Abstract
The natural alkaloid berberine has been recently described as a promising anticancer drug. In order to improve its efficacy and bioavailability, several derivatives have been designed and synthesized and found to be even more potent than the lead compound. Among the series of berberine derivatives we have produced, five compounds were identified to be able to heavily affect the proliferation of human HCT116 and SW613-B3 colon carcinoma cell lines. Remarkably, these active compounds exhibit high fluorescence emission property and ability to induce autophagy.
Introduction
Berberine chloride (BBR) (C20H18NO4+; 5,6-dihydrodibenzo[a,g]quinolizinium chloride) is an isoquinoline quaternary alkaloid (Fig. 1) isolated from many well-known medicinal plants. It has been used in Ayurvedic and Chinese medicines for hundreds of years and shows a wide range of pharmacological and biochemical effects [1–3]. This alkaloid has a definite potential to be used as drug in a wide spectrum of clinical applications because it is effective against hyperlipidemia, diabetes, metabolic syndrome, polycystic ovary syndrome, obesity, fatty liver disease, coronary artery disease, gastroenteritis, diarrhea, hypertension, neurodegeneration, and cancer [1–3]. Of note, the structure of BBR makes it an attractive natural lead compound for the introduction of various chemical modifications in appropriate positions of the skeleton [4]. In particular, arylalkyl derivatives of BBR characterized by aromatic groups bonded to the C-13 position of the parent BBR via a linker of variable length have been reported to have anticancer properties [1,2,5,6].
Molecular structure of berberine (BBR), NAX053, NAX056, NAX057, NAX080, and NAX081 Different chemical modifications at positions 7, 8, 9, and 13 in the BBR molecule are shown in red; the added groups at position 13 are drawn in blue.
Recently, we analyzed a first set of BBR derivatives with a 13-(di)arylalkyl modification, which were demonstrated to exert anticancer effects on human p53wt colon cancer cell lines [2]. In the present work, we analyze a second set of related compounds bearing either a phenyl group or a benzhydryl group linked to position 13 of the berberine skeleton through a hydrocarbon linker of variable length (Fig. 1), trying to generate a geometric propensity for additional stacking-type and non-covalent aromatic interactions, either intramolecularly or intermolecularly, with cellular targets. It is well known that aromatic interactions are ubiquitous in nature and that their geometry plays a central role in the molecular interaction with biological macromolecules [7]. These new BBR derivatives are active on both wild-type and mutated p53 cell lines and induce autophagy. Remarkably, we established a correlation between drug efficacy and the fluorescence emission properties for each compound.
Materials and Methods
Synthesis and characterization of 13-(di)arylalkyl derivatives
The 13-(di)arylalkyl berberine derivatives were synthesized starting from commercial BBR hydrate (ca. 17% H2O) purchased from Shanghai T&W Pharmaceutical Co. (Shanghai, China) and the appropriate (di)arylalkylcarboxaldehydes via a modification of an unusual enamine–aldehyde condensation performed on 7,8-dihydroberberine [8]. The aldehydes were prepared starting from commercial (di)arylalkyl alcohols following usual oxidation methods and, when requested, homologation procedures known in organic chemistry. The purity (>95%) of the derivatives was assessed by high-performance liquid chromatography on a Jasco system LC-2000 series (Jasco, Cremella, Italy) with an Agilent Eclipse XDB-C18 (4.6 mm × 150 mm × 3.5 mm) column (Agilent Technologies, Santa Clara, USA). The flow rate of the mobile phase (50% water, 50% acetonitrile plus 0.1% trifluoroacetic acid) was maintained at 1 ml/min. The absorbance was measured at 235, 265, 340, and 420 nm, and retention times (rt) were measured in minutes.
The structures of the derivatives were confirmed by 1H NMR spectra recorded on a Varian Mercury 400 MHz spectrometer (Palo Alto, USA) from CDCl3 solutions.
NAX053
[13-(4,4-diphenylbutyl)-9,10-dimethoxy-5,6-dihydrobenzo[g]-1,3-benzodioxolo[5,6-a]quinolizinium chloride]: rt = 19.6; δ: 10.80 (s, 1H), 7.70 (d, 1H), 7.51 (d, 1H), 7.30 (m, 11H), 7.00 (s, 1H), 6.90 (s, 1H), 6.10 (s, 2H), 5.30 (t, 2H), 4.35 (s, 3H), 4.10 (s, 3H), 3.31 (d, 2H), 3.15 (m, 2H).
NAX056
[13-(5-phenylpentyl)-9,10-dimethoxy-5,6-dihydrobenzo[g]-1,3-benzodioxolo[5,6-a]quinolizinium chloride]: rt = 17.0; δ: 10.75 (s, 1H), 7.76 (s, 2H), 7.35–7.25 (m, 3H), 7.10 (s, 1H), 6.90 (s, 1H), 5.30 (m, 2H), 4.35 (s, 3H), 4.10 (s, 3H), 3.31–3.20 (m, 2H), 3.15 (d, 2H), 2.65 (m, 2H), 2.00–1.50 (m, 6H).
