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Cytoskeletal filaments such as microtubules (MTs) and filamentous actin (F-actin) dynamically support cell structure and functions. In central presynaptic terminals, F-actin is expressed along the release edge and reportedly plays diverse functional roles, but whether axonal MTs extend deep into terminals and play any physiological role remains controversial. At the calyx of Held in rats of either sex, confocal and high-resolution microscopy revealed that MTs enter deep into presynaptic terminal swellings and partially colocalize with a subset of synaptic vesicles (SVs). Electrophysiological analysis demonstrated that depolymerization of MTs specifically prolonged the slow-recovery time component of EPSCs from short-term depression induced by a train of high-frequency stimulation, whereas depolymerization of F-actin specifically prolonged the fast-recovery component. In simultaneous presynaptic and postsynaptic action potential recordings, depolymerization of MTs or F-actin significantly impaired the fidelity of high-frequency neurotransmission. We conclude that MTs and F-actin differentially contribute to slow and fast SV replenishment, thereby maintaining high-frequency neurotransmission. SIGNIFICANCE STATEMENT The presence and functional role of MTs in the presynaptic terminal are controversial. Here, we demonstrate that MTs are present near SVs in calyceal presynaptic terminals and that MT depolymerization specifically prolongs the slow-recovery component of EPSCs from short-term depression. In contrast, F-actin depolymerization specifically prolongs fast-recovery component. Depolymerization of MT or F-actin has no direct effect on SV exocytosis/endocytosis or basal transmission, but significantly impairs the fidelity of high-frequency transmission, suggesting that presynaptic cytoskeletal filaments play essential roles in SV replenishment for the maintenance of high-frequency neurotransmission., (Copyright © 2020 Piriya Ananda Babu, Wang et al.)

Background: In oncology, the aim of dose-finding phase I studies is to find the maximum tolerated dose for further studies. The use of combinations of two or more agents is increasing. Several dose-finding designs have been proposed for this situation. Numerous publications have however pointed out the complexity of evaluating therapies in combination due to difficulties in choosing between different designs for an actual trial, as well as complications related to their implementation and application in practice., Methods: In this work, we propose R functions for Wang and Ivanova's approach. These functions compute the dose for the next patients enrolled and provide a simulation study in order to calibrate the design before it is applied and to assess the performance of the design in different scenarios of dose-toxicity relationships. This choice of the method was supported by a simulation study which the aim was to compare two designs in the context of an actual phase I trial: i) in 2005, Wang and Ivanova developed an empirical three-parameter model-based method in Bayesian inference, ii) in 2008, Yuan and Yin proposed a simple, adaptive two-dimensional dose-finding design. In particular, they converted the two-dimensional dose-finding trial to a series of one-dimensional dose-finding sub-trials by setting the dose of one drug at a fixed level. The performance assessment of Wang's design was then compared with those of designs presented in the paper by Hirakawa et al. (2015) in their simulation context., Results and Conclusion: It is recommended to assess the performances of the designs in the context of the clinical trial before beginning the trial. The two-dimensional dose-finding design proposed by Wang and Ivanova is a comprehensive approach that yields good performances. The two R functions that we propose can facilitate the use of this design in practice., Competing Interests: The author has declared that no competing interests exist.

Vinblastine (VLB) and its derivatives have been used for clinical first-line drugs to treat various cancers. Due to the resistance and serious side effects from using VLB and its derivatives, there is a need to discover and develop novel VLB derivatives with high activity against cancer cells. In order to better discover and develop new VLB derivatives, we need to study the structural basis of VLB's anti-cancer cytotoxicity and the mechanism of its interaction with α,β-tubulins. Based on the crystal structure of α,β-microtubule complex protein, the molecular dynamics method including the sampling PMF method was used to study the variation of dissociation free energy (Δ G ) of α,β-tubulins under different system conditions, and then from which to study the mechanism of the interaction between VLB and α,β-tubulins. The obtained results show that the dissociation of pure α,β-tubulins requires 197.8 kJ·mol -1 for Δ G . When the VLB molecule exists between the interface of α,β-tubulins, the dissociation Δ G of α,β-tubulins reaches 220.5 kJ·mol -1 , which is greater than that of pure α,β-tubulin. The VLB molecule is formed by connecting a vindoline moiety (VM) molecule with a catharanthine moiety (CM) molecule through a carbon-carbon bond, which is a larger molecule. When the CM molecule exists in the middle of α,β-tubulin interface, the dissociation Δ G of α,β-tubulins is 46.2 kJ·mol -1 , during which the CM moves with β-tubulin. When the VM molecule exists between the middle of α,β-tubulin interface, the dissociation Δ G of α,β-tubulins is 86.7 kJ·mol -1 , during which it moves with α-tubulin. Therefore, the VLB molecule is like a double-sides tape to stick α-tubulin and β-tubulin together. The VLB molecule intervenes the dynamic equilibrium between dissociation and aggregation of α-tubulin and β-tubulin by a double-sides sticking mechanism to exert high activity with toxicity against cancer cell. Besides, our results demonstrate that VLB has its structural basis for anticancer cytotoxicity due to its two compositions composed of a CM molecule and a VM molecule although they have little toxicity against cancer cell alone.

Carbon-based nanomaterials (CBNs) were previously described as regulators of plant cell division. Here, we demonstrated the ability of multi-walled carbon nanotubes (MWCNT) and graphene to enhance biomass production in callus culture of the medicinal plant Catharanthus roseus cultivated in dark conditions. Furthermore, both tested CBNs were able to stimulate biosynthesis of total produced alkaloids in CBN-exposed callus culture of Catharanthus. In one case, total alkaloids in CBN-exposed Catharanthus were double that of unexposed Catharanthus. Analysis of metabolites by HPLC revealed that production of the pharmaceutically active alkaloids vinblastine and vincristine was dramatically enhanced in callus exposed to MWCNT or graphene in both dark and light conditions of callus cultivation. In vitro assays (MTT, flow cytometry) demonstrated that total alkaloid extracts derived from Catharanthus callus treated with CBNs significantly reduced cell proliferation of breast cancer (MCF-7) and lung cancer (A549) cell lines compared to the application of extracts derived from untreated Catharanthus callus.

(1) Background: The high-grade neuroepithelial tumor of the central nervous system with BCOR alteration (HGNET-BCOR) is a highly malignant tumor. Preclinical models and molecular targets are urgently required for this cancer. Previous data suggest a potential role of insulin-like growth factor (IGF) signaling in HGNET-BCOR. (2) Methods: The primary HGNET-BCOR cells PhKh1 were characterized by western blot, copy number variation, and methylation analysis and by electron microscopy. The expression of IGF2 and IGF1R was assessed by qRT-PCR. The effect of chemotherapeutics and IGF1R inhibitors on PhKh1 proliferation was tested. The phosphorylation of IGF1R and downstream molecules was assessed by western blot. (3) Results: Phkh1 cells showed a DNA methylation profile compatible with the DNA methylation class "HGNET-BCOR" and morphologic features of cellular cannibalism. IGF2 and IGF1R were highly expressed by three HGNET-BCOR tumor samples and PhKh1 cells. PhKh1 cells were particularly sensitive to vincristine, vinblastine, actinomycin D (IC 50 < 10 nM for all drugs), and ceritinib (IC 50 = 310 nM). Ceritinib was able to abrogate the proliferation of PhKh1 cells and blocked the phosphorylation of IGF1R and AKT. (4) Conclusion: IGF1R is as an attractive target for the development of new therapy protocols for HGNET-BCOR patients, which may include ceritinib and vinblastine.

The role of Ann Arbor staging in determining treatment intensity after achieving a negative positron emission tomography (PET) has not been established in classical Hodgkin lymphoma (cHL). Patients with stage I-IV cHL, received three cycles of ABVD (doxorubicin, bleomycin, vinblastine, dacarbazine) and an interim PET scan (PET3). PET3-negative patients received no further therapy. PET3-positive patients received three additional cycles of ABVD plus involved-field radiation therapy or salvage chemotherapy, if refractory to ABVD, and were re-evaluated by PET scan (PET6). Study endpoints were 3-year progression-free survival (PFS) and overall survival (OS) rates. Two hundred and thirty-nine patients with early-stage and 138 with advanced-stage were evaluable. Overall, 260 patients (70%) were PET3-negative and had higher 3-year PFS (90% vs. 65%; P < 0·0001) and OS (98% vs. 92%; P = 0·007) rates than PET3-positive patients. All PET3-negative patients, regardless of disease stage at diagnosis, achieved similarly good PFS (90-91%; P = 0·76) and OS (97-99%). The only independent prognostic factor for PFS was PET3-negativity (Hazard ratio 3·8; 95% confidence interval 2·4-6·3; P < 0·0001). This study suggests that cHL patients who achieve a negative PET3 following ABVD have an excellent outcome, regardless of stage at diagnosis. An appropriately powered, phase III trial will be necessary to confirm these findings., (© 2019 British Society for Haematology and John Wiley & Sons Ltd.)

