Primary acute lymphoblastic leukemia cells are susceptible to microtubule depolymerization in G1 and M phases through distinct cell death pathways.

Microtubule targeting agents (MTAs) are widely used cancer chemotherapeutics which conventionally exert their effects during mitosis, leading to mitotic or postmitotic death. However, accumulating evidence suggests that MTAs can also generate death signals during interphase, which may represent a key mechanism in the clinical setting. We reported previously that vincristine and other microtubule destabilizers induce death not only in M phase but also in G1 phase in primary acute lymphoblastic leukemia cells. Here, we sought to investigate and compare the pathways responsible for phase-specific cell death. Primary acute lymphoblastic leukemia cells were subjected to centrifugal elutriation, and cell populations enriched in G1 phase (97%) or G2/M phases (80%) were obtained and treated with vincristine. We found death of M phase cells was associated with established features of mitochondrial-mediated apoptosis, including Bax activation, loss of mitochondrial transmembrane potential, caspase-3 activation, and nucleosomal DNA fragmentation. In contrast, death of G1 phase cells was not associated with pronounced Bax or caspase-3 activation but was associated with loss of mitochondrial transmembrane potential, parylation, nuclear translocation of apoptosis-inducing factor and endonuclease G, and supra-nucleosomal DNA fragmentation, which was enhanced by inhibition of autophagy. The results indicate that microtubule depolymerization induces distinct cell death pathways depending on during which phase of the cell cycle microtubule perturbation occurs. The observation that a specific type of drug can enter a single cell type and induce two different modes of death is novel and intriguing. These findings provide a basis for advancing knowledge of clinical mechanisms of MTAs.

Synthesis and Anti-Melanoma Activity of Acryloyl Pyridinone Analogues.

A major challenge for clinical management of melanoma is the prevention and treatment of metastatic disease. Drug discovery efforts over the last 10 years have resulted in several drugs that improve the prognosis of metastatic melanoma; however, most patients develop early resistance to these treatments. We designed and synthesized, through a concise synthetic strategy, a series of hybrid olefin-pyridinone compounds that consist of structural motifs from tamoxifen and ilicicolin H. These compounds were tested against a human melanoma cell line and patient-derived melanoma cells that had metastasized to the brain. Three compounds 7 b, 7 c, and 7 g demonstrated promising activity (IC50=0.4-4.3 µM). Cell cycle analysis demonstrated that 7 b and 7 c induce cell cycle arrest predominantly in the G1 phase. Both 7 b and 7c significantly inhibited migration of A375 melanoma cells; greater effects were demonstrated by 7 b. Molecular modelling analysis provides insight into a plausible mechanism of action.

Synthesis, anticancer activity and molecular docking studies of N-deacetylthiocolchicine and 4-iodo-N-deacetylthiocolchicine derivatives.

Colchicine is a plant alkaloid with a broad spectrum of biological and pharmacological properties. It has found application as an anti-inflammatory agent and also shows anticancer effects through its ability to destabilize microtubules by preventing tubulin dimers from polymerizing leading to mitotic death. However, adverse side effects have so far restricted its use in cancer therapy. This has led to renewed efforts to identify less toxic derivatives. In this article, we describe the synthesis of a set of novel double- and triple-modified colchicine derivatives. These derivatives were tested against primary acute lymphoblastic leukemia (ALL-5) cells and several established cancer cell lines including A549, MCF-7, LoVo and LoVo/DX. The novel derivatives were active in the low nanomolar range, with 7-deacetyl-10-thiocolchicine analogues more potent towards ALL-5 cells while 4-iodo-7-deacetyl-10-thiocolchicine analogues slightly more effective towards the LoVo cell line. Moreover, most of the synthesized compounds showed a favorable selectivity index (SI), particularly for ALL-5 and LoVo cell lines. Cell cycle analysis of the most potent molecules on ALL-5 and MCF-7 cell lines revealed contrasting effects, where M-phase arrest was observed in MCF-7 cells but not in ALL-5 cells. Molecular docking studies of all derivatives to the colchicine-binding site were performed and it was found that five of the derivatives showed strong ß-tubulin binding energies, lower than -8.70 kcal/mol, while the binding energy calculated for colchicine is -8.09 kcal/mol. The present results indicate that 7-deacetyl-10-thiocolchicine and 4-iodo-7-deacetyl-10-thiocolchicine analogues constitute promising lead compounds as chemotherapy agents against several types of cancer.

