Effects of sodium butyrate on growth, differentiation, and apoptosis in head and neck squamous carcinoma cell lines

BACKGROUND: Biologic agents that reverse early changes in the aerodigestive tract mucosa have potential treatment applications for patients with field cancerization of the upper aerodigestive tract. Sodium butyrate (BA) is a normal dietary constituent that induces differentiation and inhibits growth in several malignant cell types in vitro, but its effect on head and neck squamous cell carcinoma (HNSCC) has not been evaluated. METHODS: Using five HNSCC cell lines, the effects of BA on cell proliferation and apoptosis were examined by colorimetric and fluorescence-labeling methods, and the expression of differentiation markers and apoptosis-related proteins were analyzed using Western and Northern blotting, flow cytometry, and cell cycle analysis. RESULTS: BA-induced growth inhibition and apoptosis in HNSCC cells at millimolar concentrations. Apoptosis induction did not depend on the p53 status of the cell lines or on expression of members of the Bcl-2/Bax family. CONCLUSIONS: These results demonstrate that butyrate has activity against HNSCC in vitro and may have clinical applications for management of HNSCC patients.     

Control of allograft rejection by applying a novel nuclear factor-kappaB inhibitor, dehydroxymethylepoxyquinomicin

BACKGROUND: Nuclear factor (NF)-kappaB plays a crucial role in lymphocyte activation, proliferation, and survival. We examined the immunosuppressive effect of a newly developed NF-kappaB inhibitor, dehydroxymethylepoxyquinomicin (DHMEQ) in allotransplantation. METHODS: Purified C57BL/6 (H-2b) T cells were used for in vitro studies examining activation, proliferation, cytokine production and nuclear NF-kappaB and nuclear factor of activated T cells (NFAT) protein levels. A fully major histocompatibility complex incompatible BALB/c (H-2d)-to-C57BL/6 mice cardiac transplantation model was utilized for in vivo studies. DHMEQ was given intraperitoneally to transplant recipients at a various dose starting from day 0. In some, DHMEQ was administered concomitantly with tacrolimus. RESULTS: DHMEQ significantly suppressed alphaCD3 + alphaCD28 monoclonal antibody-triggered T-cell proliferation, CD25/CD69 expressions, and both interleukin-2 and interferon (IFN)-gamma production in a dose-dependent fashion. DHMEQ blocked nuclear translocation of NF-kappaB but not NFAT in activated T cells. Combined treatment with DHMEQ and tacrolimus significantly suppressed T cell activation as compared to that of mono-therapy with either agent alone. Single DHMEQ treatment moderately prolonged cardiac allograft survival. Further, combination of DHMEQ plus tacrolimus markedly prolonged graft mean survival time (MST) to 59.5 days when compared to either DHMEQ (MST: 10 days) or tacrolimus (MST: 13 days) treatment alone. Such effect was associated with inhibition of mixed lymphocyte reaction against donor antigen, IFN-gamma producing splenocytes and graft cellular infiltration as examined at 5 and 12 days posttransplantation. CONCLUSION: DHMEQ inhibits nuclear translocation of NF-kappaB but not NFAT in activated T cells, and prolongs allograft survival. Blocking both NF-kappaB and NFAT by DHMEQ and tacrolimus induces potent immunosuppression, which may become a new modality in controlling allograft rejection.     

Inducible nuclear factor-kappaB activation contributes to chemotherapy resistance in gastric cancer

