The effect of new anthracycline derivatives on the induction of apoptotic processes in human neoplastic cells

This study was an attempt to determine the effect of a selected anthracycline derivative, WP903, on apoptotic processes in human melanoma cells depending on intracellular concentrations of the compound, and to evaluate the significance of apoptosis induction for the cytotoxic effect of anthracycline antibiotics. It was found that the WP903, contrary to ADR (adriamycin) is a strong inducer of apoptotic processes in ME18 human melanoma cells regardless of their susceptibility to adriamycin and WP903. The cells were treated for 24 h with ADR (1 and 5 microg/ml) or WP903 (0.2 and 2 microg/ml). Apoptosis was detected with the use of annexin V-FITC and PI (propidium iodide) and with TUNEL assay. WP903 used at 0.2 microg/ml induced early apoptosis in 23% of ME18 cells and in 60% of ME18/R cells; at 2 microg/ml in 70% of each of cell line tested. Significant late apoptotic effect was observed in ME18 cells. In contrast, ADR was found to be a weak inducer of apoptotic events. The results suggest that apoptosis is not a mechanism directly related to the cytotoxic effect of anthracycline antibiotics.     

Redox active copper chelate overcomes multidrug resistance in T-lymphoblastic leukemia cell by triggering apoptosis

Multidrug resistance (MDR) mediated by the over expression of drug efflux protein P-glycoprotein (P-gp) is one of the major impediments to successful treatment of cancer. P-gp acts as an energy-dependent drug efflux pump and reduces the intracellular concentration of structurally unrelated drugs inside the cells. Therefore, there is an urgent need for development of new molecules that are less toxic to normal cell and preferentially effective against drug resistant malignant cells. In this preclinical study we report the apoptotic potential of copper N-(2-hydroxyacetophenone) glycinate (CuNG) on doxorubicin resistant T lymphoblastic leukaemia cells (CEM/ADR5000). To evaluate the cytotoxic effect of CuNG, we used different normal cell lines (NIH 3T3, Chang liver and human PBMC) and cancerous cell lines (CEM/ADR5000, parental sensitive CCRF-CEM, SiHa and 3LL) and conclude that CuNG preferentially kills cancerous cells, especially both leukemic cell types irrespective of their MDR status, while leaving normal cell totally unaffected. Moreover, CuNG involves reactive oxygen species (ROS) for induction of apoptosis in CEM/ADR5000 cells through the intrinsic apoptotic pathway. This is substantiated by our observation that antioxidant N-acetyle-cysteine (NAC) and PEG catalase could completely block ROS generation and, subsequently, abrogates CuNG induced apoptosis. On the other hand, uncomplexed ligand N-(2-hydroxyacetophenone) glycinate (NG) fails to generate a significant amount of ROS and concomitant induction of apoptosis in CEM/ADR5000 cells. Therefore, CuNG induces drug resistant leukemia cells to undergo apoptosis and proves to be a molecule having therapeutic potential to overcome MDR in cancer.     

IFN-beta pretreatment sensitizes human melanoma cells to TRAIL/Apo2 ligand-induced apoptosis

All human melanoma cell lines (assessed by annexin V and TUNEL assays) were resistant to apoptosis induction by TRAIL/Apo2L protein. TRAIL/Apo2L activated caspase-8 and caspase-3, but subsequent apoptotic events such as poly(ADP-ribose) polymerase cleavage and DNA fragmentation were not observed. To probe the molecular mechanisms of cellular resistance to apoptosis, melanoma cell lines were analyzed for expression of apoptosis regulators (apoptotic protease-associated factor-1, FLIP, caspase-8, caspase-9, caspase-3, cellular inhibitor of apoptosis, Bcl-2, or Bax); no correlation was observed. TRAIL/Apo2L was induced in melanoma cell lines by IFN-beta and had been correlated with apoptosis induction. Because IFN-beta induced other gene products that have been associated with apoptosis, it was postulated that one or more IFN-stimulated genes might sensitize cells to TRAIL/Apo2L. Melanoma cell lines were treated with IFN-beta for 16-24 h before treatment with TRAIL/Apo2L. Regardless of their sensitivity to either cytokine alone, >30% of cells underwent apoptosis in response to the combined treatment. Induction of apoptosis by IFN-beta and TRAIL/Apo2L in combination correlated with synergistic activation of caspase-9, a decrease in mitochondrial potential, and cleavage of poly(ADP-ribose) polymerase. Cleavage of X-linked inhibitor of apoptosis following IFN-beta and TRAIL/Apo2L treatment was observed in sensitive WM9, A375, or WM3211 cells but not in resistant WM35 or WM164 cells. Thus, in vitro IFN-beta and TRAIL/Apo2L combination treatment had more potent apoptotic and anti-growth effects when compared with either cytokine alone in melanoma cells lines.     

