The Antitumor Mechanism of the Polyphenol-enriched Ethyl Acetate Fraction Extract of Glechoma hederacea (Lamiaceae) against HepG2 Cells Involves Apoptosis Pathways


 Background: Glechoma hederacea (Lamiaceae) is a traditional Chinese herb belonging to the Labiatae family and has many biological activities. This study aimed to investigate the in vitro effects of different polar parts of the water extracts of G. hederacea (Lamiaceae) on HepG2 cells and the possible underlying mechanism.Methods: The cytotoxic effects were determined using the MTT assay. Apoptosis was quantified using annexin V-FITC/PI staining. Caspase-3 and caspase-9 activities were measured using PhiphiLux-G1D2 kit. Apoptosis-related protein expression was determined using western blot analysis. Reactive oxygen species (ROS) generation, mitochondrial transmembrane potential (MMP), and calcium ion levels were measured using specific fluorescence probes and flow cytometry. The HepG2 cell antioxidant state was determined using DAPI and comet assay, and antioxidant enzyme activities. The polyphenol contents of different polar parts of fractional extracts were determined using HPLC. Results: Our results showed that rosmarinic acid, caffeic acid, and ferulic acid were the major polyphenolic components in the ethyl acetate fraction extract (EAFE) of G. hederacea L.. EAFE could significantly inhibit HepG2 cell proliferation, induce apoptosis, and cause S phase arrest. The apoptogenic activity of EAFE involved ROS induction, Ca2+ accumulation, glutathione depletion, MMP disruption, caspases 3, 9 activation, and Bax/Bcl-2 ratio increment.Conclusion: Our findings suggest that the polyphenols in the EAFE can inhibit HepG2 cell growth and induce apoptosis through the mitochondria-mediated pathway and ROS-mediated endoplasmic reticulum stress. EAFE could be developed as a functional food or nutraceutical ingredient for chemotherapy.


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Background 1 Hepatocellular carcinoma is the fifth most common cancer type and the third leading 2 cause of cancer-related deaths worldwide, accounting for 75% to 85% of all primary 3 liver cancer cases. The major cause of carcinogenesis such as hepatocarcinogenesis is 4 a complex process that involves cell injury, inflammation, proliferation, and genomic 5 instability, leading to alterations in several oncogenic pathways [1]. The HepG2 cell 6 line was derived from the liver hepatocellular carcinoma of a Caucasian male. HepG2 7 cells retain many biological characteristics of primary hepatocytes, and protein 8 degradation is important for cellular processes such as cell cycle, antigen processing, 9 apoptosis, and lipid metabolism in the liver [2,3]. Effects of the crude extract and the three solvent fractions of HWG on the 10 proliferation of HepG2 cells were determined using the MTT assay. HepG2 cells were 11 treated with varying concentrations (50-400 g/mL) of different partition layers, i.e., 12 EAFE, NBFE, and WFE, for 24 h (Fig. 1). The results indicated that HWG, EAFE, 13 and WFE decreased the percentage of viable cells in a concentration-dependent 14 manner. Among them, EAFE was the most effective. Thus, we measured the selective 15 anticancer activity of the partition layer EAFE for further studies. 16