Effect of pomegranate fruit supplementation on performance and various markers in athletes and active subjects: A systematic review.

The aim of the study was to review recent findings on the use of POM supplements in athletes of various disciplines and physically active participants. Eleven articles published between 2010 and 2018 were included, where the total number of investigated subjects was 176. Male participants constituted the majority of the group (n = 155), as compared to females (n = 21). 45% of research described was conducted on athletes, whereas the remaining studies were based on highly active participants. Randomised, crossover, double-blind study designs constituted the majority of the experimental designs used. POM supplementation varied in terms of form (pills/juice), dosage (50 ml-500 ml) and time of intervention (7 days-2 months) between studies. Among the reviewed articles, POM supplementation had an effect on the improvement of the following: whole body strength; feeling of vitality; acute and delayed muscle fatigue and soreness; increase in vessel diameter; blood flow and serum level of TAC; reduction in the rate of increase for HR, SBP, CK and LDH; support in the recovery of post-training CK, LDH, CRP and ASAT to their baseline levels; reduction of MMP2, MMP9, hsCRP and MDA; and increased activity of antioxidant enzymes (glutathione peroxidase and superoxide dismutase). In the majority of reviewed articles POM supplementation had a positive effect on a variety of parameters studied and the authors recommended it as a supplement for athletes and physically active bodies.

Carbamate derivatives of colchicine show potent activity towards primary acute lymphoblastic leukemia and primary breast cancer cells-in vitro and ex vivo study.

Colchicine (COL) shows strong anticancer activity but due to its toxicity towards normal cells its wider application is limited. To address this issue, a library of 17 novel COL derivatives, namely N-carbamates of N-deacetyl-4-(bromo/chloro/iodo)thiocolchicine, has been tested against two types of primary cancer cells. These included acute lymphoblastic leukemia (ALL) and human breast cancer (BC) derived from two different tumor subtypes, ER+ invasive ductal carcinoma grade III (IDCG3) and metastatic carcinoma (MC). Four novel COL derivatives showed higher anti-proliferative activity than COL (IC50 = 8.6 nM) towards primary ALL cells in cell viability assays (IC50 range of 1.1-6.4 nM), and several were more potent towards primary IDCG3 (IC50 range of 0.1 to 10.3 nM) or MC (IC50 range of 2.3-9.1 nM) compared to COL (IC50 of 11.1 and 11.7 nM, respectively). In addition, several derivatives were selectively active toward primary breast cancer cells compared to normal breast epithelial cells. The most promising derivatives were subsequently tested against the NCI panel of 60 human cancer cell lines and seven derivatives were more potent than COL against leukemia, non-small-cell lung, colon, CNS and prostate cancers. Finally, COL and two of the most active derivatives were shown to be effective in killing BC cells when tested ex vivo using fresh human breast tumor explants. The present findings indicate that the select COL derivatives constitute promising lead compounds targeting specific types of cancer.

Synthesis, biological evaluation and molecular docking studies of new amides of 4-chlorothiocolchicine as anticancer agents.

Colchicine belongs to a large group of microtubule polymerization inhibitors. Although the anti-cancer activity of colchicine and its derivatives has been established, none of them has found commercial application in cancer treatment due to side effects. Therefore, we designed and synthesized a series of six triple-modified 4-chlorothiocolchicine analogues with amide moieties and one urea derivative. These novel derivatives were tested against several different cancer cell lines (A549, MCF-7, LoVo, LoVo/DX) and primary acute lymphoblastic leukemia (ALL) cells and they showed activity in the nanomolar range. The obtained IC50 values for novel derivatives were lower than those obtained for unmodified colchicine and common anticancer drugs such as doxorubicin and cisplatin. Further studies of colchicine and selected analogues were undertaken to indicate that they induced apoptotic cell death in ALL-5 cells. We also performed in silico studies to predict binding modes of the 4-chlorothiocolchicine derivatives to different ß tubulin isotypes. The results indicate that select triple-modified 4-chlorothiocolchicine derivatives represent highly promising novel cancer chemotherapeutics.

