Tubulin; Microtubule (Ki = 85 nM)

Vincristine, with a Ki of 85 nM, inhibits the net addition of tubulin dimers at the assembly ends of steady-state microtubules[1]. At low doses, Vincristine inhibits mitosis and causes metaphase arrest by stabilizing the spindle apparatus, which prevents the chromosomes from segregating. Vincristine has the potential to cause complete depolymerization of microtubules when used at higher concentrations [2]. With an IC50 of 0.1 μM, vincristine suppresses the proliferation of SH-SY5Y cells and causes apoptosis in tumor cells. Vincristine decreases the expression of cyclin D while inducing mitotic arrest and promoting the production of caspase-3, -9, and cyclin B[3]. Vincristine-induced neurotoxicity is brought on by disruption of microtubule activity, which obstructs axonal transport and ultimately causes axonal degeneration[4].

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Vinepidine, a new derivative of Vincristine, and three clinically used Catharanthus derivatives, vinblastine, vincristine, and vindesine, were examined for their abilities to inhibit net tubulin addition at the assembly ends of bovine brain microtubules at steady state. Although all four derivatives were generally similar in potency, their relative abilities to inhibit tubulin addition were distinguishable. Vinepidine and vincristine were the most potent derivatives (Ki, 0.079 +/- 0.018 (SD) microM and 0.085 +/- 0.013 microM, respectively), followed by vindesine (Ki, 0.110 +/- 0.007 microM) and vinblastine (Ki, 0.178 +/- 0.025 microM). In contrast to their relative abilities to inhibit microtubule assembly in vitro, vinblastine and its derivative, vindesine, were generally more potent than vincristine and vinepidine in inhibiting cell proliferation in culture. Vinblastine was nine times more potent than the weakest derivative, vinepidine, in B16 melanoma cells. In L-cells, vinblastine completely inhibited growth at 40 nM, whereas vincristine and vindesine caused about 25% inhibition, and vinepidine was inactive. When B16 melanoma cells were treated with drug before being injected into mice, retardation of tumor growth was best achieved with vindesine, one of the weaker of the four derivatives in vitro. The results demonstrate that chemical differences among the Catharanthus derivatives, which affect to small extents the abilities of the derivatives to inhibit microtubule assembly in vitro, result in significant differences in the order and the magnitude of the abilities of the drugs to inhibit cell growth.[1]

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Axonal ultrastructural changes induced by three Vinca alkaloids, Vincristine, vinblastine, and desacetyl vinblastine amide, were studied in vitro at concentrations of 0.01, 0.05, and 0.1 mM in the cat vagus nerve. Disruption of microtubules, appearance of paracrystalline structures, and increase in neurofilaments were induced by all three agents at 0.1 mM. A new type of paracrystal with an electron-dense central core in each subunit was also observed with each drug. Whereas all three compounds affected unmyelinated fibers (vinblastine more so than the other two), only vinblastine significantly damaged the myelinated fibers. The greater effectiveness of vinblastine in causing these in vitro ultrastructural changes contrasts strikingly with the clinical in vivo situation in which vincristine is the most neurotoxic. This suggests that clinical neurotoxicity is associated with additional factors aside from the direct interaction of the Vinca alkaloids with microtubules or tubulin.[3]

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Cytotoxic effects of Vincristine and SAHA, alone and in combination, on human leukemic MOLT-4 cells [7]
A 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay was performed to investigate the cytotoxicity of the microtubule-destabilizing agent vincristine and the HDACi vorinostat (SAHA) on human ALL MOLT-4 cells. We first tested the cytotoxic effect of SAHA and vincristine alone and in combination. As shown in Fig. 1a, there was no significant cytotoxicity at concentrations up to 500 nM of SAHA. However, SAHA had an IC50 of 840 nM for 48 h, when concentration reached the highest level (1000 nM). In addition, vincristine exhibited cytotoxicity against human leukemic MOLT-4 cells with an IC50 of 3.3 nM at 48 h (Fig. 1b). To determine whether an interaction between SAHA and vincristine took place, the cytotoxic potency of a combination assay was measured. Cells treated with 500 nM SAHA and various concentrations of vincristine (0.3 to 3 nM) significantly inhibited cell survival compared to each treatment alone (Fig. 1c).

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Effects of Vincristine in combination with SAHA on human T cell leukemic cell survival [7]
To further explore the synergistic cytotoxic effects, we determined the effects on cell cycle distribution. As compared with SAHA, treatment with vincristine induced an increase in the G2/M phase of the cell cycle. In particular, the combination of vincristine plus SAHA caused an almost complete arrest of cells in the G2/M phase following short-term treatment (24 h) and a subsequent induction in the sub-G1 phase following long-term treatment (48 h) (Fig. 2a). Figure 2b shows the statistical results. Next, the combination index (CI) method was used to evaluate the synergistic combinations [25]. A CI value of >1.0, 1.0, and <1.0 indicates an antagonistic, additive, or synergistic interaction, respectively, between the drugs. In the G2/M phase, the CI values of vincristine (0.3, 1, and 3 nM) combined with 500 nM SAHA were 1.63, 0.72, and 0.32, respectively, and the CI values in the sub-G1 phase were 0.97, 0.77, and 0.28, respectively (Fig. 2c). And this synergistic combination effect also was noted in the other T cell leukemic cell line, CCRF-CEM (Fig. 2d), rather than in acute myeloid leukemic cells (Additional file 1: Figure S2). Moreover, vincristine (1 or 3 nM) combined with various concentrations of SAHA also shows synergistic effect (Additional file 2: Figure S1). These data indicate that vincristine and SAHA synergistically induced cell arrest in the G2/M phase and subsequently in the sub-G1 phase.

