Effects of 2′,4′-Dihydroxy-6′-methoxy-3′,5′-dimethylchalcone from Syzygium nervosum Seeds on Antiproliferative, DNA Damage, Cell Cycle Arrest, and Apoptosis in Human Cervical Cancer Cell Lines

2′,4′-Dihydroxy-6′-methoxy-3′,5′-dimethylchalcone (DMC), a natural product derived from Syzygium nervosum A. Cunn. ex DC., was investigated for its inhibitory activities against various cancer cell lines. In this work, we investigated the effects of DMC and available anticervical cancer drugs (5-fluorouracil, cisplatin, and doxorubicin) on three human cervical cancer cell lines (C-33A, HeLa, and SiHa). DMC displayed antiproliferative cervical cancer activity in C-33A, HeLa, and SiHa cells, with IC50 values of 15.76 ± 1.49, 10.05 ± 0.22, and 18.31 ± 3.10 µM, respectively. DMC presented higher antiproliferative cancer activity in HeLa cells; therefore, we further investigated DMC-induced apoptosis in this cell line, including DNA damage, cell cycle arrest, and apoptosis assays. As a potential anticancer agent, DMC treatment increased DNA damage in cancer cells, observed through fluorescence inverted microscopy and a comet assay. The cell cycle assay showed an increased number of cells in the G0/G1 phase following DMC treatment. Furthermore, DMC treatment-induced apoptosis cell death was approximately three- to four-fold higher compared to the untreated group. Here, DMC represented a compound-induced apoptosis for cell death in the HeLa cervical cancer cell line. Our findings suggest that DMC, a phytochemical agent, is a potential candidate for antiproliferative cervical cancer drug development.


Introduction
The global incidence of cancer is continually increasing, with an estimated 18.1 million new patients diagnosed with cancer in 2018 [1,2]. Cervical cancer accounts for an estimated 65,620 new cases and 12,590 deaths per year [3]. In 2020, incidence and mortality rates of cervical cancer have been increased in most regions of the world. On the other hand, . Significant differences are shown at * p ≤ 0.033, ** p ≤ 0.02, and *** p ≤ 0.001; ns, not significant (p ≥ 0.05).

Isolation and Structural Identification of DMC
DMC was isolated from S. nervosum seeds by maceration using CH2Cl2 as the solvent and purified using a chromatographic technique. TLC chromatogram of crude CH2Cl2 extract from S. nervosum seeds visualized under UV 254 nm ( Figure S2). The chemical structure of DMC was verified by comparison of its spectroscopic data to previously published values, and all values were found to be identical with previous study [15,16]. The physical appearance of DMC is a yellow needle-shaped crystals obtained from crystallization using n-hexane-CH2Cl2, and its melting point is 124.2-125.6 °C. The UV-VIS spectrum of DMC in phosphate buffered saline at pH 7.4 ( Figure S3) showed and minor band of benzoyl system at wavelength of maximum absorbance (λmax) of 228 nm and major band of cinnamoyl system at λmax of 395 nm, which are characteristic of flavonoids with a structural skeleton of chalcone [26]. The methoxy group (O-CH3) showed absorption band at λmax of 314 nm [27].

Isolation and Structural Identification of DMC
DMC was isolated from S. nervosum seeds by maceration using CH 2 Cl 2 as the solvent and purified using a chromatographic technique. TLC chromatogram of crude CH 2 Cl 2 extract from S. nervosum seeds visualized under UV 254 nm ( Figure S2). The chemical structure of DMC was verified by comparison of its spectroscopic data to previously published values, and all values were found to be identical with previous study [15,16]. The physical appearance of DMC is a yellow needle-shaped crystals obtained from crystallization using n-hexane-CH 2 Cl 2 , and its melting point is 124.2-125.6 • C. The UV-VIS spectrum of DMC in phosphate buffered saline at pH 7.4 ( Figure S3) showed and minor band of benzoyl system at wavelength of maximum absorbance (λ max ) of 228 nm and major band of cinnamoyl system at λ max of 395 nm, which are characteristic of flavonoids with a structural skeleton of chalcone [26]. The methoxy group (O-CH 3 ) showed absorption band at λ max of 314 nm [27].