The nucleoside analog sangivamycin induces apoptotic cell death in breast carcinoma MCF7/adriamycin-resistant cells via protein kinase Cdelta and JNK activation

Sangivamycin has shown a potent antiproliferative activity against a variety of human cancers. However, little is known about the mechanism of action underlying its antitumor activity. Here we demonstrate that sangivamycin has differential antitumor effects in drug-sensitive MCF7/wild type (WT) cells, causing growth arrest, and in multidrug-resistant MCF7/adriamycin-resistant (ADR) human breast carcinoma cells, causing massive apoptotic cell death. Comparisons between the effects of sangivamycin on these two cell lines allowed us to identify the mechanism underlying the apoptotic antitumor effect. Fluorescence-activated cell sorter analysis indicated that sangivamycin induced cell cycle arrest in the G(2)/M phase in MCF7/ADR cells. A marked induction of c-Jun expression as well as phosphorylation of c-Jun and JNK was observed after sangivamycin treatment of MCF7/ADR cells but not MCF7/WT cells. Sangivamycin also induced cleavage of lamin A and poly(ADP-ribose) polymerase (PARP) in MCF7/ADR cells, probably via activation of caspase-6, -7, and -9. Pretreatment with a caspase-9-specific inhibitor or pan-caspase inhibitor abolished sangivamycin-induced cleavage of lamin A and PARP but not sangivamycin induction of c-Jun expression and phosphorylation. Pretreatment of MCF7/ADR cells with SP600125, a specific inhibitor of JNK, or with rottlerin, a specific inhibitor of protein kinase Cdelta (PKCdelta), significantly reduced the sangivamycin-induced apoptosis and almost completely abolished sangivamycin-induced phosphorylation of c-Jun and cleavage of lamin A and PARP. Transfection of MCF7/ADR cells with PKCdelta small interfering RNAs or PKCdelta antibody or rottlerin pretreatment significantly suppressed the phosphorylation of JNK. Taken together, our data suggest that sangivamycin induces mitochondria-mediated apoptotic cell death of MCF7/ADR cells via activation of JNK in a protein kinase Cdelta-dependent manner.     

Adaptive response towards adriamycin in vitro: circumvention with verapamil

In an attempt to identify mechanisms of adaptive response to adriamycin (ADR), we have earlier isolated ADR-resistant cell lines CHO/R and ME18/R by short-term pulse exposures of parent cell lines to this drug, followed by single-cell cloning. The results presented in this study have shown that the development of resistance to ADR was accompanied by cross-resistance to vinblastine and methotrexate. The resistance of tested cell lines towards ADR was substantially reversed by verapamil (VPL) at non-toxic concentrations. VPL abolished also the capability of these cell lines to express adaptive response after treatment of the cells with a conditioning dose of ADR. From the results of our study, we conclude that similar characteristics play a role in the mechanism of the phenomenon of adaptive response as in the mechanism of pleiotropic multidrug resistance.     

p38 Mitogen-activated protein kinases is required for counteraction of 2-methoxyestradiol to estradiol-stimulated cell proliferation and induction of apoptosis in ovarian carcinoma cells via phosphorylation Bcl-2

