Biomedical subjects

Rajesh Agarwal

Publications and source records attributed to Rajesh Agarwal.

At least 55 records · Page 3Linked to original sources

PubMed2004

Synergistic anti-cancer effects of silibinin with conventional cytotoxic agents doxorubicin, cisplatin and carboplatin against human breast carcinoma MCF-7 and MDA-MB468 cells.

Significant emphasis is being placed on combination chemotherapy of cancer using cytotoxic agents and naturally occurring chemopreventive agents, having different mechanisms of action with non-overlapping toxicity. In this regard, here we assessed whether a cancer preventive agent silibinin synergizes the therapeutic potential of doxorubicin (Dox), cisplatin or carboplatin, the chemotherapeutic drugs, in both estrogen-dependent and -independent human breast carcinoma, MCF-7 and MDA-MB468 cells, respectively. When tested alone, each of the four agents showed growth inhibition in both the cell lines in a dose- and a time-dependent manner. Based on their growth inhibitory effects, several combinations of silibinin (25-100 microM) with Dox (10-75 nM), cisplatin (0.2-2 microg/ml) or carboplatin (2-20 microg/ml) were next assessed for their synergistic, additive and/or antagonistic efficacy towards cell growth inhibition and apoptotic death. The strongest synergistic effects for cell growth inhibition [combination index (CI) 0.35 for MCF-7 and 0.45 for MDA-MB468 cells] were evident at a silibinin dose of 100 microM plus 25 nM Dox, in both the cell lines. Most of the CIs for other combinations of these three drugs with silibinin also suggested strong synergistic effects for cell growth inhibition in both MCF-7 and MDA-MB468 cells. In quantitative apoptosis studies, combination of silibinin with Dox resulted in much stronger apoptotic death compared to each agent alone in both cell lines. In case of silibinin combination with cisplatin, it showed no additional apoptotic effect in either cell line. Similarly, silibinin plus carboplatin combination showed stronger apoptotic effect only in MCF-7 cells. Together, these results suggest a possible synergism between silibinin and conventional cytotoxic agents for breast cancer treatment, and warrant further in vivo studies in pre-clinical breast cancer models.

Anticarcinogenic Agents↗

PubMed2004

Anti-angiogenic efficacy of grape seed extract in endothelial cells.

The present study is focused on the investigation of in vitro angiogenic potential of grape seed extract (GSE). Human umbilical vein endothelial cells (HUVEC) in culture were used to assess the effect of GSE on proliferation, survival, matrix metalloproteinases (MMPs) secretion and capillary tube formation. Our data show that GSE significantly inhibited cell growth (< or =91%, P<0.001) and cell viability (< or =64%, P<0.005) of HUVEC. Further studies by BrdU incorporation and annexin V staining showed that GSE strongly inhibits DNA synthesis (< or =76%, P<0.001) and induces apoptotic cell death (< or =42.8% versus control 2.6%, P<0.05) in HUVEC, respectively. Similar GSE treatment decreased secreted levels of MMP-2 from HUVEC. GSE also inhibited capillary tube formation on Matrigel by endothelial cells in a dose-dependent manner. These findings suggest that GSE possesses an anti-angiogenic potential, which is associated with its antiproliferative, proapoptotic and inhibition of MMP-2 secretion in endothelial cells. Further studies are warranted to evaluate the in vivo anti-angiogenic efficacy of GSE for its possible usefulness in the inhibition of tumor angiogenesis.

Angiogenesis Inhibitors↗

PubMed2003

Silibinin down-regulates survivin protein and mRNA expression and causes caspases activation and apoptosis in human bladder transitional-cell papilloma RT4 cells.

