Substrate Phosphorylation Rates as an In Vivo Measurement of Kinase Activity.

Measuring kinase activity in different in vivo contexts is crucial for understanding the mechanism and functions of protein kinases, such as the cyclin-dependent kinases (Cdks) and other cell cycle kinases. Here, I present the rationale and the experimental framework for an alternative approach to measure kinase activity that is based on estimating substrate phosphorylation rates in vivo. The approach presented was first developed for experiments performed to measure Cdk1 activity at different stages of the fission yeast S. pombe's cell cycle [Swaffer et al., Cell 167:1750-1761, 2016]. However, it also affords a more generalizable framework that can be adaptable to other systems and kinases, as long as specific, rapid, and reversible kinase inhibition is possible. Briefly this involves transient and reversible kinase inhibition to dephosphorylate kinase substrates in vivo, followed by quantitative measurements of phosphorylation after inhibition is removed.

Assessment of cytotoxic and genotoxic potentials of a mononuclear Fe(II) Schiff base complex with photocatalytic activity in Trigonella.

BACKGROUND: In recent times, coordination complexes of iron in various oxidation states along with variety of ligand systems have been designed and developed for effective treatment of cancer cells without adversely affecting the normal cell and tissues of various organs. METHODS: In this study, we have evaluated the mechanism of action of a Fe(II) Schiff base complex in the crop plant Trigonella foenum-graecum L. (Fenugreek) as the screening system by using morphological, cytological, biochemical and molecular approaches. Further functional characterization was performed using MCF-7 cell line and solid tumour model for the assessment of anti-tumour activity of the complex. RESULTS: Our results indicate efficiency of the Fe(II) Schiff base complex in the induction of double strand breaks in DNA. Complex treatment clearly induced cytotoxic and genotoxic damage in Trigonella seedlings. The Fe-complex treatment caused cell cycle arrest via the activation of ATM-ATR kinase mediated DNA damage response pathway with the compromised expression of CDK1, CDK2 and CyclinB1 protein in Trigonella seedlings. In cultured MCF-7 cells, the complex induces cytotoxicity and DNA fragmentation through intracellular ROS generation. Fe-complex treatment inhibited tumour growth in solid tumour model with no additional side effects. CONCLUSION: The growth inhibitory and cytotoxic effects of the complex result from activation of DNA damage response along with oxidative stress and cell cycle arrest. GENERAL SIGNIFICANCE: Overall, our results have provided comprehensive information on the mechanism of action and efficacy of a Fe(II) Schiff base complex in higher eukaryotic genomes and indicated its future implications as potential therapeutic agent.

Fipronil induces apoptosis and cell cycle arrest in porcine oocytes during in vitro maturation.

Fipronil (FPN) is a widely used phenylpyrazole pesticide that can kill pests by blocking γ-aminobutyric acid (GABA)-gated chloride channels. In addition, there are lack of studies on the effects of FPN on the female mammalian gametes. In this study, porcine oocytes were used to investigate the effects of FPN on the oocyte maturation process. The results showed that the first polar body extrusion rate significantly decreased (100 µM FPN vs. control, 18.64 ± 2.95% vs. 74.90 ± 1.50%, respectively), and oocytes were arrested at the germinal vesicle stage in 100 µM FPN group. Meanwhile, the FPN caused a significant increase in reactive oxygen species (ROS) levels and severe DNA damage inside the oocytes. Furthermore, apoptosis was enhanced along with decreases in mitochondrial membrane potential, BCL-xL, and the release of cytochrome C in FPN-treated group. Additionally, low CDK1 activity and delayed cyclin B1 degradation during germinal vesicle breakdown were found in the FPN-treated group, which resulted from the activation of ATM-P53-P21 pathway. In conclusion, FPN induces apoptosis and cell cycle arrest in porcine oocyte maturation because of increased ROS levels and DNA damage. This suggests that the FPN in the environment may have potential detrimental effects on the female mammalian reproductive system.

Long noncoding RNA OR3A4 promotes cisplatin resistance of non-small cell lung cancer by upregulating CDK1.

