Cisplatin-resistant Clones Research Articles

Cisplatin-induced activation of TGF-β signaling contributes to drug resistance.

Growing evidence suggests an association between epithelial-mesenchymal transition (EMT), a hallmark of tumor malignancy, and chemoresistance to a number of anti-cancer drugs. However, the mechanism of EMT induction in the process of acquiring anti-cancer drug resistance remains unclear. To address this issue, we obtained a number of cisplatin-resistant clones from LoVo cells and found that almost all of them lost cell-cell contacts. In these clones, the epithelial marker E-cadherin was downregulated, whereas the mesenchymal marker N-cadherin was upregulated. Moreover, the expression of EMT-related transcription factors, including Slug, was elevated. On the other hand, the upregulation of other mesenchymal marker Vimentin was weak, suggesting that the mesenchymal-like phenotypic changes occurred in these cisplatin-resistant clones. These mesenchymal-like features of cisplatin-resistant clones were partially reversed to parental epithelial-like features by treatment with transforming growth factor-β (TGF-β) receptor kinase inhibitors, indicating that TGF-β signaling is involved in cisplatin-induced the mesenchymal-like phenotypic changes. Moreover, cisplatin was observed to enhance the secretion of TGF-β into the culture media without influencing TGF-β gene transcription. These results suggest that cisplatin may induce the mesenchymal-like phenotypic changes by enhancing TGF-β secretion, ultimately resulting in drug resistance.

Role Played by Paraoxonase-2 Enzyme in Cell Viability, Proliferation and Sensitivity to Chemotherapy of Oral Squamous Cell Carcinoma Cell Lines.

Oral squamous cell carcinoma represents the most aggressive and frequent form of head and neck cancer. Due to drug resistance, the 5-year survival rate of patients with advanced disease is less than 50%. In order to identify molecular targets for effective oral cancer treatment, we focused on paraoxonase-2 enzyme. Indeed, based on data previously obtained from preliminary immunohistochemistry and Western blot analyses performed on tissue specimens, the enzyme was found to be upregulated in tumor compared with normal oral mucosa. Therefore, paraoxonase-2 gene silencing was achieved in HSC-3 and HOC621 oral cancer cell lines, and the effect on cell proliferation, viability, apoptosis induction and sensitivity to cisplatin and 5-fluorouracil treatment was evaluated. Fourier Transform InfraRed Microspectroscopy analyzed alterations of cellular macromolecules upon treatment. Enzyme level and cell proliferation were also determined in cisplatin-resistant clones obtained from HOC621 cell line, as well as in parental cells. Reported data showed that paraoxonase-2 knockdown led to a reduction of cell proliferation and viability, as well as to an enhancement of sensitivity to cisplatin, together with the activation of apoptosis pathway. Spectroscopical data demonstrated that, under treatment with cisplatin, oxidative damage exerted on lipids and proteins was markedly more evident in cells down-regulating paraoxonase-2 compared to controls. Interestingly, enzyme expression, as well as cell proliferation were significantly higher in cisplatin-resistant compared with control HOC621 cells. Taken together these results seem to candidate the enzyme as a promising target for molecular treatment of this neoplasm.

TIMP-1 Dependent Modulation of Metabolic Profiles Impacts Chemoresistance in NSCLC.

