The Structure-activity Relationship And ROS Related Mechanism Of Anti-cancer Effect Of Flavonoids

Flavonoids are a large class of polyphenolic compounds, which are ubiquitously present in the plant world and our common diet, such as vegetables, fruits and plant-derived beverages. Epidemiological investigations and laboratory studies have indicated several beneficial biological activities of flavonoids, including anti-oxidant, anti-inflammatory, anti-cancer and anti-estrogenic properties. Among these pharmacological properties, there has been an increasing scientific interest in the anti-cancer activity of flavonoids due to their potent inhibition effect on the carcinogenesis, proliferation, migration, invasion, angiogenesis and drug resistance of cancer.The pharmacological activities of flavonoids depend on their molecular structure; flavonoids with different molecular structure may have different activity. Some structure substituent groups or features may be critical or essential for their certain pharmacological activity, these structure elements are called key structure-activity elements. To analyze the relation between the molecular structure and activity, elucidating the key structure-activity elements, should be useful for revealing the mechanism of certain pharmacological effect of flavonoids, and for guiding the synthesis of potent anti-cancer compounds from flavonoids for potential clinical application. The anti-cancer activity of flavonoids has been an interesting topic around the world, yet most of the studies lay emphasis on some certain flavonoids, the difference and comparison of the anti-cancer activities of flavonoids are neglected. Furthermore, there is a lack in study of the structure-activity relationship for anti-cancer activity of flavonoids, and the key structure-activity elements are still unclear, therefore it is worthwhile to investigate the related topic.Flavonoids, the dietary phenolics, have been well known as effective anti-oxidant, yet in fact, flavonoids can be both anti-oxidative and pro-oxidative. Studies suggested that under some certain conditions (such as in the presence of O2, transition metals copper and iron), upon oxidation by peroxidase, flavonoids formed phenoxyl radicals and redox cycling of phenolics, which catalyzed GSH or NADH co-oxidation and generated ROS. The beneficial properties of flavonoids are generally believed to reflect their ability to scavenge endogenous ROS. However, the pro-oxidant action of plant-derived phenolics rather than their anti-oxidant action may be an important mechanism for their anti-cancer and apoptosis inducing properties, as ROS can mediate apoptotic DNA fragmentation. Apoptotic DNA fragmentation properties of several anti-cancer drugs, such as arsenic trioxide and cisplatin, are considered to be mediated by ROS.In normal condition, the production and scavenging of ROS in cells is a dynamic balance, cells maintain a specific redox homeostasis, which plays an important role in the life cycle of cells. ROS and GSH are the key factors influence the redox homeostasis. It has been known that slight elevation of ROS promotes cells proliferation, more high level of ROS induces apoptosis, excessive amount of ROS results in necrosis. Studies indicate that the activities of antioxidases such as SOD and CAT of cancer cells are much lower than that of normal cells, so the ability of scavenging endogenous ROS and producing GSH of cancer cells are much lower. Furthermore, the generation of ROS in cancer cells is higher. Therefore, there is severe oxidation stress in cancer cells; cancer cells are sensitive to ROS, which have selectively cytotoxicities on them. Development of anti-cancer drugs with apoptosis-inducing properties mediated by ROS has currently become a very important topic, and some certain properties of dietary phenolic compounds, such as binding and cleavage of DNA and the generation of ROS in the presence of transition metal ions, are similar to those of known anti-cancer drugs. Therefore, we could raise the hypothesis that flavonoids could induce ROS generation by pro-oxidation in cancer cells, especially in the key points such as lipid and mitochondria membrane, change the redox homeostasis and eventually induce apoptosis, it may be the important mechanism of the anti-cancer activity of flavonoids.Based on the analysis mentioned above, we used a library of 23 different flavonoids and evaluated cytotoxicity of them in human leukemia HL-60 cells, breast cancer MCF-7 cells, prostatic cancer PC3 cells et al. Structure-activity relationship and the key structure-activity elements were analyzed. Furthermore, we selected out 3 flavonoids with potent cytotoxicity and HL-60 cells to investigate the effect of these flavonoids on the ROS level, GSH level and redox potential of HL-60 cells, by flow cytometric analysis, laser confocal scanning microscopy and fluorescence recovery after photobleaching. We also investigated the effect of flavonoids on the membrane phospholipids mobility and mitochondria membrane potential of HL-60 cells, and observed the influence of NAC pre-treatment on the cytotoxicity and apoptosis induction activity.The main results and conclusions were summarized as follows:1. Cells viability assay indicated that most of the 23 flavonoids tested in this study showed significant inhibitory effect on cancer cells proliferation and the effect were enhanced along with increasing concentration. However, the intensity of their effects was different, and HL-60 cells were most sensitive to the cytotoxicity of flavonoids. 