Apoptotic Process Induced by Oxaliplatin in Rat Hippocampus Causes Memory Impairment.

Aspects of memory involved in cognitive mechanisms were investigated in rat after oxaliplatin (OX) chemotherapy using animal behavioural assessment of passive avoidance and social learning paradigms, which are both hippocampus-sensitive. Rodents, previously subjected to 2-week OX treatment, showed passive avoidance and social learning impairment and apoptotic processes in the hippocampus. Apoptosis rate significantly increased in cultured hippocampal cells exposed to OX at increasing doses, and this effect was dose-dependent. Ex vivo experiments showed that cell damage and apoptosis were blocked in the hippocampus from OX rats cotreated with copper sulphate (CS) which precludes OX transport inside the cell. In vivo, passive avoidance and social learning impairment could not be observed in OX rats co-administered with CS. Thus, a site of action of OX treatment on memory impairment appears to be located at the hippocampus. These findings strongly support that cellular damage induced by OX in rodent hippocampus underlies the weakening of some memory functions.

Involvement of c-Myc-mediated transient receptor potential melastatin 8 expression in oxaliplatin-induced cold allodynia in mice.

BACKGROUND: Oxaliplatin, a platinum-based chemotherapeutic agent, induces acute cold allodynia and dysesthesia. Cold-sensitive transient receptor potential channels (TRPM8 and TRPA1) have been implicated as candidates to mediate oxaliplatin-induced cold allodynia and hyperalgesia, but precise roles of these channels remain unclear. In this study, we investigated the role of TRPM8 in oxaliplatin-induced cold allodynia. METHODS: Oxaliplatin was injected intraperitoneally in mice. Cold allodynia was evaluated by the acetone test. Expression levels of TRPM8 mRNA and protein were measured using reverse transcription-polymerase chain reaction and Western blotting, respectively. RESULTS: Oxaliplatin-induced cold allodynia was alleviated by the TRPM8 blockers N-(2-aminoethyl)-N-[4-(benzyloxy)-3-methoxybenzyl]-N'-(1S)-1-(phenyl) ethyl] urea and TC-I 2014. Oxaliplatin increased the expression levels of TRPM8 mRNA and protein in the dorsal root ganglia and plantar skin, respectively. Prophylactic administration of the c-Myc inhibitor 10058-F4 prevented cold allodynia and the increase of TRPM8 mRNA after oxaliplatin injection. CONCLUSION: These results suggest that oxaliplatin induces cold allodynia through the increase of c-Myc-mediated TRPM8 expression in primary sensory neurons.

Role of Reactive Oxygen Species in the Abrogation of Oxaliplatin Activity by Cetuximab in Colorectal Cancer.

BACKGROUND: The antibody cetuximab, targeting epidermal growth factor receptor (EGFR), is used to treat metastatic colorectal cancer (mCRC). Clinical trials suggest reduced benefit from the combination of cetuximab with oxaliplatin. The aim of this study was to investigate potential negative interactions between cetuximab and oxaliplatin. METHODS: Thiazolyl blue tetrazolium bromide (MTT) assay and Calcusyn software were used to characterize drug interactions. Reactive oxygen species (ROS) were measured by flow cytometry and real-time polymerase chain reaction oxidative stress arrays identified genes regulating ROS production. Chromatin immunoprecipitation (ChIP) measured signal transducer and activator of transcription 1 (STAT-1) binding to dual oxidase 2 (DUOX2) promoter. SW48, DLD-1 KRAS wild-type cell lines and DLD-1 xenograft models exposed to cetuximab, oxaliplatin, or oxaliplatin + cetuximab (control [saline]; n = 3 mice per treatment group) were used. Statistical tests were two-sided. RESULTS: Cetuximab and oxaliplatin exhibited antagonistic effects on cellular proliferation and apoptosis (caspase 3/7 activity reduced by 1.4-fold, 95% confidence interval [CI] = 0.78 to 2.11, P = .003) as opposed to synergistic effects observed with the irinotecan metabolite 7-Ethyl-10-hydroxycamptothecin (SN-38). Although both oxaliplatin and SN-38 produced ROS, only oxaliplatin-mediated apoptosis was ROS dependent. Production of ROS by oxaliplatin was secondary to STAT1-mediated transcriptional upregulation of DUOX2 (3.1-fold, 95% CI = 1.75 to 2.41, P < .001). Inhibition of DUOX2 induction and p38 activation by cetuximab reduced oxaliplatin cytotoxicity. CONCLUSIONS: Inhibition of STAT1 and DUOX2-mediated ROS generation by cetuximab impairs p38-dependent apoptosis by oxaliplatin in preclinical models and may contribute to reduced efficacy in clinical settings. Understanding the rationale for unexpected trial results will inform improved rationales for combining EGFR inhibitors with chemotherapeutic agents in future therapeutic use.

