Uncovering miRNA-mRNA Regulatory Networks Related to Olaparib Resistance and Resensitization of BRCA2MUT Ovarian Cancer PEO1-OR Cells with the ATR/CHK1 Pathway Inhibitors.

Resistance to olaparib is the major obstacle in targeted therapy for ovarian cancer (OC) with poly(ADP-ribose) polymerase inhibitors (PARPis), prompting studies on novel combination therapies to enhance olaparib efficacy. Despite identifying various mechanisms, understanding how OC cells acquire PARPi resistance remains incomplete. This study investigated microRNA (miRNA) expression in olaparib-sensitive (PEO1, PEO4) and previously established olaparib-resistant OC cell lines (PEO1-OR) using high-throughput RT-qPCR and bioinformatic analyses. The role of miRNAs was explored regarding acquired resistance and resensitization with the ATR/CHK1 pathway inhibitors. Differentially expressed miRNAs were used to construct miRNA-mRNA regulatory networks and perform functional enrichment analyses for target genes with miRNet 2.0. TCGA-OV dataset was analyzed to explore the prognostic value of selected miRNAs and target genes in clinical samples. We identified potential processes associated with olaparib resistance, including cell proliferation, migration, cell cycle, and growth factor signaling. Resensitized PEO1-OR cells were enriched in growth factor signaling via PDGF, EGFR, FGFR1, VEGFR2, and TGFßR, regulation of the cell cycle via the G2/M checkpoint, and caspase-mediated apoptosis. Antibody microarray analysis confirmed dysregulated growth factor expression. The addition of the ATR/CHK1 pathway inhibitors to olaparib downregulated FGF4, FGF6, NT-4, PLGF, and TGFß1 exclusively in PEO1-OR cells. Survival and differential expression analyses for serous OC patients revealed prognostic miRNAs likely associated with olaparib resistance (miR-99b-5p, miR-424-3p, and miR-505-5p) and resensitization to olaparib (miR-324-5p and miR-424-3p). Essential miRNA-mRNA interactions were reconstructed based on prognostic miRNAs and target genes. In conclusion, our data highlight distinct miRNA profiles in olaparib-sensitive and olaparib-resistant cells, offering molecular insights into overcoming resistance with the ATR/CHK1 inhibitors in OC. Moreover, some miRNAs might serve as potential predictive signature molecules of resistance and therapeutic response.

Aggregation/Crystallization-Induced Emission in Naphthyridine-Based Carbazolyl-Modified Donor-Acceptor Boron Dyes Tunable by Fluorine Atoms.

Four donor-acceptor boron difluoride complexes based on the carbazole electron donor and the [1,3,5,2]oxadiazaborinino[3,4-a][1,8]naphthyridine acceptor were designed, synthesized, and systematically spectroscopically investigated in solutions, in dye-doped polymer films, and in the solid states. The dyes exhibit an intense blue to red solid-state emission with photoluminescence quantum yields of up to 59 % in pure dye samples and 86 % in poly(methyl methacrylate) films. All boron complexes show aggregation-induced emission and reversible mechanofluorochromism. The optical properties of these dyes and their solid state luminescence can be tuned by substitution pattern, i. e., the substituents at the naphthyridine unit. Exchange of CH3- for CF3-groups does not only increase the intramolecular charge transfer character, but also provides a crystallization-induced emission enhancement.

Targeted inhibition of the ATR/CHK1 pathway overcomes resistance to olaparib and dysregulates DNA damage response protein expression in BRCA2MUT ovarian cancer cells.

