Co-treatment With Aripiprazole and Vincristine Sensitizes P-Glycoprotein-Overexpressing Drug-resistant MCF-7/ADR Breast Cancer Cells.

BACKGROUND/AIM: Evidence supports that use of aripiprazole sensitizes drug-resistant oral cancer cells. The aim of the study was to investigate whether aripiprazole can achieve sensitization of highly drug-resistant breast cancer cells, as well as identify its relevant mechanisms of action. MATERIALS AND METHODS: MCF-7/ADR, KB, and KBV20C breast cancer cells were treated with aripiprazole, vincristine (VIC), vinorelbine, vinblastine and their combination. Cell viability assay, annexin V analyses, cellular morphology and density observation with a microscope, western-blotting, fluorescence-activated cell sorting (FACS), and analysis for P-gp inhibitory activity were performed to investigate the drugs' mechanism of action. RESULTS: We found that high drug resistance in MCF-7/ADR cells results from high P-gp inhibitory activity via overexpression of P-gp. Aripiprazole reduced cell viability, increased G2 arrest, and upregulated apoptosis when used as a co-treatment with VIC. Furthermore, we demonstrated that co-treatment with vinorelbine and vinblastine increased the sensitization of MCF-7/ADR breast cancer cells to aripiprazole. We confirmed that VIC-aripiprazole combination has much higher sensitization effects than either VIC-thioridazine or VIC-trifluoperazine co-treatment in MCF-7/ADR cells, since the previously known bipolar drugs (thioridazine and trifluoperazine) has lower P-gp inhibitory activity. However, aripiprazole-induced sensitization was not observed in VIC-treated MDA-MB-231 breast cancer cells suggesting that combination therapy with aripiprazole is specific for P-gp-overexpressing drug-resistant breast cancer cells. CONCLUSION: Co-treatment with low doses of aripiprazole sensitized MCF-7/ADR cells to VIC. Combination therapy with aripiprazole may be a valuable tool for delaying or reducing cancer recurrence by targeting P-gp-overexpressing drug-resistant breast cancer cells.

Dose-dependent bidirectional pharmacological effects of vinorelbine-based metronomic combination chemotherapy on tumor growth and metastasis and mechanisms in melanoma mouse model.

BACKGROUND: There is evidence that the empirical setting of doses and schedules of antineoplastic agents in metronomic chemotherapy (MC) might lead to undesirable outcomes, such as promoting tumor growth or metastasis at certain low doses. However, details about the dose effect of antineoplastic agents in MC have not been fully known yet. OBJECTIVES: Vinorelbine combined with cisplatin or fluorouracil (VNR/CDDP or VNR/FU) was selected to investigate its effects on tumor growth or metastasis as well as mechanisms. METHODS: Experimental techniques, including immunohistochemistry, western blot, immunofluorescence, and flow cytometry, were used to explore the mechanisms, along with cell proliferation, apoptosis, migration, and invasion. RESULTS: The results showed that VNR/CDDP or VNR/FU promoted tumor growth and metastasis at low doses and inhibited them at high ones. Except that expressions of apoptotic proteins were elevated at both low and high doses, low-dose treatments enhanced angiogenesis and promoted the mobilization and recruitment of myeloid-derived suppressor cells (MDSCs), while high-dose treatments reversed these effects. Additionally, low concentrations of VNR/CDDP or VNR/FU stimulated tumor cell functions such as anti-apoptosis, migration, and invasion, but high concentrations only suppressed cell proliferation and increased apoptosis. CONCLUSION: This study elucidated a bidirectional action mode regulated by multiple mechanisms at different doses in MC and also highlighted the risks of low-dose metronomic administration of antineoplastic agents in the clinic. More preclinical and clinical studies focusing on the dose-effect of metronomic regimens are urgently needed because an effective therapeutic regimen should be an optimal setting of drugs, doses, schedules, or combinations.

Amino acid derivative of probenecid potentiates apoptosis-inducing effects of vinblastine by increasing oxidative stress in a cancer cell-specific manner.

