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Keywords:

  • silibinin;
  • mitoxantrone;
  • docetaxel;
  • prostate cancer

Abstract

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. References

The purpose of these experiments was to assess the synergistic activity of silibinin with chemotherapy agents in clinical use against prostate cancer. Silybin-phytosome, a commercially available formulation containing silibinin, has recently been studied in a phase I clinical trial. The silibinin doses used in the present study are clinically achievable based on the preliminary phase I data. DU145, PC-3 and LNCaP prostate cancer cells were seeded in 96-well plates in triplicate. Twenty-four hours later, silibinin (10, 20 and 40 μM) and either mitoxantrone or docetaxel were added to the designated wells. Seventy-two hours post-treatment, cell viability was determined with a tetrazolium-based assay. The combination index (CI) for determination of a synergistic effect was calculated, with values of <0.9 indicating synergy and values >1.1 antagonism. Apoptosis was also assessed using a luminescent assay after 72 hr of treatment with media alone, silibinin, mitoxantrone, or silibinin plus mitoxantrone. Silibinin showed a synergistic effect with mitoxantrone, as measured by reduction in cell viability. The CI values ranged from 0.413 to 2.650 for the combination of silibinin and mitoxantrone; in contrast, treatment with docetaxel and silibinin showed little or no synergy, with CI values of 0.898–4.469. In concordance with these findings, the addition of silibinin increased the level of apoptosis compared to mitoxantrone alone, particularly in the PC-3 cells. The combination of silibinin and mitoxantrone exhibits a pattern of synergy in reducing cell viability with increased apoptosis. These data are important in the planning of future clinical applications of silibinin. © 2007 Wiley-Liss, Inc.

Prostate cancer is the most common non-skin cancer diagnosed in men. In 2005, the American Cancer Society estimates that 232,000 men were diagnosed and ∼30,000 died from prostate cancer in the United States.1 Early stage or organ confined cases may be cured with surgical prostatectomy or radiation therapy, although, some low-risk patients may also choose watchful waiting.2, 3 Disease that has spread beyond the prostate is generally first treated with hormonal ablation. The addition of cytotoxic chemotherapy is used in selected hormone-refractory patients, with this approach demonstrating a small (∼2 month) survival advantage.4, 5

Milk thistle (Silybum marianum) has a long history of use in humans and is commonly used in the treatment of liver disease.6, 7 Silymarin, the name of the crude milk thistle extract, is composed of several stereoisomers including silibinin (or silybin), silychristin and silydianin.8 Silibinin and silymarin have been shown to inhibit in vitro cell growth in several cancer models including prostate,9, 10, 11 bladder,12, 13 colon,14, 15 breast,16, 17 cervical,18 skin19, 20 and lung cancer.21, 22 Additional in vivo experiments have demonstrated an anti-cancer effect of silibinin and silymarin in models of prostate,23 skin,24, 25 bladder26 and colon cancer.27

Silibinin's anti-neoplastic actions appear to work through several different pathways.28 One of the most prominent effects seen in preclinical studies of silibinin is G1 arrest and apoptosis,29 with an increase of the cyclin dependent kinase inhibitors kip1/p27 and cip1/p21.17 Further study has shown that silibinin causes decreased phosphorylation of retinoblastoma protein leading to stability of the complex formed with E2F.9, 30 Silibinin's effect is accompanied by an inhibition of erbB131 with reduced ligand binding to this receptor demonstrated in prostate cancer cell lines.32 Using an in vivo model, silibinin increases insulin-like growth factor binding protein 3 levels after dietary feeding in a murine xenograft model of human prostate cancer.23 Transcription factor NF-κB, an important component in cell survival and chemotherapy resistance, is also inhibited by silibinin.11, 33, 34

Silibinin increases the efficacy of several chemotherapy agents both in vitro and in vivo. It acts synergistically with doxorubicin to inhibit growth via apoptosis in the human prostate cancer DU145 cells,35 and sensitizes these same cells to the anti-neoplastic effects of cisplatin and carboplatin.36 Silibinin also synergizes with doxorubicin to inhibit growth in an in vivo xenograft model of lung cancer.37

