Fasting inhibits hepatic stellate cells activation and potentiates anti-cancer activity of Sorafenib in hepatocellular cancer cells.

Hepatocellular carcinoma (HCC) has a poor outcome. Most HCCs develop in the context of liver fibrosis and cirrhosis caused by chronic inflammation. Short-term fasting approaches enhance the activity of chemotherapy in preclinical cancer models, other than HCC. Multi-tyrosine kinase inhibitor Sorafenib is the mainstay of treatment in HCC. However, its benefit is frequently short-lived. Whether fasting can alleviate liver fibrosis and whether combining fasting with Sorafenib is beneficial remains unknown. A 24 hr fasting (2% serum, 0.1% glucose)-induced changes on human hepatic stellate cells (HSC) LX-2 proliferation/viability/cell cycle were assessed by MTT and flow cytometry. Expression of lypolysaccharide (LPS)-induced activation markers (vimentin, αSMA) was evaluated by qPCR and immunoblotting. Liver fibrosis and inflammation were evaluated in a mouse model of steatohepatitis exposed to cycles of fasting, by histological and biochemical analyses. A 24 hr fasting-induced changes were also analyzed on the proliferation/viability/glucose uptake of human HCC cells exposed to Sorafenib. An expression panel of genes involved in survival, inflammation, and metabolism was examined by qPCR in HCC cells exposed to fasting and/or Sorafenib. Fasting decreased the proliferation and the activation of HSC. Repeated cycles of short term starvation were safe in mice but did not improve fibrosis. Fasting synergized with Sorafenib in hampering HCC cell growth and glucose uptake. Finally, fasting normalized the expression levels of genes which are commonly altered by Sorafenib in HCC cells. Fasting or fasting-mimicking diet diets should be evaluated in preclinical studies as a mean to potentiate the activity of Sorafenib in clinical use.

Zoledronic acid-encapsulating self-assembling nanoparticles and doxorubicin: a combinatorial approach to overcome simultaneously chemoresistance and immunoresistance in breast tumors.

The resistance to chemotherapy and the tumor escape from host immunosurveillance are the main causes of the failure of anthracycline-based regimens in breast cancer, where an effective chemo-immunosensitizing strategy is lacking.The clinically used aminobisphosphonate zoledronic acid (ZA) reverses chemoresistance and immunoresistance in vitro. Previously we developed a nanoparticle-based zoledronic acid-containing formulation (NZ) that allowed a higher intratumor delivery of the drug compared with free ZA in vivo. We tested its efficacy in combination with doxorubicin in breast tumors refractory to chemotherapy and immune system recognition as a new combinatorial approach to produce chemo- and immunosensitization.NZ reduced the IC50 of doxorubicin in human and murine chemoresistant breast cancer cells and restored the doxorubicin efficacy against chemo-immunoresistant tumors implanted in immunocompetent mice. By reducing the metabolic flux through the mevalonate pathway, NZ lowered the activity of Ras/ERK1/2/HIF-1α axis and the expression of P-glycoprotein, decreased the glycolysis and the mitochondrial respiratory chain, induced a cytochrome c/caspase 9/caspase 3-dependent apoptosis, thus restoring the direct cytotoxic effects of doxorubicin on tumor cell. Moreover, NZ restored the doxorubicin-induced immunogenic cell death and reversed the tumor-induced immunosuppression due to the production of kynurenine, by inhibiting the STAT3/indoleamine 2,3 dioxygenase axis. These events increased the number of dendritic cells and decreased the number of immunosuppressive T-regulatory cells infiltrating the tumors.Our work proposes the use of nanoparticle encapsulating zoledronic acid as an effective tool overcoming at the same time chemoresistance and immunoresistance in breast tumors, thanks to the effects exerted on tumor cell and tumor-infiltrating immune cells.

Liposome armed with herpes virus-derived gH625 peptide to overcome doxorubicin resistance in lung adenocarcinoma cell lines.

