Pharmacological inhibition of tumor anabolism and host catabolism as a cancer therapy.

The malignant energetic demands are satisfied through glycolysis, glutaminolysis and de novo synthesis of fatty acids, while the host curses with a state of catabolism and systemic inflammation. The concurrent inhibition of both, tumor anabolism and host catabolism, and their effect upon tumor growth and whole animal metabolism, have not been evaluated. We aimed to evaluate in colon cancer cells a combination of six agents directed to block the tumor anabolism (orlistat + lonidamine + DON) and the host catabolism (growth hormone + insulin + indomethacin). Treatment reduced cellular viability, clonogenic capacity and cell cycle progression. These effects were associated with decreased glycolysis and oxidative phosphorylation, leading to a quiescent energetic phenotype, and with an aberrant transcriptomic landscape showing dysregulation in multiple metabolic pathways. The in vivo evaluation revealed a significant tumor volume inhibition, without damage to normal tissues. The six-drug combination preserved lean tissue and decreased fat loss, while the energy expenditure got decreased. Finally, a reduction in gene expression associated with thermogenesis was observed. Our findings demonstrate that the simultaneous use of this six-drug combination has anticancer effects by inducing a quiescent energetic phenotype of cultured cancer cells. Besides, the treatment is well-tolerated in mice and reduces whole animal energetic expenditure and fat loss.

The nerve growth factor alters calreticulin translocation from the endoplasmic reticulum to the cell surface and its signaling pathway in epithelial ovarian cancer cells.

Ovarian cancer is the seventh most common cancer among women worldwide, causing approximately 120,000 deaths every year. Immunotherapy, designed to boost the body's natural defenses against cancer, appears to be a promising option against ovarian cancer. Calreticulin (CRT) is an endoplasmic reticulum (ER) resident chaperone that, translocated to the cell membrane after ER stress, allows cancer cells to be recognized by the immune system. The nerve growth factor (NGF) is a pro-angiogenic molecule overexpressed in this cancer. In the present study, we aimed to determine weather NGF has an effect in CRT translocation induced by cytotoxic and ER stress. We treated A2780 ovarian cancer cells with NGF, thapsigargin (Tg), an ER stress inducer and mitoxantrone (Mtx), a chemotherapeutic drug; CRT subcellular localization was analyzed by immunofluorescence followed by confocal microscopy. In order to determine NGF effect on Mtx and Tg-induced CRT translocation from the ER to the cell membrane, cells were preincubated with NGF prior to Mtx or Tg treatment and CRT translocation to the cell surface was determined by flow cytometry. In addition, by western blot analyses, we evaluated proteins associated with the CRT translocation pathway, both in A2780 cells and human ovarian samples. We also measured NGF effect on cell apoptosis induced by Mtx. Our results indicate that Mtx and Tg, but not NGF, induce CRT translocation to the cell membrane. NGF, however, inhibited CRT translocation induced by Mtx, while it had no effect on Tg-induced CRT exposure. NGF also diminished cell death induced by Mtx. NGF effect on CRT translocation could have consequences in immunotherapy, potentially lessening the effectiveness of this type of treatment.

Influence of nucleotide excision repair on mitoxantrone cytotoxicity.

Mitoxantrone (MXT) is an anticancer drug structurally related to anthracyclines, such as doxorubicin (DOX). Here we report that cells deficient in nucleotide excision repair (NER) are very sensitive to MXT. However, cells deficient in each of the NER sub-pathways - transcription coupled repair (deficient in CSB protein) and global genome repair (deficient in XPC protein) - demonstrate a difference in sensitivity from each other and also show different responses in cell cycle profile, DNA synthesis and topo II DNA complex formation upon MXT treatment. XPC-deficient cells are slightly more resistant than CSB-deficient cells, and in the same way as MRC5 NER-proficient cells, show G2/M arrest, normal DNA synthesis rate and a pattern of formation of complexes similar to proficient cells, whereas CSB-deficient cells show accumulation in S phase, reduced DNA synthesis and a more intense signal of topo II DNA complexes, indicating that they remain longer in these cells. Complementation of CSB mutant cells with CSB rescue MXT-induced sensitivity and also a decrease in the signal intensity of the complexes, suggest that resolution of these lesions would take place. Taken together, our results indicate that NER proteins are implicated in the response to MXT and that CSB protein has a key role in processing MXT-induced topo II DNA complexes.

The phytoestrogen genistein enhances multidrug resistance in breast cancer cell lines by translational regulation of ABC transporters.

