Kulpreet Bhui

Lupeol: connotations for chemoprevention.

The perception of chemoprevention lies still in its infancy. Intervention, to slow down, arrest or reverse the process of carcinogenesis, by the use of either natural or synthetic substances individually or in combination therapy has emerged as a promising and pragmatic medical approach to reduce cancer risk. Pentacyclic lupane-type triterpenes exemplified by lupeol [lup-20(29)-en-3b-ol], are principally found in common fruit plants such as olive, mango, fig, etc. Although, lupeol exhibits an array of biological activities like anti-inflammatory, anti-arthritic, anti-mutagenic and anti-malarial activity both in in vitro and in vivo systems yet, extensive exploration in regard to establish its role as chemopreventive compound is warranted. Interest in developing lupeol based potent anti-neoplastic agents, has led to the discovery of a host of highly active derivatives exhibiting greater potencies and better therapeutic indices. This review asserts on the chemopreventive prospects of lupeol and reveals potential chemoprevention drug targets, central to which are the cell cycle regulatory pathway genes and tries to explain the mechanism operating behind its action.

Resveratrol and Black Tea Polyphenol Combination Synergistically Suppress Mouse Skin Tumors Growth by Inhibition of Activated MAPKs and p53

Cancer chemoprevention by natural dietary agents has received considerable importance because of their cost-effectiveness and wide safety margin. However, single agent intervention has failed to bring the expected outcome in clinical trials; therefore, combinations of chemopreventive agents are gaining increasing popularity. The present study aims to evaluate the combinatorial chemopreventive effects of resveratrol and black tea polyphenol (BTP) in suppressing two-stage mouse skin carcinogenesis induced by DMBA and TPA. Resveratrol/BTP alone treatment decreased tumor incidence by ∼67% and ∼75%, while combination of both at low doses synergistically decreased tumor incidence even more significantly by ∼89% (p<0.01). This combination also significantly regressed tumor volume and number (p<0.01). Mechanistic studies revealed that this combinatorial inhibition was associated with decreased expression of phosphorylated mitogen-activated protein kinase family proteins: extracellular signal-regulated kinase 1/2, c-Jun N-terminal kinase 1/2, p38 and increased in total p53 and phospho p53 (Ser 15) in skin tissue/tumor. Treatment with combinations of resveratrol and BTP also decreased expression of proliferating cell nuclear antigen in mouse skin tissues/tumors than their solitary treatments as determined by immunohistochemistry. In addition, histological and cell death analysis also confirmed that resveratrol and BTP treatment together inhibits cellular proliferation and markedly induces apoptosis. Taken together, our results for the first time lucidly illustrate that resveratrol and BTP in combination impart better suppressive activity than either of these agents alone and accentuate that development of novel combination therapies/chemoprevention using dietary agents will be more beneficial against cancer. This promising combination should be examined in therapeutic trials of skin and possibly other cancers.

[6]-Gingerol induces reactive oxygen species regulated mitochondrial cell death pathway in human epidermoid carcinoma A431 cells

Since skin cancer incidence and prevalence is constantly rising up the charts despite all efforts, search for newer, better agents for protection and treatment is required. Ginger (Zingiber officinale Roscoe), a monocotyledonous herb, is widely used as a herbal medicine, given the presence of homologous phenolic ketones, of which [6]-gingerol is the major one. The quantity of [6]-gingerol in the fresh ginger rhizome was found to be 104-965 microg/g in common varieties of ginger available in Indian market. Herein, [6]-gingerol was assessed for its anti-apoptotic effects in human epidermoid carcinoma A431 cells. [6]-Gingerol treatment exhibited considerable cytotoxicity as indicated by growth inhibition of A431 cells mediated via generation of reactive oxygen species (ROS). Increase in ROS led to decrease in mitochondrial membrane potential (MMP) and subsequent induction of apoptosis. Results revealed that perturbations in mitochondrial membrane are associated with deregulation of Bax/Bcl-2 ratio at gene transcriptional level as well as protein level, where treatment with [6]-gingerol leads to up-regulation of Cytochrome-c and Apaf-1 subsequently culminating in triggering of Caspase cascade. These firmly suggest that [6]-gingerol can be effectively used for the treatment of skin cancer.

