Momordicine-I Suppresses Head and Neck Cancer Growth by Reprogrammimg Immunosuppressive Effect of the Tumor-Infiltrating Macrophages and B Lymphocytes.

Head and neck cancer (HNC) is prevalent worldwide, and treatment options are limited. Momordicine-I (M-I), a natural component from bitter melon, shows antitumor activity against these cancers, but its mechanism of action, especially in the tumor microenvironment (TME), remains unclear. In this study, we establish that M-I reduces HNC tumor growth in two different immunocompetent mouse models using MOC2 and SCC VII cells. We demonstrate that the anticancer activity results from modulating several molecules in the monocyte/macrophage clusters in CD45+ populations in MOC2 tumors by single-cell RNA sequencing. Tumor-associated macrophages (TAM) often pose a barrier to antitumor effects, but following M-I treatment, we observe a significant reduction in the expression of Sfln4, a myeloid cell differentiation factor, and Cxcl3, a neutrophil chemoattractant, in the monocyte/macrophage populations. We further find that the macrophages must be in close contact with the tumor cells to inhibit Sfln4 and Cxcl3, suggesting that these TAMs are impacted by M-I treatment. Coculturing macrophages with tumor cells shows inhibition of Agr1 expression following M-I treatment, which is indicative of switching from M2 to M1 phenotype. Furthermore, the total B-cell population in M-I-treated tumors is significantly lower, whereas spleen cells also show similar results when cocultured with MOC2 cells. M-I treatment also inhibits PD1, PD-L1, and FoxP3 expression in tumors. Collectively, these results uncover the potential mechanism of M-I by modulating immune cells, and this new insight can help to develop M-I as a promising candidate to treat HNCs, either alone or as adjuvant therapy.

Non-Coding RNAs in Oral Cancer: Emerging Roles and Clinical Applications.

Oral cancer (OC) is among the most prevalent cancers in the world. Certain geographical areas are disproportionately affected by OC cases due to the regional differences in dietary habits, tobacco and alcohol consumption. However, conventional therapeutic methods do not yield satisfying treatment outcomes. Thus, there is an urgent need to understand the disease process and to develop diagnostic and therapeutic strategies for OC. In this review, we discuss the role of various types of ncRNAs in OC, and their promising clinical implications as prognostic or diagnostic markers and therapeutic targets. MicroRNA (miRNA), long ncRNA (lncRNA), circular RNA (circRNA), PIWI-interacting RNA (piRNA), and small nucleolar RNA (snoRNA) are the major ncRNA types whose involvement in OC are emerging. Dysregulated expression of ncRNAs, particularly miRNAs, lncRNAs, and circRNAs, are linked with the initiation, progression, as well as therapy resistance of OC via modulation in a series of cellular pathways through epigenetic, transcriptional, post-transcriptional, and translational modifications. Differential expressions of miRNAs and lncRNAs in blood, saliva or extracellular vesicles have indicated potential diagnostic and prognostic importance. In this review, we have summarized all the promising aspects of ncRNAs in the management of OC.

Long noncoding RNA ELDR promotes cell cycle progression in normal oral keratinocytes through induction of a CTCF-FOXM1-AURKA signaling axis.

