Anticancer activity evaluation of the Solanum glycoalkaloid solamargine: Triggering apoptosis in human hepatoma cells

Solamargine, an herbal and molluscicidal medicine derived from Solanum incanum, is a steroidal alkaloid glycoside. To characterize the anticancer mechanism of solamargine on human hepatoma cells (Hep3B), changes of cell morphology, DNA content, and gene expression of cells after solamargine treatment were studied. The appearance in solamargine-treated cells of chromatin condensation, DNA fragmentation, and a sub-G₁ peak in a DNA histogram suggests that solamargine induces cell death by apoptosis. The maximum number of dead Hep3B cells was detected within 2 hr of incubation with constant concentrations of solamargine, and no further cell death was observed after an extended incubation with solamargine, indicating that the action of solamargine was irreversible. To determine the susceptibility of cell phases to solamargine-mediated apoptosis, Hep3B cells were synchronized at defined cell cycles by cyclosporin A, colchicine, and genistein, followed by solamargine treatment. The ₅₀ values of solamargine for control, G₀/G₁-, M-, and G₂/M-synchronized Hep3B cells were 5.0, > 10, 3.7, and 3.1 μg/mL, implying that cells in the G₂/M phases are relatively susceptible to solamargine-mediated apoptosis. In addition, a parallel up-regulation of tumor necrosis factor receptor (TNFR)-I and -II on Hep3B cells was detected after solamargine treatment, and the solamargine-mediated cytotoxicity could be neutralized with either TNFR-I or -II specific antibody. Therefore, these results reveal that the actions of TNFR-I and -II on Hep3B cells may be independent, and both are involved in the mechanism of solamargine-mediated apoptosis.     

Cytotoxic effects of cantharidin on the growth of normal and carcinoma cells

Cantharidin is isolated from Mylabris phalerata Pallas and is a potent inhibitor of hepatocellular carcinoma cells (Hep 3B cells). In the present study, the IC(50) values of cantharidin on Hep 3B cells and normal Chang liver cells were found to be 2.2 and 30.2 microM for 36 h, respectively. Furthermore, cantharidin-treated Hep 3B cells induced cell death within 1 h (IC(50)=52.8 microM), suggesting that cantharidin is an acute cytotoxic agent. We found that although cantharidin could induce cell death, it could not directly inhibit the activity of nucleic acid biosynthesis by the cellular incorporation of 3H-thymidine, 3H-uridine or 3H-leucine. Cantharidin-treated Hep 3B cells showed no evidence of major alterations in the cell cycle distribution within 1 h. However, examination of cells after treatment for 36 h showed that cantharidin regulated the cell cycle at the G(2)/M phase. Moreover, the treated Hep 3B cells had a rounded and shrunken appearance. The microvilli of treated Hep 3B cells were reduced in number and replaced by numerous blebs. Other ultrastructural changes following cantharidin treatment included the presence of lipid droplets, swelling of the mitochondria and accumulation of glycogen particles. The findings of damaged mitochondria in the cantharidin treated Hep 3B cells in this study suggest that cantharidin can induce acute and lethal toxic effects on Hep 3B cells by inhibiting the mitochondria energy system. In conclusion, this study had demonstrated that cantharidin could inhibit progression of all phases of the Hep 3B cell cycle.     

Differential regulation of P53, c-Myc, Bcl-2, Bax and AFP protein expression, and caspase activity during 10-hydroxycamptothecin-induced apoptosis in Hep G2 cells

10-Hydroxycamptothecin (HCPT), a DNA topoisomerase I (Topo I) inhibitor, exhibited a remarkable apoptosis-inducing effect on human hepatoma Hep G2 cells. We studied the effect of HCPT upon the expression of P53, c-Myc, Bcl-2, Bax and alpha-fetoprotein (AFP) proteins, and caspase (caspase-1 and caspase-3) activity of Hep G2 cells. It showed that HCPT at a dose of 0.1 microg/ml increased the expression of P53, c-Myc and Bax protein, and decreased the expression of Bcl-2 and AFP. The increase of P53, which was remarkable after only 3 h incubation with HCPT, occurred much earlier than the changes of other proteins, suggesting that the increase of P53 expression may be the upstream event in the apoptosis of Hep G2 cells induced by HCPT. Both caspase-1 and caspase-3 were activated in Hep G2 cells by HCPT treatment, suggesting that caspase-1 and caspase-3 are involved in the process of apoptosis in Hep G2 cells, and may be the main effectors of the apoptosis.     

