Autophagy Inhibition Contributes to the Synergistic Interaction between EGCG and Doxorubicin to Kill the Hepatoma Hep3B Cells

Abstract

(-)-Epigallocatechin-3-O-gallate(EGCG), the highest catechins from green tea, has promisingly been found to sensitize the efficacy of several chemotherapy agents like doxorubicin (DOX) in hepatocellular carcinoma (HCC) treatment. However, the detailed mechanisms by which EGCG augments the chemotherapeutic efficacy remain unclear. Herein, this study was designed to determine the synergistic impacts of EGCG and DOX on hepatoma cells and particularly to reveal whether the autophagic flux is involved in this combination strategy for the HCC. Electron microscopy and fluorescent microscopy confirmed that DOX significantly increased autophagic vesicles in hepatoma Hep3B cells. Western blot and trypan blue assay showed that the increasing autophagy flux by DOX impaired about 45% of DOX-induced cell death in these cells. Conversely, both qRT-PCR and western blotting showed that EGCG played dose-dependently inhibitory role in autophagy signaling, and that markedly promoted cellular growth inhibition. Amazingly, the combined treatment caused a synergistic effect with 40 to 60% increment on cell death and about 45% augmentation on apoptosis versus monotherapy pattern. The DOX-induced autophagy was abolished by this combination therapy. Rapamycin, an autophagic agonist, substantially impaired the anticancer effect of either DOX or combination with EGCG treatment. On the other hand, using small interference RNA targeting chloroquine autophagy-related gene Atg5 and beclin1 to inhibit autophagy signal, hepatoma cell death was dramatically enhanced. Furthermore, in the established subcutaneous Hep3B cells xenograft tumor model, about 25% reduction in tumor growth as well as 50% increment of apoptotic cells were found in combination therapy compared with DOX alone. In addition, immunohistochemistry analysis indicated that the suppressed tendency of autophagic hallmark microtubule-associated protein light chain 3 (LC3) expressions was consistent with thus combined usage in vitro. Taken together, the current study suggested that EGCG emerges as a chemotherapeutic augmenter and synergistically enhances DOX anticancer effects involving autophagy inhibition in HCC.

SOX2OT variant 7 contributes to the synergistic interaction between EGCG and Doxorubicin to kill osteosarcoma via autophagy and stemness inhibition

“…As expected, according to the MTT assay and the coefficient of drug interaction (CDI, shown in Table 2), EGCG and DOX yielded synergistic interactions (CDI<1). As reported previously, autophagy contributed to the synergistic antitumor effects of EGCG and DOX [3], autophagy levels were detected in various ways. Firstly, immunofluorescence staining was performed to detect the LC3 translocation in U2OS cells.…”

“…According to previous study [3], MTT assay was used to detect the absorbance at the wavelength of 570 nm as the OD value. The coefficient of drug interaction (CDI) was calculated as follows: CDI = AB/(A × B).…”

“…Therefore, developing the combination regimen with potential complementary mechanisms, especially targeting CSCs, may be a promising avenue of drug toxicity reduction and efficacy improvement.Epigallocatechin gallate (EGCG) is the highest content of catechin in green tea with many physiological and pharmacological activities. It was found that EGCG could promote the sensitivity of traditional anticancer drugs [3,6,7] and reverse multidrug resistance [8]. Similarly, EGCG was reported to exert significant inhibitory effect on osteosarcoma cells including induce apoptosis, inhibit the proliferation and invasion of osteosarcoma cells [9][10][11].…”

“…Doxorubicin (Dox) is one of the most commonly used chemotherapeutic drugs for osteosarcoma [2]. However, the intrinsic weakness of DOX severely limits its clinical efficacy: low-dose usage could not only reduce its effectiveness but also lead to drug resistance, while dose increasement would cause severe cardiotoxicity [3]. Therefore, the overall survival rate of osteosarcoma patients is only 5 to 20% which is not satisfactory [4].…”

“…Human SOX2 over lapping transcript (SOX2OT) gene can generate 8 lncRNA transcript variants (variant [1][2][3][4][5][6][7][8] which are functionally assumed to be correlated with cellular differentiation and carcinogenesis [12]. These variants show diverse expression profiles in different cell or tissue types [12].…”