Gemcitabine-resistant Cell Lines Research Articles

Abstract LB-22: Gemcitabine resistance is associated with EMT phenotype along with expression of cancer stem cell markers and a specific miRNA profile in the human pancreatic cancer cell line BxPC3

Abstract Despite new therapeutic strategies, human pancreatic ductal adenocarcinomas (PDAC) remain one of the greatest oncological challenges, as the rate of incidence almost equals the rate of mortality with a 5-year survival of less than 5%. Gemcitabine is the standard therapy for PDAC and is mostly used alone or in combination with an EGFR inhibitor. While tumors respond to Gemcitabine treatment initially, they generally develop chemoresistance. A thorough understanding of molecular events that promote resistance against Gemcitabine may identify pathways that can be efficiently targeted to reverse chemoresistance. A cell line model resistant to Gemcitabine was generated by transiently exposing BxPC3 cells to increasing concentrations of Gemcitabine. This Gemcitabine resistant cell line, BxPC3-GzR, was compared with untreated BxPC3 control cells. BxPC3-GzR cells showed properties of undergoing epithelial to mesenchymal transition (EMT) and these included a spindle shaped morphology, an increase in expression of vimentin and a decrease in expression of E-cadherin. BxPC3-GzR cells also showed an increase of expression of CD44 and in expression of phosphorylated STAT3TY705, which are characteristic of cancer stem cells. BxPC3 and BxPC3-GzR where further compared by microarray analyses for expression of micro-RNAs (miRNAs). BxPC3-GzR cells showed a specific miRNA expression profile (9 positively and 8 negatively regulated). Thus far seven of eight miRNAs identified as altered in BxPC3-GzR cells were validated by real-time RT-PCR (miR-15b, miR-21, miR-100, miRNA -125b, miRNA-155, miRNA-205 and miRNA-455-3p). These studies suggest that Gemcitabine treatment of PDAC selects for a resistant population of tumor cells that have a mesenchymal/stem cell-like phenotype and that express a distinct miRNA profile. Targeting these miRNAs may provide novel targets for restoring or enhancing anti-tumor response of Gemcitabine. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-22. doi:1538-7445.AM2012-LB-22

Abstract 798: Id1 (Inhibitor of differentiation-1) expression demonstrates important translational implications for pancreatic adenocarcinoma

Abstract Introduction: Inhibitor of differentiation-1 (Id1) demonstrates a role in tumor progression in a variety of tumors but scarce data exists for pancreatic adenocarcinoma. Previously, we demonstrated that nicotine, the addicting component of tobacco, induces Id1 expression through a Src-dependent signaling pathway promoting proliferation, invasion, and chemoresistance in pancreatic cancer cells. Conversely, silencing of Id1 re-establishes gemcitabine sensitivity in acquired and innate gemcitabine-resistant cell lines. Therefore, we determined the role of Id1 in tumor progression and chemoresistance in vivo and determined the expression status of Id1 in a large cohort of human pancreatic cancer tissues. Methods: Cell lysate analyses (protein analyses, mRNA expression) were determined in a bio-diverse sample of pancreatic cancer cells by western and RT-PCR. Metastatic pancreatic cancer cells were stably transfected with a luciferase expression vector and stable short-hairpin RNA silencing of Id1 gene expression with controls. Orthotopic nude mouse model utilized intraperitoneal injections of nicotine (1mg/kg, M/W/F) and gemcitabine (250 mg/kg, T/Th) with monitoring of in vivo growth and metastases based on luminescent signal concentration. Confirmation of primary tumor and metastases was confirmed by H&E staining. Tissue microarrays (TMAs) constructed from 100 resected pancreatic adenocarcinoma were stained for Id1, phospho-Src, and total Src using immunohistochemistry techniques. Scores based on intensity, percent of positive cells, and pathological grade (1-4) were assigned to each malignant core. Results: In vivo, nicotine significantly promoted tumor growth and metastases in vivo. While gemcitabine reduced the tumor burden in control mice, nicotine abrogates this inhibitory effect. Additionally, knockdown of Id1 results in a significant decrease in tumor burden with no identifiable liver metastases, regardless of nicotine exposure. Analyses of resected pancreatic tumor TMAs demonstrated a statistically significant correlation between Id1 and tumor grade/differentiation (p =0.0368). The correlation of protein levels of Id1 and phospho-Src was also statistically significant (p =0.0024); Comparison of overall survival and tumor grade was also significant (p = 0.05); the correlation between overall-survival and Id1 expression trended toward statistical significance (p =0.06). Conclusions: Id1, through a Src-dependent-Id1 signaling axis, promotes tumorigenesis and chemoresistance in an in vivo pancreatic cancer model. Id1 expression correlates with phospho-Src levels and tumor differentiation in human pancreatic cancer tissues. These results support the clinical assessment of Id1 expression as a biomarker of prognosis and support investigations toward targeted therapies against Id1 for pancreatic cancer patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 798. doi:1538-7445.AM2012-798

