Exosomes synergized with PIONs@E6 enhance their immunity against hepatocellular carcinoma via promoting M1 macrophages polarization.

BACKGROUND: Hepatocellular carcinoma (HCC) is easy to relapse after resection for its lack of anti-tumor immunity due to pro-tumorigenesis by promoting M2 type macrophage polarization. Recent studies have shown that exosomes are closely related to the occurrence and development of HCC. Antigenic exosomes from HCC are able to polarize into alternatively activated macrophages M2, but do not stimulate M1 macrophages polarization. Iron oxide nanoparticles (IONs) have been demonstrated to be able to promote M1 macrophages polarization. This research was to explore exosomes as vehicles to synergize with pegylated IONs loaded with chlorin e6 (PIONs@E6) to enhance their immunity against HCC via promoting M1 macrophages polarization. MATERIALS AND METHODS: PIONs@E6 was synthesized and then characterized by chemico-physical analysis, transmission electron microscope (TEM), respectively. After characterization of PIONs-contained exosomes by TEM, and then the exosomal surface specific molecules CD9 and CD63 were determined by Western Blotting assay. Markers of M1 macrophage polarization in vitro and in vivo were analyzed by enzyme linked immunosorbent assay (ELISA) and flow cytometry, respectively. Intracellular reactive oxygen species (ROS) in macrophages were analyzed using a Spectra Max fluorescence microplate reader. Inhibitory effect of PIONs-contained exosomes on HCC was evaluated by monitoring tumor growth in an in vivo xenograft mice model. RESULTS: PIONs@E6 showed good water solubility with a core diameter around 10 nm and a hydrate diameter around 37 nm. The expression of exosome specific markers CD9 and CD63 was kept at a high level. PIONs-contained exosomes can dose-dependently promote M1 macrophages polarization in vitro and in vivo. Of note, PIONs-contained exosomes could initiate a significantly higher level of ROS in macrophages and remarkably inhibit the tumor growth in mice bearing HCC xenograft. CONCLUSION: Exosomes as vehicles could be synergized with PIONs@E6 to enhance their immunity against HCC via promoting M1 macrophages polarization.

Ultrasmall iron oxide nanoparticles induced ferroptosis via Beclin1/ATG5-dependent autophagy pathway.

Iron-based nanoparticles, which could elicit ferroptosis, is becoming a promising new way to inhibit tumor cell growth. Notably, ultrasmall iron oxide nanoparticles (USIONPs) have been found to upregulate the autophagy process in glioblastoma (GBM) cells. Whether USIONPs could also elicit ferroptosis and the relationship between the USIONPs-induced autophagy and ferroptosis need to be explored. In the current study, our synthesized USIONPs with good water solubility could significantly upregulate the ferroptosis markers in GBM cells, and downregulate the expression of anti-ferroptosis genes. Interestingly,ferrostatin-1 could reverse USIONPs- induced ferroptosis, but inhibitors of apoptosis, pyroptosis, or necrosis could not. Meanwhile, autophagy inhibitor 3-methyladenine could also reverse the USIONPs-induced ferroptosis. In addition, shRNA silencing of upstream genes Beclin1/ATG5 of autophagy process could significantly reverse USIONPs-induced ferroptosis, whereas overexpression of Beclin1/ATG5 of autophagy process could remarkably promote USIONPs-induced ferroptosis. Furthermore, lysosome inhibitors could significantly reverse the USIONPs-induced ferroptosis. Collectively, these facts suggest that USIONPs-induced ferroptosis is regulated via Beclin1/ATG5-dependent autophagy pathway.

Peripheral T lymphocytes predict the severity and prognosis in patients with HBV-related acute-on-chronic liver failure.

