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Primary cilia (PC) are microtubule-based organelles that function as cellular antennae to sense and transduce extracellular signals. Nephronophthisis 3 (NPHP3) is localized in the inversin compartment of PC. Mutations in NPHP3 are associated with renal-hepatic-pancreatic dysplasia. In this study, we investigated whether vinblastine (VBL), a microtubule destabilizer, induces anticancer drug resistance through NPHP3-associated PC formation in HeLa human cervical cancer cells. A considerable increase in PC frequency was observed in HeLa cells under serum-deprived (SD) conditions, which led to the inhibition of VBL-induced cell death. VBL-resistant cells were established by repetitive treatments with VBL and showed an increase in PC frequency. NPHP3 expression was also increased by VBL treatment under serum starvation as well as in VBL-resistant cells. NPHP3 expression and PC-associated resistance were positively correlated with apoptosis-antagonizing transcription factor (AATF) and negatively correlated with inhibition of NPHP3. In addition, AATF-mediated NPHP3 expression is associated with PC formation via the regulation of intraflagellar transport protein 88 (IFT88). VBL resistance ability was reduced by treating with ciliobrevin A, a well-known ciliogenesis inhibitor. Collectively, cancer cell survival following VBL treatment is regulated by PC formation via AATF-mediated expression of IFT88 and NPHP3. Our data suggest that the activation of AATF and IFT88 could be a novel regulator to induce anticancer drug resistance through NPHP3-associated PC formation.

A biotransformation pair consisting of vinblastine: vincristine present in the Catharanthus roseus plant is of immense pharmacological significance. In this study, we successfully transformed vinblastine into vincristine outside the plant using Pseudomonas aeruginosa 8485 and Pseudomonas fluorescens 2421 and evaluated the antiangiogenic potential of thus produced vincristine through the CAM assay. The toxicity assay showed that both Pseudomonas spp. can tolerate varying concentrations (25-100 µl of 1 mg/ml) of vinblastine. The biotransformation was performed in a liquid nutrient broth medium containing vinblastine (25-100 µl), and Pseudomonas spp. inoculums (50-150 µl) by incubating at 30 °C and 37 °C, respectively for 8 days. The process was optimized for substrate and culture concentrations, pH, temperature, and rotation speed (rpm) for the highest conversion. Analysis using LC-MS/MS confirmed the presence of vincristine as a product of the vinblastine biotransformation by two Pseudomonas spp. P. fluorescens 2421 showed a faster conversion rate with 95% of vinblastine transformed within 24 h than P. aeruginosa 8485, which demonstrated a conversion rate of 92% on the 8 th day. From LC-MS/MS analysis, the optimal conditions for the reaction were determined as vinblastine (25 µl), microbial inoculums (150 µl or 200 × 10 6 and 210 × 10 6  CFU/ml), pH 7.4, rotation speed of 180 rpm, and temperatures of 30 °C and 37 °C with incubation time of 8 days. The vincristine produced exhibited potent antiangiogenic activity in the CAM assay reducing the thickness and branching of blood vessels in a dose-dependent manner. The study concludes that both Pseudomonas spp. showed promise for vincristine production from vinblastine, without compromising its antiangiogenic properties., (© 2024. The Author(s).)

In the frame of our diversity-oriented research on multitarget small molecule anticancer agents, utilizing convergent synthetic sequences terminated by Sonogashira coupling reactions, a preliminary selection of representative alkyne-tethered vindoline hybrids was synthesized. The novel hybrids with additional pharmacophoric fragments of well-documented anticancer agents, including FDA-approved tyrosine-kinase inhibitors (imatinib and erlotinib) or ferrocene or chalcone units, were evaluated for their antiproliferative activity on malignant cell lines MDA-MB-231 (triple negative breast cancer), A2780 (ovarian cancer), HeLa (human cervical cancer), and SH-SY5Y (neuroblastoma) as well as on human embryonal lung fibroblast cell line MRC-5, which served as a reference non-malignant cell line for the assessment of the therapeutic window of the tested hybrids. The biological assays identified a trimethoxyphenyl-containing chalcone-vindoline hybrid ( 36 ) as a promising lead compound exhibiting submicromolar activity on A2780 cells with a marked therapeutic window.

Microtubules, composed of αβ-tubulin heterodimers, are crucial targets for chemotherapeutic agents and possess eight binding sites. Our previous study identified cevipabulin as the only one agent capable of simultaneously binding to two different sites (Vinblastine site and The Seventh site). Binding to The Seventh site by cevipabulin induces tubulin degradation. This study aimed to investigate whether it is binding to the Vinblastine site and The Seventh site exhibited an interactive cellular effect. Surprisingly, we discovered that cevipabulin induced abnormal tubulin protofilaments polymerization, a previously undefined tubulin morphology, and we proved it was an interactive effect of Cevipabulin's binding to both Vinblastine site and The Seventh site. Immunofluorescence and transmission electron microscopy confirmed cevipabulin induced the formation of linear tubulin protofilaments and their subsequent aggregation into irregular tubulin aggregates. Competition binding assays and the αY224G mutation revealed that binding of cevipabulin to both sites was necessary for the tubulin protofilaments polymerization effect. Moreover, we found that co-treatment with a microtubule stabilization agent binding the Vinblastine site and a microtubule destabilization agent binding at the intra-dimer interface of tubulin could also induce similar tubulin protofilaments polymerization. We proposed a mechanism where a microtubule stabilization agent on the Vinblastine site enhances longitudinal interactions between tubulin dimers, while, a microtubule destabilization agent binding at the intra-dimer interface prevents the adoption of a straight conformation of the tubulin dimer and disrupts lateral interactions between tubulins, consequently leading to tubulin protofilaments polymerization. This study reported a new inhibitor-induced-tubulin-morphology-change and would provide insight into tubulin dynamic instability and also guide further study of cevipabulin., (© 2023 Wiley Periodicals LLC.)