Synthesis, biological evaluation and molecular docking studies of new amides of 4-bromothiocolchicine as anticancer agents.

Colchicine is the major alkaloid isolated from the plant Colchicum autumnale, which shows strong therapeutic effects towards different types of cancer. However, due to the toxicity of colchicine towards normal cells its application is limited. To address this issue we synthesized a series of seven triple-modified 4-bromothiocolchicine analogues with amide moieties. These novel derivatives were active in the nanomolar range against several different cancer cell lines and primary acute lymphoblastic leukemia cells, specifically compounds: 5-9 against primary ALL-5 (IC50 = 5.3-14 nM), 5, 7-9 against A549 (IC50 = 10 nM), 5, 7-9 against MCF-7 (IC50 = 11 nM), 5-9 against LoVo (IC50 = 7-12 nM), and 5, 7-9 against LoVo/DX (IC50 = 48-87 nM). These IC50 values were lower than those obtained for unmodified colchicine and common anticancer drugs such as doxorubicin and cisplatin. Further studies revealed that colchicine and selected analogues induced characteristics of apoptotic cell death but manifested their effects in different phases of the cell cycle in MCF-7 versus ALL-5 cells. Specifically, while colchicine and the studied derivatives arrested MCF-7 cells in mitosis, very little mitotically arrested ALL-5 cells were observed, suggesting effects were manifest instead in interphase. We also developed an in silico model of the mode of binding of these compounds to their primary target, ß-tubulin. We conducted a correlation analysis (linear regression) between the calculated binding energies of colchicine derivatives and their anti-proliferative activity, and determined that the obtained correlation coefficients strongly depend on the type of cells used.

Synthesis, antiproliferative activity and molecular docking of thiocolchicine urethanes.

A number of naturally occurring compounds such as paclitaxel, vinblastine, combretastatin, and colchicine exert their therapeutic effect by changing the dynamics of tubulin and its polymer form, microtubules. The identification of tubulin as a potential target for anticancer drugs has led to extensive research followed by clinical development of numerous compounds from several families. In this paper we report on the design, synthesis and in vitro evaluation of a group of thiocolchicine derivatives, modified at ring-B, labelled here compounds 4-14. These compounds have been obtained in a simple reaction of 7-deacetyl-10-thiocolchicine 3 with eleven different alcohols in the presence of triphosgene. These novel agents have been checked for anti-proliferative activity against four human cancer cell lines and their mode of action has been confirmed as colchicine binding site inhibition (CBSI) using molecular docking. Molecular simulations provided rational tubulin binding models for the tested compounds. On the basis of in vitro tests, derivatives 4-8 and 14 demonstrated the highest potency against MCF-7, LoVo and A549 tumor cell lines (IC50 values = 0.009-0.014 µM). They were more potent and characterized by a higher selectivity index than several standard chemotherapeutics including cisplatin and doxorubicin as well as unmodified colchicine. Further, studies revealed that colchicine and its several derivatives arrested MCF-7 cells in mitosis, while its selected derivatives caused microtubule depolymerization.

Activity of resveratrol triesters against primary acute lymphoblastic leukemia cells.

Resveratrol is a common polyphenol of plant origin known for its cancer prevention and other properties. Its wider application is limited due to poor water solubility, low stability, and weak bioavailability. To overcome these limitations, a series of 13 novel resveratrol triesters were synthesized previously. In this paper, we describe the synthesis of 3 additional derivatives and the activity of all 16 against primary acute lymphoblastic leukemia cells. Of these, 3 compounds were more potent than resveratrol (IC50=10.5µM) namely: resveratryl triacetate (IC50=3.4µM), resveratryl triisobutyrate (IC50=5.1µM), and resveratryl triisovalerate (IC50=4.9µM); all other derivatives had IC50 values of >10µM. Further studies indicated that the active compounds caused G1 phase arrest, increased expression of p53, and induced characteristics of apoptotic cell death. Moreover, the compounds were only effective in cycling cells, with cells arrested in G1 phase being refractory.

The mycelium of the Trametes versicolor synn. Coriolus versicolor (Turkey tail mushroom) exhibit anti-melanoma activity in vitro.

