Role of Silymarin in Cancer Treatment: Facts, Hypotheses, and Questions

Abstract

The flavonoid silymarin extracted from the seeds of Sylibum marianum is a mixture of 6 flavolignan isomers. The 3 more important isomers are silybin (or silibinin), silydianin, and silychristin. Silybin is functionally the most active of these compounds. This group of flavonoids has been extensively studied and they have been used as hepato-protective substances for the mushroom Amanita phalloides intoxication and mainly chronic liver diseases such as alcoholic cirrhosis and nonalcoholic fatty liver. Hepatitis C progression is not, or slightly, modified by silymarin. Recently, it has also been proposed for SARS COVID-19 infection therapy. The biochemical and molecular mechanisms of action of these substances in cancer are subjects of ongoing research. Paradoxically, many of its identified actions such as antioxidant, promoter of ribosomal synthesis, and mitochondrial membrane stabilization, may seem protumoral at first sight, however, silymarin compounds have clear anticancer effects. Some of them are: decreasing migration through multiple targeting, decreasing hypoxia inducible factor-1α expression, inducing apoptosis in some malignant cells, and inhibiting promitotic signaling among others. Interestingly, the antitumoral activity of silymarin compounds is limited to malignant cells while the nonmalignant cells seem not to be affected. Furthermore, there is a long history of silymarin use in human diseases without toxicity after prolonged administration. The ample distribution and easy accessibility to milk thistle—the source of silymarin compounds, its over the counter availability, the fact that it is a weed, some controversial issues regarding bioavailability, and being a nutraceutical rather than a drug, has somehow led medical professionals to view its anticancer effects with skepticism. This is a fundamental reason why it never achieved bedside status in cancer treatment. However, in spite of all the antitumoral effects, silymarin actually has dual effects and in some cases such as pancreatic cancer it can promote stemness. This review deals with recent investigations to elucidate the molecular actions of this flavonoid in cancer, and to consider the possibility of repurposing it. Particular attention is dedicated to silymarin's dual role in cancer and to some controversies of its real effectiveness.

Introduction

Research on plants and their possible curative properties is not new. It has been occurring since ancient times. In the last 200 years this search has become more scientifically oriented and led to discoveries such as curare, strychnine, atropine, salicylate, digitalis, and more recently taxanes, artemisinin, vitamins, and many others. These naturally originated molecules “have cellular targets similar to those of new drugs developed by pharmaceutical companies.” ¹ Many of these natural products were so strikingly important for human health that they swiftly entered clinical practice. Sometimes, they were favorably modified by the pharmaceutical industry and then derivatives with enhanced benefits were born. While taxane compounds are one of the best examples of a success story in oncology, other compounds, not so blatantly effective as taxanes are on the waiting list. There is also a group of natural products that were, and are, used for known diseases other than cancer. In some cases, their antitumoral effects were slowly recognized and they were repurposed. Silymarin is one of this type of products, with some recognized antitumor effects however, repurposing has not yet occurred. Seeds of Silybum marianum, ² popularly known as milk thistle, have been used since ancient times to treat diverse ailments, and more recently liver damage due to toxins, particularly Amanita phaloides poisoning (but including many others such as carbon tetrachloride, ³ metals, allylalcohol) and alcohol-induced damage, including hepatitis, cirrhosis, and jaundice.^4–6 (From a technical point, what are commonly called seeds are actually fruits, but we shall call them seeds following other publication precedents). The last 15 years have witnessed a growing interest in silymarin and the plant it comes from: Silybum marianum (L.) Gaertn (also known as Carduus marianus and wild artichoke).

Although Silymarin is probably the most thoroughly studied nutraceutical, it is looked upon with skepticism by the medical profession for multiple reasons, such as:

1. ample distribution and easy accessibility to milk thistle;

2. over the counter availability;

3. the fact that it is a weed;

4. some controversial issues regarding bioavailability, and pharmacological actions;

5. its status as a nutraceutical rather than a drug according to FDA;

6. its vulgarization through many nonscientific Internet pages dedicated to silymarin compounds;

7. the enormous number of manufacturers, many of them scarcely known (Figure 1);

8. the direct consequence of this “popularization” is that it is available over the counter at the herbalist shop or through the Internet, rather than with a prescription in the pharmacy; ⁷

9. the lack of striking effects on the disease;

10. the fact that it is not usually considered in university-level pharmacology courses.

Figure 1.

A glimpse of the multiple brands and presentations of silymarin compounds in the US and European markets with the resultant “vulgarization.” There are many “silymarins” developed in well accredited pharmaceutical laboratories, but there are also many produced by scarcely known sources. Most can be acquired through the Internet.

Definition. Silymarin is the standardized extract obtained from the dried seeds of Silybum marianum (milk thistle) containing approximately 70% to 80% of the silymarin complex and an approximately 20% to 30% chemically undefined fraction, comprising mostly other polyphenolic compounds. The main component is silybin (silibinin). Silymarin and silybin are not synonyms. However, many older reports indistinctly use one or the other term, leading to some confusion. Silymarin extract and its components may frequently differ in their effects due to differences in solubility and bioavailability.

History. Silymarin has been used in Europe since the fourth century BCE by Theophrastus of Eresus, and reappears in the year 65 of current era in Pedanius Dioscorides’ De Materia Medica. Here he proposed milk thistle for the treatment of serpent venom bite and called it silybon. ⁸ It does not seem to be part of Traditional Chinese Medicine. ⁹ It was also used in Ancient Egypt, ¹⁰ however, we do not know exactly for what purpose. ¹¹ During the Renaissance some of the therapeutic effects were discovered and published by herbalists and physicians such as Pietro Andrea Mattioli (1544) and Hieronymus Bock (1539), among others. In the seventeenth century, an English botanist, Nicholas Culpeper, suggested that milk thistle was useful for liver diseases.

Location and Habitat. This invasive annual plant was originally found in the Mediterranean basin, but now it is present in all the continents. It requires dry, warm soil and it is very competitive eliminating other plants. ¹²

Chemistry. The standardized extract obtained from the seeds of Sylibum marianum is known as silymarin which contains between 70% and 80% of silymarin flavolignans. Sylibum marianum is a mixture of 8 flavolignan structurally related isomers: silybin (or silibinin), isosilibinin, silydianin, silychristin, isosilychristin, and taxifolin.^{13, 14} The main component of silymarin is Silibinin which is a compound consisting of equal amounts of silybin A and silybin B (CAS 22888-70-6).

BOX 1: Average composition of silymarin.

Silybin 60% to 70%

Silychristin 20%

Silydianin 10%

Isosilybin 5%

Taxifolin 1%

Small amounts of the flavonoids: quercetin, kaempferol, apigenin, naringin, eriodyctiol.

In 1959, Möschlin isolated silybin, ¹⁵ and then in 1968 silymarin chemistry was described in detail by Wagner et al,^{16, 17} and Pelter and Hansel. ¹⁸ Today, more than 50 years have elapsed since the initial hepatic antitoxic and protective function of the compound was discovered and now, its antitumor activity is under scrutiny (Figures 2 to 4). Silymarin, the active principal component of milk thistle, was originally thought to be one substance, until it was discovered that it is actually composed of a group of different flavolignans (Box 1).

Figure 2.

Milk thistle and the chemical structure of silybin (C25 H22 O10), with its proprietary numbering. Of note, is the similarity between silybin and steroid hormones. The lower panel shows silibinin's structural formula where 3 different chemical groups can be identified: a taxifolin and a coniferyl alcohol united by an oxirane ring. According to Biedermann et al, ²² “the 20-OH group was established to be the most active radical scavenging moiety and also the most important group responsible for the lipoperoxidation inhibitory activity.” Positions 2-3 also play a role in antioxidant activity because these positions can be oxidized to produce 2-3 dehydrosilybin (see Figure 3). Silybin has 5 hydroxyl groups in positions 3, 5, 7, 20, and 23 which are the targets to produce silybin derivatives. Positions 7 and 20 are usual sites of glucuronidation of silybin during its conjugation in human metabolism.

Figure 4.

Nomenclature diagram of the different components of milk thistle seed and silymarin. ²⁴ Isosilybin B seems to be the most powerful anticancer fraction. ²⁵ Silymarin and Silibinin are different concepts, however some older publications use both terms interchangeably. Silibinin is the more active form of the silymarin extract. Only silymarin extracts are available in pharmacies while researchers usually use silibinin for their experiments. Standard silymarin extracts usually contain 33.5% silybin, 13% silychristin, 8.35% isosilybin, 3.5% silydianin ²⁶ (see Figure 5).

Figure 3.

2-3 dehydrosilybin has 25-fold more antioxidant activity than silybin. ²³ Taxifolin and quercetin have more antioxidant activity than 2-3 dehydrosilybin. Small amounts of 2-3 dehydrosilybin are found in silymarin.

Silybin is stable in acidic conditions but unstable under alkaline conditions. Alkaline media disrupt flavolignan's skeleton. This is important because the extracellular matrix of tumors has a low pH (approximate pH = 6.8), while intracellular tumor pH is alkaline (approximate pH = 7.5), but only slightly more alkaline than normal cell intracellular pH (approximately = 7.2). ¹⁹ Normal cells, on the other hand, have an alkaline extracellular milieu (approximate pH = 7.35). We presume, without evidence to sustain the presumption, that silybin can reach the malignant cell's acidic extracellular space without degradation. This singular feature, the acidic extracellular pH of tumors, ²⁰ may explain why silybin effects differ in normal versus malignant cells. Silymarin may be able to better access the malignant cell compared with normal cells. This theory needs experimental confirmation.

Production. Silymarin extract is obtained by compressing the seeds which leads to a loss of lipids. Then, the active principal component is extracted with acetone, methanol, ethanol, or ethyl acetate. After a second lipid and impurities extraction, what is left is a mixture of flavolignans called silymarin. ²¹ Silybin is obtained from silymarin through methanolic extraction.

Biological activity. In 1975, Desplaces et al ²⁷ showed that silymarin had a protective effect on hepatocytes against phalloidin, the toxin of Amanita phalloides, when it was administered before the poison. When it was given immediately after phalloidin, it still protected hepatocytes but when given 30 min later, this protective action was negligible. Phalloidin produces acute hemorrhagic necrosis of hepatocytes. When silymarin was administered before the poison there were no morphologic (electron microscopic level) or biochemical signs of hepatic lesions. ²⁸ Silymarin was adopted as an “hepato-protector” by lay persons and the medical profession based on sometimes controversial evidence.