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Effects of SAHA in combination with Vincristine on mitotic arrest in human leukemic MOLT-4 cells [7]
To further elucidate the synergistic effect mechanism on the G2/M phase of cell cycle progression, we investigated SAHA in combination with vincristine on tubulin polarization change and mitosis-related proteins. As shown in Fig. 3a, there were no obvious tubulin polarization changes following SAHA treatment under cell-free conditions. However, in combination with vincristine, a significant induction of microtubule depolymerization was observed (Fig. 3a). Additional file 3: Figure S3 shows a more comprehensive result, including various vincristine- and SAHA-alone in vitro tubulin polymerization assays. To understand the effects of microtubule dynamics on mitosis following drug treatment, the microtubule arrangement in human leukemic MOLT-4 cells was examined by β-tubulin staining. As shown in Fig. 3b(b), there was no significant change in microtubule distribution and cell morphology after SAHA treatment. In addition, at low vincristine concentrations, cells had accumulated at the metaphase stage of mitosis with abnormal spindles (Fig. 3b(c)). In this study, spindles with bipolar and multipolar organization, which had abnormal long astral microtubules and chromosomes, were found to be unequally distributed. Nevertheless, at a high vincristine concentration, microtubule depolymerization was observed (Fig. 3b(d)). In the present study, the vincristine and SAHA combination exerted more explicit effects than vincristine alone with regard to abnormal spindles and chromosomes (Fig. 3b and Additional file 4: Figure S4). These results suggest that SAHA potentiated the effects of vincristine due to inhibition of microtubule dynamics.

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Effects of SAHA in combination with Vincristine on the apoptotic pathway and HDAC activity in human leukemic MOLT-4 cells [7]
Mitochondria play a crucial role both in the intrinsic and extrinsic apoptotic pathways. To test whether the vincristine/SAHA-mediated apoptotic pathway was associated with mitochondrial function, a change in mitochondrial transmembrane potential (Δψm) was assessed. As shown in Fig. 4a, treatment with SAHA or vincristine alone was insufficient to affect the mitochondrial membrane potential; however, this phenomenon was enhanced by co-treatment with SAHA in a time-dependent manner. The Bcl-2 protein family plays a regulatory role in controlling the mitochondrial apoptotic pathway. The data showed that the combination treatment more effectively downregulated the expression of the pro-survival members of the Bcl-2 family, such as Bcl-2, Bcl-xl, and Mcl-1, than did either treatment alone (Fig. 4b).

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HDAC6 inhibition was involved in Vincristine/SAHA-induced apoptosis [7]
Previous findings have shown HDAC6-induced tubulin acetylation to affect the dynamics and function of microtubules [9–12]. As shown in Fig. 5a, SAHA, a pan-HDACi, induced tubulin acetylation; however, its combination with vincristine had no synergic effect. Tubastatin A, which is a specific HDAC6 inhibitor [26], was used to understand the role of HDAC6 in the vincristine/SAHA-treated cells. To evaluate the potential benefit of vincristine in combination with tubastatin A, the cytotoxicity of co-treatment was determined and the combination effects were analyzed. However, compared to tubastatin A alone, vincristine significantly enhanced the cytotoxicity of tubastatin A (Fig. 5b). Moreover, vincristine (1 and 3 nM) combined with various concentrations of tubastatin A induced cell accumulation at the G2/M phase followed by the sub-G1 phase (Fig. 5c). The CI values were <1 in combination of vincristine and tubastatin at the G2/M phase and sub-G1 phase (Fig. 5d). Co-treatment of vincristine and tubastatin revealed MPM2 and PARP activation consistent with the induction of apoptosis by western blot analysis (Fig. 5e). And vincristine and HDAC6 inhibitor combined synergism effect was further corroborated by the observation of vincristine and ACY1215 co-treatment in CCRF-CEM cells (Fig. 5f). These findings suggest that SAHA treatment may alter microtubule dynamics in cells through HDAC6 inhibition, even though the effect was insufficient to arrest cells in the G2/M phase. However, in combination with vincristine, which also had an effect on microtubules, SAHA caused extreme microtubule stress thus causing cell death.

Vincristine (3 mg/kg, i.p.) given to mice receiving bilateral subcutaneous xenografts of Rh12 or Rh18, respectively, causes a mean growth delay of more than 120 and more than 52 days and repopulates fractions of 0.06% and 5%[5].

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The antitumor activity of Vincristine and SAHA combination therapy in vivo [7]
To evaluate whether the synergistic effect of Vincristine plus SAHA could be clinically relevant, the antitumor activity of this co-treatment in severe combined immunodeficiency mice bearing established MOLT-4 tumor xenografts was investigated. Once a tumor was palpable (approximately 100 mm3), mice were randomized into vehicle control and treatment groups (n = 6 per group). All mouse tumors were allowed to reach an endpoint volume of 2000 mm3, and in vivo antitumor efficacy was expressed as tumor growth delay (TGD; Fig. 6a). There were no improvements in TGD in mice treated with vincristine (0.1 mg/kg once weekly) or SAHA (50 mg/kg once daily) alone. However, log-rank analysis showed that the co-treatment exhibited significant antitumor activity in the MOLT-4 xenograft model (P = 0.0389). In addition, Kaplan–Meier curves displayed antitumor activity for the co-treatment group (vincristine, 0.025 mg/kg once weekly; SAHA, 200 mg/kg once daily) (Fig. 6b). Notably, the mice tolerated all of the treatments without overt signs of toxicity; no significant body weight difference or other adverse side effects were observed (Fig. 6d and Additional file 5: Figure S5). To correlate the in vivo antitumor effects with the mechanisms identified in vitro, intratumoral biomarkers were assessed by western blot analysis. Consistent with in vitro results, the combined treatment markedly induced caspase 3 activation and PARP cleavage in tumors, indicating elevated apoptosis (Fig. 6e). Taken together, these findings suggest that combination of vincristine and SAHA, both in vitro and in vivo, dramatically enhanced vincristine-induced cell death.

