Sensitization of U937 leukemia cells to doxorubicin by the MG132 proteasome inhibitor induces an increase in apoptosis by suppressing NF-kappa B and mitochondrial membrane potential loss.

BACKGROUND: The resistance of cancerous cells to chemotherapy remains the main limitation for cancer treatment at present. Doxorubicin (DOX) is a potent antitumor drug that activates the ubiquitin-proteasome system, but unfortunately it also activates the Nuclear factor kappa B (NF-кB) pathway leading to the promotion of tumor cell survival. MG132 is a drug that inhibits I kappa B degradation by the proteasome-avoiding activation of NF-кB. In this work, we studied the sensitizing effect of the MG132 proteasome inhibitor on the antitumor activity of DOX. METHODS: U937 human leukemia cells were treated with MG132, DOX, or both drugs. We evaluated proliferation, viability, apoptosis, caspase-3, -8, and -9 activity and cleavage, cytochrome c release, mitochondrial membrane potential, the Bcl-2 and Bcl-XL antiapoptotic proteins, senescence, p65 phosphorylation, and pro- and antiapoptotic genes. RESULTS: The greatest apoptosis percentage in U937 cells was obtained with a combination of MG132 + DOX. Likewise, employing both drugs, we observed a decrease in tumor cell proliferation and important caspase-3 activation, as well as mitochondrial membrane potential loss. Therefore, MG132 decreases senescence, p65 phosphorylation, and the DOX-induced Bcl-2 antiapoptotic protein. The MG132 + DOX treatment induced upregulation of proapoptotic genes BAX, DIABLO, NOXA, DR4, and FAS. It also induced downregulation of the antiapoptotic genes BCL-XL and SURVIVIN. CONCLUSION: MG132 sensitizes U937 leukemia cells to DOX-induced apoptosis, increasing its anti-leukemic effectiveness.

Increase of IFN-γ and TNF-α production in CD107a + NK-92 cells co-cultured with cervical cancer cell lines pre-treated with the HO-1 inhibitor.

BACKGROUND: Natural killer (NK) cells eliminate virus-infected and tumor cells through the release of perforins and granzymes; they also produce Interferon gamma (IFN-γ) and Tumor necrosis factor alpha (TNF-α), which induce apoptosis in target cells. Many tumors express Heme oxygenase 1 (HO-1), and this expression has been associated with avoiding immunosuppression and apoptosis. In this work, HO-1+ Cervical cancer cell (CCC) lines were pre-treated with HO-1 inhibitor and we assessed whether this inhibition enhanced the sensitivity of CCC to NK cell activity. METHODS: We assessed the expression of HO-1 in HeLa, SiHa, and C-33A CCC by Flow cytometry (FC). CCC were pre-treated with SnPP or ZnPP HO-1 inhibitors. After that, NK-92 cells were co-cultured with HeLa, SiHa, and C-33A CCC pre-treated or not with HO-1 inhibitors, and the expression of IFN-γ, TNF-α, CD107a, Granzyme B, NKp44, NKp46, NKp30, and NKG2D was evaluated by FC. RESULTS: CCC lines HeLa, SiHa, and C-33A expressed HO-1. Inhibition of HO-1 in these cells increased the expression of IFN-γ and TNF-α in CD107a + NK-92 cells. We observed a reduction in the expression of NKG2D, NKp46, and NKp30 in NK cells co-cultured with HeLa and SiHa cells, and when HeLa and SiHa cells were pre-treated with the HO-1 inhibitors, the expression of NKG2D and NKp30 in NK cells was restored. We observed a similar effect in NK cells co-cultured with C-33A cells in NKp30 expression. CONCLUSION: Inhibition of HO-1 in CCC induces an increase in IFN-γ and TNF-α production in CD107a + NK-92 cells and restores NKG2D, NKp46 and NKp30 downmodulation in NK cells.

Pentoxifylline and the proteasome inhibitor MG132 induce apoptosis in human leukemia U937 cells through a decrease in the expression of Bcl-2 and Bcl-XL and phosphorylation of p65.