Hepato-protection. For example in:

1. Chronic hepatitis B and C: silymarin was able to lower transaminases but there was no change in viral load. ²⁹ However, Fried et al did not find benefits in chronic hepatitis C virus infected patients with high doses of silymarin, and did not find effective lowering of transaminases. ³⁰ No transaminase lowering was found with silymarin in hepatitis C virus infection in another study with very high doses of silymarin. ³¹ Other authors arrived to completely different results: silymarin had antiviral actions by blocking hepatitis C virus cellular entry and transmission. ³² As a first conclusion we may say that there is no clear evidence of silymarin's benefits in chronic hepatitis C.

2. Alcoholic hepatitis: Trinchet et al ³³ found no significant favorable effects of silymarin in alcoholic hepatitis in a double blind randomized study.

3. Nonalcoholic fatty liver disease: In this case, silymarin has shown favorable and less controversial results.^34–36

4. Reduction/inhibition of hepatic fibrosis: silymarin showed the ability to reduce hepatic fibrosis in the early stages of liver injuries. ³⁷

5. Cirrhosis: a large population study showed that silymarin decreased mortality in patients with hepatic cirrhosis. ³⁸

In spite of the evidence favoring its benefits in chronic liver disease, “the overall efficacy of silymarin remains unclear” according to Tighe et al. ³⁹ However, there are many, and some potentially beneficial, known biochemical effects of silymarin and silybin. For example, free radical scavenging and antioxidative properties of silybin are well known and have been thoroughly investigated. ⁴⁰ It is considered 10-fold more antioxidant than vitamin E. In 1977, Machicao and Sonnenbichler ⁴¹ showed that silybin increased RNA synthesis in rat liver cells and mainly increased the production of ribosomal RNA and polymerase A. Shriever et al ⁴² found that silymarin inhibited fatty acid synthesis in rat liver: fatty-acid-synthase and ATP-citrate-lyase, 2 of the main lipogenic enzymes, were diminished by about 50%. Fiebrich and Koch^43,44 described silymarin as a blocker of prostaglandin production in vitro through inhibition of both prostaglandin synthetase and lipoxygenase. This reduction of lipoxygenation was confirmed on liver ribosomes and mitochondria as well and probably explains silymarin's hepatoprotective actions. ⁴⁵

A few years later Sonnenbichler et al ⁴⁶ presented the first evidence that silymarin acted in a different way in noncancerous hepatic tissue and malignant cells: in the first case it stimulated DNA synthesis, in the second it did not. Silymarin is also a potent blocker of cyclic AMP breakdown (in vitro) by a phosphodiesterase preparation, ⁴⁷ an inhibitor of histamine release from human basophil leukocytes, ⁴⁸ dose-dependent downregulator of in vitro lymphocyte blastogenesis ⁴⁹ and alters the mitochondrial electron transport chain through mitochondrial calcium release, ⁵⁰ in addition to its antioxidant properties. ⁵¹ Immunostimulatory effects of silymarin were also described in experimental models, but not in the context of cancer treatment.^52,53

Silymarin and Other Diseases (Table 1)

Table 1.

Silymarin Research Beyond Hepatoprotection and Cancer: A Summary.

Type of disease	Specific disease	References
Neurologic diseases	General	62,63
	Parkinson's disease	64
	Alzheimer	65–67
	Multiple sclerosis	68,69
	Diabetic cognitive impairment	70
	Learning and memory deficits (in mice)	71
Diabetes	Diabetic complications	72–76
Hypercholesterolemia		77–81
Renal diseases	Cyclosporine nephrotoxicity	82
	Diabetic nephropathy	83,84
Ischemia/reperfusion	Damage prevention in general	85
	In heart muscle	86
	In the central nervous system	87,88
	In the kidney	89,90
	In intestine and bowel	91
	In the stomach	92
	In the lungs	93
	In the liver	94,95
	Multivisceral	96
Skin	Protection against UV radiation	97–99
	Melasma	100
	Rosacea	101
Immune system	Inhibition of UV-induced immune suppression	102
Infections: Viral	Covid infection	103,104
	Anti-Mayaro virus	105
	Anti-Chikungunya virus	106
	Anti-Zika virus	107
Infections: bacterial	Escherichia coli	108
Amiodarone	Improved effects on atrial flutter	109
	Decreased Amiodarone side-effects	110
Ulcerative colitis	Prolonged remission	111
Inflammatory bowel disease		112
Irritable bowel syndrome		113
Migraine	Reduced frequency of attacks	114
Endocrine	Hyperprolactinemia	115
	Decreased hot flashes in menopause	116
	Polycystic ovarian syndrome	117

Silymarin has been investigated and proposed for the treatment of many different diseases, from Alzheimer dementia ⁵⁴ to SARS 2 Covid-19, including diabetes, ⁵⁵ diabetic complications,^56–58 hyperlipidemia, and hypercholesterolemia,^50–61 among others. However, in the last 15 years, the main focus has been cancer.

Silymarin and Cancer

The first observation of silymarin's possible benefits in cancer is the 1991 publication by Mehta and Moon. ¹¹⁸ They showed that silymarin could act as a preventive (antipromoter) of cancer in mouse mammary glands treated with DMBA (dimethylbenzanthracene) and TPA (tetradecanoylphorbol acetate). The treatment protocol they employed made it possible to differentiate whether the chemoprevention worked at the initiation stage of carcinogenesis (DMBA phase) or during promotion (TPA phase). A 1991 review on the advances in pharmacological studies of silymarin by Rui, ¹¹⁹ did not mention anticancer activities. But in 1994, Agarwal et al ¹²⁰ performed a study on skin treated with TPA confirming the protective effect of this flavonoid against tumor promotion. Silymarin protected against induction of ornithine decarboxylase by TPA. Ornithine decarboxylase inhibition protects against tumor promotion. A protective effect of silymarin was also found in colon and small intestine adenocarcinoma cells induced by 1,2-dimethylhydrazine. ¹²¹ Silymarin and its components also inhibit beta-glucuronidase. ¹²²

Valenzuela and Garrido ¹²³ proposed 3 levels for silymarin's action in experimental animals:

• (a) as an antioxidant, by scavenging prooxidant free radicals and by increasing the intracellular concentration of the tripeptide glutathione;

• (b) through a regulatory action of cell membrane permeability and increase in its stability against xenobiotic injury;

• (c) through nuclear expression, by increasing ribosomal RNA synthesis, by stimulating DNA polymerase I, and by exerting a steroid-like regulatory effect on DNA transcription.

Silymarin also inhibits rat liver cytosolic glutathione S-transferase, ¹²⁴ although this function does not clearly hint towards anticancer activity. On the other hand, silymarin scavenges reactive oxygen species as noted above, and inhibits arachidonic acid metabolism in human cells, ¹²⁵ has antiinflammatory effects similar to those of indomethacin, ¹²⁶ protects skin against carcinogenic agents^127,128 and ultraviolet radiation.^129–131 These publications strongly suggest a cancer-preventive activity and silymarin is slowly emerging as an anticancer drug. For example, Scambia et al ¹³² tested the antiproliferative activity of silymarin on human ovarian and breast cancer cell lines and found a growth-inhibiting effect on both. Silymarin also showed synergism with the commonly used anticancer compounds doxorubicin and cisplatin.

In DU145, prostate carcinoma cells, silymarin showed inhibition of Erb1 (eukaryotic ribosome biogenesis protein 1) signaling and G1 arrest. ¹³³ In MDA-MB 486 breast cancer cells, G1 arrest was found due to increased p21 and decreased CDKs activity. ¹³⁴ In advanced human prostate carcinoma cells, silymarin decreased ligand binding to Erb1 ¹³⁵ and NF-kB expression was strongly inhibited by silymarin in hepatoma cells, ¹³⁶ as well as in histiocytic lymphoma, HeLa and Jurkat cells. ¹³⁷

According to Zi and Agarwal, low doses of silymarin inhibited ERK1 and ERK2 Map kinases in a skin cancer cell line (human epidermoid carcinoma A431) and at higher doses activated MAPK/JNK1. This means that at lower doses the effect was antiproliferative and at higher doses proapoptotic. ¹³⁸

Treating prostate carcinoma cells with silymarin the levels of PSA were significantly decreased and cell growth was inhibited through decreased CDK activity and induction of Cip1/p21 and Kip1/p27. ¹³⁹

Silymarin has also been shown to have a variety of other protective effects in various cell types, such as anti-COX2 and anti-IL-1α activity, ¹⁴⁰ antiangiogenic effects through inhibition of VEGF secretion, upregulation of Insulin like Growth Factor Binding Protein 3 (IGFBP3), ¹⁴¹ and inhibition of androgen receptors. ¹⁴² In leukemia HL-60 cells, silymarin inhibited proliferation and induced differentiation into monocytes in a dose-dependent manner. ¹⁴³ Another important effect of silymarin in cancer is the downregulation of the STAT3 pathway which was seen in many cell models. STAT3 is active in many types of cancer and is associated with poor prognosis and resistance to treatments.^144–146 Telomerase activity is another important factor in promoting carcinogenesis and evading senescense, thus inducing cancer cell immortality; silymarin has the ability to decrease telomerase activity in prostate cancer cells. ¹⁴⁷

Silymarin and Apoptosis

The apoptotic mechanism silymarin employs on cancer cells is generally p53 dependent, and follows the usual steps: increased proapoptotic proteins; decreased antiapoptotic proteins; mitochondrial cytochrome C release-caspase activation. ¹⁴⁸ Caspase inhibitors terminate silymarin apoptotic activity. Malignant p53 negative cells show only minimal apoptosis when treated with silymarin. Therefore, one conclusion is that silymarin may be useful in tumors with conserved p53.