Bladder cancer is the fourth and eighth most common cancer in men and women in the United States, respectively. Survivin, a member of inhibitor of apoptosis protein (IAP) gene family, is deregulated in a wide range of malignancies, including carcinoma of the bladder urothelium. Recent advances have identified survivin as a novel intervention target to induce apoptosis in cancer cells by phytochemicals or synthetic agents. Silibinin is a naturally occurring flavanone, isolated from milk thistle extract, and has been shown to possess cancer prevention/intervention potential against various cancers. In several animal and human studies, it is found to be safe and non-toxic. Human bladder transitional-cell papilloma RT4 cells were treated with silibinin and analyzed for survivin protein and mRNA levels by Western blotting and real-time RT-PCR, respectively. Silibinin treatment of cells for 24 h at 100 microM dose resulted in approximately 50% decrease in survivin protein level; however, treatment at 200 microM dose for 24 and 48 h showed a complete loss in survivin protein without any change in actin used as loading control. Employing RT-PCR analysis we also observed that silibinin causes a strong to complete decrease in survivin mRNA levels. In other studies, down-regulation of survivin by silibinin was associated with a very strong and prominent caspases-9 and -3 activation as well as PARP cleavage. Quantitative apoptotic assay showed that silibinin decreased survivin levels and caspases-PARP cleavages, in accord with a strong apoptotic death and growth inhibition of RT4 cells. Together, these findings suggest that more studies are needed to investigate in vivo effect of silibinin on survivin expression and associated biological effects in bladder cancer that could provide useful information for silibinin efficacy in the prevention/intervention of human bladder cancer.

Apoptosis↗

PubMed2003

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 microg/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.

Apoptosis↗

PubMed2003

Dual efficacy of silibinin in protecting or enhancing ultraviolet B radiation-caused apoptosis in HaCaT human immortalized keratinocytes.

An increasing incidence of human skin cancer and other adverse effects of solar ultraviolet (UV) radiation enhance the need for novel chemoprevention strategies. Here, we have studied the effect of silibinin on UVB-induced apoptosis in HaCaT cells. Silibinin strongly prevented lower doses (15 and 30 mJ/cm2) of UVB-induced apoptosis, as observed by a reversal in UVB-caused poly(ADP-ribose) polymerase (PARP) cleavage, caspase 9 activation and an increase in apoptotic cells. UVB-induced PARP cleavage was also abolished by all caspase inhibitor, suggesting that it is a caspase-dependent effect. In other studies, silibinin restored UVB-caused depletion of a protein inhibitor of apoptosis, survivin, concomitant with up-regulation of transcription factor nuclear factor kappaB DNA binding activity, without any noticeable effect on UVB-caused activated protein-1 activation. Further, silibinin treatment up-regulated UVB-induced extracellular signal regulated kinase 1/2 phosphorylation, suggesting a possible role as a survival event in the protective effect of silibinin. In other studies, silibinin caused a moderate increase in phospho-Bcl-2, without any noticeable changes in total Bcl-2 levels, and down-regulated bax levels moderately. Silibinin also caused a strong decrease in Bad heterodimerization with Bclx(L), which was consistent with an increased translocation of Bclx(L) to the mitochondria from the cytosol. Consistent with its protective effect on UVB-caused apoptosis, silibinin also increased S phase arrest, possibly providing a prolonged time for efficient DNA repair. Interestingly, the protective effects of silibinin in HaCaT cells were lost at a higher dose of UVB (120 mJ/cm2) and instead it further enhanced UVB-caused apoptosis together with a strong decrease in UVB-caused activated protein-1 activation. Together, these results clearly demonstrate the dual efficacy of silibinin in protecting or enhancing UVB-caused apoptosis in the same cellular system and suggest that silibinin possibly works as a UVB damage sensor to exert its biological action.

Apoptosis↗

PubMed2003

Silibinin sensitizes human prostate carcinoma DU145 cells to cisplatin- and carboplatin-induced growth inhibition and apoptotic death.