Bromelain inhibits COX-2 expression by blocking the activation of MAPK regulated NF-kappa B against skin tumor-initiation triggering mitochondrial death pathway

Chemoprevention impels the pursuit for either single targeted or cocktail of multi-targeted agents. Bromelain, potential agent in this regard, is a pharmacologically active compound, present in stems and fruits of pineapple (Ananas cosmosus), endowed with anti-inflammatory, anti-invasive and anti-metastatic properties. Herein, we report the anti tumor-initiating effects of bromelain in 2-stage mouse skin tumorigenesis model. Pre-treatment of bromelain resulted in reduction in cumulative number of tumors (CNT) and average number of tumors per mouse. Preventive effect was also comprehended in terms of reduction in tumor volume up to a tune of approximately 65%. Components of the cell signaling pathways, connecting proteins involved in cell death were targeted. Bromelain treatment resulted in upregulation of p53 and Bax and subsequent activation of caspase 3 and caspase 9 with concomitant decrease in Bcl-2. A marked inhibition in cyclooxygenase-2 (Cox-2) expression and inactivation of nuclear factor-kappa B (NF-kappaB) was recorded, as phosphorylation and consequent degradation of I kappa B alpha was blocked by bromelain. Also, bromelain treatment curtailed extracellular signal regulated protein kinase (ERK1/2), p38 mitogen-activated protein kinase (MAPK) and Akt activity. The basis of anti tumor-initiating activity of bromelain was revealed by its time dependent reduction in DNA nick formation and increase in percentage prevention. Thus, modulation of inappropriate cell signaling cascades driven by bromelain is a coherent approach in achieving chemoprevention.

Tea polyphenols enhance cisplatin chemosensitivity in cervical cancer cells via induction of apoptosis.

AIM Constitutive activation of nuclear factor-kappaB (NF-κB) and Akt has been implicated in chemoresistance as well as inhibition of apoptosis to cisplatin (CDDP), therefore, we examined whether (-) epigallocateocatechin-3-gallate (EGCG) or theaflavins (TF) could sensitize human cancer cells to CDDP via induction of apoptosis mediated by the inactivation of NF-κB and Akt signaling. MAIN METHODS Human cervical cancer cells (HeLa and SiHa cells) were treated with EGCG or TF and CDDP alone and with their combinations, further their effects on cell viability were evaluated. Western blotting was used for examining the apoptotic signaling proteins and inhibition of NF-κB activation. Levels of reactive oxygen species (ROS) production and mitochondria membrane potential (ΔΨm) were examined by flow cytometry. KEY FINDINGS The combined treatment of EGCG or TF with CDDP elicited significant inhibition of cell growth in comparison to either of them in both cell lines (p<0.05). Combinatorial treatment of both compounds potentially induced apoptosis by inhibiting the activation of Akt and NF-κB through blocking phosphorylation of inhibitor kappa Bα. These combinations acted in concert to induce increase in ROS level, release of cytochrome-c and expression of p53 and Bax, with decrease in cellular glutathione contents, ΔΨm, and Bcl-2 expression, thereby eventually resulting in the activation of caspases, poly(ADP)ribose polymerase cleavage and apoptosis of cancer cells. SIGNIFICANCE Study suggests that both EGCG and TF chemosensitize the cervical cancer cells to CDDP-induced growth inhibition and apoptosis. By these combinations ~4 folds increase in CDDP induced-apoptosis with dose advantage of ~3 times could be achieved.