Many chemotherapeutic drugs suffer from multidrug resistance (MDR). Efflux transporters, namely ATP-binding cassettes (ABCs), that pump the drugs out of the cancer cells comprise one major reason behind MDR. Therefore, ABC inhibitors have been under development for ages, but unfortunately, without clinical success. In the present study, an l-type amino acid transporter 1 (LAT1)-utilizing derivative of probenecid (PRB) was developed as a cancer cell-targeted efflux inhibitor for P-glycoprotein (P-gp), breast cancer resistant protein (BCRP) and/or several multidrug resistant proteins (MRPs), and its ability to increase vinblastine (VBL) cellular accumulation and apoptosis-inducing effects were explored. The novel amino acid derivative of PRB (2) increased the VBL exposure in triple-negative human breast cancer cells (MDA-MB-231) and human glioma cells (U-87MG) by 10-68 -times and 2-5-times, respectively, but not in estrogen receptor-positive human breast cancer cells (MCF-7). However, the combination therapy had greater cytotoxic effects in MCF-7 compared to MDA-MB-231 cells due to the increased oxidative stress recorded in MCF-7 cells. The metabolomic study also revealed that compound 2, together with VBL, decreased the transport of those amino acids essential for the biosynthesis of endogenous anti-oxidant glutathione (GSH). Moreover, the metabolic differences between the outcomes of the studied breast cancer cell lines were explained by the distinct expression profiles of solute carriers (SLCs) that can be concomitantly inhibited. Therefore, attacking several SLCs simultaneously to change the nutrient environment of cancer cells can serve as an adjuvant therapy to other chemotherapeutics, offering an alternative to ABC inhibitors.

Effect of Laetrile Vinblastine Combination on the Proliferation of the Hela Cancer Cell Line.

AIM: This study aimed to evaluate the inhibitory effect of laetrile, vinblastine, and their mixture on cervical cancer cells and probe potential synergistic consequences. METHOD: The study scrutinized the inhibitory impact of laetrile vinblastine and their mixture on the growth of human cervical cancer cells (Hela cancer cell line). The cells were incubated for 24, 48, and 72 hours with concentrations varying from 1 microgram to 10,000 micrograms of each substance. RESULT: study results showed, the combination of vinblastine and laetrile effectively reduced the viability of human cervical cancer cells. This effect was stronger than the individual cytotoxic effects of each compound. The results suggest that the cytotoxicity of the vinblastine and laetrile combination increases with higher concentrations of the compounds. Additionally, the study revealed a synergistic effect between the mixture ingredients, particularly at the lowest and highest concentrations during the 24 and 72-hour incubation periods. CONCLUSION: The antiproliferative effect of (the combination of laetrile and vinblastine) was greater than the antiproliferative effect of either compound used alone, suggesting a synergistic relationship between the two.

Single-cell impedance cytometry of anticancer drug-treated tumor cells exhibiting mitotic arrest state to apoptosis using low-cost silver-PDMS microelectrodes.

Chemotherapeutic drugs such as paclitaxel and vinblastine interact with microtubules and thus induce complex cell states of mitosis arrest at the G2/M phase followed by apoptosis dependent on drug exposure time and concentration. Microfluidic impedance cytometry (MIC), as a label-free and high-throughput technology for single-cell analysis, has been applied for viability assay of cancer cells post drug exposure at fixed time and dosage, yet verification of this technique for varied tumor cell states after anticancer drug treatment remains a challenge. Here we present a novel MIC device and for the first time perform impedance cytometry on carcinoma cells exhibiting progressive states of G2/M arrest followed by apoptosis related to drug concentration and exposure time, after treatments with paclitaxel and vinblastine, respectively. Our results from impedance cytometry reveal increased amplitude and negative phase shift at low frequency as well as higher opacity for HeLa cells under G2/M mitotic arrest compared to untreated cells. The cells under apoptosis, on the other hand, exhibit opposite changes in these electrical parameters. Therefore, the impedance features differentiate the HeLa cells under progressive states post anticancer drug treatment. We also demonstrate that vinblastine poses a more potent drug effect than paclitaxel especially at low concentrations. Our device is fabricated using a unique sacrificial layer-free soft lithography process as compared to the existing MIC device, which gives rise to readily aligned parallel microelectrodes made of silver-PDMS embedded in PDMS channel sidewalls with one molding step. Our results uncover the potential of the MIC device, with a fairly simple and low-cost fabrication process, for cellular state screening in anticancer drug therapy.

Microtubule rescue control by drugs and MAPs examined with in vitro pedestal assay.