Glutathione selectively modulates the binding of platinum drugs to human copper chaperone Cox17.

The copper chaperone Cox17 (cytochrome c oxidase copper chaperone) has been shown to facilitate the delivery of cisplatin to mitochondria, which contributes to the overall cytotoxicity of the drug [Zhao et al. (2014) Chem. Commun. 50: , 2667-2669]. Kinetic data indicate that Cox17 has reactivity similar to glutathione (GSH), the most abundant thiol-rich molecule in the cytoplasm. In the present study, we found that GSH significantly modulates the reaction of platinum complexes with Cox17. GSH enhances the reactivity of three anti-cancer drugs (cisplatin, carboplatin and oxaliplatin) to Cox17, but suppresses the reaction of transplatin. Surprisingly, the pre-formed cisplatin-GSH adducts are highly reactive to Cox17; over 90% platinum transfers from GSH to Cox17. On the other hand, transplatin-GSH adducts are inert to Cox17. These different effects are consistent with the drug activity of these platinum complexes. In addition, GSH attenuates the protein aggregation of Cox17 induced by platination. These results indicate that the platinum-protein interactions could be substantially influenced by the cellular environment.

[Total flavonoids of litsea coreana decreases the cytotoxicity of oxaliplatin in TM3 Leydig cells via enhancing the function of gap junction].

OBJECTIVE: To investigate the effects of total flavonoids of Litsea Coreana (TFLC) on the gap junction (GJ) intercellular communication in TM3 testicular Leydig cells and whether TFLC can reduce the cytotoxicity of oxaliplatin (OHP) in vitro. METHODS: We detected the effect of TFLC on the dye spread of the in vitro cultured TM3 cells by parachute assay, observed changes in the expression of connexin 43 (Cx43) total protein in the TFLC-treated TM3 cells by Western blot, and determined the effects of TFLC on the expression of Cx43 on the membrane of the TM3 cells by immunofluorescence assay and on the cytotoxicity of OHP by MTT assay. RESULTS: TFLC obviously enhanced the GJ function with the increasing of the TFLC concentration in the TM3 cells. Western blot and immunofluorescence assay confirmed that TFLC significantly enhanced the expression of Cx43 total protein and Cx43 expression on the membrane of the TM3 cells. MTT assay showed that at a high cell density (confluent with GJ formation), 20 microg/ml TFLC enhanced the GJ function of the TM3 cells and reduced the cytotoxicity of OHP (P < 0.05), while at a low density (preconfluent with no GJ formation), TFLC exhibited no effect on the cytotoxicity of OHP (P > 0.05). CONCLUSION: TFLC increases the Cx43 expression and GJ function in normal TM3 Leydig cells, and the enhancement of GJ function reduces the cytotoxicity of OHP.

Repeated administration of amitriptyline reduces oxaliplatin-induced mechanical allodynia in rats.

