Cisplatin-resistant Cancer Cell Line Research Articles

Abstract 3562: Characterization of a novel small molecule inhibitor with potent anticancer activity

Abstract A series of isoquinolineamine small molecule derivatives were synthesized and screened as potential anticancer agents. Among them, we identified one isoquinolineamine analogue with potential anticancer activity. This compound inhibited the proliferation of a variety of cancer cells derived from many human solid tumors with IC50 values ranging from 14 nM to 71 nM. The compound also was more effective against gemcitabine and cisplatin-resistant cancer cell lines when compared to the original cytotoxic cancer drugs. In mice bearing tumor xenografts, treatment with the compound significantly inhibited the growth of tumors, enhanced tumor regression, and also prevented the growth of tumors in a paclitaxel-resistant xenograft model. This compound is well tolerated and had no effects on body weight compared to control animals. Mechanistic studies showed that cancer cells treated with this compound resulted in the elevation of cytochrome c in the cytosol, the decrease of Bcl-2 and the activation of Caspase 9. Further studies showed inhibition of the expression of several signaling molecules important for cell proliferation (p-Erk, p-p38 and p-Akt) and also decreased protein levels of p-GSK3α/β and β-catenin. Therefore, our result suggests that this compound could be a promising antitumor agent. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3562.

Abstract 4525: Targeting ROS to kill cisplatin-resistant cells

Abstract Cisplatin (CP) resistance remains a major problem in the treatment of both small cell and non small cell lung cancer. We have previously shown that inhibiting mTOR can restore CP sensitivity; however, not all CP resistant cell lines are sensitive to mTOR inhibitor. Our findings have led us to search for an alternative target in these CP resistant cells. We have discovered that CP resistant cells share one common biochemical parameter which is increased reactive oxygen species (ROS) when compared to normal cells and their parental cells counterpart. Thus, further increased ROS in these CP resistant cells can push them beyond their tolerance limit which ultimately leads to cell death. We have found that increased expression of NADPH oxidase 4 (NOX4) and lower levels of thioredoxin (Trx) correlate with the high levels of ROS seen in CP resistant cell lines. Using elesclomol (N′1, N′3-dimethyl-N′1, N′3- bis [phenylcarbonothioyl] propanedihydrazide (formerly STA-4783, provided by Synta Pharmaceuticals)), an agent which is known to increase ROS, we demonstrated that elesclomol can increase H2O2 levels in CP resistant lung cancer cell lines (CP-R). Moreover, elesclomol is also significantly more cytotoxic toward CP-R with the ID50 dosage of 4-10 fold less than their parental cells (PC) counterpart and normal cells, BJ-1 (see table below). The percentage of apoptosis is also significantly higher in CP-R (65%) vs. PC (3%). The cytotoxic effect of elesclomol in CP-R is accompanied by further decrease in Trx and glutathione (GSH) antioxidant systems, while opposite results were found in PC and normal cells. This cytotoxic effect can be reversed by an antioxidant, N-acetylcysteine (NAC, 0.1mM). Furthermore, we have also found that elesclomol also increased intracellular CP and hence enhanced CP sensitivity in CP-R cells. We conclude that elesclomol is highly effective in CP resistant cells which express high basal levels of ROS and should be considered for treatment of CP resistant tumor.ID50 Dosage of Elesclomol in Parental (PC) vs. Cisplatin Resistant (CP-R) Lung Cancer Cell Lines SCLC1(PC)SCLCSR2(CP-R)SCLCB(PC)SCLCBC (CP-R)NSCLCS(PC)NSCLCSC(CP-R)BJ1Elesclomol(nM)50+/−2.55+/−0.541+/−3.89+/−1.259+/−4.610+/−2.265+/−5.5 Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4525.

Abstract 3542: Analysis of miRNA mediated Cisplatin resistant mechanisms

Abstract Cisplatin is an effective anticancer agent that is widely used in the treatment of testicular, ovarian, bladder and lung cancers. However, the development of resistance to cisplatin is a major obstacle to treatment. To understand the clinically relevant mechanisms of resistance, many studies have been aimed at clarifying the biochemical/molecular alterations of cisplatin-resistant cells. However, these studies did not conclusively identify the basis of cellular resistance to cisplatin. Recently some studies have shown that micro RNAs play an important role in cancer cell resistance to chemotherapeutic agents and cancer development. In this study, micro RNA expression profiles of cisplatin resistant non small cell lung cancer cell lines were analyzed using microarray and real time RT-PCR. We found some of micro RNAs were similarly regulated in couple of cisplatin resistant cell lines. These finding are useful for understanding the miRNA mediated cisplatin resistance mechanisms. Now, functional analyses on miRNAs involving cisplatin resistance are in progress. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3542.

