Study of multi-drug resistant mechanisms in a taxol-resistant hepatocellular carcinoma QGY-TR 50 cell line

Cancer chemotherapy with taxol often fails due to acquired resistance of cancer cells, which is frequently associated with an overexpression of P-gp and alterations of beta-tubulin. A taxol-resistant cell line, QGY-TR50, derived from a human hepatocellular carcinoma (HCC) QGY-7703 cell line was used to investigate the mechanisms of taxol-resistance. QGY-TR50 cells showed more than 250-fold resistance to taxol and exhibited cross-resistance to other drugs including actinomycin D, doxorubicin, vinblastine, and vincristine. P-gp was highly expressed in QGY-TR50 cells. Expression levels of five human beta-tubulin isotypes (betaI-, betaII-,betaIII-, betaIva, and betaIvb-tubulin) were examined by real-time semi-quantitative PCR. Comparing with QGY-7703 cells, QGY-TR50 cells did not show any significant change in the expression levels of betaI-, betaIva, and betaIvb-tubulin. While a 1.2-fold increased in betaII-tubulin and a 0.5-fold decreased in betaIII-tubulin levels were observed. All results suggest that the P-glycoprotein could be one key factor involved in enhancing drug resistance in QGY-TR50 cells.     

MKi67-siRNA可抑制QGY-7703细胞的生长

为了探讨MKI67在肝癌细胞发生发展中的作用,采用实时定量 PCR 方法检测人肝细胞癌 QGY 7703 细胞中MKI67 基因表达水平, 以及 MKI67在肝细胞癌组织和癌旁正常组织中的表达情况,设计并合成针对MKI67 的siRNA,利用脂质体转染法将其转入QGY-7703 细胞内,通过MTT和细胞集落形成实验观察MKI67-siRNA 对QGY-7703细胞生长活性和增殖能力的影响.实时定量PCR结果表明,MKI67在肝细胞癌组织中的表达水平明显高于癌旁正常组织（P< 0.01）. MTT和细胞集落形成实验结果显示,转染MKI67-siRNA 的QGY-7703细胞生长活性和集落形成率明显低于对照组（P< 0.01）.由此得出结论：MKI67 在肝癌细胞系QGY-7703细胞中的表达水平较高,且它在肝癌组织中的表达水平明显上调. 同时,MKI67-siRNA 可以有效抑制QGY-7703细胞的生长活性和增殖能力,提示MKI67可能与肝细胞癌的发生、发展相关.     

DNA methylation-regulated miR-193a-3p dictates resistance of hepatocellular carcinoma to 5-fluorouracil via repression of SRSF2 expression

Chemoresistance prevents effective cancer therapy and is rarely predictable prior to treatment, particularly for hepatocellular carcinoma (HCC). Following the chemoresistance profiling of eight HCC cell lines to each of nine chemotherapeutics, two cell lines (QGY-7703 as a sensitive and SMMC-7721 as a resistant cell line to 5-fluorouracil (5-FU) treatment) were systematically studied for mechanistic insights underpinning HCC 5-FU chemoresistance. Genomic profiling at both DNA methylation and microRNA (miR) levels and subsequent mechanistic studies illustrate a new mechanism for how DNA methylation-regulated miR-193a-3p dictates the 5-FU resistance of HCC cells via repression of serine/arginine-rich splicing factor 2 (SRSF2) expression. In turn, SRSF2 preferentially up-regulates the proapoptotic splicing form of caspase 2 (CASP2L) and sensitizes HCC cells to 5-FU. Forced changes of miR-193a-3p level reverse all of the phenotypic features examined, including cell proliferation, cell cycle progression, and 5-FU sensitivity, in cell culture and in nude mice. Importantly, the siRNA-mediated repression of SRSF2 phenocopies all of the miR-193a-3p mimic-triggered changes in QGY-7703. This newly identified miR-193a-3p-SRSF2 axis highlights a new set of companion diagnostics required for optimal 5-FU therapy of HCC, which involve assaying both the DNA methylation state of the miR-193a gene and the expression of miR-193a-3p and SRSF2 and the relative level of the proapoptotic versus antiapoptotic splicing forms of caspase 2 in clinical samples.     

