Effect of curcumin in mice model of vincristine-induced neuropathy.

CONTEXT: Curcumin exhibits a wide spectrum of biological activities which include neuroprotective, antinociceptive, anti-inflammatory, and antioxidant activity. OBJECTIVE: The present study evaluates the effect of curcumin in vincristine-induced neuropathy in a mice model. MATERIALS AND METHODS: Vincristine sulfate (0.1 mg/kg, i.p. for 10 consecutive days) was administered to mice to induce neuropathy. Pain behavior was assessed at different days, i.e., 0, 7, 10, and 14 d. Sciatic nerve total calcium, superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), reduced glutathione (GSH), nitric oxide (NO), and lipid peroxidation (LPO) were also estimated after the 14th day of study. Pregabalin (10 mg/kg, p.o.) and curcumin (15, 30, and 60 mg/kg, p.o.) were administered for 14 consecutive days. RESULTS: Curcumin at 60 mg/kg significantly attenuated the vincristine-induced neuropathic pain manifestations in terms of thermal hyperalgesia (p < 0.001) and allodynia (p < 0.001); mechanical hyperalgesia (p < 0.001); functional loss (p < 0.001); and in the delayed phase of formalin test (p < 0.001). Curcumin at 30 and 60 mg/kg exhibited significant changes (p < 0.001) in antioxidant levels and in total calcium levels in vincristine-injected mice. CONCLUSION: Curcumin at 30 and 60 mg/kg dose levels significantly attenuated vincristine-induced neuropathy which may be due to its multiple actions including antinociceptive, calcium inhibitory, and antioxidant effect.

Activation of DNA damage response by antitumor therapy counteracts the activity of vinca alkaloids.

BACKGROUND/AIM: Anthracyclines have been proven able to reduce the activity of vinca alkaloids by induction of cell-cycle arrest. The present study aims at identifying the critical initiation steps of signal transduction which transduce the inhibitory effects on the cytotoxicity of vinca alkaloids. MATERIALS AND METHODS: Several new cytostatic drug classes were evaluated together with vincristine in tumor cell lines and patients' tumor cells. RNA interference was used for molecular analyses. RESULTS: Inhibition of vincristine was observed by all cytostatic drugs, which induced cell-cycle arrest. Knockdown of proteins of the DNA damage response ascribed the inhibitory effect to a common pathway involving Chk-1, p53 and p21. Upstream of Chk-1 signal transduction depended on both ATM and ATR for all drugs except methotrexate. CONCLUSION: We have identified critical signaling steps of the DNA damage response system activated by cytostatic drugs, which reduce the anti-tumor activity of vinca alkaloids. The obtained results encourage the development of novel therapeutic strategies to prevent pathway interactions based on the molecular understanding of drug action and drug-drug interactions.

Lacosamide has protective disease modifying properties in experimental vincristine neuropathy.

Pain and paresthesias are the most common symptoms of chemotherapy induced painful neuropathy (CIPN). Current treatment and preventive strategies of CIPN are ineffective, and the neuropathy may lead to discontinuation of anti-tumor therapy. Here we used experimental vincristine-induced neuropathy in rats to evaluate the disease modifying potential of lacosamide using a sustained release formulation and the acute treatment effects of a rapid release formulation. Pain behavior was assessed by withdrawal responses to von Frey hairs, acetone drops, the Randall-Selitto device, and to radiant heat. Neuropathy was assessed using electrophysiological recordings. Preventive lacosamide treatment (30 mg/kg subcutaneously b.i.d. for 17 days) was well tolerated, and pharmacokinetic analysis revealed a peak plasma concentration 2 h post-injection with a plasma half-life of approximately 3 h. Rats treated with lacosamide, in contrast to vehicle treated rats, did not develop vincristine-induced cold allodynia. Neurophysiology showed a delayed F-wave latency in vehicle treated rats, which was not present in lacosamide treated animals. We could thus demonstrate a protective disease modifying potency of lacosamide in an animal model of CIPN. Lacosamide may be a promising candidate for preventive treatment of CIPN in patients receiving chemotherapy with vinca alkaloids or platinum drugs.