In recent efforts, several C20' urea vinblastine analogues were discovered that displayed remarkable potency against vinblastine-sensitive tumor cell lines (IC 50 50-75 pM), being roughly 100-fold more potent than vinblastine, and that exhibited decreased susceptibility to Pgp efflux-derived resistance in a vinblastine-resistant cell line. Their extraordinary activity indicate that it is not likely or even possible that their cellular functional activity is derived from stoichiometric occupancy of the intracellular tubulin binding sites. Rather, their potency indicates sub-stoichiometric or even catalytic occupancy of candidate binding sites may be sufficient to disrupt tubulin dynamics or microtubule assembly during mitosis. We detail efforts to delineate the underlying behavior responsible for the increased potency and show that the ultra-potent extended C20' ureas retain the mechanistic behavior of vinblastine, display enhanced affinity for tubulin and on-target activity approximately 100-fold both in vitro and in HeLa cells, but do not show evidence of catalytic disassembly of microtubulin. We also use the analogues to show that, in live interphase cells, the effects of the vinblastine class of drugs do not display a catastrophic effect on the microtubule skeleton, but rather a subtler insult to its dynamicity, acting as sub-stoichiometric drugs that inhibit normal microtubulin maturation and dynamics., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

In addition to their major targets, clinically effective drugs may have unknown off-targets. By identifying such off-targets it may be possible to repurpose approved drugs for new indications. We are interested in the Golgi apparatus as a novel target for cancer therapy, but there is a paucity of candidate Golgi-disrupting drugs. Here, we aimed to identify Golgi-disrupting compounds from a panel of 34 approved anticancer drugs by using HBC-4 human breast cancer cells and immunofluorescence microscopy to visualize the Golgi apparatus. The screen identified five drugs having Golgi-disrupting activity. Four of them were vinca alkaloids (vinorelbine, vindesine, vincristine and vinblastine), and the fifth drug was eribulin. This is the first study to demonstrate that vinorelbine, vindesine and eribulin possess Golgi-disrupting activity. The 5 drugs are known to inhibit tubulin polymerization and to induce microtubule depolymerization. Interestingly, a microtubule-stabilizer paclitaxel did not induce Golgi-disruption, suggesting that the three-dimensionally preserved microtubules are partly responsible for maintaining the Golgi complex. Concerning eribulin, a noteworthy drug because of its high clinical efficacy against advanced breast cancer, we further confirmed its Golgi-disrupting activity in 3 different human breast cancer cell lines, BSY-1, MDA-MB-231 and MCF-7. Golgi-disruption may contribute to anticancer efficacy of eribulin. In conclusion, the present study revealed that 4 vinca alkaloids and eribulin possessed potential Golgi-disrupting activity among a panel of 34 approved anticancer drugs. Other drugs covering various molecular-targeted drugs and classical DNA-damaging drugs showed no Golgi-disrupting effect. These results suggest that tubulin polymerization-inhibitors might be promising candidate drugs with Golgi-disrupting activity.

Objectives: Vast number of studies show the relationship between aneuploidy and cancer. Ionizing radiation in addition to induce all kinds of damages to the cells and structure of chromosomes, is also able to induce aneuploidy through direct damages to chromosome division apparatus. Also irradiation of the cells induces mutations in several genes which might be involved in cell division fidelity and play a role in reversing the effect of aneugens. Therefore, irradiation of cells and tissues might produce sensitivity to agents with aneugenic capability in irradiated cells. Methods: To investigate the persistent genomic effect of ionizing irradiation on chromosomal instability, L929 cells were gamma irradiated with the dose of 2 Gy. Cells were left to recover from the harmful effect of irradiation. They were treated with low dose of vinblastine (0.5 ng.ml-1) 72h post-gamma irradiation. Finally, the induced chromosomal abnormalities were scored using micronucleus assay in cytokinesis-blocked binucleated cells (MnBi). Results: Irradiation-recovered L929 cells treated with vinblastine showed a statistically higher frequency of MnBi compared to non-irradiated and vinblastine treated cells. Conclusion: The results indicate that gamma irradiation, in addition to direct induction of chromosomal damages, is also able to create persisting genomic sensitivity in the cells to chromosomal instability, which is detectable when exposed to the second stimulus., (Creative Commons Attribution License)

Microtubules (MTs) are protein filaments found in all eukaryotic cells which are crucial for many cellular processes including cell movement, cell differentiation, and cell division. Due to their role in cell division, they are often used as targets for chemotherapy drugs used in cancer treatment. Experimental studies of MT dynamics have played an important role in the development and administration of many novel cancer drugs; however, a complete description of MT dynamics is lacking. Here, we propose a new mathematical model for MT dynamics, that can be used to study the effects of chemotherapy drugs on MT dynamics. Our model consists of a growth-fragmentation equation describing the dynamics of a length distribution of MTs, coupled with two ODEs that describe the dynamics of free GTP- and GDP-tubulin concentrations (the individual dimers that comprise of MTs). Here, we prove the well-posedness of our system and perform a numerical exploration of the influence of certain model parameters on the systems dynamics. In particular, we focus on a qualitative description for how a certain class of destabilizing drugs, the vinca alkaloids, alter MT dynamics. Through variation of certain model parameters which we know are altered by these drugs, we make comparisons between simulation results and what is observed in in vitro studies.

Microtubule-targeting chemotherapies are linked to impaired cellular metabolism, which may contribute to skeletal muscle dysfunction. However, the mechanisms by which metabolic homeostasis is perturbed remains unknown. Tubulin, the fundamental unit of microtubules, has been implicated in the regulation of mitochondrial-cytosolic ADP/ATP exchange through its interaction with the outer membrane voltage-dependent anion channel (VDAC). Based on this model, we predicted that disrupting microtubule architecture with the stabilizer paclitaxel and destabilizer vinblastine would impair skeletal muscle mitochondrial bioenergetics. Here, we provide in vitro evidence of a direct interaction between both α-tubulin and βII-tubulin with VDAC2 in untreated single extensor digitorum longus (EDL) fibers. Paclitaxel increased both α- and βII-tubulin-VDAC2 interactions, whereas vinblastine had no effect. Utilizing a permeabilized muscle fiber bundle preparation that retains the cytoskeleton, paclitaxel treatment impaired the ability of ADP to attenuate H 2 O 2 emission, resulting in greater H 2 O 2 emission kinetics. Despite no effect on tubulin-VDAC2 binding, vinblastine still altered mitochondrial bioenergetics through a surprising increase in ADP-stimulated respiration while also impairing ADP suppression of H 2 O 2 and increasing mitochondrial susceptibility to calcium-induced formation of the proapoptotic permeability transition pore. Collectively, these results demonstrate that altering microtubule architecture with chemotherapeutics disrupts mitochondrial bioenergetics in EDL skeletal muscle. Specifically, microtubule stabilization increases H 2 O 2 emission by impairing ADP sensitivity in association with greater tubulin-VDAC binding. In contrast, decreasing microtubule abundance triggers a broad impairment of ADP's governance of respiration and H 2 O 2 emission as well as calcium retention capacity, albeit through an unknown mechanism.

Objectives: The aim of this study was to elucidate the antimitotic mechanism of zerumbone and to investigate its effect on the HeLa cells in combination with other mitotic blockers., Materials and Methods: HeLa cells and fluorescence microscopy were used to analyse the effect of zerumbone on cancer cell lines. Cellular internalization of zerumbone was investigated using FITC-labelled zerumbone. The interaction of zerumbone with tubulin was characterized using fluorescence spectroscopy. The Chou and Talalay equation was used to calculate the combination index., Results: Zerumbone selectively inhibited the proliferation of HeLa cells with an IC 50 of 14.2 ± 0.5 μmol/L through enhanced cellular uptake compared to the normal cell line L929. It induced a strong mitotic block with cells exhibiting bipolar spindles at the IC 50 and monopolar spindles at 30 μmol/L. Docking analysis indicated that tubulin is the principal target of zerumbone. In vitro studies indicated that it bound to goat brain tubulin with a Kd of 4 μmol/L and disrupted the assembly of tubulin into microtubules. Zerumbone and colchicine had partially overlapping binding site on tubulin. Zerumbone synergistically enhanced the anti-proliferative activity of vinblastine and paclitaxel through augmented mitotic block., Conclusion: Our data suggest that disruption of microtubule assembly dynamics is one of the mechanisms of the anti-cancer activity of zerumbone and it can be used in combination therapy targeting cell division., (© 2018 The Authors. Cell Proliferation Published by John Wiley & Sons Ltd.)

