RESUMO
To systematically evaluate the efficacy and safety of Yangwei Granules combined with conventional Western medicine for chronic gastritis. CNKI, SinoMed, WanFang, VIP, PubMed, Cochrane Library and EMbase database were electronically retrieved to collect randomized controlled trial(RCT) of Yangwei Granules combined with conventional Western medicine for chronic gastritis. Two reviewers independently screened out literatures according to the inclusion and exclusion criteria and extracted data, and evaluated the risk of bias of included studies. Meta-analysis was conducted by using RevMan 5.3 software. A total of 12 RCTs involving 1 164 patients were included. The results of Meta-analysis showed that:(1) The total effective rate of Yangwei Granules combined with conventional Western medicine for chronic gastritis was better than that of conventional Western medicine group, with a statistically significant difference(RR=1.24,95%CI[1.17,1.31],P<0.000 01).(2) Compared with the conventional Western medicine group, the Yangwei Granules combined with conventional Western medicine group was conducive to improving the Hp eradication rate, with a statistically significant difference(RR=1.24,95%CI[1.15,1.34],P<0.000 01).(3) The incidence of adverse reactions in Yangwei Granules combined with conventional Western medicine group was lower than that in the control group, but with no statistically significant diffe-rence(RR=0.83, 95%CI[0.39, 1.79], P=0.64).(4) Compared with the conventional Western medicine group, the Yangwei Granules combined with conventional Western medicine group was beneficial to the reduction of motilin level(MD=-17.31,95%CI[-21.83,-12.79],P<0.000 01) and endothelin level(MD=-6.60,95%CI[-10.07,-3.13],P=0.000 2), while the increase of gastrin level(SMD=0.94,95%CI[0.50,1.38],P=0.003) was related to calcitonin gene the level of peptide(MD=5.82,95%CI[4.25,7.39],P<0.000 01), with statistically significant differences.(5) Compared with conventional Western medicine group, Yangwei Granules combined with conventional Western medicine group could increase PGâ (MD=6.40,95%CI[4.26,8.54],P<0.000 01) and PGR(MD=0.89,95%CI[0.71,1.07],P<0.000 01), while decrease PGâ ¡(MD=-1.24,95%CI[-2.15,-0.33],P=0.007), with statistically significant differences. Current evidence showed that the clinical efficacy and Hp eradication rate of Yangwei Granules combined with conventional Western medicine in the treatment of chronic gastritis were better than those of the conventional Western medicine group alone, and could effectively improve the level of gastrointestinal hormones, vasoactive peptide and the pepsinogen level in patients with chronic atrophic gastritis, without increasing the incidence of adverse reactions. However, due to the limited quality and quantity of included studies, the above conclusions need to be confirmed by more large-scale and high-quality RCTs.
Assuntos
Medicamentos de Ervas Chinesas , Gastrite Atrófica , Medicamentos de Ervas Chinesas/efeitos adversos , Humanos , Resultado do TratamentoRESUMO
The phthalocyanine photosensitizer Pc 4 has been shown to bind preferentially to mitochondrial and endoplasmic reticulum membranes. Upon photoirradiation of Pc 4-loaded cells, membrane components, especially Bcl-2, are photodamaged and apoptosis, as indicated by activation of caspase-3 and cleavage of poly(ADP-ribose) polymerase, is triggered. A series of analogs of Pc 4 were synthesized, and the results demonstrate that Pcs with the aminopropylsiloxy ligand of Pc 4 or a similar one on one side of the Pc ring and a second large axial ligand on the other side of the ring have unexpected properties, including enhanced cell uptake, greater monomerization resulting in greater intracellular fluorescence and three-fold higher affinity constants for liposomes. The hydroxyl-bearing axial ligands tend to reduce aggregation of the Pc and direct it to lysosomes, resulting in four to six times more killing of cells, as defined by loss of clonogenicity, than with Pc 4. Whereas Pc 4-PDT photodamages Bcl-2 and Bcl-xL, Pc 181-PDT causes much less photodamage to Bcl-2 over the same dose-response range relative to cell killing, with earlier cleavage of Bid and slower caspase-3-dependent apoptosis. Therefore, within this series of photosensitizers, these hydroxyl-bearing axial ligands are less aggregated than is Pc 4, tend to localize to lysosomes and are more effective in overall cell killing than is Pc 4, but induce apoptosis more slowly and by a modified pathway.
