Carboplatin induces apoptotic cell death through downregulation of constitutively active nuclear factor-κB in human HPV-18 E6-positive HEp-2 cells

National Centre For Cell Science, Pune, Poona, Maharashtra, India
Biochemical and Biophysical Research Communications (Impact Factor: 2.3). 06/2004; 318(2):346-53. DOI: 10.1016/j.bbrc.2004.04.037
Source: PubMed


Because the role of nuclear factor kappaB (NF-kappaB) is in cellular growth control and neoplasia, we explored the status of NF-kappaB and investigated its role in survival of human HPV-18 E6-positive HEp-2 cells. We observed accumulation of p65 in the nucleus. Moreover, without any external stimulus constitutive NF-kappaB DNA binding and transactivation activity were detected in HEp-2 cells. Treatment with NF-kappaB inhibitor curcumin (diferuloylmethane) and transient transfection of the mutant form of IkappaBalpha, IkappaBalpha super repressor (IkappaBalpha-SR), suppressed constitutive NF-kappaB activity as well as proliferation, suggesting that constitutive NF-kappaB activity is required for the survival of HEp-2 cells. Carboplatin treatment downregulated constitutive NF-kappaB activity and prevented nuclear retention of p65. Further, carboplatin also suppressed the constitutive IkappaBalpha phosphorylation leading to stabilization of IkappaBalpha protein in the cells. Carboplatin inhibited NF-kappaB binding to its response element present in Bcl-2 promoter resulting in downregulation of antiapoptotic Bcl-2 protein. Thus, our results for the first time indicate that constitutive NF-kappaB has a significant role in the survival of HPV-18 E6-positive HEp-2 cells. Moreover, inactivation of NF-kappaB is one of the mechanisms underlying the induction of carboplatin-mediated apoptosis in HPV-18 E6-positive cancer cells.

3 Reads
  • Source
    • "There were two controls, one with only human cell culture maintained as monolayer in nutrient medium MEM supplemented with heat-inactivated fetal bovine serum (10%), penicillin (10 U/ml) and streptomycin (100 ␮g/ml) and one with this media containing solvent (DMSO), i.e. solvent control groups received the same amount of DMSO. The MTT assay, which is based on the conversion of the yellow tetrazolium salt-MTT, to purple-formazan crystals by metabolically active cells, provides a quantitative determination of viable cells (Singh and Bhat, 2004). Cells were seeded at the density of 15,000 per well into 96-well plates and allowed to adhere for 24 h at 37 • C. Next day, cells were treated with various concentrations (0, 1, 10, and 100 ␮g/ml) of bioactive molecule isolated from Eulophia nuda in DMSO for 24 h in triplicates and further incubated for 48 h in the absence of compound and then cytotoxicity was assessed by MTT assay. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Eulophia nuda L. (Orchidaceae) is a medicinally important terrestrial orchid used for the treatment of tumours and various health problems by the local healers throughout the Western Ghats region in Maharashtra (India). To isolate the active molecule from Eulophia nuda and to study its cytotoxic potential against human cancer cells. The crude methanolic extract of Eulophia nuda tubers was fractionated by stepwise gradient of the solvents-chloroform-methanol to isolate the pure compound. Isolated pure compound was assessed for its cytotoxic potential against human breast cancer cell lines, MCF-7 and MDA-MB-231 using MTT assay. Structure elucidation of the isolated active compound was carried out by extensive spectroscopic analysis including (1)H NMR, (13)C NMR, NOESY, COSY, LC-MS and IR. The isolated active molecule was identified as phenanthrene derivative 9,10-dihydro-2,5-dimethoxyphenanthrene-1,7-diol. This compound showed good antiproliferative activity against human breast cancer cell lines MCF-7 (91%) and MDA-MB-231 (85%) at 1000 microg/ml concentration. 9,10-Dihydro-2,5-dimethoxyphenanthrene-1,7-diol from Eulophia nuda tubers showed good growth suppressive effect against human cancer cell lines MCF-7 and MDA-MB-231 making it a potential biomolecule against human cancer.
    Full-text · Article · Mar 2010 · Journal of ethnopharmacology
  • Source
    • "Thereafter, effector cells were washed twice with medium and target Hep3B cells (2 × 105 cells per well) were co-plated with or without neutralizing anti-TRAIL antibody. After 72 h of growth in co-culture, trypan blue dye exclusion assay was performed as described previously [20]. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide that is particularly refractory to chemotherapy. Several studies have proposed combination chemotherapy regimen for HCC treatment. However, these therapies are not effective in regressing tumor and prolonging survival of patient's suffering from HCC. Therefore, the development of more effective therapeutic tools and new strategies for the treatment of HCC are urgently needed. Over the last decade much attention has been focused on "bystander effect" as a possible therapeutic strategy for the treatment of certain human tumors. Interest in this therapeutic approach originated from numerous reports describing the radiation induced bystander effect. However, the knowledge about chemotherapy induced bystander effect is still limited. Hence, chemotherapy induced bystander phenomenon in hepatoma cells was explored by utilizing Mitomycin C (MMC). MMC induced bystander killing was observed only in hepatoma cells and it did not occur in cervical cancer cells. MMC induced bystander killing was transferable via medium. It occurred in co-cultured cells indicating the involvement of secreted as well as membrane bound factors. FasL and TRAIL were detected in the conditioned medium from treated cells. In medium transfer experiment, pre-treatment with EDTA (a broad range protease inhibitor) diminished MMC induced bystander killing. Following drug exposure, expression of Fas and TRAIL receptors increased and treatment with neutralizing antibodies against FasL and TRAIL inhibited bystander killing. Our results highlight the therapeutic importance of MMC in the treatment of HCC and implicate role of membrane bound and secreted forms of FasL and TRAIL in MMC induced bystander killing.
    Full-text · Article · Oct 2009 · Molecular Cancer
  • Source
    • "Cisplatin appears to be superior to carboplatin in terms of therapeutic effectiveness for some tumors such as germ cell tumors, bladder cancer, as well as head and neck cancer, while in others (e.g., lung and ovarian cancer), their efficacies are comparable [50]. Carboplatin treatment downregulates constitutive NF-κB activity and prevents nuclear retention of p65 in liver cancer [51] and glioma cell lines [52]. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Platinum drugs continue to be major chemotherapy drugs for cancer treatment. Nevertheless, acquired or intrinsic resistance to these compounds is common in human tumors. One important mechanism for this resistance is the avoidance of cells entering the apoptotic pathway. Nuclear factor-kappa B (NF-kappa B, NF-kappaB) is a pleiotropic transcription factor key in determining the death threshold of human cells. This factor is important in the final response of cells to platinum drugs, as exemplified by in vitro and in vivo models showing that inhibition of NF-kappaB sensitizes cancer cells to the effects of these drugs. New approaches focusing on the inhibition of NF-kappaB could help to minimize or even eliminate intrinsic or acquired resistance to platinum drugs.
    Full-text · Article · Feb 2008 · Metal-Based Drugs
Show more