The Clustered DNA Lesions - Types, Pathways of Repair and Relevance to Human Health.

The clustered DNA lesions are a characteristic feature of ionizing radiation and are defined as two or more damage sites formed within 20 bps after the passage of a single radiation track. The clustered DNA lesions are divided into two major groups: double-stranded breaks (DSBs) and non-DSB clusters also known as Oxidatively-induced Clustered DNA Lesions (OCDLs), which could involve either two opposing strands or the same strand. As irradiation is gaining greater interest in cancer treatment as well as in imaging techniques, the detailed knowledge of its genotoxicity and the mechanisms of repair of radiation-induced DNA damage remain issues to explore. In this review we look at the ways the cell copes with clustered DNA lesions, especially with 5',8-cyclo-2'-deoxypurines. As the base excision repair deals with isolated lesions, complex damage is more difficult to repair. Depending on the number of lesions within a cluster, their types and mutual distribution, long-patch BER or NER are activated. During the repair of opposing lesions, DSBs could be generated, which are repaired either by nonhomologous end joining (NHEJ) or homologous recombination (HR). The repair of individual lesions within a cluster progresses gradually. This slower processing of particular damage might lead to severe biological consequences such as misrepair, mutations and chromosomal rearrengement as it enhances the plausibility of a cluster encountering a replication fork prior to its repair. The consequences of clustered DNA lesions on cell survival and their relevance to the efficacy and safety of radiotherapy and radiodiagnosis will also be discussed.

Actual state of knowledge in the field of diseases related with defective nucleotide excision repair.

Xeroderma pigmentosum (XP), trichothiodystrophy (TTD) and Cockayne syndrome (CS) are rare genetic diseases characterized by a large range of clinical symptoms. However, they are all associated with defects in nucleotide excision repair (NER), the system responsible for removing bulky DNA lesions such as those generated by UV light: cyclobutane pyrimidine dimers (CPDs) and pyrimidine-pyrimidone photoproducts (6-4 PPs). Over the past years, detailed structural and biochemical information on NER-associated proteins has emerged. In the first part of the article we briefly present the main steps of the NER pathway with an emphasis on the precise role of certain proteins. Further, we focus on clinical manifestations of the disorders and describe the diagnostic procedures. Then we consider how current therapy and advanced technology could improve patients' quality of life. Although to date the discussed diseases remain incurable, effective sun protection, a well thought out diet, and holistic medical care provide longer life and better health. This review summarizes the current state of knowledge regarding the epidemiology of NER-associated diseases, their genetic background, clinical features, and treatment options.

Molecular mechanism of action of oxazolinoanthracyclines in cells derived from human solid tumors. Part 2.

BACKGROUND/AIM: Oxazolinodoxorubicin (O-DOX) and oxazolinodaunorubicin (O-DAU) are derivatives of anthracyclines (DOX and DAU) with a modified daunosamine moiety. We aimed to clarify their mechanisms of action by investigating intracellular accumulation and effects on the cell cycle, phosphatidylserine externalization, and proteasome 20S activity. MATERIALS AND METHODS: Experimental model consisted of SKOV-3, A549 and HepG2 cells. Compounds were used at the concentration of 80nM. Intracellular accumulation, drug uptake, and proteasome 20S activity were evaluated by fluorimetric methods. The effects on the cell cycle and phosphatidylserine externalization were measured by flow cytometry. RESULTS: O-DOX was equivalent to DOX in terms of inducing G2/M arrest, but O-DAU was less potent in SKOV-3, HepG2, and A549 cells. O-DOX had the greatest effect on initiating apoptosis in all tested cells. Externalization of phosphatidylserine was significantly higher following O-DOX treatment compared with control cells and cells incubated with DOX. The intracellular accumulation and uptake of the derivatives were similar to those of the reference drugs. Tested compounds are able to activate proteasome 20S activity. CONCLUSION: Our results extended the understanding of the toxicity, mechanism of action, and biochemical properties of oxazoline derivatives of doxorubicin and daunorubicin, including their effects on cell cycle, apoptosis and DNA degradation.