Cancer chemotherapy increases the risk of thrombosis; however, the mechanisms underlying this thrombosis are not completely understood. Plasminogen activator inhibitor (PAI)-1 is a key molecule in the fibrinolytic system that inhibits tissue plasminogen activator and urokinase, which converts plasminogen into plasmin; therefore, excess PAI-1 increases the risk of thrombosis. In this study, we investigated whether temporary treatment of the human luminal A-type breast cancer cell line MCF-7 with antitumor drugs clinically used for breast cancer therapy promotes PAI-1 production. Treatment of MCF-7 cells with paclitaxel (PTX), a microtubule-stabilizing antitumor drug, at 1 µM for 2 h elevated the PAI-1 concentration of the conditioned medium at 48 h after treatment but not in those treated with tamoxifen and cyclophosphamide. Microtubule assembly inhibitors vinblastine (VBT) and vincristine (VCT) also increased the PAI-1 concentration in the conditioned medium. PAI-1 (SERPINE1) expression was upregulated in MCF-7 cells after PTX, VBT, and VCT treatment; this increase in expression persisted for eight days. In contrast, PAI-1 production in MDA-MB-231 cells treated with PTX, VBT, or VCT did not increase with increasing PAI-1 concentration. This study demonstrated that temporary low-dose treatment with microtubule-associated anticancer drugs increased PAI-1 release from MCF-7 cells but not from MDA-MB-231 cells. These results indicate that chemotherapy against luminal A-type breast cancer using microtubule-associated drugs may cause thrombosis through the inhibition of the fibrinolytic system by PAI-1.

Chemotherapeutic drugs such as paclitaxel and vinblastine interact with microtubules and thus induce complex cell states of mitosis arrest at the G 2 /M phase followed by apoptosis dependent on drug exposure time and concentration. Microfluidic impedance cytometry (MIC), as a label-free and high-throughput technology for single-cell analysis, has been applied for viability assay of cancer cells post drug exposure at fixed time and dosage, yet verification of this technique for varied tumor cell states after anticancer drug treatment remains a challenge. Here we present a novel MIC device and for the first time perform impedance cytometry on carcinoma cells exhibiting progressive states of G 2 /M arrest followed by apoptosis related to drug concentration and exposure time, after treatments with paclitaxel and vinblastine, respectively. Our results from impedance cytometry reveal increased amplitude and negative phase shift at low frequency as well as higher opacity for HeLa cells under G 2 /M mitotic arrest compared to untreated cells. The cells under apoptosis, on the other hand, exhibit opposite changes in these electrical parameters. Therefore, the impedance features differentiate the HeLa cells under progressive states post anticancer drug treatment. We also demonstrate that vinblastine poses a more potent drug effect than paclitaxel especially at low concentrations. Our device is fabricated using a unique sacrificial layer-free soft lithography process as compared to the existing MIC device, which gives rise to readily aligned parallel microelectrodes made of silver-PDMS embedded in PDMS channel sidewalls with one molding step. Our results uncover the potential of the MIC device, with a fairly simple and low-cost fabrication process, for cellular state screening in anticancer drug therapy.

