Proapoptotic and Antiproliferative Effects of the Desert Truffle Terfezia boudieri on Colon Cancer Cell Lines

Background Colon cancer is the second leading cause of cancer-related mortality, and ranks third among cancers in terms of prevalence. Despite advances in early detection and treatment with chemotherapy and surgery, colon cancer continues to be associated with high recurrence rates, thereby resulting in a heavy disease burden. Moreover, the effectiveness of currently available treatment modalities is limited by the occurrence of toxic side effects. Hence, there is an urgent need to develop alternative treatments. Extracts from the black desert truffle Terfezia boudieri (T. boudieri) have shown promising anticancer properties. However, the cellular mechanisms underlying this activity remain poorly understood. Methods In this study, the colon cancer cell lines HCT-116 and Caco-2 were treated with either water or ethanolic extract of T. boudieri. Cell viability and the half-maximal inhibitory concentration were determined using MTT assays. Then, the activity of the more potent water extract was further verified using crystal violet assays, and its role in inhibiting colony formation and wound healing was investigated. Protein levels of p53, B-cell lymphoma 2 (Bcl-2), Bcl-2-associated X (Bax), cyclin D1 (CCND1), and c-Myc were measured in cells treated with different doses of the water extract. Results Treatment with the water extract of T. boudieri reduced the capacity of cells for wound healing and colony formation in a dose-dependent manner. The Bax/Bcl-2 ratio and p53 expression were elevated in both cell lines. In contrast, the levels of cyclin D1 and c-Myc were suppressed. Conclusion T. boudieri water extract exerted a cytotoxic effect on colon cancer cells, and blocked colony formation and wound healing potentially through inhibition of proliferation. Mechanistically, these effects are attributed to influence the mitochondrial pathway of apoptosis, proteins involved in cellular proliferation, and the cell cycle.


Introduction
Colorectal cancer is the second leading cause of cancerrelated death worldwide and the third most common type of cancer [1]. Te incidence of colon cancer continues to increase worldwide due to unhealthy lifestyles and food habits [2,3]. Tumor cells possess innate tolerance or acquire de novo tolerance to presently available chemotherapy and radiotherapy techniques [4]. Tis ability results in high postsurgical recurrence rates, which are associated with the poor 5-year survival rates.
Similar to other types of cancer, colon cancer develops due to mutations arising in genes responsible for DNA repair and tumor suppression, such as p53 [5] (also termed as guardian of the genome), as well as several oncogenes. In turn, these mutations dysregulate important cell signaling pathways involved in cell proliferation, cell cycle progression, and apoptosis [6,7]. It has been shown that c-Myc increases the expression of proteins involved in cellular proliferation. Also, cyclin D1 induces transformation and chemoresistance. Terefore, c-Myc and cyclin D1 have attracted considerable attention as potential targets for the treatment of cancer [8,9].
As key regulators of cellular apoptosis, proteins belonging to the B-cell lymphoma 2 (Bcl-2) family may also be important targets in this setting. For example, Bcl-2-associated X (Bax) promotes this type of regulated cell death, while Bcl-2 prevents apoptosis by interfering with the activity of Bax to induce mitochondrial membrane permeability [10]. Numerous studies have proposed that the Bax/Bcl-2 ratio can serve as an important prognostic marker of clinical outcomes for patients with many types of cancers, including colon cancer [11][12][13][14][15].
Te need for treatment alternatives that may improve outcomes for patients with cancer continues to fuel the search for natural medicinal sources, particularly among plants and mushrooms [16].
Terfezia boudieri (T. boudieri) is a desert trufe that grows around the Mediterranean basin. Documentation pertaining to the usage of this desert trufe as food and medicine dates back to pre-Islamic times [17]. Many studies have analyzed the chemical composition of T. boudieri, demonstrating its high nutritional value. It is mainly composed of carbohydrates (∼60%), followed by proteins and fatty acids (particularly linoleic acid), as well as an amplitude of minerals (e.g., calcium, potassium, phosphorus, and iron). In addition, the trufe contains carotenoids, anthocyanins, favonoids, and phenolics which confer a potent antioxidant activity [18][19][20].
According to Al-Obaydi et al., the methanolic, water, nhexane, ethyl, and acetate extracts of T. boudieri contain carbohydrates, terpenoids, and phytosterols. However, tannins, alkaloids, and favonoids were only found in the methanolic and water extracts, which also contained the highest amounts of gallic acid; notably, the highest yield was obtained from the water extract [21]. Components of T. boudieri, such as phytosterols [22,23], terpenoids, [24] favonoids [25,26], and tannins, possess antioxidant and antibacterial properties [27]. Tus, it is expected that T. boudieri extracts would also possess such properties. Indeed, the antioxidant and antibacterial activities of T. boudieri have been demonstrated [28]. Furthermore, extracts of this trufe have shown an immunomodulatory capacity. Following treatment with the aforementioned extracts, lymphocytes displayed an increased rate of proliferation and secreted signifcantly higher levels of interferon-c (IFN-c) and interleukin-2 (IL-2). Similarly, macrophages responded to the exposure to T. boudieri extracts, and the highest phagocytic activity was observed in those treated with the water extract. T. boudieri extracts are also able to block angiogenesis by inhibiting the expression of vascular endothelial growth factor (VEGF) [21].