Mancozeb-induced genotoxicity and apoptosis in cultured human lymphocytes

AIMS Mancozeb is a dithiocarbamate fungicide known to be genotoxic and induces tumors in rodents at various sites. There is no report in the literature about its genotoxicity in humans. Here, we investigated the association between mancozeb exposure and induction of genotoxic and proapoptotic changes in cultured human lymphocytes (CHLs). MAIN METHODS Lymphocytes were isolated from peripheral blood of healthy non-smoking donors. Induction of micronuclei and chromosomal aberrations was recorded both by conventional and flow cytometric methods. Annexin-V FITC was used for the differentiation of apoptotic and necrotic cells by flow cytometry. KEY FINDINGS Mancozeb exposure (0.5, 2 and 5 μg/ml) to CHLs leads to significant induction in the frequency of chromosomal aberrations (CAs) and micronuclei (MN), in a dose-dependent manner. Concomitantly, pro-oxidant potential of mancozeb was also recorded, by increase in the levels of reactive oxygen species (ROS) generation. Our results demonstrated that ROS plays a critical role in the initiation of mancozeb induced apoptosis in CHLs through two ways, primarily through mitochondria-mediated pathway including induction of ROS, decrease in mitochondrial membrane potential (ΔΨm), along with cytochrome c release from mitochondria, and activation of the caspase cascade. The other pathway includes increase in ROS, which resulted in activation of NF-κB, expression of FasL and triggered FasL-dependent pathway, which also involves caspase-8. Therefore, exposure to mancozeb can lead to induction of apoptosis in CHLs through both mechanisms. SIGNIFICANCE The results of study confirm that mancozeb exposure can induce genotoxicity and apoptosis in CHLs, thus pose a potential risk to exposed human population.

Tea Polyphenols Induce Apoptosis Through Mitochondrial Pathway and by Inhibiting Nuclear Factor-κB and Akt Activation in Human Cervical Cancer Cells

Phytochemicals present in tea, particularly polyphenols, have anticancer properties against several cancer types. However, studies elucidating the role and the mechanism(s) of action of tea polyphenols in cervical cancer are sparse. In this study, we investigated the mechanism of antiproliferative and apoptotic actions exerted by tea polyphenols on human papilloma virus-18-positive HeLa cervical cancer cells. Treatment of green tea polyphenol (-)-epigallocatechin gallate (EGCG) and black tea polyphenol theaflavins (TF) in HeLa cells showed a marked concentration- and time-dependent inhibition of proliferation and induced sub-G1 phase in a dose-dependent manner after 24 h. There was an attenuation of mitochondrial membrane potential with the increase of reactive oxygen species generation, p53 expression, Bax/Bcl-2 ratio, cytochrome-c release, and cleavage of procaspase-3 and -9 and poly(ADP-ribose)-polymerase, indicating the participation of a mitochondria related mechanism. In addition, EGCG as well as TF inhibited activation of Akt and nuclear factor-kappaB (NF-kappaB) via blocking phosphorylation and subsequent degradation of inhibitor of kappaBalpha and kappaBbeta subunits, thereby downregulating cyclooxygenase-2. Additionally, the protein level of cyclin D1, a transcriptional target of NF-kappaB, was also reduced significantly. Thus, we can conclude that tea polyphenols inhibit the growth of cervical cancer cells by inducing apoptosis and regulating NF-kappaB and Akt.

Genotoxic and carcinogenic risks associated with the dietary consumption of repeatedly heated coconut oil

Repeated heating of vegetable oils at high temperatures during cooking is a very common cooking practice. Repeated heating of edible oils can generate a number of compounds, including polycyclic aromatic hydrocarbons (PAH), some of which have been reported to have carcinogenic potential. Consumption of these repeatedly heated oils can pose a serious health hazard. The objectives of the present study were to evaluate the genotoxic and carcinogenic risks associated with the consumption of repeatedly heated coconut oil (RCO), which is one of the commonly consumed cooking and frying medium. The PAH were analysed using HPLC in fresh CO, single-heated CO (SCO) and RCO. Results revealed the presence of certain PAH, known to possess carcinogenic potential, in RCO when compared with SCO. Oral intake of RCO in Wistar rats resulted in a significant induction of aberrant cells (P<0·05) and micronuclei (P<0·05) in a dose-dependent manner. Oxidative stress analysis showed a significant (P<0·05) decrease in the levels of antioxidant enzymes such as superoxide dismutase and catalase with a concurrent increase in reactive oxygen species and lipid peroxidation in the liver. In addition, RCO given alone and along with diethylnitrosamine for 12 weeks induced altered hepatic foci as noticed by alteration in positive (γ-glutamyl transpeptidase and glutathione-S-transferase) and negative (adenosine triphosphatase, alkaline phosphatase and glucose-6-phosphatase) hepatospecific biomarkers. A significant decrease in the relative and absolute hepatic weight of RCO-supplemented rats was recorded (P<0·05). In conclusion, dietary consumption of RCO can cause a genotoxic and preneoplastic change in the liver.

