N. Rajendra Prasad

Inhibitory effect of caffeic acid on cancer cell proliferation by oxidative mechanism in human HT-1080 fibrosarcoma cell line.

Caffeic acid (3,4-dihydroxy cinnamic acid) (CA) is naturally found in fruits, vegetables, olive oil, and coffee. This study was undertaken to evaluate the anticancer effect of caffeic acid on HT-1080 human fibrosarcoma cell line. The antiproliferative effect of caffeic acid was determined by MTT assay, and the oxidative stress was determined by lipid peroxidation, changes in the enzymatic, and non-enzymatic antioxidant status. To understand the mode of antiproliferative effect of CA, the authors observed intracellular ROS levels by DCFH-DA method, mitochondrial membrane potential alterations by Rh-123 staining, oxidative DNA damage by comet assay, and apoptotic morphological changes by AO/EtBr-staining method. The results show that caffeic acid enhances lipid peroxidative markers such as TBARS, CD, and LHP in HT-1080 cell line. Caffeic acid enhances the ROS levels, which is evidenced by the increased DCF fluorescence. Further, caffeic acid treatment altered the mitochondrial membrane potential in HT-1080 cells. Similarly, the authors observed increased oxidative DNA damage (% Tail DNA, % Tail length, Tail moment, and olive tail moment), and apoptotic morphological changes in caffeic acid-treated groups. These data suggest that caffeic acid exhibits potent anticancer effect in HT-1080 cell line, and that it may be used as an anticancer agent.

Effect of ursolic acid, a triterpenoid antioxidant, on ultraviolet-B radiation-induced cytotoxicity, lipid peroxidation and DNA damage in human lymphocytes

Exposure to ultraviolet B (UVB, 280-320) radiation induces the formation of reactive oxygen species (ROS) in the biological system. In this study, we examined the protective effect of ursolic acid on UVB-induced lipid peroxidation and oxidative DNA damage with reference to alterations in cellular antioxidant status in human lymphocytes. Series of in vitro tests (hydroxyl radical, superoxide, nitric oxide, DPPH and ABTS radical scavenging assays) demonstrates antioxidant property of ursolic acid in our study. Treatment of lymphocytes with ursolic acid alone (at 10 microg/mL) gave no significant change in cell viability, thiobarbituric acid reactive substances (TBARSs), lipid hydroperoxides (LHPs), % tail DNA and tail moment when compared with normal lymphocytes. UVB-exposure significantly increased TBARS, LHP and % tail DNA, tail moment; decreased % cell viability and antioxidant status in irradiated lymphocytes. Treatment with ursolic acid 30 min prior to UVB-exposure resulted in a significant decline of TBARS, LHP, % tail DNA and tail moment and increased % cell viability as ursolic acid concentration increased. Based on our results we conclude that ursolic acid, a dietary antioxidant, mediates its protective effect through modulation of UVB-induced reactive oxygen species.

Effect of Piper betle Leaf Extract on Alcoholic Toxicity in the Rat Brain

The protective effect of Piper betle, a commonly used masticatory, has been examined in the brain of ethanol-administered Wistar rats. Brain of ethanol-treated rats exhibited increased levels of lipids, lipid peroxidation, and disturbances in antioxidant defense. Subsequent to the experimental induction of toxicity (i.e., the initial period of 30 days), aqueous P. betle extract was simultaneously administered in three different doses (100, 200, and 300 mg kg(-1)) for 30 days along with the daily dose of alcohol. P. betle coadministration resulted in significant reduction of lipid levels (free fatty acids, cholesterol, and phospholipids) and lipid peroxidation markers such as thiobarbituric acid reactive substances and hydroperoxides. Further, antioxidants, like reduced glutathione, vitamin C, vitamin E, superoxide dismutase, catalase, and glutathione peroxidase, were increased in P. betle-coadministered rats. The higher dose of extract (300 mg kg(-1)) was more effective, and these results indicate the neuroprotective effect of P. betle in ethanol-treated rats.

Enhanced cytotoxicity and apoptosis-induced anticancer effect of silibinin-loaded nanoparticles in oral carcinoma (KB) cells.

Silibinin (SIL) is a plant derived flavonoid isolated from the fruits and seeds of the milk thistle (Silybum marianum). Silibinin possesses a wide variety of biological applications including anticancer activities but poor aqueous solubility and poor bioavailability limit its potential and efficacy at the tumor sites. In the present study, silibinin was encapsulated in Eudragit® E (EE) nanoparticles in the presence of stabilizing agent polyvinyl alcohol (PVA) and its anticancer efficacy in oral carcinoma (KB) cells was studied. Silibinin loaded nanoparticles (SILNPs) were prepared by nanoprecipitation technique and characterized in terms of size distribution, morphology, surface charge, encapsulation efficiency and in vitro drug release. MTT assay revealed higher cytotoxic efficacy of SILNPs than free SIL in KB cells. Meanwhile, reactive oxygen species (ROS) determination revealed the significantly higher intracellular ROS levels in SILNPs treated cells compared to free SIL treated cells. Therefore, the differential cytotoxicity between SILNPs and SIL may be mediated by the discrepancy of intracellular ROS levels. Moreover, acridine orange (AO) and ethidium bromide (EB) dual staining and reduced mitochondrial membrane potential (MMP) confirmed the induction of apoptosis with nanoparticle treatment. Further, the extent of DNA damage (evaluated by comet assay) was significantly increased in SILNPs than free SIL in KB cells. Taken together, the present study suggests that silibinin-loaded nanoparticles can be used as an effective drug delivery system to produce a better chemopreventive response for the treatment of cancer.