Long noncoding RNAs (lncRNAs) have gained widespread attention as a new layer of regulation in biological processes during development and disease. The lncRNA ELDR (EGFR long noncoding downstream RNA) was recently shown to be highly expressed in oral cancers as compared to adjacent nontumor tissue, and we previously reported that ELDR may be an oncogene as inhibition of ELDR reduces tumor growth in oral cancer models. Furthermore, overexpression of ELDR induces proliferation and colony formation in normal oral keratinocytes (NOKs). In this study, we examined in further detail how ELDR drives the neoplastic transformation of normal keratinocytes. We performed RNA-seq analysis on NOKs stably expressing ELDR (NOK-ELDR), which revealed that ELDR enhances the expression of cell cycle-related genes. Expression of Aurora kinase A and its downstream targets Polo-like kinase 1, cell division cycle 25C, cyclin-dependent kinase 1, and cyclin B1 (CCNB1) are significantly increased in NOK-ELDR cells, suggesting induction of G2/M progression. We further identified CCCTC-binding factor (CTCF) as a binding partner of ELDR in NOK-ELDR cells. We show that ELDR stabilizes CTCF and increases its expression. Finally, we demonstrate the ELDR-CTCF axis upregulates transcription factor Forkhead box M1, which induces Aurora kinase A expression and downstream G2/M transition. These findings provide mechanistic insights into the role of the lncRNA ELDR as a potential driver of oral cancer during neoplastic transformation of normal keratinocytes.

Emerging role of lncRNA ELDR in development and cancer.

Whole-genome sequencing and transcriptome analysis revealed more than 90% of the human genome transcribes noncoding RNAs including lncRNAs. From the beginning of the 21st century, lncRNAs have gained widespread attention as a new layer of regulation in biological processes. lncRNAs are > 200 nucleotides in size, transcribed by RNA polymerase II, and share many similarities with mRNAs. lncRNA interacts with DNA, RNA, protein, and miRNAs, thereby regulating many biological processes. In this review, we have focused mainly on LINC01156 [also known as the EGFR long non-coding downstream RNA (ELDR) or Fabl] and its biological importance. ELDR is a newly identified lncRNA and first reported in a mouse model, but it has a human homolog. The human ELDR gene is closely localized downstream of epidermal growth factor receptor (EGFR) gene at chromosome 7 on the opposite strand. ELDR is highly expressed in neuronal stem cells and associated with neuronal differentiation and mouse brain development. ELDR is upregulated in head and neck cancer, suggesting its role as an oncogene and its importance in prognosis and therapy. Publicly available RNA-seq data further support its oncogenic potential in different cancers. Here, we summarize all the aspects of ELDR in development and cancer, highlighting its future perspectives in the context of mechanism.

Diverse roles of bitter melon (Momordica charantia) in prevention of oral cancer.

Oral squamous cell carcinoma (OSCC) is one of the common lethal malignancies which is increasing rapidly in the world. Increasing risks from alcohol and tobacco habits, lack of early detection markers, lack of effective chemotherapeutic agents, recurrence and distant metastasis make the disease more complicated to manage. Laboratory-based studies and epidemiological studies indicate important roles of nutraceuticals to manage different cancers. The plant bitter melon (Momordica charantia) is a good source of nutrients and bio-active phytochemicals such as triterpenoids, triterpene glycosides, phenolic acids, flavonoids, lectins, sterols and proteins. The plant is widely grown in Asia, Africa, and South America. Bitter melon has traditionally been used as a folk medicine and Ayurvedic medicine in Asian culture to treat diseases such as diabetes, since ancient times. The crude extract and some of the isolated pure compounds of bitter melon show potential anticancer effects against different cancers. In this review, we shed light on its effect on OSCC. Bitter melon extract has been found to inhibit cell proliferation and metabolism, induce cell death and enhance the immune defense system in the prevention of OSCC in vitro and in vivo. Thus, bitter melon may be used as an attractive chemopreventive agent in progression towards OSCC clinical study.

Hypomethylation of LIMD1 and P16 by downregulation of DNMT1 results in restriction of liver carcinogenesis by amarogentin treatment.

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 expression of LIMD1, P16 and RBSP3 after treating cells with demethylating agent 5-aza-2-deoxycytidine indicated epigenetic modulation by amarogentin treatment.

Momordicine-I, a Bitter Melon Bioactive Metabolite, Displays Anti-Tumor Activity in Head and Neck Cancer Involving c-Met and Downstream Signaling.

