Glutamine serves as an important nutrient with many cancer types displaying glutamine dependence. Following cellular uptake glutamine is converted to glutamate in a reaction catalysed by mitochondrial glutaminase. This glutamate has many uses, including acting as an anaplerotic substrate  to replenish TCA cycle intermediates. CB-839 is a potent, selective, orally bioavailable inhibitor of glutaminase that has activity in Triple receptor-Negative Breast Cancer (TNBC) cell lines and evidence of efficacy in advanced TNBC patients.A panel of eleven breast cancer cell lines was used to investigate the anti-proliferative effects of the glutaminase inhibitors CB-839 and BPTES in different types of culture medium, with or without additional pyruvate supplementation. The abundance of the TCA cycle intermediate fumarate was quantified as a measure if TCA cycle anaplerosis. Pyruvate secretion by TNBC cultures was then assessed with or without AZD3965, a monocarboxylate transporter 1 (MCT1) inhibitor. Finally, two dimensional (2D) monolayer and three dimensional (3D) spheroid assays were used to compare the effect of microenvironmental growth conditions on CB-839 activity.The anti-proliferative activity of CB-839 in a panel of breast cancer cell lines was similar to published reports, but with a major caveat; growth inhibition by CB-839 was strongly attenuated in culture medium containing pyruvate. This pyruvate-dependent attenuation was also observed with a related glutaminase inhibitor, BPTES. Studies demonstrated that exogenous pyruvate acted as an anaplerotic substrate preventing the decrease of fumarate in CB-839-treated conditions. Furthermore, endogenously produced pyruvate secreted by TNBC cell lines was able to act in a paracrine manner to significantly decrease the sensitivity of recipient cells to glutaminase inhibition. Suppression of pyruvate secretion using the MCT1 inhibitor AZD3965, antagonised this paracrine effect and increased CB-839 activity. Finally, CB-839 activity was significantly compromised in 3D compared with 2D TNBC culture models, suggesting that 3D microenvironmental features impair glutaminase inhibitor responsiveness.This study highlights the potential influence that both circulating and tumour-derived pyruvate can have on glutaminase inhibitor efficacy. Furthermore, it highlights the benefits of 3D spheroid cultures to model the features of the tumour microenvironment and improve the in vitro investigation of cancer metabolism-targeted therapeutics. GAC splice variant encoded by the GLS gene [Cancer cells utilise glutamine to aid in the biosynthetic, bioenergetic and redox needs that are associated with proliferation –4. Many GLS gene , 6. The GLS gene , 4. ThisGLS gene . Once αKGLS gene –11.n = 27), the outcome of the Phase II study was less encouraging with ORR of 6%  [This dependence on glutamine anaplerosis renders TNBC cells at increased sensitivity to pharmacological glutaminase inhibition both in vitro and in vivo , 12, 13. cohort) , 15. A gWhile preclinical and clinical studies have confirmed the sensitivity of TNBC to glutaminase inhibition, additional reports in a variety of cancer types have uncovered a set of intrinsic and extrinsic determinants that can impair cellular sensitivity to glutaminase inhibitors. Cells derived from mouse models of non-small cell lung cancer (NSCLC) were highly dependent on glutamine for TCA cycle anaplerosis and proliferation when grown in cell culture but utilised minimal glutamine when grown in vivo, relying instead on glucose metabolism to fuel the TCA cycle . This fiSLC7A11 promotes this cystine-dependent increase in glutamine utilisation resulting in glutamine dependence [SLC7A11, including KEAP1 mutation and the subsequent NRF2 (NFE2L2)-driven antioxidant response [One possible contributor for the loss of glutamine dependence observed in vivo is the lower cystine concentration in tumours compared with cell culture medium. Growing cells in vitro in physiological concentrations of cystine 20–50 μM) suppressed the level of glutamine anaplerosis and subsequently desensitised cells to CB-839 . On the pendence . Many on–50 μM suA number of other metabolites have been identified that can reduce glutamine dependence. For example, increasing the levels of exogenous glutamate can support cell proliferation in times of glutamine deprivation or glutaminase inhibition –23. LikeIn this study we investigated a key difference in culture medium composition that can influence the sensitivity of TNBC cell lines to pharmacological glutaminase inhibition. We show that extracellular pyruvate, at physiological concentrations of 20–100 μM, can significantly impair CB-839 potency in vitro by acting as an anaplerotic substrate. Normal blood pyruvate concentration is reported in the range of 30–150 μM –28. FurtCell lines used in this study were sourced from American Type Culture Collection , with the exception of Hs578T , MDA-MB-231-luc-D3H2LN  and SUM159PT . All cell lines were tested negative for mycoplasma contamination , but have not been STR authenticated since 2013. Cells were cultured in either RPMI 1640 , αMEM  or DMEM  supplemented with 5% v/v foetal bovine serum , as indicated. SUM159PT was routinely cultured in DMEM:F12  + 5% FBS + 1 μg/mL hydrocortisone  + 5 μg/mL insulin (Sigma-Aldrich).CB-839 , Bis-2- ethyl sulphide (BPTES)  and AZD3965  were dissolved in DMSO to generate 10–25 mM stock solutions, aliquoted and stored at − 20 °C. Sterile 100 mM sodium pyruvate was acquired from ThermoFisher Scientific.3 per well) in 96-well plates in the presence of varying concentrations of inhibitor for 3 or 4 days, as indicated. 3H-thymidine  monolayer culture or 0.08 μCi per well for three dimensional (3D) spheroid culture, see below) was added (6 h for 2D monolayer culture or 16 h for 3D spheroid culture) prior to harvest, cells were then harvested on glass fiber filters using an automated TomTec harvester. Filters were incubated with Betaplate Scint and thymidine incorporation measured in a Trilux/Betaplate counter. Effects of inhibitors on the incorporation of 3H-thymidine into DNA were determined relative to the control (non-drug-treated) samples.Proliferation was measured using a thymidine incorporation assay, as described previously . Briefly5 MDA-MB-231 cells were seeded per well of 6-well plates  in 1 mL of RPMI 1640 + 5% FBS. An additional 1 mL of RPMI 1640 + 5% FBS was added containing either CB-839 to a final concentration of 1 μM or an equivalent volume of DMSO. 20 μL of 100 mM sodium pyruvate  was added to achieve a final concentration of 1 mM. After 24 h incubation culture medium was aspirated and cell monolayers were washed twice in cold saline and then lysed on ice using 0.1 mL of Fumarate Assay Buffer . Lysate was transferred into 1.5 mL microcentrfuge tubes and samples were then centrifuged for 10 min at 13,000×g at 4 °C to remove debris. The assay was conducted as detailed in the instruction booklet using 50 μL of the lysate supernatant. Sample absorbance at 450 nm was measured in an EnSpire Multimode Plate Reader (PerkinElmer) and fumarate amount (nmol) was interpolated from the standard curve. Lysate protein concentration was then quantified using bicinchoninic acid assay. The amount of fumarate (nmol) was then normalised to either initial number of cells seeded or protein (mg) to account for changes in cell number caused by drug treatment.2.5 × 105), MDA-MB-231-luc-D3H2LN (2.5 × 105) or SUM159PT (2.5 × 105) cells were seeded per well of 6-well plates in 1 mL of RPMI 1640 + 5% FBS. An additional 1 mL of RPMI 1640 + 5% FBS was added containing either AZD3965 to a final concentration of 1 μM or an equivalent volume of DMSO. After 48 h incubation, the conditioned culture medium was collected and centrifuged at 800×g to pellet floating cells and cell debris. Aliquots of the conditioned culture medium were diluted 1:3 in Pyruvate Assay Buffer  and pyruvate concentrations were quantified according to the instruction booklet. Briefly, fluorometric signal at excitation/emission 540/590 nm was measured in an EnSpire Multimode Plate Reader and pyruvate concentration was extrapolated from the standard curve.Hs578T (1.5 × 10The conditioned culture medium (or unconditioned RPMI 1640 + 5% FBS) described in the pyruvate assay method above was then supplemented with CB-839 to a final concentration of 0.01, 0.1, 1 or 10 μM. MDA-MB-231 cells were then grown in this conditioned culture medium and thymidine incorporation was assessed after 3 days growth.3 cells/well were seeded into ultra-low attachment round bottom 96-well plates  in ice-cold 50 μL RPMI 1640 + 5% FBS containing 2.5% (v/v) Geltrex LDEV-Free Reduced Growth Factor Basement Membrane Matrix . The plates were centrifuged at 800×g for 10 min and placed in the cell culture incubator to allow establishment of spheroids. On day 1, 50 μL of fresh RPMI 1640 + 5% FBS was added to the plates. On day 4, 100 μL of RPMI 1640 + 5% FBS containing CB-839 at 2× the desired final concentration was added to the spheroid plates. Images of the spheroids were captured using a 10× objective on a JuLI™ Stage Real-Time Cell History Recorder  on day 4 and day 8. On day 7 3H-thymidine was added to the cultures and thymidine incorporation was detected following a 16 h overnight incubation.On day 0, 5 × 10P < 0.05 were considered to be statistically significant. All statistical analyses presented represent data from independent studies .Statistical analysis and graphing was conducted using Prism . Comparison of two groups was done by unpaired t test. Comparison of three or more groups was done by one-way or two-way analysis of variance (ANOVA) with Dunnett’s or Tukey’s post hoc test, respectively. Correlation analysis was done using Pearson r correlation coefficient. Values of 50 analysis. The observed IC50 of 3.3 ± 0.71 μM  was > 100-fold higher than the reported value of 26 nM [3H-thymidine incorporation versus Promega Cell Titer Glo assay). To understand this discrepancy we treated MDA-MB-231 cultures with CB-839 in RPMI 1640 + 5% FBS (hereafter RPMI 1640) as used by Gross et al., 2014 and this change in culture medium resulted in an IC50 of 19.3 ± 9.3 nM (n = 3), in strong agreement with the published value   were treated with CB-839 and drug sensitivity was assessed 3 days later by IC50 values were lower in RPMI 1640 compared with αMEM culture medium in many cell lines tested, with statistically significant effects observed in four cases; BT549, MDA-MB-468, Hs578T and SKBR3, and a trend toward an effect in HCC1143 and MCF7. T47D and BT474 displayed no difference in CB-839 IC50 when cultured in either RPMI 