The experimental chemotherapeutic N6-furfuryladenosine (kinetin-riboside) induces rapid ATP depletion, genotoxic stress, and CDKN1A (p21) upregulation in human cancer cell lines

Abstract

Cytokinins and cytokinin nucleosides are purine derivatives with potential anticancer activity. N 6 -furfuryladenosine (FAdo, kinetin-riboside) displays antiproliferative and apoptogenic activity against various human cancer cell lines, and FAdo has recently been shown to suppress tumor growth in murine xenograft models of human leukemia and melanoma. In this study, FAdo-induced genotoxicity, stress response gene expression, and cellular ATP depletion were examined as early molecular consequences of FAdo-exposure in MiaPaCa-2 pancreas carcinoma, A375 melanoma, and other human cancer cell lines. FAdo, but not adenosine or N 6 -furfuryladenine, displayed potent antiproliferative activity that was also observed in human primary fibroblasts and keratinocytes. Remarkably, massive ATP depletion and induction of genotoxic stress as assessed by the alkaline comet assay occurred within 60 to 180 minutes of exposure to low micromolar concentrations of FAdo. This was followed by rapid upregulation of CDKN1A and other DNA damage/stress response genes (HMOX1, DDIT3, GADD45A) as revealed by expression array and Western analysis. Pharmacological and siRNA-based genetic inhibition of adenosine kinase suppressed FAdo cytotoxicity and also prevented ATP-depletion and p21-upregulation suggesting the importance of bioconversion of FAdo into the nucleotide form required for drug action. Taken together our data suggest that early induction of genotoxicity and energy crisis are important causative factors involved in FAdo cytotoxicity.

The Quinone Methide Aurin Is a Heat Shock Response Inducer That Causes Proteotoxic Stress and Noxa-dependent Apoptosis in Malignant Melanoma Cells

“…The RT 2 Human Stress and Toxicity Pathfinder TM PCR Expression Array (SuperArray) profiling the expression of 84 stress-related genes was run using the following PCR conditions: 95°C for 10 min followed by 40 cycles of 95°C for 15 s alternating with 60°C for 1 min (Applied Biosystems 7000 SDS). Gene-specific product was normalized to ACTB and quantified using the comparative (⌬⌬Ct) Ct method as described in the ABI Prism 7000 sequence detection system user guide as published earlier (42,45). Expression values were averaged across three independent array experiments, and standard deviation was calculated for graphing.…”

“…PCR conditions were 95°C for 10 min followed by 40 cycles of 95°C for 15 s, alternating with 60°C for 1 min using an Applied Biosystems 7000 SDS and Applied Biosystems Assays On Demand primers specific to DDIT3 (assay ID Hs00358796_g1), HSPA6 (assay ID Hs00275682_s1), HSPA1A (assay ID Hs00359163_s1), HMOX1 (assay ID Hs00157965_m1), PMAIP1 (assay ID Hs00560402_m1), and ACTB (␤-actin, assay ID Hs99999903_m1). Gene-specific product was normalized to ACTB and quantified using the comparative (⌬⌬C t ) Ct method as described before (42,45).…”

“…Immunoblot Analysis-Sample preparation, SDS-PAGE, transfer to nitrocellulose, and development occurred as described earlier (40,45). Gel percentage was 12%.…”

The topical antimicrobial zinc pyrithione is a heat shock response inducer that causes DNA damage and PARP-dependent energy crisis in human skin cells

“…Phospho-H2A.X detection by flow cytometry Treatmentinduced accumulation of nuclear phosphorylated histone variant H2A.X (γ-H2A.X) was examined in HEKs as published recently using a phospho-histone H2A.X (Ser139) monoclonal antibody (Alexa Fluor 488 conjugate, Cell Signaling, Inc., Danvers, MA, USA) followed by flow cytometric analysis (Cabello et al 2009b). …”

“…Comet assay (alkaline single cell gel electrophoresis) The alkaline Comet assay was performed according to the manufacturer's instructions (Trevigen, Gaithersburg, MD, USA) as published recently Cabello et al 2009b). Cells were seeded at 100,000 per 35-mm dish 24 h prior to treatment.…”