作用及其初步机制

目的探讨异柠檬酸脱氢酶1(IDH1)在肝内胆管癌(iCCA)细胞HuCCT1增殖与迁移中的作用及其可能的分子机制。方法采用CRISPR/Cas9基因编辑技术构建IDH1基因敲除的HuCCT1细胞(HuCCT1^(IDH1-/-));CCK-8法和克隆形成实验检测IDH1野生型HuCCT1(HuCCT1^(WT))细胞和HuCCT1^(IDH1-/-)细胞的增殖能力;细胞划痕和Transwell实验检测细胞的迁移和侵袭能力;Western blotting检测细胞上皮间质转化(EMT)相关蛋白E-钙黏蛋白(E-cadherin)、N-钙黏蛋白(N-cadherin)、波形蛋白(Vimentin)、基质金属蛋白酶-9(MMP-9)、Wnt3a、β-连环蛋白(β-catenin)的表达水平。生物信息学方法分析上述两种HuCCT1细胞的转录组测序结果,Western blotting验证转录组信息通路相关蛋白的表达。结果与HuCCT1细胞比较,HuCCT1^(IDH1-/-)细胞增殖和克隆形成数目明显减少(P<0.05),阻滞在G_(2)/M期细胞的比例明显增加(P<0.01),划痕愈合率明显降低(P<0.01),迁移细胞数目(P<0.001)和侵袭细胞数目(P<0.05)明显减少;q RT-PCR检测结果显示,HuCCT1^(IDH1-/-)细胞IDH1、Vimentin、MMP-9和调控G_(2)/M期增殖相关基因Cyclin A2、Cyclin B1及CDK1 mRNA表达水平降低(P<0.05),编码E-cadherin的CDH1 mRNA表达水平升高(P<0.01);Western blotting检测结果显示,HuCCT1^(IDH1-/-)细胞中E-cadherin表达水平升高(P<0.05),N-cadherin、Vimentin及MMP-9蛋白表达水平降低(P<0.05)。转录组测序结果显示,HuCCT1^(WT)与HuCCT1^(IDH1-/-)存在1476个差异表达基因(DEGs);基因本体论(GO)分析显示上述DEGs显著富集在炎症反应、细胞信号转导和细胞代谢等生物学过程;KEGG通路分析显示上述DEGs显著富集在Wnt、MAPK、Rap1、Hippo、TNF等与肿瘤细胞增殖和侵袭转移密切相关的信号通路。Western blotting验证结果显示,与HuCCT1^(WT)比较,HuCCT1^(IDH1-/-)细胞Wnt信号通路的Wnt3a和β-catenin蛋白表达水平降低(P<0.05)。结论IDH1基因参与调控iCCA细胞HuCCT1的迁移、侵袭及EMT过程,其机制可能与激活Wnt/β-catenin信号通路有关。

LncRNA SOX2OT靶向SIRT1/自噬通路增强胆管癌细胞5-FU耐药

目的探讨LncRNASOX2OT靶向SIRT1/自噬通路调节胆管癌细胞5-FU耐药的机制。方法将未用5-FU处理HCCC-9810细胞和不同浓度(50、100、150、200μg/mL)5-FU处理的HCCC-9810细胞分为对照组和模型组,qRT-PCR检测LncRNA SOX2OT、SIRT1 mRNA表达水平,Western blot检测SIRT1、Beclin1、LC3和p62蛋白表达。pcDNA3.1-SOX2OT和pcDNA3.1-NC转染HCCC-9810/5-FU耐药细胞,CCK-8检测细胞耐药性,划痕实验检测细胞迁移能力,qRT-PCR检测LncRNASOX2OT、SIRT1 mRNA表达水平,Western blot检测SIRT1、Beclin1、LC3和p62表达。在以上基础上做了Rescue实验,将OV-SOX2OT(2μg/孔)和si-NC(75pmol/孔)、OV-SOX2OT(2μg/孔)和si-SIRT1(75pmol/孔)分别共转染HCCC-9810/5-FU耐药细胞,分组为OV-SOX2OT+si-NC组和OV-SOX2OT+si-SIRT1组,以证明LncRNASOX2OT通过SIRT1影响自噬,并由此影响胆管癌细胞对5-FU的耐药性。RNAPulldown验证SOX2OT与SIRT1的靶向结合关系。结果不同浓度的5-FU均能抑制HCCC-9810细胞增殖(P<0.05)。与对照组相比,模型组SIRT1、Beclin1(P<0.001)和p62(P<0.01)蛋白表达、LC3Ⅱ/LC3Ⅰ比值(P<0.001)、SIRT1和LncRNA SOX2OT mRNA水平(P<0.05)均明显增加。与OV-NC组相比,OV-SOX2OT组细胞迁移能力、SIRT1、Beclin1(P<0.001)和p62(P<0.05)蛋白表达、LC3Ⅱ/LC3Ⅰ比值(P<0.001)、SIRT1和LncRNASOX2OTmRNA(P<0.05)水平均明显增加。沉默SIRT1表达导致LncRNASOX2OT过表达的HCCC-9810细胞对5-FU的耐药性显著降低。与OV-SOX2OT+si-NC组相比,OV-SOX2OT+si-SIRT1组SIRT1(P<0.001)、p62和Beclin1(P<0.01)蛋白表达、LC3Ⅱ/LC3Ⅰ比值(P<0.01)、SIRT1 mRNA(P<0.05)水平均明显降低。RNA Pulldown检测结果显示SOX2OT能够直接与SIRT1结合。结论LncRNA SOX2OT通过上调SIRT1表达促进自噬来增强胆管癌HCCC-9810细胞5-FU耐药性。