Critical role of reactive oxygen species and mitochondrial membrane potential in Korean mistletoe lectin-induced apoptosis in human hepatocarcinoma cells

Viscum album L. coloratum agglutinin (VCA), isolated from Korean mistletoe, is a strong inducer of apoptosis in a variety of tumor cells; however, the underlying molecular mechanisms responsible are not clear. Here, we show that VCA induces apoptotic killing, as demonstrated by DNA fragmentation, Hoechst 33258 staining, terminal deoxynucleotidyl transferase dUTP nick-end labeling assay, and flow cytometry analysis in hepatocarcinoma Hep3B cells. VCA treatment results in a significant increase in reactive oxygen species (ROS) and loss of mitochondrial membrane potential (DeltaPsim). Furthermore, treatment with the antioxidant N-acetyl-L-cysteine reduces ROS induction by VCA, preventing apoptosis in Hep3B cells, indicating that oxidative stress is involved in VCA-mediated cell death. Our results also show rapid changes in mitochondrial transition permeability, Bax translocation, cytochrome c release, caspase-3 activity, and poly(ADP-ribose) polymerase degradation in Hep3B cells occurring in VCA-induced apoptosis. There is much evidence that implicates c-Jun NH2-terminal kinase (JNK) activation with apoptosis in a variety of cellular and animal models. In this study, we show that VCA induces JNK phosphorylation, which is abolished with pretreatment with a JNK inhibitor. Moreover, Hep3B cells overexpressing JNK1 or stress-activated protein kinase kinase (SEK1) seem to be more susceptible to cell death from ROS and loss of DeltaPsim induced by VCA, whereas expression of dominant-negative JNK1 or SEK1 in Hep3B cells do not. These data suggest that JNK phosphorylation may be a major regulator involved in VCA-induced apoptosis. Together, these results suggest that VCA induces apoptosis by inducing ROS production and a loss of DeltaPsim, in which JNK phosphorylation plays a critical role in these events.     

Induction of apoptosis in the blue mussel Mytilus galloprovincialis by tri-n-butyltin chloride

Induction of apoptosis by tri-n-butyltin (TBT) in gill tissue of the mussel Mytilus galloprovincialis was investigated. The terminal dUTP nick-end labeling technique (TUNEL) was used to detect cells displaying DNA fragmentation within gill structures. Genomic DNA fragmentation was detected as characteristically ladder-like pattern of DNA fragments induced by single injection of different doses of TBT (1-5 microg/g) below the mantle, directly into the pallial fluid, after 24 h of incubation. DNA degradation of higher order DNA structure, as well as reduced G(0)/G(1) cell cycle region (the sub-G(1) region) was detectable after 1.5 h of TBT incubation. Presence of apoptotic cells in mussels' gills was indicated by the selective loss of G(2)/M cells concomitant with the appearance of cells with decreased DNA content in S and G(0)/G(1) cell cycle regions. The effect of the TBT on cell cycle in a mussel gill was a dose related and exposure time depending. The possible mechanism of induction of apoptosis in vivo in gill tissue of mussel treated with TBT is discussed.     

A methyl jasmonate derivative,J-7, induces apoptosis in human hepatocarcinoma Hep3B cells in vitro