NLRX1, the mitochondrial NOD-like receptor (NLR), modulates apoptosis in response to both intrinsic and extrinsic cues. Insights into the mechanism of how NLRX1 influences apoptosis remain to be determined. Here, we demonstrate that NLRX1 associates with SARM1, a protein with a toll/interleukin-1 receptor (TIR)-containing domain also found in adaptor proteins downstream of toll-like receptors, such as MyD88. While a direct role of SARM1 in innate immunity is unclear, the protein plays essential roles in Wallerian degeneration (WD), a type of neuronal catabolism occurring following axonal severing or damage. In non-neuronal cells, we found that endogenous SARM1 was equally distributed in the cytosol and the mitochondrial matrix, where association with NLRX1 occurred. In these cells, the apoptotic role of NLRX1 was fully dependent on SARM1, indicating that SARM1 was downstream of NLRX1 in apoptosis regulation. In primary murine neurons, however, Wallerian degeneration induced by vinblastine or NGF deprivation occurred in SARM1- yet NLRX1-independent manner, suggesting that WD requires the cytosolic pool of SARM1 or that NLRX1 levels in neurons are too low to contribute to WD regulation. Together, these results shed new light into the mechanisms through which NLRX1 controls apoptosis and provides evidence of a new link between NLR and TIR-containing proteins.

The antitussive agent noscapine has been shown to inhibit the proliferation of cancer cells by disruption of tubulin dynamic. However, the efficacy of several anticancer drugs that inhibit tublin dynamics (vinca alkaloids and taxanes) is reduced by the multidrug resistance phenotype. These compounds are substrates for P-glycoprotein (P-gp)-mediated extrusion from cells. Consequently, the antiproliferative activity of noscapine and a series of derivatives was measured in drug-sensitive and drug-resistant cells that overexpress P-gp. None of the noscapine derivatives displayed lower potency in cells overexpressing P-gp, thereby suggesting a lack of interaction with this pump. However, the cellular efflux of a fluorescent substrate by P-gp was potently inhibited by noscapine and most derivatives. Further investigation with purified, reconstituted P-gp demonstrated that inhibition of P-gp function was due to direct interaction of noscapine derivatives with the transporter. Moreover, coadministration of vinblastine with two of the noscapine derivatives displayed synergistic inhibition of proliferation, even in P-gp-expressing resistant cell lines. Therefore, noscapine derivatives offer a dual benefit of overcoming the significant impact of P-gp in conferring multidrug resistance and synergy with tubulin-disrupting anticancer drugs., (Copyright © 2019 by The American Society for Pharmacology and Experimental Therapeutics.)

5-Hydroxytryptophan (5-HTP), a precursor of serotonin, is therapeutically used for several psychiatric disorders such as anxiety and depression in the clinic. However, severe side effects, including abnormal mental functions, behavioral disturbances and intolerance are associated with this treatment. 5-HTP-induced elevation of plasma and brain serotonin levels may affect blood-brain barrier (BBB) breakdown, edema formation and regional cerebral blood flow (CBF) disturbances. Breakdown of BBB to serum proteins leads to vasogenic brain edema formation and cellular injuries. However, 5-HTP-neurotoxicity is still not well known. In this investigations 5-HTP induced elevation of endogenous plasma and brain serotonin levels and its effect on BBB breakdown, edema formation neuronal injuries was examined in a rat model. Furthermore, potential role of oxidative stress and nitric oxide (NO) was evaluated. In addition, several neurochemical agents such as p-CPA (5-HT synthesis inhibitor) indomethacin (prostaglandin synthase inhibitor), diazepam (ant stress drug), cyproheptadine, ketanserin (5-HT2 receptor antagonists) and vinblastine (inhibitor of microtubule function) were examined on 5-HT neurotoxicity. Our observations suggest that 4h after 5-HTP administrations, the endogenous serotonin levels increased by fourfold (150mg/kg) in the plasma and brain associated with profound hyperthermia (+3.86±0.24°C, oxidative stress and NO upregulation. Breakdown of the BBB to Evans blue albumin (EBA) in 8 brain regions and to [131] Iodine in 14 brain regions was observed. The CBF exhibited marked reduction in all the brain regions examined. Brain edema and cellular injuries are present in the areas associated with BBB disruption. Drug treatments reduced the BBB breakdown, edema formation NO production and brain pathology. These observations are the first to point out that 5-HTP-neurotoxicity caused by BBB breakdown, edema formation and NO production is instrumental in causing adverse mental and behavioral abnormalities, not reported earlier., (© 2019 Elsevier Inc. All rights reserved.)

In this work, a new sphingomyelin-cholesterol liposomal formulation (CPD100Li) for the delivery of a hypoxia activated prodrug of vinblastine, mon-N-oxide (CPD100), is developed. The optimized liposomal formulation uses an ionophore (A23187) mediated pH-gradient method. Optimized CPD100Li is characterized for size, drug loading, and stability. The in vitro toxicity of CPD100Li is assessed on different aspects of cell proliferation and apoptosis of ES2 ovarian cancer under normoxic and hypoxic conditions. The pharmacokinetics of CPD100Li in mice as well as the influence of A23187 on the retention of CPD100 are assessed. The dose limiting toxicity (DLT) and maximum tolerated dose (MTD) for CPD100Li are evaluated in nude mice. CPD100 is loaded in the liposome at 5.5 mg/mL. The sizes of CPD100Li using DLS, qNano and cryo-TEM techniques are 155.4 ± 4.2 nm, 132 nm, and 112.6 ± 19.8 nm, respectively. There is no difference between the in vitro characterization of CPD100Li with and without ionophore. Freshly prepared CPD100Li with ionophore are stable for 48 h at 4 °C, while the freeze-dried formulation is stable for 3 months under argon at 4 °C. The hypoxic cytotoxicity ratios (HCR) of CPD100 and CPD100Li are 0.16 and 0.11, respectively. CPD100Li under hypoxic conditions has a 9.2-fold lower IC 50 value as compared to CPD100Li under normoxic conditions, confirming the hypoxia dependent activation of CPD100. CPD100Li treated ES2 cells show a time dependent enhanced cell death, along with caspase production and an increase in the number of cells in G 0 /G 1 and higher cell arrest. The blood concentration profile of CPD100Li in mice without A23187 has a 12.6-fold lower area under the curve (AUC) and 1.6-fold lower circulation time compared to the CPD100Li with A23187. The DLT for both CPD100 and CPD100Li is 45 mg/kg and the MTD is 40 mg/kg in nude mice. Based on the preliminary data obtained, we clearly show that the presence of ionophore affects the in vivo stability of CPD100. CPD100Li presents a unique opportunity to develop a first-in-kind chemotherapy product based on achieving selective drug activation through the hypoxic physiologic microenvironment of solid tumors., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Efflux transporters function as vacuum cleaners of xenobiotics and therefore they hinder drugs to reach their targets at effective enough concentrations. Efflux pump inhibitors can be used to improve the cell accumulation of drugs, however all the current inhibitors lack selectivity towards cancer cells. l-Type amino acid transporter 1 (LAT1), which is expressed in many types of cancer cells can be utilized to target inhibitors of efflux transporters to these cells by converting the inhibitors into LAT1-utilizing prodrugs. In this study, we prepared 5 LAT1-utilizing prodrugs of an efflux pump inhibitor, probenecid (PRB). All novel compounds were transported into human breast cancer cells (MCF-7) mainly via LAT1. The compounds also interacted with either multiresistant proteins (MRPs), P-glycoprotein (P-gp) or breast cancer resistant protein (BCRP) and increased significantly (3-4-fold) the cellular accumulation of anti-cancer agent vinblastine (VBL). Consequently, this improved the anti-proliferative efficacy of VBL by decreasing the cell growth after 72 h from 100% (VBL treatment alone) to 48-75% (combination treatment). However, the same phenomenon was not seen with other chemotherapeutic, methotrexate (MTX). Therefore, the chemotherapeutics need to be selected carefully based on their uptake mechanism to the combinations with LAT1-utilizing prodrugs of efflux pump inhibitors to defeat effectively the multidrug resistance (MDR) of chemotherapy., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Germline BRCA1 mutations predispose women to breast and ovarian cancer. BRCA1, a large protein with multiple functional domains, interacts with numerous proteins involved in many important biological processes and pathways. However, to date, the role of BRCA1 interactions at specific stages in the progression of mammary tumors, particularly in relation to cell cycle regulation, remains elusive. Here, we demonstrate that BRCA1 interacts with cyclin B1, a crucial cell cycle regulator, and that their interaction is modulated by DNA damage and cell cycle phase. In DNA-damaged mitotic cells, BRCA1 inhibits cytoplasmic transportation of cyclin B1, which prevents cyclin B1 degradation. Moreover, restoration of cyclin B1 in BRCA1-deficient cells reduced cell survival in association with induction of apoptosis. We further demonstrate that treatment of Brca1-mutant mammary tumors with vinblastine, which induces cyclin B1, significantly reduced tumor progression. In addition, a correlation analysis of vinblastine responses and gene expression profiles in tumors at baseline revealed 113 genes that were differentially expressed between tumors that did and did not respond to vinblastine treatment. Further analyses of protein-protein interaction networks revealed gene clusters related to vinblastine resistance, including nucleotide excision repair, epigenetic regulation, and the messenger RNA surveillance pathway. These findings enhance our understanding of how loss of BRCA1 disrupts mitosis regulation through dysregulation of cyclin B1 and provide evidence suggesting that targeting cyclin B1 may be useful in BRCA1-associated breast cancer therapy.