In addition to these preclinical studies with silibinin, we have conducted a Phase I human clinical trial of high-dose oral Silybin-Phytosome™. Preliminary results of this study have been reported in abstract form, indicating that at daily doses of 5 to 20 g, peak blood concentrations of >100 μM were achieved in some patients from 30 to 60 min after dosing.38 This high-dose regimen is in contrast to a recent pharmacokinetic study of standard dose silibinin (1.4 g daily) in colorectal cancer patients, which demonstrated that measurable tissue levels were achievable at that dosing level, with a terminal plasma half-life of 3.4 hr.39

Although, preclinical data would support the use of silibinin in combination with doxorubicin, cisplatin and carboplatin, these agents are not commonly used in the treatment of prostate cancer. Docetaxel, a microtubule inhibitor, is currently a first-line chemotherapy agent in advanced prostate cancer.4, 5 Mitoxantrone, an anthracenedione and topoisomerase II inhibitor, is also Food and Drug Administration approved for prostate cancer and in clinical use. Thus, both docetaxel and mitoxantrone were selected for further study based on their clinical relevance in prostate cancer. The experimental results presented here are essential in planning for future human clinical trials of silibinin in combination with cytotoxic chemotherapy in the treatment of prostate cancer, in addition to furthering our understanding of silibinin's mechanism of action.

Material and methods

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. References

Cell culture and reagents

Human prostate cancer cells DU145, LNCaP and PC-3 (American Type Culture Collection, Manassas, VA) were grown in OptiMEM (Gibco, Grand Island, NY). Ten percent Fetal Bovine Serum (Gemini, Woodland, CA) and 1% streptomycin-penicillin sulfate (Life Technologies, Grand Island, NY) were added to the media. All cell lines were incubated at 37°C with 5% CO2. Silibinin and docetaxel (both from Sigma, Saint Louis, MO) were dissolved in Dimethyl Sulphoxide (DMSO) and ethanol respectively; mitoxantrone (Serono, Rockland, MA) was obtained in solution.

Cell viability assay

Cell viability was assessed using a tetrazolium-based assay (CellTiter 96 AQueous One Solution-Promega Corporation, Madison, WI). One thousand cells in 50 μl of media per well were plated in 96-well plates in triplicate using the 3 cell lines. Twenty-four hours after plating, the combination treatments were added with 25 μl of 4× silibinin and 25 μl of 4× chemotherapy (either docetaxel or mitoxantrone) to give a total volume of 100 μl in each well. DMSO, in equal amounts to the treatment conditions, was added to the media in the control condition. Seventy-two hours after the treatment, 20 μl of the AQueous One Solution was added to each well. Colorimetric analysis using a 96-well plate reader (Vmax Kinetic Microplate Reader, Molecular Devices, Sunnyvale, CA) was performed between 1 and 4 hr (wavelength of 490 nm), depending on cell type and cell density. Cell viability assays were performed in triplicate. Statistical analysis was prepared with the SPSS software program (13.0 for Windows, 2004-Chicago, IL).

Determination of combination index

Cell viability data were analyzed with the CalcuSyn software program (Biosoft, Ferguson, MO-version 2-2005), which utilizes the statistical method described by Chou and Talalay to calculate the combination index (CI) values.40 Using a non-constant ratio setting, the CI values were calculated for each condition.

Apoptosis assay

Using a 96-well plate system, 5,000 cells from each of the 3 cell lines were seeded in 50 μl of media, as above, in triplicate. After 24 hr, 50 μl of media, silibinin and mitoxantrone were added to create 4 treatment conditions: media alone, silibinin (20 μM), mitoxantrone (50 nM) and silibinin plus mitoxantrone (20 μM and 50 nM respectively). After 72 hr, caspase-3 and caspase-7 activity was assessed by adding 100 μl of Caspase-Glo 3/7 (Promega, Madison, WI) solution to each well. Approximately 45 min later, the plates were placed in a Victor 1420 multilevel counter (Wallac, Gaithersburg, MD) for analysis. The cell viability for each treatment condition was assessed concurrently as described, with the apoptosis results corrected for differences in viability and the corresponding loss of luminescence from the non-viable cells.