BACKGROUND: Activation of nuclear factor-kappaB (NF-kappaB) inhibits chemotherapy-induced apoptosis in some cancer cell lines. Inhibition of NF-kappaB by adenoviral delivery of an IkappaBalpha superrepressor (Ad.IkappaBalpha-SR) should potentiate 5-fluorouracil (5-FU) and irinotecan chemotherapy in gastric cancer cells. STUDY DESIGN: NCI-N87 and AGS human gastric cancer cells were studied. Chemotherapy-induced NF-kappaB activation was assessed using a luciferase reporter assay. Inhibition of NF-kappaB was assessed by luciferase reporter assay and by electrophoretic mobility shift assay. Cells were pretreated for 1 hour with Ad.IkappaBalpha (25 MOI) and incubated with 5-FU or the active metabolite of irinotecan (SN-38). Cell growth was assessed by cell proliferation assay and induction of apoptosis was determined by flow cytometry and caspase 3/7 assay. RESULTS: 5-FU and SN-38 significantly induced NF-kappaB activation as measured by luciferase reporter assay (p < 0.001). Ad.IkappaBalpha-SR treatment inhibited NF-kappaB binding as demonstrated by electrophoretic mobility shift assay and by luciferase reporter assay. In AGS cells, pretreatment with Ad.IkappaBalpha-SR followed by 5-FU (0.005 mmol/L) or SN-38 (10 ng/mL) led to increased growth inhibition of 13% and 59%, respectively (p < 0.001). Similarly, growth inhibition in NCI cells was significantly increased by pretreatment with Ad.IkappaBalpha followed by 5-FU (0.001 mmol/L) or SN-38 (0.5 ng/mL) (p < 0.001). In both cell lines, Ad.IkappaBalpha-SR enhanced apoptosis by both flow cytometry and caspase 3/7 assay as compared with chemotherapy alone. CONCLUSIONS: NF-kappaB is activated in human gastric cancer in response to chemotherapy and may result in inducible chemoresistance. Inhibition of NF-kappaB by Ad.IkappaBalpha-SR enhances the antitumor effects of chemotherapy and has potential as a novel antineoplastic strategy.     

Inhibition of cell proliferation in head and neck squamous cell carcinoma cell lines with antisense cyclin D1

Cyclin D1 and cyclin G are essential regulatory factors in the progression of the cell cycle from G0 through G1 and S phase. Aberrations in expression of these cyclins may lead to dysregulated cellular proliferation that could result in neoplasia. Amplification and overexpression of cyclin D1 have been observed in many human cancers, whereas cyclin G is a new cyclin recently described in osteosarcoma cells. This study was performed to determine whether these cyclins were amplified in head and neck squamous cell carcinoma (HNSCC) tumors. Polymerase chain reaction of DNA extracted from 22 HNSCC primary tumors and three HNSCC cell lines did not reveal amplification of cyclin D1 in any of the tumor samples. Southern blot analysis identified amplification of cyclin D1 in a single tumor. Amplification of cyclin G was not observed in any of the tumors by Southern blot hybridization with a cyclin G probe. HNSCC cell lines transfected with antisense cyclin D1 were tested for cell proliferation by the incorporation of ³ H-thymidine into cells grown in serum-free media. By 72 hours of incubation, there was a greater than 30% reduction in proliferation of cells transfected with antisense cyclin D1 as compared with nontransfected control cells. The results indicate that cyclin D1 may play an important role in the growth and proliferation of HNSCC cells. (Otolaryngol Head Neck Surg 1998;119:593-9.)     

Inhibition of NF-kappaB sensitizes non-small cell lung cancer cells to chemotherapy-induced apoptosis

BACKGROUND: Most non-small cell lung cancers (NSCLC) are chemoresistant. Identification and modulation of chemoresistance cell-signaling pathways may sensitize NSCLC to chemotherapy and improve patient outcome. The purpose of this study was to determine if chemotherapy induces nuclear factor-kappa B (NF-kappaB) activation in NSCLC in vitro and whether inhibition of NF-kappaB would sensitize tumor cells to undergo chemotherapy-induced apoptosis. METHODS: Non-small cell lung cancer cells were treated with gemcitabine, harvested, and nuclear extracts analyzed for NF-kappaB DNA binding by electrophoretic mobility shift assays. Additionally, NSCLC cells that stably expressed a plasmid encoding the superrepressor IkappaBalpha protein (H157I) or a vector control (H157V) were generated. These cells were then treated with gemcitabine and apoptosis determined by terminal deoxynucleotidyl transferase mediated nick end labeling (TUNEL) assay. RESULTS: Chemotherapy induced NF-kappaB nuclear translocation and DNA binding in all NSCLC cell lines. H157I cells had enhanced cell death compared with H157V cells, suggesting that NF-kappaB is required for cell survival after chemotherapy. The observed cell death following the loss of NF-kappaB occurred by apoptosis. CONCLUSIONS: Inhibition of chemotherapy-induced NF-kappaB activation sensitizes NSCLC to chemotherapy-induced apoptosis in vitro. Novel treatment strategies for patients with advanced NSCLC may involve chemotherapy combined with inhibition of NF-kappaB-dependent cell-survival pathways.     

