In Vitro Efficacy of Extracts and Isolated Bioactive Compounds from Ascomycota Fungi in the Treatment of Colorectal Cancer: A Systematic Review

Colorectal cancer (CRC) is the second leading cause of cancer-related deaths worldwide. Despite the advances and success of current treatments (e.g., chemotherapy), there are multiple serious side effects which require the development of new treatment strategies. In recent years, fungi have gained considerable attention as a source of extracts and bioactive compounds with antitumor capabilities because of their antimicrobial and antioxidant properties and even their anti-inflammatory and antiviral activities. In the present review, a systematic search of the existing literature in four electronic databases was carried out in which the antitumor activity against CRC cells of Ascomycota fungi extracts or compounds was tested. The systematical research in the four databases resulted in a total of 883 articles. After applying exclusion and inclusion criteria, a total of 75 articles were finally studied. The order Eurotiales was the most studied (46% of the articles), and the ethyl acetate extraction was the most used method (49% of the papers). Penicillium extracts and gliotoxin and acetylgliotoxin G bioactive compounds showed the highest cytotoxic activity. This review also focuses on the action mechanisms of the extracts and bioactive compounds of fungi against CRC, which were mediated by apoptosis induction and the arrest of the cell cycle, which induces a notable reduction in the CRC cell proliferation capacity, and by the reduction in cell migration that limits their ability to produce metastasis. Thus, the ability of fungi to induce the death of cancer cells through different mechanisms may be the basis for the development of new therapies that improve the current results, especially in the more advanced stages of the CCR.


Introduction
Colorectal cancer (CRC) is the third most common cancer type worldwide and the second deadliest malignancy for both sexes combined. In 2020, it was estimated that 935,000 deaths secondary to CRC occurred, and 1.9 million new cases were diagnosed. Specifically, the American Cancer Society had predicted that, in the United States in 2022, more than 100,000 people would be diagnosed with this type of cancer and approximately 52,000 deaths would be caused by CRC. It is known that CRC is associated with a high socioeconomic status, which explains its high incidence in European countries [1,2]. A clear correlation has been demonstrated between the development of the disease and environmental, hereditary, and lifestyle factors, including obesity, a sedentary lifestyle, smoking, processed or red meat, and alcohol. Nevertheless, certain preventive factors have A total of 151 bioactive compounds have been isolated from the different orders of Ascomycota, tested in CRC cell lines (Supplementary Table S1), and analyzed to determine antitumor action mechanisms. As shown in Figure 2, the most studied pathways are those of the Eurotiales and Hypocreales orders. A total of 151 bioactive compounds have been isolated from the different orders of Ascomycota, tested in CRC cell lines (Supplementary Table S1), and analyzed to determine antitumor action mechanisms. As shown in Figure 2, the most studied pathways are those of the Eurotiales and Hypocreales orders.