The development of chemoresistance remains a significant barrier to treating NSCLC. Alteration of cancer cell metabolism is an important mechanism for chemoresistance. This study explored the role of aberrant metabolism in TIMP-1-mediated chemoresistance. Bioinformatics analysis identified an association of high TIMP-1 with altered energy metabolism. We have defined the role of depolarized mitochondria through a reduction in lactate secretion, higher ROS levels in TIMP-1 KD cells and reduced GSH levels. TIMP-1 modulates the metabolic profile via acetylation of mitochondrial STAT3 and its interaction with CD44. Intriguingly, monomers of acetylated STAT3 were critical for altered metabolism, whereas STAT3 dimers abrogated this function. Further, the mitochondrial metabolic profile was also altered in a cisplatin-resistant clone of A549 cells. We also correlated the immunoexpression of CD44, STAT3 and TIMP-1 in patient samples. This study provided evidence that TIMP-1 alters the metabolic profile by modulating mitochondrial metabolism via the CD44-STAT3 axis through its effects on STAT3 acetylation. It also lent further support to the critical role of TIMP-1 in chemoresistance. Interrogation of the TCGA-LUAD dataset revealed perturbations in the critical modulator that can alter metabolic states in cancer cells. Higher expression of a five-gene signature, including TIMP-1, correlated with immunosuppressive cells and was found to be associated with overall survival. This study identified several metabolic mechanisms that could influence therapeutic options and prognosis in NSCLC patients.

Targeting the SREBP-1/Hsa-Mir-497/SCAP/FASN Oncometabolic Axis Inhibits the Cancer Stem-like and Chemoresistant Phenotype of Non-Small Cell Lung Carcinoma Cells.

Background: Lung cancer remains a leading cause of cancer-related death, with an annual global mortality rate of 18.4%. Despite advances in diagnostic and therapeutic technologies, non–small cell lung carcinoma (NSCLC) continues to be characterized by a poor prognosis. This may be associated with the enrichment of cancer stem cells (CSCs) and the development of chemoresistance—a double-edged challenge that continues to impede the improvement of long-term outcomes. Metabolic reprogramming is a new hallmark of cancer. Sterol regulatory element-binding proteins (SREBPs) play crucial regulatory roles in the synthesis and uptake of cholesterol, fatty acids, and phospholipids. Recent evidence has demonstrated that SREBP-1 is upregulated in several cancer types. However, its role in lung cancer remains unclear. Objective: This study investigated the role of SREBP-1 in NSCLC biology, progression, and therapeutic response and explored the therapeutic exploitability of SREBP-1 and SREBP-1-dependent oncometabolic signaling and miRNA epigenetic regulation. Methods: We analyzed SREBP-1 levels and biological functions in clinical samples and the human NSCLC cell lines H441 and A549 through shRNA-based knock down of SREBP function, cisplatin-resistant clone generation, immunohistochemical staining of clinical samples, and cell viability, sphere-formation, Western blot, and quantitative PCR assays. We conducted in-silico analysis of miRNA expression in NSCLC samples by using the Gene Expression Omnibus (GSE102286) database. Results: We demonstrated that SREBP-1 and SCAP are highly expressed in NSCLC and are positively correlated with the aggressive phenotypes of NSCLC cells. In addition, downregulation of the expression of tumor-suppressing hsa-miR-497-5p, which predictively targets SREBP-1, was observed. We also demonstrated that SREBP-1/SCAP/FASN lipogenic signaling plays a key role in CSCs-like and chemoresistant NSCLC phenotypes, especially because the fatostatin or shRNA targeting of SREBP-1 significantly suppressed the viability, cisplatin resistance, and cancer stemness of NSCLC cells and because treatment induced the expression of hsa-miR-497. Conclusion: Targeting the SREBP-1/hsa-miR-497 signaling axis is a potentially effective anticancer therapeutic strategy for NSCLC.

Clonal fitness inferred from time-series modelling of single-cell cancer genomes.

