Concentrations Of Vincristine Research Articles

Efectos del Mebendazol Sobre el Mecanismo de Apoptosis Mediado por Caspasa en Cultivos de Células Cancerosas

This study was conducted to compare Mebendazole in terms of its apoptosis–inducing and tubulin–inhibitory effects when combined with vincristine and paclitaxel, both of which are used in cancer treatment. Lung fibroblast cells (MRC–5) and small cell lung carcinoma (NCI–H209) cell lines were used in the study. Concentrations of Mebendazole, vincristine, and paclitaxel at 0.5 µM, 1 µM, 1.5 µM, and 2 µM were separately applied to these cell lines, as well as in combinations. After the cells were kept in the culture medium for 24 hours following drug administration, cell proliferation, apoptotic DNA levels, caspase 3, 8, and 9 levels, and in vitro wound healing experiments were performed. It was determined that Mebendazole suppressed cell proliferation and cell healing, increased caspase–3, caspase–8, caspase–9 levels and apoptotic DNA formation in NCI–H209 cancer lung cells. Compared to the groups given Mebendazole and vincristine alone, it was observed that cell proliferation was more suppressed and, the level of apoptosis increased in cancerous cells in the groups given the combination of the two drugs. According to the findings obtained from the present study, it was believe that Mebendazole may possess therapeutic activity against cancerous lung cells (NCI–H209) due to its apoptosis–inducing and cell proliferation–suppressive effects.

In vitro chemosensitivity testing of the feline large granular lymphocyte cell line (S87).

Feline large granular lymphocyte (LGL) lymphoma is an aggressive neoplasia characterised by short survival and poor response to chemotherapy. In this study, the effect of different chemotherapeutic agents on the growth kinetics of the feline cell line S87, a non-MHC-restricted feline LGL cell line, was investigated. Where possible, IC50 (inhibitory concentration 50) values were determined. The IC50 values of the cell line as lymphoma models can provide clues to the situation in vivo and serve as a basis for studying resistance mechanisms. Cells were incubated with various concentrations of vincristine, doxorubicin, 4-hydroperoxycyclophosphamide, prednisolone, methotrexate and L-asparaginase for 24 and 48h, respectively. The IC50 values could be determined as 14.57 (7.49-28.32) μg/mL at 24h incubation and 5.72 (4.05-8.07) μg/mL at 48h incubation for doxorubicin and 9.12 (7.72-10.76) μg/mL at 24h incubation and 4.53 (3.74-5.47) μg/mL at 48h incubation for 4-hydroperpoxycyclophosphamide. Treatment with vincristine and methotrexate resulted in relatively high cell resistance whereas L-asparaginase and prednisolone treatment led to a reduction in cell number compared to control while cell viability was not affected (cytostatic effect). Overall, the feline LGL cell line S87 proves to be relatively sensitive to doxorubicin and 4-hydroperoxycyclophosphamide and relatively resistant to treatment with vincristine, prednisolone, methotrexate and L-asparaginase. The results of this study can be used for further investigations on resistance mechanisms in feline LGL lymphoma. Doxorubicin and cyclophosphamide can be interpreted as promising candidates for the therapy of feline LGL lymphomas.

Exposure of hiPSC-Derived Mature Megakaryocytes to Vincristine Boosts Platelet Biogenesis in Vitro

Millions of healthy donor platelet (PLT) units are needed each year for therapeutic transfusions of patients with thrombocytopenia and bleeding disorders, but shortages are common. The pandemic worsened donor shortages in the US and Japan. To ensure the predictability of supply, and to mitigate clinical complications associated with conventional donor PLTs, we have developed human induced pluripotent stem cell (hiPSC)-derived megakaryocyte (MK) cell lines, imMKCLs, as a source of in vitro PLT production (iPSC-PLTs)(Nakamura et al., Cell Stem Cell, 2014; Sone et al., Stem Cell Rep, 2021), and manufactured platelets are now in clinical trials using a previously published protocol (Sugimoto et al., Blood, 2022; Sugimoto et al., Blood Adv, 2022). To optimize and improve the in vitro manufacturing protocol of iPSC-PLTs from imMKCLs, we conducted a comprehensive imaging-based compound screening which identified microtubule (MT) destabilizing agents, including the vinca alkaloid vincristine (VCR), that appear to boost proplatelet formation. Combined use of VCR at 10 μM and turbulent hydrodynamic culture conditions (Ito et al., Cell, 2018) allowed production of 23.0 PLTs per imMKCL, 2.5 fold higher than without VCR. This successful result was observed only when VCR was administered at day 3 of 6-days of the maturation phase, when imMKCLs have become polyploid, while exposure at earlier timepoints induced apoptosis. However, PLTs produced in this manner showed poor agonist-induced surface expression of P-selectin or the PAC1 epitope. Therefore, we next examined dose-dependent action of VCR on both yield of iPSC-PLTs and their function. At nanomolar concentrations (10 nM), VCR still exhibited significant PLT biogenesis-boosting activity, but without apparent impairment of PLT in vitro functionality. Interestingly, this low concentration of VCR still resulted in reduced marginal MT band staining in imMKCLs, suggesting a potential role of weakened membrane structure/status in enhanced iPSC-PLT generation. Currently, we are attempting to address if PLTs made from low-dose VCR-exposed imMKCLs have reduced marginal MT bands and whether they restore hemostasis when transfused into immunodeficient thrombocytopenic mice. Our study unveils the potential of VCR as a potent enhancer of PLT biogenesis in the imMKCL system, particularly when combined with optimized turbulent flow production systems. Our results provide a means to make production of iPSC-PLTs more cost-efficient and suggest a mechanism for the previously unexplained observation that vinca alkaloids raise PLT counts in vivo.