Genus Aspergillus
Twenty-four articles analyzed species of the genus Aspergillus, of which eight articles tested functional extracts on CRC cell lines. Ethyl acetate extracts (five articles) showed IC 50 values between 42.75 and 185.9 µg/mL [33,36,58]. Moreover, Ali et al. [36] reported that ethyl acetate extracts from nine different Aspergillus species induced death of 50.1 to 69.1% of HCT-116 cells. A similar extract obtained by Artasasta et al. [33] was reported to cause a significant reduction in the viability of WiDr cells. Asfour et al. [32] also used methanol as a mycelium extraction method, obtaining IC 50 values between 15-100 µg/mL in HCT-116 cells, while Alasmary et al. [23] obtained an ethanolic extract with higher antitumor activity (IC 50 125 µg/mL) in the same cell line. Furthermore, Abd El-Hady et al. [19] tested a sequential extract of ethyl acetate, methanol, and dichloromethane (100 µg/mL) that induced significant cytotoxicity (15.8%) in the same cell line. Finally, a crude extract obtained by sonication, centrifugation, and lyophilization showed an IC 50 value of 9.84 µg/mL in CaCo-2 cells [25]. Functional extract fractions (three articles) were tested on the HCT-116 cell line, with IC 50 values between 5.28-193.64 µg/mL [24,33] and 15.8-88% cytotoxicity [19]. Interestingly, most of the extracts obtained from the genus Aspergillus were also tested on other cancer cell lines of liver, larynx, cervix, and breast [23,32,36,58], in which, in general, a higher cytotoxic effect was noted compared to CRC cells. Only the functional extracts obtained by Ashour et al. [24] reduced the IC 50 to a greater extent in CRC (more than half the IC 50 ) than in other tumor cells, such as those derived from liver and breast cancer.
On the other hand, 49 bioactive compounds from the genus Aspergillus were described in a total of 14 articles. For example, malformin C was effective in MC-38 and HCT-116 cell lines (IC 50 0.27 and 0.18 µM, respectively), with similar results being obtained in the murine pancreatic cancer cell line PanO2 and in the human lung adenocarcinoma cell line H1975. This bioactive compound induced G2/M phase arrest, DNA damage, apoptosis, autophagy, and necrosis [40]. Two of the most promising compounds in relation to the treatment of CRC were gliotoxin and acetylgliotoxin G, which showed very low IC 50 values (0.41 and 1.06 µg/mL, respectively) against HCT-116 cells [44]. In fact, gliotoxin was also reported to have antitumor efficacy in chondrosarcoma, cervix, and glioblastoma cells [59,60]. In addition, asperphenin A showed greater activity in CRC cells than in breast cancer cells (IC 50 0.84 vs. 6.48 µM, respectively), inducing G2/M cell cycle arrest by the inhibition of tubulin polymerization, induction of apoptosis, and production of reactive oxygen species (ROS). In addition, asperphenin demonstrated a synergistic effect in combination with irinotecan and paclitaxel [20]. Other bioactive compounds, such as clavatustide B, inhibited the G1/S phase, while acetylaranotin, acetylapoaranotin, and deoxyapoaranotin induced apoptosis mediated by caspases 3, 9, and 8 [28,31,42]. Finally, isolated compounds from the genus aspergillus, such as asperphenin A, malformin C, or acetylapoaranotin have succeeded in taking a further step toward in vivo murine research, although more studies are needed [20,38,42].

Genus Penicillium
The most relevant studies in the genus Penicillium used the ethyl acetate extraction method (five out of fourteen) [22,32] or methanol and ethanol methods (two out of fourteen) [49,54,56] to develop functional extracts that showed IC 50 values between 0.2 and 102 µg/mL in CRC cells. Canturk et al. [56] and Dikmen et al. [38] showed that ethyl acetate extracts reduced the invasiveness of cancer cells by decreasing cell migration and the expression of genes related to angiogenesis and metastasis. In addition, a total of 48 bioactive compounds from different species of the genus Penicillium (nine of fourteen articles), including arenicolin A, isopenicin A, penipacids A, and norverrucosidin, were detected, showing the lowest IC 50 values against HCT-116 (7.3 µg/mL), SW-180 (0.74 µg/mL), RKO (8.4 µg/mL), and HCT-116 cells (5.7 µg/mL), respectively. Furthermore, isopenicin A induced apoptosis and modulated proteins involved in cell cycle progression from G2 to M [21,29,30,52,53,55,57]. The anti-tumor activity of some of the extracts and bioactive compounds from the genus Penicillium were tested against breast, cervix, and liver cancer cells, obtaining similar results [21,32,52,53,55,57].

Genera Cordyceps, Fusarium and Trichoderma
As shown in Table 2, the studies on the order Hypocreales (16 articles) used a wide variety of extraction methods, although methanol and ethyl acetate were the most common. Four articles focused on the genus Cordyceps, obtaining methanol extracts (two articles) that showed IC 50 values between 72.57 and 250 µg/mL against HCT-116, SW-480, and HCT-15, reducing both cell migration and cytoplasmic β-catenin [62,63]. An ethanol extract induced cell morphological changes and G2/M cell cycle arrest [64], and a butanol extract from Cordyceps militaris (sprouted soybean) induced a strong inhibition of HT-29 cell proliferation (56%) and G2/M phase arrest by blocking cyclin B1 and the expression of Cdc25c [65]. Lee et al. [64] tested this ethanol extract in a xenograft mouse model and found a significant inhibition of tumor growth and a reduction in mouse mortality. On the other hand, the genus Fusarium was studied in four articles, showing active functional extracts against CaCo-2, HCT-116, and HCT-8 cells (IC 50 between 0.3779-98.68 µg/mL) [25,[66][67][68]. In one article, standard camptothecin and camptothecin crude extract were isolated and tested against CaCo-2 cells, resulting in IC 50 values of 2.41 and 0.291 µM, respectively [68]. This compound has been used for the development of a conjugate, CT-2106, that has been studied in a clinical trial in combination with 5-fluorouracil and folic acid (NCT00291785), whose results had not been reported yet. In addition, camptothecin is the precursor of irinotecan, an antitumor drug that, in combination with other anticancer agents, has been widely used in clinical trials and its clinical use is well accepted [69]. Finally, the genus Trichoderma was analyzed in four articles, obtaining functional extracts (IC 50 between 11-100 µg/mL), fractions (IC 50 between 7.3 and 14.9 µg/mL) [24,66,70], or bioactive compounds, such as trichodermaloid A and B (IC 50 9.3 and 8.6 µM in the SW-620 cell line, respectively) [71]. All of these findings are consistent with those obtained in other forms of tumors (breast, lung, liver, and cervix cancers, among others).

