Research Article
 

Acute Anti-Cancer Activity of Crude Extracts from two Endophytic Fungi Chaetomium cochliodes and Penicillium Sp. in Cancer Cell Lines and Mice



Jingfang Xiao, Qinghua Ma, Ruili Cai, Jingya Miao, Zexuan Yan, Xiaolong Yang and Yemiao Chen
 
Facebook Twitter Digg Reddit Linkedin StumbleUpon E-mail
ABSTRACT

Background and Objective: The discovery of novel natural lead compounds is the key issue in searching for new anti-tumour drugs. Endophytic fungi have been proved to be a promising source for new bioactive compounds with potential application in medicine. This study aimed to investigate the activity of antiproliferation, cell cycle arrest, induction of apoptosis and anti-tumour growth of the crude extracts from the two endophytic fungi, Chaetomium cochliodes and Penicillium sp. Materials and Methods: The crude extracts of the fungi Chaetomium cochliodes and Penicillium sp., were obtained by enrichment and extraction in vitro. The proliferation, cell cycle and apoptosis were evaluated the killing effects of tumour cells and cytotoxicity in Huh7 and MGC-803 cell lines. The effect of the extract in vivo was studied in a subcutaneously implanted tumour model in C57BL/6 mice. Results: The cancer cell survival rate was almost less than 10% after the treatment of the crude extracts of Chaetomium cochliodes and Penicillium sp., by using a cell proliferation assay. Through the apoptosis assay, the crude extracts from Chaetomium cochliodes and Penicillium sp., significantly increased the number of apoptosis compared with the control group. The crude extract from Chaetomium cochliodes mainly induced G2/M phase arrest of MGC-803 cells, which might be contributing to the apoptosis. Importantly, these two crude extracts significantly reduced the volume and size of subcutaneous transplanted tumours after treatment in transplanted tumour models. Conclusion: The present study demonstrated that the extracts obtained from Chaetomium cochliodes and Penicillium sp., are promising materials for tumour treatment. These results expand the medicinal value of Panax notoginseng and Taxus media and provide a new direction for its clinical application.

Services
Related Articles in ASCI
Search in Google Scholar
View Citation
Report Citation

 
  How to cite this article:

Jingfang Xiao, Qinghua Ma, Ruili Cai, Jingya Miao, Zexuan Yan, Xiaolong Yang and Yemiao Chen, 2022. Acute Anti-Cancer Activity of Crude Extracts from two Endophytic Fungi Chaetomium cochliodes and Penicillium Sp. in Cancer Cell Lines and Mice. International Journal of Pharmacology, 18: 1583-1592.

DOI: 10.3923/ijp.2022.1583.1592

URL: https://scialert.net/abstract/?doi=ijp.2022.1583.1592
 
Copyright: © 2022. This is an open access article distributed under the terms of the creative commons attribution License, which permits unrestricted use, distribution and reproduction in any medium, provided the original author and source are credited.

INTRODUCTION

Natural products, with novel structures and unique activity, played a very important role in cancer treatment. During the period 1980 to 2019, there were 185 anti-tumour small molecule drugs approved worldwide, of which 120 were related to natural products1. However, in recent years, one of the biggest bottlenecks restricting the development of anti-tumour drugs is the lack of quality source molecules. Therefore, the discovery of novel natural lead compounds is the key issue in searching for new anti-tumour drugs.

Plants, including traditional Asian herbs, are a major source for the discovery of anti-tumour small molecule drugs. Plant-derived small molecule drugs such as paclitaxel, honokiol, camptothecin, vinblastine and podophyllotoxin are well-known anti-tumour drugs, some of which have been used clinically and have achieved good results2. Among them, paclitaxel has been proved curative effects on many tumours. More importantly, it is the first-line drug for the treatment of ovarian cancer and breast cancer2. However, due to the low content of these active small molecules in plants, a large number of plants was consumed to generate sufficient drugs. For example, producing 1 kg of paclitaxel needs 3,000-4,000 60-year-old trees of Taxus. Since the advent of paclitaxel, the world's natural yew has been devastated and over-exploited and the destruction of native resources is making the yew plant resources endangered. Therefore, the development of new sources of anti-cancer small molecule drugs is crucial.

