Research ArticleExperimental Studies

Total Flavonoids Isolated from Diospyros kaki L. f. Leaves Induced Apoptosis and Oxidative Stress in Human Cancer Cells

LI CHEN, YUHANG GUO, GHEDA ALSAIF and YING GAO
Anticancer Research September 2020, 40 (9) 5201-5210; DOI: https://doi.org/10.21873/anticanres.14523

Abstract

Background/Aim: Persimmon (Diospyros kaki L.) leaves are popular as a tea infusion in Asia and their main active ingredients are flavonoids. The present study aimed to explore the anticancer properties of flavonoids isolated from persimmon leaves (PLF). Materials and Methods: We investigated the in vitro anti-proliferative activity of PLF against several human cancer cell lines. Apoptosis and intracellular reactive oxygen species (ROS) induced by PLF were accessed using high-content analysis with florescent staining. The ability of PLF to scavenge free radicals was evaluated by 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay. Results: PLF demonstrated significant inhibition of proliferation of liver, breast, and colorectal cancer cells in vitro. PLF induced apoptosis and increased intracellular ROS levels in HCT116 (colorectal cancer) and HepG2 (liver cancer) cells. In addition, PLF showed strong free radical scavenging ability. Conclusion: The anti-proliferation activity of PLF against cancer cells was related to the induction of apoptosis and oxidative stress.

Cancer is a major public health problem worldwide and the second leading cause of death in the United States. In 2019, there were 1,762,450 new cancer cases and 606,880 cancer deaths in the United States (1). Current treatments for cancer, such as surgery, chemotherapy, and radiotherapy have limited curative effects and notable side effects. In recent years, various traditional Chinese medicines (TCMs) have demonstrated great promise in treating cancer as they have anti-cancer (2) and immune-regulation (3) activities with reduced toxicity compared to existing chemotherapeutic drugs and decreased damage to normal tissue or cells. TCM have also demonstrated synergies with chemotherapeutic drugs, enhancing their therapeutic effects and reducing their toxicity (4).

Persimmon leaves are the fresh or dried leaves of Diospyros kaki L. f., a species of the Ebenaceae family. Mentioned for the first time in Materia Medica of South Yunnan, a medical book written almost 600 years ago, persimmon leaves have been reported to promote the secretion of saliva, quench thirst, clear toxic heat, moisten the lung, strengthen the heart function, suppress cough, and improve clotting function (5). Because of these benefits and the absence of toxicity, users in Asian countries like China, Korea, Japan and India have developed a habit of drinking persimmon leaf tea to prevent or alleviate diseases such as cancer, hypertension, cerebral hemorrhage, and diabetes (6, 7). These uses have given persimmon leaves the reputation of “Treasure of Fitness” (8).

Studies have shown that the main active ingredients of persimmon leaves are flavonoids (9). Flavonoids are known for their anticancer (10), antioxidant (11), antibacterial (12), and hemostatic (13) activities and protective activity for the cardiovascular system (14). Zhang et al. have reported that the flavonoids in persimmon leaves were absorbed and metabolized by bacteria found in the gastrointestinal tract (15). Morel et al. have reported that flavonoids isolated from persimmon leaves are rich in quercetin, iso-quercetin, kaempferol, rutin, hyperoxide, myricetin, baicalin, etc. (16). In our previous study, we analyzed the chemical composition of flavonoids isolated from persimmon leaves (PLF) by high-performance liquid chromatography (HPLC). Remarkable antitumor and life-prolongation effects of PLF were observed on Hepatoma H22 tumor-bearing mice (17). Our subsequent study demonstrated that PLF effectively inhibited liver tumor growth in vivo by enhancing the immune function in mice. Additionally, PLF exhibited alleviated side effects in mice in contrast to the severe side effects of the chemotherapeutic drug cyclophosphamide (18).