New delivery systems including liposomes have been developed to circumvent drug resistance. To enhance the antitumor efficacy of liposomes encapsulating anti-cancer agents, we used liposomes externally conjugated to the 20 residue peptide gH625. Physicochemical characterization of the liposome system showed a size of 140 nm with uniform distribution and high doxorubicin encapsulation efficiency. We evaluated the effects of increasing concentrations of liposomes encapsulating Doxo (LipoDoxo), liposomes encapsulating Doxo conjugated to gH625 (LipoDoxo-gH625), empty liposomes (Lipo) or free Doxo on growth inhibition of either wild type (A549) or doxorubicin-resistant (A549 Dx) human lung adenocarcinoma. After 72 h, we found that the growth inhibition induced by LipoDoxo-gH625 was higher than that caused by LipoDoxo with an IC50 of 1 and 0.3 µM in A549 and A549 Dx cells, respectively. The data on cell growth inhibition were paralleled by an higher oxidative stress and an increased uptake of Doxo induced by LipoDoxo-gH625 compared to LipoDoxo, above all in A549 Dx cells. Cytometric analysis showed that the antiproliferative effects of each drug treatment were mainly due to the induction of apoptosis. In conclusion, liposomes armed with gH625 are able to overcome doxorubicin resistance in lung adenocarcinoma cell lines.

Levofolene modulates apoptosis induced by 5-fluorouracil through autophagy inhibition: clinical and occupational implications.

5-Fluorouracil (5-FU), often used in combination with levofolene (LF), can induce, as an important side effect, the hand-foot syndrome (HFS) due to toxicity on keratinocytes. This can also damage workers involved in its handling. In the present study, we investigated the mechanisms of the toxicity induced by 5-FU alone or together with LF on human keratinocytes in culture. We found that the two drugs, as expected, had potentiating activity on keratinocyte growth inhibition and that this effect was mediated by induction of apoptosis. In our experimental model, an increased autophagic vacuole accumulation was observed in keratinocytes treated with 5-FU as a significant increase of the monodansylcadaverine (MDC) labeling (marker of late autophagy vacuoles) was recorded. However, the synergism of 5-FU with LF on apoptotic occurrence was not paralleled by a similar increase in autophagic vacuoles at 72 h suggesting an antagonistic effect of LF on autophagy elicited by 5-FU. Differential effects on reactive oxygen species (ROS) elevation in cells treated with 5-FU alone or the combination between 5-FU and LF were also observed. 5-FU induced a time-dependent increase of both O2- and lipid peroxidation while the combination of 5-FU and LF caused a stronger intracellular O2- increase only at 24 h while at 48 and 72 h its effect was lower when compared with that one of 5-FU alone. On the other hand, the addition of LF to 5-FU caused a stronger increase of lipid peroxidation at 48 and 72 h, but its effects were significantly lower at 24 h. These results suggest for the first time that LF potentiates the cytotoxicity of 5-FU on keratinocytes likely through the antagonism on autophagy escape pathway and consequent apoptosis potentiation.

Self-assembling nanoparticles encapsulating zoledronic acid revert multidrug resistance in cancer cells.

The overexpression of ATP binding cassette (ABC) transporters makes tumor cells simultaneously resistant to several cytotoxic drugs. Impairing the energy metabolism of multidrug resistant (MDR) cells is a promising chemosensitizing strategy, but many metabolic modifiers are too toxic in vivo. We previously observed that the aminobisphosphonate zoledronic acid inhibits the activity of hypoxia inducible factor-1a (HIF-1a), a master regulator of cancer cell metabolism. Free zoledronic acid, however, reaches low intratumor concentration. We synthesized nanoparticle formulations of the aminobisphosphonate that allow a higher intratumor delivery of the drug. We investigated whether they are effective metabolic modifiers and chemosensitizing agents against human MDR cancer cells in vitro and in vivo. At not toxic dosage, nanoparticles carrying zoledronic acid chemosensitized MDR cells to a broad spectrum of cytotoxic drugs, independently of the type of ABC transporters expressed. The nanoparticles inhibited the isoprenoid synthesis and the Ras/ERK1/2-driven activation of HIF-1α, decreased the transcription and activity of glycolytic enzymes, the glucose flux through the glycolysis and tricarboxylic acid cycle, the electron flux through the mitochondrial respiratory chain, the synthesis of ATP. So doing, they lowered the ATP-dependent activity of ABC transporters, increasing the chemotherapy efficacy in vitro and in vivo. These effects were more pronounced in MDR cells than in chemosensitive ones and were due to the inhibition of farnesyl pyrophosphate synthase (FPPS), as demonstrated in FPPS-silenced tumors. Our work proposes nanoparticle formulations of zoledronic acid as the first not toxic metabolic modifiers, effective against MDR tumors.

