Secreted Soluble Factors from Tumor-Activated Mesenchymal Stromal Cells Confer Platinum Chemoresistance to Ovarian Cancer Cells.

Ovarian cancer (OC) ranks as the second most common type of gynecological malignancy, has poor survival rates, and is frequently diagnosed at an advanced stage. Platinum-based chemotherapy, such as carboplatin, represents the standard-of-care for OC. However, toxicity and acquired resistance to therapy have proven challenging for the treatment of patients. Chemoresistance, a principal obstacle to durable response in OC patients, is attributed to alterations within the cancer cells, and it can also be mediated by the tumor microenvironment (TME). In this study, we report that conditioned medium (CM) derived from murine and human stromal cells, MS-5 and HS-5, respectively, and tumor-activated HS-5, was active in conferring platinum chemoresistance to OC cells. Moreover, CM derived from differentiated murine pre-adipocyte (3T3-L1), but not undifferentiated pre-adipocyte cells, confers platinum chemoresistance to OC cells. Interestingly, CM derived from tumor-activated HS-5 was more effective in conferring chemoresistance than was CM derived from HS-5 cells. Various OC cells exhibit variable sensitivity to CM activity. Exploring CM content revealed the enrichment of a number of soluble factors in the tumor-activated HS-5, such as soluble uPAR (SuPAR), IL-6, and hepatocyte growth factor (HGF). FDA-approved JAK inhibitors were mildly effective in restoring platinum sensitivity in two of the three OC cell lines in the presence of CM. Moreover, Crizotinib, an ALK and c-MET inhibitor, in combination with platinum, blocked HGF's ability to promote platinum resistance and to restore platinum sensitivity to OC cells. Finally, exposure to 2-hydroxyestardiol (2HE2) was effective in restoring platinum sensitivity to OC cells exposed to CM. Our results showed the significance of soluble factors found in TME in promoting platinum chemoresistance and the potential of combination therapy to restore chemosensitivity to OC cells.

2-Hydroxyestradiol Overcomes Mesenchymal Stem Cells-Mediated Platinum Chemoresistance in Ovarian Cancer Cells in an ERK-Independent Fashion.

Ovarian cancer (OC) is the second most common type of gynecological malignancy. Platinum (Pt)-based chemotherapy is the standard of care for OC, but toxicity and acquired chemoresistance has proven challenging. Recently, we reported that sensitivity to platinum was significantly reduced in a co-culture of OC cells with MSC. To discover compounds capable of restoring platinum sensitivity, we screened a number of candidates and monitored ability to induce PARP cleavage. Moreover, we monitored platinum uptake and expression of ABC transporters in OC cells. Our results showed that 2-hydroxyestradiol (2HE2), a metabolite of estradiol, and dasatinib, an Abl/Src kinase inhibitor, were significantly effective in overcoming MSC-mediated platinum drug resistance. Dasatinib activity was dependent on ERK1/2 activation, whereas 2HE2 was independent of the activation of ERK1/2. MSC-mediated platinum drug resistance was accompanied by reduced intracellular platinum concentrations in OC cells. Moreover, MSC co-cultured with OC cells resulted in downregulation of the expression of cellular transporters required for platinum uptake and efflux. Exposure to 2HE2 and other modulators resulted in an increase in intracellular platinum concentrations. Thus, 2HE2 and dasatinib might act as sensitizers to restore platinum drug sensitivity to OC cells and thus to limit TME-mediated chemoresistance in OC.

Crizotinib acts as ABL1 inhibitor combining ATP-binding with allosteric inhibition and is active against native BCR-ABL1 and its resistance and compound mutants BCR-ABL1T315I and BCR-ABL1T315I-E255K.