Some Vinca alkaloids (eg, vinblastine, vincristine) have been widely used as antitumor drugs for a long time. Unfortunately, vindoline, a main alkaloid component of Catharanthus roseus (L.) G. Don, itself, has no antitumor activity. In our novel research program, we have prepared and identified new vindoline derivatives with moderate cytostatic activity. Here, we describe the effect of conjugation of vindoline derivative with oligoarginine (tetra-, hexa-, or octapeptides) cell-penetrating peptides on the cytostatic activity in vitro and in vivo. Br-Vindoline-(l)-Trp-OH attached to the N-terminus of octaarginine was the most effective compound in vitro on HL-60 cell line. Analysis of the in vitro activity of two isomer conjugates (Br-vindoline-(l)-Trp-Arg 8 and Br-vindoline-(d)-Trp-Arg 8 suggests the covalent attachment of the vindoline derivatives to octaarginine increased the antitumor activity significantly against P388 and C26 tumour cells in vitro. The cytostatic effect was dependent on the presence and configuration of Trp in the conjugate as well as on the cell line studied. The configuration of Trp notably influenced the activity on C26 and P388 cells: conjugate with (l)-Trp was more active than conjugate with the (d)-isomer. In contrast, conjugates had very similar effect on both the HL-60 and MDA-MB-231 cells. In preliminary experiments, conjugate Br-vindoline-(l)-Trp-Arg 8 exhibited some inhibitory effect on the tumor growth in P388 mouse leukemia tumor-bearing mice. Our results indicate that the conjugation of modified vindoline could result in an effective compound even with in vivo antitumor activity., (© 2018 European Peptide Society and John Wiley & Sons, Ltd.)

Niemann-Pick type C disease is a fatal, progressive neurodegenerative disorder caused by loss-of-function mutations in NPC1, a multipass transmembrane glycoprotein essential for intracellular lipid trafficking. We sought to define the cellular machinery controlling degradation of the most common disease-causing mutant, I1061T NPC1. We show that this mutant is degraded, in part, by the proteasome following MARCH6-dependent ERAD. Unexpectedly, we demonstrate that I1061T NPC1 is also degraded by a recently described autophagic pathway called selective ER autophagy (ER-phagy). We establish the importance of ER-phagy both in vitro and in vivo, and identify I1061T as a misfolded endogenous substrate for this FAM134B-dependent process. Subcellular fractionation of I1061T Npc1 mouse tissues and analysis of human samples show alterations of key components of ER-phagy, including FAM134B. Our data establish that I1061T NPC1 is recognized in the ER and degraded by two different pathways that function in a complementary fashion to regulate protein turnover.

Indirubin, a bis-indole alkaloid used in traditional Chinese medicine has shown remarkable anticancer activity against chronic myelocytic leukemia. The present work was aimed to decipher the underlying molecular mechanisms responsible for its anticancer attributes. Our findings suggest that indirubin inhibited the proliferation of HeLa cells with an IC 50 of 40 μM and induced a mitotic block. At concentrations higher than its IC 50 , indirubin exerted a moderate depolymerizing effect on the interphase microtubular network and spindle microtubules in HeLa cells. Studies with goat brain tubulin indicated that indirubin bound to tubulin at a single site with a dissociation constant of 26 ± 3 μM and inhibited the in vitro polymerization of tubulin into microtubules in the presence of glutamate as well as microtubule-associated proteins. Molecular docking analysis and molecular dynamics simulation studies indicate that indirubin stably binds to tubulin at the interface of the α-β tubulin heterodimer. Further, indirubin stabilized the binding of colchicine on tubulin and promoted the cysteine residue modification by 5,5'-dithiobis-2-nitrobenzoic acid, indicating towards alteration of tubulin conformation upon binding. In addition, we found that indirubin synergistically enhanced the anti-mitotic and anti-proliferative activity of vinblastine, a known microtubule-targeted agent. Collectively our studies indicate that perturbation of microtubule polymerization dynamics could be one of the possible mechanisms behind the anti-cancer activities of indirubin., (Copyright © 2018 Elsevier Masson SAS. All rights reserved.)

Indibulin, a synthetic inhibitor of tubulin assembly, has shown promising anticancer activity with a minimal neurotoxicity in preclinical animal studies and in Phase I clinical trials for cancer chemotherapy. Using time-lapse confocal microscopy, we show that indibulin dampens the dynamic instability of individual microtubules in live breast cancer cells. Indibulin treatment also perturbed the localization of end-binding proteins at the growing microtubule ends in MCF-7 cells. Indibulin reduced inter-kinetochoric tension, produced aberrant spindles, activated mitotic checkpoint proteins Mad2 and BubR1, and induced mitotic arrest in MCF-7 cells. Indibulin-treated MCF-7 cells underwent apoptosis-mediated cell death. Further, the combination of indibulin with an anticancer drug vinblastine was found to exert synergistic cytotoxic effects on MCF-7 cells. Interestingly, indibulin displayed a stronger effect on the undifferentiated neuroblastoma (SH-SY5Y) cells than the differentiated neuronal cells. Unlike indibulin, vinblastine and colchicine produced similar depolymerizing effects on microtubules in both differentiated and undifferentiated SH-SY5Y cells. The data indicated a possibility that indibulin may reduce chemotherapy-induced peripheral neuropathy in cancer patients.

Glioblastoma multiforme (GBM) is an aggressive primary brain cancer that includes focal amplification of PDGFRα and for which there are no effective therapies. Herein, we report the development of a genetically engineered mouse model of GBM based on autocrine, chronic stimulation of overexpressed PDGFRα, and the analysis of GBM signaling pathways using proteomics. We discover the tubulin-binding protein Stathmin1 (STMN1) as a PDGFRα phospho-regulated target, and that this mis-regulation confers sensitivity to vinblastine (VB) cytotoxicity. Treatment of PDGFRα-positive mouse and a patient-derived xenograft (PDX) GBMs with VB in mice prolongs survival and is dependent on STMN1. Our work reveals a previously unconsidered link between PDGFRα activity and STMN1, and highlight an STMN1-dependent cytotoxic effect of VB in GBM.