Synthesis and Characterization of Nanoparticles Extracted from <i>Catharanthus roseus</i> Plant

The Catharanthus roseus plant was extracted and converted to nanoparticles in this work. The Soxhlet method extracted alkaloid compounds from the plant Catharanthus roseus and converted them to the nanoscale. Chitosan polymer was used as a linking material and converted to Chitosan nanoparticles using Sodium TriPolyPhosphate (STPP). The extracted alkaloids were linked with Chitosan nanoparticles CSNPs by maleic anhydride to get the final product (CSNPs- Linker- alkaloids). The synthesized (CSNPs- Linker- alkaloids) was characterized using SEM spectroscopy UV–Vis., Zeta Potential, and HPLC High-Performance Liquid Chromatography. Scanning electron microscope (SEM) analysis shows that the Chitosan nanoparticles (CSNPs) have small dimensions with regular spherical and nanotube shapes of a diameter range of (49 - 70) nm. The final product (CSNPs- Linker- alkaloids) has two shapes (spherical particles and tubes) in nano dimensions and is close to each other compared to normal Chitosan. The absorption peaks for Chitosan (CS), Chitosan nanoparticles (CSNPs), Chitosan nanoparticles (CSNPs), and maleic anhydride revealed that converting Chitosan to Chitosan nanoparticles and mixing it with the plant extract, led to an increase in the absorption value and wavelength range. Also, the appearance of two peaks at 222 nm and 402 nm nano instead of the peak of Chitosan at 289.9 nm. Zeta Potential results of CSNPs- Linker- alkaloids showed that the extract of the nano-alkaloids bound to chitosan nanoparticles carries a positive charge of 54.4 mV. This surface charge is essential in maintaining the colloidal solution's stability in its natural form without changing. High-Performance Liquid Chromatography (HPLC) was used to estimate qualitative and quantitative plants extracted from Catharanthus roseus. Quantitative HPLC results show that Catharanthus roseus contains a good and acceptable concentration of Vinblastine, Vincristine, Vinorelbine, Vincamine, and Vintafolide (66.75, 242.91, 0.7, 83.77, 42.34) ppm respectively. The qualitative results show a good match for the influential groups of pure standard vincristine and alcoholic extract and dry powder of the Catharanthus roseus plant. The successful synthesis of nanoparticles from the Catharanthus roseus plant can be used in biosensors and biomedical applications.

Development of the DNA-based voltammetric biosensor for detection of vincristine as anticancer drug.

In the article presented herein, a deoxyribonucleic acid (DNA) biosensor is introduced for Vincristine determination in pharmaceutical preparations based on the modification of screen printed electrode (SPE) with double-stranded DNA (ds-DNA), polypyrrole (PP), peony-like CuO:Tb3+ nanostructure (P-L CuO:Tb3+ NS). The developed sensor indicated a wide linear response to Vincristine concentration ranged from 1.0 nM to 400.0μM with a limit of detection as low as .21nM. The intercalation of Vincristine with DNA guanine led to the response. The optimized parameters for the biosensor performance were ds-DNA/Vincristine interaction time, DNA concentration and type of buffer solution. The docking investigation confirm the minor groove interaction between guanine base at surface of or ds-DNA/PP/P-L CuO:Tb3+ NS/SPE and Vincristine. The proposed sensor could successfully determine Vincristine in Vincristine injections and biological fluids, with acceptable obtains.

The Development of New Methodology for Determination of Vincristine (VCR) in Human Serum Using LC-MS/MS-Based Method for Medical Diagnostics.

In this article, we have presented the development and validation of a rapid and sensitive reversed-phase liquid chromatography with tandem mass spectrometry (LC-MS/MS) method for the determination of vincristine (VCR) in patient serum samples. Chromatographic separation was achieved on a Kinetex® (Singapore) column using a mobile phase consisting of 25 mM acetic acid and 0.3% formic acid (A) and methanol (B) in a gradient elution mode at a flow rate of 0.3 mL/min. The VCR and internal standard (vinblastine) were monitored using the multiple reaction monitoring mode under positive electrospray ionization. The lower limit of quantification (LLOQ) was 0.67 ng/mL, and the upper limit of quantification (ULOQ) was 250 ng/mL for VCR. The calculated values of LOD and LOQ for VCR were 0.075 and 0.228 ng/mL, respectively. The calibration curve was linear over the VCR concentration range of 1.0-250 ng/mL in serum. The intra- and inter-day precision and precision were within the generally accepted criteria for the bioanalytical method (<15%). The method was successfully applied to the analysis of serum samples in clinical practice.

