Cytotoxic effects of parijoto ( Medinilla speciosa Reinw. Ex. Bl.) methanol extract combined with cisplatin on WiDr colon cancer cells through apop‐ tosis induction

Parijoto ( Medinilla speciosa Reinw. Ex. Bl.) is a medicinal plant with cytotoxic effects on cancer cells in vitro. As only a limited number of studies have reported the effect of parijoto on colon cancer cells, this study initially aimed to measure the total flavonoid levels and potential cytotoxic effects of parijoto methanol extract (PME) through cell viability assays and expression of the apoptotic protein on WiDr colon cancer cells as a model. PME cytotoxic activity was determined by conducting a cytotoxicity test on WiDr colon cancer cells using the MTT assay. The synergistic cytotoxic effects of the PME and cisplatin were tested to obtain the combination index (CI) value. Apoptosis was analyzed by flow cytometry, and the apoptotic protein expression was observed by immunocytochemical tests. Furthermore, quercetin as a major flavonoid in PME was measured using a UV–Vis spectrophotometer. The results showed that PME had a moderate cytotoxic activity with an IC 50 of 198.64±1.6 µg/mL, whereas the IC 50 of cisplatin was 2.34±0.7 µg/mL. The PME with cisplatin combination test showed a strong synergistic effect with a CI value of <1 (0.1‐0.4). The combination showed increased apoptosis properties compared to PME treatment alone. In addition, immunocytochemistry showed that PME alone or in combination with cisplatin increased the pro‐apoptosis proteins (p53 and caspase‐9) and suppressed Bcl‐2 expression. Moreover, the cell viability value increased as the PME concentration decreased. The administration of PME led to changes in cell morphology, lower cell density, and a decreasing number of living cells. Therefore, the combination of PME and cisplatin had a strong synergistic effect in inducing apoptosis.