Assuntos
Indóis/farmacologia , Lisossomos/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Fotoquimioterapia , Fármacos Fotossensibilizantes/farmacologia , Silício/química , Isoindóis , Espectroscopia de Ressonância MagnéticaRESUMO
To examine the clinical applicability of Pc 4, a promising second-generation photosensitizer, for the photodynamic treatment of lymphocyte-mediated skin diseases, we studied the A431 and Jurkat cell lines, commonly used as surrogates for human keratinocyte-derived carcinomas and lymphocytes, respectively. As revealed by ethyl acetate extraction and absorption spectrophotometry, uptake of Pc 4 into the two cell lines was linear with Pc 4 concentration and similar on a per cell basis but greater in Jurkat cells on a per mass basis. Flow cytometry showed that uptake was linear at low doses; variations in the dose-response for uptake measured by fluorescence supported differential aggregation of Pc 4 in the two cell types. As detected by confocal microscopy, Pc 4 localized to mitochondria and endoplasmic reticulum in both cell lines. Jurkat cells were much more sensitive to the lethal effects of phthalocyanine photodynamic therapy (Pc 4-PDT) than were A431 cells, as measured by a tetrazolium dye reduction assay, and more readily underwent morphological apoptosis. In a search for molecular factors to explain the greater photosensitivity of Jurkat cells, the fate of important Bcl-2 family members was monitored. Jurkat cells were more sensitive to the induction of immediate photodamage to Bcl-2, but the difference was insufficient to account fully for their greater sensitivity. The antiapoptotic protein Mcl-1 was extensively cleaved in a dose- and caspase-dependent manner in Jurkat, but not in A431, cells exposed to Pc 4-PDT. Thus, the greater killing by Pc 4-PDT in Jurkat compared with A431 cells correlated with greater Bcl-2 photodamage and more strongly to the more extensive Mcl-1 degradation. Pc 4-PDT may offer therapeutic advantages in targeting inflammatory cells over normal keratinocytes in the treatment of T-cell-mediated skin diseases, such as cutaneous lymphomas, dermatitis, lichenoid tissue reactions and psoriasis, and it will be instructive to evaluate the role of Bcl-2 family proteins, especially Mcl-1, in the therapeutic response.
Assuntos
Apoptose/efeitos dos fármacos , Indóis/farmacologia , Fotoquimioterapia , Linhagem Celular Tumoral , Humanos , Células JurkatRESUMO
Photodynamic therapy (PDT) is an efficient inducer of apoptosis in many types of cells, except in cells deficient in one or more of the factors that mediate apoptosis. Recent reports have identified autophagy as a potential alternative cell death process following PDT. Here we investigated the occurrence of autophagy after PDT with the photosensitizer Pc 4 in human cancer cells that are deficient in the pro-apoptotic factor Bax (human prostate cancer DU145 cells) or the apoptosis mediator caspase-3 (human breast cancer MCF-7v cells) and in apoptosis-competent cells (MCF-7c3 cells that stably overexpress human pro-caspase-3 and Chinese hamster ovary CHO 5A100 cells). Further, each of the cell lines was also studied with and without stably overexpressed Bcl-2. Autophagy was identified by electron microscopic observation of the presence of double-membrane-delineated autophagosomal vesicles in the cytosol and by immunoblot observation of the Pc 4-PDT dose- and time-dependent increase in the level of LC3-II, a component of the autophagosomal membrane. Autophagy was observed in all of the cell lines studied, whether or not they were capable of typical apoptosis and whether or not they overexpressed Bcl-2. The presence of stably overexpressed Bcl-2 in the cells protected against PDT-induced apoptosis and loss of clonogenicity in apoptosis-competent cells (MCF-7c3 and CHO 5A100 cells). In contrast, Bcl-2 overexpression did not protect against the development of autophagy in any of the cell lines or against loss of clonogenicity in apoptosis-deficient cells (MCF-7v and DU145 cells). Furthermore, 3-methyladenine and wortmannin, inhibitors of autophagy, provided greater protection against loss of viability to apoptosis-deficient than to apoptosis-competent cells. The results show that autophagy occurs during cell death following PDT in human cancer cells competent or not for normal apoptosis. Only the apoptosis-competent cells are protected by Bcl-2 against cell death.