Resistance remains the major clinical challenge for the therapy of Philadelphia chromosome-positive (Ph+) leukemia. With the exception of ponatinib, all approved tyrosine kinase inhibitors (TKIs) are unable to inhibit the common "gatekeeper" mutation T315I. Here we investigated the therapeutic potential of crizotinib, a TKI approved for targeting ALK and ROS1 in non-small cell lung cancer patients, which inhibited also the ABL1 kinase in cell-free systems, for the treatment of advanced and therapy-resistant Ph+ leukemia. By inhibiting the BCR-ABL1 kinase, crizotinib efficiently suppressed growth of Ph+ cells without affecting growth of Ph- cells. It was also active in Ph+ patient-derived long-term cultures (PD-LTCs) independently of the responsiveness/resistance to other TKIs. The efficacy of crizotinib was confirmed in vivo in syngeneic mouse models of BCR-ABL1- or BCR-ABL1T315I-driven chronic myeloid leukemia-like disease and in BCR-ABL1-driven acute lymphoblastic leukemia (ALL). Although crizotinib binds to the ATP-binding site, it also allosterically affected the myristol binding pocket, the binding site of GNF2 and asciminib (former ABL001). Therefore, crizotinib has a seemingly unique double mechanism of action, on the ATP-binding site and on the myristoylation binding pocket. These findings strongly suggest the clinical evaluation of crizotinib for the treatment of advanced and therapy-resistant Ph+ leukemia.

Natural Products Targeting Cancer Stem Cells for Augmenting Cancer Therapeutics.

Cancer stem cells (CSC) have been identified in several types of solid tumors. In some cases, CSC may be the source of all the tumor cells, the cause of the tumor's resistance to chemotherapeutic agents, and the source of metastatic cells. Thus, a combination therapy targeting non-CSC tumor cells as well as specifically targeting CSCs holds the potential to be highly effective. Natural products (NPs) have been a historically rich source of biologically active compounds and are known for their ability to influence multiple signaling pathways simultaneously with negligible side effects. In this review, we discuss the potential of NPs in targeting multiple signaling pathways in CSC and their potential to augment the efficacy of standard cancer therapy. Specifically, we focus on the anti-CSC activities of flavonoids, FDA-approved drugs originating from natural sources. Additionally, we emphasize the potential of NPs in targeting microRNA-mediated signaling, given the roles of microRNA in the maintenance of the CSC phenotype.

Natural Products Counteracting Cardiotoxicity during Cancer Chemotherapy: The Special Case of Doxorubicin, a Comprehensive Review.

Cardiotoxicity is a frequent undesirable phenomenon observed during oncological treatment that limits the therapeutic dose of antitumor drugs and thus may decrease the effectiveness of cancer eradication. Almost all antitumor drugs exhibit toxic properties towards cardiac muscle. One of the underlying causes of cardiotoxicity is the stimulation of oxidative stress by chemotherapy. This suggests that an appropriately designed diet or dietary supplements based on edible plants rich in antioxidants could decrease the toxicity of antitumor drugs and diminish the risk of cardiac failure. This comprehensive review compares the cardioprotective efficacy of edible plant extracts and foodborne phytochemicals whose beneficial activity was demonstrated in various models in vivo and in vitro. The studies selected for this review concentrated on a therapy frequently applied in cancer, anthracycline antibiotic-doxorubicin-as the oxidative stress- and cardiotoxicity-inducing agent.

Flavonoids Restore Platinum Drug Sensitivity to Ovarian Carcinoma Cells in a Phospho-ERK1/2-Dependent Fashion.

Ovarian cancer (OC) is the second most common type of gynecological malignancy; it has poor survival rates and is frequently (>75%) diagnosed at an advanced stage. Platinum-based chemotherapy, with, e.g., carboplatin, is the standard of care for OC, but toxicity and acquired resistance to therapy have proven challenging. Despite advances in OC diagnosis and treatment, approximately 85% of patients will experience relapse, mainly due to chemoresistance. The latter is attributed to alterations in the cancer cells and is also mediated by tumor microenvironment (TME). Recently, we reported the synthesis of a platinum (IV) prodrug that exhibits equal potency toward platinum-sensitive and resistant OC cell lines. Here, we investigated the effect of TME on platinum sensitivity. Co-culture of OC cells with murine or human mesenchymal stem cells (MS-5 and HS-5, respectively) rendered them resistant to chemotherapeutic agents, including platinum, paclitaxel and colchicine. Platinum resistance was also conferred by co-culture with differentiated murine adipocyte progenitor cells. Exposure of OC cells to chemotherapeutic agents resulted in activation of phospho-ERK1/2. Co-culture with MS-5, which conferred drug resistance, was accompanied by blockage of phospho-ERK1/2 activation. The flavonoids fisetin and quercetin were active in restoring ERK phosphorylation, as well as sensitivity to platinum compounds. Exposure of OC cells to cobimetinib-a MEK1 inhibitor that also inhibits extracellular signal-regulated kinase (ERK) phosphorylation-which resulted in reduced sensitivity to the platinum compound. This suggests that ERK activity is involved in mediating the function of flavonoids in restoring platinum sensitivity to OC co-cultured with cellular components of the TME. Our data show the potential of combining flavonoids with standard therapy to restore drug sensitivity to OC cells and overcome TME-mediated platinum drug resistance.