Microtubules are essential cytoskeletal polymers, which exhibit stochastic transitions between assembly and disassembly, known as catastrophes and rescues. Understanding of catastrophes, rescues, and their control by drugs and microtubule associated proteins (MAPs) has been informed by in vitro reconstitutions of microtubule dynamics. In such experiments microtubules are typically observed on a flat surface of the coverslip. In contrast, we have recently proposed a modified setup in which microtubules assemble from stabilized seeds, overhanging from microfabricated pedestals, so that their dynamic extensions are fully isolated from contact with the coverslip. This assay allows to eliminate potential artifacts, which may substantially affect the frequency of microtubule rescues in vitro. Here we use the pedestal assay to study the sensitivity of microtubules to paclitaxel, one of the best-known inhibitors of microtubule dynamics. By comparing observations in the conventional and the pedestal assays, we find that microtubule dynamics are substantially more sensitive to paclitaxel when the polymers can contact the coverslip. We interpret this as a consequence of the coverslip-induced microtubule assembly perturbation, leading to formation of lattice with defects, and thereby enhancing the efficiency of paclitaxel binding to microtubules in the conventional assay. To test this idea, we use vinblastine, another small-molecule inhibitor, which had been previously shown to cause microtubule growth perturbations. We find that in the pedestal assay vinblastine sensitizes microtubules to paclitaxel to the level, observed in the conventional assay. Interestingly, a minimal fragment of MAP called CLASP2, a previously characterized rescue factor, has a strong effect on microtubule rescues, regardless of the type of assay. Overall, our study underscores the role of microtubule damage in promoting rescues and highlights the utility of the in vitro pedestal assay to study microtubule dynamics modulation by tubulin inhibitors and MAPs.

Real-World Use of Positron Emission Tomography-Computed Tomography and Reported Deauville Scores in Advanced-Stage Classic Hodgkin Lymphoma: A Community Oncology Practice Perspective.

PURPOSE: To evaluate the use of interim positron emission tomography-computed tomography (PET-CT) scans and Deauville 5-point scale (5PS) score reporting for stage III/IV classic Hodgkin lymphoma (cHL) treated frontline (1L) in community oncology settings. METHODS: This retrospective, observational study included adults with stage III/IV cHL initiating 1L doxorubicin, bleomycin, vinblastine, and dacarbazine (ABVD), brentuximab vedotin, doxorubicin, vinblastine, and dacarbazine, or an escalated dosing regimen of bleomycin, etoposide, doxorubicin, cyclophosphamide, vincristine, procarbazine, and prednisone within the US Oncology Network between January 2017 and October 2019. Data were collected from electronic health records and chart reviews and summarized descriptively. RESULTS: A total of 262 patients were included; 48.9% were age 39 years or younger. Most were male (57%), White (59%), had an International Prognostic Score <4 (76%), and received 1L ABVD (74%). Forty-nine percent of patients had stage III and 51% had stage IV cHL. Of 258 patients with ≥1 PET-CT scan, 71% (n = 184) had an interim scan and 64% received ≥1 scan at an off-site location. Of patients treated 1L with ABVD who received a baseline and interim scan, Deauville 5PS scores were not documented for 45% of patients; in 90% of these cases, a standardized uptake value (SUV) was reported. CONCLUSION: In community oncology settings, under-reporting of Deauville 5PS scores for interim PET-CT scans was observed. In the absence of Deauville 5PS scores, SUV results were generally provided. These results highlight educational opportunities that exist for PET-adapted ABVD, including consistency in reporting/utilization of Deauville 5PS scores to de-escalate or escalate treatment.

Vinblastine resets tumor-associated macrophages toward M1 phenotype and promotes antitumor immune response.