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Loss of the sense of touch in fingertips and toes is one of the earliest sensory dysfunctions in patients receiving chemotherapy with anti-cancer drugs such as Vincristine. However, mechanisms underlying this chemotherapy-induced sensory dysfunction is incompletely understood. Whisker hair follicles are tactile organs in non-primate mammals which are functionally equivalent to human fingertips. Here we used mouse whisker hair follicles as a model system and applied the pressure-clamped single-fiber recording technique to explore how vincristine treatment affect mechanoreceptors in whisker hair follicles. We showed that in vivo treatment of mice with vincristine impaired whisker tactile behavioral responses. The pressure-clamped single-fiber recordings made from whisker hair follicle afferent nerves showed that mechanical stimulations evoked three types of mechanical responses, rapidly adapting response (RA), slowly adapting type 1 response (SA1) and slowly adapting type 2 response (SA2). Vincristine treatment significantly reduced SA1 responses but did not significantly affect RA and SA2 responses. Our findings suggest that SA1 mechanoreceptors were selectively impaired by vincristine leading to the impairment of in vivo whisker tactile behavioral responses. [8]

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Tumor responsiveness to Vincristine (VCR) was determined in xenografts of human rhabdomyosarcoma (RMS), in sublines of RMS selected in vivo for VCR resistance, in a KB line (KB-ChR8-5) selected in vitro for colchicine resistance, and in a colon adenocarcinoma (GC3). Sensitivity to VCR was associated with prolonged retention of VCR by the tumors after a single i.p. injection, whereas in tumors with acquired or intrinsic VCR resistance the drug was eliminated more rapidly. The sensitive tumors with prolonged retention of drug also showed increased levels of mitotic accumulation for up to 72 hr following VCR administration. There were good correlations between VCR sensitivity, VCR retention and the proposed mechanism of VCR cytotoxicity-mitotic arrest. A model has been developed consistent with data obtained that can explain the responsiveness to VCR of a series of human tumor xenografts irrespective of their tissue of origin[4].

In vitro tubulin polymerization assay [7]
To determine the microtubule polymerization of the indicated drugs in a cell-free condition, CytoDYNAMIX Screen 03 Kit was performed. General tubulin buffer, GTP stock (100 mM), and tubulin protein (10 mg/ml) were all prepared well following the protocol. A 96-well plate was placed in the spectrophotometer to prewarm at 37 °C for 30 min before detection. Then preparing the iced tubulin polymerization (TP) buffer, all mentioned processes were needed on the ice. Next, the drugs (2 μl) were added into each Eppendorf included with 85 μl TP buffer. The drugs must include DMSO (the control group), paclitaxel (10 μΜ), and Vincristine (10 μΜ). Paclitaxel and vincristine were used as positive controls. Paclitaxel would induce the microtubule polymerization; in contrast, vincristine would depolymerize the microtubules. Finally, 30 μl tubulin proteins was added into the Eppendorf and transferred to the prewarmed 96-well plate. The absorbance was measured by a spectrophotometer and recorded every 1 min for 30 min at 340 nm and 37 °C.

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VCR/Vincristine release profile in vitro from CS-ALG@TPGS-PL-GA-VCR NPs was researched using the classical dialysis bag method. Briefly, CS-ALG@TPGS-PLGA-VCR NPs (4 mg) were re-suspended in PBS (2 mL, 0.1% w/v Tween 80, pH 5.8 or pH 7.4) to simulate the cancer cytoplasmic environment (pH 5.8) and the physiological environment (pH 7.4). Subsequently, the re-suspension was transferred into a dialysis bag (molecular weight cut off 3 kDa; Millipore, Billerica, MA, USA), which was immersed in PBS (20 mL) and incubated in 37°C thermostatic water bath with 100 rpm shaking. At a designated time interval, 5 mL of the release solution was taken out to detect the concentration of VCR at 298 nm using an ultraviolet spectrophotometer. At the same time, isometric fresh PBS was added into the release medium [6].

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Mitochondrial membrane potential [7]
Rhodamine 123 was used to evaluate mitochondrial membrane potential. Rhodamine 123 is a kind of cationic fluorescent dye, which localizes in the mitochondria. Loss of mitochondrial membrane potential is associated with a lack of rhodamine 123 retention and a decrease of fluorescence intensity. Cells were treated with Vincristine, SAHA, or combination for the indicated time. Rhodamine 123 (final concentration 10 μM) was added and incubated for 30 min at 37 °C in the dark. Then, cells were harvested and rinsed with PBS. The fluorescence intensity was measured by FACScan Flow Cytometer and CellQuest.

Cell viability assay [7]
Cell viability was verified by MTT assay. Firstly, cells were seeded in a 24-well plate at a density of 4 × 105 cells/well in 1 ml culture medium and then treated with various concentrations of Vincristine or SAHA alone or a combination of both for 24 and 48 h. After treatment with drugs, 100 μl MTT solution (0.5 mg/ml in phosphate-buffered saline (PBS)) per well was added to the 24-well plate in the dark and the plate was incubated at 37 °C. The mitochondrial dehydrogenase of viable cells reduced MTT (yellow) to insoluble formazan dyes (purple). One hour later, the crystal formazan dyes were dissolved in the extraction buffer (0.1 M sodium acetate buffer, 100 μl/well). The absorbance was spectrophotometrically analyzed at 550 nm by an ELISA reader.

