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Side effects of chemotherapy are burning questions for physicians and patients involved in cancers. Ganoderma lucidum is a widely consumed traditional Chinese medicine and edible mushroom with multiple functional properties. The present study aims to investigate the potential of polysaccharides from spore of G. lucidum (SGP) on small intestinal barrier function recovery against paclitaxel (PTX) challenge in a breast cancer mice model and IEC-6 cell line. The 4T1 tumor-bearing mice were treated with PTX together with four-week daily oral administration of SGP. Results indicated that combination of PTX and SGP reversed body weight lost and remolded the histology of small intestine, accompanied with promoted proliferation but suppressed apoptosis in intestinal cells. Intestinal barrier function was enhanced by the combination as indicated by reduced endotoxemia and the up-regulation of tight junction proteins, including Zonula occludens-1 (ZO-1), E-cadherin, β-catenin and Occludin. The protection of SGP was further confirmed in IEC-6 cells affected by PTX in vitro. The combination treatment prevented PTX-induced apoptosis in IEC-6 by inhibiting microtubule polymerization, and the aforementioned tight junction proteins were also upregulated. These findings suggest a promising protective effect of SGP against small intestinal barrier injury caused by PTX, highlighting its clinical implication against the chemotherapy side effects., (Copyright © 2020 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Caffeine (1,3,7-trimethylxanthine) is a xanthine alkaloid found in a number of dietary products consumed worldwide, such as coffee, tea, and soft beverages, and is known to act as a modifying agent for cytotoxic chemotherapeutic drugs. Studies have shown that caffeine reduces the cytotoxic effects of paclitaxel and inhibits paclitaxel-induced apoptosis; however, the underlying mechanism remains unclear. Here, we investigated whether caffeine inhibits the antitumor activity of paclitaxel via down-regulation of α-tubulin acetylation. In vitro studies, involving MTT assay, wound-healing assay, cell apoptosis assay, and western blotting analysis of A549 and HeLa cells, were performed. A549 and HeLa cell-based xenografts were established, and western blotting and immunohistochemical staining were performed for in vivo studies. The results showed that caffeine promoted the growth of cancer cells treated with paclitaxel. Additionally, caffeine enhanced migration ability, inhibited apoptosis, and decreased the acetylation of α-tubulin in paclitaxel-treated cancer cells. Furthermore, caffeine decreased the inhibitory effect of paclitaxel on tumor growth through down-regulation of α-tubulin acetylation in vivo. Taken together, these findings demonstrate that caffeine inhibits the anticancer activity of paclitaxel via down-regulation of α-tubulin acetylation, suggesting that patients receiving treatment with taxanes, such as paclitaxel, should avoid consuming caffeinated beverages or foods., (Copyright © 2020 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Using DNA comet assay we found that polysaccharides from Tussilago farfara L. reduced the intensity of polychemotherapy-induced apoptosis and DNA damage in bone marrow cells and small intestinal epithelium of C57Bl/6 mice, which attested to genoprotective properties of these polysaccharides.

Given our recent evidence for the role of high mobility group box 1 (HMGB1) in chemotherapy-induced peripheral neuropathy (CIPN) in rats, we examined the origin of HMGB1 and the upstream and downstream mechanisms of HMGB1 release involved in paclitaxel-induced neuropathy in mice. Paclitaxel treatment developed mechanical allodynia in mice, as assessed by von Frey test, which was prevented by an anti-HMGB1-neutralizing antibody or thrombomodulin alfa capable of inactivating HMGB1. RAGE or CXCR4 antagonists, ethyl pyruvate or minocycline, known to inhibit HMGB1 release from macrophages, and liposomal clodronate, a macrophage depletor, prevented the paclitaxel-induced allodynia. Paclitaxel caused upregulation of RAGE and CXCR4 in the dorsal root ganglia and macrophage accumulation in the sciatic nerve. In macrophage-like RAW264.7 cells, paclitaxel evoked cytoplasmic translocation of nuclear HMGB1 followed by its extracellular release, and overexpression of CBP and PCAF, histone acetyltransferases (HATs), known to cause acetylation and cytoplasmic translocation of HMGB1, which were suppressed by ethyl pyruvate, N-acetyl-l-cysteine, an anti-oxidant, and SB203580 and PDTC, inhibitors of p38 MAP kinase (p38MAPK) and NF-κB, respectively. Paclitaxel increased accumulation of reactive oxygen species (ROS) and phosphorylation of p38MAPK, NF-κB p65 and I-κB in RAW264.7 cells. In mice, N-acetyl-l-cysteine or PDTC prevented the paclitaxel-induced allodynia. Co-culture of neuron-like NG108-15 cells or stimulation with their conditioned medium promoted paclitaxel-induced HMGB1 release from RAW264.7 cells. Our data indicate that HMGB1 released from macrophages through the ROS/p38MAPK/NF-κB/HAT pathway participates in the paclitaxel-induced peripheral neuropathy in mice, and unveils an emerging therapeutic avenue targeting a neuroimmune crosstalk in CIPN., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Drug resistance is a major problem in cancer therapy. A growing body of evidence demonstrates that the tumor microenvironment, including cancer-associated fibroblasts (CAFs), can modulate drug sensitivity in tumor cells. We examined the effect of primary human CAFs on p53 induction and cell viability in prostate cancer cells on treatment with chemotherapeutic drugs. Co-culture with prostate CAFs or CAF-conditioned medium attenuated DNA damage and the p53 response to chemotherapeutic drugs and enhanced prostate cancer cell survival. CAF-conditioned medium inhibited the accumulation of doxorubicin, but not taxol, in prostate cancer cells in a manner that was associated with increased cancer cell glutathione levels. A low molecular weight fraction (<3 kDa) of CAF-conditioned medium had the same effect. CAF-conditioned medium also inhibited induction of reactive oxygen species (ROS) in both doxorubicin- and taxol-treated cancer cells. Our findings suggest that CAFs can enhance drug resistance in cancer cells by inhibiting drug accumulation and counteracting drug-induced oxidative stress. This protective mechanism may represent a novel therapeutic target in cancer.

Paclitaxel is a chemotherapeutic agent that causes peripheral neuropathy as its major dose-limiting side effect. However, the peripheral neuropathy is difficult to manage. A study we recently conducted showed that repetitive administration of aucubin as a prophylactic inhibits paclitaxel-induced mechanical allodynia. However, the mechanisms underlying the anti-allodynic activity of aucubin, which is a major component of Plantaginis Semen, was unclear. In addition to mechanical allodynia, aucubin inhibited spontaneous and mechanical stimuli-induced firing in spinal dorsal horn neurons; however, catalpol, a metabolite of aucubin, did not show these effects. Furthermore, paclitaxel induced the expression of CCAAT/enhancer-binding protein homologous protein, a marker of endoplasmic reticulum (ER) stress, in the sciatic nerve and a Schwann cell line (LY-PPB6 cells); however, this effect was inhibited by aucubin. These results suggest that aucubin inhibits paclitaxel-induced mechanical allodynia through the inhibition of ER stress in peripheral Schwann cells.

Methylsulfinylmethane (dimethyl sulfoxide; DMSO) is widely used in clinical treatment and bioresearch. Moreover, there is bioconversion between methylsulfanylmethane (dimethyl sulfide; DMS), DMSO, and methylsulfonylmethane (DMSO2) in mammalian metabolism. Due to the real-time detection limits for volatile compounds, most research has focused on DMSO2 as a stable byproduct of DMSO. Therefore, details about the production of DMS as a byproduct of DMSO metabolism remain to be elucidated. Here, we report the characterization of trace-level volatile organic compounds (VOCs) produced following DMSO treatment of cultured human cells using an ultrasensitive vacuum ultraviolet photoionization mass spectrometer (VUV-PIMS). Using this approach, 24 h after DMSO treatment we detected 16.9 and 21 parts per billion by volume (ppbv) DMS in the atmosphere above the cells (headspace) within HeLa and 293T tissue culture flasks, respectively. When simultaneously exposed to 50 nM paclitaxel (PTX), 17.6 and 22.3 ppbv DMS were detected in the headspace of HeLa and 293T culture flasks, respectively. Nevertheless, at doses of PTX more or less than 50 nM, the detectable levels of DMS were reduced to as low as 8.4 ppbv. Our experimental results demonstrate that by co-administering 5 to 10 nM PTX with DMSO, it is possible to moderate the production of DMS considerably. However, at higher doses of PTX, increased apoptosis was observed that likely contributed to higher DMS production by cells.

