Curcumin enhances the mitomycin C-induced cytotoxicity via downregulation of MKK1/2-ERK1/2-mediated Rad51 expression in non-small cell lung cancer cells

Curcumin (diferuloylmethane), a major active component of turmeric (Curcuma longa), has been reported to suppress the proliferation of a wide variety of tumor cells. Rad51 is a key protein in the homologous recombination (HR) pathway of DNA double-strand break repair, and HR represents a novel target for cancer therapy. A high expression of Rad51 has been reported in chemo- or radio-resistant carcinomas. Therefore, in the current study, we will examine whether curcumin could enhance the effects of mitomycin C (MMC), a DNA interstrand cross-linking agent, to induce cytotoxicity by decreasing Rad51 expression. Exposure of two human non-small lung cancer (NSCLC) cell lines (A549 and H1975) to curcumin could suppress MMC-induced MKK1/2-ERK1/2 signal activation and Rad51 protein expression. Enhancement of ERK1/2 activation by constitutively active MKK1/2 (MKK1/2-CA) increased Rad51 protein levels in curcumin and MMC co-treated human lung cells. Moreover, the synergistic cytotoxic effect induced by curcumin combined with MMC was decreased by MKK1-CA-mediated enhancement of ERK1/2 activation by a significant degree. In contrast, MKK1/2 inhibitor, U0126 was shown to augment the cytotoxicity of curcumin and MMC through downregulation of ERK1/2 activation and Rad51 expression. Depletion of endogenous Rad51 expression by siRad51 RNA transfection significantly enhanced MMC and/or curcumin induced cell death and cell growth inhibition. In contrast, an overexpression of Rad51 protected lung cancer cells from synergistic cytotoxic effects induced by curcumin and MMC. We concluded that Rad51 inhibition may be an additional action mechanism for enhancing the chemosensitization of MMC by curcumin in NSCLC.     

HSP90 inhibition induces cytotoxicity via down-regulation of Rad51 expression and DNA repair capacity in non-small cell lung cancer cells

Heat shock protein 90 (HSP90) is an exciting new target in cancer therapy. Repair protein Rad51 is involved in protecting non-small cell lung cancer (NSCLC) cell lines against chemotherapeutic agent-induced cytotoxicity. This study investigated the role of Rad51 expression in HSP90 inhibitor 17-allylamino-17-demethoxygeldanamycin (17-AAG)-induced cytotoxicity in two NSCLC cell lines, A549 and H1975. The 17-AAG treatment decreased cellular Rad51 protein and mRNA levels and phosphorylated MKK1/2-ERK1/2 protein levels, and disrupted the HSP90 and Rad51 interaction. This triggered Rad51 protein degradation through the 26S proteasome pathway. The 17-AAG treatment also decreased the NSCLC cells’ DNA repair capacity, which was restored by the forced expression of the Flag-Rad51 vector. Specific inhibition of Rad51 expression by siRNA further enhanced 17-AAG-induced cytotoxicity. In contrast, enhanced ERK1/2 activation by the constitutively active MKK1/2 (MKK1/2-CA) vector significantly restored the 17-AAG-reduced Rad51 protein levels and cell viability. Arachidin-1, an antioxidant stilbenoid, further decreased Rad51 expression and augmented the cytotoxic effect and growth inhibition of 17-AAG. The 17-AAG and arachidin-1-induced synergistic cytotoxic effects and decreased DNA repair capacity were abrogated in lung cancer cells with MKK1/2-CA or Flag-Rad51 expression vector transfection. In conclusion, HSP90 inhibition induces cytotoxicity by down-regulating Rad51 expression and DNA repair capacity in NSCLC cells.     