Assuntos
Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Fotoquimioterapia , Proteínas Proto-Oncogênicas c-bcl-2/fisiologia , Animais , Células CHO , Linhagem Celular Tumoral , Cricetinae , Cricetulus , Humanos , Microscopia Eletrônica de Transmissão , Fármacos Fotossensibilizantes/farmacologiaRESUMO
The antiapoptotic Bcl-2-family proteins, Bcl-2 and Bcl-xL, are recognized phototargets of photodynamic therapy (PDT) with the mitochondrion-targeting phthalocyanine photosensitizer Pc 4. In the present study, we found that myeloid cell leukemia 1 (Mcl-1), another antiapoptotic member of the Bcl-2 family, was not photodamaged in Pc 4-PDT-treated human carcinoma cells MCF-7c3, MDA-MB468, DU145, and A431, although Mcl-1 turnover was observed after exposure of HeLa or MCF-7c3 cells to a supralethal dose of UVC. In contrast, when human lymphoma U937 and Jurkat cells were treated with Pc 4-PDT, staurosporine (STS) or UVC, Mcl-1 was cleaved to generate a 28-kDa fragment over a 2-4 h period. The cleavage of Mcl-1 was accompanied by the activation of caspases-3, -9, and -8. The broad-specificity caspase inhibitor z-VAD-fmk completely blocked Mcl-1 cleavage induced by PDT, STS or UVC, providing evidence for Mcl-1 as a substrate for caspases. Western blot analysis localized Mcl-1 to mitochondria, ER, and cytosol of both MCF-7c3 and U937 cells, suggesting that Mcl-1 protein, unlike Bcl-2 and Bcl-xL, is not a target for Pc 4-PDT, probably due to its localization to sites removed from those of Pc 4 binding. The 28-kDa cleaved fragment of Mcl-1, which has proapoptotic activity, was produced in PDT-treated lymphoid-derived cells, but not in cells of epithelial origin, suggesting that PDT-induced rapid and extensive apoptosis in lymphoma cells may result in part from the sensitivity of their Mcl-1 to caspase cleavage, removing an important negative control on apoptosis.
Assuntos
Células Epiteliais/efeitos dos fármacos , Indóis/farmacologia , Tecido Linfoide/efeitos dos fármacos , Proteínas de Neoplasias/metabolismo , Fármacos Fotossensibilizantes/farmacologia , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Caspases/metabolismo , Linhagem Celular Tumoral , Inibidores de Cisteína Proteinase/farmacologia , Células Epiteliais/citologia , Células Epiteliais/efeitos da radiação , Humanos , Tecido Linfoide/citologia , Tecido Linfoide/efeitos da radiação , Proteína de Sequência 1 de Leucemia de Células Mieloides , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-bcl-2/antagonistas & inibidores , Estaurosporina/farmacologia , Especificidade por Substrato , Raios UltravioletaRESUMO
The antiapoptotic oncoprotein Bcl-2 is now a recognized phototarget of photodynamic therapy (PDT) with the phthalocyanine Pc 4 and with other mitochondrion-targeting photosensitizers. Photodamage, observed on Western blots as the loss of the native 26-kDa Bcl-2 protein, is PDT dose dependent and occurs in multiple cell lines, in the cold, and immediately upon photoirradiation. In our initial study, no photochemical damage was observed to Bcl-xL, in spite of its similarity in size, sequence, location and function to Bcl-2. The original study used a commercial anti-Bcl-xS/L antibody. We have revisited this issue by examining Western blots developed using one of three epitope-specific anti-Bcl-xL antibodies from commercial sources, a polyclonal antibody generated to the entire protein, as well as the antibody used previously. All five Bcl-xL antibodies recognized bacterially expressed Bcl-xL, but not Bcl-2, whereas an anti-Bcl-2 antibody recognized Bcl-2 and not Bcl-xL. All five Bcl-xL antibodies recognized at least one protein migrating at approximately 30 kDa; two of the antibodies recognized an additional band, migrating at approximately 33 or approximately 24 kDa. We now observe Pc 4-PDT-induced photodamage to all Bcl-xL-related proteins, except the 33-kDa species, in several human cancer cell lines. The results indicate that, in addition to the expected quantitative differences that may reflect exposure of individual epitopes, the antibodies also detect proteins of different apparent molecular weights that may be distinct isoforms or post-translationally modified forms of Bcl-xL. No evidence for PDT-induced phosphorylation or degradation was observed. Bcl-xL localized to mitochondria was considerably more sensitive to photodamage than was Bcl-xL in the cytosol, indicating that as previously found for Bcl-2, Bcl-xL must be membrane localized to be photosensitive.