OBJECTIVE: Numerous studies have proved that long non-coding RNAs (lncRNAs) have an important role in malignant tumors, including non-small cell lung cancer (NSCLC). LncRNA olfactory receptor family 3 subfamily A member 4 (OR3A4) was explored to identify how it functions in resistance of NSCLC patients to cisplatin. MATERIALS AND METHODS: Real Time-quantitative Polymerase Chain Reaction (RT-qPCR) was utilized to detect OR3A4 expression in NSCLC patients. Then, we conducted Cell Counting Kit-8 (CCK-8) assay and flow cytometric analysis to detect the function of OR3A4 on the resistance of NSCLC cells to cisplatin. Furthermore, the potential mechanism was explored by mechanism assays. RESULTS: Compared with OR3A4 expression of paired A549 cells, OR3A4 expression of A549/DDP cells was higher. Moreover, the functional assay showed that after OR3A4 was silenced in A549/DDP cells, cell cycle arrest and cell apoptosis was induced, and resistance to cisplatin was reversed. Furthermore, it was found that CDK1 expression was suppressed in A549/DDP cells by knockdown of OR3A4. CONCLUSIONS: The present work suggests that OR3A4 participates in regulating cell cycle, cell apoptosis of NSCLC cells and the resistance to cisplatin via upregulating CDK1, indicating that OR3A4 could be identified as a potential therapeutic target for NSCLC patients.

The PERK-EIF2α-ATF4 signaling branch regulates osteoblast differentiation and proliferation by PTH.

Parathyroid hormone (PTH) and its related peptide (PTH-related peptide 1-34) are two of the Food and Drug Administration-approved bone-promoting drugs for age-related osteoporosis. Treatment with PTH stimulates bone formation. However, the molecular mechanisms of PTH-mediated osteoblast differentiation and cell proliferation are still not completely understood. In this study, we showed that PTH induced endoplasmic reticulum (ER) stress in osteoblasts through the PKR-like endoplasmic reticulum kinase (PERK)-eukaryotic initiation factor 2α (EIF2α)-activating transcription factor 4 (ATF4)-signaling pathway. After separately blocking PERK-EIF2α-ATF4 signaling with two different inhibitors [AMG'44 and integrated stress response inhibitor (ISRIB)] or specific small interfering RNA for PERK and ATF4, the following targets were all downregulated: expression of osteoblast differentiation markers [runt-related transcription factor 2 (Runx2), alkaline phosphatase (Alp), type I collagen (Col1a1), and osteocalcin (Ocn)], cell proliferation markers (CyclinE, CyclinD, and CDC2), amino acid import (Glyt1), and metabolism-related genes (Asns). Additionally, Alp-positive staining cells, Alp activity, matrix mineralization, Ocn secretion, and cell proliferation indexes were inhibited. Interestingly, we found that salubrinal enhanced PTH-induced osteoblast differentiation and proliferation by maintenance of phosphorylation of EIF2α. Furthermore, we observed that PTH increased the association between heat shock protein 90 (HSP90) and PERK and maintained PERK protein stabilization in the early stages of PTH-induced ER stress. Treatment of MC3T3-E1 cells with geldanamycin, an HSP90 inhibitor, decreased PERK protein expression and inhibited osteoblast differentiation and cell proliferation upon PTH treatment. Taken together, our data demonstrate that PTH regulates osteoblast differentiation and cell proliferation, partly by activating the HSP90-dependent PERK-EIF2α-ATF4 signaling pathway.

Elucidating the druggable interface of protein-protein interactions using fragment docking and coevolutionary analysis.

Protein-protein interactions play a central role in cellular function. Improving the understanding of complex formation has many practical applications, including the rational design of new therapeutic agents and the mechanisms governing signal transduction networks. The generally large, flat, and relatively featureless binding sites of protein complexes pose many challenges for drug design. Fragment docking and direct coupling analysis are used in an integrated computational method to estimate druggable protein-protein interfaces. (i) This method explores the binding of fragment-sized molecular probes on the protein surface using a molecular docking-based screen. (ii) The energetically favorable binding sites of the probes, called hot spots, are spatially clustered to map out candidate binding sites on the protein surface. (iii) A coevolution-based interface interaction score is used to discriminate between different candidate binding sites, yielding potential interfacial targets for therapeutic drug design. This approach is validated for important, well-studied disease-related proteins with known pharmaceutical targets, and also identifies targets that have yet to be studied. Moreover, therapeutic agents are proposed by chemically connecting the fragments that are strongly bound to the hot spots.