BACKGROUND: In Oncology, the resistance of the cancerous cells to chemotherapy continues to be the principal limitation. The nuclear factor-kappa B (NF-κB) transcription factor plays an important role in tumor escape and resistance to chemotherapy and this factor regulates several pathways that promote tumor survival including some antiapoptotic proteins such as Bcl-2 and Bcl-XL. In this study, we investigated, in U937 human leukemia cells, the effects of PTX and the MG132 proteasome inhibitor, drugs that can disrupt the NF-κB pathway. For this, we evaluated viability, apoptosis, cell cycle, caspases-3, -8, -9, cytochrome c release, mitochondrial membrane potential loss, p65 phosphorylation, and the modification in the expression of pro- and antiapoptotic genes, and the Bcl-2 and Bcl-XL antiapoptotic proteins. RESULTS: The two drugs affect the viability of the leukemia cells in a time-dependent manner. The greatest percentage of apoptosis was obtained with a combination of the drugs; likewise, PTX and MG132 induce G1 phase cell cycle arrest and cleavage of caspases -3,-8, -9 and cytochrome c release and mitochondrial membrane potential loss in U937 human leukemia cells. In these cells, PTX and the MG132 proteasome inhibitor decrease p65 (NF-κB subunit) phosphorylation and the antiapoptotic proteins Bcl-2 and Bcl-XL. We also observed, with a combination of these drugs overexpression of a group of the proapoptotic genes BAX, DIABLO, and FAS while the genes BCL-XL, MCL-1, survivin, IκB, and P65 were downregulated. CONCLUSIONS: The two drugs used induce apoptosis per se, this cytotoxicity was greater with combination of both drugs. These observations are related with the caspases -9, -3 cleavage and G1 phase cell cycle arrest, and a decrease in p65 phosphorylation and Bcl-2 and Bcl-XL proteins. As well as this combination of drugs promotes the upregulation of the proapoptotic genes and downregulation of antiapoptotic genes. These observations strongly confirm antileukemic potential.

Addition of pentoxifylline to pegylated interferon-alpha-2a and ribavirin improves sustained virological response to chronic hepatitis C virus: a randomized clinical trial.

BACKGROUND AND AIM: The commonly accepted treatment for hepatitis C virus (HCV) infection, pegylated interferon alpha (PEG INF-alpha) and ribavirin, leads to 50-60% of sustained virological response (SVR). On the other hand, pentoxifylline (PTX) possesses antiviral and hepatoprotector properties. AIM: To investigate whether the addition of PTX to conventional hepatitis C treatment increases SVR. MATERIAL AND METHODS: Seventy two patients of both genders were studied in a randomized fashion; the diagnosis of chronic HCV infection was made according to clinical and laboratory criteria and histopathologically classified according to METAVIR scoring system criteria. HCV viral load was tested by PCR, baseline, and after 6 months of treatment, as well as anti-HCV, anti-hepatitis B virus , and anti-human immunodeficiency virus antibodies by enzyme-linked immunosorbent assay. During 48 weeks, control group patients were treated with PEG INF-alpha- 2a plus ribavirin. PTX was administered to Experimental Group patients prior to the treatment. RESULTS: Demographic data were similar in both groups. Experimental- and control-group subjects were at F2 and F3 states according to the METAVIR classification. The most common HCV genotypes were 1a and 1b (39% in the control group in each case, and 42% in the experimental group in each case). At the end of the study, hepatic enzymes and viral load decreased in both groups to similar values. SVR in the experimental group increased significantly (p < 0.05) when compared with standard therapy alone. CONCLUSION: Addition of PTX to conventional chronic hepatitis C treatment may increase the percentage of patients with SVR.

Pentoxifylline sensitizes human cervical tumor cells to cisplatin-induced apoptosis by suppressing NF-kappa B and decreased cell senescence.