1640 or αMEM.To explore this relationship further, a panel of 10 additional breast cancer cell lines were treated with CB-839 in either RPMI 1640 or αMEM  to act as alternative anaplerotic substrates that can rescue cell viability during glutamine deprivation and in some cases antagonise the activity of glutaminase inhibitors . We revi50 value from 20.7 ± 8.7 nM (n = 3) in the unsupplemented RPMI 1640 to 2.4 ± 0.2 μM (n = 5) in the culture medium supplemented with 1 mM sodium pyruvate (mean ± sem). All pyruvate concentrations above 31 μM resulted in statistically significant increases in CB-839 IC50 compared with unsupplemented RPMI 1640.To test this hypothesis MDA-MB-231 cultures were treated with CB-839 in RPMI 1640 supplemented with increasing concentrations of sodium pyruvate  in the unsupplemented culture medium to 4.5 ± 2.0 μM (n = 4) in the culture medium supplemented with 1 mM sodium pyruvate (mean ± sem). CB-839 IC50 was significantly increased at pyruvate concentrations ≥100 μM. In Hs578T cultures CB-839 IC50 increased from 1.2 ± 0.2 μM in the unsupplemented culture medium to 3.3 ± 0.4 μM in the culture medium supplemented with 1 mM sodium pyruvate (n = 4). In this cell line the CB-839 IC50 was significantly increased at pyruvate concentrations ≥500 μM.BT549 and Hs578T also demonstrated pyruvate concentration-dependent changes in glutaminase inhibitor sensitivity Fig. b and c. In sensitive cells glutaminase inhibition using CB-839 was reported to decrease the concentration of many TCA cycle intermediates, including malate, citrate and fumarate , 23. To Further studies were conducted to determine whether these effects were dependent to pyruvate carboxylase (PC) activity  or SLC16A3 (MCT4). This suggests that MCT expression may be a key determinant that allows extracellular pyruvate to suppress CB-839 response.The expression of 12 genes involved in pyruvate uptake, mitochondrial transport and metabolism was compared for the 11 breast cancer cell lines using data from the Cancer Cell Line Encyclopedia . The celn = 3, Fig. The pyruvate concentration in RPMI 1640 + 5% FBS culture medium was quantified at 2.8 ± 0.2 μM  culture medium were then supplemented with various concentrations of CB-839 and used to culture drug-naïve MDA-MB-231 cells. The conditioned culture medium with the highest concentrations of pyruvate provided resistance to CB-839, in agreement with studies using sodium pyruvate supplementation , CB-839 caused a clear concentration-dependent decrease in cell proliferation in all three cell lines  was 0.88 ± 0.26 μM. This change represents a loss in sensitivity of > 100-fold compared with the 2D monolayer IC50 of 8.4 ± 0.17 nM . This 2D IC50 for a 4 day CB-839 exposure was slightly lower than the 19.3 and 20.7 nM IC50 values previously observed with 3 day drug exposure . Similarly, a 14-fold loss of CB-839 activity was observed in Hs578T with 3D IC50 of 6.6 ± 2.0 μM compared with 2D IC50 0.45 ± 0.022 μM . Again, the 4 day drug exposure produced a modestly lower IC50 than the 3 day IC50 of 1.0 and 1.2 μM observed previously  in patients treated with CB-839 in a 600 mg BID (fed) schedule was approximately 11 μM*h with C 0.65 μM . Thus, cvia pyruvate dehydrogenase complex-dependent glucose metabolism, the results from studies in clear cell renal cell carcinoma (ccRCC) display supressed glucose oxidation in the TCA cycle, more reflective of the classic “Warburg Effect” observed in most in vitro studies [1-13C-glutamine studies in VHL-deficient ccRCC tumour xenografts confirm that glutamine is a significant anaplerotic nutrient [Human studies using isotope-labelled glucose or glutamine are providing a clearer understanding of the metabolic processes, and heterogeneity therein, that occur in tumours. Whilst lung and brain cancers appear to fuel the TCA cycle  studies , 32. In nutrient . Other inutrient . Notablynutrient , 34. FurThe anaplerotic role of glutamine can be bypassed to allow cell survival in glutamine-deprived conditions by utilisation of alternative sources of αKG to replenish levels of TCA cycle intermediates, for example, exogenously acquired glutamate. This process suggests the potential for a common mechanism of resistance to pharmacological glutaminase inhibition; replenishment of TCA cycle intermediates using alternative anaplerotic substrates. Mechanistically this effect may decrease the bioenergetic stress following glutaminase inhibition and allow more of the decreased available pool of glutamate to be utilised for glutathione biosynthesis (and cystine import) and thus provide greater tolerance to oxidative stress .In preclinical tumour models of pancreatic ductal adenocarcinoma glutaminase inhibition effectively targeted proliferating tumour cells, but was ineffective against the hypoxic subpopulation of cells . The res50 studies are conducted at low cell density (1–3 × 103 cells/well of a 96-well plate in 0.12 mL of culture medium), whereas the pyruvate secretion studies were done at approximately 7.5-fold higher cell:culture medium ratio (1.5–2.5 × 105 cells/well of a 6-well plate in 2 mL of culture medium). Thus, at low cell density/high culture medium volume there will be minimal opportunity for an autocrine/paracrine effect of pyruvate to impair glutaminase inhibitor activity. This insight highlights the importance of using higher cell density 2D and 3D assays in future studies of metabolism-targeted drugs. More broadly, it also argues for the development and detailed characterisation of new cell culture technologies that closely recapitulate the in vivo tumour microenvironment.Enhanced glutaminolysis is a common feature of many TNBC cell lines that supports cell growth both in vitro and in vivo . Glutami13C-lactate into TCA cycle intermediates is extensive, suggesting a major role for lactate as an oxidative fuel [Whist the focus of this study is on pyruvate, it is also possible that lactate might also act in a similar manner. Lactate concentrations are elevated in many cancer types, including breast . Incorpoive fuel . Considevia the xCT antiporter system and excess accumulation in the extracellular compartment can impair xCT function and limit cystine uptake – a process that is required to support glutaminolysis [TNBC cells are known to produce and secrete glutamate . This exinolysis , 39. ThuTo our knowledge this is the first reported usage of 3D spheroid cell culture models as a tool to study the pharmacology of glutaminase inhibitors. Our findings demonstrate that the potency of glutaminase inhibition is impaired in 3D spheroid models compared with the 2D monolayer cultures commonly used for evaluating drug activity. There are a number of potential reasons for this change. The proportion of proliferating cells in 3D models is lower than in 2D cultures resulting in greater cellular heterogeneity within the culture , 41. TheMulticellular spheroids represent useful models of many of the microenvironmental characteristics that occur in tumours. Advanced mathematical modelling of these systems can provide key insights into cell proliferation/death, oxygen and nutrient gradients, development of necrosis, and how these compartments interact and change spatially and temporally with spheroid growth and in response to treatment . It has Another key advantage of 3D spheroids is their relative ease of use. They can be established rapidly (within a few days), grown in 96-well format to enable high-throughput studies, manipulated using conventional multichannel dispensing methods, imaged regularly to monitor growth and treatment effects and can be histologically processed at the study endpoint for more detailed microscopic analysis. Whilst 3D cell culture systems, including cell line-derived spheroids and ex vivo patient-derived organoids, are better models of solid tumour pathophysiology, there is still much work needed to characterise their metabolic alterations (using metabolomic techniques) and define appropriate culture medium compositions to model in vivo conditions. Nonetheless, cell line-derived spheroids, in their current form, represent a useful and cost-effective tool for studying inhibitors of cellular metabolism and testing how they can be combined with other therapies prior to investigations using preclinical tumour models and human subjects.Cancer cells grown in vitro display different levels of sensitivity to glutaminase inhibition depending on culture medium composition. A single metabolite, pyruvate, was particularly effective in suppressing the potency of glutaminase inhibitor by contributing to TCA cycle anaplerosis. We build upon this mechanistic observation by showing that TNBC cells themselves readily secrete pyruvate into the extracellular environment. This endogenously-derived pyruvate acted in a paracrine manner to decrease the sensitivity of drug-naïve recipient cells to subsequent treatment with CB-839. Blocking this pyruvate secretion, using an MCT1 inhibitor, prevented this process and improved CB-839 activity in the recipient cells. CB-839 potency was also significantly compromised in 3D spheroid compared with 2D monolayer culture models. These new discoveries highlight the potential influence that circulating and tumour-derived pyruvate and other microenvironmental features can have on glutaminase inhibitor efficacy. Future clinical studies to clearly define the metabolomic landscapes of human tumours will help to define which cancer types and tumour features are predictive of response to these agents.Additional file 1: Supplementary Table S1. Comparison of RPMI 1640, αMEM and DMEM culture medium formulation.Additional file 2: Supplementary Table S2. mRNA expression data  was downloaded from the Cancer Cell Line Encyclopedia for genes involved in pyruvate uptake , mitochondrial transport  and metabolism  for the 11 breast cancer cell lines used in our study. Cell lines that display increased CB-839 IC50 in the presence of pyruvate and associated P-value are also presented.Additional file 3: Supplementary Figure S1. Inhibition of Pyruvate Carboxylase using Phenylacetic acid (PAA) does not increase sensitivity to CB-839 in three of four breast cancer cell lines. Cultures were exposed to a titration of CB-839 concentrations in pyruvate-containing αMEM + 5% FBS with or without 5 mM PAA (Sigma-Aldrich). 3H-thymidine incorporation was assayed on day 3 and % signal plotted relative to CB-839-untreated samples. MDA-MB-231 (A), MCF7 (B) and Hs578T (C) did not display any increase in CB-839 sensitivity in the presence of PAA (+PAA). In contrast, BT549 (D) did demonstrate a slight decrease in the CB-839 IC50 in the presence of PAA .


