Experimental and pan-cancer genome analyses reveal widespread contribution of acrylamide exposure to carcinogenesis in humans.

Humans are frequently exposed to acrylamide, a probable human carcinogen found in commonplace sources such as most heated starchy foods or tobacco smoke. Prior evidence has shown that acrylamide causes cancer in rodents, yet epidemiological studies conducted to date are limited and, thus far, have yielded inconclusive data on association of human cancers with acrylamide exposure. In this study, we experimentally identify a novel and unique mutational signature imprinted by acrylamide through the effects of its reactive metabolite glycidamide. We next show that the glycidamide mutational signature is found in a full one-third of approximately 1600 tumor genomes corresponding to 19 human tumor types from 14 organs. The highest enrichment of the glycidamide signature was observed in the cancers of the lung (88% of the interrogated tumors), liver (73%), kidney (>70%), bile duct (57%), cervix (50%), and, to a lesser extent, additional cancer types. Overall, our study reveals an unexpectedly extensive contribution of acrylamide-associated mutagenesis to human cancers.

PI3K/Akt Pathway and miR-21 are Involved in N-Ethyl-N-Nitrosourea-Induced F1 Mouse Lung Tumorigenesis: Effect of Inositol Hexaphosphate.

The risk of cancer development in offspring due to carcinogen exposure during pregnancy is a serious issue. In this study, we explored the involvement of the phosphatidylinositol 3-kinase (PI3K)/Akt pathway and microRNA-21 (miR-21) in transplacental lung tumorigenesis and its prevention by dietary compound inositol hexaphosphate (IP6) in F1 mice. Balb/c mice were exposed to the N-ethyl-N-nitrosourea (ENU) intraperitoneally on the 17th day of gestation. After weaning, half of the litters were fed with oral 2% IP6. At the end of 30, 120, or 240 days, we did not observe any effect on fetal viability or weight between ENU-exposed and non-exposed litters and the same was true of IP6. Altered expressions of the PI3K/Akt pathway were observed in F1 mice. Further, miR-21 expressions were found to be modulated at the respective time as well, along with the activation of matrix metalloproteinase (MMP-9) and vascular endothelial growth factor expression. Akt activation also enhanced the expression of cyclin D1, cyclooxygenase-2 (COX-2), nuclear factor-κB (NF-κBp50), and mammalian target of rapamycin (mTOR). IP6-fed F1 mice showed reduced tumorigenesis along with reduced expression of the PI3K/Akt pathway miR-21 and downstream targets. The PI3K/Akt pathway and miR-21 are involved in transplacental lung tumorigenesis, whereas IP6 seemed to affect lung tumorigenesis by suppressing the expression of the PI3K/Akt pathway in F1 mice.

Preventive effects of butyric acid, nicotinamide, calcium glucarate alone or in combination during the 7, 12-dimethylbenz (a) anthracene induced mouse skin tumorigenesis via modulation of K-Ras-PI3K-AKTpathway and associated micro RNAs.

Skin cancer is among the most common cancers worldwide and identifiable molecular changes for early and late stage of skin tumorigenesis can suggest the better targets for its control. In this study, we investigated the status of K-Ras-PI3K-AKTpathway followed by NF-κB, cyclin D1, MMP-9 and regulatory micro RNA during 7, 12-dimethylbenz[a]anthracene (DMBA) induced mouse skin tumorigenesis and its prevention by butyric acid (BA), nicotinamide (NA) and calcium glucarate (CAG), individually or in combination with respect to time. DMBA upregulated the K-Ras, PI3K, Akt, NF-κB, cyclin D1 and MMP-9, but downregulated the PTEN in a time dependent manner. DMBA also reduced the levels of micoRNA let-7a but induced the levels of miR-21 and miR-20a as a function of time. BA, NA and CAG were found to prevent DMBA induced changes, but they were most effective when used together in a combination. Reduced let-7a and miR-211 were correlated with the overexpression of K-Ras and MMP-9. Overexpression of miR-21 and miR-20a was correlated with the down regulation of PTEN and overexpression of Cyclin D1. Collectively, the enhanced chemopreventive potential of natural compound in combination via regulation of K-Ras-PI3K-AKTpathway along with regulatory micro RNAs provide a newer and effective mean for cancer management.

Combinatorial chemopreventive effect of butyric acid, nicotinamide and calcium glucarate against the 7,12-dimethylbenz(a)anthracene induced mouse skin tumorigenesis attained by enhancing the induction of intrinsic apoptotic events.

We explored the basis of the combinatorial chemopreventive effect of butyric acid (BA), nicotinamide (NA) and calcium glucarate (CAG) on mouse skin exposed to 7,12-dimethylbenz(a)anthracene (DMBA). We studied the effects of topical application of DMBA in the presence or absence of BA, NA and CAG on the regulators of apoptosis. DMBA treatment suppressed Bax, Bax/Bcl-2 ratio, release of cyt c, Apaf1, caspase-9, -3 mediated apoptosis. Downregulation of p21 and upregulation of Bcl-2, mut p53 were also observed in only DMBA treated mice. Simultaneous application of BA, NA and CAG induced a mitochondria-mediated apoptosis, characterized by a rise in the Bax, Bax/Bcl-2 ratio, release of cyt c, upregulation of Apaf1 with down-stream activation of caspase-9, -3. Furthermore treatment with BA, NA and CAG demonstrated an upregulation of p21 and downregulation of Bcl-2, mut p53. But this effect was enhanced in the presence of all the three compounds together in combination. Chemoprevention by a combination of BA, NA and CAG by inducing the apoptosis, the natural cell death, suggest the importance of the potential combinational strategies capable of preventing skin tumor development.