Sustained proliferation in cancer: Mechanisms and novel therapeutic targets.

Proliferation is an important part of cancer development and progression. This is manifest by altered expression and/or activity of cell cycle related proteins. Constitutive activation of many signal transduction pathways also stimulates cell growth. Early steps in tumor development are associated with a fibrogenic response and the development of a hypoxic environment which favors the survival and proliferation of cancer stem cells. Part of the survival strategy of cancer stem cells may manifested by alterations in cell metabolism. Once tumors appear, growth and metastasis may be supported by overproduction of appropriate hormones (in hormonally dependent cancers), by promoting angiogenesis, by undergoing epithelial to mesenchymal transition, by triggering autophagy, and by taking cues from surrounding stromal cells. A number of natural compounds (e.g., curcumin, resveratrol, indole-3-carbinol, brassinin, sulforaphane, epigallocatechin-3-gallate, genistein, ellagitannins, lycopene and quercetin) have been found to inhibit one or more pathways that contribute to proliferation (e.g., hypoxia inducible factor 1, nuclear factor kappa B, phosphoinositide 3 kinase/Akt, insulin-like growth factor receptor 1, Wnt, cell cycle associated proteins, as well as androgen and estrogen receptor signaling). These data, in combination with bioinformatics analyses, will be very important for identifying signaling pathways and molecular targets that may provide early diagnostic markers and/or critical targets for the development of new drugs or drug combinations that block tumor formation and progression.

Platinum (IV)-fatty acid conjugates overcome inherently and acquired Cisplatin resistant cancer cell lines: an in-vitro study.

BACKGROUND: Platinum-based drugs are used as cancer chemotherapeutics for the last 40 years. However, drug resistance and nephrotoxicity are the major limitations of the use of platinum-based compounds in cancer therapy. Platinum (IV) complexes are believed to act as platinum prodrugs and are able to overcome some of platinum (II) limitations. METHODS: A number of previously sensitized platinum (IV) complexes were evaluated for their anti-cancer activity by monitoring ability to affect proliferation, clonigenicity and apoptosis induction of Cisplatin sensitive and resistant cancer cells. In addition, the uptake of Cisplatin and the platinum (IV) derivatives to Cisplatin sensitive and resistant cancer cells was monitored. RESULTS: The bis-octanoatoplatinum (IV) complex (RJY13), a Cisplatin derivative with octanoate as axial ligand, exhibited strong anti-proliferative effect on the Cisplatin resistant and sensitive ovarian cells, A2780cisR and A2780, respectively. Moreover, RJY13 exhibited good activity in inhibiting clonigenicity of both cells. Anti-proliferative activity of RJY13 was mediated by induction of apoptosis. Interestingly, a bis-lauratopaltinum (IV) complex (RJY6) was highly potent in inhibiting clonigenicity of both Cisplatin sensitive and Cisplatin resistant cells, however, exhibited reduced activity in assays that utilize cells growing in two dimensional (2D) conditions. The uptake of Cisplatin was reduced by 30% in A2780 in which the copper transporter-1 (Ctr1) was silenced. Moreover, uptake of RJY6 was marginally dependent on Ctr1, while uptake of RJY13 was Ctr1-independent. CONCLUSIONS: Our data demonstrated the potential of platinum (IV) prodrugs in overcoming acquired and inherited drug resistance in cancer cell lines. Moreover, our data demonstrated that the uptake of Cisplatin is partially dependent on Ctr1 transporter, while uptake of RJY6 is marginally dependent on Ctr1 and RJY13 is Ctr1-independent. In addition, our data illustrated the therapeutic potential of platinum (IV) prodrugs in cancer therapy.

Overcoming Chemoresistance in Cancer: The Promise of Crizotinib.