Peripheral neuropathy is dose limiting in paclitaxel cancer chemotherapy and can result in both acute pain during treatment and chronic persistent pain in cancer survivors. The hypothesis tested was that paclitaxel produces these adverse effects at least in part by sensitizing transient receptor potential vanilloid subtype 1 (TRPV1) through Toll-like receptor 4 (TLR4) signaling. The data show that paclitaxel-induced behavioral hypersensitivity is prevented and reversed by spinal administration of a TRPV1 antagonist. The number of TRPV1(+) neurons is increased in the dorsal root ganglia (DRG) in paclitaxel-treated rats and is colocalized with TLR4 in rat and human DRG neurons. Cotreatment of rats with lipopolysaccharide from the photosynthetic bacterium Rhodobacter sphaeroides (LPS-RS), a TLR4 inhibitor, prevents the increase in numbers of TRPV1(+) neurons by paclitaxel treatment. Perfusion of paclitaxel or the archetypal TLR4 agonist LPS activated both rat DRG and spinal neurons directly and produced acute sensitization of TRPV1 in both groups of cells via a TLR4-mediated mechanism. Paclitaxel and LPS sensitize TRPV1 in HEK293 cells stably expressing human TLR4 and transiently expressing human TRPV1. These physiological effects also are prevented by LPS-RS. Finally, paclitaxel activates and sensitizes TRPV1 responses directly in dissociated human DRG neurons. In summary, TLR4 was activated by paclitaxel and led to sensitization of TRPV1. This mechanism could contribute to paclitaxel-induced acute pain and chronic painful neuropathy. Significance statement: In this original work, it is shown for the first time that paclitaxel activates peripheral sensory and spinal neurons directly and sensitizes these cells to transient receptor potential vanilloid subtype 1 (TRPV1)-mediated capsaicin responses via Toll-like receptor 4 (TLR4) in multiple species. A direct functional interaction between TLR4 and TRPV1 is shown in rat and human dorsal root ganglion neurons, TLR4/TRPV1-coexpressing HEK293 cells, and in both rat and mouse spinal cord slices. Moreover, this is the first study to show that this interaction plays an important role in the generation of behavioral hypersensitivity in paclitaxel-related neuropathy. The key translational implications are that TLR4 and TRPV1 antagonists may be useful in the prevention and treatment of chemotherapy-induced peripheral neuropathy in humans., (Copyright © 2015 the authors 0270-6474/15/3513488-14$15.00/0.)

Background and Purpose: Paclitaxel (PAC) is associated with chemotherapy-induced neuropathic pain (CIPN) that can lead to the cessation of treatment in cancer patients even in the absence of alternate therapies. We previously reported that chronic administration of the non-psychoactive cannabinoid cannabidiol (CBD) prevents PAC-induced mechanical and thermal sensitivity in mice. Hence, we sought to determine receptor mechanisms by which CBD inhibits CIPN and whether CBD negatively effects nervous system function or chemotherapy efficacy., Experimental Approach: The ability of acute CBD pretreatment to prevent PAC-induced mechanical sensitivity was assessed, as was the effect of CBD on place conditioning and on an operant-conditioned learning and memory task. The potential interaction of CBD and PAC on breast cancer cell viability was determined using the MTT assay., Key Results: PAC-induced mechanical sensitivity was prevented by administration of CBD (2.5 - 10 mg·kg⁻¹) in female C57Bl/6 mice. This effect was reversed by co-administration of the 5-HT(1A) antagonist WAY 100635, but not the CB₁ antagonist SR141716 or the CB₂ antagonist SR144528. CBD produced no conditioned rewarding effects and did not affect conditioned learning and memory. Also, CBD + PAC combinations produce additive to synergistic inhibition of breast cancer cell viability., Conclusions and Implications: Our data suggest that CBD is protective against PAC-induced neurotoxicity mediated in part by the 5-HT(1A) receptor system. Furthermore, CBD treatment was devoid of conditioned rewarding effects or cognitive impairment and did not attenuate PAC-induced inhibition of breast cancer cell viability. Hence, adjunct treatment with CBD during PAC chemotherapy may be safe and effective in the prevention or attenuation of CIPN., (© 2013 The British Pharmacological Society.)

Objective: Peripheral neurotoxicity is a major dose-limiting side effect of many chemotherapeutic drugs. Currently there are no effective disease-modifying therapies for chemotherapy-induced peripheral neuropathies, but these side effects of chemotherapy are potentially ideal targets for development of neuroprotective therapies, because candidate drugs can be co- or preadministered before the injury to peripheral axons takes place., Methods: We used a phenotypic drug screening approach to identify ethoxyquin as a potential neuroprotective drug and carried out additional biochemical experiments to identify its mechanism of action., Results: We validated the screening results with ethoxyquin and its derivatives and showed that they prevented paclitaxel-induced peripheral neuropathy without blocking paclitaxel's ability to kill tumor cells. Furthermore, we demonstrated that ethoxyquin acts by modulating the chaperone activity of heat shock protein 90 (Hsp90) and blocking the binding of 2 of its client proteins, ataxin-2 and Sf3b2. Ethoxyquin-induced reduction in levels of both of these proteins resulted in prevention of axonal degeneration caused by paclitaxel., Interpretation: Ethoxyquin and its novel derivatives as well as other classes of small molecules that act as Hsp90 modulators may offer a new opportunity for development of drugs to prevent chemotherapy-induced axonal degeneration., (© 2013 American Neurological Association.)

Besides its prominent role in angiogenesis, the vascular endothelial growth factor (VEGF) also exerts important protective effects on neurons. In particular, mice expressing reduced levels of VEGF suffer from late-onset motor neuron degeneration, whereas VEGF delivery significantly delays motor neuron death in ALS mouse models, at least partly through neuroprotective effects. Additionally, VEGF protects dorsal root ganglion (DRG) neurons against paclitaxel-induced neurotoxicity. Here, we demonstrate that VEGF also protects DRG neurons against hyperglycemia-induced neuronal stress as a model of diabetes-induced peripheral neuropathy. Specifically, VEGF decreased expression of the stress-related gene activating transcription factor 3 (ATF3) in DRG neurons isolated from streptozotocin-induced diabetic mice (ex vivo) and in isolated DRG neurons exposed to high glucose concentrations (in vitro). In vivo, local VEGF application also protected against paclitaxel- and diabetes-induced neuropathies without causing side effects. A small synthetic VEGF mimicking pentadecapeptide (QK) exerted similar effects on DRG cultures: the peptide reduced ATF3 expression in vitro and ex vivo in paclitaxel- and hyperglycemia-induced models of neuropathy to a similar extent as the full-length recombinant VEGF protein. By using transgenic mice selectively overexpressing the VEGF receptor 2 in postnatal neurons, these neuroprotective effects were shown to be mediated through VEGF receptor 2. Overall, these results underscore the potential of VEGF and VEGF-derived peptides for the treatment of peripheral neuropathies., (Copyright © 2013 IBRO. Published by Elsevier Ltd. All rights reserved.)

Background: Peripheral neuropathy is a major dose-limiting toxicity of chemotherapy, especially after multiple courses of paclitaxel. The development of paclitaxel-induced neuropathy is associated with the activation of microglia followed by the activation and proliferation of astrocytes, and the expression and release of proinflammatory cytokines in the spinal dorsal horn. Cannabinoid type 2 (CB(2)) receptors are expressed in the microglia in neurodegenerative disease models., Methods: To explore the potential of CB(2) agonists for preventing paclitaxel-induced neuropathy, we designed and synthesized a novel CB(2)-selective agonist, namely, MDA7. The effect of MDA7 in preventing paclitaxel-induced allodynia was assessed in rats and in CB(2)(+/+) and CB(2)(-/-) mice. We hypothesized that the CB(2) receptor functions in a negative-feedback loop and that early MDA7 administration can blunt the neuroinflammatory response to paclitaxel and prevent mechanical allodynia through interference with specific signaling pathways., Results: We found that MDA7 prevents paclitaxel-induced mechanical allodynia in rats and mice in a dose- and time-dependent manner without compromising paclitaxel's antineoplastic effect. MDA7's neuroprotective effect was absent in CB(2)(-/-) mice and was blocked by CB(2) antagonists, suggesting that MDA7's action directly involves CB(2) receptor activation. MDA7 treatment was found to interfere with early events in the paclitaxel-induced neuroinflammatory response as evidenced by relatively reduced toll-like receptor and CB(2) expression in the lumbar spinal cord, reduced levels of extracellular signal-regulated kinase 1/2 activity, reduced numbers of activated microglia and astrocytes, and reduced secretion of proinflammatory mediators in vivo and in in vitro models., Conclusions: Our findings suggest an innovative therapeutic approach to prevent chemotherapy-induced neuropathy and may permit more aggressive use of active chemotherapeutic regimens with reduced long-term sequelae.