BACKGROUND: Hepatitis B virus-related acute-on-chronic liver failure (HBV-ACLF) is a life-threatening syndrome with high mortality. Biomarkers are urgently needed to predict the prognosis of HBV-ACLF. Recent evidence suggests a key role for immune system in the pathology of HBV-ACLF. Here, we analyzed the correlation between peripheral blood T lymphocytes and the severity and prognosis in HBV-ACLF patients. METHOD: Sixty-six patients with HBV-ACLF received conventional medical treatments for 4 weeks. Twenty-five healthy subjects and 20 HBV patients were enrolled for comparison. We determined white blood cell count, lymphocytes, CD3+, CD4+ and CD8+ T cells, and CD4+CD25+ Treg cells in the blood of all subjects. Their associations with laboratory parameters before or after treatments were statistically analyzed. RESULT: The results showed that compare normal subjects and chronic hepatitis B patients, HBV-ACLF patients had significantly increased white blood count, CD4+ T cells and decreased lymphocytes, CD3+ T cells, and Treg cells. Correlation analysis showed that white blood cell, lymphocytes, and peripheral T lymphocytes were correlated with prothrombin activity (PTA) and model for end-stage liver disease (MELD) scores. After treatment, white blood cell, lymphocytes, and peripheral T lymphocytes were also correlated with PTA and MELD scores. Additionally, total bilirubin (TBIL), alanine aminotransferase (ALT), international standard ratio (INR), MELD, and white blood cell count were potential prognostic criteria for HBV-ACLF patients. CONCLUSION: HBV-ACLF patients had depletion and dysfunction of immune system. Changes of peripheral T lymphocytes were closely related to the pathogenesis and prognosis of disease. Our results may contribute to predict the severity of HBV-ACLF, and provide a prognosis response to improve the treatment of HBV-ACLF.

Ultrasmall iron oxide nanoparticles inhibit the migration and invasion of human hepatocellular carcinoma HepG2 cells by enhancing autophagy / 中国肿瘤生物治疗杂志

@#[摘 要] 目的：探讨超微氧化铁纳米粒子（ultrasmall iron oxide nanoparticle，USIONP）对人肝细胞癌HepG2细胞迁移和侵袭的影响及其可能的机制。方法：采用粒径分析仪和透射电镜分别分析USIONP的水合粒径和核心粒径，Zeta电位和胶体稳定性实验分析USIONP的分散性及其稳定性以鉴定USIONP的成功制备；用不同质量浓度USIONP（0、50、100、200 μg/ml）或200 μg/ml USIONP+5 mmol/L 3-MA（自噬抑制剂）联合处理HepG2细胞，CCK-8法检测HepG2细胞的增殖活力，Transwell法检测细胞的迁移和侵袭能力，WB实验检测自噬标志物Beclin1、LC3、p62的表达，2’,7’-二氯二氢荧光素二醋酸（DCFH-DA）法测定细胞内活性氧（ROS）水平，铁离子比色法检测细胞内铁离子水平。结果：USIONP的平均水合粒径为（37.86±12.90） nm、核心粒径约10 nm，Zeta电位为–23.8 mV，有良好的水溶分散性，证实了USIONP的成功制备。随USIONP质量浓度升高和处理时间延长，HepG2细胞的增殖活力明显降低（均P<0.05）；与对照组相比，200 μg/ml USIONP处理HepG2细胞24 h后，迁移、侵袭细胞数量均显著减少（均P<0.05），而3-MA能够部分抵消上述影响（均P<0.05）。与对照组相比，100、200 μg/ml USIONP处理组的HepG2细胞中Beclin1和LC3Ⅱ蛋白相对表达水平均显著升高（均P<0.05），而p62蛋白表达水平下降（均P<0.05）；200 μg/ml USIONP可显著提高细胞内ROS水平与铁离子水平，而加入3-MA可阻断其作用（均P<0.05）。结论：USIONP能促进HepG2细胞发生自噬，而自噬通路激活后降解USIONP释放铁离子和导致细胞ROS水平升高，从而抑制HepG2细胞的迁移和侵袭。

Apoptosis selectively induced in BEL-7402 cells by folic acid-modified magnetic nanoparticles combined with 100 Hz magnetic field.

OBJECTIVE: To explore the effect of folic acid-modified magnetic nanoparticles (FA-MNPs) combined with a 100 Hz extremely low-frequency electromagnetic field (ELF-EMF) on the apoptosis of liver cancer BEL-7402 cells. MATERIALS AND METHODS: MNPs (20 nm) were prepared by coprecipitation, and then folic acid was coated onto MNPs to prepare FA-MNPs. BEL-7402 cells and HL7702 cells were selected as liver cancer cells and normal liver cells, respectively. The ELF-EMF was generated from a solenoid coil. Cellular uptake of NPs was determined by inductively coupled plasma atomic emission spectroscopy. A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay was used to evaluate cell inhibition. Apoptosis was analyzed by flow cytometry. Statistical analyses were performed using two-way analysis of variance. RESULTS: FA-MNPs combined with a 100 Hz magnetic field significantly inhibited cell proliferation and induced higher apoptosis compared to either the ELF-EMF alone or FA-MNPs alone. FA-MNPs showed a better apoptosis effect and higher iron uptake in BEL-7402 cells compared to in HL7702 cells. On the basis of the ELF-EMF, higher doses of FA-MNPs brought higher apoptosis and higher iron uptake in either BEL-7402 cells or HL7702 cells. CONCLUSION: These results suggest that FA-MNPs may induce apoptosis in a cellular iron uptake-dependent manner when combined with an ELF-EMF in BEL-7402 cells.