Chemoresistance is a major obstacle in cancer treatment, often leading to disease progression and poor outcomes. It arises through various mechanisms such as genetic mutations, drug efflux pumps, enhanced DNA repair, and changes in the tumor microenvironment. These processes allow cancer cells to survive despite chemotherapy, underscoring the need for new strategies to overcome resistance and improve treatment efficacy. Crizotinib, a first-generation multi-target kinase inhibitor, is approved by the FDA for the treatment of ALK-positive or ROS1-positive non-small cell lung cancer (NSCLC), refractory inflammatory (ALK)-positive myofibroblastic tumors (IMTs) and relapsed/refractory ALK-positive anaplastic large cell lymphoma (ALCL). Crizotinib exists in two enantiomeric forms: (R)-crizotinib and its mirror image, (S)-crizotinib. It is assumed that the R-isomer is responsible for the carrying out various processes reviewed here The S-isomer, on the other hand, shows a strong inhibition of MTH1, an enzyme important for DNA repair mechanisms. Studies have shown that crizotinib is an effective multi-kinase inhibitor targeting various kinases such as c-Met, native/T315I Bcr/Abl, and JAK2. Its mechanism of action involves the competitive inhibition of ATP binding and allosteric inhibition, particularly at Bcr/Abl. Crizotinib showed synergistic effects when combined with the poly ADP ribose polymerase inhibitor (PARP), especially in ovarian cancer harboring BRCA gene mutations. In addition, crizotinib targets a critical vulnerability in many p53-mutated cancers. Unlike its wild-type counterpart, the p53 mutant promotes cancer cell survival. Crizotinib can cause the degradation of the p53 mutant, sensitizing these cancer cells to DNA-damaging substances and triggering apoptosis. Interestingly, other reports demonstrated that crizotinib exhibits anti-bacterial activity, targeting Gram-positive bacteria. Also, it is active against drug-resistant strains. In summary, crizotinib exerts anti-tumor effects through several mechanisms, including the inhibition of kinases and the restoration of drug sensitivity. The potential of crizotinib in combination therapies is emphasized, particularly in cancers with a high prevalence of the p53 mutant, such as triple-negative breast cancer (TNBC) and high-grade serous ovarian cancer (HGSOC).

Ovarian cancer ascites confers platinum chemoresistance to ovarian cancer cells.

Ovarian cancer (OC), the second most common form of gynecologic malignancy, has a poor prognosis and is often discovered in the late stages. Platinum-based chemotherapy is the first line of therapy. Nevertheless, treatment OC has proven challenging due to toxicity and the development of acquired resistance to therapy. Tumor microenvironment (TME) has been associated with platinum chemoresistance. Malignant ascites has been used as OC tumor microenvironment and its ability to induce platinum chemoresistance has been investigated. Our results suggest that exposure to OC ascites induces platinum chemoresistance in 11 of 13 cases (85 %) on OC cells. In contrast, 75 % of cirrhotic ascites (3 of 4) failed to confer platinum chemoresistance to OC cells. Cytokine array analysis revealed that IL -6 and to a lesser extent HGF were enriched in OC ascites, whereas IL -22 was enriched in cirrhotic ascites. Pharmaceutical inhibitors targeting the IL -6/ JAK pathway were mildly effective in overcoming platinum chemoresistance induced by malignant ascites. In contrast, crizotinib, an HGF/c- MET inhibitor, and 2-hydroxyestradiol (2HE2) were effective in restoring platinum chemosensitivity to OC. Our results demonstrate the importance of OC ascites in supporting platinum chemoresistance and the potential of combination therapy to restore chemosensitivity of OC cells.

Oleylamine-carbonyl-valinol inhibits auto-phosphorylation activity of native and T315I mutated Bcr-Abl, and exhibits selectivity towards oncogenic Bcr-Abl in SupB15 ALL cell lines.

