Anticancer Effects of 1,3-Dihydroxy-2-Methylanthraquinone and the Ethyl Acetate Fraction of Hedyotis Diffusa Willd against HepG2 Carcinoma Cells Mediated via Apoptosis

Abstract

Hedyotis Diffusa Willd, used in Traditional Chinese Medicine, is a treatment for various diseases including cancer, owing to its mild effectiveness and low toxicity. The aim of this study was to identify the main anticancer components in Hedyotis Diffusa Willd, and explore mechanisms underlying their activity. Hedyotis Diffusa Willd was extracted and fractionated using ethyl acetate to obtain the H-Ethyl acetate fraction, which showed higher anticancer activity than the other fractions obtained against HepG2 cells with sulforhodamine B assays. The active component of the H-Ethyl acetate fraction was identified to be 1,3-dihydroxy-2-methylanthraquinone (DMQ) with much high inhibitory rate up to 48.9 ± 3.3% and selectivity rate up to 9.4 ± 4.5 folds (p<0.01) at 125 μmol/L. HepG2 cells treated with the fraction and DMQ visualized morphologically using light and fluorescence microscopy. Annexin V—fluorescein isothiocyanate / propidium iodide staining flow cytometry, DNA ladder and cell cycle distribution assays. Mechanistic studies showed up-regulation of caspase-3, -8, and -9 proteases activities (p<0.001), indicating involvement of mitochondrial apoptotic and death receptor pathways. Further studies revealed that reactive oxygen species in DMQ and the fraction treated HepG2 cells increased (p<0.01) while mitochondrial membrane potential reduced significantly (p<0.001) compared to the control by flow cytometry assays. Western blot analysis showed that Bax, p53, Fas, FasL, p21 and cytoplasmic cytochrome C were up-regulated (p<0.01), while Bcl-2, mitochondrial cytochrome C, cyclin E and CDK 2 were down-regulated dose-dependently (p<0.01). The reverse transcriptase-polymerase chain reaction showed that mRNA expressions of p53 and Bax increased (p<0.001) while that of Bcl-2 decreased (p<0.001). Pre-treatment with caspase-8 inhibitor Z-IETD-FMK, or caspase-9 inhibitor Z-LEHD-FMK, attenuated the growth-inhibitory and apoptosis-inducing effects of DMQ and the fraction on HepG2 cells. These results suggested that DMQ and the H-Ethyl acetate fraction of Hedyotis Diffusa Willd showed potential anticancer effects. Furthermore, the mechanisms of action may involve mitochondrial apoptotic and death receptor pathways.

The Hedyotis diffusa Willd. (Rubiaceae): A Review on Phytochemistry, Pharmacology, Quality Control and Pharmacokinetics

“…Treatment with total flavones extract from H. diffusa could reverse the invasion of MHCC97-H cells in epithelial-mesenchymal transition induced by TGF-β1 at the dose of 200µg/mL, and the effect might be carried out by decreasing the level of E-cadherin protein and increasing the expression of vimentin protein [71]. Li et al found that both 1,3-Dihydroxy-2-Methylanthraquinone (79 and 157 µmol/L) and ethyl acetate extract (100 and 200 µg/mL) induced apoptosis on HepG2 cells, resulting in upregulation of Bax, p53, Fas, FasL, p21 and cytoplasmic cytochrome C levels and caspase-3, -8, -9 proteases activities, while downregulation of Bcl-2, mitochondrial cytochrome C, cyclin E and CDK 2 in a dose-dependent manner [72]. Nine compounds from H. diffusa, namely, ethyl 13 (S)-hydroxy-chlorophyllide a, 2-methyl-3-methoxy anthraquinone, 2-hydroxymethyl anthraquinone, 2-hydroxy-3-methyl anthraquinone, 2-hydroxymethy-1-hydroxy anthraquinone, 2-hydroxy-1-methoxy anthraquinone, 2-hydroxy-3-methyl-1-methoxy anthraquinone, oleanolic acid and ursolic acid, have been researched for their anti-liver cancer effect within the concentration range from 1 to 200 µM.…”

Hedyotis�diffusa Willd inhibits proliferation and induces apoptosis of 5‑FU resistant colorectal cancer cells by regulating the PI3K/AKT signaling pathway

“…Therefore, the use of TCM promising when treating cancer cell drug-resistance and may allow for improved treatment of cancer. Hedyotis diffusa Willd (HDW) is a major component of TCM with potent anti-cancer effects on various cancers, such as ovarian, hepatocellular and cervical cancer (18)(19)(20)(21). Previous studies have also demonstrated that HDW may inhibit CRC cell proliferation (22)(23)(24)(25)(26).…”

Green coffee bean seed and their role in antioxidant–A review

“…Treatment involves surgery, and chemotherapy is the main procedure following, but it has adverse side effects with low cure rate and high secondary failures. So the scientists researched effective anticancer drugs with less toxic effects (Li et al, 2016). Phytochemicals from plants used as an alternative drug for cancer treatment along with chemotherapy (Vanitha and ., 2017) Plants sources play a great role, and many plant-derived compounds like polyphenols, lavonoids, and terpenoids are of much nutritional and health bene its and immensely studied for their potent useful effects on humans (Pan MH et al, 2008).…”