DCK is frequently inactivated in acquired gemcitabine-resistant human cancer cells

Although gemcitabine is the most effective chemotherapeutic agent against pancreatic cancer, a growing concern is that a substantial number of patients acquire gemcitabine chemoresistance. To elucidate the mechanisms of acquisition of gemcitabine resistance, we developed gemcitabine-resistant cell lines from six human cancer cell lines; three pancreatic, one gastric, one colon, and one bile duct cancer. We first analyzed gemcitabine uptake using three paired parental and gemcitabine resistant pancreatic cancer cell lines (PK-1 and RPK-1, PK-9 and RPK-9, PK-59 and RPK-59) and found that uptake of gemcitabine was rapid. However, no DNA damage was induced in resistant cells. We further examined the microarray-based expression profiles of the cells to identify genes associated with gemcitabine resistance and found a remarkable reduction in the expression of deoxycytidine kinase (DCK). DCK is a key enzyme that activates gemcitabine by phosphorylation. Genetic alterations and expression of DCK were studied in these paired parental and derived gemcitabine-resistant cell lines, and inactivating mutations were found only in gemcitabine-resistant cell lines. Furthermore, siRNA-mediated knockdown of DCK in the parental cell lines yielded gemcitabine resistance, and introduction of DCK into gemcitabine-resistant cell lines invariably restored gemcitabine sensitivities. Mutation analyses were expanded to three other different paired cell lines, DLD-1 and RDLD-1 (colon cancer cell line), MKN-28 and RMKN-28 (gastric cancer cell line), and TFK-1 and RTFK -1 (cholangiocarcinoma cell line). We found inactivating mutations in RDLD-1 and RTFK-1 and decreased expression of DCK in RMKN-28. These results indicate that the inactivation of DCK is one of the crucial mechanisms in acquisition of gemcitabine resistance.

Gene Expression Analysis In Gemcitabine Resistant Cells Derived From Human Pancreatic Cancer Cell

To analyze the expression of genes involved in gemcitabine resistance formation in gemcitabineresistant cells derived from human pancreatic cancer cell, we established several cell lines with different acquired resistance to gemcitabine from pancreatic cancer cell line MIA-PaCa2, and analyzed the expression of the genes that are considered to be associated with the resistance to gemcitabine using PCR and real-time PCR. We found that in these gemcitabine-resistant cell lines, the expression of human equilibrative nucleoside transporter 2 (hENT2) and deoxycytidylate deaminase (DCTD) was lower than that of the parental cell line MIA-PaCa2 in the earlier stage of the cells incubated with 300nM gemcitabine; and, in the later stage, the expression of hENT2 recovered, but the expression levels of ribonucletide reductase subunit M1 (RRM1) and M2 (RRM2) were increased. These findings suggest that the decreased accumulation of gemcitabine and the increased expression of RRM1 and RRM2 are involved in gemcitabine-resistance formation in MIA-PaCa2.

Emodin reverses gemcitabine resistance in pancreatic cancer cells via the mitochondrial apoptosis pathway in vitro