Background: Massive tumor-associated macrophage (TAM) infiltration is observed in many tumors, which usually display the immune-suppressive M2-like phenotype but can also be converted to an M1-like antitumor phenotype due to their high degree of plasticity. The macrophage polarization state is associated with changes in cell shape, macrophage morphology is associated with activation status. M1 macrophages appeared large and rounded, while M2 macrophages were stretched and elongated cells. Manipulating cell morphology has been shown to affect the polarization state of macrophages. The shape of the cell is largely dependent on cytoskeletal proteins, especially, microtubules. As a microtubule-targetting drug, vinblastine (VBL) has been used in chemotherapy. However, no study to date has explored the effect of VBL on TAM shape changes and its role in tumor immune response., Method: We used fluorescent staining of the cytoskeleton and quantitative analysis to reveal the morphological differences between M0, M1, M2, TAM and VBL-treated TAM. Flow cytometry was used to confirm the polarization states of these macrophages using a cell surface marker-based classification. In vivo antibody depletion experiments in tumor mouse models were performed to test whether macrophages and CD8 + T cell populations were required for the antitumor effect of VBL. VBL and anti-PD-1 combination therapy was then investigated in comparison with monotherapy. RNA-seq of TAM of treated and untreated with VBL was performed to explore the changes in pathway activities. siRNA mediated knockdown experiments were performed to verify the target pathway that was affected by VBL treatment., Results: Here, we showed that VBL, an antineoplastic agent that destabilizes microtubule, drove macrophage polarization into the M1-like phenotype both in vitro and in tumor models. The antitumor effect of VBL was attenuated in the absence of macrophages or CD8 + T cells. Mechanistically, VBL induces the activation of NF-κB and Cyba-dependent reactive oxygen species generation, thus polarizing TAMs to the M1 phenotype. In parallel, VBL promotes the nuclear translocation of transcription factor EB, inducing lysosome biogenesis and a dramatic increase in phagocytic activity in macrophages., Conclusions: This study explored whether manipulating cellular morphology affects macrophage polarization and consequently induces an antitumor response. Our data reveal a previously unrecognized antitumor mechanism of VBL and suggest a drug repurposing strategy combining VBL with immune checkpoint inhibitors to improve malignant tumor immunotherapy., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2023. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Cancer therapies use different compounds of synthetic and natural origin. However, despite some positive results, relapses are common, as standard chemotherapy regimens are not fully capable of completely eradicating cancer stem cells. While vinblastine is a common chemotherapeutic agent in the treatment of blood cancers, the development of vinblastine resistance is often observed. Here, we performed cell biology and metabolomics studies to investigate the mechanisms of vinblastine resistance in P3X63Ag8.653 murine myeloma cells. Treatment with low doses of vinblastine in cell media led to the selection of vinblastine-resistant cells and the acquisition of such resistance in previously untreated, murine myeloma cells in culture. To determine the mechanistic basis of this observation, we performed metabolomic analyses of resistant cells and resistant drug-induced cells in a steady state, or incubation with stable isotope-labeled tracers, namely, 13 C 15 N-amino acids. Taken together, these results indicate that altered amino acid uptake and metabolism could contribute to the acquisition of vinblastine resistance in blood cancer cells. These results will be useful for further research on human cell models.

Structural information about drug-receptor interactions is paramount in drug discovery and subsequent optimization processes. Drugs can bind to multiple potential targets as they contain common chemical entities in their structures. Understanding the details of such interactions offer possibilities for repurposing and developing potent inhibitors of disease pathways. Vinblastine (VLB) is a potent anticancer molecule showing multiple receptor interactions with different affinities and degrees of structural perturbations. We have investigated the multi-target binding profile of VLB with DNA and human serum albumin (HSA) in a dynamic physiological environment using spectroscopic, molecular dynamics simulations, and quantum mechanical calculations to evaluate the structural features, mode, ligand and receptor flexibility, and energetics of complexation. These results confirm that VLB prefers to bind in the major groove of DNA with some inclination toward Thymidine residue and the TR-5 binding site in HSA with its catharanthine half making important contacts with both the receptors. Spectroscopic investigation at multiple temperatures has also proved that VLB binding is entropy driven indicating the major groove and TR-5 binding site of interaction. Finally, the overall binding is facilitated by van der Waals contacts and a few conventional H-bonds. VLB portrays reasonable conformational diversity on binding with multiple receptors., (© 2022 John Wiley & Sons Ltd.)

Chronic myelogenous leukemia (CML) is characterised by the translocation of regions of the BCR and ABL genes, leading to the fusion gene BCR-ABL forming the Philadelphia (Ph) chromosome. Vinblastine (Vinb) and Vincristine (Vinc) are Vinca alkaloids and frequently used in combination chemotherapy in leukemias and lymphomas. Deubiquitinating enzyme (DUB) genes such as A20, Otubain 1 and CYLD are known as inhibitors of functional activation of immune cells mediated through the NF-κB/STAT pathway. Little is known about the regulatory role of Vinb/Vinc on the function of CML cells and the contribution of the DUBs to those effects. In the end, the gene expression profile was determined by quantitative RT-PCR, physiological properties of CML cells by flow cytometry and cytokine production by ELISA. As a result, inactivated expression of the DUBs A20, CYLD, Otubain 1 and Cezanne and enhanced activation of CD11b+ and CD4T cells were observed in CML patients. Importantly, Vinc enhanced the expression of A20 and CYLD and inhibited the proliferation and survival of CML (K562) cells. The effects were abolished in the presence of A20 siRNA, while cell proliferation only depended on the presence of CYLD. In conclusion, the up-regulation of A20 by Vinc could involve inhibitory effects on the proliferation and survival of K562 cells. The events might contribute to the anticancer effect of Vinc on A20-sensitive CML cells.

