An expedient multi-component synthesis of pyridinyl-spirooxindoles and their effect on proliferation of lung cancer A549 cells

A series of new functionalized pyridinyl-spirooxindoles have been synthesized through three-component cyclization reactions. The selected compounds were screened for their in vitro antiproliferative activity against human lung cancer cell line A549. Among the candidate structures, compound 1o demonstrated maximum inhibitory activity against A549 cells with IC₅₀ values of 28.38 μM. EdU (5-Ethynyl-2′- deoxyuridine, EdU) assay and cell colony formation test showed that cell proliferation of A549 cells was inhibited. In addition, Western blot analysis revealed that the phosphorylation levels of Akt, mTOR, 70S6, and S6 were down-regulated. Thus, these results indicated that 1o may inhibit the proliferation of A549 cells through inhibiting the phosphorylation levels of Akt, mTOR, 70S6, and S6. 1o may be developed as a potential antitumor agent for lung cancer treatment.     

Analysis of breath samples for lung cancer survival

Analyses of exhaled air by means of electronic noses offer a large diagnostic potential. Such analyses are non-invasive; samples can also be easily obtained from severely ill patients and repeated within short intervals. Lung cancer is the most deadly malignant tumor worldwide, and monitoring of lung cancer progression is of great importance and may help to decide best therapy. In this report, twenty-two patients with diagnosed lung cancer and ten healthy volunteers were studied using breath samples collected several times at certain intervals and analysed by an electronic nose. The samples were divided into three sub-groups; group d for survivor less than one year, group s for survivor more than a year and group h for the healthy volunteers. Prediction models based on partial least square and artificial neural nets could not classify the collected groups d, s and h, but separated well group d from group h. Using artificial neural net, group d could be separated from group s. Excellent predictions and stable models of survival day for group d were obtained, both based on partial least square and artificial neural nets, with correlation coefficients 0.981 and 0.985, respectively. Finally, the importance of consecutive measurements was shown.     

A therapeutic keypad lock decoded in drug resistant cancer cells

A molecular keypad lock that displays photodynamic activity when exposed to glutathione (GSH), esterase and light in the given order, is fabricated and its efficacy in drug resistant MCF7 cancer cells is investigated. The first two inputs are common drug resistant tumor markers. GSH reacts with the agent and shifts the absorption wavelength. Esterase separates the quencher from the structure, further activating the agent. After these sequential exposures, the molecular keypad lock is exposed to light and produces cytotoxic singlet oxygen. Among many possible combinations, only one ‘key’ can activate the agent, and initiate a photodynamic response. Paclitaxel resistant MCF7 cells are selectively killed. This work presents the first ever biological application of small molecular keypad locks.

Information processing therapeutics with an implemented keypad lock logic gate selects input order for activation in drug resistant cancer cells.