Other Genera
The genera Beauveria, Bionectria, Engyodontium, Metarhizium, and Myrothecium were analyzed in one article each. 1-Hydroxy-10-methoxy-dibenz[b, e]oxepin-6,11-dione, chrysazin (IC 50 > 30 µM), and globosuxanthone A (IC 50 10.7 µM) were isolated from the genus Beauveria and tested on HCT-15 cells [72]. Beauvericin, another compound from the genus Beauveria, has been used for in vivo assays in BALB/c and CB-17/SCID mice, decreasing tumor volumes and increasing necrotic areas of tumors, becoming a potentially interesting drug for the treatment of CRC [76]. Exopolysaccharides isolated from the genus Bionectria (0.45 mg/mL) significantly reduced HT-29 cell viability (15.42%) [73]. In addition, functional extracts from the genera Engyodontium and Myrothecium showed IC 50 values of 2.5 µg/mL and 380 ng/mL in HCT-116 cells, respectively. Specifically, Myrothecium extract showed higher cytotoxic activity in breast MCF-7 cells (IC 50 107 ng/mL) and lower in the liver cell line HepG2 (IC 50 711 ng/mL) [49,73]. Finally, destruxins A, B, and E from the genus Metarhizium were tested in CaCo-2 and HCT-116 cells, showing IC 50 values between 0.04 and 10 µM. However, they were also active against the KB-3-1 cell line derived from the epidermal carcinoma and A549 lung cancer cells. Furthermore, destruxin E induced ROS production and activated apoptotic caspases, even before mitochondrial membrane depolarization. The three destruxins reduced cell migration and angiogenesis, induced G0/G1 cell cycle arrest in the CaCo-2 cell line, and interfered with the MAPK and/or PI3K/Akt signaling pathways [74].

Minoritary Orders
As shown in Table 5, the antitumor activity of the order Capnodiales (three articles) was studied using the genera Cladosporium (functional extracts) and Zasmidium (bioactive compound). In fact, 8,8 -Bijuglone showed an IC 50 value of 45 µg/mL in the HCT-116 cell line. Functional extracts and bioactive compounds from Cladosporium were tested on both CRC and breast cancer cells with a significant differential effect [39,86,87]. Taxol was one of the compounds, which, due to its potent antitumor effect, has not only been tested in several clinical trials but has also come to be used in clinics against CRC [88]. In addition, the genera Sclerotinia and Lachnum (order Helotiales) were processed to obtain the exopolysaccharide LEP-2b and derivates from the genus Lachnum, which showed high antitumor activity (e.g., IC 50 of LEP-2b, 85.78 µg/mL) in the CT-26 cell line, among other tumor cells [89][90][91].
Studies on the order Diaportales (two articles) showed methanol and ethyl acetate extracts with IC 50 values ranging from 5.63 to 24.47 µg/mL in SW-480 and HCT-116 cells lines [77], and the isolation of dicerandrol A and B with significant antitumor activity in HCT-116 CRC cells with IC 50 values of 2.64 and 3.94 µM, respectively [92]. All of them were also highly effective against cell lines of other cancer types, such as breast, lung, and liver. The order Pezizales (two articles) was studied by Liu et al. [93] and Tang et al. [94]. The latter showed polysaccharides from the genus Morchella with high CRC cell cytotoxicity (IC 50 between 1.229 and 2.827 mg/mL in CaCo-2 cells). This finding was supported by results in the hepatocellular cancer line HepG2. Similarly, four different compounds were isolated from the order Xylariales, highlighting 5-methylmellein and daldinone F, which showed significant antitumor activity (IC 50 of 2 and 9.59 µM) in SW-480 and HCT-116 cells, respectively. Moreover, 5-Methylmellein showed activity against prostate and breast cancer cells. Indeed, it was encapsulated in nanoparticles, increasing the IC 50 to <0.5 µg/mL, and inducing apoptosis, ROS production, and the loss of the mitochondrial membrane potential [95,96]. Finally, other orders, such as Boliniales, Incertae sedis, Leotiales, and Venturiales were studied using ethyl acetate extracts or bioactive compounds, such as xylarenone D, greensporone C, and O-desmethyl greensporone C, which were effective against CRC cells (IC 50 1.5, 7.5 and 13.8 µM, respectively), among other cancer types (melanoma, glioblastoma, and leukemia) [34,39,49,97]. Table 3. Antitumor activity of the extracts or isolated compounds from Pleosporales order in CRC cancer cell lines.