Assuntos
Indóis/farmacologia , Luz , Fotoquimioterapia/métodos , Fármacos Fotossensibilizantes/farmacologia , Proteínas Proto-Oncogênicas c-bcl-2/química , Radiossensibilizantes/farmacologia , Western Blotting , Linhagem Celular Tumoral , Citosol/metabolismo , Relação Dose-Resposta a Droga , Epitopos , Humanos , Mitocôndrias/metabolismo , Testes de Precipitina , Isoformas de Proteínas , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteínas Recombinantes/química , Proteína X Associada a bcl-2 , Proteína bcl-XRESUMO
The human breast cancer cell line MCF-7 is deficient in procaspase-3 and in caspase-3-dependent steps in apoptosis due to deletion of the CASP-3 gene. We previously found that the cells transfected with empty vector (MCF-7v cells) were considerably less sensitive to photodynamic treatment in vitro with the phthalocyanine photosensitizer Pc 4 than were the cells stably transfected with human procaspase-3 cDNA (MCF-7c3 cells); however, overall cell killing, as determined by a clonogenic assay, was not affected by the presence of procaspase-3. The present study was undertaken to determine whether photodynamic therapy (PDT) in vivo was dependent on the ability of the cells to carry out the late steps in apoptosis that are catalyzed by this caspase. Xenografts of MCF-7 cells and the isogenic-derived MCF-7v and MCF-7c3 cells were generated in female athymic nude mice implanted with an estrogen pellet. MCF-7c3 xenografts, but not those of the other two lines, continued to express procaspase-3, as revealed by Western blots of proteins from the cells and the xenografts. When the xenografts reached 50-120 mm(3), some were treated with PDT (1mg/kg Pc 4 i.v. followed 48 h later by 150 J/cm(2) light at 672 nm and 150 mW/cm(2)), while others served as controls (no treatment, light alone, or Pc 4 alone). All Pc 4-PDT-treated tumors and none of the controls exhibited either complete or strong partial responses, and complete responses were durable for the entire observation period of 16 days. The responses were not dependent upon the presence of procaspase-3 in the xenografts. The results indicate that the rapid response of Pc 4-PDT-treated tumors in vivo is not due to their ability to carry out the major caspase-3-mediated late steps in apoptosis.
Assuntos
Neoplasias da Mama/tratamento farmacológico , Caspases/metabolismo , Indóis/uso terapêutico , Fotoquimioterapia , Fármacos Fotossensibilizantes/uso terapêutico , Animais , Western Blotting , Neoplasias da Mama/enzimologia , Caspase 3 , Feminino , Humanos , Incidência , Camundongos , Camundongos Nus , Transplante de Neoplasias , Transplante Heterólogo , Células Tumorais Cultivadas/metabolismoRESUMO
Photodynamic therapy (PDT) using the second-generation photosensitizer phthalocyanine (Pc) 4 causes mitochondrial damage and induces apoptosis through the release of cytochrome c to the cytosol. Another protein of the mitochondrial intermembrane space, Smac/DIABLO (second mitochondria-derived activator of caspase/direct inhibitor of apoptosis-binding protein with low pI), is also released to the cytosol in response to apoptotic stimuli and promotes caspase activation by binding IAP. To investigate the possible role of Smac/DIABLO in apoptosis induced by Pc 4-PDT, we transfected Smac/DIABLO (tagged at its C-terminus with green fluorescent protein [GFP]) into MCF-7c3 cells (human breast cancer MCF-7 cells stably transfected with procaspase-3) and DU-145 cells (human prostate cancer cells that express no Bax because of a frameshift insertion mutation). Confocal microscopy showed that recombinant Smac/DIABLO, like cytochrome c, localized to mitochondria and colocalized with MitoTracker Red. Three hours after exposure of MCF-7c3 cells to PDT (200 nM Pc 4 and 150 mJ/cm2 red light), Smac/DIABLO-GFP, as well as cytochrome c, was found largely in the cytosol. In contrast, for DU-145 cells, both Smac/DIABLO-GFP and cytochrome c remained in the mitochondria after PDT. By staining with Hoechst 33,342, typical apoptotic nuclei were observed in MCF-7c3 cells, but not in DU-145 cells, after Pc 4-PDT. These results suggest that the release of Smac/DIABLO from mitochondria may be regulated by a Bax-mediated mechanism and that Smac/DIABLO may cooperate with the cytochrome c-dependent apoptosis pathway. In addition, in MCF-7c3 cells transfected by Smac/DIABLO-GFP, apoptosis induced by Pc 4-PDT was greater than in cells transfected with the GFP vector alone or in untransfected cells, as determined by flow cytometry. Thus, Smac/DIABLO promotes apoptosis after Pc 4-PDT in a Bax-dependent manner and may facilitate the passage of PDT-treated cells through the late steps of apoptosis.