Melanoma is one of the most aggressive forms of skin cancer and is characterized by high metastatic potential. Despite improvements in early diagnosis and treatment, the mortality rate among metastatic melanoma patients continues to represent a significant clinical challenge. Therefore, it is imperative that we search for new forms of treatment. Trametes versicolor is a mushroom commonly used in Chinese traditional medicine due to its numerous beneficial properties. In the present work, we demonstrate T. versicolor fruiting body and mycelium ethanol extracts exhibit potent cytotoxic activity towards A375 (IC50 = 663.3 and 114.5 µg/mL respectively) and SK-MEL-5 (IC50 = 358.4 and 88.6 µg/mL respectively) human melanoma cell lines. Further studies revealed that T. versicolor mycelium extract induced apoptotic cell death and poly (ADP-ribose) polymerase cleavage, upregulated the expression of autophagy-associated marker LC3-II, increased the presentation of major histocompatibility complex II and expression of programmed death-ligand receptor, and inhibited cell migration in SK-MEL-5 cells. Therefore, our present findings highlight the therapeutic potential of T. versicolor mycelium extract for the treatment of melanoma and merit further study.

Musashi Exerts Control of Gonadotrope Target mRNA Translation During the Mouse Estrous Cycle.

The anterior pituitary controls key biological processes, including growth, metabolism, reproduction, and stress responses through distinct cell types that each secrete specific hormones. The anterior pituitary cells show a remarkable level of cell type plasticity that mediates the shifts in hormone-producing cell populations that are required to meet organismal needs. The molecular mechanisms underlying pituitary cell plasticity are not well understood. Recent work has implicated the pituitary stem cell populations and specifically, the mRNA binding proteins of the Musashi family in control of pituitary cell type identity. In this study we have identified the target mRNAs that mediate Musashi function in the adult mouse pituitary and demonstrate the requirement for Musashi function in vivo. Using Musashi RNA immunoprecipitation, we identify a cohort of 1184 mRNAs that show specific Musashi binding. Identified Musashi targets include the Gnrhr mRNA, which encodes the gonadotropin-releasing hormone receptor (GnRHR), and the Fshb mRNA, encoding follicle-stimulating hormone (FSH). Reporter assays reveal that Musashi functions to exert repression of translation of the Fshb mRNA, in addition to the previously observed repression of the Gnrhr mRNA. Importantly, mice engineered to lack Musashi in gonadotropes demonstrate a failure to repress translation of the endogenous Gnrhr and Fshb mRNAs during the estrous cycle and display a significant heterogeneity in litter sizes. The range of identified target mRNAs suggests that, in addition to these key gonadotrope proteins, Musashi may exert broad regulatory control over the pituitary proteome in a cell type-specific manner.

Anti-glioblastoma activity of monensin and its analogs in an organoid model of cancer.

Glioblastoma (GBM) remains the most frequently diagnosed primary malignant brain cancer in adults. Despite recent progress in understanding the biology of GBM, the clinical outcome for patients remains poor, with a median survival of approximately one year after diagnosis. One factor contributing to failure in clinical trials is the fact that traditional models used in GBM drug discovery poorly recapitulate patient tumors. Previous studies have shown that monensin (MON) analogs, namely esters and amides on C-26 were potent towards various types of cancer cell lines. In the present study we have investigated the activity of these molecules in GBM organoids, as well as in a host:tumor organoid model. Using a mini-ring cell viability assay we have identified seven analogs (IC50 = 91.5 ± 54.4-291.7 ± 68.8 nM) more potent than parent MON (IC50 = 612.6 ± 184.4 nM). Five of these compounds induced substantial DNA fragmentation in GBM organoids, suggestive of apoptotic cell death. The most active analog, compound 1, significantly reduced GBM cell migration, induced PARP degradation, diminished phosphorylation of STAT3, Akt and GSK3ß, increased É£H2AX signaling and upregulated expression of the autophagy associated marker LC3-II. To investigate the activity of MON and compound 1 in a tumor microenvironment, we developed human cerebral organoids (COs) from human induced pluripotent stem cells (iPSCs). The COs showed features of early developing brain such as multiple neural rosettes with a proliferative zone of neural stem cells (Nestin+), neurons (TUJ1 +), primitive ventricular system (SOX2 +/Ki67 +), intermediate zone (TBR2 +) and cortical plate (MAP2 +). In order to generate host:tumor organoids, we co-cultured RFP-labeled U87MG cells with fully formed COs. Compound 1 and MON reduced U87MG tumor size in the COs after four days of treatment and induced a significant reduction of PARP expression. These findings highlight the therapeutic potential of MON analogs towards GBM and support the application of organoid models in anti-cancer drug discovery.

