Baicalein 5, 6, 7-trimethyl ether (BTE) was efficiently isolated from Physalis Pubescens L. leaves:
In our study we isolated BTE from Physalis Pubescens L. leaves using column chromatography technique (Fig. 1). Cytotoxicity of BTE alone or its combination with epirubicin was evaluated against HebG2 hepatocellular carcinoma cells to detect if the use of BTE as adjuvant therapy could help in augmenting the cytotoxic effect of epirubicin and reducing its side effects.
Baicalein trimethyl ether isolated from Physalis pubescens L. was the flavonoid with the most cytotoxic effect against HepG2 liver cancer cells:
The percent of viability of HepG2 cancer cells after incubation with different concentration of our treatments for 48 hr were determined using MTT assay (Fig. 2). Figure (3C) demonstrated the 50% inhibitory concentration (IC50) of different treatments on HepG2 cancer cells. It was figured out that IC50 values on HepG2 cancer cells were 2.79 ± 0.626 µg/mL (epirubicin), 95.6 ± 5.849 µg/mL (BTE), 219.36 ± 9.667 µg/mL (rutin), 111.73 ± 7.445 µg/mL (quercitrin), 241 ± 10.908 µg/mL (myricitrin). The flavonoid with the most cytotoxic effect against HepG2 hepatocarcinoma was BTE, so combinations between BTE and epirubicin were carried out to assess if BTE could increase cytotoxicity of epirubicin against HepG2 and reduce its adverse effects. The first combination was BTE (95.6 µg/mL) + 0.4, 0.8, 1.2, 1.6, 2, 2.4 and 2.8 µg/mL epirubicin (EP). IC50 was more than 2.8 µg/mL (more than the IC50 of epirubicin alone) (Fig. 2C), so the second combination was prepared as BTE (200 µg/mL) + 0.4, 0.8, 1.2, 1.6, 2, 2.4 and 2.8 µg/mL epirubicin. IC50 was 0.76 ± 0.258 µg/mL of epirubicin combined with (200 µg/mL) BTE (Fig. 2D).
Baicalein trimethyl ether (200µg/mL) augmented the cytotoxic effect of epirubicin against HebG2 liver cancer cells and increased its safety profile:
Our data from MTT viability assay showed that, the combination of epirubicin with 200µg/mL BTE significantly decreased the IC50 of epirubicin against HebG2 cells from 2.79 ± 0.626 µg/mL (Fig. 2B) to 0.76 ± 0.258 µg/mL (Fig. 2D) (p = 0.040).
On the other hand, IC50 of BTE on WI-38 normal cells was 243.4 ± 11.667 µg/ mL (Fig. 3A). Moreover, IC50 of BTE in the combination of BTE 100, 200, 300, 400, 500 µg/mL and 600 + epirubicin 0.76 µg/mL on WI-38 normal cells was 570.8 ± 13.72 µg/ mL (Fig. 3B). More interestingly, combination of epirubicin (0.76 µg/mL) with BTE significantly increased the 50% inhibitory concentration (IC50) of BTE against WI-38 normal cells from 243.4 ± 11.667 µg/ mL to 570.8 ± 13.72 µg/ mL (p < 0.0001) increasing the safety profile of the combination (Fig. 3D).
Baicalein trimethyl ether and its combination with epirubicin increased DNA fragmentation and induced apoptosis in HebG2 liver cancer cells:
To determent the effect of BTE or its combination with epirubicin on DNA fragmentation of liver cancer cells, DNA fragmentation of HebG2 cells treated with epirubicin (0.76µg/mL) or BTE (95.6µg/mL) or epirubicin (0.76µg/mL) + BTE (95.6µg/mL) or epirubicin (0.76µg/mL) + BTE (200µg/mL) was analyzed by electrophoresis. Interestingly, DNA fragmentation was observed in all the treated groups compared to untreated HebG2 control cells. The present data strongly suggest the induction of apoptosis in liver cancer HebG2 cells as the possible antiproliferative mechanism of our different treatments (Fig. 4).
Baicalein trimethyl ether and its combination with epirubicin decreased the gene expression of TGF-β1 in HebG2 liver cancer cells:
RT-qPCR was used to evaluate the effect of epirubicin (0.76 µg/mL), BTE (200 µg/mL), and their combination on the gene expression of TGF-β1 (as marker of apoptosis) in HebG2 cells. Epirubicin, BTE and their combination significantly downregulated the gene expression of TGF-β1 (0.523, 0.421, and 0.457 fold respectively) compared to the control untreated HebG2 cells (1 fold) (p = 000) (Fig. 5A).
Baicalein trimethyl ether and its combination with epirubicin increased the gene expression of ATG7 in HebG2 liver cancer cells
In the current study RT-qPCR was used to evaluate the effect of epirubicin (0.76 µg/mL), BTE (200 µg/mL), and their combination on the gene expression of ATG7 (as marker of autophagy in HebG2 cells. Epirubicin, BTE and their combination significantly upregulated the gene expression of ATG7 (8.281, 10.773 and 13.653 fold respectively) compared to the control untreated HebG2 cells (1 fold) (p = 0.000). More interestingly, the gene expression of ATG7 was significantly higher in HebG2 cells treated with the combination of BTE and epirubicin than HebG2 cells treated with BTE or epirubicin alone (p = 0.001 and 0.03 respectively) (Fig. 5B).