A comparison of apoptosis and necrosis induced by hepatotoxins in HepG2 cells

7H-Dibenzo[c,g]carbazole (DBC), an N-heterocyclic aromatic hydrocarbon, is cytotoxic and carcinogenic in rodent liver. While DBC leads to necrotic lesions in the liver, the induction of apoptosis by DBC has not been investigated. The focus of this study was to determine the degree to which apoptosis and necrosis contributed to DBC cytotoxicity in a human hepatoma cell line (HepG2). To determine if these effects were unique to DBC, the results were compared to another hepatotoxin, aflatoxin B(1) (AFB(1)). DBC produced a distinct biphasic LDH release curve within 24 h of exposure. During the same time period lower concentrations of DBC (<10 microM) induced the formation of DBC-DNA adducts and increased p53 protein levels followed by apoptotic cell death. However, increasing the concentration of DBC to 80 microM led to lower DNA adduct and p53 protein levels. At this concentration, intracellular ATP levels were rapidly depleted followed by cell swelling and loss of membrane integrity consistent with necrotic cell death. In contrast to DBC, a biphasic LDH release curve was not observed for AFB(1). Instead, AFB(1) induced a concentration-dependent increase in apoptosis that reached two- to threefold higher levels than DBC. These results suggest that differences exist in the extent and type of cell death induced by DBC and AFB(1) at equimolar concentrations. Apoptosis and necrosis result from low and high concentrations of DBC, respectively, and may be dependent upon intracellular ATP levels.     

Cancer chemoprevention by dietary chlorophylls: a 12,000-animal dose-dose matrix biomarker and tumor study

Recent pilot studies found natural chlorophyll (Chl) to inhibit carcinogen uptake and tumorigenesis in rodent and fish models, and to alter uptake and biodistribution of trace (14)C-aflatoxin B1 in human volunteers. The present study extends these promising findings, using a dose-dose matrix design to examine Chl-mediated effects on dibenzo(def,p)chrysene (DBC)-induced DNA adduct formation, tumor incidence, tumor multiplicity, and changes in gene regulation in the trout. The dose-dose matrix design employed an initial 12,360 rainbow trout, which were treated with 0-4000ppm dietary Chl along with 0-225ppm DBC for up to 4weeks. Dietary DBC was found to induce dose-responsive changes in gene expression that were abolished by Chl co-treatment, whereas Chl alone had no effect on the same genes. Chl co-treatment provided a dose-responsive reduction in total DBC-DNA adducts without altering relative adduct intensities along the chromatographic profile. In animals receiving DBC alone, liver tumor incidence (as logit) and tumor multiplicity were linear in DBC dose (as log) up to their maximum-effect dose, and declined thereafter. Chl co-treatment substantially inhibited incidence and multiplicity at DBC doses up to their maximum-effect dose. These results show that Chl concentrations encountered in Chl-rich green vegetables can provide substantial cancer chemoprotection, and suggest that they do so by reducing carcinogen bioavailability. However, at DBC doses above the optima, Chl co-treatments failed to inhibit tumor incidence and significantly enhanced multiplicity. This finding questions the human relevance of chemoprevention studies carried out at high carcinogen doses that are not proven to lie within a linear, or at least monotonic, endpoint dose-response range.     

Mutagenicity and DNA adduct formation by the urban air pollutant 2-nitrobenzanthrone.

2-Nitrobenzanthrone (2-NBA) has recently been detected in ambient air particulate matter. Its isomer 3-nitrobenzanthrone (3-NBA) is a potent mutagen and suspected human carcinogen identified in diesel exhaust. The highest mutagenic activity of 2-NBA tested in Salmonella typhimurium was exhibited in strain TA1538-hSULT1A1 expressing human sulfotransferase (SULT) 1A1. 2-NBA also induced mutations in Chinese hamster lung V79 cells expressing human N-acetyltransferase 2 or SULT1A1, but no mutagenicity was observed in the parental cell line. DNA adduct formation in vitro was examined in different human cell lines by thin-layer chromatography (32)P-postlabeling. Whereas 3-NBA formed characteristic DNA adducts in lung A549, liver HepG2, colon HCT116, and breast MCF-7 cells, 2-NBA-derived DNA adducts were only observed in A549 and HepG2 cells, indicating differences in the bioactivation of each isomer. The pattern of 2-NBA-derived DNA adducts in both cell lines consisted of a cluster of up to five adducts. In HepG2 cells DNA binding by 2-NBA was up to 14-fold lower than by 3-NBA. DNA adduct formation of 2-NBA was also investigated in vivo in Wistar rats treated with a single dose of 2, 10, or 100 mg/kg body weight (bw). No DNA adduct formation was detected at doses of up to 10 mg/kg bw 2-NBA, even though 3-NBA induced DNA adducts at a dose of 2 mg/kg bw. Only after administration of one high dose of 100 mg/kg bw 2-NBA was a low level of DNA adduct formation detected, and then only in lung tissue. Density functional theory calculations for both NBAs revealed that the nitrenium ion of the 3-isomer is considerably more stable ( approximately 10 kcal/mol) than that of the 2-isomer, providing a possible explanation for the large differences in DNA adduct formation and mutagenicity between 2- and 3-NBA.     

