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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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.