Purpose: To compare healthcare resource utilization (HRU) and costs associated with dose-dense methotrexate, vinblastine, doxorubicin, cisplatin (ddMVAC) and gemcitabine, cisplatin (GC) as neoadjuvant chemotherapy for muscle-invasive bladder cancer (MIBC)., Methods: Patient treated at Dana-Farber Cancer Institute from 2010 to 2019 were identified. HRU data on chemotherapy administered, supportive medications, patient monitoring, clinic, infusion, emergency department (ED) visits and hospitalization were collected retrospectively. Unit costs for HRU components were obtained from the Centers for Medicare and Medicaid Website and HRU was compared between groups using quantile regression analysis., Results: 137 patients were included; 51 received ddMVAC and 86 GC. Baseline characteristics were similar, except lower mean age (P < 0.001) and higher proportion of ECOG-PS = 0 (P < 0.001) for ddMVAC. ddMVAC required more granulocyte-colony stimulating factor support (P < 0.001), central line placement (P = 0.017), cardiac imaging (P < 0.001), and infusion visits (P < 0.001), whereas GC required more clinic visits. ED visits were higher for ddMVAC (P = 0.048), while chemotherapy cycle delays and hospitalization days were higher for GC (P = 0.008). After adjusting for ECOG-PS and age, the cost per patient was approximately 41% lower (95%CI: 28% to 52%; P < 0.001) for GC vs. ddMVAC, which translated to a median adjusted cost savings of $7,410 (95%CI: $5,474-$9,347) per patient., Conclusions: Although excess HRU did not clearly favor one regimen, adjusting for PS and age indicated lower costs with GC vs. ddMVAC. Given the similar cumulative cisplatin delivery with both regimens, the associated values and costs supports the preferential selection of GC in the neoadjuvant setting of MIBC., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Aims: Several fungal endophytes were isolated from some medicinal plants and screened for their ability to produce the anticancer drug vinblastine., Methods and Results: An isolate was found to produce vinblastine (205·38 μg l -1 ), and the identity of the fungal vinblastine was confirmed by UV spectroscopic, high-performance liquid chromatography and electrospray ionization mass spectrometry analyses. Based on both morphological and molecular studies, the vinblastine-producing strain was identified as Alternaria alternata. Cytotoxic activities of the fungal vinblastine were evaluated against CHO-K1, MCF-7 and HepG-2 cell lines by the MTT assay. The proliferation of these cell lines was inhibited after treatment with fungal vinblastine and the recorded IC 50 values of the respective cell lines were 12·15, 8·55 and 7·48 μg ml -1 . A strain improvement programme for improving vinblastine productivity by the fungal strain was also used. In addition, 10 broth media were evaluated for further increasing the production of vinblastine. The yield of vinblastine was intensified by 3·98-fold following gamma irradiation at 1000 Gy, and a stable mutant strain was isolated. Among the screened media, M1D broth (pH 6·0) stimulated the highest vinblastine production of 1553·62 μg l -1 by the isolated mutant strain., Conclusions: The present study is the first report on the production and yield improvement of the anticancer drug vinblastine by A. alternata., Significance and Impact of the Study: These findings suggest A. alternata as a viable and potent source with excellent biotechnological potential for the production of vinblastine., (© 2021 The Society for Applied Microbiology.)

Efficacy of cytotoxic T lymphocyte (CTL)-based immunotherapy is still unsatisfactory against solid tumors, which are frequently characterized by condensed extracellular matrix. Here, using a unique 3D killing assay, we identify that the killing efficiency of primary human CTLs is substantially impaired in dense collagen matrices. Although the expression of cytotoxic proteins in CTLs remained intact in dense collagen, CTL motility was largely compromised. Using light-sheet microscopy, we found that persistence and velocity of CTL migration was influenced by the stiffness and porosity of the 3D matrix. Notably, 3D CTL velocity was strongly correlated with their nuclear deformability, which was enhanced by disruption of the microtubule network especially in dense matrices. Concomitantly, CTL migration, search efficiency, and killing efficiency in dense collagen were significantly increased in microtubule-perturbed CTLs. In addition, the chemotherapeutically used microtubule inhibitor vinblastine drastically enhanced CTL killing efficiency in dense collagen. Together, our findings suggest targeting the microtubule network as a promising strategy to enhance efficacy of CTL-based immunotherapy against solid tumors, especially stiff solid tumors., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Zhao, Zhou, Khan, Alansary, Friedmann, Yang, Schwarz, del Campo, Hoth and Qu.)

Approximately one-third of patients diagnosed with Hodgkin lymphoma presenting with Stage IV disease do not survive past 5 years. We present updated efficacy and safety analyses in high-risk patient subgroups, defined by Stage IV disease or International Prognostic Score (IPS) of 4-7, enrolled in the ECHELON-1 study that compared brentuximab vedotin plus doxorubicin, vinblastine, and dacarbazine (A + AVD) versus doxorubicin, bleomycin, vinblastine, and dacarbazine (ABVD) as first-line therapy after a median follow-up of 37.1 months. Among patients treated with A + AVD (n = 664) or ABVD (n = 670), 64% had Stage IV disease and 26% had an IPS of 4-7. Patients with Stage IV disease treated with A + AVD showed consistent improvements in PFS at 3 years as assessed by investigator (hazard ratio [HR], 0.723; 95% confidence interval [CI], 0.537-0.973; p = 0.032). Similar improvements were seen in the subgroup of patients with IPS of 4-7 (HR, 0.588; 95% CI, 0.386-0.894; p = 0.012). The most common adverse events (AEs) in A + AVD-treated versus ABVD-treated patients with Stage IV disease were peripheral neuropathy (67% vs. 40%) and neutropenia (71% vs. 55%); in patients with IPS of 4-7, the most common AEs were peripheral neuropathy (69% vs. 45%), neutropenia (66% vs. 55%), and febrile neutropenia (23% vs. 9%), respectively. Patients in high-risk subgroups did not experience greater AE incidence or severity than patients in the total population. This updated analysis of ECHELON-1 shows a favorable benefit-risk balance in high-risk patients., (© 2021 The Authors. Hematological Oncology published by John Wiley & Sons Ltd.)