Olaparib is a PARP inhibitor (PARPi) approved for targeted treatment of ovarian cancer (OC). However, its efficacy is impeded by the inevitable occurrence of resistance. Here, we investigated whether the cytotoxic activity of olaparib could be synergistically enhanced in olaparib-resistant OC cells with BRCA2 reversion mutation by the addition of inhibitors of the ATR/CHK1 pathway. Moreover, we provide insights into alterations in the DNA damage response (DDR) pathway induced by combination treatments. Antitumor activity of olaparib alone or combined with an ATR inhibitor (ATRi, ceralasertib) or CHK1 inhibitor (CHK1i, MK-8776) was evaluated in OC cell lines sensitive (PEO1, PEO4) and resistant (PEO1-OR) to olaparib. Antibody microarrays were used to explore changes in expression of 27 DDR-related proteins. Olaparib in combination with ATR/CHK1 inhibitors synergistically induced a decrease in viability and clonogenic survival and an increase in apoptosis mediated by caspase-3/7 in all OC cells. Combination treatments induced cumulative alterations in expression of DDR-related proteins mediating distinct DNA repair pathways and cell cycle control. In the presence of ATRi and CHK1i, olaparib-induced upregulation of proteins determining cell fate after DNA damage (PARP1, CHK1, c-Abl, Ku70, Ku80, MDM2, and p21) was abrogated in PEO1-OR cells. Overall, the addition of ATRi or CHK1i to olaparib effectively overcomes resistance to PARPi exerting anti-proliferative effect in BRCA2MUT olaparib-resistant OC cells and alters expression of DDR-related proteins. These new molecular insights into cellular response to olaparib combined with ATR/CHK1 inhibitors might help improve targeted therapies for olaparib-resistant OC.

Olaparib-Resistant BRCA2MUT Ovarian Cancer Cells with Restored BRCA2 Abrogate Olaparib-Induced DNA Damage and G2/M Arrest Controlled by the ATR/CHK1 Pathway for Survival.

The PARP inhibitor (PARPi) olaparib is currently the drug of choice for serous ovarian cancer (OC), especially in patients with homologous recombination (HR) repair deficiency associated with deleterious BRCA1/2 mutations. Unfortunately, OC patients who fail to respond to PARPi or relapse after treatment have limited therapeutic options. To elucidate olaparib resistance and enhance the efficacy of olaparib, intracellular factors exploited by OC cells to achieve decreased sensitivity to PARPi were examined. An olaparib-resistant OC cell line, PEO1-OR, was established from BRCA2MUT PEO1 cells. The anticancer activity and action of olaparib combined with inhibitors of the ATR/CHK1 pathway (ceralasertib as ATRi, MK-8776 as CHK1i) in olaparib-sensitive and -resistant OC cell lines were evaluated. Whole-exome sequencing revealed that PEO1-OR cells acquire resistance through subclonal enrichment of BRCA2 secondary mutations that restore functional full-length protein. Moreover, PEO1-OR cells upregulate HR repair-promoting factors (BRCA1, BRCA2, RAD51) and PARP1. Olaparib-inducible activation of the ATR/CHK1 pathway and G2/M arrest is abrogated in olaparib-resistant cells. Drug sensitivity assays revealed that PEO1-OR cells are less sensitive to ATRi and CHK1i agents. Combined treatment is less effective in olaparib-resistant cells considering inhibition of metabolic activity, colony formation, survival, accumulation of DNA double-strand breaks, and chromosomal aberrations. However, synergistic antitumor activity between compounds is achievable in PEO1-OR cells. Collectively, olaparib-resistant cells display co-existing HR repair-related mechanisms that confer resistance to olaparib, which may be effectively utilized to resensitize them to PARPi via combination therapy. Importantly, the addition of ATR/CHK1 pathway inhibitors to olaparib has the potential to overcome acquired resistance to PARPi.

The Influence of PARP, ATR, CHK1 Inhibitors on Premature Mitotic Entry and Genomic Instability in High-Grade Serous BRCAMUT and BRCAWT Ovarian Cancer Cells.

