Anti-Cancer Efficacy of Silybin Derivatives - A Structure-Activity Relationship

<div><p>Silybin or silibinin, a flavonolignan isolated from Milk thistle seeds, is one of the popular dietary supplements and has been extensively studied for its antioxidant, hepatoprotective and anti-cancer properties. We have envisioned that potency of silybin could be further enhanced through suitable modification/s in its chemical structure. Accordingly, here, we synthesized and characterized a series of silybin derivatives namely 2,3-dehydrosilybin (DHS), 7-<i>O</i>-methylsilybin (7OM), 7-O-galloylsilybin (7OG), 7,23-disulphatesilybin (DSS), 7-<i>O</i>-palmitoylsilybin (7OP), and 23-<i>O</i>-palmitoylsilybin (23OP); and compared their anti-cancer efficacy using human bladder cancer HTB9, colon cancer HCT116 and prostate carcinoma PC3 cells. In all the 3 cell lines, DHS, 7OM and 7OG demonstrated better growth inhibitory effects and compared to silybin, while other silybin derivatives showed lesser or no efficacy. Next, we prepared the optical isomers (A and B) of silybin, DHS, 7OM and 7OG, and compared their anti-cancer efficacy. Isomers of these three silybin derivatives also showed better efficacy compared with respective silybin isomers, but in each, there was no clear cut silybin A versus B isomer activity preference. Further studies in HTB cells found that DHS, 7OM and 7OG exert better apoptotic activity than silibinin. Clonogenic assays in HTB9 cells further confirmed that both the racemic mixtures as well as pure optical isomers of DHS, 7OM and 7OG were more effective than silybin. Overall, these results clearly suggest that the anti-cancer efficacy of silybin could be significantly enhanced through structural modifications, and identify strong anti-cancer efficacy of silybin derivatives, namely DHS, 7OM, and 7OG, signifying that their efficacy and toxicity should be evaluated in relevant pre-clinical cancer models in rodents.</p> </div

Oxidation of Natural Bioactive Flavonolignan 2,3-Dehydrosilybin: An Electrochemical and Spectral Study

The electrochemical oxidation of the natural antioxidant 2,3-dehydrosilybin (<b>DHS</b>) was investigated in acetonitrile. The spectral changes during two electron and two proton oxidation registered by in situ IR spectroelectrochemistry show that the electron transfer is followed by a subsequent chemical reaction with traces of water. A benzofuranone derivative (<b>BF</b>) is formed by ECEC (electron transfer–chemical reaction–electron transfer–chemical reaction) process at the potential of the first oxidation wave. A minor difference in the chemical structures of flavonolignans <b>DHS</b> and silybin, the presence of a double bond between atoms C-2 and C-3 in the <b>DHS</b> molecule, causes the formation of completely different oxidation products. <b>BF</b> was for the first time identified as the product of the oxidation of flavonolignan <b>DHS</b>. Its formation was proved by electroanalytical, chromatographic, and spectroelectrochemical techniques. Molecular orbital calculations support the experimental findings

¹H NMR (400.13 MHz for ¹H, 100.61 MHz for ¹³C, DMSO-<i>d₆</i>, 30°C).

<p>¹H NMR (400.13 MHz for ¹H, 100.61 MHz for ¹³C, DMSO-<i>d₆</i>, 30°C).</p

Effect of low doses of pure optical isomers of silybin and its derivatives on HTB9 cell growth.

<p>HTB9 cells were treated with 5 and 10 µM doses of silybin A, 2,3-dehydrosilybin A, 7-<i>O</i>-methylsilybin A, 7-<i>O</i>-galloylsilybin A or silybin B, 2,3-dehydrosilybin B, 7-<i>O</i>-methylsilybin B, 7-<i>O</i>-galloylsilybin B. After 24 or 48 h of each treatment, total cell number was determined as detailed in the ‘<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060074#s2" target="_blank">Materials and Methods</a>’. In the bar diagrams, each data-point is representative of mean ± SD of 3 samples. *p≤0.001; #p≤0.01; $p≤0.05.</p

Effect of racemic mixtures and pure optical isomers of silybin and its derivatives on colony formation by HTB9 cells.

<p>(<b>A</b>) HTB9 cells (∼1000 cells) were plated in 6 well plates and treated every 72 h with 5 and 10 µM doses of silybin (SB), 2,3-dehydrosilybin (DHS), 7-<i>O</i>-methylsilybin (7OM), 7-<i>O</i>-galloylsilybin (7OG), silybin A (SB-A), 2,3-dehydrosilybin A (DHS-A), 7-<i>O</i>-methylsilybin A (7OM-A), 7-<i>O</i>-galloylsilybin A (7OG-A) or silybin B (SB-B), 2,3-dehydrosilybin B(DHS-B), 7-<i>O</i>-methylsilybin B (7OM-B), 7-<i>O</i>-galloylsilybin B (7OG-B). On 11^th day, cells were processed and colonies with more than 50 cells were counted. (<b>B</b>) HTB9 cells (∼1000 cells) were plated in 6 well plates and treated for 2 h every 24 h with 20 µM dose of silybin (SB), silybin A (SB-A), 2,3-dehydrosilybin A (DHS-A), 7-<i>O</i>-methylsilybin A (7OM-A), 7-<i>O</i>-galloylsilybin A (7OG-A) or silybin B (SB-B), 2,3-dehydrosilybin B(DHS-B), 7-<i>O</i>-methylsilybin B (7OM-B), 7-<i>O</i>-galloylsilybin B (7OG-B). After 7 days, cells were processed and colonies with more than 50 cells were counted. In the bar diagrams, each data-point is representative of mean ± SD of 3 samples. *, p≤0.001; #, p≤0.01; $, p≤0.05.</p