Limitations of an ex vivo breast cancer model for studying the mechanism of action of the anticancer drug paclitaxel.

Paclitaxel is widely used in the treatment of breast, ovarian, lung, and other cancers. Its primary mechanism is to prevent microtubule depolymerization causing loss of dynamic instability crucial for normal microtubule function leading to mitotic arrest. Prolonged mitotic arrest results in cell death as a secondary response. The effects of paclitaxel are typically studied in cell lines which precludes assessment of the possible influence of tumor-associated cells. We therefore examined paclitaxel action ex vivo in fresh explant cultures of human breast tumors. Surprisingly, we found that paclitaxel failed to induce tumor cell death in explant culture, in contrast to several other cytotoxic agents including salinomycin and vincristine. The lack of effect was not due to defective drug uptake, and furthermore, analysis of H&E stained tumor slices indicated that paclitaxel treatment caused defective (granular) mitosis and chromosomal condensation in 5-10% of tumor cells after 72 h. These results suggest that while paclitaxel was able to penetrate into the tumor slice and disrupt mitosis in cycling tumor cells, any ensuing cell death likely occurred beyond the useful lifetime of the tumor slices. We conclude that explant culture systems may be inappropriate for the study of cytotoxic drugs where a delay exists between the drug's primary and secondary modes of action.

Single and double modified salinomycin analogs target stem-like cells in 2D and 3D breast cancer models.

Breast cancer remains one of the leading cancers among women. Cancer stem cells (CSCs) are tumor-initiating cells which drive progression, metastasis, and reoccurrence of the disease. CSCs are resistant to conventional chemo- and radio-therapies and their ability to survive such treatment enables tumor reestablishment. Metastasis is the main cause of mortality in women with breast cancer, thus advances in treatment will depend on therapeutic strategies targeting CSCs. Salinomycin (SAL) is a naturally occurring polyether ionophore antibiotic known for its anticancer activity towards several types of tumor cells. In the present work, a library of 17 C1-single and C1/C20-double modified SAL analogs was screened to identify compounds with improved activity against breast CSCs. Six single- and two double-modified analogs were more potent (IC50 range of 1.1 ± 0.1-1.4 ± 0.2 µM) toward the breast cancer cell line MDA-MB-231 compared to SAL (IC50 of 4.9 ± 1.6 µM). Double-modified compound 17 was found to be more efficacious than SAL against the majority of cancer cell lines in the NCI-60 Human Tumor Cell Line Panel. Compound 17 was more potent than SAL in inhibiting cell migration and cell renewal properties of MDA-MB-231 cells, as well as inducing selective loss of the CD44+/CD24/low stem-cell-like subpopulation in both monolayer (2D) and organoid (3D) culture. The present findings highlight the therapeutic potential of SAL analogs towards breast CSCs and identify select compounds that merit further study and clinical development.

Characterization of cannabinoid receptors expressed in Ewing sarcoma TC-71 and A-673 cells as potential targets for anti-cancer drug development.

