Abstract: Constitutively active mitogenic and prosurvival signaling cascades due to aberrant expression and interaction of growth factors and their receptors are well documented in human prostate cancer (PCa). Epidermal growth factor (EGF) and insulin-like growth factor-1 (IGF-1) are potent mitogens that regulate proliferation and survival of PCa cells via autocrine and paracrine loops involving both mitogen-activated protein kinase (MAPK)- and Akt-mediated signaling. Accordingly, here we assessed the effect of inositol hexaphosphate (IP6) on constitutive and ligand (EGF and IGF-1)-induced biological responses and associated signaling cascades in advanced and androgen-independent human PCa PC-3 cells. Treatment of PC-3 cells with 2 mM IP6 strongly inhibited both growth and proliferation and decreased cell viability; similar effects were also observed in other human PCa DU145 and LNCaP cells. IP6 also caused a strong apoptotic death of PC-3 cells together with caspase 3 and PARP cleavage. Mechanistic studies showed that biological effects of IP6 were associated with inhibition of both constitutive and ligand-induced Akt phosphorylation together with a decrease in total Akt levels, but a differential inhibitory effect on MAPKs extra cellular signal-regulated kinase 1/2 (ERK1/2), c-Jun N-terminal protein kinase (JNK1/2), and p38 under constitutive and ligand-activated conditions. Under similar condition, IP6 also inhibited AP-1 DNA-binding activity and decreased nuclear levels of both phospho and total c-Fos and c-Jun. Together, these findings for the first time establish IP6 efficacy in inhibiting aberrant EGF receptor (EGFR) or IGF-1 receptor (IGF-1R) pathway-mediated sustained growth promoting and survival signaling cascades in advanced and androgen-independent human PCa PC-3 cells, which might have translational implications in advanced human PCa control and management.
Abstract: Effective medical treatment and preventive measures for chemical warfare agent sulfur mustard (HD)-caused incapacitating skin toxicity are lacking, because of limited knowledge of its mechanism of action. The proliferating basal epidermal cells are primary major sites of attack during HD-caused skin injury. Therefore, employing mouse JB6 and human HaCaT epidermal cells, here, we investigated the molecular mechanism of HD analogue 2-chloroethyl ethyl sulfide (CEES)-induced skin cytotoxicity. As compared to the control, up to 1 mM CEES treatment of these cells for 2, 4, and 24 h caused dose-dependent decreases in cell viability and proliferation as measured by DNA synthesis, together with S and G2-M phase arrest in cell cycle progression. Mechanistic studies showed phosphorylation of DNA damage sensors and checkpoint kinases, ataxia telangiectasia-mutated (ATM) at ser1981 and ataxia telangiectasia-Rad3-related (ATR) at ser428 within 30 min of CEES exposure, and modulation of S and G2-M phase-associated cell cycle regulatory proteins, which are downstream targets of ATM and ATR kinases. Hoechst-propidium iodide staining demonstrated that CEES-induced cell death was both necrotic and apoptotic in nature, and the latter was induced at 4 and 24 h of CEES treatment in HaCaT and JB6 cells, respectively. An increase in caspase-3 activity and both caspase-3 and poly(ADP-ribose)polymerase (PARP) cleavage coinciding with CEES-caused apoptosis in both cell lines suggested the involvement of the caspase pathway. Together, our findings suggest a DNA-damaging effect of CEES that activates ATM/ATR cell cycle checkpoint signaling as well as caspase-PARP pathways, leading to cell cycle arrest and apoptosis/necrosis in both JB6 and HaCaT cells. The identified molecular targets, quantitative biomarkers, and epidermal cell models in this study have the potential and usefulness in rapid development of effective prophylactic and therapeutic interventions against HD-induced skin toxicity.
