Chrysin Attenuates Cell Viability of Human Colorectal Cancer Cells through Autophagy Induction Unlike 5-Fluorouracil/Oxaliplatin

Chemotherapeutic 5-fluorouracil (5-FU) combined with oxaliplatin is often used as the standard treatment for colorectal cancer (CRC). The disturbing side effects and drug resistance commonly observed in chemotherapy motivate us to develop alternative optimal therapeutic options for CRC treatment. Chrysin, a natural and biologically active flavonoid abundant in propolis, is reported to have antitumor effects on a few CRCs. However, whether and how chrysin achieves similar effectiveness to the 5-FU combination is not clear. In this study, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), western blotting, fluorescence microscopy, and reactive oxygen species (ROS) production were assayed. We found that chrysin exhibited similar inhibition of cell viability as the 5-FU combination in a panel of human CRC cells. Furthermore, the results showed that chrysin significantly increased the levels of LC3-II, an autophagy-related marker, in CRC cells, which was not observed with the 5-FU combination. More importantly, blockage of autophagy induction restored chrysin-attenuated CRC cell viability. Further mechanistic analysis revealed that chrysin, not the 5-FU combination, induced ROS generation, and in turn, inhibited the phosphorylation of protein kinase B (Akt) and mammalian target of rapamycin (mTOR). Collectively, these results imply that chrysin may be a potential replacement for the 5-FU and oxaliplatin combination to achieve antitumor activity through autophagy for CRC treatment in the future.


Introduction
Colorectal cancer (CRC) is one of the most common cancer types in the world, and its occurrence is still on the rise yearly. Since CRC is a heterogeneous disease, both molecular expression patterns and pathological features reflect prognostic outcomes and treatment responses [1][2][3]. Although early-stage CRC has a better prognosis than other cancer types, it is frequently found that patients

Chrysin Achieved Similar Effectiveness to 5-Fluorouracil (5-FU) Combined with Oxaliplatin in a Panel of Colorectal Cancer (CRC) Cells
Firstly, a comparison of therapeutic effectiveness on cell viability between chrysin and 5-FU combined with oxaliplatin was performed through application to a panel of CRC cells. To ensure consistent comparisons, the concentration of chrysin was made equal to the sum of 5-FU and oxaliplatin concentrations. Then, CRC cells were treated with the indicated concentration of each drug for three days, and cell viability was determined via a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. As shown in Figure 1, chrysin indeed significantly attenuated cell viability in a dose-dependent manner in CRC cell lines when compared with the control (vehicle only) group. Although chrysin had a minor effect on HT-29 cells, its effect on other CRC cell lines was dramatic. When comparing the effect between chrysin and 5-FU combined with oxaliplatin, we found that chrysin achieved similar effectiveness in these CRC cell lines. Although chrysin had a better effect than 5-FU combined with oxaliplatin in some cell lines ( Figure 1B,C) and a worse effect in others ( Figure 1D,E), the therapeutic effectiveness on the attenuation of CRC cell viability was overall equivalent between chrysin and 5-FU combined with oxaliplatin. Cell viability was determined via a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, and was statistically quantified. # p < 0.05; ## p < 0.01; ### p < 0.001 when compared with control (vehicle only) groups. * p < 0.05; ** p < 0.01; *** p < 0.001 when compared with indicated control groups. A p-value < 0.05 was considered as statistically significant. N.S.-not significant.

LC3-II Levels Were Induced in Chrysin-Treated but Not 5-Fluorouracil (5-FU) /Oxaliplatin-Treated Colorectal Cancer (CRC) Cells
Since chrysin achieved similar effectiveness in the attenuation of CRC cell viability when compared with 5-FU combined with oxaliplatin, we further analyzed how CRC cell viability was attenuated between these two groups. We examined the death pathways, apoptosis and autophagy, by detecting the expression of respective biomarkers-active caspase-3/cleaved poly-ADP-ribose polymerase (cleaved-PARP) for apoptosis and LC3-II for autophagy. As shown in Figure 2A-E, chrysin and the 5-FU combination were not much different with regards to the induction of apoptosis (upper panel) in most CRC cell lines. However, it is interesting that chrysin significantly increased LC3-II levels in all applied CRC cell lines, which was not observed in the 5-FU/oxaliplatin−treated group (lower panel). We further enforced HCT-116 cells expressing GFP-LC3, followed by chrysin treatment. During autophagy, LC3-II relocalizes to the autophagosomal membranes. Therefore, the accumulation of GFP-LC3 punctate structures provides a way of detecting autophagosomes. The results from fluorescence imaging analysis ( Figure 2F) showed that GFP-LC3 punctate structures were not obviously detected in the control group (middle panel), but exhibited a significant increase in the chrysin-treated group (right panel). Collectively, this implies the involvement of different mechanisms underlying the antitumor activities of chrysin and 5-FU/oxaliplatin. Cell viability was determined via a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, and was statistically quantified. # p < 0.05; ## p < 0.01; ### p < 0.001 when compared with control (vehicle only) groups. * p < 0.05; ** p < 0.01; *** p < 0.001 when compared with indicated control groups. A p-value < 0.05 was considered as statistically significant. N.S.-not significant.