Antiproliferative Activity of DMC on Cervical Cancer Cell Lines
The antiproliferative activities of DMC and the anticancer drugs (5-fluorouracil, cisplatin, and doxorubicin) were investigated by MTT assay. The IC 50 of DMC and the available anticancer drugs are shown in Table 1. DMC showed potential antiproliferative cervical cancer activity, with IC 50 values of 10.05 ± 0.22, 15.76 ± 1.49, and 18.31 ± 3.10 µM in HeLa, C-33A, and SiHa cells, respectively (Table 1 and Figure 1a). Similarly, cisplatin treatment in HeLa cells had an IC 50 value of 9.93 ± 0.16 µM. The IC 50 values of DMC, cisplatin and doxorubicin were significantly different, with p-values of p ≤ 0.033, p ≤ 0.02, and p ≤ 0.001, respectively, compared to every other cell model in the two-way ANOVA ( Figure 1b). Thus, DMC represents a promising specific antiproliferative cervical cancer agent in HeLa cells. literature [15].

Antiproliferative Activity of DMC on Cervical Cancer Cell Lines
The antiproliferative activities of DMC and the anticancer drugs (5-fluorouracil, cisplatin, and doxorubicin) were investigated by MTT assay. The IC50 of DMC and the available anticancer drugs are shown in Table 1. DMC showed potential antiproliferative cervical cancer activity, with IC50 values of 10.05 ± 0.22, 15.76 ± 1.49, and 18.31 ± 3.10 µ M in HeLa, C-33A, and SiHa cells, respectively (Table 1 and Figure 1a). Similarly, cisplatin treatment in HeLa cells had an IC50 value of 9.93 ± 0.16 µ M. The IC50 values of DMC, cisplatin and doxorubicin were significantly different, with p-values of p ≤ 0.033, p ≤ 0.02, and p ≤ 0.001, respectively, compared to every other cell model in the two-way ANOVA (Figure 2). Thus, DMC represents a promising specific antiproliferative cervical cancer agent in HeLa cells.

Investigation of Compound-Induced Apoptosis
However, DMC has not yet been fully studied for its biological properties in cervical cancer. Accordingly, DMC has drawn attention for further investigation as a promising anticancer drug for cervical cancer Thus, DMC induces apoptosis were investigated by comet, cell cycle, and apoptosis assays and compared to the three currently available an-

Investigation of Compound-Induced Apoptosis
However, DMC has not yet been fully studied for its biological properties in cervical cancer. Accordingly, DMC has drawn attention for further investigation as a promising anticancer drug for cervical cancer Thus, DMC induces apoptosis were investigated by comet, cell cycle, and apoptosis assays and compared to the three currently available anticervical cancer drugs.

