Search Strategy in PubMed and PICOT question

In order to elaborate the search strategy in PubMed, it was followed a research PICOT question, in which was determined the population as cancer cell lines (not specified in the search strategy); the intervention was conformed by the families, species or active substances of possible endophytic fungi with anticancer activity.

The comparator was not defined in the search strategy either, but it was expected to be rather conventional therapy or a placebo.

The outcome was defined on the basis of cancer cell mortality, and the variation of indicators of anticarcinogenic activity (inhibition of tumor growth, decrease in cell proliferation, half of the maximum inhibitory concentration, etc.); finally, the type of study considered was preclinical, those in vitro, in vivo or in silico.

The strategy in PubMed executed on April 23, 2024, ("Endophyte"[Mesh] OR endophyt*[tiab] ) AND ("Neoplasms"[Mesh] OR Neoplasm*[tiab] OR "Carcinogenesis"[Mesh] OR Carcinogenesis[tiab] OR "Cell Proliferation"[Mesh] OR "Antineoplastic Agents"[Mesh] OR Antineoplastic Agents[tiab] OR cell cycle arrest[tiab] OR metastasis inhibition[tiab] OR anticancer*[tiab] ) AND (preclinic*[tiab] OR "in vitro"[tiab] OR "in vivo"[tiab] OR "ex vivo"[tiab]) NOT (Case Reports[Publication Type]), yielded 197 results.

Inclusion and Exclusion Criteria

From the 197 results obtained, we excluded studies that were not conducted between 2015 to 2024, clinical studies, studies of anticarcinogenic secondary metabolites isolated from endophytic bacteria, those in a language other than English or Spanish, those that did not test the cytotoxicity of metabolites on cancer cells, and those that used the endophytic fungus as a chassis to confer anticarcinogenic properties to compounds.

Selection Process

In the present review, a total of 197 articles identified in Pubmed were obtained, of which those that were duplicated or not written in English or Spanish were removed, leaving 191 articles for the screening phase. The screening consisted of evaluating whether the study was potentially eligible based on the title and abstract, where they were distributed in such a way that each article had to be evaluated by a minimum of two researchers, and in case of controversy or doubt, the entire team of researchers was called to evaluate the inclusion or exclusion of the article.

After applying the aforementioned inclusion and exclusion criteria and evaluating articles for retrieval, a total of 67 eligible articles were obtained and included in the review.

Cytotoxicity

Different endophytic producing fungi were recorded, among which the genera Alternaria, Aspergillus, Fusarium, Penicillium and Phomopsis stand out. The active compounds were classified according to their cytotoxicity shown by each cell line (Tables 1 and 6). The most commonly used cell lines were the cervical cancer cell line HeLa, as well as the hepatocellular carcinoma cell line HepG2. Gastric cancer cells, hepatocellular carcinomas, colon carcinoma, melanomas, leukemia line, breast cancer line, ovarian cancer, among others, were also used.

The studies evaluated in the present review were mostly exclusively in vitro, with the exception of Hoque et al. (Hoque et al., 2022) and Da Tang et al (Tang et al., 2022), which were performed in silico, the first one with the aim of evaluating the different active compounds of the fungus Fusarium oxysporum in the colon carcinoma line HCC 2998m, Oligodendroglioma (Hs 683) among others, where it was concluded that 3β, 5α-dihydroxy -ergosta-7, 22-dien-6-one [1], 3β, 5α, 9α-trihydroxy-ergosta-7,22-dien-6-one [2] and beauvericin [5] as cytotoxic agents, however, the compounds p-hydroxybenzaldehyde [3] and 3-(R)-7-butyl-6,8-dihydroxy-3-pent-11-enylisochroman-1-one [4] were found to be mutagenic. The second one evaluated Porric acid E in HT29 cells.

Although several active compounds have shown cytotoxicity to cancer cell lines, polonicin A, polonicin B and 3,5 -hydroxydihydrofusarubins D, derived from Penicillium polonicum, did not achieve this objective according to Wen et al (Wen et al., 2020), however, they have antidiabetic activity by increasing glucose uptake.

Another fungus that did not obtain cytotoxicity from its derivatives was Aspergillus fumigatus (Astuti et al., 2020), whose compounds did not show cytotoxicity against T47D breast cancer in synergy with doxorubicin (Dox).

