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.

Application of Nanoparticles for Magnetic Hyperthermia for Cancer Treatment-The Current State of Knowledge.

Hyperthermia (HT) is an anti-cancer therapy commonly used with radio and chemotherapies based on applying heat (39-45 °C) to inhibit tumor growth. However, controlling heat towards tumors and not normal tissues is challenging. Therefore, nanoparticles (NPs) are used in HT to apply heat only to tumor tissues to induce DNA damage and the expression of heat shock proteins, which eventually result in apoptosis. The aim of this review article is to summarize recent advancements in HT with the use of magnetic NPs to locally increase temperature and promote cell death. In addition, the recent development of nanocarriers as NP-based drug delivery systems is discussed. Finally, the efficacy of HT combined with chemotherapy, radiotherapy, gene therapy, photothermal therapy, and immunotherapy is explored.

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.

New SDS-Based Polyelectrolyte Multicore Nanocarriers for Paclitaxel Delivery-Synthesis, Characterization, and Activity against Breast Cancer Cells.

The low distribution of hydrophobic anticancer drugs in patients is one of the biggest limitations during conventional chemotherapy. SDS-based polyelectrolyte multicore nanocarriers (NCs) prepared according to the layer by layer (LbL) procedure can release paclitaxel (PTX), and selectively kill cancer cells. Our main objective was to verify the antitumor properties of PTX-loaded NCs and to examine whether the drug encapsulated in these NCs retained its cytotoxic properties. The cytotoxicity of the prepared nanosystems was tested on MCF-7 and MDA-MB-231 tumour cells and the non-cancerous HMEC-1 cell line in vitro. Confocal microscopy, spectrophotometry, spectrofluorimetry, flow cytometry, and RT PCR techniques were used to define the typical hallmarks of apoptosis. It was demonstrated that PTX encapsulated in the tested NCs exhibited similar cytotoxicity to the free drug, especially in the triple negative breast cancer model. Moreover, SDS/PLL/PTX and SDS/PLL/PGA/PTX significantly reduced DNA synthesis. In addition, PTX-loaded NCs triggered apoptosis and upregulated the transcription of Bax, AIF, cytochrome-c, and caspase-3 mRNA. Our data demonstrate that these novel polyelectrolyte multicore NCs coated with PLL or PLL/PGA are good candidates for delivering PTX. Our discoveries have prominent implications for the possible choice of newly synthesized, SDS-based polyelectrolyte multicore NCs in different anticancer therapeutic applications.

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.

Newly Synthesized Melphalan Analogs Induce DNA Damage and Mitotic Catastrophe in Hematological Malignant Cancer Cells.

Myeloablative therapy with highdoses of the cytostatic drug melphalan (MEL) in preparation for hematopoietic cell transplantation is the standard of care for multiple myeloma (MM) patients. Melphalan is a bifunctional alkylating agent that covalently binds to nucleophilic sites in the DNA and effective in the treatment, but unfortunately has limited therapeutic benefit. Therefore, new approaches are urgently needed for patients who are resistant to existing standard treatment with MEL. Regulating the pharmacological activity of drug molecules by modifying their structure is one method for improving their effectiveness. The purpose of this work was to analyze the physicochemical and biological properties of newly synthesized melphalan derivatives (EE-MEL, EM-MEL, EM-MOR-MEL, EM-I-MEL, EM-T-MEL) obtained through the esterification of the carboxyl group and the replacement of the the amino group with an amidine group. Compounds were selected based on our previous studies for their improved anticancer properties in comparison with the original drug. For this, we first evaluated the physicochemical properties using the circular dichroism technique, then analyzed the zeta potential and the hydrodynamic diameters of the particles. Then, the in vitro biological properties of the analogs were tested on multiple myeloma (RPMI8226), acute monocytic leukemia (THP1), and promyelocytic leukemia (HL60) cells as model systems for hematological malignant cells. DNA damage was assessed by immunostaining γH2AX, cell cycle distribution changes by propidium iodide (PI) staining, and cell death by the activation of caspase 2. We proved that the newly synthesized derivatives, in particular EM-MOR-MEL and EM-T-MEL, affected the B-DNA conformation, thus increasing the DNA damage. As a result of the DNA changes, the cell cycle was arrested in the S and G2/M phases. The cell death occurred by activating a mitotic catastrophe. Our investigations suggest that the analogs EM-MOR-MEL and EM-T-MEL have better anti-cancer activity in multiple myeloma cells than the currently used melphalan.

