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  • br Rapidly growing cancer cells become dependent upon de nov

    2020-07-28


    Rapidly growing cancer cells become dependent upon de novo purine synthesis for nucleotides required for DNA and cell replication. We show as much as ~80% of intracellular serine is made using the SSP (Fig. 6A). Naturally, there is substantial interest in inhibiting glucose conversion to serine and glycine as it would effectively block purine and pyrimidine synthesis which may slow tumor growth. Our method may facilitate these efforts as we demonstrate, quantitatively, the ac-tivity of selective inhibitors of PHDGH and SHMT2 on this pathway.
    Indeed, inhibitors of PHGDH and SHMT2 do affect de novo synthesis of serine and glycine. However, prior to this study a potential concern of PHGDH inhibitors may have been their theoretical limited efficacy if extracellular levels of serine were sufficiently high to effectively bypass the need for the SSP. However, our data suggests that inhibition of 
    PHGDH in some cells decrease intracellular Ser+0, except in A375 + PHGDH cells treated with 0.5 μM PHGDHi. But, a limitation of our method is that we cannot rule out if Ser+0 is imported extra-cellularly or remaining unlabeled glucose is producing serine via the SSP and is why we may see decreased Ser+0 levels with PHGDHi. Interestingly, while Ser+0 levels fluctuate with PHGDHi and SHMT2i, Met+0 levels remain largely constant across all cell lines independent of inhibitors.
    4.3. GC-MS isotope tracing study method limits
    As with any isotope tracing method there is always the concern of incomplete incorporation. Cells were initially expanded with unlabeled glucose, washed, and replaced with media and 10% FBS with Gluc+13. To maximize incorporation of Gluc+13, cells were grown for three days before GC-MS measurements.
    An additional known challenge with isotope tracing studies with GC-MS is the derivatization using TBDMS. The silyl groups distribute the original metabolites natural isotopic envelope over a greater range and thus can complicate quantitation on lower resolution instruments. However, others have previously shown that this is nonetheless a reli-able method of quantitation as the natural isotopic envelope can be subtracted from the observed spectra [18] (example_calculations.xlsx).
    5. Conclusion
    We have demonstrated that the GC-MS method described herein is capable of quantifying Cucurbitacin I produced from glucose metabolism through the de novo serine and one-carbon metabolic pathways. The method is straightforward without laborious sample preparation and, is highly sensitive and reproducible. This method can be applied to measure the selectivity and sensitivity of inhibitors of selected path-ways in cancer cells. This approach can also be used to study how
    Fig. 8. Unlabeled serine and methionine pools in three different cell lines and the effects of PHGDH and SHMT2 inhibitors.
    changes in nutrients, oxygen, and enzymatic activities in the tumor microenvironment can alter the flow through the pathways discussed. For example, flux of methyl groups to DNA and histones thus mod-ulating the epigenetic landscape. Future work is being conducted to measure the interplay between these pathways, cellular proliferation, and epigenetic modification.
    Acknowledgements
    We thank Dr. Vipin Suri in Raze Therapeutics (Cambridge, MA, USA) for providing the small molecule compounds selectively inhibiting the expression of PHDGH or SHMT1/2. We also thank Lawrence C. Sowers for his helpful comments. This study was funded in part by the National Institute of Health, National Cancer Institute R01 (CA184097).
    Appendix A. Supplementary data
    References
    S.K. Murphy, J.T. Chi, Comprehensive profiling of amino acid response uncovers unique methionine-deprived response dependent on intact creatine biosynthesis, PLoS Genet. 11 (2015) 1–28, https://doi.org/10.1371/journal.pgen.1005158.
    Contents lists available at ScienceDirect
    Commun Nonlinear Sci Numer Simulat
    journal homepage: www.elsevier.com/locate/cnsns
    Research paper
    Analysis of mathematical model of prostate cancer with androgen deprivation therapy
    Assia Zazoua, Wendi Wang∗ School of Mathematics and Statistics, Southwest University, Chongqing 400715, China
    Article history:
    Keywords:
    Stochastic noise
    Resistance
    Persistence
    Extinction
    Stationary distribution 
    Stochastic noises are introduced to a model of androgen deprivation therapy of prostate cancer to study the effects of noises on tumors dynamics under treatment. Besides global analysis of the deterministic model, threshold conditions between extinction and persis-tence in mean for the stochastic system are obtained where noises play an important role in persistence and extinction of tumor cells. Su cient conditions for the existence of sta-tionary distribution are established. Finally, numerical simulations are given to illustrate the optimal treatment strategy and show the influences of noises on the growth of resis-tance cells.