*4.3. Drugs Preparation*

Proscillaridin A (Sigma-Aldrich, St. Louis, MO, USA) and Doxorubicin (EBEWE Pharma, Rome, Italy) were completely dissolved in DMSO and then prepared in 20 mM and 100 mM stock solutions, respectively. Then serial dilutions for both were prepared in DMEM medium and stored at −20 ◦C until use.

### *4.4. Cell Culture*

Two human colorectal carcinoma cell lines from the American Type Culture Collection (ATCC), namely COLO-205 (ATCC® CCL-222™) and Caco-2 (ATCC® HTB-37™), were used in this study. Additionally, normal human gingival fibroblasts (hGFs) samples prepared at the Cell Therapy Center (CTC) were used to assess the cytotoxic effects of the plant extract and drugs/treatments. All cells were cultured under the same conditions in 75 cm<sup>2</sup> tissue culture flasks and maintained in cell culture media (CCM) consisting of DMEM (Euroclone, Pero, Italy) high-glucose supplemented with 10% (*v*/*v*) heat-inactivated FBS, 1X penicillin/streptomycin, 1X non-essential amino acid, sodium pyruvate, and 1X L-Glutamine and incubated in a humidified incubator at 37 ◦C and 5% CO2. When the confluence of cells reached 70–80 percent, cells were passaged with 1X trypsin/EDTA (Euroclone, Italy) and centrifuged at 1400 rpm, 25 ◦C for five min. Different characteristics for the used cell line is listed in Table 2.


**Table 2.** Type of cells used in the study.

\*; https://www.atcc.org (accessed on 20 December 2022).

#### *4.5. Cell Viability and Proliferation Assay*

The antiproliferative effect and the median inhibitory concentration (IC50) of *D*. *maritima* bulb extract and ProA was assessed on COLO-205, Caco-2, and hGF cells using an MTT assay. In brief, cells were seeded in 96-well plates at a concentration of <sup>8</sup> × <sup>10</sup><sup>3</sup> cells/well and incubated for 24 h at 37 ◦C. The next day, cells were treated with *D*. *maritima* bulb extract and ProA at concentrations ranging from 0.122 μg to 500 to /mL and 2.46 to 5307 μg/mL, respectively, in a quadruplicate manner. The plates were incubated at 37 ◦C for 48 h. COLO-205, Caco-2, and hGF cells were also treated with a 2-fold serial dilution of Doxorubicin at concentrations ranging from 0.024 to 50 μg/mL under the same conditions. DMSO was used as a drug solvent in minimal concentrations. Furthermore, each cell line used in this study was treated with 1% DMSO as a control. Additionally, untreated cells were used as a negative control. At the end of the incubation period, DMEM media were aspirated, and 10 μL of MTT dye solution (Promega, USA) was added to each well. After 4 h of incubation at 37 ◦C, the dye solution was removed, 100 μL of DMSO was added to each well, and the absorbance was measured using a 96-well plate reader at 560 and 750 nm wavelength. The IC50 values were determined using the logarithmic trend line of the cytotoxicity graph using the GraphPad PRISM® 8.0 software (GraphPad Software, Inc., San Diego, CA, USA).

#### *4.6. Apoptosis Assay*

Cell apoptosis was determined using the Annexin V-FITC/Propidium Iodide (PI) apoptosis detection assay Kit (Invitrogen, Waltham, MA, USA). In brief, Caco-2 and COLO-205 cells were seeded in 6-well plates at a concentration of 3 × 105 cells/well, treated with different concentrations of the *D*. *maritima* bulb extract, and incubated for 24 h at 37 ◦C, 5% CO2. According to the IC50, Caco-2 cells were treated with different concentrations ranging from 0.5 to 2 μg/mL, COLO-205 cells were treated with different concentrations ranging from 0.5 to 3.5 μg/mL, and then both cells were incubated for 48 h at 37 ◦C, 5% CO2. The cells were harvested using 1X TrypLE Express, washed twice with PBS, and suspended in equal volumes of Annexin V and PI reagents (2 μL each) diluted in 100 μL of 1X binding buffer. All cells were incubated in a dark place at room temperature for 15–20 min. After that, cells were analyzed by flow cytometry BD FACSCanto software (BD FACSCanto, Wokingham, UK)

#### *4.7. Total Reactive Oxygen Species Measurement*

Seeding and treatment conditions and cell preparation were similar to apoptosis analyses. Cells were incubated with 1X ROS stain (Invitrogen, USA) (100 μL/sample) and incubated for 60 min at 37 ◦C, with 5% CO2. Then the stained cells were analyzed by flow cytometer by measuring the fluorescence emission at 520 nm.