Abstract: Inositol hexaphosphate (IP6) causes G(1) arrest and increases cyclin-dependent kinase inhibitors p21/Cip1 and p27/Kip1 protein levels in human prostate cancer (PCa) DU145 cells lacking functional p53. However, whether cyclin-dependent kinase inhibitor I induction by IP6 plays any role in its antitumor efficacy is unknown. Herein, we observed that either p21 or p27 knockdown by small interfering RNA has no considerable effect on IP6-induced G(1) arrest, growth inhibition, and death in DU145 cells; however, the simultaneous knockdown of both p21 and p27 reversed the effects of IP6. To further confirm these findings both in vitro and in vivo, we generated DU145 cell variants with knockdown levels of p21 (DU-p21), p27 (DU-p27), or both (DU-p21+p27) via retroviral transduction of respective short hairpin RNAs. Knocking down p21 or p27 individually did not alter IP6-caused cell growth inhibition and G(1) arrest; however, their simultaneous ablation completely reversed the effects of IP6. In tumor xenograft studies, IP6 (2% w/v, in drinking water) caused a comparable reduction in tumor volume (40-46%) and tumor cell proliferation (26-28%) in DU-EV (control), DU-p21, and DU-p27 tumors but lost most of its effect in DU-p21+p27 tumors. IP6-caused apoptosis also occurred in a Cip/Kip-dependent manner because DU-p21+p27 cells were completely resistant to IP6-induced apoptosis both in cell culture and xenograft. Together, these results provide evidence, for the first time, of the critical role of p21 and p27 in mediating the anticancer efficacy of IP6, and suggest their redundant role in the antiproliferative and proapoptotic effects of IP6 in p53-lacking human PCa cells, both in vitro and in vivo.
Abstract: Sulfur mustard (HD) is an alkylating and cytotoxic chemical warfare agent, which inflicts severe skin toxicity and an inflammatory response. Effective medical countermeasures against HD-caused skin toxicity are lacking due to limited knowledge of related mechanisms, which is mainly attributed to the requirement of more applicable and efficient animal skin toxicity models. Using a less toxic analog of HD, chloroethyl ethyl sulfide (CEES), we identified quantifiable inflammatory biomarkers of CEES-induced skin injury in dose- (0.05-2 mg) and time- (3-168 h) response experiments, and developed a CEES-induced skin toxicity SKH-1 hairless mouse model. Topical CEES treatment at high doses caused a significant dose-dependent increase in skin bi-fold thickness indicating edema. Histopathological evaluation of CEES-treated skin sections revealed increases in epidermal and dermal thickness, number of pyknotic basal keratinocytes, dermal capillaries, neutrophils, macrophages, mast cells, and desquamation of epidermis. CEES-induced dose-dependent increases in epidermal cell apoptosis and basal cell proliferation were demonstrated by the terminal deoxynucleotidyl transferase (tdt)-mediated dUTP-biotin nick end labeling and proliferative cell nuclear antigen stainings, respectively. Following an increase in the mast cells, myeloperoxidase activity in the inflamed skin peaked at 24 h after CEES exposure coinciding with neutrophil infiltration. F4/80 staining of skin integuments revealed an increase in the number of macrophages after 24 h of CEES exposure. In conclusion, these results establish CEES-induced quantifiable inflammatory biomarkers in a more applicable and efficient SKH-1 hairless mouse model, which could be valuable for agent efficacy studies to develop potential prophylactic and therapeutic interventions for HD-induced skin toxicity.