AFP的亚细胞定位及对肿瘤细胞生长的影响

目的观察甲胎蛋白（AFP）在不同肿瘤细胞中的亚细胞定位及对肿瘤细胞生长的影响。方法运用免疫荧光的方法观察内源性AFP在HeI。a细胞、QGY-7703细胞、MCF-7细胞中的亚细胞定位。将构建的表达AFP的质粒pcDNA3-AFP及AFP腺病毒siRNA干涉载体Adv—AFPsiRNA作用于QGY-7703细胞,MCF-7细胞,运用M1Tr,集落形成实验检测细胞增殖状况。结果免疫荧光显示,内源性的AFP在HeLa细胞、QGY-7703细胞、MCF-7细胞均只在细胞质中表达。pcDNA3-AFP使QGY-7703的细胞活性增加了2l％（P〈0．05）及集落形成能力增加了32％（P〈0．01）,MCF-7实验组比对照组细胞活性降低了30％（P〈0．01）．克隆形成能力降低82％（P〈0．01）。Adv—AFPsiRNA使QGY-7703的细胞活性降低了22％（P〈0．05）,平均克隆形成能力降低52％（P〈0．01）,MCF-7细胞活性提高了24．5％（P〈0．05）,克隆形成能力提高了89％（P〈O．01）。结论内源性的AFP只在细胞质中表达。AFP能促进QGY-7703细胞的增殖及克隆形成能力,而在MCF-7细胞中发挥相反的作用。腺病毒介导的内源性的AFP表达的下调能降低QGY-7703的增殖,却增加了MCF-7的细胞活性及克隆形成能力。     

MicroRNA-142-3p, a new regulator of RAC1, suppresses the migration and invasion of hepatocellular carcinoma cells

RAC1 regulates a diverse array of cellular events including migration and invasion. MicroRNAs (miRNAs) have a key role in the regulation of gene expression. In this study, we demonstrated that microRNA-142-3p (miR-142-3p) acted as a negative regulator of human RAC1. Overexpression of miR-142-3p decreased RAC1 mRNA and protein levels. Moreover, the overexpression of miR-142-3p suppressed, while blocking of miR-142-3p increased colony formation, migration and invasion in hepatocellular carcinoma (HCC) cell lines (QGY-7703 and SMMC-7721). RAC1 overexpression without the 3'untranslated region abolished the effect of miR-142-3p in the QGY-7703 and SMMC-7721 cells. These results demonstrated that miR-142-3p directly and negatively regulates RAC1 in HCC cells, which highlights the importance of miRNAs in tumorigenesis.     

菹草类胡萝卜素提取物对人肝癌细胞QGY-7703凋亡的影响

Human hepatoma cell line QGY-7703 was treated with the medium containing 10, 20, 40 and 60 micromol/L carotenoids extracts from Potamogoton crispus L. (CEPC) for 48 h, 96 h and 144 h, respectively. The inhibition rates to hepatoma cells were determined by MTT assay. The results showed the average inhibition rates of the three treatments varied from 0.14%-23.07%, 39.59%-70.61% and 71.65%-87.01%, respectively. After hepatoma cells were treated with the medium containing 10, 20 and 40 micromol/L of CEPC for 24 h, 48 h and 72 h, respectively, many typical morphology features of apoptotic cells were observed by Laser Scanning Confocal Microscope (LSCM), such as the distinct decrease in number and volume of cells, the shrinkage and deformation of cells, the shape of cell nucleuses appearing like crescent even piece, the decrease in area of yellow DNA in cell nuclei. The percentage of hepatoma cells in every phase of cell cycle was analyzed by Flow Cytometer (FCM) after being treated with 10 micromol/L and 20 micromol/L CEPC for 48 h. Compared with the control group, the cells in G0/G1 phase increased 23.8% (P<0.01) and 35.6% (P<0.01) respectively, in S phase decreased relevantly, while the cells in G2/M phase had no significant change. The Ca2+ concentration (fluorescence intensity) in cytoplasm of hepatoma cells was determined by LSCM after being treated with 20 micromol/L CEPC for 48 h. The Ca2+ concentration increased significantly (P<0.01) and was 1.5 times that of control group. These results demonstrated that the CEPC inhibited the proliferation of hepatoma cells QGY-7703 significantly in a dose and time-dependent manner in vitro. The hepatoma cells treated with CEPC were evidently arrested at G0/G1 phase in the cell cycle. The concentration of Ca2+ in test group cells cytoplasm increased significantly, it might be the important cause that CEPC induced the apoptosis of hepatoma cells QGY-7703. This paper provided scientific basis for further studying and developing the function and value of carotenoids in Potamogoton crispus L.     