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Flow cytometry analysis [7]
Evolution of the cell cycle histogram was performed by flow cytometry analysis to detect the changes in DNA content. Cells (1 × 106) were seeded in a 6-well plate in 2 ml fresh medium and treated with graded concentrations of Vincristine, SAHA, or combination for the indicated time. Then, cells were collected, washed with PBS and fixed with 70 % (v/v) ice cold ethanol at −20 °C for 30 min. The fixed cells were centrifuged to remove the ethanol, rinsed with PBS, resuspended in 0.1 ml DNA extraction buffer (0.2 M Na2HPO4-0.1 M citric buffer, pH 7.8) for 20 min, and subsequently stained with 500 μl PI solution (80 μg/ml propidium iodide, 100 μg/ml RNase A, and 1 % Triton X-100 in PBS) for 20 min at room temperature in the dark. Data were analyzed by FACScan Flow Cytometer and CellQuest software (Becton Dickinson).

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Immunofluorescence analysis [7]
Microtubule distribution and morphology were detected by immunofluorescence. Cover slides were placed in the 24-well plate and coated with poly-d-lysine for 1 day at least to enhance the suspension cells attached to the cover slides. Cells were seeded into the 24-well plate (8 × 105 cells/well) and treated with Vincristine, SAHA, or both drugs for 24 h. The following experiments were performed at room temperature. The cells were fixed with 8 % paraformaldehyde in PBS for 15 min. After washing with PBS for several times, the cells were permeabilized with 0.1 % Triton X-100 in PBS for 10 min. Then, the cells were rinsed with PBS for 10 min three times. For blocking, 3 % BSA in PBS was used. After 1 h, the cells were washed with PBS and incubated with a primary β-tubulin antibody (1:200) for 2 h and FITC-conjugated anti-mouse IgG antibody (1:200) for 2 h. The mounting medium, which contains DAPI stain, was dropped onto the slides, and cover slides were recovered to the slides. Images were detected and captured with the ZEISS LSM 510 META confocal microscope.

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In vitro cytotoxicity of drug-loaded NPs [6]
The cytotoxicity of free drugs, drug-loaded NPs against A549 cell, and A549/taxol cell was evaluated using the standard WST-1 assays. Briefly, A549 cell and A549/taxol cell were seeded onto 96-well plates at a density of 5.0×104 cells per well and cultured for overnight 24 h with 100 μL of DMEM medium or RPMI 1640 medium at 37°C in 5% CO2 atmosphere. Then, removing the original medium, tested cells were incubated, respectively, with fresh medium containing free DOX, free VCR/Vincristine, free (DOX plus VCR), CS-ALG@TPGS-PLGA-VCR NPs, CS-ALG-DOX@TPGS-PLGA NPs, and CS-ALG-DOX@TPGS-PLGA-VCR NPs at different concentrations. After 12 h and 24 h of incubation, 10 μL of WST-1 solution was added to each well and cultured for additional 4 h. Afterward, the cell viability was determined by measuring the absorbance at 450 nm using a microplate reader.

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The microtubule and the microfilament imaging analysis [6]
A549 cell or A549/taxol cell was seeded on a coverslip in a 6-well plate (1×105 cells/well) and incubated overnight in a 5% CO2 incubator at 37°C for attachment. Then the cells were washed and cultured with TPGS-PLGA (0.2 mg/mL) NPs, free VCR/Vincristine (5 μg/mL), and TPGS-PLGA-VCR NPs (5 μg/mL VCR-loaded NPs) for a given time at 37°C, respectively. Cells without treatment were used as control group. Tested cells were washed with PBS and treated with Hoechst 33342 (10 mg/mL) for 20 min. Subsequently, the cells were fixed with 4% paraformaldehyde at room temperature for 10 min, permeated with 1% BSA in phosphate buffered saline containing 0.1% Tween-20 (PBST) for l h. The cells were then exposed to tubulin-tracker red (1:250, diluted in PBST containing 1% BSA, for the microtubule) or actin-tracker green (8 unit/mL, diluted in PBST containing 1% BSA, for the microfilament) at 4°C for 20 min in the dark room. Then, the original medium containing tubulin-tracker red or actin-tracker green was removed and the cells were washed with ice-cold PBS. Finally, the microtubule or the microfilament of the cells was visualized under a CLSM.

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Analysis of cell cycle [6]
The cell cycle induced by empty NPs, free VCR/Vincristine, and drug-loaded NPs on A549/taxol cell was evaluated with cell cycle and apoptosis analysis kit. The cells were seeded at a density of 5×105 cells per well in 6-well plates for 24 h at 37°C, and then treated, respectively, with the fresh medium containing TPGS-PLGA NPs (0.2 mg/mL), free VCR (5 μg/mL), and TPGS-PLGA-VCR NPs (dose of VCR 5 μg/mL). The cells without treatment were used as control. After incubating for 24 h, the cells were harvested by trypsin digestion and centrifugation. Subsequently, the obtained cells were fixed with 70% cold ethanol and stored at 4°C for 24 h. Ultimately, the cells were centrifuged again, washed with cold PBS twice, and stained with 0.5 mL of the staining solution at 37°C for 30 min in the dark. The stained cells were analyzed using a flow cytometer system. Each experiment was performed in triplicates.