Preparation of T. boudieri Crude Extracts. T.boudieri
ascomycetes were purchased from a local market in Lebanon. Subsequently, they were washed by scrubbing, cut into thin slices, air-dried, and ground into fne powder. Te dried powder was homogenized in a ratio of 1 : 4 (weight/volume) in either distilled water or 90% ethanol. After 24 h of shaking at room temperature, the homogenates were fltered through Whatman flter paper and lyophilized into a soft paste. Te water extract was prepared for treatment by weighing and dissolving a stock solution of 20 mg/ml, and fltered using a 33-mm syringe flter (Sigma-Aldrich, Taufkirchen, Germany). Te stock was diluted to various concentrations using complete Dulbecco's Modifed Eagle Medium (DMEM) (Sigma-Aldrich). Te ethanolic extract stock solution was frst dissolved in dimethyl sulfoxide (DMSO) (Sigma-Aldrich), diluted with DMEM, and fltered with a 33-mm syringe flter (Sigma-Aldrich). Te percentage of DMSO in the highest treatment dose was 3%.

Maintenance of Colon Cancer Cell Lines. HCT-116 and
Caco-2 cell lines were provided by the cell culture facility of the Faculty of Science, Alexandria University (Alexandria, Egypt). Tese cells were originally purchased from the American Type Culture Collection (Manassas, VA, USA). Cells between 8 and 12 passages were used in the experiments. Tey were cultured in complete medium DMEM (Lonza, Bend, OR, USA) and supplemented with 10% fetal bovine serum (Sigma-Aldrich), 1% L-glutamine (Sigma-Aldrich, St. Louis, MO, USA), and 1% antibiotic-antimycotic (Biowest, Bradenton, FL, USA). Cells were maintained in an incubator at 37°C with 5% CO 2 and 95% humidity. Routine evaluations for the detection of mycoplasma contamination were conducted. Tere was no mycoplasma contamination detected in these cell lines.

Cell Viability Assay.
Te 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used to determine the cell viability. After reaching 70-80% confuence, the cells were incubated in 96-well plates (density: 3 × 10 4 cells per well) under standard conditions and exposed to decreasing concentrations of either water or ethanolic extracts (i.e., 10-0.75 mg/ml) for 48 h in triplicates. Following treatment, 5 mg/ml MTT (10 μl) (Termo Fisher, Waltham, MA, USA) was added to each well, the mixture was fltered through a 33-mm syringe flter (Sigma-Aldrich), and the cells were incubated for 4 h. Tereafter, isopropanol (100 μl) was added to each well, and the cells were incubated overnight. Optical density (OD) was measured at 595 nm using a microplate reader (Multiskan FC, Sigma-Aldrich). Negative controls were included containing either complete DMEM or DMEM with the same concentration of DMSO as that present in the ethanolic extract solutions. Cell viability percentage was calculated using the following formula: Lakes, NJ, USA). Data analysis was performed using the BD FACS Diva software (v 9.0; BD Biosciences).