In the current study, the surface of superparamagnetic iron oxide (SPION) was coated with dextran (DEX), and conjugated with folic acid (FA), to enhance the targeted delivery and uptake of vinblastine (VBL) in PANC-1 pancreatic cancer cells. Numerous analyses were performed to validate the prepared FA-DEX-VBL-SPION, such as field emission scanning transmission electron microscopy, high-resolution transmission electron microscopy, dynamic light scattering (DLS), Zeta Potential, Fourier transform infrared spectroscopy, and vibrating sample magnetometry (VSM). The delivery system capacity was evaluated by loading and release experiments. Moreover, in vitro biological studies, including a cytotoxicity study, cellular uptake assessment, apoptosis analysis, and real-time PCR, were carried out. The results revealed that the obtained nanocarrier was spherical with a suitable dispersion and without visible aggregation. Its average size, polydispersity, and zeta were 74 ± 13 nm, 0.080, and -45 mV, respectively. This dual functional nanocarrier also exhibited low cytotoxicity and a high apoptosis induction potential for successful VBL co-delivery. Real-time quantitative PCR analysis demonstrated the activation of caspase-3 , NF-1 , PDL-1 , and H-ras inhibition, in PANC-1 cells treated with the FA-VBL-DEX-SPION nanostructure. Close inspection of the obtained data proved that the FA-VBL-DEX-SPION nanostructure possesses a noteworthy chemo-preventive effect on pancreatic cancer cells through the inhibition of cell proliferation and induction of apoptosis.

Efflux transporter-mediated multidrug resistance (MDR) prevents chemotherapeutics to achieve therapeutically relevant concentrations within the cancer cells. Therefore, inhibitors of efflux transporters have been in demand. However, to be safe, these inhibitors are needed to be targeted into the cancer cells. For this purpose, L-type amino acid transporter 1 (LAT1), overexpressed in many different cancer cell types, can be utilized. In the present study, two LAT1-utilizing derivatives of probenecid (PRB) that can inhibit e.g., multiresistance proteins (MRPs) and organic anion transporters (OATs), were studied for their apoptosis-inducing effects in cancer cells alone and a combination with another chemotherapeutic, vinblastine (VBL). Also, their hemocompatibility and off-target toxicity were evaluated. Moreover, the brain uptake rate and extent of VBL together with the most promising LAT1-utilizing efflux inhibitor was studied after in situ rat brain perfusion and after intraperitoneal administration to mice. As a result, these targeted inhibitors increased significantly the apoptosis-inducing effects of VBL and more effectively than PRB itself. They also were hemocompatible and non-toxic in healthy cells with a concentration below 100 µM. Interestingly, the most promising compound doubled the penetration rate of VBL across the rat blood-brain barrier (BBB). This makes it a promising candidate for further studies to improve efflux transporter-related MDR of brain-targeted anti-cancer agents., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Biological activity of antisense oligonucleotides (asON), especially those with a neutral backbone, is often attenuated by poor cellular accumulation. In the present proof-of-concept study, we propose a novel delivery system for asONs which implies the delivery of modified antisense oligonucleotides by so-called transport oligonucleotides (tON), which are oligodeoxyribonucleotides complementary to asON conjugated with hydrophobic dodecyl moieties. Two types of tONs, bearing at the 5'-end up to three dodecyl residues attached through non-nucleotide inserts (TD series) or anchored directly to internucleotidic phosphate (TP series), were synthesized. tONs with three dodecyl residues efficiently delivered asON to cells without any signs of cytotoxicity and provided a transfection efficacy comparable to that achieved using Lipofectamine 2000. We found that, in the case of tON with three dodecyl residues, some tON/asON duplexes were excreted from the cells within extracellular vesicles at late stages of transfection. We confirmed the high efficacy of the novel and demonstrated that MDR1 mRNA targeted asON delivered by tON with three dodecyl residues significantly reduced the level of P-glycoprotein and increased the sensitivity of KB-8-5 human carcinoma cells to vinblastine. The obtained results demonstrate the efficacy of lipophilic oligonucleotide carriers and shows they are potentially capable of intracellular delivery of any kind of antisense oligonucleotides.

A 1 H-NMR-based metabolomic study was performed on MCF-7 cell lines treated with a novel nicotinamide derivative (DT-8) in comparison with two drugs characterized by a well-established mechanism of action, namely the DNA-metalating drug cisplatin (cis-diamminedichloridoplatinum(II), CDDP) and the antimitotic drug vinblastine (vinblastine, VIN). The effects of the three compounds, each one at the concentration corresponding to the IC 50 value, were investigated, with respect to the controls (K), by the 1 H-NMR of cells lysates and multivariate analysis (MVA) of the spectroscopic data. Relevant differences were found in the metabolic profiles of the different treatments with respect to the controls. A large overlap of the metabolic profiles in DT-8 vs. K and VIN vs. K suggests a similar biological response and mechanism of action, significantly diverse with respect to CDDP. On the other hand, DT8 seems to act by disorganizing the mitotic spindle and ultimately blocking the cell division, through a mechanism implying methionine depletion and/or S- adenosylmethionine (SAM) limitation.