Cell staining

Cells from each of the 3 cell lines (5 × 104 in 500 ml of OptiMEM, with 10% Fetal Bovine Serum and 1% streptomycin-penicillin sulfate) were plated into 12-well plates. Twenty-four hours later, the cells were treated with silibinin (20 μM final concentration), mitoxantrone (50 nM final concentration), and DMSO in the control condition. After 72 hr, the cells were stained with propidium iodide and bisbanzimide (both from Sigma, St. Louis, MO) giving a final concentration of 0.1 μg/ml to visually assess apoptosis; a Nikon Eclipse, TE 2000-S microscope (Nikon, Melville, NY) was used for visualization at 200× power.

Short duration silibinin treatment

The effect of short duration silibinin treatments was also examined. DU145, LNCaP and PC-3 cells were seeded (3,000/well) in a 96-well plate in 50 μl of media as above. Twenty-four hours later, 50 μl of media containing mitoxantrone was added to each well, for a final mitoxantrone concentration of 50 nM. After 2 hr, the media was removed and quickly replaced with either the standard media or silibinin containing media (20 μM). After an additional 2 hr, the media was again removed from all wells and replaced with fresh media. After 72 hr these treatment started, cell viability was again assessed by adding 20 μl of AQueous One Solution and utilizing the plate reader, as above.

Results

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. References

Synergy between silibinin and mitoxantrone in prostate cancer cell lines

The effect of silibinin and clinically relevant chemotherapy treatments on cell viability in prostate cancer cells was examined first. Cell viability for DU145, LNCaP and PC-3 cells treated with 50 nM mitoxantrone and varying doses of silibinin is shown in Figure 1. While both DU145 and LNCaP cells showed decreased viability with combination treatment, PC-3 cells exhibited minimal change in viability. In contrast to the effect observed with silibinin and mitoxantrone in DU145 and LNCaP cells, the combination of silibinin (10, 20 and 40 μM) and docetaxel (2.5–5 nM) showed little if any enhancement of docetaxel's efficacy in all 3 cell lines (data not shown).

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Figure 1. Treatment of cells with silibinin and mitoxantrone decreases cell viability. Cells were cultured as described in “Material and Methods” and treated with 50 nM of mitoxantrone and varying levels of silibinin (0, 10, 20 and 40 μM). After 72 hr, cell viability was determined. The control condition was defined as media alone. Each experiment was done in triplicate. Bars indicate ± standard deviation. *p <0.05, p < 0.005, p < 0.001 (Student's t-test) compared with mitoxantrone alone denoted as ‘0 condition’.

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Next, the synergistic and antagonistic effects of silibinin (doses of 10, 20 and 40 μM) in combination with docetaxel (doses of 0.62, 1.25, 2.5 and 5 nM) or mitoxantrone (doses of 25, 50, 100 and 200 nM) in DU145, LNCaP, and PC-3 were quantified. These dose ranges were selected to approximate the IC50 of docetaxel and mitoxantrone, with the silibinin dose based on the clinically achievable levels from preliminary phase I human data. Using AQueous One Solution in 96-well plates, cell viability was assessed 72 hr after the treatments. The CI values are on a continuum with respect to synergy, with values less than 0.9 consistent with synergy and values greater than 1.1 showing antagonism for a given combination. The mitoxantrone/silibinin treatment demonstrated significant synergy with CI values of 0.515–0.929, 0.521–0.967 and 0.413–2.65 for DU145, LNCaP and PC-3 cells respectively (Fig. 2). The only clear antagonism seen in the mitoxantrone combination was with low doses of mitoxantrone, 25 nM, a dose at which mitoxantrone's effect is small either with or without silibinin.

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Figure 2. CI for the combinations of mitoxantrone/silibinin and docetaxel/silibinin. Cells were cultured as described in “Material and Methods” and treated with varying levels of silibinin (10, 20 and 40 μM) and mitoxantrone (25, 50, 100 and 200 nM), or with silibinin (10, 20 and 40 μM) and docetaxel (0.62, 1.25, 2.5 and 5 nM). After 72 hr, cell viability was measured and the CI determined as described in “Material and Methods”, with CI values <0.9 indicating synergy and values >1.1 antagonism. Silibinin and mitoxantrone: (a) LNCaP, (b) DU145, (c) PC-3 cells, and silibinin and docetaxel: (d) LNCaP, (e) DU145, (f) PC-3.

