Summary of Study ST003146

This data is available at the NIH Common Fund's National Metabolomics Data Repository (NMDR) website, the Metabolomics Workbench, https://www.metabolomicsworkbench.org, where it has been assigned Project ID PR001957. The data can be accessed directly via it's Project DOI: 10.21228/M8RQ80 This work is supported by NIH grant, U2C- DK119886.

See: https://www.metabolomicsworkbench.org/about/howtocite.php

This study contains a large results data set and is not available in the mwTab file. It is only available for download via FTP as data file(s) here.

Study ID	ST003146
Study Title	Exploring The Impact of Two Novel DNA Minor Groove Binders on HCT-116 Cells: A Comprehensive Multi-Omics Analysis Using Mass Spectrometry
Study Type	LC/MS/MS
Study Summary	Colorectal cancer (CRC) poses a significant global health challenge, necessitating innovative therapeutic approaches. Despite advancements, current treatments encounter obstacles such as chemotherapy resistance and adverse effects due to non-selective targeting. DNA Minor Groove Binders (MGBs) present promising alternatives, targeting DNA structure without causing permanent damage. In this study, two novel MGB compounds were synthesized, MGB30 and MGB32, resembling distamycin, a natural DNA-binding agent. These compounds bind reversibly to the DNA minor groove, influencing DNA structure and inhibiting cancer growth-related enzymes. Our study aims to explore the unique effects of MGB30 and MGB32 on the metabolomic profiles of treated HCT-116 cells using TIMS-QTOF-UHPLC-MS. Objectives include comprehensive analysis, comparison of effects, identification of altered pathways, and insights into MGB compound mechanisms. Additionally, we established four biological replicates for each treatment condition. Advanced statistical analyses, including the two-tailed independent Student's t-test and one-way analysis of variance (ANOVA), were utilized to minimize false discoveries. Our analysis generated a comprehensive dataset from 12 samples, identifying 75 distinct metabolites. The significance of this study lies in elucidating the molecular mechanisms of action of MGB30 and MGB32, crucial for their development as CRC drug candidates.
Institute	Sharjah Institute for Medical Research
Last Name	Facility
First Name	Core
Address	M32, SIMR, College of Pharmacy, Health Sciences, University of Sharjah, Sharjah, UAE, Sharjah, 000, United Arab Emirates
Email	tims-tof@sharjah.ac.ae
Phone	+971 6 5057656
Submit Date	2024-03-27
Raw Data Available	Yes
Raw Data File Type(s)	d
Analysis Type Detail	LC-MS
Release Date	2024-07-29
Release Version	1

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Project:

Project ID:	PR001957
Project DOI:	doi: 10.21228/M8RQ80
Project Title:	Exploring The Impact of Two Novel DNA Minor Groove Binders on HCT-116 Cells: A Comprehensive Multi-Omics Analysis Using Mass Spectrometry
Project Summary:	Colorectal cancer (CRC) poses a significant global health challenge, necessitating innovative therapeutic approaches. Despite advancements, current treatments encounter obstacles such as chemotherapy resistance and adverse effects due to non-selective targeting. DNA Minor Groove Binders (MGBs) present promising alternatives, targeting DNA structure without causing permanent damage. In this study, two novel MGB compounds were synthesized, MGB30 and MGB32, resembling distamycin, a natural DNA-binding agent. These compounds bind reversibly to the DNA minor groove, influencing DNA structure and inhibiting cancer growth-related enzymes. Our study aims to explore the unique effects of MGB30 and MGB32 on the metabolomic profiles of treated HCT-116 cells using TIMS-QTOF-UHPLC-MS. Objectives include comprehensive analysis, comparison of effects, identification of altered pathways, and insights into MGB compound mechanisms. Additionally, we established four biological replicates for each treatment condition. Advanced statistical analyses, including the two-tailed independent Student's t-test and one-way analysis of variance (ANOVA), were utilized to minimize false discoveries. Our analysis generated a comprehensive dataset from 12 samples, identifying 75 distinct metabolites. The significance of this study lies in elucidating the molecular mechanisms of action of MGB30 and MGB32, crucial for their development as CRC drug candidates.
Institute:	Sharjah Institute for Medical Research
Last Name:	Facility
First Name:	Core
Address:	M32, SIMR, College of Pharmacy, Health Sciences, University of Sharjah, Sharjah, UAE, Sharjah, 000, United Arab Emirates
Email:	tims-tof@sharjah.ac.ae
Phone:	+971 6 5057656

