CisR Cell Lines Research Articles

ROLE OF PHARMACOLOGICAL NFkB INHIBITION IN CANCER STEM CELLS POPULATION OF HEAD AND NECK SQUAMOUS CELL CARCINOMA

<h3>Objectives</h3> To evaluate the effect of pharmacological inhibition of nuclear factor-kappa beta (NFkB) using emetine and CBL0137 in cancer stem cells (CSCs) of head and neck squamous cell carcinoma cell lines resistant to cisplatin (CisR). <h3>Study Design</h3> CAL27 and SCC9 cell lines were used. NFkB expression was investigated by quantitative polymerase chain reaction and Western blot. MTS and spheres formation assay were assessed to determine the inhibition doses of emetine and CBL0137. Flow cytometry was performed to quantify the CSC population. <h3>Results</h3> Both CisR cell lines presented NFkB accumulation. CAL27 CisR IC50 dose was 1.8 μM for emetine and 1.5 μM for CBL0137. Emetine IC50 dose for SCC9 CisR was 1.7 μM and 1.5 μM for CBL0137. Emetine was able to eliminate CAL27 CisR CSC at 0.5 μM and CBL0137 at 0.75 μM. For both drugs, 0.75 μM eliminated CSC of the SCC9 CisR cell line. In combination with cisplatin, emetine was more effective in reducing CSC of CAL27 CisR and CBL0137 showed better response for SCC9 CisR. Interestingly, cisplatin alone did not have any effect on CisR cell lines and emetine or CBL0137 alone was more effective in reducing the CSC population than in combination with cisplatin. <h3>Conclusions</h3> Pharmacological inhibition of NFkB is efficient in reducing the CSC population, decreasing cisplatin chemoresistance.

UNRAVELING PATHWAYS THAT DRIVE CISPLATIN CHEMORESISTANCE IN ORAL CARCINOMA CELL LINES

<h3>Objectives</h3> Investigate the mechanisms that contribute to cancer stem cells (CSCs) accumulation and chemoresistance phenotype in cisplatin chemoresistant cell lines. <h3>Study Design</h3> CAL27 and SCC9 cell lines were treated with cisplatin until the establishment of the chemoresistance (CisR). RNA-seq and gene ontology (GO) analysis were performed to identify genes and biologic processes modified by chemoresistance. CSC accumulation was assessed by flow cytometry and sphere formation. Aggressiveness was investigated by clonogenic assay, MTS assay, Ki-67 immunostaining, wound healing assay, invasion, and tumor growth in a xenograft model. Epithelial mesenchymal transition (EMT) was detected by changes in E-cadherin and vimentin. <h3>Results</h3> The transcriptome of chemoresistant cell lines revealed upregulation of genes involved with proliferation, cell migration, and stemness, including TGFβ-2 and TGFβR-2; an increase in inflammatory processes; and an association with activation of EMT. RNA-seq also revealed downregulation of genes related to adhesion and drug metabolism. Chemoresistant cells presented accumulation of CSCs as well as increased proliferation, migration, and invasion. Increases in vimentin and decreases in E-cadherin were observed in CisR cell lines, suggesting activation of EMT. For CAL27 CisR we also observed a high in vivo tumorigenic potential. <h3>Conclusions</h3> A reprogramming in the expression of genes signaling stemness and tumor aggressiveness drives chemoresistance induced by cisplatin.

Pharmacological inhibition of HDAC6 overcomes cisplatin chemoresistance by targeting cancer stem cells in oral squamous cell carcinoma.