白藜芦醇抑制HepG2细胞生长及诱导细胞凋亡的初步研究

目的　研究白藜芦醇 (Res)对人肝癌HepG2细胞生长增殖的影响 ,进而探讨Res诱导HepG2细胞凋亡的作用。方法　用不同浓度的Res处理HepG2细胞 ,MTT法检测Res对HepG2细胞生长增殖的抑制作用 ,通过HE染色、透射电子显微镜、原位末端标记法 (TUNEL)和流式细胞术观察凋亡细胞的形态结构变化以及定性、定量检测细胞凋亡。结果　Res能抑制HepG2细胞的生长增殖 ,并呈浓度依赖性 ;Res能诱导HepG2细胞凋亡。 结论　Res能够通过诱导HepG2细胞凋亡抑制HepG2细胞的增殖。     

Inhibition of Akt signaling in hepatoma cells induces apoptotic cell death independent of Akt activation status

The serine/threonine kinase Akt, a downstream effector of phosphatidylinositol 3-kinase (PI3K), is involved in cell survival and anti-apoptotic signaling. Akt has been shown to be constitutively expressed in a variety of human tumors including hepatocellular carcinoma (HCC). In this report we analyzed the status of Akt pathway in three HCC cell lines, and tested cytotoxic effects of Akt pathway inhibitors LY294002, Wortmannin and Inhibitor VIII. In Mahlavu human hepatoma cells Akt was constitutively activated, as demonstrated by its Ser473 phosphorylation, downstream hyperphosphorylation of BAD on Ser136, and by a specific cell-free kinase assay. In contrast, Huh7 and HepG2 did not show hyperactivation when tested by the same criteria. Akt enzyme hyperactivation in Mahlavu was associated with a loss of PTEN protein expression. Akt signaling was inhibited by the upstream kinase inhibitors, LY294002, Wortmannin, as well as by the specific Akt Inhibitor VIII in all three hepatoma cell lines. Cytotoxicity assays with Akt inhibitors in the same cell lines indicated that they were all sensitive, but with different IC50 values as assayed by RT-CES. We also demonstrated that the cytotoxic effect was through apoptotic cell death. Our findings provide evidence for its constitutive activation in one HCC cell line, and that HCC cell lines, independent of their Akt activation status respond to Akt inhibitors by apoptotic cell death. Thus, Akt inhibition may be considered as an attractive therapeutic intervention in liver cancer.     

Mechanisms of toxicity associated with six tyrosine kinase inhibitors in human hepatocyte cell lines