Olaparib is a poly (ADP-ribose) polymerase inhibitor (PARPi) that inhibits PARP1/2, leading to replication-induced DNA damage that requires homologous recombination repair. Olaparib is often insufficient to treat BRCA-mutated (BRCAMUT) and BRCA wild-type (BRCAWT) high-grade serous ovarian carcinomas (HGSOCs). We examined the short-term (up to 48 h) efficacy of PARPi treatment on a DNA damage response pathway mediated by ATR and CHK1 kinases in BRCAMUT (PEO-1) and BRCAWT (SKOV-3 and OV-90) cells. The combination of ATRi/CHK1i with PARPi was not more cytotoxic than ATR and CHK1 monotherapy. The combination of olaparib with inhibitors of the ATR/CHK1 pathway generated chromosomal abnormalities, independent on BRCAMUT status of cells and formed of micronuclei (MN). However, the beneficial effect of the PARPi:ATRi combination on MN was seen only in the PEO1 BRCAMUT line. Monotherapy with ATR/CHK1 inhibitors reduced BrdU incorporation due to a slower rate of DNA synthesis, which resulted from elevated levels of replication stress, while simultaneous blockade of PARP and ATR caused beneficial effects only in OV-90 cells. Inhibition of ATR/CHK1 increased the formation of double-strand breaks as measured by increased γH2AX expression at collapsed replication forks, resulting in increased levels of apoptosis. Our findings indicate that ATR and CHK1 inhibitors provoke premature mitotic entry, leading to genomic instability and ultimately cell death.

Synthesis and In Vitro Activity of Novel Melphalan Analogs in Hematological Malignancy Cells.

Despite the continuous developments in pharmacology and the high therapeutic effect of new treatment options for patients with hematological malignancies, these diseases remain a major health issue. Our study aimed to synthesize, analyze in silico, and determine the biological properties of new melphalan derivatives. We obtained three methyl esters of melphalan having in their structures amidine moieties substituted with thiomorpholine (EM-T-MEL), indoline (EM-I-MEL), or 4-(4-morpholinyl) piperidine (EM-MORPIP-MEL). These have not yet been described in the literature. The in vitro anticancer properties of the analogs were determined against THP1, HL60, and RPMI8226 cells. Melphalan derivatives were evaluated for cytotoxicity (resazurin viability assay), genotoxicity (alkaline comet assay), and their ability to induce apoptosis (Hoechst33342/propidium iodide double staining method; phosphatidylserine translocation; and caspase 3/7, 8, and 9 activity measurements). Changes in mitochondrial membrane potential were examined using the specific fluorescence probe JC-1 (5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazol carbocyanine). The EM-T-MEL derivative had the highest biological activity, showing higher cytotoxic and genotoxic properties than the parent drug. Moreover, it showed a high ability to induce apoptosis in the tested cancer cells. This compound also had a beneficial effect in peripheral blood mononuclear cells (PBMC). In conclusion, we verified and confirmed the hypothesis that chemical modifications of the melphalan structure improved its anticancer properties. The conducted study allowed the selection of the compound with the highest biological activity and provided a basis for chemical structure-biological activity analyses.

Metformin Affects Olaparib Sensitivity through Induction of Apoptosis in Epithelial Ovarian Cancer Cell Lines.

This study examined the effect of combination treatment with the poly (ADP-ribose) polymerase inhibitor olaparib and metformin on homologous recombination (HR)-proficient epithelial ovarian cancer (EOC). Ovarian cancer cell lines (OV-90 and SKOV-3) were treated with olaparib, metformin, or a combination of both. Cell viability was assessed by MTT and colony formation assays. The production of reactive oxygen species (ROS) and changes in mitochondrial membrane potential were examined using the specific fluorescence probes, DCFH2-DA (2',7'-dichloro-dihydrofluorescein diacetate) and JC-1 (5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolcarbocyanine). Apoptotic and necrotic changes were measured by double staining with Hoechst 33258 and propidium iodide, orange acridine and ethidium bromide staining, phosphatidylserine externalization, TUNEL assay, caspase 3/7 activity, and cytochrome c and p53 expression. Compared with single-drug treatment, the combination of olaparib and metformin significantly inhibited cell proliferation and colony formation in HR-proficient ovarian cancer cells. ROS production preceded a decrease in mitochondrial membrane potential. The changes in ROS levels suggested their involvement in inducing apoptosis in response to combination treatment. The present results indicate a shift towards synergism in cells with mutant or null p53, treated with olaparib combined with metformin, providing a new approach to the treatment of gynecologic cancers. Taken together, the results support the use of metformin to sensitize EOC to olaparib therapy.