LC3-II Levels Were Induced in Chrysin-Treated but Not 5-Fluorouracil (5-FU) /Oxaliplatin-Treated Colorectal Cancer (CRC) Cells
Since chrysin achieved similar effectiveness in the attenuation of CRC cell viability when compared with 5-FU combined with oxaliplatin, we further analyzed how CRC cell viability was attenuated between these two groups. We examined the death pathways, apoptosis and autophagy, by detecting the expression of respective biomarkers-active caspase-3/cleaved poly-ADP-ribose polymerase (cleaved-PARP) for apoptosis and LC3-II for autophagy. As shown in Figure 2A-E, chrysin and the 5-FU combination were not much different with regards to the induction of apoptosis (upper panel) in most CRC cell lines. However, it is interesting that chrysin significantly increased LC3-II levels in all applied CRC cell lines, which was not observed in the 5-FU/oxaliplatin−treated group (lower panel). We further enforced HCT-116 cells expressing GFP-LC3, followed by chrysin treatment. During autophagy, LC3-II relocalizes to the autophagosomal membranes. Therefore, the accumulation of GFP-LC3 punctate structures provides a way of detecting autophagosomes. The results from fluorescence imaging analysis ( Figure 2F) showed that GFP-LC3 punctate structures were not obviously detected in the control group (middle panel), but exhibited a significant increase in the chrysin-treated group (right panel). Collectively, this implies the involvement of different mechanisms underlying the antitumor activities of chrysin and 5-FU/oxaliplatin.  , as well as of LC3-II, were examined using indicated antibodies. The band images were quantified using tubulin as a calibrated control. Relative fold inductions of activated caspase-3, cleaved PARP, and LC3-II were indicated as compared to control groups. (F) The GFP-LC3 expression vector was transfected into HCT-116 cells, followed by treatment with 50 µM chrysin for 24 h. GFP-LC3 punctate structures were observed under a fluorescence microscope. White arrow represents GFP-LC3 punctate structure. The fluorescent images were displayed in a 10× multiplication.

Blockage of Autophagy Induction Restored Chrysin-Attenuated Colorectal Cancer (CRC) Cell Viability
To further confirm the significance of chrysin-induced LC3-II production, the autophagy inhibitor, 3-methyladenine (3-MA), was applied. CRC cells were pretreated with the indicated concentration of 3-MA, followed by treatment with chrysin for three days. Cell viability was then determined via a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. As shown in Figure 3, treatment with chrysin alone attenuated cell viability in a dose-dependent manner. However, this dose-dependent inhibition was reversed when these CRC cells were pretreated with 3-MA. The degree of reversal was gradually enhanced with an increase in 3-MA concentration. Supporting these results, treatment with chrysin alone induced LC3-II expression, whereas such induction was not observed in cells treated with 3-MA ( Figure 4). These results suggest that chrysin attenuates CRC cell viability at least in part through autophagy induction.

Blockage of Autophagy Induction Restored Chrysin-Attenuated Colorectal Cancer (CRC) Cell Viability
To further confirm the significance of chrysin-induced LC3-II production, the autophagy inhibitor, 3-methyladenine (3-MA), was applied. CRC cells were pretreated with the indicated concentration of 3-MA, followed by treatment with chrysin for three days. Cell viability was then determined via a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. As shown in Figure 3, treatment with chrysin alone attenuated cell viability in a dose-dependent manner. However, this dose-dependent inhibition was reversed when these CRC cells were pretreated with 3-MA. The degree of reversal was gradually enhanced with an increase in 3-MA concentration. Supporting these results, treatment with chrysin alone induced LC3-II expression, whereas such induction was not observed in cells treated with 3-MA ( Figure 4). These results suggest that chrysin attenuates CRC cell viability at least in part through autophagy induction.