DMC-Induced DNA Damage Pathways
The effect of treatment with DMC as an antiproliferative cervical cancer drug candidate on DNA damage pathways was investigated by comet assay and compared to untreated and 1% DMSO groups [28,29]. The currently available anticervical cancer drugs, including 5-fluorouracil, cisplatin, and doxorubicin, were used as positive controls. The comet assay was performed with HeLa cells treated with DMC, the available anticervical cancer drugs, 1% DMSO, or untreated cells for specific time periods (6,12,24, and 48 h) under alkaline conditions. Comet assay with alkaline conditions can explain the single-and double-strand break with DNA damage and DNA repair after treatment with anticancer drugs [30][31][32].
The DNA tails of HeLa cells after treatment with DMC for 6, 12, 24, and 48 h showed compound-induced DNA damage patterns. Similar results were observed in cells treated with the available anticervical cancer drugs for 6, 12, 24, and 48 h when compared to the 1% DMSO and untreated groups, as shown in Figures 2 and 3. Images of DNA tails were analyzed by Comet Score version 2.0, with a selected sample size (n) of 50. The analyzed parameters, including the tail moment and tail DNA%, are presented in Table 2. The DNA damage parameters in HeLa cervical cancer cells after treatment with DMC and the available anticervical cancer drugs demonstrated significant differences at * p ≤ 0.033, ** p ≤ 0.02, and *** p ≤ 0.001, as indicated in Figure 4, when compared to untreated and 1% DMSO groups. After treatment with DMC for 6, 12, 24, and 48 h, the tail DNA% of HeLa cells increased 14-, 15-, 17-, and 25-fold, respectively, compared to the untreated group. In addition, tail DNA% in the DMC group was 27-, 11-, 18-, and 30-fold higher than the 1% (w/w) DMSO group at 6, 12, 24, and 48 h, respectively. The tail moment values in cells treated with DMC were increased by 102-, 107-, 313-, and 833-fold compared to the untreated group at 6, 12, 24, and 48 h, respectively. Moreover, comparison of the percentage tail moment between DMC and 1% (w/w) DMSO groups showed 381-, 143-, 355-, and 1296-fold increases after 6, 12, 24, and 48 h of treatment, respectively. These results demonstrate increased DNA damage after treatment with DMC for the various incubation times. In addition, the tail DNA% and tail moment values of HeLa cells after treatment with DMC were similar to those observed after doxorubicin treatment. However, HeLa cells treated with 5-fluorouracil and cisplatin showed an increase in tail moment and tail DNA% after the first 6 h of treatment, but these values subsequently decreased at 12, 24, and 48 h.
The effect of treatment with DMC as an antiproliferative cervical canc date on DNA damage pathways was investigated by comet assay and co treated and 1% DMSO groups [28,29]. The currently available anticervical including 5-fluorouracil, cisplatin, and doxorubicin, were used as positive comet assay was performed with HeLa cells treated with DMC, the availab cancer drugs, 1% DMSO, or untreated cells for specific time periods (6, 12 under alkaline conditions. Comet assay with alkaline conditions can expl and double-strand break with DNA damage and DNA repair after treatm cancer drugs [30][31][32]. The DNA tails of HeLa cells after treatment with DMC for 6, 12, 24, an compound-induced DNA damage patterns. Similar results were observed with the available anticervical cancer drugs for 6, 12, 24, and 48 h when co 1% DMSO and untreated groups, as shown in Figures 2 and 3. Images of D analyzed by Comet Score version 2.0, with a selected sample size (n) of 50. parameters, including the tail moment and tail DNA%, are presented in Tab damage parameters in HeLa cervical cancer cells after treatment with DMC able anticervical cancer drugs demonstrated significant differences at *p ≤ 0 and *** p ≤ 0.001, as indicated in Figure 4, when compared to untreated a groups. After treatment with DMC for 6, 12, 24, and 48 h, the tail DNA% increased 14-, 15-, 17-, and 25-fold, respectively, compared to the untreated dition, tail DNA% in the DMC group was 27-, 11-, 18-, and 30-fold highe (w/w) DMSO group at 6, 12, 24, and 48 h, respectively. The tail moment treated with DMC were increased by 102-, 107-, 313-, and 833-fold compa treated group at 6, 12, 24, and 48 h, respectively. Moreover, comparison of tail moment between DMC and 1% (w/w) DMSO groups showed 381-, 1 1296-fold increases after 6, 12, 24, and 48 h of treatment, respectively. These r strate increased DNA damage after treatment with DMC for the various inc In addition, the tail DNA% and tail moment values of HeLa cells after t DMC were similar to those observed after doxorubicin treatment. Howev treated with 5-fluorouracil and cisplatin showed an increase in tail mo DNA% after the first 6 h of treatment, but these values subsequently decre and 48 h.

Inhibition of the G0/G1 and G2/M Phases
This work investigated the mechanism of DMC-induced cell death through the induction of cell cycle arrest after treatment with DMC at its IC50 concentration in a timedependent manner (6, 12, 24, and 48 h). HeLa cells treated with DMC, the available anticervical cancer drugs, or 1% (w/w) DMSO were analyzed by flow cytometry, and the results are presented in Figure 5 and S9. The results showed an increased population of cells in the G0/G1 phase after treatment with DMC, estimated at 63.13 ± 3.21, 64.67 ± 6.66, and results are presented in Figure 5 and Figure S9. The results showed an increased population of cells in the G 0 /G 1 phase after treatment with DMC, estimated at 63.13 ± 3.21, 64.67 ± 6.66, and 67.40 ± 3.51 after 12, 24, and 48 h, respectively, when compared to untreated cells and cells treated with 1% DMSO. After 6 h, HeLa cells treated with DMC showed no significant differences when compared to untreated cells and cells treated with 1% DMSO. In addition, the effect of DMC on the G 0 /G 1 phase of HeLa cells was similar to the effect after 12 h of doxorubicin treatment. In addition, the findings were similar to those observed for HeLa cells treated with 5-fluorouracil and cisplatin for 48 h. The G 0 /G 1 and G 2 /M phase values of HeLa cells treated with DMC and the available anticervical cancer drugs were significantly different to the untreated and 1% DMSO groups (* p ≤ 0.033, ** p ≤ 0.02, and *** p ≤ 0.001) in the two-way ANOVA. Non-significant (ns) values had a p-value ≥ 0.12. Accordingly, DMC treatment demonstrated potential antiproliferative cervical cancer activities in HeLa cells by inhibiting proliferation in the G 0 /G 1 phase of the cell cycle.