The pro-apoptotic activity of J-7, a synthetic methyl jasmonate derivative, on the Hep3B human hepatocarcinoma cell line was investigated. Treatment of Hep3B cells with J-7 resulted in growth inhibition and the induction of apoptosis as measured by trypan blue-excluding cells, MTT assay, nuclear staining, DNA fragmentation, and flow cytometry analysis. The increased apoptotic events in Hep3B cells caused by J-7 were associated with the alteration in the ratio of Bax/Bcl-2 protein expression. J-7 treatment induced the expression of death receptor-related proteins such as death receptor 5, which triggered the activation of caspase-8 and the down-regulation of the whole Bid expression. In addition, the apoptosis induction by J-7 was correlated with the activation of caspase-9 and caspase-3, down-regulation IAP family proteins such as XIAP and cIAP-1, and concomitant degradation of poly (ADP-ribose) polymerase. However, the cytotoxic and apoptotic effects induced by J-7 were significantly inhibited by z-DEVD-fmk, a caspase-3 inhibitor, which demonstrates the important role that caspase-3 plays in the process. Furthermore, blocking the extracellular signal-regulated protein kinase and c-Jun N-terminal kinase pathways showed increased apoptosis and the activation of caspases in J-7-induced apoptosis. The results indicated that J-7 induces the apoptosis of Hep3B cells through a signaling cascade of death-receptor-mediated extrinsic as well as mitochondria-mediated intrinsic caspase pathways, which are associated with the activation of the mitogen-activated protein kinases signal pathway.     

Apoptosis induction and cell cycle perturbation in human hepatoma hep G2 cells by 10-hydroxycamptothecin

10-Hydroxycamptothecin (HCPT), a DNA topoisomerase I inhibitor, is an antitumor alkaloid isolated from a Chinese tree, Camptotheca acuminata, and exhibits a remarkable antihepatoma effect. We studied HCPT to determine whether or not its anti-hepatoma activity occurs through apoptosis induction and cell cycle disturbance using the MTT method, DAPI staining, agarose gel electrophoresis and flow cytometric analysis. The results showed that HCPT inhibited proliferation of human hepatoma Hep G2, Bel-7402 and Bel-7404 cells at an optimal concentration of 0.1 microg/ml. This growth inhibition was dose and time dependent, and was accompanied by evidence of apoptotic changes and cell cycle perturbation in Hep G2 cells. Chromatin condensation and nuclear fragmentation were observed in Hep G2 cells by fluorescence microscopy. Agarose gel electrophoresis showed internucleosomal DNA fragmentation ('ladder pattern') of Hep G2 cells following treatment with HCPT, in a concentration- and time-dependent manner. Flow cytometry showed that HCPT induced a massive hypodiploid cell population and arrested cells in G2/M phase (at low dose) or in S phase (at high dose) in Hep G2 cells. The results of this study suggest that the anti-hepatoma effect of HCPT may result from apoptosis induction and cell cycle disturbance.     

p21基因对肝癌细胞的生物学影响

目的探讨逆转录病毒介导的p21基因对肝癌Hep3B细胞的杀伤作用。方法通过脂质体将有p21基因的逆转录病毒载体MMLV-p21转染肝癌Hep3B细胞系,并用G418筛选。试验分为转p21基因的Hep3B-P21组及相同遗传背景及培养过程的两对照组肝癌Hep3B系细胞组、转Hep3B-MMLV组3组。采用RT-PCR检测实验组p21基因的表达;应用流式细胞计数分别分析3组细胞周期;MTT法检测细胞增殖并比较细胞抑制率。结果野生p21基因阻止Hep3B系细胞进入S期,停滞在G1期,Hep3B-P21组细胞增殖比两对照组明显受到抑制（P〈0.05）。结论逆转录病毒介导p21基因可有效抑制肝癌Hep3B系细胞的增殖并诱导肝癌细胞的凋亡。     

Inhibition of sphingosine kinase 1 enhances cytotoxicity, ceramide levels and ROS formation in liver cancer cells treated with selenite