\n\nTumor xenograft model [7]
\nA tumor xenograft model was used to estimate the combination effect of Vincristine and SAHA in vivo. MOLT-4 cells were implanted (107 cells/ml) into severe combined immunodeficiency (SCID) mice. When the average tumor size reached 100 mm3, mice were treated with an indicated dosage of vincristine or SAHA alone or a combination of both. Mice were scarified until the average tumor size was larger than 2500 mm3. Then, tumors were resected and frozen for the western blot analysis to evaluate the effect of vincristine/SAHA combination in vivo. \n

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\n\nIn vivo Vincristine treatment and tactile behavioral assessment [8]
\nVehicle (sterile phosphate-buffered saline) or Vincristine dissolved in vehicle was i.p. administered daily for 5 consecutive days, then 2 day breaks, and daily for 5 consecutive days again. The dose of vincristine was at 0.3 mg/kg each injection. Unless otherwise indicated, whisker tactile behavioral tests were performed once every 2 or 3 days for up to 21 days. The whisker tactile test was performed in a blinded manner in that one examiner conducted vincristine injections and animal grouping, and another examiner who did not know the grouping performed in vivo whisker tactile behavioral tests. To begin the tests, mice were placed in a cage and habituated for 10 min. During habituation and subsequent experiments, the testing room only had a red light on so that animals could not see the examiner and the tactile stimulation filament. After the habituation, right whisker hairs of animals were displaced by the tactile stimulation filament in a caudal to rostral direction. The whisker tactile test was repeated 20 times with an interval of 1 min between trials. A positive whisker tactile behavioral response was considered when the testing animal exhibited an avoidance reaction to the tactile stimulation.\n

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\n\nWhisker hair follicle afferent fiber recordings [8]
\nWhisker hair follicles were harvested from animals 17–31 days following the injections of vehicle or Vincristine. Whisker hair follicle preparations were then made and whisker afferent recordings were performed using our previously described method. In brief, mice were anesthetized with isoflurane and sacrificed by decapitation. Whisker hair follicles with attached afferent fiber bundles were dissected out and anchored in a recording chamber. The whisker hair follicles were submerged and perfused in the oxygenated Krebs solution that contained (in mM): 117 NaCl, 3.5 KCl, 2.5 CaCl2, 1.2 MgCl2, 1.2 NaH2PO4, 25 NaHCO3, and 11 glucose, bubbled with 95 % O2 and 5% CO2, adjusted pH to 7.3 and osmolarity to 325 mOsm, and was maintained at 24 °C. The end of each follicle capsule was cut open to facilitate bath diffusion into the whisker hair follicle. In one set of experiments, compound action potentials evoked by mechanical stimulation and conducted on whisker afferent fibers were recorded using a suction electrode. In most of experiments, action potentials evoked by mechanical stimulation and conducted on a single whisker afferent fiber was recorded using the pressure-clamped single-fiber recording technique that we developed recently. In brief, under a 40x objective, individual fibers in the cutting end of the whisker afferent nerve bundle were separated by a positive pressure of approximately +10 mmHg delivered from the recording electrode. The end part of a single nerve fiber was then aspirated into the recording electrode by a negative pressure at approximately -10 mmHg. Once the nerve end reached approximately 10 μm in length within the recording electrode, the pressure in the recording electrode was readjusted to -5 to -1 mmHg and maintained throughout the experiment. In a different set of experiments, individual whisker hair follicles were obtained from normal animals not treated in vivo with Vincristine. The hair follicles were then in vitro incubated with 2 μM vincristine (vincristine-treated group) or vehicle (control) for 2 h, and the pressure-clamped single-fiber recordings were performed from whisker afferent fibers. In all electrophysiological recordings, nerve impulses were recorded using a Multiclamp 700A amplifier and signals sampled at 20 KHz with low pass filter set at 1 KHz. All recording experiments were performed at 24 °C.\n

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\n\nTumor-bearing mice model and administration procedure [6]
\nA549 cells of the logarithmic growth phase were harvested by centrifugation and re-suspend in PBS to form cell suspension (5×107 cells/mL). A 100 μL A549 cell (5×106 cells/mL) suspension was injected on the roots of the upper thigh of tested nude mice.\n\nThe preparation period of tumor model needs about 7–10 days. Based on tumor size and physiological state, tumor-bearing mice were randomly divided into three groups (n=5): negative control group (saline), positive control group (free DOX plus VCR/Vincristine; DOX 2 mg/kg + VCR 2 mg/kg), and tested group (CS-ALG-DOX@TPGS-PLGA-VCR NPs, dose DOX 2 mg/kg + VCR 2 mg/kg).\n\nThe day of administration was defined as day 0 (D0). Following 18 days, saline or the formulations of drugs were administered by the tail intravenous injection for 7 times in an interval of 3 days.\n

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\n\nIn vivo biodistribution of drugs [6]
\nWe also evaluated the in vivo tissue distribution of co-delivery NPs in tumor-bearing mice by quantitatively detecting the DiR fluorescence intensity in tumor tissues and some important organs. For the research, the near-infrared (IR) dye DiR was encapsulated into TPGS-PLGA NPs from TPGS-PLGA-DiR NPs. The preparation method of DiR-loaded NPs was the same as that of VCR-loaded NPs, except that VCR/Vincristine was replaced by 1 mg DiR.\n\nBased on tumor size and physiological state, tumor-bearing mice were randomly divided into DiR group and TPGS-PLGA-DiR NP group (n=3). After administration with the formulations via tail vein injection, the mice were sacrificed at 24 h to collect tumor tissues and organs (liver, kidney, spleen, heart, and tumor) for further ex vivo fluorescence density analysis with an In-Vivo Imaging System.

