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. 2013 Aug;5(8):623-36.
doi: 10.18632/aging.100593.

Berberine suppresses gero-conversion from cell cycle arrest to senescence

Hong Zhao  1 , H Dorota Halicka, Jiangwei Li, Zbigniew Darzynkiewicz
Affiliations

Berberine suppresses gero-conversion from cell cycle arrest to senescence

Hong Zhao et al. Aging (Albany NY). 2013 Aug.

Abstract

Berberine (BRB), a natural alkaloid, has a long history of medicinal use in both Ayurvedic and old Chinese medicine. Recently, available as a dietary supplement, Berberine is reported to have application in treatment of variety diseases. Previously we observed that BRB inhibited mTOR/S6 signaling concurrently with reduction of the level of endogenous oxidants and constitutive DNA damage response. We currently tested whether Berberine can affect premature, stress-induced cellular senescence caused by mitoxantrone. The depth of senescence was quantitatively measured by morphometric parameters, senescence-associated β-galactosidase, induction of p21WAF1, replication stress (γH2AX expression), and mTOR signaling; the latter revealed by ribosomal S6 protein (rpS6) phosphorylation. All these markers of senescence were distinctly diminished, in a concentration-dependent manner, by Berberine. In view of the evidence that BRB localizes in mitochondria, inhibits respiratory electron chain and activates AMPK, the observed attenuation of the replication stress-induced cellular senescence most likely is mediated by AMPK that leads to inhibition of mTOR signaling. In support of this mechanism is the observation that rhodamine123, the cationic probe targeting mitochondrial electron chain, also suppressed rpS6 phosphorylation. The present findings reveal that: (a) in cells induced to senescence BRB exhibits gero-suppressive properties by means of mTOR/S6 inhibition; (b) in parallel, BRB reduces the level of constitutive DNA damage response, previously shown to report oxidative DNA damage by endogenous ROS; (c) there appears to a causal linkage between the (a) and (b) activities; (d) the in vitro model of premature stress-induced senescence can be used to assess effectiveness of potential gero-suppressive agents targeting mTOR/S6 and ROS signaling; (e) since most of the reported beneficial effects of BRB are in age-relate diseases, it is likely that gero-suppression is the primary activity of this traditional medicine.

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Conflict of interest statement

The authors have no conflicting interest to declare.

