Assessment of Apoptosis by Immunohistochemistry to Active Caspase-3, Active Caspase-7, or Cleaved PARP in Monolayer Cells and Spheroid and Subcutaneous Xenografts of Human Carcinoma

Bressenot, Aude; Marchal, Sophie; Bezdetnaya, Lina; Garrier, Julie; Guillemin, François; Plénat, François

doi:10.1369/jhc.2008.952044

Cited by 212 publications

(159 citation statements)

References 33 publications

Supporting

Mentioning

147

Contrasting

Unclassified

Order By: Relevance

“…3G). Furthermore, immunohistochemical staining showed an increase in the number of cleaved PARP-positive tumor cells in tissue sections (18) from xenografted tumors containing KPNB1 shRNAs (Fig. 3H), further confirming that KPNB1 inhibition induces apoptosis both in vitro and in vivo, and providing a mechanism for its antitumor effect in EOC.…”

Section: Resultssupporting

confidence: 52%

In vivo loss-of-function screens identify KPNB1 as a new druggable oncogene in epithelial ovarian cancer

Kodama

Newberg

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

Epithelial ovarian cancer (EOC) is a deadly cancer, and its prognosis has not been changed significantly during several decades. To seek new therapeutic targets for EOC, we performed an in vivo dropout screen in human tumor xenografts using a pooled shRNA library targeting thousands of druggable genes. Then, in follow-up studies, we performed a second screen using a genome-wide CRISPR/Cas9 library. These screens identified 10 high-confidence drug targets that included well-known oncogenes such as ERBB2 and RAF1, and novel oncogenes, notably KPNB1, which we investigated further. Genetic and pharmacological inhibition showed that KPNB1 exerts its antitumor effects through multiphase cell cycle arrest and apoptosis induction. Mechanistically, proteomic studies revealed that KPNB1 acts as a master regulator of cell cycle-related proteins, including p21, p27, and APC/ C. Clinically, EOC patients with higher expression levels of KPNB1 showed earlier recurrence and worse prognosis than those with lower expression levels of KPNB1. Interestingly, ivermectin, a Food and Drug Administration-approved antiparasitic drug, showed KPNB1-dependent antitumor effects on EOC, serving as an alternative therapeutic toward EOC patients through drug repositioning. Last, we found that the combination of ivermectin and paclitaxel produces a stronger antitumor effect on EOC both in vitro and in vivo than either drug alone. Our studies have thus identified a combinatorial therapy for EOC, in addition to a plethora of potential drug targets.ovarian cancer | KPNB1 | loss-of-function screen | CRISPR/Cas | RNAi

show abstract

Section: Resultssupporting

confidence: 52%

In vivo loss-of-function screens identify KPNB1 as a new druggable oncogene in epithelial ovarian cancer

Kodama

Newberg

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

show abstract

“…Apoptosis was also assessed in tumor sections by immunostaining for active caspase-3, using anti-cleaved caspase-3 (Asp175) antibody (1:2,000 dilution; Cell Signaling, New England Bioloabs, UK) as previously described. 37 Apoptotic cells were quantified using a 10 Â 10 ocular graticule (total area 1 mm 2 ) using a 20Â objective in 5 random fields of view, avoiding necrotic regions. Data were expressed as the mean number of apoptotic cells/mm 2 .…”

Section: Measurement Of Tumor Cell Apoptosismentioning

confidence: 99%

Vascular effects dominate solid tumor response to treatment with combretastatin A‐4‐phosphate