Head and neck cancer (HNC) is one of the most aggressive cancers, and treatments are quite challenging due to the difficulty in early diagnosis, lack of effective chemotherapeutic drugs, adverse side effects and therapy resistance. We identified momordicine-I (M-I), a bioactive secondary metabolite in bitter melon (Momordica charantia), by performing liquid chromatography-high resolution electrospray ionization mass spectrometry (LC-HRESIMS) analysis. M-I inhibited human HNC cell (JHU022, JHU029, Cal27) viability in a dose-dependent manner without an apparent toxic effect on normal oral keratinocytes. Mechanistic studies showed that M-I inhibited c-Met and its downstream signaling molecules c-Myc, survivin, and cyclin D1 through the inactivation of STAT3 in HNC cells. We further observed that M-I was non-toxic and stable in mouse (male C57Bl/6) blood, and a favorable pharmacokinetics profile was observed after IP administration. M-I treatment reduced HNC xenograft tumor growth in nude mice and inhibited c-Met and downstream signaling. Thus, M-I has potential therapeutic implications against HNC.

Exosomes from COVID-19 Patients Carry Tenascin-C and Fibrinogen-ß in Triggering Inflammatory Signals in Cells of Distant Organ.

SARS-CoV-2 infection can cause cytokine storm and may overshoot immunity in humans; however, it remains to be determined whether virus-induced soluble mediators from infected cells are carried by exosomes as vehicles to distant organs and cause tissue damage in COVID-19 patients. We took an unbiased proteomic approach for analyses of exosomes isolated from plasma of healthy volunteers and COVID-19 patients. Our results revealed that tenascin-C (TNC) and fibrinogen-ß (FGB) are highly abundant in exosomes from COVID-19 patients' plasma compared with that of healthy normal controls. Since TNC and FGB stimulate pro-inflammatory cytokines via the Nuclear factor-κB (NF-κB) pathway, we examined the status of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and C-C motif chemokine ligand 5 (CCL5) expression upon exposure of hepatocytes to exosomes from COVID-19 patients and observed significant increase compared with that from healthy subjects. Together, our results demonstrate that TNC and FGB are transported through plasma exosomes and potentially trigger pro-inflammatory cytokine signaling in cells of distant organ.

Inhibition of Long Noncoding RNA Linc-Pint by Hepatitis C Virus in Infected Hepatocytes Enhances Lipogenesis.

BACKGROUND AND AIMS: HCV often causes chronic infection in liver, cirrhosis, and, in some instances, HCC. HCV encodes several factors' those impair host genes for establishment of chronic infection. The long noncoding RNAs (lncRNAs) display diverse effects on biological regulations. However, their role in virus replication and underlying diseases is poorly understood. In this study, we have shown that HCV exploits lncRNA long intergenic nonprotein-coding RNA, p53 induced transcript (Linc-Pint) in hepatocytes for enhancement of lipogenesis. APPROACH AND RESULTS: We identified a lncRNA, Linc-Pint, which is significantly down-regulated in HCV-replicating hepatocytes and liver specimens from HCV infected patients. Using RNA pull-down proteomics, we identified serine/arginine protein specific kinase 2 (SRPK2) as an interacting partner of Linc-Pint. A subsequent study demonstrated that overexpression of Linc-Pint inhibits the expression of lipogenesis-related genes, such as fatty acid synthase and ATP-citrate lyase. We also observed that Linc-Pint significantly inhibits HCV replication. Furthermore, HCV-mediated enhanced lipogenesis can be controlled by exogenous Linc-Pint expression. Together, our results suggested that HCV-mediated down-regulation of Linc-Pint enhances lipogenesis favoring virus replication and liver disease progression. CONCLUSIONS: We have shown that SRPK2 is a direct target of Linc-Pint and that depletion of SRPK2 inhibits lipogenesis. Our study contributes to the mechanistic understanding of the role of Linc-Pint in HCV-associated liver pathogenesis.

Long non-coding RNA ELDR enhances oral cancer growth by promoting ILF3-cyclin E1 signaling.