Abstract: Constitutive activation of phosphoinositide 3-kinase (PI3K)-Akt pathway transmits growth-regulatory signals that play a central role in promoting survival, proliferation, and angiogenesis in human prostate cancer cells. Here, we assessed the efficacy of inositol hexaphosphate (IP6) against invasive human prostate cancer PC-3 and C4-2B cells and regulation of PI3K-Akt pathway. IP6 treatment of cells suppressed proliferation, induced apoptosis along with caspase-3 and poly(ADP-ribose) polymerase (PARP) cleavage, and inhibited constitutive activation of Akt and its upstream regulators PI3K, phosphoinositide-dependent kinase-1 and integrin-linked kinase-1 (ILK1). Downstream of Akt, IP6 inhibited the phosphorylation of glycogen synthase kinase-3alpha/beta at Ser(21/9) and consequently reduced cyclin D1 expression. Efficacy studies employing PC-3 tumor xenograft growth in nude mice showed that 2% (w/v) IP6 feeding in drinking water inhibits tumor growth and weight by 52% to 59% (P < 0.001). Immunohistochemical analysis of xenografts showed that IP6 significantly reduces the expression of molecules associated with cell survival/proliferation (ILK1, phosphorylated Akt, cyclin D1, and proliferating cell nuclear antigen) and angiogenesis (platelet endothelial cell adhesion molecule-1 or CD31, vascular endothelial growth factor, endothelial nitric oxide synthase, and hypoxia-inducible factor-1alpha) together with an increase in apoptotic markers (cleaved caspase-3 and PARP). These findings suggest that, by targeting the PI3K-ILK1-Akt pathway, IP6 suppresses cell survival, proliferation, and angiogenesis but induces death in prostate cancer cells, which might have translational potential in preventing and controlling the growth of advanced and aggressive prostate cancer for which conventional chemotherapy is not effective.
Abstract: A monofunctional analog of the chemical warfare agent sulfur mustard (HD), 2-chloroethyl ethyl sulfide (CEES), induces tissue damage similar to HD. Herein we studied the molecular mechanisms associated with CEES-induced skin inflammation and toxicity in SKH-1 hairless mice. Topical CEES exposure caused an increase in oxidative stress as observed by enhanced 4-hydroxynonenal and 5,5-dimethyl-2-(8-octanoic acid)-1-pyrroline N-oxide protein adduct formation and an increase in protein oxidation. The CEES-induced increase in the formation of 8-oxo-2-deoxyguanosine indicated DNA oxidation. CEES exposure instigated an increase in the phosphorylation of mitogen-activated protein kinases (MAPKs; ERK1/2, JNK, and p38). After CEES exposure, a significant increase in the phosphorylation of Akt at Ser473 and Thr308 was observed as well as upregulation of its upstream effector, PDK1, in mouse skin tissue. Subsequently, CEES exposure caused activation of AP-1 family proteins and the NF-kappaB pathway, including phosphorylation and degradation of IkappaBalpha in addition to phosphorylation of the NF-kappaB essential modulator. Collectively, our results indicate that CEES induces oxidative stress and the activation of the transcription factors AP-1 and NF-kappaB via upstream signaling pathways including MAPKs and Akt in SKH-1 hairless mouse skin. These novel molecular targets could be supportive in the development of prophylactic and therapeutic interventions against HD-related skin injury.
Abstract: UVB exposure of skin results in various biologic responses either through direct or indirect damage to DNA and non-DNA cellular targets via the formation of free radicals, reactive oxygen species (ROS) and inflammation. Bucillamine [N-(2-mercapto-2-methylpropionyl)-l-cysteine] is a cysteine-derived compound that can replenish endogenous glutathione due to its two donatable thiol groups, and functions as an antioxidant. In this study, we investigated the effects of bucillamine on UVB-induced photodamage using the SKH-1 hairless mouse model. We have demonstrated that UVB exposure (two consecutive doses, 230 mJ cm(-2)) on the dorsal skin of SKH-1 mice induced inflammatory responses (edema, erythema, dermal infiltration of leukocytes, dilated blood vessels) and p53 activation as early as 6 h after the last UVB exposure. Bucillamine pretreatment (20 mg kg(-1) of body weight, administered subcutaneously) markedly attenuated UVB-mediated inflammatory responses and p53 activation. We have also demonstrated that the stabilization and upregulation of p53 by UVB correlated with phosphorylation of Ser-15 and Ser-20 residues of p53 protein and that bucillamine pretreatment attenuated this effect. We propose that bucillamine has potential to be effective as a photoprotective agent for the management of pathologic conditions elicited by UV exposure.