Abstract 5381: Curcumin induces chemo/radio-sensitization in ovarian cancer cells and curcumin nanoparticles inhibit ovarian cancer cell growth

Abstract Background: Chemo/radio-resistance is a major obstacle in treating advanced ovarian cancer. The efficacy of current treatments may be improved by increasing the sensitivity of cancer cells to chemo/radiation therapies. Curcumin is a naturally occurring compound with anti-cancer activity in multiple cancers; however, its chemo/radio-sensitizing potential is not well studied in ovarian cancer. Herein, we demonstrate the chemo/radio-sensitizing potential of curcumin in a cisplatin resistant ovarian cancer cell line model. Additionally, due to suboptimal pharmacokinetics and low uptake of curcumin in tumors in vivo, we developed a curcumin nanoparticle formulation to improve its therapeutic efficacy. Methods: Cisplatin resistant A2780CP ovarian cancer cells were pre-treated with curcumin followed by exposure to cisplatin or radiation and the effect on cell growth was determined by MTS and colony formation assays. The effect of curcumin pre-treatment on the expression of apoptosis related proteins and β-catenin was determined by Western blotting or Flow Cytometry. A luciferase reporter assay was used to determine the effect of curcumin on β-catenin transcription activity. The poly(lactic acid-co-glycolic acid) (PLGA) nanoparticle formulation of curcumin (Nano-CUR) was developed by a modified nano-precipitation method and physico-chemical characterization was performed by transmission electron microscopy and dynamic light scattering methods. Results: Curcumin pre-treatment considerably reduced the dose of cisplatin and radiation required to inhibit the growth of cisplatin resistant ovarian cancer cells. During the 6 hrs pre-treatment, curcumin down regulated the expression of Bcl-XL and Mcl-1 pro-survival proteins. Curcumin pre-treatment followed by exposure to low doses of cisplatin increased apoptosis as indicated by annexin V staining and cleavage of caspase 9 and Poly (ADP-ribose) polymerase (PARP). Additionally, curcumin pre-treatment lowered β-catenin expression and transcriptional activity. Physico-chemical characterization of Nano-CUR indicated an average particle size of ∼70 nm, steady release of curcumin over a period of 18 days and antibody conjugation characteristics. The Nano-CUR formulation also effectively inhibited the growth of ovarian cancer cells. Conclusion: Curcumin pre-treatment enhances chemo/radio-sensitization in A2780CP ovarian cancer cells through multiple molecular mechanisms. Therefore, curcumin pre-treatment may effectively improve ovarian cancer therapeutics. A targeted PLGA nanoparticle formulation of curcumin is feasible and may improve the in vivo therapeutic efficacy of curcumin. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5381.

FOXM1 Confers Acquired Cisplatin Resistance in Breast Cancer Cells

The transcription factor Forkhead box M1 (FOXM1) is a key regulator of cell proliferation and is overexpressed in many forms of primary cancers, leading to uncontrolled cell division and genomic instability. To address the role of FOXM1 in chemoresistance, we generated a cisplatin-resistant breast cancer cell line (MCF-7-CIS(R)), which had an elevated level of FOXM1 protein and mRNA expression relative to the parental MCF-7 cells. A close correlation was observed between FOXM1 and the expression of its proposed downstream targets that are involved in DNA repair; breast cancer-associated gene 2 (BRCA2) and X-ray cross-complementing group 1 (XRCC1) were expressed at higher levels in the resistant cell lines compared with the sensitive MCF-7 cells. Moreover, cisplatin treatment induced DNA damage repair in MCF-7-CIS(R) and not in MCF-7 cells. Furthermore, the expression of a constitutively active FOXM1 (DeltaN-FOXM1) in MCF-7 cells alone was sufficient to confer cisplatin resistance. Crucially, the impairment of DNA damage repair pathways through the small interfering RNA knockdown inhibition of either FOXM1 or BRCA2/XRCC1 showed that only the silencing of FOXM1 could significantly reduce the rate of proliferation in response to cisplatin treatment in the resistant cells. This suggests that the targeting of FOXM1 is a viable strategy in circumventing acquired cisplatin resistance. Consistently, the FOXM1 inhibitor thiostrepton also showed efficacy in causing cell death and proliferative arrest in the cisplatin-resistant cells through the downregulation of FOXM1 expression. Taken together, we have identified a novel mechanism of acquired cisplatin resistance in breast cancer cells through the induction of FOXM1.

