Diallyl Disulfide Mitigates DNA Damage and Spleen Tissue Effects After Irradiation.

BACKGROUND Several factors found in foods are beneficial to human health and they may contribute to radiation protection. Taking food factors could be an easy way to reduce the effects of radiation after nuclear accidents, as well as secondary radiation risks after cancer radiotherapy or space missions. Here, diallyl disulfide (DADS), a component of garlic oil, was studied for its ability to mitigate radiation damage. MATERIAL AND METHODS We investigated the effects of DADS on micronucleus (MN) formation and apoptosis in HepG2 cells by use of 4-Gy X-ray irradiation. We also assessed the effects of DADS on radiation damage in vivo by evaluating MN formation in bone marrow cells in mice (BALB/c, 8-week-old females) after oral intake of DADS prior to irradiation with 4 Gy. Several tissue effects were also investigated. RESULTS The presence of DADS inhibited MN formation, whereas DADS had no influence on the radiation-induced inhibition of cell cycle progression in HepG2 cells. An increase in apoptosis in HepG2 cells was induced after irradiation, and this effect was stronger in the presence of DADS than in its absence. In mice, when DADS was administered daily for 3 days prior to irradiation, MN formation in irradiated mice was decreased. The decrease in MN formation in mice was greater with 0.5% DADS compared to 1% DADS. Moreover, an increase in spleen weight observed 3 weeks after irradiation was suppressed in mice administered DADS. CONCLUSIONS DADS is a potential radiation-protective agent that effectively mitigates DNA damage, and its effects in the spleen observed after irradiation may be related to inflammation and carcinogenesis.

Evaluation of Iodine-125 Interstitial Brachytherapy Using Micro-Positron Emission Tomography/Computed Tomography with 18F-Fluorodeoxyglucose in Hepatocellular Carcinoma HepG2 Xenografts.

BACKGROUND Iodine-125 interstitial brachytherapy (125I-IBT) is a promising treatment option for unresectable hepatocellular carcinoma (HCC). This study evaluated the usefulness of micro-positron emission tomography/computed tomography (micro-PET/CT) with 18F-fluorodeoxyglucose (18F-FDG) in assessing response to 125I-IBT in HCC HepG2 xenograft. MATERIAL AND METHODS Twelve mice with bilateral HepG2 xenografts were divided into 3 equal groups implanted with iodine-125 seeds into the left xenografts with a dose of 30, 50, and 80 Gy, respectively, and the right xenografts were used as internal controls. Before and 28 days after treatment, the 18F-FDG micro-PET/CT was performed. The ratios of left to right xenografts of tumor volume (RTV), maximum standardized uptake value (RSUVmax), mean optical density of caspase-3 expression (RMODcaspase-3), and apoptosis index (RAI) were compared. RESULTS The RTV means of the 50 and 80 Gy groups were significantly lower than in the 30 Gy group after treatment (P<0.01) and the RTV means after treatment were lower than baseline in the 50 and 80 Gy groups (P<0.05). The RSUVmax mean after treatment was lower than baseline in the 80 Gy group (P<0.05). The RMODCaspase-3 and RAI means of the 80 Gy group were higher than in the 30 Gy group (P<0.05). The RSUVmax was correlated negatively to RMODcaspase-3 (r=-0.624, P<0.05) and RAI (r=-0.651, P<0.05). CONCLUSIONS This study suggest that 125I-IBT inhibits tumor growth via upregulating caspase-3 expression and prompting apoptosis in HCC HepG2 xenografts. The 18F-FDG micro-PET/CT may be a useful functional imaging modality to assess early response to 125I-IBT in HCC HepG2 xenograft.

[Killing effect and its mechanism of low-temperature plasma on different human cancer cell lines].