Gemcitabine resistance is a common problem of pancreatic cancer chemotherapy, and how to reverse it plays an important role in the treatment of pancreatic cancer. This study investigated the effect of emodin on the gemcitabine-resistant pancreatic cancer cell line SW1990/Gem, and explored the potential mechanism of its action. SW1990/Gem was obtained by culture of the pancreatic cancer cell line SW1990 in vitro by intermittently increasing the concentration of gemcitabine in the culture medium for 10 months, observing the morphology using inverted microscopy. SW1990/Gem cells were pretreated with emodin (10 μM) for different periods followed by treatment with gemcitabine (20 μM) for 48 h; cell proliferation was tested by MTT assay. SW1990/Gem cells were treated by emodin with different concentrations for 48 h, cell apoptosis was detected by flow cytometry (FCM). The expression of gene and protein, such as MDR-1 (P-gp), NF-κB, Bcl-2, Bax, cytochrome-C (cytosol), caspase-9 and -3 were measured by RT-PCR and Western blotting. The function of P-gp in SW1990/Gem cells was checked by FCM. The results showed that the SW1990/Gem cells changed greatly in morphology and the resistance index was 48.63. Emodin promoted cell apoptosis of the gemcitabine-resistant cell line SW1990/Gem in a dose-dependent manner. Emodin enhanced the SW1990/Gem cell sensitivity to gemcitabine in a time-dependent manner. Emodin monotherapy or combination with gemcitabine both decreased the gene and protein expression levels of MDR-1 (P-gp), NF-κB and Bcl-2 and inhibited the function of P-gp, but increased the expression levels of Bax, cytochrome-C (cytosol), caspase-9 and -3, and promoted cell apoptosis. This demonstrated that emodin had a reversing effect on the gemcitabine-resistant cell line SW1990/Gem, possibly via decreasing the function of P-gp and activating the mitochondrial apoptosis pathway in vitro.

Stearoyl gemcitabine nanoparticles overcome resistance related to the over-expression of ribonucleotide reductase subunit M1

Gemcitabine is a deoxycytidine analog used in the treatment of various solid tumors. However, tumors often develop resistances over time, which becomes a major issue for most gemcitabine-related chemotherapies. In the present study, a previously reported stearoyl gemcitabine nanoparticle formulation (GemC18-NPs) was evaluated for its ability to overcome gemcitabine resistance. In the wild type CCRF-CEM human leukemia cells, the IC 50 value of GemC18-NPs was 9.5-fold greater than that of gemcitabine hydrochloride (HCl). However, in the CCRF-CEM-AraC-8C cells that are deficient in the human equilibrative nucleoside transporter-1, the IC 50 of GemC18-NPs was only 3.4-fold greater than that in the parent CCRF-CEM cells, whereas the IC 50 of gemcitabine HCl was 471-fold greater than that in the parent CCRF-CEM cells. The GemC18-NPs were also more cytotoxic than gemcitabine HCl in the deoxycytidine kinase deficient (CCRF-CEM/dCK −/−) tumor cells. Similar to gemcitabine HCl, GemC18-NPs induced apoptosis through caspase activation. Another gemcitabine-resistant tumor cell line, TC-1-GR, was developed in our laboratory. In the TC-1-GR cells, the IC 50 of GemC18-NPs was only 5% of that of gemcitabine HCl. Importantly, GemC18-NPs effectively controlled the growth of gemcitabine resistant TC-1-GR tumors in mice, whereas the molar equivalent dose of gemcitabine HCl did not show any activity against the growth of the TC-1-GR tumors. Proteomics analysis revealed that the TC-1-GR cells over-expressed ribonucleotide reductase M1, which was likely the cause of the acquired gemcitabine resistance in the TC-1-GR cells. To our best knowledge, this represents the first report demonstrating that a nanoparticle formulation of gemcitabine overcomes gemcitabine resistance related to ribonucleotide reductase M1 over-expression.

Cyclopamine reverts acquired chemoresistance and down-regulates cancer stem cell markers in pancreatic cancer cell lines

The hedgehog (Hh) pathway has been implicated in the pathogenesis of cancer including pancreatic ductal adenocarcinoma (PDAC). Recent studies have suggested that Hh plays an important role in maintaining the cancer stem cell (CSCs) pool. Gemcitabine-resistant pancreatic cancer cells highly express some of the CSCs markers. However, the expression level of Hh members in gemcitabine-resistant pancreatic cancer cells remains unknown. The aim of this study was to verify the expression of HH members, such as Shh, Ptc, SMO and Gli-1 in gemcitabine-resistant PDAC cell lines, and to explore a new strategy to overcome chemoresistance in PDAC. Quantitative real-time RT-PCR (Q-PCR) and western blot were used to evaluate the relative expression level of HH members in SW1990, CFPAC-1 cells and gemcitabine-resistant SW1990, CFPAC-1 cells. The change of cancer stem cell markers and the expression level of HH members before and after cyclopamine treatment was evaluated using flow cytometry and Q-PCR, western blot, respectively. Cell apoptosis after cyclopamine treatment was measured by flow cytometry. CD44, CD133 and the expression level of HH members, including Shh, SMO, Gli-1, were found to be highly expressed in gemcitabine-resistant cells, which were significantly down-regulated by cyclopamine treatment. Flow cytometry analysis showed increased cell apoptosis after cyclopamine treatment. Gemcitabine-resistant pancreatic cancer cells highly express CSCs markers and some of the HH members, and inhibition of HH by cyclopamine is an effective method of reversing gemcitabine resistance in pancreatic cancer.