N′1,N′3-Dimethyl-N′1,N′3-bis(phenylcarbonothioyl) Propanedihydrazide (Elesclomol) Selectively Kills Cisplatin Resistant Lung Cancer Cells through Reactive Oxygen Species (ROS)

Cisplatin is an important chemotherapeutic agent in lung cancer treatment. The mechanism of drug resistance to cisplatin is complex and historically has been difficult to overcome. We report here that cisplatin resistant lung cancer cell lines possess high basal levels of reactive oxygen species (ROS) when compared to normal cells and their parental cell counterparts. These resistant cells also have low thioredoxin (TRX) levels which may be one of the contributory factors to high ROS. N′1,N′3-dimethyl-N′1,N′3-bis(phenylcarbonothioyl) propanedihydrazide (elesclomol), an agent known to increase ROS is selectively toxic to cisplatin-resistant cells, while sparing normal cells and the parental counterpart. The cytotoxic effect of elesclomol in resistant cells is accompanied by further decreases in TRX and glutathione (GSH) antioxidant systems, while opposite results were found in parental cells. The ID50 of elesclomol in cisplatin-resistant cells ranged from 5–10 nM, which is well within clinically achievable ranges. N-Acetylcysteine (NAC), which is known to neutralize ROS, can abolish the cytotoxic effect of elesclomol, suggesting that the cytotoxic effect results from increased ROS. Overall, our data suggest that elesclomol selectively kills cisplatin-resistant tumor cells through increased ROS. This agent may hold potential to overcome cisplatin resistance and should be further explored to treat patients who have failed cisplatin therapy.

Inhibition of human ovarian cancer cell lines by Devil's club Oplopanax horridus

Aim of the study To search for more effective treatment of ovarian cancer, we investigated the in vitro anti-proliferation activities of Devil's club (OH) root bark extracts, an important medicinal plant of North America, on cisplatin sensitive and resistant human ovarian cancer cell lines. Materials and methods High performance liquid chromatography was used to provide the chemical profiling of the OH extracts. The anti-proliferation activities of OH extracts alone or in combination with cisplatin or paclitaxel were determined on four human ovarian cell lines by crystal violet assay. Flow cytometry and light microscopy were employed for cell cycle analysis, and also to detect apoptosis. Results Our data showed that water, 70% ethanol, 100% ethanol, and ethyl acetate extracts of OH inhibited the proliferation of human ovarian cancer cell lines A2780, A2780CP70, OVCAR3, and OVCAR10 in vitro. The respective 50% inhibition (IC 50) was estimated at 1/256, 1/74, 1/69, 1/53; 1/4156, 1/1847, 1/1029, 1/4530; 1/25,753, 1/3310, 1/3462, 1/5049; and 1/29,916, 1/2912 1/3828, and 1/4232 dilutions. Some combinations of non-cytotoxic dilutions (<IC 50) of 70% ethanol OH extract with cisplatin and paclitaxel enhanced its anti-proliferation IC 50 on A2780 and A2780CP70 cells. Cell cycle analysis demonstrated that the effect of OH extract on cell cycle was dependent on the concentration tested, blocking cells in the S and G2/M phases. At low concentrations it induced cell death by apoptosis, while at high concentrations, it kills cells by necrosis. Conclusions Our data showed that OH extracts exhibited significant anti-proliferation effect against both cisplatin sensitive and resistant human ovarian cell lines. Further research might result in discovery of agent(s) that can potentially be useful as an adjunct therapy for ovarian cancer cells. It is one of the few North American medicinal herbs that have been tested for anti-ovarian cancer activities.