Statistical
Analysis. Data are presented as mean-± standard error of the mean. Te GraphPad Prism version 9 for Windows software (GraphPad Software, La Jolla, CA, USA; http://www.graphpad.com) was utilized to perform a one-way analysis of variance (ANOVA) with Dunnett's posttest. Statistical signifcance among the groups was determined using the one-way ANOVA. A p value <0.0001 denoted statistically signifcant diferences. Nonlinear regression analysis was used to obtain the IC 50 for the diferent extracts of T. boudieri.

Water Extract of T. boudieri Provided a Higher Yield than the Ethanolic Extract.
Te percentage yield upon extraction of 250 g of T. boudieri and lyophilization was 12.76% and 4.2% for the water and ethanolic extracts, respectively. Tis fnding demonstrated high variability in yield between the extracts.

T. boudieri Extracts Diminished the Viability of HCT-116
and Caco-2 Cells. Exposure of HCT-116 and Caco-2 cells to T. boudieri extracts resulted in cancer cell inhibition in a dose-dependent manner; the water extract was slightly more potent than the ethanolic extract. Furthermore, HCT-116 cells were more sensitive than Caco-2 cells to both extracts. Te lowest and highest IC 50 were recorded for HCT-116 cells treated with the water extract (4 mg/ml) and Caco-2 cells treated with the ethanolic extract (6.4 mg/ml), respectively ( Figure 1).

Crystal Violet Assay Confrmed the Cytotoxic Efects of T. boudieri Water Extract.
Exposure of HCT-116 and Caco-2 cells to T. boudieri water extract for 48 h resulted in a dosedependent cytotoxic efect. HCT-116 cells exhibited higher sensitivity to treatment compared with Caco-2 cells ( Figure 2).

Cytomorphological Analysis Revealed Signs of Apoptosis in
Treated Cells. Cells exposed to diferent concentrations of T. boudieri water extract showed diminished proliferation and displayed morphological changes consistent with apoptosis (e.g., shrinkage, more intense staining, and dark condensed nuclei) compared with the negative control cells which proliferated into a dense monolayer. Te magnitude of changes in treated cells appeared to be dose-dependent ( Figure 3).

Treated HCT-116 and Caco-2 Cells Showed a Signifcantly
Decreased Capacity for Colony Formation. Te T. boudieri water extract signifcantly inhibited colony formation in a dose-dependent manner in both cell lines compared with the negative control. Te inhibition of colony formation was greater in HCT-116 than in Caco-2 cells, confrming the higher sensitivity of the former cell line to the extract (Figure 4).

T. boudieri Water Extract Inhibited Wound Healing in a Dose-Dependent Manner in Treated Caco-2 and HCT-116 Cells.
Te results of the wound healing assay further revealed a dose-dependent inhibitory efect on the proliferation of treated cells. Following treatment, the cells also exhibited a diminished capacity for migration, as well as morphological changes characteristic of apoptosis. At 48 h after treatment with the respective IC 50 , the wound area had increased for both cell lines compared with that recorded at T0 (i.e., the time at which the wound was inficted on the cellular monolayers). HCT-116 demonstrated a reduction in their capacity for wound healing compared with Caco-2 ( Figure 5). Tese results supported the fndings of the previous experiments.