Breast cancer is the most frequent malignancy in women. Multidrug resistance due to overexpression of ABC drug transporters is a common cause of chemotherapy failure and disease recurrence. Genistein (GNT) is a phytoestrogen present in soybeans and hormone supplements. We investigated the effect of GNT on the expression and function of ABC transporters in MCF-7 and MDA-MB-231 breast cancer cell lines. Results demonstrated an induction at the protein level of ABCC1 and ABCG2 and of ABCC1 in MCF-7 and MDA-MB-231, respectively. MCF-7 cells showed a concomitant increase in doxorubicin and mitoxantrone efflux and resistance, dependent on ABCG2 activity. ABCC1 induction by GNT in MDA-MB-231 cells modified neither drug efflux nor chemoresistance due to simultaneous acute inhibition of the transporter activity by GNT. All inductions took place at the translational level, as no increment in mRNA was observed and protein increase was prevented by cycloheximide. miR-181a, already demonstrated to inhibit ABCG2 translation, was down-regulated by GNT, explaining translational induction. Effects were independent of classical estrogen receptors. Results suggest potential nutrient-drug interactions that could threaten chemotherapy efficacy, especially in ABCG2-expressing tumors treated with substrates of this transporter.

Novel cytotoxic isolated from Jamaican Hyptis verticillata jacq induces apoptosis and overcomes multidrug resistance.

BACKGROUND: Adult T-cell leukemia is an aggressive hematological malignancy with a poor clinical prognosis, and a rapid resistance to chemotherapy is rapid. MATERIALS AND METHODS: Cytotoxicity assay-directed fractionation identified a novel lignan-related agent, 4-methoxy-9-(3,4,5-trimethoxyphenyl)-8, 9 - dihydrofuro[3',4':6,7]naphtho[2,3-d][1,3]dioxol-6(5H)-one (4-MTDND) from the Jamaican plant Hyptis verticillata jacq, and its effects on apoptosis, cell cycle and drug resistance were elucidated. RESULTS: The novel agent, 4-MTDND, exhibited cytotoxicity against myriad cancer types, with a wide therapeutic index of 30- to 40-fold, promoted G(2)/M arrest and up-regulated expression of pro-apoptotic proteins p53 and BAX, as well as enhanced activation of caspase-3, caspase-9 and poly (ADP ribose) polymerase, consistent with apoptosis induction. Multidrug-resistant cancer cells were as susceptible to 4-MTDND as their non-resistant control counterparts, with 4-MTDND having greater efficacy compared to standard chemotherapy agents etoposide and mitoxantrone. CONCLUSION: The novel cytotoxic agent 4-MTDND induces G(2)/M arrest and apoptosis in cancer cells possibly due to direct DNA damage or interference with topoisomerase II.

Expression of multidrug resistance proteins in prostate cancer is related with cell sensitivity to chemotherapeutic drugs.

BACKGROUND: Multidrug resistance (MDR) proteins have been associated with the lack of chemotherapy response. Expression of these proteins has been described in the prostate, but there is no information about their role in the chemotherapy response of prostate cancer (PC). We studied the gene and protein expression of MDR proteins in primary cell cultures from PC tumors and PC cell lines, their relationship with chemotherapy and their effects on cell survival. METHODS: Primary cell cultures from PC were obtained from samples provided by our Institutional Hospital. Cell lines LNCaP, PC3, and DU145 were also examined. Cells were treated during 72 hr with several chemotherapeutic drugs. Protein and mRNA expressions of P-glycoprotein (P-Gp), MRP1 and LRP, before and after drug treatment, were evaluated by RT-PCR and Western blot analyses. The effect on cell survival was evaluated by proliferation assays (MTT), and cell cycle and apoptosis by flow cytometry. RESULTS: Primary PC cultures exhibited higher MDR protein expression and lower drug sensitivity than cell lines, in which P-Gp was not detected. Docetaxel and mitoxantrone displayed the highest apoptotic effect. Exposure to chemotherapeutic drugs increased apoptosis, cell cycle arrest, and MDR expression. Long-term treatment with doxorubicin diminished apoptosis elicited by all drugs examined in this study, suggesting a cross-resistance phenomenon. CONCLUSIONS: Low chemotherapy response observed in PC primary cultures could be explained, in part, by the high levels of MDR proteins (intrinsic MDR phenotype), and also, by their over-expression induced after long-term exposure to drugs (acquired MDR phenotype), which increase treatment resistance. Prostate 69: 1448-1459, 2009. (c) 2009 Wiley-Liss, Inc.

P-glycoprotein expression by cancer cells affects cell cytotoxicity and cell-cycle perturbations induced by six chemotherapeutic drugs.

BACKGROUND: P-glycoprotein (P-gp)-mediated chemoresistance plays an important role in drug resistance. METHODS: We investigated if P-gp expression by cancer cells affects cell cytotoxicity and cell-cycle perturbations induced by six commonly used chemotherapeutic agents (doxorubicin, daunorubicin, mitoxantrone, vinblastine, paclitaxel, and colchicine). For this purpose, we used KB cell lines as a model and flow cytometric cell viability and drug-induced cell-cycle perturbation-based methods. We continuously cultured KB cell lines, in the presence of various anticancer agents and measured the cell-cycle kinetics and percentage of cell viability by flow cytometry at different time intervals. RESULTS: The highly resistant cell line KB V-1 was significantly less susceptible to drug cytotoxicity than the sensitive cell line KB 3-1. KB V-1 cells cultured with different chemotherapy agents continued cell-cycle progression without any significant perturbation of cell compartment distribution. Addition of verapamil, by inhibiting P-gp, reversed resistance thereby increasing drug cytotoxicity and allowing the appearance of drug induced cell-cycle perturbations. CONCLUSIONS: We conclude that P-gp interferes with cell cytotoxicity and cell-cycle perturbations induced by anticancer agents. Flow cytometry can be used to conveniently detect P-gp-mediated chemoresistance by cancer cells as it allows the analysis of cell cytotoxicity and cell-cycle modifications after exposure to specific chemotherapeutic and reversal agents.