Metformin and epothilone A treatment up regulate pro-apoptotic PARP-1, Casp-3 and H2AX genes and decrease of AKT kinase level to control cell death of human hepatocellular carcinoma and ovary adenocarcinoma cells.

High mortality rates in ovarian and liver cancer are largely a result of resistance to currently used chemotherapy. Here, we investigated genotoxic and pro-oxidant effects of metformin (MET) and epothilone A (A) in combination with respect to apoptosis in HepG2 and SKOV-3 cancer cells. Reactive oxygen species (ROS) was studied using 2',7'-dichlorodihydrofluoresein diacetate, and samples were analyzed for the presence and absence of the N-acetylcysteine (NAC). Expression of genes involved in programmed cell death, oxidative and alkylating DNA damage was measured. Probes were analyzed in the presence of Akt or nuclear factor-κB inhibitor. Compared to either drug alone, combination of epothilone A and metformin was more potent; decreased Akt level; and elevated percentage of apoptotic cells, induced cell cycle arrest at G1 phase and elevated the sub-G1 cell population by increasing the mRNA level of caspase-3, poly (ADP-ribose) polymerase-1 and H2AX. The anticancer effect of the drug combination was partially reversed by NAC supplementation, suggesting that ROS generation is required to induce apoptosis. The present study demonstrates that novel combination such as epothilone A and MET show promise in expanding ovarian and liver cancer therapy.

Why a Combination of WP 631 and Epo B is an Improvement on the Drugs Singly - Involvement in the Cell Cycle and Mitotic Slippage.

Our previous studies clearly demonstrated that a combination of WP 631 and Epo B has higher activity against ovarian cancer cells than either of these compounds used separately. In order to fully understand the exact mechanism of action in combination, we assessed effects on the cell cycle of SKOV-3 cells. We evaluated three control points essential for WP 631 and Epo B action to determine which cell cycle-regulating proteins (CDK1/cyclin B complex, EpCAM or HMGB1) mediate activity. The effects of the drug on the cell cycle were measured based on the nuclear DNA content using flow cytometry. Expression of cell cycle-regulating genes was analyzed using real-time PCR. It was discovered that WP 631, at the tested concentration, did not affect the SKOV-3 cell cycle. Epo B caused significant G2/M arrest, whereas the drug combination induced stronger apoptosis and lower mitotic arrest than Epo B alone. This is very important information from the point of view of the fight against cancer, as, while mitotic arrest in Epo B-treated cells could be overcame after DNA damage repair, apoptosis which occurs after mitotic slippage in combination-treated cells is irreversible. It clearly explains the higher activity of the drug combination in comparison to Epo B alone. Epo B acts via the CDK1/cyclin B complex and has the ability to inhibit CDK1, which may be a promising strategy for ovarian cancer treatment in the future. The drug combination diminishes EpCAM and HMGB1 expression to a greater degree than either WP 631 and Epo B alone. Owing to the fact that the high expression of these two proteins is a poor prognostic factor for ovarian cancer, a decrease in their expression, observed in our studies, may result in improved efficacy of cancer therapy. The presented findings show that the combination of WP 631 and Epo B is a better therapeutic option than either of these drugs alone.

New potential chemotherapy for ovarian cancer - Combined therapy with WP 631 and epothilone B.

