Anticancer activity and metabolite profiling data of Penicillium janthinellum KTMT5

Fungi are ubiquitous, they proliferate even in environments with toxic pollutants that are otherwise harmful to other eukaryotes. This article presents data of fungi which were isolated from gold mine tailings and identified by DNA sequencing of their inter transcribed spacer regions 1 and 2. Five fungal isolates were identified, among which the crude extract of Penicillium janthinellum KTMT5 was investigated for anticancer activity on A549 (lung carcinoma) and UMG87 (glioblastoma) cell lines. Untargeted metabolite profiling of the crude extract of P. janthinellum KTMT5 was performed using liquid chromatography quadrupole time of flight tandem mass spectrometry (LC-QTOF-MS/MS) and a molecular network generated using the online workflow on the Global Natural Product Social molecular networking (GNPS) website. DNA sequencing showed that all fungal isolates belonged to phylum Ascomycota with the genus Penicillium representing 75% of the fungal isolates. P. janthinellum KTMT5 which was selected for further experiments showed significant anticancer activity against UMG87 cells with a calculated IC50 value of 44.23 μg/mL in the MTS assay, while the real time xCELLigence assay showed dose-dependent anticancer activity at 50 and 100 μg/mL. Metabolite profiling revealed the presence of several known metabolites in the crude extract of P. janthinellum KTMT5 and molecular networking showed the relationships among these metabolites.


a b s t r a c t
Fungi are ubiquitous, they proliferate even in environments with toxic pollutants that are otherwise harmful to other eukaryotes. This article presents data of fungi which were isolated from gold mine tailings and identified by DNA sequencing of their inter transcribed spacer regions 1 and 2. Five fungal isolates were identified, among which the crude extract of Penicillium janthinellum KTMT5 was investigated for anticancer activity on A549 (lung carcinoma) and UMG87 (glioblastoma) cell lines. Untargeted metabolite profiling of the crude extract of P. janthinellum KTMT5 was performed using liquid chromatography quadrupole time of flight tandem mass spectrometry (LC-QTOF-MS/MS) and a molecular network generated using the online workflow on the Global Natural Product Social molecular networking (GNPS) website. DNA sequencing showed that all fungal isolates belonged to phylum Ascomycota with the genus Penicillium representing 75% of the fungal isolates. P. janthinellum KTMT5 which was selected for further experiments showed significant anticancer activity against UMG87 cells with a calculated IC 50 value of 44.23 mg/mL in the MTS assay, while the real time xCELLigence assay showed dose-dependent anticancer activity at 50 and 100 mg/mL. Metabolite profiling revealed the presence of several known metabolites in the crude extract of P. janthinellum KTMT5 and molecular networking showed the relationships among these metabolites.

Value of the Data
The data provides the anticancer activity profiles of the ethyl acetate crude extract of P. janthinellum KTMT5 on A549 (lung carcinoma) cells and UMG87 (glioblastoma) human cancer cell lines and the metabolite profile acquired using LC-QTOF-MS/MS. The data is beneficial to natural product researchers in human cancer drug discovery as it provides the anticancer activity of P. janthinellum KTMT5 and the identity of compounds in its extract. The data in this article may lead to the discovery of compounds with novel mechanisms of action on UMG87 cells, leading to the development of a new drug for glioblastoma multiforme.
In addition to the anticancer and metabolite profile data for P. janthinellum KTMT5, this data article provides a reproducible experimental guideline for bioprospecting fungi from mine tailings of different minerals.