Tyrosine kinase inhibitors have revolutionized the treatment of certain cancers. They are usually well tolerated, but can cause adverse reactions including liver injury. Currently, mechanisms of hepatotoxicity associated with tyrosine kinase inhibitors are only partially clarified. We therefore aimed at investigating the toxicity of regorafenib, sorafenib, ponatinib, crizotinib, dasatinib and pazopanib on HepG2 and partially on HepaRG cells. Regorafenib and sorafenib strongly inhibited oxidative metabolism (measured by the Seahorse‐XF24 analyzer) and glycolysis, decreased the mitochondrial membrane potential and induced apoptosis and/or necrosis of HepG2 cells at concentrations similar to steady‐state plasma concentrations in humans. In HepaRG cells, pretreatment with rifampicin decreased membrane toxicity (measured as adenylate kinase release) and dissipation of adenosine triphosphate stores, indicating that toxicity was associated mainly with the parent drugs. Ponatinib strongly impaired oxidative metabolism but only weakly glycolysis, and induced apoptosis of HepG2 cells at concentrations higher than steady‐state plasma concentrations in humans. Crizotinib and dasatinib did not significantly affect mitochondrial functions and inhibited glycolysis only weakly, but induced apoptosis of HepG2 cells. Pazopanib was associated with a weak increase in mitochondrial reactive oxygen species accumulation and inhibition of glycolysis without being cytotoxic. In conclusion, regorafenib and sorafenib are strong mitochondrial toxicants and inhibitors of glycolysis at clinically relevant concentrations. Ponatinib affects mitochondria and glycolysis at higher concentrations than reached in plasma (but possibly in liver), whereas crizotinib, dasatinib and pazopanib showed no relevant toxicity. Mitochondrial toxicity and inhibition of glycolysis most likely explain hepatotoxicity associated with regorafenib, sorafenib and possibly pazopanib, but not for the other compounds investigated.     

不同浓度依托咪酯诱导人肝癌HepG2细胞体外凋亡的研究

目的探讨不同浓度的依托咪酯是否具有直接诱导人肝癌HepG2细胞凋亡的作用,寻找依托咪酯临床治疗的新途径。方法人肝癌细胞株HepG2体外扩增培养备用,选取不同浓度的依托咪酯E1、E2、E3和对照组（空白）,分别培养12h、24h、48h后观察。结果与空白对照组相比,培养12h、24h、48h后E1组、E2组人肝癌细胞株HepG2细胞凋亡率无明显差异（P〉0．05）,E3组凋亡率不断增加,具有显著性差异（P〈0．05）;随着培养时间的延长及浓度的不断增加,E1与E2组对比人肝癌细胞株HepG2细胞凋亡率无明显差异（t=1．732,P〉0．05）,E1与E3组对比人肝癌细胞株HepG2细胞凋亡率具有显著性差异（t=1．864,P〈0．05）,E2与E3组对比人肝癌细胞株HepG2细胞凋亡率具有明显差异（f=1．691,P〈0．05）。结论依托咪酯在临床安全有效的浓度下不直接诱导人肝癌HepG2细胞的体外凋亡,浓度及时间达到一定峰值时可具有直接诱导人肝癌HepG2细胞体外凋亡的作用。     

Induction of apoptosis in cells by cadmium: quantitative negative correlation between basal or induced metallothionein concentration and apoptotic rate.

Metallothionein (MT) often reduces the adverse effects of cadmium (Cd), but how it may alter Cd-induced apoptosis is unclear. The goal of this study was to define the role of MT in Cd-induced apoptosis using cell lines with widely varying sensitivity to Cd. Effects of Cd on growth of human hepatocellular carcinoma cell lines (HepG2 and PLC/PRF/5) were investigated and compared with Chang cells. These cells were cultured with 0, 5, 10, 20, 40, 80, and 120 microM of Cd for 3, 6, 12, and 24 h. Significant cytolethality was observed in HepG2 and PLC/PRF/5 cells in a time- and concentration-dependent manner, with LC(50) values of 24 microM and 13 microM, respectively. However, Chang cells were much less sensitive to Cd-induced cytotoxicity (LC(50), 64 microM). Apoptotic cell death occurring at cytolethal concentrations was demonstrated in all cell lines by DNA fragmentation on agarose gel electrophoresis or by ELISA. When MT was measured, there was a highly significant negative linear correlation between the basal cellular MT concentration or Cd-induced MT and the rate of apoptosis induced by Cd in these cell lines. Treating HepG2 cells with zinc (Zn) made the relatively sensitive HepG2 cell line resistant to Cd-induced apoptosis, likely due to Zn-induced MT. In fact, there was also a significant negative linear correlation between the amount of Zn-induced MT in HepG2 cells and the rate of Cd-induced apoptosis. These findings revealed that basal or induced MT perturbs Cd-induced apoptotic cell death in various cell lines, and a strong negative correlation exists between cellular MT content and the rate of apoptosis induced by Cd.     