Activina: A Key Factor Mediating Chemoresistance in B-Cell Type Acute Lymphoblastic Leukemia

B-Cell Acute Lymphoblastic Leukemia (B-ALL) is the most common cancer in children. Multidrug high-dose chemotherapy has increased the 5-year event free survival rate to more than 80%. However, the prognosis of refractory/relapsed (r/r) ALL remains dismal. Increasing evidence indicates that treatment failure could be linked to the protective role of leukemic bone marrow (BM) microenvironment. The identification of the molecules involved in the crosstalk between leukemic cells and the BM niche would be crucial to establish niche-targeted therapies to be combined with conventional drugs. Recently, we identified ActivinA (ActA) as a new potentially targetable leukemia-promoting factor, which is significantly overexpressed in the BM plasma of B-ALL patients. We demonstrated that ActA is able to potentiate the migratory and invasive ability of B-ALL cells in vitro and enhance both BM engraftment and metastatic potential of leukemic cells in a xenograft mouse model. Literature data indicate that ActA exerts a pro-tumoral role in several solid tumors, by promoting cancer cell migration, proliferation and chemoresistance. The aim of this work was to ascertain whether ActivinA could mediate resistance to standard chemotherapy agents routinely used in pediatric B-ALL, such as Vincristine (VCR), Dexamethasone (Dex) and Asparaginase (ASNase). To investigate the role of ActA on B-ALL cell chemoresistance, we stimulated or not 697 cells with ActA for 24h. Cells were then treated with increasing concentrations of Dex, ASNase and VCR for 72h to obtain a dose-response curve. Cell viability was evaluated by flow cytometry and used to calculate the inhibitory dose 50 (IC50). Interestingly, the IC50 of ActA stimulated 697 cells for Dex and ASNase was in both cases about ten-fold higher than NS controls. In the case of VCR-treated cells, we did not detect any difference in IC50. These data suggest that ActA is able to convey resistance to Dex and ASNase-induced cell death, while sparing VCR-based leukemic cell killing, possibly due to different mechanisms of action. Validation experiments, with selected doses of Dex and ASNase (7 µg/mL and 1 U/mL, respectively), confirmed the ability of ActA to induce chemoresistance. In detail, the median viability of ActA stimulated 697 cells after 3 days of treatment with Dex was 64.15% (range=51.95-73.75%). This percentage was significantly higher compared to NS cells (median viability=41.95%, range=31.95-60.93%, Wilcoxon test: p<0.0001). Alongside, the viability of ActA stimulated cells (median=53.40%, range=38.70-75.00%) was also significantly increased (median=31.40%, range=22.45-54.25%, p=0.001) after ASNase treatment. To understand the molecular mechanisms underlying Acta-mediated chemoprotection, we firstly evaluated in our experimental setting the percentage of 697 cells expressing the active form of Caspase-3, by flow cytometry. Our results highlight that the activation of Caspase-3 was significantly reduced in ActA pre-stimulated cells treated with Dex (mean fold change (FC) ActA/NS=0.48±0.22, p=0.0003, One-sample t test) and ASNase (mean FC=0.49±0.062, p<0.0001), compared to NS cells. Then, we focused on the impact of ActA on anti- and pro-apoptotic factors playing a key role in drug-mediated apoptosis. By western blot, we demonstrated that Bcl-2 was significantly increased upon Dex treatment in ActA pre-stimulated cells (median FC ActA+Dex/NS untreated cells=0.63, range=0.49-1.12, Wilcoxon test: p<0.05) compared to NS cells (median FC NS+Dex/NS untreated cells=0.43, range=0.26-0.74). In contrast, we did not observe any difference with ASNase. Furthermore, we demonstrated that ActA was able to decrease the drug-induced cleavage activation of pro-apoptotic factors BAX and BAK. Finally, we investigated by confocal microscopy the levels of intracellular Reactive Oxygen Species (ROS), well-known key elements of the drug-induced oxidative stress, resulting in the activation of apoptosis. Interestingly, upon Dex treatment we observed a reduction of ROS levels of 59.7%±4.6% (mean±sd) in ActA-stimulated cells compared to NS. In the case of ASNase treatment the mean reduction was 46.9%±8.8%. Overall, our data suggest that ActA plays a pivotal role in B-ALL cell chemoprotection and that its targeting may be a promising strategy to be used in combination with standard chemotherapy in r/r ALL patients.

Protective effect of selenium on vincristine-induced peripheral neuropathy in PC12 cell line.