Silymarin and Cancer Cell Migration

Enhanced cell migration is an important part of cancer progression. The antimigratory effects of silymarin in cancer cells are the result of mechanisms that ¹⁴⁹ :

1. inhibit histone deacetylase activity; ¹⁴⁹

2. increase histone acetyltransferase activity; ¹⁴⁹

3. reduce expression of the transcription factor ZEB1; ¹⁴⁹

4. increase expression of E-cadherin; ¹⁴⁹

5. increase expression of miR-203; ¹⁴⁹

6. reduce activation of sodium hydrogen isoform 1 exchanger (NHE1); ¹⁵⁰

7. target β catenin and reduce the levels of MMP2 and MMP9; ¹⁵¹

8. reduce activation of prostaglandin E2; ¹⁵²

9. suppress vimentin expression; ¹⁵³

10. inhibit Wnt signaling; ¹⁵⁴

11. modulate β1 integrin signaling. ¹⁵⁵

Silymarin and Angiogenesis

Angiogenesis is important in cancer growth because solid tumors need a blood supply to grow. Silymarin inhibits angiogenesis. There are various postulated mechanisms:

1. Decreased migration of endothelial cells. ¹⁵⁶

2. Flt1 (VEGFR1) upregulation. ¹⁵⁷ (VEGFR1 upregulation may act as a negative regulator of VEGFA that is upheld by this receptor with low protein kinase activity and therefore VGEFA is unable to bind to KDR [VEGFR2] with much higher kinase activity). ¹⁵⁸

3. VEGF downregulation. ⁴⁰

Silymarin and Epithelial–Mesenchymal Transition (EMT)

EMT is involved in tumor progression and metastatic expansion. In a transcriptome study of nonsmall cell lung cancer (NSCLC) cells, Kaipa et al ¹⁵⁹ found that silibinin had no effect on EMT. However, the opposite was found in other malignant tissues^160–162 where it showed inhibitory effects.

Silymarin and TIMP1

High expression of the tissue inactivator of metalloproteases I, or TIMP1, in cancer is a marker of poor prognosis^{163,164 54} because it is involved in tumor progression, metastasis, and shorter overall patient survival. TIMP1 also promotes accumulation of tumor-associated fibroblasts. ¹⁶⁵ Therefore, it may be considered a target in cancer treatment. Silymarin has the capacity to decrease TIMP1 expression^166–168 in mice.

Silymarin and LPAR1

LPAR1 and 3 (lisophosphatidic receptors 1 and 3) are related to cancer invasiveness.^169–172 Silymarin has the ability to downregulate LPAR1. ¹⁷³

Silymarin and TGF β2

Silibinin reduces the expression of TGF β2 in different tumors such as triple negative breast, ¹⁷⁴ prostate, and colorectal cancers. ¹⁷⁵ TGF β2 downregulation impedes the TGF β2/Smad pathway reducing cellular motility and MMP2 and MMP9 (metalloproteases) reducing invasion. In the liver, TGF β2 downregulation results in an antifibrotic effect, preventing hepatic fibrosis induced by inflammatory liver diseases.^166,176

Silymarin and Hypoxia Inducible Factor-1α (HIF-1α)

When cells are exposed to hypoxia, HIF-1α accumulates in the nucleus activating transcription of many genes and this plays an important role in tumor progression. Silymarin was found to decrease HIF-1α expression in rainbow trout brain ¹⁷⁷ and in rat lung under hypoxic conditions. ⁹³ In prostate cancer cells silibinin inhibited HIF-1α translation. ¹⁷⁸

Silymarin and CD44 and EGFR

CD44, the transmembrane receptor for hyaluronan, is increased in breast cancer and many other tumors, due to EGF (epidermal growth factor) stimulation. Silibinin decreased CD44 expression and the activation of EGFR (epidermal growth factor receptor) by EGF. ¹⁷⁹ In prostate cancer, silibinin decreased/inhibited CD44 expression as well. ¹⁸⁰ CD44 binding with hyaluronan triggers important protumoral signaling from its intracellular segment, inducing cancer cell survival, angiogenesis, migration, and invasion. The CD44 antigen (synonym HCAM) is a glycoprotein acting as an adhesion molecule ¹⁸¹ on the cell surface. Cell adhesion molecules play an important role in cell migration. In fact, CD44 has been shown to be strongly correlated with invasion^182,183 and metastasis.^184–186

Silymarin Modulation of TNFα (Tumor Necrosis Factor Alpha)

Tyagi et al ¹⁸⁷ showed that silibinin pretreatment of lung cancer cells inhibited TNFα induced “phosphorylation of STAT3, STAT1, and Erk1/2, NF-κB-DNA binding, and expression of COX2, iNOS, matrix metalloproteinases (MMP)2, and MMP9, which was mediated through impairment of STAT3 and STAT1 nuclear localization.”

Silymarin Inhibition of the Wnt/β-Catenin Signaling

The Wnt/β-catenin pathway is critical in cell proliferation, migration, and differentiation. It is a powerful regulator of embryonic development and tumorigenesis. Lu et al ¹⁸⁸ showed that silibinin inhibited the Wnt/β-catenin pathway in both prostate and breast cancer cells.

Silymarin Potentiation of TRAIL-Induced Apoptosis

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is part of the TNF superfamily. It is known to selectively induce apoptosis in cancer cells without having significant toxicity toward normal cells. Kauntz et al^189,190 found silibinin potentiated TRAIL-induced apoptosis in human colon adenocarcinoma cells. Furthermore, this potentiation was also found in TRAIL-resistant cells. Silibinin upregulated Death Receptors 4 and 5, thus increasing the number of receptors for TRAIL binding. Silibinin had the ability to induce not only the extrinsic apoptotic pathway, but also the intrinsic pathway. TRAIL sensitization by silymarin was also found in glioblastoma cells ¹⁹¹ and in hepatocarcinoma. ¹⁹²

Silymarin and Phospholipase A2

Secreted phospholipase A2 participates in inflammation and carcinogenesis. Silibinin downregulates secreted phospholipase A2 in cancer cells. ¹⁹³

Silymarin and Platelet-Derived Growth Factor (PDGF)

PDGF and its receptor are required for fibroblast proliferation and differentiation. It was found that silibinin had the ability to downregulate PDFG in fibroblasts, thus decreasing proliferation. ¹⁹⁴

Silymarin Decreases the Levels of Interleukin 8 (IL-8)

Interleukin 8 has been identified as a protumoral cytokine^195–198 and there is evidence showing that inhibition of IL-8 reduces tumorigenesis. ¹⁹⁹ Flavonoids, in general, reduce levels of IL-8. Curcumin, ²⁰⁰ apigenin, ²⁰¹ and silybin showed the ability to decrease IL-8 levels.^150,202,203

Silymarin Inhibits the Signal Transducer and Activator of Transcription 3 (STAT3) Pathway

STAT3 exists in the cytosol of cells and is a focal point of multiple oncogenic pathways. Silymarin inhibited STAT3 phosphorylation and decreased the expression of intranuclear sterol regulatory element binding protein 1 (SREBP1), decreasing lipid synthesis. The final consequences of these inhibitions were growth arrest and apoptosis. ²⁰⁴

Silibinin Acts as a Mitochondrial “Poison” in Malignant Cells

Si et al, ²⁰⁵ experimenting with 2 human breast cancer cell lines, MCF7 and MDA-MB-231, found that silibinin produced morphological and functional changes in mitochondria: decreased mitochondrial mass, condensed crests, reduced membrane potential and ATP content, and decreased mitochondrial biogenesis.

Silibinin and Metalloproteases

MMP2 and MMP9 play an important role in extracellular matrix remodeling and their levels correlate with progression of neuroblastoma tumors. ²⁰⁶ Yousefi et al ²⁰⁷ found that silibinin decreased MMP2, MMP9, and urokinase plasminogen activator receptor level (uPAR) in neuroblastoma cells. uPAR is also a marker of cell invasion.

Silymarin and COX2

COX-2 expression in cancer can stimulate angiogenesis and is associated with tumor growth, invasion, and metastasis. ²⁰⁸ Silymarin decreased the expression of COX2 in a model of chemically induced hepatocarcinoma in rats. ²⁰⁹

Silibinin and Programmed Death-Ligand 1 (PD-L1)

The programmed cell death protein and its ligand (PD-L1) complex play a key role in tumor progression being involved in growth regulation disturbance. This results in a defect in programmed cell death, apoptosis. ²¹⁰ Silibinin inhibits PD-L1 by impeding STAT5 binding in NSCLC. ²¹¹ This hints at the possible usefulness of silymarin as a complement to immune checkpoint inhibitors. A similar effect was found in nasopharyngeal carcinoma. ²¹² In renal carcinoma cells, silibinin decreased PD-L1 in murine renal cancer cells in vitro and in vivo. ²¹³

Silybin and Notch Signaling

The Notch signaling pathway is highly conserved, regulating development and is involved in angiogenesis and metastasis. ²¹⁴ Silybin inhibited Notch signaling in hepatocellular carcinoma cells showing antitumoral effects. ²¹⁵ However, dibenzazepine is much more powerful in this respect. ²¹⁶ Notch was also downregulated by silibinin in breast cancer cells impeding notch-1/ERK/Akt signaling ²¹⁷ and inducing apoptosis.

Silymarin and SIRT1

SIRT1 can deacetylate histones and other substrates and may act in a dual manner: as tumor suppressor or tumor promoter.^218,219 Silymarin has the ability to increase hepatic SIRT1 expression. ²²⁰ Silymarin can also increase SIRT1 expression in other tissues, such as hippocampus, ²²¹ articular chondrocytes, ²²² and heart muscle. ²²³ Silymarin seems to act differently in tumors: in lung cancer cells SIRT downregulated SIRT1 and exerted multiple antitumor effects such as reduced adhesion and migration and increased apoptosis. When SIRT1 was independently downregulated with siRNA the silymarin's antitumoral effects were increased. ²²⁴

Silymarin and VEGF/VEGFR

The angiogenic cytokine vascular endothelial growth factor (VEGF) and its receptor (VEGFR) play critical roles in vasculogenesis and angiogenesis. Jiang et al ²²⁵ found that adding silymarin to prostate and breast cancer cells swiftly reduced the secretion of VEGF to the medium in a dose-dependent manner. Silymarin also prevented VEGF expression in myocardial cells exposed to doxorubicin toxicity ²²⁶ as well as other manifestations of cardiotoxicity. A similar decrease in VEGF and VEGFR levels was found with silymarin in preeclamptic placenta, however the effect was very modest. ²²⁷ There is evidence showing that silymarin reduces VEGF expression at the transcription level. ²²⁸

Silymarin and Myc

C-Myc is a multifunctional master regulator transcription factor; it is activated by oncogenic pathways, drives many functions for rapid cell division, and inhibits antiproliferative pathways. ²²⁹ There is direct and indirect evidence that silymarin interacts with c-Myc, ²³⁰ in some cases increasing its expression in liver cells as a response to hepatic chemical injuries ²³¹ or decreasing it in malignancies. ²³² Rajamanickam et al^233,234 found that silymarin could prevent spontaneous tumorigenesis in an APC^min/+ mouse model (prone to develop intestinal tumors) by decreasing β-catenin, cyclin D1, c-Myc, phospho-glycogen synthase kinase-3β expression, phospho-Akt, and cyclooxygenase-2 in polyps. This report confirms silymarin's multitargeting effects in tumors and its different behavior in nonmalignant cells.

