J Breast Cancer.  2016 Dec;19(4):358-371. 10.4048/jbc.2016.19.4.358.

Alkyl Cinnamates Induce Protein Kinase C Translocation and Anticancer Activity against Breast Cancer Cells through Induction of the Mitochondrial Pathway of Apoptosis

Affiliations
  • 1Malaria Research Group, Department of Biosciences and Bioengineering, Indian Institute of Technology Guwahati, Guwahati, India. vtrivedi@iitg.ernet.in vishalash_1999@yahoo.com
  • 2Laboratory of Biological Chemistry, Department of Chemistry, Indian Institute of Technology Guwahati, Guwahati, India.

Abstract

PURPOSE
The protein kinase C (PKC) family of serine-threonine kinases plays an important role in cancer cell progression. Thus, molecules that target PKC have potential as anticancer agents. The current study aims to understand the treatment of breast cancer cells with alkyl cinnamates. We have also explored the mechanistic details of their anticancer action and the underlying molecular signaling.
METHODS
3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was performed to measure the viability of MDAMB-231 breast cancer cells to assess the anticancer activity of these compounds. In addition, flow cytometry was performed to study the effect of alkyl cinnamates on the cell cycle and apoptosis. Immunoblotting and immunofluorescence techniques were performed to study PKC translocation, cytochrome c release, and modulation of the mitochondrial membrane potential in breast cancer cells targeted with alkyl cinnamates.
RESULTS
The PKC agonist DM-2-8 translocated 16.6%±1.7% PKCα from cytosol to the plasma membrane and showed excellent anticancer activity with an half maximal inhibitory concentration (IC50) of 4.13±0.27 µg/mL against cancer cells. The treated cells had an abnormal morphology and exhibited cell cycle defects with G2/M arrest and reduced S phase. Cancer cells treated with DM-2-3, DM-2-4, or DM-2-8 underwent apoptosis as the major pathway of cell death, further confirmed by genomic DNA fragmentation. Furthermore, the mitochondrial membrane potential was perturbed, indicating involvement of the mitochondrial pathway of apoptosis. Immunolocalization studies revealed cytochrome c release from mitochondria to cytosol. Cancer cells treated with DM-2-8 and curcumin showed activation of caspase-9 and caspase-3 as downstream molecular components of the apoptotic pathway. Alkyl cinnamates also caused oxidative stress, which regulates the apoptotic machinery (DNA fragmentation), cell death, and morphological abnormalities in cancer cells.
CONCLUSION
Alkyl cinnamates specifically target cancer cells through induction of PKC translocation and the mitochondrial pathway of apoptosis, and could be promising anticancer drugs.

Keyword

Apoptosis; Caspases; Neoplasms; Oxidative stress; Protein kinase C

MeSH Terms

Antineoplastic Agents
Apoptosis*
Breast Neoplasms*
Breast*
Caspase 3
Caspase 9
Caspases
Cell Cycle
Cell Death
Cell Membrane
Cinnamates*
Curcumin
Cytochromes c
Cytosol
DNA Fragmentation
Flow Cytometry
Fluorescent Antibody Technique
Humans
Immunoblotting
Membrane Potential, Mitochondrial
Mitochondria
Oxidative Stress
Protein Kinase C*
Protein Kinases*
Protein-Serine-Threonine Kinases
S Phase
Antineoplastic Agents
Caspase 3
Caspase 9
Caspases
Cinnamates
Curcumin
Cytochromes c
Protein Kinase C
Protein Kinases
Protein-Serine-Threonine Kinases

Figure

  • Figure 1 Chemical structure of different alkyl cinnamates with their respective compound codes.

  • Figure 2 Alkyl cinnamates have potential to exhibits protein kinase C (PKC)-α translocation from cytosol to the plasma membrane in MDAMB-231 cells. (A) Immunolocalization of PKCα in MDAMB-231 treated with different alkyl cinnamates to monitor PKC translocation from cytosol to plasma membrane. MDAMB-231 cells were treated and PKCα immunolocalization was done as given in Methods. In each panel, bright field, PKCα (red), filipin to stain cell membrane (blue) and overlay. Untreated cells show a low level of PKCα at plasma membrane compared to significant high signal in alkyl cinnamate treated cells. (B, C) Immunoblotting of PKCα in MDAMB-231 treated with different alkyl cinnamates to monitor PKC translocation from cytosol to plasma membrane. MDAMB-231 cells were treated and PKCα immunolocalization was done as given in Methods. The purity of cytosolic or membrane fraction was asses by testing the presence of lactate dehydrogenase (LDH) and 5′-nucleotidase in these fractions. LDH activity assay was performed as described previously whereas presence of 5′-nucleotidase was done by immune-blotting with anti-5′-nucleotidase antibodies. Untreated cells show a basal level of PKCα at plasma membrane whereas a high level of PKCα was found in alkyl cinnamate treated cells.