Vincristine-induced peripheral neuropathy (VIPN) is the main side effect and major reason for neuropathic pain in cancer survivors treated with vincristine. Vincristine, a chemotherapeutic antimitotic drug, is used frequently in combination chemotherapy. The primary purpose of the current study was to assess the protective effect of sodium selenite (SSe) on VIPN in vitro. Cytotoxicity effects of vincristine were evaluated using PC12 cells as a neuronal model. The cell culture studies were conducted in three groups based on the various treatments, including vincristine, SSe, and co-exposure to both compositions. Cell viability and cell cycle analyses were performed using MTT assay and flow cytometry, respectively. The level of mRNA expression of Bax and Bcl-2 was determined using qRT-PCR. According tothe results, vincristine decreased the survival rate of PC12 cells. After 24 and 48h exposure to different concentrations of vincristine (0.1-20μΜ), the survival rate of PC12 cells decreased as compared to the control group. The results showed that treatment with 5μΜ of vincristine resulted in apoptosis of PC12 cells. Interestingly,co-incubation of these cells with SSe significantly reduced the cell damage induced by vincristine. Furthermore, vincristine induced the inhibition of the G2 phase in PC 12 cells, and using SSe in combination with vincristine eliminated the inhibition of the cell cycle in the G2 phase. Briefly, our in vitro preliminary study showed that SSe might protect PC12 cells from vincristine-induced peripheral neuropathy during chemotherapy.

Vincristine Promotes Transdifferentiation of Fibroblasts Into Myofibroblasts via P38 and ERK Signal Pathways.

Background: Vincristine (VCR) is used in the clinic as an anti-tumor drug. VCR can cause pulmonary fibrosis (PF), leading to respiratory failure. The transformation of fibroblasts into myofibroblasts may play a key role in PF. The present study attempted to reveal the molecular mechanism of VCR-induced PF and the possible involvement of the mitogen-activated protein kinase (MAPK) signaling pathway. Methods: Human embryonic lung fibroblasts (HELFs) were treated with different concentrations of VCR. Inhibitors of extracellular signal-regulated kinase 1/2 (ERK1/2) and p38 MAPK were added to HELFs. Cell proliferation state was assessed using cell counting kit-8 and by directly counting the number of cells. The expressions of vimentin and α-smooth muscle actin (α-SMA) were investigated using western blot and immunofluorescence analyses. Activation of ERK and P38 was estimated by the expression of phosphorylated p38 MAPK (p-p38), p38 MAPK, phosphorylated ERK1/2 (p-ERK1/2) and ERK1/2 using western blot analysis. Enzyme-linked immunosorbent assay was used to estimate the level of collagen I in cell culture supernatants. Results: Results showed that VCR promoted cellular proliferation, secretion of collagen I and the expression of vimentin and α-SMA. High expression of p-p38 and p-ERK1/2 was associated with the activation of the MAPK signaling pathway. MAPK inhibitors SB203580 and PD98059 suppressed the expression of the above proteins. Conclusion: Our study revealed that VCR could promote the differentiation of fibroblasts into myofibroblasts by regulating the MAPK signal pathway, which may be a promising way to treat VCR-induced PF.

A novel highly sensitive UHPLC-MS/MS method for monitoring vincristine in plasma and vitreous humor samples with potential application in retinoblastoma therapy

Vincristine is a chemotherapeutic agent which is effective in treating various types of cancer, including retinoblastoma, the most common malignant intraocular tumor in children. There is a clinical requirement for a highly sensitive analytical method to monitor vincristine concentration during treatment. Therefore, we developed a simple, rapid, selective, and highly sensitive UHPLC-MS/MS method to determine vincristine concentration in plasma and vitreous humor samples. This method uses an isotopically labeled internal standard. High sensitivity was achieved by specific multiple reaction monitoring (MRM) transitions using a doubly charged acetonitrile adduct of vincristine as a precursor ion in positive mode. Finally, our protocol was fully validated to demonstrate its reliability in determining vincristine in plasma and vitreous humor samples. The method showed an excellent linear range from 0.1 to 50 ng mL−1, with precision ranging from 1.8 to 11.6% (RSD), and with accuracy (relative error %) within ± 11.8%. The matrix effect and extraction recovery for vincristine ranged from 93 to 103% and 98 to 101%, respectively.

More Related Gene Pathways to Vincristine-Induced Death Events in a Human T-Acute Lymphoblastic Leukemia Cell Line.

Acute lymphoblastic leukemia (ALL) is common in children but rare in adults. Vincristine (VCR) is one of the drugs used at the beginning of treatment. Some genes are resistant to VCR in B-ALL. Here, we examined the effect of VCR on gene expression changes in a T-ALL cell line, Jurkat. The MTT method was used to determine the IC50 in Jurkat cells treated with different concentrations of VCR for 48 and 72 hours. Total RNA was isolated from the cells and cDNA was prepared. The Human Cancer Drug Target PCR Array kit was used to evaluate the 84 gene expression changes in Jurkat cells. Protein-protein interaction was analyzed by STRING software. We identified 66 differentially expressed genes as comparison to untreated cells. The response to VCR-induced apoptotic events was remarkable in the pathways of heat shock protein, topoisomerases, protein kinases, cathepsins and cell cycle. In other pathways, there were resistant genes as well as sensitive genes to VCR treatment. Some proteins like HSP90AA1 and ESR1 had determining associations with other proteins. The results suggest VCR target genes in T-ALL cells may be beneficial biomarkers for ALL treatment and can be used to select appropriate synergistic drugs for VCR.