Considering the remarkable anti-liver cancer activity of PLF as demonstrated in mice, in the present study we chose human cell lines to investigate the anticancer effects of PLF in vitro. Six human cancer cell lines HCT116 (colorectal carcinoma), A549 (lung carcinoma), HepG2 (hepatocellular carcinoma), BT20 cells (triple-negative mammary gland carcinoma), U2OS (osteosarcoma), MDA-MB-321 (triple-negative adenocarcinoma) as well as three human normal cell lines HUVEC (umbilical vein endothelial cells), MCF10A (mammary gland cells), and HPL1A (peripheral lung epithelial cells) were chosen to study the in vitro anticancer effects of PLF by observing the cell viability, apoptosis, active oxygen species (ROS) production, and DPPH scavenging activity.

Materials and Methods

Plant material. Persimmon (Diospyros kaki L.) leaves were purchased from Yaoyuan Trading Inc. (Xi'an, Shaanxi, PR China) and a voucher specimen (#20180306) was deposited in the School of Agriculture at Middle Tennessee State University. The total flavonoid extracts from the persimmon leaves (PLF) was prepared as described in our previous study (18).

Reagents. Dimethyl sulfoxide (DMSO), staurosporine, rotenone, 2,2-diphenyl-1-picrylhydrazyl (DPPH), and ascorbic acid were purchased from Sigma-Aldrich (St Louis, MO, USA). Fetal bovine serum (FBS) was purchased from Gibco (Grand Island, NY, USA). Annexin V, propidium iodide (PI), and dihydroethidium (DHE) dyes were purchased from BD BioSciences (San Jose, CA, USA). AlamarBlue Cell Viability Reagent was purchased from Invitrogen (Eugene, OR, USA).

Cell culture. The six human cancer cell lines (HCT116, U2OS, A549, BT20, MDB-MA-321, HepG2) and three normal human cell lines (HUVEC, MCF10A, HPL1A) used in this study were cultured in specific media supplemented with 10% FBS, 100 U/ml of penicillin, and 100 U/ml of streptomycin at a 37°C incubator supplied with 5% CO2. The medium for all cell lines are as follows: HCT116 and U2OS cells were cultured in McCoy's 5A medium (ATCC, Manassas, VA, USA); A549 and BT20 cells were cultured in RPMI1640 medium (Sigma); HepG2 cells were cultured in EMEM medium (ATCC); MDA-MB-321 were cultured in DMEM medium (Sigma); HUVEC cells were cultured in Vascular Cell Basal Medium (ATCC) supplemented with Endothelial Cell Growth Kit-BBE (ATCC); MCF10A cells were cultured in MEGM medium (Lonza, Walkersville, MD, USA); HPL1A cells were cultured in DME/F-12 medium (HyClone, Logan, UT, USA). The cells in logarithmic growth phase were applied in the following assays.

Cytotoxicity assay and IC50 determination. PLF was dissolved with DMSO at a concentration of 10 mg/ml (stock solution) and diluted with corresponding culture medium to the required concentration before treatment. The final DMSO concentration was below 1%. The cells were seeded in 96-well plates at a density of 5-7×103 cells/well and cultured in 5% CO2 incubator at 37°C. After 24 h incubation, the cells were attached to the plates and the culture medium was replaced by 100 μL of medium containing 100, 50, 10, 5, or 1 μg/ml PLF; each treatment group had four duplicates. After 48 h treatment, 10 μl of AlamarBlue was added into each well, and the cells were incubated for another 3 h. Subsequently, fluorescence intensity (FI) of each well at Excitation/Emission wavelength of 550/590 nm was determined by SpectraMax15 microplate reader (Molecular Devices Inc., San Jose, CA, USA). The cell growth inhibition (%) was calculated using the following formula, and IC50 values were calculated by GraphPad Prism 5.0 software (GraphPad Software Inc. San Diego, CA, USA).

Figure 1.
Figure 1.

Growth inhibition rate (%) of 100 μg/ml PLF against nine human cell lines. Cells were seeded in a 96-well plate and treated with 100 μg/ml PLF for 48 h and stained with 10% AlamarBlue dye. Fluorescence Intensity (FI) from each well of the microplate was read at Ex/Em of 550/590 nm. Values are means±S.D. (n=4).