High doses of selenite have been shown to induce cell death in acute myeloid leukemia and lung cancer cells. In this study, we combined selenite treatment with modulators of sphingolipid metabolism in the hepatocellular carcinoma cell line Huh7. Treatment with 20 μM of selenite reduced the viability of Huh7 cells by half and increased the levels of long chain C14-, C16-, C18- and C18:1- ceramides by two-fold. Inhibition of neutral sphingomyelinase with 3-O-methylsphingosine significantly reduced the cytotoxic effect of selenite. In line with this result, selenite caused a 2.5-fold increase in the activity of neutral sphingomyelinase. The sphingosine kinase 1 (SK1) inhibitor 2-(p-hydroxyanilino)-4-(p-chlorophenyl)thiazole (SK1-II) sensitized the cells to the cytotoxic effects of selenite. Preincubation with 10 μM of SK1-II prior to treatment with 10 μM of selenite caused induction of apoptosis and gave rise to a 2.5-fold increase in C14-, C16-, C18- and C18:1- ceramides. Instead, 50 μM of SK1-II combined with 10 μM of selenite caused accumulation of cells in G1/S phases, but less apoptosis and accumulation of ceramides. The formation of reactive oxygen species (ROS) after treatment with 10 μM of selenite was maximally enhanced by 1 μM of SK1-II. Moreover, combined treatment with SK1-II and 10 μM of selenite synergistically reduced the number of viable Huh7 cells, while the non-tumorigenic hepatocyte cell line MIHA remained unaffected by the same treatment. These results raise the possibility that a combination of selenite and SK1 inhibitors could be used to treat liver cancer cells, that are regarded as drug resistant, at doses that are non-toxic to normal liver cells.     

Chrysophanol-induced necrotic-like cell death through an impaired mitochondrial ATP synthesis in Hep3B human liver cancer cells

Liver cancer is the most common form of cancer in Taiwan and it usually responds to chemotherapy. However, patients often have side effects to the chemotherapeutic drugs. Thus new agents are urgently required to treat liver cancer. Chrysophanol, one of the anthraquinone derivatives, was reported to inhibit some human cancer cell growth which may be due to the induction of apoptosis similar to other anthraquinone derivatives though such actions have not been reported. In the present study, we reported that chrysophanol inhibits cell growth in Hep3B liver cancer cells based on the following observations: 1) induc cell morphological changes; 2) decreased percentage of viable cells; 3) induced S phase arrest of cell cycle progression; 4) induced DNA damage as measured by comet assay and DAPI staining. Chrysophanol-induced cell death however, seems to be related to necrotic processes rather than typical apoptosis. Chrysophanol induced reactive oxygen species and Ca(2+) production and decreased mitochondrial membrane potential (ΔΨm) and ATP levels in Hep3B cells. No effects were observed on known protein regulators of apoptosis such as Bax and Bcl-2. Chrysophanol-induced cell death took place independently of caspase-8 and -9. Based on our findings, we propose that chrysophanol reduces cellular ATP levels causing a drop in energy resulting in necrotic-like cell death.     

肉灵芝提取物调控长链非编码RNA BRCA1相邻基因2/双特异性磷酸酶4/细胞外信号调节激酶通路对肝癌细胞Hep3B增殖及凋亡的影响

目的 探讨肉灵芝提取物是否可通过长链非编码RNA BRCA1相邻基因2(LncRNA NBR2)/双特异性磷酸酶4/细胞外信号调节激酶(DUSP4/ERK)通路而影响肝癌HepB细胞增殖及凋亡。方法 体外培养肝癌HepB细胞,分为1 mg/L肉灵芝提取物组、2 mg/L肉灵芝提取物组、4 mg/L肉灵芝提取物组、si-NC组、si-LncRNA NBR2组、pcDNA-NC组、pcDNA-LncRNA NBR2组、肉灵芝提取物+si-NC组、肉灵芝提取物+si-LncRNA NBR2组;采用MTT检测肝癌HepB细胞增殖;流式细胞术检测肝癌HepB细胞凋亡;RT-qPCR检测LncRNA NBR2的表达量;蛋白质印迹法(Western blotting)检测增殖细胞核抗原(PCNA)、活化的含半胱氨酸的天冬氨酸蛋白水解酶3(Cleaved-caspase-3)、DUSP4、p-ERK蛋白表达量。结果 肉灵芝提取物可明显降低肝癌HepB细胞增殖率与PCNA、DUSP4、p-ERK蛋白水平(P<0.05),提高肝癌HepB细胞凋亡率、Cleaved-caspase-3蛋白水平及Lnc...     