Vinblastine (VB), as a chemotherapeutic agent, is widely used in treatment of different types of cancer. However, its clinical application is limited due to its low water solubility, side effects, and multidrug resistance. The aim of this study was to increase the therapeutic efficacy of VB using drug delivery systems. For this purpose, a PEGylated niosomal formulation of vinblastine (Pn-VB) was prepared by thin film hydration method and physicochemically characterized. Drug release pattern was performed by dialysis diffusion method. The cytotoxicity of Pn-VB was investigated against murine lung cancer TC-1 cells using MTT assay and its tumor inhibitory effect was evaluated in lung tumor-bearing C57BL/6 mice. Mean particle size, zeta potential, entrapment, and loading efficiency of niosomes were obtained to be about 234.3 ± 11.4 nm, -34.6 ± 4.2 mV, 99.92 ± 1.6%, and 2.673 ± 0.30%, respectively. While, the mean particle size and zeta potential for non-PEGylated niosomes were obtained about 212.4 nm and -31.4 mV, respectively. The in vitro release pattern of drug from niosomes showed a sustained release behavior. Pn-VB indicated a significant increase in toxicity against TC-l cells as compared to free VB. In animal model, Pn-VB exhibited stronger tumor inhibitory effect and longer life time in comparison to free VB. In conclusion, Pn-VB showed appropriate stability, high-entrapment efficacy, lower releasing rate, and stronger cytotoxic activity against lung cancer TC-1 cells as compared to free drug. Thus, the Pn-VB could be a promising formulation for delivery of vinblastine to tumor cells with enhanced drug bioavailability and therapeutic efficacy.

Purpose: To assess the therapeutic potential of endothelin-converting enzyme-like 1 (Ecel1) in a mouse model of optic nerve crush., Methods: Ecel1 expression was evaluated with real time quantitative (qRT)-PCR, Western blotting, and immunohistochemistry in mouse retinas after optic nerve crush. Vinblastine administration to the optic nerve and the intravitreal injection of N-methyl-d-aspartate (NMDA) were used to assess Ecel1 gene expression. Ecel1 was deleted with an adeno-associated viral (AAV) clustered regulatory interspaced short palindromic repeat (CRISPR)/Cas9 system, and retinal ganglion cell (RGC) survival was investigated with retrograde labeling, qRT-PCR, and visual evoked potential., Results: Optic nerve crush induced Ecel1 expression specifically in the RGCs, peaking on day 4 after optic nerve crush. Ecel1 gene expression was induced by the vinblastine-induced inhibition of axonal flow, but not by NMDA-induced excitotoxicity, even though both are triggers of RGC death. Knockdown of Ecel1 promoted the loss of RGCs after optic nerve crush., Conclusions: Our data suggest that Ecel1 induction is part of the retinal neuroprotective response to axonal injury in mice. These findings might provide insight into novel therapeutic targets for the attenuation of RGC damage, such as occurs in traumatic optic neuropathy.

Resin glycosides represent an important chemotaxonomic marker of the Convolvulaceae family and possess multidrug resistance (MDR) reversal activity. In our recent study, nine previously undescribed resin glycosides, Calonyctins B-J, were isolated from the seeds of Ipomoea muricata. Their structures with the absolute configuration were established on the basis of comprehensive spectroscopic analysis and chemical methods. Among these, Calonyctins F-I possessed a skeleton in which the aglycone moiety and the oligosaccharide core were linked by a 3-hydroxy-2-methylbutanoic acid moiety to form a 25-membered macrocyclic structure. Calonyctins E, J, and muricatic acid C methyl ester were non-cytotoxic but enhanced the cytotoxicity of vincristine by 2.5-407.1 fold at 25 μM in KB/VCR cells. Calonyctin E was the most active one., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Local nerve inflammation (neuritis) leads to ongoing activity and axonal mechanical sensitivity (AMS) along intact nociceptor axons and disrupts axonal transport. This phenomenon forms the most feasible cause of radiating pain, such as sciatica. We have previously shown that axonal transport disruption without inflammation or degeneration also leads to AMS but does not cause ongoing activity at the time point when AMS occurs, despite causing cutaneous hypersensitivity. However, there have been no systematic studies of ongoing activity during neuritis or noninflammatory axonal transport disruption. In this study, we present the time course of ongoing activity from primary sensory neurons following neuritis and vinblastine-induced axonal transport disruption. Whereas 24% of C/slow Aδ-fiber neurons had ongoing activity during neuritis, few (<10%) A- and C-fiber neurons showed ongoing activity 1-15 days following vinblastine treatment. In contrast, AMS increased transiently at the vinblastine treatment site, peaking on days 4-5 (28% of C/slow Aδ-fiber neurons) and resolved by day 15. Conduction velocities were slowed in all groups. In summary, the disruption of axonal transport without inflammation does not lead to ongoing activity in sensory neurons, including nociceptors, but does cause a rapid and transient development of AMS. Because it is proposed that AMS underlies mechanically induced radiating pain, and a transient disruption of axonal transport (as previously reported) leads to transient AMS, it follows that processes that disrupt axonal transport, such as neuritis, must persist to maintain AMS and the associated symptoms. NEW & NOTEWORTHY Many patients with radiating pain lack signs of nerve injury on clinical examination but may have neuritis, which disrupts axonal transport. We have shown that axonal transport disruption does not induce ongoing activity in primary sensory neurons but does cause transient axonal mechanical sensitivity. The present data complete a profile of key axonal sensitivities following axonal transport disruption. Collectively, this profile supports that an active peripheral process is necessary for maintained axonal sensitivities.

Increasing evidence indicates that the Golgi apparatus plays active roles in cancer, but a comprehensive understanding of its functions in the oncogenic transformation has not yet emerged. At the same time, the Golgi is becoming well recognized as a hub that integrates its functions of protein and lipid biosynthesis to signal transduction for cell proliferation and migration in cancer cells. Nevertheless, the active function of the Golgi apparatus in cancer cells has not been fully evaluated as a target for combined treatment. Here, we analyzed the effect of perturbing the Golgi apparatus on the sensitivity of the MDA-MB-231 breast cancer cell line to the drugs Actinomycin D and Vinblastine. We disrupted the function of ARF1, a protein necessary for the homeostasis of the Golgi apparatus. We found that the expression of the ARF1-Q71L mutant increased the sensitivity of MDA-MB-231 cells to both Actinomycin D and Vinblastine, resulting in decreased cell proliferation and cell migration, as well as in increased apoptosis. Likewise, the combined treatment of cells with Actinomycin D or Vinblastine and Brefeldin A or Golgicide A, two disrupting agents of the ARF1 function, resulted in similar effects on cell proliferation, cell migration and apoptosis. Interestingly, each combined treatment had distinct effects on ERK1/2 and AKT signaling, as indicated by the decreased levels of either phospho-ERK1/2 or phospho-AKT. Our results suggest that disruption of Golgi function could be used as a strategy for the sensitization of cancer cells to chemotherapy.

Glioblastoma (GBM) is a very aggressive tumor that has not had substantial therapeutic improvement since the introduction of temozolomide (TMZ) in combination with radiotherapy. Combining TMZ with other chemotherapeutic agents is a strategy that could be further explored for GBM. To search for molecular predictors of TMZ resistance, the TCGA (The Cancer Genome Atlas) database was utilized to assess the impact of specific genes on TMZ response. Patients whose tumors expressed low levels of FGFR3 and AKT2 responded poorly to TMZ. Combination treatment of vinblastine (VBL) plus mebendazole (MBZ) with TMZ was more effective in reducing cell number in most cultures when compared to TMZ alone, especially in cells with low expression levels of FGFR3 and AKT2. Cell cycle distribution and nuclear morphometric analysis indicated that the triple combination of TMZ, VBL and MBZ (TVM) was able to induce polyploidy and senescence, in addition to increasing the Notch3 RNA level in patient-derived gliomas. Thus, this set of data suggests that the triple combination of TMZ, VBL and MBZ may be a considerable therapeutic alternative for the TMZ-tolerant gliomas that harbor low expression of FGFR3/AKT2.

Mitotic arrest deficient-like-1 (MAD2, also known as MAD2L1) is thought to be an important spindle assembly checkpoint protein, which ensures accurate chromosome segregation and is closely associated with poor prognosis in many cancer. As a MAD2 binding protein, p31 comet counteracts the function of MAD2 and leads to mitotic checkpoint silence. In this study, we explore the function of MAD2-p31 comet axis in malignant glioma cells. Our results showed that disruption of MAD2-p31 comet axis by MAD2 knockdown or p31 comet overexpression suppressed cell proliferation, survival and migration of glioma, indicating that MAD2-p31 comet axis is required for maintaining glioma cells malignancy. It is noted that MAD2 depletion or p31 comet overexpression reduced the sensitivity of glioma cells to microtubule-interfering agents paclitaxel and vinblastine, providing clinical guidance for application of such drugs. Taken together, our findings suggest that MAD2-p31 comet axis may serve as a potential therapeutic target for glioma., (Copyright © 2018. Published by Elsevier Inc.)

