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

[email protected]

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).