Silymarin and Carbonic Anhydrases

Carbonic anhydrases (CAs) play an important role in cancer progression, particularly those associated with the cell membrane (membrane CAs), namely isoforms CA9 (CAIX) and CA12 (CAXII). These CAs intervene in acidifying the extracellular substance and, working in tandem with sodium bicarbonate cotransporters, increase the intracellular pH. Downregulating or inhibiting membrane CAs has become a valid target for cancer therapy. Silymarin has the ability to inhibit CA isoforms CA I and CA II.^235,236 However, we could not find any publications specifically addressing silymarin's role as a possible inhibitor of membrane CAs.

Silymarin and Mitochondria

This is a controversial relationship. On one side, silymarin showed ability to reduce oxygen consumption in mitochondria NAD-dependent substrates, while on the other hand stimulating respiration in mitochondria oxidizing succinate. ⁵⁰ Silymarin increases mitochondrial release of Ca⁺⁺ and lowers mitochondrial membrane potential in cancer cells ²³⁷ and increases the transmembrane potential in toxic aggressions. ²³⁸ Regarding mitochondria we may presume that silymarin has context-dependent effects.

Antimetastatic Potential

Many of the features discussed above hint towards silymarin's antimetastatic potential. In a TRAMP (Transgenic Adenocarcinoma of the Mouse Prostate) model of prostate carcinoma, when mice were fed with silibinin invasion and metastasis were reduced. ²³⁹ The antimetastatic effect was due to less invasion, less EMT, less collagen I-cancer cell adhesion, and less expression of CD44. ²⁴⁰ In a randomized clinical study with patients harboring solid tumors, silymarin was added to standard chemotherapy. Although silymarin failed to improve the results, there was a a slight—not significant—trend towards reduced metastasis. ²⁴¹ We think that this study had some flaws which included a small sample size (15 patients with silymarin and 15 with placebo), tumors being present in different organs, and very low dosage (420 mg/day). In spite of these flaws, the trend towards a decrease in metastasis is still interesting and further study with a larger sample population was suggested by the authors.

Silymarin: Decreasing Side Effects and Toxicity of Chemotherapeutic Drugs

Silymarin coadministered with chemotherapeutic drugs has the ability to reduce toxicity in normal organs^242,243:

• It protects against liver and kidney toxicities induced by methotrexate in children and adults treated for leukemia.^244,245

• Silibinin decreased cisplatin's nephrotoxicity without affecting its antitumoral effectiveness.^246,247

• There is also evidence that it protects the heart from doxorubicin toxicity, however, it is less potent than quercetin in this effect. ²⁴⁸

• Silymarin reduced docetaxel central and peripheral neurotoxicity. ²⁴⁹

• Silymarin was able to decrease diarrhea produced by irinotecan treatment. ²⁵⁰

• Silymarin reduced hepatotoxicity in patients with nonmetastatic breast cancer receiving doxorubicin /cyclophosphamide-paclitaxel. ²⁵¹

Silymarin and Resistance to Treatment

Rho et al ²⁵² found that adding silymarin to epidermal growth factor receptor tyrosine kinase inhibitors could overcome resistance produced by the T790M mutation in NSCLC xenografts. The mechanism of action seems to be impeding EGFR dimerization. It was found that bladder cancer cell lines resistant to cisplatin could be resensitized with silymarin. ²⁵³ A similar result was obtained with ovarian cancer cells resistant to paclitaxel. ²⁵⁴ The mechanism involved in resensitization to chemotherapeutic drugs is not fully known, however possible factors are: inhibition of NF-kB nuclear migration, ¹³⁶ inhibition of survivin protein levels, downregulation of Pgp (MDR 1),^255–258 and multidrug resistance-associated protein 1 (MRP 1).^258,259 Silychristin (a component of silymarin) and silychristin derivatives have shown the particular ability to inhibit Pgp activity in a concentration-dependent manner ²⁶⁰ (see Tables 2 to 12).

Table 2.

Melanoma.

Year, Ref.	Findings
2006 285	Silymarin proposed for chemoprevention of melanoma.
2007 286	Silymarin increased proapoptotic effects of anti-Fas agonistic antibody CH11 in melanoma cells.
2013 287	Silymarin for prevention for melanoma invasion.
2013 288 X	Silymarin decreased the growth of melanoma xenografts and locked MEK1/2-ERK1/2-RSK signaling that led to a reduction of NF-kB activator protein1 and STAT3, which resulted in cell cycle arrest and inhibited tumor growth in vitro and in vivo
2015 289 X	Silymarin targeted cell cycle regulators, angiogenesis, and induced apoptosis in vitro and in vivo.
2016 290	Silymarin as a modulator of the wnt/β catenin pathway.

Table 12.

Miscellaneous.

Year, Ref.	Organ/tumor	Findings
2013 361	Pharynx	Silymarin increased PTEN, reduced Akt phosphorylation, and induced apoptosis in pharynx squamous cell carcinoma cells.
2015 362	Glioma	Silibinin induced apoptosis in glioma cells through downregulation of PI3K pathway and decreased FoxM1 transcription factor expression.
2018 363 X	Oral cancer	In vivo, silymarin reduced tumor growth and volume; in vitro it produced apoptosis through induction of the DR5 (death receptor 5)-caspase 8-truncated Bid pathway.
2017 364	Salivary glands	Silymarin increased proapoptotic Bim protein in mucoepidermoid carcinoma cells, inducing apoptosis.
2012 365	Cervical	Silymarin induced apoptosis in cervical cancer cells through increase of PTEN, inhibition of Akt phosphorylation, and decreased expression of MMP9.
2007 366	Osteosarcoma	Silibinin decreased human osteosarcoma cell invasion through Erk inhibition of a FAK/ERK/uPA/MMP2 pathway.
2017 367	Rabdoid tumor	Silibinin inhibited rabdoid tumor cell migration and invasion through inhibition of the PI3K/Akt pathway.
2019 368	Human gastric cancer cells	Silymarin downregulated the MAP kinase pathway inhibiting growth and inducing apoptosis.

Table 4.

Lung NSCLC: Nonsmall Cell Lung Cancer//SCLC: Small Cell Lung Cancer.

Year, Ref.	Findings
2004 306	Silibinin impeded invasion by inhibiting expression of uPA and MMP2 and enhancing expression of TIMP2.
2013 307	Silibinin meglumine impeded the epithelial–mesenchymal transition in EGFR mutant NSCLC cells.
2010 308	Silibinin decreased NSCLC cell growth through cell cycle arrest and decreased cell cycle modulators.
2011 309	Silymarin produced apoptosis in a highly metastatic lung cancer cell line through the mitochondrial caspase pathway.
2003 310	Silibinin inhibited growth and apoptosis in NSCLC and SCLC line cells in a dose-dependent manner.
2012 311	HDAC inhibitors in combination with silibinin showed enhanced antitumor activity in NSCLC cells. There was also increased transcription of p21 through higher acetylation of its promoter. The augmented p21 was responsible for proteasomal destruction of cyclin B1.
2013 312	EGFR-mutated lung cancer cells, resistant to erlotinib and overexpressing ALDH, were resensitized by silibinin.
2009 313 X	Treating a xenograft lung cancer mouse model with silibinin resulted in a decreased tumor size through decreased angiogenesis. HIF-1α was also decreased by silibinin.
2016 314	Through downregulation of STAT3, silibinin reinstated sensitivity to crizotinib therapy in ALK rearranged lung cancer.

Table 7.

Breast.

Year, Ref.	Findings
2004 325	Silymarin, as part of a mixture of flavonoids, downregulated the Breast Cancer Resistance Protein (BCRP). The authors propose a “flavonoid cocktail” for this purpose.
2004 326	Silibinin synergized with conventional chemotherapeutic drugs in anticancer effects on breast cancer cells.
2009 327	Silibinin decreased MMP9 and VEGF expression induced by TPA through downregulation of the Raf/Mek/Erk pathway.
2013 328	Silymarin showed synergy with doxorubicin in producing MCF7 cell apoptosis
2014 329	Silymarin showed much higher proapoptotic gene induction in a lung cancer cell line than in a breast cancer cell line.
2014 330 X	Silibinin inhibited the accumulation of myeloid derived suppressor cells (MDSC) in murine breast cancer and increased overall survival. Silibinin decreased tumor volume.
2015 331	Silibinin induced autophagic death in breast cancer cells. Silibinin treatment decreased ATP levels and altered mitochondrial electric potential with increased ROS accumulation.
2015 332	Silibinin induced apoptosis in breast cancer cells. (Comment: the concentrations used were too high and are not achievable in human use).
2015 333	ERα inhibition was a key factor in silibinin-induced autophagy and apoptosis. Using ERα inhibitors with silibinin, both apoptosis and autophagia were further increased.
2016 334	Silibinin decreased BCL2 proteins in breast cancer cells and normal breast cells and ununiformly increased PTEN in different cancer cell lines.
2017 335	Silibinin sensitized breast cancer cells to doxorubicin treatment. (Comment: The concentrations used were excessively high and difficult to achieve in the clinical setting).
2017 336	Silymarin-loaded iron nanoparticles produced cell cycle arrest in triple negative breast cancer cells.
2017 337	Silymarin's anticancer effects were due to inhibition of Akt and MAPK pathway.
2021 338 X	Silymarin decreased proliferation and viability of MDA-MB-231 and MCF-7 cells in a concentration-dependent manner, inducing apoptosis. These results were obtained in vitro and in vivo.

Table 8.

Colon.