lncRNA MIR4435-2HG靶向miR-376a-3p调控胆管癌细胞生物学行为的机制研究

目的 探讨长链非编码RNA(lncRNA)MIR4435-2HG(MIR4435-2HG)对胆管癌细胞增殖、迁移、侵袭、凋亡的影响及其对微小RNA-376a-3p(miR-376a-3p)的调控作用。方法 qRT-PCR法检测人肝内胆管上皮细胞HIBEpic与人胆管癌细胞RBE中MIR4435-2HG、miR-376a-3p的表达。将si-NC、si-MIR4435-2HG、miR-NC、miR-376a-3p mimics、si-MIR4435-2HG联合anti-miR-NC、si-MIR4435-2HG联合anti-miR-376a-3p分别转染至RBE细胞,作为si-NC组、si-MIR4435-2HG组、miR-NC组、miR-376a-3p组、si-MIR4435-2HG+anti-miR-NC组、si-MIR4435-2HG+anti-miR-376a-3p组;采用MTT法、Transwell小室法及流式细胞仪分别检测细胞增殖、迁移、侵袭及凋亡情况;双荧光素酶报告基因实验验证MIR4435-2HG与miR-376a-3p的靶向关系。Western blot检测相关蛋白表达。结果 RBE细胞中MIR4435-2HG表达量升高(P<0.05),miR-376a-3p表达量降低(P<0.05)。与si-NC组比较,si-MIR4435-2HG组MIR4435-2HG表达、细胞活力及CyclinD1、MMP-2、MMP-9蛋白水平降低(P<0.05),迁移及侵袭细胞数减少(P<0.05),细胞凋亡率升高(P<0.05);与miR-NC组比较,miR-376a-3p组细胞活力及CyclinD1、MMP-2、MMP-9蛋白水平降低(P<0.05),迁移及侵袭细胞数减少(P<0.05),miR-376a-3p表达、细胞凋亡率升高(P<0.05)。MIR4435-2HG可靶向调控miR-376a-3p;与si-MIR4435-2HG+anti-miR-NC组比较,si-MIR4435-2HG+anti-miR-376a-3p组细胞活力及CyclinD1、MMP-2、MMP-9蛋白水平升高(P<0.05),迁移及侵袭细胞数增多(P<0.05),miR-376a-3p表达、细胞凋亡率降低(P<0.05)。结论 敲低MIR4435-2HG可通过靶向调控miR-376a-3p进而抑制RBE细胞增殖、迁移、侵袭,并诱导其凋亡。

基于代谢组学研究葫芦素B诱导人肝内胆管癌HuCCT1细胞凋亡的作用机制

目的探究葫芦素B(CuB)诱导人肝内胆管癌HuCCT1细胞凋亡的作用及机制。方法将HuCCT1细胞分为对照组,CuB给药低(50 nmol·L^(-1))、中(100 nmol·L^(-1))、高(200 nmol·L^(-1))剂量组,给予药物干预24 h,采用Annexin V/PI双染法和Hoechst染色法检测细胞凋亡情况。采用超高效液相色谱/四极杆飞行时间质谱(UHPLC-Q/TOF-MS)检测其主要差异代谢物,并分析其相关代谢通路。结果Annexin V/PI双染结果发现,与对照组相比,经CuB给予处理后HuCCT1细胞的凋亡比例从7.86%±0.73%增加到16.53%±0.27%,P<0.0001;Hoechst染色结果发现,与对照组相比,经200 nmol·L^(-1)CuB给予处理24 h后细胞结构被破坏,细胞破碎,边缘轮廓不规则,出现大量核固缩现象;代谢组学分析筛选出了20个显著的差异代谢物,主要涉及的代谢通路包括TCA循环、能量代谢、氨基酸代谢和脂质代谢等。结论CuB具有较好的抗肿瘤作用,可能是通过干扰HuCCT1细胞的多条代谢通路,引起细胞快速增殖的物质缺乏,最终导致细胞凋亡,而发挥药理作用。