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Araguspongine C induces autophagic death in breast cancer cells through suppression of c-Met and HER2 receptor tyrosine kinase signaling.

Akl MR, Ayoub NM, Ebrahim HY, Mohyeldin MM, Orabi KY, Foudah AI, El Sayed KA - Mar Drugs (2015)

Bottom Line: Araguspongine C-induced autophagy was associated with suppression of c-Met and HER2 receptor tyrosine kinase activation.Induction of autophagic death in BT-474 cells was also associated with decreased levels of inositol 1,4,5-trisphosphate receptor upon treatment with effective concentration of araguspongine C.In conclusion, results of this study are the first to reveal the potential of araguspongine C as an inhibitor to receptor tyrosine kinases resulting in the induction of autophagic cell death in breast cancer cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Basic Pharmaceutical Sciences, School of Pharmacy, University of Louisiana at Monroe, 1800 Bienville Drive, Monroe, LA 71201, USA. mohamedreda_pharmacy@yahoo.com.

ABSTRACT
Receptor tyrosine kinases are key regulators of cellular growth and proliferation. Dysregulations of receptor tyrosine kinases in cancer cells may promote tumorigenesis by multiple mechanisms including enhanced cell survival and inhibition of cell death. Araguspongines represent a group of macrocyclic oxaquinolizidine alkaloids isolated from the marine sponge Xestospongia species. This study evaluated the anticancer activity of the known oxaquinolizidine alkaloids araguspongines A, C, K and L, and xestospongin B against breast cancer cells. Araguspongine C inhibited the proliferation of multiple breast cancer cell lines in vitro in a dose-dependent manner. Interestingly, araguspongine C-induced autophagic cell death in HER2-overexpressing BT-474 breast cancer cells was characterized by vacuole formation and upregulation of autophagy markers including LC3A/B, Atg3, Atg7, and Atg16L. Araguspongine C-induced autophagy was associated with suppression of c-Met and HER2 receptor tyrosine kinase activation. Further in-silico docking studies and cell-free Z-LYTE assays indicated the potential of direct interaction between araguspongine C and the receptor tyrosine kinases c-Met and HER2 at their kinase domains. Remarkably, araguspongine C treatment resulted in the suppression of PI3K/Akt/mTOR signaling cascade in breast cancer cells undergoing autophagy. Induction of autophagic death in BT-474 cells was also associated with decreased levels of inositol 1,4,5-trisphosphate receptor upon treatment with effective concentration of araguspongine C. In conclusion, results of this study are the first to reveal the potential of araguspongine C as an inhibitor to receptor tyrosine kinases resulting in the induction of autophagic cell death in breast cancer cells.