Okadaic acid and dinophysis toxin 2 have differential toxicological effects in hepatic cell lines inducing cell cycle arrest, at G0/G1 or G2/M with aberrant mitosis depending on the cell line.

Okadaic acid is one of the toxins responsible for the human intoxication known as diarrhetic shellfish poisoning, which appears after the consumption of contaminated shellfish. The main diarrhetic shellfish poisoning toxins are okadaic acid, dinophysistoxin-1, -2, and -3. In vivo, after intraperitoneal injection, dinophysistoxin-2 is approximately 40% less toxic than okadaic acid in mice. The cytotoxic and genotoxic effect of okadaic acid varies very significantly in different cell lines, so similar responses could be expected for dinophysistoxin-2. In order to determine whether this was the case, we studied the effect of okadaic acid and dinophysistoxin-2 in two hepatic cell lines (HepG2 and Clone 9). The cytotoxicity of these toxins, as well as their effects on the cell cycle and its regulation on both cell lines, were determined. Okadaic acid and dinophysistoxin-2 resulted to be equipotent in clone 9 cultures, while okadaic acid was more potent than dinophysistoxin-2 in HepG2 cell cultures. Both toxins had opposite effects on the cell cycle; they arrested the cell cycle of clone 9 cells in G2/M inducing aberrant mitosis while arresting the cell cycle of HepG2 in G0/G1. When the effect of the toxins on p53 subcellular distribution was studied, p53 was detected in the nuclei of both cell types. The effect of the toxins on the gene expression of cyclins and cyclin-dependent kinases was different for both cell lines. The toxins induced an increase in gene expression of cyclins A, B, and D in clone 9 cells while they induced a decrease in cyclins A and B in HepG2 cells. They also induced a decrease in cyclin-dependent kinase 1 in HepG2 cells.

Plasma rich in quercetin metabolites induces G2/M arrest by upregulating PPAR-γ expression in human A549 lung cancer cells.

In this study, we incubated human A549 lung cancer cells with quercetin-metabolite-enriched plasma (QMP) obtained from Mongolian gerbils 2 h after quercetin feeding (100 mg/kg body wt/week). We investigated the effects of QMP on the growth of A549 cells and the possible mechanisms for these effects. We found that QMP but not control plasma (CP) reduced the cell growth in A549 cells. QMP led to cell cycle arrest at the G (2)/M phase by downregulating the expression of cdk1 and cyclin B. QMP but not CP or quercetin itself significantly increased PPAR- γ expression (p < 0.05), which was accompanied by an increase of phosphatase and tensin homologue deleted on the chromosome ten level and a decrease of phosphorylation of Akt. Furthermore, quercetin-3-glucuronide and quercetin-3'-sulfate also significantly increased PPAR- γ expression in A549 cells. GW9662, a PPAR- γ antagonist, significantly suppressed the effects of 10 % QMP on cell proliferation and on the expression of cyclin B and cdk1. Taken together, these data suggest that the activation of PPAR- γ plays an important role, at least in part, in the antiproliferative effects of quercetin metabolites.

Gambogic acid triggers DNA damage signaling that induces p53/p21(Waf1/CIP1) activation through the ATR-Chk1 pathway.