Central effect of dibutyryl cyclic AMP on the temperature in the conscious rabbit

In the rabbit, intracerebroventricular administration of dibutyryl cyclic AMP (DBC) produces fever which is selectively antagonised by phenoxybenzamine. This indicates the involvement of central alpha-adrenoceptors in DBC-induced fever. The decrease in DBC hyperthermia after 6-hydroxydopamine (6-OH-DA) supports the viewpoint that DBC-induced fever is dependent on the presence of noradrenaline (NA) in the central nervous system. The accentuation of NA hyperthermia by theophylline suggests that NA fever may be mediated by cAMP. It is unlikely that DBC-induced hyperthermia in the rabbit is mediated via prostaglandins since indomethacin does not inhibit this response to DBC.     

吡格列酮对HepG2细胞增殖和凋亡的影响及机制研究

目的观察吡格列酮对体外培养的HepG2细胞增殖和凋亡的影响,并探讨其是否通过PPARγ依赖途径发挥上述药理作用。方法将不同浓度的吡格列酮作用于体外培养HepG2细胞,以MTT比色法检测HepG2细胞增殖情况,以3H-TdR参入实验检测细胞DNA合成速率,采用RT-PCR和Western blot检测PPARγmRNA和蛋白的表达,以流式细胞术检测细胞凋亡和细胞周期;同时观察PPARγ特异性拮抗剂GW9662和(或)瞬时转染pSG5-PPARγ真核表达质粒对吡格列酮细胞增殖作用的影响;并将PPARγ小干扰RNA(pGCsi-PPARγ)表达质粒稳定转染HepG2细胞,观察PPARγ沉默后吡格列酮对HepG2细胞增殖作用的影响。结果吡格列酮作用于HepG2细胞后,导致HepG2细胞的增殖受到抑制、DNA合成速率减慢,并诱导细胞凋亡,呈一定的剂量依赖关系;在此过程中,G0/G1期细胞比例明显增加,S期细胞比例明显减少,但PPARγmRNA和蛋白的表达没有变化;GW9662部分拮抗吡格列酮的增殖抑制作用,但转染pSG5-PPARγ真核表达质粒可以逆转GW9662的作用;吡格列酮在高浓度(20μmol.L-1)时对pGCsi-PPARγ表达质粒稳定转染的HepG2细胞仍表现出增殖抑制作用。结论吡格列酮能够抑制HepG2细胞的增殖并诱导凋亡,具有潜在的抗瘤作用,这种作用与其诱导细胞G0/G1期的停滞有关,PPARγ依赖和非依赖途径参与上述过程。     

Immunohistochemical detection of melphalan-DNA adducts in colon cancer cells in vitro and human colorectal liver tumours in vivo

Melphalan is a chemotherapeutic drug that exerts its cytotoxic effect mainly through the formation of DNA adducts. We report the specific immunohistochemical detection and visualisation of melphalan-DNA adducts using the monoclonal antibody MP5/73 in cultured tumour cells and solid tumour tissue from colorectal liver metastases from patients treated with melphalan. The human colon cancer cell lines HT29, SW480 and SW1116, and the rat colon cancer cell line CC531 were exposed to different concentrations of melphalan. In addition, tumour samples from 17 patients with colorectal liver metastases treated by isolated hepatic perfusion with high dose melphalan (200mg) were collected. Cell lines and tumour samples were stained with the MP5/73 antibody against melphalan-DNA adducts and cell viability was determined by an MTT assay. Melphalan-DNA adducts could be visualised by immunohistochemistry in both cultured cells and solid tumour tissue. A correlation between melphalan exposure concentration, the subsequent melphalan-DNA adduct staining intensity, and melphalan cytotoxicity existed for each individual cell line, but the level of both parameters independently differed between cell lines. Specific staining for melphalan-DNA adducts also was feasible in the human solid tumour tissue. There was considerable variation in melphalan-DNA adduct staining, staining intensity, and distribution in the tumour stroma and the tumour epithelium among the different patients. Melphalan-DNA adducts appeared to be more intense in tumour cells at the border of the tumour nodules than in tumour cells in the centre. Thus, visualisation of melphalan-DNA adducts by immunohistochemistry allows the study of distribution of melphalan-DNA adducts in solid tumours.     

