Hypomethylation of LIMD1 and P16 by Downregulation of DNMT1 Results in Restriction of Liver Carcinogenesis by Amarogentin Treatment
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J Biosci (2021)46:53 Ó Indian Academy of Sciences DOI: 10.1007/s12038-021-00176-0 (0123456789().,-volV)(0123456789().,-volV) Hypomethylation of LIMD1 and P16 by downregulation of DNMT1 results in restriction of liver carcinogenesis by amarogentin treatment 1 2 1 3 DEBOLINA PAL ,SUBHAYAN SUR ,RITUPARNA ROY ,SUVRA MANDAL and 1 CHINMAY KUMAR PANDA * 1Department Oncogene Regulation, Chittaranjan National Cancer Institute, Kolkata 700 026, India 2Department of Pathology, Saint Louis University, Saint Louis, USA 3Department of Chemistry, National Research Institute for Ayurvedic Drug Development, Kolkata, India *Corresponding author (Email, [email protected]) MS received 28 December 2020; accepted 17 May 2021 Amarogentin (active component of Chirata) was found to prevent CCl4/NDEA-induced liver carcinogenesis at mild dysplastic stage through modulation of cell cycle, apoptosis, self-renewal pathways. The cell cycle regulatory genes LIMD1, P16 and RBSP3 were found to be upregulated in restricted liver lesions. To understand the mechanism of upregulation during restriction of cacinogenesis, the effect of amarogentin on epigenetic modification was evaluated in this study. It was also validated in vitro. Hypermethylation of LIMD1 and P16 was seen in mouse hepatocellular carcinoma (30th week carcinogen control mice); however, hypomethylation of these genes was seen in amarogentin-treated liver. In the case of RBSP3, no such change was seen. DNMT1 expression (mRNA/protein) was significantly increased in later stages of carcinogenesis, whereas its expression was comparable to normal liver in the case of amarogentin treatment. No significant change in expression (mRNA/protein) of HDAC1/2 was observed irrespective of treatment. Amarogentin treatment upregulated the expression (mRNA/protein) of LIMD1, P16 and RBSP3 in the HepG2 cell line. Here also treated cells showed LIMD1 and P16 hypomethylation with DNMT1 downregulation. Increased expres- sion of LIMD1, P16 and RBSP3 after treating cells with demethylating agent 5-aza-2-deoxycytidine indicated epigenetic modulation by amarogentin treatment. Keywords. Amarogentin; liver carcinogenesis; epigenetics; LIMD1; P16; DNMT1 1. Introduction several cellular pathways like cell cycle, apoptosis and Wnt/Hedgehog self-renewal (Pal et al. 2012; Sur et al. Amarogentin, the bitterest plant glycoside known, is 2016). During restriction of the liver carcinogenesis the active compound of Swertia chirata (chirata) which amarogentin could upregulate the expression of several had widely been used in Ayurvedic medicines since antagonists and downregulate several agonists of ancient times (Medda et al. 1999). The anticarcino- growth promoting pathways. To the best of our genic activity of this compound was first evident by its knowledge, mechanism of this expression modulation anti-proliferative and pro-apoptotic activity in a mouse by amarogentin is not yet known. skin carcinogenesis model (Saha et al. 2004). It was Thus, in this study attempts have been made to evident in our previous studies that amarogentin could understand, the mechanism of upregulation of cell restrict progression of experimental mouse liver car- cycle G1/S phase associated genes like LIMD1, P16 cinogenesis at mild dysplastic stage by modulating and RBSP3 in CCl4/NDEA-induced liver cancer mouse Supplementary Information: The online version contains supplementary material available at https://doi.org/10.1007/ s12038-021-00176-0. http://www.ias.ac.in/jbiosci 53 Page 2 of 11 Debolina Pal et al. model as well as in liver cancer cell line HepG2. It as mentioned in our previous study (Pal et al. 2012; Sur seems that amarogentin could modulate the epigenetic et al. 2016). Dose of the amarogentin for animal pathways like DNA methylation, histone deacetylation, experiment was 0.2 mg/kg body weight (supplemen- etc., as seen in case of other natural products, viz. tary table 1). EGCG, valproic acid, curcumin, etc., in their chemo- For in vitro experiment two doses of amarogentin preventive and anti-carcinogenic activities (Carlos- (i) 75 lg/ml (AM1) (30% cell death) and (ii) 100 lg/ Reyes et al. 2019; Reuter et al. 2011; Khan et al. 2015; ml (AM2) (50% cell death) were selected on the basis Lee et al. 2012; Jiang et al. 2015). of cytotoxicity assay using Sulforhodamine B (SRB) For this reason, in this study at first promoter dye (supplementary table 1) (Sur et al. 2016). methylation of LIMD1, P16 and RBSP3 genes were analyzed in CCl4/NDEA-induced mouse liver lesions with/without amarogentin treatment. Then, expression 2.3 Experimental groups for in vivo study of DNMT1 and HDACs were analyzed in the liver lesions. The in vivo results were further validated