Endophytic fungi with distinct physiological and biochemical characteristics have been proved to be a promising source for new bioactive compounds with potential application in medicine. According to a previous study, the discovery rate of new compounds in the endophytic fungi (~51%) is much higher than that of soil microorganisms (~38%)3. Many of these compounds from the endophytic fungi have anti-tumor activity, such as cell relaxin chaetoglobosin U from Chaetomium globosum IFB-E019 associated with Imperata cylindrica displaying significant inhibitory activity against human nasopharyngeal carcinoma KB cells4, a new alkaloid, 9-deacetoxyfumigaclavine C from Aspergillus fumigatus of associated with Cynodon dactylon exhibiting significant inhibitory activity against human leukemia cells K5625, a new alkaloid, IFB-E015 from Chaetomium sp., associated with Adenophora axilliflora possessing significant inhibitory activity on human leukemia cell line K562 and colon cancer SW1116 cells6, a new diterpenoid type of periconicin from \pard f5Periconia atropurpurea associated with Xylopia aromatic showing significant inhibitory activity against mammalian cell line cervical cancer (HeLa) and Chinese hamster ovary (CHO)7, a new macrolide compound brefeldin A from Acremonium sp., associated with Knema laurina having significant inhibitory activity on human tumor cells KB, BC-1 and NCI- H1878, two new polyketones compound is associated with Aegiceras corniculatum exhibiting significant inhibitory activity against human lung cancer cells and leukemia cells respectively9. Therefore, screening new anti-tumour small-molecule drugs from endophytic fungi is worth a try.

Panax notoginseng has been widely used in traditional Chinese medicine to treat stroke, ischemic heart and brain diseases. The treatment of P. notoginseng could prevent and treat ischemic cerebrovascular diseases10, promote neuronal plasticity, inhibit neuronal apoptosis11 and so on. According to recent studies, both Panax notoginseng and Taxus media have antitumor activities12,13. Taxus species are also traditional medicinal plants, the shoots and leaves have been used to treat kidney disease and rheumatism14. Multiple studies showed that Taxus species have anticancer activities, such as treating pancreatic cancer with water decoctions of Taxus cuspidate15 and treating non-small cell lung cancer with aqueous extracts16. Therefore, it’s important to explore the new anti-tumour endophytic fungi from these two medicinal plants.

In the present study, two endophytic fungi were isolated and cultured from these herbs. The anti-cancer activity of the crude extracts from two fungi was further tested by cells proliferation assay, cell cycle assay and apoptosis assay.

MATERIALS AND METHODS

Study area: This research was carried out at the Institute of Pathology and Southwest Cancer Center, Southwest Hospital, Army Medical University, Biobank and Clinical Research Center, Chongqing Public Health Medical Center, Chongqing, China in September, 2018. The crude extracts were prepared at the School of Pharmaceutical Sciences, South-Central University for Nationalities, Wuhan, China.

Fungal material: The fungal strains, XL-1325 and MPT-426, were isolated from the stems of Panax notoginseng and the roots of Taxus media, respectively. Two isolates were identified to be Chaetomium cochliodes (XL-1325) and Penicillium sp., (MPT-426) by an analysis of the ITS region of the rDNA. The voucher specimens have been deposited at the School of Pharmaceutical Sciences, South-Central University for Nationalities. Above two fungi were inoculated into the flasks with 200 mL malt extract broth (MEB) medium (sucrose 2%, malt extract 2%, peptone 0.1%) on a rotary shaker at 30°C, 180 rpm for 30 days, respectively. The harvested fermentation material was extracted three times with ethyl acetate at room temperature. Then, the crude extracts of Chaetomium cochliodes (XL-1325) and Penicillium sp. (MPT-426) were prepared from the organic solvent by a lyophilization method. Each crude extract was dissolved into the solvent of DMSO for the anticancer activity test.

Animal maintenance and treatments: The mice were purchased and cared for from the Center of Animals (Third Military Medical University, Chongqing, China). Mice studies were performed by the guidelines of the Research Council and Animal Use and Care Committee of Army Medical University. 5 weeks female C57BL/6 mice were used as the recipient mice.

Cells and culture: Human hepatocellular carcinoma cell line Huh-7, Human Glioma Cell line LN229, Human breast cancer cell line MDA231, Human gastric carcinoma cell line MGC-803 and Mouse glioma cell line GL261 were obtained from Shanghai Cell Research Institute of the Chinese Academy of Sciences (Shanghai, China). Cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM, Gibco) supplemented with 10% fetal bovine serum (Gibco) at 37°C with 5% CO2.

Dissolution and dilution of drugs: The crude extracts of Chaetomium cochliodes and Penicillium sp., were dissolved and diluted in DMSO (Sigma) at the concentration of 0.5 mg μL–1. Store working aliquots at -20°C.

Cell viability assays: Cancer cells were harvested and counted during logarithmic-phase growth and 2000 cells/per well were seeded in a 96-well plate in 100 μL of growth media. The cells were cultured overnight for 24 hrs with the medium of serum-free DMEM at 37°C in a humidified 5% CO2 atmosphere. Then, the crude extracts of Chaetomium cochliodes and Penicillium sp., were diluted in a complete medium to concentrations of 0, 0.5, 1, 2.5, 5, 10, 15, 20 and 25 μg mL–1 separately, 100 μL of these working solutions were changed in 96-well plate. The plate was then placed back in the 5% CO2 incubator at 37°C for an additional 48 hrs. Then 10 μL Cell Counting Kit-8 (Beyotime, China) was added to each well and the plates were incubated for an additional 3 hrs at 37°C. Measure the absorbance at 450 nm by a microplate reader. The IC50 value (half maximal inhibitory concentration) was determined by the sigmoidal curve.

