Actin-sequestering protein, thymosin beta-4, induces paclitaxel resistance through ROS/HIF-1alpha stabilization in HeLa human cervical tumor cells.
OBJECTIVE
We investigated whether actin-sequestering protein, thymosin beta-4 (TB4)-induced reactive oxygen species (ROS) affect the stabilization of hypoxia-inducible transcription factor (HIF)-1alpha and paclitaxel-resistance induction.


METHODS
HeLa human cervical tumor cells were used. The percentage of cell survival was determined by MTT assay. ROS production, cell cycle and hypodiploid cell formation were assessed by flow cytometry analysis. HIF-1alpha stabilization and molecular changes were analyzed by western blotting or RT-PCR. NF-kappaB activation was assessed by EMSA and western blotting.


RESULTS
TB4 protein (TB4P) significantly increased intracellular ROS level and HIF-1alpha. The increased level of HIF-1alpha by TB4P was reduced by the treatment with N-acetylcysteine (NAC), a well-known ROS scavenger. TB4P-induced ROS production was confirmed by the activation of nuclear factor kappa B. TB4P-induced Erk phosphorylation was attenuated by the treatment with NAC. In addition, tumor cell death was decreased by TB4 gene overexpression and TB4P treatment. NAC treatment attenuated tumor cell density increased by TB4P. Tumor cell death by paclitaxel was also increased by NAC treatment or the transfection with HIF-1alpha-siRNA. Paclitaxel-induced B16F10 mouse melanoma regression was physiologically inhibited in TB4-transgenic mice compared to wildtype mice.


CONCLUSIONS
These findings demonstrate that TB4-induced ROS and ROS-mediated HIF-1alpha stabilization could play a role in tumor cell resistance to anticancer agents like paclitaxel. It suggests that soluble TB4 could be a novel endogenous regulator to control intracellular ROS production in tumor cells.
Oh
Jin-Mi
JM
Department of Bioscience and Biotechnology, Sejong University, Seoul, Republic of Korea.
Moon
Eun-Yi
EY
D000818:Animals; D000972:Antineoplastic Agents, Phytogenic / Q000494:pharmacology* / Q000627:therapeutic use; D015153:Blotting, Western; D002467:Cell Nucleus / Q000187:drug effects / Q000378:metabolism; D002470:Cell Survival / Q000187:drug effects; D003600:Cytosol / Q000187:drug effects / Q000378:metabolism; D019008:Drug Resistance, Neoplasm / Q000187:drug effects* / Q000235:genetics; D024202:Electrophoretic Mobility Shift Assay; D005434:Flow Cytometry; D006367:HeLa Cells; D006801:Humans; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000096:biosynthesis*; D008546:Melanoma, Experimental / Q000188:drug therapy / Q000235:genetics / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic; D016328:NF-kappa B; D009368:Neoplasm Transplantation; D017239:Paclitaxel / Q000494:pharmacology* / Q000627:therapeutic use; D017382:Reactive Oxygen Species / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013947:Thymosin / Q000235:genetics / Q000494:pharmacology / Q000502:physiology*; D000099201:Thymosin beta(4)
D000972:Antineoplastic Agents, Phytogenic; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D016328:NF-kappa B; D017239:Paclitaxel; D017382:Reactive Oxygen Species; D013947:Thymosin; D000099201:Thymosin beta(4); C497442:HIF1A protein, human