Metabolomics highlights biochemical perturbations occurring in the kidney and liver of mice administered a human dose of colistin.

Introduction: Colistin (CMS) is used for the curation of infections caused by multidrug-resistant bacteria. CMS is constrained by toxicity, particularly in kidney and neuronal cells. The recommended human doses are 2.5-5 mg/kg/day, and the toxicity is linked to higher doses. So far, the in vivo toxicity studies have used doses even 10-fold higher than human doses. It is essential to investigate the impact of metabolic response of doses, that are comparable to human doses, to identify biomarkers of latent toxicity. The innovation of the current study is the in vivo stimulation of CMS's impact using a range of CMS doses that have never been investigated before, i.e., 1 and 1.5 mg/kg. The 1 and 1.5 mg/kg, administered in mice, correspond to the therapeutic and toxic human doses, based on previous expertise of our team, regarding the human exposure. The study mainly focused on the biochemical impact of CMS on the metabolome, and on the alterations provoked by 50%-fold of dose increase. The main objectives were i) the comprehension of the biochemical changes resulting after CMS administration and ii) from its dose increase; and iii) the determination of dose-related metabolites that could be considered as toxicity monitoring biomarkers. Methods: The in vivo experiment employed two doses of CMS versus a control group treated with normal saline, and samples of plasma, kidney, and liver were analysed with a UPLC-MS-based metabolomics protocol. Both univariate and multivariate statistical approaches (PCA, OPLS-DA, PLS regression, ROC) and pathway analysis were combined for the data interpretation. Results: The results pointed out six dose-responding metabolites (PAA, DA4S, 2,8-DHA, etc.), dysregulation of renal dopamine, and extended perturbations in renal purine metabolism. Also, the study determined altered levels of liver suberylglycine, a metabolite linked to hepatic steatosis. One of the most intriguing findings was the detection of elevated levels of renal xanthine and uric acid, that act as AChE activators, leading to the rapid degradation of acetylcholine. This evidence provides a naïve hypothesis, for the potential association between the CMS induced nephrotoxicity and CMS induced 39 neurotoxicity, that should be further investigated.

Preclinical evaluation of amiodarone for the treatment of murine leukemia P388. In vivo and in vitro investigation.

PURPOSE: The purpose of the present study was the investigation of antileukemic effect of amiodarone in leukemia P388 BDF1 bearing mice and its genotoxic and cytostatic effect in cultured normal human lymphocytes. METHODS: Leukemia P388 was used in this study. BDF1 mice were used for chemotherapy evaluation in vivo. The antitumor activity was assessed by the oncostatic parameter T/C, representing the increase of life span of drug-treated animals vs. controls. Lymphocyte cultures were used to study the genotoxic and cytostatic effect in vitro, expressed by enhanced sister chromatid exchange (SCE) and reduced proliferation rate indices (PRIS). RESULTS: Amiodarone was found to exert antileukemic potency against leukemia P388 bearing mice at all three different treatment schedules used, yielding T/C values of 155%, 163% with one cure and 230%. In the in vitro cytogenic experiments, significant increase of SCE rates by amiodarone was observed at 0.2 µM, while at the same concentration significant suppression of PRIS was achieved. CONCLUSION: According to the National Cancer Institute (NCI), a compound is characterized as potential chemotherapeutic deserving further evaluation if it produces T/C values≥125%. On the other hand the SCE assay has predictive value as a clinical assay for drugs exhibiting a strong correlation between cell killing and induction of SCEs. Further studies are warranted to clarify the structure-activity relationship of amiodarone.

Synthesis and biological evaluation of a Platinum(II)-c(RGDyK) conjugate for integrin-targeted photodynamic therapy.

A cancer-targeting conjugate 4 of a cyclometalated [N,C,N-Pt(II)] complex bearing a NˆCˆN 1,3-di(2-pyridyl)-benzene with c(RGDyK) peptide as guiding molecule was designed and synthesized for real-time drug delivery monitoring in cancer cells and photodynamic therapy (PDT). This conjugate demonstrates a mild cytostatic effect to six cancer cell lines expressing integrins at different extent, while possessing promising features for PDT. Conjugate 4 demonstrated rapid cell uptake by receptor-mediated endocytosis and efficient generation of 1O2 upon irradiation. Incubation of rat bladder cancer cells AY27 with conjugate 4 (50 µΜ) prior to blue light exposure (5 min) resulted in significant reduction (50%) of cell survival compared to control cells as measured by MTT assay post 24 h after treatment.

Combinational effect of topotecan and octreotide on murine leukemia cells in vivo and in vitro.