Year, Ref.	Findings
2002 339 X	Silymarin inhibited chemically induced carcinogenesis of the colon in mice.
2013 340 X	Silibinin blocked TNFα-induced NF-kB activation in vitro and in vivo. Tumor growth and progression were concomitantly Inhibited. Bcl2, COX2, VEGF, and MMPs levels were also diminished by silibinin feeding of xenotransplanted mice.
2015 341	Silymarin induced proteasomal degradation of cyclin D1 and inhibited growth of colon cancer cells.
2016 342	Treatment with silymarin increased the efficacy of ionizing radiation on colon cancer cells causing increased cell death.
2017 343	Silibinin inhibited proliferation and increased apoptosis in colon cancer cells.
2017 344 X	The combination of regorafenib and silybin had synergistic antiproliferative and proapoptotic effect. This combination was tested in 22 patients with metastatic colon cancer. No control group was available.
2020 345	Sylimarin, associated with other nutraceuticals, reduced intestinal polyp growth in an animal model.

Table 9.

Liver.

Year, Ref.	Findings
2005 346	Silibinin strongly inhibited growth of hepatocellular cancer cells. It also increased apoptosis with inhibition of CDK2, CDK4, and CDC2 kinases.
2006 347 X	Silymarin inhibited hepatocarcinogenesis induced by nitrosodiethylamine.
2008 348 X	Silymarin decreased the expression of MMP2 and MMP9 and decreased recruitment of mast cells in vivo in a rat liver carcinogenesis model.
2008 349	Silibinin decreased cell proliferation and migration of human hepatocellular cancer cells by inhibiting the Erk 1/2 cascade.
2009 350	Silymarin decreased growth of hepatocellular carcinoma (HCC) cells and induced apoptosis.
2009 351 X	In a xenograft mouse model of HCC, silibinin reduced growth and proliferation through reduction of Akt/Erk signaling and increased histone acetylation.
2015 352	Silibinin increased growth inhibition of hepatocarcinoma cells by either sorafenib or gefitinib.
2020 353	Silymarin showed antimetastatic and proapoptotic effects on HepG2 cells through the Slit-2/Robo-1 pathway.

Table 10.

Ovary.

Year, Ref.	Findings
2014 354	Silymarin suppressed cell growth and induced caspase-dependent apoptosis with increased p53, p21, and p27, and decreased CDK2.
2003 355 X	A silybin-phosphatidylcholine complex decreased tumor growth in xenografted mice (tumor weight inhibition of 78%).
2013 356 X	Silibinin decreased tumor growth in vitro and in vivo through downregulation of Erk and Akt signaling.

Table 11.

Hematologic.

Year, Ref.	Disease	Findings
2001 143	Promyelocytic leukemia	Silymarin inhibited proliferation and induces differentiation into monocytes. It showed synergy with vitamin D3.
2010 357	Acute myeloid leukemia	Silibinin induced differentiation of acute myeloid leukemia cells ex vivo (only in cases in which there were no chromosome aberrations).
2016 358	Lymphoma	Silibinin induced apoptosis in Alk-positive anaplastic large cell lymphoma by suppressing the phosphorylation of NPM/ALK.
2020 359	Lymphoma	Epstein-Barr positive lymphoma cell proliferation was inhibited and apoptosis induced through NF-kB inhibition by silymarin.
2016 360	Multiple myeloma	Silybin suppressed myeloma cell proliferation and induced apoptosis by inhibiting the PI3K/Akt/mTOR pathway.

Silymarin's Cancer Chemopreventive Actions

Table 5 summarizes the findings by Vinh et al that show that silymarin was able to significantly decrease the incidence of bladder neoplasms in male rats receiving the carcinogenic substance N-butyl-.N-(4-hydroxybutyl) nitrosamine. Interestingly, these results were achieved by oral administration of silymarin and were found in those animals that received silymarin not only at the initiation of carcinogenesis, but also in those of the postcarcinogenic period (for more examples on chemoprevention, see Tables 2 to 12).

Table 5.

Bladder.

Year, Ref.	Findings
2017 253	Silibinin reversed chemotherapeutic resistance in bladder cancer cells in a NF-kB signal-dependent and independent manner.
2017 315	Silibinin is an antiproliferative compound whose mechanism of action depended on p53 status (WT or mutated).
2002 316 X	In an induced bladder carcinogenesis mouse model, silymarin reduced the incidence of bladder lesions and cell proliferation. Silymarin acted as a preventive compound.
2017 317	The anticancer mechanism of silibinin in bladder cancer was through downregulation of the actin cytoskeleton, the PI3K/Akt pathway and KRAS.
2011 318 X	Oral silibinin prevented carcinogenesis, decreased proliferation, and increased apoptosis in vivo in a mouse model. Intravesical silibinin had a similar effect.
2013 319 X	Silibinin decreased bladder cancer metastasis and prolonged animal survival through downregulation of the GSK3β/β catenin pathway and Zeb1 expression. Silibinin also suppressed EMT and stemness.

Silymarin and Hormonal Receptors

Silymarin is a selective estrogen β receptor (ER-β) agonist. ²⁶¹ However, it also has some estrogenic effects through ER-α. ²⁶² Silymarin has strong binding affinity to ER-β and a mild affinity for ER-α. ²⁶³ Silymarin's estrogenic actions should be seriously considered as a problem in female hormone-dependent tumors. Furthermore, silymarin's estrogenic effects are confirmed by the observation that it produces benefits in menopausal women with hot flashes. ¹¹⁶ Contrario sensu, it may be advantageous in benign prostate hyperplasia^264,265 and prostate cancer. However, in an experiment carried out in albino rats, silymarin increased testosterone and LH. ²⁶⁶ It also increased spermatogenesis in rats. ²⁶⁷ In spite of these 2 findings, the evidence for silymarin benefits in prostate cancer is abundant (see Table 3). Our conclusion is that the possible benefits found in prostate cancer are independent of silymarin's hormonal effects.

Table 3.

Prostate.

Year, Ref.	Findings
2008 291	Silibinin had inhibitory effects on survival, motility and adhesion of highly metastatic prostate cancer cells.
2005 292	Isosylibin B was the most potent fraction of silymarin against proliferation in different prostate cancer cell lines (LNCaP, DU145, and PC3). Silymarin used in this paper contained 5% isosylibin B. Isosilybin was a suppressor of the promoter of the topoisomerase IIα gene in DU145 cells.
2001 142	Silymarin inhibited the androgen receptor (AR) by reducing its localization in the nucleus without modifying AR expression or binding ability. This action was probably related to silymarin's downregulation of FKBP51 that is a probable transporter of AR to the nucleus.
2020 293	Silymarin inhibited DU 145 cell proliferation through two mechanisms: activation of the SLIT 2 protein and inhibition of CXCR4.
1999 294	Silibinin inhibited growth by G1 cell cycle arrest in hormone-refractory human prostate carcinoma cells without apoptosis. It decreased CDKs and PSA and increased p21 and p27.
2006 295	Silymarin and silibinin produced G1 and G2-M cell cycle arrest with a decrease in CDKs and Cdc2 kinase activity, and an increase in CDK inhibitors.
2002 296	Silibinin treatment caused growth inhibition, apoptosis, and decreased viability in different prostate cancer cell lines.
2007 297	Isosylibin A and Isosylibin B caused growth arrest and apoptosis in human prostate cancer cell lines.
2004 298	Silibinin inhibited mitogenic signaling in prostate cancer cells.
2007 299	Silibinin had a synergistic effect on Mitoxantrone inhibition of cell growth arrest and apoptosis of prostate cancer cells.
2005 300 X	Dietary supplementation of silymarin in rats decreased the incidence of induced rat prostate carcinoma.
2010 301	Silibinin reversed epithelial mesenchymal transition, induced upregulation of cytokeratin-18, and downregulation of vimentin, MMP2, NF-kB nuclear translocation, and transcription factors ZEB1 and SLUG.
2010 302	Isosylibin A induced apoptosis in prostate cancer cells through phosphoAkt, NF-kB, and AR downregulation.
2003 303 X	In nude mice with xenografted prostate carcinomas, silibinin feeding increased apoptosis and reduced growth and angiogenesis.
2002 304 X	In nude mice xenografted with human prostate carcinoma cells silibinin feeding decreased tumor growth by 35% to 58%.
2002 305	In human androgen-dependent prostate cancer silibinin inhibited Rb phosphorylation and increased Rb-E2F.

Silymarin Inhibits Clathrin-Dependent Trafficking

Endocytosis is an important mechanism of cell intercommunication which acquires major relevance in cancer. This process is initiated by the invagination of the plasma membrane. The protein clathrin provides A coat to this invagination (Figure 5). The clathrin coated vesicle has the ability to select for the adequate cellular receptor. ²⁶⁸ There is also endocytosis without clathrin coating. Silymarin has the ability to inhibit clathrin-dependent trafficking at least in the case of certain viruses such as Hepatitis C virus, reovirus, influenza virus,^269,270 and Hepatitis B virus. ²⁷¹ The mechanism behind this inhibition is through interference with the clathrin endocytic pathway. Actually, silymarin interferes with all the clathrin-dependent endocytic processes. Taxifolin, a close relative of silybin, was also found to inhibit receptor-mediated endocytosis of β-hexosaminidase in normal fibroblast culture. There were similar findings with other flavonoids. ²⁷²

Figure 5.

Similarities and differences among silymarin's components.

Although there is no experimental evidence in this sense, we may presume that silymarin decreases endocytic trafficking in cancer cells too. Additionally, clathrin has protumoral effects beyond endocytosis: it switches TGF-β into a procancer role. ²⁷³

Figure 6 shows a simplified overview of the clathrin-dependent endocytosis.

Figure 6.

Silymarin impedes clathrin-dependent endocytosis. In receptor-mediated endocytosis, or clathrin-mediated endocytosis, cells incorporate hormones, proteins, and sometimes viruses in a selective manner that depends on a ligand-receptor interaction. (1) The ligand receptor interaction takes place on the cell surface. (2) An adapter protein and clathrin “coat” the internal surface of the receptors and a process of membrane invagination starts. (3) Advanced invagination process. (4) The adaptor protein and clathrin are released. Silymarin impedes this mechanism of endocytosis. Clathrin-dependent endocytosis of surface receptors participates in cellular signaling pathways. Clathrin seems to be a valid target in cancer therapy. ²⁷⁴

Silymarin and Renal Carcinoma

When targeting renal carcinoma cells with silymarin, migration and invasion were significantly decreased by inhibition of the EGFR/MMP-9 pathway: silymarin blocked phosphorylation of EGFR and ERK1/ERK2 and reduced expression of MMP-9. ²⁷⁵ This was confirmed by Liang et al ²⁷⁶ and by Chang et al in vivo ²⁷⁷ and in our unpublished observations with 2 patients with grade IV clear cell renal carcinoma, who experienced no new metastasis after they started on silymarin.