This review concerns the use of dendrimers, especially of phosphorhydrazone dendrimers, against cancers. After the introduction, the review is organized in three main topics, depending on the role played by the phosphorus dendrimers against cancers: (i) as drugs by themselves; (ii) as carriers of drugs; and (iii) as indirect inducer of cancerous cell death. In the first part, two main types of phosphorus dendrimers are considered: those functionalized on the surface by diverse organic derivatives, including known drugs, and those functionalized by diverse metal complexes. The second part will display the role of dendrimers as carriers of anticancer “drugs”, which can be either small molecules or anticancer siRNAs, or the combination of both. In the third part are gathered a few examples of phosphorhydrazone dendrimers that are not cytotoxic by themselves, but which under certain circumstances induce a cytotoxic effect on cancerous cells. These examples include a positive influence on the human immune system and the combination of bioimaging with photodynamic therapy properties. Th. Th2-Gn Ethacrynic acid has been grafted also to phosphorhydrazone dendrimers through another type of linkage, but the anti-cancer properties of these compounds have not been tested .3-Gn family , as shown in 2), affording the series of dendrimers 4a-c-Cu-Gn 4a-G , three other cancer cell lines , and two non-cancerous , and were compared in some cases with the corresponding first generation dendrimers  has a detrimental effect on the efficiency in most cases. The gold complexes are generally more efficient than the copper ones, but the difference is not large compared to what was observed with the third generation dendrimer Cu and Au complexes, but with other cell lines. The mechanisms of action are relatively similar to those elucidated with dendrimers. However, contrarily to dendrimers, the gold complexes of the dendrons are highly toxic against the non-cancerous cell line NIH-3T3 [For further expanding the scope of this study, after changing the type of ligands and of metals, the shape of the dendrimer was changed by synthesizing off-center dendrimers , which andrimers . Attempt NIH-3T3 .46-G, The usefulness of positively charged dendrimers as non-covalent carriers was recognized very early for transfection experiments . Positiv46-G has been used as carrier of 8-anilino-1-naphthalenesulfonate as a fluorescent model of drugs. Two binding sites were identified: deep inside the dendrimer, and close to or on the surface of the dendrimer. The same dendrimer was then used as carrier of the cytostatic drug cisplatin (see 46-G induced a higher cytotoxicity (74.1 ± 4.1%). Thus, this dendrimer greatly enhances the cytotoxicity of cisplatin towards a brain tumor [Dendrimer in tumor . 36-G was used to carry by electrostatic interactions the negatively charged photosensitizer Rose Bengal . A largely higher uptake was observed in the case of the RB-36-G complex. In the absence of light, the viability of these cells was not affected by the presence of RB or RB-36-G. However, upon wide range irradiation (visible light from 385–780 nm), a large difference in efficiency was observed between RB and RB-36-G for the three cell lines. For example, for a concentration of 0.5 μM in RB, in the case of ASZ cells, for RB alone the cell viability was very high (90%), whereas for RB-36-G the cell viability was decreased to only 7%, due to an enhanced 1O2 production [Dendrimer ngal RB, , used in27-G, bearing 24 carboxylic acid terminal functions, was used for this purpose , with the goal of blocking HIV infection prior to the entry of the virus into human cells [27-G complex (5 MB molecules per dendrimer) was found to generate less singlet oxygen than MB alone. However, tests with basal cell carcinoma cell lines  revealed a higher cellular uptake. Upon irradiation with visible light, phototoxicity against basal cell carcinoma cell lines was increased, accompanied with enhanced production of ROS [Methylene blue (MB) is another type of photosensitizer, but contrarily to Rose Bengal, it is positively charged. However, it is also prone to aggregation and to rapid chemical alterations in a biological environment, inducing negligible photodynamic efficiency . Its ass purpose . This dean cells . The MB-n of ROS .36-G and 46-G, PAMAM dendrimers of generations 3 and 4, and carbosilane dendrimers of generation 2 were used as carriers of three anticancer siRNAs directed against anti-apoptotic proteins of the BCL family, at a P/N (phosphates of SiRNA to ammoniums of the dendrimers) ratio of 3.33/1 in all cases. Transfection of the complexes was carried out in two cancerous cell lines HeLa and HL-60. Complexes based on dendrimers 36-G and 46-G with all the selected siRNAs were taken up by cells more efficiently than complexes based on all the other dendrimers. Experiments carried out with a mixture of the three siRNAs induced a significant decrease in the viability of the cells, in particular when using the phosphorhydrazone dendrimers as carriers [Small interfering RNAs (siRNAs) are a class of double-stranded RNA, non-coding RNA molecules, of generally 20–25 base pairs in length, which induce selective gene silencing. Depending on their structure, siRNAs can be used for the manipulation of apoptosis, to force specific cells to die, in particular cancerous cells . Phosphocarriers .36-G and 46-G and PAMAM dendrimers generations 3 and 4 have been used also as carriers of the siRNA of polo-like kinase (siPLK1) [Dendrimers (siPLK1) , as PLK1(siPLK1) , which i(siPLK1) . Indeed,(siPLK1) . 18-G [18-G was then applied as carrier of pDNA-p53 (plasmid DNA encoding both EGFP and the tumor suppressor p53 protein). A significant p53 protein expression was observed in HeLa cells. The cancer gene therapy potential of the dendriplex 18-G/pDNA-p53 was then validated through therapy of a xenografted tumor-bearing mice after intra-tumoral injection [The best generation to be used for transfection experiments remains an open question, as shown before. Generations 1 to 3, bearing as terminal functions cyclic ammonium derivatives, were tested as carriers of plasmid DNA (pDNA), encoding the enhanced green fluorescent protein (EGFP). It was shown that the first generation bearing 1-(2-aminoethyl) pyrrolidinium as terminal functions  that phosphorus dendrimers can be used for delivering either anti-cancer drugs or siRNA. An attempt has been made to combine both approaches, expecting a synergistic effect. A generation 4 phosphorhydrazone dendrimer functionalized with piperidinium terminal functions 9-G4,  has beenow doses .34a-Cu-G associated with five cytotoxic agents used in chemotherapy . Differaldehyde .In this section are gathered a few examples of phosphorhydrazone dendrimers that are not cytotoxic by themselves, but which induce a cytotoxic effect on cancerous cells.110-G, The first example concerns a first generation dendrimer capped with 12 azabisphosphonate terminal functions 10-G1, , which i211-G is also able to generate singlet oxygen under two-photon irradiation. It has been introduced in cultures of MCF-7 human breast cancer cells, in which it is internalized easily after 3 h of incubation. This dendrimer was found non-toxic in the dark, but also under daylight irradiation. On the contrary, under TPA conditions (irradiation at 760 nm for 3 × 1.57 s), this dendrimer is highly toxic for these cancerous cells. Thus, this dendrimer combines bioimaging properties with photodynamic therapy properties [A last example of indirect influence of a dendrimer concerns a second generation dendrimer having short PEG derivatives on the surface, and, inside the structure, a specially engineered fluorophore, able to be excited classically by the absorption of one photon, but also by the absorption of two photons simultaneously  Figure . Other toperties .® (SPL7013) from Starpharma [In this review, we intended to demonstrate the usefulness of phosphorhydrazone dendrimers to fight against cancers, through several types of approaches. Most of the experiments have been carried out in vitro on cancerous cell lines, but a few studies have been carried out in vivo, in particular the therapy of a xenografted tumor-bearing mice . Some ofarpharma , which iarpharma . Now, th


PUS1) gene. Using an RNA-seq dataset, we confirm the high expression of the E6 and E7 HPV18 oncogenes, show that the entire Bxv1 genome is moderately expressed, and retrieve a Bxv1 splicing event favouring expression of the env gene. Hep2 (clone 2B) is the fourth human cell line so far known to be contaminated by the Bxv1 XMLV. This contamination has to be taken into account when using the cell line in future experiments.The use of misidentified cell lines contaminated by other cell lines and/or microorganisms has generated much confusion in the scientific literature. Detailed characterization of such contaminations is therefore crucial to avoid misinterpretation and ensure robustness and reproducibility of research. Here we use DNA-seq data produced in our lab to first confirm that the Hep2 (clone 2B) cell line  is indistinguishable from the HeLa cell line by mapping integrations of the human papillomavirus 18 (HPV18) at their expected loci on chromosome 8. We then show that the cell line is also contaminated by a xenotropic murine leukemia virus (XMLV) that is nearly identical to the mouse Bxv1 provirus and we characterize one Bxv1 provirus, located in the second intron of the pseudouridylate synthase 1 ( Characterizing cell culture contamination is of prime importance as contaminants may generate flawed experimental results and considerable confusion in the scientific literature4. The catalog of cell lines misidentified or cross-contaminated with another cell line contains 529 entries as of October 20195. Among the 143 different contaminants listed, the most common is HeLa (118 entries), the first continuous human cell line ever established. HeLa derives from a cervical adenocarcinoma biopsy sampled in 1951 from Henrietta Lacks. Sequencing of the HeLa genome resolved a highly rearranged region on chromosome 8 containing multiple integrated partial genomes of the human papillomavirus 18 (HPV18), thought to have induced tumorigenesis6. In addition to standard short tandem repeat (STR) typing, known virus-HeLa cell junctions of HPV18 integrations have been used to unveil cases of cancer cell lines contamination by HeLa7.Continuous cell lines are a cornerstone of cellular and molecular biology as well as of biomedical research. A long-known problem faced by researchers using such cell lines is contamination, whereby foreign cells or microorganisms are inadvertently introduced and remain unnoticed over multiple passages10. Viral contaminations may be particularly problematic because viruses may present a risk for the persons handling cell cultures and unlike other organisms, viruses cannot be easily detected under light microscopy9. Screenings of hundreds of human cell lines have so far revealed only a relatively small proportion of them as contaminated with human viruses13. Most cases involve contamination by the Epstein Barr herpesvirus (EBV), either because primary cell cultures were established from infected patients or because EBV was intentionally used to generate transformed lymphoblastoid cell lines.Multiple microorganisms such as bacteria, fungi, viruses or yeasts have also been identified as persistent contaminants of widely-used cell lines19. MLV are gammaretroviruses found under both exogenous and endogenous  forms in wild and laboratory mouse (Mus musculus) strains, which are associated with neoplasias of hematopoietic origin20. They are classified in several groups according to their host tropism, which is determined by interactions between their envelope-encoded receptor binding domain and host receptors21. While ecotropic MLV interact with the mCAT-1 receptor and can only infect mouse, polytropic MLV interact with the XPR1 receptor and can infect both mouse and other mammalian species. The xenotropic MLV (XMLV) also interact with the XPR1 receptor but they can only infect non-mouse species. Mouse strains are immunized against XMLV thanks to the presence of several Xpr1 variants that restrict interaction with these viruses22. Most cases of human cell contaminations by MLV involve XMLV and it has been shown that contamination by XMLV often leads to important changes in cellular behavior, which can severely affect the conclusions of studies using such infected cell lines15.Non-human viruses have also been found in human cell lines, with murine leukemia viruses (MLV) being responsible for most cases of contaminations25. One of the best characterized cases of XMLV contamination is that of the so-called xenotropic murine leukemia virus-related virus (XMRV), which was originally thought to be a possible cause of prostate cancer and chronic fatigue syndrome26. XMRV was later shown to result from recombination between two distinct XMLV sequences integrated