Evaluation of a Gemcitabine‐Doxorubicin‐Paclitaxel Combination Schedule through Flow Cytometry Assessment of Apoptosis Extent Induced in Human Breast Cancer Cell Lines

Serrano, María José; Sánchez‐Rovira, Pedro; Algarra, Ignacio; Jaén, A.; Lozano, Álvaro; Gaforio, José J.

doi:10.1111/j.1349-7006.2002.tb01291.x

Cited by 18 publications

(11 citation statements)

References 20 publications

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“…52 However, it is able to stain the DNA in apoptotic and necrotic cells because of their disrupted membrane. 53 Similar to Annexin-V results, all mitochondrial p53 constructs showed significant 7-AAD intercalation with DNA compared to their MTS-EGFP controls (Figure 7A). In addition, EGFP-XL was the only construct that showed similar activity to EGFP alone (Figure 7A).…”

Section: Resultssupporting

confidence: 77%

Direct Induction of Apoptosis Using an Optimal Mitochondrially Targeted p53

et al. 2012

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Targeting the tumor suppressor p53 to the mitochondria triggers a rapid apoptotic response as efficiently as transcription-dependent p53.1, 2 p53 forms a complex with the anti-apoptotic Bcl-XL, which leads to Bak and Bax oligomerization resulting in apoptosis via mitochondrial outer membrane permeabilization.3, 4 Although p53 performs its main role in the mitochondrial outer membrane it also interacts with different proteins in the mitochondrial inner membrane and matrix.5, 6 To further investigate mitochondrial activity of p53, EGFP-p53 was fused to different mitochondrial targeting signals (MTSs) directing it to the mitochondrial outer membrane (“XL-MTS” from Bcl-XL; “TOM-MTS” from TOM20), the inner membrane (“CCO-MTS” from cytochrome c oxidase) or matrix (“OTC-MTS” from ornithine transcarbamylase). Fluorescence microscopy and a p53 reporter dual luciferase assay demonstrated that fusing MTSs to p53 increased mitochondrial localization and nuclear exclusion depending on which MTS was used. To examine if the MTSs initiate mitochondrial damage, we fused each individual MTS to EGFP (a non-toxic protein) as negative controls. We performed caspase-9, TUNEL, Annexin-V, and 7-AAD apoptosis assays on T47D breast cancer cells transfected with mitochondrial constructs. Except for EGFP-XL, apoptotic potential was observed in all MTS-EGFP-p53 and MTS-EGFP constructs. In addition, EGFP-p53-XL showed the greatest significant increase in programmed cell death compared to its non-toxic MTS control (EGFP-XL). The apoptotic mechanism for each construct was further investigated using pifithrin-α (an inhibitor of p53 transcriptional activity), pifithrin-μ (a small molecule that reduces binding of p53 to Bcl-2 and Bcl-XL), and over-expressing the anti-apoptotic Bcl-XL. Unlike the MTSs from TOM, CCO, and OTC, which showed different apoptotic mechanisms, we conclude that p53 fused to the MTS from Bcl-XL performs its apoptotic potential exclusively through p53/Bcl-XL specific pathway.

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Section: Resultssupporting

confidence: 77%

Direct Induction of Apoptosis Using an Optimal Mitochondrially Targeted p53

et al. 2012

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“…These results are also consistent with gemcitabineÕs mechanism of action where the activated molecules interfere with DNA synthesis. Others report an accumulation of cells in subG1 and apoptotic fractions [39][40][41], whereas neither subG1 fractions nor total cell recovery were different between untreated and treated cells in the hollow fiber system. It is possible that prolonging the interval between drug treatment and cell recovery beyond 2 days may result in a higher fraction of cells that have undergone apoptosis, especially since the cells grown in the bioreactor are observed to have a slower dividing time than cells grown under static conditions.…”

Section: Discussionmentioning

confidence: 92%

Characterization of an in vitro cell culture bioreactor system to evaluate anti-neoplastic drug regimens