DMC-Induced Apoptosis
HeLa cells treated with DMC and anticervical cancer agents at IC 50 concentrations for 24 and 48 h clearly showed DNA damage and inhibition of proliferation in the cell cycle. Therefore, we investigated the apoptosis pathway by flow cytometry, and the results are presented in Figure 6a. HeLa cells treated with DMC, the available anticervical cancer drugs, or 1% (w/w) DMSO were evaluated for annexin V-FITC and PI staining. After treatment with the active compounds, HeLa cells were categorized into four populations: live, early, late/dead, and dead populations (Figure 6b). The live population did not show any annexin V-FITC or PI staining. In the early population, only annexin V-FITC staining was detected. The late/dead population presented both annexin V-FITC and PI staining. Finally, the dead population showed only PI staining. The early and late/dead populations were involved in the apoptosis pathway. We then investigated the percentage of apoptosis cell death after treatment with DMC and the three anticervical cancer agents. The percentage of apoptosis cell death increased after treatment with DMC, estimated to be 29.22 ± 0.73% and 50.50 ± 4.58% after 24 and 48 h, respectively, when compared to the untreated and 1% DMSO groups. Similarly, doxorubicin treatment of HeLa cells for 48 h showed approximately 55.63 ± 2.35% apoptosis cell death. Interestingly, treatment of HeLa cells with doxorubicin for 24 h showed an increased percentage of apoptosis cell death compared to DMC treatment, with an estimated increase of 44.05 ± 7.71%, which is twofold higher than the cell death observed following DMC treatment. In addition, cisplatin and 5-fluorouracil treatment of HeLa cells for 48 h showed an estimated 25.42 ± 4.93% and 26.36 ± 0.60% apoptosis cell death, respectively. Treatment with cisplatin and 5-fluorouracil for 24 h did not increase the percentage apoptotic cell death of HeLa cells compared to the untreated and 1% DMSO groups. The percentage of apoptosis cell death observed in HeLa cells treated with DMC and the available anticervical cancer drugs demonstrated significant differences T * p ≤ 0.033, ** p ≤ 0.02, and *** p ≤ 0.001 when compared to the untreated and 1% DMSO groups in the two-way ANOVA. Non-significant (ns) differences showed p-values ≥ 0.12, as shown in Figure 7. Moreover, the death populations were considered to be an indicator of inflammatory mediators after treatment with DMC and the available anticervical cancer drugs [33]. DMC treatment of HeLa cells for 24 h showed a low percentage of inflammatory mediators, estimated to be 2.22 ± 0.65%, which was similar to that observed for doxorubicin treatment. After 48 h, HeLa cells treated with DMC showed a low percentage of inflammatory mediators (11.06 ± 3.91%), when compared to cells treated with 5-fluorouracil (33.63 ± 0.76%), similar to that observed for doxorubicin treatment. Therefore, DMC treatment demonstrated potential antiproliferative cervical cancer activity in HeLa cells involving apoptosis pathways.