Cell Cycle Analysis Detected Considerable DNA Fragmentation in Treated Cancer
Cells. Cell cycle analysis detected considerable DNA fragmentation, a hallmark of apoptosis, in all treated samples. Due to the extensive DNA fragmentation, it was difcult to determine any cell cycle arrest, and the percentage of cells beyond the G 0 -G 1 phase was greatly diminished. Te maximum percentage of cells in the G 2 -M phase was approximately 4% for both HCT-116 and Caco-2 cells exposed to the water extract ( Figure 6).

T. boudieri Water Extract Downregulated Bcl-2, cyclinD1, and c-Myc, and Upregulated p53 and Bax Proteins in Cancer
Cells. After treatment of HCT-116 and Caco-2 cells with T. boudieri water extract, there was a statistically signifcant change in the expression for all proteins analyzed compared with the negative control. Te levels of p53 increased in both treated cancer cell lines in a dose-dependent manner. Te levels of Bax were also signifcantly increased after treatment in both cell lines. In contrast, the levels of Bcl-2 (an inhibitor of Bax) were signifcantly decreased in both cell lines after treatment with T. boudieri water extract (Figure 7). In turn, these changes resulted in a signifcant rise in the Bax/Bcl-2 ratio after treatment (Figure 8). Te treatment also led to a reduction in the levels of cyclin D1 and c-Myc. Cyclin D1 is an important player in cell cycle progression into the S phase, [32] and c-Myc is a master regulator that is highly expressed in many types of cancer leading to an increase in the expression of many genes involved in cellular proliferation [9] (Figure 7).