Introduction
is one of about 375 species of Medinilla genus widespread in Africa and Asiapacific (Mabberley 2017). Parijoto fruit contains alkaloids, flavonoids, saponins, tan nins, glycosides, terpenoids, and anthocyanins (Wijayanti and Ardigurnita 2019; Niswah 2014; Sa'adah et al. 2017). According to Tusanti et al. (2014), parijoto ethanol ex tract showed moderate cytotoxicity in vitro against T47D breast cancer cells with an IC 50 value of 614.50 µg/mL. Besides possessing a potential cytotoxic effect against can cer cells, parijoto has also benefited as an antihyperlipi demic agent, antibacterial, and antioxidant (Sa'adah et al. 2017; Wahyuni et al. 2019; Wachidah 2013. Cancer is a disease with a high prevalence character ized by the uncontrolled growth of abnormal cells (Amer ican Cancer Society 2017). Based on data from the World Health Organization (2018), there are 1.8 million cases of colon cancer in the world. A total of 30,017 new cases of colon cancer occurred in Indonesia. Colon can cer is a malignant tumor growth that can damage DNA and healthy tissue around the colon and rectum (Tedja and Abdullah 2013). Colon cancer cells undergo irregu lar molecular mechanisms of cell division and apoptosis, such as inefficient control of cell proliferation, unstable genetic and chromosome structures, changes in differenti ation programs, and impaired apoptosis control (Pritchard and Grady 2011). Apoptotic function abnormalities are as sociated with colon cancer and its resistance to chemother apy and radiotherapy. Disruption of apoptosis regulation can increase tumorigenesis and lead to colon cancer resis tance (Abraha and Ketema 2016).
One of the proapoptotic proteins that act as a marker of apoptosis involving the mitochondria is caspase. Mi tochondrial pathways are activated by various cytotoxic drugs, DNA damage, deficiency of growth factors, ox idative stress, excess of Ca 2+ , and oncogenes activa tion (Mendelsohn et al. 2015). They are regulated by forming the mitochondrial permeability transition pore, which is composed of Bcl2 family members and a voltagedependent anion channel. Cytochromec (Cytc) then associates apoptosis proteaseactivating factor 1 and caspase9 to form an apoptosome complex. Activation of caspase9 and/or caspase8 leads to caspase3 cleavage, activation of endonucleases, and finally, nuclear DNA fragmentation, which characteristic of apoptosis (Redza Dutordoir and AverillBates 2016).
In colon cancer cells, Bcl2 family proteins are cen tral regulators of the intrinsic pathway, which suppresses or promotes changes in mitochondrial membrane perme ability required to release Cytc and other apoptogenic pro teins (Bostan et al. 2016). Increased expression of the anti apoptotic protein Bcl2 family proteins may lead to poor prognosis in patients with colonic adenocarcinoma, pro viding a multidrug resistance phenotype (Huang and Linda 2015). One of the apoptosis inducers in colon cancer cells is the p53 protein. The molecular mechanisms of cell death induction by p53 to suppress cancer development include transcription regulation of proapoptosis PUMA, the for mation of oxidative free radicals in mitochondrial compo nents, reduction of COX2/PGE2 synthesis, and induction of death receptors (Edagawa et al. 2014). On the other hand, p53 mutation occurs at position 273 in WiDr cells, resulting in a change in the arginine residue to histidine (Noguchi et al. 1979). However, p21 in normal WiDr cells allows cell cycle termination. Apoptosis in WiDr cells can occur through p53independent pathways (Liu et al. 2006).
Some of the therapeutic agents used in colon cancer treatment, e.g., fluoropyrimidines, cisplatin, oxaliplatin, and irinotecan, have been shown to cause resistance in killing cancer cells. The number of cancer cells may in crease by modulation of survival cell components, such as proliferative proteins or antiapoptosis factors (Haider et al. 2020). In addition, side effects, such as nausea, vom iting, hair loss, swelling, sores in the mouth and throat, drastic weight loss, and memory problems, also occur following chemotherapy with cisplatin (He et al. 2016). Thus, it is essential to find new targetbased molecules and new therapeutic approaches in colon cancer by de termining the cellular mechanisms responsible for induc ing apoptosis in cancer cells (Thornthwaite et al. 2013). It can be done through a combination of chemotherapy, which has the cytotoxicity potential for additive or syn ergistic tumors. The combination of natural compounds with anticancer drugs (cochemotherapy) can increase the effectiveness of cancer treatment, especially in highly in vasive colon cancer cells. In contrast, the use of natural compounds can reduce cytotoxic side effects in nontumor cells (Fox et al. 2012).
One of the plants that potentially be developed as a cochemotherapy candidate is parijoto (M. speciosa). Par ijoto possesses antioxidant activity. The ethyl acetate frac tion, methanol fraction, and methanol extract of the fruits display IC 50 values at 20.34, 46.65, and 48.24 µg/mL, respectively (Wachidah 2013). Forni et al. (2019) re ported a positive correlation between the antioxidant ac tivity of plants and their antiproliferative effects, such as flavonoids that exhibit various biological activities, in cluding antiinflammatory, cytoprotective activities, and some are known to act as anticancer agents. There are limited studies regarding the cytotoxic mechanism of par ijoto fruit against colon cancer cells. This study aimed to observe the cytotoxic effect of parijoto methanol (PME) extract on WiDr cancer cells, which was confirmed by the induction pattern of apoptosis through the expression of the p53, Bcl2, and caspase9 proteins, then proceed with quantification of quercetin levels in PME.

Sample collection and extract preparation
Parijoto fruits obtained from Kudus, Central Java, Indone sia, were stored in a cooler bag to transport to the labo ratory. The fruits were dried at 25°C and then blended for optimum extraction. The extraction method was con ducted based on Vifta and Advistasari (2018). The fruit powder (500 g) was extracted with 500 mL of methanol (Merck, Germany) by the maceration for 24 hours (h) at 37°C. Upon filtration, the filtrate was evaporated using a rotary evaporator at 45°C to form a thick extract. The remaining solvent in the viscous extract was evaporated using a water bath. The extract was stored at −20°C until use. This study was conducted at the Parasitology Labo ratory, Faculty of Medicine, Nursing and Public Health, Universitas Gadjah Mada (UGM), Yogyakarta, Indonesia.

Drug preparation and treatment
Cisplatin (1 mg) (Kalbe, Indonesia) was dissolved in dimethyl sulfoxide (DMSO) (0.1 mL) (Merck, Germany) and used as a comparison compound in colonic cancer cy totoxicity tests. The solvent concentration in the culture medium was not more than 0.5% in each experiment.