Abstract: Herein, for the first time, we investigated in vivo efficacy and associated molecular biomarkers and mechanisms of a chemopreventive agent, silibinin, against human colorectal carcinoma (CRC) HT29 xenograft growth. Nude mice were implanted with HT29 cells and fed with vehicle (carboxymethyl cellulose or phosphatidylcholine) or 200 mg/kg/d dose of silibinin or 100 and 200 mg/kg/d doses of silybin-phytosome (5 days per week) for 32 days. Silibinin inhibited tumor growth that accounted for 48% (P = 0.002) decrease in tumor volume and 42% (P = 0.012) decrease in tumor weight at the end of the experiment without any adverse health effect. A stronger antitumor efficacy was observed with silybin-phytosome preparation. Silibinin decreased proliferation index by 40% (P < 0.001), increased apoptotic index by approximately 2-fold (P = 0.001), and reduced microvessel density by 36% (P = 0.001) in tumors. Antiproliferative and proapoptotic effects of silibinin were associated with down-regulation of extracellular signal-regulated kinase 1/2 (ERK1/2) and Akt phosphorylation as well as cyclin D1 expression. Antiangiogenic effect of silibinin was coupled with a strong decrease in inducible nitric oxide synthase (NOS) and NOS3, cyclooxygenase-1 (COX-1) and COX-2, and hypoxia-inducing factor-1 alpha (HIF-1 alpha) and vascular endothelial growth factor (VEGF). These findings suggest in vivo antitumor efficacy of silibinin against CRC involving its antiproliferative, proapoptotic, and antiangiogenic activities. The inhibition of ERK1/2 and Akt signaling may account for antiproliferative and proapoptotic effects, whereas down-regulation of NOS, COX, HIF-1 alpha, and VEGF expression could lead to antiangiogenic effect of silibinin against CRC. Overall, potential use of silibinin against human CRC could be suggested.
Abstract: 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.
Abstract: Effective strategies are lacking for the management of urinary bladder cancer for which smoking is a potential risk factor. Herein, we evaluated chemoprevention of urinary bladder cancer by natural chemopreventive agents, silymarin and silibinin, in a preclinical animal (ICR mouse) model of bladder cancer induced by tobacco smoke carcinogen N-butyl-N-(4-hydroxybutyl) nitrosamine (OH-BBN). Mice were fed p.o. with saline or OH-BBN (0.05%, w/v) in drinking water for 6 weeks or with silymarin or silibinin (200 mg/kg body weight for both) starting 1 week before OH-BBN exposure for 51 weeks. Silymarin and silibinin strongly arrested OH-BBN-induced tumor progression at the stage of mucosal dysplasia with a striking reduction in papillary nodular dysplasia as well as invasive carcinoma. Some silymarin- or silibinin-treated mice developed no urothelial lesions in spite of OH-BBN exposure. Immunohistochemical analyses at study conclusion revealed that silymarin and silibinin decreased cell proliferation by 42% (P < 0.001) and 44% (P < 0.001) and increased apoptosis by 4-fold (P < 0.05) and 6-fold (P < 0.05) in OH-BBN-induced urothelium, respectively. Antiproliferative and apoptotic effects of silymarin and silibinin were associated with decreases in (a) cyclin D1 protein level and extracellular signal-regulated kinase-1/2 phosphorylation and in (b) protein levels of survivin and nuclear phospho-p65 (Ser(276) and Ser(536)), respectively. Together, these results suggest that silymarin and silibinin inhibit chemically induced urinary bladder tumor growth and progression possibly by inhibiting cell proliferation and enhancing apoptosis.
Abstract: UVB radiation-induced DNA damage in skin activates cellular pathways involved in DNA repair, cell cycle regulation, and apoptosis, important events that prevent conversion of damaged skin cells into cancer. We reported recently the efficacy of silibinin against photocarcinogenesis along with altered molecular events in tumors (Cancer Research, 64:6349-56, 2004). The molecular and biological events modulated by silibinin in chronically UVB-irradiated skin leading to cancer prevention, however, are not known. Herein, we describe effect of silibinin on skin 15 and 25 weeks after UVB exposure and compared them with molecular alterations in skin tumors. UVB decreased E2F1 but increased E2F2 and E2F3 protein levels in skin, and these were reversed by silibinin treatment. Silibinin-induced E2F1 was accompanied by an inhibition of apoptosis and decreases in p53 and cyclin-dependent kinase inhibitors. Silibinin-caused decrease in E2F2 and E2F3 was accompanied by reduced levels of cyclin-dependent kinases, cyclins, CDC25C, and mitogen-activated protein kinases and Akt signaling and inhibition of cell proliferation. In tumorigenesis protocols, topical or dietary silibinin significantly inhibited tumor appearance and growth. As opposed to UVB-exposed skin, UVB-induced tumors showed elevated levels of E2F1, but these were reduced in silibinin-treated tumors without any effect on E2F2 and E2F3. Contrary to the inhibition of apoptosis and p53 expression in UVB-exposed skin cells, silibinin increased these variables in tumors. These differential effects of silibinin on E2F1 versus E2F2 and E2F3 and their associated molecular alterations and biological effects in chronic UVB-exposed skin suggest their role in silibinin interference with photocarcinogenesis.