NAX057
[13-(6-phenylhexyl)-9,10-dimethoxy-5,6-dihydrobenzo[g]-1,3-benzodioxolo[5,6-a]quinolizinium chloride]: rt = 22.7; δ: 10.75 (s, 1H), 7.85 (d, 2H), 7.30–7.15 (m, 6H), 7.10 (s, 1H), 6.90 (s, 1H), 6.10 (s, 2H), 5.30 (m, 2H), 4.31 (s, 3H), 4.10 (s, 3H), 3.25 (m, 4H), 3.20 (m, 2H), 2.65 (m, 2H), 1.90–1.40 (m, 10H).
NAX080
[13-(5,5-diphenylpentyl)-9,10-dimethoxy-5,6-dihydrobenzo[g]-1,3-benzodioxolo[5,6-a]quinolizinium chloride]: rt = 25.8; δ: 10.65 (s, 1H), 7.80 (s, 2H), 7.30 (m, 10H), 7.10 (s, 1H), 6.85 (s, 1H), 6.10 (s, 2H), 5.25 (m, 2H), 4.35 (s, 3H), 4.10 (s, 3H), 3.90 (m, 1H), 3.25 (t, 2H), 3.15 (d, 2H), 2.15 (m, 4H), 1.45 (m, 2H).
NAX081
[13-(6,6-diphenylhexyl)-9,10-dimethoxy-5,6-dihydrobenzo[g]-1,3-benzodioxolo[5,6-a]quinolizinium chloride]: rt = 41.3; δ: 10.70 (s, 1H), 7.80 (s, 2H), 7.30 (m, 10H), 7.10 (s, 1H), 6.90 (s, 1H), 6.10 (s, 2H), 5.30 (m, 2H), 4.35 (s, 3H), 4.10 (s, 3H), 3.90 (m, 1H), 3.25 (t, 2H), 3.15 (d, 2H), 2.10 (m, 4H), 1.60 (m, 2H), 1.40 (m, 2H).
Cell culture and treatments
Human colon carcinoma HCT116 and SW613-B3 cell lines were obtained from Drs C.R. Boland and O. Brison, respectively, as mentioned previously [2]. Cells were grown at 37°C and 5% CO2 atmosphere in RPMI and Dulbecco's modified Eagle's medium, respectively, supplemented with 10% fetal bovine serum, 0.1 mg/ml penicillin, 100 U/ml streptomycin, 2 mM glutamine, and 2% sodium pyruvate. All reagents were from Euroclone (Milano, Italy). Twenty-four hours after seeding, cells were treated for 24 h either with etoposide [stock solution: 50 mM in dimethyl sulfoxide (DMSO); Sigma Aldrich, Milano, Italy] or 5-(N,N-hexamethylene)amiloride (HMA) (stock solution: 80 mM in DMSO; Sigma Aldrich) or with BBR and derivatives (stock solution: 10 mM in DMSO).
Morphological analysis
For bright-field microscopic observation, cells grown in 3.5 cm diameter Petri dishes (5 × 104 cells/ml) were incubated for 24 h with BBR derivatives at concentrations corresponding to their half inhibitory concentrations (IC50), followed by a 24 h recovery in drug-free medium. Thereafter, cells were observed using an Olympus IX71 microscope (Olympus, Tokyo, Japan), equipped with a 10× objective. Images were acquired with a digital camera Camedia C4040 using the MetaMorph acquisition software. Adobe Photoshop 9.0.2 was used as the elaborating software.
Viability assay
To calculate the IC50, the MTT metabolic viability assay, which measures mitochondrial activity, was applied according to a previously published protocol [2]. Briefly, cells were seeded in 96-well plates at a density of 2 × 103 cells in 100 µl per well. After 24 h of incubation, cells were treated with increasing concentrations (2.5, 5, 7.5, and 10 µM) of BBR or derivatives for 24 h. Parallel samples were incubated with 0.1% DMSO to evaluate the possible effect of the solvent. At the end of the incubation, 20 µl of Cell Titer 96 Aqueous One Solution (Promega Italia, Milano, Italy) were added to each well. The plates were then maintained at 37°C for 4 h in the dark; the absorbance of each sample was measured with a microplate reader (EZ Read 400; Biochrom, Cambridge, UK) at a wavelength of 492 nm. Data obtained from untreated cells were used as reference values (considered as 100%) to normalize the absorbance of the treated samples. Three independent experiments were carried out in quadruplicate. Statistical analysis was performed, and data were expressed as mean ± SD.
Clonogenic assay
As reported previously [2], to evaluate colony-forming ability, 2.5 × 102 cells were seeded in duplicate in 6 cm diameter Petri dishes in 2 ml of medium and grown for 24 h. Then, samples were treated with 0.5, 1, and 5 µM BBR or derivatives for 24 h, washed, incubated with 1 ml of complete medium for 10 days, and then processed according to the previous protocol [2]. Clonogenic capacity was expressed as the percentage with respect to untreated cells. Three independent experiments were performed in duplicate. Statistical analysis was applied, and data were presented as mean ± SD.