The complex nature of diseases such as cancer necessitates smarter drugs that can discriminate each disease state or regulate drug efficacy spatially and/or temporally. With this intention, activatable drugs, drugs with on demand release properties are developed with promising selectivity.^1–4 Information processing therapeutics which are based on molecular logic gate operations are another approach to solve this problem.^5–7 Molecular logic gates are small compounds using Boolean logic operations to process inputs (i.e. the analyte concentration), and give an output as a result (fluorescence, and therapeutic activity etc.).⁸ Selective drug activation, release, multiple-analyte sensing and theranostic applications of these devices have been explored by us and others.^5,9–19Among the operations that can be carried out using small molecules, keypad locks provide an alternative application in information security.²⁰ This logic operation can give a specific output when the inputs are given in the correct form and correct sequence. For the device, each input is considered as an AND logic operation where the history of the process is also considered. A pioneering example was reported by Margulies and Shanzer in 2007 where energy transfer is modulated by chelation of Fe³⁺ in a pH dependent manner.²¹ Later, various other devices were introduced with advanced properties such as more than 2 input responsiveness and error detection capability.^22–24 All-photonic logic gates to address chemical waste production is extensively studied by Gust, Andréasson and Pischel.^25,26 Beside small molecule keypad locks, enzymes, antibodies, and DNA hybrids are used to achieve the same goal.^27–30 Although their potential use in molecular cryptology is highlighted, so far, there is no solid biological application of small molecule keypad locks.In the research presented here, a molecular keypad lock is developed which displays a photodynamic therapeutic output when a molecule is exposed to analytes in the correct order and type (PS3, Fig. 1). Two inputs of the system are chosen to be the common markers of drug resistant tumours: glutathione (GSH) and esterase enzyme (E). Cancer cells develop resistance to traditional chemotherapy in time by changing the protein expression or metabolite content of the cell. This adaptation of cancer cells is an obstacle for their treatment and needs to be addressed. Glutathione is a tripeptide used in reductive biochemical synthesis and it is known to be present in elevated levels in rapidly dividing cells such as cancer cells.³¹ A high GSH level is reported to contribute to drug resistance, since GSH adducts of the drugs are exported out of the cell much more rapidly.^32,33 Likewise, esterase enzyme activity is known to be associated with drug detoxification as this enzyme contributes to the chemical conversion of the drug.^34,35 Glutathione and esterase enzyme are chosen to be the first two inputs of the molecular keypad lock, the first two digits of the password. In the research, light is used as the final input. Although trivial, light is essential for photodynamic activity and spatiotemporal control of irradiation, further improving selectivity of the therapy.Open in a separate windowFig. 1Chemical structures of model photosensitizers (PS1 and PS2) and a molecular keypad lock (PS3). Ester bonds (red) are prone to hydrolysis by the esterase enzyme. Distyryl sites of the photosensitizers (blue) can react with thiol nucleophile provided that it is bound to an electron deficient group (i.e. pyri-dinium).Keypad lock PS3 is a photodynamic therapy (PDT) agent. PDT is a non-invasive method used for the treatment of surface cancers and certain other diseases ranging from atherosclerosis to macular degeneration.^36–39 In this therapy, a photosensitizer is excited with light, and produces cytotoxic singlet oxygen (¹O₂) thereby triggering apoptosis or necrosis of the cell, initiating an immune response and blocking microvasculature.⁴⁰ In the research, a boradiazaindecene (BODIPY) photosensitizer is used to benefit from versatile chemistry and spectroscopic properties.^41–45Near-IR absorbing PS3 shown in Fig. 1 is the molecular keypad lock and it is synthesized in 13 steps (Scheme S1^†). PS3 and model compound PS2 have heavy atoms on the structure to favour intersystem crossing required for transition to the triplet state and hence ¹O₂ generation occurs.⁴³ Ester bonds on the structure of PS3 are prone to cleavage by esterase enzyme. Distryryl bonds on the PS3 (blue) tend to reduce or form an adduct with thiol nucleophiles when it is activated by the pyridinium electron withdrawing group.⁴⁶ This property lies at the heart of sequential operation of esterase and GSH. When GSH reacts with electron poor double bonds, the extended conjugated structure is broken and PS3-a is generated (Fig. 2). This structure has absorption below 550 nm, like brominated core BODIPY molecules (compound 8, Scheme S1^†), and therefore can be excited with a green light. A quencher (green) is attached to ensure that photodynamic activity is OFF until esterase cleaves the ester bond. This is because of the energy transfer from the photosensitizer to this module, until esterase separates the photosensitizer. Since PS3 lacks absorption around the 500–550 nm region, it is inactive until GSH reacts with the compound. However, the GSH reacted photosensitizer does show absorption in this region; so, in order to avoid full activation just by GSH, a quencher module is attached. Spectral overlap between the BODIPY core (see the structure of compound 8 in the ESI,^† similar to that of PS3-a in terms of conjugation) and quencher (Q) can be seen from UV-Vis absorption and fluorescence spectra (Fig. 3 and S1^†). By this way, the photosensitizer is chemically modulated by GSH to ensure excitation, and then esterase enzyme inhibits energy transfer by removing the quencher. Lastly a green light is used to excite the photosensitizer leading to generation of photodynamic action. Since light is necessary for the final excitation of the molecule, it should always be the last input. If the order of esterase and GSH changes, as shown in Fig. 2, activation is not expected to take place since cleavage of the ester bonds generates 4-hydroxybenzyl derivative on PS3, which spontaneously faces 1,4-elimination to generate pyridine (Fig. S2^†).⁴⁷ Pyridine on its own is not sufficiently electron withdrawing to favour nucleophilic attack of double bonds by GSH and to activate it as demonstrated below. Therefore, the photosensitizer preserves extended conjugation and essentially lacks absorption at the wavelength of excitation.Open in a separate windowFig. 