PARP inhibitor resistance in ovarian cancer: Underlying mechanisms and therapeutic approaches targeting the ATR/CHK1 pathway.

Ovarian cancer (OC) constitutes the most common cause of gynecologic cancer-related death in women worldwide. Despite consistent developments in treatment strategies for OC, the management of advanced-stage disease remains a significant challenge. Recent improvements in targeted treatments based on poly(ADP-ribose) polymerase (PARP) inhibitors (PARPi) have provided invaluable benefits to patients with OC. Unfortunately, numerous patients do not respond to PARPi due to intrinsic resistance or acquisition of resistance. Here, we discuss mechanisms of resistance to PARPi that have specifically emerged in OC including increased drug efflux, restoration of HR repair, re-establishment of replication fork stability, reduced PARP1 trapping, abnormalities in PARP signaling, and less common pathways associated with alternative DNA sensing and repair pathways. Elucidation of the precise mechanisms is essential for the development of novel strategies to re-sensitize OC cells to PARPi agents. Additionally, novel potential concepts for preventing and combating resistance to PARPi under development and relevant clinical reports on treatment strategies have been reviewed, with emphasis on the exploitation of the ATR/CHK1 kinase pathway in sensitization to PARPi to overcome resistance-induced vulnerability in ovarian cancer.

Current Implications of microRNAs in Genome Stability and Stress Responses of Ovarian Cancer.

Genomic alterations and aberrant DNA damage signaling are hallmarks of ovarian cancer (OC), the leading cause of mortality among gynecological cancers worldwide. Owing to the lack of specific symptoms and late-stage diagnosis, survival chances of patients are significantly reduced. Poly (ADP-ribose) polymerase (PARP) inhibitors and replication stress response inhibitors present attractive therapeutic strategies for OC. Recent research has focused on ovarian cancer-associated microRNAs (miRNAs) that play significant regulatory roles in various cellular processes. While miRNAs have been shown to participate in regulation of tumorigenesis and drug responses through modulating the DNA damage response (DDR), little is known about their potential influence on sensitivity to chemotherapy. The main objective of this review is to summarize recent findings on the utility of miRNAs as cancer biomarkers, in particular, ovarian cancer, and their regulation of DDR or modified replication stress response proteins. We further discuss the suppressive and promotional effects of various miRNAs on ovarian cancer and their participation in cell cycle disturbance, response to DNA damage, and therapeutic functions in multiple cancer types, with particular focus on ovarian cancer. Improved understanding of the mechanisms by which miRNAs regulate drug resistance should facilitate the development of effective combination therapies for ovarian cancer.

PARP Inhibition Increases the Reliance on ATR/CHK1 Checkpoint Signaling Leading to Synthetic Lethality-An Alternative Treatment Strategy for Epithelial Ovarian Cancer Cells Independent from HR Effectiveness.

Poly (ADP-ribose) polymerase inhibitor (PARPi, olaparib) impairs the repair of DNA single-strand breaks (SSBs), resulting in double-strand breaks (DSBs) that cannot be repaired efficiently in homologous recombination repair (HRR)-deficient cancers such as BRCA1/2-mutant cancers, leading to synthetic lethality. Despite the efficacy of olaparib in the treatment of BRCA1/2 deficient tumors, PARPi resistance is common. We hypothesized that the combination of olaparib with anticancer agents that disrupt HRR by targeting ataxia telangiectasia and Rad3-related protein (ATR) or checkpoint kinase 1 (CHK1) may be an effective strategy to reverse ovarian cancer resistance to olaparib. Here, we evaluated the effect of olaparib, the ATR inhibitor AZD6738, and the CHK1 inhibitor MK8776 alone and in combination on cell survival, colony formation, replication stress response (RSR) protein expression, DNA damage, and apoptotic changes in BRCA2 mutated (PEO-1) and HRR-proficient BRCA wild-type (SKOV-3 and OV-90) cells. Combined treatment caused the accumulation of DNA DSBs. PARP expression was associated with sensitivity to olaparib or inhibitors of RSR. Synergistic effects were weaker when olaparib was combined with CHK1i and occurred regardless of the BRCA2 status of tumor cells. Because PARPi increases the reliance on ATR/CHK1 for genome stability, the combination of PARPi with ATR inhibition suppressed ovarian cancer cell growth independently of the efficacy of HRR. The present results were obtained at sub-lethal doses, suggesting the potential of these inhibitors as monotherapy as well as in combination with olaparib.