Data description
This data article presents fungi isolated from gold mine tailings of an old and inactive gold mine in Springs, Johannesburg, South Africa (26 13 0 7.08 00 S, 28 29 0 8.64 00 E). These isolates were identified by sequencing of TS1 and ITS2 regions and matching them with annotated sequences in the GenBank database. Table 1 shows the identity and GenBank accession numbers of these isolates while Supplementary File 1 contains the raw data of the ITS sequences. All species belonged to phylum Ascomycota. The genus Penicillium was the predominant and represented 75% of all the isolates.
P. janthinellum isolate KTMT5 was selected for further investigations. Fig. 1 shows the data for in vitro anticancer activity screening of the crude extract of P. janthinellum KTMT5 performed using the colorimetric MTS (3-[4,5,dimethylthiazol-2-yl]-5-[3-carboxymethoxy-phenyl]-2-[4-sulfophenyl]-2Htetrazolium, inner salt) assay on two human cancer cell lines, namely A549 (lung carcinoma) cells and UMG87 (glioblastoma) cells. A significant reduction in cell viability (cell viability of 4.21 ± 0.26%) for cells treated with P. janthinellum KTMT5 crude extract in UMG87 cells was observed at 100 mg/mL. On A549 cells, no observable anti-proliferative bioactivity was observed. The highest concentration treatment of 100 mg/mL on A549 cells was observed to increase cellular metabolism as indicated by a cell viability 113 ± 3.65%. Raw data for the MTS assay can be found in Supplementary File 2. Fig. 2 shows the dose-response curve plotted for UMG87 cells to visualize the effect of the fungal crude extract on this cell line at different logarithmic concentrations. The calculated median inhibitory concentration (IC 50 ) value for fungal extract was 44.23 mg/mL and for the positive control auranofin was 5.93 mg/mL. The dose-response curve was plotted using raw data in Supplementary File 2. The secondary metabolite compounds in the crude extract of P. janthinellum KTMT5 were analysed in an untargeted approach using LC-QTOF-MS/MS. Fig. 4 shows the survey view of the detected in an analytic run of 40 minutes and the resulting base peak chromatogram.
A list of putatively identified metabolites from metabolite profiling of the crude extract of P. janthinellum KTMT5 is shown in Table 2. Supplementary File 4 contains raw spectral data generated using the LC-QTOF-MS/MS system. Fig. 5 shows the molecular network of metabolites in the crude extract of P. janthinellum KTMT5. Molecular networking resulted in 3810 detected hits, 904 identified hits and 45 unique hits. Acarbose, a compound produced by bacteria of the genus Actinoplanes was detected [1]. Synthetic contaminants which include propiconazole, diisobutyl phthalate and dioctyl phthalate commonly occur from     plasticware used during experiments [2]. Supplementary Files 5 and 6 contain data and parameters used to generate the molecular network.

Collection of mine tailing material, isolation and identification of fungi
Mine tailings were collected from an inactive tailings heap in Springs, Johannesburg, South Africa (26 13 0 7.08 00 S, 28 29 0 8.64 00 E). A disinfected auger was used to drill 30 cm deep into the mine tailings heap where about 100 g of the tailings were collected. Samples were transported to the laboratory in sterile sealable plastic bags and were processed within 24 hours of collection. Isolation of fungi was done by weighing 10 g of the mine tailings into 100 mL of sterile phosphate buffered saline (PBS) that was then mixed by vortexing. This was then serially diluted to 10 À9 and plated on potato dextrose agar. Culture plates were incubated at 25 C and for 14 days. DNA extraction was then performed using the ZR Fungal/Bacterial DNA Kit™ (Zymo Research, Irvine, CA, USA), following the manufacturer's  instructions, followed by polymerase chain reaction (PCR) amplification of the ITS region of ribosomal DNA (rDNA) using the ITS1 (5´-TCCGTAGGTGAACCTGCGG-3´) and ITS4 (5´-TCCTCCGCTTATTGATATGC-3´) primer pair. Forward and reverse direction sequencing was performed using the ABI PRISM™ 3500xl Genetic Analyzer (Thermo Fisher Scientific, Inc., Waltham, MA, USA). Purification of sequencing products was done using ZR-96 DNA Sequencing Clean-up Kit™ (Zymo Research, Irvine, CA, USA). ITS1 and ITS4 sequences of each isolated fungi were then individually analysed to identify the isolated fungi by searching for closely related fungi on GenBank database using the Nucleotide Basic Local Alignment Search (BLASTN) search tool.

