Rana P. Singh

Silibinin upregulates the expression of cyclin-dependent kinase inhibitors and causes cell cycle arrest and apoptosis in human colon carcinoma HT-29 cells

Silymarin, a defined mixture of natural flavonoid, has recently been shown to have potent cancer chemopreventive efficacy against colon carcinogenesis in rat model; however, the mechanism of such efficacy is not elucidated. Here, using pure active agent in silymarin, namely silibinin, we show its antiproliferative and apoptotic effects, and associated molecular alterations in human colon carcinoma HT-29 cells. Silibinin treatment of cells at 50–100 μg/ml doses resulted in a moderate to very strong growth inhibition in a dose- and a time-dependent manner, which was largely due to a G0/G1 arrest in cell cycle progression; higher dose and longer treatment time also caused a G2/M arrest. In mechanistic studies related its effect on cell cycle progression, silibinin treatment resulted in an upregulation of Kip1/p27 and Cip1/p21 protein as well as mRNA levels, and decreased CDK2, CDK4, cyclin E and cyclin D1 protein levels together with an inhibition in CDK2 and CDK4 kinase activities. In other studies, we observed that G2/M arrest by silibinin was associated with a decrease in cdc25C, cdc2/p34 and cyclin B1 protein levels, as well as cdc2/p34 kinase activity. In the studies assessing biological fate of silibinin-treated cells, silibinin-induced cell cycle arrest and growth inhibition were not associated with cellular differentiation, but caused apoptotic death. The quantitative apoptosis analysis showed up to 15% apoptotic cell death after 48 h of silibinin treatment. Interestingly, silibinin-induced apoptosis in HT-29 cells was independent of caspases activation, as all caspases inhibitor did not reverse silibinin-induced apoptosis. This observation was further confirmed by the findings showing a lack in caspases activity increase and caspases and PARP cleavage as well as a lack in cytochrome c release in cytosol following silibinin treatment of HT-29 cells. Additional studies conducted in mice showed that silibinin doses found effective in HT-29 cells are achievable in plasma, which increases the significance of the present findings and their possible translation in in vivo anticancer efficacy of silibinin against colon cancer. Together, these results identify molecular mechanisms of silibinin efficacy as a cell cycle regulator and apoptosis inducer in human colon carcinoma HT-29 cells, and justify further studies to investigate potential usefulness of this nontoxic agent in colon cancer prevention and intervention.

Grape seed extract inhibits advanced human prostate tumor growth and angiogenesis and upregulates insulin-like growth factor binding protein-3

Dietary intake of many fruits and vegetables has been shown to be associated with reduced risk of cancer. We investigated the in vivo efficacy of grape seed extract (GSE, patented as Traconol) against prostate cancer (PCA) and associated molecular events. Athymic nude mice were implanted with hormone‐refractory human prostate carcinoma DU145 cells and fed with 100 and 200 mg/kg/day (5 days/week) doses of GSE for 7 weeks. At the end of experiment, tumors were immunohistochemically analyzed for cell proliferation, apoptosis and angiogenesis. Our data show that GSE feeding strongly inhibited tumor growth that accounted for 59–73% (p < 0.001) inhibition in tumor volume and 37–47% (p < 0.05) decrease in tumor weight at the end of the experiment. It did not show any significant change in body weight gain profile and diet consumption. Immunohistochemical analysis of tumors showed that GSE decreases proliferation index by 51–66% (p < 0.001) and increases apoptotic index by 3–4‐fold (p < 0.001). CD31 staining for endothelial cells, showed decrease in intratumoral microvasculature in GSE‐fed group of mice. Control tumors showed 64.0 ± 1.6 CD31 positive cells/400× field compared to 23.2 ± 0.9 and 15.7 ± 0.08 (p < 0.001) CD31 positive cells in 100 and 200 mg/kg doses of GSE‐treated tumors, respectively. GSE strongly inhibited (47–70%, p < 0.05) vascular endothelial growth factor (VEGF) secretion in conditioned medium by DU145 cells. Recently, the circulating level of insulin‐like growth factor binding protein (IGFBP)‐3 is shown to inversely related with PCA risk, growth and prognosis. Consistent with this, we observed 6–7‐fold (p < 0.001) increase in tumor‐secreted levels of IGFBP‐3 after GSE feeding. In other immunohistochemical studies, compared to controls, tumor xenografts from GSE‐fed groups of mice showed a moderate decrease in VEGF but an increase in IGFBP‐3 levels. These findings suggest that GSE possesses in vivo anticancer efficacy against hormone‐refractory human PCA, which is associated with its antiproliferative, proapoptotic and antiangiogenic activities together with upregulation of IGFBP‐3.

