Summary of Study ST002731

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 PR001695. The data can be accessed directly via it's Project DOI: 10.21228/M8MB0X 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	ST002731
Study Title	Untargeted Multi-Omics of LNCaP Cell-line Treated with Novel DNA Minor Groove Binder and /or Doxorubicin Using Mass-Spectrometry
Study Summary	Prostate cancer poses a significant health risk, ranking as the second most common cancer among men in the United States. However, the effectiveness of current anti-prostate cancer drugs is limited due to increasing drug resistance and side effects. Consequently, there is a pressing need to develop new compounds and identify novel drug targets that can surpass these limitations. Due to their targeted mechanism, DNA minor groove binders (MGBs) are becoming more popular as a relatively safe and effective alternative. In our research, we employed multi-omics techniques to investigate the mechanism of action of a novel MGB compound (MGB4) through LC-MS/MS-based untargeted metabolomics combined with discovery proteomics analysis performed on LNCaP cells, which were treated with MGB4, doxorubicin, or a combination of both compounds. Through a one-way ANOVA test with a significance level of p-value < 0.05, we identified 99 metabolites and 1143 proteins associated with the treatments. Our findings indicate that treating LNCaP cells with doxorubicin or the MGB4 lead compound yielded similar effects, albeit not identical, on the cells. Both compounds deactivated the translation pathway in the cells. Furthermore, we observed alterations in sphingolipid and amino acid metabolic pathways, potentially contributing to the suppression of prostate cancer cell proliferation and division. Additionally, doxorubicin and combined treatments resulted in reduced metabolism of spermine and spermidine, likely stemming from decreased protein synthesis of key enzymes involved in their pathways. Moreover, the combined treatment exhibited a synergistic interaction between the two compounds, leading to altered purine metabolism and a more pronounced reduction in metabolite abundance compared to individual treatments. Overall, our study demonstrates the robustness of the multi-omics approach in elucidating the mechanism of action of promising drug candidates. It also suggests that MGB4 shows potential as a candidate for prostate cancer treatment.
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	2023-06-07
Raw Data Available	Yes
Raw Data File Type(s)	d
Analysis Type Detail	LC-MS
Release Date	2023-06-25
Release Version	1

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Project:

Project ID:	PR001695
Project DOI:	doi: 10.21228/M8MB0X
Project Title:	Untargeted Multi-Omics of LNCaP Cell-line Treated with Novel DNA Minor Groove Binder and /or Doxorubicin Using Mass-Spectrometry
Project Summary:	Prostate cancer poses a significant health risk, ranking as the second most common cancer among men in the United States. However, the effectiveness of current anti-prostate cancer drugs is limited due to increasing drug resistance and side effects. Consequently, there is a pressing need to develop new compounds and identify novel drug targets that can surpass these limitations. Due to their targeted mechanism, DNA minor groove binders (MGBs) are becoming more popular as a relatively safe and effective alternative. In our research, we employed multi-omics techniques to investigate the mechanism of action of a novel MGB compound (MGB4) through LC-MS/MS-based untargeted metabolomics combined with discovery proteomics analysis performed on LNCaP cells, which were treated with MGB4, doxorubicin, or a combination of both compounds. Through a one-way ANOVA test with a significance level of p-value < 0.05, we identified 99 metabolites and 1143 proteins associated with the treatments. Our findings indicate that treating LNCaP cells with doxorubicin or the MGB4 lead compound yielded similar effects, albeit not identical, on the cells. Both compounds deactivated the translation pathway in the cells. Furthermore, we observed alterations in sphingolipid and amino acid metabolic pathways, potentially contributing to the suppression of prostate cancer cell proliferation and division. Additionally, doxorubicin and combined treatments resulted in reduced metabolism of spermine and spermidine, likely stemming from decreased protein synthesis of key enzymes involved in their pathways. Moreover, the combined treatment exhibited a synergistic interaction between the two compounds, leading to altered purine metabolism and a more pronounced reduction in metabolite abundance compared to individual treatments. Overall, our study demonstrates the robustness of the multi-omics approach in elucidating the mechanism of action of promising drug candidates. It also suggests that MGB4 shows potential as a candidate for prostate cancer treatment.
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:	SU002837
Subject Type:	Human
Subject Species:	Homo sapiens
Taxonomy ID:	9606
Gender:	Male

Factors:

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

mb_sample_id	local_sample_id	Group
SA274768	4''-01-8411	Combined
SA274769	4'-02-8410	Combined
SA274770	4-02-8408	Combined
SA274771	4-01-8407	Combined
SA274772	4'-01-8409	Combined
SA274773	4''-02-8412	Combined
SA274774	1-01-8388	Control
SA274775	1-02-8389	Control
SA274776	1'-01-8390	Control
SA274777	1'-02-8391	Control
SA274778	1''-02-8393	Control
SA274779	1''-01-8392	Control
SA274780	3''-02-8406	Dox
SA274781	3''-01-8405	Dox
SA274782	3-01-8401	Dox
SA274783	3'-02-8404	Dox
SA274784	3-02-8402	Dox
SA274785	3'-01-8403	Dox
SA274786	2-02-8395	MGB4
SA274787	2-01-8394	MGB4
SA274788	2'-01-8396	MGB4
SA274789	2''-01-8398	MGB4
SA274790	2''-02-8399	MGB4
SA274791	2'-02-8397	MGB4

Showing results 1 to 24 of 24

Showing results 1 to 24 of 24

Collection:

Collection ID:	CO002830
Collection Summary:	Human prostate cancer cells LNCaP were grown in Roswell Park Memorial Institute (RPMI) 1640 Medium (Sigma Aldrich, Germany) supplemented with 10% fetal bovine serum (FBS) (Sigma Aldrich, Germany). A combination of 1 % penicillin and streptomycin antibiotics was added to RPMI/FBS growth media. Cells were maintained in humidified incubator with conditions of 37◦C temperature and 5 % CO2.
Sample Type:	LNCaP cells
Storage Conditions:	-80℃

Treatment:

Treatment ID:	TR002846
Treatment Summary:	Three biological replicates were prepared for each treatment condition in T75 cm2 ﬂasks (MGB4 and/or Doxorubicin and control group), and each replicate was seeded with approximately 2 × 106 LNCaP cells, then separately treated with MGB4 (2.15 μM) and/or Doxorubicin (1.18 μM) and incubated for 24 h. A uniform number of cells was seeded in T75 cm2 ﬂask for each sample to avoid the effect of variable cell number on the outcome of the experiment. After incubation, cells were washed twice with phosphate-buffered saline solution (PBS) and then collected by trypsinization. Finally, cells were collected as pellets by centrifugation at 1200 rounds per minute (rpm) for 10 min at room temperature and resuspended in 1 mL 1× PBS for further analysis. Cells were maintained in the same conditions during incubation, and all samples were collected simultaneously (Figure 5).

Treatment ID:

TR002846

Treatment Summary:

Three biological replicates were prepared for each treatment condition in T75 cm2 ﬂasks (MGB4 and/or Doxorubicin and control group), and each replicate was seeded with approximately 2 × 106 LNCaP cells, then separately treated with MGB4 (2.15 μM) and/or Doxorubicin (1.18 μM) and incubated for 24 h. A uniform number of cells was seeded in T75 cm2 ﬂask for each sample to avoid the effect of variable cell number on the outcome of the experiment. After incubation, cells were washed twice with phosphate-buffered saline solution (PBS) and then collected by trypsinization. Finally, cells were collected as pellets by centrifugation at 1200 rounds per minute (rpm) for 10 min at room temperature and resuspended in 1 mL 1× PBS for further analysis. Cells were maintained in the same conditions during incubation, and all samples were collected simultaneously (Figure 5).

Sample Preparation:

Sampleprep ID:	SP002843
Sampleprep Summary:	we used chloroform/methanol extraction protocol to increase the coverage of the extracted metabolites. At first, the samples (cells and buffer) were transferred into Eppendorf tubes then centrifuged at 14000 rpm for 5 min. Afterward, the buffer was discarded, and the cells were preserved. To each sample, 400 µL of the mixture containing one protease inhibitor tablet and 10 mL of lysis buffer was added. Following rest for 10 minutes, samples were transferred to 10 mL tubes, vortexed for 2–4 minutes, and sonicated with a COPLEY probe-sonicator (QSONICA SONICATOR, USA) for 30 seconds while utilizing a 30 % amplifier in an ice bath. The samples were then transferred to Eppendorf tubes and centrifuged for 5 minutes at 14000 rpm. The supernatant was then transferred to another Eppendorf, and 400 µL of methanol and 300 µL of chloroform were added. Following that, the samples were vortexed for 30 seconds and centrifuged for 5 minutes at 14000 rpm. After that, two metabolite-containing layers are obtained, after transferring the upper layer of each sample to glass vials, 400 µl of methanol was added, followed by vertexing and centrifugation. The remaining supernatant was transferred to the same glass vials used before for the drying step, with the remaining protein pellets being air-dried for proteomics. A dried metabolomics sample was resuspended in 200 µL (0.1% formic acid in water) and injected into HPLC to be analysed by Q-TOF MS.