Oxaliplatin is a key drug in the treatment of colorectal cancer, but it causes acute and chronic neuropathies in patients. Amitriptyline has widely been used in patients with painful neuropathy. In this study, we investigated the effect of amitriptyline on the oxaliplatin-induced neuropathy in rats. Repeated administration of amitriptyline (5 and 10 mg/kg, p.o., once a day) reduced the oxaliplatin-induced mechanical allodynia but not cold hyperalgesia and reversed the oxaliplatin-induced increase in the expression of NR2B protein and mRNA in rat spinal cord. These results suggest that amitriptyline is useful for the treatment of oxaliplatin-induced neuropathy clinically.

Prevention of oxaliplatin-induced mechanical allodynia and neurodegeneration by neurotropin in the rat model.

Oxaliplatin is a key drug for colorectal cancer, but it causes acute peripheral neuropathy (triggered by cold) and chronic neuropathy (sensory and motor neuropathy) in patients. Neurotropin, a non-protein extract from the inflamed rabbit skin inoculated with vaccinia virus, has been used to treat various chronic pains. In the present study, we investigated the effect of neurotropin on the oxaliplatin-induced neuropathy in rats. Repeated administration of oxaliplatin caused cold hyperalgesia from Day 5 to Day 29 and mechanical allodynia from Day 15 to Day 47. Repeated administration of neurotropin relieved the oxaliplatin-induced mechanical allodynia but not cold hyperalgesia, and inhibited the oxaliplatin-induced axonal degeneration in rat sciatic nerve. Neurotropin also inhibited the oxaliplatin-induced neurite degeneration in cultured pheochromocytoma 12 (PC12) and rat dorsal root ganglion (DRG) cells. On the other hand, neurotropin did not affect the oxaliplatin-induced cell injury in rat DRG cells. These results suggest that repeated administration of neurotropin relieves the oxaliplatin-induced mechanical allodynia by inhibiting the axonal degeneration and it is useful for the treatment of oxaliplatin-induced neuropathy clinically.

NGF protects dorsal root ganglion neurons from oxaliplatin by modulating JNK/Sapk and ERK1/2.

The involvement of the Mitogen-Activated Protein Kinases (MAPKs) family in platinum derivative-induced peripheral neuropathy has already been demonstrated. In particular, it has been evidenced that in Dorsal Root Ganglion (DRG) neurons prolonged exposure to oxaliplatin (OHP) induces early activation of p38 and ERK1/2, which mediate neuronal apoptosis, while the neuroprotective action of JNK/Sapk is downregulated by the drug treatment. In this study, the exposure of OHP-treated neurons to a neuroprotective stimulus, represented by a high dose of NGF, counteracts OHP-induced neuronal mortality. This effect was achieved by restoring the MAPK activation existing in untreated control cells. Increased viability occurred also after the administration of retinoic acid (RA), a pro-differentiative agent able to activate both JNK/Sapk and ERK1/2. The use of specific chemical inhibitors of MAPKs confirms the importance of this class of proteins for the neuroprotective pathway, since they reverse the protective effect. In summary, our findings assess the validity of MAPKs as the target of neuroprotective therapies during chemotherapeutic treatment. Moreover they also describe a double role for ERK1/2, depending on cellular stimulation, since it mediates neuronal apoptosis after OHP exposure. However, it is also important, as is JNK/Sapk, in preserving the correct cellular differentiation that is pivotal for neuronal survival.

Potential of the Akt inhibitor LY294005 to antagonize the efficacy of Cisplatin against HCT116 tumor cells in a DNA mismatch repair-dependent manner.