Immune modulator CD70 as a potential cisplatin resistance predictive marker in ovarian cancer

Objective We have used mass-spectrometry (MS) based proteomics platform to identify cell surface proteins over-expressed on a cisplatin resistant derivative of an ovarian cancer cell line A2780. Methods Membrane associated glycoproteins from A2780 and its cisplatin resistant derivative cell line, A2780cis, were processed for liquid chromatography (LC)-MS based analysis. The expression of proteins found at elevated levels in A2780cis cell line was confirmed using flow cytometry and Taqman analysis. The expression of these proteins was further evaluated in unrelated ovarian cancer cell lines using MS analysis and flow cytometry. Immunohistochemical (IHC) analysis was performed using ovarian tumor tissues to evaluate the protein density on the cell surface. Monoclonal antibodies were used in an alamar blue proliferation assay to examine the cytotoxic effects on cell proliferation in resistant cell lines. Results Six proteins were identified by LC-MS as being over-expressed on cell surface of A2780cis cell line. Mass spectrometry and flow cytometry confirmed the over-expression of CD49f, CD70 and Her-2/neu in other cisplatin resistant ovarian cancer cell lines. Immunohistochemical analysis revealed that only CD70 was expressed at moderate levels in ovarian tumors. When cisplatin resistant ovarian cell lines A2780cis and SKOV-3 were treated with antibody against CD70, there was a significant decrease in cell proliferation. Conclusion Using a MS based proteomics approach we have shown that expression of CD70 is associated with cisplatin resistance in ovarian cancer cell lines. Follow-up examination of these tumor cell line findings in clinical tumor specimens with available pathology staging and cisplatin treatment history is warranted.

ZNF93 Increases Resistance to ET-743 (Trabectedin; Yondelis®) and PM00104 (Zalypsis®) in Human Cancer Cell Lines

BackgroundET-743 (trabectedin, Yondelis®) and PM00104 (Zalypsis®) are marine derived compounds that have antitumor activity. ET-743 and PM00104 exposure over sustained periods of treatment will result in the development of drug resistance, but the mechanisms which lead to resistance are not yet understood.Methodology/Principal FindingsHuman chondrosarcoma cell lines resistant to ET-743 (CS-1/ER) or PM00104 (CS-1/PR) were established in this study. The CS-1/ER and CS-1/PR exhibited cross resistance to cisplatin and methotrexate but not to doxorubicin. Human Affymetrix Gene Chip arrays were used to examine relative gene expression in these cell lines. We found that a large number of genes have altered expression levels in CS-1/ER and CS-1/PR when compared to the parental cell line. 595 CS-1/ER and 498 CS-1/PR genes were identified as overexpressing; 856 CS-1/ER and 874 CS-1/PR transcripts were identified as underexpressing. Three zinc finger protein (ZNF) genes were on the top 10 overexpressed genes list. These genes have not been previously associated with drug resistance in tumor cells. Differential expressions of ZNF93 and ZNF43 genes were confirmed in both CS-1/ER and CS-1/PR resistant cell lines by real-time RT-PCR. ZNF93 was overexpressed in two ET-743 resistant Ewing sarcoma cell lines as well as in a cisplatin resistant ovarian cancer cell line, but was not overexpressed in paclitaxel resistant cell lines. ZNF93 knockdown by siRNA in CS-1/ER and CS-1/PR caused increased sensitivity for ET-743, PM00104, and cisplatin. Furthermore, ZNF93 transfected CS-1 cells are relatively resistant to ET-743, PM00104 and cisplatin.Conclusions/SignificanceThis study suggests that zinc finger proteins, and ZNF93 in particular, are involved in resistance to ET-743 and PM00104.

Synthesis, molecular structure and evaluation of new organometallic ruthenium anticancer agents

A number of new ruthenium compounds have been synthesised, isolated and characterised, which exhibit excellent cytotoxicity against a number of different human tumour cell lines including a defined cisplatin resistant cell line and colon cancer cell lines. Addition of hydrophobic groups to the ruthenium molecules has a positive effect on the cytotoxicity values. Evidence is provided that, after incubation of a ruthenium compound with a 46 mer oligonucleotide duplex and subsequent nuclease treatment, ruthenium is bound to a guanine residue.