MicroRNA-423-5p Promotes Autophagy in Cancer Cells and Is Increased in Serum From Hepatocarcinoma Patients Treated With Sorafenib.

Hepatocellular carcinoma (HCC) is the third cause of cancer-related deaths worldwide. Sorafenib is the only approved drug for patients with advanced HCC but has shown limited activity. microRNAs (miRs) have been involved in several neoplasms including HCC suggesting their use or targeting as good tools for HCC treatment. The purpose of this study was to identify novel approaches to sensitize HCC cells to sorafenib through miRs. miR-423-5p was validated as positive regulator of autophagy in HCC cell lines by transient transfection of miR and anti-miR molecules. miR-423-5p expression level was evaluated by real-time polymerase chain reaction (PCR) in sera collected from 39 HCC patients before and after treatment with sorafenib. HCC cells were cotreated with sorafenib and miR-423-5p and the effects on cell cycle, apoptosis, and autophagy were evaluated. Secretory miR-423-5p was upregulated both in vitro and in vivo by sorafenib treatment and its increase was correlated with response to therapy since 75% of patients in which an increase of secretory miR423-5p was found were in partial remission or stable disease after 6 moths from the beginning of therapy. HCC cells transfected with miR-423-5p showed an increase of cell percentage in S-phase of cell cycle paralleled by a similar increase of autophagic cells evaluated at both fluorescence activated cell sorter (FACS) and transmission electron microscopy. Our results suggest the miR423-5p can be used as a useful tool to predict response to sorafenib in HCC patients and is involved in autophagy regulation in HCC cells.

New Indole Tubulin Assembly Inhibitors Cause Stable Arrest of Mitotic Progression, Enhanced Stimulation of Natural Killer Cell Cytotoxic Activity, and Repression of Hedgehog-Dependent Cancer.

We designed 39 new 2-phenylindole derivatives as potential anticancer agents bearing the 3,4,5-trimethoxyphenyl moiety with a sulfur, ketone, or methylene bridging group at position 3 of the indole and with halogen or methoxy substituent(s) at positions 4-7. Compounds 33 and 44 strongly inhibited the growth of the P-glycoprotein-overexpressing multi-drug-resistant cell lines NCI/ADR-RES and Messa/Dx5. At 10 nM, 33 and 44 stimulated the cytotoxic activity of NK cells. At 20-50 nM, 33 and 44 arrested >80% of HeLa cells in the G2/M phase of the cell cycle, with stable arrest of mitotic progression. Cell cycle arrest was followed by cell death. Indoles 33, 44, and 81 showed strong inhibition of the SAG-induced Hedgehog signaling activation in NIH3T3 Shh-Light II cells with IC50 values of 19, 72, and 38 nM, respectively. Compounds of this class potently inhibited tubulin polymerization and cancer cell growth, including stimulation of natural killer cell cytotoxic activity and repression of Hedgehog-dependent cancer.

Advantages and risks of nanotechnologies in cancer patients and occupationally exposed workers.

INTRODUCTION: In recent years, different nanotechnology platforms for drug delivery in the area of medical biology have gained remarkable attention. AREAS COVERED: Nanoparticles (NPs) used as drug delivery vehicles consist of different materials such as natural or synthetic polymers, lipids or metals. They have an ultra-small size, large surface area-to-mass ratio and high reactivity. Although there are many data on the advantages in terms of both higher efficacy and less adverse effects of nanodrugs, several recent findings have reported unexpected toxicities giving origin to nanotoxicology. EXPERT OPINION: Despite the great promise that NPs show, few studies have examined the human body's reaction due to NP exposure in both patients and workers. To perform this type of evaluation, it is necessary to define an adequate index of exposure, and the measure of this index is representative of what the worker is breathing. The properties of the nanomaterials used for designing NPs, such as in the case of poorly biocompatible materials (carbon nanotubes or heavy metals), and their chemical composition (as in the case of liposomes) largely contribute in determining potential side effects. Awareness of the levels of particles, which can cause health effects, is necessary for the workers and exposed patients.