Cell proliferation assay: Cancer cells were seeded into 96-well flat-bottomed plates with the density of 2×103 cells/well for Huh-7 and 1×103 cells/well for MGC-803. The cells were suspended in the serum-free DMEM medium and cultured overnight. After 8 hrs, replace the complete medium with candidate extracts in concentrations corresponding to the IC50 value for each cell line or DMSO (control) at 37°C with 5% CO2. And continuous culture was performed for 5 days. At the indicated intervals, the 450 nm absorbance was measured by a microplate reader (Thermo Scientific). The details were proceeded according to the previous article published by the group17.

Cell cycle assay: Cancer cells Huh-7 (2×105 vcells/well) and MGC-803 (1×105 cells/well) were seeded into 6-well flat-bottomed plates. The cells were suspended with serum-free DMEM and cultured overnight at 37°C with 5% CO2. After 8 hrs, the cells were cultured with a fresh medium containing the candidate extracts in concentrations corresponding to the IC50 value for each cell line. DMSO was used as the solution control. After 24 hrs, both attached and detached cells were harvested via trypsinization, washed with PBS and fixed in 70% ethanol overnight at 4°C. The pre-treatment of cells was carried out and centrifuged for 5 min to go to the supernatant at 1000 rpm, then resuspended with cold PBS and centrifuged to go to the supernatant. The cell pellet was using Cell Cycle (Beyotime, China) following the manufacturer’s instructions. Cells were analyzed by ow cytometer BD FACS Aria II. Quantification of DNA content was performed using Flowing software and the cell cycle phase distribution was shown by histograms.

Apoptosis analysis: The Annexin V/FITC assay was performed using the Annexin V/FITC apoptosis detecting kit (Beyotime, China) to analyze the potential of tested extracts in causing apoptosis. The cells were seeded in 6-well plates at a concentration of 2×105 cells/well and incubated overnight with serum-free DMEM. On the next day, the seeded cells were treated with the IC50 value of each crude extract and incubated for 24 hrs. The cells were harvested and washed with PBS. The cells (1×105) were resuspended in 195 μL Binding solution and stained with 5 μL FITC Annexin V and 10 μL PI for 20 min at room temperature and then analyzed by a flow cytometry machine (BD FACS Aria II).

A fluorescent assay was performed using the TUNEL apoptosis kit (Beyotime, China). The cells treatment was similar to the flow cytometry tests except for seeding the cells in a 24-well plate. The cells were fixed with 4% paraformaldehyde for 20 min and washed with PBS 3 times. Fixed with PBS (with 0.3% Triton X-100) for 5 min once more. A 50 μL TUNEL working solution was added to each well and allowed to incubate for 60 min at 37°C. Then, the cells were washed with PBS 3 times and detected by microscope (Leica, DMI3000B).

In vivo anti-cancer tests: GL261 cells were harvested and resuspended in PBS. The C57BL/6 mice were divided into 3 groups of ten mice each group randomly and each mouse was subcutaneously injected with 1×106 GL261 cells. Two weeks later, the tumour size and mice weight were measured by digital callipers and a weight scale and the tumour volumes were calculated by the formula:

Image for - Acute Anti-Cancer Activity of Crude Extracts from two Endophytic Fungi Chaetomium cochliodes and Penicillium Sp. in Cancer Cell Lines and Mice

Where:

a = Tumor length
b = Tumor width

The XL-1325, MPT-426 and PBS (control) were injected into the tumour at different concentrations every 2 days when the tumour was the volume at 100 mm3 (XL-1325: 2.5 mg kg–1, MPT-426: 5 mg kg–1) and tumour size was measured every other day. All the animals were euthanized after 2 weeks. The xenograft tumours were harvested and taken pictures. The animal experiments were proceeded by the Institution Animal Care (the animal ethical statement number: AMU WEC20201059).

Statistical analysis: Statistical analysis was performed using the SPSS 18.0 software (SPSS, IL, USA) and GraphPad Prism 6 (GraphPad, La Jolla, CA, USA). Animals were randomly assigned for drug treatment. Results are expressed as mean values±standard deviation (SD). The significant differences were assessed using the student’s t-test and p<0.05 was considered significant (*p<0.05, **p<0.01 and ***p<0.001).