Abstract 1747: Biochanin A overcomes gemcitabine resistance in pancreatic cancer cells

Abstract Drug resistance is one of the major reasons for failure of chemotherapy and high mortality rate in pancreatic cancer. Gemcitabine (GEM) and 5-fluorouracil are two drugs which have shown to improve the survival of pancreatic cancer patients but intrinsic or acquired characteristics make this cancer resistant to these drugs. Previously, we have observed that biochanin A (bioA) effectively reduces the survival, signaling and progression of pancreatic cancer cells. In this study, we aim at determining the effect of bioA on drug resistant pancreatic cancer cells. To test our hypothesis, first we compared three pancreatic cancer cells line: Panc1, MiaPaCa2 and AsPC1 for their sensitivity to gemcitabine and protein expressions. We found that Panc1 cells are highly resistant to GEM with IC50 of 75µM while the same for MiaPaCa2 and AsPC1 cells was 0.04µM. Our western blot data show that the levels of NFkappaB, EGFR and CD133 were higher in Panc1 cells compared to other two cell lines suggesting the role of these factors in imparting drug resistant characteristics to pancreatic cancer cells. Our cell survival studies show that bioA is more effective in reducing the survival of Panc1 cells compared to the other two cell lines. Next, we selected GEM resistant cells (AsPC/G) by continuously exposing AsPC1 cells with increasing concentration of GEM. We generated a GEM resistant cell line that was 20-fold resistant to GEM compared to the parent cell line. AsPC/G cells had higher colony formation ability and in vivo tumorigenicity compared to the parent cells. Also, the expression of p-EGFR, NFκB, p-Akt and EMT markers were upregulated in AsPC/G cell compared to AsPC1 cells. We found that bioA was more cytotoxic to AsPC/G cells compared to AsPC1 cells suggesting that bioA affects the molecular mechanisms which impart drug resistant characteristics. To further understand this phenomenon, we analyzed the molecular mechanisms affected by bioA. We observed that bioA reduces the levels of p-EGFR, p-Akt and NFκB in AsPC1 cells. Upregulation of these proteins is known to impart drug resistant characteristics to pancreatic cancer cells; therefore, by inhibiting them, bioA reduces the survival of GEM resistant pancreatic cancer cells. Our study concludes that bioA reduces the survival of GEM resistant pancreatic cancer cells and a treatment regimen with bioA may improve the outcome of chemotherapy in pancreatic cancer patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1747. doi:10.1158/1538-7445.AM2011-1747

Abstract 1724: Inhibition of the PI3K and mTOR pathways reverses gemcitabine resistance in pancreatic cancer

Abstract BACKGROUND: Despite chemotherapy, survival for most patients with pancreatic cancer is less than one year. These dismal statistics reflect the lack of effective therapy. Understanding the molecular pathways that drive cancer growth may help target our therapy. Over 90% of pancreatic cancers are K-ras mutant which activates the PI3K/AKT pathway and signals downstream to mTOR leading to cell growth, proliferation and survival. We explored the combination of gemcitabine and BEZ235, a novel dual PI3K/mTOR inhibitor, in pancreatic cancer cell lines. METHODS: Cytotoxicity assays were performed utilizing the CellTiter-96-Aqueous-One-Solution-Cell-Proliferation-Assay-(MTS) from Promega in K-ras mutant MIA PaCa-2 pancreatic cancer cell lines. We generated gemcitabine resistant cell lines by serial passage of cells with increasing concentrations of drug. We utilized the CalcuSyn program by Biosoft to quantify synergism or antagonism with drug combinations. We also collected cells 6 hours after drug treatment for analysis of phosphorylated proteins. RESULTS: MIA PaCa-2 wild-type and gemcitabine resistant cells were treated with 100, 50 and 25 nM of either gemcitabine, BEZ235 or the combination of both. At the 100 nM dose, cell viability of the wild-type cells was 66%, 55% and 39% respectively. In the gemcitabine resistant cells, cell viability was 84%, 63% and 44%. The combination index was 0.51 in the wild-type cells and 0.46 in the gemcitabine resistant cells indicating synergy in both cell lines. In both cell lines, p-S6K and p-AKT levels increased with gemcitabine treatment which was abrogated by treatment with BEZ-235. Interestingly, in the gemcitabine resistant cells, there appeared to be significantly increased levels of p-MEK1 compared to control. CONCLUSION: Because pancreatic cancer remains resistant to current therapies despite recent advances in the development of molecular targeted therapies, there is an urgency to understand these pathways. We showed that the combination of gemcitabine and BEZ235 is synergistic in both cell lines but cell viability still remains high at 39% and 44% respectively. Combination with a MEK inhibitor may help to increase cell apoptosis and studies are on-going. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1724. doi:10.1158/1538-7445.AM2011-1724