INTRODUCTION: 2-Methoxyestradiol (2ME2), a natural endogenous product of estradiol (E2) metabolism, has been shown to be a selective apoptotic agent for cancer cells but not for normal cells. In this study, we determined that 2ME2 counteracts E2-stimulated cell growth and induces apoptosis in ovarian carcinoma cells. In addition, we demonstrate that 2ME2 induces apoptosis via p38 and phospho-Bcl2 pathway. METHODS: 2ME2 and/or E2 were administered to the OVCAR-3 (human ovarian cancer) cell line. Cell growth inhibition was analyzed by [3H] Thymidine incorporation assay and DNA fluorometric assay. Cell apoptosis was tested by DNA fragmentation analysis and FACS. The signaling pathway was determined by a series of biochemical assays. RESULTS: 2ME2 inhibited estradiol-stimulated cell growth and induced apoptosis in an ovarian carcinoma cell line. MAPK and p38, but not JNK, were found to be critical mediators in this process. Expression of a dominant negative mutant of p38 kinase or p38 specific inhibitor, SB 203580, almost completely blocked the process. Furthermore, Bcl-2 phosphorylation was required for 2ME2-induced effects. CONCLUSION: Our data suggest that 2ME2 inhibits E2-stimulated proliferation and induces apoptosis in ovarian carcinoma cells. Furthermore, activation of p38 and phosphorylation of Bcl-2 plays a critical role in the mechanism. 2ME2 therefore, may have a clinical application for the treatment of ovarian cancer.     

Resistance to CD95/Fas-induced and ceramide-mediated apoptosis of human melanoma cells is caused by a defective mitochondrial cytochrome c release

Intracellular CD95/Fas-signaling pathways have not been investigated in melanoma yet. Two different CD95 receptor-induced apoptotic pathways are presently known in other cell types: (i) direct activation of caspase-8 and (ii) induction of ceramide-mediated mitochondrial activation, both leading to subsequent caspase-3 activation. In the present study, five of 11 melanoma cell populations were shown to release cytochrome c from mitochondria, which activates caspase-3 and finally results in DNA fragmentation upon treatment with the agonistic monoclonal antibody CH-11. In contrast, this apoptotic pathway was not activated in the remaining six melanoma cell populations. Interestingly, the susceptibility of melanoma cells to CD95L/FasL-triggered cell death was clearly correlated with N-acetylsphingosine-mediated apoptosis. Our results are in line with a defect upstream of mitochondrial cytochrome c release in resistant cells.     

Killing of human melanoma cells induced by activation of class I interferon-regulated signaling pathways via MDA-7/IL-24

Restoration of the tumor-suppression function by gene transfer of the melanoma differentiation-associated gene 7 (MDA7)/interleukin 24 (IL-24) successfully induces apoptosis in melanoma tumors in vivo. To address the molecular mechanisms involved, we previously revealed that MDA7/IL-24 treatment of melanoma cells down-regulates interferon regulatory factor (IRF)-1 expression and concomitantly up-regulates IRF-2 expression, which competes with the activity of IRF-1 and reverses the induction of IRF-1-regulated inducible nitric oxide synthase (iNOS). Interferons (IFNs) influence melanoma cell survival by modulating apoptosis. A class I IFN (IFN-alpha) has been approved for the treatment of advanced melanoma with some limited success. A class II IFN (IFN-gamma), on the other hand, supports melanoma cell survival, possibly through constitutive activation of iNOS expression. We therefore conducted this study to explore the molecular pathways of MDA7/IL-24 regulation of apoptosis via the intracellular induction of IFNs in melanoma. We hypothesized that the restoration of the MDA7/IL-24 axis leads to upregulation of class I IFNs and induction of the apoptotic cascade. We found that MDA7/IL-24 induces the secretion of endogenous IFN-beta, another class I IFN, leading to the arrest of melanoma cell growth and apoptosis. We also identified a series of apoptotic markers that play a role in this pathway, including the regulation of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and Fas-FasL. In summary, we described a novel pathway of MDA7/IL-24 regulation of apoptosis in melanoma tumors via endogenous IFN-beta induction followed by IRF regulation and TRAIL/FasL system activation.     

