Synthesis and anticancer activity of new flavonoid analogs and inconsistencies in assays related to proliferation and viability measurements

Abstract

Flavonoids have been studied intensely for their ability to act as anti-carcinogenic, anti-inflammatory, anti-viral and anti-aging agents and are often marketed as supplements related to their anti-inflammatory activity. Previous studies have primarily focused on the effects of polar natural flavonoids. We examined the activity of novel hydrophobic and lipophilic flavonols against human DU-145 and PC-3 prostate cancer cell lines. All flavonol analogs were more active than the naturally occurring flavonols quercetin, kaempferol, kaempferide and galangin. The most potent analogs were 6.5-fold more active against DU-145 and PC-3 cells than quercetin and fell within the biologically relevant concentration range (low micromolar). We also evaluated the potential toxic effects of flavonol analogs on normal cells, an assessment that has frequently been ignored when studying the anticancer effects of flavonoids. During these analyses, we discovered that various metabolic and DNA staining assays were unreliable methods for assessing cell viability of flavonoids. Flavonoids reduce colorimetric dyes such as MTT and Alamar Blue in the absence of cells. We showed that flavonol-treated prostate cancer cells were stained less intensely with crystal violet than untreated cells at non-toxic concentrations. The trypan blue exclusion assay was selected as a reliable alternative for measuring cell viability.

Flavonol Regulation in Tumor Cells

“…Cautionary notes have appeared in the literature over the years warning of the difficulty of using such assays when dealing with redox reagents such as polyphenolic molecules. Forbes et al [] reviewed some of this earlier work and noted that, despite these observations, tetrazolium salt reduction assays had been used in more than a thousand studies on the action of flavonoids on cancer cells. The reduction of dye by flavonoids in the absence of cells is not restricted to tetrazolium salts and was also seen with Alomar Blue.…”

“…The reduction of dye by flavonoids in the absence of cells is not restricted to tetrazolium salts and was also seen with Alomar Blue. Forbes et al [] selected the trypan blue exclusion assay as a reliable alternative for measuring cell viability. There is a growing popularity of staining with sulforhodamine B as a technique for cytotoxicity screening [Keepers et al, ; Vichai and Kirtikara, ].…”

“…Antiproliferative activity against human leukemic cells was also increased when a flavonol was substituted with Br at the 4′ position [Burmistrova et al, ]. Forbes et al [] found that increasing the hydrophobicity and lipophilicity of a flavonol significantly lowered the effective concentration of several analogs (4′‐iodo, 3′‐phenyl, and 4′‐phenyl flavonol) into the desired low micromolar range thus enhancing their therapeutic potential.…”

Cytotoxicity of carnosine, taxifolin, linalool and their combinations with acids to the cells of human cervical carcinoma (HeLa)

“…This may have resulted from inconsistent reduction by flavonol of the metabolic compound between analyses with fluctuating variables, such as the amount of flavonols remaining in the well during incubation and the numbers of cells between analyses. Forbes et al (2014) find that flavonols used with the crystal violet assays significantly masks the effects on cell viability readings at lower concentrations, but at higher flavonol concentrations this interference appears to be minimal.…”

Synthesis, Biological Assessment, and Structure Activity Relationship Studies of New Flavanones Embodying Chromene Moieties

“…However, the quantitative structure-activity relationship (QSAR) for flavonoids as antibacterial agents has been capturing interest through the quantitative construction of associations between the molecular structures or properties with a variation in biological activities [18,33]. It is significant that anticancer activity, which is comprised of the most interesting pharmacological properties of flavonoids [34], proceeds via a unique mechanism, and aids in the prevention of cancer growth through the flavonoids' ability to function as anti-oxidants [35][36][37], enzyme inhibitors [38], and growth regulators [39]. Moreover, the biological performance of flavonoid molecules is reliant on their position and number of substitutions as well as their structures' condensation level, namely glucosides, homodimers, heterodimers, hydroxy groups, and isoprenyl units [40,41].…”