Effects of rosmarinic acid against aflatoxin B1 and ochratoxin-A-induced cell damage in a human hepatoma cell line (Hep G2)

Recent findings have suggested that oxidative damage might contribute to the cytotoxicity and carcinogenicity of aflatoxin B(1) (AFB(1)). The induction of oxidative stress also plays an important role in the toxicity of another mycotoxin: ochratoxin A (OTA). In this study, the protective effect of rosmarinic acid (Ros A) against AFB(1) and OTA-induced cytotoxicity was investigated in a human hepatoma-derived cell line (Hep G2). Rosmarinic acid, a natural phenolic compound contained in many Lamiaceae herbs such as Perilla frutescens, sage, basil and mint, inhibits complement-dependent inflammatory processes and may have therapeutic potential.The ability of Ros A to reduce radical oxygen species (ROS) production, protein and DNA synthesis inhibition and apoptosis caused by the two mycotoxins was also investigated. Our experiments proved the significant cytoprotective effect of Ros A in vitro from OTA- and AFB(1)-induced cell damage. In particular, 24-h pretreatment with 50 micro M Ros A inhibited the cytotoxicity of 10 micro M AFB(1) (by 45%) and 10 micro M OTA (by 35%) in Hep G2 cells (P < 0.001). Moreover, Ros A dose dependently attenuated ROS production and DNA and protein synthesis inhibition induced by both of the toxins. Similarly, apoptosis cell death was prevented, as demonstrated by reduction of DNA fragmentation and inhibition of caspase-3 activation (P < 0.001).     

紫杉醇诱导食管癌细胞的细胞周期阻断与细胞凋亡

目的研究紫杉醇对食管癌细胞的细胞周期阻断及诱导凋亡的作用。方法应用形态学观察、琼脂糖凝胶电泳、流式细胞术等方法对紫杉醇诱导的食管癌细胞系Eca109进行了检测和观察。结果紫杉醇可将食管癌细胞阻断于G0／G1期及G2／M期并诱导其凋亡，凋亡细胞具有典型的凋亡形态特征，流式细胞仪检测有凋亡峰出现，琼脂糖凝胶电泳显示3nmol·L-1紫杉醇作用72h便可见明显的DNA梯带。100nmol·L-1和1000nmol·L-1浓度的紫杉醇作用于细胞，在形态变化与细胞周期变化上有很大差别。紫杉醇作用短时间（＜2h）去除后再培养比持续性作用更早促使细胞凋亡。结论紫杉醇不仅可以阻断食管癌细胞的分裂，而且对于间期细胞也有很大作用。细胞周期阻断并不直接诱导细胞凋亡，甚至可能抑制细胞凋亡的进程。食管癌细胞中存在多条凋亡途径。     

Different effects of baicalein, baicalin and wogonin on mitochondrial function, glutathione content and cell cycle progression in human hepatoma cell lines

The effects of the flavonoids from Scutellaria baicalensis Georgi (baicalein, baicalin and wogonin) in cultured human hepatoma cells (Hep G2, Hep 3B and SK-Hep1) were compared by MTT assay and flow cytometry. All three flavonoids dose-dependently decreased the cell viabilities accompanying the collapse of mitochondrial membrane potential and the depletion of glutathione content. However, the influence of baicalein, baicalin or wogonin on cell cycle progression was different. All three flavonoids resulted in prominent increase of G2/M population in Hep G2 cells, whereas an accumulation of sub G1 (hypoploid) peak in Hep 3B cells was observed. In SK-Hep1 cells, baicalein and baicalin resulted in dramatic boost in hypoploid peak, but wogonin made mainly in G1 phase accumulation. These data, together with the previous findings in other hepatoma cell lines, suggest that baicalein, baicalin and wogonin might be effective candidates for inducing apoptosis or inhibiting proliferation in various human hepatoma cell lines.     

Cell cycle arrest and autoschizis in a human bladder carcinoma cell line following Vitamin C and Vitamin K3 treatment