2Sequential operation of GSH and esterase. GSH can only react with BODIPY distyryl units when the structure has electron withdrawing pyridinium, either reducing it or forming an adduct. Esterase enzyme cleaves ester bonds, liberating the photosensitizer from the quencher module (green). Initial esterase activity converts the pyridinium unit to pyridine, thereby decreasing the reactivity of double bonds with GSH.Open in a separate windowFig. 3Normalized UV-Vis absorption and fluorescence spectra of PS1–3 in 2% water in THF (a and b). Samples are excited at 600 nm. Spectral changes of PS3 (10 μM) alone (black) or PS3 upon exposure to 0.5 mM GSH (c) and 10U esterase (d) for 90 min and 60 min at 37 °C, in 2% water in THF, respectively. A new peak at 544 nm appears upon incubation with GSH which is attributed to reduced PS3 and/orthe GSH-adduct. Esterase treatment increases the relative intensity of the shoulder peak around 600 nm.In order to understand the response of the PS3 to GSH, a molecule is incubated with 0.5 mM of GSH at 37 °C for 90 min. A new peak at 544 nm appears in UV-Vis absorption spectra consistent with the hypothesis (Fig. 3c, S1 and S9^†). The formation of the GSH adduct (PS3-a) is demonstrated by Liquid Chromatography Mass spectrometry analysis (Fig. S3^†). When control module PS1 is exposed to the same conditions, this new peak is not detected indicating that the pyridine bearing structure is neither activated enough for the nucleophilic substitution by GSH nor did it display PDT activity (Fig. S4 and S5^†). On the other hand, GSH treated pyridinium bearing PS2 immediately displayed a colour change indicative of broken conjugation (Fig. S6^†). When PS3 is incubated with esterase for 1 h, a small hypsochromic shift in the absorption peak is detected as a shoulder to the parent peak which is attributed to the conversion of pyridinium to pyridine (PS3-c, Fig. 3d). The control PS3 sample which is incubated under the same conditions but lacks esterase does not show an enhancement of this peak (Fig. 3d, black). High Resolution Mass Spectrometry analysis of the esterase treated PS2 samples confirm the hydrolysis of the ester and subsequent formation of the pyridine compound (Fig. S7^†). Esterase treated samples display an increase in the emission intensity when excited at 620 nm (Fig. S8^†). This is attributed to the initial quenching of the quencher module by the pyridinium photosensitizer. Analysis of the absorption and emission spectra suggest that the quencher module of PS3 can induce energy transfer to the pyridinium photosensitizer (Fig. 3). Once separated by esterase, fluorescence of the quencher module increases. In the case of GSH treated sample, a small enhancement in emission upon excitation at 500 nm is observed (Fig. S9^†). Note that the GSH adduct (or PS3 with reduced double bonds) has higher absorption at this wavelength, which would be the reason for the increase in emission intensity. In the spectral analysis organic solvents with a low water content are used to monitor the formation of water-insoluble, neutral, pyridine-bearing intermediate species.In the project, the molecular keypad lock is aimed to unlock in the presence of drug resistant tumour markers and get activated. Activation cannot take place when the input order differs. To demonstrate this, photodynamic action in the presence of all three inputs in a different order is investigated. ¹O₂ production can be followed by using trap molecule, 1,3-diphenylisobenzofuran (DPBF).⁴⁸ This molecule reacts with ¹O₂ and loses its absorption at 418 nm. The effect of different input combinations on the PDT action are given in Fig. 4. In the first 15 min, all samples are kept in the dark. Under such conditions no ¹O₂ generation is detected, which indicates lack of dark activity. DPBF is exposed to light from a LED source (peak 505 nm) under the same experimental conditions and no decrease in the absorption is detected. This control experiment eliminates the photodegradation of DPBF in the absence of a photosensitizer. Upon irradiation before the activation of the photosensitizer by GSH and esterase, no ¹O₂ generation is observed as expected. The results show that ¹O₂ generation, and the subsequent decrease in DPBF absorption, are significantly more in the input order of glutathione, esterase enzyme and light, consistent with the proposed mode of activation.Open in a separate windowFig. 4 ¹O₂ generation ability of PS3 (0.1 μM) when three inputs are given in a different order. All samples contain 50 μM of ¹O₂ trap molecule DPBF. In the first 15 minutes samples are kept in the dark. GSH is added in 0.5 mM concentration and incubated for 90 min at 37 °C. Samples are incubated with 10U esterase for 1 h at 37 °C. An LED light is irradiated from a 30 cm distance for 45 min.To analyse the effect of PDT action in the cell, a drug resistant cell line is generated. MCF7 cells are exposed to an increased dose of traditional cancer therapeutic agent paclitaxel as described in the literature.⁴⁹ When the spindle-shaped morphology is obtained following maximum drug dose application, cells are reported to have drug resistance. At this stage, PS3 is applied to both normal and drug resistant cells. When cell viabilities at various concentrations are analysed, it has been found that the light toxicity of PS3 is significantly enhanced in drug resistant cells (Fig. 5). The IC50 values of irradiated samples are calculated to be 124.8 μM for MCF7 cells. This value is reduced to 52.5 μM in paclitaxel resistant MCF7 (Pac-MCF7) indicating improved cytotoxicity in these cells. Efficient induction of apoptosis is also proved by Annexin V and PI staining (Fig. 6). Under dark conditions, cells do not have significant loss of viability. Upon irradiation, resistant cells are more prone to apoptosis by the photosensitizer. Relative singlet oxygen generation abilities and results of cell culture experiments altogether confirm selective activation in drug resistant cells.Open in a separate windowFig. 5Change in the cell viability of normal and paclitaxel resistant MCF7 cells (Pac-MCF7) in the presence of PS3 at various concentrations. For each group, cell viability is analysed both after incubation in the dark or after irradiation with a 505 nm LED light source from a distance of 10 cm. Average values of three independent experiments are used.Open in a separate windowFig. 6Apoptosis induction by PS3 (25 μM) in normal and paclitaxel resistant MCF7 cancer cells under dark conditions and upon irradiation with a 505 nm LED light from 10 cm distance. Scale bars: 50 μm.     

