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Harnessing the lysosome-dependent antitumor activity of phenothiazines in human small cell lung cancer.

Zong D, Zielinska-Chomej K, Juntti T, Mörk B, Lewensohn R, Hååg P, Viktorsson K - Cell Death Dis (2014)

Bottom Line: We show that phenothiazines as single treatment decreased cell viability and induced cell death preferentially in small cell lung carcinoma (SCLC) over non small cell lung carcinoma (NSCLC) cell lines.Importantly, this effect in SCLC occurred despite mutation in p53 and was not influenced by intrinsic sensitivity/resistance toward conventional chemotherapeutic agents.Our data thus uncovered a novel context-dependent activity of phenothiazines in SCLC and suggest that phenothiazines could be considered as a treatment regimen of this disease, however, extended cell line analyses as well as in vivo studies are needed to make such conclusion.

View Article: PubMed Central - PubMed

Affiliation: Department of Oncology-Pathology, Karolinska Biomics Center, Karolinska Institutet, Stockholm, Sweden.

ABSTRACT
Phenothiazines are a family of heterocyclic compounds whose clinical utility includes treatment of psychiatric disorders as well as chemotherapy-induced emesis. Various studies have demonstrated that these compounds possess cytotoxic activities in tumor cell lines of different origin. However, there is considerable confusion regarding the molecular basis of phenothiazine-induced cell death. Lung cancer (LC) remains one of the most prevalent and deadly malignancies worldwide despite considerable efforts in the development of treatment strategies, especially new targeted therapies. In this work, we evaluated the potential utility of phenothiazines in human LC. We show that phenothiazines as single treatment decreased cell viability and induced cell death preferentially in small cell lung carcinoma (SCLC) over non small cell lung carcinoma (NSCLC) cell lines. Sensitivity to phenothiazines was not correlated with induction of apoptosis but due to phenothiazine-induced lysosomal dysfunction. Interestingly, the higher susceptibility of SCLC cells to phenothiazine-induced cell death correlated with an intrinsically lower buffer capacity in response to disruption of lysosomal homeostasis. Importantly, this effect in SCLC occurred despite mutation in p53 and was not influenced by intrinsic sensitivity/resistance toward conventional chemotherapeutic agents. Our data thus uncovered a novel context-dependent activity of phenothiazines in SCLC and suggest that phenothiazines could be considered as a treatment regimen of this disease, however, extended cell line analyses as well as in vivo studies are needed to make such conclusion.

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Related in: MedlinePlus

TFP induces cell death distinct from classical apoptosis. (a) SCLC (H82, H69) and NSCLC (U-1810, A549) cells were treated with TFP at the indicated concentrations for 6, 24 or 48 h; WCL was used for immunoblotting with an antibody recognizing full-length and cleaved PARP (113 and 89 kDa respectively). GAPDH antibody was used to confirm equal loading. (b) H592, U-1285, H125, H157 cells were treated with TFP at the indicated concentrations for 72 h; immunoblotting was performed as in (a). (c) H82 and U-1810 cells were pre-treated (1 h) with z-VAD-fmk (20 μM) and thereafter exposed to TFP at the indicated concentrations for 72 h; cell viability was measured by MTT. For (a and b), data shown are representative of three independent experiments. For (c), data depict mean±S.D. compiled from three independent experiments. NS, nonsignificant
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fig3: TFP induces cell death distinct from classical apoptosis. (a) SCLC (H82, H69) and NSCLC (U-1810, A549) cells were treated with TFP at the indicated concentrations for 6, 24 or 48 h; WCL was used for immunoblotting with an antibody recognizing full-length and cleaved PARP (113 and 89 kDa respectively). GAPDH antibody was used to confirm equal loading. (b) H592, U-1285, H125, H157 cells were treated with TFP at the indicated concentrations for 72 h; immunoblotting was performed as in (a). (c) H82 and U-1810 cells were pre-treated (1 h) with z-VAD-fmk (20 μM) and thereafter exposed to TFP at the indicated concentrations for 72 h; cell viability was measured by MTT. For (a and b), data shown are representative of three independent experiments. For (c), data depict mean±S.D. compiled from three independent experiments. NS, nonsignificant

Mentions: To gain insight into the mechanistic basis of phenothiazine-associated cytotoxicity, we analyzed in detail the mode by which these compounds induce cell death in human SCLC cells. While some studies have implicated apoptosis as a major cell death mode in phenothiazine-treated cells,3, 4, 9 we found that the percentage of SCLC exhibiting nuclear morphologic changes typical of apoptosis, such as chromatin condensation and fragmentation, remained low (10%) even at the highest TFP concentrations, where >98% of all cells lost the ability to exclude propidium iodide (PI; data not shown). Instead, TFP-treated SCLC cells exhibited profoundly shrunken nuclei without concomitant chromatin condensation. TFP elicited poly (ADP-ribose) polymerase (PARP) cleavage in some, but not all, LC cell lines (Figures 3a and b). Importantly, although the SCLC cells are sensitive to 10 μM TFP treatment and respond with an approximately 60% decrease in cell viability, this concentration of TFP only resulted in no or minor cleavage of PARP. Consistent with a non-essential requirement of caspase-mediated apoptotic response, the pan-caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp fluoromethylketone (z-VAD-fmk) did not significantly protect either SCLC (H82) or NSCLC (U-1810) cells from TFP-induced cell death (Figure 3c), emphasizing the involvement of non-caspase-mediated cell death in response to phenothiazines in SCLC.


