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

This article has been retracted: please see Elsevier Policy on Article Withdrawal (https://www.elsevier.com/about/our-business/policies/article-withdrawal). This article has been retracted at the request of the Editor-in-Chief. Multiple figures in this article appear to be falsified/fabricated. Figure 2A and C. The representative dot plots from the EGCG (15ug/ml)+CDDP (10ug/ml) and TF (15ug/ml) groups appear to be duplicated. Figures 3, 4 and 6. Multiple Western blot bands appear to be rotated and reused throughout Figure 3 (A and B); 4 (A and B) and 6 (A, B, C). In particular, the Cytochrome-c blot in Figure 3B is duplicated and flipped in Figure 6B as p-NFKB. The p53 blot in Figure 3B is duplicated in Figure 6C as p-NFKB. The B-actin blot in Figure 3B is shown as an unmarked control lane (flipped in Figure 6B. The p53 band in Figure 3C is very similar to the Caspase 9 blot in Figure 4B and is cropped and duplicated in Figure 6A as p-NFKB by cisplatin in SiHa cells. The Caspase 3 blot in Figure 4A is rotated and flipped and appears in Figure 6B as p-IKBa.

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.

Bromelain inhibits nuclear factor kappa-B translocation, driving human epidermoid carcinoma A431 and melanoma A375 cells through G(2)/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 G(2)/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.

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.

Tea polyphenols induce apoptosis through mitochondrial pathway and by inhibiting nuclear factor-kappaB 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.

Neoplastic alterations induced in mammalian skin following mancozeb exposure using in vivo and in vitro models.

Mancozeb, ethylene(bis)dithiocarbamate fungicides, has been well documented in the literature as a multipotent carcinogen, but the underlying mechanism remains unrevealed. Thus, mancozeb has been selected in this study with the objective to decipher the molecular mechanism that culminates in carcinogenesis. We employed two-dimensional gel electrophoresis and mass spectrometry to generate a comparative proteome profile of control and mancozeb (200 mg/kg body weight) exposed mouse skin. Although many differentially expressed proteins were found, among them, two significantly upregulated proteins, namely, S100A6 (Calcyclin) and S100A9 (Calgranulin-B), are known markers of keratinocyte differentiation and proliferation, which suggested their role in mancozeb-induced neoplastic alterations. Therefore, we verified these alterations in the human system by using HaCaT cells as an in vitro model for human skin keratinocyte carcinogenesis. Upregulation of these two proteins upon mancozeb (0.5 µg/mL) exposure in HaCaT cells indicated its neoplastic potential in human skin also. This potential was confirmed by increase in number of colonies in colony formation and anchorage-independent growth assays. Modulation of S100A6/S100A9 targets, elevated phosphorylation of extracellular signal regulated kinase (ERK1/2), Elk1, nuclear factor- kappa B and cell division cycle 25 C phosphatase, and cyclin D1 and cyclooxygenase-2 upregulation was seen. In addition, PD98059 (ERK1/2 inhibitor) reduced cell proliferation induced by mancozeb, confirming the involvement of ERK1/2 signaling. Conclusively, we herein present the first report asserting that the mechanism involving S100A6 and S100A9 regulated ERK1/2 signaling underlies the mancozeb-induced neoplastic potential in human skin.

Synergistic growth inhibition of mouse skin tumors by pomegranate fruit extract and diallyl sulfide: evidence for inhibition of activated MAPKs/NF-κB and reduced cell proliferation.

Limited outcomes from earlier chemopreventive studies have necessitated that some modifications be made to get better efficacy. It is proposed that cancer prevention is more feasible than treatment, and this could be achieved effortlessly with use of multiple agents competent of targeting multiple targets. This study was initiated to examine the chemopreventive efficacy of pomegranate fruit extract (PFE) and diallyl sulfide (DAS), alone and in combination, using 2-stage mouse skin tumorigenesis model. PFE and DAS alone delayed onset and tumor incidence by ∼55% and ∼45%, respectively, while their combination at low doses synergistically decreased tumor incidence more potentially (∼84%, p<0.01). In addition, regression in tumor volume was seen with continuous combinatorial treatment (p<0.01). Mechanistic studies revealed that this inhibition was associated with decreased expression of phosphorylated ERK1/2, JNK1 and activated NF-κB/p65, IKKα, IκBα phosphorylation and degradation in skin tissue/tumor. Histological and cell death analysis also confirmed that combined PFE and DAS inhibit cellular proliferation and markedly induce apoptosis than the single agents. Altogether, our results suggest that PFE and DAS in combination impart better suppressive activity than either of these agents alone and provide support that development of novel combination therapies/chemoprevention using dietary agents will be more beneficial against cancer.

Genotoxic and carcinogenic risks associated with the consumption of repeatedly boiled sunflower oil.