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.

Hexokinase 2 Inhibition and Biological Effects of BNBZ and Its Derivatives: The Influence of the Number and Arrangement of Hydroxyl Groups.

Hexokinase 2 (HK2), an enzyme of the sugar kinase family, plays a dual role in glucose metabolism and mediating cancer cell apoptosis, making it an attractive target for cancer therapy. While positive HK2 expression usually promotes cancer cells survival, silencing or inhibiting this enzyme has been found to improve the effectiveness of anti-cancer drugs and even result in cancer cell death. Previously, benitrobenrazide (BNBZ) was characterized as a potent HK2 inhibitor with good anti-cancer activity in mice, but the effect of its trihydroxy moiety (pyrogallol-like) on inhibitory activity and some cellular functions has not been fully understood. Therefore, the main goal of this study was to obtain the parent BNBZ (2a) and its three dihydroxy derivatives 2b-2d and to conduct additional physicochemical and biological investigations. The research hypothesis assumed that the HK2 inhibitory activity of the tested compounds depends on the number and location of hydroxyl groups in their chemical structure. Among many studies, the binding affinity to HK2 was determined and two human liver cancer cell lines, HepG2 and HUH7, were used and exposed to chemicals at various times: 24 h, 48 h and 72 h. The study showed that the modifications to the structures of the new BNBZ derivatives led to significant changes in their activities. It was also found that these compounds tend to aggregate and exhibit toxic effects. They were found to contribute to: (a) DNA damage, (b) increased ROS production, and (c) disruption of cell cycle progression. It was observed that, HepG2, occurred much more sensitive to the tested chemicals than the HUH7 cells; However, regardless of the used cell line it seems that the increase in the expression of HK2 in cancer cells compared to normal cells which have HK2 at a very low level, is a serious obstacle in anti-cancer therapy and efforts to find the effective inhibitors of this enzyme should be intensified.

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.

Two Faces of Autophagy in the Struggle against Cancer.

Autophagy can play a double role in cancerogenesis: it can either inhibit further development of the disease or protect cells, causing stimulation of tumour growth. This phenomenon is called "autophagy paradox", and is characterised by the features that the autophagy process provides the necessary substrates for biosynthesis to meet the cell's energy needs, and that the over-programmed activity of this process can lead to cell death through apoptosis. The fight against cancer is a difficult process due to high levels of resistance to chemotherapy and radiotherapy. More and more research is indicating that autophagy may play a very important role in the development of resistance by protecting cancer cells, which is why autophagy in cancer therapy can act as a "double-edged sword". This paper attempts to analyse the influence of autophagy and cancer stem cells on tumour development, and to compare new therapeutic strategies based on the modulation of these processes.

Treatment of Multiple Myeloma and the Role of Melphalan in the Era of Modern Therapies-Current Research and Clinical Approaches.

Multiple myeloma (MM) accounts for 10% of all hematological malignancies, and it is the second most common hematological neoplasm for which chemotherapy is an important pharmacological treatment. High dose melphalan followed by autologous stem cell transplantation remains the standard of treatment for transplant-eligible patients with MM. In this review, we describe aspects of the pharmacokinetics and pharmacodynamics of melphalan therapy and related compounds. In addition, we describe the use of melphalan in innovative therapies for the treatment of MM, including the development of drug carriers to reduce systemic toxicity, combination therapy to improve the effectiveness of cancer therapy, and the chemical modification of the melphalan molecule to improve antitumor activity.

Doxorubicin-transferrin conjugate alters mitochondrial homeostasis and energy metabolism in human breast cancer cells.