Chemoresistance is associated with recurrence and metastasis in oral squamous cell carcinoma (OSCC). The cancer stem cell (CSC) subpopulation is highly resistant to therapy, and they are regulated by epigenetic mechanisms. HDACs are histone deacetylase enzymes that epigenetically regulate gene expression. HDAC6 acts on several physiological processes, including oxidative stress, autophagy and DNA damage response, and its accumulation is associated with cancer. Here, we investigate the role of HDAC6 in CSC-mediated chemoresistance in oral carcinoma in addition to its application as a therapeutic target to reverse chemoresistance. Wild-type oral carcinoma cell lines (CAL27 WT and SCC9 WT), cisplatin-resistant (CAL27 CisR and SCC9 CisR), and the subpopulations of cancer stem cells (CSC+) and non-stem (CSC-) derived from CisR cells were investigated. HDAC6 accumulation was analyzed by Western blot and immunofluorescence; DNA damage was evaluated by immunofluorescence of phospho-H2A.X; the qPCR for PRDX2, PRDX6, SOD2, and TXN and ROS assay assessed oxidative stress. Apoptosis and CSC accumulation were investigated by flow cytometry. We identified the accumulation of HDAC6 in CisR cell lines and CSC. Cisplatin-resistant cell lines and CSC demonstrated a reduction in DNA damage and ROS and elevated expression of PRDX2. The administration of tubastatin A (a specific HDAC6 inhibitor) increased oxidative stress and DNA damage and decreased PRDX2. Tubastatin A as a monotherapy induced apoptosis in CisR and CSC and reduced the stemness phenotype. High levels of HDAC6 sustain CSC subpopulation and chemoresistance in OSCC, suggesting HDAC6 as a pharmacological target to overcome resistance and perhaps prevent recurrence in OSCC.

Combination of Niraparib, Cisplatin and Twist Knockdown in Cisplatin-Resistant Ovarian Cancer Cells Potentially Enhances Synthetic Lethality through ER-Stress Mediated Mitochondrial Apoptosis Pathway.

Poly (ADP-ribose) polymerase 1 inhibitors (PARPi) are used to treat recurrent ovarian cancer (OC) patients due to greater survival benefits and minimal side effects, especially in those patients with complete or partial response to platinum-based chemotherapy. However, acquired resistance of platinum-based chemotherapy leads to the limited efficacy of PARPi monotherapy in most patients. Twist is recognized as a possible oncogene and contributes to acquired cisplatin resistance in OC cells. In this study, we show how Twist knockdown cisplatin-resistant (CisR) OC cells blocked DNA damage response (DDR) to sensitize these cells to a concurrent treatment of cisplatin as a platinum-based chemotherapy agent and niraparib as a PARPi on in vitro two-dimensional (2D) and three-dimensional (3D) cell culture. To investigate the lethality of PARPi and cisplatin on Twist knockdown CisR OC cells, two CisR cell lines (OV90 and SKOV3) were established using step-wise dose escalation method. In addition, in vitro 3D spheroidal cell model was generated using modified hanging drop and hydrogel scaffolds techniques on poly-2-hydroxylethly methacrylate (poly-HEMA) coated plates. Twist expression was strongly correlated with the expression of DDR proteins, PARP1 and XRCC1 and overexpression of both proteins was associated with cisplatin resistance in OC cells. Moreover, combination of cisplatin (Cis) and niraparib (Nira) produced lethality on Twist-knockdown CisR OC cells, according to combination index (CI). We found that Cis alone, Nira alone, or a combination of Cis+Nira therapy increased cell death by suppressing DDR proteins in 2D monolayer cell culture. Notably, the combination of Nira and Cis was considerably effective against 3D-cultures of Twist knockdown CisR OC cells in which Endoplasmic reticulum (ER) stress is upregulated, leading to initiation of mitochondrial-mediated cell death. In addition, immunohistochemically, Cis alone, Nira alone or Cis+Nira showed lower ki-67 (cell proliferative marker) expression and higher cleaved caspase-3 (apoptotic marker) immuno-reactivity. Hence, lethality of PARPi with the combination of Cis on Twist knockdown CisR OC cells may provide an effective way to expand the therapeutic potential to overcome platinum-based chemotherapy resistance and PARPi cross resistance in OC.