Celastrol inhibits growth and induces apoptotic cell death in melanoma cells via the activation ROS-dependent mitochondrial pathway and the suppression of PI3K/AKT signaling

Celastrol has been reported to possess anticancer effects in various cancers; however, the precise mechanism underlying ROS-mediated mitochondria-dependent apoptotic cell death triggered by celastrol treatment in melanoma cells remains unknown. We showed that celastrol effectively induced apoptotic cell death and inhibited tumor growth using tissue culture and in vivo models of B16 melanoma. In addition to apoptotic cell death in B16 cells, several apoptotic events such as PARP cleavage and activation of caspase were confirmed. Pretreatment with caspase inhibitor modestly attenuated the celastrol-induced increase in PARP cleavage and sub-G1 cell population, implying that caspases play a partial role in celastrol-induced apoptosis. Moreover, ROS generation was detected following celastrol treatment. Blocking of ROS accumulation with ROS scavengers resulted in inhibition of celastrol-induced Bcl-2 family-mediated apoptosis, indicating that celastrol-induced apoptosis involves ROS generation as well as an increase in the Bax/Bcl-2 ratio leading to release of cytochrome c and AIF. Importantly, silencing of AIF by transfection of siAIF into cells remarkably attenuated celastrol-induced apoptotic cell death. Moreover, celastrol inhibited the activation of PI3K/AKT/mTOR signaling cascade in B16 cells. Our data reveal that celastrol inhibits growth and induces apoptosis in melanoma cells via the activation of ROS-mediated caspase-dependent and -independent pathways and the suppression of PI3K/AKT signaling.     

In vitro effects of 2-methoxyestradiol on cell morphology and Cdc2 kinase activity in SNO oesophageal carcinoma cells

The effects of 1 x 10(-6) M exogenous 2-methoxyestradiol (2 ME) were determined on cell morphology and cell division cycle (Cdc) 2 kinase activity in SNO oesophageal carcinoma cells. Mitotic indices revealed an increase in metaphase cells (11.2%) when compared to the 0.5% vehicle-treated cells after 18 h of exposure to 2 ME. Vehicle-treated control cells did not show any hallmarks of apoptosis after 18 h of exposure to dimethyl sulphoxide. Only 0.5% of 2 ME-treated cells showed characteristics of apoptosis. Conversely, increased morphological hallmarks of apoptosis were observed in SNO-treated cells after 21.5 h of 2 ME exposure. When compared to the 0.5% in vehicle-treated cells, 4.7% of cells were in apoptosis. Furthermore, 34.1% of cells were blocked in metaphase after 21.5 h of 2 ME exposure compared to 0.6% of vehicle-control cells. In addition, Cdc2 kinase activity was statistically significantly increased (1.3-fold) (p<0.005) in 2 ME-treated cells when compared to vehicle-treated controls. The present preliminary study suggests that the accumulation observed in metaphase cells and the increase in Cdc2 kinase activity caused by 2 ME are consistent with morphological hallmarks of mitotic arrest and disrupted mitotic spindle formation, thus leading to induction of apoptosis in SNO cells.     

Improved embryo development with decreased apoptosis in blastomeres after the treatment of cloned bovine embryos with beta-mercaptoethanol and hemoglobin

In preliminary experiments, the treatment of donor somatic cells with beta-mercaptoethanol (ME) or hemoglobin (Hb) improved in vitro-development of bovine cloned embryos. This study was subsequently evaluated whether the exposure to Hb and/or ME during in vitro-maturation or embryo culture could further promote the development of embryos cloned with ME-treated donor cells. A prospective, randomized study was conducted and, embryo development, cell number, and apoptosis in blastocysts were monitored. A significant (P < 0.05) effect was found after the combined treatment of cloned embryos with Hb (1 microg/ml) and ME (10 microM); the development of morulae (53 vs. 35%) was greatly improved, which resulted in enhanced blastocyst formation (38%). However, cell number and apoptosis in blastocysts were predominantly affected by ME rather than Hb; a significant increase in total cell number of blastomeres (142-154 vs. 123 cells/embryo), inner cell mass (ICM) (39-41 vs. 27), and trophectoderm (TE) (103-114 vs. 98), and the ratio of ICM to TE cell number (0.26-0.27 vs. 0.22) was found. Also, the apoptosis index indicating the ratio of apoptotic cell to normal blastomere number was greatly reduced after ME treatments (0.85 vs. 0.056-0.069). When embryos cloned with ME-treated cells were cultured in Hb + ME-containing medium, any of the treatments to recipient oocytes before enucleation did not further promote the development. In conclusion, combined treatment of cloned embryos with Hb + ME not only improved in vitro-development but also decreased blastomere apoptosis. The use of ME-treated donor cells and the culture of cloned embryos in Hb + ME-containing medium yielded the optimal results for promoting the production of blastocysts with improved quality.     