氯唑沙腙对抗HepG2肿瘤细胞的作用研究

目的研究氯唑沙腙(chlorzoxazone)对HepG2细胞存活和凋亡的影响。方法采用MTT法检测氯唑沙腙对体外培养的HepG2细胞存活率的影响,通过检测LDH释放观察氯唑沙腙对HepG2细胞的致坏死作用,通过TUNEL法评价细胞凋亡率,透射镜评价细胞的超微结构。结果氯唑沙腙在50～500μmol.L-1浓度范围内对HepG2细胞的存活率均有抑制作用,呈明显的剂量依赖效应关系。荧光显微镜和透射电镜下可见典型的肿瘤细胞凋亡改变。氯唑沙腙在100、200、300及500μmol.L-1浓度下作用48h均可诱导HepG2细胞凋亡,具有明显的剂量效应关系。氯唑沙腙100、200、300及500μmol.L-14种浓度分别作用于HepG2细胞24、48及72h,发现以上各种浓度在48h即可诱导细胞凋亡,作用时间越长,凋亡率越高,有明显的时间效应关系。结论氯唑沙腙能够抑制HepG2细胞存活和诱导细胞凋亡。     

Induction of apoptosis in HepG2 human hepatocellular carcinoma cells by a novel derivative of ursodeoxycholic acid (UDCA)

The effects of ursodeoxycholic acid (UDCA) and its novel derivative, named as HS-1030, on the proliferation of HepG2, human hepatocellular carcinoma cells were investigated. Whereas UDCA had no significant effect in a concentration range we have tested, HS-1030 inhibited the proliferation of HepG2 cells in a concentration dependent manner. Surprisingly, HS-1030 had no effect on the proliferation of Human Chang liver cell which is a normal liver cell line. We also found that proliferation-inhibitory effect of HS-1030 was due to the induction of apoptosis of HepG2 cells, which was confirmed by observing the internucleosomal DNA fragmentation and morphological changes (i.e. cell shrinkage, nuclear condensation and the formation of apoptotic bodies). These results suggest that HS-1030 may be a good candidate as a drug for the treatment of liver cancer.     

Generation of a liver targeting fusion interferon and its bioactivity analysis in vitro

The objective of this study was to generate a liver targeting fusion interferon, galactosyl-human serum albumin-interferon alpha2b (G-HSA-IFN) and to evaluate its bioactivity in vitro on HepG2.2.15 cells which express hepatitis B surface antigen (HBsAg) and hepatitis B e antigen (HBeAg). The cell proliferation was determined by Sulpho Rhodamine B (SRB) staining method and flow cytometry (FCM) assay. Hochest33342 and Propidium Iodide (PI) double staining and Western blot analysis of Bcl-2/Bax were also performed to evaluate cell lethality and apoptosis. The concentrations of HBsAg and HBeAg secreted in culture supernatant were detected using Enzyme-Linked Immunosorbent Assay (ELISA). The results demonstrated that G-HSA-IFN could inhibit the proliferation of HepG2.2.15 cells and the cell cycle was arrested at G0/G1 phase. Western blotting results showed that the expression of Bcl-2 was inhibited in a dose-dependent manner while the expression of Bax was enhanced. The expression of HBsAg was inhibited by G-HSA-IFN in a dose-dependent manner, while no significant inhibiting effect on the expression of HBeAg was observed. Conclusively, G-HSA-IFN could not only significantly inhibit the HBsAg expression and the proliferation of HepG2.2.15 cells, but also induce the apoptosis of the target cells, rendering it a promising drug candidate for hepatitis B.     

Increase the cisplatin cytotoxicity and cisplatin-induced DNA damage in HepG2 cells by XRCC1 abrogation related mechanisms

Cisplatin is one of the most potent chemotherapeutic anticancer drugs for the treatment of various cancers. The cytotoxic action of the drug is often thought to be associated with its ability to bind DNA to form cisplatin–DNA adducts. Impaired DNA repair processes including base excision repair (BER) play important roles on its cytotoxicity. XRCC1 is a key protein known to play a central role at an early stage in the BER pathway. However, whether XRCC1 contributes to decrease the cisplatin cytotoxicity and cisplatin-induced DNA damage in HepG2 still remains unknown. Hence, the purpose of this study was to explore whether abrogation of XRCC1 gene expression by short hairpin RNAs (shRNA) could reduce DNA repair and thus sensitize liver cancer cells to cisplatin. We abrogated the XRCC1 gene in HepG2 cell using shRNA transfection. Cell viability was measured by MTT assay and clonogenicity assay. Comet assay was used to detect the DNA damage induced by cisplatin. The host cell reactivation was employed to assess the DNA repair capacity of cisplatin-damaged luciferase reporter plasmid. Flow cytometry analysis was used to determine cisplatin-induced apoptosis, cell cycle and reactive oxygen species (ROS). The results showed that abrogation of XRCC1 could sensitize HepG2 cells to cisplatin. This enhanced cytotoxicity could be attributed to the increased DNA damage and reduced DNA repair capacity. Increasing cell cycle arrest and intracellular ROS production lead to more tumor cell apoptosis and then enhanced the cisplatin cytotoxicity. Our results suggested that the cisplatin cytotoxicity may increase by targeting inhibition of XRCC1.     