Patients with estrogen receptor (ER)-positive breast cancers have a better prognosis than those with ER-negative breast cancers, but often have low sensitivity to chemotherapy and a limited survival benefit. We have previously shown a combination effect of taxanes and fulvestrant and suggested that this treatment may be useful for ER-positive breast cancer. In this study, we evaluated the effects of combinations of hormone drugs and chemotherapeutic agents. In vitro, the effects of combinations of five chemotherapeutic agents (doxorubicin, paclitaxel, docetaxel, vinorelbine, and 5-fluorouracil) and three hormone drugs (fulvestrant, tamoxifen, and 4-hydroxytamoxifen) were examined in ER-positive breast cancer cell lines using CalcuSyn software. Changes in chemoresistant factors such as Bcl2, multidrug resistance-associated protein 1, and microtubule-associated protein tau were also examined after exposure of the cells to hormone drugs. In vivo, tumor sizes in mice were evaluated after treatment with docetaxel or doxorubicin alone, fulvestrant alone, and combinations of these agents. Combination treatment with fulvestrant and all five chemotherapeutic agents in vitro showed synergistic effects. In contrast, tamoxifen showed an antagonistic effect with all the chemotherapeutic agents. 4-Hydroxytamoxifen showed an antagonistic effect with doxorubicin and 5-fluorouracil, but a synergistic effect with taxanes and vinorelbine. Regarding chemoresistant factors, Bcl2 and microtubule-associated protein tau were downregulated by fulvestrant. In vivo, a combination of fulvestrant and docetaxel had a synergistic effect on tumor growth, but fulvestrant and doxorubicin did not show this effect. In conclusion, fulvestrant showed good compatibility with all the evaluated chemotherapeutic agents, and especially with docetaxel, in vitro and in vivo., (© 2011 Japanese Cancer Association.)

Loss of intraepidermal nerve fibers (IENFs) has been speculated to play a critical role in the development of various neuropathies. In this study, the density of IENFs were studied over time during the induction of Taxol (Bristol-Myers Squibb, NY, USA)-induced chemoneuropathy and compared with the changes in IENFs in animals co-treated with Taxol plus the protective agent minocycline. Rats were injected (intraperitoneally) with 2mg/kg of Taxol every other day for four injections (day 1, 3, 5, and 7). Minocycline (25mg/kg) was given in a separate group of rats 24h prior to the first dose of Taxol and every day for the next 9days (day 0 through 9). Animals were tested for mechanical paw withdrawal thresholds prior to any drug administrations and again on day 7, 14, and 30. Immunohistochemistry using the pan-neuronal marker protein gene product 9.5 was performed on glabrous skin of the hind-paw foot pad to stain for IENFs also on day 7, 14, and 30. The results show that Taxol-treated animals developed mechanical sensitivity and corresponding IENF loss. Animals receiving minocycline plus Taxol showed no hyperalgesia or loss of IENFs. This study confirms, for the first time, that a loss of IENFs occurs as a neuropathy develops, and further shows a protection against both IENF loss and hyperalgesia with minocycline treatment. The progression of Taxol-induced mechanical hypersensitivity coincides with loss of intraepidermal nerve fibers, and the hyperalgesia and nerve fiber loss were prevented with minocycline treatment., (Copyright © 2010 International Association for the Study of Pain. Published by Elsevier B.V. All rights reserved.)

The aim was to determine the potential of the allosteric mammalian target of rapamycin inhibitor, everolimus, to act in combination with cytotoxic anticancer compounds in vitro and in vivo. A concomitant combination in vitro showed no evidence of antagonism, but enhanced the antiproliferative effects (additive to synergistic) with cisplatin, doxorubicin, 5-fluorouracil, gemcitabine, paclitaxel, and patupilone. Everolimus (1-5 mg/kg/d orally) was evaluated for antitumor activity in vivo alone or in combination with suboptimal cytotoxic doses using athymic nude mice bearing subcutaneous human H-596 lung, KB-31 cervical, or HCT-116 colon tumor xenografts. Everolimus monotherapy was very well tolerated and caused inhibition of tumor growth, rather than regression, and this was associated with a dose-dependent decline in tumor pS6 levels, a key downstream protein of mammalian target of rapamycin. At the doses used, the cytotoxics inhibited tumor growth and caused tolerable body-weight loss. Concomitant combinations of cisplatin, doxorubicin, paclitaxel, or patupilone with everolimus produced cooperative antitumor effects, in some cases producing regressions without clinically significant increases in toxicity. In contrast, combinations with gemcitabine and 5-fluorouracil were less well tolerated. Alternative administration schedules were tested for cisplatin, gemcitabine, or paclitaxel combined with everolimus: these did not dramatically affect cisplatin or gemcitabine activity or tolerability but were antagonistic for paclitaxel. Everolimus showed promising maintenance activity after treatment with doxorubicin or paclitaxel ceased. Overall, the results confirm that everolimus is an effective, well-tolerated suppressor of experimental human tumor growth, and although it did not show strong potentiation of efficacy, antitumor activity in vivo was increased without marked increases in toxicity, supporting clinical use of everolimus as a partner for conventional cytotoxics.

Resveratrol, which is highly concentrated in the skin of grapes and is abundant in red wine, has been demonstrated to account for several beneficial properties, including antioxidant, anticoagulant, anti-inflammatory and anticancer effects. Taxol is a microtubule-stabilizing drug that has been extensively used as effective chemotherapeutic agents in the treatment of solid tumors. Here, we investigated whether the combination of the two compounds would yield increased antitumor efficacy in human cancer cells. Unexpectedly, resveratrol effectively prevented tumor cell death induced by taxol in 5637 bladder cancer cells. This pronounced antagonistic function of resveratrol against taxol was associated with changes in multiple signal transduction pathways, but not with tubulin polymerization. Importantly, cell cycle analysis showed that resveratrol prevented the cells from entering into mitosis, the phase in which taxol exerts its action. Furthermore, resveratrol blocked the cytotoxic effects of vinblastine but not cisplatin in 5637 cells. Interestingly, resveratrol pre-treatment followed by taxol resulted in synergistic cytotoxicity. Finally, we extended our studies to various human cancer cell lines. Taken together, our results indicate that resveratrol may have the potential to negate the therapeutic efficacy of taxol and suggest that consumption of resveratrol-related products may be contraindicated during cancer therapy with taxol.

It was reported recently that resveratrol could sensitise a number of cancer cell lines to the anticancer actions of several other cancer drugs, including paclitaxel. In the present study, we further investigated whether resveratrol could sensitise human breast cancer cells to paclitaxel-induced cell death. Unexpectedly, we found that resveratrol strongly diminished the susceptibility of MDA-MB-435s, MDA-MB-231 and SKBR-3 cells to paclitaxel-induced cell death in culture, although this effect was not observed in MCF-7 cells. Using MDA-MB-435s cells as a representative model, a similar observation was made in athymic nude mice. Mechanistically, the modulating effect of resveratrol was partially attributable to its inhibition of paclitaxel-induced G(2)/M cell cycle arrest, together with an accumulation of cells in the S-phase. In addition, resveratrol could suppress paclitaxel-induced accumulation of reactive oxygen species (ROS) and subsequently the inactivation of anti-apoptotic Bcl-2 family proteins. These observations suggest that the strategy of concomitant use of resveratrol with paclitaxel is detrimental in certain types of human cancers. Given the widespread use of resveratrol among cancer patients, this study calls for more preclinical and clinical testing of the potential benefits and harms of using resveratrol as a dietary adjuvant in cancer patients., (Copyright 2010 Elsevier Ltd. All rights reserved.)