P2.01-031 CCL Chemokines May Play an Important Role in Cisplatin Resistance

In the absence of a targetable mutation, cisplatin based chemotherapy is the backbone of NSCLC treatment. However, a diverse patient population combined with complex tumor heterogeneity is hampering its’ clinical utility. Although intrinsic and acquired resistance to cisplatin is common, the mechanisms have not yet been fully elucidated. However, some studies have suggested that inflammatory pathways may play a key role in chemo-resistance. The aim of this project is to increase our understanding of inflammatory mediated cisplatin resistance in NSCLC. A number of isogenic cell line models of NSCLC (adenocarcinoma, squamous cell carcinoma, large cell carcinoma) cisplatin resistance were utilized to assess the role of inflammation in chemo-resistance. These included a sensitive parental cell line (PT) and a matched resistant subtype (CisR). The cell lines were screened for NFKB and a number of inflammatory mediators including chemokines and TLRs at the mRNA (RT-PCR/qPCR) and protein level (Western Blot/ELISA). A specific NFKB inhibitor, DHMEQ, and recombinant chemokines were employed to further characterize inflammatory pathways in PT and CisR cells in terms of cisplatin sensitivity, proliferation (BrdU ELISA), cellular viability (Cytell Cell Imaging System) and DNA damage response (Comet). An in vivo study was also completed using DHMEQ alone and in combination with cisplatin. A number of NFKB targets and responsive pathways are deregulated in CisR cells compared with their matched sensitive PT cell line. Amongst others, CCL2 and CCL5 were altered across all NSCLC subtypes. Preliminary data suggests that DHMEQ enhances cisplatin sensitivity in both PT and CisR cells, conversely recombinant chemokines elicit a protective effect. Additionally, DHMEQ treatment resulted in opposite effects on CCL2 and CCL5 mRNA levels in the PT and CisR cell lines. This may reflect an alternative pathway hierarchy within the cells. Further characterization is ongoing assessing chemokine specific inhibitors. Although, in vivo data suggests a trend of decreased tumor growth in the DHMEQ cohorts compared with vehicle control, the data was not significant. However, tumor samples appeared more necrotic with DHMEQ and are currently being characterized using IHC for necrosis and proliferation. Targeting chemokines downstream of NFKB may provide a means to overcome inflammatory mediated acquired and intrinsic NSCLC chemo-resistance. Given the increased significance of immuno-oncology agents to harness the body’s own immune system in the fight against cancer, these agents may also prove fruitful in re-sensitizing patients to chemotherapy.

P2.03b-096 Utilization of a Novel 3D Culture Technology for the Assessment of Chemo-Resistance in Non-Small Cell Lung Cancer: Topic: Biomarkers

Elucidation of the key mechanisms underlying resistance to chemotherapy is an on-going and complex process. Owing to its suggested increased biological relevance, many are now transitioning from two-dimensional (2D) to three-dimensional (3D) cellular-based assay systems. These systems permit the formation of 3D multicellular structures (MCS). The internal micro-environment of these structures mimics closely those found in vivo. In addition they are considered to provide a more biologically relevant model of chemo-resistance. This study focuses on the utilization of a novel 3D culture technology to compare chemo-resistant models of non-small cell lung cancer (NSCLC) in 3D culture with those cultured in 2D monolayers. Critically, this will provide a valuable tool to determine the biological discrepancies which exist between the two culture methods with the aim to identify novel mechanisms of chemo-resistance in NSCLC. Isogenic NSCLC models of cisplatin resistance, which encompassed sensitive parent (PT) and matched cisplatin resistant (CisR) cell lines, were used for this study. The 3D MCS were cultured in Happy Cell Advanced Suspension Medium™ and 2D monolayers as standard. Both 2D and 3D cultures were exposed to a range of cisplatin concentrations (0 – 100 μM) for a period of 72 h. Subsequently, cellular viability, hypoxia, proteomic and morphological assays were conducted in order to compare the response of both PT and CisR cells in 2D and 3D. High content imaging has identified a central necrotic core within the 3D MCS, which is a feature of the asymmetric growth patterns observed in vivo; that being a decrease in viable cells as you move inwards from the periphery of the MCS. Preliminary data suggests that at equivalent cisplatin concentrations, H460 3D MCS exhibit increased resistance to cisplatin compared with 2D monolayers in both PT and CisR cell lines. Proteomic analysis has also identified diverse pathway modifications in 3D compared with 2D culture, with a number of proteins expressed exclusively in each. Additional cellular characterization and further bioinformatic analysis is on-going. Chemotherapeutic intervention is most frequently employed in the treatment of NSCLC, however many patients exhibit intrinsic and acquired resistance to common chemotherapy drugs, such as cisplatin and targeted agents. As it has been argued that 3D models and their micro-environment are more reflective of the in vivo situation, 3D culture may provide a more accurate in vitro model to elucidate mechanisms of chemo-resistance and possibly aid in the identification of novel targets to re-sensitize and stratify patients for therapy.