Non-aggregation based label free colorimetric sensor for the detection of Cu2+ based on catalyzing etching of gold nanorods by dissolve oxygen.

A label-free non-aggregation colorimetric sensor has been designed for the detection of Cu(2+), based on Cu(2+) catalyzing etching of gold nanorods (AuNRs) along longitudinal axis induced by dissolve oxygen in the presence of S2O3(2-), which caused the aspect ratio (length/width) of AuNRs to decrease and the color of the solution to distinctly change. The linear range and the detection limit (LD, calculated by 10 Sb/k, n=11) of this sensor were 0.080-4.8 µM Cu(2+) and 0.22 µM Cu(2+), respectively. This sensor has been utilized to detect Cu(2+) in tap water and human serum samples with the results agreeing well with those of inductively coupled plasma-mass spectroscopy (ICP-MS), showing its remarkable practicality. In order to prove the possibility of catalyzing AuNRs non-aggregation colorimetric sensor for the detection of Cu(2+), the morphological structures of AuNRs were characterized by high resolution transmission electron microscopy (HRTEM) and the sensing mechanism of colorimetric sensor for the detection of Cu(2+) was also discussed.

Ultra-sensitive non-aggregation colorimetric sensor for detection of iron based on the signal amplification effect of Fe3+ catalyzing H2O2 oxidize gold nanorods.

Fe(3+) can catalyze H2O2 to oxidize along on the longitudinal axis of gold nanorods (AuNRs), which caused the aspect ratio of AuNRs to decrease, longitudinal plasmon absorption band (LPAB) of AuNRs to blueshift (Δλ) and the color of the solution to change obviously. Thus, a rapid response and highly sensitive non-aggregation colorimetric sensor for the determination of Fe(3+) has been developed based on the signal amplification effect of catalyzing H2O2 to oxidize AuNRs. This simple and selective sensor with a wide linear range of 0.20-30.00 µM has been utilized to detect Fe(3+) in blood samples, and the results consisted with those obtained by inductively coupled plasma-mass spectroscopy (ICP-MS). Simultaneously, the mechanism of colorimetric sensor for the detection of Fe(3+) was also discussed.

Application of graphene-pyrenebutyric acid nanocomposite as probe oligonucleotide immobilization platform in a DNA biosensor.

A stable and uniform organic-inorganic nanocomposite that consists of graphene (GR) and pyrenebutyric acid (PBA) was obtained by ultrasonication, which was characterized by scanning electron microscopy (SEM) and UV-vis absorption spectra. The dispersion was dropped onto a gold electrode surface to obtain GR-PBA modified electrode (GR-PBA/Au). Electrochemical behaviors of the modified electrode were characterized by cyclic voltammetry and electrochemical impedance spectroscopy using [Fe(CN)6](3-/4-) as the electroactive probe. A novel DNA biosensor was constructed based on the covalent coupling of amino modified oligonucleotides with the carboxylic group on PBA. By using methylene blue (MB) as a redox-active hybridization indicator, the biosensor was applied to electrochemically detect the complementary sequence, and the results suggested that the peak currents of MB showed a good linear relationship with the logarithm values of target DNA concentrations in the range from 1.0×10(-15) to 5.0×10(-12) M with a detection limit of 3.8×10(-16) M. The selectivity experiment also showed that the biosensor can well distinguish the target DNA from the non-complementary sequences.

Fullerol-fluorescein isothiocyanate-concanavalin agglutinin phosphorescent sensor for the detection of alpha-fetoprotein and forecast of human diseases.