Gene expression analysis for predicting gemcitabine resistance in human cholangiocarcinoma

Gemcitabine is a promising drug for cholangiocarcinoma treatment. However, the kinetics and metabolism of this drug in cholangiocarcinoma treatment are not well defined. We aimed to investigate the potential clinical role of gemcitabine metabolism-related genes in the gemcitabine sensitivity of cholangiocarcinoma and identify and characterize novel gemcitabine resistance-related genes. Expressions of genes related to gemcitabine sensitivity and gemcitabine metabolism were measured in 10 cholangiocarcinoma cell lines, and the association between gene expression and gemcitabine sensitivity was evaluated. Furthermore, gemcitabine-resistant cell lines were established from YSCCC cells and subjected to genome-wide microarray analysis. The 2-fold upregulated and downregulated genes were then subjected to pathway analysis. p53R2 mRNA expression was significantly higher in gemcitabine-resistant cell lines (IC(50) > 1000 nM), and all subunits of ribonucleotide reductase were upregulated in the established gemcitabine-resistant cell lines. Microarray analysis revealed that the upregulated genes in the resistant cells belonged to the glutathione and pyrimidine metabolism pathways, and that the downregulated genes belonged to the N-glycan biosynthesis pathway. Increased expression of p53R2 may predict gemcitabine resistance, and upregulated RNR activity may influence gemcitabine resistance in cholangiocarcinoma cells. Glutathione pathway-related genes were induced by continuous exposure to gemcitabine and may contribute to gemcitabine resistance.

Genexol inhibits primary tumour growth and metastases in gemcitabine-resistant pancreatic ductal adenocarcinoma

IntroductionGemcitabine, the current standard of care for pancreatic ductal adenocarcinoma (PDA), has a less than 10% partial response rate. Genexol-PM, a modified form of paclitaxel, has been shown to have antitumour effects in clinical trials of metastatic breast and small-lung-cell carcinoma. The aim of the present study was to determine if Genexol would be a beneficial treatment for gemcitabine-resistant PDA. Materials and methodsWe measured the in vitro IC50s of gemcitabine and genexol in cell lines sensitive and resistant to gemcitabine. In vivo, animals with orthotopic pancreatic tumours, resistant to gemcitabine, were treated with phosphate-buffered saline (PBS), gemcitabine, Genexol or gemcitabine+Genexol. Tumour progression was monitored using red fluorescent protein imaging. ResultsWe showed equivalent IC50s for gemcitabine-sensitive and gemcitabine-resistant cell lines when treated with genexol. In vivo treatment with genexol resulted in a greater per cent reduction in tumour size, less metastatic spread and longer survival compared with treatment with gemcitabine. DiscussionGenexol proved to be an effective treatment for gemcitabine-resistant PDA. These data combined with the successful clinical use of genexol in Phase II trials of other malignancies suggests it maybe an effective treatment for pancreatic cancer, specifically for those patients resistant to gemcitabine.