The dynamic cytoskeletal network of microtubules and actin filaments can be disassembled by drugs. Cytoskeletal drugs work by perturbing the monomer-polymer equilibrium, thus changing the size and number of macromolecular crowders inside cells. Changes in both crowding and nonspecific surface interactions ("sticking") following cytoskeleton disassembly can affect the protein stability, structure, and function directly or indirectly by changing the fluidity of the cytoplasm and altering the crowding and sticking of other macromolecules in the cytoplasm. The effect of cytoskeleton disassembly on protein energy landscapes inside cells has yet to be observed. Here we have measured the effect of several cytoskeletal drugs on the folding energy landscape of two FRET-labeled proteins with different in vitro sensitivities to macromolecular crowding. Phosphoglycerate kinase (PGK) was previously shown to be more sensitive to crowding, whereas variable major protein-like sequence expressed (VlsE) was previously shown to be more sensitive to sticking. The in-cell effects of drugs that depolymerize either actin filaments (cytochalasin D and latrunculin B) or microtubules (nocodazole and vinblastine) were compared. The crowding sensor protein CrH2-FRET verified that cytoskeletal drugs decrease the extent of crowding inside cells despite also reducing the overall cell volume. The decreased compactness and folding stability of PGK could be explained by the decreased extent of crowding induced by these drugs. VlsE's opposite response to the drugs shows that depolymerization of the cytoskeleton also changes sticking in the cellular milieu. Our results demonstrate that perturbation of the monomer-polymer cytoskeletal equilibrium, for example, during natural cell migration or stresses from drug treatment, has off-target effects on the energy landscapes of proteins in the cell.

The current study was to isolate endophytic fungi producing high yields of indole alkaloids such as vinblastine analogous to their host Cathranthus roseus. Endophytic fungi were isolated from the leaves of C. roseus, identified as Curvularia verruculosa by molecular techniques, and the sequence was deposited in NCBI (MK995628). Vinblastine producing endophytic fungus was grown in 1L Vinca medium for 21 days. The extract was examined for vinblastine by chromatographic techniques. TLC plates showed purple colour spot co-migrated with authentic vinblastine and R f was calculated by HPTLC (Vin 1 vinblastine -0.75; authentic vinblastine-0.78), these results confirmed vinblastine presence in the Vin1 extract. Further, the TLC purified fungal extract was examined by LC-MS, which revealed the exact mass of vinblastine ([M + H] + m/z 811.51). The most important of the study is high yield production of vinblastine; hence, the extract analysed by HPLC revealed 182 μg/L vinblastine. The TLC purified fungal vinblastine was analysed for the cytotoxicity effect on HeLa cell line and it depicted a higher activity with IC 50 -8.5 μg/mL and apoptotic morphological changes were analysed. All the results revealed that the endophytic fungus Curvularia verruculosa produced vinblastine and for the first time in a surplus amount compared to other fungi., (Copyright © 2019 Elsevier Inc. All rights reserved.)

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.

(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.

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.)

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)

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.)

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.)

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.)

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.

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.)

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.)

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.)

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.)

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.

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.

Blood vessels in the tumor periphery have high pericyte coverage and are resistant to vascular disrupting agents (VDAs). VDA treatment resistance leads to a viable peripheral tumor rim that contributes to treatment failure and disease recurrence. Here, we provide evidence to support a hypothesis that shifting the target of VDAs from tumor vessel endothelial cells to pericytes disrupts tumor peripheral vessels and the viable rim, circumventing VDA treatment resistance. Through chemical engineering, we developed Z-GP-DAVLBH (from the tubulin-binding VDA desacetylvinblastine monohydrazide [DAVLBH]) as a prodrug that can be selectively activated by fibroblast activation protein α (FAPα) in tumor pericytes. Z-GP-DAVLBH selectively destroys the cytoskeleton of FAPα-expressing tumor pericytes, disrupting blood vessels both within the core and around the periphery of tumors. As a result, Z-GP-DAVLBH treatment eradicated the otherwise VDA-resistant tumor rim and led to complete regression of tumors in multiple lines of xenografts without producing the drug-related toxicity that is associated with similar doses of DAVLBH. This study demonstrates that targeting tumor pericytes with an FAPα-activated VDA prodrug represents a potential vascular disruption strategy in overcoming tumor resistance to VDA treatments.

