H22 Solid Tumor Research Articles

NG, a novel PABA/NO-based oleanolic acid derivative, induces Human hepatoma cell apoptosis via a ROS/MAPK-dependent mitochondrial pathway

O2-(2,4-dinitro-5-{[2-(12-en-28-β–D- galactopyranosyl-oleanolate-3-yl) -oxy-2-oxoethyl]amino}phenyl)1-(N-hydroxyethylmethylamino)diazen-1-ium-1,2- diolate (NG), a novel PABA/NO-based derivative of oleanolic acid (OA), has been found to show potent antitumor activity both in vivo and in vitro. In the present study, NG could significantly reduce tumor volume and weight in the H22 solid tumor mouse model. Meanwhile, NG showed selective effects on the HepG2 cells including NO generation, cytotoxic effect and apoptosis, which were prevented by hemoglobin (NO scavenger). Moreover, NG-induced apoptosis of HepG2 cells was characteristic of intracellular reactive oxygen species (ROS) generation, loss of mitochondrial membrane potential (Δψm) and enhanced Bax-to-Bcl-2 ratio. The release of apoptotic inducing factor (AIF) and cytochrome c (Cyt c) from mitochondria and the activation of caspase-3, 9 were also detected, indicating that NG may induce apoptosis through a mitochondrial-mediated pathway. Simultaneously, NG treatment could lead to the activation of the phosphorylation of c-Jun N-terminal kinase (JNK) and p38 MAPK but not ERK1/2. Treatment with SP600125 (an inhibitor of JNK) and SB203580 (an inhibitor of p38) prior to NG was found to reverse NG-induced apoptosis. Moreover, it was found that antioxidant N-acetylcysteine (NAC) blocked the induction of apoptosis and partly reversed the activation of JNK and p38, up-regulation of Bax, down-regulation of Bcl-2 and the activation of caspase-3 in NG-treated cells. Taking together, these findings suggest that NO can be released from NG, which induces apoptosis through a ROS/MAPK-mediated mitochondrial pathway.

Antitumor Activity of Extracts from Oxytropis falcata Bunge on human Hepatocellular Carcinoma SMMC-7721 Cells In Vitro and in H22 Bearing Mice In Vivo

Oxytropis falcata Bunge is widely used as one of the “three anti-inflamatory drugs” and is considered to be the “king of herbs” in Chinese Tibetan medicine. For thousands of years it has been used to treat inflammation, pyreticosis and bleeding [1]. In the present study, we investigated the antitumor effects of O. falcata extracts on human hepatocellular carcinoma SMMC-7721 cells in vitro and in transplanted murine H22 tumors in vivo. Cell proliferation, morphological changes, cell cycle distribution and apoptosis in SMMC-7721 cells were determined and tumor growth inhibition in H22 tumors was investigated. MTT assay revealed that essential oil fraction (EOOF) and total flavonoids fraction (TFOF) of O. falcata inhibited proliferation of SMMC-7721 cells in a dose-dependent manner. Growth inhibition in H22 solid tumors was significantly pronounced after being treated with TFOF. Hoechst 33258 staining of SMMC-7721 cells indicated that TFOF caused typical characteristics of apoptotic programmed cell death, such as cell shrinkage, apoptotic body formation and nuclear fragmentation. Cell cycle distribution was analyzed by flow cytometry with propidium iodide (PI) and annexin V-FITC/PI double staining that significant solid tumor inhibition occurred in H22 transplanted mice, cell cycle was arrested at G1 phase, and induction of apoptosis occurred in SMMC-7721 cells when subjected to TFOF. These results suggest that TFOF promotes apoptosis in SMMC-7721 cells and inhibits H22 tumor growth, resulting in a potential antitumor effect on hepatic cancer. Acknowledgements: Support for this research from the National Natural Science Foundation of China for Youth (Grant No.30902012) is gratefully acknowledged.

Preliminary Pharmacological Study of Polysaccharides from Angelica and Astragalus

Angelica and Astragalus, Chinese herbal medicine, are used as immunostimulants for enriching the blood, anti-tumor and antioxidation effects. However, the synergistic mechanisms of antioxidant and antitumor effects have long been the object of intensive study. We investigated the deferring senile effect of polysaccharides from Angelica and Astragalus (AAP) on aging mice by determination of the contents of monoamine oxidase (MAO) in brain, hydroxyproline (Hyp) in skin. In addition, KM mice with murine H22 hepatocarcinoma cell model were used to study the in vivo antitumor efficacy of AAP by measuring inhibition rate of tumor and immune organs index. The obtained results showed that AAP decreased significantly the content of MAO, and enhanced the concentrations of Hyp in aging mice. No significant differences were found as compared with those of vitamin E. The results of anti-tumor showed that inhibition rate of the higher dose of AAP was 43.8%, and it could enhance immune organs index. Our study indicates that AAP has significant deferring senile effect on the aging mice and suppressing the growth of H22 solid tumor in mice.