Genome-wide identification and characterization of long non-coding RNAs involved in acquired resistance to gefitinib in non-small-cell lung cancer

Acquired resistance is a major obstacle to the therapeutic efficacy of gefitinib in non-small-cell lung cancer (NSCLC). Current knowledge about the role of long non-coding RNAs (lncRNAs) in this phenomenon is insufficient. In this study, we searched RNA sequencing data for lncRNAs associated with acquired resistance to gefitinib in NSCLC, and constructed a functional lncRNA-mRNA co-expression network and protein-protein interaction (PPI) network to analyze their putative target genes and biological functions. The expression levels of 14 outstanding dysregulated lncRNAs and mRNA were verified using real-time PCR. Changes in the expression levels of 39 lncRNAs and 121 mRNAs showed common patterns in our two pairs of gefitinib-sensitive and gefitinib-resistant NSCLC cell lines. The co-expression network included 1235 connections among these common differentially expressed lncRNAs and mRNAs. The significantly enriched signaling pathways based on dysregulated mRNAs were mainly involved in the Hippo signaling pathway; proteoglycans in cancer; and valine, leucine, and isoleucine biosynthesis. The results show that LncRNAs play an important part in acquired gefitinib resistance in NSCLC by regulating mRNA expression and function, and may represent potential new molecular biomarkers and therapeutic targets for gefitinib-resistant NSCLC.     