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In agreement with the small or neutral effect noted in the initial viability studies, the docetaxel/silibinin treatments were largely devoid of synergy with CI values of 0.898–2.54, 0.921–2.32 and 0.895–4.47 for DU145, LNCaP and PC-3 cells respectively (Fig. 2). As evidenced by CI values of greater than 0.9, a neutral or antagonistic pattern was observed in all 3 cell lines with this combination.

Silibinin in combination with mitoxantrone induces apoptosis

Other investigations of silibinin have pointed to the induction of apoptosis as an important element of its anti-cancer efficacy. To further evaluate the mechanism of mitoxantrone/silibinin synergy, an assessment of the caspase-3 and caspase-7 levels were performed. The results of these apoptosis experiments are reported as fold-change in activity compared to control, after normalizing for differences in cell viability at the time of caspase activity assessment (Fig. 3). In all 3 cell lines, treatment with the relatively low dose of silibinin (20 μM) alone had no effect on the level of apoptosis over control. Consistent with previously published data, treatment with silibinin alone at higher doses, i.e. greater than 50 μM, did produce apoptosis (data not shown). Treatment with mitoxantrone alone did result in apoptosis in DU145 and LNCaP cells (3.84 and 1.79 times control, respectively), but not in the PC-3 cells (Fig. 3). The combination of silibinin and mitoxantrone increased the relative apoptosis in all cell lines, revealing a relative increase of 6.30, 2.18 and 2.23 fold over controls in the DU145, LNCaP and PC-3 cells, respectively (Fig. 3). To confirm silibinin's sensitization of PC-3 cells to mitoxantrone-induced apoptosis, the cells from these experiments were stained and visually observed for apoptosis. Using propidium iodide and bisbenzimide stains, the cells were assessed at 72 hr after treatment with silibinin (20 μM) and mitoxantrone (50 nM). The cell staining showed concordant visualization of the apoptosis measured by the caspase activity assay (Fig. 4).

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Figure 3. Silibinin increases apoptosis when added to mitoxantrone. Cells were cultured as described in “Material and Methods” and treated with media alone, 20 μM silibinin (Sb), 50 nM mitoxantrone (M), or the combination of silibinin and mitoxantrone (Sb+M). After 72 hr, active caspase-3 and caspase-7 cell levels were measured. Cell viability was assessed concurrently, with the apoptosis data corrected for changes in viability. Each experiment was done in triplicate. Shown here is the relative-fold increase in apoptosis (bars indicate ±standard deviation). The control condition (media alone) was defined as 1 for each cell line. (a) LNCaP; (b) DU145; (c) PC-3 cells. *p < 0.05 (Student's t test) compared with single agent mitoxantrone; p < 0.001 (Student's t test) compared with control.

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Figure 4. Propidium iodide and bisbenzimide staining shows enhanced apoptosis with silibinin and mitoxantrone combination treatment. Cells were cultured as described in “Material and Methods” and treated with (a) media alone, (b) 20 μM silibinin, (c) 50 nM mitoxantrone, or (d) both silibinin and mitoxantrone in combination. After 72 hr, the cells were stained with propidium iodine and bisbenzimide and visualized with a fluorescent microscope at 200×. Representative images are shown here for PC-3 cells and directly quantified in figure 3c.

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Short duration treatments with silibinin show enhancement of mitoxantrone's activity

Since the half-life of oral silibinin in humans is short,39 the in vitro efficacy of short duration treatments of silibinin was studied. First, the cells were treated with mitoxantrone for 2 hr, after which time the media was replaced with either standard media or media containing silibinin (20 μM). After 2 additional hours, the media was again exchanged in all wells for standard media. Treatment with 2 hr of silibinin after mitoxantrone yielded a significant reduction in cell viability in all cell lines (Fig. 5). Silibinin's enhancement of mitoxantrone's activity with this treatment regimen was most prominent in the PC-3 cell line, where a 30% decrease in cell viability was observed (p < 0.001, Student's t test).

