Keywords chrysin - oral squamous cell carcinoma - proliferation - migration - invasion - apoptosis
Introduction
Oral squamous cell carcinoma (OSCC) is the most prevalent malignant tumor of the head
and neck. These cells, which originated from stratified squamous epithelium, account
for 80 to 90% of oral cancer cases.[1 ] OSCC incidence has gradually increased.[2 ] Based on estimates from GLOBOCAN 2020 Thailand, 2.5% of all cancer cases and 2%
of cancer-related deaths were caused by oral cancer.[2 ] At present, the mainstream treatment for OSCC is surgical operation combined with
radiation therapy and/or chemotherapy, but tumors are still prone to recurrence with
a low 5-year survival rate, making patients deformed and suffered from treatment.[3 ] Therefore, alternative treatments for OSCC are required.
Chrysin is a dihydroxyflavone found in propolis, plants, and mushrooms like passion
flowers.[4 ] Propolis which is a natural resin compound in bee glue has 28 g/L of chrysin, supposedly.[5 ] Chrysin has two hydroxyl groups at positions 5 and 7 in a flavone's A ring[6 ] and has antibacterial, antiviral, anti-inflammatory, antioxidant, and anticancer
properties.[7 ] Chrysin has also demonstrated significant potential for inducing apoptosis (programmed
cell death) in cancer cells, as well as for inhibiting the proliferation of cancer
cells, cancer cell migration, and cancer cell invasion. A study of chrysin's anticancer
properties on CAL-27 human tongue carcinoma cells has been reported.[8 ] The CAL-27 cell line, on the other hand, was identified as an oral adenosquamous
carcinoma[9 ], a rare variant of OSCC which is not common.[10 ] For that reason, we focused our research on the effects of chrysin on SCC25 and
HSC4, two oral squamous cell lines that have a common histological type. Our objective
was to determine the cytotoxic effect of chrysin on OSCC cells in vitro , as well as its ability to inhibit OSCC cell proliferation, migration, and invasion.
Materials and Methods
Cell Culture
The HSC4 and SCC25 cell lines are non-human papillomavirus OSCC continuous cell lines
generated from the human tongue. Both cell lines were obtained from the American Type
Culture Collection. HSC4 cells were grown in Dulbecco's Modified Eagle's Medium (DMEM)
supplemented with 10% fetal bovine serum (FBS) in a humidified atmosphere of 5% CO2 at 37°C. SCC25 cells were grown in a 1:1 mixture of DMEM and Ham's F12 media in a
humidified atmosphere of 5% CO2 at 37°C.
Cell Viability Assay
Cell cytotoxicity was measured using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide (MTT) test.[11 ]
[12 ] OSCC cells were seeded at a density of 7 × 104 cells per well in a 96-well plate and incubated for 24 hours at 37°C, 5% CO2 , and 95% relative humidity. After that, 100 μL of negative control, positive control,
and chrysin in six different concentrations (50, 100, 200, 400, 800, and 1000 μM)
were added to each well and incubated for another 24 hours. As a negative control,
0.1% dimethyl sulfoxide (DMSO) was used. Each well received 100 μL of MTT solution.
After 2 hours of incubation at 37°C, the MTT solution was removed, and each well was
washed with 100 μL of phosphate-buffered saline (PBS) solution. Then, 100 μL DMSO
was added to each well to dissolve the formazan from the cells. The number of viable
cells was related to the amount of formazan produced. The absorbance was measured
at 570 nm using an Epoch 2 microtiter spectrophotometer (Agilent Technologies Inc.,
California, United States). The assay was performed in biological triplicate.
Cell Proliferation Assay
OSCC cells were seeded in 96-well plates at a density of 2 × 104 cells per well and incubated for 24 hours at 37°C, 5% CO2 , and 95% relative humidity. The cultured media was then withdrawn. Following that,
selected quantities of chrysin (100 and 200 μM) as well as 100 μL of DMSO-dissolved
negative and positive controls were added and incubated for 1, 3, 5, and 7 days. After
2 hours of incubation at 37°C in a humidified environment with 5% CO2 , in each well, 100 μL of DMSO was added. Finally, using an Epoch 2 microtiter spectrophotometer
(Agilent Technologies Inc.), the absorbance was measured at 570 nm. The test was performed
in biological triplicate.