Several next-generation taxanes have been reported to possess high potency against Taxol-resistant cancer cell lines overexpressing βIII-tubulin and/or P-glycoprotein (P-gp), both of which are involved in drug resistance. Using a photoaffinity Taxol analogue, 2-( m-azidobenzoyl)taxol, two potent next-generation taxanes, SB-T-1214 and SB-CST-10202, exhibited distinct inhibitory effects on photolabeling of β-tubulin from different eukaryotic sources that differ in β-tubulin isotype composition. They also specifically inhibited photolabeling of P-gp, and the inhibitory effect correlated well with the steady-state accumulation of [ 3 H]vinblastine in a multidrug resistant (MDR) cell line, SKVLB1. Several microtubule-stabilizing agents (MSAs)-resistant cell lines from the human ovarian cancer cell line Hey were isolated, and their MDR1 and βIII-tubulin levels determined. Distinct potencies of the two taxanes against different MSA-resistant cells expressing unique levels of MDR1 and βIII-tubulin were found. Cytotoxicity assays, done in the presence of verapamil, indicated that SB-T-1214 is a substrate, although not as good as Taxol, for P-gp. The mechanisms involved in drug resistance are multifactorial, and the effectiveness of new Taxol analogues depends on the interaction between the drugs and all possible targets; in this case the two major cellular targets are β-tubulin and P-gp.

Lung cancer is one of the foremost tumor-associated cause of death in the world. Most of the patients with NSCLC possesses an advanced disease at diagnosis, and are thus probable subject for systemic therapy. This study aims to evaluate the cytotoxicity of vinblastine and docetaxel combined therapy for the treatment of NSCLC, as well as verapamil (VER) enhancement of the combined therapy. We conducted P-glycoprotein (P-gp) gene expression, protein expression with RT-PCR and western blot respectively, apoptotic response of the combined therapy with VER is also determined using DAPI staining (%). Result of DAPI staining confirmed combination therapy promotes cell apoptosis to greater extent as compared to each drug alone. Real-time RT-PCR analysis revealed that mdr-1 expression level increased 6 fold with docetaxel (40 nM) and 2 fold with vinblastine (30 nM) after 24 h (p<0.001). Consequently, combination therapy reduced drug-induced up-regulation of mdr-1 significantly (p<0.05). VER with the drug combination increased P-gp expression (p<0.05). These data provide evidence showing combined therapy is a better approach to improve the efficacy of chemotherapy and decreasing drug resistance., Competing Interests: The authors have no conflict of interest in any terms or by any means during the study. All the funds were provided by research center and disbursed accordingly., (© Georg Thieme Verlag KG Stuttgart · New York.)

Ectopic pregnancies complicate 1-2 pregnancies and are a leading cause of maternal death. An effective oral drug therapy that replaces surgery might make its treatment safer, cheaper, simpler and therefore more widely accessible. The only current medical treatment offered to women is intramuscular methotrexate, but this only reliably resolves smaller ectopic pregnancies. As such, many ectopic pregnancies require surgical excision. We show that vinorelbine, an orally available chemotherapeutic agent, potently induced placental cell death but did not harm fertility in mice. Vinorelbine was 100-1000 times more potent than methotrexate in inducing placental cell death in vitro, and more potent than combination methotrexate and gefitinib (another proposed treatment for ectopic pregnancy being evaluated in phase III trials). Mechanistically, it caused microtubule condensation, blocked mitosis and activated the apoptosis cascade in placental cells. Vinorelbine was more efficacious than methotrexate±gefitinib in reducing the volume of placental cell tumors xenografted subcutaneously in SCID mice. Mice exposed to vinorelbine and allowed to breed, following a four week washout period, displayed normal fertility, however long-term fertility was not assessed. Human Fallopian tubes treated with vinorelbine did not exhibit up-regulation of apoptosis molecules. Our findings show that placental cells appear sensitive to vinorelbine and it has potential as a tablet-only approach to treat ectopic pregnancy., (Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.)

Purpose: The purpose of this report is to describe, for the first time, the pharmacokinetics of dacarbazine (DTIC) and its metabolites [5-[3-methyl-triazen-1-yl]-imidazole-4-carboxamide (MTIC), 5-[3-hydroxymethyl-3-methyl-triazen-1-yl]-imidazole-4-carboxamide (HMMTIC) and 5-aminoimidazole-4-carboxamide (AIC)] during pregnancy (n = 2) and postpartum (n = 1)., Methods: Non-compartmental DTIC, MTIC, HMMTIC, and AIC pharmacokinetics (PK) were estimated in one case at 29 week gestation and 18 day postpartum and a second case at 32 week gestation, in women receiving DTIC in combination with doxorubicin, bleomycin, and vinblastine for treatment of Hodgkin's lymphoma. Drug concentrations were measured by HPLC., Results: In the subject who completed both pregnancy and postpartum study days, DTIC area under the concentration-time curve (AUC) was 27% higher and metabolite AUCs were lower by 27% for HMMTIC, 38% for MTIC, and 83% of AIC during pregnancy compared to postpartum. At 7 and 9 year follow-up, both subjects were in remission of their Hodgkin's lymphoma., Conclusions: Based on these two case reports, pregnancy appears to decrease the metabolism of the pro-drug dacarbazine, likely through inhibition of CYP1A2 activity. Lower concentrations of active metabolites and decreased efficacy may result, although both these subjects experienced long-term remission of their Hodgkin's lymphoma.

Clinical association studies have implicated high expression of class III β-tubulin as a predictive factor for lower response rates and reduced overall survival in patients receiving tubulin binding drugs, most notably the taxanes. Because of the implications, we examined a series of key vinblastine analogs that emerged from our studies in functional cell growth inhibition assays for their sensitivity to high expression of class III β-tubulin (human non-small cell lung cancer cell line A549 vs taxol-resistant A549-T24). Unlike taxol, vinblastine and a set of key analogs 3-10 did not exhibit any loss in sensitivity toward A549-T24. The results suggest that vinblastine and related analogs are not likely prone to resistance derived from high expression of class III β-tubulin unlike the taxanes. Most significant are the results with 4-6, a subset of 20' amide vinblastine analogs. They match or exceed the potency of vinblastine and they display more potent activity against taxol-resistant A549-T24 than even wild type A549 cells (1.2-2-fold), complementing our prior observations that they also display no sensitivity to overexpression of Pgp (HCT116/VM46 vs HCT116) and are not subject to resistance derived from Pgp efflux., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Background: Throughout the history of human civilizations, cancer has been a major health problem. Despite the advancements made by modern medical sciences, complete treatment or removal of cancerous cells is still a challenging task. Vinblastine, an alkaloid obtained from Catharanthus roseus (L.) G. Don is one of the prominent antineoplastic agents that is being clinically used. To improve the biological potential and reduce sideeffects of this structurally complex molecule, several related analogues have been reported. The present article reviews recently reported structurally modified vinblastine analogues and its impact on biological activity., Methods: We carried out a comprehensive database search on recently reported vinblastine analogues. Both upper (catharanthine) and lower (vindoline) structural units have been considered. The role of functional group modification on anticancer activities has been discussed. In addition, formulations based on vinblastine being considered by NIH, USA for different types of cancers have also been discussed., Results: Around fifty papers were included in the review, including computational and experimental ones. These papers were analysed to discuss the mechanism of action of the parent vinblastine molecule and their analogues. The importance of each functionalities on its anticancer activity have been discussed. This reviewed identified the potential sites of vinblastine core where modification led to improved anticancer activity. Furthermore, several new formulations have also been discussed which are under different phases of clinical trial., Conclusion: The present article highlights the importance of vinblastine in cancer chemotherapy. Literature survey confirms that it is now possible to synthesize new molecules with activity in picomolar range. Not only the periphery of the molecule, the core structure of this magical molecule can be modified to achieve next generation antineoplastic agents., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.)