RESULTS

Crude extracts from the two endophytic fungi inhibit the proliferation of cancer cell lines: To validate the inhibitory effect of crude extracts Chaetomium cochliodes (XL-1325) on cancer cells, the IC50 values in Table 1 were obtained by using cancer cell line Hun-7 (0.62 μM), LN229 (0.67 μM), MDA231 (0.52 μM), MGC-803 (1.22 μM) and the crude extract Penicillium sp. (MPT-426) in Hun-7 (1.396 μM), LN229 (1.78 μM), MDA231 (3.424 μM), MGC-803 (1.1 μM). The ability to inhibit the proliferation of cancer cell lines was evaluated on Huh-7 and MGC-803 cell lines by CCK-8 assay. We treated Huh-7 and MGC-803 cells with the XL-1325 (2.5 μg mL–1) and MPT-426 (5 μg mL–1) or with the same dose of DMSO at different times (0, 1, 2, 3, 4 and 5 days). We observed that with crude extracts of Chaetomium cochliodes (XL-1325) and Penicillium sp. (MPT-426) treatment, the proliferation ability of cells decreased significantly on Huh-7 in Fig. 1a and b or MGC-803 cells in Fig. 1c and d compared with the control group. From day 1, the proliferation of Huh-7 and MGC-803 cells is stopped. As shown in Fig. 1a, the OD450 value (range: 0.2081-0.2551) of Huh-7 cells treated with XL-1325 on day 5 was much lower compared to the treatment with DMSO (range: 1.5244-2.0105). Similarly, the OD450 value (range: 0.2050-0.2152) of Huh-7 cells treated with MPT-426 was lower compared to the DMSO treatment (range: 0.8383-1.0370), in Fig. 1b. When the cells of MGC-803 were treated with the two crude extracts, a similar pattern was found after 5 days of culture. The OD450 value of cells treated with XL-1325 (range: 0.2021-0.2072), in Fig. 1c, MPT-426 (range: 0.2165-0.2222), in Fig. 1d was much lower compared to the control group (range: 1.0574-1.5441, 0.9685-1.1571) respectively. These results indicate that the crude extracts of Chaetomium cochliodes and Penicillium sp., can kill tumour cells effectively in vitro.

Crude extracts from the two endophytic fungi induce cell cycle arrests in cancer cell lines: The above studies confirmed that the crude extracts of Chaetomium cochliodes and Penicillium sp., can kill tumour cells in vitro and we further explored the influence on the cell cycle in both Huh-7 and MGC-803 cells. Compared with the control (DMSO) group in Fig. 2a, the cell cycle progression of MGC-803 significantly changed in Fig. 2b when treated with the crude extracts of Chaetomium cochliodes at the dose of 250 μg mL–1. But the impact of the Penicillium sp., crude extract on the cell cycle is scarce in Fig. 2c. When treated with the crude extracts of Chaetomium cochliodes, the cell population of the S phase (average: 28.69%, range: 26.89-31.76%) is reduced concurrently with the increase of the G2/M phase population (average: 47.58%, range: 46.29-49.79%) compared with the control (S phase: Average is 53.43%, ranged in 52.29-54.87%, G2/M phase: Average is 17.38%, ranged in 13.78-20.53%), on average a 30% increase in G2/M phase and a 25% reduce in S phase in Fig. 2d. When treated with the crude extracts of Penicillium sp., the average population of cells is 7.79% (range: 7.26-8.23%) in the G2/M phase and 57.14% (range: 54.62-59.2%) in the S phase in Fig. 2d. The results suggested that the Chaetomium cochliodes crude extract mainly induced G2/M phase arrest of MGC-803 cells, which might be contributing to the apoptosis.

Table 1: In vitro growth inhibitory activity (IC50 μg mL–1) of crude extracts
Crude extracts Huh-7 LN229 MDA231 MGC-803
MPT-426 1.40±0.45 1.78±0.32 3.42±0.96 1.10±0.50
XL-325 0.62±0.05 0.67±0.29 0.52±0.14 1.22±0.82


Image for - Acute Anti-Cancer Activity of Crude Extracts from two Endophytic Fungi Chaetomium cochliodes and Penicillium Sp. in Cancer Cell Lines and Mice
Fig. 1(a-d): Efficacy of the crude extracts from Chaetomium cochliodes (XL-1325) and Penicillium sp. (MPT-426) against the proliferation of human cancer cell lines, (a) Huh-7 cells treated with XL-1325, (b) Huh-7 cells treated with MPT-426, (c) MGC-803 cells treated with XL-1325 and (d) MGC-803 cells treated with MPT-426


Image for - Acute Anti-Cancer Activity of Crude Extracts from two Endophytic Fungi Chaetomium cochliodes and Penicillium Sp. in Cancer Cell Lines and Mice
Fig. 2(a-h): Effect of crude extracts XL-1325 and MPT-426 on cell cycle of human cancer cell lines, (a) Cell cycle analysis of MGC-803 cells treated with DMSO (Control), (b) Cell cycle analysis of MGC-803 cells treated with XL-1325, (c) Cell cycle analysis of MGC-803 cells treated with MPT-426, (d) Percent cell cycle distribution of MGC-803 cells, (e) Cell cycle analysis of Huh-7 cells treated with DMSO (Control), (f) Cell cycle analysis of Huh-7 cells treated with XL-1325, (g) Cell cycle analysis of Huh-7 cells treated with MPT-426 and (h) Percent cell cycle distribution of Huh-7 cells