In vitro effects of keyhole limpet hemocyanin in breast and pancreatic cancer in regards to cell growth, cytokine production, and apoptosis

BACKGROUND: We have previously shown the inhibitory effects of keyhole limpet hemocyanin (KLH) against breast and pancreatic cancer in vitro. We hypothesize that its actions in breast and pancreas cancer cells are via apoptotic or cytokine pathways. METHODS: Two breast cancer cell lines, ZR75-1 and MCF-7, and one pancreas cancer cell line, PANC-1, were treated with KLH at 500 mug, 250 mug, and 250 ng/mL. Cell viability, cytokine production, and apoptosis were measured. RESULTS: Significant growth inhibition was observed in all cell lines at all KLH concentrations tested. Significant changes in cytokine production were observed in all cell lines. An increase in early and late apoptotic activity was observed in the MCF-7, whereas a reduction in late apoptotic activity was observed in the ZR75-1 cells. CONCLUSIONS: KLH directly inhibits the growth of human breast and pancreas cancer in vitro by apoptotic and nonapoptotic mechanisms.     

Pyrrolidine dithiocarbamate exerts anti-proliferative and pro-apoptotic effects in renal cell carcinoma cell lines.

BACKGROUND: The activation of nuclear factor-kappaB (NF-kappaB) has been implicated in the development, progression and metastasis of renal cell carcinoma (RCC). This study investigates the effect of pyrrolidine dithiocarbamate (PDTC), a NF-kappaB inhibitor, on two metastatic human RCC cell lines, ACHN and SN12K1. METHODS: RCC cell lines and normal cells were exposed to 25 or 50 microM of PDTC. Apoptosis was measured by flow cytometry and TdT-mediated nick end labelling methods. Cell viability and proliferation were measured by MTT and BrdU assays, respectively. Expression of NF-kappaB subunits, IkappaBs, IkappaB Kinase (IKK) complex and apoptotic regulatory proteins were analysed by western blotting and/or immunofluorescence. DNA-binding activity of NF-kappaB subunits were measured by ELISA. RESULTS: RCC cell lines had a higher basal level expression of all the five subunits of NF-kappaB than normal primary cultures of human proximal tubular epithelial cells or HK-2 cells. PDTC decreased the viability and proliferation of RCC, but not normal cells. Of the two RCC cell lines, ACHN had a higher basal level expression of all the five NF-kappaB subunits than SN12K1 and was more resistant to PDTC. While PDTC induced an overall decrease in expression of all the five NF-kappaB subunits in both RCC cell lines, unexpectedly, it increased the nuclear expression of NF-kappaB in ACHN, but not in SN12K1. PDTC reduced the DNA-binding activity of all the NF-kappaB subunits and the expression of the IKK complex (IKK-alpha, IKK-beta and IKK-gamma) and the inhibitory units IkappaB-alpha and IkappaB-beta. PDTC induced a significant increase in apoptosis in both RCC cell lines. This was associated with a decrease in expression of the anti-apoptotic proteins, Bcl-2 and Bcl-(XL), without marked changes in the pro-apoptotic protein Bax. CONCLUSION: These data suggest that PDTC has the potential to be an anticancer agent in some forms of RCC.     

Antiproliferative and overadditive effects of everolimus and mycophenolate mofetil in pancreas and lung cancer cells in vitro