BACKGROUND: Worldwide, cervical cancer is the second most common causes of cancer in women and represents an important mortality rate. Cisplatin (CIS) is a very important antitumoral agent and can lead tumor cells toward two important cellular states: apoptosis and senescence. In some types of cancers pentoxifylline (PTX) sensitizes these cells to the toxic action of chemotherapeutics drugs such as adriamycin, inducing apoptosis. In the present work, we studied in vitro whether PTX alone or in combination with CIS induces apoptosis and/or senescence in cervix cancer HeLa and SiHa cell lines infected with HPV types 16 and 18, respectively, as well as in immortalized keratinocytyes HaCaT cells. METHODS: HeLa (HPV 18+), SiHa (HPV 16+) cervix cancer cells and non-tumorigenic immortalized HaCaT cells (control) were treated with PTX, CIS or both. The cellular toxicity and survival fraction of PTX and CIS were determinate by WST-1 and clonogenic assays respectively. Apoptosis, caspase activation and phosphorylation of ERK1/2, p38, p65 (NF-κB), Bcl-2 and Bcl-XL anti-apoptotic proteins were determinated by flow cytometry. Senescence by microscopy. Phosphorylation of IκBα and IκB total were measured by ELISA. Pro-apoptotic, anti-apoptotic and senescence genes, as well as HPV-E6/7 mRNA expression, were detected by RT-PCR. RESULTS: Our results show that after 24 hours of incubation PTX per se is toxic for cancer cells affecting cell viability and inducing apoptosis. The toxicity in HaCaT cells was minimal. CIS induces apoptosis in HeLa and SiHa cells and its effect was significantly increases when the cells were treated with PTX + CIS. In all studies there was a direct correlation with levels of caspases (-3, -6, -7, -9 and -8) activity and apoptosis. CIS induces important levels of senescence and phosphorylation of ERK1/2, p38, p65/RELA, and IκBα, and decreased the expression of anti-apoptotic protein Bcl-XL. Surprisingly these levels were significantly reduced by PTX in tumor cells, and at the same time, increases the expression of pro-apoptotic genes. CONCLUSION: PTX sensitizes cervical cancer cells to CIS-induced apoptosis and decreases the CIS-induced senescence in these cells via inhibition of NF-κB signaling pathway; diminishes expression of antiapoptotic proteins and the activation of caspases.

[Comparison of two types of anesthesia on plasma levels of inflammatory markers]. / Comparación de dos técnicas anestésicas sobre los niveles plasmáticos de marcadores inflamatorios.

BACKGROUND: The aim of this work was to determine and compare plasma levels of proinflammatory cytokines (IL-1beta, IL-6 and TNF-alpha), C-reactive protein (CRP) and lipoperoxides in patients submitted to laparoscopic cholecystectomy under general anesthesia or regional anesthesia. METHODS: Two groups of 15 patients of both sexes were submitted to laparoscopic cholecystectomy either with general or regional anesthesia. In all cases we obtained three samples of plasma. The first sample was collected immediately before surgery, whereas the other samples were collected 60 min and 24 h after the cholecystectomy. Using commercial kits, plasma levels of IL-1beta, IL-6 and TNF-alpha were quantified by ELISA, whereas lipoperoxides were determined by a colorimetric method. The ultrasensitive CRP was determined in the hospital by the Prestige 24I automated method. RESULTS: In all determinations, proinflammatory cytokines increased 24 h after surgery except plasma levels of IL-1beta in the regional anesthesia group. Comparison of the two different groups showed that the general anesthesia group had a similar or higher quantity of proinflammatory cytokines and oxidative stress when compared to the regional anesthesia group (p <0.05). In contrast, plasma levels of CRP were lower in the general anesthesia group (p <0.005). CONCLUSIONS: The type of anesthesia influences in a different manner the secretion of soluble mediators of inflammation. These observations may have important clinical repercussions.

In vivo and in vitro sensitization of leukemic cells to adriamycin-induced apoptosis by pentoxifylline. Involvement of caspase cascades and IkappaBalpha phosphorylation.