In several recent studies, we have shown that silibinin inhibits the growth of human prostate cancer cells (PCA) both in vitro and in vivo. Here, we investigated the effect of silibinin in combination with cisplatin and carboplatin on human PCA DU145 cell growth and apoptosis. Cisplatin alone at 2 microg/ml dose produced 48% cell growth inhibition, whereas a combination with 50-100 microM silibinin resulted in 63-80% (p<0.05-0.001) growth inhibition. Similarly, compared to 68% growth inhibition at 20 microg/ml carboplatin, addition of 50-100 microM doses of silibinin caused 80-90% inhibition (p<0.005-0.001). In the studies assessing the effect of these combinations on cell cycle progression, a combination of cisplatin or carboplatin with silibinin resulted in a stronger G2-M arrest, compared to these agents alone showing a moderate G2-M and G1 arrests in case of cisplatin and silibinin, and a complete S phase arrest with carboplatin, respectively. A stronger G2-M arrest by these combinations was accompanied by a substantial decrease in the levels of cdc2, cyclin B1 and cdc25C. Silibinin/platinum compound combinations were also effective in inducing apoptosis where cisplatin and carboplatin when combined with silibinin enhanced apoptosis from 8 to 15% and from 20 to 40%, respectively. Apoptosis induction was further confirmed by PARP and caspases 3, 9 and 7 whose cleaved levels were also enhanced by combination treatment. In addition, there was a significant increase in cytochrome c release in the cytosol following treatment of DU145 cells with these combinations. Together, these results show a substantial increase in the efficacy of platinum compounds on human PCA cells, when combined with silibinin, which provide a rationale for further investigations with these combinations.

Antineoplastic Agents↗

PubMed2003

Grape seed extract inhibits EGF-induced and constitutively active mitogenic signaling but activates JNK in human prostate carcinoma DU145 cells: possible role in antiproliferation and apoptosis.

A loss of functional androgen receptor and an enhanced expression of growth factor receptors and associated ligands are causal genetic events in prostate cancer (PCA) progression. These genetic alterations lead to an epigenetic mechanism where a feedback autocrine loop between membrane receptor and ligand (e.g. EGFR-TGFalpha) results in a constitutive activation of MAPK-Elk1-AP1-mediated mitogenic signaling in human PCA at an advanced and androgen-independent stage. We rationalized that inhibiting these epigenetic events could be useful in controlling advanced PCA growth. Recently, we found that grape seed extract (GSE), a dietary supplement rich in flavonoid procyanidins, inhibits advanced and androgen-independent human PCA DU145 cell growth in culture and nude mice. Here, we performed detailed mechanistic studies to define the effect of GSE on EGFR-Shc-MAPK-Elk1-AP1-mediated mitogenic signaling in DU145 cells. Pretreatment of serum-starved cells with GSE resulted in 70% to almost complete inhibition of EGF-induced EGFR activation and 50% to complete inhibition of Shc activation, which corroborated with a comparable decrease in EGF-induced Shc binding to EGFR. Conversely, EGF-induced ERK1/2 phosphorylation was inhibited only by lower doses of GSE; in fact, higher doses showed an increase. Additional studies showed that GSE alone causes a dose- and time-dependent increase in ERK1/2 phosphorylation in starved DU145 cells that is inhibited by an MEK1 inhibitor PD98059. Independent of this increase in ERK1/2 phosphorylation, GSE showed a strong inhibition of ERK1/2 kinase activity to Elk1 in both cellular and cell-free systems. GSE treatment of cells also inhibited both EGF-induced and constitutively active Elk1 phosphorylation and AP1 activation. GSE treatment also showed DNA synthesis inhibition in starved and EGF-stimulated cells as well as loss of cell viability and apoptotic death that was further increased by adding MEK1 inhibitor. Since GSE strongly induced apoptosis independent of its affect on an increase in phospho-ERK1/2, we hypothesized that apoptotic effect of GSE could be by other mechanism(s) including its effect on stress-associated MAPK, the JNK. Indeed, GSE-treated cells showed a strong and sustained increase in phospho-JNK1/JNK2 levels, JNK activity and phospho-cJun levels. An inhibition of GSE-induced JNK activation by a novel JNK inhibitor SP600125 resulted in a significant reversal of GSE-induced apoptotic death suggesting the involvement of JNK activation by GSE in its apoptosis response. Together, these results suggest that anticancer effects of GSE in PCA be mediated via impairment of EGFR-ERK1/2-Elk1-AP1-mediated mitogenic signaling and activation of JNK causing growth inhibition and apoptosis, respectively.

Adaptor Proteins, Signal Transducing↗

PubMed2003

Inositol hexaphosphate inhibits growth, and induces G1 arrest and apoptotic death of prostate carcinoma DU145 cells: modulation of CDKI-CDK-cyclin and pRb-related protein-E2F complexes.