We recently reported that primary acute lymphoblastic leukemia (ALL) cells are susceptible to the microtubule depolymerizing agent vincristine (VCR) in G1 phase. This finding prompted testing another G1 phase-active compound, palbociclib (PCB), a highly selective inhibitor of cyclin-dependent kinases 4/6 (CDK4/6), alone and in combination with VCR. PCB used alone caused G1 arrest in ALL cells with no effect on cell viability, and similar results were obtained for the retinoblastoma (RB)-proficient T98G glioblastoma cell line. In contrast, HeLa cells failed to arrest in the presence of PCB, consistent with their lack of dependence on the CDK4/6-RB pathway. When ALL cells were pretreated with PCB, they became refractory to death in G1 phase induced by VCR treatment, whereas HeLa cells retained VCR sensitivity after PCB pretreatment. Immunofluorescence microscopy showed that PCB did not disrupt the microtubule network nor prevent VCR from doing so. Furthermore, ALL cells pretreated with PCB retained susceptibility to the Bcl-2/Bcl-xL inhibitor ABT-263, indicating that downstream apoptotic signaling was unaffected. When released from PCB-enforced arrest, ALL cells reinitiated cycling and regained sensitivity to VCR. ALL cells treated with cycloheximide also arrested in G1 phase and became insensitive to VCR, independently reinforcing conclusions derived from PCB-imposed arrest. Thus, primary ALL cells advancing through G1 phase are strictly dependent on functional microtubules for survival whereas microtubules are dispensable for G1-arrested cells. These findings provide novel insight into interphase microtubule function and, from a therapy standpoint, strongly caution against combining microtubule targeting agents and CDK4/6 inhibitors for ALL.

The Pig-a assay is being used in regulatory studies to evaluate the potential of agents to induce somatic cell gene mutations and an OECD test guideline is under development. A working group involved with establishing the guideline recently noted that representative aneugenic agents had not been evaluated, and to help fill this data gap Pig-a mutant phenotype and micronucleated reticulocyte frequencies were measured in an integrated study design to assess the mutagenic and cytogenetic damage responses to vinblastine sulfate exposure. Male Sprague Dawley rats were treated for twenty-eight consecutive days with vinblastine dose levels from 0.0156 to 0.125 mg/kg/day. Micronucleated reticulocyte frequencies in peripheral blood were determined at Days 4 and 29, and mutant cell frequencies were determined at Days -4, 15, 29, and 46. Vinblastine affected reticulocyte frequencies, with reductions noted during the treatment phase and increases observed following cessation of treatment. Micronucleated reticulocyte frequencies were significantly elevated at Day 4 in the high dose group. Although a statistically significant increase in mutant reticulocyte frequencies were found for one dose group at a single time point (Day 46), it was not deemed biologically relevant because there was no analogous finding in mutant RBCs, it occurred at the lowest dose tested, and only 1 rat exceeded an upper bound tolerance interval established with historical negative control rats. Therefore, whereas micronucleus induction reflects vinblastine's well-established aneugenic effect on hematopoietic cells, the lack of a Pig-a response indicates that this tubulin-binding agent does not cause appreciable mutagenicity in this same cell type. Environ. Mol. Mutagen. 59:30-37, 2018. © 2017 Wiley Periodicals, Inc., (© 2017 Wiley Periodicals, Inc.)

Centrosomal proteins play important roles in the spindle assembly and the segregation of chromosomes in the eukaryotic cells. STARD9, a recently identified centrosomal protein, was reported to influence the spindle pole assembly. However, the role of STARD9 in maintaining the stability and organization of microtubules are not known. Here, we show that STARD9 regulates the assembly and dynamics of both interphase and mitotic microtubules. The knockdown of STARD9 in HeLa or HCT116 cells with siRNA or shRNA induced a strong depolymerization of the interphase microtubules. The over-expression of the motor domain of STARD9 stabilizes microtubules against cold and nocodazole suggesting that STARD9 stabilizes microtubules in HeLa cells. Using fluorescent recovery after photobleaching, we showed that the knockdown of STARD9 strongly reduced microtubule dynamics in the live spindles of HeLa cells. The reassembly of microtubules in the STARD9-depleted cells was strongly reduced as compared to the microtubules in the control cells implying the role of STARD9 in the nucleation of microtubules. Further, the depletion of STARD9 inhibited chromosome separation and the STARD9-depleted HeLa cells were blocked at mitosis. Interestingly, the frequency of multipolar spindle formation increased significantly in the STARD9-depleted HeLa cells in the presence of vinblastine and the STARD9-depleted cells showed much higher sensitivity towards vinblastine than the control cells indicating a new approach for cancer chemotherapy. The evidence suggests that STARD9 regulates the assembly and stability of both interphase and spindle microtubules and thereby, play important roles in the cell cycle progression.

Background: Many patients with neuropathic pain present without signs of nerve injury on routine clinical examination. Some of these patients may have inflamed peripheral nerves (neuritis). In this study, we have examined whether neuritis causes changes within the dorsal horn that may contribute to a central pain mechanism. Comparisons have been made to a model of axonal transport disruption induced using vinblastine, since neuritis disrupts such processes., Results: At the peak of cutaneous hypersensitivities, recordings from wide dynamic range neurons revealed increases in wind-up following neuritis but not vinblastine treatment. Ongoing activity from these neurons was unchanged. Vinblastine treatment caused a reduction in the responses of wide dynamic range neurons to noxious mechanical stimulation of the receptive field. The response of neurons to innocuous mechanical stimulation was also reduced in wide dynamic range neurons that were at a depth ≥550 µm following vinblastine treatment. An examination of the superficial dorsal horn revealed an increase in c-Fos-positive neurons in both groups following electrical stimulation of the sciatic nerve. The area of dorsal horn expressing substance P was also decreased following vinblastine treatment., Conclusion: These findings indicate that a minor nerve insult, such as neuritis, can lead to changes within the dorsal horn that are consistent with a central neuropathic pain mechanism.

Sensory neurons from dorsal root ganglion efficiently regenerate after peripheral nerve injuries. These neurons are widely used as a model system to study degenerative mechanisms of the soma and axons, as well as regenerative axonal growth in the peripheral nervous system. This chapter describes techniques associated to the study of axonal degeneration and regeneration using explant cultures of dorsal root ganglion sensory neurons in vitro in the presence or absence of Schwann cells. Schwann cells are extremely important due to their involvement in tissue clearance during axonal degeneration as well as their known pro-regenerative effect during regeneration in the peripheral nervous system. We describe methods to induce and study axonal degeneration triggered by axotomy (mechanical separation of the axon from its soma) and treatment with vinblastine (which blocks axonal transport), which constitute clinically relevant mechanical and toxic models of axonal degeneration. In addition, we describe three different methods to evaluate axonal regeneration using quantitative methods. These protocols constitute a valuable tool to analyze in vitro mechanisms associated to axonal degeneration and regeneration of sensory neurons and the role of Schwann cells in these processes.

Aims: In clinical practice, herbal medicines have played an important role in the modulation of drug transporters through the combination of conventional prescription drugs, which necessitates the elucidation of herb-drug interactions. The present study was designed to investigate the inhibitory effects and mechanisms of benzaldehyde, vanillin, muscone, and borneol on P-glycoprotein (P-gp)., Methods: The effects of the 4 compounds on the intracellular accumulation of rhodamine-123 (Rho-123) in vinblastine-treated Caco-2 (VB-Caco-2) cells were studied by monitoring fluorescence intensity through a flow cytometry assay, and the effects of these compounds on Rho-123 transport through VB-Caco-2 monolayers and Rho-123 intestinal absorption in the rat everted gut sac were investigated by high-performance liquid chromatography. Moreover, P-gp expression in VB-Caco-2 cells was assessed using flow cytometry and Western blot analysis, and the relative ABCB1 mRNA level was determined by Real-time RT-PCR., Key Findings: The results showed that benzaldehyde, vanillin, muscone, and borneol significantly increased Rho-123 uptake in VB-Caco-2 cells, increased the absorption rate and apparent permeability coefficient of Rho-123 in rat jejunum and ileum, and decreased the efflux ratio of Rho-123 from 6.52 to less than 2 during transport across VB-Caco-2 cell monolayers. In addition, these compounds reduced the protein and ABCB1 mRNA levels of P-gp in VB-Caco-2 cells., Conclusions: These data indicate that benzaldehyde, vanillin, muscone and borneol could effectively reverse multidrug resistance via inhibiting the P-gp function and expression pathway. The data provide fodder for further investigation into the interaction between the 4 compounds and other drugs transported by P-gp., (© 2018 S. Karger AG, Basel.)