Vincristine inhibits the proliferation of ovarian cancer cells by regulating the demethylation of RASSF2A

Objective: To investigate the effect of vincristine on the proliferation of ovarian cancer cells by regulating RASSF2A demethylation. Methods: SKOV3 cells were infected with control (LV-NC) and RASSF2A lentivirus (LV-RASSF2A) and treated with or without vincristine. Cell counting kit-8 (CCK-8) was used to detect the activity of ovarian cancer cells (SKOV3) treated with different doses of vincristine. Colony formation assay was used to detect the proliferation of SKOV3 cells. Flow cytometry was used to detect the apoptosis of SKOV3 cells. Real time polymerase chain reaction (RT-PCR) was used to examine the mRNA expression of RASSF2A in IOSE-29 and SKOV3 cells. Western blot was used to examine the protein expression of RASSF2A in IOSE-29 and SKOV3 cells. Methylation-specific PCR was used to detect methylation and demethylation levels of RASSF2A gene in IOSE-29 and SKOV3 cells. Results: The cell viabilities of SKOV3 cell treated with 6.25 nmol/L, 12.5 nmol/L, 25 nmol/L, 50 nmol/L and 100 nmol/L vincristine were (87.19±4.49)%, (73.67±8.62)%, (66.35±6.04)%, (50.32±6.00)% and (34.92±6.11)%, respectively, lower than (100.46±4.69)% of control group (P<0.05). The half maximal inhibitory concentration of vincristine at 48 hours was 50.02 nmol/L. The proliferation abilities of SKOV3 cells in vincristine 12.5 nmol/L group, 25 nmol/L group and 50 nmol/L group were (41.70±2.21)%, (32.15±1.80)% and (23.00±2.01)%, respectively, significantly lower than (100.78±5.66)% in the control group (all P<0.05). The apoptotic rates of SKOV3 cells in vincristine 12.5 nmol/L group, 25 nmol/L group and 50 nmol/L group were (3.65±0.27)%, (5.21±0.76)% and (10.46±1.00)%, respectively, significantly higher than (2.12±0.23)% in the control group (all P<0.05). Compared with the IOSE-29 group (1.00±0.07 and 0.68±0.04), the mRNA expression (0.32±0.04) and protein expression (0.24±0.02) of RASSF2A were down-regulated in SKOV3 cells (P<0.05). Compared with the LV-NC group [(101.60±4.39)%, (100.73±3.29)%, (4.06±0.30)%], over-expression of RASSF2A down-requlated cell viability (68.92±3.94)%, inhibited proliferation (16.38±2.16)%, and promoted apoptosis (8.65±0.56)%, (P<0.05). Conclusion: Vincristine can increase RASSF2A expression and inhibit ovarian cancer cell proliferation by promoting the demethylation of RASSF2A promoter.

Anticancer properties of vincristine is modulated by microRNAs in acute lymphoblastic leukemia Nalm6 cell line.

Precursor B-cell acute lymphoblastic leukemia (B-ALL), a highly diverse disease, is the most widespread pediatric malignancy characterized by cytogenetic and molecular abnormalities such as altered microRNA (miR) expression signatures. MiRs are a class of short noncoding RNAs. Dysregulation in the expression of miRs plays a crucial role in different types of cancers. Vincristine is an antineoplastic drug with a broad spectrum of activity against different hematologic malignancies and is the first-line treatment for B-ALL. Previous studies have proposed miR-17 and miR-181/b as oncomirs and miR-34/a as a tumor suppressor in Nalm6 cells, thus in the current study, we investigated the effects of vincristine treatment on the expression of miR-17, miR-34/a and miR-181/b expression levels. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide assay was conducted to estimate the optimal concentration of vincristine in the Nalm-6 cell line. Expression of miRs was calculated using real-time PCR. Our results showed significant downregulation of miR-17 (FC = 0.226; P < 0.0004) in Nalm6 cells after vincristine treatment. Conversely, miR-34/a (FC = 4.823; P < 0.0001) was significantly upregulated. Also, the expression of miR-181/b (FC = 0.156; P < 0.3465) was not significantly different between the vincristine treated group and the control group. In conclusion, it is proposed that one of the mechanisms by which vincristine improves B-ALL is by modulating the expression of specific miRs. These specific miRs will serve as good diagnostic and prognostic biomarkers.

Vincristine-Loaded Platelets Coated with Anti-CD41 MAbs: A New Proposal for the Treatment of Immune Thrombocytopenia