Repeated boiling of vegetable oils at high temperature in cooking and frying is a very common practice and leads to the formation of a class of toxic substances. Among them, polycyclic aromatic hydrocarbons (PAHs) are well-documented for their mutagenic/carcinogenic potential. The objectives of the present study were to evaluate the genotoxic and carcinogenic risks associated with the consumption of repeatedly boiled sunflower oil, which is one of the commonly consumed vegetable oils in southeast Asian countries. The presence of PAHs was analyzed using high-performance liquid chromatography (HPLC) methods in fresh, single-boiled, and repeatedly-boiled sunflower oil (FSO, SBSO, and RBSO) samples. A higher amount of known carcinogenic/mutagenic PAHs in RBSO samples were shown, as compared to FSO and SBSO. Oral administration of RBSO in Wistar rats resulted in significant induction of aberrant cells (p < 0.05) and micronuclei (p < 0.05) incidence in a dose-dependent manner. Oxidative stress analysis also showed a significant decrease in levels of antioxidant enzymes, such as superoxide dismutase and catalase, with a concurrent increase in reactive oxygen species and lipid peroxidation in animals following RBSO consumption, as compared to FSO or SBSO (p < 0.05). Additionally, RBSO administration alone and along with diethylnitrosamine for 12 weeks induced altered hepatic foci, as noticed by the alteration in positive (γ-glutamyl transpeptidase and glutathione-S-transferase) and negative (adenosine-triphosphatase, alkaline phosphatase, and glucose-6-phosphatase) liver biomarkers. A significant decrease in the relative and absolute hepatic weight in RBSO-supplemented rats was also noted (p < 0.05).

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.

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.

Polo-like kinase1 (Plk1) knockdown enhances cisplatin chemosensitivity via up-regulation of p73α in p53 mutant human epidermoid squamous carcinoma cells.

Polo-like kinase 1 (Plk1), a critical regulator of mitotic entry, progression and exit, has been shown to be involved in a variety of cancers and thus is becoming an attractive target for cancer management. In case of DNA damage, Plk1 not only inhibits p53 independent apoptosis by dysfunctioning p73α but also allows cells to recover from growth arrest. Here, we showed the effects of knocking down plk1 gene through small interference RNA (siRNA) on cell cycle progression, proliferation and chemosensitivity of p53 mutant A431 cells to cisplatin (CDDP). The expression of Plk1 was measured by RT-PCR and Western blotting. Anti-proliferative response accompanied with cell cycle arrest in G(2)/M phase and induction of cell death was recorded following Plk1 knockdown. Furthermore, cells following knockdown of Plk1, which induced increase of Cyclin B1, p-Cdc2 and p73α with a decrease in p-Cdc25C, were more sensitive to CDDP. CDDP treatment induced nuclear translocation and co-localization of Plk1 with p73α whereas combination of CDDP and Plk1siRNA upregulated the expression of p73α protein in a synergistic manner thereby leading to an increase up to ∼5 folds in CDDP-induced cell death. The increase in caspase-3 activity indicated apoptosis as a contributor in the total cell death. Conclusively, plk1 gene silencing can enhance the sensitivity of A431 cells to low doses of CDDP by upregulating p73α expression and thus can be a revolutionary approach in cancer chemotherapy.

Induction of apoptosis by [6]-gingerol associated with the modulation of p53 and involvement of mitochondrial signaling pathway in B[a]P-induced mouse skin tumorigenesis.

PURPOSE: To unravel the molecular mechanisms underlying the chemopreventive potential of [6]-gingerol, a pungent ingredient of ginger rhizome (Zingiber officinale Roscoe, Zingiberaceae), against benzo[a]pyrene (B[a]P)-induced mouse skin tumorigenesis. METHODS: Topical treatment of [6]-gingerol (2.5 muM/animal) was given to the animals 30 min prior and post to B[a]P (5 mug/animal) for 32 weeks. At the end of the study period, the skin tumors/tissues were dissected out and examined histopathologically. Flow cytometry was employed for cell cycle analysis. Further immunohistochemical localization of p53 and regulation of related apoptogenic proteins were determined by Western blotting. RESULTS: Chemopreventive properties of [6]-gingerol were reflected by delay in onset of tumorigenesis, reduced cumulative number of tumors, and reduction in tumor volume. Cell cycle analysis revealed that the appearance of sub-G1 peak was significantly elevated in [6]-gingerol treated animals with post treatment showing higher efficacy in preventing tumorigenesis induced by B[a]P. Moreover, elevated apoptotic propensity was observed in tumor tissues than the corresponding non-tumor tissues. Western blot analysis also showed the same pattern of chemoprevention with [6]-gingerol treatment increasing the B[a]P suppressed p53 levels, also evident by immunohistochemistry, and Bax while decreasing the expression of Bcl-2 and Survivin. Further, [6]-gingerol treatment resulted in release of Cytochrome c, Caspases activation, increase in apoptotic protease-activating factor-1 (Apaf-1) as mechanism of apoptosis induction. CONCLUSIONS: On the basis of the results we conclude that [6]-gingerol possesses apoptotic potential in mouse skin tumors as mechanism of chemoprevention hence deserves further investigation.

Regulation of cell growth through cell cycle arrest and apoptosis in HPV 16 positive human cervical cancer cells by tea polyphenols.

Cervical cancer is the second most common malignant neoplasm in women, in terms of both incidence and mortality rates worldwide. The polyphenolic constituents of tea (Camellia sinensis) have gained considerable attention because of its anti-cancer properties against a variety of cancers. Here we studied the effects of green and black tea polyphenols (GTP and BTP), on cellular proliferation and cell death in the SiHa cells (human cervical cancer) expressing the human papilloma virus (HPV)-16. The result showed that both GTP and BTP inhibited proliferation of cells in dose and time dependent manner. Cell cycle analysis showed anti-proliferative effect of GTP which is associated with an increase in the G2/M phase and apoptotic effect of BTP in 24 h treated SiHa cells. Further, on increase of incubation time for 48 h, GTP caused induction of apoptosis up to 20% of SiHa cells. The role GTP and BTP in apoptosis was further confirmed by reduction in mitochondrial membrane potential and increased levels of membrane phosphatidylserine. Thus, our data suggests that tea polyphenols exhibit anti-cancer potential against cervical cancer by inhibition of cell growth and induction of apoptosis.

[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.