Debolina Pal

Eugenol restricts DMBA croton oil induced skin carcinogenesis in mice: Downregulation of c-Myc and H-ras, and activation of p53 dependent apoptotic pathway

BACKGROUND Eugenol is the active component of essential oil isolated from clove (Syzigium aromaticum). Eugenol has antimutagenic, antigenotoxic, anti-inflammatory properties. The anticarcinogenic effect of eugenol was evident in different types of cell lines. However, its anticarcinogenic effect in in vivo has not yet been fully explored. OBJECTIVE The aim of this study is to evaluate the chemopreventive potential of eugenol in an experimental skin carcinogenesis mice model system. METHOD Skin tumor was induced by topical application of DMBA croton oil in Swiss mice. To assess the chemopreventive potential of eugenol, it was orally administered 15 days prior carcinogen treatment. The development of skin carcinogenesis was confirmed by histopathological analysis. Cellular proliferation and apoptosis in the skin tumor were analyzed by in situ cellular proliferation and in situ cell death assay. Expression of some proliferation and apoptosis associated genes was analyzed by RT-PCR and protein expression was analyzed by Western blot. RESULTS Reduction in incidence and sizes of skin tumors along with overall increase in survival of mice were seen due to eugenol treatment. Restriction of skin carcinogenesis at the dysplastic stage along with reduced rate of cellular proliferation and increase in apoptosis were evident in eugenol treated skin tumors. Eugenol treatment led to the downregulation of c-Myc, H-ras and Bcl2 expression along with upregulation of P53, Bax and active Caspase-3 expression in the skin lesions. CONCLUSION Restriction of skin carcinogenesis at dysplastic stage by eugenol was due to attenuation of c-Myc, H-ras and modification of some p53 associated gene expression.

Prevention of liver carcinogenesis by amarogentin through modulation of G1/S cell cycle check point and induction of apoptosis.

Amarogentin, a secoiridoid glycoside, is an active component of the medicinal plant Swertia chirata. In this study, chemopreventive and chemotherapeutic actions of amarogentin were evaluated in a carbon tetrachloride (CCl(4))/N-nitrosodiethylamine (NDEA)-induced liver carcinogenesis mouse model system during continuous and posttreatment schedule. Better survival, no toxicity and increased body weight were noted in amarogentin-treated mice. Reduction in proliferation and increase in apoptosis frequency were evident in amarogentin-treated groups. In carcinogen control group moderate dysplasia, severe dysplasia and hepatocellular carcinoma were evident at 10th, 20th and 30th week, respectively. Amarogentin was found to prevent progression of liver carcinogenesis at mild dysplastic stage. Exposure to CCl(4)/NDEA resulted in upregulation of ppRb807/811, cyclinD1 and cdc25A at 10th week and additional activation of cMyc and mdm2 along with downregulation of LIMD1, p53 and p21 at 20th week. This was followed by activation of ppRb567 and downregulation of Rbsp3 at 30th week. Prevention of carcinogenesis by amarogentin in both groups might be due to cumulative upregulation of LIMD1, RBSP3, p16 and downregulation of cdc25A at 10th week along with activation of p53 and p21 and downregulation of ppRb807/811 and ppRb567 at 20th week, followed by downregulation of cyclinD1, cMyc and mdm2 at 30th week. During carcinogenesis reduction of apoptosis was evident since 20th week. However, amarogentin treatment could significantly induce apoptosis through upregulation of the Bax-Bcl2 ratio, activation of caspase-3 and poly ADP ribose polymerase cleavage. This is the first report of chemopreventive/therapeutic role of amarogentin during liver carcinogenesis through modulation of cell cycle and apoptosis.

Tea polyphenols epigallocatechin gallete and theaflavin restrict mouse liver carcinogenesis through modulation of self-renewal Wnt and hedgehog pathways.

The aim of this study is to evaluate chemopreventive and therapeutic efficacy of tea polyphenols epigallocatechin gallete (EGCG) and theaflavin (TF) on self-renewal Wnt and Hedgehog (Hh) pathways during CCl4/N-nitosodiethylamine-induced mouse liver carcinogenesis. For this purpose, the effect of EGCG/TF was investigated in liver lesions of different groups at pre-, continuous and post initiation stages of carcinogenesis. Comparatively increased body weights were evident due to EGCG/TF treatment than carcinogen control mice. Both EGCG and TF could restrict the development of hepatocellular carcinoma at 30th week of carcinogen application showing potential chemoprevention in continuous treated group (mild dysplasia) followed by pretreated (moderate dysplasia) and therapeutic efficacy in posttreated group (mild dysplasia). This restriction was associated with significantly reduced proliferation, increased apoptosis, decreased prevalence of hepatocyte progenitor cell (AFP) and stem cell population (CD44) irrespective of EGCG/TF treatments. The EGCG/TF could modulate the Wnt pathway by reducing β-catenin and phospho-β-catenin-Y-654 expressions along with up-regulation of sFRP1 (secreted frizzled-related protein 1) and adenomatosis polyposis coli during the restriction. In case of the Hh pathway, EGCG/TF could also reduce expressions of glioma-associated oncogene homolog 1 (Gli1) and SMO (smoothened homolog) along with up-regulation of PTCH1 (patched homolog 1). As a result, in Wnt/Hh regulatory pathways decreased expressions of β-catenin/Gli1 target genes like CyclinD1, cMyc and EGFR/phospho-EGFR-Y-1173 and up-regulation of E-cadherin were seen during the restriction. Thus, the restriction of liver carcinogenesis by EGCG/TF was due to reduction in hepatocyte progenitor cell/stem cell population along with modulation of Wnt/Hh and other regulatory pathways.