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Dissolved in water; 3 mg/kg; i.p. injection

Human rhabdomyosarcoma xenografts Rh12

Absorption, Distribution and Excretion
The liver is the primary excretory organ in humans and animals. Of the injected dose of vincristine sulfate, 80% is excreted in feces and 10-20% in urine. Within 15 to 30 minutes after injection, over 90% of the drug is distributed from the bloodstream to the tissues and binds tightly, but not irreversibly. Central nervous system leukemia has occurred in patients receiving vincristine treatment in hematologic remission, which is interpreted as evidence that vincristine has difficulty crossing the blood-brain barrier. Vincristine can be infused into the arterial blood supply area of a tumor at doses several times higher than intravenous, with comparable toxicity; therefore, local absorption or destruction is very rapid. Vinca alkaloids appear to be primarily excreted into the bile via the liver. Urinary excretion is low in dogs and monkeys during the first few hours after injection. In dogs and monkeys, the drug is distributed in most tissues, but the highest concentrations are found in the lungs, kidneys, spleen, pancreas, and liver. In monkeys, vincristine and its metabolites rapidly enter the cerebrospinal fluid from the plasma, forming low concentrations of the drug that persist for several days. Absorption of vincristine sulfate in the gastrointestinal tract is unpredictable. In patients with normal renal and hepatic function, rapid intravenous injection of 2 mg of vincristine immediately results in peak serum drug concentrations of approximately 0.19–0.89 μmol/L, followed by rapid clearance from the serum. Continuous intravenous infusion of vincristine increases the area under the serum vincristine concentration-time curve (AUC) compared to rapid intravenous injection, provided the doses are comparable. The distribution of vincristine and its metabolites (and/or breakdown products) in human tissues and fluids is not fully understood, but after intravenous administration, the drug distributes rapidly and extensively. The drug distributed to tissues binds tightly to the tissues, but this binding is reversible. Vincristine and its metabolites (and/or breakdown products) rapidly and extensively distribute into the bile, reaching peak bile concentrations within 2–4 hours after rapid intravenous injection. Vincristine and its metabolites (and/or breakdown products) have difficulty crossing the blood-brain barrier after rapid intravenous injection and typically do not reach cytotoxic concentrations in the cerebrospinal fluid. For more complete data on the absorption, distribution, and excretion of vincristine (6 types), please visit the HSDB record page. Metabolism/Metabolites Hepatic metabolism. Cytochrome P450 isoenzymes of the CYP3A subfamily promote vincristine metabolism. Following intravenous injection of 3H-labeled vincristine, 69% of the radioactive material is recovered in feces and 12% in urine within 72 hours. Approximately half remains as metabolites, whose UV spectra indicate that the vincristine dimer remains intact. Patients with bile fistulas showed significant excretion of the intact drug (46.5%) and its metabolites (53.5%) via bile. Observations suggest that the bile-fecal route is the primary route of excretion. The metabolic pathway of vincristine is not fully understood; the drug appears to be extensively metabolized, likely in the liver, but the extent of metabolism is unclear due to potential breakdown within the body. The liver is involved. Cytochrome P450 isoenzymes of the CYP3A subfamily promote vincristine metabolism. Elimination pathway: The liver is the primary excretory organ in humans and animals. Of the injected dose of vincristine sulfate, 80% is excreted in feces and 10-20% in urine. Half-life: A triphasic change in serum half-life was observed when administered intravenously to cancer patients. The initial, intermediate, and terminal half-lives were 5 minutes, 2.3 hours, and 85 hours, respectively. The range of the terminal half-life in humans is 19 to 155 hours.

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Biological half-life
When administered intravenously to cancer patients, a triphasic decay pattern of serum concentration was observed. The initial half-life, intermediate half-life, and terminal half-life are 5 minutes, 2.3 hours, and 85 hours, respectively. The range of the terminal half-life in humans is 19 to 155 hours. Following intravenous injection of vincristine, a three-phase decay was observed, with half-lives of 0.85 minutes, 7.4 minutes, and 164 minutes, respectively. Following rapid intravenous injection of vincristine, serum drug concentrations appear to decrease in a three-phase manner. The terminal elimination half-life of vincristine is 19–155 hours.

Effects During Pregnancy and Lactation
◉ Overview of Medication Use During Lactation
Most data suggest that mothers should not breastfeed while receiving anti-tumor drug treatment. Due to the long half-life of vincristine, resuming breastfeeding after treatment may not be practical. Chemotherapy may adversely affect the normal microbiota and chemical composition of breast milk. ◉ Effects on Breastfed Infants
A 4-month-old infant developed neutropenia, possibly due to the mother receiving cyclophosphamide treatment 9 days prior. The mother had previously received 800 mg of cyclophosphamide intravenously once a week for 6 weeks; concurrently, she received one 2 mg intravenous injection of vincristine; and 30 mg of prednisolone orally daily. The neutropenia lasted at least 12 days and was accompanied by transient diarrhea. The effect of vincristine on neutropenia is currently undetermined.
A woman was diagnosed with B-cell lymphoma at 27 weeks of pregnancy. Labor was induced at 34 weeks and 4 days of gestation, and she began standard treatment with rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (dosage not specified), for a 21-day course, starting on day 2 postpartum. For the first 10 days of each course, she expressed and discarded breast milk, feeding the infant with donated breast milk, and then resumed breastfeeding for 10 days before the start of the next course. This 10-day breastfeeding pause was calculated based on the half-life of vincristine and was approximately equivalent to 3 half-lives. After completing 4 courses of chemotherapy, her infant was reported to be in good health, developing normally, and without any complications.
◉ Effects on lactation and breast milk
As of the revision date, no relevant published information was found.

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Protein binding rate ~75%

[1]. Comparison of the effects of vinblastine, vincristine, vindesine, and vinepidine on microtubule dynamics and cell proliferation in vitro. Cancer Res, 1985. 45(6): p. 2741-7.

[2]. Gidding, C.E., et al, Vincristine revisited. Crit Rev Oncol Hematol, 1999. 29(3): p. 267-87.

[3]. Donoso, J.A., et al, Action of the vinca alkaloids vincristine, vinblastine, and desacetyl vinblastine amide on axonal fibrillar organelles in vitro. Cancer Res, 1977. 37(5): p. 1401-7.

