Angiopreventive efficacy of pure flavonolignans from milk thistle extract against prostate cancer: targeting VEGF-VEGFR signaling.

The role of neo-angiogenesis in prostate cancer (PCA) growth and metastasis is well established, but the development of effective and non-toxic pharmacological inhibitors of angiogenesis remains an unaccomplished goal. In this regard, targeting aberrant angiogenesis through non-toxic phytochemicals could be an attractive angiopreventive strategy against PCA. The rationale of the present study was to compare the anti-angiogenic potential of four pure diastereoisomeric flavonolignans, namely silybin A, silybin B, isosilybin A and isosilybin B, which we established previously as biologically active constituents in Milk Thistle extract. Results showed that oral feeding of these flavonolignans (50 and 100 mg/kg body weight) effectively inhibit the growth of advanced human PCA DU145 xenografts. Immunohistochemical analyses revealed that these flavonolignans inhibit tumor angiogenesis biomarkers (CD31 and nestin) and signaling molecules regulating angiogenesis (VEGF, VEGFR1, VEGFR2, phospho-Akt and HIF-1α) without adversely affecting the vessel-count in normal tissues (liver, lung, and kidney) of tumor bearing mice. These flavonolignans also inhibited the microvessel sprouting from mouse dorsal aortas ex vivo, and the VEGF-induced cell proliferation, capillary-like tube formation and invasiveness of human umbilical vein endothelial cells (HUVEC) in vitro. Further studies in HUVEC showed that these diastereoisomers target cell cycle, apoptosis and VEGF-induced signaling cascade. Three dimensional growth assay as well as co-culture invasion and in vitro angiogenesis studies (with HUVEC and DU145 cells) suggested the differential effectiveness of the diastereoisomers toward PCA and endothelial cells. Overall, these studies elucidated the comparative anti-angiogenic efficacy of pure flavonolignans from Milk Thistle and suggest their usefulness in PCA angioprevention.

Role of E-cadherin in antimigratory and antiinvasive efficacy of silibinin in prostate cancer cells.

The epithelial-to-mesenchymal transition (EMT) in prostate cancer (PCA) cells is considered prerequisite for acquiring migratory/invasive phenotype, and subsequent metastasis. We hypothesized that promoting the E-cadherin expression in PCA cells by using nontoxic phytochemicals, like silibinin, would prevent EMT and consequently invasiveness. Our results showed that silibinin treatment (5-90 µmol/L) significantly inhibits migratory and invasive potential of advance human PCA PC3, PC3MM2, and C4-2B cells in in vitro assays. Importantly, the antimigratory/antiinvasive efficacy of silibinin was not due to its cytotoxicity toward PCA cells. Molecular analyses showed that silibinin increases E-cadherin level that was localized mainly at cellular membrane as evidenced by subcellular fractional and confocal analyses in PC3 cells, which might be responsible for morphologically observed shift toward epithelial character. Silibinin also decreased the levels of Slug, Snail, phospho-Akt(ser(473)), nuclear ß-catenin, phospho-Src(tyr(419)) and Hakai; together they play an important role in regulating E-cadherin expression/function and EMT. Similar silibinin effects on E-cadherin, ß-catenin, phospho-Src(tyr(419)), and Hakai levels were also observed in PC3MM2 and C4-2B PCA cells. Selective Src inhibition by dasatinib also showed increased E-cadherin expression in PC3 cells suggesting a possible involvement of Src inhibition in silibinin-caused increase in E-cadherin level. Additional studies in PC3 cells with stable knock-down of E-cadherin expression revealed that antimigratory/antiinvasive efficacy of silibinin is in-part dependent on E-cadherin expression. Together, our results showing antimigratory/antiinvasive effects of silibinin and associated mechanisms suggest that silibinin should be tested further in clinically relevant animal models toward exploiting its potential benefits against metastatic PCA.

Silibinin exerts sustained growth suppressive effect against human colon carcinoma SW480 xenograft by targeting multiple signaling molecules.