BACKGROUND: Massive tumor-associated macrophage (TAM) infiltration is observed in many tumors, which usually display the immune-suppressive M2-like phenotype but can also be converted to an M1-like antitumor phenotype due to their high degree of plasticity. The macrophage polarization state is associated with changes in cell shape, macrophage morphology is associated with activation status. M1 macrophages appeared large and rounded, while M2 macrophages were stretched and elongated cells. Manipulating cell morphology has been shown to affect the polarization state of macrophages. The shape of the cell is largely dependent on cytoskeletal proteins, especially, microtubules. As a microtubule-targetting drug, vinblastine (VBL) has been used in chemotherapy. However, no study to date has explored the effect of VBL on TAM shape changes and its role in tumor immune response. METHOD: We used fluorescent staining of the cytoskeleton and quantitative analysis to reveal the morphological differences between M0, M1, M2, TAM and VBL-treated TAM. Flow cytometry was used to confirm the polarization states of these macrophages using a cell surface marker-based classification. In vivo antibody depletion experiments in tumor mouse models were performed to test whether macrophages and CD8+ T cell populations were required for the antitumor effect of VBL. VBL and anti-PD-1 combination therapy was then investigated in comparison with monotherapy. RNA-seq of TAM of treated and untreated with VBL was performed to explore the changes in pathway activities. siRNA mediated knockdown experiments were performed to verify the target pathway that was affected by VBL treatment. RESULTS: Here, we showed that VBL, an antineoplastic agent that destabilizes microtubule, drove macrophage polarization into the M1-like phenotype both in vitro and in tumor models. The antitumor effect of VBL was attenuated in the absence of macrophages or CD8+ T cells. Mechanistically, VBL induces the activation of NF-κB and Cyba-dependent reactive oxygen species generation, thus polarizing TAMs to the M1 phenotype. In parallel, VBL promotes the nuclear translocation of transcription factor EB, inducing lysosome biogenesis and a dramatic increase in phagocytic activity in macrophages. CONCLUSIONS: This study explored whether manipulating cellular morphology affects macrophage polarization and consequently induces an antitumor response. Our data reveal a previously unrecognized antitumor mechanism of VBL and suggest a drug repurposing strategy combining VBL with immune checkpoint inhibitors to improve malignant tumor immunotherapy.

Tubulin degradation: Principles, agents, and applications.

The microtubule system plays an important role in the mitosis and growth of eukaryotic cells, and it is considered as an appealing and highly successful molecular target for cancer treatment. In fact, microtubule targeting agents, such as paclitaxel and vinblastine, have been approved by FDA for tumor therapy, which have achieved significant therapeutic effects and sales performance. At present, microtubule targeting agents mainly include microtubule-destabilizing agents, microtubule-stabilizing agents, and a few tubulin degradation agents. Although there are few reports about tubulin degradation agents at present, tubulin degradation agents show great potential in overcoming multidrug resistance and reducing neurotoxicity. In addition, some natural drugs could specifically degrade tubulin in tumor cells, but have no effect in normal cells, thus showing a good biosafety profile. Therefore, tubulin degradation agents might exhibit a better application. Currently, some small molecules have been designed to promote tubulin degradation with potent antiproliferative activities, showing the potential for cancer treatment. In this work, we reviewed the reports on tubulin degradation, and focused on the degradation mechanism and important functional groups of chemically synthesized compounds, hoping to provide help for the degradation design of tubulin.

Potentiating Biosynthesis of Alkaloids and Polyphenolic Substances in Catharanthus roseus Plant Using ĸ-Carrageenan.

Catharanthus roseus is a medicinal plant that produces indole alkaloids, which are utilized in anticancer therapy. Vinblastine and vincristine, two commercially important antineoplastic alkaloids, are mostly found in the leaves of Catharanthus roseus. ĸ-carrageenan has been proven as plant growth promoting substance for a number of medicinal and agricultural plants. Considering the importance of ĸ-carrageenan as a promoter of plant growth and phytochemical constituents, especially alkaloids production in Catharanthus roseus, an experiment was carried out to explore the effect of ĸ-carrageenan on the plant growth, phytochemicals content, pigments content, and production of antitumor alkaloids in Catharanthus roseus after planting. Foliar application of ĸ-carrageenan (at 0, 400, 600 and 800 ppm) significantly improved the performance of Catharanthus roseus. Phytochemical analysis involved determining the amount of total phenolics (TP), flavonoids (F), free amino acids (FAA), alkaloids (TAC) and pigments contents by spectrophotometer, minerals by ICP, amino acids, phenolic compounds and alkaloids (Vincamine, Catharanthine, Vincracine (Vincristine), and vinblastine) analysis uses HPLC. The results indicated that all examined ĸ-carrageenan treatments led to a significant (p ≤ 0.05) increase in growth parameters compared to the untreated plants. Phytochemical examination indicates that the spray of ĸ-carrageenan at 800 mg L-1 increased the yield of alkaloids (Vincamine, Catharanthine and Vincracine (Vincristine)) by 41.85 µg/g DW, total phenolic compounds by 3948.6 µg gallic/g FW, the content of flavonoids 951.3 µg quercetin /g FW and carotenoids content 32.97 mg/g FW as compared to the control. An amount of 400 ppm ĸ-carrageenan treatment gave the best contents of FAA, Chl a, Chl b and anthocyanin. The element content of K, Ca, Cu, Zn and Se increased by treatments. Amino acids constituents and phenolics compounds contents were altered by ĸ-carrageenan.