Discussion
Several studies have demonstrated that natural extracts have the capacity to induce cytotoxicity in cancer cells while sparing normal cells. Tis property renders them attractive candidates for the development of therapeutic agents [33][34][35][36]. A possible explanation for this diference may be the higher metabolic rates of cancer cells and the consequent increase in the demand for nutrient acquisition, which sensitize them to treatment compared with normal cells [37,38]. Africa and the Middle East countries have a rich history in the treatment of various ailments using traditional medicines derived from local plants and macrofungi. A poem found in an Egyptian temple provides us with clues regarding the reverence with which the natives beheld macrofungi: "Without leaves, without buds, without fowers: yet they form fruit; as a food, as a tonic, as a medicine: the entire creation is precious [39]." T. boudieri is a desert trufe abundantly found in the arid and semiarid areas of the Middle East, and is considered a part of the native cuisine. Studies have investigated its chemical composition and found that it is rich in numerous compounds (e.g., carbohydrates, alkaloids, tannins, favonoids, steroids, and terpenoids), linoleic acid and glutamic acid, essential amino acids (e.g., leucine and threonine), and elements (e.g., magnesium) [19,21]. Furthermore, studies have shown that this desert trufe base may have immunomodulatory, antioxidant, and anticancer properties [29].    Figure 4: (a) Te clonogenic assay showed that T. boudieri water extract signifcantly inhibits colony formation in Caco-2 and HCT-116 cell lines in a dose-dependent manner. Wells containing 1 × 10 3 cells treated in triplicates showed the most signifcant inhibition with IC 50 (4 mg/ml for HCT-116 and 5 mg/ml for Caco-2). Colony inhibition was more prominent in the HCT-116 cell line. (b) Colonies dyed with 0.5% crystal violet are shown to be higher in density in the negative control wells, followed by the wells treated with the lowest concentration of the water extract 1/4 IC 50, 1/2 IC 50, and IC 50 .
However, the mechanisms underlying this infuence on cellular physiology remains to be fully understood. In this study, HCT-116 and Caco-2 colon cancer cell lines and western blotting were used to investigate several proteins (i.e., Bax, Bcl-2, p53, CCND1, and c-Myc) that play a central role in cell apoptosis, proliferation, and cell cycle progression. Te cell viability assay revealed that both extracts exerted a comparable cytotoxic exert on the cell lines. For HCT-116 and Caco-2 cells, the IC 50 of the ethanolic extract was 4.8 mg/ml and 6.4 mg/ml, respectively, while that of the water extract was 4 mg/ml and 5.4 mg/ml, respectively (Figure 1). Tis indicates that the HCT-116 cell line was more sensitive to treatment than Caco-2 cells. Moreover, the water extract had a slightly greater efect on cells than its ethanolic counterpart. Tis could be attributed to the higher carbohydrate and phenolic content of the water extract [21]. Te crystal violet assay, conducted using the water extract, . Wound area percentages tended to be greater overall in HCT-116 cells. Wound area was measured using ImageJ (U.S. National Institutes of Health, Bethesda, Maryland, USA, https://imagej.nih.gov/ij/), and wound area percentage was measured according to the formula: % wound area � (AT48/AT0) × 100%, where AT48 hr is the area at 48 hs following treatment and AT0 hrs is the area at the time right after wound induction. Images were captured at 40x magnifcation scale bar � 500 μm.
yielded comparable results. However, the water extract showed lower IC 50 values for both cell lines (2.65 mg/ml and 3.96 mg/ml for HCT-116 and Caco-2 cells, respectively) than those of the ethanolic extract. Tese lower values are potentially important because of the use of tap water that could result in stronger detachment of treated cells (Figure 2). HCT-116 cells were more sensitive to the treatment than Caco-2 cells.
In the wound healing assay, the water extract inhibited wound healing at 48 h after treatment, even at the lowest concentrations used (i.e., 1/4 IC 50 ). In the wells where the two cell lines (HCT-116 and Caco-2) were treated with their respective IC 50 of T. boudieri water extract, the wound area was greater than that recorded at the time of wound infiction. In addition, few cells were present around the wound and some showed morphological signs of apoptosis under the microscope (e.g., shrinkage and darkening of the nucleus) ( Figure 5). Tis observation was confrmed in the cytomorphological study, in which signs of apoptosis were present in a dose-dependent manner in both the cell lines treated with the water extract ( Figure 3). Both these experiments verifed that HCT-116 cells are more sensitive to treatment than Caco-2 cells. Tese fndings suggest that the extract has a cytotoxic efect on cancer cells, and afects their migration and proliferation. Tis was supported by the results of the clonogenic assay (Figure 4), in which cells treated for 48 h showed a signifcant dose-dependent reduction in their capacity to form colonies compared with the negative control.