Cell Migration Assay
An in vitro wound healing assay was used to assess migration.[13 ] HSC4 and SCC25 cells were seeded in a 24-well plate with culture medium at a density
of 100,000 cells per well. The cells were incubated at 37°C, humidified at 5% CO2 environment for 24 hours. The cell monolayer was then scraped off in a straight line
with a 1,000 μL pipette tip. After that, 500 μL of 100 and 200 μM chrysin solution
were added and incubated for 24 hours. The plate was rinsed with PBS at the end of
the incubation period, fixed with ice-cold methanol for 10 minutes, and stained with
0.2% toluidine blue. An inverted microscope was used to photograph cell migration.
Finally, the migration zones were determined using the Image-Pro (Media Cybernetics,
Maryland, United States) program. The negative control was treated with only media,
while the positive control was treated with 100 μM cisplatin. The experiment was done
in biological triplicate.
Cell Invasion Assay
The invasion was assessed using the transwell assay.[14 ] The transwell insert contains a 24-well cell culture plate with 8 μm pores. The
upper transwell chambers were loaded with Matrigel at a density of 100,000 cells per
well and were seeded in serum-free DMEM medium. The lower chamber contains 10% FBS
in DMEM. The OSCC cells were incubated for 24 hours in 37°C, and humidified at 5%
CO2 environment after being treated with 100 μL of 100 and 200 μM chrysin. At the end
of the incubation period, invaded cells at the bottom of the membrane were fixed and
stained. Noninvading cells on the membrane's upper surface were removed by scrubbing
with a sterile cotton pellet. The transwell inserts were fixated for 10 minutes in
ice-cold ethanol before being stained for 15 minutes with 0.2% crystal violet. Methanol
was used to elute the samples, and absorbance was measured and compared with the control.
The negative control received only media, while the positive control received 100
μM cisplatin. The test was performed in biological triplicate.
Apoptosis Assay
Flow cytometry was used to examine the apoptosis of SCC25 and HSC4 cells. Approximately
100,000 cells were stained for 30 minutes at room temperature using Annexin V Alexa
Fluor 488 (BioLegend, California, United States) and propidium iodide (PI) (BioLegend).
Fluorescence-activated cell sorting buffer was added to the cells for flow cytometry
cell type quantification (LSRII, BD Biosciences, California, United States). The cell
types and frequencies were then gated using both forward scatter and side scatter.
Finally, the data was examined using the FlowJo program (Ashland, Oregon, United States).
The test was repeated three times in biological triplicate.
Statistical Analysis
PASW Statistics 18.0.0 and GraphPad Prism 6.0 were utilized for statistical analysis.
To evaluate quantitative data, the Kruskal–Wallis test was performed. The results,
which were largely measurements, were shown as mean and standard deviation. A p -value of less than 0.05 was considered statistically significant.
Results
Cytotoxicity Effect of Chrysin on OSCC Cells
The half-maximal inhibitory concentration (IC50) for any of the chrysin concentrations
used in this experiment (50, 100, 200, 400, 800, and 1,000 µM) for both HSC4 and SCC25
was not measured. Chrysin-treated HSC4 cells showed a gradual decrease in cell viability
from 50 to 1,000 µM concentration of chrysin ([Fig. 1A ]). Similarly, from 50 to 1000 µM concentration of chrysin, the percent cell viability
of chrysin-treated SCC25 cells steadily declined ([Fig. 1B ]). At 100 and 200 µM, chrysin showed that more than 75% of the cells in both cell
lines were still alive. So, 100 and 200 µM of chrysin were chosen as the best amounts
for further tests. Supplementary [Table S1 ] (available in the online version) provides more information on the data shown.
Fig. 1 Cytotoxicity effect of chrysin on cell lines in vitro using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide (MTT) cell viability assay (A ) HSC4 and (B ) SCC25. The cells were treated with six concentrations of chrysin (50, 100, 200,
400, 800, and 1,000 µM) for 24 hours. The antiproliferative effect of chrysin on cell
lines in vitro using cell proliferation assay (C ) HSC4, (D ) SCC25. The cells were treated with two concentrations of chrysin (100 and 200 µM)
for 1, 3, 5, and 7 days. The negative control contained 0.1% dimethyl sulfoxide (DMSO).