Group I (control group): Mice without any treatment. in vitro using human hepatocellular carcinoma cell line Group II (carcinogen control group): Mice were HepG2. injected intraperitoneally (i.p.) with Carbon tetrachlo- Finally, our data showed that reduced expression of ride (CCl4) successively for 4 days followed by DNMT1 might lead to upregulation of the cell cycle N-nitrosodiethylamine (NDEA) i.p. injection weekly inhibitors through promoter hypo-methylation during 75 mg/kg body weight for three successive weeks the restriction of liver carcinogenesis by amarogentin. followed by 100 mg/kg body weight for another 3 successive weeks (supplementary figure 1). Group III (pre-initiation group): Amarogentin twice a 2. Materials and methods week was administered orally for 15 days before 1st carcinogen application. Then, amarogentin treatment 2.1 Animal care was continued once a week during carcinogen appli- cation, which was continued for the rest of the exper- Adult (6 weeks) inbred female Swiss albino mice imental period. Treatment schedule of CCl4 and NDEA (average weight 25 g) were obtained from animal was same as Group II (supplementary figure 1). house of Chittaranjan National Cancer Institute, Group IV (post-initiation group): In this group Kolkata, India. Animals were maintained at 25 ± 5°C amarogentin treatment started after completion of car- temperature under alternating 12 h light/dark cycle with cinogen schedule and continued orally once a week 45–55% humid conditions. Food pellets (Lipton India throughout experimental procedure. Application and Ltd.) and drinking water was provided. Animal han- dose of amarogentin is same as Group III. Carcinogen dling and experimental protocol were undertaken as per schedule was same as Group II (supplementary guidelines of the Institutional Ethical Committee figure 1). (Registration No. 1774/GO/RBi/S/14/CPCSEA) regis- Number of mice in each group was 12. Mice from tered under CPCSEA, India (Committee for the pur- different experimental groups were sacrificed at 10th, pose of control and supervision of the experiments on 20th and 30th weeks of first carcinogen application and animals). Mice were observed regularly for their at each time point three mice were sacrificed from each wellbeing, body weight, toxicity, and survival. group. Liver tissues were dissected out and used for different experiment. Animals for experimental pur- poses were approved by institutional animal ethics 2.2 Drug solution preparation and concentrations committee. used for in vivo and in vitro studies Amarogentin 1 mg/ml stock solutions were prepared in 2.4 Sample collection 10% ethanol in water. Extraction and purification of amarogentin ([99% pure in high-performance liquid Mice from different experimental groups were sacri- chromatography analysis) were done at the National ficed at 10th, 20th and 30th week. Liver was dissected Research Institute for Ayurvedic Drug Development, out and affected liver lesions were collected and divi- Kolkata, India. The amarogentin doses for in vivo and ded for histopathological analysis, RNA, DNA and in vitro experiments were selected by toxicity analysis protein isolation. Amarogentin restricted liver carcinogenesis by epigenetic modification Page 3 of 11 53 2.5 Histopathology orthovanadate]. Equal amount of proteins was sepa- rated by 10–12% SDS–PAGE and then transferred to The formalin fixed tissue samples were processed polyvinylidine difluoride (PVDF) membrane (Milli- conventionally to prepare paraffin block. Tissue sec- pore, MA). Membranes were incubated with 3–5% tions (3–4 lm) were taken for hematoxylin-eosin (HE) nonfat dry milk for 1–2 h at room temperature followed staining and histopathological evaluation. by overnight incubation at 4°C with desired primary antibodies (Supplementary document) (1:500–1:1000 in 1% nonfat dry milk) and corresponding HRP-con- 2.6 Cell line and Amarogentin treatment condition jugated secondary antibodies (Supplementary docu- ment) (1:2000–1:5000 in 1% nonfat dry milk) for 2 h at Human liver cancer cell line HepG2 was obtained from room temperature (Pal et al. 2012). The protein bands National Centre for Cell Sciences, Pune, India. The cell were then visualized using luminol reagent and line was maintained in Dulbecco’s Modified Eagle’s autoradiographed on X-ray film (Kodak, Rochester, Medium (DMEM) supplemented with 10% FBS, 2 mM NY). All the immuno-blotting experiments were per- glutamine, 100 U Penicillin and 100 lg/ml of strepto- formed in three replicates. The band intensities were mycin. Cells were grown at 37°C in a humidified CO2 quantified using densitometric scanner (Bio Rad GS- incubator with 5% CO2. Re-feeding with fresh growth 800, Hercules, California). Peak densities of the pro- medium and subculturing (using 0.05% trypsin- teins of interest were normalized using peak density of EDTA) of cells was