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Figure 5. Short duration silibinin treatments enhance mitoxantrone's activity. LNCaP, PC-3 and DU145 cells were cultured as described in “Material and Methods” and treated with mitoxantrone (50 nM) for 2 hr, after which time the media was exchanged with either fresh media or media containing silibinin (20 μM). After 2 additional hours, the media was removed and exchanged with fresh media. Seventy-two hours after the start of the treatments, cell viability was measured, with each experiment done in triplicate. The control condition was defined as mitoxantrone followed by fresh media (without silibinin) and determined for each cell line. Bars indicate ±standard deviation. *p < 0.05, p < 0.005, p < 0.001 (Student's t test) compared with mitoxantrone alone.

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Discussion

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. References

Silibinin synergistically decreased prostate cancer cell viability in combination with mitoxantrone; this pattern of synergy was seen in all 3 human prostate cancer cell lines examined. This was observed with an increase in apoptosis, as measured by active caspase-3 and caspase-7 levels. In contrast, silibinin did not act synergistically with docetaxel in these prostate cancer cells.

These data add to our understanding of silibinin's mechanism of action. Previous studies have shown that silibinin enhances the effects of several chemotherapy agents, all of which are DNA-damaging agents.35, 36 Of these, cisplatin and carboplatin act similarly to alkylating agents, binding and cross-linking DNA, and doxorubicin is a topoisomerase II inhibitor, contributing to DNA breakage. In the present study, it is notable that mitoxantrone inhibits topoisomerase II, in a similar manner to doxorubicin, whereas docetaxel binds to microtubules and inhibits cell division, without a known DNA-damaging effect. Recent data using mouse epidermal keratinocyte JB6 cells and ultraviolet light B have given insight into silibinin's activity with DNA-damaging agents.41 In these experiments, the addition of silibinin increased the percentage of apoptotic cells when compared to the ultraviolet light B treatments alone. Silibinin's enhancement of ultraviolet light B-induced apoptosis was seen with an upregulation of DNA protein kinase, an important element in sensing genotoxic stress, with this effect abrogated by the addition of a semi-selective inhibitor of DNA protein kinase. Considering the present data in this context, consistency is seen in the synergy of silibinin with the DNA-damaging agent mitoxantrone.

Previous work has highlighted the potential importance of p53 activation in silibinin-induced apoptosis.41, 42 Of the 3 cell types examined here, LNCaP cells are considered to have wild type p53, PC-3 cells have a deletion in codon 138 with negative immunohistochemical staining for p53, and DU145 cells have 2 mutations in codons 223 and 274 of p53, but stain positive for p53 expression.43 Notably, the PC-3 cells showed little apoptosis in response to either low-dose silibinin or mitoxantrone; however, the combination demonstrated a significant increase. Silibinin therefore appears to overcome the relative resistance of PC-3 cells to mitoxantrone-induced apoptosis. The role of p53 status in silibinin's synergy in prostate cancer cells is an area worthy of additional study.

Because of the short half-life of silibinin observed in phase I testing, the effect of short duration silibinin treatment was examined, as this is believed to be clinically relevant. The short duration silibinin treatment experiments presented here were designed to better simulate the short half-life of oral silibinin in humans40 and demonstrated the potential relevance of a 2 hr silibinin exposure. Unlike the initial experiments with 72 hr of concurrent treatment (Fig. 1), PC-3 cells had decreased viability with the short duration, sequential treatment with mitoxantrone followed by silibinin (Fig. 5). One explanation for this observed finding is that mitoxantrone treatment for 2 hr induces DNA damage, but little cell death. The addition of silibinin enhances the genotoxic stress response, yielding decreased cell viability, even with a 2 hr treatment.

Most prostate cancer patients currently requiring chemotherapy are started on docetaxel therapy, as this treatment has shown a survival advantage.4, 5 The best treatment for progressive disease after docetaxel treatment is not well defined, but mitoxantrone is approved for use in prostate cancer and employed in this setting. The preclinical data presented here show significant synergy for the combination of mitoxantrone and silibinin in these in vitro models of prostate cancer. With a favorable side effect profile and long history of human use, silibinin represents a good candidate for combination treatments. If validated in further in vivo experiments, this combination could be considered in a clinical trial for patients with progressive prostate cancer, despite docetaxel treatment.

In conclusion, silibinin synergizes with mitoxantrone in prostate cancer cell lines at doses that are clinically achievable in humans. This effect was seen with increased apoptosis, especially in PC-3 cells. Additionally, a pattern of benefit from combination treatment with mitoxantrone and silibinin was also seen with short duration treatments, mimicking the silibinin levels observed in preliminary human testing. As mitoxantrone is approved for use in prostate cancer, these data suggest that additional study of this combination is warranted in the treatment of prostate cancer.