[4]. Relationships between tumor responsiveness, vincristine pharmacokinetics and arrest of mitosis in human tumor xenografts. Biochem Pharmacol, 1988. 37(20): p. 3995-4000.

[5]. Baguley, B.C., et al, Inhibition of growth of colon 38 adenocarcinoma by vinblastine and colchicine: evidence for a vascular mechanism. Eur J Cancer, 1991. 27(4): p. 482-7.

[6]. Co-delivery nanoparticles with characteristics of intracellular precision release drugs for overcoming multidrug resistance. Int J Nanomedicine. 2017 Mar 16;12:2081-2108.

[7]. The synergic effect of vincristine and vorinostat in leukemia in vitro and in vivo. J Hematol Oncol. 2015 Jul 10;8:82.

[8]. Selective impairment of slowly adapting type 1 mechanoreceptors in mice following vincristine treatment. Neurosci Lett. 2020 Nov 1:738:135355.

According to state or federal labeling requirements, vincristine sulfate may cause developmental toxicity. Vincristine sulfate is an anticancer drug. It is a white to slightly yellow amorphous or crystalline powder. It is photosensitive. It is odorless. pH (0.1% solution) 3.5-4.5. (NTP, 1992) Vincristine sulfate is a sulfate of a natural alkaloid isolated from periwinkle (Catharanthus roseus, also known as Vinca rosea L.), possessing antimitotic and antitumor activities. Vincristine irreversibly binds to microtubule and spindle proteins during the S phase of the cell cycle, interfering with the formation of the mitotic spindle, thereby arresting tumor cells in metaphase. The drug can also depolymerize microtubules and may interfere with the metabolism of amino acids, cyclic adenosine monophosphate (cAMP), and glutathione; calmodulin-dependent Ca(2+)-activated ATPase activity; cellular respiration; and the biosynthesis of nucleic acids and lipids.
Vincristine sulfate liposomes are a sphingomyelin/cholesterol liposome form of vincristine sulfate with potential antitumor activity. Vincristine is a vinca rosea alkaloid isolated from the rose of Vinca rosea that irreversibly binds to and stabilizes tubulin, thereby interfering with microtubule assembly/depolymerization kinetics and preventing the formation of the mitotic spindle, leading to cell cycle arrest at metaphase. Liposome encapsulation prolongs the bioavailability of vincristine, increases its delivery to tumor tissues, and reduces its toxicity. Compared with standard liposome delivery, sphingomyelin drug delivery further prolongs the circulation time of the drug in serum and enhances drug accumulation at tumor sites, thereby further improving efficacy.
An antitumor alkaloid isolated from the rose of Vinca rosea. (Merck, 11th edition)
See also: Vincristine (with active ingredient). Intraperitoneal injection of vincristine or colchicine into B6D2F1 mice with advanced subcutaneous colon 38 tumors significantly delayed tumor growth and induced hemorrhagic necrosis within 8 hours post-treatment. Two multidrug-resistant P388 leukemia sublines were resistant to vincristine and vinblastine in ascites cultures but sensitive to necrosis induction in subcutaneous tumor cultures. Vascular staining with two fluorescent markers showed that vincristine significantly reduced tumor blood flow within 4 hours post-treatment. The effects of vincristine, vinblastine, and colchicine were similar to those of tumor necrosis factor α, specifically: (a) inducing similar tumor necrosis and blood flow changes; (b) inhibiting tumor necrosis in combination with the 5-hydroxytryptamine antagonist cyproheptadine; and (c) increasing plasma nitrate levels, indicating stimulation of L-arginine oxidation to nitric oxide. The results showed that vinca alkaloids and colchicine act on solid tumors through host cell-mediated angiogenesis and direct tubulin-mediated cytotoxicity. [5] Combined chemotherapy has been widely accepted in clinical practice as a viable strategy to overcome multidrug resistance (MDR). In this paper, a co-delivery system called S-D1@L-D2 NPs was designed and successfully prepared, in which smaller nanoparticles (NPs) loaded with the drug doxorubicin (DOX) were loaded into larger nanoparticles loaded with another drug (vincristine [VCR]) by a water-in-oil-in-water double emulsion solvent diffusion-evaporation method. Chitosan-sodium alginate nanoparticles loaded with DOX (CS-ALG-DOX NPs) have a diameter of approximately 20 nm and constitute S-D1 NPs; vitamin E D-α-tocopherol polyethylene glycol 1000 succinate-modified polylactic acid-glycolic acid copolymer nanoparticles loaded with VCR (TPGS-PLGA-VCR NPs) have a diameter of approximately 200 nm and constitute L-D2 NPs. Some CS-ALG-DOX nanoparticles are loaded onto TPGS-PLGA-VCR nanoparticles to form CS-ALG-DOX@TPGS-PLGA-VCR nanoparticles. Under the acidic environment of the cytosol, endosomes, or lysosomes of MDR cells, CS-ALG-DOX@TPGS-PLGA-VCR nanoparticles release VCR and CS-ALG-DOX nanoparticles. VCR can inhibit microtubule polymerization in the cytoplasm, causing cell cycle arrest at metaphase. After escaping from the endosome, CS-ALG-DOX nanoparticles enter the nucleus through nuclear pores and release DOX in the alkaline environment of the nucleus. DOX interacts with DNA, thereby inhibiting the replication of MDR cells. These results indicate that S-D1@L-D2 nanoparticles are a pH-sensitive intracellular precise drug co-delivery system. S-D1@L-D2 NPs can significantly enhance the in vitro cytotoxicity and in vivo anticancer efficacy of co-delivered drugs, while reducing their adverse reactions. Overall, S-D1@L-D2 NPs can be regarded as an innovative drug co-delivery platform for clinical combination chemotherapy for the treatment of multidrug-resistant tumors. [6]