BACKGROUND: Everolimus inhibits the growth of several tumor cell lines in vitro as well as tumor growth in a rat model. Mycophenolate mofetil (MMF) inhibits growth of a Walker sarcoma in a rat model in vivo. Herein we tested the in vitro antiproliferative capacity of everolimus and MMF on a pancreatic tumor cell line Panc-1 and on a small cell lung cancer cell line ScLc. MATERIALS AND METHODS: Cells were cultured under standardized conditions. Everolimus was added in increasing doses from 0.005 to 500 microg/mL; MMF was used from 0.05 to 5000 microg/mL. For co-incubation experiments, we combined everolimus (0.005 microg/mL and 0.05 microg/mL) with five concentrations of MMF; and MMF (0.5 microg/mL and 5 microg/mL) with five concentrations of everolimus. The antiproliferative capacity was assessed by a BrdU incorporation assay. RESULTS: Everolimus and MMF inhibited BrdU incorporation into Panc-1 and ScLc in a dose-dependent fashion. A 50% inhibition was seen in Panc-1 only at 50 microg/mL everolimus, but in ScLc at 5 microg/mL everolimus. MMF was clearly more potent in Panc-1: 50% inhibition was observed at 5 microg/L. In ScLc, 40% inhibition of BrdU incorporation was seen only at 50 microg/L MMF. In co-incubation, an effective combination for both Panc-1 and ScLc was 5 microg/mL MMF with 0.005 microg/mL everolimus resulting in 50% inhibition of BrdU incorporation (P < .001). CONCLUSIONS: Everolimus and MMF showed dose-dependent antiproliferative effects in tumor cell lines in vitro both alone and in combination. The combined use of everolimus and MMF showed supra-additive effects at concentrations used for therapeutic immunosuppression in patients.     

Third place--Resident Research Competition, AAO-2000. Antisense cyclin D1 inhibits growth of head and neck cancer xenografts in nude mice.

PROBLEM: Cyclin D1 is a regulatory factor essential in the progression of the cell cycle from G1 through S phase. Amplification and overexpression of cyclin D1 have been observed in many human cancers including head and neck squamous cell carcinoma (HNSCC). We have previously transfected a HNSCC control cell line (CCL23) with an antisense cyclin D1 plasmid and demonstrated inhibition of cell proliferation in vitro. In this study, we examine whether antisense cyclin D1 could inhibit tumor growth in vivo. Methods/measures: The CCL23 and its antisense cyclin D1 transfected clone (CCL23 AS) were injected into the flanks of nude mice. Tumor growth was monitored weekly. After 5 weeks, tumors were removed and studied for tumor size, cyclin D1 expression, cyclin D1-dependent kinase activity, and retinoblastoma (Rb) phosphorylation. RESULTS: Compared with the control tumors, 11 of 19 antisense tumors were smaller, 7 tumors were of equal size, and 1 tumor was larger. Immunohistochemical analysis with an anti-cyclin D1 antibody demonstrated decreased cyclin D1 expression in CCL23 AS and the smaller antisense tumors. Cyclin D1-dependent kinase activity was reduced in CCL23 AS and the smaller antisense tumors, and this was accompanied by a relative decrease in phosphorylated Rb in these samples. CONCLUSION: Antisense cyclin D1 inhibits growth of HNSCC tumors. Cyclin D1 expression, cyclin D1-dependent kinase activity, and Rb phosphorylation are decreased in these tumors. Clinical significance: These findings lend support for the potential use of antisense cyclin D1 as gene therapy for HNSCC.     

Antitumor effects of Scutellariae radix and its components baicalein, baicalin, and wogonin on bladder cancer cell lines

OBJECTIVES: To investigate the antitumor effects of Scutellariae radix and its components baicalein, baicalin, and wogonin on human bladder cancer cell lines (KU-1 and EJ-1) and a murine bladder cancer cell line (MBT-2). METHODS: Bladder cancer cells were incubated with various concentrations of the agents. Antiproliferative activity against the bladder cancer cell lines was examined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diplenyl tetrazolium bromide assay. In an in vivo experiment, the mice were subcutaneously injected with MBT-2 cells, and Scutellariae radix was orally administered at a dose of 2 or 10 mg per mouse one time daily for 10 days from day 11 to day 20. RESULTS: All the drugs inhibited cell proliferation in a dose-dependent manner, but baicalin exhibited the greatest antiproliferative activity. The concentration of baicalin necessary to obtain 50% inhibition was 3.4 microg/mL for KU-1, 4.4 microg/mL for EJ-1, and 0.93 microg/mL for MBT-2. For KU-1 and MBT-2, the percentage of cell survival significantly decreased (P <0.05) at a baicalin concentration of 1 microg/mL. In an in vivo experiment, antitumor effects of Scutellariae radix on C3H/HeN mice implanted with MBT-2 were investigated. All the control mice showed a progressive increase in tumor volume, reaching 2.81 +/- 0.18 cm(3) on day 20 and 5.36 +/- 0.44 cm(3) on day 25. However, when Scutellariae radix was orally administered at a dose of 10 mg per mouse one time daily for 10 days from day 11 to day 20, the tumor volume was 1.99 +/- 0.19 cm(3) on day 20 and 3.86 +/- 0.26 cm(3) on day 25, a significant inhibition of tumor growth (P <0.05). Conclusions. These results suggest that Chinese herbal medicines may become an attractive and promising treatment for bladder cancer.     