Chronic myeloid leukemia (CML) is characterized by the presence of p210(Bcr-Abl) which exhibits an abnormal kinase activity. Selective Abl kinase inhibitors have been successfully established for the treatment of CML. Despite high rates of clinical response, CML patients can develop resistance against these kinase inhibitors mainly due to point mutations within the Abl protein kinase domain. Previously, we have identified oleic acid as the active component in the mushroom Daedalea gibbosa that inhibited the kinase activity of Bcr-Abl. Here, we report that the oleyl amine derivatives, S-1-(1-Hydroxymethyl-2-methyl-propyl)-3-octadec-9-enyl-urea [oleylaminocarbonyl-L-N-valinol,oroleylaminocarbonyl-S-2-isopropyl-N-ethanolamine,oleylamine-carbonyl-L-valinol] (cpd 6) and R-1-(1-Hydroxymethyl-2-methyl-propyl)-3-octadec-9-enyl-urea [oleylamineocarbonyl-D-N-valinol, oleylaminocarbonyl-R-2-isopropyl-N-ethanolamine, or oleylamine-carbonyl-D-valinol] (cpd 7), inhibited the activity of the native and T315I mutated Bcr-Abl. Furthermore, cpd 6 and 7 exhibited higher activity towards the oncogenic Bcr-Abl in comparison to native c-Abl in SupB15 Ph-positive ALL cell line.

Compounds originating from the edible mushroom Auricularia auricula-judae inhibit tropomyosin receptor kinase B activity.

Tropomyosin receptor kinase B (TrkB) serves as a pivotal factor in various cancers. To identify novel natural compounds with TrkB-inhibiting properties, a screening approach was applied using extracts from a collection of wild and cultivated mushroom fruiting bodies, and Ba/F3 cells that ectopically express TrkB (TPR-TrkB). We selected mushroom extracts that selectively inhibited proliferation of the TPR-TrkB cells. We then evaluated the ability of exogenous interleukin 3 to rescue growth inhibition by the selected TrkB-positive extracts. An ethyl acetate extract of Auricularia auricula-judae actively inhibited auto-phosphorylation of TrkB. LC-MS/MS analysis of this extract revealed substances that might be responsible for the observed activity. This screening approach demonstrates, for the first time, that extracts originating from the mushroom A. auricula-judae exhibit TrkB-inhibition properties that might hold therapeutic potential for TrkB-positive cancers.

Plant-Derived Epi-Nutraceuticals as Potential Broad-Spectrum Anti-Viral Agents.

Although the COVID-19 pandemic appears to be diminishing, the emergence of SARS-CoV-2 variants represents a threat to humans due to their inherent transmissibility, immunological evasion, virulence, and invulnerability to existing therapies. The COVID-19 pandemic affected more than 500 million people and caused over 6 million deaths. Vaccines are essential, but in circumstances in which vaccination is not accessible or in individuals with compromised immune systems, drugs can provide additional protection. Targeting host signaling pathways is recommended due to their genomic stability and resistance barriers. Moreover, targeting host factors allows us to develop compounds that are effective against different viral variants as well as against newly emerging virus strains. In recent years, the globe has experienced climate change, which may contribute to the emergence and spread of infectious diseases through a variety of factors. Warmer temperatures and changing precipitation patterns can increase the geographic range of disease-carrying vectors, increasing the risk of diseases spreading to new areas. Climate change may also affect vector behavior, leading to a longer breeding season and more breeding sites for disease vectors. Climate change may also disrupt ecosystems, bringing humans closer to wildlife that transmits zoonotic diseases. All the above factors may accelerate the emergence of new viral epidemics. Plant-derived products, which have been used in traditional medicine for treating pathological conditions, offer structurally novel therapeutic compounds, including those with anti-viral activity. In addition, plant-derived bioactive substances might serve as the ideal basis for developing sustainable/efficient/cost-effective anti-viral alternatives. Interest in herbal antiviral products has increased. More than 50% of approved drugs originate from herbal sources. Plant-derived compounds offer diverse structures and bioactive molecules that are candidates for new drug development. Combining these therapies with conventional drugs could improve patient outcomes. Epigenetics modifications in the genome can affect gene expression without altering DNA sequences. Host cells can use epigenetic gene regulation as a mechanism to silence incoming viral DNA molecules, while viruses recruit cellular epitranscriptomic (covalent modifications of RNAs) modifiers to increase the translational efficiency and transcript stability of viral transcripts to enhance viral gene expression and replication. Moreover, viruses manipulate host cells' epigenetic machinery to ensure productive viral infections. Environmental factors, such as natural products, may influence epigenetic modifications. In this review, we explore the potential of plant-derived substances as epigenetic modifiers for broad-spectrum anti-viral activity, reviewing their modulation processes and anti-viral effects on DNA and RNA viruses, as well as addressing future research objectives in this rapidly emerging field.

TRAF3 suppression encourages B cell recruitment and prolongs survival of microbiome-intact mice with ovarian cancer.