Apoptosis assay. HCT116 and HepG2 cells were seeded in a 96-well clear bottom black plate at a density of 5-7×103 cells/well and cultured in a 5% CO2 incubator at 37°C. Following attachment, cells were treated with high (100 μg/ml), medium (50 μg/ml), and low concentration (10 μg/ml) of PLF diluted with complete medium for 24 h. The negative control group was treated with complete medium only, and the positive control group was treated with 1 μM staurosporine. Each treatment group had four replicates. After treatment, 1 μl Hoechst 33342, 5 μL FITC-Annexin V, and 5 μl PI were added to each well. The plates were incubated at 37°C for 15 min. Apoptosis in each group was evaluated and analyzed by ArrayScan VTI High Content Screening (HCS) reader (Thermo Scientific, Waltham, MA, USA) and its built-in vHCS Scan software. Excitation/Emission wavelengths for Hoechst 33342, FITC-Annexin V, and PI were 350/461 nm, 494/518 nm, 535/617 nm, respectively.

Oxidative stress assay. HCT116 and HepG2 cells were seeded and treated as described in 2.4. The negative control group was treated with complete medium only and the positive control group was treated with 10 μM rotenone. The staining solution was prepared by adding 10 μl Hoechst 33342 and 13.75 μl dihydroethidium (DHE) in 5.5 ml complete medium that was warmed to 37°C before use. After treatment, 50 μl of staining solution were added to each well, and the plate was kept in a 5% CO2 incubator at 37°C for 30 min. Subsequently, 4% paraformaldehyde was warmed to 37°C, and 100 μl of the solution was slowly added to each well to fix the cells. After incubation for 30 min in a fume hood, the solution was aspirated and 100 μL PBS were applied to each well for washing. Finally, 200 μl PBS were added to each well, and the plate was scanned by ArrayScan VTI HCS reader and the reactive oxygen species (ROS) were evaluated with the build-in vHCS Scan software.

View this table:
Table I.

IC50 values of PLF (μg/ml) in the nine human cell lines.

DPPH radical scavenging activity assay. The DPPH assay (19, 20) was employed to study the free radical scavenging activity of PLF. The DPPH solution was prepared at 0.1 mM in absolute ethanol, and 100, 50, 10, 5, and 1 μg/ml PLF were prepared in DMSO. In a 96-well plate, 148 μl of 0.1 mM DPPH solution and 2 μl of different concentrations of PLF solution were added to each well. In addition, 100, 50, 10, 5, and 1 μg/ml ascorbic acid were used as positive controls, while 0.1 mM DPPH solution was used as a negative control. The blank control was made of 148 μl of absolute ethanol and 2 μl of different concentrations of PLF solution. Notably, the positive control and blank are concentration specific. The plate was stored in the dark at room temperature for 20 min, and the absorbance of the mixture was read at 517 nm. The Radical Scavenging Activity (RSA) was calculated using the following formula: Embedded Image AC is the absorbance of the negative control; AS is the absorbance of the sample; AB is the absorbance of the blank.

Statistical analysis. Data are presented as the mean±S.D. Statistical analysis was conducted using GraphPad Prism 6.0 software (GraphPad Software Inc.). One-way ANOVA and t-tests were performed to determine the difference between the treated and control groups. Data were considered significantly different when p value ≤0.05.

Results and Discussion

PLF remarkably inhibited growth of human cancer cell lines but not of normal human cell lines. First, we evaluated the cytotoxicity of PLF in six human cancer cell lines and three normal human cell lines using AlamarBlue staining. The cytotoxicity of 1-100 μg/ml PLF was examined, and IC50 value was calculated for each cell line. The growth inhibitory effect of PLF at the highest concentration (100 μg/ml) was compared between the different cell lines. As shown in Figure 1, significant cytotoxicity of PLF against human cancer cells was observed. The inhibition rates of PLF at 100 μg/ml were above 75% in all human cancer cell lines. Particularly, the inhibition rates were above 94% against human liver cancer cells HepG2 and human breast cancer cells MDB-MA-321 and BT20.