A novel form of melanoma apoptosis resistance: melanogenesis up-regulation in apoptotic B16-F0 cells delays ursolic acid-triggered cell death

Melanoma is one of the most aggressive forms of cancer with a continuously growing incidence worldwide and is usually resistant to chemotherapy agents, which is due in part to a strong resistance to apoptosis. The resistance mechanisms are complex and melanoma cells may have diverse possibilities for regulating apoptosis to generate apoptotic deficiencies. In this study, we investigated the relationship between melanogenesis and resistance to apoptosis induced by ursolic acid, a natural chemopreventive agent, in B16-F0 melanoma cells. We demonstrated that cells undergoing apoptosis are able to delay their own death. It appeared that tyrosinase and TRP-1 up-regulation in apoptotic cells and the subsequent production of melanin were clearly implicated in an apoptosis resistance mechanism; while TRP-2, a well known mediator of melanoma resistance to cell death, was repressed. Our results confirm the difficulty of treating melanomas, since, even undergoing apoptosis, cells are nevertheless able to trigger a resistance mechanism to delay death.     

Oxidation of indole-3-acetic acid by horseradish peroxidase induces apoptosis in G361 human melanoma cells

The combination of indole-3-acetic acid (IAA) and horseradish peroxidase (HRP) has recently been proposed as a novel cancer therapy. However, the mechanism underlying the cytotoxic effect involved is substantially unknown. Here, we show that IAA/HRP treatment induces apoptosis in G361 human melanoma cells, whereas IAA or HRP alone have no effect. It is known that IAA produces free radicals when oxidized by HRP. Because oxidative stress could induce apoptosis, we measured the production of free radicals at varying concentrations of IAA and HRP. Our results show that IAA/HRP produces free radicals in a dose-dependent manner, which are suppressed by ascorbic acid or (-)-epigallocatechin gallate (EGCG). Furthermore, antioxidants prevent IAA/HRP-induced apoptosis, indicating that the IAA/HRP-produced free radicals play an important role in the apoptotic process. In addition, IAA/HRP was observed to activate p38 mitogen-activated protein (MAP) kinase and c-Jun N-terminal kinase (JNK), which are almost completely blocked by antioxidants. We further investigated the IAA/HRP-mediated apoptotic pathways, and found that IAA/HRP activates caspase-8 and caspase-9, leading to caspase-3 activation and poly(ADP-ribose) polymerase (PARP) cleavage. These events were also blocked by antioxidants, such as ascorbic acid or EGCG. Thus, we propose that IAA/HRP-induced free radicals lead to the apoptosis of human melanoma cells via both death receptor-mediated and mitochondrial apoptotic pathways.     

Phorbol ester (12-O-tetradecanoylphorbol 13-acetate) prevents ornithine decarboxylase inhibition and apoptosis in L1210 leukemic cells exposed to TGF-β1