Downregulation of Rad51 participates in OTA-induced DNA double-strand breaks in GES-1 cells in vitro

Ochratoxin A (OTA), a mycotoxin produced by ubiquitous Aspergilli, is carcinogenic, teratogenic, and nephrotoxic in both humans and animals. Our previous study found that OTA induced DNA double-strand breaks (DSBs) and resulted in G2 phase arrest in human gastric epithelium immortalized (GES-1) cells. DSBs can cause genomic instability, mutations, and neoplastic transformations, and improper repair of DSBs may lead to the development of cancer. Rad51 is a key protein in the homologous recombination (HR) pathway of DSBs repair. The roles of Rad51 in the repair of DNA damage vary in response to different types of cytotoxic agents. The effect of OTA on Rad51 expression and its putative role in the OTA-induced DSBs in GES-1 cells are still not clear enough. The aim of the current study is to elucidate the role of Rad51 in OTA-induced DSBs in GES-1 cells. The results showed that OTA treatment decreased Rad51 expression in a dose- and time-dependent manner. Specific downregulation of Rad51 by siRNA induced DSBs and G2 phase arrest. Rad51 overexpression by transfection with a Rad51-expressing plasmid partly rescued the DSBs and G2 phase arrest in OTA-treated cells. The findings indicate that downregulation of Rad51 contributes to OTA-induced DNA damage in GES-1 cells. Knockdown of p53 with siRNA for 48 h effectively reversed the downregulation of Rad51, and decreased the OTA-induced DSBs in GES-1 cells. In addition, the downregulation of Rad51 induced by OTA could be significantly attenuated with specific ERK inhibitor PD98059 or specific p38 MAPK inhibitor SB203580 pre-treatment in GES-1 cells. Thus, the results suggest that downregulation of Rad51 participates in OTA-induced DNA double-strand breaks in GES-1 cells in vitro. And p53, ERK and p38 signaling pathways are all involved in the process.     

DNA damage and homologous recombination signaling induced by thymidylate deprivation

DNA damage is accepted as a consequence of thymidylate deprivation induced by chemotherapeutic inhibitors of thymidylate synthase (TS), but the types of damage and signaling responses remain incompletely understood. Thymidylate deprivation increases dUTP and uracil in DNA, which is removed by base excision repair (BER). Because BER requires a synthesis step, strand break intermediates presumably accumulate. Thymidylate deprivation also induces cell cycle arrest during replication. Homologous recombination (HR) is a means of repairing persistent BER intermediates and collapsed replication forks. There are also intimate links between HR and S-phase checkpoint pathways. In this study, the goals were to determine the involvement of HR-associated proteins and DNA damage signaling responses to thymidylate deprivation. When RAD51, which is a central component of HR, was depleted by siRNA cells were sensitized to raltitrexed (RTX), which specifically inhibits TS. To our knowledge, this is the first demonstration in mammalian cells that depletion of RAD51 causes sensitivity to thymidylate deprivation. Activation of DNA damage signaling responses was examined following treatment with RTX. Phosphorylation of replication protein A (RPA2 subunit) and formation of damage-induced foci were strikingly evident following IC₅₀ doses of RTX. Induction was much more striking following RTX treatment than with hydroxyurea, which is commonly used to inhibit replication. RTX treatment also induced foci of RAD51, γ-H2AX, phospho-Chk1, and phospho-NBS1, although the extent of co-localization with RPA2 foci varied. Collectively, the results suggest that HR and S-phase checkpoint signaling processes are invoked by thymidylate deprivation and influence cellular resistance to thymidylate deprivation.     

HSP90 inhibition induces cytotoxicity via down-regulation of Rad51 expression and DNA repair capacity in non-small cell lung cancer cells