We have previously shown that nerve inflammation (neuritis) and transient vinblastine application lead to axonal mechanical sensitivity in nociceptors innervating deep structures. We also have shown that these treatments reduce axonal transport and have proposed that this leads to functional accumulation of mechanically sensitive channels in the affected part of the axons. Though informing the etiology of mechanically induced pain, axonal mechanical sensitivity does not address the common report of ongoing radiating pain during neuritis, which could be secondary to the provocation of axonal chemical sensitivity. We proposed that neuritis and vinblastine application would induce sensitivities to noxious chemicals and that the number of chemo-sensitive channels would be increased at the affected site. In adult female rats, nerves were either untreated or treated with complete Freund's adjuvant (to induce neuritis) or vinblastine. After 3-7 days, dorsal root teased fiber recordings were taken from group IV neurons with axons within the sciatic nerve. Sciatic nerves were injected intraneurally with a combination of noxious inflammatory chemicals. Whereas no normal sciatic axons responded to this stimulus, 80% and 38% of axons responded in the neuritis and vinblastine groups, respectively. In separate experiments, sciatic nerves were partially ligated and treated with complete Freund's adjuvant or vinblastine (with controls), and after 3-5 days were immunolabeled for the histamine H 3 receptor. The results support that both neuritis and vinblastine treatment reduce transport of the histamine H 3 receptor. The finding that nociceptor axons can develop ectopic chemical sensitivity is consistent with ongoing radiating pain due to nerve inflammation. NEW & NOTEWORTHY Many patients suffer ongoing pain with no local pathology or apparent nerve injury. We show that nerve inflammation and transient application of vinblastine induce sensitivity of group IV nociceptor axons to a mixture of endogenous inflammatory chemicals. We also show that the same conditions reduce the axonal transport of the histamine H 3 receptor. The results provide a mechanism for ongoing nociception from focal nerve inflammation or pressure without overt nerve damage., (Copyright © 2017 the American Physiological Society.)

Background: VELVET Cohort 1 demonstrated the applicability of pertuzumab, trastuzumab, and vinorelbine as an alternative first-line treatment regimen for patients with HER2-positive locally advanced or metastatic breast cancer (MBC) who cannot receive docetaxel. Co-infusion of pertuzumab and trastuzumab may reduce clinic time and medical resource utilization. We report results from Cohort 2, in which pertuzumab and trastuzumab were co-infused, followed by vinorelbine., Patients and Methods: During cycle 1, patients with HER2-positive locally advanced or MBC received loading doses of pertuzumab (840 mg) and trastuzumab (8 mg/kg) on consecutive days, followed by vinorelbine (25 mg/m 2 ) on days two and nine. From cycle 2 onwards, patients received a co-infusion of pertuzumab (420 mg) and trastuzumab (6 mg/kg) on day one, followed by vinorelbine (30-35 mg/m 2 ) on days one and eight (or days two and nine). The primary endpoint was objective response rate (ORR) in patients with measurable disease. Secondary endpoints included progression-free survival (PFS) and safety., Results: Cohort 2 enrolled 107 patients. The ORR was 63.7% (95% confidence interval [CI] 53.0-73.6) in patients with measurable disease (91/107; 85.0%). Median PFS was 11.5 months (95% CI 10.3-15.8). The most common adverse events [AEs] were diarrhea (57.9%), neutropenia (57.0%), and nausea (41.1%). Grade ≥3 AEs occurred in 85 patients (79.4%) and serious AEs in 44 patients (41.1%). Eighteen patients (16.8%) had AEs suggestive of congestive heart failure., Conclusion: These results support the feasibility of pertuzumab and trastuzumab co-infusion from a safety perspective and support Cohort 1 conclusions that vinorelbine offers an alternative chemotherapy companion for pertuzumab and trastuzumab. The Oncologist 2017;22:1160-1168 IMPLICATIONS FOR PRACTICE: Combined treatment with pertuzumab, trastuzumab, and docetaxel is the standard of care for first-line HER2-positive metastatic breast cancer. However, some patients cannot, or choose not to, receive docetaxel. VELVET Cohort 2 results support the results from Cohort 1 that suggest that pertuzumab plus trastuzumab and vinorelbine is a suitable alternative for these patients. In addition to this, results from Cohort 2 support the feasibility of administering pertuzumab and trastuzumab together in a single infusion bag, which has the potential to offer greater patient convenience and reduce active health care professional time and medical resource utilization compared with administering them separately., Competing Interests: Disclosures of potential conflicts of interest may be found at the end of this article., (© 2017 The Authors. The Oncologist published by Wiley Periodicals, Inc. on behalf of AlphaMed Press.)

A series of 180 vinblastine 20' amides were prepared in three steps from commercially available starting materials, systematically exploring a typically inaccessible site in the molecule enlisting a powerful functionalization strategy. Clear structure-activity relationships and a structural model were developed in the studies which provided many such 20' amides that exhibit substantial and some even remarkable enhancements in potency, many that exhibit further improvements in activity against a Pgp overexpressing resistant cancer cell line, and an important subset of the vinblastine analogues that display little or no differential in activity against a matched pair of vinblastine sensitive and resistant (Pgp overexpressing) cell lines. The improvements in potency directly correlated with target tubulin binding affinity, and the reduction in differential functional activity against the sensitive and Pgp overexpressing resistant cell lines was found to correlate directly with an impact on Pgp-derived efflux.