Previous studies have shown that growth suppression and apoptosis of leukemic cells exposed to TGF-β₁ is associated with the inhibition of ornithine decarboxylase (ODC) — the key enzyme of polyamine pathway. The aim of the present study was to evaluate the influence of 12-O-tetradecanoylphorbol 13-acetate (TPA) — a potent ODC inducer on antiproliferative and apoptotic effects of TGF-β₁ in L1210 leukemic cells. Cells were incubated in 2%FCS/RPMI1640 medium, supplemented with TGF-β₁ (2 ng/ml), TPA (100 ng/ml) or -difluoromethyl-ornithine (DFMO) (5 mM). Cell proliferation, apoptosis and necrosis were evaluated using [methyl-³H] thymidine, electron microscopy, electrophoresis of DNA and trypan blue exclusion. Expression and activity of ODC were determinated by RT-PCR and measurement of ¹⁴CO₂ release from L-1-¹⁴C ornithine, respectively. TGF-β₁ inhibited proliferation and induced apoptotic and necrotic cell death in L1210 leukemic cells. The above effects were associated with the inhibition of ODC expression and activity, measured 2 and 4 hr after TGF-β₁ administration, respectively. The presence of DFMO, an irreversible inhibitor of ODC, led to apoptotic fragmentation of DNA, similar to that observed in TGF-β₁-treated cultures. Administration of TPA simultaneously with TGF-β₁ significantly reduced antiproliferative, apoptotic and necrotic effects of TGF-β₁, and prevented its inhibitory action on ODC expression and activity. It is concluded that: down-regulation of ODC expression may be one of the early events associated with TGF-β₁-evoked suppression of growth and apoptosis; ODC is involved in the mechanism of protective action of TPA on TGF-β₁-related growth inhibition of L1210 leukemic cells.     

Delayed reproductive death and ROS levels in the progeny of irradiated melanoma cells

The cell death and survival of proliferating (clonogenic) cells were investigated in two human melanoma cell lines to assess the optimal conditions for preparation of apoptotic bodies from melanoma cells. After 50 J/m2 UVB+UVC the maximal levels of apoptotic cells assayed by Trypan blue staining, nucleosomal DNA fragmentation, MTT, and TUNEL tests were observed within 2-3 d of radiation. In 100 Gy gamma-irradiated cultures these apoptosis indicators were delayed for up to 3 weeks. In addition, clonogenic cells were observed only in exponentially growing cultures irradiated with UV at high cell density but not in gamma-irradiated cultures. The response of melanoma cultures after high UV radiation doses contrasted to the response in lethally gamma-irradiated cultures. UV-irradiated melanoma cultures were recovered within two weeks. Most of the clonogenic cells in the recovered colonies contained micronuclei. ROS levels determined by DCF fluorescence and a modified MTT test were also normalized obviously due to the extensive antioxidant defense system of melanoma cells. UV radiation of tumor cells might be the preferential method for preparation of apoptotic bodies. The presence of clonogenic cells in the suspension of apoptotic bodies from melanoma cells used for pulsing of dendritic cells with tumor antigens might compromise this protocol for preparation of cell vaccines.     

Depletion of endogenous nitric oxide enhances cisplatin-induced apoptosis in a p53-dependent manner in melanoma cell lines

The expression of inducible nitric-oxide synthase in melanoma tumor cells was recently shown to correlate strongly with poor patient survival after combination biochemotherapy (p<0.001). Furthermore, evidence suggests that nitric oxide, a reaction product of nitric oxide synthase, exhibits antiapoptotic activity in melanoma cells. We therefore hypothesized that nitric oxide antagonizes chemotherapy-induced apoptosis. Whether nitric oxide is capable of regulating cell growth and apoptotic responses to cisplatin treatment in melanoma cell lines was evaluated. We demonstrate herein that depletion of endogenously produced nitric oxide can inhibit melanoma proliferation and promote apoptosis. Moreover, our data indicate that the depletion of nitric oxide leads to changes in cell cycle regulation and enhances cisplatin-induced apoptosis in melanoma cells. Strikingly, we observed that the depletion of nitric oxide inhibits cisplatin-induced wild type p53 accumulation and p21(Waf1/Cip1/Sdi1) expression in melanoma cells. When cisplatin-induced p53 binding to the p21(Waf1/Cip1/Sdi1) promoter was examined, it was found that nitric oxide depletion significantly reduced the presence of p53-DNA complexes after cisplatin treatment. Furthermore, dominant negative inhibition of p53 activity enhanced cisplatin-induced apoptosis. Together, these data strongly suggest that endogenously produced nitric oxide is required for cisplatin-induced p53 activation and p21(Waf1/Cip1/Sdi1) expression, which can regulate melanoma sensitivity to cisplatin.     