3,6-Dihydroxyflavone and 2′-hydroxyflavanone exhibited the most potent cytotoxic effect on all six cells. Flavonoids such as naringenin and hesperetin showed no significant cytotoxic effects on these cancer cells, gossypin and morin promoted some cancer cells viability significantly. The effects of flavonoids on the proliferation of cancer cells were arranged from strong to weak as follow based on IC50: 3,6-dihydroxyflavone > 2'-hydroxyflavanone > fisetin > 3,7-dihydroxyflavone > luteolin > apigenin > quercetin > 6-hydroxyflavone > 7-hydroxyflavone > 4'-hydroxyflavanone > galangin > geraldol > baicalein > daidzein > chrysin > myricetin > genistein > flavone > flavanone > naringenin > hesperetin > morin > gossypin.2. In order to confirm the potent cytotoxic effect of 3,6-dihydroxyflavone on cancer cells, we examined the morphological changes of cancer cells after the treatment of 3,6-dihydroxyflavone. Treated with 10μΜ3,6-dihydroxyflavone for 24h, dramatic pro-apoptotic morphological changes in comparison with the control were observed in all these six cancer cells, including cell shrinkage and cytoplasmic condensation. Cells retracted from their neighboring cells, rounded up and eventually floated into the media, which is indicative of apoptosis as well. Morphological changes in cell nuclei were determined by fluorescence microscopy after DAPI staining. After 3,6-dihydroxyflavone treatment, the stained nuclei with apparent apoptotic characteristic of condensed nuclei or nuclear fragmentation increased. The apoptosis analysis by flow cytometric assay confirmed the pro-apoptotic effects of 3,6-dihydroxyflavone in all six cancer cells.3. Structure-activity relationship analysis indicated that some structural properties associated with enhanced cytotoxicity, including appropriate hydroxyl numbers (2～4), the presence of the 2,3-double bond in ring C, ring B attached at position 2, 3-OH, 6-OH and ortho-hydroxylation in ring B, these structural elements are key structure-activity elements for anti-cancer activity of flavonoids. On the contrary, redundant or insufficient hydroxyl numbers, the absence of the 2,3-double bond in ring C, ring B attached at position 3 (iso-flavone structure), 5-OH, and mate-hydroxylation in ring B associated with decreased cytotoxicity.4. The detection of ROS and apoptosis analysis indicated that the ROS level of HL-60 cells was distinctly higher than that of the normal human lymphocytes, 3,6-dihydroxyflavone, luteolin and geraldol could induce ROS generation and apoptosis. The flavonoids with no significant effect on HL-60 cells viability, hesperetin and naringenin, showed no significant effect on ROS level and apoptosis. Further studies suggested that 3,6-dihydroxyflavone, luteolin and geraldol significantly reduced the GSH level and increased the GSSG level, inducing the elevation of redox potential. Results also indicated that these 3 flavonoids sigcificantly decreased the membrane phospholipids mobility and mitochondria membrane potential of HL-60 cells5. The addition of NAC completely prevented the generation of intracellular ROS, the decreasing of GSH, the increasing of redox potential, and the decreasing of the membrane phospholipids mobility and mitochondria membrane potential, partially suppressed apoptosis and cell viability reduction induced by 3,6-dihydroxyflavone, luteolin and geraldol. These finding suggested that ROS accumulation and the increasing of redox potential play an important role in pro-apoptosis effect of 3,6-dihydroxyflavone, luteolin and geraldol on HL-60 cells, apoptosis induction mediated by ROS was an critical mechanism of anti-cancer effect of flavonoids.In all, our study evaluated the cytotoxicity of a library of 23 different flavonoids on six different cancer cells, the findings revealed the potent anti-cancer activity of 3,6-dihydroxylflavone, and the structure-activity relationship analysis elucidated the key structure-activity elements, including appropriate hydroxyl numbers (2～4), the presence of the 2,3-double bond in ring C, ring B attached at position 2, 3-OH, 6-OH and ortho-hydroxylation in ring B. The ROS related mechanism of anti-cancer effects indicated that 3,6-dihydroxyflavone, luteolin and geraldol could significantly elevated the ROS level and redox potential, reduced the GSH level, membrane phospholipids mobility and mitochondria membrane potential, inducing apoptosis in HL-60 cells. These findings indicated that apoptosis induction by elevation of ROS and redox potential plays an important role in anti-cancer effect of flavonoids.