Doxorubicin (DOX) is considered one of the most powerful chemotherapeutic agents but its clinical use has several limitations, including cardiomyopathy and cellular resistance to the drug. By using transferrin (Tf) as a drug carrier, however, the adverse effects of doxorubicin as well as drug resistance can be reduced. The main objective of this study was to determine the exact nature and extent to which mitochondrial function is influenced by DOX-Tf conjugate treatment, specifically in human breast adenocarcinoma cells. We assessed the potential of DOX-Tf conjugate as a drug delivery system, monitoring its cytotoxicity using the MTT assay and ATP measurements. Moreover, we measured the alterations of mitochondrial function and oxidative stress markers. The effect of DOX-Tf was the most pronounced in MDA-MB-231, triple-negative breast cancer cells, whereas non-cancer endothelial HUVEC-ST cells were more resistant to DOX-Tf conjugate than to free DOX treatment. A different sensitivity of two investigate breast cancer cell lines corresponded to the functionality of their cellular antioxidant systems and expression of estrogen receptors. Our data also revealed that conjugate treatment mediated free radical generation and altered the mitochondrial bioenergetics in breast cancer cells.

Disturbance of cellular homeostasis as a molecular risk evaluation of human endothelial cells exposed to nanoparticles.

Even though application of nanoparticles in medicine seems to provide unique solutions for drug delivery and diagnosis diseases, understanding interactions between nanoscale materials and biological systems is imperative. Therefore, this study determined the effect of different types of nanoparticles (NPs) on human endothelial cells and examined the types of toxicity responses they can induce. Four different types of NPs were tested (PLA/MMT/TRASTUZUMAB, PLA/EDTMP, PLGA/MDP, and Pluronic F127 MICELLES), representing three putative areas of application: anticancer therapy, scintigraphy, and cosmetology. The experiments were performed on immortalized human umbilical vein endothelial cells (HUVEC-STs). Light contrast phase microscopy as well as cell viability assays showed that only Pluronic F127 MICELLES decreased the number of HUVEC-STs in contrast to PLA/MMT/TRASTUZUMAB, PLA/EDTMP, and PLGA/MDP NPs, which altered cell morphology, but not their confluency. The tested NPs induced not only DNA strand-breaks and alkali-labile sites, but also internucleosomal DNA fragmentation, visualized as a DNA ladder pattern typical of apoptosis. Moreover, generation of free radicals and subsequent mitochondrial membrane potential collapse showed the significance of free radical production during interactions between NPs and endothelial cells. High concentrations of NPs had different degrees of toxicity in human endothelial cells and affected cell proliferation, redox homeostasis, and triggered mitochondrial dysfunction.

Transferrin-Bound Doxorubicin Enhances Apoptosis and DNA Damage through the Generation of Pro-Inflammatory Responses in Human Leukemia Cells.

Doxorubicin (DOX) is an effective antineoplastic drug against many solid tumors and hematological malignancies. However, the clinical use of DOX is limited, because of its unspecific mode of action. Since leukemia cells overexpress transferrin (Tf) receptors on their surface, we proposed doxorubicin-transferrin (DOX-Tf) conjugate as a new vehicle to increase drug concentration directly in cancer cells. The data obtained after experiments performed on K562 and CCRF-CEM human leukemia cell lines clearly indicate severe cytotoxic and genotoxic properties of the conjugate drug. On the other hand, normal peripheral blood mononuclear cells (PBMCs) were more resistant to DOX-Tf than to DOX. In comparison to free drug, we observed that Tf-bound DOX induced apoptosis in a TRAIL-dependent manner and caused DNA damage typical of programmed cell death. These fatal hallmarks of cell death were confirmed upon morphological observation of cells incubated with DOX or DOX-Tf. Studies of expression of TNF-α, IL-4, and IL-6 at the mRNA and protein levels revealed that the pro-inflammatory response plays an important role in the toxicity of the conjugate. Altogether, the results demonstrated here describe a mechanism of the antitumor activity of the DOX-Tf conjugate.

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.