Blockage of Autophagy Induction Restored Chrysin-Attenuated Colorectal Cancer (CRC) Cell Viability
To further confirm the significance of chrysin-induced LC3-II production, the autophagy inhibitor, 3-methyladenine (3-MA), was applied. CRC cells were pretreated with the indicated concentration of 3-MA, followed by treatment with chrysin for three days. Cell viability was then determined via a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. As shown in Figure 3, treatment with chrysin alone attenuated cell viability in a dose-dependent manner. However, this dose-dependent inhibition was reversed when these CRC cells were pretreated with 3-MA. The degree of reversal was gradually enhanced with an increase in 3-MA concentration. Supporting these results, treatment with chrysin alone induced LC3-II expression, whereas such induction was not observed in cells treated with 3-MA ( Figure 4). These results suggest that chrysin attenuates CRC cell viability at least in part through autophagy induction.   Expression of LC3-II was examined using indicated antibodies. The band images were quantified using tubulin as a calibrated control. Relative fold induction of LC3-II was indicated as compared to indicated control groups.

Chrysin-Mediated Autophagy Induction Was through Protein Kinase B (Akt)/Mammalian Target of Rapamycin (mTOR) Signaling Pathway in Colorectal Cancer (CRC) Cells
Next, we investigated the mechanism underlying chrysin-mediated autophagy induction. AMP-activated protein kinase (AMPK) is a well-known energy sensor and upstream regulator of autophagy. Under starvation conditions, AMPK is activated and, in turn, inhibits mammalian target of rapamycin 1 (mTOR1) activity, followed by decreasing protein synthesis and increasing autophagy [18,19]. We first examined the activation of AMPK and mTOR. As shown in Figure 5A, chrysin significantly inhibited mTOR phosphorylation in CRC cells, which is expected to induce autophagy. AMPK phosphorylation was increased by chrysin. In addition to AMPK, Akt is reported as another upstream regulator of autophagy through the targeting of mTOR [20,21]. The results in Figure 5B show that chrysin treatment obviously attenuated Akt phosphorylation in CRC cells. These results suggest that chrysin inhibits Akt activation and, in turn, decreases mTOR phosphorylation, which eventually leads to autophagy induction in CRC cells.

Chrysin-Mediated Autophagy Induction Was through Protein Kinase B (Akt)/Mammalian Target of Rapamycin (mTOR) Signaling Pathway in Colorectal Cancer (CRC) Cells
Next, we investigated the mechanism underlying chrysin-mediated autophagy induction. AMP-activated protein kinase (AMPK) is a well-known energy sensor and upstream regulator of autophagy. Under starvation conditions, AMPK is activated and, in turn, inhibits mammalian target of rapamycin 1 (mTOR1) activity, followed by decreasing protein synthesis and increasing autophagy [18,19]. We first examined the activation of AMPK and mTOR. As shown in Figure 5A, chrysin significantly inhibited mTOR phosphorylation in CRC cells, which is expected to induce autophagy. AMPK phosphorylation was increased by chrysin. In addition to AMPK, Akt is reported as another upstream regulator of autophagy through the targeting of mTOR [20,21]. The results in Figure 5B show that chrysin treatment obviously attenuated Akt phosphorylation in CRC cells. These results suggest that chrysin inhibits Akt activation and, in turn, decreases mTOR phosphorylation, which eventually leads to autophagy induction in CRC cells. Expression of LC3-II was examined using indicated antibodies. The band images were quantified using tubulin as a calibrated control. Relative fold induction of LC3-II was indicated as compared to indicated control groups.

Chrysin-Mediated Autophagy Induction Was through Protein Kinase B (Akt)/Mammalian Target of Rapamycin (mTOR) Signaling Pathway in Colorectal Cancer (CRC) Cells
Next, we investigated the mechanism underlying chrysin-mediated autophagy induction. AMP-activated protein kinase (AMPK) is a well-known energy sensor and upstream regulator of autophagy. Under starvation conditions, AMPK is activated and, in turn, inhibits mammalian target of rapamycin 1 (mTOR1) activity, followed by decreasing protein synthesis and increasing autophagy [18,19]. We first examined the activation of AMPK and mTOR. As shown in Figure 5A, chrysin significantly inhibited mTOR phosphorylation in CRC cells, which is expected to induce autophagy. AMPK phosphorylation was increased by chrysin. In addition to AMPK, Akt is reported as another upstream regulator of autophagy through the targeting of mTOR [20,21]. The results in Figure 5B show that chrysin treatment obviously attenuated Akt phosphorylation in CRC cells. These results suggest that chrysin inhibits Akt activation and, in turn, decreases mTOR phosphorylation, which eventually leads to autophagy induction in CRC cells.  Expressions of phosphorylated AMP-activated protein kinase alpha (p-AMPKα) threonine 172, AMPKα, phosphorylated mTOR (p-mTOR) serine 2448, mTOR, phosphorylated Akt (p-Akt) serine 473, and Akt were examined using the indicated antibodies. The band images were quantified using both actin and the AMPKα, mTOR, or Akt proteins as calibrated controls. Relative fold inductions of p-AMPKα threonine 172, p-mTOR serine 2448, and p-Akt serine 473 were indicated as compared to control (vehicle alone) groups.