Sampleprep ID:

SP002843

Sampleprep Summary:

we used chloroform/methanol extraction protocol to increase the coverage of the extracted metabolites. At first, the samples (cells and buffer) were transferred into Eppendorf tubes then centrifuged at 14000 rpm for 5 min. Afterward, the buffer was discarded, and the cells were preserved. To each sample, 400 µL of the mixture containing one protease inhibitor tablet and 10 mL of lysis buffer was added. Following rest for 10 minutes, samples were transferred to 10 mL tubes, vortexed for 2–4 minutes, and sonicated with a COPLEY probe-sonicator (QSONICA SONICATOR, USA) for 30 seconds while utilizing a 30 % amplifier in an ice bath. The samples were then transferred to Eppendorf tubes and centrifuged for 5 minutes at 14000 rpm. The supernatant was then transferred to another Eppendorf, and 400 µL of methanol and 300 µL of chloroform were added. Following that, the samples were vortexed for 30 seconds and centrifuged for 5 minutes at 14000 rpm. After that, two metabolite-containing layers are obtained, after transferring the upper layer of each sample to glass vials, 400 µl of methanol was added, followed by vertexing and centrifugation. The remaining supernatant was transferred to the same glass vials used before for the drying step, with the remaining protein pellets being air-dried for proteomics. A dried metabolomics sample was resuspended in 200 µL (0.1% formic acid in water) and injected into HPLC to be analysed by Q-TOF MS.

Combined analysis:

Analysis ID	AN004428
Analysis type	MS
Chromatography type	Reversed phase
Chromatography system	Bruker Elute
Column	Hamilton Intensity Solo 2 C18 (100 x 2.1 mm, 1.8 um)
MS Type	ESI
MS instrument type	QTOF
MS instrument name	Bruker timsTOF
Ion Mode	POSITIVE
Units	AU

Chromatography:

Chromatography ID:	CH003327
Chromatography Summary:	Samples were chromatographically separated by inline reversed-phase chromatography using the Elute HPG 1300 pumps and Elute Autosampler (Bruker, Darmstadt, Germany) with solvent A 0.1% FA in HPLC grade water and solvent B 0.1% FA in ACN. A Hamilton Intensity Solo 2 C18 column (100 mm x 2.1 mm, 1.8µm beads) was maintained at 35C. For metabolomics, 10 µL was injected twice for each sample and eluted using a 30-minute gradient as follows: 1% ACN was held for 2 minutes, ramping to 99% ACN over 15 minutes, held at 99% ACN for 3 minutes before re-equilibrating to 1% ACN for 10 minutes. Flow rates were 250 µL/min for elution and 350 µL/min for re-equilibration.
Instrument Name:	Bruker Elute
Column Name:	Hamilton Intensity Solo 2 C18 (100 x 2.1 mm, 1.8 um)
Column Temperature:	35
Flow Gradient:	1%B to 99%B in 15 min
Flow Rate:	250 uL/min
Solvent A:	100% water; 0.1% formic acid
Solvent B:	100% acetonitrile; 0.1% formic acid
Chromatography Type:	Reversed phase

MS:

MS ID:	MS004175
Analysis ID:	AN004428
Instrument Name:	Bruker timsTOF
Instrument Type:	QTOF
MS Type:	ESI
MS Comments:	The MS analysis was performed using a TimsTOF (Bruker, Darmstadt, Germany) with Apollo II electrospray ionization (ESI) source. The drying gas was set to flow at 10 L/min and the drying temperature to 220C and the nebulizer pressure to 2.2 bar. The capillary voltage was 4500 V and the end plate offset 500V. For metabolomics the scan range was 20-1300 m/z. The collision energy was set to 20 eV, the cycle time to 0.5 seconds with a relative minimum intensity threshold of 400 counts per thousand and target intensity of 20,000. Sodium formate was injected as an external calibrant in the first 0.3 minutes of each LC-MS/MS run. MetaboScape 4.0 software was used for metabolite processing and statistical analysis (Bruker Daltonics). The following parameters for molecular feature identification and "bucketing" were set in the T-ReX 2D/3D workflow: For peak detection, a minimum intensity threshold of 1,000 counts is required, as well as a minimum peak duration of 7 spectra, with feature quantification determine using peak area. The file masses were recalibrated based on the external calibrant injected between 0-0.3 min.
Ion Mode:	POSITIVE