Expression of connexin 43 in ovarian cancer and its relationship with chemoresistance

To examine the expression of protein kinase C (PKC), connexin 43 (Cx43) and non-phosphorylated Cx43 in ovarian cancer, and discuss the role of phosphorylated of Cx43 in chemoresistance in ovarian cancer. We examined the expression of Cx43, non-phosphorylated Cx43 and PKC in ovarian cancer tissue by immunohistochemistry, and compared their expression in chemosensitivity group and chemoresistance group. Cisplatin resistant ovarian cancer cell line SKOV3/DDP cells were treated by staurosporine (a kind of PKC inhibitors). Then expression of Cx43, nonphosphorylated Cx43 and PKC were tested. Meanwhile, we tested chemosensitivity of SKOV3/DDP cells by ATP bioluminescence tumor chemosensitivity assay (ATP-TCA). (1) Immunohistochemically, the rates of positive expression of Cx43 and non-phosphorylated Cx43 were 54%, 14% respectively in the chemoresistance group, which were 83%, 59% in the chemosensitivity group respectively (P < 0.05). The rate of positive expression of PKC in 28 chemoresistance ovarian cancer cases (64%) was higher than that in 29 chemosensitivity cases (31%, P < 0.05). Both of them were significantly lower in chemoresistance group than in chemosensitivity group (P < 0.05). In addition, the expression of PKC was negatively correlated with the expression of Cx43 and non-phosphorylated Cx43. The correlation coefficients were -0.626 and -0.714, respectively (P < 0.05). (2) Immunohistochemically, PKC was down regulated, and Cx43 and non-phosphorylated Cx43 were up regulated in SKOV3/DDP cells after staurosporine treatment. The longer the staurosporine worked, the more expression of Cx43 was. (3) By ATP-TCA, SKOV3/DDP cells were resistant to paclitaxel and cisplatin. The tumor growth inhibition was higher in the group of paclitaxel or cisplatin combined staurosporine than in the group of paclitaxel or cisplatin alone. The sensitivity was intermediate in the group combined with low concentration staurosporine (1 x 10(-8) mol/L), and the sensitivity was high in the group combined with high concentration staurosporine (1 x 10(-7) mol/L). Phosphorylation of Cx43 caused by PKC leads to decrease in the expression of Cx43. This effect makes ovarian cancer cells less chemosensitive. Phosphorylation of Cx43 caused by PKC can be inhibited by staurosporine.

Methylation Linear Discriminant Analysis (MLDA) for identifying differentially methylated CpG islands

BackgroundHypermethylation of promoter CpG islands is strongly correlated to transcriptional gene silencing and epigenetic maintenance of the silenced state. As well as its role in tumor development, CpG island methylation contributes to the acquisition of resistance to chemotherapy. Differential Methylation Hybridisation (DMH) is one technique used for genome-wide DNA methylation analysis. The study of such microarray data sets should ideally account for the specific biological features of DNA methylation and the non-symmetrical distribution of the ratios of unmethylated and methylated sequences hybridised on the array. We have therefore developed a novel algorithm tailored to this type of data, Methylation Linear Discriminant Analysis (MLDA).ResultsMLDA was programmed in R (version 2.7.0) and the package is available at CRAN [1]. This approach utilizes linear regression models of non-normalised hybridisation data to define methylation status. Log-transformed signal intensities of unmethylated controls on the microarray are used as a reference. The signal intensities of DNA samples digested with methylation sensitive restriction enzymes and mock digested are then transformed to the likelihood of a locus being methylated using this reference. We tested the ability of MLDA to identify loci differentially methylated as analysed by DMH between cisplatin sensitive and resistant ovarian cancer cell lines. MLDA identified 115 differentially methylated loci and 23 out of 26 of these loci have been independently validated by Methylation Specific PCR and/or bisulphite pyrosequencing.ConclusionMLDA has advantages for analyzing methylation data from CpG island microarrays, since there is a clear rational for the definition of methylation status, it uses DMH data without between-group normalisation and is less influenced by cross-hybridisation of loci. The MLDA algorithm successfully identified differentially methylated loci between two classes of samples analysed by DMH using CpG island microarrays.