Chrysin Induced the Production of Reactive Oxygen Species (ROS) in Colorectal Cancer (CRC) Cells
Recently, several studies indicated that ROS induce autophagy through Akt/mTOR inhibition [22][23][24]. Notably, chrysin was reported to induce cell death through ROS production in prostate cancer and chronic lymphocytic leukemia [25,26]. Whether chrysin induces ROS production in CRC cells is not yet known. We further elucidated this issue. ROS production was analyzed using CM-H 2 DCFDA staining and flow cytometry. As shown in Figure 6A, ROS production was significantly increased in chrysin-treated CRC cells. Interestingly, such an effect was not observed in CRC cells treated with 5-fluorouracil (5-FU)/oxaliplatin. Furthermore, the level of ROS production was gradually enhanced with an increase in chrysin concentration ( Figure 6B). Taken together, these results suggest that chrysin induces ROS production and, in turn, leads to autophagy through Akt/mTOR inhibition, unlike the effect of 5-FU/oxaliplatin.

Chrysin Induced the Production of Reactive Oxygen Species (ROS) in Colorectal Cancer (CRC) Cells
Recently, several studies indicated that ROS induce autophagy through Akt/mTOR inhibition [22][23][24]. Notably, chrysin was reported to induce cell death through ROS production in prostate cancer and chronic lymphocytic leukemia [25,26]. Whether chrysin induces ROS production in CRC cells is not yet known. We further elucidated this issue. ROS production was analyzed using CM-H2DCFDA staining and flow cytometry. As shown in Figure 6A, ROS production was significantly increased in chrysin-treated CRC cells. Interestingly, such an effect was not observed in CRC cells treated with 5-fluorouracil (5-FU)/oxaliplatin. Furthermore, the level of ROS production was gradually enhanced with an increase in chrysin concentration ( Figure 6B). Taken together, these results suggest that chrysin induces ROS production and, in turn, leads to autophagy through Akt/mTOR inhibition, unlike the effect of 5-FU/oxaliplatin. h. Then, following trypsinization, cell pellets were stained with 10 μM CM-H2DCFDA at 37 °C in the dark for 30 min. Intracellular ROS level was measured using flow cytometry, and was statistically quantified. * p < 0.05; ** p < 0.01; *** p < 0.001 when compared with indicated control groups. A p-value < 0.05 was considered as statistically significant. Then, following trypsinization, cell pellets were stained with 10 µM CM-H 2 DCFDA at 37 • C in the dark for 30 min. Intracellular ROS level was measured using flow cytometry, and was statistically quantified. * p < 0.05; ** p < 0.01; *** p < 0.001 when compared with indicated control groups. A p-value < 0.05 was considered as statistically significant.