L-type calcium channel blockers reverse docetaxel and vincristine-induced multidrug resistance independent of ABCB1 expression in human lung cancer cell lines.

Multidrug resistance (MDR) of cancer cells to cytotoxic drugs significantly impedes chemotherapeutic treatment. The purpose of this study is to characterize docetaxel (DOC) or vincristine (VCR) selected A549 and H1299 non-small cell lung cancer (NSCLC) sublines that exhibit MDR phenotypes and followed by re-sensitization study. Although all drug resistant sublines showed cross-resistance to DOC, VCR, and doxorubicin (DXR), the expression of ATP-binding cassette (ABC) transporter B1 (ABCB1) gene was found to be strongly induced in DOC but not in VCR resistant A549 sublines by quantitative reverse transcription real-time polymerase chain reaction (qRT-PCR). In DOC and VCR resistant H1299 sublines, moderate expression of ABCB1 was detected. The levels of ABCB1 protein and efflux activities were further examined by immunoblotting and rhodamin-123 staining assay. The results showed that both ABC and non-ABC mediated MDR are existed. Furthermore, verapamil (VER), an inhibitor of ABCB1 and an L-type calcium channel blocker, is capable of reversing the resistance in all drug-resistant sublines independent of ABCB1 expression. Importantly, VER only sensitizes resistant sublines but has no effect on parental cancer cells. Other L-type calcium channel blockers, such as diltiazem (DIL) and nifedipine (NIF), also sensitize MDR sublines without interfering with ABCB1 activity but with lower efficacy than VER. Our data showed that in addition to ABCB1, calcium channel activity may play a crucial role in DOC- and VCR-acquired MDR. Therefore, inhibition of calcium influx may provide a new target to modulate MDR in chemotherapy.

Alpha(1) and alpha(2)-adrenoreceptor antagonists in streptozotocin- and vincristine-induced hyperalgesia.

The effect of alpha(1)- and alpha(2)-adrenoreceptor antagonists (prazosin and yohimbine, respectively) on streptozotocin (STZ)- and vincristine (VIN)-induced hyperalgesia in rats was studied. In two experimental models, yohimbine (1.0 mg/kg ip) completely abolished STZ and VIN-induced hyperalgesia. This effect was markedly prolonged in diabetic rats. Prazosin (0.3 mg/kg ip) attenuated and delayed development of STZ-induced hyperalgesia. In VIN-elicited neuropathy, the administration of prazosin not only delayed hyperalgesia but also produced antinociception. After cessation of drug administration, a significant decrease in nociceptive threshold was observed. The obtained results seem to indicate that both alpha(1)- and alpha(2)-adrenoreceptors are engaged in diabetic (STZ) and toxic (VIN) neuropathy.

Histone deacetylase inhibitors, but not vincristine, cooperate with radiotherapy to induce cell death in medulloblastoma.

BACKGROUND: Though ionising radiation (IR) is an efficient means of postoperative treatment for children with medulloblastoma, the disease is incurable in about a third of them. Thus, multimodality regimens have been introduced, typically combining IR with vincristine. MATERIALS AND METHODS: The combination of IR and vincristine was compared to the combination of IR and histone deacetylase inhibitors (HDIs) for their anticancer activity against medulloblastoma cells in vitro. Cytotoxic activities were assessed by measuring propidium iodide uptake and by cell cycle analysis. RESULTS: HDIs augmented the cytotoxic effect of IR, while the combination of vincristine and IR was significantly less cytotoxic than vincristine alone. Cell cycle analyses revealed that vincristine did not interfere with IR-induced G2/M arrest, whereas HDIs abolished the latter. CONCLUSION: These in vitro findings indicate a favourable interaction of IR and HDIs, but an unfavourable one of IR and vincristine, in medulloblastoma, and provide a rationale for comparing the combination of IR with either vincristine or HDIs in vivo.