Oral squamous cell carcinoma (OSCC) is the sixth most common cancer with a 5-year overall survival rate of 50%. Thus, there is a critical need to understand the disease process, and to identify improved therapeutic strategies. Previously, we found the long non-coding RNA (lncRNA) EGFR long non-coding downstream RNA (ELDR) induced in a mouse tongue cancer model; however, its functional role in human oral cancer remained unknown. Here, we show that ELDR is highly expressed in OSCC patient samples and in cell lines. Overexpression of ELDR in normal non-tumorigenic oral keratinocytes induces cell proliferation, colony formation, and PCNA expression. We also show that ELDR depletion reduces OSCC cell proliferation and PCNA expression. Proteomics data identifies the RNA binding protein ILF3 as an interacting partner of ELDR. We further show that the ELDR-ILF3 axis regulates Cyclin E1 expression and phosphorylation of the retinoblastoma (RB) protein. Intratumoral injection of ELDR-specific siRNA reduces OSCC and PDX tumor growth in mice. These findings provide molecular insight into the role of ELDR in oral cancer and demonstrate that targeting ELDR has promising therapeutic potential.

Bitter Melon (Momordica Charantia), a Nutraceutical Approach for Cancer Prevention and Therapy.

Cancer is the second leading cause of death worldwide. Many dietary plant products show promising anticancer effects. Bitter melon or bitter gourd (Momordica charantia) is a nutrient-rich medicinal plant cultivated in tropical and subtropical regions of many countries. Traditionally, bitter melon is used as a folk medicine and contains many bioactive components including triterpenoids, triterpene glycoside, phenolic acids, flavonoids, lectins, sterols and proteins that show potential anticancer activity without significant side effects. The preventive and therapeutic effects of crude extract or isolated components are studied in cell line-based models and animal models of multiple types of cancer. In the present review, we summarize recent progress in testing the cancer preventive and therapeutic activity of bitter melon with a focus on underlying molecular mechanisms. The crude extract and its components prevent many types of cancers by enhancing reactive oxygen species generation; inhibiting cancer cell cycle, cell signaling, cancer stem cells, glucose and lipid metabolism, invasion, metastasis, hypoxia, and angiogenesis; inducing apoptosis and autophagy cell death, and enhancing the immune defense. Thus, bitter melon may serve as a promising cancer preventive and therapeutic agent.

Establishment of a Patient-Derived Xenograft Tumor From Hepatitis C-Associated Liver Cancer and Evaluation of Imatinib Treatment Efficacy.

BACKGROUND AND AIMS: Chronic hepatitis C virus (HCV) infection is one of the major causal factors for hepatocellular carcinoma (HCC). The treatment options for HCC are limited for lack of a convenient animal model for study in HCV infection and liver pathogenesis. This study aimed to develop a patient-derived xenograft (PDX) tumor in mice by using a tumor from a patient with HCV-associated HCC and evaluating this model's therapeutic potential. APPROACH AND RESULTS: After resection of the primary tumor from the patient liver, excess viable tumor was implanted into highly immunodeficient mice. A mouse xenograft tumor line was developed, and the tumor was successfully passaged for at least three rounds in immunodeficient mice. The patient's primary tumor and the mouse xenografts were histologically similar. Genetic profiling by short-tandem-repeat analysis verified that the HCC-PDX model was derived from the HCC clinical specimen. HCV RNA present in the patient liver specimen was undetectable after passage as xenograft tumors in mice. Human albumin, α1 -antitrypsin, glypican-3, α-smooth muscle actin, and collagen type 1A2 markers were detected in human original tumor tissues and xenograft tumors. Both the patient primary tumor and the xenograft tumors had a significantly higher level of receptor tyrosine kinase (c-Kit) mRNA. Treatment of HCC-PDX xenograft tumor-bearing mice with the c-Kit inhibitor imatinib significantly reduced tumor growth and phospho-Akt and cyclin D1 expression, as compared with untreated control tumors. CONCLUSIONS: Our results demonstrated establishment of an HCV-associated HCC-PDX model as a powerful tool for evaluating candidate drugs. Information on molecular changes in cancer-specific gene expression facilitates efficient targeted therapies and treatment strategies.