BACKGROUND: Ovarian cancer (OC) is known for exhibiting low response rates to immune checkpoint inhibitors that activate T cells. However, immunotherapies that activate B cells have not yet been extensively explored and may be a potential target, as B cells that secrete immunoglobulins have been associated with better outcomes in OC. Although the secretion of immunoglobulins is often mediated by the microbiome, it is still unclear what role they play in limiting the progression of OC. METHODS: We conducted an in-vivo CRISPR screen of immunodeficient (NSG) and immune-intact wild type (WT) C57/BL6 mice to identify tumor-derived immune-escape mechanisms in a BRAC1- and TP53-deficient murine ID8 OC cell line (designated ITB1). To confirm gene expression and signaling pathway activation in ITB1 cells, we employed western blot, qPCR, immunofluorescent staining, and flow cytometry. Flow cytometry was also used to identify immune cell populations in the peritoneum of ITB1-bearing mice. To determine the presence of IgA-coated bacteria in the peritoneum of ITB1-bearing mice and the ascites of OC patients, we employed 16S sequencing. Testing for differences was done by using Deseq2 test and two-way ANOVA test. Sequence variants (ASVs) were produced in Qiime2 and analyzed by microeco and phyloseq R packages. RESULTS: We identified tumor necrosis factor receptor-associated factor 3 (TRAF3) as a tumor-derived immune suppressive mediator in ITB1 cells. Knockout of TRAF3 (TRAF3KO) activated the type-I interferon pathway and increased MHC-I expression. TRAF3KO tumors exhibited a growth delay in WT mice vs. NSG mice, which was correlated with increased B cell infiltration and activation compared to ITB1 tumors. B cells were found to be involved in the progression of TRAF3KO tumors, and B-cell surface-bound and secreted IgA levels were significantly higher in the ascites of TRAF3KO tumors compared to ITB1. The presence of commensal microbiota was necessary for B-cell activation and for delaying the progression of TRAF3KO tumors in WT mice. Lastly, we observed unique profiles of IgA-coated bacteria in the ascites of OC-bearing mice or the ascites of OC patients. CONCLUSIONS: TRAF3 is a tumor-derived immune-suppressive modulator that influences B-cell infiltration and activation, making it a potential target for enhancing anti-tumor B-cell responses in OC.

p185(BCR/ABL) has a lower sensitivity than p210(BCR/ABL) to the allosteric inhibitor GNF-2 in Philadelphia chromosome-positive acute lymphatic leukemia.

BACKGROUND: The t(9;22) translocation leads to the formation of the chimeric breakpoint cluster region/c-abl oncogene 1 (BCR/ABL) fusion gene on der22, the Philadelphia chromosome. The p185(BCR/ABL) or the p210(BCR/ABL) fusion proteins are encoded as a result of the translocation, depending on whether a "minor" or "major" breakpoint occurs, respectively. Both p185(BCR/ABL) and p210(BCR/ABL) exhibit constitutively activated ABL kinase activity. Through fusion to BCR the ABL kinase in p185(BCR/ABL) and p210(BCR/ABL) "escapes" the auto-inhibition mechanisms of c-ABL, such as allosteric inhibition. A novel class of compounds including GNF-2 restores allosteric inhibition of the kinase activity and the transformation potential of BCR/ABL. Here we investigated whether there are differences between p185(BCR/ABL) and p210(BCR/ABL) regarding their sensitivity towards allosteric inhibition by GNF-2 in models of Philadelphia chromosome-positive acute lymphatic leukemia. DESIGN AND METHODS: We investigated the anti-proliferative activity of GNF-2 in different Philadelphia chromosome-positive acute lymphatic leukemia models, such as cell lines, patient-derived long-term cultures and factor-dependent lymphatic Ba/F3 cells expressing either p185(BCR/ABL) or p210(BCR/ABL) and their resistance mutants. RESULTS: The inhibitory effects of GNF-2 differed constantly between p185(BCR/ABL) and p210(BCR/ABL) expressing cells. In all three Philadelphia chromosome-positive acute lymphatic leukemia models, p210(BCR/ABL)-transformed cells were more sensitive to GNF-2 than were p185BCR/ABL-positive cells. Similar results were obtained for p185(BCR/ABL) and the p210(BCR/ABL) harboring resistance mutations. CONCLUSIONS: Our data provide the first evidence of a differential response of p185(BCR/ABL)- and p210(BCR/ABL)- transformed cells to allosteric inhibition by GNF-2, which is of importance for the treatment of patients with Philadelphia chromosome-positive acute lymphatic leukemia.