Genistein inversely affects tubulin-binding agent-induced apoptosis in human breast cancer cells.

Genistein, a natural isoflavone phytoestrogen present in soybeans, has been extensively studied as a chemopreventive or therapeutic agent in several types of cancer. The traditional Asian diet is rich in soy products may explain in part why the incidence of breast cancer in Asian women is relatively low. To improve therapeutic benefits, we investigated the combination of genistein with chemotherapeutic agents in phenotypically dissimilar human breast cancer cells, MCF-7 and MDA-MB-231, in which estrogen receptor expression is positive and negative, respectively. In the present study, genistein significantly decreased cell apoptosis induced by tubulin-binding agents, paclitaxel and vincristine. FACScan analysis revealed that genistein also diminished the accumulation of the G2/M phase in the cell cycle caused by tubulin-binding agents. In situ staining of microtubules revealed that genistein could decrease paclitaxel-induced tubulin polymerization. However, in vivo tubulin polymerization assay revealed that simultaneous treatment of genistein did not change the tubulin/microtubule dynamic. Genistein reduced Bcl-2 phosphorylation triggered by paclitaxel and vincristine without changing Bax protein expression. p53 and p21 expression, monitored by Western blotting, was not altered by genistein. However, the expression of cyclin B1 and CDC2 kinase was markedly decreased in combination with genistein. In conclusion, genistein inversely affected tubulin-binding agent-induced apoptosis via down-regulation of cyclin B1/CDC2 kinase expression resulting in reduced Bcl-2 phosphorylation.

Effects of neurotoxic and neuroprotective agents on peripheral nerve regeneration assayed by time-lapse imaging in vivo.

A direct histological assay of axonal regeneration would have many advantages over currently available behavioral, electrophysiological, and radiometric assays. We show that peripheral sensory axons marked with the yellow fluorescent protein in transgenic mice can be viewed transcutaneously in superficial nerves. Degenerating and regenerating axons can be followed in live animals with a dissecting microscope and then, after fixation, studied at high resolution by confocal microscopy. Using this approach, we document differences in regenerative ability after nerve transection, crush injury, and crush injury after a previous "conditioning" lesion. We also show that the chemotherapeutic drug vincristine rapidly but transiently blocks regeneration and that the immunosuppressive drug FK506 modestly enhances regeneration. Moreover, FK506 nearly restores normal regenerative ability in animals treated with submaximal doses of vincristine. Because neuropathy is the major dose-limiting side effect of vincristine, we propose that its efficacy could be enhanced by coadministration of FK506 analogs that are neuroactive but not immunosuppressive.

Modulation of vinca-alkaloid induced P-glycoprotein expression by indole-3-carbinol.

The over-expression of mdr-1 gene transcript P-glycoprotein (P-gp), responsible for multiple drug resistance, is one of the major obstacles in cancer chemotherapy. In the present study, indole-3-carbinol (I3C), a well-known chemopreventive agent present in cruciferous vegetables, has been evaluated for its potential to modulate the over-expression of P-gp induced by vinblastine or vincristine, which are known inducers of mdr-1 gene. The results revealed that I3C significantly reversed the over-expression of P-gp in vinca-alkaloid induced drug resistance as evident by Western blotting using monoclonal antibody (clone JSB1). Quantization of immunostained tissue sections using image analysis technique revealed that vinblastine/ vincristine induced overexpression of P-gp was effectively reversed by I3C. The present investigation suggests that I3C can significantly inhibit the P-gp over-expression and may have utility as a dietary adjuvant in the treatment of cancer for the reversal of multiple drug resistance.

Src-regulated extracellular signal-related kinase and Syk-regulated c-Jun N-terminal kinase pathways act in conjunction to induce IL-1 synthesis in response to microtubule disruption in HL60 cells.