Purpose: Ovarian cancer recurrence with the development of paclitaxel resistance is an obstacle to long-term survival. We showed that secretory leukocyte protease inhibitor (SLPI) is a survival factor for ovarian cancer. We hypothesize that SLPI may antagonize paclitaxel injury., Experimental Design: Differential SLPI induction in response to paclitaxel and in response to stable forced expression of SLPI was shown in A2780-1A9 cells and their paclitaxel-resistant sublines, PTX10 and PTX22, and confirmed with HEY-A8 cells. SLPI-mediated survival was reduced by the MAP/extracellular signal-regulated kinase (ERK) kinase inhibitor, U0126, and a humanized neutralizing monoclonal anti-SLPI antibody, CR012. OVCAR3 xenographs tested the role of CR012 in vivo., Results: SLPI expression was lower in A2780-1A9 ovarian cancer cells than in PTX10 and PTX22, and SLPI was induced by paclitaxel exposure. Stable SLPI expression yielded a proliferation advantage (P = 0.01); expression of and response to SLPI in OVCAR3 cells were abrogated by exposure to CR012. SLPI reduced the paclitaxel susceptibility of 1A9 and HEY-A8 cells (P

Quercetin is a flavonoid with anticancer properties. In this study, we examined the effects of quercetin on cell cycle, viability, and proliferation of cancer cells, either singly or in combination with the microtubule-targeting drugs taxol and nocodazole. Although quercetin induced cell death in a dose-dependent manner, 12.5-50 μM quercetin inhibited the activity of both taxol and nocodazole to induce G2/M arrest in various cell lines. Quercetin also partially restored drug-induced loss in viability of treated cells for up to 72 h. This antagonism of microtubule-targeting drugs was accompanied by a delay in cell cycle progression and inhibition of the buildup of cyclin-B1 at the microtubule organizing center of treated cells. However, quercetin did not inhibit the microtubule targeting of taxol or nocodazole. Despite the short-term protection of cells by quercetin, colony formation and clonogenicity of HCT116 cells were still suppressed by quercetin or quercetin-taxol combination. The status of cell adherence to growth matrix was critical in determining the sensitivity of HCT116 cells to quercetin. We conclude that although long-term exposure of cancer cells to quercetin may prevent cell proliferation and survival, the interference of quercetin with cell cycle progression diminishes the efficacy of microtubule-targeting drugs to arrest cells at G2/M.

Background: Tetracyclines such as doxycycline are reported to possess cytotoxic activity against mammalian tumor cells, but the mechanism of their effects on cell proliferation remains unclear., Materials and Methods: The antitumor effect of doxycycline was investigated in human pancreatic cancer cell line, PANC-1. We also investigated the effect of doxycycline on expression of a potent proangiogenic factor, interleukin (IL)-8., Results: In excess of 20 microg/ml, cytotoxic effects of doxycycline were accompanied by G(1)-S cell cycle arrest and DNA fragmentation in PANC-1 cells. Doxycycline consistently activated transcription of p53, p21 and Fas/FasL-cascade-related genes, while reducing the expression of Bcl-xL and Mcl-1. Doxycycline (5 microg/ml) below the cytotoxic level suppressed endogenous and paclitaxel-induced IL-8 expression. In the mouse xenograft model, doxycycline treatment was shown to suppress tumor growth by 80%., Conclusion: These data suggest that doxycycline exerts its antitumor effect by activating proapoptotic genes, inhibiting IL-8 expression, and suppressing antiapoptotic genes.

Most anticancer agents activate nuclear factor kappa B (NF-kappaB), which can mediate cell survival, proliferation, and metastasis. Curcumin has been shown to inhibit the growth of various cancer cells, without toxicity to normal cells. The antitumor effects of curcumin could be due in part to the inactivation of NF-kappaB. We hypothesize that blocking NF-kappaB activity may augment paclitaxel cancer chemotherapy. In this study, we investigated whether the inactivation of NF-kappaB by curcumin would enhance the efficacy of paclitaxel for inhibiting breast cancer growth in vitro and in vivo. We confirmed that curcumin inhibited paclitaxel-induced activation of NF-kappaB and potentiated the growth inhibitory effect of paclitaxel in MDA-MB-231 breast cancer cells. The combination of curcumin with paclitaxel elicited significantly greater inhibition of cell growth and more apoptosis, compared with either agent alone. In an experimental breast cancer murine model using MDA-MB-231 cells, combination therapy with paclitaxel and curcumin significantly reduced tumor size and decreased tumor cell proliferation, increased apoptosis, and decreased the expression of matrix metalloprotease 9 compared with either agent alone. These results clearly suggest that a curcumin-paclitaxel combination could be a novel strategy for the treatment of breast cancer.

GDP inhibits paclitaxel-induced tubulin assembly without GTP when the tubulin bears GDP in the exchangeable site (E-site). Initially, we thought inhibition was mediated through the E-site, since small amounts of GTP or Mg(2+), which favors GTP binding to the E-site, reduced inhibition by GDP. We thought trace GTP released from the nonexchangeable site (N-site) by tubulin denaturation was required for polymer nucleation, but microtubule length was unaffected by GDP. Further, enhancing polymer nucleation reduced inhibition by GDP. Other mechanisms involving the E-site were eliminated experimentally. Upon finding that ATP weakly inhibited paclitaxel-induced assembly, we concluded that another ligand binding site was responsible for these inhibitory effects, and we found that GDP was not binding at the taxoid, colchicine, or vinca sites. There may therefore be a lower affinity site on tubulin to which GDP can bind distinct from the E- and N-sites, possibly on alpha-tubulin, based on molecular modeling studies.

Paclitaxel is a commonly used anticancer drug, but it frequently causes peripheral neuropathy. Neurotropin, a non-protein extract from inflamed rabbit skin inoculated with vaccinia virus, has been used to treat various chronic painful conditions. In the present study, we investigated the effect of neurotropin on the paclitaxel-induced neuropathy in rats. Repeated administration of paclitaxel induced mechanical allodynia, cold hyperalgesia, and motor dysfunction. These neuropathies were mostly reversed by the repeated administration of neurotropin. Furthermore, neurotropin ameliorated the paclitaxel-induced axonal degeneration in cultured PC12 and rat dorsal root ganglion cells, and in rat sciatic nerve. In addition, neurotropin did not affect the microtubule aggregation or anti-tumour effect induced by paclitaxel in the tumour cell lines or tumour cells-implanted mice. These results suggest that neurotropin reverses the paclitaxel-induced neuropathy without affecting anti-tumour activity of paclitaxel, and therefore may be useful for the paclitaxel-induced neuropathy in clinical settings.

Objectives: Paclitaxel is widely used in cancer chemotherapy for the treatment of solid tumors such as breast, ovarian and lung cancer. However, it sometimes induces moderate to severe muscle pain, and impairs the patients' quality of life. An appropriate method for relieving this pain is not well established. Shakuyaku-kanzo-to, a herbal medicine, is known to relieve menstrual pain, muscle spasm, and muscle pain, and its effectiveness is expected. To ascertain the effectiveness of Shakuyaku-kanzo-to on paclitaxel-induced pain, we investigated the effects of Shakuyaku-kanzo-to and its constituent herbal medicines in a mouse model., Methods: Seven-week-old male ddY mice were used. To make a mouse model of paclitaxel-induced pain, different single, intraperitoneally injected doses of this drug were tested in various groups of mice, and the optimal dose was determined. To estimate the effects of Shakuyaku-kanzo-to, the constituent herbal medicines Shakuyaku and Kanzo, and loxoprofen sodium as a non-steroidal anti-inflammatory drug on paclitaxel-induced pain, mechanical allodynia and hyperalgesia of the hind paw were assessed., Results: Paclitaxel administered at a dose of 10mg/kg or more produced allodynia and hyperalgesia; the time courses were similar to those of pain after paclitaxel administration in cancer patients. Shakuyaku-kanzo-to significantly relieved the allodynia and hyperalgesia induced by paclitaxel (10mg/kg). Shakuyaku and Kanzo inhibited the allodynia and hyperalgesia to some extent, but not significantly, while loxoprofen sodium was without effects., Conclusions: A single administration of paclitaxel (10mg/kg) produced allodynia and hyperalgesia in mice, suggesting that it could be used as an animal model resembling the painful conditions observed in humans medicated with this drug. Using this model, Shakuyaku-kanzo-to was shown to relieve paclitaxel-induced painful peripheral neuropathy.

Ornithine decarboxylase (ODC), the rate-limiting enzyme of the polyamine biosynthetic pathway, plays an important role in cell cycle, tumor promotion and anti-apoptosis. In our previous studies, overexpression of ODC prevented apoptosis induced by tumor necrosis factor-alpha and methotrexate. We further investigated the apoptotic mechanisms of the cancer chemotherapeutic drugs, including etoposide (VP-16), paclitaxel (TAX) and cisplatin (CDDP), and the influences of ODC on apoptosis and cell cycle. Our results showed that the investigated drugs induced caspase-dependent apoptosis, the generation of reactive oxygen species (ROS) and the disruption of mitochondrial membrane potential (Deltapsi m) in HL-60 cells, all of which were reversed by putrescine, glutathione or N-acetyl-l-cysteine. Overexpression of ODC prevented the cancer chemotherapeutic drugs-induced apoptosis, ROS generation and the disruption of Deltapsi m. After drug administrations, the decline of Bcl-2, cytochrome c release and caspases' activation were inhibited by ODC overexpression. In cell cycle, ODC overexpressed cells seemed to overcome the G1 arrest and G2/M arrest, caused by VP-16 and TAX, respectively, and kept on the cell cycle rolling. Overexpression of ODC increased the expression of Cyclin A, D, E and Cdk4 and the enzyme activity of Cdk1 and Cdk2 after the treatment of VP-16 and TAX, respectively. In conclusions, the cancer chemotherapeutic drugs-induced apoptosis is through ROS-related, mitochondria-mediated and caspase-dependent pathways. With higher ODC activity, cells are resistant to the cancer chemotherapeutic drugs-induced apoptosis and keep on the cell cycle rolling with the significant interference in G1/S arrest caused by VP-16 and G2/M arrest by TAX.