P2.03a-063 Small Molecule Cancer Stemness Inhibitor, BBI608, Restores Cisplatin Sensitivity in Resistant NSCLC: Topic: Clinical Trials

The cancer stem cell (CSC) hypothesis is now a well-established and widely investigated field within oncology. It hypothesizes that there is a robustly resistant stem-like population of cells that survive initial chemotherapeutic treatment. These surviving CSCs contribute to the recapitulation of a heterogeneous tumor via a combination of asymmetric and symmetric cell division, subsequently resulting in relapse and therapeutic resistance. BBI608 is a small molecule inhibitor of cancer stemness; it targets STAT3, leading to the inhibition of critical genes required for the maintenance of cancer stemness. To date, preclinical studies investigating BBI608 in in vitro and in vivo models of pancreatic and prostate cancer have shown promise. Aldefluor (Stemcell Technologies) staining and flow cytometry analysis of an isogenic panel of matched parent (PT) and cisplatin resistant (CisR) NSCLC cell lines identified the ALDH1-positive (ALDH1+ve) subpopulation of cells as a key CSC subset across cisplatin resistant NSCLC cell lines. PT and CisR cell lines were treated with BBI608 (1μM) and stemness factors investigated, the presence of the ALDH1+ve CSC population was reassessed by flow cytometry and expression of stemness factors (Nanog, Oct-4, Sox-2, Klf4 and cMyc) were examined by reverse transcriptase PCR. The functional parameters of proliferation, clonogenic survival and apoptosis were investigated with increasing concentrations of cisplatin (0-100μM) in the presence and absence of 1μM BBI608. The NSCLC CisR sublines showed a significantly greater ALDH1+ve CSC population relative to their PT counterparts. Treatment of the CisR sublines with 1μM BBI608 significantly depleted the ALDH1+ve CSC population and decreased gene expression of stemness markers. BBI608 significantly decreased the proliferative capacity and clonogenic survival of the CisR sublines when in combination with cisplatin relative to cisplatin alone. Cisplatin in combination with BBI608 significantly increased cisplatin-induced apoptosis in the CisR sublines indicating restoration of cisplatin sensitivity. To date, BBI608 has not been investigated in terms of a cisplatin resistant CSC population in lung cancer. BBI608, via the inhibition of STAT3, pharmacologically depleted the CSC subpopulation and stemness expression while simultaneously restoring cisplatin sensitivity. There are currently a number of clinical trials recruiting patients to further investigate BBI608. These data suggest a promising role for BBI608 in the treatment of non-responsive or recurrent NSCLC.

75P Inhibition and exploitation of aldehyde dehydrogenase 1 (ALDH1) as a cancer stem cell marker in cisplatin resistant NSCLC

Background The root of therapeutic resistance is hypothesized to be due to the presence of a rare CSC population within the tumour population. When challenged with chemotherapy, this tumour cell subset survives the insult and has the potential to recapitulate a heterogenic tumour. Aldehyde dehydrogenase 1 (ALDH1) is involved in the catalytic conversion of vitamin A (retinol) to retinoic acid and has been identified as a CSC marker in a number of solid malignancies. Methods FACS analysis of an isogenic panel of matched parent (PT) and cisplatin resistant (CisR) NSCLC cell lines of different histologies was used to examine ALDH1 activity. The H460 CisR subline was FACS-sorted (MoFlo®) into ALDH1-positive and ALDH1-negative fractions and subcutaneously injected into NOD/SCID mice to assess tumour initiation and growth. The effects of inhibiting ALDH1 on cell proliferation (BrdU), apoptosis (Annexin-V/propidium iodide) and colony formation (crystal violet) in PT and CisR cell lines was assessed in vitro using two ALDH1 inhibitors, DEAB (N,N-diethylaminobenzaldehyde) and disulfiram (Antabuse), alone and in combination with cisplatin. Lung cancer cells were treated with retinol and All-trans retinoic acid (ATRA) to exploit the role of ALDH1 in the vitamin A/retinoic acid axis in NSCLC. Results ALDH1 activity was significantly increased in all CisR cell lines relative to their parental counterparts in which little or no ALDH1 expression was detected. Xenograft studies using ALDH1+ and ALDH1– fractions from H460 CisR cells showed no significant difference in tumour growth in mice. ALDH1 inhibition, in combination with cisplatin, significantly decreased cell viability, proliferation and clonogenic survival of CisR sublines only and increased apoptotic cell death compared to cells treated with cisplatin alone. Similarly, treatment of cells with retinol or ATRA, in combination with cisplatin, showed similar re-sensitizing effects. Conclusions In this study, we show that inhibition of the CSC marker, ALDH1, re-sensitizes cisplatin resistant NSCLC cells to the cytotoxic effects of cisplatin. Furthermore, our data suggest a role for ALDH1 as a key enzyme in the vitamin A/retinoic acid axis.