Animal models in studies of cardiotoxicity side effects from antiblastic drugs in patients and occupational exposed workers.

Cardiotoxicity is an important side effect of cytotoxic drugs and may be a risk factor of long-term morbidity for both patients during therapy and also for staff exposed during the phases of manipulation of antiblastic drugs. The mechanism of cardiotoxicity studied in vitro and in vivo essentially concerns the formation of free radicals leading to oxidative stress, with apoptosis of cardiac cells or immunologic reactions, but other mechanisms may play a role in antiblastic-induced cardiotoxicity. Actually, some new cytotoxic drugs like trastuzumab and cyclopentenyl cytosine show cardiotoxic effects. In this report we discuss the different mechanisms of cardiotoxicity induced by antiblastic drugs assessed using animal models.

A mechanistic study on the cardiotoxicity of 5-fluorouracil in vitro and clinical and occupational perspectives.

Fluoropyrimidines are key agents for the treatment of gastrointestinal tract adenocarcinomas. The possible cardiotoxic effects in patients and occupationally exposed workers are multifactorial and remain a puzzle to solve for investigators. In the present study, we study what cell death pathways and what doses can determine direct cardiotoxic effects of 5-fluorouracil (5-FU) and doxorubicin (DOXO) on rat cardiocytes (H9c2) and a human colon adenocarcinoma (HT-29) cell line, already reported to be sensitive to 5-FU. We have found that 5-FU induced 50% growth inhibition (IC:50) at 72 h with concentrations of 400 µM and 4 µM on H9c2 and HT-29, respectively. Moreover, we have found that the addition of Levofolinic Acid (LF) to 5-FU potentiated the growth inhibition induced by 5-FU. The growth inhibition induced by 5-FU alone or in combination with LF in cardiocytes was paralleled by an increase of thiobarbituric acid-reactive species (Tbars) and end products of nitric oxide (NO) suggesting the increase of the oxidative stress status in cardiocytes. Interestingly, these effects were strongly potentiated by the addition of LF, a biochemical modulator of 5-FU activity. Our data suggest that agents such as 5-FU different from anthracyclines, conventionally related to the induction of cardiotoxic effects, can also induce cardiocyte damage paralleled by oxidative stress. The strategies based upon the use of scavengers could be used in order to prevent this effect.

Nanoparticles for the delivery of zoledronic acid to prostate cancer cells: a comparative analysis through time lapse video-microscopy technique.

Time-lapse live cell imaging is a powerful tool for studying the responses of cells to drugs. Zoledronic acid (ZOL) is the most potent aminobiphosphonate able to induce cell growth inhibition at very low concentrations. The lack of clear evidence of ZOL-induced anti-cancer effects is likely due to its unfavorable pharmacokinetic profile. The use of nanotechnology-based formulations allows overcoming these limitations in ZOL pharmaco-distribution. Recently, stealth liposomes (LIPOs) and new self-assembly PEGylated nanoparticles (NPs) encapsulating ZOL were developed. Both the delivery systems showed promising anticancer activity in vitro and in vivo. In this work, we investigated the cytostatic effect of these novel formulations (LIPOs and NPs) compared with free ZOL on 2 different prostate cancer cell lines, PC 3 and DU 145 and on prostate epithelial primary cells EPN using time lapse video-microscopy (TLVM). In PC3 cells, free ZOL showed a significant anti-proliferative effect but this effect was lower than that induced by LIPOs and NPs encapsulating ZOL; moreover, LIPO-ZOL was more potent in inducing growth inhibition than NP-ZOL. On the other hand, LIPO-ZOL slightly enhanced the free ZOL activity on growth inhibition of DU 145, while the anti-proliferative effect of NP-ZOL was not statistically relevant. These novel formulations did not induce anti-proliferative effects on EPN cells. Finally, we evaluated cytotoxic effects on DU145 where, LIPO-ZOL induced the highest cytotoxicity compared with NP-ZOL and free ZOL. In conclusion, ZOL can be transformed in a powerful anticancer agent, if administered with nanotechnology-based formulations without damaging the healthy tissues.