  • Figure 3 Alkyl cinnamates disturb cell-cycle in MDAMB-231w cells. (A) Cells were treated with different alkyl cinnamates (half maximal inhibitory concentrations [IC50]) for 24 hours in incomplete media, stained with propidium iodide as given in Methods, and immediately analyzed by flow cytometry. The cells treated with different compounds show G2/M arrest and reduction of S phase. (B) Plot of change in S phase of MDA-MB-231 cells treated with different alkyl cinnamates.

  • Figure 4 Alkyl cinnamates are causing death of MDAMB-231 following apoptosis. (A) MDAMB-231 treated with different alkyl cinnamates and distribution of healthy, dead, apoptotic and necrotic cells. Cells were treated alkyl cinnamates (IC50 for 24 hours), stained with acridine orange and propidium iodide and analyzed by flow cytometry. (B) MDAMB-231 treated with different alkyl cinnamates exhibits DNA fragmentation. Cells were treated with different alkyl cinnamates (the IC50 values of the compounds for 12 hours) in serum-free media and samples were processed for DNA laddering analysis. A laddering pattern of DNA is observed in alkyl cinnamate treated cells compared to the intact genomic DNA in untreated cells.

  • Figure 5 Loss of mitochondrial membrane potential in alkyl cinnamates treated MDAMB-231 breast cancer cells. MDAMB-231 breast cancer cells were treated with alkyl cinnamates (IC50) in serum-free media for 24 hours. After treatment, cells were stained with JC-1 dye for 20 minutes and were observed in Cytell Imaging System (GE Healthcare). Alkyl cinnamates treated cells show a decrease in mitochondrial membrane potential as indicated by loss of orange and appearance of green fluorescence (scale bar, 130 µm).

  • Figure 6 Release of cytochrome-c (cyt c) from alkyl cinnamate treated MDAMB-231 breast cancer cells. MDAMB-231 breast cancer cells were treated with alkyl cinnamates (IC50) in serum-free media for 24 hours. After treatment, cells were immune-stained with anti-cyt c antibodies and location of mitochondria in MDAMB-231 cells is determined by loading the cells with MitoTracker (BD-Biosciences; 200 nM for 45 minutes) as given in material and methods. Labelled cells were observed in a fluorescent microscope 80i (Nikon). In each panel, bright field, cyt c (green), MitoTracker Red (red) is given. High resolution fluorescence microscopy detected release of cyt c from DM2-3, DM2-4, DM2-5 and curcumin treated cells. Untreated cells seem healthy with the cyt c signal overlapping with MitoTracker signal.

  • Figure 7 Cytochrome C release activates down-stream cytosolic caspases. Caspase 9 (A) and caspase 3 (B) activity in MDAMB-231 cells treated with alkyl cinnamates. Caspase-3 activity levels increased by 90% and 65% in DM2-8 and curcumin. (C) Flow cytometric measurement of reactive oxygen species (ROS) levels in alkyl cinnamate treated breast cancer cells. Cells were treated with alkyl cinnamates (IC50) for 3 hours and DCF-DHA was used as a ROS probe. DM-2-8 treated cells show an increased level of ROS compared to untreated cells. The preincubation of cells in N-acetylcysteine (NAC) reduces ROS level in treated cell. The preincubation of cells in N-acetylcysteine (NAC) reduces ROS level and apperance of DNA fragments (D) in treated cell. Cells were treated with DM 2-8 or curcumin in the absence or presence of NAC (5 mM) and presence of NAC give intact genomic DNA with no visible appearance of DNA fragments. (E) Microscopic observation of MDAMB-231 cells treated with DM-2-8 or curcumin for 48 hours in absence or presence of NAC. Images were taken with high resolution Nikon L22 camera. Cells preincubated with NAC gives an improved cellular morphology compared to DM-2-8 or curcumin treated cells.


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