Antiproliferative Effect of Chrysin on OSCC Cells
HSC4 cells treated with 100 µM concentration of chrysin showed a gradual decrease
in absorbance after day 5 after of incubation. But after day 3, 200 µM chrysin drastically
decreased the absorbance of HSC4 cells ([Fig. 1C ]). For the SCC25 cell line, 100 and 200 µM chrysin-treated cells exhibited gradual
decreases in absorbance after day 3 with statistical significance when compared with
the control group ([Fig. 1D ]). As a consequence, chrysin at 100 and 200 µM were suggested to have the ability
to inhibit proliferation. Additional details about this data can be found in Supplementary
[Table S2 ].
Antimigratory and Anti-Invasion Effect of Chrysin on OSCC Cells
In comparison to the control group, HSC4 and SCC25 treated with chrysin 100 and 200
µM showed a concentration-dependent reduction in migratory regions ([Fig. 2A–C ], Supplementary [Table S3 ], available in the online version). The ability to reduce cell migration is comparable
to that of cisplatin-treated cells.
Fig. 2 Antimigratory effect of chrysin on oral squamous cell carcinoma (OSCC) cell lines
in vitro using wound-healing assay. HSC4 and SCC25 cells were treated with two concentrations
of chrysin (100 and 200 µM) for 24 hours, compared with the negative control (0.1%
dimethyl sulfoxide [DMSO]) and the positive control (100 µM cisplatin). (A ) Microscopic pictures. (B ) Histogram of HSC4. (C ) Histogram of SCC25.
Chrysin-treated HSC4 and SCC25 showed a concentration-dependent reduction in absorbance
and percentage of invasion as compared with the control group ([Fig. 3A–C ], Supplementary [Table S4 ]). Similar to the migratory effect, the ability to reduce cell invasion is equivalent
to that of cisplatin-treated cells
Fig. 3 Anti-invasion effect of chrysin on oral squamous cell carcinoma (OSCC) cell lines
in vitro using transwell assay. HSC4 and SCC25 cells were treated with two concentrations
of chrysin (100 and 200 µM) for 24 hours, compared with the negative control (0.1%
dimethyl sulfoxide [DMSO]) and the positive control (100 µM cisplatin). (A ) Microscopic pictures. (B ) Histogram of HSC4. (C ) Histogram of SCC25.
Chrysin Enhance Apoptotic Effect of OSCC Cells
The flow cytometry results were gated and exemplified in [Fig. 4A ] and [B ]. According to annexin-V/PI staining, SCC25 and HSC4 cells treated with chrysin had
a higher rate of apoptosis than the control and a similar level to cisplatin-treated
cells. The apoptotic rate of HSC4 cells is influenced by chrysin concentration, especially
early apoptosis. In comparison to 100 μM chrysin-treated OSCC cells, 200 μM chrysin-treated
OSCC cells have a higher percentage of early apoptosis and a similar percentage of
late apoptosis, indicating that chrysin has a significant impact on early apoptosis
([Fig. 4C–H ]). Additional information on the apoptosis data is available in Supplementary [Table S5 ], available in the online version.
Fig. 4 Apoptotic effect of chrysin on oral squamous cell carcinoma (OSCC) cell lines in vitro using flow cytometry assay. HSC4 and SCC25 cells were stained with annexin V and
propidium iodide (PI). Both cells treated with two concentrations of chrysin (100
and 200 µM) for 24 hours, compared with the negative control (0.1% dimethyl sulfoxide
[DMSO]) and the positive control (100 µM cisplatin). (A , B ) Flow cytometry gating. Histogram of (C , D ) total apoptosis, (E , F ) early apoptosis, and (G , H ) late apoptosis.
Discussion
Our study aims to explore chrysin's antiproliferative, antimigratory, anti-invasion,
and apoptotic effects on HSC4 and SCC25. The two cell lines, HSC4 and SCC25, are both
OSCC cell lines that were originally excised from the human tongue. However, chrysin
has been shown to affect the two cell lines differently. This is possibly due to the
different characteristics of the two cell lines. HSC4 is a metastasized cancer from
the cervical lymph node.[15 ] Meanwhile, SCC25 is a primary OSCC of the tongue.[16 ] Resistance to cytotoxic agents is common in metastatic tumors, which corresponds
to our findings.