Objective: Immune thrombocytopenia (ITP) is an autoimmune disorder in which platelet-reactive autoantibodies accelerate the destruction of platelets. Macrophages play a crucial role in ITP through IgG-Fcγ receptor-mediated platelet clearance. Patients responding to initial therapies often troubled with frequent relapses, severe reverse reactions and poor life quantity. Therefore, searching new effective strategies for ITP treatment is in urgent need.Methods: In this study, platelets coated with anti-CD41 antibodies, which can mimic autoantibody-binding platelets in ITP patients, were used as a smart drug delivery system to load vincristine (VCR). Vincristine-loaded platelets coated with anti-CD41 mAbs (CD41-VLP) can selective release VCR in macrophages through IgG-Fcγ receptor-mediated phagocytosis. Scanning electron microscopy (SEM), dynamic light scattering (DLS), and platelet aggregation assays were performed to evaluate the morphological and functional changes of CD41-VLP. The encapsulation efficiency, drug loading and intracellular VCR concentration were determined using high performance liquid chromatography (HPLC). Macrophages were derived from THP-1 induced by phorbol 12-myristate 13-acetate (PMA). The expression of cell differentiation antigen (CD11b and CD14) and was detected by flow cytometry (FCM). The growth inhibition of CD41-VLP on macrophages was detected by Cell Counting Kit-8 (CCK-8) assay and the macrophage apoptosis was quantified by FCM. The possible mechanism of CD41-VLP on macrophages was further investigated by western blotting.Results: platelets coated with anti-CD41 antibodies could load VCR with high drug loading and encapsulation efficiency. There is no significant difference in morphology and function between platelets and CD41-VLP. Meanwhile, VCR was released from CD41-VLP in a pH-triggered behavior and VCR would be rapidly released in an acidic condition. The THP-1 derived macrophages was detected with a strong ability of phagocytosis and a high expression of CD11b and CD14. In the group of macrophages treated with CD41-VLP, intracellular concentration of VCR was significant increased compared with other groups, which demonstrates that Cd41-VLP can selectively deliver VCR to macrophages. In the CD41-VLP group, the growth inhibition rate was also much higher than other groups and the apoptosis rate was significant increased compared with other groups. These results suggest that CD41-VLP can enhance the cytotoxicity of VCR against macrophages. Furthermore, In the CD41-VLP group, the expressions of pro-apoptotic proteins,including Bax, caspase-3 and CyclinB1, were significantly increased and the expression of Bcl-2, anti-apoptotic protein, was decreased. This finding indicates that CD41-VLP might improve the effects of VCR on macrophages through regulating the expression of apoptosis-related proteins.Conclusion: CD41-VLP can improve the cytotoxicity of VCR against macrophages, thus establishing a new targeted therapy for ITP treatment. DisclosuresNo relevant conflicts of interest to declare.

Abstract PO-117: Population pharmacokinetics of vincristine and its metabolite in Kenyan pediatric cancer patients

Abstract Purpose: Pharmacokinetic (PK) dispositions of vincristine (VCR) and its M1 metabolite were characterized through PK co-modeling for Kenyan pediatric cancer patients with acute lymphoblastic leukemia or nephroblastoma. VCR dosing strategies are largely empirical regardless of its extensive use in pediatric oncology. Little information is known about its disposition and optimal therapeutic dosing. Renbarger and associates reported that CYP3A5 enzyme metabolizes VCR to M1 more efficiently than CYP3A4 enzyme. This may be clinically significant as CYP3A5 expression varies among Kenyans (90%), African Americans (AA, 70%) and Caucasians (10-20%). Therefore, it is essential to characterize the disposition of M1 in humans to provide an insight into the inter-ethnic variability in VCR metabolism and clearance, which will be helpful for future dosing regimen optimization. Methods: Pharmacokinetic Study: Dried blood spot (DBS) samples were collected, via finger stick at various time points depending on the feasibility, from 25 Kenyan pediatric cancer patients (13 males/12 females, 1-14 years, BSA of 0.36 - 1.3 m2) after an IV dose of VCR (1.6 – 3.1 mg/m2). Concentrations of VCR and M1 from DBS were quantified using a validated LC-MS-MS assay. Pharmacokinetic Modeling: A population PK (pop PK) co-model was developed using Phoenix® NLMETM software to simultaneously capture and predict the PK of VCR and M1 concentrations. A correlation between the observed and predicted concentrations of VCR and M1 PK was established by pop PK fitting. Model discrimination was performed on data by visual inspection, the goodness of fit plots and AIC values. Results: The best fit pop PK co-model for VCR and its M1 metabolite was established. PK parameter estimates were derived for both analytes. Volumes of distribution for VCR and M1 were 0.12 L (33.55 %CV) and 249.07 L (33.42 %CV), respectively. The elimination rate constants for VCR and M1 were 5.6 hr-1 (22.81 %CV) and 0.00015 hr-1 (66.85 %CV), respectively. The conversion rate constant of VCR to M1 was 9.66 hr-1 (32.49 %CV). Finally, clearance values were 0.67 L/hr for VCR and 0.04 L/hr for M1. In conclusion, VCR and its M1 metabolite exhibit distinct PK characteristics, in that M1 distribution is substantially larger than that of VCR (249 vs 0.12 L), and clearance is slower than that of VCR (0.04 vs 0.67 L/hr). The conversion kinetics of VCR to M1 is characterized for the first time, which may potentially offer a rationale for ethnic disparity in VCR therapy. In addition, a large inter-patient variability is documented even within the same ethnic Kenyan population. After model refinement and validation, our model could potentially serve as a tool for rational VCR dosing regimen modification for Kenyan/AA pediatric cancer patients. Citation Format: Lorita Agu, Lei Wu, Jamie Renbarger, Jodi Skiles, Andi Masters, Diana S-L. Chow. Population pharmacokinetics of vincristine and its metabolite in Kenyan pediatric cancer patients [abstract]. In: Proceedings of the AACR Virtual Conference: Thirteenth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2020 Oct 2-4. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2020;29(12 Suppl):Abstract nr PO-117.