Participation of the ATR/CHK1 pathway in replicative stress targeted therapy of high-grade ovarian cancer.

Ovarian cancer is one of the most lethal gynecologic malignancies reported throughout the world. The initial, standard-of-care, adjuvant chemotherapy in epithelial ovarian cancer is usually a platinum drug, such as cisplatin or carboplatin, combined with a taxane. However, despite surgical removal of the tumor and initial high response rates to first-line chemotherapy, around 80% of women will develop cancer recurrence. Effective strategies, including chemotherapy and new research models, are necessary to improve the prognosis. The replication stress response (RSR) is characteristic of the development of tumors, including ovarian cancer. Hence, RSR pathway and DNA repair proteins have emerged as a new area for anticancer drug development. Although clinical trials have shown poly (ADP-ribose) polymerase inhibitors (PARPi) response rates of around 40% in women who carry a mutation in the BRCA1/2 genes, PARPi is responsible for tumor suppression, but not for complete tumor regression. Recent reports suggest that cells with impaired homologous recombination (HR) activities due to mutations in TP53 gene or specific DNA repair proteins are specifically sensitive to ataxia telangiectasia and Rad3-related protein (ATR) inhibitors. Replication stress activates DNA repair checkpoint proteins (ATR, CHK1), which prevent further DNA damage. This review describes the use of DNA repair checkpoint inhibitors as single agents and strategies combining these inhibitors with DNA-damaging compounds for ovarian cancer therapy, as well as the new platforms used for optimizing ovarian cancer therapy.

Chemical modification of melphalan as a key to improving treatment of haematological malignancies.

Chemical modification of known, effective drugs is one method to improve chemotherapy. Thus, the object of this study was to generate melphalan derivatives with improved cytotoxic activity in human cancer cells (RPMI8226, HL60 and THP1). Several melphalan derivatives were synthesised, modified in their two important functional groups. Nine analogues were tested, including melphalan compounds modified: only at the amino group, by replacing the amine with an amidine group containing a morpholine ring (MOR-MEL) or with an amidino group and dipropyl chain (DIPR-MEL); only at the carboxyl group to form methyl and ethyl esters of melphalan (EM-MEL, EE-MEL); and in a similar manner at both functional groups (EM-MOR-MEL, EE-MOR-MEL, EM-DIPR-MEL, EE-DIPR-MEL). Melphalan derivatives were evaluated for cytotoxicity (resazurin viability assay), genotoxicity (comet assay) and the ability to induce apoptosis (terminal deoxynucleotidyl transferase dUTP nick end labelling, TUNEL, phosphatidylserine externalisation, chromatin condensation, activity of caspases 3/7, 8 and 9 and intracellular concentration of calcium ions) in comparison with the parent drug. Almost all derivatives, with the exception of MOR-MEL and DIPR-MEL, were found to be more toxic than melphalan in all cell lines evaluated. Treatment of cultures with the derivatives generated a significant higher level of DNA breaks compared to those treated with melphalan, especially after longer incubation times. In addition, all the melphalan derivatives demonstrated a high apoptosis-inducing ability in acute monocytic and promyelocytic leukemia cells. This study showed that the mechanism of action of the tested compounds differed depending on the cell line, and allowed the selection of the most active compounds for further, more detailed investigations.

(1-4)-Thiodisaccharides as anticancer agents. Part 5. Evaluation of anticancer activity and investigation of mechanism of action.