Harnessing the lysosome-dependent antitumor activity of phenothiazines in human small cell lung cancer.

Zong D, Zielinska-Chomej K, Juntti T, Mörk B, Lewensohn R, Hååg P, Viktorsson K - Cell Death Dis (2014)

TFP induces cell death distinct from classical apoptosis. (a) SCLC (H82, H69) and NSCLC (U-1810, A549) cells were treated with TFP at the indicated concentrations for 6, 24 or 48 h; WCL was used for immunoblotting with an antibody recognizing full-length and cleaved PARP (113 and 89 kDa respectively). GAPDH antibody was used to confirm equal loading. (b) H592, U-1285, H125, H157 cells were treated with TFP at the indicated concentrations for 72 h; immunoblotting was performed as in (a). (c) H82 and U-1810 cells were pre-treated (1 h) with z-VAD-fmk (20 μM) and thereafter exposed to TFP at the indicated concentrations for 72 h; cell viability was measured by MTT. For (a and b), data shown are representative of three independent experiments. For (c), data depict mean±S.D. compiled from three independent experiments. NS, nonsignificant
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3973193&req=5

fig3: TFP induces cell death distinct from classical apoptosis. (a) SCLC (H82, H69) and NSCLC (U-1810, A549) cells were treated with TFP at the indicated concentrations for 6, 24 or 48 h; WCL was used for immunoblotting with an antibody recognizing full-length and cleaved PARP (113 and 89 kDa respectively). GAPDH antibody was used to confirm equal loading. (b) H592, U-1285, H125, H157 cells were treated with TFP at the indicated concentrations for 72 h; immunoblotting was performed as in (a). (c) H82 and U-1810 cells were pre-treated (1 h) with z-VAD-fmk (20 μM) and thereafter exposed to TFP at the indicated concentrations for 72 h; cell viability was measured by MTT. For (a and b), data shown are representative of three independent experiments. For (c), data depict mean±S.D. compiled from three independent experiments. NS, nonsignificant
Mentions: To gain insight into the mechanistic basis of phenothiazine-associated cytotoxicity, we analyzed in detail the mode by which these compounds induce cell death in human SCLC cells. While some studies have implicated apoptosis as a major cell death mode in phenothiazine-treated cells,3, 4, 9 we found that the percentage of SCLC exhibiting nuclear morphologic changes typical of apoptosis, such as chromatin condensation and fragmentation, remained low (10%) even at the highest TFP concentrations, where >98% of all cells lost the ability to exclude propidium iodide (PI; data not shown). Instead, TFP-treated SCLC cells exhibited profoundly shrunken nuclei without concomitant chromatin condensation. TFP elicited poly (ADP-ribose) polymerase (PARP) cleavage in some, but not all, LC cell lines (Figures 3a and b). Importantly, although the SCLC cells are sensitive to 10 μM TFP treatment and respond with an approximately 60% decrease in cell viability, this concentration of TFP only resulted in no or minor cleavage of PARP. Consistent with a non-essential requirement of caspase-mediated apoptotic response, the pan-caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp fluoromethylketone (z-VAD-fmk) did not significantly protect either SCLC (H82) or NSCLC (U-1810) cells from TFP-induced cell death (Figure 3c), emphasizing the involvement of non-caspase-mediated cell death in response to phenothiazines in SCLC.

Bottom Line: We show that phenothiazines as single treatment decreased cell viability and induced cell death preferentially in small cell lung carcinoma (SCLC) over non small cell lung carcinoma (NSCLC) cell lines.Importantly, this effect in SCLC occurred despite mutation in p53 and was not influenced by intrinsic sensitivity/resistance toward conventional chemotherapeutic agents.Our data thus uncovered a novel context-dependent activity of phenothiazines in SCLC and suggest that phenothiazines could be considered as a treatment regimen of this disease, however, extended cell line analyses as well as in vivo studies are needed to make such conclusion.

View Article: PubMed Central - PubMed

Affiliation: Department of Oncology-Pathology, Karolinska Biomics Center, Karolinska Institutet, Stockholm, Sweden.

ABSTRACT
Phenothiazines are a family of heterocyclic compounds whose clinical utility includes treatment of psychiatric disorders as well as chemotherapy-induced emesis. Various studies have demonstrated that these compounds possess cytotoxic activities in tumor cell lines of different origin. However, there is considerable confusion regarding the molecular basis of phenothiazine-induced cell death. Lung cancer (LC) remains one of the most prevalent and deadly malignancies worldwide despite considerable efforts in the development of treatment strategies, especially new targeted therapies. In this work, we evaluated the potential utility of phenothiazines in human LC. We show that phenothiazines as single treatment decreased cell viability and induced cell death preferentially in small cell lung carcinoma (SCLC) over non small cell lung carcinoma (NSCLC) cell lines. Sensitivity to phenothiazines was not correlated with induction of apoptosis but due to phenothiazine-induced lysosomal dysfunction. Interestingly, the higher susceptibility of SCLC cells to phenothiazine-induced cell death correlated with an intrinsically lower buffer capacity in response to disruption of lysosomal homeostasis. Importantly, this effect in SCLC occurred despite mutation in p53 and was not influenced by intrinsic sensitivity/resistance toward conventional chemotherapeutic agents. Our data thus uncovered a novel context-dependent activity of phenothiazines in SCLC and suggest that phenothiazines could be considered as a treatment regimen of this disease, however, extended cell line analyses as well as in vivo studies are needed to make such conclusion.

Show MeSH
Related in: MedlinePlus