PURPOSE: The activity of topotecan (TPT) against a number of hematological malignancies is now notably increased. TPT is a drug which inhibits the DNA enzyme topoisomerase I (topo I), thereby leading to the induction of tumor cell apoptosis. On the other hand, octreotide (OCT) is a synthetic analogue of somatostatin, which can induce apoptosis and antiproliferative effects on various human tumor cell lines, human xenografts and animal tumors, as well as on lymphoproliferative neoplasms. Hereby, we studied the effects of TPT and OCT, and their combination in the treatment of the rodent P388 lymphocytic leukemia, in vitro and in vivo. MATERIALS AND METHODS: Cell cultures of P388 lymphocytic leukemia cells, as well as BDF1 male and female mice implanted with the P388 leukemia cells, were used for the in vitro and in vivo evaluation of the antineoplastic activity of OCT and TPT. RESULTS: A significant increase of antileukemic activity of the combined treatment with both TPT and OCT was demonstrated. These results suggest that OCT enhances the effectiveness of TPT in the treatment of leukemia. CONCLUSION: Our results indicate that the combination of OCT with TPT in the treatment of hematological neoplasias is effective, and represents an interesting addition to the future therapeutic options, because os its mechanism of action and its toxicity profile.

Synergistic interaction between a mixed ligand copper (II) chelate complex and two anticancer agents in T47D human breast cancer cells in vitro.

PURPOSE: We have developed a copper(II) chelate complex with a tridentate ONN-Schiff ligand and the anion of salicylate, showing a potent cytotoxic activity against a panel of human and murine cancer cell lines. In this experiment we have explored the combination effect between Cu(SalNEt(2))salicylate (Cu-Sal) complex and two widely used drugs in cancer chemotherapy, bleomycin (BLM) and 5-fluorouracil (5-FU), against T47D human breast cancer cells. Previous theoretical quantum-chemical studies of this complex and ass adducts with biological molecules elucidated the underlying mechanism of action of this complex. MATERIALS AND METHODS: Cells grown in adherence in 96-well microplates were exposed simultaneously to both agents for 48 h. During cytotoxicity was assessed via the XTT colorimetric assay. The combined drug interaction was assessed with the median-effect analysis and the combination index (CI). RESULTS: Concurrent treatment of cells with Cu-Sal complex and the chemotherapeutic drugs BLM and 5-FU and the antioxidant agent ascorbic acid (AsA) resulted mainly in synergistic interaction for most concentration ratios. CONCLUSION: Cu-Sal complex interacts synergistically with the chemotherapeutic drugs for most schedules of administration. These findings call for prompting to search for possible interaction of this complex with other cellular elements of fundamental importance in cell proliferation.

Octreotide neutralizes dexamethasone antitumor actions on P388 murine lymphocytic leukemia in vivo.

PURPOSE: A wide variety of human malignancies, including lymphoproliferative neoplasms, express somatistatin (SS) receptors (SS-R). SS induces apoptosis and exerts pronounced antiproliferative effects on various human tumors cell lines, human xenografts, and animal tumors including P388 lymphocytic leukemia. In patients with thymoma the combination of octreotide (OCT) with corticosteroids improves the overall response rate. It has been reported that SS can increase glucocorticoid activity. Hereby, we studied the in vitro and in vivo activity of the SS analogue OCT and of the glucocorticoid dexamethasone (DEX) alone or in combination against the murine P388 lymphocytic leukemia. MATERIALS AND METHODS: Cultures of P388 lymphocytic leukemia and BDF(1) male mice implanted with P388 cells where used for the in vitro an in vivo evaluation of the antileukemic activity of SS and DEX. RESULTS: OCT induced a moderate and DEX a satisfactory cytostatic effect in vitro. OCT produced borderline antileukemic effect when administered on days 1-5 while DEX was effective in all schemes and routes of administration. However, none of the combination schemes exerted any anti-leukemic activity both in vitro and in vivo. CONCLUSION: Since both SS and glucocorticoids exert direct (via receptors) and indirect antitumor actions (regulation of growth factor activity) on several cell lines, in vitro and in vivo, it becomes obvious that further in vitro studies shall provide the molecular evidence for the signal transduction pathways which are involved in the interactions of such important anticancer drugs. Based on the results of the present study, the simultaneous use of these drugs in clinical practice should be carefully considered.

A preclinical survey on the efficacy of lactandrate in the treatment of colon carcinoma.

PURPOSE: There has been a recent and dramatic increase in the pace of drug development for colorectal cancer which holds promise to further improve curative therapy. We tested lactandrate, an alkylating ester of D-lactam androsterone, for antineoplastic activity against colon adenocarcinoma in vitro and in vivo. MATERIALS AND METHODS: The cytostatic and cytotoxic activity of lactandrate were evaluated in vitro against 9 human colon adenocarcinoma cell lines. The in vitro testing was performed with the sulforhodamine B (SRB) colorimetric assay and the mean concentrations of each drug that generated 50% (GI50) or total (100%) growth inhibition (TGI), as well as the drug concentrations that produced cytotoxicity against 50% of the cultured cells (IC50) were calculated. The in vivo antitumour effect was determined against two rodent colon carcinomas, the Colon 26 and the relatively chemoresistant Colon 38 carcinoma, as well as against the human xenograft CX-1 colon carcinoma. RESULTS: Lactandrate displayed a satisfactory activity against the 9 human colon cancer cell lines, inducing significant growth inhibition and cytotoxicity. Lactandrate induced antiproliferative activity against colon cancer cell lines linearly correlated with the carcinoembryonic antigen (CEA) production. There was a non-linear polynomial correlation between CEA production and the cytotoxic effect of lactandrate. The more differentiated cell lines DLD-1 and HCC2998 appeared more resistant to the cytostatic effect of lactandrate. In vivo, the compound produced a significant antitumour activity against Colon 26 and Colon 38, as well as a moderate antitumour effect against CX-1 colon carcinoma. CONCLUSION: Preclinical research supports the high in vitro and in vivo antitumour potential of lactandrate against colon carcinoma. Therefore, lactandrate represents an important candidate drug for further clinical development.