Despite more modern therapeutics approaches and the use of new drugs for chemotherapy, patients with ovarian cancer still have poor prognosis and therefore, new strategies for its cure are highly needed. One of the promising ways is combined therapy, which has many advantages as minimizing drug resistance, enhancing efficacy of treatment, and reducing toxicity. Combined therapy has rich and successful history in the field of ovarian cancer treatment. Currently use therapy is usually based on platinum-containing agent (carboplatin or cisplatin) and a member of taxanes (paclitaxel or docetaxel). In the mid-2000s this standard regimen has been expanded with bevacizumab, monoclonal antibody directed to Vascular Endothelial Growth Factor (VEGF). Another drug combination with promising perspectives is WP 631 given together with epothilone B (Epo B). WP 631 is a bisanthracycline composed of two molecules of daunorubicin linked with a p-xylenyl linker. Epo B is a 16-membered macrolide manifesting similar mechanism of action to taxanes. Their effectiveness against ovarian cancer as single agents is well established. However, the combination of WP 631 and Epo B appeared to act synergistically, meaning that it is much more potent than the single drugs. The mechanism lying under its efficacy includes disturbing essential cell cycle-regulating proteins leading to mitotic slippage and following apoptosis, as well as affecting EpCAM and HMGB1 expression. In this article, we summarized the current state of knowledge regarding combined therapy based on WP 631 and Epo B as a potential way of ovarian cancer treatment.

Innovative therapy of ovarian cancer based on overexpression of CD44 receptor.

Ovarian carcinoma constitutes the main cause of cancer-related death among women. The curability rates remain low despite rapid advances in medicine. Thus, the search for new and improved methods continues, with CD44-targeting as one of them. CD44 is a cell-surface glycoprotein, which binds to its ligand--hyaluronic acid (HA)--and regulates crucial processes such as cell differentiation, proliferation and migration. Overexpression of CD44, observed in many ovarian cancer cells, is used in creating carriers for selective delivery of various drugs (paclitaxel, doxorubicin, camptothecin or cisplatin) to cancer cells. In this article, we summarized the current state of knowledge regarding CD44-targeting as a new and more efficient way of ovarian cancer treatment, with high potential and promising therapeutic perspectives.

Early Activation of Apoptosis and Caspase-independent Cell Death Plays an Important Role in Mediating the Cytotoxic and Genotoxic Effects of WP 631 in Ovarian Cancer Cells.

The purpose of this study was to provide a detailed explanation of the mechanism of bisanthracycline,?WP 631 in comparison to doxorubicin (DOX), a first generation anthracycline, currently the most widely used pharmaceutical in clinical oncology. Experiments were performed in SKOV-3 ovarian cancer cells which are otherwise resistant to standard drugs such as cis-platinum and adriamycin. As attention was focused on the ability of WP 631 to induce apoptosis, this was examined using a double staining method with Annexin V and propidium iodide probes, with measurement of the level of intracellular calcium ions and cytosolic cytochrome c. The western blotting technique was performed to confirm PARP cleavage. We also investigated the involvement of caspase activation and DNA degradation (comet assay and immunocytochemical detection of phosphorylated H2AX histones) in the development of apoptotic events. WP 631 demonstrated significantly higher effectiveness as a pro-apoptotic drug than DOX. This was evident in the higher levels of markers of apoptosis, such as the externalization of phosphatidylserine and the elevated level of cytochrome c. An extension of incubation time led to an increase in intracellular calcium levels after treatment with DOX. Lower changes in the calcium content were associated with the influence of WP 631. DOX led to the activation of all tested caspases, 8, 9 and 3, whereas WP 631 only induced an increase in caspase 8 activity after 24h of treatment and consequently led to the cleavage of PARP. The lack of active caspase 3 had no outcome on the single and double-stranded DNA breaks. The obtained results show that WP 631 was considerably more genotoxic towards the investigated cell line than DOX. This effect was especially visible after longer times of incubation. The above detailed studies indicate that WP 631 generates early apoptosis and cell death independent of caspase-3, detected at relatively late time points. The observed differences in the mechanisms of the action of WP631 and DOX suggest that this bisanthracycline can be an effective alternative in ovarian cancer treatment.

Two drugs are better than one. A short history of combined therapy of ovarian cancer.