Natural plant extract tubeimoside I promotes apoptosis-mediated cell death in cultured human hepatoma (HepG2) cells

Tubeimoside I (TBMS I), an extract from Chinese herbal medicine Bolbostemma paniculatum (MAXIM.) FRANQUET (Cucurbitaceae) has been shown as a potent anti-tumor agent for a variety of human cancers, but yet to be evaluated for hepatoma that is highly prevalent in Eastern Asian countries including China. Here, we examined in vitro the cytotoxic effects of TBMS I on human hepatoma (HepG2) and normal liver (L-02) cell lines. We also investigated TBMS I-induced molecular events related to apoptosis in HepG2 cells. The results show that TBMS I inhibited the proliferation of both HepG2 and L-02 cells in a dose- and time-dependent manner, but HepG2 cells appeared more sensitive to the agent. When exposed to TBMS I for 24, 48 and 72 h, IC?? for HepG2 cells versus L-02 cells were 15.5 vs. 23.1, 11.7 vs. 16.2, 9.2 vs. 13.1 (μM, p<0.01), respectively. TBMS I induced cell shrinkage, nuclear condensation and fragmentation, cell cycle arrest at the G2/M phase, mitochondrial membrane disruption, release of cytochrome c from the mitochondria, activation of caspase 3 and 9, and shifting Bax/Bcl-2 ratio from being anti-apoptotic to pro-apoptotic, all indicative of initiation and progression of apoptosis involving mitochondrial dysfunction. Taken together, these results indicate for the first time that TBMS I potently inhibited growth in HepG2 cells by mediating a cascade of apoptosis signaling pathways. Considering its sensitivity of HepG2 cells, preferential distribution in the liver and natural product origin, TBMS I therefore may have a great potential as a chemotherapeutic drug candidate for hepatoma.     

The effect of inhibitors of phosphatidylinositol 3-kinase-related kinases on dibenzo[def,p]chrysene genotoxicity measured by γH2AX levels and neutral comet assay in HepG2 human hepatocellular cancer cells

In the study the modulating effect of inhibition of phosphatidylinositol 3-kinase-related kinases (PIKK): ATM (Ataxia Telangiectasia Mutated), ATR (Ataxia Telangiectasia and Rad3 Related) and DNA-PK (DNA-dependent protein kinase) on genotoxicity of dibenzo[def,p]chrysene (DBC) in HepG2 human hepatocellular cancer cells was investigated. The cytotoxicity of DBC was determined, also in combination with PIKK inhibitors, using the MTT reduction assay. The high cytotoxicity of DBC was observed after 72 h incubation (IC₅₀ = 0.06 μM). The PIKK inhibitors applied at non-cytotoxic concentrations: caffeine (1 mM) and KU55933 (2.5 μM) had no significant influence on the DBC cytotoxicity, however NU7026 (5 μM) caused significant increase in the cell viability by about 25%. The combinations of the inhibitors (double or triple) where NU7026 was present also caused increase in the cell viability (i.e. cytoprotective effect) compared to the effect of DBC. The level of damage to the genetic material (DNA double strand breaks, DSB) was assessed by measuring levels of phosphorylated form of H2A histone (γH2AX) and neutral comet assay. DBC induced DSB in a concentration and time-dependent manner. NU7026 considerably reduced the level of DSB level measured by γH2AX and comet assay.The obtained results confirm that DBC is cytotoxic and causes damage to the genetic material including DSB. The DNA-PK inhibitor NU7026 increases cell viability after exposure to DBC and reduces DNA damage, what indicates an important role of the sensor kinase in mediating the effect.     