Immunofluorescence experiments
In situ conversion of LC3-I to form LC3-II and the presence of active caspase 3 were visualized by the immunofluorescence assay [2]. Briefly, cells were seeded on cover slips (5 × 104 cells/ml), treated with drugs for 24 h, and then processed as follows: for LC3, cells were washed and then fixed with 2% paraformaldehyde for 15 min on ice, washed again, and permeabilized with cold 100% acetone for 5 min. After extensive wash, samples were saturated with 4% bovine serum albumin in phosphate-buffered saline (PBS) for 10 min and incubated with a polyclonal antibody against LC3-II (2775, 1:100; Euroclone) for 1 h at 37°C in a humidified chamber. For active caspase 3, cells were fixed as mentioned earlier, permeabilized with 0.1% Triton in PBS for 10 min at room temperature, washed, and then saturated for 30 min with 5% skimmed milk in PBS. Cover slips were then incubated with the polyclonal antibody against active caspase 3 (ALX-210-807, 1:50; Alexis, Firenze, Italy) for 1 h at 37°C in a humidified chamber.
Thereafter, both samples were incubated for 1 h with the Cy2-conjugated anti-rabbit secondary antibody (111-225-003, 1:50; Jackson Immuno Research, Suffolk, UK). Cover slips were then washed in the dark, incubated for 10 min with 0.2 µg/µl 4′,6-diamidino-2-phenylindole (DAPI; Sigma Aldrich), and washed four times with PBS (5 min each). Slides were finally mounted with 20 µl of antifade solution (90% glycerol, 20 mM Tris–HCl, pH 7.5, 0.1% DABCO).
As a positive control of autophagy, cells were treated for 24 h with 20 µM HMA. For the induction of apoptosis, cells were incubated with 100 µM (HCT116 cells) or 250 µM (SW613-B3 cells) etoposide. Three independent experiments were performed. Cells were observed using an Olympus BX51 fluorescence microscope (Olympus), equipped with a 60× objective. The images were acquired with a digital camera Camedia C4040. Adobe Photoshop was used as the elaborating software. The quantifications of caspase 3 and LC3-positive cells were performed.
Western blotting
Protein expression in HCT116 and SW613-B3 cells treated for 24 h with BBR derivatives was evaluated by western blotting, according to a described protocol [9]. After running and transferring proteins onto nitrocellulose, membranes were incubated overnight at 4°C for 3 h with mAbs against PARP-1 (C2-10, 1:1000; Alexis), total caspase 3 (31A1067, 1:250; Alexis), and γ-tubulin (GTU-88, 1:10,000; Sigma Aldrich). Autophagy was monitored by the detection of the marker LC3 using the polyclonal antibody 2775 (1:1000) [31]. The appropriate horseradish peroxidase-conjugated (anti-mouse or anti-rabbit) secondary antibody (1:10,000; Jackson Immuno Research) was applied for 45 min at room temperature. All antibodies were diluted in Tris-buffered saline containing 5% skimmed milk and 0.1% Tween-20. Visualization of the immunoreactive bands was achieved using a chemiluminescent substrate of the Immun-Star WesternC Chemiluminescent Kit (Bio Rad Laboratories, Segrate, Italy). Samples treated with HMA (20 µM) or etoposide (100 µM for HCT116 cells and 250 µM for SW613-B3 cells) were used as positive control of autophagy or apoptosis, respectively. Three independent experiments were performed.
Fluorescence experiments
Because BBR is fluorescent (420 nm excitation and 500–600 nm emission range), its intracellular internalization can be investigated by the fluorescence assay. Cells were seeded in a 12-well plate at 7.5 × 104 cells/ml per well on a 19 mm diameter cover slip. After 24 h, cells were treated with BBR and derivatives at their IC50 for 24 h and then processed immediately for fluorescence observation or grown in drug-free medium for another 24 h and then analyzed. Samples were then washed with PBS, and images were recorded at 40× by an Olympus phase-contrast fluorescence microscope (Olympus) equipped with a 100 W Hg excitation lamp (Osram, Berlin, Germany) and a 4.1 Megapixel digital photocamera (Camedia C-4040 zoom; Olympus). Native fluorescence emission properties of BBR and its derivatives were assessed through a microspectrofluorometric analysis of drug solutions (10 µM). The fluorescence spectra were recorded under epi-illumination by means of a microspectrograph (Leitz, Wetzlar, Germany) equipped with an Optical Multichannel Analyzer (EG&G-PAR) and a 512-element-intensified diode array detector (mod. 1420/512). Excitation light was provided by a 100 W Hg lamp, combined with KG1-BG38 anti-thermal filters, and selected by a 366 nm band-pass interference excitation filter (full width at half maximum 10 nm, T%366 1⁄4 25). A 390 nm dichroic mirror (T%366 <2) was used to select both excitation light and emission signal. This was recorded in the 400–680 nm range, through a 430 nm long pass filter, by means of an oil immersion 95 Leitz objective with incorporated iris diaphragm (NA 1.10–1.32). Ten sequential scans of 200 ms each were performed, for a total of 2 s of acquisition time. The light fluence was 6.35 mW/cm2 at the focal plane, for a total light dose of 12.70 mJ/cm2 per acquired spectrum.
Statistical analysis
The analysis of variance and Dunnett's multiple comparison tests were used. The statistical analysis was performed using GraphPad Prism 5.0.