Human colorectal adenocarcinoma sublines either deficient (HCT116+ch2) or proficient (HCT116+ch3) in the function of MLH1, one of five proteins crucial to DNA mismatch repair (MMR), were used to investigate whether the Akt-specific inhibitor LY294005 could not only increase the efficacy of platinum drugs in HCT116 cells in general but also increase the efficacy of the cisplatinum compounds Cisplatin and Lipoplatin specifically in MLH1-deficient, Cisplatin- and Lipoplatin-resistant HCT116 cells. We report that, under the conditions it increased the efficacy of Docetaxel and did not affect that of 6-thioguanine, LY294005 decreased the sensitivity of both sublines to Cisplatin, Lipoplatin, Oxaliplatin, and Lipoxal. Notably, the LY294005-imposed decrease was significantly higher in the MLH1-proficient than in the MLH1-deficient subline with Cisplatin and Lipoplatin, whereas it was nearly the same in both sublines with Oxaliplatin and Lipoxal. These LY294005-imposed changes in drug sensitivity, i.e. increase with Docetaxel and decreases with platinum compounds, were not associated with the concomitant abrogation in the levels of phospho-Aktser473. Analogous changes in drug sensitivity were also observed with the PI3-kinase inhibitor LY294002, but these changes were associated with complete abrogation of phospho-Aktser473. These observations suggest a possible relationship between MMR-mediated cisplatinum DNA damage signaling and the Akt signaling pathway, e.g. a common target for both pathways. A possibly novel property of Akt in aggravating drug sensitivity may also be proposed.

Protection against oxaliplatin acute neurosensory toxicity by venlafaxine.

Venlafaxine (Effexor; Wyeth Lederlé) has previously shown therapeutic effects for the management of chronic and neuropathic pains. We report here the efficacy of venlafaxine upon acute neurosensory symptoms secondary to oxaliplatin toxicity. A dose of 50 mg of venlafaxine was given orally at the beginning of the oxaliplatin infusion. Patients did not experience any or very low paresthesias, even in the cold. As the results were very dramatic and reproducible, we propose that venlafaxine may be of use in the daily management of oxaliplatin-related neurosensory toxicity.

Prevention by urinastatin of cis-diamminedichloroplatinum-induced nephrotoxicity in rabbits: comparison of urinary enzyme excretions and morphological alterations by electron microscopy.

Acute nephrotoxicity was produced by 3 mg/kg intramuscular administrations of cis-diamminedichloroplatinum (CDDP) in rabbits. Urinary enzyme excretions of arylamidase and gamma-glutamyl transpeptidase, and morphological alterations by electron microscopy were used as endpoints to quantitate the severity of nephrotoxicity. The preventive effect of urinastatin (US), a trypsin inhibitor, on CDDP-induced nephrotoxicity was evaluated. The alteration of urinary enzyme excretions did not parallel the severity of renal morphological changes. In proximal tubular cells, the morphological alterations consisting of nuclear irregularity, brush border disruption, and lysosomal membrane destruction were observed. In contrast, in rabbits treated concurrently with CDDP and 10,000 unit/kg of US, CDDP-induced morphological changes observed in rabbits without US were completely absent. The differences in urinary enzyme excretions and morphological alterations between US-treated rabbits and the US-nontreated group were enough to fully account for the drastic protective effect of US against CDDP nephrotoxicity.

Suppression of dexamethasone-induced metallothionein expression and cis-diamminedichloroplatinum(II) resistance by v-mos.

Metallothionein has been implicated in resistance to anticancer drugs. We examined whether transient induction of metallothionein by dexamethasone causes resistance to cis-diamminedichloroplatinum(II) (cis-DDP) in malignant and nonmalignant cells. Normal rat kidney cells (6m2) were infected with a modified v-mos oncogene construct in which expression of v-mos and consequently transformation was temperature-sensitive occurring at the permissive temperature of less than 33 degrees C and not at the nonpermissive temperature of 37 degrees C. Temperature-sensitive oncogenic transformation by v-mos attenuated induction of metallothionein by dexamethasone. No induction of metallothionein was observed in a revertant 6m2 cell line (54-5A4), which expressed v-mos and was transformed at 37 degrees C. Only nontransformed 6m2 cells displayed resistance to cis-DDP after dexamethasone pretreatment for 24 h. Dexamethasone pretreatment did not cause marked resistance to doxorubicin or melphalan in nontransformed 6m2 cells. When 6m2 cells (37 degrees C) were pretreated with dexamethasone (0.5 microM) for 24 h and then incubated in dexamethasone-free medium for 24 h, both metallothionein levels and resistance to cis-DDP decreased significantly. Thus, transient resistance to cis-DDP can be produced by a nonmetal inducer of metallothionein in nontransformed cells. Glucocorticoid-induced protection is suppressed in cells expressing v-mos and this might form the basis of future strategies to improve the therapeutic index of cis-DDP.