The results in Table I show that PLF had inhibitory effects against all human cancer cell lines, with IC50 values ranging from 12.78 to 56.85 μg/ml. Notably, PLF showed remarkable inhibition against HepG2 liver cancer cell line and MDB-MA-321 breast cancer cell line with IC50 values <20 μg/ml.

Among the three normal human cell lines that we tested, PLF showed no significant cytotoxicity against MCF10A and HPL1A; inhibition rates were lower than 16% and IC50 values were greater than 100 μg/ml. PLF showed cytotoxicity towards HUVECs with an inhibition rate of 86.0% at 100 μg/ml concentration and an IC50 of 33.71 μg/ml. HUVECs are endothelial cells isolated from the vein of the human umbilical cord that can be reprogrammed to the pluripotent state (21). The higher cytotoxicity of PLF to HUVECs compared to MCF10A and HPL1A cells, which are the fully-differentiated cells, could be due to the stemness status of HUVECs.

In summary, the cytotoxicity evaluation revealed that PLF in the dose range of 0~30 μg/ml had significant inhibitory effects on the growth of human cancer cells while having low cytotoxicity to normal human cells. These results were consistent with previous findings that showed flavonoids possessed higher cytotoxicity against the cancer cell lines than normal cells (22). The high selectivity of PLF may explain the significantly small side effects observed in the tumor-bearing mice treated with PLF compared to the severe side effects of cyclophosphamide (18). As highly selective medicine has wide clinic usage and fewer undesired adverse reactions (23), the results of our study indicate the clinical potential of PLF as an anticancer drug having few adverse effects on the functions of multiple tissues and organs of the body.

Figure 2.
Figure 2.
Figure 2.

Induction of apoptosis by PLF in HCT116 and HepG2 cells. Cells were treated with different concentrations of PLF for 24 h, and stained with a mixture of Hoechst, FITC-Annexin V and PI. The positive control was 1 μM staurosporine and negative control was vehicle only. A) Representative images of cell apoptosis imaged by an ArrayScan VTI HCS reader. Merged images are the composition of images of the same field from three different channels (Hoechst, FITC-Annexin V, and PI). B, E) Effects of various concentrations of PLF on the nuclear area of HCT116 and HepG2 cells. C) F) FITC-Annexin V staining in HCT116 and HepG2 cells treated by various concentrations of PLF. D) and G) PI staining in HCT116 and HepG2 cells treated by various concentrations of PLF. Values are means±S.D. (n=8). *p<0.05, **p<0.01 or ***p<0.001 versus the negative control group.

PLF induced apoptosis in human cancer cells. Next, we investigated whether the inhibitory effect of PLF was related to apoptosis. Kandaswami et al. have reported that the induction of apoptosis was one of the main mechanisms attributed to the anticancer activities of dietary flavonoids in addition to the suppression of protein tyrosine kinase, anti-metastasis, and anti-angiogenesis (24). Microscopic observation of cancer cells treated with PLF showed the typical morphology of apoptosis: the cells became round in shape and detached from neighboring cells. Further, we used the Hoechst 33342/FITC-Annexin V/PI triple staining assay to detect apoptosis in cancer cells. We chose the HCT116 colorectal cancer cell line and HepG2 liver cell line for the subsequent apoptosis and oxidative stress assay for two reasons. First, PLF showed remarkable cytotoxicity against both HCT116 and HepG2 cells. Second, as mentioned previously, persimmon leaves are popular as tea infusion in Asia. Therefore, it would be advantageous for us to examine the effects of PLF on gastrointestinal cancers such as colorectal and liver cancer.

Hoechst 33342 is a blue fluorescent dye that penetrates the cell membrane and stains nuclei. As shown in Figure 2A and B, after 24-h treatment with 50 or 100 μg/ml PLF, the nucleus area of HCT116 cells was significantly reduced, and the nuclei were densely stained (**p<0.01 or *p<0.05). In HepG2 cells treated with all three doses of PLF, the nucleus area was significantly decreased (Figure 2E). As nuclear condensation is a typical hallmark of apoptosis (24), the results implied that PLF induced apoptosis in HCT116 and HepG2 cancer cells.