Correction to: Inhibition of the key metabolic pathways, glycolysis and lipogenesis, of oral cancer by bitter melon extract.

Following publication of the original article [1], it was reported that Fig. 1c was not entirely readable due to overlapping Fig. 1d. The publishers apologise for this error.

miRNA-29b Inhibits Prostate Tumor Growth and Induces Apoptosis by Increasing Bim Expression.

Prostate cancer is one of the most common cancers among men. Currently available therapies improve patient survival against local prostate cancer but have shown severe side effects. Advanced prostate cancer is still incurable. Studies have suggested the involvement of non-coding RNAs, especially micro-RNAs (miRNAs), in the regulation of multiple cellular events in cancer and thus several clinical trials are ongoing using miRNAs mimics or inhibitors. We previously demonstrated that miRNA-29b-3p (miR-29b) was downregulated in prostate cancer and that the overexpression of miR-29b limited prostate cancer metastasis. However, the therapeutic potential of the miR-29b against prostate cancer remains unknown. Here, we evaluated the therapeutic role of miR-29b in in vivo prostate tumors in a mouse model. Intratumoral injection of mimic miR-29b significantly inhibited prostate cancer xenograft tumor growth in nude mice. Subsequent study demonstrated that the overexpression of miR-29b reduced prostate cancer cell PC3 proliferation in a time dependent manner and induced cell death. Mechanistic study using a cancer pathway specific transcriptomic array revealed a significant overexpression of the pro-apoptotic gene BCL2L11 (Bim) in the miR-29b overexpressed PC3 cells, which was further verified in PC3 cells overexpressing miR-29b. We also observed a significant induction of Bim protein in miR-29b treated xenograft tumors. The induction of cytosolic accumulation of cytochrome C and PARP cleavage in miR-29b overexpressed PC3 cells was observed. Thus, our results suggest that miR-29b can be used as a potential molecule for prostate cancer therapy.

Inhibition of the key metabolic pathways, glycolysis and lipogenesis, of oral cancer by bitter melon extract.

BACKGROUND: Metabolic reprogramming is one of the hallmarks of cancer which favours rapid energy production, biosynthetic capabilities and therapy resistance. In our previous study, we showed bitter melon extract (BME) prevents carcinogen induced mouse oral cancer. RNA sequence analysis from mouse tongue revealed a significant modulation in "Metabolic Process" by altering glycolysis and lipid metabolic pathways in BME fed group as compared to cancer group. In present study, we evaluated the effect of BME on glycolysis and lipid metabolism pathways in human oral cancer cells. METHODS: Cal27 and JHU022 cells were treated with BME. RNA and protein expression were analysed for modulation of glycolytic and lipogenesis genes by quantitative real-time PCR, western blot analyses and immunofluorescence. Lactate and pyruvate level was determined by GC/MS. Extracellular acidification and glycolytic rate were measured using the Seahorse XF analyser. Shotgun lipidomics in Cal27 and JHU022 cell lines following BME treatment was performed by ESI/ MS. ROS was measured by FACS. RESULTS: Treatment with BME on oral cancer cell lines significantly reduced mRNA and protein expression levels of key glycolytic genes SLC2A1 (GLUT-1), PFKP, LDHA, PKM and PDK3. Pyruvate and lactate levels and glycolysis rate were reduced in oral cancer cells following BME treatment. In lipogenesis pathway, we observed a significant reduction of genes involves in fatty acid biogenesis, ACLY, ACC1 and FASN, at the mRNA and protein levels following BME treatment. Further, BME treatment significantly reduced phosphatidylcholine, phosphatidylethanolamine, and plasmenylethanolamine, and reduced iPLA2 activity. Additionally, BME treatment inhibited lipid raft marker flotillin expression and altered its subcellular localization. ER-stress associated CHOP expression and generation of mitochondrial reactive oxygen species were induced by BME, which facilitated apoptosis. CONCLUSION: Our study revealed that bitter melon extract inhibits glycolysis and lipid metabolism and induces ER and oxidative stress-mediated cell death in oral cancer. Thus, BME-mediated metabolic reprogramming of oral cancer cells will have important preventive and therapeutic implications along with conventional therapies.