Chamaejasmine induces apoptosis in human lung adenocarcinoma A549 cells through a Ros-mediated mitochondrial pathway

In the present study, the anticancer activity of chamaejasmine towards A549 human lung adenocarcinoma cells was investigated. In order to explore the underlying mechanism of cell growth inhibition of chamaejasmine, cell cycle distribution, ROS generation, mitochondrial membrane potential (Δψ(m)) disruption, and expression of cytochrome c, Bax, Bcl-2, caspase-3, caspase-9 and PARP were measured in A549 cells. Chamaejasmine inhibited the growth of A549 cells in a time and dose-dependent manner. The IC?? value was 7.72 μM after 72 h treatment. Chamaejasmine arrested the cell cycle in the G2/M phase and induced apoptosis via a ROS-mediated mitochondria-dependent pathway. Western blot analysis showed that chamaejasmine inhibited Bcl-2 expression and induced Bax expression to desintegrate the outer mitochondrial membrane and causing cytochrome c release. Mitochondrial cytochrome c release was associated with the activation of caspase-9 and caspase-3 cascade, and active-caspase-3 was involved in PARP cleavage. All of these signal transduction pathways are involved in initiating apoptosis. To the best of our knowledge, this is the first report demonstrating the cytotoxic activity of chamaejasmine towards A549 in vitro.     

Green synthesis of zinc oxide nanoflowers using Hypericum triquetrifolium extract: characterization,antibacterial activity and cytotoxicity against lung cancer A549 cells

Zinc oxide nanoparticles have attracted significant interest in recent years due to their unique multifunctional chemical and physical properties along with their biological activities. This study demonstrated for the first time the biogenetic synthesis of zinc oxide nanoparticles by utilization of the methanolic extract of Hypericum triquetrifolium (HT). The obtained nanoparticles (HT-ZnO) were characterized by ultraviolet–visible spectroscopy (UV–Vis), Fourier-transform infrared spectroscopy (FTIR), X-ray diffraction (XRD), dynamic light scattering (DLS), scanning electron microscopy (SEM), energy dispersive X-ray spectroscopy (EDX), and transmission electron microscopy (TEM). The shape of the resulted nanoparticles is fusiform nanoflowers with an average hydrodynamic size of 275.46 ± 0.20 nm and a zeta potential of −8.23 ± 0.26 mV. SEM micrographs revealed that HT-ZnO nanoflowers have a multi-process structure in which one of the processes is large and the others have similar smaller dimensions. The synthesized nanoflowers have an average length of 312.28 ± 78.93 nm and the tip of its processes has a width of 48.69 ± 9.71 nm. The antimicrobial activity of HT-ZnO nanoflowers was performed using microbroth dilution format. It showed a bactericidal mode of action against Gram-positive Staphylococcus aureus and Enterococcus faecalis with MIC/MBC values of 20 μg/mL and 5 μg/mL, respectively. MTT assay had revealed that HT-ZnO nanoflowers caused a dose-dependent decline in the viability of A549 adenocarcinomic human alveolar basal epithelial cells with an IC₅₀ value of 20.45 μg/mL. The effect of HT-ZnO nanoflowers on the migration and colony formation abilities against the same cells was evaluated as well. In conclusion, zinc oxide nanoflowers were successfully synthesized using methanolic extract of H. triquetrifolium. The resulting particles showed a bactericidal effect against Gram-positiveS. aureus and E. faecalis and a cytotoxic activity against A549 cells.     

Cell-specific delivery of a chemotherapeutic to lung cancer cells

We report that lung cancer-targeting peptides isolated from a peptide library can be used to deliver an active chemotherapeutic in a cell-specific fashion. The peptides were removed from the context of the phage and placed on a pegylated tetrameric scaffold. The tetrameric peptides were shown to block uptake of their cognate phage. The tetrameric peptides were coupled to doxorubicin, and their cytotoxicity against a panel of different cell lines was tested. Our data demonstrate that these targeting peptides can deliver an active anticancer agent in a cell-specific fashion, resulting in an increase of the therapeutic index of the targeted drug compared to systemic delivery. The efficacy of the peptide conjugate correlates to the affinity of the targeting peptide for a particular cell line. As such, we have demonstrated that cell-specific targeted drugs can be synthesized, even when the cell surface target is unknown.     