Silibinin inhibits constitutive and TNFα-induced activation of NF-κB and sensitizes human prostate carcinoma DU145 cells to TNFα-induced apoptosis

Prostate cancer (PCA) is one of the most common invasive malignancies of men in the US, however, there have been limited successes so far in its therapy. Even most potent agents (e.g. TNFα) are ineffective in killing human PCA cells possibly due to constitutive activation of NF-κB that subsequently activates a large number of anti-apoptotic genes. In such a scenario, strong apoptotic agent TNFα, further induces NF-κB activation rather than inducing apoptosis. In several recent studies, we have demonstrated both cancer preventive and anti-cancer efficacy of silymarin and its constituent silibinin in a variety of experimental tumor models and cell culture systems. Here we examined whether silibinin is effective in inhibiting constitutive NF-κB activation in human PCA cells, which would help in overcoming TNFα-insensitivity. Our studies reveal that silibinin effectively inhibits constitutive activation of NF-κB in advanced human prostate carcinoma DU145 cells. Consistent with this, nuclear levels of p65 and p50 sub-units of NF-κB were also reduced. In the studies assessing molecular mechanism of this effect, silibinin treatment resulted in a significant increase in the level of IκBα with a concomitant decrease in phospho-IκBα. Kinase assays revealed that silibinin dose-dependently decreases IKKα kinase activity. The effect of silibinin on IKKα seemed to be direct as evidenced by the in vitro kinase assay, where immunoprecipitated IKKα was incubated with silibinin. This shows that silibinin does not necessarily need an upstream event to bring about its inhibitory effect on IKKα and downstream effectors. Additional studies showed that silibinin also inhibits TNFα-induced activation of NF-κB via IκBα pathway and subsequently sensitizes DU145 cells to TNFα-induced apoptosis. These results indicate that silibinin could be used to enhance the effectiveness of TNFα-based chemotherapy in advanced PCA.

Grape seed extract inhibits in vitro and in vivo growth of human colorectal carcinoma cells.

Purpose: Accumulating evidences suggest the beneficial effects of fruit-and-vegetable consumption in lowering the risk of various cancers, including colorectal cancer. Herein, we investigated the in vitro and in vivo anticancer effects and associated mechanisms of grape seed extract (GSE), a rich source of proanthocyanidins, against colorectal cancer. Experimental Design: Effects of GSE were examined on human colorectal cancer HT29 and LoVo cells in culture for proliferation, cell cycle progression, and apoptosis. The in vivo effect of oral GSE was examined on HT29 tumor xenograft growth in athymic nude mice. Xenografts were analyzed by immunohistochemistry for proliferation and apoptosis. The molecular changes associated with the biological effects of GSE were analyzed by Western blot analysis. Results: GSE (25-100 μg/mL) causes a significant dose- and time-dependent inhibition of cell growth with concomitant increase in cell death. GSE induced G1 phase cell cycle arrest along with a marked increase in Cip1/p21 protein level and a decrease in G1 phase–associated cyclins and cyclin-dependent kinases. GSE-induced cell death was apoptotic and accompanied by caspase-3 activation. GSE feeding to mice at 200 mg/kg dose showed time-dependent inhibition of tumor growth without any toxicity and accounted for 44% decrease in tumor volume per mouse after 8 weeks of treatment. GSE inhibited cell proliferation but increased apoptotic cell death in tumors. GSE-treated tumors also showed enhanced Cip1/p21 protein levels and poly(ADP-ribose) polymerase cleavage. Conclusions: GSE may be an effective chemopreventive agent against colorectal cancer, and that growth inhibitory and apoptotic effects of GSE against colorectal cancer could be mediated via an up-regulation of Cip1/p21.

Phytochemicals as cell cycle modulators--a less toxic approach in halting human cancers.

The multistep nature of cancer development provides a rationale for cancer prevention. Activation of oncogenes, inactivation of tumor suppressor genes and modulation of mitogenic signal transduction pathways are critical in cancer progression and present attractive targets for cancer prevention/intervention. In this respect, cell cycle regulation and its modulation by various natural (plant-derived) and synthetic agents are gaining widespread attention in recent years. A number of phytochemicals inhibit cell cycle progression in cancer cells, yet their clinical applications are still in infancy. The present review is focused on the modulatory effects of phytochemicals on critical cell cycle molecules, and discusses how they inhibit proliferation and/or induce apoptotic death in cancer cells. Key Words: Cell cycle, Cyclins, Growth factors, Oncogenes

Silibinin protects against photocarcinogenesis via modulation of cell cycle regulators, mitogen-activated protein kinases, and Akt signaling.