70P Identification of a novel microRNA signature: Potential diagnostic biomarkers and predictors of cisplatin response?

Background In addition to their involvement in mechanisms underlying cancer initiation and progression, emerging evidence has highlighted a key role for microRNAs in resistance to a number of anti-cancer therapies. Methods MicroRNA profiling and validation was carried out on a panel of age-matched parent (PT) and cisplatin resistant (CisR) NSCLC cell lines using 7th generation miRCURY LNA™ arrays (Exiqon). Transient transfections were used to either inhibit or over-express these microRNAs using antagomirs or pre-miRs, respectively. The effects of modulating these miRNAs on cell proliferation, apoptosis and clonogenic survival were examined in response to cisplatin. The translational relevance of these miRNAs in lung cancer was confirmed in a cohort of chemo-naive matched normal and tumour lung tissues and sera from NSCLC patients. MicroRNAs were also assessed in mouse xenograft FFPE tissues from H460 CisR-derived ALDH1+ and ALDH1– cancer stem cell subpopulations. Results A 5-miRNA signature consisting of members of the miR-30 family, miR-34a and miR-4286 was identified and validated. MicroRNAs were significantly up-regulated in all CisR cell lines relative to their parental counterparts, in contrast to miR-4286 which was significantly down-regulated. Inhibition of the miR-30 family and miR-34a and forced over-expression of miR-4286 significantly reduced the clonogenic survival ability of CisR cells in response to cisplatin. The miR-30 family was significantly decreased in AD and SCC tissues. MiR-34a expression was significantly lower in SCC tissues while miR-4286 levels were significantly increased in AD tissues only. A significant-fold increase in miR-4286 was found in sera from SCC lung cancer patients. Similar to that found at the in vitro level, the miR-30 family and miR-34a were up-regulated in mouse xenograft FFPE tissues derived from CisR and PT cell lines in vivo. Conclusions In this study, a 5-microRNA signature was identified using an in vitro model of cisplatin resistance. Differential expression of these microRNAs was found between matched normal and tumour lung tissues from NSCLC patients. These may offer potential as diagnostic markers in NSCLC and in predicting response to cisplatin chemotherapy.

Abstract 353: Vorinostat enhances anti-tumor effects of cisplatin in head and neck cancer cells by targeting cancer stem cells