The fact that most gastrointestinal stromal tumors (GISTs) acquire resistance to imatinib (IM)-based targeted therapy remains the main driving force to identify novel molecular targets that are capable to increase GISTs sensitivity to the current therapeutic regimens. Secondary resistance to IM in GISTs typically occurs due to several mechanisms that include hemi- or homo-zygous deletion of the wild-type KIT allele, overexpression of focal adhesion kinase (FAK) and insulin-like growth factor receptor I (IGF-1R) amplification, BRAF mutation, a RTK switch (loss of c-KIT and gain of c-MET/AXL), etc. We established and characterized the IM-resistant GIST T-1 cell line (GIST T-1R) lacking secondary c-KIT mutations typical for the IM-resistant phenotype. The resistance to IM in GIST T-1R cells was due to RTK switch (loss of c-KIT/gain of FGFR2α). Indeed, we have found that FGFR inhibition reduced cellular viability, induced apoptosis and affected the growth kinetics of the IM-resistant GISTs in vitro. In contrast, IM-naive GIST T-1 parental cells were not susceptible to FGFR inhibition. Importantly, inhibition of FGF-signaling restored the susceptibility to IM in IM-resistant GISTs. Additionally, IM-resistant GISTs were less susceptible to certain chemotherapeutic agents as compared to parental IM-sensitive GIST cells. The chemoresistance in GIST T-1R cells is not due to overexpression of ABC-related transporter proteins and might be the result of upregulation of DNA damage signaling and repair (DDR) genes involved in DNA double-strand break (DSB) repair pathways (e.g., XRCC3, Rad51, etc.). Taken together, the established GIST T-1R cell subline might be used for in vitro and in vivo studies to examine the efficacy and prospective use of FGFR inhibitors for patients with IM-resistant, un-resectable and metastatic forms of GISTs with the type of RTK switch indicated above., Competing Interests: The authors declare that they have no competing interests.

P-glycoprotein (P-gp) is an ATP-dependent drug pump that protects us from toxic agents and confers multidrug resistance. It has a tweezer-like structure with each arm consisting of a transmembrane domain (TMD) and a nucleotide-binding domain (NBD). Drug substrates bind to sites within the TMDs to activate ATPase activity by promoting a tweezer-like closing of the gap between the NBDs. The catalytic carboxylates may be critical for NBD movements because the E556Q(NBD1) or E1201Q(NBD2) mutation inhibited drug-stimulated ATPase activity. If the catalytic carboxylates were components of the mechanism to bring the NBDs together, then we predicted that insertion of a flexible cross-linker between the arms would increase ATPase activity of the mutants. We found that cross-linking (between L175C(TMD1) and N820C(TMD2)) with a short flexible cross-linker (7.8Å maximum) restored high levels of drug-stimulated ATPase activity of the E556Q or E1201Q mutants. Cross-linking with a longer cross-linker (22Å maximum) however, did not restore activity. Cross-linking could not rescue all ATPase deficient mutants. For example, cross-linking L175C/N820C with short or long cross-linkers did not activate the H-loop mutants H587A or H1232A or the Walker A K433M or K1076M mutants. The results suggest that the E556 and E1201 catalytic carboxylates are part of a spring-like mechanism that is required to facilitate movements between the open and closed conformations of P-gp during ATP hydrolysis., (Copyright © 2017 Elsevier Inc. All rights reserved.)

No standard therapy is established for metastatic uveal melanoma. Liver involvement in uveal melanoma may lead to organ impairment, which represents a common cause of death. Tumor shrinkage might improve survival by delaying hepatic failure. Since the combination of cisplatin, vinblastine, dacarbazine allowed a high response rate in metastatic cutaneous melanoma, we explored efficacy and safety of this regimen in unresectable liver metastases of uveal melanoma. In the present phase II study we administered intravenously cisplatin (80 mg/mq, day 1), dacarbazine (250 mg/mq/day, days 1-3), vinblastine (2 mg maximum, day 1) every 21 days as first line treatment for patients with unresectable metastases of uveal melanoma and BRAF wild type. Primary endpoint was objective response rate; overall survival (OS), progression-free survival and toxicity were secondary endpoints. Partial responses were observed in five (20%) patients, stable disease in 12 (48%) patients; disease control rate was 68%. Median OS of all the patients was 13 months, median progression free survival was 5.5 months. OS of responding patients was 21 months; OS of patients with disease control was 18 months, significantly longer than survival of progressing patients (7 months, P=0.0003). Five (20%) patients experienced grade 3-4 toxicity. Combination of cisplatin, vinblastine and dacarbazine was feasible and demonstrate both an interesting objective response rate and a survival benefit for patients achieving a disease control. This regimen could be considered for patients with good performance status and unresectable liver limited disease.

Nowadays, lung cancer, as a health problem in worldwide, has high mortality both in men and women. Despite advances in diagnosis and surgical techniques of lung cancer in recent decades, chemotherapy is still a fundamentally and extensively useful strategy. Vinca alkaloids are a class of important and widely used drugs in the treatment of lung cancer, targeting on the Vinca binding site at the exterior of microtubule plus ends. Either intrinsic or acquired resistance to chemotherapy of Vinca alkaloids has been a major obstacle to the treatment of lung cancer, which arose great interests in studies of understanding and overcoming resistance. In this review, we focused on the application and resistance mechanisms of the Vinca alkaloids such as vinblastine, vincristine, vinorelbine and vinflunine in lung cancer. We reviewed characteristic resistance mechanisms in lung cancer including over-expression of ATP-binding cassette (ABC) transporters P-glycoprotein and structural, functional or expression alterations of β-tubulin (βII, βIII, βIV) which may devote to the development of acquired resistance to the Vinca alkaloids; multidrug-resistance proteins (MRP1, MRP2, MRP3) and RLIP76 protein have also been identified that probably play a significant role in intrinsic resistance. Lung resistance-related protein (LRP) is contributed to lung cancer therapy resistance, but is not deal with the Vinca alkaloids resistance in lung cancer. Understanding the principle of the Vinca alkaloids in clinical application and mechanisms of drug resistance will support individualized lung cancer therapy and improve future therapies., (Copyright © 2017 Elsevier Masson SAS. All rights reserved.)

ZSTK474 is a potent phosphoinositide 3-kinase (PI3K) inhibitor that reduces cell proliferation via G 1 -arrest. However, there is little information on the susceptibility of this anticancer drug to resistance conferred by the multidrug pumps P-glycoprotein (ABCB1) and ABCG2. We have demonstrated that ZSTK474 generated cytotoxicity in cells over-expressing either pump with potency similar to that in drug sensitive cells. In addition, the co-administration of ZSTK474 with the cytotoxic anti-cancer drugs vinblastine and mitoxantrone caused a potentiated cytotoxic effect in both drug sensitive and efflux pump expressing cells. These observations suggest that ZSTK474 is unaffected by the presence of multidrug efflux pumps and may circumvent their activities. Indeed, ZSTK474 increased the cellular accumulation of calcein-AM and mitoxantrone in cells expressing ABCB1 and ABCG2, respectively. ZSTK474 treatment also resulted in reduced expression of both efflux pumps in multidrug resistant cancer cells. Measurement of ABCB1 or ABCG2 mRNA levels demonstrated that the reduction was not due to altered transcription. Similarly, inhibitor studies showed that the proteasomal degradation pathway for ABCB1 and the lysosomal route for ABCG2 degradation were unaffected by ZSTK474. Thus the mechanism underlying reduced ABCB1 and ABCG2 levels caused by ZSTK474 was due to a reduction in overall protein synthesis; a process influenced by the PI3K pathway. In summary, ZSTK474 is not susceptible to efflux by the resistance mediators ABCB1 and ABCG2. Moreover, it inhibits the drug transport function of the pumps and leads to a reduction in their cellular expression levels. Our observations demonstrate that ZSTK474 is a powerful anticancer drug., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Background: The prognosis for patients with disseminated lung cancer is poor and current treatments have limited survival benefit as resistance often occurs, and is often associated with significant toxicity. A possible strategy to improve treatment and evade chemoresistance may be to find new combinations of drugs. The aim of this study was to analyze the potential of combining proteasome inhibitors (PIs) with chemotherapeutic drugs used in the routine treatment for lung cancer patients., Results: The median-effect method was applied to the Fluorometric Microculture Cytotoxicity Assay (FMCA) to evaluate effects of combining two different PIs (bortezomib and b-AP15) with clinically used chemotherapeutic drugs representing different mechanisms of action (cisplatin, gefitinib, gemcitabine and vinorelbine) in two lung cancer cell lines (one sensitive and one resistant). Proteasome inhibition in combination with cisplatin, gemcitabine or vinorelbine had synergistic effects in at least one of the tested cell lines. Furthermore, the effect of gefitinib appeared strongly potentiated by the PI in the least resistant lung cancer cell line, although the level of synergy could not be determined with the median-effect method., Conclusions: Combining PIs with cisplatin, gefitinib, gemcitabine or vinorelbine show potential as new combination chemotherapy for the treatment of lung cancer.