Assuntos
Apoptose/efeitos dos fármacos , Fotoquimioterapia , Proteínas Proto-Oncogênicas c-bcl-2 , Apoptose/fisiologia , Proteínas Reguladoras de Apoptose , Proteínas de Transporte/metabolismo , Grupo dos Citocromos c/metabolismo , Feminino , Proteínas de Fluorescência Verde , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Proteínas Luminescentes/genética , Masculino , Proteínas Mitocondriais/metabolismo , Fotobiologia , Proteínas Proto-Oncogênicas/metabolismo , Transfecção , Células Tumorais Cultivadas , Proteína X Associada a bcl-2RESUMO
Photodynamic therapy (PDT) uses a photosensitizer, light and oxygen to produce extensive oxidative damage to organelles housing the photosensitizer. Although PDT is an efficient trigger of apoptosis, it also induces autophagy in many kinds of cells. Autophagy can serve as both a cell survival and a cell death mechanism. Our previous study indicates that autophagy contributes to cell death after PDT, especially in apoptosis-deficient cells. Here, we provide further evidence to support the role of autophagy in cell killing after PDT. Autophagy was blocked by knockdown of one essential factor, LC3 or Atg7, in MCF-7 cells. The cells were exposed to a range of doses of PDT sensitized by the phthalocyanine Pc 4; steps in autophagy were monitored by western blotting for LC3-II and by fluorescence microscopy for the uptake of monodansylcadaverine or for the distribution of transfected GFP-LC3; and overall cell death was monitored by MTT assay and by clonogenic assay. We find that blocking autophagy increased the survival of MCF-7 cells after PDT and increased the shoulder on the dose-response curve. In response to Pc 4-PDT, Atg7-deficient MCF-7 cells remained capable of robust accumulation of LC3-II, but were defective in comparison to Atg7(+) cells in the formation of autophagosomes. We conclude that apoptosis-deficient cells rely on autophagy for cell death after Pc 4-PDT and that the strong activation of LC3 maturation in response to PDT could occur even in cells with limited or no Atg7 expression.
Assuntos
Neoplasias da Mama , Linhagem Celular Tumoral/efeitos da radiação , Fotoquimioterapia , Enzimas Ativadoras de Ubiquitina/deficiência , Apoptose/fisiologia , Autofagia/fisiologia , Proteína 7 Relacionada à Autofagia , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Neoplasias da Mama/terapia , Feminino , Humanos , Indóis/uso terapêutico , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Fármacos Fotossensibilizantes/uso terapêutico , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismoRESUMO
Photodynamic therapy (PDT) with lysosome-targeted photosensitizers induces the intrinsic pathway of apoptosis via the cleavage and activation of the BH3-only protein Bid by proteolytic enzymes released from photodisrupted lysosomes. To investigate the role of Bid in apoptosis induction and the role of damaged lysosomes on cell killing by lysosome-targeted PDT, we compared the responses of wild type and Bid-knock-out murine embryonic fibroblasts toward a mitochondrion/endoplasmic reticulum-binding photosensitizer, Pc 4, and a lysosome-targeted sensitizer, Pc 181. Whereas apoptosis and overall cell killing were induced equally well by Pc 4-PDT in both cell lines, Bid(-/-) cells were relatively resistant to induction of apoptosis and to overall killing following PDT with Pc 181, particularly at low PDT doses. Thus, Bid is critical for the induction of apoptosis caused by PDT with the lysosome-specific sensitizers, but dispensable for PDT targeted to other membranes.
