Identification of heat shock protein 90 inhibitors to sensitize drug resistant side population tumor cells using a cell based assay platform

Sobhan, Praveen K.; Seervi, Mahendra; Joseph, Jeena; Chandrika, Bhavya Balan; Varghese, Saneesh; Santhoshkumar, T.R.; Pillai, M. Radhakrishna

doi:10.1016/j.canlet.2011.11.009

Cited by 13 publications

(4 citation statements)

References 35 publications

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“…The intrinsic apoptosis signaling is initiated with limited permeabilisation of mitochondria leading to the release of cytochrome c in to cytosol that triggers caspase dependent cell death through activation of procaspase 9 in the apoptosome complex. In order to analyze the role of mitochondria and cytochrome c dependency, we have used SiHa and MCF-7 cells expressing cytochrome c-EGFP fusion protein as described earlier [13]. As shown in figure 3 A and B, after 12 h of treatment with zerumbone (50 µM), cytochrome c release was initiated in few cells with a time dependent increase ( Figure 3 B ).…”

Section: Resultsmentioning

confidence: 99%

Calpain and Reactive Oxygen Species Targets Bax for Mitochondrial Permeabilisation and Caspase Activation in Zerumbone Induced Apoptosis

et al. 2013

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Fluorescent protein based signaling probes are emerging as valuable tools to study cell signaling because of their ability to provide spatio- temporal information in non invasive live cell mode. Previously, multiple fluorescent protein probes were employed to characterize key events of apoptosis in diverse experimental systems. We have employed a live cell image based approach to visualize the key events of apoptosis signaling induced by zerumbone, the active principle from ginger Zingiber zerumbet, in cancer cells that enabled us to analyze prominent apoptotic changes in a hierarchical manner with temporal resolution. Our studies substantiate that mitochondrial permeabilisation and cytochrome c dependent caspase activation dominate in zerumbone induced cell death. Bax activation, the essential and early event of cell death, is independently activated by reactive oxygen species as well as calpains. Zerumbone failed to induce apoptosis or mitochondrial permeabilisation in Bax knockout cells and over-expression of Bax enhanced cell death induced by zerumbone confirming the essential role of Bax for mitochondrial permeabilsation. Simultaneous inhibition of reactive oxygen species and calpain is required for preventing Bax activation and cell death. However, apoptosis induced by zerumbone was prevented in Bcl 2 and Bcl-XL over-expressing cells, whereas more protection was afforded by Bcl 2 specifically targeted to endoplasmic reticulum. Even though zerumbone treatment down-regulated survival proteins such as XIAP, Survivin and Akt, it failed to affect the pro-apoptotic proteins such as PUMA and BIM. Multiple normal diploid cell lines were employed to address cytotoxic activity of zerumbone and, in general, mammary epithelial cells, endothelial progenitor cells and smooth muscle cells were relatively resistant to zerumbone induced cell death with lesser ROS accumulation than cancer cells.

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Section: Resultsmentioning

confidence: 99%

Calpain and Reactive Oxygen Species Targets Bax for Mitochondrial Permeabilisation and Caspase Activation in Zerumbone Induced Apoptosis

et al. 2013

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“…In this case, the mechanism for CSC renewal involved another pathway, one involving the factor NFkB, a known factor in stemness[83]. Inhibition of Hsp90 also led to a reduction in stemness in a number of tumor types [84, 85] (Figure 4). Hsp90 is required for activity of the CSC renewal pathway involving the JAK-STAT system[86, 87].…”

Section: Hsf1 and Hsps In Cancer Stem Cells And Tumor Initiationmentioning

confidence: 99%

“…HSPs are important promoters of the CSC phenotype and such cells are highly invasive, support tumor repopulation, are profoundly metastatic and are resistant to treatment[36, 85]. Tumor hypoxia may play a part in the evolution of new cancer cell properties through its effects on Hsp90 activities in cells remote from the circulation.…”