Image for - Acute Anti-Cancer Activity of Crude Extracts from two Endophytic Fungi Chaetomium cochliodes and Penicillium Sp. in Cancer Cell Lines and Mice
Fig. 3(a-b): Effect of crude extract XL-1325 and MPT-426 on apoptosis of human cancer cell lines, (a) Treated with the crude extracts of XL-1325 and MPT-426 for 24 hrs and (b) Fluorescent image of the effect on apoptosis in two human tumour cells (scale = 100 μm)

Ratio of early and late apoptosis of Huh-7 cells and MGC-803 cells can be assessed by Annexin V-FITC/PI double-staining. Green fluorescence indicates that cells of apoptosis are increased after the treatment with crude extracts

On the contrary, compared with the DMSO treatment in Fig. 2e, both the crude extracts of Chaetomium cochliodes in Fig. 2f and Penicillium sp., Fig. 2g showed slightly infection on the cell cycle change of the Huh-7 cells, on average a 63% in G0/G1 phase (range: 59.04-66.96%), a 3.44% G2/M phase (range: 1.85-6.74%) and a 34% in S phase (range: 29.71-35.9%) in Fig. 2h. Thus, the apoptosis of Huh-7 cells might be induced mainly by another mechanism.

Crude extracts from the two endophytic fungi induce apoptosis: To elucidate whether the crude extracts of Chaetomium cochliodes and Penicillium sp., decreases cell survival through the induction of apoptosis in tumour cells, the Annexin V/PI Flow cytometry assays were conducted in Huh-7 and MGC-803 cells. As shown in Fig. 3a, the percentage of apoptotic cells increased gradually with the Chaetomium cochliodes crude extract treatment compared with the control group.

Image for - Acute Anti-Cancer Activity of Crude Extracts from two Endophytic Fungi Chaetomium cochliodes and Penicillium Sp. in Cancer Cell Lines and Mice
Fig. 4(a-f): In vivo anti-cancer tests of crude extracts XL-1325 and MPT-426, (a) Tumor growth curves for XL-1325 treatment and the control (DMSO), (b) Tumor growth curves for MPT-426 treatment and the control (DMSO), (c) Tumors excised and measurement as average±SD (mm3), (d) Representative photographs of the tumours for MPT-426 treatment, (e) XL-1325 treatment and the control (DMSO) and (f) MPT-426 treatment and the control (DMSO)

Anti-cancer activity of the crude extracts was measured in C-57 mice, quantitative graphs of tumour inhibition rate were analyzed by the volume of the subcutaneous tumour and weight of the mice (v = ab2/2), a: Tumour length, b: Tumour width and N = 8

After 12 hrs of treatment with the Chaetomium cochliodes crude extract, the early apoptotic cell population increased gradually from 4.26±1.41% in the control group to 46.58±14.19% in Huh-7 cells and 3.56±14.19% to 32.84±13.47% in MGC-803 cells. The 12 hrs treatment with the Penicillium sp., crude extract, the early apoptotic cell population increased gradually from 4.26±1.41% in the control group to 46.58±14.19% in Huh-7 cells and 3.56±14.19 -32.84±13.47% in MGC-803 cells.

Then, the apoptosis degree was evaluated with TUNEL/PI staining kit. As shown in Fig. 3b, the fluorescence microscopy images show that the crude extracts of

Chaetomium cochliodes and Penicillium sp., caused numerous positive staining of TUNEL, in comparison with the control. TUNEL/PI double-stained cells indicated that alterations have occurred in the cell morphology of apoptosis. The observation provided additional confirmation that the crude extracts of MPT-426 and XL-1325 induced the apoptosis of the cancer cells.

Crude extracts from the two endophytic fungi inhibit the growth of tumours in the mouse model: The above studies indicated that the crude extracts of Chaetomium cochliodes and Penicillium sp., inhibited cancer cell proliferation by inducing apoptosis in vitro. To further investigate whether the extract could suppress tumour growth in vivo, GL261 (the murine glioma cancer cells) was transplanted into C57BL/6 mice. The crude extracts of Chaetomium cochliodes and Penicillium sp., significantly decreased the xenograft tumour size and volume compared with the control group (the weight barely change). The tumour size increased gradually from 173 mm3 (range: 145-248 mm3) to 3832 mm3 (range: 2297-5162 mm3) in the control group, decreased gradually from 180 mm3 (range: 128-221 mm3) to 130 mm3 (range: 59-175 mm3) in XL-1325 treatment in Fig. 4a and decreased gradually from 118 mm3 (range: 73-192 mm3) to 1956 mm3 (range: 958-2467 mm3) in MPT-426 treatment in Fig. 4b. Representative photographs were shown in Fig. 4c for XL-1325 treatment and in Fig. 4d for MPT-426 treatment, respectively. Comparatively, the weight of the mice in the XL-1325 treatment (19.4 g with the range from 18.3-20.6 g, in Fig. 4e and MPT-426 treatment (18.6 g with the range from 16.7-20.7 g) in, Fig. 4f slightly decreased compared with the control group (20.4 g with the range from 18.4-25.1 g). The results suggested that the two extracts might be active anti-cancer reagents.