Repression of MicroRNA-30e by Hepatitis C Virus Enhances Fatty Acid Synthesis.

Chronic hepatitis C virus (HCV) infection often leads to end-stage liver disease, including hepatocellular carcinoma (HCC). We have previously observed reduced expression of microRNA-30e (miR-30e) in the liver tissues and sera of patients with HCV-associated HCC, although biological functions remain unknown. In this study, we demonstrated that HCV infection of hepatocytes transcriptionally reduces miR-30e expression by modulating CCAAT/enhancer binding protein ß. In silico prediction suggests that autophagy-related gene 5 (ATG5) is a direct target of miR-30e. ATG5 is involved in autophagy biogenesis, and HCV infection in hepatocytes induces autophagy. We showed the presence of ATG5 in the miR-30e-Argonaute 2 RNA-induced silencing complex. Overexpression of miR-30e in HCV-infected hepatocytes inhibits autophagy activation. Subsequent studies suggested that ATG5 knockdown in Huh7.5 cells results in the remarkable inhibition of sterol regulatory element binding protein (SREBP)-1c and fatty acid synthase (FASN) level. We also showed that overexpression of miR-30e decreased lipid synthesis-related protein SREBP-1c and FASN in hepatocytes. Conclusion: We show new mechanistic insights into the interactions between autophagy and lipid synthesis through inhibition of miR-30e in HCV-infected hepatocytes.

Depletion of PCAT-1 in head and neck cancer cells inhibits tumor growth and induces apoptosis by modulating c-Myc-AKT1-p38 MAPK signalling pathways.

BACKGROUND: Head and neck squamous cell carcinoma (HNSCC) represents one of the most common malignancies worldwide with a high mortality rate mainly due to lack of early detection markers, frequent association with metastasis and aggressive phenotype. Recently, long non-coding RNAs (lncRNAs) have been shown to have important regulatory roles in human cancers. The lncRNA prostate cancer-associated transcript 1 (PCAT-1) showed potential oncogenic roles in different cancers, however its role in HNSCC is not known. In this study, we evaluated the role of the PCAT-1 in HNSCC. METHODS: The expression of PCAT-1 was measured by quantitative real-time PCR in 23 paired human HNSCC tissues and adjacent non-tumor tissue specimens. Cell proliferation after depleting PCAT-1 was determined. Effect of PCAT-1 depletion in HNSCC cell lines was determined by qRT-PCR and Western blot analyses. Finally, JHU029 HNSCC cells was implanted subcutaneously into athymic nude mice and therapeutic potential of PCAT-1 was investigated. RESULTS: Up-regulation of PCAT-1 in TCGA dataset of HNSCC was noted. We also observed increased expression of PCAT-1 in archived HNSCC patient samples as compared to adjacent non-tumor tissues. Knockdown of PCAT-1 significantly reduced cell proliferation in HNSCC cell lines. Mechanistic study revealed significant down regulation of c-Myc and AKT1 gene in both RNA and protein levels upon knockdown of PCAT-1. We observed that c-Myc and AKT1 positively correlate with PCAT-1 expression in HNSCC. Further, we observed activation of p38 MAPK and apoptosis signal-regulating kinase 1 upon knockdown of PCAT-1 which induces Caspase 9 and PARP mediated apoptosis. Targeted inhibition of PCAT-1 regresses tumor growth in nude mice. CONCLUSION: Together our data demonstrated an important role of the PCAT-1 in HNSCC and might serve as a target for HNSCC therapy.