Induction of Drug-Resistance and Production of a Culture Medium Able to Induce Drug-Resistance in Vinblastine Untreated Murine Myeloma Cells.

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

Establishment of recombinant Catharanthus roseus stem cells stably overexpressing ORCA4 for terpenoid indole alkaloids biosynthesis.

Catharanthus roseus is a perennial herb of the Apocynaceae family, from which about 200 kinds of alkaloids have been characterized. Most alkaloids from C. roseus are terpenoid indole alkaloids (TIAs), such as vinblastine and vincristine, which are widely used in the clinic for their good antitumor activity. However, they were only biosynthesized in C. roseus, and their content in C. roseus is extremely low. The access to these valuable compounds is by plant extraction or chemical semisynthesis from their precursors catharanthine and vindoline. Since catharanthine and vindoline are also obtained from C. roseus, the supply of vinblastine and vincristine makes it difficult to meet market demands. Therefore, how to improve the yield of TIAs is an attractive issue. In this study, we compared the regulatory effect of two critical transcription factors, octadecanoid-derivative responsive Catharanthus AP2-domain protein 3 (ORCA3) and octadecanoid-derivative responsive Catharanthus AP2-domain protein 4 (ORCA4), on the biosynthesis of TIAs in C. roseus. The results showed that overexpressing both two transcription factors could increase the accumulation of TIAs. The effect was more significant when ORCA4 was overexpressed. To acquire C. roseus TIAs on a continuous and consistent basis, we then created and acquired C. roseus stem cells stably overexpressing ORCA4. This is the first time a recombinant C. roseus stem cell system with stable ORCA4 overexpression has been developed, which not only provides new ideas for future research in this area but also breaches new life into the industrial application of using plant cell culture to obtain natural products.

Conformation and energy investigation of microtubule longitudinal dynamic instability induced by natural products.

The natural products plinabulin, docetaxel, and vinblastine are microtubule targeting agents (MTAs). They have been used alone or in combination in cancer treatment. However, the exact nature of their effects on microtubule (MT) polymerization dynamics is poorly understood. To elucidate the longitudinal conformational and energetic changes during MT dynamics, a total of 140 ns molecular dynamic simulations combined with binding free energy calculations were performed on seven tubulin models. The results indicated that the drugs disrupted MT polymerization by altering both MT conformation and binding free energy of the neighboring tubulin subunits. The combination of plinabulin and docetaxel destabilized MT polymerization due to bending MT and weakening the polarity of tubulin polymerization. The new combination of docetaxel and vinblastine synergistically enhanced MT depolymerization and bending, while plinabulin and vinblastine had no synergistic inhibitory effects. The results were verified by the tubulin assembly assay. Our study obtained a comprehensive understanding of the action mechanisms of three natural drugs and their combinations on MT dynamic, provided theoretical guidance for new MTA combinations, and would promote the optimal use of MTA and contribute to developing new MTAs as anticancer agents.

Cryo-derived plants through embryogenesis showed same levels of vinblastine and vincristine (anticancer) in Catharanthus roseus and had normal genome size.