Tea polyphenols EGCG and TF restrict tongue and liver carcinogenesis simultaneously induced by N-nitrosodiethylamine in mice

The aim of this study is to understand the molecular mechanisms of N-nitrosodiethylamine (NDEA) induced multi-organ carcinogenesis in tongue and liver of the same mouse and restriction of carcinogenesis by Epigallocatechin gallate (EGCG) and Theaflavin (TF), if any. For that purpose, cellular proliferation/apoptosis, prevalence of CD44 positive stem cell population and expressions of some key regulatory genes of self renewal Wnt and Hedgehog (Hh) pathways and some of their associated genes were analyzed in the NDEA induced tongue and liver lesions in absence or presence of EGCG/TF. Chronic NDEA exposure in oral cavity could decrease mice body weights and induce tongue and liver carcinogenesis with similar histological stages (severe dysplasia up to 30thweeks of NDEA administration). Increasing mice body weights were seen in continuous and post EGCG/TF treated groups. EGCG/TF treatment could restrict both the carcinogenesis at similar histological stages showing potential chemopreventive effect in continuous treated groups (mild dysplasia) followed by pre treatment (moderate dysplasia) and therapeutic efficacy in post treated groups (mild dysplasia) up to 30thweek. The mechanism of carcinogenesis by NDEA and restriction by the EGCG/TF in both tongue and liver were similar and found to be associated with modulation in cellular proliferation/apoptosis and prevalence of CD44 positive population. The up-regulation of self renewal Wnt/β-catenin, Hh/Gli1 pathways and their associated genes Cyclin D1, cMyc and EGFR along with down regulation of E-cadherin seen during the carcinogenesis processes were found to be modulated during the restriction processes by EGCG/TF.

Association of early phase of colorectal carcinogenesis with STAT3 activation and its relevance in apoptosis regulation

Expression of STAT3/pSTAT3 in colorectal cancer (CRC) patients of Indian origin was studied to assess its significance in early detection and apoptosis regulation. Colorectal tissues with malignant lesions were STAT3/pSTAT3 positive in 66% of the cases and among these positive cases, well differentiated, moderately differentiated and poorly differentiated cancers were 86%, 60% and 0% respectively. All CRC specimens studied were immunoreactive with anti-carcinoembryonic antigen antibody. Cells purified from CRC tissues exhibited greater STAT3/pSTAT3 reactivity than peripheral blood mononuclear cells (PBMC) from healthy individuals, which served as control. apoptotic index (AI) was comparatively low in tissue specimens with STAT3/pSTAT3 expression. CRC cells with a comparatively less number of apoptotic cells, expressed a minimum number of Caspase-3 positive cells (4.73%), in comparison to healthy-PBMC (12.63%). CRC cells with high STAT3/pSTAT3 staining had cells with greater percentage of Bcl2 reactivity (23.05%), but less positivity with Caspase3 antibody (2.05%). Overall data suggests that CRC population was STAT3/pSTAT3 immunoreactive in a stage specific manner and STAT3 protects cancerous colorectal epithelial cells from apoptosis. Bcl-2, Cyclin D1 and Caspase-3 control the activity of apoptosis regulator, STAT3.

Sequential loss of cell cycle checkpoint control contributes to malignant transformation of murine embryonic fibroblasts induced by 20-methylcholanthrene

Definitive information about the number and nature of discrete steps of tumorigenesis is enigmatic. To understand the multistep nature of carcinogenesis, an in vitro model of 20‐Methylcholanthrene‐treated primary fibroblast cells CNCI‐PM‐20, from 20‐day old Swiss mouse embryo was used. Visible neoplastic changes with distinct morphological variations along with specific chromosomal aberrations like Robertsonian metacentrics, double and single‐minute chromosomes and aneuploidy were observed from Passage‐20 onwards. The cell cycle profile showed gradual increase in G2/M population till P‐32, followed by evasion of block from P‐36 onwards. Gradual increase in expression of C‐myc, CyclinD1 and a decrease in expression of P21 was observed from P‐20 onwards. CDC25A expression was significantly increased at P‐27 and remained more or less constant in subsequent passages. Additionally, an increased P16 and P53 expression were seen at P‐20 followed by their significant down‐regulation at P‐32. An increased level of phosphorylated retinoblastoma (ppRb) was observed from P‐27, probably responsible for a compromised G1/S checkpoint. The inactivation of p21 and p16 might be due to their promoter hyper‐methylation as suggested through de‐methylation experiment by 5‐aza‐deoxycytidine at P‐42. G2/M checkpoint abrogation was marked by gradual increase in expression of CyclinB1 and Cdc20, and a significant increase of Mad2 at P‐20. Interestingly, increased expression of phospho‐ATM, ATR and phospho‐Chk1 were also seen at P‐20 followed by their down‐regulation at subsequent passages, indicating a perturbation of DNA damage response pathway at early passages. Our findings therefore dramatize the multiple genetic events that can cooperate to promote tumorigenesis. J. Cell. Physiol. 224:49–58, 2010