Heat shock protein 90 (HSP90) is an exciting new target in cancer therapy. Repair protein Rad51 is involved in protecting non-small cell lung cancer (NSCLC) cell lines against chemotherapeutic agent-induced cytotoxicity. This study investigated the role of Rad51 expression in HSP90 inhibitor 17-allylamino-17-demethoxygeldanamycin (17-AAG)-induced cytotoxicity in two NSCLC cell lines, A549 and H1975. The 17-AAG treatment decreased cellular Rad51 protein and mRNA levels and phosphorylated MKK1/2-ERK1/2 protein levels, and disrupted the HSP90 and Rad51 interaction. This triggered Rad51 protein degradation through the 26S proteasome pathway. The 17-AAG treatment also decreased the NSCLC cells’ DNA repair capacity, which was restored by the forced expression of the Flag-Rad51 vector. Specific inhibition of Rad51 expression by siRNA further enhanced 17-AAG-induced cytotoxicity. In contrast, enhanced ERK1/2 activation by the constitutively active MKK1/2 (MKK1/2-CA) vector significantly restored the 17-AAG-reduced Rad51 protein levels and cell viability. Arachidin-1, an antioxidant stilbenoid, further decreased Rad51 expression and augmented the cytotoxic effect and growth inhibition of 17-AAG. The 17-AAG and arachidin-1-induced synergistic cytotoxic effects and decreased DNA repair capacity were abrogated in lung cancer cells with MKK1/2-CA or Flag-Rad51 expression vector transfection. In conclusion, HSP90 inhibition induces cytotoxicity by down-regulating Rad51 expression and DNA repair capacity in NSCLC cells.     

Guanidine-reactive agent phenylglyoxal induces DNA damage and cancer cell death

BackgroundDNA-damaging compounds (e.g., alkylating agents, cytotoxic antibiotics and DNA topoisomerase poisons) are the most widely used anticancer drugs. The inability of tumor cells to properly repair some types of DNA damage may explain why specific DNA-damaging drugs can selectively kill tumor cells. Phenylglyoxal is a dicarbonyl compound known to react with guanidine groups such as that of the DNA base guanine, therefore suggesting that phenylglyoxal could induce DNA damage and have anticancer activity.MethodsCellular DNA damage was measured by the alkaline comet assay and the γH2AX focus assay. Formation of topoisomerase I- and topoisomerase II-DNA complexes was assessed by the TARDIS assay, an immunofluorescence technique that employs specific antibodies to DNA topo I or topo II to detect the protein covalently bound to the DNA in individual cells. Cell growth inhibition and cytotoxicity were determined by XTT, MTT and clonogenic assays. Apoptosis was assessed by the Annexin V flow cytometry assay.ResultsPhenylglyoxal induced cellular DNA damage and formation of high levels of topoisomerase I- and topoisomerase II-DNA complexes in cells. These topoisomerase-DNA complexes were abolished by catalase pretreatment and correlated well with the induction of apoptosis. Phenylglyoxal-induced cell death was partially prevented by catalase pretreatment and was higher in lung cancer cells (A549) than in normal lung fibroblasts (MRC5). Mammalian cell lines defective in nucleotide excision repair (NER), homologous recombination (HR) and non-homologous end joining (NHEJ) were more sensitive to phenylglyoxal than parental cells; this suggests that phenylglyoxal may induce bulky distortions in the shape of the DNA double helix (which are repaired by the NER pathway) and DNA double-strand breaks (which are repaired by HR and NHEJ).ConclusionsThis report shows that phenylglyoxal is a new DNA-damaging agent with anticancer activity, and suggests that tumor cells with defects in NER, HR and NHEJ may be hypersensitive to the cytotoxic activity of phenylglyoxal.     

Novel tetra-acridine derivatives as dual inhibitors of topoisomerase II and the human proteasome

Acridine derivatives, such as amsacrine, represent a well known class of multi-targeted anti-cancer agents that generally interfere with DNA synthesis and inhibit topoisomerase II. But in addition, these tricyclic molecules often display secondary effects on other biochemical pathways including protein metabolism. In order to identify novel anti-cancer drugs, we evaluated the mechanism of action of a novel series of bis- and tetra-acridines. As expected, these molecules were found to interact with DNA and inhibit the topoisomerase II-mediated DNA decatenation. Interestingly when tested on human tumour cells either sensitive (HL-60) or resistant (HL-60/MX2) to topoisomerase II inhibitors, these molecules proved equicytotoxic against the two cell lines, suggesting that they do not only rely on topoisomerase II inhibition to exert their cytotoxic effects. In order to identify alternative targets, we tested the capacity of acridines 1-9 to inhibit the proteasome machinery. Four tetra-acridines inhibited the proteasome in vitro, with IC(50) values up to 40 times lower than that of the reference proteasome inhibitor lactacystin. Moreover, unlike peptide aldehydes used as reference inhibitors for the proteasome, these new acridine compounds demonstrated a good selectivity towards the proteasome, when tested against four unrelated proteases. A cellular assay based on the degradation of a proteasome protein substrate indicated that at least two of the tetra-acridines maintained this proteasome inhibition activity in a cellular context. This is the first report of tetra-acridines that demonstrate dual topoisomerase II and proteasome inhibition properties. This new dual activity could represent a novel anti-cancer approach to circumvent certain forms of tumour resistance.     