Based on the reaction of the active -OH group in fullerol (F) with the dissociated -COOH group in fluorescein isothiocyanate (FITC) to form an F-FITC and the enhanced effect of N, N-dimethylaniline (DMA) on phosphorescence signal of F-FITC, a new phosphorescent labeling reagent (DMA-F-FITC) was developed. What's more, a phosphorescent sensor for the determination of alpha-fetoprotein variant (AFP-V) has been designed via the coupling technique of the high sensitivity for affinity adsorption-solid substrate-room temperature phosphorimetry (AA-SS-RTP) with the strong specificity reaction between DMA-F-FITC-Con A and AFP-V. The DMA-F-FITC increased the number of luminescent molecules in the biological target which improved the sensitivity of phosphorescent sensor. The proposed sensor was responsive, simple, selective and sensitive, and it has been applied to the determination of trace AFP-V in human serum and the forecast of human diseases using phosphorescence emission wavelength of F or FITC, with the results agreed well with those obtained by enzyme-linked immunoassay (ELISA). Meanwhile, the mechanisms for the labeling reaction and the sensing detection of AFP-V were discussed.

Electrocatalytical oxidation and sensitive determination of acetaminophen on glassy carbon electrode modified with graphene-chitosan composite.

The electrochemical behaviors of acetaminophen (ACOP) on a graphene-chitosan (GR-CS) nanocomposite modified glassy carbon electrode (GCE) were investigated by cyclic voltammetry (CV), chronocoulometry (CC) and differential pulse voltammetry (DPV). Electrochemical characterization showed that the GR-CS nanocomposite had excellent electrocatalytic activity and surface area effect. As compared with bare GCE, the redox signal of ACOP on GR-CS/GCE was greatly enhanced. The values of electron transfer rate constant (ks), diffusion coefficient (D) and the surface adsorption amount (Γ(*)) of ACOP on GR-CS/GCE were determined to be 0.25s(-1), 3.61×10(-5) cm(2) s(-1) and 1.09×10(-9) mol cm(-2), respectively. Additionally, a 2e(-)/2H(+) electrochemical reaction mechanism of ACOP was deduced based on the acidity experiment. Under the optimized conditions, the ACOP could be quantified in the range from 1.0×10(-6) to 1.0×10(-4) M with a low detection limit of 3.0×10(-7) M based on 3S/N. The interference and recovery experiments further showed that the proposed method is acceptable for the determination of ACOP in real pharmaceutical preparations.

A sensitive DNA biosensor based on a facile sulfamide coupling reaction for capture probe immobilization.

A novel DNA biosensor was fabricated through a facile sulfamide coupling reaction. First, the versatile sulfonic dye molecule of 1-amino-2-naphthol-4-sulfonate (AN-SO3(-)) was electrodeposited on the surface of a glassy carbon electrode (GCE) to form a steady and ordered AN-SO3(-) layer. Then the amino-terminated capture probe was covalently grafted to the surface of SO3(-)-AN deposited GCE through the sulfamide coupling reaction between the amino groups in the probe DNA and the sulfonic groups in the AN-SO3(-). The step-by-step modification process was characterized by electrochemistry and attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy. Using Ru(NH3)6(3+) as probe, the probe density and the hybridization efficiency of the biosensor were determined to be 3.18×10(13) strands cm(-2) and 86.5%, respectively. The hybridization performance of the biosensor was examined by differential pulse voltammetry using Co(phen)3(3+/2+) (phen=1,10-phenanthroline) as the indicator. The selectivity experiments showed that the biosensor presented distinguishable response after hybridization with the three-base mismatched, non-complementary and complementary sequences. Under the optimal conditions, the oxidation peak currents of Co(phen)3(3+/2+) increased linearly with the logarithm values of the concentration of the complementary sequences in the range from 1.0×10(-13)M to 1.0×10(-8)M with a regression coefficient of 0.9961. The detection limit was estimated to be 7.2×10(-14)M based on 3σ.

Zr(H2O)2EDTA modulated luminescent carbon dots as fluorescent probes for fluoride detection.