Cancer chemopreventive effects of inositol hexaphosphate (IP6), a dietary constituent, have been demonstrated against a variety of experimental tumors, however, limited studies have been done against prostate cancer (PCA), and molecular mechanisms are not well defined. In the present study, we investigated the growth inhibitory effect and associated mechanisms of IP6 in advanced human PCA cells. Advanced human prostate carcinoma DU145 cells were used to study the anticancer effect of IP6. Flow cytometric analysis was performed for cell cycle progression and apoptosis studies. Western immunoblotting, immunoprecipitation and kinase assay were performed to investigate the involvement of G1 cell cycle regulators and their interplay, and end point markers of apoptosis. A significant dose- as well as time-dependent growth inhibition was observed in IP6-treated cells, which was associated with an increase in G1 arrest. IP6 strongly increased the expression of CDKIs (cyclin-dependent kinase inhibitors), Cip1/p21 and Kip1/p27, without any noticeable changes in G1 CDKs and cyclins, except a slight increase in cyclin D2. IP6 inhibited kinase activities associated with CDK2, 4 and 6, and cyclin E and D1. Further studies showed the increased binding of Kip1/p27 and Cip1/p21 with cyclin D1 and E. In down-stream of CDKI-CDK/cyclin cascade, IP6 increased hypophosphorylated levels of Rb-related proteins, pRb/p107 and pRb2/p130, and moderately decreased E2F4 but increased its binding to both pRb/p107 and pRb2/p130. At higher doses and longer treatment times, IP6 caused a marked increase in apoptosis, which was accompanied by increased levels of cleaved PARP and active caspase 3. IP6 modulates CDKI-CDK-cyclin complex, and decreases CDK-cyclin kinase activity, possibly leading to hypophosphorylation of Rb-related proteins and an increased sequestration of E2F4. Higher doses of IP6 could induce apoptosis and that might involve caspases activation. These molecular alterations provide an insight into IP6-caused growth inhibition, G1 arrest and apoptotic death of human prostate carcinoma DU145 cells.

Apoptosis↗

PubMed2003

Tumor angiogenesis: a potential target in cancer control by phytochemicals.

It is now well established that angiogenesis is an obligatory event for the growth and progression of solid tumors beyond the size limit (approximately 2 mm diameter) imposed by simple diffusion for the nutrient supply. Human tumors can remain dormant for years owing to a balance between cell proliferation and apoptosis. Several hypotheses have been articulated regarding the critical importance of tumor angiogenesis in the development and metastatic spread of tumors, and how preventive/therapeutic inhibition of angiogenesis might be exploited as a novel means of controlling cancer growth. Anti-angiogenic therapy is suggested as one of the most promising approaches to control cancer, as endothelial cells are generally non-transformed cells and are less prone to acquire drug resistance. Tumor vasculature could be an important prognostic marker, and an independent predictor of pathologic stages and malignant potential of cancer. This review is focused on recent developments and comprehensive mechanistic aspects of phytochemicals related to an interplay of angiogenic promoters and inhibitors, and associated signaling in both tumor as well as endothelial cells. Since, vascular endothelial cells constitute the first line exposure to the blood-borne agents, it is plausible that anti-angiogenic activity of phytochemicals could be associated with lowering the risk of cancer by preventing the growth and metastasis of tumor.

Angiogenesis Inhibitors↗

PubMed2003

Inhibition of NF-kappaB pathway in grape seed extract-induced apoptotic death of human prostate carcinoma DU145 cells.

The alarmingly high rate of prostate cancer (PCA) mortality as well as the limited success in the treatment of advanced PCA suggest that additional approaches are needed to control PCA growth and its metastatic potential. A constitutive activation of NF-kappaB family of transcription factors is known to play a major role in chemotherapy resistance in advanced PCA. In recent studies we showed that grape seed extract (GSE) inhibits advanced human PCA growth and induces apoptosis in cell culture and in nude mice. Accordingly, here we assessed the effect of GSE on constitutive and TNFalpha-induced NF-kappaB DNA binding activity and apoptotic death in advanced human prostate carcinoma DU145 cells. Constitutive and TNFalpha-induced NF-kappaB DNA binding activity was inhibited by GSE at doses > or =50 microg/ml and treatments for > or =12 h. This was accompanied by inhibition of IkappaBalpha phosphorylation and IKKalpha kinase activity. A strong induction of apoptosis (P<0.01) was also observed following GSE treatment, while a combination with TNFalpha strongly potentiated apoptosis induction. Our results indicate the potential of developing GSE as an effective cancer therapeutic agent, both alone and in combination with TNFalpha-based chemotherapy of advanced human prostate carcinoma that might prove to be a more effective and less toxic alternative in clinical therapy of PCA.

