ᐊ P8-47
Open Readings 2021 • P8-48

THE EFFECT OF INDOLE-CONTAINING STEROIDS ON THE MITOCHONDRIAL MEMBRANE POTENTIAL OF C6 GLIOMA CELLS

Valeriya Klopava3, Jan Panada1, 2, Tatsiana Kulahava3, 4, Nina Frolova1, Yaroslav Faletrov1, 2, Vladimir Shkumatov1, 2

1 Research Institute for Physical Chemical Problems of the Belarusian State University, Minsk, Belarus

2 Chemistry faculty of Belarusian State University, Minsk, Belarus

3 Department of Biophysics, Physics faculty of Belarusian State University, Minsk, Belarus

4 Institute for Nuclear Problems of the Belarusian State University, Minsk, Belarus

Mitochondria are key cellular organelles that play an essential role in various functions such as regulation of cellular metabolism, redox signaling, ionic homeostasis, and cell death [1]. Since tumorigenesis requires flexibility for the tumors to adapt to cellular and environmental alterations, it is not surprising that mitochondria serve a key role in this process [2].

Previously, a series of indole-containing steroids were synthesized: N-(2-(3-indolyl)ethyl)-3β-hydroxyandrost-5-en-17β-amine (IS-1), N-(2-(3-indolyl)ethyl)-3-hydroxyestra-1,3,5(10)-trien-17β-amine (IS-2) and two 20-epimers of N-(2-(3-indolyl)ethyl)-3β-hydroxypregn-5-en-20-amine (IS-3). These compounds were shown to decrease C6 glioma cell proliferation in 10 μM concentration [3], as abiraterone acetate (Abi) anticancer agent did. To examine indole-containing steroid intracellular effects, we investigated the mitochondrial membrane potential (MMP) of C6 glioma cells.

Cells were cultured in DMEM/F-12 medium at 37°C in a humidified 5% CO2 and 95% air atmosphere. On the third day of growth cells in suspension in HEPES-buffer were incubated with tetraethylbenzimidazolylcarbocyanine iodide (JC-1, 2 μM) for 30 min at 37°C, membrane potential was measured by by spectrofluorimetry. Mitochondrial inhibitors Rotenone (Rot) or Antimicina A (Ana) were added after incubation with JC-1.

We found that Abi at 10 μM decreases the MMP by $62\pm15\%$, whereas IS-3R does so by $40\pm25\%$ (Fig.1 A). IS-1, IS-2, and IS-3S did not affect the MMP. Simultaneous treatment with complex I inhibitor rotenone (Rot) and Abi, IS-1 or IS-3R led to an additional decrease of MMP (Fig.1 B). IS-1 and IS-3R lead to the minor recovery of MMP after inhibition of the third complex by Ana (Fig.1 C).

Figure 1
Fig. 1. Typical kinetics of MMP changes under the action of IS-3R and complex I (Rot) and complex III (Ana) inhibitors

Although IS-1 is an effective inhibitor of glioma cell proliferation [3], we did not detect it to influence the MMP, contrary to abiraterone, a known apoptosis inducer [4]. The observed effect of the indolesteroid IS-3R on MMP could be related to its interaction with mitochondria on complex III level. Further investigations are underway to examine if apoptosis is involved in indolesteroids' effect on C6 glioma proliferation.

[1] Lalita Guntuku, V, G.M. Naidu, Mitochondrial Dysfunction in Gliomas: Pharmacotherapeutic Potential of Natural Compounds, Curr Neuropharmacol, 14(6): 567-583 (2016).

[2] Altieri D.C., Mitochondria on the move: Emerging paradigms of organelle trafficking in tumour plasticity and metastasis, Br J Cancer. 117:301-305. (2017).

[3] Panada J., Klopava V. et al., New 3β-hydroxysteroid-indolamine conjugates: Design, synthesis and inhibition of C6 glioma cell proliferation, Steroids, 164:108728 (2020).

[4] Lin H.Y., Ko C.Y. et al., CYP17A1 maintains the survival of glioblastomas by regulating SAR-1 mediatedendoplasmic reticulum health and redox homeostasis, Cancers (Basel), 11(9): 1378 (2019).