Cryopreservation of rare plant materials is an important approach for preserving germplasms and is a good added concept to tissue banking. The preservation of embryogenic cell suspensions is even more valuable as the tissues facilitate in producing millions of embryos, plantlets and generates transgenics en masse. Catharanthus roseus is a medicinally important plant that produces a variety of anticancerous phytocompounds and needs conservation of alkaloid producing cell lines. In this study, embryogenic tissue banking has been attempted in C. roseus by the two-step cryopreservation method combining cryoprotection and dehydration. Prior to plunging into liquid nitrogen (LN), the tissues were exposed to osmotic-and cryoprotective agents. Two osmotic agents (sugar and sorbitol) and three cryoprotective compounds, polyethylene glycol (PEG), dimethyl sulfoxide (DMSO) and glycerol were used at varying concentrations to protect cells from freezing damages. Both sucrose and sorbitol increased callus biomass post-cryopreservation; the influence of sucrose was however, more prominent. Embryogenic tissue treated in medium with 0.4 M sucrose for 2 days followed by 5% PEG for 2 h showed maximum viability before (83%) and after (55%) cryopreservation, high regrowth percentage (77%) and produced an average 9 cell colonies per Petri dish. Additionally, dehydration (1-5 h) was tested to reduce water content for improving viability and regrowth of cryopreserved embryogenic cells. Among the various tested cryoprotective conditions, the highest (72%) viability was observed following the combination of treatments with 0.4 M sucrose (2 days),10% PEG (2 h) and dehydration (2 h). Maximum regrowth percentage (88%) and 12 colonies/petri dish was noted in combination of 0.4 M sucrose + 5% PEG. The cryopreserved calli differentiated into somatic embryos (52.78-54.33 globular embryos/callus mass) in NAA (0.5 mg/l) and BAP (0.5-1.0 mg/l) added media. Plantlets were successfully regenerated from cryopreserved tissue and the 2C DNA was estimated through flow cytometry. The genome size of cryopreserved regenerant was 1.51 pg/2C, which is similar to field-grown Catharanthus plants. Vinblastine and vincristine levels were nearly the same in mother plant's and frozen (cryopreserved) leaf tissue. The post cryopreservation embryogenesis protocol may be used for continuous production of plants for future applications.

Thermodynamic and structural profiles of multi-target binding of vinblastine in solution.

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

Effect of the plant probiotic bacteria on terpenoid indole alkaloid biosynthesis pathway gene expression profiling, vinblastine and vincristine content in the root of Catharanthus roseus.

BACKGROUND: Catharanthus roseus is the sole resource of vinblastine and vincristine, two TIAs of great interest for their powerful anticancer activities. Increasing the concentration of these alkaloids in various organs of the plant is one of the important goals in C. roseus breeding programs. Plant probiotic bacteria (PBB) act as biotic elicitors and can induce the synthesis of secondary products in plants. The purpose of this research is to study the effects of PBB on expression of the TIA biosynthetic pathway genes and the content of alkaloids in C. roseus. METHODS AND RESULTS: The individual and combined effects of P. fluorescens strains 169 and A. brasilense strains Ab-101 was studied for expression of the TIA biosynthetic pathway genes (G10H, DAT, T16H and CrPRX) using qRT-PCR and the content of vinblastine and vincristine using HPLC method in roots of C. roseus. P. fluorescens. This drastically increased the content of vinblastine and vincristine alkaloids, compared to the control in the roots, to 174 and 589 (µg/g), respectively. Molecular analysis showed bacterium significantly increased the expression of more genes in the TIA biosynthetic pathway compared to the control. P. fluorescens increased the expression of the final gene of the biosynthetic pathway (CrPRX) 47.9 times compared to the control. Our findings indicate the correlation between transcriptional and metabolic outcomes. The same was true for A. brasilense. CONCLUSIONS: It can be concluded that seed treatments and seedling root treatments composed of naturally occurring probiotic bacteria are likely to be widely applicable for inducing enhanced alkaloid contents in medicinal plants.

Resin Glycosides from Operculina hamiltonii and Their Synergism with Vinblastine in Cancer Cells.

Operculina hamiltonii is a vine native to the north and northeast region of Brazil, where its roots are traded as a depurative and laxative remedy with the name of Brazilian jalap in traditional medicine. Procedures for the isolation, purification by recycling HPLC, and structure elucidation of three undescribed resin glycosides are presented herein. Hamiltonin I (1) represents a macrocyclic structure of a tetrasaccharide of (11S)-hydroxyhexadecanoic acid. Additionally, two acyclic pentasaccharides, named hamiltoniosides I (2) and II (3), were also isolated, which are related structurally to the known compounds 4 and 5, macrocyclic lactone-type batatinosides. The tetrasaccharide core of 1 was diacylated by n-decanoic acid and the unusual n-hexadecanoic acid moiety, while the pentasaccharides 2-5 were esterified by one unit of n-decanoic or n-dodecanoic acid. All the isolated compounds were found to be inactive as cytotoxic agents. However, when they were evaluated (1-25 µM) in combination with a sublethal concentration of the anticancer agent vinblastine (0.003 µM), a significant enhancement of the resultant cytotoxicity was produced, especially for multidrug-resistant breast carcinoma epithelial cells. Such combined synergistic potency may be beneficial for chemotherapy, making resin glycosides potential candidates for drug repurposing of conventional chemotherapeutic drugs to reduce their side effects.