Celular Cycle arrest

Trichothecin (TCN) promotes the expression of Dehydrogenase Reductase member 2 (DHRS2) in in vitro studies with the nasopharyngeal carcinoma cell line NPC. An alteration of the cellular lipid profile associated with DHRS2 overexpression was demonstrated by gas chromatography coupled to mass spectrometry. DHRS2 evidences a close association with inhibition of cell proliferation, migration, and quiescence in the cell lines examined (Luo et al., 2019). In addition, RNA sequencing was performed to identify genes involved with the anticarcinogenic effects of TCN.

Ascomylactam A (AsA) showed growth inhibition in 6 lung cancer cell lines (L. Wang et al., 2020). These effects were seen by phase contrast microscopy after 48 h exposure at different concentration levels (IC50: 4-8 uM).

This investigation revealed two mechanisms: First, the interference of AsA in the generation of CDK4 and CDK6 kinases and cyclin D1, indispensable for the passage from G1 to S phase. Subsequently, an increase in the amounts of reactive oxygen species was observed in cells stationed in G0/G1 phase and the continuation of the cell cycle upon addition of antioxidants. This represents a particular advantage since cancer cells show greater vulnerability to cytotoxicity in in-vitro assays.

Demethylincisterol A3 (Sdy-1), on the other hand, demonstrated an anticarcinogenic effect by inducing cell apoptosis and G1-phase cell cycle arrest in human HepG2 liver carcinoma and HeLa cervical cancer lines. Both mechanisms, inhibition of Wnt/beta-catenin signaling pathways were demonstrated as beta-catenin levels and other pathway components (cyclin-dependent kinase CDK4, cyclin D1 and c-myc genes) decreased in both lines upon addition of Sdy-1. For the following mechanism, it was evidenced that Sdy-1 increases the cleavage of caspases 3 and 9, in addition, chromatin condensation, nuclear fragmentation and apoptotic debris were evidenced in the post-treatment samples (Sun et al., 2022).

Pulchranin-A demonstrated cytotoxic activity in HCT colorectal cancer, Hep-G2 liver carcinoma and MCF-7 breast cancer cell lines by sulfurodamine assay. In addition, inhibition of 3 cyclin-dependent kinases (CDK1, CDK2 and CDK4) was recorded in the MCF-7 line. This inhibition allows the interruption of the cell cycle in the G2/M and G1/S phases, and CDK4 activity is promising since, together with cyclin D1, they are the proteins most frequently affected by mutations in various types of cancer (Moussa et al., 2020).

Cell toxicity generated by Tricycloalternarene (Shen et al., 2018) was demonstrated in hepatocarcinoma cell lines SMMC-7721 and gastric cancer SGC-7901 by MTT assay, also cell cycle retention in G1 phase caused by overexpression of p27 protein was proved by Western Blot. The p27 protein interferes with the cell cycle as it prevents the formation of Cliklin-CPK complexes, and is already considered a potential tumor suppressor protein, and abnormal expression of the p27 gene is closely related to the occurrence and development of multiple malignant tumors.

Finally, a study by Zhu S et al showed that the metabolite produced after Phomosis sp, Deacetyl-mycoepoxydiene (DM), was capable of induce the reorganization and polymerization of tubulin and the G2/M cell cycle arrest of MCF-7 cell lines (Zhu et al., 2015).

Protein Inhibition

Sclerothiorin (Kabbaj et al., 2015) demonstrated activity on certain chaperones (Hsp90 inhibition, Hsp70 overexpression and Hsp40 degradation) in a progesterone receptor assay. However, this compound did not show cytotoxicity in breast cancer cell lines Hs578T, MCF7, MDA-MB-231 and MDA-MB-453, prostate cancer LNCaP and cervical cancer HeLa.

These results changed upon deacetylation of sclerostin: inhibition of Hsp90 chaperone function was reduced and cytotoxicity was evident against some cell lines (Hs578T, MDA-MB-231 and LNCaP) within 48 hours.

Penicisulfuranol A (PEN-A) is a promising anti-carcinogenic agent by inducing apoptosis in tumor cells in in vitro studies. The triple action of this compound was demonstrated: it inhibits the C-terminal polymer dimerization of Hsp90, inhibits the polymerization of Hsp90 target proteins and obstructs the co-activity of other chaperones with Hsp90 (Dai et al., 2019).