Structure and activity relationship of several novel CC-1065 analogs

Summary CC-1065 was found to cause delayed toxicity at therapeutic doses, therefore, a large number of analogs have since been synthesized. A series of analogs with simplified but closely related structures were chosen for this investigation because some were found to be superior to CC-1065 in the treatment of several experimental tumors. The inhibition of L1210 cell growth by U-68,415 was comparable to that by CC-1065. A similar situation was true in terms of their in vivo potency; however, U-68,415 was superior to CC-1065 in terms of anti-P 388 leukemia activity. At the optimal dosage, U-68,415 produced 4 out of 6 long-term (> 30 day) survivors; whereas CC-1065 produced a mere 62% increase of life span (ILS) and no long-term survivors. The order of antitumor potency and effectiveness of the CC-1065 analogs was U-68,415 > U-66,694 > U-68,819 > U-66,664, which was parallel to the inhibition of L1210 cell growth. CC-1065 and all the analogs tested here inhibited DNA synthesis approximately 10 times more than RNA synthesis. Protein synthesis was the least inhibited. On a molar basis, U-68,415 was about 6–9 times more inhibitory toward cellular DNA synthesis than CC-1065, yet the interaction and/or binding of CC-1065 to DNA determined by circular dichroism, DNA melting or differential cytotoxicity assay was much stronger than that of U-68,415. The order of binding of these analogs to calf thymus DNA was U-68,415 > U-66,694 > U-68,819 > U-66,664, and was parallel to that of DNA synthesis inhibition which was in turn parallel to cell growth inhibition and antitumor potential. These results collectively suggest that the cellular DNA is a major site of the action of CC-1065 analogs; however, time course studies reveal that the inhibition of cellular DNA synthesis could not wholly account for their cytotoxicity. Hence, the precise mechanism of action of these agents is not yet fully understood. U-68,415, which exhibited superior activity against a number of tumors and did not cause delayed death in mice, warrants further investigation. U-68,415 is a racemate and two chiral isomers were recently isolated. Therefore, further investigation of both U-68,415 and its chiral isomers is necessary.     

Synergistic effect of radon and sodium arsenite on DNA damage in HBE cells

Human epidemiological studies showed that radon and arsenic exposures are major risk factors for lung cancer in Yunnan tin miners. However, biological evidence for this phenomenon is absent. In this study, HBE cells were exposed to different concentrations of sodium arsenite, different radon exposure times, or a combination of these two factors. The results showed a synergistic effect of radon and sodium arsenite in cell cytotoxicity as determined by cell viability. Elevated intracellular ROS levels and increased DNA damage indexed by comet assay and γ-H2AX were detected. Moreover, DNA HR repair in terms of Rad51 declined when the cells were exposed to both radon and sodium arsenite. The synergistic effect of radon and sodium arsenite in HBE cells may be attributed to the enhanced DSBs and inhibited HR pathway upon co-exposure.     