Figures

Figure 1
Figure 1. Induction of premature cellular senescence of A549 cells measured by laser scanning cytometry
Human pulmonary non-small cell lung carcinoma A549 were untreated (Ctrl) or treated with 2 nM DNA topoisomerase II inhibitor mitoxantrone (Mxt) for 48 or 72 h. Panel A shows morphometric features of the cells revealed by measurement of nuclear DNA (DAPI) fluorescence reporting on the bivariate distributions (scatterplots) nuclear area versus intensity of maximal pixel of fluorescence, respectively. Intensity of maximal pixel is correlated with chromatin condensation and in the untreated cells has the highest value and marks mitotic (M) and immediately post-mitotic (pM) G1 cells, which also have low value of DAPI area [41]. In the senescing cells, while nuclear area increases, the intensity of maximal pixel decreases [39,40,64]. These morphometric changes reflect enlargement of the projected nuclear area and decreased DAPI local staining per unit area, due to “flattened” cellular appearance, the hallmark of cellular senescence [42,43]. The insets show DNA content frequency histograms of cells from the respective cultures. Panel B: Bar plots reporting mean values (+SD) of nuclear (DNA, DAPI) area, DNA (DAPI) maximal pixel, and ratio of maximal pixel to nuclear area, respectively, of cells from control and Mxt treated cultures. Panel C: Bivariate distributions (DNA content vs p21) reporting expression of p21WAF1 with respect to the cell cycle phase; the figures show the n-fold increase in mean expression of p21 of G1 and G2M cells from the Mxt-treated cultures with respect to respective cells in Ctrl. Panel D: Bivariate distributions of DNA content versus senescence-associated galactosidase (SA-β-gal) activity. Figures indicate percent of SA-β-gal positive (above the threshold marked by the horizontal lines) cells. Insets show the frequency distribution of SA-β-gal positive cells; the figures in insets show the n-fold increase in the mean activity of SA-β-gal in Mxt-treated cultures over Ctrl (1.0). Panel E: Images of cells growing in the absence (left) and presence of 2 nM Mxt for 72 h (right) stained to detect activation of SA-β-gal activity recorded by laser scanning cytometer (Research Imaging Cytometer iCys); 50 μm bars mark the length scale.
Figure 2
Figure 2. Attenuation of Mxt-induced senescence of A549 cells by berberine (BRB) as measured by cell morphometric features and SA-β-gal activity
Exponentially growing A549 cells were untreated (Ctrl) or treated with 2 nm Mxt in the absence and presence of BRB at concentration as shown, for 5 days. Top panels: Morphometric analysis, reporting changes in nuclear area (DNA-DAPI) versus maximal pixel of DAPI fluorescence. The ratio of maximal pixel to nuclear area (Mp:A) is expressed as a fraction of that of the untreated cells; shown is the percent increase in Mp:A in the BRB-treated cultures with respect to cells growing with Mxt alone, with the arrows. Bottom panels: The frequency histograms reporting SA-β-gal activity. The figures present the increase (n-fold) in the enzyme activity with respect to the Ctrl (1.0), measured as the mean intensity of SA-β-gal absorption of cells in the respective cultures.
Figure 3
Figure 3. Attenuation of Mxt-induced senescence of A549 cells by BRB as measured by reduction in expression of p21WAF1, γH2AX and rpS6P
Exponentially growing A549 cells were untreated (Ctrl) or treated with Mxt in the absence and presence of BRB at concentrations as shown, for 5 days. Top panels: bivariate distributions of p21 versus cellular DNA content; the figures (x) present the increase (n-fold) in the mean expression for all cells of p21 with respect to the untreated cells, the percent reduction in p21 in cultures with BRB with respect to Mxt alone, is shown with the arrows. Mid-panels: expression of γH2AX versus DNA content; the figures (x) represent the increase (n-fold) in the mean expression of γH2AX with respect to untreated cells (1.0); the percent reduction in expression of γH2AX in cultures grown with BRB with respect to cells growing in the presence of Mxt alone is presented with the arrows. Bottom panels: expression of rpS6P versus DNA content. The figures illustrate the change (n-fold) with respect to the untreated cells; the percent reduction in expression of rpS6P in cultures with BRB with respect to cells treated with Mxt alone is shown with the arrows.
Figure 4
Figure 4. Suppression of rpS6 phosphorylation and reduction of size of human lymphoblastoid TK6 cells grown in the presence of BRB at 5 μM - 60 μM concentration
Exponentially growing TK6 cells were untreated (Ctrl) or treated with BRB at concentrations as shown, for 24 h. Top panels: the bivariate distributions of rpS6P expression versus DNA content. Figures show percent decrease in expression of the mean rpS6P for cells at G1, S and G2M phases of the cell cycle, respectively, growing in the presence of BRB vis-à-vis to the untreated cells. Insets show the frequency histograms of rpS6P expression for all cells in culture. Bottom panels: Bivariate distributions of cellular forward light scatter (FLS) versus DNA content. Percent decrease in of mean value of FLS, considered to represent cellular size [49,50], of G1, S and G2M of cells growing in the presence of BRB with respect to the untreated cells is shown with the arrows. Insets illustrate DNA content frequency histograms of cells from the respective cultures.
Figure 5
Figure 5. Detection of BRB fluorescence in live A549 cells exposed to this isoquinoline
Exponentially growing A549 cells were treated with 5 μM BRB for 60 min, rinsed with PBS and examined under fluorescence microscopy. Immediately after illumination (the first min after exposure to UV) the BRB yellow fluorescence was localized almost exclusively in mitochondria (A). With extended time of illumination (2 min) intensity of fluorescence of mitochondria declined while nuclear and nucleolar fluorescence become more apparent (B). After 5 min exposure to UV no mitochondrial fluorescence was evident whereas intensity of nuclear and nucleolar fluorescence was distinctly increased (C). Images taken with the Nikon Microphot FXA; objective Fluor 40X.
Figure 6
Figure 6. Effect of treatment of A549 cells with Rh123 on the level of expression of phosphorylated rpS6
Exponentially growing A549 cells were treated for 6 h or 24 h with 1 μM Rh123. Expression of rpS6P in cytoplasm was detected by phosphospecific Ab and measured by the iCys laser scanning cytometry. The figures indicate percent decree in expression of RP-S6P in the Rh123 treated cells vis-à-vis the respective control (Ctrl) cells.
Figure 7
Figure 7. Schematic presentation of key pathways associated with cellular senescence and aging linking mTOR- and DNA damage- signaling
The ongoing translation particularly during perturbed cell cycle progression (replication stress), is considered to be the major factor leading to senescence. Suppression of translation that may have anti-aging effect can be achieved at several steps along the mTOR signaling pathway (marked A-g), as discussed in the text. Activation of autophagy provides an additional gero-suppressive effect. The translation requires production of ATP and thus generates ROS that cause oxidative DNA damage, which when occurs at sites of oncogenes and tumor suppressor genes, may lead to neoplasia. The damage of telomeric DNA and lipid peroxidation by ROS further contributes to the senescent phenotype. The in vitro model of stress-induced cellular senescence as presently described can be used to evaluate potential gero-suppressive agents in terms of their effect in reduction of mTOR/S6- and DNA- damage signaling. See text for further details.
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