Antitumour effect of a- and d- lactam androgen nitrogen mustards on non-small cell lung carcinoma.

PURPOSE: We tested 3 alkylating esters of D-lactam androsterone, 3 alkylating esters of A-lactam testosterone and the alkylating nitrogen mustard components of these esters, for antineoplastic activity on non-small cell lung carcinoma (NSCLC) in vitro and in vivo. MATERIALS AND METHODS: Cytostatic and cytotoxic activity was evaluated in vitro against 10 human NSCLC cell lines. The in vitro testing was performed with the MTT metabolic-colorimetric assay and the mean concentrations of each drug that generated 50% or total (100%) growth inhibition (GI50 and TGI, respectively) as well as the drug concentrations that produced cytotoxicity against 50% of the cultured cells (IC50) were calculated. Furthermore, the in vivo antitumour effect was determined against the relatively chemo-resistant Lewis lung carcinoma (LLC) on mice. The acute toxicity of the tested compounds was appointed in C57BL mice and the antitumor effect on LLC was assessed from the percent increase in median lifespan of the treated animals over the untreated (control) (T/C%). RESULTS: The lactam steroidal esters presented lower toxicity and increased antineoplastic activity in vitro and in vivo compared to their respective alkylating components. An A-lactam testosterone ester namely: 17beta-hydroxy- 3-aza-A-homo-4alpha-androsten-4-one-p-N,N-bis (2chloroethyl) amino phenoxy acetate (ALT-CAPOA) performed significantly higher anticancer activity in vitro and in vivo. This compound generated 37.5% 90-day disease free survivors (cures) against LLC. CONCLUSION: These results indicate a high antitumor potential of lactam steroid alkylating esters depended on the alkylating moiety as well as on the modified steroidal carrier. Preclinical research supports that ALT-CAPOA generates well-tolerated toxicity as well as superior antitumor activity against NSCLC. These significant results call for further clinical development.

Dexamethasone plus octreotide regimen increases anticancer effects of docetaxel on TRAMP-C1 prostate cancer model.

AIM: The aim of this study was to evaluate whether the neoadjuvant use of the dexamethasone (DEX) plus octreotide (OCT) regimen can improve the direct anticancer effects of docetaxel (DOC) in the TRAMP-C1 prostate cancer model. MATERIALS AND METHODS: TRAMP-C1 cells were first characterized for the expression of SSTR1-5 and then were inoculated onto the femur of C57Bl mice. Investigation protocols employed TRAMP-C1 cell proliferation and invasion assays, analysis of radiographic images of the bone lesions and overall survival of the diseased animals. RESULTS: The triple combination treatment scheme showed significant anticancer effects, in both proliferation and invasion assays, compared to any single agent treatment scheme. DOC treatment following the neoadjuvant administration of DEX plus OCT regimen improved significantly the anticancer effects both on the grading of the bone lesions and on the overall survival of the diseased animals. CONCLUSION: Our data suggest that the neoadjuvant administration of DEX plus OCT regimen can improve the anticancer effects of DOC on the TRAMP-C1 model.

Positive urinary cytology in patients with lung cancer in the absence of obvious urine tract metastases.

PURPOSE: To study the phenomenon of positive urine cytology in patients with lung cancer in the absence of obvious urothelial metastases. PATIENTS AND METHODS: 150 patients with small (SCLC) and non-small cell lung cancer (NSCLC) of all stages and 3 control groups were prospectively studied. Immunocytochemical study (cytokeratins 7-20, TTF1) in all positive urine specimens and chemokine profile (CXCR4, CCL21) study of the primary tumor in selected positive patients was performed. In experimental study, C57Bl/6 BALB/C mice injected with LLC lung and 4T1 mammary cancer cells were used for the detection of positive urine cytology. RESULTS: 11% of patients with NSCLC, 7% of patients with SCLC and none of the control group had positive urine cytology. In NSCLC, metastatic disease and high tumor burden positively correlated (p=0.01 and 0.03 respectively) with the phenomenon. In SCLC, correlation with extensive disease and multiple metastatic sites (p=0.02 and 0.04 respectively) was found. No correlation was found in either group with: age, gender, histology, performance status, line of chemotherapy, previous platinum-based chemotherapy, adrenal metastases, renal function, abnormal urinary sediment, response to chemotherapy and overall survival (p=0.9). Distinctive chemokine expression was identified in positive patients studied and was not observed in negative patients (×2 p=0.008). In the experimental study, only the LLC lung cancer cells were detected in the urine cytology of mice. CONCLUSION: This phenomenon, carrying undefined pathophysiological mechanisms, seems to characterize only patients with metastatic/extensive disease and high tumor burden. Further studies are needed to validate our preliminary chemokine expression results.