in the mouse genome (proviruses) and its presence in human cell lines was traced back to a contamination that occurred around 1993, when a prostate cancer cell line (CWR22Rv1) was xenotransplanted into immunocompromised mice27. Other well-characterized cases of XMLV contamination involve a virus originating from expression of a mouse chromosome 1 proviral locus called the B10 xenotropic virus 1 (Bxv1)28. This provirus is present in many severe combined immunodeficient (SCID) and nude mice strains20 and Bxv1 infection of human cell lines passaged onto these mice strains has been experimentally demonstrated24. So far, Bxv1 contamination has been detected in two prostate cancer cell lines, one B-cell line and two pancreatic β cell lines, in which it was shown to produce infectious viral particles29.Contamination by XMLV is known to occur either when a human tumor is xenografted in immunodeficient mice to establish or replicate human tumor cell lines, or through unintentional contact between contaminated and non-contaminated cell linesHere, we report a new case of a human cell line  contaminated by Bxv1 using high-throughput RNA- and DNA-seq. As part of a study aiming to characterize the potential presence of infectious agents in the cell lines of our laboratory, we set out to sequence total RNA and DNA extracted from Hep2 (clone 2B) cells. We characterized sequences from two viruses, the HPV18 papillomavirus and the Bxv1 XMLV. The presence of the former is consistent with the known contamination status of Hep2 (clone 2B) cell line by HeLa and the presence of the latter most likely results from mixing with a cell line or reagents contaminated with this virus.2 atmosphere. Upon confluence, cells contained in a T75 flask were harvested in a 15 ml Falcon tube after trypsinization, washed in Earle's balanced salt solution (EBSS) and centrifuged at 1100g for 5 min. Total RNA and DNA were then extracted using the AllPrep DNA/RNA Mini Kit (Qiagen). A paired-end DNA library (mean insert size 191 bp) was constructed with the DNA sample and a stranded RNA library was constructed with the RNA sample after rRNA depletion. Both libraries were tagged and sequenced on an Illumina HiSeq2500 platform in a 2 × 125 bp configuration in High Output mode (V4 chemistry). Raw DNA- and RNA-seq fastq reads generated for this study have been deposited in dbGaP under accession number phs001944.v1.p1. All reads mapping onto the Bxv1 (JF908815) genome are provided in Supplementary Data Hep2 (clone 2B) cells were purchased from Sigma-Aldrich  on June 19, 2013 and cultured in Dulbecco's modified Eagle medium  supplemented with 10% fetal bovine serum and 1% penicillin–streptomycin  at 37 °C in a 5% (vol/vol) CO30 and Trinity31, respectively, using default settings. The resulting contigs were used as queries to perform blastn and blastx searches on the non-redundant (NR) database of GenBank. To assess sequencing depth of the two viruses identified in this study (Bxv1 XMLV and HPV18), the reads were mapped onto the genome of these viruses (accessions number: JF908815 for Bxv1 and GQ180792 for HPV18) with Bowtie2 in “sensitive-local” mode. To identify recombination breakpoints within each of the two genomes and between the two genomes and the Hep2 (clone 2B) genome we used all reads as queries to perform separate blastn searches (megablast option) on the human genome and on the Bxv1 and HPV18 genomes. Virus-virus and cell-virus junctions were then searched within reads using the pipeline described in Ref.32. Briefly, only reads aligning over at least sixteen bp on a genome region only and over at least sixteen bp on another genome region only were retained. Reads had to align on at least 100 bp of their length. The overlap between alignment on the virus and on the host sequences was set to involve at most 20 bp and at least -5 bp .Demultiplexing was performed by the sequencing company (Genewiz), yielding 140,313,660 reads with a mean quality score of 35.15 for the DNA sample and 57,593,594 reads with a mean quality score of 35.82 for the RNA sample. Remaining adapter sequences were removed and reads were trimmed with Trimmomatic using default parameters . To identify infectious agents potentially present in the cells, we mapped paired reads onto the human genome  using Bowtie2 in “sensitive-local” mode . Reads that did not map onto the human genome were assembled for each dataset. DNA-seq and RNA-seq reads were assembled with MasurcaMus musculus (GRCm38/mm10) and Rattus norvegicus (GRSC 6.0/rn6). Using the SAMtools depth program, the mean rodent genome coverage as well as the percentage of the genome covered by the reads were determined. The nature of regions covered by more than 8000 reads was checked in the UCSC genome browser.To assess the presence of rodent DNA or RNA in our samples, reads were mapped using Bowtie2 in “end-to-end” mode on the latest genome version of −1 of each primer , 3 mM MgCl2, 0.2 mM dNTP and 0.75 unit of DreamTaq DNA polymerase (Thermo Fischer Scientific) under a 35-cycle PCR program . Then a migration of the PCR products was performed on a 1.5% agarose gel at 100 V during 25 min and bands were visualized with a Bio-Rad Transilluminator Universal Hood II under UV light. To check whether regions of the Bxv1 genome not covered by our DNA-seq dataset were in fact present in Hep2 (clone 2B) cells, we PCR-amplified and Sanger-sequenced three such regions as well as another region covered by our DNA-seq dataset using the following primer pairs: Bxv1_2-F: CCCCAGAAGAGAGAGAAGAAC; Bxv1_2-R: CATTGGTCCTTATCGAGTTGG; Bxv1_3-F: TGCCTTTGAGTGGAGAGATC; Bxv1_3-R: CTAGGGTTTGTAGAAGGGCC; Bxv1_4-F: CCTTCTCAACAACCTGGGAC; Bxv1_4-R: ACAGGGTCAGCTTGTGTTG; Bxv1_5-F: CAGGCAAGCTAACTATGGGA; Bxv1_5-R: CCCAGATTACCTCGGTTTCA.To rule out the possibility that the Bxv1 contamination occurred in our laboratory, we purchased a second batch of Hep2 (clone 2B) cells from Sigma-Aldrich in May 2018 and searched by PCR for the presence of Bxv1 in this new batch and in the Hep2 (clone 2B) cells we used for the sequencing (batch ordered at Sigma-Aldrich in 2014). Amplification reactions were performed from 5 ng of DNA extracted from Hep2 (clone 2B) cells by using 10 µmol L33. Consistent with HeLa contamination, assembling of RNA- and DNA-seq reads obtained from sequencing this cell line and not mapping on the hg19 human genome yielded various contigs that were almost identical to the HPV18 (GenBank accession number: NC_001357.1). As previously reported7 for HPV18 sequences integrated into the HeLa genome, mapping of both RNA- and DNA-seq reads onto the HPV18 genome shows that a portion of the E2 and L1 genes as well as the entire E4, E5 and L2 genes are missing   was contaminated by HeLa at some point during its propagation and they show that HPV18 sequences integrated in this subclone of HeLa have the same characteristics as in other subclones in terms of position in the genome and expression pattern34.As mentioned on the Sigma Aldrich website, the Hep2 (clone 2B) cell line  was originally derived “from tumours produced in irradiated-cortisonised weanling rats after injecting with epidermoid carcinoma tissue from the larynx of a 56-year-old male, but it was later found to be indistinguishable from HeLa by STR PCR DNA profiling.” It is a typical case of non-existent cell line that may cause important problems in cancer research29. As several studies have shown that the presence of XMLV in human cell lines could be due to contamination by mouse DNA37, we checked for such contamination by mapping both DNA-seq and RNA-seq reads onto the mouse genome. Given that the original Hep2 cells were obtained by passaging larynx carcinoma cells onto immune-compromised laboratory rats39, we also monitored the possible presence of rat DNA by mapping all reads onto the rat genome. Only ≃ 4% of the reads mapped onto the rodent genomes, with only ≃ 1% of the genome covered and a mean sequencing depth of 0.26× for the two species. Importantly, mapped regions corresponded exclusively to RNA genes  that are known to be highly similar between mammalian species and no read was found to map onto intracisternal-A particle elements, which are rodent-specific transposable elements typically used as markers of contamination37. Thus, the presence of Bxv1 is not due to contamination by rodent DNA but rather results from mixing with a contaminated cell line or reagent. To further verify that contamination did not occur in our laboratory subsequent to reception of Hep2 (clone 2B) cells on June 19 2013 we purchased a second batch from Sigma-Aldrich on May 11, 2018 and validated the presence of Bxv1 sequences by PCR  revealed no significant hit. This indicates that Bxv1 is likely absent from all or most HeLa cell lines. However, we cannot exclude that this virus was present in the very HeLa cells that contaminated Hep2 (clone 2B). Thus, whether Bxv1 was introduced into Hep2 (clone 2B) by HeLa cells, or whether it was introduced into Hep2 (clone 2B) before or after this cell line was contaminated by HeLa, remains an open question.The finding of Bxv1 in the HeLa-contaminated Hep2 (clone 2B) cell line begs the question of whether the virus is present in HeLa cells. XMLVs have been searched in a large number (> 600) of human cell lines, including HeLa and nine of its derivative subclones in which it was found to be absent17, the Bxv1 provirus characterized here lies within the second intron of the pseudouridylate synthase 1 (PUS1) gene. Furthermore, in agreement with the known propensity of MLV to preferentially target transcription start sites (TSS) and CpG islands41, the Hep2 (clone 2B) Bxv1 lies only 946 bp downstream of the nearest PUS1 TSS, well within a 1463-bp long CpG island. Altogether, these results indicate that the Bxv1 integration identified in Hep2 (clone 2B) cells is a bona fide proviral locus, confirming contamination by this virus of the Hep2 (clone 2B) cell line.To further validate the presence of Bxv1 in the HeLa-contaminated Hep2 (clone 2B) cell line, we searched for evidence of integration of the retrovirus. We identified two virus-cell junctions covered by more than one DNA-seq read and/or also supported by an RNA-seq read (Supplementary Data gag-pro-pol open reading frame (ORF) to 1955× for the env ORF . While several short segments amounting to 15% of the Bxv1 genome are not covered by DNA-seq reads Fig. A, we belORF Fig. B. HoweveORF Fig. . This is16 study, 768 RNA-seq reads were found to contain a junction between positions 587 and 5887 of Bxv1 (accession number: JF908815), which corresponds to a splicing event leading to the expression of the env ORF. Our search for virus–virus junctions unveiled a nearly identical splicing event, with 757 reads , supporting a junction between positions 587 and 5884  which is 99.98% identical to Bxv1 JF908815. However, the position of the splice acceptor site differs , which may be due to context-dependent variations in splicing. To assess whether the presence of Bxv1 in Hep2 (clone 2B) cells could result from mixing with the JY B cell line, we checked for the presence of RNA-seq and/or DNA-seq reads mapping onto the EBV genome, which is known to be present in JY B cells16. We did not find any read mapping on the EBV genome. While this suggests that the Bxv1 contamination is not due to a mix with JY B cells, we cannot exclude that a transient mix occurred between Hep2 (clone 2B) and JY B cells at some point but that JY B cells are no longer detectable. Interestingly, the RNA-seq coverage of the region corresponding to the alternative env transcript is three to five times higher than that of the rest of the Bxv1 sequence  cells, it shows that it is expressed at a moderately high level, which may impact various cellular pathways. Thus, the presence of this virus in otherwise HeLa-contaminated Hep2 (clone 2B) cells will have to be taken into consideration in future studies using this cell line to avoid erroneous interpretations of experimental results. Furthermore, this study should also encourage others using this cell line and related ones, such as the (code A) 86030501-1VL, or 85020207-1VL Hep-2C (HeLa derivative) Human Negroid cervix carcinoma (code C), to check for the presence of Bxv1 using the PCR primers we provide in the materials and methods section.In this study, we have confirmed that the Hep2 (clone 2B) cell line  is contaminated by HeLa cells based on the characterization of HPV18 integrants and expression patterns. We have further demonstrated that the cell line is also contaminated by a virus nearly identical to the Bxv1 XMLV provirus, the presence of which is likely due to direct contact between the cell line and another contaminated cell line or reagent. Based on sequencing depth, we show that the cell line might contain only one Bxv1 provirus, which we were able to map to the second intron of the Supplementary Information 1.Supplementary Information 2.Supplementary Information 3.Supplementary Information 4.Supplementary Information 5.Supplementary Legends.