Animals↗

PubMed2003

Suppression of advanced human prostate tumor growth in athymic mice by silibinin feeding is associated with reduced cell proliferation, increased apoptosis, and inhibition of angiogenesis.

Recently, we observed that dietary feeding of silibinin strongly prevents and inhibits the growth of advanced human prostate tumor xenografts in athymic nude mice without any apparent signs of toxicity together with increased secretion of insulin-like growth factor-binding protein 3 from the tumor in to mouse plasma (R. P. Singh et al., Cancer Res., 62:3063-3069, 2002). In the present study, we investigated the effect of silibinin feeding [0.05% and 0.1% (w/w) in diet for 60 days] on the prognostic biomarkers (namely, proliferation, apoptosis, and angiogenesis) in the prostate tumor xenografts of the above-reported study. Immunohistochemical analysis of the tumors for proliferating cell nuclear antigen and Ki-67 showed that silibinin decreases proliferation index by 28-60% and 30-60% (P<0.001) as compared with their controls, respectively. In situ detection of apoptosis by terminal deoxynucleotidyl transferase dUTP-mediated nick end labeling staining of tumors showed a 7.4-8.1-fold (P<0.001) increase in apoptotic cells in silibinin-fed groups over that of control group. Silibinin also increased activated caspase 3-positive cells by 2.3-3.6-fold (P<0.001). CD31 staining for tumor vasculature showed a significant decrease (21-38%; P<0.001) in tumor microvessel density in silibinin-fed groups of tumors as compared with control group of tumors. Tumor sections were also analyzed for vascular endothelial growth factor and insulin-like growth factor-binding protein 3 protein expression, and a slightly decreased and a moderately increased cytoplasmic immunostaining in silibinin-fed groups were observed as compared with the control group, respectively. Together, these results suggest that inhibition of advanced human prostate tumor xenograft growth in athymic nude mice by silibinin is associated with its in vivo antiproliferative, proapoptotic, and antiangiogenic efficacy in prostate tumor.

Angiogenesis Inhibitors↗

PubMed2003

Growth inhibitory and apoptotic effects of inositol hexaphosphate in transgenic adenocarcinoma of mouse prostate (TRAMP-C1) cells.

Inositol hexaphosphate (IP6), a dietary agent, has been extensively studied for its cancer chemopreventive and anti-cancer efficacy in several different animal models, but not in prostate cancer (PCA) possibly because the known PCA models are both expensive and highly time-consuming. One such PCA model is transgenic adenocarcinoma of mouse prostate (TRAMP), which reproduces the spectrum of benign latent, aggressive and metastatic forms of human PCA. In this study, we investigated the anti-cancer effects of IP6 in TRAMP-derived established TRAMP-C1 cell line. IP6 (0.5-4.0 mM) treatment of cells for 24-72 h resulted in 17-76% cell growth inhibition and 6-35% cell death, in a dose- and time-dependent manner. In the studies assessing whether cell growth inhibition by IP6 is associated with an alteration in cell cycle progression, IP6 treatment resulted in up to 92% cells in G0-G1 phase as compared to controls. In other studies assessing its apoptotic efficacy, IP6 induced a moderate to strong (up to 14-fold over control) apoptotic cell death. In additional studies, pretreatment of cells with all caspases inhibitor for 2 h followed by 2 mM IP6 for 48 h resulted in approximately 50% reversal in IP6-induced apoptosis suggesting a partial involvement of caspases activation in IP6-caused apoptosis. Furthermore, IP6 showed significant induction (6-fold) in caspase-3 activity compared to control suggesting the involvement of caspases activation in IP6-induced apoptosis. However, pretreatment of cells with all caspase inhibitor, which partially reversed the IP6-induced apoptosis, completely inhibited IP6-induced caspase-3 activation, providing convincing evidence of both caspase-dependent and -independent mechanisms in IP6-induced apoptotic death of TRAMP-C1 cells. Together, these results warrant further mechanistic and in vivo efficacy studies with IP6 in TRAMP and other pre-clinical models of PCA.