PURPOSE: Earlier, we reported the strong preventive efficacy of silibinin against colorectal cancer (CRC), but its usefulness against established CRC or effect of its withdrawal on CRC growth remained unknown. Present study focused on these important issues by employing two different treatment protocols in advanced human CRC SW480 xenograft in nude mice. METHODS: In the first treatment protocol, silibinin was fed for 28 days (200 mg/kg body weight, 5 days/week) to mice with growing SW480 xenograft; thereafter, tumor growth was monitored for additional 3 weeks without silibinin treatment. In the second protocol, silibinin treatment was started after 25 days of SW480 cells injection (established tumors), and tumor growth was studied 4 days, 8 days and 16 days after silibinin treatment. RESULTS: In both treatment protocols, silibinin had strong and sustained inhibitory effect on xenograft growth. Detailed xenograft analyses showed that silibinin, in both treatment protocols, exerts anti-proliferative, pro-apoptotic and anti-angiogenic effects. Further, silibinin reduced the expression of ß-catenin and phospho-GSK3ß in xenograft tissues. Silibinin also targeted signaling molecules involved in CRC proliferation and survival (cyclin D1, c-Myc and survivin) as well as angiogenesis regulators (VEGF and iNOS). CONCLUSIONS: Collectively, these findings substantiate silibinin's therapeutic efficacy against CRC, advocating its translational potential.

Anti-clastogenic activity of Azadirachta indica against benzo(a)pyrene in murine forestomach tumorigenesis bioassay.

Abstract: Present study evaluated the anti-clastogenic efficacy of Azadirchta indica (A. indica) against benzo(a)pyrene [B(a)P] in murine forestomach tumorigenesis bioassay protocol. Female Balb/c mice were divided into four groups (n = 8). Each mouse from B(a)P and B(a)P + A. indica groups received intragastric instillations of B(a)P at a dose of 40 mg/kg b. w. in 0.2 mL olive oil twice a week, starting from 3rd week to the end of 6th week of the experiment. Mice of control and A. indica groups received 0.2 mL olive oil in the same schedule as for B(a)P and B(a)P + A indica groups. Mice of A. indica and B(a)P + A. indica groups received oral doses of 100 mg/kg b. w. aqueous A. indica leaf extract (AAILE) on alternate days throughout the experiment. Two weeks after the last B(a)P instillation, mice were sacrificed and spleens were processed for micronucleus (MN) assay, while liver tissues were analyzed for lipid peroxidation (LPO), as well as antioxidant defense enzymes, namely: catalase, superoxide dismutase (SOD), glutathione reductase (GR) and glutathione peroxidase (GPx). The incidence of MN formation increased in spleen cells of mice that received only B(a)P instillations. In hepatic tissues, the extent of oxidative stress increased upon B(a)P instillations as was evidenced from enhanced LPO levels with concomitant decrease in antioxidant defense enzyme activities in mice that received only B(a)P instillations. Interestingly, A. indica treatment significantly reversed these effects as observed in mice receiving AAILE along with B(a)P when compared to only B(a)P receiving mice. Moreover, in only AAILE receiving mice, enhanced antioxidant defense with slightly decreased levels of LPO as well as MN incidences were observed. Observations of the present study suggest that A. indica exert anticlastogenic effects against B(a)P by modulating oxidative stress and antioxidant defense.

Azadirachta indica modulates carcinogen biotransformation and reduced glutathione at peri-initiation phase of benzo(a)pyrene induced murine forestomach tumorigenesis.

The present study evaluated the effects of aqueous Azadirachta indica leaf extract (AAILE) on the activities of certain phase I (cytochrome P450, cytochrome b(5) and aryl hydrocarbon hydroxylase) as well as phase II (glutathione-S-transferase and UDP-glucuronosyl transferase) biotransformation enzymes; and reduced glutathione (GSH) (in forestomach and hepatic tissues) during/after intra-gastric instillations of B(a)P in murine forestomach tumorigenesis bioassay protocol. The activities of phase I biotransformation enzymes were found to increase, whereas a decrease in GSH content as well as glutathione-S-transferase was observed in mice receiving only B(a)P during as well as 2 weeks after B(a)P instillations. The activity of UDP-glucuronosyltransferase decreased during B(a)P instillations, whereas after the latter, the activity increased when compared with the control mice. However, in mice that received AAILE along with B(a)P instillations, a decrease in phase I enzymes was accompanied by an increase in phase II enzymes as well as GSH contents. Only AAILE treatment reduced the activities of phase I biotransformation enzymes and enhanced the GSH contents as well as the activities of phase II enzymes. Observations of the present study seem to be quite significant and (when taken together with our earlier findings) provides evidence for A. indica mediated modulation of the peri-initiation phase of the process of forestomach tumorigenesis.