Progress in defining genomic fitness landscapes in cancer, especially those defined by copy number alterations (CNAs), has been impeded by lack of time-series single-cell sampling of polyclonal populations and temporal statistical models1-7. Here we generated 42,000 genomes from multi-year time-series single-cell whole-genome sequencing of breast epithelium and primary triple-negative breast cancer (TNBC) patient-derived xenografts (PDXs), revealing the nature of CNA-defined clonal fitness dynamics induced by TP53 mutation and cisplatin chemotherapy. Using a new Wright-Fisher population genetics model8,9 to infer clonal fitness, we found that TP53 mutation alters the fitness landscape, reproducibly distributing fitness over a larger number of clones associated with distinct CNAs. Furthermore, in TNBC PDX models with mutated TP53, inferred fitness coefficients from CNA-based genotypes accurately forecast experimentally enforced clonal competition dynamics. Drug treatment in three long-term serially passaged TNBC PDXs resulted in cisplatin-resistant clones emerging from low-fitness phylogenetic lineages in the untreated setting. Conversely, high-fitness clones from treatment-naive controls were eradicated, signalling an inversion of the fitness landscape. Finally, upon release of drug, selection pressure dynamics were reversed, indicating a fitness cost of treatment resistance. Together, our findings define clonal fitness linked to both CNA and therapeutic resistance in polyclonal tumours.

Metabolic features of cancer cells impact immunosurveillance

BackgroundTumors rewire their metabolism to achieve robust anabolism and resistance against therapeutic interventions like cisplatin treatment. For example, a prolonged exposure to cisplatin causes downregulation of pyridoxal kinase (PDXK), the...

Metabolic vulnerability of cisplatin-resistant cancers.

Cisplatin is the most widely used chemotherapeutic agent, and resistance of neoplastic cells against this cytoxicant poses a major problem in clinical oncology. Here, we explored potential metabolic vulnerabilities of cisplatin-resistant non-small human cell lung cancer and ovarian cancer cell lines. Cisplatin-resistant clones were more sensitive to killing by nutrient deprivation invitro and invivo than their parental cisplatin-sensitive controls. The susceptibility of cisplatin-resistant cells to starvation could be explained by a particularly strong dependence on glutamine. Glutamine depletion was sufficient to restore cisplatin responses of initially cisplatin-resistant clones, and glutamine supplementation rescued cisplatin-resistant clones from starvation-induced death. Mass spectrometric metabolomics and specific interventions on glutamine metabolism revealed that, in cisplatin-resistant cells, glutamine is mostly required for nucleotide biosynthesis rather than for anaplerotic, bioenergetic or redox reactions. As a result, cisplatin-resistant cancers became exquisitely sensitive to treatment with antimetabolites that target nucleoside metabolism.

LncRNAs are novel biomarkers for differentiating between cisplatin-resistant and cisplatin-sensitive ovarian cancer.

Cisplatin-resistant ovarian cancer occurs in patients with ovarian cancer treated with cisplatin-based chemotherapy, which results in tumor progression during treatment, or recurrence of the tumor within 6 months of the treatment. It is vital that a novel biomarker for diagnosis, or an efficient therapeutic target of cisplatin-resistant ovarian is identified. Long non-coding (lnc)RNAs were determined to serve critical functions in a variety of distinct types of cancer, including ovarian cancer; however, there is limited knowledge regarding the differential expression levels of lncRNAs in cisplatin-resistant and cisplatin-sensitive ovarian cancer. Therefore, in the present study, the expression levels were determined for these cancer types. The lncRNA expression profile in cisplatin-resistant ovarian cancer was analyzed and compared with the results for cisplatin-sensitive ovarian cancer; gene ontology and pathway analysis demonstrated that the dysregulated lncRNAs participated in important biological processes. Subsequently, it was identified that these dysregulated lncRNAs were present in other ovarian cancer tissues and in SKOV3 ovarian cancer cells, as well as its cisplatin-resistant clone, SKOV3/CDDP. In addition, it was revealed that 8 lncRNAs (Enst0000435726, Enst00000585612, Enst00000566734, Enst00000453783, NR_023915, RP11_697E22.2, uc010jub.1 and tcons_00008505) were associated with cisplatin-resistant ovarian cancer. The present study may assist in improving understanding of the initiation and developmental mechanisms underlying cisplatin-resistant ovarian cancer, which could aid future studies in discovering potential biomarkers for diagnosis or therapeutic targets that may be used in clinical treatment.