Abstract Objective: Head and neck squamous cell carcinoma (HNSCC) remains a major health care problem worldwide, comprising almost 50% of all malignancies in some developing nations. Even with extensive surgery and full course chemoradiation treatment the survival rates of HNSCC patients have not significantly improved over the last two decades. Acquisition of chemoresistance, locoregional recurrence and metastatic phenotype are the major causes of treatment failure and mortality in these patients. It is therefore imperative that we gain a better understanding of the molecular mechanisms that contribute to the aggressive tumor phenotype in order to develop novel and effective strategies for the treatment of head and neck cancer patients. Recent studies have highlighted the role of histone deacetylases (HDACs) in regulating a number of genes that are involved in cell cycle control, proliferation, survival, DNA repair and differentiation. In this study, we examined if treatment with a HDAC inhibitor, Vorinostat could reverse chemoresistance in head and neck cancer cells and enhance the therapeutic efficacy of cisplatin treatment. Methodology: HDACs expression in tumor samples from HNSCC patients was examined by real time PCR. Cell growth was examined by MTT assay and colony formation assay. Western blot analysis was used to measure changes in protein levels. Cancer stem cell phenotype was assessed by tumorsphere formation. A SCID mouse xenograft model was used to access the in vivo efficacy of Vorinostat. Results: Our results show that HDAC1, 2, and 6 levels are markedly increased in the head and neck cancer cell lines as compared to normal human oral keratinocytes. We took two cisplatin sensitive cell lines and grew them in increasing concentrations of cisplatin to mimic acquired resistance in patients. Both of these cisplatin resistant cell lines (cisR) showed further increase in HDAC1 and 2 expressions as compared to the cispaltin sensitive parental cells. Depletion of HDAC1 and 2 in these cisR cell lines by siRNA significantly enhanced cisplatin sensitivity. Next, we used an HDAC inhibitor, Vorinostat on these cell lines and observed a dose dependent inhibition of cell proliferation (MTT). In addition, Vorinostat treatment significantly decreased tumorsphere formation and the expression of stem cell marker, Nanog. Vorinostat in combination with cisplatin showed a synergistic effect on the inhibition of cell proliferation and tumorsphere formation. We then assessed the efficacy of Vorinostat treatment as a single agent or in combination with cispaltin in SCID mouse xenograft model. Similar to the in vitro results, combination treatment was most effective in reducing the tumor burden in the mice. Conclusions: Taken together, our results suggest that targeting of HDACs with Vorinostat could be an effective treatment strategy for the treatment of HNSCC patients that do not respond to currently used treatment regimens. Citation Format: Bhavna Kumar, Arti Yadav, Theodoros N. Teknos, Pawan Kumar. Vorinostat enhances anti-tumor effects of cisplatin in head and neck cancer cells by targeting cancer stem cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 353. doi:10.1158/1538-7445.AM2015-353

Suberoylanilide hydroxamic acid (SAHA) reverses chemoresistance in head and neck cancer cells by targeting cancer stem cells via the downregulation of nanog.

Acquisition of chemoresistance and metastatic phenotype are the major causes of treatment failure and mortality in head and neck squamous cell carcinoma (HNSCC) patients. Histone deacetylases (HDACs) have been shown to be overexpressed in many tumor types and directly linked to poor prognosis. In this study, we demonstrate that HDACs are markedly elevated in HNSCC. HDACs expression was further increase in cisplatin resistant cell lines (CisR). In addition, cisplatin-resistant cells showed enhanced stem cell properties and tumor metastasis. Depletion of HDAC1 and 2 in CisR cell lines significantly reversed cisplatin resistance and tumorsphere formation. Next, we tested the efficacy of Suberoylanilide hydroxamic acid (SAHA), an HDAC inhibitor, by using both in vitro and in vivo models. SAHA significantly inhibited cell proliferation and synergistically enhanced the anti-proliferative effects of cisplatin. In addition, SAHA significantly decreased tumorsphere formation by markedly reducing nanog expression. In a SCID mouse xenograft model, SAHA significantly enhanced the anti-tumor effects of cisplatin treatment with no added systemic toxicity. Furthermore, SAHA and cisplatin combination treatment significantly decreased tumor metastasis and nanog expression, in vivo. Taken together, our results suggest that targeting HDACs with SAHA could be an effective treatment strategy for the treatment of HNSCC patients.

149P - Microrna Profiling and CDDP-GPG DNA Adduct Formation Analysis in a Panel of Cisplatin Resistant Non-Small Cell Lung Cancer Cell Lines