Gambogic acid (GA) has been wildly studied to show potent anti-tumor effects in vivo and in vitro. We have confirmed that GA stabilized and activated p53 through down-regulating the expression of MDM2 in variety of cancer cell lines. However, GA-induced p53 activation could be partially reversed by caffeine, a PI3k inhibitor. Therefore, questions of whether GA induces post-translational modifications of p53 and subsequent activation of p53; and if that is the case, which upstream signaling pathway(s) is (are) responsible for that are proposed. Here, the relationship between p53 activation and its post-translational modifications was investigated in the human cancer cell lines HepG2 and A549 in response to GA or adriamycin treatment. GA induces p53 phosphorylation at sites Ser15 and Ser20 in a concentration- or time-dependent way, which was a direct result of DNA damage, as gamma-HA2X foci and 'comet' DNA fragments were detected. GA induces p53 phosphorylation through activation of an ATM- and Rad3-related pathway, and GA-induced phosphorylation of Chk1 is also involved. Upon treatment with GA, ATR activation is clearly associated with p53 phosphorylation, as well as activation of its target gene p21(Waf/CIP1). Furthermore, we found the dephosphorylation of Cdk1 at Thr161 induced by GA was abrogated, followed by a remarkable disruption of G2/M arrest when the cells were pre-incubated with caffeine. Interestingly, the sensitivity to caffeine enhanced the cytotoxicity of GA as well. Taken together, these data showed an important role of the DNA damage response mediated by ATR-Chk1 in p53/p21(Waf/CIP1) activation and downstream G2/M arrest during GA treatment.

Cyclin C and cyclin dependent kinases 1, 2 and 3 in thrombin-induced neuronal cell cycle progression and apoptosis.

The extent to which neurons proceed into the cell cycle and the mechanisms whereby cell cycle re-entry leads to apoptosis vary in response to agonists. We previously showed upregulation of early G1 regulators in thrombin-treated neurons yet neurons did not proceed to S phase but to apoptosis. The objective of this study is to explore mechanisms which might prevent S phase entry and promote apoptosis in thrombin-treated neurons. Cultured rat brain neurons are exposed to thrombin (200nM) for 30min to 4.5h and the expression of cyclin C, cyclin dependent kinases (cdk1, cdk2, cdk3, cdk8) and the cell cycle inhibitor p27 assessed. Our data show a simultaneous decrease of both cyclin C and cdk3 proteins soon after thrombin treatment. The decrease in cyclin C also correlates with decreases in cdk1 and cdk2, at both mRNA and protein levels. There is no change in expression of cdk8 or the cell cycle inhibitor p27 in response to thrombin treatment. These results suggest that decreases in G1-S regulators cyclin C and cdks 3, cdk2 and cdk1 in response to thrombin could make conditions unfavorable for S phase entry and favor neuronal apoptosis.

Oroxylin A induces G2/M phase cell-cycle arrest via inhibiting Cdk7-mediated expression of Cdc2/p34 in human gastric carcinoma BGC-823 cells.

We reported previously that oroxylin A, a natural product isolated from Scutellariae Radix, was a potent apoptosis inducer of human hepatoma HepG2 cells. In this study, cell-cycle arrest of BGC-823 human gastric carcinoma cells caused by oroxylin A has been investigated. Based on our 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT) assay and flow cytometric analysis, treatment of BGC-823 cells with growth suppressive concentrations of oroxylin A caused an irreversible arrest in the G2/M phase of the cell cycle. Western blot analysis demonstrated that oroxylin A-induced cell-cycle arrest in BGC-823 cells was associated with a significant decrease in cdc2/p34, cyclin B1 and cyclin A expression. In addition, oroxylin A-treated cells decreased the expression of Cdk7, which was responsible for the low expression of M phase promoting factor (cyclin B1/Cdc2). The results suggested that oroxylin A induced G2/M phase cell-cycle arrest via inhibiting Cdk7-mediated expression of Cdc2/p34 in human gastric carcinoma BGC-823 cells.

In vitro effects of 2-methoxyestradiol on cell morphology and Cdc2 kinase activity in SNO oesophageal carcinoma cells.