Apigenin ameliorates gamma radiation-induced cytogenetic alterations in cultured human blood lymphocytes

The aim of the present study was to assess the protective effect of apigenin, a dietary flavone, against cytogenetic alterations in human peripheral blood lymphocytes (HPBL) induced by Cobalt-60 radiation (3Gy). Results of MTT [3-(4, 5-dimethyl-2-thiaozolyl)-2,5-diphenyl-2H tetrazolium bromide] assay revealed that 37.2μM of apigenin was found to be non-toxic in HPBL. At this dose (37.2μM) of apigenin, the LD(50) radiation dose of HPBL increased from 2.9Gy to 3.4Gy, which resulted in a DMF of 1.17. Apigenin (37.2μM) treatment 1h before irradiation significantly (p<0.05) reduced DNA damage in irradiated HPBL as measured by comet assay (% tail DNA, tail length, tail moment, and olive tail moment). Moreover, apigenin treatment significantly decreased the frequencies of dicentric (DC), acentric fragments (AF), and acentric rings (AR) in irradiated HPBL. Apigenin pretreatment also reduced the radiation-induced CBMN (cytokinesis blocked micronuclei) anomalies such as micronuclei (MNi), nucleoplasmic bridges (NPB) and nuclear buds (NBUD) in HPBL. These results also showed that there was a significant correlation between NPB and DC frequencies and MNi and AF+AR. Treatment with apigenin alone had no significant effect on DNA damage and chromosomal aberrations in HPBL. Thus, the current studies indicate that apigenin protects HPBL from radiation-induced cytogenetic alterations.

Ferulic acid inhibits UVB-radiation induced photocarcinogenesis through modulating inflammatory and apoptotic signaling in Swiss albino mice.

The aim of this study was to evaluate the photochemopreventive effects of ferulic acid (FA) against chronic ultraviolet-B (290-320 nm) induced oxidative stress, inflammation and angiogenesis in the skin of Swiss albino mice. Chronic UVB exposure (180 mJ/cm(2) for 30 weeks; thrice in a week) induced tumor formation in the mice skin that showed increased expression of carcinogenic and inflammatory markers when compared with the control animals. The intraperitoneal (FAIP) and topical (FAT) administration of FA significantly reduced the incidence of UVB-induced tumor volume and tumor weight in the mice skin. Histopathological studies revealed that both FAIP and FAT administration prevented the UVB-induced hyperplasia, squamous cell carcinoma (SCC) and dysplastic feature in the mice skin. Further, it has been observed that FA treatment reverted chronic UVB-induced oxidative damage (thiobarbituric acid reactive substances, superoxide dismutase, catalase, glutathione peroxidase) accompanied with modulation of vascular endothelial growth factor (VEGF), inducible nitric oxide synthase (iNOS), TNF-α and IL-6 in the mice skin tumor. FA treatment also modulates mutated p53, Bcl-2 and Bax expressions in the UVB-induced mice skin tumor. Thus, the results of the present study indicate ferulic acid has potential against UVB-induced carcinogenesis in the Swiss albino mice.

Ferulic acid reverses ABCB1-mediated paclitaxel resistance in MDR cell lines

Multidrug resistance (MDR) remains a major obstacle in cancer chemotherapy. The use of the dietary phytochemicals as chemosensitizing agents to enhance the efficacy of conventional cytostatic drugs has recently gained the attention as a plausible approach for overcoming the drug resistance. The aim of this study was to investigate whether a naturally occurring diet-based phenolic acid, ferulic acid, could sensitize paclitaxel efficacy in ABCB1 overexpressing (P-glycoprotein) colchicine selected KB Ch(R)8-5 cell line. In vitro drug efflux assays demonstrated that ferulic acid inhibits P-glycoprotein transport function in drug resistant KB Ch(R)8-5 cell lines. However, ferulic acid significantly downregulates ABCB1 expression in a concentration dependent manner. Cytotoxicity assay reveals that ferulic acid decreased paclitaxel resistance in KBCh(R)8-5 and HEK293/ABCB1 cells, which indicates its chemosensitizing potential. Clonogenic cell survival assay and apoptotic morphological staining further confirm the chemosensitizing potential of ferulic acid in drug resistant KB Ch(R)8-5 cell lines. Ferulic acid treatment enhances paclitaxel mediated cell cycle arrest and upregulates paclitaxel-induced apoptotic signaling in KB resistant cells. Hence, it has been concluded that downregulation of ABCB1 and subsequent induction of paclitaxel-mediated cell cycle arrest and apoptotic signaling may be the cause for the chemosensitizing potential of ferulic acid in P-gp overexpressing cell lines.