References

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. References
  • 1
    Jemal A, Murray T, Ward E, Samuels A, Tiwari RC, Ghafoor A, Feuer EJ, Thun MJ. Cancer statistics, 2005. CA Cancer J Clin 2005; 55: 1030.
  • 2
    Kuban DA, Thames HD, Levy LB, Horwitz EM, Kupelian PA, Martinez AA, Michalski JM, Pisansky TM, Sandler HM, Shipley WU, Zelefsky MJ, Zietman AL. Long-term multi-institutional analysis of stage T1-T2 prostate cancer treated with radiotherapy in the PSA era. Int J Radiat Oncol Biol Phys 2003; 57: 91528.
  • 3
    Bill-Axelson A, Holmberg L, Ruutu M, Haggman M, Andersson SO, Bratell S, Spangberg A, Busch C, Nordling S, Garmo H, Palmgren J, Adami HO, et al. Radical prostatectomy versus watchful waiting in early prostate cancer. N Engl J Med 2005; 352: 197784.
  • 4
    Petrylak DP, Tangen CM, Hussain MH, Lara PN,Jr, Jones JA, Taplin ME, Burch PA, Berry D, Moinpour C, Kohli M, Benson MC, Small EJ, et al. Docetaxel and estramustine compared with mitoxantrone and prednisone for advanced refractory prostate cancer. N Engl J Med 2004; 351: 151320.
  • 5
    Tannock IF, de Wit R, Berry WR, Horti J, Pluzanska A, Chi KN, Oudard S, Theodore C, James ND, Turesson I, Rosenthal MA, Eisenberger MA. Docetaxel plus prednisone or mitoxantrone plus prednisone for advanced prostate cancer. N Engl J Med 2004; 351: 150212.
  • 6
    Saller R, Meier R, Brignoli R. The use of silymarin in the treatment of liver diseases. Drugs 2001; 61: 203563.
  • 7
    Flora K, Hahn M, Rosen H, Benner K. Milk thistle (Silybum marianum) for the therapy of liver disease. Am J Gastroenterol 1998; 93: 13943.
    Direct Link:
  • 8
    Wagner H, Horhammer L, Seitz M. [Chemical evaluation of a silymarin-containing flavonoid concentrate from Silybum marianum (L.) Gaertn]. Arzneimittelforschung 1968; 18: 6968 (in German).
  • 9
    Tyagi A, Agarwal C, Agarwal R. The cancer preventive flavonoid silibinin causes hypophosphorylation of Rb/p107 and Rb2/p130 via modulation of cell cycle regulators in human prostate carcinoma DU145 cells. Cell Cycle 2002; 1: 13742.
  • 10
    Zhu W, Zhang JS, Young CY. Silymarin inhibits function of the androgen receptor by reducing nuclear localization of the receptor in the human prostate cancer cell line LNCaP. Carcinogenesis 2001; 22: 1399403.
  • 11
    Dhanalakshmi S, Singh RP, Agarwal C, Agarwal R. Silibinin inhibits constitutive and TNFα-induced activation of NF-κB and sensitizes human prostate carcinoma DU145 cells to TNFα-induced apoptosis. Oncogene 2002; 21: 175967.
  • 12
    Tyagi A, Agarwal C, Harrison G, Glode LM, Agarwal R. Silibinin causes cell cycle arrest and apoptosis in human bladder transitional cell carcinoma cells by regulating CDKI-CDK-cyclin cascade, and caspase 3 and PARP cleavages. Carcinogenesis 2004; 25: 171120.
  • 13
    Tyagi AK, Agarwal C, Singh RP, Shroyer KR, Glode LM, Agarwal R. Silibinin down-regulates survivin protein and mRNA expression and causes caspases activation and apoptosis in human bladder transitional-cell papilloma RT4 cells. Biochem Biophys Res Commun 2003; 312: 117884.
  • 14
    Yang SH, Lin JK, Chen WS, Chiu JH. Anti-angiogenic effect of silymarin on colon cancer LoVo cell line. J Surg Res 2003; 113: 1338.
  • 15
    Agarwal C, Singh RP, Dhanalakshmi S, Tyagi AK, Tecklenburg M, Sclafani RA, Agarwal R. Silibinin upregulates the expression of cyclin-dependent kinase inhibitors and causes cell cycle arrest and apoptosis in human colon carcinoma HT-29 cells. Oncogene 2003; 22: 827182.