The effects of 1 x 10(-6) M exogenous 2-methoxyestradiol (2 ME) were determined on cell morphology and cell division cycle (Cdc) 2 kinase activity in SNO oesophageal carcinoma cells. Mitotic indices revealed an increase in metaphase cells (11.2%) when compared to the 0.5% vehicle-treated cells after 18 h of exposure to 2 ME. Vehicle-treated control cells did not show any hallmarks of apoptosis after 18 h of exposure to dimethyl sulphoxide. Only 0.5% of 2 ME-treated cells showed characteristics of apoptosis. Conversely, increased morphological hallmarks of apoptosis were observed in SNO-treated cells after 21.5 h of 2 ME exposure. When compared to the 0.5% in vehicle-treated cells, 4.7% of cells were in apoptosis. Furthermore, 34.1% of cells were blocked in metaphase after 21.5 h of 2 ME exposure compared to 0.6% of vehicle-control cells. In addition, Cdc2 kinase activity was statistically significantly increased (1.3-fold) (p<0.005) in 2 ME-treated cells when compared to vehicle-treated controls. The present preliminary study suggests that the accumulation observed in metaphase cells and the increase in Cdc2 kinase activity caused by 2 ME are consistent with morphological hallmarks of mitotic arrest and disrupted mitotic spindle formation, thus leading to induction of apoptosis in SNO cells.

Porcine SPDYA2 (RINGO A2) stimulates CDC2 activity and accelerates meiotic maturation of porcine oocytes.

RINGO, a protein with no homology to cyclin B, has been reported to be involved in activation of CDC2 and regulation of meiotic maturation in Xenopus oocytes. Although the presence of homologues of RINGO families, which are known as SPDY families, has been reported in mammals, their roles in meiotic maturation of mammalian oocytes have never been examined. In the present study, the effects of SPDY on meiotic maturation of porcine oocytes were examined. At first, Xenopus RINGO (xRINGO) mRNA was injected into immature porcine oocytes and found to significantly accelerate CDC2 activation and meiotic resumption. The CCNB (also known as cyclin B) synthesis was prematurely started at 12 h of culture, whereas it started at 18 h in normal oocytes. We next cloned RINGO A2 homologue in pig (pigSPDYA2) from total RNA of immature porcine oocytes by RT-PCR and obtained full-length cDNA that was more than 85% and 40% homologous with mammalian SPDYA2 and xRINGO, respectively. Acceleration effects similar to those by xRINGO were observed in CDC2 activation, meiotic resumption, and the start of CCNB synthesis in pigSPDYA2 mRNA-injected porcine oocytes. In clear contrast with the effects of xRINGO, which was accumulated abnormally in porcine oocytes and arrested them in the first meiotic metaphase (M1), pigSPDYA2 accelerated the meiotic progression, with about half of pigSPDYA2 mRNA-injected oocytes completing meiotic maturation within 30 h. These results suggest that pigSPDYA2 has important roles on meiotic maturation of porcine oocytes and that the rapid degradation of SPDY was necessary for the normal maturation of oocytes.

Induction of G2/M arrest and apoptosis in human gastric epithelial AGS cells by aqueous extract of Agaricus blazei.

Agaricus blazei is well known as a traditional medicinal mushroom and it has been shown to exhibit immunostimulatory and anti-cancer activity. However, the cellular and molecular mechanism of cell cycle arrest and apoptosis of cancer cells is poorly understood. In this study, we have investigated whether A. blazei extract (ABE) exerts anti-proliferative and apoptotic effects on human gastric epithelial AGS cells. It was found that ABE could inhibit cell growth in a dose-dependent manner, which was associated with the arrest of G2/M phase and the induction of apoptotic cell death. Flow cytometric analysis showed that ABE could cause an arrest at the G2/M phase of the cell cycle, which is closely correlated to decreased cyclin B1 and cdc2 levels. Furthermore, this compound induced apoptosis through up-regulation of Bax and the activation of caspases with down-regulation of XIAP and cIAP-1, but not cIAP-2, and a capase-3 inhibitor could block cell death and apoptotic body formation. These data clearly indicate that ABE-induced apoptosis is associated with caspase-3 activation. In summary, the growth inhibition of ABE is highly related to cell cycle arrest at the G2/M phase and the induction of caspase-3-dependent apoptosis in human gastric epithelial AGS cells.

Bortezomib inhibits docetaxel-induced apoptosis via a p21-dependent mechanism in human prostate cancer cells.