Objective: To investigate the killing effect of low-temperature plasma (LTP) on HepG2, A549 and HeLa cell lines and explore its possible mechanism. Methods: The inhibitory effect of LTP on the proliferation of HepG2, A549 and HeLa cells was determined by MTT assay. Transmission electron microscopy was used to observe the ultrastructural changes of HepG2, A549 and HeLa cells treated with LTP. Cell apoptosis was detected by Muse cytometry. Western blot was used to detect the expression of apoptosis-related proteins. Results: The survival rates of LTP-irradiated HepG2 cells (irradiated for 107 s), HeLa cells (irradiated for 121 s) and A549 cells (irradiated for 127 s) were 50%. LTP destroyed the ultrastructure of HepG2, A549 and HeLa cells to different degrees, showing nuclear fragmentation and organelle damages. The apoptosis rates of the three cell lines were increased at 24 h after exposure to LTP for 1/6 IC50 irradiation time. Furthermore, LTP irradiation also suppressed the protein expression of Bcl-2 and XRCC1 and increased that of Bax. Conclusions: LTP has an obvious killing effect on HepG2, A549 and HeLa cancer cell lines. This effect may be related to the induction of cell apoptosis and inhibition of DNA repair.

The synergistic radiosensitizing effect of tirapazamine-conjugated gold nanoparticles on human hepatoma HepG2 cells under X-ray irradiation.

Reductive drug-functionalized gold nanoparticles (AuNPs) have been proposed to enhance the damage of X-rays to cells through improving hydroxyl radical production by secondary electrons. In this work, polyethylene glycol-capped AuNPs were conjugated with tirapazamine (TPZ) moiety, and then thioctyl TPZ (TPZs)-modified AuNPs (TPZs-AuNPs) were synthesized. The TPZs-AuNPs were characterized by transmission electron microscopy, ultraviolet-visible spectra, dynamic light scattering, and inductively coupled plasma mass spectrometry to have a size of 16.6±2.1 nm in diameter and a TPZs/AuNPs ratio of ~700:1. In contrast with PEGylated AuNPs, the as-synthesized TPZs-AuNPs exhibited 20% increment in hydroxyl radical production in water at 2.0 Gy, and 19% increase in sensitizer enhancement ratio at 10% survival fraction for human hepatoma HepG2 cells under X-ray irradiation. The production of reactive oxygen species in HepG2 cells exposed to X-rays in vitro demonstrated a synergistic radiosensitizing effect of AuNPs and TPZ moiety. Thus, the reductive drug-conjugated TPZs-AuNPs as a kind of AuNP radiosensitizer with low gold loading provide a new strategy for enhancing the efficacy of radiation therapy.

Novel immunologic tolerance of human cancer cell xenotransplants in zebrafish.

Immune deficiency or suppression in host animals is an essential precondition for the success of cancer cell xenotransplantation because the host immune system has a tendency to reject implanted cells. However, in such animals, the typical tumor microenvironment seen in cancer subjects does not form because of the lack of normal immunity. Here, we developed a novel zebrafish (Danio rerio) model based on 2 rounds of cancer cell xenotransplantation that achieved cancer-specific immunologic tolerance without immunosuppression. We irradiated human cancer cells (PC-3, K562 and HepG2) to abolish their proliferative abilities and implanted them into zebrafish larvae. These cells survived for 2 weeks in the developing host. Three months after the first implantation, the zebrafish were implanted with the same, but nonirradiated, cell lines. These cancer cells proliferated and exhibited metastasis without immune suppression. To reveal the transcriptional mechanism of this immune tolerance, we conducted dual RNA-seq of the tumor with its surrounding tissues and identified several regulatory zebrafish genes that are involved in immunity; the expression of plasminogen activator, urokinase, and forkhead box P3 was altered in response to immunologic tolerance. In conclusion, this xenograft method has potential as a platform for zebrafish-based anticancer drug discovery because it can closely mimic human clinical cancers without inducing immune suppression.

Effect of wilfortrine on human hepatic cancer HepG2 cell proliferation potential in vitro.