Myrothecine A (Fu et al., 2019) can restrain cell proliferation induced by miR-221, a type of microRNA with oncogenic activity, in a hepatocarcinoma cell line SMMC-7721. According to the studies performed, miR-221 negatively affects the levels of p27 protein, a cyclin-dependent kinase inhibitor and a direct target molecule of miR-221 that negatively regulates cell proliferation and differentiation. Myrothecin A increased p27 levels in cultured cells by inhibiting miR-221 expression.

It is also able to induce maturation of dendritic cells in simultaneous culture with the hepatocarcinoma cell line, including an increase in CD40 and CD86 during miR-221 inhibition, which favors a better specific response against cancer cells.

Camptothecin (CPT) is a quinoline alkaloid of plant origin capable of inhibiting topoisomerase 1 in tumor cells by binding to the active site of tyrosine during the DNA replication process. Another effect observed is the activation of hypoxia-induced factor 1a (HIF1a), which causes a ribosomal blockade responsible for stopping the translational response (Dhakshinamoorthy et al., 2021).

The effects of CPT were studied in a lung carcinoma cell line A-549 in an MTT assay, in this research the cytotoxic effect generated by CPT in cancer cells was evidenced.

Apoptosis Induction

The process of cell apoptosis is favored by certain active compounds of endophytic fungi, such as rosolactone evaluated by Zhou et al (L. Zhou et al., 2017), which increases proteins involved in oxidative stress of the endoplasmic reticulum, which induces apoptosis and autophagy. This inductive capacity was evaluated in HeLa cells and it was seen how rosolactone generated an increase in ROS, which generates activation of mitochondrial apoptotic pathways, as well as increasing proteins such as BAx, caspase 3 and cytoplasmic cytochrome c.

Another of the endophytic fungi with apoptotic capacity was Aspergillus terreus (JAS-2), which according to Goutam et al (Goutam et al., 2017), cytotoxicity against HCT 116 of the active compound Terrain produced changes in the sub G1 stage of the cell cycle, in addition to producing apoptosis in the A-459 cell line. To reach this conclusion, the research required analysis of DNA bases through FACS (Fluorescence Activated Cell Sorting).

On the other hand, it has been found that the active compound of the fungus has different apoptotic metabolic pathways, such as increasing ROS expression and increasing apoptosis through the protein expression of Bax, as well as increasing the expression of cyclin-dependent p21 inhibitor kinase, evaluated through the MCF7 cell line.

Apoptotic pathways evaluated in Subban et al, a Taxol derivative, EDT, was able to induce apoptosis in HepG2 cells in the G2/M phase of the cell cycle evaluated by flow cytometry. In addition, DNA fragmentation was observed in agarose with cells with the active compound and not in controls (Subban et al., 2017).

The active compound HLP46 derived from Pestalotiopsis sp, according to Chen C et al, is able to disrupt the Gab1-Shp2 association, which was corroborated by immunoprecipitation methods. Furthermore, after evaluation by Ras Activation Assay, the derived compound was found to block EGF-induced activation of the Shp2-dependent Ras/ERK signaling pathway (C. Chen et al., 2017).

The active compound demethylincisterol A3 was able to induce G0/G1 cell stage in tumor cells, in addition to inducing necrosis of these cells, was evaluated by Zhou et al through flow cytometry (J. Zhou et al., 2018).

Li Shen et al. evaluated the endophytic fungus Alternaria sp associated with Laminaria japonica, and found that active compounds promote apoptosis in five ways. Triccycloalternarene 3a induces apoptosis in SMMC 7721 dose-dependently by overexpressing the pro-apoptotic protein Bax and decreasing the anti-apoptotic protein Bcl2, i.e. the Bcl2/bax ratio decreases with increasing dose, as evidenced in samples 48h after sample treatment (7). Caspases are affected by the active compound, since this tricycloalternarene 3a was found to be related to the receptor death pathway and the mitochondrial pathway, the samples were evaluated in Western blot to conclude this statement.

Another experiment by Wang X et al (X. Wang et al., 2015) showed the capacity of the metabolite SZ-685C to enhance the expression levels of the AKT pathway in the in vitro study of NFPA cells.

Angiogenesis Inhibition

In Liu et al., to evaluate the angiogenesis of HPV-16, E7-transfected A549 or NCI-H460 cell lines, the in vitro evaluation kit ECM 625 was used, where it was found that in addition to cytotoxic activity, angiogenesis was caused by the active compounds zj-14 , zj-17 and zj-36 inhibited microtubule formation in HPV-16 (X. Liu et al., 2017).