Here, we assessed the protective effect of silibinin on UVB-induced skin carcinogenesis in SKH-1 hairless mice. Topical application of silibinin before or immediately after UVB exposure or its dietary feeding resulted in a strong protection against photocarcinogenesis, in terms of tumor multiplicity (60–66%; P < 0.001), tumor volume per mouse (93–97%; P < 0.001) and tumor volume per tumor (80–91%; P < 0.001). Silibinin also moderately inhibited tumor incidence (5–15%; P < 0.01) and delayed tumor latency period (up to 4 weeks; P < 0.01–0.001). To investigate in vivo molecular mechanisms of silibinin efficacy, tumors and uninvolved skin from tumor-bearing mice were examined immunohistochemically for proliferation, p53, apoptosis, and activated caspase-3. Silibinin treatment showed a strong decrease (P < 0.001) in proliferating cell nuclear antigen-positive cells and an increase in p53-positive (P < 0.005–0.001), terminal deoxynucleotidyltransferase-mediated nick end labeling-positive (P < 0.005–0.001), and cleaved caspase-3–positive cells (P < 0.001). Western blot analysis of normal skin and tumor lysates showed that silibinin decreases the levels of cyclin-dependent kinase 2 and cyclin-dependent kinase 4 and associated cyclins A, E, and D1, together with an up-regulation of Cip1/p21, Kip1/p27, and p53. Silibinin also showed a strong phosphorylation of extracellular signal-regulated protein kinase 1/2, stress-activated protein kinase/c-JUN NH2-terminal kinase 1/2, and p38 mitogen-activated protein kinases but inhibited Akt phosphorylation and decreased survivin levels with an increase in cleaved caspase-3. Together, these results show a strong preventive efficacy of silibinin against photocarcinogenesis, which involves the inhibition of DNA synthesis, cell proliferation, and cell cycle progression and an induction of apoptosis. Furthermore, these results also identify in vivo molecular mechanisms of silibinin efficacy against photocarcinogenesis.

Silymarin and silibinin cause G1 and G2–M cell cycle arrest via distinct circuitries in human prostate cancer PC3 cells: a comparison of flavanone silibinin with flavanolignan mixture silymarin

Here, we assessed and compared the anticancer efficacy and associated mechanisms of silymarin and silibinin in human prostate cancer (PCA) PC3 cells; silymarin is comprised of silibinin and its other stereoisomers, including isosilybin A, isosilybin B, silydianin, silychristin and isosilychristin. Silymarin and silibinin (50–100 μg/ml) inhibited cell proliferation, induced cell death, and caused G1 and G2–M cell cycle arrest in a dose/time-dependent manner. Molecular studies showed that G1 arrest was associated with a decrease in cyclin D1, cyclin D3, cyclin E, cyclin-dependent kinase (CDK)4, CDK6 and CDK2 protein levels, and CDK2 and CDK4 kinase activity, together with an increase in CDK inhibitors (CDKIs) Kip1/p27 and Cip1/p21. Further, both agents caused cytoplasmic sequestration of cyclin D1 and CDK2, contributing to G1 arrest. The G2–M arrest by silibinin and silymarin was associated with decreased levels of cyclin B1, cyclin A, pCdc2 (Tyr15), Cdc2, and an inhibition of Cdc2 kinase activity. Both agents also decreased the levels of Cdc25B and cell division cycle 25C (Cdc25C) phosphatases with an increased phosphorylation of Cdc25C at Ser216 and its translocation from nucleus to the cytoplasm, which was accompanied by an increased binding with 14-3-3β. Both agents also increased checkpoint kinase (Chk)2 phosphorylation at Thr68 and Ser19 sites, which is known to phosphorylate Cdc25C at Ser216 site. Chk2-specific small interfering RNA largely attenuated the silymarin and silibinin-induced G2–M arrest. An increase in the phosphorylation of histone 2AX and ataxia telangiectasia mutated was also observed. These findings indicate that silymarin and silibinin modulate G1 phase cyclins–CDKs–CDKIs for G1 arrest, and the Chk2–Cdc25C–Cdc2/cyclin B1 pathway for G2–M arrest, together with an altered subcellular localization of critical cell cycle regulators. Overall, we observed comparable effects for both silymarin and silibinin at equal concentrations by weight, suggesting that silibinin could be a major cell cycle-inhibitory component in silymarin. However, other silibinin stereoisomers present in silymarin also contribute to its efficacy, and could be of interest for future investigation.