Fermentation and extraction of crude secondary metabolite compounds
The fungus P. janthinellum KTMT5 was grown in 3 L of potato dextrose broth (Potato infusion 200 g/L, dextrose 20 g/L) for 21 days at 28 C in an orbital shaking incubator (Amerex Gyromax, Temecula, CA, USA) at 150 rpm. Secondary metabolites were extracted from the broth by firstly filtering out the mycelium using a double layered muslin cloth and mixing the filtrate with an equal volume of ethyl acetate. The organic solvent phase that formed after allowing the mixture to stand for an hour was collected and concentrated under reduced pressure at 40 C. The concentrated crude extracts were then stored at e 20 C.
Initially, the wells were seeded with at 5 Â 10 4 cells/mL and incubated for 24 hours at 37 C in 5% CO 2 (v/v), then the crude fungal extract of P. janthinellum KTMT5 prepared in dimethyl sulfoxide (DMSO) was then introduced at increasing logarithmic concentrations which were as follows: 3.13, 6.25, 12.5, 25, 50 and 100 mg/mL. The treated cells were then further incubated for 96 hours under the same conditions, during which no media or fungal extract was further added. Auranofin was included in the experiment as a positive control and was tested in the same way as the fungal extract. Untreated cells (0 mg/mL) were included to serve as negative controls. At the end of the incubation period, 5 ml of MTS (Promega, Madison, WI, USA) was added to each well and the absorbance of the MTS formazan product was measured at 490 nm after 1-, 2-and 4-h incubation periods [12]. Cell viability was then calculated using the formulae below where E a is absorbance of the extract, B a is absorbance of the blank and C a is the absorbance of the negative control: The IC 50 values were then calculated in GraphPad Prism software (v. 7.05, GraphPad Software, Inc., La Jolla, CA, USA) using non-linear regression analysis of cell viability data.

xCELLigence real-time cell analyzer (RTCA) assay on U87MG cells
The xCELLigence RTCA assay was performed on 96 well electronic plates precoated with gold microelectrodes (E-Plate® 96, ACEA Biosciences Inc., San Diego, CA, USA). The UMG87 cells were initially seeded with 1 Â 10 5 cells/mL and allowed to grow for 24 hours at 37 C in 5% CO 2 (v/v). The crude extract of P. janthinellum KTMT5 and the positive control (auranofin) were dissolved in DMSO and then introduced at increasing logarithmic concentrations as described earlier: 3.13, 6.25, 12.5, 25, 50 and 100 mg/mL. Untreated cells (0 mg/mL) were included to serve as negative controls. The cells were monitored for up to 250 hours without supplementation of media or fungal extract during the incubation period. Impedance measurements taken every 15 minutes to monitor cell viability. The data was retrieved, and a graphic representation of the bioactivity was reproduced.

Metabolite profiling using LC-QTOF-MS/MS and molecular networking
Untargeted metabolite profiling of the crude extract of P. janthinellum KTMT5 was done using a liquid chromatography coupled to quadrupole time-of-flight with tandem mass spectrometry (LC-QTOF-MS/MS) system in positive mode [13]. This system has an ultra-high-performance liquid chromatography (Dionex UltiMate 3000, Thermo Scientific, Germany) connected to a QTOF (Compact™, Bruker Daltonics, Germany) with an electrospray ionization (ESI) interface. The fungal extract was prepared for analysis by dissolving it in HPLC grade methanol at 1 mg/mL (w/v) and filtering through 0.22 mm polyvinylidene fluoride (PVDF) membrane syringe filter. A set volume of 5 mL was injected into the system and chromatographic separation of analytes in reverse phase was achieved using a Raptor ARC-18 column with dimensions of 2.7 mm (particle size), 2.  Table 3 and the QTOF system parameters are shown in Table 4.
Spectral data was acquired and processed using Compass DataAnalysis software version 4.3 (Bruker Daltonics Germany). MetFrag 2.1 was used in the putative identification of metabolites by linking to three databases, namely PubChem, 2 ChemSpider 3 and KEGG Compound 4 [13]. The MetFrag settings used were as follows: Database search relative mass deviation (Search ppm) ¼ 10.0; precursor ion ¼ [MþH] þ ; fragment peak match absolute mass deviation (Mzabs) ¼ 0.01; fragment peak match relative mass deviation (Mzppm) ¼ 10; charge ¼ positive and mode ¼ [MþH] þ . Molecular networking using an online workflow at the global natural products social molecular networking platform [14]. The parameters used to generate the network are summarized in Supplementary File 1.