The IGF-1R/AKT pathway has opposing effects on Nutlin-3a-induced apoptosis

ABSTRACTNutlin-3a is a small molecule MDM2 antagonist and potent activator of wild-type p53. Nutlin-3a disrupts MDM2 binding to p53, thus increasing p53 levels and allowing p53 to inhibit proliferation or induce cell death. Factors that control sensitivity to Nutlin-3a-induced apoptosis are incompletely understood. In this study we isolated cisplatin-resistant clones from MHM cells, an MDM2-amplified and p53 wild-type osteosarcoma cell line. Cisplatin resistance in these clones resulted in part from heightened activation of the IGF-1R/AKT pathway. Interestingly, these cisplatin resistant clones showed hyper-sensitivity to Nutlin-3a induced apoptosis. Increased Nutlin-3a sensitivity was associated with reduced authophagy flux and a greater increase in p53 levels in response to Nutlin-3a treatment. IGF-1R and AKT inhibitors further increased apoptosis by Nutlin-3a in parental MHM cells and the cisplatin-resistant clones, confirming IGF-1R/AKT signaling promotes apoptosis resistance. However, IGF-1R and AKT inhibitors also reduced p53 accumulation in Nutlin-3a treated cells and increased autophagy flux, which we showed can promote apoptosis resistance. We conclude the IGF-1R/AKT pathway has opposing effects on Nutlin-3a-induced apoptosis. First, it can inhibit apoptosis, consistent with its well-established role as a survival-signaling pathway. Second, it can enhance Nutlin-3a induced apoptosis through a combination of maintaining p53 levels and inhibiting pro-survival autophagy.

Abstract AP29: BRCA1 RING DOMAIN–DEFICIENT PROTEINS PROMOTE PARP INHIBITOR AND PLATINUM RESISTANCE

Abstract Although cancers harboring BRCA1 mutations initially respond well to platinum and poly(ADP-ribose)-polymerase inhibitor (PARPi) therapy, resistance invariably arises and is a major clinical problem. The BRCA1185delAG allele is a common inherited mutation located close to the protein translation start site, thought to produce a short peptide devoid of function. In this study, we utilized the SUM1315MO2 cancer cell line that harbors a hemizygous BRCA1185delAG mutation to study PARPi and platinum resistance. SUM1315MO2 cells were initially PARPi and cisplatin sensitive but readily acquired resistance. PARPi and cisplatin resistant clones did not harbor secondary reversion mutations. Rather, increased expression of a RING domain-deficient BRCA1 protein (Rdd-BRCA1) was required for resistance. Translation initiation occurred downstream of the frameshift mutation, likely at the BRCA1-Met-297 codon. In contrast to full-length BRCA1, Rdd-BRCA1 did not require BARD1 interaction for stability. Functionally, Rdd-BRCA1 formed irradiation-induced foci and supported RAD51 foci-formation. Ectopic overexpression of Rdd-BRCA1 promoted partial PARPi and cisplatin resistance in vitro and in vivo. Furthermore, Rdd-BRCA1 protein expression was detectable in recurrent carcinomas from germline BRCA1185delAG mutation carriers. Taken together, these results indicate that RING-deficient BRCA1 proteins are hypomorphic, and when expressed at high enough levels are capable of contributing to PARPi and platinum resistance. Citation Format: Yifan Wang, John J. Krais, Andrea J. Bernhardy, Emmanuelle Nicolas, Kathy Q. Cai, Maria I. Harrell, Hyoung H. Kim, Erin George, Elizabeth M. Swisher, Fiona Simpkins and Neil Johnson. BRCA1 RING DOMAIN–DEFICIENT PROTEINS PROMOTE PARP INHIBITOR AND PLATINUM RESISTANCE [abstract]. In: Proceedings of the 11th Biennial Ovarian Cancer Research Symposium; Sep 12-13, 2016; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(11 Suppl):Abstract nr AP29.