Abstract: 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 microg/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.
Abstract: 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/cm(2) 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.
Abstract: Ultraviolet B (UVB) radiation is a complete skin carcinogen causing DNA damage as a tumor-initiating event and activating signaling cascades that play a critical role in its tumor-promoting potential. Recently we reported that a naturally occurring flavonoid, silibinin, protects UVB-induced skin damages and prevents photocarcinogenesis. Here we examined silibinin efficacy on acute and chronic UVB-caused mitogen-activated protein kinases (MAPKs) and AKT activation and associated biological responses in SKH-1 hairless mouse skin. A single UVB exposure at 180 mJ/cm2 dose resulted in varying degrees of ERK1/2, JNK1/2, MAPK/p38 and AKT phosphorylation at various time-points in mouse skin; however, topical application of silibinin prior to or immediately after UVB exposure, or its dietary feeding strongly inhibited the activation of these molecules at all the time-points examined. Stronger effects of silibinin towards inhibition of UVB-caused phosphorylation of MAPKs and AKT were also observed in a chronic UVB (180 mJ/cm2/day for 5 days) exposure protocol. Immunohistochemical analysis of chronically exposed skin sections showed that silibinin treatment in all three protocols increases UVB-induced p53-positive cells and decreases UVB-caused cell proliferation, apoptotic and sunburn cells. These findings suggest that silibinin inhibits UVB-induced MAPK and AKT signaling and increases p53 in mouse skin, and that these effects of silibinin possibly lead to a decrease in UVB-caused proliferation and apoptosis, which might, in part, be responsible for its overall efficacy against photocarcinogenesis.
Abstract: 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.
Abstract: 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.
Abstract: Non-melanoma skin cancer (NMSC) accounts for >1 million new cases each year in the US alone suggesting that more approaches are needed for its prevention and control. Earlier studies by us have shown that silymarin (a crude form of biologically active silibinin with some other isomers), isolated from milk thistle, affords strong protection against ultraviolet (UV) radiation-induced NMSC in SKH-1 hairless mice; however, the molecular mechanisms of its efficacy are not known. Here, we assessed the effect of silibinin on UV-induced DNA damage and p53-p21/Cip1 accumulation, and their roles in UV-induced cell proliferation and apoptosis in SKH-1 hairless mouse epidermis. Topical application of silibinin prior to, or immediately after, UV irradiation resulted in a very strong protective effect against UV-induced thymine dimer positive cells in epidermis accounting for 76-85% (P < 0.001) inhibition. In other studies, silibinin treatment resulted in a further up-regulation of p53 by approximately 1.6-fold (P < 0.001) together with an increase ( approximately 2-fold, P < 0.001) in p21/Cip1 protein levels. Proliferative cell nuclear antigen staining showed that silibinin pre- or post-topical application significantly inhibits (40-52 and 20-40%, respectively, P < 0.001) UV-induced epidermal cell proliferation. In addition, silibinin strongly decreased UV-caused terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling-positive apoptotic/sunburn cell formation (P < 0.001). These findings suggest that silibinin affords strong protection against UV-induced damage in epidermis by a decrease in thymine dimer positive cells and an up-regulation of p53-p21/Cip1 possibly leading to an inhibition in both cell proliferation and apoptosis. Comparable effects of silibinin following its pre- or post-UV application suggest that mechanisms other than sunscreen effect are operational in silibinin efficacy against UV-caused skin damages.
