A New Method for the Cytofluorometric Analysis of Mitochondrial Membrane Potential Using the J-Aggregate Forming Lipophilic Cation 5,5′,6,6′-Tetrachloro-1,1′,3,3′-tetraethylbenzimidazolcarbocyanine Iodide (JC-1)

Cossarizza, Andrea; Baccarani-Contri, M.; Kalashnikova, Galina; Franceschi, Claudio

doi:10.1006/bbrc.1993.2438

Cited by 976 publications

(667 citation statements)

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“…Indeed, JC-1 aggregates form when the mitochondrial membrane becomes hyperpolarized. 30 The FL1 fluorescence emission (which depends on JC-1 monomers) appeared to be very similar in the control LoVo WT and LoVo DX cells (Fig. 7a,c).…”

Section: Resultsmentioning

confidence: 72%

“…These structural alterations may be associated with functional modifications by flow cytometry on living cells previously labeled with JC-1 dye. 30,41 MMP analysis demonstrated that treatment with BSAO and spermine induced mitochondrial membrane depolarization. This effect was earlier and more pronounced in LoVo DX cells, in agreement with the clonogenic cell survival data and electron microscopy observations.…”

Section: Discussionmentioning

confidence: 99%

“…MMP was evaluated using the lipophilic cationic probe JC-1 (Molecular Probes) according to Cossarizza et al 30 LoVo cells were harvested with EDTA/trypsin, then washed, centrifuged and resuspended in PBS-1% BSA. Cells (10 5 /ml) were treated for 10 and 60 min with BSAO (6.54 ϫ 10 -3 U/ml) alone, spermine (6 M) alone or both.…”

Section: Flow Cytometrymentioning

confidence: 99%

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Enzymatic oxidation products of spermine induce greater cytotoxic effects on human multidrug‐resistant colon carcinoma cells (LoVo) than on their wild‐type counterparts

Calcabrini¹,

Arancia²,

Marra³

et al. 2002

Intl Journal of Cancer

122

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The occurrence of resistance to cytotoxic agents in tumor cells, associated with several phenotypic alterations, is one of the major obstacles to successful anticancer chemotherapy. A new strategy to overcome MDR of human cancer cells was studied, using BSAO, which generates cytotoxic products from spermine, H 2 O 2 and aldehyde(s). The involvement of these products in causing cytotoxicity was investigated in both drug-sensitive (LoVo WT) and drug-resistant (LoVo DX) colon adenocarcinoma cells. Evaluation of clonogenic cell survival showed that LoVo DX cells are more sensitive than LoVo WT cells. Fluorometric assay and treatments performed in the presence of catalase demonstrated that the cytotoxicity was due mainly to the presence of H 2 O 2 . Cytotoxicity was eliminated in the presence of both catalase and ALDH. Transmission electron microscopic observations showed more pronounced mitochondrial modifications in drug-resistant than in drug-sensitive cells. Mitochondrial functionality studies performed by flow cytometry after JC-1 labeling revealed basal hyperpolarization of the mitochondrial membrane in LoVo DX cells. After treatment with BSAO and spermine, earlier and higher mitochondrial membrane depolarization was found in LoVo DX cells than in drug-sensitive cells. In addition, higher basal ROS production in LoVo DX cells than in drug-sensitive cells was detected by flow-cytometric analysis, suggesting increased mitochondrial activity in drug-resistant cells. Our results support the hypothesis that mitochondrial functionality affects the sensitivity of cells to the cytotoxic enzymatic oxidation products of spermine, which might be promising anticancer agents, mainly against drug-resistant tumor cells.