Inhibition of homologous recombination in human cells by targeting RAD51 recombinase

The homologous recombination (HR) pathway plays a crucial role in the repair of DNA double-strand breaks (DSBs) and interstrand cross-links (ICLs). RAD51, a key protein of HR, possesses a unique activity: DNA strand exchange between homologous DNA sequences. Recently, using a high-throughput screening (HTS), we identified compound 1 (B02), which specifically inhibits the DNA strand exchange activity of human RAD51. Here, we analyzed the mechanism of inhibition and found that 1 disrupts RAD51 binding to DNA. We then examined the effect of 1 on HR and DNA repair in the cell. The results show that 1 inhibits HR and increases cell sensitivity to DNA damage. We propose to use 1 for analysis of cellular functions of RAD51. Because DSB- and ICL-inducing agents are commonly used in anticancer therapy, specific inhibitors of RAD51 may also help to increase killing of cancer cells.     

Neocarzinostatin-induced Rad51 nuclear focus formation is cell cycle regulated and aberrant in AT cells

DNA double-stranded breaks are the most detrimental form of DNA damage and, if not repaired properly, may lead to an accumulation of chromosomal aberrations and eventually tumorigenesis. Proteins of the Rad51/Rad52 epitasis group are crucial for the recombinational repair of DNA double-stranded breaks, whereas the Rad50/NBS1/Mre11 nuclease complex is involved in both the recombinational and the end-joining repair of DNA double-stranded breaks. Herein, we demonstrate that the chemotherapeutic enediyne antibiotic neocarzinostatin induced Rad51, but not NBS1, nuclear focus formation in a cell- cycle-dependent manner. Furthermore, neocarzinostatin-induced Rad51 foci formation revealed a slower kinetic change in AT cells, but not in wild-type or NBS cells. In summary, our results suggest that neocarzinostatin induces Rad51 focus formation through an ATM- and cell-cycle-dependent, but NBS1-independent, pathway.     

Activity of acyclic halogenated tubercidin analogs against human cytomegalovirus and in uninfected cells.

Novel acyclic halogenated tubercidins (4-amino-5-halo-7-[(2-hydroxyethoxy)-methyl]pyrrolo[2,3-d]pyrimidines) were examined for their ability to inhibit human cytomegalovirus (HCMV) in yield reduction assays. 5-Bromo acyclic tubercidin (compound 102) was a more potent inhibitor of virus replication than the chloro- and iodo-substituted analogs (compounds 100 and 104). At a 100 microM concentration, the bromo and chloro compounds were more potent than acyclovir but not ganciclovir. Virus titers were reduced more than 99% by compounds 102 and 104 whereas compound 100 and the equally potent acyclovir reduced titers by only 90%. Quantitation of viral DNA by DNA hybridization demonstrated strong inhibition of HCMV DNA synthesis by these compounds. The most potent inhibitor, compound 102, had a 50% inhibitory (I50) concentration (1.6 microM) comparable to that of ganciclovir (1.8 microM). Cytotoxicity in uninfected human cells was evaluated and revealed the following: cell growth rates slowed markedly in the presence of 10 microM compound 102 whereas the same concentration of compounds 100 and 104 led to only a slight prolongation of population doubling time; these compounds inhibited cellular DNA synthesis but not RNA or protein synthesis, as measured by incorporation of radiolabeled precursors into acid-precipitable macromolecules; flow cytometry indicated that compound 102 was a mid-S phase blocker, and adenosine antagonized the inhibition of [3H]dThd incorporation by compound 102. Together, these results demonstrate that compound 102 is a potent and selective inhibitor of viral and cellular DNA synthesis and that acyclic halogenated pyrrolo-pyrimidine nucleosides may have therapeutic potential.     

Expression and regulation of RAD51 mediate cellular responses to chemotherapeutics