In the present study, the cytotoxic potential of chrysin on HSC4 and SCC25 cell lines
was evaluated using the MTT cell viability assay. As a result, the viability of chrysin-treated
OSCC cells gradually decreased as the concentration of chrysin was increased. However,
cell viability of HSC4 cells treated with a 400 µM concentration of chrysin, as well
as SCC25 cells treated with 800 µM chrysin, slightly increased compared with previous
concentrations. Even though the experiment was repeated three times, errors could
have occurred due to interexperiment and intraexperiment variations. Furthermore,
because all concentrations of chrysin in this experiment were not assessed with the
half-maximal inhibitory concentration, chrysin under 1,000 μM showed no significant
inhibitory effect against both HSC4 and SCC25 cell lines from the IC50. Chrysin at
100 and 200 µM concentrations increased cell viability in both OSCC cell lines by
more than 75%. Therefore, 100 and 200 µM concentrations of chrysin were selected for
further experiments due to their low toxicity. Chrysin has been reported to suppress
proliferative activity in various cancer cells.[6 ]
[17 ] Likewise, the findings of this study revealed that chrysin has antiproliferative
effects against HSC4 and SCC25 cell lines at 100 and 200 µM concentrations, the latter
of which is superior.
OSCC has a highly invasive nature along with a high rate of lymph node metastasis.
Metastasis of cancer is a complex process in which the primary tumor disseminates
and travels to distant organs, resulting in the growing tumor within secondary sites.[18 ] Cancer cells can spread by several cellular processes, such as cell migration and
cell invasion.[19 ]
Cell migration refers to the ability of cells to move within the extracellular matrix,
and the process is recreated in vitro as the wound healing assay.[13 ]
[18 ] At 100 and 200 µM concentration, chrysin exhibited a significant antimigratory effect
on OSCC cell lines (HSC4 and SCC25), resulting in a decrease of migrated areas compared
with control within a 24-hour time interval. Moreover, the result shows that 200 µM
chrysin antimigration effects on both HSC4 and SCC25 cell lines are superior to the
positive control (100 µM cisplatin). Cell invasion refers to the ability of cells
to invade through the basement membrane. The process is recreated in vitro by the
transwell assay. The results have shown that at 100 and 200 µM concentrations, chrysin
has effectively inhibited the cell invasion of both OSCC cell lines (HSC4 and SCC25).
Furthermore, the results revealed that the anti-invasion effects of 200 µM chrysin
on both HSC4 and SCC25 cell lines are comparable to the positive control (100 µM cisplatin).
Together, chrysin may be better or comparable to cisplatin in its ability to inhibit
OSCC invasion and metastasis.
Several studies in different cancer cell lines found that chrysin increased apoptosis.[20 ]
[21 ]
[22 ]
[23 ]
[24 ] Furthermore, chrysin was discovered to significantly induce both early and late
apoptosis in cervical cancer cells[25 ] and prostate cancer cell lines.[26 ] According to our findings, chrysin has a greater impact on early apoptosis. These
findings support the idea that chrysin primarily affects tumor growth by inducing
apoptosis via death receptors and/or activating proapoptotic members of the B cell
lymphoma 2 (Bcl-2) pathway.[27 ]
[28 ]
The limitations of the study were that all experiments were conducted in vitro and on only two cell lines (HSC4 and SCC25). To determine the clinical efficacy of
chrysin on OSCC cells, additional in vitro and in vivo studies on additional OSCC cell lines are required. In addition, the precise mechanism
underlying chrysin's antimigratory and anti-invasion effects on OSCC cells should
be investigated in greater detail.
Conclusion
The current study collectively characterized the critical role of chrysin on OSCC
cells, as it could inhibit their proliferation, migration, and invasion abilities,
while enhancing cell apoptosis. And hence, 100 and 200 µM chrysin possessed the potential
to reduce the metastatic ability of OSCC. In particular, 200 μM chrysin has equivalent
antimigratory and anti-invasion effects compared with 100 μM cisplatin. And 200 µM
of chrysin is better compared with 100 µM of chrysin for antimigratory, anti-invasion,
and antiproliferative applications. As a result, chrysin 200 μM can be used in conjunction
with current OSCC treatment. Our opinion is that chrysin may be suggested for topical
application in selected circumstances.
