Abrogation of ATR function preferentially augments cisplatin-induced cytotoxicity in PTEN-deficient breast cancer cells.

Targeting replication stress response is currently emerging as new therapeutic strategy for cancer treatment, based on monotherapy and combination approaches. As a key sensor in response to DNA damage, ataxia telangiectasia and rad3-related (ATR) kinase has become a potential therapeutic target as tumor cells are to rely heavily on ATR for survival. The tumor suppressor phosphatase and tensin homolog (PTEN) plays a crucial role in maintaining chromosome integrity. Although ATR inhibition was recently confirmed to show a synergistic inhibitory effect in PTEN-deficient triple-negative breast cancer cells, the molecular mechanism needs to be further elucidated. Additionally, whether the PTEN-deficient breast cancer cells are more preferentially sensitized than PTEN-wild type breast cancer cells to cisplatin plus ATR inhibitor remains unanswered. We demonstrate PTEN dysfunction promotes the killing effect of ATR blockade through the use of RNA interference for PTEN and a highly selective ATR inhibitor VE-821, and certify that VE-821 (1.0 µmol/L) aggravates cytotoxicity of cisplatin on breast cancer cells, especially PTEN-null MDA-MB-468 cells which show more chemoresistance than PTEN-expressing MDA-MB-231 cells. The co-treatment with VE-821 and cisplatin significantly reduced cell viability and proliferative capacity compared with cisplatin mono-treatment (P < 0.05). The increased cytotoxic activity is tied to the enhanced poly (ADP-ribose) polymerase (PARP) cleavage and consequently cell death due to the decrease in phosphorylation levels of checkpoint kinases 1 and 2 (CHK1/2), the reduction of radiation sensitive 51 (RAD51) foci and the increase in phosphorylation of the histone variant H2AX (γ-H2AX) foci (P < 0.05) as well. Together, these findings suggest combination therapy of ATR inhibitor and cisplatin may offer a potential therapeutic strategy for breast tumors.

Combination of DESI2 and endostatin gene therapy significantly improves antitumor efficacy by accumulating DNA lesions, inducing apoptosis and inhibiting angiogenesis.

DESI2 is a novel pro-apoptotic gene. We previously reported that DESI2 overexpression induces S phase arrest and apoptosis by activating checkpoint kinases. This work was to test whether the combination of endostatin, an endogenous antiangiogenic inhibitor, with DESI2 could improve the therapy efficacy in vitro and in vivo. The recombinant plasmid co-expressing DESI2 and endostatin was encapsulated with DOTAP/Cholesterol cationic liposome. Mice bearing CT26 colon carcinoma and LL2 lung cancer were treated with the DNA-liposome complex. We found that, in vitro, the combination of DESI2 and endostatin more efficiently inhibited proliferation of CT26, LL2, HCT116 and A549 cancer cells via apoptosis, as assessed by MTT assay, colony-formation assays, flow cytometric analysis, hoechst staining and activation of caspase-3, respectively. In addition, DESI2 overexpression caused up-regulation of RPS7, a substrate of DESI2 deubiquitination. Furthermore, siRNA targeting RPS7 partially abrogated, whereas RPS7 overexpression enhanced DESI2-induced inhibition of cell proliferation. Importantly, the combination also caused DNA lesions accumulation, which further promotes apoptosis. Mechanistic rationale suggested that endostatin first inhibits DNA-PKcs kinase, and partly abrogated DESI2-induced phosphorylation of DNA-PKcs, leading to increase of DNA damage, then contributes to DESI2-induced apoptosis. In vivo, the combined gene therapy more significantly inhibited tumor growth and efficiently prolonged the survival of tumor bearing mice than mono therapy. The improved antitumor effect was associated with inhibition of cell proliferation via apoptosis, as analyzed by TUNEL assay and PCNA immunostaining. The combination also inhibited angiogenesis, as assessed by alginate-encapsulated tumor cell assay and CD31 staining. Our data suggest that the combined gene therapy of DESI2 and endostatin can significantly enhance the antitumor activity as a DNA lesions accumulator, apoptosis inducer and angiogenesis inhibitor. The present study may provide a novel method for the treatment of cancer.

hPNAS-4 inhibits proliferation through S phase arrest and apoptosis: underlying action mechanism in ovarian cancer cells.

