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Differential activity of caspase-3 regulates susceptibility of lung and breast tumor cell lines to Paclitaxel.

Odonkor CA, Achilefu S - Open Biochem J (2008)

Bottom Line: Previous studies suggest that cleavage of caspase-3 is not instrumental for the execution of death in tumors treated with paclitaxel, while other reports indicate that caspase-dependent pathways may be critical for paclitaxel cytotoxicity.In this study, we investigated the role of caspase-3 in breast and lung tumor cell line sensitivity to paclitaxel.Clonogenic survival and live/dead viability-assays, together with enzymatic activity and cell proliferation assays, reveal that the levels of paclitaxel-induced caspase-3 enzymatic activity in tumor cells correlate directly with tumor sensitivity to the drug.We observed a 2-fold increase in caspase-3 activity in 4T1-Luc breast tumor cells, but a 3-fold and 4-fold decrease in A549 and A427 lung tumor cell lines, respectively.

View Article: PubMed Central - PubMed

Affiliation: Department of Radiology, Washington University School of Medicine, 4525 Scott Avenue, St. Louis, MO 63110, USA.

ABSTRACT
Recent development of tumor resistance to paclitaxel presents a major problem to cancer treatment. An unsettled controversy in the cancer chemotherapy field, however, is whether caspases play a prominent role in paclitaxel-induced death in tumors. Previous studies suggest that cleavage of caspase-3 is not instrumental for the execution of death in tumors treated with paclitaxel, while other reports indicate that caspase-dependent pathways may be critical for paclitaxel cytotoxicity. In this study, we investigated the role of caspase-3 in breast and lung tumor cell line sensitivity to paclitaxel. Clonogenic survival and live/dead viability-assays, together with enzymatic activity and cell proliferation assays, reveal that the levels of paclitaxel-induced caspase-3 enzymatic activity in tumor cells correlate directly with tumor sensitivity to the drug.We observed a 2-fold increase in caspase-3 activity in 4T1-Luc breast tumor cells, but a 3-fold and 4-fold decrease in A549 and A427 lung tumor cell lines, respectively. Together, our results suggest that caspase-activation and activity levels are not only key determinants of paclitaxel-induced death in tumors but also serve as good indicators for tumor susceptibility to paclitaxel therapy. Our studies also indicate that within clinically relevant doses of paclitaxel, the ability to rid tumor populations of dormant tumor cells controls the rate of tumor recurrence.

No MeSH data available.


Related in: MedlinePlus

The extent of DNA damage observed in tumor cells was dose dependent. Arrows point to fragmented nuclei. Inserts highlight nuclear fragments and damaged DNA within cells. Scale bar indicates 90 µm. Cell morbidity was determined by Live-Dead Viability Assay. Drug treated cells were incubated with ethidium homodimer (EthD-1), which stains fragmented nuclei of dead cells. EthD-1 dye can only penetrate cells with compromised membranes and emits red fluorescence (ex/em - 495 nm/ 635 nm) on binding to nucleic acids. Exclusion of the dye by intact membrane of live cells distinguishes dead cells from live ones. (A – F) - Apoptosis in 4T1-Luc cells. Cells treated with high paclitaxel doses, 50 µM (A) and 25 µM (B), show extensive nuclear fragmentation and damaged membranes. (C) At 12.5 µM and (D) 6.25 µM, moderate nuclear fragmentation is observed. (E) Reflects background fluorescence from negative control cells. (F) Apoptotic induction in positive control (ethanol-treated) cells. Wavy black line traces boundary of damaged cell membrane.
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Figure 2: The extent of DNA damage observed in tumor cells was dose dependent. Arrows point to fragmented nuclei. Inserts highlight nuclear fragments and damaged DNA within cells. Scale bar indicates 90 µm. Cell morbidity was determined by Live-Dead Viability Assay. Drug treated cells were incubated with ethidium homodimer (EthD-1), which stains fragmented nuclei of dead cells. EthD-1 dye can only penetrate cells with compromised membranes and emits red fluorescence (ex/em - 495 nm/ 635 nm) on binding to nucleic acids. Exclusion of the dye by intact membrane of live cells distinguishes dead cells from live ones. (A – F) - Apoptosis in 4T1-Luc cells. Cells treated with high paclitaxel doses, 50 µM (A) and 25 µM (B), show extensive nuclear fragmentation and damaged membranes. (C) At 12.5 µM and (D) 6.25 µM, moderate nuclear fragmentation is observed. (E) Reflects background fluorescence from negative control cells. (F) Apoptotic induction in positive control (ethanol-treated) cells. Wavy black line traces boundary of damaged cell membrane.