Figure 3.
Figure 3.
Figure 3.

Induction of ROS by PLF in HCT116 and HepG2 cells. Cells were treated with different concentrations of PLF for 24 h, and stained with Hoechst 33342 and dihydroethidium (DHE) for 30 min. Subsequently, cells were washed with PBS and imaged by an ArrayScan VTI HCS reader. The negative control group was treated with complete medium only and the positive control group was treated with 10 μM rotenone. A) Representative images of ROS generation imaged by an Arrayscan VTI HCS reader. Blue fluorescence represents nuclei, green fluorescence represents ROS. B, D) Effects of PLF on the cell nuclear area of HCT116 and HepG2 cells. C, E) Intracellular ROS generation in HCT116 and HepG2 cells. Values are means±S.D. (n=8). *p<0.05, **p<0.01 or ***p<0.001 versus the negative control group.

In normal living cells, phosphotidylserine (PS) is located on the inner side of the cell membrane. In the early stage of cell apoptosis, PS flips from the inner side of the plasma membrane to the outer side and is exposed to the extracellular environment. Annexin V, which binds PS with high affinity, was labeled with fluorescein FITC to detect early apoptotic cells. Propidium iodide (PI) is a nucleic acid dye that cannot penetrate the intact cell membrane but can go through the cell membrane of late apoptotic and dead cells. The combination of FITC-Annexin V and PI staining can distinguish cells at different apoptotic stages. As shown in Figure 2A, C and D, after 24-h treatment with 50 or 100 μg/ml PLF, the fluorescence intensity of both FITC-Annexin V and PI was significantly increased in HCT116 cells (***p<0.001). In HepG2 cells, all three doses of PLF significantly increased the fluorescence intensity of FITC-Annexin V and PI (Figure 2A, F, and G). The results showed that PLF at 50 μg/ml and above promoted apoptosis of HCT116 cells; while PLF at 10 μg/ml and above could promote apoptosis of HepG2 cells.

PLF induced oxidative stress in human cancer cells. This assay employed the fluorescent probe dihydroethidium (DHE) to detect intracellular reactive oxygen species (ROS), and Hoechst 33342 to identify the nucleus. ROS can transform non-fluorescent DHE into fluorescent ethidium. As ethidium is embedded in DNA, its fluorescence intensity reflects the presence of intracellular ROS. As shown in Figure 3A, with the increase of the dose of PLF, the intracellular fluorescence intensity was significantly enhanced, implying that the intracellular ROS production was increased. The intracellular fluorescence intensity in HCT116 cells treated with high (100 μg/ml) or medium (50 μg/ml) dose of PLF was 1,360±48 and 1,090±26, respectively (Figure 3C), which increased significantly compared with the negative control group (954±18) (***p<0.001 or **p<0.01). In HepG2 cells, all three doses (10, 50, and 100 μg/ml) of PLF significantly increased the intracellular fluorescence intensity of cells (Figure 3E). In addition, the nucleus size of HCT116 and HepG2 cells decreased after the treatment with PLF (Figure 3B and D), consistent with the results described in the apoptosis assay. Under normal circumstances, the oxidant and antioxidant systems are in a dynamic equilibrium in cells. When cells are subjected to harmful stimuli, ROS and reactive nitrogen species (RNS) such as hydrogen peroxide, nitric oxide, peroxyl radical, peroxynitrite anion, etc., are produced in large quantities in the cells. The overproduction and accumulation of ROS and RNS exceed the cell's scavenging ability and lead to oxidative stress that is involved in the pathogenesis of several diseases including cancer (25). Oxidative stress is known to be closely related to cell apoptosis by playing dual roles: under normal physiological conditions, ROS regulate cell proliferation; when the amount of ROS exceeds a certain threshold, ROS arrests the cell cycle and causes DNA breakage. Oxidative stress thereby mediates cell apoptosis and even tissue damage through mitochondria, death receptors, endoplasmic reticulum, and other pathways (26, 27). Our results showed that PLF promoted HCT116 and HepG2 cell apoptosis by increasing intracellular ROS, causing damage to the cell membrane and rupture of the nuclear membrane.