Combined therapy of ovarian cancer has a long history. It has been applied for many years. The first drug which was commonly combined with other chemotherapeutics was cisplatin. It turned out to be effective given together with alkylating agents as well as with taxanes. Another drug which is often the basis of first-line therapy is doxorubicin. The use of traditional chemotherapy is often limited due to side effects. This is why new drugs, targeted specifically at cancer cells (e.g. monoclonal antibodies or epidermal growth factor receptor inhibitors), offer a welcome addition when used in combination with conventional anticancer agents. Drugs applied in combination should be synergistic or at least additive. To evaluate the type of interaction between drugs in a plausible sequence, isobolographic analysis is used. This method allows one to assess whether the two agents could make an efficient combination, which might improve the therapy of ovarian cancer.

Caspases and ROS - dependent mechanism of action mediated by combination of WP 631 and epothilone B.

In this article, the synergistic effects of WP 631 and epothilone B (Epo B) combination in human ovarian cancer cells (SKOV-3) cells are investigated and the reasons for the exact mechanisms of action of both drugs co-administered are explained. Compared with single drugs, the combination treatment significantly enhances apoptosis as confirmed by increases in caspases (-8, -9, -3) activity, ROS level and DNA damage and decreases in mitochondrial membrane potential. The combination of the compounds activated both caspase - 8 and -9, indicates that both pathways of apoptosis, extrinsic (induced by the effect of Epo B) and intrinsic (triggered mainly by WP 631) participate in the proposed treatment. Thus, the results of this study strongly suggest a synergistic action of the combined treatment with WP 631 and Epo B in SKOV-3 cells death induction.

WP 631 and Epo B synergize in SKOV-3 human ovarian cancer cells.

Combined therapy is one of the basic methods of treatment different types of cancer. It allows to reduce the side effects of each component while maximizing the therapeutic action. The aim of this study was to evaluate the impact of two new drugs: WP 631 (bisanthracycline) and epothilone B (Epo B), added in combination on the SKOV-3 human ovarian cancer cells. To assess the type of interaction between WP 631 and Epo B isobolografic analysis was applied based on the cytotoxicity of drugs determined by the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolinum bromide) assay. Apoptotic and necrotic cell levels were measured by double staining with Hoechst 33258 and propidium iodide, Annexin V-FITC staining and by using TUNEL assay. The combination of WP 631 and Epo B is more potent than drugs added alone. The quantitative analysis indicated that the major mode of cell death induced by the combination after 72 h treatment was early apoptosis, whereas drugs administered alone generated less intensive apoptosis. The present report demonstrates for the first time that WP 631 and Epo B co-administered synergize in SKOV-3 cell line (Z(ex)/Z(th)<1).

Pro-apoptotic activity of new analog of anthracyclines--WP 631 in advanced ovarian cancer cell line.

In this work we investigated the mode of cell death induced by WP 631, a novel anthracycline antibiotic, in the ovarian cancer cell line (OV-90) derived from the malignant ascites of a patient diagnosed with advanced disease. The effects were compared with those of doxorubicin (DOX), a first generation anthracycline. The ability of WP 631 to induce apoptosis and necrosis was examined by double staining with Annexin V and propidium iodide, measurements of the level of intracellular calcium ions and cytochrome c, PARP cleavage. We also investigated the possible involvement of the caspases activation, DNA degradation (comet assay) and intracellular reactive oxygen species (ROS) production in the development of the apoptotic events and their significance for drug efficiency. The results obtained clearly demonstrate that antiproliferative capacity of WP 631 in tested cell line was a few times greater than that of DOX. Furthermore, ovarian cancer cells treated with WP 631 showed a higher mean level of basal DNA damage in comparison to DOX. In conclusion, WP 631 is able to induce caspase - dependent apoptosis in human ovarian cancer cells. Obtained results suggested that WP 631 may be a candidate for further evaluation as chemotherapeutic agents for human cancers.