Subject:

Subject ID:	SU003263
Subject Type:	Cultured cells
Subject Species:	Homo sapiens

Factors:

Subject type: Cultured cells; Subject species: Homo sapiens (Factor headings shown in green)

mb_sample_id	local_sample_id	Treatment	Sample source
SA340716	A-01-9239	Control	HCT-116 colorectal cells
SA340717	A-02-9240	Control	HCT-116 colorectal cells
SA340718	A'''-01-9245	Control	HCT-116 colorectal cells
SA340719	A'-02-9242	Control	HCT-116 colorectal cells
SA340720	A'-01-9241	Control	HCT-116 colorectal cells
SA340721	A''-01-9243	Control	HCT-116 colorectal cells
SA340722	A'''-02-9246	Control	HCT-116 colorectal cells
SA340723	A''-02-9244	Control	HCT-116 colorectal cells
SA340724	B'-02-9251	MGB30	HCT-116 colorectal cells
SA340725	B-02-9249	MGB30	HCT-116 colorectal cells
SA340726	B-01-9248	MGB30	HCT-116 colorectal cells
SA340727	B'-01-9250	MGB30	HCT-116 colorectal cells
SA340728	B''-02-9253	MGB30	HCT-116 colorectal cells
SA340729	B'''-02-9255	MGB30	HCT-116 colorectal cells
SA340730	B'''-01-9254	MGB30	HCT-116 colorectal cells
SA340731	B''-01-9252	MGB30	HCT-116 colorectal cells
SA340732	C-01-9257	MGB32	HCT-116 colorectal cells
SA340733	C-02-9258	MGB32	HCT-116 colorectal cells
SA340734	C'-02-9260	MGB32	HCT-116 colorectal cells
SA340735	C''-01-9261	MGB32	HCT-116 colorectal cells
SA340736	C'''-01-9263	MGB32	HCT-116 colorectal cells
SA340737	C'''-02-9264	MGB32	HCT-116 colorectal cells
SA340738	C''-02-9262	MGB32	HCT-116 colorectal cells
SA340739	C'-01-9259	MGB32	HCT-116 colorectal cells

Showing results 1 to 24 of 24

Showing results 1 to 24 of 24

Collection:

Collection ID:	CO003256
Collection Summary:	The HCT-116 human colorectal cancer cells were cultured in Roswell Park Memorial Institute (RPMI) 1640 Medium, supplemented with 10% fetal bovine serum (FBS) from Sigma Aldrich, Germany. The growth media RPMI/FBS was further enriched with a 1% combination of penicillin and streptomycin antibiotics. The cells were cultured in a humidified incubator under conditions of 37°C temperature and 5% CO2.
Sample Type:	Cultured Colerectal cancer cells

Treatment:

Treatment ID:	TR003272
Treatment Summary:	Four biological replicates were established for each treatment condition in T75 cm2 flasks (MGB30, MGB32, and the control group). Each replicate was seeded with approximately 2 × 106 HCT-116 cells and treated individually with either MGB30 or MGB32. Following treatment, the cells were incubated for 24 hours. To ensure consistency, an equal number of cells were uniformly seeded in each T75 cm2 flask for every sample, minimizing the potential impact of variable cell numbers on experimental outcomes. After the incubation period, cells were washed twice with phosphate-buffered saline solution (PBS) and collected through trypsinization. The harvested cells were then pelleted by centrifugation at 1200 rounds per minute (rpm) for 10 minutes at room temperature. Finally, the cells were resuspended in 1 mL of 1× PBS for further analysis. It is essential to note that the cells were maintained under the same conditions throughout the incubation period, and all samples were collected simultaneously.