A study of human small-cell lung carcinoma (hSCLC) cell lines with different sensitivities to detect relevant mechanisms of cisplatin (CDDP) resistance.

The cisplatin(CDDP)-resistant cell line GLC4-CDDP shows a variety of differences from the parent line GLC4. The aim of this study was to determine which of the observed changes correlated with the degree of resistance and was therefore relevant to the phenomenon of CDDP resistance. For these experiments we used cells of the sensitive hSCLC cell line GLC4 and the in vitro-acquired CDDP-resistant sublines GLC4-CDDP3 and GLC4-CDDP11, with a resistance factor (RF) of 3 and 11 respectively for CDDP and of 1.8 and 7.4 respectively for carboplatin. Carboplatin was used, in addition to CDDP in seeking relevant mechanisms. No consistency was found between the RF and the growth pattern or antigen expression, cellular volume, doubling time, cellular or nuclear platinum (Pt) content or the level of Pt-non-histone chromatin protein (NHCP) binding. A correlation was found between the RF and the level of glutathione (GSH), and a trend was found for the level of Pt-DNA binding, Pt-GG adduct content and the amount of interstrand cross-links (ISC). These changes might therefore be relevant for the development of resistance. These findings are compatible with a GSH-induced reduction of the amount of reactive Pt in the resistant cell, resulting in a lower net platination and toxic Pt-DNA adduct formation.

Inhibition of cis-platinum nephrotoxicity by diethyldithiocarbamate rescue in a rat model.

The nephrotoxic effects of cis-dichlorodiammineplatinum(II) (NSC-119875) administered to male F344 rats at the median lethal dose (LD50; 7.5 mg/kg) were inhibited by treatment with sodium diethyldithiocarbamate (500 or 750 mg/kg) between 1 and 4 hr after cis-platinum administration. Those animals receiving cis-platinum alone had mean serum blood urea nitrogen levels of 234 mg/dl at the time of maximal toxicity (day 5); kidney sections revealed large areas of degeneration and necrosis. When dithiocarbamate rescue was carried out after cis-platinum treatment, mean blood urea nitrogen levels were in the range 56-95 mg/dl; kidney sections were grossly normal with a barely discernible band of degeneration at the corticomedullary junction. Gastrointestinal toxicity was observed in greater than 95% of the cis-platinum-treated rats but was totally absent in those receiving subsequent rescue treatment. A significant decrease in weight loss was also observed in the dithiocarbamate-rescued rats. Based on the chemistry of platinum-sulfur interactions and the observed time-dependence of the rescue treatment, it is suggested that dithiocarbamate exerts its effects via competitive chelation and removal of platinum coordinated to protein-bound sulfhydryl groups of the kidney tubule cells.

Thiourea reverses cross-links and restores biological activity in DNA treated with dichlorodiaminoplatinum (II).

Cis and trans dichlorodiaminoplatinum (II) compounds bind to DNA and form DNA cross-links, which are usually considered to be irreversible. Thiourea can reverse these cross-links without any apparent breakdown of the DNA. In addition, cis- and trans-Pt (II) treatment of lambda decreases its transfectivity. After suitable incubation with thiourea, full transfectivity of Pt(II)-treated lambda DNA can be restored.