Results
BBR derivatives affect cell survival and proliferation
The IC50 values obtained with the MTT assay using increasing concentrations (2.5, 5, 7.5, and 10 µM) of BBR and of each derivative (NAX053, NAX056, NAX057, NAX080, and NAX081) are illustrated in Fig. 2A and summarized in Table 1. The dose-dependent cytotoxic effect was very similar for both cell lines, irrespective of the status of p53. BBR was less effective than the derivatives, among which NAX053 and NAX080 were the most potent on both cell lines. NAX053 showed a low efficacy at 1 µM and a potent effect at 10 µM, killing the HCT116 cells completely and reducing the viability of SW613-B3 cells to 21.0% ± 1.3%. NAX056 was more efficient in inhibiting cell viability at 1 µM (26.5% ± 5.1% for HCT116 cells and 64.8% ± 4.6% for SW613-B3 cells) and even more at 10 µM (14.4% ± 5.7% for HCT116 cells and 29.2% ± 1.8% for SW613-B3 cells). NAX057 decreased the viability of HCT116 cells to 41.5 ± 2.7 at 1 µM and to 0.9 ± 0.3 at 10 µM, whereas for SW613-B3 cells, the viability was reduced to 92.3 ± 1.5 at 1 µM and 25.1 ± 1.3 at 10 µM, respectively. After the treatment with 1 µM NAX080, cell viability was lowered to 59.6 ± 3.4 for HCT116 cells and unaffected for SW613-B3 cells; the treatment with 10 µM reduced cell viability to 2.5 ± 4.5 for HCT116 cells and to 2.8 ± 4.9 for SW613-B3 cells. NAX081 decreased the viability of HCT116 cells to 63.6 ± 5.6 at 1 µM and to 1.9 ± 3.4 at 10 µM; 1 µM was ineffective on SW613-B3 cells, whereas 10 µM reduced viability to 15.9 ± 5.8.
IC50 of BBR and derivatives in HCT116 and SW613-B3 cell lines
| Drug | IC50 (µM) | |
|---|---|---|
| HCT116 | SW613-B3 | |
| BBR | 31.97 ± 1.087 | 36.63 ± 1.083 |
| NAX053 | 6.22 ± 0.269 | 6.74 ± 0.124 |
| NAX056 | 7.31 ± 1.848 | 7.19 ± 0.937 |
| NAX057 | 6.57 ± 0.488 | 7.28 ± 0.931 |
| NAX080 | 5.95 ± 0.365 | 6.18 ± 0.649 |
| NAX081 | 6.41 ± 1.129 | 6.96 ± 1.236 |
| Drug | IC50 (µM) | |
|---|---|---|
| HCT116 | SW613-B3 | |
| BBR | 31.97 ± 1.087 | 36.63 ± 1.083 |
| NAX053 | 6.22 ± 0.269 | 6.74 ± 0.124 |
| NAX056 | 7.31 ± 1.848 | 7.19 ± 0.937 |
| NAX057 | 6.57 ± 0.488 | 7.28 ± 0.931 |
| NAX080 | 5.95 ± 0.365 | 6.18 ± 0.649 |
| NAX081 | 6.41 ± 1.129 | 6.96 ± 1.236 |
The IC50 was calculated by treating cells with increasing concentrations of BBR and derivatives for 24 h treatments; the MTT assay (three independent experiments) was carried out in quadruplicate (see the legend of Fig. 2). Data obtained from untreated control cells were considered as 100% (not included in the table), and the absorbances of treated samples were elaborated with GraphPad Prism 5.0 software and are expressed in micromolar as mean ± SD.
IC50 of BBR and derivatives in HCT116 and SW613-B3 cell lines
| Drug | IC50 (µM) | |
|---|---|---|
| HCT116 | SW613-B3 | |
| BBR | 31.97 ± 1.087 | 36.63 ± 1.083 |
| NAX053 | 6.22 ± 0.269 | 6.74 ± 0.124 |
| NAX056 | 7.31 ± 1.848 | 7.19 ± 0.937 |
| NAX057 | 6.57 ± 0.488 | 7.28 ± 0.931 |
| NAX080 | 5.95 ± 0.365 | 6.18 ± 0.649 |
| NAX081 | 6.41 ± 1.129 | 6.96 ± 1.236 |
| Drug | IC50 (µM) | |
|---|---|---|
| HCT116 | SW613-B3 | |
| BBR | 31.97 ± 1.087 | 36.63 ± 1.083 |
| NAX053 | 6.22 ± 0.269 | 6.74 ± 0.124 |
| NAX056 | 7.31 ± 1.848 | 7.19 ± 0.937 |
| NAX057 | 6.57 ± 0.488 | 7.28 ± 0.931 |
| NAX080 | 5.95 ± 0.365 | 6.18 ± 0.649 |
| NAX081 | 6.41 ± 1.129 | 6.96 ± 1.236 |
The IC50 was calculated by treating cells with increasing concentrations of BBR and derivatives for 24 h treatments; the MTT assay (three independent experiments) was carried out in quadruplicate (see the legend of Fig. 2). Data obtained from untreated control cells were considered as 100% (not included in the table), and the absorbances of treated samples were elaborated with GraphPad Prism 5.0 software and are expressed in micromolar as mean ± SD.
Effect of drugs on cell viability and clonogenic capacity (A) Four increasing concentrations of BBR and derivatives NAX053, NAX056, NAX057, NAX080, and NAX081 were used for 24 h treatments in quadruplicate (three independent experiments were carried out). Data obtained from untreated control cells were considered as 100% to normalize the absorbance of treated samples. (B) The colony-forming assay was performed using increasing concentrations of BBR and derivatives, in duplicate, and repeated three times. Colonies of more than 50 cells were counted, and their number compared with control cells, which were considered as 100%. *P < 0.05, **P < 0.01, and ***P < 0.001.
