Madhulika Singh

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.

Clastogenic effects of glyphosate in bone marrow cells of swiss albino mice.

Glyphosate (N-(phosphonomethyl) glycine, C3H8NO5P), a herbicide, used to control unwanted annual and perennial plants all over the world. Nevertheless, occupational and environmental exposure to pesticides can pose a threat to nontarget species including human beings. Therefore, in the present study, genotoxic effects of the herbicide glyphosate were analyzed by measuring chromosomal aberrations (CAs) and micronuclei (MN) in bone marrow cells of Swiss albino mice. A single dose of glyphosate was given intraperitoneally (i.p) to the animals at a concentration of 25 and 50 mg/kg b.wt. Animals of positive control group were injected i.p. benzo(a)pyrene (100 mg/kg b.wt., once only), whereas, animals of control (vehicle) group were injected i.p. dimethyl sulfoxide (0.2 mL). Animals from all the groups were sacrificed at sampling times of 24, 48, and 72 hours and their bone marrow was analyzed for cytogenetic and chromosomal damage. Glyphosate treatment significantly increases CAs and MN induction at both treatments and time compared with the vehicle control (P < .05). The cytotoxic effects of glyphosate were also evident, as observed by significant decrease in mitotic index (MI). The present results indicate that glyphosate is clastogenic and cytotoxic to mouse bone marrow.

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.

Theaflavins induced apoptosis of LNCaP cells is mediated through induction of p53, down-regulation of NF-kappa B and mitogen-activated protein kinases pathways

Prostate cancer (PCA), the most frequently diagnosed malignancy in men, represents an excellent candidate disease for chemoprevention studies because of its particularly long latency period, high rate of mortality and morbidity. Infusion of black tea and its polyphenolic constituents have been shown to possess antineoplastic effects in androgen dependent PCA in both in vivo and in vitro models including transgenic animals. In the present study, we report that black tea polyphenol, Theaflavins (TF)-induced apoptosis in human prostate carcinoma, LNCaP cells is mediated via modulation of two related pathways: up-regulation of p53 and down-regulation of NF-kappa B activity, causing a change in the ratio of pro-and antiapoptotic proteins leading to apoptosis. The altered expression of Bcl-2 family member proteins triggered the release of cytochrome-C and activation of initiator capsase 9 followed by activation of effector caspase 3. Furthermore, TF also affected the protein expression of mitogen activated protein kinases (MAPK) pathways. Our results demonstrated that TF treatment resulted in down-regulation of phospho-extracellular signal-regulated protein kinase (Erk1/2) and phospho-p38 MAPK expressions. We conclude that TF induces apoptosis in LNCaP cells by shifting the balance between pro-and antiapoptotic proteins and down-regulation of cell survival pathways leading to apoptosis. Further extending this work, we also showed that TF induces apoptosis in androgen independent PCA cell line, PC-3 through caspases and MAPKs mediated pathways. Thus, effect of TF on PCA cell lines seems to be irrespective of their androgen status.

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.

Synthesis of PLGA nanoparticles of tea polyphenols and their strong in vivo protective effect against chemically induced DNA damage.

In spite of proficient results of several phytochemicals in preclinical settings, the conversion rate from bench to bedside is not very encouraging. Many reasons are attributed to this limited success, including inefficient systemic delivery and bioavailability under in vivo conditions. To achieve improved efficacy, polyphenolic constituents of black (theaflavin [TF]) and green (epigallocatechin-3-gallate [EGCG]) tea in poly(lactide-co-glycolide) nanoparticles (PLGA-NPs) were entrapped with entrapment efficacy of ~18% and 26%, respectively. Further, their preventive potential against 7,12-dimethylbenzanthracene (DMBA)-induced DNA damage in mouse skin using DNA alkaline unwinding assay was evaluated. Pretreatment (topically) of mouse skin with either TF or EGCG (100 μg/mouse) doses exhibits protection of 45.34% and 28.32%, respectively, against DMBA-induced DNA damage. However, pretreatment with TF-loaded PLGA-NPs protects against DNA damage 64.41% by 1/20th dose of bulk, 71.79% by 1/10th dose of bulk, and 72.46% by 1/5th dose of bulk. Similarly, 51.28% (1/20th of bulk), 57.63% (1/10th of bulk), and 63.14% (1/5th of bulk) prevention was noted using EGCG-loaded PLGA-NP doses. These results showed that tea polyphenol-loaded PLGA-NPs have ~30-fold dose-advantage than bulk TF or EGCG doses. Additionally, TF- or EGCG-loaded PLGA-NPs showed significant potential for induction of DNA repair genes (XRCC1, XRCC3, and ERCC3) and suppression of DNA damage responsive genes (p53, p21, MDM2, GADD45α, and COX-2) as compared with respective bulk TF or EGCG doses. Taken together, TF- or EGCG-loaded PLGA-NPs showed a superior ability to prevent DMBA-induced DNA damage at much lower concentrations, thus opening a new dimension in chemoprevention research.