Inhibitory effect of parvovirus H—1 on the formation of colonies of human hepatoma cell line in vitro and its tumors in nude mice

The inhibitory effect of parvovirus H-1 on the colonyforming ability.in vitro of QGY-7703,a cultured human hepatoma cell line,and on the formation and growth of its tumors in nude mice was studied.With higher multiplicity of infection(MOI) of H-1 given,survival of the QGY-7703 cells was found to be decreased.H-1 DNA amplification level at 30h postinfection(p.i.) was detected to be 7.4 times higher than that at 2h by dispersed cells assay,while the cells were delayed to enter into S phase.Plaques were formed in the indicator cells(new-born human kidney cell line,NBK) by progeny H-1 virus particles released from the infected QGY-7703 cells by infectious cell center assay.The formation of tumors in nude mice by QGY-7703 cells which were injected s c at 2h postinfection was observed to by prevented in 2 proups with given MOI 25 and 50.The tumor growth of MOI 10 group occurred at a lower exponential rate than that of control,after a 20d latent period.It was evident that parvovirus H-1 exhibited a direct inhibitory effect on the formation and growth of human hepatoma cells in vivo as well as in vitro.     

Mithramycin inhibits etoposide resistance in glucose-deprived HT-29 human colon carcinoma cells

Physiological cell conditions such as glucose deprivation and hypoxia play roles in the development of drug resistance in solid tumors. These tumor-specific conditions cause decreased expression of DNA topoisomerase IIalpha, rendering cells resistant to topo II target drugs such as etoposide. Thus, targeting tumor-specific conditions such as a low glucose environment may be a novel strategy in the development of anticancer drugs. On this basis, we established a novel screening program for anticancer agents with preferential cytotoxic activity in cancer cells under glucose-deprived conditions. We recently isolated an active compound, AA-98, from Streptomyces sp. AA030098 that can prevent stress-induced etoposide resistance in vitro. Furthermore, LC-MS and various NMR spectroscopic methods identified AA-98 as mithramycin, which belongs to the aureolic acid group of antitumor compounds. We found that mithramycin prevents the etoposide resistance that is induced by glucose deprivation. The etoposide-chemosensitive action of mithramycin was just dependent on strict low glucose conditions, and resulted in the selective cell death of etoposide-resistant HT-29 human colon cancer cells.     

Actin disruption inhibits endosomal traffic of P-glycoprotein-EGFP and resistance to daunorubicin accumulation

Intracellular traffic of human P-glycoprotein (P-gp), a membrane transporter responsible for multidrug resistance in cancer chemotherapy, was investigated using a P-gp and enhanced green fluorescent fusion protein (P-gp-EGFP) in human breast cancer MCF-7 cells. The stably expressed P-gp-EGFP from a clonal cell population was functional as a drug efflux pump, as demonstrated by the inhibition of daunorubicin accumulation and the conferring of resistance of the cells to colchicine and daunorubicin. Colocalization experiments demonstrated that a small fraction of the total P-gp-EGFP expressed was localized intracellularly and was present in early endosome and lysosome compartments. P-gp-EGFP traffic was shown to occur via early endosome transport to the plasma membrane. Subsequent movement of P-gp-EGFP away from the plasma membrane occurred by endocytosis to the early endosome and lysosome. The component of the cytoskeleton responsible for P-gp-EGFP traffic was demonstrated to be actin rather than microtubules. In functional studies it was shown that in parallel with the interruption of the traffic of P-gp-EGFP, cellular accumulation of the P-gp substrate daunorubicin was increased after cells were treated with actin inhibitors, and cell proliferation was inhibited to a greater extent than in the presence of daunorubicin alone. The actin dependence of P-gp traffic and the parallel changes in cytotoxic drug accumulation demonstrated in this study delineates the pathways of P-gp traffic and may provide a new approach to overcoming multidrug resistance in cancer chemotherapy. protein traffic; drug resistance in cancer; daunorubicin     