DISCUSSION

Traditional Chinese medicine books "Zhong Cao Tui Xin" and "Chinese Medicine Dictionary" have recorded the functions of Taxus on diuresis, menstrual, hypoglycemic and kidney diseases. Recent studies also showed multiple medicinal properties of Taxus on anti-inflammatory and analgesic activities, anticonvulsant, antipyretic activities, antibacterial and antifungal activities18. After the discovery of taxol (Paclitaxel), the effective anticancer drug from the bark of Pacific yew in 1971, lots of work was carried out on the crude extracts19,20 and compounds21 investigation of several yew species. However, the slow growth of Taxus and the extremely low paclitaxel content limit its long-term development. Therefore, researchers must explore new production options, such as the search for paclitaxel or similar anticancer drugs from endophytic fungi.

It was the first reported that Paclitaxel was produced by fungal entophytes associated with Pacific Yew Tree in 199322. Lots of strains of endophytic fungi were isolated from Yew trees later and found to produce Taxol23-26 or anticancer crude extracts27,28. In the present study, we successfully isolated one endophytic fungus from Taxus and proved the crude extracts have obvious cytotoxicity in cancer cells and in vivo. It was further identified as belonging to the genera of Penicillium. Except for producing the famous antibiotic-penicillin, some members of the Penicillium genus have anticancer activities29. The ethyl acetate extract of Penicillium cyaneum has anticancer activity against HepG2 cells with the IC50 of 242.24 μg mL–1. Moreover, Vasundhara et al.28 isolated an endophytic fungus Fusarium tricinctum from Taxus baccata and tested their anticancer activities in MCF-7 and HeLa cell lines and resulting in IC50 values of 225 and 220 μg mL–1, respectively. In this study, the IC50 values of the extracts from Penicillium genera in four cancer cell lines were 1.1-3.424 μg mL–1, respectively. These results showed that our endophytic fungi strains have high cancer cells cytotoxicity.

Panax notoginseng is a traditional Chinese herb. The roots, leaves, fruit and flowers were all used as medicine against bleeding, chest pain, strokes etc. Endophytic fungi from Panax notoginseng were systemically studied and showed activity in antibacterial30 and anti-inflammatory31. However, only limited evidence showed compounds or extracts from the fungal entophytes associated with Panax notoginseng have anticancer activity in vitro cancer cell lines32, with the IC50 value of 3.5-13.4 μg mL–1. In this study, the IC50 values of the crude extracts of strain XL-1325 (identified as Chaetomium cochliodes) varied from 0.52- 1.22 μg mL–1 in 4 cancer cell lines (Table 1), which showed extremely high anticancer activity both in vitro and in vivo.

Taxus media and Panax notoginseng are both widely used traditional Chinese herbs. With the development of new techniques, such as fungal culture and compound purification, more researches are focused on the fungal endophyte associated with those herbs. The two extracts from Taxus media and Panax notoginseng showed different toxicity on cancer cells and anti-cancer activity in mice in our study. The future study will be focused on the isolation of the active molecules from the extracts and their specificity on different cancer cells.

CONCLUSION

Results findings from in vivo mice model tests showed that both crude extracts of MPT-426 and XL-1325 inhibited the tumour growth but XL-1325 could inhibit the tumour growth more efficiently with a lower concentration than MPT-426. In summary, current results indicated that the crude extracts from the endophytic fungi Chaetomium cochliodes and Penicillium sp., could inhibit the cancer cell growth, induce the cell apoptosis and suppress the tumour growth in vivo.

SIGNIFICANCE STATEMENT

Overall, current research proved that the endophytic fungi strains of Chaetomium cochliodes from Taxus media and Penicillium sp., from Panax notoginseng would provide a basis for the identification of new, high-activity anticancer compounds.

ACKNOWLEDGMENT

The study is funded by the National Natural Science Foundation of China (No. 81872755), the National Civil Affairs Commission's young and middle-aged talents training program (MZR20008) and the National Key R and D Program of China (2020YFC1712703).