Limitations of the single-cell gel electrophoresis assay to monitor apoptosis in U937 and HepG2 cells exposed to 7beta-hydroxycholesterol

The single-cell gel electrophoresis (comet) assay is a method which allows the detection of DNA strand breaks in individual cells. It has been suggested that the single cell gel electrophoresis assay, as an index of DNA fragmentation during cell death, may be applied to monitor apoptosis. The aim of the present study was to determine if the pattern of DNA fragmentation determined by the single cell gel electrophoresis assay can be used to discriminate between the mode of cell death in two cell lines (U937, a human monocytic blood cell line and HepG2, a human hepatocarcinoma cell line) which were treated with 30 microM 7beta-hydroxycholesterol (7betaOHC) over a 48 hr period. The single cell gel electrophoresis assay was compared with more established methods for the determination of apoptosis such as morphological examination, flow cytometry and DNA laddering. The percentage of maximally damaged nuclei as measured by the single cell gel electrophoresis assay was found to be similar at 48 hr in both U937 and HepG2 cells when treated with 7betaOHC. However, morphological examination, flow cytometry and DNA laddering techniques showed that 7betaOHC induced apoptosis in U937 cells but not in HepG2 cells. Thus, although the alkaline single cell gel electrophoresis assay detected DNA strand breaks occurring during cell death, these breaks were observed only when the process was fairly well advanced and a major part of the cells had lost membrane permeability. Therefore the present report demonstrates that the single cell gel electrophoresis assay, used in isolation, cannot accurately be used to distinguish between the mode of cell death induced by 7betaOHC in U937 cells (apoptosis), or HepG2 cells (cell lysis).     

Preliminary studies on induction of apoptosis by genistein on HepG2 cell line.

Consumption of soy products has been linked to lower the incidence of number of cancers. Genistein, one of the principal soy isoflavones, has been shown to inhibit the growth of a number of tumor cell lines in vitro. In this study, we investigate the effects of genistein on cell growth and apoptosis in human hepatocellular carcinoma HepG2 cell by looking for the formation of nuclear apoptotic bodies and DNA ladder formation. Additionally, flow cytometry analysis with propidium iodide staining has been conducted to detect the apoptotic cells. We found inhibition of cell growth and apoptotic nuclei, DNA fragmentation and increased apoptotic cells after treatment with genistein, indicating apoptotic cell deaths. From these results we observed that genistein inhibits the growth of HepG2 cells and induce apoptosis, however, further definitive studies are needed. These results may support the potentially effective chemopreventive and/or chemotherapeutic of genistein against liver cancer.     

The role of human cytochrome P4503A4 in biotransformation of tissue-specific derivatives of 7H-dibenzo[c,g]carbazole

The environmental pollutant 7H-dibenzo[c,g]carbazole (DBC) and its derivative, 5,9-dimethylDBC (DiMeDBC), produced significant and dose-dependent levels of micronuclei followed by a substantial increase in the frequency of apoptotic cells in the V79MZh3A4 cell line stably expressing the human cytochrome P450 (hCYP) 3A4. In contrast, neither micronuclei nor apoptosis were found in cells exposed to the sarcomagenic carcinogen, N-methylDBC (N-MeDBC). A slight but significant level of gene mutations and DNA adducts detected in V79MZh3A4 cells treated with N-MeDBC, only at the highest concentration (30 μM), revealed that this sarcomagenic carcinogen was also metabolized by hCYP3A4. Surprisingly, DBC increased the frequency of 6-thioguanine resistant (6-TG^r) mutations only at the highest concentration (30 μM), while DiMeDBC failed to increase the frequency of these mutations. The resistance to 6-thioguanine is caused by the mutations in the hypoxanthine-guanine phosphoribosyltransferase (Hprt) gene. The molecular analysis of the coding region of Hprt gene showed a deletion of the entire exon 8 in DiMeDBC-induced 6-TG^r mutants, while no changes in the nucleotide sequences were identified in 6-TG^r mutants produced by DBC and N-MeDBC. Based on our results, we suggest that hCYP3A4 is involved in the metabolism of DBC and its tissue-specific derivatives. While hCYP3A4 probably plays an important role in biotransformation of the liver carcinogens, DBC and DiMeDBC, it might only have a marginal function in N-MeDBC metabolism.