A microtubule reorganization is often observed during cellular contacts that are associated to IL-1 production. Here, we show that in HL60 cells, vincristine, a microtubule-disrupting agent that induces a strong production of IL-1, triggers the activation of both extracellular signal-related kinase (ERK) and c-Jun N-terminal kinase (JNK-1). While ERK activation is rapid and transient, peaking at 10 min, the JNK1 activation is delayed and more sustained reaching a maximum at 2 h. ERK activation was blocked by CP 118556, indicating it is regulated by a Src-like kinase, while JNK1 was inhibited by piceatannol, revealing an upstream regulation by Syk. Each kind of the nonreceptor tyrosine kinase blockers efficiently inhibits the vincristine-induced IL-1 production and diminishes the level of IL-1 transcripts, indicating that the ERK and JNK pathways act coordinately to elicit the transcription of the IL-1 gene. Furthermore, we found that pertussis toxin, a blocker of Go/Gi proteins, abrogated the vincristine-induced activation of both Src and Syk. Our data support a model where the status of microtubule polymerization influences the activity of Go or Gi proteins that control, in turn, two independent Src/ERK and Syk/JNK1 cascades that are both necessary to sustain IL-1 synthesis.

Is lithium able to reverse neurological damage induced by vinca alkaloids? (Short communication).

Lithium (Li) actively antagonises the inhibiting action of vinca alkaloids on human leukocyte chemotaxis; it proved to be related to the activation of microtubular system, possibly mediated by its inhibiting effect on cyclic AMP. Vinca alkaloids induce peripheral neuropathy and muscle damage. The molecular basis of this neurotoxicity has not been fully explained, but a possible role of neurofibrillary degeneration has been reported. We studied both in animals and in humans, whether Li is able to antagonise vinca alkaloid neurotoxicity.

NGF prevention of neurotoxicity induced by cisplatin, vincristine and taxol depends on toxicity of each drug and NGF treatment schedule: in vitro study of adult rat sympathetic ganglion explants.

The simultaneous administration of nerve growth factor (NGF) has been found to prevent experimental neuropathies induced by anti-cancer drugs such as cisplatin, vincristine and taxol. However, it is clinically important to know whether NGF is beneficial once the neuropathy is already manifest. We established a bioassay system to examine the preventive effects of NGF in various treatment schedules. NGF significantly prevented the inhibition of neurite outgrowth by vincristine and taxol regardless of treatment schedules. The pre-treatment and co-treatment schedules were effective against cisplatin, but the post-treatment schedule was not. With regard to the neurite and nerve cell population densities, only the cisplatin group treated with NGF showed lower values than the control. These results indicate that NGF-treatment is effective for the toxic sympathetic nerve injury induced by vincristine and taxol regardless of the treatment schedule, but is not protective against cisplatin-induced nerve cell injury.

Modulating activity of indomethacin to vincristine cytotoxicity in various human carcinoma cells.

The modulating activity of indomethacin to vincristine (VCR) was investigated in thirty human pulmonary carcinoma cell lines (adenocarcinoma 9, large cell carcinoma 9, squamous cell carcinoma 6, small cell carcinoma 6) and five other cell lines (colon carcinoma 2, melanoma 1, teratocarcinoma 1, thymoma 1). The survival of these cell lines treated with VCR with or without indomethacin (2 microg/ml) for 72 hours were examined using MTT assay, and IC50 values were calculated. When the cutoff level of potent combined effect in clinical use was set at 2-fold increase of sensitivity, the positive rate was 100% for adenocarcinomas and large cell carcinomas, 25% for squamous cell carcinomas, 33% for small cell carcinomas. Mean modulating index was 2.91 in adenocarcinomas, 1.92 in squamous cell carcinomas, 3.06 in large cell carcinomas and 1.67 in small cell carcinomas. Of the cell lines of other tumors, three cell lines (colon carcinoma 1, melanoma 1, teratocarcinoma 1) showed the potent combined effect of VCR and indomethacin, while indomethacin was not effective in modulating activity to VCR in a thymoma cell line and fibroblast cells. In conclusion, it is considered that modulating activity of indomethacin for VCR is a general effect for various human cancer cells, and combined use with VCR and indomethacin may be a useful modulation therapy for the advanced lung cancer.