Cell culture
Human colon adenocarcinoma (WiDr) cancer and normal epithelial cell lines (Vero cells) were obtained from Par asitology Laboratory, Faculty of Medicine, Nursing and Public Health UGM. Both cell lines were cultured in the RPMI medium (Gibco, USA) supplemented with 10% heatinactivated fetal bovine serum (FBS) (Gibco, USA) and 1% penicillinstreptomycin (Gibco, USA) with 5% CO 2 supply at 37°C.

Cytotoxicity test with the MTT assay
The MTT assay (Sigma Aldrich, USA) was carried out for 24 h to measure the cytotoxic effect of parijoto fruit methanol extract (PME) and cisplatin on WiDr cancer cells and normal cells. Up to 1 × 10 4 cells/well were grown in 96well plates. The cell suspension (100 µL) was trans ferred into the wells and observed in a microscope to see the distribution of cells. Cells were then incubated in an incubator for 24 h so that the cells recovered after harvest ing. The concentration series of PME (50-400 µg/mL) and cisplatin (0-10 µg/mL) was added (100 µL) to the well in the triplicate, then reincubated in a CO 2 incubator for 24 h. At the end of the incubation time, cell conditions were documented for each treatment under an inverted micro scope. The cell media was discarded, and the cells were washed with 1× phosphate buffered saline (PBS). Then, 10 µL of MTT (0.25 mg/mL) was added to each well. Cells were reincubated for 4 h in a CO 2 incubator until for mazan crystals were formed. When the formazan crystals had formed, 10% SDS in 0.1 N HCl was added as a stop per. The absorbance was then measured using an ELISA microplate reader (BioRad microplate reader Japan) with a spectrophotometer UVVis (Genesys, Thermo Scientific, USA) at 595 nm. The log concentration and cell viability were plotted on a logarithmic graph, then used to deter mine the IC 50 value (Wulandari et al. 2021).
Based on the IC 50 value from a single cytotoxicity test, the concentration series was made of ½, ¼, and ⅛ of IC 50 for PME and cisplatin. Equation (1) is used to evaluate the combination synergism based on research conducted by Reynolds and Maurer (2005).
Dx1 and Dx2 are concentrations of one compound needed to exert an effect (IC 50 on the growth of WiDr cells), D1 and D2 are the concentrations of the two com pounds to give the same effect.

Apoptosis measurement by flow cytometry
WiDr cells in the exponential growth phase were treated with PME at ⅛ or ½ IC 50 for 72 h, then harvested by trypsinization and washed twice with icecold PBS. The cells were then resuspended with 100 µL of the binding buffer. In the next stage, 5 µL of AnnexinV FITC and 5 µL of propidium iodide (PI) were added to each tube and incubated for 10 min at room temperature in the dark. Fi nally, 400 µL of the binding buffer was added to each tube, and the data from 10,000 cells per sample were collected and analyzed on a flow cytometer (BD Accuri C6) within 1 h. The results were compared with those for untreated control cells (Haryanti et al. 2017).

Immunocytochemical test against p53, Bcl-2, and Caspase-9 proteins
Cells at a 5×10 4 cells/well density were plated on a cov erslip in a 24well plate and incubated until 70% conflu ent. The cells were treated and reincubated for 15 h. At the end of the incubation time, the cells were washed with PBS, cold methanol was added, and the cells were then incubated in the freezer for 10 min. After discard ing the methanol, the cells were washed with PBS twice, washed with distilled water twice, incubated with hydro gen peroxidase solution for 10 min, and then washed three times. Solution or cell suspension then dripped with predi luted blocking serum and incubated for 10 mins. In the treatment group, the primary antip53 monoclonal (Ther moFisher Scientific, Cat #MA512557), antiBcl2 (Ther moFisher Scientific, Cat #TA806591), and anticaspase 9 antibodies (ThermoFisher Scientific, Cat #MA116842) were added and then incubated overnight. After being washed with PBS three times, the cells were incubated with a secondary antibody conjugated with biotin (biotiny lated universal secondary antibody) (ThermoFisher Sci entific, Cat #31806) for 20 min. After being washed, a reagent containing the streptavidinhorse radish peroxi dase enzyme complex was added to the cells and incubated for 10 min. The cells were rewashed with PBS three times, dripped with DAB solution, and incubated for 10 min. Af ter being washed with distilled water, the cells were in cubated with the Mayer-Hematoxylin solution for 1 min.
The coverslip was then dipped in alcohol and xylol after washing with distilled water. After drying, the coverslip was placed on a slide and dripped with mounting media. The coverslip was closed with a slide for further observa tion with a light microscope (Noviantari et al. 2020).