Objective: Triple negative breast cancer (TNBC) is known to have aggressive clinical course and a high risk of recurrence. Given the lack of effective targeted therapy options, paclitaxel-based chemotherapy is still the primary option for TNBC patients. However, patients who fail to achieve a complete response during neoadjuvant chemotherapy may be mainly due to sensitivity and resistance to chemotherapy. Thus, we concentrated the present research on the role of PGK1 in the sensitivity to paclitaxel treatment and the possible underlying mechanisms in TNBC.Methods: After exposure to paclitaxel, a cell viability analysis was made to investigate the influence of PGK1 silencing on cell death. The effect of PGK1 on apoptosis induced by paclitaxel treatment was examined in vitro by flow cytometry cell apoptosis assays. Western blotting was performed to examine the impact of PGK1 on paclitaxel-induced apoptosis. The correlation of PGK1 with apoptosis-associated protein X-linked inhibitor of apoptosis (XIAP)-associated factor 1 (XAF1) was analyzed in 39 specimens by immunohistochemistry analysis.Results: We observed that silencing PGK1 sensitized triple-negative breast cancer (TNBC) cell lines to paclitaxel treatment as a result of increased drug-induced apoptosis. Furthermore, mechanistic investigations suggested that XAF1 was increased in PGK1-knockdown cells along with the expression of the apoptotic proteins including cleaved caspase-3 and Bax. Immunohistochemistry analysis showed that PGK1 was negatively related to XAF1. Moreover, we found that downregulation of XAF1 reduced paclitaxel-induced apoptosis in PGK1-silenced triple-negative cell lines.Conclusion: Our results identified PGK1 as a potential biomarker for the treatment of TNBC, and inhibition of PGK1 expression might represent a novel strategy to sensitize TNBC to paclitaxel treatment. Currently, breast cancer is the most prevalent cancer in women worldwide. It accounted for 268,600 30%) new cancer diagnoses and 41,760 15%) cancer-related deaths in women in the United States , as we described previously  and were validated by analysis of STR (PowerPlex 18D system). The cell lines were cultured in high-glucose Dulbecco's modified Eagle's medium (DMEM)  supplemented with 10% fetal bovine serum  at 37°C under an atmosphere of 5% COTotal RNA was isolated from differentially treated cells using a Total RNA Kit I  according to the manufacturers' instructions. The primers designed for PGK1 were as follows: forward, 5′AACCAGAGGATTAAGGCTGC3′ and reverse 5′GCCTACACAGTCCTTCAAGA3′. The primer sequences used to detect XAF1 were as follows: forward, 5′GCTCCACGAGTCCTACTGTG3′ and reverse, 5′GTTCACTGCGACAGACATCTC3′. β-actin was employed as internal reference. The primers of β-actin were designed as follows: forward, 5′CAACCGCGAGAAGATGACC3′ and reverse 5′ATCACGATGCCAGTGGTACG3′. The analysis was performed using the ABI StepOne Real-time qPCR system  and FastStart Universal SYBR Green Master (ROX) reagent  according to the protocols. Expression was relatively normalized to the endogenous control of β-actin.Western blotting analysis was performed on cells solubilized in RIPA buffer with 1% protease inhibitor. The solution was centrifuged at 12,000 g for 15 min at 4°C. The supernatant was obtained. Western blotting analysis was conducted with primary antibodies against PGK1 , XAF1 , cleaved caspase-3 , Bcl-2 , and Bax . β-tubulin  was used as a loading control.A breast cancer tissue microarray was prepared for IHC as described previously . All breLentivirus constructs specific for PGK1 and the negative control were obtained from Genechem Co. . The sequence of the shPGK1 constructs were as follows: shPGK1-1, CAAGATTGTCAAAGACCTA; and shPGK1-2, TATGAAGAACAACCAGATA. The nontargeted control sequence was 5′-TTCTCCGAACGTGTCACGT-3′. Either lentivirus constructs or controls were used to infect MDA-MB-231 and MDA-MB-468 cells. The cell lines were cultured in complete media containing 2 μg/ml puromycin to generate stable knockdown cell lines 24 h after infection. Real-time qPCR and Western blotting were performed to validate the knockdown of PGK1 mRNA  and protAll small interfering RNA (siRNA) constructs against XAF1 and the negative control plasmid were purchased from GenePharma . The sequences of the siRNAs used to suppress XAF1 expression were as follows: sense, 5′GCUCCUGAAAGGGAAAUCUTT3′ and antisense, 5′AGAUUUCCCUUUCAGGAGCTT3′. The non-targeted control sequences were as follows: sense, 5′UUCUCCGAACGUGUCACGUTT3′ and antisense, 5′ACGUGACACGUUCGGAGAATT3′. Cell lines were transfected with siRNA and negative control using Lipofectamine 2000  for 48 h according to the protocols.4 knockdown and vector cells were suspended in 100 μl medium and seeded in 96-well plates. Paclitaxel (15 and 30 nmol/L for MDA-MB-231 cells and 30 and 50 nmol/L for MDA-MB-468 cells) was added to every well in 96-well plates. The plates were incubated for 48 h. The CellTiter 96 Aqueous One Solution Cell Proliferation Assay Kit  was used according to the protocols. Then, 20 μl reagent was added to every well of the 96-well containing the cell samples and incubated in 5% CO2 at 37°C for 1 h. Microplate reader was used to measure the absorbance values at 490 nm.A total of 2 × 10The cells were concentrated and stained with a PE Annexin V Apoptosis Detection kit . Then, the cells were washed twice with cold PBS. The cells were resuspended in 100 μl binding buffer. 5 μl PE Annexin V and 5 μl 7-AAD were used to stain the samples for 15 min at room temperature in the dark. 400 μl binding buffer was added after 15 min stainning. Flow cytometry assays were performed within 1 h. The assays were performed on a BD Influx Cell Sorter . Results were analyzed using FACS software and calculated with the percentage of apoptotic cells .Total RNA was extracted from MDA-MB-231-shNC, MDA-MB-231-shPGK1-1, and MDA-MB-231-shPGK1-2 cells. Then, a sequencing library of each RNA sample was prepared. BGISEQ-500 was used to profile the transcriptional analysis, which was performed by BGI Tech . Raw sequences were aligned to the human reference genome hg38 in the UCSC genome browser. RPKM values were used for gene quantification. Gene Ontology (GO) functional enrichment analysis was performed to identify associated biological processes. Pathway analysis of differentially expressed genes was carried out to determine the important pathways based on the latest Kyoto Encyclopedia of Genes and Genomes (KEGG) database.t-test or ANOVA. The correlation between expression of PGK1 and XAF1 in TNBC tissues was analyzed by Pearson correlation test. P < 0.05 indicated statistical significance.All statistical tests were conducted using Prism 7.0 . Comparisons between groups for statistical significance were performed with Student's To analyze the possible role of PGK1 participate in the sensitivity of breast cancer cells to paclitaxel treatment, we used shPGK1-1 and shPGK1-2 lentiviruses to create stable PGK1-downregulated cell lines derived from the MDA-MB-231 (MDA-MB-231-shPGK1-1 and MDA-MB-231-shPGK1-2) and MDA-MB-468 (MDA-MB-468-shPGK1-1 and MDA-MB-468-shPGK1-2) cell lines. Compared with the control shRNA (MDA-MB-231-shNC and MDA-MB-468-shNC), the shPGK1 lentiviruses decreased the mRNA  and protAfter treatment with 15 or 30 nmol/L paclitaxel for 48 h, a dose-dependent inhibition of cell viability was detected in MDA-MB-231-knockdown cells. PGK1-knockdown cells were more sensitive to paclitaxel treatment than vector cells. In MDA-MB-468 cells, after exposure to 30 or 50 nmol/L paclitaxel, the cell viability was lower in PGK1-knockdown cell lines than in the vector cell line significantly. Statistical analysis showed that the significant differences between PGK1-knockdown and vector cell lines were presented in both MDA-MB-231 and MDA-MB-468 cell lines .Paclitaxel is well-known to play its cytotoxic role through the induction of apoptosis . TherefoThese results were further confirmed by investigation of the apoptosis markers cleaved caspase-3, Bax, and Bcl-2. The results demonstrated that the expression levels of cleaved caspase-3 and Bax in MDA-MB-231 PGK1-silenced cell lines were increased compared with those in vector cells after treatment with paclitaxel for 48 h, whereas the expression levels of the anti-apoptosis protein Bcl-2 were decreased . MDA-MB-https://www.ncbi.nlm.nih.gov/sra/PRJNA665999.To identify potential apoptosis-related molecular mechanisms of PGK1 in TNBC, we conducted RNA transcriptional sequence analysis to investigate the differential gene expression profiles between the PGK1 downregulation groups and the vector group using the MDA-MB-231 cell line. The RNA transcriptional sequencing data is accessible with the following link: By bioinformatics analysis, we found that 333 genes were differentially expressed . Among tIn our present research, we found that the silencing of PGK1 by shPGK1 increased the apoptosis level induced by paclitaxel treatment in TNBC cell lines. To elucidate the potential PGK1 target genes involved in paclitaxel-induced apoptosis in TNBC, we performed KEGG pathway analysis. The analysis indicated that the 233 differentially expressed genes were enriched in 237 pathways, and the top 20 biological processes are shown in P = 0.03).Real-time qPCR and Western blot analysis were used to measure XAF1 mRNA and protein expression in PGK1-downregulated cells and vector cells. We demonstrated that the level of XAF1 was greatly upregulated in PGK1-silenced TNBC cell lines . Taken tBecause our results indicated that PGK1 could regulate XAF1 expression and that PGK1 silencing upregulated paclitaxel-treated TNBC cell motility, we hypothesized that PGK1 modulates paclitaxel sensitivity through XAF1. In order to test the hypothesis, we transduced PGK1-silenced TNBC cell lines with siXAF1 and tested the apoptosis level by flow cytometry assays in paclitaxel-treated TNBC cell lines. We observed that the paclitaxel-induced apoptosis enhancement was significantly reversed . The numHigh aggressiveness, as well as non-susceptibility to hormone and targeted therapies, limit the number of therapeutic opportunities and make the prognosis of TNBC patients poor. Efforts have been conducted to increase the therapeutic opportunities for TNBC patients in recent years. Recent research has shown that hypoxia-associated genes are upregulated in TNBC and are associated with resistance to chemotherapy . InhibitPreviously, it has been reported that upregulation of PGK1 expression is observed in several types of cancer –18, 20. Furthermore, PGK1 has been revealed to be activated in breast cancer. High PGK1 expression presented shorter survival in breast cancer than low PGK1 expression. The depletion of PGK1 impaired cell migration and reversed the HIF-1α-mediated epithelial-mesenchymal transition (EMT) process . PGK1 waPaclitaxel is commonly used as the first-line chemotherapeutic drug for TNBC . The benIn the present study, a cell viability assay was used to show a significant decrease in cell viability after the treatment with paclitaxel in PGK1-knockdown cell lines. In addition, we revealed that knockdown of PGK1 activated the expression of the apoptotic proteins cleaved caspase-3 and Bax and inhibited the expression of the anti-apoptotic protein Bcl-2 in paclitaxel-treated TNBC cell lines. Therefore, we propose that downregulation of PGK1 may be involved in apoptosis-mediated paclitaxel sensitivity in TNBC.A promising approach, which was used to enhance the effectiveness of chemotherapy drugs or to induce apoptotic cell death, is to target the apoptotic pathway directly. On the basis of this idea, we examined the effects of PGK1 depletion on genes involved in breast cancer. RNA sequencing was used to assess the differentially expressed genes and the enrichment of functional pathways. The analysis identified 233 differentially expressed genes. Eight of the genes were involved in the breast cancer pathway. Among the 8 genes, XAF1 was upregulated by PGK1 knockdown. Upregulation of XAF1 expression was observed to suppress cancer cell growth and increase cell sensitivity to chemotherapy .XAF1 was first identified as a XIAP-interacting protein to suppress the anti-caspase activity of XIAP1 . It has In the present work, we verified the relationship between PGK1 and XAF1 expression for the first time. Immunohistochemical analysis of TNBC patient tissues confirmed the negative correlation between PGK1 and XAF1. In addition, increased expression of XAF1 occurred in parallel with increased apoptosis in PGK1-knockdown cells treated with