PLF showed superior DPPH radical scavenging activity than ascorbic acid.In addition, we investigated the ability of PLF to scavenge free radicals by the DPPH assay. The DPPH molecule is a stable nitrogen-containing free radical presenting a dark purple color in ethanol solution and a strong absorption peak at 517 nm. When DPPH meets the free radical, the single electron of the free radical binds with DPPH to produce a colorless product, which makes the solution lighter in color, and the color change can be quantitated spectrophotometrically. The ability to scavenge free radicals is presented by the inhibition rate, which is positively correlated with the antioxidant capacity.

Our study found that the DPPH inhibition rate following treatment with 5-50 μg/ml PLF was significantly higher compared with the same concentration of the positive control ascorbic acid (***p<0.001) (Figure 4). PLF in low concentration (1 μg/ml) and high concentration (100 μg/ml) showed nearly equal inhibition rate compared with that of the same concentration of ascorbic acid.

Studies have shown that the antioxidant activity of plants is strongly related to the total polyphenol flavonoids content (28, 29). Flavonoids have been found to be the main active ingredients that inhibit the proliferation of cancer cells in a wide variety of natural plants. For example, Zhan (30) and Zu (31) have shown that the extract of blueberries, which is rich in antioxidant flavones, had remarkable dose-dependent anti-proliferative activity against the cancer cells HepG2, DLD-1, and COLO205. Our study also demonstrated the in vitro antioxidant activity of PLF, which showed a greater scavenging effect free radical than ascorbic acid, indicating the PLF is a superior natural antioxidant. Consistent with other anticancer flavone studies, our results also showed the correlation of antioxidant activity and the cytotoxic effects of PLF in various human cancer cells.

Figure 4.
Figure 4.

DPPH radical scavenging activity of PLF. In a 96-well plate, 148 μl of 0.1 mM DPPH solution and 2 μl of different concentrations of PLF solution were added to each well. Ascorbic acid at the same concentration as PLF was used as positive controls, while 0.1 mM DPPH solution was used as a negative control. The plate was incubated in the dark at room temperature for 20 min and the absorbance of the mixture was read at 517 nm. Values are means±S.D. (n=4).

Conclusion

PLF demonstrated significant cytotoxicity to a variety of human cancer cells and selectivity against cancer cells over normal cells. In the non-toxic dosage range of 0~30 μg/ml, PLF inhibited the growth of the human liver, breast, and colon cancer cells. Our study demonstrated that PLF promoted the apoptosis of HCT116 colon cancer cells, possibly via regulating the redox state and increasing the intracellular ROS levels. This study presented a preliminary screening of cancer cell lines that were sensitive to PLF and the underlying mechanism through which PLF inhibits the proliferation of cancer cells. Due to the low toxicity to normal cells and high efficacy in inhibiting cancer cells, PLF could be a potential candidate of treating certain types of cancer. The effect of PLF on the signaling pathways in cancer cells and the mechanism that induces apoptosis of cancer cells is worth further study.

Acknowledgements

The Authors would like to thank Middle Tennessee State University for providing fund for this study. The authors also thank the Natural Science Foundation of Guangxi (2016GXNSFBA380080) for providing the funds for the visiting scholar L.C.

Footnotes

  • Authors' Contributions

    Y.G. conceived and designed the project. L.C. and G.A. conducted the experiments and acquired the data. L.C., G.A. and Y-H.G. analyzed and interpreted the data. Y.G. and L.C. wrote the manuscript. All the Authors have reviewed this manuscript.

  • Conflicts of Interest

    The Authors declared no conflicts of interest in relation to this study.

  • Received July 20, 2020.
  • Revision received August 1, 2020.
  • Accepted August 3, 2020.

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Total Flavonoids Isolated from Diospyros kaki L. f. Leaves Induced Apoptosis and Oxidative Stress in Human Cancer Cells
LI CHEN, YUHANG GUO, GHEDA ALSAIF, YING GAO
Anticancer Research Sep 2020, 40 (9) 5201-5210; DOI: 10.21873/anticanres.14523
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