Discussion
Colorectal cancer (CRC) is a cancer with a better prognosis than other cancer types. However, when CRC tumors progress to the advanced stage, the survival rate drops quickly. Currently, chemotherapy seems to be the only effective treatment option. Although chemotherapy is effective, the serious side effects often force patients to withdraw from treatment, and seek a better quality of life. Even when patients continue chemotherapy, acquired resistance eventually develops. These two problems severely challenge the clinical application of chemotherapy [27]. Developing alternative treatment options with similar therapeutic effectiveness but no undesirable adverse effects seems necessary. In this study, we found that a natural product, chrysin, induces similar effects on cell viability inhibition in a panel of human CRC cells when compared with a combination of 5-fluorouracil (5-FU) and oxaliplatin. Although chrysin is reported to have antitumor activity, it is not known whether it has similar effectiveness as chemotherapy in CRC treatment. Our study is the first to investigate this issue. Since chrysin was demonstrated to be harmless to normal cells, and to have multiple functions including anti-inflammation and anti-cancer progression, it is usually claimed as a health supplement to prevent disease [28,29]. Based on these studies and our results, chrysin may have significant potential for CRC treatment in the future. For example, the doses of 5-FU/oxaliplatin may be reduced to attenuate the degree of side effects by combining chrysin treatment. Alternatively, chrysin was reported as a potent inhibitor of breast cancer resistance protein (BCRP), which is one of the ATP-binding cassette (ABC) transporters, and commonly involved in the multidrug resistance (MDR) of chemotherapy [30]. This implies that chrysin may also have potential for use in interval or combination therapy to prevent the occurrence of resistance commonly observed in chemotherapy, a feature which deserves investigation. Although chrysin has potential for clinical application, as supported by in vivo results showing that chrysin efficiently attenuated tumor growth in a xenograft mouse model of several cancer types [31][32][33], chrysin was intravenously administered to mice in these studies. Chrysin was reported as having low oral bioavailability, mainly due to extensive metabolism and the efflux of metabolites back into the intestine for hydrolysis and fecal elimination [34]. Thus, improving the poor bioavailability is the first priority to address before considering clinical application of chrysin.
In the mechanistic analysis of the inhibition of CRC cell viability, we found that chrysin, not the 5-FU/oxaliplatin combination, induced reactive oxygen species (ROS) production and, in turn, led to autophagy induction, as detected by an increase in LC3-II levels, through protein kinase B (Akt)/mammalian target of rapamycin (mTOR) inhibition. This is why blockage of autophagy induction restored chrysin-attenuated CRC cell viability. This is the first time that chrysin is reported to induce ROS production and autophagy in CRC cells. Since autophagy is one mode of programmed cell death [35], our results imply that chrysin attenuates cell viability at least in part through autophagy-dependent cell death. Notably, our findings seem to conflict with studies stating that chrysin could protect rat kidneys from paracetamol-induced autophagy, and sensitize human glioblastoma cells to temozolomide by downregulating autophagy [36,37]. However, it is well known that whether autophagy induces cell death or survival depends on the cancer type and tumor microenvironment [38]. Moreover, some natural compounds from herbs are reported to potentially function as autophagy inducers for cancer therapy [39]. Therefore, the exact role of chrysin in autophagy-mediated CRC cell death awaits further investigation.

Cell Lines and Reagents
SW48, SW480, and SW620 colorectal cancer (CRC) cells were maintained in L15 medium; HT-29 and HCT-116 CRC cells were maintained in McCoy's 5A medium. The medium for the CRC cells was also supplemented with 10% fetal bovine serum (Logan, UT, USA). Chrysin, 3-methyladenine (3-MA), and the antibody against tubulin were purchased from Sigma-Aldrich (St. Louis, MO, USA). The antibodies against poly-ADP-ribose polymerase (PARP), phosphorylated protein kinase B (Akt) (p-Akt) serine 473, Akt, phosphorylated AMP-activated protein kinase alpha (AMPKα) (p-AMPKα) threonine 172, AMPKα, phosphorylated mammalian target of rapamycin (mTOR) (p-mTOR) serine 2448, and mTOR were obtained from Cell Signaling (Danvers, MA, USA). The antibody against caspase-3 was purchased from BioVision (Milpitas, CA, USA). The antibody against LC-3 was obtained from Novus (Littleton, CO, USA). The CM-H2DCFDA dye was purchased from Thermo Fisher Scientific (Waltham, MA, USA). Chrysin, and the combined 5-fluorouracil (5-FU) and oxaliplatin were dissolved in dimethylsulfoxide (DMSO), and the stock solution was prepared as 50 mM. 3-MA was dissolved in 95% ethanol with heating, and working solutions were freshly prepared prior to use. CM-H2DCFDA dye was reconstituted only as required by using DMSO, and working solutions were freshly prepared prior to use.

Cell Viability Assay
Cell viability was assessed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric assays. Cells at a density of 5 × 10 3 cells/well were seeded on 96-well plates overnight, and then treated with the indicated drugs. Three days later, relative cell amounts were determined by adding 1 µg/mL MTT to each well. Then, the medium was removed after 4 h incubation, and formazan was solubilized in 100 µL DMSO per well, followed by the measurement of absorbance at 570 nm.

Immunofluorescence Staining
The GFP-LC3 expression vector was transfected into HCT-116 cells at a ratio of 1:3 DNA to T-pro transfection reagent for two days, followed by treatment with 50 µM chrysin. Then, cells were fixed with 4% paraformaldehyde in phosphate buffered saline (PBS), followed by permeabilization using 1% Triton X-100 in PBS. The GFP-LC3 punctate structures were observed under a fluorescence microscope.

Detection of Intracellular Reactive Oxygen Species (ROS)
SW48 and SW480 cells were seeded overnight, and treated with the indicated concentration of chrysin or 5-fluorouracil (5-FU)/oxaliplatin for 24 h. Then, cells were trypsinized and centrifuged. The cell pellets were stained with 10 µM CM-H 2 DCFDA at 37 • C in the dark for 30 min. Intracellular ROS levels were measured via flow cytometry.