José Manuel Lerma-Díaz

MG132 proteasome inhibitor modulates proinflammatory cytokines production and expression of their receptors in U937 cells: involvement of nuclear factor‐κB and activator protein‐1

In response to inflammatory stimuli, monocytes/macrophages secrete greater quantities of the proinflammatory cytokines tumour necrosis factor‐α (TNF‐α), interleukin‐1β (IL‐1β) and IL‐6. The inflammatory process and the innate immune response are related to the activation of several transcription factors, such as nuclear factor κB (NF‐κB) and activator protein 1 (AP‐1). The proteasome is a multimeric protease complex, which plays a vital role in several cellular functions, including the regulation of transcription factors like NF‐κB. In this study, we used the human monocyte cell line U937 stimulated with lipopolysaccharide (LPS) and phorbol 12‐myristate 13‐acetate (PMA) as a model to investigate the in vitro effects of MG132, a proteasome inhibitor, on the release of TNF‐α, IL‐1β and IL‐6 and on the expression of their membrane and soluble receptors TNF‐R1, IL‐1R1 and IL‐6R. We also analysed the effects of MG132 on the activation of NF‐κB and AP‐1 and on the IκB molecule. MG132 significantly inhibited the secretion of those proinflammatory cytokines. MG132 increased the release of the soluble receptors TNF‐R1 and IL‐1R1 from U937 cells and decreased their cell‐surface expression. MG132 also increased IL‐6R cell‐surface expression and decreased its release. Proteasome inhibition also led to an increase in LPS+PMA‐induced AP‐1 activation and the attenuation of LPS+PMA‐induced IκB degradation, resulting in the abolition of NF‐κB activation. Our experiments strongly suggest that the proteasome is an important factor in the regulation of proinflammatory cytokines and their receptors.

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

BackgroundWorldwide, 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.MethodsHeLa (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.ResultsOur 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.ConclusionPTX 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.

Sensitization of cervix cancer cells to Adriamycin by Pentoxifylline induces an increase in apoptosis and decrease senescence

BackgroundChemotherapeutic drugs like Adriamycin (ADR) induces apoptosis or senescence in cancer cells but these cells often develop resistance and generate responses of short duration or complete failure. The methylxantine drug Pentoxifylline (PTX) used routinely in the clinics setting for circulatory diseases has been recently described to have antitumor properties. We evaluated whether pretreatment with PTX modifies apoptosis and senescence induced by ADR in cervix cancer cells.MethodsHeLa (HPV 18+), SiHa (HPV 16+) cervix cancer cells and non-tumorigenic immortalized HaCaT cells (control) were treated with PTX, ADR or PTX + ADR. The cellular toxicity of PTX and survival fraction were determinated by WST-1 and clonogenic assay respectively. Apoptosis, caspase activation and ADR efflux rate were measured by flow cytometry, senescence by microscopy. IκBα and DNA fragmentation were determinated by ELISA. Proapoptotic, antiapoptotic and senescence genes, as well as HPV-E6/E7 mRNA expression, were detected by time real RT-PCR. p53 protein levels were assayed by Western blot.ResultsPTX is toxic (WST-1), affects survival (clonogenic assay) and induces apoptosis in cervix cancer cells. Additionally, the combination of this drug with ADR diminished the survival fraction and significantly increased apoptosis of HeLa and SiHa cervix cancer cells. Treatments were less effective in HaCaT cells. We found caspase participation in the induction of apoptosis by PTX, ADR or its combination. Surprisingly, in spite of the antitumor activity displayed by PTX, our results indicate that methylxantine, per se does not induce senescence; however it inhibits senescence induced by ADR and at the same time increases apoptosis. PTX elevates IκBα levels. Such sensitization is achieved through the up-regulation of proapoptotic factors such as caspase and bcl family gene expression. PTX and PTX + ADR also decrease E6 and E7 expression in SiHa cells, but not in HeLa cells. p53 was detected only in SiHa cells treated with ADR.ConclusionPTX is a good inducer of apoptosis but does not induce senescence. Furthermore, PTX reduced the ADR-induced senescence and increased apoptosis in cervix cancer cells.

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

BackgroundThe 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.MethodsU937 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.ResultsThe 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.ConclusionMG132 sensitizes U937 leukemia cells to DOX-induced apoptosis, increasing its anti-leukemic effectiveness.

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

BackgroundIn 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.ResultsThe 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.ConclusionsThe 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.