A water soluble vitamin B12-ReI fluorescent conjugate for cell uptake screens: use in the confirmation of cubilin in the lung cancer line A549

A water soluble vitamin B(12)-rhenium conjugate was synthesized and used in concert with intrinsic factor to screen for cubilin receptor-mediated uptake in lung cancer cells. Internalization of the conjugate demonstrated that it could be used to rapidly screen for the cubilin receptor in living cells, subsequently confirmed with Western blotting and RT-PCR.     

Pyrazine based Pt(II) bis‐alkynyl organometallic complexes: Synthesis,characterization, and cytotoxic effect on A549 human lung carcinoma cells

A facile and efficient synthesis of a series of five new pyrazine‐based organometallic complexes is being reported. The complexes ( 2 – 6 ) have been fully characterized. Molecules 2 , 3 and 6 were structurally characterized by single crystal X‐ray crystallographic analyses. Additionally, anticancer properties of these organometallic complexes have been studied against A549 human lung carcinoma cells. Biological studies suggest that complex 5 (with pendant pyridine moieties) exhibited maximal growth inhibitory cytotoxic effect even at lower concentration (nearly 0.5–1 μM) whereas the other four compounds ( 2 , 3 , 4 and 6 ) were non‐toxic below 1 μM concentration. Two complexes ( 4 and 6) were effective only at higher doses (approx 30 μM) while the remaining two organometallic complexes ( 2 and 3) were able to inhibit 50% cell growth at slightly lower concentration (nearly 10 μM). As far as IC₅₀ is concerned, 5 exhibited a minimum value ranging between 3–5 μM which is comparable with cisplatin under similar conditions.     

A new 3D supramolecular Zn(II) compound: Crystal structure and important role in treatment of pulpitis

An innovative thermostable 2-D layered Zn(II) compound, chemical terms are written as [Zn(3-pysa)₂(H₂O)₂]_n (1, 3-Hpysa = 3-pyridinesulfonic acid), has been generated from the solvothermal reactions of 3-Hpysa and Zn(NO₃)₂·6H₂O. Its single crystal analysis was implemented via the single crystal X-ray diffraction analysis together with the powder X-ray diffraction, elemental analysis and thermogravimetric analysis. The crystal framework is of monoclinic P2₁/n space group and its crystal cell figures: a = 7.7499(5), b = 10.9923(6), c = 8.3430(5) Å, α = 90, β = 96.924(6), γ = 90°, V = 705.55(7) ų, Z = 4. The 2D layers were finally combined to a 3-D supramolecular conformation through intermolecular H-bonds existing between coordinated H₂O molecules and sulfonate oxygen atoms from adjacent layer. Its practical role in pulpitis treatment was estimated and the relevant mechanism was studied in the meantime.     

Proteomic and redox‐proteomic study on the role of glutathione reductase in human lung cancer cells

Glutathione reductase (GR), a cytosolic protein, plays a vital role in maintaining a correct redox status in cells. However, comprehensive investigations of GR‐modulated cellular responses, including protein level alteration and redox regulation, have yet to be performed. In this study, we cultured a human lung adenocarcinoma line transfected with empty pLKO.1 vector as a control, CL1‐0shControl, and its GR‐knockdown derivative, CL1‐0shΔGR, to evaluate differential protein level alteration and redox regulation of these two cell lines. We identified 34 spots that exhibited marked changes in intensities, and 13 proteins showing significant changes in thiol reactivity, in response to GR depletion. Several proteins involved in redox regulation, calcium signaling, cytoskeleton regulation, and protein folding showed significant changes in expression, whereas proteins involved in redox regulation, protein folding, and glycolysis displayed changes in thiol reactivity. Interestingly, GR knockdown induces peroxiredoxin‐1 overexpression in the air‐exposed tissue and high oxygen consuming tissue such as cornea and liver, but not in the low oxygen consuming tissues such as breast and uterine. In summary, we used a comprehensive lung adenocarcinoma based proteomic approach for identifying GR‐modulated protein expression alteration and redox modification. Based on our research, this is the first comprehensive proteomic and redox‐proteomic analysis used to investigate the role of GR in a mammalian cell model.     