Vitamin E and other antioxidants inhibit human prostate cancer cells through apoptosis

BACKGROUND: Many human prostate cancer cells have escaped the apoptotic effects of natural regulators of cell growth such as transforming growth factor betal (TGF beta-1) and tumor necrosis factor (TNF). METHODS: Prostate cancer cell growth was investigated by treating with antioxidants. DU-145 (androgen-unresponsive), LNCaP (androgen-responsive), and ALVA-101 (androgen moderately responsive) were grown in RPMI-1640 medium supplemented with bovine fetal calf serum and antibiotics, and were treated with various antioxidants for 1-7 days. Cell growth was then determined with the Cell Titer 96 AQ assay, and apoptosis was assessed by cell death detection ELISA, nuclear morphology, and TUNEL techniques. RESULTS: Cells treated with or without (+/-)-alpha-tocopherol (vitamin E) for 1-7 days at concentrations from 0.078-2.5 microg/ml modestly affected cell growth compared to other antioxidants tested. Tocopherol produced a significant (P < 0.01) inhibition of ALVA-101 and LNCaP (10-24% of control; 0.078-2.5 microg/ml; at 6 days; n = 6). DU-145 cells were not growth-inhibited significantly. However, pyrrolidinedithiocarbamate (PDTC) produced a significant (P < 0.01, n = 6; 17-80% of control; 2.5-20 microg/ml; 1-7 days) inhibition of DU-145 and ALVA-101 cells. A significant (P < 0.01) and maximum inhibition of LNCaP cells occurred at all concentration of PDTC (2. 5-20 microg/ml). A third compound, diethyldithiocarbamic acid (DETC), incubated for 1-7 days, produced a significant dose response suppression of cell growth of DU-145 and ALVA-101 cells (P < 0.01; 14-88% of control; 1.25-80 microg/ml; n = 6). LNCaP cells were inhibited by DETC (P < 0.01; 28% of control; 1.25-80 microg/ml; n = 6). All three antioxidants tested stimulated apoptosis in actively dividing ALVA-101, DU-145, and LNCaP cells (P < 0.01; n = 6), but confluent cells were affected less. Testosterone had additive inhibitory effects when combined with PDTC in ALVA-101 cells; however, the other cell lines were not influenced. CONCLUSIONS: These results demonstrate that antioxidants modulate human prostate cancer cell proliferation by altering apoptosis in dividing cells, and this necrosis or apoptosis in confluent cells is not as effective.     

Brefeldin A-induced apoptosis in prostatic cancer DU-145 cells: a possible p53-independent death pathway

OBJECTIVE: To investigate the growth inhibitory mechanism of brefeldin A (BFA), an antiviral antibiotic, in androgen-independent prostatic cancer DU-145 cells. MATERIALS AND METHODS: The inhibitory effects of BFA (30 ng/mL) on cell growth were monitored by cell counting and viability tests after specified exposures. Flow cytometry and western immunoblot analysis were performed to examine the effects of BFA on the cell cycle and on specific growth regulators. The possible induction of apoptosis by BFA was further assessed by in situ hybridization (ISH) assay and by qualitative DNA analysis using agarose-gel electrophoresis. RESULTS: Cell growth was completely inhibited with BFA (30 ng/mL), accompanied by 40-50% cell death. Cell cycle analysis revealed that this growth inhibition coincided with an 85% reduction in the S-phase cell population. Western blots showed that the expression of cell cycle-dependent kinases (cdk2 and cdk4), cyclin D1 and p53 was significantly reduced, while WAF1 was increased, after BFA treatment. Apoptosis was confirmed by both the ISH assay, which showed the characteristic brownish staining of BFA-treated cells, and by DNA analysis, which revealed the internucleosomal DNA ladder. CONCLUSION: BFA-induced growth inhibition in DU-145 cells is primarily due to the modulation of specific G1 cell-cycle regulators, blocking the G1-S phase progression. Such a growth arrest ultimately results in apoptosis, presumably through a p53-independent pathway.     