Bortezomib (PS-341, Velcade) is a peptide boronate inhibitor of the 20S proteasome that is currently being combined with taxanes in several clinical trials in patients with prostate cancer. Here, we report that bortezomib inhibited docetaxel-induced M-phase arrest and apoptosis in androgen-dependent LNCaP-Pro5 cells. Direct analysis of kinase activity in immune complex kinase assays revealed that docetaxel activated cyclin-dependent kinase (CDK) 1 (CDC2) and that bortezomib blocked this activation. The effects of bortezomib were associated with accumulation of p21 and mimicked by chemical CDK inhibitors or by transfecting cells with a small interfering RNA construct specific for CDK1. Transient transfection with p21 also inhibited docetaxel-induced apoptosis; conversely, p21 silencing reversed the antagonistic effects of bortezomib on docetaxel-induced apoptosis. Together, our data show that bortezomib interferes with docetaxel-induced apoptosis via a p21-dependent mechanism that is associated with CDK1 inhibition. These observations may have important implications for the ongoing bortezomib-docetaxel combination trials as well as trials using bortezomib and other cell cycle-sensitive agents.

Synthesis, anti-inflammatory, analgesic and kinase (CDK-1, CDK-5 and GSK-3) inhibition activity evaluation of benzimidazole/benzoxazole derivatives and some Schiff's bases.

A series of N-(acridin-9-yl)-4-(benzo[d]imidazol/oxazol-2-yl) benzamides has been synthesized by the condensation of 9-aminoacridine derivatives with benzimidazole or benzoxazole derivatives. Condensation of 2-hydroxy naphthaldehyde with functionalized diamines leads to the formation of Schiff's bases and not imidazole derivatives. All these compounds were characterized by correct FT-IR, (1)H NMR, MS and elemental analyses. These compounds were screened for anti-inflammatory, analgesic and kinase (CDK-1, CDK-5 and GSK-3) inhibition activities. Compounds 11 and 7e(f) showed good anti-inflammatory (35.8% at 50 mg/kg po) activity and good analgesic activity (60% at 50 mg/kg po), respectively. Compound 3b showed significant in vitro activity against CDK-5 (IC(50)=4.6 microM) and CDK-1(IC(50)=7.4 microM) and compound 3a showed moderate CDK-5 inhibitory activity (IC(50)=7.5 microM). The other compounds showed moderate anti-inflammatory and analgesic activities.

Hsp90 inhibitors cause G2/M arrest associated with the reduction of Cdc25C and Cdc2 in lung cancer cell lines.

PURPOSE: Hsp90, a molecular chaperone, is involved in folding, assembly, maturation, and stabilization of the client proteins which regulate survival of cancer cells, and thus Hsp90 inhibitors may be potential molecular targeting agents for cancer treatment. We investigated whether Hsp90 inhibitors have therapeutic value in lung cancer. METHODS: First, expression levels of Hsp90 in lung cancer cells were examined by western blotting and immunohistochemical analyses. Next, the effect of Hsp90 inhibitors, geldanamycin and 17-allylaminogeldanamycin (17-AAG), on lung cancer cell growth was examined. RESULTS: Remarkable high expression of Hsp90 protein in lung cancer cell lines and a more intense signal for Hsp90 by immunohistochemistry in males, patients with smoking index over 600, and squamous cell carcinoma were observed. Both Hsp90 inhibitors dose dependently inhibited the growth of lung cancer cell lines and induced G2/M arrest concomitant with decreased protein levels of Cdc25C and Cdc2. Moreover, combination of an Hsp90 inhibitor and irradiation had an additive effect on cell growth inhibition and reduction of Cdc25C and Cdc2 protein levels. CONCLUSION: Hsp90 inhibitor is thus a therapeutic tool for lung cancer based on its target proteins, which are involved in tumor progression and antiproliferative activity in lung cancer cells.

Mitogen requirement for cell cycle progression in the absence of pocket protein activity.