Effects of treatment with mitoxantrone in combination with novobiocin, caffeine and ara-C on human lymphocytes in culture.

Many cytotoxic mechanisms have been proposed for the antineoplastic drug mitoxantrone (MXN), among them an interaction mediated by the cleavable DNA topoisomerase II complex. Cotreatment of human peripheral blood lymphocyte cultures from six donors with MXN (12 x 10(-6) micrograms/ml) and the inhibitors of DNA repair or synthesis, caffeine (10(-3) M) and ara-C (5 x 10(-6) M) administered as 30-min pulses did not potentiate the clastogenic effect of MXN on this biological system. However, treatment with MXN and novobiocin (10 micrograms/ml), a non-specific DNA topoisomerase II inhibitor, led to a decrease in the chromosome aberrations induced by MXN in the G2 phase of the cell cycle.

In vitro evaluation of acute effects of mitoxantrone (Novantrone) in rat and guinea pig atria.

Since guinea pig and rat atria have been used as models to study acute anthracycline-induced cardiotoxicity, experiments were carried out in these preparations to evaluate possible acute cardiac effects mediated by mitoxantrone (MTX). After a latency period of approximately 90 min, MTX (10(-5)-10(-4) M) promoted a concentration-related and time-dependent decrease of spontaneous rate in guinea pig atria. A similar but less intense effect after a longer latency interval was observed in rat atria. In this preparation, MTX (10(-5)-10(-4) M) incubated up to 150 min., induced a gradual competitive beta-adrenergic blocking effect on the positive chronotropic action of isoproterenol. This was characterized by a progressive decline of pD2 values without altering Emax. A similar and stronger effect was found in isolated guinea pig atria incubated under same conditions with MTX, except that 10(-4) M exposed for 150 min. was able to depress the Emax to isoproterenol by 21.2%. In addition, MTX (10(-4) M) in this model promoted a non-competitive antagonistic effect on the chronotropic action of histamine. These data are compatible with the idea that MTX could induce cardiac acute effects qualitatively similar to but of lower potency than those produced by doxorubicin in these two models. In addition, guinea pig atria seemed to display higher sensitivity to MTX compared to rat atrial preparations.

Comparison of acute effects of mitoxantrone and doxorubicin in guinea-pig atria.

1. The acute effects of doxorubicin (DOX) and mitoxantrone (MTX) on basal rate and on positive chronotropic activity induced by 1-noradrenaline (1-NA) were investigated in isolated guinea-pig atria. 2. DOX (10(-5)-10(-4)M) progressively depressed atrial rate after a short latency period. Only 10(-4) M MTX reduced the spontaneous frequency after 120 and 180 min incubation. This effect was significantly lower to that elicited by DOX (10(-4)M). 3. Atropine (1.5 x 10(-6) M) and reserpine pretreatment did not affect the negative chronotropic action induced by DOX or MTX. 4. DOX (10(-5)-10(-4) M) produced a significant reduction of the maximal chronotropic response (Emax) to 1-noradrenaline (1-NA) after 60, 120 and 180 min of exposure. 5. MTX (10(-5)-10(-4) M) after 60 and 120 min incubation induced a beta-adrenergic, concentration- and time-dependent, competitive blocking effect. After 180 min of exposure, MTX (10(-4) M) reduced the Emax to 1-NA which was of less magnitude to that produced by DOX (10(-4) M). 6. Although both DOX and MTX depressed spontaneous and 1-NA induced chronotropic activity, MTX effects were of a slower onset and development compared to those exerted by DOX.

[Evaluation of ventricular function in patients treated with mitoxantrone and 4'-epidoxorubicin]. / Valoración de la función ventricular en pacientes tratados con mitoxantrone y 4'epidoxorubicina.

Serial assessment of ventricular function by means of radionuclide angiography was performed in previously untreated patients with lymphoproliferative diseases who received either 4'epidoxorubicin or mitoxantrone for longer than 6 months. No changes were observed in left ventricular function in patients received mitoxantrone at doses ranged 90 to 165 mg (mean 113 mg). Three patients (7%) in 4'epidoxorubicin group showed less than or equal to 10% drop in left ventricular ejection, but without clinical manifestations. The doses in this group were 420 to 810 mg (mean 610 mg). We felt that patients without high-risk factors (radiotherapy to mediastinum or previously anthracycline therapy) could be treated with high doses of both drugs and that radionuclide angiography is a useful method for monitoring cardiotoxicity of antineoplastic drugs.