Liver cancers are characterized by high morbidity and mortality owing to few effective drugs for its treatment. Wilfortrine has several pharmacological effects, including an inhibitory effect on liver cancer cell proliferation. However, whether wilfortrine can induce liver cancer cell apoptosis has not been elucidated. We investigated the role of wilfortrine on liver cancer cell HepG2 apoptosis and analyzed its possible mechanisms to provide a theoretical basis for clinical analysis of liver cancer pathogenesis. The liver cancer cell line HepG2 was treated with 40 mM wilfortrine for 48 h. Flow cytometry was applied to detect HepG2 cell apoptosis and cell cycle changes. Western blot was used to analyze Bcl-2 and Bax expression. The HepG2 cell apoptosis rate increased significantly after treatment with wilfortrine, especially the early apoptosis rate (P < 0.05). However, wilfortrine did not change the cell cycle of HepG2 cells. After wilfortrine treatment, Bcl- 2 expression decreased significantly (P < 0.05); on the contrary, Bax expression increased noticeably compared with the control group (P < 0.05). Wilfortrine can induce liver cancer cell HepG2 apoptosis, but with no effect on the cell cycle, mainly by promoting Bax expression and inhibiting anti-apoptotic protein Bcl-2 expression.

[Observation of radiobiological characteristics in a HepG2 cell line with mitochondrial DNA deletion].

OBJECTIVE: To study the radiobiological characteristics of a HepG2 cell line with mitochondrial DNA (mtDNA) deletion. METHODS: HepG2 cells were cultured in a medium containing ethidium bromide, acetylformic acid and uracil. The HepG2 cell line with mtDNA deletion (ρ(0)HepG2 cells) were acquired after 30 subcultures by limited dilution cloning. The cell survival was then observed in the absence of acetylformic acid and uracil, and the total mtDNA deletion in the cells was confirmed by PCR. The radiosensitivity of HepG2 and ρ(0)HepG2 cells was evaluated by exposure to gradient doses of 6 MV X ray irradiation. The cell apoptosis was assessed following a 2 Gy X-ray exposure with Hochest33342 staining, and the invasiveness of ρ(0)HepG2 cells was measured by Transwell assay. RESULTS: HepG2 cells could survive 30 subcultures in the presence of ethidium bromide, and massive cell death occurred after removal of acetylformic acid and uracil from the medium. PCR confirmed total mtDNA deletion from ρ(0)HepG2 cells, whose α/ß value was significantly lower than that of HepG2 cells. ρ(0)Hep-G2 cells showed an obviously lowered cell apoptosis rate following X-ray exposure with enhanced cell invasiveness. CONCLUSION: HepG2 cells can be induced by ethidium bromide into ρ(0)HepG2 cells with an increased radiation resistance, anti-apoptosis ability and cell invasiveness.

Emodin attenuates radioresistance induced by hypoxia in HepG2 cells via the enhancement of PARP1 cleavage and inhibition of JMJD2B.

Radioresistance in the tumor and radiotoxicity in the non­tumorous liver significantly restrict efficient radiotherapy of hepatocellular carcinoma (HCC). It is therefore important to study the radioresistance mechanism and development of radiosensitization to optimize the effect of irradiation on cancer cells. Emodin (1, 3, 8­trihydroxy­6­methylanthraquinone) is a plant­derived polyphenol, possessing anticancer properties. It is known to act as a radiosensitizer in human HCC cell lines. The aim of this study was to evaluate the role of emodin in radioresistance of human HCC cell lines as well as the underlying radiosensitization mechanism. The human HCC cell line (HepG2) was used in this study. Four different treatment groups, i.e., no treatment (control), irradiation (10 Gy, one fraction), emodin (10 µM), and a combination of irradiation and emodin (10 Gy+10 µM) were used for two environmental conditions: hypoxia (1% O2) and normoxia (20% O2). The cells were exposed to the respective treatments for 24 and 72 h. Following the treatment, the cell viability was determined by the 3­(4,5­dimethylthiazol­2­yl)­2,5­diphenyltetrazolium bromide (MTT) assay, and the radiosensitization mechanism was evaluated by western blotting. The proliferation of HepG2 cells was significantly suppressed in the treatment groups under hypoxic and normoxic conditions in the following order: combination of irradiation and emodin>irradiation only >emodin only. The combination of irradiation and emodin induced apoptotic signaling activities such as cleavage of poly (ADP­ribose) polymerase (PARP)­1 as well as the downregulation of epigenetic signaling such as JMJD1A and JMJD2B. Emodin attenuated radioresistance in the HepG2 cells via upregulation of the apoptotic signals and down-regulation of the proliferative signals. These results suggested that emodin is a potential candidate for the radiosensitization of HCC cells and can aid in identifying novel therapeutic strategies for HCC radiotherapy.

