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.

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

BACKGROUND: Chemotherapeutic 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. METHODS: HeLa (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. IkappaBalpha 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. RESULTS: PTX 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 IkappaBalpha 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. CONCLUSION: PTX 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.

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

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 (delta psi m) 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 delta psi m 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.

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

In response to inflammatory stimuli, monocytes/macrophages secrete greater quantities of the proinflammatory cytokines tumour necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta) and IL-6. The inflammatory process and the innate immune response are related to the activation of several transcription factors, such as nuclear factor kappaB (NF-kappaB) 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-kappaB. 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-alpha, IL-1beta 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-kappaB and AP-1 and on the IkappaB 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 IkappaB degradation, resulting in the abolition of NF-kappaB activation. Our experiments strongly suggest that the proteasome is an important factor in the regulation of proinflammatory cytokines and their receptors.

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.

In vivo adriamycin-induced apoptosis in peritoneal murine macrophages: partial participation of a caspase cascade.

BACKGROUND: Adriamycin (ADM) is a potent antitumor drug that induces apoptosis (AP) in tumor cells. AP is modulated by caspases and by mitogen-activated protein kinases (MAPK) as well as by the mitochondrial membrane potential (deltapsim). We studied the participation of these systems in peritoneal macrophages from ADM-treated mice. MATERIALS AND METHODS: Balb/c mice were either treated with ADM (5 mg/kg, i.p.) or with 0.85% NaCl solution (controls). One hour later, peritoneal cells were harvested and cultured for 28 h. AP was evaluated by ethidium bromide and acridine orange staining; deltapsim was monitored using a MitoCapture stain Kit; DNA integrity was assessed by electrophoretic analysis. Animals were treated (i.p.) 1 h before ADM administration with Z-LEHD-FMK, Z-DEVD-FMK, or Z-VAD-FMK (caspase-9, caspases-3, 7,10 and general caspase inhibitors, respectively) or with PD169316 (a MAPKp38 inhibitor). RESULTS: ADM induced a higher rate of AP and the characteristic electrophoretic DNA ladder pattern. Mice treated with caspases inhibitors plus ADM showed significant reductions in AP and DNA laddering; in contrast, no differences were observed in mice treated with PD169316 plus ADM in comparison with ADM alone. ADM also induced early loss of the deltapsim. CONCLUSION: In these experimental conditions, ADM induced AP in a mainly caspase-9-dependent manner and this was related to a reduction in the deltapsim.