PNAS-4, a novel pro-apoptotic gene, was activated during the early response to DNA damage. Previous studies have shown that hPNAS-4 can inhibit tumor growth when over-expressed in ovarian cancer cells. However, the underlying action mechanism remains elusive. In this work, we found that hPNAS-4 expression was significantly increased in SKOV3 cells when exposed to cisplatin, methyl methanesulfonate or mitomycin C, and that its overexpression could induce proliferation inhibition, S phase arrest and apoptosis in A2780s and SKOV3 ovarian cancer cells. The S phase arrest caused by hPNAS-4 was associated with up-regulation of p21. p21 is p53-dispensable and correlates with activation of ERK, and activation of the Cdc25A-Cdk2-Cyclin E/Cyclin A pathway, while the pro-apoptotic effects of hPNAS-4 were mediated by activation of caspase-9 and -3 other than caspase-8, and accompanied by release of AIF, Smac and cytochrome c into the cytosol. Taken together, these data suggest a new mechanism by which hPNAS-4 inhibits proliferation of ovarian cancer cells by inducing S phase arrest and apoptosis via activation of Cdc25A-Cdk2-Cyclin E/Cyclin A axis and mitochondrial dysfunction-mediated caspase-dependent and -independent apoptotic pathways. To our knowledge, we provide the first molecular evidence for the potential application of hPNAS-4 as a novel target in ovarian cancer gene therapy.

Enhanced gemcitabine-mediated cell killing of human lung adenocarcinoma by vector-based RNA interference against PLK1.

Specific PLK1 silencing may be an effective gene therapy modality of treating PLK1-overexpressed cancers. In this study, we first explored the anticancer efficacy of three different short hairpin-expressing plasmids targeting PLK1 in animal model, and then determined the combination therapy effect of gemcitabine with PLK1-shRNA as an adjuvant. Transfection of the PLK1-shRNAs to A549 lung cancer cells induced significant PLK1 depletion, growth inhibition and apoptosis. In vivo administration of PLK1-shRNA constructs to tumor-bearing mice resulted in xenograft regression. Moreover, the combination of PLK1-shRNA plus low-dose gemcitabine (GEM) produced an additive antitumor activity on the lung tumors owing to an inhibition of cancer cell survival and augmented apoptosis. These results indicated a feasible bio-chemotherapeutic strategy for cancer.

NOXA-induced alterations in the Bax/Smac axis enhance sensitivity of ovarian cancer cells to cisplatin.

Ovarian cancer is the most common cause of death from gynecologic malignancy. Deregulation of p53 and/or p73-associated apoptotic pathways contribute to the platinum-based resistance in ovarian cancer. NOXA, a pro-apoptotic BH3-only protein, is identified as a transcription target of p53 and/or p73. In this study, we found that genetic variants of Bcl-2 proteins exist among cisplatin-sensitive and -resistant ovarian cancer cells, and the responses of NOXA and Bax to cisplatin are regulated mainly by p53. We further evaluated the effect of NOXA on cisplatin. NOXA induced apoptosis and sensitized A2780s and SKOV3 cells to cisplatin in vitro and in vivo. The effects were mediated by elevated Bax expression, enhanced caspase activation, release of Cyt C and Smac into the cytosol. Furthermore, gene silencing of Bax or Smac significantly attenuated NOXA and/or cisplatin-induced apoptosis in chemosensitive A2780s cells, whereas overexpression of Bax or addition of Smac-N7 peptide significantly increased NOXA and/or cisplatin-induced apoptosis in chemoresistant SKOV3 cells. To our knowledge, these data suggest a new mechanism by which NOXA chemosensitized ovarian cancer cells to cisplatin by inducing alterations in the Bax/Smac axis. Taken together, our findings show that NOXA is potentially useful as a chemosensitizer in ovarian cancer therapy.

Noxa enhances the cytotoxic effect of gemcitabine in human ovarian cancer cells.

Noxa is an important proapoptotic protein in the intrinsic pathway of cell apoptosis. Experiments were carried out to investigate whether Noxa could, therefore, enhance the cytotoxic effect of gemcitabine in human ovarian cancer cell lines (A2780 and COC1). In this study, the combined treatment of Noxa and gemcitabine, in vitro, significantly inhibited the proliferation of A2780 and COC1 cells, as verified by MTT assay, Hoechst staining, and flow cytometric analysis. Moreover, the combination of Noxa and gemcitabine inhibited tumor growth and prolonged the survival of nude mice in vivo. The combined treatment also inhibited the growth of tumor xenografts through the inhibition of proliferation and the induction of apoptosis, as observed in immunohistochemical anti-PCNA staining and TdT-mediated dUTP-biotin nick-end labeling (TUNEL) assay. Our data suggest that Noxa exhibited potent proapoptotic activity against human ovarian cancer cells, and the combination of Noxa and gemcitabine showed a more significant cytotoxic effect against ovarian cancer cells in comparison with either of these agents alone. To our knowledge, we have provided the first evidence that Noxa can enhance therapeutic responses of ovarian cancer cells to gemcitabine, and that it could be potentially useful as a chemosensitizer in ovarian cancer therapy.

The p53 upregulated modulator of apoptosis (PUMA) chemosensitizes intrinsically resistant ovarian cancer cells to cisplatin by lowering the threshold set by Bcl-x(L) and Mcl-1.