Mentions: Although it is fairly well known that most tumor cells undergo paclitaxel-induced apoptotic death, it is equally well established that paclitaxel induces other non-apoptotic programmed cell death mechanisms in different tumor cell types [20,21,25]. To ascertain and distinguish between apoptotic and non-apoptotic death, we used a Live/Dead® Viability assay to evaluate the quintessential cellular features of death such as plasma membrane blebbing, DNA fragmentation, and cell shrinking. We particularly examined drug-treated cells for the presence of fragmented nuclei and compromised plasma membranes as indicators of death. We observed a dose-dependent fragmentation of nuclear DNA as well as damage to the cell membrane. This phenotype was highly pronounced in 4T1-luc tumor cells with extensive fragmentation at 50 μM and 25 μM of drug, visualized as red punctate dots in dead cells stained by EthD-1 (Fig. 2, A-F). Very few live cells were observed at these concentrations and cells had a mix of necrotic and apoptotic characteristics. Nonetheless, the extent of membrane disintegration and nuclear damage in 4T1-luc cells was more moderate at 12.5 μM and 6.25 μM of paclitaxel (Fig. 2C & D). Like 4T1-luc cells, A549 cells showed dose-dependent DNA fragmentation, but with less damaged cell membranes (Fig. 3, A-F). Moreover, dead cells had fewer and less dispersed punctate nuclear fragments (Fig. 3A, insert). Interestingly, we observed a few mitotic cells among the apoptotic A549 cell population, which were immobilized at the mitotic phase in the presence of paclitaxel. (Fig. 3A, D & F). The absence of this distinct population of cells in 4T1-luc cells clearly highlights different death response mechanisms in the two tumor cell lines. Previous studies reported that paclitaxel suppressed microtubule dynamics and disrupted the functioning of the mitotic spindle [26-29], suggesting a slow cell cycle progression at the metaphase/anaphase checkpoint that eventually induces cell death. The features in A549-cells fit perfectly with this pattern, albeit, we also observed a mix of apoptotic morphologies in this cell line. Our results demonstrate that 4T1-luc breast tumor cells respond to paclitaxel by initiating extensive nuclear fragmentation and inducing both necrotic and apoptotic forms of death. On the other hand, paclitaxel-induced death in A549 cells occurs partly through interference with cell cycle progression, thus, pointing to different phases of drug-induced apoptosis in the two tumor cell lines.


Differential activity of caspase-3 regulates susceptibility of lung and breast tumor cell lines to Paclitaxel.

Odonkor CA, Achilefu S - Open Biochem J (2008)

The extent of DNA damage observed in tumor cells was dose dependent. Arrows point to fragmented nuclei. Inserts highlight nuclear fragments and damaged DNA within cells. Scale bar indicates 90 µm. Cell morbidity was determined by Live-Dead Viability Assay. Drug treated cells were incubated with ethidium homodimer (EthD-1), which stains fragmented nuclei of dead cells. EthD-1 dye can only penetrate cells with compromised membranes and emits red fluorescence (ex/em - 495 nm/ 635 nm) on binding to nucleic acids. Exclusion of the dye by intact membrane of live cells distinguishes dead cells from live ones. (A – F) - Apoptosis in 4T1-Luc cells. Cells treated with high paclitaxel doses, 50 µM (A) and 25 µM (B), show extensive nuclear fragmentation and damaged membranes. (C) At 12.5 µM and (D) 6.25 µM, moderate nuclear fragmentation is observed. (E) Reflects background fluorescence from negative control cells. (F) Apoptotic induction in positive control (ethanol-treated) cells. Wavy black line traces boundary of damaged cell membrane.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC2627519&req=5