(1-4)-Thiodisaccharides, thiosugars with the 1-4-thio bridge, were recently shown to induce oxidative stress, as well as, apoptosis in cancer cells in the low micromolar range; however, the detailed mechanism of their anticancer action still remains unknown. In order to clarify the mechanism of (1-4)- thiodisaccharides action, we performed a series of tests including cytotoxic, clonogenic and apoptosis assays using an in vitro glioma cancer model with one ATCC cell line U87 and two novel glioma cell lines derived from cancer patients - H6PX and H7PX. We also evaluated the ability of (1-4)-thiodisaccharides to interfere with protein folding and synthesis processes, as well as, the thioredoxin system. (1-4)-thiodisaccharides induced glioma cell death, which were found to be accompanied with endoplasmic reticulum stress, inhibition of global protein synthesis, reduced overall cellular thiol level and thioredoxin reductase activity. We also performed a RT-PCR and Elisa analysis of (1-4)-thiodisaccharides-treated glioma cells to identify any changes within the pathway affected by (1-4)-thiodisaccharides. We observed a significant increase of expression in key markers of endoplasmic reticulum stress and pro-apoptotic protein, FASLG. We proposed that (1-4)-thiodisaccharides react with cellular thiols and disturb any cellular thiol-depended processes like thioredoxin system or protein folding.

Application of new lysine-based peptide dendrimers D3K2 and D3G2 for gene delivery: Specific cytotoxicity to cancer cells and transfection in vitro.

In order to enhance intracellular uptake and accumulation of therapeutic nucleic acids for improved gene therapy methods, numerous delivery vectors have been elaborated. Based on their origin, gene carriers are generally classified as viral or non-viral vectors. Due to their significantly reduced immunogenicity and highly optimized methods of synthesis, nanoparticles (especially those imitating natural biomolecules) constitute a promising alternative for virus-based delivery devices. Thus, we set out to develop innovative peptide dendrimers for clinical application as transfection agents and gene carriers. In the present work we describe the synthesis of two novel lysine-based dendritic macromolecules (D3K2 and D3G2) and their initial characterization for cytotoxicity/genotoxicity and transfection potential in two human cell line models: cervix adenocarcinoma (HeLa) and microvascular endothelial (HMEC-1). This approach allowed us to identify more cationic D3K2 as potent delivery agent, being able to increase intracellular accumulation of large nucleic acid molecules such as plasmids. Moreover, the dendrimers exhibited specific cytotoxicity towards cancer cell line without showing significant toxic effects on normal cells. These observations are promising prognosis for future clinical application of this type of nanoparticles.

Molecular Mechanisms of Antitumor Activity of PAMAM Dendrimer Conjugates with Anticancer Drugs and a Monoclonal Antibody.

Taxanes are considered fundamental drugs in the treatment of breast cancer, but despite the similarities, docetaxel (doc) and paclitaxel (ptx) work differently. For this reason, it is interesting to identify mechanisms of antitumor activity of PAMAM dendrimer conjugates that carry docetaxel or paclitaxel and monoclonal antibody trastuzumab, specifically targeted to cells which overexpressed HER-2. For this purpose, the impact on the level of reactive oxygen species, the mitochondrial membrane potential, cell cycle distribution and the activity of caspases-3/7, -8 and -9 of PAMAM-doc-trastuzumab and PAMAM-ptx-trastuzumab conjugates was determined and compared with free docetaxel and paclitaxel toward HER-2-positive (SKBR-3) and negative (MCF-7) human breast cancer cell lines. Moreover, apoptosis and necrosis were studied using flow cytometry and confocal microscopy, respectively. Our studies show the complexity of the potential mechanism of cytotoxic action of PAMAM-drug-trastuzumab conjugates that should be sought as a resultant of oxidative stress, mitochondrial activation of the caspase cascade and the HER-2 receptor blockade.

Suppression of autophagy enhances preferential toxicity of epothilone A and epothilone B in ovarian cancer cells.