Adenocarcinoma↗

PubMed2002

Antiproliferative and apoptotic effects of silibinin in rat prostate cancer cells.

BACKGROUND: The tremendous impact of prostate cancer (PCA) on the US male population has led to an increased attention on its prevention and on therapeutic intervention. Short-term models are needed to quickly screen the efficacy of promising agents against PCA. We have established recently several rat PCA cell lines from primary PCA in rats induced by a MNU-testosterone protocol, but their usefulness as a model for screening PCA preventive and therapeutic agents remains to be established. With the rationale that agents found effective in these cells could be promising for efficacy testing in long-term in vivo experiments, e.g., with MNU-testosterone-induced PCA in rats, the major goal of our study was to assess the antiproliferative and apoptotic efficacy in rat PCA cell lines of silibinin, a major active flavonoid component of silymarin, which consists of a group of flavonoid antioxidants occurring in milk thistle (Silybum marianum). METHODS: Three rat PCA cell lines, namely H-7, I-8, and I-26, were treated with silibinin or silymarin, a crude silibinin-containing preparation, at various doses for varying lengths of time. Cell growth and viability studies were carried out by using hemocytometer and Trypan blue dye exclusion methods. Cell cycle distribution studies were conducted by using PI staining and flow cytometry analysis, and DNA synthesis was assessed by bromodeoxyuridine incorporation. Apoptotic cell death was assessed as DNA damage by using an enzyme-linked immunosorbent assay method and by annexin V and PI staining followed by flow cytometry analysis. RESULTS: Silibinin resulted in a significant growth inhibition and reduction in cell viability in each cell line studied in both a dose- and a time-dependent manner. Silibinin treatment of H-7 and I-8 cells at 100 microM dose for 12 and 24 hr resulted in a G1 arrest but caused S phase arrest after a 48-hr treatment period in each cell line studied. Similar silibinin treatment of I-26 cells resulted in a slight S phase arrest at all time points studied. Consistent with these findings, silibinin showed a strong inhibition of DNA synthesis. Silibinin also induced a substantial apoptotic death in each cell line studied. Similar to silibinin, silymarin induced growth inhibition and reduced viability in a dose- and time-dependent manner. CONCLUSION: This study demonstrates that silibinin as well as silymarin induce growth inhibition and apoptosis in rat PCA cells. These results form a strong rationale for PCA prevention and therapeutic intervention studies with silibinin and silymarin in animal models, such as the MNU-testosterone rat PCA model, to establish their efficacy and to further define their mechanisms of action under in vivo conditions.

Animals↗

PubMed2002

Mechanisms of energy restriction: effects of corticosterone on cell growth, cell cycle machinery, and apoptosis.