Caffeic Acid Inhibits Chronic UVB-Induced Cellular Proliferation Through JAK-STAT3 Signaling in Mouse Skin.

Signal transducers and activators of transcription 3 (STAT3) play a critical role in inflammation, proliferation and carcinogenesis. Inhibition of JAK‐STAT3 signaling is proved to be a novel target for prevention of UVB‐induced skin carcinogenesis. In this study, chronic UVB irradiation (180 mJ cm−2; weekly thrice for 30 weeks) induces the expression of IL‐10 and JAK1 that eventually activates the STAT3 which leads to the transcription of proliferative and antiapoptotic markers such as PCNA, Cyclin‐D1, Bcl2 and Bcl‐xl, respectively. Caffeic acid (CA) inhibits JAK‐STAT3 signaling, thereby induces apoptotic cell death by upregulating Bax, Cytochrome‐C, Caspase‐9 and Caspase‐3 expression in mouse skin. Furthermore, TSP‐1 is an antiangiogeneic protein, which is involved in the inhibition of angiogenesis and proliferation. Chronic UVB exposure decreased the expression of TSP‐1 and pretreatment with CA prevented the UVB‐induced loss of TSP‐1 in UVB‐irradiated mouse skin. Thus, CA offers protection against UVB‐induced photocarcinogenesis probably through modulating the JAK‐STAT3 in the mouse skin.

Synthesis, characterization and in vitro anti-cancer evaluation of hesperetin-loaded nanoparticles in human oral carcinoma (KB) cells

Hesperetin (HET), a naturally occurring plant bioflavonoid present in citrus fruits, possesses potential anti-inflammatory and anti-carcinogenic activities but poor aqueous solubility limits its applications. To improve its applicability in cancer therapy, hesperetin was encapsulated in Eudragit® E (EE) 100 nanoparticles in the presence of polyvinyl alcohol (PVA) as a stabilizer and its anticancer efficacy in oral carcinoma (KB) cells was studied. Hesperetin-loaded nanoparticles (HETNPs) were prepared by nanoprecipitation method and characterized by dynamic light scattering (DLS), transmission electron microscopy (TEM), Fourier transform infrared (FT-IR) spectroscopy, differential scanning calorimetry (DSC), and x-ray diffraction (XRD). The results thus displayed that the prepared nanoparticles showed a particle size in the range from 55 to 180 nm. The encapsulation efficiency of hesperetin was 83.4% obtained by UV spectroscopy. The in vitro release kinetics of hesperetin under physiological condition show initial rapid release followed by slow and sustained release. 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl tetrazolium bromide (MTT) assay revealed higher cytotoxic efficacy of HETNPs than native hesperetin in KB cells. Further, it has been found that reactive oxygen species (ROS) generation, DNA damage and apoptotic indices in HETNPs treated cells are greater than those in native hesperetin treatment. Hence these findings demonstrate that HETNPs could be a potentially useful drug delivery system to produce better hesperetin therapeutics of cancers.

Apigenin prevents ultraviolet-B radiation induced cyclobutane pyrimidine dimers formation in human dermal fibroblasts

Exposure to solar ultraviolet-B (UVB) radiation leads to the formation of cyclobutane pyrimidine dimers (CPDs). We investigated the protective effect of apigenin against UVB-induced CPDs formation in human dermal fibroblasts cells (HDFa). For this purpose, HDFa cells were treated with apigenin (15μM) prior to UVB irradiation (20mJ/cm2); DNA damage and subsequent molecular end points were observed. Exposure to UVB radiation increased significant CPDs formation in HDFa cells and the frequencies of CPDs were reduced by treatment with apigenin (15μM). UVB-induced CPDs downregulates the expression of nucleotide excision repair (NER) genes such as xeroderma pigmentosum complementation group C, B, G and F (XPC, XPB, XPG and XPF), transcription factor II human (TFIIH) and excision repair cross-complementation group 1 (ERCC1) in HDFa cells. Conversely, apigenin treatment restored UVB-induced loss of NER proteins in HDFa cells, which indicates its preventive effect against CPDs formation. Besides, single low dose UVB-exposure induced nuclear fragmentation, apoptotic frequency and apoptotic proteins expression (Bax and Caspase-3) have been prevented by the apigenin pretreatment. Furthermore, apigenin exhibits strong UV absorbance property and showed 10.08 SPF value. Thus, apigenin can protect skin cells against UVB-induced CPDs formation probably through its sunscreen effect. Hence, apigenin can be considered as an effective protective agent against UV induced skin damages.