Tetrandrine partially reverses multidrug resistance of human laryngeal cancer cells.

ObjectiveStudies have demonstrated that tetrandrine reverses multidrug resistance (MDR) in animal models or cell lines derived from multiple cancer types. We examined the potential MDR reversal activity of tetrandrine in a multidrug-resistant variant of a human laryngeal cancer Hep-2 cell line and explored potential mechanisms involved.MethodsWe developed the multidrug-resistant variant cell line (Hep-2/v) by exposing Hep-2 cells to stepwise increasing concentrations of vincristine (VCR). After Hep-2 or Hep-2/v cells were treated with tetrandrine (2.52 µg/mL), MDR was measured by MTT assay, rhodamine 123 retention was measured by flow cytometry, and mRNA and protein expression of multidrug resistance 1 (MDR1), regulator of G-protein signaling 10 (RGS10), high-temperature requirement protein A1 (HTRA1), and nuclear protein 1 (NUPR1) were detected by real-time reverse transcription-PCR and western blotting, respectively.ResultsTetrandrine significantly lowered the half-maximal inhibitory concentration (IC50) of VCR in Hep-2/v cells, resulting in a 2.22-fold reversal of MDR. Treatment with tetrandrine increased rhodamine 123 retention, downregulated the mRNA and protein expression of MDR1 and RGS10, and upregulated expression of HTRA1 in Hep-2/v cells.ConclusionWe showed that tetrandrine exerts anti-MDR activity in Hep-2/v cells, possibly by inhibiting MDR1 overexpression-mediated drug efflux and by altering expression of HTRA1 and RGS10.

Fabrication of an Impedimetric Immunosensor for Screening and Determination of Vincristine in Biological Samples

Terpenoids of medicinal plants are one of the most important sources of therapeutic drugs. Among them, Vincristine (VCR) is prescribed for a wide variety of cancer types. Therefore, development of sensitive methods for VCR determination is urgently required. Here, a label-free and highly sensitive impedimetric biosensor was fabricated for electrochemical detection of VCR. First, multi-wall carbon nano tubes (MWCNTs) were dropped on the surface of a glassy carbon electrode. Then, the VCR monoclonal antibody (Ab) was covalently immobilized on the MWCNTs via carbodiimide coupling reaction using N-(3-dimethylaminopropyl)-N'-ethyl carbodiimide and N-hydroxy succinimide. The step-by-step modification process was characterized by electrochemical impedance spectroscopy and cyclic voltammetry in presence of a redox probe [Fe(CN)6]3–/4–. The VCR concentration was measured through increasing impedance values in the corresponding specific binding of VCR to the Ab. The increased electron-transfer resistance (Ret) values were proportional to the concentration of VCR and the biosensor detected concentrations varied in the range of 0.2–50 nM with a detection limit of 8 × 10–2 nM (S/N = 3). Practical analytical utility of the proposed method was demonstrated by VCR determination in medicinal plant extracts and human serum samples with satisfactory recoveries. The proposed biosensor may be a promising alternative for screening of medicinal plants to produce VCR and for the determination of drug concentration in cancerous potions.

Drug Targeting the Actin Cytoskeleton Potentiates the Cytotoxicity of Low Dose Vincristine by Abrogating Actin-Mediated Repair of Spindle Defects.

Antimicrotubule vinca alkaloids are widely used in the clinic but their toxicity is often dose limiting. Strategies that enhance their effectiveness at lower doses are needed. We show that combining vinca alkaloids with compounds that target a specific population of actin filaments containing the cancer-associated tropomyosin Tpm3.1 result in synergy against a broad range of tumor cell types. We discovered that low concentrations of vincristine alone induce supernumerary microtubule asters that form transient multi-polar spindles in early mitosis. Over time these asters can be reconstructed into functional bipolar spindles resulting in cell division and survival. These microtubule asters are organized by the nuclear mitotic apparatus protein (NuMA)-dynein-dynactin complex without involvement of centrosomes. However, anti-Tpm3.1 compounds at nontoxic concentrations inhibit this rescue mechanism resulting in delayed onset of anaphase, formation of multi-polar spindles, and apoptosis during mitosis. These findings indicate that drug targeting actin filaments containing Tpm3.1 potentiates the anticancer activity of low-dose vincristine treatment. IMPLICATIONS: Simultaneously inhibiting Tpm3.1-containing actin filaments and microtubules is a promising strategy to potentiate the anticancer activity of low-dose vincristine.