UNLABELLED: The aim of this work was to investigate whether in vivo and in vitro pentoxifylline (PTX) sensitizes hematological tumor cells to adriamycin (ADM)-induced apoptosis, and to investigate the involvement of caspase cascades and phosphorylated forms of IkappaBalpha. Balb/c mice inoculated intraperitoneally with L5178-Y murine lymphoma cells were used for in vivo experiments and for survival studies. The U937 human monocytic cell line was used for in vitro experiments. Both cell lines were treated under similar experimental conditions with PTX and/or ADM to assess their effects on apoptosis. Apoptosis was evaluated by fluorescence microscopy with ethidium bromide and acridine orange staining and confirmed by electrophoretic DNA analysis. Caspase inhibitors Z-VAD-fmk, Z-DEVD-fmk, and Z-LEHD-fmk were used to investigate the involvement of caspase cascades. C-terminally and Ser32 phosphorylated forms of IkappaBalpha were evaluated in cytoplasmic extracts in the absence or presence of TNFalpha. RESULTS: In vivo, PTX (50 mg/kg) with ADM (5 mg/kg) increased the apoptotic index relative to PTX or ADM administered alone, time- and dose-dependently. DNA laddering appeared in lymphoma cells treated with PTX+ADM at 24 h, whereas neither untreated control, PTX-, nor ADM-treated cells showed DNA fragmentation. All (100%) tumor-bearing mice treated with PTX (25 mg/kg)+ADM (2.5 mg/kg) survived for 1 year, whereas the mortality rates of mice treated with either PTX or ADM alone at the same doses were similar to that of untreated tumor-bearing mice (28+/-3 days). Caspase inhibitors inhibited apoptosis more efficiently in PTX- or ADM-treated cultures than in PTX+ADM-treated cultures. Pretreatment with TNFalpha (10 ng/mL) increased apoptosis in PTX- or ADM-treated U937 cells. However, the apoptotic index of PTX+ADM-treated cultures was significantly reduced and the expression of C-terminally and Ser32 phosphorylated IkappaBalpha was reduced. PTX sensitizes hematological malignancies to ADR-induced apoptosis. An independent caspase pathway is involved in PTX+ADM-induced apoptosis. The phosphorylation status of IkappaBalpha is closely related via TNFalpha to the possible mechanisms of drug resistance.

Gamma-irradiation induced apoptosis in peritoneal macrophages by oxidative stress. Implications of antioxidants in caspase mitochondrial pathway.

The in vivo and in vitro development of apoptosis induced by gamma-irradiation was studied in mouse peritoneal macrophages. The apoptosis index was measured by fluorescence microscopy and DNA electrophoresis. In vivo apoptosis was greatest eight days after 8 Gy total body gamma-irradiation. A DNA ladder electrophoretic pattern was only observed in the gamma-irradiated group. The participation of reactive oxygen species in apoptosis induction was investigated by pretreating mice with the antioxidants superoxide dismutase, catalase, vitamin E or lipopolysaccharide before gamma-irradiation. Measurement of serum lipoperoxides showed oxidative stress in the gamma-irradiated mice and the protection given by the antioxidants. These results were confirmed using in vitro cultures of peritoneal macrophages: gamma-irradiated groups and antioxidant-pretreated gamma-irradiation groups showed results similar to those observed with in vivo irradiation. A loss of mitochondrial membrane potential (delta psi(m)) was also observed by microscopy in the gamma-irradiated cell cultures. Experiments with caspase inhibitors confirmed the participation of caspase 3 and caspase 9.

Cervical cancer cell supernatants induce a phenotypic switch from U937-derived macrophage-activated M1 state into M2-like suppressor phenotype with change in Toll-like receptor profile.

UNLABELLED: Cervical cancer (CC) is the second most common cancer among women worldwide. Infection with human papillomavirus (HPV) is the main risk factor for developing CC. Macrophages are important immune effector cells; they can be differentiated into two phenotypes, identified as M1 (classically activated) and M2 (alternatively activated). Macrophage polarization exerts profound effects on the Toll-like receptor (TLR) profile. In this study, we evaluated whether the supernatant of human CC cells HeLa, SiHa, and C-33A induces a shift of M1 macrophage toward M2 macrophage in U937-derived macrophages. RESULTS: The results showed that soluble factors secreted by CC cells induce a change in the immunophenotype of macrophages from macrophage M1 into macrophage M2. U937-derived macrophages M1 released proinflammatory cytokines and nitric oxide; however, when these cells were treated with the supernatant of CC cell lines, we observed a turnover of M1 toward M2. These cells increased CD163 and IL-10 expression. The expression of TLR-3, -7, and -9 is increased when the macrophages were treated with the supernatant of CC cells. CONCLUSIONS: Our result strongly suggests that CC cells may, through the secretion of soluble factors, induce a change of immunophenotype M1 into M2 macrophages.