Exponentially growing cultures of human bladder tumor cells (T24) were treated with Vitamin C (VC) alone, Vitamin K(3) (VK(3)) alone, or with a VC:VK(3) combination for 1, 2, or 4hr. Flow cytometry of T24 cells exposed to the vitamins for 1h revealed a growth arrested population and a population undergoing cell death. Cells in G(1) during vitamin treatment arrested in G(1) while those in S phase progressed through S phase and arrested in G(2)/M. DNA synthesis decreased to 14 to 21% of control levels which agreed with the percent of cells in S phase during treatment. Annexin V labeling demonstrated the majority of the cells died by autoschizis, but necrosis and apoptosis also were observed. Catalase treatment abrogated both cell cycle arrest and cell death which implicated hydrogen peroxide (H(2)O(2)) in these processes. Redox cycling of VC and VK(3) increased H(2)O(2) production and decreased cellular thiol levels and DNA content, while increasing intracellular Ca(2+) levels and lipid peroxidation. Feulgen staining of treated cells revealed a time-dependent decrease in tumor cell DNA, while electrophoresis revealed a spread pattern. These results suggest that Ca(2+) disregulation activates at least one DNase which degrades tumor cell DNA and induces tumor cell death.     

Apoptotic effects of Antrodia cinnamomea fruiting bodies extract are mediated through calcium and calpain-dependent pathways in Hep 3B cells.

Antrodia cinnamomea is well known in Taiwan as a traditional medicine for treating cancer and inflammation. The purpose of this study was to evaluate the apoptotic effects of ethylacetate extract from A. cinnamomea (EAC) fruiting bodies in Hep 3B, a liver cancer cell line. EAC decreased cell proliferation of Hep 3B cells by inducing apoptotic cell death. EAC treatment increased the level of calcium (Ca2+) in the cytoplasm and triggered the subsequent activation of calpain and caspase-12. EAC also initiated the mitochondrial apoptotic pathway through regulation of Bcl-2 family proteins expression, release of cytochrome c, and activation of caspase-9 in Hep 3B cells. Furthermore, the mitochondrial apoptotic pathway amplified the calpain pathway by Bid and Bax interaction and Ca2+ translocation. We have therefore concluded that the molecular mechanisms during EAC-mediated proliferation inhibition in Hep 3B cells were due to: (1) apoptosis induction, (2) triggering of Ca2+/calpain pathway, (3) disruption of mitochondrial function, and (4) apoptotic signaling being amplified by cross-talk between the calpain/Bid/Bax and Ca2+/mitochondrial apoptotic pathways.     

Effective metabolism and long intracellular half life of the anti-hepatitis B agent adefovir in hepatic cells

Adefovir dipivoxil (ADV) is esterolytically cleaved to the 2'-deoxyadenosine monophosphate (dAMP) analog adefovir, subsequent phosphorylation leads to the formation of the anti-Hepatitis B virus (HBV) agent adefovir-DP. To better understand the mechanism of action of ADV, metabolism studies were done in Hep G2, Huh-7 and primary human hepatocytes. Separation of radiolabeled adefovir metabolites after incubation in Hep G2 cells suggested that adefovir in its mono- and di-phosphorylated forms are the only metabolites formed from adefovir. Incubation of 10 microM adefovir with hepatic cell lines and fresh monolayers of primary human hepatocytes from two donors and analysis of intracellular metabolites by liquid chromatography coupled to tandem mass spectrometry resulted in adefovir-DP levels of approximately 10 pmol/million cells. Adefovir was more efficiently phosphorylated in primary hepatocytes than cell lines with adefovir-DP accounting for 44% versus 26% of total intracellular adefovir after 24 h. Egress studies showed adefovir-DP to have a half-life of 33 +/- 3 h, 10 +/- 1 h, 48 +/- 3 h and 33 +/- 2 h in Hep G2, Huh-7, and primary hepatocytes from two separate donors, respectively. The markedly shorter half-life in Huh-7 cells was inferred to be transport dependent based on its sensitivity to the transport inhibitor MK-571. Effective phosphorylation coupled with a long intracellular half-life and small competing dATP pool sizes in primary hepatocytes forms the cellular metabolic basis for the efficacy of adefovir dipivoxil in the treatment of chronic hepatitis B.     