Treatment ID:

TR003272

Treatment Summary:

Four biological replicates were established for each treatment condition in T75 cm2 flasks (MGB30, MGB32, and the control group). Each replicate was seeded with approximately 2 × 106 HCT-116 cells and treated individually with either MGB30 or MGB32. Following treatment, the cells were incubated for 24 hours. To ensure consistency, an equal number of cells were uniformly seeded in each T75 cm2 flask for every sample, minimizing the potential impact of variable cell numbers on experimental outcomes. After the incubation period, cells were washed twice with phosphate-buffered saline solution (PBS) and collected through trypsinization. The harvested cells were then pelleted by centrifugation at 1200 rounds per minute (rpm) for 10 minutes at room temperature. Finally, the cells were resuspended in 1 mL of 1× PBS for further analysis. It is essential to note that the cells were maintained under the same conditions throughout the incubation period, and all samples were collected simultaneously.

Sample Preparation:

Sampleprep ID:	SP003270
Sampleprep Summary:	We employed a chloroform/methanol extraction protocol to enhance the comprehensiveness of the extracted metabolites. Initially, the samples (comprising cells and buffer) were transferred into Eppendorf tubes and centrifuged at 14,000 rpm for 5 minutes. Subsequently, the buffer was discarded, and the cells were preserved. For each sample, we added a mixture containing a protease inhibitor tablet, 400 µL of it, and 10 mL of lysis buffer. After a 10-minute rest, the samples were transferred to 10 mL tubes, vortexed for 2–4 minutes, and sonicated with a COPLEY probe-sonicator for 30 seconds at a 30% amplifier setting in an ice bath. The samples were then transferred back to Eppendorf tubes and centrifuged for 5 minutes at 14,000 rpm. The resulting supernatant was transferred to another Eppendorf tube, to which we added 400 µL of methanol and 300 µL of chloroform. Following a 30-second vortex and a subsequent 5-minute centrifugation at 14,000 rpm, two layers containing metabolites were obtained. After transferring the upper layer of each sample to glass vials, we added 400 µL of methanol, followed by vortexing and centrifugation. The remaining supernatant was then transferred to the initial glass vials used for the drying step. The dried metabolomics samples were resuspended in 200 µL (0.1% formic acid in water) and injected after filtration into HPLC for analysis by Q-TOF MS.

Sampleprep ID:

SP003270

Sampleprep Summary:

We employed a chloroform/methanol extraction protocol to enhance the comprehensiveness of the extracted metabolites. Initially, the samples (comprising cells and buffer) were transferred into Eppendorf tubes and centrifuged at 14,000 rpm for 5 minutes. Subsequently, the buffer was discarded, and the cells were preserved. For each sample, we added a mixture containing a protease inhibitor tablet, 400 µL of it, and 10 mL of lysis buffer. After a 10-minute rest, the samples were transferred to 10 mL tubes, vortexed for 2–4 minutes, and sonicated with a COPLEY probe-sonicator for 30 seconds at a 30% amplifier setting in an ice bath. The samples were then transferred back to Eppendorf tubes and centrifuged for 5 minutes at 14,000 rpm. The resulting supernatant was transferred to another Eppendorf tube, to which we added 400 µL of methanol and 300 µL of chloroform. Following a 30-second vortex and a subsequent 5-minute centrifugation at 14,000 rpm, two layers containing metabolites were obtained. After transferring the upper layer of each sample to glass vials, we added 400 µL of methanol, followed by vortexing and centrifugation. The remaining supernatant was then transferred to the initial glass vials used for the drying step. The dried metabolomics samples were resuspended in 200 µL (0.1% formic acid in water) and injected after filtration into HPLC for analysis by Q-TOF MS.

