Sivanandhan Dhanalakshmi

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.

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.

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.

Silibinin strongly inhibits growth and survival of human endothelial cells via cell cycle arrest and downregulation of survivin, Akt and NF-κB: implications for angioprevention and antiangiogenic therapy

Recently, we observed that suppression of tumor xenograft growth by silibinin was associated with reduction in tumor vasculature and an increased apoptosis. Here, we provide evidence for molecular events associated with antiangiogenic efficacy of pharmacologically achievable doses of silibinin in endothelial cell culture system. Our data show that silibinin almost completely (P<0.001) inhibits growth of human umbilical vein endothelial cells (HUVEC) and human microvascular endothelial cells (HMVEC-dermal origin) together with induction of cell death in a dose- and time-dependent manner. Growth inhibition was associated with a strong induction of G1 arrest accompanied by an increase in Kip1/p27, Cip1/p21 and p53. Apoptosis induction (up to 14- to 17-fold in both cell lines, P<0.001) was an underlying mechanism in silibinin-induced death of endothelial cells. In the studies elucidating the molecular events involved in apoptosis, silibinin caused loss of mitochondrial membrane potential and an increase in cytochrome c release from mitochondria. An increase in Bax and a decrease in Mcl-1 proteins were also observed. Silibinin-induced apoptosis involved both caspase-dependent and -independent mechanisms. Silibinin also decreased survivin level and inhibited Akt and NF-κB signaling. Two different PI-3K inhibitors, wortmannin and LY294002, showed Akt-independent activation of NF-κB. Further, silibinin showed a concentration-dependent strong inhibition of capillary tube formation on matrigel, retraction and disintegration of preformed capillary network, inhibition of matrigel invasion and migration, and a decrease in matrix metalloproteinase-2 secretion by HUVEC. Together, these findings identify pleiotropic mechanisms for antiangiogenic efficacy of silibinin, and suggest its usefulness in angioprevention and antiangiogenic therapy.

Oral Silibinin Inhibits Lung Tumor Growth in Athymic Nude Mice and Forms a Novel Chemocombination with Doxorubicin Targeting Nuclear Factor κB–Mediated Inducible Chemoresistance

The acute and cumulative dose-related toxicity and drug resistance, mediated via nuclear factor κB (NFκB), of anthracycline anticancer drugs pose a major problem in cancer chemotherapy. Here, we report that oral silibinin (a flavanone) suppresses human non–small-cell lung carcinoma A549 xenograft growth (P = 0.003) and enhances the therapeutic response (P < 0.05) of doxorubicin in athymic BALB/c nu/nu mice together with a strong prevention of doxorubicin-caused adverse health effects. Immunohistochemical analyses of tumors showed that silibinin and doxorubicin decrease (P < 0.001) proliferation index and vasculature and increase (P < 0.001) apoptosis; these effects were further enhanced (P < 0.001) in combination treatment. Pharmacologic dose of silibinin (60 μmol/L) achieved in animal study was biologically effective (P < 0.01 to 0.001, growth inhibition and apoptosis) in vitro in A549 cell culture together with an increased efficacy (P < 0.05 to 0.001) in doxorubicin (25 nmol/L) combination. Furthermore, doxorubicin increased NFκB DNA binding activity as one of the possible mechanisms for chemoresistance in A549 cells, which was inhibited by silibinin in combination treatment. Consistent with this, silibinin inhibited doxorubicin-caused increased translocation of p65 and p50 from cytosol to nucleus. Silibinin also inhibited cyclooxygenase-2, an NFκB target, in doxorubicin combination. These findings suggest that silibinin inhibits in vivo lung tumor growth and reduces systemic toxicity of doxorubicin with an enhanced therapeutic efficacy most likely via an inhibition of doxorubicin-induced chemoresistance involving NFκB signaling.

In Vivo Suppression of Hormone-Refractory Prostate Cancer Growth by Inositol Hexaphosphate: Induction of Insulin-Like Growth Factor Binding Protein-3 and Inhibition of Vascular Endothelial Growth Factor

Purpose: Diet composition is an important etiologic factor in prostate cancer (PCA) growth and has significant impact on clinical PCA appearance. Because inositol hexaphosphate (IP6) is a dietary phytochemical present in cereals, soy, legumes, and fiber-rich foods, we evaluated efficacy of IP6 against PCA growth and associated molecular events. Experimental Design: DU145 cells were injected into nude mice, and animals were fed normal drinking water or 1 or 2% IP6 in drinking water for 12 weeks. Body weight, diet, water consumption, and tumor sizes were monitored. Tumors were immunohistochemically analyzed for proliferating cell nuclear antigen, terminal deoxynucleotidyl transferase-mediated nick end labeling, and CD31. Tumor-secreted insulin-like growth factor binding protein (IGFBP)-3 and vascular endothelial growth factor (VEGF) were quantified in plasma by ELISA. Results: IP6 feeding resulted in suppression of hormone-refractory human prostate tumor growth without any adverse effect on body weight gain, diet, and water consumption during entire study. At the end of study, tumor growth inhibition by 1 and 2% IP6 feeding was 47 and 66% (P = 0.049–0.012) in terms of tumor volume/mouse and 40 and 66% (P = 0.08–0.003) in terms of tumor weight/mouse, respectively. Tumor xenografts from IP6-fed mice showed significantly (P < 0.001) decreased proliferating cell nuclear antigen-positive cells but increased apoptotic cells. Tumor-secreted IGFBP-3 levels were also increased up to 1.7-fold in IP6-fed groups. Additionally, IP6 strongly decreased tumor microvessel density and inhibited tumor-secreted VEGF levels. Conclusions: IP6 suppresses hormone-refractory PCA growth accompanied by inhibition of tumor cell proliferation and angiogenesis and increased apoptosis. IP6-caused increase in IGFBP-3 and decrease in VEGF might have a role in PCA growth control.

Dietary Feeding of Silibinin Prevents Early Biomarkers of UVB Radiation–Induced Carcinogenesis in SKH-1 Hairless Mouse Epidermis

Solar radiation is the causal etiologic factor in the development of nonmelanoma skin cancer (NMSC). Depletion of the stratospheric ozone layer leads to an increase in ambient UV radiation loads, which are expected to further raise skin cancer incidence in many temperate parts of the world, including the United States, suggesting that skin cancer chemopreventive approaches via biomarker efficacy studies or vice versa are highly warranted. Based on our recent study reporting strong efficacy of silibinin against photocarcinogenesis, we assessed here the protective effects of its dietary feeding on UVB-induced biomarkers involved in NMSC providing a mechanistic rationale for an early-on silibinin efficacy in skin cancer prevention. Dietary feeding of silibinin at 1% dose (w/w) to SKH-1 hairless mice for 2 weeks before a single UVB irradiation at 180 mJ/cm2 dose resulted in a strong and significant (P < 0.001) decrease in UVB-induced thymine dimer–positive cells and proliferating cell nuclear antigen, terminal deoxynucleotidyl transferase–mediated dUTP nick end labeling, and apoptotic sunburn cells together with an increase (P < 0.001) in p53 and p21/cip1-positive cell population in epidermis. These findings suggest that dietary feeding of silibinin affords strong protection against UVB-induced damages in skin epidermis by (a) either preventing DNA damage or enhancing repair, (b) reducing UVB-induced hyperproliferative response, and (c) inhibiting UVB-caused apoptosis and sunburn cell formation, possibly via silibinin-caused up-regulation of p53 and p21/cip1 as major UVB-damage control sensors.