REFERENCES

  1. Newman, D.J. and G.M. Cragg, 2020. Natural products as sources of new drugs over the nearly four decades from 01/1981 to 09/2019. J. Nat. Prod., 83: 770-803.
    CrossRef  |  Direct Link  |  


  2. Ikeda, H., 2017. Natural products discovery from micro-organisms in the post-genome era. Biosci. Biotech. Biochem., 81: 13-22.
    CrossRef  |  Direct Link  |  


  3. Martinez-Klimova, E., K. Rodríguez-Peña and S. Sánchez, 2017. Endophytes as sources of antibiotics. Biochem. Pharmacol., 134: 1-17.
    CrossRef  |  Direct Link  |  


  4. Ding, G., Y.C. Song, J.R. Chen, C. Xu, H.M. Ge, X.T. Wang and R.X. Tan, 2006. Chaetoglobosin U, a cytochalasan alkaloid from Endophytic Chaetomium globosum IFB-E019. J. Nat. Prod., 69: 302-304.
    CrossRef  |  Direct Link  |  


  5. Ge, H.M., Z.G. Yu, J. Zhang, J.H. Wu and R.X. Tan, 2009. Bioactive alkaloids from endophytic Aspergillus fumigatus. J. Nat. Prod., 72: 753-755.
    CrossRef  |  Direct Link  |  


  6. Jiao, R.H., S. Xu, J.Y. Liu, H.M. Ge and H. Ding et al., 2006. Chaetominine, a cytotoxic alkaloid produced by endophytic Chaetomium sp. IFB-E015. Org. Lett., 8: 5709-5712.
    CrossRef  |  Direct Link  |  


  7. Teles, H.L., R. Sordi, G.H. Silva, I. Castro-Gamboa and V. da Silva Bolzani et al., 2006. Aromatic compounds produced by Periconia atropurpurea, an endophytic fungus associated with Xylopia aromatica. Phytochemistry, 67: 2686-2690.
    CrossRef  |  Direct Link  |  


  8. Wang, J., Y. Huang, M. Fang, Y. Zhang, Z. Zheng, Y. Zhao and W. Su, 2002. Brefeldin A, a cytotoxin produced by Paecilomyces sp. and Aspergillus clavatus isolated from Taxus mairei and Torreya grandis. FEMS Immunol. Med. Microbiol., 34: 51-57.
    CrossRef  |  Direct Link  |  


  9. Lin, Z., T. Zhu, Y. Fang, Q. Gu and W. Zhu, 2008. Polyketides from Penicillium sp. JP-1, an endophytic fungus associated with the mangrove plant Aegiceras corniculatum. Phytochemistry, 69: 1273-1278.
    CrossRef  |  Direct Link  |  


  10. Xiao-li, D., N. San-yuan, X. Hong-bing and H. Mei-ying, 2005. Effects of hyperbaric oxygenation associated with panax notoginseng saponins therapy on hemorrhology indexes in patients with ischemic cerebrovascular disease. Chin. J. Clin. Rehabil., 9: 6-7.
    Direct Link  |  


  11. Li, H., C.Q. Deng, B.Y. Chen, S.P. Zhang, Y. Liang and X.G. Luo, 2009. Total saponins of Panax Notoginseng modulate the expression of caspases and attenuate apoptosis in rats following focal cerebral ischemia-reperfusion. J. Ethnopharmacol., 121: 412-418.
    CrossRef  |  Direct Link  |  


  12. Zhang, K., C. Sun, Y. Hu, J. Yang and C. Wu, 2021. Network pharmacology reveals pharmacological effect and mechanism of Panax notoginseng (Burk.) F. H. Chen on reproductive and genetic toxicity in male mice. J. Ethnopharmacol., Vol. 270.
    CrossRef  |  Direct Link  |  


  13. Liu, Y.H., H.Y. Qin, Y.Y. Zhong, S. Li and H.J. Wang et al., 2021. Neutral polysaccharide from Panax notoginseng enhanced cyclophosphamide antitumor efficacy in hepatoma H22-bearing mice. BMC Cancer, Vol. 21.
    CrossRef  |  Direct Link  |  


  14. Yang, W.X., Z.G. Zhao, L.H. Wang, S.J. Yu and Z.S. Liang, 2012. Control of hypertension in rats using volatile components of leaves of Taxus chinensis var. mairei. J. Ethnopharmacol., 141: 309-313.
    CrossRef  |  Direct Link  |  


  15. Qu, C. and Z. Chen, 2014. Antitumor effect of water decoctions of Taxus cuspidate on pancreatic cancer. Evidence-Based Complementary Altern. Med., Vol. 2014.
    CrossRef  |  Direct Link  |  


  16. Zhang, G., S. Dai, Y. Chen, H. Wang and T. Chen et al., 2021. Aqueous extract of Taxus chinensis var. mairei regulates the Hippo-YAP pathway and promotes apoptosis of non-small cell lung cancer via ATF3 in vivo and in vitro. Biomed. Pharmacother., Vol. 138.
    CrossRef  |  Direct Link  |  