There is evidence that RAD51 expression associates with resistance to commonly used chemotherapeutics. Our previous work demonstrated that inhibitors of thymidylate synthase (TS) induced RAD51-dependent homologous recombination (HR), and depleting the RAD51 recombinase sensitized cells to TS inhibitors. In this study, the consequences of RAD51 over-expression were studied. Over-expression of wild-type RAD51 (～6-fold above endogenous RAD51) conferred resistance to TS inhibitors. In contrast, over-expression of a mutant RAD51 (T309A) that is incapable of being phosphorylated rendered cells more chemosensitive. Moreover, over-expression of the T309A mutant acted in a dominant negative manner over endogenous RAD51 by causing the reduced localization of RAD51 foci following treatment with TS inhibitors. To measure the effect of mutant RAD51 on the cellular response to other DNA damaging chemotherapeutics, the topoisomerase poison etoposide was utilized. Cells over-expressing wild-type RAD51 showed reduced DNA strand breaks, while cells over-expressing the mutant RAD51 showed more than twice as many strand breaks, suggesting that the mutant RAD51 was actively inhibiting strand break resolution. To directly demonstrate an effect on HR, wild-type RAD51 and T309A mutant RAD51 were transiently expressed in HeLa cells that contained an HR reporter construct. HR events provoked by DNA breaks induced by the I-SceI endonuclease increased in cells expressing wild-type RAD51 and decreased in cells expressing the T309A mutant. Collectively, the data suggest that interference with the activation of RAD51-mediated HR represents a potentially useful anticancer target for combination therapies.     

Induction of apoptosis by plumbagin through reactive oxygen species-mediated inhibition of topoisomerase II

Reactive oxygen species (ROS) have been recognized as key molecules, which can selectively modify proteins and therefore regulate cellular signalling including apoptosis. Plumbagin, a naphthoquinone exhibiting antitumor activity, is known to generate ROS and has been found to inhibit the activity of topoisomerase II (Topo II) through the stabilization of the Topo II-DNA cleavable complex. The objective of this research was to clarify the role of ROS and Topo II inhibition in the induction of apoptosis mediated by plumbagin. As determined by the comet assay, plumbagin induced DNA cleavage in HL-60 cells, whereas in a cell line with reduced Topo II activity—HL-60/MX2, the level of DNA damage was significantly decreased. The onset of DNA strand break formation in HL-60 cells was delayed in comparison with the generation of intracellular ROS. In HL-60/MX2 cells, ROS were generated at a similar rate, whereas a significant reduction in the level of DNA damage was detected. The pretreatment of cells with N-acetylcysteine (NAC) attenuated plumbagin-induced DNA damage, pointing out to the involvement of ROS generation in cleavable complex formation. These results suggest that plumbagin-induced ROS does not directly damage DNA but requires the involvement of Topo II. Furthermore, experiments carried out using light spectroscopy indicated no direct interactions between plumbagin and DNA. The induction of apoptosis was significantly delayed in HL-60/MX2 cells indicating the involvement of Topo II inhibition in plumbagin-mediated apoptosis. Thus, these findings strongly suggest ROS-mediated inhibition of Topo II as an important mechanism contributing to the apoptosis-inducing properties of plumbagin.     

Synthesis and biological evaluation of new cyclic amidine analogs of chlorambucil

A number of novel cyclic amidine analogs of chlorambucil were synthesized and examined for cytotoxicity in breast cancer cell cultures and for inhibition of topoisomerases I and II. Evaluation of the cytotoxicity of these compounds employing a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and inhibition of [(3)H]-thymidine incorporation into DNA in both MDA-MB-231 and MCF-7 breast cancer cells demonstrated that these compounds were more active than chlorambucil. The degree to which these compounds inhibited cell growth breast cancer cells was directly correlated to DNA-binding affinity. These studies indicate that cyclic amidine analogs of chlorambucil are a potent catalytic inhibitor of topoisomerase II but not topoisomerase I. The highest degree of DNA binding and cytotoxicity in both MDA-MB-231 and MCF-7 breast cancer cells was observed for the compound, which possess a 4,5-dihydro-1H-imidazol moiety.     