  • 16
    Mehta RG, Moon RC. Characterization of effective chemopreventive agents in mammary gland in vitro using an initiation-promotion protocol. Anticancer Res 1991; 11: 5936.
  • 17
    Zi X, Feyes DK, Agarwal R. Anticarcinogenic effect of a flavonoid antioxidant, silymarin, in human breast cancer cells MDA-MB 468: induction of G1 arrest through an increase in Cip1/p21 concomitant with a decrease in kinase activity of cyclin-dependent kinases and associated cyclins. Clin Cancer Res 1998; 4: 105564.
  • 18
    Bhatia N, Zhao J, Wolf DM, Agarwal R. Inhibition of human carcinoma cell growth and DNA synthesis by silibinin, an active constituent of milk thistle: comparison with silymarin. Cancer Lett 1999; 147: 7784.
  • 19
    Mohan S, Dhanalakshmi S, Mallikarjuna GU, Singh RP, Agarwal R. Silibinin modulates UVB-induced apoptosis via mitochondrial proteins, caspases activation, and mitogen-activated protein kinase signaling in human epidermoid carcinoma A431 cells. Biochem Biophys Res Commun 2004; 320: 1839.
  • 20
    Dhanalakshmi S, Mallikarjuna GU, Singh RP, Agarwal R. Dual efficacy of silibinin in protecting or enhancing ultraviolet B radiation-caused apoptosis in HaCaT human immortalized keratinocytes. Carcinogenesis 2004; 25: 99106.
  • 21
    Chu SC, Chiou HL, Chen PN, Yang SF, Hsieh YS. Silibinin inhibits the invasion of human lung cancer cells via decreased productions of urokinase-plasminogen activator and matrix metalloproteinase-2. Mol Carcinog 2004; 40: 1439.
  • 22
    Sharma G, Singh RP, Chan DC, Agarwal R. Silibinin induces growth inhibition and apoptotic cell death in human lung carcinoma cells. Anticancer Res 2003; 23: 264955.
  • 23
    Singh RP, Dhanalakshmi S, Tyagi AK, Chan DC, Agarwal C, Agarwal R. Dietary feeding of silibinin inhibits advance human prostate carcinoma growth in athymic nude mice and increases plasma insulin-like growth factor-binding protein-3 levels. Cancer Res 2002; 62: 30639.
  • 24
    Lahiri-Chatterjee M, Katiyar SK, Mohan RR, Agarwal R. A flavonoid antioxidant, silymarin, affords exceptionally high protection against tumor promotion in the SENCAR mouse skin tumorigenesis model. Cancer Res 1999; 59: 62232.
  • 25
    Katiyar SK, Korman NJ, Mukhtar H, Agarwal R. Protective effects of silymarin against photocarcinogenesis in a mouse skin model. J Natl Cancer Inst 1997; 89: 55666.
  • 26
    Vinh PQ, Sugie S, Tanaka T, Hara A, Yamada Y, Katayama M, Deguchi T, Mori H. Chemopreventive effects of a flavonoid antioxidant silymarin on N-butyl-N-(4-hydroxybutyl)nitrosamine-induced urinary bladder carcinogenesis in male ICR mice. Jpn J Cancer Res 2002; 93: 429.
  • 27
    Kohno H, Tanaka T, Kawabata K, Hirose Y, Sugie S, Tsuda H, Mori H. Silymarin, a naturally occurring polyphenolic antioxidant flavonoid, inhibits azoxymethane-induced colon carcinogenesis in male F344 rats. Int J Cancer 2002; 101: 4618.
  • 28
    Singh RP, Agarwal R. Prostate cancer prevention by silibinin. Curr Cancer Drug Targets 2004; 4: 111.
  • 29
    Zi X, Agarwal R. Silibinin decreases prostate-specific antigen with cell growth inhibition via G1 arrest, leading to differentiation of prostate carcinoma cells: implications for prostate cancer intervention. Proc Natl Acad Sci USA 1999; 96: 74905.