AIMS: Characterizing cannabinoid receptors (CBRs) expressed in Ewing sarcoma (EWS) cell lines as potential targets for anti-cancer drug development. MAIN METHODS: CBR affinity and function were examined by competitive binding and G-protein activation, respectively. Cannabinoid-mediated cytotoxicity and cell viability were evaluated by LDH, and trypan blue assays, respectively. KEY FINDINGS: qRT-PCR detected CB1 (CB1R) and CB2 receptor (CB2R) mRNA in TC-71 cells. However, binding screens revealed that CBRs expressed exhibit atypical properties relative to canonical receptors, because specific binding in TC-71 could only be demonstrated by the established non-selective CB1/CB2R radioligand [3H]WIN-55,212-2, but not CB1/CB2R radioligand [3H]CP-55,940. Homologous receptor binding demonstrated that [3H]WIN-55,212-2 binds to a single site with nanomolar affinity, expressed at high density. Further support for non-canonical CBRs expression is provided by subsequent binding screens, revealing that only 9 out of 28 well-characterized cannabinoids with high affinity for canonical CB1 and/or CB2Rs were able to displace [3H]WIN-55,212-2, whereas two ligands enhanced [3H]WIN-55,212-2 binding. Five cannabinoids producing the greatest [3H]WIN-55,212-2 displacement exhibited high nanomolar affinity (Ki) for expressed receptors. G-protein modulation and adenylyl cyclase assays further indicate that these CBRs exhibit distinct signaling/functional profiles compared to canonical CBRs. Importantly, cannabinoids with the highest affinity for non-canonical CBRs reduced TC-71 viability and induced cytotoxicity in a time-dependent manner. Studies in a second EWS cell line (A-673) showed similar atypical binding properties of expressed CBRs, and cannabinoid treatment produced cytotoxicity. SIGNIFICANCE: Cannabinoids induce cytotoxicity in EWS cell lines via non-canonical CBRs, which might be a potential therapeutic target to treat EWS.

Phosphoproteomics Provides Novel Insights into the Response of Primary Acute Lymphoblastic Leukemia Cells to Microtubule Depolymerization in G1 Phase of the Cell Cycle.

Microtubule targeting agents (MTAs) have been used for the treatment of cancer for many decades and are among the most successful chemotherapeutic agents. However, their application and effectiveness are limited because of toxicity and resistance as well as a lack of knowledge of molecular mechanisms downstream of microtubule inhibition. Insights into key pathways that link microtubule disruption to cell death is critical for optimal use of these drugs, for defining biomarkers useful in patient stratification, and for informed design of drug combinations. Although MTAs characteristically induce death in mitosis, microtubule destabilizing agents such as vincristine also induce death directly in G1 phase in primary acute lymphoblastic leukemia (ALL) cells. Because many signaling pathways regulating cell survival and death involve changes in protein expression and phosphorylation, we undertook a comprehensive quantitative proteomic study of G1 phase ALL cells treated with vincristine. The results revealed distinct alterations associated with c-Jun N-terminal kinase signaling, anti-proliferative signaling, the DNA damage response, and cytoskeletal remodeling. Signals specifically associated with cell death were identified by pre-treatment with the CDK4/6 inhibitor palbociclib, which caused G1 arrest and precluded death induction. These results provide insights into signaling mechanisms regulating cellular responses to microtubule inhibition and provide a foundation for a better understanding of the clinical mechanisms of MTAs and for the design of novel drug combinations. The mass spectrometry proteomics data have been deposited to the PRIDE Archive (http://www.ebi.ac.uk/pride/archive/) via the PRIDE partner repository with the data set identifier PXD027190 and 10.6019/PXD027190.

Synthesis and Anticancer Activity of Tertiary Amides of Salinomycin and Their C20-oxo Analogues.

The polyether ionophore salinomycin (SAL) has captured much interest because of its potent activity against cancer cells and cancer stem cells. Our previous studies have indicated that C1/C20 double-modification of SAL is a useful strategy to generate diverse agents with promising biological activity profiles. Thus, herein we describe the synthesis of a new class of SAL analogues that combine key modifications at the C1 and C20 positions. The activity of the obtained SAL derivatives was evaluated using primary acute lymphoblastic leukemia, human breast adenocarcinoma and normal mammary epithelial cells. One single- [N,N-dipropyl amide of salinomycin (5 a)] and two novel double-modified analogues [N,N-dipropyl amide of C20-oxosalinomycin (5 b) and piperazine amide of C20-oxosalinomycin (13 b)] were found to be more potent toward the MDA-MB-231 cell line than SAL or its C20-oxo analogue 2. When select analogues were tested against the NCI-60 human tumor cell line panel, 4 a [N,N-diethyl amide of salinomycin] showed particular activity toward the ovarian cancer cell line SK-OV-3. Additionally, both SAL and 2 were found to be potent ex vivo against human ER/PR+ , Her2- invasive mammary carcinoma, with 2 showing minimal toxicity toward normal epithelial cells. The present findings highlight the therapeutic potential of SAL derivatives for select targeting of different cancer types.