Although HCT116 cells were more sensitive than SW613-B3 cells, the effect on the latter cell line is very important, given that the previously analyzed BBR derivatives were effective only on p53wt HCT116 cells [2].
The clonogenic capacity was affected more by BBR derivatives than by the lead compound BBR, which in fact had no impact on this parameter (Fig. 2B and Table 2). It was evident that all derivatives reduced the number of colonies in the HCT116 cell line. NAX053 residual clonogenic capacity accounted for 87.74% at 0.5 µM, 46.25% at 1 µM, and 0% at 5 µM. NAX056 was proved to be more efficient than NAX053, with the number of colonies being reduced to 57.70% at 0.5 µM, 8.53% at 1 µM, and 0% at 5 µM. NAX057 was the most efficient (29.37% at 0.5 µM, 0.81% at 1 µM, and 0% at 5 µM). The effect of NAX080 was comparable to that of NAX053 (84.17% at 0.5 µM, 39.62% at 1 µM, and 0% at 5 µM). NAX081 also showed a strong effect on HCT116 cells (75.53% at 0.5 µM, 22.67% at 1 µM, and 0% at 5 µM). On the contrary, SW613-B3 cells were more resistant to the treatment (Fig. 2B and Table 2) than HCT116 cells. The lowest concentration of all derivatives (0.5 µM) did not affect the colony number. The cytotoxic effect of all the derivatives was more pronounced at the high concentrations (1 and 5 µM). NAX053 reduces the number of colonies to 93.65% at 1 µM and 9.37% at 5 µM. NAX056 and NAX057 appeared to be effective only at 5 µM, with the number of colonies being reduced to 36.79% and 2.93%, respectively. NAX080 and NAX081 caused a decrease in the number of colonies even at 1 µM: 79.20% and 83.11%, respectively, whereas at 5 µM, NAX080 lowered the number of colonies to 5.06% and NAX081 to 11.73%.
Colony-forming ability of HCT116 and SW613-B3 cell lines
| Drug (µM) | HCT116 | SW613-B3 |
|---|---|---|
| BBR | ||
| 0.5 | 97.26 ± 3.65 | 100.37 ± 9.16 |
| 1 | 94.48 ± 5.07 | 101.57 ± 2.46 |
| 5 | 93.04 ± 0.66 | 98.06 ± 10.33 |
| NAX053 | ||
| 0.5 | 87.74 ± 13.13 | 103.64 ± 9.19 |
| 1 | 46.25 ± 0.59 | 93.65 ± 5.16 |
| 5 | 0 | 9.37 ± 0.12 |
| NAX056 | ||
| 0.5 | 57.70 ± 7.63 | 112.54 ± 15.71 |
| 1 | 8.53 ± 0.27 | 104.06 ± 22.92 |
| 5 | 0 | 36.79 ± 16.67 |
| NAX057 | ||
| 0.5 | 29.37 ± 1.98 | 112.05 ± 12.26 |
| 1 | 0.81 ± 0.304 | 101.75 ± 0.56 |
| 5 | 0 | 2.93 ± 1.11 |
| NAX080 | ||
| 0.5 | 84.17 ± 10.61 | 102.02 ± 5.73 |
| 1 | 39.62 ± 9.622 | 79.20 ± 10.72 |
| 5 | 0 | 5.06 ± 1.43 |
| NAX081 | ||
| 0.5 | 75.53 ± 13.98 | 101.80 ± 12.10 |
| 1 | 22.67 ± 4.14 | 83.11 ± 6.21 |
| 5 | 0 | 11.73 ± 3.96 |
| Drug (µM) | HCT116 | SW613-B3 |
|---|---|---|
| BBR | ||
| 0.5 | 97.26 ± 3.65 | 100.37 ± 9.16 |
| 1 | 94.48 ± 5.07 | 101.57 ± 2.46 |
| 5 | 93.04 ± 0.66 | 98.06 ± 10.33 |
| NAX053 | ||
| 0.5 | 87.74 ± 13.13 | 103.64 ± 9.19 |
| 1 | 46.25 ± 0.59 | 93.65 ± 5.16 |
| 5 | 0 | 9.37 ± 0.12 |
| NAX056 | ||
| 0.5 | 57.70 ± 7.63 | 112.54 ± 15.71 |
| 1 | 8.53 ± 0.27 | 104.06 ± 22.92 |
| 5 | 0 | 36.79 ± 16.67 |
| NAX057 | ||
| 0.5 | 29.37 ± 1.98 | 112.05 ± 12.26 |
| 1 | 0.81 ± 0.304 | 101.75 ± 0.56 |
| 5 | 0 | 2.93 ± 1.11 |
| NAX080 | ||
| 0.5 | 84.17 ± 10.61 | 102.02 ± 5.73 |
| 1 | 39.62 ± 9.622 | 79.20 ± 10.72 |
| 5 | 0 | 5.06 ± 1.43 |
| NAX081 | ||
| 0.5 | 75.53 ± 13.98 | 101.80 ± 12.10 |
| 1 | 22.67 ± 4.14 | 83.11 ± 6.21 |
| 5 | 0 | 11.73 ± 3.96 |
The colony-forming assay was performed using three increasing concentrations of BBR and derivatives, in duplicate, and repeated three times. Colonies of more than 50 cells were counted and their number compared with control cells, which were considered as 100% (not included in the table). Data are expressed as mean ± SD.