Histochemical, ultrastructural, and biochemical evidences for Azadirachta indica-induced apoptosis in benzo(a)pyrene-induced murine forestomach tumors.

The present study reports aqueous Azadirachta indica leaf extract (AAILE)-mediated induction of apoptosis in a murine forestomach tumorigenesis model. Histochemistry-based quantification of apoptosis revealed enhanced apoptotic index in the forestomach tumors of mice receiving AAILE along with benzo(a)pyrene (B(a)P). Transmission electron microscopy confirmed the presence of classical morphological features of apoptosis including chromatin condensation/marginalization, nuclear fragmentation, and formation of apoptotic bodies. Scanning electron microscopy showed surface modifications on the transformed squamous epithelial cells and certain mitotic cells among them over the forestomach tumors of mice receiving only B(a)P. In tumors of the mice receiving AAILE along with B (a)P, such mitotic cells were found to be absent; however, certain cells showing shrinkage and blebbings (characteristics of apoptosis) were observed. DNA fragmentation was observed to increase exclusively in the tumors of mice that received AAILE along with B(a)P. Lipid peroxidation (LPO) levels decreased in forestomach tissues of mice in all the groups studied when compared to control counterparts. However, levels of LPO were found to increase in the tumorous tissue of mice that received AAILE along with B(a)P when compared to mice receiving only B(a)P. Taken together, observations of the present study suggest that A. indica induces apoptosis in B(a)P-induced murine forestomach tumors.

Azadirachta indica leaf extract modulates initiation phase of murine forestomach tumorigenesis.

The effects of aqueous Azadirachta indica leaf extract (AAILE) on benzo(a)pyrene [B(a)P]-induced forestomach tumorigenesis, B(a)P-DNA adduct formation and certain parameters of carcinogen biotransformation system in mice have been reported earlier from our laboratory. In this study, the effects of AAILE on the enzymes of B(a)P biotransformation, which play crucial role in initiation of chemical carcinogenesis - aryl hydrocarbon hydroxylase (AHH) and uridinediphosphoglucuronosyltransferase (UDP-glucuronosyltransferase) have been evaluated in murine forestomach and liver. In addition, lipid peroxidation (LPO) levels in forestomach as well as liver and the activities of tissue injury marker enzymes - lactate dehydrogenase, aspartate aminotransferase and alkaline phosphatase in the serum have also been evaluated. Oral administration of AAILE (100 mg/kg body wt for 2 weeks) reduces the AHH activity and enhances the UDP-glucuronosyltransferase activity in both the tissues, suggesting its potential in decreasing the activation and increasing the detoxification of carcinogens. The LPO levels decrease upon AAILE treatment in the hepatic tissue, suggesting its antioxidative and hence anti-carcinogenic effects. Non-significant alterations have been observed in tissue injury marker enzymes upon AAILE treatment, suggesting its safety at the given dose. In conclusion, AAILE appears to modulate initiation phase of carcinogenesis and may be suggested as safe and an effective agent for chemoprevention.

Impediment of diethylnitrosamine induced hepatotoxicity in male Balb/c mice by pretreatment with aqueous Azadirachta indica leaf extract.

Considering the hepatoprotective properties of Azadirachta indica, the present study was designed to evaluate its preventive effects against diethylnitrosamine (NDEA) induced hepatotoxicity in male Balb/c mice. Exposure of NDEA caused a significant increase in micronucleated cell score, lipid peroxidation levels (LPO) and activity of lactate dehydrogenase (LDH). A significant decrease in reduced glutathione (GSH) contents and activity of glutathione-S-transferase (GST) was also observed upon NDEA treatment, whereas their activities of cytochrome P450 and cytochrome b5 showed non-significant alterations. Aqueous A. indica leaf extract (AAILE) pretreatment showed protective effects against NDEA induced toxicity by decreasing the frequency of micronucleated cell, levels of LPO and LDH activity. Also, a decreased activity of GST, cytochrome P450 and an increased activity of cytochrome b5, GSH contents was observed when AAILE pretreated mice were injected with NDEA. Only AAILE treatment caused a noticeable decrease in the frequency of micronuclei, activity of cytochrome P450 and cytochrome b5, but a significant increase in the activity of GST and GSH contents, whereas, non significant alterations were observed in the activity of LDH and levels of LPO. Significance of these observations with respect to hepatoprotective efficacy of A. indica has been discussed in the present manuscript.