BACKGROUND: Epothilones are microtubule-targeting agents that induce death in a variety of cancer cell types. Here, we focus on the cellular and molecular mechanisms underlying epothilone A (Epo A) and epothilone B (Epo B)-induced autophagy and apoptosis in ovarian cancer cells, compared to the actions of the widely used clinical chemotherapy drug paclitaxel (PTX). MATERIALS AND METHODS: Autophagy was examined in two cell lines, SKOV-3 (human ovarian adenocarcinoma) and OV-90 (human ovarian papillary serous adenocarcinoma), which differ in the levels of p-glycoprotein and drug resistance, based on the LC3 ELISA assay, fluorescence detection of autophagosome formation, morphological changes evaluated via acridine orange staining, and visualization of LC3 protein using confocal microscopy. Cell viability was detected using the 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide (MTT) assay. Apoptosis was measured via the caspase-3/7 assay and immunofluorescence labeling of caspase-3. Differences in microtubule organization in epothilone-treated cells were investigated using specific antibodies against ß-tubulin. All probes were analyzed both in the presence and absence of the autophagy inhibitor, bafilomycin A1 (Baf), and apoptosis inhibitor, Z-FA-FMK. RESULTS: Epothilone and PTX treatment induced a dose-dependent decrease in cell viability, along with increased apoptosis and disruption of microtubule dynamics. Furthermore, under conditions of inhibition of autophagy with Baf, apoptosis triggered by these compounds was significantly increased. CONCLUSION: Our collective results suggest that treatment with epothilones in combination with autophagy inhibitors present a potentially more effective chemotherapeutic approach for ovarian cancer.

Aclarubicin in subtoxic doses reduces doxorubicin cytotoxicity in human non-small cell lung adenocarcinoma (A549) and human hepatocellular carcinoma (HepG2) cells by decreasing DNA damage.

In the study, the cytotoxicity and genotoxicity of aclarubicin (ACL) against A549 (human non-small cell lung adenocarcinoma) and HepG2 (human hepatocellular carcinoma) cell lines were evaluated and compared with that of doxorubicin (DOX). The effect of both anthracyclines in combination was also investigated. In order to get a deeper insight into the effectiveness of the drugs and their combination, their effects on the DNA damage and distribution of the cell cycle of A549 and HepG2 cells were investigated. After treatment with investigated compounds, apoptotic and necrotic morphological changes were estimated by double staining cells with orange acridine and ethidium bromide. The results showed that ACL was much more cytotoxic against lung (A549) and liver (HepG2) cancer cell lines than DOX. However, the drugs affected the cell cycle differently. ACL arrested cells in the G1 phase, while DOX arrested them in the G2/M phase. DOX and ACL at high concentrations are able to trigger apoptosis in both A549 and HepG2 cells. When the drugs were used in combination, subtoxic concentrations of ACL antagonized the cytotoxic effects of doxorubicin. Pre-incubation of cells with subtoxic concentrations of ACL reduced the level of DNA damage by DOX but increased DOX genotoxicity in the presence of verapamil.

Novel oxazolinoanthracyclines as tumor cell growth inhibitors-Contribution of autophagy and apoptosis in solid tumor cells death.

Chemical modification of known, effective drugs are one method to improve the chemotherapy of tumors. We reported ability of oxazoline analogs of doxorubicin (O-DOX) and daunorubicin (O-DAU) to induce apoptosis and autophagy in ovarian and liver cancer cells. Reactive oxygen and nitrogen species (ROS and RNS, respectively), together with intracellular calcium-mediated downstream signaling, are essential for the anticancer effect of these new anthracycline analogs. The changes of mitochondrial membrane potential and induction of the ceramide pathway suggests that these compounds induce cell death by apoptosis. In addition, a significant increase of autophagosome formation was observed by fluorescence assay and acridine orange staining, indicating that the new analogs also induce autophagic cell death. Compared to free DOX- and DAU-treated cells, we observed inhibition of colony formation and migration, a time-dependency between ROS/RNS levels and a greater fall in mitochondrial membrane potential. Altogether, our research broadens the base of molecular oxazolinoanthracyclines targets and reveals that derivatives mediated oxidative stress, ceramide production and increase in intracellular calcium level by mitochondria. Furthermore, our data highlight the importance of mitochondria that simultaneously assume the role of activator of autophagy and apoptosis signals.

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.