#### *4.8. Mitochondrial Membrane Potential (*ΔΨ*m)*

Mitochondrial membrane potential (ΔΨm) was assessed in colon cancer cell lines using a Tetramethylrhodamine ethyl ester (TMRE) mitochondrial membrane potential assay kit (Abcam, Boston, MA, USA). Colon cancer cells were treated as described above, and then 0.8 μL of trifluoromethoxy carbonylcyanide phenylhydrazone (FCCP) was added to the untreated cells only (control) and incubated for 15 min at 37 ◦C, 5% CO2. Treated cells were stained with TMRE by adding 0.8 μL of TMRE/well to the cell suspensions, and then cells were re-incubated for an additional 30 min. Next, cells were harvested, centrifuged, and resuspended in PBS and then analyzed by flow cytometer and measured at 549/575 nm.

#### *4.9. Real-Time Quantitative Polymerase Chain Reaction (RT-qPCR) Amplification*

Total RNA was extracted from cell pellets using Qiagen RNeasy Mini Kit (Qiagen, Germany), according to the manufacturer's instructions. Complementary DNA (cDNA) was prepared using 1 μg RNA and PrimeScript RT Master Mix Kit (TaKaRa, Kusatsu, Japan) according to the manufacturer's instructions. The cDNA synthesis reaction was performed using veriti 96 well Thermal Cycler (Thermo Fisher Scientific, Waltham, MA, USA), under the following conditions: "37 ◦C" (reverse-transcription) for 15 min, "85 ◦C" (heat inactivation for reverse-transcription) for 5 s, and "4 ◦C" holds. Finally, the samples were stored at −20 ◦C until use.

cDNA samples were diluted in 1:10 using nuclease-free water. Samples were performed in replicate. qPCR *assay* was performed *in* a 10 μL total reaction using specific primers to amplify *CASP8*, *TNF-α*, *IL-6*, and *GADPH* genes, which are responsible for the expression of caspase-8, tumor necrosis factor-alpha, interleukin-6, and glyceraldehyde-3-phosphate dehydrogenase, as the following: "95 ◦C" for 2 min as initial denaturation cycle, then 40 cycles of 95 ◦C for 15 s (denaturation), "58 ◦C" for 60 s (annealing), and "72 ◦C" for 30 s (extension). The primers sequences are listed in Table 3. The PCR data analysis was performed using the ΔΔCt method (delta delta cycle threshold); the analysis was performed automatically according to CFX Maestro Software of Bio-Rad Company (Hercules, CA, USA). The data were normalized, across all plates, to the housekeeping gene *GAPDH*.

**Table 3.** List of primers for qPCR amplification.


#### *4.10. Statistical Analysis*

All statistical analyses were performed using GraphPad Prism software version 8.0 (GraphPad Software, San Diego, CA, USA). The comparison between different groups of numerical variables was performed using one-way or two-way ANOVA. Homogeneity of variances was tested using Dunnett's and Bonferroni's multiple comparisons tests, and a *p*-value less than 0.05 (*p* < 0.05) was considered statistically significant.

The IC50 values were determined by using log–probit analysis; log (concentration) plotted on the x-axis and inhibition percentage plotted on the y-axis; percent of inhibition was calculated, after correction of the absorbance (A) measurements for the background (blank) absorbance, according to the equation; Inhibition% = ((A control − Atreatment)/Acontrol) × 100%.

#### **5. Conclusions**

In summary, *D. maritima* bulb extract and ProA showed a selective inhibitory activity on the proliferation of colorectal cancer cell lines. Such a finding was supported by the results of the apoptosis, intracellular ROS, and mitochondrial membrane potential assays. On the molecular level, the expression of pro-inflammatory genes *TNF-α* and *IL-6* and the apoptotic initiator gene *CASP8* was also confirmative for the antiproliferative effect of the *D. maritima* bulb extract. Taken together, the findings of this study elect *D. maritima* for further investigation and validation on CRC animal cancer xenograft models.

**Author Contributions:** Conceptualization, M.Z., K.A.-A., N.A.A. and M.O.; methodology, N.A.A., S.A.H., Y.A.-A. and S.Z.; software, K.A.-A.; formal analysis, M.Z, K.A.-A.; investigation, K.A.- A., N.A.A.; supervision, A.A, N.A.A. and M.O.; project administration, M.Z., N.A.A. and A.A; funding acquisition, M.O. and A.A. All authors have read and agreed to the published version of the manuscript.

**Funding:** This study was supported by the Deanship of Scientific Research and Innovation, Al-Balqa Applied University (Grant number: 497/2020/2021).

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** All data are available upon request from the corresponding author.

**Conflicts of Interest:** The authors declare no conflict of interest.

**Sample Availability:** Samples of the compounds are available from the corresponding author upon request.

#### **References**


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