Quantification of quercetin levels in PME
The PME and a quercetin standard were plated using a capillary tube on thinlayer chromatography (TLC, Silica gel GF 254) plate, then eluted with the appropriate mo bile phase. The mobile phase used was chloroform: ace tone: formic acid (10:2:1 v/v), which was detected with a 10% FeCl 3 spray reagent. Furthermore, the determination of quercetin levels begins with determining the maximum wavelength of quercetin in the wavelength range of 400-600 nm, and the maximum wavelength was obtained at 439 nm. A calibration curve was created by dissolving 10 mg of quercetin in 10 mL of methanol and diluting it into serial concentrations of 12.5, 25, 50, 75, and 100 µg/mL. The blank solution was 0.5 mL methanol and the test solu tion used 0.5 mL PME. Each solution (0.5 mL) was added with 1.5 mL of methanol, 0.1 mL of 10% aluminum chlo ride, 0.1 mL of 1 M sodium acetate, and 2.8 mL of distilled water. After incubation at room temperature for 30 min, the absorbance of each solution was measured at 439 nm.

Analysis of cytotoxicity test data
The data obtained were in the absorbance of each well, which was then converted into cell viability. The cell via bility was calculated using equation (2) based on research conducted by Doyle and Bryan (1998). (2)

Selectivity measurement
Selectivity was determined using the Selectivity Index (SI) parameter with the following equation (3) according to Prayong et al. (2008): Artanti et al.

Apoptosis analysis
Data from flow cytometry were analyzed using Microsoft Excel 2010. The percentage of cell death, including early apoptosis, late apoptosis, and necrosis, was displayed in a bar graph. In addition, the induction of cell death by the test solution was known by comparing single and combi nation treatments with control cells.

Observation of p53, caspase-9, and Bcl-2
Protein expression was descriptively qualitatively ob served, where cells expressing the proteins would give a brown color in the nucleus, while those that did not ex press the proteins or had low expression levels would give a purple color in the nucleus.

Percentage of cells with protein expression =
Cells with protein As mentioned in equation (4), the calculation of pro tein expression based on research conducted by Zakinah et al. (2017) was carried out on a minimum of 100 cells from each point of view. Furthermore, the observation of the samples was carried out from three different points of view for each sample and documented with a camera.

Analysis of total flavonoid levels
Total flavonoid levels were calculated using equation (5) according to Chang et al. (2002) as follows: F is the total flavonoid with AlCl3 methods, c is the quercetin equivalent (µg/mL); V is the total extract volume (mL), f is the dilution factor, and m is the sample mass (mg).

Statistical analysis
The quantitative data resulting from the combination treat ment of cell viability were analyzed statistically with the Analysis of Variance (ANOVA) test followed by the Tukey HSD test using SPSS 16.