  • 30
    Tyagi A, Agarwal C, Agarwal R. Inhibition of retinoblastoma protein (Rb) phosphorylation at serine sites and an increase in Rb-E2F complex formation by silibinin in androgen-dependent human prostate carcinoma LNCaP cells: role in prostate cancer prevention. Mol Cancer Ther 2002; 1: 52532.
  • 31
    Zi X, Grasso AW, Kung HJ, Agarwal R. A flavonoid antioxidant, silymarin, inhibits activation of erbB1 signaling and induces cyclin-dependent kinase inhibitors, G1 arrest, and anticarcinogenic effects in human prostate carcinoma DU145 cells. Cancer Res 1998; 58: 19209.
  • 32
    Sharma Y, Agarwal C, Singh AK, Agarwal R. Inhibitory effect of silibinin on ligand binding to erbB1 and associated mitogenic signaling, growth, and DNA synthesis in advanced human prostate carcinoma cells. Mol Carcinog 2001; 30: 22436.
  • 33
    Yoo HG, Jung SN, Hwang YS, Park JS, Kim MH, Jeong M, Ahn SJ, Ahn BW, Shin BA, Park RK, Jung YD. Involvement of NF-κB and caspases in silibinin-induced apoptosis of endothelial cells. Int J Mol Med 2004; 13: 816.
  • 34
    Saliou C, Rihn B, Cillard J, Okamoto T, Packer L. Selective inhibition of NFκB activation by the flavonoid hepatoprotector silymarin in HepG2. Evidence for different activating pathways. FEBS Lett 1998; 440: 812.
  • 35
    Tyagi AK, Singh RP, Agarwal C, Chan DC, Agarwal R. Silibinin strongly synergizes human prostate carcinoma DU145 cells to doxorubicin-induced growth inhibition, G2-M arrest, and apoptosis. Clin Cancer Res 2002; 8: 351219.
  • 36
    Dhanalakshmi S, Agarwal P, Glode LM, Agarwal R. Silibinin sensitizes human prostate carcinoma DU145 cells to cisplatin- and carboplatin-induced growth inhibition and apoptotic death. Int J Cancer 2003; 106: 699705.
  • 37
    Singh RP, Mallikarjuna GU, Sharma G, Dhanalakshmi S, Tyagi AK, Chan DC, Agarwal C, Agarwal R. Oral silibinin inhibits lung tumor growth in athymic nude mice and forms a novel chemocombination with doxorubicin targeting nuclear factor κB-mediated inducible chemoresistance. Clin Cancer Res 2004; 10: 86417.
  • 38
    Flaig T, Agarwal R, Su L-J, Harrison GS, Gustafson DL, Glode LM. A phase I study of silibinin in hormone refractory prostate cancer. J Clin Oncol 2005; 23: 427S.
  • 39
    Hoh C, Boocock D, Marczylo T, Singh R, Berry DP, Dennison AR, Hemingway D, Miller A, West K, Euden S, Garcea G, Farmer PB, et al. Pilot study of oral silibinin, a putative chemopreventive agent, in colorectal cancer patients: silibinin levels in plasma, colorectum, and liver and their pharmacodynamic consequences. Clin Cancer Res 2006; 12: 294450.
  • 40
    Chou TC, Talalay P. Quantitative analysis of dose-effect relationships: the combined effects of multiple drugs or enzyme inhibitors. Adv Enzyme Regul 1984; 22: 2755.
  • 41
    Dhanalakshmi S, Agarwal C, Singh RP, Agarwal R. Silibinin up-regulates DNA-protein kinase-dependent p53 activation to enhance UVB-induced apoptosis in mouse epithelial JB6 cells. J Biol Chem 2005; 280: 2037583.
  • 42
    Katiyar SK, Roy AM, Baliga MS. Silymarin induces apoptosis primarily through a p53-dependent pathway involving Bcl-2/Bax, cytochrome c release, and caspase activation. Mol Cancer Ther 2005; 4: 20716.
  • 43
    van Bokhoven A, Varella-Garcia M, Korch C, Hessels D, Miller GJ. Widely used prostate carcinoma cell lines share common origins. Prostate 2001; 47: 3651.