A newly identified berberine derivative induces cancer cell senescence by stabilizing endogenous G-quadruplexes and sparking a DNA damage response at the telomere region.

The guanine-rich sequences are able to fold into G-quadruplexes in living cells, making these structures promising anti-cancer drug targets. In the current study, we identified a small molecule, Ber8, from a series of 9-substituted berberine derivatives and found that it could induce acute cell growth arrest and senescence in cancer cells, but not in normal fibroblasts. Further analysis revealed that the cell growth arrest was directly associated with apparent cell cycle arrest, cell senescence, and profound DNA damage at the telomere region. Significantly, our studies also provided evidence that Ber8 could stabilize endogenous telomeric G-quadruplexes structures in cells. Ber8 could then induce the delocalization of TRF1 and POT1 from the telomere accompanied by a rapid telomere uncapping. These results provide compelling insights into direct binding of telomeric G-quadruplexes and might contribute to the development of more selective, effective anticancer drugs.


General synthetic chemistry
The compounds ber1-ber22 were prepared in our previous synthesis except ber8 [1][2][3][4]. Reactions were carried out under nitrogen when necessary. All the material we used were purchased from commercial suppliers and used without further purification. Solvents were dried over Molecular sieves type 4A. Flash silica chromatography was performed using silica gel (200-300 mesh), dichloromethane/methanol (20/1). Reactions were routinely monitored by thin-layer chromatography on silica gel GF 254 and visualized with ultraviolet lamp (254 nm or 365 nm). 1 H and 13 C NMR spectra were determined on a BrukerAvance III 400 spectrometer using TMS as an internal standard in DMSO-d 6 solution, operating at a frequency of 400 MHz for proton and 101 MHz for carbon. Peak positions are given in parts per million (δ) from tetramethylsilane, and coupling constant value (J) are given in Hz. High-resolution mass spectra were measured on an Shimhdzu LCMS-IT-TOF instrument with an ESI mass selective detector in positive ion mode. The purities of synthesized compounds were confirmed to be higher than 95% by using analytical HPLC equipped with an Analaric C18 column (150 × 4.6 mm, 5 μm particle size) and eluted with methanol/water (70:30) containing 0.1% TFA at a flow rate of 0.4 mL/min, with calculation of the relative purity of each compound based on its absorption at 254 nm.
The scheme of synthesis of the Berberine derivative Ber8 (3) was shown in Supplementary Scheme S1.

Cellular uptake
Cells were seeded at a density of 5 × 10 6 cells / 10 cm dish in 10 mL of culture medium on day 1. On day 2, 5 μM of Ber8 or 0.1% DMSO was added into the cells. After 48 h of drug exposure, cells were collected and washed with PBS twice. 1 × 10 6 cells were lysis by 500 μL RIPA lysis buffer (Bioteke, China) and the supernatant was collected. The UV absorbance of the compound was detected at 25°C by using UV-2450 spectrophotometer (Shimadzu, Japan). The optimal wavelengths were 200-500 nm. To determine the drug concentration, calibration curves in the concentration range of 0.05-32 μM in RIPA lysis buffer were prepared.

S1 nuclease digestion assay
The 5'-FAM-labeled HTG21 (5'-FAM-GGGTTAGGGTTAGGTTAGGG-3') and non-labeled HTC21 (5'-CCCTAACCCTAACCCT-AACCC-3') were dissolved in 10 mM Tris-HCl (pH 7.4, 100 mM KCl) with a final concentration of 100 μM. 100 pmol of the HTG21 oligomer was annealed by heating to 95°C for 10 min then gradually cooled to room temperature for G-quadruplex formation, and 100 pmol of the HTG21 and HTC21 oligomers were annealed together using the same procedure for duplex formation. Samples were incubated with Ber8 at various concentrations (100, 500, and 2500 pmol) at 37°C for 1 hour. 1.5 U of S1 nuclease was then added to each sample and incubated at 37°C for 5 min. The digestion was terminated by adding 50 mM EDTA then incubated at 70°C for 10 min letting the nuclease denature. Sample was resolved in 16% native gel in 0.5 × TBE.

Telomere length assay
Long-term cultured cells were treated with Ber8. Telomere length was measured using Telo TAGGG Telomere Length Assay KIT (#2209136, Roche). Briefly, genomic DNA was digested with Hinf I/Rsa I restriction enzymes. The digested DNA fragments were separated on a 0.8% agarose gel, transferred to a nylon membrane, and fixed DNA on a wet blotting membrane by baking the membrane at 120°C for 20 min. Membrane was hybridized with a DIG-labeled hybridization probe for telomeric repeats and incubated with anti-DIG-alkaline phosphatase. TRF was performed by chemiluminescence detection.