Overcoming Bcr-Abl T315I mutation by combination of GNF-2 and ATP competitors in an Abl-independent mechanism.

BACKGROUND: Philadelphia positive leukemias are characterized by the presence of Bcr-Abl fusion protein which exhibits an abnormal kinase activity. Selective Abl kinase inhibitors have been successfully established for the treatment of Ph (+) leukemias. Despite high rates of clinical response, Ph (+) patients can develop resistance against these kinase inhibitors mainly due to point mutations within the Abl protein. Of special interest is the 'gatekeeper' T315I mutation, which confers complete resistance to Abl kinase inhibitors. Recently, GNF-2, Abl allosteric kinase inhibitor, was demonstrated to possess cellular activity against Bcr-Abl transformed cells. Similarly to Abl kinase inhibitors (AKIs), GNF-2 failed to inhibit activity of mutated Bcr-Abl carrying the T315I mutation. METHODS: Ba/F3 cells harboring native or T315I mutated Bcr-Abl constructs were treated with GNF-2 and AKIs. We monitored the effect of GNF-2 with AKIs on the proliferation and clonigenicity of the different Ba/F3 cells. In addition, we monitored the auto-phosphorylation activity of Bcr-Abl and JAK2 in cells treated with GNF-2 and AKIs. RESULTS: In this study, we report a cooperation between AKIs and GNF-2 in inhibiting proliferation and clonigenicity of Ba/F3 cells carrying T315I mutated Bcr-Abl. Interestingly, cooperation was most evident between Dasatinib and GNF-2. Furthermore, we showed that GNF-2 was moderately active in inhibiting the activity of JAK2 kinase, and presence of AKIs augmented GNF-2 activity. CONCLUSIONS: Our data illustrated the ability of allosteric inhibitors such as GNF-2 to cooperate with AKIs to overcome T315I mutation by Bcr-Abl-independent mechanisms, providing a possibility of enhancing AKIs efficacy and overcoming resistance in Ph+ leukemia cells.

Allosteric inhibition enhances the efficacy of ABL kinase inhibitors to target unmutated BCR-ABL and BCR-ABL-T315I.

BACKGROUND: Chronic myelogenous leukemia (CML) and Philadelphia chromosome-positive (Ph+) acute lymphatic leukemia (Ph + ALL) are caused by the t(9;22), which fuses BCR to ABL resulting in deregulated ABL-tyrosine kinase activity. The constitutively activated BCR/ABL-kinase "escapes" the auto-inhibition mechanisms of c-ABL, such as allosteric inhibition. The ABL-kinase inhibitors (AKIs) Imatinib, Nilotinib or Dasatinib, which target the ATP-binding site, are effective in Ph + leukemia. Another molecular therapy approach targeting BCR/ABL restores allosteric inhibition. Given the fact that all AKIs fail to inhibit BCR/ABL harboring the 'gatekeeper' mutation T315I, we investigated the effects of AKIs in combination with the allosteric inhibitor GNF2 in Ph + leukemia. METHODS: The efficacy of this approach on the leukemogenic potential of BCR/ABL was studied in Ba/F3 cells, primary murine bone marrow cells, and untransformed Rat-1 fibroblasts expressing BCR/ABL or BCR/ABL-T315I as well as in patient-derived long-term cultures (PDLTC) from Ph + ALL-patients. RESULTS: Here, we show that GNF-2 increased the effects of AKIs on unmutated BCR/ABL. Interestingly, the combination of Dasatinib and GNF-2 overcame resistance of BCR/ABL-T315I in all models used in a synergistic manner. CONCLUSIONS: Our observations establish a new approach for the molecular targeting of BCR/ABL and its resistant mutants using a combination of AKIs and allosteric inhibitors.

Inhibition of the androgen receptor activity by Coprinus comatus substances.