Establish a gemcitabine-resistant pancreatic cancer cell line SW1990/GZ and research the relationship between SW1990/GZ and pancreatic cancer stem cell

To establish a gemcitabine-resistant pancreatic cancer cell line SW1990/GZ, and to explore the relationship between drug-resistant cell line SW1990/GZ and pancreatic cancer stem cell. Gemcitabine-resistant pancreatic cancer cell line SW1990/GZ was obtained by treating parental cell line SW1990 in vitro with increasing dosage of gemcitabine in culture medium intermittently for 24 weeks. Stable cultures were obtained which were 77.2-fold increased in resistance relative to parental cells. Gene expressions of ABCB1/MDR1, ABCC1/MRP and ABCG2/BCRP were determined by real-time PCR. Tumorigenic potential was performed by nude mice xenograft transplant experiments. Side population analysis and CD24CD44 positive cells explore were determined by flow cytometry to examine cancer stem cell proportion. Gemcitabine-resistant cell line SW1990/GZ underwent obvious morphological and functional changes. Compared with the parental cell line, SW1990/GZ cell was small and turned into round shape. SW1990/GZ had a higher gene expression level of ABCB1/MDR1, ABCC1/MRP and ABCG2/BCRP than SW1990 (P < 0.01). Nude mice xenograft transplant experiments showed that only 1 × 10(5) SW1990/GZ cells were sufficient for tumor formation, whereas an injection of 1 × 10(5) SW1990 cells did not initiate tumors. Flow cytometry analysis showed that SP proportion in SW1990/GZ was (11.0 ± 1.0)%, whereas in parental SW1990 it was (4.6 ± 0.9)%, CD44CD24 positive cells was (8.73 ± 0.81)% in SW1990/GZ, whereas (1.1 ± 0.4)% in SW1990. Gemcitabine-resistant cell line SW1990/GZ has a higher proportion of pancreatic cancer stem cells compared to its parental cell line SW1990. CD44 is mainly responsible for acquired drug resistance, which can be a potential target to overcome acquired drug resistance in pancreatic cancer.

Identifying Molecular Markers for Chemosensitivity to Gemcitabine in Pancreatic Cancer

Gemcitabine is the standard chemotherapeutic agent for pancreatic cancer. Nevertheless, the prognosis of pancreatic cancer patients is still poor. Evaluating the mechanisms of chemoresistance to gemcitabine will be helpful for the improvement of the therapeutic outcome. Using 11 pancreatic cancer cell lines and global gene expression profiling, molecular markers were detected for acquired and intrinsic gemcitabine sensitivity. Acquired gemcitabine resistance in vitro was obtained by continual exposure to gradually increased concentrations of gemcitabine; however, intrinsic sensitivity is originally provided and differs between cell lines. Microarray analysis of intrinsic sensitivity showed no correlation to that of acquired resistance. Fifteen overexpressed and 49 downexpressed genes in accordance with intrinsic gemcitabine resistance were identified, and we selected those highly expressed in resected pancreatic cancer tissue. Interferon-stimulated gene 15 (ISG15), which plays a role in cellular defense from infection and carcinogenesis, was identified as the gene related to gemcitabine chemosensitivity. By inhibition of ISG15 in gemcitabine-resistant cell lines using siRNA, gemcitabine resistance was reversed. It was demonstrated that ISG15 is one of the genes associated with intrinsic gemcitabine sensitivity, having a possibility to be a candidate of molecular targeting for the improvement of chemotherapy against pancreatic cancer.

Comparative proteomic profiling identified sorcin being associated with gemcitabine resistance in non-small cell lung cancer

Although gemcitabine-based chemotherapy is one of the more effective chemotherapy regimens against NSCLC, there are still many patients who do not benefit from this therapy. The mechanism of initial or acquired resistance to gemcitabine chemotherapy remains unknown. In this study, we investigated the protein profiling in gemcitabine-resistant and gemcitabine-sensitive NSCLC cell lines by a proteomic technology in order to identify novel gemcitabine resistance associated biomarkers for NSCLC patients. The proteomic profiling of NSCLC cell line H460 and its gemcitabine-resistant subline H460/GEM were compared by an isotope-coded affinity tag technology and tandem mass spectrometry. We further validated the expression of sorcin, a gemcitabine-resistance-related protein identified by proteomics, in 62 NSCLC specimens by immunohistochemistry. Fourteen gemcitabine resistance-related proteins were identified including nine up-regulated proteins and five down-regulated proteins. Immunohistochemical results demonstrated that sorcin staining was seen in 66.1% of NSCLC tumors, and sorcin overexpression was associated with gemcitabine resistance and a poor prognosis in NSCLC patients. In conclusion, sorcin might play an important role in the resistance to gemcitabine, and it could also be a novel candidate biomarker for predicting the response of NSCLC patients to gemcitabine treatment.