Materials and Methods
A complete method was thoroughly organized with the collection of data and the steps of analysis, including the protocol registration (https://doi.org/10.17605/OSF.IO/X5KTD accessed on 10 November 2022).

Study Eligibility
Since the purpose of this review was to compile the most recent and representative knowledge of the antitumor capacities against colorectal cancer of bioactive compounds isolated from Ascomycota fungi or their functional extracts, a bibliometric analysis was carried out. A period of 10 years was established, considering older results obsolete (Burton-Kebler index for obsolescence) and including more than half of the actual disponible papers [98].

Inclusion Criteria
Research articles published in English from January 2011 to October 2021 in which extracts, or compounds isolated from Ascomycota fungi, had their antitumor activity on CRC cell lines tested were included in this systematic review. The research articles had been published in peer reviewed journals with the full text accessible.

Exclusion Criteria
Papers in which any colon cancer cell line was not used, or the bioactive compound or extract tested had no antitumor activity, were excluded. Furthermore, publications in which the bioactive compound was synthesized/purchased, or the extraction methodology was not specified, were also excluded. Finally, studies that did not exceed the minimum requirements of an in vitro study or with a low quality of the study, were excluded from the present review.

Data Sources
Four electronic databases were used to perform the systematic review: MedLars Online International Literature, via PubMed, SCOPUS, Web of Science Core Collection, and the Cochrane Library Plus. Firstly, the following Medical Subject Headings (MeSH) were defined to use as descriptors in Pubmed: "Colorectal Neoplasms", "Fungi", "Ascomycota", and "Aspergillus".

Study Selection
Two of the authors (C.L. and A.C.) performed the bibliographic search, screened the abstract of the resulting publications, and selected the adequate ones for a fully-text review. Editorials, conference papers, bibliographic and meta-analysis reviews, book chapters, epidemiological studies, and case reports were excluded. In the following stage of the selection process, the same authors analyzed and included or excluded the full-text articles. Because the aim of this study was to review the current data available relating to in vitro publications, in vivo and clinical trials were manually excluded.

Data Extraction
Once the list of the articles included in the study was obtained, the same authors independently evaluated and extracted data from the selected studies according to the Cohen kappa statistical test for agreements (more than 0.8) [99]. Any discrepancy was resolved by a consensus between C.L. and A.C. or two more authors (J.P and C.M), if necessary. All of the selected articles were analyzed for quality using a specific questionnaire for in vitro studies with a first part in which the minimum requirements of an in vitro study were determined (score > 6), and a second part in which the quality of the study was analyzed (0-6 = low; 7-14 = good; 15-20 = excellent) based on materials and methods, results, and conclusions. Publications were classified according to the order of the studied fungi and the extracted data are condensed in Tables 1-5. To facilitate the interpretation of the selected studies, reference genera of fungi studied, where the fungi were isolated from, the extraction method, isolated compounds, the cell line used, cytotoxicity activity, and the mechanism of action.

Conclusions
This systematic review focused on in vitro studies on the antitumor activity of extracts and compounds isolated from fungi of the phylum Ascomycota. Of all of the genera analyzed in the literature, Penicillium, Fusarium, and Chaetomium produced the extracts with the greatest antitumor activity in CRC. A wide variety of bioactive compounds have been isolated from different genera of the phylum, some of which are particularly interesting given their high anticancer activity against this tumor. Although current results are very promising, more research is needed on genera that have been less studied. It is also important to move towards in vivo studies and/or clinical trials of the extracts and/or bioactive compounds with the aim that they could be used as a therapy against CRC in the future.