Discussion
The majority of publications on Syzygium nervosum A. Cunn. ex DC. have focused on its phytochemical content, such as chalcone and flavanone derivatives [10,15,16]. DMC is a chalcone derivative isolated from the seeds of S. nervosum. DMC presents many biological activities, such as antioxidant, antiviral, and anticancer activities [32][33][34]. However, DMC has not been fully investigated for its effect on cell damage in cervical cancer. In our work, we investigated the biological activities of DMC from S. nervosum. Accordingly, DMC showed specific antiproliferative cervical cancer activities, with an IC 50 value of 10.05 ± 0.22 µM in an adenocarcinoma model (HeLa), which was greater than the IC 50 values for C-33A and SiHa cells. Similarly, cisplatin treatment showed an IC 50 value of 9.93 ± 0.16 µM in HeLa cells. Previously, DMC has been shown a role in compoundinduced apoptosis and G 0 /G 1 cell cycle arrest through the PI3K/AKT mitochondriadependent pathway and increased generation of reactive oxygen species (ROS) as part of the apoptosis pathways in hepatocellular carcinoma (BEL-7402 and BEL-7402/5-FU cell lines). Moreover, DMC was found to be nontoxic to human normal liver cells (L-02), human normal fetal lung fibroblast cells (HFL-1), and an ICR mouse model [24,25]. Therefore, in the current study, we investigated the biological properties of DMC as a potential antiproliferative cervical cancer agent using comet, cell cycle, and apoptosis assays.
After treatment with DMC for 6-48 h, HeLa cells showed increased DNA damage when compared to the untreated and 1% DMSO groups, similar to that observed for the available anticancer drugs. The increased tail moment and tail DNA percentage indicates DNA damage-and replication stress-induced double-strand and single-strand DNA breaks via ATM (Ataxia telangiectasia mutated)-and ATR (Ataxia telangiectasia and Rad3-related protein)-mediated pathways. The ATM and ATR signalling pathways are amplified by DNA damage signals, which activate the tumor suppressor p53 protein (p53) through the Checkpoint kinase 1 (CHK1) and Checkpoint kinase 2 (CHK2) regulator proteins, leading to cell cycle arrest and apoptosis [35]. Moreover, there was a decrease in tail moment and tail DNA percentage after treatment with cisplatin and 5-fluorouracil for 12, 24, and 48 h, indicating DNA repair. Similar to previously reports, 5-fluorouracil-and cisplatin-induced DNA damage primarily occurs through the DNA repair mechanism by nucleotide excision repair (NER) and DNA mismatch repair (MMR) pathways, which are involved in cisplatin resistance in human cervical cancer [36][37][38].
In addition, the proliferation of HeLa cells after treatment with DMC was timedependent, as observed by flow cytometry. After 6 h of treatment with DMC, HeLa cells showed no change in the number of cells in the G 0 /G 1 and G 2 /M phases. However, after 12-48 h of DMC treatment, the number of HeLa cells in the G 0 /G 1 phase was increased, which indicates the inhibition of CDK/cyclin complexes by p16 and p21 following DNA damage and p53 activation, similar to that observed after 5-fluorouracil and cisplatin treatment for 48 h [36][37][38][39].
Moreover, HeLa cells treated with DMC for 24 and 48 h were evaluated for the percentage of apoptotic cell death by flow cytometry. After DMC treatment for 24 h, the percentage apoptosis cell death of HeLa cells was increased approximately threefold when compared to untreated cells and cells treated with 1% DMSO. Cells treated with 5-fluorouracil and cisplatin did not show any percentage of apoptosis cells death. On the other hand, doxorubicin increased the percentage of apoptosis cell death fourfold when compared to the untreated group. After 48 h of DMC treatment, HeLa cells demonstrated an estimated percentage of apoptosis cell death that was increased by fourfold when compared to the untreated and 1% DMSO groups, similar to that observed for doxorubicin treatment. DMC also presented higher potential antiproliferative cervical cancer activities, estimated to be twofold higher compared to cells treated with 5-fluorouracil or cisplatin. In addition, HeLa cells treated with DMC showed low inflammatory mediators, estimated to be threefold lower than that observed for 5-fluorouracil as an anticervical cancer drug. Inflammatory mediators can promote tumor growth and metastasis through the stimulation of cell proliferation and toxicity to healthy cells [40].
After treatment with DMC, HeLa cells showed increasing DNA damage, G 0 /G 1 phases, and induced apoptosis cell death when compared to the untreated and 1% DMSO groups. These results suggest that the cell death mechanism by the ATM and ATR signalling pathways after increased DNA damage activates the p53 protein through the CHK1 and CHK2 regulator proteins, leading to cell cycle arrest and induced apoptosis cell death [41][42][43]. Accordingly, the findings of the current study suggest an antiproliferative cervical cancer effect of DMC through the induction of the cell death mechanism.