The restriction of energy intake has documented beneficial effects on numerous diseases including cancer, yet the mechanism(s) that accounts for these effects is unknown. Recently, we showed that the inhibitory activity against mammary carcinogenesis mediated by energy restriction (ER) is accompanied by an increase in the secretion of adrenal cortical steroids. However, ER caused a concomitant reduction in circulating levels of insulin-like growth factor-1, which also may be involved in inhibiting carcinogenesis. To determine what cellular and molecular effects may be because of corticosterone per se, detailed mechanistic studies were performed in vitro using a mouse mammary hyperplastic cell line (TM10). The following questions were addressed: (a) is corticosterone-mediated growth inhibition accounted for by disruption of cell cycle machinery; (b) is growth inhibition accompanied by the induction of apoptosis; and (c) is growth inhibition reversible? At doses of corticosterone (50-200 micro M for 24-72 h) that resulted in inhibition (up to 76%; P < 0.001) of growth, a dose- and time-dependent G(1) arrest in cell cycle progression was observed. In the studies analyzing cell cycle regulatory molecules, corticosterone treatment of cells resulted in a strong induction (up to approximately 10-fold over control; P < 0.01) of KIP1/P27 together with a decrease (up to 98%; P < 0.01) in cyclin-dependent kinase 4 (CDK4) and cyclin D1 protein levels. Cells treated with corticosterone also showed an increased binding (up to 2.6-fold over control; P < 0.01) of KIP1/P27 with CDK4, together with a strong decrease (up to 89%; P < 0.01) in the kinase activity of the CDK4-cyclin D1 complex. Treatment of cells with KIP1/P27 antisense oligonucleotides reversed the growth inhibitory effects of corticosterone. Treatment of cells with RU 486, a glucocorticoid receptor blocker, reversed the effects of corticosterone on cell growth and KIP/P27 protein levels suggesting the involvement of the glucocorticoid receptor in accounting for these effects. Additional studies assessing the biological fate of cells after corticosterone treatment showed that corticosterone exerted reversible growth inhibitory effects with limited apoptotic cell death. Together, these findings show a reversible cytostatic effect of corticosterone via perturbations in cell cycle regulators causing a G(1) arrest in the absence of increased levels of apoptosis. These data provide evidence for a role of corticosterone on some but not all of the cellular activities associated with ER-mediated inhibition of mammary carcinogenesis.

Animals↗

PubMed2002

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.

We have reported recently the anticancer effect of flavonoid antioxidant silymarin, the major part of milk thistle extract, against advanced human prostate carcinoma DU145 cells (X. Zi et al., Cancer Res., 58: 1920-1929, 1998) and later identified that silibinin is the main active component in silymarin responsible for its effect in cell culture studies. On the basis of these observations, here we assessed in vivo growth inhibitory potential of silibinin against advanced human prostate cancer (PCA). Dietary feeding of silibinin at 0.05 and 0.1% doses (w/w) for 60 days, 24 h after s.c. DU145 tumor xenograft implantation in athymic male nude mice, significantly inhibited tumor volume by 35 and 58% (P < 0.05), and wet weight of tumor by 29 and 40% (P < 0.05), respectively. In a second experiment where mice were fed with these test diets for 3 weeks before tumor xenograft implantation and continued on these diets for a total of 63 days, tumor volume and wet weight of tumor were reduced by 53-64% (P < 0.001-0.05) and 31-52% (P < 0.05), respectively. In both studies, animals did not show weight loss or reduced food consumption. These in vivo anticancer effects of silibinin were associated with an increased accumulation (up to 5.8 fold; P < 0.05) of human insulin-like growth factor-binding protein-3 in mouse plasma. In additional studies assessing biological availability of silibinin in nude mice and its antiproliferative activity at such doses in DU145 cells in culture, silibinin levels in plasma and prostate were found to be in the range of 7-13 microg/ml and 3.7-4.6 microg/g, respectively. At these biologically achievable silibinin concentrations, increased IGFBP-3 level in DU145 cell culture medium and a strong DU145 cell growth inhibition were observed that were irreversible in the absence of silibinin in culture medium. These findings extend and translate our observations on in vitro anticancer effect of silibinin/silymarin to an in vivo preclinical PCA model, which may form the basis for a Phase I clinical trial in PCA patients.

Animals↗

PubMed2002

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.

Animals↗

PubMed2002

Grape seed extract induces apoptotic death of human prostate carcinoma DU145 cells via caspases activation accompanied by dissipation of mitochondrial membrane potential and cytochrome c release.