Colony-forming ability of HCT116 and SW613-B3 cell lines
| Drug (µM) | HCT116 | SW613-B3 |
|---|---|---|
| BBR | ||
| 0.5 | 97.26 ± 3.65 | 100.37 ± 9.16 |
| 1 | 94.48 ± 5.07 | 101.57 ± 2.46 |
| 5 | 93.04 ± 0.66 | 98.06 ± 10.33 |
| NAX053 | ||
| 0.5 | 87.74 ± 13.13 | 103.64 ± 9.19 |
| 1 | 46.25 ± 0.59 | 93.65 ± 5.16 |
| 5 | 0 | 9.37 ± 0.12 |
| NAX056 | ||
| 0.5 | 57.70 ± 7.63 | 112.54 ± 15.71 |
| 1 | 8.53 ± 0.27 | 104.06 ± 22.92 |
| 5 | 0 | 36.79 ± 16.67 |
| NAX057 | ||
| 0.5 | 29.37 ± 1.98 | 112.05 ± 12.26 |
| 1 | 0.81 ± 0.304 | 101.75 ± 0.56 |
| 5 | 0 | 2.93 ± 1.11 |
| NAX080 | ||
| 0.5 | 84.17 ± 10.61 | 102.02 ± 5.73 |
| 1 | 39.62 ± 9.622 | 79.20 ± 10.72 |
| 5 | 0 | 5.06 ± 1.43 |
| NAX081 | ||
| 0.5 | 75.53 ± 13.98 | 101.80 ± 12.10 |
| 1 | 22.67 ± 4.14 | 83.11 ± 6.21 |
| 5 | 0 | 11.73 ± 3.96 |
| Drug (µM) | HCT116 | SW613-B3 |
|---|---|---|
| BBR | ||
| 0.5 | 97.26 ± 3.65 | 100.37 ± 9.16 |
| 1 | 94.48 ± 5.07 | 101.57 ± 2.46 |
| 5 | 93.04 ± 0.66 | 98.06 ± 10.33 |
| NAX053 | ||
| 0.5 | 87.74 ± 13.13 | 103.64 ± 9.19 |
| 1 | 46.25 ± 0.59 | 93.65 ± 5.16 |
| 5 | 0 | 9.37 ± 0.12 |
| NAX056 | ||
| 0.5 | 57.70 ± 7.63 | 112.54 ± 15.71 |
| 1 | 8.53 ± 0.27 | 104.06 ± 22.92 |
| 5 | 0 | 36.79 ± 16.67 |
| NAX057 | ||
| 0.5 | 29.37 ± 1.98 | 112.05 ± 12.26 |
| 1 | 0.81 ± 0.304 | 101.75 ± 0.56 |
| 5 | 0 | 2.93 ± 1.11 |
| NAX080 | ||
| 0.5 | 84.17 ± 10.61 | 102.02 ± 5.73 |
| 1 | 39.62 ± 9.622 | 79.20 ± 10.72 |
| 5 | 0 | 5.06 ± 1.43 |
| NAX081 | ||
| 0.5 | 75.53 ± 13.98 | 101.80 ± 12.10 |
| 1 | 22.67 ± 4.14 | 83.11 ± 6.21 |
| 5 | 0 | 11.73 ± 3.96 |
The colony-forming assay was performed using three increasing concentrations of BBR and derivatives, in duplicate, and repeated three times. Colonies of more than 50 cells were counted and their number compared with control cells, which were considered as 100% (not included in the table). Data are expressed as mean ± SD.
Morphological changes induced by BBR derivatives
At the end of the 24 h treatment, HCT116 and SW613-B3 cells were considerably decreased in number and were characterized by the presence of a large number of vacuoles, which were not visible in control samples. The two cell lines showed a different response after 24 h of recovery in drug-free medium. HCT116 cells were unable to recover and still showed vacuoles (Fig. 3), whereas vesicles disappeared in SW613-B3 cells, suggesting that the effect of all BBR derivatives could be rescued in SW613-B3 cells (Fig. 3).
Morphological changes induced by BBR and derivatives NAX053, NAX056, NAX057, NAX080, and NAX081 on HCT116 and SW613-B3 cell lines Microscopic observation was applied to samples treated with the IC50 of BBR derivatives for 24 h. Recovery in fresh drug-free medium for 24 h was also monitored. Scale bar: 50 μm.
BBR derivatives trigger autophagy
Because cytoplasmic vacuoles were detected in drug-treated cells (Fig. 3), we hypothesized that BBR derivatives induce autophagy, so we monitored the best marker of this process, that is, LC3 protein [10,11]. When autophagy is triggered, the cytosolic form of LC3 (LC3-I) is converted to its lipidated form (LC3-II), which is integrated into the autophagosome membranes (vacuoles). The status of LC3 was investigated by both immunofluorescence assay and western blotting. As a positive control, parallel samples were treated with 20 µM HMA, a proautophagic drug [9,12]. As expected, several fluorescent cells were visualized in both HMA-treated cancer cell lines, suggesting that autophagy has taken place (Fig. 4A). LC3-positive cells were also observed in both cell lines treated with BBR derivatives (Fig. 4A). Their quantification revealed that the phenomenon was more remarkable in HCT116 cells than in SW613-B3 cells (Fig. 4B). These results were confirmed by western blot analysis. Form I of LC3 was detectable as a band at 16–17 kDa in all samples (Fig. 4C), whereas LC3-II (14 kDa) was clearly visible only in HMA-treated cells and in samples treated with BBR derivatives (Fig. 4C). Overall, these results suggest that autophagy was activated by BBR derivatives.
