The extract was concentrated by dry vacuum, dissolved in methanol, and filtered to eliminate fats and hydrocarbons. (HCT 116) and pancreas (MIA PaCa). Apoptosis is induced by flavone A in better differentiated colon cancer CaCo-2 and pancreatic cancer Panc 28 cells via the intrinsic pathway by the inhibition of the activated forms of extracellular signal-regulated kinase (ERK) and pS6, and subsequent loss of phosphorylation of Bcl-2 associated death promoter (BAD) protein, while apoptosis is triggered by flavone B in poorly differentiated colon cancer HCT 116 and MIA PaCa pancreatic cancer cells through the extrinsic pathway with the concomitant upregulation of the phosphorylated forms of ERK and c-JUN at serine 73. These changes in protein levels ultimately lead to activation of apoptosis, without the involvement of AKT. Introduction The prevalence of cancer has steadily increased throughout the past decades [1]. While current therapies are effective at various levels, many of these treatments are also nonspecific with respect to their mechanism of action. This in turn increases the undesirable outcomes experienced by treated patients; harsh side effects and low rates of effectiveness are some of the many reasons why more specific treatments in oncology have been sought. Among these efforts, natural products[2] have been studied extensively in hopes of identifying new molecular entities with antineoplastic properties. Of these natural products, flavonoids, one class of polyphenolic compounds found in plants, have been shown to exert antineoplastic [3C9] properties, as well as antioxidant [10, 11], anti-inflammatory [12], antimicrobial [13] and antiviral [14, 15] activities. For the past five years our research has focused on plants from the Andean mountains largely known as and from which two active compounds were isolated, 5,7-dihydroxy 3,6,8-trimethoxy-2-phenyl-4H-chromen-4-one (5,7-dihydroxy-3,6,8-trimethoxyflavone or flavone A) [18], and 3,5-dihydroxy-6,7,8-trimethoxy-2- phenyl-4H-chromen-4-one (3,5-dihydroxy-6,7,8-trimethoxyflavone or flavone B) respectively [19]. It has been proposed in our previous work that these two flavone isomers may be relevant to the antineoplastic activities of these plants [20]. Indeed, flavones A and B demonstrated cytotoxic activity against cell lines derived from colon, pancreas, breast, and prostate cancers that have been categorized as being FGF6 highly tumorigenic, with the most promising results on cancers of the colon and pancreas. High levels of expression of aldehyde dehydrogenase (ALDH) is regarded as a very specific marker used in the detection of cancer-initiating cells as subpopulations in tumors, and has been demonstrated specifically in the cell lines studied [21C23]. Among these highly tumorigenic cell lines, the two flavone isomers show preferential antineoplastic activity on cells with dissimilar differentiation status. Flavone A induced apoptosis in the better differentiated cell lines, but not on the poorly differentiated cell lines, while flavone B was shown to be active against poorly differentiated, but not against the better differentiated cells. Moreover, these two flavone isomers do not induce apoptosis in normal cells and display significantly less apoptotic activity in less tumorigenic cell lines [20]. It is known that mitochondrial management of apoptosis can be controlled through the activity of survival factors, such as growth factors or cytokines, via extracellular receptors activating cascades of protein events that eventually lead to Caspase-3 cleavage. These pathways were probed Edoxaban (tosylate Monohydrate) by investigating the effects of the two flavone isomers on extracellular signal-regulated kinases (ERK), protein kinase B (AKT), S6 ribosomal Edoxaban (tosylate Monohydrate) protein (S6), and Bcl-2 associated death promoter (BAD). The preferential induction of apoptosis on highly tumorigenic cells with dissimilar differentiation status by flavone A and flavone B, two structurally similar compounds, suggest the activation of different cellular pathways by each compound. This study shows that flavone A exerts its cytotoxic effect on better differentiated cancer cells via an intrinsic apoptotic pathway whereas flavone B bypasses the mitochondrial pathway to induce apoptosis via an extrinsic pathway in poorly differentiated cancer cells. Materials and Methods Extraction, Purification, and Identification of Flavones The flavones were obtained as described before [20]. Briefly, flavone A was purified from 1.5 kg of dried flowers extracted with CHCl3. The extract was concentrated by dry vacuum, dissolved in methanol, and filtered to eliminate fats and hydrocarbons. It was then concentrated and dissolved in C6H6 followed by silica gel chromatography using C6H6:Me2CO (19:1) as eluent. From this, 50 mg of the flavonoid was purified from fractions 12 through 18 by crystallizations in hexane. The compound was identified by its physical and Edoxaban (tosylate Monohydrate) spectroscopic properties as 5,7 dihydroxy-3,6,8 trimethoxyflavone, mp 170 171C, 1H NMR (300MHz).