Inhibitory effect of zinc on human prostatic carcinoma cell growth.

BACKGROUND: Normal human prostate accumulates the highest levels of zinc of any soft tissue in the body. In contrast, the zinc level in prostate cancer is markedly decreased from the level detected in nonprostate tissues. Despite these relationships, the possible role of zinc in the growth of normal and malignant prostate has not been determined. METHODS: Growth inhibition and various regulatory responses were investigated in two human prostate carcinoma cell lines (LNCaP and PC-3), treated with or without zinc. RESULTS: Incubation of the prostate carcinoma cell lines with physiological levels of zinc resulted in the marked inhibition of cell growth. A lower 50% inhibition of cell growth (IC50) value for zinc (about 100 ng/ml) was detected in LNCaP cells, which are androgen-responsive, whereas androgen-independent PC-3 cells exhibited a higher IC50 for zinc (about 700 ng/ml). Incubation with 1 microg/ml zinc resulted in maximum inhibition of growth in both cell lines. These inhibitory effects of zinc correlated well with the accumulation of zinc in the cells. Simultaneously, cell flow cytometric analyses revealed a dramatic increase of the cell population in G2/M phase, in both LNCaP (2.3-fold vs. control) and PC-3 (1.9-fold vs. control), and a decreased proportion of cells in S phase (LNCaP, -51.4%; PC-3, -23%), indicating a G2/M phase arrest. The cell growth inhibition and G2/M arrest in these cells were accompanied by an increase in apoptosis, as demonstrated by the characteristic cell morphology and further confirmed by cellular DNA fragmentation. The specificity of zinc-induced apoptosis was identified by ethylenediamine-tetraacetic acid (EDTA)-chelation, which abolished the zinc effect on cellular DNA fragmentation. The zinc-induced G2/M phase arrest and apoptosis were accompanied by increased mRNA levels of p21(Waf1/Cip1/Sdi1) in both LNCaP (p53+/+) and PC-3 (p53-/-) cells. CONCLUSIONS: These results suggest that zinc inhibits human prostatic carcinoma cell growth, possibly due to induction of cell cycle arrest and apoptosis. There now exists strong evidence that the loss of a unique capability to retain high levels of zinc is an important factor in the development and progression of malignant prostate cells.     

Preexposure of murine macrophages to CpG-containing oligonucleotides results in nuclear factor kappaB p50 homodimer-associated hyporesponsiveness

BACKGROUND: DNA containing the CpG motif is associated with immunomodulation of the innate immune response. Preexposure of macrophages to CpG DNA elicits a hyporesponsiveness to subsequent lipopolysaccharide (LPS) stimulation. We tested the hypothesis that this effect is due to decreased nuclear translocation of nuclear factor kappaB (NF-kappaB). METHODS: Murine macrophage-like RAW 264.7 cells were incubated with 1.5 microg/mL CpG-containing oligonucleotides (CpG ODN) for 0.5 to 9 hours followed by restimulation with 1 microg/mL LPS for 20 minutes. Some cells were cotransfected with an NF-kappaB sensitive luciferase reporter construct and a control beta-gal plasmid. Cytoplasmic and nuclear extracts were assayed for NF-kappaB by electrophoretic mobility shift assay and supershift assays, for NF-kappaB, IkappaB and phospho-IkappaB by Western blot, for luciferase activity, and for p38, c-Jun NH(2)-terminal kinase, and extracellular signal-related kinase activity assay. RESULTS: NF-kappaB functional activity was decreased as demonstrated by luciferase activity assay in the prolonged CpG ODN pretreatment groups. Unlike endotoxin tolerance, CpG ODN preexposure increased cytoplasmic phospho-IkappaB-alpha and did not abrogate mitogen-activated protein kinase activity. CONCLUSIONS: In macrophages, exposure to CpG DNA increases expression of the inhibitory p50 NF-kappaB homodimer and decreases NF-kappaB activity without inhibition of IkappaB kinases. Mitogen-activated protein kinase activity remains intact. Understanding these interactions between different toll receptor ligands may provide insight into novel therapeutic modalities.