Embryo‐derived teratoma in vitro biological system reveals antitumor and embryotoxic activity of valproate

Abstract

Antiepileptic/teratogen valproate (VPA) is a histone deacetylase inhibitor/ epigenetic drug proposed for the antitumor therapy where it is generally crucial to target poorly or undifferentiated cells to prevent a recurrence. Transplanted rodent gastrulating embryos-proper (primitive streak and three germ layers) are the source of teratoma/teratocarcinoma tumors. Human primitive-streak remnants develop sacrococcygeal teratomas that may recur even when benign (well differentiated). To screen for unknown VPA impact on teratoma-type tumors, we used original 2-week embryoderived teratoma in vitro biological system completed by a spent media metabolome analysis. Gastrulating 9.5-day-old rat embryos-proper were cultivated in Eagle's minimal essential medium (MEM) with 50% rat serum (controls) or with the addition of 2 mM VPA. Spent media metabolomes were analyzed by FTIR. Compared to controls, VPA acetylated histones; significantly diminished overall teratoma growth, impaired survival, increased the apoptotic index, and decreased proliferation index and incidence of differentiated tissues (e.g., neural tissue). Control teratomas continued to grow and differentiate for 14 days in isotransplants in vivo, but in vitro VPA-treated teratomas resorbed. Principal component analysis of FTIR results showed that spent media metabolomes formed well-separated clusters reflecting the treatment and day of cultivation. In metabolomes of VPA-treated teratomas, we found elevation of previously described histone acetylation biomarkers [amide I a-helix and A(CH 3)/A(CH 2)]) with apoptotic biomarkers within the amide I region for b-sheets, and unordered and CH 2 vibrations of lipids. VPA may be proposed for therapy of the undifferentiated component of teratoma tumors and this biological system Abbreviations ASD, autism spectrum disorder; EC, embryonal carcinoma; GTS, growing teratoma syndrome; HDACi, histone deacetylase inhibitor; MEM, minimal essential medium; PCA, principal component analysis; PCNA, proliferating cell nuclear antigen; RMSEC, root mean square error of calibration; RMSECV, root mean square error of cross-validation; SAHA, suberoylanilidehydroxamic acid; VPA, valproate; WEC, whole rat embryo.

Teratoma Growth Retardation by HDACi Treatment of the Tumor Embryonal Source

“…A rodent teratoma in vitro system has been used to assess the impacts of various biologically active substances and therapeutics on the developmental processes important for teratoma tumorigenesis [ 15 , 16 ]. As studies show that in humans, both immature and mature teratoma may possess malignant potential [ 50 ], we performed for the first time a comprehensive histopathological analysis and an analysis of early developmental markers during the active growth phase of the mouse teratoma system in vitro.…”

“…It was proposed that the ectopic environment, through epigenetic mechanisms, induces a blockage in the differentiation and malignant transformation of the mammalian embryonal cells, which should be more important for the development of such “developmental” tumors than mutations. Additionally, the transplanted rat egg-cylinders produced mostly benign tumors cultivated in vitro, which were used to test the effects of various agents or culture conditions on the growth, differentiation and apoptosis of the resulting experimental teratoma [ 15 , 16 ]. In this research, for the first time, we treated the normal mouse embryonal tissue with epigenetic drugs to investigate the impact of histone acetylation on the source of the teratoma.…”

“…It was proposed that the ectopic environment through epigenetic mechanisms induces a blockage in differentiation and malignant transformation of the mammalian embryonal cells, and that this epigenetic regulation is more important for the development of these “developmental” tumors than mutations. Importantly, the model has been used to test the effects of various agents or culture conditions on the growth, differentiation and apoptosis of resulting experimental teratoma in vitro [ 7 , 15 , 16 ].…”

“…VPA is used clinically as an anti-epileptic agent and for the treatment of neurological disorders [ 15 , 34 , 35 , 36 ]. It inhibits class I and IIa HDACs, which increases histone acetylation and protein synthesis in cells, preventing their proliferation, and ultimately resulting in tumor cell differentiation [ 20 , 34 ].…”

“…It inhibits class I and IIa HDACs, which increases histone acetylation and protein synthesis in cells, preventing their proliferation, and ultimately resulting in tumor cell differentiation [ 20 , 34 ]. This, combined with its effect on cellular reprogramming, senescence and the promotion of apoptosis, led to its use in breast cancer adjunct therapy, and multiple clinical trials for cancer therapy [ 15 , 34 , 35 , 37 ]. The anti-tumor effect of VPA has been documented in TGCT cell lines, including choriocarcinoma [ 38 ].…”