paclitaxel. We hypothesize that depletion of PGK1 improves the sensitivity of TNBC cells to paclitaxel treatment through increased XAF1 expression. We transduced PGK1-silenced TNBC cell lines with siXAF1 and examined the apoptosis level by flow cytometry assays and apoptosis-associated proteins by Western blotting analysis in paclitaxel-treated TNBC cell lines. We observed that the paclitaxel-induced apoptosis enhancement was significantly reversed. Therefore, the paclitaxel-induced apoptosis enhanced by the silencing of PGK1 required XAF1 upregulation in TNBC.in vitro mediated via XAF1 expression in TNBC. However, several other issues also remain to be addressed, and more studies are required to analyze the precise role of PGK1. It would be of great interest to know whether other pathways are also involved in mediating the anti-apoptotic effect of PGK1 in TNBC. Thus, on the basis of our present study, we provided new insight into the role of PGK1 in TNBC. PGK1 might be a novel therapeutic target in TNBC and has important implications in the development of targeted therapeutics for improving paclitaxel sensitivity in patients receiving paclitaxel-based chemotherapy.In conclusion, our results demonstrate that PGK1 plays an important role in paclitaxel-induced apoptosis. Silencing of PGK1 confers sensitivity to paclitaxel PRJNA665999, (https://www.ncbi.nlm.nih.gov/sra/PRJNA665999).The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found at: the NCBI Sequence Read Archive, ID: The studies involving human participants were reviewed and approved by Harbin Medical University. The patients/participants provided their written informed consent to participate in this study.SS and DP designed the research. HW, XW, ZY, YJ, WW, YS, YZ, and ZC performed the research. SS, YJ, YC, and XL analyzed the data. SS drafted the manuscript and wrote the paper. All authors approved the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


KEAP1/NRF2 (27.9%) have been identified in lung squamous cell carcinoma. In this research, we explored the role of KEAP1 somatic mutations in the development of LSCC and whether a nuclear factor erythroid 2-related factor 2(NRF2) inhibitor be potential to target lung cancer carrying KEAP1/NRF2 mutations.Targeted therapy and immunotherapy have led to dramatic change in the treatment of lung cancer, however, the overall 5-year survival rate of lung cancer patients is still suboptimal. It is important to exploit new potential of molecularly targeted therapies. High-frequency somatic mutations in KEAP1 stably transfected with wild-type (WT) KEAP1 or somatic mutations in KEAP1 were used to investigate the functions of somatic mutations in KEAP1. Flow cytometry, plate clone formation experiments, and scratch tests were used to examine reactive oxygen species, proliferation, and migration of these cell lines.Lung cancer cell lines A549 and H460 with loss-of-function mutations in , and tumor cell proliferation, migration, and tumor growth were accelerated in A549 and H460 cells stably transfected with KEAP1 mutants compared to control cells with a loss-of-function KEAP1 mutation and stably transfected with WT KEAP1 in both in vitro and in vivo studies. The proliferation of A549 cell line trasfected with the R320Q KEAP1 mutant was inhibited more apparent than that of the A549 cell line trasfected with WT KEAP1 after treatment with NRF2 inhibitor ML385.The expression of NRF2 and its target genes increasedKEAP1 identified from patients with LSCC likely promote tumorigenesis mediated by activation of the KEAP1/NRF2 antioxidant stress response pathway. NRF2 inhibition with ML385 could inhibit the proliferation of tumor cells with KEAP1 mutation.Somatic mutations of Video abstract EGFR) kinase has led to a dramatic change in the treatment of patients with lung adenocarcinoma /2 [Twelve BALB/c nude mice  were randomly assigned to two groups (WT or mutant). We infected A549-(mm)2]/2 . All expP-value of 0.05 or less was considered statistically significant. These analyses were performed using SPSS 13.0 software  or GraphpadPrism 7 .All experiments were performed in quadruplicate and were repeated at least three times with similar results unless otherwise indicated. All statistical analyses were performed using unpaired two-tailed Student’s t-test and the mean ± standard error of the mean. A KEAP1 loss on activation of the KEAP1/NRF2 pathway, we collected three NSCLC cell lines with KEAP1 mutations  and four NSCLC cell lines without KEAP1/NRF2 mutations . In the present study, we have found the frequency of KEAP1/NRF2 mutation was higher in human LSCC than that in LUAD patients, thus we hope find the LSCC cell lines with KEAP1 mutation to investigate the effect of KEAP1 mutation on the function of KEAP1/NRF2 pathaway. However, after serching the literatures and cell line database, we found none LSCC cell lines carried KEAP1/NRF2 mutation. We further collected all available 7 lung cancer cell lines and found three of them carried the KEAP1 mutation , thus, in the present study, all the seven lung cancer cell lines were used to study the influence of KEAP1 mutation on the function of KEAP1/NRF2 pathway. As expected, Sanger sequencing revealed that the A549, H460, and H838 cell lines carried homozygous point mutations at D236H, G333C, and E444* in KEAP1, respectively; however, H1299, NCI-H292, 95D, and SPCA1 cell lines did not carry mutations in neither KEAP1 nor NRF2 lung cancer cells by real-time polymerase chain reaction (PCR), but the mRNA expression of NRF2 showed no significant difference between lung cancer cell lines with and without KEAP1 mutation . Given that NRF2 protein translocates into the nucleus to activate the transcription of downstream target genesin the KEAP1/NRF2 pathway, we further examined the protein expression of nuclear NRF2 and the downstream target HO-1 in these tumor cells by western blot analysis. As expected, function loss of KEAP1 significantly increased nuclear NRF2 levels and HO-1 levels incytoplasm  levels through upregulating the expression of detoxifying enzymes and antioxidant genes. Thus, we checked whether KEAP1 from five patients with LSCC. However, the role of these KEAP1 mutations in tumorigenesis is unclear. The function these five nonsynonymous mutations in KEAP1 were predicted by Polyhen2_HDIV and SIFT. The results showed that these five nonsynonymous mutations in KEAP1 were harmful, affecting protein function (Table 1).In our previous study, we identified five nonsynonymous mutations in KEAP1 influence the function of KEAP1, WT and five KEAP1 mutants were stably transfected with retroviral vectors into A549/H460 lung cancer cell lines that carry loss-of-function mutations in KEAP1. As expected, nuclear NRF2 protein levels and expression of the NRF2 target gene HO-1 were significantly decreased after A549 or H460 lung cancer cell lines were stably transfected with WT KEAP1  domain; a central intervening region (IVR); and a series of six C-terminal Kelch repeats [KEAP1 did not bind to NRF2 . Consistent with the in vitro results, the KEAP1 mutant showed significantly accelerated tumor growth in vivo. These results indicate that KEAP1 likely is a novel tumor driver gene for LSCC.Due to we have considered that R320Q mutant has a considerable effection to KEAP1’S function, the ability of the antioxidative stress or proliferation and migration was significantly increased. Therefore, we choosed the R320Q mutant for our next experiments. To further examine the effect of the somatic mutations in KEAP1, the expression levels of NRF2 and its target protein HO-1 were significantly increased in the tumor tissues from the A549 cell line transfected with the R320Q KEAP1 mutant . The mRNA levels of NRF2 and its target genes HMOX1, HO-1, GCLC, and NQO1 in the grafted tumor tissues from the A549 cell line transfected with the R320Q KEAP1 mutant were remarkably increased compared with that in grafted tumor tissues from the A549 cell line transfected with WT KEAP1.Next, we examined the expression of oxidative stress-related genes in the grafted tumor tissues from the nude mice. Compared with the expression of NRF2 in the nucleus and its target protein HO-1 in the cytoplasm in tumor tissues from the A549 cell line transfected with WT NRF2, resulting in elevated levels of intracellular ROS and increased cytotoxicity. However, it is unknown whether NRF2 inhibitors have different effects on these LSCCs with or without KEAP1 somatic mutations. Thus, we selected an effective NRF2 inhibitor, ML385, which specifically and directly interacts with NRF2 protein, blocks NRF2 transcriptional activity, and enhances the efficacy of carboplatin and other chemotherapeutic drugs in lung cancer cells [KEAP1 mutant (KEAP1-R320Q mutant) or with WT KEAP1 (KEAP1-WT) and H1299 lung cancer cells, which carry both WT KEAP1 and NRF2, were selected and treated with ML385. The number of formed colonies was decreased in all three groups in a dose-dependent manner with increased ML385 treatment  than in lung cancer cell lines without KEAP1 mutation . Interestingly, compared with the lung cancer cell lines A549 and H460, which carry KEAP1 mutation, after stable transfection with WT KEAP1, the mRNA levels of NRF2 and its target genes are significantly increased in A549 and H460 lung cancer cell lines transfected with KEAP1 mutants. Moreover, colony formation and migration were increased in A549 and H460 lung cancer cell lines transfected with KEAP1 mutants. Similarly, the grafted subcutaneous tumor sizes in nude mice were significantly larger in A549 cells transfected with the R320Q KEAP1 mutant than those inA549 cells transfected with WT KEAP1. These data suggest that the somatic mutations of KEAP1 identified in Chinese patients with LSCC likely promote the development of lung cancer through activation of the antioxidative stress response in the KEAP1/NRF2 pathway.Recently, high-frequency somatic mutations of  studies . Previoug cancer –32. In ag cancer . The los in mice , 34. Morsistance . HoweverTP53, PI3KCA, and SOX2, no clear operationable targets for the treatment of LSCC have been found thus far [KEAP1 promotes the development of lung cancer mediated by activation of the KEAP1/NRF2 pathway. Thus, it is tempting to presume that inhibitors of the KEAP1/NRF2 pathway will likely treat Lung cancer patients carrying KEAP1/NRF2 mutations. To date, some inhibitors of the KEAP1/NRF2 pathway have been identified, such as NRF2 inhibitors that inhibit this pathway: ML385 [NRF2 expression. We further explored the effect of ML385 on the development of lung cancer cell lines with or without KEAP1 mutations. Compared with A549 lung cancer cell lines trasfected with WT KEAP1, proliferation of the A549 lung cancer cell line trasfected with the R320Q KEAP1 mutant was dramatically inhibited by ML385. These preliminary datas suggest that ML385 inhibits the proliferation of lung cancer cells with KEAP1 mutations by blocking the KEAP1/NRF2 antioxidant stress response pathway. It will provide a new option for therapy targeted to the KEAP1/NRF2 pathway in patients with LSCC carrying the KEAP1 mutation.Although genomic analysis identified some high-frequency gene mutations from LSCC, such as thus far , 12, 35.thus far . Convenithus far . Additiothus far . Howevery: ML385 , Brucea y: ML385 , clonazey: ML385 , luteoliy: ML385 , 42. TheKEAP1 has been identified in Chinese patients with LSCC. The somatic nonsynonymous mutations in KEAP1 derived from patients with lung cancer likely promote tumorigenesis via activation of the KEAP1/NRF2 antioxidant stress response pathway. Notably, NRF2 inhibitor ML385 may inhibit the proliferation of tumor cells with KEAP1 mutation by enhancing the oxidative stress level of lung cancer cells.In summary, high-frequency mutation in Additional file 1: Supplementary Fig. 1. (A). Gene editing methods in A549 lung cancer cell lines with NRF2 homozygous knockout  (B). Compared with A549, expression NRF2 and its target protein HO-1 were significantly decreased in A549 with NRF2 knockout by western blot analysis. (C). The mRNA expression levels of NRF2 and its target genes were significantly decreased after the cell lines were knockout NRF2.(D) Colony-formation assay showed that the proliferation of A549 lung cancer cell lines depleted with NRF2 was significantly decreased.(E) The scratch wound-healing assay showed that the migration of A549 lung cancer cell lines depleted with NRF2 was slower at 0 h,24 h, and 48 h than that of A549 cell lines. Mean ± standard error of the mean (SEM) are reported .