ABSTRACT Introduction The outcome of cisplatin therapy in NSCLC has reached a plateau, with the development of resistance being a major obstacle in the use of this drug. Understanding the molecular mechanisms underlying this resistance phenotype may aid in the development of novel agents that enhance the sensitivity of cisplatin. In this study, we examined the microRNA profile and levels of cisplatin-DNA adduct formation in a panel of cisplatin resistant NSCLC cell lines. Methods We have previously generated a panel of cisplatin resistant (CisR) cell lines (MOR, H460, A549, SKMES-1, H1299) from original, age-matched parent (PT) cells and extensively characterised these based on a number of functional assays including proliferation (MTT), apoptosis (FACS), cell cycle arrest (PI staining), survival ability (clonogenic assay), DNA copy number gains and losses (CGH arrays) and cancer stem cell marker expression (CD44, CD133, ALDH1, Nanog, Sox-2 and Oct-4) by FACS and Western blot analysis. MicroRNA profiling was carried out using the nCounter miRNA Expression Assay consisting of 749 target miRNA's. Immunofluorescence staining and measurement of specific DNA platination products (CDDP-GpG DNA adducts) was performed at 0, 4, 12 and 24h using immunofluorescence microscopy and quantitative digital image analysis using the ACAS 6.0 CytometryAnalysis System, respectively. Results In a panel of five NSCLC cell lines exhibiting a cisplatin resistant phenotype, a 3- and 13-miRNA signature was deduced, based on differential expression of 3 miRNA's (5/5 cell lines) and 13 miRNA's (4/5 cell lines) in the CisR cell lines relative to their parent counterparts. Levels of CDDP-GpG adducts were greatly reduced in resistant cells up to 24h in response to cisplatin compared to that observed in corresponding parent cells. Conclusion We have identified a distinct miRNA fingerprint in a clinically relevant, isogenic model of cisplatin resistance that may predict resistance to cisplatin in lung cancer. Furthermore, as cisplatin-DNA adducts correlate with cisplatin resistance in vitro, these may be used as a potential predictive marker in patient response to platinum therapy in NSCLC. Disclosure All authors have declared no conflicts of interest.

Prenylation at the indole ring leads to a significant increase of cytotoxicity of tryptophan-containing cyclic dipeptides

Fourteen tryptophan-containing cyclic dipeptides 1a–14a, including all four stereoisomers of cyclo-Trp-Pro and cyclo-Trp-Ala, were converted to their C2-regularly prenylated derivatives 1b–14b in the presence of dimethylallyl diphosphate by using the purified recombinant FtmPT1 as catalyst. The enzyme products were isolated on HPLC in preparative scales and their structures were elucidated by NMR and MS analyses.The cytotoxic effects of the prenylated products and their substrates were tested with human leukemia K562 and ovarian cancer A2780 sens and A2780 CisR cell lines. Preliminary results have been clearly shown that prenylation at C2 led to a significant increase of the cytotoxicity of the tested cyclic dipeptides in all the 14 cases. The second amino acid and the stereochemistry of tryptophan moiety of the cyclic dipeptides showed less influence on the cytotoxicity of the tested compounds.

New pH-dependent complexes, from mononuclear Pd(II) monomer to heteronuclear [Pd(II),K(I)]Polymer: DNA cleavage and cytotoxicity in vitro

Two novel complexes, namely, [Pd(pdc) 2]·(dmp)·6H 2O (1) and [Pd(pdc) 2K(H 2O) 5·3H 2O]n (2) [H 2pdc = pyridine-2,3-dicarboxylate acid, dmp = 2,9-Dimethyl-1,10-phenanthroline] were synthesized and characterized as pH-dependent products. Gel electrophoresis assay demonstrated the ability of the complexes to cleave the pBR 322 plasmid DNA. Values of IC 50 calculated for complexes 1 and 2 show that the two complexes exhibit good cytotoxic activity against different cell lines tested in general, especially more effective against HL-60 and A2780 cisR cell lines.

The relative activity of cisplatin, oxaliplatin and satraplatin in testicular germ cell tumour sensitive and resistant cell lines