Novel combination of 2-methoxyestradiol and cyclophosphamide enhances the antineoplastic and pro-apoptotic effects on S-180 ascitic tumour cells

Sarcoma 180 (S-180) tumour cell line is a stable murine tumour cell line with 98–99% stumour takes capacity in Swiss albino mouse - Mus musculus. 2 Methoxyestradiol (2ME) - a promising anti-neoplastic and anti-angiogenic agent, showed toxicity to host body in higher concentration. Cyclophosphamide (CP), the anti-neoplastic agent has long been used as a chemotherapeutic drug for treatment of different cancers. Our studies have shown that the combination effect of 2ME and CP on S-180 tumour cell line is anti-proliferative and less toxic. The treatment with lower concentrations of 2ME and CP (6.5 mg 2ME/kg body weight + 75 mg CP/kg body weight) antagonistically increased the life span of tumour bearing mice and synergistically inhibited the viable cell population. 2ME or CP treatment individually induces G2/M arrest. The combination treatment of 2ME + CP (6.5 mg 2ME/kg body weight + 75 mg CP/kg body weight) produced a significant increase of cells in the G₀ which is the indication of cell arrest or apoptosis. Reduction of cell viability by 2ME + CP treatments is due to apoptotic cell death. This combination therapy produced a significant inhibitory effect of cell proliferation and augmentation of cell accumulation in the G₀ phase (i.e. apoptosis). Apoptosis is validated by Fluorescence staining of control and treated S-180 tumour cells with Acridine Orange and EtBr dye. Moreover, a steady increase in the frequency of complex chromosomal aberrations (i.e. tri-, qudri-radial translocations) in tumour cells was noted in that particular concentration of combination therapy treated series along with the increase in dead cell frequency and tumour regression pattern. It is assumed that, these chromosomal abnormalities or damages recorded in higher frequency prevent the affected metaphases to enter into the next cell cycle through apoptosis or necrosis. This study introduces a novel combination, where this particular concentration of 2ME + CP (i.e. 6.5 mg 2ME/kg body weight + 75 mg CP/kg body weight) not only enhanced the life span of tumour bearing mouse and decreased the tumour volume antagonistically but also inhibited the viable cell population synergistically, which could serve as a potential effective regimen for cancer treatment.     

Apoptotic cell death kinetics in vitro depend on the cell types and the inducers used

INTRODUCTION: In vitro exposure of cells to a fluorochrome-labeled inhibitor of caspases (FLICA) labels cells after caspase activation and arrests further progress of apoptotic cell death. The labeled apoptotic cells can be quantified in relation to time of apoptosis induction with flow cytometry. Loss of membrane integrity (late apoptosis and cell death) was measured with exposure to propidium iodide (PI). From the labeling patterns with FLICA and PI the apoptotic cell death kinetics was calculated. METHODS: HL60 cells and human umbilical vein endothelial cells (HUVECs) were incubated in the presence of the fluorescent inhibitor of caspases, FAM-VAD-FMK (20 mM, FLICA) for up to 48 h. Apoptosis was induced by Camptothecin (CPT, 0.15 microM) or by a mixture of tumour necrosis factor alpha (TNF-alpha, 3 nM)-Cycloheximide (CHX, 50 microM). Samples were counterstained with PI. RESULTS: Incubation of HL60 cells with CPT induced apoptosis in 92% of cells within the first 18 h at a rate of 5% per hour while incubation with TNF-alpha/CHX resulted in apoptosis in 76% of the cells within the first 6 h at a rate of 12% per hour. Incubation of HUVECs with TNF-alpha/CHX induced apoptosis in 65% of the cells within the first 18 h at a rate of 3.7% per hour during the first 6 h of the incubation. During incubation with TNF-alpha/CHX the remaining viable HL60 cells and HUVECs entered apoptosis within 48 h at an approximate rate of 0.2 per hour. However, on the road of the cell death, HL60 cells showed a transit from the viable (FLICA-/PI-) to early (FLICA+/PI-) and further to late apoptotic phase (FLICA+/PI+), while HUVECs entered directly from the viable to the late apoptotic stage. CONCLUSION: Apoptotic turnover rate depends on the stimulus used to induce apoptosis, while the type of the cell determines the way of the transition within the apoptotic cascade.