Purpose: Despite the efficacy of postoperative adjuvant cisplatin (CDDP)-based chemotherapy for patients who have undergone surgical resection of non-small cell lung cancer (NSCLC), few reports have presented survival data for Asian patients treated with adjuvant chemotherapy involving a combination of CDDP and vinorelbine (VNR). This study was performed to evaluate the survival of patients with NSCLC who received postoperative adjuvant chemotherapy comprising CDDP + VNR., Methods: We retrospectively evaluated patients with NSCLC who received adjuvant chemotherapy comprising CDDP + VNR at the Shizuoka Cancer Center between February 2006 and October 2011., Results: One hundred patients who underwent surgical resection of NSCLC were included in this study. The patients' characteristics were as follows: median age 63 years (range 36-74 years), female 34%, never-smokers 20%, and non-squamous NSCLC 73%. Pathological stages IIA, IIB, and IIIA were observed in 31, 22, and 47% of patients, respectively. The 5- and 2-year overall survival rates were 73 and 93%, respectively. The 5- and 2-year relapse-free survival rates were 53 and 62%, respectively. Univariate analysis of prognostic factors showed that patient characteristics (sex, histology, and pathological stage) and CDDP dose intensity were not significantly associated with survival. In 48 patients who developed NSCLC recurrence, the 5-year survival rate after recurrence was 29%, and the median survival time after recurrence was 37 months., Conclusions: Our results suggest that the prognosis after surgical resection of NSCLC and adjuvant chemotherapy comprising CDDP + VNR might be improving compared with previous survival data of adjuvant chemotherapy for NSCLC.

Background: Breast carcinoma metastasis to the gastrointestinal tract is rare and more frequently associated with lobular than ductal carcinoma (Borst and Ingold, Surg 114(4):637-641 [1]). The purpose of this article is to present a case based review of a unique gastrointestinal metastasis and literature review., Methods: A 46 year old lady with metastatic invasive ductal breast cancer was admitted to A&E with sudden onset of epigastric and left shoulder pain. She completed the first cycle of capecitabine/vinorelbine 1 week previously. Clinical examination revealed a tender epigastrium with rigidity in the upper abdomen. Free air under the diaphragm and a positive Rigler's sign was radiologically identified. A laparoscopy demonstrated a fibrinous exudate in the left upper quadrant consistent with a walled off lesser curvature gastric perforation. A subsequent oesophagogastroduodenoscopy (OGD) demonstrated a healed gastric ulcer of benign appearance; however the pathology confirmed metastatic breast carcinoma., Results: Literature review confirmed no previously reported cases of vinorelbine induced gastric perforation. Four cases of metastatic breast cancer with gastric metastasis presenting with perforation were identified; three of these cases (Fra et al., Presse Med 25(26):1215 (1996) [2], Solis-Caxaj et al., Gastroenterol Clin Biol 28(1):91-92 (2004) [3], Ghosn et al., Bull Cancer 78(11):1071-1073 (1991) [4]), were in the French medical literature, including one male patient (Fra et al., Presse Med 25(26):1215 (1996) [2]) and at least one ductal breast carcinoma (Solis-Caxaj et al., Gastroenterol Clin Biol 28(1):91-92 (2004) [3]). The fourth case (van Geel et al., Ned Tijdschr Geneeskd 144(37):1761-1763 (2000) [5]), was in the Dutch medical literature and a lobular breast carcinoma., Conclusion: This case represents a rare complication of breast cancer chemotherapy, the subsequent significant benefit the patient received from treatment is consistent with the chemosensitivity to therapy that also resulted in gastric perforation. Five years after gastric perforation she resumed palliative chemotherapy after progression on sequential hormonal therapies.

A key series of vinblastine analogs 7-13, which contain modifications to the C20' ethyl group, was prepared with use of two distinct synthetic approaches that provide modifications of the C20' side chain containing linear and cyclized alkyl groups or added functionalized substituents. Their examination revealed the unique nature of the improved properties of the synthetic vinblastine 6, offers insights into the origins of its increased tubulin binding affinity and 10-fold improved cell growth inhibition potency, and served to probe a small hydrophobic pocket anchoring the binding of vinblastine with tubulin. Especially noteworthy were the trends observed with substitution of the terminal carbon of the ethyl group that, with the exception of 9 (R=F vs H, equipotent), led to remarkably substantial reductions in activity (>10-fold): R=F (equipotent with H)>N 3 , CN (10-fold)>Me (50-fold)>Et (100-fold)>OH (inactive). This is in sharp contrast to the maintained (7) or enhanced activity (6) observed with its incorporation into a cyclic C20'/C15'-fused six-membered ring., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

The multidrug resistance (MDR) phenotype is considered as a major cause of the failure in cancer chemotherapy. The acquisition of MDR is usually mediated by the overexpression of drug efflux pumps of a P-glycoprotein. The development of compounds that mitigate the MDR phenotype by modulating the activity of these transport proteins is an important yet elusive target. Here, we screened the saponification and enzymatic degradation products from Salvia hispanica seed's mucilage to discover modulating compounds of the acquired resistance to chemotherapeutic in breast cancer cells. Preparative-scale recycling HPLC was used to purify the hydrolysis degradation products. All compounds were tested in eight different cancer cell lines and Vero cells. All compounds were noncytotoxic at the concentration tested against the drug-sensitive and multidrug-resistant cells (IC 50  > 29.2 μM). For the all products, a moderate vinblastine-enhancing activity from 4.55-fold to 6.82-fold was observed. That could be significant from a therapeutic perspective. Copyright © 2017 John Wiley & Sons, Ltd., (Copyright © 2017 John Wiley & Sons, Ltd.)