Silymarin and Pancreatic Cancer

Silymarin has not been extensively tested in pancreatic ductal adenocarcinoma (PDAC) (see Table 6). However, it does have an important antifibrotic effect. One of the major problems in PDAC is the intense stromal reaction with abundant production of stromal collagen fibers. ²⁷⁸ These impede delivery of the chemotherapeutic drug to the tumor mass and create interstitial hypertension through the strongly hydrophilic hyluronan. Therefore, silymarin's antifibrotic effects may provide an interesting complement to standard treatment. ²⁷⁹

Table 6.

Pancreas.

Year, Ref.	Findings
2011 320	Silibinin induced cell cycle arrest and apoptosis in certain pancreatic cancer cell lines.
2015 321	The combination of an HDAC inhibitor and silibinin had additive effects on growth inhibition and apoptosis of pancreatic cancer cells.
2015 322 X	In an orthotopic model of pancreatic cancer, silibinin reduced glycolytic activity of cancer cells, proliferation, and cachexia.
2013 323 X	Dose-dependent cell growth inhibition was produced by silibinin concentrations between 25 and 100 μM. In xenograft in nude mice, tumor weight was significantly decreased by dietary silibinin.
2018 324	SW1990 pancreatic cancer cells showed G1 arrest with decreased cyclins and CDKs and apoptosis with silibinin.

Desmoplastic tumors are the consequence of the intense activity of cancer-associated fibroblasts (CAFs) producing collagen fibers. PDAC and the liver have specialized CAFs known as stellate cells considered the producers of the desmoplastic reaction. Silymarin has been found to inhibit/decrease the desmoplastic reaction through 2 mechanisms:

1. it inhibits TGF β2 that induces the desmoplastic phenotype of naïve fibroblasts; ²⁸⁰

2. it increases E-cadherin expression^281,282 decreasing the invasive nature of the desmoplastic reaction.

Silymarin decreased fibrosis not only in 2 models of induced liver fibrosis^37,283 but also in lung fibrosis induced by cigarette smoke. ²⁸⁴ In this last case, this occurred by downregulation of the TGF-β1/Smad 2/3 pathway signaling.

Although we could not find any publication showing that silymarin could reduce the desmoplastic reaction in pancreatic cancer, we may assume that it has the potential to do so, because the mechanisms behind this are similar to those found in liver and lung cancers. Long et al suggested this possibility, however they did not incorporate any evidence in their review. ²⁷⁹

Tables 2 to 12: Evidence of silymarin's anticancer effects. In vivo experiments are marked with an X in column 1.

In spite of the long list of publications mentioned in Tables 2 to 12, 5 cautionary notes should be added:

1. In a mouse model of induced mammary carcinogenesis, the administration of silymarin, slightly increased mammary tumor incidence. ³⁶⁹ This may be due to silymarin's estrogenic effects,^115,261,370 however, the issue remains controversial because silymarin increases ERβ and decreases ERα expression. ²⁶⁴

2. In a model of mouse hepatic carcinogenesis (with diethylnitrosamine), silymarin showed no effects at all. ³⁷¹

3. In a mouse model of alcohol-dependent hepatocarcinoma, silibinin increased tumor progression if chronic alcohol intake continued. ³⁷²

4. Many of the in vitro experiments described in Table 2 to 12 were performed at very high concentrations that are difficult or impossible to achieve in vivo. On the other hand, in vivo experiments (marked with an X in Tables 2 to 12) were mainly conducted with oral administration of silymarin or silibinin, so those results should have a more significant impact on future clinical research.

5. Most of the published literature on silymarin and cancer does not mention the p53 status of the cells and this information is of capital importance (silymarin shows apoptotic effects on p53 positive cells but not on mutated p53).

Pharmacokinetics

Flavolignans (silymarin is a mixture of flavolignans) generally have poor bioavailability. This is the consequence of:

1. their strongly hydrophobic nature that does permit dilution to more than 50 μg/mL in water. Some organic solvents have a much better performance for this purpose. For example, ethanol shows a solubility of 225 mg/mL; ³⁷³

2. the fact that they are quickly metabolized;

3. the fact they are poorly absorbed in the intestine.

The pharmacokinetic considerations we shall make refer to the standardized form of silymarin with known amounts of silybin.

Absorption. silymarin is not soluble in water and oral administration shows poor absorption in the alimentary tract (approximately 1% in rats, ³⁷⁴ however, other authors mention a higher absorption around 30%). In spite of this low absorption, according to Janiak et al, a plasma level of 500 mg/L (500 μg/mL) is achievable 90 min after oral administration of 200 mg/kg of silymarin in mice. ³⁷⁵

Excretion. Silymarin is mainly excreted in the bile and half-life is 6 h.

Toxicity. Toxicity is almost absent ³⁷⁶ and therefore high oral doses can be administered with negligible side effects.

Dose/absorption studies in humans. A number of other studies have administered various doses and studied the plasma concentration. For example, with oral administration of 240 mg of silybin to 6 healthy volunteers the following results were obtained ³⁷⁷ :

$$\text{maximum}\text{,plasma}\text{concentration}0.34\pm 0.16\mu \text{g}/\text{m}\mathrm{L}$$

and time to maximum plasma concentration 1.32 ± 0.45 h. Absorption half life 0.17 ± 0.09 h, elimination half life 6.32 ± 3.94 h. ³⁷⁷ Beckmann-Knopp et al ³⁷⁸ also found: “Mean maximum plasma concentration after an oral dose of 700 mg silymarin, containing 254 mg of silibinin, is 317  ng/ml or 0.6 mM. Accumulation in plasma during three daily medications is negligible. Plasma protein binding is reported to reach about 90–95%.” After feeding volunteers with a smaller dose of 80 mg of a lipophilic silybin-phospatidylcholine complex (silipide) Gatti et al ³⁷⁹ found that free unconjugated silybin reached a maximum concentration of 141 ng/mL after 2.4 h. The level of conjugated silybin peaked after 3.8 h reaching 255 ng/mL. Another study on 6 healthy volunteers used a larger dose of 560 mg of silymarin and attained concentrations starting at 0.18 and going as high as 0.64 μg/mL. ³⁷⁷ These results are quite different and to some extent controversial.

Absorption studies in animals. Administration of silybin to animals also showed divergent results. In dogs, ³⁸⁰ the silybin-phosphatidylcholine complex (SPC) showed increased concentrations when compared with silymarin extract, however, the results showed a low level in general: SPC: 1.310 ± 880 ng/ml; silymarin: 383 ± 472 ng/ml. While Morazzoni et al ³⁸¹ found higher peak levels of silybin in the form of silipide when administered to rats: “After oral silipide, silybin reached peak plasma levels within 2 h, with a C_max of 9.0 ± 3.0 μg/ml for unconjugated drug and 93.4 ± 16.7 μg/ml for total (free + unconjugated drug).”

Pharmacodynamic conclusions: The above studies show that the achievable concentration in humans (with a low dose) is far lower than what was found in rodents (with a high dose). The important issue is that most of the experiments found in the literature at cellular level used a concentration around 100 μg/mL. Even in the study by Morazzoni et al, ³⁸¹ the level of 100 μg/mL was not achieved and in any case it is a peak level that cannot be sustained. Therefore, is the experimental level of 100 μg/mL achievable at the bedside?

We think that there is no evidence that it can be. Oral administration of silymarin in humans achieves nanogram, but not microgram levels. Furthermore, we should not extrapolate Morazzoni's findings in rats to humans as their pharmacokinetics may differ.

Therefore, the evidence based on these high concentration experiments should be viewed with caution. On the other hand, experiments with xenograft models are more reliable (Tables 2 to 12, xenograft results are marked with an X).

Tissue concentration. For cancer treatment purposes the important data to know are the concentrations achievable in tissues. Zhao and Agarwal ³⁸² found the following results in mice 30 min after administration:

• Liver: 8.8 μg per gram of tissue

• Lung: 4.3 μg per gram of tissue

• Stomach: 123 μg per gram of tissue

• Pancreas: 5.8 μg per gram of tissue

• Prostate: 2.5 μg per gram of tissue after 1 h.

After an oral intake, silipide (the lipophilic SPC), achieved a maximum concentration of silybin in bile within 4 h and then declined with a mean time of approximately 10 h. ³⁸³ Silibinin complexed with the amino-sugar meglumine is water soluble and can reach a tissue concentration high enough to show clear antigrowth effects in NSCLC xenografts. ³⁰⁷ The distribution in different tissues also varies widely according to the type of tissue considered. It is higher in the liver and dimishes in lungs, pancreas, and prostate. ³⁸² A relatively high concentration is achievable in colorectal muscosa (20-141 nmol/g of tissue). ³⁸⁴

The tissue levels obtainable compare unfavorably with those used in cell studies. To achieve apoptosis in cell studies, a concentration of more than 20 μM was necessary, ³⁸⁵ and this concentration does not seem easy to achieve by oral intake of standard preparations. It was also necessary to use a concentration of 100 μg/mL to induce apoptosis in Ramos cells (B lymphocytes). ³⁸⁶ Kamrani et al ³⁸⁷ used concentrations between 50 and 100 μg/mL to induce apoptosis in colon cancer cells. Therefore, while only a nanomolar concentration can be attained in tissues, micromolar concentrations were needed to induce apoptosis in these studies (the molecular weight of silybin is 482, 100 μg/ml = 207 μM).

In spite of this difficulty, Sing and Agarwal ²⁹⁸ found an important decrease in tumor volume in xenografted mice with human prostate carcinoma cells when the mice were orally fed with silymarin.

There are also different requirements for effects on cell migration versus proliferation. For endothelial cells, it was necessary to use a concentration of 48.1 μg/mL of silymarin to achieve a 20% reduction in proliferation and 16.1 μg/mL to achieve the same reduction in proliferation of LoVo colon cancer cells ¹⁵⁶ to achieve a reduction of migration of 50%, it is necessary a concentration of 1.15 μg/mL on endothelial cells (with silibinin instead of silymarin 0.66 μg/mL were enough to achieve the same). ¹⁵⁶ Our conclusion is that, from a bioavailability standpoint, it is much easier to achieve migration inhibition, than proliferative reduction.