Assuntos
Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/fisiologia , Sistemas de Liberação de Medicamentos , Muramidase/efeitos da radiação , Fotoquimioterapia , Apoptose/efeitos dos fármacos , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/genética , Western Blotting , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Técnicas de Inativação de Genes , Mitocôndrias/efeitos dos fármacos , Estrutura Molecular , Muramidase/efeitos dos fármacos , Fármacos Fotossensibilizantes/efeitos da radiaçãoRESUMO
We are developing and evaluating choline molecular imaging with positron emission tomography (PET) for monitoring tumor response to photodynamic therapy (PDT) in animal models. Human prostate cancer (PC-3) was studied in athymic nude mice. A second-generation photosensitizer Pc 4 was used for PDT in tumor-bearing mice. MicroPET images with (11)C-choline were acquired before PDT and 48 h after PDT. Time-activity curves of (11)C-choline uptake were analyzed before and after PDT. For treated tumors, normalized choline uptake decreased significantly 48 h after PDT, compared to the same tumors pre-PDT (p < 0.001). However, for the control tumors, normalized choline uptake increased significantly (p < 0.001). PET imaging with (11)C-choline is sensitive to detect early tumor response to PDT in the animal model of human prostate cancer.
RESUMO
Photodynamic therapy (PDT) induces apoptosis in many cell types. Recent reports identified autophagy as an alternative cell-death process following PDT. Here we investigated the occurrence of autophagy after PDT with the photosensitizer Pc 4 in human cancer cells that are deficient in the pro-apoptotic factor Bax (human prostate cancer DU145) or the apoptosis mediator caspase-3 (human breast cancer MCF-7v) and in apoptosis-competent cells (MCF-7c3 stably overexpressing human pro-caspase-3 and Chinese hamster ovary CHO 5A100). Further, each cell line was also studied with and without stably overexpressed Bcl-2. By electron microscopy and immunoblot analysis, autophagy was observed in all cells studied, whether or not they were capable of typical apoptosis or overexpressed Bcl-2. Bcl-2 overexpression protected against PDT-induced apoptosis and loss of clonogenicity in apoptosis-competent cells (MCF-7c3 and CHO); however, it did not protect against the development of autophagy or against loss of clonogenicity in apoptosis-deficient cells (MCF-7v and DU145). The results show that autophagy may be the dominant cell death pathway following PDT in cells that are incapable of undergoing normal apoptosis. In such cells, Bcl-2 does not protect against autophagic death.
Assuntos
Apoptose/fisiologia , Autofagia/fisiologia , Fotoquimioterapia , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Animais , Células CHO , Linhagem Celular Tumoral , Cricetinae , Cricetulus , Humanos , Fagossomos/metabolismo , Fármacos Fotossensibilizantes/metabolismoRESUMO
To test the role of caspase 3 in apoptosis and in overall cell lethality caused by the protein kinase inhibitor staurosporine, we compared the responses of MCF-7c3 cells that express a stably transfected CASP-3 gene to parental MCF-7:WS8 cells transfected with vector alone and lacking procaspase-3 (MCF-7v). Cells were exposed to increasing doses (0.15-1 microM) of staurosporine for periods up to 19 h. Apoptosis was efficiently induced in MCF-7c3 cells, as demonstrated by cytochrome c release, processing of procaspase-3, procaspase-8, and Bid, increase in caspase-3-like DEVDase activity, cleavage of the enzyme poly(ADP-ribose) polymerase, DNA fragmentation, changes in nuclear morphology, and TUNEL assay and flow cytometry. For all of these measures except cytochrome c release, little or no activity was detected in MCF-7v cells, confirming that caspase-3 is essential for efficient induction of apoptosis by staurosporine, but not for mitochondrial steps that occur earlier in the pathway. MCF-7c3 cells were more sensitive to staurosporine than MCF-7v cells when assayed for loss of viability by reduction of a tetrazolium dye. However, the two cell lines were equally sensitive to killing by staurosporine when evaluated by a clonogenic assay. A similar distinction between apoptosis and loss of clonogenicity was observed for the cancer chemotherapeutic agent VP-16. These results support our previous conclusions with photodynamic therapy: (a) assessing overall reproductive death of cancer cells requires a proliferation-based assay, such as clonogenicity; and (b) the critical staurosporine-induced lethal event is independent of those mediated by caspase-3.