Assessment of WiDr cell viability following treatment with PME, cisplatin, or their combination
The relationship between cisplatin concentration and cell viability is presented in Figure 1 and Figure 2. The de termination of cisplatin activity through linear regression obtained a linear equation of y = −2.3562× + 55.518 with an Rvalue of 0.9752. Based on the linear regression, the IC 50 values of cisplatin and PME were 2.34 ± 0.7 µg/mL and 198.64 ± 1.6 µg/mL, respectively. Therefore, the lin ear equation obtained for PME cytotoxicity activity was y = −0.3868x + 127.47 with an Rvalue of 0.9505. The IC 50 value is a value that indicates the death of 50% of the cell population so that its potential cytotoxicity can be known (Mazumder et al. 2020).
Viable WiDr cells have a polygonal shape and attach to the bottom of the well, while dead WiDr cells are round and smaller, scattered, and do not attach to the bottom of the well (Anandani et al. 2018). PME selectively inhib ited colon cancer cells compared that of to normal cells (Vero cells). The results showed that there were differ ences between control cells and PMEtreated cells. The living WiDr cells were polygonal in the control group, and  the cell density was higher. In addition, cell viability value was increased with the high concentration of PME admin istration. The administration of PME affects the cell mor phology, decreases cell density, and decreases the num ber of living cells with increasing concentrations ( Figure  1), but had less effects on Vero cells (Figure 3). Based on the test results, the IC 50 value of PME was 198.64±1.6 µg/mL. Moreover, our results showed that the IC 50 value in the Vero cell was 5,258.42 µg/mL. According to Weer apreeyakul et al. (2012), natural material is categorized as having very strong cytotoxicity if it has an IC 50 value of <10 µg/mL; strong cytotoxicity if it has an IC 50 value be tween 10-100 µg/mL; and moderate cytotoxicity when it has an IC 50 value of 100-500 µg/mL.
The safety level of an anticancer compound against normal cells is confirmed by determining the value of its selectivity compared to normal cells. Extracts are classi fied as selective if they have an SI value of ≥3 and as less selective if the SI value is <3 (Sutejo et al. 2016). The re quired SI value is ≥3, which indicates that the extract has cytotoxic activity against cancer cells with minimal effect on normal cells and can be further developed as a chemo prevention agent (Sutejo et al. 2016). As shown in Table 1, the selectivity analysis showed that PME selectively toxic to WiDr colon cancer cells and had minimal effect on Vero cells with an SI value of 26.47 (SI ≥ 3). The IC 50 PME in WiDr cells was higher than that of normal Vero cells. This indicates that PME cyto toxicity in WiDr cancer cells is stronger than Vero cells. The SI value after PME treatment and after treatment with cisplatin was 26.46 and 9, respectively. A compound with a cytotoxic effect and high selectivity can be devel oped as a chemoprevention agent because the compound is able to distinguish between cancer cells and normal cells (Sholikhah et al. 2018). We next tested the cytotoxicity of PME and cisplatin combination through the cytotoxicity test PME and cis platin combination. Combination treatment of PME and cisplatin has a synergistic effect (Reynolds and Maurer 2005) at cisplatin concentration of 200 nM and all PME concentrations (24.8, 49.6, and 99.3 µg/mL), with CI val ues of 0.14-0.97 (Table 2). These results revealed that PME could increase the sensitivity of WiDr cells toward cisplatin.

Effect of PME, cisplatin, and its combination on apoptosis
The confirmation of the cytotoxicity test results was car ried out through the apoptosis induction test using flow cy tometry. Cell detection using AnnexinV and PI showed the living cells and dead cells in early apoptosis, late apop tosis, and necrosis ( Figure 4). PME induces less cell death than cisplatin. The higher cisplatin cytotoxic effect by PME through apoptosis induction mechanisms. The flow cytometry results of PME in WiDr cells are present in Fig  ure 4 (n=3).
The PME apoptosis induction test on WiDr cells showed that untreated cells (control) showed more living cells (95.61%) than dead cells (4.39%). Meanwhile, cells treated with PME experienced death up to 25.8%, and af ter the combination treatment of ⅛ IC 50 PME and 7/8 IC 50 cisplatin, the cell death raised to 60.6%. The percentage of cell mortality due to cisplatin treatment was 96.7%. The reduction in cell death after combination was 36.1% com pared to cisplatin alone. Cell necrosis in the combina tion treatment increased by 18.3% compared to the cis platin treatment alone. PME induces less cell death than cisplatin. Therefore, the high cytotoxicity of cisplatin by PME probably occurs through apoptosis induction mech anisms. The apoptosis induction was confirmed through immunocytochemical tests by looking at the expression profiles of Bcl2, p53, and caspase9.

FIGURE 4
Analysis of the percentage of cell death after treatment with PME, cisplatin, and the combination of both. WiDr cells were treated with PME, cisplatin, and the combination for 72 h using Annexin-V FITC and propidium iodide staining. Significant difference (p <0.05) compared with untreated WiDr cells is indicated by the increase of apoptotic The observation was conducted for three times (n=3).

Effect of PME, cisplatin, and their combination on Bcl-2, p53, and caspase-9 expressions in WiDr cell
The antiapoptotic protein Bcl2 was highly expressed in WiDr cells, and it was correlated with the low effect of the chemotherapy agent ( Figure 5 7). Therefore, we se lect PME in this study because of the quercetin compound. Quercetin increased the expression of p53 and caspase 9 proteins while suppressing the expression of Bcl2.

