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. 2017 Jul 20;18(1):26.
doi: 10.1186/s12860-017-0141-0.

Cladosporol A triggers apoptosis sensitivity by ROS-mediated autophagic flux in human breast cancer cells

Mytre Koul  1   2 , Ashok Kumar  1   2 , Ramesh Deshidi  3   2 , Vishal Sharma  4   2 , Rachna D Singh  5 , Jasvinder Singh  1   2 , Parduman Raj Sharma  1   2 , Bhahwal Ali Shah  6   7 , Sundeep Jaglan  4   2 , Shashank Singh  8   9
Affiliations

Cladosporol A triggers apoptosis sensitivity by ROS-mediated autophagic flux in human breast cancer cells

Mytre Koul et al. BMC Cell Biol. .

Erratum in

Abstract

Background: Endophytes have proven to be an invaluable resource of chemically diverse secondary metabolites that act as excellent lead compounds for anticancer drug discovery. Here we report the promising cytotoxic effects of Cladosporol A (HPLC purified >98%) isolated from endophytic fungus Cladosporium cladosporioides collected from Datura innoxia. Cladosporol A was subjected to in vitro cytotoxicity assay against NCI60 panel of human cancer cells using MTT assay. We further investigated the molecular mechanism(s) of Cladosporol A induced cell death in human breast (MCF-7) cancer cells. Mechanistically early events of cell death were studied using DAPI, Annexin V-FITC staining assay. Furthermore, immunofluorescence studies were carried to see the involvement of intrinsic pathway leading to mitochondrial dysfunction, cytochrome c release, Bax/Bcl-2 regulation and flowcytometrically measured membrane potential loss of mitochondria in human breast (MCF-7) cancer cells after Cladosporol A treatment. The interplay between apoptosis and autophagy was studied by microtubule dynamics, expression of pro-apoptotic protein p21 and autophagic markers monodansylcadaverine staining and LC3b expression.

Results: Among NCI60 human cancer cell line panel Cladosporol A showed least IC50 value against human breast (MCF-7) cancer cells. The early events of apoptosis were characterized by phosphatidylserine exposure. It disrupts microtubule dynamics and also induces expression of pro-apoptotic protein p21. Moreover treatment of Cladosporol A significantly induced MMP loss, release of cytochrome c, Bcl-2 down regulation, Bax upregulation as well as increased monodansylcadaverine (MDC) staining and leads to LC3-I to LC3-II conversion.

Conclusion: Our experimental data suggests that Cladosporol A depolymerize microtubules, sensitize programmed cell death via ROS mediated autophagic flux leading to mitophagic cell death. The proposed mechanism of Cladosporol A -triggered apoptotic as well as autophagic death of human breast cancer (MCF-7) cells. The figure shows that Cladosporol A induced apoptosis through ROS mediated mitochondrial pathway and increased p21 protein expression in MCF-7 cells in vitro.

Keywords: Apoptosis; Breast cancer; Cladosporium cladosporioides; Cladosporol a; Endophytes; Reactive oxygen species.