Multidrug resistance (MDR), which is a significant impediment to the success of cancer chemotherapy, is attributable to the overexpression of membrane transport proteins, such as P-glycoprotein (P-gp), resulting in an increased drug efflux. In this study, we show that the histone deacetylase (HDAC) inhibitor apicidin leads to resistance of HeLa cells to paclitaxel through the induction of P-gp expression. Furthermore, apicidin dramatically increases the release of a fluorescent P-gp substrate, rhodamine 123, from cells. In parallel, apicidin resistance to the apoptotic potential of paclitaxel is associated with induction of P-gp expression in HeLa cells, as evidenced by specific inhibition of P-gp function using either the pharmacological inhibitor verapamil or RNA silencing. We also demonstrate the contribution of apicidin-induced functional P-gp expression to drug resistance using KB cells. Failure of P-gp induction by apicidin does not reverse paclitaxel-induced cytotoxicity in the cells. Although HDAC inhibitors are widely appreciated as a new class of anti-tumor agent, our findings clearly demonstrate that apicidin treatment may lead to P-gp-mediated resistance to other anti-tumor agents, suggesting a need for careful design of clinical applications using HDAC inhibitors.

The marked clinical anticancer activity of the paclitaxel (PTX) and gemcitabine (GEM) combination has suggested that the two drugs may interact more than additively. We have analyzed the in vitro growth and molecular interactions of the two chemotherapy drugs in a panel of human breast cancer cells. We evaluated cell viability in four breast cancer cell lines (i.e., MCF-7, MDA-MB-231, MDA-MB-468, and SKBR3) that were treated with PTX and GEM combined either simultaneously (PTX + GEM) or sequentially (PTX --> GEM; GEM --> PTX). PTX-GEM interactions at the cellular level were assessed mathematically employing both the isobologram analysis (Berenbaum) and the combination index (Chou-Talalay) method. PTX-GEM molecular interactions on the apoptotic markers PARP, Bcl-2 and Bax were analyzed by immunoblotting procedures. Apoptosis was detected using a DNA ladder assay. We observed significant synergistic growth inhibitory interactions when PTX was administered before GEM. Additive interactions were observed when both the simultaneous regimen and the GEM followed by PTX regimen were used. DNA ladder and Western blotting results in the PTX followed by GEM sequence revealed a significant increase in the apoptotic cell death of breast cancer cells related to the Bax/Bcl-2 apoptotic pathway. In summary, the occurrence of clinically relevant synergism between PTX and GEM suggests a sequence-dependent nature in human breast cancer cells. This synergistic interaction on the PTXright curved arrow GEM schedule appears to be related to an increase in the Bcl-2-related mitochondrial apoptotic pathway. The synergism that we have observed may explain the favorable clinical responses that have been achieved in clinical studies, in which patients are administered PTX first, and then GEM.

The combination of carboplatin and paclitaxel is widely used to treat multiple solid tumors including ovarian, lung and breast cancer. Usually these drugs are given simultaneously with little regard to the importance of scheduling to obtain a maximal response. To investigate the importance of sequencing, the human breast Bcap37 and ovarian OV2008 cancer cell lines were exposed to carboplatin and paclitaxel in three different sequences: (1) pretreatment with paclitaxel followed by carboplatin, (2) pretreatment of carboplatin followed by paclitaxel and (3) simultaneous treatment with these two agents. The combination of carboplatin and paclitaxel resulted in antagonistic interactions when tumor cells were exposed to carboplatin prior to paclitaxel or exposed to the two drugs simultaneously, but there was little antagonistic interaction observed when paclitaxel was administered before carboplatin. Biochemical examination revealed that pretreatment or cotreatment of carboplatin inhibited paclitaxel-induced IkappaBalpha degradation and bcl-2 phosphorylation. Further analyses demonstrated that carboplatin could significantly interfere with the cytotoxic effects of paclitaxel on both mitotic arrest and apoptotic cell death unless paclitaxel was administered before carboplatin. These results indicate that the interaction between paclitaxel and carboplatin is highly schedule dependent. The optimal schedule for this combination is sequential exposure of paclitaxel followed by carboplatin.

Paclitaxel is used frequently for the treatment of patients with non-small cell lung cancer. Hypersensitivity reactions remain one of the major adverse events in the clinical use of paclitaxel. Glucocorticoids are used to prevent these adverse events. This study was carried out in order to clarify the effect of glucocorticoids on paclitaxel-induced cytotoxity of cancer cells. Pretreatment with 10 microM of dexamethasone inhibited ERK activation and subsequent retinoblastoma protein (pRB) phosphorylation, and reduced sensitivity to paclitaxel in A549 cells. Then, we utilized ERK (PD98059) and AKT (LY294002) inhibitors. PD98059 and LY294002 effectively suppressed pRB phosphorylation in A549 cells. Dexamethasone (10 microM) suppressed ERK activity as well as PD98059, although it did not affect AKT activity. Furthermore, the combinations of paclitaxel with PD98059 or LY294002 were similarly antagonistic. Our observation in this study raised the possibility that dexamethasone pretreatment antagonizes paclitaxel-induced cytotoxicity through ERK suppression and pRB dephosphorylation. These observations support the development of new generation taxane-based chemotherapy without glucocorticoid premedication.

Introduction: Overexpression of the apoptosis-related protein clusterin is associated with breast cancer development and tumor progression. We describe the use of clusterin-specific antisense oligonucleotides and antibodies to sensitize breast carcinoma cells to anticancer drugs routinely used in breast cancer therapy., Methods: MCF-7 and MDA-MB-231 cells were treated with the oligonucleotide or antibody, chemotherapeutic agents (doxorubicin or paclitaxel), tamoxifen, or with combinations of these., Results: Treatments that include antisense clusterin oligonucleotide or antibody to clusterin have been shown to reduce the number of viable cells more effectively than treatment with the drugs alone. We also demonstrate that dexamethasone pretreatment of breast cancer cell lines inhibits chemotherapy-induced cytotoxicity and is associated with the transcriptional induction of clusterin. However, anticlusterin treatment increases chemotherapy-induced cytotoxicity, even in the presence of glucocorticoids, suggesting a possible role for these proteins in glucocorticoid-mediated survival., Conclusion: These data suggest that combined treatment with antibodies to clusterin or antisense clusterin oligodeoxynucleotides and paclitaxel, doxorubicin, or tamoxifen could be a novel and attractive strategy to inhibit the progression of breast carcinoma by regulation of the clusterin function. Moreover, glucocorticoid activation in breast cancer cells regulates survival signaling by the direct transactivation of genes like clusterin which encode proteins that decrease susceptibility to apoptosis. Given the widespread clinical administration of dexamethasone before chemotherapy, understanding glucocorticoid-induced survival mechanisms is essential for achieving optimal therapeutic responses.