Combined analysis:

Analysis ID	AN005162
Analysis type	MS
Chromatography type	Reversed phase
Chromatography system	Bruker Elute
Column	Hamilton Intensity Solo 2 C18 (100 x 2.1mm, 1.8um)
MS Type	ESI
MS instrument type	QTOF
MS instrument name	Bruker timsTOF
Ion Mode	POSITIVE
Units	AU

Chromatography:

Chromatography ID:	CH003907
Chromatography Summary:	The detection of metabolites involved the use of an Elute UHPLC system, coupled with a Q-TOF Mass Spectrometer (Bruker, Bremen, Germany). The system included Elute HPG 1300 pumps, an Elute Autosampler (Bruker, Bremen, Germany), and a Hamilton® Intensity Solo 2 C18 column (100 mm × 2.1 mm, 1.8 um beads) for reversed-phase chromatography. Solvent A consisted of 0.1% FA in LC grade water, while solvent B comprised 0.1% FA in ACN. Metabolites extracts underwent duplicate analyses. The column temperature was maintained at 35 °C, and samples were injected twice with a 10 µL volume. A 30-minute gradient elution involved using 1% ACN for 2 minutes, followed by a ramp to 99% ACN within 15 minutes. Subsequently, 99% ACN was held for 3 minutes, and re-equilibration with 1% ACN was carried out for 10 minutes. The flow rate was set at 0.25 mL/min for 20 minutes, 0.35 mL/min for 8.3 minutes, and 0.25 mL/min for 1.7 minutes. For metabolomics, a 10 µL aliquot from each sample was combined to create the quality control (QC) sample.
Instrument Name:	Bruker Elute
Column Name:	Hamilton Intensity Solo 2 C18 (100 x 2.1mm, 1.8um)
Column Temperature:	35 ◦C
Flow Gradient:	A 30-minute gradient elution involved using 1% ACN for 2 minutes, followed by a ramp to 99% ACN within 15 minutes. Subsequently, 99% ACN was held for 3 minutes, and re-equilibration with 1% ACN was carried out for 10 minutes.
Flow Rate:	0.25 mL/min for 20 minutes, 0.35 mL/min for 8.3 minutes, and 0.25 mL/min for 1.7 minutes
Solvent A:	100% water; 0.1% Formic Acid
Solvent B:	100% ACN; 0.1% Formic Acid
Chromatography Type:	Reversed phase

MS:

MS ID:	MS004898
Analysis ID:	AN005162
Instrument Name:	Bruker timsTOF
Instrument Type:	QTOF
MS Type:	ESI
MS Comments:	The data underwent processing through MetaboScape ® 4.0 software (Bruker, Bremen, Germany). In the T-ReX 2D/3D workflow, specific settings were applied for molecular feature detection, including a minimum intensity threshold of 1,000 counts, a maximum peak duration of seven spectra, and a maximum peak area. Mass recalibration was conducted within a retention time range of 0 to 0.3 minutes, and only features present in at least six of the 24 samples per cell type were considered. The MS/MS import method was set to be averaged. Bucketing parameters for the data were defined with a retention duration of 0.3 to 25 minutes and a mass range of 50 to 1,000 m/z. Metabolite identification involved comparing combined MS/MS, precursor m/z values, and isotope pattern scores to the human metabolome database (HMDB) 4.0. The annotation quality score (AQ score) played a crucial role in selecting the best matching feature when multiple features corresponded to a given database entry. For further analysis, metabolite data were saved as CSV files and integrated into the comprehensive metabolomics platform MetaboAnalyst 6.0 software (https://www.metaboanalyst.ca, Accessed: 1st of February 2024). The identification of significantly altered metabolites in the group treated with MGB compounds, in comparison to the standard control group, was accomplished using a two-tailed independent Student's t-test. This process led to the creation of a volcano plot, visually illustrating the statistical significance and fold change (p<0.05*, FC=1.25), thereby highlighting the alteration of cellular metabolites under each condition. Furthermore, each medication underwent one-way analysis of variance (ANOVA) to pinpoint significantly different metabolites from the control group. Additionally, box plots illustrating statistically altered cellular metabolites were generated. Multiple groups were compared using ANOVA, with the same significance threshold set at p-value < 0.05 and fold change = 1.25. MetaboAnalyst 6.0 software was also utilized for enrichment analysis, joint pathway analysis, and Partial Least Squares-Discriminant Analysis (PLS-DA) to compare the two groups. False discovery rate was implemented to eliminate false positives and address multiple hypothesis testing (FDR).
Ion Mode:	POSITIVE