Ovarian cancer is the number one cause of death from gynecologic malignancy. A defective p53 pathway is a hallmark of ovarian carcinoma. The p53 mutation correlates significantly with resistance to platinum-based chemotherapy, early relapse and shortened overall survival in ovarian cancer patients. PUMA (p53 upregulated modulator of apoptosis), a BH3-only Bcl-2 family protein, was recently identified as a transcriptional target of p53 and a potent apoptosis inducer in various cancer cells. In this study, we showed that the induction of PUMA by cisplatin was abolished in p53-deficient SKOV3 cells. Elevated expression of PUMA-induced apoptosis and sensitized A2780s and SKOV3 ovarian cancer cells to cisplatin, and the combination of PUMA and low-dose cisplatin, significantly suppressed xenograft tumor growth in vivo through enhanced induction of apoptosis compared with treatment with PUMA or cisplatin alone. The effects of PUMA were mediated by enhanced caspase activation and release of cytochrome c and Smac (second mitochondria-derived activator of caspase) into the cytosol. Furthermore, PUMA chemosensitized intrinsically resistant SKOV3 cells to cisplatin through downregulation of B-cell lymphoma-extra large (Bcl-x(L)) and myeloid cell leukemia sequence 1 (Mcl-1). PUMA-mediated Bcl-x(L) downregulation mainly happened at the transcription level, whereas PUMA-induced Mcl-1 down-regulation was associated with caspase-dependent cleavage and proteasome-mediated degradation. To our knowledge, these data suggest a new mechanism by which overexpression of PUMA enhances sensitivity of SKOV3 cells to cisplatin by lowering the threshold set simultaneously by Bcl-x(L) and Mcl-1. Taken together, our findings indicate that PUMA is an important modulator of therapeutic responses of ovarian cancer cells and is potentially useful as a chemosensitizer in ovarian cancer therapy.

Induction of tumor inhibition and apoptosis by a candidate tumor suppressor gene DRR1 on 3p21.1.

Down-regulated in renal cell carcinoma gene (DRR1) is one of the candidate tumor suppressor genes (TSGs) on human 3p21.1. This study was performed to validate the expression status of DRR1 gene in cancer cells and the expression pattern of the protein in clinical specimens of human lung cancer and to examine its potential as a molecular target for treatment of lung cancer in vivo. DRR1 expression was analyzed in 7 human lung cancer cell lines. DRR1 protein expression was also examined in clinical non-small cell lung cancer (NSCLC) specimens. Furthermore, effects of DRR1 re-expression on A549 cells in vitro and A549 xenograft tumors in nude mice were evaluated. Loss of DRR1 mRNA expression was detected in 6 of the 7 human cancer cell lines, the exception was the renal cancer cell line OS-RC-2. DRR1 protein expression was absent in 15 of 20 (75%) human NSCLC specimens by immunostaining. Transfection of DRR1 gene into DRR1-negative-expressing A549 cells resulted in significant cell growth suppression and apoptosis. Plasmids containing DRR1 cDNA complexed with DOTAP:Chol liposomes were administered intravenously via tail vein to nude mice bearing A549 xenograft tumors resulting in tumor growth inhibition and elevation of apoptosis compared with the controls. DRR1 is a potent growth suppressor of NSCLC, acting through apoptosis pathway in vivo and it may be a potential therapeutic gene for human lung cancer.

Formaldehyde at low concentration induces protein tau into globular amyloid-like aggregates in vitro and in vivo.

Recent studies have shown that neurodegeneration is closely related to misfolding and aggregation of neuronal tau. Our previous results show that neuronal tau aggregates in formaldehyde solution and that aggregated tau induces apoptosis of SH-SY5Y and hippocampal cells. In the present study, based on atomic force microscopy (AFM) observation, we have found that formaldehyde at low concentrations induces tau polymerization whilst acetaldehyde does not. Neuronal tau misfolds and aggregates into globular-like polymers in 0.01-0.1% formaldehyde solutions. Apart from globular-like aggregation, no fibril-like polymerization was observed when the protein was incubated with formaldehyde for 15 days. SDS-PAGE results also exhibit tau polymerizing in the presence of formaldehyde. Under the same experimental conditions, polymerization of bovine serum albumin (BSA) or alpha-synuclein was not markedly detected. Kinetic study shows that tau significantly misfolds and polymerizes in 60 minutes in 0.1% formaldehyde solution. However, presence of 10% methanol prevents protein tau from polymerization. This suggests that formaldehyde polymerization is involved in tau aggregation. Such aggregation process is probably linked to the tau's special "worm-like" structure, which leaves the epsilon-amino groups of Lys and thiol groups of Cys exposed to the exterior. Such a structure can easily bond to formaldehyde molecules in vitro and in vivo. Polymerizing of formaldehyde itself results in aggregation of protein tau. Immunocytochemistry and thioflavin S staining of both endogenous and exogenous tau in the presence of formaldehyde at low concentrations in the cell culture have shown that formaldehyde can induce tau into amyloid-like aggregates in vivo during apoptosis. The significant protein tau aggregation induced by formaldehyde and the severe toxicity of the aggregated tau to neural cells may suggest that toxicity of methanol and formaldehyde ingestion is related to tau misfolding and aggregation.