In Europe one of the most used brands of silymarin is Legalon® L (silybin 3,23-O-bis-hemisuccinate) that comes in capsules of 150 mg. It also comes in vials containing 350 mg of silibinin for intravenous use. In the United States, silymarin is considered a nutritional supplement. ³⁸⁸ The intake of 5 of these capsules in 6 human volunteers, showed no adverse events. The concentration in plasma correlated with the dose and only 10% of it was unconjugated silymarin. A half life of 6 h was estimated. ³⁸⁹

In experimental conditions, many researchers dilute silybin in DMSO, a polar solvent in which silymarin is highly soluble. Unfortunately, this is not possible at the bedside.

Pharmaceutical Methods to Increase Bioavailability

Silymarin's low solubility, rapid metabolism, and quick excretion, led researchers and pharmaceutical industry to develop methods that could solve these very important drawbacks. Therefore, many compounds have been formulated mainly using nanotechnology. These compounds include nanosuspensions, solid dispersions, complexes with cyclodextrins and phospholipids, microemulsions, nanoemulsions, liposomes, polymer nanocarriers, solid-lipid nanoparticles and nanostructured lipid carriers, and polymer-based nanocarriers. ³⁹⁰ We shall discuss only a few of them.

• ► Combination with succinate: is available on the market under the trade mark Legalon® (bis hemisuccinate silybin).

• ► Combination with phosphatidylcholine: this was the first system developed for a better bioavailability: it consists of the combination of 2 molecules of phosphatidylcholine with one of sylibin. It has been registered under the name Siliphos®, but is also known as Idb1016, silipide, or phytosome.^391–394 This method increased bioavailability 10-fold. ³⁹⁵

• ► Silybin-cyclodextrin complex: adding cyclodextrin considerably enhances silymarin's water solubility.

• ► Other combinations with: meglumine, 23-O-phosphate.

• The problem with silybin combinations is that although they increase water solubility at the same time, they may reduce other effects such as antioxidant properties. ²³

• ► Nanosuspensions: are colloidal dispersions of drug particles with surfactants on the surface or other kind of synthetic stabilizers. This method improves dissolution and prolongs drug half life. ³⁹⁶

• ► Polymeric micelles: are nano-sized particles in which a hydrophobic substance is fully covered by a hydrophilic external layer. Wu et al ³⁹⁷ developed a silybin core included in amphiphilic chitosan micelles.

• ► Self micro-emulsifying drug delivery systems (SMEDDS): are mixtures of oil and surfactants. Liu et al ³⁹⁸ developed a silybin SMEDD that significantly increased its bioavailability.

• ► Liposomes: are lipid bilayer structures with a silybin core. This composition substantially improves bioavailability. ³⁹⁹

• ► Inclusion in polymeric matrices that carry and protect the drug. ⁴⁰⁰

• There are many other mechanisms based on nano-particles that increase absorption, prolong half life, and improve water solubility of silym arin, that escape the scope of this article. For a review of the issue, see Di Costanzo et al ⁴⁰¹ and Piazzini et al ⁴⁰² (Figure 7).

Figure 7.

Methods to increase silymarin's bioavailability.

Dosage and side Effects

A phase I study of silymarin in prostate cancer patients showed that 13 g daily per os divided into 3 doses was well tolerated. The most frequent adverse event was asymptomatic liver toxicity. ⁴⁰³ Side effects, although rare, were mainly related to the gastrointestinal tract, such as diarrhea, bloating, and nausea. ⁴⁰⁴ Abenavoli et al ⁴⁰⁵ found that daily doses beyond 1500 mg had laxative effects and increased bile flow. The usual dose of 400 or 800 mg a day is probably insufficient to achieve anticancer effects. It may be necessary to administer 800 mg 4 times a day because the half-life is short. However, the dose of silymarin for cancer treatment remains controversial. In one study, a high dose of silybinin was administered to patients prior to prostatectomy (13 g daily). They achieved high plasma concentrations, but nevertheless, low levels of silibinin were found in prostate tissue. ⁴⁰⁶ In an attempt to circumvent some of these problems one group used a silymarin-phosphatidylcholine compound administered orally as a daily dose of 2.8 g for 4 weeks prior to surgery. They achieved high levels in human breast cancer tissue. ⁴⁰⁷ This high bioavailability in this breast cancer study is an encouraging signal for a phase II clinical trial. It should also be noted that silymarin constituents have different anticancer abilities, ²² therefore a formulation of the strongest combination would represent a fundamental step in order to incorporate this flavonoid into standard treatments.

The Main Problems with Silymarin

Problem 1: Bioavailability. The evidence gathered in Tables 2 to 12 clearly shows that silymarin should have a place in cancer treatment. The main problem is its bioavailability. Many of the in vitro investigations have used concentrations that are very difficult to achieve at the bedside. The combination of silymarin with phosphatidylcholine (silipide) has a better bioavailability, however this combination is not available for clinical use.

Problem 2: Dual nature of silymarin's effects. Silymarin has protumoral and antitumoral effects. For example, in pancreatic cancer it promotes growth arrest and apoptosis (see Table 6) and decreases CD44 signaling. However, Lee et al ⁴⁰⁸ found that in addition to the antitumoral actions, silymarin also upregulated cancer stemness-related genes, namely TWIST1, Snail, and c-Jun. At the same time, it decreased p53 wild type and increased Ki-67 (a marker of proliferation). This is a powerful call for caution. On the other hand, in bladder cancer, silymarin seems to decrease stemness through inhibition of the β-cathenin/ZEB1 signaling (Wu 178). In pancreatic tumors (PANC1), it was also found that silymarin targeted stem cells decreasing proliferation and increasing apoptosis, ⁴⁰⁹ and had similar effects in breast cancer cells.^410,411 These controversies on silymarin prostemness or antistemness effects may be due to context or tumor dependency. The question remains unsolved.

Problem 3: DNA intercalation. In 2020, Pawar and Jaldappagari ⁴¹² reported that flavolignans had the ability to intercalate into the DNA double helix with moderate binding affinity. Other authors have vehemently contradicted this finding. ⁴¹³ However, if this silymarin effect on DNA is confirmed, it may have unthought consequences which are favorable (modulating gene activities against cancer) or undesirable (genotoxicity and or mutations). The issue is important enough to encourage further basic research in this area.

Clinical Trials

The United States Clinical Trials web-page lists the following trials for silymarin in cancer:

1. NCT03130634: The Efficacy of Silymarin as Adjuvant Therapy on Colorectal Cancer patients Undergoing FOLFIRI Treatment.

   State: recruiting since 2017.

   Kaohsiung Medical University Chung-Ho Memorial Hospital. Taiwan.

   Study Design: This is an open-label, randomized, comparative, double arm, single center study to assess efficacy of Silymarin (150 mg 3 times a day) as adjuvant therapy on metastatic colorectal cancer patients undergoing FOLFIRI chemotherapy in Taiwan.

2. NCT00487721: The Effect of High-dose Silybin-phytosome in Men With Prostate Cancer. (A Pilot Biomarker Study of Oral Silybin-Phytosome Followed by Prostatectomy in Patients With Localized prostate cancer).

   State: completed 2014

   University of Colorado. Denver

   Subjects will take Silibin-Phytosome for 2 to 10 weeks. The dose of Silibin-Phytosome is 13 g daily, in 3 divided doses.

   Outcome: To determine if measurable silibinin tissue levels are detectable in the prostate glands of men treated with Silybin-Phytosome administered according to the protocol.

   Results: low concentration of silymarin in prostatic tissue. ¹¹⁷

3. NCT01829178: Evaluation of Effects of Silymarin on Cisplatin Induced Nephrotoxicity in Upper Gastrointestinal Adenocarcinoma.

   State: completed 2015

   University of Tehran

   This study looked for possible protective effects of silymarin on kidney injury in patients receiving cisplatin.

   No results posted.

4. NCT00055718: Silymarin (Milk Thistle Extract) in Treating Patients with Acute Lymphoblastic Leukemia Who Are Receiving Chemotherapy.

   State: completed 2013

   Miami Children's Hospital, Winthrop University Hospital, Mount Sinai Medical School.

   This study looked for hepatoprotective effects of silymarin in patients receiving chemotherapy.

   No results posted.

5. NCT02146118: A Phase II Study to Assess Efficacy of Combined Treatment with Erlotinib (Tarceva) and Silybin-phytosome (Siliphos) in Patients With EGFR Mutant Lung Adenocarcinoma.

   State: unknown

   Gosin University. Busan, Korea.

   No results.

6. NCT01402648: Estrogen Receptor Beta Agonists (Eviendep) and Polyp Recurrence

   State: completed 2011

   Ospedale Policlinico Consorziale—Gastroenterology Unit. Bari, Italy

   No results.

The conclusion we reach regarding clinical trials is:

1. There were only 2 clinical trials (4 and 5) to determine therapeutic possibilities of silymarin against cancer. Neither have published results.

2. The dose used in the clinical trials showed differences of up to 1000% which clearly means that there is no standard dose.

Schröder et al ⁴¹⁴ conducted a randomized double-blind, cross-over placebo-controlled trial (with 2 periods of 10 weeks with a wash out period in the middle) with 49 patients that showed rising PSA levels after radical prostatectomy (34) or radiotherapy (15). They received a supplement containing soy, different isoflavones, silymarin, vitamins, minerals, and antioxidants. While receiving the treatment the doubling time for PSA was 1150 days compared with 445 days with the placebo. The fact that the supplement contained many other components besides the silymarin makes it impossible to draw conclusions about this compound. But it is evident that the supplement modified the biochemical evolution of the disease, delaying PSA progression.

Four Clinical Cases

Four clinical case reports are available, which though they cannot in themselves constitute a proof for the efficacy of silymarin, are nonetheless interesting and suggest a need for further studies. Hsu et al ⁴¹⁵ describe the case of a 66-year-old Taiwanese patient with a regression of an 11 cm diameter hepatocellular carcinoma. The patient was receiving 450 mg of silymarin daily, and no other medication. Even if we cannot consider this regression as a consequence of silymarin treatment, the fact that spontaneous regression of hepatomas is quite infrequent, makes us think of some intervention of silymarin in this unusual event. Moroni and Zanlorenzi ⁴¹⁶ published another case of complete regression of an advanced unresectable hepatocellular carcinoma treated with sorafenib and silymarin. Additionally, Bosch-Barrera et al ⁴¹⁷ presented 2 cases of brain metastases from lung cancer in which the treatment with silymarin decreased edema and the size of metastases, without improvement of the primary tumor.