Suppression of vincristine-mediated cytotoxic activity by mitoxantrone in human cell lines.

In the present study, we used different cell lines to determine the anticellular effect of a combination of mitoxantrone (MXT) and vincristine (VCR). In all the cell lines tested, most cells (approximately 90%) in cultures with VCR (0.01-1 microM) alone died in the 3 days following exposure, while those with VCR and MXT (0.1-1 microM) invariably survived much longer (6-9 days). Based on the MTT and the 3H-thymidine uptake assays, it was shown that the antagonistic effect of MXT was optimal at 0.1-1 microM and when applied simultaneously. Our results showed that neither modulation of drug accumulation nor inhibition of tubulin assembly could account for the antagonistic effect of MXT. Furthermore, the cytotoxic effects of VCR and/or MXT had no correlation with c-myc gene expression and DNA fragmentation was not observed. Flow cytometry revealed that while most cells (> 90%) exposed to VCR alone for 16-24 h were arrested at the G2/M phase, a fraction of cells were able to escape mitotic arrest when MXT was also present. These results suggest that the use of MXT in conjugation with VCR for the treatment of cancers should be applied with caution.

Therapy of streptozotocin induced diabetes with a bifunctional antibody that delivers vinca alkaloids to IL-2 receptor positive cells.

IVA039.1 is a bifunctional antibody with specificity for the murine IL-2 receptor and vinca alkaloids. Biodistribution studies show that IVA039.1 can target and deliver vinca alkaloids to tissues that contain IL-2 receptor positive cells. Vinca alkaloids are lymphocytotoxic. Therapy of diabetic mice with IVA039.1 plus vincristine results in a significant decrease in the glucose levels of diabetic compared to untreated mice. The therapeutic effect of IVA039.1 plus vincristine therapy was additive but surprisingly not synergistic. The binding of IVA039.1 to vincristine has moderate affinity with a slow off rate. In vitro studies suggest that, when bound to IVA039.1, the vincristine is inactivated. We attribute the lack of an enhanced therapeutic response to bifunctional antibody therapy using IVA039.1 plus vincristine to the inaccessibility of the drug to the target cells.

Nerve growth factor prevents neurotoxic effects of cisplatin, vincristine and taxol, on adult rat sympathetic ganglion explants in vitro.

Anti-cancer drugs, cisplatin, vincristine and taxol clinically induce toxic sensory as well as autonomic neuropathy. Administration of nerve growth factor (NGF) has been found to prevent experimental sensory neuropathies induced by these anti-cancer drugs, but the information about autonomic neuropathy is lacking. We developed an adult rat superior cervical ganglion (SCG) explant culture, which we treated with cisplatin, vincristine and taxol either in the presence or absence of NGF. The maximum length of regenerated neurites was shortened by cisplatin, vincristine and taxol in a dose-dependent manner. However cotreatment with NGF significantly promoted the regeneration of neurites in all drug-treated explants. The effect of NGF was clearly blocked by the anti-NGF antibody. These findings suggest that cotreatment of NGF prevents and reverses the toxic effects of the anti-cancer drugs on the sympathetic neurons.

Gangliosides attenuate vincristine neurotoxicity on dorsal root ganglion cells.

We investigated the effects of bovine brain gangliosides on the neurotoxicity of vincristine in dissociated cultures of dorsal root ganglion cells from 10-day chick embryos. The effects of the drugs were quantified as the numbers of neurite-bearing cells or total neurite length in individual neurite-bearing cells. The administration of vincristine (1 to 1000 pg/mL) inhibited neurite outgrowth from the cells, whereas gangliosides (10 to 1000 micrograms/mL) protected them against this inhibition in a concentration-dependent manner. Electron microscopy revealed vincristine-induced fragmentation and longitudinal disorientation of microtubules in neurites and showed the protection by gangliosides against such damaging effects. Our results show that exogenous administration of gangliosides attenuates the neurotoxicity of vincristine in vitro.