Contrasting effects of microtubule destabilizers versus stabilizers on induction of death in G1 phase of the cell cycle.

Microtubule targeting agents (MTAs) have been reported to manifest their cytotoxic effects not only in mitosis but also in interphase. However, the relationship between phase-specific susceptibility and MTA concentration, especially with respect to microtubule integrity, remains poorly defined. In addition, whether microtubule stabilizers and destabilizers act similarly or differ in the ability to induce interphase death is unclear. In order to resolve these uncertainties, we report here the results of a systematic comparison of primary acute lymphoblastic leukemia (ALL) and HeLa cells treated with three different MTAs, namely the microtubule stabilizer paclitaxel and two microtubule destabilizers, vincristine, and eribulin. Both types of cells were sensitive to each MTA, with IC50 values in the sub-nanomolar to low nanomolar range. Primary ALL cells arrested in mitosis when treated with paclitaxel at all tested concentrations, whereas the effects of vincristine or eribulin were concentration-dependent; low (<30 nM) concentrations induced mitotic death whereas higher concentrations (>100 nM) induced death directly in G1 phase. G1 phase death in response to higher concentrations of the destabilizers was associated with complete loss of interphase microtubule structure. In contrast, HeLa cells were only susceptible in M phase regardless of drug type or concentration. These results represent an important advance in our understanding and appreciation of microtubule function, and indicate that susceptibility to MTAs in G1 phase is both cell type- and drug type-restricted. The findings have important implications for the clinical use of MTAs especially in the context of drug combinations.

The response of phyllodes tumor of the breast to anticancer therapy: An in vitro and ex vivo study.

Phyllodes tumors of the breast (PTB) are uncommon stromal-epithelial neoplasms, with the main recommended treatment being surgical removal. However, even with adequate resection, the risk of recurrence in the malignant form remains as high as 40%, and there is no recognized consensus on the most effective drugs for PTB. In the present study, an ex vivo model of malignant phyllodes and derived primary cell cultures were used to evaluate the effectiveness of a panel of different drugs, including the Bcl-2/Bcl-xL inhibitor ABT-263, salinomycin (SAL), doxorubicin (DOX), paclitaxel (TAX), vincristine (VCR), colchicine (COL) and cisplatin (CIS). ABT-263, SAL and DOX were highly effective towards phyllodes spindle cells when assessed in the ex vivo model, contributing to ~98% tumor cell death. Furthermore, ABT-263 was highly selective for tumor cells in this system, and exhibited little toxic effect on adjacent normal epithelial cells. Furthermore, consistent with findings in the ex vivo model, ABT-263 was significantly less toxic towards MCF 10A non-tumorigenic breast epithelial cells compared with SAL and DOX. A conditional reprogramming strategy was subsequently used, involving Rho kinase inhibition, to successfully generate primary phyllodes tumor cells that could be cultured for several passages. The primary cells were sensitive to DOX with an IC50 of 0.40±0.07 µM in a standard viability assay and the preliminary results were obtained indicating sensitivity to ABT-263 and SAL. The present study demonstrated the feasibility of using explants and primary cells for drug discovery, selectively targeting PTB cells.

Salinomycin derivatives exhibit activity against primary acute lymphoblastic leukemia (ALL) cells in vitro.

Salinomycin (SAL) and monensin (MON) are polyether ionophore antibiotics commonly used in veterinary medicine. They are known from their anti-cancer activity against various types of cancer cells, including those that display multi-drug resistance as well as cancer stem cells. In order to increase the biological activity profile and reduce toxicity against normal cells, while retaining the activities in the micromolar range, a library of ester and amide derivatives of SAL was synthesized and previously reported. In this paper, we examined the activity of SAL, its ten derivatives, and MON on primary acute lymphoblastic leukemia cells. MON and six SAL derivatives were more potent than SAL in cell viability assays. Further, selected active SAL analogs induced characteristics of apoptotic cell death and increased expression of p53. Moreover, SAL acted synergistically with the Bcl-2 inhibitor ABT-263, whereas 2,2,2-trifluoroethyl ester, the most active analog of SAL, antagonized ABT-263, suggesting possible differences in molecular mechanism.