Cellular Migration Inhibition

The research by Vasarri et al evaluated the anti-migratory capacity of dihydroauroglaucine (DAG) and detetrahydroauroglaucine (TAG) in the SH-SY5Y cell line through wound healing assay obstructs cell migration, where it was demonstrated that DAG presents anti-migratory, plus or cytotoxic capacity; however, this research provides grounds for further in vitro and in vivo investigations (Vasarri et al., 2022).

Antioxidant Activity

According to Pan F. et al (Pan et al., 2019), the active compound of Fusarium sp. A14, Exopolysaccharides, has antioxidant capacity, compounds A14EPS-1 and A14EPS-2 were evaluated at low concentrations (0.1e4.0 mg mL1 ), which showed cell carving activities of 47.22 2.30% and 23.74 4.15% respectively.

Autophagy Induction:

Co-cilioquinone B (CoB1) derivatives demonstrated cytostatic autophagy-inducing activity in the lung carcinoma cell line A-549, the experiments were performed in an MTT assay and the results were verified by western blot (Xu et al., 2021).

The postulated mechanism is related to the blockade of the PI3K/Akt1/mTOR signaling pathway and at the same time to the activation of the TAK1/MKK4/JNK/Smad pathway and the consequent expression of miR125b which reduces Foxp3 levels.

Trichodermal acid (TDA) demonstrated induction of autophagy in colorectal cancer cell lines as studies showed that TDA inhibited the proliferation of these cells. TDA generates cellular stress at the endoplasmic reticulum level which generates apoptosis in the IRE1α/XBP1 and PERK/ATF4/CHOP pathways. During the investigation it was also evidenced that cell autophagy in the tumor line was a defense mechanism, proof of this was the joint treatment with TDA and an autophagy inhibitor where the inhibited proliferation was even higher. The cell cycle could be retained in the G0/G1 phase with TDA treatment (Qu et al., 2021).

Endophytic fungi have been developed over the years in order to find better drugs for anticancer management; however, it requires both pharmacokinetic knowledge and an understanding of the mechanisms of action of each of the extracted metabolites. Our review details the IC50 and possible proposed mechanisms of action of different endophytic fungal metabolites.

According to the IC50 values of current antimitotics, Paclixatel has an IC50 of 2.5 and 7.5 nM in human tumor cells (Liebmann et al., 1993); Vincristine on the other hand, managed to inhibit SH-SY5Y cells at O.1 μM (Donoso et al., 1977) and Vinblastine to MCF-7 cells at 0.68μM (Sobottka & Berger, 1992); according to the in vitro investigations included in our literature search, metabolites such as Alternaria, Aspergillus, Fusarium or Penicillium may become a future equivalent in that sense, so the cell type they inhibit should be taken into account, in addition to the concentration used, this requires further investigations to complement these initial results.

Alternaria was one of the most frequently used in the studies described. Gao et al (Gao et al., 2020) evaluated 3 novel (1-3) and 3 known (4-6) alterchothecenes along with 9 additional compounds (7-15), which were evaluated for cytotoxicity in human colon carcinoma cell lines HT-29 and breast cancer cell line MCF-7. A higher cytotoxicity was demonstrated in groups 4-6 for both cell lines. Also, a larger group of compounds evaluated (1-6) showed synergistic activity with TNF-a related apoptosis inducing ligands (TRAILs), such finding is relevant since it favors the apoptotic activity of the ligands in a context where a large number of neoplasms showed to develop resistance to cytotoxicity induced only by TRAILs. On the other hand, Palanichamy et al. performed the purification, crystallization and evaluation of the anticancer activity of alternariol-methyl-ether present in Alternaria alternata. Cell lines of hepatocellular carcinoma HUH-7 and lung cancer A549 were used. The compound evaluated showed induction of apoptotic activity when apoptotic bodies were found in the cells cultured for more than 24h in the compound-generated extract.

Fusarium was employed as well in several studies, Dar et al (Dar et al., 2016) evaluated anticancer properties of the extract generated from Fusarium in several cell lines (colon cancer Caco-2, prostate cancer PC-3, lung cancer A549, leukemia THP-1, neuroblastoma IMR-32 and breast cancer MCF-7) and in murine models, however, despite demonstrating tumor growth inhibition, it was outperformed by extracts from other endophytic fungal species which demonstrated dose-dependent cell growth inhibition in all cell lines. On the study of Ibrahim et al (Ibrahim et al., 2018) determined the cytotoxic activity of fusarithionamide in ovarian, epidermoid, malignant melanoma, mammary adenocarcinoma, colorectal and ductal adenocarcinoma cancer lines, demonstrating cytotoxicity with IC50 comparable to doxorubicin.