LC3 protein autophagic conversion (A) Immunofluorescence analysis. The localization of LC3 (green fluorescence) was visualized by fluorescence microscopy (60×) in cells treated with BBR derivatives (NAX053, NAX056, NAX057, NAX080, and NAX081) or HMA for 24 h. DNA was stained with DAPI (blue fluorescence). Scale bar: 50 µm. (B) Quantification of LC3-positive green fluorescent cells. Black columns: HCT116 cells; gray columns: SW613-B3 cells. *P < 0.05, **P < 0.01, and ***P < 0.001. (C) Western blot analysis of LC3-I and LC3-II in the extracts from cells treated for 24 h with the IC50 concentration of BBR derivatives. Samples were processed as described. HMA treatment (20 µM, 24 h) represents the positive control for autophagy in both cell lines. γ-tubulin was used as the loading control.
On the contrary, no apoptotic hallmarks were detected in immunofluorescence assay or western blotting, as demonstrated by the loss of caspase 3 activation (Fig. 5A,B) and PARP-1 cleavage (Fig. 5C), which occurred only in cells treated with the pro-apoptotic drug etoposide. This body of evidence indicates that modifications on the structure of BBR improved its ability to affect cell viability and to trigger cell death possibly through the activation of the autophagic process.
Detection of caspase activation (A) Visualization of active caspase 3 by immunofluorescence in cells treated for 24 h with BBR derivatives (NAX053, NAX056, NAX057, NAX080, and NAX081) or etoposide (100 µM for HCT116 cells and 250 µM for SW613-B3 cells). Caspase 3: green fluorescence and DNA (stained with DAPI): blue fluorescence. Scale bar: 50 µm. (B) Quantification of green fluorescent caspase 3-positive cells. Black columns: HCT116 cells; gray columns: SW613-B3 cells. (C) PARP-1 cleavage monitored by western blot analysis in the same samples. Etoposide treatment (100 µM for HCT116 cells and 250 µM for SW613-B3 cells for 24 h) was used as positive control for apoptosis. 113 kDa: intact PARP-1 protein; 89 kDa: cleaved apoptotic fragment of PARP-1. γ-tubulin was used as the loading control.
Fluorescence properties of berberine
To exploit the fluorescence properties of BBR, we performed imaging fluorescence analysis and observed BBR green fluorescence within the cells as diffuse staining in the cytosol until the first 4 h of incubation (Fig. 6A). Later on (until 24 h), the fluorescence appeared to be concentrated in specific spots in the cytosol. The observation that the nucleus was not stained could suggest that the cytotoxic properties of BBR are not mediated by a direct effect on nuclear factors. Then, we extended our analysis to the three BBR derivatives NAX053, NAX056, and NAX057, which were demonstrated to be potentially active in vitro. Their fluorescence spectra clearly demonstrated that their emission amplitudes were much higher than BBR (Fig. 6B).
Berberine fluorescence properties (A) SW613-B3 cells were treated with 10 µM BBR for increasing times. Samples were analyzed by phase-contrast (upper) and fluorescence microscopy (lower). Scale bar: 50 µm. (B) Emission fluorescence spectra of BBR and derivatives NAX053, NAX056, and NAX057, are expressed as intensity with arbitrary units (a.u.).
The fluorescence properties of the new BBR derivatives prompted us to investigate their intracellular distribution in comparison with BBR. In fact, all derivatives entered both cancer cell lines and remained in the cytoplasm for up to 24 h, without any degradation or extrusion (Fig. 7). The absence of fluorescent signal in the nucleus suggested that in our experimental conditions, the drug effects were not mediated by a direct interaction with nuclear macromolecules. Of note, given that the lead compound BBR emitted a fluorescent signal lower than its derivatives (Fig. 7), we could affirm that the addition of aromatic groups at position 13 of BBR, that is a typical feature of these derivatives, was essential to increase fluorescence emission yield. In both cell lines recovered for 24 h in drug-free medium, the fluorescence was decreased. However, HCT116 cells retained more of the derivatives than SW613-B3 cells, which could explain that SW613-B3 cells can recover by discarding the drug more rapidly than HCT116 cells (data not shown).
Emission fluorescence properties of BBR and derivatives NAX053, NAX056, NAX057, NAX080, and NAX081 HCT116 and SW613-B3 cells were treated for 24 h with the IC50 concentration of drugs. Samples were observed by phase-contrast (left) and fluorescence microscopy (right). Scale bar: 50 µm.
Discussion
Several studies on different cancer cells treated with BBR reported its ability to regulate (up/down) different proteins that trigger apoptosis or autophagy [5]. We previously found that some BBR derivatives (NAX012, NAX014, and NAX018, characterized by arylalkyl groups bonded to the C-13 position) are able to induce DNA damage and cell cycle arrest, inhibit cellular proliferation, and promote cell death through apoptosis in a p53-dependent manner [2].