A novel fluorescent probe (Zr(CDs-COO)(2)EDTA) has been designed for fluoride ion (F(-)) content detection based on the competitive ligand reactions carried out between the carboxylate groups (-COOH) on the surface of the luminescent carbon dots (CDs) and F(-) coordinated to Zr(H(2)O)(2)EDTA. The strong and stable fluorescence signal of this probe was quenched upon the addition of F(-) as a result of the formation of the non-fluorescent complex Zr(F)(2)EDTA, due to the stronger affinity of F(-) than the -COOH in the CDs to Zr(IV). The fluorescence change (ΔF) in this process was linear with respect to the content of F(-), ranging from 0.10 µM to 10 µM. The probe has been applied to F(-) detection in toothpaste and water samples with satisfactory results. Moreover, the mechanism of this Zr(H(2)O)(2)EDTA modulated fluorescent probe for the detection of F(-) was also discussed.

Catalytic solid substrate-room temperature phosphorimetry for the determination of residual perphenazine based on the electronic effect of rhodamine 6G.

The rhodamine 6G(+) -perphenazine (Rhod 6G(+) -PPH) compound is formed in the ester-exchange reaction between -OH of PPH and -COOC2 H5 of Rhod 6G(+) . PPH was oxidized to a red compound (PPH') in the presence of K2 S2 O8 . Interestingly, the room temperature phosphorescence (RTP) of Rhod 6G(+) was quenched because the -OH of PPH' reacted with -COOC2 H5 of Rhod 6G(+) -PPH to form Rhod 6G(+) -PPH' and PPH, which decreased the π-electron density (δ) of the carbon atom in the Rhod 6G(+) -PPH' conjugated system and enhanced the nonradiation energy loss of the excited Rhod 6G(+) of the triplet state. The PPH content was directly proportional to the ΔIp of the system. Thus, a new catalytic solid-substrate room temperature phosphorimetry (SSRTP) method was established for the determination of PPH. The method had high sensitivity (the limit of detection was 0.019 fg/spot, corresponding to a concentration of 4.8 × 10(-14) g/mL; the sampling quantity was 0.40 µL/spot), good selectivity, convenience and speed. The analytical results were in accordance with those of high-performance liquid chromatography (HPLC). The structures of Rhod 6G(+) , PPH and Rhod 6G(+) -PPH were characterized by infrared spectra. The reaction mechanism by which PPH was determined is discussed.

Ultra-sensitive complex as phosphorescence probe for the determination of trace protein.

ß-CD-HMTA-L-Tyr complex, formed in the host guest inclusion reaction carried out between host molecule ß-cyclodextrin (ß-CD) in ß-CD-HMTA (HMTA is methenamine) and guest molecule L-tryptophan (L-Tyr), possessing the characteristic of room temperature phosphorescence (RTP). Bovine serum albumin (BSA) reacted with L-Tyr to form a complex of cage structure bringing in the sharply RTP signal quenching of L-Tyr. Based on the above facts, a new ultra-sensitive solid substrate room temperature phosphorimetry (SSRTP) for the determination of trace protein has been established using ß-CD-HMTA-L-Tyr complex as a phosphorescence probe. Under the optimum conditions, the linear range of this method was 0.0040-0.56 agspot(-1) with a detection limit (D.L.) as 0.92 zgspot(-1), and the regression equations of working curve was ΔI(p)=0.8239+162.5 m(BSA) (agspot(-1), n=8) with the correlation coefficient (r) of 0.9994. The relatively standard deviation (RSD) and the recovery of SSRTP were 4.8-3.3% and 96.7-102%, respectively, indicating that this method had good repeatability. The proposed phosphorescence probe has been applied in the detection of protein in real samples and the results agreed well with those obtained with SSRTP using methylene blue-sodium tetraphenylborate as phosphorescence probe. Meanwhile, the reaction mechanism for the determination of trace protein with ß-CD-HMTA-L-Tyr complex as phosphorescence probe has been discussed.

A specific Tween-80-Rhodamine S-MWNTs phosphorescent reagent for the detection of trace calcitonin.