Lunt

Akerman

Hill

et al. 2011

Intl Journal of Cancer

View full text Add to dashboard Cite

Vascular‐targeted therapeutics are increasingly used in the clinic. However, less is known about the direct response of tumor cells to these agents. We have developed a combretastatin‐A‐4‐phosphate (CA4P) resistant variant of SW1222 human colorectal carcinoma cells to examine the relative importance of vascular versus tumor cell targeting in the ultimate treatment response. SW1222Res cells were generated through exposure of wild‐type cells (SW1222WT) to increasing CA4P concentrations in vitro. Increased resistance was confirmed through analyses of cell viability, apoptosis and multidrug‐resistance (MDR) protein expression. In vivo, comparative studies examined tumor cell necrosis, apoptosis, vessel morphology and functional vascular end‐points following treatment with CA4P (single 100 mg/kg dose). Tumor response to repeated CA4P dosing (50 mg/kg/day, 5 days/week for 2 weeks) was examined through growth measurement, and ultimate tumor cell survival was studied by ex vivo clonogenic assay. In vitro, SW1222Res cells showed reduced CA4P sensitivity, enhanced MDR protein expression and a reduced apoptotic index. In vivo, CA4P induced significantly lower apoptotic cell death in SW1222Res versus SW1222WT tumors indicating maintenance of resistance characteristics. However, CA4P‐induced tumor necrosis was equivalent in both lines. Similarly, rapid CA4P‐mediated vessel disruption and blood flow shut‐down were observed in both lines. Cell surviving fraction was comparable in the two tumor types following single dose CA4P and SW1222Res tumors were at least as sensitive as SW1222WT tumors to repeated dosing. Despite tumor cell resistance to CA4P, SW1222Res response in vivo was not impaired, strongly supporting the view that vascular damage dominates the therapeutic response to this agent.

show abstract

“…It had been reported that apoptotic pathways may be initiated [9][10][11][12], however, autophagy or necrosis [13] may prevail -especially if high PDT doses are applied [14].…”

Section: Introductionmentioning

confidence: 99%

“…Apparently, effects may not only include activation of caspase cascades [10,11] but also acute-phase response processes [15], and expression changes of heat-shock proteins [16], hypoxiamarkers [17], matrix metalloproteinases [18] or cytokines [19]. Since some of these cellular responses may modify, delay or even counteract cell death mechanisms, detailed analyses are highly needed.…”

Section: Introductionmentioning

confidence: 99%

Cellular and molecular effects of the liposomal mTHPC derivative Foslipos in prostate carcinoma cells in vitro

Gyenge

Hiestand

Graefe

et al. 2011

Photodiagnosis and Photodynamic Therapy

View full text Add to dashboard Cite

BACKGROUND: Meso-tetra-hydroxyphenyl-chlorine (mTHPC) is among the most powerful photosensitizers available for photodynamic therapy (PDT). However, the mechanisms leading to cell death are poorly understood. We here focused on changes at DNA and RNA levels after treatment with the liposomal mTHPC derivative Foslipos in vitro. METHODS: After determination of darktoxicity, laser conditions and uptake kinetics, PC-3 prostate carcinoma cells were subjected to PDT with Foslipos, followed by assessment of cell numbers directly (TP0) or 1h (TP1), 2h (TP2), 5h (TP5) and 24h (TP24) after illumination. Nucleic acids had been extracted for evaluation of RNA amounts and integrity as well as for estimation of abasic sites as a measure for DNA damage. Furthermore, expression changes of 84 genes related to oxidative stress were investigated by quantitative polymerase chain reaction. RESULTS: Already at TP0, the number of dead cells was significantly higher after PDT versus controls and at TP24 more than 90% of cells had been destroyed. PDT resulted in a severe damage of both RNA and DNA. Gene expression analyses revealed an impact of PDT on pathways for oxidative and metabolic stress, heat shock, proliferation and carcinogenesis, growth arrest, inflammation, DNA repair and apoptosis signaling. CONCLUSIONS: Mechanisms of Foslipos-mediated PDT comprise a combination of acute and delayed lethal effects in PC-3 cells. The latter may include death processes initiated by nucleic acid damage, activation of stress and growth arrest genes in combination with a reduced capability to adequately cope with oxidative toxicity. Our results will help to better understand molecular photodynamic effects. AbstractBackground: Meso-tetra-hydroxyphenyl-chlorine (mTHPC) is among the most powerful

show abstract

Assessment of Apoptosis by Immunohistochemistry to Active Caspase-3, Active Caspase-7, or Cleaved PARP in Monolayer Cells and Spheroid and Subcutaneous Xenografts of Human Carcinoma

Cited by 212 publications

References 33 publications

In vivo loss-of-function screens identify KPNB1 as a new druggable oncogene in epithelial ovarian cancer

In vivo loss-of-function screens identify KPNB1 as a new druggable oncogene in epithelial ovarian cancer

Vascular effects dominate solid tumor response to treatment with combretastatin A‐4‐phosphate

Cellular and molecular effects of the liposomal mTHPC derivative Foslipos in prostate carcinoma cells in vitro

Contact Info

Product

Resources

About