Paclitaxel is a widely used naturally occurring antineoplastic agent that has shown great promise in the treatment of a variety of human solid tumors, particularly for advanced breast and ovarian cancers. Recent studies in our laboratory discovered that glucocorticoids could selectively inhibit paclitaxel-induced apoptosis in a number of human solid tumor cell lines in vitro. Since glucocorticoids (such as dexamethasone) are routinely used as a premedication in the clinical application of paclitaxel to prevent hypersensitivity reactions and other adverse effects, the inhibitory effect of glucocorticoids on paclitaxel-induced apoptosis has raised a clinically relevant question as to whether the pretreatment with glucocorticoids might interfere with the therapeutic efficacy of paclitaxel. In the present study, through development of animal models bearing human breast and ovarian xenograft tumors, we evaluated the potential influence of dexamethasone on antitumor activity of paclitaxel in vivo. The results demonstrated that pretreatment of dexamethasone significantly attenuated therapeutic efficacy of paclitaxel against human breast and ovarian xenograft tumors. The inhibition rate of 20 mg paclitaxel/kg on the growth of breast and ovarian xenograft tumors was around 20-25% less when the animals were pretreated with 1 mg dexamethasone/kg. Further analyses with histological examination and TdT-mediated dUTP nick end labeling assay indicate that pretreatment with dexamethasone clearly interferes with the cytotoxic effects of paclitaxel on both morphological alterations and induction of cell death. Additionally, immunohistochemical staining of proliferation marker Ki-67 indicates that the percentage of proliferating cells in xenograft tumors with pretreatment of dexamethasone is much higher than that in the tumors treated with paclitaxel alone. Put together, the results obtained from the animal experiments show that pretreatment of xenograft tumors with dexamethasone results in significant inhibition of the therapeutic efficacy of paclitaxel in vivo. This finding may have a potential implication on the clinical practice of paclitaxel-based chemotherapy., (Copyright (c) 2006 Wiley-Liss, Inc.)

Paclitaxel (Taxol) is a widely used chemotherapeutic agent in the treatment of several tumors. However, its use is often associated with the generation of peripheral neuropathic pain expressed as mechanical allodynia and thermal hyperalgesia. The molecular mechanism behind this debilitating side effect is obscure, and efficient drugs for its prevention are required. We sought to clarify the cellular changes in the involved nociceptor types underlying paclitaxel-induced neuropathic pain and to test for an alleviating effect of gabapentin treatment in a murine model of paclitaxel-induced neuropathic pain. We found that a single treatment with paclitaxel (4 mg/kg i.p.) led to a decrease in both thermal and mechanical nociceptive thresholds as well as a reduction in the thresholds for 250-Hz (A delta-fiber) and 2000 Hz (A beta-fiber) but not 5-Hz (C-fiber) sine wave electrical stimuli-induced paw withdrawal. The paclitaxel-induced neuropathic pain was completely abrogated by gabapentin (30 mg/kg i.p.) treatment. Furthermore, we found that mRNA and protein levels of the voltage-gated calcium channel (alpha 2)delta-1 subunit (Ca(alpha 2)delta-1), one of the putative targets for gabapentin, was up-regulated in dorsal root ganglions (DRGs), as well as increased expression of Ca(alpha 2)delta-1 protein in medium/large-sized DRG neurons by immunohistochemistry, following paclitaxel treatment. This suggests that paclitaxel induces A-fiber-specific hypersensitization, which may contribute to the functional mechanical allodynia and hyperalgesia, and that gabapentin could be a potential therapeutic agent for paclitaxel-induced neuropathic pain.

Non-small cell lung cancer (NSCLC) demonstrates a strong etiologic association with smoking. Although nicotine is not carcinogenic, it can induce cell proliferation and angiogenesis and suppress apoptosis induced by certain agents. Here we show that nicotine inhibits apoptosis induced by the drugs gemcitabine, cisplatin, and taxol, which are used to treat NSCLCs. This protection correlated with the induction of XIAP and survivin by nicotine in a panel of human NSCLC cell lines, and depletion of XIAP and survivin ablated the protective effects of nicotine. The antiapoptotic effects of nicotine were mediated by dihydro beta-erythroidine-sensitive alpha3-containing nicotinic acetylcholine receptors and required the Akt pathway. Chromatin immunoprecipitation assays demonstrated that nicotine stimulation caused an increased recruitment of E2F1 and concomitant dissociation of retinoblastoma tumor suppressor protein (Rb) from survivin promoter in A549 cells. Moreover, ablation of E2F1 levels caused abrogation of the protective effects of nicotine against cisplatin-induced apoptosis in A549 cells whereas ablation of signal transducer and activator of transcription 3 levels had no effect. These studies suggest that exposure to nicotine might negatively impact the apoptotic potential of chemotherapeutic drugs and that survivin and XIAP play a key role in the antiapoptotic activity of nicotine.

The chemotherapeutic drug paclitaxel induces microtubular stabilization and mitotic arrest associated with increased survivin expression. Survivin is a member of the inhibitor of apoptosis (iap) family which is highly expressed in during G2/M phase where it regulates spindle formation during mitosis. It is also constitutively overexpressed in most cancer cells where it may play a role in chemotherapeutic resistance. MCF-7 breast cancer cells stably overexpressing the sense strand of survivin (MCF-7(survivin-S) cells) were more resistant to paclitaxel than cells depleted of survivin (MCF-7(survivin-AS) despite G2/M arrest in both cell lines. However, survivin overexpression did not protect cells relative to control MCF-7(pcDNA3) cells. Paclitaxel-induced cytotoxicity can be enhanced by retinoic acid and here we show that RA strongly reduces survivin expression in MCF-7 cells and prevents paclitaxel-mediated induction of survivin expression. Mitochondrial release of cytochrome c after paclitaxel alone or in combination with RA was weak, however robust Smac release was observed. While RA/paclitaxel-treated MCF-7 (pcDNA3) cultures contained condensed apoptotic nuclei, MCF-7(survivin-S) nuclei were morphologically distinct with hypercondensed disorganized chromatin and released mitochondrial AIF-1. RA also reduced paclitaxel-associated levels of cyclin B1 expression consistent with mitotic exit. MCF-7(survivin-S) cells displayed a 30% increase in >2N/<4N ploidy while there was no change in this compartment in vector control cells following RA/paclitaxel. We propose that RA sensitizes MCF-7 cells to paclitaxel at least in part through survivin downregulation and the promotion of aberrant mitotic progression resulting in apoptosis. In addition we provide biochemical and morphological data which suggest that RA-treated MCF-7(survivin-S) cells can also undergo catastrophic mitosis when exposed to paclitaxel.

In previous studies we demonstrated that resveratrol acts in an antiapoptotic manner on the paclitaxel-treated human neuroblastoma (HN) SH-SY5Y cell line inhibiting the apoptotic pathways induced by the antineoplastic drug. In the present study we evaluated the antiapoptotic effect of resveratrol, studying its activity on cell cycle progression. We determined the mitotic index of cultures exposed to resveratrol and paclitaxel alone or in combination, the cell cycle distribution by flow cytometric analysis (FACS), and the modulation of some relevant cell cycle regulatory proteins. Resveratrol is able to induce S-phase cell arrest and this interference with the cell cycle is associated with an increase of cyclin E and cyclin A, a downregulation of cyclin D1, and no alteration in cyclin B1 and cdk 1 activation. The resveratrol-induced S-phase block prevents SH-SY5Y from entering into mitosis, the phase of the cell cycle in which paclitaxel exerts its activity, explaining the antiapoptotic effect of resveratrol.

Studies were conducted to evaluate the potential mechanism-based inactivation of recombinant and human liver microsomal CYP2C8 by clinically used drugs. Several tricyclic antidepressants, calcium channel blockers, monoamine oxidase inhibitors, and various other known CYP3A4 inhibitors exhibited greater inhibition of CYP2C8 (paclitaxel 6alpha-hydroxylation) following preincubation, consistent with mechanism-based inactivation. Inactivation of recombinant CYP2C8 by phenelzine, amiodarone, verapamil, nortriptyline, fluoxetine, and isoniazid was of the pseudo-first order type and was characterized by respective inactivation kinetic constants (KI and kinact) of 1.2 microM and 0.243 min(-1), 1.5 microM and 0.079 min(-1), 17.5 microM and 0.065 min(-1), 49.9 microM and 0.036 min(-1), 294 microM and 0.083 min(-1), and 374 microM and 0.042 min(-1). Spectral scanning of recombinant CYP2C8 demonstrated the formation of metabolite-intermediate complexes with verapamil, nortriptyline, fluoxetine, and isoniazid, but not amiodarone. In contrast, inactivation by phenelzine resulted from heme destruction by free radicals. Studies with human liver microsomes (HLMs) revealed that nortriptyline, verapamil, and fluoxetine were not mechanism-based inactivators (MBIs) of CYP2C8. Simultaneous inactivation of CYP2C8 and CYP3A4 (paclitaxel 3'-phenyl-hydroxylation) was observed using amiodarone, isoniazid, and phenelzine with the efficiency of inactivation greater for the CYP3A4 pathway. With the exception of phenelzine, glutathione and superoxide dismutase failed to protect CYP2C8 (recombinant and HLMs) or CYP3A4 from inactivation by MBIs. However, the alternate CYP2C8 substrate, torsemide, prevented CYP2C8 inactivation in all cases. These data are consistent with mechanism-based inactivation of CYP2C8 by a range of commonly prescribed drugs, several of which have been implicated in clinically important drug-drug interactions.