Isolation and Structural Identification of 2 ,4 -Dihydroxy-6 -methoxy-3 ,5 -dimethylchalcone
The isolation of DMC was achieved as previously reported [15,16]. The voucher specimen of the plant (BKF no. 187213) was deposited at the Forest Herbarium, Department of National Parks, Wildlife, and Plant Conservation, Ministry of Natural Resources and Environment, Bangkok, Thailand. Briefly, 5 kg of dried seed powder of S. nervosum was macerated with 10 L of CH 2 Cl 2 (performed three times, each time for 3 days). The seed residues were filtered out, and the filtrate was collected and further concentrated under reduced pressure to produce crude CH 2 Cl 2 extract as a dark green viscous liquid using a rotary evaporator (Hei-Vap Value Digital, Heidolph Instruments GmbH & Co., Schwabach, Germany). The extract was subjected to flash column chromatography on a silica gel, eluted with gradient concentrations of n-hexane-EtOAc from 100:0 to 80:20. The DMC-containing fractions were collected and further purified by crystallization with CH 2 Cl 2 -n-hexane to yield 2.8645 g of DMC as yellow needle-shaped crystals with high purity. The isolated DMC was verified by comparison to previously reported 1 H-and 13

Determination of Antiproliferative Activity
The antiproliferative cervical cancer activity of DMC were investigated in the human cervical cancer cell lines C-33A (ATCC ® HTB-31™), HeLa (ATCC ® CCL-2™), and SiHa (ATCC ® HTB-35™) using the MTT assay. The cell lines (5 × 10 3 cells/mL) were seeded in 96-well plates and incubated for 24 h at 37 • C in 5% CO 2 atmosphere. DMC and the three available anticancer drugs (5-fluorouracil, cisplatin, and doxorubicin) at concentrations of 0-50 µM were fixed in 1% DMSO and incubated for 48 h. The culture medium was removed and cells were washed with phosphate-buffered saline (PBS, pH 7.4). A 5 mg/mL aliquot of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide solution (MTT) was added, and the plate was incubated for 4 h at 37 • C in 5% CO 2 atmosphere. The purple formazan crystals produced were dissolved in DMSO and measured using a SpectraMax i3x multi-mode microplate reader at 540 and 620 nm. The percentages of cell viability and half maximal inhibitory concentration (IC 50 ) were calculated by nonlinear regression (curve fit) using GraphPad Prism version 8.0. Finally, data were analyzed for statistical significance by two-way ANOVA for active compound classification for potential anticervical cancer drugs.

DNA Damage Assessed by the Comet Assay
DNA damage of DMC and the available anticancer drugs was investigated. Human cervical cancer cells (HeLa) at a concentration of 2.5 × 10 5 cells/mL were incubated in 6-well plates for 24 h at 37 • C in a 5% CO 2 atmosphere. DMC and the available anticancer drugs (5-fluorouracil, cisplatin, and doxorubicin) were applied to HeLa cells at their IC 50 concentration for 6, 12, 24, and 48 h. Untreated cells and cells treated with 1% (v/v) DMSO were used as the negative controls. After treatment with DMC, the cell line was investigated for DNA damage using the modified comet assay [28,29]. For cell slide preparation, glass slides were covered with 1.0% (w/v) agarose gel at 4 • C until the gel had solidified. PBS (pH 7.4) was added to the human cervical cancer cell lines (HeLa), then cell suspensions (80 µL) were fixed in low-melting agarose gel to give a final concentration of 2 × 10 4 cells/gel. The mixture solution (330 µL) was seeded on agarose gel (1.0%) and the cover glass was applied. Then, preparation of the cell slides was performed. Cell slides were incubated overnight in lysis buffer (Tris-base 0.001 M, NaCl 2.5 M, EDTA 0.01 M, DMSO 10% (v/v), Triton-X100 1% (v/v); pH 10.0). The cell slides were neutralized with neutralizing buffer (Tris-base pH 7.5, 0.40 M) for 20 min, then DNA was separated by electrophoresis in an alkaline running buffer (Tris-base 0.90 M, NaOH 0.125 M; pH 13.0) at 20 V for 25 min. Next, the cell slides were incubated with neutralizing buffer for 20 min and stained with propidium iodide (PI; 10 µg/mL) dye for 20 min in a dark room. Cell slides were destained with deionized water (DI) three time for 20 min to remove all background PI dye. DNA damage was determined by fluorescence microscopy with a digital camera (DP70; Olympus, Tokyo, Japan) at maximum excitation and emission wavelengths of 535 and 617 nm, respectively. Data analysis was performed using Comet Score version 2.0 software. The computational parameters included the percentage tail DNA (tail DNA%) and the tail moment. Tail DNA% is the tail DNA content as a percentage of the comet DNA content. Tail moment is the tail length multiplied by the tail DNA%.