Cisplatin selects short forms of the mitochondrial DNA OriB variant (16184\u201316193 poly-cytosine tract), which confer resistance to cisplatin

A number of alternations in mitochondrial DNA (mtDNA) have been reported in different types of cancers, and the role of mtDNA in cancer has been attracting increasing interest. In order to investigate the relationship between mtDNA alternations and chemosensitivity, we constructed cybrid (trans-mitochondrial hybrid) cell lines carrying a HeLa nucleus and the mtDNA of healthy individuals because of the presence of somatic alternations in the mtDNA of many cancer cells. After a treatment with 1.0 μg/mL cisplatin for 10 days, we isolated 100 cisplatin-resistant clones, 70 of which carried the shorter mtDNA OriB variant (16184–16193 poly-cytosine tract), which was located in the control region of mtDNA. Whole mtDNA sequencing of 10 clones revealed no additional alternations. Re-construction of the HeLa nucleus and mtDNA from cisplatin-resistant cells showed that cisplatin resistance was only acquired by mtDNA alternations in the control region, and not by possible alternation(s) in the nuclear genome.

RING domain-deficient BRCA1 promotes PARP inhibitor and platinum resistance.

Patients with cancers that harbor breast cancer 1 (BRCA1) mutations initially respond well to platinum and poly(ADP-ribose) polymerase inhibitor (PARPi) therapy; however, resistance invariably arises in these patients and is a major clinical problem. The BRCA1185delAG allele is a common inherited mutation located close to the protein translation start site that is thought to produce a shortened, nonfunctional peptide. In this study, we investigated the mechanisms that lead to PARPi and platinum resistance in the SUM1315MO2 breast cancer cell line, which harbors a hemizygous BRCA1185delAG mutation. SUM1315MO2 cells were initially sensitive to PARPi and cisplatin but readily acquired resistance. PARPi- and cisplatin-resistant clones did not harbor secondary reversion mutations; rather, PARPi and platinum resistance required increased expression of a really interesting gene (RING) domain-deficient BRCA1 protein (Rdd-BRCA1). Initiation of translation occurred downstream of the frameshift mutation, probably at the BRCA1-Met-297 codon. In contrast to full-length BRCA1, Rdd-BRCA1 did not require BRCA1-associated RING domain 1 (BARD1) interaction for stability. Functionally, Rdd-BRCA1 formed irradiation-induced foci and supported RAD51 foci formation. Ectopic overexpression of Rdd-BRCA1 promoted partial PARPi and cisplatin resistance. Furthermore, Rdd-BRCA1 protein expression was detected in recurrent carcinomas from patients who carried germline BRCA1185delAG mutations. Taken together, these results indicate that RING-deficient BRCA1 proteins are hypomorphic and capable of contributing to PARPi and platinum resistance when expressed at high levels.

Synthesis, characterization and anticancer activity of gold(III) complexes with (1R,2R)-(−)-1,2-diaminocyclohexane

Two gold(III) complexes, [(DACH)AuCl2]Cl (1) and [(DACH)2Au]Cl3 (2), derived from (1R,2R)-(−)-1,2-diaminocyclohexane and sodium tetrachloroaurate dihydrate, NaAuCl4·2H2O, were synthesized and fully characterized using elemental analysis and different physical methods, such as FTIR, 1H NMR, 13C NMR, solid-state NMR and X-ray crystallography. The data confirm the coordination of the DACH ligand to the gold(III) metal centre through its nitrogen atoms. According to in vitro cytotoxic studies, complex 2 was more effective against the three human tumor cell lines evaluated (the ovarian cancer cell line A2780, its cisplatin-resistant clone A2780cis and the classical Hodgkin lymphoma cell line L-540). The fold-resistance of the A2780 and A2780cis cells to cisplatin was about twofold lower for both gold(III) complexes.