A synergistic interaction between lapatinib and topoisomerase I inhibitors in cisplatin sensitive and resistant cancer cell lines

14618 Background: Lapatinib is a HER1 and 2 tyrosine kinase inhibitor (TKI). Little is know about its interaction with other chemotherpy drugs. In this work investigate interactions between lapatinib and cisplatin or SN-38 (the active metabolite of irinotecan) in cisplatin sensitive (testis cancer) and resistant cancer cell lines. Methods: We obtained 3 cancer cells lines (833K, GCT27, SUSA) all of which had cisplatin resistant daughter lines (833Kr, GCT27r, SUSAr). These resistant lines were established by exposing the sensitive lines to increasing doses of cisplatin. Western Blot analysis was used to estimate HER 1–4 levels and expression of proteins in the PI3K and MAPK pathways. The IC 50s for lapatinib, cisplatin and SN-38 were calculated in these cell lines. These drugs were then given in combination, and the effect was estimated using calcusin plots. Flow cytomery was used to estimate the effect on the cell cycle and apoptosis. Identical experiments were carried out with Pazopanib, instead of lapatinib, which was used as a control. Results: The IC50 for lapatinib in the resistant and sensitive cell lines were similar (4.0–5.0 micromolar and 3.6–4.4 micromolar respectively). Sensitivity was not related to HER 1–4 expression. Synergistic interactions were observed between SN-38 and lapatinib in all 6 cell lines (calculated combination indices <0.7 in all lines). No synergistic interaction was seen between cisplatin and lapatinib. Cell cycle data revealed the synergistic combination was associated with an increase in apoptosis and reduces cell cycle arrest. Pazopanib was not associated with a synergistic interaction in any of the cell lines with either cisplatin or SN-38. Conclusions: Lapatinib appears equally efficacous in both cisplatin sensitive and resistant cell lines. A synergistic interaction occurs between lapatinib and SN-38 in this model which is associated with increased apoptosis. Author Disclosure Employment or Leadership Consultant or Advisory Role Stock Ownership Honoraria Research Expert Testimony Other Remuneration GlaxoSmithKline

In vitro growth inhibition of cisplatin-resistant human lung cancer cell lines by recombinant human tumor necrosis factor and/or recombinant human interferon-gamma by virtue of collateral sensitivity.

The colony-inhibitory effects of recombinant human tumor necrosis factor (rH-TNF) and recombinant human interferon-gamma (rH-IFN-gamma) were evaluated in four human lung cancer cell lines and their cisplatin-resistant sublines. The cell lines tested were PC-7 and PC-9 (adenocarcinoma), H69 and N231 (small cell lung cancer) and four cisplatin-resistant sublines, PC-7/1.0, PC-9/0.5, H69/0.2 and N231/0.2, which were 20.0, 7.1, 4.8 and 8.4 fold resistant to cisplatin, respectively, compared to the respective parental cell line in terms of IC50 in a soft agar colony assay. All parental cell lines were resistant to rH-TNF and rH-IFN-gamma, alone or in combination. However, two resistant sublines showed sensitivity to rH-TNF and rH-IFN-gamma. Colony formation by PC-9/0.5 was significantly inhibited, in the absence or presence of cisplatin, by 10(2) U/ml of rH-TNF (less than 50% of control) and the inhibition was synergistic with that produced by 10(3) or 10(4) U/ml of rH-IFN-gamma. RH-IFN-gamma inhibited the colony formation of H69/0.2 only at the highest concentration tested (10(4) U/ml) (less than 50% of control) and the combined effect with rH-TNF was additive. These results suggest that rH-TNF and rH-IFN-gamma may have some potential in overcoming cisplatin resistance by virtue of collateral sensitivity.

Gene expression profiling of the response of esophageal carcinoma cells to cisplatin

Cisplatin is the most common chemotherapeutic agent used in esophageal cancer. However, sensitivity to cisplatin varies greatly between patients. It is important to identify the gene(s) that are related to the sensitivity to cisplatin in esophageal cancer patients. The IC50 for cisplatin was measured for 15 esophageal cancer cell lines (TE1-5, TE8-15, KYSE140, and KYSE150). RNA was extracted from each of these cell lines and a normal esophageal epithelial cell line, namely, Het1A, and gene expression profiles were analyzed using an oligonucleotide microarray consisting of 34 594 genes. TE4 was highly resistant and TE12, 14, and 15 were sensitive to cisplatin. Thirty-seven genes were differentially expressed in the cisplatin-resistant esophageal cancer cell line. Our investigation provides a list of candidate genes that may be associated with resistance to cisplatin in esophageal cancer cells, which may serve as a basis for additional functional studies.