Preventive effects of Azadirachta indica on benzo(a)pyrene-DNA adduct formation in murine forestomach and hepatic tissues.

In the present investigation, the effects of aqueous Azadirachta indica leaf extract (AAILE) on (3)H-benzo(a)pyrene-DNA [(3)H-B(a)P-DNA] adduct formation, the status of biotransformation enzymes and reduced glutathione (GSH) content were evaluated in the forestomach and liver of Balb/c mice. Two weeks of AAILE treatment reduced the (3)H-B(a)P-DNA adduct levels by 31.6% in forestomach tissue. Similarly, (3)H-B(a)P-DNA adduct levels were decreased by 34.7% in the liver of AAILE treated mice compared with their control counterparts. After AAILE treatment, the cytochrome P450 content decreased, whereas the GSH content increased significantly in the hepatic tissue. In the forestomach as well as in the liver, the cytochrome b5 content declined, whereas an increase in glutathione-S-transferase (GST) activity was observed in both tissues. These observations suggested that AAILE may have reduced the metabolic activation of (3)H-B(a)P with enhanced detoxification of its active metabolites, hence the observed decrease in the levels of (3)H-B(a)P-DNA adducts. These molecular and biochemical modulations observed at the initiation phase of carcinogenesis seems to be significant and could be correlated with the chemopreventive effects of A. indica against B(a)P induced forestomach tumorigenesis.

Modulatory effects of Azadirachta indica on benzo(a)pyrene-induced forestomach tumorigenesis in mice.

AIM: To evaluate the chemopreventive effects of aqueous Azadirachta indica (A indica) leaf extract (AAILE) against benzo(a)pyrene [B(a)P]-induced forestomach tumorigenesis in Balb/c mice. METHODS: Female Balb/c mice were divided into four groups of 10-12 animals each. For induction of forestomach tumors, starting from d 14 of the experi-ment, mice of B(a)P and B(a)P+A indica groups were given intra-gastric instillations of B(a)P (40 mg/kg), twice a week for four weeks. Mice of A indica and B(a)P+A indica groups were orally administered with AAILE (100 mg/kg), two weeks prior to B(a)P instillations till the end of the experiment. After 22 wk of the first B(a)P instillation, mice were sacrificed and the forestomachs were analyzed for development of tumors, scanning electron microscopy (SEM) and histopathology. RESULTS: Tumor incidence was observed to be 100% in mice that received only B(a)P. However, treatment with AAILE reduced the tumor incidence by 58.4% as observed in mice of B(a)P+A indica group when compared to that of B(a)P group. Similarly, the tumor burden and multiplicity were seen to decrease by 87.3% and 69.6% respectively in mice of B(a)P+A indica group when compared to those of B(a)P group. Scanning electron microscopy analysis showed that AAILE treatment itself did not cause any abnormalities on the surface architecture of forestomach epithelium. In tumorous forestomach, surface disruption was observed. Over the forestomach tumors of B(a)P group of mice certain rounded structures were seen in addition to closely placed tongue-shaped squamous cells. Interestingly, these rounded structures were not observed in B(a)P + A indica group of mice. Histopathalogically, the tumors were identical and diagnosed to be papillomas. Mice from control and A indica groups of mice did not develop any forestomach tumors and showed normal histo-architecture. CONCLUSION: The present data suggest that A indica exerts chemopreventive effects against B(a)P-induced forestomach tumors in murine model. Because of lack of toxicity and ubiquitous bioavailability, A indica may play a promising role in future drug discovery and development as far as chemoprevention of cancer is concerned.

Chemomodulatory effects of Azadirachta indica on the hepatic status of skin tumor bearing mice.