Inhibition of vincristine-induced retinal impairments by a specific PAF antagonist.

The alkaloid vincristine is widely used for its anti-leukemic and anti-tumor activity. However, the drug also displays considerable toxicity, particularly for the retina. Indeed, vincristine has been shown to induce alteration of photoreceptor outer segments in animals and impairment of scotopic vision in man. This type of retinopathy is an inflammatory disease in which PAF may be implicated and for which specific PAF antagonist may have a therapeutic role. Thus, we measured the effects of a new hetrapezine derived PAF antagonist, BN 50730, on a vincristine-induced retinopathy in the rat. Retinal impairments were established by recording several parameters of the electroretinogram (ERG) obtained from isolated retina. Our results indicate that, first, the increase in PIII duration induced by vincristine is significantly reduced by BN 50730 administration and, second, the decrease in the value of the PIII/b wave ratio caused by vincristine is partially inhibited by treatment with BN 50730. These experiments suggest that PAF is implicated in vincristine retinopathy and demonstrate the therapeutic effect of a specific antagonist of the mediator.

Expression of the mdr1 gene in human colorectal carcinomas: relationship with multidrug resistance inferred from analysis of human colorectal carcinoma cell lines.

To investigate whether mdr1 gene products are involved in conferring the chemoresistant phenotype to human colorectal carcinomas (HCCs), we determined the mdr1 mRNA expression level (mdr1 EL) in surgical specimens from 29 pharmacologically untreated patients and analyzed the relationship between mdr1 EL and drug resistance in an in vitro experimental model. This consisted of 7 HCC cell lines chosen to cover the range of mdr1 ELs detected in the neoplastic specimens. No relationship was observed between the mdr1 EL of the HCC cell lines and the degree of chemosensitivity found for each drug tested, regardless of whether mdr1 gene products may [doxorubicin (DOX), vincristine (VCR), and actinomycin-D (ACT-D)] or may not affect [cis-diamminedichloroplatinum (CDDP)] drug-transmembrane equilibria. Conversely, a direct relationship was found between the mdr1 EL of HCC cell lines and the number of drug-resistant (DR) colonies arising from each parent cell line treated in continuous culture with high DOX concentrations. In addition, the chemoresistance index and mdr1 EL of the DR cell variants were roughly proportional to the mdr1 EL of the parent cell line. Our findings suggest that primary HCCs derive multidrug resistance from biochemical mechanism(s) other than mdr1 gene products. However, the mdr1 EL might be indicative of a predisposition to develop DR cell variants after chemotherapeutic treatment.

Human multi-drug-resistant cancer cells exhibit a high degree of selectivity for stereoisomers of verapamil and quinidine.

An in vitro cell proliferation assay was developed to measure the capacity of substances to overcome multi-drug resistance (MDR). The assay is a modification of the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) method. The inclusion of cell titration curves for each concentration of the resistance modifier (RM) allows the IC50 of the RM to be calculated and provides empirical correction of the cell survival curves for the effect of the RM when it is combined with a standard cytotoxic drug, vincristine. The resistance modification index (RMI) is defined as the ratio of the IC50 of vincristine obtained in control cultures divided by that measured in the presence of RM and is linearly related to the dose of RM. The RMI0.1, the RMI at a one-tenth the IC50 of the RM, provides a relative comparison between the activities of different RMs at non-toxic doses. The results obtained using the MDR cell line, KB-8-5, show that l-(-)-verapamil is approximately 4 times more active than d-(+)-verapamil in modifying MDR. The racemic mixture has an intermediate activity. A similar comparison between the epimers quinidine and quinine shows that, at equimolar doses, quinine has a higher RMI but, because it is more toxic, the RMI0.1 is about one-half of that of quinidine. These results demonstrate the importance of comparing the resistance-modifying activities of different compounds at doses relative to their own toxicity.