Abstract: Several recent studies by us have shown the strong chemopreventive efficacy of silibinin against both ultraviolet B (UVB) radiation and chemical carcinogen-induced tumorigenesis in mouse skin models. The molecular mechanisms underlying silibinin protective efficacy, however, are not completely known. Here, we examined the effect of silibinin on UVB-caused apoptosis in human epidermoid carcinoma A431 cells. Irradiation of cells with different doses of UVB (5-100 mJ/cm2) and different time periods (0.5-24h) resulted in a dose- and time-dependent increase in apoptosis (P < 0.05-0.001). Silibinin (100-200 microM) pre-treatment, however, resulted in an increase in UVB-induced apoptosis (P < 0.05-0.001); interestingly, its post-treatment caused a decrease in UVB-induced apoptosis (P < 0.05-0.001). A similar pattern in the activation of caspases-9, -3, and -7 was observed with these silibinin treatments. Further, silibinin treatment prior to or immediately after UVB exposure altered Bcl-2, Bax, Bak, and cytochrome c levels in mitochondria and cytosol in favor of or against apoptosis, respectively. Silibinin treatment prior to UVB also increased the activation of mitogen/stress activated protein kinases Erk1/2, JNK, and p38 kinase as compared to its post-treatment. Together, for the first time, our results demonstrate the role of mitochondrial apoptotic machinery and MAPK signaling cascade in silibinin-caused increase as well as protection in UVB-induced apoptosis in A431 cells, and suggest that similar mechanisms might be involved in preventive efficacy of silibinin against UVB-induced skin tumorigenesis.
Abstract: 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.
Abstract: The acute and cumulative dose-related toxicity and drug resistance, mediated via nuclear factor kappaB (NFkappaB), 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 mumol/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 NFkappaB 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 NFkappaB 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 NFkappaB signaling.
Abstract: The present study was conducted to ascertain the modulatory influences of Ferula asafoetida L. (asafoetida, flavoring agent) on the mammary epithelial tissue differentiation, hepatic drug metabolizing enzymes, antioxidant profiles and N-methyl-N-nitrosourea (MNU)-induced mammary carcinogenesis in Sprague-Dawley rats. Feeding with two doses of asafoetida (1.25 and 2.5% w/w in diet) showed a remarkable increase in the development and differentiation of ducts/ductules (p < 0.01-0.005), lobules (p < 0.005) and a decrease in terminal end buds (p < 0.05-0.005) as compared to both normal and MNU-treated control animals. To assess the biochemical parameters, effect of asafoetida on drug-metabolizing enzymes was evaluated in the liver of rats. Asafoetida treatment significantly reduced (p < 0.05) the levels of cytochrome P450 and b5. There was an enhancement in the activities of glutathione S-transferase (p < 0.05-0.005), DT-diaphorase (p < 0.05-0.01), superoxide dismutase (p < 0.01-0.005) and catalase (p < 0.05-0.005) and in the level of reduced glutathione (p < 0.05-0.005), followed by asafoetida treatment. Also, asafoetida significantly restored the level of antioxidant system, depleted by MNU-treatment. The strengthening of antioxidant system by the lower and higher doses of asafoetida in the presence and absence of MNU was further substantiated by a significant inhibition (p < 0.005) in lipid peroxidation as measured by thiobarbituric acid-reactive substances (TBARS) in the liver of rat. Further, in long-term animal studies, where MNU was used to induce mammary carcinogenesis, asafoetida treatment resulted in a significant reduction in the multiplicity (p < 0.001) and size of palpable mammary tumors (p < 0.005-0.001) and a delay in mean latency period of tumor appearance (p < 0.005). Together, these findings indicate the chemopreventive potential of asafoetida against MNU-induced mammary carcinogenesis. Thus, asafoetida needs further investigation with regard to identification and characterization of its active principle(s) and mechanism of action, for this compound to be developed as a potential chemopreventive agent for human cancers.