Glucose deprivation increases nuclear DNA repair protein Ku and resistance to radiation induced oxidative stress in human cancer cells

Recent studies have indicated that nutrient deprivation particularly glucose may play a major role in tumor cell tolerance to a generally oxidative stress environment in solid tumors. Here, we studied the impact of glucose deprivation on the response of human colon (HT29) and prostate (DU145) cancer cells to γ radiation. A significant decrease in intracellular glucose level was observed in glucose deprived cells as measured by bioreductive assay. The survival of HT29 and DU145 were increased by 30 and 100% respectively when these cells were exposed to γ radiation in the absence of glucose compared to that in the presence of glucose. In glucose depleted medium, glutathione (GSH), a free radical scavenger, content remained the same, and showed no correlation with the radiation resistance induced by glucose deprivation. Glucose regulated protein78 (GRP78), a stress response survival protein, was not significantly increased in cells deprived of glucose for 4 h compared to those cells in glucose. DNA repair protein Ku, which is known to play a major role in cellular resistance to radiation, was significantly increased in glucose deprived cancer cells that showed enhanced radiation resistance. These results have demonstrated, for the first time, that glucose deprivation mediated stress increased the expression of nuclear Ku and resistance to radiation induced oxidative stress in human cancer cells. The additional resistance caused by glucose deprivation in cancer cells has clinical significance since solid tumors are known to have low level of glucose due to diffusion limited blood supply and higher metabolic activity. Copyright © 2009 John Wiley & Sons, Ltd.     

grp75对细胞缺糖损伤的保护作用

为研究ｇｒｐ７５的功能,对过表达ｇｒｐ７５的ＣＨＬ细胞进行了无糖培养以施加能量代谢应激,运用台盼蓝染色计数、ＬＤＨ释放测定和流式细胞术等方法评估其损伤程序。结果显示,无糖培养５ｈ,过表达ｇｒｐ７５细胞和对照组细胞比较,细胞活率、亚二倍体细胞率均无明显差别;无糖培养１０ｈ,过表达ｇｒｐ７５细胞的活率高于对照组（Ｐ〈０．０１）,亚二倍体细胞率低于对照组（Ｐ〈０．０５）;无糖培养至２０ｈ,两组细胞活率和     

Elevated Pretherapy Serum IL17 in Primary Hepatocellular Carcinoma Patients Correlate to Increased Risk of Early Recurrence after Curative Hepatectomy

Background and Aims

Primary hepatocellular carcinoma (HCC) is usually presented in inflamed fibrotic/cirrhotic liver with extensive lymphocyte infiltration. We examined the associations between the HCC early recurrence and alterations in serum levels of inflammatory cytokines.

Methods

A cohort of 105 HCC patients with chronic hepatitis B virus infection were included. Pre-therapy, we quantified their serum concentrations of Th1-, Th2-, Th17-, Treg-related, and other cytokines that have been reported to be associated with poor prognosis in human cancers. IL17-producing T-cells were generated in vitro from HCC patients and co-cultured with HCC cell lines separated by a 0.4 µM transwell.

Results

All the 105 cases of HCC patients had liver cirrhosis. The patients who suffered from HCC early recurrence had higher pre-therapy serum levels of IL17 and lower levels of IL10 than those who did not suffer from recurrence after curative hepatectomy. After adjustment for general tumor clinicopathological factors, elevated serum levels of IL17 (≥0.9 pg/ml) was found to be an independent risk factor for HCC early recurrence with a hazard ratio of 2.46 (95%CI 1.34–4.51). Patients with bigger tumors (>5 cm in diameter) and elevated serum levels of IL17 had the highest risk of early recurrence as compared to those with only one of these factors (P = 0.009) or without any (P<0.001). These factors showed similar effects on the HCC patient overall survival. Intrahepatic infiltrated T-cells in HCC patients were identified as the major IL17-producing cells. Proliferation of HCC cells, QGY-7703, was augmented QGY-7703, was augmented in the presence of IL17-producing T-cells. This effect diminished after neutralizing antibody against human IL17A or TNFα was included.

Conclusion

Both tumors and IL17 from liver infiltrated T-cells contributed to HCC early recurrence and progression after curative resection. Pre-therapy serum IL17 levels may serve as an additional indicator for predicting high-risk patients.     