The present study proposed a simple sensitive and specific immunoassay for the quantification of calcitonin (CT) in human serum with water-soluble multi-walled carbon nanotubes (MWNTs). The COOH group of MWNTs could react with the NH group of rhodamine S (Rhod.S) molecules to form Rhod.S-MWNTs, which could emit room temperature phosphorescence (RTP) on acetate cellulose membrane (ACM) and react with Tween-80 to form micellar compound. Tween-80-Rhod.S-MWNTs (TRM), as a phosphorescent labelling reagent, could dramatically enhance the RTP signal of the system. The developed TRM phosphorescent reagent was used to label anti-calcitonin antibody (Ab(CT)) to form the TRM-Ab(CT) labelling product, which could take high specific immunoreaction with CT, and the ΔI(p) (= I(p2)-I(p1), I(p2) and I(p1) were the phosphorescence intensity of the test solution and the blank sample, respectively) of the system was linear to the content of CT. Hence, a new solid substrate room temperature phosphorescence immunoassay (SSRTPIA) was established for the determination of CT in human serum. This sensitive (limit of quantification (LOQ) was 8.0×10(-14) g mL(-1)), accurate, selective and precise method has been applied to determine CT in human serum and predict primary osteoporosis and fractures, with the results in good agreement with those obtained by chemiluminescence immunoassay (CLIA). Simultaneously, the structure of MWNTs was characterized with scanning electron microscopy (SEM) and infrared spectroscopy (IR), and the reaction mechanisms of both labelling Ab(CT) with TRM and SSRTPIA for the determination of trace CT were discussed.

Coupling technique of self-ordered ring and phosphorimetry for the determination of alkaline phosphatase and diseases prediction.

Rhodamine S could emit strong and stable room temperature phosphorescence (RTP) on polyamide membrane (PAM) in the presence of heavy atom perturber Pb(2+). When Rhodamine S-piperidine solution was dropped on PAM, the red (Rhod.S)(n)-P-SOR (Rhod.S, (Rhod.S)(n), P and SOR refer to alizarin red S, multiple Rhod.S molecules, piperidine and self-ordered ring, respectively) formed on PAM, leading to the enhancement of room temperature phosphorimetry (RTP) intensity (I(p), 117.2) of (Rhod.S)(n)-P-SOR system, which was 2.4 times higher than that without SOR (I(p), 48.1). Wheat germ agglutinin (WGA) was labelled with (Rhod.S)(n)-P-SOR by the -NH- of Rhod.S reacting with the -COOH of WGA to form WGA-(Rhod.S)(n)-P-SOR. The formation of WGA-AP-WGA-(Rhod.S)(n)-P-SOR in the affinity adsorption (AA) reaction carried out between the -COOH of WGA in WGA-(Rhod.S)(n)-P-SOR and the -NH(2) of alkaline phosphatase (AP) caused the RTP intensity (ΔI(p)) of the WGA-AP-WGA-(Rhod.S)(n)-P-SOR system 7.8 times larger than that without (Rhod.S)(n)-P-SOR. Therefore, the coupling technique of SOR and solid substrate-room temperature phosphorimetry (SS-RTP) for the determination of trace AP has been established. This method possessed good selectivity, high sensitivity (Detection limit (L.D) was 3.4×10(-16)gmL(-1)) and accuracy, and it has been applied to the determination of trace AP in human serum and the forecast of human diseases, and the results agreed well with those obtained by enzyme-linked immunoassay (ELISA). Besides, the mechanism of the coupling technique for the determination of AP was discussed.

The effect of 100 Hz magnetic field combined with X-ray on hepatoma-implanted mice.

Our previous cellular experiments demonstrated that 100 Hz magnetic field (MF) was effective at enhancing apoptosis of liver cancer cells BEL-7402 induced by X-ray irradiation. This study was performed to further explore the possible synergism between 100 Hz MF and X-ray in treatment of hepatoma-implanted Balb/c mice. 100 Hz MF exposure with a mean flux density of 0.7 mT was performed inside an energized solenoid coil. Six MV X-ray irradiation was generated using a linear accelerator. Tumor growth and survival of mice implanted with H22 cells were evaluated by measuring the tumor diameters and overall days of survival. Six groups treated with 100 Hz MF or X-ray alone or a combination of MF and X-ray were examined. Furthermore, the effects of different numbers of MF exposure periods on tumor growth and mice survival were examined when combined with 4 Gy X-ray. Data referring to overall survival days and tumor diameters of the above groups were compared using log-rank test and Student's t-test. Our results showed that five periods of combined 100 Hz MFs and 4 Gy X-ray could significantly extend the overall days of survival and reduce the tumor size compared to MF or X-ray alone. Also, a greater number of 100 Hz MF exposure periods could further improve the survival and inhibit tumor growth in hepatoma-implanted mice when combined with 4 Gy X-ray. In conclusion, these findings suggested that 100 Hz MF could possibly synergize with 4 Gy X-ray in terms of survival improvement and tumor inhibition in hepatoma-implanted mice.