Flow cytometry for cell cycle analysis after treatment with IC 50 and 1/2 IC 50 of water extract for 48 h showed extensive fragmentation (preG 0 was approximately 40-65%, with higher percentages observed for cells treated with IC 50 ). Tis is a hallmark of apoptosis that aligns with the fndings of the previous experiments. However, given the high percentages of the preG 0 state, it was difcult to determine the efect of the water extract on the cell cycle. Notably, the highest and lowest percentages of cells were found in the G 0 -G 1 and G 2 phases, respectively. Moreover, it was shown that cyclin D1 is a major driver of the cell cycle beyond G 1 [32]. Te western blotting results showed a remarkable decrease in the levels of cyclin D1 in cells treated with the IC 50 of the water extract (Figure 7). Terefore, it is reasonable to conclude that few treated cells would transition into the S phase.
p53 is involved in a wide range of cellular processes (e.g., diferentiation, DNA repair, cellular senescence, cell death, and cell cycle) [40]. Dysregulation of p53 through mutation or deactivation of its signaling capacities is important for cancer development [5]. Tis suppression of p53, found in   [41]. Te present results showed a signifcant increase in the levels of p53 in both colon cancer cell lines in a dose-dependent manner following treatment with the water extract of T. boudieri (Figure 7). It has been previously shown that favonoids present in the aqueous/water extracts of T. boudieri [21] induce apoptosis and cell cycle arrest through p53 accumulation [42]. Te results of the western blotting analysis  revealed a rise in the levels of another protein that plays a central role in the mitochondrial or intrinsic pathway of apoptosis, namely Bax. When activated, Bax enters into the mitochondrial membrane, thereby inducing its permeabilization, the release of cytochrome c into the cellular cytoplasm, and the formation of the apoptosome which leads to the induction of apoptosis [43]. Dysregulation of apoptosis results in the disruption of tissue turnover, and this process is associated with the occurrence of neoplasms [44]. Bcl-2 plays an important role in suppressing apoptosis by inhibiting the binding of Bax to the mitochondrial membrane [43]. Te antiapoptotic activity of Bcl-2 confers resistance to chemotherapy in patients with cancer [45]. Terefore, Bcl-2 has been identifed as a target for cancer therapy [46,47]. Moreover, the Bax/Bcl-2 ratio has a clinical signifcance in the prognosis of patients with cancer and the development of resistance to chemotherapeutic agents [11,48]. Treatment of HCT-116 and Caco-2 cells with their respective IC 50 of T. boudieri water extract led to a signifcant decrease in the protein levels of Bcl-2 ( Figure 7) and a signifcant rise in the Bax/Bcl-2 ratio (Figure 8). Te canonical Wnt pathway or Wnt/β catenin pathway is another important pathway in cancer development. Tis pathway is commonly overactivated in colon cancer [49][50][51]. Te endpoint of this pathway is the activation of the transcription factor/lymphoid enhancer binding factor (TCF/ LEF) family [52]. A characteristic of this activation is the expression of the c-Myc gene, a target of TCF4. c-Myc is a master regulator that can directly repress p21, thereby causing hyperproliferation [53][54][55]. Increased Wnt signaling leads to overexpression of the CCDN1 gene. cyclin D1 is an important regulator of the cell cycle, promoting the transition from the G1 phase to the S phase [56]. It is also implicated in promoting the invasion and dissemination of cancer cells [32]. Furthermore, a coordinated efect between c-Myc and cyclin D1 may drive tumor aggressiveness and accelerate disease progression [8]. Tus, the roles of c-Myc, cyclin D1, and the Wnt pathway in the treatment of cancer have been extensively studied [50,57,58]. Similar to Bax and Bcl-2, c-Myc and cyclin D1 have been suggested as prognostic factors in cancer [59]. In this study, the protein levels of cyclin D1 and c-Myc were signifcantly decreased in HCT-116 and Caco-2 cells after treatment with their respective IC 50 of the water extract; this reduction was more notable in Caco-2 cells.
Collectively, the results of this study provide a more indepth description of the processes underlying the cytotoxic efect of T. boudieri. Al-Obaydi et al. [21] previously demonstrated the apoptotic efect of T. boudieri extracts through a caspase 3 (CASP3) assay. In addition, Dahham et al. demonstrated the cytotoxic efect of various extracts of T. boudieri on diferent cell lines using the MTT cytotoxicity assay, through loss of the mitochondrial membrane potential [29].

Conclusions
T. boudieri water and ethanolic extracts exerted comparable cytotoxic efects on colon cancer cells, HCT-116 and Caco-2. Te slightly more potent water extract was able to reduce cancer cell stemness. Te treatment reduced the capacity of cells for proliferation and migration. Te morphological characteristics of apoptosis were also evident following treatment. Further analysis revealed extensive DNA fragmentation after treatment, a hallmark of apoptosis. Te protein levels of p53 and Bax were elevated in treated cells, whereas those of Bcl-2, c-Myc, and CCND1 were reduced. Tis suggests that T. boudieri water extract exerts a proapoptotic efect on colon cancer cells through multiple signaling pathways. In addition, the extract also demonstrated an antiproliferative efect. Te extract simultaneously affected the levels of various proteins considered as important targets in cancer therapy. Further investigation is warranted to determine the usefulness of T. boudieri and its constitutive elements as an adjunct to current chemotherapy for the treatment of cancer.

Data Availability
Te data generated during and/or analyzed during the current study are available from the corresponding author upon reasonable request.