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BT-474 breast cancer cells show characteristic vacuoles upon treatment with araguspongine C associated with cytotoxic effects. (A) Phase-contrast microscopy of BT-474 cells after araguspongine C treatment. BT-474 cells were treated with vehicle or araguspongine C at 10 μM for 24 h. The morphology of the cells was observed under an inverted phase contrast microscope. Magnification 100×; (B) Soft agar assay shows inhibition of BT-474 cell anchorage-independent growth by ARG C. BT-474 cells were cultured for 8 days in the absence (left) or presence (right) of 10 μM araguspongine C according to assay protocol and colony formation was observed under light microscope; (C) Western blot analysis of relative levels of c-PARP after araguspongine C treatment for 48 h in BT-474 breast cancer cells. Cells were plated at a density of 1 × 106 cells/100 mm culture plate and maintained in RPMI-1640 media supplemented with 10% FBS and allowed to adhere overnight. The next day, cells were divided into different treatment groups and then given various treatments in RPMI-1640 medium containing 40 ng/mL HGF for 48 h. At the end of treatment period, cells were lysed and equal amounts of whole cell extracts were fractionated on SDS-PAGE gels and immunoblotted as described in Materials and Methods. The visualization of β-actin was used as a loading control. Representative blots are from one of the three experiments; (D) Flow cytometry analysis. Cells were plated at a density of 1 × 106 cells/100 mm culture plates, allowed to attach overnight. Afterwards, cells were incubated in the respective control or araguspongine C-treated RPMI-1640 medium containing 40 ng/mL HGF for 48 h. Analysis of annexin V was determined using Annexin V-FITC Early Apoptosis Detection Kit as described in the Methods section. (−)-Oleocanthal was used as a positive control known to induce apoptosis at the dose used in this experiment. * p < 0.05 indicates values significantly different from non-treated cells. ARG C: Araguspongine C, c-PARP: cleaved Poly (ADP-ribose) polymerase, OC: (−)-Oleocanthal.
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marinedrugs-13-00288-f004: BT-474 breast cancer cells show characteristic vacuoles upon treatment with araguspongine C associated with cytotoxic effects. (A) Phase-contrast microscopy of BT-474 cells after araguspongine C treatment. BT-474 cells were treated with vehicle or araguspongine C at 10 μM for 24 h. The morphology of the cells was observed under an inverted phase contrast microscope. Magnification 100×; (B) Soft agar assay shows inhibition of BT-474 cell anchorage-independent growth by ARG C. BT-474 cells were cultured for 8 days in the absence (left) or presence (right) of 10 μM araguspongine C according to assay protocol and colony formation was observed under light microscope; (C) Western blot analysis of relative levels of c-PARP after araguspongine C treatment for 48 h in BT-474 breast cancer cells. Cells were plated at a density of 1 × 106 cells/100 mm culture plate and maintained in RPMI-1640 media supplemented with 10% FBS and allowed to adhere overnight. The next day, cells were divided into different treatment groups and then given various treatments in RPMI-1640 medium containing 40 ng/mL HGF for 48 h. At the end of treatment period, cells were lysed and equal amounts of whole cell extracts were fractionated on SDS-PAGE gels and immunoblotted as described in Materials and Methods. The visualization of β-actin was used as a loading control. Representative blots are from one of the three experiments; (D) Flow cytometry analysis. Cells were plated at a density of 1 × 106 cells/100 mm culture plates, allowed to attach overnight. Afterwards, cells were incubated in the respective control or araguspongine C-treated RPMI-1640 medium containing 40 ng/mL HGF for 48 h. Analysis of annexin V was determined using Annexin V-FITC Early Apoptosis Detection Kit as described in the Methods section. (−)-Oleocanthal was used as a positive control known to induce apoptosis at the dose used in this experiment. * p < 0.05 indicates values significantly different from non-treated cells. ARG C: Araguspongine C, c-PARP: cleaved Poly (ADP-ribose) polymerase, OC: (−)-Oleocanthal.

Mentions: Araguspongine C exerted antiproliferative effects when applied to multiple breast cancer cell lines in vitro. Five breast cancer cell lines with different phenotypes and molecular characteristics were used for the evaluation of araguspongine C treatment over two days in culture. Results showed that it effectively suppressed the growth of all breast cancer cell lines used in a dose-dependent manner (Figure 3). However, growth inhibition of breast cancer cell lines showed a range of IC50 values (Table 2). MDA-MB-231 and MCF-7 cells were the most sensitive while T-47D cells were the least sensitive to the antiproliferative activity of araguspongine C (Table 2). Araguspongine C was not toxic in the non-tumorigenic MCF10A mammary epithelial cells at the used treatment doses. Interestingly, araguspongine C-induced antiproliferative activity in BT-474 cells was associated with a characteristic change of cellular phenotype indicated by vacuole accumulation in all colonies of BT-474 cells exposed to the compound treatment as compared to their vehicle-treated control group (Figure 4A). Vacuole accumulation in BT-474 cells was observed with araguspongine C concentration of 10 µM within three hours of treatment exposure. Furthermore, these alterations in cancer cell phenotype were exclusively noticed upon araguspongine C treatment of BT-474 cells, but not with other breast cancer cell lines concomitantly treated with the compound or with other oxaquinolizidine alkaloids previously used in BT-474 cell treatment.