Germ cell tumours (GCT) can become resistant to cisplatin, which is associated with a relatively poor prognosis. Oxaliplatin and satraplatin have been developed to overcome cisplatin resistance in other cancers, but their effect in cisplatin resistant (cisR) GCTs is unclear. In this work we address this issue by comparing their efficacy in three paired sensitive and cisR GCT cell lines. Three established cisplatin sensitive (cisS) and resistant cell line pairs were used (GCT27, GCT27r: SUSA, SUSAr: 833k, 833kr). Viability was assessed using a luciferase based ATP assay and EC(50) and EC(80) concentrations were calculated. Western blot analysis and flow cytometry was used for further assessment. Sensitivity to the three platinum compounds was broadly similar in the three cisS lines GCT cell lines (EC(50) = 0.27-0.51 microM for cisplatin, 0.52-0.79 microM for oxaliplatin, 0.31-1.26 microM for satraplatin). EC(50) values for cisplatin in the three cisR sub lines were 1.8- to 3.8-fold higher than in the sensitive parental lines. Cross resistance to satraplatin and oxaliplatin occurred in all three cisR cell lines (resistance factor 1.9-4.4), with the exception of oxaliplatin in the 833Kr (resistance factor 0.9). Differences in the effect of specific drugs on cell cycle distribution, p53, p21 and MDM2 were observed. These data suggest that satraplatin and oxaliplatin could theoretically be used in chemo-naive GCTs and support the further clinical evaluation of these agents in this setting. The mechanism of cross resistance to these drugs appears multifactorial.

Studies on activities, cell uptake and DNA binding of four trans-planaramineplatinum(II) complexes of the form: trans-PtL(NH 3)Cl 2, where L=2-hydroxypyridine, imidazole, 3-hydroxypyridine and imidazo(1,2-α)pyridine

Four trans-planaramineplatinum(II) complexes code named YH9, YH10, YH11 and YH12 each of the form trans-PtL(NH 3)Cl 2, where L=2-hydroxypyridine and 3-hydroxypyridine, imidazole, and imidazo(1,2-α)pyridine for YH9, YH10, YH11 and YH12, respectively, have been synthesized and the activity of the compounds against human cancer cell lines, cell uptake, DNA-binding and nature of interaction with pBR322 plasmid DNA have been studied. The compound having imidazo(1,2-α)pyridine ligand as one the carrier ligands in the trans-configuration is found to be significantly more active than cis-platin against ovarian A2780 cisR cancer cell line corresponding with higher Pt-DNA binding. All other compounds have resistance factors less than that for cis-platin in the A2780 and A2780 cisR cell lines. A greater prevention of BamH1 digestion with increasing concentration of the compounds indicates that as the compounds bind with nucleobases in DNA, the DNA conformation is changed sufficiently so as to prevent BamH1 digestion at the specific GG site. Gel electrophoresis results also indicate that as the compounds bind to DNA, unwinding of supercoiled form I DNA takes place to change it from the negatively supercoiled form I through relaxed circular form I to the positively supercoiled form I.

Anti-proliferative activity and mechanism of action of titanocene dichloride.

Development of resistance to cytotoxic agents is a major limitation to their clinical use. Novel compounds are synthesized with a view to develop non-cross-resistant, less toxic and more potent activity. The detection of the anti-tumour properties of the inorganic compound cisplatin stimulated a broad search for other metal-containing complexes. Titanocene dichloride was synthesized on this basis and has shown potent anti-neoplastic activity in experimental animals. We have examined the in vitro activity of titanocene dichloride in two pairs of platinum-sensitive and resistant human ovarian carcinoma cell lines, A2780/2780CP and CH1/CH1cisR, and in mutated p53- and bcl-2-transfected clones of A2780 cells. A time- and concentration-dependent anti-proliferative effect was observed in all cell lines treated with titanocene dichloride. The drug was found to significantly overcome platinum resistance in the 2780CP and the CH1 cisR cell lines and in the bcl-2 and the mutant p53 transfectants of A2780 cells. Titanocene dichloride induced a block in late S/early G2 phase of the cell cycle; however apoptotic cell death occurred from any phase of cycle. Titanium-DNA adducts were detected in A2780 cells treated with titanocene dichloride using atomic absorption spectrometry, suggesting that DNA may be a target for this drug. In agreement with this finding, p53 accumulated rapidly in drug-treated A2780 cells, indicative of a role for titanocene dichloride as a DNA-damaging agent. We have also performed studies to determine whether titanocene dichloride could demonstrate synergy with other cytotoxic agents in vitro. Isobologram analysis of cytotoxicity data obtained suggests that the combination of titanocene dichloride and 5-fluorouracil (5-FU) is synergistic. The potent in vivo anti-tumour activity of this compound, supported by the encouraging results from two phase I clinical trials, suggests that titanocene dichloride could be a promising novel chemotherapeutic agent.