New Enlightenment of Skin Cancer Chemoprevention through Phytochemicals: In Vitro and In Vivo Studies and the Underlying Mechanisms

Skin cancer is still a major cause of morbidity and mortality worldwide. Skin overexposure to ultraviolet irradiations, chemicals, and several viruses has a capability to cause severe skin-related disorders including immunosuppression and skin cancer. These factors act in sequence at various steps of skin carcinogenesis via initiation, promotion, and/or progression. These days cancer chemoprevention is recognized as the most hopeful and novel approach to prevent, inhibit, or reverse the processes of carcinogenesis by intervention with natural products. Phytochemicals have antioxidant, antimutagenic, anticarcinogenic, and carcinogen detoxification capabilities thereby considered as efficient chemopreventive agents. Considerable efforts have been done to identify the phytochemicals which may possibly act on one or several molecular targets that modulate cellular processes such as inflammation, immunity, cell cycle progression, and apoptosis. Till date several phytochemicals in the light of chemoprevention have been studied by using suitable skin carcinogenic in vitro and in vivo models and proven as beneficial for prevention of skin cancer. This revision presents a comprehensive knowledge and the main molecular mechanisms of actions of various phytochemicals in the chemoprevention of skin cancer.

PLGA-encapsulated tea polyphenols enhance the chemotherapeutic efficacy of cisplatin against human cancer cells and mice bearing Ehrlich ascites carcinoma.

The clinical success of the applicability of tea polyphenols awaits efficient systemic delivery and bioavailability. Herein, following the concept of nanochemoprevention, which uses nanotechnology for enhancing the efficacy of chemotherapeutic drugs, we employed tea polyphenols, namely theaflavin (TF) and epigallocatechin-3-gallate (EGCG) encapsulated in a biodegradable nanoparticulate formulation based on poly(lactide-co-glycolide) (PLGA) with approximately 26% and 18% encapsulation efficiency, respectively. It was observed that TF/EGCG encapsulated PLGA nanoparticles (NPs) offered an up to ~7-fold dose advantage when compared with bulk TF/EGCG in terms of exerting its antiproliferative effects and also enhanced the anticancer potential of cisplatin (CDDP) in A549 (lung carcinoma), HeLa (cervical carcinoma), and THP-1 (acute monocytic leukemia) cells. Cell cycle analysis revealed that TF/EGCG-NPs were more efficient than bulk TF/EGCG in sensitizing A549 cells to CDDP-induced apoptosis, with a dose advantage of up to 20-fold. Further, TF/EGCG-NPs, alone or in combination with CDDP, were more effective in inhibiting NF-κB activation and in suppressing the expression of cyclin D1, matrix metalloproteinase-9, and vascular endothelial growth factor, involved in cell proliferation, metastasis, and angiogenesis, respectively. EGCG and TF-NPs were also found to be more effective than bulk TF/EGCG in inducing the cleavage of caspase-3 and caspase-9 and Bax/Bcl2 ratio in favor of apoptosis. Further, in vivo evaluation of these NPs in combination with CDDP showed an increase in life span (P<0.05) in mice bearing Ehrlich’s ascites carcinoma cells, with apparent regression of tumor volume in comparison with mice treated with bulk doses with CDDP. These results indicate that EGCG and TF-NPs have superior cancer chemosensitization activity when compared with bulk TF/EGCG.

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.

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.