A genomic approach to identify mechanisms associated with chemotherapy resistance

2534 Background: Gene expression profiling has shown an ability to predict chemotherapeutic response (Potti et al. Nature Medicine 2006). Building on this work, we used a genomic strategy to explore the biology associated with the development of chemotherapy resistance and sought to determine if disease context impacted results. Methods: Gene set enrichment analysis (GSEA) was performed on expression data for NCI60 cell lines sensitive and resistant to specific chemotherapeutic agents (adriamycin, cyclophosphamide, docetaxel, etoposide, 5-fluoruracil, paclitaxel, topotecan). GSEA was additionally performed on a series of lung cancer cell lines with a defined sensitivity to cisplatin and docetaxel. Sensitive and resistant cell lines with individual mean and confidence intervals greater than 1SD from the mean across all samples were included. Adjusting for multiple hypothesis testing, gene sets with a false discovery rate (FDR) &lt;0.25 were deemed statistically significant. In the discovery mode, gene sets with a nominal p-value &lt;0.05 were also of interest. Finally, overlapping gene sets between agents were assessed. Results: Statistically-significant gene sets, representing biologic pathways associated with resistance, were identified for the various chemotherapeutic agents (i.e., cell death, erbb3, and bad pathways associated with docetaxel resistance). No gene sets with FDR &lt;0.25 or nominal p-value &lt;0.05 were common to all drugs. In assessing disease specific resistance, 22 lung cancer cell lines for cisplatin (15 sensitive, 7 resistant) and 14 lung cancer cell lines for docetaxel (10 sensitive, 4 resistant) were analyzed. GSEA identified the bcl-2 pathway (p&lt;0.002) to be associated with cisplatin resistance. In contrast, the proteosome (p=0.01) and akt (p=0.02) pathways were associated with docetaxel resistance. Pathways involved in the production of V-H+-ATPase were enriched in cisplatin and docetaxel resistant lung cancer cell lines suggesting a global mechanism of resistance. Conclusions: These results support the use of a genomic approach to identify unique drug-specific and global therapeutic targets associated with the development of chemotherapy resistance. Interestingly, disease context appears important in identifying novel targets. No significant financial relationships to disclose.

Bridge linkage role played by CD98hc of anti-tumor drug resistance and cancer metastasis on cisplatin-resistant ovarian cancer cells

Anti-tumor drug resistance and cancer metastasis are always clinically coincidental, which are conducted by different molecules such as P-glycoprotein (P-gp) and CD147, respectively. P-gp and CD147/CD98hc complex are both found highly expressed on cisplatin resistant ovarian cancer cell line SKOV3/DDP but only slightly expressed on its parent cell SKOV3. RNAi targeting CD98hc or CD147 both reduce their own and P-gp expression as well as cisplatin IC50 of drug-resistant tumor cells. CD147 interference only reduced membrane CD98hc rather than its intracellular forms. Stop of CD98hc also diminished CD147 translation. Cloned potential CD98hc promoter region showed promoter activity in luciferase assay under cisplatin pressure. Intracellular cisplatin accumulation was found increased in RNAi groups and the efflux ability of cisplatin resistant SKOV3 cells was disrupted as well. CD147 has been reported to play an important role in tumor metastasis1, 2. Taken together, CD98hc was for the first time revealed to be a bridge between MDR phenotype and tumor metastasis.

547 POSTER Novei small-molecule inhibitors of STAT3 that selectively induce antitumor cell activity

ZD0473 is a new generation platinum agent that, in preclinical studies, shows evidence of an extended spectrum of anti-tumor activity and overcomes platinum resistance mechanisms. The drug contains a bulky methylpyridine ligand at its platinum center, which is responsible for its ability to overcome platinum resistance. We examined the growth inhibitory effects of ZD0473 in human lung cancer cell lines resistant to cisplatin in vitro. Four cisplatin resistant human lung cancer cell lines (PC-14/CDDP, SBC-3/CDDP, PC-9/CDDP, H69/CDDP) showed the expected resistance to cisplatin but were non-cross, or much less, resistant to ZD0473, as determined by an MTT assay. A reduction in the intracellular accumulation of cisplatin, but not of ZD0473, was observed in the PC-14/CDDP cells compared with the levels in PC-14 parental cells. The reduction in cisplatin accumulation is considered a major mechanism of the acquired cisplatin resistance in PC-14/CDDP cells. Therefore, the increase in platinum accumulation is considered a possible mechanism underlying the activity of ZD0473 in cisplatin-resistant cells. Glutathione-mediated resistance to cisplatin was also overcome by ZD0473 in PC-14/CDDP cells. In addition, we showed that the intraperitoneal administration of ZD0473 at its maximum tolerable dose in mice produced a marked in vivo antitumor activity against cisplatin-resistant PC-14/CDDP tumors. These results suggest that ZD0473 may be a potent agent in human lung cancer cells with multifactorial cisplatin resistance.