This library of compounds includes [(C^N^C)PtII(L)] (L=dimethylsulfoxide (DMSO) 2 and triphenylphosphine (PPh3) 3) and [((C^N^C)PtII)2(L‘)] (where L‘=N‐heterocycles (pyrazine (pyr) 4, 4,4‘‐bipyridine  5 or diphosphine butane (dppb) 6). Their cytotoxicity was assessed against four cancerous cell lines and one normal cell line, with results highlighting significantly increased antiproliferative activity for the dinuclear complexes (4–6), when compared to the mononucleated species (2 and 3). Complex 6 is the most promising candidate, displaying very high selectivity towards cancerous cells, with selectivity index (SI) values >29.5 (A2780) and >11.2 (A2780cisR), and outperforming cisplatin by >4‐fold and >18‐fold, respectively.A series of cyclometallated mono‐ and di‐nuclear platinum(II) complexes and the parent organic ligand, 2,6‐diphenylpyridine  A study of the cytotoxicity of a library of cyclometallated mono‐ and di‐nuclear platinum(II)‐complexes, against a range of ovarian and breast cancer cell lines, identifies the importance of both ligand type and nuclearity on their antiproliferative activity. The lead candidate, a di‐nuclear platinum(II)‐phosphine complex, exhibits very high selectivity towards cancerous cells, with a selectivity index (SI) value >11.2 against cisplatin‐resistant ovarian cancer cells and outperforming monodentate analogues by >11‐fold and cisplatin by >18‐fold. Since the approval of the chemotherapeutic drug cisplatin, by the FDA in 1978, platinum(II)‐based complexes have become integral in the clinical treatment of a range of different cancers.Cyclometallated platinum(II) complexes have emerged as attractive alternatives to existing clinical antiproliferative platinum drugs, and several compounds were reported to possess moderate to potent cytotoxicity against cancer cell lines that are resistant to current platinum(II) anticancer drugs.5, 6 TN‐heterocycles and thiolates in the fourth coordination site, against a panel of human cancer cell lines including two human ovarian carcinomas, A2780 and A2780cisR.II(L)], wherein C^N^C is a tridentate dianionic cyclometallating motif bearing a range of aryl groups; including phenyl, naphthyl and dibenzoacridine derivatives and L=DMSO or acetonitrile (MeCN), displayed good to moderate cytotoxicity against human cell lines; colorectal adenocarcinoma (HT‐29) and breast adenocarcinoma (MDA‐MB‐231).Early reports from Lowe and co‐workers outlined the potent cytotoxicity of cyclometallated platinum(II)‐complexes incorporating a N^N^N terpyridine (terpy) ligand scaffold, and a range of 2(μ‐NHC)]+ platinum(II) complexes where HC^N^N=6‐phenyl‐2,2‘‐bipyridyl and μ‐NHC=a bridging N‐heterocyclic carbene ligand. They showed that through increasing the length of the linker between the two metal centres, up to a 2‐fold increase in cytotoxicity was induced against a panel of human cell lines, including cervix epithelioid adenocarcinoma (HeLa), liver hepatocellular carcinoma (HepG2) and nasopharyngeal carcinoma (SUNE1).+ displayed significantly higher antiproliferative activity, with nanomolar to sub‐micromolar potency (IC50=0.057–1.3 μm), compared to the dinuclear [((C^N^N)Pt)2(μ‐NHC)]+ platinum(II) complexes, (IC50=3.9–9.4 μm) against three tested cancer cell lines .2(bipy)] (where N^N^N=terpy and bipy is a range of pyridine substituted derivatives connected by different linker groups) against several human ovarian cell lines including those resistant to cisplatin and doxorubicin; CH1, CH1cis, CH1dox, A2780 and A2780cisR. Generally, they found that the presence of short rigid linkers, for example, 4,4‘‐bipyridine , between the metal centres generated complexes with potent cytotoxicity against the tested cancer cell lines including nanomolar IC50 values against the doxorubicin resistant ovarian cell line CH1dox.Polynuclear platinum(II) complexes represent an important growing class of anticancer agents with potential clinical significance.24, 25II(L)] (L=dimethylsulfoxide (DMSO) 2 and triphenylphosphine (PPh3) 3) and [((C^N^C)PtII)2(L‘)] (L‘=N‐heterocycles (pyrazine (pyr) 4, 4,4‘‐bipyridine  5 or diphosphine butane (dppb) 6) respectively, (where HC^N^CH=diphenylpyridine), against a range of cancer cell lines. This series of cyclometallated platinum(II) complexes were designed to consider the influence of three important structural features; 1) changes to the nature of the donor atom at the fourth coordination site of the metal centre  , 2) size of ligand spacer between the two metal centres for two dinuclear complexes (from pyrazine (pyr) to 4,4‘‐bipy) and 3) nuclearity of the complex (from mono‐ and di‐nuclear). We also report chemosensitivity studies against a normal cell type, showing that the most promising candidate, [((C^N^C)PtII)2(dppb)], is non‐toxic towards normal cells, unlike cisplatin, which demonstrates high cytotoxicity.In this study, we investigate the SARs for a series of mono‐ and di‐nuclear cyclometallated C^N^C platinum(II) compounds Figure ,  2 (where DMSO=dimethylsulfoxide) and [(C^N^C)Pt(PPh3)] 3 (where PPh3=triphenylphosphine), and three dinuclear platinum(II) complexes [((C^N^C)Pt)2(pyr)] 4, [((C^N^C)Pt)2] 5 and [((C^N^C)Pt)2(dppb)] 6  and is due to the two phosphines coupling with the platinum centres in this symmetric molecule.The library of compounds consists of a ligand, 2,6‐diphenylpyridine ocedures.33, 346 suitable for X‐ray diffraction were obtained through the slow evaporation of a concentrated chloroform solution at room temperature. The complex crystallizes in a monoclinic crystal system and solution refinement was performed in the space group P21/c (Table S3 in Supporting Information). The molecular structure of 6 is shown in Figure pseudo square‐planar geometry expected of d8 PtII complex (Tables S4 and S5).6, there are intermolecular, edge‐to‐face, π–π stacking interactions present between the phenyl rings of dppb and aromatics of the C^N^C pincer ligand .Bright yellow crystals of 1, complexes 2–6, cisplatin (CDDP), oxaliplatin (OXA) and carboplatin (CARB) were all screened for their cytotoxicity against human cell lines: cisplatin‐sensitive ovarian carcinoma (A2780), cisplatin‐resistant ovarian carcinoma (A2780cisR) and breast adenocarcinomas (MCF‐7 and MDA‐MB‐231). The IC50 values were obtained via the MTT assay after a 96 h incubation period of each compound with the cells at 37 °C and 5 % CO2  complexes, 2 and 3, have significant differences in their cytotoxicity. The potency of complex 2 increases by up to 10‐fold against MCF‐7 in comparison to ligand 1 (p<0.05), and exhibits similar potency (IC50=4.4±0.5 μm) to CDDP (IC50=3.07±0.02 μm) against MDA‐MB‐231 (p=0.01). Importantly, 2 is the only compound in this library which has noticeable toxicity towards MCF‐7 (IC50=10±1 μm), however, it remains >6‐fold less cytotoxic than CDDP (p<0.05). A recent report by Klein and co‐workers determined that 2 has a IC50 value against MDA‐MB‐231 of 12.12±1.84 μm, which is within experimental error.2 is more active towards the cisplatin‐resistant ovarian cell line A2780cisR than the parental cisplatin‐sensitive A2780 cells. On replacing the monodentate DMSO ligand in 2 for PPh3 in 3, the activity significantly decreases, and 3 is non‐toxic against all cell lines tested (IC50>100 μm) (Table Ligand 4 was designed to study the cytotoxicity effects of a dinuclear system with the short pyrazine linker. The cytotoxicity of 4 increases by up to 27‐fold and 5‐fold against A2780 (p<0.05), in comparison to mononuclear complexes 2 and 3, respectively. Upon extending the linker to 4,4‘‐bipy (5), the compound remains equitoxic to that of 4 against A2780, however, the toxicity decreases against all other cell lines, with up to a 2‐fold decrease against A2780cisR and an 11‐fold decrease against MDA‐MB‐231 (p<0.05). Harper et al. showed that the related [((N^N^N)Pt)2] (N^N^N=terpy) analogue of 5 has been reported to have GI50 values of 2.7±1.3 μm against A2780 and 5.6±0.4 μm against A2780cisR.50 values of >25 μm against A2780 and 9.6 μm against A2780cisR.50 values of 5 and the [((N^N^N)Pt)2] (N^N^N=terpy) analogue, highlights that the nature of the pincer ligand is also an important factor on determining the cytotoxicity of these cyclometallated platinum(II) complexes. On modification of the linker to a flexible butyl substituent, in the dinuclear diphosphine complex 6, the cytotoxicity against MDA‐MB‐231 decreases even further, with >4‐fold decrease when compared to 5 and >52‐fold decrease when compared to 4. Moreover, increasing the nuclearity of the platinum(II)‐phosphine compounds from mononuclearity in 3 to dinuclearity in 6 results in significantly increased cytotoxicity values against the ovarian carcinoma cell lines, with up to >29‐fold and >11‐fold increases observed against A2780 and A2780cisR, respectively. As the IC50 values of 3 against both ovarian cancer cell lines, A2780 and A2780cisR, is greater than the tested threshold concentration of 100 μm, this observed increase in cytotoxicity between these two platinum(II)‐phosphine ligands could be greater than reported here. As for 3, complex 6 is non‐toxic against MCF‐7 and MDA‐MB‐231 (IC50>100 μm).Complex 2 increasing the potency in all test cancer cells lines by up to 25‐fold compared to the analogous phosphine‐platinum(II) complex 3 , ii) the addition of a second platinum centre and linker unit increases the potency of the compounds by up to 29‐fold (e.g. 3 versus 6 against A2780), iii) the shorter pyrazine linker in complex 4 is the optimal linker of the N‐heterocyclic dinuclear species exhibiting up to sub‐micromolar potency against MDA‐MB‐231 (IC50=1.9±0.5 μm). A notable result is that of complex 6, which is non‐toxic