Figure 2: The extent of DNA damage observed in tumor cells was dose dependent. Arrows point to fragmented nuclei. Inserts highlight nuclear fragments and damaged DNA within cells. Scale bar indicates 90 µm. Cell morbidity was determined by Live-Dead Viability Assay. Drug treated cells were incubated with ethidium homodimer (EthD-1), which stains fragmented nuclei of dead cells. EthD-1 dye can only penetrate cells with compromised membranes and emits red fluorescence (ex/em - 495 nm/ 635 nm) on binding to nucleic acids. Exclusion of the dye by intact membrane of live cells distinguishes dead cells from live ones. (A – F) - Apoptosis in 4T1-Luc cells. Cells treated with high paclitaxel doses, 50 µM (A) and 25 µM (B), show extensive nuclear fragmentation and damaged membranes. (C) At 12.5 µM and (D) 6.25 µM, moderate nuclear fragmentation is observed. (E) Reflects background fluorescence from negative control cells. (F) Apoptotic induction in positive control (ethanol-treated) cells. Wavy black line traces boundary of damaged cell membrane.
Mentions: Although it is fairly well known that most tumor cells undergo paclitaxel-induced apoptotic death, it is equally well established that paclitaxel induces other non-apoptotic programmed cell death mechanisms in different tumor cell types [20,21,25]. To ascertain and distinguish between apoptotic and non-apoptotic death, we used a Live/Dead® Viability assay to evaluate the quintessential cellular features of death such as plasma membrane blebbing, DNA fragmentation, and cell shrinking. We particularly examined drug-treated cells for the presence of fragmented nuclei and compromised plasma membranes as indicators of death. We observed a dose-dependent fragmentation of nuclear DNA as well as damage to the cell membrane. This phenotype was highly pronounced in 4T1-luc tumor cells with extensive fragmentation at 50 μM and 25 μM of drug, visualized as red punctate dots in dead cells stained by EthD-1 (Fig. 2, A-F). Very few live cells were observed at these concentrations and cells had a mix of necrotic and apoptotic characteristics. Nonetheless, the extent of membrane disintegration and nuclear damage in 4T1-luc cells was more moderate at 12.5 μM and 6.25 μM of paclitaxel (Fig. 2C & D). Like 4T1-luc cells, A549 cells showed dose-dependent DNA fragmentation, but with less damaged cell membranes (Fig. 3, A-F). Moreover, dead cells had fewer and less dispersed punctate nuclear fragments (Fig. 3A, insert). Interestingly, we observed a few mitotic cells among the apoptotic A549 cell population, which were immobilized at the mitotic phase in the presence of paclitaxel. (Fig. 3A, D & F). The absence of this distinct population of cells in 4T1-luc cells clearly highlights different death response mechanisms in the two tumor cell lines. Previous studies reported that paclitaxel suppressed microtubule dynamics and disrupted the functioning of the mitotic spindle [26-29], suggesting a slow cell cycle progression at the metaphase/anaphase checkpoint that eventually induces cell death. The features in A549-cells fit perfectly with this pattern, albeit, we also observed a mix of apoptotic morphologies in this cell line. Our results demonstrate that 4T1-luc breast tumor cells respond to paclitaxel by initiating extensive nuclear fragmentation and inducing both necrotic and apoptotic forms of death. On the other hand, paclitaxel-induced death in A549 cells occurs partly through interference with cell cycle progression, thus, pointing to different phases of drug-induced apoptosis in the two tumor cell lines.

Bottom Line: Previous studies suggest that cleavage of caspase-3 is not instrumental for the execution of death in tumors treated with paclitaxel, while other reports indicate that caspase-dependent pathways may be critical for paclitaxel cytotoxicity.In this study, we investigated the role of caspase-3 in breast and lung tumor cell line sensitivity to paclitaxel.Clonogenic survival and live/dead viability-assays, together with enzymatic activity and cell proliferation assays, reveal that the levels of paclitaxel-induced caspase-3 enzymatic activity in tumor cells correlate directly with tumor sensitivity to the drug.We observed a 2-fold increase in caspase-3 activity in 4T1-Luc breast tumor cells, but a 3-fold and 4-fold decrease in A549 and A427 lung tumor cell lines, respectively.

View Article: PubMed Central - PubMed

Affiliation: Department of Radiology, Washington University School of Medicine, 4525 Scott Avenue, St. Louis, MO 63110, USA.

ABSTRACT
Recent development of tumor resistance to paclitaxel presents a major problem to cancer treatment. An unsettled controversy in the cancer chemotherapy field, however, is whether caspases play a prominent role in paclitaxel-induced death in tumors. Previous studies suggest that cleavage of caspase-3 is not instrumental for the execution of death in tumors treated with paclitaxel, while other reports indicate that caspase-dependent pathways may be critical for paclitaxel cytotoxicity. In this study, we investigated the role of caspase-3 in breast and lung tumor cell line sensitivity to paclitaxel. Clonogenic survival and live/dead viability-assays, together with enzymatic activity and cell proliferation assays, reveal that the levels of paclitaxel-induced caspase-3 enzymatic activity in tumor cells correlate directly with tumor sensitivity to the drug.We observed a 2-fold increase in caspase-3 activity in 4T1-Luc breast tumor cells, but a 3-fold and 4-fold decrease in A549 and A427 lung tumor cell lines, respectively. Together, our results suggest that caspase-activation and activity levels are not only key determinants of paclitaxel-induced death in tumors but also serve as good indicators for tumor susceptibility to paclitaxel therapy. Our studies also indicate that within clinically relevant doses of paclitaxel, the ability to rid tumor populations of dormant tumor cells controls the rate of tumor recurrence.

No MeSH data available.


Related in: MedlinePlus