5-Fluorouracil induces apoptosis in rat cardiocytes through intracellular oxidative stress.

BACKGROUND: Cardiotoxicity is a major complication of anticancer drugs, including anthracyclines and 5-fluorouracil(5-FU) and it can have detrimental effects both in patients and workers involved in the preparation of chemotherapy. METHODS: Specifically, we have assessed the effects of increasing concentrations of 5-FU and doxorubicin (DOXO) on proliferation of H9c2 rat cardiocytes and HT-29 human colon adenocarcinoma cells by MTT assay. Cells were treated for 24, 48 and 72 h with different concentrations of the two drugs alone or with 5-FU in combination with 10(-4) M of levofolene (LF). RESULTS: 5-FU induced a time- and dose-dependent growth inhibition in both cell lines. The 50% growth inhibition (IC:50) was reached at 72 h with concentrations of 4 µM and 400 µM on HT-29 and H9c2, respectively. The addition of LF to 5-FU enhanced this effect. On the other hand, the IC:50 of DOXO was reached at 72 h with concentrations of 0.118 µM on H9c2 and of 0.31 µM for HT-29. We have evaluated the cell death mechanism induced by 50% growth inhibitory concentrations of 5-FU or DOXO in cardiocytes and colon cancer cells. We have found that the treatment with 400 µM 5-FU induced apoptosis in 32% of H9c2 cells. This effect was increased by the addition of LF to 5-FU (38% of apoptotic cells). Apoptosis occurred in only about 10% of HT-29 cells treated with either 5-FU or 5-FU and LF in combination. DOXO induced poor effects on apoptosis of both H9c2 and HT-29 cells (5-7% apoptotic cells, respectively). The apoptosis induced by 5-FU and LF in cardiocytes was paralleled by the activation of caspases 3, 9 and 7 and by the intracellular increase of O(2-) levels. CONCLUSIONS: These results suggest that cardiotoxic mechanism of chemotherapy agents are different and this disclose a new scenario for prevention of this complication.

Chondroitin sulphate enhances the antitumor activity of gemcitabine and mitomycin-C in bladder cancer cells with different mechanisms.

Non-muscle invasive bladder cancer is the most common type of bladder cancer in Western countries. The glycosaminoglycan (GAG) layer at the bladder surface non-specifically blocks the adherence of bacteria, ions and molecules to the bladder epithelium and bladder cancer cells express CD44 that binds GAG. Currently, there are few options other than cystectomy for the management of non-muscle invasive bladder cancer with intravesical chemotherapy using several drugs such as gemcitabine (GEM) and mitomycin-C (MMC) with poor prophylactic activity. In this study, we investigated the effects of the GAG chondroitin sulphate (CS) on the growth inhibition of human bladder cancer cell lines HT-1376 and effects of the combination between GEM or MMC with CS. We have found that CS, MMC and GEM induced 50% growth inhibition at 72 h. Therefore, we have evaluated the growth inhibition induced by different concentration of CS in combination with MMC or GEM, respectively, at 72 h. We have observed, at Calcusyn analysis, a synergism when HT-1376 cells were treated with CS in combination with MMC or GEM, respectively, if used at an equimolar ratio. We have also found that CS/GEM combination induced a strong potentiation of apoptosis with the consequent activation of caspases 9 and 3. On the other hand, HT-1376 cells were necrotic if exposed to the CS/MMC combination and no signs of caspase activation was observed. In conclusion, in the human bladder cancer cell line HT-1376 pharmacological combination of CS with GEM or MMC resulted in a strong synergism on cell growth inhibition.