towards either of the two human breast cancer cell lines , has significantly increased cytotoxicity against the human ovarian cancer cell lines, >29‐fold against A2780  and >11‐fold against A2780cisR .On analysis of these results, no definitive SARs can be determined, however, some important structural features can be highlighted; i) the nature of the ligand plays a significant role in determining the cytotoxicity of the mononuclear complexes, with the presence of the sulfoxide ligand in CDDP, OXA and CARB), which exhibit high potency towards normal cell types, this library of compounds was screened against normal prostate cell line, PNT2 , 8.5±0.4 μm (CDDP) and 27±2 μm (CARB). Ligand 1 and complex 3 are non‐toxic towards PNT2 (IC50 >100 μm). Complexes 2, 4 and 5 all remain moderately cytotoxic against the normal cell line, and even though complex 4 is >4‐fold more cytotoxic against MDA‐MB‐231 than PNT2, it remains relatively cytotoxic against normal cells (IC50=8.3±0.6 μm). The most promising result is observed for 6, which remains non‐toxic against PNT2 (IC50>100 μm) yet is cytotoxic towards human ovarian carcinomas.Due to the current issues of the clinical platinum compounds  and 6.7‐fold (A2780cisR) , however, 6 exhibits a SI value of 29.5, which is 4‐fold higher than that of CDDP (p<0.05). Importantly, the SI values observed for complex 6 against A2780cisR show a higher degree of selectivity than the clinical platinum compounds, with SI values >18‐fold (CDDP and OXA) and >42‐fold (CARB).The selectivity index (SI) values were calculated for all compounds, using the ICne Table . Whereby1–6 to target the ovarian cisplatin‐resistance cell line A2780cisR, the IC50 values were compared with those from the ovarian cisplatin‐sensitive cell line A2780. Resistance factors (RF)were obtained by dividing the IC50 values of A2780 by A2780cisR, where values >1 indicate a preference for the cisplatin resistant cell line A2780cisR. The RF values could not be calculated for ligand 1 and complex 3, as IC50 values are >100 μm against both ovarian cell lines. As with the clinical platinum drugs, complexes 4–6 are all more cytotoxic towards A2780. However, complex 2 has a slightly higher selectivity for A2780cisR, with a RF value of 1.4 .As many cancers become resistance to drugs, including the clinical platinum drugs, there is an urgent need to address the issues of drug resistance, by designing new and effective drugs. To address the potential of compounds 1–6 in comparison with the clinical drugs, the sensitivity factors (SF) were calculated by dividing the IC50 values of the clinical drugs by the IC50 values of compounds 1–6. An SF >1 indicates a selectivity for our library of compounds over the clinical drugs  and A2780cisR (4 and 6) whilst the rest of the library displays SF<1 against the other tested cell lines. When the biological performance of 1–6 is compared to OXA  are the most complex and aggressive types of breast cancer,2, 4–6 all have increases activity towards one or more of the ovarian carcinomas, when compared to CARB, with significantly higher activity against A2780 displaying SF ranging from 4.3 (5) to 5 (6) and, notably, against the cisplatin‐resistance ovarian cell line A2780cisR, with SF ranging from 5.4 (5) to 11.4 (4). Additionally, complex 2 has significantly higher activity when compared to CARB against the breast adenocarcinoma MCF‐7, displaying an IC50 value at least 10x higher than this clinical drug.To address the activity of compounds P Figure , complexA Figure , only cos Figure . In part4 and 6. Although complex 4 is moderately cytotoxic towards the normal cell line, its increased selectivity towards the TNBC line MDA‐MB‐231 is promising and should be investigated in further studies. Complex 6 is non‐toxic towards the normal cell line and has the highest cancer cell selectivity for this library, warranting further modifications and in‐depth in vitro studies. On analysis of these results, it is possible that these complexes have mechanism of actions which differs from that of the clinical platinum drugs. As it has previously been shown that cyclometallated N^N^N platinum(II) cationic complexes form strong intercalations with DNA, with binding constants from 0.8×105 m−1 to 2.0×107 m−1, and [((C^N^C)PtII)2(L‘)] respectively, where H C^N^CH is 2,6‐diphenylpyridine, L is a monodentate ligand (DMSO 2 or PPh33) and L‘ is a bidentate ligand . All compounds were screened against human ovarian carcinomas (A2780 and A2780cisR), human breast adenocarcinomas (MCF‐7 and MDA‐MB‐231) and normal prostate cell line (PNT2). There are no definite conclusions which can be drawn on the SARs however, some general trends can be observed.In conclusion, we have synthesized a library of mono‐ and di‐nuclear, cyclometallated platinum(II) complexes, of the general formula, + (100 %); HR‐ESIMS (+ve): calcd 232.1121 found 232.1115 for C17H14N. Elemental Analysis: Anal. Found: C: 88.2, H: 5.7, N: 6.1 %. Anal. Calculated: C: 88.3, H: 5.7, N: 6.1 %.2: To a solution of H2L  in acetic acid (55 mL) was added a solution of K2PtCl4  in H2O (3 mL) in a dropwise manner to generate a light rose coloured solution. The mixture was refluxed for 18 h, after which time, the red colour of the Pt salt had disappeared. The yellow precipitate that had formed was filtered off and washed with H2O (4 mL), acetone (4 mL), Et2O (4 mL) and pet. ether (2 mL). The yellow solid was then dissolved in DMSO (3 mL) and K2CO3  and H2O (2 mL) were added and the mixture heated to 90 °C for 1 h. After this time, the reaction mixture was allowed to cool to room temperature and upon the addition of H2O (10 mL), the product precipitated out as a bright yellow solid. This crude product was subjected to flash column chromatography (CH2Cl2). Single bright yellow crystals suitable for X‐ray diffraction were grown through the slow evaporation of chloroform from a saturated solution of 2. . 1H NMR : δ 7.81 =29.6 Hz, ArH), 7.63 , 7.49 , ArH), 7.33–7.27 , 7.13 , 3.69 =27.2 Hz, 2 × CH3); IR (solid) νmax 3034 (w), 2920 (w), 1598 (m), 1577 (m), 1562 (m) cm−1; LR‐ESIMS (+ve) m/z (%): 503 [M+H]+(100 %); HR‐ESIMS (+ve): calcd 503.0751 found 503.0745 for C19H18NO2NaPtS. Elemental Analysis: Anal. Found: C: 45.6, H: 3.3, N: 2.8 %. Anal. Calculated: C: 45.4, H: 3.4, N: 2.8 %.3: To a solution of 2  in CH2Cl2 (2 mL) was added PPh3  and the resultant pale‐yellow solution was stirred at ambient temperature for 1 min. After this time, the excess solution was removed under reduced pressure and the resulting yellow residue was subjected to flash column chromatography  to yield a bright yellow solid. Single crystals suitable for X‐ray crystallography were grown through the slow diffusion of di‐ethyl ether into a saturated solution of 3 in chloroform. . 1H NMR : δ 7.94 , 7.85–7.80 , 7.73 , 7.62 , 7.60–7.51 , 6.93 , 6.62 , 6.18 =32.4 Hz, ArH); IR (solid) νmax 3040 (w), 1597 (m), 1563 (m), 1508 (w) cm−1; LR‐ESIMS (+ve) m/z (%): 687 [M+H]+(100 %); Elemental Analysis: Anal. Found: C: 61.1, H: 3.7, N: 2.1 %. Anal. Calculated: C: 61.2, H: 3.8, N: 2.0 %.4: To a solution of 2  in CH2Cl2 (2 mL) was added pyrazine  and the resultant pale‐yellow solution was stirred at ambient temperature overnight. After this time, the red precipitate that had formed was filtered off, washed with CH2Cl2 (2 mL) to give the desired product as a bright red solid. . 1H NMR : δ 8.65 , 7.84 , 7.76 , 7.65–7.63 , 7.19 , 7.08 ; IR (solid) νmax 3043 (w), 1697 (w), 1598 (m), 1575 (m), 1560 (m) cm−1; LR‐ESIMS (+ve): 929 [M+H]+ (100 %); Elemental Analysis: Anal. Found: C: 49.0, H: 2.8, N: 6.0 %. Anal. Calculated: C: 49.1, H: 2.8, N: 6.0 %.5: To a solution of 2  in CH2Cl2 (2 mL) was added 4,4‘‐bipyridine  and the resultant pale‐yellow solution was stirred at ambient temperature overnight. After this time, the red precipitate that had formed was filtered off, washed with CH2Cl2 (2 mL) to give the desired product as a bright orange solid. . 1H NMR : δ 8.73 , 7.84–7.82 , 7.77 , 7.66–7.63 , 7.20 , 7.08 ; IR (solid) νmax 3041 (w), 1597 (m), 1575 (m), 1558 (m), 1541 (m) cm−1; LR‐ESIMS (+ve): 1005 [M+H]+; Elemental Analysis: Anal. Found: C: 51.9, H: 3.0, N: 5.4 %. Anal. Calculated: C: 52.6, H: 3.0, N: 5.6 %.6: To a solution of 2  in CH2Cl2 (2 mL) was added dppb  and the resultant pale‐yellow solution was stirred at ambient temperature for 1 h. After this time, the pale‐yellow precipitate that had formed was filtered off and yellow crystals suitable for X‐ray diffraction were grown through the slow diffusion of pet. ether into a saturated solution of chloroform . m.p. 310–311 °C; 1H NMR : δ 7.70–7.65 , 7.44 , 7.38–7.28 , 6.93 , 6.70 , 6.55 , 2.62–2.55 , 2.01–1.99 ; 13C{1H} NMR : δ 165.7 (JC‐Pt=104 Hz), 150.2, 139.6, 138.0 (JC‐Pt=116 Hz), 132.6, 132.5 (JC‐Pt=100 Hz), 129.5, 129.1, 127.5, 127.4, 123.2, 122.7, 114.2, 25.7, 25.3; 31P NMR : δ 20.3 (JP‐Pt=4960 Hz); IR (solid) νmax 3047 (w), 2936 (w), 2918 (w), 1597 (m), 1578 (m), 1565 (m), 1546 (m); LR‐ESIMS (+ve): 1275 [M+H]+; 1297 [M+Na]+; HRESI (+ve): calcd 1275.2823 found 1275.2803 for C62H51N2P2Pt2; calcd 1297.2642 found 1297.2637 for C62H50N2NaP2Pt2.Deposition Number(s) 1998373, 1998377 and 1998378 contain(s) the supplementary crystallographic data for this paper. These data are provided free of charge by the joint Cambridge Crystallographic Data Centre and Fachinformationszentrum Karlsruhe Access Structures service www.ccdc.cam.ac.uk/structures.The authors declare no conflict of interest.As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer reviewed and may be re‐organized for online delivery, but are not copy‐edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors.SupplementaryClick here for additional data file.