Bromelain inhibits nuclear factor kappa-B translocation, driving human epidermoid carcinoma A431 and melanoma A375 cells through G2/M arrest to apoptosis

Bromelain, obtained from pineapple, is already in use clinically as adjunct in chemotherapy. Our objective was to test its ability to act as a sole anti‐cancer agent. Therefore, we describe its anti‐proliferative, anti‐inflammatory and subsequent anti‐cancer effects in vitro, against human epidermoid carcinoma‐A431 and melanoma‐A375 cells. Bromelain exhibited reduction in proliferation of both these cell‐lines and suppressed their potential for anchorage‐independent growth. Further, suppression of inflammatory signaling by bromelain was evident by inhibition of Akt regulated‐nuclear factor‐kappaB activation via suppression of inhibitory‐kappaBα phosphorylation and concomitant reduction in cyclooxygenase‐2. Since, the inflammatory cascade is well‐known to be closely allied to cancer; we studied the effect of bromelain on events/molecules central to it. Bromelain caused depletion of intracellular glutathione and generation of reactive oxygen‐species followed by mitochondrial membrane depolarization. This led to bromelain‐induced cell‐cycle arrest at G2/M phase which was mediated by modulation of cyclin B1, phospho‐cdc25C, Plk1, phospho‐cdc2, and myt1. This was subsequently followed by induction of apoptosis, indicated by membrane‐blebbing, modulation of Bax‐Bcl‐2 ratio, Apaf‐1, caspase‐9, and caspase‐3; chromatin‐condensation, increase in caspase‐activity and DNA‐fragmentation. Bromelain afforded substantial anti‐cancer potential in these settings; hence we suggest it as a potential prospect for anti‐cancer agent besides only an additive in chemotherapy.

Pineapple bromelain induces autophagy, facilitating apoptotic response in mammary carcinoma cells

Bromelain, from pineapple, possesses potent anticancer effects. We investigated autophagic phenomenon in mammary carcinoma cells (estrogen receptor positive and negative) under bromelain treatment and also illustrated the relationship between autophagy and apoptosis in MCF‐7 cells. MCF‐7 cells exposed to bromelain showed delayed growth inhibitory response and induction of autophagy, identified by monodansylcadaverine localization. It was succeeded by apoptotic cell death, evident by sub‐G1 cell fraction and apoptotic features like chromatin condensation and nuclear cleavage. 3‐Methyladenine (MA, autophagy inhibitor) pretreatment reduced the bromelain‐induced autophagic level, also leading to decline in apoptotic population, indicating that here autophagy facilitates apoptosis. However, addition of caspase‐9 inhibitor Z‐LEHD‐FMK augmented the autophagy levels, inhibited morphological apoptosis but did not prevent cell death. Next, we found that bromelain downregulated the phosphorylation of extracellular signal‐regulated kinase ½ (ERK½), whereas that of c‐jun N‐terminal kinase (JNK) and p38 kinase were upregulated. Also, MA had no influence on bromelain‐suppressed ERK½ activation, yet, it downregulated JNK and p38 activation. Also, addition of mitogen‐activated protein kinase (MAPK) inhibitors enhanced the autophagic ratios, which suggested the role of MAP kinases in bromelain‐induced autophagy. All three MAPKs were seen to be constantly activated over the time. Bromelain was seen to induce the expressions of autophagy‐related proteins, light chain 3 protein B II (LC3BII), and beclin‐1. Using ERK½ inhibitor, expressions of LC3BII and beclin‐1 increased, whereas p38 and JNK inhibitors decreased this protein expression, indicating that bromelain‐induced autophagy was positively regulated by p38 and JNK but negatively regulated by ERK½. Autophagy‐inducing property of bromelain can be further exploited in breast cancer therapy.