Enhancing Apoptosis and Overcoming Resistance of Gemcitabine in Pancreatic Cancer with Bortezomib: A Role of Death-Associated Protein Kinase-Related Apoptosis-Inducing Protein Kinase 1

To investigate the role of the apoptosis gene, DAP (death-associated protein) kinase-related apoptosis-inducing protein kinase 1 (DRAK1), which is involved in enhancing cell sensitivity and overcoming cell resistance to gemcitabine in pancreatic cancer cells by the proteasome inhibitor bortezomib. Cultured human pancreatic cancer gemcitabine-sensitive cell lines (bxpc-3) and gemcitabine-resistant (panc-1) cell lines were divided into four groups: control, treatment with bortezomib, treatment with gemcitabine, and the two-drug combination. Expression of DRAK1 genes in each group was detected by using reverse transcription-polymerase chain reaction and western blot. Apoptosis in the pancreatic cancer cell lines was measured by flow cytometry. We found that the effects of growth inhibition and apoptosis of gemcitabine on both pancreatic cancer cell lines were enhanced by bortezomib. Treatment of panc-1 and bxpc-3 cells with bortezomib (100 nM) and gemcitabine (50 microg/ml and 0.05 microg/ml, respectively) induced an increase in the levels of DRAK1 mRNA compared with the control and single-agent treatment. Furthermore, immunblotting analysis in panc-1 but not bxpc-3 cells showed similar changes in the expression of DRAK1 protein produced by combination therapy. Our results demonstrated that bortezomib enhanced cell sensitivity and overcame cell resistance to gemcitabine in pancreatic cancer cells, which may be attributed to DRAK1 induced by bortezomib and the combination with gemcitabine.

The difference between multi-drug resistant cell line A549/Gem and its parental cell A549

Objective To discuss the difference between multi-drug resistant cell line A549/Gem and its parental cell A549 on the basis of establishment of human gemcitabine-resistant cell line A549/Gem so as to elaborate the possible mechanisms of gemcitabine resistance.

Chemosensitization of gemcitabine‐resistant human bladder cancer cell line both in vitro and in vivo using antisense oligonucleotide targeting the anti‐apoptotic gene, clusterin

To characterize changes in clusterin (sCLU-2) expression in bladder cancer cells after continuous treatment with gemcitabine and to determine whether knockdown of sCLU-2 can re-introduce sensitivity of gemcitabine-resistant cells to treatment with gemcitabine. A human bladder cancer cell line, UM-UC-3, was continuously exposed to increasing doses of gemcitabine in vitro, and a gemcitabine-resistant cell line UM-UC-3R was developed. The role of sCLU-2 in chemoresistant phenotype acquired in both in vitro and in vivo was then analysed using antisense oligonucleotide targeting the sCLU-2 gene (OGX-011). Treatment of parental UM-UC-3 cells (UM-UC-3P) with gemcitabine induced transient up-regulation of sCLU-2 protein. There was a sustained increase in sCLU-2 expression levels in UM-UC-3R compared with UM-UC-3P cells (6.4-fold). Treatment of UM-UC-3R cells with OGX-011 resulted in a dose-dependent and sequence- specific inhibition in sCLU-2 expression. Furthermore, OGX-011 chemo-sensitized UM-UC-3R cells to gemcitabine in vitro with a reduction in the concentration that reduces the effect by 50% (IC50) from 100 nm to 10 nm. Tumour volume and the incidence of metastasis in nude mice injected with UM-UC-3R cells was significantly greater than those of nude mice injected with UM-UC-3P cells; however, systemic administration of OGX-011 plus a low dose of gemcitabine significantly suppressed tumour volume and the incidence of metastasis in both groups. These findings suggest that sCLU-2 plays a significant role in the acquisition of chemoresistant phenotype in bladder cancer cells and the knockdown of sCLU-2 using OGX-011 combined with a chemotherapeutic agent could be an attractive approach for advanced bladder cancer through the enhancement of chemosensitivity.

The difference between multi-drug resistant cell line H460/Gem and its parental cell NCI-H460

Objective To discuss the difference between multi-drug resistant cell line H460/Gem and its parental cell NCI-H460 on the basis of establishment of human gemcitabine-resistant cell line H460/Gem so as to elaborate the possible mechanisms of gemcitabine resistance.

