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Chloroquine promotes apoptosis in melanoma cells by inhibiting BH3 domain-mediated PUMA degradation.

Lakhter AJ, Sahu RP, Sun Y, Kaufmann WK, Androphy EJ, Travers JB, Naidu SR - J. Invest. Dermatol. (2013)

Bottom Line: The Bcl homology-3 (BH3)-only protein p53 upregulated modulator of apoptosis (PUMA) counters Bcl-2 family anti-apoptotic proteins and promotes apoptosis.Fusion of the BH3 domain to a heterologous protein led to its rapid turnover that was inhibited by CQ.Although both CQ and inhibitors of lysosomal proteases stalled autophagy, only CQ stabilized PUMA protein and promoted apoptosis.

View Article: PubMed Central - PubMed

Affiliation: Department of Dermatology, Indiana University School of Medicine, Indianapolis, IN, USA.

ABSTRACT
The Bcl homology-3 (BH3)-only protein p53 upregulated modulator of apoptosis (PUMA) counters Bcl-2 family anti-apoptotic proteins and promotes apoptosis. Although PUMA is a key regulator of apoptosis, the post-transcriptional mechanisms that control PUMA protein stability are not understood. We show that a lysosome-independent activity of chloroquine (CQ) prevents degradation of PUMA protein, promotes apoptosis, and reduces the growth of melanoma xenografts in mice. Compared with wild-type PUMA, a BH3 domain-deleted PUMA protein showed impaired decay in melanoma cells. Fusion of the BH3 domain to a heterologous protein led to its rapid turnover that was inhibited by CQ. Although both CQ and inhibitors of lysosomal proteases stalled autophagy, only CQ stabilized PUMA protein and promoted apoptosis. Our results reveal a lysosomal protease-independent activity of CQ that selectively promotes apoptosis in melanoma cells.

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Chloroquine reduces growth of human melanoma cells in micea) NOD-SCID mice were implanted with SK-MEL23 cells, 21 days post-implantation, mice were divided in to two groups for vehicle and CQ treatment. Tumor volumes of vehicle or CQ treated mice were measured at indicated days and the growth kinetics of tumor volume shown on the graph (p = 0.0001). b) Dissected tumors from vehicle and CQ treated mice were weighed (p=<0.05).
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Figure 2: Chloroquine reduces growth of human melanoma cells in micea) NOD-SCID mice were implanted with SK-MEL23 cells, 21 days post-implantation, mice were divided in to two groups for vehicle and CQ treatment. Tumor volumes of vehicle or CQ treated mice were measured at indicated days and the growth kinetics of tumor volume shown on the graph (p = 0.0001). b) Dissected tumors from vehicle and CQ treated mice were weighed (p=<0.05).

Mentions: To test the effects of CQ in an in vivo model, we implanted SK-MEL23 melanoma cells in NOD-SCID mice and evaluated the effects of CQ on subcutaneous tumor growth. Melanoma cells were allowed to establish an average volume of 100 mm3 at which point the mice were divided into two groups. A set of four mice served as vehicle control and six mice were treated with a low dose of CQ (25 mg/kg body weight) on an every three-day treatment schedule for 21 days. Tumor volume measurements showed that chloroquine treatment reduced the tumor volume in the experimental group (p = 0.0001) compared to the vehicle controls (Fig 2a). There were no differences in total body weight between the treatment and control groups. At the end of the study, xenografts were excised and gross weight values of tumors were recorded (Fig 2b). Consistent with tumor volume measurements, we found a statistically significant (p=<0.05) reduction in tumor mass from this low dose of CQ-treated animals as compared to the vehicle group.


Chloroquine promotes apoptosis in melanoma cells by inhibiting BH3 domain-mediated PUMA degradation.

Lakhter AJ, Sahu RP, Sun Y, Kaufmann WK, Androphy EJ, Travers JB, Naidu SR - J. Invest. Dermatol. (2013)

Chloroquine reduces growth of human melanoma cells in micea) NOD-SCID mice were implanted with SK-MEL23 cells, 21 days post-implantation, mice were divided in to two groups for vehicle and CQ treatment. Tumor volumes of vehicle or CQ treated mice were measured at indicated days and the growth kinetics of tumor volume shown on the graph (p = 0.0001). b) Dissected tumors from vehicle and CQ treated mice were weighed (p=<0.05).
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3675185&req=5

Figure 2: Chloroquine reduces growth of human melanoma cells in micea) NOD-SCID mice were implanted with SK-MEL23 cells, 21 days post-implantation, mice were divided in to two groups for vehicle and CQ treatment. Tumor volumes of vehicle or CQ treated mice were measured at indicated days and the growth kinetics of tumor volume shown on the graph (p = 0.0001). b) Dissected tumors from vehicle and CQ treated mice were weighed (p=<0.05).
Mentions: To test the effects of CQ in an in vivo model, we implanted SK-MEL23 melanoma cells in NOD-SCID mice and evaluated the effects of CQ on subcutaneous tumor growth. Melanoma cells were allowed to establish an average volume of 100 mm3 at which point the mice were divided into two groups. A set of four mice served as vehicle control and six mice were treated with a low dose of CQ (25 mg/kg body weight) on an every three-day treatment schedule for 21 days. Tumor volume measurements showed that chloroquine treatment reduced the tumor volume in the experimental group (p = 0.0001) compared to the vehicle controls (Fig 2a). There were no differences in total body weight between the treatment and control groups. At the end of the study, xenografts were excised and gross weight values of tumors were recorded (Fig 2b). Consistent with tumor volume measurements, we found a statistically significant (p=<0.05) reduction in tumor mass from this low dose of CQ-treated animals as compared to the vehicle group.

Bottom Line: The Bcl homology-3 (BH3)-only protein p53 upregulated modulator of apoptosis (PUMA) counters Bcl-2 family anti-apoptotic proteins and promotes apoptosis.Fusion of the BH3 domain to a heterologous protein led to its rapid turnover that was inhibited by CQ.Although both CQ and inhibitors of lysosomal proteases stalled autophagy, only CQ stabilized PUMA protein and promoted apoptosis.

View Article: PubMed Central - PubMed

Affiliation: Department of Dermatology, Indiana University School of Medicine, Indianapolis, IN, USA.

ABSTRACT
The Bcl homology-3 (BH3)-only protein p53 upregulated modulator of apoptosis (PUMA) counters Bcl-2 family anti-apoptotic proteins and promotes apoptosis. Although PUMA is a key regulator of apoptosis, the post-transcriptional mechanisms that control PUMA protein stability are not understood. We show that a lysosome-independent activity of chloroquine (CQ) prevents degradation of PUMA protein, promotes apoptosis, and reduces the growth of melanoma xenografts in mice. Compared with wild-type PUMA, a BH3 domain-deleted PUMA protein showed impaired decay in melanoma cells. Fusion of the BH3 domain to a heterologous protein led to its rapid turnover that was inhibited by CQ. Although both CQ and inhibitors of lysosomal proteases stalled autophagy, only CQ stabilized PUMA protein and promoted apoptosis. Our results reveal a lysosomal protease-independent activity of CQ that selectively promotes apoptosis in melanoma cells.

Show MeSH
Related in: MedlinePlus