  17. Yang, S.X., J.F. Xiao, T.K. Liu, Z.D. Huang, X. Li, Y.M. Chen and X.L. Yang, 2020. A 3D screening approach identifies the compound epitajixanthone hydrate as a new inhibitor of cancer cell growth and invasion. Anti-Cancer Drugs, 31: 890-899.
    CrossRef  |  Direct Link  |  


  18. Juyal, D., V. Thawani, S. Thaledi and M. Joshi, 2014. Ethnomedical properties of Taxus Wallichiana Zucc. (Himalayan Yew). J. Traditional Complementary Med., 4: 159-161.
    CrossRef  |  Direct Link  |  


  19. Durak, Z.E., S. Buber, E. Devrim, H. Kocaoglu and I. Durak, 2014. Aqueous extract from taxus baccata inhibits adenosine deaminase activity significantly in cancerous and noncancerous human gastric and colon tissues. Phcog. Mag., 10: 214-216.
    CrossRef  |  Direct Link  |  


  20. Shang, W., J. Qiao, C. Gu, W. Yin and J. Du et al., 2011. Anticancer activity of an extract from needles and twigs of Taxus cuspidata and its synergistic effect as a cocktail with 5-fluorouracil. BMC Complementary Altern. Med., Vol. 11.
    CrossRef  |  Direct Link  |  


  21. Zheng, Z.Q., Y.Y. Fu, B.H. Li, M.L. Zhang, X.L. Yang et al., 2014. PSY-1, a Taxus chinensis var. mairei extract, inhibits cancer cell metastasis by interfering with MMPs. Nat. Prod. Commun., 9: 241-245.
    CrossRef  |  Direct Link  |  


  22. Talbot, N.J., 2015. Plant immunity: A little help from fungal friends. Curr. Biol., 25: R1074-R1076.
    CrossRef  |  Direct Link  |  


  23. Kasaei, A., M. Mobini-Dehkordi, F. Mahjoubi and B. Saffar, 2017. Isolation of taxol-producing endophytic fungi from Iranian yew through novel molecular approach and their effects on human breast cancer cell line. Curr. Microbiol., 74: 702-709.
    CrossRef  |  Direct Link  |  


  24. Heinig, U., S. Scholz and S. Jennewein, 2013. Getting to the bottom of taxol biosynthesis by fungi. Fungal Divers., 60: 161-170.
    CrossRef  |  Direct Link  |  


  25. Wang, X., C. Wang, Y.T. Sun, C.Z. Sun, Y. Zhang, X.H. Wang and K. Zhao, 2015. Taxol produced from endophytic fungi induces apoptosis in human breast, cervical and ovarian cancer cells. Asian Pac. J. Cancer Prev., 16: 125-131.
    CrossRef  |  Direct Link  |  


  26. Zaiyou, J., M. Li and H. Xiqiao, 2017. An endophytic fungus efficiently producing paclitaxel isolated from Taxus wallichiana var. mairei. Medicine, Vol. 96.
    CrossRef  |  Direct Link  |  


  27. Fatima, N., T.P. Kondratyuk, E.J. Park, L.E. Marler and M. Jadoon et al., 2016. Endophytic fungi associated with Taxus fuana (West Himalayan Yew) of Pakistan: Potential bio-resources for cancer chemopreventive agents. Pharm. Biol., 54: 2547-2554.
    CrossRef  |  Direct Link  |  


  28. Vasundhara, M., M. Baranwal and A. Kumar, 2016. Fusarium tricinctum, an endophytic fungus exhibits cell growth inhibition and antioxidant activity. Indian J. Microbiol., 56: 433-438.
    CrossRef  |  Direct Link  |  


  29. Wajima, T., R. Kinugawa, T. Yamada, H. Ikoshi and N. Noguchi, 2019. Panax Notoginseng extract possesses significant antibacterial activity against pathogenic streptococci. Pharmacology, 103: 221-227.
    CrossRef  |  Direct Link  |  


  30. Ma, L.J., F. Liu, Z.F. Zhong and J.B. Wan, 2017. Comparative study on chemical components and anti-inflammatory effects of Panax notoginseng flower extracted by water and methanol. J. Sep. Sci., 40: 4730-4739.
    CrossRef  |  Direct Link  |  


  31. Li, G.Y., B.G. Li, T. Yang, J.F. Yan, G.Y. Liu and G.L. Zhang, 2006. Chaetocochins A−C, Epipolythiodioxopiperazines from Chaetomium cochliode. J. Nat. Prod., 69: 1374-1376.
    CrossRef  |  Direct Link  |  


  32. Phonkerd, N., S. Kanokmedhakul, K. Kanokmedhakul, K. Soytong, S. Prabpai and P. Kongsearee, 2008. Bis-spiro-azaphilones and azaphilones from the fungi Chaetomium cochliodes VTh01 and C. cochliodes CTh05. Tetrahedron, 64: 9636-9645.
    CrossRef  |  Direct Link  |  


©  2023 Science Alert. All Rights Reserved