Recently, survival benefit by chemotherapy using paclitaxel (PTX) and the induction of thymidine phosphorylase (TP) by PTX have been reported in several solid tumors. On the other hand, TP confers antiapoptotic effect on tumor cells through inhibition of caspase-8 activation in vitro. On the basis of these previous observations, we hypothesized that (a) TP can be induced after PTX treatment in human prostate cancer (PC) and (b) blockade of PTX-induced TP expression can enhance the apoptotic processes in human PC cells. PTX was used to find TP expression in all eight hormone-refractory PC cases after chemotherapy; however, cleaved caspase-8 was not expressed after chemotherapy in the six hormone-refractory PC cases with strong TP expression. In PC cell lines (PC-3, DU 145, and LNCaP), TP expression after PTX treatment was clearly up-regulated in a dose-dependent manner. Cell viability of PC cell lines treated with PTX and TP antisense was significantly reduced in a time-dependent and dose-dependent manner compared with the PTX treatment alone. Likewise, apoptotic index of PC cells treated with PTX and TP antisense was significantly increased in comparison with PTX alone. After complete blockade of PTX-induced TP translation by TP antisense transfection, cleaved form of caspase-3 and poly(ADP-ribose) polymerase was increased, and this exaggeration of apoptosis also ran parallel with caspase-8 activation in a PTX dose-dependent manner. However, in PC cell lines treated with TP antisense alone, neither caspase-3 nor poly(ADP-ribose) polymerase was cleaved despite caspase-8 activation. These results indicate that PTX-induced TP up-regulation is associated with decreased caspase-8 activation. This study is the first report showing that blockade of PTX-induced TP expression could exaggerate the processing of apoptosis in PC cells treated with PTX. Our results provide preclinical evidence that TP could be a new molecular target for enhancing the potency of PTX-mediated apoptosis in PC cells.

The functional significance of the cyclin-dependent kinase inhibitor (CDKI) p21(Cip1/WAF1) in paclitaxel-mediated lethality was examined in p53-null human leukemia cells (U937 and Jurkat). In these cells, paclitaxel exposure failed to induce p21(Cip1/Waf1) expression. Nevertheless, stable expression of U937 cells with a p21(Cip1/WAF1) antisense construct blocked paclitaxel-induced G(2)M arrest and increased mitochondrial injury, caspase activation, apoptosis, and loss of clonogenic potential. Consistent with these results, enforced expression of p21(Cip1/WAF1) in Jurkat cells increased the percentage of cells arrested in G2M and attenuated paclitaxel-mediated mitochondrial injury and apoptosis. Unexpectedly, enforced expression of p21(Cip1/WAF1) diminished paclitaxel-mediated inactivation of ERK, and reduced paclitaxel-induced activation of JNK as well as Bcl-2 phosphorylation. Together, these findings suggest that p21(Cip1/WAF1) partially protects p53-null human leukemia cells from paclitaxel-mediated lethality, and raise the possibility that p21(Cip1/WAF1)-associated perturbations in signal transduction pathways as well as Bcl-2 phosphorylation status may play a role in this phenomenon.

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.

We investigated cytochrome P450 (P450)-catalyzed metabolism of the important cancer drugs paclitaxel and docetaxel in rat, pig, minipig, and human liver microsomes and cDNA-expressed P450 enzymes. In rat microsomes, paclitaxel was metabolized mainly to C3'-hydroxypaclitaxel (C3'-OHP) and to a lesser extent to C2-hydroxypaclitaxel (C2-OHP), di-hydroxypaclitaxel (di-OHP), and another unknown monohydroxylated paclitaxel. In pig and minipig microsomes, this unknown hydroxypaclitaxel was the main metabolite, whereas C3'-OHP was a minor product. In minipigs, C2-OHP was the next minor product. In human liver microsomes, 6 alpha-hydroxypaclitaxel (6 alpha-OHP) was the main metabolite, followed by C3'-OHP and C2-OHP. Among different cDNA-expressed human P450 enzymes (CYP1A2, 1B1, 2A6, 2C9, 2E1, and 3A4), only CYP3A4 enzyme formed C3'-OHP and C2-OHP. Docetaxel was metabolized in pig, minipig, rat, and human liver microsomes mainly to hydroxydocetaxel (OHDTX), whereas CYP3A-induced rat microsomes produced primarily diastereomeric hydroxyoxazolidinones. Human liver microsomes from 10 different individuals formed OHDTX at different rates correlated with CYP3A4 content. Troleandomycin as a selective inhibitor of CYP3A inhibited the formation of C3'-OHP, C2-OHP, and di-OHP, as well as the unknown OHP produced in rat, minipig, and pig microsomes. In human liver microsomes, troleandomycin inhibited C3'-OHP and C2-OHP formation, and a suitable inhibitor of human CYP2C8, fisetin, strongly inhibited the formation of 6 alpha-OHP, known to be catalyzed by human CYP2C8. In conclusion, the metabolism of docetaxel is the same in all four species, but metabolism of paclitaxel is different, and 6 alpha-OHP remains a uniquely human metabolite. Pigs and minipigs compared with each other formed the same metabolites of paclitaxel.

Paclitaxel (Taxol), a naturally occurring antimitotic agent, has shown significant cell-killing activity against tumor cells through induction of apoptosis. The mechanism by which paclitaxel induces cell death is not entirely clear. Recent studies in our laboratory discovered that glucocorticoids selectively inhibited paclitaxel-induced apoptosis without affecting the ability of paclitaxel to induce microtubule bundling and mitotic arrest. This finding implies that apoptotic cell death induced by paclitaxel may occur via a pathway independent of mitotic arrest. Through analyses of a number of apoptosis-associated genes or regulatory proteins, we found that glucocorticoids and paclitaxel possess opposite regulatory role in the NF-kappa B/Ikappa Balpha signaling pathway. Further studies indicate that paclitaxel activates Ikappa B Kinase (IKK), which in turn causes degradation of Ikappa Balpha and activation of NF-kappa B, whereas glucocorticoids antagonize paclitaxel-mediated NF-kappa B activation through induction of Ikappa Balpha synthesis. These results suggest that the NF-kappa B/Ikappa Balpha signaling pathway might play a critical role in the mediation or regulation of paclitaxel-induced cell death. On the other hand, since glucocorticoids (such as dexamethasone) are routinely used in the clinical application of paclitaxel to prevent hypersensitivity reactions and other adverse effects, the inhibitory action of glucocorticoids on paclitaxel-induced apoptosis also raises a clinically relevant question as to whether the pretreatment with glucocorticoids might interfere with the therapeutic efficacy of paclitaxel.

Background: Paclitaxel, a naturally occurring antineoplastic agent, can cause both mitotic arrest and apoptotic cell death, but the relationship between these two events is not entirely clear. The focus of this study was to determine whether apoptosis induced by paclitaxel may occur independent of mitotic arrest and to examine the underlying molecular mechanisms of paclitaxel-induced apoptosis., Materials and Methods: Two paclitaxel-sensitive tumor cell lines, human breast cancer BCap37 and human epidermoid carcinoma KB cells, were pulsed exposed to various concentrations of paclitaxel. Multiple methods were used to analyze the possible correlation between paclitaxel-induced apoptosis and paclitaxel-induced mitotic arrest. A series of assays were also performed in which we utilized parthenolide, a specific inhibitor of NF-kappaB, to analyze the possible role that the NF-kappaB/IkappaB signaling pathway has in mediating paclitaxel-induced apoptosis., Results: Both tumor cell lines treated with pulsed paclitaxel exposures exhibited a significant number of cells undergoing apoptosis, however many fewer cells were arrested at the G2/M-phase of the cell cycle when compared to the continuous paclitaxel exposures. Short exposures to paclitaxel also induced the phosphorylation and degradation of IkappaB-alpha, which in turn caused the activation of NF-kappaB in both cell lines. Parthenolide was found to inhibit paclitaxel-induced activation of the NF-kappaB/IkappaB signal pathway as well as apoptotic cell death., Conclusion: These findings suggest that paclitaxel-induced apoptosis might occur independent of a prior G2/M-phase arrest and be mediated or regulated by the NF-kappaB/IkappaB signal pathway.