Nucleoside analog inhibitors of hepatitis C virus replication

Of the 30 compounds currently marketed in the United States for treatment of viral infections, 15 are nucleoside analogs, demonstrating the utility of this class of compound as a source of antiviral drugs. The success of nucleoside analogs in treating other viral infections provides a compelling rationale for the significant effort that is currently being devoted to the discovery and development of nucleoside analogs to treat infection by hepatitis C virus (HCV) that may lead to improvements in response rates compared to currently available therapies. Several different approaches have been adopted to identify promising analogs, including the use of surrogate viruses in cell culture assays, screening in the cell-based bicistronic HCV replicon assay, and screening nucleoside triphosphates for the ability to inhibit the activity of the HCV RNA-dependent RNA polymerase in vitro. Several classes of ribonucleoside analogs with modifications of the ribose inhibit HCV replication. Nucleoside analogs incorporating a 2'-C-methyl modification are potent inhibitors in the replicon assay in the absence of cytotoxicity, and appear to exert their inhibition by acting as functional chain terminators of RNA synthesis. NM283, a prodrug of 2'-C-methylcytidine, has entered clinical trials and demonstrated viral load reductions in subjects infected with genotype 1 HCV, a genotype known to be difficult to treat effectively with currently approved therapies. Overall, results to date offer encouragement that improved therapies to treat HCV infection including newly developed nucleoside analogs may become available within the next few years.     

Substrate-Selective Inhibition of Cyclooxygenase-2: Development and Evaluation of Achiral Profen Probes

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Modulation of Rad51, ERCC1, and thymidine phosphorylase by emodin result in synergistic cytotoxic effect in combination with capecitabine

Thymidine phosphorylase (TP) is the rate-limiting enzyme for the activation of capecitabine (pro-drug of fluorouracil), and as a useful predictor of tumor response to capecitabine-based chemotherapy. Overexpression of Rad51 and ERCC1 induce resistance to chemotherapeutic agents. Emodin, one of the main bioactive anthraquinone derivatives in the roots and rhizomes of numerous plants, possesses potent antitumor effects. Accordingly, we aimed to explore the molecular mechanism of emodin enhances the capecitabine-induced cytotoxicity through controlling Rad51, ERCC1, and TP expression in human non-small cell lung cancer (NSCLC). The results show that capecitabine increases the phosphorylation of MKK1/2–ERK1/2 and protein levels of Rad51 and ERCC1 through enhancing the protein stability. Depletion of endogenous Rad51 or ERCC1 expression by specific small interfering RNA transfection significantly increases capecitabine-induced cell death and growth inhibition. Emodin enhances the capecitabine-induced cytotoxic effects through ERK1/2 inactivation and decreasing the Rad51 and ERCC1 protein levels induced by capecitabine. Enhancement of ERK1/2 signaling by constitutively active MKK1/2 (MKK1/2-CA) results in increasing Rad51 and ERCC1 protein levels and cell viability in NSCLC cell lines treated with emodin and capecitabine. Interestingly, emodin enhances TP mRNA and protein expression in capecitabine treated NSCLC cell lines, and depletion of the TP expression decreases the cytotoxic effects induced by capecitabine and emodin. We conclude that enhancing the cytotoxicity to capecitabine by emodin is mediated by down-regulation the expression of Rad51 and ERCC1 and up-regulation TP expression.     

Synthesis of microtubule-interfering halogenated noscapine analogs that perturb mitosis in cancer cells followed by cell death

We have previously identified the naturally occurring non-toxic antitussive phthalideisoquinoline alkaloid, noscapine as a tubulin-binding agent that arrests mitosis and induces apoptosis. Here we present high-yield efficient synthetic methods and an evaluation of anticancer activity of halogenated noscapine analogs. Our results show that all analogs display higher tubulin-binding activity than noscapine and inhibit proliferation of human cancer cells (MCF-7, MDA-MB-231 and CEM). Surprisingly, the bromo-analog is ∼40-fold more potent than noscapine in inhibiting cellular proliferation of MCF-7 cells. The ability of these analogs to inhibit cellular proliferation is mediated by cell cycle arrest at the G₂/M phase, in that all analogs except 9-iodonoscapine, caused selective mitotic arrest with a higher efficiency than noscapine followed by apoptotic cell death as shown by immunofluorescence and quantitative FACS analyses. Furthermore, our results reveal the appearance of numerous fragmented nuclei as evidenced by DAPI staining. Thus, our data indicate a great potential of these compounds for studying microtubule-mediated processes and as chemotherapeutic agents for the management of human cancers.