EAFE induced S-phase arrest and apoptosis in HepG2 cells 18
The cytotoxicity results are shown in Fig. 2A. EAFE dose-dependently inhibited the 19 proliferation in HepG2 cells. The 50% inhibitory concentration (IC50) of EAFE was 20 350 μg/mL ( Fig. 2A). 21 Cell cycle phase G0/G1 is the cells at the resting phase, S is the synthesis phase 22 where DNA replication starts, and G2/M is where cells prepare to enter mitotic 23 division. Cancer cells show uncontrolled proliferation and have the potential to resist 24 apoptotic death, both of which are beneficial for cancer progression. Therefore, the 25 inhibition of cell cycle progression and activation of apoptotic pathways are valuable 26 therapeutic targets [21]. To determine the effect of EAFE on cell cycle arrest in 1 HepG2 cells, we investigated the cell cycle distribution by using flow cytometry. The 2 results revealed that EAFE significantly disrupted cell cycle progression and induced 3 cell cycle arrest at S phase in HepG2 cells in a time-dependent manner (Fig. 2B). 4 Analysis of the phase of apoptosis that was mainly induced by EAFE revealed that the 5 number of cells in late apoptosis had mainly increased (Fig. 2C). 6 7 EAFE induced apoptosis and DNA damage in HepG2 cells: Analysis using DAPI 8 staining and comet assay 9 DAPI staining was conducted to detect any apoptotic changes induced by EAFE in 10 HepG2 cells. Comet assay (single cell gel electrophoresis), which detects breaks in 11 the single-and double-stranded naked supercoiled DNA, was used to evaluate the 12 DNA damage induced by chemical oxidants. 13 EAFE induced apoptosis, as was revealed by the light staining of nuclei with DAPI, 14 indicating an increase in apoptotic cells (Fig. 3A). EAFE-induced DNA damage was 15 noted by the extension of the comet tail in EAFE-treated cells (Fig. 3B)

HepG2 cells 25
The DCFH-DA probe was used to measure the level of ROS in HepG2 cells after 26 treatment with EAFE. Fluo-3 AM fluorescent probes were used to detect the levels of 1 Ca 2+ . The endoplasmic reticulum is the main storage site for intracellular Ca 2+ . During 2 stress, Ca 2+ is released from the endoplasmic reticulum. Mitochondria, which can 3 regulate the frequency of oscillatory Ca 2+ signals and blunt the spread of cytosolic 4 Ca 2+ waves, acted as a firewall that control the Ca 2+ concentration in the cells and in 5 cytoplasmic microdomains on the mitochondria-mediated death pathway [22]. 6 HepG2 cells treated with 350 μg/mL of EAFE for 1-48 h were harvested for ROS, 7 Ca 2+ , and MMP determinations. EAFE promoted ROS and Ca 2+ production as well as 8 decreased the levels of MMP in HepG2 cells; these effects were time-dependent ( Fig.  9 4A-4C). 10 11 EAFE induces caspase-3 and caspase-9 activity in HepG2 cells 12 Caspase-3 and caspase-9 are key proteins in the mitochondrial apoptosis pathway [23]. 13 HepG2 cells treated with 350 μg/mL of EAFE for 12, 24, and 48 h were harvested for 14 caspase-3 and caspase-9 activity determination. EAFE increased the staining of cells, 15 as revealed using the PhiPhilux kit. EAFE promoted caspase-3 and caspase-9 activity 16 in HepG2 cells in a time-dependent manner ( Fig. 5A and 5B). 17