Chemical structure of silybin and its derivatives.

<p>(<b>a–h</b>) Chemical structures of silybin, silybin A, silybin B, 2,3-dehydrosilybin, 7-<i>O</i>-methylsilybin, 7-<i>O</i>-galloylsilybin, 7,23-disulphate silybin, 7-<i>O</i>-palmitoylsilybin, and 23-<i>O</i>-palmitoylsilybin.</p

¹³C NMR (400.13 MHz for ¹H, 100.61 MHz for ¹³C, DMSO-<i>d₆</i>, 30°C).

<p>¹³C NMR (400.13 MHz for ¹H, 100.61 MHz for ¹³C, DMSO-<i>d₆</i>, 30°C).</p

Effect of silybin and its derivatives on HTB9, HCT116, and PC3 cancer cell growth.

*<p>p≤0.001;</p>#<p>p≤0.01;</p>$<p>p≤0.05.</p

Effect of pure optical isomers of silybin, 2,3-dehydrosilybin, 7-<i>O</i>-methylsilybin, and 7-<i>O</i>-galloylsilybin on HTB9, HCT116, and PC3 cancer cell growth.

*<p>p≤0.001;</p>#<p>p≤0.01;</p>$<p>p≤0.05.</p

DataSheet1.PDF

<p>The inherited cardiomyopathies, hypertrophic cardiomyopathy (HCM) and dilated cardiomyopathy (DCM) are relatively common, potentially life-threatening and currently untreatable. Mutations are often in the contractile proteins of cardiac muscle and cause abnormal Ca²⁺ regulation via troponin. HCM is usually linked to higher myofilament Ca²⁺-sensitivity whilst in both HCM and DCM mutant tissue there is often an uncoupling of the relationship between troponin I (TnI) phosphorylation by PKA and modulation of myofilament Ca²⁺-sensitivity, essential for normal responses to adrenaline. The adrenergic response is blunted, and this may predispose the heart to failure under stress. At present there are no compounds or interventions that can prevent or treat sarcomere cardiomyopathies. There is a need for novel therapies that act at a more fundamental level to affect the disease process. We demonstrated that epigallocatechin-3 gallate (EGCG) was found to be capable of restoring the coupled relationship between Ca²⁺-sensitivity and TnI phosphorylation in mutant thin filaments to normal in vitro, independent of the mutation (15 mutations tested). We have labeled this property “re-coupling.” The action of EGCG in vitro to reverse the abnormality caused by myopathic mutations would appear to be an ideal pharmaceutical profile for treatment of inherited HCM and DCM but EGCG is known to be promiscuous in vivo and is thus unsuitable as a therapeutic drug. We therefore investigated whether other structurally related compounds can re-couple myofilaments without these off-target effects. We used the quantitative in vitro motility assay to screen 40 compounds, related to C-terminal Hsp90 inhibitors, and found 23 that can re-couple mutant myofilaments. There is no correlation between re-couplers and Hsp90 inhibitors. The Ca²⁺-sensitivity shift due to TnI phosphorylation was restored to 2.2 ± 0.01-fold (n = 19) compared to 2.0 ± 0.24-fold (n = 7) in wild-type thin filaments. Many of these compounds were either pure re-couplers or pure desensitizers, indicating these properties are independent; moreover, re-coupling ability could be lost with small changes of compound structure, indicating the possibility of specificity. Small molecules that can re-couple may have therapeutic potential.</p><p>HIGHLIGHTS</p><p>- Inherited cardiomyopathies are common diseases that are currently untreatable at a fundamental level and therefore finding a small molecule treatment is highly desirable.</p><p>- We have identified a molecular level dysfunction common to nearly all mutations: uncoupling of the relationship between troponin I phosphorylation and modulation of myofilament Ca²⁺-sensitivity, essential for normal responses to adrenaline.</p><p>- We have identified a new class of drugs that are capable of both reducing Ca²⁺-sensitivity and/or recouping the relationship between troponin I phosphorylation and Ca²⁺-sensitivity.</p><p>- The re-coupling phenomenon can be explained on the basis of a single mechanism that is testable.</p><p>- Measurements with a wide range of small molecules of varying structures can indicate the critical molecular features required for recoupling and allows the prediction of other potential re-couplers.</p><p></p