Oxaliplatin activity in selected and unselected human ovarian and colorectal cancer cell lines

Oxaliplatin is used for treatment of colon cancer in combination with 5-fluorouracil or irinotecan. Oxaliplatin has similar, but also different resistant mechanisms as cisplatin. We studied the activity of oxaliplatin in ovarian and colon cancer cells with different resistance patterns to cisplatin. The 40-fold cisplatin-resistant cell line ADDP was only 7.5-fold resistant to oxaliplatin. The gemcitabine-resistant AG6000 cell line, 9-fold resistant to cisplatin, was not cross-resistant. LoVo-175X2, with mutant p53 showed no resistance compared to the empty vector control. However, LoVo-Li, with inactive p53, was 3.6-fold resistant corresponding to decreased accumulation and Pt adducts. Accumulation and DNA adducts formation showed no significant correlation with oxaliplatin sensitivity. Cell cycle distribution after exposure to oxaliplatin showed arrest in G2/M (A2780) or in S-phase (LoVo-92) for wt-p53 cells. ADDP and LoVo-Li showed G1 arrest followed by S-phase arrest and no changes in distribution, respectively. The cell cycle related proteins Cyclins A and B1 and (p)CDC25C were marginally affected by oxaliplatin. Expression of hCTR1 was decreased in ADDP, LoVo-Li and AG6000, OCT1 decreased in ADDP and AG6000 and OCT3 in LoVo-175X2, compared to the parental cell lines. In ADDP and LoVo-175X2 ATP7A and B were decreased but were increased in AG6000. From this study it can be concluded that changes in cell cycle distribution were cell line dependent and not related to changes in expression of Cyclin A or B1. Oxaliplatin accumulation was related to hCTR1 and, at low concentration, ATP7A to DNA adducts formation while the retention was related to hCTR1, OCT2 and ATP7B.

Establishment and biological characteristics of a multi-drug resistant cell line A549/Gem.

Multi-drug resistance is one of the most important reason why the survival time of non-small cell lung cancer patients is so short. The aim of this study is to establish multi-drug resistant cell line A549/Gem and discuss its biological characters so as to elaborate the possible mechanisms of gemcitabine resistance. Human gemcitabine-resistant non-small cell lung cancer cell line A549/Gem was established by repeated clinical serous peak concentration then low but gradually increasing concentration of gemcitabine from its parental cell human lung adenocarcinoma cell line A549 which is sensitive to gemcitabine. During the course of inducement, monitored its morphology, checked its resistance index and resistant pedigree by MTT method, gathered its growth curve and calculated its doubling time, examined its DNA contents and cell cycles by flow cytometry; at the same time, measured its expression of P53, EGFR, c-erb-B-2, PTEN, PCNA, c-myc, VEGF, MDR-1, Bcl-2, nm23, MMP-9, TIMP-1, CD44v6 Proteins, and RRM1 mRNA. The resistance index of A549/Gem' to gemcitabine was 163.228, and the cell line also exhibited cross-resistance to vinorelbine, taxotere, fluorouraci, etoposide and cisplatin, but kept sensitivity to paclitaxol and oxaliplatin. The doubling time of it was shorter and figures in G0-G1 phase were increased than A549. Compared with A549, A549/Gem' achieved EGFR and c-myc protein expression, nm23 protein expression enhanced, p53, Cerb-B-2 and bcl-2 protein expression reduced, PTEN, PCNA and MDR-1 protein expression vanished, but that of MMP-9, VEGF, CD44v6 and TIMP-1 protein changed trivially. Meanwhile, the expression of RRM1 mRNA was augmented markedly. The resistance index of A549/Gem to gemcitabine was 129.783, and the cell line also held cross-resistance to vinorelbine, taxotere, etoposide, cisplatin and sensitivity to paclitaxol. But the resistance to fluorouracil and sensitivity to oxaliplatin vanished. And the expression of RRM1 mRNA decreased visibly. The doubling time of A549/Gem was longer and figures in G0-G1 phase were decreased than A549/Gem'. It's expressions of P53, EGFR, PCNA and MDR-1 protein was same to that of A549/Gem'. A549/Gem achieved TIMP-1 and PTEN protein expression, Cerb-B-2, MMP-9, cmyc and bcl-2 protein expression enhanced, nm23 protein expression vanished, but the expression of VEGF and CD44v6 protein changed trivially. Furthermore, Compared with its parental cell, A549/Gem was mixed with giant cells of different sizes that were larger and more irregular. The multi-drug resistant non-small cell lung cancer cell line A549/Gem has multi-drug resistance and great change of biological character compared with its parental cell. And the change can participate in the formation of multidrug resistance.