Prostatic adenocarcinoma is the second leading cause of death from cancer in Western men. The common prostate cancer treatments are effective in the early stages; however, advanced prostate cancer is resilient to most of these treatments. Altered androgen receptor (AR) activity caused by point mutations or signaling mechanisms that regulate AR function has been proposed as a key mechanism in the transition to the androgen-independent stage. Our previous results demonstrated that hexane extract prepared from Coprinus comatus (C. comatus) strain 734 was able to interfere with AR activity. The current study was made to further evaluate the antiandrogenic activity of the C. comatus mushroom strain 734. Activity-guided chromatography was conducted and 2 active fractions, F-32-and F-33, were found to contain substances that were able to inhibit AR-mediated reporter activity and reduce the levels of AR and prostate-specific antigen (PSA) transcripts in LNCaP cells. Fraction F-32 also inhibited the proliferation and clonigenicity of LNCaP cells. Furthermore, F-32 was able to inhibit the binding of AR to the PSA enhancer region and to inhibit Akt-mediated AR phosphorylation at Ser 213. This study illustrated the potential of substances from the C. comatus mushroom to serve as natural antiandrogenic modulators for the treatment of prostatic disorders.

The Shaggy Inc Cap medicinal mushroom, Coprinus comatus (O.F.Mull.: Fr.) Pers. (Agaricomycetideae) substances interfere with H2O2 induction of the NF-kappaB pathway through inhibition of Ikappaalpha phosphorylation in MCF7 breast cancer cells.

Breast cancer is the most commonly diagnosed cancer among women. Currently, there is no effective therapy for malignant estrogen-independent breast cancer. In our study, we used hydrogen peroxide, a well-known strong oxidative reagent capable of activating the nuclear factor kappa B (NF-kappaB) transcription factor. The IC50 value of the culinary-medicinal Shaggy Inc Cap mushroom Coprinus comatus culture liquid crude extract on MCF7 cell viability was found to be as low as 76 microg/mL, and the IC50 value of C. comatus ethyl acetate extract was only 32 microg/ mL. Our results also showed that both extracts significantly affected IkappaBalpha phosphorylation in a dose-dependent manner. The effect of ethyl acetate extract was comparable to the effect of curcumin, a known NF-kappaB pathway inhibitor, and seemed to be the most active inhibitor of H2O2-dependent IkappaBalpha phosphorylation. In addition, the data obtained showed that only ethyl acetate extract inhibited the activity of IKK complex, at close to 90% as compared to the control of the untreated sample. These results suggest that C. comatus contains potent compounds capable of inhibiting NF-kappaB function and also possibly acts as an antitumor agent.

Inhibition of TNFα-induced iNOS expression in HSV-tk transduced 9L glioblastoma cell lines by Marasmius oreades substances through NF-κB- and MAPK-dependent mechanisms.

Nitric oxide (NO) is a gaseous, radical molecule that plays a role in various physiological processes. Previously, we reported that transduction of murine colon cancer cells (MC38) with herpes simplex virus thymidine kinase (HSV-tk) gene resulted in a significant over-expression of cyclooxygenase-2 (COX-2) and activation of NF-kB pathway. In this study we show that TNFα, but not LPS, was significantly able to stimulate the production of NO in HSV-tk transduced 9L glioblastoma cell lines, mediated by the up-regulation of iNOS transcript and iNOS protein. The TNFα-induced up-regulation of iNOS expression was mediated by MAPK and NF-κB signaling pathways as revealed by using selective pharmaceutical inhibitors. A culture liquid extract of the edible and medicinal mushroom Marasmius oreades that was previously shown to inhibit iNOS expression in MCF-7 was utilized to prepare fractions and evaluate their ability to affect TNFα-induced iNOS expression in HSV tk transduced 9L cell lines. While most of the tested fractions were shown to inhibit TNFα-induced iNOS expression, they targeted different signaling pathways in a selective fashion. Here, we report that fraction SiSiF1 interfered with IKBα phosphorylation and consequently interfered with NF-κB activation pathway. SiSiF1 showed minimal interference with the phosphorylation of p38 and JNK proteins. In contrast, fraction SiSiF3 selectively inhibited the phosphorylation of p38 and fractions SiSiF4 and SiSiF5 selectively inhibited the phosphorylation of JNK with no observed effect against IKBα and p38 phosphorylation. Our data illustrate the complexity of iNOS regulation in HSV tk transduced 9L cell lines and also the richness of natural products with bioactive substances that may act synergistically through different signaling pathways to affect iNOS gene expression.