Baohuoside I Inhibits the Proliferation of Hepatocellular Carcinoma Cells via Apoptosis Signaling and NF-kB Pathway

Baohuoside I is a flavonoid isolated from Epimedium koreanum Nakai and has many pharmacological activities. However, its role in liver cancer remains unclear. This study aimed to investigate the inhibitory effect of Baohuoside I on the Human Hepatocellular Carcinoma (HCC) cell lines QGY7703, and underlying mechanisms. QGY7703 cells were used as the model to assess the function of Baohuoside I in vitro. The effects of Baohuoside I on QGY7703 cells’ growth, proliferation, and invasiveness were confirmed by CCK-8, lactate dehydrogenase release, and invasion assays. Cell apoptosis was analyzed by flow cytometry, and the levels of cleaved Caspase-3, Bax, and Bcl-2 were quantified by western blot. Western blot analysis, nuclear translocation of NF-κB, and Q-PCR were used to measure the expression of affected molecules. In QGY7703 cells, Baohuoside I induced the expression of molecules related to NF-κB pathway. The toxicity of Baohuoside I on QGY7703 cells was also confirmed in vivo, in a tumor model. Baohuoside I had a significant toxic effect on QGY7703 cells from a concentration of 10 μM. This compound significantly inhibited the proliferation of QGY7703 cells by inducing apoptosis and downregulating NF-κB signaling pathway. Thus, Baohuoside I is a novel candidate drug and opens new possibilities of clinical strategies for HCC treatment.     

p27 modulates cell cycle progression and chemosensitivity in human malignant glioma.

The cell cycle regulatory protein p27, an inhibitor of cyclin-dependent kinases (CDK), has been attributed a role in (i) prognosis in breast and colon cancer, (ii) induction of apoptosis in cancer cells, and (iii) resistance to cancer chemotherapy. Here we report that p27 is widely expressed in human malignant gliomas in vivo and in glioma cell lines in vitro. Serum deprivation or confluency promotes p27 protein accumulation in vitro. Neither baseline p27 levels nor p27 levels induced by confluency or serum deprivation correlate with p53 status or drug sensitivity of human glioma cell lines. Expression of antisense p27 mRNA increased the doubling times in T98G glioma cells, whereas sense p27 mRNA had no such effect. There was a density-dependent and drug-specific modulation of chemosensitivity by sense or antisense mRNA expression in T98G cells. Taken together, these data define a strong p27 response to altered growth conditions and suggest a role for p27 in modulating response to chemotherapy in human malignant glioma cells.     

Resistance to hypoxia-induced necroptosis is conferred by glycolytic pyruvate scavenging of mitochondrial superoxide in colorectal cancer cells

Cancer cells may survive under oxygen and nutrient deprivation by metabolic reprogramming for high levels of anaerobic glycolysis, which contributes to tumor growth and drug resistance. Abnormally expressed glucose transporters (GLUTs) are colocalized with hypoxia (Hx) inducible factor (HIF)1α in peri-necrotic regions in human colorectal carcinoma. However, the underlying mechanisms of anti-necrotic resistance conferred by glucose metabolism in hypoxic cancer cells remain poorly understood. Our aim was to investigate signaling pathways of Hx-induced necroptosis and explore the role of glucose pyruvate metabolite in mechanisms of death resistance. Human colorectal carcinoma cells were Hx exposed with or without glucose, and cell necroptosis was examined by receptor-interacting protein (RIP)1/3 kinase immunoprecipitation and ³²P kinase assays. Our results showed increased RIP1/3 complex formation and phosphorylation in hypoxic, but not normoxic cells in glucose-free media. Blocking RIP1 signaling, by necrostatin-1 or gene silencing, decreased lactodehydrogenase (LDH) leakage and plasma membrane disintegration. Generation of mitochondrial superoxide was noted after hypoxic challenge; its reduction by antioxidants inhibited RIP signaling and cell necrosis. Supplementation of glucose diminished the RIP-dependent LDH leakage and morphological damage in hypoxic cells, whereas non-metabolizable sugar analogs did not. Hypoxic cells given glucose showed nuclear translocation of HIF1α associated with upregulation of GLUT-1 and GLUT-4 expression, as well as increase of intracellular ATP, pyruvate and lactate levels. The glucose-mediated death resistance was ablated by iodoacetate (an inhibitor to glyceraldehyde-3-phosphate dehydrogenase), but not by UK5099 (an inhibitor to mitochondrial pyruvate carrier), suggesting that glycolytic pathway was involved in anti-necrotic mechanism. Lastly, replacing glucose with cell-permeable pyruvate derivative also led to decrease of Hx-induced necroptosis by suppression of mitochondrial superoxide in an energy-independent manner. In conclusion, glycolytic metabolism confers resistance to RIP-dependent necroptosis in hypoxic cancer cells partly through pyruvate scavenging of mitochondrial free radicals.     