High effectiveness of platinum(IV) complex with adamantylamine in overcoming resistance to cisplatin and suppressing proliferation of ovarian cancer cells in vitro

[( OC-6-43)-bis(acetato)(1-adamantylamine)amminedichloroplatinum(IV)], coded as LA-12, is an octahedral platinum(IV) complex containing a bulky hydrophobic ligand – adamantylamine. The use of bulky hydrophobic amines as non-leaving ligands, may increase uptake of the compound by the cancer cells. Therefore, the effects of LA-12 on sensitive (A2780) and cisplatin resistant (A2780cis) ovarian cancer cell lines were investigated and compared to those of cisplatin. IC 50 and IC 90 concentrations of LA-12 were 6- (A2780) or 18-fold (A2780cis) lower than those for cisplatin (MTT assay). Equitoxic concentrations (IC 50 or IC 90) of both compounds caused a significant and similar time- and dose-dependent inhibition of cell proliferation and an increase in the number of floating cells which corresponded to the decrease of total cell viability. A different type and dynamics of cell cycle perturbation after cisplatin and LA-12 treatment were detected. Exposure to LA-12 resulted in transient accumulation of A2780 and A2780cis cells in S phase, while cisplatin caused G 2/M arrest in sensitive and S phase arrest in resistant cells. A relatively low rate of apoptosis after exposure to IC 50 or IC 90 of both complexes was observed, markedly higher in resistant A2780cis cells. Western blot analysis indicated a concentration-dependent p53 level increase in both lines (higher after cisplatin treatment). PARP cleavage was observed only in A2780cis cells. In conclusion, LA-12 was found to be significantly more efficient than cisplatin, and it was able to overcome the acquired cisplatin resistance (showing resistance factor 2.84-fold lower than those for cisplatin). In spite of the low rate of apoptosis, LA-12 caused increase of p53 level and cell cycle perturbations in the ovarian cancer cell lines studied.

Effect of lung resistance-related protein on the resistance to cisplatin in human ovarian cancer cell lines

The mechanisms of drug-resistance in human ovarian cancer cells have not been entirely clarified. The purpose of this study was to investigate whether LRP is involved in the resistance of ovarian cancer cell lines to cisplatin and its molecular mechanism. Human ovarian cisplatin-resistant cancer cell lines (A2780/DDP and COC1/DDP) and their parental cisplatin-sensitive cell lines (A2780 and COC1), alone or transfected with antisense LRP-specific oligonucleotides (ODN) or sense ODN, were treated with cisplatin to induce differentiation. Expression of LRP was examined by RT-PCR and Western blot analysis. The sensitivities of cells to cisplatin were assessed using sulforhodamine B (SRB) assay and flow cytometry, and the accumulation and efflux of cisplatin in the cells and isolated nuclei were examined by high performance liquid chromatographic (HPLC) assay. The expressions of LRP in A2780/DDP and COC1/DDP cells were higher than those in A2780 and COC1 cells and conferred resistance to cisplatin. Transfection of LRP AsODN into A2780/DDP and COC1/DDP cells down-regulated LRP expression and reversed the resistance phenotype. Levels of cisplatin accumulating in cells were increased by LRP-specific AsODN and anti-LRP monoclonal antibody. Isolated nuclei from A2780 and COC1 cells or A2780/DDP and COC1/DDP cells incubated with anti-LRP antibody contained more cisplatin than the nuclei of A2780/DDP and COC1/DDP cells not treated with anti-LRP antibody. Efflux of cisplatin was greater from the nuclei of A2780/DDP and COC1/DDP cells than those of A2780 and COC1 cells, and was inhibited by anti-LRP monoclonal antibody. Thus, LRP was involved in the resistance of ovarian cancer cells to cisplatin and has an important role in the transport of cisplatin both in exocytotic vesicles and between the nucleus and cytoplasm.