SirT1 confers hypoxia-induced radioresistance via the modulation of c-Myc stabilization on hepatoma cells.

Intratumoral hypoxia is an important contributory factor to tumor cell resistance to radiotherapy. SirT1, a nicotinamide adenine dinucleotide (NAD(+))-dependent histone/protein deacetylase, has been linked to the decrease of radiation-induced DNA damage and seems to be critical for cancer therapy. The purpose of this study was to investigate the role of SirT1 in hypoxia-induced radiation response on hepatoma cells. It was found that the administration with resveratrol, a putative SirT1 activator, enhanced the resistance of HepG2 cells against radiation-induced DNA damage of MN formation under hypoxia condition; while nicotinamide, a well-known SirT1 inhibitor, sensitized this radiation damage. Nevertheless, pretreatment of cells with 10058-F4, a specific inhibitor of c-Myc, almost eliminated the nicotinamide-induced radiosensitive effect. Further studies revealed that resveratrol inhibited c-Myc protein accumulation via up-regulation of SirT1 expression and deacetylase activity, and this loss of c-Myc protein was abolished by inhibiting its degradation in the presence of MG132, a potent inhibitor of proteasome. In contrast, nicotinamide attenuated c-Myc protein degradation induced by radiation under hypoxia through inhibition of SirT1 deacetylase activity. Our findings suggest that SirT1 could serve as a novel potent target of radiation-induced DNA damage and thus as a potential strategy to advance the efficiency of radiation therapy in hepatoma entities.

The mechanism of the G0/G1 cell cycle phase arrest induced by activation of PXR in human cells.

CONTEXT: Pregnane X receptor (PXR) is an important transcriptional regulator that plays important roles in the cell metabolism and cell growth by regulating the transcriptional of a sort of metabolizing enzymes. OBJECTIVE: To investigate whether rifampicin effected HepG2 cells growth and the inhibition was due to the G0/G1 phase arrest. METHODS: PXR-knockdown experiments using RNAi showed that the cell cycle phase arrest mediated by rifampicin based on activation of PXR. The results also indicated that cell phase arrest by rifampicin could protect cells form UVB-induced DNA damage. Retinoid X receptor alpha (RXRα) expression level in cells is another key factor for cell cycle phase arrest mediated by rifampicin. Both over expression and lacking expression of RXRα in cell reduced the cell arrest efficiency mediated by rifampicin. In the study, we found that rifampicin inhibited HepG2 cells growth and demonstrated that the inhibition is due to the G0/G1 phase arrest through flow cytometry analysis. CONCLUSION: The results showed that RXRα promote cell cycle phase transition rate of HepG2. Competitive bind of rifampicin-activated PXR with RXRα is one main reason to arrest cell cycle phase through inhibiting combination of RXRα with other partners. Rifampicin could promote cell growth rate when RXRα expressed more excessively than PXR in cells.

[Study on absorption spectra of cell substrate for Hep-2 cell after being radiated by X-ray].

UV-absorption spectra of the Hep-2 cell's culture medium RPMI1640 (10% Foetal Calf Serum) were collected by UV-3101 spectrophotometer after the Hep-2 cell was radiated by X-ray and cultivated for 24, 48 and 72 h, and the absorbability of the proteins in the substrate was analyzed. From these results it was found that there were visible differences among these absorption spectra. In particular, the absorption peaks of the RPMI1640 culture medium during the cultivation shifted from 233 to 235 nm, while the absorption peak at 278 nm became more and more smooth and even finally disappeared with the cultivation time. On the other hand, the absorption intensity of the different-dose groups rose greatly with the time, and were all lower than the control group until the cells were cultivated for 72 h after being radiated by X-ray. It was showed that the content of each amino acid has already changed. That is, during the growing course of the cancer cells, the tryptophan and casein were not depleted equivalently. And there were some important relations between the absorption spectra and the cells' apoptosis and necrosis induced by X-ray. This will be a foundation for the study of the best X-ray dose for the larynx carcinoma.