Abstract 5467: BRCA1 N-terminal-deficient proteins provide PARP inhibitor and platinum resistance

Abstract Introduction: Tumors harboring BRCA1 mutations initially respond well to platinum and PARP inhibitor therapy; however, resistance invariably arises and is a major clinical problem. The BRCA1 185delAG allele is a common founder mutation located close to the protein translation start site, thought to produce a short peptide devoid of function. Experimental procedures: In this study, we utilized the SUM1315MO2 breast cancer cell line that harbors a BRCA1 185delAG mutation to study mechanisms of PARP inhibitor and platinum resistance. SUM1315MO2 cells were cultured in the presence of increasing concentrations of the PARP inhibitor rucaparib or cisplatin until rucaparib resistant (RR) and cisplatin resistant (CR) clones emerged. Results: DNA sequencing revealed that no BRCA1 gene reversion mutations were present in resistant cells. We next measured BRCA1 protein levels by Western blotting with antibodies specific for the N- and C-terminal domains of BRCA1. As a control, the wild-type BRCA1 protein expressed in MDA-MB-231 cells was detectable with both N- and C-terminal antibodies. In contrast, BRCA1 protein was undetectable in both SUM1315MO2 parental and resistant clones using the N-terminal specific antibody. However, the C-terminal specific antibody identified a more quickly migrating band of low abundance in parental cells, but with elevated expression in both RR and CR clones. We inferred that this protein was devoid of the extreme N-terminal RING domain-containing region that mediates interaction with BARD1. To investigate the functionality of the N-terminal truncated BRCA1 protein (Nt-BRCA1), we measured BRCA1 and RAD51 irradiation-induced focus formation by immunofluorescence. RR and CR clones demonstrated 2.25 - 2.75-fold increase (P = 0.024) in the number of cells with BRCA1 foci compared to parental cells. Similarly, we detected a 2 - 2.85-fold increase (P = 0.0325) in RAD51 foci formation in resistant clones compared to parental cells. Additionally, BRCA1 siRNA treated cells were 12- and 9-fold more sensitive to rucaparib compared to scrambled siRNA-treated control cells (P = 0.0002). Similarly, CR-1 cells treated with BRCA1 siRNAs were 1.56- and 1.7-fold more sensitive to cisplatin (P = 0.0346) compared to scrambled siRNA treated cells. Furthermore, N-terminal truncated BRCA1 proteins were detectable in a primary tumor from a germline BRCA1185delAG mutation carrier. Conclusions: Taken together, these results provide evidence for a novel, mutation location-dependent mechanism of PARP inhibitor and platinum resistance. Citation Format: Yifan Wang, Andrea Bernhardy, Emmanuelle Nicolas, Ryan Winters, Kathy Cai, Kelly Duncan, James Duncan, Maria Harrell, Elizabeth Swisher, Neil Johnson. BRCA1 N-terminal-deficient proteins provide PARP inhibitor and platinum resistance. [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 5467. doi:10.1158/1538-7445.AM2015-5467

Resistance to therapy in BRCA2 mutant cells due to loss of the nucleosome remodeling factor CHD4.

Hereditary cancers derive from gene defects that often compromise DNA repair. Thus, BRCA-associated cancers are sensitive to DNA-damaging agents such as cisplatin. The efficacy of cisplatin is limited, however, by the development of resistance. One cisplatin resistance mechanism is restoration of homologous recombination (HR), which can result from BRCA reversion mutations. However, in BRCA2 mutant cancers, cisplatin resistance can occur independently of restored HR by a mechanism that remains unknown. Here we performed a genome-wide shRNA screen and found that loss of the nucleosome remodeling factor CHD4 confers cisplatin resistance. Restoration of cisplatin resistance is independent of HR but correlates with restored cell cycle progression, reduced chromosomal aberrations, and enhanced DNA damage tolerance. Suggesting clinical relevance, cisplatin-resistant clones lacking genetic reversion of BRCA2 show de novo loss of CHD4 expression in vitro. Moreover, BRCA2 mutant ovarian cancers with reduced CHD4 expression significantly correlate with shorter progression-free survival and shorter overall survival. Collectively, our findings indicate that CHD4 modulates therapeutic response in BRCA2 mutant cancer cells.