Grape seed extract (GSE), rich in the bioflavonoids commonly known as procyanidins, is one of the most commonly consumed dietary supplements in the United States because of its several health benefits. Epidemiological studies show that many prostate cancer (PCA) patients use herbal extracts as dietary supplements in addition to their prescription drugs. Accordingly, in recent years, we have focused our attention on assessing the efficacy of GSE against PCA. Our studies showed that GSE inhibits growth and induces apoptotic death of human PCA cells in culture and in nude mice. Here, we performed detailed studies to define the molecular mechanism of GSE-induced apoptosis in advanced human PCA DU145 cells. GSE treatment of cells at various doses (50-200 micro g/ml) for 12-72 h resulted in a moderate to strong apoptotic death in a dose- and time-dependent manner. In the studies assessing the apoptotic-signaling pathway induced by GSE, we observed an increase in cleaved fragments of caspases 3, 7 and 9 as well as PARP in GSE-treated cells after 48 and 72 h of treatment. Pre-treatment of cells with general caspases inhibitor, z-Val-Ala-Asp(OMe)-FMK or caspase 3-like proteases inhibitor [z-Asp(OMe)-Glu(OMe)-Val-Asp(OMe)-FMK], almost completely (approximately 90%) inhibited the GSE-induced apoptotic cell death. In a later case, GSE-induced caspase-3 activity was completely inhibited. Selective caspase 9 inhibitor [z-Leu-Glu(OMe)-His-Asp(OMe)-FMK] showed only partial inhibition of GSE-induced apoptosis whereas GSE-induced protease activity of caspase 9 was completely inhibited. Upstream of caspase cascade, GSE showed disappearance of mitochondrial membrane potential and an increase in cytochrome c release in cytosol. Together, these results suggest that GSE possibly causes mitochondrial damage leading to cytochrome c release in cytosol and activation of caspases resulting in PARP cleavage and execution of apoptotic death of human PCA DU145 cells. Furthermore, GSE-caused caspase 3-mediated apoptosis also involves other pathway(s) including caspase 9 activation.

Adenocarcinoma↗

PubMed2002

Silymarin inhibits growth and causes regression of established skin tumors in SENCAR mice via modulation of mitogen-activated protein kinases and induction of apoptosis.

This study reports in vivo therapeutic efficacy of silymarin against skin tumors with mechanistic rationale. 7,12-Dimethylbenz[a]anthracene-12-O-tetradecanoyl-phorbol-13-acetate (DMBA-TPA)-induced established skin papilloma (tumor)-bearing SENCAR mice were fed with 0.5% silymarin in AIN-93M-purified diet (w/w), and both tumor growth and regression were monitored during 5 weeks of feeding regimen. Silymarin feeding significantly inhibited (74%, P < 0.01) tumor growth and also caused regression (43%, P < 0.01) of established tumors. Proliferating cell nuclear antigen and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling immunohistochemical staining of tumors showed that silymarin decreases proliferation index by 48% (P < 0.001) and increases apoptotic index by 2.5-fold (P < 0.001), respectively. Skin tumor growth inhibition and regression by silymarin were also accompanied by a strong decrease (P < 0.001) in phospho-ERK1/2 levels in tumors from silymarin-fed mice compared with controls. In the studies evaluating bioavailability and physiologically achievable level of silymarin (as silibinin) in plasma, skin tumor, skin, liver, lung, mammary gland and spleen, we found 10, 6.5, 3.1, 13.7, 7.7, 5.9 and 4.4 microg silibinin/ml plasma or per gram tissue, respectively. In an attempt to translate these findings to human skin cancer and to establish biological significance of physiologically achievable level, effect of plasma concentration of silibinin was next examined in human epidermoid carcinoma A431 cells. Silibinin treatment of cells in culture at 12.5, 25 (plasma level) and 50 microM doses resulted in 30-74% (P < 0.01-0.001) growth inhibition and 7-42% death of A431 cells in a dose- and time-dependent manner; apoptosis was identified as a cell death response by silibinin. Similar silibinin treatments also resulted in a significant decrease in phospho-mitogen-activated protein kinase/extracellular signal-regulated protein kinase 1/2 (MAPK/ERK1/2) levels, but an up-regulation of stress-activated protein kinase/jun NH(2)-terminal kinase (SAPK/JNK1/2) and p38 mitogen-activated protein kinase (p38 MAPK) activation in A431 cells. The use of MEK1 inhibitor, PD98059, showed that inhibition of ERK1/2 signaling, in part, contributes to silibinin-caused cell growth inhibition. Together, the data suggest that an inhibition of ERK1/2 activation and an increased activation of JNK1/2 and p38 by silibinin could be possible underlying molecular events involved in inhibition of proliferation and induction of apoptosis in A431 cells. These data suggest that silymarin and/or its major active constituent silibinin could be an effective agent for both prevention and intervention of human skin cancer.

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