Kirstein

Brundage

Elmquist

et al. 2006

Breast Cancer Res Treat

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A dynamic 3-dimensional tissue culture system has been developed that will allow for control of gemcitabine exposure to mimic concentration-time profiles measured from biologic samples. Gemcitabine was infused into a central reservoir. Media is mixed and delivered through hollow fiber capillaries, where it diffuses into the extracapillary space containing anchorage-dependent MDA-231 cells. To test for control of gemcitabine concentration-time profiles, drug was first infused through bioreactors without cells, and gemcitabine concentrations were measured with HPLC. Concentrations could be controlled to simulate 30-min and 2.5 h infusions, and were similar in both the lumen and extracapillary space. MDA-231 cells were then seeded into control (n = 4) and gemcitabine treatment (n = 4) groups, and maintained in culture for 2 weeks. Gemcitabine (5.3 mg) was infused over 30 min to the treatment group, and blank media to the control group. Accuracy of measured gemcitabine maximum concentration (Cmax) was 83.4%, and area under the curve (AUC), 106.2%, relative to pre-experimental theoretical values. With cells present, gemcitabine AUC in the extracapillary space was 32% of the value in the lumen. For the control group, 21.2 million cells (94.3% viable) were recovered, and for the gemcitabine-treated group, 16.8 million cells (87.1 % viable). Flow cytometry showed that 13.3 % of cells in the control group were in S-phase and 34.3 % in the gemcitabine-treated group were in S-phase (p = 0.003). In conclusion, gemcitabine concentration-time profiles could be accurately controlled through dosage, infusion rate, and pump flow rate, and cells could be recovered afterward to evaluate drug treatment.

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“… 12 , 14 As we have shown previously 12 , 15 , p53-CC is capable of inducing cell death in this as well as other cancer cell lines, regardless of endogenous p53 status. Figure 2 illustrates the superior tumor suppressor function of p53-CC over wt-p53 in a 7-AAD viability assay which stains apoptotic and necrotic cells 16 , 17 (compare Figure 2A vs 2B ). Wt-p53 activity is not significantly different from that achieved by the negative control Ad-ZsGreen1 ( Figure 2B vs 2C ).…”

Section: Resultsmentioning

confidence: 99%

Re-engineered p53 activates apoptosis in vivo and causes primary tumor regression in a dominant negative breast cancer xenograft model

Okal

Matissek

et al. 2014

Gene Ther

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Inactivation of p53 pathway is reported in more than half of all human tumors and can be correlated to malignant development. Missense mutation in the DNA binding region (DBD) of p53 is the most common mechanism of p53 inactivation in cancer cells. The resulting tumor-derived p53 variants, similar to wild-type (wt) p53, retain their ability to oligomerize via the tetramerization domain (TD). Upon hetero-oligomerization, mutant p53 enforces a dominant negative effect over active wt-p53 in cancer cells. To overcome this barrier, we have previously designed a chimeric superactive p53 (p53-CC) with an alternative oligomerization domain capable of escaping transdominant inhibition by mutant p53 in vitro. In this report, we demonstrate the superior tumor suppressor activity of p53-CC and its ability to cause tumor regression of the MDA-MB-468 aggressive p53-dominant negative breast cancer tumor model in vivo. In addition, we illustrate the profound effects of the dominant negative effect of endogenous mutant p53 over wt-p53 in cancer cells. Finally, we investigate the underlying differential mechanisms of activity for p53-CC and wt-p53 delivered using viral-mediated gene therapy approach in the MDA-MB-468 tumor model.

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Evaluation of a Gemcitabine‐Doxorubicin‐Paclitaxel Combination Schedule through Flow Cytometry Assessment of Apoptosis Extent Induced in Human Breast Cancer Cell Lines

Cited by 18 publications

References 20 publications

Direct Induction of Apoptosis Using an Optimal Mitochondrially Targeted p53

Direct Induction of Apoptosis Using an Optimal Mitochondrially Targeted p53

Characterization of an in vitro cell culture bioreactor system to evaluate anti-neoplastic drug regimens

Re-engineered p53 activates apoptosis in vivo and causes primary tumor regression in a dominant negative breast cancer xenograft model

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