FG020326 sensitized multidrug resistant cancer cells to docetaxel-mediated apoptosis via enhancement of caspases activation

Apoptotic resistance is the main obstacle for treating cancer patients with chemotherapeutic drugs. Multidrug resistance (MDR) is often characterized by the expression of P-glycoprotein (P-gp), a 170-KD ATP-dependent drug efflux protein. Functional P-gp can confer resistance to activate caspase-8 and -3 dependent apoptosis induced by a range of different stimuli, including tumor necrosis and chemotherapeutic drugs such as docetaxel and vincristine. We demonstrated here that comparison of sensitive KB cells, P-gp positive (P-gp(+ve)) KBv200 cells were extremely resistant to apoptosis induced by docetaxel. FG020326, a pharmacological inhibitor of P-gp function, could enhance concentration-dependently the effect of docetaxel on cell apoptosis and sensitize caspase-8, -9 and -3 activation in P-gp overexpressing KBv200 cells, but not in KB cells. Therefore, the enhancement of caspase-8, -9 and -3 activation induced by docetaxel may be one of the key mechanisms of the reversal of P-gp mediated docetaxel resistance by FG020326.     

Modelling the role of dual specificity phosphatases in herceptin resistant breast cancer cell lines

BackgroundBreast cancer remains the most lethal type of cancer for women. A significant proportion of breast cancer cases are characterised by overexpression of the human epidermal growth factor receptor 2 protein (HER2). These cancers are commonly treated by Herceptin (Trastuzumab), but resistance to drug treatment frequently develops in tumour cells. Dual-specificity phosphatases (DUSPs) are thought to play a role in the mechanism of resistance, since some of them were reported to be overexpressed in tumours resistant to Herceptin.ResultsWe used a systems biology approach to investigate how DUSP overexpression could favour cell proliferation and to predict how this mechanism could be reversed by targeted inhibition of selected DUSPs. We measured the expression of 20 DUSP genes in two breast cancer cell lines following long-term (6 months) exposure to Herceptin, after confirming that these cells had become resistant to the drug. We constructed several Boolean models including specific substrates of each DUSP, and showed that our models correctly account for resistance when overexpressed DUSPs were kept activated. We then simulated inhibition of both individual and combinations of DUSPs, and determined conditions under which the resistance could be reversed.ConclusionsThese results show how a combination of experimental analysis and modelling help to understand cell survival mechanisms in breast cancer tumours, and crucially enable us to generate testable predictions potentially leading to new treatments of resistant tumours.     

Up-regulation of BLT2 is critical for the survival of bladder cancer cells

The incidence rates of urinary bladder cancer continue to rise yearly, and thus new therapeutic approaches and early diagnostic markers for bladder cancer are urgently needed. Thus, identifying the key mediators and molecular mechanisms responsible for the survival of bladder cancer has valuable implications for the development of therapy. In this study, the role of BLT2, a receptor for leukotriene B((4)) (LTB((4))) and 12(S)-hydroxyeicosatetraenoic acid (HETE), in the survival of bladder cancer 253J-BV cells was investigated. We found that the expression of BLT2 is highly elevated in bladder cancer cells. Also, we observed that blockade of BLT2 with an antagonist or BLT2 siRNA resulted in cell cycle arrest and apoptotic cell death, suggesting a role of BLT2 in the survival of human bladder cancer 253J-BV cells. Further experiments aimed at elucidating the mechanism by which BLT2 mediates survival revealed that enhanced level of reactive oxygen species (ROS) are generated via a BLT2-dependent up-regulation of NADPH oxidase members NOX1 and NOX4. Additionally, we observed that inhibition of ROS generation by either NOX1/4 siRNAs or treatment with an ROS-scavenging agent results in apoptotic cell death in 253J-BV bladder cancer cells. These results demonstrated that a 'BLT2-NOX1/4-ROS' cascade plays a role in the survival of this aggressive bladder cancer cells, thus pointing to BLT2 as a potential target for anti-bladder cancer therapy.     