Araguspongine C induces autophagic death in breast cancer cells through suppression of c-Met and HER2 receptor tyrosine kinase signaling.

Akl MR, Ayoub NM, Ebrahim HY, Mohyeldin MM, Orabi KY, Foudah AI, El Sayed KA - Mar Drugs (2015)

BT-474 breast cancer cells show characteristic vacuoles upon treatment with araguspongine C associated with cytotoxic effects. (A) Phase-contrast microscopy of BT-474 cells after araguspongine C treatment. BT-474 cells were treated with vehicle or araguspongine C at 10 μM for 24 h. The morphology of the cells was observed under an inverted phase contrast microscope. Magnification 100×; (B) Soft agar assay shows inhibition of BT-474 cell anchorage-independent growth by ARG C. BT-474 cells were cultured for 8 days in the absence (left) or presence (right) of 10 μM araguspongine C according to assay protocol and colony formation was observed under light microscope; (C) Western blot analysis of relative levels of c-PARP after araguspongine C treatment for 48 h in BT-474 breast cancer cells. Cells were plated at a density of 1 × 106 cells/100 mm culture plate and maintained in RPMI-1640 media supplemented with 10% FBS and allowed to adhere overnight. The next day, cells were divided into different treatment groups and then given various treatments in RPMI-1640 medium containing 40 ng/mL HGF for 48 h. At the end of treatment period, cells were lysed and equal amounts of whole cell extracts were fractionated on SDS-PAGE gels and immunoblotted as described in Materials and Methods. The visualization of β-actin was used as a loading control. Representative blots are from one of the three experiments; (D) Flow cytometry analysis. Cells were plated at a density of 1 × 106 cells/100 mm culture plates, allowed to attach overnight. Afterwards, cells were incubated in the respective control or araguspongine C-treated RPMI-1640 medium containing 40 ng/mL HGF for 48 h. Analysis of annexin V was determined using Annexin V-FITC Early Apoptosis Detection Kit as described in the Methods section. (−)-Oleocanthal was used as a positive control known to induce apoptosis at the dose used in this experiment. * p < 0.05 indicates values significantly different from non-treated cells. ARG C: Araguspongine C, c-PARP: cleaved Poly (ADP-ribose) polymerase, OC: (−)-Oleocanthal.
© Copyright Policy
Related In: Results  -  Collection

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marinedrugs-13-00288-f004: BT-474 breast cancer cells show characteristic vacuoles upon treatment with araguspongine C associated with cytotoxic effects. (A) Phase-contrast microscopy of BT-474 cells after araguspongine C treatment. BT-474 cells were treated with vehicle or araguspongine C at 10 μM for 24 h. The morphology of the cells was observed under an inverted phase contrast microscope. Magnification 100×; (B) Soft agar assay shows inhibition of BT-474 cell anchorage-independent growth by ARG C. BT-474 cells were cultured for 8 days in the absence (left) or presence (right) of 10 μM araguspongine C according to assay protocol and colony formation was observed under light microscope; (C) Western blot analysis of relative levels of c-PARP after araguspongine C treatment for 48 h in BT-474 breast cancer cells. Cells were plated at a density of 1 × 106 cells/100 mm culture plate and maintained in RPMI-1640 media supplemented with 10% FBS and allowed to adhere overnight. The next day, cells were divided into different treatment groups and then given various treatments in RPMI-1640 medium containing 40 ng/mL HGF for 48 h. At the end of treatment period, cells were lysed and equal amounts of whole cell extracts were fractionated on SDS-PAGE gels and immunoblotted as described in Materials and Methods. The visualization of β-actin was used as a loading control. Representative blots are from one of the three experiments; (D) Flow cytometry analysis. Cells were plated at a density of 1 × 106 cells/100 mm culture plates, allowed to attach overnight. Afterwards, cells were incubated in the respective control or araguspongine C-treated RPMI-1640 medium containing 40 ng/mL HGF for 48 h. Analysis of annexin V was determined using Annexin V-FITC Early Apoptosis Detection Kit as described in the Methods section. (−)-Oleocanthal was used as a positive control known to induce apoptosis at the dose used in this experiment. * p < 0.05 indicates values significantly different from non-treated cells. ARG C: Araguspongine C, c-PARP: cleaved Poly (ADP-ribose) polymerase, OC: (−)-Oleocanthal.
Mentions: Araguspongine C exerted antiproliferative effects when applied to multiple breast cancer cell lines in vitro. Five breast cancer cell lines with different phenotypes and molecular characteristics were used for the evaluation of araguspongine C treatment over two days in culture. Results showed that it effectively suppressed the growth of all breast cancer cell lines used in a dose-dependent manner (Figure 3). However, growth inhibition of breast cancer cell lines showed a range of IC50 values (Table 2). MDA-MB-231 and MCF-7 cells were the most sensitive while T-47D cells were the least sensitive to the antiproliferative activity of araguspongine C (Table 2). Araguspongine C was not toxic in the non-tumorigenic MCF10A mammary epithelial cells at the used treatment doses. Interestingly, araguspongine C-induced antiproliferative activity in BT-474 cells was associated with a characteristic change of cellular phenotype indicated by vacuole accumulation in all colonies of BT-474 cells exposed to the compound treatment as compared to their vehicle-treated control group (Figure 4A). Vacuole accumulation in BT-474 cells was observed with araguspongine C concentration of 10 µM within three hours of treatment exposure. Furthermore, these alterations in cancer cell phenotype were exclusively noticed upon araguspongine C treatment of BT-474 cells, but not with other breast cancer cell lines concomitantly treated with the compound or with other oxaquinolizidine alkaloids previously used in BT-474 cell treatment.