Primary mouse embryonic fibroblasts lacking expression of all three retinoblastoma protein family members (TKO MEFs) have lost the G1 restriction point. However, in the absence of mitogens these cells become highly sensitive to apoptosis. Here, we show that TKO MEFs that survive serum depletion pass G1 but completely arrest in G2. p21CIP1 and p27KIP1 inhibit Cyclin A-Cdk2 activity and sequester Cyclin B1-Cdk1 in inactive complexes in the nucleus. This response is alleviated by mitogen restimulation or inactivation of p53. Thus, our results disclose a cell cycle arrest mechanism in G2 that restricts the proliferative capacity of mitogen-deprived cells that have lost the G1 restriction point. The involvement of p53 provides a rationale for the synergism between loss of Rb and p53 in tumorigenesis.

Cyclin A-associated kinase activity is needed for paclitaxel sensitivity.

Cyclin A-associated kinases, such as cyclin-dependent kinase 2 (CDK2), participate in regulating cellular progression from G(1) to S to G(2), and CDK2 has also been implicated in the transition to mitosis. The antitumor properties of CDK inhibitors, alone or in combination with taxanes, are currently being examined in clinical trials. Here, we examined whether the activity of kinases associated with cyclin A (such as CDK2) is important in determining cellular sensitivity to paclitaxel, a taxane and mitotic inhibitor used in chemotherapy for breast and ovarian cancer. We used adenoviral suppression or overexpression to manipulate the expression of CDK2 and cyclin A in one breast cancer and three ovarian cancer cell lines with different sensitivities to paclitaxel and assessed protein expression, kinase activity, cell cycle distribution, and sensitivity to paclitaxel. Transfection of a dominant-negative (DN)-CDK2 evoked resistance to paclitaxel by preventing cellular progression to mitosis through loss of CDK1 activity. Reexpression of wild-type CDK2 in DN-CDK2-transfected cancer cells restored CDK2 activity but not paclitaxel sensitivity. However, expression of cyclin A in DN-CDK2-transfected cells restored their sensitivity to paclitaxel. Although CDK2 activity was not directly involved in paclitaxel sensitivity, cyclin A-associated kinases did up-regulate CDK1 via phosphorylation. We conclude that cyclin A-associated kinase activity is required for these cells to enter mitosis and undergo paclitaxel-induced cell death. Combining taxane chemotherapy with any drug targeting cyclin A-associated kinases (e.g., pure CDK2 inhibitors) should be done with caution, if at all, because of the potential for enhancing taxane resistance.

Sensitization of taxol-induced apoptosis by curcumin involves down-regulation of nuclear factor-kappaB and the serine/threonine kinase Akt and is independent of tubulin polymerization.

Taxol is the best anticancer agent that has ever been isolated from plants, but its major disadvantage is its dose-limiting toxicity. In this study, we report with mechanism-based evidence that curcumin, a nontoxic food additive commonly used by the Indian population, sensitizes tumor cells more efficiently to the therapeutic effect of Taxol. A combination of 5 nm Taxol with 5 microm curcumin augments anticancer effects more efficiently than Taxol alone as evidenced by increased cytotoxicity and reduced DNA synthesis in HeLa cells. Furthermore, our results reveal that this combination at the cellular level augments activation of caspases and cytochrome c release. This synergistic effect was not observed in normal cervical cells, 293 cells (in which Taxol down-regulates nuclear factor-kappaB (NF-kappaB)), or HeLa cells transfected with inhibitor kappaBalpha double mutant (IkappaBalpha DM), although the transfection itself sensitized the cells to Taxol-induced cytotoxicity. Evaluation of signaling pathways common to Taxol and curcumin reveals that this synergism was in part related to down-regulation of NF-kappaB and serine/threonine kinase Akt pathways by curcumin. An electrophoretic mobility shift assay revealed that activation of NF-kappaB induced by Taxol is down-regulated by curcumin. We also noted that curcumin-down-regulated Taxol induced phosphorylation of the serine/threonine kinase Akt, a survival signal which in many instances is regulated by NF-kappaB. Interestingly, tubulin polymerization and cyclin-dependent kinase Cdc2 activation induced by Taxol was not affected by curcumin. Altogether, our observations indicate that Taxol in combination with curcumin may provide a superior therapeutic index and advantage in the clinic for the treatment of refractory tumors.