Section: Hsf1 and Hsps In Cancer Stem Cells And Tumor Initiationmentioning

confidence: 99%

Heat Shock Proteins Promote Cancer: It's a Protection Racket

Calderwood

Gong

2016

Trends in Biochemical Sciences

320

251

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Heat Shock Proteins (HSP) are expressed at high levels in cancer and form a fostering environment essential for tumor development. We have reviewed the recent data in this area, concentrating mainly on Hsp27, Hsp70 and Hsp90. The overriding role of the HSPs in cancer is to stabilize the active functions of overexpressed and mutated cancer gene. Elevated HSPs are thus required for many of the traits that underlie the morbidity of cancer, including increased growth, survival and formation of secondary cancers. In addition, HSPs participate in the evolution of cancer treatment resistance. HSPs are also released from cancer cells and influence malignant properties by receptor- mediated signaling. Current data strongly support efforts to target HSPs in cancer treatment.

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“…Cells stably expressing cytochrome c -EGFP have been used as a marker for mitochondrial permeabilization and apoptosis detection in image-based drug screening platforms [31] , [32] . To study the correlation of cytochrome c release and mitochondrial ROS, cytochrome c -EGFP stable cells stained with mitochondrial ROS specific dye MitoSOX red was employed.…”

Section: Resultsmentioning

confidence: 99%

A high-throughput real-time in vitro assay using mitochondrial targeted roGFP for screening of drugs targeting mitochondria

et al. 2019

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Most toxic compounds including cancer drugs target mitochondria culminating in its permeabilization. Cancer drug-screening and toxicological testing of compounds require cost-effective and sensitive high-throughput methods to detect mitochondrial damage. Real-time methods for detection of mitochondrial damage are less toxic, allow kinetic measurements with good spatial resolution and are preferred over end-stage assays.Cancer cell lines stably expressing genetically encoded mitochondrial-targeted redox-GFP2 (mt-roGFP) were developed and validated for its suitability as a mitochondrial damage sensor. Diverse imaging platforms and flow-cytometry were utilized for ratiometric analysis of redox changes with known toxic and cancer drugs. Key events of cell death and mitochondrial damage were studied at single-cell level coupled with mt-roGFP. Cells stably expressing mt-roGFP and H2B-mCherry were developed for high-throughput screening (HTS) application.Most cancer drugs while inducing mitochondrial permeabilization trigger mitochondrial-oxidation that can be detected at single-cell level with mt-roGFP. The image-based assay using mt-roGFP outperformed other quantitative methods of apoptosis in ease of screening. Incorporation of H2B-mCherry ensures accurate and complete automated segmentation with excellent Z value. The results substantiate that most cancer drugs and known plant-derived antioxidants trigger cell-death through mitochondrial redox alterations with pronounced ratio change in the mt-roGFP probe.Real-time analysis of mitochondrial oxidation and mitochondrial permeabilization reveal a biphasic ratio change in dying cells, with an initial redox surge before mitochondrial permeabilization followed by a drastic increase in ratio after complete mitochondrial permeabilization. Overall, the results prove that mitochondrial oxidation is a reliable indicator of mitochondrial damage, which can be readily determined in live cells using mt-roGFP employing diverse imaging techniques. The assay described is highly sensitive, easy to adapt to HTS platforms and is a valuable resource for identifying cytotoxic agents that target mitochondria and also for dissecting cell signaling events relevant to redox biology.

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Identification of heat shock protein 90 inhibitors to sensitize drug resistant side population tumor cells using a cell based assay platform

Cited by 13 publications

References 35 publications

Calpain and Reactive Oxygen Species Targets Bax for Mitochondrial Permeabilisation and Caspase Activation in Zerumbone Induced Apoptosis

Calpain and Reactive Oxygen Species Targets Bax for Mitochondrial Permeabilisation and Caspase Activation in Zerumbone Induced Apoptosis

Heat Shock Proteins Promote Cancer: It's a Protection Racket

A high-throughput real-time in vitro assay using mitochondrial targeted roGFP for screening of drugs targeting mitochondria

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