Bottom Line: Araguspongine C-induced autophagy was associated with suppression of c-Met and HER2 receptor tyrosine kinase activation.Induction of autophagic death in BT-474 cells was also associated with decreased levels of inositol 1,4,5-trisphosphate receptor upon treatment with effective concentration of araguspongine C.In conclusion, results of this study are the first to reveal the potential of araguspongine C as an inhibitor to receptor tyrosine kinases resulting in the induction of autophagic cell death in breast cancer cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Basic Pharmaceutical Sciences, School of Pharmacy, University of Louisiana at Monroe, 1800 Bienville Drive, Monroe, LA 71201, USA. mohamedreda_pharmacy@yahoo.com.

ABSTRACT
Receptor tyrosine kinases are key regulators of cellular growth and proliferation. Dysregulations of receptor tyrosine kinases in cancer cells may promote tumorigenesis by multiple mechanisms including enhanced cell survival and inhibition of cell death. Araguspongines represent a group of macrocyclic oxaquinolizidine alkaloids isolated from the marine sponge Xestospongia species. This study evaluated the anticancer activity of the known oxaquinolizidine alkaloids araguspongines A, C, K and L, and xestospongin B against breast cancer cells. Araguspongine C inhibited the proliferation of multiple breast cancer cell lines in vitro in a dose-dependent manner. Interestingly, araguspongine C-induced autophagic cell death in HER2-overexpressing BT-474 breast cancer cells was characterized by vacuole formation and upregulation of autophagy markers including LC3A/B, Atg3, Atg7, and Atg16L. Araguspongine C-induced autophagy was associated with suppression of c-Met and HER2 receptor tyrosine kinase activation. Further in-silico docking studies and cell-free Z-LYTE assays indicated the potential of direct interaction between araguspongine C and the receptor tyrosine kinases c-Met and HER2 at their kinase domains. Remarkably, araguspongine C treatment resulted in the suppression of PI3K/Akt/mTOR signaling cascade in breast cancer cells undergoing autophagy. Induction of autophagic death in BT-474 cells was also associated with decreased levels of inositol 1,4,5-trisphosphate receptor upon treatment with effective concentration of araguspongine C. In conclusion, results of this study are the first to reveal the potential of araguspongine C as an inhibitor to receptor tyrosine kinases resulting in the induction of autophagic cell death in breast cancer cells.

Show MeSH
Related in: MedlinePlus