Amyloid-like aggregates of neuronal tau induced by formaldehyde promote apoptosis of neuronal cells.

BACKGROUND: The microtubule associated protein tau is the principle component of neurofibrillar tangles, which are a characteristic marker in the pathology of Alzheimer's disease; similar lesions are also observed after chronic alcohol abuse. Formaldehyde is a common environmental contaminant and also a metabolite of methanol. Although many studies have been done on methanol and formaldehyde intoxication, none of these address the contribution of protein misfolding to the pathological mechanism, in particular the effect of formaldehyde on protein conformation and polymerization. RESULTS: We found that unlike the typical globular protein BSA, the natively-unfolded structure of human neuronal tau was induced to misfold and aggregate in the presence of ~0.01% formaldehyde, leading to formation of amyloid-like deposits that appeared as densely staining granules by electron microscopy and atomic force microscopy, and bound the amyloid-specific dyes thioflavin T and Congo Red. The amyloid-like aggregates of tau were found to induce apoptosis in the neurotypic cell line SH-SY5Y and in rat hippocampal cells, as observed by Hoechst 33258 staining, assay of caspase-3 activity, and flow cytometry using Annexin V and Propidium Iodide staining. Further experiments showed that Congo Red specifically attenuated the caspase-3 activity induced by amyloid-like deposits of tau. CONCLUSION: The results suggest that low concentrations of formaldehyde can induce human tau protein to form neurotoxic aggregates, which could play a role in the induction of tauopathies.

Changes in conformation of human neuronal tau during denaturation in formaldehyde solution.

Human neuronal tau was incubated in formaldehyde solution at low concentrations and the intensity of light scattering of tau-40 solution at 480 nm increased markedly. Then potassium iodide was used to quench the intrinsic fluorescence of tau. The fluorescent quenching constants decreased as formaldehyde concentrations increased. 8-anilino-1-naphthalenesulfonic acid (ANS) binding assay showed that a putative hydrophobic core formed in tau polymers during incubation with formaldehyde. Native tau was hydrolyzed by immobilized earthworm fibrinolytic enzyme-II (EFE-II), producing a digested fragment (36-37 kDa). However, formaldehyde-treated tau could not be digested under the same conditions, suggesting that aggregated protein was relatively rigidly deposited.

Neuronal tau induces DNA conformational changes observed by atomic force microscopy.

Neuronal tau associates with chromosome scaffold and localizes in the nuclear and the nucleolar organization regions in neuronal and some non-neuronal cells. Observing the interaction of neuronal tau with DNA under AFM shows that tau binds to DNA as a monomer, and tau-DNA complex forms a beads-on-a-string structure when the mass ratio is 1:10 (molar ratio of tau/DNA approximately 1:700 bp). A beads-on-a-coil structure, in which tau is as polymers, will appear when the mass ratio is up to 1:5 (molar ratio of tau/DNA approximately 1:350 bp). The present observation that neuronal tau bends the DNA double strands indicates that the appearance of the tau-DNA complex is dependent upon the mass (or molar) ratio of tau and DNA.

Chaperone-like manner of human neuronal tau towards lactate dehydrogenase.

In our experiments, inactivation of lactate dehydrogenase (LDH, EC1.1.1.27) in the presence of human microtubule-associated tau is observably suppressed during thermal and guanidine hydrochloride (GdnHCl) denaturation. Kinetic studies show tau can prevent LDH from self-aggregation monitored by light scattering during thermal denaturation. On the other hand, neuronal tau promotes reactivation of LDH and suppresses self-aggregation of non-native LDH when GdnHCl solution is diluted. Furthermore, the reactivation yield of LDH decreases significantly with delayed addition of tau. All experiments were completed in the reducing buffer with 1 mM DTT to avoid between tau and LDH forming the covalent bonds during unfolding and refolding. Thus, Tau prevents proteins from misfolding and aggregating into insoluble, nonfunctional inclusions and assists them to refold to reach the stable native state by binding to the exposed hydrophobic patches on proteins instead of by forming or breaking covalent bonds. Additionally, tau remarkably enhances reactivation of GDH (glutamic dehydrogenase, EC 1.4.1.3), another carbohydrate metabolic enzyme, also showing a chaperone-like manner. It suggests that neuronal tau non-specifically functions a chaperone-like protein towards the enzymes of carbohydrate metabolism.