Berberine suppresses gero-conversion from cell cycle arrest to senescence

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.

Geroconversion: irreversible step to cellular senescence

“…38,[46][47][48] In particular, hypoxia, starvation and contact inhibition all suppress geroconversion. 38,[47][48][49] As we will discuss, this can explain how quiescent cells remain quiescent in the organism for so long.…”

Attenuation of Replication Stress–Induced Premature Cellular Senescence to Assess Anti‐Aging Modalities

“…Such treatment causes persistent DNA replication stress. Berberine (BRB), a naturally occurring alkaloid with a long history of use in traditional Chinese and Ayurvedic medicine, is used as an example of a potential anti-aging agent (Lee et al, 2006; Halicka et al, 2012; Zhao et al, 2013). A549 cultures are treated with Mxt with or without BRB, and the depth of cellular senescence is assessed and compared between BRB-treated and untreated cultures.…”

“…This methodology can also be used to measure senescence-associated changes in other cell types (e.g., WI-38), as well as changes in other biomarkers, such as induction of SA-β-gal or γH2AX and activation of ATM (Zhao et al, 2010, 2013; Halicka et al, 2012; McKenna et al, 2012; Zhao and Darzynkiewicz, 2013). Activation of SA-β-gal is measured using the Chemicon Cellular Senescence Assay Kit (Millipore) and the capability of LSC to measure both light absorption and intensity of fluorescence (Bedner et al, 1999; Darzynkiewicz et al, 1999; Henriksen et al, 2011; Zhao et al, 2013).…”

“…Activation of SA-β-gal is measured using the Chemicon Cellular Senescence Assay Kit (Millipore) and the capability of LSC to measure both light absorption and intensity of fluorescence (Bedner et al, 1999; Darzynkiewicz et al, 1999; Henriksen et al, 2011; Zhao et al, 2013). …”

“…In Ctrl cells, intensity of the mean value of maximal pixels is highest in mitotic (M) and immediately post-mitotic (pM) G 1 cells; nuclei of these cells also have a small area of DAPI fluorescence, consistent with their high degree of chromatin condensation (Bedner et al, 1999; Darzynkiewicz et al, 1999; Henriksen et al, 2011). In senescing cells, nuclear area increases while maximal pixel intensity decreases (Zhao et al, 2010, 2013; McKenna et al, 2012). These morphometric changes reflect enlargement of the projected nuclear area and decreased intensity of DAPI local staining per unit area due to a flattened cellular appearance, the hallmark of cellular senescence (Dimri et al, 1995; Itahana et al, 2007).…”