Flavonoid Antioxidant Silymarin and Skin Cancer

Oxidative stress is one of the key players in skin carcinogenesis, and therefore identifying nontoxic strong antioxidants to prevent skin cancer is an important area of research. In both animal and cell culture studies, we have shown that silymarin, a naturally occurring polyphenolic flavonoid antioxidant, exhibits preventive and anticancer effects against skin cancer. For example, silymarin strongly prevents both photocarcinogenesis and skin tumor promotion in mice, in part, by scavenging free radicals and reactive oxygen species and strengthening the antioxidant system. We also found that this effect of silymarin is by inhibiting endogenous tumor promoter tumor necrosis factor alpha in mouse skin, a central mediator in skin tumor promotion. In mechanistic studies, silymarin inhibits mitogenic and cell survival signaling and induces apoptosis. Furthermore, silymarin effectively modulates cell-cycle regulators and check points toward inhibition of proliferation, and growth arrest in G0-G1 and G2-M phases of the cell cycle. Thus, due to its mechanism-based chemopreventive and anticancer effects in experimental models, silymarin is an important candidate for the prevention and/or therapy of skin cancer, as well as other cancers of epithelial origin in humans.

Silibinin inhibits inflammatory and angiogenic attributes in photocarcinogenesis in SKH-1 hairless mice.

Sunscreens partially filter UVB and, therefore, could partially prevent skin cancer; however, efficient approaches are desired to effectively prevent photocarcinogenesis. It is hypothesized that nontoxic pharmacologically active natural compounds can increase photoprotective effects. Our completed studies suggest that silibinin, a bioactive phytochemical, strongly prevents photocarcinogenesis; however, its mechanism is not fully understood. Herein, for the first time, we used a clinically relevant UVB dose (30 mJ/cm(2)/day) to examine the photoprotective effect and associated mechanisms of silibinin in SKH1 hairless mice. Topical or dietary silibinin treatment caused a strong protection against photocarcinogenesis in terms of delay in tumor appearance, multiplicity, and volume. Analyses of normal skin, uninvolved skin from tumor-bearing mice, and skin tumors showed a statistically significant decrease (P < 0.05-0.001) in inducible nitric oxide synthase (iNOS) and cyclooxygenase 2 (COX-2) levels by silibinin. Concomitantly, phospho-signal transducers and activators of transcription 3 (Tyr(705)) and phospho-p65(Ser(536)) were also decreased by silibinin, which are potential up-stream regulators of iNOS and COX-2. Simultaneously, silibinin also decreased UVB-caused increase in cell proliferation and microvessel density. In tumors, hypoxia-inducible factor 1alpha (HIF-1alpha) and vascular endothelial growth factor protein levels were decreased by silibinin. Further analysis showed that silibinin inhibited UVB-caused phosphorylation and nuclear translocation of STAT3 and p65, as well as nuclear factor kappaB (NF-kappaB) DNA binding activity. Together, these results suggest that silibinin causes a strong protective effect against photocarcinogenesis via down-regulation of inflammatory and angiogenic responses, involving HIF-1alpha, STAT3, and NF-kappaB transcription factors, as well as COX2 and iNOS.

PROSTATE CANCER CHEMOPREVENTION BY SILIBININ: BENCH TO BEDSIDE

Prostate cancer (PCA) is the most invasive malignancy and second leading cause of cancer deaths in American males. One approach to reduce PCA incidence, growth and metastasis is prevention and intervention targeted towards mitogenic and survival signaling and cell‐cycle regulation. This approach is based on the rationale that overexpression of receptor tyrosine kinases (RTKs) and/or non‐receptor tyrosine kinases leads to persistent autocrine stimulation of malignant cells for deregulated cell‐cycle progression and uncontrolled growth. PCA progression has also been associated with transition from a paracrine to an autocrine relationship between receptors and growth ligands as this malignancy progresses to an advanced androgen‐independent aggressive stage. Together, these studies suggest that targeting RTK‐mediated signaling pathways along with cell‐cycle regulators could be a practical and translational approach for PCA prevention and intervention. Here, we provide evidence that a naturally occurring nontoxic flavanoid, silibinin, targets the epidermal growth factor receptor (EGFR), insulin‐like growth factor‐1 receptor (IGF‐1R) and NF‐κB (nuclear factor‐kappa B) pathways in PCA. Furthermore, it modulates cell‐cycle regulators, including cyclin‐dependent kinases (CDKs), Cip/Kip and cyclins for its anticancer efficacy against PCA. Silibinin inhibits growth of PCA cells from human, mouse, and rat origins, and also suppresses human prostate tumor xenograft growth in nude mice. Silibinin also inhibits PCA growth in the transgenic adenocarcinoma of mouse prostate (TRAMP) mouse model. Now, silibinin has been entered into phase I/II clinical trials in human PCA patients where preliminary observations were suggestive of its further study in a larger base of the patient population.