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Figures

Fig. 1
Fig. 1
Morphology of isolate MRCJ-314 (Cladosporium cladosporioides); Appearance of colonies on PDA plate. The obverse (a) and reverse (b) view
Fig. 2
Fig. 2
Neighbor-Joining tree of fungal endophyte MRCJ-314 (Cladosporium cladosporioides) based on ITS1–5.8S–ITS2 rDNA sequences. Confidence values above 50% obtained from a 1000-replicate bootstrap analysis are shown at the branch nodes
Fig. 3
Fig. 3
Structure of Cladosporol A
Fig. 4
Fig. 4
Clonogenic ability was assessed in human breast cancer (MCF-7) cells, after Cladosporol A treatment. The representative images are shown. a Reduction in colony forming capability was determined in MCF-7 cells. Cells (1 × 103/ml/well) were seeded in six well plates and treated with different concentrations of Cladosporol A (5, 10, 20 μM). The number of crystal violet stained colony were counted randomly after seven days, quantified and photographed. b Data are presented as mean ± S.D., statistical analysis was done with **p < 0.01 and ***p < 0.001
Fig. 5
Fig. 5
a Cell cycle distribution in human breast cancer (MCF-7) cells treated with 5, 10, 20 μM of Cladosporol A. Data are expressed as the mean ± SD of three similar experiments. b Cladosporol A disrupts the microtubules of human breast cancer (MCF-7) cells. Cells were treated with indicated concentrations of Cladosporol A for 24 h. Immunocytochemical staining was conducted using anti α-tubulin antibody and Alexa Flour-555-labelled secondary antibody and nuclei were stained with DAPI (c). Analysis of p21 protein expression of human breast cancer (MCF-7) cells after 24 h of Cladosporol A (5, 10, 20 μM) treatment by immunofluorescence microscopy (d). Western blot indicating the increase in p21 protein expression after Cladosporol A treatment in concentration dependent manner
Fig. 6
Fig. 6
a Nuclear morphology analysis of human breast cancer (MCF-7) cells (2 × 105/ml/well) using DAPI after treatment with different concentrations of Cladosporol A (5, 10, 20 μM) for 24 h and examined using fluorescence microscopy (40X). Paclitaxel (1 μM) was used as positive control. With increase in concentration of Cladosporol A there is significant increase in nuclear condensation and formation of apoptotic bodies (b). The effect of Cladosporol A on the exposure of phosphatidylserine (PS) in human breast cancer (MCF-7) cells after 24 h treatment was analysed. Phosphatidylserine exposure was assessed by the annexin-V/ propidium iodide assay, as described in methodology and analyzed by confocal microscopy (c). The percentage of cells in early and late stages of apoptosis obtained by analysis of the cell images (mean ± SD, 3 experiments), p*< 0.05 and **p < 0.01
Fig. 7
Fig. 7
a Intracellular ROS level was measured by flow cytometry analysis using DCFH- DA after 24 h. Human breast cancer (MCF-7) cells were treated with indicated concentrations of Cladosporol A and 0.05% H2O2 and incubated with 5 μM DCFH-DA. b Detection of ROS by fluorescence microscope. After incubation with DCFH-DA and 0.05% H2O2, MCF-7 cells (2 × 105/ml/well) were washed and examined by fluorescence microscope (40X). c MCF-7 cell growth inhibition in the presence of ROS scavenger (NAC) was also determined. The cells were treated with indicated concentrations of Cladosporol A in the presence or absence of NAC (150 μM)
Fig. 8
Fig. 8
Cladosporol A induces loss of mitochondrial transmembrane potential. Human breast cancer (MCF-7) cells were incubated with different concentrations of Cladosporol A for 24 h. Thereafter, cells were stained with Rh-123 (1 μM) for 20 min and analyzed by (a) Flow cytometer (b). Confocal microscopy (c)). Histogram showing the effect of Cladosporol A on Δψm measured with laser scanning confocal microscope by staining with Rhodamine 123. Data are presented as mean ± S.D., statistical analysis was done with **p < 0.01
Fig. 9
Fig. 9
Colocalization of cytochrome c and mitochondria was determined by confocal microscopy. Human breast cancer (MCF-7) cells were immunostained for cytochrome c release (green) and the mitochondria of the cells were stained with MitoTracker (red). MCF-7 cells were treated with different concentrations of Cladosporol A and doxorubicin (500 nM) for 24 h and stained with anti-cytochrome c antibody and Alexa Fluor 488-labeled secondary antibody
Fig. 10
Fig. 10
Effects of Cladosporol A and doxorubicin treatment on the expression of apoptosis related proteins. (a) Representative images of immunofluorescence analysis of effects of the expression of; antiapoptotic Bcl-2 and pro-apoptotic Bax by confocal microscopy (using 40× oil immersion lens) in human breast cancer (MCF-7) cells treated with Cladosporol A for 24 h (b). Statistical analysis of the expression of Bcl-2 and Bax. The relative fluorescence intensity was compared to the control group. Data are mean ± S.D. of three similar experiments; statistical analysis was done with **p < 0.01 and ***p < 0.001. c Western blot indicating the increase in bax and decrease bcl-2 protein expression after Cladosporol A treatment in concentration dependent manner
Fig. 11
Fig. 11
Cladosporol A induces autophagy in human breast cancer (MCF-7 cells) (a). The autophagic vacuoles were observed under fluorescence microscope (40×) with MDC staining. The treatment of Cladosporol A and BEZ235 (positive control group) induced concentration-dependent formation of autophagic vacuoles in MCF-7 cells after 24 h. b Detection of autophagy with LC3b antibody using confocal microscopy. Immunocytochemical staining was conducted using anti-LC3b antibody and Alexa Flour-555-labelled secondary antibody. Nuclei were stained with DAPI. c Detection of autophagy by western blot analysis after Cladosporol A treatment in concentration dependent manner
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