Hepatocarcinogenesis is a complicated process including several steps, with a plenty of signals, leading to a diversified molecular profile.26
DEN generates ROS which result in DNA damage, mutations and cancer.27 Appling of DEN is accompanied by over expression of inflammatory cytokines such as TNF which is the rate limiting step of carcinogenesis cascade. The oxidative stress produced upon DEN application can activate NOX1 (NADPH oxidase) axis which subsequently activate Kupffer cells and newly recruited macrophages to express TNF in abnormal concentration to start the inflammation and carcinogenesis.28
In the present study the Ch compound exerts an ability to inhibit TNF-α molecules. Two docking studies were carried out to deduce that chromen-4-one nucleus has special behavior to inhibit TNF-α. They mentioned that the TNF-α molecule has pharmacophoric features can act as hydrogen bond acceptors as the chromen-4-one has aromatic rings and (C = O) in aromatic ring which can donate electrons to the residues Leu 120 and Gly 121 in the TNF-α in addition the two aromatic rings can donate electrons to leu 57 and Gly 122 and Ile 58.29–30
The data of the present study revealed that the level of TNF- α was increased in the DEN induced HCC but decreased upon Chromene treatment our finding was supported by Afzal et al. 31 who mentioned that, once the expression of TNF- α increased, the cascade of inflammation starts to activate tumor promotion, angiogenesis, proliferation and survival via activation of NF-kB.
The angiogenesis is initiated by up regulation of VEGF as a step next to NF-kB activation 31 that, VEGF is known as an angiogenic agent which has a motivated facility for angiogenesis and growth of tumors and metastasis in the conditions like HCC.32
Saleem et al.33 observed that levels of VEGF in the DEN group were significantly elevated implying advanced pace of angiogenesis which supports our results of increasing VEGF level in the group treated with DEN.
Also, the activation of NF-kB initiates over expression of other tumor promotors such as MMP-9 and COX-2 .34
In the present study MMP-9 level is increased in the group treated with DEN while the treatment with Chromene derivative decrease the MMP-9 level.
Among them, MMP-2 (72 kDa) and MMP-9 (92 kDa) are found to regulate tumor invasion and metastasis. Researches in malignant tumors reported the overexpression and enhanced activity of both of these MMPs .35 Enhanced metastasis is evidently linked with enhanced MMP-2 and MMP-9 expressions as a tumor progressor. On the other hand, COX-2 enzyme enhances tumor through over production of MDA as a biproduct of COX-2 catalyzed breaking downof PGH 2 (prostaglandin H2).36
Malondialdehyde is considered as a mutagen in mammalian cells and carcinogen for rats 37 due to its affinity toward DNA to form adducts with deoxyguanosine to yield pyrimidopurinone – deoxyguanosine both MDA carbonyls react with nitrogen, forming the pyrimido [1,2-α]purine-10(3H)-one-2'-deoxyribose, or malondialdehyde-2'-deoxyguanosine adduct (M1dG) 38 and N6-(3-oxoprenyl) deoxyadenosine (M1dA) and N4-(3-oxoprenyl) deoxycytidine (M1dC).39 Although, formation of DNA adducts is a fateful step in cancer development, that it can be exerted by another pathway in addition to the effect of MDA. That, DEN itself is not a carcinogen but it exerts its carcinogenic effect after metabolism in hepatic cytochrome P450 (CYP) enzymes to yield alkylated guanine 40 while, Kang et al.41 revealed that, the CYP deficient mice show less tumor incidence in comparison with the wild type. Also, as the CYP enzymes decrease, the bioactivation of DEN is declined leading to less availability of formation of DNA adduct and less tumorigenesis. 42 As a normal defense response, the hepatic cells start to express the protein p53 to obligate the mutated cells for entrance the apoptotic pathway .15 The mode of action of p53 involves stimulation of reactive oxygen species so that the over production of p53 leads to more inflammatory response, accumulation of ROS and initiation of tumorigenesis in the surrounding environment as mentioned by43 who recorded the accumulation of p53 in wild type rates treated with DEN with histopathological evidence of malignant hepatic tumors while less p53 filtration in hepatic p53+/− rats with lower risk of tumor formation.
Chiu et al. 44 studied two groups of humans with HCC, the first group is negative immune staining of p53 and the second group is positive immune staining p53. They found an increase in the level of Bax in both groups but the expression of Bcl2 decreased in the first group and increased in the second group with more advanced histopathological of tumor grade. They explain their results by finding out the ratio of anti-apoptotic and pro apoptotic proteins Bcl2 and Bax where, this ratio decreased in the first group immune negative p53 leading to apoptotic fate of the tumor cells but increased in the second group immune positive p53 to exceeds the percentage 88% with respect to controls that means that the tumor cells have more tendency to an anti-apoptotic behavior in group 2.
It has been demonstrated extensively that translocation of Bax from cytosol to mitochondria facilitates the release of the cytochrome c45 because Bax can form channels, which allow direct cytochrome c release.46
The Bcl2 itself binds to pro apoptotic members such as Bax, preventing pore formation and cytochrome c release .47 In contrast, increase in expression of Bax, induces cell death eliminating tumor cells.48
It has been suggested that a high ratio of Bax to Bcl2 can lead to collapse of mitochondrial membrane potential, resulting in release of cytochrome c and consequently causes cell apoptosis .49 Our data also confirm that decreased Bcl2 protein expression, its inhibitory effect on Bax and caspase-9 was removed and leads to over expression of Bax and finally activation of caspase-9. Although, activation of caspase-9 also leads to loss of mitochondrial membrane potential by cleaving anti apoptotic members of Bcl2 family including Bcl-xL and Bcl2.50
However, it has been recently reported that ethyl 2-amino-6-bromo-4-(1-cyano-2-ethoxy-2-oxoethyl)-4H-Chromene-3-carboxylate (HA14-1) binds to Bcl2 protein and blocks its anti-apoptotic function in HL-60 cells.51
Our results revealed that increase in the Bcl2 in the DEN treated group accompanied by drop in the level of the Bax leading to anti-apoptotic behavior appears in pathological observation while the treated group with DEN + Ch shows rebalanced Bcl2 and Bax levels near the control group.
The previous events are concerned with the cell proliferation which appeared as elevation in alpha fetoprotein (AFP) to develop the proliferation in HCC .52 This finding supports our data where the treatment with DEN leads to elevation of AFP parallel to over expression of p53. In addition, after the inflammatory event and DNA disturbance affected by DEN administration, the inflammatory cascade reaching HCC, the hepatic function is disturbed as shown in elevation of liver function enzymes ALT and AST 43 as well as, increase in the expression of CRP in the HCC induced group because, hepatocyte is the main origin of the CRP and its synthesis is increased due to inflammatory stimulation .53–54
The administration of Ch derivative treats with the inflammatory event by deactivation of NF-кB which in turn controls the cascade of inflammation this finding is supported by .4