EAFE induces HepG2 cell apoptosis and regulates the levels of apoptosis-related 19
proteins 20 MMP is mainly regulated by the interaction between members of the Bcl-2 family, 21 and the ratio of Bax to Bcl-2 is considered an important upstream checkpoint for the 22 mitochondrial pathway. HepG2 cells treated with 350 μg/mL of EAFE for 12, 24, and 23 48 h were harvested. We then examined the levels of apoptosis-associated molecules 24 in HepG2 cells by using western blotting. The data revealed that the protein levels of 25 Bax, AIF, endonuclease G (Endo G), and cytochrome c were significantly increased, 26 whereas that of Bcl-2 significantly decreased in EAFE-treated HepG2 cells compared 1 to those in the control group ( Fig. 6A and B). EAFE promoted the release of AIF, 2 Endo G, and cytochrome c from the mitochondria in a time-dependent manner (Fig.  3   6). Taken together, the above data suggest that EAFE probably induces HepG2 cell 4 apoptosis through caspase-dependent and caspase-independent mitochondrial 5 pathways. Our results suggest that EAFE is an effective apoptosis-inducing agent for 6 HepG2 cells, which causes S-phase arrest and ER stress as well as induces 7 mitochondria-mediated apoptotic pathways. The HPLC chromatogram of the three active partitioned fractional extracts from 2 HWG exhibited a total of 12 peaks, which were identified by comparing the retention 3 times with those of standard reference compounds ( The concentrations of phenolic compounds in EAFE, NBFE, and WFE are shown 12 in with consequent inhibition of cell growth. Second, such drugs trigger the apoptotic 4 pathway in cells. In fact, the S-phase of the cell cycle is associated with cell apoptosis 5 [25]. 6 Oxidative stress is considered to be important for the promotion of apoptosis in 7 response to various apoptotic stimuli. ROS play an essential role in the oxidative 8 stress response. Mitochondria are involved in several events leading to apoptosis, 9 such as generation of ROS, loss of m, release of apoptotic factors, and regulation 10 of Bcl-2 family proteins. Excess ROS results in potentially cytotoxic "oxidative 11 stress." Activation of the mitochondria-mediated intrinsic apoptotic pathway is also a 12 key mechanism involved in the function of antitumor drugs. In our study, we determined the cytotoxic effects of EAFE on HepG2 cells and 26 15 found that EAFE inhibited HepG2 cell proliferation ( Fig. 1 and Fig. 2). We also 1 showed that treatment of HepG2 cells with EAFE could increase ROS generation and 2 induce apoptosis and arrest the cell cycle in the S phase ( Fig. 2 and Fig. 4). In 3 addition, EAFE treatment promoted the induction of apoptosis and DNA damage in 4 HepG2 cells (Fig. 3). EAFE treatment also increased caspase-3 and caspase-9 5 activities (Fig. 5). Calcium ion is one of the key regulators of apoptosis induction via 6 ER stress. Higher level of Ca +2 , as observed in our study, could be a consequence of 7 mitochondrial membrane damage after EAFE exposure (Fig. 4). Western blot analysis 8 revealed that the upregulation of Bax, AIF, Endo G, and cytochrome c and the 9 corresponding downregulation of Bcl-2 proteins observed in our study may be one of 10 the critical mechanisms through which EAFE induces apoptosis in HepG2 cells (Fig.  11   6). Therefore, we indicated that EAFE inhibited HepG2 cell growth and induced 12 apoptosis through ROS-dependent endoplasmic reticulum stress. Our data revealed that treatment with EAFE significantly enhanced intracellular 14 ROS production and MDA levels; decreased GSH level; and increased GSH-Px, SOD, 15 and catalase activities in HepG2 cells (Fig. 7). This is an important finding since 16 HepG2 cells express high intracellular levels of GSH, which facilitates the growth of 17

HepG2 cells and confers them resistance to current therapies. Considering that cells 18
can undergo apoptosis in response to GSH depletion, EAFE treatment might trigger 19 events leading to the accumulation of intracellular free radicals and initiation of 20 apoptotic cascades, which may contribute, at least in part, to the reduction in HepG2 In our study, we evaluated the phenolic compound contents of EAFE, NBFE, and 12 WFE by using HPLC. The highest polyphenol content in EAFE was RA and CA (Fig.  13   8 and Table 1). In structure-activity relationships, RA is an ester of caffeic acid and have shown that RA has certain protective effects against lung cancer, colon cancer, 26 breast cancer, gastric carcinoma, and liver cancer. Furthermore, RA remarkably 1 suppresses cancer cell proliferation, induces cell apoptosis, and arrests the cell cycle, 2 which are characteristics associated with drugs that effectively prevent tumors [44]. 3 CA (3,4-dihydroxycinnamic acid), is a polyphenol produced through the secondary 4 metabolism of vegetables, including olives, coffee beans, fruits, potatoes, carrots, and 5 propolis, and constitutes the main hydroxycinnamic acid found in the diet of humans 6 [45]. The mitochondrial membrane potential was found to be reduced in the colon 7 cancer cell line HCT-15 when cells were exposed to CA [46]. CA also exhibits an 8 anti-cancer effect, and a previous study showed that CA promoted NSCLC A549 cell   Different letters (a-f) denote significant difference among groups (p < 0.05). 16 An asterisk indicates significant difference (p < 0.05) from 0 h. 17  Different letters (a-d) denote significant difference among groups (p < 0.05). 23 Each value represents mean ± S.D. (n = 50). 24