Involvement of EZH2, SUV39H1, G9a and associated molecules in pathogenesis of urethane induced mouse lung tumors: potential targets for cancer control.

In the present study, we showed the correlation of EZH2, SUV39H1 or G9a expression and histone modifications with the urethane induced mouse lung tumorigenesis in the presence or absence of antitumor agent, inositol hexaphosphate (IP6). Tumorigenesis and the molecular events involved therein were studied at 1, 4, 12 or 36 weeks after the exposure. There were no tumors at 1 or 4 weeks but tumors started appearing at 12 weeks and grew further till 36 weeks after urethane exposure. Among the molecular events, upregulation of EZH2 and SUV39H1 expressions appeared to be time dependent, but G9a expression was altered significantly only at later stages of 12 or 36 weeks. Alteration in miR-138 expression supports the upregulation of its target, EZH2. H3K9me2, H3K27me3 or H4K20me3 was found to be altered at 12 or 36 weeks. However, ChIP analysis of p16 and MLH1 promoters showed their binding with H3K9me2 and H3K27me3 which was maximum at 36 weeks. Thus, histone modification and their interactions with gene promoter resulted in the reduced expression of p16 and MLH1. IP6 prevented the incidence and the size of urethane induced lung tumors. IP6 also prevented the urethane induced alterations in EZH2, SUV39H1, G9a expressions and histone modifications. Our results suggest that the alterations in the histone modification pathways involving EZH2 and SUV39H1 expressions are among the early events in urethane induced mouse lung tumorigenesis and could be exploited for cancer control.

Involvement of epigenetics and microRNA-29b in the urethane induced inception and establishment of mouse lung tumors.

Epigenetic changes are correlated with tumor development showing aberrations in DNA methylation and histone modifications. To find the early changes, we evaluated the epigenetic events from early to late stage of the urethane induced lung tumor development in mouse model and tried to correlate the molecular events with the progression of tumor. We addressed the hypothesis by examining the tumor development, status of DNMTs, HDACs and MBDs, DNA methylation and expression of microRNA-29b during 1 to 36 weeks after urethane exposure that included the period before and after the tumor appearance. Tumors did not appear after 1 or 4 weeks but well defined tumors appeared after 12 weeks and larger tumors appeared at 36 weeks which was prevented by IP6. DNMT1, DNMT3a and DNMT3b were upregulated after urethane exposure at the time of no tumor till the tumor developed and showed its upregulated functional activity. DNMTs are shown to be the targets of microRNA-29b and we showed that microRNA-29b was downregulated in the line of DNMT upregulation. HDAC, the histone modifier, also showed progressive upregulation. Periodic increase in methyl binding proteins, MBD2, supported the expression of gene silencing pathways in terms of the downregulation of tumor suppressor genes, p16 and MLH1. All these molecular alterations were protected in the presence of IP6. Our results showed that the key steps of epigenetics, DNMTs, mir29b, and HDAC1, are altered both before and after the development of tumors.

Involvement of STAT3, NF-κB and associated downstream molecules before and after the onset of urethane induced lung tumors in mouse.

Here we have shown the alteration of transcription factors STAT3, NF-κB and downstream associated molecules much before the appearance of lung tumor and their response to antitumor agent, inositol hexaphosphate. Histological examination revealed the pathophysiology of the lung tissues and the onset or progression of tumor from 4 or 9 to 24 weeks in terms of tumor volume and the number. Over expression of NF-κB (p50/Rel A), COX-2, STAT3, pSTAT3 (Tyr 705), IL-6 and cyclin D1 also progressed from the time of no tumor to the time of tumor appearance and was reduced in mice drinking 2%IP6. We suggest that the alterations of STAT3, NF-κB and downstream associated molecules are critical in the development of lung tumors and can be exploited as possible mechanisms after the exposure. Status of these altered genes before the tumor development suggests their possible use as targets for the tumor control in the predisposed conditions.

Epigenetics, an early event in the modulation of gene expression by inositol hexaphosphate in ethylnitrosourea exposed mouse lungs.

Mechanisms of anticancer effects of inositol hexaphosphate are not fully understood. Epigenetic changes are the early changes in tumorigenesis. DNA methyl transferases, methyl CpG binding proteins, methyl CpG DNA binding domain protein, and histone deacetylases are the major molecules involved in epigenetics. We have shown the effects of IP6 at the molecular level in mouse lungs before the tumor is developed. After 3 mo of ENU exposure, there was no tumor formation, but there was hyperplasia and lymphocytic infiltration in the lungs. Inflammation and DNA damage repair enzymes COX-2 and MLH1 appear to be upregulated, whereas tumor suppressor gene p16 was downregulated by ENU. On the other hand, ENU exposure more or less upregulated the epigenetic events such as the expressions of DNMT1, MeCP2, MBD1, and HDAC1. This alteration was reduced by IP6 administration. Results were supported by modulation of global DNA methylation and the modulation of promoter CpG methylation of p16, MLH1, and COX-2 genes. Hence, this study indicates the possible role of epigenetics at the early stage of tumor development and in the regulation of gene expression by IP6 before the onset of ENU-induced lung tumors.