Methylglyoxal-bis(guanylhydrazone), a polyamine analogue, sensitized γ-radiation-induced cell death in HL-60 leukemia cells Sensitizing effect of MGBG on γ-radiation-induced cell death

Methylglyoxal-bis(guanylhydrazone) (MGBG), a polyamine analogue, has been known to inhibit the biosynthesis of polyamines, which are important in cell proliferation. We showed that MGBG treatment significantly affected γ-radiation-induced cell cycle transition (G(1)/G(0)→S→G(2)/M) and thus γ-radiation-induced cell death. As determined by micronuclei and comet assay, we showed that it sensitized the cytotoxic effect induced by γ-radiation. One of the reasons is that polyamine depletion by MGBG treatment did not effectively protect against the chemical (OH) or physical damage to DNA caused by γ-radiation. Through in vitro experiment, we confirmed that DNA strand breaks induced by γ-radiation was prevented more effectively in the presence of polyamines (spermine and spermidine) than in the absence of polyamines. MGBG also blocks the cell cycle transition caused by γ-radiation (G(2) arrest), which helps protect cells by allowing time for DNA repair before entry into mitosis or apoptosis, via the down regulation of cyclin D1, which mediates the transition from G(1) to S phase of cell cycle, and ataxia telangiectasia mutated, which is involved in the DNA sensing, repair and cell cycle check point. Therefore, the abrogation of G(2) arrest sensitizes cells to the effect of γ-radiation. As a result, γ-radiation-induced cell death increased by about 2.5-3.0-fold in cells treated with MGBG. However, exogenous spermidine supplement partially relieved this γ-radiation-induced cytotoxicity and cell death. These findings suggest a potentially therapeutic strategy for increasing the cytotoxic efficacy of γ-radiation.     

Influence of a novel platinum compound--cis-dichloro (dimethylsulphoxide) (1-beta-D-rybofuranosyl-1,2,4-triazolo-3-arboxyamide) platinum (II)--"Pt-rib-1"--on cell cycle and apoptosis in ClS91 and B16 mouse melanoma in vitro

Cisplatin has a significant role in the treatment of selected human tumors including advanced melanoma, but new platinum compounds are still in focus of search for better properties. Modern drug design is often based on studies detecting the abilities of tested drug to induce apoptosis and disturb cell cycle. Aim of the study was to establish the influence of a platinum complex Pt-rib-1 on cell cycle and apoptosis occurrence in mouse melanoma B16 and ClS91 cells. Pt-rib-1 is a ribavirin derivative. previously characterized and described as cytotoxic to B16 and ClS91 mouse melanoma cells in vitro. The new platinum complex (Pt-rib-1); cis- dichloro (dimethylsulphoxide) (1- beta- D-ribofuranosyl- 1,2,4-triazolo -3- carboxyamide) platinum (II) was supplied. Cisdiaminodichloroplatinum (II), (cisplatin) was used in control groups. To detect apoptotic and necrotic cells, Annexin V- conjugated with fluorescein isothiocyanate (Annexin V-FITC, Immunotech) and propidium iodide (IP, Immunotech) were used. Apoptosis detection were done using fluorescence microscopy and flow cytometry. The total DNA content within the cell indicated phase of the cell cycle. DNA content was measured using flow cytometry. Values given represent the mean from three determinations. Results were presented as mean +/- standard deviation (SD). Statistical analysis was done using t-Student test. There were 70.4% of apoptotic cells in the ClS91 culture after 24 h incubation with Pt-rib-1 at a concentration of 2.04 x 10(-3) M (4 x IC50). In B16 group, 83.2 per cent of apoptotic cells was found after 24h incubation with Pt-rib-1 at high concentration (2.30 x 10(-3) M). A 24-h experiment shows a threshold at a concentration higher than 3 x IC50 responsible for apoptosis induction in B16 and ClS91 cells. After 48 h incubation with Pt-rib-1 the per cent of apoptotic cells increased gradually with rising concentrations of Pt-rib-1 up to a final concentration of 2.04 x 10(-3) M and 0.92 x 10(-3) M in ClS91 and B16 groups, respectively. Cell accumulation was observed in S phase after 48 h incubation with Pt-rib-1. The per cent of cells in S phase increased from 31 to 51.1% and 38.8 to 50.0% in ClS91 and B16 culture, respectively. There were no B16 and ClS91 cells in G2/M phase after incubation with higher concentrations of Pt-rib-1 (from 0.2 to 2.0 x 10(-5) M/dm3). Pt-rib-1 partially exhibits action of cisplatin. which has no specific influence on cell cycle and ribavirin. probably responsible for DNA synthesis delay.