Quantification of the quercetin content in PME
The quantification results show that PME contains the sec ondary metabolite quercetin (Figure 8, Table 3). PME has a total flavonoid content of 21.86 ppm or 2.73% g/g of flavonoids which is equivalent to quercetin. The data used were in the form of five serial concentrations, which then the linear regression calculation was carried out with ab sorbance. The linear regression obtained from quercetin standards was y = 0.0074x − 0.0324 with an Rvalue of 0.9999 (Figure 9). Quercetin standard calibration curve equation was used to determine the total flavonoid levels contained in PME. The absorbance value obtained from the PME was then calculated with linear regression. Quercetin levels measurement was carried out using UV-Vis spectrophotometry at 400-600 nm (Fawwaz et al. 2017). Determination of the maximum wavelength en sures the wavelength required to produce maximum ab sorption. The maximum absorption will also produce maximum sensitivity and minimize errors. The results showed that the maximum wavelength of the quercetin standard was 439 nm and quercetin was used as a standard as it is a flavonoid (Azizah et al. 2014). The results also showed that the total flavonoid concentration was 21.81 ppm or 2.73% g/g of flavonoids calculated as quercetin. Stevens et al. (1994) stated that quercetin was able to in hibit the growth of WiDr cells with an IC 50 value of 56 µg/mL.