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Background: Combination therapy is a key strategy for reducing drug resistance, which is a common problem in cancer treatment. Vincristine, a microtubule depolymerizing agent, is widely used to treat acute leukemia. To reduce the toxicity and resistance of vincristine, this study investigated the effects of vincristine combined with vorinostat (salinomycin A (SAHA), a pan-histone deacetylase inhibitor) on human acute T-cell lymphoblastic leukemia cells. Methods: Cell viability was assessed using the MTT assay, and cell cycle distribution and mitochondrial membrane potential were analyzed by flow cytometry. Microtubule assembly was detected using an in vitro microtubule polymerization assay, and microtubule distribution and morphology were analyzed by immunofluorescence. The in vivo efficacy of this combination therapy was evaluated using a MOLT-4 xenograft tumor model. Statistical analysis was performed using the Bonferroni t test. Results: The combination of vincristine and SAHA showed stronger cytotoxicity, with an IC50 value of 0.88 nM, while the IC50 values of vincristine or SAHA alone were 3.3 nM and 840 nM, respectively. This combination therapy synergistically induced G2/M phase arrest, followed by an increase in the number of cells in the sub-G1 phase and caspase activation. In addition, the results of combining vincristine with an HDAC6 inhibitor (tubastatin A, an inhibitor of acetylated α-tubulin) were consistent with those of combining vincristine with SAHA, suggesting that SAHA may alter microtubule dynamics by inhibiting HDAC6. Conclusion: These findings suggest that combining vincristine with SAHA can alter microtubule dynamics in T-cell leukemia cells, leading to M-phase arrest and subsequently inducing apoptosis. These data suggest that the combined effect of vincristine and SAHA may provide an important preclinical basis for future clinical trials. [7]

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In our study, SA1 mechanoreceptor function was impaired after acute exposure of isolated beard hair follicles to vincristine in vitro. On the other hand, beard tactile behavioral responses were also impaired several days after vincristine treatment in vivo. The delayed impairment of sensory behavioral responses may be due to the slow accumulation of vincristine in beard hair follicles during vincristine treatment in vivo. In addition, the SA1 mechanoreceptor, or Merkel disc, is located deep within the hair follicle and is protected by a glassy membrane, which may hinder the spread of vincristine to the Merkel disc area during in vivo vincristine treatment. Alternatively, when the damage to the SA1 mechanoreceptor is not severe in the early stages of in vivo vincristine treatment, RA and SA2 may compensate for the beard tactile response. Although the in vivo vincristine treatment regimen used in this study did not damage the RA and SA2 mechanoreceptors, it would be meaningful to investigate whether long-term in vivo vincristine treatment will eventually damage these two mechanoreceptors. Furthermore, it would be meaningful to investigate whether and how long vincristine-induced mechanoreceptor damage can recover after vincristine treatment ends. [8]

C46H58N4O14S

923.04

922.367

C, 66.97; H, 6.84; N, 6.79; O, 19.39

2068-78-2

57-22-7; 2068-78-2 (sulfate); Vincristine-d3-ester sulfate;1217854-24-4;Vincristine-d3 sulfate;1246817-10-6;Vincristine-d6 sulfate

249332

White to off-white solid powder

273-281 °C

300 °C

4.522

5

16

10

65

1830

9

CC[C@@]1(C[C@@H]2C[C@@](C3=C(CCN(C2)C1)C4=CC=CC=C4N3)(C5=C(C=C6C(=C5)[C@]78CCN9[C@H]7[C@@](C=CC9)([C@H]([C@@]([C@@H]8N6C=O)(C(=O)OC)O)OC(=O)C)CC)OC)C(=O)OC)O.OS(=O)(=O)O

AQTQHPDCURKLKT-JKDPCDLQSA-N

InChI=1S/C46H56N4O10.H2O4S/c1-7-42(55)22-28-23-45(40(53)58-5,36-30(14-18-48(24-28)25-42)29-12-9-10-13-33(29)47-36)32-20-31-34(21-35(32)57-4)50(26-51)38-44(31)16-19-49-17-11-15-43(8-2,37(44)49)39(60-27(3)52)46(38,56)41(54)59-6;1-5(2,3)4/h9-13,15,20-21,26,28,37-39,47,55-56H,7-8,14,16-19,22-25H2,1-6H3;(H2,1,2,3,4)/t28-,37+,38-,39-,42+,43-,44-,45+,46+;/m1./s1

(3aR,3a1R,4R,5S,5aR,10bR)-methyl 4-acetoxy-3a-ethyl-9-((3S,5S,7S,9S)-5-ethyl-5-hydroxy-9-(methoxycarbonyl)-2,4,5,6,7,8,9,10-octahydro-1H-3,7-methano[1]azacycloundecino[5,4-b]indol-9-yl)-6-formyl-5-hydroxy-8-methoxy-3a,3a1,4,5,5a,6,11,12-octahydro-1H-indolizino[8,1-cd]carbazole-5-carboxylate sulfate.

Leurocristine; NSC-67574 sulfate; 22-Oxovincaleukoblastine sulfate; leurocristine sulfate; NSC67574; NSC 67574; Vincristine sulfate; 2068-78-2; Kyocristine; Vincristine sulphate; Vincrisul; Oncovin; Novopharm; Leurocristine sulfate; Vincasar PFS; Oncovin; VCR

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture and light.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Solubility in Formulation 1: ≥ 2.08 mg/mL (2.25 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.08 mg/mL (2.25 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.08 mg/mL (2.25 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 4: Saline:30 mg/mL

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Solubility in Formulation 5: 100 mg/mL (108.34 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication (<60°C).

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 (Please use freshly prepared in vivo formulations for optimal results.)