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

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.

In vitro Induction of Apoptosis in U937 Cells by Perillyl Alcohol with Sensitization by Pentoxifylline: Increased BCL-2 and BAX Protein Expression

Background: Chemotherapy is effective against a wide variety of tumor cells, although its use is limited by side effects. In vitro experiments and phase I and II trials have shown that phytochemicals such as perillyl alcohol (P-OH) have antitumor effects. Pentoxifylline (PTX), a synthetic methylxanthine used mainly to treat pathologies associated with hematological diseases, sensitizes tumor cells to chemotherapy. The aim of this study was to determine whether PTX amplifies the antitumor effects of P-OH in U937 human myelomonocytic leukemia cells. Methods: Apoptosis was measured by the loss of mitochondrial membrane potential determined by flow cytometry using dihexyloxacarbocyanine iodide (DiOC6) and propidium iodide. Bcl-2 and Bax protein expression was also assessed by Western blot analysis. Results: P-OH and PTX induced loss of the mitochondrial membrane potential in U937 cells in vitro. Culturing the cells in the presence of both compounds caused a significant increase (p < 0.001) in apoptosis and expression of anti-apoptotic Bcl-2 and pro-apoptotic Bax proteins. However, despite their coexistence, Bax expression prevailed in our experiments. These data suggest that the effects of PTX might be attributable to changes in the mitochondrial membrane potential. Conclusion: PTX sensitizes tumor cells to the anti-neoplastic action of P-OH. These observations may have clinical relevance in the treatment of cancer patients.

MEIS1, PREP1, and PBX4 Are Differentially Expressed in Acute Lymphoblastic Leukemia: Association of MEIS1 Expression with Higher Proliferation and Chemotherapy Resistance

BackgroundThe Three-amino acid-loop-extension (TALE) superfamily of homeodomain-containing transcription factors have been implicated in normal hematopoiesis and in leukemogenesis and are important survival, differentiation, and apoptosis pathway modulators. In this work, we determined the expression levels of TALE genes in leukemic-derived cell lines, in blood samples of patients with Acute lymphoblastic leukemia (ALL), and in the blood samples of healthy donors.ResultsHere we show increased expression of MEIS1, MEIS2, and PREP1 genes in leukemia-derived cell lines compared with blood normal cells. High levels of MEIS1 and PREP1, and low levels of PBX4 expression were also founded in samples of patients with ALL. Importantly, silencing of MEIS1 decreases the proliferation of leukemia-derived cells but increases their survival after etoposide treatment. Etoposide-induced apoptosis induces down-regulation of MEIS1 expression or PREP1 up-regulation in chemotherapy-resistant cells.ConclusionsOur results indicate that up-regulation of MEIS1 is important for sustaining proliferation of leukemic cells and that down-regulation of MEIS1 or up-regulation of PREP1 and PBX genes could be implicated in the modulation of the cellular response to chemotherapeutic-induced apoptosis.

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.

BackgroundNatural 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-γ), 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.MethodsWe 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-γ, CD107a, Granzyme B, NKp44, NKp46, NKp30, and NKG2D was evaluated by FC.ResultsCCC 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.ConclusionInhibition 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.

Gossypol induced apoptosis of polymorphonuclear leukocytes and monocytes: involvement of mitochondrial pathway and reactive oxygen species.

Abstract The aim of this study was to determine how gossypol affects the viability and activity of polymorphonuclear leukocytes and monocytes in blood obtained from healthy donors. Loss of mitochondrial membrane potential (δψm) and apoptosis was maximized in human polymorphonuclear leukocytes and monocytes after incubation with gossypol. Pretreatment with a caspase-9 inhibitor or antioxidants (superoxide dismutase or Trolox) inhibited gossypol-induced loss of the δψm and apoptosis. Likewise, we observed participation of caspase -3, -7, and -10 in gossypol-induced apoptosis. Expression of the proapoptotic genes bax, bak, bad and p53/Tp53 increased in polymorphonuclear leukocytes exposed to gossypol. The expression of the anti-apoptotic genes bcl-XL and mcl-1 was reduced when polymorphonuclear leukocytes and monocytes were treated with gossypol. Gossypol treatment also inhibited yeast phagocytosis by these cells. We concluded that gossypol induces apoptosis in phagocytic cells and that this effect was dose-dependent. The findings in this report may be important to consider in light of possible gossypol use in clinical strategies for cancer treatment.