Aspergillus is described as well, Goutam et al (Goutam et al., 2017) evaluated the anticarcinogenic activity of “Terrein” compound extracted from Aspergillus terreus in lung cancer cell line A-549. The extract showed antiproliferative activity in the cell model evaluated, nevertheless, further analysis is required in order to determine the possible mechanism of action. The study by Moussa et al (Moussa et al., 2020) reported the activity of pulchranin A from Aspergillus evaluated in MCF-7 breast cancer, Hep-G2 hepatocarcinoma and HCT colorectal cancer cell lines. Cell growth restriction occurs through inhibition of cyclin-dependent kinases CDK1, CDK2 and CDK4. These results were further supported by an in-silico model. Finally, Ukwatta (Ukwatta, Lawrence, & Wijayarathne, 2019) et al demonstrated cytotoxicity of cowabenzophenone A extracted from Aspergillus terreus in HCT116 colon cancer cells, with the IC50 being higher than the control (doxorubicin).

Penicillium was the subject of different studies, Hasan et al (Hasan et al., 2022), for instance, evaluated the anticarcinogenic capacity of Penicillium ethyl extract “Sir-SM2” on HeLa cervical cancer cells. Cytotoxicity and growth inhibition were demonstrated by observing a compromised cell morphology under microscopy. Furthermore, “Sir-SM2” showed very low toxicity levels against Chang’s normal human cells, therefore, can be a potential natural anticancer. Wen et al (Wen et al., 2020) , on the other hand, studied the anticarcinogenic properties of ethanol extract belonging to Penicillium polonicum in hepatocellular carcinoma HepG2 cells. Nine compounds were determined and four of them showed cytotoxic activity against the cell line used, even one compound showed a lower IC50 than cisplatin which was used as a control, this is a big step for developing more anticarcinogenic treatment options derived from metabolites.

In spite of showing promising anticancer potential in the last studies, an exact mechanism of action was not determined. Subsequent research could be helpful to fully understand cytotoxic activity and enhance our current knowledge about this endophytic fungus species.

In comparison with previous research, the review by Gupta et al. (S. Gupta et al., 2020) mentions that recently discovered active compounds from endophytic fungi can be as effective as conventional antimitotics, which is a similar result compared to our literature review and the IC50s described. The review performed by Kharwar et al., (2011) between 1990 and 2010, recorded more than 100 metabolites, of which compounds with alkaloid structures such as chaetomugilide A, B were considered effective, as well as Xanthones, Peroxides and Quinones. On the other hand; the review by Chen L. et al., (2016) focused on active compounds reported from 2010 to 2013, made a count of about 100 potentially antitumor metabolites and 8 discovered in that time frame. Both investigations demonstrate how relevant endophytic fungi will be for the development of antitumor drugs in the coming years that can match taxol and other antimitotics.

On the other hand, our review details different proposed mechanisms of action that may support the use of endophytic fungi in anticancer management in the near future, the methods necessary for metabolite extraction require constant updating. A review by Kumar et al (Kumar et al., 2021) details the mechanisms of conventionally employed antitumor drugs, as is the case of taxol, where its intervention in stabilizing microtubules and preventing depolymerization is highlighted. In addition, its antiangiogenic activity continues to be studied.

A previous literature review by Hridoy et al. (Hridoy et al., 2022) details in depth the mechanisms of action of endophytic compounds, where it is emphasized that alkaloid compounds such as Camptothecin (CPT) and terpenes such as paclitaxel (Taxol) were the most used in the last thirty years. CPT was shown to be related to DNA replication arrest by inhibiting topoisomerase I; however, another alkaloid such as vincristine was associated with cell cycle arrest in metaphase. Taxol, on the other hand, is related to the induction of apoptosis.

Our review coincides with the presence of these mechanisms of action and it is hoped that the routes proposed by previous research may favor the search for anticancer treatments in the near future. Although it is true that we were able to extract exhaustively the articles related to the subject, studies with more databases that can complete the vision of endophytic fungi in anticancer management are required.