Discussion
Cisplatin is a chemotherapy agent in the treatment of colon cancer. In this study, cisplatin was used as a positive con trol. To overcome resistance, cisplatin is commonly used with some other drugs in treating colon cancer, ovarian cancer, colorectal cancer, biliary tract cancer, lung cancer, gastric cancer, pancreatic cancer cell lines, and urothelial bladder and cervical cancer. Cisplatin enters the cells by passive diffusion through the plasma membrane and by ac tive transport mediated by several membrane transporters (Spreckelmeyer et al. 2014; Hall et al. 2008. Combina tions of cisplatin with compounds that interfere with spe cific cisplatin resistance factors have been tested in var ious preclinical cancer models. Notably, p53 and p53 mediated DNA damage responses might be used to target biochemical modulators in colorectal cancer cells. Effects of chemotherapeutic drugs are also influenced by the ef ficiency of DNA repair. Exploiting DNA repair might be another strategy in cancer therapy (Herůdková et al. 2017; Rawlinson and Massey 2014; Shen et al. 2013. Based on that, the exploration of PME and cisplatin apoptotic mechanism, either alone or in combination, were directed to inhibit Bcl2 expression. The qualitative ob  servation showed the decrease of Bcl2 expression after a single treatment of 198.64 µg/mL PME, 2.34 µg/mL cis platin, and the combination compared with control cells (Figure 3). The decrease in Bcl2 expression following combination treatment was more than a single treatment of PME and cisplatin, which was shown by the low in tensity of brown color in the cytoplasm compared with treatment with cisplatin alone. Therefore, both PME and cisplatin contributed to Bcl2 expression in combination treatment. Furthermore, we also observed the control treatment of PME, combination of PME + cisplatin, and cisplatin against Vero cells (Figure 2). Each treatment showed changes in cell morphology. Vero cells were polygonal in shape and had higher cell density in the con trol cells. The morphology of Vero cells was round when compared to the treatment group. We selected WiDr cells based on a previous study that demonstrated expression of the p53 in vitro by fermented black rice bran extract (Ashcroft and Vousden 1999). Im munocytochemistry was used to detect the p53 expression level in treated WiDr cells and control cells. The p53 pro tein detection was performed to confirm the apoptosis in duction mechanisms by PME, cisplatin, and their com bination. The p53 protein is a known potent tumor sup pressor and is maintained at low levels in unstressed cells (Ashcroft and Vousden 1999). In stimulated cells, p53 is increased and acts as a transcription factor to upregulate the expression of many genes, including cellgrowtharrest and apoptosisassociated genes (Roos and Kaina 2013). The present study found that PME significantly increased p53 expression in WiDr cells. This may be due to the differences in p53 expression levels, the qualitative sta tus of p53, and other cellular contexts, which have been suggested to influence p53 to stimulate cell cycle arrest or apoptosis (Haupt et al. 2003; Zuckerman et al. 2009).
We analyzed anticancer against proteins that oppose the proliferation of colon cancer cells, namely, p53, from the chemical composition of PME and to determine the apoptotic of colon cancer cells. p53 protein plays a cru cial role in response to cellular stress, such as exposure to carcinogens. This protein inhibited the proliferation of abnormal cells to prevent the development of neoplasms. On the other hand, protein inactivity can cause malignancy until the cancer is malignant (Hu et al. 2003). Besides, p53 also regulates apoptosis, inhibits angiogenesis, and regu lates DNA repairment. p53 is a mutation in cancer and can be in the form of degradation of p53, loss of the abil ity of p53 induces cell cycle arrest or apoptosis, and loss affinity of p53 to bind damaged DNA. These conditions in crease p53 and decrease COX2 on the cancer cell cycle. Therefore, the growth and development of colon cancer cells (WiDr) can be inhibited (Zhu et al. 2015; Zambetti et al. 1992. The intensity of the brown color indicated the pres ence of p53 expression both in the cytoplasm and in the nucleus. An increased level of ROS characterizes the mechanism of cancer cell death treated with quercetin and p53 expression, decreased Bcl2 expression, mitochon drial membrane depolarization, caspase3 cleavage, and DNA fragmentation (Edagawa et al. 2014; Abraha and Ketema 2016; SanchezGonzalez et al. 2011). New drug compounds may induce apoptosis by activating caspases, Fas, Bax, Bid, APC, or molecules that promote colon can cer cell survival (mutant p53, Bcl2, or COX2). Caspase proteins are used as markers of apoptosis through mito chondrial regulation (Mendelsohn et al. 2015). Activation of caspase9 and/or caspase8 causes caspase3 cleavage, endonuclease activation, and nuclear DNA fragmentation, which are characteristic of apoptosis (RedzaDutordoir and AverillBates 2016). The immunocytochemical tests showed that PME treatment alone or a combination with cisplatin could increase the expression of the proapoptotic p53 and caspase9 proteins and suppress Bcl2 expression. The decrease in Bcl2 expression in the PME single treat ment was probably due to the presence of quercetin in PME. Quercetin binds directly to the BH3 domain of Bcl 2 and BclxL proteins, thereby inhibiting their activity and promoting cancer cell apoptosis (Primikyri et al. 2014).
The induction of apoptosis by PME is thought to in volve secondary metabolites contained in PME; one of them is quercetin. Quercetin is a secondary metabolite in volved in suppressing cancer cell activity, oxidative stress, proliferation, and metastasis. Another bioactive com pound in PME is tannin. At the present study, we focus on quercetin, which is a proapoptotic compound specif ically inhibits the growth of colon cancer cells compared to that of to normal cells (SanchezGonzalez et al. 2011). The antitumor effect found on SW480 colon cancer cells is associated with inhibition of cyclin D and surviving ex pression, as well as the Wnt/betacatenin signaling path way (Mabberley 2017; Tai et al. 2014). Quercetin has antiproliferative effects against breast, ovarian, and colon cancer cells in vitro (Neuhouser 2004). Quercetin also in duced inhibition of Akt phosphorylation was coupled with a significant decrease of Bcl2 and BclXL because ac tive or unphosphorylated BAD is known to induce apop tosis by inhibiting antiapoptotic Bcl2 family members allowing proapoptotic proteins to be aggregate and ini tiate cytochrome c release and subsequent caspase9 ac tivation (Cheng et al. 2001). Moreover, Akt is known to inhibit caspase9 activity and inhibit the expression of death ligands on the cells. Quercetin may exert its anti cancer effect through several mechanisms, including act ing as an antioxidant, inducing apoptosis, acting as an anti inflammatory agent, and modulating signaling pathways (Inoue et al. 2004).

Conclusions
PME has moderate cytotoxic activity with an IC 50 value of 198.64 ± 1.6 µg/ml. The combination of PME with cis platin showed a strong synergistic effect with a CI value of <1. Flow cytometry test results showed that the com bination of PME and cisplatin increased apoptosis induc tion compared to PME treatment alone. The immunocyto chemical tests showed that cisplatin increased the expres sion of the proapoptosis p53 and caspase9 proteins and suppressed Bcl2 expression compared to PME. The quan tification of total flavonoids in PME was 21.86 ppm or 2.73% g/g of flavonoids, calculated as quercetin.