Purpose: Antineoplastic drugs belonging to platinum or taxane families are severely neurotoxic, inducing the onset of disabling peripheral neuropathies with different clinical signs. Acetyl-L-carnitine (ALC) is a natural occurring compound with a neuroprotective activity in several experimental paradigms. In this study we have tested the hypothesis that ALC may have a protective role on cisplatin and paclitaxel-induced neuropathy., Experimental Design: Sensory nerve conduction velocity (SNCV) was measured in rats before, at end, and after an additional follow-up period from treatments with cisplatin, paclitaxel, or with the respective combination with ALC. In addition, serum from treated animals was collected to measure the levels of circulating NGF, and left sciatic nerves were processed for light and electron microscope observations. ALC interference on cisplatin and paclitaxel antitumor activity and protective mechanisms were investigated using several in vitro and in vivo models., Results: ALC cotreatment was able to significantly reduce the neurotoxicity of both cisplatin and paclitaxel in rat models, and this effect was correlated with a modulation of the plasma levels of NGF in the cisplatin-treated animals. Moreover, experiments in different tumor systems indicated the lack of interference of ALC in the antitumor effects of cisplatin and paclitaxel. The transcriptional profile of gene expression in PC12 cells indicated that ALC, in the presence of NGF, was able to positively modulate NGFI-A expression, a gene relevant in the rescue from tissue-specific toxicity. Finally, the transcriptionally ALC-mediated effects were correlated to increase histone acetylation., Conclusion: In conclusion, our results indicate that ALC is a specific protective agent for chemotherapy-induced neuropathy after cisplatin or paclitaxel treatment without showing any interference with the antitumor activity of the drugs.

Paclitaxel is an important, recently introduced anti-neoplastic drug. Paclitaxel metabolites are virtually inactive in comparison with the parent drug. The study investigated whether phenolic antioxidants could inhibit metabolic inactivation sufficiently to increase paclitaxel effects. Cytochrome p450 (CYP)-catalysed metabolism of paclitaxel was investigated in rat and human liver microsomes. In rat microsomes, paclitaxel was metabolised mainly to C3'-hydroxypaclitaxel (C3'-OHP), less to C2-hydroxypaclitaxel (C2-OHP), di-hydroxypaclitaxel (di-OHP) and another monohydroxylated paclitaxel. In human liver microsomes, 6alpha-hydroxypaclitaxel (6alpha-OHP), formed by CYP2C8, was the main metabolite, while C3'-OHP, C2-OHP and another product different from di-OHP were minor metabolites, formed by CYP3A4. In individual human livers 6alpha-OHP was formed at 1.8-fold to 13-fold higher rates than C3'-OHP. Kinetic parameters (K(m) and V(max)) of production of various metabolites in rat and human liver microsomes revealed differences between species as well as human individual differences. Nine phenolic antioxidants ((+)-catechin, (-)-epicatechin, fisetin, gallic acid, morin, myricetin, naringenin, quercetin and resveratrol) were tested for inhibition of paclitaxel metabolism. In rat microsomes, resveratrol was more inhibitory than fisetin; the other phenolic antioxidants were without effect. In human microsomes, the inhibiting potency decreased in the order fisetin >quercetin >morin >resveratrol, while the other phenolic antioxidants were not inhibitory; the formation of 6alpha-OHP (CYP2C8) was generally more inhibited than that of C3'-OHP. The inhibition was mostly mixed-type. The results suggest that oral administration of some phenolic substances might increase paclitaxel blood concentrations during chemotherapy.

The neuroprotective effect of BGP-15 against peripheral sensory neuropathy was studied in rats that were exposed to short-term cisplatin or taxol administration. The changes of nerve conduction velocity were determined in situ after treating the Wistar rats with BGP-15 (50, 100, and 200 mg/kg po daily doses throughout the experiment), cisplatin (1.5 mg/kg ip daily dose for 5 days), or taxol (5.0 mg/kg ip daily dose every other day in a 10-day interval) alone or giving the test compound in combination with cisplatin or taxol. Electrophysiological recordings were carried out in vivo by stimulating the sciatic nerve at both sciatic notch and ankle site. Neither motor nor sensory nerve conduction velocity was altered by any dose level of BGP-15 tested. Both anticancer drugs decreased the sensory nerve conduction velocity (SNCV). BGP-15 treatment prevented the impairment of SNCV either in part or totally in the cisplatin- or taxol-treated groups. This neuroprotective potential of BGP-15 could be well correlated with its recently described poly(ADP-ribose) polymerase- inhibitory effect and its ability to protect against the damages induced by the increased level of reactive oxygen species in response to anticancer treatment.

Objective: In vitro studies have revealed that treatment of various human cancer cell lines with specific cyclo-oxygenase 2 (COX-2) inhibitors induces apoptotic cell death. It is currently proposed that the combination of COX-2 inhibitors with chemotherapeutic agents improves the efficacy of cancer treatment., Materials and Methods: In this study we sought to determine the effects of combining paclitaxel and the COX-2 inhibitor NS398 on apoptosis of epithelial ovarian cancer (EOC) cells. Two EOC cell lines, SKOV3 and MDAH2774, were exposed to increasing concentrations of paclitaxel (0.1, 10, and 100 microM) and NS398 (10, 100 microM) as well as a combination of both drugs. Apoptosis was evaluated by the Tunel assay. The fluorescein-labeled DNA was visualized directly by fluorescence microscopy and quantitated by flow cytometry., Results: While NS398 did not significantly alter apoptosis of either EOC cell lines after 24 h of continuous exposure, treatment of both cell lines with paclitaxel resulted in a significant increase in the rate of apoptosis (60-70%). Concomitant treatment of both SKOV3 and MDAH2774 cells with paclitaxel and NS398 resulted in marked impairment of paclitaxel-induced apoptosis. Similarly, sequential treatment during which both cell lines were treated with NS398 for 4 h, triple-washed, and then exposed to paclitaxel for 24 h resulted in a significant inhibition of paclitaxel-induced apoptosis. Similar inhibition was seen when NS398 was replaced by aspirin., Conclusions: Combining COX-2 inhibitors and paclitaxel does not have an additive or synergistic tumoricidal effect. On the contrary, NS398 treatment markedly inhibited the apoptotic effects of paclitaxel in each of these two EOC cell lines.

This study was performed to investigate how caffeine modifies the cytotoxic effects of paclitaxel on a human lung carcinoma cell line. Caffeine doses up to 5mM had less effect on clonogenic survival. The cell killing effect, due to paclitaxel, increased in a dose-dependent manner up to 50 nM. For combined treatment with caffeine and paclitaxel, added caffeine reduced the cytotoxic effect of paclitaxel not only in dose-response but also in time-response curves. Caffeine combined with paclitaxel clearly suppressed cell proliferation in a dose-dependent manner. In the cell cycle analysis, caffeine alone caused early G1 accumulation, whereas paclitaxel alone caused an early increase in G2-M and a decrease in G1. As for the effect of caffeine on paclitaxel, caffeine suppressed the effect of paclitaxel on cell cycle distribution, where a dose-dependent early increase in G2-M and a decrease in G1 were not clear. We suggest that cell cycle modifying agents, such as caffeine, potentially diminish the cytotoxic activity of paclitaxel, and one should be careful when combining such agents.

Receptors for activated C kinase (RACKs) are a group of PKC binding proteins that have been shown to mediate isoform-selective functions of PKC and to be crucial in the translocation and subsequent functioning of the PKC isoenzymes on activation. RACK1 cDNA from the shrimp Penaeus japonicus was isolated by homology cloning. The hepatopancreas cDNA from this shrimp was found to encode a 318-residue polypeptide whose predicted amino acid sequence shared 91% homology with human G(beta2)-like proteins. Expression of the cDNA of shrimp RACK1 in vitro yielded a 45-kDa polypeptide with positive reactivity toward the monoclonal antibodies against RACK1 of mammals. The shrimp RACK1 was biotinylated and used to compare the effects of geranylgeranyl pyrophosphate and farnesyl pyrophosphate on its binding with PKCgamma in anti-biotin-IgG precipitates. PKCgammas were isolated from shrimp eyes and mouse brains. Both enzyme preparations were able to inhibit taxol-induced tubulin polymerization. Interestingly, when either geranylgeranyl pyrophosphate or farnesyl pyrophosphate was reduced to the submicrogram level, the recruitment activity of RACK1 with purified PKCgamma was found to increase dramatically. The activation is especially significant for RACK1 and PKCgamma from different species. The observation implies that the deprivation of prenyl pyrophosphate might function as a signal for RACK1 to switch the binding from the conventional isoenzymes of PKC (cPKC) to the novel isoenzymes of PKC (nPKC). A hydrophobic binding pocket for geranylgeranyl pyrophosphate in RACK1 is further revealed via prenylation with protein geranylgeranyl transferase I of shrimp P. japonicus., (Copyright 2003 Wiley-Liss, Inc.)