Reversal of P-glycoprotein expressed in Escherichia coli leaky mutant by ascorbic acid

It has been reported that functional expression of the multidrug resistance protein P-glycoprotein (P-gp) in E. coli is useful for screening P-gp substrates and inhibitors. In the present study, we have constructed by nitrosoguanidine and UV mutagenesis 28 leaky mutants of E. coli UT5600. These mutants are significantly susceptible to the toxic effect of known P-gp substrates and lipophilic cancer drugs. Mouse mdr1 was functionally expressed in the most permeable E. coli mutant (UTP17). Expression of P-gp in this mutant confers cross-resistance to mitomycin C, tegafur, daunorubicin, rhodamine 6G, tetraphenylphosphonium bromide and ciprofloxacin. To examine the reversal of P-gp expressed in this heterologous system, UTP17 cells expressing mouse mdr1 or lac permease as negative control were treated with various concentrations of mitomycin C with or without ascorbic acid. We found that ascorbic acid abrogated P-gp mediated multidrug resistance, suggesting that ascorbic acid might be used in combination with anticancer drugs to reduce emergence of multidrug resistance. We also demonstrated that tomato lectin antagonized the inhibitory action of ascorbic acid. This study provide a heterologous system for mdr1 expression in E. coli leaky mutant that can be used as a system for the screening of P-gp inducers and inhibitors, since it is quick and simple.     

Anti-Tumor Effect of a Novel Soluble Recombinant Human Endostatin: Administered as a Single Agent or in Combination with Chemotherapy Agents in Mouse Tumor Models

Background

Angiogenesis has become an attractive target in cancer treatment. Endostatin is one of the potent anti-angiogenesis agents. Its recombinant form expressed in the yeast system is currently under clinical trials. Endostatin suppresses tumor formation through the inhibition of blood vessel growth. It is anticipated that combined therapy using endostatin and cytotoxic compounds may exert an additive effect. In the present study, we expressed and purified recombinant human endostatin (rhEndostatin) that contained 3 additional amino acid residues (arginine, glycine, and serine) at the amino-terminus and 6 histidine residues in its carboxyl terminus. The recombinant protein was expressed in E. Coli and refolded into a soluble form in a large scale purification process. The protein exhibited a potent anti-tumor activity in bioassays. Furthermore, rhEndostatin showed an additive effect with chemotherapy agents including cyclophosphamide (CTX) and cisplatin (DDP).

Methods

rhEndostatin cDNA was cloned into PQE vector and expressed in E. Coli. The protein was refolded through dialysis with an optimized protocol. To establish tumor models, nude mice were subcutaneously injected with human cancer cells (lung carcinoma A549, hepatocellular carcinoma QGY-7703, or breast cancer Bcap37). rhEndostatin and/or DDP was administered peritumorally to evaluate the rate of growth inhibition of A549 tumors. For the tumor metastasis model, mice were injected intravenously with mouse melanoma B16 cells. One day after tumor cell injection, a single dose of rhEndostatin, or in combination with CTX, was administered intravenously or at a site close to the tumor.

Results

rhEndostatin reduced the growth of A549, QGY-7703, and Bcap37 xenograft tumors in a dose dependent manner. When it was administered peritumorally, rhEndostatin exhibited a more potent inhibitory activity. Furthermore, rhEndostatin displayed an additive effect with CTX or DDP on the inhibition of metastasis of B16 tumors or growth of A549 tumors.

Conclusion

Soluble rhEndostatin exhibits a potent anti-tumor activity in mouse xenograft models and it also has an additive effect with CTX and DDP, implying possible applications in clinical settings.