The effect of 2-methoxyestradiol liposome on growth inhibition, angiogenesis and expression of VEGF and Ki67 in mice bearing H22 hepatocellular carcinoma

2-methoxyestradiol (2-ME), an endogenous metabolite of estrogen, has very low water solubility. It is currently in phase II clinical trials as both a chemopreventive and chemotherapeutic agent and has been orally administered to cancer patients. However, the poor oral absorption of the compound is one of the major obstacles for 2-ME development. Based on the molecular features of 2-ME, liposome can be considered an attractive formulation approach. Our purpose in this study is to research the antitumor efficacy of 2-methoxyestradiol liposome (2-ME-L) in mice bearing H 22 tumors. Murine H22 hepatocarcinoma served as an ectopic solid tumor model. The effects of antitumor therapy were evaluated by testing tumor growth, measuring the tumor inhibition rates in terms of weight and volume, and staining the tissues by hematoxylin and eosin. The synergistic mechanism of 2-ME-L therapy was elucidated by detecting changes in the expression of pathognostic factors in the tumor microenvironment. 2-ME-L significantly suppressed tumor growth. The morphological changes in the tumors indicated that the tumors in the treatment groups were effectively confined with little surrounding angiogenesis. Tumor cells of the treatment groups had abundant areas of necrosis with few nuclei in the mitotic phase. It was found that there was less immunohistochemical expression of vascular endothelial growth factor (VEGF), Ki67 and CD31 in the treatment groups and the efficacy of 2-ME-L was better than that of 2-ME solution (2-ME-S). This research demonstrated that 2-ME-L inhibited the growth of H 22 tumors in a concentration-dependent manner and was more effective than 2-ME-S. 2-ME-L can suppress the growth of H22 solid tumors and has antiproliferative, proapoptotic and antiangiogenic activity. 2-ME-L could be of potential use in the treatment of hepatocellular carcinoma.

Oridonin nanosuspension enhances anti-tumor efficacy in SMMC-7721 cells and H22 tumor bearing mice

The aim of the present study was to evaluate both the in vitro and in vivo antitumor activity of an oridonin nanosuspension (ORI-N) relative to efficacy of bulk oridonin delivery. ORI-N with a particle size of 897.2±14.2 nm and a zeta potential of -21.8±0.8 mV was prepared by the high-pressure homogenization (HPH) technique. The in vitro cytotoxicity of ORI-N against SMMC-7721 cells was evaluated by MTT[3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay, the effects of ORI-N on cell cycle and cell apoptosis was analyzed by flow cytometry; the in vivo anti-tumor activity was observed in H22 tumor bearing mice. ORI-N effectively inhibited the proliferation of SMMC-7721 cells. Flow cytometric analysis demonstrated that ORI-N arrested SMMC-7721 cells in the G2/M cycle, and furthermore, that ORI-N induced a higher apoptotic rate than the bulk ORI solution. In vivo studies ORI-N also showed higher antitumor efficacy as measured by reduced tumor volume and tumor weight, as well as lower toxicity in H22 solid tumor bearing mice compared to free ORI at the same concentration. These results suggest that the delivery of ORI-N as a nanosuspension is a promising approach for treating tumors.

ZL11n is a novel nitric oxide-releasing derivative of farnesylthiosalicylic acid that induces apoptosis in human hepatoma HepG2 cells via MAPK/mitochondrial pathways

ZL11n is a novel furoxan-based nitric oxide (NO)-releasing derivative of farnesylthiosalicylic acid. In this study, we examined the anticancer effects and the potential mechanism of action of ZL11n in vitro and in vivo. It was found that ZL11n exhibited a favorable, selective cytotoxic effect in the HepG2 cell line. The yield of NO in the ZL11n treated HepG2 cells was much higher than in the control group and the normal human liver L-02 cells. Furthermore, the NO concentration was correlated to the degree of cytotoxicity observed. The ZL11n-induced apoptosis was assessed by Annexin V-FITC/propidium iodide flow cytometry assay. ZL11n triggered the mitochondrial/caspase apoptotic pathway by decreasing mitochondrial membrane potential, cytochrome c release from mitochondrial, and reducing the Bcl-2-to-Bax ratio, in addition to activating the caspase cascade. Simultaneously, we found that ZL11n treatment led to an increase in JNK and ERK1/2 phosphorylation. Furthermore, treatment with SP600125 (a JNK inhibitor) and PD98059 (an ERK1/2 inhibitor) prior to ZL11n treatment was found to significantly reverse ZL11n-induced apoptosis. The in vivo findings also revealed that ZL11n significantly reduced tumor volume and weight in the H22 solid tumor mouse model examined. In short, our findings suggest that ZL11n induced apoptosis through the coordination of the mitochondrial apoptotic pathway (activated by NO) and MAPKs signaling pathway (triggered by JNK or ERK).