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Section: Resultsmentioning

confidence: 72%

Section: Discussionmentioning

confidence: 99%

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Enzymatic oxidation products of spermine induce greater cytotoxic effects on human multidrug‐resistant colon carcinoma cells (LoVo) than on their wild‐type counterparts

Calcabrini¹,

Arancia²,

Marra³

et al. 2002

Intl Journal of Cancer

122

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“…At higher concentrations, as those which are attained in the mitochondria of healthy cells, JC-1 forms red-emitting "J-aggregates." Accordingly, the ratio of red-to-green JC-1 fluorescence can be used as a sensitive measure of the m , that is relatively independent of putative interfering factors, including mitochondrial mass, shape or density [85][86][87]. DiOC 6 (3) has become the most commonly used carbocyanine fluorochrome for the cytofluorometric measurement of m [88], due to its rapid mitochondrial equilibration and negligible quenching effects, at least at low concentrations (recommended dose 10-20 nM) [77,89,90].…”

Section: Detection Of Im Permeabilizationmentioning

confidence: 99%

Methods for the assessment of mitochondrial membrane permeabilization in apoptosis

et al. 2007

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Mitochondrial membrane permeabilization (MMP) is considered as the "point-of-no-return" in numerous models of programmed cell death. Indeed, mitochondria determine the intrinsic pathway of apoptosis, and play a major role in the extrinsic route as well. MMP affects the inner and outer mitochondrial membranes (IM and OM, respectively) to a variable degree. OM permeabilization culminates in the release of proteins that normally are confined in the mitochondrial intermembrane space (IMS), including caspase activators (e.g. cytochrome c) and caspase-independent death effectors (e.g. apoptosis-inducing factor). Partial IM permeabilization disrupts mitochondrial ion and volume homeostasis and dissipates the mitochondrial transmembrane potential ( m ). The assessment of early mitochondrial alterations allows for the identification of cells that are committed to die but have not displayed yet the apoptotic phenotype. Several

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“…To this purpose, the potential antiproliferative effects of VLM and HyVLMs were evaluated on four different cancer cell lines, namely, rat C6 glioma cells, MCF-7 human breast carcinoma cells, A2780 human ovarian carcinoma cells, and HepG2 liver hepatocellular carcinoma cells. In all experiments, cancer cell lines were treated with concentrations of compounds 1−4, ranging from 0.0001 to 10 μM, at different incubation times (24,48, and 72 h), and cell viability was determined quantitatively by the MTT conversion assay. 19 Table 1 collects the IC 50 values obtained after 72 h of incubation, while the histogram in Figure 1 shows the timedependent trend of C6 cells survival upon exposure to 0.01 μM 1−4 and is representative for the other cases examined.…”

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confidence: 99%

Antitumor Potential of Conjugable Valinomycins Bearing Hydroxyl Sites: In Vitro Studies

Iacobazzi

Annese

Azzariti

et al. 2013

ACS Med. Chem. Lett.

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Following our pioneering studies on the direct and efficient introduction of derivatizable hydroxyl handles into the valinomycin (VLM, 1) structure, a K + -ionophore with potent antitumor activity, the ensuing conjugable analogues (HyVLMs 2, 3, and 4) have herein been compared to the parent macrocycle for their potential antiproliferative effects on a panel of cancer cell lines, namely, human MCF-7, A2780, and HepG2, as well as rat C6 cells. On the basis of IC 50 values, we find that hydroxyl analogues 3 and 4 are only moderately less active than 1, while analogue 2 experiences a heavily diminished activity. Cytofluorimetric analyses of MCF-7 cells treated with HyVLMs suggest that the latter depolarize mitochondria, thus retaining the typical VLM behavior. It is likely that C6 cells, for which the exceptionally potent cytotoxicity of VLM has never reported previously, follow the same fate, as evidenced by alteration of mitochondrial morphology upon incubation with each ionophore.

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A New Method for the Cytofluorometric Analysis of Mitochondrial Membrane Potential Using the J-Aggregate Forming Lipophilic Cation 5,5′,6,6′-Tetrachloro-1,1′,3,3′-tetraethylbenzimidazolcarbocyanine Iodide (JC-1)

Cited by 976 publications

References 0 publications

Enzymatic oxidation products of spermine induce greater cytotoxic effects on human multidrug‐resistant colon carcinoma cells (LoVo) than on their wild‐type counterparts

Enzymatic oxidation products of spermine induce greater cytotoxic effects on human multidrug‐resistant colon carcinoma cells (LoVo) than on their wild‐type counterparts

Methods for the assessment of mitochondrial membrane permeabilization in apoptosis

Antitumor Potential of Conjugable Valinomycins Bearing Hydroxyl Sites: In Vitro Studies

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