In vitro survival of nonsmall cell lung cancer cells following combined treatment with ionizing radiation and photofrin-mediated photodynamic therapy

It has been suggested that combination high dose rate (HDR) intraluminal brachytherapy and photodynamic therapy (PDT) in nonsmall cell lung cancer (NSCLC) may improve efficacy of treatment, reduce toxicity and enhance quality of life for patients. To provide a cellular basis for this we examined the in vitro sensitivity of MRC5 normal lung fibroblasts and four NSCLC cell lines following HDR radiation, PDT and combined HDR radiation and PDT. HDR radiation was cobalt-60 gamma rays (1.5–1.9 Gy min⁻¹). For PDT treatment, cells were exposed to 2.5 μg mL⁻¹ Photofrin for 18–24 h followed by light exposure (20 mW cm⁻²). For combined treatment cells were exposed to Photofrin and then either exposed to light and 15–30 min later exposed to HDR radiation or exposed to HDR radiation and 15–30 min later exposed to light. D₃₇ values calculated from clonogenic survival curves indicated a six-fold difference in HDR radiation sensitivity and an eight-fold difference in PDT sensitivity. The effect of combined treatment was not significantly different from an additive effect of the individual treatment modalities for the NSCLC cells, but was significantly less than additive for the MRC5 cells. These results suggest an equivalent tumor cell kill may be possible at reduced systemic effects to patients.     

An important role of intramolecular free radical reactions in antimalarial activity of artemisinin and its analogs

The kinetic schemes of intramolecular reactions of five analogs of artemisinin were built. The method of intersecting parabolas was used for the calculation of activation energies and rate constants of each elementary step of these schemes. The competition between monomolecular and bimolecular free radicals was taken into account. It was evidenced that the intramolecular oxidation of these compounds proceeds as a cascade of consecutive free radical reactions with the formation of hydroperoxide groups. The latter decompose via reactions with the Fe(II) complexes generating free radicals. Among the radicals formed, the hydroxyl radical was proved to play the key role. A correlation between the yield of hydroxyl radicals n(OH) and antimalarial activity of compounds (IC(50)) was observed. The dependence of index IC(50) on n(OH) is linear in the logarithmic coordinates: ln[IC(50)(Artemisinin)/IC(50)(Compound)] = -14.10 + 3.85 ×n(OH). The proposed scheme explains and demonstrates a strong dependence of the antimalarial effectiveness of a drug on the chemical structure.     

Lysosomal signaling enhances mitochondria-mediated photodynamic therapy in A431 cancer cells: role of iron

In photodynamic therapy (PDT), light activates a photosensitizer added to a tissue, resulting in singlet oxygen formation and cell death. The photosensitizer phthalocyanine 4 (Pc 4) localizes primarily to mitochondrial membranes in cancer cells, resulting in mitochondria-mediated cell death. The aim of this study was to determine how lysosomes contribute to PDT-induced cell killing by mitochondria-targeted photosensitizers such as Pc 4. We monitored cell killing of A431 cells after Pc 4-PDT in the presence and absence of bafilomycin, an inhibitor of the vacuolar proton pump of lysosomes and endosomes. Bafilomycin was not toxic by itself, but greatly enhanced Pc 4-PDT-induced cell killing. To investigate whether iron loading of lysosomes affects bafilomycin-induced killing, cells were incubated with ammonium ferric citrate (30 μM) for 30 h prior to PDT. Ammonium ferric citrate enhanced Pc 4 plus bafilomycin-induced cell killing without having toxicity by itself. Iron chelators (desferrioxamine and starch-desferrioxamine) and the inhibitor of the mitochondrial calcium (and ferrous iron) uniporter, Ru360, protected against Pc 4 plus bafilomycin toxicity. These results support the conclusion that chelatable iron stored in the lysosomes enhances the efficacy of bafilomycin-mediated PDT and that lysosomal disruption augments PDT with Pc 4.