Discussion

The concept of a tumor as a consequence of the mutation of one gene, and with one driver signaling or metabolic pathway, is flawed in most cases with the exception of cases such as chronic myeloid leukemia. Usually many genes and pathways are involved. The approach of attacking only one of the many hallmarks of cancer is also flawed. ⁴¹⁸ Recent evidence suggests that multiple genes are usually involved along with many signaling pathways, all interconnected, and interdependent and generating an extraordinary ability of tumor cells to survive and resist internal and external threats. ⁴¹⁹ This is one of the reasons why treatments made up of many different drugs are implemented in most treatment protocols.

Silymarin and its derivatives, through its multipronged attacks, allow one drug to reach many targets at the same time. Of course, we cannot expect silymarin to “cure” cancer all by itself, and it cannot replace any conventional chemotherapeutic treatment, but it is rather a privileged companion to therapeutic schemes in which it may develop useful complementary activity. This activity entails 3 concepts:

1. cancer prevention;

2. synergy with some treatment protocols;

3. decrease of collateral damage induced by chemotherapeutic drugs.

Silymarin's clinically achievable concentration in serum and at the tumor site, with the possible exception of the liver, seems insufficient for inducing apoptosis. However, xenograft model experiments showed that even with this low bioavailability drawback, silymarin could stop tumor growth.^288,351,356

The first studies on silymarin activity in cancer were performed in hepatic cells showing some characteristics that cannot be really considered antitumoral such as increased ribosomal synthesis and RNA polymerase I activation. This did not happen in hepatoma cells or in other malignant cells (Figure 8).

Figure 8.

Different effects of silymarin according to context.

Antiproliferative activity was found against almost all types of tumors, whether solid or nonsolid (Tables 2 to 12). These findings were confirmed not only at cellular level but also in vivo .

Silymarin has many other antitumor effects that can complement mainstream treatment protocols, such as:

• reduction of cell motility and invasion through TGF-β2 inhibition;

• inhibition of HIF-1α translation;

• decreased TIMP1 expression, thus decreasing metalloproteases activation;

• inhibition of the EGFR-MMP9 pathway;

• decreasing the accumulation of MDSCs in the tumor;

• inhibition of ERK and AKT signaling;

• protection against off-target toxicity of chemotherapeutic drugs;

• synergistic or added effects with some chemotherapeutics;

• reduction of extracellular fibronectin production.

To this short list we must add that there is evidence sustaining clear benefits in clinical cases such as hepatocarcinoma and clear cell renal carcinoma.

However, silymarin also has some effects that work against classical chemotherapy. For example, its ability as an antioxidant reduces ROS production. Many of the drugs currently used against cancer are precisely based on the creation of an oxidative stress with increased ROS that induces apoptosis of malignant cells.

Therefore, we must ask: why is silymarin a useful complement to chemotherapy?

Evidence indicates that there may be 2 possible answers:

1. silymarin has context-dependent effects: its behavior is different in normal and malignant cells as can be seen in BOX 2.

2. Its anticancer effects overwhelm those that seem favorable for cancer cell survival.

Box 2.

built on references.^420–423

The dual effects of silymarin can be clearly seen in an experiment by Su et al. ⁴²⁴ Here nasopharyngeal carcinoma cells were exposed to low concentrations of silymarin and this showed protumoral effects: “Silymarin increased the expressions of superoxide dismutase 1, catalase, and glutathione peroxidase. Consequently, the cell apoptosis was reduced markedly. An increase of Bcl-2 expression and a decrease of activated caspase-3….” Additionally, there is the problem of increased stemness induced by silymarin in pancreatic adenocarcinoma cells.

In spite of these isolated discouraging report, ⁴²⁴ the overwhelming sum of experimental evidence shows that silymarin has clear anticancer effect, and at the same time protects normal cells from the collateral damage of chemotherapy. There is reasonable doubt that the concentration that can be reached in vivo is enough to induce apoptosis. However, xenografted tumors were reduced in size suggesting that somehow silymarin induces tumor apoptosis. In a small cohort prospective study there were no benefits found when silymarin was added to standard chemotherapy. On the other hand, a slight tendency to lower metastatic behavior was seen in the cohort receiving silymarin. ⁴²⁵

As already mentioned, silymarin is not a stand-alone compound against cancer and it should be used alongside other medications. On this point, it is important to note that silymarin is an inhibitor of the Cytochrome P450 system, particularly silychristin. Therefore, caution should be exercised in this respect. ⁴²⁶

Silybin could be used as a scaffold or structure that can be modified improving its antitumoral effects. For example, Manivannan et al ⁴²⁷ have synthesized silybin analogues with increased anticancer capacity. One of these compounds named “15k,” was very potent and selective for ovarian cancer cells, where it bound to tubulin with high affinity. Subsequent experiments found that 15k induced growth arrest and apoptosis of ovarian cancer cells at a much lower concentration than silymarin. Furthermore, it showed no toxicity in animals. ⁴²⁸

Finally, it is important to note that many of silymarin's multipronged antitumoral actions are equally, or sometimes even better conveyed by other flavonoids such as genistein and epigallocatechin gallate.^429,430

What Remains to be Done?

In the first place, some of silymarin's protumoral effects demand further research with the objective of ascertaining if they need to be counteracted. Then, the precise silymarin concentrations required for the different antitumoral effects need to be established. And finally, the tumor concentration achievable with the different pharmaceutical preparations has to be determined. Once these 3 pieces of information are combined silymarin will be ready for serious clinical trials as a complement to classical chemotherapeutic schedules.

Conclusions

Silymarin compounds have considerable antitumoral effects. Well-planned clinical trials should be necessary to finally asses its bedside indications. Its dual, antitumoral and protumoral, effects merit further research.

Silymarin should be used at very high doses because low concentrations may induce protumoral effects. There is no toxicity even with very high doses. Silymarin's low absorption and bioavailability make it preferable to use modified pharmaceutical forms, such as nanoparticles or conjugated with compounds that increase its water solubility. These combinations already exist even if they have not been marketed as yet. The abundant existing evidence shows that silymarin has a definite place in cancer treatment. It has the ability to interfere with the expression of proteins related to cell cycle regulation, apoptosis, angiogenesis, and multidrug resistance. These characteristics define an anticancer drug. On the other hand, its strong antioxidant activity makes it a useful drug in cancer prevention.

Silymarin's lack of toxicity, even at very high doses, and the lack of effects on normal cells are important reasons for its further development.

Is there any other drug, that with no toxicity at all that can:

1. Inhibit EGFR signaling

2. Upregulate CDK inhibitors such as p21 and p27

3. Downregulate CDKs

4. Induce growth arrest by interfering MAPkinases cascade

5. Induce apoptosis

6. Inhibit TGF-alpha

7. Reduce the expression of VEGF and VEGFR

8. Inhibit the AKT axis

9. Decrease tumoral fibrosis

10. and have many other antitumoral effects?

Probably no other drug can achieve all these results without adverse events or high toxicity. We cannot expect that such a nontoxic pharmaceutical work as a stand-alone drug against cancer. But it can be an important factor in a multidrug anticancer schedule. Having mentioned this, the reports of increased stemness problem remain an unsolved issue which needs further investigation.

The inability to patent the compound is no doubt a drawback for the pharmaceutical industry and will restrict investment in these types of compounds. In its bioavailable formulations, silymarin deserves to be tested on clinical grounds, not as a stand-alone pharmaceutical, but as part of a treatment schedule.

Finally, the low concentrations that can be achieved with silymarin extracts at the bedside (in the order of ng/mL) hints to a serious bias in much of the past and present research at the cellular level where the average concentration range used was between 50 and 100 μg/mL. As a precondition for repurposing silymarin, newer pharmaceutical formulations should be screened in order to establish whether they can reach the necessary therapeutic concentrations.

Abbreviations

AR

androgen receptor

BCRP

Breast cancer resistance protein.

CDK

cyclin dependent kinase

CA

carbonic anhydrase

CAF

cancer associated fibroblast

COX2

cyclooxygenase 2

CXCR4

C-X-C chemokine receptor type 4

DMBA

dimethylbenzanthracene

EGF

epidermal growth factor

EGFR

epidermal growth factor receptor

EMT

epithelial–mesenchymal transition

ER

estrogen receptor.

ERB1

eukaryotic ribosome biogenesis protein 1

ERK

extracellular signal-regulated kinases

FDA

Food and Drug Administration (USA)

FKBP5

FK506 binding protein 5

HCC

hepatocellular carcinoma

HIF-1 alpha

hypoxia inducible factor 1 alpha

HDAC

histone deacetylase

IGFBP3

insulin like growth factor binding protein 3

IL

interleukin

MDSC

myeloid derived suppressor cell

MAPK

mitogen-activated protein kinase

MMP

metallopreoteases

MDR

multidrug resistance protein

MRP1

multidrug resistance-associated protein 1

NF-kB

nuclear factor-kappa B

NSCLC

nonsmall cell lung cancer

PDAC

pancreatic ductal adenocarcinoma

PDGF

platelet derived growth factor

PD-L1

programmed death ligand 1

PSA

prostate specific antigen

Rb

retinoblastoma protein

ROS

reactive oxygen species

SCLC

small cell lung cancer

SIRT1

NAD-dependent deacetylase sirtuin-1

SLIT2

slit homolog 2 protein

SMEDDS

self micro-emulsifying drug delivery systems

SPC

silybin phosphatidyl coline

SREBP1

sterol regulatory element binding protein 1

STAT3

signal transducer and activator of transcription 3

TGF

transforming growth factor

TPA

tetradecanoylphorbol acetate

TRAIL

tumor necrosis factor (TNF)-related apoptosis-inducing ligand

TRAMP

transgenic adenocarcinoma of the mouse prostate

UPAR

urokinase plasminogen activator receptor

VEGF

vascular endothelial growth factor

VEGFR

vascular endothelial growth factor receptor

ZEB1

zinc finger E-box-binding homeobox 1