The liver plays an important role in the modulation of the process of carcinogenesis, as it is the primary site for the biotransformation of xenobiotics including carcinogens as well as anticancer drugs. The present study was designed to evaluate the biochemical alterations occurring in the liver of 7,12-dimethylbenz(a)anthracene (DMBA) induced skin tumor bearing male Balb/c mice and their modulation by aqueous Azadirachta indica leaf extract (AAILE). It was observed that skin tumor induction caused hepatic damage characterized by a decreased hepatosomatic index and significantly increased (p < 0.001) activities of the hepatic tissue injury marker enzymes, namely alkaline phosphatase, alanine aminotransferase and aspartate aminotransferase. However, upon treatment with AAILE, the above-mentioned alterations, including the increased activities of hepatic tissue injury marker enzymes, were significantly reversed, which signified the hepato-protective efficacy of Azadirachta indica. Increased oxidative stress was also observed in the hepatic tissue of skin tumor bearing mice as revealed by a significant increase (p < 0.001) in lipid peroxidation levels and a decrease in reduced glutathione contents and activities of various antioxidant enzymes studied, namely glutathione-S-transferase, glutathione peroxidase and glutathione reductase. The AAILE treatment reduced oxidative stress by decreasing lipid peroxidation levels and enhancing the reduced glutathione contents and activities of various antioxidant enzymes. The activities of the xenobiotic biotransformation enzymes, namely cytochrome P450, cytochrome b5 and glutathione-S-transferase, were found to be decreased in the hepatic tissue of tumor bearing mice. Treatment with AAILE further caused a decrease in the activity of cytochrome P450 and cytochrome b5, whereas it up-regulated the activity of glutathione-S-transferase. The significance of these observations with respect to the progress of the process of carcinogenesis is explained in the present research article.

Inhibitory effects of Azadirachta indica on DMBA-induced skin carcinogenesis in Balb/c mice.

Male Balb/c mice were divided into four groups on the basis of their respective treatments wherein mice of Group I served as controls. For induction of skin tumors, mice of Group II and IV were injected sub-cutaneously with 7,12-dimethylbenz(a)anthracene (DMBA). Mice of Group III and IV were administered aqueous Azadirachta indica leaf extract (AAILE) thrice a week throughout the experiment. After 14 weeks of the first DMBA injection, Group II and IV mice developed tumors. In the tumor-bearing mice that received AAILE (Group IV), a significant reduction in mean tumor burden and tumor volume was observed. The tumors were confirmed to be papillomas and interestingly, the extent of hyper-chromatia was observed to be much more in skin tumors of Group II mice vis a vis the mice receiving AAILE. An increase in the extent of lipid peroxidation was observed in tumorous tissue of Group IV when compared to that of Group II mice. Glutathione (GSH) content and the activities of GSH-based antioxidant enzymes viz. glutathione peroxidase (GPx) and glutathione reductase (GR) increased significantly in the skin tissues of all the groups of mice when compared to control counterparts. Catalase activity was found to decrease significantly in the skin of mice, which received AAILE treatment only (Group III). Activity of super-oxide dismutase (SOD) decreased significantly in all the tumorous tissues (Group II and IV mice). In light of the above observations, the role of AAILE in inhibition of DMBA-induced skin carcinogenesis is discussed in the present study.

Modulatory effects of different doses of alpha-tocopherol on benzo(a)pyrene-DNA adduct formation in the pulmonary tissue of cigarette smoke inhaling mice.

Cigarette smoke (CS) has been established as one of the major risk factors for many pathologies including lung cancer in humans and experimental animals. In view of the discrepancy about the role of alpha-tocopherol (AT) in carcinogenesis, the present study was designed to investigate the effects of different doses of AT on benzo(a)pyrene-DNA [B(a)P-DNA] adduct formation in lungs of CS inhaling mice. Extent of carcinogen-DNA adduct formation has been considered as an index for carcinogenesis. Feeding of 35 IU AT/kg body weight increased B(a)P-DNA adducts formation significantly whereas feeding of 5 IU AT/kg body weight did not altered much the B(a)P-DNA adduct levels when both were compared to the control counterparts. With CS inhalation, the B(a)P-DNA adducts formation increased in all the groups when compared to their respective sham counterparts. Interestingly, in CS exposed groups, there was least increase in B(a)P-DNA adducts formation in 5 IU AT/kg fed animals followed by the control and 35 IU AT/kg body weight fed groups respectively. The results suggest that higher doses of AT accentuate DNA adduct formation in CS inhaling mice.