PLA nanoparticles loaded with an active lactone form of hydroxycamptothecin: Development, optimization, and in vitro–in vivo evaluation in mice bearing H22 solid tumor

AbstractHydroxycamptothecin (HCPT)‐loaded PLA nanoparticles were prepared by a one‐step method using the direct dialysis technique, and were examined for particle characteristics, in vitro drug release, and cytotoxicity, as well as antitumor efficiency. Three main influential factors based on the results of a single‐factor test, i.e., PLA concentration, ratio of HCPT to PLA (wt/wt), and dialysis bags with different molecule weight cutoffs, were evaluated using an orthogonal design, giving nanoparticles an average diameter of ∼226.8 nm with smooth surface, modest drug entrapment efficiency (65.15%), and fine drug‐loading content (5.16%, w/w). HCPT was in a crystalline state within the particles. In vitro drug release studies exhibited a slow and prolonged release profile over a period of 30 days. The cytotoxicity test on BEL‐7402 cells indicated that the HCPT‐PLA nanoparticles were more cytotoxic than commercially available HCPT injection. When the antitumor effect was examined by i.v. administration to mice bearing H22 solid tumor, HCPT‐PLA nanoparticles showed a significant suppression of tumor growth without loss of body weight. These results suggest that HCPT‐PLA nanoparticles prepared by the dialysis technique present desirable characteristics for sustained drug delivery and are potentially useful to enhance the antitumor efficacy of HCPT. Drug Dev Res 72: 337–345, 2011. © 2011 Wiley‐Liss, Inc.

Study on treatment of H22 liver cancer using 10-HCPT-Ioaded microbubbles and ultrasound-targeted destruction

Objective To prepare lipid-coated ultrasound microbubbles containing 10-HCPT(HLM) and explore the antitumor effects on mice xenografed H22 solid tumor using the technique of ultrasound-mediated HLM destruction. Methods Sixty-four tumor-bearing mice were radomly divided into A and B groups. Each group was divided into four groups again and administered respectively by tail vein with HI.M, non-drug-loaded microbubbles,10-HCPT and saline once a day. Ultrasound irradiation was applied on the tumor sites immediately after injection. After 7 days of consecutive treatment, all mice in group A were sacrificed and the tumors were harvested to measure weights. The tumor inhibition rate was calculated by weights. The tumor microvessel density (MVD) was detected by immunohistochemical staining. The tumor growth curve was depicted according to volumes. The survival time of mice in group B was recorded. Results The tumor inhibition rate was the highest in HLM group while this group's MVD was the lowest. Survival time in HLM group and 10-HCPT group were obviously longer compared with the control group,while no statistic difference was observed between the two groups. There was no statistic difference between the group of non-drug-loaded microbubbles and the control group. Conclusions Ultrasound irradiation mediates HLM destruction so that the drug is released from the vihicles at the same time, which can significantly enhance the tumor inhibition effect of 10-HCPT on the H22 tumor. This technique is expected to be adopted as a novel tool for liver cancer chemotherapy. Key words: Ultrasonography; Microbubbles ; Liver neoplasms; experimental ; Topotecan

Experimental Studies of Anti-tumor Effect of Baoganyannian Injection on Liver Cancer

目的 研究保肝延年注射液的抗肝癌作用.方法选择小鼠肝癌H22癌模型、MTT法和集落形成法,观察保肝延年注射液对荷瘤鼠和人肝癌SMMC-7721细胞株的作用.结果腹腔注射保肝延年注射液80mg/kg、160mg/kg、320mg/kg,肿瘤受到明显抑制,抑制率分别为72.5%、90.1%和94.6%(P＜0.05,P＜0.01);小鼠生存时间显著延长;在体外保肝延年注射液对人肝癌细胞有明显的细胞毒作用,其IC50为2.04 μg/ml;同时保肝延年注射液可抑制人肝癌SMMC-7221克隆原细胞形成集落,其IC50为2.35μg/ml.结论保肝延年注射液在体内和体外均具有抗肝癌作用。