Cell Cycle Assay by Flow Cytometry
Cell cycle assay was performed using modified protocol from Darzynkiewicz et al., 1999;Rajamanikyam et al., 2017 andKantapan et al., 2020 [44-46]. HeLa human cervical cancer cells (2.5 × 10 5 cells) were seeded in 6-well plates and cultured for 24 h. DMC and the available anticancer drugs (5-fluorouracil, cisplatin, and doxorubicin) were applied to HeLa cells at their IC 50 concentrations for 6, 12, 24, and 48 h. Untreated cells and cells treated with 1% (v/v) DMSO were used as the negative controls. After treatment with DMC or the anticancer drugs, human cervical cancer cells (HeLa) were fixed overnight in 70% (v/v) ethanol. Next, the cervical cancer cells were washed with PBS (pH 7.4) and centrifuged at 11,000 rpm for 1 min. PBS (pH 7.4; 11.25 µL) and Triton X-100 (0.5% v/v) were then added to the cell lines. RNase (0.5 mg/mL) and PI dye (200 ng/mL) were mixed with the human cervical cancer cells and incubated at 37 • C in a dark room for 30 min. PBS (pH 7.4; 50 µL) was then added to the cells. The DNA content, indicated by PI staining, was examined by imaging flow cytometry (FlowSight, Seattle, WA, USA) with an excitation wavelength of 595 nm and maximum emission of 642 nm. Data were analyzed using IDEAS version 6.2 (Amnis, Seattle, WA, USA).

Annexin V-FITC and Propidium Iodide Staining to Evaluate Apoptosis
Apoptosis assay was performed using modified protocol from Bian et al., 2019 [47]. HeLa human cervical cancer cells (2.5 × 10 5 cells) were seeded in 6-well plates for 24 h. DMC and the available anticancer drugs (5-fluorouracil, cisplatin, and doxorubicin) were applied to HeLa cells at their IC 50 concentrations for 24 and 48 h. Untreated cells and cells treated with 1% (v/v) DMSO were used as the negative controls. Treated and untreated HeLa cells were stained with annexin V-fluorescein isothiocyanate (FITC) using the ApopNexin Annexin V-FITC Apoptosis Kit (APT750, Millipore, Temecula, CA, USA). The fluorescence intensity of annexin V-FITC and PI staining was examined using imaging flow cytometry (FlowSight, Seattle, WA, USA). The fluorescence intensity of annexin V-FITC-positive cells was quantified at excitation and emission wavelengths of 505 and 560 nm, respectively. The fluorescence intensity of PI-stained dead cells was quantified at excitation and emission wavelengths of 595 and 642 nm, respectively. Data were analyzed using IDEAS version 6.2 (Amnis, Seattle, WA, USA).

Statistical Analysis
All the results were obtained in triplicate and reported as the mean ± standard deviation. Data were analyzed by two-way analysis of variance (ANOVA) using GraphPad prism version 8 statistical software. A p-value of ≤0.05 was considered statistically significant at the 5% significance level.

Conclusions
2 ,4 -Dihydroxy-6 -methoxy-3 ,5 -dimethylchalcone, a chalcone derivative that is isolated from the seed of Syzygium nervosum A.Cunn. ex DC. presented high antiproliferative cervical cancer activity in human cervical cancer cells (HeLa). HeLa cells treated with DMC demonstrated DNA damage, inhibition of cell proliferation, and induced apoptosis cell death.