Increased Adenovirus Type 5 Mediated Transgene Expression Due to RhoB Down-Regulation

Adenovirus type 5 (Ad5) is a non-enveloped DNA virus frequently used as a gene transfer vector. Efficient Ad5 cell entry depends on the availability of its primary receptor, coxsackie and adenovirus receptor, which is responsible for attachment, and integrins, secondary receptors responsible for adenovirus internalization via clathrin-mediated endocytosis. However, efficacious adenovirus-mediated transgene expression also depends on successful trafficking of Ad5 particles to the nucleus of the target cell. It has been shown that changes occurring in tumor cells during development of resistance to anticancer drugs can be beneficial for adenovirus mediated transgene expression. In this study, using an in vitro model consisting of a parental cell line, human laryngeal carcinoma HEp2 cells, and a cisplatin-resistant clone CK2, we investigated the cause of increased Ad5-mediated transgene expression in CK2 as compared to HEp2 cells. We show that the primary cause of increased Ad5-mediated transgene expression in CK2 cells is not modulation of receptors on the cell surface or change in Ad5wt attachment and/or internalization, but is rather the consequence of decreased RhoB expression. We propose that RhoB plays an important role in Ad5 post-internalization events and more particularly in Ad5 intracellular trafficking. To the best of our knowledge, this is the first study showing changed Ad5 trafficking pattern between cells expressing different amount of RhoB, indicating the role of RhoB in Ad5 intracellular trafficking.

Preclinical evaluation of a new liposomal formulation of cisplatin, lipoplatin, to treat cisplatin-resistant cervical cancer

ObjectiveCisplatin-based chemotherapy has been shown to improve survival in cervical cancer; however, treatment is associated with tumor resistance and significant toxicity. Lipoplatin is a new liposomal formulation of cisplatin, developed to reduce cisplatin toxicity, to improve drug accumulation at tumor sites and to overcome drug resistance.The aim of this study is to analyze the antitumoral activity of lipoplatin against cisplatin-resistant cervical cancer cells and to investigate its mechanism of action. MethodsThe activity and mechanism of action of lipoplatin were studied in the ME-180 cervical cancer cell line and its cisplatin-resistant clone R-ME-180 and HeLa cells using cell proliferation assays, flow cytometry, ELISA assay, cell migration, spheroids and tumor xenograft. ResultsWe demonstrated that lipoplatin exhibited a potent antitumoral activity on HeLa, ME-180 cells and its cisplatin-resistant clone R-ME-180. Lipoplatin inhibited cell proliferation in a dose-dependent manner and was more active than the reference drug cisplatin in R-ME-180 cells and induced apoptosis, as evaluated by Annexin-V staining and DNA fragmentation, caspases 9 and 3 activation, Bcl-2, and Bcl-xL down-regulation, but Bax up-regulation inhibited thioredoxin reductase (TrxR) enzymatic activity and increased reactive oxygen species (ROS) accumulation; reduced EGFR expression and inhibited both migration and invasion. R-ME-180, but not ME-180 cells, generated three-dimensional (3D)-multicellular spheroids expressing the cancer stem cell marker ALDH. The ability of R-ME-180 cells to form spheroids in vitro and tumors in nude mice was also remarkably decreased by lipoplatin. ConclusionsOverall, our results suggest that lipoplatin has potential for the treatment of cisplatin-resistant cervical cancer.