Up-regulation of GLI1 in vincristine-resistant rhabdomyosarcoma and Ewing sarcoma

BackgroundThe clinical significance of GLI1 expression either through canonical Hedgehog signal transduction or through non-canonical mechanisms in rhabdomyosarcoma (RMS) or Ewing sarcoma (EWS) is incompletely understood. We tested a role for Hedgehog (HH) signal transduction and GL11 expression in development of vincristine (VCR) resistance in RMS and EWS.MethodsWe characterized baseline expression and activity of HH pathway components in 5 RMS (RD, Rh18, Ruch-2, Rh30, and Rh41) and 5 EWS (CHLA9, CHLA10, TC32, CHLA258, and TC71) cell lines. We then established VCR-resistant RMS and EWS cell lines by exposing cells to serially increasing concentrations of VCR and determining the IC50. We defined resistance as a ≥ 30-fold increase in IC50 compared with parental cells. We determined changes in gene expression in the VCR-resistant cells compared with parental cells using an 86-gene cancer drug resistance array that included GLI1 and tested the effect of GLI1 inhibition with GANT61 or GLI1 siRNA on VCR resistance.ResultsWe found evidence for HH pathway activity and GLI1 expression in RMS and EWS cell lines at baseline, and evidence that GLI1 contributes to survival and proliferation of these sarcoma cells. We were able to establish 4 VCR-resistant cell lines (Ruch-2VR, Rh30VR, Rh41VR, and TC71VR). GLI1 was significantly up-regulated in the Rh30VR, Rh41VR, and TC71VR cells. The only other gene in the drug resistance panel that was significantly up-regulated in each of these VCR-resistant cell lines compared with their corresponding parental cells was the GLI1 direct target and multidrug resistance gene, ATP-binding cassette sub-family B member 1 (MDR1). We established major vault protein (MVP), which was up-regulated in both vincristine-resistant alveolar RMS cell lines (Rh30VR and Rh41VR), as another direct target of GLI1 during development of drug resistance. Treatment of the VCR-resistant cell lines with the small molecule inhibitor GANT61 or GLI1 siRNA together with VCR significantly decreased cell viability at doses that did not reduce viability individually.ConclusionsThese experiments demonstrate that GLI1 up-regulation contributes to VCR resistance in RMS and EWS cell lines and suggest that targeting GLI1 may benefit patients with RMS or EWS by reducing multidrug resistance.

Abstract C008: Pharmacokinetic analysis of vincristine and its m1 metabolite in Kenyan pediatric cancer patients through co-modeling

Abstract Objectives: The aim of this study was to develop a pharmacokinetic (PK) model that can determine the PK of vincristine (VCR) and its M1 metabolite using concentration data from Kenyan pediatric cancer patients. VCR is one of the most widely used anticancer agents in treating a variety of malignancies in pediatric oncology. Regardless of its extensive pediatric use, dosing strategies for VCR are largely empirical as there is little information about its disposition and optimal therapeutic dosing. Our collaborator, Dr. Jamie Renbarger and her group have reported that CYP3A5 enzyme generates the major VCR metabolite, M1, more efficiently than CYP3A4 enzyme. This finding maybe clinically significant because CYP3A5 expression varies with up to 90%, 70% and 10-20% of expressions in Kenyans, African Americans and Caucasians, respectively. Therefore, it is crucial to monitor M1 and characterize its disposition in humans to provide an insight of inter-ethnic variability in VCR metabolism and clearance. Methods: Kenyan pediatric cancer patients (8 males:8 females, age range: 1-13 years, weight range: 7.0 - 36.6 kg, body surface area: 0.36 - 1.24 m2) were intravenously administered with VCR (delivered dose, 0.4 - 2.5 mg). Blood samples were collected using human dried blood spot (DBS) collection paper via finger prick at various time points depending on the feasibility and duration of patient stay for care in the hospital. Using a developed LC-MS-MS assay, the concentrations of VCR and M1 were measured from patient DBS samples. A PK compartmental model was developed through co-modeling of VCR and M1 using Phoenix (version 8.0). The model was selected based on comparison of quality of fit plots and on the likelihood ratio test on the difference of criteria (-2LL). Results: The best fit structural model for VCR and its M1 metabolite was established. The model consists of a one-way metabolite formation transfer from VCR to M1 compartments. Good correlations were observed between observed and predicted values of VCR and M1 in the subjects used. The best-fit PK parameter estimates were derived from the PK compartmental model. In one subject, values estimated for PK parameters that only our study could derive include: conversion rate constant from VCR to M1 of 0.04 1/hr, M1 metabolite volume of distribution of 51.49 L, and M1 metabolite elimination rate constant of 0.44 1/hr. Conclusion: For the first time, a compartmental PK model that could determine the PK parameters of VCR and its M1 metabolite from DBS samples through co-modeling was developed. The model resulted in a good fit for the subjects used. The model will be validated after further adjustments and could potentially be used to rationally modify future VCR dosing regimen for Kenyan pediatric patients. Further, this modification could be possibly extrapolated for African American pediatric patients. Additionally, using our validated model, we will characterize and predict the population PK of VCR and its M1 metabolite among different ethnic groups. Citation Format: Lorita Agu, Jamie Renbarger, Diana S-L. Chow. Pharmacokinetic analysis of vincristine and its m1 metabolite in Kenyan pediatric cancer patients through co-modeling [abstract]. In: Proceedings of the Twelfth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2019 Sep 20-23; San Francisco, CA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2020;29(6 Suppl_2):Abstract nr C008.