Amarogentin regulates self renewal pathways to restrict liver carcinogenesis in experimental mouse model.

Amarogentin, a secoiridoid glycoside isolated from medicinal plant Swertia chirata, was found to restrict CCl4/N‐nitrosodiethyl amine (NDEA) induced mouse liver carcinogenesis by modulating G1/S cell cycle check point and inducing apoptosis. To understand its therapeutic efficacy on stem cell self renewal pathways, prevalence of CD44 positive cancer stem cell (CSC) population, expressions (mRNA/protein) of some key regulatory genes of self renewal Wnt and Hedgehog pathways along with expressions of E‐cadherin and EGFR were analyzed during the liver carcinogenesis and in liver cancer cell line HepG2. It was observed that amarogentin could significantly reduce CD44 positive CSCs in both pre and post initiation stages of carcinogenesis than carcinogen control mice. In Wnt pathway, amarogentin could inhibit expressions of β‐catenin, phospho β‐catenin (Y‐654) and activate expressions of antagonists sFRP1/2 and APC in the liver lesions. In Hedgehog pathway, decreased expressions of Gli1, sonic hedgehog ligand, and SMO along with up‐regulation of PTCH1 were seen in the liver lesions due to amarogentin treatment. Moreover, amarogentin could up‐regulate E‐cadherin expression and down‐regulate expression of EGFR in the liver lesions. Similarly, amarogentin could inhibit HepG2 cell growth along with expression and prevalence of CD44 positive CSCs. Similar to in vivo analysis, amarogentin could modulate the expressions of the key regulatory genes of the Wnt and hedgehog pathways and EGFR in HepG2 cells. Thus, our data suggests that the restriction of liver carcinogenesis by amarogentin might be due to reduction of CD44 positive CSCs and modulation of the self renewal pathways.

Regular black tea habit could reduce tobacco associated ROS generation and DNA damage in oral mucosa of normal population.

Tobacco and tea habit are very common in world wide. In the present study, an attempt was made to evaluate the effect of regular drinking of black tea on reactive oxygen species (ROS) generation and DNA damage in buccal cells of normal subjects with or without tobacco habit. Expression of ROS associated proteins IκB, NF-κB as well as DNA repair associated proteins p53, MLH1 were also analyzed. Exfoliated buccal cells were collected from 308 healthy individuals and classified according to age, tobacco and tea habits. In all age groups, comparatively high ROS level and significantly high DNA damage frequency were seen in individuals with tobacco habit than the subjects without tea and tobacco habits. Tea habit effectively lowered ROS level and restrict DNA damage in tobacco users irrespective of ages. The DNA damage seen in the subjects was not associated with apoptosis. Moreover, tea habit effectively lowered the expression of IκB, NF-κB, p53 and MLH1 in tobacco users in all age groups. It seems that regular black tea habit could have anti-genotoxic effect as revealed by reduced tobacco associated ROS generation and DNA damage in buccal cells.

Epigallocatechin gallate in combination with eugenol or amarogentin shows synergistic chemotherapeutic potential in cervical cancer cell line: PAL et al.

In this study, antitumor activity of epigallocatechin gallate (EGCG; major component of green tea polyphenol), eugenol (active component of clove), and amarogentin (active component of chirata plant) either alone or in combination were evaluated in Hela cell line. It was evident that EGCG with eugenol–amrogentin could highly inhibit the cellular proliferation and colony formation than individual treatments. Induction of apoptosis was also higher after treatment with EGCG in combination with eugenol–amrogentin than individual compound treatments. The antiproliferative effect of these compounds was due to downregulation of cyclinD1 and upregulation of cell cycle inhibitors LIMD1, RBSP3, and p16 at G1/S phase of cell cycle. Treatment of these compounds could induce promoter hypomethylation of LimD1 and P16 genes as a result of reduced expression of DNA methyltransferase 1 (DNMT1). Thus, our study indicated the better chemotherapeutic effect of EGCG in combination with eugenol–amarogentin in Hela cell line. The chemotherapeutic effect might be due to the epigenetic modification particularly DNA hypomethylation through downregulation of DNMT1.