Cisplatin causes cell death via TAB1 regulation of p53/MDM2/MDMX circuitry

The interdependence of p53 and MDM2 is critical for proper cell survival and cell death and, when altered, can lead to tumorigenesis. Mitogen-activated protein kinase (MAPK) signaling pathways function in a wide variety of cellular processes, including cell growth, migration, differentiation, and death. Here we discovered that transforming growth factor β-activated kinase 1 (TAK1)-binding protein 1 (TAB1), an activator of TAK1 and of p38α, associates with and inhibits the E3 ligase activity of MDM2 toward p53 and its homolog, MDMX. Depletion of TAB1 inhibits MDM2 siRNA-mediated p53 accumulation and p21 induction, partially rescuing cell cycle arrest induced by MDM2 ablation. Interestingly, of several agents commonly used as DNA-damaging therapeutics, only cell death caused by cisplatin is mitigated by knockdown of TAB1. Two mechanisms are required for TAB1 to regulate apoptosis in cisplatin-treated cells. First, p38α is activated by TAB1 to phosphorylate p53 N-terminal sites, leading to selective induction of p53 targets such as NOXA. Second, MDMX is stabilized in a TAB1-dependent manner and is required for cell death after cisplatin treatment. Interestingly TAB1 levels are relatively low in cisplatin-resistant clones of ovarian cells and in ovarian patient's tumors compared with normal ovarian tissue. Together, our results indicate that TAB1 is a potential tumor suppressor that serves as a functional link between p53-MDM2 circuitry and a key MAPK signaling pathway.

Abstract 3288: Isolation of anti-neoplastic drug candidates against metastatic breast, lungs and ovarian carcinoma cells.

Abstract Cancer is a leading cause of death in man and women worl-wide. In recent years, wold-wide increase of obesity and diabetes, consumption of food additives and pollution are affecting quality of life and the incidense of all types of cancers are on the rise. Although new treatment regimens are also introduced into clinic war against cancer is still not won. Treatment of cancer with synthetic chemoterapics come with a unfavorable side effects which often ends therapy. Instead of investing in synthetic compounds, OZES BIOPHARMA is engaged in discovery of anti-neoplastic drug candidates from endemic plants of TURKEY. We have been extracting and analyzing organic compounds from variety of plants consumed as a food. Our search in past two years yielded very enchoraging results and we have discovered 6 novel compounds named OZS 1-6. Our compounds show significant levels of anti-tumor and anti-metastatic activities against human metastatic and non-metastatic breast cancer cells(MDA-231,MCF7), lung cancer cells (A549), Ovarian cancer cells (A2780) and its cisplatin resistant clone. Currently, we are idientifying the pathways inhibited or activated by our compounds. Citation Format: Osman N. Ozes. Isolation of anti-neoplastic drug candidates against metastatic breast, lungs and ovarian carcinoma cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3288. doi:10.1158/1538-7445.AM2013-3288

Two 4N Cell-Cycle Arrests Contribute to Cisplatin-Resistance

Cisplatin is a platinum-based drug that is used for the treatment of a wide-variety of primary human cancers. However, the therapeutic efficacy of cisplatin is often limited by intrinsic or acquired drug resistance. An important goal, therefore, is to identify mechanisms that lead to cisplatin resistance in cancer, and then use this information to more effectively target resistant cells. Cisplatin-resistant clones of the HCT116 cell line underwent a prolonged G2 arrest after cisplatin treatment while sensitive clones did not. The staurosporine analog UCN-01 abrogated this G2 arrest and sensitized the resistant clones to cisplatin. At later time points, 4N arrested cells assumed a tetraploid G1 state that was characterized by depletion of Cyclin A, Cyclin B, and CDC2, and increased expression of p53 and p21, in 4N cells. siRNA-mediated knockdown of p21 abrogated the tetraploid G1 arrest and induced killing that was dependent on p53. The results identify two targetable 4N arrests that can contribute to cisplatin resistance: First, a prolonged G2 arrest that can be targeted by UCN-01, and second, a tetraploid G1 arrest that can be targeted by siRNA against p21.