Pongapin and Karanjin, furanoflavanoids of Pongamia pinnata, induce G2/M arrest and apoptosis in cervical cancer cells by differential reactive oxygen species modulation, DNA damage, and nuclear factor kappa-light-chain-enhancer of activated B cell signaling.

In this study, the antitumor activity of two furanoflavanoid derivatives, Pongapin and Karanjin, was evaluated in comparison with Plumbagin, a plant-derived polyphenol with proven antitumor activity. The compounds differentially inhibit the growth of different cancer cell lines (most effective on HeLa cells), with very low inhibitory effect on the growth of normal mouse embryonic fibroblast cell line. Pongapin like Plumbagin could significantly increase the intracellular reactive oxygen species (ROS) in the HeLa cells by stabilization of nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor (I-κB) expression and reduction of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) expression. In contrast, Karanjin could decrease ROS level by inhibition of I-κB degradation resulting restriction of NF-κB nuclear translocation. Pongapin and Plumbagin significantly increased DNA damage-induced p53 expression and p21 nuclear expression. However, Karanjin treatment showed low DNA damage with increased p53 expression. The compounds induced G2/M arrest and increase in SubG1 population, indicating induction of apoptosis. Apoptosis was further validated by acridine orange/ethidium bromide dual staining and terminal deoxynucleotidyl transferase dUTP nick-end labeling assay in HeLa cells after treatment with the compounds. The compounds induced caspase-dependent apoptosis through induction of Bax/Bcl-2 ratio either through increased expression of Bax by Pongapin and Plumbagin or low expression of Bcl-2 by Karanjin. Thus, Pongapin and Karanjin may be potential natural anticancer agents in the future, like Plumbagin.

Association between MicroRNA-373 and Long Noncoding RNA NORAD in Hepatitis C Virus-Infected Hepatocytes Impairs Wee1 Expression for Growth Promotion.

Chronic hepatitis C virus (HCV) infection may lead to end-stage liver disease, including hepatocellular carcinoma (HCC). We have shown previously that microRNA-373 (miR-373) is upregulated in HCV-infected human liver biopsy specimens. To gain insight into the role of miR-373 in HCV-mediated pathogenesis, we investigated its interacting partner for hepatocyte growth regulation. Transcriptome sequencing (RNA-seq) data revealed that Wee1 is associated with miR-373 and is a direct target. Interestingly, higher expression of Wee1 was noted in HCV-infected hepatocytes than in uninfected hepatocytes, suggesting that other factors may block miR-373-mediated Wee1 inhibition. We subsequently found an association between the long noncoding RNA NORAD (LINC00657) and miR-373, and we demonstrated that NORAD binds to miR-373 and Wee1 independently. However, the high level of Wee1 expression in HCV-infected hepatocytes suggested that miR-373 forms a complex with NORAD. Depletion of miR-373 or the inhibitor Wee1 reduces the growth of Huh7.5 cells harboring the HCV genome as well as reducing Wee1 expression. Taken together, our data demonstrate a novel mechanism of hepatocyte growth promotion during HCV infection involving a miR-373-NORAD-Wee1 axis, which may be a target for future therapy against HCV-associated HCC.IMPORTANCE The mechanism of HCV-mediated liver pathogenesis is poorly understood. In this study, we observed that HCV infection upregulates miR-373 and Wee1, a pivotal player in the G2 checkpoint in the cell cycle, although Wee1 is a direct target for miR-373. Subsequent investigation demonstrated that miR-373 forms a complex with the long noncoding RNA NORAD, resulting in the release of their common target, Wee1, in HCV-infected cells, which, in turn, favors uncontrolled cell growth. Our study suggested a previously unknown mechanism for hepatocyte growth promotion following HCV infection, and this pathway can be targeted for future therapy against HCV-mediated liver pathogenesis.

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

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.