Emodin Sensitizes Cervical Cancer Cells to Vinblastine by Inducing Apoptosis and Mitotic Death.

In recent years, studies on the effects of combining novel plant compounds with cytostatics used in cancer therapy have received considerable attention. Since emodin sensitizes tumor cells to chemotherapeutics, we evaluated changes in cervical cancer cells after its combination with the antimitotic drug vinblastine. Cellular changes were demonstrated using optical, fluorescence, confocal and electron microscopy. Cell viability was assessed by MTT assay. The level of apoptosis, caspase 3/7, Bcl-2 protein, ROS, mitochondrial membrane depolarization, cell cycle and degree of DNA damage were analyzed by flow cytometry. The microscopic image showed indicators characteristic for emodin- and vinblastine-induced mitotic catastrophe, i.e., multinucleated cells, giant cells, cells with micronuclei, and abnormal mitotic figures. These compounds also increased blocking of cells in the G2/M phase, and the generated ROS induced swelling and mitochondrial damage. This translated into the growth of apoptotic cells with active caspase 3/7 and inactivation of Bcl-2 protein and active ATM kinase. Emodin potentiated the cytotoxic effect of vinblastine, increasing oxidative stress, mitotic catastrophe and apoptosis. Preliminary studies show that the combined action of both compounds, may constitute an interesting form of anticancer therapy.

Pyrimidines-Based Heterocyclic Compounds: Synthesis, Cytoxicity Evaluation and Molecular Docking.

A variety of structurally different pyrimidines were synthesized. Elemental analysis, FT-IR, 1H NMR, and 13C NMR spectroscopy were used to confirm the chemical structures of all prepared compounds. The synthesized pyrimidines were screened against the growth of five human cancer cell lines (prostate carcinoma PC3, liver carcinoma HepG-2, human colon cancer HCT-116, human breast cancer MCF-7, human lung cancer A-549), and normal human lung fibroblasts (MRC-5) using MTT assay. Most of the screened pyrimidines have anti-proliferative activity on the growth of the PC3 cell line. Compounds 3b and 3d were more potent than the reference vinblastine sulfate (~2 to 3 × fold) and they can be considered promising leads for treating prostate cancer disease. Moreover, the screened compounds 3b, 3f, 3g, 3h, and 5 were assessed according to the values of their selectivity index (SI) and were found to be more selective and safer than vinblastine sulfate. Furthermore, using in silico computational tools, the physicochemical properties of all pyrimidine ligands were assessed, and the synthesized compounds fall within the criteria of RO5, thus having the potential to be orally bioavailable.

Vinca alkaloid binding to P-glycoprotein occurs in a processive manner.

A mechanistic understanding of how P-glycoprotein (Pgp) is able to bind and transport its astonishing range of substrates remains elusive. Pharmacological data demonstrated the presence of at least four distinct binding sites, but their locations have not been fully elucidated. The combination of biochemical and structural data suggests that initial binding may occur in the central cavity or at the lipid-protein interface. Our objective was to define the binding sites for two transported substrates of Pgp; the anticancer drug vinblastine and the fluorescent probe rhodamine 123. A series of mutations was generated in positions proximal to previously defined drug-interacting residues on Pgp. The protein was purified and reconstituted into styrene-maleic acid lipid particles (SMALPs) to measure the apparent drug binding constant or into liposomes for assessment of drug-stimulated ATP hydrolysis. The biochemical data were reconciled with structural models of Pgp using molecular docking. The data indicated that the binding of rhodamine 123 occurred predominantly within the central cavity of Pgp. In contrast, the significantly more hydrophobic vinblastine bound to both the lipid-protein interface and within the central cavity. The data suggest that the initial interaction of vinca alkaloids with Pgp occurs at the lipid interface followed by internalisation into the central cavity, which also provides the transport conduit. This model is supported by recent structural observations with Pgp and early biophysical and cross-linking approaches. Moreover, the proposed model illustrates that the broad substrate profile for Pgp is underpinned by a combination of multiple initial interaction sites and an accommodating transport conduit.