We focussed here on five new derivatives, designated as NAX053, NAX056, NAX057, NAX080, and NAX081, which are characterized by the presence of either phenyl or diphenylmethyl (benzhydryl) groups linked to position 13 of the BBR skeleton through hydrocarbon linkers of varying length (Fig. 1). We evaluated the antiproliferative effect of these BBR derivatives on colon carcinoma HCT116 and SW613-B3 cell lines, showing that NAX053, NAX056, NAX057, NAX080, and NAX081 are more effective than BBR, with an IC50 very similar for both cell lines, providing the evidence that the aforementioned modifications of the lead BBR resulted in derivative compounds with improved efficacy also on drug-resistant SW613-B3 cells [13–15].
Remarkably, we provided the interesting evidence that these new potent derivatives kill the cells through the induction of autophagy. Although few studies have described the ability of other BBR derivatives to induce autophagy, our data are in agreement with the results obtained with BBR in lung [16], liver [17], and hepatoma [18] cancer cell lines as well as in macrophages [19], adding a piece of information to the field of research aiming at investigating the proautophagic power of BBR [16–19]. The impact of autophagy on the response of cancer cells to BBR derivatives (and on their cytotoxicity) is still under investigation, in order to define whether this process could ensure cancer cell survival or act as a form of death, given that two opposite roles have been attributed to it [11,12,20,21].
Interestingly, NAX053, NAX056, NAX057, NAX080, and NAX081 were not able to induce apoptosis, in contrast to the derivatives previously analyzed (NAX012, NAX014, and NAX018). To explain why some BBR derivatives activate apoptosis or autophagy or both, we hypothesize that the length of the hydrocarbon linker at position 13 could be responsible for the diverse responses we observed. In fact, NAX012, NAX014, and NAX018 have C2 and C3 linkers, whereas NAX053, NAX056, NAX057, NAX080, and NAX081 have C4–C6 linkers. Thus, variations on the BBR structure can improve its antiproliferative effect, suggesting that the addition of (di)-arylalkyl groups is the most effective modification.
It has recently been reported that the lead compound BBR possesses fluorescence properties [22] as we confirmed in this study. Our new derivatives also possess fluorescence as proved by emission absorption spectra, and once entered the cell, remained localized only in the cytoplasm and did not reach the nucleus. The detection of BBR and its derivatives at the cytoplasmic level opens a new working hypothesis, different from the dogma that BBR binds DNA [23] and induces direct DNA damage [24,25], suggesting that cytoplasmic targets should also be investigated. In fact, preliminary assays monitoring the phosphorylation of the H2AX in cells treated with NAX053, NAX056, and NAX057 did not reveal a significant amount of γH2AX after a 24 h drug treatment (data not shown), suggesting that in our conditions the cytotoxicity is not related to a direct DNA damage, despite the reported effect of the lead compound BBR [26,27].
In conclusion, the present work demonstrates the following issues. (i) Compared with the lead compound BBR, BBR derivatives are more cytotoxic for both p53wt (HCT116) and p53mutated (SW613-B3) colon cancer cell lines with IC50 values similar for both cell lines. This result is relevant because it opens new perspectives for the treatment of SW613-B3 cells, given that they are generally characterized by an intrinsic resistance to antiproliferative and proapoptotic drugs [13–15]. Of course, this observation has to be validated by extending the analysis to other cell lines representative of each p53 condition. (ii) All derivatives trigger autophagy but not apoptosis. Thus, in our experimental system, autophagy could represent a genuine form of cell death, also known as PCD type II [12]. (iii) Fluorescence emission properties of BBR and derivatives revealed their localization within the cytoplasm of the cell, suggesting the existence of cytoplasmic targets governing their cytotoxic and autophagic potential. This is an original observation. Comparing viability results with fluorescence analysis of BBR derivatives, we could conclude that the intensity of fluorescence is correlated with the cytotoxic effect. More fluorescent is a derivative, more active it is. Because we did not observe a net fluorescence inside the nuclei of cells, we can hypothesize that these derivatives not necessarily induce DNA damage to activate cell death in a p53-dependent manner, but could induce other pathways such as autophagy [28] or parthanatos [29], which are mainly regulated by cytoplasmic factors.
Further experiments are required to confirm the correlation between the fluorescence properties and the cytotoxicity. Preliminary emission fluorescence data of our BBR derivatives characterized by arylamidealkyl (and not arylalkyl) substitution at position 13 (NAX027, NAX028, NAX029, and NAX030; data not shown) indicated that the fluorescence property depends on the nature of the added group. The correlation between the biological effects of BBR derivatives and its fluorescence is intriguing, because the detection of emission yield might be used as a preliminary assay to anticipate drug cytotoxicity of new synthesized compounds.
Funding
The work was supported by a grant from Regione Lombardia, Italy (Project Plant Cell, grant No. 13810040 to A.I.S. and Naxospharma).
Acknowledgements
The technical assistance of Cristian Israel Bastidas Vélez (UTPL, Ecuador) is acknowledged. L.M.G.O. was a PhD student (Dottorato in Genetica, Biologia Cellulare e Molecolare, University of Pavia, Italy) supported by SENESCYT (Quito, Ecuador) and Universidad Técnica Particular de Loja (Loja, Ecuador). F.A. is a PhD student (Dottorato in Genetica, Biologia Cellulare e Molecolare, University of Pavia, Italy).
