Qingfeng Wu

Role of autophagy in high linear energy transfer radiation-induced cytotoxicity to tumor cells

Heavy‐ion radiotherapy has a potential advantage over conventional radiotherapy due to improved dose distribution and a higher biological effectiveness in cancer therapy. However, there is a little information currently available on the cellular and molecular basis for heavy‐ion irradiation‐induced cell death. Autophagy, as a novel important target to improve anticancer therapy, has recently attracted considerable attention. In this study, the effect of autophagy induced by high linear energy transfer (LET) carbon ions was examined in various tumor cell lines. To our knowledge, our study is the first to reveal that high‐LET carbon ions could induce autophagy in various tumor cells effectively, and the autophagic level in the irradiated cells increased in a dose‐ and LET‐dependent manner. The ability of carbon ions to inhibit the activation of the PI3K/Akt pathway rose with increasing their LET. Moreover, modulation of autophagy in tumor cells could modify their sensitivity to high‐LET radiation, and inhibiting autophagy accelerated apoptotic cell death, resulting in an increase in radiosensitivity. Our data imply that targeting autophagy might enhance the effectiveness of heavy‐ion radiotherapy.

Different Roles of CHOP and JNK in Mediating Radiation-Induced Autophagy and Apoptosis in Breast Cancer Cells.

Unfolded protein response (UPR) is comprised of complex and conserved stress pathways that function as a short-term adaptive mechanism to reduce levels of unfolded or misfolded proteins and maintain homeostasis in the endoplasmic reticulum (ER). UPR can be triggered by prolonged or persistent ER stress under many physiological or pathological conditions, including radiation exposure. Radiation-induced ER stress elicits autophagy and apoptosis in cancer cells, where C/EBP homologous protein (CHOP) and c-Jun NH2-terminal kinase (JNK) may play crucial roles. However, the specific mechanisms that regulate autophagy and apoptosis through CHOP and JNK after radiation exposure and how the balance of these activities determines the cellular radiosensitivity remain largely unclear. In this study, we found that exposure to X-ray radiation induced ER stress, UPR and high expression of CHOP and JNK. Furthermore, autophagy and apoptosis occurred in sequential order when breast cancer MDA-MB-231 and MCF-7 cells were exposed to X-ray radiation. CHOP gene knockdown with RNA interference inhibited autophagy and enhanced radiosensitivity in MDA-MB-231 cells, while impacting apoptosis and subsequently increasing radioresistance in MCF-7 cells. However, treatment with JNK inhibitor decreased autophagy while promoting apoptosis, thereby leading to radiosensitivity in both cell lines. Our results indicate that CHOP mediates radiation-induced autophagy and apoptosis in a cellular environment. Importantly, the functional consistency of regulating apoptosis and autophagy in these two irradiated breast cancer cell lines suggests that JNK may be more useful as a potential target for maximizing the efficacy of radiation therapy for breast cancers.

The antioxidant mechanism of nitroxide TEMPO: scavenging with glutathionyl radicals

A rhodamine-nitroxide probe (R-NO˙), combining rhodamine fluorophore with a 2,2,6,6-tetramethylpiperidinyl-1-oxy (TEMPO) receptor unit was introduced to probe glutathionyl radicals (GS˙) with high sensitivity and selectivity. The R-NO˙ probe could effectively scavenge GS˙ radicals with fluorescence enhancement since the nitroxide group restored the fluorescence properties. In this work, horseradish peroxidase (HRP)-catalyzed and metal-catalyzed oxidation systems were selected as the model of simulating the generation of GS˙, and we found that the metal-catalyzed system had the same experimental results with the HRP-catalyzed system, which provided a new approach to demonstrate the strong oxidant ability of the hydroxyl radical (˙OH) to initiate toxic GS˙. Furthermore, we confirmed that the production of GS˙ abided by a radical-initiated peroxidation mechanism of GSH with the mass spectrometry (MS) analysis and fluorescence spectroscopy. By using combined high-performance liquid chromatography (HPLC) detection and MS analysis, we also demonstrated that the R-NO˙ was converted into fluorescent secondary amine derivative (R-NH). The application of the probe in biological system was explored to monitor GS˙ in HL-60 cells and secondary amine fluorescence was observed upon stimulation by hydrogen peroxide and phenol. Development of fluorescence was prevented via preincubation with the thiol-blocking reagent N-ethylmaleimide (NEM).

Inhibiting Survivin Expression Increases the Radiosensitivity of Human Hepatoma HepG2 Cells to High-LET Carbon Ions

In this study, we investigated whether survivin plays a direct role in mediating high-LET radiosensitivity. We designed small interfering RNA (siRNA) targeting survivin mRNA, in vitro chemo-synthesized and transfected into HepG2 human hepatoma cells. Real-time PCR assay and western bolting were used to determine survivin expressions at transcriptional and post-transcriptional levels, respectively. The survivin expressions decreased at both the transcriptional and post-transcriptional levels after treatment with survivinspecific siRNA. This observation indicates that survivinspecific siRNA could make survivin gene silent effectively. After exposure to high-LET carbon ion radiation, a reduced clonogenic survival effect was observed in cells treated with siRNA compared with those untreated with siRNA. Furthermore, Annexin V assay was used to determine apoptotic rate. Compared with siRNA mismatch and irradiation alone, siRNA treated cells showed increased apoptotic rates. We also found that transfection with survivin siRNA increased levels of G2/M arrest. These results suggest that the radiosensitivity of HepG2 cells to high-LET carbon ions is increased after inhibiting survivin expression and gene therapy for constitutive survivin may be effective for therapeutic improvement in radioresistant solid tumors by high-LET radiation.

Mutagenic Research on Ciliary Neurotrophic Factor (CNTF) in Escherichia coli After Heavy Ions Irradiation

Based on the natural human CNTF gene, Ying et al. acquired CNTF-T mutant and constructed Escherichia coli expressing CNTF. In order to increase the yield and enhance the protein expression in the constructed E. coli, the strains were irradiated with high-LET heavy ions. Some mutants were obtained in this work and the mechanisms underlying the enhanced expression of CNTF in E. coli after exposure to heavy ions are discussed in this paper.